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Cancer of the psyche: antenatal maternal stress accentuates functional brain responses and neuronal endangerment during conditioned fear in adult offspring
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Cancer of the psyche: antenatal maternal stress accentuates functional brain responses and neuronal endangerment during conditioned fear in adult offspring
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Content
CANCER OF THE PSYCHE: ANTENATAL MATERNAL STRESS ACCENTUATES
FUNCTIONAL BRAIN RESPONSES AND NEURONAL ENDANGERMENT
DURING CONDITIONED FEAR IN ADULT OFFSPRING
by
Theodore R. Sadler
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
(PATHOBIOLOGY)
May 2009
Copyright 2009 Theodore R. Sadler
ii
Epigraph
“The public is wonderfully tolerant. It forgives everything except genius.”
-- Oscar Wilde
iii
Dedication
You know not why, but you’re compelled.
Your name is known, but not to all.
Your home is here and there.
You’re present, yet invisible.
Life is your struggle.
Hope is what we have.
Our dedication is our drive.
One day the archaic rituals will happily come to pass.
We ask what has been done and pursue what has not.
To families and friends who understand these words, this dissertation I dedicate.
--Theodore R. Sadler
iv
Acknowledgements
Aimless are we, the nascent graduate student, upon the landscape of our work if it were not
for our guides to help us negotiate the dense boscage of our field. As the geography
transformed before us, so did we change with it from a hopeful researcher to an experienced
scientist. Yet, if it was not for the patience of our guides—our mentors, committee
members, faculty, family, and friends that provided us with direction when confused,
support when despondent, and solace when exhausted we would not have succeeded in our
accomplishments to become guides ourselves. And so, as I transformed throughout this
journey, I wish to personally acknowledge those who have graciously guided and supported
me along the way.
The author wishes to express his sincere gratitude and appreciation to Drs David R.
Hinton and Daniel P. Holschneider for their leadership as excellent mentors throughout my
Ph.D. studies and in the assistance in the preparation of this manuscript. Further, I would
like to extend my deepest thanks to Dr. Alan Epstein for introducing me to the Pathology
Department Graduate Program at the USC Keck School of Medicine, to Dr. Gabrielle
Obrocea for introducing me to Dr. Holschneider, and Dr. Lynne Meyer for her kindness
and friendship over the years. To my committee chairman, Dr. Hinton I cannot thank him
enough for his great humor, optimism, and support through this challenging project.
Additionally, I would like to extend my indebtedness to my other committee members Drs
Clive Taylor, Cheng-Ming Chuong, and Peter Conti for posing queries that have enabled me
to reach my goal. I would also like to thank Dr. Ani C. Khodavirdi for her support,
encouragement, and piquant critiques of my writings and ideas. I would be sorely remiss if I
did not also express my gratitude toward my friends both here at USC and afar who have
v
provided me with distractions and entertainment when required. Those who I wish to pay
tribute, in no particular order, are Mr. Gregory Williams, SFC Roderic Haworth MNARNG,
Deputy Michael Wahl, Mr. Edward Krekeler III, Dr. Keith Bayha, Mr. Tejpal Chawla, JD,
AUSA DOJ, and Cpt. Patrick Ross, JD, JAG US ARMY. Lastly, and most importantly, I
would like to thank my parents, David and Nancy Sadler, my brother’s, Nicholas and
Thomas, as well as my cousin Michael Odenbach and his family for all their support and
encouragement throughout the years in addition to being a wonderful source of inspiration.
vi
Table of Contents
Epigraph ii
Dedication iii
Acknowledgements iv
List of Tables viii
List of Figures ix
List of Abbreviations x
Abstract xiii
Chapter I: Introduction 1
Overview 1
Animal Models: Early Life and Adult Emotionality 4
The Neuroanatomy of Fear 7
Death of a Neuron 9
Imaging 11
Hypothesis 12
Chapter II: Antenatal Maternal Stress Alters Functional Brain Responses and
Neuronal Survival during Fear Conditioning in Adult Offspring 13
Abstract 13
Introduction 13
Materials and Methods 16
Results 25
Discussion 36
Conclusions 38
Chapter III: Preliminary Study: Persistent Exposure to Exaggerated Maternal Stress
Response Throughout Gestation Alters Fetal Mass and Nnat Promoter
Methylation In Utero 40
Abstract 40
Introduction 41
Materials and Methods 44
Results 47
Discussion 50
Conclusions 52
Chapter IV: Conclusions and Future Directions 53
vii
Bibliography 56
References 70
Appendices 82
Appendices: Introduction 82
Appendix A: Snap-Frozen Brain Tissue Sections Stored With Desiccant At
Ambient Laboratory Conditions Without Chemical Fixation
Are Resistant To Degradation For A Minimum Of Six Months 83
Appendix B: Provisional United States Patent For Ambient Storage For
Fresh/Frozen Tissue Sections Via Desiccation 99
viii
List of Tables
Table 1: Intra- and intergroup statistical comparisons of behaviors during
conditioned fear training and recall 33
Table 2: MSP Primers for genomic fetal rat brains 47
Table 3: Assay matrix studying the effects of different storage conditions on
brain tissue samples over 6 months. 86
ix
List of Figures
Figure 1: Brain regions and connections implicated in auditory conditioned fear
paradigm 9
Figure 2: Photograph of implantable microbolus infusion pump, MIP 17
Figure 3: Schematic overview illustrating MIP location when implanted in the adult
male rat. 18
Figure 4: Intergroup comparison of pathophysiological responses 27
Figure 5: Intra- and intergroup SPM maps 32
Figure 6: Representative Western blot of free- and phosphorylated-Tau from the
amygdala and ventral hippocampus 34
Figure 7: Representative photomicrographs of immunoflourescent and TUNEL
stains of ventral hippocampus 35
Figure 8: Molecular analysis of NMS and MS animals 36
Figure 9: Maternal and fetal changes in mass throughout psychophysiological
stressors 48
Figure 10: Quantification of TUNEL(+) cells from fetal amygdala and hippocampus
as well as TUNEL stains of fetal hippocampus 49
Figure 11: MSP from fetal brain tissue 50
Figure 12: Chart of normalized protein concentration 91
Figure 13: Image comparison of SDS-PAGE gels stained with silver 88
Figure 14: Western blot for presence of TH 92
Figure 15: Various tissue stains of archived brain sections 95
Figure 16: Front side view of a device of the invention 101
Figure 17: Top view of a device of the invention 102
Figure 18: Side view of a desiccant cartridge of the invention 102
x
List of Abbreviations
3D: Thee dimensions
11-β-HSD-2: 11-β-hydroxysteroid dehydrogenase type 2
ABC: Avidin-Biotinylated-Peroxidase complex
AEC: 3-amino-9-ethylcarbazole
AMYG: Amygdala
BC: Blood corticosterone
CBF: Cerebral blood flow
CBF-TR: Cerebral blood flow-tracer radioactivity
CF: Conditioned fear
CNS: Central nervous system
CON: Control
CR: Conditioned response
CRH: Corticotropin releasing hormone
CS: Conditioned stimulus
DAPI: 4’,6-diamidino-2-phenylindole
DNA: Deoxyribose nucleic acid
DPI: Dots per square inch
FITC: Fluorescein isothiocyannate
FMRI: Function magnetic resonance imaging
GAPDH: Glyceraldehyde-3-phosphate dehydrogenase
GC: Glucocorticoid
xi
GR: Glucocorticoid receptor type II
GFAP: Glial fibillary acidic protein
HE: Hematoxylin and eosin
HPA: Hypothalamic-pituitary-adrenal axis
HPC: Hippocampus
IF: Immunofluorescent
IHC: Immunohistochemistry
IP: Immunoperoxidase
MAP: Microtubule associated protein
MAP5B: Microtubule associate protein-5 –b
MCT: Microcentrifuge tube
MIP: Microbolus infusion pump
MS: Maternal stress/ severe antenatal maternal stress
MSP: Methylation-specific PCR (polymerase chain reaction)
NeuN: Neuronal Nuclei
NFM: Non-fat milk
NMS: No maternal stress
Nnat: Neuronatin
OCT: Optimal cutting temperature (solution)
PBS: Phosphate buffered saline
PBS-T: Phosphate buffered saline-tween
PEB: Protein extraction buffer
PET: Positron emission tomography
xii
PVDF: Polyvinyldifluoride
RT: Room temperature (~20˚C)
SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEM: Standard error of the mean
SFHD: Snap-frozen, heat dried
SPECT: Single-photon emission computed tomography
SPM: Statistical parametric mapping
TBS: Tris-buffered saline
TBS-T: Tris-buffered saline-tween
TH: Tyrosine hydroxylase
TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end-labeling
UR: Un-conditioned response
US: Un-conditioned stimulus
WB: Western blot
xiii
Abstract
Profound, long-term behavioral and physiological consequences have been identified with,
and attributed to prior exposure to severe early life stressors vis-à-vis altered maternal
responses. To date, questions remain unanswered regarding potential changes of both
functional brain activity and molecular expression of progeny that corresponds with known
behavioral and physiological alterations in adulthood. Furthermore, questions regarding
potential molecular changes during gestation, particularly related to neuro-development have
remained elusive. For instance, given the potency of stress hormones, do fœtuses, when
exposed to excess quantities, have an aberrant rate in programmed cell death, or alterations
of epigenetic markers to an important gene related to proper brain and neuron formation?
Here, for the first time, we show localization of functional brain activity and it’s
corresponding molecular profile, programmed cell death, during the conditioned fear
paradigm of adult animals with and without prior history of antenatal maternal stress. Also,
we offer preliminary data that indicates the epigenetic profile of neuronatin, a critical gene
for proper brain and neuron development, was changed from chronic exposure to
gestational stress.
1
Chapter I
Introduction
“Do not seek to follow in the footsteps of the wise. Seek what they sought.”
--Matsuo Basho
Overview
As cancerous cells can invade and erode normal tissue, so too, can invasive thoughts or
feelings germinated in the mind precipitate the deterioration of healthy behavior leading to
psychiatric illness. According to the American Cancer Society for 2008 an estimated 0.47%
of the American population will be diagnosed, while another 0.20% will die from cancer this
year—a trend that continues to decline [1]. This decline, in part, is attributed to a better
understanding of the disease mechanism, which improves prevention and treatment
measures. Conversely, according to a recent report from the Archives of General Psychiatry
6 – 8% of the adult American population (~20 million people) suffer from a severe,
debilitating mental illness—a trend that has remained stable over the years [2]. Though
cancer has become synonymous with treatment and struggle to overcome death, the
development of a severe psychiatric illness can lead to the loss of a familiar, well-loved
personality with the formation of a new, and often times, unfamiliar one. Over the course of
a lifetime, this requires an enormous amount of flexibility for both family and friends in
order to adapt. What causes this? How do illnesses of the mind translate into
pathophysiology of the brain for severe mental illnesses? Is there a common cause to the
development of these disorders? Work has speculated that one primary factor could be
attributed to changes forged during early life development.
2
Superstitions regarding an unborn child’s well-being date back to antiquity, where
maternal thoughts or fears could bring about physical abnormalities in her baby. Such
classically specious associations that have been passed down through the ages include the
formation of a harelip that resulted from a mother startled by a rabbit, or ichthyosis that
developed on a child when the mother bathed in a stream filled with fish [3]. Since the
1950’s however, it has become axiomatic that offspring exposed to severe antenatal maternal
stress (MS) will develop abnormalities, of interest, those affecting emotional processing [3-
12]. MS may act as a double-edged sword, first augmenting the offspring’s normal
development in utero, then second via reduced postnatal maternal care. Specifically, MS first
exposes the developing offspring to the responses of the mother in utero to the
consequences of a severe, chronic stressor(s). Such stressors whether emotional
(fear/anxiety), immunological (illness), or survival (shelter/starvation/thirst), precipitate the
release of a potent class of steroid hormones, specifically glucocorticoids (GC), via the
hypothalamic-pituitary-adrenal (HPA) axis [7, 8, 10, 13, 14].
Readily crossing the cell membrane to participate in genomic and nongenomic roles,
steroid hormones have the capacity to activate signaling pathways and gene expression [14-
18]. These hormones released during severe maternal stress may alter fœtal development by
1.) reducing blood flow to the fœtus, 2.) elevating maternal cortisol (corticosterone in
rodents), or 3.) placental release of corticotropin releasing hormone into the intrauterine
environment [3, 19, 20]. Normally present in the placenta during gestation, 11-β-
hydroxysteroid dehydrogenase type 2 (11-β-HSD-2) coverts GC into an inert metabolite,
which maintains a low GC content in fœtal circulation [20, 21]. However, during the middle
of the second trimester, fœtal brain 11-β-HSD-2 gene expression is silenced and enzymatic
3
levels are acutely reduced for a brief period, allowing an acute surge of GC that enhances the
development of the brain as well as other organs [14, 20, 21]. Clearly, a persistent exposure
to excess GC as a consequence of increased stress hormone levels (i.e., GC) throughout
gestation could pose a threat to the normal maturation of the brain by sabotaging critical
growth signals [3, 22].
Adding humoral insult to developmental injury, the second impact, altered maternal
care poses an additional threat to the neonate. Animal studies have illustrated that two
factors, nursing and grooming, are both significantly diminished after MS [10, 23, 24]. This
decreased care during postnatal period has equally demonstrated its ability to affect the
offspring. It is during this time that the newborn imprints to its mother, and under normal
conditions, forms strong bonds that solidifies neuroprocesses and tempers
neuroendocrinological responses [7, 10, 24].
Indeed, both retrospective and prospective clinical studies have made a convincing
argument correlating devastating psychiatric illnesses, such as mood disorders, depression,
and schizophrenia to prior MS exposure. One ongoing prospective study entitled Project Ice
Storm recruited 150 mothers who were exposed to a severe stressor for over 40 days at
different trimesters. Their study illustrated that children had different Bayley Mental
Development Index (MDI) scores depending on the timing of the stress exposure.
Specifically, children born from mothers exposed to psychological stressors during the first
and second trimesters had lower MDI scores than mothers stressed during the third
trimester [25]. But how and why does this occur?
Well established as the emotional region of the brain, the limbic system has been the
suspected culprit driving these underlying differences [26-31]. As with most psychiatric
4
ailments, a disrupted limbic system often leads to altered emotionality [9, 27-30, 32-35].
However, unlike the discovery of plaques within the brains of Alzheimer’s patients suffering
from dementia, neuroanatomists have failed to find a conclusive pathological marker from
the brains of patients afflicted with debilitating psychological disorders [35, 36]. Only in the
past two decades, with the development of finer tools and techniques, have investigators
begun to more clearly understand the impact MS has on the psychological development of
the offspring.
