Vesicular Acetylcholine Transporter Immunoreactivity Research Articles

Применение антител к везикулярному транспортеру ацетилхолина и ацетилхолинэстеразе в исследованиях пренатального развития парасимпатической иннервации поджелудочной железы человека

Introduction. Parasympathetic fibers innervating the pancreas are involved in the regulation of both exo-crine and endocrine function, in the regulation of endocrine cell proliferation, and are also implicated in the pathogenesis of type 1 diabetes. Nonetheless, data concerning the distribution of parasympathetic fibers within the human pancreas in prenatal development are absent in the literature. Our aim was to evaluate the possibility of using the markers of cholinergic neurons and nerve fibers, namely vesicular acetylcholine transporter (VAChT) and acetylcholinesterase (AChE) in studies of prenatal develop-ment of parasympathetic innervation of the human pancreas. Materials and methods. The study was performed on 10 autopsies of the fetal pancreas (gestational age 10-34 weeks) using immunoperoxidase labeling with antibodies to VAChT and AChE. Results. Immunopositive reaction to AChE was detected in bundles of nerve fibers of various diameters, networks of thin nerve fibers as well as in individual neurons of the intramural ganglia. The structures of the nervous system were immunonegative to VAChT. In the exocrine pancreas, that is, in the interlobular connective tissue, near the ducts and inside the forming lobules, thin cholinergic fibers prevailed on the studied developmental periods. In pancreatic islets, cholinergic fibers were detected less frequently and were located at the periphery.Immunopositive reaction with antibodies to AChE and mouse monoclonal antibodies to VAChT was also detected in some endocrine cells in the pancreatic islets. Conclusion. We have shown that antibodies to AChE detect cholinergic neurons and nerve fibers in the developing human pancreas. We have also demonstrated that in the fetal pancreas thin cholinergic fibers prevail in the exocrine part and rarely are detected in the pancreatic islets, which is typical in adults. The results showing the VAChT and AChE immunoreactivity in the endocrine cells of fetal pancreatic islets are in agreement with data obtained in the adult human pancreas and suggest that the endocrine cells can be a source of acetylcholine. Keywords: pancreas, human development, parasympathetic innervation, VAChT, AChE

Electron microscopic localization of M2-muscarinic receptors in cholinergic and noncholinergic neurons of the laterodorsal tegmental and pedunculopontine nuclei of the rat mesopontine tegmentum.

Muscarinic m2 receptors (M2Rs) are implicated in autoregulatory control of cholinergic output neurons located within the pedunculopontine (PPT) and laterodorsal tegmental (LTD) nuclei of the mesopontine tegmentum (MPT). However, these nuclei contain many noncholinergic neurons in which activation of M2R heteroceptors may contribute significantly to the decisive role of the LTD and PPT in sleep-wakefulness. We examined the electron microscopic dual immunolabeling of M2Rs and the vesicular acetylcholine transporter (VAchT) in the MPT of rat brain to identify the potential sites for M2R activation. M2R immunogold labeling was predominately seen in somatodendritic profiles throughout the PPT/LTD complex. In somata, M2R immunogold particles were often associated with Golgi lamellae and cytoplasmic endomembrannes, but were rarely in contact with the plasma membrane, as was commonly seen in dendrites. Approximately 36% of the M2R-labeled somata and 16% of the more numerous M2R-labeled dendrites coexpressed VAchT. M2R and M2R/VAchT-labeled dendritic profiles received synapses from inhibitory- and excitatory-type axon terminals, over 88% of which were unlabeled and others contained exclusively M2R or VAchT immunoreactivity. In axonal profiles M2R immunogold was localized to plasmalemmal and cytoplasmic regions and showed a similar distribution in many VAchT-negative glial profiles. These results provide ultrastructural evidence suggestive of somatic endomembrane trafficking of M2Rs, whose activation serves to regulate the postsynaptic excitatory and inhibitory responses in dendrites of cholinergic and noncholinergic neurons in the MPT. They also suggest the possibility that M2Rs in this brain region mediate the effects of acetylcholine on the release of other neurotransmitters and on glial signaling. J. Comp. Neurol. 524:3084-3103, 2016. © 2016 Wiley Periodicals, Inc.

