Lateral regions of the rodent striatum reveal elevated glutamate decarboxylase 1 mRNA expression in medium‐sized projection neurons

Abstract

The GABA-synthesizing enzymes glutamate decarboxylase (GAD)1 and GAD2 are universally contained in GABAergic neurons in the central nervous system of the mouse and rat. The two isoforms are almost identically expressed throughout the brain and spinal cord. By using in situ hybridization, we found that the mouse lateral striatum concentrates medium-sized projection neurons with high-level expression of GAD1, but not of GAD2, mRNA. This was confirmed with several types of riboprobe, including those directed to the 5'-noncoding, 3'-noncoding and coding regions. Immunohistochemical localization of GAD1 also revealed predominant localization of the enzyme in the same striatal region. The lateral region of the mouse striatum, harboring such neurons, is ovoid in shape and extends between interaural +4.8 and +2.8, and at lateral 2.8 and dorsoventral 2.0. This intriguing region corresponds to the area that receives afferent inputs from the primary motor and sensory cortex that are presumably related to mouth and forelimb representations. The lateral striatum is included in the basal ganglia-thalamocortical loop, and is most vulnerable to various noxious stimuli in the neurodegeneration processes involving the basal ganglia. We have confirmed elevated expression of GAD1 mRNA, but not of GAD2 mRNA, also in the rat lateral striatum. Image analysis favored the view that the regional increase is caused by elevated cellular expression, and that the greatest number of medium-sized spiny neurons were positive for GAD1 mRNA. The GAD1 mRNA distribution in the mouse lateral striatum partially resembled those of GPR155 and cannabinoid receptor type 1 mRNAs, suggesting functional cooperation in some neurons.

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

“…Similar findings for subcellular localization of the two GADs were obtained when using the GABA antisera [22,58,59]. In marked contrast to the immunohistochemical methods, cell labeling by ISH assay is primarily confined to the cytoplasm of neuronal cell bodies, with little or no staining of punctate structures [35,36,57,60], in congruence with our present observations. The clear labeling of cell bodies by ISH techniques allows us to more precisely locate neurons and clarify their neuronal phenotype, in particular for GABAergic neurons.…”

“…The clear labeling of cell bodies by ISH techniques allows us to more precisely locate neurons and clarify their neuronal phenotype, in particular for GABAergic neurons. Given the differences in subcellular localization, regional expression level, biochemical characteristics, and roles in the regulation of multiple behaviors [36,53–55,60], it is necessary to determine the extent of colocalization of the two GAD isoforms in individual GABAergic cells. Consistent with previous ISH studies showing high percentage of colocalization of the two GADs in the same neurons in several brain regions [35,61], we found the overwhelming majority of GABAergic neurons in the mouse LS contain mRNAs encoding both GAD65 and GAD67, suggesting that the LS GABAergic neurons have the potential to synthesize GABA via both GADs.…”

“…To overcome the limitations of immunohistochemical assay, conventional nonradioactive/radioactive ISH study using sections labeled for GAD65 and GAD67 mRNAs was undertaken [35,36,57,61]. Despite the high sensitivity of visualization, radioactive ISH methods did not allow unambiguous identification of the cell bodies of the majority of GABAergic neurons due to scattering of the emitted radiation and associated silver grains beyond the boundaries of the cell bodies, making a precise assessment difficult [60,61].…”

“…More importantly, earlier nonradioactive ISH studies using riboprobes specific for each of the two GAD mRNAs allows detection of the cell bodies of the vast majority of GABAergic neurons, thus making accurate quantification of GABAergic neurons possible [35,36]. Fluorescence ISH (FISH) with tyramide signal amplification (TSA) has been established to be a useful means of detecting low abundance target mRNAs and colocalization of overlapping genes in a single cell based on its dramatically enhanced sensitivity of detection and high cellular resolution [37–41].…”

Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain

“…GAD exists as two isoforms, GAD1 and GAD2, having different molecular weights of 67 kDa and 65 kDa, respectively. They are encoded by separate genes [ 3 – 5 ] and are coexpressed in most of the GABA-containing neurons, but often in variable ratios [ 3 , 6 – 9 ]. GAD isoforms share enormous sequence homology at protein level, but have different affinity to the cofactor pyridoxal 5′-phosphate and distinct intracellular localizations, which suggests that they might be involved in the synthesis of different pools of GABA with distinct functions [ 1 , 4 , 7 , 10 , 11 ].…”