A Possible Role of the Non-GAT1 GABA Transporters in Transfer of GABA From GABAergic to Glutamatergic Neurons in Mouse Cerebellar Neuronal Cultures

Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, β-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC₅₀ 142 μM), β-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC₅₀ 0.8 μM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 μM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of β-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also GAT2 or GAT3 (HUGO nomenclature) could play a role.     

猫腰髓中GABA能神经元的分布及年龄相关变化

目的观察和比较GABA能神经元在青年猫和老年猫L6段脊髓的分布,探讨GABA能神经元在脊髓中分布的年龄相关变化及意义.方法免疫组织化学ABC法.结果青年猫与老年猫L6段脊髓灰质内,GABA能神经元及神经纤维分布广泛,各个Rexed板层均可见GABA-IR细胞,其中背侧灰质阳性最强,其次是腹侧灰质.标记的GABA能神经元胞体为卵圆形、三角形、多角形和星形,可分为大、中、小三种类型.经比较,老年组GABA能神经元的数量及免疫反应性均明显低于青年组.结论老年动物脊髓调节功能的减弱可能与GABA能神经元减少有少.     

Antisera to gamma-aminobutyric acid. II. Immunocytochemical application to the central nervous system

An antiserum to gamma-aminobutyric acid (GABA) was tested for the localization of GABAergic neurons in the central nervous system using the unlabeled antibody enzyme method under pre- and postembedding conditions. GABA immunostaining was compared with glutamate decarboxylase (GAD) immunoreactivity in the cerebellar cortex and in normal and colchicine-injected neocortex and hippocampus of cat. The types, distribution, and proportion of neurons and nerve terminals stained with either sera showed good agreement in all areas. Colchicine treatment had little effect on the density of GABA-immunoreactive cells but increased the number of GAD-positive cells to the level of GABA-positive neurons in normal tissue. GABA immunoreactivity was abolished by solid phase adsorption to GABA and it was attenuated by adsorption to beta-alanine or gamma-amino-beta-hydroxybutyric acid, but without selective loss of immunostaining. Reactivity was not affected by adsorption to glutamate, aspartate, taurine, glycine, cholecystokinin, or bovine serum albumin. The concentration (0.05-2.5%) of glutaraldehyde in the fixative was not critical. The antiserum allows the demonstration of immunoreactive GABA in neurons containing other neuroactive substances; cholecystokinin and GABA immunoreactivities have been shown in the same neurons of the hippocampus. In conclusion, antisera to GABA are good markers for the localization of GABAergic neuronal circuits.     

Disruption of postsynaptic GABA receptor clusters leads to decreased GABAergic innervation of pyramidal neurons

We have used RNA interference (RNAi) to knock down the expression of the gamma2 subunit of the GABA(A) receptors (GABA(A)Rs) in pyramidal neurons in culture and in the intact brain. Two hairpin small interference RNAs (shRNAs) for the gamma2 subunit, one targeting the coding region and the other one the 3'-untranslated region (UTR) of the gamma2 mRNA, when introduced into cultured rat hippocampal pyramidal neurons, efficiently inhibited the synthesis of the GABA(A) receptor gamma2 subunit and the clustering of other GABA(A)R subunits and gephyrin in these cells. More significantly, this effect was accompanied by a reduction of the GABAergic innervation that these neurons received. In contrast, the gamma2 shRNAs had no effect on the clustering of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, postsynaptic density protein 95 (PSD-95) or presynaptic glutamatergic innervation. A gamma2-enhanced green fluorescent protein (EGFP) subunit construct, whose mRNA did not contain the 3'-UTR targeted by gamma2 RNAi, rescued both the postsynaptic clustering of GABA(A)Rs and the GABAergic innervation. Decreased GABA(A)R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex was also observed after in utero transfection of these neurons with the gamma2 shRNAs. The results indicate that the postsynaptic clustering of GABA(A)Rs in pyramidal neurons is involved in the stabilization of the presynaptic GABAergic contacts.     

