Functional Significance of Angiotensin Receptor Type 2 in the Neuroplasticity of Autonomic Ganglia in (mRen2)27 Transgenic Hypertensive Rats.

ABSTRACT: The over-expression of Ren -2 d gene in (mRen2)27 rats leads to development of hypertension mediated by the renin-angiotensin-system axis and exaggerated sympathetic nerve activity. Exogenously applied angiotensin II (AngII) on the superior cervical ganglion evokes ganglionic compound action potentials (gCAP) and ganglionic long-term potentiation (gLTP). We studied the functional role of angiotensin receptors and expression of reactive oxygen species marker, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) proteins in AngII-induced postganglionic transmission. Bath-applied AngII revealed that the indices of ganglionic transmission, synaptic strength of gCAP, and decay time for gLTP are remarkably prolonged in (mRen2)27 rats and were abolished by an angiotensin receptor blocker (ARB), suggesting postganglionic AngII Type 1 (AT 1 ) receptor localization and mediation. Receptor density for AT 1 was similar in (mRen2)27 and control animals, and quantitative reverse transcription polymerase chain reaction revealed that it is consistent with the mRNA profile. Furthermore, immunocytochemistry analysis showed similar AT 1 receptor distribution and signals. However, assessment of Type 2 (AT 2 ), Ang-(1-7)-MAS and NOX4-specific proteins showed that AT 2 receptor protein expression was 4-fold lower, consistent with a low mRNA profile. MAS receptor expression was 10-fold lower and NOX4 protein was 2-fold lower. Despite similarity in the densities of AT 1 receptor, the low levels of the components of the protective arm of the renin-angiotensin system at the ganglia may contribute to the differential superior cervical ganglion sensitivity to AngII. The lower NOX4 affects reactive oxygen species balance and possibly results in activation of downstream pathways to promote increased sympathetic nerve activity. We speculate that the significant diminution in AT 2, MAS, and NOX4 protein expressions may play an indirect role in the alteration and efficacy of gCAP and gLTP in hypertension.

Novel Cell-Based Assay for Alpha-3 Nicotinic Receptor Antibodies Detects Antibodies Exclusively in Autoimmune Autonomic Ganglionopathy.

BACKGROUND AND OBJECTIVES: Autoantibodies against α3-subunit-containing nicotinic acetylcholine receptors (α3-nAChRs), usually measured by radioimmunoprecipitation assay (RIPA), are detected in patients with autoimmune autonomic ganglionopathy (AAG). However, low α3-nAChR antibody levels are frequently detected in other neurologic diseases with questionable significance. Our objective was to develop a method for the selective detection of the potentially pathogenic α3-nAChR antibodies, seemingly present only in patients with AAG. METHODS: The study involved sera from 55 patients from Greece, suspected for autonomic failure, and 13 patients from Italy diagnosed with autonomic failure, positive for α3-nAChR antibodies by RIPA. In addition, sera from 52 patients with Ca2+ channel or Hu antibodies and from 2,628 controls with various neuroimmune diseases were included. A sensitive live cell-based assay (CBA) with α3-nAChR-transfected cells was developed to detect antibodies against the cell-exposed α3-nAChR domain. RESULTS: Twenty-five patients were found α3-nAChR antibody positive by RIPA. Fifteen of 25 patients were also CBA positive. Of interest, all 15 CBA-positive patients had AAG, whereas all 10 CBA-negative patients had other neurologic diseases. RIPA antibody levels of the CBA-negative sera were low, although our CBA could detect dilutions of AAG sera corresponding to equally low RIPA antibody levels. No serum bound to control-transfected cells, and none of the 2,628 controls was α3-CBA positive. DISCUSSION: This study showed that in contrast to the established RIPA for α3-nAChR antibodies, which at low levels is of moderate disease specificity, our CBA seems AAG specific, while at least equally sensitive with the RIPA. This study provides Class II evidence that α3-nAChR CBA is a specific assay for AAG. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that an α3-nAChR cell-based assay is a more specific assay for AAG than the standard RIPA.

Somatostatin immunoreactivity within the urinary bladder nerve fibers and paracervical ganglion urinary bladder projecting neurons in the female pig.

