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α2 Receptors in the lateral parabrachial nucleus generates the pressor response of the cardiovascular chemoreflex, effects of GABAA receptor
Brain Research Bulletin 140 (2018) 190–196
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α2 Receptors in the lateral parabrachial nucleus generates the pressor response of the cardiovascular chemoreflex, effects of GABAA receptor Nafiseh Mirzaei-Damabi, Gholam Reza Namvar, Fahimeh Yeganeh, Masoumeh Hatam
T
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Dept. of Physiology, Shiraz University of Medical Sciences, Shiraz, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: LPBN Peripheral chemoreflex Bicuculline Yohimbine
The lateral parabrachial nucleus (LPBN) is a pontine area involved in cardiovascular chemoreflex. This study was performed to find the effects of reversible synaptic blockade of the LPBN on the chemoreflex responses, and to find the roles of GABAA receptor and α2-adenoreceptor (α2-AR) in chemoreflex. It also aimed to seek possible interaction between GABA and noradrenergic systems of the LPBN in urethane-anesthetized male rats. Cardiovascular chemoreflex was activated by intravenous injection of potassium cyanide (KCN, 80 μg/kg). The cardiovascular responses of chemoreflex were evaluated before (control), 5 and 15 min after microinjection of each drug (100 nl) into the LPBN. Microinjections of cobalt chloride (5 mM), a reversible synaptic blocker, into the LPBN greatly attenuated the chemoreflex pressor and bradycardic responses indicating that the LPBN plays a main role in chemoreflex. Local injection of yohimbine (10 nmol), an α2-AR antagonist, attenuated the pressor response with no effect on bradycardic response, suggesting that α2-adrenoreceptors are involved in producing the pressor response of the chemoreflex. Microinjection of bicuculline methiodide (BMI, 100 pmol), a GABAA antagonist, into the LPBN augmented the pressor response and attenuated the bradycardic response, indicating that GABA inhibits the sympathetic output to the heart and vasculature. Sequential injection of yohimbine and BMI had no significant effect on the pressor response but attenuated the bradycardia. In conclusion, the LPBN is essential for the chemoreflex responses. The pressor response of the chemoreflex, at least partly, is produced by α2- adenoreceptors. GABA in the LPBN inhibits the cardiovascular system. Finally, there is no interaction between GABAergic and adrenergic neurons of the LPBN in producing the cardiovascular chemoreflex.
1. Introduction The chemoreflex is an important neural mechanism involved in cardiovascular and respiratory control. Peripheral chemoreceptors, which in rats are located mainly in the carotid body glomus cells respond to changes in blood PO2 within milliseconds, contributing to ventilatory and cardiovascular regulation (Menani and Johnson 1995; Colombari et al., 1996; Lahiri et al., 2001). Activation of chemoreceptors can be performed by systemic administration of potassium cyanide (KCN), which produces a cytotoxic hypoxia (Barros et al., 2002). It is characterized by an increase in respiratory frequency, an increase in arterial pressure and a decrease in heart rate in awake (Barros et al., 2002; Haibara et al., 2002; Fernandes et al., 2005; Roncari et al., 2011) and in anesthetized rats (Ciriello and Moreau, 2013). These responses are essentially dependent on the stimulation of carotid chemoreceptors, because bilateral ligature of the carotid body ⁎
Corresponding author. E-mail address: [email protected] (M. Hatam).
https://doi.org/10.1016/j.brainresbull.2018.05.009 Received 20 October 2017; Received in revised form 24 April 2018; Accepted 8 May 2018 Available online 21 May 2018 0361-9230/ © 2018 Elsevier Inc. All rights reserved.
arteries (Franchini and Krieger 1993; Haibara et al., 1995; Barros et al., 2002) or deafferentation of the carotid sinus nerve bifurcation (Franchini and Krieger 1993) abolishes both cardiorespiratory and behavioral responses to KCN injection. The cardiovascular responses to KCN result from the activation of the following two independent autonomic mechanisms: (1) a sympathetic pathway related to the pressor response, which is blocked by intravenous injection of prazosin, an α1adrenoceptor antagonist; and (2) a parasympathetic pathway related to the bradycardic response, which is abolished by intravenous injection of atropine (Franchini and Krieger 1993; Haibara et al., 1995; Fernandes et al., 2005; Braga et al., 2008). The parabrachial nucleus (PBN) is a brain structure located in the dorsolateral pons surrounding the superior cerebellar peduncle. The PBN consists of three major subdivisions: the lateral parabrachial nucleus (LPBN), the medial parabrachial nucleus (MPBN) and the ventrolateral Kolliker- Fuse nucleus (Fulwiler and Saper 1984). Several anatomical (Herbert and Flügge, 1990) and physiological
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AR antagonist yohimbine or the GABAA-receptor antagonist bicuculline methiodide (BMI). For interaction experiment one barrel contained yohimbine, the second barrel contained bicuculline methiodide. Drugs were microinjected using a pressurized air pulse applicator. The injection volume was measured by direct observation of the fluid meniscus in the micropipette using a microscope fitted with an ocular micrometer that allowed a 2-nl resolution (U.W.O, Canada). The injection volume for drugs was100 nl for each side. Blood pressure and heart rate were rerecorded using a ML T844 pressure transducer coupled to a pre-amplifier (FE221 Bridge amplifier, AD instruments) connected to a power lab 4/35 data acquisition system (model PL3504 CE instruments). The chemoreflex was activated by systemic intravenous injection of 80 μg/Kg/0.1 ml of KCN (Sigma, USA) [9,10]. The cardiovascular responses to chemoreflex activation were evaluated before (control), 5 and 15 min after microinjection of each drug. The vehicle groups received bilateral microinjection of saline or DMSO with the same experimental protocol. All drugs were dissolved in saline except yohimbine, which was dissolved in DMSO. Experiments performed on both sides of the LPBN.
