Role of cuneiform nucleus in regulation of sympathetic vasomotor tone in rats

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1 Pathophysiology 19 (2012) Role of cuneiform nucleus in regulation of sympathetic vasomotor tone in rats Mohammad Naser Shafei a,, Ali Nasimi b, Hojatallah Alaei b, Ali Asghar Pourshanazari b, Mahmoud Hosseini a a Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran b Department of Physiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Received 2 September 2010; received in revised form 16 February 2011; accepted 2 November 2011 Abstract The cuneiform nucleus (CnF) is a sympathoexcitatory area involved in the central cardiovascular regulation. Its role in the maintaining vasomotor tone has, however, not yet been clarified. In the present study the effects of cobalt chloride (CoCl 2 ) a nonselective synapse blocker and NMDA and non-nmda glutamate receptors on resting mean arterial blood pressure and heart rate of CnF have been evaluated. CoCl 2, AP5 (an NMDA receptor antagonist) and CNQX (an AMPA/kinase receptor antagonist) (100 nl) were microinjected into the CnF of anesthetized rats. The blood pressure and heart rate were recorded throughout the experiment. The responses of blood pressure and heart rate were compared with the pre-injection (paired t-test) and control (independent t-test) values. Microinjection of CoCl 2, AP5 and CNQX did not change the basal blood pressure and heart rate. In conclusion, our present study indicates that the CnF is not important in the regulation of cardiovascular tone Published by Elsevier Ireland Ltd. Keywords: Cuneiform nucleus (CnF); Vasomotor tone; Rats; NMDA; AMPA/Kainite receptor; CoCl 2 1. Introduction In recent years the studies in the central cardiovascular regulation have focused on the origins of sympathetic vasomotor activity under resting conditions (sympathetic vasomotor tone) and its neurocircuitry [1 3]. Previous studies in humans and animals have shown that under resting conditions, sympathetic vasomotor tone and arterial blood pressure are driven by descending inputs originating from supraspinal areas [4]. These inputs that directly project to sympathetic preganglionic neurons in the spinal cord (premotor neurons) are largely confined to five discrete regions within the brainstem (ventrolateral medulla (RVLM), rostral ventromedial medulla, caudal raphe nuclei, A5 area Group), and hypothalamus (paraventricular nucleus). Of these five nuclei, the RVLM appear to have a principal role in vasomotor regulation of cardiovascular tone and arterial blood pressure [3 5]. Corresponding author. Tel.: ; fax: address: Shafeimn1@mums.ac.ir (M.N. Shafei). In addition, RVLM receives several inputs from of different nuclei in the brain including cuneiform nucleus (CnF) [6,7]. The CnF is a sympathoexcitatory area of the midbrain and is involved in the central cardiovascular regulation [1,6 8]. Both chemical and electrical stimulation of the CnF evoked an increase in arterial blood pressure and excitation of the lumbar sympathetic vasomotor neurons [6,7]. Neuroanatomical studies have showed that CnF is connected with regions involved in cardiovascular regulation, such as the periaqueductal gray (PAG) matter, the parabrachial/kollikerfuse nuclei complex, locus coeruleus, nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus and RVLM [1,6,9]. It has been shown that cardiovascular effects of CnF are partly due to activation of sympathoexcitatory neurons in the RVLM [1,6]. Glutamate is a major excitatory receptor in the CNS and via its two N-methyl-d-aspartic acid (NMDA), and non-nmda receptors regulate cardiovascular functions. The role of glutamate and its NMDA and non-nmda receptors in maintaining vasomotor tone in several areas such as lateral tegmental files and RVLM has been shown [4,10]. In /$ see front matter 2011 Published by Elsevier Ireland Ltd. doi: /j.pathophys

