Stuart J. McDougall

Primary afferent activation of thermosensitive TRPV1 triggers asynchronous glutamate release at central neurons.

TRPV1 receptors feature prominently in nociception of spinal primary afferents but are also expressed in unmyelinated cranial visceral primary afferents linked to homeostatic regulation. Cranial visceral afferents enter the brain at the solitary tract nucleus (NTS) to control the heart, lungs, and other vital organs. Here we identify a role for central TRPV1 in the activity-dependent facilitation of glutamatergic transmission from solitary tract (ST) afferents. Fast, synchronous ST-NTS transmission from capsaicin-sensitive (TRPV1+) and -insensitive (TRPV1-) afferents was similar. However, afferent activation triggered long-lasting asynchronous glutamate release only from TRPV1+ synapses. Asynchronous release was proportional to synchronous EPSC amplitude, activity, and calcium entry. TRPV1 antagonists and low temperature blocked asynchronous release, but not evoked EPSCs. At physiological afferent frequencies, asynchronous release strongly potentiated the duration of postsynaptic spiking. This activity-dependent TPRV1-mediated facilitation is a form of synaptic plasticity that brings a unique central integrative feature to the CNS and autonomic regulation.

Thermally Active TRPV1 Tonically Drives Central Spontaneous Glutamate Release

Central synapses spontaneously release neurotransmitter at low rates. In the brainstem, cranial visceral afferent terminals in caudal solitary tract nucleus (NTS) display pronounced, activity-dependent, asynchronous release of glutamate and this extra release depends on TRPV1 receptors (TRPV1+). Asynchronous release is absent for afferents lacking TRPV1 (TRPV1−) and resting EPSC frequency was greater in TRPV1+. Here, we studied this basal activity difference by assessing thermal sensitivity of spontaneous and miniature synaptic events in TRPV1+ and TRPV1− second-order NTS neurons. The spontaneous EPSC rate decreased when temperature was decreased, increased steeply between 30 and 42°C only in TRPV1+ neurons, and was calcium-dependent. TRPV1-specific antagonist SB366791, but not TTX, strongly attenuated thermal responses. Temperature changes failed to alter EPSC frequency in TRPV1− neurons. EPSC amplitudes and decay kinetics changed little with temperature. IPSCs in these second-order NTS neurons were unaltered by temperature. Such results suggest that activated, presynaptic TRPV1+ receptors trigger continuous resting release of glutamate vesicles at physiological temperatures only in capsaicin-responsive terminals. In mechanically isolated individual neurons harvested from medial NTS, increases in temperature increased the rate of glutamate release only in TRPV1+ neurons, whereas IPSC rates were unaffected. Cadmium failed to block thermal increases in glutamate release, suggesting that calcium entry through TRPV1 channels may trigger glutamate release independently of voltage-activated calcium channels. Together, our findings indicate a new form of afferent signaling in which TRPV1 channels within central terminals of peripheral afferents tonically generate glutamate release in NTS at 37°C in the absence of afferent action potentials.

Differential cardiovascular responses to stressors in hypertensive and normotensive rats

The aim of this study was to determine to what extent stress‐induced cardiovascular responses depend upon rat strain and/or stressor. Spontaneously hypertensive rats (SHRs), Wistar‐Kyoto rats (WKYs) and Sprague‐Dawley (SD) rats were implanted with telemetry probes in order to measure heart rate and blood pressure changes when exposed to a stressor. The stress protocols employed included handling, air‐jet and restraint, where each stressor was repeated over 10 consecutive days. In addition, a heterologous protocol was established whereby the experimental groups having experienced 10 days of air‐jet stress were then immediately exposed to 10 consecutive days of restraint. Each stressor caused graded tachycardic and pressor responses in all strains. For all strains, the magnitude and duration of heart rate and blood pressure increases were greatest in the restraint‐based protocols while handling and air‐jet caused submaximal changes. A comparison between strains indicated that SHRs exhibited prolonged pressor responses to each of the stressor types tested as compared to the normotensive strains. In addition, repeated exposure over 10 days to handling and air‐jet in SHRs caused tachycardic and/or pressor responses to adapt to ‘normotensive‐like’ levels. Heterologous restraint stress caused sensitization of cardiovascular responses upon first exposure, predominantly in normotensive strains. Collectively these data show that the magnitude and duration of the tachycardia and pressor responses evoked by the stressors were different within the strains and were also modified by prior experience. In addition, the cardiovascular profiles presented in this study demonstrate that, within each strain, the heart rate response during stress is graded according to the type of stressor encountered.

