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Cardiovascular and single-unit responses to microinjection of angiotensin II into the bed nucleus of the stria terminalis in rat
Neuroscience 300 (2015) 418–424
CARDIOVASCULAR AND SINGLE-UNIT RESPONSES TO MICROINJECTION OF ANGIOTENSIN II INTO THE BED NUCLEUS OF THE STRIA TERMINALIS IN RAT M. KAFAMI AND A. NASIMI *
et al., 1985), solitary tract nucleus (Holstege et al., 1985) and amygdala (Krettek and Price, 1978; Weller and Smith, 1982), suggesting its role in cardiovascular control. It has been shown that chemical stimulation of the BST with glutamate decreased the mean arterial pressure (MAP) and heart rate (HR) in rats (Ciriello and Janssen, 1993; Hatam and Nasimi, 2007). In addition, the GABAergic system of the BST decreases arterial pressure by inhibiting vasopressin release and HR by sympathetic inhibition (Hatam et al., 2009). In addition, it was found that microinjection of acetylcholine into the BST increased MAP and had no effect on HR (Nasimi and Hatam, 2011). Furthermore, blood pressure increased after microinjection of noradrenaline (Crestani et al., 2007) or carbachol (Alves et al., 2007) into the BST. Angiotensin II (Ang II) is a peptide that plays various functions in the body. The key role of this peptide is regulation of blood volume and pressure. Ang II acts through various receptors, including AT1, AT2 and AT4. It has been established that there is a local renin– angiotensin system in the brain. Angiotensinogen, the precursor molecule for angiotensins I, II and III, and the enzyme renin, angiotensin-converting enzyme, and aminopeptidases A and N, are all synthesized within the brain. Angiotensin AT1, AT2 and AT4 receptors are also plentiful in the brain (see McKinley et al., 2003, and von Bohlen und Halbach and Albrecht, 2006 for review). These findings support the hypothesis that angiotensin may act as a neurotransmitter within the brain. Enzymatic pathway for the formation of the local Ang of the brain is relatively clear (McKinley et al., 2003), however the exact neuronal circuits are still unknown. AT1 receptors were found in several regions known to regulate the cardiovascular system. The highest density of AT1 has been found in the nucleus tractus soliterius (NTS), PVN, rostral ventrolateral medulla (RVLM), caudal ventrolateral medulla (CVLM), amygdala and BST (Allen et al., 2000; McKinley et al., 2003). In addition it has been shown that angiotensinogen, the protein from which Ang II is generated, is present in the BST of the rat fetus (Mungall et al., 1995). Furthermore, neuronal cell bodies exhibiting Ang-like immunoreactivity have been observed in the BST (Chappell et al., 1987, 1989). Injection of Ang II into the cerebral ventricles (Onitsuka et al., 2012; Clayton et al., 2013), amygdala (Brown and Gray, 1988), arcuate nucleus (Arakawa et al., 2011), PVN (Bains et al., 1992), and RVLM (Andreatta et al., 1988) resulted in pressor responses,
Department of Physiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Abstract—The bed nucleus of the stria terminalis (BST) is part of the limbic system located in the rostral forebrain. BST is involved in behavioral, neuroendocrine and autonomic functions, including cardiovascular regulation. The angiotensin II (Ang II) receptor, AT1, was found in the BST, however its effects on the cardiovascular system and on single-unit responses have not been studied yet. In the present study, Ang II was microinjected into the BST of anesthetized rats and cardiovascular and single-unit responses were recorded simultaneously. Furthermore the responses were re-tested after the microinjection of a blocker of the AT1 receptor, losartan, into the BST. We found that microinjection of Ang II into the BST produced a pressor response of 11 ± 1 mmHg for a duration of 2–8 min. Ang II had no consistent effect on heart rate. It also produced two types of single-unit responses in the BST, short excitatory and long inhibitory. Blockade of AT1 receptors abolished both the cardiovascular and single-unit responses, indicating that the responses were mediated through AT1 receptors. These findings imply that Ang II may be utilized as a neurotransmitter and may play a role in returning blood pressure toward normal during hypotension. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: angiotensin II, BST, bed nucleus of the stria terminalis, single-unit responses, blood pressure.
INTRODUCTION The bed nucleus of the stria terminalis (BST) is part of the limbic system located in the rostral forebrain involved in behavioral, neuroendocrine and autonomic functions (see Crestani et al., 2013 for review). BST is connected to some major cardiovascular centers, including the paraventricular nucleus (PVN) of the hypothalamus (Swanson et al., 1983), ventrolateral medulla (Holstege *Corresponding author. Tel: +98-031-3792484; fax: +98-03136688597. E-mail address: [email protected] (A. Nasimi). Abbreviations: Ang II, angiotensin II; BST, bed nucleus of the stria terminalis; BSTMA, BST, medial division, anterior part; CVLM, caudal ventrolateral medulla; HR, heart rate; MAP, mean arterial pressure; NTS, nucleus tractus soliterius; PVN, paraventricular nucleus; RVLM, rostral ventrolateral medulla. http://dx.doi.org/10.1016/j.neuroscience.2015.05.050 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 418
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while injection of Ang II into CVLM produced a depressor response (Sasaki and Dampney, 1990). Injection of Ang II into NTS produced both pressor and depressor responses (Rettig et al., 1986). In spite of the evidence showing the presence of AT1 receptor in the BST, there is no study investigating the role of Ang II of the BST in cardiovascular regulation. This study was conducted to find the cardiovascular and single-unit responses to microinjection of Ang II into the BST. We also investigated the Ang II receptor involved in these responses.
