Fos expression in the NTS in response to peripheral chemoreflex activation in awake rats

Autonomic Neuroscience: Basic and Clinical 152 (2010) 27–34

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Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u

Fos expression in the NTS in response to peripheral chemoreflex activation in awake rats Josiane de Campos Cruz a, Leni G.H. Bonagamba a, Javier E. Stern b, Benedito H. Machado a,⁎ a b

Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, 14049-900, Ribeirão Preto, SP, Brazil School of Medicine, Department of Physiology, Medical College of Georgia, Augusta, GA, USA

a r t i c l e

i n f o

Article history: Received 2 July 2009 Received in revised form 1 August 2009 Accepted 25 August 2009 Keywords: Chemoreflex Baroreflex Fos protein Nucleus tractus solitarii Cardiovascular regulation

a b s t r a c t Chemoreflex afferent fibers terminate in the nucleus tractus solitarii (NTS), but the specific location of the NTS neurons excited by peripheral chemoreflex activation remains to be characterized. Here, the topographic distribution of chemoreflex sensitive cells at the commissural NTS was evaluated. To reach this goal, Fosimmunoreactive neurons (Fos-ir) were accounted in rostro-caudal levels of the intermediate and caudal commissural NTS, after intermittent chemoreflex activation with intravenous injection of potassium cyanide [KCN (80 μg/kg) or saline (0.9%, vehicle), one injection every 3 min during 30 min]. In response to intermittent intravenous injections of KCN, a significant increase in the number of Fos-ir neurons was observed specifically in the lateral intermediate commissural NTS [LINTS (82 ± 9 vs. 174 ± 16, cell number mean per section)] and lateral caudal commissural NTS [LCNTS (71 ± 9 vs. 199 ± 18, cell number mean per section)]. To evaluate the influence of baroreceptor-mediated inputs following the increase in blood pressure during intermittent chemoreflex activation, we performed an intermittent activation of the arterial baroreflex by intravenous injection of phenylephrine [1.5 μg/kg iv (one injection every 3 min during 30 min)]. This procedure induced no change in Fos-ir in LINTS (64 ± 6 vs. 62 ± 12, cell number mean per section) or LCNTS (56 ± 15 vs. 77 ± 12, cell number mean per section). These data support the involvement of the commissural NTS in the processing of peripheral chemoreflex, and provide a detailed characterization of the topographical distribution of activated neurons within this brain region. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The arterial chemosensitive cells located in the carotid body, at the bifurcation of the carotid arteries, are very sensitive to arterial PO2 reduction (Biscoe and Dunchen, 1990). The carotid sinus nerves convey primary chemoreflex afferent to the nucleus of the solitary tract [NTS (Spyer, 1990)] where the information is integrated through local neuronal circuits producing cardiovascular, respiratory and behavioral adjustment. Anatomical studies evidenced that the peripheral chemoreceptor afferent from carotid body fibers conveys to the commissural regions of the NTS (Finley and Katz, 1992) and several functional studies have shown chemosensitive cell distribution along the commissural NTS. In this regard, Fos immunohistochemistry has been widely used as a metabolic marker for neuronal transynaptic activation, to map neurons at the NTS level activated by hypoxiahypoxic approach. Studies performed by Erickson and Millhorn (1991) have shown a Fos-like protein expression in the commissural NTS in response to stimulation of afferent fibers of the carotid sinus nerve (CSN), as well as to different physiological carotid body stimulation,

⁎ Corresponding author. Tel.: +55 16 602 3015; fax: +55 16 633 0017. E-mail address: [email protected] (B.H. Machado). 1566-0702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2009.08.016

such as moderate hypoxia (Erickson and Millhorn, 1991, Berquim et al., 2000ab; Hirooka et al., 1997, Teppema et al., 1997), chronic intermittent hypoxia (Greenberg et al., 1999), anoxia [100% N2 (Miura et al., 1994)] and/or after carbon monoxide inhalation (Bodineau and Larnicol, 2001). The involvement of the commissural NTS in the processing of chemoreflex responses has been studied by different methodological techniques and potassium cyanide (KCN, iv) has been used as a tool for peripheral chemoreflex activation (Haibara et al 1995, 1999; Braga et al., 2007, Cruz et al., 2008, Cruz and Machado, 2009, Granjeiro and Machado, 2009). KCN (iv) promotes a cytotoxic-hypoxia at the glomus cells, producing prominent cardiorespiratory adjustments and behavioral responses, which are similar to those evoked by hypoxic-hypoxia activation in awake rats (Bao et al., 1997; Barros et al. 2002). Although, KCN (iv) has been widely used in studies involving peripheral chemoreflex neurotransmission at the NTS level, the functional mapping of the neurons in the NTS activated after cytotoxic-hypoxia remains to be characterized. In the present study, we used Fos immunohistochemical labeling to characterize the distribution of the NTS subnuclei excited by peripheral chemoreflex activation using intermittent injection of KCN (iv), similar to the paradigm used by Cruz et al. (2008) who evaluated chemoreflex induced Fos-ir in the various paraventricular nucleus (PVN) subnuclei.

