Nitric Oxide Synthase Positive Neurons Research Articles

Neuronal nitric oxide synthase positive neurons in the human corpus callosum: a possible link with the callosal blood-oxygen-level dependent (BOLD) effect.

Brain functions have been investigated in the past decades via the blood-oxygen-level dependent (BOLD) effect using functional magnetic resonance imaging. One hypothesis explaining the BOLD effect involves the Nitric Oxide (NO) gaseous neurotransmitter, possibly released also by cells in the corpus callosum (CC). The eventual presence of NO releasing neurons and/or glial cells in the CC can be assessed by immunohistochemistry. Serial sections both from paraffin-embedded and frozen samples of CC obtained from adult human brains autopsy were studied with immunohistochemistry and immunofluorescence analysis, using an antibody against the neuronal isoform of Nitric Oxide Synthase (nNOS), the enzyme synthetizing the NO. The staining revealed the presence of many nNOS-immunopositive cells in the CC, shown to be neurons with immunofluorescence. Neuronal NOS-positive neurons presented different morphologies, were more numerous 4mm apart from the midline, and displayed a peak in the body of the CC. In some cases, they were located at the upper boundary of the CC, more densely packed in the proximity of the callosal arterioles. The significant presence of nNOS-immunopositive neurons within the commissure suggests their probable role in the CC neurovascular regulation in the adult brain and could explain the BOLD effect detected in human CC.

New Subregions of the Mouse Entopeduncular Nucleus Defined by the Complementary Immunoreactivities for Substance P and Cannabinoid Type-1 Receptor Combined with Distributions of Different Neuronal Types

The entopeduncular nucleus (EPN) and substantia nigra pars reticulata (SNr) constitute the output nuclei of the basal ganglia, but studies on the EPN are limited compared with those on the SNr. Both nuclei receive projections from the striatum with axons containing substance P (SP) and cannabinoid type-1 receptor (CB1R), and immunoreactivities for these substances show complementary patterns in the striatum and SNr. In this study, we revealed a similar complementarity in the mouse EPN, combined it with region-specific neuronal distributions, and defined subregions of the EPN. First, the EPN was divided into two areas, one showing low SP and high CB1R (lSP/hCB1R) immunoreactivities, and the other showing high SP and low CB1R (hSP/lCB1R). The former received inputs from the dorsolateral striatum that are innervated by sensorimotor cortices, whereas the latter received inputs from the medial striatum that are innervated by limbic/association cortices. Then, the lSP/hCB1R area was further divided into the dorsolateral subregion in the rostral EPN and the core subregion in the caudal EPN, the latter characterized by the concentration of parvalbumin-positive neurons targeting the ventral anterior–ventral lateral thalamic nucleus. The hSP/lCB1R area was divided into the ventromedial subregion in the rostral EPN and the shell subregion in the caudal EPN, the former characterized by the concentration of nitric oxide synthase-positive neurons targeting the lateral habenula (LHb). Somatostatin-positive neurons targeting the LHb were located diffusely in three subregions other than the core. These findings illuminate structural organization inside the basal ganglia, suggesting mechanisms for sorting diverse information through parallel loops with differing synaptic modulation by CB1R.

Quantitative relationship between cerebrovascular network and neuronal cell types in mice.

SUMMARYThe cerebrovasculature and its mural cells must meet brain regional energy demands, but how their spatial relationship with different neuronal cell types varies across the brain remains largely unknown. Here we apply brain-wide mapping methods to comprehensively define the quantitative relationships between the cerebrovasculature, capillary pericytes, and glutamatergic and GABAergic neurons, including neuronal nitric oxide synthase-positive (nNOS+) neurons and their subtypes in adult mice. Our results show high densities of vasculature with high fluid conductance and capillary pericytes in primary motor sensory cortices compared with association cortices that show significant positive and negative correlations with energy-demanding parvalbumin+ and vasomotor nNOS+ neurons, respectively. Thalamo-striatal areas that are connected to primary motor sensory cortices also show high densities of vasculature and pericytes, suggesting dense energy support for motor sensory processing areas. Our cellular-resolution resource offers opportunities to examine spatial relationships between the cerebrovascular network and neuronal cell composition in largely understudied subcortical areas.

