GCH1 attenuates cardiac autonomic nervous remodeling in canines with atrial-tachypacing via tetrahydrobiopterin pathway regulated by microRNA-206.

BACKGROUND/AIMS: Cardiac autonomic nerve remodeling (ANR) is an important mechanism of atrial fibrillation (AF). GTP cyclohydrolase I, encoded by GCH1, is the rate-limiting enzyme in de novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide (NO) synthesis. Previous studies reported that increased BH4 and NO content negatively regulated nerve regeneration. This study investigated the effects of GCH1 on ANR via BH4 pathway, regulated by microRNA-206 (miR-206). METHODS AND RESULTS: In canines, atrial tachypacing (A-TP), together with miR-206 overexpression, increased PGP9.5 level and inhibited GCH1 expression by quantitative real-time polymerase chain reaction and western blot analysis. GCH1 was validated to be a direct target of miR-206 by luciferase assays. Meanwhile, miR-206 overexpression by lentiviruses infection into right superior pulmonary vein fat pad decreased GCH1 expression to ∼40% and further reduced BH4 and NO content compared with the control canines. After infection of GCH1 overexpression lentiviruses for two weeks, atrial effective refractory period was increased compared with the control group (105.8 ± 1.537 ms vs 99.17 ± 2.007 ms, P < 0.05). Moreover, GCH1 overexpression attenuated canines' atrial PGP9.5 level to ∼56% of the controls. In myocardial cells, transfection of GCH1 overexpression lentiviruses also decreased PGP9.5 expression to 26% of the control group. In patients, plasma was collected and miR-206 expression was upregulated in AF patients (n = 18) than the controls (n = 12). CONCLUSIONS: Our findings suggested that GCH1 downregulation exacerbated ANR by decreasing atrial BH4 and NO content modulated by miR-206 in A-TP canines. This indicates that GCH1 may prevent the initiation of AF through inhibiting ANR.

Human brain aminopeptidase A: biochemical properties and distribution in brain nuclei.

Aminopeptidase A (APA) generated brain angiotensin III, one of the main effector peptides of the brain renin angiotensin system, exerting a tonic stimulatory effect on the control of blood pressure in hypertensive rats. The distribution of APA in human brain has not been yet studied. We first biochemically characterized human brain APA (apparent molecular mass of 165 and 130 kDa) and we showed that the human enzyme exhibited similar enzymatic characteristics to recombinant mouse APA. Both enzymes had similar sensitivity to Ca(2+). Kinetic studies showed that the K(m) (190 mumol/L) of the human enzyme for the synthetic substrate-l-glutamyl-beta-naphthylamide was close from that of the mouse enzyme (256 mumol/L). Moreover, various classes of inhibitors including the specific and selective APA inhibitor, (S)-3-amino-4-mercapto-butyl sulfonic acid, had similar inhibitory potencies toward both enzymes. Using (S)-3-amino-4-mercapto-butyl sulfonic acid, we then specifically measured the activity of APA in 40 microdissected areas of the adult human brain. Significant heterogeneity was found in the activity of APA in the various analyzed regions. The highest activity was measured in the choroids plexus and the pineal gland. High activity was also detected in the dorsomedial medulla oblongata, in the septum, the prefrontal cortex, the olfactory bulb, the nucleus accumbens, and the hypothalamus, especially in the paraventricular and supraoptic nuclei. Immunostaining of human brain sections at the level of the medulla oblongata strengthened these data, showing for the first time a high density of immunoreactive neuronal cell bodies and fibers in the motor hypoglossal nucleus, the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the Roller nucleus, the ambiguus nucleus, the inferior olivary complex, and in the external cuneate nucleus. APA immunoreactivity was also visualized in vessels and capillaries in the dorsal motor nucleus of the vagus and the inferior olivary complex. The presence of APA in several human brain nuclei sensitive to angiotensins and involved in blood pressure regulation suggests that APA in humans is an integral component of the brain renin angiotensin system and strengthens the idea that APA inhibitors could be clinically tested as an additional therapy for the treatment of certain forms of hypertension.

