Sensory and autonomic innervation of the local tissues at traditional acupuncture point locations GB14, ST2 and ST6.

OBJECTIVE: To visualize and compare the sensory and autonomic innervation of the local tissues at the sites of different traditional acupuncture points in the rat forehead and face by histochemical examination. METHODS: GB14 (Yangbai), ST2 (Sibai) and ST6 (Jiache) were selected as the representative traditional acupuncture points in this study, and the local tissues at these sites were dissected in rats after perfusion followed by double or triple fluorescent histochemical staining. Here, calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT) were used to label the sensory, sympathetic and parasympathetic nerve fibers, respectively. RESULTS: The CGRP+ sensory, TH+ sympathetic and VAChT+ parasympathetic nerve fibers were simultaneously demonstrated in the local tissues at GB14, ST2 and ST6. Although the three kinds of nerve fibers ran in parallel or intermingled with each other, by the analysis from the view of three-dimensional reconstruction, it was clear that each of them distributed in an independent pattern to their corresponding target tissues including the blood vessels, hair follicles, arrector pili and subcutaneous muscles, as well as sebaceous glands. CONCLUSION: Our study demonstrated the sensory and autonomic innervation of the local tissues at GB14, ST2 and ST6, providing neurochemical evidence indicating that the CGRP+ sensory, TH+ sympathetic and VAChT+ parasympathetic nerve fibers form a neural network at these point locations that may respond to acupuncture stimulation.

The organization of cholinergic projections in the visual thalamus of the mouse.

Cholinergic projections from the brainstem serve as important modulators of activity in visual thalamic nuclei such as the dorsal lateral geniculate nucleus (dLGN). While these projections have been studied in several mammals, a comprehensive examination of their organization in the mouse is lacking. We used the retrograde transport of viruses or cholera toxin subunit B (CTB) injected in the dLGN, immunocytochemical labeling with antibodies against choline acetyltransferase (ChAT), brain nitric oxide synthase (BNOS), and vesicular acetylcholine transporter (VAChT), ChAT-Cre mice crossed with a reporter line (Ai9), as well as brainstem virus injections in ChAT-Cre mice to examine the pattern of thalamic innervation from cholinergic neurons in the pedunculopontine tegmental nucleus (PPTg), laterodorsal tegmental nucleus (LDTg), and the parabigeminal nucleus (PBG). Retrograde tracing demonstrated that the dLGN receives input from the PPTg, LDTg, and PBG. Viral tracing in ChAT-Cre mice and retrograde tracing combined with immunocytochemistry revealed that many of these inputs originate from cholinergic neurons in the PBG and PPTg. Most notable was an extensive cholinergic projection from the PBG which innervated most of the contralateral dLGN, with an especially dense concentration in the dorsolateral shell, as well as a small region in the dorsomedial pole of the ipsilateral dLGN. The PPTg was found to provide a sparse somewhat diffuse innervation of the ipsilateral dLGN. Neurons in the PPTg co-expressed ChAT, BNOS, and VAChT, whereas PBG neurons expressed ChAT, but not BNOS or VAChT. These results highlight the presence of distinct cholinergic populations that innervate the mouse dLGN.

Remodeling of cholinergic input to the hippocampus after noise exposure and tinnitus induction in Guinea pigs.

Here, we investigate remodeling of hippocampal cholinergic inputs after noise exposure and determine the relevance of these changes to tinnitus. To assess the effects of noise exposure on the hippocampus, guinea pigs were exposed to unilateral noise for 2 hr and 2 weeks later, immunohistochemistry was performed on hippocampal sections to examine vesicular acetylcholine transporter (VAChT) expression. To evaluate whether the changes in VAChT were relevant to tinnitus, another group of animals was exposed to the same noise band twice to induce tinnitus, which was assessed using gap-prepulse Inhibition of the acoustic startle (GPIAS) 12 weeks after the first noise exposure, followed by immunohistochemistry. Acoustic Brainstem Response (ABR) thresholds were elevated immediately after noise exposure for all experimental animals but returned to baseline levels several days after noise exposure. ABR wave I amplitude-intensity functions did not show any changes after 2 or 12 weeks of recovery compared to baseline levels. In animals assessed 2-weeks following noise-exposure, hippocampal VAChT puncta density decreased on both sides of the brain by 20-60% in exposed animals. By 12 weeks following the initial noise exposure, changes in VAChT puncta density largely recovered to baseline levels in exposed animals that did not develop tinnitus, but remained diminished in animals that developed tinnitus. These tinnitus-specific changes were particularly prominent in hippocampal synapse-rich layers of the dentate gyrus and areas CA3 and CA1, and VAChT density in these regions negatively correlated with tinnitus severity. The robust changes in VAChT labeling in the hippocampus 2 weeks after noise exposure suggest involvement of this circuitry in auditory processing. After chronic tinnitus induction, tinnitus-specific changes occurred in synapse-rich layers of the hippocampus, suggesting that synaptic processing in the hippocampus may play an important role in the pathophysiology of tinnitus.

