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Differential baroreceptor modulation mediated by the ventrolateral medulla
Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 156 – 162 www.elsevier.com/locate/autneu
Differential baroreceptor modulation mediated by the ventrolateral medulla Ca´ssia de Toledo Bergamaschi a, Bruno de Arruda Carillo a, Henrique Azevedo Futuro Neto b, Ruy Ribeiro de Campos a,* a
Departamento de Fisiologia, Universidade Federal de Sa˜o Paulo—Escola Paulista de Medicina, Rua Botucatu, 862, CEP 04023-060, Sa˜o Paulo, SP, Brazil b Universidade Federal do Espı´rito Santo and Escola Superior de Cieˆncias da Santa Casa de Miserico´rdia de Vito´ria (EMESCAM)—ES, Brazil Received 28 October 2005; received in revised form 7 February 2006; accepted 20 February 2006
Abstract Previous studies have shown that pharmacological stimulation of a region denominated caudal pressor area (CPA), located in the caudal end of the ventrolateral medulla, induces increases in arterial blood pressure (BP). The aim of this study was to compare the responses on renal sympathetic nerve activity (rSNA) and BP responses mediated by stimulation of CPA or rostral ventrolateral medulla (RVLM), in intact or sino-aortic barodenervated rats. Male Wistar rats (300 – 350g, n = 15) were anesthetized (urethane 1.2 to 1.4g/kg, i.v.) and artificially ventilated. The mean arterial pressure (MAP) and rSNA were measured during bilateral glutamate microinjection (10 nmo/100 nl) into the CPA or into the RVLM. Glutamatergic stimulation of the RVLM increased MAP (46 T 7 mm Hg) and rSNA (82 T 21%); during CPA stimulation, MAP and rSNA increased 60 T 7 mm Hg and 93 T 9%, respectively. However, despite the similarity of responses mediated by both regions, the duration of rSNA and blood pressure responses mediated by the CPA were significantly longer than the duration of the responses mediated by the RVLM. After barodenervation, there was an increase in the time-course and magnitude of sympathetic response only in response to RVLM stimulation but not in response to CPA. The results suggest a differential baroreceptor modulation on rSNA mediated by the ventrolateral medulla neurons. Glutamatergic activation of CPA neurons can cause large increases in the rSNA and BP with a weaker baroreceptor modulation when compared to responses mediated by the RVLM neurons. D 2006 Elsevier B.V. All rights reserved. Keywords: Baroreceptor; Caudal pressor area; Rostral ventrolateral medulla; Glutamate; Sympathetic activity
1. Introduction It is well known that the ventrolateral medulla (VLM) contains neurons involved in the tonic and reflex control of the cardiovascular system. Two regions within the VLM were initially identified: the rostral ventrolateral medulla (RVLM), which raises arterial blood pressure, as well as sympathetic nerve activity when stimulated, and the caudal ventrolateral medulla (CVLM), which acts in the opposite way. Both VLM regions are involved in the baroreceptor and chemoreceptor reflex integration (Guertzenstion and Silver, 1974; Guertzenstein and Lopes, 1984; Dampney, 1994a,b). * Corresponding author. Tel.: +55 11 5573 7820; fax: +55 11 5573 7820. E-mail address: [email protected] (R.R. de Campos). 1566-0702/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2006.02.012
The RVLM premotor neurons project directly to sympathetic preganglionic neurons and are involved in the maintenance of resting sympathetic vasomotor tone (Dampney et al., 2003). A significant proportion of tonic activity in the RVLM sympathetic premotor neurons is driven by neurons located in a third region of the VLM denominated caudal pressor area (CPA) (Campos and McAllen, 1999). The CPA is located caudal to the CVLM depressor neurons in the caudal end of VLM and, when activated, increases arterial blood pressure in anesthetized and in conscious rats (Gordon and McCann, 1988; Campos et al., 1994; Silva et al., 2001). Inhibition of neuronal activity in the CPA by glycine microinjection induces hypotension and decreases the activity of the RVLM neurons by approximately 40–
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45% while glutamatergic stimulation causes increase in the RVLM activity, sympathetic nerve activity and blood pressure (Campos and McAllen, 1999; Natarajan and Morrison, 2000). However, there is no information about the characteristic of sympathetic activation mediated by the CPA neurons. This raises the following question: the activation of the CPA or RVLM neurons by microinjections of excitatory amino acid into the CPA or into the RVLM may produce differential actions on sympathetic nerve activity? In the present study, we investigated the pattern of renal sympathetic activation mediated by the CPA compared to the RVLM mediated responses. To excite cell bodies within the CPA and RVLM, we used local microinjections of glutamate, which have been shown to produce pressor responses when injected in both regions (McAllen, 1986a; Gordon and McCann, 1988). There is no information regarding how the baroreceptor reflex can modulate the sympathetic nerve activity during the increase in arterial blood pressure in response to glutamatergic stimulation of the CPA neurons. One hypothesis to explain the sympathoexcitatory actions of CPA is an inhibitory pathway from the CPA to the CVLM. According to this hypothesis, during CPA stimulation there is an inhibition of the baroreceptor reflex, causing an acute increase in the sympathetic nerve activity and blood pressure (Campos et al., 1994). In this case, it is reasonable to suppose that the sympathetic activation mediated by the CPA is less dependent of baroreceptor modulation compared to the RVLM actions. Thus, we tested this hypothesis, comparing the sympathoexcitatory and hypertensive actions mediated by the CPA with the responses mediated by the RVLM in intact and in barodenervated rats. The first aim of the present study (first series of experiments) was to determine the extent by which glutamatergic stimulation of CPA neurons can activate the renal sympathetic nerve activity (rSNA) in comparison to the RVLM mediated responses. The second aim (second series of experiments) was to determine the effects of acute sino-aortic barodenervation on sympathetic and blood pressure responses mediated by the CPA and RVLM glutamatergic stimulation.
