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Endocrinology Vol. 139, No. 6 2734-2740
Copyright © 1998 by The Endocrine Society

ARTICLES

Luteinizing Hormone Releasing Hormone (LHRH) Neurons Maintained in Nasal Explants Decrease LHRH Messenger Ribonucleic Acid Levels after Activation of GABAA Receptors

S. M. Fueshko, S. Key and S. Wray

Laboratory of Neurochemistry, National Institute of Neurological Disease and Stroke, National Institutes of Health, Bethesda, Maryland 20892-4130

Address all correspondence and requests for reprints to: Dr. S. Wray, Lab of Neurochemistry, National Institutes of Health, Building 36, Rm 4D-12 Bethesda, Maryland 20892. E-mail: swray{at}codon.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibition of the LHRH system appears to play an important role in preventing precocious activation of the hypothalamic-pituitary-gonadal axis. Evidence points to {gamma}-aminobutyric acid (GABA) as the major negative regulator of postnatal LHRH neuronal activity. Changes in LHRH messenger RNA (mRNA) levels after alterations of GABAergic activity have been reported in vivo. However, the extent to which GABA acts directly on LHRH neurons to effect LHRH mRNA levels has been difficult to ascertain. The present work evaluates the effect of GABAergic activity, via GABAA receptors, on LHRH neuropeptide gene expression in LHRH neurons maintained in olfactory explants generated from E11.5 mouse embryos. These explants maintain large numbers of primary LHRH neurons that migrate from bilateral olfactory pits in a directed manner. Using in situ hybridization histochemistry and single cell analysis, we report dramatic alterations in LHRH mRNA levels. Inhibition of spontaneous synaptic activity by GABAA antagonists, bicuculline (10-5 M) or picrotoxin (10-4 M), or of electrical activity by tetrodotoxin (TTX, 10-6 M) significantly increased LHRH mRNA levels. In contrast, LHRH mRNA levels decreased in explants cultured with the GABAA receptor agonist, muscimol (10-4 M), or KCl (50 mM). The observed responses suggest that LHRH neurons possess functional pathways linking GABAA receptors to repression of neuropeptide gene expression and indicate that gene expression in embryonic LHRH neurons, outside the CNS, is highly responsive to alterations in neuronal activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LHRH NEURONS play a pivotal role in reproductive function. Investigations on the trans-synaptic activation of LHRH neurons in vivo are extremely difficult due to the small number of LHRH neurons and dispersed distribution of the population within the forebrain. However, several in vivo studies indicate that the activity of the LHRH neuronal population is regulated, in part, through inhibition by {gamma}-aminobutyric acid (GABA; 1–4). The mechanism underlying the effect(s) of GABA on LHRH neurons is still poorly understood. GABA and/or experimental manipulations of GABAergic receptors can regulate neuropeptide activity by altering the level of neuropeptide transcription (5, 6, 7) and/or neuropeptide biosynthesis (8, 9, 10). This is certainly true in adult LHRH neurons where GABAergic input has been shown to regulate gene expression (11, 12, 13, 14, 15) as well as LHRH release (1, 2, 3, 4, 16, 17). However, both enhanced (3, 11, 18) and suppressed (3, 4, 12, 13, 14, 15, 16, 17, 18) LHRH responses have been documented. Certainly the disparate responses reported could reflect activation of either direct (19, 20) and/or indirect (21) inputs to LHRH neurons, with those activated being directly linked to the experimental paradigm chosen.

To minimize many of the complexities encountered in vivo, our laboratory has developed two in vitro systems in which large numbers of primary LHRH neurons are maintained: one from CNS tissue containing postnatal LHRH neurons (22, 23, 24, 25) and one from olfactory tissue (26, 27) containing prenatal LHRH neurons. Both models maintain approximately 25% of the primary LHRH neuronal population (22, 26). However, in the CNS slice explants, LHRH neurons are maintained in different anatomical regions, within approximately four slice explants (22), whereas in the olfactory explants, the LHRH neuronal population is maintained in one explant. Both models exhibit characteristics that make them powerful alternatives to in vivo studies: in CNS slice explants, the LHRH neurons are exposed to different interneuronal environments (22), whereas LHRH neurons in olfactory explants are devoid of CNS influences (26).