Diurnal Variation of the Peripheral Cholinergic Antiinflammatory Function in Mice.

AIMS: Cholinergic antiinflammatory (CAI) pathway functions importantly in inflammation via α7 nicotinic acetylcholine receptors (α7nAChR). The present work tested circadian rhythm in peripheral CAI activity and validities of CAI activity and glucocorticoids in chronotherapy for lipopolysaccharide (LPS)-induced shock. METHODS: Vesicular acetylcholine transporter (VAChT) expressed in liver and kidney was examined every 3 h in C57BL/6 mice. Proinflammatory cytokines in serum and survival time in shock were monitored after LPS injection every 3 h. Mifepristone, antagonist of glucocorticoid receptors, and methyllycaconitine (MLA), antagonist of α7nAChR, were administrated before LPS to block antiinflammatory function of endogenous glucocorticoids and acetylcholine. RESULTS: Both levels of tumor necrosis factor α, interleukin 1ß, and interleukin 6 and mortality exhibited diurnal variations with prominent peaks when LPS was given at 15:00, and the minimum mortality occurred at 00:00. Expression of VAChT increased during resting period. MLA increased serum proinflammatory cytokines slightly, but not affected survival rate. Both differences in cytokines and in survival times between LPS injection at 15:00 and 00:00 were eliminated by mifepristone, but not by MLA. CONCLUSION: Peripheral CAI pathway exerts more powerful antiinflammatory effect during resting period. Glucocorticoids appear to be efficient in chronotherapy for septic shock.

Neuregulin 1 confers neuroprotection in SOD1-linked amyotrophic lateral sclerosis mice via restoration of C-boutons of spinal motor neurons.

INTRODUCTION: Increasing evidence implicates the role of the cell types surrounding motor neurons, such as interneurons and glial cells, in non-cell autonomous neurodegeneration of amyotrophic lateral sclerosis (ALS). C-boutons, the large cholinergic synapses that innervate spinal α-motor neurons to control their excitability, are progressively lost from motor neurons in both human ALS and mutant Cu/Zn superoxide dismutase 1 (SOD1)-ALS mice. Neuregulin-1 (NRG1), a trophic factor implicated in neural development, transmission, and synaptic plasticity, has been reported to localize in the synapse of C-boutons. However, the roles of NRG1 in maintenance of motor neuron health and activity, as well as the functional consequences of its alteration in motor neuron disease, are not fully understood. RESULTS: NRG1 was localized to the post-synaptic face of C-boutons and its expression was significantly lost in SOD1-ALS mice and human ALS patients. Losses of NRG1 expression and C-boutons occurred almost contemporaneously in SOD1-ALS mice. In addition, expressions of ErbB3 and ErbB4, receptors for NRG1, were reduced in the motor neurons of SOD1-ALS mice. Furthermore, viral-mediated delivery of type III-NRG1 to the spinal cord restored the number of C-boutons and extended the survival time of SOD1-ALS mice. CONCLUSIONS: These results suggest that maintenance of NRG1-ErbB4/3 axis by supplementation of NRG1 confers neuroprotection in motor neuron disease, partly through the maintenance of C-boutons of spinal motor neurons.

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.

A major external source of cholinergic innervation of the striatum and nucleus accumbens originates in the brainstem.

