Michael P. Gilbey

Cardioprotection evoked by remote ischaemic preconditioning is critically dependent on the activity of vagal pre-ganglionic neurones

Aims Innate mechanisms of inter-organ protection underlie the phenomenon of remote ischaemic preconditioning (RPc) in which episode(s) of ischaemia and reperfusion in tissues remote from the heart reduce myocardial ischaemia/reperfusion injury. The uncertainty surrounding the mechanism(s) underlying RPc centres on whether humoral factor(s) produced during ischaemia/reperfusion of remote tissue and released into the systemic circulation mediate RPc, or whether a neural signal is required. While these two hypotheses may not be incompatible, one approach to clarify the potential role of a neural pathway requires targeted disruption or activation of discrete central nervous substrate(s). Methods and results Using a rat model of myocardial ischaemia/reperfusion injury in combination with viral gene transfer, pharmaco-, and optogenetics, we tested the hypothesis that RPc cardioprotection depends on the activity of vagal pre-ganglionic neurones and consequently an intact parasympathetic drive. For cell-specific silencing or activation, neurones of the brainstem dorsal motor nucleus of the vagus nerve (DVMN) were targeted using viral vectors to express a Drosophila allatostatin receptor (AlstR) or light-sensitive fast channelrhodopsin variant (ChIEF), respectively. RPc cardioprotection, elicited by ischaemia/reperfusion of the limbs, was abolished when DVMN neurones transduced to express AlstR were silenced by selective ligand allatostatin or in conditions of systemic muscarinic receptor blockade with atropine. In the absence of remote ischaemia/reperfusion, optogenetic activation of DVMN neurones transduced to express ChIEF reduced infarct size, mimicking the effect of RPc. Conclusion These data indicate a crucial dependence of RPc cardioprotection against ischaemia/reperfusion injury upon the activity of a distinct population of vagal pre-ganglionic neurones.

Sympathetic rhythms and nervous integration.

1 The present review focuses on some of the processes producing rhythms in sympathetic nerves influencing cardiovascular functions and considers their potential relevance to nervous integration. 2 Two mechanisms are considered that may account for rhythmic sympathetic discharges. First, neuronal elements of peripheral or central origin produce rhythmic activity by phasically exciting and/or inhibiting neurons within central sympathetic networks. Second, rhythms arise within central sympathetic networks. Evidence is considered that indicates the operation of both mechanisms; the first in muscle and the second in skin sympathetic vasoconstrictor networks. 3 Sympathetic activity to the rat tail, a model for the nervous control of skin circulation, is regulated by central networks involved in thermoregulation and those associated with fear and arousal. In an anaesthetized preparation, activity displays an apparently autonomous rhythm (T‐rhythm; 0.4–1.2 Hz) and the level of activity can be manipulated by regulating core body temperature. This model has been used to study rhythm generation in central sympathetic networks and possible functional relevance. 4 A unique insight provided by the T rhythm, into possible physiological function(s) underlying rhythmic sympathetic discharges is that the activity of single sympathetic post‐ganglionic neurons within a population innervating the same target can have different rhythm frequencies. Therefore, the graded and dynamic entrainment of the rhythms by inputs, such as central respiratory drive and/or lung inflation‐related afferent activity, can produce graded and dynamic synchronization of sympathetic discharges. The degree of synchronization may influence the efficacy of transmission in a target chain of excitable cells. 5 The T‐rhythm may be generated within the spinal cord because the intrathecal application of 5‐hydroxytryptamine at the L1 level of the spinal cord of a rat spinalized at T10–T11 produces a T‐like rhythm. Thus, induction and modulation of spinal cord oscillators may be mechanisms that influence ganglionic and neuroeffector transmission. 6 The study of sympathetic rhythms may not only further understanding of sympathetic control, but may also inform on the relevance of rhythmic nervous activities in general.