Localization and neurochemical features of the sympathetic trunk ganglia neurons projecting to the urethral muscle. An experimental study in a porcine animal model

The striated perineal urethral muscle (UM) is involved in the voluntary control of the micturition requiring complex interactions between afferent and efferent (autonomic and somatic) pathways to store and periodically eliminate urine. Our aim was to define the site, cross sectional area and phenotype of sympathetic trunk ganglia (STG) neurons projecting to the porcine UM, combining retrograde neuronal tracer Fast Blue (FB) and double immunohistochemical labelling methods. The research was carried out on 3 male intact pigs, in which we counted a total number of 4992.67 ± 834.35 (mean ± S.E.M., n = 3) FB+ neurons distributed in the bilateral T12-S3 STG. These neurons were significantly larger in lumbar STG than in the sacral ones. Moreover we highlighted the presence of Dopamine β hydroxylase (DβH), Vesicular Acetylcholine Transporter (VAChT), neuronal Nitric Oxyde Sinthase (n-NOS), Calcitonine Gene Related Peptide (CGRP), Leu-Enkephaline (LENK), Neuropeptide Y (NPY), Substance P (SP), Vasoactive Intestinal Polypeptide (VIP) and Somatostatine (SOM) and their eventual co-existence with Tyrosine Hydroxylase(TH) in both lumbar and sacral FB+ neurons. In particular, lumbar and sacral STG neurons expressed similar percentages of immunoreactivity for TH, SP and CGRP, but showed significantly different levels of immunoreactivity for NPY, VIP, VAChT, LENK, nNOS, DβH and SOM. Taken together, these data indicate a different contribution of lumbar and sacral pathways in the sympathetic transmission to the boar UM.

Single dose exposure of sarin and physostigmine differentially regulates expression of choline acetyltransferase and vesicular acetylcholine transporter in rat brain

Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are the key components of cholinergic system apart from acetylcholinesterase. Effects of subcutaneous exposures of 0.25 and 0.5 LD50 sarin and 0.75mg/kg physostigmine on immunoreactivity levels of these two proteins (ChAT and VAChT) were studied. Immunoreactivity levels of ChAT decreased significantly after 1 and 3days in cortex and 3days of 0.25 LD50 sarin administration in cerebellum. While 0.5 LD50 sarin exposure caused significant down regulation after 2.5h to 7days in cortex and 1 and 3days in cerebellum with respect to controls. Physostigmine at 0.75mg/kg dose showed enhanced levels of ChAT after 1day which decreased significantly after 3 and 7days both in cortex and cerebellum compared to controls. VAChT level decreased significantly after 1day in cortex and 3 and 7days in cerebellum after 0.25 LD50 sarin administration, while 0.5 LD50 sarin significantly lowered VAChT immunoreactivity level after 2.5h and 7days in cortex and 2.5h and 1day in cerebellum. Physostigmine at 0.75mg/kg dose showed significant enhanced immunoreactivity levels of VAChT after 1, 3, and 7days in cortex and 3days in cerebellum. Results show that acetylcholinesterase inhibition by sarin caused reduction in cholinergic neurotransmission at cholinergic proteins expression levels, while physostigmine caused differential expression of key cholinergic proteins. Moreover, cortex, which receives greater cholinergic innervations, is more susceptible to anticholinesterase effect on cholinergic gene expression. These changes can explain delayed neurocognitive changes during anticholinesterases induced chronic neurotoxicity.

