Downstream projection of Barrington's nucleus to the spinal cord in mice.

Barrington's nucleus (Bar), which controls micturition behavior through downstream projections to the spinal cord, contains two types of projection neurons, BarCRH and BarESR1, that have different functions and target different spinal circuitry. Both types of neurons project to the L6-S1 spinal intermediolateral (IML) nucleus, whereas BarESR1 neurons also project to the dorsal commissural nucleus (DCN). To obtain more information about the spinal circuits targeted by Bar, we used patch-clamp recording in spinal slices from adult mice in combination with optogenetic stimulation of Bar terminals. Recording of opto-evoked excitatory postsynaptic currents (oEPSCs) in 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (DiI)-labeled lumbosacral preganglionic neurons (LS-PGNs) revealed that both Bar neuronal populations make strong glutamatergic monosynaptic connections with LS-PGNs, whereas BarESR1 neurons also elicited smaller-amplitude glutamatergic polysynaptic oEPSCs or polysynaptic opto-evoked inhibitory postsynaptic currents (oIPSCs) in some LS-PGNs. Optical stimulation of BarCRH and BarESR1 terminals also elicited monosynaptic oEPSCs and polysynaptic oIPSCs in sacral DCN neurons, some of which must include interneurons projecting to either the IML or ventral horn. Application of capsaicin increased opto-evoked firing during repetitive stimulation of Bar terminals through the modulation of spontaneous postsynaptic currents in LS-PGNs. In conclusion, our experiments have provided insights into the synaptic mechanisms underlying the integration of inputs from Bar to autonomic circuitry in the lumbosacral spinal cord that may control micturition.NEW & NOTEWORTHY Photostimulation of BarCRH or BarESR1 axons in the adult mouse spinal cord elicits excitatory or inhibitory postsynaptic responses in multiple cell types related to the autonomic nervous system including preganglionic neurons (PGNs) in the lumbosacral intermediolateral nucleus and interneurons in the lumbosacral dorsal commissure nucleus. Integration of excitatory inputs from Bar and from visceral primary afferents in PGNs may be important in the regulation of micturition behavior.

Spinal cord neural network interactions: implications for sympathetic control of the porcine heart.

Inherent and acquired factors determine the integrated autonomic response to cardiovascular stressors. Excessive sympathoexcitation to ischemic stress is a major contributor to the potential for sudden cardiac death. To define fundamental aspects of cardiac-related autonomic neural network interactions within the thoracic cord, specifically as related to modulating sympathetic preganglionic (SPN) neural activity. Adult, anesthetized Yorkshire pigs (n = 10) were implanted with penetrating high-density microarrays (64 electrodes) at the T2 level of the thoracic spinal cord to record extracellular potentials concurrently from left-sided dorsal horn (DH) and SPN neurons. Electrical stimulation of the T2 paravertebral chain allowed for antidromic identification of SPNs located in the intermediolateral cell column (57 of total 1,760 recorded neurons). Cardiac stressors included epicardial touch, occlusion of great vessels to transiently alter preload/afterload, and transient occlusion of the left anterior descending coronary artery (LAD). Spatial/temporal assessment of network interactions was characterized by cross-correlation analysis. While some DH neurons responded solely to changes in preload/afterload (8.5 ± 1.9%) or ischemic stress (10.5 ± 3.9%), the majority of cardiovascular-related DH neurons were multimodal (30.2 ± 4.7%) with ischemia sensitivity being one of the modalities (26.1 ± 4.7%). The sympathoexcitation associated with transient LAD occlusion was associated with increased correlations from baseline within DH neurons (2.43 ± 0.61 to 7.30 ± 1.84%, P = 0.04) and between SPN to DH neurons (1.32 ± 0.78 to 7.24 ± 1.84%, P = 0.02). DH to SPN network correlations were reduced during great vessel occlusion. In conclusion, increased intrasegmental network coherence within the thoracic spinal cord contributes to myocardial ischemia-induced sympathoexcitation.NEW & NOTEWORTHY In an in vivo pig model, we demonstrate using novel high-resolution neural electrode arrays that increased intrasegmental network coherence within the thoracic spinal cord contributes to myocardial ischemia-induced sympathoexcitation.

On the presence and functional significance of sympathetic premotor neurons with collateralized spinal axons in the rat.

