GAD67 mRNA Levels Research Articles

Increased glutamate decarboxylase mRNA levels in the striatum and pallidum of MPTP-treated primates

The mRNA levels encoding for the enzyme glutamate decarboxylase (GAD67) were measured by computerized image analysis after in situ hybridization histochemistry and radioautography in the striatum and pallidum of normal squirrel monkeys (Saimiri sciureus), or after treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). All MPTP-injected monkeys exhibited profound motor deficits including akinesia. The dopaminergic innervation, as visualized and quantified on x-ray films after 3H-mazindol binding on tissue sections, was uniformly lost throughout the striatum of MPTP-treated monkeys. Brain sections processed with a probe synthesized from a feline or human GAD67 cDNA exhibited intense radioautographic labeling throughout the striatum. When measured on x-ray films, the intensity of GAD67 mRNA labeling was increased in the striatum of MPTP-treated versus control monkeys. Increased labeling reached statistical significance in the dorsolateral sector of the rostral putamen and throughout the putamen and the caudate at the caudal, postcommissural, level. Analysis of emulsion radioautographs demonstrated that the increase in GAD67 mRNA labeling in MPTP-treated monkeys occurred in individual neurons of the striatum. In the external and internal segments of the pallidum, numerous neurons labeled with the GAD67 cRNA probe were visualized on emulsion radioautographs. The intensity of GAD67 mRNA labeling in single neurons of both pallidal segments was increased in MPTP-treated versus control monkeys. Construction of the histograms of frequency distribution of labeling indicated that this increase occurred in a majority of labeled neurons. The present study demonstrates that GAD67 mRNA levels are significantly altered in the striatum and pallidum of parkinsonian monkeys. The preferential increase of GAD67 mRNA labeling in the dorsolateral putamen, which receives afferents from the sensorimotor cortex, provides further evidence of the involvement of GABAergic transmission in the expression of the motor deficits elicited after MPTP. In addition, increased GAD67 mRNA levels in the internal segment of the pallidum support the hypothesis of an increased activity of GABAergic neurons in the output structures of the basal ganglia in parkinsonism.

Localization of mRNAs encoding two forms of glutamic acid decarboxylase in the rat hippocampal formation

The mRNAs for two forms of glutamic acid decarboxylase (GAD65 and GAD67) were localized in the rat hippocampal formation by nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes. Some neurons in all layers of the hippocampus and dentate gyrus were readily labeled for each GAD mRNA, and the patterns of labeling for GAD65 and GAD67 mRNAs were very similar. All major groups of previously described GAD- and GABA-containing neurons appeared to be labeled for each GAD mRNA. Such findings suggest that most GABA neurons in the hippocampal formation contain both GAD mRNAs. When the labeling of neurons in the hippocampal formation and cerebral cortex was compared in the same sections, the intensity of neuronal labeling for GAD67 mRNA was generally similar in the two regions. However, the intensity of labeling for GAD65 mRNA was generally stronger for many neurons in the hippocampal formation than for most neurons in the cerebral cortex. Neurons in the hilus of the dentate gyrus were particularly well labeled for GAD65. The nonradioactive labeling for the GAD mRNAs was confined to the cytoplasm of neuronal cell bodies, and this allowed a clear visualization of the relative number and location of labeled neurons. Several distinct patterns of GAD mRNA-containing neurons were observed among different regions of the hippocampal formation. In the hilus of the dentate gyrus, GAD mRNA-containing neurons were numerous in the regions deep to the granule cell layer as well as in more central parts of the hilus. Within CA3, the densities (quantities) of labeled neurons varied among the regions. In the inner or hilar segment of CA3, the density of labeled neurons was often lower than that in the outer part of CA3 where numerous labeled neurons were distributed throughout all layers. In CA1, GAD mRNA-labeled neurons were distributed in a relatively laminar pattern with the highest density in stratum pyramidale and moderate densities in stratum oriens and at the interface between strata radiatum and lacunosum-moleculare. Lower densities were found within the latter two layers. The prominent localization of the two GAD mRNAs in the hippocampal formation suggests that a dual system for GABA synthesis is necessary for normal GABAergic function in this brain region. Most putative GABA neurons contain relatively high levels of GAD67 mRNA as might be expected if this GAD form is responsible for the synthesis of GABA for metabolic and baseline synaptic function.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutamate decarboxylase (GAD65) mRNA levels in the striatum and pallidum of MPTP-treated monkeys

