Expression Of Gamma-aminobutyric Acid Research Articles

GABA immunoreactivity in the developing rat thalamus and Otx2 homeoprotein expression in migrating neurons

We investigated the expression of gamma-aminobutyric acid (GABA) in the developing rat thalamus by immunohistochemistry, using light, confocal and electron microscopy. We also examined the relationship between the expression of the homeoprotein Otx2, a transcription factor implicated in brain regionalization, and the radial and non-radial migration of early generated thalamic neurons, identified by the neuronal markers calretinin (CR) and GABA. The earliest thalamic neurons generated between embryonic days (E) 13 and 15 include those of the reticular nucleus, entirely composed by GABAergic neurons. GABA immunoreactivity appeared at E14 in immature neurons and processes laterally to the neuroepithelium of the diencephalic vesicle. The embryonic and perinatal periods were characterized by the presence of abundant GABA-immunoreactive fibers, mostly tangentially oriented, and of growth cones. At E15 and E16, GABA was expressed in radially and non-radially oriented neurons in the region of the reticular thalamic migration, between the dorsal and ventral thalamic primordia, and within the dorsal thalamus. At these embryonic stages, some CR- and GABA-immunoreactive migrating-like neurons, located in the migratory stream and in the dorsal thalamus, expressed the homeoprotein Otx2. In the perinatal period, the preponderance of GABAergic neurons was restricted to the reticular nucleus and several GABAergic fibers were still detectable throughout the thalamus. The immunolabeling of fibers progressively decreased and was no longer visible by postnatal day 10, when the adult configuration of GABA immunostaining was achieved. These results reveal the spatio-temporal features of GABA expression in the developing thalamus and suggest a novel role of Otx2 in thalamic cell migration.

Long-term effects of recurrent seizures in neonate period on gamma-aminobutyric acid A receptor alpha1 and beta2 subunits expression in adult brain: experiment with rats

To investigate the long-term effects of recurrent seizures in neonate period on the expression of gamma-aminobutyric acid A receptor (GABAAR) alpha1 and beta2 subunits in brain and spatial memory and seizure susceptibility in adult period. Thirty-two 7-day-old SD rats were randomly divided into 2 equal groups: seizure group, inhaling flurothyl to induce seizure daily for 6 days, and control group. On days 61 - 65 after birth Morris water maze test was used to record the escape latency. On day 75 after birth pentylenetetrazol (PTZ) was injected intraperitoneally to induce seizure so as to record the latency. Then the rats were killed to take their brains, 8 in each group used to undergo immunohistochemistry to examine the protein expression of the GABAAR alpha1 and beta2 subunits, and the other 8 in each group used to examine the mRNA expression of the GABAAR alpha1 and beta2 subunits in the brains using RT-PCR. On day 64 the escape latency of the seizure group was 82,424 ms +/- 35,622 ms, significantly longer than that of the control group (40,712 ms +/- 29,468 ms, P = 0.001). On day 75 the frequency of crossing target within 120 s in the water maze of the seizure group was 1.2 times +/- 0.9 times, significantly less than that of the control group (3.1 times +/- 1.3 times, P < 0.01). The seizure latency after the PTZ injection of the seizure group was (1487 +/- 662) s, not significantly different from that of the control group (1841 s +/- 648 s, P = 0.133). In comparison with the control group the accumulated optical density (AOD) of GABAAR alpha1 subunit protein immunoactivity in the parietal cortex, and hippocampal CA1-2 and CA4 regions of the seizure group decreased significantly (all P < 0.05), and was not significantly different in the frontal cortex, dentate gyrus, and hippocampal CA3 region (all P > 0.05). In comparison with the control group the accumulated optical density (AOD) of GABAAR beta2 subunit protein immunoactivity in the thalamus, and hippocampal CA1-4 regions of the seizure group decreased significantly (all P < 0.05), and was not significantly different in the frontal cortex and parietal cortex (both P > 0.05). In comparison with the control group the mRNA expression of GABAAR alpha1 subunit and the mRNA expression of GABAAR beta2 subunit of the seizure group were significantly lower in the hippocampus (both P < 0.05) and not significantly different in the cerebral cortex (both P > 0.05). Recurrent seizures in neonate period modify the expression of GABAAR alpha1 and beta2 subunits in the cerebral cortex and hippocampus in adult period which may be related to cognitive deficit.