Medial prefrontal cortical integration of psychological stress in rats

The present study aimed to determine whether the medial prefrontal cortex (mPFC) (prelimbic and infralimbic regions) is implicated in the integration of a stress response. Sprague‐Dawely rats were implanted with telemetry probes and guide cannulae so that either muscimol or vehicle could be administered locally within the mPFC or dorsomedial hypothalamus (DMH). The heart rate and blood pressure of rats was continuously recorded as either muscimol or vehicle was administered centrally and rats were either exposed to restraint stress or left alone in their home cages. After the stress challenge, or equivalent period, rats that had received intra‐mPFC injections were processed for immunohistochemical detection of Fos throughout the neuraxis. Bilateral microinjection of muscimol into the mPFC had no effect upon either baseline cardiovascular parameters or restraint stress‐induced tachycardia or pressor responses whereas, in the DMH, pretreatment with muscimol attenuated the cardiovascular stress response. Analysis of Fos expression throughout the CNS of nonstressed rats showed no effect of muscimol injections into the mPFC on baseline expression in the nuclei examined. In contrast, rats that had received muscimol injections into their mPFC and were subsequently restrained exhibited an increase in the number of Fos‐positive cells in the DMH, medial amygdala, and medial nucleus tractus solitarius as compared to vehicle‐injected rats that experienced restraint stress. These results indicate that, during acute psychological stress, the mPFC does not modulate the cardiovascular system in rats but does inhibit specific subcortical nuclei to exert control over aspects of an integrated response to a stressor.

Convergence of Cranial Visceral Afferents within the Solitary Tract Nucleus

Primary afferent axons within the solitary tract (ST) relay homeostatic information via glutamatergic synapses directly to second-order neurons within the nucleus of the solitary tract (NTS). These primary afferents arise from multiple organ systems and relay multiple sensory modalities. How this compact network organizes the flow of primary afferent information will shape central homeostatic control. To assess afferent convergence and divergence, we recorded ST-evoked synaptic responses in pairs of medial NTS neurons in horizontal brainstem slices. ST shocks activated EPSCs along monosynaptic or polysynaptic pathways. Gradations in shock intensity discriminated multiple inputs and stimulus recruitment profiles indicated that each EPSC was unitary. In 24 pairs, 75% were second-order neurons with 64% receiving one direct ST input with the remainder receiving additional convergent ST afferent inputs (22% two; 14% three monosynaptic ST-EPSCs). Some (34%) second-order neurons received polysynaptic EPSCs. Neurons receiving only higher-order inputs were uncommon (13%). Most ST-EPSCs were completely independent, but 4 EPSCs of a total of 81 had equal thresholds, highly correlated latencies, and synchronized synaptic failures consistent with divergence from a single source ST axon or from a common interneuron producing a pair of polysynaptic EPSCs. We conclude that ST afferent inputs are remarkably independent with little evidence of substantial shared information. Individual cells receive highly focused information from the viscera. Thus, afferent excitation of second-order NTS neurons is generally dominated by single visceral afferents and therefore focused on a single afferent modality and/or organ region.

