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.

Cholinergic neurons of the pelvic autonomic ganglia and uterus of the female rat: distribution of axons and presence of muscarinic receptors

“…c is focused on the plane of the emulsion and shows the paucity of the [ 3 H]QNB binding (nonspecific binding) following preincubation in the muscarinic antagonist atropine and d is focused on the plane of the tissue in the same section as in c. Bar 50 µm. ×165 1996; Arvidsson et al 1997). Further, the exclusive presence of VAChT in small clear synaptic vesicles suggests that VAChT is a specific marker for synaptic vesicles containing ACh (Gilmor et al 1996;Weihe et al 1996;Varoqui and Erickson 1997).…”

“…Recently this difficulty was overcome with the development of a ChAT antibody that reliably immunostains peripheral cholinergic neurons (Schemann et al 1993;Keast et al 1995). Also, newly generated antibodies against the ACh vesicular transporter molecule (VAChT) have become available and they also immunostain cholinergic nerves in the peripheral nervous system (Weihe et al 1996;Arvidsson et al 1997;Li et al 1998a,b). Furthermore, ACh influences not only nicotinic, but also muscarinic, transmission in various parasympathetic ganglia including the PG (Somogyi and de Groat 1993;van Koppen et al 1987;Fischer et al 1998).…”

Neurons with choline acetyltransferase immunoreactivity and mRNA are present in the human cerebral cortex

“…A standard RNA blot analysis failed to detect ChAT mRNA in the rat cerebral cortex (Ibáñez et al 1991), but a very sensitive method, reverse transcriptase-polymerase chain reaction, revealed a low level of ChAT expression in the rat cortex (Cavicchioli et al 1991). Vesicular acetylcholine transporter (VAChT), a new marker of cholinergic neurons, has been identified in small ovoid or fusiformshaped neurons in layers II and III of the rat cerebral cortex (Arvidsson et al 1997;Weihe et al 1996). Taken together, these observations indicate that it is likely that a small number of rat cortical neurons are cholinergic.…”

Reduced neuronal co-localisation of nardilysin and the putative α-secretases ADAM10 and ADAM17 in Alzheimer’s disease and Down syndrome brains

“…To prevent any cross-reaction between the two different polyclonal antibodies or the subsequently applied secondary antisera, the sections were incubated with non-immunised normal rabbit IgG (1:500, dilution in 2% BSA-PBS) between the incubation steps (Arvidsson et al 1997). …”