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Contributions of the cerebellum and neural pathways in the extinction of discrete motor responses

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Contributions of the cerebellum and neural pathways in the extinction of discrete motor responses

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CONTRIBUTIONS OF THE CEREBELLUM AND NEURAL PATHWAYS
IN THE EXTINCTION OF DISCRETE MOTOR RESPONSES

by

Karla Robleto

____________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PSYCHOLOGY)

August 2006

Copyright 2006 Karla Robleto
UMI Number: 3238305
3238305
2007
Copyright 2006 by
Robleto, Karla
UMI Microform
Copyright
All rights reserved. This microform edition is protected against
unauthorized copying under Title 17, United States Code.
ProQuest Information and Learning Company
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P.O. Box 1346
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by ProQuest Information and Learning Company.
ii

Dedication

To my parents, Marta and Mario, who always believed in me and whose incessant
devotion, encouragement and unconditional love made this possible.
And to my sister Daniela, who never ceases to amaze me and from
whom I have much to learn.

iii
Acknowledgments

First, I would like to thank my advisor Dr. Richard F. Thompson who throughout
these years has been a constant source of support and extraordinary mentorship. He
is not only an exceptional researcher but a person with a tremendous amount of
kindness and generosity. To him I owe the invaluable gift of knowledge and the true
priviledge of having been part of his outstanding lab. His success as a pioneer
neuroscientist is a lesson of discipline, perseverance, strong ethics and arduous work.
He certainly excels in every single one of those qualities and will undoubtedly be a
role-model wherever I go. I would also like to thank the members of my committee,
Dr. Larry Swanson, Dr. Stephen Madigan and Dr. Frank Manis for their kind help
and guidance. I am truly grateful to have such a wonderful advisory group and
tremendously appreciate them taking the time to be part of my committee.

I would like to especially thank Judith Thompson for not only providing me with an
enormous amount of guidance but for all her advice and comfort when it was much
needed. Her ascertiveness, kindheartedness, intelligence and strength are nothing but
enviable. She is truly an example to follow and I will always be grateful for having
known such an exceptional woman. Thank you for making the lab such an enjoyable
place and for being like a second mom to me.

iv
I would also like to thank the past and present members of the Thompson Lab
Andrea Scicli, Richard Hinchliffe, Benjamin Tran, Ingrid Liu, Yu Zheng, Andrew
Poulos, Kim Christian, Ka Hung Lee and Narawut Pakaprot. Andrea Scicli and
Richard Hinchliffe provided a significant amount of help and taught me many
fundamental techniques that greatly facilitated my research when I was first starting.
Ingrid will always touch my life with her incredible achivements as a scientist and
mother and I will always value her friendship regardless of where she is. To Kim I
thank for the countless hours she devoted helping me out whenever I needed it -in
spite of how busy she always was- and for being a truly good friend and such a great
example throughout my years at the lab. I would also like to thank Andrew for all his
help and invaluable frienship and for the long hours spent brainstorming about
putative neural models of learning I lost count of how many times we found the
holy grail of memory! Michael McClelland was an incredible help and I always
knew I could count on him to solve any problem at the lab no matter how big or
small. He truly deserves all the credit for making our work at the lab so much easier.

I would also like to thank the excellent undergraduates I had the fortune of
mentoring throughout my graduate years, Hiroko Nobuta, Jasmin Persch, Jennilee
Tuazon and Teresa Sanchez who were truly dependable and who in one way or
another helped make my work more manageable.

Finally, I would like to thank Rob for holding my hand all along this journey and for
providing me with nothing but unconditional support and love.
v
Table of Contents

DEDICATION......................................................................................................... ii

ACKNOWLEDGEMENTS .................................................................................... iii

LIST OF FIGURES ............................................................................................... vii

ABSTRACT ............................................................................................................ x

CHAPTER 1
GENERAL INTRODUCTION ................................................................................ 1
Eyeblink Conditioning as a Model....................................................................... 9
Neuronal Substrates of Extinction...................................................................... 10
The Conditioned Response Pathway .................................................................. 13
The Inferior Olive-Climbing Fiber Reinforcement System................................. 19
The Locus of Preserved Memories..................................................................... 23
Context, the Hippocampus and Extinction ......................................................... 26
Overview of Experiments .................................................................................. 31

CHAPTER 2
ROLE OF RESPONSE-PRODUCED AFFERENT FEEDBACK IN THE
EXTINCTION OF THE EYELID CONDITIONED RESPONSE
Introduction ........................................................................................................ 34
Methods.............................................................................................................. 39
Table 2.1: Parameters used in periorbital stimulation study 43
Results................................................................................................................ 44
Discussion .......................................................................................................... 50

CHAPTER 3
MAPPING OF NUCLEI INVOLVED IN EXTINCTION
Introduction ........................................................................................................ 54
Methods.............................................................................................................. 60
Results................................................................................................................ 66
Discussion .......................................................................................................... 84

CHAPTER 4
EFFECTS OF ELECTRICAL STIMULATION OF THE RED NUCLEUS DURING
INACTIVATIONS OF THE INTERPOSITUS IN EXTINCTION
Introduction ........................................................................................................ 92
Methods.............................................................................................................. 98
Results.............................................................................................................. 103
Discussion ........................................................................................................ 116

vi
CHAPTER 5
LEARNING RELATED CHANGES DURING EXTINCTION: THE ROLE OF
THE NMDA RECEPTOR
Introduction ...................................................................................................... 123
Methods............................................................................................................ 130
Results.............................................................................................................. 135
Discussion ........................................................................................................ 152

CHAPTER 6
GENERAL CONCLUSION................................................................................. 158

REFERENCES .................................................................................................... 167

vii
List of Figures

Figure 1.1 Acquisition, extinction and spontaneous recovery of a conditioned
response ................................................................................................................... 6

Figure 1.2 Simplified schematic of the cerebellum and associated brain circuitry
essential for eyeblink conditioning.......................................................................... 12

Figure 1.3 Schematic representation of essential neural circuitry involved in
eyeblink conditioning ............................................................................................. 15

Figure 2.1 Schematic of eyeblink reflex pathways and associated cerebellar circuitry .. 36

Figure 2.2 Mean percentage conditioned responses during each treatment
conditioned (Tetracaine-Control)............................................................................ 45

Figure 2.3 Mean percentage conditioned responses during each training day
(Tetracaine-Control) ............................................................................................... 46

Figure 2.4 Mean percentage conditioned response amplitudes of each extinction
session (Tetracaine-Control) ................................................................................... 48

Figure 2.5 Mean amplitude unconditioned responses to stimulation thresholds
(Tetracaine-Control) ............................................................................................... 49

Figure 3.1 Schematic illustration of the GABA
A
receptor structure .............................. 57

Figure 3.2 Schematic representation of infusion cannulae in the interpositus nucleus .. 63

Figure 3.3 Mean percentage of conditioned responses during each day of training
(1ul-0.1ul-Control) ................................................................................................. 69

Figure 3.4 Mean percentage of conditioned responses during each treatment phase
(1ul-0.1ul-Control) ................................................................................................. 70

Figure 3.5 Mean conditioned responses percentage during the reacquisition session
(1ul-0.1ul-Control) ................................................................................................. 71

Figure 3.6 Mean conditioned response peak latencies within the 250ms inter-
stimulus interval (1ul-0.1ul-Control)....................................................................... 73

Figure 3.7 Mean conditioned response onset latencies within the 250ms inter-
stimulus interval (1ul-0.1ul-Control)....................................................................... 74

viii
Figure 3.8 Histological reconstruction of cannulae placements targeting the
interpositus............................................................................................................. 75

Figure 3.9 Mean conditioned response percentages of groups during each training
day (RN-Control) ................................................................................................... 78

Figure 3.10 Mean conditioned response percentages during each treatment condition
(RN-Control).......................................................................................................... 80

Figure 3.11 Mean conditioned response peak latencies within the 250ms inter-
stimulus interval (RN-Control) ............................................................................... 81

Figure 3.12 Mean conditioned response onset latencies within the 250ms inter-
stimulus interval ..................................................................................................... 82

Figure 3.13 Histological reconstruction of cannulae placements targeting the RN ........ 83

Figure 4.1 Schematic of experimental procedure used in the study ............................... 97

Figure 4.2 Behavioral graphs of conditioned response frequencies during each
session (RN/IP-Control) ....................................................................................... 106

Figure 4.3 Mean percentage of conditioned responses during each day of training
(RN/IP-Control).................................................................................................... 107

Figure 4.4 Mean percentage of conditioned responses during each treatment phase
(RN/IP-Control).................................................................................................... 108

Figure 4.5 Mean percentage of conditioned responses during the reacquisition
session (RN/IP-Control). ...................................................................................... 111

Figure 4.6 Mean conditioned response onset latencies within the 250ms inter-
stimulus interval ................................................................................................... 112

Figure 4.7 Mean conditioned response peak latencies within the 250ms inter-
stimulus interval ................................................................................................... 113

Figure 4.8 Histological reconstruction of cannulae tip targeting the interpositus......... 114

Figure 4.9 Histological reconstruction of electrode tips targeting the RN.................... 115

Figure 5.1 Schematic illustration of the NMDA receptor............................................. 127

ix
Figure 5.2a Mean conditioned response percentages of groups during each training
day (AP5-Control)................................................................................................. 138

Figure 5.2b Mean CR% of AP5 group during each block in the session (extinction
day 1) ................................................................................................................... 138

Figure 5.3a Mean conditioned response percentages during each treatment
condition (AP5-Control) ....................................................................................... 139

Figure 5.3b Mean conditioned response percentages during reacquisition (AP5-
Control)................................................................................................................ 139

Figure 5.4 Mean conditioned response onset latencies within the 250ms inter-
stimulus interval (AP5-Control)............................................................................ 141

Figure 5.5 Mean conditioned response peak latencies within the 250ms inter-
stimulus interval (RN-Control) ............................................................................. 142

Figure 5.6 Histological reconstruction of cannulae placements targeting the
interpositus........................................................................................................... 143

Figure 5.7 Mean conditioned response percentages of groups during each training
day (CPP-Control)................................................................................................ 146

Figure 5.8 Mean percentage of conditioned responses during each treatment phase
(CPP-Control) ...................................................................................................... 147

Figure 5.9 Mean conditioned response onset latencies within the 250ms inter-
stimulus interval (CPP-Control)............................................................................ 149

Figure 5.10 Mean conditioned response peak latencies within the 250ms inter-
stimulus interval (CPP-Control)............................................................................ 150

Figure 5.11 Histological reconstruction of cannulae placements targeting the
interpositus........................................................................................................... 151

x
Abstract

It is well established that the cerebellum and its associated circuitry are essential for
classical conditioning of the eyeblink response and other discrete motor responses
(e.g. limb flexion, head turn etc) learned with an aversive unconditioned stimulus
(US). However, brain mechanisms underlying extinction of these responses are still
relatively unclear. Extinction, as a form of inhibitory learning, entails the
suppression of excitatory conditioned responses to a former stimulus. The original
formation of these excitatory associations is created when a conditioned stimulus
(CS) is paired with an unconditioned stimulus (US). In extinction, the CS is
presented alone and consequently losses its ability to evoke responding. One of the
core questions in the study of extinction is whether the learned association simply
fades away, is destroyed or unlearned, or whether it involves a more active process
were a new inhibitory association is formed concurrent with the original one.
Fundamentally, extinction, unlike forgetting, does not involve a passive mechanism
of decay as a consequence of passage of time. It rather entails an active process of
learning in which due to CS alone presentations after excitatory training, the
organism learns that the CS no longer predicts the US. Indeed, behavioral studies
have demonstrated extinction to be an active learning process distinct from
acquisition. This current understanding of extinction has guided neural studies,
which have tried to identify possible brain structures that could support this new
learning. Whether extinction engages the same brain sites necessary for acquisition is
xi
not yet clear. This posses an overriding problem for understanding brain
mechanisms necessary for extinction since such analysis cannot be done without first
identifying brain sites and pathways involved in this phenomenon. Here, we explore
issues of anatomical and physiological specificity related to extinction and
demonstrate the important role of cerebellar structures in the extinction of discrete
motor responses.

1
CHAPTER ONE

GENERAL INTRODUCTION

One of the central goals in the study of learning and memory has been the
development of a comprehensive model of memory storage with mechanisms that
link, in a flowing manner, molecular, cellular and systems-level changes to
behavior. Although that level of understanding has not yet been achieved, animal
models that allow for experimental manipulations have proved to be extremely
valuable in bridging the gap between brain and learned behavior. Realization of this
goal would seem to require working with an all-inclusive model that is fully testable,
experimentally controlled and characterized by a well defined anatomy of its brain
structures and neural circuits.

Classical conditioning, one of the simplest models of associative learning, has been
studied extensively in a wide variety of preparations that have ranged from
invertebrates to mammals, including humans (Carew & Sahley, 1986; Lovibond,
Davis, & O'Flaherty, 2000). Pioneered by Pavlov (1927), it has arguably been one
the most useful paradigms in the study of the substrates underlying learning and
memory. As one example of classical conditioning, eyeblink conditioning is
2
arguably the best-understood mechanism of learning in vertebrates. Decades of
theoretical models and experimental evidence by Thompson and colleagues have
resulted in the detailed mapping of the neural circuits involved in conditioning of the
eyeblink response (R. E. Clark, Gohl, & Lavond, 1997; R. E. Clark & Lavond,
1993a, 1996; R. E. Clark, Zhang, & Lavond, 1997; R. F. Thompson & Krupa, 1994).
Recording, lesion, stimulation and anatomical tracing studies have resulted in the
development of a testable model which identifies in detail the pathways, nuclei and
possible sites of plasticity involved in eyeblink conditioning and discrete motor
responses (R. F. Thompson, 1986, 1990).

As two fundamental processes, the formation of excitatory and inhibitory
associations have permeated the understanding of this learning paradigm. Among
one of the training procedures, extinction, as a form of inhibitory learning, entails the
suppression of excitatory conditioned responses to a former stimulus. The original
formation of these excitatory associations is created when a conditioned stimulus
(CS) is paired with an unconditioned stimulus (US). After several pairings the CS
comes to excite or evoke a representation of the US resulting in execution of a
conditioned response (CR) in anticipation to the presentation of the actual US. In
extinction, the CS is presented alone and consequently losses its ability to evoke
responding.

3
One of the core questions in the study of extinction is whether the learned
association simply fades away, is destroyed or unlearned, or whether extinction
involves a more active process where a new inhibitory association is formed
concurrent with the original one. Based on a large body of experimental evidence
(see below), contemporary theories have been forming a consensus that extinction
does not involve the destruction or loss of the original learning. Nonetheless, models
that champion the unlearning view still influence the conceptualization of this
phenomenon (McClelland and Rumelhart, 1985; Rescorla and Wagner, 1972;
McCloskey and Cohen, 1989). Genesis of the notion of extinction as unlearning or
forgetting of the original association can be found in early theories of psychology
that tried to explain how old learning is affected by the interpolation of new material
(McGeoch, 1932, McGeoch & Irion, 1952; Barnes & Underwood, 1959, Melton, &
Von Lackum, 1941). Most of the early observations were based on the learning of
word list associations from which dominant extinction models were derived to
explain the deficits seen during recall of the original list association.

In recent years the conceptualization of extinction as the loss of the original
association has fallen into disfavor due to the emergence of extensive behavioral
evidence that has challenged unlearning theories. Most of the observations seen by
researchers have caused a shift towards an alternative view of extinction. In fact, it
has seemed clear since Pavlovs (1927) original observations that extinction is not
merely the fading away of a memory. The classical phenomena of spontaneous
4
recovery and disinhibition, as well as re-exposure to the reinforcer (M. E. Bouton,
1984; Rescorla & Heth, 1975), change in context (M. E. Bouton & Swartzentruber,
1991), sensitivity to changes in reinforcer value (Rescorla, 1995) and marked savings
in reacquisition (Macrae & Kehoe, 1999; Napier, Macrae, & Kehoe, 1992; Scavio &
Thompson, 1979) all argue that the originally learned associations are in some
manner preserved during extinction training (see also Falls, 1998). The common
explanation for these phenomena is that extinction induces new learning that
somehow interferes with performance.

A consistent observation among a number of experimental manipulations that can be
conducted after extinction is the predictable return of the extinguished response.
Moreover, all of them point towards context as being an important modulator of
extinction. Spontaneous recovery (Figure 1.1), for instance, has been consistently
observed in a variety of conditioning protocols (Courville & Brodal, 1966 ). Bouton
(2002) explained this recovery as the effects of ambiguity caused by memory of the
excitatory and inhibitory association. Likewise, time could provide a form of
temporal context to which extinction is highly sensitive. Thus, spontaneous recovery
is seen when the CS is tested in a different temporal context and the subject cannot
retrieve the memory of extinction. In a series of experiments using appetitive
conditioning, Brooks and colleagues (Brooks 2000, Brooks and Bowker 2001;
Brooks et al. 1999) confirmed this hypothesis by observing that cues presented
during extinction training attenuate spontaneous recovery if presented during the
5
recovery test. This would indicate a failure of retrieval of the inhibitory memory
outside the extinction context, given that delay would result in a new temporal
context. In what is referred to as the renewal effect, the capacity of the CS to
evoke responses is renewed after extinction if the context is changed during CS
testing, supporting Boutons hypothesis.

6

rest period acquisition extinction extinction
CS-US pairings CS alone
strength of the response
spontaneous
recovery
CS alone
rest period acquisition extinction extinction
CS-US pairings CS alone
strength of the response
spontaneous
recovery
CS alone
rest period acquisition extinction extinction
CS-US pairings CS alone
strength of the response
spontaneous
recovery
CS alone

Figure 1.1 Acquisition, Extinction and Spontaneous Recovery of a conditioned response.
Spontaneous recovery can be observed when sufficient time elapses between extinction training
and when the CS is tested again. Behaviorally, it is evidenced by a return of the conditioned
response even after succesful extinction has occurred. This effect has been observed consistently
across conditioning paradigms.
7
Rapid reacquisition following extinction, noted above, provides strong support for
preservation of the original responses. Recent studies on long-term memory of
conditioned responses have further shown that reacquisition after long extinction
testing intervals (i.e. 1, 2, 3 and 6 months), is still very rapid, also suggesting that the
original memory does not fade away and is instead, to a great degree, retained
(Schreurs, 1993). Further proof can be seen in experiments that show a high degree
of transfer to a novel CS even after conditioned responses to the original CS have
been extinguished (Kehoe, Morrow and Holt 1984). In other words, acquisition is
faster to a novel CS in subjects that have acquired an original CS-US association, an
effect that can even be seen after extinction training of the original CS.

Experimental evidence has shown that a CS can enter concurrently into an inhibitory
and excitatory association with the same (Barnet & Miller, 1996; M. Smith &
Gormezano, 1965). Strong evidence comes from an early experiment by Smith and
Gormezano (1965) demonstrating that a single CS can simultaneously support robust
conditioning and extinction. In this experiments rabbits were trained to delay
eyeblink conditioning using a tone CS. Following robust acquisition, rabbits received
11 alternating sessions of conditioning and extinction. Although CR expression
during conditioning sessions remained robust, responding during CS alone sessions
yielded a within session extinction function that continued to decline across sessions.
Also, in an elegant experiment, Tait and Saladin (1986) trained rabbits to a tone CS
and a periorbital shock US. They subsequently trained them for lick suppression
8
conditioned responses in a backward conditioning protocol where the US preceded
the CS. Backward conditioning is considered a form of inhibitory learning since it
retards acquisition when the pairings are subsequently done in a forward manner
(CS-US). They observed that rabbits showed more lick suppression to the tone than
control animals, indicating that the CS acted as an excitor. Training was
subsequently resumed by switching to a forward conditioning protocol (CS-US)
presenting the same stimuli but now training for eyeblink conditioned responses.
The animals were found to be retarded in their acquisition of the conditioned
response compared to control animals that had not been exposed to backward
training, indicating that the CS acted as an inhibitor. These results are important
because they suggest that a single CS can simultaneously have inhibitory and
excitatory associations with a US.

At a behavioral level there is thus considerable evidence that extinction training
somehow results in inhibition of at least expression of the original learned response.
But the term inhibition used in this context does not necessarily imply neuronal-
synaptic processes of inhibition. It is a behavioral descriptor.

This current understanding of extinction has guided neural studies, which have tried
to identify possible brain structures that could support this new learning. Likewise,
as a form of learning, additional plastic changes, different from those involved in
acquisition, may occur in extinction. Whether extinction engages the same brain sites
9
necessary for acquisition is not yet clear. This posses an overriding problem for
understanding brain mechanisms necessary for extinction since such analysis cannot
be done without first identifying brain sites and pathways involved in this
phenomenon.

Eyeblink Conditioning as a Model
As one of the most thorougly characterized neural circuits mediating a learned
response in the mammalian brain, eyeblink conditioning provides an extremely
exploitable model for the study of extinction. Indeed, as a cerebellar dependant type
of learning, classical conditioning of the eyelid has already proved to be a most
useful system for analysis of both behavioral and neural aspects of acquisition and
retention of learned responses. One of the factors that have made possible the
detailed study of this system is the exquisite regularity that characterizes the
anatomical architecture of the cerebellum. At the center of its functional circuits, for
instance, are Purkinje cells and deep cerebellar neurons which provide the sole
output from the cerebellar cortex and the cerebellum itself respectively. Furthermore,
in eyeblink conditioning (and other forms of classical conditioning) there exists a
precise understanding of the neural pathways that convey the behavioral signals (e.g.
CS and US) as well as the benefit that the stimuli involved are well defined and
controlled, providing an important advantage over other more complex forms of
learning.

10
Neuronal Substrates of Extinction
There are at least two key issues in terms of understanding the neuronal bases of
extinction of learned responses: 1) identification of the neuronal circuits and
mechanisms that result in the new learning in extinction and inhibition of the
original learned response, and 2) the loci in the brain where the original associations
are still to some significant degree preserved.

The neuronal circuitry essential for classical conditioning of the eyeblink and other
discrete responses learned with an aversive unconditioned stimulus has been largely
identified (Figure 1.2), at least for acquisition and retention, and the most likely sites
of neuronal plasticity (memory traces) have been to a degree localized (Christian
& Thompson, 2003; J.F. Medina, Nores, Ohyama, & Mauk, 2000; Steinmetz, 2000;
R. F. Thompson et al., 1997; R. F. Thompson & Krupa, 1994; C.H. Yeo & Hesslow,
1998). Consequently it would seem an ideal system in which to analyze neuronal
processes of extinction.

