Electrophysiological study of paraventricular nucleus neurons projecting to the dorsomedial medulla and their response to baroreceptor stimulation in rats

In male rats anesthetized with urethane, extracellular recordings were made from 415 neurons in the paraventricular nucleus (PVN) and adjacent areas. Of these neurons 64 were excited antidromically by stimulation of the dorsomedial medulla but not by stimulation of the pituitary stalk (first group). Seventy-three neurons were antidromically excited by stimulation of the pituitary stalk but not of the dorsomedial medulla (second group, neurosecretory cells). The other 2 neurons were antidromically excited by stimulation of both the dorsomedial medulla and the pituitary stalk (third group). Latencies of antidromically evoked action potentials by stimulation of the dorsomedial medulla and of the pituitary stalk ranged between 8 and 60 ms (mean +/- S.D., 38.5 +/- 9.8, n = 66) and from 7 to 24 ms (mean +/- S.D., 13.0 +/- 3.6, n = 75), respectively, suggesting unmyelinated fiber projections in both instances. PVN neurons of these 3 groups were found to be dispersed throughout the PVN and no difference in specific locations between the neuron groups existed. Their characteristics, however, were different. The first group of neurons discharged at a slower rate and showed no phasic pattern of firing, while 28% of the second group of neurons ('identified' neurosecretory cells) showed phasic patterns of firing and their rates of discharge were higher than those of the first group of neurons. The two neurons belonging to the third group showed irregular spontaneous discharges. The areas within the dorsomedial medulla stimulation of which evoked antidromic excitation of PVN neurons were located within and adjacent to the nucleus of the tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMV). Among PVN neurons which were antidromically excited by stimulation of dorsomedial medulla, 51 cells were examined for their responses to excitation of baroreceptors. An increase in pressure of the 'isolated' carotid sinus excited 2 neurons, and inhibited 7 (14%). On the other hand, 27% (11 out of 41) of neurosecretory cells (second group) were inhibited by baroreceptor stimulation. From these results, it was concluded that essentially separate populations of PVN neurons project to the neurohypophysis and to the NTS, DMV and their vicinities, and that some of the caudally-projecting PVN neurons receive synaptic input from carotid baroreceptor reflex pathway, suggesting the possible involvement of these PVN neurons in central cardiovascular regulation.     

Cholecystokinin activates catecholaminergic neurons in the caudal medulla that innervate the paraventricular nucleus of the hypothalamus in rats

Stimulation of gastric vagal afferents by systemic administration of cholecystokinin octapeptide (CCK) inhibits gastric motility, reduces food intake, and stimulates pituitary secretion of oxytocin and adrenocorticotropic hormone in rats. To characterize further the central neurol circuits responsible for these effects, the present study used triple-labeling immunocytochemical methods to determine whether or not exogenous CCK activates cFos expression in catecholaminergic neurons in the caudal medulla that project to the paraventricular nucleus of the hypothalamus (PVN). To identify these neurons, the retrograde tracer fluorogold (FG) was iontophoresed into the PVN of anesthetized rats under stereotaxic guidance. After 2 weeks, rats were injected with CCK (100 μg/kg, i. p.) and then anesthetized and killed 1 hour later by perfusion fixation. Medullary sections were processed for triple immunocytochemical localization of cFos, retrogradely transported FG, and tyrosine hydroxylase (TH). In rats with FG injections centered in the PVN (n = 10), approximately 70% of the FG-labeled neurons in the caudal nucleus of the solitary tract (NST) and ventrolateral medulla (VLM) expressed cFos. Of these activated PVN-projecting neurons, approximately 78% in the NST and 89% in the VLM were catecholaminergic (TH positive). These results indicate that PVN-projecting catecholaminergic neurons within the caudal medulla are activated by periph eral administration of CCK, further implicating these ascending catecholaminergic path ways in the neuroendocrine, physiological, and behavioral effects produced by gastric vagal stimulation. © 1995 Wiley-Liss, Inc.     

