Benedito H. Machado

Increased sympathetic outflow in juvenile rats submitted to chronic intermittent hypoxia correlates with enhanced expiratory activity

Chronic intermittent hypoxia (CIH) in rats produces changes in the central regulation of cardiovascular and respiratory systems by unknown mechanisms. We hypothesized that CIH (6% O2 for 40 s, every 9 min, 8 h day−1) for 10 days alters the central respiratory modulation of sympathetic activity. After CIH, awake rats (n= 14) exhibited higher levels of mean arterial pressure than controls (101 ± 3 versus 89 ± 3 mmHg, n= 15, P < 0.01). Recordings of phrenic, thoracic sympathetic, cervical vagus and abdominal nerves were performed in the in situ working heart–brainstem preparations of control and CIH juvenile rats. The data obtained in CIH rats revealed that: (i) abdominal (Abd) nerves exhibited an additional burst discharge in late expiration; (ii) thoracic sympathetic nerve activity (tSNA) was greater during late expiration than in controls (52 ± 5 versus 40 ± 3%; n= 11, P < 0.05; values expressed according to the maximal activity observed during inspiration and the noise level recorded at the end of each experiment), which was not dependent on peripheral chemoreceptors; (iii) the additional late expiratory activity in the Abd nerve correlated with the increased tSNA; (iv) the enhanced late expiratory activity in the Abd nerve unique to CIH rats was accompanied by reduced post‐inspiratory activity in cervical vagus nerve compared to controls. The data indicate that CIH rats present an altered pattern of central sympathetic–respiratory coupling, with increased tSNA that correlates with enhanced late expiratory discharge in the Abd nerve. Thus, CIH alters the coupling between the central respiratory generator and sympathetic networks that may contribute to the induced hypertension in this experimental model.

Microinjection ofl-glutamate into the nucleus tractus solitarii increases arterial pressure in conscious rats

Microinjection of L-glutamate into the nucleus tractus solitarii (NTS) of anesthetized rats produces a fall in mean arterial pressure (MAP) similar to that observed during activation of baroreceptor afferents. In the present study we examined the effect of bilateral microinjections of L-glutamate through chronically implanted cannulae in the NTS of conscious freely moving rats. Group I (n = 6) was studied under conscious conditions and 24 h later the rats were anesthetized with urethane and the effects of L-glutamate re-examined. In conscious rats, L-glutamate (30 pmol to 5 nmol/100 nl) produced dose-dependent increases in MAP (+37 +/- 7 mmHg, 5 nmol), whereas under urethane anesthesia falls in MAP were observed (-11 +/- 3 mmHg, 5 nmol). Group II (n = 7) was studied under conscious conditions and 1 h later the rats were anesthetized with chloralose and the effects of L-glutamate re-examined. In this group of conscious rats L-glutamate (300 pmol to 5 nmol/100 nl) also produced dose-dependent increases in MAP (+37 +/- 5 mmHg, 5 nmol), whereas under chloralose anesthesia a dose-dependent depressor response was observed (-33 +/- 6 mmHg, 5 nmol). Saline microinjections into the NTS of conscious and anesthetized rats produced negligible effects. These data demonstrate that microinjection of L-glutamate into the NTS of rats produces a pressor response in conscious animals in contrast to depressor responses in animals anesthetized with chloralose or urethane.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased sympathetic activity in rats submitted to chronic intermittent hypoxia

Long‐term exposure to intermittent hypoxia may lead to important cardiovascular dysfunctions, such as hypertension. Rodent models of chronic intermittent hypoxia (CIH) have been used to study the mechanisms underlying the increase in mean arterial pressure (MAP) observed after exposure to CIH. Several studies suggest that the hypertension of rats submitted to CIH is associated with an increase in sympathetic activity. However, there are no studies documenting the direct measurement of sympathetic activity in conscious freely moving rats exposed to CIH. Therefore, the present study aimed to evaluate whether or not the increase of MAP in rats exposed to CIH is associated with an increase in sympathetic activity. To reach this goal, we analysed the effect of ganglionic blockade on baseline MAP as well as the plasma levels of catecholamines. Rats submitted to CIH (fractional inspired O2 of 6%, for 40 s in every 9 min, 8 h day−1) for 35 days (n= 31) exhibited a significant increase in MAP compared with control rats (n= 28) maintained under normoxia (112 ± 2 versus 103 ± 1 mmHg, P= 0.0003). The injection of the ganglionic blocker hexamethonium resulted in a similar fall in MAP in CIH and control groups (−46 ± 2 versus−41 ± 3 mmHg). However, hexamethonium after previous antagonism of the angiotensin II type 1 (AT1) receptors with losartan produced a larger decrease in MAP in the CIH than in the control group (−58 ± 2 versus−50 ± 2 mmHg, P= 0.0165). The injection of losartan itself produced no major changes in the baseline MAP in both groups. The measurement of plasma catecholamines showed an increase in plasma noradrenaline (10.12 ± 0.90 versus 4.74 ± 0.32 ng ml−1, P= 0.0042) in rats exposed to CIH compared with control rats. These data provide strong evidence to support the concept that rats submitted to CIH exhibit an increase in sympathetic activity, which seems to be determinant in the maintenance of hypertension in this experimental model.