In olfactory explants, LHRH neurons can be identified in situ. This important aspect of olfactory explants led to studies that revealed that prenatally, LHRH cells exhibit spontaneous activity and display a variety of ion channels characteristic of mature neurons, including functional GABAA receptors (27). The presence of large numbers of primary LHRH neurons in a single culture, maintained in an environment outside the CNS, and exhibiting function GABAA receptors, provided an unique opportunity to study the mechanisms underlying GABAergic regulation of this critical neuroendocrine cell population. Thus, using this in vitro system, we examined whether GABAergic signals could directly alter LHRH gene expression. LHRH neurons maintained in cultures treated with GABAA receptor antagonists showed increased LHRH messenger RNA (mRNA) levels, whereas treatment with the GABAA receptor agonist, muscimol, dramatically decreased LHRH mRNA levels. Similar increases in LHRH mRNA levels vs. decreases in LHRH mRNA levels were seen in cultures treated with TTX or KCl, respectively, indicating that LHRH gene expression, outside the CNS, is highly susceptible to the state of LHRH neuronal activity. The observed neurotransmitter-specific responses suggest that LHRH neurons possess functional pathways linking GABAA receptors to down-regulation of neuropeptide gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Olfactory explants
Olfactory explants were prepared as previously described (26). Briefly, E11.5 mouse embryos were obtained from timed pregnant NIH Swiss females according to NIH guidelines. The olfactory epithelium was removed and plated on glass coverslips (12 mm x 24 mm; Gold Seal) coated with 10 µl chicken plasma (Cocalico, Philadelphia, PA). Thrombin (10 µl, Sigma, St. Louis, MO) was then added to adhere (thombin/plasma clot) the explant to the coverslip. To eliminate unknown serum constituents, as well as the possibility that effects of pharmacological agents on LHRH cell movement would be masked under serum-containing conditions, olfactory explants were maintained in a defined, serum-free media (SFM, 24) for 7 days at 37 C in a humidified atmosphere with 5% CO2. The media was changed twice a week, and one dose of fluorodeoxyuridine (FdUR; 8 x 10-5 M, Sigma) was given at day 3 for 3 days. This treatment was found to be effective in decreasing the number of nonneuronal cells, without affecting either the general health of the explant or LHRH cell number (26).

Explants in experimental groups were maintained in SFM containing agents that 1) block spontaneous synaptic GABAergic activity [bicuculline (bicuculline methochloride, 10-5 M) and picrotoxin (10-4 M), both obtained from Sigma)], or 2) activate GABAA receptors [muscimol (10-4 M, Sigma). Potassium (KCl, 50 mM) and KCl + tetrodotoxin (TTX; 50 mM, 10-6 M, respectively), was used as a general depolarizing agent, while TTX (10-6 M), either alone or with muscimol (muscimol + TTX; 10-4 M, 10-6 M, respectively) was used as a general inhibitor of electrical activity (28, 29), including that generated via GABAergic input. Drug concentrations were based on effectiveness in prior electrophysiological studies (27). Chronic treatments were initiated at 1 day in vitro (div), before the emergence of LHRH cells from the olfactory pits (26) and replenished at 3 and 6 div. Control cultures were maintained in SFM that was changed, as in the treatment groups, at 1, 3 and 6 div. After 7 div, cultures were processed for in situ hybridization histochemistry (ISHH).

Probes and ISHH
ISHH was performed using a synthetic antisense oligonucleotide probe, 48 nucleotides in length, complementary to the coding region of the mouse LHRH complementary DNA (cDNA), covering bases 1653–1700 (30). Explant cultures were processed for ISHH by modifying a procedure previously described (30). Cultures were fixed (30 min, 4% formaldehyde), rinsed in PBS (2 x 5 min), acetylated (10 min, 0.1 M triethanolamine hydrochloride-0.9% NaCl, pH 8.0, containing 0.25% acetic anhydride), rinsed in 2 x SSC (sodium chloride, sodium citrate, 2 x 1.5 min), sequentially dehydrated in ethanol, and delipidated in chloroform (7 min). The cultures were then rehydrated to 95% ethanol, air dried, and hybridized with a [35S]dATP-labeled probe.

