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GONADAL STEROID REGULATION OF CONNEXIN
EXPRESSION IN THE RAT BRAIN
Arawn A. Therrien
A thesis submitted with the requirements for the degree of Master of Science Graduate Department of Physiology
University of Toronto
O Copyright by Arawn A. Therrien (1997)
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GONADAL STEROID REGULATION OF CONNEXIN EXPRESSION IN THE RAT BRAIN
Master of Science, 1997 Arawn A. Therrien
Department of Physiology, University of Toronto
ABSTRACT
Intercellular gap junctions are fonned by specialized proteins, termed connexins
(Cxs). In addition to effects on neuronal electrical excitability, gonadal hormones have
been implicated in the modulation of comexins in the brain. The objective of this thesis
was to document the expression patterns of two principal comexins in the brain, Cx43
and Cx32, in cycling female rats, castrate or hormone-treated adult female and male rats,
and in neonatal rats. Androgen and/or estrogen exposure do not induce large-scale
changes in brain connexin mRNA expression. Zn situ hybridization demonstrates that
estradiol up-regulates Cx43 expression only slightly in a few, restricted regions of the
brain, including the bed nucleus of the stria terminalis, the dorso-lateral septum, and the
periventricular hypothalamus. A possible explanation for the restricted nature of the few
significant connexin responses observed is that effects of gonadal steroids may be
dependent on coincident or sequential electrical activation of the pathways involved.
A L N I U VV L P I U l r l V l l ' r l Y 1 3
Several people have been cited for having made contributions to this thesis: Grace
Erb was responsible for the sequencing of the connexi1143 in situ hybridization probe;
Deborah Bowlby rneasured the serum hormone profiles reported in Figure 5; Libertad Puy
performed in situ hybridization of pup brains as shown in Figure 19; Heather Edwards has
been cited for her unpublished data of the regulation of connexin expression in
epileptically kindled rats.
In addition, there are a number of people who 1 wish to acknowledge for their
invaluable contributions to this thesis. First and foremost, 1 owe a debt of gratitude to my
supervisor, Dr. Neil. J MacLusky, Ph.D., for his skillfùl teaching and constant guidance,
without which this thesis could not have been accomplished. 1 would also like to give
special thanks to Dr. Theodore J. Brown, Ph.D. for his generosity of time, support, and
mostly for his unparalleled sense of humour. 1 also thank Dr. Steven J. Lye, Ph. D. for
helpfiil advice and suggestions throughout my research, and the mernbers his laboratory,
with special thanks to Angela Orsino and Grace Erb. To Julie Kim and Andreas
Evangelou, 1 owe thanks for their lab assistance, support, and friendship. In particular, 1
must thank Deboleena Roy for her 3-dimensional insight into science, life and my own
insanity. A special thanks is also due to Robert "B." Bradizza, who has helped me
through the final stretch of this thesis, and has given me al1 the support, laughter and De
Cecco pasta necessary to complete it. Furtherrnore, 1 thank my parents, Olwen and Ross
Therrien, for their unflagging support and encouragement, and for having instilled in me
the drive to achieve. A final thank you is extended to Margaret Wise Brown for her
practical wisdorn and unfailing guidance, that only she c m give.
TABLE OF CONTENTS
. . ABSTRACT ............................................................................................................ -11
... .................................................................................. ACKNOWLEDGMENTS iii
.................................................................................... TABLE OF CONTENTS iv
.............................................................................................. LIST OF FIGUIRES vi
... ............................................................................................... LIST OF TABLES vm
............................................................................ LIST OF ABBREVIATIONS ix
CHAPTER 1 INTRODUCTION
....................................... ROLE OF GONADAL STEROIDS IN THE CNS 1 Localization of Gonadal Steroid Receptors in the CNS ..................................... 2
Estrogen receptors ................................................................................ -2 ............................................................................... Androgen receptors -3
The Role of Testosterone and Estrogen in the CNS ........................................... 4 .......................... Androgen and estrogen action on the developing CNS 4
.................................................... Sexual dimorphic nuclei in the brain -5 Activational eflects ofgonadal steroids on the C M .............................. 7
GAP JUNCTIONS ......................................................................................... 10 ..................................................................................... Structure of Connexins 12
....................................................................... Connexin gene structure 12 Connexin protein structure ................................................................... 14
Structure and Modulation of Gap Junctions ................................................... 16 .................................................................. Formation of gap junctions 16
Compatibiliîy of interactions between connexins ................................ -19 ................................................... Con trol of gap junction permeability 2 0
Gap Junctions in the Brain ................................................................................ 25 Functional Roles of Gap Junctions ................................................................... 26
.............. GONADAL STEROID REGULATION OF GAP JUNCTIONS 28 Gap Junctions in the Uterus ............................................................................. 28 Gap Junctions in the CNS ................................................................................. 30
Possible role of gap juncfions in rhe regulation o f synchronous elecfricul act ivity ................................................................................... 31
HYPOTHESIS .............................................................................................. -33 ................................................................................................... OBJECTIVE -33
UUIY AUAL 3 1 Lnwv K L ~ U L A 1 MJ~Y VF
CONNEXIN EXPRESSION IN THE RAT BRAIN
MATEMALS AND METHODS ...................................................................... 34 RESULTS ........................................................................................................ -45 DISCUSSION .................................................................................................. -70 FUTURE DIRECTIONS ................................................................................. -78
LlS'l' 0 F FIGUKES
........................................................ FIGURE 1 Illustration of gap junction plaque 11
FIGURE 2 Structure and topology of connexins ................................................... 15
FIGURE 3 Composite graph illustrating Cx32 transcript levels in specific brain regions of cycling female rats ...................................................... -47
FIGURE 4 Composite graph illustrating Cx43 transcript levels in specific brain regions of cycling female rats ....................................................... 48
FIGURE 5 Hormone profiles during the estrous cycle of the female rat ................. 49
FIGURE 6 Northern analysis of Cx43 transcript levels in hypothalamus/ preoptic area of intact and gonadectomized male rats ........................... 5 1
FIGURE 7 Graphs illustrating Cx32 transcript levels in specific brain regions of intact and gonadectomized male rats .................................... 52
FIGURE 8 Graphs illustrating Cx43 transcript levels in specific brain .................................... regions of intact and gonadectomized male rats 53
FIGURE 9 Graphs illustrating Cx32 transcript levels in specific brain regions of ovariectomized female rats + estrogen treatment.. ............... .54
FIGURE 10 Graphs illustrating Cx43 transcript levels in specific brain regions of ovariectomized female rats -L estrogen treatrnent .................. 55
FIGURE 11 Atlas diagrams illustrating the location and orientation of specific regions of interest in the rat brain ................................ .., ......... 57
FIGURE 12 In situ hybridization of Cx43 transcript labelling in intact and gonadectornized male rat at different levels of the brain ..................... . .58
FIGURE 13 Composite graph showing Cx43 transcript levels, as quantified by in situ hybridization, in intact and gonadectomized male rat brains.. .................................................................................................. .60
FIGURE 14 Composite graph showing Cx43 transcript levels, as quantified by in situ hybridization, in brains of ovariectomized femaIe rats f estrogen treatment. ..................................................................... .6 1
FIGURE 15 Photomicrographs of in situ hybridization for Cx43 mRNA in dorso-lateral septum of ovariectomized + estradiol-treated female
FIGURE 16 Photomicrographs of in situ hybridization for Cx43 mRNA in dorsal preoptic area of ovariectomized + estradiol-treated female rats.. .................................................................................................... .64
FIGURE 17 In situ hybridization of Cx43 transcript Iabelling in male pup brain illustrating regions used for autoradiographic quantification.. ..................................................................................... -67
FIGURE 18 Composite graph showing Cx43 transcript levels, as quantified .................. by in situ hybridization, in fernale and male rat pup brains 68
FIGURE 19 In situ hybridization of Cx43 transcript labelling in the .... periventricular preoptic area of neonatal male and female rat pups. .69
vii
U I U l W1' I n Y Y m u
TABLE 1 Sexual dimorphisms in the central nervous systern ................................ 6
. . ............................................................. TABLE 2 Farnily of connexins in rodents 13
... V l l l
A
AFP
AMY
AR
AVPN-POA
BC
~ P S
BST
CAMP
cDNA
CMTX
CNS
CTX
Cx
Da, kDa
Sa-DHT
El, E2
E2
ER
ERE
GDX
GnRH
HIP
H P 0
IP3
kb
LA
LH
Angstrom unit
a-fetoprotein
arnygdala
androgen receptor
anteroventral periventricular nucleus of the preoptic area
bulbocavernosus muscles
base pairs
bed nucleus of the stria teminalis
cyclic adenosine monophosphate
complimentary deoxyribonucleic acid
Charcot-Marie-Tooth disease
central nervous system
cortex
connexin
dalton, kilodalton
5a-di hydrotestosterone
extracellular domains of connexin protein
estradiol
estrogen receptor
estrogen response element
gonadectornized
gonadotropin releasing hormone
hippocampus
hypothalamus + preoptic area
inositol 1,4,5-trisphophate
kilobase
levator ani muscles
luteinizing hormone
Lnnn
LSD
Ml-M4
MBH
mRNA
OVX
Pe
PKA
PKC
PO A
PRE
PVCP
PVN
rER
SDN-POA
SNB
SON
TPA
VAH
VMN
1 U ~ G ~ I I I L I I I ~ I i u r m w m : rclcasing norrnone
dorso-lateral septum
transmembrane domains of connexin protein
mediobasal hypothalamus
messenger ribonucleic acid
ovariectomized
periventricular hypothalarnic nucleus
protein kinase A
protein kinase C
preoptic area
progesterone response element
periventricular caudate putamen
paraventricular nucleus of the hypothalamus
rough endoplasrnic reticuhm
sexually dimorphic nucleus of the preoptic area
spinal nucleus of bulbocavernosus
supraoptic nucleus
phorbol ester tumour prornoter
visceroatrial heterotaxia syndrome
ventromedial nucleus of the hypothalamus
bUlYAUAL 3 1 L K V l U KhbULA 1 lVlY Vi! LVlYlYLAllY
EXPRESSION IN THE RAT BRAIN
Arawn A. Therrien
CHAPTER 1 INTRODUCTION
Gap junctions are intercellular communication charnels that are forrned by
specialized proteins, terrned connexins. Gap junctional coupling is dynamically regulated
by a variety of factors in both neural and non-neural tissues 16,186,225 . Despite the
extensive Iiterature on hormonal regulation of gap junctions in the uterus 210,218 , relatively
little is known about how gonadal steroids affect connexins in the central nervous system
(CNS). What information there is, however, suggests that CNS gap junctions may also be
horrnonally regulated '59. Supporting this view are recent studies that propose a possible I0,192 role for electrical gap junctional coupling in the etiology of epileptic seizures , and
fùrther clinical observations of changes in epileptic seizure activity with alterations in
gonadal steroid levels 9. The influence of gonadal steroids on epileptiform activity in both
humans and animals is well docurnented, revealing the epileptogenic potential of
estrogen, while progesterone appears to produce opposing, seizure-protective effects 5,34,296 . The objective of this thesis was to investigate whether gonadal steroids could
potentially affect neuronal activity by altering the expression of the two principal
connexins found in the brain, connexin43 and connexin32. The following introduction is
composed of three sections, summarizing the role of gonadal steroids in the central
nervous system, the structure and regulation of gap junctions, and finally the gonadai
steroid regulation of gap junctions.
ROLE OF GONADAL STEROIDS IN THE CNS
Gonadal steroid hormones play important roles in the organization of the CNS
during fetal development, postnatal maturation, and adulthood. In addition to the
reproductively oriented effects on the CNS, such as the control of sex behaviour and
gonadotropin release, these hormones also demonstrate extensive actions on non-
l V ~ l V U I i L V L 1 V V I I I U ~ I I L l l L . C l 1 U l I U W I I U V I W L L I , l L L V I U U 1 1 1 6 I V C U I l l L l ~ ~ IbbUlll5 L U I U 3 b b l l C L l l U L ~ l l l C ) .
Estrogens, progestins and androgens exert widespread effects on the brain at the cellular
and molecular levels, which are believed to be involved in mediating the behavioural and
neuroendocrine effects of these steroids.
Hormonal effects on sex-steroid sensitive neuronal structures have traditionally
been categorized as either "organizational" or "activational" 4. Organizational effects
include estrogen and testosterone influences on neuronal development and neurocircuitry
formation, which in the adult CNS, lead to permanent structural differences between the
male and female. These sexual dimorphisms exist at rnany rnorphological levels 5,109,264 9
and include such hormone-dependent differences as nuclear volume, synaptic formation,
and neuronal connectivity. Activational effects are neuronal responses which are
impermanent, reversible, and dependent on the presence or absence of gonadal steroids in
the animal. Traditionally, activational effects have been associated with adulthood, in
which hormones exert effects on previously established neural circuits. However, it has
now been established that the rat can show at least some activational responses to gonadal
steroids early in life, while it remains sensitive to the organizational effects of those
steroids 76,290,29 1 . Activational effects in the CNS encornpass a wide range of
rnorphological as well as neurochemical parameters, including nuclear volume 42, neuro- 66 glial connections and synaptic remodeling . The key difference between activational
and organizational effects on neural architecture is that changes associated with
activational effects are relatively short-terni and dependent on the presence or absence of
circulaing hormones.
LOCALIZATION OF GONADAL STEROID RECEPTORS IN THE CNS
Estrogen receptors
Immunohistochemical and in vitro autoradiography studies have identified
estrogen-containing neurons in specific brain regions which are known to regulate
gonadotropin secretion and reproductive behaviour, namely the preoptic area (POA),
liypothalarnus and midbrain 82,229,234,249 In addition, in situ hybridization analyses have
demonstrated the wide-spread distribution of neurons containing estrogen receptor (ER)
1 1 1 1 1 1 ~ ~ III L I ~ C ~ ~ U U I L la1 U I ~ ~ I I I , a11u I ~ ~ ; U I I ~ L ~ I mi L I Y ~ . i ne uisrri~ur~on
pattern of ER mRNA in the adult brain is consistent with the distribution of estrogen-
concentrating cells and ER immunoreactive cells. No differences were observed in the
overall pattern of estrogen binding in fetal and neonatal brains as revealed by
autoradiography, but in situ hybridization profiles showed that on post-natal day 2 in rats,
ER mRNA is increased in the female POA, whereas the ER mRNA in males does not
change with age 53. There are several reports 53,l 17,118 that suggest that sex differences in
both preoptic area and hypothalamic ER density are at least in part the result of early
androgen secretion in the male.
Androgen receptors
Autoradiographic studies and in situ hybridization analysis have demonstrated an
extensive distribution of androgen-concentrating cells and androgen receptor (AR)
mRNA-containing cells, respectively, within the marnrnalian nervous system 230,23 1,237
Many areas of the CNS express high levels of AR mRNA, including the bed nucleus of
the stria terminalis, the arnygdaloid complex, the hypothalamic anterior periventricular
and arcuate nuclei, and the media1 POA 237. Certain regions of the CNS show parallel
expression patterns of both AR and ER, particularly in the hippocarnpus, amygdala, bed
nucleus of the stria terminalis, media1 POA, periventricular nucleus, arcuate nucleus,
ventromedial nucleus, and periaqueductal grey matter.
The intracellular cytochrome P450 enzyme aromatase converts testosterone to 17P-
estradiol, the metabolite which rnediates the majority of testosterone's effects within the
male CNS. Testosterone-derived estradiol is crucial for the sexual differentiation of the
central nervous system. Areas with the highest density of neurons containing both AR
inRNA and ER mRNA also express aromatase 22'. Imrnun~hi~tochemical studies reveal
aromatase-containing neurons in the media1 and periventricular preoptic nuclei, bed
nucleus of the stria terminalis, and amygdaloid nucleus during the late prenatal and early 235,236 neonatal phase . Maturation of the rat is paralleled by increased aromatase activity
101 in the lateral septum, bed nucleus of the stria terminalis and amygdaloid nucleus .