The mRNA levels encoding for the enzyme glutamate decarboxylase (GAD65) were measured by computerized image analysis after in situ hybridization histochemistry in the striatum and pallidum of normal and MPTP-treated squirrel monkeys. At striatal level, GAD65 mRNA labeling in MPTP-treated monkeys was primarily increased in the dorsolateral sector of the putamen. At pallidal level, the intensity of GAD65 mRNA labeling in single neurons was increased in the internal but not the external segment of the pallidum of MPTP-treated monkeys. The regulation of GAD65 mRNA levels in the striatum and internal segment of the pallidum suggest an important role for this enzyme in the regulation of GABAergic functions in basal ganglia.

Differential regulation of glutamate decar☐ylase and preproenkephalin mRNA levels in the rat striatum

The mRNA levels encoding for the enzyme glutamate decar☐ylase (GAD67) and the peptide enkephalin were measured in the striatum of adult and 15 day-old rats by in situ hydridization histochemistry and radioautography after neonatal injections of 6-hydroxydopamine or after acute pharmacological blockade of dopamine receptors with haloperidol or sulpiride. In adult rats injected as neonates with 6-hydroxydopamine or treated with the D 1/D 2 dopamine receptors antagonist, haloperidol, an increase in preproenkephalin and GAD67 mRNA levels was measured in the striatum. The D 2 dopamine receptor antagonist, sulpiride, did not change the mRNA levels of either GAD67 or PPE in the striatum. In 15-day-old rats, neonatal 6-hydroxydopamine or haloperidol treatment resulted in increased preproenkephalin but unchanged GAD67 mRNA levels compared to controls. In these 15-day-old rats, however, sulpiride produced an increase in GAD67 but not preproenkephalin mRNA levels. Intrastriatal injections to adult rats of pertussis toxin which uncouples Gi/Go proteins from their receptors resulted in a dramatic increase in preproenkephalin without concomitant change in GAD67 mRNA levels. Altogether, these results show that GAD67 and preproenkephalin mRNA levels are modulated in parallel in adult but not in 15 day-old rats after 6-hydroxydopamine injections or dopaminergic blockade. In keeping with evidence of a co-localization of GAD67 and preproenkephalin mRNAs in some striatal neurons, the results indicate that these two mRNAs can be differentially regulated in the same neurons. In addition, the differential effect of haloperidol, sulpiride or pertussis toxin on GAD67 and preproenkephalin mRNA levels suggests that these two mRNAs are regulated through different dopamine receptor subtypes.

Glutamic acid decarboxylase gene expression in thalamic reticular neurons transplanted as a cell suspension in the adult thalamus

The goal of the present study was to determine whether alterations in neuronal morphology and connections in thalamic grafts were accompanied by changes in the expression of mRNA encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of GABA, the normal neurotransmitter of neurons of the thalamic reticular nucleus. Cell suspensions of rat fetal tissue containing both thalamic reticular nucleus and ventrobasal primordia were transplanted into the excitotoxically lesioned somatosensory thalamus of adult rats. Levels of messenger RNA (mRNA) encoding GAD (Mr 67,000; GAD67) were measured 7 days to 4 months following transplantation via quantitative in situ hybridization with 35S-radiolabeled antisense RNAs. Expression of GAD67 mRNA in the thalamic reticular nucleus was analyzed in parallel in rat pups between 0 and 30 days postnatally, and in adult animals. As already observed with immunohistochemistry, transplanted neurons of the thalamic reticular nucleus did not group in specific clusters but rather mingled with unlabeled (putatively ventrobasal) neurons. Levels of labelling for GAD67 mRNA per neuron increased over time and reached adult levels during the third week post-grafting, i.e. 2 weeks after the theoretical birthdate of the neurons (grafted at embryonic days 15–16). Similar values were observed and a plateau was reached at similar time points during normal ontogeny. The results suggest that, in contrast to morphology and size of the neuronal cell bodies, gene expression of GAD67 develops normally despite the ectopic location of neurons of the thalamic reticular nucleus in the somatosensory thalamus, the abnormal connectivity and the lack of segregation from non-GABAergic neurons. Therefore, dissociation and transplantation of the tissue, resulting in disruption of the extracellular matrix and alteration of cellular interactions, differentially affects various aspects of neuronal maturation. Because neuronal activity in the graft is similar to that in the adult thalamic reticular nucleus, the results further suggest that, as shown in other experimental systems, GAD67 mRNA levels in the graft are regulated in relation to the functional activity of GABAergic neurons.