Differential expression of γ-aminobutyric acid receptor A (GABAA) and effects of homocysteine

gamma-Aminobutyric acid (GABA) is a known inhibitory neurotransmitter in the mammalian central nervous system, and homocysteine (Hcy) behaves as an antagonist for GABA(A) receptor. Although the properties and functions of GABA(A) receptors are well studied in mouse neural tissue, its presence and significance in non-neural tissue remains obscure. The aim of the present study was to examine the expression of GABA(A) receptor and its subunits in non-neural tissue. The mice were analyzed. The presence of GABA(A) receptor and its subunits was evaluated using Western blot and reverse transcription polymerase chain reaction. We report that GABA(A) receptor protein is abundant in the renal medulla, cortex, heart, left ventricle, aorta and pancreas. Low levels of GABA(A) receptor protein were detected in the atria of the heart, right ventricle, lung and stomach. The mRNA protein expression of GABA(A) receptor subunit shows that alpha1, beta1, beta3 and gamma1 subunits are present only in brain. The mRNA protein expression levels of GABA(A) receptor alpha2, alpha6, beta2 and gamma3 subunits were highly expressed in brain compared to other tested tissue, while GABA(A) receptor gamma2 subunit was expressed only in brain and kidney. Treatment of microvascular endothelial cells with Hcy decreased GABA(A) receptor protein level, which was restored to its baseline level in the presence of GABA(A) receptor agonist, muscimol. The distribution of GABA(A) and GABA(B) receptors in wild type mice was determined and tissue-specific expression patterns were found showing that several receptor subtypes were also expressed in the central nervous system. Hcy, a GABA(A) agonist, was found to decrease GABA(A) expression levels. These data enlarge knowledge on distribution of GABA receptors and give novel ideas of the effects of Hcy on different organs.

Dark‐rearing‐induced reduction of GABA and GAD and prevention of the effect by BDNF in the mouse retina

Gamma-aminobutyric acid (GABA) is an important retinal neurotransmitter. We studied the expression of GABA, glutamate decarboxylase 65 (GAD65) and GAD67 by immunocytochemistry and Western blot, in the retinas of control and dark-reared C57BL/6J black mice. This study asked three questions. First, is visual input necessary for the normal expression of GABA, GAD65 and GAD67? Second, can the retina recover from the effects of dark-rearing if returned to a normal light-dark cycle? Third, does BDNF prevent the influence of dark-rearing on the expression of GABA and GAD? At postnatal day 10 (P10), before eye opening, GABA immunoreactivity was present in the ganglion cell layer (GCL), in the innermost rows of the inner nuclear layer (INL) and throughout the inner plexiform layer (IPL) of control and dark-reared retinas. In P30 control retinas, GABA immunoreactivity showed similar patterns to those at P10. However, in P30 dark-reared retinas, the density of GABA-immunoreactive cells was lower in both the INL and GCL than in control retinas. In addition, visual deprivation retarded GABA immunoreactivity in the IPL. Western blot analysis showed corresponding differences in the levels of GAD65 but not of GAD67 expression between control and dark-rearing conditions. In our study, dark-rearing effects were reversed when the mice were put in normal cyclic light-dark conditions for 2 weeks. Moreover, dark-reared retinas treated with BDNF showed normal expression of both GABA and GAD65. Our data indicate that normal expression of GABA and GAD65 is dependent on visual input. Furthermore, the data suggest that BDNF controls this dependence.

Possible expression of a particular gamma-aminobutyric acid transporter isoform responsive to upregulation by hyperosmolarity in rat calvarial osteoblasts

