INTERMITTENT ACTIVATION OF PERIPHERAL CHEMORECEPTORS IN AWAKE RATS INDUCES Fos EXPRESSION IN ROSTRAL VENTROLATERAL MEDULLA-PROJECTING NEURONS IN THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS

Despite the well-established sympathoexcitation evoked by chemoreflex activation, the specific sub-regions of the CNS underlying such sympathetic responses remain to be fully characterized. In the present study we examined the effects of intermittent chemoreflex activation in awake rats on Fos-immunoreactivity (Fos-ir) in various subnuclei of the paraventricular nucleus of the hypothalamus (PVN), as well as in identified neurosecretory preautonomic PVN neurons. In response to intermittent chemoreflex activation, a significant increase in the number of Fos-ir cells was found in autonomic-related PVN subnuclei, including the posterior parvocellular, ventromedial parvocellular and dorsal-cap, but not in the neurosecretory magnocellular-containing lateral magnocellular subnucleus. No changes in Fos-ir following chemoreflex activation were observed in the anterior PVN subnucleus. Experiments combining Fos immunohistochemistry and neuronal tract tracing techniques showed a significant increase in Fos-ir in rostral ventrolateral medulla (RVLM)-projecting (PVN-RVLM), but not in nucleus of solitarii tract (NTS)-projecting PVN neurons. In summary, our results support the involvement of the PVN in the central neuronal circuitry activated in response to chemoreflex activation, and indicate that PVN-RVLM neurons constitute a neuronal substrate contributing to the sympathoexcitatory component of the chemoreflex. Published by Elsevier Ltd on behalf of IBRO.

Inhibition of spontaneous neurotransmission in the nucleus of solitary tract of the rat by the cannabinoid agonist WIN 55212-2 is not via CB1 or CB2 receptors

Cannabinoids have been shown to modulate central autonomic regulation and baroreflex control of blood pressure. Both CB1 and CB2 cannabinoid receptors have been described in the nucleus tractus solitarius (NTS), which receives direct afferent projections of cardiovascular reflexes. in the present study we evaluated the effects of WIN 55212-2 (WIN), a cannabinoid agonist, on fast neurotransmission in the NTS. We recorded spontaneous post-synaptic currents using the whole-cell configuration in NTS cells in brainstem slices from young rats (25-30 days old). Application of 5 mu M WIN inhibited the frequency of both glutamatergic and GABAergic sPSCs, without affecting their amplitudes. Effects of WIN were not blocked by application of the CB1 antagonist AM251, the CB2 antagonist AM630 or the varmiloid receptor TRPV1 antagonist AMG9810, suggesting that the effect of WIN is via a non-CB1 non-CB2 receptor. Neither the CB1/CB2 agonist HU210 nor the CB1 agonist ACPA affected the frequency of sPSCs. We conclude WIN inhibits the neurotransmission in the NTS of young rats via a receptor distinct from CB1 or CB2. (c) 2008 Elsevier B.V. All rights reserved.

Interaction of purinergic and nitrergic mechanisms in the caudal nucleus tractus solitarii of rats

The interaction of purinergic and nitrergic mechanisms was evaluated in the caudal nucleus tractus solitarii (cNTS) using awake animals and brainstem slices. In awake animals, ATP (1.25 nmol/50 nL) was microinjected into the cNTS before and after the microinjection of a selective neuronal nitric oxide synthase (nNOS) inhibitor N-propyl-L-arginine (NPLA, 3 pmoles/50 nL, n=8) or vehicle (saline, n=4), and cardiovascular and ventilatory parameters were recorded. In brainstem slices from a distinct group of rats, the effects of ATP on the NO concentration in the cNTS using the fluorescent dye DAF-2 DA were evaluated. For this purpose brainstem slices (150 pm) containing the cNTS were pre-incubated with ATP (500 mu M; n=8) before and during DAF-2 DA loading. Microinjection of ATP into the cNTS increases the arterial pressure (AP), respiratory frequency (f(R)) and minute ventilation (V(E)), which were significantly reduced by pretreatment with N-PLA, a selective nNOS inhibitor (AP: 39 +/- 3 vs 16 +/- 14 mm Hg; f(R): 75 +/- 14 vs 4 +/- 3 cpm; V(E): 909 159 vs 77 39 mL kg(-1) m(-1)). The effects of ATP in the cNTS were not affected by microinjection of saline. ATP significantly increased the NO fluorescence in the cNTS (62 +/- 7 vs 101 +/- 10 AU). The data show that in the cNTS: a) the NO production is increased by ATP; b) NO formation by nNOS is involved in the cardiovascular and ventilatory responses to microinjection of ATP. Taken together, these data suggest an interaction of purinergic and nitrergic mechanisms in the cNTS. (C) 2009 Elsevier B.V. All rights reserved.