Involvement of l‐glutamate and ATP in the neurotransmission of the sympathoexcitatory component of the chemoreflex in the commissural nucleus tractus solitarii of awake rats and in the working heart–brainstem preparation

Peripheral chemoreflex activation with potassium cyanide (KCN) in awake rats or in the working heart–brainstem preparation (WHBP) produces: (a) a sympathoexcitatory/pressor response; (b) bradycardia; and (c) an increase in the frequency of breathing. Our main aim was to evaluate neurotransmitters involved in mediating the sympathoexcitatory component of the chemoreflex within the nucleus tractus solitarii (NTS). In previous studies in conscious rats, the reflex bradycardia, but not the pressor response, was reduced by antagonism of either ionotropic glutamate or purinergic P2 receptors within the NTS. In the present study we evaluated a possible dual role of both P2 and NMDA receptors in the NTS for processing the sympathoexcitatory component (pressor response) of the chemoreflex in awake rats as well as in the WHBP. Simultaneous blockade of ionotropic glutamate receptors and P2 receptors by sequential microinjections of kynurenic acid (KYN, 2 nmol (50 nl)−1) and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonate (PPADS, 0.25 nmol (50 nl)−1) into the commissural NTS in awake rats produced a significant reduction in both the pressor (+38 ± 3 versus+8 ± 3 mmHg) and bradycardic responses (−172 ± 18 versus−16 ± 13 beats min−1; n= 13), but no significant changes in the tachypnoea measured using plethysmography (270 ± 30 versus 240 ± 21 cycles min−1, n= 7) following chemoreflex activation in awake rats. Control microinjections of saline produced no significant changes in these reflex responses. In WHBP, microinjection of KYN (2 nmol (20 nl)−1) and PPADS (1.6 nmol (20 nl)−1) into the commissural NTS attenuated significantly both the increase in thoracic sympathetic activity (+52 ± 2%versus+17 ± 1%) and the bradycardic response (−151 ± 17 versus−21 ± 3 beats min−1) but produced no significant changes in the increase of the frequency of phrenic nerve discharge (+0.24 ± 0.02 versus+0.20 ± 0.02 Hz). The data indicate that combined microinjections of PPADS and KYN into the commissural NTS in both awake rats and the WHBP are required to produce a significant reduction in the sympathoexcitatory response (pressor response) to peripheral chemoreflex activation. We conclude that glutamatergic and purinergic mechanisms are part of the complex neurotransmission system of the sympathoexcitatory component of the chemoreflex at the level of the commissural NTS.

Pressor response to microinjection of orexin/hypocretin into rostral ventrolateral medulla of awake rats

Orexin A (or hypocretin 1)-immunoreactive neurons in the rat lateral hypothalamus project to several areas of the medulla oblongata that are closely associated with cardiovascular regulation. The present study was undertaken to further strengthen the hypothesis that orexin A accelerates cardiovascular response by activating sympathoexcitatory neurons in the rat rostral ventrolateral medulla (RVLM). First, immunohistochemical studies revealed the presence of orexin A-immunoreactive fibers in the RVLM. Double labeling the sections with orexin A- and tyrosine hydroxylase (TH)-antisera further showed that orexin A-immunoreactive fibers are in close proximity with TH-immunoreactive neurons, some of which may be barosensitive, bulbospinal neurons in the RVLM. Second, microinjection of orexin A (6.35, 12.7 and 38.1 microM) into the RVLM, which was verified later by histological examination, caused a significant increase of mean arterial pressure (MAP) and a moderate increase of heart rate (HR) in awake rats. L-glutamate (33.3 mM) injected into the same sites, caused a larger increase in MAP, but a decrease in HR; whereas, saline injection was without significant effect. Results from this study suggest that orexin A, which may be released from the nerve fibers originating from the neurons in the lateral hypothalamus, acting on RVLM neurons in the medulla, increases sympathetic outflow targeted to the heart and blood vessels in awake animals.

