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

Somatostatin 2 Receptors in the Spinal Cord Tonically Restrain Thermogenic, Cardiac and Other Sympathetic Outflows

The anatomical and functional characterization of somatostatin (SST) and somatostatin receptors (SSTRs) within the spinal cord have been focused in the dorsal horn, specifically in relation to sensory afferent processing. However, SST is also present within the intermediolateral cell column (IML), which contains sympathetic preganglionic neurons (SPN). We investigated the distribution of SSTR2 within the thoracic spinal cord and show that SSTR2A and SSTR2B are expressed in the dorsal horn and on SPN and non-SPN in or near the IML. The effects of activating spinal SSTR and SSTR2 were sympathoinhibition, hypotension, bradycardia, as well as decreases in interscapular brown adipose tissue temperature and expired CO2, in keeping with the well-described inhibitory effects of activating SSTR receptors. These data indicate that spinal SST can decrease sympathetic, cardiovascular and thermogenic activities. Unexpectedly blockade of SSTR2 revealed that SST tonically mantains sympathetic, cardiovascular and thermogenic functions, as activity in all measured parameters increased. In addition, high doses of two antagonists evoked biphasic responses in sympathetic and cardiovascular outflows where the initial excitatory effects were followed by profound but transient falls in sympathetic nerve activity, heart rate and blood pressure. These latter effects, together with our findings that SSTR2A are expressed on GABAergic, presumed interneurons, are consistent with the idea that SST2R tonically influence a diffuse spinal GABAergic network that regulates the sympathetic cardiovascular outflow. As described here and elsewhere the source of tonically released spinal SST may be of intra- and/or supra-spinal origin

Maps of cardiovascular and respiratory regions of rat ventral medulla : focus on the caudal medulla

The ventral medulla oblongata is critical for cardiorespiratory regulation. Here we review previous literature relating to sites within the ventral medulla that have been identified as having a ‘cardiovascular’ or ‘respiratory’ function. Together with the maps generated here, of sites from which cardiovascular and respiratory responses were evoked by glutamate microinjection, specific ‘cardiovascular’ regions have been defined and delineated. Commonly investigated regions, including the vasopressor rostral ventrolateral medulla (RVLM) and vasodepressor caudal ventrolateral medulla (CVLM), or areas only described by others, such as the medullary cerebral vasodilator area, are included for completeness. Emphasis is given to the caudal medulla, where three pressor regions, the caudal pressor area (CPA), the intermediate pressor area (IPA) and the medullo-cervical pressor area (MCPA), caudal to the vasodepressor CVLM were defined in the original data provided. The IPA is most responsive under pentobarbitone rather than urethane anaesthesia clearly delineating it from both the rostrally located CPA and the caudally located MCPA. The description of these multiple pressor areas appears to clarify the confusion that surrounds the identification of the ‘CPA’. Also noted is a vasopressor region adjacent to the vasodepressor CVLM. Apart from the well described ventral respiratory column, a region medial to the pre-Bötzinger is described, from which increases in both phrenic nerve frequency and amplitude were evoked. Limitations associated with the technique of glutamate microinjection to define functionally specific regions are discussed. Particular effort has been made to define and delineate the regions with respect to ventrally located anatomical landmarks rather than the commonly used ventral surface or dorsal landmarks such as the obex or calamus scriptorius that may vary with the brain orientation or histological processing. This should ensure that a region can easily be defined by all investigators. Study of defined regions will help expedite the identification of the role of the multiple cell groups with diverse neurotransmitter complements that exist even within each of the regions described, in coordinating the delivery of oxygenated blood to the tissues.13 page(s

