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Volume 16, Number 16,
Issue of August 15, 1996
pp. 4872-4880
Copyright ©1996 Society for Neuroscience
Plasticity in GABAA Receptor Subunit mRNA Expression
by Hypothalamic Magnocellular Neurons in the Adult Rat
Valérie S. Fénelon and
Allan E. Herbison
Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge
CB2 4AT, United Kingdom
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The magnocellular hypothalamic neurons exhibit a substantial degree
of structural and functional plasticity over the time of pregnancy,
parturition, and lactation. This study has used in situ
hybridization techniques to examine whether the content of
 1, 2,
 2, and  2
GABAA receptor subunit mRNAs expressed by these
cells fluctuates over this period. A process of regional, followed by
cellular and then topographical, analyses within the supraoptic (SON)
and posterior paraventricular (PVN) nuclei revealed that an increase in
magnocellular 1 subunit mRNA content occurred
during the course of pregnancy up to day 19, after which a decline in
expression was detected on the day of parturition. Significant
fluctuations of this nature were observed only in the oxytocin
neuron-enriched regions of the SON and PVN. The expression of
2, 2, and
2 subunit mRNAs in the SON and PVN and of all
subunit mRNAs in the cingulate cortex did not change over this period.
During lactation, 2 subunit mRNA content
within the PVN increased significantly on day 14 of lactation as
compared with day 7, and topographical analysis suggested that it
involved principally magnocellular vasopressin neurons.
These results demonstrate the cell- and subunit-specific regulation of
GABAA receptor mRNA expression within the
hypothalamic magnocellular system. In particular, they suggest that
fluctuations in 1 subunit expression may
contribute to the marked variations in electrical activity exhibited by
magnocellular oxytocin neurons at the time of parturition. More
generally, they provide evidence in support of
GABAA receptor plasticity within a physiological
context in the adult rat brain.
Key words:
GABAA receptor subunit;
in
situ hybridization;
lactation;
oxytocin;
paraventricular nucleus;
parturition;
pregnancy;
supraoptic nucleus;
vasopressin
INTRODUCTION
The aminobutyric acidA
(GABAA) receptor is responsible for the majority
of fast synaptic inhibition in the mammalian forebrain. Molecular
cloning studies now have revealed the existence of a large
GABAA receptor gene family encoding six , four
, three , one  , and two  subunits, which are thought to
combine as heteropentamers to form pharmacologically distinct receptor
isoforms (Macdonald and Olsen, 1994 ; Sieghart, 1995). Although the
exact composition and stoichiometry of native
GABAA receptors remains unknown, photolabeling
studies and the identification within the brain of different patterns
of GABAA receptor ligand binding (Bureau and
Olsen, 1993) and receptor subunit expression (Wisden et al., 1992)
indicate that a wide variety of GABAA receptor
isoforms is likely to be expressed.
The activity of neuronal networks in the forebrain is thought to be
critically dependent on GABAergic transmission and substantial interest
has focused on the regulatory control of GABAA
receptors. To date, such studies have concentrated on developmental
regulation, including changes during aging (MacLennan et al., 1991;
Poulter et al., 1993; Fritschy et al., 1994; Gutierrez et al., 1994;
Mathews et al., 1994), and examined the response of
GABAA receptor expression and function in adults
to a variety of allosteric GABAA receptor
modulators (Kang and Miller, 1991; Montpied et al., 1991; Primus and
Gallager, 1992; Mhatre et al., 1993; Tseng et al., 1994) as well as
excitatory and inhibitory amino acids (Kim et al., 1993; Mhatre and
Ticku, 1994; Zhu et al., 1995; Fénelon and Herbison, 1996).
Further work has examined GABAA receptor
expression in pathological states such as epilepsy (Kokaia et al.,
1994; Kamphuis et al., 1995), Huntington's disease (Faull et al.,
1993), and chronic stress (Montpied et al., 1993). Although these
studies clearly have relevance in understanding the developmental and
pathophysiological roles of GABA transmission in the brain,
surprisingly little evidence exists for changes in
GABAA receptor expression within a physiological
context in the adult brain.
The hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei
contain the cell bodies of the magnocellular oxytocin neurons, which
project to the posterior pituitary gland and play a critical role in
enabling parturition and lactation to occur. It is now well established
that these cells exhibit marked, reversible changes in their
morphology, synaptic connectivity, and electrical activity with each
cycle of pregnancy and lactation in the female rat (Hatton, 1990;
Theodosis and Poulain, 1993). As such, they provide an excellent
example of an adult neuronal population that exhibits a substantial
degree of neuronal plasticity within a defined physiological context.
We have determined the complement of GABAA
receptor subunits expressed by these hypothalamic magnocellular neurons
and shown that, regardless of whether they synthesize oxytocin or
vasopressin, they all express mRNA and protein for the
1, 2,
2, and 2 subunits
(Fénelon and Herbison, 1995; Fénelon et al., 1995). Because
evidence suggests a critical role for GABA in regulating the activity
of adult hypothalamic magnocellular neurons via the
GABAA receptor (Moos, 1995; Voisin et al., 1995),
we questioned in this study whether these magnocellular neurons may
represent a neural population in which fluctuations in
GABAA receptor subunit expression may occur
within a physiological context in the adult rat brain.
MATERIALS AND METHODS
Animals and tissue preparation. Parturient, pregnant,
lactating, and virgin (200-250 gm) Wistar female rats from the
Babraham colony were maintained in a controlled environment (lights on
from 5:00 A.M. to 7:00 P.M.; 22°C) with food and water freely
available. Although gonadal steroids have been shown to influence
GABAA receptor subunit expression in other
regions of the hypothalamus (Herbison and Fénelon, 1995),
ovariectomy has no effect on the subunit mRNA content of magnocellular
neurons (V. S. Fénelon and A. E. Herbison, unpublished data). In
this study, virgin female rats were ovariectomized under Avertin
anesthesia (2% tribromoethanol, 1 ml/100 gm body weight, i.p.) and
used as our nonpregnant control group. Six groups of five animals were
used; nonpregnant rats, day 10 and day 19 pregnant rats (day 0 of
pregnancy equals day after overnight mating), parturient rats 1-2 hr
after the delivery of the first pup, and day 7 and day 14 lactating
rats (day 1 of lactation equals day of parturition). All rats were
killed by cervical dislocation and decapitated; brains were removed
quickly and frozen on dry ice. Parturient animals were killed
throughout the afternoon, whereas all other rats were killed between
10:00 A.M. and 12:00 A.M.
In situ hybridization histochemistry. Detection of
GABAA receptor 1,
2, 2, and
2 subunit mRNAs was undertaken by using
in situ hybridization procedures reported previously
(Fénelon and Herbison, 1995; Fénelon et al., 1995).
Briefly, fresh-frozen sections (15 µm thick) were cut in the coronal
plane through the hypothalamus (plates 22-26 of Swanson, 1992) and
thaw-mounted onto Vectabond-coated slides. Antisense oligonucleotides
(42-45 mer) complementary to the coding regions for amino acids
342-356, 340-354, 325-339, and 338-352 of the rat
GABAA receptor 1,
2, 2, and
2 subunits, respectively, were synthesized and
3 end-labeled with [35S]dATP (1000-1500
Ci/mmol; DuPont NEN, Boston, MA) by using terminal deoxynucleotidyl
transferase (50 U, Pharmacia) and resulting in a specific activity of
~5 × 108 dpm/mg. Fixed sections were
dehydrated with alcohol, and 250 µl of hybridization buffer (4× SSC,
50% deionized formamide, 10% dextran sulfate, 1× Denhardt's
solution, 250 µg/ml sheared salmon testis DNA, and 0.3% mercaptoethanol) containing one of each of the
35S-labeled 1,
2, 2, and
2 subunit probes (28 fmol/ml equivalent to
2 × 109 cpm/ml) was applied to different
series of slides containing 6-8 coronal sections.