The study was designed to examine the distribution and chemical coding of somatostatin-immunoreactive (SOM-IR) nerve fibers supplying the urinary bladder wall and to establish the distribution and immunohistochemical characteristics of the subpopulation of paracervical ganglion (PCG) SOM-IR neurons projecting to this organ in female pigs. The PCG-urinary bladder projecting neurons (PCG-UBPN) were visualized with retrograde neuronal tracer Fast Blue (FB). Double-labeling immunohistochemistry performed on cryostat sections from the urinary bladder wall revealed that the greatest density of SOM-IR nerve fibers was found in the muscle layer and around blood vessels, a moderate number of these nerve terminals supplied the submucosa and only single SOM-IR axons were encountered beneath the urothelium. In all the investigated sections the vast majority of SOM-IR nerve fibers were immunopositive to vesicular acetylcholine transporter (VAChT) and many SOM-IR axons contained immunoreactivity to neuropeptide Y (NPY). Approximately 65 % of FB-positive (FB+) PCG-UBPN were immunoreactive to SOM. Moreover, PCG FB+/SOM + nerve cells were simultaneously immunoreactive to choline acetyltransferase (ChAT; 64.6 ± 0.6 %), NPY (59.7 ± 1.2 %), neuronal nitric oxide synthase (nNOS; 46.1 ± 0.7 %), vasoactive intestinal polypeptide (VIP; 29.9 ± 2.2 %), Leu5-enkephalin (L-ENK; 19.5 ± 6.3 %), dopamine ß-hydroxylase (DßH; 14.9 ± 1.9 %) or pituitary adenylate cyclase-activating polypeptide (PACAP; 14.8 ± 2.4 %). The present study reveals the extensive expression of SOM in both the nerve fibres supplying the porcine urinary bladder wall and the PCG neurons projecting to this organ, indicating an important regulatory role of SOM in the control of the urinary bladder function.

The role of the tripartite synapse in the heart: how glial cells may contribute to the physiology and pathophysiology of the intracardiac nervous system.

One of the major roles of the intracardiac nervous system (ICNS) is to act as the final site of signal integration for efferent information destined for the myocardium to enable local control of heart rate and rhythm. Multiple subtypes of neurons exist in the ICNS where they are organized into clusters termed ganglionated plexi (GP). The majority of cells in the ICNS are actually glial cells; however, despite this, ICNS glial cells have received little attention to date. In the central nervous system, where glial cell function has been widely studied, glia are no longer viewed simply as supportive cells but rather have been shown to play an active role in modulating neuronal excitability and synaptic plasticity. Pioneering studies have demonstrated that in addition to glia within the brain stem, glial cells within multiple autonomic ganglia in the peripheral nervous system, including the ICNS, can also act to modulate cardiovascular function. Clinically, patients with atrial fibrillation (AF) undergoing catheter ablation show high plasma levels of S100B, a protein produced by cardiac glial cells, correlated with decreased AF recurrence. Interestingly, S100B also alters GP neuron excitability and neurite outgrowth in the ICNS. These studies highlight the importance of understanding how glial cells can affect the heart by modulating GP neuron activity or synaptic inputs. Here, we review studies investigating glia both in the central and peripheral nervous systems to discuss the potential role of glia in controlling cardiac function in health and disease, paying particular attention to the glial cells of the ICNS.

Paracervical ganglion in the female pig during prenatal development: Morphology and immunohistochemical characteristics.

The present study investigated the development of the paracervical ganglion in 5-, 7- and 10-week-old porcine foetuses using double labelling immunofluorescence method. In 5-week-old foetuses single PGP-positive perikarya were visible only along the mesonephric ducts. They contained DßH or VAChT, and nerve fibres usually were PGP/VAChT-positive. The perikarya were mainly oval. In 7-week-old foetuses, a compact group of PGP-positive neurons (3144±213) was visible on both sides and externally to the uterovaginal canal mesenchyme of paramesonephric ducts. Nerve cell bodies contained only DßH (36.40±1.63%) or VAChT (17.31±1.13%). In the 10-week-old foetuses, the compact group of PGP-positive neurons divided into several large and many small clusters of nerve cells and also became more expanded along the whole uterovaginal canal mesenchyme reaching the initial part of the uterine canal of the paramesonephric duct. The number of neurons located in these neuronal structures increased to 4121±259. Immunohistochemistry revealed that PGP-positive nerve cell bodies contained DßH (40.26±0,73%) and VAChT (30.73±1.34%) and were also immunoreactive for NPY (33.24±1,27%), SOM (23.6±0,44%) or VIP (22.9±1,13%). Other substances studied (GAL, NOS, CGRP, SP) were not determined at this stage of the development. In this study, for the first time, the morphology of PCG formation in the porcine foetus has been described in three stages of development. Dynamic changes in the number of neurons and their sizes were also noted, as well as the changes in immunochistochemical coding of maturing neurons.

