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Molecular mechanisms underlying the bi-directional control of presynaptic homeostatic plasticity

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Molecular mechanisms underlying the bi-directional control of presynaptic homeostatic plasticity

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Content Molecular mechanisms underlying the bi-directional
control of presynaptic homeostatic plasticity
by
Xiling Li
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
NEUROSCIENCE
May 2020
© Copyright 2020 Xiing Li
ii
DEDICATION
To my beloved grandfather, Yunsen Li
who always supported me and believed in me.
iii
ACKNOWLEDGMENTS
I wish to express my sincere gratitude for all the support that I have received for the past
six years. I feel incredibly fortunate to be able to attend USC for my graduate study and join
Dickman’s lab. I would like to give my sincerest thanks to my advisor, Dr. Dion Dickman, for
giving me excellent mentorship, inspiration in science, and always being supportive. And special
thanks for the understanding and caring when I have a broken leg. Also, I would like to thank my
committee members, and all the excellent guidance and suggestions from Dr. Chien-Ping Ko,
Dr. Karen Chang, Dr. Bruce Herring, Dr. Robert Chow, and Dr. Andrew Hires.
I feel extremely lucky to meet all the current and former lab members in Dickman’s lab,
that fulfill my six years with a lot of fun and memorable experiences. Special thanks to Pragya
Goel for performing all the quantitative imaging in chapter 3, and for many other collaborations
in my graduate research. Many thanks to another major collaborator in the lab, Koto Kikuma,
who is very collaborative and easy to talk to; Xun Chen, my elder brother, who has the same
hometown and attended the same college as I do, and he is very knowledgeable and
trustworthy; Beril Kiragasi, for encouraging me ‘keep pushing’ and good life attitude; Yifu Han,
who is always generous and ‘hardcore in electronics’; Jerry Chien, who is like my younger
brother, very energetic and also very caring; Sarah Perry, for being patient with me and
teaching me genetics; Zihan Sun, as a undergrad, who did a great job contributing to my last
project in chapter 4; Catherine Chen, for helping me on the staining and quantification in chapter
2; Nancy Tran; Manisha Sajnani… Thank you all for making my best graduate student
experience.
Many thanks to my loving mom, Ying Zheng and dad, Bin Li, who love me the most in
this world; my boyfriend, Biao He, who understands me the most and always supports my work
and life; Jing Zou, my best roommate, who saved me from the ground (and put teeth paste on
iv
my wound) when I broke my leg, and she is very passionate about science and always inspiring
me on new ideas… tons of thanks to all my friends and family. And in the end, I would like to
thank my former mentor, Jianyuan Sun, who inspired me got fascinated by the miracles in
synapses and inspired me to pursue my life career on understanding the fundamental
mechanisms that driving our everyday life.
v
TABLE OF CONTENTS
Dedication………………………………………………………………………... ii
Acknowledgments………………………………………………………………. iii
Table of Contents……………………………………………………………….. v
List of Tables………………..………………………………………………….. vii
List of Figures ………………………………………………………………..... viii
Abstract………………………………………………………………….……...... x
Chapter 1: General introduction of homeostatic plasticity………….…. 1
1.1 Homeostatic signaling stabilizes neural function………………………... 1
1.2 Homeostatic regulations of postsynaptic receptor scaling and intrinsic
excitability……………………………………………………………………....... 2
1.3 Presynaptic homeostatic potentiation of neurotransmitter release……. 3
1.4 Presynaptic homeostatic depression in neurotransmitter release…..… 9
1.5 Glutamate homeostasis and excitotoxicity…………………………….... 10
Chapter 2: Synapse-specific and compartmentalized expression of
presynaptic homeostatic potentiation
2.1 Abstract…………………………………………..……………………....… 12
2.2 Introduction………………………………………………………………… 13
2.3 Results……………………………………………………………………... 16
2.4 Discussion……………………………………………………………...….. 25
2.5 Materials and Methods………………………………………..………….. 31
vi
Chapter 3: A Glutamate Homeostat Controls the Presynaptic
Inhibition of Neurotransmitter Release
3.1 Abstract………………………………………..………………………….... 50
3.2 Introduction………………………………………………………………… 51
3.3 Results……………………………………………………………………... 54
3.4 Discussion……………………………………………………………...….. 64
3.5 Materials and Methods………………………………………..………….. 68
Chapter 4: Autocrine inhibition by the Glutamate-Gated Chloride
channel controls presynaptic homeostatic depression
4.1 Abstract…………………………………………..………………………… 91
4.2 Introduction………………………………………………………………… 92
4.3 Results……………………………………………………………………... 95
4.4 Discussion……………………………………………………………....... 104
4.5 Materials and Methods………………………………………..……….... 107
Chapter 5: General Conclusion and Discussion……………………….123
References…………………………………………………………………... 126
vii
LIST OF TABLES
Chapter 2
Supplementary Table 1: Absolute values for all data and additional statistics. 35
Chapter 3
Supplementary Table 1: Absolute values for normalized data and additional statistics. 73
viii
LIST OF FIGURES
Chapter 2
Figure 1: GluRIIA knock down phenocopies GluRIIA mutants. 40
Figure 2: GluRIIA-containing receptors can be knocked down specifically on muscle 6 using
M6>Gal4. 41
Figure 3: Presynaptic homeostatic potentiation can be induced and expressed exclusively at
synapses innervating muscle 6. 42
Figure 4: PHP can be induced and expressed acutely on muscle 7 following chronic GluRIIA
knock down on M6. 43
Figure 5: Homeostatic modulation of the readily releasable vesicle pool is restricted to synapses
innervating muscle 6 at M6>GluRIIA
RNAi
NMJs. 44
Figure 6: The number of functional release sites are specifically enhanced in synapses
innervating muscle 6 at M6>GluRIIA
RNAi
NMJs. 45
Figure 7: Compartmentalized changes in CaMKII activity at postsynaptic densities of
M6>GluRIIA
RNAi
NMJs. 46
Figure 8: Overexpression of constitutively active CaMKII inhibits retrograde PHP signaling at
M6>GluRIIA
RNAi
NMJs. 47
Supplemental Figure 1: Synaptic growth is mildly reduced at NMJs of GluRIIA mutants and
G14>GluRIIA
RNAi
. 48
Supplemental Figure 2: Biased Gal-4 expression on muscle 6 does not alter synaptic growth or
function. 49
Chapter 3
Figure 1: Presynaptic homeostatic potentiation and depression can be induced, expressed, and
balanced. 78
Figure 2: Conventional mechanisms are required for the acute expression of PHP in vGlut-OE.
79
Figure3: Characterization of Ca
2+
cooperativity, failure analysis, and readily releasable vesicle
pools in GluRIIA+vGlut -OE. 80
Figure 4: BRP and Cac abundance at active zones are enhanced in GluRIIA mutants and are
unchanged in vGlut-OE at Ib boutons. 82
Figure 5: In vivo Ca
2+
imaging reveals both Ib and Is boutons express PHD. 83
ix
Figure 6: pCaMKII levels are reduced specifically at Ib postsynaptic densities in GluRIIA
mutants and unchanged in vGlut-OE. 84
Figure 7: Enhanced quantal size triggered by postsynaptic overexpression of GluRIIA does not
induce or modulate PHD expression. 85
Supplemental Figure 1: GluRIIA-containing receptors are absent and vGlut intensity is
increased in GluRIIA+vGlut-OE, related to Figure 1. 86
Supplemental Figure 2: The total releasable vesicle pool is unchanged in vGlut-OE, related to
Figure 3. 87
Supplemental Figure 3: vGlut-OE induces a selective increase in Is bouton number, related to
Figure 4. 88
Supplemental Figure 4: GluRIIA overexpression does not impact NMJ growth or structure,
related to Figure 7. 89
Supplemental Figure 5: Failure to homeostatically tune presynaptic glutamate release at
GluRIIA-OE NMJs, related to Figure 7. 90
Chapter 4
Figure 1: A screen of Drosophila glutamate receptors identifies the Glutamate-Gated Chloride
Channel to be necessary for PHD. 114
Figure 2: The Glutamate-Gated Chloride channel GluCl is necessary for PHD expression. 115
Figure 3: Characterization of Ca2+ cooperativity, failure analysis and PPF/PPD in GluCl + vGlut-
OE. 116
Figure 4: GluCl is expressed exclusively in the nervous system and required presynaptically for
PHD. 117
Figure 5: Acute application of the GluCl agonist IVM induces a local GluCl-dependent inhibition
in neurotransmitter release. 118
Figure 6: GluCl localizes and traffics with synaptic vesicles. 119
Figure 7: Extracellular chloride ion is required to express PHD. 120
Supplemental Figure 1: Additional screening for metabotropic signaling finds no evidence for
roles in PHD. 121
Supplemental Figure 2: GluCl is dispensable for PHP expression. 122
x
ABSTRACT
Homeostatic plasticity adaptively modulates neuronal activity to ensure the functionality
of the nervous system in front of various destabilizing signaling during learning, development,
and diseases. Synaptic homeostatic plasticity is employed to maintain synaptic efficacy when
there are perturbations of synaptic function and believed to counterbalance with Hebbian
plasticity (Davis, 2013; Turrigiano, 2012). Bi-directional homeostatic regulations of presynaptic
neurotransmitter release have been demonstrated at the Drosophila neural muscular junction
(NMJ). Disruptions of postsynaptic receptor activity lead to a compensatory increase in the
presynaptic release, which precisely restores synaptic strength through a retrograde signal. In
the opposite process, enhancing glutamate release by increasing synaptic vesicle content leads
to homeostatic inhibition of presynaptic release. Despite the recent progress on understanding
the mechanisms underlying these two homeostatic modulations of synaptic efficacy,
fundamental properties and molecular mechanisms remain largely unknown. In this thesis work,
I have interrogated the synaptic dialog that enables the bi-directional, homeostatic control of
presynaptic neurotransmitter release, and further identified a novel molecular mechanism that
drives the presynaptic homeostatic depression.
In chapter 2 we provide evidence that the retrograde homeostatic potentiation is
achieved through highly specific and compartmentalized adaptions. The enhancement of
presynaptic efficacy can be induced and expressed in a subset of synapses from the same
motor neuron that bifurcates innervations on two adjacent muscle fibers. Homeostatic
modulations to CaMKII, readily releasable pool size, and functional releasable sites are
compartmentalized and restricted without spreading to neighboring boutons, which enables the
target-specific homeostatic control of neurotransmitter release.
xi

In chapter 3 we examined the interface between the conflict homeostatic adaptions
induced at the same synapses. We found that homeostatic potentiation and depression utilize
distinct genetic, induction, and expression mechanisms and able to be balanced in acute and
chronic time scales to fulfill homeostatic tuning of presynaptic glutamate release. Further, we
show that these two homeostatic modulations are expressed at separate neuronal subtypes at
the NMJ. Finally, unlike homeostatic potentiation, homeostatic depression operates with
complete disregard to the postsynaptic excitability, and only adaptively react to excess
glutamate release. Therefore, we propose that homeostatic depression of presynaptic release
functions cell-autonomously to balance glutamate release through an autocrine mechanism.
This model indicates the existence of a presynaptic glutamate receptor that senses the excess
glutamate release and initiate the presynaptic inhibition of neurotransmitter release.
In chapter 4, to determine the glutamate receptor that functions as the autocrine receptor
to mediate homeostatic depression expression, we performed a candidate screen of 11
glutamate receptors in the Drosophila genome and identified the glutamate-gated chloride
channel (GluCl) to be required in the homeostatic depression. We showed that GluCl is
exclusively expressed in the nervous system, and it is present and function at motor neuron
terminal to drive the presynaptic depression through an activity-dependent localize chloride
conductance, which leads to a reduction in calcium influx and in turn reduces presynaptic
release in response to excess glutamate release. Thus, in this work, we identify the molecular
mechanism underlying presynaptic homeostatic depression and gain mechanistic insights into
the contributions of chloride conductance in presynaptic inhibition and the prevention of
excitotoxicity.

1
Chapter 1
General introduction of homeostatic plasticity
1.1 Homeostatic signaling stabilizes neural function
Tremendous changes are going on continuously in every living organism. However, a stable
range of fluctuations is required for the optimal functionality of a system. Homeostasis is a
fundamental and ubiquitous form of biological regulation for the maintenance of a relatively
constant condition of various physiological processes in the face of destabilizing challenges and
perturbations. It has long been established that body temperature, blood glucose level,
osmolality, and other key biological parameters are all under homeostatic regulations, in which
deviations from failures of homeostatic control would lead to pathological consequences.
Theoretically, four key factors are shared within all homeostatic systems: a set point level for
optimal function, a sensor, a control center, and an effector. The sensor senses the imbalance
and sends an error signal to the control center, and then the control center further executes the
change to restore the setpoint level through the effectors. And more recently, in the past several
decades, it has become clear that the electrical activity that mediates the information transfer in
the nervous system is also regulated by homeostatic signaling (Davis, 2013; Pozo and Goda,
2010; Turrigiano, 2012).
With the wire of billions of neurons in the brain, the complex network, from a single
connection to circuits, is undergoing changes all the time to mediate different behaviors and
enable the achievement of learning and memory. The crosstalk of neurons happens at the
junctional compartments between neurons, which called synapses. The strength of the electric
signal at the synapses can be enhanced or decreased in response to changes in activity levels,
2
which provides a synaptic basis for information storage and learning (Abbott and Nelson, 2000).
This process is referred to as Hebbian plasticity. However, this form of plasticity is inherently
destabilizing for the following reasons. First, it allows unconstrained strengthening of the
synapses; second, with the intrinsic property of “neurons that fire together, wire together,”
unlimited enhancing of synaptic strength could result in a chaos of activity in neuronal circuits
and finally lose the ability to store or process meaningful information. To stabilize neural activity
within a physiologically appropriate range, the nervous system is endowed with the ability to
adapt the homeostatic signaling to modulate and restore the synaptic strength at a set point
level, which allows future adaptions. It is believed that the homeostatic plasticity coexists and
interface with Hebbian plasticity to ensure the dynamic, yet stable neural function, and enable
coherent behaviors (Fox and Stryker, 2017; Turrigiano, 2012; Yee et al., 2017). Despite
counterbalancing learning-related plasticity, synaptic homeostatic plasticity is adopted following
other destabilizing forces during development and diseases.
Emerging evidence illustrates that homeostatic modulations of synaptic activity are
achieved through the modulations of ion channel abundance, receptor trafficking and ion
channel abundance (Davis, 2013; Frank, 2014; Parrish et al., 2014; Turrigiano, 2011; Turrigiano
et al., 1998). Three subdivision of homeostatic plasticity have been described in the field,
including homeostatic regulations of postsynaptic receptor scaling, intrinsic excitability, and
synaptic transmission.
1.2 Homeostatic regulations of postsynaptic receptor scaling and intrinsic
excitability
Synaptic scaling that bi-directionally modulates the neurotransmitter receptor abundance in the
postsynaptic membrane in response to abolished or elevated activity has been first reported in
3
cultured neurons (Turrigiano et al., 1998). Later findings demonstrate the presence of synaptic
scaling in manipulating synaptic strength in the visual cortex in vivo (Desai et al., 2002; Keck et
al., 2013). Notably, as it termed synaptic “scaling,” the levels of the receptor modulations are
proportional to the original strength of the synaptic function, thereby promote the balancing of
overall synaptic weights in a circuit (Turrigiano, 2012; Turrigiano et al., 1998). Later on, a
localized and more rapid form of homeostatic upregulation of the postsynaptic receptor has
been shown by TTX treatment in combination with transmission blockage (Sutton et al., 2006;
Sutton et al., 2004). This form of plasticity is achieved through local protein synthesis but
expressed without the involvement of transcription (Sutton et al., 2006; Sutton and Schuman,
2006; Sutton et al., 2004). Retinoic acid (RA) has been identified as a key modulator that
mediating the homeostatic signaling in the postsynaptic cell (Aoto et al., 2008).
Compensatory mechanisms that regulate the intrinsic excitability of a neuron stabilize
circuit activity. This form of homeostatic plasticity is accomplished by alteration of ion channels
that contribute to the neuronal firing rate (Marder and Goaillard, 2006). The ion channel number,
type, expression pattern, and distribution together determine neuron’s electric properties. A
specific firing pattern of a neuron is required in a functional circuit. Change of the expression of
one channel could be homeostatically compensated by the function of another ion channel to
maintain the intrinsic firing pattern, which enables the meaningful functionality of the biological
system (MacLean et al., 2003). Most strikingly, a rhythmically active neuron is able to
regenerate the intrinsic firing properties by regulating its conductance during long-term isolation
(Turrigiano et al., 1994). Therefore, homeostatic regulations of intrinsic excitability is a robust
mechanism that stabilizes the function of a nervous system.
1.3 Presynaptic homeostatic potentiation of neurotransmitter release
4
Bi-directional homeostatic regulations of presynaptic neurotransmitter release have been shown
to maintain normal synaptic function in the face of different perturbations. Retrograde signaling
is employed to mediate presynaptic homeostatic plasticity (Jakawich et al., 2010). One
established synaptic homeostatic modulation called presynaptic homeostatic potentiation (PHP).
Disruption of postsynaptic receptor functionality leads to a homeostatic enhancement of
presynaptic release through retrograde signaling, which precisely compensates for the reduction
in postsynaptic sensitivity to neurotransmitters (Frank et al., 2020; Petersen et al., 1997).
Parallel similar process has been observed in both periphery and central nervous system from
Drosophila to mammals (Burrone et al., 2002; Davis and Muller, 2015; Delvendahl et al., 2019;
Wang et al., 2016b), as well as in the neuromuscular junction (NMJ) of patients with myasthenia
gravis syndrome (Cull-Candy et al., 1980). The molecular and genetic mechanisms underlying
PHP have been intensively investigated at Drosophila NMJ for over the past two decades.
The Drosophila NMJ is a glutamatergic synapse with innate advantages in genetic
manipulation, easy accessibility for in vivo electrophysiology in combination with imaging, and
simple stereotype neuronal network. Therefore, it provides a powerful model to dissect the
fundamental mechanisms that drive synaptic transmission and homeostatic synaptic plasticity.
The most characterized muscles at Drosophila larval NMJ are adjacent muscle 6 and muscle 7,
which receive standardized innervations from two motor neurons, including MN-Ib and MN-Is
that drive tonic and phasic activities respectively (Lnenicka and Keshishian, 2000). MN-Ib forms
lb boutons with comparably larger size on the targeted muscle fiber that surrounded by more
elaborated subsynaptic reticulum (SSR), while MN-Is forms “smaller” Is boutons with less SSR
structure at postsynaptic cell. Postsynaptic glutamate receptors at Drosophila NMJ can be
divided into two subtypes, which are constituted with three common subunits: GluRIIC, GluRIID,
GluRIIE, and one alternative subunit: either GluRIIA or GluRIIB, that mediate the electric
signaling in the muscle fibers (DiAntonio et al., 1999; Featherstone et al., 2005; Han et al.,
5
2015; Marrus et al., 2004). Loss of GluRIIA leads to significantly reduced miniature response
amplitudes due to a reduction in the postsynaptic sensitivity to glutamate. However, the evoked
amplitude is similar to wild type level, which indicates a homeostatic retrograde signaling
precisely and robustly potentiates presynaptic neurotransmitter release to restore the synaptic
strength to set point levels (Petersen et al., 1997). Remarkably, this retrograde potentiation can
be induced rapidly within 10 min through acutely blockages of GluRIIA receptors by a toxin
derived from wasp’s venom, philanthotoxin (PhTx; (Frank et al., 2006)). PHP at Drosophila
larval NMJ will be one of the major topics for the studies in this thesis that will be mainly
described in chapter 2 and 3.
1.3.1 Presynaptic mechanisms
Multiple mechanisms are uncovered at the presynaptic motor neuron terminals to potentiate
release under PHP conditions. Two major components for enhancing presynaptic vesicle
release that are necessary for the PHP expression includes an increase in calcium influx (Muller
and Davis, 2012) and increased readily releasable pool (RRP) sizes (Li et al., 2018a; Li et al.,
2018b; Muller et al., 2015; Weyhersmuller et al., 2011). Forward genetic screen in combination
with candidates test on known synaptic molecules reveal numerous genetic molecular
mechanisms for the expression of PHP at the presynaptic motor neurons. Mutations that disrupt
cacophony (cac; a voltage-gated calcium channel; (Frank et al., 2006) or α2δ-3 auxiliary subunit
of the CaV2.1 calcium channel block PHP by preventing the increase in calcium influx following
postsynaptic receptor perturbations (Wang et al., 2016a). Homeostatic regulations of RRP size
could be a secondary mechanism after the change in calcium influx to process homeostatic
signaling. In the synapses, Rab3 interaction molecule (RIM) and RIM-binding protein are
required in the modulations of the pool of synaptic vesicles for PHP expression. And it has been
shown that a protein encoded from the schizophrenia-linked gene dysbindin functions to
6
regulate the pool of synaptic vesicles to allow the potentiation of presynaptic release (Dickman
and Davis, 2009). Additionally, activities of presynaptic ion channels contribute to enhancing the
single action potential induced calcium influx during the expression of PHP (Kiragasi et al.,
2017; Younger et al., 2013).
Further investigations of the expression mechanisms in presynaptic neuron detected a
homeostatic remodeling of active zone proteins. Increase in the size of the active zone
scaffolding protein from the ELKS family Bruchpilot (BRP), along with the increase in cac
intensity have been observed in acute and chronic PHP (Bohme et al., 2019; Goel et al., 2017;
Gratz et al., 2019; Weyhersmuller et al., 2011). And later experiment shows this homeostatic
remodeling is input-specific, indicating the expression of PHP in a specific neuron subtype (Li et
al., 2018a; Newman et al., 2017). However, a extracellular calcium dependent target-wide
induction of PHP in both neuron subtypes has been argued by a recent research (Genc and
Davis, 2019).
1.3.2 Postsynaptic induction mechanisms
Unlike the presynaptic expression mechanisms, the induction mechanisms at the postsynaptic
muscle fiber remain largely unknown. Although it is clear that PHP is induced by perturbations in
postsynaptic receptor functionality, the signaling system for the induction of PHP in the
postsynaptic compartment is critical, yet, mysterious. A reduction in Ca
2+
/calmodulin-dependent
protein kinase II (CaMKII) activity level has been proposed to drive the induction of the
retrograde signaling in the postsynaptic side (Haghighi et al., 2003). The levels of
phosphorylated CaMKII at the postsynaptic density are specifically reduced at the Ib boutons
following the disruption of postsynaptic GluRs (Goel et al., 2017; Li et al., 2018a; Newman et al.,
2017). One hypothesis is that the activity of the CaMKII could serve as a sensor for PHP
7
induction by sensing postsynaptic Ca
2+
influx. Is there existence of other signaling in addition to,
or downstream of CaMKII modulation to ultimately control the trans-cellular communication to
increase presynaptic release? A recent forward genetic screen identified insomniac (inc)
function to be required at the postsynaptic muscle cells to enable the PHP processing (Kikuma
et al., 2019). inc encodes a potential adaptor for the Cullin-3 (Cul3) E3 ubiquitin ligase, and it is
a hit from a sleep deficit screen, where mutations in inc or knockdown of Cul3 lead to severe
malfunction in sleep behavior in Drosophila (Kikuma et al., 2019; Stavropoulos and Young,
2011). Disruptions in either Inc or Cul3 function abolish the expression of PHP. Therefore, it
provides a molecular link between PHP and sleep regulations. Interestingly, Inc and Cul3 rapidly
accumulate specifically at postsynaptic density following PhTx application in 10 min to mediate
a mono-ubiquitination related signaling that drives the retrograde homeostatic potentiation
(Kikuma et al., 2019). Further investigations of Inc/Cul3 interaction proteins found that Peflin, a
Ca
2+
-regulated Cul3 co-adaptor, is necessary for PHP signaling, suggesting a relationship
between Ca2+ signaling and control of Cul3/Inc activity in the postsynaptic compartment
(Kikuma et al., 2019). Notably, both the regulations of CaMKII activity or the Cul3/Inc dependent
signaling is compartmentalized to the postsynaptic density, which support the hypothesis of
synapse-specific PHP expression.
1.3.3 Retrograde signaling
Following the PHP induction initiated at the postsynaptic cell, a retrograde signaling is required
to allow the trans-cellular communication to occur. Two putative retrograde signals to mediate
PHP at presynaptic terminals have been indicated in recent work, including Endostatin and
Semaphorine 2B (Sema2B). The Endostatin is a proteolytic cleavage product of Drosophila
Multiplexin, which is the Drosophila homolog of mammalian Collagens XV and XVIII. This study
shows Endostatin function as a trans-synaptic signaling molecule to modulate presynaptic
8

calcium channels to enhance Ca
2+
influx during PHP expression, and mutations in multiplexin
block PHP (Wang et al., 2014). Intriguingly, genetic interaction experiments suggest a related
function of Cul3/Inc to Multiplexin, and a plausible model has been proposed that the Cul3/Inc
dependent mono-ubiquitination targets ER substrates at postsynaptic compartments to boost
the release of Multiplexin (Kikuma et al., 2019). A subsequent study shows that a synaptic
signaling molecule associated with axon guidance and synapse formation, Semaphorin 2B
(Sema2B) and its receptor Plexin B (PlexB), operates as a retrograde signal to enhance
presynaptic release in PHP (Orr et al., 2017). Mutants of sema2B abolish PHP expression, and
the application of exogenous purified Sema2B protein rescues the failure in PHP expression.
However, whether additional retrograde signals exist to promote the expression of PHP and
how these retrograde signals interact with each other to process the PHP signaling remain
enigmatic. Finally, most recent work shows evidence for the role of epigenetic signaling from
Glia that stabilizes the extracellular matrix to facilitate the homeostatic control of presynaptic
release (Wang et al., 2020). This work suggests the participation of another composition, glia, in
PHP processing, which indicates the complex intercellular signaling from all three cells at the
NMJ to collaboratively drive PHP expression.

1.3.4 Acute and chronic PHP
The retrograde homeostatic potentiation of presynaptic release can operate over distinct
temporal scales to maintain overall synaptic strength at Drosophila NMJ. Although both acute
and chronic PHP enhances presynaptic release, the induction and expression mechanisms that
underlying these PHP may vary. It has been shown that genes that are necessary for GluRIIA-
dependent chronic PHP are dispensable for acute PhTx induced PHP expression (Frank et al.,
2009; Kauwe et al., 2016; Penney et al., 2016; Spring et al., 2016; Tsurudome et al., 2010).
Additionally, robust PHP is observed in conditions of blocking translation along with PhTx
9

incubation, indicates that acute PHP is translational independent (Frank et al., 2006; Goel et al.,
2017). However, loss of the translational regulator, target of rapamycin (Tor), block chronic PHP
in GluRIIA mutant while leave the PhTx-induced PHP intact (Goel et al., 2017; Penney et al.,
2012). Overexpression of Tor in the postsynaptic muscle, which leads to ubiquitous increases in
protein synthesis (Chen and Dickman, 2017) artificially triggers a retrograde signaling to
potentiate presynaptic release (Goel et al., 2017; Penney et al., 2012). Although the activity of
CaMKII is reduced following receptor perturbations in GluRIIA or PhTx conditions, the reduction
is not observed in Tor-OE NMJs (Goel et al., 2017). On the other hand, the same presynaptic
molecular mechanism is engaged to potentiate neurotransmitter release, and overexpression of
Tor in acute or chronic PHP did not further increase the presynaptic release (Goel et al., 2017).
Together, distinct induction mechanisms in the postsynaptic cell converge on the same
retrograde signaling system to drive PHP through PHP application, loss of GluRIIA and Tor-OE.

1.4 Presynaptic homeostatic depression in neurotransmitter release
The inverse regulation of presynaptic neurotransmitter release at the Drosophila NMJ referred
to as presynaptic homeostatic depression (PHD). Previous work shows that overexpression of
the glutamate transporters (vGlut-OE) increases synaptic vesicle size and excess glutamate
emitted from each vesicle that leads to enlarged quantal size in both Drosophila NMJ and
mammalian hippocampal neurons (Daniels et al., 2004; Wojcik et al., 2004). If no presynaptic
release regulation is employed in the vGlut-OE condition, enlarged evoked responses should be
detected in the postsynaptic cell. However, the evoked amplitude stays the same as wild type
animals, suggesting a homeostatic inhibition of presynaptic release in vGlut-OE animals
(Daniels et al., 2004). Parallel evidence of PHD is reported in mutants with endocytic deficiency,
which results in increases in synaptic vesicle sizes (Chen et al., 2014; Dickman et al., 2005;
Marie et al., 2004; Verstreken et al., 2002). This homeostatic downregulation of presynaptic
10

release is achieved through a reduction in presynaptic release probability (Pr; (Daniels et al.,
2004), and reductions in Ca
2+
influx measured at the presynaptic terminals might contribute to
the decreased Pr (Gavino et al., 2015). Although remodeling of active zone proteins happens in
PHP expression, no obvious modulations of BRP and Cac are found at the motor neuron
terminals in vGlut-OE animals (Gavino et al., 2015; Gratz et al., 2019). Additionally, enlarging of
RRP size mediates the enhancing of synaptic vesicle release in PHP, but the opposite
regulations of RRP size is not observed in PHD condition (Gavino et al., 2015). Different from
PHP, PHD is induced by a presynaptic mechanism and expressed presynaptically, therefore
whether a trans-synaptic communication participates in this homeostatic signaling is unclear. It
is also unknown whether PHD is responding to excess glutamate release or deviations in
synaptic strength.

1.5 Glutamate homeostasis and excitotoxicity
Glutamate homeostasis commonly exists at all glutamatergic systems to regulate glutamate
levels and avoid excitotoxicity induced by the presence of excess glutamate. Glutamate is a
major neurotransmitter in the brain which mediates the transmission in over 90% of the synaptic
connections. However, excess glutamate poses a serious threat to the nervous system, in which
misregulations of glutamate release can cause severe consequences. Glutamate excitotoxicity
has been implicated in seizures, stroke, traumatic brain injury, and neurodegenerative diseases
(Doble, 1999; Lai et al., 2014; Murphy-Royal et al., 2017; Olloquequi et al., 2018; Verma et al.,
2015). To avoid this toxicity, the nervous system evolved numerous clearance mechanisms to
homeostatically control glutamate levels and prevent the damage. The most well-characterized
mechanism is through the excitatory amino acid transporter (EAAT) on the membrane of the
presynaptic neuron and glia cells to uptake excess extracellular glutamate (Oliet et al., 2001;
Sun et al., 2014). There is only one EAAT that transport glutamate encoded in Drosophila
11

genome, dEAAT1 (Besson et al., 2000; Rival et al., 2004), which present and function at adult
NMJ in flies. Disruption of dEAAT1 leads to flight and locomotion deficits in adult flies (Rival et
al., 2006). However, dEAAT1 is absent in the embryonic or larval periphery system (Rival et al.,
2006; Rival et al., 2004). Therefore, the mechanisms that regulating glutamate homeostasis at
the embryonic and larval NMJ remain obscure. Thus, the larval NMJ is a unique and powerful
model to investigate fundamental questions into how glutamate imbalance, occurring in the
absence of classical glutamate transporters.
Other than the conventional mechanism for glutamate homeostasis, there is evidence
showing that presynaptic inhibition of glutamate release plays a role in balancing glutamate
signaling to promote neuronal survival (Li et al., 2009; Oliet et al., 2001). One hypothesis is that
PHD is a presynaptic inhibition mechanism that utilized to homeostatically regulate glutamate
signaling at Drosophila larval NMJ, and this topic will be discussed in more detail in chapter 4.