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Hence, Fos-ir positive neurons in response to intermittent chemoreflex activation (KCN, every 3 min) during 30 min in awake rats were quantified in six antero-posterior sections of the intermediate commissural NTS (starting at −13.59 mm caudal to the bregma) and in six antero-posterior sections of the caudal commissural NTS (starting at bregma −13.14 mm caudal to the bregma) apart by 90 μm. 2. Methods 2.1. Animals Wistar male rats weighing 270–310 g were used in the present study. All animals were kept in a normal 12-h light (6 AM to 6 PM) to 12-h dark (6 PM to 6 AM) cycle. All experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals and Ethical Principles for Animal Experimentation established by the Brazilian Committee for Animal Experimentation and approved by the Animal Care and Ethics Committee of the School of Medicine of Ribeirão Preto, University of São Paulo (Protocol #057/ 2003). The number of animals used, as well as their suffering, was minimized as much as possible. 2.2. Artery and vein catheterization One day before the experiments, under tribromoethanol anesthesia [(i.p.) Aldrich Chemical Co., Milwaukee, WI, USA] a catheter [PE-10 connected to PE-50 (Clay Adams, Parsippany, NJ, USA)] was inserted into the right femoral artery for measurement of pulsatile arterial pressure (PAP), mean arterial pressure (MAP), and heart rate (HR). A second catheter was inserted into the right femoral vein for systemic administration of drugs. The catheters were tunneled subcutaneously and exteriorized through the back of the neck to be connected to the pressure transducer on the subsequent day. 2.3. Cardiovascular records PAP was measured with a pressure transducer (model CDX III, Cobe Laboratories, Lakewood, CO) connected to a polygraph (Narcotrace 80, Narco Bio-Systems, Austin, TX). MAP was derived from the PAP using a universal coupler (model 7179, Narco Bio-Systems), and HR was quantified from the PAP using a Biotachometer Coupler (model 7302, Narco Bio-Systems). 2.4. Experimental procedures for chemoreflex intermittent activation Twenty-four hours after the catheter implantation, the cardiovascular parameters were recorded simultaneously in two awake rats with free movement and submitted to the same environmental conditions. In one rat an intermittent intravenous injection of KCN [160 µg/kg in a volume of 50 µL/dissolved in saline (0.9%)] was performed for stimulation of the chemoreflex and the other received an intermittent injection of the saline (0.9%) as vehicle control. Before the onset of the experimental protocols the rats of both groups received an intravenous injection of KCN to verify if the venous catheter was patent and to check the magnitude of the cardiovascular chemoreflex response. In case the magnitude of the cardiovascular responses was not similar to the pattern usually observed in our experiments (Cruz et al., 2008; Cruz and Machado, 2009) the animal was not considered in the data analyses. It is important to mention that no changes in NTS Fos-ir were observed following a single injection of KCN used to test the venous catheter in both saline and KCN groups (data not shown). Ten minutes after testing the catheter with KCN, each animal received a bolus intravenous injection of KCN or saline every 3 min during 30 min, a protocol previously demonstrated to be effective to increase the number of FOS positive neurons after intermittent activation of the chemoreflex (Cruz et al., 2008).

