Elsevier

Neuroscience

Volume 84, Issue 2, 11 February 1998, Pages 331-359
Neuroscience

Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. I. Central nervous system

https://doi.org/10.1016/S0306-4522(97)00516-2Get rights and content

Abstract

Antibodies directed against the C-terminus of the rat vesicular acetylcholine transporter mark expression of this specifically cholinergic protein in perinuclear regions of the soma and on secretory vesicles concentrated within cholinergic nerve terminals. In the central nervous system, the vesicular acetylcholine transporter terminal fields of the major putative cholinergic pathways in cortex, hippocampus, thalamus, amygdala, olfactory cortex and interpeduncular nucleus were examined and characterized. The existence of an intrinsic cholinergic innervation of cerebral cortex was confirmed by both in situ hybridization histochemistry and immunohistochemistry for the rat vesicular acetylcholine transporter and choline acetyltransferase. Cholinergic interneurons of the olfactory tubercle and Islands of Calleja, and the major intrinsic cholinergic innervation of striatum were fully characterized at the light microscopic level with vesicular acetylcholine transporter immunohistochemistry. Cholinergic staining was much more extensive for the vesicular acetylcholine transporter than for choline acetyltransferase in all these regions, due to visualization of cholinergic nerve terminals not easily seen with immunohistochemistry for choline acetyltransferase in paraffin-embedded sections. Cholinergic innervation of the median eminence of the hypothalamus, previously observed with vesicular acetylcholine transporter immunohistochemistry, was confirmed by the presence of vesicular acetylcholine transporter immunoreactivity in extracts of median eminence by western blotting. Cholinergic projections to cerebellum, pineal gland, and to the substantia nigra were documented by vesicular acetylcholine transporter-positive punctate staining in these structures. Additional novel localizations of putative cholinergic terminals to the subependymal zone surrounding the lateral ventricles, and putative cholinergic cell bodies in the sensory mesencephalic trigeminal nucleus, a primary sensory afferent ganglion located in the brainstem, are documented here. The cholinergic phenotype of neurons of the sensory mesencephalic trigeminal nucleus was confirmed by choline acetyltransferase immunohistochemistry.

A feature of cholinergic neurons of the central nervous system revealed clearly with vesicular acetylcholine transporter immunohistochemistry in paraffin-embedded sections is the termination of cholinergic neurons on cholinergic cell bodies. These are most prominent on motor neurons of the spinal cord, less prominent but present in some brainstem motor nuclei, and apparently absent from projection neurons of the telencephalon and brainstem, as well as from the preganglionic vesicular acetylcholine transporter-positive sympathetic and parasympathetic neurons visualized in the intermediolateral and intermediomedial columns of the spinal cord. In addition to the large puncta decorating motor neuronal perikarya and dendrites in the ventral horn, vesicular acetylcholine transporter-positive terminal fields are distributed in lamina X surrounding the central canal, where additional small vesicular acetylcholine transporter-positive cell bodies are located, and in the superficial layers of the dorsal horn.

Components of the central cholinergic nervous system whose existence has been controversial have been confirmed, and the existence of new components documented, with immunohistochemistry for the vesicular acetylcholine transporter. Quantitative visualization of terminal fields of known cholinergic systems by staining for vesicular acetylcholine transporter will expand the possibilities for documenting changes in synaptic patency accompanying physiological and pathophysiological changes in these systems.

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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