The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla/Bötzinger/pre-Bötzinger complexes (RVLM/BötC/pre-BötC) on the respiratory modulation of sympathoexcitatory response to peripheral chemoreflex activation (chemoreflex) was evaluated in the working heart-brain stem preparation of juvenile rats. We identified different types of baro- and chemosensitive presympathetic and respiratory neurons intermingled within the RVLM/BötC/pre-BötC. Bilateral microinjections of kynurenic acid (KYN) into the rostral aspect of RVLM (RVLM/BötC) produced an additional increase in frequency of the phrenic nerve (PN: 0.38 ± 0.02 vs. 1 ± 0.08 Hz; P < 0.05; n = 18) and hypoglossal (HN) inspiratory response (41 ± 2 vs. 82 ± 2%; P < 0.05; n = 8), but decreased postinspiratory (35 ± 3 vs. 12 ± 2%; P < 0.05) and late-expiratory (24 ± 4 vs. 2 ±1%; P < 0.05; n = 5) abdominal (AbN) responses to chemoreflex. Likewise, expiratory vagal (cVN; 67 ± 6 vs. 40 ± 2%; P < 0.05; n = 5) and expiratory component of sympathoexcitatory (77 ± 8 vs. 26 ± 5%; P < 0.05; n = 18) responses to chemoreflex were reduced after KYN microinjections into RVLM/BötC. KYN microinjected into the caudal aspect of the RVLM (RVLM/pre-BötC; n = 16) abolished inspiratory responses [PN (n = 16) and HN (n = 6)], and no changes in magnitude of sympathoexcitatory (n = 16) and expiratory (AbN and cVN; n = 10) responses to chemoreflex, producing similar and phase-locked vagal, abdominal, and sympathetic responses. We conclude that in relation to chemoreflex activation 1) ionotropic glutamate receptors in RVLM/BötC and RVLM/pre-BötC are pivotal to expiratory and inspiratory responses, respectively; and 2) activation of ionotropic glutamate receptors in RVLM/BötC is essential to the coupling of active expiration and sympathoexcitatory response.
- sympathetic–respiratory coupling
- respiratory control
the eupneic respiratory pattern consists of three phases: inspiration, expiratory phase 1 or postinspiration (post-I), and expiratory phase 2 (E2) (Abdala et al. 2009; Ramirez and Richter 1996; Richter 1982; Smith et al. 2007). The well-coordinated three-phase activity of cranial and spinal respiratory motor outputs is generated by a complex brain stem respiratory network, which includes interactions of neurons of Bötzinger (BötC) and pre-Bötzinger complexes (pre-BötC) located in the ventral respiratory column (VRC) (Ezure 1990; Rybak et al. 2007; Smith et al. 2007; Tian et al. 1999). The activities of these neurons are coordinated by inputs from respiratory pontine nucleus, raphe nucleus, and nucleus of the solitary tract (NTS) (Alheid et al. 2004; Dutschmann and Herbert 2006; Rybak et al. 2004a, 2008). On the other hand, it is well known that sympathetic activity, generated mainly by presympathetic neurons located in the rostral ventrolateral medulla (RVLM) (Llewellyn-Smith et al. 1992; Mills et al. 1988; Ross et al. 1984a,b), is markedly modulated by the three phases of central respiratory activity both during baseline and chemoreflex activation (Boczek-Funcke et al. 1992; Costa-Silva et al. 2010; Häbler et al. 1994; Koshiya and Guyenet 1996; Miyawaki et al. 1995, 1996a; Pilowsky 1995; Zoccal et al. 2008). During baseline, the predominant pattern of respiratory modulation of sympathetic activity is a ramping pattern of discharge during the inspiratory phase, reaching a peak during late-inspiration or beginning of post-I (Costa-Silva et al. 2010; Miyawaki et al. 1996a; Zoccal et al. 2008), whereas during chemoreflex activation the increase in sympathetic activity occurs mainly during the expiration (postinspiration) (Costa-Silva et al. 2010; Dick et al. 2004; Koshiya and Guyenet 1994, 1996; Mandel and Schreihofer 2009), to allow appropriate integrative cardiovascular and respiratory adjustments for the maintenance of body homeostasis. Therefore, for a better understanding of this complex interrelationship it is important to know what are the neuronal mechanisms and mainly the neurotransmitter responsible for generating baseline and chemoreflex-induced respiratory modulation of sympathetic activity. For this reason the main focus of the present study is on the coupling of sympathoexcitatory response and active expiration in response to peripheral chemoreflex activation.
In the pre-BötC are located inspiratory neurons considered the kernel of generation of inspiratory rhythm in vivo, in situ, and in vitro (Rybak et al. 2007; Smith et al. 1991, 2007; St-John et al. 2009). On the other hand, the expiratory activity during baseline seems to be generated mainly by expiratory neurons of BötC (Ezure et al. 2003a,b,c; Tian et al. 1999) while during high respiratory drive, such as in conditions of hypercapnia/hypoxia, a distinct expiratory generator located at the retrotrapezoid/parafacial respiratory group (RTN/pFRG) is activated and plays a critical role in the generation of active expiration (Abdala et al. 2009; Molkov et al. 2010, 2011). Intermingled with the BötC and pre-BötC neurons are the presympathetic neurons of the RVLM, which anteroposterior distribution is as long as 700 μm from the caudal end of the facial nucleus to the caudal ventrolateral medulla (CVLM) (Dobbins and Feldman 1994; Wang et al. 2002, 2009). There is evidence that stimulation of neurons in the RVLM/BötC and RVLM/pre-BötC produces distinct pattern of sympathetic and respiratory responses in anesthetized (Monnier et al. 2003) and unanesthetized rats (Moraes et al. 2011). With respect to respiratory responses to stimulation of pre-BötC, there is evidence that the patterns in anesthetized cats are different (Solomon 2002; Solomon et al. 1999).
