Brain stem control of arterial pressure in chronic arterial baroreceptor-denervated rats

Interruption of the baroreceptor reflex by transection of afferent nerves (sinoaortic denervation; SAD) or lesions of nucleus tractus solitarius (NTS) elevates sympathetic nerve activity (SNA) and arterial pressure (AP). However, within 1 wk, mean AP returns to normal despite the absence of baroreflexes. In this study, we examine central mechanisms that control AP in chronic baroreceptor-denervated rats. In urethane-anesthetized rats (1.5 g/kg i.v.) after autonomic ganglionic blockade (5 mg/kg i.v. chlorisondamine), alpha1-adrenergic-mediated pressor responses (1-100 microg/kg i.v. phenylephrine) were not altered by chronic lesions of NTS, indicating vascular reactivity to sympathetic stimulation is normal. Transection of the spinal cord at T1 profoundly decreased AP and was not further reduced by chlorisondamine in control or denervated rats. Inhibition of the rostral ventrolateral medulla (RVLM) by microinjections of muscimol (100 pmol/side) decreased AP to levels not further reduced by chlorisondamine in control rats, rats with SAD, and rats with NTS lesions. Blockade of GABA(A) receptors in the RVLM (50 pmol/side bicuculline) increased AP similarly in control rats and denervated rats. In agreement, inhibition of the caudal ventrolateral medulla (CVLM) by microinjections of muscimol or blockade of glutamatergic inputs (2.7 nmol/side kynurenate) produced comparable increases in AP in control and denervated rats. These data suggest the RVLM continues to drive the SNA that regulates AP in the chronic absence of baroreceptor inputs. In addition, despite the absence of a tonic excitatory input from NTS, in chronic baroreceptor-denervated rats glutamatergic inputs drive the CVLM to tonically inhibit the RVLM. Baroreceptor-independent regulation of the ventrolateral medulla may underlie central mechanisms contributing to the long-term control of AP.     

Exercise training attenuates increases in lumbar sympathetic nerve activity produced by stimulation of the rostral ventrolateral medulla.

Exercise training (ExTr) has been associated with blunted activation of the sympathetic nervous system in several animal models and in some human studies. Although these data are consistent with the hypothesis that ExTr reduces the incidence of cardiovascular diseases via reduced sympathoexcitation, the mechanisms are unknown. The rostral ventrolateral medulla (RVLM) is important in control of sympathetic nervous system activity in both physiological and pathophysiological states. The purpose of the present study was to test the hypothesis that ExTr results in reduced sympathoexcitation mediated at the level of the RVLM. Male Sprague-Dawley rats were treadmill trained or remained sedentary for 8-10 wk. RVLM microinjections were performed under Inactin anesthesia while mean arterial pressure, heart rate, and lumbar sympathetic nerve activity (LSNA) were recorded. Bilateral microinjections of the GABA(A) antagonist bicuculline (5 mM, 90 nl) into the RVLM increased LSNA in sedentary animals (169 +/- 33%), which was blunted in ExTr animals (100 +/- 22%, P < 0.05). Activation of the RVLM with unilateral microinjections of glutamate (10 mM, 30 nl) increased LSNA in sedentary animals (76 +/- 13%), which was also attenuated by training (26 +/- 2%, P < 0.05). Bilateral microinjections of the ionotropic glutamate receptor antagonist kynurenate (40 mM, 90 nl) produced small increases in mean arterial pressure and LSNA that were similar between groups. Results suggest that ExTr may reduce increases in LSNA due to reduced activation of the RVLM. Conversely, we speculate that the relatively enhanced activation of LSNA in sedentary animals may be related to the increased incidence of cardiovascular disease associated with a sedentary lifestyle.     

