Effects of nigrostriatal lesions on the levels of messenger RNAs encoding two isoforms of glutamate decarboxylase in the globus pallidus and entopeduncular nucleus of the rat.

The neurotransmitter gamma-aminobutyric acid (GABA) is present in efferent neurons of the striatum and of the pallidum, one of the main striatal target areas. Dopaminergic nigrostriatal neurons play a critical role in the regulation of GABAergic neurotransmission in the striatum. In the present study, we investigated their role in the regulation of glutamate-decarboxylase (GAD) mRNA expression in two divisions of the pallidum in rats: the globus pallidus and entopeduncular nucleus, equivalent to the external and internal pallidum, respectively, of primates. Dopaminergic neurons were lesioned by unilateral injections of 6-hydroxydopamine (6-OHDA) in the substantia nigra of adult rats. Two or 3 weeks after the lesion, frontal cryostat-cut sections of the brain were processed for in situ hybridization histochemistry with 35S-labeled RNA probes synthesized from cDNAs encoding two distinct isoforms of GAD of respective molecular weight 67,000 (GAD67) and 65,000 (GAD65). The number of labeled cells was determined, and intensity of labeling in individual cells was analyzed by computerized image analysis on emulsion radioautographs. In the globus pallidus, the number of labeled neurons and intensity of labeling per cell were increased on the side ipsilateral to the lesion as compared with control rats in sections hybridized with the GAD67 RNA probe. No changes were detected on the side contralateral to the lesion or in the levels of labeling for GAD65 mRNA. Confirming previous data, the level of labeling for GAD65 mRNA was much higher than for GAD67 mRNA in the entopeduncular nucleus of control rats. In rats with a 6-OHDA lesion, labeling for both GAD67 and GAD65 mRNAs was decreased on the side contralateral, but not ipsilateral, to the lesion, as compared with control rats. The results show that lesions of the nigrostriatal pathway in rats affect the levels of mRNAs encoding two distinct isoforms of GAD in neurons of the globus pallidus and entopeduncular nucleus differently. In addition, results in the entopeduncular nucleus further support a bilateral effect of unilateral dopaminergic lesions.     

Glutamatergic pallidothalamic projections and their implications in the pathophysiology of Parkinson's disease

GABAergic projections emitted from the entopeduncular nucleus (ENT) and the substantia nigra pars reticulata (SNr) innervate different thalamic nuclei and they are known to be hyperactive after dopaminergic depletion. Here we show that isoform 2 of the vesicular glutamate transporter (VGLUT2) is expressed by neurons in the ENT nucleus but not in the SNr. Indeed, dual in situ hybridization demonstrated that the ENT nucleus contains two different subpopulations of projection neurons, one single-expressing GAD65/67 mRNAs and another one that co-expresses either of the GAD isoforms together with VGLUT2 mRNA. Unilateral dopaminergic depletion induced marked changes in pallidothalamic-projecting neuron gene expression, resulting in increased expression of GAD65/67 mRNAs together with a clear down-regulation of VGLUT2 mRNA expression. Our results indicate that the increased thalamic inhibition typical of dopamine depletion might be explained by a synergistic effect of increased GABA outflow coupled to decreased glutamate levels, both neurotransmitters coming from ENT neurons.     

Disappearance of GAD-mRNA and tyrosine hydroxylase in substantia nigra following striatal ibotenic acid lesions: evidence for transneuronal regression.

Transneuronal regression in substantia nigra reticulata (SNR) and substantia nigra compacta (SNC) neurons was studied in Fischer 344 male rats by immunocytochemistry and by in situ hybridization. Three months after striatal lesioning by ibotenic acid, there was a shrinkage (30%) of the SNR region cross-sectional area and a 50% disappearance of neurons that contain glutamic acid decarboxylase (GAD)-mRNA, but only in the ventromedial portion of this nucleus. Loss of dopaminergic neurons, as recognized by tyrosine hydroxylase immunoreactivity, occurred only in caudal portions of the SNC and SNR. These findings suggest that lesions in reciprocally connected pathways, like the nigrostriatal and striatonigral systems, may produce a vicious cycle (feedforward cascade) of neurodegeneration due to interference with retrograde ana anterograde influences.     

Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey.

In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin-dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non-GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate. This suggests that the kinase may be differentially engaged in pre- and postsynaptic functions at certain synapses.     

Changes in [3H]muscimol binding in substantia nigra, entopeduncular nucleus, globus pallidus, and thalamus after striatal lesions as demonstrated by quantitative receptor autoradiography

A quantitative autoradiographic technique for measuring the binding of [3H]muscimol to central nervous system GABA receptors is described using tritium-sensitive film. [3H]Muscimol binding was studied in primary and secondary striatal projection areas of rat brain following kainic acid lesions of the striatum. Seven days after the lesion, binding affinities in the striatum and its projection areas were not altered significantly. There was a loss of [3H]muscimol receptors in the striatum. Receptors increased in numbers in the ipsilateral globus pallidus (19%), entopeduncular nucleus (22%), and substantia nigra pars reticulata (38%). [3H]Muscimol binding was decreased in the ipsilateral anteroventrolateral and ventromedial (8%) thalamic nuclei. [3H]Muscimol binding in other brain areas (layer IV of the cerebral cortex, central gray, superior colliculus, and stratum moleculare of hippocampus) was not affected. The findings suggest that a loss of striatal innervation resulted in increased numbers of GABA receptors in striatal projection sites. It is further suggested that loss of inhibitory striatal inputs to neurons in the entopeduncular nucleus and substantia nigra pars reticulata may activate GABAergic projections to thalamus and thus result in decreased numbers of thalamic GABA receptors.     

Pattern of expression of the serotonin2C receptor messenger RNA in the basal ganglia of adult rats

The distribution of the serotonin (5-HT) receptor 5-HT_2C mRNA was examined at the single-cell level with in situ hybridization histochemistry and emulsion autoradiography in the basal ganglia and mesolimbic system of adult rats, with focus on the pallidum and the substantia nigra, which receive striatal inputs and play a critical role in basal ganglia function. 5-HT_2C receptor mRNA expression was always restricted to a subpopulation of neurons in the regions examined. In the neostriatum, labeled neurons were more numerous in the rostral nucleus accumbens than in the caudal nucleus accumbens and were more numerous in the ventral and ventrolateral caudate-putamen than in the dorsal caudate-putamen, where labeled neurons were restricted to isolated clusters. In striatal target areas, dense labeling in the entopeduncular nucleus (internal pallidum, direct striatal output pathway) contrasted with an absence of labeling in the globus pallidus (external pallidum, indirect striatal output pathway). Double-label in situ hybridization in the substantia nigra revealed coexpression of 5-HT_2C receptor mRNA with glutamic acid decarboxylase but not with tyrosine hydroxylase mRNA, indicating that it was restricted to γ-aminobutyric acid (GABA)ergic neurons. In this region, dense labeling for 5-HT_2C mRNA was found in half of the neurons at middle and caudal levels of both the pars compacta and the pars reticulata, with little labeling rostrally. The data suggest that drugs acting on the 5-HT_2C receptor could selectively affect discrete neuronal populations in the basal ganglia and mesolimbic systems and indicate a new level of neurochemical heterogeneity among GABAergic neurons of the substantia nigra. J. Comp. Neurol. 384:233-247, 1997. © 1997 Wiley-Liss, Inc.     

Dopaminergic regulation of glutamic acid decarboxylase mRNA expression and GABA release in the striatum: A review

Lindefors Nils: Dopaminergic Regulation of Glutamic Acid Decarboxylase mRNA Expression and GABA Release In the Striatum: A Minireview. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1993, 17(6): 887–903.

