Parasympathetic Neurons Research Articles

The identification and neurochemical characterization of central neurons that target parasympathetic preganglionic neurons involved in the regulation of choroidal blood flow in the rat eye using pseudorabies virus, immunolabeling and conventional pathway tracing methods.

The choroidal blood vessels of the eye provide the main vascular support to the outer retina. These blood vessels are under parasympathetic vasodilatory control via input from the pterygopalatine ganglion (PPG), which in turn receives its preganglionic input from the superior salivatory nucleus (SSN) of the hindbrain. The present study characterized the central neurons projecting to the SSN neurons innervating choroidal PPG neurons, using pathway tracing and immunolabeling. In the initial set of studies, minute injections of the Bartha strain of the retrograde transneuronal tracer pseudorabies virus (PRV) were made into choroid in rats in which the superior cervical ganglia had been excised (to prevent labeling of sympathetic circuitry). Diverse neuronal populations beyond the choroidal part of ipsilateral SSN showed transneuronal labeling, which notably included the parvocellular part of the paraventricular nucleus of the hypothalamus (PVN), the periaqueductal gray, the raphe magnus (RaM), the B3 region of the pons, A5, the nucleus of the solitary tract (NTS), the rostral ventrolateral medulla (RVLM), and the intermediate reticular nucleus of the medulla. The PRV+ neurons were located in the parts of these cell groups that are responsive to systemic blood pressure signals and involved in systemic blood pressure regulation by the sympathetic nervous system. In a second set of studies using PRV labeling, conventional pathway tracing, and immunolabeling, we found that PVN neurons projecting to SSN tended to be oxytocinergic and glutamatergic, RaM neurons projecting to SSN were serotonergic, and NTS neurons projecting to SSN were glutamatergic. Our results suggest that blood pressure and volume signals that drive sympathetic constriction of the systemic vasculature may also drive parasympathetic vasodilation of the choroidal vasculature, and may thereby contribute to choroidal baroregulation during low blood pressure.

Inhibitory effects of endomorphin-2 on excitatory synaptic transmission and the neuronal excitability of sacral parasympathetic preganglionic neurons in young rats.

The function of the urinary bladder is partly controlled by parasympathetic preganglionic neurons (PPNs) of the sacral parasympathetic nucleus (SPN). Our recent work demonstrated that endomorphin-2 (EM-2)-immunoreactive (IR) terminals form synapses with μ-opioid receptor (MOR)-expressing PPNs in the rat SPN. Here, we examined the effects of EM-2 on excitatory synaptic transmission and the neuronal excitability of the PPNs in young rats (24–30 days old) using a whole-cell patch-clamp approach. PPNs were identified by retrograde labeling with the fluorescent tracer tetramethylrhodamine-dextran (TMR). EM-2 (3 μM) markedly decreased both the amplitude and the frequency of the spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) of PPNs. EM-2 not only decreased the resting membrane potentials (RMPs) in 61.1% of the examined PPNs with half-maximal response at the concentration of 0.282 μM, but also increased the rheobase current and reduced the repetitive action potential firing of PPNs. Analysis of the current–voltage relationship revealed that the EM-2-induced current was reversed at −95 ± 2.5 mV and was suppressed by perfusion of the potassium channel blockers 4-aminopyridine (4-AP) or BaCl2 or by the addition of guanosine 5′-[β-thio]diphosphate trilithium salt (GDP-β-S) to the pipette solution, suggesting the involvement of the G-protein-coupled inwardly rectifying potassium (GIRK) channel. The above EM-2-invoked inhibitory effects were abolished by the MOR selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), indicating that the effects of EM-2 on PPNs were mediated by MOR via pre- and/or post-synaptic mechanisms. EM-2 activated pre- and post-synaptic MORs, inhibiting excitatory neurotransmitter release from the presynaptic terminals and decreasing the excitability of PPNs due to hyperpolarization of their membrane potentials, respectively. These inhibitory effects of EM-2 on PPNs at the spinal cord level may explain the mechanism of action of morphine treatment and morphine-induced bladder dysfunction in the clinic.

Targeting prostate cancer cells with neurotransmission modulating drugs.

e16093 Background: Innervation of prostate tissue by sympathetic and parasympathetic neurons is critical for prostate cancer (PCa) initiation and progression. Both, adrenergic and cholinergic recep...