Neuronal and Non‐neuronal Cell Type Mapping in the Mouse Brain

The brain contains multiple neuronal and non‐neuronal cell types with distinct spatial relationship that serve as a basic building block in the brain. I previously developed high resolution mapping methods to visualize and to quantify these individual brain cell types across the entire mouse brain. In this talk, I will present my lab’s effort to understand spatial arrangement of several major cell types in the mouse brain across lifetime. I previously applied my mapping methods to examine GABAergic neuronal cell types, c‐Fos‐labeled cells representing functional network activation, and long‐range anatomical connectivity of oxytocin neurons. Moreover, I used the method to trace the complete network of cerebrovasculature and pericytes that showed striking correlation with cortical parvalbumin and neuronal nitric oxide synthase (nNOS) positive neurons. Recently, my lab established a new online anatomical atlas for the adult mouse brain that combines the segmentation of the Allen Mouse Brian Atlas and Paxinos Mouse Brain Atlas, and developed new atlas framework for early postnatal brains. Using these resources, I have mapped developmental trajectories of oxytocin receptor‐expressing neurons during early postnatal development, unveiling spatial temporal patterns of the brain cell type. In summary, high resolution anatomical mapping of cell types helps to understand overall anatomical and functional organization of the brain.

CNS-Sparing Histamine H3 Receptor Antagonist as a Candidate to Prevent the Diabetes-Associated Gastrointestinal Symptoms.

Among the histamine receptors, growing evidence points to the histamine H3 receptor as a pharmacological candidate to counteract the autonomic neuropathy associated with diabetes. The study aimed to evaluate the effect of PF00868087 (also known as ZPL-868), a CNS-sparing histamine H3 receptor antagonist, on the autonomic neuropathy of the intestinal tract associated with diabetes. Diabetes was induced in male BALB/c mice by a single high dose of streptozotocin (150 mg/kg). Colorectal specimens from control and diabetic mice, randomized to vehicle or PF0086087 (10, 30, 100 mg/kg/day by oral gavage for 14 days), were processed for morphological and immunohistochemical analysis. A significant overproduction of mucus in the intestinal mucosa of diabetic mice compared to the controls was observed. PF0086087 at the highest dose prevented mucin overproduction. The immunohistochemistry analysis demonstrated that diabetes causes a decrease in the inhibitory component of enteric motility, measured as the percentage of neuronal nitric oxide synthase-positive neurons (p < 0.05) and a parallel increase in the excitatory component evaluated as substance P-positive fibres (p < 0.01). PF0086087 dose-dependently prevented these pathophysiological events. In conclusion, PF0086087 may be an essential tool in preventing nitrergic dysfunction in the myenteric plexus of the distal colon and diabetes-induced gastrointestinal complications.

Analgesic Activity of Synaptamide in a Rat Sciatic Nerve Chronic Constriction Injury Model

At present, there is a growing interest in the study of the neurotropic activity of polyunsaturated fatty acid ethanolamides (N-acylethanolamines). N-docosahexaenoylethanolamine (DHEA), or synaptamide, an endogenous metabolite of docosahexaenoic acid, is a promising compound with anti-inflammatory activity. The results of this study demonstrate that synaptamide, when administered subcutaneously (4 mg/kg/day, 35 days), promotes a decrease in cold allodynia and mechanical hyperalgesia in a rat sciatic nerve chronic constriction injury (CCI) model. After CCI, synaptamide treatment enhanced the remyelination process in the site of sciatic nerve injury (33.4 ± 1.1% in the CCI+Syn group, compared to 28.4 ± 0.9% in the CCI group). Further, synaptamide suppressed the CCI-induced increase in the activity of microglia (13.1 ± 0.5% in CCI+Syn, compared to 15.3 ± 0.7% in the CCI group) and the number of nitric oxide synthase-positive neurons (58,307 ± 5,206 cells/mm3 in CCI+Syn, compared to 80,288 ± 4,287 cells/mm3 in the CCI group) in the dorsal horns of the spinal cord, and also reduced the concentration of interleukin 1 beta in the spinal cord (169.3 ± 4 pg/mg of protein in CCI+Syn, compared to 236.9 ± 9.3 pg/mg of protein in CCI group) 35 days after surgery. Synaptamide treatment resulted in decrease of reactive astrogliosis in the spinal cord dorsal horns to 20.8 ± 1.3%, which occurred simultaneously with a decrease in the substance P (SP) level (9.8 ± 0.5%) compared to vehicle-treated animals (30.2 ± 2.2% and 13.4 ± 0.9% of astroglia and SP staining area, respectively). In addition, synaptamide increased superoxide dismutase activity up to 68.6 ± 0.8% (control 50.6 ± 0.9%) in astrocyte culture. Thus, synaptamide provides anti-inflammatory and neuroprotective effects in both peripheral and central nervous system after sciatic nerve injury.

