Bilateral Microinjections Of Muscimol Research Articles

CHARACTERIZING THE ROLE OF THE DORSOMEDIAL HYPOTHALAMIC NUCLEUS IN THE CARDIOGENIC SYMPATHETIC AFFERENT REFLEX IN RATS

Myocardial ischemia causes the production and release of metabolites such as bradykinin (BK), which stimulates cardiac spinal sensory afferents causing chest pain and an increase in sympathetic activity. This sympathetic reflex, which increases heart rate and blood pressure, is referred to as the cardiogenic sympathetic afferent reflex (CSAR). During myocardial ischemia, this sympathetic reflex activity is important for the maintenance of blood pressure and perfusion of vital organs. However, the increased sympathetic activity may also be detrimental because it further increases the oxygen demand of the ischemic myocardium. While the brain stem nuclei, such as the nucleus tractus solitarius and rostral ventrolateral medulla have been shown to play an important role in the CSAR, the role of other supramedullary nuclei and the sources that generate sympathetic activity in the CSAR are still not clear. We have previously characterized the role of the paraventricular nucleus (PVN) of the hypothalamus in the CSAR and shown that acute chemical inhibition of the PVN attenuates the CSAR. However, we also have shown that the CSAR recovers in rats after chronic ibotenic acid lesion of the PVN suggesting that other supramedullary sites are involved. The dorsomedial hypothalamic nucleus (DMH) is another important hypothalamic site that plays an important role in cardiovascular sympathetic regulation. However, little is known regarding the role of the DMH in the CSAR. In this study, we tested the hypothesis that “inhibition of the DMH attenuates the CSAR in rats”. Renal sympathetic nerve activity (RSNA) and blood pressure were recorded in anesthetized rats during epicardial application of BK (10 μg/ml). Bilateral microinjection of muscimol, a GABAA agonist (0.5 nmol in 50 nl), was used to inhibit the DMH. Vehicle injection into the DMH had no significant effect (n=6, P > 0.05) on blood pressure or RSNA. In vehicle‐injected rats, epicardial BK application significantly increased (P < 0.05) RSNA to 204.4 ±35.08% of baseline and mean arterial pressure from 103.4 ±2.02 mmHg to 118.1 ±4.73 mmHg. In a sperate group of rats (n=8) muscimol injection into the DMH significantly (P < 0.05) reduced basal blood pressure from 98.0 ±4.66 mmHg to 77.1 ±7.33 mmHg but had no significant effect (P > 0.05) on baseline RSNA. In this group of rats, epicardial BK application significantly increased (P < 0.05) RSNA to 193.9 ±28.00% of baseline and increased mean arterial pressure from 77.1 ±7.33 mmHg to 89.29 ±8.48 mmHg. Whereas muscimol injection into the DMH appears to attenuate the overall magnitude of the blood pressure response it had no effect on the RSNA response to epicardial bradykinin. These results suggest that in the presence of intact spinal and medullary circuits inhibition of DMH does not play an important role in the generation of sympathetic activity in the CSAR. Whereas inhibition of DMH does not appear to impair the CSAR there is good evidence to show that disinhibition of DMH increases basal vasomotor activity and augment reflex sympathetic responsiveness. Thus, our next objective is to determine if activation of DMH augments the CSAR and if so to determine the medullary connections of these DMH neurons.Support or Funding InformationNHLBI R15HL145645‐01