Diabetes induces GABA receptor plasticity in murine vagal motor neurons.

Autonomic dysregulation accompanies type-1 diabetes, and synaptic regulation of parasympathetic preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV) is altered after chronic hyperglycemia/hypoinsulinemia. Tonic gamma-aminobutyric acid A (GABAA) inhibition prominently regulates DMV neuron activity, which contributes to autonomic control of energy homeostasis. This study investigated persistent effects of chronic hyperglycemia/hypoinsulinemia on GABAA receptor-mediated inhibition in the DMV after streptozotocin-induced type-1 diabetes using electrophysiological recordings in vitro, quantitative (q)RT-PCR, and immunohistochemistry. Application of the nonspecific GABAA receptor agonist muscimol evoked an outward current of significantly larger amplitude in DMV neurons from diabetic mice than controls. Results from application of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP), a δ-subunit agonist, suggested that GABAA receptors containing δ-subunits contributed to the enhanced inducible tonic GABA current in diabetic mice. Sensitivity to THIP of inhibitory postsynaptic currents in DMV neurons from diabetic mice was also increased. Results from qRT-PCR and immunohistochemical analyses indicated that the altered GABAergic inhibition may be related to increased trafficking of GABAA receptors that contain the δ-subunit, rather than an expression change. Overall these findings suggest increased sensitivity of δ-subunit containing GABAA receptors after several days of hyperglycemia/hypoinsulinemia, which dramatically alters GABAergic inhibition of DMV neurons and could contribute to diabetic autonomic dysregulation.

Purinergic signalling in the urinary bladder

It is well established that in most species, exocytotic vesicular release of ATP from parasympathetic neurons contributes to contraction of the bladder. However, ATP is released not only from parasympathetic nerves, but also from the urothelium. During bladder filling, the urothelium is stretched and ATP is released from the umbrella cells thereby activating mechanotransduction pathways. ATP release can also be induced by various mediators present in the urine and and/or released from nerves or other components of the lamina propria. Urothelial release of ATP is mainly attributable to vesicular transport or exocytosis and, to a smaller extent, to pannexin hemichannel conductive efflux. After release, ATP acts on P2X3 and P2X2/3 receptors on suburothelial sensory nerves to initiate the voiding reflex and to mediate the sensation of bladder filling and urgency. ATP also acts on suburothelial interstitial cells/myofibroblasts generating an inward Ca(2+) transient that via gap junctions could provide a mechanism for long-distance spread of signals from the urothelium to the detrusor muscle. ATP release can be affected by urological diseases, e.g., interstitial cystitis and both the mechanisms of release and the receptors activated by ATP may be targets for future drugs for treatment of lower urinary tract disorders.

Muscarinic receptor-mediated excitation of rat intracardiac ganglion neurons

Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of −60 mV. Removal of extracellular Ca2+ markedly increased the OxoM-induced current (IOxoM). The inward IOxoM in the absence of extracellular Ca2+ was fully inhibited by removal of extracellular Na+, indicating the involvement of non-selective cation channels. The IOxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The IOxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M1 receptor, produced partial inhibition of the IOxoM. In the presence of MT-7, concentration–inhibition curve of the M3-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M1 and M3 receptors to the OxoM response. The IOxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP3 receptor blocker xestospongin C also inhibited the IOxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the IOxoM, while KN-62, a blocker of Ca2+/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca2+ from intracellular Ca2+ stores and calmodulin.The cation channels activated by muscarinic receptors may play an important role in neuronal membrane depolarization in rat intracardiac ganglion neurons.

Is the Atrial Neural Plexis a Therapeutic Target in Atrial Fibrillation?