2 152 M.N. Shafei et al. / Pathophysiology 19 (2012) addition, glutamate-containing neurons have been shown in the CnF in pharmacological and immunocytochemical studies [11,12]. Although role of the CnF in cardiovascular regulation and its effect on sympathetic vasomotor neurons has been indicated in previous studies, its effect on maintaining vasomotor tone has not yet been clarified. Therefore, in the present study the role of CnF in resting mean arterial blood pressure and heart rate after blocked by cobalt chloride (CoCl 2 ) (a nonselective synapse blocker) in anesthetized rats has been evaluated. In addition, role of NMDA and non-nmda of glutamate receptors on the resting mean arterial blood pressure and heart rate was also evaluated. 2. Materials and methods 2.1. Animals Experiments were done in 32 male Wistar rats ( g; Razi Institute, Tehran, Iran) in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). The rats were anesthetized with urethane (1.4 g/kg, i.p.), and supplementary doses (0.7 g/kg). The trachea was intubated for ease ventilation and temperature was kept at 37.5 C with a thermostatically controlled heating pad. After induction of anesthesia a polyethylene catheter (PE- 50) were inserted in the left femoral artery for recording of blood pressure (BP) and heart rate (HR). The BP and HR were continuously recorded by both a Harvard polygraph and a computer program written in this laboratory Drugs The drugs used in the experiments were urethane (Sigma Chemical Co., MO, USA), CNQX (6-cyano-7- nitroquinoxaline-2-3-dione, an AMPA receptor antagonist), AP5 (dl-2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist) and CoCl 2 (all from Sigma). All drugs were dissolved in saline (0.9% NaCl). CNQX was first solubilized with dimethyl sulfoxide (DMSO) (Aldrich, USA) then dissolved in saline Microinjection Drug microinjections into CnF were performed by a single barreled micropipette with internal diameter ranging m. The animals were placed in a stereotaxic apparatus (Stoelting, USA). The scalp was longitudinally incised and skull was leveled between lambda and bregma and a small hole drilled in the skull. The stereotaxic coordinates of CnF were mm caudal to bregma, mm lateral to the midline suture and mm ventral from the bregma according to the atlas of Paxinos and Watson [13]. The micropipette connected through PE-10 tube to an injection syringe and was carefully introduced into the CnF and injection was done. Volume injection in all of groups was 100 nl Experimental groups In this experiment the following groups were used: - The control group: injection of 100 nl of saline into CnF. - AP5 group: injection of 100 nl of AP5 (5 mm) into CnF. - CNQX group: injection of 100 nl of CNQX (1 mm) into CnF. - CoCl 2 group: injection of 100 nl of cobalt chloride (CoCl 2, 1 mm) into CnF Histological procedure At the end of each experiment, the rats were anesthetized with a high dose of the urethane, chest was surgically opened and the brain perfused transcardially with 100 ml of 0.9% saline, followed by 100 ml of 10% formalin. The brain was removed and stored in 10% formalin for at least 24 h at 4 C. Frozen serial transverse sections (50 m) of brain stem were cut using a cryostat at 20 C. Brain sections were stained with cresyl violet 1% and the injection sites were determined according to the rat brain atlas of Paxinos and Watson under the light microscope [13,14] Data analysis The data of the blood pressure and heart rate were expressed as means ± SEM. The course of changes in the heart rate and arterial pressure were plotted. The maximum change was compared with the pre-injection (paired t-test) and control (un paired t-test) values. The criterion for a statistical significance was a P < Results 3.1. Cardiovascular responses to microinjections of saline into the CnF on blood pressure and heart rate Injection of the saline (100 nl, n = 12) into the CnF had no significant effect on mean arterial pressure (MAP; before: 92.7 ± 2.3 vs. after: ± 2.23 mm Hg) or heart rate (HR; before: ± 8.8 vs. after: ± 7.9 beats/min) Cardiovascular responses to microinjections of CoCl 2 into the CnF on blood pressure and heart rate In this group the CnF was blocked by microinjection 100 nl of CoCl 2. A sample of the arterial pressure and heart rate tracings before and after the injection are shown in Fig. 1. Microinjection of CoCl 2 into the CnF decreased MAP (92.3 ± 2.45 mm Hg vs ± 1.7 mm Hg) and increased HR (343.8 ± 4.6 beats/min vs ± 8.32 beats/min).

M.N. Shafei et al. / Pathophysiology 19 (2012) 151 155 153 Compared to the control group and pre-injection, microinjection of CoCl 2 caused no significant effect on MAP and HR (t-test, P > 0.

