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Cardiovascular responses to microinjection of l -glutamate into the NTS in AV3V-lesioned rats
Brain Research 1025 (2004) 106 – 112 www.elsevier.com/locate/brainres
Research report
Cardiovascular responses to microinjection of l-glutamate into the NTS in AV3V-lesioned rats Alexandre Antonio Vieira, Eduardo Colombari, Laurival A. De Luca Jr., De´bora Simo˜es de Almeida Colombari, Jose´ V. Menani* Department of Physiology and Pathology, School of Dentistry, Paulista State University (UNESP), Araraquara, SP 14801-903, Brazil Department of Physiology, UNIFESP-EPM, Sa˜o Paulo, SP, Brazil Accepted 10 August 2004 Available online 11 September 2004
Abstract The excitatory amino acid l-glutamate injected into the nucleus of the solitary tract (NTS) in unanesthetized rats similar to peripheral chemoreceptor activation increases mean arterial pressure (MAP) and reduces heart rate. In this study, we investigated the effects of acute (1 day) and chronic (15 days) electrolytic lesions of the preoptic-periventricular tissue surrounding the anteroventral third ventricle (AV3V region) on the pressor and bradycardic responses induced by injections of l-glutamate into the NTS or peripheral chemoreceptor activation in unanesthetized rats. Male Holtzman rats with sham or electrolytic AV3V lesions and a stainless steel cannula implanted into the NTS were used. Differently from the pressor responses (28F3 mm Hg) produced by injections into the NTS of sham-lesioned rats, l-glutamate (5 nmol/ 100 nl) injected into the NTS reduced MAP ( 26F8 mm Hg) or produced no effect (2F7 mm Hg) in acute and chronic AV3V-lesioned rats, respectively. The bradycardia to l-glutamate into the NTS and the cardiovascular responses to chemoreflex activation with intravenous potassium cyanide or to baroreflex activation with intravenous phenylephrine or sodium nitroprusside were not modified by AV3V lesions. The results show that the integrity of the AV3V region is essential for the pressor responses to l-glutamate into the NTS but not for the pressor responses to chemoreflex activation, suggesting dissociation between the central mechanisms involved in these responses. D 2004 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Baroreceptor; Chemoreceptor; Solitary tract; Hypothalamus; Hypertension
1. Introduction The excitatory amino acid l-glutamate is considered one of the main neurotransmitters released in the nucleus of the solitary tract (NTS) by the cardiovascular afferent projections [26,27,29,30], but the central mechanisms involved in the cardiovascular responses to l-glutamate injected into the NTS are not completely understood. * Corresponding author. Departamento de Fisiologia e Patologia, Faculdade de Odontologia de Araraquara, UNESP, Rua Humaita´, 1680, Araraquara, 14801 903, SP, Brazil. Tel.: +55 16 201 6486; fax: +55 16 201 6488. E-mail address: [email protected] (J.V. Menani). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.08.006
Injections of l-glutamate into the NTS in anesthetized rats similar to baroreflex activation produce hypotension and bradycardia [26,27]. However, injections of l-glutamate into the NTS in awake rats usually produce pressor responses and bradycardia similar to peripheral chemoreceptor activation [7,8,11,16,17,20]. A medullary circuitry essential for cardiovascular regulation that receives influences from peripheral cardiovascular receptors involves the NTS, nucleus ambiguus, and the ventrolateral medulla (caudal and rostral) [6,9,10,16,25]. After the first synapse into the NTS, the signals from peripheral baroreceptors and chemoreceptors can reach different medullary areas that control autonomic projections to the cardiovascular system [6,9,10,16,25]. In addition,
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signals from peripheral receptors can also ascend to the forebrain after a relay in the NTS [5,13,15,22,24]. Although the information that arises from peripheral receptors to the medullary circuitry provides a basic control of cardiovascular functions, descending signals from different encephalic areas, especially from the forebrain, might influence the activity of the medullary circuitry and the autonomic discharges to the cardiovascular system allowing more complex cardiovascular adjustments [13]. Several studies have shown the importance of the preoptic-periventricular tissue surrounding the anteroventral third ventricle (AV3V region) for the cardiovascular regulation and the control of fluid–electrolyte balance [2– 4,12–14,19]. Among other specific nuclei, the AV3V region includes the organum vasculosum of the lamina terminalis, an area lacking blood–brain barrier and rich in angiotensin II (ANG II) receptors [2–4,12–14]. In rats, AV3V lesions impair the development of experimental hypertension, abolish the pressor responses to central ANG II and central cholinergic activation, reduce vasopressin secretion and the pressor responses to bilateral common carotid occlusion, and increase baroreflex sensitivity to ANG II without changing the baroreflex responses to intravenous (i.v.) phenylephrine and sodium nitroprusside [1–4,12–14,18,19,28]. In spite of the importance of the AV3V region for the cardiovascular regulation modulating sympathetic activity and vasopressin secretion [2–4,12–14] and the existence of neural connections between the AV3V region and hindbrain areas related to cardiovascular regulation, especially the NTS [5,13,15,22–24], a possible involvement of the AV3V region on the cardiovascular responses to chemoreflex or direct activation of hindbrain areas has not been investigated. Therefore, in this study, we investigated the effects of electrolytic AV3V lesions on the pressor and bradycardic responses to l-glutamate injected into the NTS and to peripheral chemoreflex activation in awake rats. To compare with results from a previous study [1], baroreflex responses were also tested. As already shown for daily water ingestion and pilocarpine-induced salivation, the effects of acute (1 to 7 days) and chronic (more than 10 days) AV3V lesions can be totally different [2,3,21]. Thus, in this study, we tested lglutamate responses and the cardiovascular reflexes in rats with acute (1 day) and chronic (15 days) AV3V lesions.
