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Department of Physiology (T.S.P.-S., K.A.G., J.P.Y.K., M.M.M.), University of Maryland School of Medicine, Baltimore, Maryland 21201; Department of Biomedical Sciences (A.M.D.), E. K. Shriver Center, Waltham, Massachusetts 02452; Department of Obstetrics and Gynecology (K.A.G.), University of Maryland School of Medicine, Baltimore, Maryland 21201; and Medical Biotechnology Center (J.P.Y.K.), University of Maryland Biotechnology Institute, Baltimore, Maryland 21201
Address all correspondence and requests for reprints to: Tara Perrot-Sinal, Ph.D., Department of Physiology, BRB 5-020, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201. E-mail: tperr001{at}umaryland.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several lines of evidence suggest that gammabutyric acid (GABA) can have excitatory, stimulatory effects early during development and then gradually switch to mediate inhibitory processes in the adult brain. For example, activation of the GABAergic system during early development stimulates PRL release in female rats, whereas this same activation inhibits PRL release in rats near puberty (4, 5). GABA is excitatory in primary neuronal cultures from the developing hypothalamus (6), hippocampus (7), neocortex (8), thalamus (9), cerebellum (10), and spinal cord (11) and in adult brain after injury (12). Activity of specific chloride cotransporters (13) results in the Cl- reversal potential being more positive than the resting membrane potential. Under these conditions, activation of GABAA receptors enables Cl- fluxes that cause membrane depolarization, which, in turn, opens voltage-gated Ca2+ channels (6, 9). By causing depolarization and subsequently elevating intracellular calcium, excitatory GABA influences neuronal survival (9, 10), neurite outgrowth (14), neuroblast motility (15), expression of neurogenesis-related proteins (16, 17), and synapse formation (18). Many of these same parameters are also influenced by estradiol.
It has been known, for some time, that estrogen-receptive neurons in the preoptic/anterior hypothalamic area of the rat brain are also GABAergic (19). Because of this anatomical colocalization and the convergence in their effects, GABA is implicated as a mediator of estradiol’s actions during the process of sexual differentiation (20). GABA concentrations and messenger RNA (mRNA) levels for the rate-limiting enzyme in GABA synthesis, glutamic acid decarboxylase (GAD), are almost twice as high in the newborn male mediobasal hypothalamus and CA1 of hippocampus, relative to female (21, 22). These sex differences are developmentally and hormonally regulated (21, 22). More importantly, the presence of functional GABA during the critical period is essential for the process of sexual differentiation. Using antisense oligonucleotides against GAD mRNA to reduce GABA levels, around the day of birth, results in deficient adult male sexual behavior (23) and disrupted estradiol-mediated glial differentiation (24).
In summary, we have demonstrated the ability of estradiol to enhance GABA synthesis early in development and have established the functional significance of GABA in the process of sexual differentiation. In the present study, we used an in vitro model system to examine the mechanism of GABA action at the cellular level and its modulation by estradiol. To achieve this end, dispersed neonatal hypothalamic neurons were loaded with the calcium indicator, fura-2. [Ca2+]i (free intercellular level of calcium) was then measured during application of the GABAA receptor agonist, muscimol. The results demonstrate that estradiol pretreatment significantly enhances the frequency and magnitude of Ca2+ transients evoked by muscimol, implicating a potential role for excitatory GABA in estradiol-mediated sexual differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hormonal treatments
Treatment groups are summarized in Table 1
. Cells imaged on DIV 4 were incubated
in SCM containing 10-10
M estradiol benzoate (Sigma) in
dimethylsulfoxide (DMSO; Sigma) or DMSO alone at the time
of plating (DIV 0). Final DMSO concentration was 0.01%. Treated SCM
was replaced with fresh treated SCM each subsequent day for 3 DIV.
Cells that were imaged on DIV 7 were treated in the same manner, except
that cells were incubated with untreated SCM from DIV 0–DIV 2 and then
treated for 4 DIV beginning on DIV 3. Treated SCM was always removed
and replaced with untreated SCM before imaging. Hormone treatment from
DIV 0–DIV 3 and DIV 3–DIV 6 encompassed times when in vivo
levels of aromatizable testosterone (25) and hypothalamic
levels of estradiol (26), respectively, are elevated in
male rat fetuses.