Circumferential pulmonary vein isolation is the mainstay of atrial fibrillation (AF) ablation, but alternative approaches and techniques have been developed to improve the outcomes. One of these additional ablation targets are ganglionated plexi of the intrinsic cardiac autonomic system that contain a variety of sympathetic and parasympathetic neurons that communicate with the extrinsic cardiac autonomic nervous system. The ganglionated plexi of the heart do not serve as a simple relay station but could modulate the autonomic interaction between the extrinsic and intrinsic cardiac autonomic system. Intrinsic cardiac autonomic nerve activity is an invariable trigger of paroxysmal atrial tachyarrhythmia, including atrial fibrillation. Although multiple studies have shown that ganglionated plexi play an important role in initiating atrial fibrillation, there is no consensus on a standardized protocol for selecting target sites and determining how ganglionated plexi ablation can best be accomplished. Recent clinical trials have demonstrated the feasibility and efficacy of ganglionated plexi ablation in addition to pulmonary vein isolation, but novel technologies and strategies are necessary to improve the current ablation techniques in managing patients with atrial fibrillation. This review focuses on the relationship between atrial ganglionated plexi and atrial fibrillation and the potential benefits and limitations of ganglionated plexi ablation in the management of atrial fibrillation.

Olanzapine Reduces Neuronal Activity in the Dorsal Motor Nucleus of the Vagus

Olanzapine, a widely prescribed atypical antipsychotic, relieves the symptoms of schizophrenia and bipolar disorder. Despite its positive impact on mental health, patients treated with olanzapine experience side effects related to weight gain and impaired glucose homeostasis. A variety of mechanisms and brain areas underlying the metabolic side effects have been proposed; however, the involvement of the brainstem remained to be elucidated. Parasympathetic neurons in the dorsal motor nucleus of the vagus (DMV) set the vagal tone and thus control visceral functions including feeding, digestion, and glucose homeostasis. In this study, whole‐cell patch‐clamp recordings were performed to test the hypothesis that olanzapine modulates neuronal activity in the DMV of mice. Acute application of olanzapine (10 µM) hyperpolarized the majority (~70 %) of DMV neurons in male mice by an average of ‐7.77 ± 1.17 mV (n=20). This effect was concentration‐dependent (EC50 = 16.02 ± 1.78 µM), and was associated with a reduction of input resistance. Similarly, hyperpolarization was observed in female mice (n=9). The hyperpolarization also occurred in the presence of tetrodotoxin, suggesting a postsynaptic mechanism of action. Next, stomach‐ and liver‐projecting DMV neurons were identified with transsynaptic retrograde viral labeling. Acute olanzapine application resulted in hyperpolarization of the majority of both liver‐ and stomach‐related DMV neurons. In summary, our data demonstrate that in slices olanzapine administration hyperpolarizes the majority of DMV neurons via postsynaptic mechanisms. These data suggest that olanzapine has the potential to modulate vagal outflow to subdiaphragmatic organs.Supported by: Pilot Project Grant Tulane University School of Medicine

Identification of autonomic neuronal chains innervating gingiva and lip.

The major goals of this present study were 1) to further clarify which parasympathetic ganglion sends postganglionic fibers to the lower gingiva and lip that may be involved in the inflammatory processes besides the local factors; 2) to separately examine the central pathways regulating sympathetic and parasympathetic innervation; and 3) to examine the distribution of central premotor neurons on both sides. A retrogradely transported green fluorescent protein conjugated pseudorabies virus was injected into the lower gingiva and lip of intact and sympathectomized adult female rats. Some animals received virus in the adrenal medulla which receive only preganglionic sympathetic fibers to separately clarify the sympathetic nature of premotor neurons. After 72-120h of survival and perfusion, the corresponding thoracic part of the spinal cord, brainstem, hypothalamus, cervical, otic, submandibular and trigeminal ganglia were harvested. Frozen sections were investigated under a confocal microscope. Green fluorescence indicated the presence of the virus. The postganglionic sympathetic neurons related to both organs are located in the three cervical ganglia, the preganglionic neurons in the lateral horn of the spinal cord on ipsilateral side; premotor neurons were found in the ventrolateral medulla, locus ceruleus, gigantocellular and paraventricular nucleus and perifornical region in nearly the same number on both sides. The parasympathetic postganglionic neurons related to the gingiva are present in the otic and related to the lip are present in the otic and submandibular ganglia and the preganglionic neurons are in the salivatory nuclei. Third order neurons were found in the gigantocellular reticular and hypothalamic paraventricular nuclei and perifornical area.