2. Material and methods 2.1. Animals Male Holtzman rats weighing 280 to 320 g were used. The animals were housed individually in stainless steel cages in a room with controlled temperature (23F2 8C) and humidity (55F10%). Lights were on from 7:00 a.m. to 7:00 p.m. Standard Purina chow and tap water were available ad libitum. The Ethical Committee for Animal
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Care and Use from Dentistry School of Araraquara, UNESP approved the experimental protocols used in this study. 2.2. Electrolytic AV3V lesions Rats were anesthetized by injection of ketamine (100 mg/ kg of body weight) intraperitoneally (i.p.) and placed in a two-arm stereotaxic frame (model 900, David Kopf Instruments). The skull was positioned to have bregma and lambda at the same level. A tungsten wire electrode (0.4 mm in diameter), bared at the tip (0.5 mm), was inserted into the brain using the following coordinates: 0.0 mm from bregma, in the midline and 7.0 mm below the dura mater. The electrolytic lesion was performed using a cathode current (2 mA for 10 sec). A clip attached to the tail was used as the indifferent electrode. The sham-lesioned rats had the electrode placed along the same coordinates, except that no current was passed. 2.3. Intracerebral cannulas Immediately after the AV3V lesion, using the second arm of the stereotaxic, a stainless steel cannula (140.6 mm o.d.) was implanted into the NTS using the coordinates 14.5 mm caudal to bregma, 0.5 mm lateral to the midline, and 7.5 mm below the dura mater. The cannulas were fixed to the cranium using dental acrylic resin and jeweler screws. The chronic AV3V-lesioned and sham-lesioned rats received a prophylactic dose of penicillin (30,000 IU) given intramuscularly postsurgically. Besides water and food pellets, the chronic AV3V-lesioned rats had access to 10% sucrose during 1 week following the lesions to compensate for the acute adipsia that follows AV3V lesions. 2.4. Arterial pressure and heart rate recordings Mean arterial pressure (MAP) and heart rate (HR) were recorded in unanesthetized rats. Under ketamine (100 mg/kg of body weight, i.p.) anesthesia, a polyethylene tubing (PE10 connected to a PE-50) was inserted into the abdominal aorta through the femoral artery on the day before the experiments. At the same time, a second polyethylene tubing was inserted in the femoral vein for drug administration. Both tubings were tunneled subcutaneously and exposed on the back of the rat to allow access in unrestrained, freely moving rats. To record pulsatile arterial pressure, MAP and HR, the arterial catheter was connected to a Stathan Gould (P23 Db) pressure transducer coupled to a preamplifier (model ETH-200 Bridge Bio Amplifier) that was connected to a Powerlab computer data acquisition system (model Powerlab 16SP, ADInstruments). In the acute group, the arterial and venous cannula implant was performed immediately after the cerebral surgery. In the chronic group, the arterial and venous cannulas were implanted 14 days after the cerebral surgery.
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2.8. Experimental protocol One day after the surgical implant of the arterial and venous cannulas, the arterial cannula was connected to the recording system. About 20 min after starting the recordings, saline (100 nl) was injected into the NTS. Ten minutes later, the rats received injection of l-glutamate (5 nmol/100 nl) into the NTS. Fifteen minutes after l-glutamate injection, the chemoreflex was tested with i.v. injection of potassium cyanide (KCN, 40 Ag/0.1 ml/rat), and the baroreflex was tested by i.v. injection of pressor dose of phenylephrine (5 Ag/kg of body weight) and depressor dose of sodium nitroprusside (30 Ag/kg of body weight). The interval between each i.v. injection to test the reflexes was 5 min.
3. Results 3.1. Effects of AV3V lesions on the cardiovascular responses induced by L-glutamate into the NTS
Fig. 1. Changes in MAP and HR induced by l-glutamate (5 nmol/100 nl) into the NTS in acute (1 day) or chronic (15 days) sham- and AV3Vlesioned rats. The results are represented by meansFstandard error of means (S.E.M.). The number of rats is indicated in parenthesis.
2.5. Injections into the NTS The injections into the NTS were made using 5-Al Hamilton syringes connected by polyethylene tubing (PE10) to the injector needle 2 mm longer than the guide cannula implanted in the brain. The injection volume into the NTS was 100 nl.
Baseline MAP and HR were similar in acute (1 day) sham- (116F3 mm Hg and 364F14 bpm, respectively) and AV3V-lesioned rats (115F4 mm Hg and 366F8 bpm, respectively). Injection of l-glutamate (5 nmol/100 nl) in acute sham-lesioned rats increased MAP (28F3 mm Hg), while in acute AV3V-lesioned rats, l-glutamate into the NTS reduced MAP [ 26F8 mm Hg; F(1, 16)=34.5; pb0.0001; Figs. 1 and 2]. l-glutamate into the NTS induced similar bradycardia in acute sham- ( 76F13 bpm) and AV3V-lesioned rats [ 65F26 bpm; F(1, 16)=0.14; pN0.05; Figs. 1 and 2). Baseline MAP and HR were similar in chronic (15 days) sham- (114F4 mm Hg and 360F14 bpm, respectively) and AV3V-lesioned rats (118F6 mm Hg and 352F10 bpm,
2.6. Histology At the end of the experiments, 2% methylene blue solution (100 nl) was injected into the NTS. Immediately after the dye injection, the animals were deeply anesthetized with sodium thiopental (70 mg/kg of body weight, i.p.). Saline followed by 10% buffered formalin was perfused through the heart. The brains were frozen, cut coronally into 50-Am sections, stained with Giemsa stain, and analyzed by light microscopy to confirm the injections into the NTS and the AV3V lesions. 2.7. Statistical analysis The results are reported as meansFstandard error of means (S.E.M.). One-way analysis of variance (ANOVA) and Newman Keuls tests were used for comparisons. Differences were considered significant at pb0.05.