Diverse populations of intrinsic cholinergic interneurons in the mouse olfactory bulb

Cholinergic activities affect olfactory bulb (OB) information processing and associated learning and memory. However, the presence of intrinsic cholinergic interneurons in the OB remains controversial. As a result, morphological and functional properties of these cells are largely undetermined. We characterized cholinergic interneurons using transgenic mice that selectively mark choline acetyltransferase (ChAT)-expressing cells and immunolabeling. We found a significant number of intrinsic cholinergic interneurons in the OB. These interneurons reside primarily in the glomerular layer (GL) and external plexiform layer (EPL) and exhibit diverse distribution patterns of nerve processes, indicating functional heterogeneity. Further, we found these neurons express ChAT and vesicular acetylcholine transporter (VAChT), but do not immunoreact to glutamatergic, GABAergic or dopaminergic markers and are distinct from calretinin-expressing interneurons. Interestingly, the cholinergic population partially overlaps with the calbindin D28K-expressing interneuron population, revealing the neurotransmitter identity of this sub-population. Additionally, we quantitatively determined the density of VAChT labeled cholinergic nerve fibers in various layers of the OB, as well as the intensity of VAChT immunoreactivity within the GL, suggesting primary sites of cholinergic actions. Taken together, our results provide clear evidence showing the presence of a significant number of cholinergic interneurons and that these morphologically and distributionally diverse interneurons make up complex local cholinergic networks in the OB. Thus, our results suggest that olfactory information processing is modulated by dual cholinergic systems of local interneuron networks and centrifugal projections.

Immunoreactivity for high-affinity choline transporter colocalises with VAChT in human enteric nervous system

Cholinergic nerves are identified by labelling molecules in the ACh synthesis, release and destruction pathway. Recently, antibodies against another molecule in this pathway have been developed. Choline reuptake at the synapse occurs via the high-affinity choline transporter (CHT1). CHT1 immunoreactivity is present in cholinergic nerve fibres containing vesicular acetylcholine transporter (VAChT) in the human and rat central nervous system and rat enteric nervous system. We have examined whether CHT1 immunoreactivity is present in nerve fibres in human intestine and whether it is colocalised with markers of cholinergic, tachykinergic or nitrergic circuitry. Human ileum and colon were fixed, sectioned and processed for fluorescence immunohistochemistry with antibodies against CHT1, class III beta-tubulin (TUJ1), synaptophysin, common choline acetyl-transferase (cChAT), VAChT, nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). CHT1 immunoreactivity was present in many nerve fibres in the circular and longitudinal muscle, myenteric and submucosal ganglia, submucosa and mucosa in human colon and ileum and colocalised with immunoreactivity for TUJ1 and synaptophysin confirming its presence in nerve fibres. In nerve fibres in myenteric ganglia and muscle, CHT1 immunoreactivity colocalised with immunoreactivity for VAChT and cChAT. Some colocalisation occurred with SP immunoreactivity, but little with immunoreactivity for VIP or NOS. In the mucosa, CHT1 immunoreactivity colocalised with that for VIP and SP in nerve fibres and was also present in vascular nerve fibres in the submucosa and on epithelial cells on the luminal border of crypts. The colocalisation of CHT1 immunoreactivity with VAChT immunoreactivity in cholinergic enteric nerves in the human bowel thus suggests that CHT1 represents another marker of cholinergic nerves.

The quantitative evaluation of cholinergic markers in spatial memory improvement induced by nicotine–bucladesine combination in rats

We previously showed that post-training intra-hippocampal infusion of nicotine–bucladesine combination enhanced spatial memory retention in the Morris water maze. Here we investigated the role of cholinergic markers in nicotine–bucladesine combination-induced memory improvement. We assessed the expression of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) in CA1 region of the hippocampus and medial septal area (MSA) of the brain. Post-training bilateral infusion of a low concentration of either nicotine or bucladesine into the CA1 region of the hippocampus did not affect spatial memory significantly. Quantitative immunostaining analysis of optical density in CA1 regions and evaluation of immunopositive neurons in medial septal area of brain sections from all combination groups revealed a significant increase (P < 0.001) in the ChAT and VAChT immunoreactivity. The maximum increase was observed with combination of 10-µM/side bucladesine and 0.5 µg/side nicotine and in a concentration dependent manner. Also, increase in the optical density and amount of ChAT and VAChT immunostaining correlated with the decrease in escape latency and traveled distance in rats treated with nicotine and low dose of bucladesine. Taken together, these results suggest that significant increases of ChAT and VAChT protein expressions in the CA1 region and medial septal area are the possible mechanisms of spatial memory improvement induced by nicotine–bucladesine combination.