2.5. Experimental procedure for intermittent activation of baroreflex This protocol was performed to verify whether Fos-ir in the NTS in response to chemoreflex activation was, at least in part, secondary to the baroreflex activation due to a large increase in MAP produced after chemoreflex activation. Using an α1-adrenoceptor agonist, phenylephrine [Sigma, St Louis, MO, USA (6 µg/50 µL, dissolved in saline)], the baroreflex was activated every 3 min during 30 min, similarly to the chemoreflex protocol. The dose of phenylephrine used was sufficient to increase mean arterial pressure to levels similar to those produced by chemoreflex activation. Control rats, recorded simultaneously, received an intravenous injection of the same volume of saline (50 µL, iv) every 3 min during 30 min. 2.6. Experimental procedure for continuous activation of the baroreflex An experimental protocol related to the continuous activation of the arterial baroreceptors was performed in order to compare its effect with the intermittent activation of the arterial baroreceptors on the Fos-ir in the NTS. In this case, baroreflex activation was performed by continuous infusion of phenylephrine (0.75 μg/0.03 ml/min) during 30 min by using an infusion pump in order to keep MAP approximately 50 mmHg above the baseline for 30 min. Volume control was performed by saline 0.9% (0.03 ml/min) infusion during 30 min. 2.7. Tissue processing Previous studies showed that Fos-ir assessed 60 min after the last chemoreflex activation (i.e. 90 min following the first activation) produces a similar Fos-ir when compared to 30 min [i.e. 60 min following the first activation (Cruz et al, 2008)]. Thus, in the present study Fos immunohistochemistry protocols were assessed 30 min after the last chemoreflex activation. The rats were deeply anesthetized with thionembutal® (0.1 ml/100 mg i.p., Abbott Laboratories, North Chicago, USA) and transcardially perfused with 0.1 M phosphate buffered saline (PBS, 50 ml) followed by 4% paraformaldehyde (PFA, 500 ml). The brains were post fixed in sucrose 20% dissolved in 4% PFA solution overnight, and then cryoprotected with 0.1 M PBS containing 30% sucrose, for at least 48 h. 2.8. Immunocytochemistry Coronal brain sections (30 μm) were cut using a cryostat (Micron, International GmbH, Waldorf, Germany), and sequentially divided into three groups: experimental, control (omission of the primary antibody), and Nissl staining (for better identification of NTS anteroposterior levels). The brain sections were incubated in normal goat serum [(1:250), Jackson Immunoresearch Laboratories, West Grove MO, USA] for 2 h. The sections were then incubated overnight in the presence of a rabbit anti-Fos antibody [(1:4000), sc-52, Santa Cruz Biotechnology, Santa Cruz, CA], followed by a 2 h incubation in the presence of a goat anti-rabbit biotinylated antibody [(1:500), Jackson Immunoresearch Laboratories, West Grove MO, USA]. The sections were then processed with the avidin–biotin complex (ABC-Standart, 1:50; Vector Laboratories, Burlingame, CA, USA), and the immunoreactivity visualized by H2O2 30% and the diaminobenzidine reaction (DAB, Sigma, St. Louis, MO, USA) for 5 min. 2.9. Image analysis The immunoreactivity in the brain sections was visualized using a microscope [Axioskop (Carl Zeiss, Germany)] connected to a digital camera Axiocam HRc (Carl Zeiss, Vision GmbH, Germany). Images were captured using a computerized AxioVision 3.0 system (Carl Zeiss, Vision GmbH, Germany). Fos-ir neurons were identified by the

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Fig. 1. Tracings showing changes in heart rate (HR), pulsatile arterial pressure (PAP) and mean arterial pressure (MAP) in response to the first (at the time 0) and the last (at 30 min) injection of saline (iv), potassium cyanide (KCN, iv) and phenylephrine (phenyl, iv) in awake rats representative of their respective groups. The injections (iv) were performed every 3 min during 30 min.

presence of dark, nuclear staining, and were manually counted under 5 times magnification. Fos-ir neurons were systematically quantified within specific rostro-caudal coordinates at a 90 μm interval from the area postrema (intermediate commissural NTS) and calamus scriptorium levels [caudal commissural NTS (Paxinos and Watson, 1986)]. Therefore, in the intermediate commissural NTS (lateral aspect) the Fos-ir was quantified in six rostro-caudal levels (from −13.6 to 14.05 mm caudal to the bregma). In the lateral caudal commissural NTS (lateral aspect), Fos-ir neurons were quantified in six rostrocaudal levels (from −14.13 to −14.58 mm caudal to the bregma). On average, 7–12 sections (30 μm) were counted on each rostrocaudal bregma level of the intermediate commissural and caudal NTS, and, the mean number of positive Fos-ir per section per subnucleus region was calculated within each experimental group for quantitative purposes (Cruz et al., 2008).