Fig. 2. Tracings of a representative rat of each group tested showing the typical changes recorded in pulsatile arterial pressure (PAP, mm Hg), mean arterial pressure (MAP, mm Hg), and heart rate (HR, bpm) in response to lglutamate (5 nmol/100 nl—arrows) injected into the NTS in (A) sham rats, (B) acute (1 day) AV3V-lesioned rats, and (C) chronic (15 days) AV3Vlesioned rats.
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respectively). Chronic AV3V lesions abolished the pressor response induced by l-glutamate into the NTS [2F8 mm Hg vs. sham 28F3 mm Hg; F(1, 17)=10.2; pb0.01], without changing bradycardia [ 90F29 bpm vs. sham 90F15 bpm; F(1, 17)=0.002; pN0.05; Figs. 1 and 2]. 3.2. Chemoreflex responses in AV3V-lesioned rats The pressor response to i.v. KCN (40 Ag/rat) was not altered by acute [ F(1, 16)=0.0002; pN0.05] or chronic AV3V lesions [ F(1, 17)=0.005; pN0.05; Fig. 3]. The bradycardia to KCN was also not altered by acute [ F(1, 16)=0.007; pN0.05] or chronic AV3V lesions [ F(1, 17)=0.16; pN0.05; Fig. 3]. 3.3. Baroreflex responses in AV3V-lesioned rats The pressor response produced by i.v. phenylephrine (5 Ag/kg of body weight) was not modified by acute [ F(1, 14)=3.8; pN0.05] or chronic AV3V lesions [ F(1, 16)=0.17; pN0.05; Fig. 4]. The reflex bradycardia to phenylephrine was also not modified by acute [ F(1, 14)=0.8; pN0.05] or chronic AV3V lesions [ F(1, 16)=1.0; pN0.05; Fig. 4]. The hypotensive response to i.v. sodium nitroprusside (30 Ag/kg of body weight) was not altered by acute [ F(1, 14)=0.8; pN0.05] or chronic AV3V lesions [ F(1, 16)=0.006; pN0.05; Fig. 5]. The reflex tachycardia to i.v. sodium nitroprusside was also not altered in acute [ F(1, 14)=0.002; pN0.05] or chronic AV3V-lesioned rats [ F(1, 16)=0.28; pN0.05; Fig. 5].
Fig. 3. Changes in MAP and HR induced by i.v. KCN (40 Ag/rat) in acute (1 day) or chronic (15 days) sham- and AV3V-lesioned rats. The results are represented by meansFstandard error of means (S.E.M.). The number of rats is indicated in parenthesis.
Fig. 4. Changes in MAP and HR induced by i.v. phenylephrine (5 Ag/kg of body weight) in acute (1 day) or chronic (15 days) sham- and AV3Vlesioned rats. The results are represented by meansFstandard error of means (S.E.M.). The number of rats is indicated in parenthesis.
Fig. 5. Changes in MAP and HR induced by i.v. sodium nitroprusside (30 Ag/kg of body weight) in acute (1 day) or chronic (15 days) sham- and AV3V-lesioned rats. The results are represented by meansFstandard error of means (S.E.M.). The number of rats is indicated in parenthesis.
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Fig. 6. Photomicrograph showing the typical AV3V lesion (arrow).
3.4. Histological analysis Fig. 6 shows the typical AV3V lesion in one rat representative of the lesioned groups. The AV3V lesions were located between the anterior commissure and the floor of the third ventricle with bilateral periventricular damage from the lamina terminalis to the preoptic area and anterior hypothalamus, never extending caudally to the arcuate nucleus or medial hypothalamus [2,3,19]. The brain structures that were consistently destroyed by AV3V lesions were the preoptic periventricular nuclei, the ventral part of median preoptic nucleus, and the anterior wall of the third ventricle with the associated organum vasculosum of the lamina terminalis. Partial destruction of the medial preoptic nuclei and the medial region of the anterior hypothalamic nuclei was also observed in some rats. Fig. 7 shows the typical injection site into the medial NTS, laterally to the area postrema, where the injection of lglutamate in unanesthetized rats usually induces pressor and bradycardic responses [7,17]. From a total of 74 rats submitted to AV3V lesions and microinjections into the NTS, 19 rats had positive histology in both targets. A total of 41 rats with sham lesions were submitted to microinjections into the NTS, and 18 of them had positive histology in the NTS.