Respiratory modulation during baseline or chemoreflex activation produces changes in sympathetic activity with distinct effects during inspiration and expiration, but the neurochemical mechanisms involved remain to be fully understood (Costa-Silva et al. 2010; Koshiya and Guyenet 1996; Mandel and Schreihofer 2009; Zoccal et al. 2008). The RVLM contains presympathetic neurons that exhibit post-I activity and may be responsible for the generation of the post-I peak seen in the sympathetic activity during baseline and chemoreflex activation (Haselton and Guyenet 1989; Miyawaki et al. 1995). There is also evidence that in the RVLM, glutamatergic neurotransmission is important for the post-I activity during baseline in the thoracic and lumbar sympathetic activities in anaesthetized rats (Guyenet et al. 1990; Miyawaki et al. 1996a). It is important to note that the origin of the respiratory modulation of sympathetic activity during baseline and the subregion of RVLM involved in this modulation are not yet fully understood. Regarding chemoreflex activation, the increase in sympathetic activity occurs by two distinct components: a respiratory-related oscillation, dependent on inhibitory connections between RVLM-CVLM, and another tonic, respiratory-independent component, whose changes are probably mediated by direct glutamatergic projections from NTS to RVLM presympathetic neurons (Koshiya and Guyenet 1996; Koshiya et al. 1993; Mandel and Schreihofer 2009). However, in these previous studies the evaluation of an index of the expiratory and inspiratory outflows was restricted to the phrenic nerve recordings. Therefore, the precise mechanisms and origin of expiratory and inspiratory modulation on sympathetic activity in response to chemoreflex activation were not yet evaluated. In the present study we hypothesized that the mechanisms generating expiratory and inspiratory outflows during baseline or in the challenges produced by chemoreflex activation, as well as the baseline and chemoreflex-induced respiratory modulation on sympathetic activity, involve glutamatergic neurotransmission in different subregions of the RVLM/BötC/pre-BötC. To test these hypotheses we used the in situ working heart-brain stem preparation (WHBP) of juvenile rats to study the respiratory network and respiratory–sympathetic coupling before and after the antagonism of ionotropic glutamate receptors in RVLM/BötC/pre-BötC, while recording sympathetic and respiratory nerves simultaneously.
The experiments were performed on male Wistar rats (60–90 g) provided by the Animal Care Facility of the University of São Paulo, campus of Ribeirão Preto, Brazil, and kept on a 12-h light–dark cycle, with food and water administered without restriction. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and Ethical Principles for Animal Experimentation established by the Brazilian Committee for Animal Experimentation and approved by the Ethics Committee on Animal Experimentation of the School of Medicine of Ribeirão Preto, University of São Paulo (protocol #064/2010).
Decerebrated arterially perfused in situ preparation.
Rats were deeply anesthetized with halothane (Astra Zeneca, Cotia, SP, Brazil) such that the withdrawal responses to noxious pinching of the tail and paw were absent. The animals were then transected caudal to the diaphragm, exsanguinated, submerged in a cooled Ringer solution, decerebrated at the precollicular level to make insentient, skinned, and had descending aorta isolated. To expose the ventral medullary surface, the preparations were placed supine and the head was fixed on a silicon elastomer cushion using insect pins that ensured that the brain stem was orientated similarly in all preparations. The trachea and esophagus were removed. All muscles and connective tissues covering to the basilar surface of occipital bone were removed. The basilar portion of the atlantooccipital membrane was cut and the bone was removed carefully using a micro-Rongeur (D.L. Micof, São Paulo, Brazil) to expose the ventral surface of the medulla in the anteroposterior extension from the vertebral arteries to the pontine nuclei. Preparations were then transferred to a recording chamber, the descending aorta was cannulated and perfused retrogradely with Ringer solution (containing, in mM: 125 NaCl, 24 NaHCO3, 3 KCl, 2.5 CaCl2, 1.25 MgSO4, 1.25 KH2PO4, 2 lactate, and 10 glucose) containing 1.25% poly(ethylene glycol) (an oncotic agent; Sigma, St Louis, MO) using a roller pump (Watson-Marlow 502s; Falmouth, Cornwall, UK) via a double-lumen cannula. The perfusion pressure was maintained in the range of 50–70 mmHg by adjusting the rate flow to 21–25 mL/min and by adding vasopressin to the perfusate (600–1.200 pM, Sigma) as previously described (Costa-Silva et al. 2010; Zoccal et al. 2008). The perfusate was gassed continuously with 5% CO2-95% O2, warmed to 31–32°C, and filtered using a nylon mesh (pore size: 25 μm; Millipore, Billerica, MA). A neuromuscular blocker (vecuronium bromide, 3–4 μg/mL; Cristália, Itapira, SP, Brazil) was added to the perfusate to avoid contractions of the respiratory muscles.
Simultaneous recordings of phrenic nerve (PN), cervical vagus nerve (cVN), hypoglossal nerve (HN), lumbar abdominal nerve (AbN; T13–L1), and thoracic sympathetic nerve (tSN; T8–T12) activities were obtained using bipolar suction electrodes mounted on separate 3D micromanipulators (Narishige, Tokyo, Japan). Single-unit recordings of BötC, pre-BötC, and RVLM neurons were made using glass microelectrodes filled with 3 M NaCl (10–30 MΩ). In some experiments the microelectrodes were filled with 2% methylene blue dye, in 0.5 M sodium acetate, to mark the recording sites by ionophoretic deposition of this dye (10 μA; 10 min). Microelectrodes were mounted in a 3D manipulator (Narishige, Tokyo, Japan) and positioned into the ventral surface of medulla under visual control (binocular microscope; Carl Zeiss, Jena, Germany) using surface landmarks (trapezoid body, rootlets of the HN, and basilar artery) for orientation. All signals were amplified, band-pass filtered (0.05–5 kHz), and digitized (3–10 kHz; CED Micro1401; Cambridge Electronic Design [CED], Cambridge, UK) to a computer using Spike2 software (version 5, CED).
Microinjections into the RVLM/BötC and RVLM/pre-BötC.
The coordinates used for microinjections into the RVLM/BötC and RVLM/pre-BötC were determined in accordance with the location in which different respiratory and presympathetic neurons are co-localized: RVLM/BötC: 350–500 μm beneath the ventral surface, 800–1,100 μm caudal to the caudal end of the trapezoid body and 1,500–1,700 μm lateral from the midline (aligned with the rootlets of the HN); RVLM/pre-BötC: 350–500 μm beneath the ventral surface, 1,200–1,500 μm caudal to the caudal end of the trapezoid body, and 1,500–1,700 μm lateral from the midline (aligned with the rootlets of the HN). Drugs were applied unilaterally or bilaterally, in accordance with the experimental protocol, via glass micropipette connected to a picopump (Picospritzer II; Parker Instruments, Cleveland, OH) and the injected volume was approximately 20 nL.
Microinjections into the RTN/pFRG and CVLM.
For microinjections into the RTN/pFRG or CVLM the coordinates used were in accordance with the location of late-E neurons and sympathoinhibitory neurons, respectively (data not shown): RTNpFRG (50–100 μm beneath the ventral surface, 500 μm caudal to the caudal end of the trapezoid body, and 1,700 μm lateral from the midline), CVLM (350–500 μm beneath the ventral surface, 1,700 to 1,800 μm caudal to the caudal end of the trapezoid body, and 1,500 to 1,700 μm lateral from the midline; aligned with the rootlets of the HN). Drugs were applied unilaterally or bilaterally, in accordance with the experimental protocol, via glass micropipette connected to a picopump (Picospritzer II; Parker Instruments) and the injected volume was approximately 20 nL.
Chemo- and baroreflex activation.