Cholinergic transmission in the striatal complex is critical for the modulation of the activity of local microcircuits and dopamine release. Release of acetylcholine has been considered to originate exclusively from a subtype of striatal interneuron that provides widespread innervation of the striatum. Cholinergic neurons of the pedunculopontine (PPN) and laterodorsal tegmental (LDT) nuclei indirectly influence the activity of the dorsal striatum and nucleus accumbens through their innervation of dopamine and thalamic neurons, which in turn converge at the same striatal levels. Here we show that cholinergic neurons in the brainstem also provide a direct innervation of the striatal complex. By the expression of fluorescent proteins in choline acetyltransferase (ChAT)::Cre(+) transgenic rats, we selectively labeled cholinergic neurons in the rostral PPN, caudal PPN, and LDT. We show that cholinergic neurons topographically innervate wide areas of the striatal complex: rostral PPN preferentially innervates the dorsolateral striatum, and LDT preferentially innervates the medial striatum and nucleus accumbens core in which they principally form asymmetric synapses. Retrograde labeling combined with immunohistochemistry in wild-type rats confirmed the topography and cholinergic nature of the projection. Furthermore, transynaptic gene activation and conventional double retrograde labeling suggest that LDT neurons that innervate the nucleus accumbens also send collaterals to the thalamus and the dopaminergic midbrain, thus providing both direct and indirect projections, to the striatal complex. The differential activity of cholinergic interneurons and cholinergic neurons of the brainstem during reward-related paradigms suggest that the two systems play different but complementary roles in the processing of information in the striatum.

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.

Analysis on interrelation between electroacupuncture-induced cumulative analgesic effect and hypothalamic cholinergic activities in chronic neuropathic pain rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

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.

Transgenic mice as a model of pre-clinical Alzheimer's disease.

At diagnosis, Alzheimer's disease (AD) brains are extensively burdened with plaques and tangles and display a degree of synaptic failure most likely beyond therapeutic treatment. It is therefore crucial to identify early pathological events in the progression of the disease. While it is not currently feasible to identify and study early, pre-clinical stages of AD, transgenic (Tg) models offer a valuable tool in this regard. Here we investigated cognitive, structural and biochemical CNS alterations occurring in our newly developed McGill-Thyl-APP Tg mice (over-expressing the human amyloid precursor protein with the Swedish and Indiana mutations) prior to extracellular plaque deposition. Pre-plaque, 3-month old Tg mice already displayed cognitive deficits concomitant with reorganization of cortical cholinergic pre-synaptic terminals. Conformational specific antibodies revealed the early appearance of intracellular amyloid ß (Aß)-oligomers and fibrillar oligomers in pyramidal neurons of cerebral cortex and hippocampus. At the same age, the cortical levels of insulin degrading enzyme -a well established Aß-peptidase, were found to be significantly down-regulated. Our results suggest that, in the McGill-Thy1-APP Tg model, functional, structural and biochemical alterations are already present in the CNS at early, pre-plaque stages of the pathology. Accumulation of intraneuronal neurotoxic Aß-oligomers (possibly caused by a failure in the clearance machinery) is likely to be the culprit of such early, pre-plaque pathology. Similar neuronal alterations might occur prior to clinical diagnosis in AD, during a yet undefined 'latent' stage. A better understanding of such pre-clinical AD might yield novel therapeutic targets and or diagnostic tools.

Coenzyme Q10 diminishes ischemia-reperfusion induced apoptosis and nerve injury in rabbit urinary bladder.

AIMS: Ischemia/reperfusion (I/R) can significantly change the nerve function of the bladder, thus resulting in detrusor weakness and overactivity. CoQ10 is a lipid-soluble cofactor found naturally in the mitochondria and has been reported to have neuroprotective and antiapoptosis effects. The aim of this study is to determine if CoQ10 can protect bladders subjected to I/R injury. METHODS: Four groups of male New Zealand White rabbits (N = 4) were treated with CoQ10 (3 mg/kg body weight/day) (groups 1 and 2) or vehicle (groups 3 and 4). In groups 2 and 4 (I/R groups), bilateral vesicular ischemia was induced for 2 hr and the rabbits allowed to recover for 2 weeks. Groups 1 and 3 were controls and given sham surgery. The cholinergic nerve marker, vesicular acetylcholine transporter (VAChT), was examined by western blotting. Nerve density and cell apoptosis were calculated by immunohistochemistry. RESULTS: I/R significantly decrease bladder innervation; CoQ10 has significant neuroprotective effects, which are evidenced by increased VAChT expression and neurofilament immunostaining. Detrusor cells apoptosis increase significantly by I/R. CoQ10 control and I/R groups both show significantly lower apoptosis than vehicle groups. CONCLUSIONS: The current study clearly demonstrated that these CoQ10 supplement provides significant bladder protection against I/R injury. This protective effect is in part by protecting damage to cholinergic innervation.

