B R A I N R E S E A R C H 1 3 9 3 ( 2 0 1 1 ) 3 1 – 4 3 ava i l ab l e a t www.sc i enced i r ec t . com www.e l sev i e r . com/ loca te /b ra i n res Research Report Hypothalamic supraoptic but not paraventricular nucleus is involved in cardiovascular responses to carbacholmicroinjected into the bed nucleus of stria terminalis of unanesthetized rats Fernando H.F. Alvesa,⁎,1, Carlos C. Crestanib,1, Cristiane Busnardoa,1, José Antunes-Rodriguesc, Felipe V. Gomesa, Leonardo B.M. Resstela, Fernando M.A. Corrêaa aDepartment of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil bDepartment ofNatural Active Principles andToxicology, School of Pharmaceutical Sciences, São Paulo StateUniversity, UNESP,Araraquara, SP, Brazil cDepartment of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil A R T I C L E I N F O ⁎ Corresponding author at: University of São P 3900, 14049–900, Ribeirão Preto, São Paulo, B E-mail address: fer_farmacia@yahoo.com Abbreviations: aCSF, Artificial cerebrospina rate; MAP, Mean arterial pressure; PAP, Puls nucleus of the hypothalamus 1 The authors contributed equally to this w 0006-8993/$ – see front matter © 2011 Elsevi doi:10.1016/j.brainres.2011.03.067 A B S T R A C T Article history: Accepted 29 March 2011 Available online 3 April 2011 Microinjection of the cholinergic agonist carbachol into the bed nucleus of the stria terminalis (BST) has been reported to cause pressor response in unanesthetized rats,whichwas shown to be mediated by an acute release of vasopressin into the systemic circulation and followed by baroreflex-mediated bradycardia. In the present study, we tested the possible involvement of the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei in the pressor response evoked by carbachol microinjection into the BST of unanesthetized rats. For this, cardiovascular responses following carbachol (1 nmol/100 nL) microinjection into the BST were studied before and after PVN or SON pretreatment, either ipsilateral or contralateral in relation to BST microinjection site, with the nonselective neurotransmission blocker cobalt chloride (CoCl2, 1 mM/100 nL). Carbacholmicroinjection into the BST evokedpressor response. Moreover, BST treatment with carbachol significantly increased plasma vasopressin levels, thus confirming previous evidences that carbacholmicroinjection into the BST evokes pressor response due to vasopressin release into the circulation. SON pretreatment with CoCl2, either ipsilateral or contralateral in relation to BSTmicroinjection site, inhibited the pressor response to carbachol microinjection into the BST. However, CoCl2 microinjection into the ipsilateral or contralateral PVN did not affect carbachol-evoked pressor response. In conclusion, our results suggest that pressor response to carbachol microinjection into the BST is mediated by SON magnocellular neurons, without significant involvement of those in the PVN. The results also indicate that responses to carbachol microinjection into the BST are mediated by a neural pathway that depends on the activation of both ipsilateral and contralateral SON. © 2011 Elsevier B.V. All rights reserved. Keywords: Cholinergic neurotransmission BST Hypothalamus Vasopressin Heart rate and arterial pressure aulo, School of Medicine of Ribeirão Preto, Department of Pharmacology, Av. Bandeirantes razil. Fax: +55 16 3633 2301. .br (F.H.F. Alves). l fluid; CBH, Carbachol; BST, Bed nucleus of stria terminalis; CoCl2, Cobalt chloride; HR, Heart atile arterial pressure; PVN, Paraventricular nucleus of the hypothalamus; SON, Supraoptic ork. er B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2011.03.067 mailto:fer_farmacia@yahoo.com.br http://dx.doi.org/10.1016/j.brainres.2011.03.067 32 B R A I N R E S E A R C H 1 3 9 3 ( 2 0 1 1 ) 3 1 – 4 3 1. Introduction The bed nucleus of the stria terminalis (BST) is a structure of the limbic system that is involved in behavioral, neuroendo- crine and autonomic responses (Alves et al., 2007; Crestani et al., 2007, 