Glutamatergic and purinergic transmitters and astrocyte modulation in the synaptic transmission in the NTS of rats exposed to short-term sustained hypoxia.

There is evidence that astrocytes modulate synaptic transmission in the nucleus tractus solitarius (NTS) interacting with glutamatergic and purinergic mechanisms. Here, using in situ working heart-brainstem preparations, we evaluated the involvement of astrocyte and glutamatergic/purinergic neurotransmission in the processing of autonomic and respiratory pathways in the NTS of control and rats exposed to sustained hypoxia (SH). Baseline autonomic and respiratory activities and the responses to chemoreflex activation (KCN) were evaluated before and after microinjections of fluorocitrate (FCt, an astrocyte metabolic inhibitor), kynurenic acid, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (PPADS) (nonselective antagonists of glutamatergic and purinergic receptors) into the rostral aspect of the caudal commissural NTS. FCt had no effects on the baseline parameters evaluated but reduced the bradycardic response to chemoreflex activation in SH rats. FCt combined with kynurenic acid and PPADS in control rats reduced the baseline duration of expiration, which was attenuated after SH. FCt produced a large increase in PN frequency discharge in control rats, which was reduced after SH, indicating a reduction in the astrocyte modulation after SH. The data show that 1) the bradycardic component of the peripheral chemoreflex is reduced in SH rats after astrocytes inhibition, 2) the inhibition of astrocytes in the presence of double antagonists in the NTS affects the modulation of baseline duration of expiration in control but not in SH rats, and 3) the autonomic and respiratory responses to chemoreflex activation are mediated by glutamatergic and purinergic receptors in the rostral aspect of the caudal commissural NTS.NEW & NOTEWORTHY Our findings indicate that the neurotransmission of autonomic and respiratory components of the peripheral chemoreflex in the nucleus tractus solitarius (NTS) is mediated by glutamatergic and purinergic mechanisms and reveal a selective involvement of NTS astrocytes in controlling the chemoreflex parasympathetic response in rats exposed to sustained hypoxia (SH) and the baseline duration of expiration mainly in control rats, indicating a selective role for astrocytes modulation in the NTS of control and SH rats.

Enhancement of the Evoked Excitatory Transmission in the Nucleus Tractus Solitarius Neurons after Sustained Hypoxia in Mice Depends on A2A Receptors.

The first synapses of the afferents of peripheral chemoreceptors are located in the Nucleus Tractus Solitarius (NTS) and there is evidence that short-term sustained hypoxia (SH - 24 h, FiO2 0.1) facilitates glutamatergic transmission in NTS neurons of rats. Adenosine is an important neuromodulator of synaptic transmission and hypoxia contributes to increase its extracellular concentration. The A2A receptors mediate the excitatory actions of adenosine and are active players in the modulation of neuronal networks in the NTS. Herein, we used knockout mice for A2A receptors (A2AKO) and electrophysiological recordings of NTS neurons were performed to evaluate the contribution of these receptors in the changes in synaptic transmission in NTS neurons of mice submitted to SH. The membrane passive properties and excitability of NTS neurons were not affected by SH and were similar between A2AKO and wild-type mice. The overall amplitude of spontaneous glutamatergic currents in NTS neurons of A2AKO mice was lower than in Balb/c WT mice. SH increased the amplitude of evoked glutamatergic currents of NTS neurons from WT mice by a non-presynaptic mechanism, but this enhancement was not observed in NTS neurons of A2AKO mice. Under normoxia, the amplitude of evoked glutamatergic currents was similar between WT and A2AKO mice. The data indicate that A2A receptors (a) modulate spontaneous glutamatergic currents, (b) do not modulate the evoked glutamatergic transmission in the NTS neurons under control conditions, and (c) are required for the enhancement of glutamatergic transmission observed in the NTS neurons of mice submitted to SH.

Cardiovascular and respiratory evaluation in adenosine A2A receptor knockout mice submitted to short-term sustained hypoxia.