Electrophysiological identification of neurons in ventrolateral medulla sending collateral axons to paraventricular and supraoptic nuclei in the cat

In recent studies, it has been reported that high-frequency stimulation restricted to A alpha beta fibers in man can be perceived as painful and evoke a nociceptive flexion reflex. These results would indicate that some patterns of activity in low-threshold mechanoreceptors can lead to painful sensations. Because of the theoretical importance of this question, the above studies were extended by recording the evoked neural activity with the technique of percutaneous microneurography. Painful sensations and the nociceptive reflex did not appear unless the evoked nerve response contained activity in A delta fibers. The results support the theory that painful sensations occur in normal man only when nociceptor afferents are activated.     

A comparison of hypotensive and non-hypotensive hemorrhage on Fos expression in spinally projecting neurons of the paraventricular nucleus and rostral ventrolateral medulla

The protein, Fos, detected immunohistochemically, was used to identify neurons in the brain that were activated after hemorrhage in the conscious rat. Spinally projecting neurons in the paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) were identified by the presence of rhodamine-labeled latex beads which had been previously injected into the upper thoracic spinal cord. On the experimental day, conscious rats underwent either (1) withdrawal of 4 ml of blood from a carotid cannula (n = 8) which reduced mean arterial pressure from 96.6 ± 2.7 to 42.7 ± 7.1mmHg, (2) withdrayl of 2 ml of blood (n = 4) which did not affect mean arterial pressure. Animals that were not hemorrhaged were used as controls (n = 6). After the 4 ml hemorrhage, dense concentrations of Fos-positive cells nuclei were found in the lamina terminalis, supraoptic nuclei (SON), PVN and in the medulla. In contrast, the density of Fos-positive cells in 2 ml-hemorrhaged rats was not different from controls except in the SON and in the medial PVN in 2 of 4 rats. After the 4 ml hemorrhage 14.4 ± 1.2% of the spinally projecting neurons in the PVN and 22.7 ± 6.1% in the RVLM expressed Fos (P < 0.001 compared to control). After the 2 ml hemorrhage the proportion was 12.2 ± 3.1% in the PVN (P < 0.001 compared control) but only 5.4 ± 2.2% in the RVLM (P > 0.05 compared to control) . The results suggest that spinally projecting neurons in the PVN and RVLM participate in the reflex responses to hemorrhage. PVN-spinal neurons may respond to changes in blood volume even when arterial pressure does not alter.     

Neurons in the hypothalamic paraventricular nucleus that project to the rostral ventrolateral medulla are activated by haemorrhage

The retrogradely-transported tracer, rhodamine-tagged microspheres, was injected into the pressor region of the rostral ventrolateral medulla (RVLM) to identify paraventricular neurons in the hypothalamus that project to the RVLM. The protein, Fos, was detected immunohistochemically and used to highlight neurons that were activated by a hypotensive haemorrhage. Compared to controls, Fos production was increased by approximately 3-fold in the paraventricular nucleus (P<0.009) and there was a significant increase in the number of retrogradely-labelled cells that expressed Fos. These represented 5% of the retrogradely-labelled cell population. The results suggest that a small subpopulation of PVN neurons projecting to the RVLM are activated by haemorrhage and may be involved in the reflex responses initiated by that stimulus.     

Neurons in the hypothalamic paraventricular nucleus that project to the rostral ventrolateral medulla are not activated by hypotension

The retrogradely-transported tracer, rhodamine-tagged microspheres was injected into the pressor region of the rostral ventrolateral medulla (RVLM) to enable detection of paraventricular neurons in the hypothalamus that project to the RVLM. The protein, Fos, was detected immunohistochemically and used to highlight neurons that were activated by hypotension (−16±5 mmHg) induced by diazoxide (30 mg/kg s.c.). Compared to controls, Fos production was increased by three-fold in the parvocellular paraventricular nucleus but there was no significant increase in the number of retrogradely-labelled cells that expressed Fos. The results suggest paraventricular nucleus (PVN) neurons projecting to the RVLM are not activated by hypotension.     