Collateral axonal projections from rostral ventromedial medullary nitric oxide synthase containing neurons to brainstem autonomic sites.

The magnocellular reticular nucleus and adjacent lateral paragigantocellular nucleus have been shown to contain a large population of nitric oxide synthase (NOS) immunoreactive neurons. However, little is known about the projections of these neurons within the central nervous system. Retrograde tract-tracing techniques combined with immunohistochemistry were used in this study to investigate whether NOS neurons in this rostral ventromedial medullary (RVMM) region send collateral axonal projections to autonomic sites in the nucleus of the solitary tract (NTS) and in the nucleus ambiguus (Amb). Fluorogold and/or rhodamine labeled latex microspheres were microinjected into the NTS and Amb at sites that elicited bardycardia and/or depressor responses (l-glutamate; 0.25 M; 10 nl). After a survival period of 10-14 days, the rats were sacrificed and tissue sections of the brainstem were processed immunohistochemically for the identification of NOS containing neuronal perikarya. After unilateral injection of the tract-tracers into the NTS and Amb, retrogradely labeled neurons were observed bilaterally throughout the RVMM region. Of the number of RVMM neurons retrogradely labeled from the NTS (684+/-143), 9% were found to be immunoreactive to NOS. Similarly, of those RVMM neurons retrogradely labeled from the Amb (963+/-207), 7% also contained NOS immunoreactivity. Neurons with collateral axonal projections to NTS and Amb (14% and 10%, respectively) were observed predominantly within a region of RVMM that extended co-extensively with approximately the rostrocaudal extent of the facial nucleus. Of these double labeled neurons, 36.4+/-20 (39%) were also found to be immunoreactive to NOS. These data indicate that the RVMM contains at least three population of NOS neurons that send axons to innervate functionally similar cardiovascular responsive sites in the NTS and Amb. Although the function of these NOS containing medullary pathways in cardiovascular control is not known, it is likely that those with collateral axonal projections represent the anatomical substrate by which the RVMM may simultaneously coordinate cardiovascular responses during physiological changes associated with respiration and/or motor movements.

Influence of age-related changes in nitric oxide synthase-expressing neurons in the rat supraoptic nucleus on inhibition of salivary secretion.

Age-related inhibition of salivary secretion has been demonstrated in rats, and the nitric oxide (NO) present in the supraoptic nucleus (SON) and the medial septal area has been reported to play an inhibitory role in the regulation of salivary secretion. In the present study, we investigated the age-related changes occurring in the NO synthase (NOS)-expressing neurons in the SON, which is related to the production of NO, and discussed the interrelation between the age-related changes in the NOS-expressing neurons and the age-related inhibition of salivary secretion. Nissl staining and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry were performed for young adult and aged rats. Quantitative analysis was also performed using the Nissl-stained and NADPH-d-positive neurons. Although the numbers of the Nissl-stained neurons did not change, significant age-related increases were detected in cell number, cell size and reactive density of the NADPH-d-positive neurons. Therefore, the production of NO in the SON neurons increased with age. We concluded that the age-related increase in the NO in the SON might be a factor that contributes to the age-related inhibition of salivary secretion.

Upregulation of GAD65 mRNA in the medulla of the rat model of metabolic syndrome.