2. Materials and methods 2.1. First series of experiments 2.1.1. General procedures Experiments were performed in male Wistar rats (300 – 350 g, n = 9). The experiments were approved by the Animal Experimental Ethics Committee of the Federal University of Sa˜o Paulo—Escola Paulista de Medicina. The rats were initially anesthetized with halothane (2%) and instrumented with femoral venous and arterial catheters for
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blood pressure (BP) and heart rate (HR) recordings. The animals were then anesthetized very slowly with urethane (1.4 g/kg, i.v.) and artificially ventilated with oxygen enriched air with a respiratory pump at a level that maintained end-tidal CO2 close to 3.5 –4%. Rectal temperature was maintained at 37 T 0.5 C by a servo-controlled electric blanket. The rat was then mounted prone in a stereotaxic frame with the head mildly ventroflexed (bite bar at 11 mm). The occipital bone was opened over the dorsal medulla and caudal cerebellum, and the dura was retracted. The anesthetic level was tested during the experiments (withdrawal reflexes to noxious pinching) and extra urethane (0.1 –0.5 g/kg, i.v.) was given when it was needed. A pneumatic cuff was placed around the lower thoracic aorta, via a small left intercostal incision in order to artificially increase BP and activate baroreceptor reflex. 2.1.2. Renal sympathetic nerve recording The left renal sympathetic nerve was exposed through a left retroperitoneal flank incision placed on bipolar recording electrodes and was covered with mineral oil. The signal from the renal nerve was displayed on an oscilloscope and monitored by means of an audio amplifier. The nerve activity was also amplified (Neurolog, 10 to 20K) and filtered by a band-pass filter (50 –1000 Hz) and was rectified and integrated every 1 s. At the end of the experiments, the baseline noise level of renal sympathetic nerve activity (rSNA) was determined after the administration of hexamethonium bromide (30mg/kg, i.v.). The change in nerve discharge during the experiment was expressed by a percentage from the basal value subtracting the background noise. The mean arterial pressure (MAP) and HR signals were derived from the pulsatile BP. All signals were recorded on a computer-based data acquisition system (Power Lab system, AD Instruments, NSW, Australia). 2.1.3. Drugs intramedullary microinjections Glass micropipettes of tip diameter of approximately 20 Am were used to pressure injected l-glutamate (monosodium salt, 10nmol/100 nl) into the CPA or into the RVLM in a random way. The solution was prepared in normal saline (pH 7.0 – 7.4). Glutamate was injected in 5 to 10 s sequentially in one side and in the contraleral region; interval between injection sides was around of 10 s. The pipettes were held in a vertical position and the CPA was located 1 mm caudal to the Obex, 1.5 –1.6 mm lateral to the midline, and 2mm deep to the dorsal medullary surface (Campos and McAllen, 1999). The RVLM was located 3 mm rostral to Obex, 1.7 – 1.8 mm lateral to midline, and 3mm deep. Injections sites were marked at the end of the experiments by subsequently injecting 50 to 100 nl of Evans blue. In three experiments, the vehicle (saline) was injected into the RVLM or CPA as a control injection.
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2.2. Second series of experiments
2.4. Statistical analysis
In six rats, before to start the surgical procedures as above described for rSNA recordings and intramedullary microinjections, a sino-aortic denervation (SAD) was performed according to standard methods (Krieger, 1970). A pneumatic cuff was placed around the lower thoracic aorta and the baroreceptor activation in response to a brief aortic occlusion did not cause any decrease in the rSNA or HR in barodenervated rats. After the end of the surgical preparation, the animals were positioned in the streotaxic frame and glutamate microinjections into the RVLM or CPA were performed in this group in a random way, using the same dose of the first series of experiments. The responses on BP and rSNA mediated by glutamate microinjections into the two regions were then recorded for further off-line analysis.
All values are expressed as mean T S.E.M. The changes in MAP, HR and percentage of rSNA responses during RVLM or CPA activation were analyzed in intervals of 1 s after microinjection in relation to basal condition by paired and unpaired t-test. The differences on MAP, rSNA and HR between CPA and RVLM mediated responses were assessed by one-way analysis of variance (ANOVA) followed by the Student – Newman – Keuls method. Differences were considered significant at P < 0.05.
2.3. Histology At the end of the experiments, animals were killed by an intravenous overdose of urethane. The brain stem was then removed and fixed by immersion for at least 24h in 10% paraformaldehyde solution, after which coronal sections (40 Am) were cut on a freezing microtome, the sections were mounted on glass slices and stained with neutral red. Microinjection sites were identified by deposition of Evans blue. Fig. 1 shows the distribution of the centers of sites at which glutamate (10 nmol/100 nl) was injected into the RVLM or CPA. A positive location for the RVLM was considered when the dye was deposited ventral to the nucleus ambiguous and lateral to the inferior olive nucleus. The CPA was located in the caudal end of the lateral reticular nucleus of the ventrolateral medulla as described in previous investigations (Campos and McAllen, 1999; Possas et al., 1994).
Fig. 1. Distribution of the centers of sites of glutamate microinjections (10 nmol/100nl) in the caudal pressor area (CPA) 1 mm caudal to the Obex or in the rostral ventrolateral medulla (RVL) 3mm rostral to the Obex. Abbreviations: X, nucleus ambiguous (compact formation); V, spinal trigeminal tract; Gr, nucleus gracilis; Pyx, pyramidal decussation.
3. Results First, the rSNA was tested in relation to baroreceptor reflex activation. A brief aortic constriction increased MAP from 107 T 3.5 to 154 T 5mm Hg and decreased HR (from 382 T 18 to 375 T 21bpm) and rSNA by 78 T 9% in the barointac group (n = 9). In the acute baroreceptor denervated rats (n = 6), a similar increase in MAP in response to aortic constriction did not cause any change in the rSNA or HR. 3.1. Effect of bilateral microinjection of glutamate into the CPA or RVLM on rSNA and arterial blood pressure in nonbarodenervated rats Glutamate microinjections were performed at random into the RVLM or CPA in the same animal (n = 9) with an interval time of at least 30 min, when the sympathetic nerve activity and blood pressure had returned to the basal preinjection level. Following bilateral microinjection of glutamate into the CPA, there was an immediate increase in MAP and rSNA by 60 T 7.3mm Hg (basal 100 T 3.5 mm Hg, n = 9, P < 0.05) and 93 T 9.3% ( P < 0.05) respectively, compared to the preinjection control levels. The increase in rSNA was immediate, reaching a peak around 7 s and remained increased above basal level for approximately 1 min, as shown in Fig. 2A. The arterial blood pressure increased in response to microinjection and reached the peak approximately 10 s after injection and gradually returned to the basal level (Fig. 2B). There was no significant change in HR (from 392 T 15 to 416 T 16 bpm). When glutamate was microinjected into the RVLM, there was an immediate increase in MAP and rSNA of 46 T 7 mm Hg, P < 0.05 (basal 108 T 7 mm Hg) and 82 T 21% ( P < 0.05) respectively, compared to control values. The peak of rSNA response was in the fifth second followed by an abrupt reduction in the nerve activity after the seventh second of injection (from 81 T12% to 38 T 15% in relation to basal level), as shown Fig. 2A. During RVLM injections it was observed no significant change in HR (from 379 T 19 to 394 T 20bpm).
