Increased arrhythmia susceptibility in type 2 diabetic mice related to dysregulation of ventricular sympathetic innervation.

Patients with type 2 diabetes mellitus (T2DM) have a greater risk of developing life-threatening cardiac arrhythmias. Because the underlying mechanisms and potential influence of diabetic autonomic neuropathy are not well understood, we aimed to assess the relevance of a dysregulation in cardiac autonomic tone. Ventricular arrhythmia susceptibility was increased in Langendorff-perfused hearts isolated from mice with T2DM (db/db). Membrane properties and synaptic transmission were similar at cardiac postganglionic parasympathetic neurons from diabetic and control mice; however, a greater asynchronous neurotransmitter release was present at sympathetic postganglionic neurons from the stellate ganglia of db/db mice. Western blot analysis showed a reduction of tyrosine hydroxylase (TH) from the ventricles of db/db mice, which was confirmed with confocal imaging as a heterogeneous loss of TH-immunoreactivity from the left ventricular wall but not the apex. In vivo stimulation of cardiac parasympathetic (vagus) or cardiac sympathetic (stellate ganglion) nerves induced similar changes in heart rate in control and db/db mice, and the kinetics of pacing-induced Ca2+ transients (recorded from isolated cardiomyocytes) were similar in control and db/db cells. Antagonism of cardiac muscarinic receptors did not affect the frequency or severity of arrhythmias in db/db mice, but sympathetic blockade with propranolol completely inhibited arrhythmogenicity. Collectively, these findings suggest that the increased ventricular arrhythmia susceptibility of type 2 diabetic mouse hearts is due to dysregulation of the sympathetic ventricular control.NEW & NOTEWORTHY Patients with type 2 diabetes mellitus have greater risk of suffering from sudden cardiac death. We found that the increased ventricular arrhythmia susceptibility in type 2 diabetic mouse hearts is due to cardiac sympathetic dysfunction. Sympathetic dysregulation is indicated by an increased asynchronous release at stellate ganglia, a heterogeneous loss of tyrosine hydroxylase from the ventricular wall but not apex, and inhibition of ventricular arrhythmias in db/db mice after ß-sympathetic blockade.

The role of the paravertebral ganglia in human sympathetic neural discharge patterns.

KEY POINTS: The mechanisms affecting recruitment patterns of postganglionic sympathetic nerves remain unclear. The divergent and convergent preganglionic innervation patterns of postganglionic neurons and the presence of differently sized postganglionic nerves suggest that the ganglia may participate in modifying the discharge patterns of single sympathetic postganglionic neurons innervating the skeletal muscle circulation. Whether the ganglia affect the ordered behaviour of varying sized postganglionic sympathetic neurons in humans has not been studied. Trimethaphan infusion produced an ordered pattern of action potential (AP) de-recruitment whereby the firing of larger, low probability APs present at baseline was abolished first, followed by progressive decreased probability of smaller APs. Although integrated sympathetic bursts were no longer detected after several minutes of trimethaphan, firing of the smallest APs was detected. These data suggest the ganglia affect the distribution of firing probabilities exhibited by differently sized sympathetic neurons. The ganglia may contribute to sympathetic neural emission patterns involved in homeostatic regulation. ABSTRACT: Do the ganglia contribute to the ordered behaviour of postganglionic neuronal discharge within the sympathetic nervous system? To further understand the functional organization of the sympathetic nervous system we employed the microneurographic approach to record muscle sympathetic nerve activity (MSNA) and a continuous wavelet transform to study postganglionic action potential (AP) behaviour during nicotinic blockade at the ganglia (trimethaphan camsylate, 1-7 mg min-1 ) in seven females (37 ± 5 years). Trimethaphan elicited a progressive reduction in sympathetic outflow characterized by fewer integrated bursts with decaying amplitude. Underlying trimethaphan-mediated attenuations in integrated MSNA were reductions in AP incidence (186 ± 101 to 29 ± 31 AP (100 beats)-1 ) and AP content per integrated burst (7 ± 2 to 3 ± 1 APs burst-1 ) (both P < 0.01) in the final minute of detectable bursting activity in the trimethaphan condition, compared to baseline. We observed an ordered de-recruitment of larger to smaller AP clusters active at baseline (14 ± 3 to 8 ± 2 active AP clusters, P < 0.01). Following cessation of integrated bursts in the trimethaphan condition, the smallest 6 ± 2 sympathetic AP clusters persisted to fire in an asynchronous pattern (49 ± 41 AP (100 beats)-1 ) in all participants. Valsalva's manoeuvre did not increase the incidence of these persistent APs (60 ± 42 AP (100 beats)-1 , P = 0.52), or recruit any larger APs in six of seven participants (6 ± 1 total AP clusters, P = 0.30). These data suggest that the ganglia participate in the ordered recruitment of differently sized postganglionic sympathetic nerves.

Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons.

The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation.

Localization of the autonomic, somatic and sensory neurons innervating the cranial tibial muscle of the pig.

The location of sympathetic, somatic and sensory neurons projecting to the cranial tibial muscle of the pig hindlimb was studied with the neuronal non-transynaptic tracer Fast Blue. Additionally, the number and the size of these neurons were determinated. The Fast blue, randomly applied to the cranial tibial muscle belly of 3 pigs, labelled sympathetic neurons in the ipsilateral L5-S3 and contralateral S1 sympathetic trunk ganglia and in the prevertebral caudal mesenteric ganglia of both sides. The somatic motoneurons were identified in the ipsilateral ventral horn of the S1 segment of spinal cord, while the sensory neurons were located in the ipsilateral L7-S1 spinal ganglia. The diameter of the multipolar sympathetic neurons oscillated between 26 and 46 microm in the sympathetic trunk ganglia and between 18 and 42 microm in the caudal mesenteric ganglia. The size of the multipolar spinal motoneurons oscillated between 33 and 102 microm. The size of the pseudounipolar sensory neurons oscillated between 23 and 67 microm. In all ganglia, the labelled neurons were localized at random and did not show a somatotopic distribution. Our results document a conspicuous autonomic innervation projecting to the "classic" skeletal cranial tibial muscle. Probably this innervation is destined to the muscle vessels.

Age-associated alterations in sympathetic noradrenergic innervation of primary and secondary lymphoid organs in female Fischer 344 rats.

Normal aging processes, as well as, psychological stress affect the immune system; each can act alone, or interact with each other, to cause dysregulation of immune function substantially altering physical and mental health. The sympathetic nervous system (SNS), a major mediator of stress effects on immune function, is significantly affected by normal aging process, and stress can affect aging of the SNS. Previously, we have shown age-associated changes in sympathetic noradrenergic (NA) innervation of lymphoid organs in male rodents that affect immune regulation. The purpose of this study was to investigate sympathetic innervation of lymphoid organs and associated alterations in immune responses in young and aging female Fischer 344 (F344) rats. Histofluorescence and immunocytochemistry for NA innervation, and neurochemistry for norepinephrine (NE) levels were performed in the thymus, spleen, and mesenteric lymph nodes (MLN) isolated from 3-month-old young (normal estrous cycle), 8- to 9-month-old (onset of irregular estrous cycling), and 24-25 month, and 30-31 month female F344 rats (acyclic) at diestrus based on vaginal smears. Age-related alterations in natural killer (NK) cell activity, interleukin-2 (IL-2) and interferon-γ (IFN-γ) production, T and B lymphocyte proliferation were examined in splenocytes. Sympathetic NA innervation and NE levels increased with aging in the thymus, declined in spleen and MLN, and was accompanied by significant reductions in NK cell activity, IL-2 and IFN-γ production, and T and B cell proliferation in old female rats. In 8-9 mo rats, NE levels in the hilar region of the spleen and IFN-γ production were unaltered, while NE levels in the end region of the spleen and IL-2 production were reduced. Collectively, these results suggest that aging is characterized by significant alterations in sympathetic NA innervation in the thymus, spleen, and MLN associated with immunosuppression, and that there is a marked shift in NA activity and immune reactivity occurring during middle-aged female rats.

ß-adrenoceptor blockers increase cardiac sympathetic innervation by inhibiting autoreceptor suppression of axon growth.