Hybridization was performed in humidified chambers at 37°C overnight
and washed in 1× SSC at room temperature, three times in 1× SSC at
55°C (30 min each), and again in 1× SSC for 1 hr at room
temperature. Slides were dipped in Ilford K-5 nuclear track emulsion
and exposed for 6-9 weeks in dark, tight boxes. At the appropriate
exposure time, as determined by test slides, all slides were
photodeveloped with Ilford Phenisol and counterstained lightly with
methylene blue. Signal specificity was assessed by use of competition
experiments in which radiolabeled probes were hybridized to sections in
the presence of an excess (50- to 100-fold) unlabeled probe. Also,
specificity was confirmed by reference to previous reports of the
distribution of the rat transcripts of the 1,
2, 2, and
2 subunits of the GABAA
receptor (MacLennan et al., 1991; Zhang et al., 1991; Araki et al.,
1992; Wisden et al., 1992).
Data analysis. Using a Joyce-Loebl Magiscan analyzer
coupled to a Leica Orthoplan microscope, we undertook an initial
quantitative analysis of GABAA receptor subunit
mRNA expression by determining silver-grain density (silver
grains/µm2) overlying the whole SON in its midportion in the coronal
plane (plates 23-24 of Swanson, 1992), the posterior magnocellular
division of the PVN (pPVN; plates 25-26), as well as a 200 × 500 µm rectangle overlying layers 2 and 3 of the cingulate cortex. For
each animal, four measurements of the SON and pPVN and three
measurements of the cortex were taken from two to four sections and
averaged to provide mean values for each subunit. Values from each
animal were combined to give experimental group means (± SEM). For
those subunits and regions in which statistical differences were
evident, a subsequent cellular analysis of subunit mRNA expression was
undertaken by analyzing 18-25 cells from two to four sections in each
region.
Because these results suggested the possibility that subpopulations
within the SON and pPVN expressed subunit mRNAs on a differential
basis, we reanalyzed these sections on a detailed topographical basis.
Despite their potential for oxytocin/vasopressin coexpression, the
great majority of magnocellular neurons synthesizes only oxytocin or
vasopressin (Jirikowski, 1992) and resides, in general, within
different subregions of the PVN and SON (Rhodes et al., 1981; Sawchenko
and Swanson, 1982). In this reanalysis, care was taken to identify and
analyze, in two to three sections from each animal, a minimum of 40 magnocellular neurons equally from the oxytocin neuron-enriched dorsal
half of the SON (Fig. 1A) and
anteroventromedial pPVN (Fig. 1B1) and the vasopressin
neuron-enriched ventral SON (Fig. 1A) and
posterodorsolateral pPVN (Fig. 1B2). In all single-cell
silver-grain analyses, the numbers of silver grains overlying cells in
the excess unlabeled-probe control sections were determined, and, in
experimental sections, only those cells expressing numbers of silver
grains greater than five times that of controls were used for analysis.
For each rat and each region, an average silver-grain count per cell
was determined, and these values were combined to give experimental
group means (±SEM). The same procedure was applied to the
determination of the median when population shifts in silver-grain
numbers per cell were analyzed. For each brain region, nonparametric
Mann-Whitney U tests were performed between each temporally
contiguous animal group (nonpregnant rats were considered as time point
0 and thus compared with day 10 pregnancy, then day 10 pregnancy versus
day 19 pregnancy, and so forth); p  0.05 was considered
statistically significant.
Fig. 1.