Studies carried out by Beyer and Hutchison 94195 have dernonstrated that dispersed ce11
~ U L L U L U J ~ ~ U L L U I U L ~ U 1 1 ~ 1 1 1 L A L L ~ 1 ~ 1 1 1 3 VI L I I L U L ~ U I I I L IIIILC L a n c u vil LIIC I J L I I uily VI ~ G S L ~ ~ L I U I I ,
before the presurned critical period for sexual differentiation, show a drarnatic sex
diffcrcnce in their subsequent development of aromatase activity. Hypotlialarnic cultures
derived from males show significantly higher levels of aromatase activity, as compared to
similsu: cultures from the female brain. This sex difference was not observed in cultures
derived from the cerebral cortex.
THE ROLE OF TESTOSTERONE AND ESTROGEN IN THE CNS
Antlrogen und estrogen action on the developing CNS
In the developing rat, the central nervous system is highly sensitive to the
organizational effects of estrogen and testosterone. Organizational effects are primarily
exerted during what is commonly referred to as the "critical period" for CNS sexual
differentiation. The "critical period" is broadly defined as the species-specific
developmental period during which the CNS is most sensitive to organizational effects of
testosterone exposure. Exposure to the hormone either earlier or later may require
supraphysiological sex steroid levels, or may be ineffective in terms of eliciting a
permanent response 40,75,292
The second key concept for the current understanding of C M sexual
differentiation is the mechanism of androgen action within the brain. Testosterone is the
principal sex steroid in males, and exerts cellular effects through three pathways: direct
binding to AR; the enzymatic reduction of testosterone by Sa-reductase, to form 5cc-
dihydrotestosterone (Sa-DHT) which binds to AR; and aromatization of the testosterone
A-ring, by aromatase, to yield 17P-estradiol which can bind to ER 139,140,191 . A critical
component of testosterone action on the brain is mediated through testosterone-derived
estrogen biosynthesis within neurons 165,191 . Blockade of either estrogen biosynthesis or
estrogen action interferes with normal defeminization of the brain in the male rat 164.165
However, the role of AR-mediated responses in the sexual differentiation of the brain
cannot be discounted. Despite the importance of locally-formed estrogen in rats and
mice, in other species estrogen may not be absolutely required for sexual differentiation
VI LIIC L L Y ~ . 1 VI C A U I I I ~ I G , 111 l c x l i a l c guwca p1g3 auu I L I U L ~ C Y ~ , LIIC C A ~ I C ~ > I U ~ I UI
masculine sex behaviour is mimicked by prenatal treatment with the non-aromatizable
androgen Sa-DHT 76.
One apparent contradiction to this estrogenic theory arises from the female
developmental pattern. Despite high levels of circulating estrogen, the female CNS does
not experience the sarne estrogen-induced masculinizing effect that is seen in the male. In
fact, CNS development in the female rat requires only relatively low levels of estrogens,
which contrasts with the high levels of estrogens required in the male to induce masculine 76 differentiation, gonadotropin secretion and sexual behaviour . In rats and mice, a
specific mechanism exists that "protects" the female fetus fiom the effects of circuIating
estrogen. Circulating a-fetoprotein (AFP), an estrogen-binding protein, is believed to
protect the female CNS fi-om these masculinizing effects of estrogen. Although AFP
exhibits a high affinity for estrogen, it does not associate with androgens, thereby
selectively preventing the access of estradiol to the female brain.
Sexual dimorphic nuclei in the brain
Structural differences between adult male and female nervous systems are sex-
steroid sensitive, and have been reported in many species including the rat, zebra finch
and human (Table 1) 2 1,113,253 . In particular, three sexually dimorphic nuclei have been
the focus of much investigation: the sexually dimorphic nucleus of the POA; the
anteroventral periventricular nucleus of the POA; and the spinal nucleus of
bulbocavernosus.
Gorski et ul. (1978) demonstrated a five-fold greater nuclear volume of the
sexually dimorphic nucleus of the POA (SDN-POA) in the male compared to the female.
Increased neuronal numbers in the male SDN-POA are attributed to the presence of
testosterone in the rat, during perinatal development. The aromatization of testosterone to
17p-estradiol seems to mediate the majority of the morpliological differentiation of the
nucleus 262. Prolonged administration of testosterone to fernale rats (embryonic day 16
tllrough postnatal day 10) increases the volun~e of the adult SDN-POA to Ievels of the
Avian Song rclatled nuclei Higher vocal center Robust nucleus of the archistriatum
Neocortex Primary motor cortex Sensorimotor cortex Visual cortex
Accessory olfactory bulb Bed nucleus of the stria terminalis Amygdaloid nucleus Hippocampus and dentate gyrus Anterior commissure Corpus callosum
Preoptic area Anteroventral periventricular nu. of POA Sexually dimorphic nu. of POA
Hypothalamus Interstitial nu. of anterior hypothalamus Suprachiasmatic nucleus Supraoptic nucleus Ventromediaf nucleus Arcuate nucleus
Massa intermedia
Substantia nigra
Locus coeruleus
Intermediolateral nucleus Spinal nucleus of bulbocavernosus Onuf s nucleus
TABLE 1. Regions of sexual dimorphism in the central nervous system. Al1 the indicated sex differcnces have been identified in mammals except those in the avian Song related nuclei which are found in avian song-bird species, such as the zebra finch. (Adapted from Kawata, 1995)
male rat, and similarly, castration of neonatal males reduces the volume of the adult
nricleus 74,78.98 . The SDN-POA is first appvent on embryonic day 20, yet the sex
difference in SDN-POA volume is only detected on postnatal day 1 'OD. Over the course
of the next ten days, the nuclear volume of the male SDN-POA continues to increase, 50,99 until reaching its adult size . Females undergo insignificant increases in nuclear
volume.
The anteroventral periventricular nucleus of the POA (AVPN-POA) is one of the
few sexually dimorphic structures that is larger in the female than in the male. The
female AVPN-POA contains more cholecystokinin-, thyroid hormone releasing factor-,
corticotropin-releasing factor-, and ER-containing neurons than males I 62,173,237,239
Males, however, have more enkephalin neurons in the AVPN-POA as compared to
fernales 238. Neurogenesis of the AVPN-POA extends fiom embryonic day 13 to 18 in
both sexes 197, and testosterone-derived estradiol may be the driving force for an apoptotic 3,252 demise of the AVPN-POA in males .
In adult male rats, the spinal nucleus of bulbocavernosus (SNB) is three to four
fold larger than in the female 26. In the neonatal female rat, the bulbocavernosus (BC) and
levator ani (LA) muscles that are imervated by motoneurons of the SNB undergo
atrophy, thereby resulting in a significant loss of SNB motoneurons 24.198 . The adult
female rat is completely devoid of either BC or LA muscles. Breedlove and Arnold
(1980) reported that androgen exposure during the critical period is important for normal
SNB development, by acting directly on the muscles to prevent atrophy. In the SNB of
mature rats, testosterone continues to exert activational effects on neuronal somata and
dendrites of motoneurons 26,120 , as well as the size and frequency of gap junctional
plaques between motoneurons 160,161
Activational effects of gonadal steroids on the CNS
In ternis of sexually differentiated neuroendocrine functions and behaviours, the
activational effects of circulating gonadal steroids have long been recognized '49. In the
adult male rat, testosterone-derived estrogeii is the primary activator of masculine sex
behaviours and aggression 44,165,179 . LocaIly arornatized androgens stimulate full
bupuIaLuLy UCl lav luuL 111 illç Illalc, a 1 1 c I I G L L WI1lC;Il 13 UIIIIlIllsIl~U Uy 1I1I110111UI1 U1 CSLrUgCIl
synthesis in the brain '79, or treatment with anti-estrogens. In female rats, estrogens also
exert a variety of effects on the CNS, including reguiation of gonadotropin and prolactin
secretion, activation of sex behaviour and the stimulation of neurogenesis and 149 synaptogenesis .
Accurnulating evidence suggests that the synaptogenic effects of gonadal steroids
are not restricted to the perinatal period, but also exist in adulthood 22. These activational
effects of testosterone and estrogen on neuronal connectivity and structure differ fiom the
earlier organizational effects in that they are transient and dependent on the current
hormonal status of the animal. The neural actions of steroids can be classified into two
distinct mechanisms. In the non-genomic mechanism, steroids effect a rapid, short-term
response, which lasts seconds to minutes, and may be mediated through direct interactions
with neural membranes 151,163 . Administration of gonadal steroids to brain tissue in vitro
or in vivo, can trigger rapid changes in electrical membrane properties, synaptic
transmission, or ion channel activity 151,181,294 . Alternatively, gonadal steroids can act via
the classical genome mechanism, to exert long-term effects, with a time course lasting
hours to days. This hormonal mechanism is thought to involve steroid activation of
intracellular receptors that regulate transcription and protein synthesis. For instance,
synaptic remodeling in the hypothalarnic arcuate nucleus has been shown to occur in
parallel with the estrous cycle of the fernale rat 1 89,20 1 . Further studies using deafferented
arcuate nuclei demonstrated an estrogenic induction of axonal sprouting and dendritic
spine formation in the adult as well as the aged brain 157,158 . In the adult male rat,
castration reduces the number of synaptic inputs on motoneurons of the spinal nucleus of 156 bulbocavernosus (SNB), which is prevented with testosterone replacement .
Androgens have been reported to induce synaptic rernodeling in the SNB 136,160 , as well as 26,120 inducing growth of perikarya and dendrites of SNB neurons .
In addition to their neuronal effects, gonadal steroids may exert part of their effect
through astroglia. It is clear that glial cells are also influenced by changes in the sex
steroid environinent '! II vvirro studies have dernonstrated the presence of receptors for
gonadal hormones in glial cells 105,106.127 . In fact, astroglial sexual dimorphisms have also
U C C I ~ SIIUWII I I I scvclal ura111 arcas, wiin uiiicrcnces in giiai rnorpnoiogy,
immunoreactivity and gene expression in vitro and in vivo 20,45,67,26s . Furthemore, glial
cells participate in the synthesis of endogenous steroids by the nervous system 104,171
Due to the close interactions between neurons and glial cells, it is reasonable to expect a
close communication between ce11 types via intercellular channels and/or secretion of
soluble factors. Several steps during the development of sexually dimorphic neuronal
networks may be regulated by glial cells, including proliferation, survival, migration and
functional maturation of neurons. Glial cells, in turn, may be affected by hormonally-
driven changes in neuronal activity 66.
In adult female rats, astroglia participate in the phasic synaptic remodelling that
takes place during the estrous cycle in the arcuate nucleus of the hypothalamus under the
influence of estradiol 20'. In adult primates, there is a horrnonally-induced astrocytic
ensheathing of hypothalamic neurons associated with modification of the number of
synaptic inputs to hypothalamic neurons 190,293 . Furthermore, astroglia appear to be
involved in the development of gender differences in synaptic connectivity. Sexual
dimorphisms in astroglial organization in the rat arcuate nucleus in situ are evident by 66 postnatal day 20, and occur following the exposure to perinatal androgens . By this
point of development, males already show a higher astrogliaI coverage of neuronal
surface than in females, a difference which may be related to the reduced nurnber of
synaptic contacts associated with neuronal soma.
It is abundantly clear that gonadal steroids exert extensive effects throughout the
central nervous system from the embryonic period through to senescence. In addition to
the regulation of rnorphology, comectivity and remodeling of neurons and glia, gonadal
steroids can also modulate intercellular communication between these cells. Gap
junctions are a potential target for gonadal steroids in certain sex-steroid sensitive neurons
and glia of the CNS 4 1.159.209 . Changes in cell-ce11 cominuilication processes may
represent an important component of the mechanisms by which gonadal steroids exert
their effects within the nervous system. The next section considers the structure and
fùi~ction of gap junctions in greater detail. Subsequent sections will then return
GAP JUNCTIONS
Gap junctions are specialized membrane channels that serve as sites of
intercellular communication, by forrning an aqueous passage for small ions and molecules
between neighbouring cells Iz6. Aside fiom a few terminally differentiated structures (e.g.
erythrocytes, lymphocytes), gap junctions have been identified in virtually all ce11 types in
both vertebrates and non-vertebrates. In fact, structures homologous to the mamrnalian
gap junction have been observed in higher plant cells, which use a similar communication
mechanism via plasmodesmata. Gap junction membrane channels possess a high degree
of syrnrnetry, and are structurally composed of two hemichannels, or "connexons". As
one connexon cornes into contact with a connexon fiom an adjacent ce11 membrane, a
continuous aqueous passage forms which enables small molecules and ions to move
between cells 143. The connexon itself is formed by a hexamer of "connexin" proteins
arranged around a central pore 270.27 1 . Current nomenclature for specific connexins is
designated by the abbreviation "Cx" followed by the moIecular mass in kilodaltons @Da)
as predicted by analysis of the cloned genes (e.g. Cx43).
The accepted mode1 of the connexon has been produced by a variety of imaging 3 5 techniques including thin section , negative staining 1 1,242,271 , low-dose cryo-electron
88 microscopy 270, X-ray diffraction ls3, and atomic force microscopy (Figure 1). Al1
nlethods consistently show the connexon to be a cylindrical water-filled structure
rneasuring 6-6.5 nm in diarneter with a pore 1.5-2 nm in diarneter, and a channel 1-1.7 nm
in length with less than 1 .O nrn protruding into the cytoplasmic space 269,270
Since the first description more than 30 years ago 49, gap junctional intercellular
channels have been implicated in numerous fundamental physiological processes, 3 6 including tissue homeostasis and ce11 growth '42, embryonic neural organization , and
the initiation of labour and parturition 177. AS well, aberrations in connexin expression
have been implicated in human pathological and disease States, such as cardiac
FIGURE 1. Illustration of a gap junction plaque formed between two apposing ce11 membranes. Each plaque is composed of a cluster of intrarnembranous channels. The intercellular channel forms a continuous aqueous passage extending between adjacent ce11 cytoplasm. Each connexon is a hexarneric structure composed of six connexin proteins. (Adapted from Beyer, E.C., 1993)
rnauurrnauons , oncogenesis , a aemyeiinaring aisoraer , ana more recentiy,
female infertility in mice 240. The following chapter serves as a general overview of the
structure, regulation and potential roles of gap junctions and their connexin subunits.
STRUCTURE OF CONNEXINS
Connexin gene structure
The structure of the comexin gene is remarkably similar in al1 members of the
connexin family, and is composed of one intron of variable size, flanked by an exon
sequence on either side 222. Exon 1 comprises the 5' untranslated region and is usually a
short sequence, approximately 100 base pairs (bps) long. The second exon encodes the
uninterrupted open reading frame, with the translational start codon characteristically
Iocated within the first 100 bps of exon 2.
Until relatively recently, the wide diversity of connexin proteins (i.e. at least 13
rodent connexin isoforrns, Table 2) was believed not to be associated with alternative 222 splicing, but rather was attributed to each comexin being encoded by one gene .
Recently however, O'Brien et al. (1996) reported the presence of an intron in the coding
region of skate Cx35. In addition, the promoters of mouse Cx26, rat and mouse Cx32 and
human Cx43 have been found to contain some consensus motifs characteristic of
transcriptional control. Furthermore, the Cx32 gene appears to undergo alternative
splicing and uses three promoters that are activated in a ce11 type-specific manner l94,24 1
In combination, these results suggest that the same connexin, expressed in different ce11
types, c m be regulated differently.
Chromosomal mapping has been perforrned on several connexin genes 63,92,289 7
from which it was concluded that genes for Cx37 and Cx40 are CO-localized on human
chromosome 1, Cx26 and Cx46 on human chromosome 13, and Cx32 on chromosome X.