Peripheral facial nerve axotomy in mice causes sprouting of motor axons into perineuronal central white matter: Time course and molecular characterization

Generation of new axonal sprouts plays an important role in neural repair. In the current study, we examined the appearance, composition and effects of gene deletions on intrabrainstem sprouts following peripheral facial nerve axotomy. Axotomy was followed by the appearance of galanin+ and calcitonin gene-related peptide (CGRP)+ sprouts peaking at day 14, matching both large, neuropeptide+ subpopulations of axotomized facial motoneurons, but with CGRP+ sprouts considerably rarer. Strong immunoreactivity for vesicular acetylcholine transporter (VAChT) and retrogradely transported MiniRuby following its application on freshly cut proximal facial nerve stump confirmed their axotomized motoneuron origin; the sprouts expressed CD44 and alpha7beta1 integrin adhesion molecules and grew apparently unhindered along neighboring central white matter tracts. Quantification of the galanin+ sprouts revealed a stronger response following cut compared with crush (day 7–14) as well as enhanced sprouting after recut (day 8 + 6 vs. 14; 14 + 8 vs. 22), arguing against delayed appearance of sprouting being the result of the initial phase of reinnervation. Sprouting was strongly diminished in brain Jun-deficient mice but enhanced in alpha7 null animals that showed apparently compensatory up-regulation in beta1, suggesting important regulatory roles for transcription factors and the sprout-associated adhesion molecules. Analysis of inflammatory stimuli revealed a 50% reduction 12–48 hours following systemic endotoxin associated with neural inflammation and a tendency toward more sprouts in TNFR1/2 null mutants (P = 10%) with a reduced inflammatory response, indicating detrimental effects of excessive inflammation. Moreover, the study points to the usefulness of the facial axotomy model in exploring physiological and molecular stimuli regulating central sprouting. J. Comp. Neurol. 518:699–721, 2010. © 2009 Wiley-Liss, Inc.

The vanadium (IV) compound rescues septo-hippocampal cholinergic neurons from neurodegeneration in olfactory bulbectomized mice

The bilateral olfactory bulbectomy (OBX) mouse exhibits neurodegeneration of cholinergic neurons in the medial septum with concomitant cognitive deficits. Consistent with our previous observations, choline acetyltransferase (ChAT) protein levels in the medial septum decreased by 43.5% 2 weeks after OBX without changes in glutamic acid decarboxylase-65 (GAD65) levels. Interestingly, levels of the vesicular acetylcholine transporter (VAChT), which is localized at cholinergic neuron terminals, decreased both in hippocampal CA1 and CA3 regions following OBX. Confocal microscopy showed that VAChT expression was more severely reduced in CA3 14 days after OBX compared with CA1. Intriguingly, chronic treatment with a vanadium (IV) compound, VO(OPT) [bis(1-N-oxide-pyridine-2-thiolato)oxovanadium(IV)] (0.5–1 mg as vanadium (V)/kg/day, i.p.), significantly rescued cholinergic neurons in the medial septum in a dose-dependent manner. VO(OPT) treatment also prevented decreased VAChT immunoreactivity both in CA1 and CA3 regions in the hippocampus. Consistent with these findings, an impaired hippocampal long-term potentiation (LTP) and memory deficits seen in OBX mice were significantly prevented by VO(OPT) treatment. Taken together, OBX induces neurodegeneration of septo-hippocampal cholinergic neurons and impairment of memory-related behaviors. The neuroprotective effect of VO(OPT) could lead to novel therapeutic strategies to ameliorate cognitive deficits associated with cholinergic neuron degeneration in Alzheimer's disease and other neurodegenerative disorders.