Metabolic syndrome is characterized by obesity, elevated blood pressure (BP), insulin resistance, and hypercholesterolemia. Recently an animal model of this disorder has been proposed in rats selectively bred based on their performance on a treadmill-running task. Accordingly, low capacity runner (LCR) rats exhibited all of the diagnostic criteria for metabolic syndrome, including elevated BP, as compared to their high capacity runner (HCR) counterparts [U. Wisløff, S.M. Najjar, O. Ellingsen, P.M. Haram, S. Swoap, Q. Al-Share, M. Fernstrom, K. Rezaei, S.J. Lee, L.G. Koch, S.L. Britton, Cardiovascular risk factors emerge after artificial selection for low aerobic capacity, Science 307 (2005) 418-420]. Previous studies have highlighted the importance of GABAergic neurotransmission in the medullary cardiovascular-regulatory areas in the central control of BP. Thus, we hypothesized a dysregulation in GABAergic transmission in the medullary cardiovascular-regulatory nuclei of LCR rats. To begin testing this hypothesis we carried out experiments examining expression of the GABA synthetic enzymes, GAD65 and GAD67, mRNAs in the two rat strains via radioactive in situ hybridization. Our results showed GAD65 and GAD67 mRNAs were widely expressed throughout the brainstem; quantification revealed increased GAD65 mRNA expression in LCR animals in the caudal nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (VLM) as compared to HCR rats. Conversely, no differences in the expression of GAD67 were detected in these regions. These data are consistent with the notion of altered GABAergic neurotransmission in the NTS and VLM in metabolic syndrome, and point to the importance of these regions in cardiovascular regulation.

Localization of NADPH diaphorase in the lumbosacral spinal cord and dorsal root ganglia of the cat.

The distribution of NADPH-d activity in the spinal cord and dorsal root ganglia of the cat was studied to evaluate the role of nitric oxide in lumbosacral afferent and spinal autonomic pathways. At all levels of the spinal cord NADPH-d staining was present in neurons and fibers in the superficial dorsal horn and in neurons around the central canal and in the dorsal commissure. In addition, the sympathetic autonomic nucleus in the rostral lumbar segments exhibited prominent NADPH-d cellular staining whereas the parasympathetic nucleus in the sacral segments was not well stained. The most prominent NADPH-d activity in the sacral segments occurred in fibers extending from Lissauer's tract through laminae I along the lateral edge of the dorsal horn to lamina V and the region of the sacral parasympathetic nucleus. These fibers were very similar to VIP-containing and pelvic nerve afferent projections in the same region. They were prominent in the S1-S3 segments but not in adjacent segments (L6-L7 and Cx1) or in thoracolumbar and cervical segments. NADPH-d activity and VIP immunoreactivity in Lissauer's tract and the lateral dorsal horn were eliminated or greatly reduced after dorsal-ventral rhizotomy (S1-S3), indicating the fibers represent primary afferent projections. A population of small diameter afferent neurons in the L7-S2 dorsal root ganglia were intensely stained for NADPH-d. The functional significance of the NADPH-d histochemical stain remains to be determined; however, if NADPH-d is nitric oxide synthase then this would suggest that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic efferent pathways and in sacral parasympathetic afferent pathways in the cat.

Nitric oxide synthase in the autonomic and sensory ganglia innervating the submandibular salivary gland.

This article reviews the neuroanatomical studies on the distribution of nitric oxide synthase (NOS) in neurons and nerve fibers innervating the submandibular gland. Specificity of NADPH-diaphorase activity as a histochemical marker of neuronal NOS is discussed in light of corresponding NOS immunoreactivity. Anatomical data suggest that nitric oxide may affect neural regulation of the submandibular gland through both sympathetic, parasympathetic and sensory divisions of the autonomic nervous system. NOS-containing nerve terminals in the gland parenchyme are mainly vascular and either parasympathetic and/or sensory in nature, while sympathetic terminals lack NOS. Most postganglionic parasympathetic neurons are intensely NOS-immunoreactive. Some of the preganglionic parasympathetic neurons show vague reactivity, while their terminals in the submandibular ganglia stain heavily. The postganglionic sympathetic neurons normally show only barely visible reactivity, while manipulations interrupting axonal continuity increase neuronal NOS content. A subpopulation of the preganglionic sympathetic neurons and their terminals are intensely reactive. The observations summarized here suggest that nitric oxide participates in the control of blood flow through the gland, while direct effect on secretion is unlikely.

Acute sarin exposure causes differential regulation of choline acetyltransferase, acetylcholinesterase, and acetylcholine receptors in the central nervous system of the rat.