The proximity of different connexin genes may suggest similarities in expression patterns
and functions 80.8 1.288 , however some CO-localization experiments on CO-expressed 189 connexin genes have indicated the contrary . There are not yet consistent results
Cx26 Hepatocytes, pancreatic acinar cells, mammary gland alveolar cells, keratinocytes, kidney, leptomeninges, pinealocytes, intestine
Cx30 Brain, skin Cx30.3Kidney, skin, preimplantation embryo Cx31 Kidney, keratinocytes, trophectodemi, preimplantation blastocyst Cx3f .l Keratinocytes, preimplantation blastocyst, squarnous epithelia Cx32 Hepatocytes, pancreatic acinar cells, mammary gland alveolar cells,
neurons, oligodendrocytes, Schwann cells, thyroid fo!licular cells, proximal tubules
Cx33 Testes Cx37 Endothelium, keratinocytes, heart, stomach, testes, cortical neuroblasts Cx40 Endotheliurn, conductive myocardiurn (His bundle, Purkinje fibers),
preirnplantation blastocysts Cx43 Cardiac myocytes, smooth muscle, endothelial cells, marnmary gland
myoepithelium, testes, lens and corneal epithelium, pancreatic p-cells, keratinocytes, preimplantation blastocyst, thyroid follicular cells, astrocytes, granulosa, fibroblasts, osteocytes
Cx45 Heart, intestine, kidney, lung, skin, preimplantation blastocyst, embryonic brain
Cx46 Heart, kidney, lens fibers, Schwann cells Cx50 Lens fibers, corneal epithelium, atrioventricular valves
TABLE 2. Distribution of connexins in various rodent tissues (Adapted from White and Bruzzone, 1996; Bruzzone et al., 1996)
regulation of expression.
Connexin protein structure
Membrane protection assays using protease and antibody labelling techniques
have illustrated the topological similarities of connexin isofonns, forming the basis of the
folding structure s h o w by al1 connexins. The connexin protein traverses the membrane
four times and c m be broadly divided into three functional domains: the hydrophobic
transmembrane domains thought to form the intercellular channel; two extracellular loops
that fùnction as cell-ce11 recognition and docking sites; and the cytoplasmic domain that
seerns to modulate the physiological properties of the channel (Figure 2).
Within the connexin molecule, there are both highly variable and conserved
regions. The divergent regions comprise a major part of the cytoplasmic loop and
carboxy tail, both in ternis of sequence and length. X-ray diffraction measurements of
Cx32 and Cx26 show that this cytoplasrnic surface extends out to about 90 A fiom the
middle of the gap 152.154 . These variable regions may confer specific channel 19 conductances and distinct regulatory properties to different connexins . For instance,
the cytoplasmic loop ranges fiom 37 amino acids in Cx31.1 to 70 amino acids in Cx45,
while the cytoplasmic tail ranges from 18 arnino acids in Cx26 to 21 1 amino acids in
(2x50 288. This variability in length of the carboxy terminus may result in part from point
mutations inserting or removing a stop codon. Sequences near the end of the carboxy
terminals contain serine and basic amino acids which are putative phosphorylation sites 223
In contrast to the variable structure of cytoplasmic domains, transmembrane
regions are highly conserved regions of the connexin molecule. Each of the four
transmembrane domains (M 1 -M4) are organized in an a-helical fashion of approximately
20 ainino acids in length, as suggested by their hydrophobicity profiles. In fact, it is
likely tliat some of the a-helical structure extends past the bilayer into the extracelluiar 26 1 176 domains . The current mode1 based on connexin sequencing , suggests
FIGURE 2. Structural topoloçy of a connexin protein within the plasma membrane. Each connexin protein contains 4 transmembrane, 7 extracellular, and a short N-terminal domain which are highly conserved. The cytoplasmic loop (A) and C-terminal domain (B) are variable in sequence and size among different connexins. (Adapted frorn Beyer, E.C., 1993)
iiiai m e pu~aiivt: l v u arnpnipainiç neilces nom eacn or rne six connexin suwnxrs
contribute to form the wall of the channel pore.
The two extracellular loops (El-E2) are the most highly conserved regions of the
connexin molecule, each being approximately 40 amino acids in length. Within each
extracellular domain, there is a strictly conserved set of three cysteines, which forrn
disulfide bonds within individual connexins 57,103 . Extracellular loops are postulated to
play a key role in cell-ce11 recognition and docking of connexon hemichannels in
neighbouring membranes 222. Specific conserved amino acid sequences in the 277 extracellular domains have also been implicated in voltage sensitivity , and in the
binding of calcium ions for connexon docking 208.
STRUCTURE AND MODULATION OF GAP JUNCTIONS
Formation of gap junctions
Despite over three decades of research, the regulation of gap junction formation,
stability and removal is still far from clear. There are two features of gap junction
proteins, however, that contrast with other integral membrane proteins. First, connexon
assembly appears to occur within the tram-Golgi network I s 8 , a process which for other 93 integral membrane proteins, usually takes place within the endoplasmic reticulum .
61,123 Second, gap junctions display a remarkably rapid rate of turnover . The assembly of connexins into gap junctions involves a lengthy and complex
sequence of events: connexin oligomerization into connexons, post-translational
modification, and finally transport and insertion into the plasma membrane. Then, the
connexon of one ce11 joins in mirror symmetry with a connexon in the adjacent ce11
membrane to form an intercelldar channel, and finally the connexons aggregate into a
gap junctional plaque. Although connexin oligomerization into connexons has been
clarified as an event localized to the tram-Golgi network I s 8 , most of the late events
involving connexon trafficking, docking and the formation of gap junction plaques still
remain obscure.
Integration into the rough endoplasmic reticulum (rER) membrane is commonly
the first event in the biosynthesis of an integral membrane protein, such as a connexin
. ~ U W G V G I , ln w r u siuuics usirig uug paric;rt;aiic; r~iic;rusu~riai rricrnoraries suggesl inai
the insertion of connexin into the rER is accompanied by additional processing by a signal
peptidase 60. This processing is believed to occur as a result of a cryptic signal peptidase
cIeavage site within the cornexin sequence that is not normally used in the cell. Another
key difference between gap junction proteins and other integral membrane proteins is the
occurrence of post-rER oligomerization in the tram-Golgi network. In general, folding
and assembly of rnost integral proteins occurs in the rER 93. Connexins, however, seem 188 to accumulate in the Golgi apparatus at least in vitro and possibly in vivo . Late
assembly of gap junction proteins may be a mechanism to avoid accidental pairing of two
connexons that could lead to the formation of intra-reticular gap junction channels.
The process of connexon transport from the Golgi complex to the membrane is
still largely unclear. Post-translational modification of the connexon protein is believed
to occur en route to the membrane surface. Puranam et al. (1993) used rat cardiomyocyte
cultures to investigate the sequence of Cx43 phosphorylation. The initial phosphorylation
of Cx43 occurs in the rER or at the cis-media1 Golgi fiom the 40-kDa to the 4 1 -kDa forrn,
and eventually undergoes more extensive phosphorylation to 42-kDa and 44-kDa forrn at
locations distal to the trans-Golgi cisternae. It is not clear whether phosphorylation of
Cx43 occurs this early in al1 cell types, nor whether the reaction is critical for trafficking
or correct assembly. Phosphorylation is not essential for al1 connexins as there are
instances of functional non-phosphorylated connexin isoforrns (Le. Cx26).
Both docking and aggregation of connexons require communication between
adjacent cells. In order for docking of connexons to occur, the membrane of each
interacting ce11 must be very close together before intercellular gap junction channels c m
form. Furthermore, the docking and aggregation of connexons in one ce11 membrane
should be matched with comparable events occurring in the apposed cell membrane.
Thus, there must be some form of interaction across the intervening extracellular space.
Accumulating evidence indicates that cell adhesion molecules (e.g. cadherins) have an
important influence on gap junction formation and communication between cells. Kanno
el (11. (1984) reported that treatment of teratocarcinorna cells with antibodies against E-
cadherin inhibits dye transfer between the ceIls. Alternatively, transfection of a poorly
L U U ~ K U C ; ~ H iint: wirn c-cauncrin grcauy inçreaseu çoupiing , as aemonstrarea i>y rne
appearance of phosphorylated connexons in gap junction plaques in regions of cell-to-ce11
contact "'. In regenerating hepatocytes, an E-cadherih-catenin complex provides the
close intercellular contact necessary for docking of connexon hemichannels, and can act
to generate an intracellular signal to initiate and regulate the synthesis, oligomerization
and translocation of connexins 65.
Following connexon docking in the membrane, most connexons remain closed
until they form a complete intercellular channel with a partnered connexon Ig8. In fact,
after arriva1 at the ce11 surface, there is a rapid formation of intercellular channels upon
celi contact. There have been reports, however, of connexons that are functionally
competent to switch between open and closed states without joining to a juxtaposed 204,285 connexon , though this is not a common occurrence. Stability of gap junction
channels is achieved when several channels cluster to form a gap junction plaque, the size
of which can be over 1 pm in diarneter. Plaque formation has also been correlated with
extensive phosphorylation of Cx43 18'. Oh et 02. (1993) showed that incompletely
phosphorylated species of Cx43 could also assemble into gap junction plaques although
these channels were not functional.
The removal mechanisms of gap junctions from the ce11 surface are poorly
understood. Several studies indicate that gap junctions are removed frorn the membrane
by internalization of the entire junction within one of the coupled cells, thereby forming
"annular" gap junctions 128,130 , which eventually degrade in lysosomes 129,274 . Fujimoto et
al. (1 997) present further findings from regenerating mouse hepatocytes that suggest that
during the disappearance of gap junctions, most of the actual plaques are also broken up
into smalIer aggregates, internalized and finally degraded. Alternatively, degradation may
occur via a non-lysosomal mechanism. such as an endopeptidase 12.
The other key feature that sets gap junction channels apart from other integral
membrane proteins, is their remarkably higli rate of turnover 300. Gap junctions are not
static long-lived structures but actually undergo a continua1 process of assembly and
degradation, which serves as a regulatory mechanism of intercellular communication. In
V L V U SLUUIGS WILII LL 1 vIuruurli1ic laociirng revcarcu nair-iize rimes or rive nours ror 6 1 mouse liver gap junctions . Pulse-chase experiments revealed that Cx32 and Cx43 have
half-lives as short as 1.5-3.5 hours 122,123,267
Compatibiiity of interactions between connexhs
The multiplicity of connexins that exist within each species suggests that the
number of physiologically distinct channel types that could theoretically exist is immense,
in the magnitude of the hundreds. In fact, most cells express more than one type of
connexin, giving rise to ei-iher heteromeric (composed of two or more connexins) or
homomeric (single connexin species) connexons. Further, each gap junction channel c m
be classified as either heterotypic (joining of two homomeric connexons made from
different connexins), homotypic (two homomeric connexons made from the sarne
connexin), or heteromeric (heteromeric charinel associated with either another
heteromeric connexon or homomeric connexon). Selectivity of channel formation is
displayed by a11 members of the connexin family, and may play a critical role in the
establishment of communication boundaries.
Despite the enormous potential for heterogenous gap junctions, compatibility of
connexins within a channel is a limiting factor. The Xenoptrs oocyte expression system is
often a mode1 of choice to elucidate the properties of individual connexins. Several
studies show that juxtapositioned cells that express different comexins have little
probability of assernbling gap junctions 284. Most connexins are able to form channels
with approximately half of their tested partners, although some extreme exarnples of
compatibility exist: Cx3 1 only establishes homotypic channels, while Cx46 interacts with
five out of six partners tested. The pattern of compatibility of any given connexin,
however, is as yet unpredictable.
The structural locus that controls connexin compatibility has been identified as the
extracellular E2 domain 28! For example, Cx43 functionally interacts with Cx46 but not
Cx50. Swapping the E2 domains of Cx50 with that of Cx46 inverts the ability to form
heterotypic channels with Cx43, such that the Cx50 chimera with the E2 region from
Cx46 is able to interact with Cx43, and the opposite is true for the Cx46 chimera.
w ~ l t t i l l t ; ~ ail S G I ~ ~ C L I V C ; CUIUIGXIIIS U I S C I I I I I I I I ~ L ~ : U ~ ~ L W G G I I U I ~ L G I G I I L p a ~ i ~ i e r s U I ~ L M D a s i s 0 1
their E2 sequences remains to be determined.
The ability of connexins to discriminate between one another rnay have profound
biological consequences in normal physiological and pathological situations. Multiple
connexin expression may provide a powerfùl mechanism to limit or segregate, rather than
facilitate comrnunication, such as that seen in "comrnunication compartrnents" 29. These
compartments are characterized by clusters of cells that communicate within, but not
between, adjacent groups of cells, and are frequently observed during developmental
differentiation. The expression of incompatible connexins could achieve the
compartrnentalization required without comprornising cell-to-ce11 contacts. Gap junctions
expressing different connexin components demonstrate unique properties of unitary
conductance, ionic permeability, size cutoff and voltage gating 27,247,275
Control of gap junction permeabil@
Traditionally, gap junctions have been thought of as relatively non-selective
channels, allowing the bidirectional transfer of most ions and molecules smaller than
1000-1200 Da. However, recent evidence indicates that size selectivity is a connexin-
specific feature. The molecular basis of selective ionic perrneabilities between connexins
has been hypothesized to be the first extracellular loop which displays differences in net 13,3 1,287 charge between comexins . Steinberg et al. (1994) compared intercellular
communication in two ce11 lines of osteoblastic origin, and deterrnined that despite
equivalent levels of electrical coupling, the transfer of microinjected Lucifer Yellow (457
Da) differed between cells expressing Cx43 or Cx45. Another series of experiments 27h
utilized a communication-deficient ce11 line transfected with Cx37, Cx40, Cx43 or Cx45,
in order to restore corninunication. Differential permeabilities were observed between
cells transfected with different connexins, in relation to both 6-carboxyfluorescein (376
Da) and to chloride ions. Together these resuIts imply that intercellular channels may
exhibit inuch grcater selectivity for smail molecules and ions than previously thought.
One consequence
connexons nlay display
of permeability differences is that cells expressing heteromeric
the dominant phenotype imposed by one connexin. Thus,
U ~ I ~ ~ ~ I I I I C : G I I ~ I I ~ G S III LIIG IGVGIS UI LUIUIGXIII G X ~ I G S S I V I I III UIK ~ I V U ~ UI GGIIS may r~prcs~ri i
an additional means to modulate their communication. Gap junction perrneability has
also been shown to be reversibly altered by a variety of independent factors, including
intracellular calcium, pH, cyclic AMP, and connexin phosphorylation.
Intracellular Calcium:
Calcium (ca2+) rnay play a dual role with respect to gap junctions, both in gating
of the channels, as well as being transferred by diffùsion through the channel. Junctional
uncoupling occurs following elevations in intracelluiar calcium concentration ([ca2+ J)
from IO-' to IO-' M 142,219 . However, while an increase in [ca2'] has been correlated with
reduced gap junctional communication, this occurs at high levels of [ca2+], that are
probably observed only in some coupled cells, such as heart myocytes.
There have also been numerous reports that alterations in [ca2+] and intercellular
propagating waves of free ca2' may play a role in a number of other important
physiological activities. Several ce11 types can propagate ca2+ waves across
communicating cells, which appears to rely on intercellular perrneability to inositol 1,4,5-
trisphosphate (IP,), the ca2+-releasing messenger 224,228 . These oscillatory changes in
[ca2+] may serve to coordinate neuron-glial ce11 interactions. Nedergaard (1994)
observed a direct gap junctional communication of ca2+ between astrocytes and neurons,
as dernonstrated by the ability of altered levels of intracellular [ca2+] to be transferred
between cells. One inconsistency however, is the apparent incompatibility between
astrocytic Cx43 and neuronal Cx32, as illustrated in the Xenopus oocyte system 287. The
lack of interaction between Cx32 and Cx43 as reported by White and colleagues,
contradicted earlier reports of heterotypic channel formation by these two connexins 254,283 . The major difference between these studies was the elimination of endogenous
Xenopzts Cx38 by White et al. (1995), a step which prevents coupling of (2x43-injected
oocytes to uninjected cells. When Xenopus Cx38 is eliminated by injecting oocytes with
antisense oligonucleotides, cells expressing Cx43 do not demonstrate electrical coupling
with cells expressing Cx32 242.