2008a; Dunn and Williams, 1995). Moreover, it has been proposed that the BST is an important center for the regulation of cardiovascular activity (Crestani et al., 2009a; Gelsema et al., 1987; Ulrich-Lai andHerman, 2009). The electric stimulation of the BST has been reported to evoke pressor as well as depressor responses in anesthetized rats (Dunn and Williams, 1995). Cardiovascular responses was also observed after chemical stimulation of the BST using either glutamate, D,L-homocysteic acid or noradrenaline (Ciriello and Janssen, 1993; Crestani et al., 2007; Gelsema et al., 1987, 1993; Hatam and Nasimi, 2007). In addition, there is evidence that the BST tonically modulates cardiac baroreflex activity (Alves et al., 2009; Crestani et al., 2006, 2008b; Li and Dampney, 1994; McKitrick et al., 1992). Cholinergic synaptic terminals as well as muscarinic and nicotinic cholinergic receptors have been identified in the BST (Clarke et al., 1985; Ruggiero et al., 1990; Wamsley et al., 1984), thus providing evidence of a cholinergic neurotransmission in the BST. We have previously reported that microinjection of carbachol, a cholinergic agonist, into the BST of unanesthe- tized rats caused an increase in arterial pressure that was followed by a baroreflex-mediated reduction of the heart rate (HR) (Alves et al., 2007). These responses were inhibited by systemic pretreatment with a V1-vasopressinergic receptor antagonist (Alves et al., 2007), thus suggesting a mediation by acute vasopressin release into the systemic circulation. Moreover, cardiovascular responses to carbachol microinjec- tion into the BST were mediated by activation of local M2- cholinergic receptors (Alves et al., 2007). These results suggested the existence of a cholinergic mechanism in the BST that integrates cardiovascular and neuroendocrine con- trol and could take part in fluid balance adjustments. However, the neural pathway involved in cardiovascular responses to carbachol microinjection into the BST is yet unknown. Vasopressin, also known as antidiuretic hormone, is a nonapeptide with a potent vasoconstrictor action (Altura and Altura, 1984; Barer, 1961). This peptide is synthesized by magnocellular neurons located in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus and stored in the posterior hypophysis to further release into the systemic circulation (Swaab et al., 1975). Because cardiovas- cular responses following carbachol microinjection into the BST were shown to be mediated by an acute release of vasopressin into the systemic circulation (Alves et al., 2007), the magnocellular neurons in the SON and/or PVN must be involved in their mediation. Considering this, it is relevant to study which hypothalamic magnocellular nucleus mediates the cardiovascular response evoked by carbachol microinjec- tion into the BST. Taking that into consideration, we evaluated the hypoth- esis that PVN and/or SON neurons are part of the neural pathway related to cardiovascular responses following carba- chol microinjection into the BST of unanesthetized rats. For this, we investigated cardiovascular responses evoked by carbachol microinjection into the BST before and after PVN or SON pretreatment, either ipsilateral or contralateral in relation to BST microinjection site, with the nonselective neurotransmission blocker cobalt chloride (CoCl2). 2. Results 2.1. Cardiovascular responses to aCSF or carbachol microinjection into the bed nucleus of the stria terminalis of unanesthetized rats Microinjection of aCSF into the BST (n=5) did not affect either MAP (99±2 vs. 98±3 mmHg, t=0.2, P>0.05) or HR (379±11 vs. 352±9 bpm, t=1.3, P>0.05) baseline values. However, micro- injection of carbachol into the BST caused significant pressor and bradycardiac responses in unanesthetized rats (Fig. 1). Photomicrography of a coronal brain section showing a representative microinjection site into the BST is presented in Fig. 2. Diagrammatic representation of the BST indicating microinjection sites into the BST of all animals used in the present study is also shown in Fig. 2. 