Sustained hypoxia (SH) in mice induces changes in the respiratory pattern and increase in the parasympathetic tone to the heart. Among adenosine G-protein-coupled receptors (GPCRs), the A2A receptors are especially important in mediating adenosine actions during hypoxia due to their expression in neurons involved with the generation and modulation of the autonomic and respiratory functions. Herein, we performed an in vivo evaluation of the baseline cardiovascular and respiratory parameters and their changes in response to SH in knockout mice for A2A receptors (A2A KO). SH produced similar and significant reductions in mean arterial pressure and heart rate in both wild-type (WT) and A2A KO mice when compared to their respective normoxic controls. Mice from WT and A2A KO groups submitted to normoxia or SH presented similar cardiovascular responses to peripheral chemoreflex activation (KCN). Under normoxic conditions A2A KO mice presented a respiratory frequency (fR ) significantly higher in relation to the WT group, which was reduced in response to SH. These data show that the lack of adenosine A2A receptors in mice does not affect the cardiovascular parameters and the autonomic responses to chemoreflex activation in control (normoxia) and SH mice. We conclude that the A2A receptors play a major role in the control of respiratory frequency and in the tachypnoeic response to SH in mice. NEW FINDINGS: What is the central question of this study? Are cardiovascular and respiratory parameters and their changes in response to sustained hypoxia (SH) altered in adenosine A2A receptor knockout mice? What is the main finding and its importance? Cardiovascular parameters and their changes in response to SH were not altered in A2A KO mice. The respiratory frequency in A2A KO was higher than in WT mice. In response to SH the respiratory frequency increased in WT, while it was reduced in A2A KO mice. A2A receptors play a major role in the modulation of respiratory frequency and in the tachypnoeic response to SH in mice.

Evidences That Sympathetic Overactivity and Neurogenic Hypertension Correlate with Changes in the Respiratory Pattern in Rodent Models of Experimental Hypoxia.

The main question of this chapter is as follows: What is the contribution of changes in the sympathetic-respiratory coupling to the hypertension observed in some experimental models of hypoxia? Although there is evidence supporting the concept that sympathetic-respiratory coupling is increased in different models of experimental hypoxia [chronic intermittent hypoxia (CIH) and sustained hypoxia (SH)], it was also observed that in some strains of rats and in mice, these experimental models of hypoxia do not affect the sympathetic-respiratory coupling and the baseline arterial pressure. The data from studies performed in rats (different strains, male and female, and in the natural sleep cycle) and mice submitted to chronic CIH or SH are critically discussed. The main message from these studies performed in freely moving rodents and in the in situ working heart-brainstem preparation is that experimental hypoxia changes the respiratory pattern, which correlates with increased sympathetic activity and may explain the hypertension observed in male and female rats previously submitted to CIH or SH.

Sustained hypoxia in mice increases parasympathetic but not sympathetic tone.

The autonomic profile of mice submitted to sustained hypoxia (SH) was not yet fully evaluated. Herein, we characterized the cardiovascular and autonomic profile of conscious freely moving mice submitted to SH using two sequential experimental protocols to evaluate the parasympathetic and sympathetic tone to the heart and the sympathetic tone to the vascular resistance. In the first protocol the sequence of antagonists was methyl-atropine followed by propranolol and then by prazosin, while in the second protocol the sequence was propranolol followed by methyl-atropine and then by prazosin. In SH the baseline heart rate was significantly lower than in control mice and the antagonism of the parasympathetic and sympathetic tone to the heart in both experimental protocols indicated an increased parasympathetic tone in SH mice and no changes in the sympathetic tone. Antagonism of the sympathetic tone to the vascular resistance with prazosin produced similar changes in arterial pressure in control and SH mice. Altogether these findings support the concept that mice submitted to SH present a significant increase in the parasympathetic but not in the sympathetic tone, which may explain why the baseline arterial pressure was not increased in SH mice.

Intrinsic and synaptic mechanisms controlling the expiratory activity of excitatory lateral parafacial neurones of rats.

