Three-Dimensional Spatial Analyses of Cholinergic Neuronal Distributions Across The Mouse Septum, Nucleus Basalis, Globus Pallidus, Nucleus Accumbens, and Caudate-Putamen.

Neuronal networks are regulated by three-dimensional spatial and structural properties. Despite robust evidence of functional implications in the modulation of cognition, little is known about the three-dimensional internal organization of cholinergic networks in the forebrain. Cholinergic networks in the forebrain primarily occur in subcortical nuclei, specifically the septum, nucleus basalis, globus pallidus, nucleus accumbens, and the caudate-putamen. Therefore, the present investigation analyzed the three-dimensional spatial organization of 14,000 cholinergic neurons that expressed choline acetyltransferase (ChAT) in these subcortical nuclei of the mouse forebrain. Point process theory and graph signal processing techniques identified three topological principles of organization. First, cholinergic interneuronal distance is not uniform across brain regions. Specifically, in the septum, globus pallidus, nucleus accumbens, and the caudate-putamen, the cholinergic neurons were clustered compared with a uniform random distribution. In contrast, in the nucleus basalis, the cholinergic neurons had a spatial distribution of greater regularity than a uniform random distribution. Second, a quarter of the caudate-putamen is composed of axonal bundles, yet the spatial distribution of cholinergic neurons remained clustered when axonal bundles were accounted for. However, comparison with an inhomogeneous Poisson distribution showed that the nucleus basalis and caudate-putamen findings could be explained by density gradients in those structures. Third, the number of cholinergic neurons varies as a function of the volume of a specific brain region but cell body volume is constant across regions. The results of the present investigation provide topographic descriptions of cholinergic somata distribution and axonal conduits, and demonstrate spatial differences in cognitive control networks. The study provides a comprehensive digital database of the total population of ChAT-positive neurons in the reported structures, with the x,y,z coordinates of each neuron at micrometer resolution. This information is important for future digital cellular atlases and computational models of the forebrain cholinergic system enabling models based on actual spatial geometry.

Intrinsic cholinergic innervation in the human sigmoid colon revealed using CLARITY, three-dimensional (3D) imaging, and a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum.

BACKGROUND: We previously reported the specificity of a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum for immunostaining of cholinergic neuronal cell bodies and fibers in the human colon. In this study, we investigate 3D architecture of intrinsic cholinergic innervation in the human sigmoid colon and the relationship with nitrergic neurons in the enteric plexus. METHODS: We developed a modified CLARITY tissue technique applicable for clearing human sigmoid colon specimens and immunostaining with hpChAT antiserum and co-labeling with neuronal nitric oxide synthase (nNOS) antibody. The Z-stack confocal images were processed for 3D reconstruction/segmentation/digital tracing and computational quantitation by Imaris 9.2 and 9.5. KEY RESULTS: In the mucosa, a local micro-neuronal network formed of hpChAT-ir fibers and a few neuronal cell bodies were digitally assembled. Three layers of submucosal plexuses were displayed in 3D structure that were interconnected by hpChAT-ir fiber bundles and hpChAT-ir neurons were rarely co-labeled by nNOS. In the myenteric plexus, 30.1% of hpChAT-ir somas including Dogiel type I and II were co-labeled by nNOS and 3 classes of hpChAT-ir nerve fiber strands were visualized in 3D images and videos. The density and intensity values of hpChAT-ir fibers in 3D structure were significantly higher in the circular than in the longitudinal layer. CONCLUSIONS AND INFERENCES: The intrinsic cholinergic innervation in the human sigmoid colon was demonstrated layer by layer for the first time in 3D microstructures. This may open a new venue to assess the structure-function relationships and pathological alterations in colonic diseases.

Cortical VIP+ /ChAT+ interneurons: From genetics to function.

