QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (55) Citing Articles via Google Scholar Google Scholar Articles by Liu, J. Articles by Lauder, J. M. Search for Related Content PubMed PubMed Citation Articles by Liu, J. Articles by Lauder, J. M.

 Previous Article  |  Next Article 

Volume 17, Number 7, Issue of April 1, 1997 pp. 2420-2428
Copyright ©1997 Society for Neuroscience

GABA_A Receptors Mediate Trophic Effects of GABA on Embryonic Brainstem Monoamine Neurons In Vitro

Jiangping Liu¹, A. Leslie Morrow², Leslie Devaud², Dennis R. Grayson³, and Jean M. Lauder¹

Departments of ¹ Cell Biology and Anatomy and ² Psychiatry and Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, and ³ Department of Psychiatry, Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 15212

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

REFERENCES

---

ABSTRACT

The inhibitory neurotransmitter GABA may act as a trophic signal for developing monoamine neurons in embryonic rat brain, because GABA neurons and their receptors appear in brainstem during generation of monoamine neurons. To test this hypothesis, we used dissociated cell cultures from embryonic day 14 rat brainstem, which contains developing serotonin (5-HT), noradrenaline (tyrosine hydroxylase; TH), and GABA neurons. Immunocytochemistry and reverse transcription-PCR (RT-PCR) revealed the presence of multiple , , , and  subunits in these cultures. Competitive RT-PCR demonstrated high levels of 3 subunit transcripts. Expression of functional GABA_A receptors was demonstrated using ³⁶Cl flux assays. To investigate GABAergic regulation of neuronal survival and growth, cultures were treated for 1-3 d in vitro with 10 µM GABA and/or GABA_A antagonist (bicuculline or the pesticide dieldrin). The effects of treatments were quantified by analysis of immunoreactive 5-HT, TH, and GABA neurons. GABA_A receptor ligands differentially regulated neuronal survival and growth depending on neurotransmitter phenotype. GABA exerted positive effects on monoamine neurons, which were countered by bicuculline (and dieldrin, 5-HT neurons only). By itself, bicuculline produced inhibitory effects on both 5-HT and TH neurons, whereas dieldrin potently inhibited 5-HT neurons only. GABA neurons responded positively to both antagonists, but more strongly to bicuculline. Taken together, these results demonstrate that the activation/inhibition of GABA_A receptors produces opposite effects on the development of embryonic monoamine and GABA neurons. This suggests that these neurotransmitter phenotypes may express GABA_A receptors that differ in fundamental ways, and these differences determine the developmental responses of these cells to GABAergic stimuli.

Key words: GABA_A receptor; 5-HT; tyrosine hydroxylase; survival; neurite outgrowth; rat; bicuculline; organochlorine pesticides; dieldrin; RT-PCR; chloride influx; embryonic; brainstem

---

INTRODUCTION

GABA is present in the mammalian brain during early stages of development, where it may act as a trophic signal for developing neurons. In the embryonic rat, GABA axons project through the brainstem when serotonergic (5-HT) and noradrenergic neurons are being generated (Lauder et al., 1986). This raises the possibility that GABA could exert trophic influences on developing monoamine neurons, if they express appropriate receptors. In adult rat brain, these neurons do express functional GABA_A receptors (Smith and Gallager, 1987; Fritschy et al., 1992; Nicholson et al., 1992), but embryonic receptor expression has yet to be investigated. Trophic actions of GABA on other types of neurons are well documented (Spoerri and Wolff, 1981; Eins et al., 1983; Meier et al., 1984, 1991; Spoerri, 1988; Prasad and Barker, 1990; Hansen et al., 1991; Wolff et al., 1993; Abraham et al., 1994; Behar et al., 1994; Belhage et al., 1997) and seem to involve GABA_A receptors coupled to Cl (Meier et al., 1985, 1991; Mehta and Ticku, 1988) and Ca²⁺ channels (Connor et al., 1987; Reichling et al., 1994; Obrietan and van den Pol, 1995; Xian et al., 1995) .

GABA_A receptors are pentomeric complexes consisting of several subunits (1-6, 1-4, 1-3, , 1-2). Molecular cloning has identified a family of GABA_A isoreceptors (subtypes) formed by several subunits from these classes (for review, see MacDonald and Olsen, 1994). These receptors are targeted by benzodiazepines, barbiturates, neurosteroids (Schofield et al., 1987; Barnard, 1988; Olsen and Tobin, 1990; for review, see Morrow, 1995), and organochlorine pesticides (Abalis et al., 1986; Gant et al., 1987; Costa, 1988; Bloomquist, 1992).

[View Larger Version of this Image (65K GIF file)]

GABA_A receptors develop in approximate spatiotemporal coincidence with GABAergic neurons and axons (Cobas et al., 1991; Schlumpf et al., 1989), suggesting that GABA may modulate expression of these receptors. In situ hybridization has revealed transient expression patterns of GABA_A subunit transcripts in developing rat brain (Olsen and Tobin, 1990; Gambarana et al., 1991; Bovolin et al., 1992; Laurie et al., 1992; Poulter et al., 1992; Zheng et al., 1993; Ma and Barker, 1995). These subunits form functional GABA_A receptors that can be activated by specific agonists (Hebebrand et al., 1988; Kellogg and Pleger, 1989; Fiszman et al., 1990; Schlumpf et al., 1992; Ma et al., 1993).

