Daniela Accorsi-Mendonça

The balloon catheter induces an increase in contralateral carotid artery reactivity to angiotensin II and phenylephrine

The effects of balloon injury on the reactivity of ipsilateral and contralateral carotid arteries were compared to those observed in arteries from intact animals (control arteries). Carotid arteries were obtained from Wistar rats 2, 4, 7, 15, 30 or 45 days after injury and mounted in an isolated organ bath. Reactivity to angiotensin II (Ang II), phenylephrine (Phe) and bradykinin (BK) was studied. Curves were constructed in the absence or presence of endothelium or after incubation with 10 μM indomethacin, 500 μM valeryl salicylate or 0.1 μM celecoxib. Phe, Ang II and BK maximum effects (Emax) were decreased in ipsilateral arteries when compared to control arteries. No differences were observed among pD2 or Hill coefficient. Emax to Phe (4 and 7 days) and to Ang II (15 and 30 days) increased in the contralateral artery. In addition, Phe or Ang II reactivity was not significantly different in aorta rings from control or carotid‐injured animals. The increased responsiveness of contralateral artery was not due to changes in carotid blood flow or resting membrane potential. The endothelium‐dependent inhibitory component is not present in the contraction of contralateral arteries and it is not related to superoxide anion production. Indomethacin decreased contralateral artery responsiveness to Phe and Ang II. Valeryl salicylate reduced the Ang II response in contralateral and control arteries. Celecoxib decreased the Phe Emax of contralateral artery. In conclusion, decreased endothelium‐derived factors and increased prostanoids appear to be responsible for the increased reactivity of contralateral arteries after injury.

Synaptic transmission of baro- and chemoreceptors afferents in the NTS second order neurons.

Second order neurons in the nucleus tractus solitarius (NTS) process and integrate the afferent information from arterial baroreceptors with high fidelity and precise timing synaptic transmission. Since 2nd-order NTS neurons receiving baroreceptors inputs are relatively well characterized, their electrophysiological profile has been accepted as a general characteristic for all 2nd-order NTS neurons involved with the processing of different sensorial inputs. On the other hand, the synaptic properties of other afferent systems in NTS, such as the peripheral chemoreceptors, are not yet well understood. In this context, in previous studies we demonstrated that in response to repetitive afferents stimulation, the chemoreceptors 2nd-order NTS neurons also presented high fidelity of synaptic transmission, but with a large variability in the latency of evoked responses. This finding is different in relation to the precise timing transmission for baroreceptor 2nd-order NTS neurons, which was accepted as a general characteristic profile for all 2nd order neurons in the NTS. In this brief review we discuss this new concept as an index of complexity of the sensorial inputs to NTS with focus on the synaptic processing of baro- and chemoreceptor afferents.

Glial cells modulate the synaptic transmission of NTS neurons sending projections to ventral medulla of Wistar rats

There is evidence that sympathoexcitatory and respiratory responses to chemoreflex activation involve ventrolateral medulla‐projecting nucleus tractus solitarius (NTS) neurons (NTS‐VLM neurons) and also that ATP modulates this neurotransmission. Here, we evaluated whether or not astrocytes is the source of endogenous ATP modulating the synaptic transmission in NTS‐VLM neurons. Synaptic activities of putative astrocytes or NTS‐VLM neurons were recorded using whole cell patch clamp. Tractus solitarius (TS) stimulation induced TS‐evoked excitatory postsynaptic currents (TS‐eEPSCs) in NTS‐VLM neurons as well in NTS putative astrocytes, which were also identified by previous labeling. Fluoracetate (FAC), an inhibitor of glial metabolism, reduced TS‐eEPSCs amplitude (−85.6 ± 16 vs. −39 ± 7.1 pA, n = 12) and sEPSCs frequency (2.8 ± 0.5 vs. 1.8 ± 0.46 Hz, n = 10) in recorded NTS‐VLM neurons, indicating a gliomodulation of glutamatergic currents. To verify the involvement of endogenous ATP a purinergic antagonist was used, which reduced the TS‐eEPSCs amplitude (−207 ± 50 vs. −149 ± 50 pA, n = 6), the sEPSCs frequency (1.19 ± 0.2 vs. 0.62 ± 0.11 Hz, n = 6), and increased the paired‐pulse ratio (PPR) values (~20%) in NTS‐VLM neurons. Simultaneous perfusion of Pyridoxalphosphate‐6‐azophenyl‐2′,5′‐disulfonic acid (iso‐PPADS) and FAC produced reduction in TS‐eEPSCs similar to that observed with iso‐PPADS or FAC alone, indicating that glial cells are the source of ATP released after TS stimulation. Extracellular ATP measurement showed that FAC reduced evoked and spontaneous ATP release. All together these data show that putative astrocytes are the source of endogenous ATP, which via activation of presynaptic P2X receptors, facilitates the evoked glutamate release and increases the synaptic transmission efficacy in the NTS‐VLM neurons probably involved with the peripheral chemoreflex pathways.