12

Chapter 2
Synapse-specific and compartmentalized expression of presynaptic
homeostatic potentiation

2.1 Abstract
Postsynaptic compartments can be specifically modulated during various forms of synaptic
plasticity, but it is unclear whether this precision is shared at presynaptic terminals. Presynaptic
Homeostatic Plasticity (PHP) stabilizes neurotransmission at the Drosophila neuromuscular
junction, where a retrograde enhancement of presynaptic neurotransmitter release
compensates for diminished postsynaptic receptor functionality. To test the specificity of PHP
induction and expression, we have developed a genetic manipulation to reduce postsynaptic
receptor expression at one of the two muscles innervated by a single motor neuron. We find that
PHP can be induced and expressed at a subset of synapses, over both acute and chronic time
scales, without influencing transmission at adjacent release sites. Further, homeostatic
modulations to CaMKII, vesicle pools, and functional release sites are compartmentalized and
do not spread to neighboring pre- or post-synaptic structures. Thus, both PHP induction and
expression mechanisms are locally transmitted and restricted to specific synaptic
compartments.

13

2.2 Introduction
Synaptic strength can be modulated with a remarkable degree of specificity to enable the
flexibility necessary for learning and memory, where compartmentalized changes in dendritic
spines tune responses to neurotransmitter release during information transfer in the nervous
system. Such plasticity mechanisms require compartmentalized trafficking and insertion of
glutamate receptors (GluRs) into postsynaptic densities at specific locations in response to
correlated activity (Herring and Nicoll, 2016; Malinow, 2003). However, these processes of
Hebbian plasticity are inherently destabilizing, and homeostatic mechanisms have been
proposed to adaptively counteract such forces to maintain synaptic strength within
physiologically stable levels (Davis, 2013; Pozo and Goda, 2010; Turrigiano, 2012). Although
the induction and expression of various forms of plasticity can clearly be restricted to individual
postsynaptic compartments, it is less certain that such plasticity can be similarly
compartmentalized at the presynaptic terminals of a single neuron. There is evidence for
heterogeneity in presynaptic efficacy (Branco et al., 2008; Dobrunz and Stevens, 1997;
Holderith et al., 2012; Trommershauser et al., 2003; Vitureira et al., 2011), and release
probability can vary considerably along a single axon (Murthy et al., 1997; Paul et al., 2015;
Peled and Isacoff, 2011). Further, target-specific differences in presynaptic function (Frank,
1973; Katz et al., 1993; Scanziani et al., 1998) and homeostatic plasticity (Davis and Goodman,
1998) have been demonstrated. However, how presynaptic terminals are modulated by Hebbian
and homeostatic forces and whether these adaptations can occur without “spreading” to
adjacent synapses remains enigmatic.
The Drosophila neuromuscular junction (NMJ) is a powerful model system to interrogate
the mechanisms governing homeostatic synaptic plasticity. At this glutamatergic synapse,
genetic and pharmacological perturbations to postsynaptic receptors initiates a retrograde,
trans-synaptic signaling system that homeostatically increases presynaptic neurotransmitter
14

release to maintain stable levels of synaptic strength (Davis and Muller, 2015; Frank, 2014).
This form of plasticity is achieved through an increase in presynaptic efficacy, and is therefore
referred to as Presynaptic Homeostatic Potentiation (PHP). Parallel forms of homeostatic
regulation are conserved at NMJs of rodents (Wang et al., 2016b) and human (Cull-Candy et
al., 1980). PHP initiates a single retrograde signaling system that triggers two key expression
mechanisms to enhance presynaptic glutamate release: increases in both presynaptic calcium
influx and the number of synaptic vesicles participating in the readily releasable pool (Goel et
al., 2017; Kiragasi et al., 2017; Muller and Davis, 2012; Weyhersmuller et al., 2011). Several
genes necessary for PHP expression have been identified that function as putative retrograde
signals (Orr et al., 2017; Wang et al., 2014) and presynaptic effectors in the motor neuron
(Bruckner et al., 2017; Dickman and Davis, 2009; Dickman et al., 2012; Kiragasi et al., 2017;
Muller et al., 2015; Muller et al., 2012; Tsurudome et al., 2010; Younger et al., 2013), but the
postsynaptic induction mechanisms that initiate PHP signaling remain enigmatic (Chen and
Dickman, 2017; Goel et al., 2017). Indeed, PHP induction and expression at a subset of
synapses within a single motor neuron has never been demonstrated at the Drosophila NMJ.
PHP is expressed exclusively at one of two motor neuron subtypes that innervate most
muscles at the Drosophila NMJ, Type Is and Type Ib. Type Is motor inputs exhibit smaller
boutons, less subsynaptic reticulum (SSR), higher basal release probability, and do not
participate in PHP adaptation over chronic time scales (Lnenicka and Keshishian, 2000;
Newman et al., 2017). In contrast, Type Ib motor neurons have larger boutons, more elaborate
SSR, and lower basal release probability, which is enhanced after loss of postsynaptic GluRs
(Newman et al., 2017), demonstrating that PHP is expressed exclusively at Type Ib synapses.
Further, a reduction in phosphorylated (active) levels of CaMKII, presumably related to PHP
inductive signaling, occurs specifically in the postsynaptic density of Ib boutons (Goel et al.,
2017; Newman et al., 2017), suggesting a possible mechanism for the specificity of retrograde
15

PHP signaling to the Ib motor neuron. Despite these insights, it is not known whether PHP can
be expressed at a subset of Type Ib boutons within a single motor terminal, nor whether PHP
modulations at individual boutons influence neighboring synapses.
We have developed a genetic manipulation that enables the reduction of postsynaptic
GluR expression on one of the two muscles innervated by a single Type Ib motor neuron at the
Drosophila NMJ. We have used this system to test whether PHP signaling is synapse specific
and to determine to what extent the postsynaptic induction and presynaptic expression of PHP
is compartmentalized. This analysis has revealed highly specific and compartmentalized PHP
adaptations that are restricted and target specific without influencing neurotransmission at
neighboring synapses within the same motor neuron.

16

2.3 Results
Reduced expression of the glutamate receptor subunit GluRIIA specifically on muscle 6
at the Drosophila NMJ
The postsynaptic response to glutamate release at the Drosophila NMJ is mediated by two
types of GluRs. Both types contain the essential subunits GluRIIC, GluRIID, and GluRIIE, but
differ in containing either GluRIIA or GluRIIB subunits (DiAntonio et al., 1999; Featherstone et
al., 2005; Han et al., 2015; Marrus et al., 2004). Although null mutations in the GluRIIA subunit
have been studied for decades (Petersen et al., 1997), RNAi knock-down of this receptor has
not been reported or characterized. We obtained an RNAi transgene targeting the GluRIIA
subunit (see Methods) and compared the impact of postsynaptic knock-down of GluRIIA by the
muscle driver G14-Gal4 (G14>GluRIIA
RNAi
) to GluRIIA null mutants (Figure 1). First, we
immunostained the NMJ of wild type, GluRIIA mutants, and G14>GluRIIA
RNAi
with antibodies
against the GluRIIA subunit as well as the common subunits GluRIIC and GluRIID (Figure 1A).
This revealed an absence of GluRIIA signals from GluRIIA mutants, as expected, with signals
from GluRIIC and GluRIID persisting due to the remaining GluRIIB-containing receptors (Figure
1A,B). Similarly, GluRIIA expression is almost completely absent in G14>GluRIIA
RNAi
, with no
significant difference in fluorescence intensity compared to GluRIIA mutants (Figure 1A,B).
Indeed, quantitative PCR analysis revealed a dramatic reduction in the level of transcripts
encoding the GluRIIA subunit in G14>GluRIIA
RNAi
, while levels of the other four subunits were
not significantly changed (Figure 1C). In addition, we quantified synaptic growth in these
genotypes, finding a small reduction in bouton number in both GluRIIA mutants and
G14>GluRIIA
RNAi
compared to controls (Figure 1-figure supplement 1), as suggested previously
(Choi et al., 2014; Schmid et al., 2006; Sigrist et al., 2002). Finally, we examined synaptic
physiology, which revealed a large reduction in mEPSP amplitude in both GluRIIA mutants and
G14>GluRIIA
RNAi
compared to wild type and G14-Gal4/+ (Figure 1D,E), while EPSP amplitudes
17

were not significantly changed between these genotypes because of a homeostatic
enhancement in presynaptic glutamate release (quantal content; Figure 1D,H). Together, this
demonstrates that postsynaptic knock down of the GluRIIA subunit effectively phenocopies
GluRIIA mutants and induces the robust expression of PHP.
Next, we sought to specifically knock down the GluRIIA subunit on one of the two
muscles innervated by a single Ib motor neuron at the NMJ by selectively biasing expression of
Gal4. At the muscle 6/7 NMJ, a single Ib and a single Is motor neuron bifurcates to innervate
both muscle 6 and 7, with ~60% of the boutons from each motor neuron subtype innervating the
larger muscle 6, and ~40% innervating the smaller muscle 7 (Figure 2-figure supplement
2A,B,C). To bias Gal4 expression selectively on muscle 6, we modified a genetic manipulation
using the H94-Gal4 driver, which expresses transiently on muscle 6 early in development (Davis
and Goodman, 1998). Gal4 expression on muscle 6 is amplified and converted into constitutive
expression by utilizing a cassette in which a flippase is co-expressed to excise a stop codon
between the strong and ubiquitous Tubulin-promotor and Gal4 (see Methods; (Choi et al., 2014;
Roy et al., 2007)). Thus, this manipulation enables Gal4 to be strongly and consistently
expressed specifically on muscle 6 (Choi et al., 2014). We validated this approach by visualizing
UAS-GFP selectively on muscle 6, which demonstrated strong expression on muscle 6 and no
detectable expression in either the motor neuron or the adjacent muscle 7 (Figure 2-figure
supplement 2A). Importantly, we confirmed that M6>Gal4 driving a control RNAi
(M6>mCherry
RNAi
) did not have any significant impact on muscle surface area, synaptic growth,
active zone numbers, or synaptic physiology compared to wild type (Figure 2-figure supplement
2A-H). Thus, M6>Gal4 enables strong and biased expression of Gal4 on muscle 6 without
impacting synaptic growth or function.
Finally, we evaluated this M6>Gal4 system to determine whether the GluRIIA subunit
could be specifically knocked down on muscle 6, as observed using pan-muscle knock down in
Figure 1 (see schematic in Figure 2A). First, we performed immunocytochemistry at the larval
18

NMJ with antibodies that label the neuronal membrane (HRP) and the GluRIIA subunit in wild
type and following knock down of GluRIIA on muscle 6 (M6>GluRIIA
RNAi
). We observed a near-
absence of the GluRIIA signal specifically on muscle 6, while GluRIIA expression on the
adjacent muscle 7 was unperturbed (Figure 2B,C). Quantification of synaptic growth on muscles
6 and 7 in M6>GluRIIA
RNAi
revealed no significant change on muscle 7, while a small but
significant reduction was observed on muscle 6, as expected (Figure 2D). Finally, quantification
of GluRIIA and GluRIID fluorescence levels at muscles 6 and 7 confirmed a large reduction of
GluRIIA expression on muscle 6 and no significant change on muscle 7 in M6>GluRIIA
RNAi

(Figure 2C,E). Thus, M6>GluRIIA
RNAi
effectively and specifically eliminates GluRIIA expression
on muscle 6 without altering glutamate receptor expression on the adjacent muscle 7.

Presynaptic homeostatic potentiation can be exclusively expressed at synapses
innervating muscle 6 in M6>GluRIIA
RNAi

A single Ib motor neuron (RP3) bifurcates to innervate both muscles 6 and 7 at the Drosophila
NMJ (Broadie and Bate, 1993) (Figure 3A). Having established strong and selective knock down
of the GluRIIA subunit on muscle 6 in M6>GluRIIA
RNAi
, we next characterized synaptic function
and homeostatic plasticity. As expected, mEPSP amplitudes on muscle 6 were diminished at
M6>GluRIIA
RNAi
NMJs, while mEPSP amplitudes were not affected on the adjacent muscle 7
(Figure 3A-C). We considered three possibilities for how presynaptic neurotransmitter release
sites may be modulated within the single Ib terminal in response to GluRIIA knock down
exclusively on muscle 6. First, if PHP signaling is communicated to synapses in the Ib motor
neuron innervating muscle 6, but the entire motor neuron undergoes PHP adaptations, then
quantal content would be enhanced on both muscles 6 and 7. Second, PHP signaling may be
communicated to synapses innervating only muscle 6, but PHP expression may be occluded
without simultaneous signaling also received from muscle 7, leading to no change in quantal
content on either muscle. Finally, if PHP signaling is target-specific and compartmentalized,
19

then quantal content should be selectively enhanced on synapses innervating muscle 6 in
response to reduced GluRIIA expression, while synaptic function at synapses innervating the
adjacent muscle 7 would be unchanged. Results of electrophysiological recordings were
consistent with this last model: EPSP amplitude was similar on both muscles 6 and 7 in
M6>GluRIIA
RNAi
and not significantly different compared to wild type (Figure 3A,D). Indeed,
quantal content was selectively enhanced only at synapses innervating muscle 6, while quantal
content at synapses innervating muscle 7 was unaffected (Figure 3B,E). Thus, PHP can be
induced and expressed exclusively at a subset of synapses within the same motor neuron
without influencing neurotransmitter release at neighboring sites.
The results above suggest that while PHP was chronically induced and expressed
specifically on synapses innervating muscle 6, the adjacent synapses within the same motor
neuron that innervate muscle 7 were apparently not affected. One possibility is that PHP
adaptations are induced throughout the entire motor neuron innervating both muscle 6 and 7,
but that negative regulators are active that repress or occlude the expression of PHP on
synapses innervating muscle 7. Indeed, such a model has been proposed (Muller et al., 2011).
In this case, PHP should not be capable of being induced or expressed at the synapses
innervating muscle 7 in M6>GluRIIA
RNAi
. PHP can be acutely induced and expressed through a
pharmacological blockade of the postsynaptic GluRs using a 10 min incubation in the presence
of the antagonist philanthotoxin-433 (PhTx; (Frank et al., 2006)). This results in an acute
reduction in mEPSP amplitude due to blockade of GluRIIA-containing receptors in the
postsynaptic muscle, but EPSP amplitudes are maintained because of PHP expression. We
reasoned that acute application of PhTx to M6>GluRIIA
RNAi
NMJs would enable us to determine
whether PHP could be induced and expressed at synapses innervating muscle 7 following
chronic expression of PHP on the adjacent synapses innervating muscle 6.
We therefore applied PhTx to wild type and M6>GluRIIA
RNAi
synapses. As expected, this
caused a large reduction in mEPSP amplitudes at wild-type muscles 6 and 7, as well as muscle
20

7 in M6>GluRIIA
RNAi
, while a small reduction in mEPSP amplitude was observed at muscle 6
(Figure 4A-C). Interestingly, EPSP amplitudes at both muscles 6 and 7 in wild type and
M6>GluRIIA
RNAi
NMJs were maintained at similar levels (Figure 4A,D) due to the homeostatic
enhancement of quantal content (Figure 4B,E). Together, this demonstrates that PHP can be
acutely induced and chronically expressed at distinct presynaptic release sites within the same
neuron according to the state of GluR functionality at postsynaptic compartments opposing
these sites.

Homeostatic modulations to the synaptic vesicle pool and functional release sites are
compartmentalized at presynaptic terminals
One important presynaptic expression mechanism that enables the enhanced efficacy
necessary for PHP expression is an increase in the readily releasable synaptic vesicle pool
(RRP; (Kiragasi et al., 2017; Muller et al., 2015; Weyhersmuller et al., 2011)). The RRP is
defined as the pool of vesicles that are primed and available for immediate release upon strong
synaptic stimulation (Rosenmund and Stevens, 1996). Although PHP expression appears to be
compartmentalized, it is unknown whether conventional homeostatic modulations to the
presynaptic terminal are similarly compartmentalized, or rather whether novel mechanisms are
utilized in M6>GluRIIA
RNAi
. In particular, synaptic vesicles are highly mobile and can rapidly
traffic between adjacent boutons at presynaptic terminals (Darcy et al., 2006; Kahms and
Klingauf, 2018), and vesicle pools can span multiple presynaptic terminals (Staras et al., 2010).
Hence, it is possible that changes in the RRP following PHP expression at a subset of
presynaptic terminals may influence vesicle pools at neighboring synapses that do not directly
experience local PHP signaling. We therefore measured the size of RRP in wild type and
M6>GluRIIA
RNAi
synapses separately innervating muscle 6 and 7. To determine RRP size, we
performed two electrode voltage clamp (TEVC) measurements using high frequency stimulation
(60 Hz) in elevated external calcium concentrations (3 mM) and measured the cumulative EPSC
21

(Figure 5A-D; (Kiragasi et al., 2017; Muller et al., 2015; Weyhersmuller et al., 2011). We
observed a ~65% increase in the estimated RRP size that was restricted to boutons innervating
muscle 6 in M6>GluRIIA
RNAi
, similar in magnitude to what has been reported for muscle 6
synapses in which PHP is expressed following PhTx application (Muller et al., 2015) and by loss
of GluRIIA (Kiragasi et al., 2017; Weyhersmuller et al., 2011). However, no significant change in
RRP size was observed at synapses innervating the adjacent muscle 7 (Figure 5A,E). Thus, the
homeostatic modulation of the RRP is restricted to presynaptic terminals that oppose
postsynaptic compartments with reduced GluR functionality, and does not “spread” to influence
vesicle pools at adjacent release sites.
Next, we examined whether a change in the number of functional release sites (N)
accompanies the compartmentalized expression of PHP. N is defined as the number of
functional release sites and is one of the three basic parameters used to describe synaptic
transmission, where quantal content (QC) is the product of N, P (release probability), and Q
(quantal size). Although there is no major difference in the anatomical number of active zones at
NMJs of wild type and GluRIIA mutants (Frank et al., 2006; Goel et al., 2017; Penney et al.,
2012; Schmid et al., 2006), an increase in the fraction of active zones that participate in release
has been reported following the expression of PHP (Davis and Muller, 2015; Newman et al.,
2017; Penney et al., 2016). Indeed, the value of N is significantly increased following PhTx
application and in GluRIIA-mutant synapses (Muller et al., 2012; Weyhersmuller et al., 2011).
We determined the number of functional release sites at NMJs of muscles 7 and 6 in wild type,
GluRIIA mutants, and M6>GluRIIA
RNAi
using a variance-mean plot analysis (Bohme et al., 2016;
Clements and Silver, 2000). We performed TEVC recordings over a range of increasing
extracellular calcium concentrations, from 0.5 mM to 6 mM (Figure 6B,F). The variance in the
amplitude of repeated evoked responses fluctuates across different calcium concentrations in
relation to the proportion of total release sites that participate in these responses. At low calcium
concentrations, the number of release sites that participate in the evoked response is low, and
22

the variance is therefore small in this condition. As the extracellular calcium concentration is
elevated, the variance then increases with increasing release during repeated evoked
responses due to an increase in the number of release sites that participate in synaptic
transmission. At very high extracellular calcium concentrations, the variance is then reduced
due to saturation of the total number of releasable sites.
We plotted the variance of EPSC responses across increasing calcium conditions
against the mean of the EPSC amplitude at each individual calcium conditions from recordings
at both muscle 7 and muscle 6 (Figure 6C,G). This analysis resulted in a parabolic behavior of
variance-mean plot due to the binomial nature of the fluctuation (Clements and Silver, 2000;
Weyhersmuller et al., 2011). The number of functional release sites (N) was determined by
fitting a parabola to the variance-mean plot (see Methods). Based on this result, the value of N
increased in GluRIIA-mutant NMJs compared to wild type at both muscles 7 and 6 (Figure
6D,H), consistent with what was reported following acute PHP expression and indicating that
chronic PHP expression requires the recruitment of additional functional release sites to
participate in presynaptic neurotransmitter release. Further, a similar increase in N was
observed at muscle 6 in M6>GluRIIA
RNAi
(Figure 6H), while no significant change was found at
muscle 7 (Figure 6D), suggesting that the biased induction of PHP results in the
compartmentalized expression of the same mechanisms observed in GluRIIA mutants (Figure
6A,E). Therefore, retrograde PHP expression is achieved by elevating the RRP and recruiting
additional functional release sites to participate in transmission, with specificity according to the
excitability state of their postsynaptic partners.

Compartmentalized reductions in postsynaptic CaMKII activity are required for PHP
induction in M6>GluRIIA
RNAi

The postsynaptic induction mechanisms that drive PHP retrograde signaling are unclear.
However, reductions in postsynaptic CaMKII activity have been proposed to mediate the
23

induction of retrograde PHP signaling (Haghighi et al., 2003). Indeed, modulations to
postsynaptic CaMKII phosphorylation has been demonstrated to occur in a synapse-specific
and activity-dependent manner at the Drosophila NMJ (Hodge et al., 2006). Consistent with this
idea, reductions in the level of phosphorylated (active) CaMKII were observed specifically at
postsynaptic densities of Type Ib boutons following PhTx application and in GluRIIA mutants
(Goel et al., 2017; Newman et al., 2017). To determine whether this same signaling system
mediates PHP induction and is restricted to specific targets at M6>GluRIIA
RNAi
NMJs, we
examined pCaMKII levels at postsynaptic densities of Is and Ib boutons on both muscles 6 and
7. Ib and Is boutons were distinguished by differential areas and intensity signals of the
postsynaptic scaffold Discs large (Dlg). We observed a significant reduction in the mean
intensity of pCaMKII in Ib postsynaptic densities only on muscle 6 of M6>GluRIIA
RNAi
, while no
significant difference was observed at Ib synapses in the adjacent muscle 7, nor were any
changes found at Is synapses on either muscle (Figure 7A-E). Thus, postsynaptic pCaMKII
levels are diminished and compartmentalized at Ib boutons specifically on muscle 6 without
impacting pCaMKII levels on neighboring Is boutons or at the adjacent muscle 7 in
M6>GluRIIA
RNAi
.
Finally, we tested whether a reduction in postsynaptic CaMKII activity was
required for retrograde PHP signaling. In particular, if PHP signaling is induced at Ib
postsynaptic densities through diminished pCaMKII levels, as suggested by immunostaining,
then postsynaptic overexpression of a constitutively active, phospho-mimetic form of CaMKII,
CaMKII
T287D
, should inhibit or occlude PHP induction and expression (Haghighi et al., 2003). We
first expressed CaMKII
T287D
alone on muscle 6 (M6>CaMKII
T287D
) to determine if baseline
synaptic function was influenced by constitutively active CaMKII (Figure 8A). We found no
significant difference in synaptic physiology on muscles 6 or 7 in this condition (Figure 8A-D).
Next, we expressed constitutively active CaMKII in combination with GluRIIA knock down on
muscle 6 (M6>GluRIIA
RNAi
+CaMKII
T287D
). If reduced CaMKII activity were functionally required
24

for retrograde PHP signaling, and not just a compartmentalized biomarker of GluRIIA levels
and/or activity at Ib boutons, then constitutively active CaMKII should disrupt PHP expression at
M6>GluRIIA
RNAi
synapses. Indeed, EPSP amplitude was not maintained at baseline levels due
to a failure to homeostatically increase quantal content in M6>GluRIIA
RNAi
+CaMKII
T287D
(Figure
8E-H). In this condition, quantal content was significantly increased (134±8% increase for
M6>GluRIIA
RNAi
+CaMKII
T287D
compared to M6>CaMKII
T287D
) but was below the level necessary
to maintain synaptic strength (166±9% increase in quantal content for M6>GluRIIA
RNAi

compared to wild type). This data is consistent with reduced CaMKII activity, compartmentalized
at Ib postsynaptic densities, being required for retrograde PHP signaling. We propose a model
schematized in Figure 8E.

25

2.4 Discussion
Although the genes and mechanisms that mediate retrograde homeostatic potentiation have
been intensively investigated, whether this process can be expressed and restricted to a subset
of synapses within a single neuron has not been determined. We have developed a
manipulation that enables the loss of GluRs on only one of the two postsynaptic targets
innervated by a Type Ib motor neuron at the Drosophila NMJ. Our analysis of synaptic structure
and function in this condition has revealed the spectacular degree of compartmentalization in
postsynaptic signaling and presynaptic expression that ultimately orchestrate the synapse-
specific modulation of presynaptic efficacy.

Compartmentalization of postsynaptic PHP signaling
GluRs are dynamically trafficked in postsynaptic compartments where they mediate the
synapse-specific expression of Hebbian plasticity such as LTP (Herring and Nicoll, 2016;
Matsuzaki et al., 2004) and homeostatic plasticity, including receptor scaling (Hou et al., 2008;
Pozo and Goda, 2010; Sutton et al., 2006). In contrast, homeostatic plasticity at the human,
mouse, and fly NMJ is expressed through a presynaptic enhancement in neurotransmitter
release, but is induced through a diminishment of postsynaptic neurotransmitter receptor
functionality (Cull-Candy et al., 1980; Frank et al., 2006; Petersen et al., 1997; Wang et al.,
2016b). Using biased expression of Gal4 to reduce GluR levels on only one of the two muscle
targets innervated by a single motor neuron, we demonstrate that the inductive signaling
underlying PHP is compartmentalized at the postsynaptic density, and does not influence
activity at synapses innervating the adjacent muscle. Postsynaptic changes in CaMKII function
and activity have been associated with PHP retrograde signaling (Goel et al., 2017; Haghighi et
al., 2003; Newman et al., 2017). Consistent with this compartmentalized inductive signaling, we
observed pCaMKII levels to be specifically reduced at postsynaptic densities of Ib boutons in
26

which GluR expression is perturbed, while pCaMKII was unchanged at postsynaptic
compartments opposite to Is boutons and at NMJs in the adjacent muscle with normal GluR
expression. Further, postsynaptic overexpression of the constitutively active CaMKII occludes
the expression of PHP. Similar synapse-specific control of postsynaptic CaMKII
phosphorylation, modulated by activity, has been previously observed (Hodge et al., 2006). As
noted in other studies (Goel et al., 2017; Newman et al., 2017), this localized reduction in
pCaMKII provides a plausible mechanism for the inductive PHP signaling restricted to and
compartmentalized at Ib synapses.
How does a perturbation to GluR function lead to a reduction in CaMKII activity that is
restricted to postsynaptic densities opposing Type Ib boutons? Recent evidence suggests that
distinct mechanisms regulate pCaMKII levels during retrograde PHP signaling depending on
pharmacologic or genetic perturbation to glutamate receptors and the role of protein synthesis
(Goel et al., 2017). Scaffolds at postsynaptic densities are associated in complexes with GluRs
and CaMKII (Gillespie and Hodge, 2013; Hodge et al., 2006; Koh et al., 1999; Lu et al., 2003;
Mullasseril et al., 2007). Intriguingly, the scaffold dCASK is capable of modulating CaMKII
activity at specific densities in an activity-dependent fashion (Hodge et al., 2006; Malik et al.,
2013). Further, CaMKII activity can regulate plasticity with specificity at subsets of synapses in
Drosophila and other systems (Griffith, 2004; Hodge et al., 2006; Merrill et al., 2005). Although
we cannot rule out intra-cellular “cross talk” between Is and Ib boutons, as GluRIIA is reduced at
postsynaptic sites of both neuronal subtypes, it is striking that reductions in pCaMKII are
restricted to Ib postsynaptic compartments. An attractive model, therefore, is that the
postsynaptic density isolates calcium signaling over chronic time scales to compartmentalize
PHP induction. The membranous complexity and geometry of the SSR at the Drosophila NMJ
may be the key to restricting calcium signaling at these sites, as this structure can have major
impacts on ionic signaling during synaptic transmission (Nguyen and Stewart, 2016; Teodoro et
al., 2013). These properties, in turn, may lead to local modulation of CaMKII function (Goel et
27

al., 2017; Griffith, 2004; Haghighi et al., 2003; Newman et al., 2017). Interestingly, Drosophila
mutants with defective SSR elaboration and complexity have been associated with defects in
PHP expression (Koles et al., 2015). In the mammalian central nervous system, it is well
established that dendritic spines function as biochemical compartments that isolate calcium
signaling while enabling propagation of voltage changes (Svoboda et al., 1997; Yuste and Denk,
1995), and it is tempting to speculate that the SSR may subserve similar functions at the
Drosophila NMJ to enable synapse-specific retrograde signaling.

Compartmentalization of presynaptic PHP expression
The homeostatic modulation of presynaptic neurotransmitter release is compartmentalized at
the terminals of Type Ib motor neurons. It was previously known that PHP can be acutely
induced and expressed without any information from the cell body of motor neurons (Frank et
al., 2006). Our data suggests that the signaling necessary for PHP expression is even further
restricted to specific postsynaptic densities and presynaptic boutons, demonstrated through
several lines of evidence. First, quantal content is specifically enhanced at boutons innervating
muscle 6 in M6>GluRIIA
RNAi
without measurably impacting transmission on the neighboring
boutons innervating muscle 7. In addition, PHP can be acutely induced at synapses innervating
muscle 7 despite PHP having been chronically expressed at muscle 6. Finally, the homeostatic
modulation of the RRP and enhancement of the functional number of release sites is fully
expressed regardless of whether PHP is induced at all Type Ib boutons or only a subset. Thus,
PHP signaling is orchestrated at specific boutons according to the state of GluR functionality of
their synaptic partners and does not influence neighboring boutons within the same motor
neuron.
Although the compartmentalized expression of PHP was not unexpected, there was
precedent to suspect inter-bouton crosstalk during homeostatic signaling. In the dynamic
propagation of action potentials along the axon, the waveform could, in principle, change
28

following PHP expression to globally modulate neurotransmission at all release sites in the
same neuron. However, voltage imaging did not identify any change in the action potential
waveform at individual boutons following PHP signaling (Ford and Davis, 2014; Gavino et al.,
2015), and we did not observe any impact on neighboring boutons despite PHP being induced
at a subset of synapses in the same motor neuron. Further, mobilization of an enhanced readily
releasable synaptic vesicle pool is necessary for the expression of PHP (Davis and Muller,
2015; Kiragasi et al., 2017; Muller et al., 2015; Weyhersmuller et al., 2011), and synaptic
vesicles and pools are highly mobile within and between presynaptic compartments (Darcy et
al., 2006; Kahms and Klingauf, 2018; Staras et al., 2010). Hence, it was conceivable that a
mobilized RRP, induced at some presynaptic compartments, may be promiscuously shared
between other boutons. However, while we observed a large enhancement in the RRP at
synapses innervating muscle 6 in M6>GluRIIA
RNAi
, this adaptation had no impact on the RRP at
adjacent presynaptic compartments innervating muscle 7. Thus, PHP signaling is constrained to
boutons innervating one of two postsynaptic targets and does not “spread” to synapses
innervating the adjacent target despite sharing common cytosol, voltage, and synaptic vesicles.
What molecular mechanisms mediate the remarkable specificity of PHP expression at
presynaptic compartments? One attractive possibility is that active zones themselves are
fundamental units and act as substrates for the homeostatic modulation of presynaptic function.
The active zone scaffold BRP remodels during both acute and chronic PHP expression (Goel et
al., 2017; Weyhersmuller et al., 2011), and other active zone proteins are likely to participate in
this remodeling (Gratz et al., 2019). Indeed, many genes encoding active zone components are
required for PHP expression, including the calcium channel cac (Frank et al., 2006) and
auxiliary subunit α2- δ (Wang et al., 2016a), the piccolo homolog fife (Bruckner et al., 2017), the
scaffolds RIM (Rab3-interacting Molecule; (Muller et al., 2012)) and RIM-binding protein (RBP;
(Muller et al., 2015)), and the kainite receptor DKaiR1D (Kiragasi et al., 2017). If individual
active zones can undergo the adaptations necessary and sufficient for PHP expression, this
29

would imply that PHP can be induced and expressed with specificity at individual active zones.
Indeed, the BRP cytomatrix stabilizes calcium channel levels at the active zone (Kittel et al.,
2006), and also controls the size of the RRP (Matkovic et al., 2013), two key presynaptic
expression mechanisms that drive PHP. Further, we and others have observed the recruitment
of new functional release sites following both chronic and acute PHP expression (Davis and
Muller, 2015; Newman et al., 2017; Weyhersmuller et al., 2011), suggesting that previously
silent active zones become “awakened” and utilized to potentiate presynaptic neurotransmitter
release. Interestingly, presynaptic GluRs, localized near active zones, are necessary for PHP
expression and have the capacity to modulate release with specificity at individual active zones
(Kiragasi et al., 2017). Thus, active zones have the capacity to remodel with both the specificity
and precision necessary and sufficient for compartmentalized PHP expression.
If each active zone operates as an independent homeostat to adjust release efficacy in
response to target-specific changes, how is information transfer at individual sites integrated to
ensure stable and stereotypic “global” levels of neurotransmission? One speculative possibility
is that active zones at terminals of each neuron are endowed with a total abundance of material
that is tightly controlled and sets stable global levels of presynaptic neurotransmitter release.
Such active zone material may be “sculpted” with considerable heterogeneity within presynaptic
terminals, varying in number, size, and density. Consistent with such a possibility, mutations in
the synaptic vesicle component Rab3 exhibit extreme changes in active zone size, number, and
density, but stable global levels of neurotransmission (Graf et al., 2009). Within this global
context, plasticity mechanisms may operate at individual active zones, superimposed as
independent homeostats to adaptively modulate synaptic strength. In addition, there is intriguing
evidence for the existence of “nanocolumns” between presynaptic active zones and
postsynaptic GluRs that form structural and functional signaling complexes (Biederer et al.,
2017; Tang et al., 2016). One particularly appealing possibility, therefore, is that a dialogue
traversing synaptic nanocolumns functions to convey the retrograde signaling and active zone
30

remodeling necessary for PHP expression at individual release sites. Studies in mammalian
neurons have revealed parallel links between the functional plasticity of active zones, including
their structure and size, and the homeostatic modulation of neurotransmitter release (Glebov et
al., 2017; Matz et al., 2010; Murthy et al., 2001; Schikorski and Stevens, 2001). Such
intercellular signaling systems are likely to modify synaptic structure and function to not only
establish precise pre- and post-synaptic apposition during development, but also to maintain the
plasticity necessary for synapses to persist with the flexibility and stability to last a lifetime.