KEY POINTS: Spinally-projecting neurons of the rostral ventrolateral medulla (RVLM) determine sympathetic outflow to different territories of the body. Previous studies suggest the existence of RVLM neurons with distinct functional classes, such as neurons that target sympathetic nerves bound for functionally-similar tissue types (e.g. muscle vasculature). The existence of RVLM neurons with more general actions had not been critically tested. Using viral tracing, we show that a significant minority of RVLM neurons send axon collaterals to disparate spinal segments (T2 and T10 ). Furthermore, optogenetic activation of sympathetic premotor neurons projecting to lumbar spinal segments also produced activation of sympathetic nerves from rostral spinal segments that innervate functionally diverse tissues (heart and forelimb muscle). These findings suggest the existence of individual RVLM neurons for which the axons branch to drive sympathetic preganglionic neurons of more than one functional class and may be able to produce global changes in sympathetic activity. ABSTRACT: We investigate the extent of spinal axon collateralization of rat rostral ventrolateral medulla (RVLM) sympathetic premotor neurons and its functional consequences. In anatomical tracing experiments, two recombinant herpes viral vectors with retrograde tropism and expressing different fluorophores were injected into the intermediolateral column at upper thoracic and lower thoracic levels. Histological analysis revealed that ∼21% of RVLM bulbospinal neurons were retrogradely labelled by both vectors, indicating substantial axonal collateralization to disparate spinal segments. In functional experiments, another virus with retrograde tropism, a canine adenovirus expressing Cre recombinase, was injected into the left intermediolateral horn around the thoracolumbar junction, whereas a Cre-dependent viral vector encoding Channelrhodopsin2 under LoxP control was injected into the ipsilateral RVLM. In subsequent terminal experiments, blue laser light (473 nm × 20 ms pulses at 10 mW) was used to activate RVLM neurons that had been transduced by both vectors. Stimulus-locked activation, at appropriate latencies, was recorded in the following pairs of sympathetic nerves: forelimb and hindlimb muscle sympathetic fibres, as well as cardiac and either hindlimb muscle or lumbar sympathetic nerves. The latter result demonstrates that axon collaterals of lumbar-projecting RVLM neurons project to, and excite, both functionally similar (forelimb and hindlimb muscle) and functionally dissimilar (lumbar and cardiac) preganglionic neurons. Taken together, these findings show that the axons of a significant proportion of RVLM neurons collateralise widely within the spinal cord, and that they may excite preganglionic neurons of more than one functional class.

Adrenergic agonist induces rhythmic firing in quiescent cardiac preganglionic neurons in nucleus ambiguous via activation of intrinsic membrane excitability.

Cholinergic vagal nerves projecting from neurons in the brain stem nucleus ambiguus (NAm) play a predominant role in cardiac parasympathetic pacemaking control. Central adrenergic signaling modulates the tone of this vagal output; however, the exact excitability mechanisms are not fully understood. We investigated responses of NAm neurons to adrenergic agonists using in vitro mouse brain stem slices. Preganglionic NAm neurons were identified by ChAT-tdTomato fluorescence in young adult transgenic mice, and their cardiac projection was confirmed by retrograde dye tracing. Juxtacellular recordings detected sparse or absent spontaneous action potentials (AP) in NAm neurons. However, bath application of epinephrine or norepinephrine strongly and reversibly activated most NAm neurons regardless of their basal firing rate. Epinephrine was more potent than norepinephrine, and this activation largely depends on α1-adrenoceptors. Interestingly, adrenergic activation of NAm neurons does not require an ionotropic synaptic mechanism, because postsynaptic excitatory or inhibitory receptor blockade did not occlude the excitatory effect, and bath-applied adrenergic agonists did not alter excitatory or inhibitory synaptic transmission. Instead, adrenergic agonists significantly elevated intrinsic membrane excitability to facilitate generation of recurrent action potentials. T-type calcium current and hyperpolarization-activated current are involved in this excitation pattern, although not required for spontaneous AP induction by epinephrine. In contrast, pharmacological blockade of persistent sodium current significantly inhibited the adrenergic effects. Our results demonstrate that central adrenergic signaling enhances the intrinsic excitability of NAm neurons and that persistent sodium current is required for this effect. This central balancing mechanism may counteract excessive peripheral cardiac excitation during increased sympathetic tone. NEW & NOTEWORTHY Cardiac preganglionic cholinergic neurons in the nucleus ambiguus (NAm) are responsible for slowing cardiac pacemaking. This study identified that adrenergic agonists can induce rhythmic action potentials in otherwise quiescent cholinergic NAm preganglionic neurons in brain stem slice preparation. The modulatory influence of adrenaline on central parasympathetic outflow may contribute to both physiological and deleterious cardiovascular regulation.