EXPERIMENTAL PROCEDURES Animals and surgery Experiments were performed on male Wistar rats (200– 300 g). Experiments were approved by the Ethics Committee of Animal Use of the Isfahan University of Medical Science. Rats were anesthetized with urethane (Sigma, 1.4 g/kg, ip) and supplementary doses (0.7 g/kg) were given if necessary. The animal’s temperature was maintained at 37°C with a thermostatically controlled heating pad. The trachea was intubated to ease ventilation. A polyethylene catheter (PE-50) was inserted into the left femoral artery for blood pressure recording. A hole was drilled above BST at coordinates of 0.12 to 0.36 mm caudal, 1.1–1.7 mm lateral and 6.4–7.2 mm ventral to bregma according to the atlas of Paxinos and Watson (2005).
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pressure was seen, we waited for 30 min to make sure that the effect of injected Ang II disappeared, then losartan was microinjected by the second barrel and 2– 4 min later, Ang II was microinjected again. For the control group the same volume of the vehicle (normal saline) was microinjected in the BST. If the animal was healthy, another set of injections was done on the contralateral side. Data analysis Blood pressure and HR values were expressed as mean ± SE. The course of changes of HR and arterial pressure was plotted and the maximum changes were compared with those of the pre-injection (paired t-test) and the control (independent t-test) values. A P < 0.05 was used to indicate statistical significance. After data recording, single-unit spikes were isolated from the background, and a peristimulus time histogram (PSTH) was generated from the spike times. Then the cardiovascular response pattern and the cell-firing patterns of each injection were aligned and compared. Histology At the end of each experiment the animal was sacrificed by a high dose of the anesthetic, and then was 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. Frozen serial transverse sections (60 lm) of the forebrain were cut and stained with Cresyl Violet 1%. The injection sites
Experimental protocol A three-barreled micropipette was used to inject Ang II by one of them, losartan (an AT1 antagonist) by the next and to record extracellular action potentials by the other. Ang II (100 lM, 100–150 nl) (Albrecht et al., 2000) or losartan (100 lM, 200 nl) (Albrecht et al., 2000) was microinjected into the BST using a micropipette with an internal diameter of 35–45 lm using a pressurized air pulse applicator. The volume of injection was measured by direct observation of the fluid meniscus in the micropipette by using an ocular micrometer. Arterial pressure and HR were recorded continuously, using a pressure transducer connected to a polygraph (HSE Germany) and a computer program written in this laboratory by A. Nasimi. Extracellular action potentials were recorded simultaneously using a glass microelectrode pulled to a fine-tip diameter (1–3 lm) and filled with NaCl solution (2 M). Extracellular action potentials were amplified (10,000) and filtered (0.3–3 kHz) by a preamplifier (WPI, DAM 80) and displayed continuously on an oscilloscope. Then single-unit firings were digitized, saved in multiunit mode and isolated by a program written in this lab by A. Nasimi. The program does multiple unit recordings then segregates each single unit exactly similar to the ordinary ‘‘window discriminators’’, with more precision. When blood pressure and firing were stable, both blood pressure and spontaneous activity of the neurons were recorded simultaneously for 5–8 min. Then, Ang II was microinjected into the BST. If a change in blood
Fig. 1. A sample of arterial pressure (a), heart rate (b) and single-unit responses(c) to vehicle (saline) injection into the BST. The arrow shows the injection time of saline.
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were determined according to a rat brain atlas (Paxinos and Watson, 2005) under the light microscope.
RESULTS Cardiovascular responses to vehicle microinjected into the BST Microinjection of vehicle (saline, 100–150 nl) did not affect arterial pressure (DMAP = 0.3 ± 0.2 mmHg) or HR (DHR = 0.5 ± 0.2 beats/min) or firing rate of the neurons (n = 6 rats, Fig. 1).
medial division, anterior part (BSTMA) with a maximum change of 37 mmHg, which did not completely return to the baseline even after 20 min. These two responses were not included in the calculation of the mean. As a group, Ang II injection caused a significant increase (paired t-test, P < 0.01) in MAP of 11 ± 1 mmHg. The peak was also significantly (t-test, P < 0.01) different from that of the control (Fig. 3). In response to Ang II injection, HR did not change more than five beats/min in 57% (Figs. 2a, 3b) and showed a small decrease ( 13 ± 1 beats/min) in 43% of the cases (Fig. 4a).