Active expiration is essential for increasing pulmonary ventilation during high chemical drive (hypercapnia). The lateral parafacial (pFL ) region, which contains expiratory neurones, drives abdominal muscles during active expiration in response to hypercapnia. However, the electrophysiological properties and synaptic mechanisms determining the activity of pFL expiratory neurones, as well as the specific conditions for their emergence, are not fully understood. Using whole cell electrophysiology and single cell quantitative RT-PCR techniques, we describe the intrinsic electrophysiological properties, the phenotype and the respiratory-related synaptic inputs to the pFL expiratory neurones, as well as the mechanisms for the expression of their expiratory activity under conditions of hypercapnia-induced active expiration, using in situ preparations of juvenile rats. We also evaluated whether these neurones possess intrinsic CO2 /[H+ ] sensitivity and burst generating properties. GABAergic and glycinergic inhibition during inspiration and expiration suppressed the activity of glutamatergic pFL expiratory neurones in normocapnia. In hypercapnia, these neurones escape glycinergic inhibition and generate burst discharges at the end of expiration. Evidence for the contribution of post-inhibitory rebound, CaV 3.2 isoform of T-type Ca2+ channels and intracellular [Ca2+ ] is presented. Neither intrinsic bursting properties, mediated by persistent Na+ current, nor CO2 /[H+ ] sensitivity or expression of CO2 /[H+ ] sensitive ion channels/receptors (TASK or GPR4) were observed. On the other hand, hyperpolarisation-activated cyclic nucleotide-gated and twik-related K+ leak channels were recorded. Post-synaptic disinhibition and the intrinsic electrophysiological properties of glutamatergic neurones play important roles in the generation of the expiratory oscillations in the pFL region during hypercapnia in rats. KEY POINTS: Hypercapnia induces active expiration in rats and the recruitment of a specific population of expiratory neurones in the lateral parafacial (pFL ) region. Post-synaptic GABAergic and glycinergic inhibition both suppress the activity of glutamatergic pFL neurones during inspiratory and expiratory phases in normocapnia. Hypercapnia reduces glycinergic inhibition during expiration leading to burst generation by pFL neurones; evidence for a contribution of post-inhibitory rebound, voltage-gated Ca2+ channels and intracellular [Ca2+ ] is presented. pFL glutamatergic expiratory neurones are neither intrinsic burster neurones, nor CO2 /[H+ ] sensors, and do not express CO2 /[H+ ] sensitive ion channels or receptors. Post-synaptic disinhibition and the intrinsic electrophysiological properties of glutamatergic neurones both play important roles in the generation of the expiratory oscillations in the pFL region during hypercapnia in rats.

Sustained Hypoxia Reduces GABAergic Modulation on NTS Neurons Sending Projections to Ventral Medulla of Rats.

Peripheral chemoreflex is activated during short-term sustained hypoxia (SH), and the first synapse of these afferents is located in Nucleus Tractus Solitarius(NTS). NTS neurons projecting to the ventral lateral medulla (NTS-VLM) are part of the respiratory pathways of the chemoreflex. SH increases the magnitude of basal respiratory parameters in rats from Wistar-Hannover strain. In this study, we hypothesized that the observed changes in the respiratory pattern in response to SH were due to changes in the GABAergic modulation of the synaptic transmission of NTS-VLM neurons. We used an electrophysiological approach to record the synaptic activity of NTS neurons labeled with a retrograde tracer previously microinjected into VLM of Wistar-Hannover rats submitted to 24 h SH. The data are showing that: (a) the amplitude of evoked inhibitory currents in NTS-VLM neurons of SH rats was reduced and not accompanied by any change in rise-time and decay-time; (b) the 1/CV2 and the number of failures in response to evoked currents were also affected by SH; (c) the frequency of spontaneous inhibitory currents was reduced by SH without changes in amplitude and half-width. These effects of SH were observed in NTS-VLM neurons located in caudal and intermediate NTS, but not in NTS-VLM neurons located in the rostral NTS. We conclude that SH causes a reduction in inhibitory modulation onto NTS-VLM neurons by pre-synaptic mechanisms, which may contribute to the observed changes in the respiratory pattern of Wistar-Hannover rats submitted to SH.

Changes in the autonomic and respiratory patterns in mice submitted to short-term sustained hypoxia.