In addition to the development, and subsequent use of electrophysiological and
pharmacological studies, imaging tools such as functional magnetic resonance imaging
(fMRI) and positron emission tomography (PET) have allowed for the visualization of the
active brain of patients afflicted with diverse mental illnesses. Classically, affected brain
regions have predominately involved the amygdala and hippocampus as well as the
prefrontal and cingulate cortices [26, 28, 30, 33-42]. Due to ethical reasons, human studies
replicating severe stress on expectant mothers to observe their effects on the unborn child
cannot be performed. In lieu of this, various animal models have been developed to examine
the impact of early life stressors and their corresponding clinical responses. The availability
of these animal models allows better molecular analysis with improved insight into how and
why emotional processing is altered.
Animal Models: Early Life and Adult Emotionality
A substantial quantity of literature has shown that MS results in abnormalities in behavior as
well as brain biochemistry and structure in adult life [5, 7, 43-45]. Abnormalities include
aberrant social activity, anxiety, and maternal behavior/care [5, 7, 45, 46], altered levels and
distribution of regulatory neurotransmitters (norepinephrine, dopamine, serotonin,
5
acetycholine) [5, 7, 8, 47], alterations of hormones and receptors of the HPA axis[5, 7, 8, 47],
and reduction in cell size and counts in regions of the limbic system (e.g. amygdala and
hippocampus) [5, 7, 45, 48]. In part limited merely by imagination, several animal models of
early life stressors have been developed to correspond with potential real-life scenarios.
These include exposing dams to the following distressing situations: environment (crowding,
noise, lights, smells), survival (predatory odors or images), physical (restraint), and
uncontrollable/unpredictable (nociceptive foot-shocks). Yet, an elegant paradigm was
introduced in order to evaluate potential modification to emotional processing in adulthood
from early life stressors.
The conditioned fear (CF) paradigm, a method developed to examine altered
emotionality by acute exposure to a negative emotional stimulus, has become utilitarian.
While CF is an animal model of human anxiety and emotion, fear and anxiety must be
differentiated, where fear is a response to a known, external stimuli and anxiety is a negative
feeling toward an unknown, vague internal threat [35, 36]. Whereas the immediate response
to a traumatic event is fear, the long-term outcome of such exposure can be anxiety. Stress
itself results in a state of threatened homeostasis that produces adaptive changes, both
physiological and psychological [49-53]. Substantial evidence indicates that repeated stress
affects the hormonal and behavioral activity of adult animals, including learning and
memory, while it promotes anxious, avoidant traits [27, 54-57]. Chronic stress has been
found to alter baseline and stress-induced responsivity of the HPA axis [58-63]. Recent
evidence indicates that exposure of subjects to MS may impact behavior in adult life, that is
they exhibit increased risk for the development of affective disorders among which are
anxiety disorders, autism, bipolar disorder, and schizophrenia [3, 5, 7, 45, 46]. While it
6
appears unlikely that MS acts as the sole etiological agent in the expression of such disorders,
MS may act as a risk factor for establishing susceptibility in individuals predisposed to
disease by their genes or environment [3, 5-7, 41, 46, 64, 65].
Based on Pavlov’s renowned conditioning experiments, the cue CF paradigm was
developed to study responses to negative emotions [66-69]. Here, this model intimately
mates two dissimilar stimuli in the lateral nucleus of the amygdala to create an associative
memory in a temporal-dependent manner. Traditionally, a benign conditioning stimulus
(CS), for instance a tone, is paired within a few seconds to an often nociceptive
unconditioned stimulus (US), which elicits a robust constellation of psychophysiological
responses (unconditioned response, UR) from the animal. Behaviorally, during this fear
response locomotor activity is suppressed (termed ‘freezing’), which can been readily
monitored and quantified.
Initial Training: Environment 1, Day 1
CS + US UR
Some time after (hours to days) the association is forged in one context, assessment of the
CS fear-memory (anxiety) is evaluated in another. Subsequent re-exposure to the CS, even in
the absence of the US, in a new setting evokes a response with re-suppression of locomotor
activity (conditioned response, CR).
Recall: Environment 2, Day 2
CS CR
Mechanistically and temporally, streams of information are formed and augmented by
various cortical and sub-cortical nodes throughout both the training and recall phases.
7
The Neuroanatomy of Fear
Organisms as primitive as invertebrates can produce a fear response to a threatening
stimulus [27], which in itself is a normal and necessary reaction for survival and evolution.
Over the millennia, the central nervous system has evolved and developed specific neuronal
circuits in the brain to perform this precise task. Where, within the brain, there are circuits
exclusively constructed to form the memory of a threatening event and those to consolidate
the multiple sensory information about the experience. However, when fear and anxiety
perpetuate upon themselves without legitimate external stimulation, pathology can ensue in
the form of psychiatric disorders.
Canonized as the epicenter of emotional processing, the amygdala plays a key role in
the acquisition, consolidation, and formation of CF processing [27, 30, 57, 69]. An almond-
shaped, multinucleated structure within the medial temporal lobes, the amygdala can be
divided into two components [27, 57, 69]. First, the basolateral complex (BLA) comprised of
the lateral (LA), basolateral (BL), as well as basomedical (BM) nuclei and second, the central
(CE) nucleus [27, 57, 69]. However, it is the BLA that intimately mates two dissimilar stimuli
(CS + US) to create an associative memory in a temporal-dependent manner [41]. A variety
of sensory sources are received and integrated within the BLA. For instance, in regard to CF
this includes auditory information (thalamic medial geniculate nucleus and primary auditory
cortex), visual (perirhinal cortex), somatosensory (thalamic posterior intralaminar nucleus,
insular and somatosensory cortex), as well as spatial memory (hippocampal formation,
entorhinal cortex, piriform cortex, and ventral subiculum) [57]. Then, the intra-amygdaloid
circuitry conveys the association to the CE where divergent projections to the hypothalamus
and brain stem mediate fear responses (absence of locomotor activity, tachycardia,
8
hyperpnoea, and glucocorticoid release) [57]. Current data strongly implicates the amygdala
in the control of the responses and expression of emotions, in particular fearful ones, while
the hippocampus stores the memory [28, 57, 69]. Yet, the control and modulation of
emotional stimuli add a layer of complexity.
Different stimuli elicit different emotional response; analogous to a rheostat, there
exists within the brain a sub-system that modulates responses to emotional-provoking
stimuli. Hence, depending on experience and perception, some stimuli will elicit a mild
response, for example seeing a small Gartner snake several feet away on your lawn. While
another, may elicit tremendous distress, such as being inches away from a hungry 20-foot
long anaconda in the Amazonian rainforest. This underlying neuropsychological processing
of emotions is only now being evaluated and understood. Yet, its potential implication in the
etiology of psychiatric disorders is clear. An erosion of this mechanism could lead to
aberrant emotional responses that could lead to the development disease states. From
animal, human lesion, and neuroimaging studies, two distinct systems that comprise this
emotional rheostat are the ventral and dorsal system [33]. Important for how MS can affect
CF processing, the ventral system includes the amygdala, insula, ventral striatum, ventral
anterior cingulate gyrus, and ventral prefrontal cortex [33]. In particular, it is the cingulate
gyrus that helps inhibit limbic responses from the amygdala and prefrontal cortex when
challenged by a negative emotional stressor (Fig. 1).
9
Figure 1. Regions and connections implicated in processing CF. 3D emotional processing centers and streams,
presented in 2D. AC = Association cortex, Am = Amygdala, Au = Auditory cortex, BS = Brain stem, CC =
Cingulate cortex (cingulate gyrus), dH = Dorsal hippocampus, Hy = Hypothalamus, LS = Lateral septum, SS =
Somatosensory cortex, Th = Thalamus, and vH = Ventral hippocampus.
Death of a Neuron
Though dysregulated apoptosis has been a focal area of research in neurodegenerative
disorders, these efforts have not extended to evaluate the effects of psychological stressors
on apoptotic processes in the brain. Programmed cell death (PCD), or apoptosis, is an
energy-dependent mechanism that in a highly coordinated fashion eliminates unwanted cells
to facilitate proper development, tissue homeostasis, or removal of damaged cells by a
dedicated set of gene products [48, 64, 65, 70, 71]. Although initiated by several routes, PCD
generally commences by either extrinsic or intrinsic signals. Extrinsic signals may include
withdrawal of growth hormones, injury from toxins, or receptor-ligand interactions. Intrinsic
signals result from intracellular damage such as DNA damage by free-radical formation and
oxidative damage. A commonly accepted feature of cells undergoing PCD is chromosome
10
condensation with subsequent dissolution of DNA into 50 – 200 base pair units. The DNA
fragments are readily detected on an agrose gel as a ‘ladder’ or by hybridizing small broken
DNA from a tissue sample with a fluorescent-tagged oligonucleotide [13, 71-73].
Incidentally, studies have shown decreases in hippocampal cell numbers in adult
animals subjected to chronic, and more recently, acute stress, however, the role of apoptosis
specifically has not yet been investigated [71, 72, 74, 75]. Questions regarding a possible link
between MS and neuronal apoptosis in the brains of the offspring have had limited
consideration. Recent work suggests that antenatal exposure to high levels of glucocorticoids
[7, 64, 76, 77], cortisol in humans and corticosterone in rodents, can increase susceptibility of
nerve cells to stress-induced cell death [16, 32, 71, 74, 78], but more extensive work is
required.
Lastly, two independent, yet interrelated factors, Tau and GC may determine the
survivability of neurons as well, particularly those within the hippocampus, during negative
stress. First, ubiquitous in the CNS, the microtubule associated protein (MAP) Tau plays a
critical role in neuronal cell polarity, axonal outgrowth, axonal transport, and maintenance of
axonal morphology [79]. Regulated in part by the glycogen synthase kinase 3 (GSK3)
signaling pathway, Tau’s primary function is to stabilize axonal microtubules via
phosphorylation [79, 80]. In pathological states, Tau has been implicated in several
neurodegenerative diseases such as Alzheimer and Pick disease [79, 80]. Most of these
“Tauopathies” have been associated with neuronal cell death. Second, Tau has been
positively correlated with serum GC levels [17, 79-81]. Key to emotional and memory
processing as well as attenuating the HPA response, neurons within the hippocampus
contain a high concentration of GC type II receptors (GR), shown to be diminished with
11
prior exposure to MS [16, 82]. Finally, excessive levels of GC have been found to be
excitotoxic to neurons [14-16, 21]. This form of neurotoxicity has been confirmed by in vivo
and in vitro experiments as well as by clinical studies examining victims of torture where
high basal levels of GC in conjunction with reduced hippocampal volume have been noted
[16-18, 37, 82].
Imaging
A missing link between abnormalities in behavior, biochemistry, and brain structure
attributable to MS has been the ability to monitor neuronal activity in adult offspring during
stress exposure. Specifically, little is known if the brains of animals exposed to MS process
information using different neural circuits from those reared during normal pregnancy. This
is relevant not only for subjects at rest, but also during exposure to an acute stressor, which
typically elicits a characteristic abnormal behavioral response in affected individuals.
One primary problems encountered in the monitoring of neural functions in non-
restrained, freely-moving subjects has been motion artifacts (Holschneider and Maarek, 2004
[83]). Traditional methods of functional neuroimaging, such as PET or fMRI, have been
limited to studies in immobilized subjects, which extinguishes all but the simplest behaviors
[83-87]. Our laboratory has recently developed a method that allows a ‘naturalistic’
assessment of brain function in freely-moving animals, to address the problem of
immobilization. A self-contained implantable, miniature infusion pump (MIP) is used for the
administration of a cerebral blood flow (CBF) radiotracer [83-85, 88-90]. Our method has
been validated acquiring functional brain maps from rats during auditory challenge and while
walking on a rotarod [83-85, 88, 89].
12
Hypothesis
With the advent of our novel high resolution imaging technique that can monitor functional
brain activation in un-tethered, freely-moving small animals and the potential molecular
consequences related to neuronal endangerment from exposure to psychological antenatal
maternal stress, we proposed the following two hypotheses:
First, we hypothesized that normal male adult rats exposed to an auditory CF
paradigm will exhibit increase in regional CBF (rCBF) in the amygdala and ventral
hippocampus during acute recall of the CF cue, a response that will be exaggerated by prior
exposure to chronic MS.
The second concurrent hypothesis is that male adult rats previously exposed to MS
will have abnormal activation of PCD in limbic structures potentially due to excessive GC
and/or Tau upon exposure to an acute stressor (CF). Specifically, MS rats compared to rats
developed and reared under normal conditions will exhibit a greater number of TUNEL-
positive neurons in the ventral hippocampus and amygdala and a PCD profile exhibiting
activation of signaling cascade caspase-3 and –7.
13
Chapter II
Antenatal Maternal Stress Alters Functional Brain Responses And
Neuronal Survival During Fear Conditioning In Adult Offspring
Abstract
Early life stress has been shown to result in altered behavior and stress hormone response in
humans, as well as animals, yet little is know about its effects on functional brain activation.
We present evidence that antenatal maternal stress exposure in rats sets off a cascade of
neural changes that in adult offspring culminates in a state of amygdala hyperresponsivity to
fear conditioned stimuli, inadequate top-down control by medial prefrontal cortical regions,
heightened fear responsivity, exaggerated and prolonged corticosterone release, and
increased levels of the apoptotic markers caspase-3 and -7 as well as brain microtubule-
associated protein Tau. Dysregulation of corticolimbic circuits and increased vulnerability to
programmed cell death may represent risk factors in the future development of anxiety
disorders and associated alterations in cognition and brain structure.
Introduction
It is axiomatic that the behavior, growth, and developmental trajectories are markedly altered
in progeny formerly exposed to severe antenatal maternal stress (MS) [4-7, 9, 11-13, 20, 25,
45-47, 65, 76, 77, 91-94]. Psychological stress of the mother during pregnancy has been
implicated as a potential etiology of anxiety disorders, mood disorders and schizophrenia in
the offspring [5-7, 13]. Debate continues as to whether problems arise during early
formation and maturation of neural circuits (“hardwiring problem”), or if MS exposure
establishes a vulnerability for progressive circuit dysfunction in the face of future stress
challenges (“functional vulnerability”). With regards to the first mechanism, stress during
14
pregnancy is well known to elicit release of a potent class of steroid hormones, specifically
glucocorticoids (GC), via the hypothalamic-pituitary-adrenal (HPA) axis [7, 8, 13, 14].
Readily crossing the cell membrane to participate in genomic and nongenomic roles, steroid
hormones have the capacity to activate signaling pathways and alter gene transcription [14-
18, 37]. These hormones released during severe maternal stress may alter fœtal development
in several ways: 1.) reduction of blood flow to the fœtus, 2.) excess maternal cortisol crossing
the placenta, or 3.) placental release of corticotropin releasing hormone into the intrauterine
environment [3]. Normally at high levels in the placenta during gestation, 11-β-
hydroxysteroid dehydrogenase type 2 (11-β-HSD-2) converts GC into an inert metabolite,
which maintains a low GC content in fœtal circulation [19-21]. Yet, during the middle of the
second trimester, fœtal brain 11-β-HSD-2 gene expression is silenced and enzymatic levels
acutely reduced for a brief period, allowing a normal acute surge of GC to enter the fœtal
bloodstream and enhance development of the brain, as well as other organs [13, 14, 19-21,
94, 95]. Exposure to excessive levels of glucocorticoids during this time may result in
widespread acute effects upon neuronal structure and synapse formation and may
permanently alter brain structure [3, 22, 96, 97].