Cardiovascular responses to microinjection of Ang II into the BST
Single-unit responses to microinjection of Ang II into the BST
In the Ang II group, the baseline values of MAP and HR were 74 ± 5 mmHg and 430 ± 8 beats/min, respectively. Microinjection of Ang II (100 lM, 100– 150 nl) into the BST produced a pressor response after 20 s that returned to the baseline after 8 min. MAP changed from 6 to 18 mmHg, with a mean of 11 ± 1 mmHg (n = 13 rats, 40 injections). A sample response was shown in Fig. 2, with a maximum pressor response of 8 mmHg, returning to the baseline in 8 min. A stronger pressor response is shown in Fig. 4. We saw two exceptionally strong responses in the BST,
Figs. 2 and 4 show the simultaneous cardiovascular and single-unit responses to microinjection of Ang II into the BST. Ang II caused no response in 29% of the neurons and produced two types of single-unit responses, short excitatory and long inhibitory, in 71% of the cases. These responses and their subgroups are summarized in Table 1. Excitatory response was seen in 57.5% of the cases, from which 44.5% had a short excitatory
Fig. 2. A sample of simultaneous recording of cardiovascular and single-unit responses to Ang II injection into BST. Ang II caused short excitatory (d) and long inhibitory single-unit responses (e). Arrow shows the injection time of Ang II into the BST. a: HR change, b: MAP change, c: arterial pressure change, d and e: single-unit responses showed as PSTH.
Fig. 3. Time courses of MAP (a) and HR (b) responses to microinjection of Ang II into the BST (n = 13 rats, 40 injections) compared to the control group (n = 6 rats). Ang II significantly increased MAP compared to the pre-injection (paired t-test, P < 0.01) and to the control value (t-test, P < 0.01).
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Fig. 4. A sample of simultaneous recording of cardiovascular and single-unit responses to Ang II and losartan injection into the BST. First, Ang II, then after 30 min, 200–250 nl of losartan, an AT1 receptor antagonist, and then Ang II (100–150 nl) were microinjected into the same site of the BST. Losartan did not affect the cardiovascular or single-unit responses. As seen, losartan abolished the responses to Ang II. a: HR change, b: MAP change, c: arterial pressure change, d: a single-unit response showed as PSTH.
(onset, up to 30 s, Figs. 2d, 4d) and 13% had an excitatory response longer than 100 s. The long inhibitory response was seen in 14.1% of the cases; an example is shown in Fig. 2e. As shown in Fig. 2, in response to some Ang II injections, both types of excitatory and inhibitory singleunit responses were observed. Cardiovascular and single-unit responses to losartan microinjected into the BST To determine whether the Ang II effect is mediated by activation of AT1 receptor, first Ang II was microinjected into the BST, then after 30 min 200 nl of losartan (100 lM), an AT1 receptor antagonist, then Ang II were microinjected into the same site of the BST. An example Table 1. Type and number of single-unit responses Response
Response subtype
Number of neurons
Example
No
No
44 (28.4%)
Excitation
Short Long (P100 s)
69 (44.5%) 20 (13%)
Figs. 2d, 4d
Inhibition
Long Total
22 (14.1%) 155 (100%)
Fig. 2e
is shown in Fig. 4. Injection of losartan (n = 9 rats, 25 injections) had no significant effect on MAP, HR (Fig. 5a, b; paired t-test, P > 0.05) or firing rate (Figs. 4d, 5c, d) of the neurons (n = 57 neurons, paired t-test, P > 0.05). Injection of Ang II after losartan also had no significant effect on MAP, HR or firing rate of the neurons (Figs. 4 and 5). In other words, losartan abolished the responses to Ang II. Histology The distribution of the injection sites is shown in Fig. 6. All injections outside the BST and some of the injections inside the BST produced no responses. Also, the distribution of various response types in different parts of the BST is shown in Table 2. Best cardiovascular responses to Ang II injection were observed in BSTMA, BST, lateral division, dorsal part (BSTLD) and BST, lateral division, posterior part (BSTLP).
DISCUSSION In this study the cardiovascular effects of microinjection of Ang II into BST, the involved receptor and simultaneous single-unit responses were explored. Results showed that microinjection of Ang II into BST produced a pressor response of 11 ± 1 mmHg (Fig. 2). The
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Fig. 6. Schematic coronal section of rat brain adopted from an atlas (Paxinos and Watson, 2005). The injection sites of Ang II were shown as filled squares. BSTLD: BST, lateral division, dorsal part; BSTLI: BST, lateral division, intermediate part; BSTLJ: BST, lateral division, juxtacapsular part; BSTLP: BST, lateral division, posterior part; BSTLV: BST, lateral division, ventral part; BSTMA: BST, medial division, anterior part; BSTMV: BST, medial division, ventral part.