ephrine. KCN and phenylephrine injection induced pressor and bradycardic responses, while injection of saline produced no cardiovascular changes. As summarized in Table 1, the magnitude of the pressor response to intermittent phenylephrine injection was similar to that produced by intermittent injection of KCN and the magnitude of the pressor response to the first (0 min) injection of KCN or phenylephrine was similar to the responses produced by the last injection at 30 min. Table 1 also shows the baseline values of MAP and HR before the first and after the last intravenous injection of KCN, phenylephrine or saline in the respective experimental groups. 3.2. Fos-ir in the lateral intermediate and caudal commissural NTS of rats submitted to intermittent chemoreflex activation Fig. 2 shows representative examples of Fos-ir in the LINTS and LCNTS of rats submitted to intermittent injection of saline (panels A1 and B1) or KCN (panels A2 and B2) injections. Two-way ANOVA indicated significant increase in the number of Fos-ir cells in the NTS as a function of the treatment [saline × KCN (F1.36 =63, p b 0.0001)] but not in the interaction (F1.36 =1.7, p =0.2) or sub-region (F1.36 =0.2, p= 0.6). As summarized in Fig. 3, Bonferroni post-hoc indicated a similar and significant increase in the number of Fos-ir in the LINTS [82±9 vs. 174±16, pb 0.001 (n= 10)] and LCNTS [71 ±8 vs. 199 ±19, p b 0.001 (n=10)] after the intermittent chemoreflex activation.

2.10. Statistics analysis To compare changes in Fos-ir according to experimental group (i.e. saline- vs. KCN-treated) and topographical distribution within the NTS, the mean number of positive Fos-ir per section per subnucleus region was evaluated within each experimental group by two-way ANOVA, followed by Bonferroni post-hoc test. The magnitude of the cardiovascular changes obtained in the first and in the last activation of chemoreflex or the baroreflex was also compared using the twoway ANOVA followed by Bonferroni post-hoc test (p b 0.05). All values are reported as means ± SEM.

3.3. Fos-ir in the lateral intermediate and caudal commissural NTS of rats submitted to intermittent baroreflex activation

3. Results Fig. 4 shows representative examples of Fos-ir in the LINTS and LCNTS of rats submitted to intermittent saline (panels A1 and B1) or phenylephrine (panels A2 and B2) injections. Two-way ANOVA indicated no significant changes to Fos-ir cells in the NTS in relation to treatment [saline × phenyl (F1,17 =1.2, p =0.3)], interaction (F1,17 = 1.6, p =0.2) or sub-region (F1,17 = 0.1, p = 0.7). As summarized in Fig. 5,

3.1. Cardiovascular responses elicited by intermittent chemoreflex and baroreflex activation in awake rats Fig. 1 shows tracings of three rats representative of the respective groups submitted to intermittent injection of saline, KCN or phenyl-

Table 1 Baseline MAP, HR and pressor and bradycardic responses in the first (0 min) and the last (30 min) intravenous injection of KCN, phenylephrine and their respective controls (saline) performed every 3 min during 30 min. First injection (0 min)

KCN Saline Phenyl Saline

Last injection (30 min)

N

Basal MAP

ΔMAP

Basal HR

ΔHR

Basal MAP

ΔMAP

Basal HR

ΔHR

10 10 5 5

106 ± 3 102 ± 4 97 ± 3 103 ± 3

56 ± 5* 2±1 50 ± 5* 3±4

390 ± 19 378 ± 16 368 ± 25 362 ± 15

− 247 ± 31* 1±1 − 70 ± 13* 1±1

104 ± 3 101 ± 4 95 ± 4 106 ± 6

46 ± 5* 1±1 55 ± 4* 1±1

402 ± 6 374 ± 14 400 ± 21 385 ± 12

− 270 ± 12* 1±1 − 65 ± 12* 8±8

Values are means ± SE; n = no. of rats. *Significantly different from the respective control (saline), p b 0.05; two-way ANOVA and Bonferroni post-hoc test.

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Fig. 2. Photomicrographs of the sections (30 μm) of the brainstem at the intermediate (A1–A2) and caudal commissural NTS levels [B1–B2 (objective magnification, 5×)]. Panels A1 and B1 are from rats submitted to intermittent injection of saline (iv) whereas panels A2 and B2 are from rats submitted to intermittent chemoreflex activation with KCN (iv). AP: area postrema; cc— central canal; cs: calamus scriptorium; XII: hypoglossal nucleus; DMX: dorsal motors nucleus of vagus, Sol DM: nu. solitary tract., dorsal medial part; Sol C: commissural nu. solitary tract.; Sol L: nu. solitary tract., lateral part. Bar (panel A1): 100 μm. The areas correspondent to the NTS subnuclei were outlined in accordance with the atlas of Paxinos and Watson (2007).