2.2. Effect of aCSF or carbachol microinjection into the bed nucleus of the stria terminalis in plasma vasopressin levels Microinjection of carbachol (n=6) into the BST significantly increased plasma vasopressin content (aCSF: 2.3±0.5 pg/mL vs. carbachol: 21.3±3.6 pg/mL, t=5, P<0.005), when compared to the control group that received vehicle (aCSF) injection into the BST (n=6). 2.3. Effect of supraoptic nucleus pretreatment with aCSF or CoCl2 in cardiovascular responses to carbachol microinjection into the bed nucleus of the stria terminalis 2.3.1. Ipsilateral supraoptic nucleus Microinjection of aCSF into the ipsilateral SON (n=7) did not affect either MAP (98±2 vs. 101±3 mmHg, t=0.5, P>0.05) or HR (352±7 vs. 367±11 bpm, t=1.5, P>0.05) baseline values. Pretreatment of the ipsilateral SON with aCSF also did not affect the pressor (43±2 vs. 38±2 mmHg, t=2.3, P>0.05) and bradycardiac (−67±7 vs. −64±8 bpm, t=0.2, P>0.05) response to carbachol microinjection into the BST (Fig. 1A). Microinjection of CoCl2 into the ipsilateral SON (n=7) did not affect either MAP (102±2 vs. 100±2 mmHg, t=0.6, P>0.05) or HR (351±6 vs. 356±8 bpm, t=0.7, P>0.05) baseline values. However, ipsilateral SON pretreatment with CoCl2 significant- ly reduced the pressor (44±2 vs. 6±1 mmHg, t=16, P<0.0001) and bradycardiac (−74±6 vs. −12±1 bpm, t=10, P<0.0001) response to carbachol microinjection into the BST (Fig. 1A). Time-course analysis indicated a significant effect of SON pretreatment with CoCl2 in carbachol cardiovascular effects (ΔMAP: F(1,456)=468, P<0.0001 and ΔHR: F(1,456)=111, P<0.0001), a significant effect over time (ΔMAP: F(37,456)=23, P<0.0001 and ΔHR: F(37,456)=11, P<0.0001), and an interaction between treatment and time (ΔMAP: F(37,456)=20, P<0.0001 and ΔHR: F(37,456)=4, P<0.0001) (Fig. 1B). Fig. 1 – A) Changes in MAP (ΔMAP) and HR (ΔHR) in response to carbachol (CBH; 1 nmol/100 nL) microinjection into the BST before (filledcolumns) andafter (open columns)pretreatmentof ipsilateral or contralateral SON, in relation toBSTmicroinjectionsite,with vehicle (aCSF) or CoCl2 (cobalt). Columns represent the mean and bars the SEM. *P<0.05, paired Student's t-test. B) Time-course of the effect of CBHmicroinjection into the BST onMAP and HR before (filled circles) or after (open circles) pretreatment of the ipsilateral SON (left) or contralateral SON (right)withCoCl2. CBH injectionsweremadeat time 0. Circles represent themean andbar the SEM. Data were analyzed using two-way ANOVA. 33B R A I N R E S E A R C H 1 3 9 3 ( 2 0 1 1 ) 3 1 – 4 3 2.3.2. Contralateral supraoptic nucleus Microinjection of aCSF into the contralateral SON (n=6) did not affect either MAP (100±3 vs. 102±4 mmHg, t=0.5, P>0.05) or HR (353±11 vs. 372±6 bpm, t=1.6, P>0.05) baseline values. Pretreatment of the contralateral SON with aCSF also did not affect both the pressor (44±4 vs. 37±3 mmHg, t=2.2, P>0.05) and bradycardiac (−67±8 vs. −74±8 bpm, t=0.5, P>0.05) response to carbachol microinjection into the BST (Fig. 1A). Fig. 2 – A) Photomicrograph of a coronal brain section showing a microinjection site in the BST of one representative animal. B) Diagrammatic representation, modified from the rat brain atlas of Paxinos and Watson (1997), indicating carbachol and aCSFmicroinjection sites into the BST. ac, anterior commissure; f, fornix; IA, interaural coordinates (mm); ic, internal capsule; LSV, lateral septal ventricle; LV, lateral ventricle; PS, parastrial nuclei; and st, stria terminalis. 