Fig. 5. Summary data for responses to Ang II before and after losartan injection into the BST. Injection of Ang II after losartan had no significant effect on MAP, HR or firing rate of the neurons i.e. losartan abolished the responses to Ang II. a: MAP change, b: HR change, c and d: single-unit responses shown as histograms.
strongest responses to Ang II were seen in the BSTMA compared to the other subdivisions of the BST. Although no similar study for comparison exists in the literature, this finding is comparable to the results of injection of Ang II into cerebral ventricles (Onitsuka
et al., 2012; Clayton et al. 2013), amygdala (Brown and Gray, 1988), arcuate nucleus (Arakawa et al., 2011), PVN (Bains et al., 1992), and RVLM (Andreatta et al., 1988). But injection of Ang II into the CVLM produced a depressor response (Sasaki and Dampney, 1990), and microinjections of Ang II into the NTS resulted in a monophasic depressor response, while higher doses were characterized by a biphasic response (Rettig et al., 1986). Ang II caused no response in 28.4% of the neurons and the rest produced two types of single-unit responses, short excitatory and long-inhibitory (Table 1, Figs. 2 and 4). These responses are comparable to the responses observed to iontophoretic application of Ang II in the BST (Lienard et al., 1996). At least some of these responses might be secondary to Ang II-evoked arterial
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Table 2. Distribution of various response types in different parts of the BST Location
BSTMA BSTLI BSTLD BSTMV BSTLJ BSTLP Total number
Cardiovascular responses
Single-unit responses
>10 mmHg
<10 mmHg
Excitatory
Inhibitory
6 (20.6%)
6 2 3 4
49 (44.14%) 10 (9.009) 12 (10.81) 5 (4.5) 2 (1.8) 11 (9.9) 89 (80.1)
22 (19.81) 0 0 0 0 0 22 (19.81)
2 (6.89) 1 (3.44) 2 (6.89) 11 (37.93)
(20.6%) (6.89) (10.34) (13.79)
3 (10.34) 18 (62.06)
BSTLD: BST, lateral division, dorsal part; BSTLI: BST, lateral division, intermediate part; BSTLJ: BST, lateral division, juxta-capsular part; BSTLP: BST, lateral division, posterior part; BSTLV: BST, lateral division, ventral part; BSTMA: BST, medial division, anterior part; BSTMV: BST, medial division, ventral part.
pressure change, as it was shown that changes in arterial pressure alter the activity of single units in the BST (Wilkinson and Pittman, 1995). However we found a simultaneous change in single-unit activity and in blood pressure, supporting the primary effect of Ang II on neurons. It is possible that Ang II in the BST affects cardiovascular function through both sympathoexcitation and vasopressin release. Excitatory single-unit responses might represent excitatory effect on the sympathetic system. It is likely that sympathoexcitation in responses to Ang II is mediated through the cholinergic system of the BST, as it was shown that microinjection of Ach into the BST produced a pressor response with no concomitant effect on HR (Nasimi and Hatam, 2011). Another possibility is that the sympathoexcitatory effect of Ang II is mediated through the adrenergic system of the BST, since a significant subpopulation of AT1 receptors was identified on noradrenergic terminals of the BST (Grove et al., 1998). It was also shown that NEP microinjected in the BST produced long-lasting dose-related pressor and bradycardiac responses (Crestani et al., 2007). In most autonomic controls regions, Ang II and Lglutamate affect arterial pressure in similar directions. For example in the CVLM, both Ang II (Sasaki and Dampney, 1990) and glutamate (Willette et al., 1983; Smith and Barron, 1990) produced a depressor response. However, in the BST, Ang II increases arterial pressure while L-glutamate decreases it (Hatam and Nasimi, 2007). Based on the present knowledge, it could be said that the possible interaction between angiotensinergic and glutamatergic neurons in the BST must be different. Long inhibitory single-unit response might represent inhibition of GABAergic neurons of the BST, resulting in disinhibition of vasopressin release by VPN. In support of this finding it has been demonstrated that the BST sends GABAergic neurons to the PVN (Boudaba et al., 1996) that in turn, decrease the release of vasopressin (Choi et al., 2007). Also it was shown that blocking the V1 receptors of vasopressin abolished the pressor responses of bicuculline (Hatam et al., 2009), indicating that bicuculline disinhibited vasopressin release. We found that blockade of AT1 receptor of Ang II in the BST abolished the pressor response to microinjection of Ang II (Fig. 4), indicating that pressor response is mediated through AT1 receptors.
Microinjection of AT1 antagonist, losartan, into the BST did not affect the blood pressure or HR (Fig. 4), indicating that in our experimental condition there was little or no intrinsic release of Ang II, so one can speculate that Ang II might release during hypotension to return blood pressure to normal as circulating Ang II does. It has been reported that circulating Ang II activates central pathways involved in blood pressure regulations such as RVLM, PVN, OVLT and BST (Potts et al., 1999; McMullan et al., 2007). It was also shown that circulating Ang II activates neurons in the circumventricular organs of the lamina terminalis that project to the BST (Sunn et al., 2003). The effect of circulating Ang II on the central pathways may synchronize the actions of the peripheral and central renin–angiotensin systems. Simultaneous actions of circulating Ang II as a hormone, and CNS Ang II as a neurotransmitter, may make the renin–angiotensin system an effective feedback mechanism for blood pressure regulation.
CONCLUSION Microinjection of Ang II into the BST produced a pressor response through AT1 receptors. These findings imply that Ang II may be utilized as a neurotransmitter and may play a role in returning blood pressure toward normal during hypotension. Acknowledgment—This study was supported by a grant from Vice-Chancellery of Research of the Isfahan University of Medical Sciences, Iran.