Exercise training preserves vagal preganglionic neurones and restores parasympathetic tonus in heart failure.

KEY POINTS: Heart Failure (HF) is accompanied by reduced ventricular function, activation of compensatory neurohormonal mechanisms and marked autonomic dysfunction characterized by exaggerated sympathoexcitation and reduced parasympathetic activity. With 6 weeks of exercise training, HF-related loss of choline acetyltransferase (ChAT)-positive vagal preganglionic neurones is avoided, restoring the parasympathetic tonus to the heart, and the immunoreactivity of dopamine ß-hydroxylase-positive premotor neurones that drive sympathetic outflow to the heart is reduced. Training-induced correction of autonomic dysfunction occurs even with the persistence of abnormal ventricular function. Strong positive correlation between improved parasympathetic tonus to the heart and increased ChAT immunoreactivity in vagal preganglionic neurones after training indicates this is a crucial mechanism to restore autonomic function in heart failure. ABSTRACT: Exercise training is an efficient tool to attenuate sympathoexcitation, a hallmark of heart failure (HF). Although sympathetic modulation in HF is widely studied, information regarding parasympathetic control is lacking. We examined the combined effects of sympathetic and vagal tonus to the heart in sedentary (Sed) and exercise trained (ET) HF rats and the contribution of respective premotor and preganglionic neurones. Wistar rats submitted to coronary artery ligation or sham surgery were assigned to training or sedentary protocols for 6 weeks. After haemodynamic, autonomic tonus (atropine and atenolol i.v.) and ventricular function determinations, brains were collected for immunoreactivity assays (choline acetyltransferase, ChATir; dopamine ß-hydroxylase, DBHir) and neuronal counting in the dorsal motor nucleus of vagus (DMV), nucleus ambiguus (NA) and rostroventrolateral medulla (RVLM). HF-Sed vs. SHAM-Sed exhibited decreased exercise capacity, reduced ejection fraction, increased left ventricle end diastolic pressure, smaller positive and negative dP/dt, decreased intrinsic heart rate (IHR), lower parasympathetic and higher sympathetic tonus, reduced preganglionic vagal neurones and ChATir in the DMV/NA, and increased RVLM DBHir. Training increased treadmill performance, normalized autonomic tonus and IHR, restored the number of DMV and NA neurones and corrected ChATir without affecting ventricular function. There were strong positive correlations between parasympathetic tonus and ChATir in NA and DMV. RVLM DBHir was also normalized by training, but there was no change in neurone number and no correlation with sympathetic tonus. Training-induced preservation of preganglionic vagal neurones is crucial to normalize parasympathetic activity and restore autonomic balance to the heart even in the persistence of cardiac dysfunction.

Partial restoration of cardiovascular function by embryonic neural stem cell grafts after complete spinal cord transection.

High-level spinal cord injury can lead to cardiovascular dysfunction, including disordered hemodynamics at rest and autonomic dysreflexia during noxious stimulation. To restore supraspinal control of sympathetic preganglionic neurons (SPNs), we grafted embryonic brainstem-derived neural stem cells (BS-NSCs) or spinal cord-derived neural stem cells (SC-NSCs) expressing green fluorescent protein into the T4 complete transection site of adult rats. Animals with injury alone served as controls. Implanting of BS-NSCs but not SC-NSCs resulted in recovery of basal cardiovascular parameters, whereas both cell grafts alleviated autonomic dysreflexia. Subsequent spinal cord retransection above the graft abolished the recovery of basal hemodynamics and reflexic response. BS-NSC graft-derived catecholaminergic and serotonergic neurons showed remarkable long-distance axon growth and topographical innervation of caudal SPNs. Anterograde tracing indicated growth of medullar axons into stem cell grafts and formation of synapses. Thus, grafted embryonic brainstem-derived neurons can act as functional relays to restore supraspinal regulation of denervated SPNs, thereby contributing to cardiovascular functional improvement.