Diagnostic Algorithm in Hirschsprung's Disease: Focus on Immunohistochemistry Markers.

BACKGROUND/AIM: Hirschsprung disease (HD) is caused by the congenital absence of ganglion cells in the distal bowel (aganglionosis). Rectal biopsy is considered important for its diagnosis. The aim of this study was to apply immunohistochemical staining using a minimal set of antibodies and develop an algorithm that will assist in the diagnosis of HD. PATIENTS AND METHODS: Rectal or colonic biopsies were performed in patients with HD (n=26) or patients treated for other bowel diseases (n=34). Immunohistochemical staining was performed for MAP1b, peripherin, S-100, calretinin, NSE, bcl-2 and CD56 proteins. RESULTS: Staining for CD56, S-100, peripherin and calretinin facilitated the identification of ganglion cells. The use of CD56 and S-100 antibodies together resulted in the highest rate of ganglion cell staining intensity (94%). CONCLUSION: We propose a practical diagnostic algorithm with the application of CD56 and S-100 antibodies that can be used in clinical practice in children suspected of Hirschsprung's disease.

An improved animal model for herpesvirus encephalitis in humans.

Herpesviral encephalitis caused by Herpes Simplex Virus 1 (HSV-1) is one of the most devastating diseases in humans. Patients present with fever, mental status changes or seizures and when untreated, sequelae can be fatal. Herpes Simplex Encephalitis (HSE) is characterized by mainly unilateral necrotizing inflammation effacing the frontal and mesiotemporal lobes with rare involvement of the brainstem. HSV-1 is hypothesized to invade the CNS via the trigeminal or olfactory nerve, but viral tropism and the exact route of infection remain unclear. Several mouse models for HSE have been developed, but they mimic natural infection only inadequately. The porcine alphaherpesvirus Pseudorabies virus (PrV) is closely related to HSV-1 and Varicella Zoster Virus (VZV). While pigs can control productive infection, it is lethal in other susceptible animals associated with severe pruritus leading to automutilation. Here, we describe the first mutant PrV establishing productive infection in mice that the animals are able to control. After intranasal inoculation with a PrV mutant lacking tegument protein pUL21 and pUS3 kinase activity (PrV-ΔUL21/US3Δkin), nearly all mice survived despite extensive infection of the central nervous system. Neuroinvasion mainly occurred along the trigeminal pathway. Whereas trigeminal first and second order neurons and autonomic ganglia were positive early after intranasal infection, PrV-specific antigen was mainly detectable in the frontal, mesiotemporal and parietal lobes at later times, accompanied by a long lasting lymphohistiocytic meningoencephalitis. Despite this extensive infection, mice showed only mild to moderate clinical signs, developed alopecic skin lesions, or remained asymptomatic. Interestingly, most mice exhibited abnormalities in behavior and activity levels including slow movements, akinesia and stargazing. In summary, clinical signs, distribution of viral antigen and inflammatory pattern show striking analogies to human encephalitis caused by HSV-1 or VZV not observed in other animal models of disease.

Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.

BACKGROUND: The presence of calcitonin gene-related peptide and its receptors in multiple brain areas and peripheral tissues previously implicated in migraine initiation and its many associated symptoms raises the possibility that humanized monoclonal anti-calcitonin gene-related peptide antibodies (CGRP-mAbs) can prevent migraine by modulating neuronal behavior inside and outside the brain. Critical to our ability to conduct a fair discussion over the mechanisms of action of CGRP-mAbs in migraine prevention is data generation that determines which of the many possible peripheral and central sites are accessible to these antibodies - a question raised frequently due to their large size. MATERIAL AND METHODS: Rats with uncompromised and compromised blood-brain barrier (BBB) were injected with Alexa Fluor 594-conjugated fremanezumab (Frema594), sacrificed 4 h or 7 d later, and relevant tissues were examined for the presence of Frema594. RESULTS: In rats with uncompromised BBB, Frema594 was similarly observed at 4 h and 7 d in the dura, dural blood vessels, trigeminal ganglion, C2 dorsal root ganglion, the parasympathetic sphenopalatine ganglion and the sympathetic superior cervical ganglion but not in the spinal trigeminal nucleus, thalamus, hypothalamus or cortex. In rats with compromised BBB, Frema594 was detected in the cortex (100 µm surrounding the compromised BBB site) 4 h but not 7 d after injections. DISCUSSION: Our inability to detect fluorescent (CGRP-mAbs) in the brain supports the conclusion that CGRP-mAbs prevent the headache phase of migraine by acting mostly, if not exclusively, outside the brain as the amount of CGRP-mAbs that enters the brain (if any) is too small to be physiologically meaningful.