Briefly, in so far as acquisition is concerned, CR pathway exits from the interpositus
nucleus ipsilateral to the trained eye via the superior cerebellar peduncle (scp) to the
red nucleus and subsequently via descending rubral pathways to the premotor and
motor nuclei generating the CR. The CS pathway includes sensory relay nuclei and
their projections via the pontine nuclei and other sources as mossy fibers to the
cerebellum. The US pathway includes sensory (trigeminal) relays to the inferior
11
olive and from there via climbing fibers to the cerebellum. The unconditioned
response (UR) pathway includes both direct and indirect projections from the
trigeminal nucleus to the motor nuclei generating the behavioral response and need
not involve the cerebellum.

12

Figure 1.2 Highly simplified schematic of the cerebellum and associated brain circuitry essential
for eyeblink conditioning. Shadowed boxes illustrate loci that have been previously inactivated
during acquisition training. (a) Motor nuclei including facial (seventh), accessory (sixth) and
adjacent reticular regions. (b) Magnocellular red nucleus. (c) Dorsal anterior interpositus. (d)
Superior cerebellar peduncle. (Modified from Thompson and Krupa 1994)
13
The Conditioned Response Pathway
The strongest evidence for localization of the putative sites of memory storage
within this circuit has come from studies using methods of reversible inactivation.
Inactivations of the motor nuclei, an essential region for generations of the UR and
CR, result in complete abolition of both the CR and UR (Zhang and Lavond, 1991;
Thompson et al. 1993, Krupa,Weng and Thompson 1996). However, release of this
inactivation results in inmediate exhibition of asymptotic CR performance and
normal UR display. Evidently, expression of the CR and UR are not necessary for
the formation of the memory trace and are completely efferent from the it. Lesions of
the red nucleus, a major target for IP axons, abolish expression of the CR yet do not
interfere with learning of the eyeblink responses, leaving the learning related
neuronal activity observed in the IP unaffected (Krupa et al. 1993; Clark and
Lavond, 1993) the nuclei are completely efferent from the memory trace.

Inactivations of the cerebellar nuclei, specifically the IP, completely prevent
eyeblink conditioning (R. E. Clark, Zhang, & Lavond, 1992; Hardiman, Ramnani, &
Yeo, 1996; Krupa, Thompson, & Thompson, 1993; Nordholm, Thompson,
Dersarkissian, & Thompson, 1993). an effect that can also be produced by very
localized kainic acid lesions in this cerebellar region (Lavond, Hembree, &
Thompson, 1985). In subsequent post-activation training, animals learned normally
as though completely naïve, they showed no savings at all relative to non-inactivated
control animals. None of the methods of inactivation had any effect at all on the
14
performance of the UR on US alone trials (but see Bracha et al. 1994). Krupa and
Thompson (1995) demonstrated that injections of tetrodoxin (TTX) into the
brachium conjunctivum, which block cerebellar outflow, allowed for normal learning
even though no conditioned responses (CRs) were present during inactivation.
Collectively, these data clearly show the essential plasticity for eyeblink conditioning
to be located within the cerebellum (see Christian and Thompson 2003 for a more
detailed review).

The role of the cerebellar cortex in acquisition is not yet clear. All workers
agree that it is extremely important, indeed critical, for normal learning but whether
or not it is essential remains controversial (see detailed discussion by Christian and
Thompson, 2003; see also Yeo et al. 2002). In brief, generally two cortical regions
have been implicated to play an important role in eyeblink conditioning: Larsells
hemispheric lobule VI (HVI) and the anterior lobe. HVI, the area of the cortex
immediately dorsal to the interpositus, has been found to be involved in rate of
eyeblink conditioning (Logan 1991) and possibly in maintaining the frequency of CR
expression in well trained animals (Yeo et al. 1984; Hardiman et al. 1988; Woodruff-
Pak et al. 1985; Lavond et al. 1987; Clark et al. 1990). Conversely, lesions of the
anterior lobe have been found to affect timing and amplitude of the CR (Perret and
Mauk 1995; Garcia et al. 1999) yet this view still remains in dispute.

15

Figure 1.3 Schematic representation of the essential neural circuitry involved in classical eyeblink
conditioning. Reflex pathways from the sensory trigeminal nerve to the motor nuclei (accessory
6th and 7th nerves) are also identified. CS information is relayed through the pontine nuclei to the
cerebellum and US information is relayed through the inferior olive. Efferent motor pathways from
the cerebellum include the red nucleus and final effector motor nuclei. Sites of sensory integration
and proposed learning-related plasticity in the interpositus nucleus and cerebellar cortex are marked
by stars. (Modified from Thompson, 1986).
16
Several lines of evidence argue strongly for learning-induced processes that develop
in the interpositus, as opposed to developing elsewhere and being relayed to the
interpositus. Infusion of muscimol localized entirely to the interpositus nucleus
completely prevents learning and expression of the CR (Krupa & Thompson, 1997a).
While it is true that this treatment will block Purkinje GABAergic action on nuclear
cells, the fact that animals show no savings at all after the inactivation has been
removed demonstrates that no part of the memory trace has been formed in
cerebellar cortex independent of the interpositus; otherwise there would be savings.
Indeed, other than the direct projections from the interpositus to the cerebellar
cortex, all normal mossy and climbing fiber projections to the cortex are intact and
functional (muscimol does not inactivate fibers). Projections from the interpositus to
the cortex, either direct or indirect, would seem to play a key role in establishing
plasticity in cortex.

In a recent study Krupa & Thompson (2003) inactivated the relevant motor nuclei
during CS alone extinction training of the conditioned eyeblink response in rabbits.
Results from this study indicated that inactivation of the motor nuclei that generate
the eyeblink CR and UR during CS alone extinction training, given over 3 days or 6
days, completely prevented extinction, measured either by percent CR or CR
amplitude. In post-inactivation extinction training, extinction performance of the
inactivation groups was identical to the initial extinction performance of the saline
control groups; there is no savings in extinction. Consequently, inactivation of the
17
motor nuclei during extinction training completely prevents the occurrence of
extinction. Because complete inactivation of these same motor nuclei during
acquisition training has no deleterious effect at all on learning of the CR, (Krupa,
Weng, & Thompson, 1996), these results provide additional evidence that acquisition
and extinction processes differ, not only in behavioral properties but also in neuronal
substrates in eyeblink conditioning.

Pavlov (1927), Rescorla (1997) and others have emphasized the possibility that
extinction results in an inhibitory association between a stimulus and a particular
response, which would disrupt performance despite the continued presence of the
original associations (see e. g. Rescorla & Wagner, 1972). As Rescorla (1997) notes,
one line of evidence favoring this view comes from the correlation between the
amount of responding that occurs in extinction and the amount of deterioration that
the extinction experience produces. Procedures that reduce responding during
extinction reduce performance loss (Holland & Rescorla, 1975) and procedures that
augment responding during extinction amplify the loss resulting from non-
reinforcement (Rescorla & Skucy, 1969; Wagner, 1971). Our finding that
inactivation of the motor nuclei prevents extinction would seem to be supportive of
this response-extinction hypothesis.

Yeo and associates (Hardiman et al. 1966; Ramnani and Yeo 1996) reported that
reversible inactivation of the interpositus nuclear region with muscimol during CS
18
alone extinction training completely prevented extinction (and completely prevented
expression of the CR). We have replicated this result using very low doses of
muscimol (1µg in 0.1 µl) infused in the interpositus. (Robleto and Thompson 2003).
Based on earlier studies (Krupa 1993) actions of these very doses do not appear to
spread beyond the interpositus. These results would seem to implicate the
interpositus in extinction. The fact that inactivation of the motor nuclei prevented
extinction of the CR suggested an alternative interpretation, namely that inactivation
of the interpositus prevents extinction simply because it prevents performance of the
CR, consistent with the response-extinction hypothesis.

To further test this hypothesis, we have inactivated with muscimol the region of the
red nucleus critical for expression of the CR (Robleto and Thompson, unpublished
observations). After having been conditioned to asymptotic levels, animals were
presented with 8 daily sessions of tone alone presentation. Infusions of muscimol or
saline were administered 1 hour prior to extinction training and were discontinued
after day 4 of training. Using the same low dose of muscimol we used for
interpositus inactivation during CS alone extinction training, these infusions
completely prevented performance of the CR. Yet extinction occurred during red
nucleus inactivation! This result is consistent with the fact that red nucleus
inactivation does not prevent learning of the CR but would seem not to support the
response-extinction hypothesis (see Chapter 3 for a more detailed discussion).

19
In sum, these results demonstrate clearly that the neuronal bases of acquisition and
extinction differ. Consistent with this view, there is considerable evidence using the
conditioned fear paradigm that the neuronal substrates of acquisition and extinction
may differ at both systems and molecular levels. In particular, the medial prefrontal
cortex (rat) seems to play a more important role in extinction than in acquisition
(Herry & Garcia, 2002; Milad & Quirk, 2002; Morgan, Romanski, & LeDoux, 1993;
Quirk, Russo, Barron, & Lebron, 2000) (but see R. Garcia, Vouimba, Baudry, &
Thompson, 1999; Gewirtz, Falls, & Davis, 1997). It appears that L-type voltage
gated calcium channels are required for extinction but not for acquisition or
expression of conditioned fear to context of cue (Cain, Blouin, & Barad, 2002). It
also appears that although NMDA receptors in the amygdala may be necessary for
acquisition and long-term extinction (Falls, Miserendino, & Davis, 1992; H. Lee &
Kim, 1998) they may not be necessary for short term extinction (systemic
administration) (Santini, Muller, & Quirk, 2001). Finally, although protein synthesis
in the amygdala is necessary for acquisition of conditioned fear to both context and
cue stimuli (Bailey, Kim, Sun, Thompson, & Helmstetter, 1999) it does not appear
necessary for extinction of fear to context, at least with systemic injections of an
inhibitor (Lattal & Abel, 2001).

The Inferior Olive-Climbing Fiber Reinforcement System
Several lines of evidence support the view that the inferior olive (IO) and its
climbing fiber projections to the cerebellum serve as the essential reinforcement
20
system for motor learning (Albus, 1971; Eccles, 1977; Ito, 1982; R.F. Thompson,
1989; R. F. Thompson, Thompson, Kim, Krupa, & Shinkman, 1998). The critical
region of the IO for eyeblink conditioning is the dorsal accessory olive (DAO),
which receives predominately somatosensory input relayed from the spinal cord and
appropriate cranial nuclei, including nociceptive input (Brodal & Brodal, 1981).

At the beginning of training, the onset of the US evokes short latency unit responses
by neurons in the face region of the DAO. Over the course of training this evoked
unit response gradually disappears inversely with the development of behavioral CRs
(Sears & Steinmetz, 1991). Lesions of the critical region of the inferior olive, the
face representation in the DAO, completely prevent learning if made before training
and result in extinction of the CR with continued paired training made after training
(McCormick, Steinmetz, & Thompson, 1985; Mintz, Lavond, Zhang, Yun, &
Thompson, 1994; Voneida, Christie, Bogdanski, & Chopko, 1990). Neurons in this
critical DAO region do not respond to auditory stimuli (CS), respond only to US
onset and show no learning-related activity. Electrical microstimulation of this
region serves as a very effective US (Mauk et al. 1986; Steinmetz et al. 1989).
Whatever response is elicited by stimulation of the IO-climbing fibers is easily
learned to any neutral stimulus. Immediate abolition of the CR has been observed in
large IO lesion and inactivation studies (Welsh & Harvey, 1998; C. H. Yeo,
Hardiman, & Glickstein, 1986) perhaps partially due to significant increases in
Purkinje cell simple spike activity (Montarolo, Palestini, & Strata, 1982; Savio &
21
Tempia, 1985). Increased inhibition of Purkinje cells to the IP would prevent CR
expression which depends on sufficiently high activity levels in the nuclei in order to
occur. Eyeblink conditioning employing cerebellar stimulation without a peripheral
US does not require such involvement of the IO (Shinkman, Swain, & Thompson,
1996b).

The interpositus nucleus sends direct GABAergic projections to the DAO (Nelson,
Adams, Barmack, & Mugnaini, 1989). Hence, as learning-induced increases in
interpositus neuron activity develop, inhibition of the DAO neurons will increase
(Hesslow & Ivarsson, 1996). This accounts for the fact that US evoked activity in the
DAO decreases as learning develops, consistent with the Rescorla and Wagner
(1972) formulation. This also appears to serve as a part of the neural circuit essential
for the behavioral learning phenomenon of blocking, where prior training to one
CS, e.g. tone, prevents subsequent learning to a light CS when it is then presented
together with the tone in paired compound stimulus training (Kamin 1969). Infusion
of picrotoxin in the DAO to block the GABA inhibition from the interpositus during
compound stimulus training completely blocks the development of behavioral
blocking (Kim et al. 1998). In other words, blocking interpositus inhibition of the IO
greatly facilitates new learning.

In the context of extinction, preventing US activation of the IO-climbing fiber
system by IO lesions results in extinction, even in the presence of continued CS-US
22
paired training, whereas blocking inhibition of the IO greatly facilitates learning and
would be expected to counteract normal CS alone extinction training. Both these
effects have been replicated and extended in important studies by Mauk and
associates. Medina et al. (2002) examined the role of inhibitory and excitatory inputs
to the climbing fibers in extinction by administering infusions of picrotoxin, a
GABA A antagonist, and the AMPA receptor antagonist NBQX. It was found that
infusions of picrotoxin to block inhibitory transmission from the interpositus to the
IO prevented the extinction of CRs. Conversely, infusions of NBQX (which block
excitatory input to climbing fibers) during paired CS-US presentation resulted in a
gradual disappearance of CRs that was not different from extinction behavior
observed during tone-alone presentations under normal conditions or with paired
training following lesions of the DAO.

These studies indicate that changes in activity of the IO greatly contribute to the
acquisition and extinction of the conditioned response. It has been previously shown
that the IO intrinsically maintains an activity level of approximately 1 Hz (De
Zeeuw et al., 1998; Hesslow & Ivarsson, 1996). At this self-regulatory state, IO
activity seems to be non-conductive for learning (Medina et al. 2000; Medina, Garcia
and Mauk 2000. Acquisition and extinction would therefore require that this balance
be shifted either by an overall increase or decrease in IO activity (Medina et al.
2002). In extinction, IP inhibition would promote IO activity decreases below
baseline levels considering that the omission of the US would cease to excite
23
climbing fibers. The strong IP inhibitory output that results during performance of
the conditioned response without a US would then cause IO activity to decrease
signaling changes that would ultimately allow extinction to occur.

All these results argue that the IO-climbing fiber system is the critical reinforcing or
teaching system for motor learning and that it plays a key role in extinction.

The Locus of Preserved Memories
A fundamental question concerning brain substrates of extinction is the locus or loci
of the preserved memory. Current evidence would seem to suggest either the
cerebellar cortex, the interpositus nucleus, or both as candidates. Gould and
Steinmetz (1996) reported that multi-unit activity in the interpositus correlated
closely with the behavioral CR over both acquisition and extinction, showing
complete absence of increased activity when the CR was fully extinguished. In
contrast, multi-unit activity in cerebellar cortex increased over acquisition but did not
decrease much during extinction of the CR. Although several different patterns of
responses were seen for identified single Purkinje neurons, some 13 Purkinje cells
showed increased responses over training and did not decrease over extinction. Such
increased Purkinje neuron activity could promote inhibition of interpositus activity in
extinction.

24
Perrett and Mauk (1995) reported that lesions of the anterior lobe of cerebellar cortex
in well-trained animals prevented extinction of the behavioral CR. They separated
two groups of such lesioned animals on the basis of response timing. Short latency
CR animals did not show extinction but normal latency CR animals did. More
recently, Garcia et al. (1999) reported that anterior lobe lesioned animals that
displayed short-latency CRs showed no evidence at all of extinction in terms of
frequency of CRs or amplitudes over 15 days of extinction training. However, there
was no significant difference in percent or frequency of CRs between these animals
and a control group with lobule HVI lesions. The control lesion animals did show
significant extinction in terms of response amplitude but the anterior lesion animals
did not. In an earlier study, lesions of HVI, although retarding acquisition
somewhat, had no effect on rate of extinction, which occurred over several days in
both lesion and control animals (Weiss, Logan, & Thompson, 1991). However, in
the Garcia et al. (1999a) study, the CR amplitudes of the anterior lesioned animals
were extremely low, perhaps a basement effect, whereby the already low amplitude
responses cannot be reduced any further and therefore no extinction can be observed.

Medina et al. (2001) reported that picrotoxin infusion in the interpositus nucleus
revealed short latency CRs during training and also revealed the presence of such
short latency CRs after 45 days of extinction training, when no CRs occurred in the
absence of picrotoxin, suggesting that the memory is preserved to some degree in the
interpositus nucleus. However, they did not give untrained animals 45 days of tone-
25
alone exposures to control for non-associative effects. This is critically important
since it has recently been shown that under some conditions, infusion of picrotoxin
in the interpositus nucleus can result in the performance of short-latency 95 dB tone-
evoked eyeblink responses in untrained animals (Poulos, Nobuta & Thompson,
2003). It is also the case that if the memory is indeed preserved in the interpositus
nucleus, then unit recordings there should reveal some trace of it and they do not
(Gould and Steinmetz 1996). But it is always possible that the residual memory
trace following extinction is below threshold for increased probability of action
potentials.

We used the Purkinje cell deficient (pcd) mice to explore the essential role of the
cerebellar cortex in acquisition and extinction of the conditioned eyeblink responses.
In this mutant, cerebellar development proceeds normally until about 2 weeks after
birth, at which time all Purkinje neurons in the cerebellar cortex die. Hence the
cerebellar cortex becomes completely non-functional. These animals show a slower
rate of acquisition and reach a lower asymptotic of CR performance but do show
very substantial and highly significant learning of the eyeblink CR (Chen et al.
1996). However, if the interpositus nucleus is lesioned in these mutants prior to
training they are completely unable to learn the eyeblink CR, arguing that the
learning occurs in the interpositus nucleus (Chen et al. 1999). These results seem to
contradict other cerebellar cortical studies (i.e. Perret and Mauk 1995). Such
conflicting results are more likely due to a variability in the lesions. Complete
26
removal of the cortex is extremely difficult and such large lesions likely result in
damage to the deep nuclei.

Using a different methodological approach, these studies of the pcd mouse argue
strongly that the cerebellar cortex is not essential for acquisition of the eyeblink CR -
although it is clearly necessary for normal learning. Importantly, after learning the
eyeblink CR, pcd mice show normal, rapid extinction of the CR, arguing that the
cortex is not essential for extinction either. However, this does not rule out the
possibility suggested by Gould and Steinmetz (1996) that a memory trace remains
in the cerebellar cortex after extinction of the CR. The critical test would be to
retrain the pcd animals after extinction. If they showed no savings in reacquisition
over initial acquisition it would support their view.

Context, the Hippocampus and Extinction
The role of the hippocampus in the area of learning and memory has been widely
studied (see Jarrard, 1995, for review). Over time, researchers have linked its
function to the ability to form spatial relationships, remember facts and events
(declarative memory), and have further associated hippocampal damage to deficits in
learning tasks that demand inhibition of responses (Akase, Alkon, & Disterhoft,
1989; Eichenbaum, 1997; Squire & Zola, 1996; Benoit, Davidson, Chan, Trigilio &
Jarrard, 1999; Olton, Becker & Handelmann).

27
Although the hippocampus does not appear to play a critical role in standard delay
eyeblink conditioning, it does play a key role in trace conditioning, where a period of
no stimulation intervenes between CS offset and US onset. Specifically,
hippocampal lesions made prior to training essentially prevent subsequent learning
(anterograde amnesia) (Moyer, Deyo, & Disterhoft, 1990; Solomon et al., 1986). If
the lesions are made immediately after training, trace but not delay CRs are
abolished. However, if the lesions are made a month after training, the trace CR is
not impaired at all (time-limited retrograde amnesia) (Kim et al. 1995). These are of
course the hallmarks of medial temporal lobe-hippocampal lesion impairment of
declarative memories in humans (Squire, 1987). One possible explanation of the key
role of the hippocampus in trace conditioning concerns context. Perhaps the
hippocampus somehow codes the context in order to bridge the gap between CS and
US.

Indeed, Penick and Solomon (1991) showed that the hippocampus appears to code
context in eyeblink conditioning. After partial training, animals were given
continued training in the same or different context. Context shift impaired CR
performance in normal rabbits but did not impair performance at all in hippocampal
lesioned animals. In fear conditioning, hippocampal lesions prevent learned fear to
context but not to tone cue (Kim and Fanselow 1992).

28
Several studies indicate that CS alone extinction is markedly impaired by lesions of
the hippocampus, regardless of whether CRs were established with delay or trace
conditioning procedures. Whether these deficits are due to an increase in nonspecific
behavioral activation or to deficits in forming an inhibitory association once an
excitatory association has been learned is still a matter of debate. Experimental data
from several studies seem to render the view of a general increase in activity
unlikely. For example experiments that have measured behavioral activity in an open
field have found no differences in activity between hippocampal and control animals
(Vianna, Igaz, Coitinho, Medina, & Izquierdo, 2003). Moreover, it seems that
behavioral deficits in extinction are more profound and increase more as a function
of the number of exposures to the US. This phenomenon can be observed in the
results obtained by Benoit et al. (1999) and in other studies were several training
conditions have been used (Akase et al., 1989). Consistent with an involvement of
the hippocampus in the formation of inhibitory learning, Schmaltz and Theios (1972)
demonstrated that bilateral hippocampal lesions made prior to delay conditioning
were associated with a higher level of CR expression through much of extinction
training then cortical lesioned control animals. More recently, Alkase et al. (1989)
trained eyeblink conditioning using the standard delay procedure in
hippocampectomized rabbits. In well-trained animals, there was essentially no
extinction in a subsequent CS alone training session. Even more dramatic, Moyer et
al. (1990) showed that hippocampal lesioned rabbits given short interval (300 ms)
trace conditioning learned normally but showed no evidence of extinction over three
29
sessions, in contrast to sham and neocortical control lesions, who showed normal
extinction.

There is growing evidence that contextual cues other than the CS can exert control of
the CR in extinction. Bouton and colleagues (2000) have demonstrated in Pavlovian
fear conditioning that following extinction, presentation of the US alone reinstates
the CR to the CS. However, if the post extinction presentation of the US occurs in a
different context reinstatement of the CR does not occur. Along similar lines, if the
context in which CR acquisition occurs is followed by extinction in a second context,
return to the original context will renew the expression of the CR to the once
extinguished tone. Interestingly, lesions of the hippocampus specifically disrupt CR
reinstatement, while resulting in no discernable effects on CR renewal.