Acute neurotoxic effects of sarin (O:-isopropylmethylphosphonoflouridate) in male Sprague-Dawley rats were studied. The animals were treated with intramuscular (im) injections of either 1 x LD(50) (100 microg/kg), and sacrificed at 0. 5, 1, 3, 6, 15, or 20 h after treatment, or with im injections of either 0.01, 0.1, 0.5, or 1 x LD(50) and sacrificed 15 h after treatment. Plasma butyrylcholinesterase (BChE) and brain regional acetylcholinesterase (AChE) were inhibited (45-55%) by 30 min after the LD(50) dose. BChE in the plasma and AChE in cortex, brainstem, midbrain, and cerebellum remained inhibited for up to 20 h following a single LD(50) treatment. No inhibition in plasma BChE activity was observed 20 h after treatment with doses lower than the LD(50) dose. Midbrain and brainstem seem to be most responsive to sarin treatment at lower doses, as these regions exhibited inhibition (approximately 49% and 10%, respectively) in AChE activity following 0.1 x LD(50) treatment, after 20 h. Choline acetyltransferase (ChAT) activity was increased in cortex, brainstem, and midbrain 6 h after LD(50) treatment, and the elevated enzyme activity persisted up to 20 h after treatment. Cortex ChAT activity was significantly increased following a 0.1 x LD(50) dose, whereas brainstem and midbrain did not show any effect at lower doses. Treatment with an LD(50) dose caused a biphasic response in cortical nicotinic acetylcholine receptor (nAChR) and muscarinic acetylcholine receptor (m2-mAChR) ligand binding, using [(3)H]cytisine and [(3)H]AFDX-384 as ligands for nAChR and mAChR, respectively. Decreases at 1 and 3 h and consistent increases at 6, 15, and 20 h in nAChR and m2-mAChR were observed following a single LD(50) dose. The increase in nAChR ligand binding densities was much more pronounced than in mAChR. These results suggest that a single exposure of sarin, ranging from 0.1 to 1 x LD(50), modulates the cholinergic pathways differently and thereby causes dysregulation in excitatory neurotransmission.

Reduced number of intrinsic pulmonary nitrergic neurons in Fawn-Hooded rats as compared to control rat strains.

The Fawn-Hooded rat (FHR) strain reveals a congenital predisposition to primary (idiopathic) pulmonary hypertension (PPH), and can therefore be regarded as an animal model in which to study possible mechanisms underlying an inherited susceptibility to pulmonary hypertension. Pulmonary hypertension can be induced in FHRs after a short exposure to mild hypoxia, presumably because of an altered peripheral oxygen sensitivity. Given the presence of pulmonary nitrergic neurons in rat lungs, the observed link between airway hypoxia and the expression of pulmonary neuronal nitric oxide synthase (nNOS), and the fact that nNOS appears to be involved in peripheral chemoreceptor sensitivity, we examined the intrinsic pulmonary nitrergic innervation in the FHR. In the present study the number of intrapulmonary nitrergic nerve cell bodies, detected by NADPH diaphorase (NADPHd) histochemistry, was quantified in the FHR and three control rat strains. Compared to the control rat strains, the FHR lungs revealed a highly significantly lower number of intrinsic nitrergic neurons, while no apparent differences were found in the number of enteric nitrergic neurons in the esophagus. In conclusion, the possible links between neuronal NO, hypersensitivity to airway hypoxia, and the development of PPH clearly deserve further investigation.

Ultrastructural localization of acetylcholinesterase and choline acetyltransferase in oligodendrocytes, glioblasts and vascular endothelial cells in the external cuneate nucleus of the gerbil.