Herpes Simplex Virus 2 in Autonomic Ganglia: Evidence for Spontaneous Reactivation.

Herpes simplex virus 2 (HSV-2) can be transmitted in the presence or absence of lesions, allowing efficient spread among the general population. Recurrent HSV genital lesions are thought to arise from reactivated latent virus in sensory cell bodies of the dorsal root ganglia (DRG). However, HSV-2 has also been found latent in autonomic ganglia. Spontaneous reactivation or a low level of chronic infection could theoretically also occur in these peripheral nervous tissues, contributing to the presence of infectious virus in the periphery and to viral transmission. Use of a recently described, optimized virus with a monomeric mNeonGreen protein fused to viral capsid protein 26 (VP26) permitted detection of reactivating virus in explanted ganglia and cryosections of DRG and the sacral sympathetic ganglia (SSG) from latently infected guinea pigs. Immediate early, early, and late gene expression were quantified by droplet digital reverse transcription-PCR (ddRT-PCR), providing further evidence of viral reactivation not only in the expected DRG but also in the sympathetic SSG. These findings indicate that viral reactivation from autonomic ganglia is a feature of latent viral infection and that these reactivations likely contribute to viral pathogenesis.IMPORTANCE HSV-2 is a ubiquitous important human pathogen that causes recurrent infections for the life of its host. We hypothesized that the autonomic ganglia have important roles in viral reactivation, and this study sought to determine whether this is correct in the clinically relevant guinea pig vaginal infection model. Our findings indicate that sympathetic ganglia are sources of reactivating virus, helping explain how the virus causes lifelong recurrent disease.

Alarm pheromone and kairomone detection via bitter taste receptors in the mouse Grueneberg ganglion.

BACKGROUND: The mouse Grueneberg ganglion (GG) is an olfactory subsystem specialized in the detection of volatile heterocyclic compounds signalling danger. The signalling pathways transducing the danger signals are only beginning to be characterized. RESULTS: Screening chemical libraries for compounds structurally resembling the already-identified GG ligands, we found a new category of chemicals previously identified as bitter tastants that initiated fear-related behaviours in mice depending on their volatility and evoked neuronal responses in mouse GG neurons. Screening for the expression of signalling receptors of these compounds in the mouse GG yielded transcripts of the taste receptors Tas2r115, Tas2r131, Tas2r143 and their associated G protein α-gustducin (Gnat3). We were further able to confirm their expression at the protein level. Challenging these three G protein-coupled receptors in a heterologous system with the known GG ligands, we identified TAS2R143 as a chemical danger receptor transducing both alarm pheromone and predator-derived kairomone signals. CONCLUSIONS: These results demonstrate that similar molecular elements might be used by the GG and by the taste system to detect chemical danger signals present in the environment.

Neurite outgrowth in cultured mouse pelvic ganglia - Effects of neurotrophins and bladder tissue.

Neurotrophic factors regulate survival and growth of neurons. The urinary bladder is innervated via both sympathetic and parasympathetic neurons located in the major pelvic ganglion. The aim of the present study was to characterize the effects of the neurotrophins nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) on the sprouting rate of sympathetic and parasympathetic neurites from the female mouse ganglion. The pelvic ganglion was dissected out and attached to a petri dish and cultured in vitro. All three factors (BDNF, NT-3 and NGF) stimulated neurite outgrowth of both sympathetic and parasympathetic neurites although BDNF and NT-3 had a higher stimulatory effect on parasympathetic ganglion cells. The neurotrophin receptors TrkA, TrkB and TrkC were all expressed in neurons of the ganglia. Co-culture of ganglia with urinary bladder tissue, but not diaphragm tissue, increased the sprouting rate of neurites. Active forms of BDNF and NT-3 were detected in urinary bladder tissue using western blotting whereas tissue from the diaphragm expressed NGF. Neurite outgrowth from the pelvic ganglion was inhibited by a TrkB receptor antagonist. We therefore suggest that the urinary bladder releases trophic factors, including BDNF and NT-3, which regulate neurite outgrowth via activation of neuronal Trk-receptors. These findings could influence future strategies for developing pharmaceuticals to improve re-innervation due to bladder pathologies.