Axotomy of tributaries of the pelvic and pudendal nerves induces changes in the neurochemistry of mouse dorsal root ganglion neurons and the spinal cord

Using immunohistochemical techniques, we characterized changes in the expression of several neurochemical markers in lumbar 4-sacral 2 (L4-S2) dorsal root ganglion (DRG) neuron profiles (NPs) and the spinal cord of BALB/c mice after axotomy of the L6 and S1 spinal nerves, major tributaries of the pelvic (targeting pelvic visceral organs) and pudendal (targeting perineum and genitalia) nerves. Sham animals were included. Expression of cyclic AMP-dependent transcription factor 3 (ATF3), calcitonin gene-related peptide (CGRP), transient receptor potential cation channel subfamily V, member 1 (TRPV1), tyrosine hydroxylase (TH) and vesicular glutamate transporters (VGLUT) types 1 and -2 was analysed seven days after injury. L6-S1 axotomy induced dramatic de novo expression of ATF3 in many L6-S1 DRG NPs, and parallel significant downregulations in the percentage of CGRP-, TRPV1-, TH- and VGLUT2-immunoreactive (IR) DRG NPs, as compared to their expression in uninjured DRGs (contralateral L6-S1-AXO; sham mice); VGLUT1 expression remained unaltered. Sham L6-S1 DRGs only showed a small ipsilateral increase in ATF3-IR NPs (other markers were unchanged). L6-S1-AXO induced de novo expression of ATF3 in several lumbosacral spinal cord motoneurons and parasympathetic preganglionic neurons; in sham mice the effect was limited to a few motoneurons. Finally, a moderate decrease in CGRP- and TRPV1-like-immunoreactivities was observed in the ipsilateral superficial dorsal horn neuropil. In conclusion, injury of a mixed visceral/non-visceral nerve leads to considerable neurochemical alterations in DRGs matched, to some extent, in the spinal cord. Changes in these and potentially other nociception-related molecules could contribute to pain due to injury of nerves in the abdominopelvic cavity.

Autonomic cardiac nerves: literature review.

The aim of this paper was to summarise the knowledge about the autonomic cardiac innervation. It is generally known, that the cardiac nervous system consists of nerve plexoganglionic structures located mostly around the strategic regions of the heart. They consist of two main types of components: parasympathetic neurons, which exert an inhibitory effect, and sympathetic postganglionic nerve fibres, which stimulate the cardiac conduction system, and myocardial cells. However, many authors describe that cardiac ganglia contain various populations of neurons. The largest group are classical cholinergic neurons. The second group of cardiac neurons are cells of dual, cholinergic-adrenergic character. There is also subpopulation of small intensely fluoroscent cells of typically adrenergic phenotype. Moreover, many authors indicated the presence of various neurotransmitters in various combinations. In this way, the neurons in cardiac ganglia are a neurochemical complex beyond the classical vision of parasympathetic ganglia.

The Effects of Nerve Growth Factor on Nicotinic Synaptic Transmission in Mouse Airway Parasympathetic Neurons.

In autonomic ganglia, acetylcholine (ACh) is released from preganglionic nerve terminals and binds to nicotinic ACh receptors (nAChRs) on postganglionic neurons, resulting in a brief, short-lived synaptic potential (fast excitatory postsynaptic potential [fEPSP]). Although nerve growth factor (NGF) is known to affect sensory and sympathetic nerves, especially during development, little is known regarding its effect on parasympathetic nerves, especially on adult neurons. Elevated levels of NGF and NGF-mediated neural plasticity may have a role in airway diseases, such as asthma and chronic obstructive pulmonary disease. In this study, we characterize the composition and response of nAChRs in parasympathetic neurons located in lower airways of mice, and note the effects of NGF on fEPSPs and on nicotinic currents. Based on immunohistochemical staining, nAChRs are made up of α-3 and β-4 subunits; in addition, tropomyosin-related kinase A, the receptor for NGF, is also expressed by the neurons. Vagus nerve evoked fEPSPs and inward currents evoked by a nicotinic receptor agonist (1,1-dimethyl-4-phenylpiperazinium) were increased by NGF. NGF also affected the action potential after hyperpolarization. These studies were done in mice, which are routinely used to study airway diseases, such as asthma, where the allergen-induced contraction of airway smooth muscle has a well-defined parasympathetic cholinergic component.