This study reports the reactivities of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in some of the nonneuronal elements in the external cuneate nucleus (ECN) of gerbils. AChE reaction products were localized in some oligodendrocytes in their cisternae of rough endoplasmic reticulum, nuclear envelope and Golgi saccules. The basal lamina lining the capillary endothelia also displayed AChE reactivity. In ChAT immunocytochemistry, the reaction products were found to be associated with the vascular basal lamina as well as the endothelial plasma membrane facing the lumen. The most remarkable finding was the localization of ChAT immunoreactivity in some oligodendrocytes and occasional glioblasts (small glial precursor cells containing a thin rim of cytoplasm surrounding an irregular nucleus with homogeneous chromatin materials). The ChAT-positive oligodendrocytes consisted of two types, medium-dense and dark cells, either associated with blood vessels or ChAT-stained neuronal elements. It is suggested from these new findings that at least some of the oligodendrocytes and glioblasts in the ECN of gerbils may be involved in the synthesis, storage, release and degradation of acetylcholine.

Centrally administered bombesin activates COX-containing spinally projecting neurons of the PVN in anesthetized rats.

The paraventricular nucleus (PVN) of the hypothalamus has a heterogenous structure containing different types of output neurons that project to the median eminence, posterior pituitary, brain stem autonomic centers and sympathetic preganglionic neurons in the spinal cord. Presympathetic neurons in the PVN send mono- and poly-synaptic projections to the spinal cord. In the present study using urethane-anesthetized rats, we examined the effects of centrally administered bombesin (a homologue of the mammalian gastrin-releasing peptide) on the mono-synaptic spinally projecting PVN neurons pre-labeled with a retrograde tracer Fluoro-Gold (FG) injected into T8 level of the spinal cord, with regard to the immunoreactivity for cyclooxygenase (COX) isozymes (COX-1/COX-2) and Fos (a marker of neuronal activation). FG-labeled spinally projecting neurons were abundantly observed in the dorsal cap, ventral part and posterior part of the PVN. The immunoreactivity of each COX-1 and COX-2 was detected in FG-labeled spinally projecting PVN neurons in the vehicle (10 µl of saline/animal, i.c.v.)-treated group, while bombesin (1 nmol/animal, i.c.v.) had no effect on the number of these immunoreactive neurons for each COX isozyme with labeling of FG. On the other hand, the peptide significantly increased the number of double-immunoreactive neurons for Fos and COX-1/COX-2 with FG-labeling in the PVN (except triple-labeled neurons for FG, COX-2 and Fos in the dorsal cap of the PVN), as compared to those of vehicle-treated group. These results suggest that centrally administered bombesin activates spinally projecting PVN neurons containing COX-1 and COX-2 in rats.

Organisation of the catecholaminergic system in the vagal motor nuclei of pigs: a retrograde fluorogold tract tracing study combined with immunohistochemistry of catecholaminergic synthesizing enzymes.

The vagal motor system is involved in the regulation of cardiorespiratory and gastrointestinal functions. Vagal motor neurons are localized near or adjacent to catecholaminergic neurons, but their co-localisation seems species dependent, present in the cat but absent in the rabbit. In pig, a species commonly used as an experimental model in humans brain disorders (sudden infant death syndrome, hypoxia), the relationship is poorly understood. We aimed at describing the distribution of vagal motor neurons and tyrosine hydroxylase-immunoreactive (-ir) neurons by using a double staining method in combination with retrograde tracing of vagal efferent neurons. After fluorogold impregnation of the central part of the sectioned left cervical vagal trunk, two main vagal motor neuronal populations were located in the dorsal motor nucleus of the vagus nerve (DMX) and in the area of the nucleus ambiguus (Amb). Like in the human, the DMX was composed of different subpopulations of neurons with the same morphological characteristics. Immunohistochemistry of catecholaminergic synthesizing enzymes differentiated two main sites containing vagal motor populations: the dorsomedial and the ventrolateral medulla. TH-ir was rarely seen in vagal motor neurons of the DMX, but TH-ir neurons were present around the two main vagal motor neuronal populations that contained TH-ir fibres. The anatomical organisation of the vagal motor and the catecholaminergic neuronal systems are similar to those described in humans and suggest that the involvement of the catecholamines in the control of the vagal motor system may be similar in pigs and in humans.

Cyclooxygenase-1 and -2 in spinally projecting neurons are involved in CRF-induced sympathetic activation.