Presence of a non-neuronal cholinergic system and occurrence of up- and down-regulation in expression of M2 muscarinic acetylcholine receptors: new aspects of importance regarding Achilles tendon tendinosis (tendinopathy)

Limited information is available concerning the existence of a cholinergic system in the human Achilles tendon. We have studied pain-free normal Achilles tendons and chronically painful Achilles tendinosis tendons with regard to immunohistochemical expression patterns of the M(2) muscarinic acetylcholine receptor (M(2)R), choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT). M(2)R immunoreactivity was detected in the walls of blood vessels. As evidenced via parallel staining for CD31 and alpha-smooth muscle actin, most M(2)R immunoreactivity was present in the endothelium. M(2)R immunoreactivity also occured in tenocytes, which regularly immunoreact for vimentin. The degree of M(2)R immunoreactivity was highly variable, tendinosis tendons that exhibit hypercellularity and hypervascularity showing the highest levels of immunostaining. Immunoreaction for ChAT and VAChT was detected in tenocytes in tendinosis specimens, particularly in aberrant cells. In situ hybridization revealed that mRNA for ChAT is present in tenocytes in tendinosis specimens. Our results suggest that autocrine/paracrine effects occur concerning the tenocytes in tendinosis. Up-regulation/down-regulation in the levels of M(2)R immunoreactivity possibly take place in tenocytes and blood vessel cells during the various stages of tendinosis. The presumed local production of acetylcholine (ACh), as evidenced by immunoreactivity for ChAT and VAChT and the detection of ChAT mRNA, appears to evolve in response to tendinosis. These observations are of importance because of the well-known vasoactive, trophic, and pain-modulating effects that ACh is known to have and do unexpectedly establish the presence of a non-neuronal cholinergic system in the Achilles tendon.

Cholinergic projections to the suprachiasmatic nucleus and lower subparaventricular zone of diurnal and nocturnal rodents

In nocturnal species cholinergic agonists alter circadian rhythm phase when injected intraventricularly or directly into the suprachiasmatic nucleus (SCN), but the phase shifts obtained differ depending upon the site being injected. Cholinergic projections reach the SCN of nocturnal laboratory rats, however, nothing is known about these projections in diurnal rodents. The first objective of this study was to evaluate the hypothesis that cholinergic projections to the SCN are only present in nocturnal species. The second objective was to evaluate the hypothesis that the lower part of the subparaventricular zone (LSPV) is a candidate for being a site that mediates the phase shifts observed when cholinergic agonists are injected intraventricularly. These hypotheses were tested in the diurnal unstriped Nile grass rat ( Arvicanthis niloticus) and the nocturnal laboratory rat. Additionally, we evaluated if the light–dark (LD) cycle had an effect on the expression of the vesicular acetylcholine transporter (VAChT) in the SCN, LSPV, and in two control areas. Animals were kept in a 12:12 LD cycle and perfused at six time points. VAChT immunoreactivity was observed in the SCN, LSPV, and in the control areas of both species. The SCN and LSPV showed a differential distribution and density of cholinergic projections between the two species, but similar temporal patterns of VAChT expression were found across species. These results provide evidence for a differential cholinergic stimulation of the SCN between grass rats and laboratory rats that may reflect a rewiring of neural projections brought about by the adoption of a diurnal activity profile.