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(Accepted 20 May 2015) (Available online 27 May 2015)
CARDIOVASCULAR AND SINGLE-UNIT RESPONSES TO MICROINJECTION OF ANGIOTENSIN II INTO THE BED NUCLEUS OF THE STRIA TERMINALIS IN RAT M. KAFAMI AND A. NASIMI *
et al., 1985), solitary tract nucleus (Holstege et al., 1985) and amygdala (Krettek and Price, 1978; Weller and Smith, 1982), suggesting its role in cardiovascular control. It has been shown that chemical stimulation of the BST with glutamate decreased the mean arterial pressure (MAP) and heart rate (HR) in rats (Ciriello and Janssen, 1993; Hatam and Nasimi, 2007). In addition, the GABAergic system of the BST decreases arterial pressure by inhibiting vasopressin release and HR by sympathetic inhibition (Hatam et al., 2009). In addition, it was found that microinjection of acetylcholine into the BST increased MAP and had no effect on HR (Nasimi and Hatam, 2011). Furthermore, blood pressure increased after microinjection of noradrenaline (Crestani et al., 2007) or carbachol (Alves et al., 2007) into the BST. Angiotensin II (Ang II) is a peptide that plays various functions in the body. The key role of this peptide is regulation of blood volume and pressure. Ang II acts through various receptors, including AT1, AT2 and AT4. It has been established that there is a local renin– angiotensin system in the brain. Angiotensinogen, the precursor molecule for angiotensins I, II and III, and the enzyme renin, angiotensin-converting enzyme, and aminopeptidases A and N, are all synthesized within the brain. Angiotensin AT1, AT2 and AT4 receptors are also plentiful in the brain (see McKinley et al., 2003, and von Bohlen und Halbach and Albrecht, 2006 for review). These findings support the hypothesis that angiotensin may act as a neurotransmitter within the brain. Enzymatic pathway for the formation of the local Ang of the brain is relatively clear (McKinley et al., 2003), however the exact neuronal circuits are still unknown. AT1 receptors were found in several regions known to regulate the cardiovascular system. The highest density of AT1 has been found in the nucleus tractus soliterius (NTS), PVN, rostral ventrolateral medulla (RVLM), caudal ventrolateral medulla (CVLM), amygdala and BST (Allen et al., 2000; McKinley et al., 2003). In addition it has been shown that angiotensinogen, the protein from which Ang II is generated, is present in the BST of the rat fetus (Mungall et al., 1995). Furthermore, neuronal cell bodies exhibiting Ang-like immunoreactivity have been observed in the BST (Chappell et al., 1987, 1989). Injection of Ang II into the cerebral ventricles (Onitsuka et al., 2012; Clayton et al., 2013), amygdala (Brown and Gray, 1988), arcuate nucleus (Arakawa et al., 2011), PVN (Bains et al., 1992), and RVLM (Andreatta et al., 1988) resulted in pressor responses,
Department of Physiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Abstract—The bed nucleus of the stria terminalis (BST) is part of the limbic system located in the rostral forebrain. BST is involved in behavioral, neuroendocrine and autonomic functions, including cardiovascular regulation. The angiotensin II (Ang II) receptor, AT1, was found in the BST, however its effects on the cardiovascular system and on single-unit responses have not been studied yet. In the present study, Ang II was microinjected into the BST of anesthetized rats and cardiovascular and single-unit responses were recorded simultaneously. Furthermore the responses were re-tested after the microinjection of a blocker of the AT1 receptor, losartan, into the BST. We found that microinjection of Ang II into the BST produced a pressor response of 11 ± 1 mmHg for a duration of 2–8 min. Ang II had no consistent effect on heart rate. It also produced two types of single-unit responses in the BST, short excitatory and long inhibitory. Blockade of AT1 receptors abolished both the cardiovascular and single-unit responses, indicating that the responses were mediated through AT1 receptors. These findings imply that Ang II may be utilized as a neurotransmitter and may play a role in returning blood pressure toward normal during hypotension. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: angiotensin II, BST, bed nucleus of the stria terminalis, single-unit responses, blood pressure.
INTRODUCTION The bed nucleus of the stria terminalis (BST) is part of the limbic system located in the rostral forebrain involved in behavioral, neuroendocrine and autonomic functions (see Crestani et al., 2013 for review). BST is connected to some major cardiovascular centers, including the paraventricular nucleus (PVN) of the hypothalamus (Swanson et al., 1983), ventrolateral medulla (Holstege *Corresponding author. Tel: +98-031-3792484; fax: +98-03136688597. E-mail address: [email protected] (A. Nasimi). Abbreviations: Ang II, angiotensin II; BST, bed nucleus of the stria terminalis; BSTMA, BST, medial division, anterior part; CVLM, caudal ventrolateral medulla; HR, heart rate; MAP, mean arterial pressure; NTS, nucleus tractus soliterius; PVN, paraventricular nucleus; RVLM, rostral ventrolateral medulla. http://dx.doi.org/10.1016/j.neuroscience.2015.05.050 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 418
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while injection of Ang II into CVLM produced a depressor response (Sasaki and Dampney, 1990). Injection of Ang II into NTS produced both pressor and depressor responses (Rettig et al., 1986). In spite of the evidence showing the presence of AT1 receptor in the BST, there is no study investigating the role of Ang II of the BST in cardiovascular regulation. This study was conducted to find the cardiovascular and single-unit responses to microinjection of Ang II into the BST. We also investigated the Ang II receptor involved in these responses.
