Cristiane Busnardo

Paraventricular nucleus modulates autonomic and neuroendocrine responses to acute restraint stress in rats

The paraventricular nucleus of the hypothalamus (PVN) has been implicated in several aspects of neuroendocrine and cardiovascular control. The PVN contains parvocellular neurons that release the corticotrophin release hormone (CRH) under stress situations. In addition, this brain area is connected to several limbic structures implicated in defensive behavioral control, as well to forebrain and brainstem structures involved in cardiovascular control. Acute restraint is an unavoidable stress situation that evokes corticosterone release as well as marked autonomic changes, the latter characterized by elevated mean arterial pressure (MAP), intense heart rate (HR) increases and decrease in the tail temperature. We report the effect of PVN inhibition on MAP and HR responses, corticosterone plasma levels and tail temperature response during acute restraint in rats. Bilateral microinjection of the nonspecific synaptic blocker CoCl(2) (1 mM/100 nL) into the PVN reduced the pressor response; it inhibited the increase in plasma corticosterone concentration as well as the fall in tail temperature associated with acute restraint stress. Moreover, bilateral microinjection of CoCl(2) into areas surrounding the PVN did not affect the blood pressure, hormonal and tail vasoconstriction responses to restraint stress. The present results show that a local PVN neurotransmission is involved in the neural pathway that controls autonomic and neuroendocrine responses, which are associated with the exposure to acute restraint stress.

N-Methyl-D-aspartate glutamate receptors in the hypothalamic paraventricular nucleus modulate cardiac component of the baroreflex in unanesthetized rats

In the present study, we investigated the role played by the hypothalamic paraventricular nucleus (PVN) in the modulation of cardiac baroreflex activity in unanesthetized rats. Bilateral microinjections of the nonselective neurotransmission blocker CoCl(2) into the PVN decreased the reflex bradycardic response evoked by blood pressure increases, but had no effect on reflex tachycardia evoked by blood pressure decreases. Bilateral microinjections of the selective NMDA glutamate receptor antagonist LY235959 into the PVN caused effects that were similar to those observed after microinjections of CoCl(2), decreasing reflex bradycardia without affecting tachycardic response. The microinjection of the selective non-NMDA glutamate receptor antagonist NBQX into the PVN did not affect the baroreflex activity. Also, the microinjection of L-glutamate into the PVN increased the reflex bradycardia, an effect opposed to that observed after PVN treatment with CoCl(2) or LY235959, and this effect of L-glutamate was blocked by PVN pretreatment with LY235959. LY235959 injected into the PVN after i.v. treatment with the selective beta(1)-adrenoceptor antagonist atenolol still decreased the reflex bradycardia. Taken together, our results suggest a facilitatory influence of the PVN on the bradycardic response of the baroreflex through activation of local NMDA glutamate receptors and a modulation of the cardiac parasympathetic activity.

Role of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors in the cardiovascular effects of L-glutamate microinjection into the hypothalamic paraventricular nucleus of unanesthetized rats

We report on the cardiovascular effects of L‐glutamate (L‐glu) microinjection into the hypothalamic paraventricular nucleus (PVN) as well as the mechanisms involved in their mediation. L‐glu microinjection into the PVN caused dose‐related pressor and tachycardiac responses in unanesthetized rats. These responses were blocked by intravenous (i.v.) pretreatment with the ganglion blocker pentolinium (PE; 5 mg/kg), suggesting sympathetic mediation. Responses to L‐glu were not affected by local microinjection of the selective non‐NMDA receptor antagonist NBQX (2 nmol) or by local microinjection of the selective NMDA receptor antagonist LY235959 (LY; 2 nmol). However, the tachycardiac response was changed to a bradycardiac response after treatment with LY235959, suggesting that NMDA receptors are involved in the L‐glu heart rate response. Local pretreatment with LY235959 associated with systemic PE or dTyr(CH2)5(Me)AVP (50 μg/kg) respectively potentiated or blocked the response to L‐glu, suggesting that L‐glu responses observed after LY235959 are vasopressin mediated. The increased pressor and bradycardiac responses observed after LY + PE was blocked by subsequent i.v. treatment with the V1‐vasopressin receptor antagonist dTyr(CH2)5(Me)AVP, suggesting vasopressin mediation. The pressor and bradycardiac response to L‐glu microinjection into the PVN observed in animals pretreated with LY + PE was progressively inhibited and even blocked by additional pretreatment with increasing doses of NBQX (2, 10, and 20 nmol) microinjected into the PVN, suggesting its mediation by local non‐NMDA receptors. In conclusion, results suggest the existence of two glutamatergic pressor pathways in the PVN: one sympathetic pathway that is mediated by NMDA receptors and a vasopressinergic pathway that is mediated by non‐NMDA receptors.