34 B R A I N R E S E A R C H 1 3 9 3 ( 2 0 1 1 ) 3 1 – 4 3 Microinjection of CoCl2 into the contralateral SON (n=6) did not affect either MAP (101±3 vs. 100±4 mmHg, t=0.1, P>0.05) or HR (362±9 vs. 359±10 bpm, t=0.3, P>0.05) baseline values. However, contralateral SON pretreatment with CoCl2 signifi- cantly reduced the pressor (42±5 vs. 9±2 mm Hg, t=5, P<0.005) and bradycardiac (−74±6 vs. −13±2 bpm, t=10, P<0.0001) response to carbachol microinjection into the BST (Fig. 1A). Time-course analysis indicated a significant effect of SON pretreatment with CoCl2 in carbachol cardiovascular effects (ΔMAP: F(1,380)=215, P<0.0001 and ΔHR: F(1,380)=141, P<0.0001), a significant effect over time (ΔMAP: F(37,380)=16, P<0.0001 and ΔHR: F(37,380)=8, P<0.0001), and an interaction between treatment and time (ΔMAP: F(37,380)=11, P<0.0001 and ΔHR: F(37,380)=3, P<0.0001) (Fig. 1B). Cardiovascular responses to carbachol microinjection into the BST of animals that received CoCl2 in the ipsilateral or contralateral SON were not significantly different (MAP: t=2, P>0.05; HR: t=1, P>0.05) (Fig. 1). Representative recordings showing the cardiovascular re- sponses to carbachol microinjection into the BST before and after ipsilateral or contralateral SON pretreatment with CoCl2 is presented in Fig. 3. Moreover, photomicrography of coronal brain section showing the microinjection site in the ipsilateral and contralateral SON of representative animals are presented in Figs. 4 and 5, respectively. Diagrammatic representation showingmicroinjection sites of CoCl2 and aCSF in the ipsilateral andcontralateralSONisalso showninFigs. 4 and5, respectively. 2.4. Effect of paraventricular nucleus pretreatment with aCSF or CoCl2 in cardiovascular responses to carbachol microinjection into the bed nucleus of the stria terminalis 2.4.1. Ipsilateral paraventricular nucleus Microinjection of aCSF into the ipsilateral PVN (n=7) did not affect either MAP (99±3 vs. 102±2 mmHg, t=0.6, P>0.05) or HR (357±7 vs. 364±10 bpm, t=0.5, P>0.05) baseline values. Ipsilateral PVN treatment with aCSF also did not affect the pressor (43±3 vs. 40±2 mmHg, t=0.7, P>0.05) and bradycar- diac (−78±6 vs. −73±5 bpm, t=0.8, P>0.05) response following carbachol microinjection into the BST (Fig. 6A). image of Fig.�2 Fig. 3 – Typical recordings showing pulsatile arterial pressure (PAP),mean arterial pressure (MAP) and heart rate (HR) changes in response to carbachol (CBH; 1 nmol/100 nL) microinjection into the BST of unanesthetized rats before (before cobalt) and after (after cobalt) pretreatment of ipsilateral or contralateral SON, in relation to BST microinjection site, with CoCl2. The panels represent segments of recording in different animals. 35B R A I N R E S E A R C H 1 3 9 3 ( 2 0 1 1 ) 3 1 – 4 3 Microinjection of CoCl2 into the ipsilateral PVN (n=7) did not affect either MAP (99±3 vs. 100±3 mmHg, t=0.8, P>0.05) or HR (366±9 vs. 374±9 bpm, t=0.5, P>0.05) baseline values. Moreover, ipsilateral PVN pretreatment with CoCl2 did not affect the pressor (41±3 vs. 38±2 mmHg, t=0.9, P>0.05) and bradycardiac (−76±8 vs. −73±6 bpm, t=0.3, P>0.05) response to carbacholmicroinjection into the BST (Fig. 6A). Time-course analysis did not show a significant effect of PVN pretreatment with CoCl2 in carbachol cardiovascular responses (ΔMAP: F(1,456)=2, P>0.05; ΔHR: F(1,456)=2, P>0.05), but indicated a significant effect over time on the MAP (F(37,456) = 45, P<0.0001) and HR (F(37,456)=18, P<0.0001) (Fig. 6B). 2.4.2. Contralateral paraventricular nucleus Microinjection of aCSF into the contralateral PVN (n=6) did not affect either MAP (101±3 vs. 98±2 mmHg, t=0.5, P>0.05) or HR Fig. 4 – A) Photomicrograph of a coronal brain section showing a microinjection site, of one representative animal. B) Diagramma Paxinos and Watson (1997), indicating aCSF (open circles) and Co SON. IA, interaural coordinates (mm); LH, lateral hypothalamus; (353±11 vs. 361±7 bpm, t=0.5, P>0.05) baseline values. Contralateral PVN treatment with aCSF also did not affect the pressor (43±4 vs. 39±3 mm Hg, t=0.8, P>0.05) and bradycardiac (−76±9 vs. −68±6 bpm, t=0.8, P>0.05) response to carbachol microinjection into the BST (Fig. 6A). Microinjection of CoCl2 into the contralateral PVN (n=6) did not affect either MAP (99±3 vs. 104±4 mm Hg, t=1.5, P>0.05) or HR (359 ± 9 vs. 372± 12 bpm, t = 0.9, P>0.05) baseline values. Moreover, contralateral PVN pretreatment with CoCl2 did not affect the