The habenula-targeting neurons in the mouse entopeduncular nucleus contain not only somatostatin-positive neurons but also nitric oxide synthase-positive neurons

The entopeduncular nucleus (EPN) in rodents is one of the two major output nuclei of the basal ganglia and corresponds to the internal segment of the globus pallidus in primates. Previous studies have shown that the EPN contains three types of neurons that project to different targets, namely, parvalbumin (PV)-, somatostatin (SOM)-, and choline acetyltransferase-positive neurons. However, we have recently reported that neurons lacking immunoreactivities for these substances are present in the EPN. Here, we demonstrate that 27.7% of all EPN neurons showed immunoreactivity for nitric oxide synthase (NOS). Among them, NOS-only positive and NOS/SOM double-positive neurons accounted for 20.1% and 6.8%, respectively, whereas NOS/PV double-positive neurons were rarely observed. NOS-containing neurons were distributed in a shell region surrounding the thalamus-targeting, PV-rich core region of the EPN, especially in the ventromedial part of the shell. The retrograde tracer fluoro-gold (FG) was injected into several target regions of EPN neurons. Among FG-labeled EPN neurons after injection into the lateral habenula (LHb), NOS-only positive, NOS/SOM double-positive, and SOM-only positive neurons accounted for 25.7%, 15.2%, and 59.1%, respectively. We conclude that NOS-positive neurons are the second major population of LHb-targeting EPN neurons, suggesting their possible involvement in behaviors in response to aversive stimuli.

Dual effect of chitosan activated platelet rich plasma (cPRP) improved erectile function after cavernous nerve injury

The intracavernosal (IC) injection of chitosan activated platelet rich plasma (cPRP) has shown to improve the erectile dysfunction in cavernous nerve injury rat model. However, the action target of PRP in improving neurogenic erectile dysfunction remains unclear. We aimed to determine the effect of cPRP action at early stage that further mediates its effect on erectile function (EF) recovery in the bilateral cavernous nerve crushing (BCNC) injury rat model. Fifty-four rats were randomly divided into two equal groups: intracavernosal ( IC) injection of saline after BCNC (group 1) and IC injection of cPRP after BCNC (group 2). Five animals in each group were euthanized at 3, 7 and 14 day (d) post-injection, and the tissues were harvested to conduct transmission electron microscopy and histological assays. Six animals in each group were used to determine the recovery of EF at 14 and 28d post-injury. IC injections of cPRP increased all EF parameters at 28d and 14d post-injury (p<0.05). cPRP injections simultaneously prevented the loss of neuronal nitric oxide synthase-positive neurons (p<0.05) and nerve fibers (p<0.05) in the major pelvic ganglion and cavernous nerve (CN), respectively, compared with saline injections. This simultaneous accelerated the regeneration of myelinated axons of the CN, reduced apoptosis, and enhanced the proliferation of the corporal smooth muscle cells at an earlier stage. These results suggest that the application of cPRP was beneficial to restore EF via neuroprotective and tissue-protective effects at early stage.

Calcium-sensing receptor (CaSR) agonist R568 inhibits small intestinal motility of mice through neural and non-neural mechanisms.