Hydrogen sulfide selectively potentiates central preganglionic fast nicotinic synaptic input in mouse superior mesenteric ganglion.

Hydrogen sulfide (H2S) plays important roles in the enteric system in the wall of the gastrointestinal tract. There have been no studies on whether H2S is endogenously generated in peripheral sympathetic ganglia and, if so, its effect on synaptic transmission. In this study, we examined the effect of H2S on cholinergic excitatory fast synaptic transmission in the mouse superior mesenteric ganglion (SMG). Our study revealed that NaHS and endogenously generated H2S selectively potentiated cholinergic fast EPSPs (F-EPSPs) evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. The H2S-producing enzyme cystathionine-γ-lyase (CSE) was expressed in both neurons and glial cells. The CSE blocker PAG (dl-propargylglycine) significantly reduced the amplitude of F-EPSPs evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. Inhibiting the breakdown of endogenously generated H2S with stigmatellin potentiated the amplitude of F-EPSPs evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. Splanchnic F-EPSPs but not colonic F-EPSPs were reduced in CSE knock-out (KO) mice. Functional studies showed that NaHS enhanced the inhibitory effect of splanchnic nerve stimulation on colonic motility. Colonic motility in CSE-KO mice was significantly higher than colonic motility in wild-type mice. We conclude that endogenously generated H2S acted selectively on presynaptic terminals of splanchnic nerves to modulate fast cholinergic synaptic input and that this effect of H2S modulates CNS control of gastrointestinal motility. Our results show for the first time that the facilitatory effect of endogenous H2S in the mouse SMG is pathway specific.

Mapping the cellular electrophysiology of rat sympathetic preganglionic neurones to their roles in cardiorespiratory reflex integration: a whole cell recording study in situ.

Sympathetic preganglionic neurones (SPNs) convey sympathetic activity flowing from the CNS to the periphery to reach the target organs. Although previous in vivo and in vitro cell recording studies have explored their electrophysiological characteristics, it has not been possible to relate these characteristics to their roles in cardiorespiratory reflex integration. We used the working heart-brainstem preparation to make whole cell patch clamp recordings from T3-4 SPNs (n = 98). These SPNs were classified by their distinct responses to activation of the peripheral chemoreflex, diving response and arterial baroreflex, allowing the discrimination of muscle vasoconstrictor-like (MVC(like), 39%) from cutaneous vasoconstrictor-like (CVC(like), 28%) SPNs. The MVC(like) SPNs have higher baseline firing frequencies (2.52 ± 0.33 Hz vs. CVC(like) 1.34 ± 0.17 Hz, P = 0.007). The CVC(like) have longer after-hyperpolarisations (314 ± 36 ms vs. MVC(like) 191 ± 13 ms, P < 0.001) and lower input resistance (346 ± 49 MΩ vs. MVC(like) 496 ± 41 MΩ, P < 0.05). MVC(like) firing was respiratory-modulated with peak discharge in the late inspiratory/early expiratory phase and this activity was generated by both a tonic and respiratory-modulated barrage of synaptic events that were blocked by intrathecal kynurenate. In contrast, the activity of CVC(like) SPNs was underpinned by rhythmical membrane potential oscillations suggestive of gap junctional coupling. Thus, we have related the intrinsic electrophysiological properties of two classes of SPNs in situ to their roles in cardiorespiratory reflex integration and have shown that they deploy different cellular mechanisms that are likely to influence how they integrate and shape the distinctive sympathetic outputs.

Long-term reproducibility of Edinger-Westphal stimulated accommodation in rhesus monkeys.