Blocking systemic nitric oxide production alters neuronal activation in brain structures involved in cardiovascular regulation during polymicrobial sepsis

In a previous study, we concluded that overproduction of nitric oxide (NO) by inducible nitric Oxide synthase (iNOS) in the late phase of sepsis prevents hypothalamic activation, blunts vasopressin secretion and contributes to hypotension, irreversible shock and death. The aim of this follow-up study was to evaluate if the same neuronal activation pattern happens in brain structures related to cardiovascular functions. Male Wistar rats received intraperitoneal injections of aminoguanidine, an iNOS inhibitor, or saline 30 min before cecal ligation and puncture (CLP) or sham surgeries. The animals were perfused 6 or 24 h after the surgeries and the brains were removed and processed for Fos immunocytochemistry We observed an increase (P < 0.001) in c-fos expression 6 h after CLP in the area postrema (AP), nucleus of he tractus solitarius (NTS), ventral lateral medulla (VLM), locus coeruleus (LC) and parabrachial nucleus (PB). At 24 h after CLP, however, c-fos expression was strongly decreased in all these nuclei (P < 0.05), except for the VLM. Aminoguanidine reduced c-fos expression in the AP and NTS at 6 h after CLR but showed an opposite effect at 24 h, with an increase in the AP, NTS, and also in the VLM. No such effect was observed in the LC and PB at 6 or 24 h. In all control animals, c-fos expression was minimal or absent. We conclude that in the early phase of sepsis iNOS-derived NO may be partially responsible for the activation of brain structures related to cardiovascular regulation. During the late phase, however, this activation is reduced or abolished. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Adenosine receptor type 2a is differently modulated by nicotine in dorsal brainstem cells of Wistar Kyoto and spontaneously hypertensive rats

Hypertension can result from neuronal network imbalance in areas of central nervous system that control blood pressure, such as the nucleus tractus solitarius (NTS). There are several neurotransmitters and neuromodulatory substances within the NTS, such as adenosine, which acts on purinoreceptors A(2a) (A(2a)R). The A(2a)R modulates neurotransmission in the NTS where its activation may induce decrease in blood pressure by different mechanisms. Nicotine is a molecule that crosses the hematoencephalic barrier and acts in several areas of central nervous system including the NTS, where it may interact with some neurotransmitter systems and contributes to the development of hypertension in subjects with genetic predisposition to this disease. In this study we first determined A(2a)R binding, protein, and mRNA expression in dorsomedial medulla oblongata of neonate normotensive (WKY) and spontaneously hypertensive rats (SHR). Subsequently, we analyzed the modulatory effects of nicotine on A(2a)R in cell culture in order to evaluate its possible involvement in the development of hypertension. Data showed a decreased A(2a)R binding and increased protein and mRNA expression in tissue sample and culture of dorsal brainstem from SHR compared with those from WKY rats at basal conditions. Moreover, nicotine modulated A(2a)R binding, protein, and mRNA expression in cells from both strains. Interestingly, nicotine decreased A(2a)R binding and increased protein levels, as well as, induced a differential modulation in A(2a)R mRNA expression. Results give us a clue about the mechanisms involved in the modulatory effects of nicotine on A(2a)R as well as hypothesize its possible contribution to the development of hypertension. In conclusion, we demonstrated that A(2a)R of SHR cells which differ from WKY and nicotine differentially modulates A(2a)R in dorsal brainstem cells of SHR and WKY.

Adenosine modulates alpha2-adrenergic receptors through a phospholipase C pathway in brainstem cell culture of rats

Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A(1) adenosine receptor (A(1R)) activation promotes an increase on alpha(2)-adrenoceptor (Alpha(2R)) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca(2+)-dependent, IP(3) receptor and intracellular calcium were shown to participate in A(1R)/Alpha(2R) interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control. (C) 2009 Elsevier B.V. All rights reserved.