---

Fig. 1. Representative 5-HT (A), TH (B), and GABA (C) immunoreactive neurons from E14 brainstem cultures. Cells were cultured for 1 d in DMEM + 10% FCS and then switched to serum-free medium (DMEM + ITS + 0.1% BSA) for 48 hr. Scale bar, 50 µM.
[View Larger Version of this Image (62K GIF file)]

---

---

Fig. 2. Expression of GABA_A receptor subunit proteins in E14 brainstem cultures. Immunocytochemistry with anti-GABA_A 1 rabbit polyclonal antibody (A) and anti-GABA_A 2/3 monoclonal antibody (B). C, Background control with 1 primary antibody omitted. D, Background control with 2/3 primary antibody omitted. Cells were cultured for 1 d in DMEM + 10% FCS and then switched to serum-free medium (DMEM + ITS + 0.1% BSA) for 48 hr. Scale bar, 50 µM.
[View Larger Version of this Image (132K GIF file)]

---

---

Fig. 3. RT-PCR analysis of GABA_A receptor subunit mRNAs in E14 rat brainstem cultures. Cells were cultured for 1 d in DMEM + 10% FCS and then for 2 d in serum-free medium (DMEM + ITS + 0.1% BSA). RT-PCR revealed expression of mRNAs encoding most of the known GABA_A receptors, except 6.
[View Larger Version of this Image (20K GIF file)]

---

---

Fig. 4. A-C, Representative gels for GABA_A receptor subunit mRNAs from cultured E14 rat brainstem analyzed by competitive RT-PCR using internal standards. A series of concentrations of internal standard cRNAs were added to each tube containing 1 µg of total RNA. The PCR products from each tube are shown in triplicate for each subunit. Top bands, PCR products of target mRNA. Bottom bands, BglII-digested internal standard PCR products. Note that increasing concentrations of internal standards compete with target mRNA for amplification. The point of equivalence was determined by linear regression analysis of the ratio of counts incorporated into the target PCR product across the series of concentrations of internal standards. The point of equivalence (when the ratio is 1) is the absolute concentration of GABA_A receptor subunit mRNA/microgram of total RNA. D, Quantification of GABA_A receptor subunit mRNA levels assayed in this study. Note that 3 is the most abundant subunit compared with 1 and 1. Cells were cultured for 1 d in DMEM + 10% fetal calf serum and then for 2 d in serum-free medium (DMEM + ITS + 0.1% BSA).
[View Larger Version of this Image (33K GIF file)]

---

---

Fig. 5. GABA_A receptor-mediated Cl uptake into cultured E14 brainstem cells. Addition of exogenous GABA enhanced Cl uptake from basal levels of 3.3 nmol/mg protein in a dose-dependent manner to 13.6 nmol/mg protein (at 10 µM GABA). Addition of 10 µM bicuculline lowered Cl uptake stimulated by 10 µM GABA to 4.3 nmol/mg protein.
[View Larger Version of this Image (13K GIF file)]

---

---

Fig. 6. Effects of GABA receptor ligands on survival of 5-HT, TH, and GABA neurons in E14 rat brainstem cultures (number of immunoreactive neurons/mm², expressed as percentage control). Cells were cultured for 1 d in DMEM + 10% FCS and then switched to serum-free medium (DMEM + ITS + 0.1% BSA) plus ligand for 48 hr. Cultures were then fixed, stained with antibodies to 5-HT, TH, or GABA, and immunoreactive neurons were counted. Individual data from three separate experiments (n = 60 cells/treatment group) were converted to percentage control by dividing individual data points by the overall mean control value. Statistical analysis was performed by ANOVA followed by Dunnet's multiple comparison test when ANOVA was significant (p < 0.05).
[View Larger Version of this Image (29K GIF file)]

---

GABA_A receptors have been suggested to mediate trophic effects of GABA during neuroembryogenesis (Ma and Barker, 1995). In the present study, we have used dissociated cell cultures from embryonic rat brainstem to study the effects of GABA on the growth and survival of developing monoamine and GABA neurons and have used GABA_A antagonists to assess the involvement of GABA_A receptors.

---

MATERIALS AND METHODS

Cell cultures Primary dissociated cell cultures were prepared from embryonic day 14 (E14) rat brainstem as described previously (Liu and Lauder, 1991). Cells were plated in 12-well plates in complete medium [DMEM + 10% fetal calf serum (FCS) + penicillin/streptomycin/dextrose] at a density of 10⁶ cells/milliliter on polylysine-coated coverslips. At 1 d in vitro (1 DIV), cultures were switched to serum-free medium [DMEM + insulin, transferrin, selenium (ITS) + 0.1% bovine serum albumin (BSA)]. To determine the effects on neuronal survival and growth, cultures were treated with 10 µM GABA and/or GABA_A antagonist (bicuculline or dieldrin) in serum-free medium (to avoid GABA from serum-containing medium; Loscher, 1979) daily for 48 hr beginning at 1 DIV.

Immunocytochemistry and cell counts At 3 DIV, cultures were rinsed with HBSS and fixed with 4% paraformaldehyde in 70 mM phosphate buffer. Cultures then were rinsed in PBS, permeabilized with 0.2% Triton X-100, and immunostained using the avidin-biotin peroxidase method (Vector Labs, Burlingame, CA) with specific rabbit polyclonal antisera against 5-HT-hemocyanin conjugates (Wallace et al., 1982), tyrosine hydroxylase (TH) (Boehringer Mannheim, Indianapolis, IN), GABA-hemocyanin conjugates (Lauder et al., 1986), GABA_A receptor 1 subunits (Sato and Neale, 1989; gift of Dr. Joseph Neale), or a monoclonal antibody to GABA_A receptor 2-3 subunits (bd-17, Boehringer Mannheim). To determine the effects of treatments on neuronal survival, the number of 5-HT, TH, and GABA immunoreactive neurons was counted in three cultures/treatment group using an ocular grid. Twenty grid areas (4.2 mm²) were counted per culture. Data were expressed as the mean number of cells/mm²/culture, and then converted to percentage control by dividing individual data points by the overall control mean. Data were statistically analyzed using one-way ANOVA followed by the post hoc Dunnet's multiple comparison test (p < 0.05).

Morphometry Cell size, shape, and complexity of neurite outgrowth were measured for 5-HT, TH, and GABA neurons as indices of morphological changes in these neurons in response to treatments. Measured neurites consisted mainly of dendrites, because long, thin processes (presumably axons) were eliminated from the analysis. Morphometry was performed using a custom-designed computer imaging system, which automatically measured soma area (SA), field area (FA; area covered by a neurite arbor), number of neurites (NN), and number of terminal neurite segments (NTS), and then stored the images for further analysis. Details of this system and the morphometric parameters measured have been described previously (Lieth et al., 1990; Liu and Lauder, 1991; Lieth, 1992). In this experiment, 20 randomly selected neurons were analyzed from each of three cultures per treatment group. Data from these three experiments (total of 60 cells per group) were averaged to obtain means for each parameter ± SEM. Data (n = 60) were analyzed using one-way ANOVA followed by post hoc Bonferroni multiple comparisons.