Neurokinin-1 receptors in cholinergic neurons of the rat ventral pallidum have a predominantly dendritic distribution that is affected by apomorphine when combined with startle-evoking auditory stimulation.

Cholinergic neurons of the basal forebrain are implicated in startle reflex inhibition by a prior weak stimulus often referred to as prepulse inhibition (PPI) and used as an index of sensorimotor gating deficits in schizophrenia. Gating deficits can be produced in rodent models by acute systemic administration of apomorphine, a non-selective dopamine D1 and D2 receptor agonist that also affects trafficking of neurokinin-1 (NK(1)) receptors induced by startle evoking auditory stimulation (AS) in midbrain neurons. We used electron microscopic immunolabeling of NK(1) receptors and the vesicular acetylcholine transporter (VAchT) to test the hypothesis that the subcellular distributions of these receptors in cholinergic neurons of the rat ventral pallidum are subject to a similar regulation. In vehicle controls, NK(1) immunogold was often seen near cytoplasmic endomembranes in somata and large dendrites, but was more equally distributed in cytoplasmic and plasmalemmal compartments of medium dendrites, and principally located on the plasma membrane of small dendrites. These labeling patterns appeared to be largely independent of whether the NK(1) receptor was co-expressed with VAchT, however only the medium and small VAchT-labeled dendrites showed significant treatment-specific differences in NK(1) immunogold distributions. The NK(1) receptor immunogold particle density on the plasma membrane of medium cholinergic dendrites was significantly enhanced by combined apomorphine and AS, while neither alone affected either the plasmalemmal density or the equality of the plasmalemmal and cytoplasmic distributions of NK(1) receptors in these dendrites. Small cholinergic dendrites showed a significant AS-induced increase in both the plasmalemmal and cytoplasmic density of NK(1) gold particles, and an apomorphine-induced disruption of the preferential plasmalemmal targeting of the NK(1) receptors. These results provide ultrastructural evidence that NK(1) receptors in cholinergic neurons of the ventral pallidum have subcellular locations and plasticity conducive to active involvement in dopamine-dependent sensorimotor processing.

SEC14-like protein 1 interacts with cholinergic transporters.

Trafficking of the vesicular acetylcholine transporter (VAChT) to synaptic vesicles has the potential to regulate storage and release of acetylcholine. We used the C-terminal tail of the vesicular acetylcholine transporter as bait for the screening of a brain cDNA library by yeast-two hybrids. Here we report an interaction uncovered in this screening with SEC14L1, a mammalian SEC14-like protein that may function as a phospholipid transfer protein. The interaction of VAChT and SEC14L1 occurred through the GOLD domain found in the latter and was confirmed in mammalian cells. In addition, we also found that SEC14L1 co-immunoprecipitates with the high affinity choline transporter (CHT1), but not with synaptophysin or synaptotagmin. In cultured cells SEC14L1 was predominantly found in the cytosol with little or no localization in defined organelles. In contrast, overexpression of VAChT or CHT1 with SEC14L1 recruited the latter to large intracellular organelles similar to vesicles or vesicle aggregates. Finally, we find that overexpression of SEC14L1 modestly decreases high affinity choline transport activity. We suggest that interaction of cholinergic transporters with proteins containing the GOLD domain may be relevant for transporter function.

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.

The organization of the brainstem and spinal cord of the mouse: relationships between monoaminergic, cholinergic, and spinal projection systems.