One of the urgent tasks of neuroscience is to understand how neuronal circuits operate, what makes them fail, and how to repair them when needed. Achieving this goal requires identifying the principal circuitry elements and their interactions with one another. However, what constitutes 'an atom' of a neuronal circuit, a neuronal type, is a complex question. In this review we focus on a class of cortical neurons that are exclusively identified by the expression of vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT). The genetic profile of these VIP+ /ChAT+ interneurons suggests that they can release both γ-aminobutyric acid (GABA) and acetylcholine (ACh). This hints to a specific potential role in the cortical circuitry. Yet the VIP+ /ChAT+ interneurons are sparse (a mere 0.5% of the cortical neurons), which raises questions about their potential to significantly affect the circuit function. In view of recent developments in genetic techniques that allow for direct manipulation of these neurons, we provide a thorough and updated picture of the properties of the VIP+ /ChAT+ interneurons. We discuss their genetic profile, their physiological and structural properties, and their input-output mapping in sensory cortices and the medial prefrontal cortex (mPFC). Then, we examine possible amplification mechanisms for mediating their function in the cortical microcircuit. Finally, we discuss directions for further exploration of the VIP+ /ChAT+ population, focusing on its function during behavioral tasks as compared to the VIP+ /ChAT- population.

Hippocampal Cholinergic Neurostimulating Peptide Suppresses LPS-Induced Expression of Inflammatory Enzymes in Human Macrophages.

Hippocampal cholinergic neurostimulating peptide (HCNP) is a secreted undecapeptide produced through proteolytic cleavage of its precursor protein, HCNPpp. Within hippocampal neurons, HCNP increases gene expression of choline acetyltransferase (ChAT), which catalyzes acetylcholine (ACh) synthesis, thereby modulating neural activity. HCNPpp also appears to be expressed in various immune cells. In the present study, we observed that HCNPpp is expressed in U937 human macrophage-like cells and that HCNP exposure suppresses lipopolysaccharide (LPS)-induced gene expression of ChAT. The opposite action is also seen in T lymphocytes, which suggest that HCNP appear to suppress cholinergic system in immune cells. In addition, HCNP suppresses LPS-induced gene expression of inflammatory enzymes including cyclooxygenase 2 (COX2) and inducible nitric oxide (NO) synthase (iNOS). The suppressive effect of HCNP may reflect suppression of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling activated by LPS. Thus, HCNP may have therapeutic potential as an anti-inflammatory drug.

Decreased density of cholinergic interneurons in striatal territories in Williams syndrome.

Williams syndrome (WS) is a rare neurodevelopmental disorder caused by the hemideletion of approximately 25-28 genes at 7q11.23. Its unusual social and cognitive phenotype is most strikingly characterized by the disinhibition of social behavior, in addition to reduced global IQ, with a relative sparing of language ability. Hypersociality and increased social approach behavior in WS may represent a unique inability to inhibit responses to specific social stimuli, which is likely associated with abnormalities of frontostriatal circuitry. The striatum is characterized by a diversity of interneuron subtypes, including inhibitory parvalbumin-positive interneurons (PV+) and excitatory cholinergic interneurons (Ch+). Animal model research has identified an important role for these specialized cells in regulating social approach behavior. Previous research in humans identified a depletion of interneuron subtypes associated with neuropsychiatric disorders. Here, we examined the density of PV+ and Ch+ interneurons in the striatum of 13 WS and neurotypical (NT) subjects. We found a significant reduction in the density of Ch+ interneurons in the medial caudate nucleus and nucleus accumbens, important regions receiving cortical afferents from the orbitofrontal and ventromedial prefrontal cortex, and circuitry involved in language and reward systems. No significant difference in the distribution of PV+ interneurons was found. The pattern of decreased Ch+ interneuron densities in WS differs from patterns of interneuron depletion found in other disorders.

Distinct gradients of various neurotransmitter markers in caudate nucleus and putamen of the human brain.

The caudate nucleus (CN) and the putamen (PUT) as parts of the human striatum are distinguished by a marked heterogeneity in functional, anatomical, and neurochemical patterns. Our study aimed to document in detail the regional diversity in the distribution of dopamine (DA), serotonin, γ-aminobuturic acid, and choline acetyltransferase within the CN and PUT. For this purpose we dissected the CN as well as the PUT of 12 post-mortem brains of human subjects with no evidence of neurological and psychiatric disorders (38-81 years old) into about 80 tissue parts. We then investigated rostro-caudal, dorso-ventral, and medio-lateral gradients of these neurotransmitter markers. All parameters revealed higher levels, turnover rates, or activities in the PUT than in the CN. Within the PUT, DA levels increased continuously from rostral to caudal. In contrast, the lowest molar ratio of homovanillic acid to DA, a marker of DA turnover, coincided with highest DA levels in the caudal PUT, the part of the striatum with the highest loss of DA in Parkinson's disease (N. Engl. J. Med., 318, 1988, 876). Highest DA concentrations were found in the most central areas both in the PUT and CN. We observed an age-dependent loss of DA in the PUT and CN that did not correspond to the loss described for Parkinson's disease indicating different mechanisms inducing the deficit of DA. Our data demonstrate a marked heterogeneity in the anatomical distribution of neurotransmitter markers in the human dorsal striatum indicating anatomical and functional diversity within this brain structure.

