Caron Dean

Differential control of sympathetic activity to kidney and skeletal muscle by ventral medullary neurons

A rise in arterial blood pressure can be evoked by microinjections of D,L-homocysteic acid into localized regions of the ventrolateral medulla of the cat. Three patterns of sympathetic discharge can be identified during the pressor response. A differential pattern consisting of an increase in renal nerve activity and no change in sympathetic activity to skeletal muscle vasculature can be elicited from sites ventromedial to the caudal pole of the facial nucleus. From more lateral and caudal sites, a generalized sympatho-excitation is evoked in the outflow to both the kidney and hindlimb muscle vasculature. A third response consisting of a differential increase in muscle sympathetic activity simultaneous with a small decrease in renal nerve activity could be evoked from caudal sites, lateral to the inferior olives and superficial to the ventral surface. The results show that ventral medullary neurons can selectively activate sympathetic outflow to control specific vascular beds. These data may support the hypothesis that the ventrolateral medulla contains discrete groups of topographically arranged neurons that can differentially control sympathetic tone to various end-organs.

Serotonergic projections to the rostroventrolateral medulla from midbrain and raphe nuclei

Double-label fluoresence immunohistochemistry was performed to define serotonergic projections from the raphe and midbrain to the sympathoexcitatory region of the rostroventrolateral medulla (RVLM). Immunolabelling of cholera toxin B subunit retrogradely transported from the pressor region of the RVLM was combined with serotonin (5-HT) immunohistochemistry. Major sources of serotonergic input to the RVLM were shown to include the raphe obscurus, raphe pallidus and raphe magnus with a minor contribution from the ventrolateral, lateral and ventral regions of the periaqueductal gray matter, and the dorsal raphe nucleus. Serotonergic modulation of sympathoexcitatory neurons may establish patterns of sympathetic nerve activity evident in many aspects of cardiovascular regulation.

Expression of c-fos protein in the nucleus tractus solitarius in response to physiological activation of carotid baroreceptors

This study has utilized unilateral physiological pressure stimulation f a vascularly isolated carotid sinus combined with c-fos immunohistochemistry to locate neurons of the nucleus tractus solitarius which are activated by carotid baroreceptors in the anesthetized, vagotomized dog. Carotid baroreceptor stimulation primarily activated neurons in the ipsilateral commissural and medial subnuclei of the caudal nucleus tractus solitarius. In the intermediate and rostral nucleus tractus solitarius, carotid baroreceptor stimulation activated neurons in the dorsal and medial subnuclei. Results from this study also suggested that different subgroups of nucleus tractus solitarius neurons may be activated by baroreceptors with different pressure thresholds. The use of c-fos immunohistochemistry in this study has enabled the definition of populations of dorsal medullary neurons in the carotid baroreflex pathway. The results also suggest a different projection of carotid baroreceptors with different pressure thresholds.

Role of glutamate receptors in transmission of vagal cardiac input to neurones in the nucleus tractus solitarii in dogs

1 Vagal afferent input from cardiac mechanoreceptors excites neurones in the nucleus tractus solitarii (NTS), but discharge patterns evoked by physiological activation of pressure‐sensitive cardiac mechanoreceptors have not been studied in vivo. The role of glutamate receptor subtypes in transmission of afferent activity to the NTS neurones has not been determined. The present study therefore has two aims: first, to characterise the discharge patterns of neurones in the NTS that receive pressure‐sensitive vagal cardiac receptor input and second, to determine the roles of ionotropic glutamate receptor subtypes in the transmission of this putative cardiac mechanoreceptor‐related activity to NTS neurones. 2 Pulse‐synchronous activity of neurones in the NTS evoked by vagal afferent input was recorded extracellularly in an anaesthetised dog model using multibarrel glass electrodes, which allowed picoejection of the glutamate receptor antagonists NBQX or AP5 to block either non‐NMDA or NMDA receptors, respectively, during the neuronal recording. Pressure sensitivity of the recorded neurones was examined by monitoring their response to a small increase in arterial blood pressure. Selective pressure activation of carotid sinus baroreceptors in an isolated sinus or selective denervation of aortic baroreceptors were used to test for convergent excitation of the neurones by arterial baroreceptors. 3 Pulse‐synchronous cardiac‐related neuronal activity recorded from neurones in both the right and left NTS was eliminated following section of the left (n= 17) or right (n= 1) vagus nerves. No spontaneous, non‐pulsatile activity was observed in these neurones before or after vagotomy. Activity transmitted via left vagal afferents was found to be sensitive to changes in arterial blood pressure. In these neurones, activity was blocked in 13 of 17 neurones by picoejection of NBQX, with the remainder requiring both NBQX and AP5. None of the cardiac‐related neurones responded to activation of carotid baroreceptors or denervation of aortic baroreceptors, indicating no convergence of activity from carotid baroreceptors or aortic baroreceptors with pressure thresholds of approximately 130 mmHg or less. 4 The results suggest that vagal pressure‐sensitive afferent input from cardiac mechanoreceptors is transmitted primarily by left vagal afferent fibres via non‐NMDA receptors to neurones in both the ipsilateral and contralateral NTS. NMDA receptors were also found to have a role in the activation of a small subpopulation of neurones.