Paraventricular nucleus of the hypothalamus glutamate neurotransmission modulates autonomic, neuroendocrine and behavioral responses to acute restraint stress in rats

In the present study, the involvement of paraventricular nucleus of the hypothalamus (PVN) glutamate receptors in the modulation of autonomic (arterial blood pressure, heart rate and tail skin temperature) and neuroendocrine (plasma corticosterone) responses and behavioral consequences evoked by the acute restraint stress in rats was investigated. The bilateral microinjection of the selective non-NMDA glutamate receptor antagonist NBQX (2 nmol/ 100 nL) into the PVN reduced the arterial pressure increase as well as the fall in the tail cutaneous temperature induced by the restraint stress, without affecting the stress-induced tachycardiac response. On the other hand, the pretreatment of the PVN with the selective NMDA glutamate receptor antagonist LY235959 (2 nmol/100 nL) was able to increase the stress-evoked pressor and tachycardiac response, without affecting the fall in the cutaneous tail temperature. The treatment of the PVN with LY235959 also reduced the increase in plasma corticosterone levels during stress and inhibited the anxiogenic-like effect observed in the elevated plus-maze 24h after the restraint session. The present results show that NMDA and non-NMDA receptors in the PVN differently modulate responses associated to stress. The PVN glutamate neurotransmission, via non-NMDA receptors, has a facilitatory influence on stress-evoked autonomic responses. On the other hand, the present data point to an inhibitory role of PVN NMDA receptors on the cardiovascular responses to stress. Moreover, our findings also indicate an involvement of PVN NMDA glutamate receptors in the mediation of the plasma corticosterone response as well as in the delayed emotional consequences induced by the restraint stress.

Cardiovascular effects of L-glutamate microinjection in the supraoptic nucleus of unanaesthetized rats.

We report on the cardiovascular effects of L-glutamate (L-glu) microinjection in the hypothalamic supraoptic nucleus (SON) as well as possible receptor and mechanisms involved. Microinjection of L-glu in 100 nL in the SON caused dose-related pressor and bradycardic responses in unanesthetized rats. Responses were markedly reduced in urethane-anesthetized rats. The response to L-glu 10 nmol was blocked by local pretreatment with 2 nmol of the non-NMDA-receptors antagonist NBQX and not affected by 2 nmol of the selective NMDA-receptor antagonist LY 235959, suggesting that non-NMDA receptors mediate these responses. The pressor and bradycardic response to L-glu was potentiated by intravenous pretreatment with the ganglion blocker pentolinium and was blocked by intravenous pretreatment with the V1-vasopressin receptor antagonist dTyr(CH2)5(Me)AVP, suggesting involvement of circulating vasopressin in this response. Additionally L-glu microinjection into the SON increased plasma vasopressin levels (control: 1.3 +/- 0.2 pg/mL, n = 6; L-glu: 14.7+/-2.3 pg/mL, n=6). In conclusion the results suggest that pressor responses to SON microinjection of L-glu are caused by activation of non-NMDA glutamate receptors and mediated by vasopressin release into systemic circulation.