GABA Neurons in the Ventral Tegmental Area Responding to Peripheral Sensory Input

Dopamine (DA) neurons in the ventral tegmental area (VTA) not only participate in reward processing, but also respond to aversive stimuli. Although GABA neurons in this area are actively involved in regulating the firing of DA neurons, few data exist concerning the responses of these neurons to aversive sensory input. In this study, by employing extracellular single-unit recording and spectral analysis techniques in paralyzed and ventilated rats, we found that the firing pattern in 44% (47 of 106) of GABA cells in the VTA was sensitive to the sensory input produced by the ventilation, showing a significant ventilation-associated oscillation in the power spectra. Detailed studies revealed that most ventilation-sensitive GABA neurons (38 of 47) were excited by the stimuli, whereas most ventilation-sensitive DA neurons (11 of 14) were inhibited. When the animals were under anesthesia or the sensory pathways were transected, the ventilation-associated oscillation failed to appear. Systemic administration of non-competitive N-methyl-D-aspartase (NMDA) receptor antagonist MK-801 completely disrupted the association between the firing of GABA neurons and the ventilation. Interestingly, local MK-801 injection into the VTA dramatically enhanced the sensitivity of GABA neurons to the ventilation. Our data demonstrate that both GABA and DA neurons in the VTA can be significantly modulated by sensory input produced by the ventilation, which may indicate potential functional roles of VTA in processing sensation-related input.     

γ-Aminobutyric acid type B (GABAB) receptor expression is needed for inhibition of N-type (Cav2.2) calcium channels by analgesic α-conotoxins

α-Conotoxins Vc1.1 and RgIA are small peptides isolated from the venom of marine cone snails. They have effective anti-nociceptive actions in rat models of neuropathic pain. Pharmacological studies in rodent dorsal root ganglion (DRG) show their analgesic effect is mediated by inhibition of N-type (Ca(v)2.2) calcium channels via a pathway involving γ-aminobutyric acid type B (GABA(B)) receptor. However, there is no direct demonstration that functional GABA(B) receptors are needed for inhibition of the Ca(v)2.2 channel by analgesic α-conotoxins. This study examined the effect of the GABA(B) agonist baclofen and α-conotoxins Vc1.1 and RgIA on calcium channel currents after transient knockdown of the GABA(B) receptor using RNA interference. Isolated rat DRG neurons were transfected with small interfering RNAs (siRNA) targeting GABA(B) subunits R1 and R2. Efficient knockdown of GABA(B) receptor expression at mRNA and protein levels was confirmed by quantitative real time PCR (qRT-PCR) and immunocytochemical analysis, respectively. Whole-cell patch clamp recordings conducted 2-4 days after transfection showed that inhibition of N-type calcium channels in response to baclofen, Vc1.1 and RgIA was significantly reduced in GABA(B) receptor knockdown DRG neurons. In contrast, neurons transfected with a scrambled nontargeting siRNA were indistinguishable from untransfected neurons. In the HEK 293 cell heterologous expression system, Vc1.1 and RgIA inhibition of Ca(v)2.2 channels needed functional expression of both human GABA(B) receptor subunits. Together, these results confirm that GABA(B) receptors must be activated for the modulation of N-type (Ca(v)2.2) calcium channels by analgesic α-conotoxins Vc1.1 and RgIA.     

咖啡因对大鼠背根神经节急性分离神经元GABA-激活电流的抑制作用

应用全细胞膜片钳记录技术,在大鼠新鲜分离背根神经节(dorsal root ganglion,DRG)神经元上,观察预加咖啡因对GABA-激活电流(IGABA)的调制作用。实验中,大部分受检细胞(97．4％,l13／116)对外加GABA敏感。1-1000μmol／L GABA引起一剂量依赖性、有明显上敏感作用的内向电流。在受检的108个DRG细胞中,约有半数(53．7％,58／108)对胞外加咖啡因(0．1-100μmol／L)敏感．产生一幅值很小的内向电流。倾加咖啡因(0．1～100μmol／L)30s后再加GABA能明显抑制GABA(100μmol／L)激活电流的幅值。预加咖啡因后GABA量效曲线明显下移;GABA-激活电流的最人值较之对照下降约57％;而Kd值(30μmol／L)几乎不变,表示此种抑制为非竞争性的。预加安定(diazepam,1μmol／L)对GABA(100μmol／L)激活电流有增强作用,而预加咖啡因(10μmol／L)有拈抗安定增强IGABA的作用。胞内透析H-8后,几乎可以完全消除咖啡因对,IGABA的抑制作用。已知GABA作用于初级感觉神经元能引起初级传入去极化,因而实验结果提示,咖啡因有可能在初级传入末梢产生对抗突触前抑制的效应。     

Ethanol potentiates the effect of gamma-aminobutyric acid on medial vestibular nucleus neurons responding to horizontal rotation

Electrophysiological studies were performed to determine whether or not ethanol potentiates the inhibitory effects of gamma-aminobutyric acid (GABA) on medial vestibular nucleus (MVN) neurons responding to horizontal sinusoidal rotation using alpha-chloralose anesthetized cats. The MVN neurons were classified into types I, II, III and IV neurons according to the responses to the horizontal rotation of the animal placed on the turntable in directions ipsilateral and contralateral to the recording site. In addition, the effects of ethanol and GABA on type I neurons were also examined. Micro-osmotic application of ethanol up to 100 nA did not affect the spontaneous firing or the rotation-induced increase in firing of type I neurons. However, the inhibitory effects of GABA up to 50 nA on the rotation-induced increase in firing were potentiated during simultaneous application of ethanol up to 100 nA. This potentiated inhibition was blocked by iontophoretic application of bicuculline (25-150 nA) and picrotoxin (45-150 nA). These results suggest that ethanol potentiates the inhibitory effects of GABA on MVN type I neurons by acting on the GABA receptor and/or receptor-coupled chloride ion channel.     

Drosophila pacemaker neurons require g protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms

Intercellular signaling is important for accurate circadian rhythms. In Drosophila, the small ventral lateral neurons (s-LN(v)s) are the dominant pacemaker neurons and set the pace of most other clock neurons in constant darkness. Here we show that two distinct G protein signaling pathways are required in LN(v)s for 24?hr rhythms. Reducing signaling in LN(v)s via the G alpha subunit Gs, which signals via cAMP, or via the G alpha subunit Go, which we show signals via Phospholipase 21c, lengthens the period of behavioral rhythms. In contrast, constitutive Gs or Go signaling makes most flies arrhythmic. Using dissociated LN(v)s in culture, we found that Go and the metabotropic GABA(B)-R3 receptor are required for the inhibitory effects of GABA on LN(v)s and that reduced GABA(B)-R3 expression in?vivo lengthens period. Although no clock neurons produce GABA, hyperexciting GABAergic neurons disrupts behavioral rhythms and s-LN(v) molecular clocks. Therefore, s-LN(v)s require GABAergic inputs for 24?hr rhythms.     

猫小脑皮质GABA能神经元年龄相关性变化

为探讨青年猫和老年猫小脑皮质GABA能神经元及其表达的年龄相关性变化,利用Nissl染色显示小脑皮质结构及神经元,免疫组织化学ABC法标记GABA免疫阳性神经元。光镜下观察,采集图像,并利用图像分析软件对分子层、蒲肯野细胞层和颗粒层神经元及GABA免疫阳性神经元及其灰度值进行分析统计。结果显示,GABA免疫阳性神经元、阳性纤维及终末在青年猫和老年猫小脑皮质各层均有分布。与青年猫相比,老年猫分子层、蒲肯野细胞层神经元和GABA免疫阳性神经元密度及其GABA免疫阳性反应强度均显著下降(P<0.01),颗粒层神经元密度和GABA免疫阳性强度也显著下降(P<0.01),但其GABA免疫阳性神经元密度无显著变化(P>0.05);蒲肯野细胞的胞体萎缩,阳性树突分枝减少。因此认为,衰老过程中猫小脑皮质GABA能神经元的丢失和GABA表达的下降,可能是老年个体运动协调、精确调速和运动学习等能力下降的重要原因之一。     

Physiological and pharmacological properties of responses to GABA and ACh by abdominal motor neurons in Manduca sexta