Methylene blue-counterstained sections of the
supraoptic (A) and posterior paraventricular (B)
nuclei from an ovariectomized rat after hybridization with
35S-labeled GABAA receptor
1 subunit oligonucleotide illustrating the
different regions in which cellular silver-grain analysis was
undertaken. The dashed line in A divides
the dorsal from the ventral half of the supraoptic nucleus.
B1, The dashed rectangle indicates the
region of the anteroventromedial posterior paraventricular nucleus in
which magnocellular cells were sampled, whereas the dashed
circle in B2 indicates the position of
magnocellular cells sampled from the posterodorsolateral posterior
paraventricular nucleus. The anteroposterior level of B1 is
rostral to that of B2. oc, Optic chiasm;
3V, third ventricle. Scale bars: A, 50 µm;
B1, B2, 100 µm.
[View Larger Version of this Image (143K GIF file)]
RESULTS
The distribution of the four GABAA receptor
subunit transcripts in the coronal brain sections was the same as that
reported in our previous studies with the same oligonucleotide probes
(Fénelon and Herbison, 1995; Fénelon et al., 1995).
Magnocellular neurons of the SON and PVN displayed hybridization
signals for 1, 2,
2, and 2 subunit
mRNAs.
Regional silver-grain density analysis
Silver-grain density analysis of the whole SON, pPVN, and
layers 2 and 3 of the cingulate cortex detected three significant
changes in receptor subunit expression throughout pregnancy,
parturition, and lactation (Fig. 2, Table
1). In terms of
1 subunit mRNA, a 40-60% decrease in
silver-grain density was observed in both the SON (p < 0.05) and pPVN (p < 0.01) on the day of parturition as
compared with day 19 of pregnancy (Fig. 2). Before parturition, a
nonsignificant increase in 1 subunit mRNA
content was observed as pregnancy progressed within both the SON and
pPVN (Fig. 2). No significant differences in silver-grain density were
detected for the 1 subunit in the cingulate
cortex; overall, 1 subunit mRNA content was
lower in the pPVN as compared with the SON (Fig. 2). The second
significant change was observed with the 2
subunit signal in which a significant (p < 0.05)
~40% increase in silver-grain density was observed in the pPVN alone
on day 10 of pregnancy as compared with nonpregnant ovariectomized rats
(Fig. 2). Expression of 2 subunit mRNA did not
change at any time in the SON or cingulate cortex or at other times in
the pPVN (Fig. 2). The final significant alteration in subunit
expression was found with the 2 subunit
hybridization signal, in which a significant (p < 0.01) 160% increase in silver-grain density was noted only within the
pPVN of day 14 lactating rats as compared with day 7 animals (Table 1).
A nonsignificant (p = 0.06) increase in
2 mRNA content was noted within the SON
between day 19 of pregnancy and the day of parturition (Table 1). Apart
from the day 14 lactating rats, 2 subunit mRNA
expression was consistently lower in the pPVN as compared with the SON.
No significant changes were detected at other time points, and no
differences in 2 subunit mRNA expression were
found at any time over the course of pregnancy, parturition, and
lactation (Table 1). Again, 2 subunit mRNA
expression was consistently lower in the pPVN as compared with the SON
(Table 1.).
Fig. 2.
Quantitative silver-grain density analysis of
1 (top) and
2 (bottom)
GABAA receptor subunit mRNA expression in the
supraoptic nucleus (SON), posterior paraventricular nucleus
(PVN), and cingulate cortex (CTX)
in nonpregnant, ovariectomized (NP) pregnant day 10 (P10) and 19 (P19), parturient (PA),
and lactating day 7 (L7) and 14 (L14)
rats. Data are expressed as relative numbers of silver
grains/µm2. Each bar represents the mean ± SEM of five animals. *p < 0.05; **p < 0.01.
[View Larger Version of this Image (47K GIF file)]
Cellular silver-grain analysis
Single-cell silver-grain analysis of 1
subunit mRNA hybridizations in the SON and pPVN of pregnant day 10 and
day 19 and parturient rats enabled the construction of
silver-grain-per-cell distribution histograms (Fig. 3).