Inhibition of Medial Preoptic Area Improves Physical Performance in Trained Rats

The medial preoptic area (MPO) plays a key role in the regulation of autonomic thermoeffectors and this area seems to be more activated after physical training, as evidenced by greater number of exercise‐induced c‐Fos immunoreactive neurons in trained rats. This observation raised the question as to whether inhibition of MPO can affect physical performance and thermoregulatory responses in trained rats. Following 2 weeks of high‐intensity interval training (HIIT), Wistar rats were submitted to an incremental‐speed exercise on a motor‐driven treadmill until fatigue after bilateral microinjection of muscimol (80 pmol/100 nl) to evaluate their aerobic capacity and thermoregulatory responses. The core body temperature (Tc) and the maximal oxygen uptake (VO2 max) were recorded continuously during exercise. Physical training increased the time to fatigue (43 ± 2 min vs. 64 ± 2 min; p=0.0009; n=6); the maximum speed attained during the incremental exercise (24 ± 1 m/min vs. 31 ± 1 m/min; p=0.0013), the distance traveled (675 ± 36 m vs. 1261 ± 68 m; p=0.0008); and resulted in a higher oxygen uptake (38 ± 1 ml/min/kg vs. 42 ± 1 ml/min/kg; p=0.01). Interestingly, at fatigue, no difference in the absolute Tc was found between the groups (38.28 ± 0.2 °C vs. 38.78 ± 0.1 °C; p=0.1). Bilateral blockade of MPO using muscimol improved physical performance, as observed by the increased time to fatigue (71 ± 6 min muscimol vs. 58 ± 2 min ‐ control, p=0.004; n=3); distance traveled (1528 ± 188 m muscimol vs. 1094 ± 84 m control; p=0.05); and maximum oxygen uptake (49 ± 1 ml/min/kg muscimol vs. 44 ± 2 ml/min/kg control; p=0.04). Regarding the thermoregulatory adjustments, MPO inhibition tended to increase Tc at fatigue (40.37 ± 0.5 °C muscimol vs. 39.35 ± 0.2 °C ‐ control; p=0.08). There was an increase in the Tc of the control animals during exercise but this temperature remained lower than that of the muscimol group (p<0.001). Our findings show that neurons in the MPO are involved in the control of the aerobic capacity of trained rats, however, the improved physical performance of these animals caused by MPO inhibition did not result from changes in thermoregulatory adjustments.Support or Funding InformationConselho Nacional de Desenvolvimento Científico e Tecnlógico ‐ CNPq; grant 434646/2016‐4This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role played by periaqueductal gray neurons in parasympathetically mediated fear bradycardia in consciousrats.

Freezing, a characteristic pattern of defensive behavior elicited by fear, is associated with a decrease in the heart rate. Central mechanisms underlying fear bradycardia are poorly understood. The periaqueductal gray (PAG) in the midbrain is known to contribute to autonomic cardiovascular adjustments associated with various emotional behaviors observed during active or passive defense reactions. The purpose of this study was to elucidate the role played by PAG neurons in eliciting fear bradycardia. White noise sound (WNS) exposure at 90 dB induced freezing behavior and elicited bradycardia in conscious rats. The WNS exposure‐elicited bradycardia was mediated parasympathetically because intravenous administration of atropine abolished the bradycardia (P < 0.05). Moreover, WNS exposure‐elicited bradycardia was mediated by neuronal activation of the lateral/ventrolateral PAG (l/vlPAG) because bilateral microinjection of muscimol, a GABAA agonist, into the l/vlPAG significantly suppressed the bradycardia. It is noted that muscimol microinjected bilaterally into the dorsolateral PAG had no effect on WNS exposure‐elicited bradycardia. Furthermore, retrograde neuronal tracing experiments combined with immunohistochemistry demonstrated that a number of l/vlPAG neurons that send direct projections to the nucleus ambiguus (NA) in the medulla, a major origin of parasympathetic preganglionic neurons to the heart, were activated by WNS exposure. Based on these findings, we propose that the l/vlPAG‐NA monosynaptic pathway transmits fear‐driven central signals, which elicit bradycardia through parasympathetic outflow.

GABA and glycine receptors in the nucleus ambiguus mediate tachycardia elicited by chemical stimulation of the hypothalamic arcuate nucleus.