Reactive plasticity in the dentate gyrus following bilateral entorhinal cortex lesions in cynomolgus monkeys

Hippocampal structural plasticity induced by entorhinal cortex (EC) lesions has been studied extensively in the rat, but little comparable research has been conducted in primates. In the current study we assessed the long-term effects of bilateral aspiration lesions of the EC on multiple markers of circuit organization in the hippocampal dentate gyrus of young adult monkeys (Macaca fascicularis). Alternate histological sections were processed for the visualization of somatostatin and vesicular acetylcholine transporter (VAChT) immunoreactivity and acetylcholinesterase histochemistry (AChE). The markers revealed the distinct laminar organization of dentate gyrus circuitry for stereology-based morphometric quantification. Consistent with findings in rats, the volume of the somatostatin-immunopositive outer molecular layer (OML), innervated by projections from the EC, was decreased by 42% relative to control values. The inner molecular layer (IML) displayed a corresponding volumetric expansion in response to denervation of the OML as measured by AChE staining, but not when visualized for quantification by VAChT immunoreactivity. Nonetheless, stereological estimation revealed a 36% increase in the total length of VAChT-positive cholinergic fibers in the IML after EC damage, along with no change in the OML. Together, these findings suggest that despite substantial species differences in the organization of hippocampal circuitry, the capacity for reactive plasticity following EC damage, previously documented in rats, is at least partly conserved in the primate dentate gyrus.

γ‐Aminobutyric acid‐containing sympathetic preganglionic neurons in rat thoracic spinal cord send their axons to the superior cervical ganglion

Gamma-aminobutyric acid (GABA)-containing fibers have been observed in the rat superior cervical ganglion (SCG) and, to a lesser extent, in the stellate ganglion (STG). The aim of present study is to clarify the source of these fibers. No cell body showed mRNAs for glutamic acid decarboxylases (GADs) or immunoreactivity for GAD of 67 kDa (GAD67) in the cervical sympathetic chain. Thus, GABA-containing fibers in the ganglia are suggested to be of extraganglionic origin. GAD67-immunoreactive fibers were found not in the dorsal roots or ganglia, but in the ventral roots, so GABA-containing fibers in the sympathetic ganglia were considered to originate from the spinal cord. Furthermore, almost all GAD67-immunoreactive fibers in the sympathetic ganglia showed immunoreactivity for vesicular acetylcholine transporter, suggesting that GABA was utilized by some cholinergic preganglionic neurons. This was confirmed by the following results. 1) After injection of Sindbis/palGFP virus into the intermediolateral nucleus, some anterogradely labeled fibers in the SCG were immunopositive for GAD67. 2) After injection of fluorogold into the SCG, some retrogradely labeled neurons in the thoracic spinal cord were positive for GAD67 mRNA. 3) When the ventral roots of the eighth cervical to the fourth thoracic segments were cut, almost all GAD67- and GABA-immunoreactive fibers disappeared from the ipsilateral SCG and STG, suggesting that the vast majority of GABA-containing fibers in those ganglia were of spinal origin. Thus, the present findings strongly indicate that some sympathetic preganglionic neurons are not only cholinergic but also GABAegic.

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.

Palisade Endings in Extraocular Muscles of the Monkey are Immunoreactive for Choline Acetyltransferase and Vesicular Acetylcholine Transporter

To analyze palisade endings in extraocular muscles (EOMs) of a primate species and to examine our previous findings in cat that palisade endings are putative effector organs. Eleven monkeys (Macaca fascicularis) of both sexes, between 4 and 6 years of age were analyzed. Whole EOM myotendons were immunostained with four combinations of triple-fluorescent labeling and examined by confocal laser scanning microscopy. Labeling included antibodies against choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), neurofilament, and synaptophysin. Muscle fibers were counterstained with phalloidin. Palisade endings were observed in all monkey EOMs. Nerve fibers extended from the muscle into the tendon and looped back to divide into a terminal arborization (palisade ending) around a single muscle fiber tip. In approximately 30% of the cases, nerve fibers supplying palisade endings often established motor terminals outside the palisade complex. Nerve fibers forming palisade endings were ChAT-neurofilament positive. Axonal branches of palisade endings were ChAT-neurofilament positive as well. All palisade nerve terminals exhibited ChAT-synaptophysin immunoreactivity. Within the palisade complex, palisade nerve terminals exhibited VAChT immunoreactivity. All palisade nerve terminals were VAChT-synaptophysin immunoreactive. The results confirm that in the monkey, palisade endings contain acetylcholine and are therefore most likely effector organs. Palisade endings are also present in human EOMs and because of their location at the myotendinous junction, these organs are of crucial interest for strabismus surgery.