Corticotropin-releasing factor (CRF) in the brain has been shown to stimulate sympathetic activity, leading to elevations of blood pressure, heart rate and plasma catecholamine levels and neuronal activation of the sympathetic ganglia and adrenal medulla. We previously reported that brain cyclooxygenase (COX), the rate-limiting enzyme in the synthesis of prostanoids, is involved in centrally administered CRF-induced sympathetic activation in rats. Therefore, the present study was designed to reveal the effect of centrally administered CRF (1.5 nmol/animal) on the expression of COX isozymes, COX-1 and COX-2, in spinally projecting neurons until 6h after the administration, using rats microinjected with a monosynaptic retrograde tracer into the intermediolateral cell column of the thoracic spinal cord. Retrogradely labeled neurons were detected in the paraventricular hypothalamic nucleus (PVN), locus coeruleus (LC), raphe pallidus nucleus and rostral ventrolateral medulla. Centrally administered CRF significantly increased the number of spinally projecting PVN neurons expressing COX-1 throughout the experimental period and those expressing COX-2 during only the late phase. CRF also increased the number of spinally projecting LC neurons expressing COX-2 throughout the experimental period. In other regions, the CRF administration had no effect on COXs expression in spinally projecting neurons. These results suggest that COX-1 and COX-2 in the PVN and COX-2 in the LC play roles in the CRF-induced sympathetic regulation in rats.

Morphological and quantitative study of ganglionated plexus of Calomys callosus trachea.

Calomys callosus is a wild, native forest rodent found in South America. In Brazil, this species has been reported to harbour the parasitic protozoan Trypanosoma cruzi. The ganglionated plexus of this species was studied using whole-mount preparations of trachea that were stained using histological and histochemical methods. The histological methods were used to determine the position of the ganglia with respect to the trachea muscle and to determine the presence of elastic and collagen fibers. The histochemical method of NADH-diaphorase was used for morphometric evaluations of the plexus. The tracheal plexus lies exclusively over the muscular part of the organ, dorsal to the muscle itself. It varies in pattern and extent between animals. The average number of neurons was 279 and the cellular profile area ranged from 38.37 microm2 to 805.89 microm2. Acetylcholinesterase (AChE) histochemistry verified that both ganglia and single neurons lie along nerve trunks and are reciprocally interconnected with the plexus. Intensely AChE-reactive neurons were found to be intermingled with poorly reactive ones. Two longitudinal AChE-positive nerve trunks were also observed and there was a diverse number of ganglia along the intricate network of nerves interconnecting the trunks. A ganglion capsule of collagen and elastic fibers surrounding the neurons was observed. Under polarized light, the capsule appeared to be formed by Type I collagen fibers.

Activation of Rho-kinase in the brainstem enhances sympathetic drive in mice with heart failure.

Rho-kinase is involved in the pathogenesis of hypertension and left ventricular remodelling after myocardial infarction (MI). In an earlier study, we had demonstrated that Rho-kinase in the brainstem contributes to hypertensive mechanisms via the sympathetic nervous system; however, it is not known whether Rho-kinase in the brainstem also contributes to sympathetic nerve activation after MI. Male Institute of Cancer Research mice (8-10 weeks old) were used for the study. Two days before coronary artery occlusion (MI group), the left ventricular function was estimated by echocardiography. Following this, Y-27632 (0.5 mM, 0.25 microL/h), a specific Rho-kinase inhibitor, or a vehicle was intracisternally infused in the mice using an osmotic mini-pump. Nine days after coronary artery occlusion, we evaluated the 24-hour urinary norepinephrine excretion (U-NE) as a marker of sympathetic nerve activity. Ten days after coronary artery occlusion, we measured organ weight and evaluated Rho-kinase activity in the brainstem by measuring the amount of phosphorylated ezrin/radixin/moesin proteins, one of the substrates of Rho-kinase. The control group underwent a sham operation. Rho-kinase activity, U-NE, and lungs and liver weight were significantly greater in the MI group compared with the control group. Left ventricular size increased and percent fractional shortening decreased in the MI group compared with the control group. Y-27632 significantly decreased Rho-kinase activity and attenuated the increase in U-NE after MI. These results demonstrate that Rho-kinase is activated in the brainstem after MI and that the activation of this pathway is involved in the resulting enhanced sympathetic drive.