1. 1. The majority of neurons in the striatum (caudate-putamen, dorsal striatum; nucleus accumbens, ventral striatum) and in striatal projection regions (the pallidum, the entopeduncular nucleus and substantia nigra reticulata) use γ-aminobuturic acid (GABA) as transmitter and express glutamic acid decarboxylase (GAD; rate limiting enzyme) in the synthesis of GABA. GABA is the major inhibitory transmitter in the mammlian brain.

2. 2. GAD in brain is present as two isoenzymes, GAD₆₅ and GAD₆₇. GAD₆₅ is largely present as an inactive apoenzyme, which can be induced by nerve activity, while most GAD₆₇ is present as a pyridoxal phosphate-bound permanently active holoenzyme. Thus GAD₆₅ and GAD₆₇ seem to provide a dual system for the control of neuronal GABA synthesis.

3. 3. GAD mRNA expression can be visualised and quantified using in situ hybridisation, and GABA release can be quantified using in vivo microdialysis.

4. 4. Different populations of GABA neurons can be distinguished in both dorsal and ventral striatum as well as in other parts of the basal ganglia.

5. 5. Inhibition of dopaminergic transmission in the striatum by lesion of dopamine neurons or by neuroleptic treatment is followed by an increased release of GABA and increased expression of GAD₆₇ mRNA in a subpopulation of striatal medium-sized neurons which project to the globus pallidus, and increased striatal GAD enzyme activity.

6. 6. Increased dopaminergic transmission by repeated but not single doses of amphetamine is followed by decreased striatal GABA release and decreased GAD₆₇ mRNA expression in a subpopulation of medium-sized neurons in the striatum.

7. 7. Two populations of medium-sized GABA neurons in the striatum seem to be under tonic dopaminergic influence. The majority of these GABA neurons are under inhibitory influence, whereas a small number seem to be stimulated by dopamine.

8. 8. Specific changes in activity in subpopulations of striatal GABA neurons probably mediate the dopamine-dependent hypokinetic syndrome seen in Parkinson's disease and following neuroleptic treatment.

Lesions of the Cerebral Cortex and Caudate-Putamen Enhance GABA Function in the Rat Superior Colliculus

Unilateral lesions of the rat frontal cortex were made either alone or in combination with the caudate-putamen in order to examine (a) their morphological influence on the substantia nigra and (b) their neurochemical influence on GABA function in the superior colliculus. One to two months following the combined lesion, neuronal somata in the ipsilateral pars reticulata of the substantia nigra were clearly hypertrophied (+ 30%). Morphological changes in the substantia nigra were not evident contralaterally or in animals bearing only cortical lesions. One to two months following cortex-only lesions, no significant alterations in tectal GABA concentration were observed. However, the combined lesion induced elevations of GABA within both the medial and lateral sectors of the intermediate and deep layers of the superior colliculus. This effect was restricted to the ipsilateral side and was most pronounced in lateral sectors. The vast majority of GABA released from superfused control tectal slices by a depolarizing stimulus (35 mM KCl) was calcium-dependent. Such evoked GABA release from ipsilateral tectal slices was significantly reduced (- 25%) by unilateral lesions of the substantia nigra, a structure that is known to provide GABA-containing inputs to the tectum. In contrast, cortical lesions alone significantly enhanced the evoked tectal GABA release (+ 66%), although their influence was again confined to the ipsilateral side. Combined lesions of the cerebral cortex and caudate-putamen significantly enhanced the evoked GABA release from tectal slices in both hemispheres but the changes were most marked ipsilaterally (+ 147%). It is suggested that the hypertrophy of GABA-containing nigrotectal somata seen after removal of corticostriatal, corticotectal and in particular GABA-containing striatonigral fibres may reflect concomitant increases in GABA synthesis within and/or sprouting of nigrotectal terminals.     