Bonferroni post-hoc indicated no changes in the Fos-ir in the LINTS [64 ± 6 vs. 68± 12, p N 0.05 (n = 5)] or in the CINTS [53 ± 14 vs. 52± 11, p N 0.05 (n = 5)].

Fig. 3. Mean number of Fos-ir neurons per section in the LINTS and LCNTS subnuclei following intermittent chemoreflex activation [i.v. (black bars)] or saline intermittent injection [i.v. (white bars)]. *p b 0.001, when compared to saline group. Two-way ANOVA and Bonferroni post-hoc test. LINTS: lateral intermediate commissural NTS; LCNTS: lateral caudal commissural NTS. In my powerpoint the Y axis reads as “number mean per section” If this is like this, it should read: “mean number per section”.

3.4. Fos-ir in the lateral intermediate and caudal commissural NTS of rats submitted to phenylephrine infusion Fig. 6 shows tracings of one rat representative of the group submitted to phenylephrine infusion during 30 min. As clearly illustrated in Fig. 6, phenylephrine infusion elicited a pressure and bradycardic response. Saline infusion produced negligible effect on MAP or HR (not shown). Fig. 7 shows representative examples of Fos-ir in the LINTS and LCNTS of two rats submitted to saline (panels A1 and B1) or phenylephrine (panels A2 and B2) infusion. Two-way ANOVA indicated no changes in Fos-ir cells in the NTS in relation to treatment [saline × phenyl (F1,14 = 1.2, p = 0.56)], interaction (F1,14 = 0.9, p = 0.0006) or sub-region (F1,14 = 0.3, p = 1.18.). As summarized in Fig. 8 (panel A), Bonferroni post-hoc indicated no changes in the Fosir neither in the LINTS [10 ± 5 vs. 15 ± 4, p N 0.05 (n = 5)] nor in the CINTS [18 ± 5 vs. 21 ± 10, p N 0.05 (n = 5)]. On the other hand, significant changes were observed in Fos-ir positive cells in the dorsomedial aspect of the intermediate commissural NTS [at solitary tract level (Fig. 7, panels A1–A2)] and in the midline region of the caudal commissural NTS [at calamus scriptorium level (Fig. 7, panels B1–B2)] in response to phenylephrine infusion. Two-way ANOVA indicated significant increase to Fos-ir cells in the NTS in relation to treatment [saline × phenylephrine (F1,12 = 23, p = 0.0005)], but not changes in the interaction (F1,12 = 1.1, p = 0.3) or sub-region (F1,12 = 0.11, p = 2.9). As summarized in Fig. 8 (panel B), Bonferroni

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Fig 4. Photomicrographs of the sections (30 μm) of the brainstem at the intermediate (A1–A2) and caudal commissural NTS levels [B1–B2 (objective magnification, 5×)]. Panels A1 and B1 are from rats submitted to intermittent injection of saline (iv) whereas panels A2 and B2 are from rats submitted to intermittent baroreflex activation with phenyl (iv). AP: area postrema; cc— central canal; cs: calamus scriptorium; XII: hypoglossal nucleus; DMX: dorsal motors nucleus of vagus; Gr: nucleus gracile; Sol DM : nu. solitary tract., dorsal medial part; Sol C: commissural nu. solitary tract.; Sol L: nu. solitary tract., lateral part. Bar (panel A1): 100 μm. The area correspondent to the NTS subnuclei was outlined in accordance with the atlas of Paxinos and Watson (2007).

post-hoc indicated changes in the Fos-ir in the dorsomedial aspect of the intermediate commissural NTS [at solitary tracts level (12 ± 4 vs. 42 ± 7, p b 0.01, n = 5)] and in the midline region of the caudal commissural NTS [at calamus scriptorium level (8 ± 1 vs. 28 ± 6, p b 0.005, n = 5)].

Fig. 5. Mean number of Fos-ir neurons per section in the LINTS and LCNTS subnuclei following intermittent baroreflex activation [i.v. (black bars)] or saline intermittent injection [i.v. (white bars)]. *p b 0.001, when compared to saline group. Two-way ANOVA and Bonferroni post-hoc test. LINTS: lateral intermediate commissural NTS; LCNTS: lateral caudal commissural NTS.