TRPV1 Marks Synaptic Segregation of Multiple Convergent Afferents at the Rat Medial Solitary Tract Nucleus

TRPV1 receptors are expressed on most but not all central terminals of cranial visceral afferents in the caudal solitary tract nucleus (NTS). TRPV1 is associated with unmyelinated C-fiber afferents. Both TRPV1+ and TRPV1- afferents enter NTS but their precise organization remains poorly understood. In horizontal brainstem slices, we activated solitary tract (ST) afferents and recorded ST-evoked glutamatergic excitatory synaptic currents (ST-EPSCs) under whole cell voltage clamp conditions from neurons of the medial subnucleus. Electrical shocks to the ST produced fixed latency EPSCs (jitter<200 µs) that identified direct ST afferent innervation. Graded increases in shock intensity often recruited more than one ST afferent and ST-EPSCs had consistent threshold intensity, latency to onset, and unique EPSC waveforms that characterized each unitary ST afferent contact. The TRPV1 agonist capsaicin (100 nM) blocked the evoked TRPV1+ ST-EPSCs and defined them as either TRPV1+ or TRPV1- inputs. No partial responses to capsaicin were observed so that in NTS neurons that received one or multiple (2–5) direct ST afferent inputs – all were either blocked by capsaicin or were unaltered. Since TRPV1 mediates asynchronous release following TRPV1+ ST-evoked EPSCs, we likewise found that recruiting more than one ST afferent further augmented the asynchronous response and was eliminated by capsaicin. Thus, TRPV1+ and TRPV1- afferents are completely segregated to separate NTS neurons. As a result, the TRPV1 receptor augments glutamate release only within unmyelinated afferent pathways in caudal medial NTS and our work indicates a complete separation of C-type from A-type afferent information at these first central neurons.

Characterisation of vasopressin V1A, angiotensin AT1 and AT2 receptor distribution and density in normotensive and hypertensive rat brain stem and kidney: effects of restraint stress1

In the present study, we have examined neurochemical correlates that may be involved in the differential cardiovascular responses observed in normotensive and hypertensive rats during stress. Using a restraint stress paradigm, both normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive rats (SHR) underwent acute (1 h restraint in a perspex tube), chronic (1 h restraint for ten consecutive days) or no restraint (control) stress. Following cessation of restraint, rats were processed by incubating sections of brain stem and kidney with [125I]-HO-LVA (0.03 nM) or [125I]Sar(1)Ile(8)-AngiotensinII (0.5 nM), in the presence of PD123319 (10 microM) or losartan (10 microM), to determine the distribution and density of vasopressin V(1A), angiotensin AT(1) and AT(2) receptors, respectively. Analysis of autoradiograms indicated changes in the density of radioligand binding in acutely and chronically-stressed rats, as compared to controls. For example, V(1A) binding in the medial nucleus tractus solitarius (SolM) decreased in the WKY but increased in the SHR. AT(1) binding in SolM did not significantly change in the WKY but decreased in the SHR with repeated restraint. In kidney slices, AT(1) binding decreased with stress in the WKY (-17%) but increased in SHR (+10-15%). AT(2) binding in the kidney showed a pattern similar to that of AT(1) binding in SHR, but not WKY. Graded increases in V(1A) binding were measured in kidney medulla and cortex of both strains (+50-60% with chronic restraint). These results suggest that physiological adaptation to restraint is associated with specific changes in V(1A), AT(1) and AT(2) receptor density within brain nuclei and kidney.

Nucleus incertus promotes cortical desynchronization and behavioral arousal.

Arousal and vigilance are essential for survival and relevant regulatory neural circuits lie within the brainstem, hypothalamus and forebrain. The nucleus incertus (NI) is a distinct site within the pontine periventricular gray, containing a substantial population of GABAergic neurons with long-range, ascending projections. Existing neuroanatomical data and functional studies in anesthetized rats, suggest the NI is a central component of a midline behavioral control network well positioned to modulate arousal, vigilance and exploratory navigation, yet none of these roles have been established experimentally. We used a chemogenetic approach—clozapine-N-oxide (CNO) activation of virally delivered excitatory hM3Dq-DREADDs—to activate the NI in rats and examined the behavioral and physiological effects, relative to effects in naïve rats and appropriate viral-treated controls. hM3Dq activation by CNO resulted in long-lasting depolarization of NI neurons with action potentials, in vitro. Peripheral injection of CNO significantly increased c-Fos immunoreactivity in the NI and promoted cortical electroencephalograph (EEG) desynchronization. These brain changes were associated with heightened arousal, and increased locomotor activity in the homecage and in a novel environment. Furthermore, NI activation altered responses in a fear conditioning paradigm, reflected by increased head-scanning, vigilant behaviors during conditioned fear recall. These findings provide direct evidence that the NI promotes general arousal via a broad behavioral activation circuit and support early hypotheses, based on its connectivity, that the NI is a modulator of cognition and attention, and emotional and motivated behaviors.