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the brain, but widely distributed in different peripheral organs. We have previously shown the functional expression of GABA B receptors required for GABAergic signal input by cultured rat calvarial osteoblasts. This study focused on the possible functional expression of the machinery required for GABAergic signal termination such as GABA transporters. In rat calvarial osteoblasts cultured for 7 days, [ 3H]GABA accumulation was observed in a temperature-, sodium- and chloride-dependent manner, consisting of a single component with a K m value of 789.6 ± 9.0 μM and a V max value of 4.4 ± 0.1 nmol/min/mg protein, respectively. Both nipecotic and l-2,4-diaminobutyric acids significantly inhibited [ 3H]GABA accumulation in a concentration-dependent manner. Constitutive expression was seen with mRNA for the betaine/GABA transporter-1 (BGT-1) and taurine transporter (TauT), while hyperosmotic cultivation led to significant increases in both [ 3H]GABA accumulation and BGT-1 mRNA expression without affecting TauT mRNA expression. Highly immunoreactive cells were detected for the BGT-1 isoform at the surface of trabecular bone of neonatal rat tibias. Sustained exposure to GABA significantly inhibited alkaline phosphatase (ALP) activity, but not cellular viability, at concentrations above 0.1 mM in osteoblasts cultured for 3 to 28 days. Nipecotic acid not only decreased ALP activity alone, but also further decreased ALP activity in osteoblasts cultured in the presence of GABA. These results suggest that the BGT-1 isoform may be functionally expressed by rat calvarial osteoblasts to play a hitherto unidentified role in mechanisms underlying hyperosmotic regulation of osteoblastogenesis.

Long-term alteration of gamma-aminobutyric acid B receptor subunits in immature rats after recurrent febrile seizures

Febrile seizure (FS) is closely related to an altered transmission of gamma-aminobutyric acid (GABA). GABA exerts its effects through ionotropic receptors (GABA(AR) and GABA(CR)) and metabotropic receptors (GABA(BR)). GABA(BRs) are located at pre- and postsynaptic sites. Stimulation of postsynaptic receptors generates long-lasting inhibitory postsynaptic potentials (IPSPs) that are important for the fine-tuning of inhibitory neurotransmission and caused by an increase in K(+) conductance. At presynaptic sites, GABA(BRs) mediate a suppression on the release of neurotransmitters such as of GABA or glutamate by inhibiting voltage-sensitive Ca(2+) channels. The present study aimed to explore the long-term changes of GABA(B) receptor subunits in immature rats after recurrent febrile seizures. Rats were randomly divided into control group and hyperthermia treatment group. The control rats (n = 64) were put into 37 degrees C water for 5 minutes. Rats with hyperthermia treatment were put into 44.8 degrees C water for 5 minutes. If a rat in hyperthermia treatment group showed seizure within 5 min, the rat was taken out of the water as soon as the seizure occurred. Water-immersion was carried out 10 times, once every 2 days. Rats showing 10 seizures (FS(10), n = 64) were studied. Rats exposed to hyperthermia for 10 times without seizure were also studied as hyperthermia-only (H, n = 64) group. Rats showing one seizure at the last time of 10 times of hyperthermia treatment were studied as one-seizure group (FS(1), n = 64). The other rats were studied for other research. The changes of GABA(B)R(1) and GABA(B)R(2) co-localization were detected by double fluorescence;the quantitative alteration of GABA(B)R(1) and GABA(B)R(2) were detected by quantitative RT-PCR; the binding of GABA(B)R(2) to GABA(B)R(1) was detected by immunoprecipitation/Western blot. GABA(B)R(1), GABA(B)R(2), and the binding of GABA(B)R(2) to GABA(B)R(1) decreased after the last febrile seizure in FS(10) group, the expression of GABA(B)R(1) returned to normal in later phase while GABA(B)R(2) and the binding of them did not. Recurrent FS down-regulated the expression of GABA(B)R subunits in a long term.

Developmental changes in the expression of GABA A receptor subunits α1, α2, and α3 in brain stem nuclei of rats

Gamma-aminobutyric acid (GABA) A receptor subunit switching is a suggested postnatal mechanism for changes in GABA transmission from depolarization to hyperpolarization. Previously, we found an apparent switch between GABA A α3 and α1 subunit expression in the rat pre-Bötzinger complex (PBC) on postnatal day (P) 12, a presumed peak critical period of respiratory nuclei development. The present study aimed at determining if GABA A subunit switching occurred in another respiratory nucleus, the ventrolateral subnucleus of the solitary tract nucleus (NTS VL), and in a non-respiratory cuneate nucleus (CN) of P0 to P21 rats. In both nuclei: (1) the expression of GABA A α1 subunit was relatively low at birth but increased with development; (2) that of GABA A α3 was relatively high at birth but declined with age; and (3) GABA A α2 remained relatively low and constant throughout development. However, the specific patterns differed between the two nuclei, but were similar between the NTS VL and the PBC. In the NTS VL, GABA A α1 expression gradually increased from birth and peaked at P12, whereas that in the CN sharply rose from P7 and peaked at P10. GABA A α3 expression had a prominent decrease from P11 to P12 in the NTS VL, whereas that in the CN only gradually declined from P10 to P12. The developmental trends of α1 and α3 in the NTS VL intersected close to P12, whereas those in the CN intersected at P10. Despite differences in timing, GABA A α subunit switching may be a common theme in the brain stem that may mediate different functional properties of GABA transmission.