4. Discussion 4.1. Involvement of the NTS specific subnuclei in the central chemoreflex pathway In the present study the Fos immunohistochemistry approach was used to characterize the different subnuclei of the commissural NTS involved in the cardiovascular and respiratory responses to peripheral chemoreflex activation produced by intermittent injection of potassium cyanide (KCN; 80 μg/kg) in conscious freely moving rats. The data show that intermittent KCN injection produced a significant increase in the number of Fos-ir specifically on the lateral aspect of the intermediate (area postrema level) and lateral caudal commissural NTS (calamus scriptorius level). Previous studies by Finley and Katz (1992) showed that those commissural aspects of the NTS are densely innerved by carotid sinus nerve afferent fibers. Besides, a similar Fos distribution in the commissural NTS was also found after direct electrical stimulation of the carotid sinus nerve (Erickson and Millhorn, 1991) as well as after stimulation of carotid bodies in rats exposed to moderate hypoxia [10–11% O2 (Berquim et al., 2000a; Hirooka et al., 1997)], severe ambient hypoxia [9% O2 (Teppema et al, 1997)] or after carbon monoxide inhalation (Bodineau and Larnicol, 2001). Studies by Sun and Reis (1994) suggest that hypoxic-hypoxia can also activate central neurons. The authors showed that in rats with denervated chemoreceptors, severe hypoxia can produce an

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Fig. 6. Tracings showing changes in heart rate (HR), pulsatile arterial pressure (PAP) and mean arterial pressor (MAP) in response to infusion during 30 min of phenylephrine in one awake rat representative of the group.

increase in sympathetic activity, due to a direct excitatory action on RVLM sympatho-excitatory neurons. Chemoreflex activation with KCN (cytotoxic-hypoxia) activated mainly arterial peripheral chemoreceptors because the ligature of carotid body arteries abolished cardiovascular response to chemoreflex activation with KCN (Barros et al., 2002). For this reason in the present study we assume that Fos-ir in the commissural NTS after intermittent activation of the chemoreflex was essentially due to carotid body cell activation.

In spite of different studies showing Fos-ir in the commissural NTS after different hypoxia-hypoxic stimulus situations, the present study brings a full characterization of the location of the cells excited by peripheral chemoreceptors in the antero-posterior aspect [bregma −13.6 to −14.58 mm (see Methods)] of the commissural NTS of conscious freely moving rats. Furthermore, this is the first study using the intermittent activation of peripheral chemoreflex with KCN in awake rats in order to identify the localization of the chemosensitive

Fig. 7. Photomicrographs of the sections (30 μm) of the brainstem at the intermediate (A1–A2) and caudal commissural NTS levels [B1–B2 (objective magnification, 5×)]. Panels A1 and B1 are from rats submitted to saline (iv) infusion during 30 min whereas panels A2 and B2 are from rats submitted to baroreflex activation by phenyl (iv) infusion during 30 min. AP: area postrema; cc— central canal; cs: calamus scriptorium; XII: hypoglossal nucleus; DMX: dorsal motors nucleus of vagus; Gr: nucleus gracile; Sol DM : nu. solitary tract., dorsal medial part; Sol C: commissural nu. solitary tract.; Sol L: nu. solitary tract., lateral part. Bar (panel A1): 100 μm. The areas correspondent to the NTS subnuclei were outlined in accordance with the atlas of Paxinos and Watson (2007).

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protocol (Cruz et al., 2008). Given that NTS neurons provide the major visceroceptive afferent input to the PVN (Ricardo and Koh, 1978), it would be important to determine in the future whether the KCNactivated NTS and PVN neurons are indeed interconnected, as part of the same central neuronal circuitry mediating autonomic adjustments to chemoreflex activation. 4.2. Baroreflex control

Fig. 8. Mean number of Fos-ir neurons per section in the dorsomedial aspect of the commissural intermediate and caudal midline NTS subnuclei after continuous infusion of phenylephrine during 30 min [i.v. (black bars)] or saline [i.v. (white bars)]. *p b 0.001, when compared to saline group. Two-way ANOVA and Bonferroni post-hoc test.