Distinct pattern of enteric phospho-alpha-synuclein aggregates and gene expression profiles in patients with Parkinson's disease.

Phosphorylated alpha-synuclein (p-α-syn) containing Lewy bodies (LBs) and Lewy neurites (LNs) are neuropathological hallmarks of Parkinson's disease (PD) in the central nervous system (CNS). Since they have been also demonstrated in the enteric nervous system (ENS) of PD patients, the aim of the study was to analyze enteric p-α-syn positive aggregates and intestinal gene expression. Submucosal rectal biopsies were obtained from patients with PD and controls and processed for dual-label-immunohistochemistry for p-α-syn and PGP 9.5. p-α-syn positive aggregates in nerve fibers and neuronal somata were subjected to a morphometric analysis. mRNA expression of α-syn and dopaminergic, serotonergic, VIP (vaso intestinal peptide) ergic, cholinergic, muscarinergic neurotransmitter systems were investigated using qPCR. Frequency of p-α-syn positive nerve fibers was comparable between PD and controls. Although neuronal p-α-syn positive aggregates were detectable in both groups, total number and area of p-α-syn positive aggregates were increased in PD patients as was the number of small and large sized aggregates. Increased expression of dopamine receptor D1, VIP and serotonin receptor 3A was observed in PD patients, while serotonin receptor 4 and muscarinic receptor 3 (M3R) were downregulated. M3R expression correlated negative with the number of small sized p-α-syn positive aggregates. The findings strengthen the hypothesis that the CNS pathology of increased p-α-syn in PD also applies to the ENS, if elaborated morphometry is applied and give further insights in altered intestinal gene expression in PD. Although the mere presence of p-α-syn positive aggregates in the ENS should not be regarded as a criterion for PD diagnosis, elaborated morphometric analysis of p-α-syn positive aggregates in gastrointestinal biopsies could serve as a suitable tool for in-vivo diagnosis of PD.

Uterine Cervical Neurotransmission and Cervical Remodeling.

Cervical remodeling (CR) is a complex process, which, in part, is believed to be induced by physiological inflammation. Even though the female reproductive tissues are richly innervated by nerves from the parasympathetic pelvic autonomic ganglia, sensory dorsal root and nodose ganglia, their roles (neuronal factors) in this process (CR) has been largely attributed to sex steroid hormones, until recently. Here, we discuss the interaction between neuropeptides derived from peripheral nerves associated with uterine cervix and estrogen, and their likely impact on cervical remodeling. It is likely that these neuronal factors, under the influence of estrogen, could induce physiological inflammation during cervical remodeling by promoting the expression of vascular endothelial growth factor, among other factors.

Characterization of glutamatergic neurons in the rat atrial intrinsic cardiac ganglia that project to the cardiac ventricular wall.

The intrinsic cardiac nervous system modulates cardiac function by acting as an integration site for regulating autonomic efferent cardiac output. This intrinsic system is proposed to be composed of a short cardio-cardiac feedback control loop within the cardiac innervation hierarchy. For example, electrophysiological studies have postulated the presence of sensory neurons in intrinsic cardiac ganglia (ICG) for regional cardiac control. There is still a knowledge gap, however, about the anatomical location and neurochemical phenotype of sensory neurons inside ICG. In the present study, rat ICG neurons were characterized neurochemically with immunohistochemistry using glutamatergic markers: vesicular glutamate transporters 1 and 2 (VGLUT1; VGLUT2), and glutaminase (GLS), the enzyme essential for glutamate production. Glutamatergic neurons (VGLUT1/VGLUT2/GLS) in the ICG that have axons to the ventricles were identified by retrograde tracing of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injected in the ventricular wall. Co-labeling of VGLUT1, VGLUT2, and GLS with the vesicular acetylcholine transporter (VAChT) was used to evaluate the relationship between post-ganglionic autonomic neurons and glutamatergic neurons. Sequential labeling of VGLUT1 and VGLUT2 in adjacent tissue sections was used to evaluate the co-localization of VGLUT1 and VGLUT2 in ICG neurons. Our studies yielded the following results: (1) ICG contain glutamatergic neurons with GLS for glutamate production and VGLUT1 and 2 for transport of glutamate into synaptic vesicles; (2) atrial ICG contain neurons that project to ventricle walls and these neurons are glutamatergic; (3) many glutamatergic ICG neurons also were cholinergic, expressing VAChT; (4) VGLUT1 and VGLUT2 co-localization occurred in ICG neurons with variation of their protein expression level. Investigation of both glutamatergic and cholinergic ICG neurons could help in better understanding the function of the intrinsic cardiac nervous system.