Expression of gamma‐aminobutyric acid and glutamic acid decarboxylases in rat descending colon and their relation to epithelial differentiation

To detect the expression of gamma-aminobutyric acid (GABA) and glutamic acid decarboxylases (GADs; including two isoforms GAD65 and GAD67) in the epithelial growth zones of the descending colon in rats, and to investigate their relation to epithelial differentiation and proliferation. The expression of GABA and GADs in rat descending colon was investigated by immunofluorescent staining and confocal laser scanning techniques, and goblet cells were further investigated by wheat-germ agglutinin histochemistry. In addition, GAD65 and GAD67 mRNAs were also detected by reverse transcription-polymerase chain reaction. Furthermore, evaluation of cell kinetics in colonic epithelia was conducted by ABC immunostaining using a monoclonal antibody against proliferating cell nuclear antigen (PCNA). Immunoreactive GABA and GADs were distributed in the upper third of the crypts and at the luminal surface in the rat descending colon. Strong staining for GABA and GADs was localized mainly in the cytoplasm of epithelial cells near the neck of the crypts and along the luminal surface. In addition, GABA and GAD65 were also detected at the lamina propria in colonic mucosa. No staining for GABA or GADs was found in goblet cells. GAD65 and GAD67 mRNAs were identified in homogenates of rat descending colon. PCNA labeled nuclei were found in the lower two-thirds of the crypts. The expression of GABA and GADs in the maturation and function zones of rat descending colon suggests that GABA may be involved in the differentiation of colonic epithelial cells.

Expression of gamma-aminobutyric acid B receptor subunits in hypothalamus of male and female developing rats

GABA and its receptors show particular ontogenic distributions in different rat brain areas. Recently, GABA B receptors (GBR) have been described to assemble as heterodimers formed by a GBR1a/b and a GBR2 subunit. Here, the ontogeny of rat GBRs and the pattern of subunit expression in both sexes were determined in the hypothalamus, a critical area for homeostatic regulation. Male and female rats were sacrificed at 1, 4, 12, 20, 28, 38 days of life and at adulthood and hypothalami were removed and frozen. Western blots analysis for GBR1 and GBR2 subunits showed that both were expressed in male and female hypothalamic membranes from day 1 to adulthood. In females, both GBR1a and GBR1b were maximally expressed in newborns and decreased towards adulthood. At birth, expression of GBR1a was significantly higher than GBR1b, while at 38 days, GBR1b was more abundant. In males, GBR1a and GBR1b expression was higher in young animals and decreased gradually showing adult levels between the second and third weeks of age without differences between isoforms. Comparing GBR1 variants levels in hypothalamus between sexes, GBR1a was significantly more abundant in females at birth while at 38 days its expression was higher in males; GBR1b showed no sex differences along development. GBR2 was detected in hypothalami of females and males at all ages; maximum levels were observed at 12 days and adult levels were attained at 38 days, without sex differences. This is the first report on the ontogeny of hypothalamic GABA B receptors in male and female rats, with a particular developmental pattern of subunit and isoform expression and presenting some sex differences.