IlIuaLcllulal VI 1.
Gap junctional sensitivity to intracellular pH varies according to the system
studied. Most connexons close in response to intracellular acidiflcation, resulting in a
reduction in gap junctional intercellular communication at pH 6.5-6.8 220. This sensitivity
to intracellular pH is, however, connexin dependent. The length and sequence of the
middle cytoplasmic loop and carboxy terminal tail play a critical role in this form of
chemical gating. Embryonic cells are most sensitive in the region of pH 7, while liver gap
junctions only respond when the pH falls below 6.5 245. Further, rat Cx32 is weakly
sensitive to acidification, whereas Xenopus Cx38 rapidly closes with decreasing
intracellular pH. The physiological significance of these responses to changes in pH is
indeterminable however, since the intracellular pH is thought to be tightly regulated at pH
7.0-7.4 91.
Intracellular CvcIic AMP (CAMP);
The intracellular second rnessenger, CAMP, plays an integral tissue-specific role in
the regulation of ce11 coupling. Increased levels of [CAMP] c m either resuit in an increase 33,227 in junctional conductance, as in rat hepatocytes and heart myocytes , or else reduced
conductance, as in rat myometrial cells, retinal horizontal cells, and Sertoli cells The
reason for this range of responses to cAMP in different tissues has yet to be elucidated.
Junctional permeability may be modulated by cAMP via the phosphorylation of connexin
proteins through a CAMP-dependent protein kinase.
Phos~horvlation of Connexins;
Phosphorylation on serine, threonine and tyrosine residues is a comrnon
mechanism used by extracellular signals to gate ion channels. Phosphorylation represents
a post-translational mechanism that specifically targets these residues in the cytoplasmic
tail of connexin proteins, eventually resulting in a modification of intercellular
communication. The mechanism by which this type of modification alters Sap junctional
intercellular communication is still unclear. Although phosphorylation-induced structural 186 modifications of connexin may function to open or close gap junction cl~annels , the
p I V b C i 3 3 V I ~ I L V ~ ~ L L W L J L U L I W L I I l l U Y U 1 3 U b A b l L R b y L l l C C l L S ClL LllC l C V C l buIIllCAVI1 da>CII luIY,
intracellular transport or degradation processes ls5.
Evidence for a role of phosphorylation stems from studies demonstrating that
agonists or antagonists of CAMP-dependent protein kinase (PKA), protein kinase C
(PKC), calmodulin/~a~'-dependent kinase, and tyrosine kinases rnodulate gap junctional 186 communication . The activation of these protein kinases is kinase- and connexin-
dependent 121,180,255 . Several connexins have been identified as phosphoproteins (e.g.
Cx32, Cx43, Cx46, Cx50) 43.62.225 . Phosphorylation-induced changes in permeability and
unitary conductance are also connexin-specific, demonstrating a positive correlation
between frequency of lower conductance States and decreased permeability 121,180
Serine threonine phosphorylation
The phosphorylation of connexin was first demonstrated by the metabolic 226 labelling of Cx32 with [P'~] orthophosphate in primary hepatocyte cultures . The
addition of 8-bromo-CAMP increased gap junctional conductance, and enhanced the
incorporation of [P"] into Cx32 serine residues, though without a simultaneous increase 267 in levels of Cx32 protein . Phosphorylation of serine residues in Cx32 is also
modulated by CAMP-dependent protein kinase (PKA) in isolated rat liver gap junctions 226 . Taken together, this evidence suggests that the phosphorylation of Cx32 by PKA may
play a further role in gap junctional intercellular communication. In rat hepatocytes,
Cx32 has also been shown to be phosphorylated by another serinelthreonine kinase,
protein kinase C (PKC) 257. The significance of PKC-rnediated phosphorylation of Cx32
is questionable, however, as the issue of cell-ce11 coupling was not addressed.
Cx43 is another ubiquitous connexin species that is post-translationally serine
phosphorylated 43,184 . The serine kinase(s) that may be responsible for Cx43
phosphorylation events has not yet been identified, although there are several candidates,
including protein kinase C. A number of studies have demonstrated that the activation of
PKC by phorbol esters tumour promoters (TPA) is correlated with a reduction of gap
junctional communication as well as the serine phosphorylation of Cx43 223.
The importance of the post-translational conversion of Cx43 to its fully
phosphorylated form lies partly in its ability to provide junctional competence. Musil et
al. (1990) performed studies using a communication incompetent ce11 line that
constitutively synthesized Cx43, yet failed to accumulate it on the ce11 surface or
phosphorylate it to its mature forrn. Transfection of the ce11 line with the ce11 adhesion
rnolecule, E-cadherin, led to phosphorylation of Cx43 to the mature forrn, expression of 185 ce11 surface gap junctions, and renewed communicational abilities . Contradictory
studies by Musil et al. (1991) later reported that non-phosphorylated Cx43 was, in fact,
able to be exported to the ce11 surface.
Further evidence for a role of serine phosphorylation of Cx43 comes from genetic
mutations in patients with visceroatrial heterotaxia syndrome (VAH), a disease associated
with cardiac abnorrnalities. Analysis of the Cx43 gene fiom these patients indicate point
mutations in serine and threonine residues believed to be involved in c o ~ e x i n
phosphorylation 28.
Tyrosine phosphorylation
Cx43 is specifically phosphorylated on tyrosine residues in the presence of pp60v- src (v-Src), a 60,000 MW plasma-membrane associated tyrosine protein kinase encoded by
a viral oncogene. This method of tyrosine phosphorylation inhibits junctionai
communication 617. Overexpression of activated forms of the cellular Src protein in N1H
3T3 cells also results in reduced gap junctional communication ', and is accompanied by
the rapid accumulation of phosphotyrosine on Cx43 43162. Furthermore, this loss of gap
junctional communication was neither due to a major loss of Cx43 gap junction plaques,
nor to a reduction of Cx43 expression. In fact, elevated levels of Cx43 mRNA and 72,246 protein were observed in v-Src-transformed cells . The inhibition of gap junctional
communication in Xenopus oocytes, CO-expressing v-Src and Cx43 mRNA was shown to
be dependent upon the phosphorylation of Cx43 on tyrosine 265 255 . Another rnernber of
the Src farnily of tyrosine kinases, v-Fps, also disrupts gap junctional communication and I l9 stimulates the phosphorylation of Cx32 on tyrosine . These studies suggest that the
b u 1 L b l U L L W L L U b L V V b b L L V - U A - ~ L L U J ~ L L V L J L U L L W L L V A LJ L V J I L L W L W J L U U b L I V I 1 U A 7 - I U L L U L L l W
disruption of junctional communication, rnay be due to altered intercellular gating 13'.
GAP JUNCTIONS IN THE BRAIN
Evidence for gap junctional coupling between the diverse cellular compartments
of brain tissues has been documented by a variety of different techniques 47, including
structural mapping, functional analyses, and dye coupling. Results suggest that the
degree of coupling is not a static phenomenon, but is subject to plasticity regulated either
by developrnental or functional factors, such as neurotransmitter effects. Gap junctions
and dye coupling have been documented in many brain regions including the
hippocampus 10,150 , cerebral cortex 77, and retina '67. There are at least six different
connexins expressed and widely distributed in the marnmalian brain with Cx43 expressed
abundantly in astrocytes 48,174.299 , and Cx32 found at lower levels in neurons and
oligodendrocytes 48. Gap junctions can be dynarnically regulated in the CNS. Calcium
ions can reduce intercellular coupling 216, as can arachidonic acid 178, diacylglycerol 14, 244 phorbol esters '24, and acidosis . The neurotransmitter, dopamine, uncoupled
electrotonic junctions in the retina lb7. Astroglial gap junctional communication is
increased by glutamate or high potassium ion concentration 59.
The physiological relevance of gap junctions within the CNS is poorly understood
47. Gap junctions between neuronal cells are believed to mediate synchrony among active
cells or rapidly relaying signals from pre- to postsynaptic elements 17,54,14 1 . Neuronal
junctional communication may also be important in second-messenger exchange between
coupled cells, as indicated by the diffbsional ability of ca2+, CAMP, and IP, Yang et al.
(1 990) proposed that such a mechanism could modulate presynaptic neurotransmitter
release by postsynaptic second messenger molecules. Astrocytic gap junctions probably
provide a direct pathway to the site of potassium ion (K') disposa1 into the perivascular 68,1 16,195 compartments . However, astrocytic coupling may also increase the volume of the
buffer sink, thereby allowing the astrocytic coillpartnlent to sliare the load of a liighly
active adjacent neuron 11 1 , I 16 . Further studies demonstrate both induced and spontaneous
VH V I U V VY Y y i I I V U U I . A b C I I L V U & S A V U & LllV UU'- IVVJ L I V I L U C V V V L A \ 7 I I I U L w u L a V b a sllu1
communication pathway that may play an active role in neuromodulation 272,273
206 Neural development is associated with increased electrotonic coupling , and
exhibits temporai changes in the expression of neuronal connexins, starting with Cx26
prenatally and ending with (2x32 postnatally 48. Also, neuropathological conditions such
as ischernia 90 and spreading depression, which is associated with epilepsy 84, have been
associated with changes in gap junctional protein levels. A direct intercellular 150 communication pathway was identified in hippocampal CA3 neurons . Baimbridge et
al. (1991) demonstrated that epileptiform bursts from depolarizing currents in CA1
neurons were seen only in dye coupled neurons. As well as the presence of junctional
communication in neurons, there is evidence of gap junctions in interneurons in the 114 hippocarnpal CA1 region 'O8 and the dentate gyms . Electrotonic coupling of
interneurons could be critical for epileptogenesis. Naus et al. (1991) demonstrated the
increased expression of Cx43 mRNA in sarnples of temporal lobe neocortex obtained
from patients with intractable seizure disorder. Significantly lower levels of Cx43 mRNA
were present in the peritumoral tissue. Similar changes were observed in Cx32 mRNA
levels, although the changes were not as drarnatic.
FUNCTIONAL ROLES OF GAP JUNCTIONS
The wide distribution and modulation of connexins throughout the rnajority of
cells and species, is suggestive of their fundamental and wide-spread importance for the
basic physiology of the species. Gap junctions have important roles throughout the body,
including: metabolic cooperation and tissue homeostasis 73,2 12,222. , electrical coupling 69,268. , ernbryogenesis 278; tissue response to hormones 133,222, , growth 142,170,222. , and labour
39.177 initiation and parturition . Clearly, gap junctional intercellular communication is an
essential element of most animals, and recently, interest has grown as to the possible
implications of aberrations in the gap junctional system.
The first creation of a connexin-deficient rnouse lacked the gene for Cx43 2 1 7 .
Surprisingly, the mice embryos were found to survive to term but died shortly after birth.
The animals exhibited anatomical defects in the heart, where the right ventricular outflow
tract was severely enlarged and the ventricular charnber was filled with septa that
prevented the normal cardiac output to the pulmonary circulation. As a result, passage of
blood from the right ventricle to the pulmonary arteries was irnpeded, thereby preventing
postnatal oxygenation. This putative link between junctional communication and cardiac
development is supported by certain human familial cardiac anomalies, which are also
associated with connexin mutations. Cx43 mutations have been found in patients with
visceroatrial heterotaxia syndrome (VAH) 28, a disease characterized by defects of
laterality, including atrial and bronchopulmonary isomerism, transposition of the great
arteries, and often asymmetries of the abdominal viscera. Analysis of the Cx43 gene from
patients with VAH identified point mutations in serine and threonine residues is believed
to be involved in connexin phosphorylation 28.
More recently, Simon et al. (1997) were able to engineer a Cx37 knock-out
mouse, which exhibits female infertility. Oocytes in (2x37" animals were found to lack
both recognizable gap junctions and intercellular communication with granulosa cells.
The homozygous Cx37-deficient mice failed to develop mature (Graffian) follicles, were
anovulatory, and showed an inappropriate formation of corpora lutea. Furthermore, lack
of junctional communication caused an arrest of oocyte development before meiotic
competence was achieved. Taken together, these observations suggest that gap junctional
communication may be critical for successful oogenesis and ovulation.
Abnormalities of the Cx32 gene has been associated with the X-linked form of
Charcot-Marie-Tooth disease (CMTX), which is a demyelinating disorder of the 18 peripheral nervous system . The Cx32 gene in CMTX patients exhibits a total of 42
mutations, and analysis using Xenopus oocytes indicate that these mutations are
responsible for channel inactivity 32. Despite the wide expression pattern of Cx32 203, the
clinical manifestations of CMTX are restricted to the peripheral nervous system. It is
possible that, except in myelinating Schwann cells, alternative connexins rnay compensate
for the Ioss of function due to Cx32 mutations. Some of these mutations, however, act as
seleclive dominant negativc inhibitors, when coexpressed in paired &nopus oocytes with
compatible connexins 32. If tliis situation occurs in vivo, the ability of Cx32-expressing
GONADAL STEROID REGIJLATION OF GAP JUNCTIONS
GAP JUNCTIONS IN THE UTERUS
Gap junctions in the uterus are generally undetectable with the exception of those 177 that are up-regulated during late gestation and parturition . For this reason, most
investigations of gap junction reguiation in the uterus have focused on their role in the
synchronization of muscular contractions during labour. Elevated levels of gap junctions
accommodate the greater need for electric activity and synchronized uterine contractions
for an effective expulsion of the fetus. Recently, Risek et al. (1995) exarnined gap
junction regulation by estrogen and progesterone in the immature rat uterus, thereby
enabling a separation of the regulatory actions of estrogen and progesterone. Risek
reported an estradiol up-regulation of Cx43 expression in the myometrium and
endometrial stroma, which was entirely suppressed by progesterone . Gap junctions occur in the uterus during pregnancy in a number of species,
including the rat, sheep, and hurnan 70,145,256 During most of pregnancy, gap junctions are
undetectable. However, approximately 24-48 hours prior to labour in the rodent, gap
junction Cx43 mRNA and protein levels increase dramatically in the myometrium, with
maximal levels occurring during delivery itself 2'0. In fact, parturition does not
commence in rats until Cx43 protein is assembled into functional gap junctions in the
myometrial plasma membrane. Further studies in sheep 147 and humans 39,256
dernonstrated similar increases in Cx43 mRNA and protein with the onset of labour. In
both pregnant and non-pregnant animals, there is also evidence that Cx43 synthesis and/or
trafficking is regulated positively by estrogen and negatively by progesterone 39,148.2 10.21 8
Similarly, Cx43 protein levels are also modulated by estrogen and progesterone in rat
myometrium. Western analysis suggests that estrogen treatment is correlated with a shifi
from the un- and mono-phosphorylated forms of Cx43 protein, to the di-phosphorylated 37 form . The administration of progesterone in conjunction with estrogen reduces the
estrogen-induction of Cx43 phosphorylation, and switches the banding pattern back to the
unphosphorylated state.
The exact mechanism of gonadal steroid action on myometrial cells to induce
increased contractility and excitability during labour is unknown. Estrogen is known to
directly increase transcription of the Cx43 gene as a result of interactions between the
dimers of the ligand bound receptor and the putative estrogen response element (ERE)
within the promoter region of the (2x43 gene 13*. However, estrogen could also act
indirectly by inducing the synthesis of nuclear transcription factors (e.g. c-myc, c-fos and c-jun) 183,280,282 . These transcription factor protein products may in turn act through the
137 putative activator protein-1 (AP-1) sites on the promoter region of the Cx43 gene , to
increase transcription of Cx43. The c-fos and c-jun genes are of particular interest since
Lefebvre et al. (1995) identified an AP-1 site within the murine Cx43 promoter. The c-
fos protein has a leucine zipper motif that promotes dimerization with other oncogene
products, most comrnonly with the jun farnily (c-jun, jun B, and jun D). The AP-1 factor
is cornprised of specific combinations of the Jun and Fos protein families "". Binding of
these protein dimers occurs at a specific DNA AP-1 binding site, also called the TPA
response element (TRE) which is present in many genes 2, including the rnurine Cx43 137 gene .