Reciprocal nerve staining (RNS) for the concurrent detection of choline acetyltransferase and myelin basic protein on paraffin-embedded sections.

BACKGROUND: Objective of our work was to develop a sequential double nonfluorescent immunostaining method which allows the selective identification of myelinated motor fibers in paraffin-embedded samples of peripheral nerves. Motor recovery after a nerve gap-lesion repaired by artificial nerve-guides ("conduits") is often less complete and slower than sensory recovery. The mechanism for this is not fully understood. NEW METHOD: Incubation in sheep polyclonal choline acetyltransferase antibody (Abcam 18,736) at dilution of 1:150 was followed by incubation in mouse monoclonal anti-myelin basic protein antibody (Abcam 62,631) at a dilution of 1:5000. Counterstaining was performed with hematoxylin QS (Vector Labs H-3404). RESULTS: Immunostaining of choline acetyltransferase and myelin basic protein can be combined together and results show a good contrast between the light brown of the choline acetyltransferase reaction product and the green of myelin basic protein reaction product. Cell nuclei are stained blue. This new protocol retains the advantages of paraffin embedded sections such as (i) having a relatively simple methodology, (ii) years-long storage life, and (iii) easy sharing among laboratories. Comparison with existing method. This specific combinatorial protocol has never been used before on paraffin embedded sections. It has been named "reciprocal nerve staining" (RNS). CONCLUSIONS: Routine combination of choline acetyltransferase and myelin basic protein immunostaining provides a highly specific, highly contrasted paraffin-embedded sections where optical differentiation of myelinated motor fibers is easy and straightforward. This method will likely simplify and speed-up the routine histological study of nerve regeneration and will contribute a better identification of the nerve motor component.

Clinical Effects of "Selective Drug" Regulating Vagus Nerve Signal Pathway in Vagally-Mediated Atrial Fibrillation.

BACKGROUND The cardiac autonomic nervous system plays a crucial role in genesis and development of atrial fibrillation (AF) through the G protein signal transduction pathway. Therefore, intervening in the G protein signal transduction pathway may be a new "selective drug" method to regulate autonomic nerve activity to prevent vagally-mediated AF. MATERIAL AND METHODS Seventeen adult beagles were randomized into 3 groups: shame-operation control group (group A, n=5), empty vector gene control group (group B, n=6), and Gαi2ctp gene experimental group (group C, n=6). Group A was injected with normal saline into the anterior atrial wall, and group B and group C animals were injected with recombinant adenovirus with empty vector or Gαi2ctp vector in the same region. AF was induced by the method of rapid atrial pacing in groups B and C. To determine the clinical effect of vagal modulation, the effective refractory periods (ERP) and field action potential duration (FAPD) were evaluated by electrophysiological study. The expression levels of tyrosine hydroxylase (TH) and choline acetyl transferase (CHAT) in different parts were determined with immunohistochemistry. RESULTS After successful Gai2ctp gene transfer, in group B, the ERP and FAPD significantly decreased (P<0.05), and TH and CHAT expression observably increased (P<0.05), while those differences were absent between groups A and C (P>0.05). CONCLUSIONS Recombinant adenovirus-mediated overexpression of Gαi2ctp in canine myocardial cells can interfere with the activity of the vagus nerve, reverse the development and progression of electrical remodeling, and reduce the incidence of AF.

The cholinergic system in the basal forebrain of the Atlantic white-sided dolphin (Lagenorhynchus acutus).