31

2.5 Materials and Methods
Fly stocks and genetics: Drosophila stocks were raised at 25°C on standard molasses food.
The w
1118
strain is used as the wild type control unless otherwise noted, as this is the genetic
background of the transgenic lines and other genotypes used in this study. The following fly
stocks were used: G14-Gal4 (Aberle et al., 2002); GluRIIA
RNAi
(p{TRiP.JF02647}attP2};
Bloomington Drosophila Stock Center (BDSC)); UAS-CaMKII
T287D
(BDSC); mCherry
RNAi

(p{VALIUM20-mCherry}attP2}; BDSC); GluRIIA
SP16
(Petersen et al., 1997); M6-Gal4 (Tub-FRT-
STOP-FRT-Gal4, UAS-FLP, UAS-CD8-GFP; H94-Gal4, nSyb-Gal80) (Choi et al., 2014).

Immunocytochemistry: Third-instar larvae were dissected in ice cold 0 Ca
2+
modified HL-3
saline and immunostained as described (Kikuma et al., 2017). Briefly, larvae were fixed in
Bouin's fixative (Sigma, HT10132-1L) for 2 min and washed with PBS containing 0.1% Triton X-
100 (PBST) for 30 min, then blocked for 1 hour in 5% Normal Donkey Serum (NDS). Following
overnight incubation in primary antibodies at 4°C, preparations were washed in PBST,
incubated in secondary antibodies for 2 hours, washed and mounted in VectaShield (Vector
Laboratories). The following antibodies were used: guinea pig anti-vGlut (1:2000; (Chen et al.,
2017); rabbit anti-DLG (1:5000; (Pielage et al., 2005)); mouse anti-GluRIIA (8B4D2; 1:100;
Developmental Studies Hybridoma Bank (DSHB)); rabbit anti-GluRIIC (1:1000; (Marrus et al.,
2004)); guinea pig anti-GluRIID (1:1000; (Perry et al., 2017)); mouse anti-pCaMKII (1:100; MA1-
047; Invitrogen); mouse anti-GFP (8H11; 1:100; DSHB); Tetramethylrhodamine (TRITC)-
conjugated phalloidin (R415; Thermo Fisher); Alexa Fluor 647-conjugated goat anti-HRP (1:200;
Jackson ImmunoResearch). Donkey anti-mouse, anti-guinea pig, and anti-rabbit Alexa Fluor
488-, DyLight 405-, and Cyanine 3 (Cy3)-conjugated secondary antibodies (Jackson
ImmunoResearch) were used at 1:400.

32

Confocal imaging and analysis: Samples were imaged using a Nikon A1R Resonant
Scanning Confocal microscope equipped with NIS Elements software and a 100x APO 1.4NA
oil immersion objective using separate channels with four laser lines (405 nm, 488 nm, 561 nm,
and 637 nm) as described (Chen et al., 2017). All genotypes were immunostained in the same
tube with identical reagents, then mounted and imaged in the same session. z-stacks were
obtained using identical settings for all genotypes with z-axis spacing between 0.15 µm to 0.2
µm within an experiment and optimized for detection without saturation of the signal. Both Type
Ib and Is boutons were counted using vGlut and HRP-stained NMJ terminals on muscle 6/7 of
segment A3, considering each vGlut puncta to be a bouton. Muscle surface area was calculated
by creating a mask around the phalloidin channel that labels the entire muscle. The general
analysis toolkit in the NIS Elements software was used to quantify GluRIIA/C/D and pCamKII
intensity levels by applying intensity thresholds and filters to binary layers for each the channels
of the maximum intensity projection images. To quantify GluR intensity levels, the total
fluorescence intensity of each GluRIIA, GluRIIC, or GluRIID puncta was averaged over the NMJ
area covered by muscle 6 or muscle 7 separately to determine mean fluorescence intensity; this
value was then normalized as a percentage of wild type for the corresponding muscle. For
analysis of pCaMKII levels, Ib and Is regions were identified using DLG and HRP on muscle 6/7
of segment A2 and A3, and only the pCamKII signal that co-localized with DLG was summated
and divided by the bouton area under consideration to obtain average pCamKII.

quantitative PCR: quantitative PCR (qPCR) was performed using the Luna® Universal One-
Step RT-qPCR Kit (NEB, E3005S) according to the manufacturer’s instructions. RNA was
isolated and prepared from body wall tissue as described previously (Chen et al., 2017). 20 ng
of total RNA was used as the template for each reaction. Three biological replicates were
performed for each sample and the comparative Ct method was used for qPCR data analysis.
The following primers were used (fwd; rev: 5’-3’):
33

GluRIIA: TCCTCAACTTGGAACTGGAAAG; CGTACTTTTCCCTGCCTCTG.
GluRIIB: GCGAATACAGATGAATGGGATG; TGCATGAAGGGTACAGTGAAG.
GluRIIC: CGGAAAACTGGACAAGGAAAC; AGCTGCATAAAGGGCACTG.
GluRIID: CCCAAGCTGTCAACTTCAATG; CCATAACCCTGGAACTGATTGT.
GluRIIE: CGGTGCAAAGAAAACTGGATC; GTCTTAACTCGATTCACTCCCTC.
αTub84D (control): CTACAACTCCATCCTAACCACG; CAGGTTAGTGTAAGTGGGTCG.

Electrophysiology: All dissections and recordings were performed in modified HL-3 saline
(Dickman et al., 2005; Kiragasi et al., 2017; Stewart et al., 1994) at room temperature containing
(in mM): 70 NaCl, 5 KCl, 10 MgCl2, 10 NaHCO3, 115 Sucrose, 5 Trehelose, 5 HEPES, and 0.4
CaCl2 (unless otherwise specified), pH 7.2. Neuromuscular junction sharp electrode recordings
were performed on muscles 6 or 7 of abdominal segments A2 or A3 in wandering third-instar
larvae. Biased Gal4 expression was verified by verifying GFP fluorescence on the particular
muscle before experimentation, and recordings were performed at only the GFP-positive muscle
6 and the adjacent muscle 7. Muscle input resistance (Rin) and resting membrane potential
(Vrest) were monitored during each experiment (Supplementary Table 1). To acutely block
postsynaptic receptors, larvae were incubated with or without philanthotoxin-433 (20 μM;
Sigma) and resuspended in HL-3 for 10 mins, as described (Dickman and Davis, 2009; Frank et
al., 2006).
The readily releasable pool (RRP) size was estimated by analyzing cumulative EPSC
amplitudes while recording using a two-electrode voltage clamp (TEVC) configuration. Muscles
were clamped to -70 mV and EPSCs were evoked with a 60 Hz, 60 stimulus train while
recording in HL-3 supplemented with 3 mM Ca
2+
. A line fit to the linear phase (stimuli # 18-30) of
the cumulative EPSC data was back-extrapolated to time 0. The RRP value was estimated by
determining the extrapolated EPSC value at time 0 and dividing this value by the average
mEPSC amplitude. Data used in the variance-mean plot was obtained from TEVC recordings
34

using an initial 0.5 mM Ca
2+
concentration, which was subsequently increased to 1.5, 3.0, and
6.0 mM through saline exchange using a peristaltic pump (Langer Instruments, BT100-2J).
EPSC amplitudes were monitored during the exchange, and 30 EPSC (0.5 Hz stimulation rate)
recordings were performed in each calcium condition. To obtain the variance-mean plot, the
variance (squared standard deviation) and mean (averaged evoked amplitude) were calculated
from the 30 EPSCs at each individual Ca
2+
concentration. The variance was then plotted against
the mean for each specific calcium condition using MATLAB software (MathWorks, USA). One
additional data point, in which variance and mean are both theoretically at 0, was used for Ca
2+
-
free saline. Data from these five conditions were fit with a standard parabola (variance = Q*Ī -
Ī
2
/N), where Q is the quantal size, Ī is the mean evoked amplitude (x-axis), and N is the
functional number of release sites. N, as a parameter of the standard parabola, was directly
calculated for each cell by best parabolic fit.

Statistical analysis: All data are presented as mean +/-SEM. Data was compared using either
a one-way ANOVA followed by Tukey’s multiple comparison test, or using a Student’s t-test
(where specified). Data was analyzed using Graphpad Prism or Microsoft Excel software, with
varying levels of significance assessed as p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****),
ns: not significant. M6>GluRIIA
RNAi
results were compared to M6>mCherry
RNAi
(Figure 2-figure
supplement 2) and G14/+, in addition to w
1118
; in no case did the control change the statistically
significant result. See Supplementary Table 1 for further statistical details and values.

35

Supplementary Table 1: Absolute values for all data and additional statistics. The figure
and panel, genotype, and conditions used are noted (muscle segment, external calcium
concentration, PhTx application). All electrophysiological recordings were performed in 0.4 mM
external calcium unless specified otherwise. Average values for mEPSP, EPSP, quantal
content, resting potential, input resistance, number of data samples (n), p values, and
significance are shown. Average values for confocal imaging intensity levels are also indicated.
Standard error values are noted in parentheses.

Figure 1 Genotype

GluR puncta intensity (% WT)

n

p value (significance)
(GluRIIA, GluRIIC,
GluRIID)
GluRIIA GluRIIC GluRIID
1B w
1118
100
(4.171)
100
(2.460)
100
(2.184)
10
1B

w; GluRIIA
SP16

15.89
(1.090)
79.48
(2.161)
86.12
(1.990)
12
GluRIIA: <0.0001 (****);
GluRIIC: <0.0001 (****);
GluRIID: 0.00014 (***)
compare to w
1118

1B
w; G14-Gal4/+;UAS-
GluRIIA
RNAi
/+
19.73
(1.074)
74.00
(1.463)
82.08
(1.885)
12
GluRIIA: <0.0001 (****);
GluRIIC: <0.0001 (****);
GluRIID: <0.0001 (****)
compare to w
1118

Figure 1 Genotype

Transcript level (% G14-Gal4/+)

n

p value (significance)
(GluRIIA, GluRIIB,
GluRIIC, GluRIID,
GluRIIE) GluRIIA GluRIIB GluRIIC GluRIID GluRIIE
1C w; G14-Gal4/+

100
(3.030)
100
(6.816)
100
(19.388)
100
(6.023)
100
(11.099)
3
1C
w; G14-Gal4/+;
UAS-GluRIIA
RNAi
/+
18.757
(2.150)
105.857
(4.012)
99.889
(20.860)
96.0166
(5.518)
94.188
(1.326)
3
GluRIIA: <0.0001 (****);
GluRIIB: 0.477 (ns);
GluRIIC: 0.997 (ns);
GluRIID: 0.618 (ns);
GluRIIE: 0.514 (ns)
compare to w; G14-
Gal4/+
Figure 1 Genotype PhTX
mEPSP
(mV)
EPSP
(mV)
QC
Rinput
( M Ω )
Vmrest
(mV)
n

p value (significance)
(mEPSP, EPSP, QC)
1E,F,G,H w
1118
-
0.931
(0.042)
31.523
(1.825)
34.030
(1.889)
11.940
(0.665)
-66.536
(1.655)
8

1E,F,G,H w;GluRIIA
SP16
-
0.455
(0.019)
29.702
(1.171)
65.562
(1.819)
10.270
(0.928)
-67.867
(2.057)
11
<0.0001 (****);
0.390 (ns);
<0.0001 (****)
compare to w
1118

1E,F,G,H w; G14-Gal4/+
0.956
(0.045)
30.248
(0.753)
32.467
(1.778)
9.667
(0.762)
-67.057
(0.842)
12
0.705 (ns);
0.473 (ns);
0.566 (ns)
compare to w
1118

36

1E,F,G,H
w; G14-
Gal4/+;UAS-
GluRIIA
RNAi
/+
-
0.4485
(0.023)
28.070
(1.923)
64.837
(5.828)
12.360
(1.065)
-65.659
(3.736)
13
<0.0001 (****);
0.317 (ns);
<0.0001 (****)
compare to w;G14-
Gal4/+
Figure 2 Genotype Muscle Bouton number n

p value (significance)

2D

w
1118
7
48.111
(2.690)
12

2D

w;M6-Gal4/UAS-
GluRIIA
RNAi

7
47.125
(3.324)
13
0.822 (ns)
compare to w
1118
muscle 7

2D

w
1118
6
74.889
(3.221)
12

2D
w;M6-Gal4/UAS-
GluRIIA
RNAi

6
62.375
(3.693)
13
0.019 (*)
compare to w
1118

muscle 6
Figure 2 Genotype Muscle

GluR intensity (% WT)

n

p value (significance)
(GluRIIA, GluRIIC,
GluRIID)
GluRIID GluRIIA
2E w
1118
7

100
(2.193)
100
(3.061)
13
2E
w;M6-Gal4/
UAS-GluRIIA
RNAi

7
99.227
(5.014)
86.718
(7.523)
15
GluRIID: 0.903 (ns);
GluRIIA: 0.213 (ns)
compare to w
1118
muscle 7
2E w
1118

6

100
(1.926)
100
(2.523)
13
2E
w;M6-Gal4/
UAS-GluRIIA
RNAi

6
81.146
(5.227)
25.266
(2.212)
15
GluRIID: 0.002 (**);
GluRIIA: <0.0001 (****)
compare to w
1118
muscle 6
Figure 3 Genotype Muscle PhTx
mEPSP
(mV)
EPSP
(mV)
QC
Rinput
( M Ω )
Vmrest
(mV)
n

p value (significance)
(mEPSP, EPSP, QC)
3B,C,D,E w
1118
7 -
1.046
(0.062)
32.708
(1.134)
33.234
(1.992)
13.345
(0.646)
-65.586
(2.515)
9

3B,C,D,E
w;M6-Gal4/
UAS-
GluRIIA
RNAi

7 -
1.083
(0.037)
32.926
(0.982)
29.189
(1.334)
12.877
(1.923)
-65.867
(1.862)
9
0.985 (ns);
0.523 (ns);
0.528 (ns)
compare to w
1118
muscle 7
3B,C,D,E w
1118
6 -
1.035
(0.084)
34.485
(1.374)
33.320
(2.175)
8.622
(0.648)
-64.235
(1.985)
9

3B,C,D,E
w;M6-Gal4/
UAS-
GluRIIA
RNAi
6 -
0.563
(0.032)
31.116
(1.941)
55.705
(3.036)
9.262
(1.659)
-67.253
(2.615)
9
<0.0001 (****);
0.418 (ns);
<0.0001 (****)
compare to w
1118

muscle 6
37

Figure 4 Genotype Muscle PhTx
mEPSP
(mV)
EPSP
(mV)
QC
Rinput
( M Ω )
Vmrest
(mV)
n

p value (significance)
(mEPSP, EPSP, QC)
4B,C,D,E w
1118
7 -
1.084
(0.049)
32.708
(1.134)
30.769
(2.052)
12.025
(1.622)
-65.256
(3.024)
10

4B,C,D,E w
1118
7 +
0.492
(0.031)
31.034
(2.138)
64.436
(5.965)
13.135
(2.315)
-64.216
(1.625)
9
<0.0001 (****);
0.587 (ns);
<0.0001 (****)
compare to w
1118
muscle 7
4B,C,D,E
w;M6-Gal4/
UAS-
GluRIIA
RNAi
7 +
0.512
(0.021)
30.082
(1.401)
58.930
(2.109)
12.362
(1.035)
-62.427
(1.076)
8
<0.0001 (****);
0.256 (ns);
<0.0001 (****)
compare to w
1118
muscle 7
4B,C,D,E w
1118
6 -
1.008
(0.049)
33.578
(2.148)
33.578
(2.148)
8.065
(1.054)
-66.042
(2.145)
10

4B,C,D,E w
1118
6 +
0.501
(0.027)
34.930
(1.281)
69.023
(1.730)
7.356
(1.953)
-66.487
(2.541)
9
<0.0001 (****);
0.606 (ns);
<0.0001 (****)
compare to w
1118

muscle 6
4B,C,D,E
w;M6-Gal4/
UAS-
GluRIIA
RNAi
6 +
0.436
(0.023)
31.993
(1.311)
75.432
(5.608)
8.142
(1.014)
-66.805
(2.565)
8
<0.0001 (****);
0.298 (ns);
<0.0001 (****)
compare to w
1118

muscle 6
Figure 5 Genotype Muscle
[Ca
2+
]
(mM)

mEPSC
(nA)
Cumulative
EPSC
(nA)
Estimated
RRP size
n

p value (significance)
(mEPSC, Cum EPSC,
RRP size)
5C,D,E w
1118
7 3
0.748
(0.022)
542.200
(91.901)
637.200
(46.990)
9

5C,D,E
w;M6-Gal4/
UAS-GluRIIA
RNAi

7 3
0.697
(0.029)
439.757
(32.810)
719.100
(122.000)
8
0.176 (ns);
0.334 (ns);
0.522 (ns)
compare to w
1118
muscle
7
5C,D,E w
1118
6 3
0.810
(0.022)
850.604
(75.953)
1039.000
(75.680)
9

5C,D,E
w;M6-Gal4/
UAS-GluRIIA
RNAi
6 3
0.354
(0.019)
679.684
(45.304)
1681.700
(158.200)
8
<0.0001 (****);
0.081 (ns);
0.0002 (***)
compare to w
1118
muscle
6
Figure 6 Genotype
Muscl
e
N n

p value (significance)

6C w
1118
7
405.750
(22.420)
8

6C w;GluRIIA
SP16
7
675.857
(28.412)
7

<0.0001 (****)
compare to w
1118

muscle 7
6C
w;G14-Gal4/+;
UAS-GluRIIA
RNAi
/+
7
394.000
(14.731)
7

0.679 (ns)
compare to w
1118

muscle 7
6F w
1118
6
473.076
(32.645)
11

38

6F w;GluRIIA
SP16
6
791.669
(61.599)
7

0.00013 (***)
compare to w
1118
muscle 6
6F
w;G14-Gal4/+;
UAS-GluRIIA
RNAi
/+
6 796.125
(52.551)
8
<0.0001 (****)
compare to w
1118
muscle 6
Figure 7 Genotype
Muscl
e

pCaMKII intensity (% WT)

n

p value (significance)

Ib Is
7C,E w
1118
7

100
(10.087)
100
(9.030)
11
7C,E
w;M6-Gal4/UAS-
GluRIIA
RNAi
7
111.480
(8.816)
99.262
(11.717)
13
Ib: 0.412 (ns);
Is: 0.935 (ns)
compare to w
1118
muscle 7
7C,E w
1118

6

100
(10.696)
100
(12.584)
11
7C,E
w;M6-Gal4/UAS-
GluRIIA
RNAi
6
56.431
(5.649)
106.833
(9.926)
13
Ib: 0.0006 (***);
Is: 0.616 (ns)
compare to w
1118
muscle 6
Figure 8 Genotype Muscle PhTx
mEPSP
(mV)
EPSP
(mV)
QC
Rinput
( M Ω )
Vmrest
(mV)
n

p value (significance)
(mEPSP, EPSP, QC)
8B,C,D w
1118
7 -
1.033
(0.043)
35.192
(2.192)
34.708
(3.167)
9.143
(0.595)
-67.983
(3.633)
7

8B,C,D
w;UAS-
CaMKII-
T287D/+;M6-
Gal4/+
7 -
1.090
(0.030)
34.895
(1.640)
32.076
(1.421)
8.300
(1.165)
-68.992
(1.295)
10
0.275 (ns);
0.913 (ns);
0.413 (ns)
compare to w
1118
muscle 7
8B,C,D w
1118
6 -
0.994
(0.059)
36.266
(1.415)
37.590
(3.294)
7.571
(0.997)
-72.023
(2.796)
7

8B,C,D
w;UAS-
CaMKII-
T287D/+;M6-
Gal4/+
6 -
1.034
(0.058)
34.987
(1.494)
34.414
(1.733)
7.200
(0.629)
-71.688
(2.195)
10
0.651 (ns);
0.561 (ns);
0.369 (ns)
compare to w
1118

muscle 6
8F,G,H
w;M6-Gal4/
UAS-
GluRIIA
RNAi
7 -
1.085
(0.033)
34.503
(1.600)
31.945
(1.470)
9.200
(1.047)
-65.554
(1.965)
11

8F,G,H
w; UAS-
CaMKII-
T287D/+;M6-
Gal4/
UAS-
GluRIIA
RNAi
7 -
1.055
(0.039)
36.742
(0.871)
35.217
(1.144)
10.083
(0.753)
-66.599
(0.930)
12
0.569 (ns);
0.222 (ns);
0.091 (ns)
compare to w;M6-
Gal4/
UAS-GluRIIA
RNAi
muscle 7
8F,G,H
w;M6-Gal4/
UAS-
GluRIIA
RNAi
6 -
0.560
(0.028)
34.625
(2.082)
62.871
(4.061)
8.625
(0.596)
-70.999
(1.648)
11

8F,G,H
w; UAS-
CaMKII-
T287D/+;M6-
Gal4/
UAS-
GluRIIA
RNAi
6 -
0.510
(0.021)
23.308
(1.573)
46.025
(2.918)
9.583
(1.048)
-64.079
(1.162)
12
0.162 (ns);
0.00026 (***);
0.00262 (**)
compare to w;M6-
Gal4/
UAS-GluRIIA
RNAi

muscle 6
39

Supplemental
Figure 1
Genotype Muscle Bouton number n

p value (significance)

S1B

w
1118
7
33.5
(2.036)
12

S1B

w; GluRIIA
sp16

7
27.667
(1.994)
12
0.053 (*),
compare to w
1118

muscle 6

S1B

w;G14-Gal4/+;
UAS-GluRIIA
RNAi
/+
7
27.583
(1.9001)
12
0.045 (*),
compare to w
1118

muscle 6

S1B

w
1118
6
60.000
(2.874)
12

S1B

w; GluRIIA
sp16

6
50.833
(1.744)
12
0.012 (*)
compare to w
1118
muscle 7

S1B

w;G14-Gal4/+;
UAS-GluRIIA
RNAi
/+
6
51.750
(1.615)
12
0.020 (*)
compare to w
1118
muscle 7
Supplemental
Figure 2
Genotype Muscle
muscle
surface area
( μm
2
)

Bouton #/muscle

BRP puncta
#/muscle
n
p value (significance)
(muscle surface area,
bouton #, BRP puncta #)
S2B,C,D w
1118
7

1151.023
(41.576)
39.363
(1.725)
185.000
(14.431)
10
S2B,C,D
w;M6-
Gal4/UAS-
mCherry
RNAi
7
1069.876
(46.821)
37.179
(2.080)
189.250
(5.795)
9
0.211 (ns),
0.4268 (ns),
0.7963 (ns),
compare to w
1118
muscle 7
S2B,C,D w
1118
6
1916.771
(123.294)
54.137
(2.870)
259.556
(14.715)
10
S2B,C,D
w;M6-Gal4/
UAS-
mCherry
RNAi
6

1723.829
(35.631)
54.667
(2.552)
274.167
(17.857)
9
0.170 (ns),
0.8929 (ns),
0.533 (ns),
compare to w
1118
muscle 6
Supplem
ental
Figure 2
Genotype
Muscl
e
PhT
x
mEPSP
(mV)
EPSP
(mV)
QC
Rinput
( M Ω )
Vmrest
(mV)
n

p value (significance)
(mEPSP, EPSP, QC)
S2F,G,H w
1118
7 -
1.084
(0.049)
32.708
(1.134)
30.769
(2.052)
11.825
(0.974)
-65.105
(1.128)
9

S2F,G,H
w;M6-
Gal4/UAS-
mCherry
RN
Ai
7 -
1.046
(0.062)
33.725
(1.532)
33.234
(1.992)
10.867
(0.576)
-66.201
(1.195)
15
0.668 (ns),
0.645 (ns),
0.425 (ns)
compare to w
1118
muscle 7
S2F,G,H w
1118
6 -
1.008
(0.062)
32.926
(0.982)
33. 587
(2.148)
8.865
(1.754)
-63.689
(0.949)
9

S2F,G,H
w;M6-
Gal4/
UAS-
mCherry
RN
Ai
6 -
1.035
(0.084)
34.485
(1.374)
33.320
(2.175)
7.185
(1.002)
-67.055
(1.978)
15
0.825 (ns),
0.430 (ns),
0.612 (ns)
compare to w
1118