Reverse transcription-polymerase chain reaction (RT-PCR) RNA purification. Total RNA was extracted as described previously (Morrow et al., 1992). Culture media was removed from each well, and the cells were washed with ice-cold, sterile PBS. Prechilled 4 M guanidine thiocyanate was added to each of the wells, and then the solution from the combined six wells was transferred to centrifuge tubes, followed by homogenization on ice using a Polytron. RNA was purified by ultracentrifugation over a 5.7 M CsCl₂ cushion and resolubilized in 0.2 M sodium acetate (with 0.1% SDS). Extraneous protein was removed by consecutive extraction with equal volumes of Tris-buffered phenol and chloroform/isoamyl alcohol (49:1), followed by precipitation with 100% ethanol overnight. The yield of total RNA was determined by measuring the absorbance of an aliquot of the resuspended stock at 260/280 nm.

General RT-PCR. Aliquots of total RNA (1.0 µg) were reverse-transcribed in First Strand buffer (50 mM Tris, pH 8.3, 3 mM MgCl₂, 75 mM KCl) with 10 mM dithiothreitol, 1 mM deoxynucleotide triphosphates (dNTPs), 25 µM random hexamers, and 200 U Moloney Murine Leukemia Virus reverse transcriptase (BRL, Bethesda, MD) at 37°C for 60 min. The resulting cDNA was heat-denatured at 95°C for 10 min, and then tubes were put on ice until they were ready for PCR. The PCR reaction was conducted in PCR buffer (50 mM Tris, pH 9.0, 20 mM ammonium sulfate, 1.5 mM MgCl₂) with 50 µM each of 5 (sense) and 3 (antisense) primers, 200 µM dNTP, and 1 U Hot Tub Polymerase (Amersham, Arlington Heights, IL). The PCR reaction was performed with 26 cycles using a DNA Thermal Cycler, each cycle consisting of 94°C for 45 sec, 60°C for 45 sec, and 72°C for 1 min, followed by a final elongation step (72°C for 15 min). Aliquots of PCR products were run on a 1.8% agarose gel in 0.5 × TBE buffer.

Competitive RT-PCR reaction using internal standards. Competitive RT-PCR reactions using internal standards specific for 1, 3, and 1 subunits were conducted according to methods described previously (Bovolin et al., 1992; Grayson et al., 1993; Devaud et al., 1995). The cloned amplification products were sequenced to verify authenticity, and the primers were complementary to unique sequences within corresponding cDNAs (Bovolin et al., 1992). Various amounts of the internal standard cRNA (containing a BglII restriction site) were added to a constant amount of the total RNA of interest to generate a competitive PCR amplification curve. Each mixture was reverse-transcribed and run through PCR as in the general protocol, except for the addition of 1-2 µCi [³²P]dCTP/tube and the magnesium concentration, which was optimized for each primer pair and determined to be 1.5 mM for the  subunit mRNAs, 2.0 mM for  subunit mRNAs, and 3.0 mM for  subunit mRNAs (Devaud et al., 1995). Aliquots of PCR products were digested overnight with BglII and separated on a 1.8% agarose gel. The gels were dried and exposed to a phosphorimaging screen for 1-2 hr. The signal intensity for both the native RNA products (~300 bp) and the cRNA products (~150 bp after digestion) was quantified using a phosphorimager (Molecular Dynamics, Sunnyvale, CA). Data were presented as the ratio of amounts incorporated into the amplified cRNA internal standards to amounts incorporated into the corresponding subunit mRNA amplification product versus the known amounts of internal standard cRNA added to the test sample to generate a competitive PCR linear regression curve. The amount of target RNA was calculated directly from this curve (Grayson et al., 1993).

GABA_A receptor-mediated ³⁶Cl influx into cultured cells Chloride influx was measured in cultured brainstem cells by modification of a previously described method (Mehta and Ticku, 1988, 1992). Briefly, after culture of cells for 1 DIV in DMEM + 10% FCS followed by 2 DIV in DMEM + ITS + 0.1% BSA, coverslips were removed from culture wells and rinsed for 3-4 sec at room temperature in assay buffer (HEPES-buffered saline containing 20 mM HEPES, 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, and 2.5 mM CaCl_2, adjusted to pH 7.4 with Tris-base, with 10 mM glucose added just before use). Immediately after this rinse, the coverslips were drained rapidly on tissue paper and transferred to 3 ml HEPES-buffered saline containing 2 µCi ³⁶Cl/ml (specific activity 13.38 Ci/gm), with or without various concentrations of GABA (5-60 µM) ± bicuculline (10 µM). Influx was terminated after 5 sec by transfer of the coverslip to 1000 ml ice-cold HEPES-buffered saline (with 100 mg/l picrotoxin) and then rinsed for 7 sec in another beaker containing 1000 ml ice-cold HEPES-buffered saline. Coverslips were then drained and transferred to scintillation vials containing 1 ml 0.2N NaOH. The vials were vortexed and then allowed to sit at room temperature for 1 hr. Aliquots (100 µl) were removed for protein determination using the Bradford method (Bradford, 1976). Retained radioactivity was determined by liquid scintillation spectroscopy. Values for ³⁶Clinflux were expressed as nanomol/milligram protein. Background levels of ³⁶Cl influx (without added GABA) were determined and subtracted from all values.