If longitudinal studies of accommodation or accommodation restoration procedures are undertaken in rhesus monkeys, the methods used to induce and measure accommodation must remain reproducible over the study period. Stimulation of the Edinger-Westphal (EW) nucleus in anesthetized rhesus monkeys is a valuable method to understand various aspects of accommodation. A prior study showed reproducibility of EW-stimulated accommodation over 14 months after chronic electrode implantation. However, reproducibility over a period longer than this has not been investigated and therefore remains unknown. To address this, accommodation stimulation experiments in four eyes of two rhesus monkeys (13.7 and 13.8 years old) were evaluated over a period of 68 months. Carbachol iontophoresis stimulated accommodation was first measured with a Hartinger coincidence refractometer (HCR) two weeks before electrode implantation to determine maximum accommodative amplitudes. EW stimulus-response curves were initially measured with the HCR one month after electrode implantation and then repeated at least six times for each eye in the following 60 months. At 64 months, carbachol iontophoresis induced accommodation was measured again. At 68 months, EW stimulus-response curves were measured with an HCR and photorefraction every week over four consecutive weeks to evaluate the short-term reproducibility over one month. In the four eyes studied, long-term EW-stimulated accommodation decreased by 7.00 D, 3.33 D, 4.63 D, and 2.03 D, whereas carbachol stimulated accommodation increased by 0.18 D-0.49 D over the same time period. The short-term reproducibility of maximum EW-stimulated accommodation (standard deviations) over a period of four weeks at 68 months after electrode implantation was 0.48 D, 0.79 D, 0.55 D and 0.39 D in the four eyes. Since the long-term decrease in EW-stimulated accommodation is not matched by similar decreases in carbachol iontophoresis stimulated accommodation, the decline in accommodation cannot be due to the progression of presbyopia but is likely to result from variability in EW electrode position. Therefore, EW-stimulated accommodation in anesthetized monkeys is not appropriate for long-term longitudinal studies of age-related loss of accommodation or accommodation restoration procedures.

Changes in contractile activity of rabbit colon under stress conditions and during post-stress period before and after blockade of muscarinic and nicotinic cholinergic receptors.

Psychogenic stress in rabbits induced by fixation of the animal to a frame was accompanied by an increase in contractile activity of the initial portion of the distal colon, which was abolished by blockade of muscarinic and nicotinic cholinergic receptors. Increased contractile activity of the colon was due to centrogenic stimulation of preganglionic neurons of the parasympathetic nervous system followed by the involvement of the effector cholinergic neurons of the enteric nervous system into excitation.

Immediate and sustained increases in the activity of vagal preganglionic neurons during exercise and after exercise training.

AIMS: The brain controls the heart by dynamic recruitment and withdrawal of cardiac parasympathetic (vagal) and sympathetic activity. Autonomic control is essential for the development of cardiovascular responses during exercise, however, the patterns of changes in the activity of the two autonomic limbs, and their functional interactions in orchestrating physiological responses during exercise, are not fully understood. The aim of this study was to characterize changes in vagal parasympathetic drive in response to exercise and exercise training by directly recording the electrical activity of vagal preganglionic neurons in experimental animals (rats). METHODS AND RESULTS: Single unit recordings were made using carbon-fibre microelectrodes from the populations of vagal preganglionic neurons of the nucleus ambiguus (NA) and the dorsal vagal motor nucleus of the brainstem. It was found that (i) vagal preganglionic neurons of the NA and the dorsal vagal motor nucleus are strongly activated during bouts of acute exercise, and (ii) exercise training markedly increases the resting activity of both populations of vagal preganglionic neurons and augments the excitatory responses of NA neurons during exercise. CONCLUSIONS: These data show that central vagal drive increases during exercise and provide the first direct neurophysiological evidence that exercise training increases vagal tone. The data argue against the notion of exercise-induced central vagal withdrawal during exercise. We propose that robust increases in the activity of vagal preganglionic neurons during bouts of exercise underlie activity-dependent plasticity, leading to higher resting vagal tone that confers multiple health benefits associated with regular exercise.

Identification of the spinal pathways involved in the recovery of baroreflex control after spinal lesion in the rat using pseudorabies virus.

Neurons in the rostroventrolateral medulla (RVLM) mediate baroreflex regulation (BR) of spinal sympathetic preganglionic neurons. Previously, our laboratory has shown that recovery of BR occurs in the rat after spinal hemisection. (Zahner MR, Kulikowicz E, and Schramm LP. Am J Physiol Regul Integr Comp Physiol 301: R1584-R1590, 2011). The goal of these experiments was to determine whether the observed recovery of BR is mediated by the reorganization of ipsilateral pathways or by compensation by spared contralateral pathways. To determine this, we infected the left kidney in rats with the retrograde transynaptic tracer, pseudorabies virus (PRV), either 1 or 8 wk after left spinal hemisection at either T(3) or T(8), or after a sham lesion. In sham-lesioned rats, PRV infection of RVLM neurons was bilateral. In all rats with a left hemisection, regardless of the location of the lesion (T(3) or T(8)) or postlesion recovery time (1 or 8 wk), PRV infection of left RVLM neurons was significantly reduced compared with sham-lesioned rats (P < 0.05). In a separate group of rats, we performed BR tests by measuring responses of left renal sympathetic nerve activity to pharmacologically induced decreases and increases in arterial pressure. In rats with T(8) left hemisection and 8-wk recovery, BR was robust, and acute right upper thoracic hemisection abolished all BR of left renal sympathetic nerve activity. Collectively, these data suggest that the recovery of BR is not mediated by reorganization of ipsilateral bulbospinal connections, but instead by improved efficacy of existing contralateral pathways.