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Cells were imaged individually at the soma and were selected using the following criteria: 1) clearly distinguishable as a neuron, not an astrocyte; 2) healthy oval shape and at least one identifiable process; and 3) a cell body that was relatively isolated (i.e. not in contact with another cell body). Cells from estradiol and control-treated plates were imaged alternately. All pharmacological agents were diluted to working strength on the day of experimentation, perfused over cultures in 50-sec pulses, and allowed to wash out. The responses to 10 µM muscimol (Sigma), a specific GABAA receptor agonist, and to 10 µM glutamate (Sigma) were assessed in all neurons. The involvement of L-type Ca2+ channels in the response to muscimol was investigated by perfusing some cells with a 100-sec preliminary perfusion of the L-type Ca2+ channel blocker, nimodipine (1 µM; RBI/Sigma), followed by 10 µM muscimol in the continued presence of 1 µM nimodipine.
At the end of experimentation on each cell, 20 µM ionomycin, a Ca2+ ionophore (Calbiochem, San Diego, CA), was applied to the perfusion chamber to initiate massive Ca2+ influx, which served as a positive control. A cell’s ability to recover rapidly from the ionomycin-induced rise in [Ca2+]i also gave evidence of viability. Computations and calibrations were performed as previously described (27, 28). Briefly, all fura-2 fluorescence records were corrected for background fluorescence by subtracting the light intensity measured from neurons depleted of cytosolic fura-2 by permeabilization with 20–40 µM digitonin. Values of [Ca2+]i were derived using the ratio method (29): [Ca2+]i = Kd x [(R - Rmin)/(Rmax - R)] x [Sf2/Sb2], where R is the ratio F340/F380 (F340 is the fluorescence emitted by fura-2 when excited at 340 nm and F380 is the fluorescence emitted by fura-2 when excited at 380 nm), and Rmin and Rmax are the minimum and maximum values of the ratio, attained at zero and saturating Ca2+ concentrations, respectively. Sf2/Sb2 is the ratio of fluorescence intensities for Ca2+-free and Ca2+-bound indicator measured with 380 nm excitation.
Reagent delivery by perfusion
When measuring cellular Ca2+ responses,
pharmacological agents in solution were delivered to neurons via a
gravity-flow perfusion system. The delivery time of the system (time
for drug solution from a reservoir to reach the neuron) was determined
by monitoring the delivery of carboxyfluorescein dye. The delivery time
is defined to be the time elapsed between reservoir opening and the
time at which the reagent concentration at the cell reaches 10% of the
peak value. For the present experiments, delivery time was 8.2 ±
0.7 sec (n = 17).
Analysis of Ca2+ transients
The amplitude of a Ca2+ transient was
calculated by subtracting the resting
[Ca2+]i (average of five
time points preceding the transient) from the peak
[Ca2+]i (average of five
points at the apex of the transient) attained in response to each
agonist. The latency to respond to each agonist was the time from
agonist application to the first detectable rise in
[Ca2+]i. All latencies
reported have been corrected for the delivery time of the perfusion
system. The kinetics of the recovery from agonist challenge were
analyzed by fitting the declining phase of the
Ca2+ transient to an exponential decay. The time
required to reach 50% recovery (t1/2) was
calculated from the exponential decay time constant (
) such that
t1/2 = -ln (0.5) x . Curve
fitting and analysis were performed with Origin software (Microcal
Software, Northampton, MA).
Immunocytochemistry (MAP-2 immunoreactivity)
To examine the possible contributions of neuronal morphology to
any effects of estradiol treatment on calcium signaling, we used
standard immunocytochemistry techniques to stain for the neuronal
marker, MAP-2. Cover slips were prepared as above using hypothalamic
cells collected from at least ten ED-15 rat embryos. Cells were seeded
at a density of 150,000 per coverslip and treated each day from DIV 0
to DIV 3 with DMSO or
10-10 M
estradiol, as described above. Briefly, cells were fixed,
permeabilized, and then incubated with monoclonal anti-MAP-2 mouse
primary antibody (1:1000 dilution; Sigma) in 10% goat
serum in PBS, overnight, at 4 C. The following day, cells were rinsed,
incubated with secondary antibody (goat antimouse biotinylated IgG;
Vector Laboratories, Inc., Burlingame, CA; 1:200
dilution), and (following a PBS wash) an avidin-horseradish peroxidase
complex (Vectastain ABC, Elite kit; Vector Laboratories, Inc.; 1:500 dilution) was applied for 1 h at
room temperature. Cells were visualized with 0.05%
3,3'-diaminobenzidine tetrahydrochloride (DAB; Polysciences,
Warrington, PA) and 0.001%
H2O2 for 10 min, rinsed
again, mounted, and coverslipped.