We have previously reported that stimulation of the hypothalamic arcuate nucleus (ARCN) by microinjections of N-methyl-d-aspartic acid (NMDA) elicits tachycardia, which is partially mediated via inhibition of vagal inputs to the heart. The neuronal pools and neurotransmitters in them mediating tachycardia elicited from the ARCN have not been identified. We tested the hypothesis that the tachycardia elicited from the ARCN may be mediated by inhibitory neurotransmitters in the nucleus ambiguus (nAmb). Experiments were done in urethane-anesthetized, artificially ventilated, male Wistar rats. In separate groups of rats, unilateral and bilateral microinjections of muscimol (1 mM), gabazine (0.01 mM), and strychnine (0.5 mM) into the nAmb significantly attenuated tachycardia elicited by unilateral microinjections of NMDA (10 mM) into the ARCN. Histological examination of the brains showed that the microinjections sites were within the targeted nuclei. Retrograde anatomic tracing from the nAmb revealed direct bilateral projections from the ARCN and hypothalamic paraventricular nucleus to the nAmb. The results of the present study suggest that tachycardia elicited by stimulation of the ARCN by microinjections of NMDA is mediated via GABAA and glycine receptors located in the nAmb.

The dorsomedial hypothalamus (DMH) plays a strain-dependent role in vascular responses to cocaine but not cold stress in conscious rats

s / Autonomic Neuroscience: Basic and Clinical 163 (2011) 1–133 56 P.039 The dorsomedial hypothalamus (DMH) plays a strain-dependent role in vascular responses to cocaine but not cold stress in conscious rats M.M. Knuepfer, M. Busauskas (St. Louis University School of Medicine Pharmacol. & Physiol. Science, USA) We demonstrated that cocaine or cold water stress (CWS) elicits systemic vasoconstriction in Brown-Norway (BN) rats but an increase in cardiac output in Dahl salt sensitive (DSS) rats. Since the DMH plays an important role in stress responsiveness, we sought to determine the role of the DMH to these disparate stressors on regional vascular hemodynamics. We hypothesized that inhibition of the DMH would attenuate greater hindquarters vasoconstriction to either stress in BN compared to DSS rats. BN and DSS rats were instrumented for determination of arterial pressure, heart rate, andwith pulsed Doppler flowprobes on the superiormesenteric and abdominal aorta. Bilateral guide cannulaswere implanted above the DMH. After recovery, we observed that cocaine (5 mg/kg, iv) elicited greater hindquarters andmesenteric vasoconstriction and greater bradycardia in BN rats compared toDSS rats. In contrast, CWS (1 cm deep for 1 min) elicited tachycardia in all rats and greater increases in mesenteric resistance in BN rats but hindquarters vasodilation that was greater in BN compared to DSS rats. Therefore, visceral and skeletalmuscle vascular responses differed between strains and stressors. We examined the dependence of these responses on the DMH with bilateral microinjections of muscimol (80 pmol in 100 nl) in theDMH.Muscimol attenuated thegreaterhindquarters andmesenteric vasoconstriction and tachycardia in BN rats without affecting responses in DSS rats in response to cocaine. Responses to CWSwere unaffected by muscimol except that tachycardia was selectively reduced in BN rats. Kainate (10 pmol) in the DMH elicited a similar response pattern as CWS. These studies demonstrate that the DMH plays a strainand stimulus-dependent role in mediating hemodynamic responses to stress. Our data suggest that there are significant variations in central neural pathways mediating stress that vary in different rat strains.

Asymmetrical changes in lumbar sympathetic nerve activity following stimulation of the sciatic nerve in rat

Noxious stimulation of the leg increases hind limb blood flow (HBF) to the ipsilateral side and decreases to the contralateral in rat. Whether or not this asymmetrical response is due to direct control by sympathetic terminals or mediated by other factors such as local metabolism and hormones remains unclear. The aim of this study was to compare responses in lumbar sympathetic nerve activity, evoked by stimulation of the ipsilateral and contralateral sciatic nerve (SN). We also sought to determine the supraspinal mechanisms involved in the observed responses. In anesthetized and paralyzed rats, intermittent electrical stimulation (1 mA, 0.5 Hz) of the contralateral SN evoked a biphasic sympathoexcitation. Following ipsilateral SN stimulation, the response is preceded by an inhibitory potential with a latency of 50 ms ( N = 26). Both excitatory and inhibitory potentials are abolished following cervical C1 spinal transection ( N = 6) or bilateral microinjections of muscimol ( N = 6) in the rostral ventrolateral medulla (RVLM). This evidence is suggestive that both sympathetic potentials are supraspinally mediated in this nucleus. Blockade of RVLM glutamate receptors by microinjection of kynurenic acid ( N = 4) selectively abolished the excitatory potential elicited by ipsilateral SN stimulation. This study supports the physiological model that activation of hind limb nociceptors evokes a generalized sympathoexcitation, with the exception of the ipsilateral side where there is a withdrawal of sympathetic tone resulting in an increase in HBF.