---

RESULTS

Embryonic brainstem cultures contain GABA and monoamine neurons, and express multiple GABA_A receptor subunits

Immunocytochemistry demonstrated that 5-HT, TH, and GABA-immunoreactive neurons were present in E14 brainstem cultures (Fig. 1) and revealed extensive expression of ₁ and 2-3 GABA_A subunit proteins, where dense immunoreactivity was localized to the outer membranes of neuronal somata and neurites (Fig. 2). General RT-PCR demonstrated the presence of mRNA transcripts encoding multiple  (1-4, but not 6),  (1-3),  (1, 2S,2L, 3), and  subunits of GABA_A receptors (Fig. 3). Many of these subunits may be expressed by glial cells as well as by neurons in these cultures, because detectable levels of 1-3, 2-3, 1, 2S, and  transcripts have also been found in purified glial cultures from E14 brainstem (J. Liu, R. Haberman, and J. Lauder, unpublished results). Quantitative competitive RT-PCR was performed for 1 and 3 subunits, because 1 and 2-3 antibodies had been used to characterize cultures immunocytochemically. The 1 subunit was chosen for comparison. Competitive RT-PCR revealed that 3 subunit transcripts were highly expressed (272.5 ± 26.1 pg/µg total RNA) compared with 1 (1.9 ± 0.06 pg/µg total RNA) and 1 (6.9 ± 0.3 pg/µg total RNA) transcripts (Fig. 4).

GABA_A ligands regulate Cl influx, indicating the presence of functional GABA_A receptors

GABA produced concentration-dependent and saturable effects on Cl influx in E14 brainstem cells at 3 DIV, with maximal enhancement at 10 µM GABA to ~13.6 nmol/mg protein (Fig. 5), which increased Cl influx over basal values by 400%. This effect was blocked by 10 µM bicuculline.

Neuronal survival and growth are differentially affected by GABA_A ligands

Survival The effects of GABA agonist and antagonist treatments (10 µM GABA and/or bicuculline or dieldrin in serum-free medium for 48 hr) on the density of 5-HT, TH, and GABA neurons are shown in Figure 6. GABA significantly stimulated survival of 5-HT and TH neurons but did not affect the survival of GABA neurons. The classical GABA_A receptor antagonist bicuculline significantly reduced the survival of 5-HT and TH neurons, whereas the pesticide dieldrin reduced the survival of 5-HT neurons only. On the contrary, both GABA_A antagonists significantly enhanced survival of GABA neurons. GABA completely blocked the effects of bicuculline on all three neuronal phenotypes, but did not reverse the effects of dieldrin on 5-HT and GABA neurons.

Growth The effects of treatments (described above) on the growth of cell bodies and neurites are shown in Table 1.

SA: The size of cell somas (soma area) of TH neurons was significantly increased by GABA. When added together with GABA, bicuculline and dieldrin inhibited this growth effect but had no significant effect when given alone. Other treatments had no significant effects on cell somas of these neurons. Cell somas of GABA and 5-HT neurons were not affected significantly by any treatment.

FA: The surface area covered by neurites (field area) of 5-HT neurons was increased after treatment with GABA, whereas GABA had no significant effects on the FA of TH or GABA neurons. Dieldrin significantly reduced the FA in 5-HT neurons, an effect that was not overcome by co-administration of GABA, whereas bicuculline had no significant effect; however, bicuculline did decrease the FA in TH neurons, which was reversed by GABA. Although GABA itself had no significant effect on the FA of GABA neurons, bicuculline stimulated this parameter, which was blocked by co-administration of GABA. Dieldrin also seemed to increase the FA of these neurons to some extent, but this effect did not reach significance using the Bonferroni test (although it was significant by the less conservative post hoc t test).

NN: The number of primary neurites of TH neurons was increased significantly by GABA, which was reduced to nonsignificant levels by bicuculline. Although the one-way ANOVA for treatment effects on NN of 5-HT neurons was highly significant (p < 0.001), and post hoc t tests indicated significant growth-promoting effects of GABA and inhibitory effects of dieldrin, no treatment effect reached significance when it was analyzed by the more conservative Bonferroni test. No significant effects on the NN of GABA neurons were seen after any treatment.

NTS: GABA did not significantly affect the number of terminal neurite segments of 5-HT and TH neurons, but both bicuculline and dieldrin significantly decreased this parameter in TH neurons, effects that were reversed by GABA. The NTS of GABA neurons were unaffected by any treatment.

---

DISCUSSION

GABA acts as a trophic signal for monoamine neurons but negatively regulates development of GABA neurons

GABA stimulated survival and growth of 5-HT and TH neurons in embryonic brainstem cultures, and these effects were reversed by GABA_A antagonists. These cultures expressed multiple GABA_A receptor subunit mRNA transcripts, as measured by RT-PCR, and exhibited functional GABA-gated Cl channels, as determined by ³⁶Cl influx. The amount of ³⁶Cl influx stimulated by 10 µM GABA was comparable to values reported previously in whole fetal rat brain (Kellogg and Pleger, 1989), suggesting that GABA-gated channels are highly expressed in these cultures. Taken together, these results suggest that GABA_A receptors mediate the trophic effects of GABA on brainstem monoamine neurons in these cultures. Although it could be argued that GABA-stimulated release of 5-HT from serotonin neurons (Becquet et al., 1993) could account for some of the positive effects of GABA on 5-HT and TH neurons, two points argue against this possibility. First, 5-HT does not promote survival of brainstem monoamine neurons, although it does stimulate their growth (Lauder, 1990; Liu and Lauder, 1991). Second, the GABA_A antagonist bicuculline reversed the positive effects of GABA on monoamine neurons and inhibited growth and survival of these cells when added alone. For these reasons, we believe it is unlikely that released 5-HT played a significant role in the positive effects of GABA on monoamine neurons.

Although GABA itself did not have significant effects on GABA neurons, both bicuculline and dieldrin stimulated survival of these cells, and bicuculline promoted areal growth of neurites (FA). The positive effects of these GABA_A antagonists suggest that GABA may negatively autoregulate development of the GABAergic neuronal population in embryonic brainstem in vivo. The lack of significant effects of GABA on GABA neurons in our cultures could be attributable to the presence of endogenous GABA (released by GABA neurons) (Barbin et al., 1993; Becquet et al., 1993), which produced maximal inhibitory effects on these cells before treatment. GABA breaks down within hours after being added to culture medium. Therefore, it is possible that if muscimol had been used instead of GABA, significant negative effects on GABA neurons would have been found; however, because GABA was added daily to the culture medium, and significantly affected growth and survival of monoamine neurons, we think it unlikely that degradation of GABA contributed significantly to the lack of effects on GABA neurons.