muscle 6
100 msec 10 mV
2 mV
quantal content
0
20
60
40
80
EPSP (mV)
0
10
30
20
35
mEPSP (mV)
0
0.4
0.8
D E
200 msec
0.6
1.0
0.2
****
ns
****
B
GluR puncta
intensity (% WT)
GluRIID
0
GluRIIA
50
GluRIIC
****
100
C
A
C
D
E
B
C
D
E
****
****
20 μm 20 μm
GluRIIA wild type
A
G14>GluRIIA
RNAi
F G
GluRIIC
GluRIIA
GluRIID
2 µm
transcript level
(% G14/+)
0
50
100
A B C D E
H
% wild type
0
50
100
200
****
****
150
mEPSP quantal
content
****
****
ns
***
****
****
****
****
****
ns
ns
ns
Figure 1: GluRIIA knock down phenocopies GluRIIA mutants. (A) Representative images of
NMJs on muscle 6 immunostained with antibodies that recognize GluRIIA, GluRIIC, and GluRIID
receptor subunits in wild type (w
1118
), GluRIIA mutants (w
1118
;GluRIIA
SP16
), and G14>GluRIIA
RNAi
(w
1118
;G14-Gal4/+;UAS-GluRIIA
RNAi
/+). (B) Quantification of the mean fluorescence intensity of
individual GluR puncta reveals GluRIIA subunits are virtually undetectable at NMJs of both GluRIIA
mutants and G14>GluRIIA
RNAi
, while the essential subunits GluRIIC and GluRIID are moderately
reduced, reflecting expression of the remaining GluRIIB-containing receptors. (C) Left: Schematic
illustrating the composition of GluRIIA-containing and GluRIIB-containing postsynaptic receptor
subtypes at the Drosophila NMJ. Right: Quantitative PCR analysis of GluR transcript levels for
GluRIIA/B/C/D/E subunits in G14>GluRIIA
RNAi
normalized to G14/+. (D) Representative
electrophysiological traces of EPSP and mEPSP recordings in the indicated genotypes. (E-G)
Quantification of mEPSP amplitude (E), EPSP amplitude (F), and quantal content (G) in the
indicated genotypes. Note that while mEPSP amplitudes are reduced to similar levels in GluRIIA
mutants and G14>GluRIIA
RNAi
, EPSP amplitudes remain similar to wild type because of a
homeostatic increase in presynaptic glutamate release (quantal content). (H) Quantification of
mEPSP amplitude and quantal content values of the indicated genotypes normalized to wild type
values.
40
wild type M6>GluRIIA
RNAi
A
HRP
GluRIIA
6
M6
M7 M6 M7 M6
D E
GluRIID puncta
intensity (% WT)
0
50
100
bouton #/muscle
0
40
M6
100
80
60
20
M7
*
ns
M7 M6 M7 M6 C
GluRIID
2 µm
GluRIIA
M7 M6
**
ns
20 μm
M7 B M6 M7 M6 M7 M6 M7
0
50
100
****
M7 M6
ns
GluRIIA puncta
intensity (% WT)
Figure 2: GluRIIA-containing receptors can be knocked down specifically on muscle 6 using
M6>Gal4. (A) Schematic of the Type Ib motor neuron innervating both muscle 6 and 7 at the
Drosophila NMJ. Gal4 is expressed specifically on muscle 6 in combination with UAS-GluRIIA
RNAi
using M6>Gal4 (M6>GluRIIA
RNAi
: w
1118
;Tub-FRT-STOP-FRT-Gal4, UAS-FLP, UAS-CD8-GFP/+;
H94-Gal4, nSyb-Gal80/UAS-GluRIIA
RNAi
). GluRIIA expression is reduced on muscle 6, while
expression of GluRIIA on muscle 7 is unperturbed. (B) Representative images of muscle 6/7 NMJs
immunostained with antibodies that recognize the neuronal membrane (HRP) and the postsynaptic
GluR subunit GluRIIA in wild type and M6>GluRIIA
RNAi
. (C) Representative images of individual Ib
synaptic boutons immunostained with anti-GluRIIA and anti-GluRIID on the indicated muscles in
wild type and M6>GluRIIA
RNAi
. (D) Quantification of bouton numbers on muscle 6 and 7 in wild type
and M6>GluRIIA
RNAi
. (E) Quantification of mean fluorescence intensity of GluRIIA and GluRIID
puncta normalized to wild type on the indicated muscles.
41
A wild type M6>GluRIIA
RNAi
C
100 msec
10 mV
2 mV
6
M7 M6 M7
M6
mEPSP (mV)
0
0.5
1.0
M6 M7
EPSP (mV)
0
M6 M7
10
30
****
20
40 ns
ns
ns
quantal content
0
20
M6 M7
40
60 ****
ns
200 msec
B
M6>GluRIIA
RNAi
(% wild type)
0
50
M6 M7
100
200
****
ns
ns
****
150
mEPSP quantal
content
wild type
M6>
GluRIIA
RNAi
D E
M6>
GluRIIA
RNAi
Figure 3: Presynaptic homeostatic potentiation can be induced and expressed exclusively
at synapses innervating muscle 6. (A) Schematic and electrophysiological traces of recordings
from muscles 7 and 6 of wild type and M6>GluRIIA
RNAi
NMJs. Note that mEPSPs are reduced
only on muscle 6 of M6>GluRIIA
RNAi
, while EPSP amplitudes are similar across all muscles. Thus,
the expression of PHP is restricted to synapses innervating muscle 6 of M6>GluRIIA
RNAi
and does
not impact neurotransmission at neighboring synapses within the same motor neuron. (B)
Quantification of mEPSP amplitude and quantal content values of M6>GluRIIA
RNAi
normalized to
wild type values. (C-E) Quantification of mEPSP amplitude (C), EPSP amplitude (D), and quantal
content (E) in the indicated muscles and genotypes.
42
200 msec
10 mV
2 mV
200 msec
M6>GluRIIA
RNAi
+PhTx
M7
M6
A wild type+PhTx
M7 M6
C
EPSP (mV)
0
M6 M7
10
30
20
40
ns
quantal content
0
20
M6 M7
60
80
wild type
M6>
GluRIIA
RNAi
+PhTx
M6>GluRIIA
RNAi
+PhTx (% baseline)
0
100
B
150
mEPSP quantal
content
****
****
mEPSP (mV)
0
0.5
M6 M7
1.0
40
**
**
ns
**** ****
**** ****
wild type+PhTx
200
D E
M6 M7
50
M6>
GluRIIA
RNAi
+PhTx
****
****
****
****
Figure 4: PHP can be induced and expressed acutely on muscle 7 following chronic
GluRIIA knock down on M6. (A) Schematic and representative traces illustrating the acute
application of PhTx on wild type and M6>GluRIIA
RNAi
NMJs. The acute expression of PHP was
observed on previously non-potentiated muscle 7 synapses in M6>GluRIIA
RNAi
NMJs. mEPSP
amplitudes are diminished at wild type and M6>GluRIIA
RNAi
NMJs on both muscles following
PhTx application, while EPSP amplitudes are maintained at baseline levels due to a homeostatic
increase in presynaptic neurotransmitter release. (B) Quantification of mEPSP and quantal
content values normalized to baseline values (-PhTx) at muscle 6 and 7 NMJs of M6>GluRIIA
RNAi
larvae. (C-E) Quantification of mEPSP amplitude (C), EPSP amplitude (D), and quantal content
(E) values in the indicated genotypes.
4 3
B
cum. EPSC (nA)
1 s
0
1000
2000
3000
100 nA
cum. EPSC (nA)
D
M6 M7
ns
0
200
1000
800
400
600
ns
estimated RRP size
M6 M7
0
500
1000
2000
1500
***
E
ns
A wild type
M6>GluRIIA
RNAi
200 ms
mEPSC (nA)
C
M6 M7
****
0
0.2
0.8
0.4
0.6
ns
M7 M6 M7 M6
Figure 5: Homeostatic modulation of the readily releasable vesicle pool is restricted to
synapses innervating muscle 6 at M6>GluRIIA
RNAi
NMJs. (A) Schematic illustrating the size of
the RRP is enhanced specifically at the terminals innervating muscle 6 in M6>GluRIIA
RNAi
.
Representative traces of two-electrode voltage clamp recordings (30 stimuli at 60 Hz in 3 mM
extracellular Ca
2+
) from muscles 7 or 6 in the indicated genotypes. (B) Averaged cumulative
EPSC amplitude plotted as a function of time. A line fit to the 18-30
th
stimuli was back-
extrapolated to time 0. (C-E) Quantification of mEPSC amplitude (C), average cumulative EPSC
values (D), and estimated readily releasable pool (RRP) sizes (E) for the indicated muscles and
genotypes. Note that RRP size is significantly increased at muscle 6 NMJs, but no change is
observed at muscle 7.
4 4
muscle 7
C A
E
B
N # (muscle 6)
0
1200
1000
WT GluRIIA
***
0.5 1.5 3
EPSC (nA)
50
100
150
0
200
6
WT
GluRIIA
calcium concentration (mM)
****
WT
GluRIIA
C
M6>GluRIIA
RNAi
M6>
GluRIIA
RNAi
M6>GluRIIA
RNAi
0
variance (nA
2
)
0
100
10
20
40
30
200
EPSC (nA)
G F
N # (muscle 7)
0
200
800
600
400
M6>
GluRIIA
RNAi
WT
ns
0
5
15
25
20
10
0 100 200 150 50
EPSC (nA)
variance (nA
2
)
50
100
150
0
200
EPSC (nA)
0.5 1.5 3 6
WT
GluRIIA
M6>GluRIIA
RNAi
GluRIIA
****
WT
GluRIIA
M6>GluRIIA
RNAi
calcium concentration (mM)
800
600
400
200
M6>GluRIIA
RNAi
D
H
M6>GluRIIA
RNAi
muscle 6
Figure 6: The number of functional release sites are specifically enhanced in synapses
innervating muscle 6 at M6>GluRIIA
RNAi
NMJs. (A) Schematic illustrating the number of
functional release sites (marked as red triangles) at synapses innervating muscle 7 of
M6>GluRIIA
RNAi
NMJs. (B) Scatter plot EPSC distribution of recordings derived from muscle 7 of
wild type, GluRIIA mutants, and M6>GluRIIA
RNAi
in the indicated extracellular Ca
2+
concentrations. (C) Variance-mean plots for the indicated genotypes. Variance was plotted
against the mean amplitude of 30 EPSCs recorded at 0.2 Hz from the four Ca
2+
concentrations
detailed in (B). Dashed lines are the best fit parabolas to the data points. (D) Estimated number of
functional release sites (N) obtained from the variance-mean plots in (C). Note that the number of
functional release sites are enhanced only at muscle 7 NMJs of GluRIIA mutants. (E) Schematic
illustrating the number of functional release sites (marked as red triangles) at synapses
innervating muscle 6 of M6>GluRIIA
RNAi
NMJs. (F) Scatter plot EPSC distribution of recordings
derived from muscle 6 of wild type, GluRIIA mutants, and M6>GluRIIA
RNAi
in the indicated
extracellular Ca
2+
concentrations. (G) Variance-mean plots for the indicated genotypes. (H)
Estimated number of functional release sites (N) obtained from the variance-mean plots in (G).
Both GluRIIA mutants and M6>GluRIIA
RNAi
showed a significant enhancement of functional
release site number at muscle 6 NMJs compared to wild type.
4 5
6
E
% wild type
0
50
Is pCaMKII
intensity
100
150
ns
ns
C
% wild type
0
50
Ib pCaMKII
intensity
100
150
ns
***
Is
Ib
Is
pCaMKII
HRP
20 μm
wild type M6> GluRIIA
RNAi
A
B
muscle 7 muscle 6 muscle 7
D muscle 7 muscle 6 muscle 7
2 µm
pCaMKII
DLG
pCaMKII
DLG
Is
Ib
Ib
Ib
Is
muscle 6
muscle 6
Ib Is
M7 M6 M7 M6
Figure 7: Compartmentalized changes in CaMKII activity at postsynaptic densities of
M6>GluRIIA
RNAi
NMJs. (A) Representative images of muscle 6/7 NMJs from wild type and
M6>GluRIIA
RNAi
immunostained with antibodies against with the active (phosphorylated) form of
CaMKII (pCaMKII) and the presynaptic neuronal membrane marker HRP. Note that pCaMKII
intensity is specifically reduced at Ib postsynaptic densities on muscle 6 of M6>GluRIIA
RNAi
(dashed areas). (B,C) Images (B) and quantification (C) of pCaMKII fluorescence intensity at Ib
boutons on muscles 7 and 6 normalized to wild type values. A reduction in pCaMKII intensity is
observed on muscle 6 of M6>GluRIIA
RNAi
, while no significant change is observed at synapses
innervating the adjacent muscle 7. (D,E) Images (D) and quantification (E) of pCaMKII intensity at
Is boutons shows no significant difference between wild type and M6>GluRIIA
RNAi
at NMJs of
either muscle.
4 6
250 ms
2 mV
50 ms
10 mV
B
mEPSP (mV)
0
0.5
1.0
M6 M7
ns
ns
EPSP (mV)
0
M6 M7
10
30
20
40
ns ns
quantal content
0
20
M6 M7
30
40
ns
ns
wild type
M6>CaMKII
T287D
C D
10
F G H
mEPSP (mV)
0
0.5
1.0
M6 M7
ns
ns
EPSP (mV)
0
M6 M7
10
30
20
40 ns
***
quantal content
0
40
M6 M7
60
**
ns
20
M6>GluRIIA
RNAi
M6>GluRIIA
RNAi
+CaMKII
T287D
A
M7
M6
E
M7 M6
wild type M6>CaMKII
T287D
M6>GluRIIA
RNAi
M6>GluRIIA
RNAi
+CaMKII
T287D
pCaMKII pCaMKII pCaM
KII
pCaMKII
pCaMKII pCaMKII
M6>CaMKII
T287D
M6>GluRIIA
RNAi
+CaMKII
T287D
Figure 8: Overexpression of constitutively active CaMKII inhibits retrograde PHP signaling
at M6>GluRIIA
RNAi
NMJs. (A) Schematic and representative traces illustrating that
overexpression of a constitutively active form of CaMKII only on muscle 6 (M6>CaMKII
T287D
) has
no significant impact on neurotransmission. (B-D) Quantification of mEPSP amplitude (B), EPSP
amplitude (C), and quantal content (D) in the indicated muscles. (E) Schematic and
representative traces illustrating that expression of constitutively active CaMKII with GluRIIA
knock down (M6>GluRIIA
RNAi
+CaMKII
T287D
) on muscle 6 inhibits the retrograde potentiation of
presynaptic glutamate release. (F-H) Quantification of mEPSP amplitude (F), EPSP amplitude
(G), and quantal content (H) in the indicated muscles.
4 7
B
bouton #/muscle
0
40
*
GluRIIA wild type A
G14>GluRIIA
RNAi
20
60
20 µm
vGlut
HRP
M6 M7
* *
*
Figure 1-figure supplement 1: Synaptic growth is mildly reduced at NMJs of GluRIIA
mutants and G14>GluRIIA
RNAi
. (A) Representative images of muscle 6/7 NMJs of wild type,
GluRIIA mutants, and G14>GluRIIA
RNAi
immunostained with antibodies against with the neuronal
membrane (HRP) and the synaptic vesicle marker vGlut. (B) Quantification of bouton number per
muscle segment in the indicated genotypes demonstrates that synaptic growth is reduced by
~18% in both GluRIIA mutants and G14>GluRIIA
RNAi
. Error bars indicate ±SEM. Asterisks
indicate statistical significance using one-way analysis of variance (ANOVA), followed by T uke y’ s
multiple-comparison test: (*) p<0.05.
4 8
F
B C D
0
20
40
60
M6 M7
bouton #/muscle
ns
BRP puncta #/muscle
0
100
200
300
M6 M7
0
5
10
15
M6 M7
20
muscle surface area (x100
µm
2
)
ns
ns
ns
ns
ns
mEPSP (mV)
0
0.5
1.0
M6 M7
EPSP (mV)
0
10
30
M6 M7
20
40
quantal content
0
10
30
M6 M7
20
40
ns
ns
ns
ns
ns ns
wild type M6>mCherry
RNAi
10 mV
200 msec
2 mV
100 msec
E
20 μm
HRP
wild type M6>mCherry
RNAi
A
M7 M6 M7 M6 M7 M6 M7 M6
Phalloidin
GFP
G H
M7 M6 M7 M6
Figure 2-figure supplement 2: Biased Gal-4 expression on muscle 6 does not alter synaptic
growth or function. (A) Representative images of muscle 6/7 NMJs of wild type and
M6>mCherry
RNAi
(w
1118
;Tub-FRT-STOP-FRT-Gal4, UAS-FLP, UAS-CD8-GFP/+; H94-Gal4, nSyb-
Gal80/UAS-mCherry
RNAi
) labeled with phalloidin (actin marker), GFP, and the neuronal membrane
marker HRP. (B-D) Quantification of muscle surface area (B), bouton number (C), and BRP
puncta number per NMJ (D). No significant differences in any of these parameters were
observed. (E) Schematic and electrophysiological traces of recordings from wild type and
M6>mCherry
RNAi
NMJs. (F-H) Quantification of mEPSP amplitude (F), EPSP amplitude (G), and
quantal content (H) from the indicated genotypes finds no significant difference in synaptic
physiology.
4 9
50
Chapter 3
A Glutamate Homeostat Controls the Presynaptic Inhibition of
Neurotransmitter Release
3.1 Abstract
We have interrogated the synaptic dialogue that enables the bi-directional, homeostatic control
of presynaptic efficacy at the glutamatergic Drosophila neuromuscular junction (NMJ). We find
that homeostatic depression and potentiation utilize disparate genetic, induction, and expression
mechanisms. Specifically, homeostatic potentiation is achieved through reduced CaMKII activity
postsynaptically and increased abundance of active zone material presynaptically at one of the
two neuronal subtypes innervating the NMJ, while homeostatic depression occurs without these
adaptations and is expressed at both neuronal subtypes. Further, homeostatic depression is
only induced through excess presynaptic glutamate release and operates with complete
disregard to the postsynaptic response. We propose that two independent homeostats modulate
presynaptic efficacy at the Drosophila NMJ: one is an inter-cellular signaling system that
potentiates synaptic strength following diminished postsynaptic excitability, while the other
stabilizes presynaptic glutamate release through an autocrine mechanism without feedback
from the postsynaptic compartment.
51
3.2 Introduction
Synapses have the remarkable ability to adaptively modulate synaptic strength when confronted
with diverse challenges that destabilize neurotransmission, yet the mechanisms controlling the
integration of these responses remain enigmatic. Homeostatic mechanisms operate to stabilize
synaptic activity in nervous systems of varied organisms ranging from invertebrates to humans
(Pozo and Goda, 2010). In these physiologic systems, destabilizing perturbations to
neurotransmission are offset by compensatory adaptations to postsynaptic neurotransmitter
receptors (synaptic scaling) and/or presynaptic efficacy that maintain normal levels of
functionality (Davis and Muller, 2015; Turrigiano, 2012). This phenomenon, termed homeostatic
synaptic plasticity, is thought to interface with Hebbian plasticity mechanisms to ensure stable
yet flexible ranges in synaptic strength (Turrigiano, 2017). While adaptive responses to
individual destabilizing perturbations have been characterized in significant detail, far less is
known about how homeostatic signaling systems integrate reactions to concurrent challenges,
particularly when these are in conflict.
The Drosophila NMJ is a powerful model system to study the bi-directional, homeostatic
control of synaptic strength. At this glutamatergic synapse, acute pharmacological and chronic
genetic manipulations that reduce postsynaptic glutamate receptor (GluR) function activates a
retrograde, trans-synaptic signaling system that triggers a compensatory increase in presynaptic
glutamate release, restoring baseline levels of synaptic strength (Frank, 2013). Because the
expression of this form of plasticity requires a presynaptic increase in neurotransmitter release,
this process is referred to as presynaptic homeostatic potentiation (PHP). Multiple lines of
evidence have established that the homeostat that governs PHP is exquisitely sensitive to
diminished postsynaptic excitability and operates through a retrograde enhancement of
presynaptic efficacy, stabilizing overall synaptic strength (Frank et al., 2006; Petersen et al.,
1997). Parallel phenomena have been observed at cholinergic NMJs in rodents and humans
52
(Cull-Candy et al., 1980), suggesting this is a fundamental and conserved form of synaptic
plasticity that does not depend on the neurotransmitter system.
In contrast to PHP, far less is known about the homeostat that governs an inverse
process at the Drosophila NMJ referred to as presynaptic homeostatic depression (PHD). The
first evidence for PHD, while not appreciated as such, was discovered while characterizing
mutations in synaptic vesicle endocytosis genes, where increased synaptic vesicle size was
found to result from defects in vesicle re-formation mechanisms (Chen et al., 2014; Dickman et
al., 2005; Marie et al., 2004; Verstreken et al., 2002). Independently, evidence for PHD was
found using a separate manipulation that also increased synaptic vesicle size through
overexpression of the vesicular glutamate transporter (vGlut; vGlut-OE) (Daniels et al., 2004).
Both defective endocytosis and vGlut-OE result in enlargement of individual synaptic vesicles,
leading to excess glutamate emitted from each synaptic vesicle and enhanced postsynaptic
responsiveness (quantal size). However, normal levels of synaptic strength (EPSP amplitude)
were observed due to a homeostatic reduction in the number of synaptic vesicles releasing
glutamate (quantal content). When the phenomenon of PHD was initially defined, one
hypothesis put forward was that PHD may be induced as an adaptive response to excess
glutamate itself (Daniels et al., 2004). However, more recently, PHD has been considered to be
a mechanism that stabilizes synaptic strength in the same way that PHP operates, implying that
PHD is calibrated as a homeostat that responds to overall synaptic strength (Gavino et al.,
2015). Despite these studies, the nature of the homeostat that controls PHD, as well as the
genes and mechanisms involved, remain much less understood relative to PHP. Indeed, it is not
even clear whether trans-synaptic communication is required to induce, express, or modulate
PHD.
We have characterized the adaptations to synaptic physiology, growth, structure, and
plasticity when PHP and PHD are induced and expressed alone and in conjunction at an
individual synapse. Several lines of evidence demonstrate that PHP and PHD are independent
53
processes that utilize distinct mechanisms to modulate presynaptic efficacy in opposing
directions and, notably, operate at separate neuronal subtypes. However, PHP and PHD are not
simply independent signaling systems that each tune presynaptic efficacy to maintain stable
levels of overall synaptic strength. Rather, our data indicates that PHP is indeed a homeostat
dedicated to maintaining overall synaptic strength, induced through retrograde signaling in the
postsynaptic compartment. However, PHD operates with complete indifference to the state of
the postsynaptic cell and is oblivious to overall synaptic strength, instead functioning cell
autonomously in the presynaptic neuron as a negative feedback system to homeostatically
modulate glutamate release itself.
54
3.3 Results
Bi-directional, homeostatic control of presynaptic efficacy at the Drosophila NMJ
Homeostatic regulation of presynaptic glutamate release can be induced and expressed at the
Drosophila NMJ. To characterize the mechanisms underlying PHD alone and when PHP and
PHD are combined at an individual synapse, we utilized four distinct conditions (schematized in
Figure 1A). Genetic mutations in the postsynaptic GluR subunit GluRIIA was used to assess the
chronic expression of PHP. In this mutant, reduced mEPSP amplitude but normal EPSP
amplitude is observed due to a homeostatic increase in presynaptic glutamate release (quantal
content; Figure 1A-E). To induce PHD, we overexpressed vGlut in motor neurons (vGlut-OE).
This increases mEPSP amplitude but synaptic strength is similar to wild type levels because of
a homeostatic reduction in quantal content (Figure 1A-E). Thus, in both PHP and PHD, quantal
content is inversely adjusted relative to quantal size, maintaining constant levels of synaptic
strength.
We next probed how a synapse adapts to a combination of genetic manipulations that
individually induce PHP or PHD expression. When we combined GluRIIA and vGlut-OE
(GluRIIA+vGlut-OE), quantal size was intermediate to either manipulation alone (Figure 1C),
consistent with reduced postsynaptic GluR expression but increased glutamate released per
vesicle. Nonetheless, EPSP amplitude and quantal content are maintained at levels similar to
control values (Figure 1A,D,E). We confirmed the expected GluRIIA and vGlut expression in all
four genotypes (Supplemental Figure 1). Finally, we observed robust scaling of quantal content
as a function of mEPSP amplitude in all genotypes, including GluRIIA+vGlut-OE (Figure 1F),
consistent with sensitivity of the homeostat to presynaptic glutamate release and/or
postsynaptic responsiveness. Thus, presynaptic glutamate release is under exquisite, bi-
directional homeostatic modulation and can be induced, expressed, and balanced at an
individual synapse, maintaining stable levels of synaptic strength over chronic time scales.
55
Conventional mechanisms drive the acute expression of PHP in a chronically depressed
synapse
PHP can be rapidly induced and expressed using an acute pharmacological method to block
postsynaptic GluRs with the antagonist philanthotoxin-433 (PhTx) (Frank et al., 2006). A recent
study demonstrated that PHP can be acutely expressed at vGlut-OE NMJs (Gavino et al.,
2015), as we have shown over chronic time scales (GluRIIA+vGlut-OE). We first confirmed this
result by applying PhTx to vGlut-OE NMJs, which resulted in the expected ~50% reduction in
mEPSP amplitude and a robust increase in quantal content, maintaining the homeostatic control
of EPSP amplitude (Figure 2A-C). Thus, presynaptic neurotransmitter release can be acutely
potentiated despite chronic depression induced by vGlut-OE, balancing overall synaptic
strength.
To assess whether conserved genetic mechanisms underlie PHP and PHD, we asked
whether the schizophrenia susceptibility gene dysbindin (dysb), necessary in motor neurons for
both acute and chronic forms of PHP expression (Dickman and Davis, 2009), is required for
PHD expression. We observed no difference in quantal content when vGlut-OE was combined
with dysb mutations (dysb+vGlut-OE; Figure 2D-F), suggesting that dysb plays no role in PHD
induction or expression, consistent with separate genetic mechanisms driving PHP and PHD
(Gavino et al., 2015; Kiragasi et al., 2017). We also probed whether conventional PHP
expression mechanisms remain utilized when PHP is induced at a homeostatically depressed
synapse. We applied PhTx to dysb+vGlut-OE synapses and observed a failure to
homeostatically potentiate quantal content (Figures 2D-2F), while PHD was normally expressed.
Therefore, conventional genetic mechanisms are required for the acute induction of PHP at a
synapse expressing PHD, while PHP and PHD utilize separate genetic expression mechanisms
that are superimposed when balanced at an individual synapse.
56
Ca
2+
cooperativity, release probability, and vesicle pools are balanced in GluRIIA+vGlut-
OE
PHP and PHD utilize shared but inverse changes in Ca
2+
influx at presynaptic terminals (Gavino
et al., 2015; Muller and Davis, 2012), but only PHP appears to involve modulation of the readily
releasable vesicle pool (RRP) when examined individually (Gavino et al., 2015; Muller et al.,
2012). However, it is unclear how these expression mechanisms are integrated when combined
at an individual synapse. We first measured the apparent Ca
2+
cooperativity of
neurotransmission by examining quantal content over a range of external Ca
2+
concentrations.
We observed no significant difference in the apparent Ca
2+
cooperativity in GluRIIA+vGlut-OE
(Figure 3A), which showed a similar slope compared to wild type, GluRIIA, and vGlut-OE.
Further, we found significant difference in EPSC amplitudes at elevated Ca
2+
concentrations
across all genotypes (Figure 3B). Together, this demonstrates presynaptic glutamate release is
properly tuned across a range of Ca
2+
conditions following individual or simultaneous
expressions of PHP and PHD at a single synapse.
We next performed a series of experiments to assess release probability (Pr) in GluRIIA
mutants, vGlut-OE, and GluRIIA+vGlut-OE. Failure analysis assesses presynaptic function
independently of mEPSP amplitude, which has been used to show that GluRIIA mutants exhibit
fewer failures (Petersen et al., 1997), while vGlut-OE displays increased failure rates (Daniels et
al., 2004). We observed the expected decrease in failure rate in GluRIIA mutants (consistent
with an increased Pr) and increase in failures in vGlut-OE relative to wild type (consistent with
reduced Pr; Figure 3C). Failure rate in GluRIIA+vGlut-OE was intermediate and not significantly
different from wild type, consistent with a balancing of PHP and PHD modulations (Figure 3C).
In addition, paired pulse ratios have been used to gauge Pr, where depression or facilitation is
observed in the second stimulus evoked shortly after an initial stimulus. At physiologic Ca
2+

conditions (1.5mM), paired pulse depression (PPD) is observed at the Drosophila NMJ (Bohme
et al., 2016). We found enhanced PPD in GluRIIA mutants, reduced PPD in vGlut-OE, and no
57
significant difference in GluRIIA+vGlut-OE compared to wild type (Figure 3D,E). Similarly, no
change in paired pulse facilitation (PPF) is observed in reduced extracellular Ca
2+
(0.3mM) in
GluRIIA+vGlut-OE, while reduced and enhanced PPF is observed in GluRIIA and vGlut-OE
respectively (Figure 3F,G). Hence, the results of failure analysis, PPD, and PPF are consistent
with PHP exhibiting increased release probability, vGlut-OE showing reduced Pr, and Pr being
similar to wild type when these opposing modulations are simultaneously expressed.
An increase in the readily releasable pool (RRP) is induced at synapses following acute
and chronic expressions of PHP (Kiragasi et al., 2017; Muller et al., 2012; Weyhersmuller et al.,
2011), but no changes in the vesicle pool are observed at synapses expressing PHD (Gavino et
al., 2015). We therefore measured the RRP in GluRIIA+vGlut-OE, a situation in which we would
predict an increase in the RRP as part of PHP expression, and no change contributed by PHD.
We performed two-electrode voltage clamp (TEVC) recordings in 3mM external Ca
2+
,
stimulating at 60 Hz and measuring the cumulative EPSC (Figure 3H-K). We observed the
expected increase in the RRP in GluRIIA mutants, no change in vGlut-OE, and an increase in
GluRIIA+vGlut-OE (Figure 3I). Thus, one key expression mechanism underlying PHP, an
increase in the RRP, can be fully induced at synapses also expressing PHD, demonstrating that
PHD induction does not occlude the mechanisms necessary for PHP expression.
Finally, vGlut-OE enlarges synaptic vesicle size (Daniels et al., 2004), which may have
secondary impacts on vesicle biogenesis, membrane trafficking, and/or mobility that could alter
synaptic physiology. We therefore measured the entire releasable synaptic vesicle pool size in
vGlut-OE using a temperature-sensitive allele of shibire (shi), the fly homolog of the endocytic
gene dynamin (Kidokoro et al., 2004). In this mutation, all forms of synaptic vesicle endocytosis
are blocked at the restrictive temperature (32°C) due to a failure of vesicle scission from the
membrane. In shi-mutant synapses, release ceased after ~350 s, demonstrating that all the
releasable synaptic vesicles had been depleted (Supplemental Figure 2A). We found that the
total releasable vesicle pool was equivalent in shi compared to shi;GluRIIA, shi;vGlut-OE, and
58
shi;GluRIIA+vGlut-OE NMJs (~88,000 total quanta; Supplemental Figure 2B). Thus, the total
releasable synaptic vesicle pool is unchanged despite the enlargement of individual vesicles
due to vGlut-OE.
The active zone scaffold BRP and Ca
2+
channel Cac are enhanced specifically at Ib
boutons in GluRIIA mutants but unchanged in vGlut-OE
It is unknown whether an anatomical change contributes to the stabilization of synaptic strength
in vGlut-OE alone or in GluRIIA+vGlut-OE. Indeed, synaptic growth may be impacted by
enlarged vesicle size, perhaps altering the balance of constitutive vs. regulated membrane
trafficking during development (Kikuma et al., 2017). Most muscles in Drosophila receive input
from two motor neurons exhibiting morphologically distinct terminals, Type Is (small) and Ib (big)
(Atwood et al., 1993), but synaptic growth at these terminals has not been defined in GluRIIA
mutants or vGlut-OE. We found a small reduction in the number of Ib boutons in GluRIIA
mutants, which was also observed in vGlut-OE and GluRIIA+vGlut-OE (Supplemental Figure
3A-D). Surprisingly, we observed an increase in Is boutons in both vGlut-OE and
GluRIIA+vGlut-OE with no change in GluRIIA mutants (Supplemental Figure 3A-D), leading to
no difference in total bouton number per NMJ (Supplemental Figure 3E). The reason for
enhanced synaptic growth at Is boutons in vGlut-OE is unclear, but may be related to the
enlarged synaptic vesicles and possibly enhanced membrane conferred at this high Pr neuronal
subtype. We also observed no significant difference in the density of active zones labeled by the
active zone scaffold bruchpilot (BRP) in either Ib or Is terminals (Supplemental Figure 3B-F) nor
in active zone-GluR apposition (Table S1). Thus, PHP and PHD expression have distinct
influences on synaptic growth at the Ib and Is motor neuron subtypes.
The intensity of the active zone scaffold BRP increases following both acute and chronic
PHP induction (Goel et al., 2017; Gratz et al., 2019; Weyhersmuller et al., 2011). However, this
remodeling has not been defined at Is vs. Ib boutons. We therefore imaged individual BRP
59
puncta and observed a significant increase in both the mean and sum intensity of puncta in
GluRIIA mutants specifically at terminals of Ib boutons, with no significant change at Is (Figure
4A-C,F-H). Further, we found no significant change in BRP intensity at Ib or Is terminals in
vGlut-OE, consistent with a previous study (Gavino et al., 2015). In GluRIIA+vGlut-OE, BRP
intensity was enhanced at Ib terminals, with no change at Is, as expected (Figure 4A-C,F-H).
These results are consistent with PHP signaling and BRP remodeling happening specifically at
Ib boutons and confirm that no obvious changes in the intensity of the BRP scaffold occurs as
part of PHD expression.
Next, we examined levels of the Ca
2+
channel Cacophony (Cac) at active zones of Is vs
Ib boutons following both PHP and PHD expression. Although presynaptic Ca
2+
influx increases
following PHP induction (Muller and Davis, 2012), and mutations in cac disrupt PHP expression
(Frank et al., 2006), levels of Cac itself have not been assessed. We overexpressed Cac-GFP
(Cac-GFP-OE) in neurons and quantified fluorescence intensity at active zones. First, we
verified that Cac-GFP-OE has no major impact on synaptic transmission in wild type or when
overexpressed in GluRIIA mutants (Supplemental Figure 4A-D). We found a significant increase
in Cac intensity (both mean and sum) at Ib boutons in both GluRIIA mutants and in
GluRIIA+vGlut-OE (Figure 4A,D,E), while no change was found at Is boutons (Figure 4F,I,J).
These results are consistent with a recent report that levels of Cac-GFP tagged at genomic loci
are enhanced following the acute induction of PHP with PhTx application (Gratz et al., 2019).
Thus, both Ca
2+
channels and BRP levels are enhanced specifically at Ib active zones in
GluRIIA mutants, presumably contributing to the increase Ca
2+
influx and release probability that
drives PHP expression.
Finally, we assessed the impact of PHD on Cac levels at Ib and Is boutons when Cac-
GFP is overexpressed with vGlut-OE. A previous study found a reduction in presynaptic Ca
2+