Peripheral chemoreceptors were stimulated by injections of potassium cyanide (KCN 0.05%, 50 μL) via descending aorta in the in situ preparation via the perfusion cannula as previously described (Braga et al. 2007; Costa-Silva et al. 2010). The stimulation of the peripheral chemoreflex by KCN produced consistent sympathetic and respiratory responses, which present low variability within and among experiments. In the present study, the magnitude of the inhibitory response of RVLM presympathetic neurons after electrical stimulation of the aortic depressor nerve (ADN; 0.2 ms, 3 pulses at 400 Hz) was used as an index of their barosensitivity. The stimulus intensity was set at threefold the threshold required to elicit a fall in the perfusion pressure >10 mmHg in response to ADN stimulation at 50 Hz (0.2-ms duration) for 5 s, as described by Miyawaki et al. (1995).
Spinal cord stimulation and collision test.
Spinally projecting axons of RVLM presympathetic neurons were identified by recording antidromic responses to stimulation of spinal segment T8–T12 (0.2 ms and 1 V). Two criteria were considered to characterize the spinally projecting axons of RVLM presympathetic neurons: 1) an action potential was evoked with constant latency, and 2) the collision test was positive.
Extracellular recordings from presympathetic and six distinct types of respiratory neurons were performed to identify the anteroposterior and dorsoventral extension of the ventral medulla encompassing RVLM/BötC/pre-BötC (n = 42 rats). Concentration–response curves to microinjections of l-glutamate were obtained in the RVLM/BötC (n = 8) and RVLM/pre-BötC (n = 6) in separate groups of animals. Different concentrations of l-glutamate (0.5, 2, 10, 20, 40, and 100 mM) were microinjected unilaterally (interval of 15 min between microinjections) in a random sequence into these subregions and changes in PN, cVN, AbN, HN, and tSN were evaluated. The same volume of vehicle (154 mM NaCl; 20 nL) was unilaterally microinjected into the RVLM/BötC (n = 6) or RVLM/pre-BötC (n = 6) as control experiments. The concentration of l-glutamate corresponding to the EC50 was used to determine the effective concentration of the antagonist of ionotropic glutamate receptors, kynurenic acid (KYN, IC50), in the RVLM/BötC (n = 5), and RVLM/pre-BötC (n = 4). This concentration of KYN was used in all experimental protocols involving bilateral microinjections into the RVLM/BötC/pre-BötC and activation of the peripheral chemoreflex (n = 40) or into the CVLM and RTN/pFRG regions (control experiments for misplaced microinjections; n = 8). The same volume of vehicle (154 mM NaCl; 20 nL) was bilaterally microinjected into the RVLM/BötC (n = 6) or RVLM/pre-BötC (n = 6) as control experiments. The solutions of l-glutamate, KYN (Sigma Chemical, St. Louis, MO) and KCN (Merck, Darmstadt, Germany) were freshly dissolved in saline and the pH was adjusted to 7.4 using sodium bicarbonate. The pH was determined using a pH indicator (Spezialindikator, pH 6.4–8.0; Merck).
All analyses were performed offline in rectified and integrated (τ = 50 ms) signals using Spike2 software with custom-written scripts. cVN, HN, AbN, and tSN recordings were subtracted from the electrical noise obtained at the end of each experiment after the death of the preparation.
Baseline and responses to chemoreflex activation analysis.
Baseline PN activity was assessed by their burst frequency (Hz) and duration (s). To analyze the effects of microinjections into the RVLM/BötC/pre-BötC in the average activities of respiratory and sympathetic nerves, we determined a percentage scale for each nerve (AbN, cVN, HN, and tSN), considering the peak activity observed during ischemia (pump-off; 100%) and the noise level (0%). At the end of the experiments, the noise level was determined 10–20 min after stopping arterial perfusion. To analyze the pattern of respiratory and sympathetic nerves, cycle-triggered averages (CTAs) were generated for AbN, cVN, HN, and tSN, using the PN as the trigger or reference point (50 respiratory cycles). The CTAs were divided into three parts in accordance with each nerve: inspiratory (coincident with inspiratory PN discharge), postinspiratory (first half of expiratory phase), and expiratory phase 2 (E2; second half of expiratory phase). To assess the activities in the three phases of AbN, cVN, HN, and tSN, the areas under the curves were quantified in each part and normalized by the total area (i.e. sum of areas obtained in inspiratory, post-I, and E2). This analysis was applied for each preparation and the data obtained were pooled together and compared before and after bilateral microinjection of KYN or vehicle into the RVLM/BötC/pre-BötC.
The quantification of the sympathetic and respiratory responses to chemoreflex activation was performed in accordance with a previous description by Costa-Silva et al. (2010). PN response to chemoreflex activation was assessed by the difference between baseline PN burst frequency and the peak of response observed after the stimulus (ΔPN, expressed in Hz). The tSN, AbN, cVN, and HN responses were determined as the percentage of change in relation to the respective baseline activity before chemoreflex activation.
At the end of each experiment, the brains were removed and stored in buffered formalin for 7 days and then serial transverse sections (30-μm thickness) were cut and stained with cresyl violet using the Nissl method. The site of microinjection was confirmed by the tracks of pipettes in the brain tissue. Only the rats in which the tracks of pipettes were centered in the RVLM/BötC, RVLM/pre-BötC, RTN/pFRG, or CVLM were considered for data analysis in the correspondent experimental protocol. In the experiments in which methylene blue was used to identify the location of the recorded neurons in the ventral medulla, the center of the spreading area of this dye was drawn on an outline of a medulla coronal section. In this case, the caudal tip of the facial nucleus was used as an anteroposterior reference (from 0 to 700 μm caudal) and the distance from the ventral surface (dorsoventral) and the midline (lateral) also measured.
The results are expressed as means ± SE and compared using one-way ANOVA followed by Newman–Keuls posttest. Differences were considered significant when P < 0.05.
Using the caudal tip of facial nucleus as a reference, we verified that RVLM extended approximately 700 μm caudal to this point. This characterization was performed by serial histologic sections of each brain stem (n = 137; total number of rats used in all experimental protocols) and the presence of presympathetic neurons (n = 25), which were recorded throughout this region (Fig. 1A). Regarding the anteroposterior extension of BötC, we recorded expiratory neurons: postinspiratory (post-I; n = 11) and augmenting expiratory neurons (aug-E; n = 8) between 0 and 300 μm caudal to the caudal tip of facial nucleus. With respect to the pre-BötC, similar recordings were performed of inspiratory neurons (n = 30): preinspiratory (pre-I), early-inspiratory (early-I), ramp-inspiratory (ramp-I), and late-inspiratory neurons (late-I) between 400 and 700 μm caudal to the caudal tip of facial nucleus (Fig. 1A). Based on the pattern of evoked responses to microinjections of l-glutamate (see the following text), the presence of different presympathetic and respiratory neurons and the histologic verification of the center of microinjections and recordings, we subdivided the RVLM in two functional subregions: 1) rostral aspect of RVLM (RVLM/BötC) in which respiratory neurons of BötC are colocalized with presympathetic neurons; and 2) caudal aspect of RVLM (RVLM/pre-BötC) containing mostly respiratory neurons of pre-BötC and presympathetic neurons. These findings are in accordance with our previous findings in awake rats (Moraes et al. 2011) and studies from other laboratories performed in anaesthetized rats (Alheid and McCrimmon 2008; Dobbins and Feldman 1994; Monnier et al. 2003; Wang et al. 2002).