pressor (42±4 vs. 41±2 mm Hg, t=0.1, P>0.05) and bradycardiac (−70±9 vs. −65±8 bpm, t=0.5, P>0.05) response to carbachol microinjection into the BST (Fig. 6A). Time-course analysis did not show a signifi- cant effect of contralateral PVN pretreatment with CoCl2 in carbachol cardiovascular responses (ΔMAP: F(1,380) = 2, P>0.05; ΔHR: F(1,380) =0.2, P>0.05) (Fig. 6B), but indicated a microinjection site in the ipsilateral SON, in relation to BST tic representation, modified from the rat brain atlas of Cl2 (filled black circles) microinjection sites in the ipsilateral and ox, optic chiasm. image of Fig.�3 image of Fig.�4 Fig. 5 – A) Photomicrograph of a coronal brain section showing a microinjection site in the contralateral SON, in relation to BST microinjection site, of one representative animal. B) Diagrammatic representation, modified from the rat brain atlas of Paxinos andWatson (1997), indicating aCSF (open circles) and CoCl2 (filled black circles) microinjection sites in the contralateral SON. IA, interaural coordinates (mm); LH, lateral hypothalamus; and ox, optic chiasm. 36 B R A I N R E S E A R C H 1 3 9 3 ( 2 0 1 1 ) 3 1 – 4 3 significant effect over time on the MAP (F(37,380) = 44, P<0.0001) and HR (F(37,380) =11, P<0.0001). Photomicrography of coronal brain section showing the microinjection site in the ipsilateral and contralateral PVN of representative animals is presented in Figs. 7 and 8, respec- tively. Diagrammatic representation showing microinjection sites of CoCl2 and aCSF in the ipsilateral and contralateral PVN is also shown in Figs. 7 and 8, respectively. 3. Discussion The BST is localized in the rostral prosencephalon, and is associated with autonomic and neuroendocrine functions (Dunn, 1987; Dunn andWilliams, 1995; Ulrich-Lai andHerman, 2009). Cholinergic synaptic terminals were indentified in the BST (Ruggiero et al., 1990). Moreover, binding studies described the presence of muscarinic and nicotinic receptors in the BST (Clarke et al., 1985; Wamsley et al., 1984). Electrophysiological studies reported that BST neurons showed an increase in firing rate in response to local administration of acetylcholine through activation of local muscarinic cholinergic receptors (Casada and Dafny, 1993a, 1993b). We have previously reported that microinjection of carbachol, a cholinergic agonist, into the BST of unanesthetized rats evoked a pressor response that was followed by a baroreflex-mediated brady- cardia (Alves et al., 2007). These cardiovascular effects were blocked after local pretreatment with an M2-muscarinic receptor antagonist as well as after systemic pretreatment with a V1-vasopressinergic receptor antagonist (Alves et al., 2007), thus suggesting that activation of M2-muscarinic receptor activation within BST evokes cardiovascular changes that are mediated by acute vasopressin release into the circulation. In the present study, we show results indicating that carbachol microinjection into the BST increases circulat- ing vasopressin levels, thus confirming previous evidence that carbachol microinjection into the BST evokes pressor re- sponse due to vasopressin release. Vasopressin is a potent vasoconstrictor agent (Altura and Altura, 1984; Barer, 1961). This nonapeptide is synthesized by magnocellular neurons of the PVN and SON (Swaab et al., 1975). Each neuron gives rise to a single axon into the posterior pituitary gland, where its neurosecretory endings release vasopressin (Swaab et al., 1975). Because the capillaries within the pituitary gland do not have a blood-brain barrier, vasopres- sin released in closeproximity to the capillaries easily enters the bloodstream (Leng et al., 1999). To verify if SON and/or PVN synapses mediate the pressor response to the carbachol microinjection into the BST, we pretreated both nuclei with the nonselective neurotransmission blocker CoCl2. The use of CoCl2 is a common approach to investigate a possible involve- ment of specific brain areas in a functional neural pathway. The