1. GABA, ACh, and other agents were applied by pressure ejection to the neuropil of the third abdominal ganglion in the isolated nerve cord of Manduca sexta. Intersegmental muscle motor neurons with dendritic arborizations in the same hemiganglion were inhibited by GABA (Fig. 2) and excited by ACh (Fig. 5).
2. Picrotoxin was a potent antagonist of GABA (Fig. 4A). Bicuculline reduced GABA responses in some motor neurons (Fig. 4C), but had no effect on many other motor neurons. Curare reduced ACh responses (Fig. 6A). Bicuculline was an effective ACh antagonist in most motor neurons tested (Fig. 6B).
3. Motor neurons with dendrites across the ganglion from the ejection pipette exhibited different responses to GABA and ACh. Contralateral motor neurons often showed smaller, delayed hyperpolarizing GABA responses (Fig. 7). On two occasions, contralateral motor neurons had excitatory responses (Fig. 8). Contralateral motor neurons were hyperpolarized by ACh (Fig. 9). The inhibitory responses had only slightly longer latencies than ipsilateral excitatory ACh responses (Fig. 10A). The contralateral inhibitory ACh responses, but not the ipsilateral excitatory ACh responses, were eliminated by TTX (Fig. 10B).
4. A model, which includes inhibitory interneurons that cross the ganglionic midline to inhibit their contralateral homologs and motor neurons (Fig. 11), is proposed to account for contralateral responses to GABA and ACh and antagonistic patterns of activity of motor neurons during mechanosensory reflex responses.

     

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

Summary The localization of -aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [³H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [³H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.     

Concurrent acetylcholinesterase staining and gamma-aminobutyric acid uptake of cortical neurons in culture

Gamma-aminobutyric acid (GABA) uptake and acetylcholinesterase (AChE) content were demonstrated concurrently in cortical neurons grown in tissue culture. Positive reactions either for GABA uptake or for AChE content were encountered in pyramidal and stellate, as well as spindle-shaped neurons. Neither reaction was confined to a specific morphological subtype. Nearly half the neurons were negative for either reaction. Most of the remaining neurons were positive only for GABA or only for AChE. However, a subpopulation of neurons showed not only a high AChE content, but also an avid GABA uptake. Thus, four types of neurons could be identified on the basis of these two reactions. The high AChE content in some of the cortical neurons that also showed GABA uptake indicates that there are at least two distinct types of GABAergic neurons.     

γ-Aminobutyric acid system in isolated dorsal and ventral horn neurons from bovine spinal cord

In order to search for the relationship between the structure and the function of the nervous system, the spinal cord provides suitable material. We devised a procedure for isolation of large ventral and small dorsal horn neurons. Then we examined the -aminobutyric acid (GABA) system in both neurons isolated using our procedure. Glutamic decarboxylase (GAD) activity in dorsal horn neurons was much higher than that in ventral horn neurons. Further, GABA uptake activity by the dorsal horn neurons was also somewhat higher than that by the ventral horn neurons, although some properties of GABA uptake were found to be almost the same in both neuronal fractions. However, we could not find any difference of GABA--ketoglutarate transaminase (GABA-T) activity between the dorsal and ventral horn neurons. These results suggest that GAD and GABA uptake may be indicators for cell specificity to some extent.     

Neuronal and glial localization of GABA transporter immunoreactivity in the myenteric plexus

The neurotransmitter gamma-aminobutyric acid (GABA) is removed from the extracellular space by sodium and chloride dependent high affinity plasma membrane transporters. In the rat central nervous system, three GABA transporters, GAT1, GAT2 and GAT3, have been cloned and localized by immunohistochemistry. The purpose of this study was to examine the distribution of these transporters within the myenteric plexus of the rat gastrointestinal tract. We investigated their cellular locations using GAT1-3 specific antisera in lightly fixed segments of rat duodenum, ileum and colon. Immunohistochemistry revealed a large number of GAT2-immunoreactive structures that surrounded neurons within each ganglion of the myenteric plexus. GAT2 was colocalized in these structures with the glial cell marker p75(NTR), suggesting that the predominant high affinity GABA transporter within enteric glia is GAT2. GAT3 immunoreactivity was localized within many nerve cell bodies, and no labeling for GAT1 was detected, although it was present in retina, which was used as a control. Double labeling for calretinin and nitric oxide synthase (NOS) revealed colocalization of GAT3 with approximately 75% of calretinin-immunoreactive neurons and 15% of NOS-immunoreactive neurons. This suggests that a small proportion of inhibitory motor neurons and at least some putative intrinsic primary afferent neurons within the rat gastrointestinal tract express GAT3. Thus NOS neurons, which appear to utilize GABA as a transmitter, and calretinin-immunoreactive neurons, which do not appear to be GABAergic, both express immunoreactivity for GABA transporters.     