present data suggest a significant control of the AV3V region over the sympathetic pressor mechanisms activated by l-glutamate injected in the NTS. Because AV3V lesions reduced the pressor responses to l-glutamate into the NTS, but had no effect on the responses to peripheral chemoreflex activation, the present results suggest dissociation between the mechanisms activated by l-glutamate into the NTS and the chemoreflex. Moreover, AV3V lesions affected the pressor responses to l-glutamate into the NTS, but not the bradycardia, which also suggests dissociation between the mechanisms activated by lglutamate into the NTS to increase sympathetic and parasympathetic activity. Although studies in anesthetized rats suggest a possible involvement of l-glutamate as the neurotransmitter of chemoreflex into the NTS [29,30], results from awake rats have shown that the treatment with glutamate antagonists into the NTS abolished the bradycardia, but not the pressor responses to peripheral chemoreflex activation with KCN, which raises the possibility that different neurotransmitters into the NTS may mediate the sympathetic and parasympathetic component of the chemoreflex [11,16]. The AV3V lesions impair the pressor responses to carotid occlusion and to forebrain angiotensinergic and cholinergic activation and prevent the development of neurogenic hypertension produced by sinoaortic denervation or by ablation of the medial NTS and the development of many other experimental models of hypertension [2–4,18,19,28]. However, there are cardiovascular responses, like baroreflex responses to i.v. phenylephrine or sodium nitroprusside [1], and peripheral chemoreflex (present results) that escape from the control of AV3V region. Therefore, the bradycardia to lglutamate into the NTS that is not changed by AV3V lesion may be related to activation of baro- or chemoreflex mechanisms. On the other hand, the pressor responses to l-glutamate into the NTS that is abolished by AV3V lesions seem to involve a more complex and still unknown
4. Discussion Injections of l-glutamate into the NTS, similar to chemoreflex activation in awake rats, increase sympathetic and parasympathetic activity causing pressor and bradycardic responses [7,8,16,17]. The pressor response to lglutamate injected into the NTS of unanesthetized rats was either reversed to hypotension by acute (1 day) or abolished by chronic (15 days) AV3V lesions. However, the bradycardia that resulted from l-glutamate injected into the NTS and the pressor and bradycardic responses to chemoreflex activation were not affected by the AV3V lesions. The
Fig. 7. Photomicrograph showing the typical site of injection into the NTS (arrow).
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mechanism than only the activation of the chemoreflex pathways. The direct connections among the NTS and the main hindbrain areas of the medullary circuitry involved in cardiovascular regulation may provide the basic mechanisms for sympathetic activation [10,25]. However, a previous study has shown that bilateral lesions of the paraventricular nucleus of the hypothalamus abolish the pressor response to chemoreflex activation [20], and the present results are showing that AV3V lesions abolish the pressor responses to l-glutamate injected into the NTS. Thus, forebrain mechanisms are also involved in the pressor responses produced by these stimuli and the sole integrity of the medullary circuitry is not sufficient to induce the sympathetic activation necessary for these pressor responses. Anatomical connections between the NTS and the forebrain, including nuclei in the AV3V region, have been described [5,13,22,23]. In addition, the AV3V region is also reciprocally connected with different brain areas and may receive and send signals to modulate sympathetic activity and vasopressin secretion that result in pressor responses [2–4,12– 14]. Therefore, signals produced by l-glutamate injected into the NTS could ascend to the AV3V region, which in turn activates the sympathetic system. Another possibility is that the AV3V region may send descending signals to the NTS or to other areas of the medullary circuitry that tonically facilitate the pressor mechanisms activated by lglutamate into the NTS. l-glutamate injected into the NTS induced hypotension in acute AV3V-lesioned rats and induced neither hypertension nor hypotension in chronic AV3V-lesioned rats. These differences may be the consequence of a partial recovery of the forebrain influence due to neural plasticity in chronic AV3V-lesioned rats, which may allow some activation of the pressor mechanisms enough to counterbalance the depressor effects produced by glutamate into the NTS in acute AV3V-lesioned rats. The recovery of a function in chronic AV3V-lesioned rats has been reported for daily water ingestion and pilocarpine-induced salivation. Both responses are strongly reduced in acute and completely recovered in chronic AV3V-lesioned rats [2,21]. The depressor responses to l-glutamate into the NTS in acute AV3V-lesioned rats are similar to those produced by the injections of l-glutamate into the NTS in anesthetized rats, which simulate the activation of baroreflex [26,27]. Therefore, the depressor responses to l-glutamate into the NTS in anesthetized rats is perhaps due to the blockade or reduction of AV3V mechanisms by the anesthesia. The importance of the AV3V region for cardiovascular regulation and also for fluid–electrolyte balance controlling sympathetic activity and vasopressin secretion has been related mainly to ANG II receptors and osmoreceptors present in this region and to its connections with a forebrain circuitry, especially within the lamina terminalis and with specific hypothalamic nuclei [2–4,12–14]. In addition, the
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AV3V region also connects with the medullary circuitry related to cardiovascular regulation [5,15,22–24], and, according to the present results, the AV3V region is involved in the pressor response produced by direct NTS activation with l-glutamate. Thus, the results may provide information for better understanding the antihypertensive effects of the AV3V lesion, especially on neurogenic hypertension that directly depends on NTS mechanisms. They also reinforce the importance of the links between forebrain and hindbrain in the control of cardiovascular function and the importance of the AV3V region as one of the main areas of the brain involved in cardiovascular regulation.
Acknowledgements We thank Silas Pereira Barbosa, Reginaldo da Conceic¸a˜o Queiro´z, and Silvia Fo´glia for the expert technical assistance, Silvana A.D. Malavolta for secretarial assistance, and Ana L.V. de Oliveira for animal care. This research was supported by public funding from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Conselho Nacional de Pesquisa (CNPq), and PRONEX.