These histograms indicated a nonsignificant shift toward higher values
of silver grains per cell in pregnancy day 19 rats as compared with day
10 pregnant animals in both the SON (medians: day 10 = 125 ± 8 and day 19 = 146 ± 8 silver grains/cell) and pPVN
(medians: day 10 = 67 ± 5 and day 19 = 90 ± 13).
A return toward lower values in parturient animals as compared with day
19 pregnant rats was found in both the SON (medians: day 19 = 146 ± 8 and parturition = 105 ± 7;
p < 0.01) and pPVN (medians: day 19 = 90 ± 13 and parturition = 62 ± 13; Fig. 3). Although the
histograms from pregnant day 10 rats were suggestive of an
approximately normal distribution of silver grains per cell, those
constructed from pregnancy day 19 rats, and in particular the SON, were
suggestive of the appearance of a second population of high-expressing
cells with a mean of ~190 silver grains per cell (arrow,
Fig. 3). This high-expressing population did not exist in parturient
rats (Fig. 3).
Fig. 3.
Frequency distribution histograms of silver grains
per magnocellular cell after 1 subunit
hybridizations in the supraoptic nucleus (SON, left
column) and paraventricular nucleus (PVN,
right column) from pregnant rats on day 10 (P10) and 19 (P19) and parturient rats
(PA). Median ± SEM values are given in
parentheses for each group (n = 5).
Arrow in SON P19 indicates a population
of relatively high-expressing cells not detected at other time points.
**p < 0.01 as compared with P19.
[View Larger Version of this Image (38K GIF file)]
Reanalysis of the SON and pPVN on a more detailed topographical
basis revealed that a significant increase and decrease in
1 subunit mRNA expression over pregnancy was
observed only in magnocellular neurons located within the oxytocin
neuron-enriched regions of these nuclei. A significant 41-85%
increase (p < 0.05) and then 30-40% decrease
(p < 0.05) in the number of silver grains per
magnocellular cell was found as pregnancy progressed from day 10 to day
19 and then to parturition in only the dorsal SON and
anteroventromedial pPVN (Fig. 4A,B). A
significant increase (p < 0.05) in the number of
silver grains per cell also was found from day 10 to 19 of pregnancy in
the ventral SON, but no decreases were detected at the time of
parturition in either of the vasopressin neuron-enriched subregions:
the ventral SON and posterodorsolateral pPVN (Fig. 4C,D). On
day 10 of pregnancy, the number of silver grains per magnocellular cell
did not differ in oxytocin- and vasopressin-enriched regions of either
the SON or pPVN (Fig. 4).
Fig. 4.
Cellular silver-grain analysis after
1 subunit mRNA hybridization in the dorsal
(SON oxytocin region) and ventral (SON
vasopressin region) parts of the supraoptic nucleus
and in the anteroventromedial (PVN oxytocin
region) and posterodorsolateral (PVN vasopressin
region) parts of the posterior paraventricular nucleus in
pregnant day 10 (P10) and 19 (P19) and parturient
(PA) rats (n = 5). Bars represent mean
number of silver grains per cell ± SEM. *p < 0.05; **p < 0.01.
[View Larger Version of this Image (70K GIF file)]
Histogram analysis of the number of silver grains per cell
resulting from the 2 subunit hybridization in
the pPVN of nonpregnant and day 10 pregnant rats revealed a normally
distributed pattern of silver grains per cell at both times (data not
shown) and a nonsignificant shift to the right in the median (medians:
213 ± 5 in nonpregnant animals and 259 ± 14 silver
grains/cell in day 10 pregnant rats). The mean number of silver grains
per cell also was not significantly different (226 ± 8 in
nonpregnant rats and 264 ± 14 silver grains/cell in day 10 pregnant animals; p = 0.095).