SYNAPTIC PROFILE OF NUCLEUS TRACTUS SOLITARIUS NEURONS INVOLVED WITH THE PERIPHERAL CHEMOREFLEX PATHWAYS

The glomus cells in the carotid bodies (CB) detect alterations in pH and pCO₂ and low pO₂ level in arterial blood. The carotid sinus nerve conveys the information related to the oxygen level to 2nd-order neurons in the nucleus tractus solitarius (NTS) via tractus solitarius (TS), which is part of the chemoreflex pathways. It has been demonstrated that in 2nd-order NTS neurons receiving inputs from the aortic depressor nerve (ADN), the TS stimulation presents high temporal fidelity. However, the temporal properties of synaptic activity in NTS neurons receiving inputs from CB were not yet fully investigated. Herein using patch-clamp recordings in NTS brainstem slices, we studied TS-evoked excitatory postsynaptic currents (TS-eEPSCs) on morphologically identified 2nd-order NTS neurons that receive afferent inputs from the CB and compared with 2nd-order ADN-NTS neurons recorded in the same experimental conditions. The amplitudes of TS-eEPSCs were similar in both groups, but the latencies and standard deviation (SD) of latency were significantly higher in the CB-NTS neurons (latency: 4±0.2 ms, SD: 0.49±0.03 ms) than in ADN-NTS neurons (latency: 3.3±0.3 ms, SD: 0.19±0.02 ms; P=0.049 for latency and P<0.001 for SD of latency). In a series of double-labeling experiments, we confirmed that some CB-NTS 2nd-order neurons send direct projections to the rostral ventrolateral medulla (RVLM). We conclude that: (a) CB-NTS 2nd-order neurons present temporally distinct postsynaptic currents when compared with ADN-NTS 2nd-order neurons; (b) low SD of latency of TS-eEPSCs is not necessarily a characteristic of all 2nd-order neurons in the NTS; and (c) the presence of direct connections between these 2nd-order neurons in the NTS and RVLM is indicative that these synaptic properties of CB-NTS neurons are relevant for the processing of respiratory and autonomic responses to chemoreflex activation.

Are l-glutamate and ATP cotransmitters of the peripheral chemoreflex in the rat nucleus tractus solitarius ?

Peripheral chemoreflex activation in awake rats or in the working heart–brainstem preparation (WHBP) produces sympathoexcitation, bradycardia and an increase in the frequency of phrenic nerve activity. Our focus is the neurotransmission of the sympathoexcitatory component of the chemoreflex within the nucleus of the tractus solitarius (NTS), and recently we verified that the simultaneous antagonism of ionotropic glutamate and purinergic P2 receptors in the NTS blocked the pressor response and increased thoracic sympathetic activity in awake rats and WHBP, respectively, in response to peripheral chemoreflex activation. These previous data suggested the involvement of ATP and L‐glutamate in the NTS in the processing of the sympathoexcitatory component of the chemoreflex by unknown mechanisms. For a better understanding of these mechanisms, here we used a patch‐clamp approach in brainstem slices to evaluate the characteristics of the synaptic transmission of NTS neurons sending projections to the ventral medulla, which include the premotor neurons involved in the generation of the sympathetic outflow. The NTS neurons sending projections to the ventral medulla were identified by previous microinjection of the membrane tracer dye, 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI), in the ventral medulla and the spontaneous (sEPSCs) and tractus solitarius (TS)‐evoked excitatory postsynaptic current (TS‐eEPSCs) were recorded using patch clamp. With this approach, we made the following observations on NTS neurons projecting to the ventral medulla: (i) the sEPSCs and TS‐eEPSCs of DiI‐labelled NTS neurons were completely abolished by 6,7‐dinitroquinoxaline‐2,3(1H,4H)‐dione (DNQX), an antagonist of ionotropic non‐NMDA glutamatergic receptors, showing that they are mediated by L‐glutamate; (ii) application of ATP increased the frequency of appearance of spontaneous glutamatergic currents, reflecting an increased exocytosis of glutamatergic vesicles; and (iii) ATP decreased the peak of TS‐evoked glutamatergic currents. We conclude that L‐glutamate is the main neurotransmitter of spontaneous and TS‐evoked synaptic activities in the NTS neurons projecting to the ventral medulla and that ATP has a dual modulatory role on this excitatory transmission, facilitating the spontaneous glutamatergic transmission and inhibiting the TS‐evoked glutamatergic transmission. These data also suggest that ATP is not acting as a cotransmitter with L‐glutamate, at least at the level of this subpopulation of NTS neurons studied.