There is recent evidence that prolonged exposure to the training context can result in
extinction-like reductions of conditioned eyeblink responses (Kehoe, Weidemann, &
Dartnall, 2004). In these studies rabbits were trained to both tone and light CSs and
then exposed solely to the training context for 6 days. The following day, animals
were presented with either CS alone and showed virtually no CRs over the course of
4 days. Subsequent CS-US reacquisition yielded CRs at a rate similar to tone alone
extinction controls. Such results demonstrating that context exposure produces
reductions in previously learned behaviors have been previously reported in cued
avoidance and lick suppression tasks (Gabriel 1970; Marlin 1981). However, to date,
30
experiments examining hippocampal involvement in this context dependent
phenomenon have yet to be carried out.

As a form of new learning, extinction has been hypothesized to induce changes in
synaptic connections that require gene expression and protein synthesis. Some of the
widely studied receptors and proteins that have been found to be involved in synaptic
organization as a result of new learning have been NMDA (N-methyl-D-aspartate)
glutamatergic receptors and downstream molecules and proteins such as mitogen-
activated protein kinase (MAPK), Protein Kinase A (PKA) and calcium/calmodulin-
dependent protein kinase II (CAMKII). Szapiro, Vianna, McGaugh, Medina, and
Izquierdo (2003) found that infusions of NMDA, PKA, CAMKII and PKA
antagonists into the hippocampus prevented the extinction to a fear conditioning
task. Infusions of these antagonists into the CA 1 region of the hippocampus have
previously been shown to result in retrograde amnesia for inhibitory avoidance tasks
(Izquierdo & McGaugh, 2000; Izquierdo & Medina, 1997). Apparently these
treatments have detrimental effects on the formation of new memories, which
involves the activation of these proteins and receptors. The authors concluded that as
in the acquisition of the original memory, blockage of extinction was observed by
the applications of these four antagonists. In other words, it seem that, at least in fear
conditioning, the molecular mechanisms underlying formation of the original
memory are the same or similar to those involved in the extinction of the memory.
31
Moreover, in both acquisition and extinction of this task, these molecular
mechanisms seem to be co localized to the hippocampus.

Overview of Experiments
Mapping of the circuitry involved in classical conditioning of discrete behavioral
responses has contributed enormously to the understanding of mechanisms of
learning and memory. Understanding the process of how memories form and, in
particular, how they contribute to inhibition or suppression of learned behavior will
arguably make possible the prediction of the necessary and required neural processes
that may be involved in a variety of learning contexts which demand extinction of
acquired responses. Moreover, as a complimentary phenomenon to acquisition,
extinction is a very important component of adaptation. Certainly, due to changes in
environmental contingencies an organism might be better off learning to supress a
previously learned response than continue a behavior that is no longer adaptively
relevant. It becomes evident then, that acquisition as well as extinction are essential
components in learning and memory. However, although there has been a great deal
of understanding of the basic mechanisms underlying acquisition of conditioned
responses, at both behavioral and neural levels the processes involved in extinction
are not as well understood.

These experiments are designed to answer yet unknown issues of anatomical and
physiological specificity within the cerebellum and related brain regions during
32
extinction of the previously acquired conditioned response. Given that classical
conditioning of the eyeblink is arguably one of the most understood mechanisms in
the study of learning and memory, eyeblink conditioning is used here as a working
model to study brain mechanisms involved in the extinction of these responses.

In the first experiment, it is hypothesized that sensory reafference from the cornea
plays a role in extinction. Previous data have shown sensory reafference to be
involved in acquisition. It has been found that, although UR amplitude and latency
remained unaffected, anesthetization of the orbital region does have a detrimental
effect on acquisition of the eye-blink response when using a periorbital shock US.
This phenomenon has not been investigated in extinction.

In Chapter 3, functional disruption of nuclei will be examined with the goal of
mapping brain structures that might play an important role in extinction. Specifically,
pharmacological inactivation of two important nuclei involved in eyeblink
conditioning will be studied: 1.The interpositus (IP), which has been extensively
demonstrated to be a structure where learning-induced processes develop and 2.The
red nucleus, a necessary structure for the expression of the CR.

Chapter 4 seeks to further investigate the response-extinction hypothesis which, as
previously discussed, suggests that the loss of learned performance that results from
extinction training is correlated with the amount of responding that occurs during
33
extinction. One limitation of previous inactivation studies that result in abolition of
the CR however, is that they cannot discriminate between response-induced and
neuronal plasticity related effects. This study tries to override this problem by
performing inactivations of the IP and at the same time inducing a CR through
electrical stimulation of the red nucleus during extinction. This manipulation strives
to better understand the role of the IP and the RN as well as answer whether response
production is an essential component in the extinction of responses.

Finally, Chapter 5 investigates the role of the NMDA receptor in extinction by
microinjecting the IP with NMDA antagonists. While previous studies have found
NMDA receptors to be involved in the acquisition and retention of a number of
learning tasks, including eyeblink conditioning, their role in extinction of these
responses has not been tested directly. The specific hypothesis tested in this
experiment is that as a form of new learning, extinction should recruit neuronal
changes in specific loci in order to occur. A likely candidate modulating these
changes is the NMDA receptor given its widely known involvement in learning and
memory.

34
CHAPTER TWO

ROLE OF RESPONSE-PRODUCED AFFERENT FEEDBACK IN
THE EXTINCTION OF THE EYELID CONDITIONED RESPONSE
1
____________________________________________________________________

Introduction
The eyeblink reflex is a rapid and brief contraction of the eyelid produced by the
orbicularis oculi muscle, bulbi motor system as well as the contraction of extraocular
muscles. Specifically, the rabbits eyeblink CR as well as reflexive UR consists of 2
discrete and highly correlated movements: 1) external closure of the eyelid, and 2)
concurrent eyeball retraction and nictitating membrane (NM) extension
(McCormick, Lavond, & Thompson, 1982). In brief, innervations from motor
neurons in the facial nucleus (FN) send projections via the VIIth nerve to orbicularis
oculi muscles responsible for external eyelid closure. Eyeball retraction is in turn
effected by projections originating from the accesory abducens nucleus (ACC) which
ultimately innervate the retractor bulbi muscle via the VIth nerve (Cegavske,
Harrison and Torigoe, 1987; (Marek, McMaster, Gormezano, & Harvey, 1984)). In
addition, reflexive eyeblinks can also be evoked by mechanically activating receptors
in the cornea, eyelashes and other cutaneous places of the face or by electrical

1
Results published in Behavioral Neuroscience, 118(6): 1433.
35
stimulation of the trigeminal nerve (Evinger et al., 1984; Baker, McCrea and
Spencer, 1980; Gruart, Zamora and Delgado-Garcia, 1993).

As a form of associative learning, the reflexive eyeblink response is widely used in
classical conditioning given that it proves to be a highly controlled and accurate
indicator of learning. In the rabbit preparation using an airpuff, eyeblink reflexes are
evoked by the US through the activation of corneal and periorbital receptors which
send information about the stimuli to both the cerebellar cortex and deep cerebellar
nuclei. These responses are then conditioned (CR) through presentation of the CS-
US association to occur at the time the CS is presented; in anticipation of the US. At
the level of the brainstem, expression of both the CR and UR is ultimately effected
by the motor nuclei which receive both information about the reflexive and
conditioned response resulting in the observable eyeblink.

36

Figure 2.1 Simplified schematic of the eyeblink reflex pathways and associated cerebellar
circuitry. Trigeminal blink area (Vp) is represented with shaded neurons. Input from the
cerebellum is conveyed by the red nucleus to this area and motor nuclei (OOcVII: orbicularis
oculi, AccVI: abducens and LPIII: palpebrae). Trigeminal neurons, in turn, send information
about the US to the cerebellum via the Dorsal Accesory Olive. Dotted lines to the cerebellum
represent modulatory input from spinal trigemina nuclei (Vo: pars oralis,Vi: pars interpolaris and
Vc: pars caudalis). Other abbrevitions used: Ret, Reticular formation; C1-C4, spinal segments.
(Adapted from van Ham and Yeo 1996).
37
Relevant to the study of extinction remains the question of how behavior/responses
affect this phenomenon (extinction). As previously discussed, several researchers in
the field have stressed the importance of the amount of responding that occurs in
extinction and agree in the existence of a strong correlation between responding and
the resulting loss of the behavior that is observed (Rescorla, 1997; Rescorla and
Skucy, 1969, Wagner, 1971). What component of responding results in the
successful extinction of a response is still unknown. Perhaps feedback mechanisms
that convey information about response performance are essential in response
supression. In fact, if we look at an important mechanism shared by sensory systems,
reafference could very well be a likely candidate. Certainly, as an element of
sensory input that an organism receives as a consequence of its own movements,
sensory reafference could be hypothesized to be the component of behavior
triggering a signal for extinction. Previous data have found an involvement of
sensory reafference in the acquisition of the eyelid response and in particular
indicate that, in eyeblink conditioning, blockage of sensory feedback from the cornea
can affect learning of the association (Kettlewell & Papsdorf, 1971). Indeed, as an
important part of motor control and adaptive behavior (Wolpert and Ghahramani,
2000; Kawato, 1999), the role of response-produced afferent feedback in extinction
would seem to need to be explored in more detail.

Thus far, the response-extinction hypothesis has previously been directly
investigated through inactivations of nuclei involved in the external eyelid closure.
38
Krupa, Weng and Thompson (1996) demonstrated that reversible inactivation
(muscimol) of the motor nuclei generating the eyeblink-NM response during training
did not prevent acquisition at all. Indeed, following six days of tone-corneal airpuff
training with infusions of muscimol in FN the ACC and adjacent reticular formation
ipsilateral to the trained eye, such that no behavioral responses at all were given with
the CS or the US, these animals showed asymptotic learned performance following
removal of inactivation. However, if these same motor nuclei were inactivated
during CS alone extinction training, no extinction at all occurred ((Krupa &
Thompson, 2003)). These findings are consistent with the widely accepted view that
extinction is not simply a fading away of learning but rather involves other processes
(e.g., Bouton, 1994; Pavlov, 1927; Rescorla, 1997; Wagner and Rescorla, 1972).

There are at least two possible and not mutually exclusive explanations for this
inactivation-extinction result: 1) response-produced afferent feedback from the eye is
important for extinction, or 2) reafference feedback from the motor nuclei and
higher motor systems to other brain structures is important for extinction. Yet,
another alternative account for the effect obtained could be that unlike acquisition,
extinction of the eyeblink response may require plastic changes specific to the motor
nuclei. However, required neural mechanisms of extinction still need further
investigation.

39
Concerning the scope of the present study, it is clear that at least response-produced
afferent feedback plays no critical role in acquisition (Krupa, Weng and Thompson,
1996). We test this afferent feedback hypothesis here, giving extinction training with
local anesthetization of the cornea.

Method
Subjects:
New Zealand albino rabbits (n=22), weighing 2.0 to 2.4 kg were used for the
experiment. Animals were individually housed with ad-lib food and water.
Temperature and a 12 hr light/ dark cycle were continuously controlled.

Behavioral Training Procedures:
Rabbits were habituated to a Plexiglas restraint and the training chamber for two
daily sessions of one hour in duration. At the end of the second day of habituation, a
nylon suture was surgically placed in the apex of the animals left nictitating
membrane. Training began on the following day.

Behavioral training consisted of three phases. Phase I comprised the acquisition
phase in which animals were presented with four daily sessions of tone-airpuff
pairings. Each acquisition session consisted of 100 trials divided into ten blocks of
ten trials. Each block contained one tone alone trial, one airpuff alone trial and eight
tone-airpuff pairings. Intertrial intervals ranged randomly between 20 and 40 s.
40
Tone-airpuff pairings consisted of a tone CS presented for 350 msec (1 kHz, 85 dB)
with a coterminating corneal airpuff US (100 msec, 3psi). Conditioned responses
were defined as nictitating membrane extensions of at least 0.5mm occurring
between 35 ms and 250 ms after CS onset. On tone alone trials the critical CR range
was between 35 ms and 750 ms after CS onset.
In phase I, rabbits were required to meet the following criteria: 1) perform at least
eight conditioned responses (conditioned responses) within nine consecutive trials by
the end of the third session, and 2) give at least 80% conditioned responses during
the final acquisition session. This ensured that all rabbits had reached the same level
of performance by the end of the fourth session. At the end of this phase animals
were randomly assigned to either a control or an experimental group.

Phase II consisted of four tone alone extinction sessions. Each session in phases II
and III consisted of 100 trials of CS alone presentations. The tone was presented for
350 msec with an intertrial interval that varied randomly between 20 and 40 s.
Before each session an artificial tear solution (Phoenix Pharmaceutical Inc.) was
administered to the cornea of animals in the control group. The experimental group
was given Tetracaine Hydrochloride (0.5%), which effectively anesthetized the
rabbits cornea for more than an hour. The dosage each animal received before each
session consisted of two drops of solution given in intervals of five minutes during a
period of ten minutes (a total of three applications). Phase III consisted of three tone
41
alone extinction sessions with no solution administered to either group before each
session.

Animals responses (NM extensions) were recorded using a minitorque
potentiometer attached to a nylon suture loop in the apex of the rabbits left
nictitating membrane. Data were saved on an IBM PC for later analysis.

Periorbirtal Stimulation Experiment:
To control for the possibility that the local anesthetic might act directly on motor
nerves or nuclei, a group of 4 animals were implanted with stimulating electrodes
periorbitally. Four different current ranges were established according to the
amplitude of the eyeblink that resulted from the delivery of the shock during a
baseline test (see Table 2.1): High range levels consisted of shocks in the 3mA range
which resulted in eyeblinks of more than 10mm in amplitude. Mid range levels
evoked eyeblinks that were 5mm in amplitude. A shock of 2mA was used. Threshold
levels included eyeblinks that were between .5 and 1mm in amplitude and were
generated by a shock of 1mA. Subthreshold levels consisted of a shock of .5 mA
which would not evoke any visible response.

All rabbits were presented with 4 daily sessions of periorbital shock stimulation.
Animals were administered tetracaine or saline before the beginning of each test
session and each treatment condition was counterbalanced for all 4 animals. A
42
session consisted of delivery of all four current ranges presented in the following
alternating order: 3 Threshold pulses 3 Subthreshold pulses 3 Mid pulses 3
Subthreshold pulses 3 High pulses 3 Subthreshold pulses. This sequence was
repeated 3 times during each session. Possible effects of the local anesthetic on
performance of the response elicited by periorbital electrical stimulation were
subsequently determined.

43

Range Current Eyeblink Amplitude
High 3mA 10mm+
Mid 2mA 5mm
Threshold 1mA 0.5-1mm
Sub 0.5mA -

Table 2.1 Depicts parameters used for the periorbital stimulation study. Four different current
ranges were used based on the amplitude of the eyeblink that resulted from delivery of the shock
during a baseline test.
44
Results
Of the 22 rabbits used in the experiment, five did not reach learning criteria and were
thus excluded from further training and analysis. Three rabbits failed to perform
eight conditioned responses out of nine consecutive trials by the end of the third
acquisition session and two reached less than 80% conditioned responses on the last
day of acquisition. As a result, 6 control animals and 7 experimental or Tetracaine
animals were included in the statistical analyses.

Corneal desensitization was effectively achieved in rabbits administered tetracaine.
Gentle corneal taps at the end of each extinction session showed that tetracaine
completely suppressed eyeblinks normally elicited by the presentation of the eye tap.
Figure 2.2 shows a summary of the learning performance as measured by the
percentage of conditioned responses given by both groups during each treatment
condition (refer to Figure 2.3 for CR performance on each day). A repeated measures
analyses of variance (ANOVA) showed that all animals attained similar rates of
conditioned responses during the acquisition phase of the experiment, indicating that
both groups reached the same level of learning before being given extinction
training, (F (1,11)= 0.012, p < 0.91). In addition, no differences in percent CRs were
found between the groups as a result of drug administration, F (1,11)= 0.31, p=0.58.
Both the experimental and control groups gave similar extinction response
frequencies, which continued to decline in subsequent no drug extinction sessions, F
(1,11)=0.47, p < 0.83
45

0
10
20
30
40
50
60
70
acq drug nodrug
Experimental phase
Percent CRs
control
tetracaine

Figure 2.2 Mean (± SEM) percentage conditioned responses (CRs) for both groups during
each treatment condition. Each data point shows the overall learning performance for each
phase in the experiment. Acq: Average CR performance during the four days of acquisition.
drug: Average CR performance during the four days of extinction with drug administration.
nodrug: Average CR performance during the 3 days of extinction without drug
administration. reacq: Average CR performance during reacquisition.

46

0
10
20
30
40
50
60
70
80
90
100
acq1
acq2
acq3
acq4
ext1(drug)
ext2(drug)
ext3(drug)
ext4(drug)
ext5
ext6
ext7
Session
Percent CR'S
control
tetracaine

Figure 2.3 Mean (± SEM) percentage conditioned responses (CRs) for both groups during
each day of training.

47
Although there were no differences in the rate of conditioned responses throughout
extinction sessions between the groups, it is possible that corneal desensitization
could have produced significant changes in the amplitude of these conditioned
responses while leaving CR rate intact. This was examined by normalizing the
amplitudes of each extinction session with the average conditioned response
amplitude of the last acquisition session. This yielded a measurement that reflected
changes in the strength of the response between acquisition and extinction training
(Figure 2.4).

Figure 2.4 shows evidence of suppression in the amplitude of the conditioned
responses of the tetracaine group during treatment days. Although conditioned
response amplitudes were not significantly different between both groups during
extinction training there does seem to be a trend. Particularly, on day two of
extinction training, the tetracaine group presented a more pronounced decrease of
amplitudes (SEM ± 0.216) compared to controls (SEM ± 0.639). It should be noted
however, that this difference was not significant, F (1,11)=2.512, p=.141.

In addition, tetracaine anesthetization of the cornea had no effect at all on the
amplitudes of the NM responses elicited by periorbital electrical stimulation. Using a
just suprathreshold stimulus of intensity that varied between 1 and 3 mA, the mean
NM response amplitude for the control condition was 1.54 mm and for the tetracaine
condition was 1.26mm, (t=0.799, p< .483) (see Figure 2.5)
48

Figure 2.4 Mean (± SEM) percentage conditioned response amplitudes of each extinction
session. Percentages represent the difference of the amplitudes of each extinction day with
those of the last day of acquisition (see Results section). A trend towards amplitude
suppression can be seen in the tetracaine group during treatment days. This effect was
significant early in extinction.

49

0
2
4
6
8
10
12
14
Tetracaine mean Control mean
amplitude (mm)
High range
Mid range
Threshold
Subthreshold

Figure 2.5 Mean amplitude of UR to stimulation thresholds between control and tetracaine
animals.

50
Discussion
The results of this study indicate that blockage of corneal sensation does not disrupt
the extinction process. Animals in the tetracaine group show a gradual decrement in
percent CR performance that is no different from controls even after drug
administrations are stopped (Phase III). Further, the fact that the experimental group
extinguished in a manner that was not significantly different from the control group
seems to rule out the possibility that drug administrations may have also affected CR
performance due to motor nerve damage, as does the lack of tetracaine effect on
eyeblinks elicited by periorbital stimulation. It should be noted, however, that
animals in the experimental group showed lower response amplitudes than those in
the control group.

Taken together, data from the present study suggest that proprioception related to the
cornea is not critical during extinction of the NM. While these findings seem to
indicate that blockage of corneal sensory feedback does not interfere with extinction,
a different effect has previously been reported during acquisition. Kettlewell and
Papsdorf (1971) found that, although UR amplitude and latency remained
unaffected, anesthetization of the orbital region does have a detrimental effect on
acquisition of the eye-blink response when using a periorbital shock US. The
different effects seen in acquisition and extinction due to blockage of sensory
feedback could be interpreted in various ways. For example, it could be argued that
during acquisition, cutaneous deafferentation would decrease the aversive or
51
intensive value of the US resulting in poor learning of the CS-US association.
Extinction, on the other hand, would not be affected by this manipulation given the
nonreinforcing nature of the process itself. That is, blockage of sensory feedback
during CS alone presentations would still permit the formation of what has been
previously described as an inhibitory CS-no US relation (Pearce, 1987) thus allowing
extinction to occur uninterrupted.

Although the role of sensory input to motor movements in eye-blink extinction is not
well understood, various experiments have focused on effects of responding during
extinction training, most reporting a positive relationship between the frequency of
responding during extinction and degree of extinction (Holland & Rescorla, 1975;
Rescorla & Skucy, 1969). Together with the present data, these results may indicate
that expression of the response may be an important component in the formation of
inhibitory associations even in the absence of sensory reafference.

It could be argued, however, that administrations of tetracaine into the animals
cornea did not completely block sensory feedback. Indeed, peripheral areas that may
contribute to the US in NM acquisition, such as the eyelids, were probably not
inactivated during extinction. It is known that eyelid responses involve activity of
facial, oculomotor and bulbi systems (Evinger, Manning, & Sibony, 1991; Gruart,
Blazquez, & Delgado-Garcia, 1995). Corneal proprioception, consequently, may
only be partially involved in conditioned blink responses. However, it should be
52
noted that except for cutaneous receptors, orbicularis oculi motor units that
contribute to lid position and movement, do not receive proprioceptive signals from
the eyelid (Trigo, Gruart, & Delgado-Garcia, 1999). Nonetheless, sensory feedback
from other eyeblink related areas might still be important in eyeblink conditioning.

Taking into account the limitations of the current findings, the effect of corneal
anesthetization on the amplitude of the conditioned NM response during CS alone
extinction training may indicate that corneal sensation plays some role in
performance of the CR and possibly on acquisition, as do the results of the
Kettlewell and Papsdorf (1971) study. This finding needs further investigation. On
the other hand, corneal sensation seems to play no role in performance of the reflex
response elicited by periorbital stimulation.

With growing evidence indicating that extinction does not involve erasure of the
original memory (Bouton, 1993, 1994), the underlying neural mechanisms mediating
extinction are not well understood. It has previously been shown that muscimol
infusions into the cranial motor nuclei, which do not block acquisition (Krupa, Weng
and Thompson, 1996), effectively prevent extinction of the conditioned NM
response in the rabbit (Krupa and Thompson, 2003). Lesions or inactivation of the
cerebellum have also been reported to prevent extinction in eyeblink conditioning
(Perret & Mauk, 1995; Hardiman, Ramnani and Yeo, 1996; Robleto and Thompson,
2003). However, to the extent that CRs were prevented from occurring in these
53
studies, results can be reinterpreted simply as the consequence of non-responding,
independent of necessary cerebellar involvement.