NEW FINDINGS: What is the central question of this study? Do mice submitted to sustained hypoxia present autonomic and respiratory changes similarly to rats? What is the main finding and its importance? Arterial pressure in the normal range, reduced baseline heart rate and tachypnoea were observed in behaving sustained hypoxia mice. Recordings in the in situ preparation of mice submitted to sustained hypoxia show an increase in cervical vagus nerve activity and a simultaneous reduction in thoracic sympathetic nerve activity correlated with changes in the respiratory cycle. Therefore, mice are an important model for studies on the modulation of sympathetic activity to the cardiovascular system and the vagus innervation of the upper airways due to changes in the respiratory network induced by sustained hypoxia. ABSTRACT: Short-term sustained hypoxia (SH) in rats induces sympathetic overactivity and hypertension due to changes in sympathetic-respiratory coupling. However, there are no consistent data about the effect of SH on mice due to the different protocols of hypoxia and difficulties associated with the handling of these rodents under different experimental conditions. In situ recordings of autonomic and respiratory nerves in SH mice have not been performed yet. Herein, we evaluated the effects of SH ( FiO2  = 0.1 for 24 h) on baseline mean arterial pressure (MAP), heart rate (HR), respiratory frequency (fR ) and responses to chemoreflex activation in behaving SH mice. A characterization of changes in cervical vagus (cVN), thoracic sympathetic (tSN), phrenic (PN) and abdominal (AbN) nerves in SH mice using the in situ working heart-brainstem preparation was also performed. SH mice presented normal MAP, significant reduction in baseline HR, increase in baseline fR , as well as increase in the magnitude of bradycardic response to chemoreflex activation. In in situ preparations, SH mice presented a reduction in PN discharge frequency, and increases in the time of expiration and incidence of late-expiratory bursts in AbN activity. Nerve recordings also indicated a significant increase in cVN activity and a significant reduction in tSN activity during expiration in SH mice. These findings make SH mice an important experimental model for better understanding how changes in the respiratory network may impact on the modulation of vagal control to the upper airways, as well as in the sympathetic activity to the cardiovascular system.

Distinct cardiovascular and respiratory responses to short-term sustained hypoxia in juvenile Sprague Dawley and Wistar Hannover rats.

Short-term sustained hypoxia (SH) elicits active expiration, augmented late-expiratory (late-E) sympathetic activity, increased arterial pressure and ventilation, and amplified sympathetic and abdominal expiratory responses to chemoreflex activation in rats of the Wistar-Ribeirão Preto (WRP) strain. Herein, we investigated whether SH can differentially affect the cardiovascular and respiratory outcomes of Sprague-Dawley (SD) and Wistar Hannover (WH) rats and compared the results with previous data using WRP rats. For this, we exposed SD and WH rats to SH (FiO2 = 0.1) for 24 h and evaluated arterial pressure, sympathetic activity, and respiratory pattern. SD rats presented increased arterial pressure, respiratory rate and tidal volume, as well as augmented late-E expiratory motor output and increased sympathetic outflow due to post-inspiratory and late-E sympathetic overactivity. WH rats presented reduced changes, suggesting lower responsiveness of this strain to this SH protocol. The magnitudes of changes in sympathetic and abdominal expiratory motor activities to chemoreflex activation in SD rats were reduced by SH. Pressor responses to chemoreflex activation were shown to be blunted in SD and WH rats after SH. The data are showing that SD, WH, and WRP rat strains exhibit marked differences in their cardiovascular, autonomic and respiratory responses to 24-h SH and draw attention to the importance of rat strain for studies exploring the underlying mechanisms involved in the neuronal changes induced by the experimental model of SH.

Centrally acting adrenomedullin in the long-term potentiation of sympathetic vasoconstrictor activity induced by intermittent hypoxia in rats.

NEW FINDINGS: What is the central question of this study? Adrenomedullin in the rostral ventrolateral medulla (RVLM) increases sympathetic activity; given that adrenomedullin is released during hypoxia, what are the effects of its agonism and antagonism in the RVLM after chronic intermitent hypoxia (CIH) exposure? What is the main finding and its importance? CIH exposure sensitizes adrenomedullin-dependent mechanisms in the RVLM, supporting its role as a sympathoexcitatory neuromodulator. A novel mechanism was identified for the generation of sympathetic overdrive and hypertension associated with hypoxia, providing potential guidance on new therapeutic approaches for controlling sympathetic hyperactivity in diseases such as sleep apnoea and neurogenic hypertension. ABSTRACT: Adrenomedullin in the rostral ventrolateral medulla (RVLM) has been shown to increase sympathetic activity whereas the antagonism of its receptors inhibited this autonomic activity lowering blood pressure in conditions of hypertension. Given that hypoxia is a stimulant for releasing adrenomedullin, we hypothesized that the presence of this peptide in the RVLM associated with chronic intermittent hypoxia (CIH) would cause sympathetic overdrive. Juvenile male rats (50-55 g) submitted to CIH (6% oxygen every 9 min, 8 h day-1 for 10 days) were studied in an arterially perfused in situ preparation where sympathetic activity was recorded. In control rats (n = 6), exogenously applied adrenomedullin in the RVLM raised baseline sympathetic activity when combined with episodic activation of peripheral chemoreceptors (KCN 0.05%, 5 times every 5 min). This sympathoexcitatory response was markedly amplified in rats previously exposed to CIH (n = 6). The antagonism of adrenomedullin receptors in the RVLM caused a significant reduction in sympathetic activity in the CIH group (n = 7), but not in controls (n = 8). The transient reflex-evoked sympathoexcitatory response to peripheral chemoreceptor stimulation was not affected by either adrenomedullin or adrenomedullin receptor antagonism in the RVLM of control and CIH rats. Our findings indicate that CIH sensitizes the sympathoexcitatory networks within the RVLM to adrenomedullin, supporting its role as an excitatory neuromodulator when intermittent hypoxia is present. These data reveal novel state-dependent mechanistic insights into the generation of sympathetic overdrive and provide potential guidance on possible unique approaches for controlling sympathetic discharge in diseases such as sleep apnoea and neurogenic hypertension.