Chemical coding for cardiovascular sympathetic preganglionic neurons in rats.

Cocaine and amphetamine-regulated transcript peptide (CART) is present in a subset of sympathetic preganglionic neurons in the rat. We examined the distribution of CART-immunoreactive terminals in rat stellate and superior cervical ganglia and adrenal gland and found that they surround neuropeptide Y-immunoreactive postganglionic neurons and noradrenergic chromaffin cells. The targets of CART-immunoreactive preganglionic neurons in the stellate and superior cervical ganglia were shown to be vasoconstrictor neurons supplying muscle and skin and cardiac-projecting postganglionic neurons: they did not target non-vasoconstrictor neurons innervating salivary glands, piloerector muscle, brown fat, or adrenergic chromaffin cells. Transneuronal tracing using pseudorabies virus demonstrated that many, but not all, preganglionic neurons in the vasoconstrictor pathway to forelimb skeletal muscle were CART immunoreactive. Similarly, analysis with the confocal microscope confirmed that 70% of boutons in contact with vasoconstrictor ganglion cells contained CART, whereas 30% did not. Finally, we show that CART-immunoreactive cells represented 69% of the preganglionic neuron population expressing c-Fos after systemic hypoxia. We conclude that CART is present in most, although not all, cardiovascular preganglionic neurons but not thoracic preganglionic neurons with non-cardiovascular targets. We suggest that CART immunoreactivity may identify the postulated "accessory" preganglionic neurons, whose actions may amplify vasomotor ganglionic transmission.

Differential activation of adrenal, renal, and lumbar sympathetic nerves following stimulation of the rostral ventrolateral medulla of the rat.

Under acute and chronic conditions, the sympathetic nervous system can be activated in a differential and even selective manner. Activation of the rostral ventrolateral medulla (RVLM) has been implicated in differential control of sympathetic outputs based on evidence primarily in the cat. Although several studies indicate that differential control of sympathetic outflow occurs in other species, only a few studies have addressed whether the RVLM is capable of producing varying patterns of sympathetic activation in the rat. Therefore, the purpose of the present study was to determine whether activation of the RVLM results in simultaneous and differential increases in preganglionic adrenal (pre-ASNA), renal (RSNA), and lumbar (LSNA) sympathetic nerve activities. In urethane-chloralose anesthetized rats, pre-ASNA, RSNA, and LSNA were recorded simultaneously in all animals. Microinjections of selected concentrations and volumes of glutamate increased pre-ASNA, RSNA, and LSNA concurrently and differentially. Pre-ASNA and RSNA (in most cases) exhibited greater increases compared with LSNA on a percentage basis. By varying the volume or location of the glutamate microinjections, we also identified individual examples of differential and selective activation of these nerves. Decreases in arterial pressure or bilateral blockade of RVLM GABA(A) receptors also revealed differential activation, with the latter having a 3- to 4-fold greater effect on sympathetic activity. Our data provide evidence that activation of the rat RVLM increases renal, lumbar, and preganglionic adrenal sympathetic nerve activities concurrently, differentially, and, in some cases, selectively.

Ganglionic transmission in a vasomotor pathway studied in vivo.