ß-Adrenoceptor antagonists are used widely to reduce cardiovascular sympathetic tone, but withdrawal is accompanied by sympathetic hyperactivity. Receptor supersensitivity accounts for some but not all aspects of this withdrawal syndrome. Therefore, we investigated effects of ß-blockers on sympathetic innervation. Rats received infusions of adrenergic receptor blockers or saline for 1 week. The nonselective ß-blocker propranolol and the ß(1)-antagonist metoprolol both increased myocardial sympathetic axon density. At 2 d after propranolol discontinuation, ß-receptor sensitivity and responsiveness to isoproterenol were similar to controls. However, tyramine-induced mobilization of norepinephrine stores produced elevated ventricular contractility consistent with enhanced sympathetic neuroeffector properties. In addition, rats undergoing discontinuation showed exaggerated increases in mean arterial pressure in response to air puff or noise startle. In sympathetic neuronal cell cultures, both propranolol and metoprolol increased axon outgrowth but the ß(2)-blocker ICI 118551 did not. Norepinephrine synthesis suppression by α-methyl-p-tyrosine also increased sprouting and concurrent dobutamine administration reduced it, confirming that locally synthesized norepinephrine inhibits outgrowth via ß(1)-adrenoceptors. Immunohistochemistry revealed ß(1)-adrenoceptor protein on sympathetic axon terminations. In rats with coronary artery ligation, propranolol reversed heart failure-induced ventricular myocardial sympathetic axon depletion, but did not affect infarct-associated sympathetic hyperinnervation. We conclude that sympathetic neurons possess ß(1)-autoreceptors that negatively regulate axon outgrowth. Chronic ß-adrenoceptor blockade disrupts this feedback system, leading to ventricular sympathetic axon proliferation and increased neuroeffector gain, which are likely to contribute to ß-blocker withdrawal syndrome.

Localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers.

Superoxide anion (O(2)(-*)) production was previously reported to be increased in celiac ganglia (CG) during DOCA-salt hypertension, possibly via activation of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. This suggested a role for neuronal NADPH oxidase in autonomic neurovascular control. However, the expression and localization of NADPH oxidase in the peripheral neurons are not fully known. The purpose of this study was to examine the subcellular localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers. In rat CG, p22(phox) and neuropeptide Y (NPY) were colocalized in all neurons. P22(phox) was also localized to dorsal root ganglia (DRG) neurons that contain calcitonin gene related peptide (CGRP). In mesenteric arteries, p22(phox) and p47(phox) were colocalized with NPY or CGRP in perivascular nerve terminals. A similar pattern of nerve terminal staining of p22(phox) and p47(phox) was also found in cultured CG neurons and nerve growth factor (NGF)-differentiated PC12 cells. These data demonstrate a previously uncharacterized localization of NADPH oxidase in perivascular nerve fibers. The presence of a O(2)(-*)-generating enzyme in close vicinity to the sites of neurotransmitter handling in the nerve fibers suggests the possibility of novel redox-mediated mechanisms in peripheral neurovascular control.

Extracellular signals regulating sympathetic neuron survival and target innervation during development.

The comparative ease with which paravertebral sympathetic neurons are studied in vitro and in vivo at stages throughout their development has facilitated major advances in our understanding of several key aspects of neuronal development. Detailed anatomical descriptions of the in vivo development of these neurons, studies of the effects of various extracellular signalling molecules on these neurons in vitro and analysis of the sympathetic phenotype of relevant transgenic mice have provided an in-depth understanding of how different extracellular signals orchestrate sequential steps in the establishment and refinement of sympathetic innervation. In this review, I will document the roles of neurotrophic factors, cytokines and other extracellular signals in regulating sympathetic neuron survival and target innervation at sequential stages of development.

Transgenic mice expressing nerve growth factor in smooth muscle cells.