Bladder outlet obstruction causes up-regulation of nicotinic acetylcholine receptors in bladder-projecting pelvic ganglion neurons

Pelvic ganglion (PG) neurons relay sympathetic and parasympathetic signals to the lower urinary tract, comprising the urinary bladder and bladder outlet, and are thus essential for both storage and voiding reflexes. Autonomic transmission is mediated by activation of the nicotinic acetylcholine receptor (nAChR) in PG neurons. Previously, bladder outlet obstruction (BOO), secondary to benign prostatic hyperplasia, was found to increase soma sizes of bladder-projecting PG neurons. To date, however, it remains unknown whether these morphological changes are accompanied by functional plasticity in PG neurons. In the present study, we investigated whether BOO alters acetylcholine receptor (nAChR) transcript expression and current density in bladder PG neurons. Partial ligation of the rat urethra for six weeks induced detrusor overactivity (DO), as observed during cystometrical measurement. In rats exhibiting DO, membrane capacitance of parasympathetic bladder PG neurons was selectively increased. Real-time PCR analysis revealed that BOO enhanced the expression of the transcripts encoding the nAChR α3 and β4 subunits in PG neurons. Notably, BOO significantly increased ACh-evoked current density in parasympathetic bladder PG neurons, whereas no changes were observed in sympathetic bladder and parasympathetic penile PG neurons. In addition, other ligand-gated ionic currents were immune to BOO in bladder PG neurons. Taken together, these data suggest that BOO causes upregulation of nAChR in parasympathetic bladder PG neurons, which in turn may potentiate ganglionic transmission and contribute to the development of DO.

微細加工を用いた副交感神経細胞-心筋細胞共培養系の構築

The sympathetic neurons (SN) and the parasympathetic neurons (PN) have important roles in regulation of the cardiac activity. Imbalance of the SN and the PN activities results in abnormal cardiac electrophysiology and can cause several diseases such as atrial fibrillation. While activities of SN and cardiomyocytes have been studied using microfabrication technique, relationship between activities of PN and cardiomyocytes remains unclear. In this study, we developed a device for co-culture of intracardiac ganglion (ICG) neurons which are parasympathetic neurons and atrial myocytes (AMs) using microfabrication technique. Morphological connections between ICG neurons and AMs were observed by immunostaining. Inter-beat-intervals (IBI) were modulated after electrical stimulation of ICG neurons. This modulation completely blocked after applying atropine, a muscarinic receptor antagonist. These results suggest that the co-culture system can be utilized in the research of cell-cell interactions between parasympathetic neurons and cardiomyocytes.

The Intestinal Barrier in Air Pollution-Associated Neural Invol vement in Mexico City Residents: Mind the Gut, the Evolution of a Chan ging Paradigm Relevant to Parkinson Disease Risk

Objective: Braak et al proposal in 2003 “a putative environmental pathogen capable of passing the gastric epithelial lining might induce α-synuclein misfolding and aggregation” could indeed be particulate matter gaining access through the most vulnerable section of the GI tract: the small bowel. This study is focused on the electron microscopy examination of tight junctions in duodenum of healthy dogs residing in one of the most polluted megacities in our continent, Mexico City Metropolitan Area (MCMA)with high concentrations of fine particulate matter (PM 2.5) and nanosize PM versus low-air pollution controls and to measure serum antibodies to tight junctions (TJ) and neural proteins in MCMA versus low air pollution exposed children.The small intestine would be a prime PM target: it has a single unattached mucus layer, particles have easy access to epithelial cells and Peyer’s patches, altering epithelial integrity and accessing the enteric nervous system.Autopsies in MCMA children v controls show extensive brainstem oxidative stress, microglial activation, and accumulation of α-synuclein, from the dorsal motor nucleus of the vagus to the substantianigrae.Air pollution targets the dorsal vagal complex in mice exposed to the polluted MCMA atmosphere. Methods: A pilot observational case-control dogs and children study of high versus low PM 2.5 exposures.We counted and evaluated the integrity of TJ’s in duodenal electron micrographs from 6 MCMA dogs (5.01 ± 1.36 years) and 4 control dogs (5.87 ± 1.50 years) and we measured by ELISA serum antibodies to tight junctions (TJ) and neural proteins in 95 MCMA versus controls (11.02 ± 3.6 years). Results: Disruption of epithelial integrity with TJ structural changes in MCMA v control dogs (p<0.0001), the major determinant of paracellular permeability characterized the MCMA dogs’ small bowel architecture. MCMA children had higher occludin-zonulin, actin, transglutaminase 3 and 6, and glutamic acid decarboxylase autoantibodies (p<0.01).Conclusion: The integrity of the gastrointestinal (GI) barrier is significantly compromised in MCMA dogs and could be altered in MCMA children as evidenced by the autoimmune response to TJ and neural proteins. The GI breakdown likely impacts neuronal enteric populations and PM could reach the vagus and the brainstem. In the setting of urban air pollution, the evolution of a changing paradigm favoring a pathogen penetrating an epithelial lining and via transsynaptic transmission reaching preganglionic parasympathetic motor neurons of the vagus nerve has to entertain particles as a potential culprit.Defining the linkage and the health consequences of the brain/ gut/ immune system interactions in urban children showing already the early hallmarks of Parkinson’s disease ought to be of pressing importance for public health, may provide a fresh insight into Parkinson disease pathogenesis and open opportunities for pediatric neuroprotection.