Analysis of cellular morphology was performed under a Nikon 100 X oil-immersion objective using the Neurolucida system (MicroBrightField Inc., Colchester, VT). Five to nine cells from two cover slips of each treatment (estradiol, DMSO) were examined. The same criteria used for cell selection for calcium imaging were used to select cells for morphological analysis. The experimenter was blind to the treatment group during the analysis. The following morphological features were measured: 1) somal area (µm2), calculated using the formula for a polygon; 2) somal perimeter (µm); 3) somal roundness (value from 0–1, as defined in Neurolucida software program manual, v.3.22); 4) total dendritic length (µm) (includes all processes of a cell); 5) primary dendritic length (µm) (length of primary dendrites only); and 6) number of primary dendrites per soma.
Statistical analyses
Student’s t test was used to assess differences
between estradiol- and control-treated cells for each dependent
variable. ANOVA was used to assess differences in levels of dependent
variables among estradiol- and control-treated cells imaged on DIV 4
vs. DIV 7. Where appropriate, repeated-measures ANOVA was
used to investigate differences in dependent measurements within the
same cell. Percentage data were analyzed using a 
-square test. All
statistical tests used 
< 0.05 as the criterion for
significance.
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Results |
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Effect of estradiol exposure from DIV 0–DIV 3 on GABA-induced
Ca2+ flux on DIV 4
The percentage of cells responding to muscimol on DIV 4 was 85%
(see Fig. 1) and is similar to
percentages reported previously using embryonic hypothalamic cultures
(6). There was no difference in the number of cells
responding on DIV 4, between control (24/30 neurons) and
estradiol-treated cultures (24/27 neurons). However, estradiol
treatment from DIV 0–DIV 3 altered several parameters of the cellular
response. Representative traces of Ca2+
transients from one individual estradiol-treated and one
control-treated neuron are illustrated in Fig. 2. After 10 µM muscimol
perfusion, Ca2+ transient amplitudes in
estradiol-treated cells averaged 94 ± 9 nM (n =
21), an increase of 68%, relative to control cells (56 ± 6
nM; n = 21) (P < 0.002; Fig. 3A). After muscimol application, the
increase in [Ca2+]i was
detectable within 4.3 ± 0.7 sec, almost 2 sec faster than in
control cells (6.2 ± 0.6 sec; P < 0.05; Fig. 3B). Once [Ca2+]i was
elevated in response to muscimol, there was a tendency for recovery to
basal [Ca2+]i to take
approximately 1.5-fold longer in estradiol-treated neurons
(P = 0.15; data not shown).
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). Furthermore, estradiol-treated cells
displayed significantly larger Ca2+ transients
(54 ± 12 nM; n = 8) after muscimol, relative to
those in control cells, which averaged 21 ± 5 nM
(n = 7; P < 0.05; Fig. 3C). This was an increase
of 61%, which is very similar in magnitude to the
estradiol-enhancement of the muscimol response observed in cells imaged
on DIV 4. Nonetheless, we observed the normal developmental decrease in
the magnitude of the Ca2+ transient amplitudes,
which was about 50% lower than those in cells imaged on DIV 4
(P < 0.01), regardless of treatment. There was no
significant difference in the latency to respond to muscimol in
control- and estradiol-treated neurons (Fig. 3D).
Involvement of L-type Ca2+ channels in the
muscimol response
To determine whether L-type Ca2+
channels were involved in the response to muscimol with or without
estradiol, some cells imaged on DIV 4 were perfused with the
L-type Ca2+ channel blocker,
nimodipine. A mean Ca2+ transient amplitude of
71 ± 12 nM after muscimol in control cells, was
reduced to 13 ± 5 nM in the presence of 1
µM nimodipine, a reduction of 82% (n = 4;
P < 0.001; Fig. 4).
Similarly, a mean Ca2+ transient amplitude of
104 ± 16 nM in estradiol-treated cells was
reduced to13 ± 5 nM in the presence of 1
µM nimodipine, a reduction of 89% (n = 4;
P < 0.001; Fig. 4). The magnitude of the reduction is
consistent with a previous study in which nimodipine greatly attenuated
GABA-induced Ca2+ transients in embryonic
hypothalamic cultures (6). Our present results demonstrate
no difference between estradiol and control cells in the extent to
which nimodipine attenuated GABA-evoked Ca2+
transients, suggesting that estradiol does not exert its effects by
recruiting new routes of Ca2+ entry.