Cardiovascular responses to l-glutamate microinjection into the hypothalamic paraventricular nucleus are mediated by a local nitric oxide-guanylate cyclase mechanism

It has been reported that L-glutamate (L-glu) microinjection into the hypothalamic paraventricular nucleus (PVN) evokes pressor and tachycardiac responses in unanesthetized rats. In the present study the hypothesis was tested that a local nitric oxide (NO)-guanylate cyclase interaction mediates cardiovascular effects of L-glu microinjection into the PVN of rats. The cardiovascular responses evoked by 10 nmol/100 nL of L-glu microinjected into the PVN were measured before and 10 min after PVN treatment with vehicle, the selective neuronal NO-synthase (nNOS) inhibitor N(omega)-Propyl-L-arginine (N-Propyl, 0.04 nmol or 4 nmol/100 nL), the NO scavenger carboxy-PTIO (C-PTIO, 1 nmol/100 nL) or the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolol [4,3-a]quinoxalin-1-one (ODQ, 1 nmol/100 nL). In a final experiment, different doses of the NO donor sodium nitroprusside (SNP; 9, 27 or 45 nmol/100 nL) were microinjected into the PVN. Cardiovascular responses evoked by L-glu microinjection into the PVN were abolished by local pretreatment with N-Propyl in both anesthetized and unanesthetized rats. PVN treatment with either C-PTIO or ODQ also reduced L-glu cardiovascular responses. The microinjection of SNP into the PVN caused pressor and tachycardiac responses in unanesthetized rats, whereas depressor and bradycardiac responses were observed in anesthetized rats. The present results suggest that cardiovascular responses evoked by L-glu microinjection into the PVN involve a local production of NO and activation of guanylate cyclase.

Non-N-methyl-D-aspartate glutamate receptors in the paraventricular nucleus of hypothalamus mediate the pressor response evoked by noradrenaline microinjected into the lateral septal area in rats.

The lateral septal area (LSA) is a part of the limbic system and is involved in cardiovascular modulation. We previously reported that microinjection of noradrenaline (NA) into the LSA of unanesthetized rats caused pressor responses that are mediated by acute vasopressin release. Magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) of the hypothalamus synthesize vasopressin. In the present work, we studied which of these nuclei is involved in the pressor pathway activated by unilateral NA injection into the LSA as well as the local neurotransmitter involved. Chemical ablation of the SON by unilateral injection of the nonspecific synapses blocker cobalt chloride (1 mM/100 nl) did not affect the pressor response evoked by NA (21 nmol/200 nl) microinjection into the LSA. However, the response to NA was blocked when cobalt chloride (1 mM/100 nl) was microinjected into the PVN, indicating that this hypothalamic nucleus is responsible for the mediation of the pressor response. There is evidence in the literature pointing to glutamate as a putative neurotransmitter activating magnocellular neurons. Pretreatment of the PVN with the selective non‐N‐methyl‐D‐asparate (NMDA) antagonist NBQX (2 nmol/100 nl) blocked the pressor response to NA microinjected into the LSA, whereas pretreatment with the selective NMDA antagonist LY235959 (2 nmol/100 nl) did not affect the response to NA. Our results implicate the PVN as the final structure in the pressor pathway activated by the microinjection of NA into the LSA. They also indicate that local glutamatergic synapses and non‐NMDA glutamatergic receptors mediate the response in the PVN.

Involvement of hypothalamic paraventricular nucleus non-N-methyl-d-aspartate receptors in the pressor response to noradrenaline microinjected into the bed nucleus of the stria terminalis of unanesthetized rats

Microinjection of noradrenaline into the bed nucleus of the stria terminalis (BST) has been reported to cause a pressor response in unanesthetized rats, which was shown to be mediated by acute vasopressin release into the systemic circulation. In the present study we verified the involvement of magnocellular neurons of the hypothalamic paraventricular (PVN) or supraoptic (SON) nuclei and the local neurotransmitter involved in the pressor response to noradrenaline microinjection into the BST. The PVN pretreatment with the non‐selective neurotransmission blocker CoCl2 (1 nmol/100 nL) inhibited the noradrenaline‐evoked pressor response. However, responses were not affected by SON treatment with CoCl2. Further experiments were carried out to test if glutamatergic neurotransmission in the PVN mediates the pressor response evoked by noradrenaline microinjection into the BST. Pretreatment of the PVN with the selective N‐methyl‐d‐aspartate (NMDA) receptor antagonist LY235959 (2 nmol/100 nL) did not affect the noradrenaline‐evoked pressor response. However, PVN pretreatment with the selective non‐NMDA receptor antagonist NBQX (2 nmol/100 nL) significantly reduced the pressor response to noradrenaline microinjection into the BST. In conclusion, our results suggest that pressor responses to noradrenaline microinjection into the BST are mediated by PVN magnocellular neurons without involvement of SON neurons. They also suggest that a glutamatergic neurotransmission through non‐NMDA glutamate receptors in the PVN mediates the response.