Ectopic expression of nerve growth factor (NGF) in transgenic mice leads to site-specific sympathetic sprouting. Smooth muscle cells in the intestines, urinary bladder, and arteries have been shown to express NGF. To address whether enhanced NGF production among these different organ systems stimulates comparable patterns of sympathetic collateral growth, we generated transgenic mice that express NGF under the control of the smooth muscle alpha-actin promoter. In response to elevated levels of NGF protein in the colon, bladder, and arteries/arterioles, sympathetic axons displayed robust sprouting only in the colon and bladder. These data reveal that, unlike most other peripheral tissues, sympathetic efferents in adult mammalian arteries/arterioles do not undergo collateral growth in response to increased levels of smooth muscle-derived NGF.

The developing left superior cervical ganglion of Pacas (Agouti paca).

In this study the main question investigated was the number and size of both binucleate and mononucleate superior cervical ganglion (SCG) neurons and, whether post-natal development would affect these parameters. Twenty left SCGs from 20 male pacas were used. Four different ages were investigated, that is newborn (4 days), young (45 days), adult (2 years), and aged animals (7 years). By using design-based stereological methods, that is the Cavalieri principle and a physical disector combined with serial sectioning, the total volume of ganglion and total number of mononucleate and binucleate neurons were estimated. Furthermore, the mean perikaryal (somal) volume of mononucleate and binucleate neurons was estimated using the vertical nucleator. The main findings of this study were a 154% increase in the SCG volume, a 95% increase in the total number of mononucleate SCG neurons and a 50% increase in the total volume of SCG neurons. In conclusion, apart from neuron number, different adaptive mechanisms may coexist in the autonomic nervous system to guarantee a functional homeostasis during ageing, which is not always associated with neuron losses.

Asymmetric post-natal development of superior cervical ganglion of paca (Agouti paca).

Functional asymmetry has been reported in sympathetic ganglia. Although there are few studies reporting on body side-related morphoquantitative changes in sympathetic ganglion neurons, none of them have used design-based stereological methods to address this issue during post-natal development. We therefore aimed at detecting possible asymmetry-related effects on the quantitative structure of the superior cervical ganglion (SCG) from pacas during ageing, using very precise design-based stereological methods. Forty (twenty left and twenty right) SCG from twenty male pacas were studied at four different ages, i.e. newborn, young, adult and aged animals. By using design-based stereological methods the total volume of ganglion and the total number of mononucleate and binucleate neurons were estimated. Furthermore, the mean perikaryal volume of mononucleate and binucleate neurons was estimated, using the vertical nucleator. The main findings of this study were: (1) the right SCG from aged pacas has more mononucleate and binucleate neurons than the left SCG in all other combinations of body side and animal age, showing the effect of the interaction between asymmetry (right side) and animal age, and (2) right SCG neurons (mono and binucleate) are bigger than the left SCG neurons (mono and binucleate), irrespective of the animal age. This shows, therefore, the exclusive effect of asymmetry (right side). At the time of writing there is still no conclusive explanation for some SCG quantitative changes exclusively assigned to asymmetry (right side) and those assigned to the interaction between asymmetry (right side) and senescence in pacas. We therefore suggest that forthcoming studies should focus on the functional consequences of SCG structural asymmetry during post-natal development. Another interesting investigation would be to examine the interaction between ganglia and their innervation targets using anterograde and retrograde neurotracers. Would differences in the size of target organs explain ganglia structural asymmetry?

Experimental study to localize the neurons projecting to the lamb retractor penis muscle.

Aim of the present study was to locate the neurons projecting to the lamb retractor penis muscle, a smooth muscle associated to the penis. The retrograde neuronal tracer Fast Blue was injected into the bulbopenile portion of the left retractor penis muscle. Labelled cells were found bilaterally in the S2-S4 spinal ganglia, from the last two lumbar (L5-L6 or L6-L7) to S4 sympathetic trunk ganglia and in the hypogastric and pelvic plexuses. Fast blue-positive (FB+) neurons were also found in the intermediate gray substance in the S1-S4 segments of the spinal cord. Our research enables us to describe the organization of the innervation of the lamb retractor penis muscle, highlighting the site of the primary afferent, postganglionic efferent and presumably preganglionic parasympathetic neurons projecting to the muscle.

Intrinsic neuronal control of the pyloric sphincter of the lamb.