influx as well as an apparent decrease in the abundance of overexpressed Cac-GFP at both Is
and Ib active zones (Gavino et al., 2015). We repeated this experiment and while we did
60
observe a ~50% reduction in Cac-GFP-OE intensity at Is boutons of both vGlut-OE and
GluRIIA+vGlut-OE, we did not find a significant difference at Ib boutons (Figure 4A,D-F,I,J).
Electrophysiological recordings of vGlut-OE+Cac-GFP-OE revealed reduced EPSP amplitude in
this manipulation (Supplemental Figure 4E,F), suggesting that vGlut co-overexpression with
cac-GFP disrupts Ca
2+
channels at Ib active zones and inhibits synaptic transmission in a way
that may not reflect the behavior of endogenous Cac channels. Consistent with this idea, no
change in Cac-GFP levels were found at Is or Ib terminals of vGlut-OE when Cac abundance
was assessed using an alternative version of Cac-GFP tagged at genomic locus (Gratz et al.,
2019). Together, this data suggests that active zone structures are remodeled exclusively at Ib
boutons in GluRIIA mutants as part of chronic PHP signaling, consistent with functional studies
(Newman et al., 2017). However, we find no evidence for PHD-related adaptations in BRP at
either Is or Ib in vGlut-OE, nor were differences found in levels of endogenous Cac-GFP (Gratz
et al., 2019), consistent with a distinct expression mechanism.
Ca
2+
imaging reveals PHD is expressed at both Is and Ib terminals
It is an open question whether PHD is expressed at terminals of Ib and/or Is neuronal subtypes.
A Ca
2+
indicator localized to postsynaptic densities of the Drosophila NMJ was recently
developed and used to show that PHP is expressed exclusively at Ib boutons (Newman et al.,
2017). This indicator, SynapGCaMP6f, targets GCaMP6f to the postsynaptic density and
enables robust quantal imaging at Ib and Is bouton terminals. Active zones of Is boutons have
release probabilities 2-3 fold higher than those of Ib boutons, and a larger proportion of Ib active
zones become functional during repeated trains of activity (Newman et al., 2017). Large Ib
boutons were morphologically identified and functionally defined by facilitating during a 2 Hz
train (Figure 5B,E). In contrast, anatomically small Is boutons were functionally defined by
assaying the depression characteristic of this motor neuron following stimulation (Figure 5M).
Using these criteria, we observed the expected increase in the amplitude of spontaneous
61
quantal events at both Ib and Is terminals of NMJs in vGlut-OE compared with wild type (Figure
5C,K). However, although quantal size was enhanced in vGlut-OE, the Ca
2+
signal following
evoked release was significantly reduced compared with wild type at both Ib and Is terminals
(Figure 5D,E,L,M), demonstrating reduced quanta released/bouton (Figure 5G,O). This
demonstrates that vGlut-OE induces PHD at both Ib and Is motor subtypes, in contrast to PHP,
which is expressed only at Ib synapses. Thus, only PHD signaling is expressed at Is boutons,
while both PHP and PHD are simply superimposed when simultaneously expressed at a Ib
terminal.
Postsynaptic CaMKII levels are unaffected by PHD signaling
Many lines of evidence indicate that perturbation or loss of GluRIIA-containing postsynaptic
receptors induces PHP expression (Frank et al., 2006; Petersen et al., 1997). Although little is
known about the induction mechanism triggering PHP downstream of receptor perturbation,
levels of phosphorylated (active) CaMKII (pCaMKII) are reduced specifically at postsynaptic
densities of Ib boutons in GluRIIA mutants and after PhTx application (Goel et al., 2017;
Newman et al., 2017). CaMKII activity is bi-directionally regulated in opposing forms of Hebbian
plasticity, LTP and LTD, in mammalian systems (Coultrap et al., 2014; Pi et al., 2010). We
therefore considered that if PHD were a homeostat governing synaptic strength, similar to PHP
but in the opposite direction, then perhaps postsynaptic pCaMKII levels might be elevated
following PHD expression or otherwise altered when PHP and PHD are combined at an
individual NMJ. First, we confirmed a ~50% reduction in pCaMKII intensity specifically at Ib
postsynaptic densities of GluRIIA mutants (Figure 6A,B) and no change in pCaMKII levels in Is
boutons (Figure 6C,D). Excess glutamate release and increased mEPSP amplitude could, in
principle, enhance pCaMKII levels and induce retrograde PHD signaling, in a manner inverse to
PHP signaling. However, we observed no significant difference in pCaMKII levels at Ib or Is
postsynaptic densities in vGlut-OE compared to wild type (Figure 6A-D), revealing that
62
postsynaptic pCaMKII is not modulated during PHD signal transduction. Finally, pCaMKII levels
were reduced at Ib and unchanged at Is synapses in GluRIIA+vGlut-OE (Figure 6A-D),
consistent with PHP induction being superimposed on, but not influenced by, PHD expression
when both homeostatic processes are triggered at the same NMJ. Thus, a key inductive signal
involved in PHP expression in the postsynaptic compartment, pCaMKII, is not regulated by PHD
signaling.
PHD is not induced by or responsive to increased postsynaptic excitability
A variety of manipulations that disrupt postsynaptic GluRIIA-containing receptors induce
retrograde signaling from the muscle to enhance presynaptic glutamate release and stabilize
overall synaptic strength (Frank et al., 2006; Petersen et al., 1997). In contrast, the only process
known to be capable of inducing PHD at the Drosophila NMJ are mutations or manipulations in
the presynaptic motor neuron that increase synaptic vesicle size and presynaptic glutamate
release (Chen et al., 2014; Daniels et al., 2004; Dickman et al., 2005; Marie et al., 2004;
Verstreken et al., 2002). In our final set of experiments, we considered that if PHD was
controlled by a homeostat that stabilized overall synaptic strength similar to PHP, but inverse in
direction, then a converse perturbation, increased postsynaptic expression of GluRIIA-
containing receptors, might induce or modulate PHD expression. However, while GluRIIA
overexpression was reported to increase mEPSP amplitude, no compensatory change in
presynaptic neurotransmitter release was observed, resulting in increased EPSP amplitude
(DiAntonio et al., 1999; Petersen et al., 1997). We revisited this finding in light of our
characterization of PHD by vGlut-OE.
First, we overexpressed the GluRIIA subunit in the muscle (GluRIIA-OE), which
increased mEPSP amplitudes to levels similar to vGlut-OE (Figure 7A,B). We confirmed that
GluRIIA-OE enhanced GluRIIA-containing receptor abundance without impacting synaptic
growth (Figure S5A-D). However, no change in presynaptic neurotransmitter release was
63
observed, leading to a maladaptive, non-homeostatic increase in EPSP amplitude (Figure 7A-
C). Thus, while vGlut-OE and GluRIIA-OE each induce the same enhancement in miniature
activity in the postsynaptic cell, only excess presynaptic glutamate release, and not increased
postsynaptic responsiveness to glutamate, is capable of inducing PHD expression. Indeed, we
observed no scaling of quantal content as a function of mEPSP amplitude in GluRIIA-OE
(Figure S6).
Next, we applied PhTx to GluRIIA-OE synapses, which reduced mEPSP amplitude by
~60% (Figure 7A-C), as expected. This diminution of mEPSP activity now induced an
enhancement in presynaptic glutamate release (Figure 7A-C), demonstrating that PHP can be
induced and expressed when elevated postsynaptic GluR levels are acutely perturbed. Hence,
we find no evidence that retrograde communication modulates presynaptic efficacy when
muscle excitability is elevated.
Finally, we asked whether increasing GluR levels has any influence on PHD expression.
We combined both GluRIIA-OE and vGlut-OE manipulations (GluRIIA-OE+vGlut-OE), which led
to an enhancement in mEPSP amplitude above either manipulation alone (Figure 7D,E).
Although EPSP amplitude was also increased in this genotype, quantal content remained the
same as vGlut-OE alone (Figure 7D-F), demonstrating that PHD induction and expression is not
influenced by GluRIIA-OE. In addition, application of PhTx to GluRIIA-OE+vGlut-OE NMJs
reduced mEPSP amplitude and increased quantal content (Figure 7D-F).
These results illuminate two important points. First, there is no evidence for the
existence of a retrograde homeostatic signaling system that stabilizes synaptic strength when
muscle excitability is elevated. Second, PHD operates with complete obliviousness with regard
to the state of the postsynaptic cell and to overall synaptic strength, emitting and scaling
presynaptic glutamate release independently of the muscle response. This is in marked contrast
to PHP, which is exquisitely sensitive to reduced excitability in the muscle and extremely
responsive to reductions in synaptic strength.
64
3.4 Discussion
We have characterized the induction and expression mechanisms driving PHD and PHP alone
and defined how synaptic strength is balanced when these two processes are induced at the
same synapse. While the independent homeostats controlling PHP and PHD result in balanced
neurotransmission when induced at the same synapse, this is actually the result of a
superimposition of separate signaling systems operating at disparate yet overlapping motor
inputs, rather than a deliberate integrated response in the same neuron to stabilize synaptic
strength.
Independent expression mechanisms control PHP and PHD
Clearly, distinct genetic mechanisms underlie PHP and PHD signaling, as genes necessary for
PHP have no role in PHD. This is illustrated by loss of the gene dysbindin, which is absolutely
required for PHP expression (Dickman and Davis, 2009) but has no impact on PHD, consistent
with findings from other genes that function in PHP (Gavino et al., 2015; Kiragasi et al., 2017).
Thus, while PHP and PHD appear to be parallel processes that modulate presynaptic
neurotransmitter release in inverse directions, they do not employ overlapping genetic
machinery.
PHP and PHD also utilize distinct physiological expression mechanisms. Our study and
others have found PHP adaptations involve an increase in presynaptic release probability
mediated through increased Ca
2+
influx, active zone scaffolding, Ca
2+
channel abundance, and
enhancement of the readily releasable synaptic vesicle pool (Goel et al., 2017; Gratz et al.,
2019; Muller and Davis, 2012; Muller et al., 2012; Weyhersmuller et al., 2011). In contrast, PHD
reduces release probability through reduced Ca
2+
influx yet without a change in BRP, Cac, or
the size of the RRP (Gavino et al., 2015; Gratz et al., 2019). Indeed, remodeling of active zones
appears to be unique to PHP and to terminals of Ib boutons. The BRP scaffold controls the size
65
of the RRP (Matkovic et al., 2013) and stabilizes Cac channels (Kittel et al., 2006), so an
attractive hypothesis is that PHP requires enhancements in Cac and BRP to promote both Ca
2+

influx and increase RRP size. Interestingly, Ca
2+
channels and active zone scaffolds are also
homeostatically regulated to control presynaptic neurotransmitter release in mammalian
neurons (Thalhammer et al., 2017), suggesting such plasticity mechanisms may be
evolutionarily conserved. In contrast, PHD expression is orchestrated at both Ib and Is motor
neurons, but the active zone is not obviously remodeled, indicating the inhibition of presynaptic
efficacy is driven through a distinct mechanism that remains to be elucidated.
Distinct induction mechanisms gate PHP and PHD signaling
The postsynaptic induction mechanisms that orchestrate PHP signaling are enigmatic (Chen
and Dickman, 2017; Goel et al., 2017). However, it is clear that PHP signaling is extremely
sensitive to reductions in postsynaptic excitability which triggers an inter-cellular signaling
system that originates in the postsynaptic muscle to potentiate neurotransmitter release in the
presynaptic neuron. If PHD were a homeostat designed to stabilize synaptic strength in a way
that parallels PHP, then enhanced muscle excitability should induce a retrograde signaling
system to depress presynaptic glutamate release. However, our data and work from other
groups show that homeostatic depression is not induced when quantal size is increased
(Petersen et al., 1997). Rather, excess glutamate release from the motor neuron itself appears
to be necessary and sufficient to induce and express PHD. This suggests that an autocrine
mechanism triggers PHD signaling, where excess glutamate is sensed and transduced into a
reduction in presynaptic release. Such an autocrine mechanism was astutely discussed as a
possibility in the original vGlut-OE study (Daniels et al., 2004).
If an autocrine mechanism mediates PHD induction, this would imply the existence of
presynaptic GluRs that can sense excess glutamate and initiate presynaptic inhibition in
response. Presynaptic autoreceptors exist at both cholinergic and glutamatergic NMJs of
66
invertebrates and vertebrates that modulate presynaptic function (Kiragasi et al., 2017; Lerma
and Marques, 2013; Takayanagi-Kiya et al., 2016). One attractive candidate is the lone
metabotropic GluR encoded in the Drosophila genome, mGluRA. Interestingly, mGluRA is
present at presynaptic terminals of motor neurons at the larval NMJ and depresses presynaptic
release following excess glutamate released during high frequency stimulation (Bogdanik et al.,
2004). Another possibility is presynaptic NMDA receptors, which have been reported to exist at
the Drosophila NMJ (Schuster, 2006), homologs of which mediate presynaptic inhibition in
response to excess glutamate release in the mammalian hippocampus (Padamsey et al., 2017).
The nature of the glutamate sensor and autocrine signaling system that governs the induction
and expression of PHD remains to be defined.
Glutamate homeostasis at the Drosophila NMJ
Why doesn’t a homeostat governing synaptic strength exist at the NMJ that is responsive to
enhanced postsynaptic excitability? Proper control of muscle contraction is essential to life, and
NMJs in many systems utilize a safety factor that ensures neurotransmitter is released in
excess to induce muscle contraction. Hence, given this safety factor, it is not clear that a
postsynaptic signaling system at the NMJ is necessary to detect and respond to heightened
neurotransmitter release or sensitivity. Indeed, pharmacologic perturbations to cholinergic NMJs
that inhibit the enzymatic breakdown of neurotransmitter lead to rapid paralysis and death in
worms and mammals, and there is no evidence that homeostatic retrograde signaling systems
are induced to inhibit presynaptic neurotransmitter release during these challenges. Thus, a
retrograde homeostatic signaling system to depress presynaptic efficacy in response to
increased postsynaptic excitability may not have developed due to a lack of evolutionary
pressure.
Why, then, does a process like PHD exist at the Drosophila NMJ, designed to inhibit glutamate
release through an autocrine presynaptic signaling system? One attractive possibility is that
67
PHD may actually be a process that maintains stable extracellular glutamate concentrations at
the larval NMJ of Drosophila in lieu of classical glutamate reuptake mechanisms. In the central
nervous system, there are a variety of clearance mechanisms that homeostatically maintain
extracellular glutamate levels to prevent excitotoxicity (Murphy-Royal et al., 2017), and
excitatory GluRs are present at presynaptic terminals of the larval Drosophila NMJ (Kiragasi et
al., 2017). The major mechanism for glutamate clearance in the mammalian brain requires
glutamate transporter proteins in the plasma membrane of both glial cells and neurons. In
Drosophila, there a single excitatory amino acid transporter specific for glutamate reuptake
encoded in the genome, dEAAT1. dEAAT1 is expressed in glial processes at the adult NMJ,
where it is involved in glutamate clearance (Rival et al., 2006). However, dEAAT1 is not
expressed at the embryonic or larval NMJ (Rival et al., 2006; Rival et al., 2004), and it is unclear
how glutamate concentrations are controlled at this synapse. Accordingly, PHD may serve as
an adaptive autocrine mechanism that responds to excess glutamate and inhibits release to
maintain extracellular glutamate homeostasis, a process that may be conserved in the
mammalian central nervous system (Padamsey et al., 2017).
68
2.5 Materials and Methods
Fly Stocks: Drosophila stocks were raised at 25°C on standard molasses food. The w
1118
strain
is used as the wild type control unless otherwise noted, as this is the genetic background of the
transgenic lines and other genotypes used in this study. The following fly stocks were used:
MHC-GluRIIA and GluRIIA
SP16
(Petersen et al., 1997), dysb
1
(Dickman and Davis, 2009);
OK371-Gal4 (Mahr and Aberle, 2006), UAS-cac-GFP (Kawasaki et al., 2004), SynapGCaMP6f
(MHC-CD8-GCaMP6f-Sh; (Newman et al., 2017)), and UAS-vGlut (Daniels et al., 2004). All
other stocks were obtained from the Bloomington Drosophila Stock Center.
Immunocytochemistry: Third-instar larvae were dissected in ice cold 0 Ca
2+
HL-3 and fixed in
either Bouin's fixative for 2 min, 100% ethanol for 5 min, or 4% paraformaldehyde for 10 min as
described (Perry et al., 2017). Briefly, larvae were washed with PBS containing 0.1% Triton X-
100 (PBST) for 30 min, incubated overnight in primary antibodies at 4°C with 5% normal donkey
serum, washed, and equilibrated in 70% glycerol. Blocking was done with 5% normal donkey
serum in PBST. Samples were mounted in VectaShield (Vector Laboratories). The following
antibodies were used: mouse anti-Synapsin (1:10; 3C11; Developmental Studies Hybridoma
Bank; DSHB); mouse anti-Bruchpilot (BRP; nc82; 1:100; DSHB); mouse anti-GluRIIA (1:100;
8B4D2; DSHB); rabbit anti-GluRIII (1:2000; (Marrus et al., 2004)); rabbit anti-DLG (1:5000;
(Pielage et al., 2005)); mouse anti-DLG (1:100; 4F3; DSHB); mouse anti-pCaMKII (1:100; MA1-
047; Invitrogen); guinea pig anti-vGlut (1:2000; (Chen et al., 2017)). Donkey anti-mouse, anti-
guinea pig, and anti-rabbit Alexa Fluor 488-, DyLight 405-, and Cyanine 3 (Cy3)-conjugated
secondary antibodies (Jackson Immunoresearch) were used at 1:400. Alexa Fluor 647
conjugated goat anti-HRP (Jackson ImmunoResearch) was used at 1:200.
69
Imaging and analysis: Samples were imaged using a Nikon A1R Resonant Scanning Confocal
microscope equipped with NIS Elements software and a 100x APO 1.4NA oil immersion
objective, using separate channels with four laser lines (405 nm, 488 nm, 561 nm, and 637 nm)
as described (Kikuma et al., 2017). For fluorescence quantifications of Cac-GFP, BRP, and
pCaMKII intensities, all genotypes were immunostained in the same tube with identical
reagents, then mounted and imaged in the same session. z-stacks were obtained using
identical settings for all genotypes with z-axis spacing between 0.15 µm to 0.2 µm within an
experiment and optimized for detection without saturation of the signal. Maximum intensity
projections were used for quantitative image analysis with the NIS Elements software General
Analysis toolkit. Type Ib and Is boutons were distinguished based on size and intensity of the
HRP and DLG signal on muscle 6/7 and muscle 4 of segments A2 and A3. Boutons were
counted by considering each DLG and HRP pair to be a bouton. Synapse surface area was
calculated by creating a mask around the HRP channel that labels the neuronal membrane. Ib
and Is boutons were separated at individual NMJs and Cac-GFP, BRP, and pCaMKII
quantifications were performed separately on these two bouton types. For analysis of pCaMKII
levels, only the pCaMKII signal that localized within DLG and HRP areas were quantified at Ib
and Is boutons to obtain mean pCaMKII intensity levels. BRP puncta number, area, mean and
sum intensity were quantified by applying intensity thresholds and filters to binary layers on the
BRP channel. For quantification of Cac-GFP, GFP mean intensity was calculated by quantifying
the average GFP signal localized with individual BRP puncta, considering only signals in which
GFP overlapped with BRP. GFP sum intensity was calculated by quantifying the total GFP
signal per individual BRP puncta.
Ca
2+
imaging and analysis: Third-instar larvae were dissected and incubated in HL-3
containing 1.5mM Ca
2+
. The larval central nervous system was removed, and Type Ib and Is
boutons were identified on muscle 6/7 of abdominal segments A2 and A3 by observing the
70
basal GCaMP fluorescence level in the postsynaptic density as described (Newman et al.,
2017). Live imaging was performed using a Nikon A1R Resonant Scanning Confocal
microscope equipped with NIS Elements software and a 60x APO 1.0NA water immersion
objective. Band scanning at a resonant frequency of 113 fps (512 x 86 pixels) was performed
across the field of view. 4-8 individual Ib or Is boutons located at distal terminals were imaged
during each 90 sec session. Miniature events were recorded for the first 30 secs followed by
evoking a response using a Master 9 stimulator (A.M.P.I) and ISO-Flex stimulus isolator at 0.2
Hz. Average evoked amplitude for each recording was calculated from the response to 5 stimuli.
To functionally confirm the bouton subtype, a train of 10 stimuli at 2 Hz was given to ensure
facilitation was observed for Ib boutons and depression at Is boutons. Measurements based on
Ca
2+
imaging were taken from at least two different animals (approximately 14-16 of each Ib and
Is boutons).
Fluorescence events were detected using identically sized regions of interest drawn around
individual boutons. Ib and Is regions were defined based on the baseline SynapGCaMP6f
fluorescence levels, which was 2-3 fold higher at Ib synapses compared to their Is counterparts
at a particular muscle. ΔF for a spontaneous event was calculated by subtracting the basal
GCaMP fluorescence level from the peak intensity of the GCaMP signal at a particular bouton.
For each bouton under consideration, these spontaneous ΔF values were averaged for all
events in the 30 sec time frame to obtain the mean quantal size for each bouton. The evoked
ΔF value was obtained by calculating the increase in fluorescence after a 1 msec stimulus and
averaged for 5 stimuli to get the mean evoked ΔF for that bouton. The evoked ΔF was divided
by the spontaneous ΔF to calculate the quanta released per bouton per stimulation. This
analysis was done identically but separately for Ib and Is bouton types for wild type and vGlut-
OE NMJs.
71
Electrophysiology: All dissections and recordings were performed in modified HL-3 saline
(Dickman et al., 2005; Kiragasi et al., 2017; Stewart et al., 1994) at room temperature containing
(in mM): 70 NaCl, 5 KCl, 10 MgCl2, 10 NaHCO3, 115 Sucrose, 5 Trehelose, 5 HEPES, and 0.4
CaCl2 (unless otherwise specified), pH 7.2. Neuromuscular junction sharp electrode (electrode
resistance between 10-35 MΩ) recordings were performed on muscles 6 and 7 of abdominal
segments A2 and A3 in wandering third-instar larvae. Recordings were performed on an
Olympus BX61 WI microscope using a 40x/0.80NA water-dipping objective, and acquired using
an Axoclamp 900A amplifier, Digidata 1440A acquisition system and pClamp 10.5 software
(Molecular Devices). Electrophysiological sweeps were digitized at 10 kHz and filtered at 1 kHz.
Data were analyzed using Clampfit (Molecular devices), MiniAnalysis (Synaptosoft), Excel
(Microsoft), and SigmaPlot (Systat) software. Miniature excitatory postsynaptic potentials
(mEPSPs) or currents (mEPSCs) were recorded in the absence of any stimulation for 1 min and
cut motor axons were stimulated to elicit an evoked response. An ISO-Flex stimulus isolator
(A.M.P.I.) was used to modulate the amplitude of stimulatory currents. Average EPSP or EPSC
amplitudes for each recording was calculated from the average response to 20 stimuli. Quantal
content was determined for each genotype as indicated in Table S1. Muscle input resistance
(Rin) and resting membrane potential (Vrest) were monitored during each experiment (Table S1).
To acutely block postsynaptic receptors, larvae were incubated with or without philanthotoxin-
433 (PhTx; Sigma; 20 μM) and resuspended in HL-3 for 10 mins, as described (Dickman and
Davis, 2009; Frank et al., 2006).
The readily releasable pool (RRP) size was estimated by examining cumulative EPSC
amplitudes while recording using a two-electrode voltage clamp configuration. Muscles were
clamped to -70 mV and EPSCs were evoked with a 60 Hz, 60 stimulus train while recording in
3mM Ca
2+
HL-3. EPSC amplitudes were calculated as the difference between peak and
baseline before stimulus onset of a given EPSC. A line fit to the linear phase (stimuli # 18-30) of
the cumulative EPSC data was back-extrapolated to time 0. The RRP value was estimated by
72
normalizing the ratio of the EPSC value at time 0 and the relative change in mEPSP/mEPSC
amplitude between respective genotypes and wild type. Finally, for shibire experiments,
preparations were maintained between 32-34°C using a TC-324B Automatic Temperature
Controller (Warner Instruments) mounted on the electrophysiology stage to control the
temperature of the preparation for the duration of the experiment. Recordings in which Vrest
drifted to become more depolarized below -60 mV during the recording were discarded.
Statistical Analysis: All data are presented as mean +/-SEM. Data were compared using
either a one-way ANOVA followed by Tukey’s multiple comparison test, or using a Student’s t-
test (where specified), analyzed using Graphpad Prism or Microsoft Excel software, with varying
levels of significance assessed as p<0.05 (*), p<0.01 (**), p<0.001 (***),p<0.0001 (****), ns=not
significant. A Kruskal-Wallis test in Graphpad prism was used to assess significance between a
set of distributions. See Table S1 for further statistical details and values.
73
Table S1: Absolute values for normalized data and additional statistics, related to Figure
1-7, and Supplemental Figure 1-5. The figure and panel, genotype, and experimental
conditions are noted (external calcium concentration, PhTx application). Average values for
mEPSP, EPSP, quantal content (QC), resting potential, input resistance, and number of data
samples (n), p values, and significance are shown, with standard error values noted in
parentheses.
Figure 2 Genotype
[Ca
2+
]
(mM)
PhTx
mEPSP
(mV)
EPSP
(mV)
QC
Rin
( M Ω )
Vmrest
(mV)
n
p value
(significance)
(mEPSP, EPSP,
QC)
2B,C,E,F w
1118
0.4 -
1.0056
(0.0762)
31.0911
(2.2608)
30.9406
(2.0372)
7.8000
(1.6064)
-63.7875
(1.9471)
8
2B,C,E,F w
1118
0.4 +
0.5162
(0.0234)
32.6537
(1.4819)
64.3294
(3.7935)
10.0100
(1.1235)
-66.9797
(1.2416)
10
<0.0001 (****),
0.5784 (ns),
<0.0001 (****)
compare to w
1118

2B,C
w;OK371-
Gal4/UAS-vGlut
0.4 -
1.4435
(0.0439)
30.0122
(0.9845)
20.7913
(1.0472)
8.9200
(1.0472)
-67.6596
(1.7346)
15
2B,C
w;OK371-
Gal4/UAS-vGlut
0.4 +
0.8319
(0.0693)
28.6139
(1.7661)
35.8490
(2.5770)
9.1300
(0.8546)
-66.3501
(2.7433)
12
<0.0001 (****),
0.5967 (ns),
<0.0001 (****)
compare to
w;OK371-
Gal4/UAS-vGlut
2E,F
w;UAS-
vGlut/OK371-
Gal4;dysb
1

0.4 -
1.5425
(0.0674)
33.0431
(1.3528)
21.4218
(1.0165)
11.9200
(0.9413)
-70.5135
(2.3614)
12
2E,F
w;UAS-
vGlut/OK371-
Gal4;dysb
1

0.4 +
0.8368
(0.0376)
15.9261
(2.3748)
19.0321
(2.0988)
9.7600
(1.2165)
-67.4717
(1.9417)
12
<0.0001 (****),
<0.0001 (****),
0.8647 (ns)
compare to
w;UAS-
vGlut/OK371-
Gal4;dysb/dysb
Figure 3 Genotype
[Ca
2+
]
(mM)
Corrected QC
{Ca
2+
coop slope}
Rin
( M Ω )
Vmrest
(mV)
n
p value
(significance)
(slope)
3A w
1118
0.2
15.3939 (1.4097)
{slope: 1.677
0.09278}
7.9335
(0.6421)
-68.3254
(1.2878)
11
Figure 1 Genotype
[Ca
2+
]
(mM)
PhTx
mEPSP
(mV)
EPSP
(mV)
QC
Rinput
( M Ω )
Vmrest
(mV)
n
p value
(significance)
(mEPSP, EPSP,
QC)
1B,C,D,E w
1118
0.4 -
0.9914
(0.0393)
33.4924
(1.0977)
33.9533
(1.0483)
11.3400
(0.6494)
-65.5340
(0.5151)
20
1B,C,D,E w;GluRIIA
SP16
0.4 -
0.5080
(0.0480)
32.1150
(1.4294)
64.6108
(3.8744)
12.2700
(0.9281)
-65.8668
(1.0574)
10
<0.0001 (****),
0.1285 (ns),
<0.0001 (****)
compare to w
1118

1B,C,D,E
w;OK371-
Gal4/UAS-vGlut
0.4 -
1.3991
(0.0440)
32.6430
(0.9841)
20.9300
(1.0470)
12.2600
(1.2514)
-67.6596
(1.7340)
15
<0.0001 (****),
0.8494 (ns),
<0.0001 (****)
compare to w
1118

1B,C,D,E
w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

0.4 -
0.7802
(0.0621)
31.8259
(0.9583)
40.7920
(2.4600)
11.8100
(0.8211)
-64.8231
(0.5483)
8
0.0080 (**),
0.0677 (ns),
0.0055 (**)
compare to w
1118

74
3A w;GluRIIA
SP16
0.2
23.1698 (2.1979)
{slope: 1.735
0.1171}
8.8623
(0.9332)
-65.8173
(1.0870)
11
0.7020 (ns)
3A
w;OK371-Gal4/UAS-
vGlut
0.2
8.71597 (2.4949)
{slope: 1.849
0.07422}
8.0923
(0.9214)
-65.7876
(1.1433)
8
0.1909 (ns)
3A
w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

0.2
13.7856 (1.8541)
{slope: 1.905
0.1069}
10.1215
(1.0027)
-65.7276
(1.4353)
13
0.1284 (ns)
3A w
1118
0.3 30.2856 (3.7410) 9.9245
(0.8187)
-68.2932
(1.2387)
12
3A w;GluRIIA
SP16
0.3 44.6716 (7.0051) 8.4188
(0.9335)
-66.4821
(1.3557)
12
3A w;OK371-Gal4/UAS-
vGlut
0.3 23.8395 (2.7002) 10.1794
(0.7581)
-67.8732
(1.0677)
12
3A w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

0.3 38.6108
(8.7470)
11.1523
(1.2154)
-66.4898
(1.3648)
10
3A w
1118
0.4 66.7824
(3.7664)
10.0355
(0.7422)
-69.3164
(1.3878)
25
3A w;GluRIIA
SP16
0.4 101.8281
(13.9240)
11.1921
(0.6214)
-66.7276
(1.5430)
10
3A w;OK371-Gal4/UAS-
vGlut
0.4 39.4838
(5.1183)
8.9428
(0.9322)
-66.2867
(1.3659)
8
3A w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

0.4 82.3515
(10.3752)
9.2669
(0.8621)
-68.4715
(1.2883)
8
3A w
1118
0.5 81.6493
(8.4052)
9.1204
(1.0860)
-68.4276
(0.8474)
10
3A w;GluRIIA
SP16
0.5 119.8757
(10.4457)
11.3495
(0.8516)
-66.4265
(0.6474)
10
3A w;OK371-Gal4/UAS-
vGlut
0.5 58.4299
(3.5823)
10.0326
(1.1644)
-70.2404
(1.2421)
11
3A w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

0.5 101.4982
(4.7526)
8.8962
(0.9197)
-67.4165
(0.6570)
10
Figure 3 Genotype
[Ca
2+
]
(mM)
EPSC
(nA)
leak current
(nA)
Vckamp
(mV)
n
p value
(significance)
3B w
1118
0.3
-23.6975
(2.2113)
-3.0453
(0.4523)
-70 10
3B w;GluRIIA
SP16
0.3
-17.9278
(1.0473)
-3.3151
(0.3012)
-70 7
0.0576 (ns)
compare to w
1118

3B
w;OK371-Gal4/UAS-
vGlut
0.3
-21.9627
(3.3927)
-2.8944
(0.2688)
-70 10
0.6735 (ns)
compare to w
1118

3B
w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

0.3
-18.7370
(1.0260)
-2.5212
(0.4532)
-70 8
0.0795 (ns)
compare to w
1118

3B w
1118
1.5
122.2934
(8.7934)
-3.4066
(0.8096)
-70 13
3B w;GluRIIA
SP16
1.5
103.9607
(8.3797)
-3.3510
(0.7313)
-70 11
0.1499 (ns)
compare to w
1118

3B
w;OK371-Gal4/UAS-
vGlut
1.5
110.5388
(7.6059)
-4.1504
(0.7644)
-70 12
0.3261 (ns)
compare to w
1118

3B
w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

1.5
110.2926
(11.2848)
-3.9586
(0.6133)
-70 10
0.4036 (ns)
compare to w
1118

3B w
1118
3.0
223.4777
(18.3938)
-5.4874
(0.7790)
-70 8
3B w;GluRIIA
SP16
3.0
231.5789
(12.5643)
-5.0085
(0.8141)
-70 9
0.7159 (ns)
compare to w
1118

3B
w;OK371-Gal4/UAS-
vGlut
3.0
218.5739
(21.4239)
-5.1151
(0.8012)
-70 8
0.8646 (ns)
compare to w
1118

3B
w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

3.0
221.0977
(12.1318)
-6.2996
(0.7622)
-70 10
0.9123 (ns)
compare to w
1118

75
Figure 3 Genotype
[Ca
2+
]
(mM)
Failure rate (%)
Rin
( M Ω )
Vmrest
(mV)
n
p value
(significance)
(failure rate)
3C w
1118
0.05
33.6500
(5.0000)
7.8498
(0.5241)
-65.2097
(1.7753)
17
3C w;GluRIIA
SP16
0.05
19.2500
(5.1900)
8.6184
(1.0351)
-62.0841
(1.2026)
16
0.0454 (*),
compare to w
1118

3C
w;OK371-Gal4/UAS-
vGlut
0.05
56.0833
(5.0350)
8.3468
(0.6841)
-64.6915
(1.6429)
15
0.0037 (**),
compare to w
1118

3C
w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

0.05
35.4267
(6.8331)
11.5468
(1.0035)
-64.8286
(0.8485)
22
0.8436 (ns),
compare to w
1118

Figure 3 Genotype
[Ca
2+
]
(mM)
paired-pulse rate (%)
Vclamp
(mV)
n
p value
(significance)
3E w
1118
1.5
0.7724
(0.0201)
-70 11
3E w;GluRIIA
SP16
1.5
0.6699
(0.0159)
-70 11
0.0007 (***),
compare to w
1118

3E
w;OK371-Gal4/UAS-
vGlut
1.5
0.8972
(0.0116)
-70 19
< 0.0001 (****),
compare to w
1118

3E
w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

1.5
0.7765
(0.0111)
-70 12
0.8570 (ns),
compare to w
1118

3G w
1118
0.3
1.8863
(0.0697)
-70 15
3G w;GluRIIA
SP16
0.3
1.5865
(0.0332)
-70 13
< 0.0001 (****),
compare to w
1118

3G
w;OK371-Gal4/UAS-
vGlut
0.3
2.2133
(0.0933)
-70 16
0.0095 (**),
compare to w
1118