Similar distribution analysis for the 2
subunit showed a significant (p < 0.01) shift to the
right in lactating day 14 rats as compared with day 7 lactating rats in
only the pPVN (medians: 116 ± 9 on day 7 of lactation and
170 ± 12 silver grains/cell on day 14; Fig.
5A). When reanalyzed on a topographical
basis, the significant increase in the number of silver grains per cell
on day 14 of lactation was found to be restricted to those
magnocellular cells within the vasopressin neuron-enriched
posterodorsolateral pPVN (p < 0.01; Fig.
5B). The number of silver grains per magnocellular cell in
the oxytocin- and vasopressin-enriched regions of the pPVN were not
different on parturition and day 7 of lactation (Fig.
5B).
Fig. 5.
A, Frequency distribution histograms of
silver grains per magnocellular cell after 2
subunit hybridizations in the posterior paraventricular nucleus of
parturient (PA), day 7 (L7), and day 14 (L14) lactating rats (n = 5). Median ± SEM values are given in parentheses for each group.
B, Cellular silver-grain analysis after
2 subunit mRNA hybridization in the
anteroventromedial (PVN oxytocin region) and
posterodorsolateral (PVN vasopressin region)
parts of the posterior paraventricular nucleus in parturient
(PA), day 7 (L7), and day 14 (L14)
lactating rats. Bars represent mean number of silver grains per
cell ± SEM. **p < 0.01 as compared with
L7.
[View Larger Version of this Image (32K GIF file)]
DISCUSSION
These results provide evidence for the cell- and subunit-specific
regulation of GABAA receptor mRNA expression
within the hypothalamic magnocellular system over the course of
pregnancy, parturition, and lactation. Most important, we have shown
here with regional silver-grain density analyses that
1 subunit mRNA expression falls between day 19 of pregnancy and the day of parturition in both the SON and pPVN,
whereas other subunit mRNAs remain constant. Frequency histogram
analysis of cellular silver-grain numbers in these animals indicated
the possibility that a specific population of high-expressing
1 subunit mRNA-containing cells existed only
on day 19 of pregnancy. A cellular reanalysis of the SON and pPVN on a
more detailed topographical basis revealed that a 40-80% increase in
1 subunit mRNA expression between day 10 and
19 of pregnancy, followed by a 30-40% fall at the time of
parturition, was found only in the oxytocin neuron-enriched regions of
both nuclei. A similar process of analysis revealed a substantial
increase in the 2 subunit mRNA content of
putative pPVN vasopressin neurons during late lactation. To the best of
our knowledge, these findings represent the first demonstration of
plasticity in GABAA receptor subunit mRNA
expression within a physiological context in the adult rat brain.
We confirm here our earlier demonstration of
1, 2,
2, and 2 subunit mRNA
expression by hypothalamic magnocellular neurons and note that this
correlates well with immunocytochemical evidence for the synthesis of
only 1, 2,
2/3, and 2 subunit
proteins by all of these cells (Fénelon and Herbison, 1995;
Fénelon et al., 1995). Because the GABAA
receptor subunits expressed by oxytocin and vasopressin neurons do not
differ in any qualitative sense, we have taken advantage of the
topographical distribution of oxytocin and vasopressin neurons within
the SON and pPVN (Rhodes et al., 1981; Sawchenko and Swanson, 1982) to
try to differentiate receptor subunit mRNA expression in these two cell
types. The partially overlapping topography of these cells within the
SON means that we can claim only to be examining oxytocin neuron- and
vasopressin neuron-enriched cell populations in this nucleus; indeed,
the significant increase in 1 subunit mRNA
detected in the ventral SON between day 10 and 19 of pregnancy may
reflect this incomplete differentiation between oxytocin and
vasopressin neurons. However, our careful attention to analyzing only
those cells within the core of the posterodorsolateral and ventral
aspect of the anteroventromedial pPVN (Fig. 1) should have ensured that
we dealt with almost pure vasopressin and oxytocin neural populations
(Sawchenko and Swanson, 1982), respectively, in our subregional pPVN
analysis.