studies (Braga et al., 2008) indicated a significant role for the LPBN in regulating cardiovascular activity. Electrical and chemical stimulation of the LPBN subnuclei, dorsal, central and the external, caused a significant increase in mean arterial pressure (MAP), in heart rate (HR) and in sympathetic nerve activity (Chamberlin and Saper 1992) whereas bradycardia was reported from stimulation of the dorsal LPBN (Chamberlin and Saper 1992). Reversible bilateral blockade of the LPBN by injections of lidocaine induced an immediate pressor response (Saleh and Connell 1997). Thus, it seems that the LPBN tonically attenuates MAP. Furthermore, it has been shown that stimulation of the LPBN modulates baroreflex elicited by stimulation of aortic depressor nerve (Hayward and Felder 1998). Microinjection of lidocaine, a reversible blocker of neuronal activity and fibers of passage, into the dorsolateral part of the PBN reduced pressor response to chemoreflex activation with potassium cyanide in awake rat with no significant change in the bradycardic responses (Haibara et al., 2002). Immunohistochemical studies have shown the presence of α2adrenergic sites (Herbert and Flügge, 1995; Talley et al., 1996; Andrade et al., 2011) and GABAA receptors (Callera et al., 2005; Roncari et al., 2011) in the LPBN. Several studies indicated that activation of LPBN α2-adrenergic receptors (Andrade et al., 2015) and GABA receptors enhanced NaCl and water intake in satiated or sodium depleted rats (Callera et al., 2005; de Oliveira et al., 2007; De Oliveira et al., 2008; De Oliveira et al., 2011). Despite evidence showing the presence of GABAA and alpha 2adrenergic receptors in the LPBN there is no study investigating cardiovascular responses of these receptors and possible interaction between them. Also, there is no study showing the involvement of these two receptors in the chemoreflex activation. Thus, this study was performed to find:
2.3. Experimental groups The experiments consisted of the following groups:
• The vehicle group: 100 nl of the vehicle (saline: n = 8 rats, DMSO: n = 4 rats) was microinjected into each side of LPBN. • Microinjection of a reversible synaptic blocker cobalt chloride • •
– The effects of microinjection of yohimbine, an α2-adenoreceptor (AR) antagonist, into the LPBN on chemoreflex activation. – The effects of microinjection of bicuculline, a GABAA receptor antagonist, in the LPBN on the chemoreflex activation. – The possible interaction between α2-adenoreceptor and GABAA receptor in the LPBN – The effect of acute inhibition of the LPBN by CoCl2 microinjection on chemoreflex activation.
•
(CoCl2, 5 mM/100 nl, Sigma, USA) (Rasoulpanah et al., 2014, Hatam et al., 2015) Microinjection of anα2-AR antagonist, yohimbine (10 nmol/100 nl; n = 13 rats; Sigma, USA) (Yeganeh et al., 2017) Microinjection of a GABAA antagonist, bicuculline methiodide (BMI, 100 pmol/100 nl, n = 9 rats, Sigma, USA) (Rasoulpanah et al., 2014, Hatam et al., 2015). Sequential microinjections of bicuculline (100 pmol) and yohimbine (10nmol; n = 8 rats) in a final volume of 100 nl.
2.4. Histological verification of injection sites At the end of each experiment, the animal was sacrificed using a high dose of the anesthetic (urethane) and perfused transcardially with100 ml of 0.9% saline followed by100 ml of 10% formalin. The brain was removed and stored in 10% formalin for at least 24 h. Frozen serial transverse sections (40 mm) of the forebrain were cut and stained with cresyl violet 1%. The injection sites were determined according to brain atlas ((Paxinose and Watson, 2007), using a light microscope.
2. Experimental procedures 2.1. General procedures The experiments were performed on 50 male Sprague Dawley rats (250–300 g, 10–11 weeks old), approved by the Animal Use and Care Committee of Shiraz University of Medical Sciences. The rats had free access to food and water under a 12 h light/dark cycle with a room temperature of ∼25 °C. Animals were anesthetized with urethane (1.4 g/kg, ip), and supplementary doses (0.7 g/kg) were given if needed. The trachea was cannulated to ease the ventilation. The body temperature was maintained at 37 ± 1C°, using a controlled heat pad. For recording of blood pressure, the right femoral artery was cannulated with a Polyethylene catheter (PE-50) filled with heparinized saline. Two holes were drilled above the LPBN at coordinates 9.0–9.6 mm caudal to bregma, 2–2.2 mm lateral to the midline and 6.2–6.6 mm ventral to the cortical surface of bregma according to a stereotaxic atlas of the rat brain (Paxinose and Watson, 2007).
2.5. Statistical analysis The mean arterial pressure (MAP) and heart rate (HR) values were expressed as mean ± SE. The mean of the maximum changes of MAP or HR was compared with those of the vehicle group using independent t-test and with the pre-injection values using paired t-test. A P < 0.05 was considered as the statistical significance. 3. Results 3.1. Chemoreflex responses to bilateral microinjection of vehicle (Saline or DSMO) into the LPBN In this group, the baseline MAP was 78.8 ± 6.7 and HR was 395 ± 8.4. Bilateral microinjection of vehicle (100 nl each side) did not affect MAP or HR (saline: ΔMAP = 3.3 ± 2.1 mmHg, ΔHR = 2.6 ± 1.2 beats/min; n=8 rats; DMSO: ΔMAP = .6 ± 0.8 mmHg, ΔHR = − 3.0 ± 1.0 beats/min; paired t-
2.2. Microinjection and chemoreflex activation To inject drugs into the LPBN, a single or double barreled micropipette with a tip diameter of 30–35 μwas made using capillary tubing (Stoleting, USA). One barrel of the pipette contained saline or the α2191
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significant effect on the baseline values of MAP and HR (MAP = 59.5 ± 4.9 vs. 62.7 ± 6.3 mmHg and HR = 378.5 ± 15.0 vs. 378.7 ± 14.8 bpm, paired t-test, P > 0.05, n = 7 rats, Fig. 1). Microinjections of cobalt chloride into the LPBN significantly attenuated the pressor and bradycardic responses of the chemoreflex (before: ΔMAP 22.8 ± 3.2, after: ΔMAP = 6.2 ± 2.3 mmHg and ΔHR: before −33.5 ± 7.0, after:−12.1 ± 8.1bpm, paired t-test, P < 0.05, Figs. 3 and 4). 3.3. Chemoreflex responses to bilateral microinjection of yohimbine, an α2 adrenoceptor (AR) blocker, into the LPBN Bilateral microinjection of yohimbine (10 nmol/100 nl, each side, Sigma) into the LPBN had no significant effect on baseline MAP and HR (MAP = 78.3 ± 7.3 vs. 71.6 ± 8.1 mmHg and HR = 402.0 ± 11.0 vs. 400.3 ± 12.8 bpm, paired t-test, P > 0.05, n = 13 rats, Fig. 1).Yohimbine significantly attenuated the pressor response of chemoreflex (before: ΔMAP = 23 ± 2.4, after: ΔMAP = 11 ± 2.5 mmHg, paired t-test, P < 0.01, n = 13 rats) and had no significant effect on bradycardic response (ΔHR: before: −52.2 ± 7.5, after: −74.8 ± 15.2.1 bpm, paired t-test, P > 0.05, Figs. 3 and 5). The effect of yohimbine was reversible, since the response returned to normal level 15 min after the microinjection. 3.4. Chemoreflex responses to microinjection of bicuculline methiodide, a GABAA antagonist, into the LPBN Bilateral or unilateral microinjection of BMI (100 pmol/100nl) into the LPBN produced an increase in baseline MAP (MAP = 70.8 ± 5.5 vs. 87.3 ± 3.4 mmHg, n = 7 rats, paired t-test, P < 0.5) and HR (HR = 342.5 ± 12.1 vs. 379.5.6 ± 15.5 bpm, n = 7 rats, paired t-test, P < 0.5, Fig. 1). BMI significantly augmented the chemoreflex pressor response (before: ΔMAP = 9.6 ± 2.5, after: ΔMAP = 14.3 ± 1.9 mmHg, paired t-test, P < 0.01) and attenuated the chemoreflex bradycardic response (before: ΔHR = −38.6 ± 8.6, after: ΔHR = − 21.6 ± 2.8 bpm, paired t-test, P < 0.05). The effect of BMI lasted more than 1 h and in some cases did not return until the end of the experiment (Figs. 3 and 6).