Cardiovascular and respiratory profiles during the sleep-wake cycle of rats previously submitted to chronic intermittent hypoxia.

NEW FINDINGS: What is the central question of this study? Chronic intermittent hypoxia (CIH) causes increased arterial pressure (AP), sympathetic overactivity and changes in expiratory modulation of sympathetic activity. However, changes in the short-term sleep-wake cycle pattern after CIH and their potential impact on cardiorespiratory parameters have not been reported previously. What is the main finding and its importance? Exposure to CIH for 10 days elevates AP in wakefulness and sleep but does not cause major changes in short-term sleep-wake cycle pattern. A higher incidence of muscular expiratory activity was observed in rats exposed to CIH only during wakefulness, indicating that active expiration is not required for the increase in AP in rats submitted to CIH. ABSTRACT: Chronic intermittent hypoxia (CIH) increases arterial pressure (AP) and changes sympathetic-respiratory coupling. However, the alterations in the sleep-wake cycle after CIH and their potential impact on cardiorespiratory parameters remain unknown. Here, we evaluated whether CIH-exposed rats present changes in their short-term sleep-wake cycle pattern and in cardiorespiratory parameters. Male Wistar rats (∼250 g) were divided into CIH and control groups. The CIH rats were exposed to 8 h day-1 of cycles of normoxia (fraction of inspired O2  = 0.208, 5 min) followed by hypoxia (fraction of inspired O2  = 0.06, 30-40 s) for 10 days. One day after CIH, electrocorticographic activity, cervical EMG, AP and heart rate were recorded for 3 h. Plethysmographic recordings were collected for 2 h. A subgroup of control and CIH rats also had the diaphragm and oblique abdominal muscle activities recorded. Chronic intermittent hypoxia did not alter the time for sleep onset, total time awake, durations of rapid eye movement (REM) and non-REM (NREM) sleep and number of REM episodes in the 3 h recordings. However, a significant increase in the duration of REM episodes was observed. The AP and heart rate were increased in all phases of the cycle in rats exposed to CIH. Respiratory frequency and ventilation were similar between groups in all phases, but tidal volume was increased during NREM and REM sleep in rats exposed to CIH. An increase in the incidence of active expiration during wakefulness was observed in rats exposed to CIH. The data show that CIH-related hypertension is not caused by changes in the sleep-wake cycle and suggest that active expiration is not required for the increase in AP in freely moving rats exposed to CIH.

Enhancement of excitatory transmission in NTS neurons projecting to ventral medulla of rats exposed to sustained hypoxia is blunted by minocycline.