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Fig. 2. Grouped data showing the time courses of changes in renal sympathetic nerve activity (rSNA) in A and mean arterial pressure (MAP) in B, following bilateral microinjection of glutamate (10nmol, 100nl) into the CPA or RVLM. *P < 0.05. Comparison between CPA and RVLM mediated responses (n = 9).
When the peak of maximal changes of the MAP and rSNA in response to glutamate injection into the CPA or RVLM was compared, there was no difference between regions. Nevertheless, the sympathetic increase (percentage of increase) mediated by CPA remained elevated, above basal preinjection levels whose duration was longer than the duration of the responses mediated by the RVLM, as shown in Fig. 2A. The sympathetic response mediated by the RVLM had an abrupt decrease after the seventh second of microinjection; this was not observed when CPA was activated. Time-course of changes in the arterial blood pressure in response to stimulation of CPA or RVLM was also compared and it was observed that CPA activation caused a significant longer lasting response on MAP when compared to the MAP increase mediated by the RVLM, as shown in Fig. 2B. In three experiments, saline was bilaterally injected into the CPA or RVLM, no responses on sympathetic activity or arterial blood pressure were observed. 3.2. Effect of bilateral microinjection of glutamate into the CPA or RVLM on rSNA and arterial blood pressure in sinoaortic barodenervated rats In a group of six sino-aortic barodenervated rats, bilateral glutamate microinjection into the CPA increased MAP and rSNA in 78 T 9 mm Hg, P < 0.05 (basal, 113 T 12 mm Hg) and 112 T 24% ( P < 0.05), respectively. The response of sympathetic activity was immediate and remained above basal preinjection level for approximately 1 min (Fig. 3A).
When the responses on MAP and rSNA during CPA stimulation between intact and barodenervated rats were compared, there was no significant difference in the peak of changes for both variables. There was also no change in the time course of rSNA increase in response to glutamate into the CPA after barodenervation (Fig. 3A). However, in relation to the arterial blood pressure increase in response to CPA stimulation in barodenervated rats, there was a longer lasting response in relation to control group, with statistical significance (see Fig. 3B). In this group, the MAP remained elevated above basal level for approximately 4 min and gradually returned to the preinjection level. In the control non-barodenervated group, MAP returned to the basal level in 2 min approximately. Bilateral microinjection of glutamate into the RVLM in these barodenervated rats, caused an increase in MAP and rSNA in 55 T 6 mm Hg ( P < 0.05) and 159 T 32% ( P < 0.05), respectively (Fig. 4). The increase in sympathetic activity was immediate and reached a peak in 7 s and gradually returned to the basal level. In contrast to what had been observed in baro-intact rats, there was no abrupt reduction in the rSNA after the peak of sympathetic activation after barodenervation (see Figs. 2A and 4A), indicating that the sympathetic reduction was mediated by baroreceptor reflex. When the peak of changes on rSNA and MAP– in response to glutamate injection into the RVLM between intact and barodenervated rats – was compared, it was noticed that there was a larger increase in the rSNA after barodenervation (rSNA: barointact 90 T 15.6% and barode-
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Fig. 3. Grouped data showing the time courses of changes in renal sympathetic nerve activity (rSNA) in A and mean arterial pressure (MAP) in B, following bilateral microinjection of glutamate (10 nmol, 100 nl) into the CPA in control (n = 9) or barodenervated rats (n = 6). *P < 0.05 control versus barodenervated.
nervated 159 T 32%, P = 0,05). By contrast, a not significant difference in the peak of MAP responses was observed (MAP: barointact 46 T 7 and barodenervated 53 T 10 mm Hg see Fig. 4B). However, there was a statistical difference in the duration of MAP increase in response to glutamate into the RVLM in the barodenervated group when compared with the control group. The response’s duration was longer than the one observed in barointact rats (see Fig. 4B), the MAP pressure remained elevated above basal preinjection level for approximately 6 min in the barodenervated group.
In the control group, pressure returned to the basal level approximately in 1 min.
4. Discussion The present series of experiments showed that: 1) both ventrolateral regions of the medulla, CPA and RVLM, can increase arterial blood pressure and the renal sympathetic nerve activity with similar intensity; 2) despite the similarity
Fig. 4. Grouped data showing the time courses of changes in renal sympathetic nerve activity (rSNA) in A and mean arterial pressure (MAP) in B, following bilateral microinjection of glutamate (10 nmol, 100 nl) into the RVLM in control (n = 9) or in barodenervated rats (n = 6). *P < 0.05 control versus barodenervated.