The radiolabeled probe (5 pmol) was 3'-end labeled in a final volume of 10–15 µl by incubation (37 C, 10 min) with [35S]dATP (1 mM; 1, 300 Ci/mmol; New England Nuclear, Boston, MA) and terminal deoxyribonucleotidyl transferase (TdT, 100U; BRL, Gaithersburg, MD) to a specific activity of 7,000–15,000 Ci/mmol (30). Isotopic probe (5 x 105 cpm) was applied to individual cultures in 10 µl of hybridization buffer (30). Cultures were coverslipped and hybridized for 16 h at 37 C.

The following day, parafilm coverslips were removed in 1 x SSC containing 75 mM dithiothreitol. Cultures were washed (4 x 15 min) in 2 x SSC/50% formamide containing 75 mM DTT at 40 C, and rinsed at room temp in 1 x SSC (3 x 10 min), H2O (2 x 1 min), 70% EtOH (2 min), 95% EtOH (5 min) and then allowed to air dry. After drying, the coverslips were dipped in NTB3 emulsion (Eastman Kodak, Rochester, NY) and exposed for appropriate times. Frozen adult mouse brain sections were used as positive controls, and these were treated by identical ISHH procedures on the same day.

Immunocytochemistry
A polyclonal antibody against pro-LHRH [SW-1 (1:2500), 22] was used to detect LHRH. Single label immunocytochemistry was performed as previously described (30). Briefly, cultures were fixed (4% formaldehyde), rinsed in PBS and blocked by incubating in 10% normal goat serum (NGS)/0.3% Triton X-100. Following two PBS washes, cultures were incubated in primary antiserum overnight at 4 C; negative controls were incubated in 10% NGS without primary antibody. On day 2, cultures were rinsed, incubated in biotinylated secondary antibody (1:500, Vector, Burlingame, CA) followed by avidin-biotin-conjugated horseradish peroxidase complex (1:600, Vectastain Elite ABC-peroxidase; Vector). Staining was visualized using 3'3-diaminobenzidine (DAB, Sigma) and glucose oxidase. Cultures were counterstained with 0.05% methyl green, dehydrated, cleared with xylene, and mounted in Permount (Fisher Scientific, Pittsburgh, PA).

Statistical analysis
Quantitation of mRNA was performed as previously described (24). Briefly, LHRH mRNA levels within single cells were examined by obtaining integrated densities of areas of silver grains over individual labeled cells, and the cell areas enclosing these grains, using an image analysis system (NIH Image). Silver grains were digitized under brightfield microscopy and mean optical density (OD) measurements (15% above background) per cell area, expressed as OD/µm2, were calculated for single cells and local background. The value was then multiplied by the highlighted cell area to obtain a total LHRH mRNA level per cell (OD/cell). Local background, multiplied by the measured background area, was subtracted from each cell measurement to obtain a corrected LHRH mRNA level per single cell:

This value was taken as a reflection of the relative level of mRNA/cell. The thickness of the tissue and the close association of cells located on the main tissue mass made it extremely difficult to discern the boundaries of single cells. Thus, relative cellular levels of LHRH mRNA were obtained only for labeled cells that were located off of the main tissue mass of each culture. Values were then compared between treatment groups.

Statistical analysis of the OD values from the seven treatment groups was made using two approaches. In the first approach, a single mean OD/cell value was obtained for each culture by averaging the OD/cell of all the cells in that culture. The mean OD/cell values of an experimental group were then averaged to give a mean OD/cell value per treatment. A one-way ANOVA was applied to the data and N equals the number of cultures in each treatment group (i.e. control, bicuculline, picrotoxin, etc.) and not the number of cells analyzed. This approach allowed analysis of variation among cultures (both within and between treatment groups). However, in this approach data are lost because individual values (up to 200 cells/culture) are pooled. Thus, the second approach used all of the OD/cell values, and N equals the total number of cells in an experimental group, irrespective of the number of explant cultures in which these cells were located. In this analysis, all values were treated independently, grouped according to treatment, and analyzed using the nonparametric Kolmogorov-Smirnov (KS) test (a significance level of P < 0.001 was chosen). For diagramatic purposes, the OD/cell data were divided into 10 equal bins covering the entire range of measured OD/cell values (mRNA levels) and frequency histograms were generated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Large numbers of LHRH cells, labeled by ICC or by ISHH using an LHRH mRNA specific probe, were detectable under all experimental conditions (Fig. 1Go). However, quantitation of relative LHRH mRNA levels within treatment groups revealed pronounced differences from controls.