Modulation of the carotid baroreceptor reflex by substance P in the nucleus tractus solitarius.

Previous studies have shown that administration of substance P (SP) into the nucleus tractus solitarius (NTS) can evoke a depressor response similar to that produced by activation of the arterial baroreceptors. In addition, some studies have suggested that SP increases the reflex responses to activation of baroreceptor input. The present study was performed to determine the effects of SP on the carotid sinus baroreceptor reflex at the level of the NTS by examining the effects of both exogenous SP microinjected into different rostrocaudal locations in the NTS and blockade of the effects of endogenous SP, through the microinjection of a substance P antagonist (SPa; [D-Pro, D-Trp]-substance P). Changes in pressure in an isolated carotid sinus in anesthetized dogs were used to evoke baroreflex changes in arterial blood pressure (BP) before and after microinjection of SP (0.5 microM) or SPa (10 microM) into barosensitive regions of the NTS. Microinjection of SP or its antagonist did not alter baseline, resting BP but did produce significant changes in baroreflex sensitivity. Microinjection of SP into different rostrocaudal regions of the NTS produced different responses, with rostral and caudal NTS microinjections producing significant increases in sensitivity. No effects on baroreflex sensitivity were obtained in response to SP microinjections into the intermediate NTS. Unlike SP, microinjection of the SPa significantly decreased baroreflex sensitivity at all rostrocaudal levels of the NTS. These data demonstrated that SP has the capability to modulate the carotid baroreflex at the level of the NTS and support a physiological role for endogenously released SP.

Distribution and co-localization of 5-hydroxytryptamine, thyrotropin-releasing hormone and substance p in the cat medulla

This study demonstrates the co-existence of three neurochemicals in ventral medullary neurons of the cat utilizing fluorescence immunohistochemistry. Neurons containing 5-hydroxytryptamine, thyrotropin-releasing hormone and substance P were identified within the rostrocaudal extent of the medulla, specifically within the raphe pallidus and raphe magnus and in the reticular formation of the ventrolateral medulla in the nucleus paragigantocellularis lateralis. Within the raphe pallidus the majority of 5-hydroxytryptamine-containing neurons were co-localized with thyrotropin-releasing hormone and substance P. However, in the raphe magnus the majority of stained neurons contained 5-hydroxytryptamine and thyrotropin-releasing hormone but were devoid of substance P. In the ventrolateral medulla two major populations of neurons were identified rostral to the inferior olivary nuclei, one containing 5-hydroxytryptamine and thyrotropin-releasing hormone, while a more lateral group contained substance P alone. More caudally, at the level of the inferior olives, the majority of 5-hydroxytryptamine-containing cells also displayed immunoreactivity for thyrotropin-releasing hormone and substance P. A consistent finding in both the ventromedial and ventrolateral regions of the medulla was a population of 5-hydroxytryptamine-containing cells which did not stain for either thyrotropin-releasing hormone or substance P. The functional role of co-localized neurochemicals remains unknown but co-existence of neurotransmitter substances in medullary neurons may allow for specific and multiple actions in the spinal cord.

Pontine μ-opioid receptors mediate bradypnea caused by intravenous remifentanil infusions at clinically relevant concentrations in dogs