54
CHAPTER THREE

MAPPING OF NUCLEI INVOLVED
IN EXTINCTION

Introduction
Much of the knowledge gained in the last decades regarding the neural circuits of
conditioned discrete motor responses has been acquired through the employment of
lesioning techniques (G. A. Clark, McCormick, Lavond, & Thompson, 1984; Oakley
& Russell 1977; Krupa et al., 1996). Central to this approach is the view that by
means of exclusion and inclusion, neural structures critical for learning and
maintenance of a particular behavior can be identified. The assumption is that lesions
targeting a particular structure involved in the system subserving a given form of
learning, will result in deficits or blockage of the acquisition or expression of the
learned behavior. Permanent lesions (e.g. electrolytic, kainic acid, aspirations),
although useful in determining whether a particular structure is involved in a learned
response, generally permit limited conclusions about the particular role played by
that brain region. This is specially a matter of concern when the learned behavior is
no longer observed after the lesion is performed and it is difficult to determine
whether a site of plastic changes or memory has been damaged or simply whether
expresion of the response has been blocked.
55
Reversible lesions, on the other hand, allow for the continued study of the system
after the structure has been restored to normal functioning. This affords the valuable
comparison of the effects observed during inactivation and after the inactivation has
been released therefore allowing a better understanding of the role the targeted
structure plays in that type of learning. For instance, if a subject is trained while an
essential structure for a particular learning task (i.e. eyeblink conditioning) is
inactivated no learned responses will be observed. This is true whether an output
pathway essential for expression of the behavior (e.g. motor nuclei), or a structure
critical for formation of the memory (e.g. interpositus) is inactivated. The same
effect can result with inactivations of a pathway critical in relaying information
about the stimuli (e.g. pons, inferior olive). However, with the employment of a
reversible lesion, once the inactivation is removed further conclusions can be made
based on the following scenarios: 1) If once the inactivation is removed there is an
immediate display of learned responses then it can be concluded that the subject has
learned the response and i) the structure inactivated is not the site of memory
formation (the locus of memory must be afferent to the targeted structure) ii) the site
of memory formation does not receive critical input from the inactivated structure. 2)
If, on the other hand, removal of the inactivation results in the absency of any
learned behavior and the animal subsequently learns as naïve then if can be deduced
that i) the structure inactivated is the locus of memory or ii) the targeted region
provides critical afferent projections to the site of memory formation.

56
Among the many techniques used to reversibly inactivate a specific structure, use of
the GABA agonist, muscimol, has several advantages. Unlike anesthetics of the
caine family or the paralytic substance Tetrodoxin (TTX) which affect fibers of
passage (Hille, 1966), muscimol blockage only affects somata and dendrites leaving
fiber bundles intact (Somogyi, Takagi, Richards, & Mohler, 1989; Velazquez,
Thompson, Barnes, & Angelides, 1989). Indeed, TTX and local anesthetics are
selective sodium (Na+) channel blockers and thus block action potentials through the
fibers. Conversely, muscimol binds to GABA
A
receptors which are only found in
somata and dendrites therefore rendering a much more localized effect. More
specifically, muscimol acts by binding to the receptor which opens the Cl
-
channel
allowing Cl
-
to enter the cell resulting in hyperpolarization and therefore inactivation
of the cell. This effect can be highly localized to the targeted region and can have a
duration of several hours which is specially advantageous in learning protocols that
require long training sessions. Indeed, because of its properties, muscimol has
proven to be a great tool in the identification and role of brain structures subserving
the acquisition of a learning task. It could therefore be of extremely valuable use as
well in the identification of structures that might play a critical part in the extinction
of conditioned eyelid responses.

57

Figure 3.1 Schematic illustration of the GABA
A
receptor structure containing two α and β
subunits and a single γ subunit to form an intrinsic Cl- ion channel. When GABA (or
muscimol) binds to the receptor the Cl- channel is opened, allowing Cl- influx. Other
putative ligands and drugs known to positively or negatively modulate GABA-gated Cl- ion
conductance are also illustrated. (From Paul, 1995 and Cooper, Bloom and Roth 1996)

58
Localization of Essential Brain Structures through Lesions:
Although the localization of sites of memory formation and storage have consistently
been demonstrated to reside in the cerebellum, little is known about the loci and
brain circuitry involved in extinction of these memories. Given the overwhelming
behavioral evidence demonstrating that extinction involves the formation of a new
inhibitory association, learning-specific neuronal changes within the necessary
circuitry should also take place. As cited earlier, previously it has been demonstrated
that inactivation of the motor nuclei during tone alone training completely prevent
extinction from occurring (Krupa & Thompson 2003). Other studies that have
employed cerebellar aspirations or muscimol microinfusions have shown that
inactivation of the cerebellum also prevent extinction (Perret & Mauk 1995;
Ramnani & Yeo 1996) however, the exact involvement of a specific cerebellar
region cannot be determined from such data given the inconsistent extent of the
lesions or the uncertain spread of the infusions in the targeted area.

As previously discussed, involvement of the cerebellum and related structures in
acquisition of the response has been extensively demonstrated. This study was
designed to investigate the role of specific regions known to be involved in eyeblink
conditioning by performing reversible inactivations during extinction training using
muscimol as an agent. As a new competitive form of learning, extinction would be
expected to require plasticity in the same cerebellar circuits. We hypothesized that
disruption of actively engaged structures involved in inhibition of the response
59
would severely impair extinction or prevent it from occurring. Extinction training
during inactivation of a targeted nuclei might result in the immediate presence of
CRs once the block has been removed. In this scenario the structure inactivated
during training would be actively involved in extinction as evidenced by the lack of
performance loss once inactivation is removed. If, however, CRs are absent after
inactivation is removed, then the targeted structure would not be critically involved
in extinction and extinction would occur uninterrupted. However, non-performance
of CRs could also be due to deficits (i.e. motor) related to drug administration. The
reacquisition phase of the experiment is intended to clarify this finding. Ultimately,
this experiment seeks the identification of the neuronal circuits and mechanisms that
result in the new learning in extinction and inhibition of the original learned
response.

Two of the nuclei that will be targeted are the interpositus nucleus; which has been
extensively shown to be an essential structure in acquisition and maintenance of the
response as well as a putative location for storage of the associative memory (see
Chapter 1), and the red nucleus, which constitutes an important efferent of the CR
and is known to share direct and indirect connections with the interpositus (Courville
& Brodal, 1966 ). Prior studies have further shown through electrophysilogical
techniques changes in neuronal activity of magnocellular red nucleus cells that
precede the CR in a time locked fashion (Desmond & Moore, 1991; Haley,
Thompson, & Madden, 1988). Inactivations with lidocaine or cooling of the
60
interpositus nucleus reversibly abolish neural activity in the red nucleus (Chapman,
Steinmetz, Sears, & Thompson, 1990; R. E. Clark et al., 1992). Also, lesions of the
contralateral red nucleus completely prevent expression of the conditioned response;
yet unlike inactivations of the interpositus nucleus, have no effect on the ability to
learn the CS-US association (R. E. Clark & Lavond, 1993a). Finally, Chapman,
Steinmentz and Thompson (1988) showed that electrical stimulation of the essential
region of the red nucleus induces and eyeblink of the contralateral eye. Together
these studies leave no doubt as to the involvement of the red nucleus in acquisition of
the response. Its role in extinction of conditioned responses remains an open question
and is here investigated.

Methods
Subjects:
Twenty and fourteen male New Zealand White rabbits weighing between 2.2-3.0 kg
at the time of surgery were used in the interpositus inactivation and red nucleus
experiment respectively. Animals were housed individually and maintained on a 12
hours light/dark cycle with ad-lib access to water and food.

Rabbits in the interpositus experiment were divided into three groups. One group
received infusions of muscimol that targeted the IP consisting of 1µL (0.01M
solution). The second group received muscimol infusions into the IP consisting of
0.1 µL (0.01M), a much smaller dose that was less likely to spread to the cortex or
61
HVI. The final group received infusions of saline into the IP. For the red nucleus
inactivation study, animals in the experimental group were infused with 0.1 µL
(0.01M solution) of muscimol into the red nucleus. The control group was infused
with saline.

Surgery:
All animals were anesthetized with subcutaneous injections of ketamine (60mg/kg)
and xylazine (8mg/kg) and placed on a stereotaxic frame with a constant flow of
1.5%-2% of halothane in oxygen throughout the length of the surgical procedure.
Under asceptic surgical conditions an anterior to posterior incision was made in the
scalp in order to retract the skin and periostium and expose the skull. A small hole of
approximately 1.5mm in diameter was drilled through the bone at the
anterior/posterior and laterla/medial coordinates of the target structure. A guide
cannulae (26 gauge) 25mm in length was implanted into the target nuclei with
bregma 1.5mm dorsal to lambda. The guide cannulae was lowered with an insulated
stainless steel stylet (Elephant brand 000 insect pin) inserted and extending 1.5mm
below the tip. The outer cannulae tip was sharpened in order to minimize damage to
brain tissue during its implantation. These cannulae assembly was lowered slowly
with a stereotaxic manipulator that targeted the following coordinates based on the
stereotaxic atlas of McBride and Klemm: 1) interpositus nucleus (from lambda):
0.7mm anterior, 5.1mm lateral and 14.5mm ventral. 2) red nucleus (from bregma):
8.5mm posterior, 1.0mm lateral and 15.5mm ventral. Subsequently four holes were
62
drilled to fit surgical stainless steel screws that anchored a headstage constructed
from dental acrylic. A plexiglas mount built to hold a minitorque potentiometer
during behavioral training was also cemented to the headstage. Skin surrounding the
dental acrylic was asceptically cleaned with antibiotic ointment (iodine). Animals
were allowed to recover for a period of 1week before the beginning of behavioral
training. During the first two post-operative days animals were given two daily
injections of analgesic (buprenorphine hydrochloride) and were monitored
throughout the length of their recovery. All procedures and animal care were
approved by the USC Institutional Animal Care and Use Committee in accordance
with NIH guidelines.

63

Figure 3.2 Schematic representation of the infusion cannulae in the interpostitus nucleus.
Same procedure and apparatus was used in the red nucleus study. The assembly consisted of
a guide cannula with an inner stylet inserted and protruding 1.5mm from the tip. Cannulae
assembly was chronically implated targeting the brain structure of interest. The inner
infusion cannula was inserted into the guide cannula at the time of infusion. A polyethelene
tube was attached to the inner infusion cannula leading to the infusion pump. (Adapted from
Krupa 1993).
64
Apparatus:
Animals were habituated and trained in a padded pexiglas restraint and placed into a
behavioral recording chamber. During training nictitating membrane (NM)
movements were measured with a minitorque potentiometer temporarily attached to
the animals headstage. Voltage changes due to movement of the NM were measured
via mechanical movement of a bar attached to the potentiometer and connected by a
thread lead hook to a suture loop implanted in the left NM. The voltage signal was
stored and recorded on a computer using custom software written in Forth. The
software also controlled delivery of the stimuli during training. Data was
subsequently analyzed offline. Infusions were delivered to the targeted structure
using a 1µl and a 10µl syringe (Hamilton instruments) for infusions of a volume of
0.1 µl and 1µl respectively. Syringes were connected to a polyethelene tube attached
to an injection cannula (31 gauge) 26.5mm in length. Drug was delivered by
compression of the syringe using an infusion pump (Harvard Apparatus) at a rate of
0.38 µl per minute.

Behavioral Training Procedures:
The same behavioral procedure described in Chapter 1 were used with the following
exceptions:
Phase II: Following acquisition animals received 4 daily sessions of tone alone
training. One hour before the beginning of each training session rabbits received
infusions of muscimol or saline targeting the structure of interest. Infusions were
65
administered by removal of the inner stylet and insertion through the guide cannula
of the injection cannula. Delivery of muscimol was followed by a waiting period of 3
minutes in order to ensure absorption of the drug before into the brain before the
infusion cannula was removed and replaced with the stylet.
Phase III: Extinction training without drug infusions were given to rabbits for 4
consecutive days or until CR levels dropped to 15% or less.
Phase IV: In order to measure the ability of animals to reacquire the response, the
association was presented again to animals on the day following the conclusion of
extinction training.

Histology:
Following training an insulated electrode with 250µ m of tip exposure was lowered
through the guide cannula. A 1µ A current was subsequently passed through the
electrode for 10 seconds in order to create a marking lesion at the site of injection.
Animals were then given an intravenous overdose of sodium pentobarbital and
perfused intracardially with saline and 10% formaline. Brains were then extracted
and preserved for later histological reconstruction in 10% formalin for a minimum of
3 days. Brains were then embedded in an albumin and gelatin mixture, sectioned at
80µ m and mounted for later staining. Mounted sections were nissl stained with
Cresyl violet and counterstained with Prussian blue to label iron deposits left by the
tip of the electrode at the time of the marking lesion. Injection sites were then
recorded using a dissecting microscope.
66
Results
Inactivations of the interpositus nucleus:
Of the twenty animals that began the experiment, two in the group infused with
0.1uL muscimol and one animal in the control group were not included in the
analyses. One of the animals in the 0.1ul group developed a brain infection during
the drug infusion phase of extinction training. The second rabbit showed high CR
frequencies during IP inactivation sessions. Later histological verification confirmed
misplacement of the cannula tip in the white matter dorsal to the IP and close to the
paramedian lobe. The animal excluded in the control group failed to reach learning
criteria during the acquisition phase. As a result, data shown is for 7 animals in the
1uL group, 5 rabbits in the 0.1uL group and 5 in the control group.

Figure 3.3 shows the mean percentage CRs during each session of acquisition and
extinction training for all three groups. Animals acquired the association and met
learning critieria for Phase I of the experiment with no overall significant differences
in performance (average CR% on the last day of acquisition = 93.3% ± 1.5 SEM).
There were significant effects of days (sessions) consistent with increases in CR
frequency indicative of acquisition of the association, F(3, 42) = 80.6, p< 0.01. A
significant main effect was found on day 3 between the control group and the 1uL
group (p = 0.042), however no significant interactions were found confirming that all
groups attained similar CR rates before entering the extinction phase of the
67
experiment, F(2, 14) = 2.23, p = 0.144 (see figure 3.4 for overall group
performances).

During Phase II (extinction days 1-4), infusions of muscimol targeting the
interpositus nucleus effectively abolished the CR in both experimental groups
indicating succesful inactivation of the nucleus during extinction training, F(2, 14) =
16.01, p< 0.01. Differences were significant between controls and the 1uL group (p<
0.01) as well as the 0.1uL group (p< 0.01). On average, 1uL and 0.1uL groups
performed at levels of 3.9% ± 6.2 SEM and 1.5% ± 7.3 SEM respectively, whereas
controls displayed much higher CR frequecies (M = 52.5% ± 7.3 SEM). There was
also a Group x day effect found for the control group, F(3, 12) = 4.8, p = 0.02,
indicating a significant gradual loss of response performance due to tone alone
training.

Analyses of CR performance during extinction training with no drug (Phase III)
revealed that subjects in the experimental groups showed significantly high CR
frequencies during the first extinction session (1uL group: M = 66.8 ± 9.1 SEM;
0.1uL group: M = 63.2 ± 10.8 SEM) compared to controls ( M = 12.1 ± 10.8 SEM),
F(2, 14) = 8.5, p< 0.004, who were already performing at almost baseline levels.
There was a main group effect during this phase of training, F(2, 14) = 10.38, p<
0.002) confirming that extinction levels of experimental subjects were different from
controls. Although no differences were found between overall performance of both
68
experimental groups (p< 0.298) there does seem to be a trend with subjects in the
0.1uL group showing higher extinction levels than the 1uL group. Also, there was a
highly significant difference between overall extinction levels of the 1uL and
controls (p< 0.001) compared to 0.1uL and controls (p< 0.38). With continued
training animals in both experimental groups extinguished the response to baseline
levels that were no different than controls by day 9, F(2, 14) = 3.29, p = 0.067.
Moreover, experimental subjects extinguished the conditioned response during post-
infusion tone alone training (days 5-9) at rates that were identical to those of the
control group during days 1-5, F(1,14) = 0.70, p = 0.511.

Figure 3.5 illustrates group performance during reintroduction of the CS-US
association after the end of extinction training. In spite of almost no production of
CRs on the last day of extinction training all groups were able to reacquire the
response at levels that were different from naïve training ( M= 77.1% ± 2.7 SEM).
However, there was a significant group effect, F(2, 14) = 9.6, p< 0.002. Controls
displayed significanlty lower levels of CRs compared to the 1uL group (p< 0.014)
and the 0.1uL group (p< 0.002). No difference in CR reacquisition wass observed
between the experimental groups (p< 0.441).

69

0
10
20
30
40
50
60
70
80
90
100
acq1 acq2 acq3 acq4 ext1 ext2 ext3 ext4 ext5 ext6 ext7 ext8 ext9
Session
CR%
(1uL)
(0.1uL)
control

Figure 3.3 Mean (±SEM) percentage of conditioned responses for all three groups during
each day of training. Animals in the experimental group received infusions of muscimol
targeting the IP before extinction sessions 1-4. Starting on day 5 of extinction animals were
trained without the drug treatment.
70

0
10
20
30
40
50
60
70
80
90
100
ACQ-TOT DRUG-TOT NO DRUG-TOTAL
phase
CR%
1UL
0.1UL
CONTROLS

Figure 3.4 Mean CR (±SEM) percentages for all groups during each treatment phase. All
animals acquired the association at similar levels (ACQ-TOT). CRs were blocked to
baseline levels during the infusion phase (DRUG-TOT). Once infusions were stopped (NO
DRUG-TOTAL) animals in the experimental group showed significantly high levels of CRs
compared to controls.
71

Reacquisition
0
10
20
30
40
50
60
70
80
90
100
Group
CR%
1UL
0.1UL
CONTROLS

Figure 3.5 Mean CR (±SEM) percentages for all groups during the reacquisition session.
Experimental groups showed higher levels of performance compared to controls.
72
The effects of IP inactivations on timing of the CR were analyzed during and after
treatment. Figure 3.6a depicts CR peak latencies (milliseconds) of each session
during treatment and after treatment. Figure 3.6b shows overall CR peak latencies
during the treatment, no treatment and reacquisition phase of the experiment. During
the treatment phase (Phase II) control animals displayed longer peak latencies (M=
200ms ± 9.2 SEM) than the 0.1uL (M= 89.6ms ± 32 SEM) or the 1uL group (M=
131.1ms ± 13.1 SEM), F(2, 14) = 7.45, p = 0.006. However,disruption of adaptively
timed CRs is expected given that inactivations of the IP severely block CR
expression. No significant differences were found in peak latencies between all 3
groups during extinction after infusions were discontinued, F(2, 14) = 0.701, p =
0.513. Animals in all groups showed similar peak latencies during reacquisition
(1uL: M= 231.4ms ± 7.1 SEM; 0.1uL: M= 225.7ms ± 8.5 SEM; Controls: M=
210.5ms ± 8.5 SEM), F(2,14) = 0.921, p = 0.421. CR Onset latencies for each
session during and after drug infusions are depicted in Figure 3.7a. Figure 3.7b
illustrates onset latencies during the treatment, no treatment and reacquisition phase
of the experiment. Again, as expected a main group effect was found in onset
latencies during the drug treatment phase, F(2,14) = 6.96, p< 0.008. All animals
showed similar onset latencies during the extinction phase with no drug infusions,
F(2,14) = 0.85, p = 0.45. No differences were found between groups when the CS-
US association was reintroduced, F(2, 14) = 1.32, p = 0.299.

73
a)

0
50
100
150
200
250
300
ex1-D ex2-D ex3-D ex4-D ex5 ex6 ex7 ex8 ex9
Extinction day
CR Peak (ms)
CONTROL
0.1UL
1UL

b)

0
50
100
150
200
250
Ext-D E xt Reacq
Phase
CR Peak (ms)
control
0.1uL
1uL

Figure 3.6 Mean CR peak latencies (±SEM ) within the 250ms Inter-Stimulus Interval (ISI)
(a) Average time of peak response for all groups during each extinction day. Infusions were
administered during the first 4 days of extinction training. (b) Average time of peak response
during each treatment condition. Ext-D: Phase of extinction with drug infusions. Ext:
Extinction phase without drug infusions. Reacq: Reacquisition session.
74
a)

0
50
100
150
200
250
ex1-D ex2-D ex3-D ex4-D ex5 ex6 ex7 ex8 ex9
Extinction day
CR Onset (ms)
CONTROL
0.1UL
1UL

b)

0
50
100
150
200
250
Ext-D Ext Reacq
Phase
CR Ons e t
control
0.1uL
1uL

Figure 3.7 Mean CR onset latencies (±SEM) within the 250ms ISI. (a) Average time of
response onset during each extinction session. Infusions were given during the first 4 days of
extinction and stopped on day 5. (b) Average time of response onset during each treatment
phase. Note that onsets were shorter for the experimental groups only during the drug
treatment phase (Ext-D). Ext: Extinction with no infusions. Reacq: Reacquisition session.
75

Figure 3.8 Histological reconstruction of cannulae placements targeting the IP. Circles: 1uL
group; Squares: 0.1uL group. Numerals above plates represent distance in milimiteres rostral
from lambda.
76
Inactivations of the Red Nucleus:
All but 2 animals in the control group were included in the experiment for analyses.
One animal failed to reach learning criteria and was thus excluded from further
training. A second animal had post-surgery complications and could not be included
in the study. Final statistical analyses was done for 7 animals in the experimental
group and 5 in the control group. Figure 3.9 shows performance across training days
for both the experimental and control groups. By day 4 of acquisitioin training both
groups were performing at asymptotic levels (M= 85.4% ± 2.3 SEM). Also, both
groups attained similar levels of learning before extinction training, F(1, 10) = 0.017,
p = 0.90 (refer to Figure 3.10). There was also a significant day effect indicating
learning during training, F(3, 30) = 48.2, p< 0.00.

During phase II of the study, animals in the experimental group showed lower
response frequencies (M= 2.9% ± 6.7 SEM) compared to controls (M= 54% ± 7.9
SEM). A main group effect was found during this phase of the study, F(1, 10) =
24.02, p< 0.001, confirming that inactivations targeting the red nucleus were
effective. Control subjects showed declining levels of CRs during extinction sessions
typical of tone alone training, F(3, 12) = 7.04, p< 0.006. Release of the inactivations
during extinction training (Phase III) revealed no differences in CR frequencies
between groups, F(1, 10) = 0.329, p = 0.579. A significant day effect during Phase
III was found indicative of loss of performance with tone alone training, F(3, 30) =
3.89, p = 0.018. Figure 3.10 shows CR performance during reacquisition training
77
(M= 78.5% ± 4.04 SEM). Animals in the experimental group were able to reacquire
the association and showed response frequencies that were no different from
controls, F(1, 10) = 0.22, p = 0.87.