Changes in Interstitial Cells of Cajal at the Deep Muscular Plexus Are Associated with Loss of Distention-Induced Burst-Type Muscle Activity in Mice Infected by Trichinella spiralis

The physiology and pathophysiology of the network of interstitial cells of Cajal associated with the deep muscular plexus (ICC-DMP) of the small intestine are still poorly understood. The objectives of the present study were to evaluate the effects of inflammation associated with Trichinella spiralis infection on the ICC-DMP and to correlate loss of function with structural changes in these cells and associated structures. We used immunohistochemistry, electron microscopy, and assessment of distention-inducing electrophysiological parameters in vitro. Damage to ICC-DMP was associated with a loss of distention-induced patterns of electrical activity normally associated with distention-induced peristalsis. Consistently, the timing of recovery of ICC-DMP paralleled the timing of recovery of the distention-induced activity. Nerve varicosities associated with ICC-DMP including cholinergic nerves, assessed by immunoelectron microscopy and whole mount double labeling, paralleled injury to ICC-DMP thus contributing to impaired excitatory innervation of smooth muscle cells. Major additional changes included a remodeling of the inner circular muscle layer, which may affect long-term sensitivity to distention after infection. In conclusion, transient injury to ICC-DMP in response to T. spiralis infection is severe and associated with a complete lack of distention-induced burst-type muscle activity.

A part of cholinergic fibers in mouse superior cervical ganglia contain GABA or glutamate

The localizations and functions of glutamate and GABA, the major amino acid neurotransmitters in the central nervous system, are still unclear in the peripheral nervous system. We immunohistochemically double-stained mouse superior cervical ganglia with antibodies for the vesicular acetylcholine transporter (VAchT), GAD65, the vesicular glutamate transporters 1–3 (VGluTs1–3), the marker of the sympathetic preganglionic neuron (SPN), GABAergic, and glutamatergic terminals, respectively. All GAD65-positive terminals showed VAchT immunoreactivity, indicating that GABAergic fibers originate from SPNs. VGluT2-immunoreactive terminals showing colocalization with VAchT were observed, but VGluT1 and 3 immunoreactive terminals were not. Colocalization of GAD65 and VGluT2 was rarely found. All VGluT2-immunopositive terminals were also immunopositive for neuronal nitric oxide synthase (nNOS), a marker for the subpopulation of the SPNs, while about half of the GABA-immunopositive fibers were immunopositive for nNOS. The origin of these fibers was discussed.

Expression of the cholinergic gene locus in the rat placenta.

High amounts of acetylcholine (ACh) and its synthesising enzyme choline acetyltransferase (ChAT) have been detected in the placenta. Since the placenta is not innervated by extrinsic or intrinsic cholinergic neurons, placental ACh and ChAT originate from non-neuronal sources. In neurons, cytoplasmic ACh is imported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT), and released through vesicular exocytosis. In view of the coordinate expression of VAChT and ChAT from the "cholinergic gene locus" in neurons, we asked whether VAChT is coexpressed with ChAT in rat placenta, and investigated this issue by means of RT-PCR, in situ hybridisation, western blot and immunohistochemistry. Messenger RNA and protein of the common type of ChAT (cChAT), its splice variant peripheral ChAT (pChAT), and VAChT were detected in rat placenta with RT-PCR and western blot. ChAT in situ hybridisation signal and immunoreactivity for cChAT and pChAT were observed in nearly all placental cell types, while VAChT mRNA and immunolabelling were detected in the trophoblast, mesenchymal cells and the visceral yolk sac epithelial cells. While ChAT is nearly ubiquitously expressed in rat placenta, VAChT immunoreactivity is localised cell type specifically, implying that both vesicular and non-vesicular ACh release machineries prevail in placental cell types.