3G
w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

0.3
1.9058
(0.0421)
-70 12
0.8240 (ns),
compare to w
1118

Figure 3 Genotype
[Ca
2+
]
(mM)
First EPSC
(nA)
Cumulative EPSC
(nA)
RRP size
Vmclamp
(mV)
n
p value
(first EPSC, Cum
EPSC, RRP size)
3I,K w
1118
3
223.4777
(18.3938)
832.6545
(56.1079)
1027.9280
(69.5400)
-70 10
3I,K w;GluRIIA
SP16
3
231.5789
(12.5643)
688.1722
(40.4953)
1609.0820
(94.6859)
-70 7
0.7172 (ns),
0.0129 (*),
< 0.0001 (****),
compare to w
1118

3I,K
w;OK371-Gal4/UAS-
vGlut
3
218.5739
(21.4239)
973.6326
(94.2264)
1012.9820
(90.7341)
-70 8
0.2429 (ns),
0.0528 (ns),
0.9155 (ns),
compare to w
1118

3I,K
w;UAS-
vGlut,GluRIIA
SP16

/OK371-
Gal4,GluRIIA
SP16

3
221.0977
(12.1318)
813.8876
(68.9567)
1390.8961
(87.1653)
-70 14
0.8128 (ns),
0.2750 (ns),
0.0008 (***),
compare to w
1118

0.1090 (ns), RRP
compare to
GluRIIA
Figure 4 Genotype
BRP puncta mean
intensity (%WT)
BRP puncta area
( μm
2
)
BRP puncta sum
intensity (%WT)
n
p value (significance)
(mean-Ib, mean-Is,
area-Ib, area-Is, sum-Ib,
sum-Is)
Ib Is Ib Is Ib Is
4B,D,G,H w
1118

100
(3.79)
100
(4.65)
0.29
(0.01)
0.30
(0.01)
100
(7.80)
100
(10.22)
1
5
4B,D,G,H w;GluRIIA
SP16

134.50
(4.74)
108.70
(5.73)
0.38
(0.006)
0.26
(0.01)
234.30
(19.66)
103.00
(12.34)
1
5
<0.0001 (****), 0.9768
(ns), <0.0001 (****),
0.3557 (ns), <0.0001
(****), 0.9990 (ns)
76
Figure 4 Genotype
Cac-GFP-OE per BRP
puncta mean intensity
(%WT)
Cac-GFP-OE per BRP
puncta sum intensity
(%WT)
n
p value (significance)
(Cac mean-Ib, Cac
mean-Is, Cac-sum-Ib,
Cac sum-Is) Ib Is Ib Is
4D,E,I,J
w;OK371-Gal4/+;UAS-
Cac-GFP/+
100
(5.49)
100
(2.87)
100
(10.11)
100
(7.72)
1
5
4D,E,I,J
w;OK371-Gal4,
GluRIIA
SP16

/GluRIIA
SP16
;UAS-Cac-
GFP/+
123.20
(5.39)
102.30
(8.12)
152.80
(12.11)
93.95
(16.14)
1
5
0.0041 (**), 0.9860 (ns),
0.0006 (***), 0.9561 (ns)
Figure 5 Genotype
quantal size ( F)
evoked
amplitude ( F)
quanta/bouton
n
p value (significance)
(quantal size-Ib, quantal
size-Is, evoked-Ib,
evoked-Is,
quanta/bouton-Ib,
quanta/bouton-Is)
Ib Is Ib Is Ib Is
5C,D,G,H,K,L,
O,P
w; MHC-CD8-GCaMP6f-
Sh

4.925
(0.278)
7.66
(0.192)
32.5
(2.318)
56.97
(5.784)
6.723
(0.479)
7.433
(0.764)
16
5C,D,G,H,K,L,
O,P
w; UAS-vGlut/OK371-
Gal4;MHC-CD8-
GCaMP6f-Sh/+
6.893
(0.416)
10.77
(0.611)
23.64
(3.398)
31.37
(3.76)
3.41
(0.409)
3.053
(0.414)
14
0.0007 (***), 0.0002 (***),
0.0417 (*), 0.0011 (**),
<0.0001 (****), <0.0001
(****)
Figure 6 Genotype
pCamKII intensity (%WT)
n p value (significance)
(CamKII-Ib, CamKII-Is)
Ib Is
6B, D w
1118
100
(7.43)
100
(9.29)
22
6B, D w;GluRIIA
SP16
53.50
(4.34)
84.82
(7.31)
20 <0.0001 (****), 0.4419 (ns)
6B, D
w;OK371-Gal4/UAS-
vGlut
84.69
(5.84)
88.27
(4.56)
21 0.2223 (ns), 0.6314 (ns)
6B, D
w;UAS-vGlut,
GluRIIA
SP16
/OK371-
Gal4, GluRIIA
SP16

49.89
(3.85)
86.22
(6.72)
19 <0.0001 (****), 0.5579 (ns)
Figure 7 Genotype
[Ca
2+
]
(mM)
PhT
x
mEPSP
(mV)
EPSP
(mV)
QC
Rin
( M Ω )
Vmrest
(mV)
n
p value
(significance)
(mEPSP, EPSP,
QC)
7B,C w
1118
0.3 -
1.0090
(0.0704)
23.3404
(1.3601)
23.1322
(2.6673)
8.0500
(1.3546)
-67.7731
(2.5120)
8
7B,C
MHC-GluRIIA-
myc
0.3 -
1.5706
(0.0690)
34.9010
(1.4183)
22.5767
(1.0605)
10.7800
(0.8400)
-67.7776
(1.385)
13
<0.0001 (****),
<0.0001 (****),
0.8244 (ns)
compare to w
1118

7B,C
MHC-GluRIIA-
myc
0.3 +
0.5628
(0.0305)
22.1535
(1.9125)
35.1170
(2.4024)
8.1300
(1.1650)
-62.8511
(0.9267)
12
<0.0001 (****),
<0.0001 (****),
<0.0001 (****)
compare to
baseline
7E,F
w;OK371-
Gal4/UAS-
vGlut
0.3 -
1.3341
(0.0305)
23.7235
(1.6910)
17.8793
(1.0450)
8.7765
(0.8971)
-69.6677
(1.3209)
12
7E,F
MHC-GluRIIA-
myc; OK371-
Gal4/UAS-
vGlut
0.3 -
1.8911
(0.0706)
30.9118
(0.7626)
16.5933
(0.5467)
9.8100
(0.9027)
-66.2703
(1.4246)
17
<0.0001 (****),
0.0002 (***),
0.2481 (ns)
compare to OK371-
Gal4/UAS-vGlut
7E,F
MHC-GluRIIA-
myc; OK371-
Gal4/UAS-
vGlut
0.3 +
0.8727
(0.0662)
21.7119
(1.7391)
24.8790
(1.4060)
10.2600
(0.9681)
-72.4116
(1.1653)
10
<0.0001 (****),
<0.0001 (****),
<0.0001 (****),
compare to
baseline
77
Supplemental
Figure 1
Genotype
GluRIIA puncta
#/NMJ
vGlut intensity (%WT)
n
p value (significance)
(GluRIIA #, vGlut intensity-Ib,
vGlut intensity-Is)
Ib Is
S1C, D w
1118
114.90
(6.81)
100
(7.97)
100
(8.62)
11
S1C, D w;GluRIIA
SP16
7.80
(0.60)
121.76
(13.03)
115.76
(11.45)
9
<0.0001 (****), 0.8620 (ns),
0.9647 (ns)
S1C, D w;OK371-Gal4/UAS-vGlut
125.90
(6.47)
170.57
(12.41)
160.57
(14.77)
11
0.9981 (ns), 0.0031 (**), 0.0042
(**)
S1C, D
w;UAS-vGlut,
GluRIIA
SP16
/OK371-Gal4,
GluRIIA
SP16

8.70
(0.54)
195.99
(14.87)
178.14
(18.98)
12
0.0001 (***), <0.0001 (****),
0.0005 (***)
Supplemental
Figure 2
Genotype Total quanta released n
p value
S2B w
1118

407658.8000
(46502.1700)
5
S2B shi
80506.5436
(13272.3407)
6
S2B shi;GluRIIA
SP16

92050.7435
(14730.8351)
4
0.5846 (ns),
compare to shi
S2B shi;OK371-Gal4/UAS-vGlut
66406.4506
(6721.4480)
8
0.3264 (ns),
compare to shi
S2B
shi;UAS-vGlut,GluRIIA
SP16
/OK371-
Gal4,GluRIIA
SP16