Presynaptic and postsynaptic D1 dopamine receptors in the nigrostriatal system of the rat brain: a quantitative autoradiographic study using the selective D1 antagonist [3H]SCH 23390

Ibotenic acid lesions of the caudate-putamen in rat brain resulted in dramatic reductions in [3H]SCH 23390 binding in both the ipsilateral caudate-putamen and substantia nigra reticulata as assessed by quantitative autoradiography. Nigral ibotenic acid and 6-hydroxydopamine lesions did not significantly alter the binding in either structure. This indicates that D1 receptors in the caudate-putamen are postsynaptic on striatal neurons, while those in the substantia nigra reticulata are presynaptic on nerve terminals originating in the caudate-putamen.     

5-HT1D receptors in the guinea pig brain: pre- and postsynaptic localizations in the striatonigral pathway

The caudate-putamen, globus pallidus and substantia nigra pars reticulata of the guinea pig contain high densities of the 5-HT1D receptor subtype. The cellular localization of these sites in the striatonigral pathway was investigated using receptor autoradiography and selective neurotoxin lesions. In guinea pigs with unilateral 6-hydroxydopamine lesions of the nigral dopaminergic cells, no significant decrease was observed in any of the components of the striatonigral pathway. In contrast, when quinolinic acid was injected in the caudate-putamen, marked reductions in [3H]5-HT binding were seen in the caudate-putamen, the globus pallidus and the substantia nigra pars reticulata, on the side ipsilateral to the lesion. These data, which are comparable to previous results in human pathologies where similar cell populations are known to degenerate (Parkinson disease and Huntington's chorea), indicate a presynaptic localization of 5-HT1D receptors on the terminals of the striatal neurons projecting to the pars reticulata of the substantia nigra. In addition, these receptors could be located on the cell bodies or dendrites of these neurons in the striatum, postsynaptically to serotoninergic fibers.     

Presynaptic and postsynaptic D1 dopamine receptors in the nigrostriatal system of the rat brain: a quantitative autoradiographic study using the selective D1 antagonist [H]SCH 23390

Ibotenic acid lesions of the caudate-putamen in rat brain resulted in dramatic reductions in [³H]SCH 23390 binding in both the ipsilateral caudate-putamen and substantia nigra reticulata as assessed by quantitative autoradiography. Nigral ibotenic acid and 6-hydroxydopamine lesions did not significantly alter the binding in either structure. This indicates that D₁ receptors in the caudate-putamen are postsynaptic on striatal neurons, while those in the substantia nigra reticulata are presynaptic on nerve terminals originating in the caudate-putamen.     

Amygdala-kindling does not induce a persistent loss of GABA neurons in the substantia nigra pars reticulata of rats

GABAergic inhibition of the substantia nigra pars reticulata (SNR) has been shown to suppress seizures in most models of epilepsy, including the amygdala-kindling model of temporal lobe epilepsy (TLE). A dysfunction of this seizure gating mechanism of the SNR may lead to facilitation of seizure propagation in such models. In post-status epilepticus models of TLE, GABAergic neurons in the SNR are damaged, but it is not known whether such damage also occurs in kindling. By using stereological techniques for cell counting in amygdala-kindled rats, we determined the density of SNR neurons that were labeled for GABA by immunohistochemistry or for the two isoforms of the GABA-synthesizing enzyme glutamate decarboxylase (GAD), GAD65 and GAD67, by in situ hybridization (ISH). In addition, GABA neurons in the basolateral amygdala (BLA) were counted. While there was a significant reduction of GAD65 mRNA expressing neurons in the BLA of kindled rats, no alteration in the density of neurons was observed in the anterior or posterior SNR when cells were counted 6 weeks after the last kindled seizure. Our previous finding of reduced GAD and GABA levels in synaptosomes isolated from the SN of kindled rats together with the present observation of unchanged density of SNR neurons in such rats suggest that kindling affects the GABAergic projections from the striatum or globus pallidus to the SNR rather than directly affecting GABA neurons in the SNR.     