Nicotinic Acetylcholine Receptors and Cardiac Vagal activity in Rats with Type 2 Diabetes

Reduced cardiac parasympathetic activity is involved in sudden cardiac death and is responsible for high mortality in patients with type 2 diabetes mellitus (T2DM). Nicotinic acetylcholine receptors (nAChRs) mediate synaptic transmission in the intracardiac ganglia (ICG) and regulate the excitability of postganglionic parasympathetic neurons. We hypothesized that changes in the functional responsiveness of postsynaptic nAChRs to nicotine (a nAChR agonist) might be involved in attenuated cardiac parasympathetic (vagal) activity in type 2 diabetic rats. A rat model of T2DM was induced by a combination of both high-fat diet and injection of low-dose streptozotocin (30 mg/kg, i.p.). As an index of cardiac vagal function, changes of heart rate in response to graded vagal efferent nerve stimulation were blunted in T2DM rats, compared to sham rats. In isolated Langendorff-perfused hearts, there was no significant difference on sensitivity of the heart to acetylcholine in sham and T2DM rats. Whole-cell patch-clamp data showed that T2DM decreased nAChR currents and sensitivity of the nAChR channel to nicotine in ICG neurons. However, the data from immunofluorescence staining showed that there is no significant difference in the protein expression of ?3 and ?4 nAChR subunits in ICG neurons of sham and T2DM rats. These results suggest that the low sensitivity of nAChR channels to nicotine might contribute to impairment of the cardiac vagal activity in T2DM.

Neurochemical characterization of extrinsic nerves in myenteric ganglia of the guinea pig distal colon.

Extrinsic nerves to the gut influence the absorption of water and electrolytes and expulsion of waste contents, largely via regulation of enteric neural circuits; they also contribute to control of blood flow. The distal colon is innervated by extrinsic sympathetic and parasympathetic efferent and spinal afferent neurons, via axons in colonic nerve trunks. In the present study, biotinamide tracing of colonic nerves was combined with immunohistochemical labeling for markers of sympathetic, parasympathetic, and spinal afferent neurons to quantify their relative contribution to the extrinsic innervation. Calcitonin gene-related peptide, vesicular acetylcholine transporter, and tyrosine hydroxylase, which selectively label spinal afferent, parasympathetic, and sympathetic axons, respectively, were detected immunohistochemically in 1 ± 0.5% (n = 7), 15 ± 4.7% (n = 6), and 24 ± 4% (n = 7) of biotinamide-labeled extrinsic axons in myenteric ganglia. Immunoreactivity for vasoactive intestinal polypeptide, nitric oxide synthase, somatostatin, and vesicular glutamate transporters 1 and 2 accounted for a combined maximum of 14% of biotinamide-labeled axons in myenteric ganglia. Thus, a maximum of 53% of biotinamide-labeled extrinsic axons in myenteric ganglia were labeled by antisera to one of these eight markers. Viscerofugal neurons were also labeled by biotinamide. They had distinct morphologies and spatial distributions that correlated closely with their immunoreactivity for nitric oxide synthase and choline acetyltransferase. As reported for the rectum, nearly half of all extrinsic nerve fibers to the distal colon lack the key immunohistochemical markers commonly used for their identification. Their abundance may therefore have been significantly underestimated in previous immunohistochemical studies.