88929.1299
(15369.2675)
5
0.6864 (ns),
compare to shi
Supplemental
Figure 3
Genotype
neuronal
surface
area ( μm
2
)
Bouton #/NMJ
BRP density (#/
μm
2
)
n
P value
(significance)
(neuronal surface
area, bouton # (Ib),
bouton # (Is), bouton
# (total), BRP
density (Ib), BRP
density (Is)
Ib Is
Total
Ib Is
S3C,D,E,F w
1118
287.70
(13.55)
35.68
(2.04)
25.16
(1.71)
62.95
(2.398)
1.06
(0.09)
0.60
(0.09)
19
S3C,D,E,F w;GluRIIA
SP16
285.20
(33.20)
26.75
(1.20)
30.50
(2.66)
57.25
(2.887)
0.95
(0.08)
0.65
(0.03)
14
0.9998 (ns), 0.0105
(*), 0.4924 (ns),
0.6518 (ns), 0.9739
(ns), 0.4727 (ns)
S3C,D,E,F
w;OK371-
Gal4/UAS-
vGlut
290.80
(21.59)
29.73
(1.89)
48.40
(5.07)
73.45
(4.625)
0.90
(0.06)
0.58
(0.05)
15
0.9996 (ns), 0.0421
(*), <0.0001 (****),
0.1704 (ns), 0.9957
(ns), 0.6622 (ns)
S3C,D,E,F
w;UAS-vGlut,
GluRIIA
SP16
/O
K371-
Gal4,GluRIIA
SP16
257.80
(17.59)
26.42
(2.53)
40.50
(3.72)
68.58
(5.064)
0.89
(0.04)
0.65
(0.04)
15
0.7397 (ns), 0.0074
(**), 0.0009 (***),
0.6596 (ns), 0.9513
(ns), 0.9424 (ns)
Supplemental
Figure 4
Genotype bouton #/NMJ
GluR intensity (%WT)
n
P value (significance)
(bouton #, GluRIIA, GluRIIB,
GluRIID) GluRIIA GluRIIB
GluRII
D
S4B, D w
1118
105.30
(4.03)
100
(7.81)
100
(5.78)
100
(8.62)
8
S4B, D MHC-GluRIIA-myc
116.00
(5.48)
157.70
(18.00)
30.00
(5.00)
96.00
(8.00)
9
0.1262 (ns), 0.0047 (**),
<0.0001 (****), 0.9914 (ns)
mEPSP (mV)
0
0.5
1.0
C
1.5
D
EPSP (mV)
0
10
40
30
20
quantal content
0
20
40
80 E
**
****
60
****
****
****
**
A
200 ms
10 mV
2 mV
100 ms
% wild type
0
50
100
200
B
150
mEPSP quantal
content
ns
wild type GluRIIA vGlut-OE GluRIIA+vGlut-OE
wild type
vGlut-OE
GluRIIA
GluRIIA+vGlut-OE
F
0
50
100
150
200
quantal
content
0.0 0.5 1.0 1.5 2.0 2.5
mEPSP (mV)
****
****
**
****
****
**
Figure 1: Presynaptic homeostatic potentiation and depression can be induced,
expressed, and balanced. (A) Schematic of genetic manipulations to the Drosophila NMJ that
induce bi-directional, homeostatic changes in presynaptic neurotransmitter release over chronic
time scales. Presynaptic homeostatic potentiation (PHP) is observed when mEPSP amplitudes
are reduced due to genetic loss of the postsynaptic GluR subunit GluRIIA (GluRIIA). Presynaptic
homeostatic synaptic depression (PHD) is observed when mEPSP amplitudes are enhanced
following overexpression of the vesicular glutamate transporter in motor neurons (vGlut-OE).
Synaptic strength (EPSP amplitude) is maintained at baseline levels when PHP and PHD are
individually expressed or combined at an individual NMJ in GluRIIA+vGlut-OE. Inset:
Representative electrophysiological traces in the indicated genotypes, each showing similar
EPSP amplitudes despite differences in mEPSP amplitudes. (B) Quantification of mEPSP
amplitude and quantal content in the indicated genotypes (coded by color), normalized to wild-
type values. Note that a homeostatic increase in presynaptic glutamate release (quantal content)
is observed in GluRIIA mutants, while a homeostatic decrease is observed in vGlut-OE. When
combined, GluRIIA+vGlut-OE show similar mEPSP and quantal content values compared to wild
type. (C,D,E) Quantification of mEPSP amplitude (C), EPSP amplitude (D), and quantal content
(E) in the indicated genotypes. (F) All genotypes show a homeostatic tuning of quantal content
over varying average quantal sizes, in effect maintaining stable levels of synaptic strength. The
black curve represents ideal homeostatic tuning, derived by solving the function of
EPSP= QC· mEPSP.
78
mEPSP (mV)
B
0
0.5
1.0
EPSP (mV)
0
30
10
40
quantal content
0
40
20
60
1.5
F
% baseline (-PhTx)
0
100
50
150
mEPSP quantal
content
****
20
A
200 ms
2 mV
10 mV
wild type
+PhTx
vGlut-OE
+PhTx
dysb dysb
dysb+
vGlut-OE
dysb+vGlut-OE
+PhTx
****
****
****
+ - + -
100 ms
E D
mEPSP (mV)
0
0.5
1.0
EPSP (mV)
0
30
10
40
quantal content
0
20
30
1.5
20
****
****
10
ns
WT vGlut-OE
PhTx + - + -
WT vGlut-OE
+ - + -
WT vGlut-OE
PhTx - + -
WT
dysb +
vGlut-OE
% baseline (-PhTx)
C
0
100
50
150
mEPSP quantal
content
WT vGlut-OE
WT
dysb +
vGlut-OE
200
****
****
****
****
200
****
****
****
ns
- + -
WT
dysb +
vGlut-OE
- + -
WT
dysb +
vGlut-OE
ns
ns
Figure 2: Conventional mechanisms are required for the acute expression of PHP in vGlut-
OE. (A) Schematic and representative traces of wild type and vGlut-OE NMJs following the acute
application of PhTx. Diminished mEPSP amplitudes are observed in both wild-type and vGlut-OE
NMJs after PhTx application, while EPSP amplitudes are maintained at baseline levels due to a
homeostatic increase in quantal content. (B) Quantification of mEPSP amplitude, EPSP
amplitude, and quantal content values in the indicated genotypes and conditions. (C) mEPSP and
quantal content values following PhTx application normalized as a percentage of baseline values
(no PhTx treatment). (D) Schematic and representative traces of vGlut-OE in a dysb mutant
background (dysb+vGlut-OE) in baseline conditions and following application of PhTx. While loss
of dysb has no impact on PHD expression, PHP expression is blocked following application of
PhTx to dysb+vGlut-OE. (E) Quantification of mEPSP amplitude, EPSP amplitude, and quantal
content values in the indicated genotypes and conditions. (F) Quantification of mEPSP and
quantal content values normalized to baseline values.
79
I
estimated RRP size
2000
1000
1500
500
0
B
300
0
100 EPSC (nA)
200
H
1000
500
cum. EPSC
K
0
31.8 31.6 31.4 31.2 31 30.8 30.6 30.4 30.2
Tim e (s)
IN 0
(mV)
0
IN 1
(nA)
-320
-300
-280
-260
-240
-220
-200
-180
-160
-140
-120
-100
-80
-60
-40
-20
0
20
40 nA
200 ms
WT GluRIIA vGlut-OE GluRIIA+
vGlut-OE
C
quantal content
external [Ca
2+
] (mM)
0.3
1
10
100
0.2 0.5 0.4
A
GluRIIA
WT
GluRIIA+vGlut-OE
failure rate (%)
40
60
80
20
0
*
**
WT
vGlut-OE
GluRIIA
GluRIIA
+vGlut-OE
vGlut-OE
ns
ns
ns
*
ns
ns
***
****
ns
50 nA
EPSC2/EPSC1 (%)
40
60
100
20
0
***
****
12 nA
EPSC2/EPSC1 (%)
50
100
0
****
**
D E
80
ns
ns
F G
wild type GluRIIA vGlut-OE GluRIIA+vGlut-OE
20 ms
1.5 mM Ca
2+
0.3 mM Ca
2+
150
200
250
J
cum. EPSC
1000
2000
3000
200 ms
0
ns
ns
external [Ca
2+
] (mM)
0.3 1.5 3.0
80
Figure 3: Characterization of Ca
2+
cooperativity, failure analysis, and readily releasable
vesicle pools in GluRIIA+vGlut-OE. (A) Quantal content plotted as a function of extracellular
Ca
2+
concentration on a log/log scale, with best fit lines shown, for the indicated genotypes. No
significant differences in the slopes were observed (Table S1). (B) EPSC amplitude following
homeostatic challenge remains similar to wild type in the indicated genotypes across a range of
extracellular Ca
2+
conditions. (C) Failure analysis reveals a decrease in failure rate in GluRIIA
mutants, while an increase in failure rate is observed in vGlut-OE, as expected. An intermediate
failure rate is observed in GluRIIA+vGlut-OE, consistent with a balancing of adaptations resulting
from both PHP and PHD expression. Recordings were performed in 0.05mM extracellular Ca
2+
.
(D) Representative paired-pulse EPSC traces at 1.5mM extracellular Ca
2+
with an interstimulus
interval of 16.7 msec in the indicated genotypes. Enhanced paired-pulse depression (PPD) was
observed in GluRIIA mutants, while reduced PPD was found in vGlut-OE, consistent with
increased and reduced probability of release. (E) Quantification of the paired-pulse ratio
(EPSC2/EPSC1) in the indicated genotypes. (F) Representative paired-pulse EPSC traces at
0.3mM extracellular Ca
2+
in the indicated genotypes. Reduced paired-pulse facilitation (PPF) was
observed in GluRIIA mutants, while enhanced PPF was found in vGlut-OE, consistent with
increased and reduced probability of release. (G) Quantification of the paired-pulse ratio
(EPSC2/EPSC1) in the indicated genotypes. (H) Representative EPSC recordings of 30 stimuli at
3mM extracellular Ca
2+
during a 60 Hz stimulus train in the indicated genotypes. (I) Estimated
size of the readily releasable pool in the indicated genotypes demonstrating the RRP is increased
in both GluRIIA mutants and GluRIIA+vGlut-OE. (J) Average cumulative EPSC amplitude plotted
as a function of time. A line fit to the 18-30
th
stimuli was back extrapolated to time 0. (K) Average
cumulative EPSC values for the indicated genotypes.
81
0
150
100
50
% wild type
Is BRP
mean
Is BRP
sum
ns ns
D
cumulative
frequency
50
100
0
Ib BRP intensity (a.u.)
0 1 2 3 4
cumulative
frequency
50
100
0
Is BRP intensity (a.u.)
0 1 2 3
B
A
H G I J
F
cumulative
frequency
50
100
0
Is Cac intensity (a.u.)
0 1 2 3 4
****
****
ns
Ib BRP
mean
% wild type
0
200
100
****
****
ns
Ib BRP
sum
***
*
ns
**** ****
ns
Ib Cac
mean
% wild type
0
150
50
100
Ib Cac
sum
C
E
cumulative
frequency
50
100
0
0 1 2 3 4
Ib Cac intensity (a.u.)
***
**
ns
****
****
ns
% wild type
0
50
100
Is Cac
mean
Is Cac
sum
Ib Cac
GluRIIA vGlut-OE GluRIIA+vGlut-OE
Ib BRP
wild type
2
μm
GluRIIA vGlut-OE GluRIIA+vGlut-OE
Is Cac
Is BRP
2 μm
wild type
Figure 4: BRP and Cac abundance at active zones are enhanced in GluRIIA mutants and
are unchanged in vGlut-OE at Ib boutons. (A) Representative images of Ib boutons
immunostained with anti-GFP and anti-BRP at NMJs transgenically overexpressing the Ca
2+
channel Cac-GFP in the indicated genotypes. (B-E) Quantification of mean and sum
immunofluorescence intensity of BRP (B) and Cac-GFP-OE (D) puncta, and cumulative
frequency distribution of mean BRP (C) and Cac-GFP-OE (E) puncta at Ib boutons reveals a
significant increase in these values in GluRIIA mutants and GluRIIA+vGlut-OE, while no change
is found in vGlut-OE. (F) Representative images of Is boutons with the same immunolabeling in
the same genotypes as (A). (G-J) No changes in BRP mean or sum puncta intensity (G) nor
cumulative frequency distribution (H) are observed at Is boutons in any genotype. However, Cac-
GFP-OE intensity is reduced at Is boutons in vGlut-OE and GluRIIA+vGlut-OE (I,J).
82
0
% wild type
50
150
****
100
quantal
size
t=60 ms t=300 ms t=0 ms
1μm
t=60 ms t=400 ms t=0 ms
A
C
0
8
ΔF
I
K
2
0
quantal size (ΔF)
4
8
***
6
10
0
evoked
amplitude (ΔF)
10
*
20
30
0
quanta/bouton
2
****
6
4
8
WT vGlut-OE WT vGlut-OE
WT vGlut-OE WT vGlut-OE
0
quanta/bouton
2
****
6
10
4
8
D
E G H
L
M
N O P
ΔF=5
10 sec
ΔF=5
ΔF=20
10 sec
0.5 sec
0.5 sec
ΔF=20
F
quanta/
bouton
*
*
2
0
quantal size (ΔF)
4
8
***
6
10
12
0
% wild type
50
150
****
100
quantal
size
quanta/b
outon
0
40
ΔF
0
14
ΔF
0
70
ΔF
0
evoked
amplitude (ΔF)
20
**
40
60
Ib spontaneous Ib evoked
Is spontaneous
Is evoked
t=60 ms t=200 ms t=0 ms
B
1μm
t=60 ms t=300 ms t=0 ms
J
1μm
1μm
Figure 5: In vivo Ca
2+
imaging reveals both Ib and Is boutons express PHD. (A)
Representative traces of spontaneous Ca
2+
transients imaged at Ib boutons in the indicated
genotypes. Consistent with electrophysiological recordings, vGlut-OE synapses show enhanced
miniature Ca
2+
signals compared to WT. (B) Representative images of spontaneous GCaMP
signals coded as a heatmap at individual Ib boutons shown at three time points. The change in
fluorescence ( ΔF) during a spontaneous event is larger at vGlut-OE Ib boutons. (C,D)
Quantification of quantal size ( ΔF of the spontaneous Ca
2+
transient event) (C) and evoked Ca
2+
transient event (D) at individual boutons. (E) ΔF traces of a single evoked event followed by a 2
Hz stimulus train to functionally define Ib boutons based on their characteristic facilitation. Note
the reduced evoked response at Ib boutons in vGlut-OE. (F) Representative images of evoked
GCaMP signals. (G,H) Reduced quanta released per bouton per evoked stimulus at vGlut-OE
NMJs compared to wild type, indicative of PHD expression. (I) Ca
2+
transients during
spontaneous events at Is boutons. vGlut-OE boutons show enhanced Ca
2+
signals compared to
WT. (J) Representative images of spontaneous GCaMP signals at individual Is boutons. (K,L)
Quantal size is enhanced at Is boutons in vGlut-OE (K) and the evoked Ca
2+
transient event per
individual bouton is significantly reduced (L). (M) ΔF traces of an individual bouton stimulus
followed by a 2 Hz train functionally defines Is boutons by their characteristic depression. (N)
Representative images of evoked GCaMP signals at Is boutons shown at three different time
points. (O,P) Reduced quanta is released per bouton in vGlut-OE, indicative of PHD expression.
83
50
100
0
% wild type
B
D
ns
GluRIIA vGlut-OE GluRIIA+vGlut-OE A wild
type
2 μm
pCaMKII DLG GluRIID
Ib pCaMKII intensity
Ib boutons
50
100
% wild type
****
****
0
ns
Is pCaMKII intensity
GluRIIA vGlut-OE GluRIIA+vGlut-OE
C
1 μm
Is boutons
pCaMKII DLG GluRIID
wild type
Figure 6: pCaMKII levels are reduced specifically at Ib postsynaptic densities in GluRIIA
mutants and unchanged in vGlut-OE. (A) Representative images of NMJs immunostained with
antibodies that recognize the active (phosphorylated) form of CaMKII (pCaMKII), the postsynaptic
scaffold Discs Large (DLG), and the essential postsynaptic GluR subunit GluRIID. (B)
Quantification of total pCaMKII intensity at muscle 6/7 Ib boutons reveals a reduction in GluRIIA
and GluRIIA+vGlut-OE, and no significant change in vGlut-OE. (C) Representative images of Is
boutons immunostained in the same conditions and genotypes as in (A). (D) Quantification of
pCaMKII intensity at Is boutons shows no significant change in any genotype, consistent with
PHP not operating at Is boutons and PHD having no impact on postsynaptic pCaMKII levels.
84
2 mV
10 mV
200 ms
100 ms
A GluRIIA-OE
+PhTx
GluRIIA-OE+vGlut-OE
+PhTx
GluRIIA-OE
+vGlut-OE
D
GluRIIA-OE
mEPSP (mV)
E
0
0.5
1.0
1.5
2.0
quantal content
0
10
30
10
20
EPSP (mV)
0
30
20
40
mEPSP (mV)
B
0
0.5
1.0
1.5
2.0
quantal content
0
20
40
10 10
****
30
EPSP (mV)
0
30
20
40
****
ns
****
****
****
****
- + - PhTx
GluRIIA-OE
+vGlut-OE
- + -
vGlut-OE
GluRIIA-OE
+vGlut-OE
- + -
- + - PhTx
WT GluRIIA-OE
- + -
WT GluRIIA-OE
- + -
WT GluRIIA-OE
% baseline (-PhTx)
0
100
200
50
150
mEPSP
quantal
content
% baseline (-PhTx)
0
100
200
50
150
mEPSP quantal
content
C
F
WT GluRIIA-OE
vGlut-OE
GluRIIA-OE
+vGlut-OE
vGlut-OE
GluRIIA-OE
+vGlut-OE
vGlut-OE
****
****
****
***
ns
****
****
****
****
****
****
****
****
wild type
vGlut-OE
Is Ib Is Ib Is Ib
Is Ib Is Ib Is Ib
Figure 7: Enhanced quantal size triggered by postsynaptic overexpression of GluRIIA does
not induce or modulate PHD expression. (A) Schematic and representative traces of
postsynaptic overexpression of the GluRIIA subunit (GluRIIA-OE) at baseline conditions and after
PhTx application. Although GluRIIA-OE increases mEPSP amplitude, no change in presynaptic
neurotransmitter release is observed, leading to enhanced EPSP amplitude and no change in
quantal content. Hence, enhanced mEPSP amplitude through postsynaptic mechanisms does not
induce retrograde PHD signaling (schematized by red dashed arrow). After PhTx application to
GluRIIA-OE, mEPSP amplitude is reduced, while retrograde PHP signaling is now induced,
leading to a homeostatic increase in quantal content. (B) Quantification of average mEPSP
amplitude, EPSP amplitude, and quantal content values for the indicated genotypes and
conditions. (C) mEPSP and quantal content values normalized to baseline conditions (-PhTx). (D)
Schematic and representative traces of vGlut-OE combined with GluRIIA-OE alone and after
PhTx application. In this condition, GluRIIA-OE+vGlut-OE, mEPSP amplitudes are further
enlarged, but PHD is normally expressed, resulting in no change in quantal content compared
with vGlut-OE alone. An autocrine mechanism requiring excess glutamate release to induce PHD
is schematized by the red arrow. Acute PhTx application induces PHP expression in GluRIIA-
OE+vGlut-OE. (E) Quantification of mEPSP amplitude, EPSP amplitude, and quantal content
values in the indicated genotypes and conditions. (F) Quantification of mEPSP and quantal
content values normalized to baseline values (-PhTx).
85
C
D
GluRIIA vGlut-OE wild type GluRIIA+vGlut-OE
10 µm
GluRIIA
HRP
0
GluRIIA puncta
#/NMJ
50
100
150
**** ****
ns
B
Ib vGlut
2 µm
Is vGlut
A
% wild type
0
200
100
Ib vGlut intensity
**
****
**
***
ns
ns
Is vGlut intensity
Supplemental Figure 1: GluRIIA-containing receptors are absent and vGlut intensity is
increased in GluRIIA+vGlut-OE, related to Figure 1. (A) Representative images of NMJs
immunostained with antibodies against the neuronal membrane marker HRP and the
postsynaptic glutamate receptor subunit GluRIIA. Note that the GluRIIA signal is completely
absent in GluRIIA and GluRIIA+vGlut-OE NMJs, as expected. (B) Representative images of Ib
and Is boutons labeled with an antibody against the vesicular glutamate transporter vGlut. Both
bouton types show enhanced vGlut expression at vGlut-OE and GluRIIA+vGlut-OE NMJs, as
expected. (C) Quantification of GluRIIA puncta number per NMJ in the indicted genotypes. (D)
vGlut intensity is significantly increased at Ib and Is motor neuron inputs in vGlut-OE and
GluRIIA+vGlut-OE NMJs. Detailed statistical information for represented data (mean values,
SEM, n, p) is shown in Table S1.
86
2D Graph 1
Col 1 vs Col 2
Col 1 vs Col 4
Col 1 vs Col 6
Col 1 vs Col 8
Col 1 vs Col 10
15 Hz, 32°C
300
40
20
60
200 0 400 500
0
% starting QC
100
Time (sec)
A
total quanta released
(×10
3
)
0
500
100
50
ns
shi;GluRIIA
shi
shi;GluRIIA
+vGlut-OE
shi;vGlut-OE
B
WT
shi
shi;GluRIIA
shi;vGlut-OE
shi;GluRIIA
+vGlut-OE
300
400
WT
Supplemental Figure 2: The total releasable vesicle pool is unchanged in vGlut-OE, related
to Figure 3. (A) The following genotypes were stimulated at 15 Hz for 8 minutes in 5mM
extracellular Ca
2+
at 32-34°C: WT (w
1118
); shi (shi
ts1
); shi;GluRIIA (shi
ts1
;GluRIIA
SP16
); shi;vGlut-OE
(shi
ts1
;OK371-Gal4/UAS-vGlut); and shi;GluRIIA+vGlut-OE (shi
ts1
;OK371-Gal4,GluRIIA
SP16
/UAS-
vGlut,GluRIIA
SP16
). EPSP amplitudes eventually deplete to zero in shi mutant backgrounds
because all endocytosis is blocked in this condition. EPSP amplitudes were averaged (binning
1.5 sec responses for each time point), converted to quantal content, normalized to pre-stimulus
values, and plotted as a function of time. (B) Quantification of total quanta released during the 8
min stimulation protocol determines the entire releasable synaptic vesicle pool in each genotype.
Detailed statistical information for represented data (mean values, SEM, n, p) is shown in Table
S1.
87
BRP
GluRIII
B
C E
0
neuronal surface
area (μm
2
)
100
200
300
ns D
F
2 µm
BRP puncta
density (#/μm
2
)
0.0
1.0
0.5
Ib Is
ns
ns
GluRIIA vGlut-OE
SYN
HRP
10 µm
wild type
A
GluRIIA+vGlut-OE
bouton #/NMJ
0
60
40
20
Ib Is
*
****
*
**
***
ns
0
total bouton #/NMJ
20
ns
40
60
80
Supplemental Figure 3: vGlut-OE induces a selective increase in Is bouton number, related
to Figure 4. (A) Representative images of muscle 6/7 NMJs immunostained with antibodies that
recognize the synaptic vesicle marker Synapsin (SYN) and the neuronal membrane marker HRP.
(B) Representative images of individual synaptic boutons immunostained with antibodies that
recognize the presynaptic active zone scaffold Bruchpilot (BRP) and the postsynaptic glutamate
receptor subunit GluRIII. (C) Quantification of the total neuronal membrane surface area (labeled
by HRP) in the indicated genotypes. (D) Quantification of Ib and Is bouton numbers reveals a
slight but significant reduction in Ib boutons in all genotypes compared to wild type, but a
selective increase in the number of Is boutons in vGlut-OE and GluRIIA+vGlut-OE. (E-F)
Quantification of total bouton number (both Is and Ib) per NMJ (E) and BRP puncta density at Ib
and Is boutons (F) in the indicated genotypes. Detailed statistical information for represented data
(mean values, SEM, n, p) is shown in Table S1.
88
A B
bouton #/NMJ
0
50
100
ns
GluRIIA GluRIIB GluRIID
WT GluRIIA-OE
1 μm
WT GluRIIA-OE
D C
Tor-
OE
10 µm
SYN
HRP
% wild type
0
50
100
150
ns
**
****
GluRIIA GluRIIB GluRIID
intensity
Supplemental Figure 4: GluRIIA overexpression does not impact NMJ growth or structure,
related to Figure 7. (A) Representative images of NMJs immunostained with antibodies that
recognize the postsynaptic glutamate receptor subunits GluRIIA, GluRIIB, and GluRIID. (B)
GluRIIA-OE results in increased levels of GluRIIA and a near loss of GluRIIB compared to wild
type. (C) Representative images of muscle 6/7 NMJs immunostained with antibodies that
recognize the synaptic vesicle marker Synapsin (SYN) and the neuronal membrane marker HRP.
(D) Quantification of bouton numbers showing no significant difference between wild type and
GluRIIA-OE. Detailed statistical information for represented data (mean values, SEM, n, p) is
shown in Table S1.
89
wild type
vGlut-OE
0.0 0.5 1.0 1.5 2.0 2.5
mEPSP (mV)
0
40
60
80
100
quantal content
20
GluRIIA-OE
Supplemental Figure 5: Failure to homeostatically tune presynaptic glutamate release at
GluRIIA-OE NMJs, related to Figure 7. Quantal content is plotted as a function of mEPSP
amplitude in the indicated genotypes. Note that wild type and vGlut-OE show the expected
homeostatic tuning of quantal content over varying mEPSP amplitudes, maintaining stable levels
of synaptic strength, while no scaling is observed in GluRIIA-OE. The black curve represents
ideal homeostatic tuning, derived by solving the function of EPSP= QC· mEPSP.
90
91
Chapter 4
Autocrine inhibition by the Glutamate-Gated Chloride channel
controls presynaptic homeostatic depression
4.1 Abstract
Presynaptic inhibition of neurotransmitter release is a fundamental mechanism that
modulates synaptic function in the nervous system. A homeostatic inhibition of
presynaptic release is employed to prevent excess glutamate release at Drosophila
larval NMJ. To unveil the molecular mechanism that necessary to drive the presynaptic
homeostatic depression (PHD), we did a candidate screen of 11 GluRs and identified
the glutamate-gated chloride channel (GluCl) to be required to sense the exuberance
glutamate and inhibit presynaptic vesicle release. Mutation in GluCl block PHD
expression, without altering the baseline function. Next, we reported that GluCl is
exclusively expressed in the nervous system, and pharmacological activation of the
GluCl determined its presence and function at the presynaptic motor neuron terminals.
Interestingly, we find that GluCl may associate with synaptic vesicles and function to
reduce presynaptic efficacy in an activity-dependent manner. Finally, we show that
extracellular chloride is essential to control the presynaptic inhibition. Altogether, these
results imply a novel mechanism of a release-dependent presynaptic autocrine
signaling through a ligand-gated chloride channel that controls the homeostatic
downregulations of presynaptic neurotransmitter release.
92
4.2 Introduction
Conserved homeostatic modulations of synaptic function are adapted in the face of stress and
perturbations to stabilize the normal function of nervous systems in various organisms (Davis,
2013; Turrigiano, 2012). Particularly, bi-directional homeostatic regulations of presynaptic
neuro-transmitter release have been established in different physiological conditions.
Disruptions of postsynaptic receptors function lead to a retrograde signal to potentiate
presynaptic release that precisely compensates for the reduction in postsynaptic sensibility to
neurotransmitters (Cull-Candy et al., 1980; Delvendahl et al., 2019; Petersen et al., 1997),
which referred to as presynaptic homeostatic potentiation (PHP). The inverse regulation of
neurotransmitter release, termed as presynaptic depression (PHD), has been characterized in
manipulations when synaptic vesicle (SV) size and con-tent are increased by vGlut
overexpression, which leads to enlarged spontaneous miniature postsynaptic activities (quantal
size; (Daniels et al., 2004)). In this process, the evoked response amplitude is maintained at
baseline levels due to a homeostatic downregulation of the presynaptic-tic release of SVs
(Daniels et al., 2004). Unlike destabilizing synaptic strength triggers PHP expression, PHD has
been proposed as an adaptive response to excess glutamate (Daniels et al., 2004; Gavino et
al., 2015; Li et al., 2018b).
Present evidence shows that increasing SV diameter and content is the sole condition
that induces PHD expression. Independent with vGlut overexpression, PHD has also been
implicated in mutants with enlarged SV sizes caused by defects in SV endocytosis and vesicle
reformation mechanisms (Chen et al., 2014; Dickman et al., 2005; Marie et al., 2004; Verstreken
et al., 2002). Normal synaptic strength due to a reduction in presynaptic release probability (Pr)
is observed in PHD conditions (Daniels et al., 2004). It has been shown that the reduction in Pr
in PHD resulted from a decreasing in Ca
2+
influx during a single action potential without
changing active zones (AZs) or Ca
2+
channel abundance (Gavino et al., 2015; Gratz et al.,
93
2019), however, the mechanisms underlying this process remain enigmatic. Either endocytic
deficits or vGlut overexpression leads to excess neurotransmitter release from each SV and
enhanced postsynaptic responsiveness. Nevertheless, increasing the miniature responses by
increasing postsynaptic sensitivity to the neurotransmitter does not induce PHD (DiAntonio et
al., 1999; Li et al., 2018b; Petersen et al., 1997). PHD is responsive to excess glutamate
release and operates in ignorance of postsynaptic excitability rather than balancing synaptic
strength (Li et al., 2018b). Therefore, a model has been proposed that PHD is governed by
glutamate homeostasis through an autocrine inhibition of presynaptic release, which meditated
by a presynaptic glutamate autocrine receptor to balance overall synaptic glutamate release (Li
et al., 2018b).
Presynaptic inhibition has been studied for a long time in different species that believed
to mediate synaptic depression and prevent excessive transmitter release (Dudel and Kuffler,
1961; Rudomin and Schmidt, 1999; Wu and Saggau, 1997). The downregulations of
presynaptic release are mainly achieved through two major mechanisms. First, extensive
evidence on the metabotropic pathways suggests that the G-protein coupled signaling induced
by presynaptic receptors mediates presynaptic inhibition in mammalian central neurons
(Blackmer et al., 2001; Gerachshenko et al., 2005; Straiker et al., 2002; Takago et al., 2005;
Takahashi et al., 1998). Not only metabotropic receptors (Alexander and Godwin, 2005; Bocchio
et al., 2018; Scanziani et al., 1997; Takahashi et al., 1996; Wang et al., 2005) but also common
excitatory ionotropic glutamate receptors (iGluRs), including AMPA, NMDA, and KAINATE
receptors are demonstrated to engage in the metabotropic pathways to inhibit presynaptic
release (Kamiya, 2002; Negrete-Diaz et al., 2007; Padamsey et al., 2017; Takago et al., 2005).
Second, the presynaptic chloride conductance that drives a shunting current at the axon
terminals has been implicated in presynaptic inhibition (Rudomin and Schmidt, 1999;
Takayanagi-Kiya et al., 2016). Notably, modulations in Ca
2+
influx has been reported in both
94
mechanisms to achieve the reduction in presynaptic neurotransmitter release (Blackmer et al.,
2001; Rudomin and Schmidt, 1999; Takahashi et al., 1996).
To elucidate the molecular mechanisms underlying PHD expression, we systematically
screened 11 GluRs encoded in the Drosophila genome with electrophysiology recordings at the
neuromuscular junction (NMJ). Surprisingly, we identified the Drosophila glutamate-gated
chloride channel (GluCl) to be required for PHD expression. Here we provide evidence of the
presence of GluCl at motor neuron terminals that localize and traffic with SVs to induce an
activity-dependent autocrine inhibition of presynaptic release when excess glutamate release is
triggered by vGlut overexpression. Our findings reveal a release-dependent chloride
conductance contributes to the rapid presynaptic inhibition in PHD expression.
95
4.3 Results
Electrophysiology-based candidate screen for PHD identifies GluCl
To identify the presynaptic autocrine glutamate sensor that mediates PHD (Figure 1A), we
systematically screened all 11 known glutamate receptors that potentially presented in the
Drosophila CNS (excluding olfactory GluRs). These glutamate receptors include AMPA, kainite,
NMDA, metabotropic receptors, and a ligand-gated chloride channel that fall into five
evolutionarily distinct families (Figure 1B). In most cases, the conventional ionotropic receptors
are mediating excitatory synaptic transmission in the nervous system, while existing evidence
has shown a link of metabotropic function to the ionotropic receptors (Kamiya, 2002; Takago et
al., 2005). Particularly, it has been reported that a presynaptic NMDAR mediating an autocrine
inhibition of presynaptic release in the presence of excess glutamate in mammalian
hippocampal neurons (Padamsey et al., 2017). We generated a collection of 24 known and
putative genetic mutations and RNAi lines that targeting these GluRs from public resources to
screen for the defects in PHD expression. We removed the mutants that failed to survive to
third-instar larval stages. To carefully exam the function of NMDARs in PHD, we created crisper
mutants against NMDAR1 and NMDAR2 separately. Together we screened 20 mutants/RNAi
lines through electrophysiology recordings.
We induce PHD by overexpression vGlut in the motor neurons (Daniels et al., 2004).
vGlut-OE leads to a significant increase in miniature excitatory postsynaptic potential (mEPSP)
amplitude, but the evoked amplitude stays at the baseline level due to a homeostatic reduction
of presynaptic glutamate release (Figure 1C-G). In this screen, we investigated the synaptic
transmission of GluR disruptions alone for baseline function in the mutants and RNAi lines
driven by a neuronal driver (OK371-Gal4) and in combination with vGlut-OE to compare
baseline and PHD conditions. Lines of evidence have shown that the metabotropic glutamate
96
receptors mediate presynaptic inhibition of neurotransmitter release in the mammalian nervous
system (Alexander and Godwin, 2005; Bocchio et al., 2018; Oliet et al., 2001). Consistently, the
null mutation of Drosophila mGluRA exhibits activity-dependent synaptic augmentations at the
larval NMJs (Bogdanik et al., 2004). However, the vGlut-OE induced PHD is intact in mGluR
mutants. There’s a homeostatic decrease in the quantal content of mGluR mutants when the
mEPSP is increased due to vGlut overexpressing (Figure 1D-G). To further test the role of
metabotropic pathways in PHD, we also screened seven G protein-coupled signaling and found
no evidence for a function in PHD (Supplemental Figure 1).
After the screening of all 11 GluRs, we identified one mutant in GluCl that abolished
PHD expression. Similar levels of increase in the mEPSP amplitudes were observed across all
genotypes in vGlut-OE background, and the homeostatic reduction of quantal content was
detected in all genotypes except for the GluCl homozygous mutant with vGlut-OE (Figure 1C).
Thus, disruption of GluCl is sufficient to block PHD expression.
The glutamate-gated chloride channel GluCl is required for PHD expression
Phylogenetic analysis reveals that GluCl is far distinct from the other four families of GluRs in
Drosophila genome (Figure 1B). GluCl is a member of a Cys-loop LGICs, which is highly
conserved through most of the invertebrates, and their pore-lining transmembrane region of this
family has extensive homology to GABA- and glycine-gated chloride channels in mammals.
Based on the crystal structure analysis of C. elegance GluCl, each GluCl subunit contains four
transmembrane domains and forms homo-pentamer receptor (Figure 2B) (Cull-Candy et al.,
1980; Hibbs and Gouaux, 2011). Glutamate binding leads to a conformational change in the
GluCl receptors and open the central pore that mediate an intrinsic chloride conductance.
Ivermectin has been proven to be an effective agonist that irreversibly open the chloride channel
with low concentrations (Cully et al., 1994; Hibbs and Gouaux, 2011; Kane et al., 2000). The
97
GluCl mutant allele from the screen contained a Minos transposon insertion into an intronic
region near the 3’ UTR of the putative transcript of GluCl (Figure 2A).
Disruption of GluCl leads to locomotor and other behavior deficits (Collins et al., 2012;
Cook et al., 2006; Dent et al., 1997; Pemberton et al., 2001). However, the role of endogenous
GluCl in synaptic function has not been studied at the Drosophila NMJ. GluCl may mediate
feedback inhibition in baseline transmission, or GluCl could function as a “stress response” to
excess glutamate release, with no significant role in baseline neurotransmission. We therefore
further characterized baseline synaptic transmission and PHD condition in GluCl mutant as
homozygous and in trans-heterozygous with a deficiency. We observed no significant change in
both miniature and evoked responses amplitudes in GluCl or GluCl/DF (Figure 2C-F). However,
following overexpression of vGlut, both miniature and evoked amplitudes were increased
significantly without changing the quantal release in GluCl or GluCl/DF (Figure 2C-F), indicating
no PHD expression was detected in either mutant allele alone, or in mutant allele in trans to a
deficiency that removes the entire GluCl locus (Figure 2A). Thus, GluCl expression has no
major contribution to basal synaptic transmission, but GluCl function is required for the
expression of PHD when the stress (vGlut-OE) is induced.
The presynaptic release can be regulated bi-directionally at the Drosophila NMJ. Except
for PHD, presynaptic homeostatic potentiation (PHP) can be induced by disruption of
postsynaptic receptors by acute application of philanthotoxin (PhTx). Although GluCl is
necessary for PHD, whether GluCl plays a role in PHP is unknown. Hence, we tested the PHP
in GluCl mutant. Following PhTx application, we observed a significant reduction in mEPSC, but
no change in EPSC in GluCl due to a homeostatic potentiation of presynaptic neurotransmitter
release (Supplemental Figure 2). Therefore, GluCl is required for PHD but not necessary for
acute PHP expression.
98
The reduction in release probability is canceled in GluCl + vGlut-OE
To thoroughly investigate PHD expression in GluCl mutants, we assessed calcium cooperativity
and short-term plasticity in GluCl mutant and GluCl + vGlut-OE in comparison to wild type and
vGlut-OE alone. It has been shown that there is no change in apparent Ca
2+
cooperativity in
vGlut-OE (Li et al., 2018b). Whether GluCl leads to a change in the Ca
2+
cooperativity in
baseline and PHD conditions is unclear. We first measured the apparent Ca
2+
cooperativity of
synaptic transmission by testing quantal content (QC) over a range of external Ca
2+
concentrations. No significant difference in the apparent Ca
2+
cooperativity was observed in
GluCl mutant and GluCl + vGlut-OE (Figure 3A-B), which showed similar slopes of QC over
Ca
2+
compare to wild type and vGlut-OE. In addition, the depression of QC in vGlut-OE was
similarly abolished in GluCl + vGlut-OE across different Ca
2+
conditions (Figure 3A-B). Thus,
presynaptic glutamate release is appropriately tuned across a range of Ca
2+
concentrations in
GluCl alone and in PHD condition.
The presynaptic neurotransmitter release probability (Pr) is reduced in vGlut-OE with the
evidence of increased failure rate, reduced paired-pulse depression (PPD) and enhanced
paired-pulse facilitation (PPF) (Daniels et al., 2004; Li et al., 2018b). We next examined whether
the reduction of Pr in vGlut-OE is blocked in GluCl. Failure analysis was performed to assess
presynaptic function independent with the mEPSC amplitude. We detected the expected
increase in failures in vGlut-OE compare to wild type, while the failure rates in both GluCl and
GluCl + vGlut-OE were not significantly different from wild type (Figure 3C), which suggested
that the reduction of Pr in vGlut-OE is prohibited in GluCl + vGlut-OE. Additionally, measuring of
paired-pulse facilitation and depression ratios were utilized to estimate Pr in 0.3 mM and 1.8
mM separately (Li et al., 2018b). In reduced extracellular Ca
2+
concentration (0.3mM), PPF is
observed at the drosophila NMJ. vGlut-OE exhibited enhanced PPF as expected, and no
significant change was observed in GluCl and GluCl + vGlut-OE compare to wild type (Figure
99
3D-E). Consistently, at physiological Ca
2+
conditions (1.8 mM), we found no change of PPD in
GluCl, reduced PPD in vGlut-OE, and the reduction was abolished in GluCl + vGlut-OE (Figure
3F-G). Together, the results of failure analysis, PPF, and PPD indicate that GluCl plays no role
for maintenance of baseline Pr, however, GluCl is required for the down regulation of Pr in PHD
expression. And the requirement of GluCl in PHD expression is uniformly across low to elevated
extracellular Ca
2+
conditions.
GluCl is exclusively expressed in the nervous system to mediate PHD
Electrophysiology examinations suggests GluCl is essential for PHD expression, but where its
function is required for the process of PHD remained undefined. Early studies show the present
of GluCls in both insect muscle cells and neurons, including larval lateral neurons in D.
melanogaster which mediate rhythmic light avoidance behaviors (Cleland, 1996; Collins et al.,
2012; Cull-Candy, 1976; Gration et al., 1979). However, GluCl has not been investigated at the
fly NMJ and the expression pattern is unclear. We therefore generated a GluCl promoter Gal4 to
express a GFP reporter and determine the GluCl expression by the GFP signal. GFP signal is
broadly observed in the CNS including the motor neuron, but no GFP signal is detected in the
muscle, which demonstrate that the GluCl is exclusively expressed in the nervous system
including the presynaptic motor neuron at the NMJ (Figure 4A). This finding was repeated and
confirmed with GluCl-Gal4 obtained from other resources. This result fits with the hypothesis
that a presynaptic autocrine receptor that sensing excess glutamate release and inhibit
presynaptic release. If the GluCl function is only required in the motor neurons, disruption of the
expression of GluCl specifically in the neurons with RNAi should also block PHD expression. To
further test this idea, we assessed PHD in GluCl presynaptic knockdown with GluCl-RNAi.
Consistent with the block of PHD in GluCl mutant, presynaptic knockdown of GluCl exhibited
enhanced mEPSC and EPSC in vGlut-OE background suggesting no PHD was observed, while
no baseline defect was found in presynaptic GluCl knockdown (Figure 4B-C). Thus, GluCl is
10 0
exclusively expressed in the neurons, and functioning at the presynaptic neurons to mediate
PHD expression at Drosophila larval NMJs.
Gain of function of GluCl decreases synaptic efficacy
GluCls were demonstrated to mediating an inhibitory role in synaptic function (Chalasani et al.,
2007; Dent et al., 1997; Ohnishi et al., 2011; Strycharz et al., 2011). Next, we sought to
characterize the gain of function of GluCl at the NMJ. First, overexpression of GluCl by the
neuronal driver leads to a reduction in EPSC amplitude without changing the mEPSC,
suggesting a depression in presynaptic efficacy (Figure 5A-D). Second, to probe the PHD
function in GluCl-OE, we assessed the synaptic transmission in GluCl-OE in combination with
vGlut-OE. mEPSC amplitude was increased significantly in GluCl-OE + vGlut-OE, while the
EPSC amplitude kept the same compare to GluCl-OE alone, indicating a further reduction in the
quantal content (Figure 5A-D). Thus, GluCl overexpression leads to a reduction in presynaptic
release, and PHD can be expressed when presynaptic release is tuned down by GluCl-OE.
Acute Pharmacological activation of GluCl induces local inhibition of neurotransmitter
release at Drosophila NMJs
GluCl regulates synaptic transmission at the NMJ, however where this chloride channel function
at the motor neuron remains unclear. It may present and function at the motor neurons
dendrites, cell body, or the axon terminals (Figure 5E). Heterologous expression of Drosophila
GluCl has shown that GluCl forms homomeric channels, which can be activated by L-
glutamate, and interestingly, GluCl can be irreversibly activated by an active ingredient of a
commonly used pesticide, ivermectin (IVM), as a pharmacological agonist (Hibbs and Gouaux,
2011; Kane et al., 2000). First, we tested whether IVM could acutely activate GluCl and
modulate synaptic function in vivo using the semi-intact Drosophila larval NMJ preparation.
1 01
Notably, to perform the electrophysiology recordings, we cut the motor nerve from the cell body
prior to the acute application of IVM. Hence, the acute activation of GluCl by IVM would provide
evident insights into whether GluCl presents and functions at the motor neuron terminals during
baseline synaptic transmission and under PHD condition. If GluCl only performs in the cell body
or dendrites, the acute application of IVM should have no effect on synaptic transmission at the
NMJ, and vice versa. Although both mEPSC and EPSC amplitudes remained unchanged, a
significant reduction in the EPSC decay time constant but not the rise time was observed in wild
type synapses after acute IVM application compares to the EPSCs of the same cell before the
drug application (Figure 5F- H). And this accelerated decay after IVM was not detected in GluCl
mutant (Figure 5F, H). Therefore, this validates GluCl activity at the presynaptic terminals and
verifies the GluCl mutant as a loss-of-functional allele.
Next, to examine whether activation of the chloride channel by IVM modify PHD
expression at the NMJ, EPSCs were recorded in vGlut-OE animal with severed motor nerve
before and after IVM diffusion. Surprisingly, significant reductions in both EPSC amplitude and
decay were found in vGlut-OE following IVM application without changing mEPSC (Figure 5F-
H), suggesting IVM induced a more robust GluCl-dependent inhibition of neurotransmitter
release in vGlut-OE compare to control NMJs. One preferred hypothesis for this strengthened
impact is that vGlut-OE leads to enhanced sensitivity to IVM through elevated GluCl expression
at the presynaptic terminals when SVs are enlarged. Altogether, this demonstrates that GluCl
localizes to motor neuron terminals, and acute IVM application mediates a local GluCl-
dependent inhibition in synaptic transmission. So far, all experimental evidence favored our
proposed model that a glutamate receptor presents at presynaptic terminals and mediates
autocrine signaling to tune down presynaptic release (Figure 1A).
However, if GluCl constitutively presents and functions at the presynaptic terminal
membrane, and the hemolymph glutamate in Drosophila larval is high (Augustin et al., 2007;
102
Chen et al., 2009), which is far enough from activating the chloride channel based on the
heterologous expression examinations (Kane et al., 2000), then why GluCl is dispensable for
baseline transmission is still not understood. One possibility could be that the level of GluCl
protein is low on the presynaptic terminal membrane surface in wild type.
GluCl localizes and traffics with synaptic vesicles
To further dissect the function of GluCl in synaptic function at the NMJ, we first resolved its
subcellular expression pattern by neuronal expression of a UAS- construct of GluCl with a smFP
tag (UAS-GluCl-smFP; see Methods). Interestingly, imaging of GluCl-smFP and synaptic vesicle
markers (vGlut and synaptotagmin (Syt)) reveals a strong co-localization of the GluCl-smFP
signal to the vesicle markers (Figure 6A), leading to a hypothesis that GluCl associates and
traffics with synaptic vesicles during synaptic transmission. If GluCl presents on synaptic
vesicles, depleting the releasable vesicles in combination with the absence of endocytosis
should lead to a redistribution of the protein to the presynaptic membrane surface. Therefore, to
test the hypothesis, we utilize a temperature-sensitive mutant shibire (shi
ts1
), which encodes
Dynamin, to prevent endocytosis at the non-permissive temperature (34 ℃) and stimulate the
preparation with high K
+
bath solution in the meantime (Roos and Kelly, 1998; Yao et al., 2009).
The non-permeabilized antibody staining of GluCl-smFP reveals that little GluCl is present on
the surface of the presynaptic terminal membrane in shibire background at the permissive
temperature (22 ℃) without stimulation (Figure 6B left panel). Therefore, with low level of GluCl
presence, loss of GluCl barely affects baseline transmission. After 10 min stimulation in high K
+