Electrophysiological identification of neurons in the parabrachial nucleus projecting directly to the hypothalamus in the rat

Experiments were done in urethane anesthetized rats to identify single units in the region of the parabrachial nucleus (PBN) projecting directly to ‘cardiovascular’ responsive sites in either the paraventricular nucleus of the hypothalamus (PVH) or the supraoptic commissure and nucleus (SOC-SON) region. Fifty-five single units were antidromically activated in the ipsilateral PBN by electrical stimulation of either the PVH (n = 27) or SOC-SON region (n = 28) with latencies corresponding to conduction velocities of 0.3–5.1 m/s. The axons of PBN units projecting to the PVH conducted at significantly slower velocities (0.5 ± 0.04m/s) than those projecting to the SOC-SON region (1.6 ± 0.25m/s). These data suggest that aacending fibers from the PBN to the PVH are unmyelinated, whereas those to the SOC-SON region are primarily a little myelinated. In addition, since the PBN is known to receive cardiovascular and visceral afferent inputs, it is suggested that these neurons likely function in relaying this afferent information to hypothalamic areas involved in autonomic regulation.     

Direct pathway from neurons in the ventrolateral medulla relaying cardiovascular afferent information to the supraoptic nucleus in the cat

In chloralose anesthetized cats experiments were done to electrophysiologically identify neurons in the ventrolateral medulla (VLM) which relay cardiovascular afferent information directly to the supraoptic nucleus (SON). Action potentials elicited antidromically by electrical stimulation of the SON were recorded from 69 histologically verified single units in the VLM. Single units responded with latencies corresponding to conduction velocities of 7.8 ± 0.6m/s. Of these units 26 were excited orthodromically by stimulation of the buffer nerves; 12 responded to stimulation of only the carotid sinus nerve, 7 responded to stimulation of only the aortic depressor nerve, and 7 responded to both buffer nerves. The axons of VLM units that responded to buffer nerves conducted at a significantly slower velocity than those of non-responsive units (5.7 ± 0.4and9.1 ± 0.8m/s, respectively). These data provide electrophysiological evidence of two different populations of VLM neurons which project directly to the SON, and suggest that the direct pathway from the VLM to the SON is involved in the release of vasopressin by SON neurons during activation of baroreceptor and chemoreceptor afferent fibers.     

Differential responses of barosensitive neurons of rostral ventrolateral medulla to hypoxia in rats

We examined the responses to hypoxia of 48 spontaneously active barosensitive neurons of the rostral ventrolateral medulla (RVL) of anesthetized rats. Twenty-nine projected to the spinal cord while 19 did not. All spinal barosensitive neurons increased their discharges in advance of an elevation of arterial pressure in the presence or absence of arterial chemoreceptors. In contrast, 18/19 of the non-spinal barosensitive neurons were not excited by hypoxia. The results indicate that barosensitive RVL neurons consist of two populations differing in efferent pathway and responsivity to hypoxia and that the spinal barosensitive RVL neurons are functionally discrete and selectively sensitive to hypoxia.     

The ventrolateral medulla as a source of synaptic drive to rhythmically firing neurons in the cardiovascular nucleus tractus solitarius of the rat

We sought to determine whether the caudal ventrolateral medulla (cVLM), at the level of area postrema, influences the rhythmically beating neurons found within the dorsomedial NTS in rat brainstem slices. Intra- or extracellular recordings of neurons firing rhythmically at around 5 Hz were characterized as either auto-active (i.e. pacemaker; AA) or synaptically driven (SD) by pharmacological interventions. The nature of inputs evoked from the ipsilateral cVLM were orthodromic and the majority were excitatory (latency 3-20 ms). Further, this excitatory influence was found to be tonically active in 25/47 cells studied since inactivating the ipsilateral cVLM by localized cooling reduced the firing rate by 0.5-3.0 Hz (23% on average). Neuronal characterization showed that the most consistent and pronounced effect occurred on SD rather than AA cells. Control experiments that cooled other areas of the slice closer to the recording site proved ineffective. Additional studies showed that most rhythmically firing cells in the NTS received an excitatory synaptic input from the solitary tract (ts; latency 3-30 ms). This input was reduced or blocked by inactivating the cVLM in neurons in which the ts latency of activation was greater than 8 ms in half of the neurons tested. Subsequent pharmacological tests revealed that these neurons were predominantly SD. Identified AA neurons received an input from the ts at a shorter latency, typically less than 8 ms, and this was unperturbed by cooling the cVLM in all cases. Further, there was no obvious difference in the baseline discharge rates between cells in the hemi-slice and those recorded in an intact slice. In a hemi-coronal slice cooling the cVLM also produced a 20% decrease in firing rate in identified SD neurons but no consistent change in AA cells. We conclude that (1) the ipsilateral cVLM contributes principally tonic excitatory drive to rhythmically active neurons in the dorsomedial NTS in vitro and this preferentially effects SD neurons; (2) other excitatory drives other than those from the ipsilateral cVLM impinge upon SD cells, the origin of which are relatively local and likely to be in the NTS; (3) in the slice the projection from the cVLM to the NTS appears to be present but the reciprocal connection is absent.     

