Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms

Abstract

Two isoforms of glutamic acid decarboxylase (GAD67 and GAD65) and their mRNAs were localized in the rat brain by immunohistochemistry and nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes. In most brain regions, both GAD isoforms were present in neuronal cell bodies as well as axon terminals. A few populations of neurons, such as those in the reticular nucleus of the thalamus, exhibited similar cell body labeling for both GADs. However, in many brain regions, the cell bodies that were immunoreactive for GAD67 were often more numerous than those that were immunoreactive for GAD65. In contrast, the density (quantity) of GAD65-immunoreactive axon terminals was higher than that of GAD67-immunoreactive terminals. Strong parallels were observed between the intensity of immunohistochemical labeling of cell bodies and the levels of mRNA labeling for both GAD isoforms. Many groups of GAD-containing cell bodies were distinctly labeled for GAD67, and these same groups of neurons were heavily labeled for GAD67 mRNA. Such neurons included Purkinje cells of the cerebellar cortex, nonpyramidal cells in the cerebral cortex, and neurons of the reticular nucleus of the thalamus. Similar parallels in labeling were observed for GAD65 and its mRNA. Distinct cell body labeling for the protein and associated high levels of GAD65 mRNA were found in neurons of the reticular nucleus of the thalamus and periglomerular cells in the olfactory bulb. However, many cell bodies were not readily labeled for GAD65 with immunohistochemical methods. Such absence or weakness of cell body labeling for the protein was associated with low or moderate levels of GAD65 mRNA. Even though light cell body staining was frequently observed for GAD65 and its mRNA, strong axon terminal labeling for GAD65 was present. Thus, in the deep cerebellar nuclei to which the Purkinje cells of the cerebellar cortex project, strong terminal labeling was observed for both GAD isoforms even though only light cell body labeling of the Purkinje cells was obtained for GAD65 and its mRNA. The findings suggest that the two isoforms of GAD are present in most classes of GABA neurons but that they are not similarly distributed within the neurons. GAD67 is present in readily detectable amounts in many GAD-containing cell bodies whereas GAD65 is particularly prominent in many axon terminals. In addition, neurons that express either form of GAD mRNA also express the corresponding protein. Levels of labeling for the GAD mRNAs suggest that, under normal conditions, the synthesis of GAD65 is frequently lower than that of GAD67.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurochemical characterization of tyrosine hydroxylase-immunoreactive interneurons in the developing rat cerebral cortex

“…To confirm this phenotype in developing TH-IR neurons, we examined the coproduction of GAD by double-labeled for TH and GAD67, the isoform of GAD prevalent in somata of cortical neurons (Erlander et al, 1991;Feldblum et al, 1993;Esclapez et al, 1994). Neurons and fibers immunoreactive for GAD67 were distributed throughout the cortex as expected.…”

“…Furthermore, some cortical TH-IR neurons colocalized with GAD67, the isoform of the GABA-synthesizing enzyme most common in cortical neuron somata (Erlander et al, 1991;Feldblum et al, 1993;Esclapez et al, 1994). The intensity of GAD67 immunoreactivity in many double-labeled cell profiles was faint, suggesting that the number of cell profiles scored for TH and GAD colocalization was an underestimate.…”

CRF2 Receptor Deficiency Eliminates the Long-Lasting Vulnerability of Motivational States Induced by Opiate Withdrawal

“…To further investigate the neural substrates underlying the CRF 2 receptor-mediated vulnerability, in situ hybridization studies are also carried out. In particular, expression of the key catecholamine and γ-aminobutyric acid (GABA) synthesis enzymes tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD 67 ) is assessed in stress-and drug dependence-relevant brain regions, such as the ventral tegmental area (VTA), the locus coeruleus (LC), the central (CeA), and the basolateral (BLA) nucleus of the amygdala (Esclapez et al, 1994;Ingallinesi et al, 2012;Koob, 2008;Papaleo et al, 2007;Phelps and LeDoux, 2005;Vrana et al, 1993).…”

GAD isoforms exhibit distinct spatiotemporal expression patterns in the developing mouse lens: Correlation with Dlx2 and Dlx5

“…GABA is synthesized from glutamate in an enzymatic reaction catalyzed by members of the glutamic acid decarboxylase (GAD) family in most, if not all GABAϩ cells. Two known molecular isoforms of GAD, GAD65 and GAD67, are coexpressed at variable ratios in GABAergic neurons (Erlander et al, 1991;Martin and Rimvall, 1993;Esclapez et al, 1994;Martin et al, 2000;Varju et al, 2001). The GAD isoforms are highly homologous at the protein level, but show different affinity to the cofactor pyridoxalphosphate (PLP) and have distinct subcellular localizations.…”