Addressing the second mechanism, nursing and grooming by the mother, are both
significantly diminished after exposure to MS [10, 23, 24]. This decreased care during the
postnatal period powerfully affects the offspring. It is during this time that the newborn
imprints to its mother, and under normal conditions, forms a strong bond that solidifies
neuroprocesses and tempers endocrinological responses. In fact, when exposed to decreased
antenatal maternal care, adult offspring display altered behavior and stress hormone
responses [8, 13, 20, 46, 92]. In addition, they display permanent decreases in glucocorticoid
15
receptor density in the hippocampus and prefrontal cortex, a change anticipated to attenuate
feedback sensitivity of the hypothalamic-pituitary-adrenal axis, with resultant increases in the
stress-hormone responses [8, 13, 20, 46, 92]. The possible neurotoxic effects of such
excessive stress hormone responses [15, 16, 81, 95], has led to a proposal that changes in
maternal care may suppress cell proliferation in the early developing brain [98], and may
modulate the relationship between prenatal risk and hippocampal volume later in life [99].
When adults have “suffered the slings and arrows” of this two-fold manipulation, they
display altered behavior and neuroendocrinological responses [3, 7, 10, 23, 24].
Two independent, yet interrelated factors, Tau and GC may determine the
survivability of neurons, particularly those within the hippocampus during negative stress.
Ubiquitous in the CNS, the microtubule associated protein (MAP) Tau plays a critical role in
neuronal cell polarity, axonal outgrowth, axonal transport, and maintenance of axonal
morphology [79]. Regulated, in part, by the glycogen synthase kinase 3 (GSK3) signaling
pathway, Tau primarily stabilizes axonal microtubules by phosphorylation and in
pathological states has been implicated in several neurodegenerative diseases [79, 80]. Most
of these “tauopathies” have been associated with neuronal cell death [79]. Tau has been
positively-correlated with serum GC levels [17, 79-81] though the exact role Tau plays in
neurotoxic effects of GCs remain to be explained.
While there is strong evidence that MS alters behavior and neurohormonal responses
in adult offspring, little is known about its effects on functional brain activation. In our
study, we examined adult male rats with or without a prior history of MS and evaluated their
functional brain activation during exposure to an auditory stimulus previously fear
conditioned to delivery of a foot-shock. Regional cerebral blood flow related tissue
16
radioactivity (rCBF) was analyzed by statistical parametric mapping (SPM) from
autoradiographic images of the three-dimensionally reconstructed brains. These maps were
used to assess activation of canonical regions reported to be associated with fear
conditioning and/or emotional processing. To evaluate the degree of change in emotional
reactivity, we monitored anxiety-like behavior and the stress hormone response, specifically
corticosterone, throughout the fear conditioning and recall. Tissue sections at the level of the
amygdala and hippocampus were either stained or dissected to examine status of DNA
fragmentation (TUNEL), Tau (free and phosphorylated), and the apoptotic markers Caspase
3/7.
Materials and Methods
Generating male offspring exposed to antenatal maternal stress
Eight pregnant Wistar dams (200g, Harlan Sprague-Dawley Labs, Indianapolis, IN) were
exposed to daily scrambled electric foot-shocks starting from embryonic day 1 until delivery.
Control dams were similarly exposed in the absence of the foot-shock. After 20 days of
gestation, pups were born and left undisturbed with their mothers. Upon weaning on
postnatal day 21, male rats exposed to MS and male offspring not exposed to maternal stress
(NMS) were separately placed in social groups of 4, and housed until 14 weeks of age under
standard vivaria conditions. At 14 weeks, MS and NMS animals, were implanted with
external jugular vein catheters. A subgroup of these (MS: n= 14, NMS: n = 17) was retained
for serial blood analysis of corticosterone levels during fear conditioning, and brain tissue
collection for molecular analysis. Remaining rats (MS: n= 25, NMS: n = 17) were implanted
with a subcutaneous minipump for functional brain mapping during fear-conditioned recall.
This self-contained minipump, developed by our group, allows for bolus administration of
17
radiotracers by remote activation for functional neuroimaging applications in freely moving,
nontethered animals [88, 89].
External Jugular Vein Catheterization
One week prior to examining the effects of CF (brain mapping behavior or blood
corticosterone and molecular assays), rats were deeply anesthetized with isoflurane (2.0%
induction and 1.5% maintenance). Using aseptic surgical techniques, the ventral skin of the
neck was prepared, and the right external jugular vein was catheterized with a 5 French
silastic catheter, which was advanced 3.5cm into the superior vena cava. The catheter was
tunneled through the subcutaneous space to the back, where it was either connected to the
MIP or capped and used as an access port for blood withdrawals to evaluate blood
corticosterone concentration.
Implantation of MIP
Prior to implantation of the sterilized MIP (Fig. 2), a 5cm incision was made in the mid-
infrascapular region parallel to the spine.
Figure 2. Photograph of implantable microbolus infusion pump, MIP. (1) Drug reservoir. (2) Electronics
module. (3) Valve. (4) Ejection chamber, radioisotope. US quarter shown for size comparison.
18
Here, employing blunt dissection techniques, the skin was separated from the underlying
tissue in order to form a small pouch for the MIP. The MIP was placed in the pouch and the
skin was sutured over the implant (Fig. 3), except around the MIP percutaneous access port
(a 2cm silastic tubing capped with stainless steel plug).
Figure 3. Schematic overview illustrating MIP location when implanted in the adult male rat.
Immediately following surgery, the animals received a broad-spectrum antibiotic (i.v.
Ceflazoin 75mg/kg) and a non-steroidal anti-inflammatory (s.c. Flunixin 2.5mg/kg). The
percutaneous port allowed for flushes of the catheter every two days post-operatively
ensuring catheter patency (0.8mL of 0.9% saline, followed by 0.1mL of 20U/mL heparin in
0.9% saline). The access port was also employed for the loading of the radiotracer
approximately 1 hour prior to imaging.
19
Conditioned Fear Paradigm
Four days postsurgery, evaluation of emotional processing was conducted using standard
methods [27, 68]. Animals were habituated to the experimental room for 40 minutes in a
stainless steel transport cage. Thereafter, rats were placed in the test chamber, which
consisted of a Plexiglas/stainless steel box (30cm
3
) with a floor of stainless steel rods of
2mm diameter and 8mm separation. The chamber was illuminated with the indirect ambient
fluorescent light from a ceiling panel and was subjected to background ambient sound level
of 57dB. After a 3 minute baseline, the CF animals were subjected to the training phase in
which they received 8 tone/footshock pairings, each separated by a 1 minute silent interval.
In each pairing, presentation of a tone (30s, 72dB, 1000Hz/8000Hz continuous, alternating
sequence of 250ms pulses) was followed immediately by a footshock (1mA, 1s). Control
animals received identical exposure to the tone but without the footshock. One minute after
the final footshock, rats were returned to their home cages. Rats were individually trained;
animals awaiting their turn were placed in a separate room, separated by 3 doors, while in the
presence of a low level white noise generator to minimize the possibility of detection of the
ultrasonic vocalization, which may occur during CF training.
Injection of CBF radiotracer
Five days after implantation of the MIP, rats were briefly immobilized (less than 5 minutes)
in a soft plastic rodent restrainer (Decapicone, Braintree Scientific, Braintree, MA) and the
radiotracer (
14
C-iodoantipyrine, 100µCi/kg in 300µL of 0.9% saline, American Radiolabeled
Chemicals, St. Louis, MO), followed by a euthanasia compound (1mL of 50mg/mL
pentobarbital, 3M KCl), was loaded into the pump through the percutaneous port. Animals
subsequently rested undisturbed for 40 minutes in a transport cage next to the novel-testing
20
chamber (round opaque Plexiglas cylinder with paper floor) prior to exposure to the
behavioral paradigm, recall of CS.
The MIP was triggered transcutaneously by an infrared light source above the testing
cage. This resulted in a bolus injection of the radiotracer, followed immediately by infusion
of the euthanasia solution. Cardiac arrest followed within approximately 10 seconds,
accompanied by a precipitous fall of arterial blood pressure, termination of brain perfusion,
and death [89]. Brains were rapidly removed, flash frozen in dry ice/methylbutane (-50˚C),
covered in optimum cutting temperature solution (OCT, Sakura Finetek, Torrance, CA) and
stored at –20˚C until sectioned into coronal segments for autoradiography.
Autoradiography
Cerebral blood flow related tissue radioactivity (CBF-TR) was measured by the
14
C-
iodoantipyrine method [100-102]. With this method, there is a strict linear proportionality
between tissue radioactivity and CBF when measurements are made within a brief period
(roughly 10 seconds) after injection [103, 104]. Brains were sectioned in a cryostat at –18˚C
in 20µm sections, with an intersection spacing of 300µm. Sections were heat-dried on glass
microscope slides and exposed to Kodak Ektascan film for 15 days at room temperature
with 16
14
C standards (Amersham Biosciences). Images (autoradiographs) of brain sections
were digitized on an 8-bit gray-scale with a ChromaPro 45 IAIS “Dumas” film illumination
system and a Phillips charged-coupled device monochrome imaging module couple to a
Flashpoint 128 digitizing board on a microcomputer.
21
Statistical Parametric Mapping
Image Alignment
Our earlier work has described in detail the process of aligning the slices (approximately 67
coronal sections) in order to reconstruct the three dimensional (3D) brain, the spatial
normalization and the smoothing of the individual brains [105]. For brevity a short review is
provided, the digitized coronal sections (Bregma +4.2mm to –11.2mm) were selected and
stored as two-dimensional arrays of 72 µm
2
pixels. Adjacent sections were aligned both
manually and using TurboReg [106], an automated pixel-based alignment algorithm [105].
Creation of Rat Brain Template
Employing a 12-parameter, non-linear spatial normalization into a standard space defined by
a single, ‘artifact-free’ rat brain, individual 3D images were spatially normalized. This was
followed by a non-linear spatial normalization using a low-frequency basis functions of the
three-dimensional discrete cosine transformation (7x7x8 in each direction for 1176
parameters), plus a linear intensity transformation (4 parameters) [105].
SPM Analysis
First introduced by Friston, et al. [107, 108], statistical parametric mapping (SPM) is a
collection of tools available in the public domain for basic visualization and analysis of
neuroimages (http://www.fil.ion.ucl.ac.uk/spm/). Initially developed for analysis of human
imaging data, it has recently been adapted by our laboratory, as well as others, for use in rat
brain autoradiographs [105, 109, 110]. Critically, adapted for the use in freely-moving un-
tethered small animals, our tracer injection precluded the direct sampling of arterial blood
necessary for obtaining quantitative measures of flow in mL/min-grams of brain tissue.
Hence, normalization was necessary to minimize intersubject variability and, most
22
importantly, differences in the total amount of tracer received by each animal. Global
differences in the absolute amount of radiotracer delivered to the brain were adjusted by the
SPM software in each animal by scaling the voxel (3D pixel element) intensities so that the
mean intensity for each brain was the identical (proportional scaling). Voxels for each brain
failing to reach a specific threshold (80%) were masked out to eliminate the background and
ventricular spaces.
We implemented a Student’s t-test (unpaired) at each voxel (72x72x300µm
3
), testing
the null hypothesis that there was no effect of group, i.e. no difference between
14
C-
iodoantipyrine CBF tracer distributions. Map of positive and negative t were separately
analyzed. A significance threshold was set a p<0.05 (uncorrected for multiple comparisons)
for individual voxels within clusters of contiguous voxels, and a minimum cluster size of 100
contiguous voxels (extent threshold). We then evaluated the corrected significance of
individual voxels, clusters of contiguous voxels exceeding threshold, and the number of
clusters detected in the entire SPM. Regions determined to be significant at the voxel level
were required to show significance in two or more autoradiographic sections. Group
differences in the distribution of the rCBF were displayed as color-coded statistical
parametric maps superimposed on the brain coronal sections. Brain regions were identified
using anatomical atlas of the rat brain atlas [111].
Behavior Analysis
The duration of the animal’s freezing response (in seconds) was recorded on videotape and
analyzed with the Observer (Version 4.1, Noldus, Inc., Sterling, VA), a software package for
the analysis of animal behavior. Freezing was defined as the absence of all visible
movements of the body and vibrissae aside from respiratory movement, as scored by an
23
observer blind to the experimental conditions. The freezing scores were expressed as the
percentage of time spent freezing within each 30-second interval. The dataset was exported
and analyzed. Repeated measures analysis of variance was used to compare groups on
average responses. SAS Proc Mixed was used for these analyses.
Molecular Assays
Stress Hormones
Four days after catheterization of the external right Jugular vein the animal was placed in a
transport cage adjacent to the testing chamber and allowed to rest for 40 minutes. With
exception to recall, 300µL of venous blood was removed via externalized catheter at times 0
(initially placed into testing cage), 4 (one minute after tone-shock pairing or tone alone), 12
(just prior to end of training), and 120 minutes (two hours after end of training). During
recall (twenty-four hours after training), blood samples were only taken at time 0 and 4
minutes. Immediately following recall the animals were infused with a 1mL bolus of
euthanasia solution (50mg/mL pentobarbital, 3M KCl). As described elsewhere, brains were
rapidly removed, snap-frozen, covered OCT, and stored at –20˚C. Corticosterone levels
were determined by ELISA using a rabbit polyclonal antibody (Neogen Corp, Lexington,
KY). All blood corticosterone levels were determined on the day the sample was acquired
and compared to a new calibration curve each time. Results were analyzed via Student’s t-
test testing the null hypothesis there is no difference between groups.
Brain Tissue Samples
Brains obtained from blood corticosterone assays were sectioned in a cryostat at –18˚C in
alternating 10 and 20µm sections, with an intersection spacing of 100µm. Sections were air-
dried and stored at –80˚C until needed.
24
Dual Stains
Brain tissue sections of 10µm thickness corresponding to the amygdala (~Bregma –3.36mm)
and the hippocampus (~Bregma –5.30mm) were stained with TUNEL, structural markers
neuron nucleus (NeuN, Chemicon, Inc., Temecula, CA) or glial (GFAP, Chemicon, Inc.),
and the nucleus (DAPI). Fluorescent-tagged (FITC) oligonucleotides were hybridized in
accordance to a commercial TUNEL kit (FragEL DNA Fragmentation Detection Kit, EMD
Bioscience, La Jolla, CA). Immediately following TUNEL staining, samples were covered
with a blocking solution (10% normal goat serum and 1% bovine serum albumin in 1x PBS)
in the dark for two hours at room temperature. After blocking, samples were covered with
the appropriate diluted mouse monoclonal antibody (1:500 1x PBS) and incubated (4˚C)
overnight in the dark. After incubation the samples were rinsed for five minutes, three times
in the dark. Next, they were treated with horse anti-mouse Rhodamine (Vector Laboratories,
Inc., Burlingame, CA) secondary antibody (diluted 1:40 in PBS) for 60 minutes. The signal
was detected with 4', 6-diamidino-2-phenylindole (DAPI) fluorescent mounting media
(Vector Shield, Vector Laboratories, Inc.). Samples were kept at 4˚C in the dark until
reading. Images were acquired with a Zeiss LSM-510 laser scanning confocal microscope
(Carl Zeiss, Thornwood, NY) using a plan-neofluar 40x oil immersion lens NA 1.3. Slides
were scanned under the same conditions for magnification, laser intensity, brightness, gain,
and pinhole size. Images were processed using the LSM 510 software version 3.2 SP2.