Isoflurane Differentially Modulates Inhibitory and Excitatory Synaptic Transmission to the Solitary Tract Nucleus

Background:Isoflurane anesthesia produces cardiovascular and respiratory depression, although the specific mechanisms are not fully understood. Cranial visceral afferents, which innervate the heart and lungs, synapse centrally onto neurons within the medial portion of the nucleus tractus solitarius (NTS). Isoflurane modulation of afferent to NTS synaptic communication may underlie compromised cardiorespiratory reflex function. Methods:Adult rat hindbrain slice preparations containing the solitary tract (ST) and NTS were used. Shocks to ST afferents evoked excitatory postsynaptic currents with low-variability (SEM <200 &mgr;s) latencies identifying neurons as second order. ST-evoked and miniature excitatory postsynaptic currents as well as miniature inhibitory postsynaptic currents were measured during isoflurane exposure. Perfusion bath samples were taken in each experiment to measure isoflurane concentrations by gas chromatography–mass spectrometry. Results:Isoflurane dose-dependently increased the decay-time constant of miniature inhibitory postsynaptic currents. At greater than 300 &mgr;m isoflurane, the amplitude of miniature inhibitory postsynaptic currents was decreased, but the frequency of events remained unaffected, whereas at equivalent isoflurane concentrations, the frequency of miniature excitatory postsynaptic currents was decreased. ST-evoked excitatory postsynaptic current amplitudes decreased without altering event kinetics. Isoflurane at greater than 300 &mgr;m increased the latency to onset and rate of synaptic failures of ST-evoked excitatory postsynaptic currents. Conclusions:In second-order NTS neurons, isoflurane enhances phasic inhibitory transmission via postsynaptic &ggr;-aminobutyric acid type A receptors while suppressing excitatory transmission through presynaptic mechanisms. These results suggest that isoflurane acts through multiple distinct mechanisms to inhibit neurotransmission within the NTS, which would underlie suppression of homeostatic reflexes.

Paired Assessment of Volatile Anesthetic Concentrations with Synaptic Actions Recorded In Vitro

The volatile anesthetic isoflurane poses a number of experimental challenges in the laboratory. Due to its rapid evaporation, the open conditions of most in vitro electrophysiological recording systems make the determination of actual isoflurane concentrations a challenge. Since the absolute anesthetic concentration in solution is directly related to efficacy, concentration measurements are important to allow comparisons between laboratory and clinical studies. In this study we quantify the sources of isoflurane loss during experimentation and describe a method for the measurement of isoflurane concentrations using gas chromatography and mass spectrometry simultaneous to in vitro electrophysiological measurements. Serial samples of perfused bath solution allowed correlation of isoflurane concentrations with ongoing biological effects. Saturated physiological solutions contained 13.4±0.2 mM isoflurane and were diluted to desired “nominal” concentrations for experiments. The perfusion system established stable isoflurane concentrations within the bath by 2 minutes. However, bath isoflurane concentrations varied substantially and unpredictably between experiments. The magnitudes of such discrepancies in isoflurane concentrations spanned clinically important levels. Our studies suggest that, despite countermeasures, solution handling significantly impacted the isoflurane content in the tissue bath. The magnitude of these discrepancies appears to necessitate systematic direct measurement of bath isoflurane concentrations during most in vitro conditions.