Gastrointestinal motility (GI) disorder causes symptoms such as dyspepsia, abdominal distention, and constipation and severely affects quality of life. The calcium (Ca2+)-sensing receptor (CaSR) expressed in the digestive tract can be activated by amino acids and participates in GI motility regulation. This study is designed to explore the effect and underlying mechanism of CaSR agonist R568 on the small intestine motility of mice in vivo and ex vivo. R568 was given to male C57BL/6 mice by gavage or incubated with isolated jejunum and ileum segments to observe its effects on GI motility and the involved neurons, neurotransmitters and hormones were detected by fluorescence immunohistochemistry and enzyme-linked immunosorbent assays. The in vivo results showed that the intestinal propulsive rate reduced in response to oral intake of R568. R568 treatment increased the numbers of nitric oxide synthase-positive neurons and nitric oxide release but decreased the choline acetyl transferase-positive neurons and acetylcholine release in the myenteric plexuses. R568 increased the secretion of cholecystokinin in the intestinal tissues and serum but had no effect on the secretion of glucagon like peptide-1. Ex vivo results showed that R568 inhibited the contractility of intestinal strips from the jejunum and ileum. Nitric oxide synthase (NOS) inhibitor L-nitroarginine methyl ester (L-NAME), M-receptor antagonist-atropine, and tetrodotoxin (TTX) failed to block the effect of R568. CaSR co-expressed with interstitial cells of Cajal (ICCs) in the myenteric plexus suggests the possibility that ICCs mediated the effect of R568. Our findings demonstrate that CaSR activation inhibited intestinal motility, and both the enteric nervous system and non-neural mechanism are involved in this process.

Architecture of vagal motor units controlling striated muscle of esophagus: Peripheral elements patterning peristalsis?

Little is known about the architecture of the vagal motor units that control esophageal striated muscle, in spite of the fact that these units are necessary, and responsible, for peristalsis. The present experiment was designed to characterize the motor neuron projection fields and terminal arbors forming esophageal motor units. Nucleus ambiguus compact formation neurons of the rat were labeled by bilateral intracranial injections of the anterograde tracer dextran biotin. After tracer transport, thoracic and abdominal esophagi were removed and prepared as whole mounts of muscle wall without mucosa or submucosa. Labeled terminal arbors of individual vagal motor neurons (n=78) in the esophageal wall were inventoried, digitized and analyzed morphometrically. The size of individual vagal motor units innervating striated muscle, throughout thoracic and abdominal esophagus, averaged 52 endplates per motor neuron, a value indicative of fine motor control. A majority (77%) of the motor terminal arbors also issued one or more collateral branches that contacted neurons, including nitric oxide synthase-positive neurons, of local myenteric ganglia. Individual motor neuron terminal arbors co-innervated, or supplied endplates in tandem to, both longitudinal and circular muscle fibers in roughly similar proportions (i.e., two endplates to longitudinal for every three endplates to circular fibers). Both the observation that vagal motor unit collaterals project to myenteric ganglia and the fact that individual motor units co-innervate longitudinal and circular muscle layers are consistent with the hypothesis that elements contributing to peristaltic programming inhere, or are "hardwired," in the peripheral architecture of esophageal motor units.

Tenuigenin ameliorates learning and memory impairments induced by ovariectomy

Estrogen deficiency is associated with cognitive impairment. Hormone replacement therapy (HRT) has proven to be effective in preventing and reversing the memory and learning deficiencies. However, conventional estrogenic treatment could increase the risks of breast cancer and venous thromboembolism. Tenuigenin (TEN) is putatively believed as the active component extracted from a Chinese herb Polygala tenuifolia root. Although TEN has been shown to enhance learning and memory in healthy mice, it remains unknown whether or not TEN could ameliorate learning and memory impairments. In the present study, mice were divided into four groups: sham-operated (sham), ovariectomized (OVX), OVX+estradiol benzoate (EB) and OVX+TEN groups. Step-through passive avoidance and Y-maze tests were used to assess learning and memory abilities, and the number of nitric oxide synthase (NOS) positive neurons and the synaptic measurement of hippocampal CA1 area were examined. The results showed that TEN was given orally to OVX mice, leading to the improvement of learning and memory in step-through passive avoidance and Y-maze tests. TEN could reduce the loss of NOS positive neurons and prevent the synaptic morphological changes induced by ovariectomy. Our results suggest that TEN may exert a potential therapeutic value for menopause cognitive dysfunction.