Caspase 3/7 Activity
Brain tissue sections of 20µm thickness corresponding to the amygdala (~Bregma –3.36mm)
and the lower 1/3 of hippocampus (~Bregma –5.30mm), i.e. ventral hippocampus, were
dissected and protein extracted as described elsewhere [112]. Active caspase-3 and -7
25
(‘caspase-3/7’) was measured using corresponding Caspase-Glo Assay Kits (Promega,
Madison, WI) as previously described [113], where 20µg of total protein in a volume of
200µL was analyzed.
Western Blot
As described from the previous section, protein extracts were acquired from 20µm thick
brain tissue sections corresponding to the amygdala and ventral hippocampus. Brain protein
extracts were resolved by SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad,
Inc., Hercules, CA) following Otter et al. protocol [114]. The membranes were blotted for
free- or phospho-Tau activity with a mouse anti-rat monoclonal antibody (Cell Signaling
Technologies, Inc., Danvers, MA). GAPDH (Chemicon, Inc.) was used to demonstrate even
loading of samples. Expression of both Tau and GAPDH was detected with HRP-
conjugated secondary antibody (Vector Laboratories, Inc.) and the corresponding signals
were detected with a chemiluminescent solution (Super Signal West Pico, Pierce
Biotechnology, Inc., Rockford, IL) per manufacturer’s instructions.
Results
Antenatal maternal stress heightened psychophysiological responses
NMS and MS rats were randomly selected to either be exposed to a classic conditioned fear
(CF) paradigm (tone cue associated with foot-shock) or to serve as controls, exposed only to
tones (CON) [68].
During the 3 minute baseline, prior to receiving tone/foot-shock pairings, NMS and
MS rats were actively engaged in exploratory behavior in the training chamber with no
significant group difference in freezing behavior (range 0 – 15%). Exposure to the
tone/foot-shock pairings (minutes 4 – 15) produced significant increase in the animals’
26
freezing response (40 – 90%). Animals with prior exposure to MS showed greater anxiety-
like responses (percent time in immobile or ‘freezing’ postures) compared to NMS animals
during the CF training phase. Histograms plotting the number of events of the percent
observed freezing behavior clearly demonstrate a separation in the MS and NMS
populations. However, for rats exposed to the CF stimulus, a ceiling effect was noted, with
no group statistical significance differences between MS/CF and NMS/CF (p=0.13, Fig.
4D). Surprisingly, the effects of MS were most apparent in control animals that had not
received foot-shocks (MS/CON vs. NMS/CON, average 70±15% vs. 35±14%, P<0.001,
Fig. 4E). This increased response may be attributed to the tone, which itself may have been
interpreted as an unfamiliar, fearful stimulus.
27
Figure 4. Psychophysiological responses of adult male rats, with or without previous exposure to MS. (A) Intergroup behavioral responses of
NMS/CF (n=9) and MS/CF (n=14) rats during CF training. (B) Intergroup behavioral responses of NMS/CON (n=8) and MS/CON (n=11) rats
when exposed only to CS during training. (C) Intergroup behavioral responses of NMS/CF and MS/CF rats conditioned to fear a tone during recall of
CS. (D) Intergroup behavioral of NMS/CON and MS/CON rats previously exposed to tone-only during recall of CS. (E) Blood corticosterone levels
during CF training of all groups: NMS/CF (n=7), NMS/CON (n=10), MS/CF (n=7), and MS/CON (n=7). (F) Blood corticosterone levels during CF
recall of all groups: NMS/CF, NMS/CON, MS/CF, and MS/CON. (A – D) Left Panel: Graph % Freeze over Time, with each point representing the
group average over 30s. Right Panel: Histogram plotting number of events per 30s interval by percent of observed freezing behavior. Horizontal arrow
indicates time and duration of tone played. Vertical dashed bar represent time MIP was activated. Errors bars represent +/- SEM. Data that was
statistically significantly different as calculated by repeated measures analysis of variance (SAS Proc Mixed) is represented by an asterick, *. Solid red
circle and line for behavioral graphs represent MS group. Solid blue circle and line for behavioral graphs represent NMS group.
28
Twenty-four hours later, upon being placed into a novel context, and exposed to the
auditory cue, CF-trained animals had elevated freezing behavior compared to their respective
group controls (MS/CF vs. MS/CON, p<0.02; NMS/CF vs. NMS/CON, p<0.001).
NMS/CF animals gradually increased their freezing response to near maximal levels (~97%)
over two minutes, whereas MS/CF animals were 100% motionless within the first 30
seconds of tone playback, remaining so throughout the entire recall (Fig. 4E left panel).
Comparison of MS/CF to NMS/CF animals showed statistically significant differences in
freezing between these groups (p=0.002).
Results of the corticosterone assays paralleled those noted in the behavioral scoring. At
baseline, prior to CF training, NMS rat had a mean BC level of 20 – 50ng/mL, while MS
animals had a mean BC level of 20 – 70ng/mL, with no group statistical differences.
Nominal basal BC values that have been reported at 60ng/mL +/-15ng/mL [115, 116].
After the first tone/foot-shock pairing, BC levels increased to 68ng/mL +/-16 in NMS/CF
rats, while levels in MS/CF animals dramatically increased to 235ng/mL +/-70 (MS/CF vs.
NMS/CF, p < 0.05) (Fig. 4C). Significant differences in BC levels between MS/CF and
NMS/CF rats persisted during the duration of tone exposure (MS/CF 230 – 280ng/mL,
NMS 68-120ng/mL), with continued elevation even 120 minutes for MS/CF rats (150±
63ng/mL) after they had been returned to their home cages. As noted in the behavioral
measures, exposure to ‘tone alone’ elicited significant differences between MS/CON and
NMS/CON animals (MS/CON 200± 50ng/mL, NMS/CON 50± 12ng/mL, p<0.05),
suggesting that the auditory stimulus was perceived as being more stressful by animals with
prior MS exposure. Overall these results support prior reports that MS results in a
dysregulated HPA axis [20].
29
Functional brain mapping was accentuated by antenatal maternal stress
Functional activation of the brain was assessed on exposure to the auditory cue during the
recall phase of the CF paradigm in rats used in the behavioral studies. After a 2 minute
continuous exposure to the conditioned tone, the perfusion tracer [
14
C]-iodoantipyrine [89],
was injected as an intravenous bolus by remote activation of the implanted minipumps.
Regional CBF (rCBF) was analyzed postmortem in autoradiographic images of the three-
dimensionally reconstructed brain by SPM [105]. Group subcortical differences in the
distribution of rCBF are shown as color-coded statistical parametric maps superimposed on
the brain coronal and transverse slices (Fig. 5). Lists of cortical, subcortical, and white matter
track regions of interest for which group differences were significant (p < 0.05) for the SPM
[105] analysis are shown in Table 1.
Amygdala:
Exposure to the conditioned tone cue (MS/CF vs. MS/CON and NMS/CF vs.
NMS/CON) resulted in a significant increase in rCBF in the lateral nucleus of amygdala, a
region considered the primary sensory interface for unimodal, sensory processes associated
with auditory stimuli in the acquisition and expression of CF [57, 117, 118]. Significant
decrease in rCBF were noted in anterior portions of the basolateral and basomedial
amygdala, a sensory interface for multimodal, complex, configural, conditioned stimuli [119,
120]. Regional CBF was decreased in the amygdala’s central nucleus (CE), which was
surprising in so far as this structure is felt to control the animal’s motor and autonomic
response via the midbrain, hypothalamus, medulla, and extended amygdala. CE deactivation
may have been the result of the fact that measurements of rCBF were performed during later
stages of recall, rather than during acquisition. Similarly in human subjects during CF
30
exposure, it has been suggested that learning-related activation occurs only during early
acquisition, whereas deactivation is seen during later stages of retention and extinction [121-
123], possibly due to inhibitory modulation arising from the mPFC [124].
Maternal stress accentuated functional activation of the amygdala during exposure to
the conditioned tone (MS/CF vs. NMS/CF), as well as during exposure to the tone in the
absence of prior conditioning (MS/CON vs. NMS/CON). Increases in rCBF noted in MS
rats were broadly expressed and noted, not only in the lateral amygdala but also in the
medial, basal and central nuclei.
Hippocampus:
In the hippocampus (HPC), rats conditioned to the tone cue compared to controls
(NMS/CF vs. NMS/CON and MS/CF vs. MS/CON) showed significant increased of rCBF
in the posterior ventral HPC (CA1, CA2), the fimbria and the ventral subiculum, while a
decrease was noted in the anterior, dorsal HPC (CA1 – 3), dentate gyrus and the dorsal
subiculum. MS compared to NMS animals showed significantly greater increases of rCBF in
the fimbria and significantly greater decreases in rCBF in the dorsal HPC, dentate gyrus and
dorsal subiculum (MS/CF vs. NMS/CF and MS/CON vs. NMS/CON).
Prefrontal Cortex:
The conditioned tone cue elicited greater activation in ventral structures and greater
deactivation in dorsal structures of the prefrontal cortex in MS compared to NMS rats. Both
NMS and MS rats showed significant increases in rCBF in the anterior, ventral cingulate
(Cg2) in response to the conditioned tone (NMS/CF vs. NMS/CON and MS/CF vs.
MS/CON). This activation, however, was significantly greater in MS than in NMS animals
(MS/CF vs. NMS/CF) and was noted in particular in the ventral cingulate overlying the
31
induseum griseum. Fear conditioning MS rats, but not NMS rats, showed significant
deactivation of the posterior, dorsal cingulate (Cg1) compared to controls. The accentuated
functional deactivation of the dorsal cingualte in MS rats was noted during exposure to the
conditioned tone (MS/CF vs. NMS/CF), as well as during exposure to the tone in the
absence of prior conditioning (MS/CON vs. NMS/CON). In response to the tone, rCBF
increased in the infralimbic (IL) in both the MS and NMS animals, though this response was
significantly greater in MS animals (MS/CF vs. NMS/CF and MS/CON vs. NMS/CON).
Regional CBF in orbital cortex was increased in MS compared to NMS rats in response to
the conditioned tone and to the control tone.
Other:
MS compared to NMS exposure also was associated with increased rCBF in the
insula, the reticular nucleus of the thalamus, as well as broad deactivation of deep cerebral
white matter, and deactivation of the dorsal nucleus of the thalamus (MS/CF vs. NMS/CF
and MS/CON vs. NMS/CON).
32
Figure 5. Statistical parametric maps of functional brain activation during recall of a CS 24-hours after CF training, a comparison amongst four
different groups. The rat brain autoradiograph obtained were digitized, aligned, and template created as described elsewhere [83, 105]. Group
differences in distribution of rCBF were displayed as color-coded SPM superimposed on the brain coronal slice. Red to white represents statistically
significant increases (hyperæmia) and blue to green represents statistically significant decreases (hypoæmia). (A) Coronal sections from anterior to
posterior highlighting cortical and sub-cortical regions associated with emotional processing. (B) Transverse sections from dorsal to ventral
highlighting white matter tracks at two different levels. Landmarks indicated are per the Paxinos and Watson Rat Brain Atlas [111]. Scale indicates
significance (p-values) of superimposed colors. A = anterior, P = posterior, D = dorsal, V = ventral. Horizontal/vertical magenta lines indicate
approximate level transverse sections aligns to coronal section in B, left column. Horizontal/vertical green line indicates approximate level transverse
sections aligns to coronal section in B, right column. Numbers of animals per group are identical to those listed in the behavioral group of Figure 4.
33
Table 1. Summary of functional brain activity related to cognition, feat and emotional processing. Landmarks
indicated per the Paxinos and Watson Rat Brain Atlas [111]. -- = no change, up arrow = hyperæmia/activation,
down arrow = hypoæmia/deactivation.
NMS MS CF CON
CF vs. CON CF vs.
CON
MS vs.
NMS
MS vs.
NMS
CORTEX
Cingulate, dorsal posterior (Cg1) -- ↓/↓ ↓/↓ ↓/↓
Cingulate, ventral (Cg2) ↑ ↓/↓ ↓/↓ ↓/↓
Entorhinal, perrhinal (Ent, PR) ↑/↑ -- /↑ ↑/↑
Infralimbic (IL) ↑ ↑/↑ ↑/↑ ↑/↑
Insula ↑/↑ ↑/↑ ↑/↑
Orbital, lateral (LO) -- ↑/↑ ↑/↑ --
Orbital, ventral (VO) -- ↑/↑ ↑/↑ ↑/↑
Prelimbic (PrL) ↑ -- -- --
SUBCORTEX
Amygdala, central n. (Ce) ↓ -- ↑/↑ ↑/↑
Amygdala, lateral n. (La) ↑ ↑/↑ ↑/↑ ↑/↑
Amygdala, medial, basal (Me, BMA, BLA) ↓ ↓/↓ ↑/↑ ↑/ , --
Amygdala anterior area, cortical anterior,
cortical posterior (AA, ACo, PLCo)
↓/↓ ↓/↓ -- --
Hippocampus, ventral ↑/↑ ↑/↑ ↓/↓ --
Hippocampus, dorsal ↓/↓ ↓/↓post ↓/↓post ↓/↓
Striatum, ventral anterior ≠≠ ≠≠ ≠≠ (≠≠)
WHITE MATTER TRACKS
Corpus callosum ≠≠ ≠≠ ≠≠ ≠≠
Anterior Commisure ↑ ↑/↑ ↑/↑ ↑/↑
External capsule ↑ ↑/↑ ↑/↑ ↑/↑
Deep cerebral white matter ↑ ↑ ↑/↑ ↑/↑
Hippocampus, fimbria ≠≠ ≠≠ ↑/↑ ↑/↑
OTHER
Indusium griseum ≠≠ ≠≠ ↑/↑ ≠≠
Septal nucleus, lateral ↑/↑ ↑/↑ ↓/↓ ≠≠
Molecular differences noted between animals with and without prior exposure to MS
Utilizing the SPM maps as a guide, tissue sections of the amygdala and hippocampus were
obtained from animals used to monitor BC levels. Briefly, immediately after recall animals
were euthanized (i.v. pentobarbital 75 mg/kg/3M KCl) and brains were removed, snap-
frozen in methylbutane at –55˚C, covered in OCT and sectioned on a cryostat. Serial
34
sections were taken, where the amygdala as well as hippocampus were dissected and
processed as previously described [112], and all tissue sections were stored at -80˚C.