3 M.N. Shafei et al. / Pathophysiology 19 (2012) Compared to the control group and pre-injection, microinjection of CoCl 2 caused no significant effect on MAP and HR (t-test, P > 0.05, n = 10, Fig. 2). Time courses of the changes in MAP and HR are shown in Fig Effects of blockade of glutamate receptor subtypes in the CnF on the cardiovascular responses Fig. 1. Tracings of blood pressure and heart rate, showing the responses to microinjection of CoCl 2 into the CnF of urethane anesthetized male rats. The vertical lines show the time of injection. In this experiment we used AP5 or CNQX as antagonists for NMDA and non-nmda receptors, respectively. In AP5 group, baseline MAP and HR were 92.3 ± 03 mm Hg and ± 6.98 beats/min (n = 12), respectively. Microinjection AP5 in the CnF changed MAP by ± 3.17 mm Hg and HR by ± 4.13 beats/min. However, these responses were not significant compared to the control group and preinjection (t-test, P > 0.05, Fig. 4). The time course of changes in both MAP and HR after the injection of AP5 is shown in Fig. 5. In CNQX group, baseline MAP and HR were ±.07 mm Hg and ± 8.82 beats/min (n = 10), respectively. Microinjection CNQX in the CnF changed MAP by ± 1.5 mm Hg and HR by ± 7.6 beats/min. However, these responses were not statistically significant compared to control group and pre-injection levels (t-test, Fig. 2. The responses to microinjection of CoCl 2 into the CnF (n = 10) in urethane anesthetized male rats compared to the control group (n = 12). Microinjection CoCl 2 has no significant effect on MAP (a) and HR (b). Fig. 3. Time courses of the changes to microinjection of CoCl 2 into the CnF (n = 10) in urethane anesthetized male rats. Compared to the control group (n = 12). Microinjection of CoCl 2 produced no significant effect on MAP (a) and HR (b).

154 M.N. Shafei et al. / Pathophysiology 19 (2012) 151 155 Fig. 4.

4 154 M.N. Shafei et al. / Pathophysiology 19 (2012) Fig. 4. The responses to microinjection of AP5 (n = 12) and CNQX (n = 10) into the CnF in urethane anesthetized male rats compared to the control group (n = 12). Microinjection AP5 and CNQX had no significant effect on MAP (a) and HR (b). P > 0.05, Fig. 4). The time course of changes in both MAP and HR after the injection of CNQX is shown in Fig Discussion The results of this study showed that blockade of the CnF by CoCl 2 or its inhibition by antagonists of glutamate receptors had no significant effect on baseline blood pressure and HR. This result suggests that CnF is not involved in tonic control of cardiovascular system. Maintaining cardiovascular tone has a complicated mechanism and is not completely understood. The role of several supraspinal regions such as RVLM in tonic control of cardiovascular system, have been shown [3,4]. However, the role of other areas such as CnF is uncertain. In this study effect of CnF on basal cardiovascular examined. In first experiment, maintenance effect of CnF on MAP and HR by its acute ablation was evaluated. In this procedure a specific region was blocked by microinjection of compound for a given period of time [15]. CoCl 2 is one of common compounds that non-selectively blocks pre-synaptic Ca 2+ influx and inhibits the neurotransmitter release, without affecting the passage of fibers of [15,16]. Blocked of the CnF by CoCl 2 did not effect on basal MAP and HR. This result suggested that in resting time synapses in the CnF maybe are not involved in maintenance of cardiovascular tone. In second experiment, we investigated the role of glutamate receptor subtypes of the CnF in tonic control of cardiovascular. AP5, an NMDA receptor antagonist, and CNQX, an antagonist of non-nmda receptors, were used. Results demonstrated that blockade of glutamate NMDA or non-nmda receptors caused no significant changes in the baseline of MAP or HR. Similar findings have also been shown in the other nuclei such as bed nucleus of the stria terminalis [14] and diagonal band of Broca [17]. These results suggest that under the resting conditions, the glutamatergic neurons of the CnF might be quiescent or have too little activity to be detected by the microinjection of antagonists. However, decrease of the arterial pressure or other stresses such as pain or fear might activate them. There are many findings that several nuclei are involved in cardiovascular regulation and are subject to the tonic inhibition [4,18]. One of the important inhibitory areas is CVLM that contain GABA A receptor [19,20] and its input to the CnF has been shown [8]. Therefore, it is conceivable that effect of CnF on tonic cardiovascular strongly inhibited by GABAergic neurons in CVLM. Previous studies have reported that the CnF is involved in the autonomic and behavioral components of the defense reaction [1,21,22]. This reaction is accompanied by activation sympathetic system, elicits blood pressure and heart rate [23,24]. The CnF receives projections from superior colliculus, dpag, dorsomedial hypothalamus which play important role in mediating defensive reaction [8,21,25]. Electrical and chemical stimulation of the CnF produces physiological and behavioral responses such as excitation of Fig. 5. Time courses of the changes to microinjection of AP5 (n = 12), and CNQX (n = 10) into the CnF in rats co urethane anesthetized male compared to the control group (n = 12). Microinjection of AP5 and CNQX produced no significant effect on MAP (a) and HR (b).