Overexpression of Bcl-2 promotes survival and differentiation of neuroepithelial stem cells after transplantation into rat aganglionic colon

IntroductionNeural stem cell transplantation is a promising tool for the restoration of the enteric nervous system in a variety of motility disorders. However, limited cell viability after transplantation has restricted its regenerative capacity. The aim of this study was to evaluate the effect of transplantation of neuroepithelial stem cell (NESC) overexpressing anti-apoptotic gene Bcl-2 on the survival, differentiation and function of grafted cells in rat aganglionic colon.MethodsNESCs were isolated from neural tube of embryonic rat (embryonic day 11.5) and manipulated to overexpress the Bcl-2 gene. After transplantation into the benzalkonium chloride-induced rat aganglionic colon, grafted cells were visualized in colonic sections. Apoptosis and differentiation of the implanted cells were assessed 1, 4 and 8 weeks post transplantation, respectively. Eight weeks post transplantation, neuronal function of the colon was assessed by measuring the response of muscle strips to electrical field stimulation.ResultsTransplantation with Bcl-2-NESCs reduced apoptosis within the transplant at 1 week compared with the vector-NESC grafted group. Our findings also indicated that overexpression of Bcl-2 in the transplanted NESCs enhanced differentiation into PGP9.5-positive and neuronal nitric oxide synthase-positive neurons at 8-week assessment. Moreover, electrical field stimulation-induced relaxation of colonic strips was also significantly increased in the Bcl-2-NESC grafted group.ConclusionTransplantation of NESCs genetically modified to overexpress Bcl-2 may have value for enhancing survival and neurogenesis of grafted cells in the adult gut environment and for improving the efficacy of stem cell therapy following a broad range of gastrointestinal motility disorders.

Sleep-Active Neuronal Nitric Oxide Synthase-Positive Cells of the Cerebral Cortex: A Local Regulator of Sleep?

Our recent report demonstrated that a small subset of GABAergic interneurons in the cerebral cortex of rodents expresses Fos protein, a marker for neuronal activity, during SWS [1]. The population of sleep-active neurons consists of strongly immunohistochemically-stained cells for the enzyme neuronal nitric oxide synthase (Type I cells). By virtue of their widespread localization within the cerebral cortex and their widespread projections to other cortical cell types, cortical neuronal nitric oxide synthase-positive neurons are positioned to play a central role in the local regulation of sleep waveforms within the cerebral cortex. Here, we review the possible functions of neuronal nitric oxide synthase and its diffusible gas product, nitric oxide, in regulating neuronal activity, synaptic plasticity and cerebral blood flow within the context of local sleep regulation in the cerebral cortex. We also summarize what is known, in addition to their expression of neuronal nitric oxide synthase, about the biochemical phenotype, synaptic connectivity and electrophysiological properties of this novel sleep-active population of cells. Finally, we raise some critical unanswered questions about the role of this population in local sleep regulation within the cerebral cortex and describe some experimental approaches that might be used to address those questions.

Nicotinamide adenine dinucleotide phosphate/neuronal nitric oxide synthase-positive neurons in the trigeminal subnucleus caudalis involved in tooth pulp nociception