大鼠头端延髓腹外侧区前交感神经元的电生理研究

采用细胞外记录方法,研究氨基甲酸乙酯麻醉大鼠头端延髓腹外侧区前交感神经元的电生理特性。观察到１２５个ＲＶＬＭ神经元中有２４个的自发放电被电刺激主动脉神经和静脉注射苯肾上腺素后抑制,同时其放电具有心必节律,以上２４个中有１８个单位能被 脊髓侧索电刺激逆行激活。     

Difference in distribution of glutamate-immunoreactive neurons projecting into the subretrofacial nucleus in the rostral ventrolateral medulla of SHR and WKY: a double-labeling study

Glutamate immunoreactivity was found in 19% and 21% of the neurons of the central autonomic nuclei projecting into the subretrofacial nucleus (SRF) in the rostral ventrolateral medulla of Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR), respectively, using a double-labeling technique in combination with glutamate immunocytochemistry. Double-labeled neurons were distributed in 22 nuclei or subnuclei in the limbic system, hypothalamus, midbrain, pons and medulla. The average number of glutamate-immunoreactive neurons per thousand in SHR was significantly higher in the ipsilateral lateral parabrachial nucleus (P < 0.05) and Koelliker-Fuse nucleus (P < 0.01) than in WKY, while it was significantly lower in the ipsilateral medial subnucleus (P < 0.05) and the commissure subnucleus (P < 0.05) of the nucleus tractus solitarii in SHR than in WKY. The results indicate that: (1) glutamate-immunoreactive neurons (possibly glutamatergic) in many central autonomic nuclei project into the sympathetic vasomotor control neurons in the SRF; (2) the large population of glutamate-immunoreactive neurons in the lateral parabrachial nucleus and the Koelliker-Fuse nucleus of SHR is likely to increase excitatory inputs to the SRF vasomotor control neurons, while the smaller population of glutamate-immunoreactive neurons in the medial and commissure subnuclei of the nucleus tractus solitarii is likely to decrease excitatory inputs to the GABAergic neurons intrinsic to the SRF.     

Evidence for involvement of the lateral parabrachial nucleus in mediation of cholinergic inputs to neurons in the rostral ventrolateral medulla of the rat

We examined whether sites in the lateral parabrachial nucleus (PBN) where

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-glutamate produced increases in arterial pressure were involved in mediation of cholinergic inputs to neurons in the rostral ventrolateral medulla (RVLM). Male Wistar rats were anesthetized, paralyzed and artificially ventilated. Unilateral microinjection of

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-glutamate into the lateral PBN produced a pressor response. Microinjection of the muscarinic receptor antagonist scopolamine into the unilateral RVLM inhibited the pressor response to

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-glutamate injected ipsilaterally into the lateral PBN, whereas microinjection of the cholinesterase inhibitor physostigmine into the RVLM enhanced it. PBN microinjection of

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-glutamate also enhanced the firing rate of RVLM sympathoexcitatory neurons and the enhancement of the firing rate was inhibited by scopolamine iontophoretically applied on neurons. PBN injection of

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-glutamate produced a tetrodotoxin (TTX)-sensitive release of ACh in the RVLM. Unilateral microinjection of TTX into the lateral PBN inhibited the pressor response induced by RVLM microinjection of physostigmine. These results provide evidence that neurons in the pressor sites of the lateral PBN are involved in mediation of cholinergic inputs responsible for pressor responses in the RVLM.     