Role of Dopaminergic D2 Receptors in the Regulation of Glutamic Acid Decarboxylase Messenger RNA in the Striatum of the Rat

Levels of messenger RNA (mRNA) encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot and in situ hybridization analyses in the striatum of the rat, after chronic injections of two neuroleptics, sulpiride and haloperidol. The Northern blot analysis showed that the chronic injection of sulpiride at high doses (80 mg/kg, twice a day, 14 days) increased striatal GAD and PPE mRNA levels by 120% and 78% respectively, when compared to vehicle-injected rats. Haloperidol injections at relatively low doses (1 mg/kg, once a day, 14 days) produced parallel increases in GAD (40%) and PPE (52%) mRNA levels. After in situ hybridization densitometric measurements were performed on autoradiograms from rats treated with sulpiride, haloperidol or vehicle. The distribution of GAD and PPE mRNA signals in control rats was homogeneous along the rostrocaudal extension of the striatum. A similar increase was found along this axis after sulpiride (20%) and haloperidol (30%) treatments. The cellular observation of hybridization signals showed that grain density for GAD mRNA was increased in a majority of striatal cells after both treatments. By contrast, the PPE mRNA hybridization signal only increased in a subpopulation of neurons. The effects of such treatments were also analysed by measuring GAD activity in the striatum and in its output structures, the globus pallidus and the substantia nigra. After the administration of sulpiride, GAD activity was not modified in the striatum but increased in the globus pallidus (by 17%). After haloperidol treatment, GAD activity was increased in the globus pallidus (20%) and the substantia nigra (17%). It is concluded that the interruption of dopaminergic transmission, more precisely the D2 receptor blockade, promotes in striatopallidal neurons an increase in GAD mRNA accompanied by an increase in GAD activity and PPE mRNA. A possible regulation of GAD mRNA and GAD activity in striatonigral neurons is also discussed.     

Blockade of A2A receptors plus l-DOPA after nigrostriatal lesion results in GAD67 mRNA changes different from l-DOPA alone in the rat globus pallidus and substantia nigra reticulata

Studies in animal models of Parkinson's disease (PD) suggest the potential utility of adenosine A(2A) antagonists in the treatment of this disease. In the present study, unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats received chronic intermittent treatment with the adenosine A(2A) antagonist SCH58261 (5 mg/kg) plus l-DOPA (3 mg/kg) or l-DOPA (6 mg/kg) alone, at doses producing the same intensity of contralateral turning on first administration. Three days after discontinuation of treatments, GABA synthesizing enzyme glutamic acid decarboxylase (GAD67) mRNA was evaluated at cellular level in the globus pallidus (GP) and substantia nigra pars reticulata (SNr) by in situ hybridization. 6-OHDA lesion significantly increased GAD67 mRNA levels in both the GP and SNr ipsilateral to the lesion. Chronic l-DOPA (6 mg/kg), in contrast to SCH58261 plus l-DOPA (3 mg/kg), produced a sensitized contralateral turning indicative of dyskinetic potential and further increased GAD67 mRNA in the GP. In the SNr, a significant decrease in GAD67 mRNA was observed after either treatments. However, while l-DOPA (6 mg/kg) decreased SNr GAD67 mRNA below the intact side, SCH58261 plus l-DOPA (3 mg/kg) brought GAD67 mRNA to the same level of the intact SNr. l-DOPA (3 mg/kg) or SCH58261 (5 mg/kg) alone failed to modify GAD67 mRNA. Results suggest that an increase in GAD67 mRNA in GP and a decrease in SNr might underlie dyskinetic movements induced by chronic l-DOPA. In contrast, the lack of GAD67 mRNA changes in the GP and a less marked inhibition of SNr might correlate with the absence of dyskinetic potential observed after SCH58261 plus l-DOPA.     