GABA and enkephalin tonically alter sympathetic outflows in the rat spinal cord

GABA and enkephalin provide significant innervation of sympathetic preganglionic neurons. Despite some investigation as to the identity of premotor sources of these innervations no comprehensive analyses have been conducted. Similarly, although data describing the cardiovascular effects of blockade of GABAA receptors in the spinal cord is available, the effects at other sympathetic outflows are unknown. In contrast the sympathetic effects of opioid blockade in the spinal cord are unclear. The aims of this study were to identify potential sympathetic premotor sources of GABAergic and enkephalinergic input to the spinal cord and to describe the sympathetic and cardiovascular effects of spinal GABAA receptor and delta/mu opioid receptor blockade in urethane anaesthetised rats. Glutamic acid decarboxylase (GAD67) and preproenkephalin (PPE) mRNA were found in all regions containing sympathetic premotor neurons, with the medullary raphe and RVMM providing the major GABAergic projections, while the PVN, RVMM and medullary raphe provided the major enkephalinergic projections. Intrathecal injection of bicuculline, a GABAA antagonist, elicited large and prolonged increases in all outflows measured, confirming previous work describing a tonic GABAergic influence on vasomotor tone, and revealing a tonic GABAergic inhibition of interscapular brown adipose tissue temperature. Intrathecal naloxone elicited transient small inhibitory effects only on MAP and HR. Thus GABA acting in the spinal cord plays an important role in the tonic suppression of sympathetic outflows while enkephalin appears to play only a minor role.8 page(s

Differential role of kinases in brain stem of hypertensive and normotensive rats

Spontaneously hypertensive rats (SHR) are characterized by extreme elevations of blood pressure. The genetic factors underlying this are yet to be identified. Here we demonstrate, in vivo, that in SHR and normotensive Wistar-Kyoto rats (WKY), injection of the mitogen-activated protein kinase inhibitor PD 098,059 bilaterally into the rostral ventrolateral medulla (RVLM) dramatically lowers arterial pressure. PD 098,059 does not alter the responses evoked by microinjection of glutamate into the RVLM or brief apnea. Wortmannin (phosphatidylinositol-3 kinase inhibitor) bilaterally into the RVLM causes a 35±4% fall in arterial pressure in SHR but has no effect in WKY. Furthermore, wortmannin reduces the pressor response evoked by microinjection of angiotensin (Ang) II in the RVLM of SHR compared with WKY. The response to Ang II microinjection into the RVLM of WKY was unaffected by wortmannin. Simultaneous bilateral injections of PD 098,059 and wortmannin into the RVLM abolished the response to exogenous Ang II in the RVLM but did not affect the response evoked by glutamate in either SHR or WKY. Thus, it appears that PD 098,059– and/or wortmannin-sensitive mechanisms are not involved in the responses evoked by glutamate in the RVLM and that these kinase inhibitors are not neurotoxic. We conclude that a PD 098,059–sensitive pathway in the RVLM of SHR and WKY tonically regulates arterial pressure and that a wortmannin-sensitive pathway in the RVLM is important in the maintenance of hypertension in SHR. This may be related to a phosphatidylinositol-3 kinase–dependent mechanism involved in the action of Ang II on the Ang II type 1 receptor.6 page(s

Insight into autonomic nervous system control of heart rate in the rat using analysis of heart rate variability and baroreflex sensitivity

Heart rate variability and baroreflex sensitivity are two readily accessible methods available to assess control of heart rate by the autonomic nervous system. Heart rate variability can provide information regarding neural (parasympathetic, sympathetic, reflex) and nonneural (hormones, thermoregulation) control of heart rate. Baroreflex sensitivity provides information on how effective the autonomic nervous system is in rapidly changing heart rate in response to changes in blood pressure. Methods are presented on how to calculate heart rate variability using frequency domain analysis and how to calculate baroreflex sensitivity using the sequence method and α-coefficient method of analysis. Data is presented from a variety of rat strains (control, hypertensive, kidney disease, depressed) to indicate the utility of these methods for comparing autonomic regulation of the heart in both anaesthetised animals via intra-arterial recordings and conscious animals using telemetry.21 page(s