The viscerosympathetic response in rabbits is mediated by GABAergic and glutamatergic inputs into the sympathetic premotor neurons of the rostral ventrolateral medulla

Neurons in the rostral ventrolateral medulla (RVLM) receive inputs from various sources, including baroreceptors, and then regulate the activity of sympathetic preganglionic neurons in the spinal cord. Whether RVLM neurons mediate the viscerosympathetic reflex has yet to be clarified. In the present study, we investigated the role of RVLM neurons in the viscerosympathetic reflex in anaesthetized and vagotomized rabbits. Electrical stimulation of the greater splanchnic nerve (SplN) evoked reflex responses in renal sympathetic activity that were composed of inhibitory and/or excitatory components. Bilateral microinjection of muscimol, a GABA(A) receptor agonist, into the RVLM blocked the reflex responses. Bilateral microinjection of bicuculline, a GABA(A) receptor antagonist, largely attenuated the inhibitory component, whereas kynurenic acid, a glutamate receptor antagonist, eliminated the excitatory component. The activities of 21 RVLM barosensitive bulbospinal neurons were recorded. Twenty of the neurons responded to the SplN stimulation. The responses also consisted of inhibitory and/or excitatory components. The excitatory component of these neurons preceded that of the renal sympathetic nerve activity by about 100 ms. This latency difference was almost the same as that of the inhibitory responses evoked by aortic nerve stimulation. Therefore, the renal sympathetic reflex responses evoked by SplN stimulation are mediated by RVLM neurons, and GABAergic and glutamatergic transmission in the RVLM are related to this reflex.

The hypothalamic paraventricular nucleus is required for insulin's action to increase baroreflex gain of lumbar sympathetic nerve activity

During pregnancy, low brain insulin levels are associated with impaired baroreflex control of heart rate (HR) and sympathetic nerve activity (SNA), and intracerebral insulin infusion normalizes baroreflex gain (BRG). The specific site of insulin action is unknown, though a likely candidate is the paraventricular nucleus (PVN). To test this hypothesis, BRG of lumbar SNA (LSNA) and HR were obtained in chloralose anesthetized female rats before and at 30min intervals during iv infusion of insulin (15mU/kg/min, n=5) or saline (n=5). Insulin increased LSNA (P&lt;0.05; in % baseline) to 166±18 (30min), 224±43 (60min), 219±46 (90min), and 227±49 (120min). Similarly, insulin increased LSNA BRG (P&lt;0.05; in %LSNA/mmHg) from 2.6±0.8 to 2.9±0.7 (30min), 4.5±0.9 (60min), 4.3±0.5 (90min), and 4.7±0.6 (120min). Insulin did not affect basal HR or HR BRG. No changes in LSNA, HR or BRG were observed with saline infusion. In another group (n=4), the effect of insulin to increase LSNA and LSNA gain was reversed by blockade of PVN via bilateral microinjection of muscimol (M; 60nL of 1mM). LSNA (% baseline) increased to 223±37 after insulin and fell to 126±11 after M (P&lt;0.05). LSNA gain (%LSNA/mmHg) increased from 2.8±1.3 to 4.4±1.2, and decreased to 2.4±0.7 after M (P&lt;0.05). In summary, iv insulin increased LSNA and LSNA gain. This action was reversed by PVN blockade, suggesting that insulin acts via the PVN to improve BRG. Support: the EMBL/GenBank Data Libraries under Accession No. HL088552

Neural circuits underlying the inhibition of sympathetically‐mediated thermogenesis in brown adipose tissue (BAT) induced by atypical antipsychotic drugs