Sensory information on facial structures, including teeth pulp, periodontium, and gingiva, is relayed in the trigeminal complex. Tooth pulp inflammation constitutes a common clinical problem, and this peripheral injury can induce neuroplastic changes in trigeminal nociceptive neurons. There is considerable evidence that the trigeminal subnucleus caudalis (Vc) is the principal relay for trigeminal nociceptive information as well as modulation of the painful stimuli. Glutamatergic primary afferents innervating the tooth pulp project to the most superficial laminae of the Vc. N-methyl-D-aspartate receptor stimulation leads to the activation of the enzyme nitric oxide synthase (NOS), which synthesizes the free radical nitric oxide (NO). This enzyme is expressed mainly in lamina II interneurons, and in a small number of cells in lamina I as well as in deep laminae projection neurons of Vc. In the present study, we analyzed the temporal changes in neuronal NOS (nNOS) in Vc local circuitries after unilateral intermediate molar pulp injury. Our results demonstrate that a peripheral dental pulp injury leads to neuroplastic changes in the relative amount and activity of nNOS enzyme. Moreover, after a period of time, the nitrergic system shifts to the initial values, independently of the persistence of inflammation in the pulp tissues.

Functional and in situ hybridization evidence that preganglionic sympathetic vasoconstrictor neurons express ghrelin receptors

Agonists of ghrelin receptors can lower or elevate blood pressure, and it has been suggested that the increases in blood pressure are caused by actions at receptors in the spinal cord. However, this has not been adequately investigated, and the locations of neurons in the spinal cord that express ghrelin receptors, through which blood pressure increases may be exerted, are not known. We investigated the effects within the spinal cord of two non-peptide ghrelin receptor agonists, GSK894490 and CP464709, and two peptide receptor agonists, ghrelin and des-acyl ghrelin, and we used polymerase chain reaction (PCR) and in situ hybridization to examine ghrelin receptor expression. I.v. application of the non-peptide ghrelin receptor agonists caused biphasic changes in blood pressure, a brief drop followed by a blood pressure increase that lasted several minutes. The blood pressure rise, but not the fall, was antagonized by i.v. hexamethonium. Application of these agonists or ghrelin peptide directly to the spinal cord caused only a blood pressure increase. Des-acyl ghrelin had no significant action. The maximum pressor effects of agonists occurred with application at spinal cord levels T9 to T12. Neither i.v. nor spinal cord application of the agonists had significant effect on heart rate or the electrocardiogram. Ghrelin receptor gene expression was detected by PCR and in situ hybridization. In situ hybridization localized expression to neurons, including autonomic preganglionic neurons of the intermediolateral cell columns at all levels from T3 to S2. The numbers of ghrelin receptor expressing neurons in the intermediolateral cell columns were similar to the numbers of nitric oxide synthase positive neurons, but there was little overlap between these two populations. We conclude that activation of excitatory ghrelin receptors on sympathetic preganglionic neurons increases blood pressure, and that decreases in blood pressure caused by ghrelin agonists are mediated through receptors on blood vessels.

Effects of distilled fluid of olfactory ensheathing cells by intracerebreventricular injection on treatment of Aizheimer's disease rat

Objective To probe the effects of distilled fluid of olfactory ensheathing cells(DFOECs)by intracerebreventricular injection on treatment of Alzheimer's disease(AD)rat.Methods The model of AD rats were builded by intracerebreventficular injection of Aβ1-40.The rats were divided into the normal control groups,the model of AD ones,the manpower cerebral spinal fluid(MCSF)injected ones and the DFOECs injected ones at random,and there were eight rats every group.Four weeks alter being injected,the change of memory recovery was observed by Y maze test,the total number of nitric oxide synthase positive neurons and senile plaques of hippocampus were observed by NADPH-d and Congo red histochemical staining.Results ①Y msze test：TN.EN，D，TRT of DFOECs injected group obviously decreased compared to the model of AD group(P=0.000).The former was separately(18.82±2.64)times,(4.81±1.85)times,(5.18±4.86)s,(1236.78±97.26)s,the latter was (48.33±5.10)times,(10.83±2.98)times，(11.32±12.23)s，(2264.32±244.60)s.② NADPH-d histochemical staining:the total number of NOS positive neurons of DFOECs injected group were higher than that of the model of AD group(P=0.000),they were separately(16.08±2.51)entries/eye shot,(5.08±0.89)entries/eye shot.③Congo red histochemical staining：compared to the model of AD group and the DFOECs injected ones were significantly changed in SP-IA(P=0.000)，they were separately(481.15±144.18)，(74.39±9.14).④ There was not significant differences between the model of AD group and MCSF injected group.Conclusion The distilled fluid of olfactory ensheathing cells can greatly improve the effects on treatment of Alzheimer's disease rat by intracerebreventricular injection. Key words: Alzheimer's disease; Olfactory ensheathing cells; Distilled fluid