Sympathetic-mediated hypertension of awake juvenile rats submitted to chronic intermittent hypoxia is not linked to baroreflex dysfunction.

In the present study, we evaluated the mechanisms underpinning the hypertension observed in freely moving juvenile rats submitted to chronic intermittent hypoxia (CIH). Male juvenile Wistar rats (20–21 days old) were submitted to CIH (6% O2 for 40 s every 9 min, 8 h day−1) for 10 days while control rats were maintained in normoxia. Prior to CIH, baseline systolic arterial pressure (SAP), measured indirectly, was similar between groups (86 ± 1 versus 87 ± 1 mmHg). After exposure to CIH, SAP recorded directly was higher in the CIH (n= 28) than in the control group (n= 29; 131 ± 3 versus 115 ± 2 mmHg, P < 0.05). This higher SAP of CIH rats presented an augmented power of oscillatory components at low (10.05 ± 0.91 versus 5.02 ± 0.63 mmHg2, P < 0.05) and high (respiratory‐related) frequencies (12.42 ± 2.46 versus 3.28 ± 0.61 mmHg2, P < 0.05) in comparison with control animals. In addition, rats exposed to CIH also exhibited an increased cardiac baroreflex gain (−3.11 ± 0.08 versus−2.1 ± 0.10 beats min−1 mmHg−1, P < 0.0001), associated with a shift to the right of the operating point, in comparison with control rats. Administration of hexamethonium (ganglionic blocker, i.v.), injected after losartan (angiotensin II type 1 receptor antagonist) and [β‐mercapto‐β,β‐cyclopenta‐methylenepropionyl1, O‐Me‐Tyr2, Arg8]‐vasopressin (vasopressin type 1a receptor antagonist), produced a larger depressor response in the CIH (n= 8) than in the control group (n= 9; −49 ± 2 versus−39 ± 2 mmHg, P < 0.05). Fifteen days after the cessation of exposure to CIH, the mean arterial pressure of CIH rats returned to normal levels. The data indicate that the sympathetic‐mediated hypertension observed in conscious juvenile rats exposed to CIH is not secondary to a reduction in cardiac baroreflex gain and exhibits a higher respiratory modulation, indicating that an enhanced respiratory–sympathetic coupling seems to be the major factor contributing to hypertension in rats exposed to CIH.

Multiple pontomedullary mechanisms of respiratory rhythmogenesis

Mammalian central pattern generators producing rhythmic movements exhibit robust but flexible behavior. However, brainstem network architectures that enable these features are not well understood. Using precise sequential transections through the pons to medulla, it was observed that there was compartmentalization of distinct rhythmogenic mechanisms in the ponto-medullary respiratory network, which has rostro-caudal organization. The eupneic 3-phase respiratory pattern was transformed to a 2-phase and then to a 1-phase pattern as the network was physically reduced. The pons, the retrotrapezoid nucleus and glycine mediated inhibition are all essential for expression of the 3-phase rhythm. The 2-phase rhythm depends on inhibitory interactions (reciprocal) between Bötzinger and pre-Bötzinger complexes, whereas the 1-phase-pattern is generated within the pre-Bötzinger complex and is reliant on the persistent sodium current. In conditions of forced expiration, the RTN region was found to be essential for the expression of abdominal late expiratory activity. However, it is unknown whether the RTN generates or simply relays this activity. Entrained with the central respiratory network is the sympathetic nervous system, which exhibits patterns of discharge coupled with the respiratory cycle (in terms of both gain and phase of coupling) and dysfunctions in this coupling appear to underpin pathological conditions. In conclusion, the respiratory network has rhythmogenic capabilities at multiple levels of network organization, allowing expression of motor patterns specific for various physiological and pathophysiological respiratory behaviors.