Consequences of the Evolution of the GABAA Receptor Gene Family

1. This paper reviews the evolution of the family of genes present in mammals and other vertebrates that encode gamma-aminobutyric acid (GABA) type A (GABA(A)) receptors, which are the major inhibitory neurotransmitter receptors in the central nervous system (CNS). In mammals, 16 different polypeptides (alpha1-alpha6, beta1-beta3, gamma1-gamma3, delta, epsilon, pi, and theta) have been identified, using recombinant DNA techniques, each of which is encoded by a distinct gene. The products of these genes assemble in diverse combinations to form a variety of receptor subtypes that have different sensitivities to a number of clinically relevant compounds, such as the benzodiazepines (BZs). 2. Based on a number of chromosomal mapping techniques, the majority of the GABA(A) receptor genes have been localized, in man, in four clusters on chromosomes 4, 5, 15, and the X. Furthermore, the genes that are present within these clusters have a conserved transcriptional orientation. It has, therefore, been proposed that the clusters arose largely as a consequence of two whole-genome doublings that occurred during chordate evolution, and that the ancestral cluster contained an "alpha-like," a "beta-like," and a "gamma-like" subunit gene. 3. Our laboratory has identified two additional GABA(A) receptor polypeptides (the beta4 and gamma4 subunits) in a number of vertebrate species; these do not appear to be present in mammals. We discuss here the relationship of the corresponding genes to other GABA(A) receptor genes, and conclude that their products are orthologous to the mammalian theta and epsilon subunits, respectively. 4. The GABA(A) receptor has a number of binding sites for compounds such as BZs, barbiturates, neurosteroids, and certain volatile anaesthetics. However, the only site at which endogenous compounds are thought to be active is the steroid site; this binds steroids such as certain metabolites of progesterone and deoxycorticosterone, which are synthesized in the periphery and CNS. Since the in vivo functional relevance, if any, of binding sites for other classes of compounds (such as the BZs) is unknown, the significance of differences in primary sequence, between different receptor subunits, is uncertain. We suggest that a possibly more important consequence of gene duplication is that it permitted greater flexibility in the level, pattern and regulation of expression of GABA(A) receptor genes.

Differential expression of GABAA receptor π subunit in cultured rat alveolar epithelial cells

Although type A gamma-aminobutyric acid (GABA) receptors (ligand-gated Cl(-) channels) have been extensively studied in the central nervous system, no information is available on this receptor in lung cells. We have examined the expression of GABA(A) receptor pi-subunit (GABRP) during the trans-differentiation between rat alveolar epithelial type II cells and type I cells. Rat alveolar type II cells, when cultured on plastic plates, gradually trans-differentiated into type-I-like cells and lost their GABRP mRNA expression. However, the GABRP mRNA was partially retained in the type II cells cultured on Matrigel. Keratinocyte growth factor (a mitogen of type II cells) increased GABRP expression. A detached collagen gel maintained the GABRP mRNA to a level close to that of the freshly isolated type II cells. An air-liquid interface culture system, mimicking in vivo conditions in the lung, significantly up-regulated the expression of GABRP mRNA and protein. mRNAs of the GABA(A) receptor alpha1-, alpha3-, beta2-, gamma2-, and gamma3-subunits were also detected in rat type II cells. These results suggest that GABRP expression is differentially regulated by culture substrata, growth factor, detached gel, and an air-apical surface.

Developmental changes in expression of GABAA receptor‐channels in rat intrinsic cardiac ganglion neurones

The effects of gamma-aminobutyric acid (GABA) on the electrophysiological properties of intracardiac neurones were investigated in the intracardiac ganglion plexus in situ and in dissociated neurones from neonatal, juvenile and adult rat hearts. Focal application of GABA evoked a depolarizing, excitatory response in both intact and dissociated intracardiac ganglion neurones. Under voltage clamp, both GABA and muscimol elicited inward currents at -60 mV in a concentration-dependent manner. The fast, desensitizing currents were mimicked by the GABA(A) receptor agonists muscimol and taurine, and inhibited by the GABA(A) receptor antagonists, bicuculline and picrotoxin. The GABA(A0) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methyl phosphonic acid (TPMPA), had no effect on GABA-induced currents, suggesting that GABA(A) receptor-channels mediate the response. The GABA-evoked current amplitude recorded from dissociated neurones was age dependent whereby the peak current density measured at -100 mV was approximately 20 times higher for intracardiac neurones obtained from neonatal rats (P2-5) compared with adult rats (P45-49). The decrease in GABA sensitivity occurred during the first two postnatal weeks and coincides with maturation of the sympathetic innervation of the rat heart. Immunohistochemical staining using antibodies against GABA demonstrate the presence of GABA in the intracardiac ganglion plexus of the neonatal rat heart. Taken together, these results suggest that GABA and taurine may act as modulators of neurotransmission and cardiac function in the developing mammalian intrinsic cardiac nervous system.