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Discussion |
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We have proposed previously that excitatory GABA is a major mediator of brain sexual differentiation (20), based on our observations of sex differences in both GAD mRNA and GABA levels in the developing hypothalamus (21, 22). We have now established a causal relationship between increased GABA levels and masculinization (23, 24). Sex differences in synaptic patterning, mediated by neonatal estradiol, are found throughout the hypothalamus, including the medial preoptic, ventromedial, and arcuate nuclei (2). Sexual differentiation of the brain is mediated primarily by estrogens aromatized from testicularly-derived androgens, released in two separate surges on ED 18 and day of birth. The observation that cultured hypothalamic neurons exhibit enhanced responses to excitatory GABA after exposure to estradiol implicate this as a potential mechanism in the differentiation process. By increasing cytosolic [Ca2+]i and the length of time that [Ca2+]i is elevated, excitatory GABA could mediate various estradiol-induced actions in the developing brain, including neurite extension, dendritic branching (30), and neuronal migration (31). Our present results also have implications for understanding estrogen’s actions in the adult brain, in which GABA becomes excitatory after injury (12). Whether this phenotypic shift is protective or damaging is unknown. The possibility exists for estradiol enhancing adult excitatory GABA and may be a potential contributor to the well-known neuroprotective effects of estrogens (32, 33, 34).
The maintenance of excitatory GABA in the presence of estradiol, compared with its developmental loss in the absence of steroid, results in two highly dimorphic phenotypes. Excitatory GABA rapidly converts to inhibitory GABA after even slight shifts in the transmembrane chloride gradient caused by shunting inhibition (35). Inhibitory GABA results in low continuous intracellular Ca2+ (36), as opposed to the acute Ca2+ transients induced by excitatory GABA. These opposing calcium profiles can produce unique patterns of phosphorylation and dephosphorylation of proteins within the cell, as observed in response to electrophysiological paradigms producing LTP vs. LTD (37). Thus, excitatory vs. inhibitory GABA may serve as a major divergence point in estradiol-mediated sexual differentiation of the brain.
The mechanism by which estradiol enhances excitatory GABA is unknown but can be inferred from the present data. We have investigated previously the potential for hormonal influence of GABAA receptors in the developing brain, and we found no effect on receptor binding (38) or expression levels of two developmentally regulated receptor subunits (39), making it unlikely that estradiol modulates responses to muscimol in neonatal hypothalamus by altering GABAA receptors. It is equally unlikely that estradiol alters L-type calcium channels directly, for example, by increasing the number of channels. Various signaling molecules alter membrane excitability by activating voltage-gated Ca2+ channels; and thus, this is a promiscuous response that is not selective to GABA action. Opening L-type calcium channels requires sufficient depolarization (40), which, for excitatory GABA, is dependent on the chloride reversal potential. This gradient is the primary determinant of excitatory vs. inhibitory GABA action and is the result of differential expression of at least two chloride cotransporters. In adult brain, where GABA is hyperpolarizing (i.e. inhibitory), an electroneutral K+–Cl- cotransporter, KCC-2, is widely expressed and maintains a mature chloride gradient (13). In contrast, BSC-2 transports Cl- into immature neurons in which GABA is depolarizing (i.e. excitatory) and is heavily expressed in brain during the first postnatal week, declining thereafter (41). Several features of our results are consistent with estradiol modulating expression of these cotransporters to retain an immature chloride gradient profile. In particular, estradiol maintained an excitatory response to muscimol as development proceeded, suggesting that these neurons had a chloride reversal potential characteristic of neonates, allowing a depolarizing response to GABAA receptor activation. Furthermore, we found that the enhancing effect of estradiol on excitatory GABA was mediated by L-type Ca2+ channels and did not involve recruitment of an additional mechanism. This further implicates changes in the transmembrane Cl- gradient as a potential mechanism by which estradiol enhances excitatory GABA.
By increasing intracellular calcium after depolarization (10, 42), GABA exerts well-documented trophic actions in the nervous system (9, 10, 14, 43). Calcium influx is uniquely suited to couple changes in cellular excitability and gene expression because Ca2+ signals generated by electrical activity can activate signal transduction pathways, inducing various transcription factors (44, 45). The immediate early gene, c-fos, is rapidly transcribed in response to increases in intracellular Ca2+ (46, 47), and c-fos may stimulate the synthesis of neurotrophins like nerve growth factor and brain-derived neurotrophic factor and their receptors, which may be involved in sexual differentiation of the brain.
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Footnotes |
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Received November 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4, ß1, 
1
subunits in the proliferative zone of the embryonic rat central nervous
system. J Neurosi 15:2547–2560[Abstract]
1 and 2 in the
hypothalamus and limbic system of the rat. Dev Brain Res 119:127–138[CrossRef][Medline]
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