GABAA receptor changes within the adult brain
It is clear that the modulation of inhibition in the brain has
important implications for normal physiological processes as well as
for the generation of pathological states. In principle, changes in
GABAA receptor-mediated transmission may result
from alterations in either the profile of synaptic GABA concentrations
and/or the functioning of the GABAA receptors
themselves. It seems likely that most synaptic
GABAA receptors in the brain are saturated fully
by spontaneous GABA bombardment, with near-maximal numbers of open
channels, and as such, it has been suggested that a more effective
means of modulating GABAergic transmission in the brain may result from
altering the number and/or pharmacodynamics of
GABAA receptors (Mody et al., 1994). Although
changes in GABAA receptor expression are known to
occur in a variety of pathological situations (see introductory
remarks), it is not known whether such alterations also occur in the
normal adult brain.
One possibility for a physiologically significant regulation of
GABAA receptor functioning in the adult brain
would be via receptor phosphorylation (Macdonald and Olsen, 1994;
Sieghart, 1995). Another mechanism, suggested by the present studies,
would involve postsynaptic neurons regulating their own level and type
of GABAA receptor expression. In this respect, it
is interesting to note recent work in the primate, in which monocular
deprivation has been shown to reduce GABAA
receptor subunit mRNA expression in a selective manner within the
visual cortex (Huntsman et al., 1994). In that system, reduced mRNA
expression is thought to result from a reduction in activity-dependent
subunit gene transcription (Huntsman et al., 1994). A similar mechanism
seems unlikely within the hypothalamic magnocellular neurons, because
an inverse relationship between 1
subunit mRNA expression and electrical activity exists (see below).
Furthermore, the changes reported here are unlikely to result from
autoregulation of GABAA receptor mRNA expression,
because increases in endogenous GABA concentrations do not influence
subunit mRNA expression within the SON or PVN, or the
2 subunit in the PVN (Fénelon and
Herbison, 1996).
Although the present study has not evaluated whether the fluctuations
in 1 subunit mRNA expression observed here
during pregnancy are translated into 1 subunit
protein, previous studies have indicated a positive correlation between
1 subunit mRNA expression and
1 subunit protein in vitro (Zheng
et al., 1994) and in vivo (Huntsman et al., 1994). It is not
unreasonable, therefore, to suggest that the 30-80% variations in
mRNA expression observed here may result in changes in functional
subunit protein. More difficult, perhaps, is the speculation as to what
effects single subunit changes may have on GABAA
receptor functioning in magnocellular oxytocin neurons. All of these
cells express the 1,
2, 2, and
2 subunits of the GABAA
receptor (Fénelon and Herbison, 1995; Fénelon et al.,
1995), and current wisdom would suggest that these subunits form
pentamers composed of 1,
2, 2 or
2, 2,
2, and/or all four subunits (Sieghart, 1995).
Hence, in the absence of significant changes in the expression of the
other subunits over pregnancy, an increase in
1 subunit abundance before parturition may not
change the total number of receptors but, rather, result in a greater
proportion of 1 subunit-containing receptors
on magnocellular oxytocin neurons. This would alter the overall
pharmacodynamics of GABA responses in these cells by moving them toward
the benzodiazepine type 1 profile, which shows enhanced benzodiazepine
facilitation (Pritchett et al., 1989) and increased allopregnanolone
sensitivity (Shingai et al., 1991) as compared with
2 subunit-containing receptor isoforms.
GABAA receptor changes with respect to magnocellular
neuron physiology
Approximately 40% of the terminals synapsing on hypothalamic
magnocellular neurons contain GABA (Decavel and Van den Pol, 1990; Gies
and Theodosis, 1994), and electrophysiological studies indicate that
GABA acts via the GABAA receptor to hyperpolarize
these cells (Randle and Renaud, 1987). In vivo studies
examining the role of GABA in influencing the activity of SON oxytocin
neurons in the lactating rat show that occupancy of the
GABAA receptor is critical for these neurons to
exhibit the intermittent, synchronized bursts of electrical activity
necessary for milk ejection (Voisin et al., 1994, 1995; Moos, 1995).