Information regarding the organization of the CNS in terms of neurotransmitter systems and spinal connections in the mouse is sparse, especially at the level of the brainstem. An overview is presented of monoaminergic and cholinergic systems in the brainstem and spinal cord that were visualized immunohistochemically in inbred C57BL/6 and outbred CD-1 mice. This information is complemented with data on spinal cord-projecting systems that were characterized using retrograde tracing, spinal hemisections, and double labeling techniques. Attention is given to differences in these systems related to spinal levels. The data are discussed with reference to studies in the rat, and to standardized information as provided in the atlas of the mouse brain. Although the overall organization of these systems in the mouse is largely similar to those in the rat, species differences are present in relative location, size and/or connectivity of cell groups. For example, catecholaminergic neurons in the (ventro)lateral pons (A5 and A7 cell groups) in the mouse project to the spinal cord mainly via contralateral, and not ipsilateral, pathways. The data further supplement information as provided in standardized brainstem sections of the C57BL/6 mouse [Paxinos, G., Franklin, K.B.J., 2001. The mouse brain in stereotaxic coordinates. Academic Press, San Diego], especially with respect to the size and/or location of the catecholaminergic retrorubral field (A8 group), A5, A1, and C1 cell groups, and the serotonergic B4 group, reticulotegmental nucleus (B9 group), lateral paragigantocellular nucleus and raphe magnus nucleus (B3 group). Altogether this study provides a comprehensive overview of the spatial relationships of neurochemically and anatomically defined neuronal systems in the mouse brainstem and spinal cord.

Transgenic AD model mice, effects of potential anti-AD treatments on inflammation and pathology.

The extracellular deposition of amyloid (A) peptides in plaques, and neurofibrillary tangles are the two characteristic pathological features of Alzheimer's disease (AD). Plaques are surrounded by activated astrocytes and microglia, to study the relation between amyloid neuropathology and inflammation, we examined the changes in amyloid pathology in the hippocampus following three different treatments aimed at reducing the amyloid burden. (1) To investigate the effects of long-term cholinergic deafferentation, we lesioned the fimbria-fornix pathway in our AD-model mice at 7 months of age, and 11 months post-lesion the mice were sacrificed for histopathological analysis. The fimbria-fornix transection resulted in a substantial depletion of cholinergic markers in the hippocampus, but the lesion did not result in an alteration in hippocampal A deposition and inflammation (i.e., numbers or staining density of astrocytes and microglia). (2) To investigate the effects of estrogen, we ovariectomized mice and treated them with estrogen (sham-lesion, zero dose, low dose, and high dose) and studied the pathology at different postsurgery intervals. Estrogen depletion (i.e., ovariectomy) or estrogen replacement did not affect A deposition or inflammation at any time point. (3) In the final studies, we treated mice with flurbiprofen and an NO-donating derivative of flurbiprofen (HCT 1026) for several months (from 6 till 14 months of age), and studied the A pathology and inflammation in the brain. Sham treatment, flurbiprofen, and the low-dose HCT 1026 did not affect pathology; however, a higher dose of HCT 1026 reduced both A load and amount of microglial activation surrounding plaques.

Aging and subcellular localization of m2 muscarinic autoreceptor in basalocortical neurons in vivo.

By using immunohistochemical approaches at the light and electron microscopic levels, we have shown that aging modifies the subcellular distribution of the m2 muscarinic autoreceptor (m2R) differentially at somato-dendritic postsynaptic sites and at axonal presynaptic sites in cholinergic basalocortical neurons, in vivo. In cholinergic perikarya and dendrites of the nucleus basalis magnocellularis (NBM), aging is associated with a decrease of the density of m2R at the plasma membrane and in the cytoplasm, suggesting a decrease of the total number of m2R in the somato-dendritic field. In contrast, the number of substance P receptors per somato-dendritic surface was not affected. In the frontal cortex (FC), we have shown a decrease of cytoplasmic m2R density also leading to a decrease of the number of m2R per surface of varicosities but with no change of the density of m2R at the membrane. Our results suggest that the decrease of m2R in the somato-dendritic field of the NBM, but not a modification of the number of presynaptic m2 autoreceptors at the plasma membrane in the FC, could contribute to the decrease of the efficacy of cholinergic transmission observed with aging in the rat.