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Research report
Cardiovascular responses to microinjection of l-glutamate into the NTS in AV3V-lesioned rats Alexandre Antonio Vieira, Eduardo Colombari, Laurival A. De Luca Jr., De´bora Simo˜es de Almeida Colombari, Jose´ V. Menani* Department of Physiology and Pathology, School of Dentistry, Paulista State University (UNESP), Araraquara, SP 14801-903, Brazil Department of Physiology, UNIFESP-EPM, Sa˜o Paulo, SP, Brazil Accepted 10 August 2004 Available online 11 September 2004
Abstract The excitatory amino acid l-glutamate injected into the nucleus of the solitary tract (NTS) in unanesthetized rats similar to peripheral chemoreceptor activation increases mean arterial pressure (MAP) and reduces heart rate. In this study, we investigated the effects of acute (1 day) and chronic (15 days) electrolytic lesions of the preoptic-periventricular tissue surrounding the anteroventral third ventricle (AV3V region) on the pressor and bradycardic responses induced by injections of l-glutamate into the NTS or peripheral chemoreceptor activation in unanesthetized rats. Male Holtzman rats with sham or electrolytic AV3V lesions and a stainless steel cannula implanted into the NTS were used. Differently from the pressor responses (28F3 mm Hg) produced by injections into the NTS of sham-lesioned rats, l-glutamate (5 nmol/ 100 nl) injected into the NTS reduced MAP ( 26F8 mm Hg) or produced no effect (2F7 mm Hg) in acute and chronic AV3V-lesioned rats, respectively. The bradycardia to l-glutamate into the NTS and the cardiovascular responses to chemoreflex activation with intravenous potassium cyanide or to baroreflex activation with intravenous phenylephrine or sodium nitroprusside were not modified by AV3V lesions. The results show that the integrity of the AV3V region is essential for the pressor responses to l-glutamate into the NTS but not for the pressor responses to chemoreflex activation, suggesting dissociation between the central mechanisms involved in these responses. D 2004 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Baroreceptor; Chemoreceptor; Solitary tract; Hypothalamus; Hypertension
1. Introduction The excitatory amino acid l-glutamate is considered one of the main neurotransmitters released in the nucleus of the solitary tract (NTS) by the cardiovascular afferent projections [26,27,29,30], but the central mechanisms involved in the cardiovascular responses to l-glutamate injected into the NTS are not completely understood. * Corresponding author. Departamento de Fisiologia e Patologia, Faculdade de Odontologia de Araraquara, UNESP, Rua Humaita´, 1680, Araraquara, 14801 903, SP, Brazil. Tel.: +55 16 201 6486; fax: +55 16 201 6488. E-mail address: [email protected] (J.V. Menani). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.08.006
Injections of l-glutamate into the NTS in anesthetized rats similar to baroreflex activation produce hypotension and bradycardia [26,27]. However, injections of l-glutamate into the NTS in awake rats usually produce pressor responses and bradycardia similar to peripheral chemoreceptor activation [7,8,11,16,17,20]. A medullary circuitry essential for cardiovascular regulation that receives influences from peripheral cardiovascular receptors involves the NTS, nucleus ambiguus, and the ventrolateral medulla (caudal and rostral) [6,9,10,16,25]. After the first synapse into the NTS, the signals from peripheral baroreceptors and chemoreceptors can reach different medullary areas that control autonomic projections to the cardiovascular system [6,9,10,16,25]. In addition,
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signals from peripheral receptors can also ascend to the forebrain after a relay in the NTS [5,13,15,22,24]. Although the information that arises from peripheral receptors to the medullary circuitry provides a basic control of cardiovascular functions, descending signals from different encephalic areas, especially from the forebrain, might influence the activity of the medullary circuitry and the autonomic discharges to the cardiovascular system allowing more complex cardiovascular adjustments [13]. Several studies have shown the importance of the preoptic-periventricular tissue surrounding the anteroventral third ventricle (AV3V region) for the cardiovascular regulation and the control of fluid–electrolyte balance [2– 4,12–14,19]. Among other specific nuclei, the AV3V region includes the organum vasculosum of the lamina terminalis, an area lacking blood–brain barrier and rich in angiotensin II (ANG II) receptors [2–4,12–14]. In rats, AV3V lesions impair the development of experimental hypertension, abolish the pressor responses to central ANG II and central cholinergic activation, reduce vasopressin secretion and the pressor responses to bilateral common carotid occlusion, and increase baroreflex sensitivity to ANG II without changing the baroreflex responses to intravenous (i.v.) phenylephrine and sodium nitroprusside [1–4,12–14,18,19,28]. In spite of the importance of the AV3V region for the cardiovascular regulation modulating sympathetic activity and vasopressin secretion [2–4,12–14] and the existence of neural connections between the AV3V region and hindbrain areas related to cardiovascular regulation, especially the NTS [5,13,15,22–24], a possible involvement of the AV3V region on the cardiovascular responses to chemoreflex or direct activation of hindbrain areas has not been investigated. Therefore, in this study, we investigated the effects of electrolytic AV3V lesions on the pressor and bradycardic responses to l-glutamate injected into the NTS and to peripheral chemoreflex activation in awake rats. To compare with results from a previous study [1], baroreflex responses were also tested. As already shown for daily water ingestion and pilocarpine-induced salivation, the effects of acute (1 to 7 days) and chronic (more than 10 days) AV3V lesions can be totally different [2,3,21]. Thus, in this study, we tested lglutamate responses and the cardiovascular reflexes in rats with acute (1 day) and chronic (15 days) AV3V lesions.