EXPERIMENTAL PROCEDURES Animals and surgery Experiments were performed on male Wistar rats (200– 300 g). Experiments were approved by the Ethics Committee of Animal Use of the Isfahan University of Medical Science. Rats were anesthetized with urethane (Sigma, 1.4 g/kg, ip) and supplementary doses (0.7 g/kg) were given if necessary. The animal’s temperature was maintained at 37°C with a thermostatically controlled heating pad. The trachea was intubated to ease ventilation. A polyethylene catheter (PE-50) was inserted into the left femoral artery for blood pressure recording. A hole was drilled above BST at coordinates of 0.12 to 0.36 mm caudal, 1.1–1.7 mm lateral and 6.4–7.2 mm ventral to bregma according to the atlas of Paxinos and Watson (2005).
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pressure was seen, we waited for 30 min to make sure that the effect of injected Ang II disappeared, then losartan was microinjected by the second barrel and 2– 4 min later, Ang II was microinjected again. For the control group the same volume of the vehicle (normal saline) was microinjected in the BST. If the animal was healthy, another set of injections was done on the contralateral side. Data analysis Blood pressure and HR values were expressed as mean ± SE. The course of changes of HR and arterial pressure was plotted and the maximum changes were compared with those of the pre-injection (paired t-test) and the control (independent t-test) values. A P < 0.05 was used to indicate statistical significance. After data recording, single-unit spikes were isolated from the background, and a peristimulus time histogram (PSTH) was generated from the spike times. Then the cardiovascular response pattern and the cell-firing patterns of each injection were aligned and compared. Histology At the end of each experiment the animal was sacrificed by a high dose of the anesthetic, and then was 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. Frozen serial transverse sections (60 lm) of the forebrain were cut and stained with Cresyl Violet 1%. The injection sites
Experimental protocol A three-barreled micropipette was used to inject Ang II by one of them, losartan (an AT1 antagonist) by the next and to record extracellular action potentials by the other. Ang II (100 lM, 100–150 nl) (Albrecht et al., 2000) or losartan (100 lM, 200 nl) (Albrecht et al., 2000) was microinjected into the BST using a micropipette with an internal diameter of 35–45 lm using a pressurized air pulse applicator. The volume of injection was measured by direct observation of the fluid meniscus in the micropipette by using an ocular micrometer. Arterial pressure and HR were recorded continuously, using a pressure transducer connected to a polygraph (HSE Germany) and a computer program written in this laboratory by A. Nasimi. Extracellular action potentials were recorded simultaneously using a glass microelectrode pulled to a fine-tip diameter (1–3 lm) and filled with NaCl solution (2 M). Extracellular action potentials were amplified (10,000) and filtered (0.3–3 kHz) by a preamplifier (WPI, DAM 80) and displayed continuously on an oscilloscope. Then single-unit firings were digitized, saved in multiunit mode and isolated by a program written in this lab by A. Nasimi. The program does multiple unit recordings then segregates each single unit exactly similar to the ordinary ‘‘window discriminators’’, with more precision. When blood pressure and firing were stable, both blood pressure and spontaneous activity of the neurons were recorded simultaneously for 5–8 min. Then, Ang II was microinjected into the BST. If a change in blood
Fig. 1. A sample of arterial pressure (a), heart rate (b) and single-unit responses(c) to vehicle (saline) injection into the BST. The arrow shows the injection time of saline.
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were determined according to a rat brain atlas (Paxinos and Watson, 2005) under the light microscope.
RESULTS Cardiovascular responses to vehicle microinjected into the BST Microinjection of vehicle (saline, 100–150 nl) did not affect arterial pressure (DMAP = 0.3 ± 0.2 mmHg) or HR (DHR = 0.5 ± 0.2 beats/min) or firing rate of the neurons (n = 6 rats, Fig. 1).
medial division, anterior part (BSTMA) with a maximum change of 37 mmHg, which did not completely return to the baseline even after 20 min. These two responses were not included in the calculation of the mean. As a group, Ang II injection caused a significant increase (paired t-test, P < 0.01) in MAP of 11 ± 1 mmHg. The peak was also significantly (t-test, P < 0.01) different from that of the control (Fig. 3). In response to Ang II injection, HR did not change more than five beats/min in 57% (Figs. 2a, 3b) and showed a small decrease ( 13 ± 1 beats/min) in 43% of the cases (Fig. 4a).