Development of the human motor-related thalamic nuclei during the first half of gestation, with special emphasis on GABAergic circuits.

This study analyzed the expression of differentiation markers (Calbindin D28K: CaBP; parvalbumin: PARV; calretinin: CalR), gamma-aminobutyric acid (GABA) markers (GABA, glutamic acid decarboxylases: GAD65, GAD67; and GABA transporters: GAT1, GAT3), and other markers (neurotensin: NT, and neurofilament-specific protein: SMI32) in the human thalamus at 8-23 gestation weeks (g.w.), focusing on the motor-related nuclei. From 8-13 g.w. mainly CaBP was expressed in the cells while fiber bundles traversing the thalamus in addition to CaBP expressed all GABA markers except GAD67. CaBP and PARV expression patterns in different nuclei changed over the time course studied, whereas NT was expressed consistently along the anterior-lateral curvature of the thalamus. CalR and SMI were detectable at 23 g.w. in the ventral parts of the dorsal thalamus. Most remarkably, punctate GAD65 immunoreactivity in the neuropil was confined to the nigro- and pallidothalamic afferent receiving nuclei from 16 to about 21 g.w., overlapping with that of CaBP in some of these nuclei (subdivisions of the ventral anterior and mediodorsal nuclei) and with PARV in others (centromedian nucleus). During this period, GAD65 immunoreactivity can be considered a marker of the basal ganglia afferent receiving territory in the motor thalamus. GAD67-positive local circuit neurons were first detected at 12-13 g.w. in the thalamic nuclei outside the basal ganglia afferent receiving territory. In the ventral anterior and centromedian nuclei, GAD-containing local circuit neurons were not conspicuous even at 22-23 g.w. The cells of the reticular nucleus expressed GAD67 and PARV from 12 g.w. on starting in the lateral-posterior regions. By 23 g.w., both markers were expressed in about two-thirds of the nucleus except for its most medial-anterior part. The results imply spatially and temporally differential expression of GABA and differentiation markers in the developing human thalamus.

Hetero-oligomerization between GABAA and GABAB Receptors Regulates GABAB Receptor Trafficking

The neurotransmitter gamma-aminobutyric acid (GABA) mediates inhibitory signaling in the brain via stimulation of both GABA(A) receptors (GABA(A)R), which are chloride-permeant ion channels, and GABA(B) receptors (GABA(B)R), which signal through coupling to G proteins. Here we report physical interactions between these two different classes of GABA receptor. Association of the GABA(B) receptor 1 (GABA(B)R1) with the GABA(A) receptor gamma2S subunit robustly promotes cell surface expression of GABA(B)R1 in the absence of GABA(B)R2, a closely related GABA(B) receptor that is usually required for efficient trafficking of GABA(B)R1 to the cell surface. The GABA(B)R1/gamma2S complex is not detectably functional when expressed alone, as assessed in both ERK activation assays and physiological analyses in oocytes. However, the gamma2S subunit associates not only with GABA(B)R1 alone but also with the functional GABA(B)R1/GABA(B)R2 heterodimer to markedly enhance GABA(B) receptor internalization in response to agonist stimulation. These findings reveal that the GABA(B)R1/gamma2S interaction results in the regulation of multiple aspects of GABA(B) receptor trafficking, allowing for cross-talk between these two distinct classes of GABA receptor.

Characteristic expression of gamma-aminobutyric acid and glutamate decarboxylase in rat jejunum and its relation to differentiation of epithelial cells.

To investigate the expression between gamma-aminobutyric acid (GABA) and glutamate decarboxylase and its relation with differentiation and maturation of jejunal epithelial cells in rat jejunum. Immunohistochemical expression of GABA and glutamate decarboxylase (GAD, including two isoforms, GAD65 and GAD67) was investigated in rat jejunum. Meanwhile, double staining was performed with GAD65 immunohistochemistry, followed by lectin histochemistry of fluorescent wheat germ agglutinin. Furthermore, evaluation of cell kinetics in jejunum was conducted by (3)H-thymidine autoradiography and immunohistochemistry using a monoclonal antibody to proliferating cell nuclear antigen (PCNA). The cells showing positive immunoreactivity GABA and GAD65 were mainly distributed in the villi in rat jejunum, while jejunal epithelial cells were negative for GAD67. Positive GABA or GAD65 staining was mainly located in the cytoplasm and along the brush border of epithelial cells in the middle and upper portions. In addition, a few GABA and GAD65 strongly positive cells were scattered in the upper two thirds of jejunal villi. Double staining showed that GAD65 immunoreactivity was not found in goblet cells. (3)H-thymidine-labeled nuclei were found in the lower and middle portions of jejunal crypts, which was consistent with PCNA staining. Therefore, GABA and GAD65 were expressed in a maturation or functional zone. The characteristic expression of GABA and GAD suggests that GABA might be involved in regulation of differentiation and maturation of epithelial cells in rat jejunum.