The basal forebrain (BFB) cholinergic neurotransmitter system is important in a number of brain functions including attention, memory, and the sleep-wake cycle. The size of this region has been linked to the increase in encephalization of the brain in a number of species. Cetaceans, particularly those belonging to the family Delphinidae, have a relatively large brain compared to its body size and it is expected that the cholinergic BFB in the dolphin would be a prominent feature. However, this has not yet been explored in detail. This study examines and maps the neuroanatomy and cholinergic chemoarchitecture of the BFB in the Atlantic white-sided dolphin (Lagenorhynchus acutus). As in some other mammals, the BFB in this species is a prominent structure along the medioventral surface of the brain. The parcellation and distribution of cholinergic neural elements of the dolphin BFB was comparable to that observed in other mammals in that it has a medial septal nucleus, a nucleus of the vertical limb of the diagonal band of Broca, a nucleus of the horizontal limb of the diagonal band of Broca, and a nucleus basalis of Meynert. The observed BFB cholinergic system of this dolphin is consistent with evolutionarily conserved and important functions for survival.

Increase of Autonomic Nerve Factors in Epicardial Ganglionated Plexi During Rapid Atrial Pacing Induced Acute Atrial Fibrillation.

BACKGROUND The cardiac autonomic nervous system plays an essential role in epicardial ganglionated plexi (GP) regulation of atrial fibrillation onset and progression. To date, the activity of GP and the function of the cardiac autonomic nervous system are not well understood. The aim of this study was to determine alterations in epicardial GP cholinergic nerve, adrenergic nerve, and nerve growth factor expression using rapid atrial pacing to induce atrial fibrillation in canines. MATERIAL AND METHODS Nine healthy adult beagles were divided into two groups: the pacing experimental group (n=6) and the sham-operation control group (n=3). For the pacing group, high frequency pacing of the left atrial appendage was performed for eight hours. In the control group, electrodes were implanted without rapid atrial pacing. Immunocytochemistry was used to identify neurons positively expressing tyrosine hydroxylase, choline acetyl transferase, nerve growth factor and neurturin. RESULTS After successfully establishing a rapid atrial pacing of the left atrial appendage induced atrial fibrillation model, we found that expression of choline acetyl transferase, tyrosine hydroxylase, nerve growth factor, and neurturin was significantly higher in the rapid atrial pacing group than the control group (p<0.05). CONCLUSIONS In our model, incremental excitability of both the adrenergic and cholinergic nerves led to frequent incidents of atrial fibrillation, which were possibly due to an imbalance of autonomic nerve factors in the epicardial GP during acute atrial fibrillation.

The distribution of cholinergic neurons and their co-localization with FMRFamide, in central and peripheral neurons of the spider Cupiennius salei.

The spider Cupiennius salei is a well-established model for investigating information processing in arthropod sensory systems. Immunohistochemistry has shown that several neurotransmitters exist in the C. salei nervous system, including GABA, glutamate, histamine, octopamine and FMRFamide, while electrophysiology has found functional roles for some of these transmitters. There is also evidence that acetylcholine (ACh) is present in some C. salei neurons but information about the distribution of cholinergic neurons in spider nervous systems is limited. Here, we identify C. salei genes that encode enzymes essential for cholinergic transmission: choline ACh transferase (ChAT) and vesicular ACh transporter (VAChT). We used in-situ hybridization with an mRNA probe for C. salei ChAT gene to locate somata of cholinergic neurons in the central nervous system and immunohistochemistry with antisera against ChAT and VAChT to locate these proteins in cholinergic neurons. All three markers labeled similar, mostly small neurons, plus a few mid-sized neurons, in most ganglia. In the subesophageal ganglia, labeled neurons are putative efferent, motor or interneurons but the largest motor and interneurons were unlabeled. Groups of anti-ChAT labeled small neurons also connect the optic neuropils in the spider protocerebrum. Differences in individual cell labeling intensities were common, suggesting a range of ACh expression levels. Double-labeling found a subpopulation of anti-VAChT-labeled central and mechanosensory neurons that were also immunoreactive to antiserum against FMRFamide-like peptides. Our findings suggest that ACh is an important neurotransmitter in the C. salei central and peripheral nervous systems.

Honeybees Produce Millimolar Concentrations of Non-Neuronal Acetylcholine for Breeding: Possible Adverse Effects of Neonicotinoids.

