Immunocytochemistry of Central Histaminergic Neurons

Grzanna, Reinhard

Abstract

The existence of histaminergic (HA) neurons in the mammalian central nervous system (CNS) has been established on the basis of biochemical, pharmacological, physiological and anatomical experiments. These neurons have not yet been characterized because no histologic staining method is as yet available for their selective visualization. The objective of the proposed studies is to prepare specific antibodies against histidine decarboxylase (HDC), the biosynthetic enzyme of histamine, for immunocytochemical studies of HA neurons in the rat CNA. Histidine decarboxylase will be purified in an 8-step procedure involving salt precipitation, ion-exchange, adsorption and affinity chromatography, gel filtration, preparative isoelectric focusing and preparative SDS-electrophoresis. The molecular weight of the native enzyme and its subunit composition will be determined by gel filtration and by polyacrylamide gel electrophoresis of the native and reduced enzyme. Monoclonal antibodies will be produced against HDC using partially purified enzyme preparations for immunization of mice. These antibodies will be used to purify HDC by affinity chromatography for production of a high titer antiserum in guinea pigs. Antisera against HDC will be used to analyze by immunocytochemistry the anatomic organization of HA neurons in the rat brain. The PAP method will be employed to identify and localize cell bodies of HA neurons in 100 Mu thick Vibratome sections and to describe their dendritic morphology. The distribution of HA axons and axon terminals will be examined at the light microscopic level with the indirect immunofluorescence method. This method will also be used in combination with retrograde transport of a fluorescent dye to determine the topographic organization of HA neurons projecting to different areas of the forebrain. To obtain information about a possible localization of histamine and HDC in a non-neuronal compartment, discrete lesions of identified HA cell groups will be made and biochemical changes of HA markers in their projection fields will be correlated with ICC staining results in these regions. These neuroanatomic studies should contribute new information about the functional role of central HA neurons, which are affected by widely used drugs, and which have been implicated in arousal mechanisms and in psychiatric disorders, especially depression.