Experimental Biology 2000 Symposium on Differential Control of Sympathetic Outflow MULTIPLE OSCILLATORS, DYNAMIC SYNCHRONIZATION AND SYMPATHETIC CONTROL

1. Intermittent bursts of activity are a robust feature of the discharges of sympathetic nerves. There are at least two major mechanisms producing such discharges: (i) phasic inputs influencing sympathetic circuits; and (ii) oscillators embedded within sympathetic networks. The functional significance of patterned and synchronized activity underlying bursts of population activity may reside in their influence on information transfer between excitable cells. At the level of the single neuron, firing pattern appears to be an important determinant of synaptic/neuroeffector function (e.g. the probability of transmitter release, the types of transmitter released, the types of receptor activated and plasticity). Synchronization of inputs at a target favours summation and, therefore, may influence response (short term and long term).

Generation of a physiological sympathetic motor rhythm in the rat following spinal application of 5-HT

When applied in vitro to various CNS structures 5‐HT and/or NMDA have been observed to generate rhythmic nervous activity. In contrast, reports of similar in vivo actions are relatively rare. Here we describe a physiological sympathetic motor rhythm regulating the thermoregulatory circulation of the rat tail (T‐rhythm; 0.40–1.20 Hz) that can be elicited following intrathecal (i.t.) application of 5‐HT to an in situ‘isolated’ spinal cord preparation (anaesthetized rats spinalized at T10–T11 and cauda equina cut). i.t. injections were delivered to L1 as sympathetic neuronal activity to the tail (SNAT) arises from preganglionic neurones at T11–L2. SNAT was abolished after spinal transection (n= 18) and it did not return spontaneously. The administration of 5‐HT (250 nmol) generated rhythmic sympathetic discharges (n= 6). The mean frequency of the T‐like rhythm during the highest level of activity was 0.88 ± 0.04 Hz which was not significantly different from the T‐rhythm frequency observed in intact animals (0.77 ± 0.02 Hz; P > 0.05 n= 16). In contrast, NMDA (1 μmol) generated an irregular tonic activity, but it failed to generate a T‐like rhythm (n= 9), even though the mean levels of activity were not significantly different to those produced by 5‐HT. However, 5‐HT (250 nmol) applied after NMDA generated a T‐like rhythm (0.95 ± 0.11 Hz, n= 6). Our observations support the idea that 5‐HT released from rostral ventromedial medullary neurones, known to innervate sympathetic preganglionic neurones, can induce sympathetic rhythmic activity.

Coherent rhythmic discharges in sympathetic nerves supplying thermoregulatory circulations in the rat

1 In anaesthetised rats, activity recorded from sympathetic postganglionic neurones innervating the tail circulation has characteristic rhythmicity (0·4‐1·2 Hz). At the population level this rhythmicity can be seen as a peak (T‐peak) in autospectra of sympathetic activity recorded from ventral collector nerves (VCNs). 2 Here we investigated whether nerves supplying thermoregulatory circulations share common rhythmic discharges at T‐peak frequency. Activity was recorded from nerve pairs consisting of left ventral collector nerve (LVCN) and one of the following: right ventral collector nerve (RVCN), left dorsal collector nerve (DCN), left saphenous nerve (SN) or left renal nerve (RN). 3 During central apnoea, T‐peak frequencies in RVCN autospectra were similar to those of simultaneously recorded LVCN and these activities were coherent. Similar observations were made for nerve pairs involving LVCN‐DCN and LVCN‐SN. In contrast, autospectra of RN activity did not contain T‐peaks. 4 In comparison to the peaks in autospectra of RN activity, when the frequency of rhythmic phrenic nerve activity was manipulated T‐peaks in VCN, DCN and SN autospectra did not show obligatory 1:1 locking. 5 We conclude that T‐peaks are a robust feature of autospectra of sympathetic discharges supplying thermoregulatory circulation but not those influencing the kidney. The high coherence demonstrated between the T‐peak discharges is consistent with the view that common/coupled oscillators located within the CNS influence cutaneous vasoconstrictor sympathetic activity.