Differential role of ionotropic glutamatergic mechanisms in responses to NTS P(2x) and A(2a) receptor stimulation

Activation of ATP P(2x) receptors in the subpostremal nucleus tractus solitarii (NTS) via microinjection of alpha,beta-methylene ATP (alpha,beta-MeATP) elicits fast initial depressor and sympathoinhibitory responses that are followed by slow, long-lasting inhibitory effects. Activation of NTS adenosine A(2a) receptors via microinjection of CGS-21680 elicits slow, long-lasting decreases in arterial pressure and renal sympathetic nerve activity (RSNA) and an increase in preganglionic adrenal sympathetic nerve activity (pre-ASNA). Both P(2x) and A(2a) receptors may operate via modulation of glutamate release from central neurons. We investigated whether intact glutamatergic transmission is necessary to mediate the responses to NTS P(2x) and A(2a) receptor stimulation. The hemodynamic and neural (RSNA and pre-ASNA) responses to microinjections of alpha,beta-MeATP (25 pmol/50 nl) and CGS-21680 (20 pmol/50 nl) were compared before and after pretreatment with kynurenate sodium (KYN; 4.4 nmol/100 nl) in chloralose-urethan-anesthetized male Sprague-Dawley rats. KYN virtually abolished the fast responses to alpha,beta-MeATP and tended to enhance the slow component of the neural responses. The depressor responses to CGS-21680 were mostly preserved after pretreatment with KYN, although the increase in pre-ASNA was reduced by one-half following the glutamatergic blockade. We conclude that the fast responses to stimulation of NTS P(2x) receptors are mediated via glutamatergic ionotropic mechanisms, whereas the slow responses to stimulation of NTS P(2x) and A(2a) receptors are mediated mostly via other neuromodulatory mechanisms.     

Purinergic and glutamatergic interactions in the hypothalamic paraventricular nucleus modulate sympathetic outflow

P2X receptors are expressed on ventrolateral medulla projecting paraventricular nucleus (PVN) neurons. Here, we investigate the role of adenosine 5′-triphosphate (ATP) in modulating sympathetic nerve activity (SNA) at the level of the PVN. We used an in situ arterially perfused rat preparation to determine the effect of P2 receptor activation and the putative interaction between purinergic and glutamatergic neurotransmitter systems within the PVN on lumbar SNA (LSNA). Unilateral microinjection of ATP into the PVN induced a dose-related increase in the LSNA (1 nmol: 38 ± 6 %, 2.5 nmol: 72 ± 7 %, 5 nmol: 96 ± 13 %). This increase was significantly attenuated by blockade of P2 receptors (pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid, PPADS) and glutamate receptors (kynurenic acid, KYN) or a combination of both. The increase in LSNA elicited by L-glutamate microinjection into the PVN was not affected by a previous injection of PPADS. Selective blockade of non-N-methyl-D-aspartate receptors (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, CNQX), but not N-methyl-D-aspartate receptors (NMDA) receptors (DL-2-amino-5-phosphonopentanoic acid, AP5), attenuated the ATP-induced sympathoexcitatory effects at the PVN level. Taken together, our data show that purinergic neurotransmission within the PVN is involved in the control of SNA via P2 receptor activation. Moreover, we show an interaction between P2 receptors and non-NMDA glutamate receptors in the PVN suggesting that these functional interactions might be important in the regulation of sympathetic outflow.     

Sympathoinhibitory pathway from caudal midline medulla to RVLM is independent of baroreceptor reflex pathway

Glutamate stimulation of the caudal midline medulla (CMM) causes profound sympathoinhibition due to GABAergic inhibition of presympathetic neurons in the rostral ventrolateral medulla (RVLM). We investigated whether the sympathoinhibitory pathway from CMM to RVLM, like the central baroreceptor reflex pathway, includes a glutamatergic synapse in the caudal ventrolateral medulla (CVLM). In pentobarbital sodium-anesthetized rats, the RVLM on one side was inhibited by a muscimol microinjection. Then the response evoked by glutamate microinjections into the CMM or by baroreceptor stimulation was determined before and after 1) microinjection of the GABA receptor antagonist bicuculline into the RVLM on the other side or 2) microinjections of the glutamate receptor antagonist kynurenate bilaterally into the CVLM. Bicuculline in the RVLM greatly reduced both CMM- and baroreceptor-evoked sympathoinhibition. Compared with the effect of vehicle solution, kynurenate in the CVLM greatly reduced baroreceptor-evoked sympathoinhibition, whereas its effect on CMM-evoked sympathoinhibition was not different from that of the vehicle solution. These findings indicate that the output pathway from CMM sympathoinhibitory neurons, unlike the baroreceptor and other reflex sympathoinhibitory pathways, does not include a glutamatergic synapse in the CVLM.     