Connection between the Striatal Neurokinin‐1 Receptor and Nitric Oxide Formation during Methamphetamine Exposure

Methamphetamine (METH) is a widely used "club drug" that produces neural damage in the brain, including the loss of some neurons. METH-induced striatal neuronal loss has been attenuated by pretreatment with the neurokinin-1 receptor antagonist WIN-51,708 in mice. Using a histologic method, we have observed the internalization of the neurokinin-1 receptor into endosomes in the striatal somatostatin/NPY/nitric oxide synthase interneurons. To investigate the role of this interneuron in the striatal cell death induced by METH, we assessed by immunohistochemistry the number of striatal nitric oxide synthase-positive neurons in the presence of METH at 8 and 16 hours after systemic injection of a bolus of METH (30 mg/kg, i.p.). We found the number of striatal nitric oxide synthase-positive neurons unchanged at these time points after METH. In a separate experiment we measured the levels of striatal 3-nitrotyrosine (3-NT) by HPLC (high-pressure liquid chromatography) as an indirect index of nitric oxide synthesis. METH increased the levels of 3-nitrotyrosine in the striatum and this increase was significantly attenuated by pretreatment with a selective neurokinin-1 receptor antagonist. These observations suggest a causal relationship between the neurokinin-1 receptor and the activation of neuronal nitric oxide synthase that warrants further investigation.

Visceral Neuropathy and Intestinal Pseudo-Obstruction in a Murine Model of a Nuclear Inclusion Disease

Intestinal dysmotility is a component of many neurodegenerative disorders, including some characterized by neuronal intranuclear inclusions. PrP-SCA7-92Q transgenic mice phenocopy many aspects of the human polyglutamine neurodegenerative disorder spinocerebellar ataxia type 7 (SCA7). The enteric neuropathology of PrP-SCA7-92Q mice was investigated after observing that they develop signs of intestinal pseudo-obstruction. Gastrointestinal transit of radio-opaque pellets through presymptomatic and symptomatic PrP-SCA7-92Q mice and nontransgenic littermates was compared. Gross, microscopic, and ultrastructural studies were conducted, along with histologic and whole mount immunohistochemistry, to identify intranuclear inclusions and quantify subsets of enteric neurons. Immunoblot analysis was performed to confirm selective loss of particular neuronal populations. A subset of cholinergic enteric ganglion cells in PrP-SCA7-92Q mice harbor nuclear inclusions composed of transgene-derived ataxin-7, which contains a pathogenic polyglutamine expansion. These animals die between 15 and 20 weeks of age with intestinal distension and enterocolitis. Signs of disease are preceded by selective loss of nitric oxide synthase-positive neurons (which lack nuclear inclusions), loss of nerve fibers in the myenteric nerve plexus, and delayed gastrointestinal transit. Cholinergic neurons, including those with inclusions, are spared. PrP-SCA7-92Q mice may be useful models for human intestinal pseudoobstruction, particularly visceral neuropathies with neuronal intranuclear inclusions. Loss of inclusion-free inhibitory neurons supports the hypothesis that inclusions may be neuroprotective or coincidental, as opposed to harbingers of neuron death. Because enteric neuropathology in PrP-SCA7-92Q animals is easily missed by routine histopathology, quantitative immunohistochemical approaches may be required to recognize analogous forms of human enteric neuropathy.