cell in the intermediate and caudal commissural NTS. Cyanide (iv) has been widely used in many studies since Heymans et al (1931) to activate the chemoreflex as a reliable tool (Franchini and Krieger, 1993; Haibara et al., 1995, 1999; Braga et al, 2007; Cruz et al., 2008; Granjeiro and Machado, 2009). For example, previous studies from our laboratory (Haibara et al, 1999) showed the involvement of ionotropic glutamate receptors in the bradycardic response to KCNmediated chemoreflex activation at the lateral intermediate NTS level and Braga et al. (2007) showed the involvement of ionotropic glutamate and purinergic receptors in the processing of the sympatho-excitatory component of the chemoreflex at the commissural caudal NTS level. These previous studies supported the involvement of the intermediate commissural NTS in the parasympathetic component of the chemoreflex, as well as, the involvement of caudal commissural NTS in the sympathetic component of the peripheral chemoreflex. Moreover, recent studies from our laboratory showed that the respiratory component of the chemoreflex activation involves nitric oxide produced by neuronal nitric oxide-synthase at the level of caudal commissural NTS (Granjeiro and Machado, 2009). All these previous studies suggested the involvement of the glutamate and purinergic receptors in the cardiovascular and the nitric oxide in the respiratory components of the chemoreflex in the commissural NTS. The identification of Fos-ir neurons in response to intermittent chemoreflex activation in specific subnuclei of the lateral intermediate and caudal commissural NTS will be important for a series of new experiments to be performed in NTS sub regions involved in the chemoreflex pathways. In a recent work, we demonstrated that a subpopulation of PVN neurons, likely involved in the control of sympathetic outflow from the PVN, was also activated by the same chemoreflex activation

In the present study, the intermittent chemoreflex activation produced a significant increase in the Fos-ir in the NTSLI or NTSLC. Nevertheless, the Fos-ir increasing could be due to baroreflex activation following a large increase in MAP produced by intermittent chemoreflex activation. Although different findings have shown that systemic hypoxia or chronic intermittent hypoxia produced a significant increase in the baseline MAP (Greenberg et al, 1999; Hirooka et al., 1997), no one has addressed the possible contribution of baroreceptor inputs inducing Fos-ir in the NTS in response to hypoxia activation protocols. Hence, we analyzed Fos-ir in the NTS after intermittent baroreflex activation produced by phenylephrine (iv), which is a vasoconstrictor (α1-adrenoceptors agonist) widely used to assess arterial baroreflex function (Chan and Sawchenko, 1998, Chan et al., 1998, 2000; Weston et al., 2003, Antunes and Machado, 2003). Our results showed that intermittent phenylephrine injection (iv) produced no changes in Fos-ir in the NTSLI or NTSLC, indicating that Fos-ir increase after the chemoreflex activation was not secondary to baroreflex activation due to intermittent increase in the baseline MAP produced by KCN. These results support the concept that the Fos-ir in the NTS after intermittent KCN (iv) is mainly due to carotid body cell activation, and is in agreement with our recent findings in the PVN (Cruz et al., 2008). However, it is well known that the NTS receives monosynaptic inputs from baroreceptor afferents (Spyer, 1990; Chan et al., 1998, 2000), and that Fos-ir depends on intensity of the stimulus, as well as, the time between onset stimulus and euthanasia and we considered the possibility that intermittent phenylephrine (iv) might not have produced a stimulation enough to produce Fos expression in the NTS. For this reason we also analyzed Fos-ir in the intermediate and caudal commissural NTS after continuous phenylephrine infusion during 30 min, in order to keep the arterial baroreceptors continuously activated. Our results showed that baroreflex activation after phenylephrine infusion evoked Fos-ir in the dorsomedial intermediate commissural NTS (at solitary tract level) and in the midline caudal commissural NTS, corresponding to NTS location of primary baroreceptor afferents from carotid sinus and aortic depressor nerves (Erickson and Milhorn, 1994; Chan et al., 1998). Besides, similar Fos-ir distribution in the NTS was shown by Chan et al. (2000) and Weston et al. (2003) after phenylephrine infusion for 25 min. Studies by Chan et al. (1998) also showed that sinoaortic denervation dramatically reduced phenylephrine-induced Fos expression in the NTS, suggesting that phenylephrine-induced Fos in the NTS is essentially dependent on the baroreceptor afferent integrity. The data of the present study support the concept that the baroreflex and chemoreflex sensitive neurons are located in distinct areas within the intermediate and caudal commissural NTS. 4.3. Methodological considerations Some methodological aspects of this work deserve special attention. In the present study the chemoreflex was intermittently activated using cytotoxic-hypoxia evoked by KCN (iv). It is questioned whether KCN (iv) evoking cytotoxic-hypoxia reproduces a physiological stimulus such as hypoxic-hypoxia. In this regard, studies from our laboratory showed that bilateral carotid body artery ligature abolished the cardiovascular responses to chemoreflex activation with KCN (iv), indicating that KCN is an appropriate and selective tool to activate arterial chemoreceptor cells (Barros et al., 2002).