Innervation and neurotransmitter localization in the lung of the Nile bichir Polypterus bichir bichir.

Anatomical and functional studies of the autonomic innervation in the lung of dipnoan fishes and the bichirs are lacking. The present immunohistochemical studies demonstrated the presence of nerve fibers in the muscle layers of the lung of the bichir, Polypterus bichir bichir, and identified the immunoreactive elements of this innervation. Tyrosine hydroxylase, acetylcholinesterase, and peptide immunoreactivity was detected in the intramural nerve fibers. Extensive innervation was present in the submucosa where adenylatecyclase/activating polypeptide 38, substance P, P(2)X(2), and 5-hydroxytryptamine (5-HT)-immunoreactive nerve fibers mainly supplied blood vessels. A collection of monopolar neurons located in the submucosal and the muscular layers of the glottis expressed a variety of various transmitters. These neurons may be homologous to ganglion cells in the branchial and pharyngeal rami of the vagus in fishes. Nerves containing 5-HT and P(2)X(2) receptor immunoreactivity projected to the lung epithelium. Associated with neuroepithelial cells in mucociliated epithelium, were neuronal nitric oxide synthase-immunopositive axons. The physiological function of this innervation is not known. The present study shows that the pattern of autonomic innervation of the bichir lung may by similar in its elements to that in tetrapods.

Localization of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the gastrointestinal sphincters in the guinea pig.

The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS), in the nerves of gastrointestinal sphincters in guinea pigs was investigated to throw some light on the role of nitric oxide (NO) in these sphincteric regions. The nerve fibres with NADPH-d activity were observed chiefly in the circular muscle layer of the wall of gastrointestinal sphincters. Compared with the adjacent non-sphincteric regions, the proportions of NADPH-d positive myenteric neurons in the lower esophageal sphincter (LES), pyloric sphincter (PS) and internal anal sphincter (IAS) were higher (P < 0.05). The densities of NADPH-d-positive fibres in the circular muscle of above sphincteric regions were also higher (in LES P < 0.01; in PS and IAS P < 0.05). Within the circular muscle of LES, some discrete NADPH-d-positive intrinsic nerve cell bodies, usually smaller than their outlying myenteric neurons, were also observed. The dense distribution of NADPH-d-positive fibres within the circular muscles of gastrointestinal sphincteric regions suggests that NO, as a non-adrenergic non-cholinergic (NANC) inhibitory neurotransmitter, might play an important role in the neuronal regulation of the guinea-pig gastrointestinal sphincters.

Development of nerves containing nitric oxide synthase in the human male urogenital organs.

OBJECTIVE: To determine the spatial and temporal distribution of nitric oxide synthase (NOS) in the urogenital organs of a series of human male fetuses, using an immunohistochemical technique. MATERIAL AND METHODS: Thirteen pre-natal specimens ranging in gestational age from 13 to 30 weeks were acquired following abortion or miscarriage. The distribution of NOS, which catalyses the production of nitric oxide (NO), was revealed using an indirect immunolabelling technique and compared with the overall innervation of each specimen visualized using the general nerve-marker protein gene product 9.5 (PGP). RESULTS: At 13 weeks of gestation the majority of nerves supplying the developing prostate gland expressed NOS while similar nerves formed a very minor proportion of the total innervation to the urinary bladder and intramural ureters. With increasing gestational age, NOS-containing nerves became more numerous in the lower urinary tract, the majority occurring at the bladder neck and around the prostatic urethra. In contrast, NOS-containing nerves were not detected in the muscle coat of the vas deferens and seminal vesicle until 23 weeks of gestation and at 30 weeks still only formed a small proportion of the intramuscular nerves. From 23 weeks onwards NOS-containing nerves were present occasionally in the dense subepithelial nerve plexuses which developed in the bladder, prostate, vas deferens and seminal vesicle. Also from 23 weeks onwards, many of the epithelial cells lining the vas deferens, seminal vesicle and ejaculatory ducts showed immunoreactivity to NOS but no immunoreactivity was observed in the epithelial lining of the urinary bladder and the intramural ureters. CONCLUSION: Based on the comparative density of NOS-containing nerves and the difference in their temporal development among the various urogenital organs it is apparent that NO plays an increasingly important role in the autonomic control of the lower urinary tract during fetal development but that its involvement in the functional control of the vas deferens and seminal vesicle is relatively minor before birth.