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of the magnitude of responses on MAP and rSNA mediated by both regions, the time-course of changes was differential, long-lasting responses on rSNA and MAP were mediated by the CPA when compared to RVLM stimulation; 3) A baroreceptor mediated sympathetic inhibition was observed only when glutamate was injected into the RVLM, the inhibition disappeared after barodenervation; 4) The barodenervation caused larger increase in rSNA following glutamate microinjection into the RVLM but not into the CPA. The results suggest that the activation of CPA can cause large increases in the sympathetic activity and arterial blood pressure with a weaker baroreceptor modulation when compared to responses mediated by the RVLM neurons. The existence of pressor neurons in the caudal end of the caudal ventrolateral medulla was first demonstrated in the cat by Feldberg and Guertzenstein (1986). Subsequently, in experiments in rats, Gordon and McCann (1988) localized a pressor region in the caudal part of CVLM that was referred to as the CPA, from which significant pressor responses could be evoked by glutamate microinjections. Later, Campos et al. (1994) and Possas et al. (1994) showed that inhibition of CPA neurons causes a large fall in arterial pressure, indicating that the pressor neurons in the CPA are tonically active. More recently, Horiuchi and Dampney (2002) demonstrated for the first time in rabbits that the inhibition of CPA neurons by muscimol not only decreased arterial blood pressure but also almost abolished the rSNA. The purpose of the present study was to compare the actions mediated by the CPA and RVLM on rSNA and arterial blood pressure in the rat. We showed for the first time that CPA stimulation increases rSNA and MAP with a significant longer duration in comparison to RVLM actions. The barodenervation changed the sympathetic pattern of response only when the RVLM was stimulated but not when the CPA was activated, suggesting a differential baroreceptor sympathetic modulation in response to stimulation of CPA or RVLM neurons. Our results are in agreement with the hypothesis that CPA neurons may inhibit CVLM inhibitory neurons involved in the baroreceptor modulation (Campos et al., 1994). In this case, the sympathetic response to CPA stimulation is not greatly modulated by the baroreceptor reflex. In contrast, RVLM neurons may still be powerfully inhibited by GABAergic inputs, which have been activated by baroreceptor stimulation, even when glutamate is applied exogenously. However, only direct neuronal activity recording within CVLM barosensitive neurons during CPA activation or inhibition will be able to characterize the pathways and the way by which CPA neurons can influence the activity of RVLM premotor neurons. The first direct demonstration of the sympathetic effects of manipulation of the neurons activity in the CPA was related by Natarajan and Morrison (2000). They showed that unilateral microinjection of the excitatory amino acid dlhomocysteic acid (DLH) caused an increase in splancnic sympathetic nerve activity (peak + 25% of control). In our experiments, we found that the stimulation of CPA by
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glutamate increased sympathetic renal nerve discharge by 100% approximately, with a duration of around 60 s. It is possible that the difference between our results and the ones obtained by Natarajan and Morrison on the intensity of sympathetic responses, may be related to the differences in the sympathetic recorded bed (splancnic versus renal) or even in the way by which CPA was activated. They used DLH in a volume of 15 nl while we used a larger volume (100 nl) of glutamate. Another possibility is that CPA neurons can produce a differential sympathetic discharge, with its response is larger on renal than it is in the splancnic territory. In the present series of experiments, we observed that the renal sympathetic responses mediated by CPA were longer in relation to RVLM actions. The brief renal sympathetic activation in response to glutamate microinjection into the RVLM was observed previously; the increase in arterial blood pressure had a longer duration than the increase in the sympathetic activity followed by RVLM activation (McAllen, 1986b). We also showed that the short lasting response of sympathetic activation during RVLM stimulation is due to the baroreceptor modulation during the increase in arterial pressure. The difference in the time course of sympathetic activation and blood pressure responses between regions is probably not due to a larger sensitivity of CPA to lglutamate compared to RVLM. In a few experiments, glutamate was injected in a smaller volume and dose (5 nmol, 20nl) into the CPA or into the RVLM and the same pattern of activation was observed (data not shown). The pattern observed using small doses of glutamate also suggests that the short lasting responses mediated by 10 nmol of glutamate microinjection into the RVLM are not due to inhibition of the RVLM as a consequence of depolarization blockade. In the present study, no significant changes in HR were observed during baroreceptor activation by aortic constriction. The absence of bradycardia is probably due to the effects of urethane anesthesia. Shimokawa et al. (1998) showed in rats that urethane decreases the gain of baroreceptor reflex control of HR but does not change the reflex control of rSNA. The barodenervation increased the duration of MAP responses not only after the stimulation of the RVLM, but also in response to CPA stimulation. This is probably related to the fact that the arterial blood pressure responses during ventrolateral medulla stimulation are caused not only by sympathetic activation but also by the secretion of adrenal catecholamines and vasopressin (Ross et al., 1984). In this case, to differentiate the actions of the two VLM regions on cardiovascular regulation, the sympathetic responses are probably a more sensitive approach than the arterial blood pressure responses. The mechanisms by which CPA neurons affect sympathetic vasomotor tone are not very well understood. However, it is documented that the CPA actions are dependent on RVLM integrity. A previous study by Campos
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and McAllen (1999) in rats demonstrated that unilateral or bilateral inhibition of the CPA results in a significant inhibition of the firing rate of RVLM presympathetic neurons. Those observations therefore indicate that a significant proportion of the tonic activity of RVLM presympathetic neurons is dependent upon the tonic activity of CPA neurons. Recently, Horiuchi and Dampney (2002) in agreement with a previous study (Campos et al., 1994) found that microinjection of kynurenic acid into the RVLM, had no effect on the hypotension or on sympathoinhibition evoked by bilateral inhibition of CPA. The response, however, was blocked by previous microinjection of bicuculline into the RVLM, suggesting that bilateral inhibition of CPA results in a GABA-receptor-mediated inhibition of RVLM presympathetic neurons. Other evidence that CPA actions are mediated through the RVLM and CVLM neurons was the study of Sun and Panneton (2002) that showed anatomical projections from CPA to these regions. Our hypothesis is that CPA inhibits GABAergic interneurons within the CVLM neurons that receive excitatory inputs from baroreceptors. During CPA activation the baroreceptor reflex is less effective. 4.1. Perspectives Neurons in the CPA are not only a strong candidate for one of the sources of the synaptic drive that supports the resting activity of RVLM neurons, but they also may be involved in the organization of specific sympathetic responses. Specific cardiovascular adjustments as an increase in blood pressure and sympathetic activation, without a strong baroreceptor modulation such as observed in the emotional stress, can be in part organized by the actions of the CPA neurons. Projections from CPA to the caudal ventrolateral and to the rostral ventrolateral medulla can change the net sympathetic response and the baroreceptor reflex during acute stress.