solution in 34 ℃, the surface level of GluCl-smFP on the presynaptic terminal membrane is
obviously increased, which suggests that GluCl localizes and traffics with SVs and function at
the plasma membrane of motor neuron terminals upon SV fusion.
Extracellular Chloride is required for the expression of PHD
1 03
Heterologous expression of GluCl in Xenopus oocytes showed selective response to glutamate
and conductance to chloride (Kane et al., 2000). We therefore investigated whether the chloride
conductance through GluCl is essential for driving PHD expression. To achieve this, we
performed electrophysiology recordings in chloride-free saline, which methanesulphonate was
chosen as a chloride substitute (see Methods). PHD is consistently observed in vGlut-OE under
condition of regular modified HL3, where mEPSC is enhanced with no change in the EPSC
amplitude compare to control, due to a significant reduction in quantal release (Figure 7A-E).
Nevertheless, recordings of vGlut-OE animals in chloride-free saline exhibited increased
mEPSC amplitude as well as the evoked amplitude compare to wild type in the same saline
condition, resulting from no regulation of presynaptic release (quantal content; Figure 7A-E).
Hence, PHD is not observed in vGlut-OE in chloride-free saline, which suggests that the
extracellular chloride ion is essential for the expression of PHD.
1 04
4.4 Discussion
In this study, we have systematically assessed 11 GluRs of their function in PHD, and
identified that GluCl to be necessary for the autocrine inhibition of presynaptic release in
vGlut-OE. Our data suggest that GluCl is expressed exclusively in the nervous system,
and function at the presynaptic terminals of the NMJs. Additionally, we find that GluCl
co-localizes with synaptic vesicle markers, and operates in an activity-dependent
manner to modulate presynaptic release in PHD conditions. At Drosophila larval NMJ,
GluCl senses excess glutamate release, and in turn, decreases presynaptic
neurotransmitter release with a Cl
-
dependent mechanism. Altogether, we revealed the
molecular mechanism for PHD expression and provided evidence for a model of a
presynaptic anion-selective ion channel that controls release-dependent autocrine
signaling to homeostatically regulate glutamate release.
GluCl-dependent mechanisms mediate PHD expression
The release-dependent GluCl function is dispensable for baseline synaptic transmission, yet, it
is essential for PHD expression (Figure 2). In gain-of-function conditions by GluCl
overexpression or acute application of GluCl agonist IVM reduce presynaptic efficacy.
Particularly, applications of IVM to wild type animals shortened the decay time constant but not
the rising phase (Figure 5), suggesting that upon synaptic fusion and glutamate release,
activating of GluCl inhibits further SV release in the late phase of an action potential triggered
release. Intriguingly, IVM mediates a stronger effect on synapse with vGlut-OE, and this
increase in the ligand sensitivity of GluCl after IVM could be explained by one favored
hypothesis that more GluCl is operating at the presynaptic terminal membrane during synaptic
activity due to an elevated number of GluCl is correspond to the enlarged SV size in vGlut-OE.
1 05
Results from motor neuron RNA-seq suggested this increase of GluCl in vGlut-OE could be
resulted from an increase in GluCl expression level (data not shown).
How does this glutamate-gated chloride channel contributes to PHD expression? It is
known that PHD is achieved through a significant down regulation of Ca
2+
influx following a
single action potential at the NMJ (Gavino et al., 2015). However the calcium channel
abundance is unperturbed in vGlut-OE animals (Gratz et al., 2019). Therefore the molecular
mechanism that regulate the level of Ca
2+
influx remain obscure. Early research on vertebrate
spinal cord suggested that the reduction in presynaptic Ca
2+
currents which contributes to
presynaptic inhibition is achieved through a GABA receptor dependent Cl
-
shunting of
presynaptic activity (Rudomin and Schmidt, 1999). One hypothesis is that, the activity-
dependent local Cl
-
conductance through elevated GluCl at the presynaptic terminals in PHD
condition dampens the potential near active zoon leading to an inhibition of Ca
2+
influx, which in
turn reduces presynaptic release.
Presynaptic autocrine signaling in glutamate homeostasis
The homeostatic decrease in presynaptic efficacy has been proposed to be regulated in
glutamate homeostasis (Li et al., 2018b). Homeostatic regulation of glutamate signaling
is vital for a nervous system, and excitotoxicity due to excess glutamate could cause
severe consequences. Glutamate excitotoxicity has been implicated in seizures, stroke,
traumatic brain injury, and neurodegenerative diseases (Doble, 1999; Lai et al., 2014;
Murphy-Royal et al., 2017; Olloquequi et al., 2018; Verma et al., 2015). To avoid this
toxicity, the nervous system evolved numerous clearance mechanisms to
homeostatically control glutamate levels and prevent the damage. Other than the
conventional mechanism for glutamate homeostasis based on excitatory amino acid
transporters (EAATs) in glia cells and presynaptic neurons, existing evidence suggests
1 06
that presynaptic inhibition of glutamate release plays a role in balancing glutamate
signaling to promote neuronal survival (Li et al., 2009; Oliet et al., 2001). In rat central
nervous system, blocking of the glutamate transporter, which exclusively expressed in
astrocytes, triggers inhibition of glutamate release through a presynaptic metabotropic
GluR receptor reversibly (Oliet et al., 2001).
At Drosophila larval NMJ, there is no expression of the sole EAAT for glutamate
that encoded in the Drosophila genome (Rival et al., 2004). Therefore, we proposed that
PHD is a presynaptic inhibition mechanism that utilized to homeostatically regulate
glutamate signaling at Drosophila larval NMJ. And this presynaptic inhibition is achieved
through a glutamate-gate chloride channel that autocrine modulate presynaptic
glutamate release. Additionally, our data suggest that presynaptic GluCl operates as
both the sensor and the effector for PHD that senses the excess glutamate signal and
modulate presynaptic release. The function of this auto-receptor enables superefficient
feedback inhibition to homeostatically control glutamate release and avoid glutamate
toxicity. Interestingly, GluCl is a close homolog to mammalian glycine/GABA receptors,
and there is evidence that glutamate and glycine/GABA co-release in mammalian CNS
(Gillespie et al., 2005; Shabel et al., 2014). Thus, our work could shed light on the
understandings of the novel autocrine mechanism of presynaptic inhibition in glutamate
homeostasis in other systems.
1 07
4.5 Materials and Methods
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Drosophila stocks were raised at 25°C on standard molasses food in an incubator with a 12-hr
light/dark cycle. All experimental flies were collected at the wandering third-instar larval stage.
No significant sex difference in the results observed in this study. The w
1118
strain was used as
the wild-type control unless otherwise noted because this is the genetic background in which all
genotypes are bred.
METHOD DETAILS
PHD screen
We identified 11 glutamate receptors that potentially present at drosophila CNS. We used a
combination of previously characterized genetic and/or transposon mutations or RNA-
interference transgenes targeting these genes to obtain a stock collection to screen. To
cautiously exam the role of NMDARs in PHD, we generate crisper mutants targeting both
NMDARs separately. We assessed the baseline synaptic transmission of homozygous mutants
and RNAi lines driven by neuronal drivers. And PHD function was tested in vGlut
overexpression combined with the mutations or RNAi knockdowns.
Fly stocks
All Drosophila stocks were obtained from the Bloomington Drosophila Stock Center unless
otherwise specified. The following fly stocks were used in this study for the characterization of
GluCl in PHD: OK371-Gal4 (Mahr and Aberle, 2006); UAS-vGlut (Daniels et al., 2004); UAS-
GluCl-RNAi (P{KK109167}VIE-260B; Vienna Drosophila Resource Center); UAS-CD4-td-eGFP;
UAS-Dcr2; GluCl
MI02890
.
1 08
Molecular biology
There are 11 predicted GluCl isoforms in Drosophila bases on expressed sequence tags
(http://flybase.org/reports/FBgn0024963.html). Among the isoforms, GluCl-RM appears to be
the major isoform based on expression profiling. To generate UAS-GluCl, we subcloned the full-
length GluCl cDNA from the expressed sequence tag (Drosophila Genomics Resources Center;
Bloomington, IN) into the pACU2 vector (31223; Addgene, Cambridge, MA). Separately, a
spaghetti monster FLAG tag33 (10xFLAGsmFP) was PCR- amplified and placed in-frame
before the stop codon of the GluCl open reading frame. The Gibson Assembly Cloning Kit (New
England Biolabs Inc., E5510S) was used to generate the final construct. Constructs were
sequence verified and injected into the w
1118
strain using the VK18 insertion site on the second
chromosome by BestGene Inc. (Chino Hill, CA). GluCl-Gal4 contains GluCl sequences,
extending from 4.2 kb upstream of the transcription start site to the endogenous start codon,
fused to GAL4.
We generated NMDAR1 and NMDAR2 mutants by using a CRISPER/Cas-9 genome
editing strategy (Gratz et al., 2013). Briefly, we selected a target Cas-9 cleavage site in the
earliest coding exon of NMDAR1 without obvious off-target sequences in the Drosophila
genome. NMDAR2 mutants were generated using triple gRNA. DNA sequences covering these
target sequences were synthesized and subcloned into the pU6-BbsI-chiRNA plasmid (45946;
Addgene). To generate the triple gRNA, pU6-BbsI-chiRNA was PCR amplified and cloned into
the pattB vector. The constructs were separately injected to create the gRNA animal lines. The
NMDAR1 triple gRNA and NMDAR2 triple gRNA lines were separately crossed with a stock
expressing Cas-9 under control of vas regulatory sequences, which led to mutations in the open
reading frame of the targeted gene. PCR followed by sequencing screening of 20 lines with
each active CRISPR mutagenesis led to 4 independent deletions or insertions with predicted
frame shift mutations in the NMDAR1 open reading frame, and 2 independent mutations in the
NMDAR2 open reading frame.
1 09
Immunocytochemistry
Larvae were dissected in ice-cold 0 Ca
2+
-modified HL3 saline (Dickman et al., 2005; Stewart et
al., 1994) containing 70 mM NaCl, 5 mM KCl, 10 mM MgCl2, 10 mM NaHCO3, 115 mM sucrose,
5 mM trehelose, 5 mM HEPES, pH 7.2, and immunostained as described (Li et al., 2018b).
Briefly, dissected larvae were washed three times with modified HL3 saline, and fixed in Bouin’s
fixative (HT10132-1L; Sigma Chemical, St. Louis, MO) for 3 min. Larvae were washed with PBS
containing 0.1% Triton X-100 (PBST) and blocked with 5% normal donkey serum followed by
primary antibodies incubation at 4 ⁰C overnight. Then three 10 min washes in PBST and
incubate in secondary antibodies at room temperature for 2 hr. Samples were transferred in
VectaShield (Vector Laboratories, Burlingame, CA) and mounted on glass cover slides. The
following antibodies were used: guinea pig anti-vGlut (1:2000; (Chen et al., 2017)); rabbit anti-
SYT1 (1:2,500; (Mackler et al., 2002)), mouse anti-GFP (1:1000, 3e6; Invitrogen, Carlsbad, CA).
Donkey anti-mouse, anti-guinea pig, and anti-rabbit Alexa Fluor 488- (715-545-150, 706-545-
148, 711-545-152; Jackson Immunoresearch), DyLight 405- (715-475-150, 706-475-148, 711-
475-152; Jackson Immunoresearch), and Cyanine 3 (Cy3)- (715-165-150, 706-165-148, 711-
165-152; Jackson Immunoresearch) conjugated secondary antibodies were used at 1:400.
Alexa Fluor 647 conjugated goat anti-HRP (123-605-021; Jackson ImmunoResearch) was used
at 1:200.
Confocal imaging and analysis
Samples were imaged using a Nikon A1R Resonant Scanning Confocal microscope equipped
with NIS Elements software and a 100x APO 1.4NA or 20x 1.4NA oil immersion objective using
separate channels with four laser lines (405 nm, 488 nm, 561 nm, and 637 nm) at room
temperature. For fluorescence quantifications of SV markers and GluCl-smFP signal, all
genotypes within a dataset were immunostained in the same tube with identical reagents and
110
then mounted and imaged in the same session. z-stacks were obtained using identical settings
for all genotypes, with z-axis spacing between 0.15 and 0.25 μm within an experiment and
optimized for detection without saturation of the signal. Maximum intensity projections were
used for quantitative image analysis with the NIS Elements software General Analysis toolkit. All
quantifications were performed for Type Ib boutons on muscle 6/7 and muscle 4 of segments A2
and A3. Measurements were taken from at least ten synapses acquired from at least six
different animals. For all images, fluorescence intensities were quantified by applying intensity
thresholds to the relevant channel, and the mean puncta intensity was calculated as the total
fluorescence intensity signal of the puncta divided by the area of the puncta.
Synaptic vesicle depletion experiments were done as previously described (Roos and
Kelly, 1998). Briefly, Shibire
ts1
;OK371-Gal4/UAS-GluCl-smFP flies were dissected and
incubated at 34 °C for 2 min in prewarmed Ca
2+
- HL3 saline and then incubated in pre-warmed
high K
+
saline (15 mM NaCl, 115 mM sucrose, 60 mM KCl, , 10 mM MgCl2, 1.5 mM CaCl2, 10
mM NaHCO3, 5 mM Trehalose, 5 mM HEPES, pH 7.2) at 34 °C for 10 min. After stimulation,
the samples were transferred to prewarmed calcium-free saline shortly prior to fixation.
shibire
ts1
-depleted preparations were fixed in bouins and labeled with α- vGlut, α- GFP, α- syt
and HRP primary antibodies. The redistributions of synaptic vesicle marker signals were
quantified using NIS Elements software.
Electrophysiology
All dissections and recordings were performed in modified HL-3 saline (Dickman et al., 2005;
Kiragasi et al., 2017; Stewart et al., 1994) at room temperature containing (in mM): 70 NaCl, 5
KCl, 10 MgCl2, 10 NaHCO3, 115 Sucrose, 5 Trehelose, 5 HEPES, and 0.4 CaCl2 (unless
otherwise specified), pH 7.2. Recordings were performed on an Olympus BX61 WI microscope
using a 40x/0.80NA water-dipping objective, and acquired using an Axoclamp 900A amplifier,
Digidata 1440A acquisition system and pClamp 10.5 software (Molecular Devices).
111
Electrophysiological sweeps were digitized at 10 kHz and filtered at 1 kHz. In the PHD
screening, sharp electrode (electrode resistance between 10–25 MΩ) recordings were
performed on muscles 6 or 7 of abdominal segments A2 or A3 in wandering third-instar larvae
(Li et al., 2018). Muscle input resistance (Rin) and resting membrane potential (Vrest) were
monitored during each experiment. Recordings were rejected if the Vrest was more depolarized
than −60 mV and if the Rin was less than 5MΩ. Two-electrode voltage clamp (TEVC) recordings
were performed on muscles 6 of abdominal segments A2 and A3 as described (Goel et al.,
2019; Li et al., 2018a). In all voltage clamp recordings, muscles were clamped at −70 mV, with a
leak current absolute value below 5 nA.
Miniature excitatory postsynaptic potentials (mEPSPs) or currents (mEPSCs) were
recorded for 1 min from each muscle cell in the absence of stimulation. 20 evoked responses
(EPSPs or EPSCs) were acquired for each cell under stimulation at 0.5 Hz, using 0.5 ms
stimulus duration and with stimulus intensity adjusted with an ISO-Flex Stimulus Isolator
(A.M.P.I.). Averaged mEPSPs (or mEPSCs) and EPSPs (or EPSCs) were calculated for each
recording, and quantal content was determined for each genotype. To acutely block
postsynaptic receptors, larvae were incubated with or without philanthotoxin-433 (PhTx; 20 μM;
Sigma) in HL-3 for 10 min before recordings. Data were analyzed using Clampfit (Molecular
devices), MiniAnalysis (Synaptosoft), and Excel (Microsoft) software.
To test the function of acute activation of GluCl channel in vivo, EPSCs were recorded
from 50 continuous stimuli that delivered to the cut motor axons with 0.5 Hz. The bath saline
was replaced by 0.4 mM Ca
2+
modified HL3 containing 10µM ivermectin (IVM, Millipore Sigma,
I8898) upon the time of the 20
th
stimulation with pipetting. Average EPSCs for each genotype
before the drug were calculated from the 1-20
th
stimuli, and the average EPSCs after IVM
treatment were assessed from the 30-50
th
stimuli. The recordings acquired in the chloride-free
saline were done as described above. The NaCl, KCl, MgCl2 and CaCl2 in modified HL3 were
replaced with Methyl sulfate sodium salt (Sigma-Aldrich 318183), Potassium methaesulfonate
112
(Sigma-Aldrich 83000), Magnesium nitrate hexahydrate (Sigma-Aldrich 63084) and calcium
nitrate tetrahydrate (Sigma-Aldrich C1396) respectively in the same final concentration. Pipette
offset increases dramatically with the pipette in the chloride-free solution.
EM
EM analysis was performed as described previously (Atwood et al., 1993). Larvae were
dissected in Ca
2+
-free HL-3 and then fixed in 1.25% glutaraldehyde/0.1 M phosphate buffer at
4°C. Larvae were then washed three times for 10 min in ddH2O. Samples were then placed in
1% osmium tetroxide in ddH2O for 90 min at room temperature. After washing the larva three
times with ddH2O, larvae were then dehydrated in ethanol. Samples were then cleared in 0.5%
uranyl acetate in ddH2O at 4°C overnight and washed three times with ddH2O in the following
day. Then samples were dehydrated in graded acetone with 15 min in each concentration.
Larva were infiltrated in Spurr’s in acetone. After infiltration, samples were polymerized in
catalyzed Spurr’s at 60°C for 24 hr. The following day, samples were embedded in fresh
Eponate 12. EM sections were obtained on a Morgagni 268 transmission electron microscope
(FEI). NMJs were serial sectioned at a 60- to 70-nm thickness. The sections were mounted on
Formvar-coated single slot grids and viewed at a 23,000 magnification and were recorded with a
Megaview II CCD camera. Synaptic vesicle size were analyzed blind to genotype using the
general analysis toolkit in the NIS Elements software and ImageJ software.
Phylogenetic Sequence Analysis
Phylogenetic analysis was conducted using amino acid sequence alignments for 11 Drosophila
GluR sequences culled from Drosophila melanogaster genome in MEGAX as described in
protocol (Hall, 2013). If more than one isoform was present, the shorted isoforms with essential
functional domains were chosen for the analysis to avoid discrepancy rises from different
isoforms. The 11 sequences were aligned with MUSCLE (Edgar, 2004). The maximum
113
likelihood (ML) fits of 56 different amino acid substitution models were tested (data not shown).
With the best model estimated by MEGAX, GluR amino acid sequences were performed using
the maximum likelihood (ML) tree reconstruction method and Le-Gascuel model (Le and
Gascuel, 2008). Initial tree(s) for the heuristic search were obtained by applying the Neighbor-
Joining method to a matrix of pairwise distances estimated using a JTT model. A discrete
Gamma distribution was used to model evolutionary rate differences among sites (5 categories
(+G, parameter = 2.0465)). The tree is drawn to scale, with branch lengths measured in the
number of substitutions per site.
B
D
50 msec
200 msec
5 mV
2 mV
wild type + vGlut-OE mGluRA
GluR
A
% baseline (-vGlut-OE)
wild type CG11155 KaiR1D
200
150
100
50
0
quantal content mEPSP
clumsy KaiR1C Ekar GluRIA GluRIB mGluRA GluCl NMDAR1 NMDAR2
***
**
****
**
***
****
***
****
**
****
**
ns
****
C
mEPSP (mV)
0
0.5
1.0
EPSP (mV)
0
10
1.5
20
****
30
ns E
quantal content
0
20
10
30 ****
** ****
+ vGlut-OE
Drosophila glutamate receptors
SV size
vGlut-OE
ns
F G
**
****
****
****
****
****
**
** ****
****
**
KAINATE
AMPA
NMDA
metabotropic
chloride channel
KaiR1C
KaiR1D
Ekar
clumsy
CG11155
GluRIA
GluRIB
NMDAR2
NMDAR1
GluCl
mGluRA
wild type
PHD
wild type
vGlut-OE
mGluRA
mGluRA+
vGlut-OE
Figure 1: A screen of Drosophila glutamate receptors identifies the Glutamate-Gated
Chloride Channel to be necessary for PHD. (A) Schematic illustrating enlarged synaptic vesicle
size and excess glutamate release following vGlut-OE (OK371-Gal4/UAS-vGlut). And a
presynaptic glutamate receptor mediating autocrine inhibition of presynaptic release in synapses
overexpression of vGlut. (B) Phylogenic analysis of potential presynaptic glutamate receptors of
D. melanogaster. The Maximum likelihood topology tree is constructed by GluRs including AMPA
(GluRIA, GluRIB), KAINATE (CG11155, Ekar, clumsy, KaiR1C, KaiR1D), NMDA (NMDAR1,
NMDAR2), metabotropic (mGluRA) receptors, and an ionotropic glutamate-gated chloride
channel (GluCl). (C) Quantification of mEPSP amplitude and quantal content in the indicated
genotypes, normalized to baseline (-vGlut) values (n ≥ 8). Note that the mEPSP amplitudes
enhances following vGlut overexpression in each mutant and a homeostatic decrease in
presynaptic glutamate release (quantal content) is observed in screened mutants with vGlut-OE
except for GluCl. The homeostatic inhibition of release induced by vGlut-OE is abolished in GluCl
mutants. (D) Schematic and representative traces of wild type and mGluRA baseline and +vGlut-
OE NMJs. Increased mEPSP amplitudes are observed in both wild-type and mGluRA NMJs after
vGlut overexpression, while EPSP amplitudes are maintained at baseline levels due to a
homeostatic decrease in quantal content. (E, F, G) Quantification of mEPSP amplitude (E), EPSP
amplitude (F), and quantal content (G)in the indicated genotypes.
114
C
****
**
ns
mEPSC
quantal
content
% baseline (-vGlut-OE)
150
100
50
0
****
D G
****
****
****
**
mEPSC (nA)
0
0.2
0.4
0.6
EPSC (nA)
0
20
40
60
80
quantal content
0
50
100
GluCl
A B
wild type
GluCl + vGlut-OE + vGlut-OE
ns
ns
E F
C
N
20 ms
200 ms
1 nA
10 nA
456 A.A.
GluCl TM TM TM TM
****
**
ns
****
ns
MI02890 (m25)
RNAi
1000 bp
wild type vGlut-OE GluCl
GluCl+
vGlut-OE
GluCl/DF
GluCl/DF
+vGlut-OE
Figure 2: The Glutamate-Gated Chloride channel GluCl is necessary for PHD expression.
(A) Schematic of the Drosophila GluCl locus, with one transposon insertion and an RNAi target
are shown. (Bottom) Structure and transmembrane domains of GluCl. (B) Diagram of speculated
protein structure of GluCl, and a homopentamer GluCl receptor. (C) Schematic and
representative traces of wild type and GluCl baseline and +vGlut-OE (OK371-Gal4/UAS-vGlut;
GluCl) NMJs. Increased mEPSC amplitudes are observed in both wild-type and GluCl NMJs
following vGlut overexpression. The evoked amplitude of vGlut-OE NMJ is the same as baseline,
meanwhile GluCl + vGlut-OE EPSC amplitude is enhanced significantly with unchanged quantal
content. (D- F) Quantification of mEPSC amplitude (D), EPSC amplitude (E), and quantal content
(F) in the indicated genotypes (baseline: wild type, n = 18; GluCl, n = 11; GluCl/DF, n = 11; +
vGlut: wild type, n =19; GluCl, n = 10; GluCl/DF, n = 10). (G) Quantification of mEPSC and
quantal content values normalized to baseline values.
115
EPSC2/EPSC1 (%)
50
100
0
****
*
G
150
200
250
F
ns
ns
10 nA
0.3 mM Ca
2+
10 ms
A
40
60
20
0
failure rate (%)
**
ns
ns
C B
quantal content
external [Ca
2+
] (mM)
0.4
10
50
100
0.3 0.5
WT
vGlut-OE
GluCl
vGlut-OE+GluCl
quantal content
external [Ca
2+
] (mM)
0.4
10
50
100
0.3 0.5
80
100
wild type + vGlut-OE + vGlut-OE GluCl
D
E
60 nA
10 ms
1.8 mM Ca
2+
EPSC2/EPSC1 (%)
40
60
100
20
0
**
80
ns
ns
Figure 3: Characterization of Ca
2+
cooperativity, failure analysis and PPF/PPD in GluCl +
vGlut-OE. (A, B) Quantal content plotted as a function of extracellular Ca
2+
concentration on a
log/log scale, with best fit lines shown, for wild type and vGlut-OE (A), and for GluCl and GluCl +
vGlut-OE (B). No significant differences in the slopes were observed in both graphs (Table S2).
(C) Failure analysis reveals a significant increase in failure rate in vGlut-OE, as expected. This
increase in failure analysis is blocked in GluCl mutant with vGlut overexpression, consistent with
a blockage of PHD expression. Recordings were performed in 0.05 mM extracellular Ca
2+
. (D)
Representative paired-pulse EPSC traces at 0.3mM extracellular Ca
2+
with an interstimulus
interval of 16.7 msec in the indicated genotypes. Enhanced paired-pulse facilitation (PPF) was
observed in vGlut-OE NMJ, consistent with decrease in release probability, while the increase in
PPF was not observed in GluCl + vGlut-OE. (E) Quantification of the paired-pulse ratio
(EPSC2/EPSC1) in the indicated genotypes (baseline: wild type, n = 8; GluCl, n = 9; + vGlut: wild
type, n =10; GluCl, n = 6). (F) Representative paired-pulse EPSC traces at 1.8 mM extracellular
Ca
2+
in the indicated genotypes. Ameliorate paired-pulse depression (PPD) was observed in
vGlut-OE, however the same PPD was shown for GluCl + vGlut-OE. (G) Quantification of the
paired-pulse ratio (EPSC2/EPSC1) in the indicated genotypes (baseline: wild type, n = 11; GluCl,
n = 10; + vGlut: wild type, n =7; GluCl, n = 12).
116
0.5 nA
200 ms
10 ms
10n A
B + vGlut-OE wild type pre>GluCl RNAi GluCl>CD4-tdGFP
200 µm
CNS
20 µm 200 µm
GluCl > CD4-tdGFP
CNS
nerve
NMJ
GFP
F-actin
A
****
***
****
ns
mEPSC
quantal
content
C
% baseline (-vGlut-OE)
150
100
50
0
muscle
+ vGlut-OE
20 µm
Figure 4: GluCl is expressed exclusively in the nervous system and required
presynaptically for PHD. (A) Representative images of central nervous system (CNS, left) and
muscle 6/7 NMJ images (right) of GFP expression driven by the GluCl promoter (GluCl-
Gal4/UAS-CD4-tdeGFP). Anti-GFP and anti-phalloidin (actin marker) are shown. GluCl is
exclusively expressed in CNS including presynaptic motor neurons. (B) Schematic and
representative EPSC and mEPSC traces in wild type and presynaptic GluCl knockdown by RNAi
(OK371-Gal4/pvGlut, GluCl-RNAi;UAS-Dcr2/+) baseline and with vGlut-OE. Presynaptic GluCl
knockdown is sufficiently inhibit PHD expression. (C) Quantification of mEPSC and quantal
content values in the indicated genotypes relative to baseline (baseline: wild type, n =10;
pre>GluCl-RNAi, n = 8; + vGlut-OE: wild type, n = 13 pre>GluCl-RNAi, n = 20).
117
10 ms
10 nA
wild type + IVM GluCl + IVM vGlut-OE + IVM F
- IVM
+ IVM
10 ms
100 ms
1 nA
10 nA
wild type GluCl-OE
A
0
10
20
30
40
50
EPSC (nA)
0
50
100
quantal content
0.0
0.2
0.4
0.6
mEPSC (nA)
ns
**
*
B
**
**
**
C D
E
% baseline (-ivermectin)
100
50
0
quantal content mEPSC
ns
*
ns
ns
ns
ns
n=4
n=4
% baseline (-ivermectin)
100
50
0
**
ns
*
decay tau
G H
wild type
GluCl-OE
vGlut-OE+
GluCl-OE
Figure 5: Acute application of the GluCl agonist IVM induces a local GluCl-dependent
inhibition in neurotransmitter release. (A) Schematic and representative EPSC and mEPSC
traces in wild type, GluCl-OE (OK371-Gal4/UAS-GluCl), and GluCl-OE+vGlut-OE (OK371-
Gal4/UAS-GluCl,UAS-vGlut). (B- D) Quantification of mEPSC amplitude (B), EPSC amplitude
(C), and quantal content (D) in the indicated genotypes (wild type, n = 10; GluCl-OE, n = 13;
GluCl-OE+vGlut-OE, n=10). The synaptic strength is reduced in GluCl-OE by decreasing
presynaptic release. And PHD is observed in GluCl-OE. (E) Schematic illustrating the model of
subcellular localizations of GluCl at the motor neuron dendrites and axon terminals. And only the
GluCl that presents at the axon terminals were tested for the IVM-dependent function by cutting
the motor neuron axon. (F) Schematic and representative EPSC traces in wild type, GluCl, and
vGlut-OE before and after IVM application from the same cell separately. (G) Quantification of
mEPSC and quantal content values in the indicated genotypes, and data was presented as the
average of mEPSC and quantal content of individual cell after IVM application relative to the
baseline before IVM application (wild type, n =5; GluCl, n = 4; vGlut-OE, n = 4). (F) Quantification
of the average decay time constant of individual cell normalized to its own baseline before IVM
application in the indicated genotypes.
118
GFP Syt vGlut
shi
ts1
(34 °C) shi
ts1
(22 °C)
GluCl-smFP + vGlut A
B
GFP GFP HRP
5 µm
-Triton
119
Figure 6: GluCl localizes and traffics with synaptic vesicles. (A) Representative images of
NMJs immunostained with antibodies that recognize the synaptic vesicle markers vGlut,
synaptotagmin (syt), and GFP at boutons with presynaptic GluCl-smFP overexpression (OK371-
Gal4/UAS-GluCl-smFP) and along with vGlut-OE (OK371-Gal4/UAS-GluCl-smFP, UAS-vGlut).
The GluCl-smFP signal co-localize very well with synaptic vesicle markers (vGlut and syt). (B)
Representative images of NMJs non-permeabilized immunostaining with antibodies that
recognize HRP and GFP at boutons with presynaptic GluCl-smFP overexpression in shibire
ts1
mutant background (Shi
ts1
;OK371-Gal4/UAS-GluCl-smFP) in permissive temperature(22 ⁰C) in
rest and non-permissive temperature (34 ⁰C) with high K+ stimulations. Little GluCl-smFP signal
is observed in the non-permissive temperature in rest, but increased GluCl-smFP signal is
detected to be associated with neuronal membrane in 34 ⁰C with high K
+
stimulations.
2 µm
***
**
ns
mEPSC
quantal
content
% baseline (-vGlut-OE)
150
100
50
0
****
B E
***
****
*
***
mEPSC (nA)
0
0.2
0.4
0.6
EPSC (nA)
0
20
40
60
quantal content
0
50
100
ns
ns
C D
***
20 ms
10 nA
A
wild type wild type (Cl
-
free) vGlut-OE vGlut-OE (Cl
-
free)
1 nA
200 ms
150
Figure 7: Extracellular chloride ion is required to express PHD. (A) Representative traces of
mEPSC and EPSC of indicated genotypes in specified saline conditions. Although vGlut-OE
increases mEPSC amplitude in both saline conditions, no change in presynaptic neurotransmitter
release is observed in the vGlut-OE NMJs recorded in Cl
-
free saline, leading to enhanced EPSC
amplitude and no change in quantal content. Hence, PHD is inhibited in Cl
-
free condition. (B-D)
Quantification of average mEPSC amplitude (B), EPSC amplitude (C), and quantal content (D)
values for the indicated genotypes and conditions (wild type, n=15 ; vGlut-OE, n=17 ; Cl
-
free:
wild type, n=12 ; vGlut-OE, n=14). (E) Quantification of mEPSC and quantal content values
normalized to baseline values (-vGlut-OE).
120
+ vGlut-OE
% baseline (-vGlut-OE)
150
100
50
0
quantal content mEPSP
wild type OK371>
pertussis toxin
***
****
****
****
OK371>
G αo
RNAi
***
****
50 msec
200 msec
5 mV
2 mV
ok371>pertussis toxin
B A
***
**
200
OK371>
G αi
RNAi
Supplemental Figure 1: Additional screening for metabotropic signaling finds no evidence
for roles in PHD. (A) Schematic and representative traces of ok371>pertussis toxin (OK371-
Gal4/+; UAS-pertussis toxin/+) baseline and +vGlut-OE (OK371-Gal4/UAS-vGlut; UAS-pertussis
toxin/+) NMJs. Pertussis toxin is shown to disrupt the function of G
αi
and G
αo
signals. Increased
mEPSP amplitudes are observed in the NMJs following vGlut overexpression, while a reduction
in presynaptic release maintains the EPSP as baseline level due to PHD expression. (B)
Quantification of mEPSC and quantal content values normalized to baseline values (n ≥ 8 for all
genotypes in this analysis).
121
10 ms
100 ms
1 nA
10 nA
wild type
+ PhTx GluCl + PhTx
****
****
mEPSC
% baseline (-vGlut-OE)
150
100
50
0
****
B
200
****
quantal
content
A
Supplemental Figure 2: GluCl is dispensable for PHP expression. (A) Representative traces
of wild type and GluCl baseline and after PhTx incubations. mEPSC amplitudes are diminished
following PhTx application in both wild-type and GluCl NMJs, with constant EPSC amplitudes
compare to the baseline transmission due to a retrograde enhancement of presynaptic release.
Therefore, PHP is adequately expressed in both wild type and GluCl mutant. (B) Quantification of
mEPSC and quantal content values normalized to baseline values genotypes (baseline: wild type,
n = 11; GluCl, n = 12; + PhTx: wild type, n =6; GluCl, n = 8).
122
123
Chapter 5
General Conclusion and Discussion
By interrogation of the bi-directional homeostatic modulations of presynaptic neurotransmitter
release, our results suggest two independent homeostats co-exist at Drosophila larval NMJ to
adaptively stabilize synaptic strength and control glutamate release respectively.
We first determined the specificity of PHP expression by a genetic manipulation that
specifically disrupts the postsynaptic glutamate receptors on one of the two adjacent muscle
fibers, which are innervated by the same motor neuron. And we find that the induction and
expression of PHP are compartmentalized to the synapses on the perturbed muscle while
leaving the synapses on the other muscle unperturbed. Therefore, the retrograde potentiation of
presynaptic release operates with high specificity, where it can be exclusively induced and
expressed at a subset of the synapses from the same neuron and enable flexible homeostatic
modulation of presynaptic efficacy with precise spatial resolution.
Interestingly, here we talked about two adjacent postsynaptic cells receiving the same
input, on the other hand, each of these muscles forms synapses with two specific glutamatergic
inputs, type Is (small, with less SSR) and type Ib (big, with elaborated SSR). Distinct electric
properties are defined in MN-Is and MN-Ib. Is inputs exhibit higher release probability, and tune
to depress following high-frequency stimulation; while Ib terminals have lower release
probability and facilitate during high-frequency stimulations. Both motor neurons operate
coordinately to mediate tonic and phasic activities in locomotion. Recent work has shown that in
chronic PHP expression varies at Is and Ib synapses depending on the extracellular calcium
concentration (Genc and Davis, 2019; Newman et al., 2017). In contrast, acute PHP expresses
124
in both synapses cross different external Ca
2+
conditions (Genc and Davis, 2019).
Unfortunately, with the limitations on quantification of miniature events specifically from Is and Ib
synapses, the quantal content assessed by EPSP(C)/mEPSP(C) is less accurate. In these two
distinct inputs, it is unclear whether the same molecular and cellular mechanisms are utilized for
PHP induction and expression regards to the dramatic differences of the postsynaptic
compartment (postsynaptic density scaffold) and presynaptic release properties in Is and Ib
synapses. Further, it also remains ambiguous whether Ib and Is function independently or with
crosstalk for the synaptic development, baseline transmission, and PHP expression.
We next investigated the inverse homeostatic modulation of presynaptic release, PHD,
and the interface between PHP and PHD. We have shown that these two homeostatic
signalings independently contribute to the bi-directional control of presynaptic efficacy. Although
the loss of GluRIIA exclusively triggers the expression of PHP in Ib boutons in low external Ca
2+

concentration, vGlut-OE induces PHD adequately expressed in both inputs. And most
interestingly, we find that PHD is only induced in synapses with excess glutamate packaged in
single vesicles and operates regardless of postsynaptic activity, it is not induced or influenced
by changing postsynaptic excitability. Therefore, we hypothesized that PHD is a homeostatic
mechanism that is aiming to balance glutamate release instead of stabilizing synaptic strength.
In this work, we proposed a model that a presynaptic receptor that senses the excess glutamate
release and autocrinally decrease glutamate release.
Finally, we performed a candidate screen of 11 GluRs in the Drosophila melanogaster
genome and identified the glutamate-gated chloride channel to be needed for PHD expression.
We find that GluCl is exclusively expressed in the CNS and operates at the presynaptic
terminals at the larval NMJs. And pharmacological applications of IVM suggest that the IVM-
dependent glutamate sensitivity is significantly enhanced in vGlut-OE animals, indicating
increased GluCl function in PHD condition. Strikingly, we found that GluCl associates with SVs,
125
and a low level of GluCl present on the surface of the presynaptic terminal membrane in resting
state. Therefore, GluCl could function to modulate presynaptic efficacy only upon vesicle fusion.
Besides, we provide evidence that external chloride is required for mediating PHD expression.
Thus, we proposed that GluCl senses the excess glutamate release upon vesicle fusion during
single action potentials and reduces presynaptic glutamate release through its intrinsic chloride
conductance to regulate glutamate signaling and avoid excitotoxicity. Intriguingly, it has been
shown that a chloride conductance is required to facilitate glutamate filling of synaptic vesicles
(Chang et al., 2018; Martineau et al., 2017). It would be of great interest to test whether GluCl
serves as the chloride conductance that contributes to vesicle filling and whether this
mechanism correlates with the hypothesis that increased GluCl is expressed in synapses
overexpress the glutamate transporter.
The larval NMJ is a tripartite synapse, and the glial cells is a critical player in glutamate
homeostasis in many cases. Recent work indicates the epigenetic factor from glia controls the
retrograde potentiation of presynaptic release (Wang et al., 2020). It would be provocative to
investigate the role of glia in PHD. Although the classic mechanism for glutamate clearance,
EAAT is missing in the neuron and glia at the larval NMJ, we could not rule out the possibility of
additional PHD mechanisms involve glia function.
Altogether, these studies elucidate the mechanisms of how bi-directional homeostatic
modulations behave under various challenges that with differential temporal and spatial
resolutions to adaptively and honestly enable normal synaptic function and avoid toxic
threatening. Furthermore, these results could advance the understandings of the homeostatic
regulations in the mammalian and human nervous systems that contribute to our everyday life.
126
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Nature 375, 682-684.

Abstract (if available)

Abstract Homeostatic plasticity adaptively modulates neuronal activity to ensure the functionality of the nervous system in front of various destabilizing signaling during learning, development, and diseases. Synaptic homeostatic plasticity is employed to maintain synaptic efficacy when there are perturbations of synaptic function and believed to counterbalance with Hebbian plasticity (Davis, 2013

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Li, Xiling (author)

Core Title Molecular mechanisms underlying the bi-directional control of presynaptic homeostatic plasticity

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 04/26/2020

Defense Date 03/13/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Drosophila NMJ,homeostasis,OAI-PMH Harvest,synaptic plasticity

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Herring, Bruce (committee chair), Chang, Karen (committee member), Dickman, Dion (committee member)

Creator Email xilingli@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-290112

Unique identifier UC11664166

Identifier etd-LiXiling-8347.pdf (filename),usctheses-c89-290112 (legacy record id)

Legacy Identifier etd-LiXiling-8347.pdf

Dmrecord 290112

Document Type Dissertation

Rights Li, Xiling

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA