Recovery from monocular deprivation in the monkey. II. Reversal of morphological effects in the lateral geniculate nucleus

The cross-sectional area was measured of neurons in the lateral geniculate nucleus (l.g.n.) of monkeys (Erythrocebus patas) subjected to monocular deprivation by unilateral eyelid suture, and of others in which the closed lids had been subsequently opened (either alone, "reopening', or together with closure of the previously open eye, "reverse suture'). Monocular deprivation for the first month of the monkey's life retards l.g.n. cell growth such that neurons in the laminae innervated by the closed eye are about 15% smaller in cross-sectional area than those in normally innervated laminae. This failure of normal growth can be countered by reverse suture for even short periods of time, the size difference between laminae being abolished within 6 days after reverse suture performed at the age of 1 month. Simply reopening the closed eye has little or no effect on l.g.n. neuronal recovery. These morphological results in the l.g.n. correlate closely with studies on the width of ocular dominance "stripes' in layer IVc of the visual cortex of the same animals: the stripes, narrower than normal after monocular deprivation, "expand' with a time course similar to that of l.g.n. cell recovery, as judged by single unit recording and by autoradiography in the cortex after transneuronal transport of labelled tracers injected in an eye.     

Cellular distribution ofl-glutamate decarboxylase (GAD) andγ-aminobutyric acidA (GABAA) receptor mRNAs in the retina

1. Gamma-aminobutryic acid (GABA), a major inhibitory transmitter of the vertebrate retina, is synthesized from glutamate by L-glutamate decarboxylase (GAD) and mediates neuronal inhibition at GABAA receptors. GAD consists of two distinct molecular forms, GAD65 and GAD67, which have similar distribution patterns in the nervous system (Feldblum et al., 1990; Erlander and Tobin, 1991). GABAA receptors are composed of several distinct polypeptide subunits, of which the GABAA alpha 1 variant has a particularly extensive and widespread distribution in the nervous system. The aim of this study was to determine the cellular localization patterns of GAD and GABAA alpha 1 receptor mRNAs to define GABA- and GABAA receptor-synthesizing neurons in the rat retina. 2. GAD and GABAA alpha 1 mRNAs were localized in retinal neurons by in situ hybridization histochemistry with 35S-labeled antisense RNA probes complementary to GAD67 and GABAA alpha 1 mRNAs. 3. The majority of neurons expressing GAD67 mRNA is located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL). Occasional GAD67 mRNA-containing neurons are present in the inner plexiform layer. Labeled neurons are not found in the distal INL or in the outer nuclear layer (ONL). 4. GABAA alpha 1 mRNA is expressed by neurons distributed to all regions of the INL. Some discretely labeled cells are present in the GCL. Labeled cells are not observed in the ONL. 5. The distribution of GAD67 mRNA demonstrates that numerous amacrine cells (conventional, interstitial, and displaced) and perhaps interplexiform cells synthesize GABA. These cells are likely to employ GABA as a neurotransmitter. 6. The distribution of GABAA alpha 1 mRNA indicates that bipolar, amacrine, and perhaps ganglion cells express GABAA receptors having an alpha 1 polypeptide subunit, suggesting that GABA acts directly upon these cells.     

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。     

Chronic antidepressants and GABA "B" receptors: a GABA hypothesis of antidepressant drug action

Amitryptyline (10 mg/kg), desipramine (5 mg/kg), citalopram (10 mg/kg) and viloxazine (10 mg/kg) were administered to rats either acutely (decapitation 1 hr after i.p. injection) or subacutely (by subcutaneous minipump implantation for 18 days followed by decapitation 24 hr after removal). After acute administration there was not any consistent alteration in GABA levels, GAD activity, 3H GABA "A" or 3H-GABA "B" receptor binding or 3H-nipecotic acid binding to the recognition site for GABA uptake in the frontal cortex or hippocampus. Upon subacute antidepressant drug infusion, GABA levels, GAD activity and 3H-GABA-"A" binding showed only scattered differences in drug treated animals as compared to saline treated rats. However, 3H-GABA "B" binding in the frontal cortex was consistently elevated after all drug treatments (in % of control: amitryptyline = 155%; desipramine = 151%; citalopram = 173%; viloxazine = 189%). Scatchard analysis showed that this was due to a Bmax increase without an effect in Kd. These findings were reproduced by subacute administration of pargyline, a MAO inhibitor. These data suggest that GABA "B" receptors may be involved in the mechanism of action of antidepressant drugs and provide a link between GABAergic and monoaminergic hypotheses of depression.     

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.     

Activation of a wide-spread network of inhibitory neurons in barrel cortex

Abstract The one-to-one correspondence of whiskers to barrels in layer IV of rodent somatosensory cortex can be demonstrated by a precise match between columns of heavy 2-deoxyglucose (2DG) label in layer IV barrels and other layers which correspond to stimulated whiskers. While there is specificity of peripheral-to-central mapping, the extent to which integration and/or modulation are generated by circuitry within or interactions between the barrel-defined whisker columns is not clear. Following stimulation of selected whiskers, large cells at the layer IV-V boundary throughout the barrel field are heavily labeled by 2-deoxyglucose (2DG) at high resolution. Many of these cells are outside the barrel columns of the stimulated whiskers. Further, the number of cells labeled is not directly related to the number of activated barrel columns. These neurons are not labeled in animals anesthetized before 2DG injection and are not as heavily labeled in barrel fields of somnolent animals. Most of the heavily labeled neurons immunolabel for glutamate decarboxylase (GAD) and are presumed to be inhibitory, while a smaller number of labeled neurons, presumed to be excitatory, immunolabel for glutamate (Glu). Similar populations of large, heavily 2DG-labeled neurons are found in other cortical areas. These relatively few neurons are exceptionally active and may modulate integrative functions of cerebral cortex.     

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.     

Neonatal monosodium glutamate treatment modifies glutamic acid decarboxylase activity during rat brain postnatal development

Monosodium glutamate (MSG) produces neurodegeneration in several brain regions when it is administered to neonatal rats. From an early embryonic age to adulthood, GABA neurons appear to have functional glutamatergic receptors, which could convert them in an important target for excitotoxic neurodegeneration. Changes in the activity of the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD), have been shown after different neuronal insults. Therefore, this work evaluates the effect of neonatal MSG treatment on GAD activity and kinetics in the cerebral cortex, striatum, hippocampus and cerebellum of the rat brain during postnatal development. Neonatal MSG treatment decreased GAD activity in the cerebral cortex at 21 and 60 postnatal days (PD), mainly due to a reduction in the enzyme affinity (K(m)). In striatum, the GAD activity and the enzyme maximum velocity (V(max)) were increased at PD 60 after neonatal MSG treatment. Finally, in the hippocampus and cerebellum, the GAD activity and V(max) were increased, but the K(m) was found to be lower in the experimental group. The results could be related to compensatory mechanisms from the surviving GABAergic neurons, and suggest a putative adjustment in the GAD isoform expression throughout the development of the postnatal brain, since this enzyme is regulated by the synaptic activity under physiological and/or pathophysiological conditions.     

Neurotransmitter synthesis in poststroke cortical neurogenesis in adult rats

Neurogenesis occurs in the cerebral cortex of adult rats after focal cerebral ischemia. Whether or not the newborn neurons could synthesize neurotransmitters is unknown. To elucidate such a possibility, a photothrombotic ring stroke model with spontaneous reperfusion was induced in adult male Wistar rats. The DNA duplication marker BrdU was repeatedly injected, and the rats were sacrificed at various times after stroke. To detect BrdU nuclear incorporation and various neurotransmitters, brain sections were processed for single/double immunocytochemistry and single/double/triple immunofluorescence. Stereological cell counting was performed to assess the final cell populations. At 48 h, 5 days, 7 days, 30 days, 60 days and 90 days after stroke, numerous cells were BrdU-immunolabeled in the penumbral cortex. Some of these were doubly immunopositive to the cholinergic neuron-specific marker ChAT or GABAergic neuron-specific marker GAD. As analyzed by 3-D confocal microscopy, the neurotransmitters acetylcholine and GABA were colocalized with BrdU in the same cortical cells. In addition, GABA was colocalized with the neuron-specific marker Neu N in the BrdU triple-immunolabeled cortical cells. This study suggests that the newborn neurons are capable of synthesizing the neurotransmitters acetylcholine and GABA in the penumbral cortex, which is one of the fundamental requisites for these neurons to function in the poststroke recovery.     

Immunocytochemistry of GABA and glutamic acid decarboxylase in the thoracic ganglion of the crab Eriphia spinifrons

We have used specific antisera against protein-conjugated -aminobutyric acid (GABA) and rat-brain glutamic acid decarboxylase (GAD) in immunocytochemical preparations to study the distribution of putatively GABAergic neurons in the fused thoracic ganglion of the crab Eriphia spinifrons. In the thoracic neuromeres, about 2000 neurons with somata arranged in clusters or located singly in the cell cortex exhibited both GABA-like and GAD-like immunoreactivity. In addition, more than a hundred cells showed only GABA-like immunoreactivity. Fibrous immunoreactive staining to GAD and GABA was distributed throughout the neuropil of the thoracic ganglion, and several fiber tracts contained immunoreactive processes. Sets of serially homologous neurons exhibited GABA-like and GAD-like immunoreactivity in the thoracic neuromeres. Especially prominent were one medial and four ventro-lateral clusters of somata, together with thirteen individually recognized cells in each neuromere. Six of these cells in the ventro-medial cell cortex may be the somata of inhibitory motoneurons. The leg nerves contained three immunoreactive fibers, corresponding to the previously described common inhibitory motoneuron and the two specific inhibitors. The results present further evidence for GABA being the neurotransmitter of all inhibitory leg motorneurons, and suggest its presence and role as a neurotransmitter in a considerable number of interneurons in the thoracic ganglion of the crab.     

Glutamic acid decarboxylase of embryonic avian retina cells in culture: Regulation byγ-aminobutyric acid (GABA)

1. Retina-cell aggregate cultures expressed glutamate decarboxylase activity (L-glutamate 1-carboxylase; EC 4.1.1.15) as a function of culture differentiation. 2. Glutamic acid decarboxylase (GAD) activity was low in the initial phases of culture and increased eight-fold until culture day 7, remaining high up to day 13 (last stage studied). 3. The addition of GABA to the culture medium 24 h after cell seeding almost totally prevented the expression of GAD activity. 4. In association with decreased enzyme activity, aggregates exposed to GABA did not display immunoreactivity for GAD, suggesting that GAD molecules were either lost from GABAergic neurons or significantly altered with GABA treatment. 5. Control, untreated aggregates showed intense GAD immunoreactivity in neurons. Positive cell bodies were characterized by a thin rim of labeled cytoplasm with thickest labeling at the emergence of the main neurite. 6. Heavily labeled patches were also observed throughout the aggregates, possibly reflecting regions enriched in neurites. 7. The GABA-mediated reduction of GAD immunoreactivity was a reversible phenomenon and could be prevented by picrotoxin.     

Recovery from monocular deprivation in the monkey. I. Reversal of physiological effects in the visual cortex

This is a study of the effects of monocular deprivation, reverse suturing (opening the deprived eye with closure of the other) and reopening of the deprived eye alone (without closing the other) on the physiological organization of the primary visual cortex in monkeys (Erythrocebus patas). All animals were initially monocularly deprived by suture of the lids of the right eye from soon after birth until about 4 weeks of age (24-29 days). In a monocularly deprived animal, recordings were taken from area 17 at 24 days. Already most neurons recorded outside layer IVc, were strongly or completely dominated by functional input from the left eye. The Non-oriented cells of layer IVc, where the bulk of the afferent input terminates, were also mainly dominated by the left eye. Although segregation of input from the two eyes was not complete, large areas of layer IVc were already monocularly dominated by the left eye. Four animals were reverse-sutured at about 4 weeks and recorded 3, 6, 15 and 126 days later. In each animal the pattern of ocular dominance was fairly similar within and outside layer IVc. Even with only 3 days of forced usage of the initially deprived right eye, about half of all cells recorded had become dominated by it, and the process of "recapture' of cortical cells by the initially deprived eye was apparently complete within 15 days. In layer IVc, the recovery took the form of an expansion of zones dominated by the deprived eye, as if the originally shrunken stripes of afferent termination had become enlarged. Binocularly driven neurons were rare at all stages, in all layers, but when present and orientation-selective, they had similar preferred orientations in the two eyes. Likewise the "columnar' sequences of preferred orientation continued without obvious disruption on shifting from regions dominated by one eye to those dominated by the other. Simply reopening the deprived eye at about 4 weeks, for 15 to 96 days caused no detectable change in the overall ocular dominance of cortical cells and, on average, no expansion of right-eye dominance columns in layer IVc. Therefore the recovery seen after reverse suturing depends not just on the restoration of normal activity to axons carrying information from the right eye, but on the establishment of a competitive advantage, through the right eye being made more active than the left.     

