Comparative analyses of the cholinergic locus of ChAT and VAChT and its expression in the silkworm Bombyx mori.

The cholinergic locus, which encodes choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), is specifically expressed in cholinergic neurons, maintaining the cholinergic phenotype. The organization of the locus is conserved in Bilateria. Here we examined the structure of cholinergic locus and cDNA coding for ChAT and VAChT in the silkworm, Bombyx mori. The B. mori ChAT (BmChAT) cDNA encodes a deduced polypeptide including a putative choline/carnitine O-acyltransferase domain and a conserved His residue required for catalysis. The B. mori VAChT (BmVAChT) cDNA encodes a polypeptide including a putative major facilitator superfamily domain and 10 putative transmembrane domains. BmChAT and BmVAChT cDNAs share the 5'-region corresponding to the first and second exon of cholinergic locus. Polymerase chain reaction analyses revealed that BmChAT and BmVAChT mRNAs were specifically expressed in the brain and segmental ganglia. The expression of BmChAT was detected 3 days after oviposition. The expression level was almost constant during the larval stage, decreased in the early pupal stage, and increased toward eclosion. The average ratios of BmChAT mRNA to BmVAChT mRNA in brain-subesophageal ganglion complexes were 0.54±0.10 in the larvae and 1.92±0.11 in adults. In addition, we examined promoter activity of the cholinergic locus and localization of cholinergic neurons, using a baculovirus-mediated gene transfer system. The promoter sequence, located 2kb upstream from the start of transcription, was essential for cholinergic neuron-specific gene õexpression. Cholinergic neurons were found in several regions of the brain and segmental ganglia in the larvae and pharate adults.

Unusual regulation of splicing of the cholinergic locus in Caenorhabditis elegans.

The essential neurotransmitter acetylcholine functions throughout the animal kingdom. In Caenorhabditis elegans, the acetylcholine biosynthetic enzyme [choline acetyltransferase (ChAT)] and vesicular transporter [vesicular acetylcholine transporter (VAChT)] are encoded by the cha-1 and unc-17 genes, respectively. These two genes compose a single complex locus in which the unc-17 gene is nested within the first intron of cha-1, and the two gene products arise from a common pre-messenger RNA (pre-mRNA) by alternative splicing. This genomic organization, known as the cholinergic gene locus (CGL), is conserved throughout the animal kingdom, suggesting that the structure is important for the regulation and function of these genes. However, very little is known about CGL regulation in any species. We now report the identification of an unusual type of splicing regulation in the CGL of C. elegans, mediated by two pairs of complementary sequence elements within the locus. We show that both pairs of elements are required for efficient splicing to the distal acceptor, and we also demonstrate that proper distal splicing depends more on sequence complementarity within each pair of elements than on the sequences themselves. We propose that these sequence elements are able to form stem-loop structures in the pre-mRNA; such structures would favor specific splicing alternatives and thus regulate CGL splicing. We have identified complementary elements at comparable locations in the genomes of representative species of other animal phyla; we suggest that this unusual regulatory mechanism may be a general feature of CGLs.

Analysis on interrelation between electroacupuncture-induced cumulative analgesic effect and hypothalamic cholinergic activities in chronic neuropathic pain rats.

OBJECTIVE: To observe the effects of repeated electroacupuncture (EA) of Zusanli (ST36)- Yanglingquan (GB34) on hypothalamic acetylcholinesterase (AchE) and vesicular acetylcholine (ACh) transporter (VAChT) activities and choline acetyltransferase (ChAT) mRNA and muscarinic M1 receptor (M1R) mRNA expression in chronic constrictive injury (CCI) and/or ovariectomy (OVX) rats so as to reveal its underlying mechanism in cumulative analgesia. METHODS: A total of 103 female Wistar rats were randomly divided into normal control (n =15), CCI (n =15), CCI+EA2d (n =15), CCI+EA2W (n =15), OVX+CCI =13), OVX+CCI+EA2d (n =15), and OVX+CCI+EA2W groups (n =15). CCI model was established by ligature of the unilateral sciatic nerve with surgical suture. Memory impairment model was established by removal of the bilateral ovaries. Morris water test was conducted to evaluate the OVX rats' memory learning ability, and the thermal pain threshold (PT) of the bilateral paws was detected the next morning after EA. EA (2/15 Hz, 1 mA) was applied to bilateral ST36-GB34 for 30 min, once daily for 2 days or 2 weeks, respectively. Hypothalamic AChE activity was detected by histochemistry, VAChT immunoactivity was determined by immunohistochemistry, and ChAT mRNA and M1R mRNA expressions were assayed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In comparison with the normal control group, the AChE activity in hypothalamic arcuate nucleus (ARC) and supraoptic nucleus (SON) regions of CCI group, AChE activity in paraventricular nucleus (PVN), ARC, and SON regions of OVX+CCI group, and hypothalamic muscarinic M1R mRNA expression levels in both CCI and OVX+CCI groups were down-regulated significantly (P <0.05). Compared with the CCI group, the AChE activities in hypothalamic ARC and SON regions of CCI+EA2d and CCI+EA2W groups and PVN region of CCI+EA2W group and hypothalamic ChAT mRNA and M1R mRNA expression levels in CCI+EA2W group were up-regulated considerably (P <0.05). In comparison with the OVX+CCI group, the AChE activities in PVN, ARC, and SON regions and the expressions of hypothalamic ChAT mRNA and VAChT in ARC region of OVX+CCI+EA2W group were up-regulated remarkably (P <0.05). The effects in rats of CCI+EA2W group were evidently superior to those of OVX+CCI+EA2d group in up-regulating AChE activities in PVN, ARC, and SON regions, VAChT immunoactivity in ARC region, and expression levels of hypothalamic ChAT mRNA and M1R mRNA (P <0.05). Similar situations were found in OVX+CCI rats after EA2W. It suggested a cumulative effect after repeated EA of ST36-GB34. Comparison between CCI+EA2W and OVX+CCI+EA2W groups showed that the effects in rats of the former group were evidently better than those of the latter group in up-regulating AChE activity in ARC and SON regions and the expressions of hypothalamic ChAT mRNA and M1 mRNA (P <0.05), suggesting a reduction of EA2W effects after OVX. CONCLUSION: Repeated EA can significantly up-regulate AChE and VAChT activities and ChAT mRNA and M1R mRNA expressions in the hypothalamus of CCI and OVX+CCI rats, which may contribute to the cumulative analgesic effects of repeated EA and be closely related to the animals' neuromemory ability.