KEY POINTS: Rats subjected to sustained hypoxia (SH) present increases in arterial pressure (AP) and in glutamatergic transmission in the nucleus tractus solitarius (NTS) neurons sending projections to ventrolateral medulla (VLM). Treatment with minocycline, a microglial inhibitor, attenuated the increase in AP in response to SH. The increase in the amplitude of glutamatergic postsynaptic currents in the NTS-VLM neurons, induced by postsynaptic mechanisms, was blunted by minocycline treatment. The number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS of minocycline-treated rats. The data show that microglial recruitment/proliferation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the observed increase in AP. ABSTRACT: Short-term sustained hypoxia (SH) produces significant autonomic and respiratory adjustments and triggers activation of microglia, the resident immune cells in the brain. SH also enhances glutamatergic neurotransmission in the NTS. Here we evaluated the role of microglial activation induced by SH on the cardiovascular changes and mainly on glutamatergic neurotransmission in NTS neurons sending projections to the ventrolateral medulla (NTS-VLM), using a microglia inhibitor (minocycline). Direct measurement of arterial pressure (AP) in freely moving rats showed that SH (24 h, fraction of inspired oxygen ( FI,O2 ) 0.1) in vehicle and minocycline (30 mg/kg i.p. for 3 days)-treated groups produced a significant increase in AP in relation to control groups under normoxic conditions, but this increase was significantly lower in minocycline-treated rats. Whole-cell patch-clamp recordings revealed that the active properties of the membrane were comparable among the groups. Nevertheless, the amplitudes of glutamatergic postsynaptic currents, evoked by tractus solitarius stimulation, were increased in NTS-VLM neurons of SH rats. Changes in asynchronous glutamatergic currents indicated that the observed increase in amplitude was due to postsynaptic mechanisms. These changes were blunted in the SH group previously treated with minocycline. Using immunofluorescence, we found that the number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS neurons of minocycline-treated rats. Our data support the concept that microglial activation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the increase in AP observed in this experimental model.

Astrocytic modulation of glutamatergic synaptic transmission is reduced in NTS of rats submitted to short-term sustained hypoxia.

Sustained hypoxia (SH) activates chemoreceptors to produce cardiovascular and respiratory responses to bring the arterial partial pressure of O2 back to the physiological range. We evaluated the effect of SH (fraction of inspired O2 = 0.10, 24 h) on glutamatergic synaptic transmission and the interaction neuron-astrocyte in neurons of the nucleus tractus solitarii (NTS). Tractus solitarius (TS) fiber stimulation induced glutamatergic currents in neurons and astrocytes. SH increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) [-183 ± 122 pA (n = 10) vs. -353 ± 101 pA (n = 10)] and N-methyl-d-aspartate (NMDA) current amplitude [61 ± 10 pA (n = 7) vs. 102 ± 37 pA (n = 10)]. To investigate the effects of SH, we used fluoroacetate (FAC), an astrocytic inhibitor, which revealed an excitatory modulation on AMPA/kainate current and an inhibitory modulation of NMDA current in control rats. SH blunted the astrocytic modulation of AMPA [artificial cerebrospinal fluid (aCSF): -353 ± 101 pA vs. aCSF + FAC: -369 ± 76 pA (n = 10)] and NMDA currents [aCSF: 102 ± 37 pA vs. aCSF + FAC: 108 ± 32 pA (n = 10)]. SH increased AMPA current density [control: -6 ± 3.5 pA/pF (n = 6) vs. SH: -20 ± 12 pA/pF (n = 7)], suggesting changes in density, conductance, or affinity of AMPA receptors. SH produced no effect on astrocytic resting membrane potential, input resistance, and AMPA/kainate current. We conclude that SH decreased the neuron-astrocyte interaction at the NTS level, facilitating the glutamatergic transmission, which may contribute to the enhancement of cardiovascular and respiratory responses to baro- and chemoreflexes activation in SH rats. NEW & NOTEWORTHY Using an electrophysiological approach, we have shown that in nucleus tractus solitarii (NTS) from control rats, astrocytes modulate the AMPA and NMDA currents in NTS neurons, changing their excitability. Sustained hypoxia (SH) increased both glutamatergic currents in NTS neurons due to 1) a reduction in the astrocytic modulation and 2) an increase in the density of AMPA receptors. These new findings show the importance of neuron-astrocyte modulation in the excitatory synaptic transmission in NTS of control and SH rats.

Hyperexcitability and plasticity induced by sustained hypoxia on rectus abdominis motoneurons.