Ultrastructural evidence for mu-opioid modulation of cholinergic pathways in rat dentate gyrus

Within the rat hippocampal formation, cholinergic afferents and μ-opioid receptors (MORs) are involved in many crucial learning processes, including those associated with drug reward. Pharmacological data, and the overlapping distributions of cholinergic and μ-opioid systems, particularly in the dentate gyrus, suggest that MOR activation is a potential mechanism for endogenous opioid modulation of cholinergic activity. To date, anatomical evidence supporting this has not been reported. To delineate the relationship between cholinergic afferents and MOR-containing processes in the dentate gyrus, hippocampal sections were dually immunolabeled for vesicular acetylcholine transporter (VAChT) and MOR-1 and examined by electron microscopy. VAChT immunoreactivity was in unmyelinated axons and axon terminals, and was most often associated with small synaptic vesicles. MOR immunoreactivity was found in axons, axon terminals and, to a lesser extent, perikarya, which resembled GABAergic basket cells. Semi-quantitative ultrastructural analysis revealed that from 5% to 13% (depending on laminar location) of VAChT-immunoreactive (ir) presynaptic profiles contained MOR immunoreactivity. Additionally, 7% of VAChT-ir presynaptic profiles directly apposed MOR-ir axons and terminals, and there were almost no appositions to MOR-ir dendrites. These data suggest that opioids may directly and indirectly modulate acetylcholine release and/or reuptake. In the hilus and molecular layer, 4% of VAChT-ir terminals contacted dendritic shafts that were also contacted by MOR-ir terminals. This suggests that cholinergic afferents and MOR-containing afferents can converge on granule cell dendrites (which are restricted to the molecular layer) and on interneuron dendrites in the hilus. The results of this study provide ultrastructural evidence for direct and indirect modulation of cholinergic systems by μ-opioids in the hippocampal formation.

Neurochemical characterization of extrinsic innervation of the guinea pig rectum.

The presence of markers for parasympathetic, sympathetic, and glutamatergic or peptidergic sensory innervation was investigated by using in vitro tracing with biotinamide, combined with immunohistochemistry, to characterise quantitatively extrinsic axons to myenteric ganglia of the guinea pig rectum. Of biotinamide-filled varicose axons, 3.6 +/- 1.3% were immunoreactive for tyrosine hydroxylase (TH) and 16.0 +/- 4.8% for vesicular acetylcholine transporter (VAChT). TH and vesicular monoamine transporter (VMAT1) showed high coexistence (83-100%), indicating that varicosities lacking TH immunoreactivity also lacked VMAT1. VAChT was detectable in 77% of choline acetyltransferase (ChAT)-immunoreactive varicosities. Calcitonin gene-related peptide (CGRP) was detected in 5.3 +/- 1.6% of biotinamide-labeled varicosities, the vesicular glutamate transporter (VGluT) 1 in 2.8 +/- 0.8%, and VGluT2 in 11.3 +/- 4.2% of varicosities of extrinsic origin. Varicosities from the same axon showed consistent immunoreactivity. A novel type of nerve ending was identified, with branching, flattened lamellar endings, similar to the intraganglionic laminar endings (IGLEs) of the proximal gut. Rectal IGLEs were frequently immunoreactive for VGluT1 and VGluT2. Thus most varicose axons of extrinsic origin, which innervate rectal myenteric ganglia, lack detectable levels of immunoreactivity for TH, VMAT1, VAChT, ChAT, VGluT1/2, or CGRP, under conditions in which these markers are readily detectable in other axons. Although some unlabeled varicosities may belong to afferent axons that lack detectable CGRP or VGluT1/2 in the periphery, this suggests that a large proportion of axons do not release any of the major autonomic or sensory transmitters. We speculate that this may vary under particular circumstances, for example, inflammation or obstruction of the gut.