AT(1) and glutamatergic receptors in paraventricular nucleus support blood pressure during water deprivation

Water deprivation activates sympathoexcitatory neurons in the paraventricular nucleus (PVN); however, the neurotransmitters that mediate this activation are unknown. To test the hypothesis that ANG II and glutamate are involved, effects on blood pressure (BP) of bilateral PVN microinjections of ANG II type 1 receptor (AT1R) antagonists, candesartan and valsartan, or the ionotropic glutamate receptor antagonist, kynurenate, were determined in urethane-anesthetized water-deprived and water-replete male rats. Because PVN may activate sympathetic neurons via the rostral ventrolateral medulla (RVLM) and because PVN disinhibition increases sympathetic activity in part via increased drive of AT1R in the RVLM, candesartan was also bilaterally microinjected into the RVLM. Total blockade of the PVN with bilateral microinjections of muscimol, a GABA(A) agonist, decreased BP more (P < 0.05) in water-deprived (-29 +/- 8 mmHg) than in water-replete (-7 +/- 2 mmHg) rats, verifying that the PVN is required for BP maintenance during water deprivation. PVN candesartan slowly lowered BP by 7 +/- 1 mmHg (P < 0.05). In water-replete rats, however, candesartan did not alter BP (1 +/- 1 mmHg). Valsartan also produced a slowly developing decrease in arterial pressure (-6 +/- 1 mmHg; P < 0.05) in water-deprived but not in water-replete (-1 +/- 1 mmHg) rats. In water-deprived rats, PVN kynurenate rapidly decreased BP (-19 +/- 3 mmHg), and the response was greater (P < 0.05) than in water-replete rats (-4 +/- 1 mmHg). Finally, as in PVN, candesartan in RVLM slowly decreased BP in water-deprived (-8 +/- 1 mmHg; P < 0.05) but not in water-replete (-3 +/- 1 mmHg) rats. These data suggest that activation of AT(1) and glutamate receptors in PVN, as well as of AT1R in RVLM, contributes to BP maintenance during water deprivation.     

Modulation of respiratory responses to chemoreflex activation by L-glutamate and ATP in the rostral ventrolateral medulla of awake rats

Presympathetic neurons in the different anteroposterior aspects of rostral ventrolateral medulla (RVLM) are colocalized with expiratory [B?tzinger complex (B?tC)] and inspiratory [pre-B?tzinger complex (pre-B?tC)] neurons of ventral respiratory column (VRC), suggesting that this region integrates the cardiovascular and respiratory chemoreflex responses. In the present study, we evaluated in different anteroposterior aspects of RVLM of awake rats the role of ionotropic glutamate and purinergic receptors on cardiorespiratory responses to chemoreflex activation. The bilateral ionotropic glutamate receptors antagonism with kynurenic acid (KYN) (8 nmol/50 nl) in the rostral aspect of RVLM (RVLM/B?tC) enhanced the tachypneic (120 ± 9 vs. 180 ± 9 cpm; P < 0.01) and attenuated the pressor response (55 ± 2 vs. 15 ± 1 mmHg; P < 0.001) to chemoreflex activation (n = 7). On the other hand, bilateral microinjection of KYN into the caudal aspect of RVLM (RVLM/pre-B?tC) caused a respiratory arrest in four awake rats used in the present study. Bilateral P2X receptors antagonism with PPADS (0.25 nmol/50 nl) in the RVLM/B?tC reduced chemoreflex tachypneic response (127 ± 6 vs. 70 ± 5 cpm; P < 0.001; n = 6), but did not change the chemoreflex pressor response. In addition, PPADS into the RVLM/B?tC attenuated the enhancement of the tachypneic response to chemoreflex activation elicited by previous microinjections of KYN into the same subregion (188 ± 2 vs. 157 ± 3 cpm; P < 0.05; n = 5). Our findings indicate that: 1) L-glutamate, but not ATP, in the RVLM/B?tC is required for pressor response to peripheral chemoreflex and 2) both transmitters in the RVLM/B?tC are required for the processing of the ventilatory response to peripheral chemoreflex activation in awake rats.     