78

0
10
20
30
40
50
60
70
80
90
100
acq1
acq2
acq3
acq4
ex1
ex2
ex3
ex4
ex5
ex6
ex7
ex8
reacq
days
%CR
RED NUCLEUS
C ONTROLS

Figure 3.9 Mean CR percentages (±SEM) of groups during each training day. Animals in
the experimental group showed no CR expression during muscimol infusions (ex1-ex4)
confirming succesful inactivation of the red nucleus. Infusions were stopped on day 5 of
extinction training.
79
Effects of red nucleus inactivations on timing of the learned response were analyzed
during and after drug treatment. Figure 3.11a shows CR peak latencies across
training days. Subjects in the experimental group showed a marked decrease in CR
peak latencies during Phase II of the study (M= 108ms ± 21.7 SEM) compared to
controls (M= 202.1ms ± 25.7 SEM), F(1, 10) = 7.7, p = 0.20, consistent with
disruption of CR performance due to inactivations of the nucleus (refer to Figure
3.11b for illustration of overall peak latencies during each treatment phase). Peak
latencies in the experimental group during release of the inactivation (Phase III)
showed to be no different than controls (overall mean latency 140.1ms ± 21.1 SEM),
F(1,10) = 0.56, p = 0.470. Figure 3.12a illustrates CR onset latencies of the groups
throughout extinction training as well as reacquisition. During Phase II of training,
onset of the response was significantly shorter in experimental animals (M= 94.5ms
± 20.8 SEM) compared to controls (M= 157.2ms ± 24.7), F(1, 10) = 3.8, p = 0.081.
These difference was not evident after inactivations were released indicating that
infusions had no long term effects on timing of the learned response, F(1, 10) =
0.724, p = 0.415. Figure 3.12b shows CR onset during reacquisition training. No
significant difference between the groups was found for this session, F (1, 10) =
2.01, p = 0.186.

80

Performance across treatment
0
10
20
30
40
50
60
70
80
90
100
ACQUISITION EXT-DRUG EXT-NODRUG REACQUISTION
Treatment
%CR
RED NUCLEUS
CONTROLS

Figure 3.10 Mean CR percentages(±SEM) during each treatment condition. Both groups
attained similar levels of learning before entering the extinction phase. EXT-DRUG shows
the effects of CR blockage due to inactivations of the nuclei. Release of the inactivation did
not result in differences of CR frequencies compared to controls. Both groups reacquired the
response at high CR levels.
81
a)

0
50
100
150
200
250
ex1-D ex2-D ex3-D ex4-D ex5 ex6 ex7 ex8
session
CR Peak (ms)
CONTROL
RED NUCLEUS

b)

0
50
100
150
200
250
EXT-DRUG EXT-NO DRUG REACQ
CR Peak (ms)
CONTROL
RED NUCLEUS

Figure 3.11 Mean CR peak latencies (±SEM) within the 250ms ISI. (a) Average time of
peak response during each extinction session. Infusions were given during the first 4 days of
extinction and stopped on day 5. (b) Average time of peak response during each treatment
phase. Note that onsets were shorter for the experimental groups only during the drug
treatment phase (EXT-DRUG). EXT-NO DRUG: Extinction with no infusions. REACQ:
Reacquisition session.
82

a)

0
50
100
150
200
250
ex1 ex2 ex3 ex4 ex5 ex6 ex7 ex8
session
CR Onset (ms)
CON TROL
RED NUCLEUS

b)

0
50
100
150
200
250
EXT-DRUG EXT-NO DRUG RE ACQ
Phase
CR Onset (ms)
CONTROL
RED NUCLEUS

Figure 3.12 Mean CR onset latencies (±SEM) within the 250ms ISI. (a) Average time of
response onset during each extinction session. Infusions were given during the first 4 days of
extinction and stopped on day 5. (b) Average time of response onset during each treatment
phase. Note that onsets were shorter for the experimental groups only during the drug
treatment phase (EXT-D). EXT-NO DRUG: Extinction with no infusions. REACQ:
Reacquisition session.
83

Figure 3.13 Histological reconstruction of cannulae placements targeting the red nucleus.
Numerals above the plates correspond to distances (mm) caudal from bregma. IN:
interpeduncular nucleus; MGN: medial geniculate nucleus; PAG: periaqueductal grey; RN
red nucleus; SN: substantia nigra.
84
Discussion
The results of the experiments described above are compelling. Inactivations with
muscimol of the interpositus nucleus completely block extinction (and expression) of
the conditioned response. Subsequent post-infusion extinction training revealed that
animals were able to extinguish at rates that were identical to those of the control
group, indicating that the effects observed were not due to motor deficits or the result
of damage to the nucleus due to repeated drug infusions. This effect was seen even
with very small doses of muscimol (1 µg in 0.1uL) which, based on radiolabeling
studies (Krupa et al., 1993) and histological reconstruction, are very likely to have
only diffused to the deep nuclei and not the cerebellar cortex ruling out
confounding effects of cortical regions. Moreover, animals in the experimental group
were able to reacquire the response at almost asymptotic levels after just one session
of CS-US training. Controls on the other hand, although able to reacquire the
association, showed much lower levels of learned responses. This makes a stronger
case for blockage of extinction learning due to inactivation of the nucleus and not to
detrimental effects of the drug. Indeed, it seems that controls received extinction
training for nine consecutive days whereas experimental animals showed no
evidence of extinction upon release of the inactivation indicating that extinction
learning for these animals did not begin until day 5 of tone alone training. In fact, as
mentioned above, experimental animals extinguished at the same rate as controls
extinguished during the first days of extinction training (Figure 3.3). In sum,
inactivation of the interpositus nucleus prevents extinction from occurring while
85
having no effect upon the ability to learn to inhibit the response after the inactivation
has been removed. These results are entirely consistent with previous findings
showing that inactivations of the cerebellum using higher doses of muscimol prevent
extinction (Ramnani & Yeo, 1996).

Conversely, inactivations with muscimol of the contralateral red nucleus had no
effect at all upon the ability to extinguish the response even though infusions
completely prevented expression of the response. Indeed, after inactivations were
stopped, animals showed identical levels of extinction compared to controls
indicating that inactivation of the nucleus did not prevent learning of the inhibitory
association during treatment days. In fact, not only response frequency but timing of
the conditioned response was no different than controls throughout post-infusion
extinction training. This effect was seen with very small doses of muscimol (1µg in
0.1µL) that were very unlikely to diffuse to neighboring structures. Moreover, unlike
higher doses which affect motor ability and can influence contextual cues during
extinction training, the dose of muscimol used in this experiment caused very
minimal motor effects in animals. The ability to reacquire the response was also the
same as controls, showing high levels of response performance after tone alone
training. Overall, inactivations of this structure block response expression but, unlike
inactivations of the interpositus nucleus, do not have any detrimental effects on
extinction of the conditioned learned response.

86
From the present findings several general explanations can be proposed: 1) The
essential memory trace for extinction of the CR is not located in the red nucleus. If
this was the case, then inactivations of the nucleus during tone alone training would
have prevented extinction learning. However, animals were able to extinguish the
CR normally; in spite of inactivations of the nucleus. It is important to note
nonetheless, that this does not exclude the possibility that this structure might still
play a role in extinction. 2) The interpositus nucleus may be an essential structure (or
part of the essential circuit) involved in extinction. Possibly plastic changes need to
happen in this structure that allow extinction to occur. The fact that inactivations of
this region completely prevented extinction suggest an important, maybe critical,
involvement of this structure in suppression of the response.

However, as mentioned in Chapter 1, the fact that inactivations of the motor nucleus
block extinction put forward an alternative interpretation of these results. Perhaps,
inactivations of the interpositus block extinction simply because they prevent CR
expression. How can this interpretation be reconciled with the effects of red nucleus
inactivations? Possibly the presence of motor effects induced by the drug might be
an explanation. Actually, the red nucleus infusions elicited small, tonic alterations in
behavior such as tilting of the head in a direction contralateral to the infusion site.
The presence of these drug-induced behavioral responses could have concurrently
activated brain pathways associated with the eyeblink response resulting in an
amount of responding sufficient to produce extinction; as studies on response-
87
inhibition have shown. This scenario, although unlikely, would still allow the
implication of the interpositus nucleus as a site where plastic changes, due in this
case to feedback from response activity, occur. Another possible explanation
concerns context. Muscimol inactivation of the relevant motor nuclei on one side
results in complete paralysis of the ipsilateral face and head musculature, which
could provide a very different context for the animal. In marked contrast,
inactivation of the relevant region of the red nucleus simply prevents performance of
the eyeblink CR and little else other than a slight turning of the head; in short a much
less pronounced change in context. If so, the hippocampus may be involved in the
motor nuclei inactivation effect on extinction (see Hippocampus section in Chapter
1).

As reviewed in earlier chapters, the essential CR pathway of discrete motor
responses includes the interpositus, its efferent projections to critical regions of the
contralateral red nucleus, and the descending rubral connections to motor nuclei cells
and reticular formation. Inactivation or permanent lesions within this pathway,
completely block expression of the CR and prevent acquisition of the response if
made in the interpositus (Chapman et al., 1990; R. E. Clark et al., 1992; Krupa et al.,
1996). Results of the experiments presented here are in agreement with these
observations yet seem to be in variance of the response-extinction hypothesis (i.e.
Rescorla 1997). However, recent data using genetic and pharmacological techniques
strongly challenge this hypothesis and propose instead that memory retrieval and not
88
conditioned responding is critical in extinction, at least for eyeblink conditioning.
One of the major findings has stemmed from the discovery that norepinephrine (NE)
signaling is critical for contextual memory retrieval during a transient period after
training (Murchison et al., 2004). In a series of elegant experiments Thomas and
collegues (Ouyang & Thomas, 2005; Thomas, Marck, Palmiter, & Matsumoto, 1998)
were able to dissociate memory retrieval from behavior by using genetically altered
mice that lacked the ability to synthesize NE. More importantly, the gene can be
rescued by treatment with L-threo-3,4-dihydroxyphenyl serine (L-DOPS) allowing
for the specific manipulation of NE signaling and thus retrieval. It was found that
extinction of fear conditioning is only blocked during the NE-dependant retrieval
period. During the time period when NE is critical for memory retrieval, extinction
can only be rescued by treatment with L-DOPS. Moreover, local infusions of the
hippocampus showed that β
1
-adrenergic receptor (required in NE signaling) agonists
rescued extinction during this critical period only when it was applied to a specific
region of the hippocampus, regardless of whether animals showed responding or not.
Also supporting dissociation of CR performance and extinction are data using
Drosophila showing that blocking conditioned responding by inactivation of
mushroom body Kenyon cells does not prevent extinction (Schwaerzel, Heisenberg,
& Zars, 2002).

These new findings are in conflict with response-extinction theories and raise
important questions regarding the role played by response expression in this
89
phenomenon. Based on these data, interpretation of the the motor nucleus study
would then point towards an effect of context (described above) rather than response
suppression; implicating the hippocampus as a modulatory structure. Indeed, results
of hippocampal lesions and recordings have shown that the hippocampus is
important in coding the conditioning context (Penick & Solomon 1991) and that it
develops patterns of neuronal activity highly correlated with the behavioral CR
(Hoehler & Thompson, 1980).

Of particular interest in the studies presented here are the differences in performace
between IP inactivation animals and controls when the association was
reintroduced.The fact that experimental animals achieved significantly higher levels
of CR suggests these animals were only able to start extinguishing the response with
the nucleus intact and were therefore exposed to less days of extinction training
compared to controls hence, more easily reacquiring the association. This is
important, because it suggests an essential role of the cerebellum in the extinction of
conditioned responses. Perret and Mauk (1995) found that aspirations of the anterior
lobe of the cerebellar cortex block extinction. Further, blockage of inhibitory input to
climbing fibers, which shares afferent and efferent connections with the cerebellum,
prevent extinction as well (J. F. Medina, Nores, & Mauk, 2002). More recently it
was reported that inactivations of the brachium conjunctivum, a major output of the
cerebellum, also prevent extinction (Nilaweera, Zenitsky, & Bracha, 2005). Gould
and Steinmetz (1996) described multiple unit patterns of activity in the IP that
90
closely matched decreases of CR production due to extinction training, yet, in the
cortex activity remained relatively unchanged. Finally Poulos & Thompson (2004)
found that stimulation thresholds of the IP increase during extinction compared to
acquisition levels, yet, remain significantly lower than baseline levels. This is
indicative of residual plasticity which would also account for savings observed
during reacquistion. Altogether these studies suggest an important signal for
extinction that probably involves inhibition of IO activity through GABAergic inputs
from the interpositus (Nelson et al., 1989).

Perhaps, the cerebellar cortex and interpositus nucleus work synergistically resulting
in changes that lead to extinction. Long-Term Depression (LTP) for instance, has
been reported to occur at granule-Purkinje cell synapses when active in the absence
of climbing fiber activity such as in tone alone training (Ito, 1989; Linden,
Dickinson, Smeyne, & Connor, 1991; Sakurai, 1987). This phenomenon would
increase Purkinje cell input into interpositus cells, inhibiting the nucleus from firing;
behaviorally resulting in CR loss. Moreover, mossy fiber-interpositus nucleus
synapses can undergo a generalized form of hebbian plasticity (Brown, Kairiss, &
Keenan, 1990; Racine, Wilson, Gingell, & Sunderland, 1986). Assuming that co
activation of IO and mossy fiber input to the IP induces LTP at the mossy fiber-IP
synapse, CS alone presentations may weaken this long term potentiation at the
synapse so that repeated activation of mossy fibers becomes less able to depolarize
nuclei cells resulting in the disappearance of CRs.
91
Clearly the experiments presented in this chapter demonstrate that inactivating the
interpositus completely blocks brain mechanisms that lead to extinction learning and
suggest that plastic changes, perhaps even the memory for extinction, is located
upstream of the red nucleus. Moreover, unlike acquisition using delay conditioning,
the hippocampus is likely to play an essential modulatory role in the extinction of
responses. More importantly, these experiments seem to argue against performance
of the CR being essential for extinction learning. Without a doubt, this brings forth
important questions as to the validity of response-extinction theories that certainly
need to be investigated further.

92
CHAPTER FOUR

EFFECTS OF ELECTRICAL STIMULATION OF THE RED NUCLEUS
DURING INACTIVATIONS OF THE INTERPOSITUS IN EXTINCTION

Introduction
One of the fundamental tenets of contemporary learning theories is based on the idea
that learning occurs as a result of a mismatch between experience and expectations
the delta rule (Pearce & Hall, 1980; Schmajuk & DiCarlo, 1992). Acquisition of
learning in the naïve animal may occur due to the great difference that exists
between the lack of expectations (no relevant memories to retrieve) and experience.
In extinction the learned association or expected event no longer occurs resulting in a
mismatch between expectation and experience that leads to extinction of the
response. It is thus proposed that memory retrieval of the expected event must occur
during extinction training. However, as discussed in the previous chapter, as of this
writing no concensus exists on whether response performance is critical for
extinction to occur. Supporting retrieval only and response performance theories is
evidence demonstrating that introduction of a second conditioned excitor during CS
alone presentations results in greater responding and a larger decrement of
responding to the CS during subsequent extinction training (Rescorla, 2000).
Similarly, introduction of a conditioned inhibitor during CS alone presentations
93
leads to less responding and a lesser response suppresion to the CS during
subsequent extinction training (Rescorla, 2003). Whether these findings are due
specifically to the amount of responding or the degree of difference between
expectation and experience is unclear. Recent evidence (Schwaerzel et al., 2002)
seems to argue against response-extinction theories and rather suggests that
expectation of the US is sufficient to drive extinction. Further supporting this view
are data presented in chapter three showing that inactivations of the red nucleus,
which prevent CR expression, do not prevent extinction.

Results presented in the previous chapter and by others (Ramnani & Yeo, 1996)
clearly demonstrate that inactivations of the IP completely prevent extinction of the
response. Whether extinction is blocked due to the disruption of intrinsic changes
within the nucleus or simply because expression of the response is interrupted cannot
be determined with certainty. In acquisition, reversible lesions of CR and UR output
pathways such as the IP, red nucleus and motor nucleus (R. E. Clark & Lavond,
1993b; Krupa & Thompson, 1997b; Krupa et al., 1996) cause complete abolition of
the CR yet, only lesions of the IP prevent learning of the original association clearly
expression of the response is not necessary in learning the original association.
Conversely, given the hypothesized role of response performance in extinction, it is
far more difficult to asses the interaction between this phenomenon and retrieval
because it is hard to dissociate both experimentally. In eyeblink conditioning, one of
the effects that result from inactivation of the CR pathway is the inevitable abolition
94
of CR expression. Given that reversible lesions of the IP block response performance
another technique or experimental manipulation is needed to better understand the
contributions of this nuclei in extinction.

Electrical Brain Stimulation as a Valuable Tool in Eyeblink Conditioning:
One of the techniques that has proved extremely useful in the identification of the
neuroanatomical substrates of eyeblink conditioning is that of electrical stimulation
of brain structures. Pioneering this technique, Brodgen and Gantt (1942) were the
first ones to show that the cerebellum could support basic associative learning by
passing current through electrodes implanted in the cerebellar cortex. They were not
only able to induce discrete motor movements such as limb flexion and eyeblink
responses but more importantly, they showed that by pairing these movements with a
neutral stimulus these responses could be conditioned. More recently, brain
stimulation studies have further shown the cerebellum to be a structure were
information about the CS and the US converge (Shinkman, Swain, & Thompson,
1996a; Swain, Shinkman, Nordholm, & Thompson, 1992; Swain, Shinkman,
Thompson, Grethe, & Thompson, 1999; R. F. Thompson et al., 1998). Stimulation of
the pontine nuclei, for instance, has been found to serve as an effective CS using the
standard delay conditioning paradigm (Solomon et al., 1986). Furthermore,
stimulation of the dorsal accesory nucleus of the IO produces an eyeblink which can
be effectively used as a US (Mauk, Steinmetz, & Thompson, 1986). More
importantly, stimulation of the IP as a CS has been found to induce eyeblinks that
95
can be effectively conditioned and that result in fast and robust learning when paired
with a US (Poulos & Thompson, 2004). Interestingly, standard delay training using
IP stimulation as a US under subthreshold levels has been found to show substantial
savings when stimulations are substituted for an airpuff US (Chapman, Steinmetz, &
Thompson, 1988). Conversely, Champman et al. (1988) also reported that
stimulation of the red nucleus, which receives information from the IP about the CR,
does not support conditioning (but see Nowak et al., 1997). Indeed, the red nucleus
seems to be part of the essential pathway for expression of the CR yet, unlike the IP,
is not essential in memory formation and storage of the conditioned response.

In light of a suspected involvement of response expression in extinction one way in
which the role of the IP could be better understood is by means of a technique in
which responding is mantained during inactivations of this brain region -possibly by
activation of one of its efferent pathways. As a recipient of interpositus output, the
red nucleus would appear to be a suitable structure in the experimental induction of
an eyeblink via electrical brain stimulation. Indeed, stimulation of the relevant region
of the red nucleus has been demonstrated to elicit eyeblinks or movements of the
NM (Chapman, Steinmetz & Thompson, 1988; Nowak et al., 1997). Moreover,
lesions of the red nucleus selectively abolish the CR while leaving the UR unaffected
ruling out its involvement in production of the reflexive response (Haley, Lavond &
Thompson, 1983; Rosenfiel & Moore, 1983).

96
Here we report the results of inactivating the IP while using electrical stimulation of
the red nucleus as a CR (see Figure 4.1 for experimental manipulation). We examine
the role of both the interpositus and red nucleus taking into consideration current
theories of extinction and putative brain processes that may account for the effects
obtained.

97

Cerebellar cortex
IP
RN
IO PN
eyeblink
Cerebellar cortex
IP
RN
IO PN
eyeblink

Figure 4.1 Experimental procedure used in the study. Shaded boxes illustrate nuclei
manipulated during extinction training. The interpositus (IP) was inactivated via
administrations of muscimol which blocked response expression. Induction of an eyeblink
was achieved through a bipolar electrode targeting the red nucleus (RN). PN: Pontine
nucleus; IO: Inferior olive.
98
Methods
Subjects
Twenty-two New Zealand White rabbits weighing between 2.2-3.0 kg were
implanted with a bipolar stimulating electrode targeting the red nucleus and a
cannula in the interpositus. Animals were housed individually and maintained on a
12 hour light/dark cycle with ad-lib access to water and food.

Rabbits in the experimental group received infusions of muscimol that targeted the
IP consisting of 0.1 µL (0.01M). Animals in the control group received infusions of
saline.

Surgery:
All animals were anesthetized with subcutaneous injections of ketamine (60mg/kg)
and xylazine (8mg/kg) and placed on a stereotaxic frame with a constant flow of
1.5%-2% of halothane in oxygen throughout the length of the surgical procedure.
Under asceptic surgical conditions an anterior to posterior incision was made in the
scalp in order to retract the skin and periostium and expose the skull. A small hole of
approximately 1.5mm in diameter was drilled through the bone at the
anterior/posterior and laterla/medial coordinates of the target structure. A guide
cannulae (26 gauge) 25mm in length was implanted into the ipsilateral interpositus
nucleus with bregma 1.5mm dorsal to lambda. The guide cannulae was lowered with
an insulated stainless steel stylet (Elephant brand 000 insect pin) inserted and
99
extending 1.5mm below the tip. The outer cannulae tip was sharpened in order to
minimize damage to brain tissue during its implantation. These cannulae assembly
was lowered slowly with a stereotaxic manipulator that targeted the following
coordinates based on the stereotaxic atlas of McBride and Klemm (from lambda):
0.7mm anterior, 5.1mm lateral and 14.5mm ventral. Subsequently a set of bipolar
electrodes was slowly implanted into the red nucleus. The electrodes were custom-
designed and consisted of two Number 000 stainless steel insect pins with several
layers of epoxylite insulation and a tip exposure of 250-300µm. Electrodes were
stereotaxically placed into the red nucleus based on the following coordinates (from
bregma): 8.5mm posterior, 1.0mm medial and 15.5mm ventral. Final placement of
electrodes in all animals was determine upon observable movement of the NM or
eyelids when current was passed. Four holes were then drilled to fit surgical stainless
steel screws that anchored a headstage constructed from dental acrylic. A plexiglas
mount built to hold a minitorque potentiometer during behavioral training was also
cemented to the headstage. Skin surrounding the dental acrylic was asceptically
cleaned with antibiotic ointment (iodine). Animals were allowed to recover for a
period of 1week before the beginning of behavioral training. During the first two
post-operative days animals were given two daily injections of analgesic
(buprenorphine hydrochloride) and were monitored throughout the length of their
recovery. All procedures and animal care were approved by the USC Institutional
Animal Care and Use Committee in accordance with NIH guidelines.