Free- and phospho-Tau as determined by Western blot displayed higher levels in the
ventral HPC of MS compared to NMS animals (Fig 6A, MS/CF vs. NMS/CF and
MS/CON vs. NMS/CON, p<0.05). A similar trend for free-, though nonsignificant, for
phospho-Tau was observed in the amygdala (Fig 6B).
Figure 6. Representative Western blot of free- and phosphorylated-Tau from the amygdala and ventral
hippocampus. (A) Free-Tau Western blot with a bar graph below displaying optical density group differences.
(B) Phosphorylated-Tau Western blot with a bar graph below displaying optical density group differences.
Amyg = Amygdala. vHPC = ventral hippocampus. GAPDH = Glyceraldehyde-3-phosphate dehydrogenase,
served as internal protein loading control standard. N = 3 for all groups.
35
Dual immunofluorescent stains of free-Tau in the ventral HPC recapitulated the Western
blot with NMS animals had free-Tau at lower levels and in a relatively structured pattern,
whereas, MS animals had higher free-Tau in a less structured pattern in the ventral
hippocampus (Fig 7A, left and middle panel).
Figure 7. Representative photomicrographs of immunoflourescent and TUNEL stains of ventral hippocampus.
(A) NMS, vHPC f-Tau, green. Neurofilament, red. Cell nucleus, blue. (B) MS, vHPC f-Tau, green.
Neurofilament, red. Cell nucleus, blue. (C) MS, vHPC TUNEL(+), green. NeuN, red. Cell nucleus, blue. All
representative images were taken with a 40x objective. White arrow points to neuron soma positive for
apoptosis.
Correlated with Tau, increases in GC have been found to be excitotoxic to neurons [15-17].
In support of this, both NMS/CF and MS/CF animals compared with their respective
controls showed a small significant increase in TUNEL(+) cells within the amygdala and
ventral hippocampus (Fig 7C and 8A, ~0.07 – 0.10% vs. ~0.04%, p<0.05). This was
coincided with a modest increase in active caspase-3 and –7 in the ventral HPC and
amygdala (Fig. 8B, NMS/CF vs. NMS/CON and MS/CF vs. MS/CON, p< 0.05).
36
Figure 8. Molecular analysis of NMS and MS animals. (A) Quantification of TUNEL(+) cell from amygdala
and ventral hippocampus (n=4/group). (B) Quantification of active caspase-3/7 from amygdala and ventral
hippocampus (n=4/group).
Discussion
This study demonstrated for the first time clear differences in functional brain activation in
animals previously exposed to antenatal MS. Prior exposure to MS accentuated changes in
the amygdala, where increases appeared broadly in the lateral, central, medial, and
basolateral/basomedial nuclei. Maternal stress exposure also was associated with increased
rCBF in the insula, parahippocampal regions, ventromedial striatum, orbitofrontal cortex as
well as infralimbic cortex. Such changes were noted both in CF exposed and control (no CF)
rats. In addition, MS exposed rats compared to NMS rats, showed relative decreases in rCBF
in the posterior, dorsal cingulate (Cg1) and prelimbic cortex, areas thought to exert an
inhibitory effect on the amygdala [39]. Based on their functional and structural
characteristics, many of these rat brain regions have homologues in the human brain [125,
126]. Several of these homologues have recently been identified as critical nodes in a central
network responsible for processing of fearful and negative emotional stimuli [127]. This
cortico-limbic network includes regulatory regions such as the supragenual cingulate (Human
Broadmann Area, BA 32, rat prelimbic cortex) and posterior cingulate (BA 23, rat posterior
37
Cg1), as well as emotional integrative regions such as the subgenual cingulate (BA 25, rat
infralimbic cortex), orbitofrontal cortex (BA 11, rat orbital frontal cortex), dorsolateral
prefrontal cortex (BA 46, rat prelimbic cortex), amygdala, insula, and parahippocampal gyrus
including HPC [39].
Our results are also consistent with the idea proposed by Phillips et al. [33] of the
existence of a ventral and a dorsal stream of emotional cognition. The ventral stream,
consisting of the amygdala, insula, ventral striatum, and ventral regions of the anterior
cingulate gyrus and prefrontal cortex, is posited to appraise emotional behavior and produce
an affective state. The dorsal stream, consisting of dorsal regions of the anterior cingulate
gyrus and prefrontal cortex, as well as the hippocampus, acts as a regulatory mechanism for
the ventral stream. Our study shows clear effects of MS in these two neural systems in the
rat, with a predominant activation of the ventral system and deactivation of the dorsal
system in MS exposed animals during CF recall. Similar functional changes have been
suggested in a number of psychiatric disorders including posttraumatic stress disorder and
depression [128], whose risk appears to be increased by the presence of early life stressors
[129].
Prior exposure to MS decreased the rCBF response in the dorsal HPC (CA1) and
dentate gyrus. Though the implications of this remain to be determined, these findings are
consistent with reports suggesting that MS produces learning deficits as well as inhibition of
neurogenesis in the HPC [46]. A further interesting observation was the broad increase in
rCBF noted in the deep white matter of animals exposed to MS which provide correlate to
structural changes observed in humans exposed to early life stress [130, 131].
38
Guided by the functional maps, we next evaluated molecular changes in the ventral
HPC and amygdala. Ubiquitous and critical within the CNS, Tau provides microtubule
stabilization but also flexibility to the distal portion of the axon. Fear conditioning increased
free-Tau levels in the HPC (but not in the amygdala) of MS and NMS animals. Phospho-Tau
increased in response to fear conditioning only in the NMS animals. Alterations in Tau-
phosphorylation in response to fear conditioning have been previously reported in the HPC
[132]. Increases in Tau in the HPC were accompanied also by increases in BC levels in
agreement with earlier work [15-17]. In the ventral HPC, both free- and phospho-Tau were
significantly increased in MS compared to NMS animals (MS/CF vs. NMS/CF and
MS/CON vs. NMS/CON, p<0.05), with a similar trend noted in the amygdala. Tau in MS
animals appeared also less structured in appearance by immunofluorescent staining
compared to NMS animals. Consistent with the neurotoxic effects associated both with
elevated levels of BC and Tau, we noted a modest increase in programmed cell death 24
hours after fear conditioning in both MS and NMS animals.
Conclusions
This study demonstrated for the first time clear differences in functional brain
activation in animals previously exposed to antenatal MS. Maternal stress elicits a
dysregulation of cortico-limbic circuits and heightened fear response that was apparent in
the adult offspring. Stress hormone levels that were normal at baseline were greatly elevated
in MS compared to NMS rats in response to a threat-related stimulus. This was associated
with a small but significant increase in apoptotic markers (TUNEL and caspase-3 and –7)
and the microtubule-associated protein Tau. These findings point to the potential
importance of early life stress in determining how traumatic events are processed in adult
39
life, the longevity of associated symptoms, and the risk of developing brain-based alterations
in structure and function.
In the realm of molecular psychiatry, as we move closer to bridging “bench to
bedside”, the changes observed in functional brain mapping and molecular biology may
expand our understanding of how MS could affect the behavior of the progeny in adulthood
when exposed to such conditions or other antenatal manipulations. For example, the current
administration and use of prednisone to ensure proper organ growth for a fœtus in crisis
may require further vigilance. The results reported here have underscored the delicate
environment the fœtus resides, which, if manipulated, must be done so with great care, lest
we alter the trajectory of its normal development.
40
Chapter III
Preliminary Study: Persistent Exposure To Exaggerated Maternal Stress
Response Throughout Gestation Alters Nnat Promoter Methylation In
Utero
Abstract
Prolonged exposure to exaggerated maternal physiological responses from a chronic stressor
(i.e., antenatal stress) has long been suspected as the etiology of severe psychiatric disorders,
such as schizophrenia. Animal research and clinical studies (retrospective and prospective)
have demonstrated altered behavior and neuroendocrine responses in adulthood with prior
exposure to maternal stress. The objective of our study was to examine differences to fœtal
rat brains after nineteen days, with and without, exposure to chronic maternal stress.
Specifically, we examined the immature amygdala and hippocampus for increased apoptosis
as well as globally for alteration in the methylation profile of the promoter for
neuronatin—key for both brain and neuron development. One dam was randomly selected
to receive a daily stressor while the other served as control. Rat fœtuses were collected after
either exposure to a persistent psychophysiological stressor (n=15) or without (n=15).
Number of fœtuses and mass was recorded after removal. Brains were removed for either
TUNEL stains or genomic DNA extraction to compare Nnat promoter methylation
profiles. Maternal mass was reduced by 10% after exposure to persistent psychophysiological
stressor throughout nineteen days of gestation. Consequently, fœtal mass was reduced by
approximately 10% compared to non-stressed fœtuses. Percentage of apoptotic cells in the
amygdala were higher than controls while numbers in the hippocampus were
indistinguishable. DNA methylation of Nnat promoter region was reduced after persistent
MS exposure. Maternal mass decreased throughout exposure to severe stress, while fœtal
41
exposure to MS had pronounced effects on the developing fœtus. Growth, as measured by
fœtal mass, was reduced. Apoptosis of key limbic structures was enhanced slightly and DNA
methylation of the promoter for a critical gene controlling brain and neuron development
was reduced.
Introduction
Since the mid-20
th
Century, animal experiments and human studies have mutually made a
convincing argument that both the growth and developmental trajectory of offspring
exposed to chronic exaggerated maternal stress response (MS) are distinctly altered [4-7, 11,
12]. Specifically, changes in physiological responses (hormones and neuropeptides),
structural malformations (cardiac defects), as well as behavior and memory, most
importantly, have all been noted [3, 4, 65, 92, 133-137]. Mechanistically, MS exposes the
developing offspring to the responses of the mother in utero to the consequences of a
severe, chronic stressor(s). Such stressors could be emotional (fear/anxiety), immunological
(illness), or survival (shelter/starvation/thirst) [3, 5-7, 14]. All the aforementioned scenarios,
as succinctly illustrated Koenig et al. [14], precipitate the release of a potent class of steroid
hormones, namely glucocorticoids (GC), via the hypothalamic-pituitary-adrenal (HPA) axis.
Readily crossing the cell membrane to participate in genomic and nongenomic roles, steroid
hormones have the capacity to activate signaling pathways and alter gene transcription [13,
20, 94, 138]. These hormones released during severe maternal stress may alter fœtal
development in several ways: 1.) reduction of blood flow to the fœtus, 2.) excess maternal
cortisol crossing the placenta, or 3.) placental release of corticotropin releasing hormone into
the intrauterine environment [3]. Normally at high levels in the placenta during gestation, 11-
β-HSD-2 converts GC into an inert metabolite, which maintains a low GC content in fœtal
42
circulation [13, 14, 21, 94]. Yet, during the middle of the second trimester, fœtal brain11-β-
HSD-2 gene expression is silenced and enzymatic levels acutely reduced for a brief period,
allowing a normal acute surge of GC to enter the fœtal bloodstream and enhance
development of the brain, as well as other organs [13, 21, 45, 76, 77, 94]. Clearly, persistent
exposure to excess GC throughout gestation could pose a threat to the normal maturation of
the brain by sabotaging critical growth signals [13, 21, 45, 76, 77, 94].
Known as “timetables of neurogenesis,” a predictable feature of the central nervous
system (CNS) is that each neuronal population is generated during a specific temporal
window [31]. Furthermore, careful examinations have demonstrated that rat neurogenetic
timetables can be extrapolated to human neurogenetic timetables as well [31, 139]. Given the
gestation period of the rat fœtus to be 21 – 23 days, crucial regions for emotional processing
all terminate their development at the end of neurogenesis, E19 [38, 139, 140]. These regions
include the amygdala, hippocampus, neocortex, and limbic cortex, among others [31, 38].
Concurrently, a key factor whose gene expression peaks during neurogenesis, neuronatin
(Nnat) participates in multiple unique roles as well.
A straightforward gene, Nnat has been implicated for both accurate brain and
neuron development in humans as well as rats [141, 142]. Homologous to humans, the rat
Nnat gene has one promoter and three exons that generates two alternative splice forms
[142]. The α-isomer is produced by all three exons and appears early in brain development,
E7 – 10. The β-isomer is formed only from exons one and three, which appears later in
development, E11 – 14. Overall, total Nnat activity has been noted to be elevated during
neurogenesis, then declining postnatally to adult levels [141, 142]. Previous reports have
43
indicated Nnat is an imprinted gene [140]. That is, containing a high concentration of CpG
islands surrounding its promoter region.
Demonstrated to have enormous implications in molecular psychiatry, epigenetics
(aka “imprinting”) bridges the gap between the external environments an organism resides
and the internal control of its gene expression [140, 143-145]. In mammals it is estimated
that several hundred gene are under epigenetic control, the greatest proportion identified are
expressed in the CNS [140]. Rather through changes to its underlying DNA, epigenetics
controls gene expression via unique chemical modifications to either the histones or the
DNA upon which the histones are intertwined [93, 140, 146, 147]. These chemical
modifications include acetylation, methylation, or ubiquitination. Among the most
recognizable epigenetic modifications is the methylation of cytosine to the dinucleotide
repeat of cytosine and guanine that is linked by phosphodiester bonds. Colloquially known
as “CpG Islands” and repeated in vast quantities, this dinucleotide sequence is found
punctuated around the promoter region of certain genes [140, 147]. Generally, analogous to
a household light switch, gene expression is “off” by methylation of the cytosines. That is,
inhibited via contraction of the chromatin. And conversely, it is “on” without the
methylation, where the chromatin is in a relaxed configuration allowing for binding of
eukaryotic initiation factors thereby permitting gene expression. Given the potency for
altering the correct developmental trajectory as well as epigenetic elements [138, 144], might
antenatal exposure to excessive stress hormones via MS also affect apoptosis, or PCD?
As a key mechanism to maintaining homeostasis within the brain parenchyma,
programmed cell death (PCD) removes superfluous neuronal and glial cells during
development in a controlled manner [18, 30, 32, 35, 148]. Essential for proper maturation
44
during fœtal and neonatal periods, apoptosis has been suspected in being altered from
exposure to MS [5, 6, 14, 149]. Any dysregulation of this process could have potential
consequences later in life, which remains unclear. Of particular importance, would be
alteration to either the amygdala or hippocampus. Critical in emotional processing, both the
amygdala and hippocampus have been suspected in the genesis of mental illnesses, such as
mood disorders, depression, and schizophrenia.
In the present study, we exposed fœtuses either to chronic stress hormones via MS
during the first nineteen days of gestation or not at all. The dam’s masses were record
throughout the experimental period until the fœtuses were removed and collected. Fœtuses
were counted and weighed upon removal. Brains were subsequently removed, snap-frozen,
and either sectioned for histological staining or genomic DNA was extracted to determine
changes, if any, of the methylation of the Nnat promoter.