Electrophysiological effects of GABA on cat pancreatic neurons

In mammalian peripheral sympathetic ganglia GABA acts presynaptically to facilitate cholinergic transmission and postsynaptically to depolarize membrane potential. The GABA effect on parasympathetic pancreatic ganglia is unknown. We aimed to determine the effect of locally applied GABA on cat pancreatic ganglion neurons. Ganglia with attached nerve trunks were isolated from cat pancreata. Conventional intracellular recording techniques were used to record electrical responses from ganglion neurons. GABA pressure microejection depolarized membrane potential with an amplitude of 17.4 +/- 0.7 mV. Electrically evoked fast excitatory postsynaptic potentials were significantly inhibited (5.4 +/- 0.3 to 2.9 +/- 0.2 mV) after GABA application. GABA-evoked depolarizations were mimicked by the GABA(A) receptor agonist muscimol and abolished by the GABA(A) receptor antagonist bicuculline and the Cl(-) channel blocker picrotoxin. GABA was taken up and stored in ganglia during preincubation with 1 mM GABA; beta-aminobutyric acid application after GABA loading significantly (P < 0.05) increased depolarizing response to GABA (15.6 +/- 1.0 vs. 7.8 +/- 0.8 mV without GABA preincubation). Immunolabeling with antibodies to GABA, glial cell fibrillary acidic protein, protein gene product 9.5, and glutamic acid decarboxylase (GAD) immunoreactivity showed that GABA was present in glial cells, but not in neurons, and that glial cells did not contain GAD, whereas islet cells did. The data suggest that endogenous GABA released from ganglionic glial cells acts on pancreatic ganglion neurons through GABA(A) receptors.     

γ-Aminobutyric Acid Function in the Rat Striatum Is Under the Double Influence of Nigrostriatal Dopaminergic and Thalamostriatal Inputs: Two Modes of Regulation?

Glutamic acid decarboxylase (GAD), gamma-[3H]-aminobutyric acid [( 3H]GABA) high-affinity uptake into synaptosomes, and endogenous GABA content were measured in the rat striatum 2-3 weeks following 6-hydroxydopamine injection in the ipsilateral substantia nigra to destroy the nigrostriatal dopaminergic pathway and after kainic acid injection into the centromedial-parafascicular complex of the ipsilateral thalamus to lesion the thalamostriatal input. Both lesions resulted in apparent GAD increase concomitant with a decreased [3H]GABA uptake into striatal synaptosomes. GABA content was increased selectively following the dopaminergic lesion. Kinetic analysis of the uptake process for [3H]GABA showed selectively a decreased Vmax following the dopaminergic lesion; in animals with thalamic lesion, however, the change only concerned the Km, which showed a decreased affinity of the transport sites for [3H]GABA. Determination of Km and Vmax for GAD action on its substrate glutamic acid showed an increased affinity of GAD for glutamic acid in the case of the dopaminergic lesion without any change in Vmax, whereas the thalamic lesion resulted in GAD increase concomitant with a selective increase in Vmax. These data suggest that striatal GABA neurons are under the influence of nigrostriatal dopaminergic neurons which may reduce the GABA turnover, whereas the exact nature of the powerful control also revealed on these neurons following thalamic lesion remains to be determined. Both lesions induced adaptive neurochemical responses of striatal GABA neurons, possibly reflecting in the case of the dopaminergic deprivation an increased GABA turnover.     

EFFECTS OF AMINOOXYACETIC ACID AND l-GLUTAMIC ACID-γ-HYDRAZIDE ON GABA METABOLISM IN SPECIFIC BRAIN REGIONS

Abstract— Aminooxyacetic acid (AOAA) administration produced an increase in γ-aminobutyric acid (GABA) levels in regions of cerebral cortex, subcortex and cerebellum. In some cortical areas studied, the maximal effect was observed with 25 mg/kg AOAA; in other regions GABA levels were increased further with 50 and 75 mg/kg AOAA. Pretreatment with 25 mg/kg AOAA effectively inhibited GABA:2-oxoglutarate aminotransferase (GABA-T) and partially inhibited glutamic acid decarboxylase (GAD) activity in regions of cerebral cortex. However, this dose did not affect GAD activity in substantia nigra while GABA-T in the nigra and in the cerebellum was only partially inhibited. In both cortical and subcortical areas, the increase in GABA produced by 25 mg/kg of AOAA was linear. In contrast, l -glutamic acid-hydrazide (GAH) had no effect in the pyriform and cingulate cortex for the first 60 min after injection, and produced a biphasic GABA increase in caudate and substantia nigra over a 4 h period. Results suggest that GAH and AOAA affect regional GABA metabolism differentially and that there are several problems associated with estimating absolute GABA synthesis rates by measuring the rate or GABA accumulation after inhibition of GABA catabolism with these agents. This approach, however, may provide an easily obtainable indication of whether drugs or other manipulations are altering GABA synthesis in a given region.     