Sympathoexcitatory neurotransmission of the chemoreflex in the NTS of awake rats

Cardiovascular responses to chemoreflex activation by potassium cyanide (KCN, 20 microgram/rat iv) were analyzed before and after the blockade of ionotropic or metabotropic receptors into the nucleus of the solitary tract (NTS) of awake rats. Microinjection of ionotropic antagonists [6,7-dinitroquinoxaline-2,3-dione or kynurenic acid (Kyn)] into the lateral commissural NTS (NTSlat), the midline commissural NTS (NTSmid), or into both (NTSlat+mid), produced a significant increase in basal mean arterial pressure, and the pressor response to chemoreflex activation was only partially reduced, whereas microinjection of Kyn into the NTSmid produced no changes in the pressor response to the chemoreflex. The bradycardic response to chemoreflex activation was abolished by microinjection of Kyn into the NTSlat or into NTSlat+mid but not by Kyn microinjection into the NTSmid. Microinjection of alpha-methyl-4-carboxyphenylglycine, a metabotropic receptor antagonist, into the NTSlat or NTSmid produced no changes in baseline mean arterial pressure or heart rate or in the chemoreflex responses. These results indicate that 1) the processing of the parasympathetic component (bradycardia) of the chemoreflex seems to be restricted to the NTSlat and was blocked by ionotropic antagonists and 2) the pressor response of the chemoreflex was only partially reduced by microinjection of ionotropic antagonists and not affected by injection of metabotropic antagonists into the NTSlat or NTSmid or into NTSlat+mid in awake rats.

Electrophysiological Properties of Rostral Ventrolateral Medulla Presympathetic Neurons Modulated by the Respiratory Network in Rats

The respiratory pattern generator modulates the sympathetic outflow, the strength of which is enhanced by challenges produced by hypoxia. This coupling is due to the respiratory-modulated presympathetic neurons in the rostral ventrolateral medulla (RVLM), but the underlining electrophysiological mechanisms remain unclear. For a better understanding of the neural substrates responsible for generation of this respiratory-sympathetic coupling, we combined immunofluorescence, single cell qRT-pCR, and electrophysiological recordings of the RVLM presympathetic neurons in in situ preparations from normal rats and rats submitted to a metabolic challenge produced by chronic intermittent hypoxia (CIH). Our results show that the spinally projected cathecholaminergic C1 and non-C1 respiratory-modulated RVLM presympathetic neurons constitute a heterogeneous neuronal population regarding the intrinsic electrophysiological properties, respiratory synaptic inputs, and expression of ionic currents, albeit all neurons presented persistent sodium current-dependent intrinsic pacemaker properties after synaptic blockade. A specific subpopulation of non-C1 respiratory-modulated RVLM presympathetic neurons presented enhanced excitatory synaptic inputs from the respiratory network after CIH. This phenomenon may contribute to the increased sympathetic activity observed in CIH rats. We conclude that the different respiratory-modulated RVLM presympathetic neurons contribute to the central generation of respiratory-sympathetic coupling as part of a complex neuronal network, which in response to the challenges produced by CIH contribute to respiratory-related increase in the sympathetic activity.

Intermittent hypoxia-induced sensitization of central chemoreceptors contributes to sympathetic nerve activity during late expiration in rats.

Hypertension elicited by chronic intermittent hypoxia (CIH) is associated with elevated activity of the thoracic sympathetic nerve (tSN) that exhibits an enhanced respiratory modulation reflecting a strengthened interaction between respiratory and sympathetic networks within the brain stem. Expiration is a passive process except for special metabolic conditions such as hypercapnia, when it becomes active through phasic excitation of abdominal motor nerves (AbN) in late expiration. An increase in CO(2) evokes late-expiratory (late-E) discharges phase-locked to phrenic bursts with the frequency increasing quantally as hypercapnia increases. In rats exposed to CIH, the late-E discharges synchronized in AbN and tSN emerge in normocapnia. To elucidate the possible neural mechanisms underlying these phenomena, we extended our computational model of the brain stem respiratory network by incorporating a population of presympathetic neurons in the rostral ventrolateral medulla that received inputs from the pons, medullary respiratory compartments, and retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG). Our simulations proposed that CIH conditioning increases the CO(2) sensitivity of RTN/pFRG neurons, causing a reduction in both the CO(2) threshold for emerging the late-E activity in AbN and tSN and the hypocapnic threshold for apnea. Using the in situ rat preparation, we have confirmed that CIH-conditioned rats under normal conditions exhibit synchronized late-E discharges in AbN and tSN similar to those observed in control rats during hypercapnia. Moreover, the hypocapnic threshold for apnea was significantly lowered in CIH-conditioned rats relative to that in control rats. We conclude that CIH may sensitize central chemoreception and that this significantly contributes to the neural impetus for generation of sympathetic activity and hypertension.