Life-threatening side effects such as profound bradypnea or apnea and variable upper airway obstruction limit the use of opioids for analgesia. It is yet unclear which sites containing μ-opioid receptors (μORs) within the intact in vivo mammalian respiratory control network are responsible. The purpose of this study was 1) to define the pontine region in which μOR agonists produce bradypnea and 2) to determine whether antagonism of those μORs reverses bradypnea produced by intravenous remifentanil (remi; 0.1-1.0 μg·kg(-1)·min(-1)). The effects of microinjections of agonist [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO; 100 μM) and antagonist naloxone (NAL; 100 μM) into the dorsal rostral pons on the phrenic neurogram were studied in a decerebrate, vagotomized, ventilated, paralyzed canine preparation during hyperoxia. A 1-mm grid pattern of microinjections was used. The DAMGO-sensitive region extended from 5 to 7 mm lateral of midline and from 0 to 2 mm caudal of the inferior colliculus at a depth of 3-4 mm. During remi-induced bradypnea (~72% reduction in fictive breathing rate) NAL microinjections (~500 nl each) within the region defined by the DAMGO protocol were able to reverse bradypnea by 47% (SD 48.0%) per microinjection, with 13 of 84 microinjections producing complete reversal. Histological examination of fluorescent microsphere injections shows that the sensitive region corresponds to the parabrachial/Kölliker-Fuse complex.

Discharge patterns of baroreceptor-modulated neurons in the nucleus tractus solitarius

Activity of baroreceptor-modulated neurons in the nucleus tractus solitarius (NTS) was recorded extracellularly during selective pressure stimulation of carotid baroreceptors, using an isolated carotid sinus preparation in anesthetized dogs. One of two different patterns of activity was recorded from individual baro-sensitive neurons in response to slow ramp increases in carotid sinus pressure. The cause of these two distinct firing patterns is not known but preliminary results indicate that it may be due in part to input from different functional types of baroreceptors. These results suggest that some differentiation in blood pressure control may be encoded in the responses of central baro-sensitive neurons in the NTS.

Effects of endocannabinoids on discharge of baroreceptive NTS neurons

Previously, we have shown that microinjection of endocannabinoids (ECBs) into the nucleus tractus solitarius (NTS) can modulate baroreflex control of blood pressure (BP), prolonging pressor-induced inhibition of renal sympathetic nerve activity. This suggests that ECBs can modulate excitability of baroreceptive neurons in the NTS. Studies by others have shown that neural cannabinoid (CB1) receptors are present on fibers in the NTS, suggesting that some presynaptic modulation of transmitter release could occur in this region which receives direct afferent projections from arterial baroreceptors and cardiac mechanoreceptors. This study, therefore, was performed to determine the effects of ECBs on NTS baroreceptive neuronal discharge. Picoinjection of the ECB anandamide (AEA) was found to significantly increase discharge of baroreceptive neurons (20 of 23). Picoinjection of the ECB uptake inhibitor, AM404, which enhances endogenous ECB activity, also significantly increased discharge of baroreceptive neurons (8 of 10 neurons). To determine if effects of ECBs involved a GABAA mechanism, the neuronal responses to AEA and AM404 were tested after prior blockade of postsynaptic GABAA receptors by bicuculline (BIC) or SR 95531 hydrobromide (gabazine--SR 95531), which would eliminate any effects due to modulation of GABA input. The increase in neuronal discharge to both AEA and AM404 was significantly attenuated following BIC or SR 95531, which alone significantly increased discharge of baroreceptive neurons tested. These results support the hypothesis that ECBs enhance baroreflex function through increases in NTS baroreceptive neuronal activity, due in part to modulation of GABAergic inhibitory effects at the neuronal level.

Differential activation of medullary vagal nuclei during different phases of swallowing in the cat

The aim of this study was to identify the medullary vagal nuclei involved in the different phases of swallowing activated physiologically in a species with an esophagus similar to human. In decerebrate cats, the pharyngeal (0.5-1.0 ml water in pharynx (N=6)) or esophageal (1-3 ml air in esophagus (N=5)) phases of swallowing were stimulated separately once per minute for 3 h, and we compared the resulting c-fos immunoreactivity within neuronal cell nuclei of the dorsal motor nucleus (DMN), nucleus tractus solitarius (NTS) and nucleus ambiguus (NA) with a sham control group (N=5). We found that the pharyngeal phase was associated with an elevated number of c-fos positive neurons in the intermediate (NTSim), interstitial (NTSis), ventromedial (NTSvm) subnuclei of the NTS, caudal DMN, and dorsal NA; and the esophageal phase was associated with an elevated number of c-fos positive neurons in the central (NTSce), ventral, dorsolateral, ventrolateral subnuclei of the NTS, rostral DMN, and ventral NA. We concluded that the pharyngeal and esophageal phases of swallowing are associated with different sets of NTS subnuculei; and the DMN and NA may contain functionally different populations of motor neurons situated rostrocaudally and dorsoventrally associated with the different phases of swallowing. The central pattern generator (CPG) for swallowing probably receives significant peripheral feedback, and the NTSvm may participate in the transition of the pharyngeal to the esophageal phase of swallowing.