Fig. 1. Histograms summarizing the effects of various drugs microinjected into the lateral part of the PBN on the baseline MAP (a) and HR (b). Data were compared to the preinjection values (paired t-test, *P < 0.5, ***P < 0.001).
test, P > 0.05, n = 4 rats; Fig. 1). Microinjections of vehicle into the LPBN produced no change in the pressor (saline: ΔMAP: 11.5 ± 2 vs. 11.8 ± 2 mmHg; DMSO: ΔMAP = 16.0 ± 2.0 mmHg vs. 15.6 ± 1.45 mm Hg, paired t-test, P > 0.05) or bradycardic response (saline: ΔHR: −59.5 ± 9.5 vs −61.6 ± 8.8 beats/min; DMSO: ΔHR: −26.6 ± 4.9 vs −32.6 ± 2.3 beats/min, paired t-test, P > 0.05) to chemoreflex activation by KCN (Figs. 2 and 3).
3.5. Chemoreflex responses to sequential microinjection of BMI and yohimbine into the LPBN
3.2. Chemoreflex responses after bilateral microinjection of cobalt chloride into the LPBN
Bilateral sequential microinjection of yohimbine (10 nmol/50 nl) and BMI (100 pmol/50 nl) into the LPBN did not elicit a significant change in the baseline values of MAP and HR (MAP: 63.1 ± 8.5 vs.
Bilateral microinjection of a reversible synaptic blocker, cobalt chloride (CoCl2: 5 mM/100 nl, each side; Sigma), into the LPBN had no
Fig. 2. Tracings of a representative experiment showing the chemoreflex responses before and 5 min after bilateral microinjection of saline (100 nl, n = 8 rats) into the lateral part of the PBN. The arrow shows the injection time. a: arterial pressure, b: MAP, c: HR. 192
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Fig. 3. Histograms summarizing chemoreflex responses, before and 5 min after various drugs microinjected into the lateral part of the PBN (paired t-test, *P < 0.5, ***P < 0.001). a: ΔMAP, b: ΔHR.
Fig. 6. Tracings of a representative experiment showing the chemoreflex responses before and 5 min after bilateral microinjection of bicuculline (100 pmol/100 nl; n = 7 rats) into the lateral part of the PBN. a: arterial pressure, b: MAP, c: HR.
Fig. 4. Tracings of a representative experiment showing the chemoreflex responses before and 5 min after bilateral microinjection of CoCl2 (5 mM/100 nl; n = 7 rats) into the lateral part of the PBN. a: arterial pressure, b: MAP, c: HR.
response (before: ΔHR = − 61.6 ± 9.8, after: ΔMAP = −32.2 ± 7 bpm, paired t-test, P < 0.05, Figs. 3 and 7). 3.6. Histology Distribution of the injection sites is shown in Fig. 8. Data of the injection sites outside the LPBN were not included in the analysis. 4. Discussion The peripheral chemoreflex is an important mechanism in oxygen sensing during hypoxic states such as obstructive sleep apnea (Prabhakar et al., 2007; Semenza and Prabhakar 2007) and sudden infant death syndrome (Gauda et al., 2007). In this study we investigated the roles of GABAA and alpha 2-adrenergic receptors of the LPBN in the chemoreflex cardiovascular responses and the possible interaction between them. The present study contributes to the advance in the understanding of chemoreflex network by showing that the LPBN is the main (if not the only) producer of the cardiovascular chemoreflex responses, in which the pressor response of the chemoreflex, is produced by α2- adenoreceptors, and GABA in the LPBN inhibits the cardiovascular system. KCN was used to stimulate peripheral
Fig. 5. Tracings of a representative experiment showing the chemoreflex responses before and 5 min after bilateral microinjection of yohimbine (10 nmol/ 100 nl; n = 13 rats) into the lateral part of the PBN. a: arterial pressure, b: MAP, c: HR.
74.0 ± 12.4 mm Hg; HR: 401 ± 8.2 vs. 408 ± 11 bpm, paired t-test, P > 0.05, n = 11 rats, Fig. 1). Sequential microinjection of yohimbine and BMI had no significant effect on the chemoreflex pressor response (before: ΔMAP = 19.9 ± 2.7, after: ΔMAP = 17.4 ± 4.9 mmHg, paired t-test, P > 0.05) but significantly attenuated the bradycardic 193
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Fig. 7. Tracings of a representative experiment showing the chemoreflex responses before and 5 min after bilateral sequential microinjection of bicuculline (100 pmol) and yohimbine (10 nmol; n = 11 rats) into the lateral part of the PBN. a: arterial pressure, b: MAP, c: HR.
