Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. I. Central nervous system
Section snippets
Characteristics of specific antisera against the vesicular acetylcholine transporter and the vesicular monoamine transporter isoform 2
Rabbit antisera no. 80259 raised against the C-terminal peptide CEDDYNYYSRS of rat VAChT and no. 80182, raised against human vesicular monoamine transporter isoform 2 (VMAT2) C-terminal peptide and cross-reacting with rat VMAT2, were employed at final dilutions of 1:1000–1:2000 in immunohistochemistry as described below. Rabbit polyclonal antibody against rat ChAT used at a working dilution 1:1000 was a generous gift of Dr M. Schemann.
Generation of specific complementary RNA probes for vesicular acetylcholine transporter and choline acetyltransferase
Specific riboprobes for rat VAChT and rat ChAT mRNA were
Distribution of vesicular acetylcholine transporter immunoreactivity in intrinsic and projection systems in the rat brain
The specificity of the VAChT antipeptide antiserum no. 80259 is demonstrated in Fig. 1 by both western blotting and adsorption of immunohistochemical staining with excess unlabelled antigen. VAChT immunoreactivity appears in a western blot as an approximately 65,000 mol. wt protein, consistent with the reports of Gilmor et al. using a polyclonal antibody raised against a rat VAChT fusion protein.[30]The specificity of the antibody in western blotting is demonstrated by the greater than 100-fold
Discussion
Anti-VAChT immunohistochemistry can be used to visualize both cell bodies and terminal fields of cholinergic neurons in routinely paraffin-embedded tissues especially when picric acid-containing formaldehyde fixation solutions such as the common Bouin's or Bouin Hollande fixatives are used. The anatomy of the cholinergic nervous system in mammals has been extensively characterized with immunohistochemistry for the cytosolic cholinergic marker ChAT, and the less specific but highly sensitive
Conclusions
Comprehensive staining of cholinergic elements of the CNS with VAChT reveals terminal fields consistent with the many previous reports in which ChAT immunohistochemistry has been employed, yet far more extensive than previously detected. Sensitivity of detection of VAChT by both immunohistochemistry and immunoblotting is such that recently described VAChT-immunoreactive cholinergic terminal fields in median eminence by immunohistochemistry have been confirmed with western blotting, areal and
Acknowledgements
This work was supported by the Volkswagen-Stiftung and the German Research Foundation. For excellent technical work we thank T. Henke, P. Lattermann, H. Preibsch, E. Rodenberg, S. Roscher, and J. Schmidt. Western blot analysis was supported by Dr M. Bette. For expert photographic assistance we are indebted to H. Schneider. For generous supply of ChAT antibody we thank Dr Schemann (Hannover, Germany).
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