Acknowledgements This study was sponsored by FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo). We also thank Dr. Robin M. McAllen for the helpful discussions concerning this study.
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Differential baroreceptor modulation mediated by the ventrolateral medulla Ca´ssia de Toledo Bergamaschi a, Bruno de Arruda Carillo a, Henrique Azevedo Futuro Neto b, Ruy Ribeiro de Campos a,* a
Departamento de Fisiologia, Universidade Federal de Sa˜o Paulo—Escola Paulista de Medicina, Rua Botucatu, 862, CEP 04023-060, Sa˜o Paulo, SP, Brazil b Universidade Federal do Espı´rito Santo and Escola Superior de Cieˆncias da Santa Casa de Miserico´rdia de Vito´ria (EMESCAM)—ES, Brazil Received 28 October 2005; received in revised form 7 February 2006; accepted 20 February 2006
Abstract Previous studies have shown that pharmacological stimulation of a region denominated caudal pressor area (CPA), located in the caudal end of the ventrolateral medulla, induces increases in arterial blood pressure (BP). The aim of this study was to compare the responses on renal sympathetic nerve activity (rSNA) and BP responses mediated by stimulation of CPA or rostral ventrolateral medulla (RVLM), in intact or sino-aortic barodenervated rats. Male Wistar rats (300 – 350g, n = 15) were anesthetized (urethane 1.2 to 1.4g/kg, i.v.) and artificially ventilated. The mean arterial pressure (MAP) and rSNA were measured during bilateral glutamate microinjection (10 nmo/100 nl) into the CPA or into the RVLM. Glutamatergic stimulation of the RVLM increased MAP (46 T 7 mm Hg) and rSNA (82 T 21%); during CPA stimulation, MAP and rSNA increased 60 T 7 mm Hg and 93 T 9%, respectively. However, despite the similarity of responses mediated by both regions, the duration of rSNA and blood pressure responses mediated by the CPA were significantly longer than the duration of the responses mediated by the RVLM. After barodenervation, there was an increase in the time-course and magnitude of sympathetic response only in response to RVLM stimulation but not in response to CPA. The results suggest a differential baroreceptor modulation on rSNA mediated by the ventrolateral medulla neurons. Glutamatergic activation of CPA neurons can cause large increases in the rSNA and BP with a weaker baroreceptor modulation when compared to responses mediated by the RVLM neurons. D 2006 Elsevier B.V. All rights reserved. Keywords: Baroreceptor; Caudal pressor area; Rostral ventrolateral medulla; Glutamate; Sympathetic activity
1. Introduction It is well known that the ventrolateral medulla (VLM) contains neurons involved in the tonic and reflex control of the cardiovascular system. Two regions within the VLM were initially identified: the rostral ventrolateral medulla (RVLM), which raises arterial blood pressure, as well as sympathetic nerve activity when stimulated, and the caudal ventrolateral medulla (CVLM), which acts in the opposite way. Both VLM regions are involved in the baroreceptor and chemoreceptor reflex integration (Guertzenstion and Silver, 1974; Guertzenstein and Lopes, 1984; Dampney, 1994a,b). * Corresponding author. Tel.: +55 11 5573 7820; fax: +55 11 5573 7820. E-mail address: [email protected] (R.R. de Campos). 1566-0702/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2006.02.012
The RVLM premotor neurons project directly to sympathetic preganglionic neurons and are involved in the maintenance of resting sympathetic vasomotor tone (Dampney et al., 2003). A significant proportion of tonic activity in the RVLM sympathetic premotor neurons is driven by neurons located in a third region of the VLM denominated caudal pressor area (CPA) (Campos and McAllen, 1999). The CPA is located caudal to the CVLM depressor neurons in the caudal end of VLM and, when activated, increases arterial blood pressure in anesthetized and in conscious rats (Gordon and McCann, 1988; Campos et al., 1994; Silva et al., 2001). Inhibition of neuronal activity in the CPA by glycine microinjection induces hypotension and decreases the activity of the RVLM neurons by approximately 40–
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45% while glutamatergic stimulation causes increase in the RVLM activity, sympathetic nerve activity and blood pressure (Campos and McAllen, 1999; Natarajan and Morrison, 2000). However, there is no information about the characteristic of sympathetic activation mediated by the CPA neurons. This raises the following question: the activation of the CPA or RVLM neurons by microinjections of excitatory amino acid into the CPA or into the RVLM may produce differential actions on sympathetic nerve activity? In the present study, we investigated the pattern of renal sympathetic activation mediated by the CPA compared to the RVLM mediated responses. To excite cell bodies within the CPA and RVLM, we used local microinjections of glutamate, which have been shown to produce pressor responses when injected in both regions (McAllen, 1986a; Gordon and McCann, 1988). There is no information regarding how the baroreceptor reflex can modulate the sympathetic nerve activity during the increase in arterial blood pressure in response to glutamatergic stimulation of the CPA neurons. One hypothesis to explain the sympathoexcitatory actions of CPA is an inhibitory pathway from the CPA to the CVLM. According to this hypothesis, during CPA stimulation there is an inhibition of the baroreceptor reflex, causing an acute increase in the sympathetic nerve activity and blood pressure (Campos et al., 1994). In this case, it is reasonable to suppose that the sympathetic activation mediated by the CPA is less dependent of baroreceptor modulation compared to the RVLM actions. Thus, we tested this hypothesis, comparing the sympathoexcitatory and hypertensive actions mediated by the CPA with the responses mediated by the RVLM in intact and in barodenervated rats. The first aim of the present study (first series of experiments) was to determine the extent by which glutamatergic stimulation of CPA neurons can activate the renal sympathetic nerve activity (rSNA) in comparison to the RVLM mediated responses. The second aim (second series of experiments) was to determine the effects of acute sino-aortic barodenervation on sympathetic and blood pressure responses mediated by the CPA and RVLM glutamatergic stimulation.