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  		Figure 1. Cells expressing LHRH mRNA, and LHRH protein product, were observed under all experimental conditions. A, Low magnification, darkfield photomicrograph of a control olfactory explant maintained for 7 days in vitro (div), then processed for ISHH. B, Low magnification, brightfield photomicrograph of an olfactory explant maintained in SFM in the presence of muscimol for 7 div then processed for ICC. Note that the number of neurons containing LHRH mRNA (arrows in A) is similar to that obtained by ICC (arrowheads in B). *, Olfactory pit region; bar, 100 µm in A and B.

 
LHRH mRNA levels are affected by spontaneous electrical activity
To evaluate levels of gene expression in the absence of spontaneous activity in LHRH neurons, LHRH mRNA levels were determined in TTX-treated cultures. The results of a one-way ANOVA on the effect of TTX on LHRH gene expression is shown in Table 1Go, with representative darkfield micrographs shown in Fig. 2Go, a–b. Blocking spontaneous electrical activity significantly increased relative LHRH mRNA levels above those observed for control cultures (15133 ± 710) to 17308 ± 810 for TTX treated cultures (F = 24.363; P < 0.001). As indicated by the frequency distributions (Fig. 2cGo), TTX increased the proportion of neurons exhibiting intense labeling as compared with controls [TTX: Max. Diff. (MD) = 0.2 for n1 = 1550 and n2 = 1069; P < 0.0001]. These findings reveal that, in vitro, in defined media, LHRH gene expression is down-regulated.


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  		Table 1. Mean LHRH mRNA levels in olfactory explants

 


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  		Figure 2. Inhibition of electrical activity by TTX significantly increased LHRH gene expression. In the absence of spontaneous activity, or synaptic activity induced by GABAergic input, in LHRH neurons, LHRH mRNA levels were up-regulated. Darkfield photomicrographs show representative LHRH neurons from control (A) and TTX-treated (B) cultures. Cells were treated as independent values and analyzed by the KS test (analysis II in Materials and Methods). C, Frequency distributions of density/cell of individual labeled LHRH cells maintained in TTX revealed a rightward shift in the LHRH population, indicating an up-regulation of LHRH mRNA levels with respect to controls. Bin 1 contains LHRH neurons with the lowest LHRH mRNA levels, whereas bin 10 contains LHRH neurons with the highest LHRH mRNA levels. Note: cells circled in (A) had mRNA levels within bins 1–4, whereas cells circled in (B) had mRNA levels in bins 6–8. Solid bars, control; hatched bars, TTX treatment. Bar, 20 µm in A and B.

 
LHRH mRNA levels are regulated by GABAergic input
To determine whether GABAergic systems, present in the explant (31), were responsible for the inhibition of LHRH neuronal gene expression, olfactory explants were treated with the GABAA receptor antagonists, picrotoxin and bicuculline, or with the receptor agonist, muscimol (Fig. 3Go). Both antagonists significantly increased LHRH mRNA levels above control values (F = 24.363; P = 0.0001). Analysis indicated that there was no significant difference between cultures treated with either GABAA antagonist; thus, these two groups were combined for subsequent analysis. As shown in Table 1Go, addition of GABAA antagonists raised the mean mRNA level/cell to 18318 ± 620: a level significantly different from the control group but comparable to that observed for TTX-treated cultures. The corresponding darkfield micrographs are shown in Fig. 3Go, a–b. As indicated by the frequency distribution in Fig. 3dGo, GABAA antagonists significantly increased the proportion of neurons exhibiting intense labeling as compared with controls (GABAA antagonists: MD = 0.18 for n1 = 1550 and n2 = 1556; P < 0.0001). Interestingly, as indicated above, the GABAA antagonist group was not significantly different from TTX-treated cultures by the ANOVA. However, a significant difference between these experimental conditions was observed with the KS analysis. This indicates that, although mean OD values/cell were statistically identical for GABAA antagonists and TTX-treated groups, the distribution of the population around those means differed significantly (MD = 0.12 for n1 = 1556 and n2 = 1069; P < 0.0001).