Vasopressin and sympathetic system mediate the cardiovascular effects of the angiotensin II in the bed nucleus of the stria terminalis in rat.

The bed nucleus of the stria terminalis (BST) is involved in cardiovascular regulation. The angiotensin II (Ang II) receptor (AT1), and angiotensinogen were found in the BST. In our previous study we found that microinjection of Ang II into the BST produced a pressor response. This study was performed to find the mechanisms mediating this response in anesthetized rats. Ang II was microinjected into the BST and the cardiovascular responses were re-tested after systemic injection of a blocker of autonomic or vasopressin V1 receptor. The ganglionic nicotinic receptor blocker, hexamethonium dichloride, attenuated the pressor response to Ang II, indicating that the cardiovascular sympathetic system is involved in the pressor effect of Ang II. A selective vasopressin V1 receptor antagonist greatly attenuated the pressor effect of Ang II, indicating that the Ang II increases the arterial pressure via stimulation of vasopressin release as well. In conclusion, in the BST, Ang II as a neurotransmitter increases blood pressure by exciting cardiovascular sympathetic system and directly or indirectly causing vasopressin to release into bloodstream by VPN. This is an interesting new finding that not only circulating Ang II but also brain Ang II makes vasopressin release.

[Age-related expression of calcium-binding proteins in autonomic ganglionic neurons].

Calbindin 28 kDa (CB), calretinin (CR) and parvalbumin (PB) are belonged to calcium-binding proteins which are widely distributed in the nervous system and selectively expressed in certain population of neurons. These proteins are expressed not only in the central nervous system, but also in the autonomic ganglia. CB and PB are found in the sympathetic ganglia of rodents, CB and CR are found in metasympathetic intramural ganglia. Their functions are poor understood but one can suggest their important role in regulation of the Ca2+ level in the cell. Сalcium-binding proteins are also play an important role in the development of autonomic neurons. There is an increasing of the percentage of CB and CR in the metasympathetic intramural ganglia of small intestine in the early postnatal development, whereas in sympathetic ganglia the percentage of CB is decreased. Possibly, the functional meaning of such changes can be explained by the role of calcium currents in the development of neurons and the synaptic transmission.

Mechanism of the cardiovascular effects of the GABAA receptors of the ventral tegmental area of the rat brain.

The ventral tegmental area (VTA) contains GABA terminals involved in the regulation of the cardiovascular system. Previously, we demonstrated that blocking GABAA but not GABAB receptors produced a pressor response accompanied by marked bradycardia. This study was performed to find the possible mechanisms involved in these responses by blocking ganglionic nicotinic receptors, peripheral muscarinic receptors or peripheral V1 vasopressin receptors. Experiments were performed on urethane anesthetized male Wistar rats. Drugs were microinjected unilaterally into the VTA (100 nl). The average changes in mean arterial pressure (MAP) and heart rate (HR) were compared between pre- and post-treatment using paired t-test. Injection of bicuculline methiodide (BMI), a GABAA antagonist, into the VTA caused a significant increase in MAP and a decrease in HR. Administration (i.v.) of the nicotinic receptor blocker, hexamethonium, enhanced the pressor response but abolished the bradycardic response to BMI, which ruled out involvement of the sympathetic nervous system. Blockade of the peripheral muscarinic receptors by homatropine (i.v.) abolished the bradycardic effect of BMI, but had no effect on the pressor response, indicating that bradycardia was produced by the parasympathetic outflow to the heart. Both the pressor and bradycardic responses to BMI were blocked by V1 receptor antagonist (i.v.), indicating that administration of BMI in the VTA disinhibited the release of vasopressin into the circulation. In conclusion, we demonstrated that GABAergic mechanism of the VTA exerts a tonic inhibition on vasopressin release through activation of GABAA receptors. The sympathetic system is not involved in the decrease of blood pressure by GABA of the VTA.