2. Material and methods 2.1. Animals Male Holtzman rats weighing 280 to 320 g were used. The animals were housed individually in stainless steel cages in a room with controlled temperature (23F2 8C) and humidity (55F10%). Lights were on from 7:00 a.m. to 7:00 p.m. Standard Purina chow and tap water were available ad libitum. The Ethical Committee for Animal
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Care and Use from Dentistry School of Araraquara, UNESP approved the experimental protocols used in this study. 2.2. Electrolytic AV3V lesions Rats were anesthetized by injection of ketamine (100 mg/ kg of body weight) intraperitoneally (i.p.) and placed in a two-arm stereotaxic frame (model 900, David Kopf Instruments). The skull was positioned to have bregma and lambda at the same level. A tungsten wire electrode (0.4 mm in diameter), bared at the tip (0.5 mm), was inserted into the brain using the following coordinates: 0.0 mm from bregma, in the midline and 7.0 mm below the dura mater. The electrolytic lesion was performed using a cathode current (2 mA for 10 sec). A clip attached to the tail was used as the indifferent electrode. The sham-lesioned rats had the electrode placed along the same coordinates, except that no current was passed. 2.3. Intracerebral cannulas Immediately after the AV3V lesion, using the second arm of the stereotaxic, a stainless steel cannula (140.6 mm o.d.) was implanted into the NTS using the coordinates 14.5 mm caudal to bregma, 0.5 mm lateral to the midline, and 7.5 mm below the dura mater. The cannulas were fixed to the cranium using dental acrylic resin and jeweler screws. The chronic AV3V-lesioned and sham-lesioned rats received a prophylactic dose of penicillin (30,000 IU) given intramuscularly postsurgically. Besides water and food pellets, the chronic AV3V-lesioned rats had access to 10% sucrose during 1 week following the lesions to compensate for the acute adipsia that follows AV3V lesions. 2.4. Arterial pressure and heart rate recordings Mean arterial pressure (MAP) and heart rate (HR) were recorded in unanesthetized rats. Under ketamine (100 mg/kg of body weight, i.p.) anesthesia, a polyethylene tubing (PE10 connected to a PE-50) was inserted into the abdominal aorta through the femoral artery on the day before the experiments. At the same time, a second polyethylene tubing was inserted in the femoral vein for drug administration. Both tubings were tunneled subcutaneously and exposed on the back of the rat to allow access in unrestrained, freely moving rats. To record pulsatile arterial pressure, MAP and HR, the arterial catheter was connected to a Stathan Gould (P23 Db) pressure transducer coupled to a preamplifier (model ETH-200 Bridge Bio Amplifier) that was connected to a Powerlab computer data acquisition system (model Powerlab 16SP, ADInstruments). In the acute group, the arterial and venous cannula implant was performed immediately after the cerebral surgery. In the chronic group, the arterial and venous cannulas were implanted 14 days after the cerebral surgery.
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2.8. Experimental protocol One day after the surgical implant of the arterial and venous cannulas, the arterial cannula was connected to the recording system. About 20 min after starting the recordings, saline (100 nl) was injected into the NTS. Ten minutes later, the rats received injection of l-glutamate (5 nmol/100 nl) into the NTS. Fifteen minutes after l-glutamate injection, the chemoreflex was tested with i.v. injection of potassium cyanide (KCN, 40 Ag/0.1 ml/rat), and the baroreflex was tested by i.v. injection of pressor dose of phenylephrine (5 Ag/kg of body weight) and depressor dose of sodium nitroprusside (30 Ag/kg of body weight). The interval between each i.v. injection to test the reflexes was 5 min.
3. Results 3.1. Effects of AV3V lesions on the cardiovascular responses induced by L-glutamate into the NTS
Fig. 1. Changes in MAP and HR induced by l-glutamate (5 nmol/100 nl) into the NTS in acute (1 day) or chronic (15 days) sham- and AV3Vlesioned rats. The results are represented by meansFstandard error of means (S.E.M.). The number of rats is indicated in parenthesis.
2.5. Injections into the NTS The injections into the NTS were made using 5-Al Hamilton syringes connected by polyethylene tubing (PE10) to the injector needle 2 mm longer than the guide cannula implanted in the brain. The injection volume into the NTS was 100 nl.
Baseline MAP and HR were similar in acute (1 day) sham- (116F3 mm Hg and 364F14 bpm, respectively) and AV3V-lesioned rats (115F4 mm Hg and 366F8 bpm, respectively). Injection of l-glutamate (5 nmol/100 nl) in acute sham-lesioned rats increased MAP (28F3 mm Hg), while in acute AV3V-lesioned rats, l-glutamate into the NTS reduced MAP [ 26F8 mm Hg; F(1, 16)=34.5; pb0.0001; Figs. 1 and 2]. l-glutamate into the NTS induced similar bradycardia in acute sham- ( 76F13 bpm) and AV3V-lesioned rats [ 65F26 bpm; F(1, 16)=0.14; pN0.05; Figs. 1 and 2). Baseline MAP and HR were similar in chronic (15 days) sham- (114F4 mm Hg and 360F14 bpm, respectively) and AV3V-lesioned rats (118F6 mm Hg and 352F10 bpm,
2.6. Histology At the end of the experiments, 2% methylene blue solution (100 nl) was injected into the NTS. Immediately after the dye injection, the animals were deeply anesthetized with sodium thiopental (70 mg/kg of body weight, i.p.). Saline followed by 10% buffered formalin was perfused through the heart. The brains were frozen, cut coronally into 50-Am sections, stained with Giemsa stain, and analyzed by light microscopy to confirm the injections into the NTS and the AV3V lesions. 2.7. Statistical analysis The results are reported as meansFstandard error of means (S.E.M.). One-way analysis of variance (ANOVA) and Newman Keuls tests were used for comparisons. Differences were considered significant at pb0.05.