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  		Figure 3. Perturbation of GABAA receptors significantly altered LHRH mRNA levels. A–C, Representative, high-magnification, darkfield photomicrographs of ISHH-labeled cells from (A) control, (B) GABAA antagonist-treated, and (C) muscimol-treated cultures. D, Frequency distributions of density/cell of individual labeled LHRH cells indicate that GABAA receptor antagonists dramatically increased relative LHRH mRNA levels, whereas the GABAA receptor agonist, muscimol, significantly decreased relative LHRH mRNA levels with respect to controls. As in Fig. 2Go, the total range of density/cell values were divided into 10 bins, where bin 1 contains cells with the lowest LHRH mRNA levels and bin 10 contains cells with the highest LHRH mRNA levels. Cells depicted in (A) had mRNA levels within bins 4–6, in (B), mRNA levels in bins 7–10, and in (C), mRNA levels in bins 1–4. Solid bars, control; stippled bars, GABAA antagonist treatment; hatched bars, muscimol treatment. Bar, 20 µm in A–C.

 
The GABAA receptor agonist, musicmol, produced an inverse effect on LHRH gene expression as compared with the antagonist group; a decrease in LHRH mRNA levels was observed in cultures treated with muscimol. The results of a one-way ANOVA on the observed effect of muscimol is shown in Table 1Go (F = 24.363; P < 0.0001), with a representative darkfield micrograph shown in Fig. 3cGo. Receptor activation by muscimol produced a significant decrease in LHRH mRNA levels as compared with control cultures, with a mean OD/cell for the muscimol-treated cultures of 11961 ± 1100. In contrast to the observed effect of the GABAA antagonists, activation of GABAA receptors by muscimol decreased the proportion of labeled neurons exhibiting intense labeling (Fig. 3dGo; muscimol: MD = 0.18 for n1 = 1550 and n2 = 367; P < 0.0001). The results of these experiments clearly demonstrate that release of GABA in olfactory explants negatively regulates LHRH gene expression.

Down-regulation of LHRH gene expression is augmented by KCl
GABAergic input was shown to cause depolarization of prenatal LHRH neurons, in vitro, via functional GABAA receptors (27). To determine whether the repression of LHRH mRNA levels by GABA was a direct result of depolarization, per se, explant cultures were treated with the general depolarizing agent, KCl. The results of a one-way ANOVA are shown in Table 1Go (F = 24.363; P < 0.0001). Depolarization with KCl ± TTX (analysis indicated that no significant difference existed between cultures treated with KCl ± TTX; thus, these two groups were combined for subsequent analysis) produced a dramatic decrease in LHRH mRNA levels, depressing the mean OD/cell to 9507 ± 360. Consistent with the ANOVA, the KS test indicated that KCl-induced depolarization significantly decreased the proportion of neurons exhibiting intense labeling with respect to controls (MD = 0.46 for n1 = 1550 and n2 = 1287 P < 0.0001). Although the effect of the two depolarizing stimuli was similar, LHRH mRNA levels in the KCl group were significantly lower than those of the muscimol group (MD = 0.20 for n1 = 1287 and n2 = 367; P < 0.0001).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present work evaluates the effect of GABAergic activity, via GABAA receptors, on LHRH neuropeptide gene expression in primary LHRH neurons maintained in olfactory explants. Using in situ hybridization histochemistry and single cell analysis, we report alterations in LHRH mRNA levels; treatment with the GABAA agonist, muscimol, significantly reduced LHRH mRNA levels, whereas GABAA antagonists (picrotoxin and bicuculline) significantly increased LHRH mRNA levels. The observed neurotransmitter-specific responses indicate that, prenatally, LHRH neurons possess functional pathways linking GABAA receptors to a repression of neuropeptide gene expression.