Synaptic contacts of substance P-immunoreactive axon terminals in the nucleus tractus solitarius onto neurons projecting to the caudal ventrolateral medulla oblongata in the rat

The possibility that substance P (SP)-immunoreactive axon terminals in the nucleus tractus solitarius (NTS) make synaptic contacts onto NTS neurons projecting to the catecholaminergic cell region in the caudal ventrolateral medulla oblongata (CVLM) was examined in the rat using a retrograde tract-tracing method combined with immunohistochemistry. After injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex (WGA-HRP-gold), into the CVLM region where tyrosine hydroxylase-immunoreactive neurons were situated, many retrogradely labeled neurons were detected in the dorsal parts of the NTS, especially at levels between 1.0 mm caudal and 0.5 mm rostral to the obex. Immunoelectron microscopy revealed synaptic contacts between SP-immunoreactive axon terminals and WGA-HRP-gold-labeled neurons in the NTS. These findings indicated that SP regulates NTS neurons which project to the catecholaminergic cell region of the CVLM. ©1997 Elsevier Science B.V. All rights reserved.     

皮质酮对大鼠头端延髓腹外侧区伤害调制性神经元的作用

在以氨基甲酸乙酯麻醉的SD大鼠上,用多管微电极细胞外记录头端髓腹外侧区（RVLM）神经元的自发放电,在RVLM内共记录到87个自发放电的神元。伤害刺激（有齿镊钳夹同侧后肢皮肤）后放电频率发生迅速变化的57个神经元被认为是伤害调制性神经元放电快速抑制,而4个（11%）神经元兴奋,余7个（20%）神经元没有反应,在伤害刺激所抑制的22个伤害抑制性神经元。微电泳CORT后3个（14%）神经元放电抑制14个（64%）神经元兴奋,余5个（22%0神经元没有反应。另30个非伤害调制性神经元中,微电泳CORT引起12个（40%）神经元兴奋,10个（32%）神经元抑制,余8个（28%）的神经元没有反应,以上结果证明CORT可能通过非基因组机制快速影响RVLM神经元的活动,提示在应激等情况下CORT的快速作用机制在一行性的伤害抑制、应激性麻醉等抗伤害活动的整合中具有重要意义。     

Monosynaptic connection from caudal to rostral ventrolateral medulla in the baroreceptor reflex pathway

Experiments were done to test the hypothesis that caudal ventrolateral medulla (CVLM) neurons excited by activation of arterial baroreceptors and by stimulation of depressor sites in the nucleus tractus solitarii (NTS) project monosynaptically to the rostral ventrolateral medulla (RVLM). In urethan anaesthetized and artificially ventilated rats we recorded extracellular activity from 46 spontaneously firing units in the CVLM. Twenty of these units were excited by baroreceptor activation (1–3 μg phenylephrine i.v.) and of these 6 were excited (mean latency of9.8 ± 2.3ms) by single pulses (0.1 ms,30 ± 8.3 μA) delivered once per second to a depressor site in the ipsilateral NTS. These 6 units were also antidromically activated with a latency of4.1 ± 0.12ms by stimulation of a pressor region in the ipsilateral RVLM. These results provide evidence for the existence of an excitatory projection from the NTS to the CVLM which, in turn, projects monosynaptically to sympathoexcitatory neurons in the RVLM.     

Connections of neurons in the region of the nucleus tractus solitarius with the hypothalamic paraventricular nucleus: Their possible involvement in neural control of the cardiovascular system in rats

Extracellular recordings were made from 607 spontaneously firing neurons within the nucleus tractus solitarius (NTS) and its vicinity in urethane-anesthetized male rats. Following electrical stimulation of the hypothalamic paraventricular nucleus (PVN) area, 21% of the neurons were orthodromically excited, 6% were inhibited and 2.5% were antidromically activated. The antidromic spike latencies were 22-64 ms. Among those orthodromically responding neurons, 81 neurons were tested by pressure pulse stimulation of the isolated carotid sinus. The pressure stimulation produced excitation in 7 and inhibition in 13 neurons. Of the 8 tested neurons which were antidromically activated, one neuron was excited and another neuron inhibited by the pressure pulse stimulation. These results provide electrophysiological evidence for reciprocal connections between neurons in the NTS region and the PVN, and give support to the hypothesis that the PVN is involved in the neural control of the cardiovascular system.     