Evidence for glycinergic respiratory neurons: Bötzinger neurons express mRNA for glycinergic transporter 2

B?tzinger (BOTZ) neurons in the rostral ventrolateral medulla fire during the late expiratory phase of the respiratory cycle. These cells inhibit phrenic motor neurons and several types of respiratory neurons in the medulla oblongata. BOTZ cells produce a fast, chloride-mediated inhibition of their target neurons, but the neurotransmitter used by these cells has not been determined. In the present study, we examine whether gamma-aminobutyric acid (GABA) or glycine could be the inhibitory neurotransmitter of BOTZ cells. In chloralose-anesthetized rats, we individually filled 20 physiologically characterized BOTZ neurons with biotinamide by using a juxtacellular labeling method. Medullary sections containing the labeled BOTZ neurons were processed for in situ hybridization by using digoxigenin-labeled riboprobes for glutamic acid decarboxylase isoform 67 (GAD67), a marker for GABAergic neurons, or for glycine transporter 2 (GLYT2), a marker for glycinergic neurons. All BOTZ cells examined contained GLYT2 mRNA (n = 10), whereas none had detectable levels of GAD67 mRNA (n = 10). For a positive control, 12 GABAergic neurons in the substantia nigra pars reticulata also were recorded and filled with biotinamide in vivo. Most of these cells, as expected, had detectable levels of GAD67 mRNA (11 out of 12). These results demonstrate that the juxtacellular labeling method can be combined with in situ hybridization to identify physiologically characterized cells with probable GABAergic or glycinergic phenotypes. Furthermore, these data suggest that BOTZ neurons use the neurotransmitter glycine and not GABA to provide widespread inhibition of respiratory-related neurons.     

Topographical study of the distribution of gamma-aminobutyric acid (GABA) in the human substantia nigra. A case study.

The topographical distribution of gamma-aminobutyric acid (GABA) in the substantia nigra of a 28-year-old male 4 h after death was investigated. In a preliminary study the entire substantia nigra was dissected from transverse sections. The results showed that there was no correlation between the GABA concentration and the number of melanin-rich nigral cell bodies. This was especially so in the rostral third of the substantia nigra. Using the method of Miyata and Otsuka, transverse sections (150 mum) of the rostral, middle and caudal substantia nigra were cut into 500 mum X 500 mum square blocks which were assayed for GABA by an enzymatic method. In the rostral substantia nigra the GABA distribution was markedly uneven. The highest concentration of GABA was found in the pars reticulata. Within the pars reticulata the highest levels of GABA clearly occurred in two separate regions, a medial and a lateral. In the middle and caudal substantia nigra the GABA distribution was again uneven; however, the highest GABA levels were divided between the pars reticulata and the pars compacta. The results support the view that in the substantia nigra the greatest part of the GABA content is due to the presence of striato-nigral nerve terminals which are known to synapse with the dendrites of the substantia nigra dopamine neurons. In the rostral substantia nigra the concentration of GABA within the pars reticulata is in keeping with the presence of dendrites of such neurons in this region. Presumably on this basis it can be assumed that in the middle and caudal substantia nigra the dendrites are oriented in a more rostro-caudal direction.     

Comparative distribution of messenger RNAs encoding glutamic acid decarboxylases (Mr 65,000 and Mr 67,000) in the basal ganglia of the rat.