100
Apparatus:
Animals were habituated and trained in a padded pexiglas restraint and placed into a
behavioral recording chamber. During training nictitating membrane (NM)
movements were measured with a minitorque potentiometer temporarily attached to
the animals headstage. Voltage changes due to movement of the NM were measured
via mechanical movement of a bar attached to the potentiometer and connected by a
thread lead hook to a suture loop implanted in the left NM. The voltage signal was
stored and recorded on a computer using custom software written in Forth. The
software also controlled delivery of the stimuli during training. Data was
subsequently analyzed offline. Infusions were delivered to the targeted structure
using a 1µl syringe (Hamilton instruments). The syringe was connected to a
polyethelene tube attached to an injection cannula (31 gauge) 26.5mm in length.
Drug was delivered by compression of the syringe using an infusion pump (Harvard
Apparatus) at a rate of 0.38 µl per minute (0.1 µL; 0.01M). Electrical stimulation
during inactivation of the interpositus was delivered using a Grass Intruments
(Model S48) stimulator and a constant current isolation unit.

Behavioral Training Procedures:
Same behavioral procedure described in Chapter 1 with the following exceptions:
Phase II: Following acquisition animals received 4 daily sessions of tone alone
training. One hour before the beginning of each training session rabbits received
infusions of muscimol or saline targeting the interpositus nucleus. Infusions were
101
administered by removal of the inner stylet and insertion through the guide cannula
of the injection cannula. Delivery of muscimol was followed by a waiting period of 3
minutes in order to ensure absorption of the drug into the brain before the infusion
cannula was removed and replaced with the stylet. An hour after infusions were
administered, animals were put inside each training chamber where the stimulator
was connected to the electrodes. Before the beginning of training eyelid responses
elicited by red nucleus stimulation were determined. Parameters for each animals
varied slightly according to the minimum amount of current necessary to evoke a
response. Intensities varied from 400-700µA and were delivered at a rate of 200 Hz
with pulses of 0.2ms in duration. Animals were only used if the stimulation
successfully evoked an observable eyeblink. Following threshold measurements
animals were then given a baseline tone alone test to confirm the absence of response
expression and thus successful inactivation of the interpositus. If responses were
observed during this baseline trial animals were brought back a few hours later and
infusions were administered again. Failure of the infusions to supress response
expression this second time suggested an inaccurate cannula placement and
exclusion of these animals from the study followed. Rabbits that met stimulation and
inactivation criteria were then given 100 trials of tone-alone training (1 kHz, 85 dB)
during each extinction session (8 extinction sessions in total). Stimulation timing
during training was determined by taking the average response onset of 6 randomly
selected well-trained animals. This yielded an average onset of 165ms after tone
102
presentation. Based on this calculation, responses were induced by applying the
stimulation at exactly this time during the trial.
Phase III: Extinction training without drug infusions or electrical brain stimulation
was given to rabbits for 4 consecutive days or until CR levels dropped to 15% or
less.
Phase IV: In order to measure the ability of animals to reacquire the response, the
association was presented again to animals on the day following the conclusion of
extinction training.

Histology:
Following training an insulated electrode with 250µ m of tip exposure was lowered
through the guide cannula. A 1µ A current was subsequently passed through the
electrode for 10 seconds in order to create a marking lesion at the site of injection.
The same amount of current was applied through the electrodes to confirm
placement. Animals were then given an intravenous overdose of sodium
pentobarbital and perfused intracardially with saline and 10% formaline. Brains were
then extracted and preserved for later histological reconstruction in 10% formalin for
a minimum of 3 days. Brains were then embedded in an albumin and gelatin mixture,
sectioned at 80µ m and mounted for later staining. Mounted sections were nissl
stained with Cresyl violet and counterstained with Prussian blue to label iron
deposits left by the tip of the electrode at the time of the marking lesion. Injection
103
sites and electrode placements were then analyzed and recorded using a dissecting
microscope.

Results
Of the twenty-two rabbits that began the study, four in the experimental group and
five in the control group were included for further analyses. Animals excluded in the
control group had post-surgery complications that prevented subsequent training.
Rabbits not included in the experimental group either showed CRs during the
baseline IP inactivation test or failed to exhibit an observable eyeblink via electrode
stimulation. Also, several animals did not learn the CS-US association and were thus
excluded early during training. Histological analyses of these animals showed
cannulae and/or electrode placements that penetrated most of the nuclei (interpositus
and/or red nucleus) suggesting lesioning of the critical region of the structure.

Figure 4.3 shows the overall CR performance for both groups in the experiment
(refer to Figure 4.2 for individual performances throughout training). During the
acquisition phase of the study a significant day effect was found consisted with
increases in CR frequency indicative of learning of the association, F(3,21) = 73.4,
p< 0.00. Furthermore, both the experimental and control group acquired the
association at asymptotic levels (average CR% on the last day of acquisition:
controls = 88.1% ± 3.2; experimental = 90.3% ± 3.6) with no significant differences
104
in performance between the groups before entering Phase II of the study, F(1,7) =
1.6, p< 0.249.

During Phase II of extinction training (days 1-4) animals in the control group showed
a gradual decrease of CRs due to tone alone training (M = 70.3% ± 6.03) that was
found to be significant, F(3,12) = 15.89, p< 0.00. By the last day of this phase (day
4), the average CR performance for controls was down to 35% (SEM ± 13.1). In
contrast, experimental animals trained under IP inactivation paired with brain
stimulation displayed high CR levels throughout this phase (M = 95.4% ± 1.5) up to
day 4 (M = 95.7% ± 2.7). No significant day effects were found, F(3,9) = 0.10, p<
0.95, indicative of successful induction of the response via stimulation of the red
nucleus in spite of IP activity blockage. As it would be expected, differences were
significant between controls and experimental groups during this phase of the study,
F(1,7) = 12.9, p< 0.009 (see Figure 4.4 for overall performance during each phase).

Analyses of CR performance after experimental manipulations were stopped (Phase
III) revealed that during the first extinction session of this phase (day 5) animals in
the experimental group showed a great loss in CR frequency (M = 19% ± 7.6) that
was no different than controls (M = 23.2% ± 7.6), F(1,7) = 0.13, p< 0.72. Although
there seems to be a trend of lower levels of performance for experimental subjects
there were no significant overall differences between the groups during this phase,
F(1,7) = 1.44, p< 0.27. By the last day of extinction training both groups were
105
performing at almost baseline levels (experimental: M = 6.5% ± 3.5; controls: M =
5.8% ± 3.1).

106
a)

RN/IP
0
10
20
30
40
50
60
70
80
90
100
ACQ1
ACQ2
ACQ3
ACQ4
EXT1
EXT2
EXT3
EXT4
EXT5
EXT6
EXT7
EXT8
days
CR%

b)

Controls
0
10
20
30
40
50
60
70
80
90
100
ACQ1
ACQ2
ACQ3
ACQ4
EXT1
EXT2
EXT3
EXT4
EXT5
EXT6
EXT7
EXT8
days
CR%

Figure 4.2 Behavioral graphs depicting CR frequencies (%) during each training session. (a)
Individual results of each animal in the IP inactivation-RN stimulation (experimental) group.
(b) Individual performance of animals in the control group. ACQ: Acquisition; EXT1-4:
Extinction training during IP inactivation-RN stimulation; EXT5-8: Extinction training with
no experimental manipulation.
107

0
10
20
30
40
50
60
70
80
90
100
ACQ1
ACQ2
ACQ3
ACQ4
EXT1
EXT2
EXT3
EXT4
EXT5
EXT6
EXT7
EXT8
days
CR%
IP /RN
CON TROLS

Figure 4.3 Mean (±SEM) percentage of conditioned responses for both groups during each
day of training. Animals in the experimental group received infusions of muscimol targeting
the IP paired with electrical brain stimulation to the RN on extinction sessions 1-4. Starting
on day 5 of extinction animals were trained without the drug treatment and brain stimulation.
108

0
10
20
30
40
50
60
70
80
90
100
ACQ-TOT D /S-TOT ND-TOTAL
Phase
CR%
IP /RN
CONTROLS

Figure 4.4 Mean (±SEM) percentage of conditioned responses for both groups during each
treatment phase. Both groups acquired the association at similar levels (ACQ-TOT). D/S-
TOT: Phase in which animals received infusions targeting the IP paired with RN
stimulation. Note that throughout this phase of extinction stimulation successfully elicited an
eyeblink in spite of inactivations of the IP. ND-TOTAL: Phase of extinction in which no
experimental manipulations were used. Both groups extinguished the response at levels that
were statistically identical.
109
Figure 4.5 shows CR performance of both groups during the session when the
original association was reintroduced. Despite the significant performance loss
observed for both groups during the last day of extinction training, both experimental
and control animals achieved high levels of CR frequency (M = 75.6% ± 5.3) that
were evidently different from naïve (see performance on acquisition day 1). No
significant diffences were found in CR reacquisition between the groups, F(1,7) =
1.17, p= 0.00.

The effects of stimulation and inactivation of the targeted nuclei on timing of the
response were analyzed during and after the experimental manipulation. Figure 4.6a
shows the CR peak onset during each day of extinction training. There were no
significant differences between the groups in response onset during extinction with
brain stimulation (Phase II), F(1, 7) = 0.62, p< 0.457. Moreover, no Group x Day
interaction was observed during this phase of the experiment, F(3,21) = 1.4, p<0.27.
Extinction without the experimental manipulation (Phase III) showed that CR onset
for animals in the experimental group was no different than controls, F(1,7) = 0, p<
0.99. Also, no Group x Day interaction was revealed during this last phase of
extinction, F(3, 21) = 1.3, p< 0.29. Analyses of the CR onset during the reacquisition
phase revealed that brain stimulation resulted in onsets of the CR that were
significantly longer (M = 172.8ms ± 6.9) compared to controls (M = 136.9ms ± 6.2),
F(1, 7) = 14.76, p< 0.006. Interestingly, a closer inspection of the results revealed
that CR onsets for the experimental group did not change significantly across each
110
phase, F(2, 6) = 0.15, p< 0.86, whereas controls CR onsets were significantly shorter
during reacquisition compared to performance during the other phases, F(2, 8) =
6.67, p< 0.02 (see Figure 4.6b for average CR onset during each phase). Figure 4.7a
illustrates average latencies of the CR peak for both groups across training. No
differences were observed between groups during extinction days with experimental
manipulation (days 1-4), F(1, 7) = 0.173, p< 0.14. Similarly, during extinction
training without manipulation (days 5-8) no differences in CR peak latencies were
observed between control and experimental animals, F(1,7) = 2.78, p< 0.14. During
reintroduction of the CS-US association the experimental group showed longer peak
latencies (M = 336ms ± 6.9) compared to controls (M = 212ms ± 6.25). This
difference proved to be significant, F(1, 7) = 6.59, p< 0.03 (see Figure 4.7b for
average peak onset during each phase).

111

Reacquisition
0
10
20
30
40
50
60
70
80
90
100
Group
CR%
IP /RN
CONTROLS

Figure 4.5 Mean (±SEM) percentage of conditioned responses for both groups during the
reacquisition session. There were no significant differences between the groups in the ability
to reacquire the association.
112
a)

0
50
100
150
200
250
ex1-DS ex2-DS ex3-DS ex4-DS ex5 ex6 ex7 ex8
Extinction session
CR Onset(ms)
IP /R N
CONTROL

b)

0
50
100
150
200
250
E xt-D/S E xt Reacq
Phase
CR Onset
IP /RN
CONTROL

Figure 4.6 Mean CR onset latencies (±SEM) within the 250ms Inter-Stimulus Interval
(ISI). (a) Average time of response onset during each extinction session. Infusions were
paired with brain stimulation during the first 4 days of extinction training (ex #-DS) and
were stopped on day 5. Starting on day 5 animals received extinction training without any
experimental manipulations (ex 5-8). (b) Average time of response onset during each
treatment phase. Note that onsets were shorter for the control group during the reacquisition
session (Reacq). Ext-D/S: Extinction with infusions and brain stimulation. Ext: Extinction
with no experimental manipulations.
113
a)
0
50
100
150
200
250
ex1-DS ex2-DS ex3-DS ex4-DS ex5 ex6 ex7 ex8
Extinction session
Peak(ms)
IP /RN
C ONTROL

b)
0
50
100
150
200
250
300
E xt-D/S E xt Reacq
Phase
Peak(ms)
IP /RN
CONTROL

Figure 4.7 Mean CR peak latencies (±SEM) within the 250ms ISI. (a) Average time of peak
response for both groups during each extinction day. Infusions and brain stimulations were
administered during the first 4 days of extinction training (ex #-DS). Starting on day 5 of
extinction no experimental manipulation were used (ex5-8). (b) Average time of peak
response during each treatment condition. Note that peak latencies were longer for the
experimental group during the reacquisition session (Reacq). Ext-D/S: Extinction with drug
infuisions and brain stimulations. Ext: extinction training with no experimental
manipulation.
114

Figure 4.8 Histological reconstruction of cannulae tip placements targeting the IP. Numerals
above plates represent distance in millimeters rostral from lambda.
115

Figure 4.9 Histological reconstruction of electrode tip placement targeting the red nucleus.
Numeral above plates represent distances in milimeters caudal to bregma. IN:
interpeduncular nucleus; MGN: medial geniculate nucleus; PAG: periaqueductal grey; RN:
red nucleus; SN: substantia nigra.
116
Discussion
The results of the present study are the first to demonstrate that inactivations of the
IP do not block extinction when paired with electrical stimulation of the red nucleus.
In fact, post-treatment extinction training clearly showed that in spite of IP
inactivations, brain stimulation of the red nucleus seemed to have resulted in
extinction rates that were not only much lower than asymptotic acquisition levels but
in rates that were actually identical to those of controls. Moreover, just as the control
group, CR performance of experimental animals gradually decreased to almost
baseline levels with further tone alone training indicating that brain stimulation had
no effect on the ability to extinguish the response. Data from experimental animals
further show that when the CS-US association was reintroduced after extinction
training, animals were able to reacquire the response at high levels just as controls.
This is important because it demonstrates that the low response performance of
experimental animals observed during Phase III of extinction (days 5-8) was not due
to damage of the red nucleus caused by the applied stimulation. Intriguingly, we
found that stimulation of the red nucleus affects onset and peak latencies of the CR
during reacquisition pointing towards a possible contribution of the red nucleus in
timing aspects of the learned response. Finally, this experiment is also consistent
with previous findings showing that intrinsic activity in the red nucleus is capable of
generating an eyeblink response (Chapman, Steinmetz & Thompson, 1988; Nowak
et al., 1997) on the contralateral eye as we showed through direct stimulation of the
structure. Such evidence supports extensive reports indicating that the red nucleus is
117
essentially involved in the performance of learned responses (Chapman et al., 1988;
Rosenfield, Dovydaitis, & Moore, 1985; A. M. Smith, 1970).

Given the open debate regarding the validity of existing theories of extinction, the
present findings raise the question of whether the results obtained are in fact mainly
due to the presence of CRs elicited through brain stimulation during extinction
training. Supporting response-extinction theories this explanation of the results
would also lead to the conclusion that IP inactivations block extinction simply
because response expression is blocked. However, interpreting the present data under
the response-extinction premise markedly contradicts recent findings that dismiss the
importance of response performance (see Chapter 3), including evidence presented
previously which clearly shows that red nucleus inactivations do not disrupt
extinction even though response expression is supressed. Such results support what
is emerging as a different conceptualization of extinction. Namely, that extinction
learning might involve mechanisms that do not rely on response performance. In
fact, prior findings suggesting that blocking the expression of CRs through
inactivations of the motor nucleus (Krupa & Thompson, 2003) prevents extinction
should be considered carefully. Of particular concern is the fact that in that study
experimental animals were performing at relatively high CR levels up to the last day
of tone alone training. It is not clear then, whether animals were capable of
extinction at all given that training was discontinued before this could be determined.
More elusive is what might have resulted in the observed high levels of responding
118
since it is unlikely that damage to the nucleus would be the cause.This certainly
hinders any attempts to favor a singular theory or mechanism of extinction and
grants the question of whether other factors other than lack of response expression
might have come into play.

Of great importance in the study presented here is the fact that the effect of blockage
of extinction, due to inactivation of the IP, is completely removed with stimulation of
the red nucleus. This puts forth the possibility of the existence of an alternative
pathway via the red nucleus through which extinction can occur successfully.
Moreover, based on more recent findings, it could be further proposed that this
mechanism can successfully result in extinction without dependance on CR
production. This interpretation would most likely require the involvement of brain
structures that have been previously demonstrated to be critically involved in
acquisition and extinction of conditioned responses. As discussed in previous
chapters, a great body of evidence has demonstrated the essential role of the
cerebellum in the acquisition of the response (Chapman et al., 1990; R. E. Clark et
al., 1992; Krupa et al., 1996) and several lines of evidence consistently indicate that
the cerebellar cortex and/or interpositus are likewise importantly involved in the
extinction of these responses (Perret & Mauk, 1995; Ramnani & Yeo, 1996; Gould
& Steinmetz, 1996). Furthermore, as a a source of climbing fiber input to the
cerebellum, the inferior olive (IO) has been shown to play a key role in providing the
signal that drives extinction (J. F. Medina et al., 2002).
119
If the proposed interpretation of the existence of an alternative pathway is true then
direct stimulation of the red nucleus should have affected those critical brain
structures thought to be necessarily involved in extinction. Based on this premise, a
likely recipient of the applied red nucleus stimulation could very well be a structure
that is also capable of conveying important information to the cerebellum, namely
the IO. Indeed, evidence of the existence of a rubro-olivary tract has been
extensively reported (Horn, Hamm, & Gibson, 1998; Kennedy, 1979; Weiss, Houk,
& Gibson, 1990) and has been shown to be able to modulate changes in the IO. For
instance, Weiss and colleagues (1990) reported a marked inhibition of cells in the IO
to peripheral stimuli after electrical stimulation of the magnocellular red nucleus.
Similarly, Horn et al. (1998) confirmed the existence of red nucleus mediated IO
inhibition by using stimulation of the ventral funiculus (VF) of the spinal cord
instead of peripheral stimulation.

It is very possible that direct stimulation of the red nucleus strongly inhibited activity
within the inferior olive promoting changes responsible for extinction. Consistent
with this explanation are data showing that inhibition of climbing fibers below
equilibrium (1 Hz) provides the teaching signal that drives extinction (Medina &
Mauk, 2002). Activity of the IO below baseline levels would happen under
conditions in which inhibition to this structure is not counteracted by excitatory input
(i.e. airpuff) such as in tone alone training. For instance, in the well trained animal,
the US would provide excitatory inputs that activate climbing fibers, however, IP
120
output that generates the conditioned responses would also inhibit climbing fibers
(Kim, Krupa, & Thompson, 1998) maintaining their activity near equilibrium levels.
Conversely, during extinction the absence of excitatory inputs due to removal of the
US would result in a strong inhibitory input from the IP that would shift climbing
fiber activity well below baseline levels providing the signal necessary for the
induction of extinction. Considering that the IP was inactivated during extinction
training, the results of this study suggest that the strong stimulation applied in this
experiment resulted in an inhibitory input from the red nucleus that was sufficient to
tilt the balance of climbing fibers below background levels.

Can this signal, conveyed by climbing fibers, modulate changes in the cerebellum?
More importantly, can these changes result in extinction? Electrophysiological
studies support the hypothesis that olivary activity can greatly influence patterns of
cell firing and strenght of inputs in the cerebellum. Rawson and Tilokskulchai (1981)
showed that high rates of climbing fiber firing can completely inhibit Purkinje cell
simple spikes. A different effect has been reported under conditions of low-rate
climbing fiber discharge whereby Purkinje cell sensitivity to peripheral input is
heightened after a complex spike (Ebner & Bloedel, 1981). This suggests that olivary
activity may also importantly determine mossy fiber influence onto the cerebellar
cortex. Indeed, co activation of mossy and climbing fiber inputs induces LTD at
granule-Purkinje cell synapses (Linden & Connor, 1993), whereas activation of
mossy fibers in the absence of climbing fiber input leads to LTP (Sakurai, 1987).
121
During mossy fiber activation, LTP would increase Purkinje cell input into the
interpositus, inhibiting depolarization of the the nuclei and thus expression of the CR
(see chapter 3).

In sum, the present results indicate that stimulation of the red nucleus can support
changes conducive of extinction. Moreover, the fact that extinction occurred in spite
of inactivation of the IP puts forth the possibility that the cerebellar cortex may be a
site of plastic changes necessary for extinction learning. As a structure thought to be
involved in timing of the CR (Perret and Mauk, 1995; Garcia et al., 1999),
significant effects on peak and onset of the CR observed here, during reacquisition,
might further point towards an engagement of the cerebellar cortex through brain
stimulation. It should be noted however, that under natural conditions, the IP might
play a main role in providing the essential inhibitory input to the IO (Kim, Krupa &
Thompson, 1998) that ultimately results in the signal that drives extinction.
Supporting this view are results presented in the previous chapter where it was found
that inactivations of the red nucleus did not have any detrimental effects on
extinction of the response yet, inactivations of the IP result in complete blockage of
extinction.

Unfortunately, due to the feedback nature of red nucleus connections no strong
conclusions can be made as to the validity of the response-extinction argument.
Indeed, a limitation of this study is that the possible engagement of intrinsic changes
122
due to stimulation cannot be dissociated from the fact that responses were being
elicited. In an attempt to further explore this issue, it would be interesting to test the
importance of CR expression by employing the experimental manipulation used here
while also inactivating the motor nucleus. Although potentially a challenging
experiment, this would ensure the blockage of response expression while still being
able to test the effects of red nucleus stimulation on extinction of the response.

What is clear from the present findings is that the cerebellum is critically involved in
the induction of changes that lead to extinction. Moreover, GABAergic input to the
IO seems to be an essential factor in driving these changes. These results are
consistent with models of cerebello-olivary feedback and provide further evidence
that extinction requires mechanisms that are different from those of acquisition.

123
CHAPTER 5

LEARNING RELATED CHANGES DURING EXTINCTION: THE
ROLE OF THE NMDA RECEPTOR
____________________________________________________________________

Introduction
The importance of the NMDA receptor (NMDAR) in excitatory synaptic
transmission and other related cellular processes in the brain has been well
demonstrated (for reviews see Dudai, 1989; Nicoll & Malenka, 1999). As a widely
distributed site of glutamate binding, the NMDAR is commonly expressed in the
central nervous system (CNS) and has been linked with processes as diverse as
neural development and perception of pain (Dickenson & Sullivan, 1991).
Relevant to learning and memory, it is widely believed that NMDARs play an
important role in the induction of long-term changes in neuronal function such as
LTP and LTD (Bliss & Collingridge, 1993; Kemp & Bashir, 2001). More
importantly, a great body of research indicates that NMDARs are involved in a wide
variety of learning tasks that include experimental conditions such as fear
conditioning, taste-potentiated odor conditioning, spatial discrimination (Fanselow &
Kim, 1994 (Fanselow & Kim, 1994; Hatfield & Gallagher, 1995; Mathis, de Barry,
& Ungerer, 1991) as well as eyeblink conditioning (G. Chen & Steinmetz, 2000; L.
T. Thompson & Disterhoft, 1997).
124
NMDARs are heteromers compossed of at least four subunits surrounding an inner
water filled pore (see Figure 5.1). Although a type of ligand-gated ionotropic
receptor, NMDARs exhibit unique properties that distinguish them from other types
of ionotropic receptors. First, extracellular Mg
2+
creates a voltage-dependent block
in the channel that is only removed when the membrane is depolarized allowing the
influx of Ca
2+
and Na
+
into the cell. This mechanism confers NMDARs the capacity
to act as molecular coincidence detectors in that maximal ion flow is only possible
when pre and postsynaptic cells are active simultaneously. Second, NMDAR
channels are highly permeable to Ca
2+
which contributes to a cascade of intracellular
changes that are thought to be responsible for the induction of long-lasting
modifications in the cell such as LTP and LTD. Third, opening of the NMDAR
channel depends on the presence not only of glutamate, but also glycine as a
cofactor. Moreover, NMDAR channel properties are determined by its subunit
composition creating a large variation in NMDAR functionality.