Enhanced Firing in NTS Induced by Short-Term Sustained Hypoxia Is Modulated by Glia-Neuron Interaction

Humans ascending to high altitudes are submitted to sustained hypoxia (SH), activating peripheral chemoreflex with several autonomic and respiratory responses. Here we analyzed the effect of short-term SH (24 h, FIO210%) on the processing of cardiovascular and respiratory reflexes using an in situ preparation of rats. SH increased both the sympatho-inhibitory and bradycardiac components of baroreflex and the sympathetic and respiratory responses of peripheral chemoreflex. Electrophysiological properties and synaptic transmission in the nucleus tractus solitarius (NTS) neurons, the first synaptic station of afferents of baroreflexes and chemoreflexes, were evaluated using brainstem slices and whole-cell patch-clamp. The second-order NTS neurons were identified by previous application of fluorescent tracer onto carotid body for chemoreceptor afferents or onto aortic depressor nerve for baroreceptor afferents. SH increased the intrinsic excitability of NTS neurons. Delayed excitation, caused by A-type potassium current (IKA), was observed in most of NTS neurons from control rats. The IKA amplitude was higher in identified second-order NTS neurons from control than in SH rats. SH also blunted the astrocytic inhibition of IKA in NTS neurons and increased the synaptic transmission in response to afferent fibers stimulation. The frequency of spontaneous excitatory currents was also increased in neurons from SH rats, indicating that SH increased the neurotransmission by presynaptic mechanisms. Therefore, short-term SH changed the glia-neuron interaction, increasing the excitability and excitatory transmission of NTS neurons, which may contribute to the observed increase in the reflex sensitivity of baroreflex and chemoreflex in in situ preparation.

Immunoreactivity for neuronal NOS and fluorescent indication of NO formation in the NTS of juvenile rats submitted to chronic intermittent hypoxia.

Exposure to chronic intermittent hypoxia (CIH) leads to significant autonomic and respiratory changes, similar to those observed in obstructive sleep apnea. The hypertension associated with CIH is due to sympathoexcitation triggered by long-term exposure to intermittent hypoxia. However, the mechanisms underlying these effects are unknown. Changes in central regulation of sympathetic activity may underlie CIH-induced hypertension. Since NO appears to be mainly sympathoinhibitory in the nucleus of the solitary tract (NTS), we hypothesized that CIH augments sympathetic activity, in part by reducing neuronal nitric oxide synthase (nNOS) expression and consequently nitric oxide (NO) production in this brain region. To test our hypothesis, juvenile male Wistar rats were exposed to CIH for 8 h/day for 10 days and sections of perfused brainstem were either stained to reveal nNOS-immunoreactivity or loaded with DAF 2-DA to label neurons containing NO. CIH rats showed a significant increase in mean arterial pressure and heart rate compared to controls. However, there was no significant difference in the distribution, staining intensity or numbers of nNOS-immunoreactive neurons in the NTS between experimental and control rats. We also found no significant change in NO content in the DAF 2-DA-loaded sections of NTS from CIH rats. Our data show that NO is not altered in the NTS of juvenile CIH rats, suggesting that nitrergic mechanisms, at least in the NTS, are unlikely to be involved in the sympathetic excitation that generates the hypertension observed after 10 days of CIH.

Evolution and physiology of neural oxygen sensing

Major evolutionary trends in animal physiology have been heavily influenced by atmospheric O2 levels. Amongst other important factors, the increase in atmospheric O2 which occurred in the Pre-Cambrian and the development of aerobic respiration beckoned the evolution of animal organ systems that were dedicated to the absorption and transportation of O2, e.g., the respiratory and cardiovascular systems of vertebrates. Global variations of O2 levels in post-Cambrian periods have also been correlated with evolutionary changes in animal physiology, especially cardiorespiratory function. Oxygen transportation systems are, in our view, ultimately controlled by the brain related mechanisms, which senses changes in O2 availability and regulates autonomic and respiratory responses that ensure the survival of the organism in the face of hypoxic challenges. In vertebrates, the major sensorial system for oxygen sensing and responding to hypoxia is the peripheral chemoreflex neuronal pathways, which includes the oxygen chemosensitive glomus cells and several brainstem regions involved in the autonomic regulation of the cardiovascular system and respiratory control. In this review we discuss the concept that regulating O2 homeostasis was one of the primordial roles of the nervous system. We also review the physiology of the peripheral chemoreflex, focusing on the integrative repercussions of chemoreflex activation and the evolutionary importance of this system, which is essential for the survival of complex organisms such as vertebrates. The contribution of hypoxia and peripheral chemoreflex for the development of diseases associated to the cardiovascular and respiratory systems is also discussed in an evolutionary context.

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 microM 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 vanniloid 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.

Pacemaking Property of RVLM Presympathetic Neurons.

Despite several studies describing the electrophysiological properties of RVLM presympathetic neurons, there is no consensus in the literature about their pacemaking property, mainly due to different experimental approaches used for recordings of neuronal intrinsic properties. In this review we are presenting a historical retrospective about the pioneering studies and their controversies on the intrinsic electrophysiological property of auto-depolarization of these cells in conjunction with recent studies from our laboratory documenting that RVLM presympathetic neurons present pacemaking capacity. We also discuss whether increased sympathetic activity observed in animal models of neurogenic hypertension (CIH and SHR) are dependent on changes in the intrinsic electrophysiological properties of these cells or due to changes in modulatory inputs from neurons of the respiratory network. We also highlight the key role of INaP as the major current contributing to the pacemaking property of RVLM presympathetic neurons.