Intracellular recordings were made in vivo from 40 spontaneously active cells in the third lumbar sympathetic ganglion of urethane-anaesthetized rats. In 38/40 cells ongoing action potentials showed strong cardiac rhythmicity (93.4 +/- 1.9% modulation) indicating high barosensitivity and probable muscle vasoconstrictor (MVC) function. Subthreshold excitatory postsynaptic potentials (EPSPs) showed the same pattern. The 38 barosensitive neurons fired action potentials at 2.9 +/- 0.3 Hz. All action potentials were triggered by EPSPs, most of which were unitary events. Calculations indicated that <5% of action potentials were triggered by summation of otherwise subthreshold EPSPs. 'Dominant' synaptic inputs with a high safety factor were identified, confirming previous work. These were active in 24/38 cells and accounted for 32% of all action potentials; other ('secondary') inputs drove the remainder. Inputs (21 dominant, 19 secondary) attributed to single preganglionic neurons fired at 1.38 +/- 0.16 Hz. An average of two to three preganglionic neurons were estimated to drive each ganglion cell's action potentials. When cells were held hyperpolarized to block spiking, a range of spontaneous EPSP amplitudes was revealed. Threshold equivalent was defined as the membrane potential value that was exceeded by spontaneous EPSPs at the same frequency as the cell's original firing rate. In 10/12 cells examined, a continuum of EPSP amplitudes overlapped threshold equivalent. Small changes in cell excitability could therefore raise or lower the percentage of preganglionic inputs triggering action potentials. The results indicate that vasoconstrictor ganglion cells in vivo mostly behave not as 1:1 relays, but as continuously variable gates.

Targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced sustained ventricular tachycardia in conscious rats.

The Cardiac Arrhythmia Suppression Trial demonstrated that antiarrhythmic drugs not only fail to prevent sudden cardiac death, but actually increase overall mortality. These findings have been confirmed in additional trials. The "proarrhythmic" effects of most currently available antiarrhythmic drugs makes it essential that we investigate novel strategies for the prevention of sudden cardiac death. Targeted ablation of cardiac sympathetic neurons may become a therapeutic option by reducing sympathetic activity. Thus cholera toxin B subunit (CTB) conjugated to saporin (a ribosomal inactivating protein that binds to and inactivates ribosomes; CTB-SAP) was injected into both stellate ganglia to test the hypothesis that targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced, sustained ventricular tachycardia in conscious rats. Rats were randomly divided into three groups: 1) control (no injection); 2) bilateral stellate ganglia injection of CTB; and 3) bilateral stellate ganglia injection of CTB-SAP. CTB-SAP rats had a reduced susceptibility to ischemia-induced, sustained ventricular tachycardia. Associated with the reduced susceptibility to ventricular arrhythmias were a reduced number of stained neurons in the stellate ganglia and spinal cord (segments T(1)-T(4)), as well as a reduced left ventricular norepinephrine content and sympathetic innervation density. Thus CTB-SAP retrogradely transported from the stellate ganglia is effective at ablating cardiac sympathetic neurons and reducing the susceptibility to ventricular arrhythmias.

Structural neuroplasticity following T5 spinal cord transection: increased cardiac sympathetic innervation density and SPN arborization.

When the spinal cord is injured at or below thoracic level 5 (T5), cardiovascular control is markedly unbalanced as the heart and blood vessels innervated by upper thoracic segments remain under brain stem control, whereas the vasculature of the lower body is affected by unregulated spinal reflexes. Importantly, the regulation of heart rate and cardiac function is abnormal after spinal cord injury (SCI) at T5 because sympathetic outflow to the heart is increased. An increase in tonic sympathetic outflow may be attributable to multiple mechanisms, such as increases in cardiac sympathetic innervation density, altered morphology of stellate ganglia neurons, and/or structural neuroplasticity of cardiac sympathetic preganglionic neurons (SPNs). Furthermore, these neuroplastic changes associated with SCI may be mediated by nerve growth factor (NGF). NGF is a neurotrophin that supports the survival and differentiation of sympathetic neurons and enhances target innervation. Therefore, we tested the hypothesis that T5 spinal cord transection (T5X) is associated with an increased left ventricular (LV) NGF content, LV sympathetic innervation density, and cardiac SPN arborization. In intact and paraplegic (9 wk posttransection) rats, LV NGF content (ELISA), LV sympathetic innervation density (tyrosine hydroxylase immunohistochemistry), and cardiac SPN arborization (cholera toxin B immunohistochemistry and Sholl Analysis) were determined. Paraplegia, compared with intact, significantly increased LV NGF content, LV sympathetic innervation density, and cardiac SPN arborization. Thus, altered autonomic behavior following SCI is associated with structural neuroplastic modifications.

Evidence for functional ghrelin receptors on parasympathetic preganglionic neurons of micturition control pathways in the rat.