Firing properties and location of sympathetic and respiratory neurons.
This analysis is based on 74 extracellular recordings from presympathetic and six distinct types of respiratory neurons located within the RVLM/BötC/pre-BötC. Presympathetic neurons presented spontaneous activity and were: 1) responsive to chemoreflex activation; 2) inhibited by ADN stimulation; and 3) activated antidromically from the T8 to T12 spinal segment (Fig. 1, B1–B4; n = 25). These neurons showed a largely irregular pattern of activity with the firing frequency ranging from 5 to 45 spikes/s. We did not find an on–off pattern of discharge of these neurons in relation to the respiratory cycle. In spite of other types of respiratory modulation on presympathetic neurons (Miyawaki et al. 1995), we identified mainly neurons with an inspiratory followed by a postinspiratory peak (17 of 25 neurons). Figure 1A shows that the majority of recording points of presympathetic neurons were ventral or ventromedial to the nucleus ambiguus (350–500 μm dorsal to the ventral surface) in the entire anteroposterior extension of RVLM (700 μm).
The characterization of respiratory neurons types was based on their phase relationship to respiratory cycle and Fig. 1, C1–C6 shows representative tracings of each group. BötC post-I neurons discharged action potentials immediately after inspiration (n = 11; Fig. 1C5), whereas BötC aug-E neurons fired in augmenting fashion and terminated abruptly at the onset of inspiration (n = 8; Fig. 1C6). Regarding pre-BötC inspiratory neurons, pre-I neurons displayed an augmenting pattern with peak frequency 50–100 ms before the onset of inspiration (PN; n = 11; Fig. 1C1). The firing of early-I (n = 9) occurred coincident with the onset of PN activity and terminated midway through this phase, displaying a declining pattern of discharge (Fig. 1C2). Neurons ramp-I (n = 5) and late-I (n = 5) displayed an augmenting pattern, which terminated abruptly at the end of inspiration (Fig. 1, C3 and C4). All expiratory and inspiratory neurons recorded were excited by peripheral chemoreflex activation as illustrated in Fig. 2, A–F (pre-I: +22 ± 3; early-I: +41 ± 4; ramp-I: +34 ± 5; late-I: +65 ± 3; post-I: +81 ± 5; aug-E: 73 ± 6 spikes/s; n = 49). Histologic section of brain stem of a rat (Fig. 1D1), representative of the group, shows the recording site of a presympathetic neuron (Fig. 1, D1 and D2) located lateral to an aug-E neuron in the RVLM/BötC (Fig. 1, D1s and D3). In addition, as shown in Fig. 1A, the recording sites of presympathetic and respiratory neurons overlapped; rostrally RVLM presympathetic intermixed with BötC expiratory neurons (RVLM/BötC), whereas caudally RVLM presympathetic intermixed with pre-BötC inspiratory neurons (RVLM/pre-BötC).
Concentration–response curves to unilateral microinjections of l-glutamate into the RVLM/BötC or RVLM/pre-BötC.
Figure 3, A1 and A2 shows photomicrographs of the ventral surface of medulla and a sagittal section of the medulla, respectively, of two rats, representative of the groups, showing the center of unilateral microinjections sites in the RVLM/BötC and RVLM/pre-BötC. Figure 3, A3 and A4 shows photomicrographs of coronal sections of the brain stem of two rats, representative of the groups, showing the sites of microinjections bilaterally in the RVLM/BötC and RVLM/pre-BötC, respectively. Figure 3B is a schematic drawing showing location of the center of microinjections in the RVLM/BötC/pre-BötC of all rats used in these experimental protocols.
The typical patterns of PN, tSN, HN, cVN, and AbN activities recorded are shown in the working heart-brain stem preparation in Fig. 3, C1 to C4 and Fig. 5A1. These patterns recorded in in situ preparations resembled those recorded in vivo during generation of a normal three-phase respiratory pattern (inspiratory, post-I, and E2 phases) (Janczewski and Feldman 2006; Koshiya and Guyenet 1996; Miyawaki et al. 1996a; St-John and Paton 2003) and exhibited the following characteristics: 1) an augmenting inspiratory discharge pattern in PN that is also present in the cVN; 2) an incrementing inspiratory discharge of HN that often starts slightly before PN discharge (50–100 ms); 3) a decrementing postinspiratory discharge in cVN that terminates in the middle of expiration; 4) low-amplitude expiratory AbN discharge during post-I and E2 phases; and 5) a phasic increase of tSN activity coincident with PN burst, reaching a peak during late inspiration or the initial part of the expiratory phase (post-I).
With respect to the experiments involving microinjections of l-glutamate and KYN into the RVLM/BötC/pre-BötC, we selected the sites in which the presympathetic neurons were colocalized with the expiratory and inspiratory neurons, as illustrated in Fig. 4, A–D. We observed that respiratory and sympathetic responses to microinjections of increasing concentrations of l-glutamate into the RVLM/BötC and RVLM/pre-BötC followed a concentration-dependent pattern (Fig. 4, A–D). The concentration of 10 mM of l-glutamate produced changes in PN and tSN corresponding to 50% of the maximal responses (EC50). Microinjections of this concentration (10 mM) produced similar increases in tSN, either in the RVLM/BötC or in the RVLM/pre-BötC (65 ± 13 vs. 57 ± 13%; n = 14; Fig. 3, C1 and C3). However, l-glutamate microinjected into the RVLM/BötC and RVLM/pre-BötC elicited different patterns of motor respiratory responses. Microinjections of l-glutamate (10 mM) into the RVLM/BötC produced a decrease in the inspiratory PN (−0.17 ± 0.04 Hz; n = 8) and HN (−0.15 ± 0.02 Hz; n = 4) frequencies (Fig. 3, C1 and C2; Fig. 4B) and increase in the expiratory cVN (63 ± 11%; n = 4) and AbN activities (69 ± 16%; n = 4). In the RVLM/pre-BötC, l-glutamate produced an increase in the inspiratory PN (0.19 ± 0.01; n = 6) and HN frequencies (0.15 ± 0.03 Hz; n = 4) (Fig. 3, C3 and C4; Fig. 4D) but no changes in the average expiratory cVN (n = 4) and AbN activities (n = 4). Unilateral microinjection of the same volume of saline (vehicle; 20 nL) into RVLM/BötC (n = 6) or RVLM/pre-BötC (n = 6) produced negligible changes in the PN (0.04 ± 0.001 Hz), tSN (2 ± 0.2%), cVN (2 ± 0.3%), HN (0.03 ± 0.002 Hz), and AbN activities (3 ± 0.2%). Considering that the baseline values of respiratory and sympathetic variables were not altered by uni- or bilateral microinjections of vehicle into the RVLM/BötC and RVLM/pre-BötC (see the following text), in the subsequent experimental protocols we considered, as control condition, the values immediately before bilateral microinjections of KYN to evaluate the effects of this antagonist on baseline and chemoreflex-induced changes on respiratory and sympathetic variables.