Materials and Methods
Maternal Stress
Experiments were carried out under a protocol approved by the Animal Research
Committee of the University of Southern California. Two Wistar dams (Harlan Sprague-
Dawley, Indianapolis, IN) were randomly selected to either be exposed to an acute stressor,
twice daily until the nineteenth day of gestation (maternal stress, MS) or were not (no
maternal stress, NMS). Animals arrived from the vendor one day after being tested plug-
positive; this day was established as E1. Rats were housed individually in a rodent colony on
a 12hr(light):12hr(dark) cycle where food and water were available ad libitum. The dams
were transported to the testing room in a rodent transport cage and were allowed to
acclimate in a quiet environment for 30 minutes. Each morning the dam’s masses were
45
recorded. Twice daily, once in the morning and once in the afternoon, for twenty minutes
the MS dam was exposed to the stressor, which consisted of scrambled electric footshocks
(1mA, 1s duration) in an ambulatory-restrictive environment with a floor of stainless steel
rods (2mm diameter and 8mm separation). After stressor, the dam was returned to its home
cage via rodent transport cage. The NMS dam was only exposed to the handling stress and
normal routines of animal husbandry facility, i.e. cage cleaning.
Tissue Acquisition
On the nineteenth day of gestation both dams were deeply anesthetized with an inhalation
anesthetic (Isoflurane, 4% induction and maintenance) and rapidly decapitated. All fœtuses
were collected via standard method [150], counted, and weighed. After weighing, brains were
removed and snap-frozen at –50˚C in a mixture of dry ice and t-methylbutane for either
genomic DNA extraction or histological stains.
TUNEL Stain
Fœtal brain tissue sections were collected sequentially in triplicate on microscope slides
(Superfrost Plus, VWR, Bativa, IL) with an interslice distance of 20µm and a section
thickness of 10µm on a cryostat (Microm HM550 microtome/cryostat, Microm
International, Walldorf, Germany). Using a rat embryonic brain atlas as a guide [139], tissue
sections containing developing amygdala and hippocampus were stained for fragmented
DNA oligonucleotides, a hallmark of apoptosis. Briefly, samples were fixed in 4% buffered
paraformaldehyde for 15 minutes at room temperature (RT, ~20˚C). Samples were then
immersed in 1x TBS for an additional 15 minutes. Afterward, the samples were permeablized
with 0.01% Triton X
100
for 10 minutes and rinsed three times in 1x TBS. Samples were
subsequently stained via commercial TUNEL kit (FragEL DNA Fragmentation Detection
46
Kit, EMD Bioscience, Inc., La Jolla, CA) following manufacture’s protocol and
counterstained with DAPI (Vector Laboratories, Inc.). Images were acquired with a Zeiss
LSM-510 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY) using a plan-
neofluar 40x oil immersion lens NA 1.3. Total percentage of TUNEL-positive cells was
calculated within observed region (fixed field) comparing fetuses from MS and NMS dams
(n=6 for both groups).
Methylation-Specific PCR (MSP)
Known for its ability to distinguish the difference of one nucleotide mismatch, methylation-
specific PCR is a sensitive techniques used to detect changes in CpG methylation status [147,
151]. Generally, three sets of DNA primers are designed. One matched to amplify the
original DNA (O), another to amplify unprotected cytosines (M), and finally, one to amplify
a sequence corresponding to all cytosines converted to uracil via bisulfite treatment (U)
[147]. Here, fœtal brain genomic DNA was extracted via common methods and analyzed. A
total of 200ng of genomic DNA was used in each bisulfite conversion reaction, where
unprotected cytosines are chemically converted to uracil using EZ DNA-Methylation Gold
kit (Zymo Research, Orange, CA) according to the manufacturer’s instructions. Each PCR
reaction consists of 2µL of eluted DNA, 1 µL of primer pair (10 pmol), 9.5 µL of water and
12.5 µL of ZymoTaq Premix (Zymo Research). Methylation-specific primers for MSP
amplification were designed to test for DNA methylation status of Nnat promoter along
with primers for GAPDH (loading control). MSP primers are noted in Table 2.
47
Table 2. List of primers used for methylation-specific PCR. Nnat = neuronatin, GAPDH = glyceraldehyde-3-
phosphate dehydrogenase, O = original, M = methylated, U = un-methylated + = positive control.
Statistics
All statistical analysis were performed using Student’s t-test, alpha=0.05, unless otherwise
noted. Histograms were plotted to visualize distribution of data in determining population
differences.
Results
Both maternal and fœtal mass decreased by ~10%, respectively, following exposure for
nineteen days to a severe psychophysiological stressor (Fig. 9). After gestation day ten (G10),
maternal masses began to noticeably deviate from one another. Compared to non-stressed
control animal, mass 382g, the MS dam mass was ~12% less than (338g) the NMS at the
time the fetuses were collected. On average, the MS dam from G10 until fœtal collection
(G19) remained 10% less in mass than NMS dam (Fig. 9A). This decrease in maternal mass
was carried over to a decrease in fœtal mass. The range for MS-fetuses (n=15) was from 0.8g
to 1.8g (SEM +/- 0.06g) with a mean value of 1.3g. Conversely, NMS-fœtuses (n=15) that
received no stress had a mass range from 1.2g to 1.6g (SEM +/-0.03) with a mean value of
1.4g. This ~10% decrease in mass was calculated to be statistically significantly different
(p<0.05) from the control animals (Fig. 9B). Surprisingly, there was no difference between
the numbers of fœtuses each dam carried, i.e. both had a total of 15 fœtuses.
MSP Primer Forward (5’ 3’) Reverse (3’ 5’) bp
Nnat (O) GAAGGGTGTTAGAGCGGACTCAGGAGGTGG ACGGGAACGGGCGGGGACAAATAGT 500
Nnat (M) TTTTTTATTTTATATTTTAGAGTTACGA GCATACGCATAATTCCACCATCC 231
Nnat (U) TTTTTTATTTTATATTTTAGAGTTATGA ACACATACACATAATTCCACCATCC 231
GAPDH (O) CACTTACCCCAGCCTTCTCC TTCCCGTTCCGATTTCCAGT 998
GAPDH (U) GTGAAGTTTATTTTTTGGTATGTGG CCTATAACTCTCATTCTCTCTCCAA 249
(+) CONTROL CTCTCGAGAAAATATCGTATTAGGCGTTATTCGTT GCCAATTGCGCGCCCCTCTCCGCCAACCTAGGGC 466
48
Figure 9. Comparison of dam and fœtal mass with and without exposure to chronic MS. (A) Dam change in
mass over eighteen days of gestation. Red region highlights period of consistent divergence. (B) Histogram
plotting frequency (number of occurrences) by fœtal mass from two different maternal environments.
Statistical significant differences indicated by *.
Apoptosis in the undeveloped amygdala and hippocampus from stressed fœtal brains (n=6)
demonstrated a slight increase, but were not statistically significantly different from non-
stress fœtuses (n=6), p=0.07 and 0.8 respectively (Fig. 10A). The largest change observed
49
was between the NMS and MS amygdala, whereas the developing hippocampus had very
little variation in quantity per area observed (Fig. 10A – C).
Figure 10. Quantification of TUNEL(+) cells from immature rat amygdala and hippocampus as well as
TUNEL stain of fatal hippocampus. (A) Bar graph displaying percentage of TUNEL(+) cells per group and
region examined. (B) TUNEL stain from fœtal brain exposed to MS. Left Panel: TUNEL(+) cells, Middle
Panel: no stain, Right Panel: DAPI nuclear stain. (C) TUNEL stain from NMS fœtal brain. Left Panel:
TUNEL(+) cells, Middle Panel: no stain, Right Panel: DAPI nuclear stain. AMYG = amygdala, HPC =
hippocampus, fHPC = fetal hippocampus, Vt = ventricle. White arrows point to TUNEL(+) cells.
Methylation status of the Nnat promoter between the two fœtal groups (n=3 each)
was different as evidenced by Nnat MSP. Fœtal brains from MS dams readily amplified un-
methylated DNA of the Nnat promoter (Fig. 11A), whereas NMS showed no amplified
50
products. Amplification of the un-methylated DNA from the Nnat promoter region
correspond to all cytosines having undergone a chemical conversion to uracil, including
one’s on CpG sequences purportedly protected by methylation. Further, amplification of our
GAPDH loading controls appeared even from all samples (Fig. 11B).
Figure 11. Comparison of Nnat promoter methylation. (A) MSP of Nnat promoter region O = primers for
original, unadulterated, genomic rat DNA. M = primers corresponding to DNA protected by methylation on
CpG islands only. U = primers for amplifying DNA completely unprotected by methylation. (B) MSP rat
GAPDH. O = primers for orignal, unadulterated, genomic rat DNA. U = primers for amplifying DNA
completely unprotected by methylation. + = positive control, - = negative control, B = blank, bp = base pairs.
Discussion
Negative stress is well known to have a detrimental impact on adult physiology, more so
within the delicate and dynamic milieu of the fœtus [3, 5, 7, 16, 17, 20, 45, 46, 65, 93].
51
Congruent with these previous reports, the dam that was exposed to chronic stress after
approximately day ten of pregnancy consistently remained 10% less than the NMS dam in
mass until collection of the fœtuses. Here too, we saw the translation of reduced maternal
mass by stress to reduced fœtal mass via exaggerated physiological responses, i.e. stress
hormones.
A critical mechanism employed during development, PCD helps maintain tissue
homeostasis not only in adulthood but also during organogenesis. Given GC’s potency, it
was hypothesized that exaggerated levels from MS would alter apoptosis in fœtal brains,
particularly the amygdala and hippocampus regions critical for emotional processing as well
as attenuating the HPA response in adulthood. This was not observed, however quantities
were increased in MS fœtuses and close to statistical significance. At a minimum suggesting
evidence PCD could still be a factor in potentially altering developmental dynamics.
In contrast to no-effect in the quantity of apoptotic cells in underdeveloped limbic
structures, we found that the methylation status of the fœtal brain Nnat promoter region to
be altered compared to controls. We readily amplified genomic DNA corresponding to de-
methylation in the promoter region of neuronatin—an indication that Nnat expression could
be continuing beyond its normal endpoint compared to controls. While previous studies
have indicated that Nnat expression is highly active between E16 – 19 [141, 142], we did not
detect un-methylated DNA from NMS fœtuses on E19. Suggesting that Nnat expression for
NMS fœtuses at this point in time was attenuated or completely inhibited. Generally, for MS
fœtuses this could be interpreted as a relaxed chromatin structure, which would be more
susceptible to gene expression. Additionally, studies have shown that Nnat to be protective
of neuronal cells from toxic elements [152]. These results appear analogous with previous
52
work indicating that epigenetic mechanisms may control expression of GC receptors, and
therefore the life-long ability to modulate HPA response appropriately [13, 93, 138, 140, 143,
153], which themselves be altered by antenatal exposure to excess GC. Might elevated levels,
as suggested from our MSP data, be a proxy measure of excess stress hormones, or other
potentially harmful products, during development?
Conclusions
In conclusion, we demonstrated that in addition to a reduction in mass, when fœtuses are
exposed to chronic MS that globally and temporally the methylation status of the Nnat
promoter region was transformed from one that was inhibited to one that was potentially
expressing when compared to non-stressed controls. Also, while there was no statistical
significant difference between quantity of apoptotic cells, that there was a generalized
increase with animals prior exposure to MS. Future work will investigate, with a larger
population size, not only apoptosis in developing limbic structures and methylation status of
the Nnat promoter, but also to quantify mRNA as well as immunoblots for Nnat protein as
well. This study further underscores the delicate milieu in which the fetus resides. Which, if
manipulated, must be done so with great care least we alter its proper developmental
trajectory.
53
Chapter IV
Conclusions and Future Directions
As imaging combined with molecular biology helped improve our understanding of cancer,
so too has this combined technique help us better understand the impact early life stressors
has on the brain. The investigation of the role of prior exposure to antenatal maternal stress
has in augmenting functional brain activation, behavior, psychophysiological responses, and
neuronal apoptosis in adult life has lead to several conclusions. First, prior exposure to MS
in adult animals, when confronted by an acute stressor, produced striking differences in
cortical and sub-cortical activations. For the first time in a small animal model, we confirmed
previous clinical reports of cingulate cortex activation with exaggerated amygdala activation
[33]. Second, congruent with increased activity in the ventral hippocampus as well as the
amygdala, two nodes critical for processing CF stimuli, we detected robust changes in free-
and phosphorylated-Tau—a protein essential for proper neuron formation and implicated in
serious neurodegenerative disease, such as Alzheimer. Previously identified to be positively
correlated with a stress hormone, both Tau and GC in excess can lead to programmed cell
death. While neurons within the amygdala and hippocampus were positive for fragmented
DNA, a hallmark of apoptosis, their overall quantities were less than 0.10% per region
observed within 0.9mm. Nonetheless, these differences were significant for the
hippocampus more so then the amygdala. This appeared to be in agreement with Sapolsky’s
observation that over the course of a lifetime, one could be more susceptible to neuronal cell
death, i.e. endangerment to the neuron. Further, this may be one explanation as to why two
different individuals can have two completely different responses to an identical stressor as
well as potentially help us better understand how hippocampal volumes decrease when
54
exposed to repeated severe stress. Third, from our preliminary study, persistent exposure to
MS during gestation displayed a decrease in the methylation status of the promoter for
neuronatin. Recently implicated as a potential barometer for neuron exposure to toxic
elements, Nnat has also been established as a gene that is crucial for proper brain and
neuron development as well. This gene, established as one of several imprinted genes in the
CNS is typically silenced after neurogenesis via hypermethylated. Our discovery of
demethylation in the promoter region after chronic exposure to MS from fœtal brains may
correspond to a potentially harmful environment, but more importantly it may demonstrates
a possible injury that could have life-long psychiatric consequences. Fourth, during the
course of studying the molecular profile of various brain tissue sections left at ambient room
temperature it became evident that protein degradation may not be as robust as we once
thought. A novel investigation (see Appendix A) determined that protein degradation from
brain tissue sections, as visualized on a denaturing gels and epitope detected via Western blot
and tissue stains, did not show signs of decomposition after six months left at ambient
conditions. This appears to contradict the established dogma that tissues are sensitive to
decay if left at room temperature. Though this observation clearly may not be true of all
proteins, e.g. phopho-protein and the like. This discovery may help to alleviate storage issues
found in the common molecular biology laboratory.
There are several points that can be investigated further from the results presented
here. First, regarding the timing of exposure to a severe antenatal psychological stressor.
Behavioral studies indicate that greater impact can be generated by acute exposure during the
period when fœtal brain 11-β-HSD-2 is at its lowest point, i.e. the middle of the second
trimester. From the functional brain maps, comparison could be made in conjunction with
55
behavior in order to better evaluate this claim as well as examining Tau protein and
programmed cell death cascade. Second, the impact of the postnatal environment related to
neonatal care. Again, studies have indicated the importance of maternal care during neonatal
development and its long-term impact on behavioral and neuroendocrinological responses.
