Vesicular acetylcholine transporter (VAChT) protein: A novel and unique marker for cholinergic neurons in the central and peripheral nervous systems

Abstract

Acetylcholine (ACh) is synthesized in nerve terminals from choline and acetyl coenzyme A by the cytoplasmic enzyme choline acetyltransferase (ChAT). The neurotransmitter is thereafter transported into synaptic vesicles, where it is stored until release. cDNA clones encoding a vesicular ACh transporter (VAChT) were recently isolated. In this paper, we report on the generation of highly specific goat polyclonal antisera to the rat VAChT protein by using a synthetic carboxy-terminal 20-amino-acid peptide sequence as an immunogen. Characterization of the antisera revealed recognition of VAChT, but not vesicular monoamine transporter (VMAT) protein, in transfected CV-1 cells. VAChT immunoreactivity was also detected in cells that endogenously express the protein, such as in PC12 cells and in primary cultures of spinal motoneurons. Absorption controls showed that the VAChT antisera could be completely blocked at the 10(-5) M concentration by cognate peptide used for immunization. The antisera cross-reacted with the VAChT protein in rat and mouse but not in guinea pig, rabbit, or cat. Immunohistochemistry and confocal laser microscopy, using the goat VAChT antisera, showed strong immunoreactivity in discrete fibers and neuronal cell bodies of the central and peripheral nervous systems. Within cell bodies and axonal nerve terminals, as well as in dendrites, the staining appeared granular, presumably representing labeling of synaptic vesicles containing ACh. In the rat central nervous system, VAChT-positive cell bodies were demonstrated in the cerebral cortex, striatum, septum, nucleus basalis, medial habenula, mesopontine complex, cranial, and autonomic and spinal motor nuclei and in the intermediomedial region near the central canal. High densities of VAChT-immunoreactive axonal fibers were encountered in areas such as the olfactory bulb, cerebral cortex, striatum, basal forebrain, amygdala, thalamus, hypothalamus including median eminence, hippocampal formation, superior colliculus, interpeduncular nucleus, and pedunculopontine and laterodorsal tegmental nuclei. In cranial and spinal motor nuclei, particularly large varicosities were seen in close proximity to the motoneuron cell somata and their proximal dendrites. In the peripheral nervous system, VAChT immunoreactivity was also detected in motor endplates of skeletal muscle as well as in fibers of sympathetic and parasympathetic abdominal ganglia, heart atrium, respiratory tract, gastrointestinal tract, pancreas, adrenal medulla, male genitourinary tract, and salivary and lacrimal glands. Direct double labeling revealed colocalization of VAChT and ChAT immunoreactivity in neurons. The results show that VAChT antisera represent novel and unique tools for the study of cholinergic neurons in the central and peripheral nervous systems.

Central cholinergic regulation of respiration: nicotinic receptors

“…The terminals that make synaptic contact with XII motoneurons contain both choline acetyltransferase(ChAT) and acetylcholinesterases (AChEs) [46,47] . The soma and proximal dendrites of XII motoneurons are covered by a plexus of large puncta expressing a vesicular acetylcholine transporter (VAChT) [45,[48][49][50] and a high-affinity choline transporter [51] . Premotor neurons in the caudal medullary intermediate reticular (IRt) region provide inspiratory drive to XII motoneurons [37][38][39]52] .…”

“…Cholinergic synapses are found on the cell bodies and dendrites of spinal ventral horn motoneurons probably including phrenic motoneurons [41,53] , and these synaptic terminals contain VAChT [45,[48][49][50] and high-affinity choline transporter [51] . These synaptic terminals most likely derive from cholinergic neurons in the spinal cord with some from axon collaterals of motoneurons within the motor nuclei [41,53] .…”

Localization of neuropeptide Y1 receptor immunoreactivity in the rat retina and the synaptic connectivity of Y1 immunoreactive cells

“…Mouse monoclonal antibodies directed against calbindin (CaBP; Sigma, St. Louis, MO; Katsetos et al, 1994) and choline acetyltrans-ferase (ChAT; Chemicon, Temecula, CA; Eckenstein and Thoenen, 1982) were used to identify horizontal cells and cholinergic amacrine cells, respectively. A guinea pig polyclonal antibody to vesicular acetylcholine transporter (VAChT; Chemicon; Arvidsson et al, 1997) was also used to confirm Y1 localization to cholinergic amacrine cell processes. Secondary antibodies were Alexa Fluorconjugated affinity-purified antibodies (Molecular Probes, Eugene, OR).…”

Spinal cholinergic interneurons regulate the excitability of motoneurons during locomotion

“…The somata and proximal dendrites of MNs are contacted by large cholinergic varicosities named ''C boutons'' (3)(4)(5)(6)(7)(8)(9)(10)(11). It has been known since 1972 (12) that C boutons originate from spinal cord neurons, but the location of these cells remains unknown (10).…”