Gamma-amino-butyric acid immunoreactivity in intramucosal colonic tumors.

The level of gamma-amino-butyric acid (GABA) is reported to be increased in colon cancer. Moreover, data suggests that GABA plays a role in the proliferation or maturation of some types of cells. We examined the expression of GABA in intramucosal colonic tumors to clarify the relation between GABA and the degree of atypia. Paraffin sections were prepared from 56 protruded-type colonic neoplasms, which were classified as intramucosal adenocarcinoma (AC), adenoma with severe atypia (ASA), or adenoma with mild to moderate atypia (AMA). Expression of GABA was investigated immunohistochemically, and GABA immunoreactivity was compared to the staining patterns of carcinoembryonic antigen (CEA) and cancer-associated antigen (CA19-9) which were classified into three categories. Intense GABA immunoreactivity was observed in 73.7%, 54.6%, 13.3%, and 5.4% of AC, ASA, AMA, and normal mucosa specimens, respectively. Kendall's correlation coefficient between GABA immunoreactivity and the degree of atypia was 0.447. Strong, positive CEA staining (pattern 3) was observed in 57.9%, 36.3%, and 13.3% of AC, ASA, and AMA specimens, respectively. Strong, positive CA19-9 staining was observed: 26.3%, 9.1% and 0%, respectively. In AC and ASA, the proportion of glands with strong GABA immunoreactivity was greater than the proportion of glands that were strongly positive for CA19-9. GABA may be useful as a tumor marker in combination with other tumor markers such as CEA and CA19-9.

Brain-derived neurotrophic factor mitigates chronic ethanol-induced attenuation of gamma-aminobutyric acid responses in cultured cerebellar granule cells.

This study examined the effect of chronic exposure to ethanol and brain-derived neurotrophic factor (BDNF) on the responsiveness of cerebellar granule cells to gamma-aminobutyric acid (GABA). Cerebellar granule cell cultures were chronically exposed to ethanol (100 mM), BDNF (20 ng/ml), or the combination of ethanol and BDNF. Whole-cell current responses of granule cells to exogenously applied GABA were monitored following at least 5 days of chronic exposure. In the ethanol-treated cultures, granule cell responsiveness to GABA was attenuated. Concomitant exposure of cultures to ethanol and BDNF mitigated the ethanol-induced attenuation of GABA response, although BDNF, by itself, did not affect responsiveness to GABA. BDNF increased the expression of the GABA(A) receptor alpha6 subunit, whereas ethanol had no effect, in chronically treated granule cell cultures. In addition, concomitant treatment with BDNF and ethanol did not increase the expression of the GABA(A) receptor alpha6 subunit, so the subunit expression alone could not account for the mitigating effect of BDNF. We propose that different mechanisms regulating responsiveness to GABA underlie the effects induced by ethanol and BDNF, with the former influencing the expression of functional GABA(A) receptors and the latter involving the activation of the TrkB receptor and its downstream signaling pathways.

Dlx1, Dlx2, Pax6, Brn3b, and Chx10 homeobox gene expression defines the retinal ganglion and inner nuclear layers of the developing and adult mouse retina.