Administration of atypical antipsychotic drugs suppresses sympathetic activation of metabolism in BAT, which may contribute to the weight gain observed with maintenance on these medications. This study investigated the neural circuitry responsible for the antipsychotic drug‐evoked inhibition of the sympathetic outflow to BAT. In urethane/chloralose anesthetized rats, the increase in BAT sympathetic nerve activity (SNA) evoked by cooling the core temperature to ~35.5°C was completely inhibited by the atypical antipsychotic drug, Clozapine (Cloz; 3mg/kg, iv). Bilateral microinjection of muscimol (2mM, 120nl/side) into the ventrolateral medulla (VLM) reversed or prevented the Cloz‐evoked inhibition of BAT SNA. In contrast to the effect of Cloz on the cooling response, the increase in BAT SNA evoked by blockade of GABAA receptors within the rostral raphe pallidus (RPa) was not inhibited by Cloz (3–6mg/kg, iv). These results suggest that neurons within the VLM play a role in the inhibition of BAT SNA evoked by atypical antipsychotic drugs, possibly by activating a GABAergic input to the RPa. Supported by NIH grant DK082558 and an American Heart Association SDG.

Cardiovascular function of a glutamatergic projection from the hypothalamic paraventricular nucleus to the nucleus tractus solitarius in the rat

Experiments were done in urethane-anesthetized, barodenervated, male Wistar rats. Chemical stimulation of the hypothalamic paraventricular nucleus (PVN) by unilateral microinjections of N-methyl- d-aspartic acid (NMDA) elicited increases in mean arterial pressure (MAP) and greater splanchnic nerve activity (GSNA). The increases in the MAP and GSNA induced by chemical stimulation of the PVN were significantly exaggerated by bilateral microinjections of d(−)-2-amino-7-phosphono-heptanoic acid ( d-AP7) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydro-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) (ionotropic glutamate receptor antagonists) into the medial subnucleus of the nucleus tractus solitarius (mNTS). These results were confirmed by single unit recordings; i.e. excitation of mNTS barosensitive neurons caused by chemical stimulation of the ipsilateral PVN was blocked by application of d-AP7 and NBQX to these neurons. Bilateral microinjections of d-AP7 and NBQX into the mNTS elicited pressor responses which were significantly attenuated by inhibition of PVN neurons by bilateral microinjections of muscimol. Unilateral microinjections of fluorogold into the mNTS resulted in bilateral retrograde labeling of the PVN neurons. Unilateral microinjections of biotinylated dextran amine into the PVN resulted in anterograde labeling of axons and terminals in the mNTS bilaterally and the labeled terminals exhibited vesicular glutamate transporter-2 immunoreactivity. These results indicated that 1) a tonically active glutamatergic bilateral projection from the PVN to the mNTS exists; 2) bilateral blockade of ionotropic glutamate receptors in the mNTS exaggerates the increases in MAP and GSNA, but not heart rate, to the chemical stimulation of the PVN; and 3) this projection may serve as a restraint mechanism for excitatory cardiovascular effects of PVN stimulation.

Unreinforced spatial (latent) learning is mediated by a circuit that includes dorsal entorhinal cortex and fimbria fornix

The relationship of the entorhinal cortex (EC) and fimbria-fornix (FF) in unreinforced spatial (latent) learning was studied using the conditioned-cue-preference task on an eight-arm radial maze. The maze was turned before every trial to eliminate the use of local cues. During three pre-exposure sessions, food-deprived rats explored the center platform and two adjacent arms of the maze. Since most of the same cues were visible from both arm locations, discriminating them required spatial learning. The rats were then alternately confined to the end of each arm over several days: one arm always contained food, the other was empty. Finally, the rats were allowed free access to both arms with no food present. Normal rats spent more time in their food-paired than in their unpaired arms showing that they learned to discriminate between the arm locations. Bilateral micro-injections of muscimol into the dorsal, but not into the ventral EC, given before the pre-exposure sessions only, impaired the discrimination. The discrimination was also impaired in rats with unilateral lesions of FF and contralateral injections of muscimol into the dorsal EC given before the pre-exposure sessions. Ipsilateral FF lesions and entorhinal inactivation had no effect. These results indicate that the acquisition of information during unreinforced exploration of a novel environment requires an intact circuit involving the dorsal EC and fimbria fornix. Together with previous reports, that this form of learning does not require a functional hippocampus, (Gaskin et al. (2005) Hippocampus 15:1085-1093) the findings also suggest that the acquisition of certain kinds of unreinforced information by this circuit is independent of the hippocampus.