1. Previous work indicates that agonists of ghrelin receptors can act within the spinal cord to stimulate autonomic outputs to the colorectum and to blood vessels. Because of the close relationship between colorectal and urinary bladder control, we have investigated whether ghrelin receptor agonists also stimulate spinal centres that influence the bladder. 2. The ghrelin receptor agonist capromorelin (10 mg/kg), injected intravenously in anaesthetized male rats, disrupted the ongoing cycle of micturition reflexes and caused phasic oscillations in pressure that averaged approximately 20 mmHg. Fluid output from the bladder was diminished. The effects of capromorelin were inhibited by hexamethonium (10 mg/kg bolus followed by 4 mg/kg per h infusion, i.v.) and were further reduced by atropine (5 mg/kg bolus followed by 2.5 mg/kg per h infusion, i.v.). Capromorelin (250 microg) injected directly into the spinal cord at the lumbosacral level also increased contractile activity of the bladder. However, capromorelin, up to 0.1 mmol/L, had no effect on the tension of isolated muscle strips from the bladder. Effects of intravenous capromorelin (10 mg/kg) on bladder pressure were still observed after the descending pathways in the spinal cord were disrupted at the thoracic level. 3. In situ hybridization studies revealed ghrelin receptor gene expression in neurons of the autonomic intermediolateral (IML) cell columns. Following a series of micturition reflexes elicited by infusion of saline into the bladder, the immediate early gene product c-Fos was observed in neurons of the lumbosacral IML and approximately 20% of these also expressed ghrelin receptor gene transcripts. 4. It is concluded that ghrelin receptors are expressed by lumbosacral autonomic preganglionic neurons of the micturition reflex pathways and that ghrelin receptor agonists stimulate these neurons.

Enteric nervous system: sensory transduction, neural circuits and gastrointestinal motility.

The gastrointestinal tract is the only internal organ to have evolved with its own independent nervous system, known as the enteric nervous system (ENS). This Review provides an update on advances that have been made in our understanding of how neurons within the ENS coordinate sensory and motor functions. Understanding this function is critical for determining how deficits in neurogenic motor patterns arise. Knowledge of how distension or chemical stimulation of the bowel evokes sensory responses in the ENS and central nervous system have progressed, including critical elements that underlie the mechanotransduction of distension-evoked colonic peristalsis. Contrary to original thought, evidence suggests that mucosal serotonin is not required for peristalsis or colonic migrating motor complexes, although it can modulate their characteristics. Chemosensory stimuli applied to the lumen can release substances from enteroendocrine cells, which could subsequently modulate ENS activity. Advances have been made in optogenetic technologies, such that specific neurochemical classes of enteric neurons can be stimulated. A major focus of this Review will be the latest advances in our understanding of how intrinsic sensory neurons in the ENS detect and respond to sensory stimuli and how these mechanisms differ from extrinsic sensory nerve endings in the gut that underlie the gut-brain axis.

Tonic GABAergic inhibition of sympathetic preganglionic neurons: a novel substrate for sympathetic control.

The sympathetic tone is primarily defined by the level of activity of the sympathetic preganglionic neurons. We report a novel inhibitory influence on sympathetic activity, that of tonic GABAergic inhibition which could have a profound global effect on sympathetic outflow. Recording from identified SPNs in the intermediolateral cell column (IML) of rat spinal cord slices, application of the GABA receptor antagonist bicuculline, but not gabazine, elicited a change in voltage that lasted for the duration of application. This response was mediated by a direct effect on SPNs since it persisted in tetrodotoxin and low Ca(2+)/high Mg(2+) and the amplitude of responses were related to Cl(-) concentration in patch solutions. Such tonic inhibitory responses were not observed in interneurons, the other neuronal type in the IML, although ongoing IPSPs were antagonized in these neurons. The effects of bicuculline were enhanced by diazepam but not zolpidem or the GABA modulators THIP and THDOC suggesting a role for alpha5 subunits. PCR using primers for the alpha5 and delta subunits indicated the presence of alpha5, but not delta subunits in the IML. Firing rates of SPNs were enhanced by bicuculline and decreased by diazepam indicating that this tonic inhibition has a profound effect on the excitability of SPNs. These data indicate a novel influence for controlling the activity of SPNs regardless of their function.