As outlined previously, the ability of estradiol to enhance the expression of
myometrial Cx43 and its importance in parturition has been well documented 39,148.2 1 O
While the 5' Cx43 promoter region displays several potential EREs and is responsive to 3 02 estrogen , these response elements do not conform to consensus sequences, and
cornparisons between rat and human 5' regions illustrate that these EREs are not well 46,302 conserved . In contrast, there is one AP-1 binding site within the Cx43 promoter
withi.n 200 base pairs of the transcription start site that is highly conserved among mouse,
rat and human genes 38,46,25 1,302 . This is of interest because estrogen has been reported to
increase the expression of the mRNA- and protein-encoding fos and jun in the non- 1 12,280 pregnant uterus . Petrocelli and Lye (1993) provide associative evidence of an
estrogenic up-regulation of Cx43 gene expression through a protein cascade initiated by
W J L L V ~ W L Z , I W L ~ U L L L L L ~ L A A b u r O J ~ L L L L ~ L ~ L ~ ~ V L L LA - 1 I U U L V L ~ ~ V V A A L W L L U W L L A I I V U ~ L L b~7-1 n 1 - 1 J ~ L ~ O LU
stimulate transcription. Further data consistent with this hypothesis was provided by
Piersanti and Lye (1995), who demonstrated that estradioi administration to
ovariectomized rats resulted in a significant increase in Cx43 mRNA, which was preceded
by elevated expression of c-fos and c-jun.
The expression of c-fos and c-jun may also be subject to regulation by
progesterone. The presence of putative progesterone response elements (Pm) in the
mouse gene 302 may be important since estrogen-induced expression of Cx43 and the 210 normal prepartum increase in Cx43 mRNA can be blocked by progesterone .
Progesterone blocked the estrogen-induced increase in c-fos mRNA in whole rat uteri '12,
inhibited basal c-jun expression in the chick oviduct 250, but had no effect on c j u n
expression in whole rat uteri 281. Thus, the ability of the Cx43 gene to respond to both
estrogen and progesterone could be conferred through the AP-I cis-acting element.
Estrogen rnay also regulate Cx43 expression by enhancing the stability for its mRNA 163,
as suggested by the repeated expression of a sequence on the 3' region of Cx43 mRNA,
which is known to increase mRNA instability. In addition to the possibility of a direct
action via PREs, progesterone may also influence Cx43 gene transcription by decreasing
the synthesis of estrogen receptors, or preventing the binding of stimulatory agents to the
Cx43 promoter.
GAP JUNCTIONS IN THE CNS
By cornparison with the extensive literature on hormonal regulation of connexin
expression in the uterus, relatively little is known about how gonadal steroids affect gap
junctions in the CNS. The first demonstration of hormonal regulation of connexin mRNA
expression in the central nervous system was by Matsumoto el al. (1991), who examined
the androgenic regulation of gap junction mRNA expression in the androgen-sensitive
motoneurons of the spinal nucleus of the bulbocavernosus (SNB). Castration of male rats
was found to reduce the density of signals for hybridizable Cx32 nlRNA in the SNB
motoneurons, a change that could be reversed by treatment with testosterone. This
elevation of gap junction transcript levels was paralleled by an androgen-induced increase
in the number and size ot junctional plaques between SNB motoneurons '"". More
recently, Micevych et al. (1996) demonstrated a dramatic up-regulation of Cx32 mRNA
expression in the rat supraoptic nucleus (SON) of the hypothalamic magnocellular
nucleus, during late pregnancy and lactation, both periods characterized by increased
neuronal membrane apposition and dye coupling. ImmediateIy after birth, Cx32
transcript levels decreased and were similar to levels in non-pregnant rats. On postpartum
day 13, when dye-coupIing between magnocellular neurons is high, Cx32 transcript levels
were again very high.
Further evidence for hormonal regulation of gap junctions in the brain was
demonstrated by Pérez et al. (1990), who reported that estradiol replacement in
ovariectomized rats resulted in an up-regulation of gap junctions in the arcuate nucleus.
This observation suggests that estrogen-sensitive neurons of the arcuate nucleus may forrn
gap junctions in response to estradiol in order to prepare for synchronous activity, which
occurs in this nucleus under the influence of estrogen. Cobbett et al. (1987) demonstrated
that castration profoundly lowers the incidence of dye coupling among magnocellular
paraventricular nucleus neurons in male rats, and that testosterone replacement reverses
these effects.
Possible role of gap jurzctions in the regulation of synchrurtous electrical activity
Changes in neuroendocrine function and behaviour ultimately reflect integrate
alterations in the electrical activity of individual neurons. Not surprisingly, therefore,
numerous studies have documented changes in neuronal activity associated with gonadal
steroid exposure. Most of the rapid, short-term effects of steroids are due to direct
interactions with neural membranes and in some cases have been shown to involve
specific membrane receptors. For example, a progesterone metabolite has beeii shown to
bind the GABA, receptor to potentiate the GABA-activated chloride current in CA1 151 neurons . Accumulating evidence demonstrates that naturally occurring fluctuations in
estradiol during the estrous cycle affect the excitability of brain tissue 115,182
Administration of estradiol 96,259 5 8 and testosterone has also been demonstrated to alter
electrical activity in the limbic system as well as hippocampal pyramidal cells in vitro 260.
In addition to the reports supporting an association of normal neuronal excitability
with gonadal steroids, there is considerable evidence that suggests that epileptic seizure
activity may also be modulated by gonadal steroids 1(6,85), 145 . A variety of experimental
models in several species support the epileptogenic potential of estrogen 155,248,296 9
especially in the kindling mode1 of secondarily generalized seizures 71,168,232 . The
exogenous administration of estradiol in rabbits induces an increase in spontaneous
electrical spike discharges 144, while estradiol replacement to female rats facilitates dorsal
hippocampal kindled seinire acquisition 34. Conversely, progesterone reduces
spontaneous and induced epileptifonn discharges 125,196,243 . Relatively little is known
about the influence of testosterone on neuronal excitability, although the effect does not
appear unifom 243,258,260,297
The influence of gonadal steroids on epileptiforrn activity in humans is well
documented, revealing an estrogen seizure-activating effect, while progesterone appears
to produce opposing, seizure-protective effects. During catarnenial epilepsy, seizure
frequency varies within the menstrual cycle s6983, showing a positive correlation with the 9 serum estradio1:progesterone ratio . The ratio is highest during the days prior to
ovulation and menstruation and is lowest during the early and rnidluteal phase. Some
patients experience a midcycle exacerbation of seizures, which may be attributed to the
preovulatory surge of estrogen 83, while the premenstrual exacerbation of seinires has
been attributed to the withdrawal of the antiseizure effects of progesterone. Seizures are
least common during the midluteal phase when progesterone levels are highest. In many
patients with catarnenial epilepsy, seizures can be significantly reduced or eliminated by
adrninistering gonadotropin releasing hormone (GnRH) agonists, which act to "shut
down" ovarian function 15. Taken as a whole, the results provide experimental and
clinical evidence of a role for estradiol in the catarnenial exacerbation of seizures.
n Y r u 1 nbùm
We hypothesize that the known effects of gonadal steroids, in particular estrogen,
on physiological and pathological changes in electrical excitability within the brain may
involve changes in gap junction formation. These changes rnay involve alterations in the
biosynthesis of the principal neural gap junctions proteins, Cx43 and Cx32.
OBJECTIVE
The immediate objective of the work embodied in this thesis was to determine
whether gonadal steroid exposure, either during development or in adulthood, might alter
expression of the mRNAs encoding Cx43 and Cx32 in the brain.
LHAY 1 LK L bUlYAUAL 3 1 LKUIU KhbULA 1 l V l Y VF b A Y
JUNCTION EXPRESSION IN THE BRAIN
As outlined above, gap junctional intercellular communication can be dynarnically
regulated by a variety of factors in both neural and non-neural tissues. For example,
accumulating evidence suggests that the initiation of Iabour is closely associated with a
marked increase in comexin expression in the myometriurn. The up-regulation of
fùnctional gap junctions in the myometriurn is thought to lead to the effective termination
of pregnancy by allowing the propagation of electrical impulses throughout the uterus,
thereby facilitating the synchronous muscle contractility of labour. Despite the extensive
literature on hormonal regulation of gap junctions in the uterus, however, relatively little
is known about how gonadal steroids affect comexins in the central nervous system. The
current state of information suggests that gonadal hormones may also modulate electrical
gap junctional coupling between cells in the brain, with clinical implications for epileptic
seizure activity; but it remains unknown whether there are direct effects of gonadal
steroids on neural connexin biosynthesis, comparable to the effects observed in the
reproductive tract.
MATERIALS & METHODS
ANIMAL PREPARATION
Female and male Wistar rats (Charles River Co., St. Constance, PQ, Quebec;
250g) were housed under standard environmental conditions (14hr light/lO hr dark cycle,
lights on at 0700 hrs) and fed Purina Rat Chow and water ad libirum. Rats were assigned
to six different treatment groups, of which the latter three required surgical procedures:
(1) cycling adult females; (2) male and female 18-hour postpartum rat pups; (3) intact
adult males; (4) males one week post-gonadectorny (GDX); (5) ovariectomized (OVX)
females; (6) OVX females afier three days of 5% estradiol treatment. Al1 anirnals were
killed by decapitation, after which the uteri, heart and liver were removed and snap frozen
in Iiquid nitrogen. The brain was quickly removed and either frozen over liquid nitrogen,
V I YldV r l y Y Y A A l V L Y 6 L V A L ~ \ W L 1 1 6 U A U L U U V 4 L G A 7 4 l l ~ ~ V U L L L I L ~ U J ) 11 J ~ J W L l L U I C U L l U 3 ' ~ L b U ~ L l b U b U 7
arnygdala) were isolated and snap frozen in liquid nitrogen, depending on whether the
tissue was to be used for in situ hybridization or Northern analysis, respectively. Al1
tissues were stored at -80°C.
1) Adult female cycling rats (tissues for Northerns): Adult female Wistar rats were
obtained and maintained as previously described. The rats were followed with daily
vaginal smears to determine the stage of the estrous cycle of the animal. After at least
three consecutive 4-day cycles, the animals were sacrificed by decapitation at 10 am on
the momings of estrus, metestrus, diestrus and proestrus.
2) Female and male rat pups (tissues for ISE?): Female and male rat pups delivered to
female Wistar rats were sexed and decapitated, 18 hours post-delivery. The intact head
was allowed to fieeze over dry ice and was stored at -80°C. The pup heads were later
cryo-sectioned for the purposes of in situ hybridization.
3) Intact male rats (tissues for Northerns and ISH): Rats were not given any
anaesthetic treatment or subjected to any surgery. On the morning that castrated males
were sacrificed, the intact males were also killed, and tissues collected.
Surgical Procedures - Prior to surgery, each animal in the following three groups was
anaesthetized by an i.p. injection (O.lml/lOOg body weight) of a ketamine (100mg/ml,
MTC Phmaceuticals) and xylazine (20mg/ml, Miles Canada) mixture, in the ratio of
2:l.
4 ) Ovariectontized fentale rats (tissues for Nortizerns and ISH): The ovaries were
removed from rats through bilateral flank incisions. On the morning of the fifth day
following ovariectomy, rats were lightly anaesthetized using carbon dioxide, as a sharn
control for estrogen capsule implantation, and promptly returned to their cages. After
another tliree days, al1 animals were killed, and tissues harvested.
3) uvarrecromrzea, esrraarol-rrearea jemaie raïs (trssues jor rvormerns ana 13n):
Ovaries were removed through bilateral flank incisions. On the morning of the fifth day
following surgery, rats were anaesthetized using carbon dioxide and given a S.C. placed
Silastic capsule (id, 1.47 mm; od, 1.96 mm; 1.0 cm Iength; Dow-Corning, Midland, MI)
containing E2 diluted with 9 vol cholesterol. The rats were left another three days, afler
which the animals were sacrificed. OVX female rats treated with 5% E, Silastic capsules
for three days experience semm Eî levels of 80 pglrnl 303, which approximates hormonal
levels observed in cycling female rats at 6pm on proestrus.
6 ) Castrated male rats (tissues for Nartherns und ISH): Testes were removed through a
midline, lower abdominal incision. Afier five days, rats were killed by decapitation and
tissues collected.
NORTHERN ANALYSIS
Brain dissection
Brain regions (cingulate cortex, hippocampus, hypothalamus + preoptic area,
amydala) were dissected freehand using a sterile razor blade, as described by Luine et al.
(1 974).
Total RNA isolation
Al1 tissues (brain regions, heart, liver and uteri) were homogenized in 1 ml of
chilled TRIzol Reagent (Gibco BRL). Total RNA was extracted from tissues according to
the procedure outlined in the product specifications. After homogenization of the tissue
in Trizol solution, the mixture was left at RT for 5 min, after which 200pl of chloroform
was added. The mixture was gently vortexed, and allowed to incubate at RT for 2-3 min.
The RNA, DNA and protein phases were separated by centrifugation at 12,000xg at 4OC
for 15 min, and the RNA phase was transferred to a new microfuge tube, along with
5 0 0 ~ 1 of isopropanol, then incubated at RT for 15 min. The reaction mixture was
centrifuged at 12,000xg at 4OC for 15 min, after which the pellet was reconstituted in 1 ml
of 75% ethanol, and stored at -80°C overnight. The following day, the mixture was
b b l l L l 1 L U & b U U L I ,JUVAS U L -r b L U 1 I V 111111, L I I b 3 U ~ b I 1 l U L b l b l l 1 V V b U ~ U1U L l 1 L P b I I C i L Q l 1 - U A l b U
for 5-10 min. The RNA pellet was redissolved in DEPC-treated water, and stored at -
80°C.
RNA electrophoresis and Northern trailsfer
Optical density readings were taken, in order to detennine the concentration and
purity of the RNA, immediately prior to loading the RNA samples onto an agarose gel.
Total RNA samples (10pg) were combined with 5p1 of 6X RNA tracking dye in a total
volume of 2 5 ~ 1 , and heated to 65OC for 5 min. Each gel was run with liver and heart
samples which served as positive and negative controls for both Cx32 and Cx43 Northern
blots. Total RNA was loaded ont0 a 1.2% agarose-0.66M fonnaldehyde denaturing gel
and electrophoresed for 1-2 hours at 120-130 V. The RNA was transferred ont0 a
Genescreen membrane (Dupont Canada Inc., Lachine, PQ) in 0.1M sodium phosphate for
at least 16 hours. RNA was fixed ont0 the membrane by UV crosslinking (UV
Stratalinker 1800, Stratagene), followed by baking at 80°C for 2 hours. The membrane
was then stored in a heat-sealable bag at -20°C, prior to hybridization with labelled probe.
Nortltern lzybridization
The membrane was incubated 2 1 hour in pre-hybridization solution (1% BSA
(wlv), 0.35M sodium phosphate, 7% SDS and 30% deionized forrnarnide) at 55OC to
block non-specific binding. Hybridization was carried out using 1) Cx43 (1.3kb cDNA)
probe (rat; a gift from Dr. David Paul, Department of Ce11 Biology, Harvard University,
Boston, Ma.), 2) Cx32 (rat; 386 bp PCR product) and 3) 18s ribosomal RNA cDNA
probe (a gifi frorn Dr. D. Denliardt, Rutgers University, Piscataway, NJ). In between
hybridizations with these different probes, each blot was stripped from the previous probe
in O. 1 % SDS, O. 1 % SSC at 95°C for 30 mins.
Approximately 50ng of each probe was radio-labelled with f 2 p ] - d c ~ p by randorn
priming (Multiprime DNA Labelling System, RPN 160 12, Amersham) to a specific
activity of 1 ~ % ~ r n / p . ~ . Ammonium acetate precipitation of the radio-labelled probe was
baiilbu u u r uy uuulii5 I d w p i u A L \ I UIIIIVA A 115, W L U L Y A LU A n , W . J /u U U U I , L V V ~ ~ I Y I
ammonium acetate, 1 Opg tRNA and lm1 95% ethanol to the labelled mixture. Following
precipitation, the labelled probe was redissolved in the hybridization solution (final
concentration of approxirnately 106 cpm/ml) and incubated with the separated RNA
samples on Genescreen membranes at 55OC for 16-24 hours. Membranes were washed to
a final stringency of 30mM sodium phosphate/O.l% SDS at 5S°C and then exposed to X-
ray film (Dupont Canada Inc., Lachine, PQ) with an intensifier screen at -80°C. Signals
were visualized autoradiographically and quantified by computer-assisted densitometry.