KEY POINTS: Acute hypoxia induces active expiration in rectus abdominis (RA) muscles in conscious freely moving rats, although its overall contribution is smaller than in internal oblique (IO) muscles. Tonically active and silent RA motoneurons were identified in in vitro preparations of rat spinal cords. Sustained hypoxia (SH) increased the synaptic strength and induced morphological changes in tonically active RA motoneurons. Expiratory RA motoneurons were recorded in the in situ preparation and SH enhanced both the excitability and the synaptic transmission in those firing during the stage 2 expiration. The present study contributes to a better understanding of the mechanisms involved in SH recruitment of RA motoneurons to induce active expiration in rats. ABSTRACT: Rectus abdominis (RA) motoneurons translate the complex respiratory brainstem inputs into effective muscle contractions. Despite their fundamental role in respiration, their functional and morphological properties are not fully understood. In the present study, we investigated for the first time the contribution of RA muscle to active expiration and characterized RA motoneurons regarding their electrical, molecular and morphological profiles in control rats and in rats submitted to sustained hypoxia (SH), which induces chronic recruitment of abdominal muscles. Electromyographic experiments in conscious freely moving control rats and SH rats showed that RA contributes to active expiration induced by acute hypoxia, although its contribution is smaller than in internal oblique muscles. in vitro whole-cell patch clamp recordings from RA motoneurons revealed two populations of cells: tonically active and silent. SH induced hyperexcitability in the tonically active cells by changing their action potential properties, and EPSCs. Three-dimensional morphological reconstructions of these cells showed that SH increased the dendritic complexity, stimulated the appearance of dendrite spines, and increased the somatic area and volume. Physiologically identified RA motoneurons, firing in two distinct phases of expiration, were recorded in the brainstem-spinal cord in situ preparation of rats. SH increased the firing frequency and EPSCs of neurons firing during stage 2 expiration. Taken together, our results show that RA motoneurons reconfigure their biophysical properties, morphology and synaptic strength to produce an appropriate expiratory drive in response to SH in rats.

Firing properties of ventral medullary respiratory neurons in sino-aortic denervated rats.

NEW FINDINGS: What is the central question of this study? After sino-aortic denervation (SAD), rats present normal levels of mean arterial pressure (MAP), high MAP variability and changes in breathing. However, mechanisms involved in SAD-induced respiratory changes and their impact on the modulation of sympathetic activity remain unclear. Herein, we characterized the firing frequency of medullary respiratory neurons after SAD. What is the main finding and its importance? Sino-aortic denervation-induced prolonged inspiration was associated with a reduced interburst frequency of pre-inspiratory/inspiratory neurons and an increased long-term variability of late inspiratory neurons, but no changes were observed in the ramp-inspiratory and post-inspiratory neurons. This imbalance in the respiratory network might contribute to the modulation of sympathetic activity after SAD. ABSTRACT: In previous studies, we documented that after sino-aortic denervation (SAD) in rats there are significant changes in the breathing pattern, but no significant changes in sympathetic activity and mean arterial pressure compared with sham-operated rats. However, the neural mechanisms involved in the respiratory changes after SAD and the extent to which they might contribute to the observed normal sympathetic activity and mean arterial pressure remain unclear. Here, we hypothesized that after SAD, rats present with changes in the firing frequency of the ventral medullary inspiratory and post-inspiratory neurons. To test this hypothesis, male Wistar rats underwent SAD or sham surgery and 3 days later were surgically prepared for an in situ experiment. The duration of inspiration significantly increased in SAD rats. During inspiration, the total firing frequency of ramp-inspiratory, pre-inspiratory/inspiratory and late-inspiratory neurons was not different between groups. During post-inspiration, the total firing frequency of post-inspiratory neurons was also not different between groups. Furthermore, the data demonstrate a reduced interburst frequency of pre-inspiratory/inspiratory neurons and an increased long-term variability of late-inspiratory neurons in SAD compared with sham-operated rats. These findings indicate that the SAD-induced prolongation of inspiration was not accompanied by alterations in the total firing frequency of the ventral medullary respiratory neurons, but it was associated with changes in the long-term variability of late-inspiratory neurons. We suggest that the timing imbalance in the respiratory network in SAD rats might contribute to the modulation of presympathetic neurons after removal of baroreceptor afferents.

Locus Coeruleus as a vigilance centre for active inspiration and expiration in rats.