Inhibition of LHRH mRNA levels in olfactory explants
Inhibition of electrical activity by TTX increased LHRH mRNA levels (14.4% above control mean value), indicating that LHRH gene expression, in vitro, is repressed. GABAergic neurons, hypothesized to supply the major inhibitory input to mature LHRH neurons (2), are also present during prenatal development in nasal regions in vivo (27, 32) and in nasal explants in vitro (27). However, whether GABAergic activity is coupled to down-regulation of LHRH gene expression in embryonic LHRH neurons, and thus could account for the change in LHRH mRNA levels recorded after TTX treatment, was unknown. In this report, increases in LHRH mRNA levels (21% above control mean value) were found after treatment of embryonic olfactory explants with the GABAA receptor antagonists, bicuculline or picrotoxin. Statistically, although the overall frequency distributions of the TTX-treated group and antagonist-treated groups were different, the mean mRNA OD levels were not. These results indicate that down-regulation of LHRH mRNA levels in LHRH neurons maintained in control is likely to be due to GABAergic signals. Consistent with this premise, muscimol treatment reduced LHRH mRNA levels; exogenous application of muscimol significantly reduced LHRH mRNA levels from those measured in LHRH neurons in cultures grown in SFM alone (21% below control mean value).

GABAergic modulation of LHRH mRNA levels
In the current study, muscimol treatment resulted in a decrease in LHRH mRNA levels, a response that might have been predicted for postnatal LHRH neurons, where GABA appears inhibitory. On mature LHRH neurons, GABAergic synapses are present (19, 20) and inhibitory responses to GABA have been observed at both the level of LHRH mRNA (12, 13, 14, 15) and LHRH secretion (for review, see Ref. 4; 2, 16, 17). However, prenatally GABA can cause excitation of a postsynaptic cell due to an immature chloride equilibrium (27, 33, 34, 35). Indeed, in nasal explants, GABA is known to depolarize LHRH neurons (27). Terasawa et al. (36) have shown that depolarization triggers LHRH secretion from LHRH neurons in olfactory explants. Certainly, the secretory rate of a neuroendocrine peptide is generally coordinated with its biosynthetic rate in a positive manner (5, 6, 7, 8, 9, 37). Thus, an agent responsible for depolarization and, presumably, neuropeptide release may initiate a compensatory mechanism to replenish depleted supplies of peptide, e.g. increased transcription and/or translation. This was not observed in olfactory explants following muscimol treatment. Thus, the alterations in LHRH mRNA levels observed after perturbation of GABAergic signals may have resulted from 1) an autocrine mechanism, whereby secretion from LHRH neurons down-regulates LHRH mRNA and/or 2) a mature GABA receptor intracellular signaling pathway(s) that is linked to LHRH gene expression/mRNA turnover.

Secretion-coupled mechanisms
To examine the interplay between depolarization/secretion and mRNA levels in LHRH neurons, cultures were treated with the general depolarizing agent, KCl. KCl treatment, like muscimol treatment, depressed LHRH mRNA levels. Thus, a second depolarizing agent produced a down-regulation of LHRH mRNA levels, though the decrease observed after KCl treatment was greater than that measured after muscimol treatment (26% below muscimol mean value; 38% lower than control values). Although a strong stimulus, it is unlikely that KCl depolarization augmented GABA release and increased the endogenous GABA concentration beyond that which was obtained by exogenous application of muscimol. Several other possibilities exist for the observed augmented decrease in LHRH mRNA levels after KCl treatment.

KCl treatment may have caused an autocrine response via release of: 1) LHRH, which negatively regulates its own gene expression, and/or 2) another modulatory neuropeptide coexpressed in LHRH neurons, which through feedback mechanisms results in down-regulation of LHRH gene expression. In support of the possibility that LHRH transcription is down-regulated (or degradation of LHRH mRNA altered) as a consequence of LHRH secretion, it has been shown that GT1 cells express autoreceptors (38). Upon exposure to LHRH agonist, these cells exhibit a transient elevation of LHRH release, followed by suppressed basal secretion (38). In addition, in immortalized LHRH cells, rapid stimulation of LHRH release by secretagogues (forskolin, ionomycin and PMA) depresses LHRH mRNA (39), suggesting that negative feedback in these cells occurs, at least in part, at the level of transcription and/or posttranscriptional modification. During development, primary LHRH neurons may regulate a secretory response using a similar mechanism; autoregulation would result in repression/down-regulation of LHRH gene expression and possibly, an inhibition of additional secretion. However, an obstacle to the acceptance of negative autoregulation as an explanation for mRNA repression is that the presence of LHRH receptor mRNA in primary LHRH neurons has yet to be documented (personal observation, S. Wray).