Selenium induces cholinergic motor neuron degeneration in Caenorhabditis elegans.

Selenium is an essential micronutrient required for cellular antioxidant systems, yet at higher doses it induces oxidative stress. Additionally, in vertebrates environmental exposures to toxic levels of selenium can cause paralysis and death. Here we show that selenium-induced oxidative stress leads to decreased cholinergic signaling and degeneration of cholinergic neurons required for movement and egg-laying in Caenorhabditis elegans. Exposure to high levels of selenium leads to proteolysis of a soluble muscle protein through mechanisms suppressible by two pharmacological agents, levamisole and aldicarb which enhance cholinergic signaling in muscle. In addition, animals with reduction-of-function mutations in genes encoding post-synaptic levamisole-sensitive acetylcholine receptor subunits or the vesicular acetylcholine transporter developed impaired forward movement faster during selenium-exposure than normal animals, again confirming that selenium reduces cholinergic signaling. Finally, the antioxidant reduced glutathione, inhibits selenium-induced reductions in egg-laying through a cellular protective mechanism dependent on the C. elegans glutaredoxin, GLRX-21. These studies provide evidence that the environmental toxicant selenium induces neurodegeneration of cholinergic neurons through depletion of glutathione, a mechanism linked to the neuropathology of Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease.

VAChT knock-down mice show normal prepulse inhibition but disrupted long-term habituation.

The neurotransmitter acetylcholine (ACh) plays a crucial role in both the central and peripheral nervous system. Central cholinergic transmission is important for cognitive functions and cholinergic disruptions have been associated with different neural disorders. We here tested the role of cholinergic transmission in basic cognitive functions, i.e. in prepulse inhibition (PPI) and short-term habituation (STH) as well as long-term habituation (LTH) of startle using mice with a 65% knockdown (KD) of the vesicular ACh transporter (VAChT). These mice are slow in refilling cholinergic synaptic transmitter vesicles, leading to a reduced cholinergic tone. Prepulse inhibition has been assumed to be mediated by cholinergic projections from the midbrain to the reticular formation. Surprisingly, PPI and STH were normal in these mice, whereas LTH was disrupted. This disruption could be rescued by pre-testing injections of the ACh esterase inhibitor galantamine, but not by post-testing injections. The lack of a PPI deficit might be because of the fact that VAChT KD mice show disruptions mainly in prolonged cholinergic activity, therefore the transient activation by prepulse processing might not be sufficient to deplete synaptic vesicles. The disruption of LTH indicates that the latter depends on a tonic cholinergic inhibition. Future experiments will address which cholinergic cell group is responsible for this effect.

Hypothalamic proopiomelanocortin (POMC) neurons have a cholinergic phenotype.

Neuronal networks originating in the hypothalamic arcuate nucleus play fundamental roles in the control of energy balance. Neuropeptide Y (NPY)-producing neurons in the arcuate nucleus stimulate food intake, whereas arcuate nucleus neurons that release the proopiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) potently reduce food intake. Relatively little attention has been focused on classical neurotransmitters in regulation of food intake. Here, we have investigated the potential presence of acetylcholine (ACh) in NPY- and POMC-containing neuronal populations of the arcuate nucleus. Antisera to proteins required for cholinergic neurotransmission, including choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT), were employed in double-labeling immunohistochemical experiments. In colchicine-treated rats, ChAT- and VAChT-immunopositive cell bodies were located in the ventral aspect of the arcuate nucleus. ChAT and VAChT immunoreactivities were demonstrated in alpha-MSH- and cocaine- and amphetamine-regulated transcript (CART)-containing cell bodies of the arcuate nucleus, whereas cell bodies containing NPY or agouti-related peptide (AGRP) were distinct from VAChT-immunoreactive neuronal perikarya. VAChT immunoreactivity was also present in a large number of alpha-MSH-containing nerve fiber varicosities throughout the central nervous system. In the commissural part of the nucleus tractus solitarius, no alpha-MSH-containing cell bodies were found to have ChAT or VAChT immunoreactivity. The presence of markers for cholinergic neurotransmission in a subpopulation of hypothalamic POMC/CART neurons suggests co-release of ACh with peptides derived from the POMC precursor and CART. The results indicate a role for ACh in control of energy balance, mediating the effects of peripheral hormones such as leptin and insulin.