several days of intermittent hypoxia showed hypertension by increased sympathetic nerve activity and elevated urinary catecholamines similar to the phenotype reported in sleep apnea patients that exhibit elevated circulating and urinary catecholamines (both norepinephrine and epinephrine), which were attributed to increased sympathetic nerve activity (Fletcher 1995; Prabhakar 2016). The present study was also performed to find the role of GABAA receptors of LPBN in peripheral chemoreflex activation. GABAergic nerve terminals and receptors have been found in the LPBN (Callera et al., 2005; de Oliveira et al., 2007; Domingos-Souza et al., 2016). The present work demonstrated that GABAA receptor inhibition by microinjection of bicuculline into the LPBN increased baseline MAP and HR (Fig. 1), suggesting that GABA tonicly inhibits cardiovascular system through GABAA receptors in the LPBN. We showed that microinjection of GABAA antagonist, BMI, into the LPBN significantly augmented the chemoreflex pressor response (Figs. 3a and 6) and significantly attenuated the bradycardia (Fig. 3b and 6), Some previous reports indicated that the chemoreflex bradycardia response following blood pressure increase is predominantly due to an increase in cardiac vagal activity since it was prevented by i.v. treatment with the cholinergic muscarinic receptor antagonist methyl atropine and was not affected by i.v. treatment with the selective β1adrenoceptor antagonist, atenolol (Head and McCarty, 1987; Braga et al., 2008). However, our data indicates that chronotropic sympathetic output to the heart must be involved too. Since when GABAA receptors are blocked, the bradycardia is attenuated. If GABA was inhibiting the parasympathetic output to the heart, GABAA receptors blockade should has been resulted in augmentation of bradycardia. In another series of experiments, we found that sequential microinjection of yohimbine and BMI caused small non-significant increase in basal levels of blood pressure with no change in HR (Fig. 1a and b). Microinjection of yohimbine and BMI, 5 min before chemoreflex activation by KCN, did not significantly affect the chemoreflex pressor response and significantly attenuated the bradycardia (Fig. 3). In other words, we saw the effects of both GABA and yohimbine, indicating that there is no interaction between GABAergic and adrenergic neurons. If there was an interaction, the effect of one of them should be eliminated. Attenuation of the bradycardic response was similar to the effect of microinjection of BMI alone on the bradycardia. In other words, yohimbine alone had no effect on the bradycardia, so the resulted attenuation of bradycardia by BMI and yohimbine must be the effect of BMI. In conclusion, the LPBN is essential for the chemoreflex responses. The pressor response of the chemoreflex is produced by α2-
chemoreceptors located in the carotid bodies. Our observations similar to the previous findings showed that the first response to KCN-induced activation of peripheral chemoreceptors was a rapid rise in blood pressure. This is attributable to an increase in sympathetic drive to the vasculature since pretreatment with α1-adrenergic receptor antagonist, prazosin, prevented this component of the response (Braga et al., 2008). As previously reported (Braga et al., 2008) and we saw in our experiments, this rapid elevation of blood pressure was immediately followed by a significant hypotension (Figs. 4–7). This hypotension is at least partly dependent on the bradycardia produced by parasympathetic activation, since pretreatment with the muscarinic receptor antagonist, atropine, attenuated both responses (Braga et al., 2008). However it cannot rule out the effect of cyanide acting directly on vasculature producing additional hypotension or the effect of activation of baroreflex during pressor response resulting in a transient sympatho-inhibition. Microinjection of a reversible synaptic blocker, cobalt chloride, into the LPBN strongly attenuated both pressor and bradycardic responses of chemoreflex, indicating that the LPBN is a major site for producing cardiovascular chemoreflex. We found that bilateral microinjections of yohimbine (an α2 adrenergic receptor blocker) into the LPBN caused no statistically significant change in basal levels of blood pressure and heart rate (Fig. 1), suggesting that there is no or little tonic activity of α2 adrenoceptors in the LPBN. Approximately 5 min following blockade of α2 adrenergic receptors in LPBN, chemoreflex was activated by KCN and we observed a marked attenuation in chemoreflex pressor response with no change in bradycardia (Figs. 3 and 5). Reflex responses returned to control within 1 h. This finding suggests that α2-adrenoceptors of LPBN are involved in the pressor response, independent of parasympathetic component of chemoreflex bradycardia (Braga et al., 2008). Hypoxia-activated afferent inputs from chemoreceptors are relayed to the Kölliker-Fuse/parabrachial complex directly via the NTS and indirectly via ventrolateral medulla (VLM) and A5 region (Song et al., 2011). Immunohistochemical studies have shown that brief exposure to hypoxia in rats activates noradrenergic neurons in the A1 and A5 regions (Linda 2001). These inputs directly or indirectly stimulate the adrenergic neuron, and adrenergic neuron via α2 receptors stimulates the sympathoexcitatory neuron to the RVLM (Agarwal and Calaresu 1993) producing the pressor response. Enhancement of sympathetic component of peripheral chemoreflex response by α2-AR may be especially important in exaggerated pressor response in obstructive sleep apnea. It was found that rats exposed to 194
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Fig. 8. Schematic coronal section of rat brain adopted from an atlas (Paxinose and Watson, 2007). The injection sites of CoCl2 were shown as star symbol, DMSO as filled circles, bicuculline as filled squares, yohimbine as open squares and yohimbine + bicuculline as filled triangle: 4 V: the 4th ventricle, KF: the Kȍlliker-Fuse nucleus, LPB: the lateral parabrachial nucleus, mcp: the middle cerebellar peduncle, MPB: the medial parabrachial nucleus, scp: the superior cerebellar peduncle.
adenoreceptors. GABA in the LPBN inhibits the cardiovascular system. Finally, there is no interaction between GABAergic and adrenergic neurons of the LPBN in producing the cardiovascular chemoreflex. Conflict of interest The authors declare that they have no conflict of interest with anyone or with any organization. Acknowledgments This manuscript was extracted from a M.S. thesis by Gholam Reza Namvar sponsored by the Vice Chancellery of Research of Shiraz 195
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Song, G., Xu, H., Wang, H., MacDonald, S.M., Poon, C.-S., 2011. Hypoxia-excited neurons in NTS send axonal projections to Kölliker-Fuse/parabrachial complex in dorsolateral pons. Neuroscience 175, 145–153. Talley, E.M., Rosin, D.L., Lee, A., Guyenet, P.G., Lynch, K.R., 1996. Distribution of (2Aadrenergic receptor-like immunoreactivity in the rat central nervous system. J. Comp. Neurol. 372 (1), 111–134. Yeganeh, F., Nasimi, A., Hatam, M., 2017. Interaction of GABA and norepinephrine in the lateral division of the bed nucleus of the stria terminals in anesthetized rat, correlating single-unit and cardiovascular responses. Neuroscience. de Oliveira, L.B., Callera, J.C., De Luca, L.A., Colombari, D.S.A., Menani, J.V., 2007. GABAergic mechanisms of the lateral parabrachial nucleus on sodium appetite. Brain Res. Bull. 73 (4), 238–247.