Resetting of sympathetic rhythm by somatic afferents causes post‐reflex coordination of sympathetic activity in rat

We have proposed previously that graded synchronous activity is produced by periodic inputs acting on weakly coupled or uncoupled oscillators influencing the discharges of a population of cutaneous vasoconstrictor sympathetic postganglionic neurones (PGNs) in anaesthetized rats. Here we investigated the effects of somatic afferent (superficial radial nerve, RaN) stimulation, on the rhythmic discharges of this population. We recorded (1) at the population level from the ventral collector nerve and (2) from single PGNs focally from the caudal ventral artery of the tail. Following RaN stimulation we observed an excitatory response followed by a period of reduced discharge and subsequent rhythmical discharges seemingly phase‐locked to the stimulus. We suggest that the rhythmical discharges following the initial excitatory response (conventional reflex) result from a resetting of sympathetic rhythm generators such that rhythmic PGN activity is synchronized transiently. We also demonstrate that a natural mechanical stimulus can produce a similar pattern of response. Our results support the idea that in sympathetic control, resetting of multiple oscillators driving the rhythmic discharges of a population of PGNs may provide a mechanism for producing a sustained and coordinated response to somatic input.

Cyclical modulation of human ventricular repolarization by respiration

Background: Respiratory modulation of autonomic input to the sinus node results in cyclical modulation of heart rate, known as respiratory sinus arrhythmia (RSA). We hypothesized that the respiratory cycle may also exert cyclical modulation on ventricular repolarization, which may be separately measurable using local endocardial recordings. Methods and Results: The study included 16 subjects with normal ventricles undergoing routine clinical electrophysiological procedures for supraventricular arrhythmias. Unipolar electrograms were recorded from 10 right and 10 left ventricular endocardial sites. Breathing was voluntarily regulated at 5 fixed frequencies (6, 9, 12, 15, and 30 breaths per min) and heart rate was clamped by RV pacing. Activation-recovery intervals (ARI: a surrogate for APD) exhibited significant (p < 0.025) cyclical variation at the respiratory frequency in all subjects; ARI shortened with inspiration and lengthened with expiration. Peak-to-peak ARI variation ranged from 0–26 ms; the spatial pattern varied with subject. Arterial blood pressure also oscillated at the respiratory frequency (p < 0.025) and lagged behind respiration by between 1.5 s and 0.65 s from slowest to fastest breathing rates respectively. Systolic oscillation amplitude was significantly greater than diastolic (14 ± 5 vs. 8 ± 4 mm Hg ± SD, p < 0.001). Conclusions: Observations in humans with healthy ventricles using multiple left and right ventricular endocardial recordings showed that ARI action potential duration (APD) varied cyclically with respiration.

Immunohistochemical detection of connexin36 in sympathetic preganglionic and somatic motoneurons in the adult rat.

Gap junctional communication in the adult CNS plays an important role in the synchronization of neuronal activities. In vitro studies have shown evidence of electrotonic coupling through gap junctions between sympathetic preganglionic motoneurons and between somatic motoneurons in the neonatal and adult rat spinal cord. Electrotonic transmission of membrane oscillations might be an important mechanism for recruitment of neurons and result in the generation of rhythmic sympathetic and somato-motor activity at the population level. Gap junctions in the adult spinal cord are constituted principally by connexin36 (Cx36). However, the distribution of Cx36 in specific neuronal populations of the spinal cord is unknown. Here, we identify Cx36-like immunoreactivity in sympathetic preganglionic and somatic motoneurons in thoracic spinal cord segments of the adult rat. For this purpose, double immunostaining against Cx36 and choline acetyltransferase (ChAT) was performed on transverse sections (20 μm) taken from spinal segments T6–T8. Cx36 punctate immunostaining was detected in the majority of ChAT-immunoreactive (-ir) neurons from lamina VII [intermediolateral cell column (IML) and intercalated cell group (IC)], lamina X [central autonomic nucleus (CA)] and in ventral horn neurons from laminae VIII and IX. Cx36 puncta were distributed in the neuronal somata and along dendritic processes. The presence of Cx36 in ChAT-ir neurons is consistent with electrical coupling between sympathetic preganglionic motoneurons and between somatic motoneurons through gap junctions in the adult spinal cord.