Putative role of the NTS in alterations in neural control of the circulation following exercise training in rats

Exercise training (ExTr) has been associated with alterations in neural control of the circulation, including effects on arterial baroreflex function. The nucleus tractus solitarius (NTS) is the primary termination site of cardiovascular afferents and critical in the regulation of baroreflex-mediated changes in heart rate (HR) and sympathetic nervous system outflow. The purpose of the present study was to determine whether ExTr is associated with alterations in neurotransmitter regulation of neurons involved in control of cardiovascular function at the level of the NTS. We hypothesized that ExTr would increase glutamatergic and reduce GABAergic transmission in the NTS and that, collectively, these changes would result in a greater overall sympathoinhibitory drive from the NTS in ExTr animals. To test these hypotheses, male Sprague-Dawley rats were treadmill trained or maintained under sedentary conditions for 8-10 wk. NTS microinjections were performed in Inactin-anesthetized animals instrumented to record mean arterial pressure (MAP), HR, and lumbar sympathetic nerve activity (LSNA). Generalized activation of the NTS with unilateral microinjections of glutamate (1-10 mM, 30 nl) produced dose-dependent decreases in MAP, HR, and LSNA that were unaffected by ExTr. Bilateral inhibition of NTS with the GABAA agonist muscimol (1 mM, 90 nl) produced increases in MAP and LSNA that were blunted by ExTr. In contrast, pressor and sympathoexcitatory responses to bilateral microinjections of the ionotropic glutamate receptor antagonist, kynurenate (40 mM, 90 nl), were similar between groups. Bradycardic responses to bilateral microinjections of the GABAA antagonist bicuculline (0.1 mM, 90 nl) were attenuated by ExTr. These data indicate that alterations in neurotransmission at the level of the NTS contribute importantly to regulation of HR and LSNA in ExTr animals. In addition to alterations at NTS, these experiments suggest indirectly that changes in other cardiovascular nuclei contribute to the observed alterations in neural control of the circulation following ExTr.     

Glutamate Transmission in the Rostral Ventrolateral Medullary Sympathetic Premotor Pathway

1. The aim of these studies was to test the hypothesis that glutamate is the principal excitatory neurotransmitter in the sympathetic premotor pathway from the rostral ventrolateral medulla (RVLM) to the sympathetic preganglionic neurons (SPNs) in the thoracic spinal cord.2. Iontophoretic and pressure ejection of glutamate receptor agonists and antagonists was made onto antidromically identified splanchnic and adrenal SPNs before and during electrical stimulation of the RVLM in urethane/chloralose-anesthetized, artificially ventilated rats.3. SPNs were excited by both NMDA and non-NMDA glutamate receptor agonists. Blockade of glutamate receptors in the IML interrupted the ability of electrical activation of sympathetic premotor neurons in the RVLM to excite SPNs. Within the IML, antergradely labeled terminals of RVLM neurons were found to contain glutamate immunoreactivity and to make asymmetric synapses on local dendrites.4. These data support a significant role for glutamate neurotransmission in mediating the tonic and phasic excitation of SPNs by the sympathetic premotor pathway from the RVLM. It seems likely that stimulation of the RVLM produces glutamate release from both C1 and non-PNMT-containing axon terminals in the IML.     

Differential role of nitric oxide in regional sympathetic responses to stimulation of NTS A2a adenosine receptors

Our previous studies showed that preganglionic adrenal (pre-ASNA), renal (RSNA), lumbar, and postganglionic adrenal sympathetic nerve activities (post-ASNA) are inhibited after stimulation of arterial baroreceptors, nucleus of the solitary tract (NTS), and glutamatergic and P2x receptors and are activated after stimulation of adenosine A1 receptors. However, stimulation of adenosine A2a receptors inhibited RSNA and post-ASNA, whereas it activated pre-ASNA. Because the effects evoked by NTS A2a receptors may be mediated via activation of nitric oxide (NO) mechanisms in NTS neurons, we tested the hypothesis that NO synthase (NOS) inhibitors would attenuate regional sympathetic responses to NTS A2a receptor stimulation, whereas NO donors would evoke contrasting responses from pre-ASNA versus RSNA and post-ASNA. Therefore, in chloralose/urethane-anesthetized rats, we compared hemodynamic and regional sympathetic responses to microinjections of selective A2a receptor agonist (CGS-21680, 20 pmol/50 nl) after pretreatment with NOS inhibitors Nomega-nitro-L-arginine methyl ester (10 nmol/100 nl) and 1-[2-(trifluoromethyl)phenyl]imidazole (100 pmol/100 nl) versus pretreatment with vehicle (100 nl). In addition, responses to microinjections into the NTS of different NO donors [40 and 400 pmol/50 nl sodium nitroprusside (SNP); 0.5 and 5 nmol/50 nl 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA NONOate, also known as NOC-18), and 2 nmol/50 nl 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA NONOate, also known as NOC-15)], the NO precursor L-arginine (10-50 nmol/50 nl), and sodium glutamate (500 pmol/50 nl) were evaluated. SNP, DETA NONOate, and PAPA NONOate activated pre-ASNA and inhibited RSNA and post-ASNA, whereas l-arginine and glutamate microinjected into the same site of the NTS inhibited all these sympathetic outputs. Decreases in heart rate and depressor or biphasic responses accompanied the neural responses. Pretreatment with NOS inhibitors reversed the normal depressor and sympathoinhibitory responses to stimulation of NTS A2a receptors into pressor and sympathoactivatory responses and attenuated the heart rate decreases; however, it did not change the increases in pre-ASNA. We conclude that NTS NO mechanisms differentially affect regional sympathetic outputs and differentially contribute to the pattern of regional sympathetic responses evoked by stimulation of NTS A2a receptors.     