Role of the basal forebrain cholinergic projection in somatosensory cortical plasticity.

Trimming all but two whiskers in adult rats produces a predictable change in cortical cell-evoked responses characterized by increased responsiveness to the two intact whiskers and decreased responsiveness to the trimmed whiskers. This type of synaptic plasticity in rat somatic sensory cortex, called "whisker pairing plasticity," first appears in cells above and below the layer IV barrels. These are also the cortical layers that receive the densest cholinergic inputs from the nucleus basalis. The present study assesses whether the cholinergic inputs to cortex have a role in regulating whisker pairing plasticity. To do this, cholinergic basal forebrain fibers were eliminated using an immunotoxin specific for these fibers. A monoclonal antibody to the low-affinity nerve growth factor receptor 192 IgG, conjugated to the cytotoxin saporin, was injected into cortex to eliminate cholinergic fibers in the barrel field. The immunotoxin reduces acetylcholine esterase (AChE)-positive fibers in S1 cortex by >90% by 3 wk after injection. Sham-depleted animals in which either saporin alone or saporin unconjugated to 192 IgG is injected into the cortex produces no decrease in AChE-positive fibers in cortex. Sham-depleted animals show the expected plasticity in barrel column neurons. In contrast, no plasticity develops in the ACh-depleted, 7-day whisker-paired animals. These results support the conclusion that the basal forebrain cholinergic projection to cortex is an important facilitator of synaptic plasticity in mature cortex.

Autonomic nerve changes in the mouse pancreas after pancreatic duct ligation.

INTRODUCTION: The mouse pancreas exhibits distinct atrophy of the exocrine tissue following pancreatic duct ligation. AIM: To investigate changes of innervation in the whole pancreas after pancreatic duct ligation. METHODOLOGY: The mouse pancreatic duct was ligated at 6 weeks of age. Pancreatic tissues were removed 7 days and 14 days after the ligation, fixed by perfusion and immersion with Zamboni solution, and embedded in gelatin. The whole organ was serially sectioned at a thickness of 100 microm, histochemically stained for cholinesterase, and observed by light microscopy. The number and volume of intrapancreatic ganglia, number of ganglion cells, and volume of each ganglion cell in the whole pancreas were quantitated. Some sections were analyzed using transmission electron microscopy after histochemically staining for cholinesterase. RESULTS: In the normal pancreas, ganglia were often situated on the outer surface of the islets of Langerhans. Thick nerve bundles ran along the arteries and emanated thin nerve fibers that surrounded the arterioles. In the atrophied pancreas following pancreatic duct ligation, ganglia remained on the islets of Langerhans as in normal mice, while the nerve fibers appeared dense, bending and curling in a more complex manner. The thin nerves also crossed each other in a complex network. Using morphometry in the pancreas following pancreatic duct ligation, the total ganglion cell number was found to decrease from normal levels. The mean ganglion cell volume in the ligated pancreas was significantly smaller than that in normal mice. As observed by transmission electron microscopy, some ganglion cells in the ligated pancreas were negative for cholinesterase activity but were surrounded by positive staining around the surface. CONCLUSIONS: These results suggest that the function of pancreatic ganglion cells changes with organ atrophy after pancreatic duct ligation.