A non-peptidyl neurotrophic small molecule for midbrain dopaminergic neurons

Abstract A small organic molecule (CUR-162590) that selectively enhances survival of midbrain dopaminergic neurons was identified by screening small molecule compound libraries. In embryonic midbrain cultures, CUR-162590 increased dopamine uptake and the number of dopaminergic neurons without altering the number of total neurons or astroglia or the uptake of GABA or serotonin. CUR-162590 reduced apoptosis of cultured dopaminergic neurons and protected against death induced by toxins such as MPP(+). Several synthetic analogs of CUR-162590 also had similar bioactivities. CUR-162590 thus represents a new class of neurotrophic small molecules that may have utility in the treatment of Parkinson's disease, which is marked by degeneration of midbrain dopaminergic neurons.     

Structures with GABA-like and GAD-like immunoreactivity in the cervical sympathetic ganglion complex of adult rats

Summary The distribution of gamma-aminobutyric acid (GABA)-like and glutamate decarboxylase (GAD)-like immunoreactivity was studied in the cervical sympathetic ganglion complex of rats, including the intermediate and inferior cervical ganglia and the uppermost thoracic ganglion. GABA-positive axons may enter the ganglion complex via its caudal end. Others apparently arise from small GABA-positive cell bodies which are scattered among principal neurons, within clusters of SIF cells and in bundles of GABA-negative axons. The majority of these cells is located in the lower half of the ganglion complex. Principal neurons did not react with antibodies against GABA or GAD. An unevenly distributed meshwork of GABA-immunoreactive axons was seen in each of the ganglia. Immunoreactive axons formed numerous varicosities. Some of them were aggregated in a basket-like form around a subpopulation of GABA-negative principal ganglion cell bodies. Electron-microscopic immunocytochemistry revealed that GABA-positive nerve fibers establish asymmetric synaptic junctions with dendritic and somatic spines of principal neurons, whereas postsynaptic densities are inconspicous or absent on dendritic shafts and somata. The results suggest that in the cervical sympathetic ganglion complex principal neurons are not GABAergic, but are innervated by axons which react with both antibodies against GAD and/ or GABA antibodies and originate from a subpopulation of small neurons.     

Maitotoxin-Evoked γ-Aminobutyric Acid Release Is Due Not Only to the Opening of Calcium Channels

The effects of maitotoxin (MTX) on endogenous amino acid release were tested on highly purified striatal neurons differentiated in primary culture. MTX induced a large and concentration-dependent release of gamma-aminobutyric acid (GABA). This effect was abolished when experiments were performed in the absence of external Ca2+, and restored when Ca2+ ions were added after removing the MTX-containing Ca2+-free solution. MTX-induced amino acid release was not affected by 1 microM nifedipine and only slightly inhibited by 1 mM Co2+. MTX also induced a massive accumulation of 45Ca2+ in the neurons which, in contrast to the MTX-evoked GABA release, was totally blocked in the presence of 1 mM Co2+. Whereas 500 nM tetrodotoxin was without significant effect, MTX-evoked GABA release was dependent on the presence of external Na+ and sensitive to nipecotic acid, a GABA uptake inhibitor. It is concluded that, on striatal neurons, MTX induced Na+ influx only in the presence of external Ca2+. The increase in cytoplasmic Na+ ions then triggers the release of GABA.     

Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system

Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.     

Distinct patterns of expression and regulation of GABA receptors containing the delta subunit in cerebellar granule and hippocampal neurons

Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter receptors such as the type A receptor (GABA(A)R) for gamma-aminobutyric acid. We now show that two different rat neuronal populations in culture manifest distinct patterns of GABA(A)R plasticity in response to identical stimuli. Whereas prolonged exposure to ethanol had no effect on expression of the delta subunit of GABA(A)Rs at the mRNA or protein level in cerebellar granule neurons, it increased the abundance of delta subunit mRNA and protein in hippocampal neurons. Subsequent ethanol withdrawal transiently down-regulated delta subunit expression in cerebellar granule neurons and gradually normalized that in hippocampal neurons. These effects of ethanol exposure and withdrawal were accompanied by corresponding functional changes in GABA(A)Rs. GABA(A)Rs containing the delta subunit were also distributed differentially in the cerebellar and hippocampal neurons. These findings reveal complex and distinct mechanisms of regulation of the expression of GABA(A)Rs that contain the delta subunit in different neuronal types.