Modulation of the cough reflex by antitussive agents within the caudal aspect of the nucleus tractus solitarii in the rabbit

We have previously shown that ionotropic glutamate receptors in the caudal portion of the nucleus tractus solitarii (NTS), especially in the commissural NTS, play a prominent role in the mediation of tracheobronchial cough and that substance P potentiates this reflex. This NTS region could be a site of action of some centrally acting antitussive agents and a component of a drug-sensitive gating mechanism of cough. To address these issues, we investigated changes in baseline respiratory activity and cough responses to tracheobronchial mechanical stimulation following microinjections (30-50 nl) of centrally acting antitussive drugs into the caudal NTS of pentobarbitone-anesthetized, spontaneously breathing rabbits. [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and baclofen decreased baseline respiratory frequency because of increases in the inspiratory time only at the higher concentration employed (5 mM and 1 mM, respectively). DAMGO (0.5 mM) and baclofen (0.1 mM) significantly decreased cough number, peak abdominal activity, peak tracheal pressure, and increased cough-related total cycle duration. At the higher concentrations, these agents suppressed the cough reflex. The effects of these two drugs were counteracted by specific antagonists (10 mM naloxone and 25 mM CGP-35348, respectively). The neurokinin-1 (NK1) receptor antagonist CP-99,994 (10 mM) abolished cough responses, whereas the NK2 receptor antagonist MEN 10376 (5 mM) had no effect. The results indicate that the caudal NTS is a site of action of some centrally acting drugs and a likely component of a neural system involved in cough regulation. A crucial role of substance P release in the mediation of reflex cough is also suggested.     

Is visceral sympathoexcitation to heat stress dependent on activation of ionotropic excitatory amino acid receptors in the rostral ventrolateral medulla?

Acute heat stress activates visceral sympathetic nerve discharge (SND) in young rats, and the functional integrity of the rostral ventrolateral medulla (RVLM) is required for sustaining visceral sympathoexcitation during peak increases in internal body temperature (T(c)). However, RVLM mechanisms mediating SND activation to hyperthermia remain unknown. In the present study, we investigated the role of RVLM ionotropic excitatory amino acid receptors in mediating visceral SND activation to heat stress in anesthetized, young rats. The effects of bilateral RVLM kynurenic acid (Kyn; 2.7 and 5.4 nmol), saline, or muscimol (400-800 pmol) microinjections on renal SND and splenic SND responses to heat stress were determined at peak hyperthermia (T(c) 41.5°C), during progressive hyperthermia (T(c) 40°C), and at the initiation of heating (T(c) increased from 38 to 38.5°C). RVLM Kyn microinjections did not reduce renal and splenic SND recorded during progressive or peak hyperthermia and did not attenuate SND activation at the initiation of heating. In fact, renal and splenic SND tended to be or were significantly increased following RVLM Kyn microinjections at the initiation of heating and during hyperthermia (40 and 41.5°C). RVLM muscimol microinjections at 39, 40, and 41.5°C resulted in immediate reductions in SND. These data indicate that RVLM ionotropic glutamate receptors are required for mediating visceral sympathoexcitation to acute heating and suggest that acute heating activates an RVLM ionotropic excitatory amino acid receptor dependent inhibitory input, which reduces the level of visceral SND to heating.     