Analysis of Cx43 and Cx32 expression was conducted by determining the relative
signal intensity for these probes and that of 18s ribosomal RNA for the corresponding
lane. The ratio of the optical density for the connexin and 18s signals (i.e. Cx43:1 8S,
Cx32: 18S), was calculated in order to control for possible differences in RNA loading.
Statistical analysis
Data are expressed as mean f SEM. Data for Cx32 and Cx43 transcripts were
subjected to a two-way analysis of variance (ANOVA) followed by a multiple
comparison test (Student-Newrnan-Keuls Method) to determine between group
differences. Natural log transformations of the data were performed when necessary, to
correct for inhomogeneity of variance. Data for Cx32 mRNA and Cx43 mRNA
transcripts in cycling female brains were subjected to a one-way ANOVA followed by the
Student-Newrnan-Keuls test to determine differences between days of the estrous cycle.
Data that could not be subjected to pararnetric analysis, because of residual
inhomogeneity of variance that was not correctable by mathematical transformation, were
analysed non-parametrically using Kruskal-Wallis One-Way ANOVA.
IN SITU HYBRIDIZATION
Glrrss slide su bbing protocol
Frosted end, pre-cleaned glass slides (VWR Canlab) were dipped in ethanol and
placed in a sterile rnetal staining rack. Slides were baked overnight at 280°C, cooled to
room temperature, and dipped in 0.02mglml poly-L-lysine (Sigma, High MW) in
autoclaved 0.1 % diethyl pyrocarbonate (DEPC) water. The subbed slides were dried
overnight at 37OC, stored at 4OC and used within one week.
Tissue preparation and frxation
Tissue was removed from -80°C and allowed to equilibrate to the cryostat
temperature of -26°C for at least 30 minutes. Each brain or pup head was sectioned at a
thickness of 14pm in a cryostat (Reichert Jung Frigocut 2800E), and sections were fi-eeze-
thaw mounted ont0 poly-L-lysine coated slides. Rat heart and liver were also
cryosectioned as a positive and negative control, respectively, for the Cx43 probe. Al1
solutions for tissue fixation were made up with autoclaved O. 1% DEPC water to minimize
RNA degradation. Afier sections were collected, slides were irnrnersed in a Wheaton
staining dish filled with 4% paraformaldehyde in IXPBS, pH 7.4 (0.14M NaCl, 0.003M
KCl, 0.01M Na2HP04, 0.001 8M KH2P04), for 15 min at 4OC. The slide rack was
transferred to a new dish filled with 3XPBS, pH 7.4 (0.41M NaCl, 0.008M KCl, 0.03M
Na2HP04, 0.005M KH2P04) and incubated at 4OC for 5 min. This step is necessary to
stop the paraforrnaldehyde fixation. Slides were transferred to a new dish containing
IXPBS, pH 7.4 at RT for 5 min. Sections were dehydrated through a series of 3 min
incubations in 50% ethanol, 70% ethanol, 95% ethanol and 100% ethanol twice. Sections
were allowed to air dry for 10-15 min, afier which they were kept at RT for 2 hours-
overnight in a vacuum desiccator. Slides were stored in the desiccator at RT for up to 2
days; for longer periods of time, they were stored at -80°C.
Generatioït of coïrnexin43 polymerase cltain reactiopt product
Complernentary DNA (cDNA) frorn rat myometrium was generated in a 20pI
reaction mixture containing lpg of total RNA, 5ngIp1 random hexamers (Pharmacia),
5UIp1 MMLV reverse transcriptase (Gibco BRL), 5mM MgCl, (Perkin Elmer, Cetus), 1 X
PCR buffer (10mM Tris-HC1, 50mM KCI, Perkin Elmer, Cetus), ImM each dNTP
(dATP, dCTP, dGTP, dTTP, Pharmacia), 1 U/p1 RNase inhibitor (B~ehringer Mannheim),
illlu 1 UIUV~ u 1 1 ~UIVLU DU). 1111s revcrst: iranucripiion (K 1 ) mixture was incuoarea ar
25°C for 10 min, followed by 42°C for 30 mins, and finally heated to 99OC for 5 min.
Primers were created to specifically ampli@ a 294bp segment of cornexin43
cDNA from rat myometriurn (Primer 1: 5'-TAC CAC GCC ACC ACC GGC CCA-3'
Primer 2: 3'-AAC GGA CTG CTG TCG GTT TTA CGG-5', obtained from ACGT
Corporation, Toronto, Ontario). A polymerase chain reaction was performed using the
total RT mixture (20yl) in a total volume of lOOpl including 200pM of each
deoxynucleoside triphosphate, 0.5pM of each primer, 2.5U Taq polymerase (Boehringer
Mannheim) in PCR buffer (2mM MgC12, 50mM KCI, lOmM Tris-HC1, pH 8.3).
Preincubation proceeded at 95OC for 5 min, denaturation at 95OC for 1 min, primer
annealing at 60°C for 90 sec, and extension at 72°C for 1 min for 40 cycles. Cycling was
completed by 7 min at 72OC to fùlly extend the DNA. The PCR product was
electrophoresed in a 1.5% agarose gel in order to visualize amplification products. The
Cx43 PCR product was sequenced by another member of the laboratory (G. Erb) in order
to confirrn the identity of the Cx43 PCR product. To further validate the specificity of the
Cx43 probe, the Cx43 PCR product was used to probe a Northern blot of rat heart and
liver tissue. The presence of a single transcript Cx43 signal in heart tissue and a lack of
signal in liver tissue, indicates the Cx43 in situ hybridization probe is highly specific.
DNA preparation for riboprobe
The 294bp connexin43 PCR product was subcloned into the phagemid vector,
pBluescript II SK (+/-) (Stratagene) and transfomed into competent DHSa cells, as
follows. Linearized vector product was generated from the PCR product in a 2 0 ~ 1
reaction mixture by adding 15p1 pBluescript II, 1U/p1 of EcoRI (Gibco BlZL), and l/lOM
of React III at 37OC for 2 hours, afler which the enzyme was heat inactivated at 65OC for
15min. In order to prevent re-ligation of the linear vector, the linearized pBluescript
vector was dephosphorylated with 0.005UIpl alkaline phosphatase (Pharmacia) at 37OC
for 30 min. The alkaline phosphatase was heat inactivated at 85OC for 15 min. Following
this treatment, 4 % of the vector DNA was presumed to retain terminal phosphate
b L V U y U . A.& V A U V A C V V V A A I I A A A A A I I A W U I A L > U C I V . A ) & L A W U A a V Y C V U ) U V , d L & V Y , J A I V I J I U C V U f i l U A U V U V A A y L
product was electrophoresed on a 1% agarose DNA gel.
The connexin43 PCR product (294 bp) was subsequently ligated into the purified,
linearized vector. Equal amounts of Cx43 insert and vector DNA (0.1 pg) were combined
in a final volume of 7.5~1, which was heated to 45°C for 5 min, and promptly chilled to
0°C. For ligation of cohesive ends of the vector, the final reaction mix contained 1X
bacteriophage T4 DNA ligase buffer (20mM Tris-CI, pH 7.6; 5mM MgCl*; 5mM DTT;
50pglml BSA), 0.1 Weiss unit of bacteriophage T4 DNA ligase, and 0.5mM ATP. The
reaction was incubated at lS°C for 15 hours.
The ligation product was transformed into DH5a bacterial cells. Competent
DHSa bacteria (100pl) were added to the total volume of Cx43lpBluescript ligation
reaction volume ( 1 0 ~ 1 ) ~ and lefi on ice for 30 min. The solution was heat shocked at
42°C for 2 min. Sterile LB media (400~1) was immediateiy added to the transformation
mixture, and allowed to incubate for 1 hour at 37OC. On a premade LB-Amp plate treated
with Xgal and IPTG, lOOpl of the transformation reaction was spread and allowed to
incubate at 37OC overnight. The folfowing morning, white colonies were selected and
grown overnight in LB media treated with O.l25mg/ml of ampicillin at 37OC.
DNA was isolated from the bacteria using a mini-prep procedure. 1.5 ml of the
culture was microfuged for 20 sec at 10,000rpm. The pellet was resuspended in 3 5 0 ~ 1 of
sucrose solution (8% sucrose, 5% Triton X-100, 1 OrnM Tris-HC1, pH 8, 50mM EDTA),
and 25ul of lOmg/ml of lysozyme (in 1 OmM Tris HCI, pH a), which was incubated at RT
for 5 min. The sample was boiled for 40 sec, and spun immediately for 15 min at RT. An
equal volume of isopropanol was added to the supernatant, and allowed to precipitate for
at least 10 min at -20°C. The sample was centrifuged for 15 min at 4OC and the pellet
was resuspended in sterile water.
An endonuclease digest using EcoRI to excise the Cx43 insert was performed on
the mini-prep product to confirm if colonies containing DNA insert had been selected.
The digest product was run on a 2% agarose gel. Subject to confirmation, more colonies
were cultured, and a maxi-prep procedure was performed using the Qiagen protocol.
V U I I L Y L V U "1 I L L U L \ . Y L V p J / L V U U V L V I W L W Y A Y V L L V ~ I X V L V L > U U W L I C U L UIIUIJ L L U U A 1 I V U&UIVJk E j b l i > U I L U
optical density measurements were performed on the DNA.
Directional antisense and sense Cx43 probes were prepared, the orientation of
which was based on previous sequencing reactions. The Cx43 probe was linearized for
later use as the antisense probe using Spel, while linearization for a sense probe was
generated using EcoRV. A reaction volume of 20p1 included 5pg of maxi-prepped Cx43
DNA, 1 U/p1 restriction endonuclease enzyme and 1 /l OM Reaction buffer. The reaction
mixture was incubated at 37°C for 1.5 hours, afier which the contents were treated with
10pg of proteinase K in order to destroy any contarninating proteins. Samples were
electrophoresed on a 2% agarose gel in order to confirm completion of linearization, and
purified using Geneclean.
cRNA transcription reaction
Transcription of Cx43 DNA into a [s"] radiolabelled riboprobe (RNA
Transcription Kit, Stratagene) was performed using corresponding RNA primer
polymerase sites on either side of the Cx43 insert according to the Bluescript vector map.
A reaction volume of 2 5 ~ 1 included final concentrations of 1X transcription buffer, 1pg
restricted, proteinase-K treated Cx43 DNA template, 0.4mM each of rNTPs (rATP, rCTP,
rGTP), 0.003M DTT, 4pCi/pl [s3']-~UTP (Amersham), and 0.4U/p1 of RNA polymerase
(T3 polymerase for sense and T7 polymerase for antisense). After incubating the reaction
mixture at 37°C for 1 hour, unincorporated nucleotides were removed using a Sephadex
G-50 Quick Spin Colurnn (Boehringer Mannheim).
Pretrentntertt of in situ Izybridizatiurt slides
Sections that had been stored at -80°C after fixation, were removed from the
freezer and kept in a vacuum desiccator at 4OC until completely thawed and dry (1 hour).
Slides were immersed in a solution of 0.1% Triton and 2XSCC (0.3M NaCl, 0.003M
sodium citrate) at RT for 30 min. Slides were washed twice in 2XSSC, for 5 min and 3
min respectively, followed by washes in 4% PFA in lXPBS (pH 7.4) for 5 min, and
1 XPBS for 5 min. Acetylation was performed using 0.1M triethanolamine + 0.25% acetic
anhydride solution for 10 min to reduce non-specific binding of probe, followed by two
washes for 2 min in 2XSSC. The tissue was dehydrated in ascending concentrations of
fresh ethanol for 3 min each of 50% ethanol, 70% ethanol, 95% ethanol and 100% ethanol
twice. Slides were allowed to air dry for 10 min and stored in a vacuum desiccator for 2-
16 hours.
Probe application and hybridization
Hybridization solution was prepared by combining the riboprobe mixture and
hybridization buffer in the ratio of 1 :4 respectively. The riboprobe mixture contained 0.5
mg/ml tRNA, lOmM DTT, and [s'~]-labelled Cx43 riboprobe (adjusted to 10,000,000
cprnfmI of hybridization solution). The hybridization buffer contained 62.5% (wlv)
redistilled formamide, 12.5% (wlv) Dextran sulfate, 0.375M NaCl, 1.25X Denhardt's
solution (50X stock: l g each Ficoll, polyvinylpyrrolidone, BSA Fraction V, made up to
100ml), 0.0125M Tris, pH 8 and 0.0025M EDTA, pH 8. The hybridization solution
mixture was heated to 65OC for 10 min to rnelt any annealed strands, and 75p1 was
applied directly to a 50 x 22 mm coverslip, ont0 which the slide bearing the tissue
sections was gently applied. This method of application minimized air bubbles trapped
over tissue sections. The slides were incubated overnight in a 58°C incubator, in a well
humidified tray containing SXSSC + 50% formamide.
Post-hybridization treatment
After 16-20 hours in the 58°C incubator, slides were removed and allowed to
equilibrate to RT for 30 min. Slides were incubated in 4XSSC (0.6M NaCl, O.OGM
sodium citrate) for 20-30 min with gentle agitation, in order to soak off the coverslips,
then washed for 4 X 5 min in fresh 4XSSC. An RNAse digestion (RNAse A 20pg/rnl,
0.5M NaCl, O.OlM Tris, 0.001M EDTA) at 37°C for 30 min removed most of the
nonspecifically bound probe. Slides were then dipped in: 2XSSC/lOmM DTT twice for 5
min; 1 XSSCIl OmM DTT for 10 min; 0.5XSSCll CmM DTT for 10 min; O. 1 XSSC/l OmM
LJ 1 1 lur JU min al 2 -L; mu ci mai mer rinse in u. lnrssuiumivi u r 1. sections were
dehydrated through a series of 3 min incubations in 50% ethanol, 70% ethanol, 95%
ethanol and 100% ethanol twice, allowed to air dry 10 min and stored in a vacuum
desiccator for at least 20 min.
The slides were exposed to Kodak Biomax MR High Resolution Autoradiography
Film at RT for 2-3 days. Cx43 riboprobe signals were detected by autoradiography, and
quantified by cornputer-assisted densitometry. Analysis of Cx43 mRNA expression was
conducted by determining the relative optical density of various brain regions
Emulsion Autorudiography
For higher resolution analysis to distinguish individual cells, Kodak NTB-2 liquid
autoradiography emulsion was used. Under a sodium vapour lamp, the emulsion was
diluted with 600 mM ammonium acetate, and slides dipped while the emulsion was
maintained at 42°C. Slides were dried at RT, and stored at 4°C in a light-proof container
for 15 days. Development of the emulsion dipped slides was carried out for 3 min at
20°C in Kodak D-19 developer, diluted 1:3 with water. Slides were then washed for 30
sec in 2% acetic acid and fixed for 4 min in 5% sodium thiosulfate. Slides were washed
in 20°C water for 20 min, and dehydrated by 2 min dips in 50%, 70%, 95%, and 100%
ethanols. The slides were left in another 100% ethanol dip for 1 hour, followed by
immersion in cresyl violet stain for 8 minutes, and a brief dip in 95% and 100% ethanol.
The slides were then cleared in 3X xylene and coverslipped with Permount (Fisher).
Statistica l analysis
Al1 data are presented as niean + SEM. Data were subjected to a two-way analysis
of variance (ANOVA) foIlowed by a multiple cornparison test (Student-Newman-Keuls
Method) to determine between group differences. Natural log transformations of the data
were performed, where necessary, to correct for inhomogeneity of variance. Differences
were considered significant at a level of p<0.05 (two-tailed).