Antagonism of ionotropic glutamate receptors with KYN reduced the sympathetic and respiratory responses to unilateral microinjection of l-glutamate into the RVLM/BötC and RVLM/pre-BötC.
The effects of previous unilateral microinjections of KYN (200 mM) into the RVLM/BötC and RVLM/pre-BötC on sympathoexcitatory and respiratory responses to unilateral microinjections of l-glutamate (EC50) into the same subregions were evaluated. Unilateral microinjection of KYN (200 mM) into the RVLM/BötC (PN: 0.36 ± 0.06 vs. 0.42 ± 0.05 Hz; tSN: 13.5 ± 2 vs. 10.5 ± 1.5%; n = 5) or RVLM/pre-BötC (PN: 0.46 ± 0.05 vs. 0.32 ± 0.04 Hz; tSN: 10.5 ± 1.5 vs. 11.75 ± 2.4%; n = 4) produced no significant changes on baseline tSN and PN activities. However, this concentration of KYN (200 mM) was effective in antagonizing the excitatory amino acid receptors because the sympathoexcitatory and respiratory responses to microinjection of l-glutamate into the RVLM/BötC (65 ± 5 vs. 21 ± 4%; −0.24 ± 0.03 vs. −0.12 ± 0.05 Hz; P < 0.05; n = 5) and RVLM/pre-BötC (59 ± 3 vs. 20 ± 6%; P < 0.05; 0.17 ± 0.04 vs. 0.12 ± 0.05 Hz; n = 4) were almost abolished.
Effects of bilateral ionotropic glutamate receptor antagonism in the RVLM/BötC and RVLM/pre-BötC on baseline respiratory activity.
Figure 5, A1 and A2 shows tracings of one rat, representative of the group, in which are illustrated the changes on baseline respiratory parameters (PN, cVN, and AbN) in response to bilateral microinjections of KYN (200 mM) into the RVLM/BötC. These data are summarized in Fig. 5, B1–B6 and Fig. 6, A–C. We observed that bilateral microinjections of KYN into the center of RVLM/BötC region (100–200 μm caudal to the caudal tip of facial nucleus) produced a large increase on baseline PN frequency (0.43 ± 0.01 vs. 0.86 ± 0.07 Hz; Fig. 5, A1 and A2; Fig. 6, A and B; P < 0.05; n = 18/20) and a decrease in the duration of inspiration (DI: 0.89 ± 0.07 vs. 0.25 ± 0.06 s; Fig. 6C) and expiration (DE: 2.2 ± 0.09 vs. 1.3 ± 0.06 s; Fig. 6C).
The antagonism of ionotropic glutamate receptors in the RVLM/BötC also produced changes in the cranial respiratory motor outflows, as observed in the representative CTAs showing PN, cVN, and AbN activities before and after bilateral microinjections of KYN (Fig. 5, A3 and A4). In the control condition, PN and cVN exhibited a ramp inspiratory discharge pattern, whereas AbN exhibited low-amplitude expiratory discharge during post-I and E2 phases (Fig. 5A3). Figure 5A4 shows that the pre-I activity recorded from the cVN was longer after KYN in the RVLM/BötC (0.13 ± 0.02 vs. 0.64 ± 0.06 s; P < 0.05; Fig. 5, A4 and B3; n = 5). Likewise, KYN microinjected into the RVLM/BötC produced an increase in the magnitude (7.5 ± 2.5 vs. 40 ± 5%; P < 0.05; Fig. 5B1) and duration of pre-I HN activity (0.17 ± 0.03 vs. 0.46 ± 0.05; P < 0.05; Fig. 5B2; n = 8). In addition, CTA shows that the onset of this cVN pre-I activity is coincident with the increase in the pre-I HN discharge and a nonramping squared inspiratory PN profile, suggesting that the increase in PN frequency correlates with a positive effect on pre-I HN and cVN activities and a negative effect in the ramp-I PN activity. In contrast, the post-I cVN (47.5 ± 2.5 vs. 17 ± 5%; P < 0.05; Fig. 5, A3, A4, and B4; n = 5) and AbN activities (38.5 ± 6.5 vs. 12.5 ± 0.5%; P < 0.05; Fig. 5, A3, A4, and B5; n = 5) were reduced after bilateral microinjections of KYN into the RVLM/BötC, producing a reduction in the DE. To verify that the effects of KYN in the RVLM/BötC were primarily due to these effects on RVLM presympathetic and BötC expiratory neurons and not due to spreading of the microinjected volume to other adjacent areas involved in the control of expiratory activity, we also performed intentional misplaced microinjections of KYN into the RTN/pFRG (control experiments for misplaced microinjections). With these microinjections into the RTN/pFRG we verified opposite changes in the PN activity and duration of expiration (PN: 0.34 ± 0.02 vs. 0.12 ± 0.01 Hz; DE: 2.1 ± 0.3 vs. 3.7 ± 0.4 s; P < 0.05; n = 4), indicating that the observed effects of KYN in the RVLM/BötC were not due to its spreading to the neighboring RTN/pFRG.
At the center of RVLM/pre-BötC (500–600 μm caudal to the caudal tip of facial nucleus), bilateral microinjections of KYN abolished all three inspiratory outflows recorded: PN (n = 16), HN (n = 6), and cVN (n = 5) activities (Fig. 6A). Regarding the average AbN activity (lumbar), no significant changes (30 ± 5 vs. 39 ± 3%; Fig. 8, B1 and B2; n = 5) were observed after KYN in the RVLM/pre-BötC, but the pattern was converted from biphasic expiratory bursts to a prolonged monophasic discharge. Bilateral microinjections of saline into the RVLM/BötC (PN: 0.38 ± 0.02 vs. 0.4 ± 0.03 Hz; cVN: 30 ± 1 vs. 28 ± 2%; AbN: 26 ± 3 vs. 30 ± 4%; HN: 32 ± 2 vs. 27 ± 4%; tSN: 10 ± 2 vs. 12 ± 3%; n = 6) or RVLM/pre-BotC (PN: 0.41 ± 0.03 vs. 0.37 ± 0.03 Hz; cVN: 32 ± 2 vs. 34 ± 3%; AbN: 31 ± 2 vs. 33 ± 4%; HN: 29 ± 2 vs. 32 ± 3%; tSN: 12 ± 2 vs. 9 ± 3%; n = 6) produced no significant changes on baseline sympathetic and respiratory parameters.
Effects of bilateral ionotropic glutamate receptor antagonism in the RVLM/BötC and RVLM/pre-BötC on the baseline sympathetic–respiratory coupling.