AT1 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.

Cardiovascular effects produced by activation of GABA receptors in the rostral ventrolateral medulla of conscious rats

The rostral ventrolateral medulla (RVLM) has been proposed as a region playing a major role in the tonic and reflex control of sympathetic vasomotor activity and blood pressure. Pharmacological activation of GABA A receptors with muscimol in the RVLM of anesthetized rats results in a large fall in mean arterial pressure (MAP), heart rate (HR) and sympathetic activity. In this study we evaluated the effects of activation of GABA receptors in the RVLM of conscious, freely moving rats. Bilateral microinjections of muscimol into the RVLM of conscious rats produced a large fall in MAP (−38±4 mm Hg, n=7) when compared with saline injections (NaCl 0.9%, 7±1 mm Hg, n=4). The decrease in MAP evoked by muscimol was accompanied by a significant increase in HR (muscimol 69±13 bpm vs. vehicle −33±12 bpm, P<0.05), an effect that was completely abolished by beta1 adrenergic receptor blockade. Conversely, bilateral microinjections of GABA B agonist, baclofen, evoked a pressor response, but in this case, the increase was not significantly different from that evoked by vehicle injections. These results 1) indicate that GABA A receptors have a powerful influence on the resting activity of RVLM neurons in conscious rats; 2) indicate that a compensatory sympathetic-mediated tachycardia is present after inhibition of RVLM neurons in conscious rats; 3) confirm and extend previous findings showing that RVLM neurons are critical for blood pressure maintenance even in normal non-anesthetized conditions.

Central efferent pathways mediating skin cooling-evoked sympathetic thermogenesis in brown adipose tissue

Control of thermoregulatory effectors by the autonomic nervous system is a critical component of rapid cold-defense responses, which are triggered by thermal information from the skin. However, the central autonomic mechanism driving thermoregulatory effector responses to skin thermal signals remains to be determined. Here, we examined the involvement of several autonomic brain regions in sympathetic thermogenic responses in brown adipose tissue (BAT) to skin cooling in urethane-chloralose-anesthetized rats by monitoring thermogenic [BAT sympathetic nerve activity (SNA) and BAT temperature], metabolic (expired CO(2)), and cardiovascular (arterial pressure and heart rate) parameters. Acute skin cooling, which did not reduce either rectal (core) or brain temperature, evoked increases in BAT SNA, BAT temperature, expired CO(2), and heart rate. Skin cooling-evoked thermogenic, metabolic, and heart rate responses were inhibited by bilateral microinjections of bicuculline (GABA(A) receptor antagonist) into the preoptic area (POA), by bilateral microinjections of muscimol (GABA(A) receptor agonist) into the dorsomedial hypothalamic nucleus (DMH), or by microinjection of muscimol, glycine, 8-OH-DPAT (5-HT(1A) receptor agonist), or kynurenate (nonselective antagonist for ionotropic excitatory amino acid receptors) into the rostral raphe pallidus nucleus (rRPa) but not by bilateral muscimol injections into the lateral/dorsolateral part or ventrolateral part of the caudal periaqueductal gray. These results implicate the POA, DMH, and rRPa in the central efferent pathways for thermogenic, metabolic, and cardiac responses to skin cooling, and suggest that these pathways can be modulated by serotonergic inputs to the medullary raphe.