Automatic classification of interference patterns in driven event series: application to single sympathetic neuron discharge forced by mechanical ventilation

This study proposes a method for the automatic classification of nonlinear interactions between a strictly periodical event series modelling the activity of an exogenous oscillator working at a fixed and well-known rate and an event series modelling the activity of a self-sustained oscillator forced by the exogenous one. The method is based on a combination of several well-known tools (probability density function of the cyclic relative phase, probability density function of the count of forced events per forcing cycle, conditional entropy of the cyclic relative phase sequence and a surrogate data approach). Classification is reached via a sequence of easily applicable decision rules, thus rendering classification virtually user-independent and fully reproducible. The method classifies four types of dynamics: full uncoupling, quasiperiodicity, phase locking and aperiodicity. In the case of phase locking, the coupling ratio (i.e. n:m) and the strength of the coupling are calculated. The method, validated on simulations of simple and complex phase-locking dynamics corrupted by different levels of noise, is applied to data derived from one anesthetized and artificially ventilated rat to classify the nonlinear interactions between mechanical ventilation and: (1) the discharges of two (contemporaneously recorded) single postganglionic sympathetic neurons innervating the caudal ventral artery in the tail and (2) arterial blood pressure. Under central apnea, the activity of the underlying sympathetic oscillators is perturbed by means of five different lung inflation rates (0.58, 0.64, 0.76, 0.95, 1.99 Hz). While ventilation and arterial pressure are fully uncoupled, ventilation is capable of phase locking sympathetic discharges, thus producing 40% of phase-locked patterns (one case of 2:5, 1:1, 3:2 and 2:2) and 40% of aperiodic dynamics. In the case of phase-locked patterns, the coupling strength is low, thus demonstrating that this pattern is sliding. Non-stationary interactions are observed in 20% of cases. The two discharges behave differently, suggesting the presence of a population of sympathetic oscillators working at different frequencies.

A comparison of simultaneously recorded muscle and skin vasoconstrictor population activities in the rat using frequency domain analysis

In anaesthetized rats, an apparently autonomous sympathetic rhythm (T-rhythm, frequency range 0.4-1.2 Hz), has been observed in nerve activity controlling thermoregulatory circulations but not renal nerves. To further explore the differential control of sympathetic activity here, we investigate whether the so-called T-rhythm is a feature of muscle vasoconstrictor (MVC) population activity. Population activity was studied in vagotomised anaesthetised rats (alpha-chloralose or urethane maintenance, after barbiturate or halothane induction, respectively). Some rats were additionally sino-aortic denervated (SAD) and/or given a pneumothorax and neuromuscular blocked. Animals were held in central (hypocapnic) apnoea (ventilated at 2 Hz, tidal volume<or=2 ml) so that the T-rhythm could be studied without the confounding influence of central respiratory drive. In all animals (34; 17 with SAD) a peak in autospectra at T-rhythm frequency (T-peak: approximately 0.75 Hz) was a characteristic feature of activity supplying a thermoregulatory circulation (hind foot cutaneous vasoconstrictor activity, CVC), but not of simultaneously recorded MVC (gastrocnemius) activity. Percentage power at T-peak frequency was 4-5 times greater in CVC than MVC autospectra and at heart rate frequency approximately 14 fold greater in MVC than CVC autospectra: no peak was present at heart rate frequency in CVC autospectra. No peaks were present in MVC autospectra in SAD preparations. MVC-CVC coherence at both frequencies was low (approximately 0.2) in all types of preparation; i.e., most of the activity recorded from the two nerves was not linearly related. We conclude that under the experimental conditions of this study the T-rhythm is not a robust feature of MVC activity and SAD does not increase MVC-CVC coherence: observations which are consistent with fundamentally different neural substrates regulating MVC and CVC activities under the conditions of these experiments.