Certainly, release of other modulatory neuropeptide(s) from LHRH neurons could feedback to inhibit LHRH transcription. To date, only a few substances have been colocalized in LHRH neurons; including delta sleep-inducing peptide (40, 41), GABA (32), NPY (42), and galanin (43, 44). Of these three substances, only galanin is known to be colocalized in LHRH neurons in nasal regions (45) and in CNS (43, 44). Whether galanin (or another colocalized substance) participates in the regulation of LHRH gene expression in prenatal LHRH neurons is currently unknown.

In addition to secretion from LHRH neurons, KCl treatment may have caused release of an unidentified molecule from non-LHRH neurons, or may have resulted in desensitization of LHRH neurons, either of which could have down-regulated LHRH gene expression. Alternatively, KCl depolarization may have induced changes in GABAA receptor subunit composition or expression level in LHRH neurons. KCl depolarization up-regulates acetylcholine receptor subunits in sympathetic neurons, resulting in more ligand binding sites (46), and increases transcription of genes encoding NMDAR2A subunit mRNA (47), as well as {alpha}1 and {alpha}5 GABAA receptor subunits (48) in cerebellar granular cells. Further studies are needed to determine whether a similar phenomenon occurs with respect to a GABAA receptor subunit(s) in LHRH neurons. However, changes in the GABAA receptor subunit composition or expression level, together with KCl-induced release of GABA, could appear as an increased activation of GABAA receptors/GABA concentration and thus an enhanced response (lower LHRH mRNA levels) as compared with muscimol.

Mature GABAA receptor signaling-coupled mechanisms
Although excitatory (causing depolarization), the action of GABA on embryonic LHRH mRNA levels is negative, similar to the response predicted for mature LHRH neurons. This suggests that the signal transduction mechanism for the GABAA receptor may be appropriately coupled to transcriptional/RNA stability regulation shortly after differentiation of the LHRH neuronal phenotype. It is clear that the molecular and pharmacological properties of GABAA receptors vary with receptor subunit composition. Relatively few subunit combinations make up a large fraction of the GABAA receptors found in the CNS; one of the prevalent combinations appears to be composed of {alpha}1, ß2/3 and {gamma}2 (49). While studies by Petersen et al. (50) demonstrated that postnatal LHRH neurons have the capacity to synthesize ß3 GABAA receptor subunit, they were unable to demonstrate the {alpha}1 or ß2 receptor subunit in postnatal LHRH cells, suggesting a rather unique subunit combination. To date, the GABAA receptor subunit composition in prenatal LHRH cells is unknown. However, our laboratory has not detected the presence of the {alpha}1 subunit in LHRH neurons maintained in nasal explants (personal observation, S. Wray), a result that is in agreement with that reported for postnatal LHRH neurons. Thus, although far from complete, the composition the GABAA receptor in prenatal LHRH neurons is consistent with the premise that this receptor, its signal transduction machinery and transcriptional/RNA stability regulation are appropriately coupled early in LHRH neuronal development.

The importance of altering LHRH neuropeptide levels in neurons outside the CNS remains to be determined. Recently, it was shown that modifying GABAergic activity alters the distance LHRH neurons migrate in mouse olfactory explants, with the GABAA receptor agonist, muscimol, inhibiting LHRH cell movement (51). No differences were observed in LHRH cell survival or morphology. It was proposed (51) that GABAergic signals provided a migratory stop signal for LHRH neurons, thereby regulating their entrance into the CNS. The results of the present study provide evidence that GABA also represses LHRH mRNA levels. Thus, it appears that developmentally, LHRH transcription/RNA stability may be linked to migratory behavior via functional GABAA receptors.

In summary, this study demonstrates that embryonic LHRH neurons outside of the CNS are competent to regulate gene expression in response to neuronal activity, with GABAergic signaling, via GABAA receptors, inducing down-regulation of LHRH gene expression. In addition, the studies described in this investigation indicate that nasal explants provide an alternative model in which, isolated from many of the complexities encountered in the postnatal in vivo milieu, gene expression in primary LHRH neurons can be examined.

Received December 3, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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