5 M.N. Shafei et al. / Pathophysiology 19 (2012) the lumbar sympathetic vasomotor neurons, increased HR and blood pressure, freezing, darting and fast running that are associated with behavioral reactions to fear and stressful stimuli [1,9,21]. Korte et al. have reported a circuit in the brain that is activated by painful or threatening stimuli and is accompanied by bradycardiac and pressor responses [9]. The CnF is placed in the center of this circuit and mediates specific autonomic response to stress [9,22]. Based on previous studies and our current results, we suggest that the CnF neurons might be not involved in basal cardiovascular regulation. However, threatening or other stressor conditions such as pain or fear might activate them. The CnF also involved in pain modulation and its relation with descending pain modulatory system has been shown [11]. It is well known that RVLM, PAG, raphe and paraberachial nuclei are important sites for integration of pain and cardiovascular response [26]. The fact the CnF has projection to these areas; raise the possibility that the CnF may be implicated in the cardiovascular responses to painful stimuli. In summary, results of our study demonstrated that the CnF is not involved in tonic regulation of cardiovascular system. But it may be involved in organization of cardiovascular responses to stressor stimuli such as pain or fear. Acknowledgement This study was financially supported by Research Department of Isfahan University of Medical Sciences. References [1] A.J. Verberne, W. Lam, N.C. Owens, D. Sartor, Supramedullary modulation of sympathetic vasomotor function, Clin. Exp. Pharmacol. Physiol. 24 (1997) [2] J.P. Chalmers, V. Kapoor, I.J. Llewellyn-Smith, J.B. Minson, P.M. Pilowsky, Central control of blood pressure, Eur. Heart J. 13 (1992) 2 9. [3] R.A.L. Dampney, Functional organization of central pathways regulating the cardiovascular system, Physiol. Rev. 74 (1994) [4] R.A.L. Dampney, J. Horiuchi, T. Tagawa, M.A.P. Fontes, P.D. Potts, J.W. Polson, Medullary and supramedullary mechanisms regulating sympathetic vasomotor tone, Acta Physiol. Scand. 177 (2003) [5] R.R. Campos, B.A. Carillo, E.B. Oliveira-Sales, A.M. Silva, N.F. Silva, H.A. Futuro Neto, C.T. Bergamaschi, Role of the caudal pressor area in the regulation of sympathetic vasomotor tone, Braz. J. Med. Biol. Res. 41 (2008) (Review). [6] A.J. Verberne, Cuneiform nucleus stimulation produces activation of medullary sympathoexcitatory neurons in rats, Am. J. Physiol. 268 (1995) R752 R758. [7] W. Lam, A.J. Verberne, Cuneiform nucleus stimulation-induced sympathoexcitation: role of adrenoceptors, excitatory amino acid and serotonin receptors in rat spinal cord, Brain Res. 757 (1997) [8] W. Lam, A.L. Gundlach, A.J. Verberne, Increased nerve growth factor inducible-a gene and c-fos messenger RNA levels in the rat midbrain and hindbrain associated with the cardiovascular response to electrical stimulation of the mesencephalic cuneiform nucleus, Neuroscience 71 (1996) [9] S.M. Korte, D. Jaarsma, P.G. Luiten, B. Bohus, Mesencephalic cuneiform nucleus and its ascending and descending projections serve stress-related cardiovascular responses in the rat, J. Auton. Nerv. Syst. 41 (1992) [10] S.M. Barman, G.L. Gebber, H.S. Orer, Medullary lateral tegmental field: an important source of basal sympathetic nerve discharge in the cat, Am. J. Physiol. Regul. Integr. Comp. Physiol. 