Fig. 2. Tracings of a representative rat of each group tested showing the typical changes recorded in pulsatile arterial pressure (PAP, mm Hg), mean arterial pressure (MAP, mm Hg), and heart rate (HR, bpm) in response to lglutamate (5 nmol/100 nl—arrows) injected into the NTS in (A) sham rats, (B) acute (1 day) AV3V-lesioned rats, and (C) chronic (15 days) AV3Vlesioned rats.
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respectively). Chronic AV3V lesions abolished the pressor response induced by l-glutamate into the NTS [2F8 mm Hg vs. sham 28F3 mm Hg; F(1, 17)=10.2; pb0.01], without changing bradycardia [ 90F29 bpm vs. sham 90F15 bpm; F(1, 17)=0.002; pN0.05; Figs. 1 and 2]. 3.2. Chemoreflex responses in AV3V-lesioned rats The pressor response to i.v. KCN (40 Ag/rat) was not altered by acute [ F(1, 16)=0.0002; pN0.05] or chronic AV3V lesions [ F(1, 17)=0.005; pN0.05; Fig. 3]. The bradycardia to KCN was also not altered by acute [ F(1, 16)=0.007; pN0.05] or chronic AV3V lesions [ F(1, 17)=0.16; pN0.05; Fig. 3]. 3.3. Baroreflex responses in AV3V-lesioned rats The pressor response produced by i.v. phenylephrine (5 Ag/kg of body weight) was not modified by acute [ F(1, 14)=3.8; pN0.05] or chronic AV3V lesions [ F(1, 16)=0.17; pN0.05; Fig. 4]. The reflex bradycardia to phenylephrine was also not modified by acute [ F(1, 14)=0.8; pN0.05] or chronic AV3V lesions [ F(1, 16)=1.0; pN0.05; Fig. 4]. The hypotensive response to i.v. sodium nitroprusside (30 Ag/kg of body weight) was not altered by acute [ F(1, 14)=0.8; pN0.05] or chronic AV3V lesions [ F(1, 16)=0.006; pN0.05; Fig. 5]. The reflex tachycardia to i.v. sodium nitroprusside was also not altered in acute [ F(1, 14)=0.002; pN0.05] or chronic AV3V-lesioned rats [ F(1, 16)=0.28; pN0.05; Fig. 5].
Fig. 3. Changes in MAP and HR induced by i.v. KCN (40 Ag/rat) in acute (1 day) or chronic (15 days) sham- and AV3V-lesioned rats. The results are represented by meansFstandard error of means (S.E.M.). The number of rats is indicated in parenthesis.
Fig. 4. Changes in MAP and HR induced by i.v. phenylephrine (5 Ag/kg of body weight) in acute (1 day) or chronic (15 days) sham- and AV3Vlesioned rats. The results are represented by meansFstandard error of means (S.E.M.). The number of rats is indicated in parenthesis.
Fig. 5. Changes in MAP and HR induced by i.v. sodium nitroprusside (30 Ag/kg of body weight) in acute (1 day) or chronic (15 days) sham- and AV3V-lesioned rats. The results are represented by meansFstandard error of means (S.E.M.). The number of rats is indicated in parenthesis.
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Fig. 6. Photomicrograph showing the typical AV3V lesion (arrow).
3.4. Histological analysis Fig. 6 shows the typical AV3V lesion in one rat representative of the lesioned groups. The AV3V lesions were located between the anterior commissure and the floor of the third ventricle with bilateral periventricular damage from the lamina terminalis to the preoptic area and anterior hypothalamus, never extending caudally to the arcuate nucleus or medial hypothalamus [2,3,19]. The brain structures that were consistently destroyed by AV3V lesions were the preoptic periventricular nuclei, the ventral part of median preoptic nucleus, and the anterior wall of the third ventricle with the associated organum vasculosum of the lamina terminalis. Partial destruction of the medial preoptic nuclei and the medial region of the anterior hypothalamic nuclei was also observed in some rats. Fig. 7 shows the typical injection site into the medial NTS, laterally to the area postrema, where the injection of lglutamate in unanesthetized rats usually induces pressor and bradycardic responses [7,17]. From a total of 74 rats submitted to AV3V lesions and microinjections into the NTS, 19 rats had positive histology in both targets. A total of 41 rats with sham lesions were submitted to microinjections into the NTS, and 18 of them had positive histology in the NTS.
