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A monoclonal antibody generated against purified acetylcholine receptor from Torpedo electric organ was used to immunohistochemically localize a neuronal nicotinic acetylcholine receptor. Regions of the rat brain stained with this antibody paralleled those areas of the brain exhibiting [3H]nicotine binding sites and corresponded to areas in which mRNAs encoding for a subunits of the neuronal nicotinic acetylcholine receptor are present. Thus, the anteroventral thalamus, cortex, hippocampus, medial habenula, interpeduncular nucleus, and substantia nigra/ventral tegmental area exhibited significant immunoreactivity. Neurons of the medial habenula and substantia nigra were densely stained, and processes were prominently delineated. Furthermore, in the projection areas of the medial habenula (interpeduncular nucleus and median raphe) axons were strongly immunoreactive and were distributed to distinct subdivisions of the target sites. The present data suggest that there are several discrete neuronal systems in which nicotinic acetylcholine receptors have functional importance. These immunohistochemical studies delineate at the single-cell level the localization within the mammalian central nervous system of certain nicotinic acetylcholine receptors.Although acetylcholine exerts some of its effects within the central nervous system (CNS) through interaction with nicotinic acetylcholine receptors (nAChRs), the identity, localization, and functional significance of these receptors have yet to be clearly resolved. The demonstration that curaremimetic neurotoxins derived from snake venom, such as a-bungarotoxin (a-BGTX), produce neuromuscular blockade by binding to the muscle form of the nAChR greatly facilitated the characterization of this receptor. While a-BGTX binding sites are also present within the CNS, these binding sites appear to be distinct from the neuronal nAChR (1). Lack of a reliable probe for neuronal nAChR, in addition to relatively low levels of nAChR within the CNS, has hampered biochemical identification and characterization of CNS nAChR and has prevented its localization to single neurons.Recent studies using a cDNA clone encoding the a-subunit of the mouse muscle nAChR have suggested that a family of genes is related to the muscle nAChR (2, 3). The screening of genomic libraries prepared from rat neural tissue with a cDNA clone to the muscle nAChR has identified at least two a-subunit-related genes (a3, a4) as members of this family (3). Furthermore, studies utilizing in situ hybridization histochemistry (isHH) indicate that different CNS regions vary considerably in the expression of mRNA that cross-hybridizes with the a3 and a4 cDNAs. These studies offer evidence that different nAChR subtypes exist within the CNS and identify the brain areas in which these gene products are expressed.Immunohistochemical localization of receptors or other antigens offers greater anatomical resolution than do current autoradiographic binding techniques and, in contrast to isHH methods, also allows the determi...

The developmental regulation of neurotransmitter synthesis has been extensively studied and appears in many cases to depend on electrical activity. The central nervous system of the Xenopus embryo and young larva is an attractive subject for such studies, since action potentials first elicited from Xenopus spinal neurons at the time of closure of the neural tube are long in duration and calcium-dependent. Moreover, cells exhibit spontaneous elevations of intracellular calcium during this early period as a consequence of calcium influx through voltage-dependent channels, which induces calcium release from intracellular stores. Since the early differentiation of Xenopus spinal neurons in dissociated cell culture parallels development in vivo, we have examined the maturation of gamma-aminobutyric acid (GABA) immunoreactivity in cultured neurons and explored its dependence on spontaneous calcium influx at early stages of development. We find that specific GABA immunoreactivity develops in spinal neurons in dissociated cell culture with the same time course previously defined in vivo. Additionally, this process requires calcium influx that occurs spontaneously through voltage-dependent channels. The appearance of GABA immunoreactivity is blocked by transcriptional inhibitors. The early appearance of GABA raises the possibility that it may play additional roles at early stages of development.

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