The worldwide use of neonicotinoid pesticides has caused concern on account of their involvement in the decline of bee populations, which are key pollinators in most ecosystems. Here we describe a role of non-neuronal acetylcholine (ACh) for breeding of Apis mellifera carnica and a so far unknown effect of neonicotinoids on non-target insects. Royal jelly or larval food are produced by the hypopharyngeal gland of nursing bees and contain unusually high ACh concentrations (4-8 mM). ACh is extremely well conserved in royal jelly or brood food because of the acidic pH of 4.0. This condition protects ACh from degradation thus ensuring delivery of intact ACh to larvae. Raising the pH to ≥5.5 and applying cholinesterase reduced the content of ACh substantially (by 75-90%) in larval food. When this manipulated brood was tested in artificial larval breeding experiments, the survival rate was higher with food supplemented by 100% with ACh (6 mM) than with food not supplemented with ACh. ACh release from the hypopharyngeal gland and its content in brood food declined by 80%, when honeybee colonies were exposed for 4 weeks to high concentrations of the neonicotinoids clothianidin (100 parts per billion [ppb]) or thiacloprid (8,800 ppb). Under these conditions the secretory cells of the gland were markedly damaged and brood development was severely compromised. Even field-relevant low concentrations of thiacloprid (200 ppb) or clothianidin (1 and 10 ppb) reduced ACh level in the brood food and showed initial adverse effects on brood development. Our findings indicate a hitherto unknown target of neonicotinoids to induce adverse effects on non-neuronal ACh which should be considered when re-assessing the environmental risks of these compounds. To our knowledge this is a new biological mechanism, and we suggest that, in addition to their well documented neurotoxic effects, neonicotinoids may contribute to honeybee colony losses consecutive to a reduction of the ACh content in the brood food.

Differentiation of Cholinergic Neurons in Rat Spinal Cord Under Conditions of Allotransplantation into a Peripheral Nerve and In Situ Development.

The method of ectopic transplantation of embryonic anlages of CNS allows studying histoblastic potencies of progenitor cells developing under conditions of changed microenvironment. Some progenitor cells in the transplants of rat embryonic spinal cord retained their ability to express choline acetyltransferase after transplantation into the sciatic nerve of adult animals. Comparative analysis of cholinergic neurons in the neurotransplants and neurons formed in rat spinal cord during normal ontogeny showed that choline acetyltransferase-positive cells after transplantation into the nerve reached morphological differentiation of motor neurons at later terms than cells developing in situ. They were scattered one by one and did not form nuclear nerve centers. We did not fi nd structures similar to presynaptic cholinergic buds typical of intact spinal cord near these cells throughout the observation period. Solitary cholinergic neurons survived in the transplants for 19 months.

Immunostaining to visualize murine enteric nervous system development.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Enteric plexuses of two choline-acetyltransferase transgenic mouse lines: chemical neuroanatomy of the fluorescent protein-expressing nerve cells.

We studied cholinergic circuit elements in the enteric nervous system (ENS) of two distinct transgenic mouse lines in which fluorescent protein expression was driven by the choline-acetyltransferase (ChAT) promoter. In the first mouse line, green fluorescent protein was fused to the tau gene. This construct allowed the visualization of the fiber tracts and ganglia, however the nerve cells were poorly resolved. In the second mouse line (ChATcre-YFP), CRE/loxP recombination yielded cytosolic expression of yellow fluorescent protein (YFP). In these preparations the morphology of enteric neurons could be well studied. We also determined the neurochemical identity of ENS neurons in muscular and submucous layers using antibodies against YFP, calretinin (CALR), calbindin (CALB), and vasoactive intestinal peptide (VIP). Confocal microscopic imaging was used to visualize fluorescently-conjugated secondary antibodies. In ChATcre-YFP preparations, YFP was readily apparent in somatodendritic regions of ENS neurons. In the myenteric plexus, YFP/CALR/VIP staining revealed that 34% of cholinergic cells co-labeled with CALR. Few single-stained CR-positive cells were observed. Neither YFP nor CALR co-localized with VIP. In GFP/CALB/CALR staining, all co-localization combinations were represented. In the submucosal plexus, YFP/CALR/VIP staining revealed discrete neuronal populations. However, in separate preparations, double labeling was observed for YFP/CALR and CALR/VIP. In YFP/CALR/CALB staining, all combinations of double staining and triple labeling were verified. In conclusion, the neurochemical coding of ENS neurons in these mouse lines is consistent with many observations in non-transgenic animals. Thus, they provide useful tools for physiological and pharmacological studies on distinct neurochemical subtypes of ENS neurons.