Age-dependent changes in adenosine A(1) receptor distribution and density within the nucleus tractus solitarii of normotensive and hypertensive rats

This study shows the distribution and density of adenosine A1 receptor (A(1)R) within the nucleus tractus solitarii (NTS) of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) from birth to adulthood (1, 15, 30 and 90 days old). The NTS shows heterogeneous distribution of A(1)R in dorsomedial/dorsolateral, subpostremal and medial/intermediate subnuclei. A(1)R decrease from rostral to caudal within dorsomedial/dorsolateral subnucleus in 15-, 30- and 90-day-old WKY and SHR. A(1)R increase from rostral to caudal subpostremal subnucleus in 30- and 90-day-old WKY, and in 15-, 30- and 90-day-old SHR. Furthermore, A(1)Rs are increased in SHR as compared with WKY within dorsomedial/dorsolateral in 30- and 90-day-old and within subpostremal of 15-, 30- and 90-day-old rats. Finally, A(1)Rs increase from 1- to 30-day-old rats. Medial/intermediate did not show any changes in A(1)R from rostral to caudal levels, age or strain. In summary, our result highlights the importance of A1 adenosine system regarding the neural control of blood pressure and the development of hypertension.

Brainstem oxytocinergic modulation of heart rate control in rats: effects of hypertension and exercise training

Oxytocinergic brainstem projections participate in the autonomic control of the circulation. We investigated the effects of hypertension and training on cardiovascular parameters after oxytocin (OT) receptor blockade within the nucleus tractus solitarii (NTS) and NTS OT and OT receptor expression. Male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were trained (55% of maximal exercise capacity) or kept sedentary for 3 months and chronically instrumented (NTS and arterial cannulae). Mean arterial blood pressure (MAP) and heart rate (HR) were measured at rest and during an acute bout of exercise after NTS pretreatment with vehicle or OT antagonist (20 pmol of OT antagonist (200 nl of vehicle)-1). Oxytocin and OT receptor were quantified (35S-oligonucleotide probes, in situ hybridization) in other groups of rats. The SHR exhibited high MAP and HR (P < 0.05). Exercise training improved treadmill performance and reduced basal HR (on average -11%) in both groups, but did not change basal MAP. Blockade of NTS OT receptor increased exercise tachycardia only in trained groups, with a larger effect on trained WKY rats (+31 +/- 9 versus +12 +/- 3 beats min-1 in the trained SHR). Hypertension specifically reduced NTS OT receptor mRNA density (-46% versus sedentary WKY rats, P < 0.05); training did not change OT receptor density, but significantly increased OT mRNA expression (+2.5-fold in trained WKY rats and +15% in trained SHR). Concurrent hypertension- and training-induced plastic (peptide/receptor changes) and functional adjustments (HR changes) of oxytocinergic control support both the elevated basal HR in the SHR group and the slowing of the heart rate (rest and exercise) observed in trained WKY rats and SHR.

Adenosine Modulatesa alpha(2)-Adrenergic Receptors within Specific Subnuclei of the Nucleus Tractus Solitarius in Normotensive and Spontaneously Hypertensive Rats

Adenosine Is known to modulate neuronal activity within the nucleus tractus solitarius (NTS). The modulatory effect of adenosine A, receptors (A(1R)) on alpha(2)-adrenoceptors (Adr(2R)) was evaluated using quantitative radioautography within NTS subnuclei and using neuronal culture of normotensive (WKY) and spontaneously hypertensive rats (SHR). Radioautography was used in a saturation experiment to measure Adr2R binding parameters (B(max), K(d)) In the presence of 3 different concentrations of N(6)-cyclopentyladenosine (CPA), an A(1R) agonist. Neuronal culture confirmed our radioautographic results. [(3)H]RX821002, an Adr(2R) antagonist, was used as a ligand for both approaches. The dorsomedial/dorsolateral subnucleus of WKY showed an increase in B(max) values (21%) Induced by 10 nmol/L of CPA. However, the subpostremal subnucleus showed a decrease in Kd values (24%) induced by 10 nmol/L of CPA. SHR showed the same pattern of changes as WKY within the same subnuclei; however, the modulatory effect of CPA was induced by I nmol/L (increased B(max), 17%; decreased K(d), 26%). Cell culture confirmed these results, because 10(-5) and 10(-7) mol/L of CPA promoted an Increase in [3H]RX821002 binding of WKY (53%) and SHR cells (48%), respectively. DPCPX, an AIR antagonist, was used to block the modulatory effect promoted by CPA with respect to Adr2R binding. In conclusion, our study shows for the first time an interaction between A(1R) that increases the binding of Adr2R within specific subnuclei of the NTS. This may be important In understanding the complex autonomic response induced by adenosine within the NTS. In addition, changes in interactions between receptors might be relevant to understanding the development of hypertension. (Hypertens Res 2008; 31: 2177-2186)