Differential effects of receptor ligands suggest heterogeneous expression of GABA_A receptor subunits by monoamine and GABA neurons

The differential effects of GABA_A receptor ligands on cultured monoamine and GABA neurons raise the interesting possibility that these neurotransmitter phenotypes express different amounts of GABA_A receptors and/or distinct GABA_A isoreceptors (subtypes) with differing pharmacological properties. These findings are consistent with evidence that (1) GABA promotes differentiation of different types of neurons to varying degrees in other culture systems (Hansen et al., 1984; Meier et al., 1985; Spoerri, 1988; Michler, 1990; Belhage et al., 1997) and (2) adult GABA neurons and 5-HT neurons express different complements of particular GABA_A receptor subunits (Gao et al., 1993; Gao and Fritschy, 1994).

Differential effects of GABA_A receptor ligands on growth of cell bodies and neurites (Table 1) raises the further possibility that GABA_A receptors may be differentially distributed on cultured 5-HT, TH, and GABA neurons. This view is supported by previous evidence that (1) high affinity GABA_A receptors are situated on cell bodies of cerebellar granule cells, whereas low affinity GABA_A receptors are preferentially located on cell processes (Hansen et al., 1991); (2) 2, 5, 3, and 2 subunit transcripts are expressed by cell somas, neurites, and growth cones of cortical neurons, whereas 3 and 3 subunits are confined to cell somas (Poulter et al., 1994); and (3) GABA_A subunit expression on cell bodies and neurites of developing cerebellar granule cells is differentially regulated by the GABA_A agonist THIP (Gaboxadol), suggesting that sorting and targeting of newly synthesized receptors may undergo maturational changes that can be influenced by agonists (Elster et al., 1995).

Organochlorine pesticides affect neuronal growth and survival and alter trophic effects of GABA

Certain pesticides exert their neurotoxic actions by selectively blocking ion channels in the insect nervous system. Organochlorine pesticides such as dieldrin interact with specific sites on the GABA_A-Cl channel complex to block GABA-induced Cl flux in both insect and mammalian cells (Abalis et al., 1986; Gant et al., 1987; Bloomquist, 1992; Pomés et al., 1994a,b). The GABA recognition site has been reported to be targeted by dieldrin (Eldefrawi and Eldefrawi, 1987; Ogata et al., 1988; Tokutomi et al., 1994). Other evidence, however, suggests that dieldrin binds to the picrotoxin site on the Cl channel (Nagata and Narahashi, 1994) and suppresses GABA-induced Cl currents in a noncompetitive manner (Nagata et al., 1994).

Data from the present study indicate that dieldrin, like bicuculline, has opposite effects on growth and survival of cultured monoamine and GABA neurons. This could be explained by different binding characteristics and sensitivity of these GABA_A receptors to dieldrin. If this is the case, then it would suggest that GABA_A receptors expressed by embryonic monoamine and GABA neurons may have different subunit compositions, because subunit composition is known to affect the binding characteristics and sensitivity of GABA_A receptors to particular receptor ligands (Pritchett et al., 1989; Malherbe et al., 1990; Pritchett and Seeburg, 1990; Burt and Kamatchi, 1991).

Summary

The present study has revealed evidence that GABA acts as a trophic signal for embryonic brainstem monoamine neurons by activating GABA_A receptors, but it may be a negative autoregulatory signal for developing GABA neurons. Consistent with this interpretation, bicuculline and dieldrin had negative effects on monoamine neurons but exerted positive effects on GABA neurons. These differential effects of GABA_A receptor ligands on neurons of serotonergic, noradrenergic, and GABAergic phenotypes raise the possibility that prenatal exposure to pesticides or drugs acting as GABA_A antagonists could interfere with the positive and negative regulatory influences of GABA, thereby producing imbalances in monoaminergic and GABAergic neurotransmission in the developing brain. If long-lasting, these effects could have functional and behavior consequences in offspring. The differential effects of GABA_A receptor ligands on the growth of cell bodies and neurites suggest further that particular cellular compartments may express different levels and/or distinct populations of GABA_A receptor subtypes.

---

FOOTNOTES

Received Dec. 23, 1996; accepted Jan. 13, 1997.

  

This work was supported by National Institute of Environmental Health Sciences Grant R01 ES07017 to J.M.L. We are grateful to Dr. Joseph Neale for the gift of 1 antibodies, to Mary Beth Wilkie for editorial assistance, and to Drs. Cindy Lawler and Josephine Johns for consultation on the statistical analyses.

Correspondence should be addressed to Dr. Jean M. Lauder, Department of Cell Biology and Anatomy, CB 7090, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7090.