Physiol. Neurobiol. 157 (1), 148–153. Prabhakar, N.R., 2016. Carotid body chemoreflex: a driver of autonomic abnormalities in sleep apnoea. Exp. Physiol. 101 (8), 975–985. Rasoulpanah, M., Kharazmi, F., Hatam, M., 2014. Evaluation of GABA receptors of ventral tegmental area in cardiovascular responses in rat. Iran. J. Med. Sci. 39 (4), 374. Roncari, C., David, R.B., De Paula, P.M., Colombari, D.S.A., De Luca, L.A., Menani, J.V., 2011. Importance of central AT 1 receptors for sodium intake induced by GABAergic activation of the lateral parabrachial nucleus. Neuroscience 196, 147–152. Saleh, T.M., Connell, B.J., 1997. Modulation of the cardiac baroreflex following reversible blockade of the parabrachial nucleus in the rat. Brain Res. 767 (2), 201–207. Semenza, G.L., Prabhakar, N.R., 2007. HIF-1–dependent respiratory, cardiovascular, and redox responses to chronic intermittent hypoxia. Antioxid. Redox Signal. 9 (9), 1391–1396.
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Research report
α2 Receptors in the lateral parabrachial nucleus generates the pressor response of the cardiovascular chemoreflex, effects of GABAA receptor Nafiseh Mirzaei-Damabi, Gholam Reza Namvar, Fahimeh Yeganeh, Masoumeh Hatam
T
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Dept. of Physiology, Shiraz University of Medical Sciences, Shiraz, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: LPBN Peripheral chemoreflex Bicuculline Yohimbine
The lateral parabrachial nucleus (LPBN) is a pontine area involved in cardiovascular chemoreflex. This study was performed to find the effects of reversible synaptic blockade of the LPBN on the chemoreflex responses, and to find the roles of GABAA receptor and α2-adenoreceptor (α2-AR) in chemoreflex. It also aimed to seek possible interaction between GABA and noradrenergic systems of the LPBN in urethane-anesthetized male rats. Cardiovascular chemoreflex was activated by intravenous injection of potassium cyanide (KCN, 80 μg/kg). The cardiovascular responses of chemoreflex were evaluated before (control), 5 and 15 min after microinjection of each drug (100 nl) into the LPBN. Microinjections of cobalt chloride (5 mM), a reversible synaptic blocker, into the LPBN greatly attenuated the chemoreflex pressor and bradycardic responses indicating that the LPBN plays a main role in chemoreflex. Local injection of yohimbine (10 nmol), an α2-AR antagonist, attenuated the pressor response with no effect on bradycardic response, suggesting that α2-adrenoreceptors are involved in producing the pressor response of the chemoreflex. Microinjection of bicuculline methiodide (BMI, 100 pmol), a GABAA antagonist, into the LPBN augmented the pressor response and attenuated the bradycardic response, indicating that GABA inhibits the sympathetic output to the heart and vasculature. Sequential injection of yohimbine and BMI had no significant effect on the pressor response but attenuated the bradycardia. In conclusion, the LPBN is essential for the chemoreflex responses. The pressor response of the chemoreflex, at least partly, is produced by α2- adenoreceptors. GABA in the LPBN inhibits the cardiovascular system. Finally, there is no interaction between GABAergic and adrenergic neurons of the LPBN in producing the cardiovascular chemoreflex.
1. Introduction The chemoreflex is an important neural mechanism involved in cardiovascular and respiratory control. Peripheral chemoreceptors, which in rats are located mainly in the carotid body glomus cells respond to changes in blood PO2 within milliseconds, contributing to ventilatory and cardiovascular regulation (Menani and Johnson 1995; Colombari et al., 1996; Lahiri et al., 2001). Activation of chemoreceptors can be performed by systemic administration of potassium cyanide (KCN), which produces a cytotoxic hypoxia (Barros et al., 2002). It is characterized by an increase in respiratory frequency, an increase in arterial pressure and a decrease in heart rate in awake (Barros et al., 2002; Haibara et al., 2002; Fernandes et al., 2005; Roncari et al., 2011) and in anesthetized rats (Ciriello and Moreau, 2013). These responses are essentially dependent on the stimulation of carotid chemoreceptors, because bilateral ligature of the carotid body ⁎
Corresponding author. E-mail address: [email protected] (M. Hatam).
https://doi.org/10.1016/j.brainresbull.2018.05.009 Received 20 October 2017; Received in revised form 24 April 2018; Accepted 8 May 2018 Available online 21 May 2018 0361-9230/ © 2018 Elsevier Inc. All rights reserved.
arteries (Franchini and Krieger 1993; Haibara et al., 1995; Barros et al., 2002) or deafferentation of the carotid sinus nerve bifurcation (Franchini and Krieger 1993) abolishes both cardiorespiratory and behavioral responses to KCN injection. The cardiovascular responses to KCN result from the activation of the following two independent autonomic mechanisms: (1) a sympathetic pathway related to the pressor response, which is blocked by intravenous injection of prazosin, an α1adrenoceptor antagonist; and (2) a parasympathetic pathway related to the bradycardic response, which is abolished by intravenous injection of atropine (Franchini and Krieger 1993; Haibara et al., 1995; Fernandes et al., 2005; Braga et al., 2008). The parabrachial nucleus (PBN) is a brain structure located in the dorsolateral pons surrounding the superior cerebellar peduncle. The PBN consists of three major subdivisions: the lateral parabrachial nucleus (LPBN), the medial parabrachial nucleus (MPBN) and the ventrolateral Kolliker- Fuse nucleus (Fulwiler and Saper 1984). Several anatomical (Herbert and Flügge, 1990) and physiological
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AR antagonist yohimbine or the GABAA-receptor antagonist bicuculline methiodide (BMI). For interaction experiment one barrel contained yohimbine, the second barrel contained bicuculline methiodide. Drugs were microinjected using a pressurized air pulse applicator. The injection volume was measured by direct observation of the fluid meniscus in the micropipette using a microscope fitted with an ocular micrometer that allowed a 2-nl resolution (U.W.O, Canada). The injection volume for drugs was100 nl for each side. Blood pressure and heart rate were rerecorded using a ML T844 pressure transducer coupled to a pre-amplifier (FE221 Bridge amplifier, AD instruments) connected to a power lab 4/35 data acquisition system (model PL3504 CE instruments). The chemoreflex was activated by systemic intravenous injection of 80 μg/Kg/0.1 ml of KCN (Sigma, USA) [9,10]. The cardiovascular responses to chemoreflex activation were evaluated before (control), 5 and 15 min after microinjection of each drug. The vehicle groups received bilateral microinjection of saline or DMSO with the same experimental protocol. All drugs were dissolved in saline except yohimbine, which was dissolved in DMSO. Experiments performed on both sides of the LPBN.