Typical patterns of PN and tSN activities from a rat, representative of the group, are shown in Fig. 7, A and C. As shown in the CTA in Fig. 7, inset A1, under control conditions, the integrated activity in the tSN expressed an augmenting inspiratory modulation with the activity profile slowly increasing during inspiration, reaching a peak at the post-I period, and rapidly falling at the beginning of expiration. Figure 7, B and inset B1 show that, similar to post-I cVN and AbN activities, bilateral microinjections of KYN into the RVLM/BötC produced a decrease in the post-I tSN activity (31 ± 2 vs. 9.5 ± 0.5%; P < 0.05; Fig. 5B6; n = 18). In addition, the inspiratory tSN activity decreased after bilateral microinjections of KYN into RVLM/BötC (62.5 ± 2.5 vs. 32 ± 3%; P < 0.05; Fig. 5B6; Fig. 7, insets A1 and B1; n = 18). Figure 7, C and D shows the PN and tSN activities before and after bilateral microinjection of KYN into the RVLM/pre-BötC. No significant changes were observed in the average baseline tSN after KYN in the RVLM/pre-BötC (9 ± 2 vs. 12 ± 1%; Fig. 7, C and D; n = 16). However, it was not possible to examine the effect of antagonism of ionotropic glutamate receptors in the RVLM/pre-BötC on the sympathetic–respiratory coupling, since bilateral microinjection of KYN into the RVLM/BötC abolished the PN activity (Fig. 7D). Bilateral microinjections of saline (vehicle) into the RVLM/BötC (inspiratory: 60 ± 2 vs. 64 ± 4%; post-I: 30 ± 4 vs. 28 ± 4%; E2: 27 ± 3 vs. 25 ± 4%; n = 6) or RVLM/pre-BötC (inspiratory: 58 ± 4 vs. 62 ± 5%; post-I: 29 ± 4 vs. 32 ± 3%; E2: 25 ± 5 vs. 21 ± 6%; n = 6) produced no changes on the sympathetic–respiratory coupling. To verify that the effects of KYN in the RVLM/pre-BötC were primarily due to these effects on RVLM presympathetic and pre-BötC inspiratory neurons and not due to spreading to adjacent areas, we also performed intentional misplaced microinjections of KYN into the CVLM (control experiments for misplaced microinjections). In spite of the fact that microinjection of KYN into the CVLM produced respiratory arrest similar to that observed when the microinjection was performed into the RVLM/pre-BötC, we observed a significant increase in the average tSN activity (11 ± 3 vs. 40 ± 4%; P < 0.05; n = 4), indicating that the observed effects of KYN in the RVLM/pre-BötC were not due to its spreading to the neighboring CVLM.
Effects of bilateral microinjections of KYN into the RVLM/BötC and RVLM/pre-BötC on sympathoexcitatory and respiratory responses to chemoreflex activation.
Figure 8 shows tracings of one rat, representative of the group, illustrating the changes in the PN, AbN, and tSN activities in response to chemoreflex activation before (Fig. 8A1) and after (Fig. 8A2) bilateral microinjections of KYN into the RVLM/BötC. These data are summarized in Fig. 9. Figure 8A3 shows typical sympathetic and respiratory responses to chemoreflex activation characterized by tachypnea, sympathoexcitation, and active expiration. In addition, it is possible to evaluate two different components in the expiratory AbN response to chemoreflex activation: post-I and late-expiratory (late-E) components during postinspiration and E2 phases, respectively. Bilateral microinjections of KYN into the RVLM/BötC reduced the post-I (35 ± 3 vs. 12 ± 2%; P < 0.05) and late-expiratory (24 ± 4 vs. 2 ± 1%; P < 0.05; n = 5) AbN responses to chemoreflex activation (Fig. 8, A3 and A4; Fig. 9D). Likewise, the expiratory cVN response was reduced after KYN microinjected into the RVLM/BötC (67 ± 6 vs. 40 ± 2%; P < 0.05; Fig. 9E; n = 5). KYN microinjected into the RVLM/BötC also reduced the expiratory component of the sympathoexcitatory response (77 ± 8 vs. 26 ± 5%; P < 0.05; Fig. 8, A3 and A4; Fig. 9B; n = 18), but enhanced the inspiratory [PN (n = 18) and HN (n = 8)] response to chemoreflex activation (0.38 ± 0.02 vs. 1 ± 0.08 Hz; 41 ± 2 vs. 82 ± 2%; P < 0.05; Fig. 8, A1 and A2; Fig. 9, A and C).
In relation to RVLM/pre-BötC, Fig. 8 shows the typical sympathetic and respiratory responses (PN, AbN, and tSN) to chemoreflex activation before (Fig. 8B1) and after (Fig. 8B2) bilateral microinjections of KYN into this subregion. We observed that KYN into the RVLM/pre-BötC abolished the inspiratory [PN (n = 16) and HN (n = 6)] response but did not change the average expiratory (cVN : 69 ± 3 vs. 67 ± 4%; AbN: 66 ± 3 vs. 77 ± 4%; n = 10) and sympathoexcitatory (80 ± 4 vs. 69 ± 12%; n = 16) responses to chemoreflex activation (Fig. 8, B1 and B2), producing similar and phase-locked vagal, abdominal, and sympathoexcitatory chemoreflex responses. Bilateral microinjections of saline (vehicle) into either RVLM/BötC (PN: 0.35 ± 0.01 vs. 0.41 ± 0.06 Hz; tSN: 93 ± 4 vs. 79 ± 8%; cVN: 74 ± 6 vs. 63 ± 4%; HN: 39 ± 2 vs. 38 ± 3; AbN: 71 ± 1 vs. 68 ± 3%; n = 6) or RVLM/pre-BötC (PN: 0.43 ± 0.04 vs. 0.45 ± 0.07 Hz; tSN: 93 ± 5 vs. 82 ± 7%; cVN: 72 ± 5 vs. 69 ± 7%; HN: 33 ± 5 vs. 39 ± 6%; AbN: 63 ± 4 vs. 65 ± 4%; n = 6) produced no significant changes on chemoreflex responses.
This study reveals an essential role for the glutamatergic neurotransmission in the RVLM/BötC/pre-BötC in the control of expiratory and inspiratory activities on basal and during chemoreflex activation, but mainly in the expiratory and inspiratory modulation of sympathoexcitatory response to peripheral chemoreflex activation. In the in situ preparations, ionotropic glutamate receptors antagonism in the RVLM/BötC decreased baseline and chemoreflex-evoked post-I cVN and AbN activities and also the expiratory component of tSN response, showing that l-glutamate in this subregion is essential for generation of the expiratory response as well for expiratory modulation of the tSN response to chemoreflex activation. In the RVLM/pre-BötC, the antagonism of ionotropic glutamate receptors abolished baseline and chemoreflex-evoked inspiratory PN and HN motor outflows and the inspiratory modulation of tSN response to chemoreflex activation, revealing a critical role for l-glutamate in this subregion in generation of the inspiratory activity. Altogether, these data indicate distinct and important roles for glutamatergic neurotransmission at the RVLM/BötC/pre-BötC on expiratory and inspiratory drives and mainly in the coupled modulation of sympathetic activity, during peripheral chemoreflex activation.