Neurogenic differentiation of dental pulp stem cells to neuron-like cells in dopaminergic and motor neuronal inductive media.

BACKGROUND/PURPOSE: Dental pulp stem cells (DPSCs) have been proposed as a promising source of stem cells in nerve regeneration due to their close embryonic origin and ease of harvest. The aim of this study was to evaluate the efficacy of dopaminergic and motor neuronal inductive media on transdifferentiation of human DPSCs (hDPSCs) into neuron-like cells. METHODS: Isolation, cultivation, and identification of hDPSCs were performed with morphological analyses and flow cytometry. The proliferation potential of DPSCs was evaluated with an XTT [(2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)] assay. Media for the induction of dopaminergic and spinal motor neuronal differentiation were prepared. The efficacy of neural induction was evaluated by detecting the expression of neuron cell-specific cell markers in DPSCs by immunocytochemistry and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: In the XTT assay, there was a 2.6- or 2-fold decrease in DPSCs cultured in dopaminergic or motor neuronal inductive media, respectively. The proportions of ßIII-tubulin (ßIII-tub), glial fibrillary acidic protein (GFAP), and oligodendrocyte (O1)-positive cells were significantly higher in DPSCs cultured in both neuronal inductive media compared with those cultured in control media. Furthermore, hDPSC-derived dopaminergic and spinal motor neuron cells after induction expressed a higher density of neuron cell markers than those before induction. CONCLUSION: These findings suggest that in response to the neuronal inductive stimuli, a greater proportion of DPSCs stop proliferation and acquire a phenotype resembling mature neurons. Such neural crest-derived adult DPSCs may provide an alternative stem cell source for therapy-based treatments of neuronal disorders and injury.

Distribution of the P2X2 receptor and chemical coding in ileal enteric neurons of obese male mice (ob/ob).

AIM: To investigate the colocalization, density and profile of neuronal areas of enteric neurons in the ileum of male obese mice. METHODS: The small intestinal samples of male mice in an obese group (OG) (C57BL/6J ob/ob) and a control group (CG) (+/+) were used. The tissues were analyzed using a double immunostaining technique for immunoreactivity (ir) of the P2X2 receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT) and calretinin (Calr). Also, we investigated the density and profile of neuronal areas of the NOS-, ChAT- and Calr-ir neurons in the myenteric plexus. Myenteric neurons were labeled using an NADH-diaphorase histochemical staining method. RESULTS: The analysis demonstrated that the P2X2 receptor was expressed in the cytoplasm and in the nuclear and cytoplasmic membranes only in the CG. Neuronal density values (neuron/cm(2)) decreased 31% (CG: 6579 ± 837; OG: 4556 ± 407) and 16.5% (CG: 7796 ± 528; OG: 6513 ± 610) in the NOS-ir and calretinin-ir neurons in the OG, respectively (P < 0.05). Density of ChAT-ir (CG: 6200 ± 310; OG: 8125 ± 749) neurons significantly increased 31% in the OG (P < 0.05). Neuron size studies demonstrated that NOS, ChAT, and Calr-ir neurons did not differ significantly between the CG and OG groups. The examination of NADH-diaphorase-positive myenteric neurons revealed an overall similarity between the OG and CG. CONCLUSION: Obesity may exert its effects by promoting a decrease in P2X2 receptor expression and modifications in the density of the NOS-ir, ChAT-ir and CalR-ir myenteric neurons.