Central pathways mediating skin cooling‐evoked sympathetic activation of brown adipose tissue thermogenesis

In the regulation of body temperature in homeothermic animals, thermal afferents from the skin provide an important signal allowing rapid autonomic responses in peripheral thermoregulatory effectors to changes in ambient temperature. The preoptic area (POA) receives thermal information from the skin and sends efferent signals that alter peripheral effector activity to maintain body temperature. However, the central pathways mediating such autonomic responses to skin thermal information remain to be determined. In this study, we examined electrophysiologically the involvement of several autonomic brain regions in sympathetic thermogenic responses in brown adipose tissue (BAT) to skin cooling in urethane-chloralose-anesthetized rats. Cooling the skin (32–35°C) with a water jacket for 1–2 min, which did not affect rectal (core) or brain temperature, evoked increases in BAT sympathetic nerve activity (SNA). The skin cooling-evoked activation of BAT SNA was blocked by bilateral microinjections of muscimol, a GABAA receptor agonist, into the dorsomedial hypothalamic nucleus (DMH), but not by those into the ventrolateral part of the caudal periaqueductal gray. Furthermore, injection of muscimol or 8-OH-DPAT, a 5-HT1A receptor agonist, into the rostral raphe pallidus nucleus (rRPa) also blocked the skin cooling-evoked BAT SNA. These results suggest that the DMH and rRPa are involved in BAT sympathetic thermogenic responses to skin cooling, which are mediated by efferent neuronal pathways probably from the POA, and that these pathways can be modulated by serotonergic inputs to the medullary raphe. Supported by NIH grant NS40987.

Direct pyrogenic input from prostaglandin EP3 receptor‐expressing preoptic neurons to the dorsomedial hypothalamus

Fever is induced by a neuronal mechanism in the brain. Prostaglandin (PG) E2 acts as a pyrogenic mediator in the preoptic area (POA) probably through the EP3 subtype of PGE receptor expressed on GABAergic neurons, and this PGE2 action triggers neuronal pathways for sympathetic thermogenesis in peripheral effector organs including brown adipose tissue (BAT). To explore pyrogenic efferent pathways from the POA, we determined projection targets of EP3 receptor-expressing POA neurons with a special focus on rat hypothalamic regions including the dorsomedial hypothalamic nucleus (DMH), which is known as a center for autonomic responses to stress. Among injections of cholera toxin b-subunit (CTb), a retrograde tracer, into hypothalamic regions at the rostrocaudal level of the DMH, injections into the DMH, lateral hypothalamic area (LH) and dorsal hypothalamic area (DH) resulted in EP3 receptor immunolabelling in substantial populations of CTb-labeled neurons in the POA. Bilateral microinjections of muscimol, a GABA(A) receptor agonist, into the DMH and a ventral region of the DH, but not those into the LH, inhibited thermogenic (BAT sympathetic nerve activity, BAT temperature, core body temperature and expired CO2) and cardiovascular (arterial pressure and heart rate) responses to an intra-POA PGE2 microinjection. Further immunohistochemical observations revealed a close association of POA-derived GABAergic axon swellings with DMH neurons projecting to the medullary raphe regions where sympathetic premotor neurons for febrile and thermoregulatory responses are localized. These results suggest that a direct projection of EP3 receptor-expressing POA neurons to the DMH/DH region mediates febrile responses via a GABAergic mechanism.

Medullary lateral tegmental field: control of respiratory rate and vagal lung inflation afferent influences on sympathetic nerve discharge

We used spectral analysis and event-triggered averaging to determine the effects of chemical inactivation of the medullary lateral tegmental field (LTF) on 1) the relationship of intratracheal pressure (ITP, an index of vagal lung inflation afferent activity) to sympathetic nerve discharge (SND) and phrenic nerve activity (PNA) and 2) central respiratory rate in paralyzed, artificially ventilated dial-urethane-anesthetized cats. ITP-SND coherence value at the frequency of artificial ventilation was significantly (P<0.05; n=18) reduced from 0.73+/-0.04 (mean+/-SE) to 0.24+/-0.04 after bilateral microinjection of muscimol into the LTF. Central respiratory rate was unexpectedly increased in 12 of these experiments (0.28+/-0.03 vs. 0.95+/-0.25 Hz). The ITP-PNA coherence value was variably affected by chemical inactivation of the LTF. It was unchanged when central respiratory rate was also not altered, decreased when respiratory rate was increased above the rate of artificial ventilation, and increased when respiratory rate was raised from a value below the rate of artificial ventilation to the same frequency as the ventilator. Chemical inactivation of the LTF increased central respiratory rate in four of six vagotomized cats but did not significantly affect the PNA-SND coherence value. These data demonstrate that the LTF 1) plays a critical role in mediating the effects of vagal lung inflation afferents on SND but not PNA, 2) helps maintain central respiratory rate in the physiological range, but 3) is not involved in the coupling of central respiratory and sympathetic circuits.