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The use of Fos as a marker of neuronal activation has been extensively characterized (Dragunow and Faull, 1989; Berquim et al., 2000a, Berquim et al., 2000b; Hirooka et al., 1997; Teppema et al., 1997; Greenberg et al., 1999; Cruz et al., 2008) because it allows the identification of populations of neurons that are activated by a specific stimulus in awake rats. However, it is possible that the Fos could be expressed by a non-specific stimulus associated with the surgical or any other stressful condition related to the experimental protocol. For these reasons, the present study is based upon comparison between experimental and control rats placed under the same experimental and environmental conditions. In the present study we verified that surgical procedures and anesthesia (ip) produced no significant changes in Fos-ir in the NTS (data not shown). The Fos protein expression in the brainstem and hypothalamus starts from 15 to 30 min after the first stimulus and reaches a maximum expression 60 to 90 min after the beginning of the stimulus (Hoffman and Murphy, 2000; Chan et al., 1998, 2000; Weston et al., 2003; Cruz et al., 2008). We tested the maximum expression 30 and 60 min after the last KCN (iv) stimulus which corresponds respectively to 60 and 90 min in relation to the first activation of the chemoreflex with KCN and similar NTS Fos expression in both periods was observed (data not shown). Studies from Hirooka et al. (1997), after a moderate hypoxia (10% CO2) stimulus, showed a Fos increase in NTS neurons 30 min after the end of the protocol. Our results showed that Fos-ir depends also on the frequency of the stimulus because both saline and KCN animals receive a first KCN injection control but Fos-ir increased only in rats submitted to intermittent chemoreflex activation every 3 min during 30 min (11 KCN bolus injection/rat). Acknowledgements In Brazil these studies were funded by FAPESP (2004/03285-7) and CNPQ (522150/95-0). J.C.C. was supported by a CAPES fellowship. In USA, funding was provided by NIH HL68725 (JES). References Antunes, V.R., Machado, B.H., 2003. Antagonism of glutamatergic metabotropic receptors in the NTS of awake rats does not affect the gain of the baroreflex. Auton. Neurosc. 103 (1–2), 65–71. Bao, G., Randhawa, P.M., Fletcher, E.C., 1997. Acute blood pressure elevation during repetitive hypocapnic and eucapnic hypoxia in rats. J. Appl. Physiol. 82 (4), 1071–1078. Barros, R.C.H., Bonagamba, L.G.H., Okamoto-Canesin, R., Oliveira, M., Branco, L.G.S., Machado, B.H., 2002. Cardiovascular response to chemoreflex activation with potassium cyanide or hypoxic hypoxia in awake rats. Auton. Neurosci. 97 (2), 100–105. Berquim, P., Bodineau, L., Gros, F., Larnicol, N., 2000a. Brainstem and hypothalamic areas involved in respiratory chemoreflex: a Fos study in adults rats. Brains Res. 857 (1–2), 30–40. Berquim, P., Cayetanot, F., Gros, F., Larnicol, N., 2000b. Postnatal changes in Fos-like immunoreactivity evoked by hypoxia in the in the rat brainstem and hypothalamus. Brains Res. 877 (2), 48–159. Biscoe, T.J., Dunchen, M.R., 1990. Cellular basis of transduction in the carotid chemoreceptors. Am. J. Physiol. 258 (6 Pt 1), L 271–L 278. Bodineau, L., Larnicol, N., 2001. Brainstem and hypothalamic areas activated by tissue hypoxia: Fos-like immunoreactivity induced by carbon monoxide inhalation in the rat. Neuroscience 108 (4), 643–653. Braga, V.A., Soriano, R.N., Braccialli, A.L., de Paula, P.M., Bonagamba, L.G., Paton, J.F., Machado, B.H., 2007. Involvement of l-glutamate and ATP in the neurotransmission of the sympathoexcitatory component of the chemoreflex in the commissural nucleus

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