KLS U L 1.3
CYCLING FEMALE RATS
Northern nnniysis of Cx43 and Cx32 transcript Ievels
To address the possibility of changes in connexin mRNA expression in the female
rat during the four day estrous cycle, Cx32 and Cx43 transcript levels were quantified.
Northern blots were perforrned on the cingulate cortex, hippocarnpus, arnygdala, and
hypothalarnuslpreoptic area dissected fiom female rats during estrus, metestrus, diestms
and proestrus. Data are s h o w in Figures 3 and 4. To facilitate cornparison across
different stages of the cycle, data are expressed relative to the values for the Cx43:18S
ratio at metestrus. Cx32 transcript levels demonstrated no significant changes in any of
the brain regions (hippocampus: F(,, , ,) = 1.28; p=0.3 1 8 amygdala: F(,,,, =3.27; p=0.043
hypothalamuslpreoptic area: FVTl7) =O. 173; p=O.9 13). However, Northern analysis of
Cx43 transcript levels demonstrated small, but statistically significant differences in the
amygdala of the cycling female rat (Student-Newman-Keuls, pC0.05). There was a
significant reduction of Cx43 mRNA expression during estrus, diestrus and proestrus,
with respect to metestrus. There were no other significant differences observed in other
tissues during the estrous cycle (cortex: Fu,, ,) =O.87 1 ; p=0.475 hippocampus: Fu,, = 1.2;
p=O.3 3 9 hypothalarnuslpreoptic area: F(3,2) =O.65 5; p=OS 89)
These preliminary data suggested that there might be small regional changes in
connexin43 expression in the brain, associated with the fluctuating levels of gonadal
steroid hormones experienced during the female reproductive cycle. There are three
principal hormonally-active steroids secreted by the ovary of the rat during the estrous
cycle: estradiol, progesterone and testosterone. The concentrations of these steroids
fluctuate during the estrous cycle in a highly predictable fashion. Figure 5 shows the
serum levels of estradiol, progesterone and testosterone in this strain of rats, during the 4-
day estrous cycle, measured previously by another member of this laboratory (D.
Bowlby). Estradiol rises sharply over diestnis and proestrus, falling to undetectable levels
at estrus. Progesterone rises at estrus, peaking at metestrus, while testosterone follows a
pattern resembling that of estradiol. We hypothesized that the preliminary findings
U U L ~ I I K U 111 L Y L I I I I ~ L L I I I I ~ I ~ L I > I I I I ~ I I L ICXICLL I E ; ~ I U I I ~ ~ I G I I ~ I I ~ G S 111 GUILI~CKIII IIIIUY A c x p r c s s I u 1 1
induced by one, or more, of the ovarian steroids. To investigate this hypothesis, further
studies were performed using Northern analysis and in situ hybridization, in GDX rats
treated with replacement doses of gonadal steroids. In addition, in view of the much
higher circulating levels of testosterone experienced by males as compared to fernale rats,
and the previously cited evidence for an effect of testosterone in males on spinal cord
connexin expression Is9, we also compared intact and GDX males.
HIP
I Estrus V7A Metestrus
Diestrus Proestrus
AMY
FIGURE 3. Composite graph showing mean (2) SEM of connexin32 transcript leveIs in different regions of cycling female rats, as determined by Northern analysis (n=4-6). Values are expressed as a percentage of the mean metestrus Cx32: 18s. Cx32 transcript levels are not significantly different over the four day cycle.
CTX
I I Estrus Metestrus
FSSJ Diestrus Proestrus
HIP AMY
FIGURE 4. Composite graph showing mean (1) SEM of connexin43 transcript levels in different regions of cycling female rats, as determined by Northern analysis (n=4-6). Values are expressed as a percentage of the mean metestrus Cx43 : 1 8s. * indicates significant difference verszrs metestrus (Kruskall-Wallis one way ANOVA; p<0.05).
FIGURE 5. Semm estradiol, testosterone and progesterone concentrations in cyclinç female rats sacrificed at 10 am on diestms (D), proestrus (P), estrus (E), and metestms (M). Results represent means f SEM in 4-5 observations in each case. * indicates significant difference verszts metestnis (Newman-Keuls Multiple Range test; p<0.05).
- - - - - - - -* .----- * - * .- a.-----
Northern analysis of Cx43 mRNA and Cx32 mRNA
in order to investigate gonadal hormonal influences on comexins in male and
female rats, Northern analyses were perfomed on animais in the presence (OVX females
with E2 treatment; intact males) and absence of hormones (GDX males and females).
Figure 6 demonstrates a typical Northern blot hybridized with Cx32 cDNA, Cx43 cDNA,
and 18s ribosomal RNA, which is used to dernonstrate equal loading of mRNA. Figures
7-10 demonstrate connexin transcript levels in the cingulate cortex, hippocarnpus,
amygdala and hypothalamus/preoptic area in female and male rats. Cornparison between
treatment groups did not reveal any statistically signifiant differences in individual
tissues, in either males or fernales. The ability to make reliable comparisons is limited to
different hormonal treatments within each individual tissue, due to the fact that Northern
blots were exposed to autoradiographic film for varying periods of time, in order to
optimize visualization. However, control and treated samples from each tissue were run
on the sarne RNA gel, thereby eliminating inter-gel variability for the treatment
comparisons.
FIGUlRE 6 . Northern blot hybridization of total RNA from hypothalamus/preoptic area of intact vs. gonadectomized (GDX) male rats (10 pg RNA/lane). Panel A: Blot probed with 32 P-labelled Cx32, present in rat liver ( ositive control) and absent in rat P* heart (negative control). Panel B: Blot probed with P-labelled Cx43. Cx43 transcript is present in rat heart (positive control tissue) and absent from liver (negative control tissue). Panel C : The same blot reprobed with the house-keeping gene 18s ribosomal RNA to demonstrate equal loading of samples.
Cortex ' .O 1 tlippocampus
Amygdala
FIGURE 7. Individual graphs showing mean (+ SEM) Cx32 transcript levels in different regions of the intact (empty bar) and gonadectomized (hatched Sar) male rat brain. Northern analysis reveals high connexin mRNA expression in al1 brain regions from GDX male rats although levels were not significantly altered by steroid exposure.
Cortex 1 ,O Hippocampus 0.9 0.9 1
r
',O 1 Amygdala i." 1 HypothalamudPOA
FIGURE 8. Individual graphs showing mean (f SEM) Cx43 transcript levels in different regions of the intact (empty bar) and gonadectomized (hatched bar) male rat brain. Northern analysis reveals high connexin mRNA expression in al1 brain regions from GDX male rats although levels were not significantly altered by steroid exposure.
Ratio Cx32: 18s Ratio Cx32: 18s
Ratio Cx32: 18s Ratio Cx32: 18s
0 o., - 7 &j 0.6 -
3 0.5 - O 0 0.4 - .- w (I1 0.3- lY
Cortex
Amygdala
Hippocampus
FIGURE 10. Individual graphs showing mean (f SEM) Cx43 transcript levels in different regions of the OVX (empty bar) and OVX + E, (hatched bar) fernale rat brain. Northern analysis reveals high connexin mRNA expression in al1 brain regions from OVX female rats although levels were not significantly altered by steroid exposure.
IIC DCLLI ~YU~CUCZULLUIZ UJ LX43 LfUIZYCfl~C 1LiVLIlY
Effects of gonadal hormones on brain Cx43 expression are shown in Figures 12-
16. Figure 11 illustrates the location and orientation of distinct brain regions in which
connexin expression was measured by densitometry. The densitometric data for each
region were analysed on a section-by-section basis, plotting the average regional
densitometric data for al1 sections corresponding to similar hormonal treatments in the
male and fernale rats. Figure 12 demonstrates in situ hybridization for Cx43 mRNA in
intact and gonadectomized male rat brains at different levels of the brain. Visual
inspection suggests no significant differences in connexin mRNA expression between
treatment groups. Figure 13 demonstrated no significant differences in Cx43 transcript
levels based on hormonal status of distinct regions of intact and gonadectornized male
rats. However, the in situ hybridization assay demonstrated estradiol-induced increases in
Cx43 transcript levels in the dorso-lateral septum, bed nucleus of the stria terrninalis and
periventricular hypothalamic nucleus of the female rat (Figure 14). Dark- and bright-field
photomicrographs (Figure 15 and 16) show in situ hybridization results for Cx43 rnRNA,
manifest in grains over cells, in the dorso-lateral septum and dorsal preoptic area of the
control and estradiol-treated female rat. There is a small increase in Cx43 mRNA
expression in sections from hormone replaced rats.
FIGURE 11. Atlas diagram illustrating the location and orientation of regions in which connexin transcript levels were analysed densitometrically (Figure 12 and 13). PVCP: periventricular caudate putamen; LSD: dorso-lateral septal nucleus; BST: bed nucleus of stria terminalis; Pe; periventricular hypothalamic nucleus; CTX: cingulate cortex; H P : hippocampus; MBH: mediobasal hypothalamic nucleus; AMY: amygdaloid nucleus (Adapted from Paxinos and Watson, 1986)
FIGUW 12. In situ hybridization of Cx43 mRNA in intact (A, C) and gonadectomized (B, D) male rats at different levels of the brain reveals no regional labelling differences between treatment groups. Antisense [)' SI-labelled riboprobe corresponding to a 294bp fragment specific Cx43 was used. A section hybridized with Cx43 sense riboprobe (E) serves as the negative control for specific probe interaction.
I I Intact
FIGURE 13. Composite graph showing mean (+ SEM) Cx43 transcript levels, as measured by in situ hybridization, in different regions of the intact (empty bars) and gonadectomized (hatched bars) male rat. Histograms represent the average regional densitometric data for al1 sections corresponding to respective hormonal treatment groups (intact, n=4; GDX, n=4) (F(1,480) = 0.467; p=1.495)
I ovx OVX + E2
FIGURE 14. Composite graph showing mean Cx43 transcript levels, as measured by in situ hybridization, in different regions of the control (empty bars) and estradiol-treated (hatched bars) ovariectomized female rats. Histograrns represent the average regional densitometric data for al1 sections corresponding to respective hormonal treatinent groups. Vertical bars represent the SEM for the OVX E2 group (F (1,330) =1.27; p=0.260); filled circles represent average data points for each animal in the OVX group. (OVX, 11=2; OVX + Es, n=3).
FIGURE 15. Photomicrographs of in situ hybridization for Cx43 mRNA in the dorso- lateral septum of OVX (a,b) and estradiol-treated OVX (c,d) rats. Photomicrographs were taken under dark-field illumination (a,c) to de ict the distribution of reduced silver P, grains (white dots) resulting from exposure to the [ SI-labelled cRNA probe. Bright - field illumination photomicrographs (b,d) demonstrate the location of ce11 nuclei and labelled Cx43 riboprobe (black dots). LV: Iateral ventricle
FIGURE 16. Photomicrographs of in situ hybridization for Cx43 mRNA in the dorsal preoptic area of OVX (a,b) and estradiol-treated OVX (c,d) rats. Photomicrographs were taken under dark-field illumination (a+) to depict the distribution of reduced silver grains (white dots) resulting from exposure to the [35~]-labelled cRNA probe. Bright - field illumination photomicrographs (b,d) demonstrate the location of ce11 nuclei and labelled Cx43 riboprobe (black dots). ac: anterior commissure; 3V: third ventricle
m' m > L v f i n u u AI Y U l v f i n u u fi- L 1 v 1 u
In Situ Hybriiiization of Cx43 transcript ieveis
No significant differences in connexin expression were observed between intact and
GDX adult males. We were also interested in the possibility, however, that there might be
effects of androgen on connexin expression at other stages of CNS development. The
rationale for this hypothesis is based on the observation that there are significant sex
differences in the migration patterns of neurons in the rostral hypothalamus and preoptic
area, during perinatal development, resulting from androgen action in the male 5 1,52,99,263 . In
the female rat, the ovary is quiescent during early life and this lack of sex steroid secretion
allows a different pattern of neuronal migration to occur. Since astrocytes, which express
Cx43, are vitally important components of the mechanism controlling neuronal migration 2 15 away from the neuroepithelial layers lining the ventricular walls , and the growth of
265 astrocyte processes has been shown to be sensitive to gonadal steroids in vitro , we
considered the possibility that neonatal androgen production in males rnight affect astrocytic
Cx43 expression, as part of the mechanism of sexual differentiation.
To address the possibility of differences in connexin mRNA expression in neonatal
female and male rat pups, levels of transcript were measured using in situ hybridization.
Figure 17 depicts the sarnpling windows for the distinct brain regions of interest, including,
cingulate cortex, hippocarnpus, hypothalarnus/preoptic area, amygdala and periventricular
hypothalamus. Cx43 transcript levels showed no significant differences between the female
and male rat pups (Figure 18). However, despite this lack of large-scale change in connexin
expression, examination of the sections under higher resolution revealed a smaller-scale
regulatory effect of hormones on Cx43 mRNA levels, expressed in the form of regional
differences in grain density around the apex of the third ventricle (Figure 19).
FIGURE 17. In situ hybridization of Cx43 mRNA in an 18-hours postnatal male pup at different levels of the brain. Cx43 labelling was analyzed on a section-by section basis, using sampling windows as shown - A: cingulate cortex; B: hippocampus; C: amygdala; D: Iiypothalamus/preoptic area. Window D also corresponds to the periventricular hypothalamus in brain sections from a more frontal plane.
CTX
1 EZBl Male 1
HIP HP0 AMY
FIGURE 18. Composite graph showing mean (f SEM) Cx43 transcript levels, as measured by in situ hybridization, in 18-hour postnatal female and male rat pups. Histograms represent the average regional densitometric data for al1 sections corresponding to female or male pups (n=3) (F( ,,,,,, = 1.03; p=0.3 12)
FIGURE 19. Photomicrographs taken under bright-field illumination of female (A) and male (B) pup brain to depict the location of nuclei and labelled Cx43 riboprobe (black dots). Arrows indicate the neuroepithelial layer at the apex of the third ventricle. (L. Puy, unpub lished observations)
U1DL U L 3 L 3 1 U l ' l
The influence of gonadal steroids on the electrical activity of the brain of both
animals and humans is well documented. In animals, changes in estrogen Ievels are
associated with drarnatic changes in the electrical and neurochemical excitability of
neurons, in several regions of the brain 18 1,294,298 . Pathological changes in electrical
activity associated with epileptic motor seizures are also highly sensitive to gonadal 34,83 steroids. Estrogen potentiates seizures in the central nervous system , while
progesterone seems to produce opposing, seizure-protective effects 125,196,243. R~~~~~
evidence suggesting that gonadal steroids might modulate connexin expression in the
brain, coupled with studies indicating a possible role for electrical gap junctional coupling
in the etiology of epileptic seimres 843192, led to our hypothesis that gonadal hormones
could potentially affect neuronal activity by altering expression of the two principal
connexins found in the brain, Cx43 and Cx32.
Gonadal steroids have been shown to strongly regulate connexin biosynthesis in
non-neural target tissues, such as the uterus, in both the pregnant and non-pregnant state 218. Elevated levels of uterine gap junctions accornrnodate the need for the
synchronization of muscular contractions during the process of labour. Currently, it is
thought that the onset of labour is driven by the dramatic up-regulation of Cx43 mRNA
and protein, a process which is associated with an increase in the materna1 plasma
estrogen:progesterone ratio 14'. In the myometrium of both pregnant and non-pregnant
rats, there is also evidence that Cx43 synthesis and/or trafficking is regulated positively
by estrogen and negatively by progesterone. The mechanism through which gonadal
steroids modulate Cx43 synthesis is not yet clear, although two principal pathways have
been proposed. Yu et al. (1994) demonstrated that the Cx43 gene is responsive to
estrogen, despite the lack of a palindrornic sequence homologous to the estrogen response
element within the Cx43 promoter. The 5' promoter region of the Cx43 gene does,
liowever, exhibit a consensus activator protein-l (AP-1) binding site. Within the uterus,
estrogen is known to induce the expression of the AP-1 proteins, efos and e j u n 1 12,280
Piersanti and Lye (1 995) demonstrated an increase in myometrial Cx43 mRNA following
w-*i---vr r-.yiu-.riirrirr -v v i r r iu-u) ui i u r r v v b v.iiiuii i w u o y i v v u u v u UJ C L A U U.k V U L U U
expression of mRNA encoding c-fos and c-jun. Spontaneous and premature labour was
also associated with a coincident increased expression of Cx43 and c-fos within the
rnyornetriurn of pregnant rats 2' l .