Distribution of nitric oxide synthase positive neurons in the substantia nigra of rats with liver cirrhosis

Background Nitrogen monoxide plays an important role in the physiological activity and pathological process of striatum in substantia nigra, and the nitric oxide synthase in substantia nigra may have characteristic changes after liver cirrhosis. Objective To observe the distribution and forms of nitric oxide synthase (NOS) positive neurons and fibers in substantia nigra of rats with liver cirrhosis. Design A comparative observational experiment. Settings Beijing Friendship Hospital; Capital Medical University. Materials Twenty 4-month-old male Wistar rats (120–150 g) of clean grade, were maintained in a 12-hour light/dark cycle at a constant temperature with free access to standard diet and water. Cryostat microtome (LEICA, Germany); All the reagents were purchased from Sigma Company. Methods The experiment was carried out in the Department of Anatomy (key laboratory of Beijing city), Capital Medical University from July 2000 to March 2002. The rats were randomly divided into normal group ( n =10) and liver fibrosis group ( n =10). Rats in the liver fibrosis group were subcutaneously injected with 60% CCl 4 oil at a dose of 5 mL/kg for the first time, and 3 mL/kg for the next 14 times, twice a week, totally 15 times. Liver fibrosis of grades 5–6 was taken as successful models. Whereas rats in the normal group were not given any treatment. Four months after CCl 4 treatment, all the rats were anesthetized to remove brain, and frontal frozen serial sections were prepared. The expressions of nitric oxide synthase positive neurons in substantia nigra of rats were observed under inverted microscope. The number and gray scale of cell body of nitric oxide synthase positive neurons in substantia nigra were detected with NADPH-diaphorase staining. Main outcome measures ▪ Number and gray scale of cell body of nitric oxide synthase positive neurons in substantia nigra; ▪ Expressions of nitric oxide synthase positive neurons in substantia nigra. Results All the 20 rats were involved in the analysis of results. ▪ The nitric oxide synthase positive neurons in substantia nigra were obviously fewer in the liver cirrhosis group than in the normal group ( P < 0.01), and the gray scale of the positive cell body was higher in the liver cirrhosis group than in the normal group ( P < 0.05). ▪ Abundant nitric oxide synthase positive neurons were observed in substantia nigra of normal rats, the cell body of nitric oxide synthase positive neurons was clear and transparent, with short own cloudy processes. In substantia nigra of rats with liver cirrhosis, the body of nitric oxide synthase positive neurons were observed shrink obviously, less fibrin than normal. Conclusion Rats with liver cirrhosis may suffer from the physiological dysfunction of neurons due to lack of fibers. The nitric oxide synthase positive neurons in substantia nigra can shrink and reduce.

Effects of chronic heroin treatment on nitric oxide synthase positive neurons of brain in rats

目的 观察不同浓度海洛因处理对大鼠伏隔核(Nac)、中央灰质背侧(CGd)一氧化氮合酶(NOS)阳性神经元数量和形态的影响.方法 将32只SD大鼠随机分成4组,海洛因慢性处理后,采用还原型辅酶Ⅱ-黄递酶(NADPH-d)组织化学方法 染色,观察不同浓度海洛因处理对大鼠Nac、CGd区NOS阳性神经元表达的影响.结果 0.25mg·kg-1、1.0mg·kg-1海洛因慢性处理组Nac区(38.2±3.8)(36.8±1.9)、CGd区(65.3±1.3)(61.8±1.8)和0.05mg·kg-1海洛因慢性处理组NAC区(44.3±1.4)NOS阳性神经元细胞数较对照组Nac区(29.8±1.9)、CGd区(52.4±2.7)明显增多(P＜0.05);0.05mg·kg-1海洛因慢性处理组CGd区(33.9±1.3)和1.0mg·kg-1海洛因慢性处理组CGd区(32.1±1.1)、Nac区(31.0±1.6)NOS阳性神经元胞体面积较对照组(40.8±0.8)有明显缩小(P＜0.05);0.05mg·kg-1、1.0mg·kg-1组大鼠CGd区(49.2±2.1)(43.5±1.4)NOS阳性神经元轴突长度较对照组(58.2±3.6)明显变短(P＜0.05),而三个处理组Nac区NOS阳性神经元轴突长度差异无显著性(P＞0.05).结论 海洛因慢性处理能够引起CGd、Nac区NOS阳性神经元数量增多、胞体缩小、轴突长度变短,提示海洛因慢性处理对NOS阳性神经元有损伤作用,NOS参与了海洛因的依赖过程。