2. Materials and methods 2.1. First series of experiments 2.1.1. General procedures Experiments were performed in male Wistar rats (300 – 350 g, n = 9). The experiments were approved by the Animal Experimental Ethics Committee of the Federal University of Sa˜o Paulo—Escola Paulista de Medicina. The rats were initially anesthetized with halothane (2%) and instrumented with femoral venous and arterial catheters for
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blood pressure (BP) and heart rate (HR) recordings. The animals were then anesthetized very slowly with urethane (1.4 g/kg, i.v.) and artificially ventilated with oxygen enriched air with a respiratory pump at a level that maintained end-tidal CO2 close to 3.5 –4%. Rectal temperature was maintained at 37 T 0.5 C by a servo-controlled electric blanket. The rat was then mounted prone in a stereotaxic frame with the head mildly ventroflexed (bite bar at 11 mm). The occipital bone was opened over the dorsal medulla and caudal cerebellum, and the dura was retracted. The anesthetic level was tested during the experiments (withdrawal reflexes to noxious pinching) and extra urethane (0.1 –0.5 g/kg, i.v.) was given when it was needed. A pneumatic cuff was placed around the lower thoracic aorta, via a small left intercostal incision in order to artificially increase BP and activate baroreceptor reflex. 2.1.2. Renal sympathetic nerve recording The left renal sympathetic nerve was exposed through a left retroperitoneal flank incision placed on bipolar recording electrodes and was covered with mineral oil. The signal from the renal nerve was displayed on an oscilloscope and monitored by means of an audio amplifier. The nerve activity was also amplified (Neurolog, 10 to 20K) and filtered by a band-pass filter (50 –1000 Hz) and was rectified and integrated every 1 s. At the end of the experiments, the baseline noise level of renal sympathetic nerve activity (rSNA) was determined after the administration of hexamethonium bromide (30mg/kg, i.v.). The change in nerve discharge during the experiment was expressed by a percentage from the basal value subtracting the background noise. The mean arterial pressure (MAP) and HR signals were derived from the pulsatile BP. All signals were recorded on a computer-based data acquisition system (Power Lab system, AD Instruments, NSW, Australia). 2.1.3. Drugs intramedullary microinjections Glass micropipettes of tip diameter of approximately 20 Am were used to pressure injected l-glutamate (monosodium salt, 10nmol/100 nl) into the CPA or into the RVLM in a random way. The solution was prepared in normal saline (pH 7.0 – 7.4). Glutamate was injected in 5 to 10 s sequentially in one side and in the contraleral region; interval between injection sides was around of 10 s. The pipettes were held in a vertical position and the CPA was located 1 mm caudal to the Obex, 1.5 –1.6 mm lateral to the midline, and 2mm deep to the dorsal medullary surface (Campos and McAllen, 1999). The RVLM was located 3 mm rostral to Obex, 1.7 – 1.8 mm lateral to midline, and 3mm deep. Injections sites were marked at the end of the experiments by subsequently injecting 50 to 100 nl of Evans blue. In three experiments, the vehicle (saline) was injected into the RVLM or CPA as a control injection.
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2.2. Second series of experiments
2.4. Statistical analysis
In six rats, before to start the surgical procedures as above described for rSNA recordings and intramedullary microinjections, a sino-aortic denervation (SAD) was performed according to standard methods (Krieger, 1970). A pneumatic cuff was placed around the lower thoracic aorta and the baroreceptor activation in response to a brief aortic occlusion did not cause any decrease in the rSNA or HR in barodenervated rats. After the end of the surgical preparation, the animals were positioned in the streotaxic frame and glutamate microinjections into the RVLM or CPA were performed in this group in a random way, using the same dose of the first series of experiments. The responses on BP and rSNA mediated by glutamate microinjections into the two regions were then recorded for further off-line analysis.
All values are expressed as mean T S.E.M. The changes in MAP, HR and percentage of rSNA responses during RVLM or CPA activation were analyzed in intervals of 1 s after microinjection in relation to basal condition by paired and unpaired t-test. The differences on MAP, rSNA and HR between CPA and RVLM mediated responses were assessed by one-way analysis of variance (ANOVA) followed by the Student – Newman – Keuls method. Differences were considered significant at P < 0.05.
2.3. Histology At the end of the experiments, animals were killed by an intravenous overdose of urethane. The brain stem was then removed and fixed by immersion for at least 24h in 10% paraformaldehyde solution, after which coronal sections (40 Am) were cut on a freezing microtome, the sections were mounted on glass slices and stained with neutral red. Microinjection sites were identified by deposition of Evans blue. Fig. 1 shows the distribution of the centers of sites at which glutamate (10 nmol/100 nl) was injected into the RVLM or CPA. A positive location for the RVLM was considered when the dye was deposited ventral to the nucleus ambiguous and lateral to the inferior olive nucleus. The CPA was located in the caudal end of the lateral reticular nucleus of the ventrolateral medulla as described in previous investigations (Campos and McAllen, 1999; Possas et al., 1994).
Fig. 1. Distribution of the centers of sites of glutamate microinjections (10 nmol/100nl) in the caudal pressor area (CPA) 1 mm caudal to the Obex or in the rostral ventrolateral medulla (RVL) 3mm rostral to the Obex. Abbreviations: X, nucleus ambiguous (compact formation); V, spinal trigeminal tract; Gr, nucleus gracilis; Pyx, pyramidal decussation.
3. Results First, the rSNA was tested in relation to baroreceptor reflex activation. A brief aortic constriction increased MAP from 107 T 3.5 to 154 T 5mm Hg and decreased HR (from 382 T 18 to 375 T 21bpm) and rSNA by 78 T 9% in the barointac group (n = 9). In the acute baroreceptor denervated rats (n = 6), a similar increase in MAP in response to aortic constriction did not cause any change in the rSNA or HR. 3.1. Effect of bilateral microinjection of glutamate into the CPA or RVLM on rSNA and arterial blood pressure in nonbarodenervated rats Glutamate microinjections were performed at random into the RVLM or CPA in the same animal (n = 9) with an interval time of at least 30 min, when the sympathetic nerve activity and blood pressure had returned to the basal preinjection level. Following bilateral microinjection of glutamate into the CPA, there was an immediate increase in MAP and rSNA by 60 T 7.3mm Hg (basal 100 T 3.5 mm Hg, n = 9, P < 0.05) and 93 T 9.3% ( P < 0.05) respectively, compared to the preinjection control levels. The increase in rSNA was immediate, reaching a peak around 7 s and remained increased above basal level for approximately 1 min, as shown in Fig. 2A. The arterial blood pressure increased in response to microinjection and reached the peak approximately 10 s after injection and gradually returned to the basal level (Fig. 2B). There was no significant change in HR (from 392 T 15 to 416 T 16 bpm). When glutamate was microinjected into the RVLM, there was an immediate increase in MAP and rSNA of 46 T 7 mm Hg, P < 0.05 (basal 108 T 7 mm Hg) and 82 T 21% ( P < 0.05) respectively, compared to control values. The peak of rSNA response was in the fifth second followed by an abrupt reduction in the nerve activity after the seventh second of injection (from 81 T12% to 38 T 15% in relation to basal level), as shown Fig. 2A. During RVLM injections it was observed no significant change in HR (from 379 T 19 to 394 T 20bpm).