Glutamate receptors in RVLM modulate sympathetic baroreflex in conscious rabbits

In this study, we examined the effect of excitatory amino acid (EAA) receptor blockade in the rostral ventrolateral medulla (RVLM) on the renal sympathetic baroreflex in conscious rabbits. Rabbits were implanted with guide cannulas for bilateral microinjections into the RVLM (+2 to +3 mm from the obex, n = 8) or into the intermediate ventrolateral medulla (IVLM; 0 to +1 mm from the obex, n = 5) and with an electrode for measuring renal sympathetic nerve activity (RSNA). After 7 days of recovery, microinjection of the EAA receptor antagonist kynurenate (10 nmol) into the RVLM did not affect resting RSNA or arterial pressure. Kynurenate decreased the gain of the RSNA baroreflex by 53% but did not change the reflex range. By contrast, injection of kynurenate into the IVLM increased resting arterial pressure and RSNA by 27 mmHg and 88%, respectively, but did not alter the RSNA baroreflex gain or range. Pentobarbital sodium anesthesia attenuated the gain and range of the RSNA baroreflex by 78 and 40%, respectively. Under these conditions, microinjection of kynurenate into the RVLM did not cause any further change in the gain of this reflex. These results suggest that endogenous EAA neurotransmitters in the RVLM are important in modulating the sympathetic baroreflex in conscious rabbits. Anesthesia can mask the functional significance of EAAs in the RVLM in modulating the baroreflexes, which may explain why previous studies in anesthetized animals found no effect of blocking EAA receptors in the RVLM on sympathetic baroreflexes.     

Modulation of ATP-induced LTP by cannabinoid receptors in rat hippocampus

Cannabinoids exert powerful action on various forms of synaptic plasticity. These retrograde messengers modulate GABA and glutamate release from presynaptic terminals by acting on presynaptic CB1 receptors. In particular, they inhibit long-term potentiation (LTP) elicited by electrical stimulation of excitatory pathways in rat hippocampus. Recently, LTP of the field excitatory postsynaptic potential (fEPSP) induced by exogenous ATP has been thoroughly explored. The present study demonstrates that cannabinoids inhibit ATP-induced LTP in hippocampal slices of rat. Administration of 10 μM of ATP led to strong inhibition of fEPSPs in CA1/CA3 hippocampal synapses. Within 40 min after ATP removal from bath solution, robust LTP was observed (fEPSP amplitude comprised 130.1 ± 3.8% of control, n = 10). This LTP never appeared when ATP was applied in addition to cannabinoid receptor agonist WIN55,212-2 (100 nM). Selective CB1 receptor antagonist, AM251 (500 nM), completely abolished this effect of WIN55,212-2. Our data indicate that like canonical LTP elicited by electrical stimulation, ATP-induced LTP is under control of CB1 receptors.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-012-9296-5) contains supplementary material, which is available to authorized users.     

Depressor and bradycardic responses to microinjections of endomorphin-2 into the NTS are mediated via ionotropic glutamate receptors

The presence of endomorphin-like immunoreactivity has been reported in the nucleus tractus solitarius (NTS). It was hypothesized that endomorphins may play a role in cardiovascular regulation in the medial subnucleus of the NTS (mNTS). Endomorphin-2 (E-2, 0.1-4 mmol/l) was microinjected (100 nl) into the mNTS of urethane-anesthetized, artificially ventilated, adult male Wistar rats. E-2 (0.2 mmol/l) elicited decreases in mean arterial pressure (40 +/- 3.5 mmHg) and heart rate (50 +/- 7.0 beats/min). These responses were blocked by prior microinjections of naloxonazine (1 mmol/l) into the mNTS. Responses to microinjections of E-2 into the mNTS were abolished by prior combined microinjections of d-2-amino-7-phosphonoheptanoic acid (an NMDA receptor antagonist, 5 mmol/l) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (a non-NMDA receptor antagonist, 2 mmol/l) into the mNTS. These results were confirmed by extracellular neuronal recordings. Blockade of GABA receptors in the mNTS by prior combined microinjections of gabazine (a GABA(A) receptor antagonist, 2 mmol/l) and 2-hydroxysaclofen (a GABA(B) receptor antagonist, 100 mmol/l) also blocked the responses to E-2. It was concluded that 1) the depressor and bradycardic responses to microinjections of E-2 into the mNTS are mediated via micro(1)-opioid receptors as well as ionotropic glutamate receptors, 2) GABAergic neurons in the mNTS, which may inhibit the release of glutamate from nerve terminals, are inhibited by E-2 via micro(1)-opioid receptors, and 3) disinhibition caused by the inhibition of GABAergic neurons by E-2 may result in an increase in the glutamate release from nerve terminals, which, in turn, may elicit depressor and bradycardic responses.     