To better understand the local neuronal network of the gastro-duodenal junction in ruminants, we identified the components of the enteric nervous system (ENS) innervating the pyloric sphincter (PS) of the lamb abomasum. The neurons were labelled after injecting the tracer Fast Blue (FB) into the wall of the PS, and the phenotype of the FB-labelled neurons was immunohistochemically investigated using antibodies against nitric oxide synthase (NOS), choline acetyltransferase (ChAT), tachykinin (substance P) and tyrosine hydroxylase (TH). The FB-labelled abomasal myenteric plexus (MP) neurons, observed up to 14cm from the PS, were NOS-immunoreactive (IR) (82+/-12%), ChAT-IR (51+/-29%), SP-IR (61+/-33%), and also TH-IR (2%). The descending nitrergic neurons were also SP-IR (64%) and ChAT-IR (21%); the cholinergic descending neurons were SP-IR (3%). The FB-labelled duodenal neurons were located only in the MP, up to 8cm from the sphincter and were ChAT-IR (79+/-16%), SP-IR (32+/-18%), NOS-IR (from 0 to 2%), and also TH-IR (4+/-3%). The cholinergic ascending neurons were also SP-IR (60%) whereas no ChAT-IR cells were NOS-IR. The findings of this research indicate that the sheep PS is innervated by long-projecting neurons of the abomasal and duodenal ENS.

Sympathetic nerve fibers sprout into rat odontoblast layer, but not into dentinal tubules, in response to cavity preparation.

This study was designed to determine if sympathetic nerve fibers exist in dentinal tubules in rat normal dental pulp, and if they sprout into the dentinal tubules in response to artificial cavity preparation in dentin. Sympathetic nerve fibers in rat molar dental pulp were labeled using an anterograde axonal transport technique involving injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the superior cervical ganglion (SCG). They were then observed using light and electron microscopes. In normal dental pulp (control), scattered WGA-HRP reaction products were observed in unmyelinated nerve endings in the odontoblast layer and subodontoblastic region. In injured pulp 3 weeks after cavity preparation, reaction products were about 1.8-times more plentiful in the above areas (versus control pulp). However, no labeled nerve fibers were observed in the dentinal tubules in either control or injured dental pulp. These results indicate that although sympathetic nerve fibers do indeed sprout in rat dental pulp in response to cavity preparation, they do not penetrate into the dentinal tubules in which postganglionic nerve endings derived from the SCG were not originally present.

Analysis of gene expression during neurite outgrowth and regeneration.

BACKGROUND: The ability of a neuron to regenerate functional connections after injury is influenced by both its intrinsic state and also by extrinsic cues in its surroundings. Investigations of the transcriptional changes undergone by neurons during in vivo models of injury and regeneration have revealed many transcripts associated with these processes. Because of the complex milieu of interactions in vivo, these results include not only expression changes directly related to regenerative outgrowth and but also unrelated responses to surrounding cells and signals. In vitro models of neurite outgrowth provide a means to study the intrinsic transcriptional patterns of neurite outgrowth in the absence of extensive extrinsic cues from nearby cells and tissues. RESULTS: We have undertaken a genome-wide study of transcriptional activity in embryonic superior cervical ganglia (SCG) and dorsal root ganglia (DRG) during a time course of neurite outgrowth in vitro. Gene expression observed in these models likely includes both developmental gene expression patterns and regenerative responses to axotomy, which occurs as the result of tissue dissection. Comparison across both models revealed many genes with similar gene expression patterns during neurite outgrowth. These patterns were minimally affected by exposure to the potent inhibitory cue Semaphorin3A, indicating that this extrinsic cue does not exert major effects at the level of nuclear transcription. We also compared our data to several published studies of DRG and SCG gene expression in animal models of regeneration, and found the expression of a large number of genes in common between neurite outgrowth in vitro and regeneration in vivo. CONCLUSION: Many gene expression changes undergone by SCG and DRG during in vitro outgrowth are shared between these two tissue types and in common with in vivo regeneration models. This suggests that the genes identified in this in vitro study may represent new candidates worthy of further study for potential roles in the therapeutic regrowth of neuronal connections.

Neural mechanism of acupuncture-modulated gastric motility.