278 (2000) R995 R1004. [11] A. Haghparast, I.P. Gheitasi, R. Lashgari, Involvement of glutamatergic receptors in the nucleus cuneiformis in modulating morphine-induced antinociception in rats, Eur. J. Pain 11 (2007) [12] M.N. Shafei, A. Nasimi, H. Alaei, A.A. Pourshanazari, The role of non- NMDA receptor of glutamate in cuneiform nucleus on cardiovascular response in anaesthetized rats, Pharmacology Online 1 (2009) [13] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 5th ed., Elsevier Academic Press, San Diego, 2005, pp [14] M. Hatam, A. Nasimi, Glutamatergic systems in the bed nucleus of the stria terminalis, effects on cardiovascular system, Exp. Brain Res. 178 (2007) [15] R. Kretz, Local cobalt injection: a method to discriminate presynaptic axonal from postsynaptic neuronal activity, J. Neurosci. Methods 11 (1984) [16] S.G. Lomber, The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function, J. Neurosci. Methods 86 (1999) (Review). [17] A. Nasimi, M. Hatam, GABA and glutamate receptors in the horizontal limb of diagonal band of Broca (hdb): effects on cardiovascular regulation, Exp. Brain Res. 167 (2005) [18] D.A. Mandel, A.M. Schreihofer, Glutamatergic inputs to the CVLM independent of the NTS promote tonic inhibition of sympathetic vasomotor tone in rats, Am. J. Physiol. Heart Circ. Physiol. 295 (2008) H1772 H1779. [19] S.L. Cravo, S.F. Morrison, The caudal ventrolateral medulla is a source of tonic sympathoinhibition, Brain Res. 621 (1993) [20] C.M. Heesch, J.D. Laiprasert, L. Kvochina, RVLM glycine receptors mediate GABA A and GABA B independent sympathoinhibition from CVLM in rats, Brain Res (2006) [21] I.J. Mitchell, P. Dean, P. Redgrave, The projection from superior colliculus to cuneiform area in the rat. II. Defence-like responses to stimulation with glutamate in cuneiform nucleus and surrounding structures, Exp. Brain Res. 72 (1988) [22] B. Bohus, J.M. Koolhaas, S.M. Korte, B. Roozendaal, A. Wiersma, Forebrain pathways and their behavioural interactions with neuroendocrine and cardiovascular function in the rat, Clin. Exp. Pharmacol. Physiol. 23 (2) (1996) [23] E. Farkas, A.S. Jansen, A.D. Loewy, Periaqueductal gray matter input to cardiac-related sympathetic premotor neurons, Brain Res. 792 (1998) [24] J. Horiuchi, L.M. McDowall, R.A. Dampney, Role of 5-HT(1A) receptors in the lower brainstem on the cardiovascular response to dorsomedial hypothalamus activation, Auton. Neurosci. 142 (2008) [25] W. Lam, A.L. Gundlach, A.J.M. Verberne, Neuronal activation in the forebrain following electrical stimulation of the cuneiform nucleus in the rat: hypothalamic expression of c-fos and NGFI-A messenger RNA, Neuroscience 78 (1997) [26] G.A. Karlsson, C.V. Preuss, K.A. Chaitoff, T.J. Maher, A. Ally, Medullary monoamines and NMDA-receptor regulation of cardiovascular responses during peripheral nociceptive stimuli, Neurosci. Res. 55 (2006)

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