Characterization of GABAergic Neurons in the Mouse Lateral Septum: A Double Fluorescence In Situ Hybridization and Immunohistochemical Study Using Tyramide Signal Amplification

Gamma-aminobutyric acid (GABA) neurotransmission in the lateral septum (LS) is implicated in modulating various behavioral processes, including emotional reactivity and maternal behavior. However, identifying the phenotype of GABAergic neurons in the CNS has been hampered by the longstanding inability to reliably detect somal immunoreactivity for GABA or glutamic acid decarboxylase (GAD), the enzyme that produces GABA. In this study, we designed unique probes for both GAD65 (GAD2) and GAD67 (GAD1), and used fluorescence in Situ hybridization (FISH) with tyramide signal amplification (TSA) to achieve unequivocal detection of cell bodies of GABAergic neurons by GAD mRNAs. We quantitatively characterized the expression and chemical phenotype of GABAergic neurons across each subdivision of LS and in cingulate cortex (Cg) and medial preoptic area (MPOA) in female mice. Across LS, almost all GAD65 mRNA-expressing neurons were found to contain GAD67 mRNA (approximately 95-98%), while a small proportion of GAD67 mRNA-containing neurons did not express GAD65 mRNA (5-14%). Using the neuronal marker NeuN, almost every neuron in LS (> 90%) was also found to be GABA-positive. Interneuron markers using calcium-binding proteins showed that LS GABAergic neurons displayed immunoreactivity for calbindin (CB) or calretinin (CR), but not parvalbumin (PV); almost all CB- or CR-immunoreactive neurons (98-100%) were GABAergic. The proportion of GABAergic neurons immunoreactive for CB or CR varied depending on the subdivisions examined, with the highest percentage of colocalization in the caudal intermediate LS (LSI) (approximately 58% for CB and 35% for CR). These findings suggest that the vast majority of GABAergic neurons within the LS have the potential for synthesizing GABA via the dual enzyme systems GAD65 and GAD67, and each subtype of GABAergic neurons identified by distinct calcium-binding proteins may exert unique roles in the physiological function and neuronal circuitry of the LS.     

Postnatal expression of glutamate decarboxylases in developing rat cerebellum

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD₆₇, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD₆₇ mRNA predominates in the cerebellum. The mRNA for GAD₆₅, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD₆₅ is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD₆₇ mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.Special issue dedicated to Dr. Eugene Roberts.     

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.     

Brain damage and global stereopsis.

When a single object lies in front of or beyond the plane of fixation its retinal image lies on disparate positions in the two eyes. This 'local' retinal disparity is an excellent cue to depth, and retinal disparties of a few seconds of arc are detectable by people and monkeys. However, most visual scenes produce a complex array of contours in each eye and we can detect the disparity in the arrays despite the ambiguous nature of the disparities, i.e. each contour in one eye could be related to any of several similar contours in the other eye. This ability, known as 'global' stereopsis, may be selectively impaired following brain damage in man. Global stereopsis was measured in rhesus monkeys before and after removing a different cortical visual area in different groups of animals. Only removal of the inferotemporal cortex impaired global stereopsis. The result is related to the findings with human patients and to receptive field properties of neurons in the inferotemporal cortex of monkeys.     

Glutamate Decarboxylase Activities in Single Vertebrate Neurons

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to a degree yielding high sensitivity and low blank. Single cell bodies of anterior horn cells and dorsal root ganglion cells were dissected out from the freeze-dried sections of rabbit and chicken spinal cords and Purkinje cell bodies from those of rabbit cerebellum. A minute amount of GABA, present in single neurons or synthesized by GAD in single neurons, was enzymatically converted to NADPH. The NADPH was amplified 10,000-350,000-fold and measured, using an enzymatic amplification reaction (NADP cycling). GAD was contained in all Purkinje cell bodies and its average activity was four- to fivefold higher than those of the molecular and granular layers of rabbit cerebellum. The GABA concentration was threefold higher in Purkinje cell bodies than in these layers. GAD activity, at a level similar to that in the cerebellar layers, was found in almost all the cell bodies of anterior horn cells from rabbit and chicken. GABA was detected in 40% of rabbit neurons and not in chicken neurons. Dorsal root ganglion cells from both species contained no measurable GAD or GABA.