---

REFERENCES

• Abalis IM, Eldefrawi ME, Eldefrawi AT (1986) Effects of insecticides on GABA-induced chloride influx into rat brain microsacs. J Toxicol Environ Health 18:13-23 . [ISI][Medline]
• Abraham JH, Seiler N, Schousboe A (1994) Induction of low-affinity GABA_A receptor by the GABA-agonist THIP in cultured rat cerebellar granule cells is prevented by inhibition of polyamine biosynthesis. J Neurosci Res 39:656-662 . [ISI][Medline]
• Barbin G, Pollard H, Gaiarsa JL, Ben-Ari Y (1993) Involvement of GABA_A receptors in the outgrowth of cultured hippocampal neurons. Neurosci Lett 152:150-154 . [ISI][Medline]
• Barnard EA (1988) The structure of the GABA/benzodiazepine receptor complex with its gated ion channel. In: GABA and benzodiazepine receptors (Squires RP, ed), pp 103-122. Boca Raton, FL: CRC.
• Becquet D, Hery M, Francois-Bellan AM, Giraud P, Deprez P, Faudon M, Fache MP, Hery F (1993) Glutamate, GABA, glycine and taurine modulate serotonin synthesis and release in rostral and caudal rhombencephalic raphe cells in primary cultures. Neurochem Int 23:269-283 . [ISI][Medline]
• Behar TN, Schaffer AE, Colton CA, Somogy R, Olah Z, Lehel C, Barker JL (1994) GABA-induced chemokinesis and NGF-induced chemotaxis of embryonic spinal cord neurons. J Neurosci 14:29-38 . [Abstract]
• Belhage B, Hansen GH, Elster L, Schousboe A (1997) Effects of -aminobutyric acid (GABA) on synaptogenesis and synaptic function. Persp Dev Neurosci, in press.
• Bloomquist JR (1992) Intrinsic lethality of chloride-channel-directed insecticides and convulsants in mammals. Toxicol Lett 60:289-298 . [ISI][Medline]
• Bovolin P, Santi MR, Memo M, Costa E, Grayson DR (1992) Distinct developmental patterns of rat 1, 5, 2S and 2L -aminobutyric acid receptor subunit mRNAs in vivo and in vitro. J Neurochem 59:62-72 . [ISI][Medline]
• Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal Biochem 72:248-254 . [ISI][Medline]
• Burt DR, Kamatchi GL (1991) GABA_A receptor subtypes: from pharmacology to molecular biology. FASEB J 5:2916-2923 . [Abstract]
• Cobas A, Fairen A, Alvarez-Bolado G, Sanchez MP (1991) Prenatal development of the intrinsic neurons of the rat neocortex: a comparative study of the distribution of the GABA-immunoreactive cells and the GABA_A receptor. Neuroscience 40:375-397 . [ISI][Medline]
• Connor JA, Tseng HY, Hockberger PE (1987) Depolarization- and transmitter-induced changes in intracellular Ca²⁺ of rat cerebellar granule cells in explant culture. J Neurosci 7:1384-1400 . [Abstract]
• Costa LG (1988) Interactions of neurotoxicants with neurotransmitter systems. Toxicology 49:359-366 . [ISI][Medline]
• Devaud LL, Smith FD, Grayson DR, Morrow AL (1995) Chronic ethanol consumption differentially alters the expression of -aminobutyric acid_A receptor subunit mRNAs in rat cerebral cortex: competitive, quantitative reverse transcriptase-polymerase chain reaction analysis. Mol Pharmacol 48:861-868 . [Abstract]
• Eins S, Spoerri PE, Heyder E (1983) GABA or sodium bromide-induced plasticity of neurites of mouse neuroblastoma cells in culture: a quantitative study in culture. Cell Tissue Res 229:457-460 . [ISI][Medline]
• Eldefrawi AT, Eldefrawi ME (1987) Receptors for -aminobutyric acid and voltage-dependent chloride channels as targets for drugs and toxicants. FASEB J 1:262-271 . [Abstract]
• Elster L, Hansen GH, Belhage B, Fritschy JM, Möhler H, Schousboe A (1995) Differential distribution of GABA_A receptor subunits in soma and processes of cerebellar granule cells: effects of maturation and a GABA agonist. Int J Dev Neurosci 13:417-428 . [ISI][Medline]
• Fiszman ML, Novotny EA, Lange GD, Barker JL (1990) Embryonic and early postnatal hippocampal cells respond to nanomolar concentrations of muscimol. Dev Brain Res 53:186-193 . [Medline]
• Fritschy JM, Benke D, Mertens S, Oertel WH, Bachi T, Möhler H (1992) Five subtypes of type A gamma-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies. Proc Natl Acad Sci USA 89:6726-6730 . [Abstract/Free Full Text]
• Gambarana C, Beattie CE, Rodriguez ZR, Siegel RE (1991) Region-specific expression of messenger RNAs encoding GABA_A receptor subunits in the developing rat brain. Neuroscience 45:423-432 . [ISI][Medline]
• Gant DB, Eldefrawi ME, Eldefrawi AT (1987) Cyclodiene insecticides inhibit GABA_A receptor-regulated chloride transport. Toxicol Appl Pharmacol 88:313-321 . [ISI][Medline]
• Gao B, Fritschy JM (1994) Selective allocation of GABA_A receptors containing the 1 subunit to neurochemically distinct subpopulations of rat hippocampal neurons. Eur J Neurosci 6:837-853 . [ISI][Medline]
• Gao B, Fritschy JM, Benke D, Möhler H (1993) Neuron-specific expression of GABA_A receptor subtypes: differential association of the 1- and 3-subunits with serotonergic and GABAergic neurons. Neuroscience 54:881-892 . [ISI][Medline]
• Grayson DR, Bovolin P, Santi MR (1993) Absolute quantitation of -aminobutyric acid A receptor subunit mRNAs by competitive polymerase chain reaction. Methods Neurosci 12:191-208.
• Hansen GH, Meier E, Schousboe A (1984) GABA influences the ultrastructure composition of cerebellar granule cells during development in culture. Int J Dev Neurosci 2:247-257.
• Hansen GH, Belhage B, Schousboe A (1991) Effect of a GABA agonist on the expression and distribution of GABA_A receptors in the plasma membrane of cultured cerebellar granule cells: an immunocytochemical study. Neurosci Lett 124:162-165 . [ISI][Medline]
• Hebebrand J, Hofmann D, Reichelt R, Scnarr S, Knapp M, Propping P, Födisch HJ (1988) Early ontogeny of the central benzodiazepine receptor in human embryos and fetuses. Life Sci 43:2127-2136 . [ISI][Medline]
• Kellogg CK, Pleger GL (1989) GABA-stimulated chloride uptake and enhancement by diazepam in synaptoneurosomes from rat brain during prenatal and postnatal development. Brain Res Dev Brain Res 49:87-95 . [Medline]
• Lauder JM (1990) Ontogeny of the serotonergic system in the rat: serotonin as a developmental signal. Ann NY Acad Sci 600:296-314.
• Lauder JM, Han VKM, Henderson P, Verdoorn T (1986) Prenatal ontogeny of the GABAergic system in the rat brain: an immunocytochemical study. Neuroscience 19:465-493 . [ISI][Medline]
• Laurie DJ, Wisden W, Seeburg PH (1992) The distribution of thirteen GABA_A receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development. J Neurosci 12:4151-4172 . [Abstract]
• Liu J, Lauder JM (1991) Serotonin and nialamide differentially regulate survival and growth of cultured serotonin and catecholamine neurons. Dev Brain Res 60:59-67.
• Lieth E (1992) Computerized morphometric analysis of neurons in culture. Methods Neurosci 10:391-405.
• Lieth E, McClay DR, Lauder JM (1990) Neuronal-glial interactions: complexity of neurite outgrowth correlation with substrate adhesive of serotonergic neurons. Glia 3:169-170 . [ISI][Medline]
• Loscher W (1979) GABA in plasma and cerebrospinal fluid of different species: effects of gamma-acetylenic GABA, gamma-vinyl GABA and sodium valproate. J Neurochem 32:1587-1591 . [ISI][Medline]