Cardiovascular responses to microinjection of Ang II into the BST
Single-unit responses to microinjection of Ang II into the BST
In the Ang II group, the baseline values of MAP and HR were 74 ± 5 mmHg and 430 ± 8 beats/min, respectively. Microinjection of Ang II (100 lM, 100– 150 nl) into the BST produced a pressor response after 20 s that returned to the baseline after 8 min. MAP changed from 6 to 18 mmHg, with a mean of 11 ± 1 mmHg (n = 13 rats, 40 injections). A sample response was shown in Fig. 2, with a maximum pressor response of 8 mmHg, returning to the baseline in 8 min. A stronger pressor response is shown in Fig. 4. We saw two exceptionally strong responses in the BST,
Figs. 2 and 4 show the simultaneous cardiovascular and single-unit responses to microinjection of Ang II into the BST. Ang II caused no response in 29% of the neurons and produced two types of single-unit responses, short excitatory and long inhibitory, in 71% of the cases. These responses and their subgroups are summarized in Table 1. Excitatory response was seen in 57.5% of the cases, from which 44.5% had a short excitatory
Fig. 2. A sample of simultaneous recording of cardiovascular and single-unit responses to Ang II injection into BST. Ang II caused short excitatory (d) and long inhibitory single-unit responses (e). Arrow shows the injection time of Ang II into the BST. a: HR change, b: MAP change, c: arterial pressure change, d and e: single-unit responses showed as PSTH.
Fig. 3. Time courses of MAP (a) and HR (b) responses to microinjection of Ang II into the BST (n = 13 rats, 40 injections) compared to the control group (n = 6 rats). Ang II significantly increased MAP compared to the pre-injection (paired t-test, P < 0.01) and to the control value (t-test, P < 0.01).
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Fig. 4. A sample of simultaneous recording of cardiovascular and single-unit responses to Ang II and losartan injection into the BST. First, Ang II, then after 30 min, 200–250 nl of losartan, an AT1 receptor antagonist, and then Ang II (100–150 nl) were microinjected into the same site of the BST. Losartan did not affect the cardiovascular or single-unit responses. As seen, losartan abolished the responses to Ang II. a: HR change, b: MAP change, c: arterial pressure change, d: a single-unit response showed as PSTH.
(onset, up to 30 s, Figs. 2d, 4d) and 13% had an excitatory response longer than 100 s. The long inhibitory response was seen in 14.1% of the cases; an example is shown in Fig. 2e. As shown in Fig. 2, in response to some Ang II injections, both types of excitatory and inhibitory singleunit responses were observed. Cardiovascular and single-unit responses to losartan microinjected into the BST To determine whether the Ang II effect is mediated by activation of AT1 receptor, first Ang II was microinjected into the BST, then after 30 min 200 nl of losartan (100 lM), an AT1 receptor antagonist, then Ang II were microinjected into the same site of the BST. An example Table 1. Type and number of single-unit responses Response
Response subtype
Number of neurons
Example
No
No
44 (28.4%)
Excitation
Short Long (P100 s)
69 (44.5%) 20 (13%)
Figs. 2d, 4d
Inhibition
Long Total
22 (14.1%) 155 (100%)
Fig. 2e
is shown in Fig. 4. Injection of losartan (n = 9 rats, 25 injections) had no significant effect on MAP, HR (Fig. 5a, b; paired t-test, P > 0.05) or firing rate (Figs. 4d, 5c, d) of the neurons (n = 57 neurons, paired t-test, P > 0.05). Injection of Ang II after losartan also had no significant effect on MAP, HR or firing rate of the neurons (Figs. 4 and 5). In other words, losartan abolished the responses to Ang II. Histology The distribution of the injection sites is shown in Fig. 6. All injections outside the BST and some of the injections inside the BST produced no responses. Also, the distribution of various response types in different parts of the BST is shown in Table 2. Best cardiovascular responses to Ang II injection were observed in BSTMA, BST, lateral division, dorsal part (BSTLD) and BST, lateral division, posterior part (BSTLP).
DISCUSSION In this study the cardiovascular effects of microinjection of Ang II into BST, the involved receptor and simultaneous single-unit responses were explored. Results showed that microinjection of Ang II into BST produced a pressor response of 11 ± 1 mmHg (Fig. 2). The
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Fig. 6. Schematic coronal section of rat brain adopted from an atlas (Paxinos and Watson, 2005). The injection sites of Ang II were shown as filled squares. BSTLD: BST, lateral division, dorsal part; BSTLI: BST, lateral division, intermediate part; BSTLJ: BST, lateral division, juxtacapsular part; BSTLP: BST, lateral division, posterior part; BSTLV: BST, lateral division, ventral part; BSTMA: BST, medial division, anterior part; BSTMV: BST, medial division, ventral part.