Effects of neonatal 6-hydroxydopamine injections on glutamate decarboxylase, preproenkephalin and dopamine D 2 receptor mRNAs in the adult rat striatum

The effects of neonatal 6-hydroxydopamine injections on the levels and cellular distribution of glutamate decarboxylase (GAD67), preproenkephalin and dopamine D 2 receptor messenger RNAs were studied in the striatum of adult rats. Cerebroventricular injections of 150 μg or 100 μg of 6-hydroxydopamine to 3-day-old neonate rats resulted in the total disappearance of neurons labeled with a tyrosine hydroxylase probe in sections of the substantia nigra and ventral tegmental area. In the striatum of adults, both doses of 6-hydroxydopamine induced an increase in GAD67 and preproenkephalin mRNA levels compared to controls. A smaller but consistent increase in dopamine D 2 receptor mRNA levels was also found on adjacent sections of the striatum only in animals injected with 150 μg of 6-hydroxydopamine. Regional analysis of labeling showed that the increased GAD67, preproenkephalin or dopamine D 2 receptor mRNA levels occured in all striatal sectors examined. Emulsion radioautographs confirmed the increased GAD67, preproenkephalin and dopamine D 2 receptor mRNA labeling at cellular level. The present study demonstrates that bilateral 6-hydroxydopamine lesions of dopamine neurons in neonate rats are able to induce a long-term and widespread alteration in the expression of genes encoding for GAD67, preproenkephalin and dopamine D 2 receptor in the striatum. In view of previous results after 6-hydroxydopamine lesions in adults, it appears that the behavioral differences observed after adult or neonatal 6-hydroxydopamine lesions are accompanied by a similar alteration of GAD67, preproenkephalin and dopamine D 2 receptor gene expression in presumed striatal projection neurons.

Effects of Increased γ‐Aminobutyric Acid Levels on GAD67 Protein and mRNA Levels in Rat Cerebral Cortex

Rats were injected with saline or the gamma-aminobutyric acid (GABA) transaminase inhibitor gamma-vinyl-GABA for 7 days and the effects on GABA content and glutamic acid decarboxylase (GAD) activity, and the protein and mRNA levels of the two forms of GAD (GAD67 and GAD65) in the cerebral cortex were studied. gamma-Vinyl-GABA induced a 2.3-fold increase in GABA content, whereas total GAD activity decreased by 30%. Quantitative immunoblotting showed that the decline in GAD activity was attributable to a 75-80% decrease in GAD67 levels, whereas the levels of GAD65 remained unchanged. RNA slot-blotting with a 32P-labeled GAD67 cDNA probe demonstrated that the change in GAD67 protein content was not associated with a change in GAD67 mRNA levels. Our results suggest that GABA specifically controls the level of GAD67 protein. This effect may be mediated by a decreased translation of the GAD67 mRNA and/or a change in the stability of the GAD67 protein.

Quantitative in situ hybridization analysis of glutamic acid decarboxylase messenger RNA in developing rat cerebellum

The appearance and relative amounts of GAD mRNA in rat cerebellar neurons during postnatal development was studied by in situ hybridization. GAD mRNA content within all GABAergic neurons increased during the first month of postnatal development, but the degree and time course of the increase varied among different neuronal types. In newborn rats, GAD mRNA was present only in the prenatally-formed Purkinje and Golgi cells. GAD mRNA in Golgi cells had reached adult levels by postnatal day 14, while GAD mRNA levels in Purkinje cells reached adult levels one week later. Most basket cells expressed GAD mRNA by postnatal day 14, and final levels were attained one week later. Stellate cells in the bottom two-thirds of the molecular layer attained their final GAD mRNA content by postnatal day 21 whereas stellate cells in close proximity to the pial surface were not yet mature at this age. No GAD mRNA was detected within the external granular layer at any time during development. In adult rat, approximately 40% of cerebellar GAD mRNA was contained within the Purkinje cell population, 38% within the stellate cells, 17% within the basket cells, and only 5% within the Golgi cells. Increases in GAD mRNA within GABAergic neurons during cerebellar development correlated with the timing of neuronal maturation and synaptogenesis in these cell populations, suggesting that synaptic activity affects GAD gene expression in developing cerebellum.