AIM: To investigate the acupuncture-modulated gastric motility and its underlying neural mechanism. METHODS: Intragastric pressure and/or waves of gastric contraction in rats were recorded by intrapyloric balloon and changes of gastric motility induced by acupuncture stimulation were compared with the background activity before any stimulation. Gastro-vagal or splanchnic-sympathetic nerves were recorded or cut respectively for investigating the involvement of autonomic nerve pathways. Spinalization experiment was also performed. RESULTS: Acupuncture-stimulation by exciting A delta and/or C afferent fibers, could only modulate gastric motility. Acupuncture-stimulation on fore- and hind-limbs evoked a moderate gastric motility followed by increased vagus discharges with unchanged sympathetic activity, while the same stimulus to the acupoints in abdomen resulted in reversed effects on gastric motility and autonomic nervous activities. The inhibitory gastric response was completely abolished by splanchnic denervation, but the facilitative gastric response to stimulation of acupoints in limbs was not influenced, which was opposite to the effect when vagotomy was performed. The similar depressive effects were produced by the stimulation at the acupoints homo-segmental to the gastric innervation in the animals with or without spinalization. However, the facilitation induced by the stimulation at the acupoints hetero-segmental to the gastric innervation was not observed in the spinalized animals. CONCLUSION: Facilitative effects of stimulating hetero-segmental acupoints are involved in the intact preparation of vagal nerves and spinal cord, while the inhibitory response induced by stimulating homo-segmental acupoints is involved in the intact preparation of sympathetic nerves. Only the acupuncture-stimulation with intensity over the threshold of A delta and/or C afferent fibers can markedly modulate gastrointestinal motility.

Global expression of NGF promotes sympathetic axonal growth in CNS white matter but does not alter its parallel orientation.

Axonal regeneration is normally limited after injuries to CNS white matter. Infusion of neurotrophins has been successful in promoting regenerative growth through injured white matter but this growth generally fails to extend beyond the infusion site. These observations are consistent with a chemotropic effect of these factors on axonal growth and support the prevailing view that neurotrophin-induced axonal regeneration requires the use of gradients, i.e., gradually increasing neurotrophin levels along the target fiber tract. To examine the potential of global overexpression of neurotrophins to promote, and/or modify the orientation of, regenerative axonal growth within white matter, we grafted nerve growth factor (NGF) responsive neurons into the corpus callosum of transgenic mice overexpressing NGF throughout the CNS under control of the promoter for glial fibrillary acidic protein. One week later, glial fibrillary acidic protein and chondroitin sulfate proteoglycan immunoreactivity increased within injured white matter around the grafts. NGF levels were significantly higher in the brains of transgenic compared with non-transgenic mice and further elevated within injury sites compared with the homotypic region of the non-injured side. Although there was minimal outgrowth from neurons grafted into non-transgenic mice, extensive parallel axonal regeneration had occurred within the corpus callosum up to 1.5 mm beyond the astrogliotic scar (the site of maximum NGF expression) in transgenic mice. These results demonstrate that global overexpression of neurotrophins does not override the constraints limiting regenerative growth to parallel orientations and suggest that such factors need not be presented as positive gradients to promote axonal regeneration within white matter.

Different subcellular distributions of the vesicular monoamine transporter, VMAT2, in subclasses of sympathetic neurons.

A subpopulation of neurons in the rat superior cervical ganglion (SCG) was found to lack immunostaining for VMAT2, an isoform of the vesicular monoamine transporter that loads catecholamines into vesicles for release at the synapse. Double labeling with neuropeptide Y (NPY), a marker for vasomotor neurons, revealed selective cellular colocalization of NPY together with intense perinuclear staining for VMAT2. This implied that VMAT2-negative neurons were likely to have secretomotor and pilomotor phenotypes. We tested this by identifying peripheral noradrenergic axons by their expression of immunoreactivity for tyrosine hydroxylase (TH) and determining whether they also expressed NPY and VMAT2. This analysis revealed the presence of VMAT2-positive, non-vasomotor sympathetic axons in the submandibular gland and at the base of piloerector hairs. Together the results confirm earlier indications that virtually all sympathetic neurons in the rat SCG express VMAT2 and they show for the first time that functional subclasses of cells can be distinguished by different somatic levels of immunoreactivity for VMAT2.