Regarding the control of respiratory parameters, our findings support the concept that the antagonism of ionotropic glutamate receptors in the RVLM/BötC and RVLM/pre-BötC produces distinct patterns of alterations, as previously described in awake rats (Moraes et al. 2011). Bilateral microinjections of KYN into the RVLM/BötC produced a decrease in the post-I activity of two respiratory outflows (cVN and AbN), an increase in the pre-I activity (HN and cVN) and in the PN frequency. KYN microinjected into the RVLM/pre-BötC produced a silencing of all three inspiratory nerves (PN, cVN, and HN), but no changes in the average AbN expiratory activity, albeit its pattern has changed. Previous studies suggested that both the pons and RTN/pFRG provide excitatory drive to post-I and aug-E neurons in the BötC (Dutschmann and Herbert 2006; Rybak et al. 2004b). These excitatory drives seems to be essential for the generation of the eupneic respiratory pattern (Rybak et al. 2007; Smith et al. 2007), by shaping the respiratory pattern and controlling the durations of expiration and inspiration (Alheid et al. 2004; Cohen and Shaw 2004; Okazaki et al. 2002; St-John and Paton 2003). Considering the increase in the respiratory frequency after KYN microinjected into the RVLM/BötC, we suggest that bilateral microinjections of KYN into the BötC decreased pontine/RTN excitatory inputs to post-I and aug-E BötC neurons, changing the basal respiratory pattern to the observed abnormal pattern characterized by an increase in the respiratory frequency due to a decrease in the duration of inspiration and expiration. This change probably occurred because the pre-I neurons received inhibitory inputs from aug-E neurons (controlling the pre-I activity), whereas ramp-I received inhibitory inputs from post-I (controlling the duration of inspiration and expiration) (Hayashi et al. 1996; Molkov et al. 2011; Rybak et al. 2008). Microinjections of KYN into the RVLM/BötC decreased the inhibitory tonus from BötC post-I and aug-E neurons to pre-BötC inspiratory neurons, allowing a decrease in the duration of expiration and an increase in the activities of pre-I neurons. This major change in the respiratory network causes: 1) an increment of baseline pre-I HN and cVN outflows, 2) an increase in the PN frequency, and 3) an enhancement of the PN and HN inspiratory outflows in response to chemoreflex activation after KYN in the RVLM/BötC. We suggest that glutamatergic pontine/RTN inputs to medullary circuits contribute significantly to expiratory activity through activation of inhibitory BötC post-I and aug-E neurons, important for generation of the eupneic respiratory pattern.
With respect to the RVLM/pre-BötC, previous studies performed in rats showed that the antagonism of ionotropic glutamate receptors at this level produced respiratory arrest in anesthetized (Koshiya et al. 1993) and unanesthetized rats (Moraes et al. 2011). The data of the present study clearly demonstrate in the unanesthetized condition, that three inspiratory neural outflows (PN, cVN, and HN) were abolished at baseline and not reestablished in response to peripheral chemoreflex activation after antagonism of ionotropic glutamate receptors in the RVLM/pre-BötC. Therefore, our results support the concepts that ionotropic glutamate receptors in the RVLM/pre-BötC critically contribute to the generation of inspiratory activity and are also required for the processing of inspiratory response to chemoreflex activation. The elimination of respiratory activity by microinjections of KYN into the RVLM/pre-BötC clearly indicates that the inspiratory pattern (PN, cVN, and HN) originates in this area. This hypothesis is in agreement with the working model proposed by Rybak et al. (2008), indicating that the generation of eupneic inspiratory activity requires a tonic drive from Raphe, pons, or other brain stem regions to the pre-BötC. In this regard, we hypothesize that part of this tonic drive may rely on glutamatergic neurotransmission, a very important issue that deserves further experiments to be fully elucidated. In addition, we may not rule out the possibility that l-glutamate contributes to generation of inspiratory pattern through the excitatory synaptic interactions within VRC. In this regard, previous studies showed that pre-BötC inspiratory neurons with voltage-dependent, pacemaker-like oscillatory properties, which are considered the main candidates for the inspiratory-rhythm–generating cells (Smith et al. 1991; St-John et al. 2009), receive excitatory synaptic inputs that appear to be critical for regulation of their bursting behavior (Funk et al. 1993). Taking into account all these findings, we suggest that excitatory synaptic interactions would be required to synchronize bursting within the inspiratory rhythm generators neurons in the pre-BötC. Alternatively, glutamatergic neurotransmission may modulate excitability of inspiratory pre-BötC neurons via pontomedullary interactions. However, it is worthy to note that all these hypotheses require further investigation. With respect to the baseline and the magnitude of chemoreflex-evoked AbN expiratory activity after microinjection of KYN into the RVLM/pre-BötC no significant changes were observed. These results are consistent with the previous studies performed in vivo showing that suppression of the inspiratory activity did not change the overall expiratory activity (Janczewski and Feldman 2006). Similarly to working models presented in different studies (Abdala et al. 2009; Molkov et al. 2010), suppression of the inspiratory activity (in the present study after microinjection of KYN into the RVLM/pre-BötC) eliminates inspiratory inhibition of expiratory neurons, which converts the biphasic activity of AbN during baseline to a prolonged monophasic discharge. These findings suggest a separate generator of expiratory activity, which interacts with the pre-BötC inspiratory oscillator and seems to be essential for respiratory pattern generation.
The main core of the coupling between the sympathetic and respiratory activity apparently is within the RVLM/BötC/pre-BötC (Guyenet et al. 1990; Haselton and Guyenet 1989; Miyawaki et al. 1995, 1996a). Our data show that the post-I tSN activity is dependent on ionotropic glutamate receptors in the RVLM/BötC, suggesting that the post-I peak is generated by respiratory related inputs that converge onto presympathetic neurons. These findings are in agreement with the previous studies in anesthetized rats showing that the antagonism of ionotropic glutamate receptors in the rostral aspect of RVLM (RVLM/BötC) markedly attenuates the post-I activity seen in lumbar and thoracic sympathetic nerve discharge (Guyenet et al. 1990; Miyawaki et al. 1996a). In the present study, we identified 17 presympathetic neurons with an inspiratory/post-I peak. However, the sources of the respiratory-related excitatory input to the RVLM neurons that generate the inspiratory/post-I activity remain unknown. Considering the significant reduction of inspiratory/post-I tSN activities after bilateral microinjections of KYN into the RVLM/BötC and that KYN also reduced the presumably pontine-dependent post-I activity in the cVN, we suggest that RVLM presympathetic neurons receive direct excitatory projections from inspiratory–expiratory pontine neurons as well as receiving inputs from respiratory neurons of post-I BötC, whose activity critically depends on pontine inputs (Baekey et al. 2008). However, further studies are required to analyze whether these pontine projections are targeting directly to the presympathetic neurons or depend on post-I neurons in the BötC.