Neuronal damage, central cholinergic dysfunction and oxidative damage correlate with cognitive deficits in rats with chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion has been identified to be a risk factor for cognitive decline in aging, vascular dementia, and Alzheimer's disease. Substantial evidence has shown that chronic cerebral hypoperfusion may cause cognitive impairment, but the underlying neurobiological mechanism is poorly understood so far. In this study, we used a rat model of chronic cerebral hypoperfusion by permanent bilateral common carotid artery occlusion (BCCAO) to investigate the alterations of neuronal damage, glial activation oxidative stress and central cholinergic dysfunction, and their causal relationship with the cognitive deficits induced by chronic cerebral hypoperfusion. We found that BCCAO rats exhibited spatial learning and memory impairments and working memory dysfunction 12 weeks after BCCAO compared with sham-operated rats, simultaneously accompanied by significantly increased neuronal damage and glial cell activation in the cerebral cortex and hippocampus. Twelve weeks of BCCAO treatment in rats resulted in central cholinergic dysfunction and increased oxidative damage compared with sham-operated rats. Correlational analyses revealed that spatial learning and memory impairments and working memory dysfunction were significantly correlated with the measures of neuronal damage, central cholinergic dysfunction and oxidative damage in the cerebral cortex and hippocampus of rats with BCCAO. Moreover, the measures of neuronal damage and central cholinergic dysfunction were significantly correlated with the indexes of oxidative damage in rats with BCCAO. Collectively, this study provides novel evidence that neuronal damage and central cholinergic dysfunction is likely due to increased oxidative stress under the condition of chronic cerebral hypoperfusion. Furthermore, the results of the present study suggest that neuronal damage, central cholinergic dysfunction and oxidative damage in the brain following the reduction of cerebral blood flow could be involved in cognitive deficits induced by chronic cerebral hypoperfusion.

Alterations in TH- and ChAT-immunoreactive neurons in the DMV and gastric dysmotility in an LPS-induced PD rat model.

To study movement disorder in Parkinson's disease (PD), an animal model of PD can be created by injecting lipopolysaccharide (LPS) into the substantia nigra of rats. In addition to body movement disorders, patients with PD often experience gastrointestinal (GI) dysfunction, such as gastroparesis. However, the underlying mechanism of these disorders remains unclear. The dorsal motor nucleus of vagus (DMV) is a well-known visceral nucleus that regulates GI function. The present study investigated alterations in DMV neurons and gastric motility in rats with LPS-induced PD (LPS-PD rats). Gastric motility was recorded using a strain gauge force transducer in vivo. The distributions of tyrosine hydroxylase (TH)- and choline acetyltransferase (ChAT)-positive neurons in the DMV were determined using immunofluorescence and confocal laser microscopy. Our results indicated that in LPS-PD rats, the number of neurons in the substantia nigra, including neurons with TH immunoreactivity, was markedly reduced, although glial cell proliferation was clearly observed. However, enhanced TH immunoreactivity and decreased ChAT immunoreactivity were found in the DMV. Furthermore, weakened gastric motility was recorded in anesthetized LPS-PD rats. In conclusion, rats with LPS-induced PD exhibited gastric dysmotility with an alteration in DMV neurons. This PD model may be used to study autonomic nervous system disorders that are often observed in patients with early-stage PD.

Morphological demonstration of a vagal inhibitory pathway to the lower esophageal sphincter via nitrergic neurons in the rat esophagus.

BACKGROUND: The involvement of vagal parasympathetic efferents in esophageal myenteric neurons in vagal inhibitory pathways to the lower esophageal sphincter (LES) is not clear. Thus, this study was performed to demonstrate morphologically the presence of vagal inhibitory pathways to the LES via esophageal neurons. METHODS: Fast Blue (FB) was injected into the LES of Wistar rats, and 3 days after injection, the animals were subjected to electrical stimulation of the vagus nerve. The esophagus was processed for immunohistochemistry for Fos that was an immediate-early gene as a marker of neuronal activity, nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT). The immunoreactivities were then compared with the FB labeling in esophageal neurons. KEY RESULTS: Fast Blue-labeled neurons were observed within an esophageal area of 30 mm oral to the LES, with the highest frequency in the esophagus just above the LES. Most of the FB-labeled neurons were positive for NOS and VIP, but a few for ChAT. Following vagal-electrical stimulation, one fourth of the FB-labeled neurons presented nuclei expressing Fos and most of these Fos/FB neurons were NOS-positive. CONCLUSIONS & INFERENCES: A majority of the FB-labeled esophageal neurons appeared to be descending motor neurons innervating the LES. Moreover, the colocalization of VIP and NOS in most of the LES-projecting neurons suggests that VIP and NO released from these neurons induce LES relaxation, and the innervation of the vagal efferents to the LES-projecting esophageal neurons in the distal esophagus implies a vagal inhibitory pathway responsible for LES relaxation.