present data suggest a significant control of the AV3V region over the sympathetic pressor mechanisms activated by l-glutamate injected in the NTS. Because AV3V lesions reduced the pressor responses to l-glutamate into the NTS, but had no effect on the responses to peripheral chemoreflex activation, the present results suggest dissociation between the mechanisms activated by l-glutamate into the NTS and the chemoreflex. Moreover, AV3V lesions affected the pressor responses to l-glutamate into the NTS, but not the bradycardia, which also suggests dissociation between the mechanisms activated by lglutamate into the NTS to increase sympathetic and parasympathetic activity. Although studies in anesthetized rats suggest a possible involvement of l-glutamate as the neurotransmitter of chemoreflex into the NTS [29,30], results from awake rats have shown that the treatment with glutamate antagonists into the NTS abolished the bradycardia, but not the pressor responses to peripheral chemoreflex activation with KCN, which raises the possibility that different neurotransmitters into the NTS may mediate the sympathetic and parasympathetic component of the chemoreflex [11,16]. The AV3V lesions impair the pressor responses to carotid occlusion and to forebrain angiotensinergic and cholinergic activation and prevent the development of neurogenic hypertension produced by sinoaortic denervation or by ablation of the medial NTS and the development of many other experimental models of hypertension [2–4,18,19,28]. However, there are cardiovascular responses, like baroreflex responses to i.v. phenylephrine or sodium nitroprusside [1], and peripheral chemoreflex (present results) that escape from the control of AV3V region. Therefore, the bradycardia to lglutamate into the NTS that is not changed by AV3V lesion may be related to activation of baro- or chemoreflex mechanisms. On the other hand, the pressor responses to l-glutamate into the NTS that is abolished by AV3V lesions seem to involve a more complex and still unknown
4. Discussion Injections of l-glutamate into the NTS, similar to chemoreflex activation in awake rats, increase sympathetic and parasympathetic activity causing pressor and bradycardic responses [7,8,16,17]. The pressor response to lglutamate injected into the NTS of unanesthetized rats was either reversed to hypotension by acute (1 day) or abolished by chronic (15 days) AV3V lesions. However, the bradycardia that resulted from l-glutamate injected into the NTS and the pressor and bradycardic responses to chemoreflex activation were not affected by the AV3V lesions. The
Fig. 7. Photomicrograph showing the typical site of injection into the NTS (arrow).
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mechanism than only the activation of the chemoreflex pathways. The direct connections among the NTS and the main hindbrain areas of the medullary circuitry involved in cardiovascular regulation may provide the basic mechanisms for sympathetic activation [10,25]. However, a previous study has shown that bilateral lesions of the paraventricular nucleus of the hypothalamus abolish the pressor response to chemoreflex activation [20], and the present results are showing that AV3V lesions abolish the pressor responses to l-glutamate injected into the NTS. Thus, forebrain mechanisms are also involved in the pressor responses produced by these stimuli and the sole integrity of the medullary circuitry is not sufficient to induce the sympathetic activation necessary for these pressor responses. Anatomical connections between the NTS and the forebrain, including nuclei in the AV3V region, have been described [5,13,22,23]. In addition, the AV3V region is also reciprocally connected with different brain areas and may receive and send signals to modulate sympathetic activity and vasopressin secretion that result in pressor responses [2–4,12– 14]. Therefore, signals produced by l-glutamate injected into the NTS could ascend to the AV3V region, which in turn activates the sympathetic system. Another possibility is that the AV3V region may send descending signals to the NTS or to other areas of the medullary circuitry that tonically facilitate the pressor mechanisms activated by lglutamate into the NTS. l-glutamate injected into the NTS induced hypotension in acute AV3V-lesioned rats and induced neither hypertension nor hypotension in chronic AV3V-lesioned rats. These differences may be the consequence of a partial recovery of the forebrain influence due to neural plasticity in chronic AV3V-lesioned rats, which may allow some activation of the pressor mechanisms enough to counterbalance the depressor effects produced by glutamate into the NTS in acute AV3V-lesioned rats. The recovery of a function in chronic AV3V-lesioned rats has been reported for daily water ingestion and pilocarpine-induced salivation. Both responses are strongly reduced in acute and completely recovered in chronic AV3V-lesioned rats [2,21]. The depressor responses to l-glutamate into the NTS in acute AV3V-lesioned rats are similar to those produced by the injections of l-glutamate into the NTS in anesthetized rats, which simulate the activation of baroreflex [26,27]. Therefore, the depressor responses to l-glutamate into the NTS in anesthetized rats is perhaps due to the blockade or reduction of AV3V mechanisms by the anesthesia. The importance of the AV3V region for cardiovascular regulation and also for fluid–electrolyte balance controlling sympathetic activity and vasopressin secretion has been related mainly to ANG II receptors and osmoreceptors present in this region and to its connections with a forebrain circuitry, especially within the lamina terminalis and with specific hypothalamic nuclei [2–4,12–14]. In addition, the
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AV3V region also connects with the medullary circuitry related to cardiovascular regulation [5,15,22–24], and, according to the present results, the AV3V region is involved in the pressor response produced by direct NTS activation with l-glutamate. Thus, the results may provide information for better understanding the antihypertensive effects of the AV3V lesion, especially on neurogenic hypertension that directly depends on NTS mechanisms. They also reinforce the importance of the links between forebrain and hindbrain in the control of cardiovascular function and the importance of the AV3V region as one of the main areas of the brain involved in cardiovascular regulation.
Acknowledgements We thank Silas Pereira Barbosa, Reginaldo da Conceic¸a˜o Queiro´z, and Silvia Fo´glia for the expert technical assistance, Silvana A.D. Malavolta for secretarial assistance, and Ana L.V. de Oliveira for animal care. This research was supported by public funding from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Conselho Nacional de Pesquisa (CNPq), and PRONEX.
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