Glutamic acid decarboxylase, the enzyme required for GABA synthesis, exists as distinct isoforms, which have recently been found to be encoded by different genes. The relative expression of messenger RNAs encoding two isoforms of glutamic acid decarboxylase (Mr 67,000 and Mr 65,000) was measured at the single-cell level in neurons of the rat basal ganglia with in situ hybridization histochemistry. Both messenger RNAs were expressed in neurons of the striatum, pallidum, and substantia nigra pars reticulata, but marked differences in the relative level of labelling were observed with the two probes. In striatum, efferent neurons were more densely labelled for the messenger RNA encoding glutamic acid decarboxylase (Mr 65,000) than for the messenger RNA encoding glutamic acid decarboxylase (Mr 67,000), whereas the reverse was observed for GABA-ergic interneurons. Neurons of the entopeduncular nucleus were much more densely labelled for messenger RNA encoding glutamic acid decarboxylase (Mr 65,000) than for messenger RNA encoding glutamic acid decarboxylase (Mr 67,000). In addition, labelling for messenger RNA encoding glutamic acid decarboxylase (Mr 65,000) was higher in the entopeduncular nucleus (internal pallidum) than in the globus pallidus (external pallidum), a structure which expressed similar levels of both mRNAs. In contrast to neurons of the internal pallidum, efferent neurons of the substantia nigra pars reticulata expressed slightly more messenger RNA encoding glutamic acid decarboxylase (Mr 67,000) than that encoding the other isoform of the enzyme. The results suggest a differential expression of the messenger RNAs encoding the two isoforms of glutamic acid decarboxylase in subpopulations of basal ganglia neurons in rats.     

Gabaergic nerve terminals decrease in the substantia nigra following hemitransections of the striatonigral and pallidonigral pathways

Glutamic acid decar☐ylase (GAD), the enzyme that synthesizes the neurotransmitter, GABA, was immunocytochemically localized in axon terminals as well as in small and medium-sized neurons of the rat substantia nigra. The pattern formed by GAD-containing axon terminals with the dendrites and somata of neurons in the substantia nigra was altered following ipsilateral hemitransections of the striatonigral and pallidonigral pathways. A marked reduction of GAD-positive terminals occurred throughout this brain region, but the ventral fifth of the pars reticulata showed a nearly normal pattern of GAD-positive axon terminals.The results of this investigation are consistent with results from biochemical studies which have indicated that the striatonigral and/or pallidonigral pathways are GABAergic. In addition, these results suggest that the residual GABAergic terminals remaining after hemitransection are derived from intrinsic neurons of the substantia nigra.     

Origin and distribution of glutamate decarboxylase in substantia nigra of the cat

The topographical distribution of glutamate decar?ylase (GAD) in substantia nigra in unoperated and operated cats was studied in samples microdissected from freeze-dried tissue sections.The concentration of GAD, the enzyme synthesizing γ-aminobutyric acid (GABA), was highest in the medial part of pars reticulata, and decreased in the mediolateral direction. In pars compacta, on the other hand, the highest enzyme activity was found in the lateral part which merges with pars reticulata, and it decreased gradually in the latero-medial direction. The activity of GAD was always lower in the medial part of pars compacta, which contains the highest concentration of cell bodies. GAD in substantia nigra decreased after lesions in putamen, nucleus caudatus, globus pallidus and nucleus entopeduncularis. The loss of enzyme activity was strictly localized and was related to the site of termination of the degenerating striato-nigral fibers. The reduction of GAD in substantia nigra following lesions of globus pallidus or nucleus entopeduncularis may be ascribed to the interruption of striato-nigral fibers passing through these regions. The results thus indicate that the fibers of the GAD-containing axon terminals in substantia nigra of the cat originate in putamen and nucleus caudatus.Subcellular fractionation showed that about 85% of GAD and about 25% of lactate dehydrogenase were present in particles (probably synaptosomes) from substantia nigra in unoperated animals. Electron microscopic examination revealed that 11.5% of the tissue volume of pars reticulata was occupied by boutons compared to 5.9% for pars compacta. The concentration of GABA in pars reticulata was found to be 9 mM. From these data the intraterminal concentration of GABA was estimated to be at least 60 mM, probably over 100 mM.DOPA decar?ylase was mainly found in pars compacta. Acetylcholin-esterase showed a very high activity in substantia nigra, the highest concentration being found in the medial part of pars reticulata. In contrast, the concentration of choline acetyltransferase was very low. The ratio of acetylcholinesterase activity to choline acetyltransferase activity was 1000. DOPA decar?ylase and the cholinergic enzymes were little affected by the above described lesions.