Previous studies demonstrated that the respiratory and sympathetic responses to peripheral chemoreflex activation are tightly entrained, and the increase in sympathetic activity occurs in bursts mainly during the expiratory phase (Costa-Silva et al. 2010; Dick et al. 2004; Mandel and Schreihofer 2009). However, there is no previous study showing a clear relationship between active expiration and sympathoexcitatory response during chemoreflex activation. Our findings in the in situ working heart-brain stem preparation are in agreement with previous studies in awake (Moraes et al. 2011) and anaesthetized rats (Kubo et al. 1993; Miyawaki et al. 1996b; Sun and Reis 1995) showing that the ionotropic glutamate receptors in the presympathetic neurons at the rostral aspect of RVLM (from 0 to 200 μm caudal to the caudal tip of facial nucleus), but not in the caudal aspect of RVLM (from 500 to 600 μm caudal to the caudal tip of facial nucleus), as observed by Koshiya et al. (1993) and Moreira et al. (2006), are important for the processing of sympathoexcitatory response to chemoreflex activation. In the present study, we enlightened the degree of knowledge of this important field by exploring the consequences of the antagonism of excitatory amino acid receptors in the RVLM/BötC on the coupling of sympathoexcitation and active expiration in response to chemoreflex activation. We verified that the two expiratory components of AbN response (post-I and late-E) and the post-I component of cVN to chemoreflex activation were attenuated after microinjection of KYN into the RVLM/BötC. Studies by Abdala et al. (2009) showed that the pons and RTN are essential for expression of late-E AbN component induced by hypercapnia/anoxia, supporting the concept that generation of active expiration also involves these brain stem areas. Considering our data showing that microinjections of KYN into the RVLM/BötC abolished the expiratory component of tSN and attenuated the AbN and cVN expiratory responses to chemoreflex activation, it is possible that the pons and RTN are the sources of excitatory drives to RVLM/BötC required for generation of post-I and late-E activities in the tSN, cVN, and AbN responses to chemoreflex activation. However, microinjection of KYN into BötC did not abolish the AbN response to chemoreflex activation. To explain why the AbN response to chemoreflex activation was not abolished after bilateral microinjections of KYN into the RVLM/BötC, we must take into account the findings by Fortuna et al. (2008) showing that some RTN neurons send projections to the retroambiguus region, the site of premotor bulbospinal expiratory neurons. We observed that the average increase in the magnitude of tSN in response to chemoreflex activation was not affected by bilateral microinjections of KYN into the RVLM/pre-BötC. The most important change produced by KYN was the abolishment of the classic respiratory oscillations in the tSN response to chemoreflex activation. Previous studies suggested that CVLM neurons are not necessary for the tonic excitation of SN produced by acute hypoxia, but it may play a role in the respiratory-related changes in SN activity observed under this condition (Koshiya and Guyenet 1996; Mandel and Schreihofer 2009). Our data strongly support the concept that the increase in tSN by chemoreflex activation involves two components in the context of RVLM/BötC/pre-BötC: 1) excitatory projections to presympathetic RVLM neurons dependent of the expiratory activity and; and 2) inhibitory projections to presympathetic RVLM neurons modulated by inspiratory activity. However, it is important to note that the neural pathways involved in the expiratory and inspiratory modulation of the tSN response to chemoreflex activation remain to be determined.
In addition to a possible direct pathway from the NTS to presympathetic neurons in the ventral medulla involved in the sympathoexcitatory response to chemoreflex activation (Koshiya and Guyenet 1996), we must consider interactions between expiratory neurons in the BötC and presympathetic neurons in the RVLM in the expiratory modulation of the sympathoexcitatory response, because the tSN and AbN expiratory responses to chemoreflex activation were comparable and phase-locked after microinjections of KYN into the RVLM/pre-BötC. Considering that inhibition of the RVLM/pre-BötC (present study) or the CVLM (Koshiya and Guyenet 1996; Mandel and Schreihofer 2009) produced a overall silencing of baseline and chemoreflex-evoked inspiratory outflows and a monophasic sympathoexcitatory response, it is possible that during chemoreflex activation inspiratory neurons in the pre-BötC, in charge of the inspiratory response to chemoreflex activation, produce an inhibitory modulation of the tSN response. In this context, we suggest that the inspiratory neurons in the pre-BötC are more important in the classic respiratory oscillations in the tSN response to chemoreflex activation than the activation of inhibitory barosensitive neurons in the CVLM, as suggested by Mandel and Schreihofer (2009) and Miyawaki et al. (1996a). Nevertheless, the neural pathways between the chemoreflex activated inhibitory inspiratory neurons of the pre-BötC and the presympathetic neurons of the RVLM also required further investigation.
The findings of the present study reveal that the baseline and peripheral chemoreflex control of expiratory and inspiratory activities as well as the respiratory modulation of baseline and peripheral chemoreflex tSN activity are entirely dependent of l-glutamate and its ionotropic receptors in the RVLM/BötC/pre-BötC. These results extend, in a very important direction, our understanding about the central neural control of baseline and peripheral chemoreflex sympathetic–respiratory coupling. Considering that in a recent study we demonstrated that active expiration, evidenced by late-E component of the AbN activity, is present during eupnea in rats submitted to chronic intermittent hypoxia (Zoccal et al. 2008), the findings of the present study bring to this field new fundamental concepts about the neurochemical mechanisms involved in this coupling and open new and interesting perspectives for a better understanding about possible dysfunction in the glutamatergic neurotransmission, which might contribute to the enhancement of sympathetic–respiratory coupling and consequently to the hypertension observed in rats submitted to chronic intermittent hypoxia and in patients suffering of obstructive sleep apnea.
This work was supported by São Paolo Research Foundation (FAPESP) Grant 2009/50113-0, National Council for Scientific and Technological Development (CNPq, Barzil) Grants 502098/2008-2 and 301147/2008-6), and a FAPESP Fellowship Grant 2010/09805-03 (to D.J.A.M.).
No conflicts of interest, financial or otherwise, are declared by the author(s).
D.J.M., D.B.Z., and B.H.M. conception and design of research; D.J.M. and D.B.Z. performed experiments; D.J.M., D.B.Z., and B.H.M. analyzed data; D.J.M., D.B.Z., and B.H.M. interpreted results of experiments; D.J.M. prepared figures; D.J.M. drafted manuscript; D.J.M., D.B.Z., and B.H.M. edited and revised manuscript; D.J.M., D.B.Z., and B.H.M. approved final version of manuscript.
Present address of D. B. Zoccal: Department of Physiological Sciences, Center of Biological Sciences, Federal University of Santa Catarina, Florianópolis, SC, Brazil.
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