studies (Braga et al., 2008) indicated a significant role for the LPBN in regulating cardiovascular activity. Electrical and chemical stimulation of the LPBN subnuclei, dorsal, central and the external, caused a significant increase in mean arterial pressure (MAP), in heart rate (HR) and in sympathetic nerve activity (Chamberlin and Saper 1992) whereas bradycardia was reported from stimulation of the dorsal LPBN (Chamberlin and Saper 1992). Reversible bilateral blockade of the LPBN by injections of lidocaine induced an immediate pressor response (Saleh and Connell 1997). Thus, it seems that the LPBN tonically attenuates MAP. Furthermore, it has been shown that stimulation of the LPBN modulates baroreflex elicited by stimulation of aortic depressor nerve (Hayward and Felder 1998). Microinjection of lidocaine, a reversible blocker of neuronal activity and fibers of passage, into the dorsolateral part of the PBN reduced pressor response to chemoreflex activation with potassium cyanide in awake rat with no significant change in the bradycardic responses (Haibara et al., 2002). Immunohistochemical studies have shown the presence of α2adrenergic sites (Herbert and Flügge, 1995; Talley et al., 1996; Andrade et al., 2011) and GABAA receptors (Callera et al., 2005; Roncari et al., 2011) in the LPBN. Several studies indicated that activation of LPBN α2-adrenergic receptors (Andrade et al., 2015) and GABA receptors enhanced NaCl and water intake in satiated or sodium depleted rats (Callera et al., 2005; de Oliveira et al., 2007; De Oliveira et al., 2008; De Oliveira et al., 2011). Despite evidence showing the presence of GABAA and alpha 2adrenergic receptors in the LPBN there is no study investigating cardiovascular responses of these receptors and possible interaction between them. Also, there is no study showing the involvement of these two receptors in the chemoreflex activation. Thus, this study was performed to find:
2.3. Experimental groups The experiments consisted of the following groups:
• The vehicle group: 100 nl of the vehicle (saline: n = 8 rats, DMSO: n = 4 rats) was microinjected into each side of LPBN. • Microinjection of a reversible synaptic blocker cobalt chloride • •
– The effects of microinjection of yohimbine, an α2-adenoreceptor (AR) antagonist, into the LPBN on chemoreflex activation. – The effects of microinjection of bicuculline, a GABAA receptor antagonist, in the LPBN on the chemoreflex activation. – The possible interaction between α2-adenoreceptor and GABAA receptor in the LPBN – The effect of acute inhibition of the LPBN by CoCl2 microinjection on chemoreflex activation.
•
(CoCl2, 5 mM/100 nl, Sigma, USA) (Rasoulpanah et al., 2014, Hatam et al., 2015) Microinjection of anα2-AR antagonist, yohimbine (10 nmol/100 nl; n = 13 rats; Sigma, USA) (Yeganeh et al., 2017) Microinjection of a GABAA antagonist, bicuculline methiodide (BMI, 100 pmol/100 nl, n = 9 rats, Sigma, USA) (Rasoulpanah et al., 2014, Hatam et al., 2015). Sequential microinjections of bicuculline (100 pmol) and yohimbine (10nmol; n = 8 rats) in a final volume of 100 nl.
2.4. Histological verification of injection sites At the end of each experiment, the animal was sacrificed using a high dose of the anesthetic (urethane) and perfused transcardially with100 ml of 0.9% saline followed by100 ml of 10% formalin. The brain was removed and stored in 10% formalin for at least 24 h. Frozen serial transverse sections (40 mm) of the forebrain were cut and stained with cresyl violet 1%. The injection sites were determined according to brain atlas ((Paxinose and Watson, 2007), using a light microscope.
2. Experimental procedures 2.1. General procedures The experiments were performed on 50 male Sprague Dawley rats (250–300 g, 10–11 weeks old), approved by the Animal Use and Care Committee of Shiraz University of Medical Sciences. The rats had free access to food and water under a 12 h light/dark cycle with a room temperature of ∼25 °C. Animals were anesthetized with urethane (1.4 g/kg, ip), and supplementary doses (0.7 g/kg) were given if needed. The trachea was cannulated to ease the ventilation. The body temperature was maintained at 37 ± 1C°, using a controlled heat pad. For recording of blood pressure, the right femoral artery was cannulated with a Polyethylene catheter (PE-50) filled with heparinized saline. Two holes were drilled above the LPBN at coordinates 9.0–9.6 mm caudal to bregma, 2–2.2 mm lateral to the midline and 6.2–6.6 mm ventral to the cortical surface of bregma according to a stereotaxic atlas of the rat brain (Paxinose and Watson, 2007).