Peripheral neurons innervating the extrinsic smooth penile musculature of the pig: experimental study by retrograde transport and immunohistochemistry.

Peripheral autonomic and sensitive neurons projecting to the extrinsic smooth penile musculature of the pig were studied by means of retrograde tracing and single-labelling immunofluorescence methods. The fluorescent retrograde tracer Fast Blue was injected into the left retractor penis muscle, that was taken as an experimental model of the male genital smooth musculature, of 4 castrated pigs. After a 7 day survival time, the ipsilateral paravertebral ganglion S1, the caudal mesenteric ganglion and the dorsal root ganglion S2 were collected. In these ganglia, the presence and the distribution of immunoreactivities to cathecolamine- (Tyrosine Hydroxylase), acetylcholine- (Vesicular Acetylcholine Transporter), or nitric oxide-synthesizing (neuronal Nitric Oxide Synthase) enzymes and to some biologically active peptides (Calcitonine Gene-Related Peptide, Leu-Enkephaline, Neuropeptide Y, Substance P and Vasoactive Intestinal Peptide) were studied. In paravertebral ganglion S1, Tyrosine Hydroxylase and Neuropeptide Y were the most frequently present substances. Also Leu-Enkephaline and neuronal Nitric Oxide were present quite frequently, while there was scarce immunoreactivity for the other antisera (in decreasing order Substance P, Vasoactive Intestinal Peptide, Vesicular Acetylcholine Transporter, Calcitonine Gene-Related Peptide). In caudal mesenteric ganglion, in addition to Tyrosine Hydroxylase- and Neuropeptide Y-immunoreactivity, Substance P-, Vesicular Acetylcholine Transporter-, Vasoactive Intestinal Peptide-, Leu-Enkephaline- immunoreactivity were also frequently present, followed by neuronal Nitric Oxide- and Calcitonine Gene-Related Peptide- immunoreactivity. In dorsal root ganglion S2, Calcitonine Gene-Related Peptide and neuronal Nitric Oxide resulted to be the most frequently present neurotransmitters, followed by Vasoactive Intestinal Peptide, Vesicular Acetylcholine Transporter, Leu-Enkephaline, Substance P, Tyrosine Hydroxylase and Neuropeptide Y.

Sympathetic sprouting drives hippocampal cholinergic reinnervation that prevents loss of a muscarinic receptor-dependent long-term depression at CA3-CA1 synapses.

Degeneration of septohippocampal cholinergic neurons results in memory deficits attributable to loss of cholinergic modulation of hippocampal synaptic circuits. A remarkable consequence of cholinergic degeneration is the sprouting of noradrenergic sympathetic fibers from the superior cervical ganglia into hippocampus. The functional impact of sympathetic ingrowth on synaptic physiology has never been investigated. Here, we report that, at CA3-CA1 synapses, a Hebbian form of long-term depression (LTD) induced by muscarinic M1 receptor activation (mLTD) is lost after medial septal lesion. Unexpectedly, expression of mLTD is rescued by sympathetic sprouting. These effects are specific because LTP and other forms of LTD are unaffected. The rescue of mLTD expression is coupled temporally with the reappearance of cholinergic fibers in hippocampus, as assessed by the immunostaining of fibers for VAChT (vesicular acetylcholine transporter). Both the cholinergic reinnervation and mLTD rescue are prevented by bilateral superior cervical ganglionectomy, which also prevents the noradrenergic sympathetic sprouting. The new cholinergic fibers likely originate from the superior cervical ganglia because unilateral ganglionectomy, performed when cholinergic reinnervation is well established, removes the reinnervation on the ipsilateral side. Thus, the temporal coupling of the cholinergic reinnervation with mLTD rescue, together with the absence of reinnervation and mLTD expression after ganglionectomy, demonstrate that the autonomic-driven cholinergic reinnervation is essential for maintaining mLTD after central cholinergic cell death. We have discovered a novel phenomenon whereby the autonomic and central nervous systems experience structural rearrangement to replace lost cholinergic innervation in hippocampus, with the consequence of preserving a form of LTD that would otherwise be lost as a result of cholinergic degeneration.