To date, there are three main NMDAR subunits that have been identified: NR1, a
family of NR2 subtypes (NR2A, -2B, -2C and -2D) and two NR3 subunits (NR3A
and NR3B) (Hollman, 1999). Functional receptors include assemblies of both NR1
and NR2 subunits which bind glycine and glutamate, respectively. More importantly,
it is thought that NR2 subunits are critical in modulating NMDAR functionality that
determine important properties of the receptor including single-channel conductance,
glutamate affinity, deactivation time, sensitivity to Mg
2+
, receptor gating and other
125
important functional and pharmacological properties (Brickley, Farrant, Swanson, &
Cull-Candy, 2001; Misra, Brickley, Farrant, & Cull-Candy, 2000; Stern, Cik,
Colquhoun, & Stephenson, 1994; Wyllie, Behe, & Colquhoun, 1998). Accordingly,
NR2 subunits have been proposed to play an essential role in the induction of long
term changes (Bear & Abraham, 1996; Huang et al., 2001; H. K. Lee, Kameyama,
Huganir, & Bear, 1998; Rossi et al., 2002) thought to be linked to learning and
memory.

In the cerebellum, expression of NMDARs has been observed in both the cerebellar
cortex and deep nuclei (Akazawa, Shigemoto, Bessho, Nakanishi, & Mizuno, 1994;
Audinat, Gahwiler, & Knopfel, 1992). Cerebellar granule cells, for instance,
predominantly express different complements of NMDAR subunits NR2A, 2B and
2C. Moreover, deep nuclei have been found to express subunits NR2A, NR2B,
NR2D and little NR2C (Audinat, Knopfel, & Gahwiler, 1990; Watanabe, Mishina, &
Inoue, 1994). As for Purkinje cells, evidence is still inconclusive as to whether
expression of NMDAR exists in these cells. In situ hybridization studies found that
NR1 is expressed in Purkinje cells whereas NR2 subunits seem to not be expressed
in them (Akasawa et al., 1994; Watanabe, Mishina & Inoue, 1994). Other studies
using pharmacological and electrophysiological techniques have failed to find
evidence of NMDAR functionality in Purkinje cells and thus are in conflict with data
reporting that NMDARs are expressed in these cells (Audinat, Gahwiler & Knopfel,
1992; Audinat, Knopfel, Gahwiler, 1990). However, it has been proposed that these
126
findings can be reconciled by the fact that functional receptors require the
coexpression of both NR1 and NR2 subunits (G. Chen & Steinmetz, 2000).

127

Figure 5.1 Schematic illustration of the NMDA receptor. The receptor contains a cation
channel permeable to Na
+
and Ca
2+
and blocked in a voltage-dependant fashion by Mg
2+
.
PCP and MK-801 block the NMDA channel whereas the competitive antagonist AP5 and
CPP bind to the glutamate site. (From Cooper, Bloom and Roth 1996)

128
In an attempt to associate NMDAR function with memory processes, many studies
have investigated the effects of NMDAR manipulation on the acquisition and
retention of different learning tasks. Engineering of mutant mice in which the
NMDAR gene is ablated in CA3 pryramidal cells of adult mice revealed an
impairment in associated memory recall when tested in a Morris water maze task
(Nakazawa et al., 2002). Moreover, Caramanos & Shapiro (1994) found that
peripheral administrations of the non-competitive NMDAR blocker MK-801and
ventricular infusions of d-2-amino-5-phosphonovaleric acid (AP5), a competitive
NMDA antagonist, impair acquisition of spatial working memory and reference
memory in rats tested in a radial maze task. Similarly, various studies have shown
that intra-amygdala and ventricular infusions of APV block acquisition of auditory
and contextual fear conditioning while having no effect on expression of the
response (Campeau, Miserendino, & Davis, 1992; Kim, DeCola, Landeira-
Fernandez, & Fanselow, 1991; Miserendino, Sananes, Melia, & Davis, 1990).

The involvement of NMDARs has also been demonstrated in eyeblink conditioning.
Data using gene knockout techniques have shown that mice lacking subunit NR2A
are much slower at acquiring eyeblink conditioning compared to wild-type yet can
achieve asymptotic levels of performance just as controls (Kishimoto et al., 1997).
Also, systemic administrations of the NMDA antagonists dizocilpine (MK-801),
phencyclidine (PCP) and CGP-39551 retarded conditioning of the eyeblink response
in both rabbits and rats (Robinson & Crawley, 1993; Servatius & Shors, 1996; L. T.
129
Thompson & Disterhoft, 1997) without affecting retention of the learned response.
Interestingly, infusions of memantine, a NMDA antagonist with higher affinity for
the cerebellum than the forebrain was also found to cause deficits in eyeblink
conditioning when tested in humans (Schugens et al., 1997). Chen and Steinmetz
further (2000) showed that cerebellar infusions of NMDA receptor antagonist AP5
into the interpositus nucleus severely attenuate the acquisition of conditioned
eyeblink responses.

NMDARs have been more recently implicated in extinction of conditioned eyeblink
responses. Scavio et al. (1992) reported that post-training intravenous injections of
the NMDA receptor antagonist ketamine decreased the rate of extinction. Kehoe et
al. (1996) further elaborated the involvement of NMDA receptors by demonstrating
that pre-training injections of MK-801 completely prevent eyeblink extinction (but
see Takatsuki, Kawahara, Takehara, Kishimoto, & Kirino, 2001). Rabbits were
administered MK-801 or saline prior to six days of extinction during which MK-801
treated animals expressed virtually no CRs. However, when MK-801 administration
was suspended, CRs reappeared immediately and showed no evidence of the
previous extinction treatment. Likewise, Thompson and Disterhoft (1997) showed
that injections of high doses of MK-801 or another NMDA receptor antagonist
(phencyclidine) failed to affect the expression of trace or delay CRs, yet completely
prevented extinction.

130
All together these studies suggest that NMDARs are essentially involved in a wide
array of learning and memory mechanisms including extinction of conditioned
eyeblink responses. However, while the role of the NMDAR in acquisition of
eyeblink conditioning has been directly investigated in the brain, in eyeblink
extinction relevant substances have only been administered systemically so it is not
possible to localize their effects on brain structures. We reasoned that if extinction
involved long-term neuronal changes in the interpositus that require NMDARs,
blockage of NMDAR functioning would affect extinction of the conditioned
response. Here we used intra-cerebellar infusions targeting the IP of the two
competitive NMDA antagonist AP5 and 3-(RS)-2-carboxypiperazin-4-
yl)propyl-1-phosphonic acid (CPP). Each of these blockers have been found to have
different effects on the induction of LTP and LTD (see discussion section) given
their differential affinity to each the NR2 receptor subunits (Hrabetova & Sacktor,
1997). Thus, they were used in an effort to better understand the role that NMDAR
dependant long-term changes play in extinction of the conditioned response.

Methods
Subjects:
Fifteen and fourteen male New Zealand White rabbits weighing between 2.2-3.0 kg
at the time of surgery were used in the AP5 inactivation and CPP experiment,
respectively. Animals were housed individually and maintained on a 12 hours
light/dark cycle with ad-lib access to water and food.
131
Rabbits in the AP5 group received infusions that targeted the IP consisting of a
concentration of 2.5µg/µl. Animals in the CPP group were infused with a
concentration of the drug consisting of 0.07µg/µl (see behavioral training procedure
for volumes).

Surgery:
All animals were anesthetized with subcutaneous injections of ketamine (60mg/kg)
and xylazine (8mg/kg) and placed on a stereotaxic frame with a constant flow of
1.5%-2% of halothane in oxygen throughout the length of the surgical procedure.
Under asceptic surgical conditions an anterior to posterior incision was made in the
scalp in order to retract the skin and periostium and expose the skull. A small hole of
approximately 1.5mm in diameter was drilled through the bone at the
anterior/posterior and laterla/medial coordinates of the target structure. A guide
cannulae (26 gauge) 25mm in length was implanted into the target nuclei with
bregma 1.5mm dorsal to lambda. The guide cannulae was lowered with an insulated
stainless steel stylet (Elephant brand 000 insect pin) inserted and extending 1.5mm
below the tip. The outer cannulae tip was sharpened in order to minimize damage to
brain tissue during its implantation. These cannula assemblies were lowered slowly
with a stereotaxic manipulator that targeted the following coordinates based on the
stereotaxic atlas of McBride and Klemm (from lambda): 0.7mm anterior, 5.1mm
lateral and 14.5mm ventral. Subsequently four holes were drilled to fit surgical
stainless steel screws that anchored a headstage constructed from dental acrylic. A
132
plexiglas mount built to hold a minitorque potentiometer during behavioral training
was also cemented to the headstage. Skin surrounding the dental acrylic was
asceptically cleaned with antibiotic ointment (iodine). Animals were allowed to
recover for a period of 1week before the beginning of behavioral training. During the
first two post-operative days animals were given two daily injections of analgesic
(buprenorphine hydrochloride) and were monitored throughout the length of their
recovery. All procedures and animal care were approved by the USC Institutional
Animal Care and Use Committee in accordance with NIH guidelines.

Apparatus:
Animals were habituated and trained in a padded pexiglas restraint and placed into a
behavioral recording chamber. During training nictitating membrane (NM)
movements were measured with a minitorque potentiometer temporarily attached to
the animals headstage. Voltage changes due to movement of the NM were measured
via mechanical movement of a bar attached to the potentiometer and connected by a
thread lead hook to a suture loop implanted in the left NM. The voltage signal was
stored and recorded on a computer using custom software written in Forth. The
software also controlled delivery of the stimuli during training. Data was
subsequently analyzed offline. Infusions of both AP5 and CPP were delivered to the
targeted structure using a 10µl syringe (Hamilton instruments). Syringes were
connected to a polyethelene tube attached to an injection cannula (31 gauge) 26.5mm
133
in length. Drug was delivered by compression of the syringe using an infusion pump
(Harvard Apparatus).

Behavioral Training Procedures:
The same behavioral procedure described in Chapter 1 were used with the following
exceptions:
Phase II: Following acquisition animals received 4 daily sessions of tone alone
training. Infusions were administered by removal of the inner stylet and insertion
through the guide cannula of the injection cannula. Infusion procedures varied
according to the drug used due to differences in duration and potency of the agents
infused. Drug infusions of AP5 were administered 5 minutes prior to extinction
training and continued for the total duration of the session at a rate of 3.8ul/h. CPP
was infused 5 minutes before the beginning of extinction training and continued to
be delivered for the total duration of the session at a rate of 2.7ul/h. Infusions were
discontinued after day 4 of extinction.
Phase III: Extinction training without drug infusions were given to rabbits for 4
consecutive days (day 5-8 of extinction training) or until CR levels dropped to 15%
or less.
Phase IV: In order to measure the ability of animals to reacquire the response, the
association was presented again to animals on the day following the conclusion of
extinction training.
134
Muscimol test: Following completion of all training sessions, animals were infused
with muscimol (0.01ul of a 0.01M solution) and were then given several trials of CS-
US pairings to confirm accurate cannulae placement. Performance of more than 10%
CRs during 2 consecutive blocks was considered to indicate an ineffective cannula
placement and exclusion of the animal from further analysis.

Histology:
Following training an insulated electrode with 250µ m of tip exposure was lowered
through the guide cannula. A 1µ A current was subsequently passed through the
electrode for 10 seconds in order to create a marking lesion at the site of injection.
Animals were then given an intravenous overdose of sodium pentobarbital and
perfused intracardially with saline and 10% formaline. Brains were then extracted
and preserved for later histological reconstruction in 10% formalin for a minimum of
3 days. Brains were then embedded in an albumin and gelatin mixture, sectioned at
80µ m and mounted for later staining. Mounted sections were nissl stained with
Cresyl violet and counterstained with Prussian blue to label iron deposits left by the
tip of the electrode at the time of the marking lesion. Injection sites were then
recorded using a dissecting microscope.

135
Results
Infusions of AP5 targeting the IP:
Of the fifteen animals that began the experiment, four in the experimental group
were excluded from further analyses. Two of the animals not included failed the
muscimol test indicating an ineffective cannulae placement. Later histological
reconstruction of cannulae locations indicated misplacement close to the white
matter or in close proximity to the paraflocculus. The other two rabbits developed
post-surgery complications during the course of the experiment. In total, data shown
is for 5 animals in the AP5 group and 6 rabbits in the control group.

Figure 5.2 depicts the mean percentage CR for both groups during each training day
of acquisition and extinction. Rabbits in both groups acquired the conditioned
response with no significant differences in performance, F(1,9) = 0.62, p< 0.810, and
met learning criteria for Phase I before entering the extinction phase of the
experiment (average CR% on the last day of acquisition: AP5= 89.9% ± 3.2;
controls= 88% ± 2.9). As expected, there was a significant day effect consistent with
a gradual increase in CR frequency indicative of learning of the association, F(3,
27)= 87.3, p= 0.00 (refer to figure 5.3a for overall group performances).

During Phase II of training (extinction days 1-4) infusions of AP5 targeting the IP
caused a significantly higher CR loss compared to performance of controls,
indicative of enhancement of extinction during treatment, F(1, 9) = 8.2, p= 0.018.
136
These responses declined gradually during the session as it would be expected during
tone alone training. On average, experimental animals showed CR levels of 16% ±
10.3 SEM, whereas controls CR frequencies were much higher throughout Phase II
(M= 57% ± 9.4 SEM). By the last day of this phase (day 4), average CR performance
of the AP5 group was down to 7.8% ± 9.9 SEM. In contrast, control animals showed
a less pronounced CR loss at the end of Phase II of extinction training (M= 28.5% ±
0.09 SEM. Moreover, a significant day effect was found for the control group,
F(3,15) = 6.4, p< 0.010, but not for the AP5 group, F(3, 12) = 3.2, p< 0.06; most
likely the \result of a basement effect. Furthermore Figure 5.2 shows that infusions
did not interfere with response performance. Indeed, a block by block analyses of CR
frequency during day 1 of AP5 infusion revealed that animals in the experimental
group showed on average 46% CRs on block 1 and that responses gradually
decreased throughout the session.

Continuation of extinction training without infusions of AP5 (Phase III) revealed that
animals in the experimental group still displayed low CR levels during the first
session (extinction day 5: M= 9.8% ± 6.9 SEM). These low CR frequency levels
were evident during subsequent extinction sessions and maintained until the last day
of tone alone training reaching almost baseline levels by the last extinction session
(M= 1.6% ± 2 SEM). Although overall CR levels were slightly higher for controls
(M=11.4 ± 3.8 SEM) compared to the experimental group (M=7.5% ± 4.2 SEM), no
significant differences were found between both groups, F(1,9) = 0.47, p< 0.5.
137
CR performance during reacquisition training is shown in Figure 5.3b. After almost
complete behavioral extinction of the CR, experimental animals were able to
reacquire the CS-US association at levels that were no different from controls, F(1,9)
= 0.063, p< 0.8.

138
a)

0
10
20
30
40
50
60
70
80
90
100
acq1 acq2 acq3 acq4 ext1 ext2 ext3 ext4 ext5 ext6 ext7 ext8
Training Day
CR%
AP5
C ONTROL

b)
Infusion day 1
AP5
0
10
20
30
40
50
60
70
80
90
100
BLOCK
1
BLOCK
2
BLOCK
3
BLOCK
4
BLOCK
5
BLOCK
6
BLOCK
7
BLOCK
8
BLOCK
9
BLOCK
10
CR%

Figure 5.2 a) Mean (±SEM) percentage of conditioned responses for both groups during
each day of training. Animals in the experimental group received infusions of AP5 targeting
the IP on extinction sessions 1-4. Starting on day 5 of extinction animals were trained
without the drug treatment. Note that animals in the AP5 group showed a higher rate of
extinction compared to the control group b) Average CR% performance of animals in the
experimental group during each block in the session (Extinction day 1) . Note that infusions
of AP5 do not appear to interfere with response production.

139
a)

0
10
20
30
40
50
60
70
80
ACQUISITION EXT-DRUG EXT-NO DRUG
Treatment
CR%
AP5
CON TROL

b)

0
10
20
30
40
50
60
70
80
90
100
Group
CR%
AP5
CONTROL

Figure 5.3 a) Mean CR (±SEM) percentages for all groups during each treatment phase. All
animals acquired the association at similar levels. Admininstration of AP5 significantly
enhanced extinction during the first 4 days of tone alone training (EXT-DRUG).
Continuation of tone alone training without drug infusions (EXT-NO DRUG) revealed that
animals still maintained baseline levels of performance. b) Mean CR (±SEM) percentages
for all groups during the reacquisition session. Experimental animals were able to reacquire
the association at levels similar to controls.
140
Effects of AP5 infusions targeting the IP on timing of the CR were analyzed during
and after drug treatment. Figure 5.4 a shows CR onset latencies across training days.
Although there was a significant group effect on day 3 of extinction, F(1, 9) = 17.6,
p< 0.002, overall CR onsets during extinction training with AP5 (Phase II) were no
different between both groups, F(1,9) = 0.251, p< 0.63. In addition, no Group x Day
interaction was found during this phase of training, F(3, 27) = 0.78, p< 0.51.
Removal of infusions during subsequent extinction sessions (Phase III) also showed
no differences in CR onset of experimental animals compared to controls, F(1, 9) =
0.473, p< 0.5, and no significant day x group interactions, F(3, 27) = 0.65, p< 0.58.
Conversely, analyses of CR onsets during the reacquisition phase revealed that IP
infusions of AP5 resulted in onsets of the CR that were significantly longer (M=
204.8ms ± 9.1 SEM) than those of controls (M= 153ms ± 8.33), F(1, 9 ) = 17.5, p<
0.002 (see Figure 5.4b for average onset latencies during each phase). Figure 5.5a
depicts the average CR peak latencies for both groups across extinction training. No
differences were observed between the groups during extinction sessions with AP5
infusions (extinction days 1-4), F(1, 9) = 0.29, p< 0.6, and subsequent extinction
sessions with no AP5 administration, F(1, 9) = 1.7, p< 0.22. Similarly, reintroduction
of the CS-US association resulted in peak latencies for experimental animals that
were no different than those of controls, F(1, 9) = 3.6, p< 0.09.

141
a)

0
50
100
150
200
250
ex1-D ex2-D ex3-D ex4-D ex5 ex6 ex7 ex8
Extinction day
C R Onse t (m s )
CONTROL
AP5

b)

0
50
100
150
200
250
Ext-D E xt Reacq
Phase
CR Onset (ms)
CONTROL
AP5

Figure 5.4 Mean CR onset latencies (±SEM) within the 250ms ISI. (a) Average time of
response onset during each extinction session. Infusions were given during the first 4 days of
extinction and were stopped on day 5 (b) Average time of response onset during each
treatment phase. Onsets were longer for the AP5 group during the reacquisition phase of the
experiment (Reacq). Ext-D: extinction training with AP5 infusions; Ext: extinction training
without drug administrations.

142
a)

0
50
100
150
200
250
ex1-D ex2-D ex3-D ex4-D ex5 ex6 ex7 ex8
Extinct ion Day
CR Peak (ms)
CONTR OL
AP5

b)

0
50
100
150
200
250
E xt-D Ext Reacq
Phase
CR Peak(ms)
CONTROL
AP5

Figure 5.5 Mean CR peak latencies (±SEM) within the 250ms Inter-Stimulus Interval (ISI).
(a) Average time of peak response for all groups during each extinction day. Infusions were
administered during the first 4 days of extinction training. (b) Average time of peak response
during each treatment condition. ). Ext-D: extinction training with AP5 infusions; Ext:
extinction training without drug administrations; Reacq: Reacquisition session..
143

Figure 5.6 Histological recontruction of cannulae placements targeting the IP. Numeral
above plates represent distance in millimeters rostral from lambda.

144
Infusions of CPP targeting the IP:
Of the animals included in the CPP experiment, all but one animal were included in
the experiment for further analyses. The animal excluded developed post-surgery
complications during training. Final statistical analyses was done on 7 experimental
animals and 6 controls. Figure 5.7 shows performance of both the CPP and control
group across training days. By the end of acquisition training (day 4) both groups
were performing at asymptotic levels (M= 89.5% ± 2.1 SEM) and attained similar
levels of learning before entering the extinction phase of the study, F(1,11) = 0.61,
p< 0.45. There was also a significant day effect indicating learning throughout
acquisition training, F(3, 33) = 39.1, p= 0.00.

Figure 5.7 illustrates CR percentages of animals infused with CPP and controls
during each day of training. During Phase II of the study, animals receiving infusions
of CPP showed lower CR frequencies (M= 34.5% ± 11.9 SEM) than controls (M=
57% ± 12.8 SEM). However there was no group effect found indicating that animals
in both groups showed similar extinction levels in spite of drug infusions, F(1, 11)
=1.6, p< 0.227. There was a significant day effect found during this phase with both
groups showing declining levels of CRs during extinction sessions typical of tone
alone training, F(3, 33) = 7.58, p< 0.001. Continuation of extinction training without
CPP infusions (Phase III) revealed no differences in CR frequencies between the
groups, F(1, 11) = 0.038, p< 0.85 (see Figure 5.8 for overall performances during
each phase of the study). Figure 5.8 depicts CR performance during reintroduction of
145
the CS-US association. Animals in the experimental group were able to reacquire the
association and showed response frequencies that were no different from those of the
control group, F(1, 11) = 0.083, p< 0.778.