Third, a more thorough investigation of the molecular genetic consequences from exposure
to chronic gestational stress, in particular with focus on neuronatin mRNA and protein
levels. Fourth, further dissection of the molecular pathways of how GC and Tau protein are
related. Fifth, culturing adult neurons with and without prior exposure to MS to perform in
vitro assays related to GC excitotoxicity and Tau protein. Sixth, carefully dissecting the
functional brain activation in a temporal manner, i.e. collect functional brain activation data
from specific time points to help us better understand how the brain responds to an acute
stressor. Time points could be gathered from 10 seconds, 30 seconds, and 1 minute.
Seventh, and last, investigation of behavior extinction from a functional brain perspective.
56
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Appendices: Introduction
In the course of acquiring molecular data from brain tissue sections left at ambient
laboratory conditions for several weeks, observations were made that indicated tissue
proteins were more robust than previously thought. An analysis was conducted in order to
evaluate brain tissue protein stability over a period of six months at predetermined time
points and at various storage conditions. The results of this study are presented in Appendix
A. From the results of this study a new storage device was designed and patented, this
information is given in Appendix B.
83
Appendix A
Snap-Frozen Brain Tissue Sections Stored With Desiccant At Ambient
Laboratory Conditions Without Chemical Fixation Are Resistant To
Degradation For A Minimum Of Six Months
Abstract
Cryo-sectioned tissues from snap-frozen samples offer the advantage of preserving proteins
at the cellular and sub-cellular levels and maintaining overall cell integrity in the tissue of
interest without the use of chemical fixatives. To prevent specific or nonspecific degradation
of proteins by autolytic and/or proteolytic processes, it is common practice to immediately
store frozen tissue sections obtained from a cryostat under cryogenic conditions, e.g. -80˚C.
Our laboratory recently challenged this widely held belief by extracting proteins from brain
tissue samples that were archived for 1 day, 1 week, 1 month, and 6 months at various
storage conditions (frozen, ambient, or desiccated) without the use of chemical fixatives.
Our results from immunofluorescent stains, immunoperoxidase stains, silver stains, and
Western blot analyses demonstrated that snap-frozen, heat-dried tissue sections stored
desiccated at ambient laboratory conditions are comparable to frozen samples stored up to 6
months.
Introduction
The study design and analysis of ex vivo tissue samples determines the method by
which the specimen is processed and preserved. Tissues used for morphological or
immunohistochemical analyses are frequently fixed with a common chemical fixative, such
as formalin, and are embedded in paraffin. While formalin-fixed paraffin-embedded
specimens are well preserved and conveniently stored at ambient room temperatures, the
84
cross-linking and sulfide bond formation caused by the fixative make them less compatible
with current molecular techniques [154-156]. Hence, epitope retrieval methodologies are
used to recover the antigens; but recovery in some instances can be less than optimal [154,
157, 158]. An alternative to the use of fixatives is the century-old method of freezing the
tissue at sub-zero temperatures. Freezing the tissue provides a snapshot of the cells as they
would appear at the time the sample was removed from the organism, while avoiding
degradation of intracellular molecules via autolysis or similar mechanism [159-161]. The vox
populi among pathologists and histologists is to store snap-frozen tissue samples, which have
been sectioned and desiccated, at -80˚C. Typically, sectioned tissues are placed onto
microscope slides (subbed or with electrostatic charge), dehydrated to remove moisture, and
immediately stored at cryogenic temperatures.
The widely accepted belief that snap-freezing any tissue adequately preserves protein
integrity at cellular and sub-cellular levels has served as the gold standard for molecular
analysis [154-156, 162]. Ever since Altmann [160] described cellular degradation in the form
of proteolysis, the conventional method of cryogenic storage of frozen tissues has been
used, and few deviations from this practice have been reported. However, the question
remains whether sectioned tissue samples need to be maintained at freezing temperatures to
preserve protein integrity. Challenging conventional practice, our laboratory asked whether
proteins can be extracted from tissues, specifically brain sections, that were previously snap-
frozen and stored at various conditions for a month or longer.
85
Materials and Methods
Tissue Acquisition
Brain tissue was harvested from rats (n=5) immediately after intravenous euthanasia (1.0 cc
Euthasol™ I.C., Delmarva Laboratories, Inc., Greenland, NH). Tissues were snap-frozen at
-55˚C in methylbutane cooled with dry ice. The tissue samples were submerged in the
cryogenic solution for 14 seconds, removed, and embedded in Optimal Cutting Temperature
(OCT) solution (Sakura Finetek, Torrance, CA). Brains were sectioned (Microm HM550
microtome/cryostat, Microm International, Walldorf, Germany) at 20µm (used for protein
extraction) and 10µm (used for tissue staining) at -18˚C. Three consecutive sections were
placed onto electrostatic-charged microscope slides (Superfrost, VWR Scientific, West
Chester, PA), and the slides were either dried on a microscope slide warmer (Lablyne,
Melrose Park, IL) at 50˚C for 45 minutes (noted as “With Heat,” or “w/h”) or merely air
dried at room temperature for 15 minutes (noted as “Without Heat,” or “w/oh”). Samples
were tested after being stored at the following conditions: Frozen (-80˚C), Ambient (20˚C
and 45% relative humidity, RH), and Desiccated (20˚C and <10% relative humidity, RH) for
1 day, 1 week, 1 month, and 6 months. The assay matrix is summarized in Table 3, where all
tests were performed in triplicate.
86
Table 3. Assay matrix studying the effects of different storage conditions on brain tissue samples over 6
months.
Tissue Preparation
Samples of tissue sections (20µm) were excised under a dissecting microscope (Olympus,
Tokyo, Japan) with a disposable 1.0 cc syringe and 27ga 1/2” needle (Becton Dickinson Co.,
Franklin Lakes, NJ). Specifically, the area of tissue to be removed (i.e. striatum) was outlined
with the needle, then a minute quantity of protein extraction buffer (PEB, T-PER, Pierce
Biotechnology, Inc., Rockford, IL) was pipetted (Finnpipette, ThermoScientific, Waltham,
MA) in 2.0mL increments on the outlined section until the buffer immersed approximately
half of the inscribed area. To both minimize tissue loss and ease transfer into a
microcentrifuge tube (USA Scientific, Inc., Ocala, FL), the tissue was removed from the slide
using the 27ga needle and combined with PEB. The tissue/PEB was carefully transferred to
Assays
Sample
Storage
Condition Metrics
Tissue Stains
(IHC and HE) Silver Stain Western Blot
Fresh √ √ √ √
Frozen
Ambient 1 Day
Desiccated
Frozen
Ambient 1 Week
Desiccated
Frozen
Ambient 1 Month
Desiccated
Frozen
Ambient 6 Months
Desiccated
Frozen
Ambient 1 Day
Desiccated
Frozen
Ambient 1 Week
Desiccated
Frozen
Ambient 1 Month
Desiccated
Frozen
Ambient 6 Months
Desiccated
87
a 1.5mL tube. Approximately 150mL of PEB without protease inhibitors was added to the
semi-dried sample, and the tube was placed on ice. Immediately after sample preparation the
protein was extracted.
Protein Extraction
To minimize sample loss, the same 27ga needle used to excise the tissue was used to aspirate
the mixture ten times, while cautiously avoiding introduction of excess air in the mixing
process. This series was repeated using a 30ga needle (Becton Dickinson Co.) with the same
syringe. The sample was agitated (Maxi Mix, Barnstead International, Dubuque, IA) for 60
seconds, then centrifuged (Eppendorf model 5415C, Hamburg, Germany) for ten minutes at
10,000 x G at room temperature (RT, 20˚C). The supernatant was transferred into a clean
microfuge tube, and the pellet was stored at -20˚C. Supernatant total protein concentration
was quantified with a microplate reader (Benchmark Plus, Bio-Rad, Inc., Hercules, CA), via
the Bradford method (minimal protein detection size of 4.5kDa), and normalized by area
removed.
Silver Stain
Protein samples (total loading mass = 10.0µg) were resolved by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% Tris-HCl gel (Bio-Rad, Inc.) at
constant voltage (120V) for approximately 45 minutes. The gel was silver stained as
previously described, dried overnight using a commercial gel drying kit (Owl, Inc.,
Portsmouth, NH), and imaged with a conventional flatbed scanner at 600dpi on the
following day.
88
Western Blot
Protein samples were resolved by SDS-PAGE on a 10% Tris-HCl gel at constant voltage
(120V) for approximately 45 minutes. Proteins were transferred onto a PVDF membrane
(Bio-Rad, Inc.) overnight at 40 volts and 4˚C. A 5% non-fat milk (NFM) blocking solution
(Bio-Rad, Inc.) containing Tris-buffered saline with 0.01% Tween-20 (TBS-T) was applied to
the membranes for 60 minutes at RT. The membranes were subsequently washed three
times for 5 minutes each in TBS-T. The membranes were then examined for tyrosine
hydroxylase (TH) activity with a mouse anti-rat monoclonal antibody (Chemicon, Inc.,
Temecula, CA) in a 5% NFM/TBS blocking solution (1:1000) overnight at 4˚C with mild
agitation (Orbit Shaker, Barnstead International). To normalize sample loading the
ubiquitous enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Chemicon, Inc.)
blot was performed. Briefly, the membranes were incubated in mouse anti-rat GAPDH and
5% NFM/TBS-T at a 1:300 dilution overnight at 4˚C with mild agitation. Expression of
both TH and GAPDH was detected with 1:10,000 and 1:5,000 dilution horseradish
peroxidase-conjugated secondary antibody (Vector Laboratories, Inc., Burlingame, CA) and
chemiluminescent solution (Super Signal West Pico, Pierce Biotechnology, Inc., Rockford,
IL) following the manufacturer’s instructions.
H&E Stain
OCT-embedded, fresh and archived brain sections were fixed in acetone and stained with
hematoxylin and eosin as previously described [163].
Immunoperoxidase (IP) Stains
OCT-embedded, fresh and archived brain sections were fixed with 4% paraformaldehyde for
15 minutes, and washed twice for five minutes in phosphate-buffered saline (PBS) with
89
Tween-20 (PBS-T) washing solution. To block endogenous peroxidases, samples were
incubated in 0.1% H
2
O
2
in 1x PBS for 20 minutes at room temperature. After blocking for
60 minutes with serum blocking solution (10% normal goat serum and 1% bovine serum
albumin in 1x PBS), the sections were incubated overnight at 4˚C with TH diluted at 1:500
in 1x PBS. Samples were then washed three times for 5 minutes each in 1x PBS, and
incubated with a secondary antibody, Biotinylated Anti-Mouse (Vector Laboratories, Inc.,
1:100) for 30 minutes at room temperature. While the secondary antibody incubated, the
Avidin-Biotinylated-Peroxidase Complex (ABC) solution was prepared and stored at 4˚C for
30 minutes prior to use. The secondary antibody was washed off with 1X PBS three times
for 5 minutes each, and subsequently incubated with ABC solution for an additional 30
minutes at room temperature. After a wash with 1x PBS, a solution of 3-amino-9-
ethylcarbazole (AEC) was applied to the samples for a maximum of 6 minutes, or until red
reaction product developed. The reaction was terminated by a 1x PBS wash. Finally, samples
were counterstained with hematoxylin and mounted with aquamount and cover slipped.
Immunofluorescent (IF) Stains
Brain tissue samples were either dual labeled (fluorescent) with TH and MAP5B or labeled
individually for GFAP with a DAPI counterstained to evaluate epitope recognition. OCT-
embedded brain sections (fresh and archived) were fixed with 4% paraformaldehyde for 15
minutes, and washed twice in phosphate-buffered saline (PBS) with Tween-20 (PBS-T)
washing solution for five minutes. After blocking for 60 minutes with serum blocking
solution (10% normal goat serum and 1% bovine serum albumin in 1x PBS), the sections
were incubated overnight at 4˚C with TH diluted at 1:500 in 1x PBS. Next, the sections were
treated with horse anti-mouse Rhodamine red (Vector Laboratories, Inc.) secondary
90
antibody (diluted 1:40 in PBS) for 60 minutes. Following three PBS-T rinses, the tissues were
blocked for 120 minutes with serum blocking solution (10% normal goat serum and 1%
bovine serum albumin in 1x PBS), and incubated overnight at 4˚C with MAP5B (a stable
structural protein found in neurons) diluted at 1:500 in 1x PBS. Next, they were treated with
horse anti-mouse FITC (Vector Laboratories, Inc.) secondary antibody (diluted 1:40 in PBS)
for 60 minutes. Samples tested for GFAP (stable structural protein found in glial, non-
neuronal, cells) activity were treat similarly, where samples were fixed, rinsed, and blocked as
noted above then incubated overnight at 4˚C diluted at 1:500 in 1x PBS. GFAP sampled
were treated with horse anti-mouse FITC (Vector Laboratories, Inc.) secondary antibody
(diluted 1:40 in PBS) for 60 minutes. The signal was detected with 4', 6-diamidino-2-
phenylindole (DAPI) fluorescent mounting media (Vector Shield, Vector Laboratories, Inc.).
Images were acquired with a Zeiss LSM-510 laser scanning confocal microscope (Carl Zeiss,
Thornwood, NY) using a plan-neofluar 40x oil immersion lens NA 1.3. Slides were scanned
under the same conditions for magnification, laser intensity, brightness, gain, and pinhole
size. Images were processed using the LSM 510 software version 3.2 SP2.
Results
Protein extraction was possible from brain tissue sections after six months without
cryogenic storage or chemical fixatives. Tissue sections stored with or without desiccant at
ambient temperatures provide protein concentrations comparable to fresh samples after six-
months (Figure 12), though ambient and desiccated samples provided statistically
significantly less protein per given volume as calculated against a normalized region
removed. This reduction in protein concentration can be attributed to the technical difficulty
in isolating tissues from the slide by hand, which resulted in minor loss of sample.
91
Specifically, some pieces of the ambient and desiccated tissue sections possessed an
electrostatic charge that at times were repelled off the needle tip by the minor electrostatic
charge on the plastic MCT.
Figure 12. Comparison of normalized protein concentration (µg/mL) extract from 20µm murine brain tissue
sections stored at various conditions over 6-months period. Frozen refers to tissue sections stored at –80˚C,
ambient refers to sections stored at room temperature (RT, ~20˚C), and desiccated refers to tissue sections
stored at RT with low RH. Slides that were not placed on a slide warmer are noted as “wo/h” and those that
were are noted as “w/h”. Error bars represent +/- SEM. Statistically significantly difference, p<0.05 (Student’s
t-test), represented by *. RH indicates relative humidity; w/h, with heat; wo/h without heat.
Proteins from tissues stored at ambient conditions after six months, particularly
those desiccated, are similar to both fresh and frozen samples. Silver stain images from 1-
day-, 1-week-, and 1-month-old brain sections stored at ambient conditions are almost
indistinguishable from their fresh or frozen counterparts as they exhibit similar appearances
in fractionation patterns while displaying the least change in band definition (Figure 13). The
most observable change in band profile was detected with the 6-month old specimens,
92
which displayed only a minor loss in band resolution as compared to fresh and frozen
samples. Overall, sample bands appear sharp with minimal blurring, uncharacteristic of
tissue that is undergoing proteolysis or complete degradation. These observations appear
congruent with the results shown in Figure 12, demonstrating relatively identical protein
detection and concentration over time at various storage conditions.