ANGIOTENSINERGIC NEUROTRANSMISSION IN THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS MODULATES THE PRESSOR RESPONSE TO ACUTE RESTRAINT STRESS IN RATS

We tested the hypothesis that the angiotensinergic neurotransmission, specifically in the paraventricular nucleus of the hypothalamus (PVN), is involved in the cardiovascular modulation during acute restraint stress (RS) in rats. The intravenous pretreatment with the angiotensin AT1 receptor antagonist losartan (5mg/kg) inhibited the pressor response to RS, but did not affect the concomitant RS-evoked tachycardiac response. Because similar effects were observed after the PVN pretreatment with CoCl2, and considering the high density of angiotensin receptors reported in the PVN, we studied the effect of the pretreatment of the PVN with either losartan or the angiotensin-converting enzyme (ACE) inhibitor lisinopril on the RS-evoked cardiovascular response. The bilateral microinjection of losartan (0.5 nmol/100 nL) or lisinopril (0.5 nmol/100nL) into the PVN inhibited the RS-related pressor response without affecting the tachycardiac response, suggesting that the PVN angiotensinergic neurotransmission modulates the vascular component of the stress response. Finally, to exclude the possibility that centrally injected drugs could be leaking to the circulation and acting on peripheral vascular receptors, we tested the effect of the intravenous pretreatment with either losartan (0.5 nmol/animal) or lisinopril (0.5 nmol/animal), assuming the hypothesis of a total spread of drugs from the CNS to the peripheral circulation. When animals were pretreated with such doses of either losartan or lisinopril, the cardiovascular RS-evoked response was not affected, thus indicating that even if there were a complete leakage of the drug to the periphery, it would not affect the cardiovascular response to RS. This observation favors the idea that the effect of the intravenous injection of 5mg/kg of losartan on the RS-related cardiovascular response would be explained by an action across the blood-brain barrier, possibly in the PVN. In conclusion, the results suggest that an angiotensinergic neurotransmission in the PVN acting on AT1-receptors modulates the vascular component of the RS-evoked cardiovascular response.

The ventral hippocampus NMDA receptor/nitric oxide/guanylate cyclase pathway modulates cardiovascular responses in rats

The hippocampus is a limbic structure that is involved in the expression of defensive reactions and autonomic changes in rats. The injection of L-glutamate (L-glu) into the ventral hippocampus (VH) decreases blood pressure and heart rate in anesthetized rats. Activation of NMDA receptors in the VH increases the production of nitric oxide (NO), leading to guanylate cyclase activation. The hypothesis of the present study was that a local NMDA receptor-NO-guanylate cyclase interaction mediates the cardiovascular effects of microinjection of L-glu into the VH. Microinjection of increasing doses of L-glu (30, 60 and 200 nmol/200 nL) into the VH of conscious rats caused dose-related pressor and tachycardiac responses. The cardiovascular effects of L-glu were abolished by local pretreatment with: the glutamate receptor antagonist AP-7 (0.4 nmol); the selective neuronal NO synthase (nNOS) inhibitor N(ω)-Propyl-L-arginine (0.04 nmol); the NO scavenger C-PTIO (2 nmol) or the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolol [4,3-a]quinoxalin-1-one (2 nmol). Moreover, these cardiovascular responses were blocked by intravenous pretreatment with: the ganglionic blocker mecamylamine (2mg/Kg); the nonselective β-adrenergic receptor antagonist propranolol (2mg/Kg); the β1-adrenergic receptor selective antagonist atenolol (1mg/kg). However, pretreatment with the selective α1-adrenergic receptor antagonist prazosin (0,5mg/kg) caused only a small reduction in the pressor response, without affecting the L-glu evoked tachycardia. In conclusion, our results suggest that cardiovascular responses caused by L-glu microinjection into the VH are mediated by NMDA glutamate receptors and involve local nNOS and guanylate cyclase activation. Moreover, these cardiovascular responses are mainly mediated by cardiac sympathetic nervous system activation, with a small involvement of the vascular sympathetic nervous system.