Assessment of DNA synthesis in Islet-1+ cells in the adult murine heart

Rationale: Islet-1 positive (Islet-1+) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1+ cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart. Methods and results: DNA synthesis was analyzed by the incorporation of tritiated thymidine (3H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of 3H-thymidine for 5days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1+ cells. Whereas Islet− non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. Conclusions: Islet-1+ cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes.

Recent advances in research on nitrergic nerve-mediated vasodilatation.

Cerebral vascular resistance and blood flow were widely considered to be regulated solely by tonic innervation of vasoconstrictor adrenergic nerves. However, pieces of evidence suggesting that parasympathetic nitrergic nerve activation elicits vasodilatation in dog and monkey cerebral arteries were found in 1990. Nitric oxide (NO) as a neurotransmitter liberated from parasympathetic postganglionic neurons decreases cerebral vascular tone and resistance and increases cerebral blood flow, which overcome vasoconstrictor responses to norepinephrine liberated from adrenergic nerves. Functional roles of nitrergic vasodilator nerves are found also in peripheral vasculature, including pulmonary, renal, mesenteric, hepatic, ocular, uterine, nasal, skeletal muscle, and cutaneous arteries and veins; however, adrenergic nerve-induced vasoconstriction is evidently greater than nitrergic vasodilatation in these vasculatures. In coronary arteries, neurogenic NO-mediated vasodilatation is not clearly noted; however, vasodilatation is induced by norepinephrine released from adrenergic nerves that activates β1-adrenoceptors. Impaired actions of NO liberated from the endothelium and nitrergic neurons are suggested to participate in cerebral hypoperfusion, leading to brain dysfunction, like that in Alzheimer's disease. Nitrergic neural dysfunction participates in impaired circulation in peripheral organs and tissues and also in systemic blood pressure increase. NO and vasodilator peptides, as sensory neuromediators, are involved in neurogenic vasodilatation in the skin. Functioning of nitrergic vasodilator nerves is evidenced not only in a variety of mammals, including humans and monkeys, but also in non-mammals. The present review article includes recent advances in research on the functional importance of nitrergic nerves concerning the control of cerebral blood flow, as well as other regions, and vascular resistance. Although information is still insufficient, the nitrergic nerve histology and function in vasculatures of non-mammals are also summarized.

Chronic intermittent hypoxia alters neurotransmission from lateral paragigantocellular nucleus to parasympathetic cardiac neurons in the brain stem.

Patients with sleep-related disorders, including obstructive sleep apnea (OSA), have an increased risk of cardiovascular diseases. OSA events are more severe in rapid eye movement (REM) sleep. REM sleep further increases the risk of adverse cardiovascular events by diminishing cardioprotective parasympathetic activity. The mechanisms underlying REM sleep-related reduction in parasympathetic activity likely include activation of inhibitory input to cardiac vagal neurons (CVNs) in the brain stem originating from the lateral paragigantocellular nucleus (LPGi), a nucleus that plays a role in REM sleep control. This study tests the hypothesis that chronic intermittent hypoxia and hypercapnia (CIHH), an animal model of OSA, inhibits CVNs because of exaggeration of the GABAergic pathway from the LPGi to CVNs. GABAergic neurotransmission to CVNs evoked by electrical stimulation of the LPGi was examined with whole cell patch-clamp recordings in an in vitro brain slice preparation in rats exposed to CIHH and control rats. GABAergic synaptic events were enhanced after 4-wk CIHH in both male and female rats, to a greater degree in males. Acute hypoxia and hypercapnia (H/H) reversibly diminished the LPGi-evoked GABAergic neurotransmission to CVNs. However, GABAergic synaptic events were enhanced after acute H/H in CIHH male animals. Orexin-A elicited a reversible inhibition of LPGi-evoked GABAergic currents in control animals but evoked no significant changes in CIHH male rats. In conclusion, exaggerated inhibitory neurotransmission from the LPGi to CVNs in CIHH animals would reduce cardioprotective parasympathetic activity and enhance the risk of adverse cardiovascular events.