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Fig. 2. Grouped data showing the time courses of changes in renal sympathetic nerve activity (rSNA) in A and mean arterial pressure (MAP) in B, following bilateral microinjection of glutamate (10nmol, 100nl) into the CPA or RVLM. *P < 0.05. Comparison between CPA and RVLM mediated responses (n = 9).
When the peak of maximal changes of the MAP and rSNA in response to glutamate injection into the CPA or RVLM was compared, there was no difference between regions. Nevertheless, the sympathetic increase (percentage of increase) mediated by CPA remained elevated, above basal preinjection levels whose duration was longer than the duration of the responses mediated by the RVLM, as shown in Fig. 2A. The sympathetic response mediated by the RVLM had an abrupt decrease after the seventh second of microinjection; this was not observed when CPA was activated. Time-course of changes in the arterial blood pressure in response to stimulation of CPA or RVLM was also compared and it was observed that CPA activation caused a significant longer lasting response on MAP when compared to the MAP increase mediated by the RVLM, as shown in Fig. 2B. In three experiments, saline was bilaterally injected into the CPA or RVLM, no responses on sympathetic activity or arterial blood pressure were observed. 3.2. Effect of bilateral microinjection of glutamate into the CPA or RVLM on rSNA and arterial blood pressure in sinoaortic barodenervated rats In a group of six sino-aortic barodenervated rats, bilateral glutamate microinjection into the CPA increased MAP and rSNA in 78 T 9 mm Hg, P < 0.05 (basal, 113 T 12 mm Hg) and 112 T 24% ( P < 0.05), respectively. The response of sympathetic activity was immediate and remained above basal preinjection level for approximately 1 min (Fig. 3A).
When the responses on MAP and rSNA during CPA stimulation between intact and barodenervated rats were compared, there was no significant difference in the peak of changes for both variables. There was also no change in the time course of rSNA increase in response to glutamate into the CPA after barodenervation (Fig. 3A). However, in relation to the arterial blood pressure increase in response to CPA stimulation in barodenervated rats, there was a longer lasting response in relation to control group, with statistical significance (see Fig. 3B). In this group, the MAP remained elevated above basal level for approximately 4 min and gradually returned to the preinjection level. In the control non-barodenervated group, MAP returned to the basal level in 2 min approximately. Bilateral microinjection of glutamate into the RVLM in these barodenervated rats, caused an increase in MAP and rSNA in 55 T 6 mm Hg ( P < 0.05) and 159 T 32% ( P < 0.05), respectively (Fig. 4). The increase in sympathetic activity was immediate and reached a peak in 7 s and gradually returned to the basal level. In contrast to what had been observed in baro-intact rats, there was no abrupt reduction in the rSNA after the peak of sympathetic activation after barodenervation (see Figs. 2A and 4A), indicating that the sympathetic reduction was mediated by baroreceptor reflex. When the peak of changes on rSNA and MAP– in response to glutamate injection into the RVLM between intact and barodenervated rats – was compared, it was noticed that there was a larger increase in the rSNA after barodenervation (rSNA: barointact 90 T 15.6% and barode-
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Fig. 3. Grouped data showing the time courses of changes in renal sympathetic nerve activity (rSNA) in A and mean arterial pressure (MAP) in B, following bilateral microinjection of glutamate (10 nmol, 100 nl) into the CPA in control (n = 9) or barodenervated rats (n = 6). *P < 0.05 control versus barodenervated.
nervated 159 T 32%, P = 0,05). By contrast, a not significant difference in the peak of MAP responses was observed (MAP: barointact 46 T 7 and barodenervated 53 T 10 mm Hg see Fig. 4B). However, there was a statistical difference in the duration of MAP increase in response to glutamate into the RVLM in the barodenervated group when compared with the control group. The response’s duration was longer than the one observed in barointact rats (see Fig. 4B), the MAP pressure remained elevated above basal preinjection level for approximately 6 min in the barodenervated group.
In the control group, pressure returned to the basal level approximately in 1 min.
4. Discussion The present series of experiments showed that: 1) both ventrolateral regions of the medulla, CPA and RVLM, can increase arterial blood pressure and the renal sympathetic nerve activity with similar intensity; 2) despite the similarity
Fig. 4. Grouped data showing the time courses of changes in renal sympathetic nerve activity (rSNA) in A and mean arterial pressure (MAP) in B, following bilateral microinjection of glutamate (10 nmol, 100 nl) into the RVLM in control (n = 9) or in barodenervated rats (n = 6). *P < 0.05 control versus barodenervated.