2.5. Statistical analysis The mean arterial pressure (MAP) and heart rate (HR) values were expressed as mean ± SE. The mean of the maximum changes of MAP or HR was compared with those of the vehicle group using independent t-test and with the pre-injection values using paired t-test. A P < 0.05 was considered as the statistical significance. 3. Results 3.1. Chemoreflex responses to bilateral microinjection of vehicle (Saline or DSMO) into the LPBN In this group, the baseline MAP was 78.8 ± 6.7 and HR was 395 ± 8.4. Bilateral microinjection of vehicle (100 nl each side) did not affect MAP or HR (saline: ΔMAP = 3.3 ± 2.1 mmHg, ΔHR = 2.6 ± 1.2 beats/min; n=8 rats; DMSO: ΔMAP = .6 ± 0.8 mmHg, ΔHR = − 3.0 ± 1.0 beats/min; paired t-
2.2. Microinjection and chemoreflex activation To inject drugs into the LPBN, a single or double barreled micropipette with a tip diameter of 30–35 μwas made using capillary tubing (Stoleting, USA). One barrel of the pipette contained saline or the α2191
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significant effect on the baseline values of MAP and HR (MAP = 59.5 ± 4.9 vs. 62.7 ± 6.3 mmHg and HR = 378.5 ± 15.0 vs. 378.7 ± 14.8 bpm, paired t-test, P > 0.05, n = 7 rats, Fig. 1). Microinjections of cobalt chloride into the LPBN significantly attenuated the pressor and bradycardic responses of the chemoreflex (before: ΔMAP 22.8 ± 3.2, after: ΔMAP = 6.2 ± 2.3 mmHg and ΔHR: before −33.5 ± 7.0, after:−12.1 ± 8.1bpm, paired t-test, P < 0.05, Figs. 3 and 4). 3.3. Chemoreflex responses to bilateral microinjection of yohimbine, an α2 adrenoceptor (AR) blocker, into the LPBN Bilateral microinjection of yohimbine (10 nmol/100 nl, each side, Sigma) into the LPBN had no significant effect on baseline MAP and HR (MAP = 78.3 ± 7.3 vs. 71.6 ± 8.1 mmHg and HR = 402.0 ± 11.0 vs. 400.3 ± 12.8 bpm, paired t-test, P > 0.05, n = 13 rats, Fig. 1).Yohimbine significantly attenuated the pressor response of chemoreflex (before: ΔMAP = 23 ± 2.4, after: ΔMAP = 11 ± 2.5 mmHg, paired t-test, P < 0.01, n = 13 rats) and had no significant effect on bradycardic response (ΔHR: before: −52.2 ± 7.5, after: −74.8 ± 15.2.1 bpm, paired t-test, P > 0.05, Figs. 3 and 5). The effect of yohimbine was reversible, since the response returned to normal level 15 min after the microinjection. 3.4. Chemoreflex responses to microinjection of bicuculline methiodide, a GABAA antagonist, into the LPBN Bilateral or unilateral microinjection of BMI (100 pmol/100nl) into the LPBN produced an increase in baseline MAP (MAP = 70.8 ± 5.5 vs. 87.3 ± 3.4 mmHg, n = 7 rats, paired t-test, P < 0.5) and HR (HR = 342.5 ± 12.1 vs. 379.5.6 ± 15.5 bpm, n = 7 rats, paired t-test, P < 0.5, Fig. 1). BMI significantly augmented the chemoreflex pressor response (before: ΔMAP = 9.6 ± 2.5, after: ΔMAP = 14.3 ± 1.9 mmHg, paired t-test, P < 0.01) and attenuated the chemoreflex bradycardic response (before: ΔHR = −38.6 ± 8.6, after: ΔHR = − 21.6 ± 2.8 bpm, paired t-test, P < 0.05). The effect of BMI lasted more than 1 h and in some cases did not return until the end of the experiment (Figs. 3 and 6).
Fig. 1. Histograms summarizing the effects of various drugs microinjected into the lateral part of the PBN on the baseline MAP (a) and HR (b). Data were compared to the preinjection values (paired t-test, *P < 0.5, ***P < 0.001).
test, P > 0.05, n = 4 rats; Fig. 1). Microinjections of vehicle into the LPBN produced no change in the pressor (saline: ΔMAP: 11.5 ± 2 vs. 11.8 ± 2 mmHg; DMSO: ΔMAP = 16.0 ± 2.0 mmHg vs. 15.6 ± 1.45 mm Hg, paired t-test, P > 0.05) or bradycardic response (saline: ΔHR: −59.5 ± 9.5 vs −61.6 ± 8.8 beats/min; DMSO: ΔHR: −26.6 ± 4.9 vs −32.6 ± 2.3 beats/min, paired t-test, P > 0.05) to chemoreflex activation by KCN (Figs. 2 and 3).
3.5. Chemoreflex responses to sequential microinjection of BMI and yohimbine into the LPBN
3.2. Chemoreflex responses after bilateral microinjection of cobalt chloride into the LPBN
Bilateral sequential microinjection of yohimbine (10 nmol/50 nl) and BMI (100 pmol/50 nl) into the LPBN did not elicit a significant change in the baseline values of MAP and HR (MAP: 63.1 ± 8.5 vs.
Bilateral microinjection of a reversible synaptic blocker, cobalt chloride (CoCl2: 5 mM/100 nl, each side; Sigma), into the LPBN had no
Fig. 2. Tracings of a representative experiment showing the chemoreflex responses before and 5 min after bilateral microinjection of saline (100 nl, n = 8 rats) into the lateral part of the PBN. The arrow shows the injection time. a: arterial pressure, b: MAP, c: HR. 192
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Fig. 3. Histograms summarizing chemoreflex responses, before and 5 min after various drugs microinjected into the lateral part of the PBN (paired t-test, *P < 0.5, ***P < 0.001). a: ΔMAP, b: ΔHR.
Fig. 6. Tracings of a representative experiment showing the chemoreflex responses before and 5 min after bilateral microinjection of bicuculline (100 pmol/100 nl; n = 7 rats) into the lateral part of the PBN. a: arterial pressure, b: MAP, c: HR.
Fig. 4. Tracings of a representative experiment showing the chemoreflex responses before and 5 min after bilateral microinjection of CoCl2 (5 mM/100 nl; n = 7 rats) into the lateral part of the PBN. a: arterial pressure, b: MAP, c: HR.
response (before: ΔHR = − 61.6 ± 9.8, after: ΔMAP = −32.2 ± 7 bpm, paired t-test, P < 0.05, Figs. 3 and 7). 3.6. Histology Distribution of the injection sites is shown in Fig. 8. Data of the injection sites outside the LPBN were not included in the analysis. 4. Discussion The peripheral chemoreflex is an important mechanism in oxygen sensing during hypoxic states such as obstructive sleep apnea (Prabhakar et al., 2007; Semenza and Prabhakar 2007) and sudden infant death syndrome (Gauda et al., 2007). In this study we investigated the roles of GABAA and alpha 2-adrenergic receptors of the LPBN in the chemoreflex cardiovascular responses and the possible interaction between them. The present study contributes to the advance in the understanding of chemoreflex network by showing that the LPBN is the main (if not the only) producer of the cardiovascular chemoreflex responses, in which the pressor response of the chemoreflex, is produced by α2- adenoreceptors, and GABA in the LPBN inhibits the cardiovascular system. KCN was used to stimulate peripheral
Fig. 5. Tracings of a representative experiment showing the chemoreflex responses before and 5 min after bilateral microinjection of yohimbine (10 nmol/ 100 nl; n = 13 rats) into the lateral part of the PBN. a: arterial pressure, b: MAP, c: HR.