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.

Role of paraventricular nucleus in the cardiogenic sympathetic reflex in rats.

Myocardial ischemia stimulates cardiac spinal afferents to initiate a sympathoexcitatory reflex. However, the pathways responsible for generation of increased sympathetic outflow in this reflex are not fully known. In this study, we determined the role of the paraventricular nucleus (PVN) in the cardiogenic sympathetic reflex. Renal sympathetic nerve activity (RSNA) and blood pressure were recorded in anesthetized rats during epicardial application of 10 microg/ml bradykinin. Bilateral microinjection of muscimol (0.5 nmol), a GABA(A) receptor agonist, was performed to inhibit the PVN. In 10 vehicle-injected rats, epicardial bradykinin significantly increased RSNA 178.4 +/- 48.5% from baseline, and mean arterial pressure from 76.9 +/- 2.0 to 102.3 +/- 3.3 mmHg. Microinjection of muscimol into the PVN significantly reduced the basal blood pressure and RSNA (n = 12). After muscimol injection, the bradykinin-induced increases in RSNA (111.6 +/- 35.9% from baseline) and mean arterial pressure (61.2 +/- 1.3 to 74.5 +/- 2.7 mmHg) were significantly reduced compared with control responses. The response remained attenuated even when the basal blood pressure was restored to the control. In a separate group of rats (n = 9), bilateral microinjection of the ionotropic glutamate antagonist kynurenic acid (4.82 or 48.2 nmol in 50 nl) had no significant effect on the RSNA and blood pressure responses to bradykinin compared with controls. These results suggest that the tonic PVN activity is important for the full manifestation of the cardiogenic sympathoexcitatory response. However, ionotropic glutamate receptors in the PVN are not directly involved in this reflex response.

Central integration of muscle reflex and arterial baroreflex in midbrain periaqueductal gray: roles of GABA and NO.

It has been suggested that the midbrain periaqueductal gray (PAG) is a neural integrating site for the interaction between the muscle pressor reflex and the arterial baroreceptor reflex. The underlying mechanisms are poorly understood. The purpose of this study was to examine the roles of GABA and nitric oxide (NO) in modulating the PAG integration of both reflexes. To activate muscle afferents, static contraction of the triceps surae muscle was evoked by electrical stimulation of the L7 and S1 ventral roots of 18 anesthetized cats. In the first group of experiments (n = 6), the pressor response to muscle contraction was attenuated by bilateral microinjection of muscimol (a GABA receptor agonist) into the lateral PAG [change in mean arterial pressure (DeltaMAP) = 24 +/- 5 vs. 46 +/- 8 mmHg in control]. Conversely, the pressor response was significantly augmented by 0.1 mM bicuculline, a GABAA receptor antagonist (DeltaMAP = 65 +/- 10 mmHg). In addition, the effect of GABAA receptor blockade on the reflex response was significantly blunted after sinoaortic denervation and vagotomy (n = 4). In the second group of experiments (n = 8), the pressor response to contraction was significantly attenuated by microinjection of L-arginine into the lateral PAG (DeltaMAP = 26 +/- 4 mmHg after L-arginine injection vs. 45 +/- 7 mmHg in control). The effect of NO attenuation was antagonized by bicuculline and was reduced after denervation. These data demonstrate that GABA and NO within the PAG modulate the pressor response to muscle contraction and that NO attenuation of the muscle pressor reflex is mediated via arterial baroreflex-engaged GABA increase. The results suggest that the PAG plays an important role in modulating cardiovascular responses when muscle afferents are activated.