Distal-less homeobox genes are expressed in the developing forebrain. We assessed Dlx gene expression in the developing and adult mouse retina. Dlx1 and Dlx2 are detected in retinal neuroprogenitors by embryonic day (E) 12.5 (Eisenstat et al. [1999] J. Comp. Neurol. 217-237). At E13.5, the expression of four homeodomain proteins, DLX2, BRN3b, PAX6, and CHX10, define distinct yet overlapping domains in the retinal neuroepithelium. By postnatal day (P) 0, DLX2 is expressed in the neuroblastic layer and the ganglion cell layer (GCL) consisting of ganglion and displaced amacrine cells. DLX1 expression resembles DLX2 to P0 but decreases postnatally. In the adult, DLX2 is localized to ganglion, amacrine, and horizontal cells as determined by coexpression with retinal cell-specific markers. There is coincident expression of DLX2 with gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD)65, and GAD67 in the inner nuclear layer (INL) and GCL. In the adult, DLX2 is coexpressed with BRN3b in ganglion cells; PAX6 in amacrine, horizontal, and ganglion cells; and Chx10 in some bipolar cells. We predict that a combinatorial code of these homeobox genes and others specify retinal cell fate. Our results support a possible role for Dlx1 and Dlx2 in inner retinal development and in the terminal differentiation and/or maintenance of INL interneurons and ganglion cells in the adult. The correlation of DLX2 with GABA expression in the mouse retina closely mirrors the relationship of DLX2 to GABAergic neuronal differentiation in the embryonic forebrain, including neocortex, olfactory bulb and hippocampus, signifying a conservation of function of Dlx genes in the developing central nervous system.

In developing Drosophila neurones the production of gamma-amino butyric acid is tightly regulated downstream of glutamate decarboxylase translation and can be influenced by calcium.

The presented work pioneers the embryonic Drosophila CNS for studies of the developmental regulation and function of gamma-amino butyric acid (GABA). We describe for the first time the developmental pattern of GABA in Drosophila and address underlying regulatory mechanisms. Surprisingly, and in contrast to vertebrates, detectable levels of GABA occur late during Drosophila neurogenesis, after essential neuronal proliferation and growth have taken place and synaptogenesis has been initiated. This timeline is almost unchanged when the GABA synthetase glutamate decarboxylase (GAD) is strongly misexpressed throughout the nervous system suggesting a tight post-translational regulation of GABA expression. We confirmed such GABA control mechanisms in an independent model system, i.e. primary Drosophila cell cultures raised in elevated [K+]. The data suggest that, in both systems, GABA suppression occurs via control of GAD activity. Using developing embryos and cell cultures as parallel assay systems for pharmacological and genetic studies we show that the negative regulation of GAD can be overridden by drugs known to elevate intracellular free [Ca2+]. Our results provide the basis for investigations of genetic mechanisms underlying the observed phenomenon, and we discuss the potential implications of this work for Drosophila neurogenesis but also for a general understanding of GAD regulation.

Differential Expression of GABA and Glutamate‐receptor Subunits and Enzymes Involved in GABA Metabolism between Electrophysiologically Identified Hippocampal CA1 Pyramidal Cells and Interneurons

The balance between synaptic excitation and inhibition within the hippocampus is critical for maintaining normal hippocampal function. Even mild reduction in inhibition or enhancement of excitation can produce seizures. Synaptic excitation is produced by pyramidal cells and granule cells, whereas inhibition is produced by a smaller number of interneurons. To understand how two subpopulations of these excitatory and inhibitory neurons are regulated at the molecular level, we analyzed specific mRNA expression profiles for receptors that are significantly involved in synaptic transmission and in the synthesis and storage of the principal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). Our hypothesis was that differences in gene expression between inhibitory and excitatory neurons in the rat hippocampus might point to specific new targets for seizure pharmacotherapy. We combined the techniques of (a) whole-cell patch clamping in rat hippocampal slices, (b) biocytin staining for cell identification, (c) single-cell mRNA amplification, and (d) small-scale cDNA microarray analysis to allow us to obtain expression profiles for candidate genes from identified CA1 pyramidal neurons and interneurons. Electrophysiologic and morphologic data and expression profiles were obtained from 12 stratum pyramidale and seven stratum radiatum cells. Presumed inhibitory neurons expressed significantly more GAD65, GAD67, vGAT, GABA(A)-receptor alpha3, and N-methyl-d-aspartate (NMDA)-receptor IIB mRNA, and presumed excitatory neurons expressed more GABA(A)-receptor alpha1, and NMDA-receptor I mRNA. Differential expression of candidate neurotransmitter-receptor subunits distinguished CA1 pyramidal neurons from interneurons. These differences may indicate potential new targets for altering the balance of inhibition and excitation in the treatment of epilepsy.