74.0 ± 12.4 mm Hg; HR: 401 ± 8.2 vs. 408 ± 11 bpm, paired t-test, P > 0.05, n = 11 rats, Fig. 1). Sequential microinjection of yohimbine and BMI had no significant effect on the chemoreflex pressor response (before: ΔMAP = 19.9 ± 2.7, after: ΔMAP = 17.4 ± 4.9 mmHg, paired t-test, P > 0.05) but significantly attenuated the bradycardic 193
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Fig. 7. Tracings of a representative experiment showing the chemoreflex responses before and 5 min after bilateral sequential microinjection of bicuculline (100 pmol) and yohimbine (10 nmol; n = 11 rats) into the lateral part of the PBN. a: arterial pressure, b: MAP, c: HR.
several days of intermittent hypoxia showed hypertension by increased sympathetic nerve activity and elevated urinary catecholamines similar to the phenotype reported in sleep apnea patients that exhibit elevated circulating and urinary catecholamines (both norepinephrine and epinephrine), which were attributed to increased sympathetic nerve activity (Fletcher 1995; Prabhakar 2016). The present study was also performed to find the role of GABAA receptors of LPBN in peripheral chemoreflex activation. GABAergic nerve terminals and receptors have been found in the LPBN (Callera et al., 2005; de Oliveira et al., 2007; Domingos-Souza et al., 2016). The present work demonstrated that GABAA receptor inhibition by microinjection of bicuculline into the LPBN increased baseline MAP and HR (Fig. 1), suggesting that GABA tonicly inhibits cardiovascular system through GABAA receptors in the LPBN. We showed that microinjection of GABAA antagonist, BMI, into the LPBN significantly augmented the chemoreflex pressor response (Figs. 3a and 6) and significantly attenuated the bradycardia (Fig. 3b and 6), Some previous reports indicated that the chemoreflex bradycardia response following blood pressure increase is predominantly due to an increase in cardiac vagal activity since it was prevented by i.v. treatment with the cholinergic muscarinic receptor antagonist methyl atropine and was not affected by i.v. treatment with the selective β1adrenoceptor antagonist, atenolol (Head and McCarty, 1987; Braga et al., 2008). However, our data indicates that chronotropic sympathetic output to the heart must be involved too. Since when GABAA receptors are blocked, the bradycardia is attenuated. If GABA was inhibiting the parasympathetic output to the heart, GABAA receptors blockade should has been resulted in augmentation of bradycardia. In another series of experiments, we found that sequential microinjection of yohimbine and BMI caused small non-significant increase in basal levels of blood pressure with no change in HR (Fig. 1a and b). Microinjection of yohimbine and BMI, 5 min before chemoreflex activation by KCN, did not significantly affect the chemoreflex pressor response and significantly attenuated the bradycardia (Fig. 3). In other words, we saw the effects of both GABA and yohimbine, indicating that there is no interaction between GABAergic and adrenergic neurons. If there was an interaction, the effect of one of them should be eliminated. Attenuation of the bradycardic response was similar to the effect of microinjection of BMI alone on the bradycardia. In other words, yohimbine alone had no effect on the bradycardia, so the resulted attenuation of bradycardia by BMI and yohimbine must be the effect of BMI. In conclusion, the LPBN is essential for the chemoreflex responses. The pressor response of the chemoreflex is produced by α2-
chemoreceptors located in the carotid bodies. Our observations similar to the previous findings showed that the first response to KCN-induced activation of peripheral chemoreceptors was a rapid rise in blood pressure. This is attributable to an increase in sympathetic drive to the vasculature since pretreatment with α1-adrenergic receptor antagonist, prazosin, prevented this component of the response (Braga et al., 2008). As previously reported (Braga et al., 2008) and we saw in our experiments, this rapid elevation of blood pressure was immediately followed by a significant hypotension (Figs. 4–7). This hypotension is at least partly dependent on the bradycardia produced by parasympathetic activation, since pretreatment with the muscarinic receptor antagonist, atropine, attenuated both responses (Braga et al., 2008). However it cannot rule out the effect of cyanide acting directly on vasculature producing additional hypotension or the effect of activation of baroreflex during pressor response resulting in a transient sympatho-inhibition. Microinjection of a reversible synaptic blocker, cobalt chloride, into the LPBN strongly attenuated both pressor and bradycardic responses of chemoreflex, indicating that the LPBN is a major site for producing cardiovascular chemoreflex. We found that bilateral microinjections of yohimbine (an α2 adrenergic receptor blocker) into the LPBN caused no statistically significant change in basal levels of blood pressure and heart rate (Fig. 1), suggesting that there is no or little tonic activity of α2 adrenoceptors in the LPBN. Approximately 5 min following blockade of α2 adrenergic receptors in LPBN, chemoreflex was activated by KCN and we observed a marked attenuation in chemoreflex pressor response with no change in bradycardia (Figs. 3 and 5). Reflex responses returned to control within 1 h. This finding suggests that α2-adrenoceptors of LPBN are involved in the pressor response, independent of parasympathetic component of chemoreflex bradycardia (Braga et al., 2008). Hypoxia-activated afferent inputs from chemoreceptors are relayed to the Kölliker-Fuse/parabrachial complex directly via the NTS and indirectly via ventrolateral medulla (VLM) and A5 region (Song et al., 2011). Immunohistochemical studies have shown that brief exposure to hypoxia in rats activates noradrenergic neurons in the A1 and A5 regions (Linda 2001). These inputs directly or indirectly stimulate the adrenergic neuron, and adrenergic neuron via α2 receptors stimulates the sympathoexcitatory neuron to the RVLM (Agarwal and Calaresu 1993) producing the pressor response. Enhancement of sympathetic component of peripheral chemoreflex response by α2-AR may be especially important in exaggerated pressor response in obstructive sleep apnea. It was found that rats exposed to 194
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Fig. 8. Schematic coronal section of rat brain adopted from an atlas (Paxinose and Watson, 2007). The injection sites of CoCl2 were shown as star symbol, DMSO as filled circles, bicuculline as filled squares, yohimbine as open squares and yohimbine + bicuculline as filled triangle: 4 V: the 4th ventricle, KF: the Kȍlliker-Fuse nucleus, LPB: the lateral parabrachial nucleus, mcp: the middle cerebellar peduncle, MPB: the medial parabrachial nucleus, scp: the superior cerebellar peduncle.
adenoreceptors. GABA in the LPBN inhibits the cardiovascular system. Finally, there is no interaction between GABAergic and adrenergic neurons of the LPBN in producing the cardiovascular chemoreflex. Conflict of interest The authors declare that they have no conflict of interest with anyone or with any organization. Acknowledgments This manuscript was extracted from a M.S. thesis by Gholam Reza Namvar sponsored by the Vice Chancellery of Research of Shiraz 195
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