Figure 13. Examination of protein degradation from 20mm brain tissue sections at defined time points from
various storage conditions. Extracted samples were resolved on a denaturing gel and stained by silver method.
(A) One-day old sample. (B) One-week old sample. (C) One-month old sample. (D) Six-month old sample. S =
Protein calibration standard. 1 = Fresh sample. 2 = Frozen brain tissue section (without heat). 3 = Ambient
brain tissue section (without heat). 4 = Desiccated brain tissue section (without heat). 5 = Frozen brain tissue
section (with heat). 6 = Ambient brain tissue section (with heat). 7 = Desiccated brain tissue section (with
heat).
93
Epitopes from tissue that have been desiccating for over six months are comparable
to fresh and frozen samples. All brain tissue sections display equal levels of TH over time
(Figure 14). Two bands were identified as possible rat-specific isoforms of TH [164].
Furthermore, equal loading between all samples was verified by GAPDH detection.
Figure 14. Demonstration of epitope detection of target protein extracted from 20µm brain tissue sections at
defined time points from various storage conditions. Samples were resolved on a denaturing gel and Western
blot performed for tyrosine hydroxylase (TH). GAPDH served as internal loading control, 1 month sample
shown as representative. A = One-day old sample. B = One-week old sample. C = One-month old sample. D
= Six-month old sample. 1 = Fresh brain tissue. 2 = Frozen brain tissue section (without heat). 3 = Ambient
brain tissue section (without heat). 4 = Desiccated brain tissue section (without heat). 5 = Frozen brain tissue
section (with heat). 6 = Ambient brain tissue section (with heat). 7 = Desiccated brain tissue section (with
heat). GAPDH = indicates glyceraldehydes-3-phophate dehydrogenase.
Immunofluorescent stains for TH and MAP5B or GFAP proteins remain unchanged
across various aged brain sections. Immunoperoxidase stains were equally readily
identifiable, and displayed minimal differences between fresh and desiccated samples. H&E
stains display minimal changes after six-months as well. Dual TH and MAP5B or GFAP
staining was readily detectable after six-months desiccation compared to fresh sample
94
(Figure 15A-H). Here, even astrocytic processes were easily seen after six-months of
desiccated storage. A negative control slide (only secondary antibodies added) displayed no
activity (data not shown). After six-months, eosin staining was reduced compared to fresh
sample, yet basophilic structures, in particular the cell nucleus had comparable hematoxylin
staining. Additionally, nuclear morphology appeared consistent between samples (Figure 15I
and J). Similarly observed with immunofluorescent stains, immunoperoxidase staining
revealed little change (Figure 15K and L).
95
Figure 15. Photomicrographs of brain tissue sections with hematoxylin and eosin (H&E), immunperoxidase, or immunoflourescence stains. Using a rat
stereotaxtic atlas as a guide [111], all samples were taken at approximately bregma 0.72mm (AP), at a junction between the striatum and cortex. (A,C,
and E) Fresh brain tissue section dual stain against TH and MAP5B, nuclear counter stain. (B, D, and F) Tissue section from 6-month-old desiccated
samples with identical stains. (G) Fresh brain GFAP stain. (H) GFAP stain of sample after stored in desiccated condition for 6 months. (I) Fresh brain
H&E stain. (J) H&E stain of sample after stored in desiccated condition for 6 months. (K) Fresh brain immunoperoxidase stain fro TH. (L)
Immunoperoxidase stain for TH of sample stored in desiccated condition for 6 months. TH = tyrosine hydroxylase, MAP5B = neuron, GFAP = glia
(non-neuronal), DAPI = nuclear stain. Photomicrographs were taken at 2 different magnifications, unless otherwise noted, one at 10x and another
40x (shown as inset).
96
Discussion
Concentration of protein from the various samples, though calculated to be
statistically different between ambient and desiccated samples, may not have reached a
statistically significant difference if the tissue collection processes did not prove to be as
difficult. Dissimilarity in calculated protein concentration notwithstanding, the integrity of
proteins extracted from brain tissue sections stored at ambient condition stored with or
without desiccant tissues over six-months appeared similar to fresh and frozen samples as
evidenced by the silver stain, a method understood to be more sensitive than Coomassie
[165]. . Minor loss of definition was observed after one month of storage, yet desiccated at
room temperature was well-defined and comparable to either fresh or frozen samples. Only,
after six months of storage at ambient temperatures was degradation evident, yet if products
of complete proteolysis had been present the lane in question would have displayed a smear,
which it did not.
Western blot analysis was performed to determine whether the epitopes of interest
remained recognizable. We selected an enzyme as the protein of interest because enzymes
are known to be potentially sensitive to decomposition or degradation at ambient conditions,
i.e. room temperature. The chosen epitope target was tyrosine hydroxylase, a critical enzyme
with various levels of activity present in all catecholaminergic cells, especially in tissues that
react to the sympathetic response, such as brain, heart, and kidneys [166]. Additionally, it is
worth noting that the different molecular weight bands observed for TH can be explained by
the recent evidence that two isoforms of TH exist in the rat while only one isoforms is
known to be produced sub primate species [164, 166, 167]. To normalize sample loading, the
membranes were blotted for GAPDH, a ubiquitous housekeeping protein found in almost
97
all tissues. Additionally, signal intensities of all tissues from fresh to 6-month appear
equivalent, with no detectable loss from archived specimens.
Immunofluorescent results demonstrated no difference in signal intensity over time.
Both MAP5B and TH stains/signals counterstained with DAPI remained consistent, as well
as easily recognizable. As compared to fresh samples, astrocytic processes of GFAP stained
specimens were clearly identifiable even after 6 months desiccated. To explain why the
investigated proteins from heat-dried, snap-frozen tissue sections stored at ambient
condition, in particular desiccated, do not appear degraded and epitopes of interest remain
detectable, we suggest the following. First, proteosome-based degradation is interrupted,
where either the prolonged exposure to 50˚C or air-drying for 15 minutes may have
potentially affected the ability of proteosome/ubiquitin complex to efficiently degrade
proteins, instead of mere denaturation. Second, cellular desiccation due to a combination of
prolonged elevated temperatures and a thin tissue section impedes cellular processes,
including action of degradative enzymes. Third, a combination of attenuated degradative
process and the absence of water have altered the functional structures of various enzymes,
which typically produces irreversible damage. Fourth, proteins in general, may simply be
more stable at ambient conditions than they have been previously perceived.
Conclusions
From the present experimental results, we can draw several conclusions that appear to
contradict the central dogma of SFHD tissue section storage. First, it is possible to extract
proteins from brain tissue section with minor indication of degradation after six-month, as
evidenced by silver stains, regardless of storage conditions. Next, the investigated epitopes
(TH, MAP5B, GFAP, and GAPDH) were successfully detectable in the target tissues as
98
shown by immunofluorescence, immunoperoxidase, and Western blots. With the preceding
statements in mind, we suggest that SFHD tissue sections may be stored at ambient,
desiccated conditions for up to 6 months at minimum without significant damage to protein
integrity. Albeit these results may be inapplicable to all proteins due to their variable
stabilities as well as the limited number examined, proteins of interest should be tested
individually to verify the suitability of this method. Future studies include examination of
samples stored for more than a year using the same techniques, assessment of competition
assays, and investigation of less stable macromolecules.
99
Appendix B
Provisional United States Patent For Ambient Storage For Fresh/Frozen
Tissue Sections Via Desiccation
Abstract
The present invention (US Provisional Application 61/103,750) discloses a device for
ambient storage of fresh/frozen tissue sections via desiccation. Also disclosed are methods
for making and using such a device.
Funding
This invention was made with support in part by grants from Doheny Core (EY03040) and
NCCAM (5R24AT002681). Therefore, the U.S. government has certain rights.
Field Of The Invention
The present invention relates in general to a device for storing fresh/frozen tissue
samples. More specifically, the invention provides a device for ambient storage of
fresh/frozen tissue sections via desiccation. Also disclosed are methods for making and
using such a device.
Background Of The Invention
The study design and analysis of ex vivo tissue samples determines the method by
which the specimen is processed and preserved. Tissues used for morphological or
immunohistochemical analyses are frequently fixed with a common chemical fixative, such
as formalin, and are embedded in paraffin. While formalin-fixed paraffin-embedded
specimens are well preserved and conveniently stored at ambient room temperatures, the
cross-linking and sulfide bond formation caused by the fixative make them less compatible
with current molecular techniques [154-156]. Hence, epitope retrieval methodologies are
100
used to recover the antigens; but recovery in some instances can be less than optimal [154,
157, 158]. An alternative to the use of fixatives is the century-old method of freezing the
tissue at sub-zero temperatures. Freezing the tissue provides a snapshot of the cells as they
would appear at the time the sample was removed from the organism, while avoiding
degradation of intracellular molecules via autolysis or similar mechanism [159-161]. The vox
populi among pathologists and histologists is to store snap-frozen tissue (aka fresh/frozen)
samples, which have been sectioned and desiccated, at -80°C. Typically, sectioned tissues are
placed onto microscope slides (subbed or with electrostatic charge), dehydrated to remove
moisture, and immediately stored at cryogenic temperatures.
The widely accepted belief that snap-freezing any tissue adequately preserves protein
integrity at cellular and sub-cellular levels has served as the gold standard for molecular
analysis [154-156, 162]. Ever since Altmann [160] described cellular degradation in the form
of proteolysis, the conventional method of cryogenic storage of frozen tissues has been
used, and few deviations from this practice have been reported. However, the question
remains whether sectioned tissue samples need to be maintained at freezing temperatures to
preserve protein integrity.
Summary Of The Invention
A device of the invention is designed to store fresh/frozen tissue sections on
microscope slides at ambient laboratory temperatures (~20°C, 45%RH) with the addition of
a replaceable desiccant cartridge and hygrometer to monitor conditions within the container.
Under these conditions, tissue degradation is no more than that seen with samples stored
under cryogenic (-80°C) conditions and comparable to fresh tissue specimens.
101
The above-mentioned and other features of this invention and the manner of
obtaining and using them will become more apparent, and will be best understood, by
reference to the following description, taken in conjunction with the accompanying
drawings. The drawings depict only typical embodiments of the invention and do not
therefore limit its scope.
Brief Description Of The Figures
Figure 16. Front side view of a device of the invention.
102
Figure 17. Top view of a device of the invention.
Figure 18. . Side view of a desiccant cartridge of the invention.
Detailed Description Of The Invention
Challenging conventional practice, our laboratory asked whether proteins can be
extracted from tissues specifically brain sections that were previously snap-frozen and stored
103
at various conditions for a month or longer. Our recent work has demonstrated that brain
tissue could produce data (i.e., valid molecular data could be obtained via Western Blots,
Immunohistochemistry, or Chromgen-based staining) from samples stored desiccated at
ambient laboratory conditions for a minimum of six months with minor change when
compared to fresh or frozen samples. Based on this discovery a newly designed microscope
slide storage box was designed and prototyped.
Two key important advantages for the use of this device would be the reduction
and/or elimination of the use of harmful or toxic chemicals typically employed to fix tissue
samples for assays and second, a reduction of energy demanding storage units (freezers).
This storage device would only require normal shelf space and replacement of its desiccant
cartridge when necessary.
The following example is intended to illustrate, but not to limit, the scope of the
invention. While such example is typical of those that might be used, other procedures
known to those skilled in the art may alternatively be utilized. Indeed, those of ordinary skill
in the art can readily envision and produce further embodiments, based on the teachings
herein, without undue experimentation.
Example
A storage device of the invention would consist of three plastic injection molded
parts (see Figures 12-14):
The bottom half (~210 mm long, 160 mm wide, and 21 mm high with a wall
thickness of ~4 mm, of which a 1 mm centered-groove [width and depth] would inscribe the
top rim to hold a silicone rubber o-ring) that would hold the microscope slide vertical along
its shortest side in three rows - each row would be approximately 6 mm in length. Each
104
column of rows would hold approximately 25 slides for a total capacity of 75 slides. The
floor of the container would contain a soft material (e.g., cork or silastic) to prevent damage.
The top half would have the same dimension as described above with an opening in
the middle of the approximately 7 mm by 3 mm. Both the top and bottom half would be
formed with a “living hinge” between the two components to assist in closing the container
appropriate and a push button locking mechanism to secure the two halves. Also, in the
upper corner of the top portion would be a 1.5 mm diameter hole to secure a hygrometer
that will indicate the appropriate moisture content inside the container.
The replaceable desiccant cartridge makes up the third part that would snap-fit into
the opening of the top of the container. This portion would be approximately 7x3 mm with
a depth of 1.5mm. This cartridge would have a simple cage for support and a one-way
hydrophobic membrane ultrasonically welded onto the cage after the cartridge is filled with
desiccant.
Abstract (if available)
Abstract
Profound, long-term behavioral and physiological consequences have been identified with, and attributed to prior exposure to severe early life stressors vis-à-vis altered maternal responses. To date, questions remain unanswered regarding potential changes of both functional brain activity and molecular expression of progeny that corresponds with known behavioral and physiological alterations in adulthood. Furthermore, questions regarding potential molecular changes during gestation, particularly related to neuro-development have remained elusive. For instance, given the potency of stress hormones, do fœtuses, when exposed to excess quantities, have an aberrant rate in programmed cell death, or alterations of epigenetic markers to an important gene related to proper brain and neuron formation? Here, for the first time, we show localization of functional brain activity and it’s corresponding molecular profile, programmed cell death, during the conditioned fear paradigm of adult animals with and without prior history of antenatal maternal stress. Also, we offer preliminary data that indicates the epigenetic profile of neuronatin, a critical gene for proper brain and neuron development, was changed from chronic exposure to gestational stress.
Linked assets
University of Southern California Dissertations and Theses
Asset Metadata
Creator
Sadler, Theodore R.
(author)
Core Title
Cancer of the psyche: antenatal maternal stress accentuates functional brain responses and neuronal endangerment during conditioned fear in adult offspring
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Pathobiology
Publication Date
05/06/2009
Defense Date
11/25/2008
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
amygdala,apoptosis,behvaior,conditioned fear,freely-moving untethered animal,functional brain activation,glucocoirtcoids,hippocampus,microbolus infusion pump,neuronatin,OAI-PMH Harvest,small animal model,statistical parametric mapping,tau protein
Language
English
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Advisor
Hinton, David R. (
committee chair
), Chuong, Cheng-Ming (
committee member
), Conti, Peter (
committee member
), Holschneider, Daniel P. (
committee member
), Taylor, Clive R. (
committee member
)
Creator Email
nervousgyrus@hotmail.com,tsadler@usc.edu
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https://doi.org/10.25549/usctheses-m2182
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Tags
amygdala
apoptosis
behvaior
conditioned fear
freely-moving untethered animal
functional brain activation
glucocoirtcoids
hippocampus
microbolus infusion pump
neuronatin
small animal model
statistical parametric mapping
tau protein