At rest, inspiration is an active process while expiration is passive. However, high chemical drive (hypercapnia or hypoxia) activates central and peripheral chemoreceptors triggering reflex increases in inspiration and active expiration. The Locus Coeruleus contains noradrenergic neurons (A6 neurons) that increase their firing frequency when exposed to hypercapnia and hypoxia. Using recently developed neuronal hyperpolarising technology in conscious rats, we tested the hypothesis that A6 neurons are a part of a vigilance centre for controlling breathing under high chemical drive and that this includes recruitment of active inspiration and expiration in readiness for flight or fight. Pharmacogenetic inhibition of A6 neurons was without effect on resting and on peripheral chemoreceptors-evoked inspiratory, expiratory and ventilatory responses. On the other hand, the number of sighs evoked by systemic hypoxia was reduced. In the absence of peripheral chemoreceptors, inhibition of A6 neurons during hypercapnia did not affect sighing, but reduced both the magnitude and incidence of active expiration, and the frequency and amplitude of inspiration. These changes reduced pulmonary ventilation. Our data indicated that A6 neurons exert a CO2-dependent modulation of expiratory drive. The data also demonstrate that A6 neurons contribute to the CO2-evoked increases in the inspiratory motor output and hypoxia-evoked sighing.

Purinergic plasticity within petrosal neurons in hypertension.

The carotid bodies are peripheral chemoreceptors and contribute to the homeostatic maintenance of arterial levels of O2, CO2, and [H+]. They have attracted much clinical interest recently because of the realization that aberrant signaling in these organs is associated with several pathologies including hypertension. Herein, we describe data suggesting that sympathetic overactivity in neurogenic hypertension is, at least in part, dependent on carotid body tonicity and hyperreflexia that is related to changes in the electrophysiological properties of chemoreceptive petrosal neurons. We present results showing critical roles for both ATP levels in the carotid bodies and expression of P2X3 receptors in petrosal chemoreceptive, but not baroreceptive, terminals in the etiology of carotid body tonicity and hyperreflexia. We discuss mechanisms that may underlie the changes in electrophysiological properties and P2X3 receptor expression in chemoreceptive petrosal neurons, as well as factors affecting ATP release by cells within the carotid bodies. Our findings support the notion of targeting the carotid bodies to reduce sympathetic outflow and arterial pressure, emphasizing the potential clinical importance of modulating purinergic transmission to treat pathologies associated with carotid body dysfunction but, importantly, sparing physiological chemoreflex function.

Previous exposure to chronic intermittent hypoxia blunts the development of one-kidney, one-clip hypertension in rats.

NEW FINDINGS: What is the central question of this study? Chronic intermittent hypoxia (CIH) and one-kidney, one-clip experimental models lead to sympathetic overactivity and hypertension. The present study explored the impact of previous exposure to CIH on one-kidney, one-clip renal hypertension; we hypothesized that CIH potentiates its development. What is the main finding and its importance? The development of one-kidney, one-clip renal hypertension was attenuated by previous exposure to CIH, and this protective effect was eliminated by carotid body denervation. These findings indicate that inputs from peripheral chemoreceptors in CIH-preconditioned rats play a role in preventing the increase in sympathetic activity and arterial pressure induced by one-kidney, one-clip renal hypertension. ABSTRACT: Chronic intermittent hypoxia (CIH) and one-kidney, one-clip (1K, 1C) experimental models lead to sympathetic overactivity and hypertension. We hypothesized that previous exposure to CIH potentiates the development of 1K, 1C renal hypertension. Male rats were divided into the following four groups: Control-1K, 1C, maintained under normoxia followed by 1K, 1C surgery (n = 19); Control-Sham, maintained under normoxia, followed by sham surgery (n = 19); CIH-1K, 1C, exposed to CIH (10 days) and 1K, 1C surgery (n = 19); and CIH-Sham, exposed to CIH and sham surgery (n = 18). Animals were catheterized 8 days after 1K, 1C or Sham surgeries and cardiovascular and respiratory parameters recorded on the following day. Baseline mean arterial pressure was higher in Control-1K, 1C than in Control-Sham rats (P < 0.05) and was higher in CIH-1K, 1C than in CIH-Sham rats (P < 0.05). However, the increase in mean arterial pressure in CIH-1K, 1C animals was significantly blunted in comparison to Con-1K, 1C rats (P < 0.05), indicating that previous exposure to CIH attenuates the development of renal hypertension. Systemic administration of hexamethonium, a ganglionic blocker, promoted a larger hypotensive response in Con-1K, 1C compared with CIH-1K, 1C rats (P < 0.05), suggesting that sympathetic activity was attenuated in rats previously exposed to the CIH protocol. In addition, removal of the carotid bodies before 1K, 1C renal hypertension eliminated the protective effect of CIH preconditioning on the development of the 1K, 1C hypertension. We conclude that previous exposure to CIH attenuates the development of renal hypertension via a carotid body-dependent mechanism.