• Ma W, Barker JL (1995) Complementary expression of transcripts encoding GAD₆₇ and GABA_A receptor alpha 4, beta 1, and gamma 1 subunits in the proliferative zone of the embryonic rat central nervous system. J Neurosci 15:2547-2560 . [Abstract]
• Ma W, Saunders PA, Somogyi R, Poulter MO, Barker JL (1993) Ontogeny of GABA_A receptor subunit mRNAs in rat spinal cord and dorsal root ganglia. J Comp Neurol 338:357-359.
• MacDonald RL, Olsen RW (1994) GABA_A receptor channels. Annu Rev Neurosci 17:569-602 . [ISI][Medline]
• Malherbe P, Sigel E, Baur R, Persohn E, Richards JG, Möhler H (1990) Functional expression and sites of gene transcription of a novel  subunit of the GABA_A receptor in rat brain. FEBS Lett 260:261-265 . [ISI][Medline]
• Mehta AK, Ticku MK (1988) Developmental aspects of benzodiazepine receptors and GABA-gated chloride channels in primary cultures of spinal cord neurons. Brain Res 454:156-163 . [ISI][Medline]
• Mehta AK, Ticku MK (1992) Chronic GABA exposure down-regulates GABA- benzodiazepine receptor-ionophore complex in cultured cerebral cortical neurons. Mol Brain Res 16:29-36 . [Medline]
• Meier E, Drejer J, Schousboe A (1984) GABA induces functionally active low-affinity GABA receptors on cultures cerebellar granule cells. J Neurochem 43:1737-1744 . [ISI][Medline]
• Meier H, Hansen GH, Schousboe A (1985) The trophic effect of GABA on cerebellar granule cells is mediated by GABA receptors. Int J Dev Neurosci 3:401-407.
• Meier H, Hertz L, Schousboe A (1991) Neurotransmitters as developmental signals. Neurochem Int 19:1-15.
• Michler A (1990) Involvement of GABA receptors in the regulation of neurite growth in cultured embryonic chick tectum. Int J Dev Neurosci 8:463-472 . [ISI][Medline]
• Morrow AL (1995) Regulation of GABA_A receptor function and gene expression in the central nervous system. Int Rev Neurobiol 38:1-41 . [ISI][Medline]
• Morrow AL, Herbert JS, Montpied P (1992) Differential effects of chronic ethanol administration of GABA_A receptor 1 and 6 subunit mRNA levels in rat cerebellum. J Mol Cell Neurosci 3:251-258.
• Nagata K, Narahashi T (1994) Dual action of the cyclodiene insecticide dieldrin on the -aminobutyric acid receptor-chloride channel complex of rat dorsal root ganglion neurons. J Pharmacol Exp Ther 269:164-172 . [Abstract/Free Full Text]
• Nagata K, Hamilton BJ, Carter DB, Narahashi T (1994) Selective effects of dieldrin on the GABA_A receptor-channel complex subunits expressed in human embryonic kidney cells. Brain Res 645:19-26 . [ISI][Medline]
• Nicholson LFB, Faull RLM, Waldvogel HJ, Dragunow M (1992) The regional, cellular and subcellular location of GABA_A/benzodiazepine receptors in the substantia nigra. Neuroscience 50:355-370. [ISI][Medline]
• Obrietan K, van den Pol AN (1995) GABA neurotransmission in the hypothalamus: developmental reversal from Ca²⁺ elevating to depressing. J Neurosci 15:5065-5077 . [Abstract]
• Ogata N, Vogel SM, Narahashi T (1988) Lindane but not deltamethrin blocks a component of a GABA-activated chloride channel. FASEB J 2:2895-2900 . [Abstract]
• Olsen RW, Tobin AJ (1990) Molecular biology of GABA_A receptors. FASEB J 4:1469-1480 . [Abstract]
• Pomés A, Rodriguez-Farré E, Suñol C (1994a) Disruption of GABA-dependent chloride flux by cyclodienes and hexachlorocyclohexanes in primary cultures of cortical neurons. J Pharmacol Exp Ther 271:1616-1623 . [Abstract/Free Full Text]
• Pomés A, Frandsen AA, Suñol C, Sanfeliu E, Rodriguez-Farré E, Schousboe A (1994b) Lindane cytotoxicity in cultured neocortical neurons is ameliorated by GABA and flunitrazepam. J Neurosci Res 39:663-668 . [ISI][Medline]
• Poulter MO, Barker JL, O'Carroll A-M, Lolait SJ, Mahan LC (1992) Transient and differential expression of GABA receptor subunit mRNAs during development of the rat CNS. J Neurosci 12:2888-2900 . [Abstract]
• Poulter MO, Larmet Y, Pruss R, Feltz P (1994) Preferential and transient expression of GABA_A receptor subunit mRNAs in growing neurites of early postnatal cortical cells. Soc Neurosci Abstr 20:659.
• Prasad A, Barker JL (1990) Functional GABA_A receptor are critical for survival and process outgrowth of embryonic chick spinal cord cells. Soc Neurosci Abstr 16:647.
• Pritchett DB, Seeburg PH (1990) Gamma-aminobutyric acid A receptor 5-subunit creates novel type II benzodiazepine receptor pharmacology. J Neurochem 54:1802-1804 . [ISI][Medline]
• Pritchett DB, Luddens H, Seeburg PH (1989) Type I and type II GABA_A benzodiazepine receptors produced in transfected cells. Science 245:1389-1392 . [Abstract/Free Full Text]
• Reichling DB, Kyrozis A, Wang J, Macdermott AB (1994) Mechanisms of GABA and glycine depolarization-induced calcium transient in rat dorsal horn neurons. J Physiol (Lond) 476:411-421 . [Abstract/Free Full Text]
• Sato TN, Neale JH (1989) Immunological identification of multiple 1-like subunits of the -aminobutyric acid_A receptor complex purified from neonatal rat cortex. J Neurochem 53:1089-1095 . [ISI][Medline]
• Schlumpf M, Ramseier H, Abriel H, Youmbi M, Baumann JB, Lichtensteiger W (1989) Diazepam effects on the fetus. Neurotoxicology 10:501-516 . [ISI][Medline]
• Schlumpf M, Parmar R, Schreiber A, Ramseier HR, Bütikofer E, Abriel H, Barth M, Rhyner T, Lichtensteiger W (1992) Nervous and immune systems as targets for developmental effects of benzodiazepines. Dev Pharmacol Ther 18:145-158 . [ISI][Medline]
• Schofield PR, Darlison MG, Fujita N, Burt DR, Stephenson FA, Rodriguez H, Rhee LM, Ramachandran J, Reale V, Glencorse TA, Seeburg PH, Barnard EA (1987) Sequence and function expression of the GABA_A receptor shows a ligand-gated receptor super-family. Nature 328:221-227 . [Medline]
• Smith D, Gallager D (1987) GABA, benzodiazepine and serotonergic receptor development in the dorsal raphe nucleus: electrophysiological studies. Dev Brain Res 35:191-198.
• Spoerri PE (1988) Neurotrophic effects of GABA in culture of embryonic chick brain and retina. Synapse 2:11-12 . [ISI][Medline]
• Spoerri PE, Wolff JR (1981) Effect of GABA-administration on murine neuroblastoma cells in culture. Cell Tissue Res 218:567-579 . [ISI][Medline]
• Tokutomi N, Ozoe Y, Katayama N, Akaike N (1994) Effects of lindane (-BHC) and related convulsants on GABA_A receptor-operated chloride channels in frog dorsal root ganglion neurons. Brain Res 643:66-73 . [ISI][Medline]
• Wallace JA, Petrusz P, Lauder JM (1982) Serotonin immunocytochemistry in the adult and developing rat brain: methodological and pharmacological considerations. Brain Res Bull 9:117-129 . [ISI][Medline]
• Wolff JR, Joo F, Kasa P (1993) Modulation by GABA of neuroplasticity in the central and peripheral nervous system. Neurochem Res 18:453-461 . [ISI][Medline]
• Xian H, Maric D, Maric I, Barker JL (1995) GABA elevates cytoplasmic calcium in fractionated embryonic rat cortical cells. Soc Neurosci Abstr 21:1288.
• Zheng T, Santi MR, Bovolin P, Marlier LNJ-L, Grayson DR (1993) Developmental expression of the 6 GABA_A receptor subunit mRNA occurs only after cerebellar granule cell migration. Dev Brain Res 75:92-103.