Glutamatergic inputs to the CVLM independent of the NTS promote tonic inhibition of sympathetic vasomotor tone in rats

GABAergic neurons in the caudal ventrolateral medulla (CVLM) are driven by baroreceptor inputs relayed via the nucleus tractus solitarius (NTS), and they inhibit neurons in rostral ventrolateral medulla to reduce sympathetic nerve activity (SNA) and arterial pressure (AP). After arterial baroreceptor denervation or lesions of the NTS, inhibition of the CVLM continues to increase AP, suggesting additional inputs also tonically activate the CVLM. This study examined whether the NTS contributes to baroreceptor-independent drive to the CVLM and whether glutamate promotes baroreceptor- and NTS-independent activation of the CVLM to tonically reduce SNA. In addition, we evaluated whether altering central respiratory drive, a baroreceptor-independent regulator of CVLM neurons, influences glutamatergic inputs to the CVLM. Splanchnic SNA and AP were measured in chloralose-anesthetized, ventilated, paralyzed rats. The infusion of nitroprusside decreased AP below threshold for baroreceptor afferent firing (<50 mmHg) and increased SNA to 209+/-22% (P<0.05), but the subsequent inhibition of the NTS by microinjection of the GABA(A) agonist muscimol did not further increase SNA. In contrast, after inhibition of the NTS, blockade of glutamatergic inputs to CVLM by microinjection of kynurenate increased SNA (274+/-54%; P<0.05; n=7). In vagotomized rats with baroreceptors unloaded, inhibition of glutamatergic inputs to CVLM evoked a larger rise in SNA when central respiratory drive was increased (219+/-16% vs. 271+/-17%; n=5; P<0.05). These data suggest that baroreceptor inputs provide the major drive for the NTS-mediated excitation of the CVLM. Furthermore, glutamate tonically activates the CVLM to reduce SNA independent of the NTS, and this excitatory input appears to be affected by the strength of central respiratory drive.     

Leptin in nucleus of the solitary tract alters the cardiovascular responses to aortic baroreceptor activation

Recent data suggests that neurons expressing the long form of the leptin receptor form at least two distinct groups within the caudal nucleus of the solitary tract (NTS): a group within the lateral NTS (Slt) and one within the medial (Sm) and gelantinosa (Sg) NTS. Discrete injections of leptin into Sm and Sg, a region that receives chemoreceptor input, elicit increases in arterial pressure (AP) and renal sympathetic nerve activity (RSNA). However, the effect of microinjections of leptin into Slt, a region that receives baroreceptor input is unknown. Experiments were done in the urethane-chloralose anesthetized, paralyzed and artificially ventilated Wistar or Zucker obese rat to determine leptin's effect in Slt on heart rate (HR), AP and RSNA during electrical stimulation of the aortic depressor nerve (ADN). Depressor sites within Slt were first identified by the microinjection of l-glutamate (Glu; 0.25 M; 10 nl) followed by leptin microinjections. In the Wistar rat leptin microinjection (50 ng; 20 nl) into depressor sites within the lateral Slt elicited increases in HR and RSNA, but no changes in AP. Additionally, leptin injections into Slt prior to Glu injections at the same site or to stimulation of the ADN were found to attenuate the decreases in HR, AP and RSNA to both the Glu injection and ADN stimulation. In Zucker obese rats, leptin injections into NTS depressor sites did not elicit cardiovascular responses, nor altered the cardiovascular responses elicited by stimulation of ADN. Those data suggest that leptin acts at the level of NTS to alter the activity of neurons that mediate the cardiovascular responses to activation of the aortic baroreceptor reflex.     

Differential cardiorespiratory control elicited by activation of ventral medullary sites in mice.