Fig. 5. Summary data for responses to Ang II before and after losartan injection into the BST. Injection of Ang II after losartan had no significant effect on MAP, HR or firing rate of the neurons i.e. losartan abolished the responses to Ang II. a: MAP change, b: HR change, c and d: single-unit responses shown as histograms.
strongest responses to Ang II were seen in the BSTMA compared to the other subdivisions of the BST. Although no similar study for comparison exists in the literature, this finding is comparable to the results of injection of Ang II into cerebral ventricles (Onitsuka
et al., 2012; Clayton et al. 2013), amygdala (Brown and Gray, 1988), arcuate nucleus (Arakawa et al., 2011), PVN (Bains et al., 1992), and RVLM (Andreatta et al., 1988). But injection of Ang II into the CVLM produced a depressor response (Sasaki and Dampney, 1990), and microinjections of Ang II into the NTS resulted in a monophasic depressor response, while higher doses were characterized by a biphasic response (Rettig et al., 1986). Ang II caused no response in 28.4% of the neurons and the rest produced two types of single-unit responses, short excitatory and long-inhibitory (Table 1, Figs. 2 and 4). These responses are comparable to the responses observed to iontophoretic application of Ang II in the BST (Lienard et al., 1996). At least some of these responses might be secondary to Ang II-evoked arterial
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Table 2. Distribution of various response types in different parts of the BST Location
BSTMA BSTLI BSTLD BSTMV BSTLJ BSTLP Total number
Cardiovascular responses
Single-unit responses
>10 mmHg
<10 mmHg
Excitatory
Inhibitory
6 (20.6%)
6 2 3 4
49 (44.14%) 10 (9.009) 12 (10.81) 5 (4.5) 2 (1.8) 11 (9.9) 89 (80.1)
22 (19.81) 0 0 0 0 0 22 (19.81)
2 (6.89) 1 (3.44) 2 (6.89) 11 (37.93)
(20.6%) (6.89) (10.34) (13.79)
3 (10.34) 18 (62.06)
BSTLD: BST, lateral division, dorsal part; BSTLI: BST, lateral division, intermediate part; BSTLJ: BST, lateral division, juxta-capsular part; BSTLP: BST, lateral division, posterior part; BSTLV: BST, lateral division, ventral part; BSTMA: BST, medial division, anterior part; BSTMV: BST, medial division, ventral part.
pressure change, as it was shown that changes in arterial pressure alter the activity of single units in the BST (Wilkinson and Pittman, 1995). However we found a simultaneous change in single-unit activity and in blood pressure, supporting the primary effect of Ang II on neurons. It is possible that Ang II in the BST affects cardiovascular function through both sympathoexcitation and vasopressin release. Excitatory single-unit responses might represent excitatory effect on the sympathetic system. It is likely that sympathoexcitation in responses to Ang II is mediated through the cholinergic system of the BST, as it was shown that microinjection of Ach into the BST produced a pressor response with no concomitant effect on HR (Nasimi and Hatam, 2011). Another possibility is that the sympathoexcitatory effect of Ang II is mediated through the adrenergic system of the BST, since a significant subpopulation of AT1 receptors was identified on noradrenergic terminals of the BST (Grove et al., 1998). It was also shown that NEP microinjected in the BST produced long-lasting dose-related pressor and bradycardiac responses (Crestani et al., 2007). In most autonomic controls regions, Ang II and Lglutamate affect arterial pressure in similar directions. For example in the CVLM, both Ang II (Sasaki and Dampney, 1990) and glutamate (Willette et al., 1983; Smith and Barron, 1990) produced a depressor response. However, in the BST, Ang II increases arterial pressure while L-glutamate decreases it (Hatam and Nasimi, 2007). Based on the present knowledge, it could be said that the possible interaction between angiotensinergic and glutamatergic neurons in the BST must be different. Long inhibitory single-unit response might represent inhibition of GABAergic neurons of the BST, resulting in disinhibition of vasopressin release by VPN. In support of this finding it has been demonstrated that the BST sends GABAergic neurons to the PVN (Boudaba et al., 1996) that in turn, decrease the release of vasopressin (Choi et al., 2007). Also it was shown that blocking the V1 receptors of vasopressin abolished the pressor responses of bicuculline (Hatam et al., 2009), indicating that bicuculline disinhibited vasopressin release. We found that blockade of AT1 receptor of Ang II in the BST abolished the pressor response to microinjection of Ang II (Fig. 4), indicating that pressor response is mediated through AT1 receptors.
Microinjection of AT1 antagonist, losartan, into the BST did not affect the blood pressure or HR (Fig. 4), indicating that in our experimental condition there was little or no intrinsic release of Ang II, so one can speculate that Ang II might release during hypotension to return blood pressure to normal as circulating Ang II does. It has been reported that circulating Ang II activates central pathways involved in blood pressure regulations such as RVLM, PVN, OVLT and BST (Potts et al., 1999; McMullan et al., 2007). It was also shown that circulating Ang II activates neurons in the circumventricular organs of the lamina terminalis that project to the BST (Sunn et al., 2003). The effect of circulating Ang II on the central pathways may synchronize the actions of the peripheral and central renin–angiotensin systems. Simultaneous actions of circulating Ang II as a hormone, and CNS Ang II as a neurotransmitter, may make the renin–angiotensin system an effective feedback mechanism for blood pressure regulation.
CONCLUSION Microinjection of Ang II into the BST produced a pressor response through AT1 receptors. These findings imply that Ang II may be utilized as a neurotransmitter and may play a role in returning blood pressure toward normal during hypotension. Acknowledgment—This study was supported by a grant from Vice-Chancellery of Research of the Isfahan University of Medical Sciences, Iran.
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(Accepted 20 May 2015) (Available online 27 May 2015)