Codistribution of GABA‐ with acetylcholine‐synthesizing neurons in the basal forebrain of the rat

Abstract

In recent years, GABAergic neurons have been identified in the basal forebrain where cholinergic cortically projecting neurons are located and known to be important in mechanisms of cortical activation. In the present study in the rat, the relationship of the GABA-synthesizing neurons to the acetylcholine-synthesizing neurons was examined by application of a sequential double staining immunohistochemical procedure involving the peroxidase-antiperoxidase technique for glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT). In these double and adjacent single immunostained series of sections, the GAD+ and ChAT+ cells were mapped, counted and measured with the aid of a computerized image analysis system. Through the entire basal forebrain, there was no evidence for colocalization of GAD and ChAT in the same neurons. Instead, a large population of GAD-immunoreactive neurons is codistributed with ChAT-immunoreactive neurons and outnumbers them by a factor of two: approximately 39,000 GAD+ cells to 18,000 ChAT+ cells. Although the GAD+ and ChAT+ neurons lie intermingled within fascicles of the major longitudinal and transverse forebrain fiber systems in subregions of the basal forebrain, the GAD+ cells are more highly concentrated within different sectors of the pathways and regions than the ChAT+ cells. Although GAD+ neurons resemble ChAT+ neurons in certain regions, both being bi- or multipolar and, on average, medium-sized cells, the GAD+ neurons are, in the majority (51%), small-sized cells (< 15 microns in length) and as a population significantly smaller than the ChAT+ neurons. These results suggest that many GABAergic neurons may represent interneurons in the basal forebrain and potentially exert an inhibitory influence on adjacent cortically projecting cholinergic neurons. Medium- to large GAD+ cells, which resemble similar ChAT+ cells, are also present and represent the majority of the GAD+ cells in the nucleus of the diagonal band of Broca, magnocellular preoptic nucleus, and olfactory tubercle, but represent the minority in the anterior and posterior substantia innominata and globus pallidus. Given their prominent size, such GABAergic cells may also exert an inhibitory influence outside the basal forebrain as projection neurons and potentially in parallel with cholinergic neurons, to certain regions of the cerebral cortex. Accordingly, GABAergic cells may be considered as constituents of the magnocellular basal nucleus and potentially important elements within the ventral extrathalamic relay from the brainstem reticular formation to the cerebral cortex.

Parvalbumin, calbindin, or calretinin in cortically projecting and GABAergic, cholinergic, or glutamatergic basal forebrain neurons of the rat

“…For morphologic and numerical assessments of CBP-immunopositive (ϩ) and CT ϩ cells in the BF, cells were drawn or plotted through the basal forebrain cholinergic cell area. For this purpose, a previously designed and published computer atlas (Gritti et al, 1993) was used that allowed fitting of each section to the atlas and plotting of cells into the contours of the cytoarchitectonically and immunohistochemically delineated nuclei of the BF at six levels (separated by 800 m) from ϳA10.9 to ϳA6.9 (with reference to Paxinos and Watson, 1986). For mapping, all profiles of nerve cell bodies that were immunopositive were plotted (by using a 40ϫ objective) through the entire area of each nucleus at each level (Gritti et al, 1993).…”

“…After a brief rinse with phosphate buffered saline (0.1 M, containing 20 mM MgCl 2 , pH 7.4), the animals were perfused through the ascending aorta with 1,000 ml of 3% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4 over ϳ30 minutes), followed by 10% sucrose in phosphate buffer (pH 7.4, over ϳ15 minutes), as previously described (Gritti et al, 1993(Gritti et al, , 1997. The brains were removed and immersed overnight in 30% sucrose in phosphate buffer for cryoprotection.…”

“…In addition to cholinergic cells, other neurons constitute important elements of the BF cell population. Among these, ␥-amino butyric acidergic (GABAergic) neurons are twice as numerous and more heterogeneous than the cholinergic neurons (Brashear et al, 1986;Kiss et al, 1990a;Gritti et al, 1993). Relatively small GABAergic neurons project caudally and most probably locally (Gritti et al, 1994) to the diencephalon and brainstem, and relatively large GABAergic neurons project in parallel with the cholinergic neurons to the cerebral cortex (Gritti et al, 1997).…”

“…With immunoperoxidase staining, we examined the distribution of all Parv-, Calb-, and Calret-immunostained neurons and the distribution of those projecting to the prefrontal or parietal cortex visualized by retrograde transport of cholera toxin (CT). Given unanticipated larger numbers of CBP-containing neurons as compared with GABAergic neurons (Gritti et al, 1993(Gritti et al, , 1997 in estimated total and cortically projecting cell contingents, we subsequently examined the colocalization of the CBPs with the synthetic enzyme for GABA, glutamic acid decarboxylase (GAD), by dual-immunofluorescent staining. We also investigated whether Calb might be colocalized with choline acetyl transferase (ChAT) in the rat brain, as documented in the monkey brain (Chang and Kuo, 1991).…”

Hypoglycemia activates arousal-related neurons and increases wake time in adult rats

“…Diagrams of atlas levels used for counting Fos staining in selected basal forebrain cholinergic nuclei: medial septum (MS), diagonal band of Broca (DBB), magnocellular preoptic nucleus (MCPO), based on ref. 23. Diagrams are modified from a rat brain atlas [48].…”