146

0
10
20
30
40
50
60
70
80
90
100
acq1
acq2
acq3
acq4
ex1
ex2
ex3
ex4
ex5
ex6
ex7
ex8
training day
CR%
CP P
CONTROL

Figure 5.7 Mean (±SEM) percentage of conditioned responses for both groups during each
day of training. Animals in the experimental group received infusions of AP5 targeting the
IP on extinction sessions 1-4. Starting on day 5 of extinction animals were trained without
the drug treatment. Note that animals in the CPP group extinguished at a rate that was not
significantly different from controls.
147

0
10
20
30
40
50
60
70
80
90
100
Acquisition Ext-Drug Ext Reacquistion
Phase
CR%
CP P
CONTROL

Figure 5.8 Mean CR (±SEM) percentages for all groups during each treatment phase. All
animals acquired the association at similar levels. Admininstration of CPP during the first 4
days of tone alone training had no effect on extinction (EXT-DRUG). Continuation of tone
alone training without drug infusions (EXT-NO DRUG) revealed that animals given CPP
extiguished the response just as controls. During reacquisition, experimental animals were
able to reacquire the association at levels similar to controls.

148
Changes in CR timing due to CPP infusions were analyzed during and after drug
treatment. Figure 5.9a shows CR onset latencies across training days. Infusions of
CPP during extinction training (Phase II) did not cause any significant changes on
onset of the CR compared to controls, F(1, 11) = 0.04, p< 0.851. Similarly, no
significant differences were observed after infusions were stopped, F(1, 11) = 0.015,
p< 0.905. Figure 5.9b shows CR onset during reacquisition training. Animals in the
CPP group showed onset latencies that were significantly longer (M= 192.3ms ± 9.6
SEM) compared to controls (M=139ms ± 10.4 SEM), F(1, 11) =14.09, p< 0.003.
Figure 5.10 illustrates the overall CR peak latencies across extinction training and
reacquisition. There were no significant differences found between groups during
CPP infusions, F(1, 11) = 0.036, p< 0.85, and during extinction with no drug
treatment, F(1, 11) = 0.20, p< 0.66. However, during reacquisition, peak latencies for
experimental animals were longer (M= 241ms ± 6.3 SEM) compared to controls (M=
205.9ms ± 6.8 SEM). This difference proved to be significant, F(1, 11) = 14.8, p<
0.003.

149
a)
0
50
100
150
200
250
ex1-D ex2-D ex3-D ex4-D ex5 ex6 ex7 ex8
session
onset (ms)
CON TROL
CP P

b)
0
50
100
150
200
250
Ext-D Ext Reacq
Phase
Onset (ms)
CONTROL
CPP

Figure 5.9 Mean CR onset latencies (±SEM) within the 250ms ISI. (a) Average time of
response onset during each extinction session. Infusions were given during the first 4 days of
extinction and were stopped on day 5 (b) Average time of response onset during each
treatment phase. Onsets were longer for the CPP group during the reacquisition phase of the
experiment (Reacq). Ext-D: extinction training with AP5 infusions; Ext: extinction training
without drug administrations.

150
a)

0
50
100
150
200
250
ex1-D ex2-D ex3-D ex4-D ex5 ex6 ex7 ex8
session
Peak (ms)
CONTROL
CPP

b)

0
50
100
150
200
250
300
Ext-D E xt R eacq
Phase
peak (ms)
C ONTROL
CP P

Figure 5.10 Mean CR peak latencies (±SEM) within the 250ms Inter-Stimulus Interval
(ISI). (a) Average time of peak response for all groups during each extinction day. Infusions
were administered during the first 4 days of extinction training. (b) Average time of peak
response during each treatment condition. ). Ext-D: extinction training with CPP infusions;
Ext: extinction training without drug administrations. Note that peak latencies of
experimental animals were longer during the reacquisition phase of the experiment (Reacq).
151

Figure 5.11 Histological reconstruction of cannulae placements targeting the interpositus.
152
Discussion
The results of the experiments described above can be summarized as follows: 1) IP
infusions of the NMDA receptor antagonist AP5 significantly facilitate extinction of
the acquired response 2) Although IP infusions of the NMDA antagonist CPP seem
to enhance extinction slightly, this effect is not significant compared to controls 3)
Administration of both AP5 and CPP during extinction training have significant
effects on timing of the CR once the CS-US association has been reintroduced.

Experimental evidence using systemic injections has shown that blockage of NMDA
receptor functioning can result in facilitation of a learning task. Scavio and collegues
(1992) found that systemic infusions of the NMDA antagonist ketamine accelerate
acquisition of the eyeblink response. Also, Mondadori and Weiskrantz (1993) trained
animals to a passive avoidance task where stepping down from a platform with all
four feet on a grid would result in delivery of a shock. They found that animals given
intraperitonial injections of the NMDA receptor blockers CGP 37849 and MK 801
showed much longer step-down latencies compared to controls, indicating that
NMDA receptor blockage facilitated acquisition of the behavioral task. However, it
is important to note that these studies should be interpreted with caution given the
mode of administration which does not permit determination of a specific site of
action. Moreover, the observed behavioral effects are most likely the result of action
of the infused drug on more than one brain structure.

153
Interestingly, certain NMDA blockers have been found to render a different effect
depending on the learning task. In tasks that require passive avoidance for instance,
it has been found that although NMDA receptor blockers facilitate step-down passive
avoidance the same blockers impair acquisition of step-through dark avoidance
(Mondadori & Weiskrantz, 1993). In eyeblink conditioning, Scavio et al. (1992)
found that while ketamine accelerated acquisition, administration of the drug during
extinction training actually retarded extinction of the response. Similarly, Chen and
Steinmetz (2000) showed that cerebellar infusions of NMDA receptor antagonist
AP5 into the IP nucleus severely attenuate the acquisition of conditioned eyeblink
responses while in this study we found that IP infusions of AP5 result in an
enhancement of extinction. Based on these results it seems likely that the effects of
an NMDA receptor blockade might vary depending on the involvement of
glutaminergic activity that the task requires. Furthermore, it would seem that NMDA
receptor antagonists facilitate and impair learning by acting on different underlying
processes. Supporting this view are data demonstrating that steroids can reverse the
facilitating effects of NMDA blockers in step-down passive avoidance whereas the
negative effects on acquisition of step-through dark avoidance are steroid-insensitive
(Mondadori & Weiskrantz, 1993). This would indicate that NMDA antagonists
improve learning and memory through mechanisms that are different from those
through which they impair it.

154
Also of interest, is the different effect on extinction that resulted from infusions of
AP5 and CPP. It has been reported that AP5 and CPP affect NMDA receptor kinetics
differently and are therefore not equally efficient in blocking the induction of LTP
and LTD (Hrabetova & Sacktor, 1997). In vivo, for instance, CPP has been found to
be unable to block associative LTD in the hippocampus (Christie & Abraham, 1992).
AP5, on the other hand, has been demonstrated to block both LTP and LTD in a
wide array of brain areas such as the hippocampus, neocortex and visual cortex
(Hrabetova & Sacktor, 1997; Castro-Alamancos, Donoghue, & Connors, 1995;
Kirkwood & Bear, 1994; Kirkwood, Dudek, Gold, Aizenman, & Bear, 1993). This
differential effect on LTP and LTD is mainly due on the particular binding properties
of each of the agents infused. CPP binds differentially to specific NMDA receptor
combinations (Beaton, Stemsrud, & Monaghan, 1992; Buller et al., 1994; Monaghan,
Olverman, Nguyen, Watkins, & Cotman, 1988), with high affinity for receptors
containing NR1 coupled with NR2A and NR2B subunits that subserve LTP and
subtantially low affinity to NMDA receptors containing NR2C and NR2D that
subserve LTD (Hrabetova & Sacktor, 1997; Hrabetova et al., 2000) but see Massey
et al. 2004. On the other hand, AP5 has equal binding affinity to subunits NR2A,
NR2B and NR2D. The finding that CPP has no effect on extinction but AP5 does is
therefore important because it suggests that extinction most likely recruits unique
cellular changes and the activation of specific NMDA receptor subtypes that are
different from those blocked by CPP administration.

155
An important issue addressed in this study is the direct determination of a site in the
brain where NMDA functioning is importantly involved in mechanisms that lead to
extinction. Previously, NMDA receptor blockers have only been administered
sistemically prohibiting the identification of a site of action in the brain. Here, we
administered infusions of the two NMDA antagonists that directly targeted the IP
and have therefore identified the cerebellum as an important site of glutaminergic
influence on extinction. Moreover, Krupa et al. (1993) showed that injections of
muscimol, using similar cannulae placements as the ones used in this study, did not
spread outside the cerebellum but rather were localized in the IP area with little
diffusion to portions of the overlying cerebellar cortex. Although we used a larger
volume of the infused agents, rates of administration were significantly slower and
so probably spread into similar areas as the ones identified in Krupas study.
Accordingly, it seems that the IP or the cerebellar cortex -or both, could be possible
candidates for the location of NMDA receptor blockage action. Current models of
eyeblink conditioning have proposed both the IP and cerebellar cortex to be
importantly involved in the extinction of these responses (see previous chapters and
Medina & Mauk, 2002 (L. Chen, Bao, Lockard, Kim, & Thompson, 1996; J. F.
Medina et al., 2002; Perrett & Mauk, 1995). It is certainly possible that the effects of
NMDA blockage on timing of the conditioned response observed during
reacquisition (see Figures 5.4b, 5.5b, 5.9b and 5.10b) were the result of the infused
NMDA receptor blocker affecting cortical areas in the cerebellum.

156
Of concern is the question of whether the facilitation of extinction observed in this
study was not caused by the effects of AP5 on learning processes but rather the result
of impairment of performance caused by the infused agent. The fact that throughout
post-infusion training experimental animals maintained baseline levels of CRs makes
the performance deficit explanation very unlikely. Certainly, if this was the case
animals would have shown a significant increase in responding once drug
administrations were stopped. Moreover, reacquisition training demonstrated that
these animals were able to reacquire the association just as controls further indicating
that infusions of AP5 did not cause any detrimental effects on performance of the
CR. It should be noted however, that these results should be conservatively
interpreted in conjunction with findings showing that AP5 seems to have a transitory
effect on CR production in some well-trained animals (Chen & Steinmetz, 2000).
Nonetheless, the authors reported that this effect on CR production was not observed
during the first and last days of infusion. Furthermore, no overall significant effects
were found between the groups during the infusion phase of the experiment.

Specifically what changes are at play in extinction remains to be determined. One
possibility is mechanisms affected by AP5 but not CPP namely, LTD or the activity
of the NR2C receptor subunit. It would be interesting to investigate the effects on
extinction of a NMDA receptor antagonist with high affinity to subunits NR2C and
NR2D which have been found to subserve LTD. Until recently, however, no such
agent had been synthesized. Presently, (2S*,3R*)-1-(phenanthrene-2-carbonyl)
157
piperazine-2,3-dicarboxylic acid, PPDA, has been found to be the most selective
compound for NR2C and NR2D (Feng et al., 2004). Unfortunately, as of this
writing, PPDA is not commercially available and its selectivity for these subunits is
only moderate (D. Monaghan, personal communication).

These data show that AP5 seems to have opposite effects on acquisition and
extinction of eyeblink responses. Furthermore, they suggest that at least in eyeblink
conditioning glutamate related changes are not recruited in the same way during
acquisition and extinction of conditioned responses. All together these findings
indicate that extinction involves mechanisms that recruit functioning of NMDA
receptors. More importantly, they suggest that plastic long-term changes participate
in the extinction of conditioned responses.

158
CHAPTER 6

GENERAL CONCLUSION

____________________________________________________________________

Although the holy grail of relating cellular phenomena to behavior through the
intermediate levels of cerebellar synapses and circuits is not yet at hand, the good
news is that it seems possible. Hansel 2001

The fact that extinction is a highly complex phenomenon cannot be refuted. Indeed,
the more we immerse ourselves in the investigation of this phenomenon the more
aware we become of our shortcomings in understanding its processes and
mechanisms. Ample experimental evidence has yielded information about what
extinction is not that is, the destruction of the original learning. And yet, although in
recent years there has been an upsurge of interest in extinction, the delineation of the
neural basis of this phenomenon remains rather limited. These series of experiments
have been an attempt to answer important fundamental questions of anatomical and
physiological specificy involved in the extinction of conditioned responses. More
importantly, the findings presented here should aid in the understanding of the
neurobiological foundations of extinction and contribute to a more in-depth analysis
of this not well understood phenomenon.

159
One of the main issues explored in the experiments summarized above was the
validity of the response-extinction hypothesis. Results of Chapter 2 confirmed that if
response production is indeed essential in the extinction of conditioned eyelid
responses, corneal proprioception is not an important component. However, there
are at least two explanations for the lack of effect obtained 1) other components of
the response are critical in extinction 2) extinction does not require production of
responses. Support for the latter explanation was found from results of the
experiments in Chapter 3. Indeed, response expression was completely blocked both
during inactivations of the IP and the Red Nucleus yet only blocking IP activity
prevented extinction of the response. These results are consistent with recent
findings demonstrating that extinction can still take place regardless of whether
responding occurs or not (Ouyang & Thomas, 2005; Schwaerzel et al., 2002). Yet, as
mentioned before, Krupa (2003) previously reported that extinction is affected by
preventing the expression of a classically conditioned behavior in the animal. The
most serious problem in Krupas study however, is the lack of extinction observed in
the experimental groups (3 day and 6 day muscimol group). In particular, the group
that received infusions of muscimol for 6 days during tone alone training showed
frequencies of responding higher than 50% up until the last day of extinction without
treatment. Unfortunately, extinction days without drug treatment were discontinued
before significant CR loss could be observed and so it is not certain whether
experimental animals were capable of extinction or not. Given the high rate of
responding observed once infusions were stopped, damage to the nuclei can be ruled
160
out as an explanation for the lack of extinction observed. However, persistence of the
significant motor deficits observed during infusions to the motor nuclei could have
resulted in a change of context that would have prevented behavioral loss of the CR
during tone alone training. Alternatively, even the presence of motor deficits during
extinction training with infusions alone could have provided a very different context
for the animal. The high performance rate observed right after release of the
inactivation could have very well been the result of a renewal effect and not merely
due to blockage of response expression. It therefore seems that firm conclusions
cannot be drawn from the results of the motor nuclei study rendering the response-
expression explanation somewhat unconvincing. In any case, it is still premature to
confirm with certainty the validity of a specific theory of extinction. What is
important however, is that the findings presented here together with recent results of
others support the need for a reevaluation of any particular theory of extinction that
sustains response expression as a premise.

A key and greatly important issue addressed in these experiments is the identification
of neuronal circuits and mechanims recruited in extinction of the learned response.
Inactivations of important nuclei involved in acquisition of the CR (Chapter 3)
demonstrated that the essential memory trace for extinction is most likely afferent to
the red nucleus. In fact, inactivations of the red nucleus did not interfere or prevent
extinction from occurring at all. In marked contrast IP inactivations completely
blocked extinction of the response from occurring without affecting the ability of
161
animals to extinguish the learned response once the inactivation was removed. This
is important because it identifies the localization of an essential structure for
extinction to the cerebellum and specifically suggests that the IP may very well be
part of the essential circuit recruited during extinction.

Results from Chapter 4 provided further insights into the role of the IP in extinction
and further revealed the existence of an important mechanism or component
necessary for the induction of this phenomenon. In particular it was found that
stimulations of the red nucleus can remove the effects of blockage of extinction
caused by functional disruption of the IP. Indeed in spite of effective inactivations of
the interpositus, brain stimulation of the red nucleus resulted in a loss of CR
performance that was no different than controls. This would seem to favor the
response hypothesis yet it is also likely that the stimulation effect was the result of
input activity to other brain structures. In fact, it is possible, based on these results
and the findings of Chapter 3, that stimulation of the red nucleus could have
provided an efficient substitution for a signal of extinction normally conveyed by the
interpositus through a specific brain structure. The most likely candidate would be a
target that is not only capable of providing the teaching signal for extinction to the
cerebellum but one which receives inputs from both the IP and the red nucleus.
Based on electrophysiological and anatomical evidence the IO seems to be a
structure that meets all these requirements. In fact, changes in activity levels of the
IO have not only been found to greatly influence cell activity in the cerebellum but
162
the IP and Red nucleus both send inhibitory inputs to this structure. More
importantly, the sum of these findings indicates that GABAergic input to the IO is
key in driving the signal necessary for the induction of extinction. Furthermore,
based on findings from the IP and Red nucleus inactivation studies it seems that in
the intact brain, the IP would normally provide the essential inhibitory input to the
IO that ultimately results in the signal that drives extinction.

The identification of an inhibitory input to the IO as an important mechanism for the
induction of extinction further indicates that the cerebellar cortex may be importantly
involved in the extinction of conditioned responses. The fact that extinction still
occurred with red nucleus stimulation in spite of inactivation of the IP suggests that
the cerebellar cortex may very well be a site were necessary plastic changes happen
during extinction. Also, it has been previously demonstrated that changes in IO
activity can significantly alter Purkinje cell firing and strenght of synapses in the
cerebellar cortex. As mentioned in previous chapters, parallel fiber LTD requires
paired input from granule cells and climbing fibers whereas LTP has been found to
occur at the parallel fiber synapse in the absence of climbing fiber activation.
Moreover, while parallel fiber-Purkinje cell LTD has been found to be induced
postsynaptically, LTP at this synapse is presynaptically induced. Cerebellar LTD and
LTP at this level would not actually reverse each other, but would rather be an
additional, separate occurrence. Such synaptic properties would support observed
experimental evidence that reject the notion of extinction as a result of unlearning
163
and instead consider it as an active learning process in which the subject learns a new
inhibitory association concurrent with the original one. Under this hypothesis, at
least at some level, the original association is not truly wiped out but is inhibited by
an active mechanism; in this case parallel fiber LTP. This prediction would also
assume that cortical lesions might affect observed savings during reacquisition
training. Unfortunately, data from reacquisition studies using cortical lesions are
rather inconsistent (Woodruff-Pak, Lavond, Logan, Steinmetz, & Thompson, 1993;
C. H. Yeo, Hardiman, & Glickstein, 1984). It would therefore be interesting to study
reacquisition data based on more reliable cerebellar cortex removal techniques.
Probably, studies using pcd mice (see Chapter 1) would prove very helpful in this
quest.

Of great importance, results from Chapter 5 are the first to directly address the
effects of NMDA functioning on the extinction of conditioned eyeblink responses.
Interestingly, IP infusions of the NMDA antagonist AP5 significantly facilitate
extinction whereas CPP has no effect on the ability to extinguish the CR. A
parsimonious explanation for this effect would point towards the differential action
of both antagonists on NR2 subunits and consequently LTP and LTD. That is,
unlike CPP which selectively binds to NR2A and 2B, AP5 has high binding affinity
to all NR2 subunits except NR2C and has been demonstrated to block both LTP and
LTD. It would therefore be likely that the facilitating effects observed by AP5
infusions involves mechanisms not affected by CPP namely, NR2C and NR2D
164
functionality or blockage of LTD. Of particular interest is the fact that AP5 does not
effectively bind to NR2C. It is possible that induction of LTD via NR2C was able to
occur more readily in the absence of competitive activity from receptor subunits
NR2A and NR2B which have been reported to subserve LTP. Furthermore, NR2D
blockage by AP5 might have further facilitated this effect. One of the differences
between NR2C and NR2D subunits relates to the decay of the response or
deactivation time to a glutamate pulse (Cathala, Misra, & Cull-Candy, 2000; Vicini
et al., 1998; Wyllie et al., 1998). In general, NR2C displays much shorter
deactivation times compared to NR2D subunits. How this might contribute to a more
efficient induction of LTD is not clear as of this writing but it is plausible that NR2D
with its slower kinetic properties might result in a slower induction of plastic
changes as well. What is clear from these data is that extinction of eyelid responses
involves mechanisms that recruit long term changes in the cerebellum. Given the
volume of the infusions, these changes could be specifically localized to the IP or the
cerebellar cortex -or both.

Although, plasticity in the cortex seems to be important in extinction, it is possible
that plasticity of IP synapses may also modulate firing in the nuclei and thus CR
expression. Electrophysiological studies have found LTP at mossy fiber-IP synapses
and both LTP and LTD at GABAergic Purkinje cell-IP synapses (Aizenman, Manis,
& Linden, 1998). Assuming that co activation of IO and mossy fiber input to the IP
induces LTP at the mossy fiber-IP synapse, CS alone presentations may weaken this
165
long term potentiation at the synapse so that repeated activation of mossy fibers
becomes less able to depolarize nuclei cells resulting in the disappearance of CRs.
However, it is likely that intrinsic excitability of the IP may remain relatively high
even after degradation of mossy fiber LTP which would mean then that these nuclei
may show changes that are not found in naïve animals even after extinction training.
Such changes may be analogous to residual plasticity suggesting that, as in the
cerebellar cortex, the IP may be one location in the cerebellum where a trace of the
original memory can be found after extinction. Moreover, LTP and LTD at Purkinje
cell-IP synapses may serve a homeostatic role by way of a feedback mechanism
where overall excitability of the IP is regulated. Based on this assumption, as
extinction training proceeds, LTP at parallel fibers begins to override depression of
Purkinje cells so that inhibitory output to the IP is gradually restored, contributing to
the further suppression of CRs. The cerebellar cortex would then help maintain the
IP under threshold levels decreasing the likelihood of CR expression. This prediction
would account for the labile nature of extinction given that excitable associations
seem to be more resistant to degradation. Consequently, any disruption of the
balance established during extinction training would unmask such excitatory
memory traces resulting in CR expression.

Taken together the results presented here suggest that extinction of conditioned
eyelid responses depends on mechanisms that involve cerebellar functioning.
However, unlike acquisition, the cerebellar cortex is capable of supporting extinction
166
independent of IP functioning but only under conditions that engage GABAergic
input to the IO. It appears that under non-experimental conditions, nonetheless, the
IP is the main source of this inhibitory input and is therefore importantly involved in
the induction of extinction. Moreover, plastic changes in the IP, perhaps at a synaptic
level (e.g. mossy fiber-IP synapses), may also be involved in this phenomenon.
Finally, data suggest that extinction of eyelid conditioning may also involve
contribution from extracerebellar areas such as the hippocampus. Indeed, attention
and other changes in contextual cues have been found to significantly influence the
degree of extinction.

Evidently, extinction is a complex phenomenon that cannot simply be explained as
the fading away of acquisition related changes. In an effort to further understand
this phenomenon thoroughly characterized models of learning and memory can
prove to be invaluable tools in this quest. Considering the great advances
accomplished in the area of associative memory at both a behavioral and anatomical
level, use of this knowledge in the investigation of mechanisms of extinction can
prove extremely fruitful. Here, we have addressed previously unexplored issues
essential for the understanding of the neuronal bases of extinction of conditioned
eyelid responses. In this respect, attention must now be turned to a more in-depth
understanding of the relative roles of the interpositus and the cerebellar cortex as
well as the investigation of molecular mechanisms critical in the induction of
extinction.
167
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Core Title Contributions of the cerebellum and neural pathways in the extinction of discrete motor responses

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