In addition to the crucial role of gonadal hormones within the term uterus, steroids
exert a variety of morphological, biochemical and electrophysiological effects in a
number of different regions in marnmalian brain 1639166. AS previously discussed, estrogen
is one of the most potent steroids, and exerts a range of effects, including the modulation
of key enzymes for newotransmission, neuro-glial connectivity, and synaptic
remodelling. Such genomic effects are mediated by intracellular estrogen receptors
present in both neurons and glia 106,127,237 . Alternatively, gonadal steroids can exert rapid
and reversible effects on their target cells, ofien via interactions with neural membrane
activity 260,294 . It is well documented that estrogen modulates neuronal activity within the
CNS, specifically in estrogen-sensitive regions important in the regulation of different
aspects of reproductive h c t i o n of the animal 1 10,233,295 . Estrogen priming, for example,
has the ability to alter neuronal excitability and sensitivity 55,233,295 . Within the CNS, the
expression of c-fos by individual neurons is ofien used as a marker for ce11 activation 85987.
Following estradiol administration to OVX rats, Insel (1 990) reported an increase in
staining for c-fos-like protein in a variety of regions rich in estradiol-concentrating cells,
including the anterior media1 POA, the media1 POA, the media1 amygdala nucleus, and
the ventromedial nucleus of the hypothalamus. Estrogen priming has also been shown to
increase the expression of c-fos in the CA1 region 'O2.
Thus, the above evidence suggests that like the uterus, the brain may also possess
the requisite mechanisms for a hormonal regulation of connexin expression, potentially
mediated through the steroidal induction of proto-oncogenes, which act on connexin
promoter AP-1 sites to regulate transcription. However, the current state of knowledge
with respect to gonadal hormone regulation of gap junctions in the central nervous system
is poor, and furthemore, possible mechanisms for such effects remain unexplored.
Evidence consistent with the potential for hormonal regulation of connexin inRNA
expression in the central nervous system was first described by Matsumoto e l al. (1991),
W I ~ U ç A a u l u l C u LAJL LIIIUYL-I C A ~ C ~ ~ I U I ~ 111 LUC Q I I U I U ~ C I I - ~ C I I S I L L V C ILIULUIICULUIIS UI LIIC
spinal nucleus of the bulbocavernosus. Castration of male rats was shown to drarnatically
reduce the expression of Cx32 mRNA in the SNB motoneurons, a change which was
counteracted by treatment with testosterone. More recently, Micevych et al. (1996)
demonstrated a drarnatic up-regulation of Cx32 mRNA expression in the rat supraoptic
nucleus (SON) of the hypothalamic magnocellular nucleus, during late pregnancy and
lactation, both periods characterized by increased neuronal membrane apposition and dye-
coupling. These results suggest that connexons may form between SON neurons in
response to increased activation. The increased expression of Cx32 mRNA on the day
before parturition, may mediate perinatal activation, resulting in the release of
approximately 60% of the oxytocin and vasopressin from the neurohypophysis 64. On
postpartum day 13, there are once again high levels of Cx32 mRNA, a time when the
SON is in a continua1 state of activation induced by suckling stimuli and the homeostatic
maintenance of matemal water levels. Estrogen has also been shown to modulate dye
coupting within the female rat SON and may also influence ex32 mRNA levels in virgins
and in postpartum lactating dams 79. Although this proposed mechanism of connexin
mRNA regulation may be consistent with observations of elevated Cx32 mRNA and dye
coupling during the lactating period when there is low estrogen, the hypothesis breaks
down in the prepartum condition, when there is high levels of both matemal estrogen and
Cx32 mRNA.
Further evidence consistent with a possible hormonal regulation of gap junctions
in the brain, was demonstrated by Pérez et al. (1990), who reported an estradiol-induction
of gap junctions in the arcuate nucleus. This observation suggests that estrogen-sensitive
neurons of the arcuate nucleus may form gap junctions in response to estradiol in order to
prepare for synchronous activity, which occurs in this nucleus under the influence of
estrogen. Cobbett et al. (1987) demonstrated that castration profoundly lowers the
incidence of dye coupling among magnocellular paraventricular nucleus neurons in male
rats, and that testosterone replacement reverses these effects.
In this study, we demonstrate that the presence or absence of gonadal steroids in
the systemic circulation of rats is not associated with large-scale changes in connexin
mmuA expression, a resuir cunirary iu our iniiiai nypumesis. r ne expressron or connexin
transcripts was examined in four groups of rats: cycling female rats; control and estradiol-
treated ovariectomized adult female rats; gonadectomized and intact adult male rats, and;
male and female rat pups, 18 hours post-partum. Al1 Northern analyses and in situ
hybridization assays focused on the regulation of connexin mRNA expression in areas of
the brain reported in the literature to exhibit high levels of high estradiol binding.
Northern analyses of several estrogen-sensitive regions of the cycling female rat brain
demonstrated a small, but significant reduction of Cx43 mRNA levels in the amygdala on
the days of estrus, diestrus and proestrus, with respect to metestrus. Northern analyses of
brain tissue following hormone exposure (in the form of either testicular secretions in the
male, or estradiol treatrnent in the female) fiom adult female and male rats exhibited no
change in Cx43 or Cx32 transcript levels.
Further examination of several experimental groups (i.e. adult female and male
rats; female and male rat pups) were perfomed using in situ hybridization with a [s'~]- labelled Cx43 riboprobe. Measurements of connexin mRNA labelling were performed by
taking the average densitometric value over various estradiol-sensitive regions. Within
the female ovariectomized rat, treatment with estradiol induced a small up-regdation of
Cx43 mRNA expression in only a few, restricted regions, including the bed nucleus of the
stria terminalis, the dorso-lateral septum, and the periventricular hypothalamus. No other
gonadal steroid effects were observed in the adult male rats, female pups or male rat pups.
However, visual inspection of riboprobe labelled sections after autoradiographic emulsion
dipping demonstrate hormone-induced changes of connexin mRNA expression on a
small-scale, very regionalized manner. Such small, region-specific alterations in
connexin mRNA expression were indistinguishable when densitometrically analysed over
relatively large areas. In order to investigate these highly regionalized regulatory effects
of hormones, emulsion dipped brain sections of a greater number of anirnaIs should be
quantified for the number reduced silver grains per unit area. Where connexin expression
is regulated by hormones in the brain, the changes are highly localized and therefore
undetectable by analysis on a large-scale.
lnis rernaritamy srnaii normonai reguiarion 01 connexin expression in ail
treatment groups, differs from the regulation observed in uterine tissues, in which
hormone-indrrced changes in electrical activity involve dramatic changes in gap junction
mRNA and protein synthesis 39,148,218 . There are several possible interpretations. One
explanation for the discrepancy between steroidal effects in non-neural and neural tissue
is that, in fact, gonadal hormones may not regulate connexin transcript levels within the
central nervous system. This explanation is at best only partially correct, as previously
demonstrated in studies by Matsumoto et al. (1991), Micevych et al. (1 996), Cobbett et
al. (1987), and Pérez et al. (1990). These studies have provided the precedent for
steroidal regulation of gap junctions, dye coupling and connexin mRNA expression in
regions of the CNS; but it remains possible that these may be highly region-specific
responses, that are not observed in other gonadal sensitive areas of the brain.
A second interpretation is that a large-scale change in connexin mRNA expression
may occur at a different time point than was investigated in this study. Comparison of
data arnong cycling females demonstrated only negligible changes in Cx43 and Cx32
transcript levels, when the animals were sacrificed and brain tissue collected in the
morning hours (9am to 1 1 am) of the respective days of the estrous cycle. We chose this
time of day very specifically in an attempt to minimize the potential confounding effects
of alterations in neuronal activity, which could indirectly driving changes in connexin
expression via altered removal or astrocytic c-fos synthesis. We were interested initially
in this study in the specific effects of the steroids on Cx43 mRNA levels, as a potential
causative factor, rather than as the result of changes in the electrical activity of the brain
induced by steroid action. In female cycling rats, a sharp rise in estradiol and
progesterone that occurs in proestrus stirnulates the surge in luteinizing hormone (LH) in
the late afternoon hours of proestrus. This LH surge is a result of a sudden
synchronization of electrical activity within LHRH neurons dispersed in the preoptic area
and hypothalamus. During the afiernoon of proestrus, when luteinizing hormone
releasing hormone (LHRH) output is significantly increased, cTfos expression rises 135 dramatically in LHRPI neurons around the time of the LH surge . Thus, it is possible
that, while we saw very little evidence of altered connexin mFWA levels in the present
study, Cx43 andor Cx32 expression might change later in the day, closer to the expected
time of alterations in neuronal electrical activity associated with the estrogen-induced LH
surge. Future studies should investigate the time course of connexin expression in
ovariectomized rats with estradiol replacement to test this hypothesis.
The third, related interpretation for the lack of steroidal regulation of connexin
expression in the brain, may lie in the status of electrical activation of the CNS.
Regulation of connexin expression may only be apparent when the exposure to gonadal
horrnones is concomitant to a change in CNS electrical activity. The results reported
here are consistent with the assurnption that in the absence of altered electrical activity,
the administration of gonadal hormones is insufficient to induce a change in connexin
mRNA expression. This hypothesis is also consistent with results previously reported in
the literature. Micevych et al. (1996) reported an up-regulation of Cx32 mRNA levels in
the SON prior to parturition and during lactation, as well as a down-regulation of
transcript on post-parturn day 1 of the rat. On the day prior to the onset of labour when
there is increased Cx32 mRNA in the SON, high levels of circdating estrogen coincide
with synchronized high-fiequency bursts of activity within oxytocin neurons. Postpartum
day 1 is characterized by a sudden reduction in Cx32, consistent with Iow levels of both
estrogen and SON neuronal activity. On postpartum day 13, there are once again high
levels of Cx32 mRNA, a time when the SON is in a continua1 state of activation induced
by suckling stimuli.
This interpretation of electrical activation as a necessary coincident stimulus for
comexin regulation by horrnones could also be consistent with studies in uterine tissue.
The onset of coordinated uterine contractions during the process of labour is correlated
with both an increased ratio of estrogen:progesterone, as well as increased levels of Cx43
expression. Full induction of Cx43 expression and the assembly of functional Cx43 gap
junctions may require both estrogen action and increased myocyte electrical activity.
Piersanti and Lyc (1995) found that prernature, delayed, and spontaneous term labour
were associated with a coincident increase in Cx43 and c-fos mRNA. The increase in
estradiol prior to parturition induces a dramatic increase in electrical excitability and
contractility of the uterus. Estradiol is also known to increase the electrical excitability of
me uuxus in non-pregnan~ rxs, wnicn may accounr ror rne increase In c-jos prior ro me 212 elevation in Cx43 mRNA expression . The question of whether a change in Cx43
mRNA expression would be observed in the uterus if electrical activity was blocked
following estrogen exposure remains to be answered experimentally. If a quiescent utems
exhibited a much smaller rise in gap junction expression following estrogen exposure than
one in which alterations in membrane conductivity were allowed to occur, this might
indicate convergence of the mechanisms regulating connexin expression. The same
hypothesis could also be consistent with the known interactions between the effects of
estrogen and stretch 279.
The most compelling support for the hypothesis that electrical activity coupled
with estrogen action is required for altered connexin expression in the brain cornes from
recent unpublished data from Our laboratory (H. Edwards). Based on the hypothesis that
there might be synergistic effects of estrogen and electrical activation on gap junction
expression, perhaps contributing to epileptic seinire activity, experiments were initiated
using the kindled rat mode1 of epilepsy. Kindling involves delivery of a small electrical
stimulus to regions of the limbic system on a daily basis, until eventually the stimulus
resuits in a spreading after-discharge that recruits a full-scaIe motor seizure that resembles
epilepsy in humans. Kindling was combined with varying steroid treatment regimens.
Preliminary results suggest that there is a drarnatic up-regulation of Cx43 mRNA in
estrogen target regions of the female rat brain, when estradiol treatment is provided to
OVX epileptically kindled animals, but not when the estrogen is given to non-kindled
animals, confirrning the data 1 have obtained. Furthermore, progesterone treatment seerns
to yield an opposing, down-regulatory effect on Cx43 transcripts as demonstrated by in
situ hybridization. Similar effects were found whether the rat had been given the kindling
stimulus from the dorsal hippocampus, or fiom the dorso-lateral arnygdala. Thus, large-
scale electrical activation of the brain may be necessary for hormonal induction of
connexin expression.
This hypothesis, if correct, has major clinical implications in the field of
epileptiform activity in the brain. Recent evidence suggests a possible role for electrical
gap junctional coupling in the etiology of epileptic seizures 84,192 . Patients with
caLameniai cpiiepsy experieric;e a r1uc;Luaiiori III scizure 1requerrc;y wriic;ri varies wlrnin uie
menstrual cycle, showing a positive correlation with the ratio of estradio1:progesterone 9.
Furtherrnore, estrogen priming is known to stimulate the expression of c-fos in the CA1 102 , which is a marker correlated with seizure-like activity. This combination of evidence
suggests that the epileptiform activity which is known to be induced by estrogen 9*34, may
also be correlated with changes in connexin expression.
Although the data reported here indicates that there are no large scale changes in
comexin transcript levels as a function of hormonal exposure, there are many other steps
that may be horrnonally infiuenced during the synthetic processes of connexins and gap
junctions. Hormones may alternatively influence the production of connexin proteins,
connexin assembly, phosphorylation, trafficking, or docking. Thus further studies should
examine not only the gonadal steroid regulation of connexin transcript levels, but also
protein levels, phosphorylation, and the presence of functional gap junctions. Further
studies may also reveal a gonadal steroid regulation of CNS connexins other than those
studied within this thesis.
In surnmary, these studies have docurnented the expression patterns of Cx43
and/or Cx32 mRNA in cycling female rats, castrate or hormone treated female and male
rats and in neonatal rat pups. Changes in the brain induced by androgen or estrogen
exposure are not associated with large-scale changes in comexin expression. In siru
hybridization techniques demonstrate that estradiol up-regulates expression only in a few,
restricted regions of the brain, including the bed nucleus of the stria terrninalis, the dorso-
lateral septum, and the periventricular hypothalamus. There were no other significant
changes in Cx43 or Cx32 mRNA in the other experimental groups investigated. It
remains possible that for regulation of connexin expression in the brain by gonadal
steroids to occur, there rnust first be electricai excitation in order to "prime" or accentuate
the hormonal effects.
r u 1 Ul\U JJlL\UL 1 1Vl\fl
The work presented here clearly represents only a first step in atternpting to
elucidate the effects of gonadal steroids on connexin expression in the CNS. A
considerable amount of additional work remains to be done. In particular, there are two
specific additional studies that should be performed:
Time course experiment: Examine the time course of connexin expression in the
OVX rat with estradiol replacement, and during the 4-day estrous cycle of the female
rat; this may reveal a potential association between connexin expression and neuronal
electrical activity associated with fluctuating levels of gonadal hormones.
Mechanisrn of hormonal modulation of connexins: Investigate a possible cascade
mechanism of estrogen regulation of connexin expression, via estrogenic induction of
the proto-oncogenes, c-fos and c-jun, which rnay subsequently alter connexin gene
transcription. This may be achieved through ce11 culture experiments using tissues
removed from specific regions of the brain. Connexin expression levels could be
manipulated through treatment with hormones, and could be compared with patterns
of expression following administration of antisense oligonucleotides that targets AP-1
expression on the connexin gene.
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