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of the magnitude of responses on MAP and rSNA mediated by both regions, the time-course of changes was differential, long-lasting responses on rSNA and MAP were mediated by the CPA when compared to RVLM stimulation; 3) A baroreceptor mediated sympathetic inhibition was observed only when glutamate was injected into the RVLM, the inhibition disappeared after barodenervation; 4) The barodenervation caused larger increase in rSNA following glutamate microinjection into the RVLM but not into the CPA. The results suggest that the activation of CPA can cause large increases in the sympathetic activity and arterial blood pressure with a weaker baroreceptor modulation when compared to responses mediated by the RVLM neurons. The existence of pressor neurons in the caudal end of the caudal ventrolateral medulla was first demonstrated in the cat by Feldberg and Guertzenstein (1986). Subsequently, in experiments in rats, Gordon and McCann (1988) localized a pressor region in the caudal part of CVLM that was referred to as the CPA, from which significant pressor responses could be evoked by glutamate microinjections. Later, Campos et al. (1994) and Possas et al. (1994) showed that inhibition of CPA neurons causes a large fall in arterial pressure, indicating that the pressor neurons in the CPA are tonically active. More recently, Horiuchi and Dampney (2002) demonstrated for the first time in rabbits that the inhibition of CPA neurons by muscimol not only decreased arterial blood pressure but also almost abolished the rSNA. The purpose of the present study was to compare the actions mediated by the CPA and RVLM on rSNA and arterial blood pressure in the rat. We showed for the first time that CPA stimulation increases rSNA and MAP with a significant longer duration in comparison to RVLM actions. The barodenervation changed the sympathetic pattern of response only when the RVLM was stimulated but not when the CPA was activated, suggesting a differential baroreceptor sympathetic modulation in response to stimulation of CPA or RVLM neurons. Our results are in agreement with the hypothesis that CPA neurons may inhibit CVLM inhibitory neurons involved in the baroreceptor modulation (Campos et al., 1994). In this case, the sympathetic response to CPA stimulation is not greatly modulated by the baroreceptor reflex. In contrast, RVLM neurons may still be powerfully inhibited by GABAergic inputs, which have been activated by baroreceptor stimulation, even when glutamate is applied exogenously. However, only direct neuronal activity recording within CVLM barosensitive neurons during CPA activation or inhibition will be able to characterize the pathways and the way by which CPA neurons can influence the activity of RVLM premotor neurons. The first direct demonstration of the sympathetic effects of manipulation of the neurons activity in the CPA was related by Natarajan and Morrison (2000). They showed that unilateral microinjection of the excitatory amino acid dlhomocysteic acid (DLH) caused an increase in splancnic sympathetic nerve activity (peak + 25% of control). In our experiments, we found that the stimulation of CPA by
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glutamate increased sympathetic renal nerve discharge by 100% approximately, with a duration of around 60 s. It is possible that the difference between our results and the ones obtained by Natarajan and Morrison on the intensity of sympathetic responses, may be related to the differences in the sympathetic recorded bed (splancnic versus renal) or even in the way by which CPA was activated. They used DLH in a volume of 15 nl while we used a larger volume (100 nl) of glutamate. Another possibility is that CPA neurons can produce a differential sympathetic discharge, with its response is larger on renal than it is in the splancnic territory. In the present series of experiments, we observed that the renal sympathetic responses mediated by CPA were longer in relation to RVLM actions. The brief renal sympathetic activation in response to glutamate microinjection into the RVLM was observed previously; the increase in arterial blood pressure had a longer duration than the increase in the sympathetic activity followed by RVLM activation (McAllen, 1986b). We also showed that the short lasting response of sympathetic activation during RVLM stimulation is due to the baroreceptor modulation during the increase in arterial pressure. The difference in the time course of sympathetic activation and blood pressure responses between regions is probably not due to a larger sensitivity of CPA to lglutamate compared to RVLM. In a few experiments, glutamate was injected in a smaller volume and dose (5 nmol, 20nl) into the CPA or into the RVLM and the same pattern of activation was observed (data not shown). The pattern observed using small doses of glutamate also suggests that the short lasting responses mediated by 10 nmol of glutamate microinjection into the RVLM are not due to inhibition of the RVLM as a consequence of depolarization blockade. In the present study, no significant changes in HR were observed during baroreceptor activation by aortic constriction. The absence of bradycardia is probably due to the effects of urethane anesthesia. Shimokawa et al. (1998) showed in rats that urethane decreases the gain of baroreceptor reflex control of HR but does not change the reflex control of rSNA. The barodenervation increased the duration of MAP responses not only after the stimulation of the RVLM, but also in response to CPA stimulation. This is probably related to the fact that the arterial blood pressure responses during ventrolateral medulla stimulation are caused not only by sympathetic activation but also by the secretion of adrenal catecholamines and vasopressin (Ross et al., 1984). In this case, to differentiate the actions of the two VLM regions on cardiovascular regulation, the sympathetic responses are probably a more sensitive approach than the arterial blood pressure responses. The mechanisms by which CPA neurons affect sympathetic vasomotor tone are not very well understood. However, it is documented that the CPA actions are dependent on RVLM integrity. A previous study by Campos
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and McAllen (1999) in rats demonstrated that unilateral or bilateral inhibition of the CPA results in a significant inhibition of the firing rate of RVLM presympathetic neurons. Those observations therefore indicate that a significant proportion of the tonic activity of RVLM presympathetic neurons is dependent upon the tonic activity of CPA neurons. Recently, Horiuchi and Dampney (2002) in agreement with a previous study (Campos et al., 1994) found that microinjection of kynurenic acid into the RVLM, had no effect on the hypotension or on sympathoinhibition evoked by bilateral inhibition of CPA. The response, however, was blocked by previous microinjection of bicuculline into the RVLM, suggesting that bilateral inhibition of CPA results in a GABA-receptor-mediated inhibition of RVLM presympathetic neurons. Other evidence that CPA actions are mediated through the RVLM and CVLM neurons was the study of Sun and Panneton (2002) that showed anatomical projections from CPA to these regions. Our hypothesis is that CPA inhibits GABAergic interneurons within the CVLM neurons that receive excitatory inputs from baroreceptors. During CPA activation the baroreceptor reflex is less effective. 4.1. Perspectives Neurons in the CPA are not only a strong candidate for one of the sources of the synaptic drive that supports the resting activity of RVLM neurons, but they also may be involved in the organization of specific sympathetic responses. Specific cardiovascular adjustments as an increase in blood pressure and sympathetic activation, without a strong baroreceptor modulation such as observed in the emotional stress, can be in part organized by the actions of the CPA neurons. Projections from CPA to the caudal ventrolateral and to the rostral ventrolateral medulla can change the net sympathetic response and the baroreceptor reflex during acute stress.
Acknowledgements This study was sponsored by FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo). We also thank Dr. Robin M. McAllen for the helpful discussions concerning this study.
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