[CHANGES IN THE PANCREATIC ISLETS AND NERVOUS ELEMENTS IN RATS DURING AGING (AN IMMUNOHISTOCHEMICAL STUDY)].

The neural apparatus and the endocrine part of the pancreas was studied in Wistar rats aged 3-4 and 19 months (n = 24) using the immunohistochemical reactions for synaptophysin (Syn), tyrosine hydroxylase (TH) and protein gene product 9.5 (PGP 9.5). Since Syn and PGP 9.5 are highly selective in detection of pancreatic islet (PI) endocrinocytes, it was possible to examine their topography and density in all parts of the pancreas. It was found that in rats aged 19 months, the total number of PI was decreased as compared to that in young animals. The study of PI size distribution has shown that the number of large islets decreased with age. Young animals showed rich innervation of the pancreas which was represented by three nerve plexuses: the first was a broadly-looped one, formed by small nerve trunks and bundles of unmyelinated and myelinated nerve fibers, the second consisted of thin bundles of postganglionic axons and microganglia, and the third (main terminal plexus) was formed by axons with varicosities and synapses of "en passant" type. In aged rats, marked degenerative changes in the neurons of intramural ganglia, nerve trunks and bundles were noted together with the reduction or complete absence of Syn- and TH-positive efferent parasympathetic and sympathetic terminals around blood vessels, excretory ducts, denervation of the exocrine and endocrine parts of the pancreas. Innervation disturbances in some lobules were accompanied by small inflammatory perivascular infiltrates.

Developmental markers of ganglion cells in the enteric nervous system and their application for evaluation of Hirschsprung disease.

Hirschsprung disease (HSCR) is a congenital disease resulting from failure of neural crest-derived ganglion cells to colonize the colon. Conventional diagnostic methods are insufficient for evaluating the 'functional' prognosis of HSCR. In order to elucidate the maturation of ganglion cells, 17 immunohistochemical markers were examined. We examined the digestive tracts of 2 human early delivery patients, 2 miniature swine fetuses, 4 little infants, 3 infants, 3 children, 6 adults, and 3 aged individuals. With increasing age, the labeling index (LI) for both calretinin and tyrosine hydroxylase (TH) increased, whereas that for SOX10 decreased. We then examined the 'transitional zone' of HSCR in 21 affected patients and 18 controls for these three markers. The LI of calretinin and TH were significantly lower than in the controls (median: 3.7 in HSCR and 8.2 in controls, P < 0.001, median: 27.9 in HSCR and 44.4 in controls, P < 0.001, respectively). In contrast, the LI for SOX10 showed no significant difference (median: 33.7 in HSCR and 29.2 in controls, P = 0.666) however, hierarchical cluster analysis was able to divide HSCR patients into two groups. These results suggest that immature ganglion cells are present in the transitional zone of HSCR, and that HSCR may have two different pathophysiological processes.

Spinal cord stimulation suppresses focal rapid firing-induced atrial fibrillation by inhibiting atrial ganglionated plexus activity.

OBJECTIVE: This study was designed to demonstrate that spinal cord stimulation (SCS) could suppress high-frequency stimulation (HFS)-induced focal atrial fibrillation (AF) at atrial and pulmonary vein (PV) sites by inhibiting atrial ganglionated plexus (GP) activity. METHODS: Multielectrode catheters were attached to atria and all PV sites. SCS was performed at the T1-T5 spinal region for 1 hour. At the baseline state and the end of 1 hour of SCS, 40 milliseconds of HFS was delivered 2 milliseconds after atrial pacing to determine the AF threshold at each site. One electrode was attached to the superior left GP so that HFS to this site induced sinus rate slowing. Microelectrodes inserted into the anterior right GP recorded neural firing. RESULTS: SCS induced a significant increase in AF threshold at all sites (all P < 0.05). The sinus rate slowing response induced by superior left GP stimulation was blunted by SCS (17% ± 3.6% vs. 39% ± 3.8%, P < 0.05). The frequency (32 ± 4 vs. 87 ± 6 impulses per minute, P < 0.05) and amplitude (0.16 ± 0.02 vs. 0.42 ± 0.04 mv, P < 0.05) of the neural activity recorded from the anterior right GP were markedly inhibited by SCS. CONCLUSIONS: SCS may prevent episodic AF caused by rapid PV and non-PV firing through modulating GP activity.