Although similar studies have not, as yet, been undertaken in pregnant
or parturient animals, it is highly likely that a powerful GABAergic
influence also exists on magnocellular oxytocin neurons at this time.
On the day of parturition, oxytocin neurons exhibit an increase in
basal firing rate, which is followed later by the onset of
intermittent, synchronized bursts of electrical activity leading to
birth of the pups (Summerlee, 1981; Jiang and Wakerley, 1995). The
mechanisms involved in changing the electrical and biosynthetic
activity of magnocellular oxytocin neurons over this period remain
unclear.
Our present findings of an increase in putative oxytocin neuron
1 subunit mRNA content leading up to day 19 of
pregnancy, followed by a fall at the time of parturition, promotes
speculation that these magnocellular cells may regulate their
GABAA receptor expression to maintain an enhanced
level of inhibitory input in late pregnancy. This inhibitory influence
then may be relaxed at the time of parturition to enable either the
increased basal firing rate and/or the high-frequency discharges
required of oxytocin neurons at this time. The neighboring
magnocellular vasopressin neurons show relatively little variation in
their phasic pattern of electrical activity over this time (Summerlee,
1981), and, accordingly, we have detected no similar significant
changes in subunit mRNA profile in putative vasopressin neurons over
pregnancy and parturition. As noted above, the changes involving
oxytocin neurons may not necessarily involve the appearance of more
GABAA receptors within the synapse but, instead,
involve more subtle changes in receptor dynamics. The increased
sensitivity of 1 subunit-containing receptors
to facilitation by progesterone metabolites (Shingai et al., 1991) may
be particularly important because progesterone levels are at their
highest in late pregnancy, and the GABAA
receptors located on the terminals of magnocellular neurons, at least,
have been shown to be sensitive to allopregnanolone (Zhang and Jackson,
1994).
Also, we have found evidence for a substantial increase in
2 subunit mRNA expression at late pregnancy,
and our topographic analysis strongly suggests that this change is
restricted to the vasopressin neurons of the pPVN. There is a clear
reduction in the responsiveness of oxytocin and vasopressin neurons to
osmotic stimuli in lactation (Higuchi et al., 1988; Koehler et al.,
1993), and although this may be explained for oxytocin by a reduction
in pituitary stores in late lactation, vasopressin content remains
unchanged (Koehler et al., 1993). Reduced vasopressin responsiveness
could, conceivably, arise from enhanced GABAA
receptor-mediated inhibition at this time.
In conclusion, we present here evidence indicating that
GABAA receptor mRNA expression fluctuates under
physiological circumstances within the hypothalamic magnocellular
system of the adult female rat. Such findings support further the
highly plastic nature of this neuronal system and suggest that
neuron-specific variations in GABAA receptor
expression may be relevant to the regulation of GABAergic transmission
within the normal adult brain.
FOOTNOTES
Received March 18, 1996; revised May 15, 1996; accepted May 15, 1996.
V.S.F. was a Wellcome Trust European Traveling Fellow. A.E.H. is a
Lister Institute-Jenner Fellow. We thank Drs. S. Augood and R. J. Bicknell for critical review of this manuscript and Mr. I. King for
performing the emulsion autoradiography.
Correspondence should be addressed to Dr Allen E. Herbison at the above
address.
Dr. Fénelon's current address: Centre National de la Recherche
Scientifique, Unité de Recherche Associée 1126, Laboratoire
de Neurobiologie et Physiologie Comparées, Université de
Bordeaux I, Place du Dr Peyneau, 33120 Arcachon,
France.
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