Bidirectional cardiovascular connections between ventrolateral medulla and nucleus of the solitary tract

Single-unit recording experiments were done in chloralose-anesthetized, paralyzed and artificially ventilated cats to identify neurons in ventrolateral medulla (VLM) that send efferent axons directly to the region of the nucleus of the solitary tract (NTS) and receive cardiovascular afferent inputs from the carotid sinus (CSN) and aortic depressor (ADN) nerves and the NTS. Units in VLM were identified by antidromic excitation to stimulation of functionally and histologically verified sites in the NTS complex. Antidromic potentials were recorded from 34 units in VLM. Units responded with a mean antidromic latency of 4.37 +/- 0.32 ms corresponding to a mean conduction velocity of 0.93 +/- 0.07 m/s. Of these 34 units, 18 were excited orthodromically by stimulation of the CSN and/or ADN. Furthermore, 10 of the 18 units responding to stimulation of the buffer nerves were also orthodromically excited by stimulation of NTS. An additional 76 units were identified in VLM that only responded orthodromically to stimulation of NTS with a mean latency of 9.75 +/- 2.93 ms, of which 33 also responded orthodromically to stimulation of the buffer nerves. These data provide electrophysiological evidence of a bidirectional connection between neurons in VLM that receive and integrate peripheral cardiovascular afferent inputs and send efferent axons directly back to the region of NTS. These results suggest that neurons in the VLM may be part of a medullary feedback reflex loop through which afferent information from cardiovascular receptors exerts an influence on NTS neurons involved in the control of the circulation.     

Cardiovascular afferent inputs to neurons in the ventrolateral medulla projecting directly to the central autonomic area of the thoracic cord in the cat

Experiments were done in chloralosed, paralyzed and artificially ventilated cats to identify single units in the ventrolateral medulla (VLM) projecting directly to the central autonomic area of the thoracic cord (CA) and responding to peripheral and central inputs carrying cardiovascular information. Forty-three single units were antidromically activated in the VLM to stimulation of either ipsilateral or contralateral CA with latencies corresponding to conduction velocities of 27.5 ± 2.0m/s. Of these 43 units, only 14 (33%) responded orthodromically to stimulation of either the carotid sinus nerve (CSN) or of pressor sites in the paraventricular nucleus of the hypothalamus (PVM) or both. These experiments have demonstrated a bilateral projection of VLM neurons to the CA and have provided evidence for their role in integrating and mediating cardiovascular information from the CNS and PVH directly to spinal sympathetic centers.     

Membrane and synaptic properties of nucleus tractus solitarius neurons projecting to the caudal ventrolateral medulla

Projections from the nucleus of tractus solitarius (NTS) to the caudal ventrolateral medulla (CVLM) are important in mediating autonomic reflexes. However, little is known about the cellular properties of the CVLM-projecting NTS neurons. In this study, the CVLM-projecting NTS neurons were retrogradely labeled by fluorescent microspheres injected into the CVLM. Whole cell voltage- and current-clamp recordings were performed on labeled NTS neurons in coronal brainstem slices. Compared with unlabeled neurons, the labeled NTS neurons had more depolarized resting membrane potentials, larger input resistance, and higher firing activity in response to depolarizing currents. Bath application of an ionotropic glutamate receptor antagonist kynurenic acid and a non-NMDA receptor antagonist CNQX significantly decreased the firing activity in the majority of labeled NTS neurons. In contrast, an NMDA receptor antagonist AP5 failed to alter the firing activity in labeled neurons tested. While the glycine receptor antagonist strychnine had no effect on the firing activity, blockade of GABA(A)receptors with bicuculline significantly increased the firing rate in the majority of labeled NTS neurons. Furthermore, CNQX blocked the majority of spontaneous excitatory postsynaptic currents (EPSCs) and evoked EPSCs elicited by stimulation of the tractus solitarius. The residual spontaneous and evoked EPSCs were abolished by the nicotinic receptor antagonist mecamylamine and the purinergic P2X receptor antagonist iso-PPADS. Finally, while bicuculline completely blocked the miniature inhibitory postsynaptic currents (IPSCs), the spontaneous and evoked IPSCs were abolished by a combination of bicuculline and strychnine in labeled NTS neurons. Collectively, these data suggest that the CVLM-projecting neurons are a population of neurons with distinctive membrane properties.