We studied the respiratory and blood pressure responses to chemical stimulation of two regions of the ventral brainstem in mice: the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). Stimulation of the RVLM by microinjections of the excitatory amino acid L-glutamate induced increases in diaphragm activity and breathing frequency, elevation of blood pressure (BP), and a slight increase in heart rate (HR). However, activation of the CVLM induced a decrease in breathing frequency, mainly due to prolongation of expiratory time (TE), and hypotension associated with a slight slowing of HR. Because adrenergic mechanisms are known to participate in the control of respiratory timing, we examined the role of alpha(2)-adrenergic receptors in the RVLM region in mediating these inhibitory effects. The findings demonstrated that blockade of the alpha(2)-adrenergic receptors within the RVLM by prior microinjection of SKF-86466 (an alpha(2)-adrenergic receptor blocker) significantly reduced changes in TE induced by CVLM stimulation but had little effect on BP responses. These results indicate that, in mice, activation of the RVLM increases respiratory drive associated with an elevation of BP, but stimulation of CVLM induces prolongation of TE via an alpha(2)-adrenergic signal transduction pathway.     

Activation of NTS A1 adenosine receptors inhibits regional sympathetic responses evoked by activation of cardiopulmonary chemoreflex

Previously we have shown that adenosine operating via the A(1) receptor subtype may inhibit glutamatergic transmission in the baroreflex arc within the nucleus of the solitary tract (NTS) and differentially increase renal (RSNA), preganglionic adrenal (pre-ASNA), and lumbar (LSNA) sympathetic nerve activity (ASNA>RSNA≥LSNA). Since the cardiopulmonary chemoreflex and the arterial baroreflex are mediated via similar medullary pathways, and glutamate is a primary transmitter in both pathways, it is likely that adenosine operating via A(1) receptors in the NTS may differentially inhibit regional sympathetic responses evoked by activation of cardiopulmonary chemoreceptors. Therefore, in urethane-chloralose-anesthetized rats (n = 37) we compared regional sympathoinhibition evoked by the cardiopulmonary chemoreflex (activated with right atrial injections of serotonin 5HT(3) receptor agonist phenylbiguanide, PBG, 1-8 μg/kg) before and after selective stimulation of NTS A(1) adenosine receptors [microinjections of N(6)-cyclopentyl adenosine (CPA), 0.033-330 pmol/50 nl]. Activation of cardiopulmonary chemoreceptors evoked differential, dose-dependent sympathoinhibition (RSNA>ASNA>LSNA), and decreases in arterial pressure and heart rate. These differential sympathetic responses were uniformly attenuated in dose-dependent manner by microinjections of CPA into the NTS. Volume control (n = 11) and blockade of adenosine receptor subtypes in the NTS via 8-(p-sulfophenyl)theophylline (8-SPT, 1 nmol in 100 nl) (n = 9) did not affect the reflex responses. We conclude that activation of NTS A(1) adenosine receptors uniformly inhibits neural and cardiovascular cardiopulmonary chemoreflex responses. A(1) adenosine receptors have no tonic modulatory effect on this reflex under normal conditions. However, when adenosine is released into the NTS (i.e., during stress or severe hypotension/ischemia), it may serve as negative feedback regulator for depressor and sympathoinhibitory reflexes integrated in the NTS.     

Parabrachial nucleus induces suppression of baroreflex bradycardia by the release of glutamate in the rostral ventrolateral medulla of the rat

The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla (RVLM) in the suppression of baroreflex bradycardia by the parabrachial nucleus (PBN) was investigated. Repeated electrical activation of the PBN increased the concentration of glutamate in the dialysate collected from the RVLM. The same stimulation also suppressed baroreflex bradycardia in response to transient hypertension evoked by phenylephrine (5 microg/kg, intravenously). Microinfusion of L-glutamate (10, 50 or 100 microM) via the microdialysis probe into the RVLM dose-dependently elicited a significant inhibition of baroreflex bradycardia that paralleled the concentration and time course of the PBN-elicited elevation in extracellular glutamate in the RVLM. The suppression of baroreflex bradycardia elicited by microinjection of L-glutamate (1 nmol) into the RVLM was appreciably reversed by coinjection of the NMDA receptor antagonist, dizocilpine (500 pmol), or the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione (50 pmol). These results suggest that an increase in the extracellular concentration of glutamate and activation of both NMDA and non-NMDA receptors in the RVLM may mediate the suppression of baroreflex bradycardia by activation of the PBN.