---

Copyright ©1997 Society for Neuroscience   0270-6474/1997/172420-09$05.00/0

This article has been cited by other articles:

				J. R. Richardson, W. M. Caudle, M. Wang, E. D. Dean, K. D. Pennell, and G. W. Miller Developmental exposure to the pesticide dieldrin alters the dopamine system and increases neurotoxicity in an animal model of Parkinson's disease FASEB J, August 1, 2006; 20(10): 1695 - 1697. [Abstract] [Full Text] [PDF]

				L.-L. Zhang, H. R. Pathak, D. A. Coulter, M. A. Freed, and N. Vardi Shift of Intracellular Chloride Concentration in Ganglion and Amacrine Cells of Developing Mouse Retina J Neurophysiol, April 1, 2006; 95(4): 2404 - 2416. [Abstract] [Full Text] [PDF]

				L. G. Costa, L. Steardo, and V. Cuomo Structural Effects and Neurofunctional Sequelae of Developmental Exposure to Psychotherapeutic Drugs: Experimental and Clinical Aspects Pharmacol. Rev., March 1, 2004; 56(1): 103 - 147. [Abstract] [Full Text] [PDF]

				V. C. Moser, T. J. Shafer, T. R. Ward, C. A. Meacham, M. W. Harris, and R. E. Chapin Neurotoxicological Outcomes of Perinatal Heptachlor Exposure in the Rat Toxicol. Sci., April 1, 2001; 60(2): 315 - 326. [Abstract] [Full Text] [PDF]

				B.-S. Li, W. Ma, L. Zhang, J. L. Barker, D. A. Stenger, and H. C. Pant Activation of Phosphatidylinositol-3 Kinase (PI-3K) and Extracellular Regulated Kinases (Erk1/2) Is Involved in Muscarinic Receptor-Mediated DNA Synthesis in Neural Progenitor Cells J. Neurosci., March 1, 2001; 21(5): 1569 - 1579. [Abstract] [Full Text] [PDF]

				Q.-Y. Liu, A. E. Schaffner, Y. H. Chang, D. Maric, and J. L. Barker Persistent Activation of GABAA Receptor/Cl- Channels by Astrocyte-Derived GABA in Cultured Embryonic Rat Hippocampal Neurons J Neurophysiol, September 1, 2000; 84(3): 1392 - 1403. [Abstract] [Full Text] [PDF]

				S. T. Sinkkonen, M. C. Hanna, E. F. Kirkness, and E. R. Korpi GABAA Receptor epsilon and theta Subunits Display Unusual Structural Variation between Species and Are Enriched in the Rat Locus Ceruleus J. Neurosci., May 15, 2000; 20(10): 3588 - 3595. [Abstract] [Full Text] [PDF]

				S. Soares, Y. von Boxberg, M. Ravaille-Veron, J.-D. Vincent, and F. Nothias Morphofunctional Plasticity in the Adult Hypothalamus Induces Regulation of Polysialic Acid-Neural Cell Adhesion Molecule through Changing Activity and Expression Levels of Polysialyltransferases J. Neurosci., April 1, 2000; 20(7): 2551 - 2557. [Abstract] [Full Text] [PDF]

				B. G. Condie, G. Bain, D. I. Gottlieb, and M. R. Capecchi Cleft palate in mice with a targeted mutation in the gamma -aminobutyric acid-producing enzyme glutamic acid decarboxylase 67 PNAS, October 14, 1997; 94(21): 11451 - 11455. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services