technique is based on the administration of circumscribed microinjections of compounds that reversibly block neuronal activity over a given period of time. Themicroinjection of CoCl2 into discrete brain areas has been used for the functional inactivation of synapses (Crestani et al., 2006, 2009b; Giancola et al., 1993; Scopinho et al., 2008). The CoCl2 reduces presynaptic Ca+2 influx, leading to an inhibition of neurotransmitter release and a consequent synaptic blockade (Kretz, 1984), without influence on passage fibers. The inhibition caused by this compound, whenmicroinjected in volumes and concentrations that were similar to those presently used, was reported to spread over an area up to 1 mm2 (Lomber, 1999). Pretreatment of the PVN with CoCl2, either injected ipsilateral or contralateral in relation to BST microinjection image of Fig.�5 Fig. 6 –A) Changes inMAP (ΔMAP) andHR (ΔHR) in response to carbachol (CBH; 1 nmol/100 nL)microinjection into the BST before (filled columns) and after (open columns) pretreatment of the ipsilateral and contralateral PVN, in relation to BSTmicroinjection site, with vehicle (aCSF) or CoCl2 (cobalt). Columns represent the mean and bars the SEM. *P<0.05, paired Student's t-test. B) Time-course of the effect of CBH microinjection into the BST on MAP and HR before (filled circles) or after (open circles) pretreatment of the ipsilateral or contralateral PVNwith CoCl2. CBH injections were made at time 0. Circles represent the mean and bar the SEM. Data were analyzed using two-way ANOVA. 37B R A I N R E S E A R C H 1 3 9 3 ( 2 0 1 1 ) 3 1 – 4 3 site, did not affect the cardiovascular response to the microinjection carbachol into the BST. The absence of effect was not due to an insufficient dose of CoCl2, because in previous studies it has been reported that the microinjection of such dose into the PVNwas effective to inhibit vasopressin- mediated pressor responses observed after the injection of image of Fig.�6 Fig. 7 – A) Photomicrograph of a coronal brain section showing a microinjection site in the ipsilateral PVN, in relation to BSTmicroinjection site, of one representative animal. B) Diagrammatic representationmodified from the rat brain atlas of Paxinos andWatson (1997), indicating aCSF (open circles) and CoCl2 (filled black circles)microinjection sites in the ipsilateral PVN. 3V, third ventricle; IA, interaural coordinates (mm); LH, lateral hypothalamus; ox, optic chiasm; and sox, supraoptic decussation. 38 B R A I N R E S E A R C H 1 3 9 3 ( 2 0 1 1 ) 3 1 – 4 3 noradrenaline into either the BST or the lateral septal area (Crestani et al., 2009b; Scopinho et al., 2008). Pretreatment of either the ipsilateral or the contralateral SON with CoCl2 blocked the pressor and bradycardiac responses caused by the microinjection carbachol into the BST. Together, these results suggest that synapses in the SON, but not in the PVN, mediate the cardiovascular responses to the microinjection of carba- chol into the BST. There is neuroanatomic evidence that the BST projects to the SON and that such projections aremainly ipsilateral (Dong and Swanson, 2003, 2004, 2006). The existence of bilateral neural connections between the two SON was suggested by electrophysiological and in vivo studies, thus supporting our results that both SON are involved in the mediation of the cardiovascular response to the microinjection of carbachol into the BST. Takano et al. (1990) reported that one-third of the vasopressin-containing neurons tested in the SON were excited by electric stimulation of the contralateral SON. In the same study, those authors reported that vasopressin neurons tested in the SON were not antidromically activated by a contralateral SON stimulation, thus suggesting that neural connections between the bilateral SON are mainly polysynaptic. It was also reported that antidiuretic effect associated with noradrenaline microinjection into the SON was inhibited either by a lesion of the contralateral SON or its pretreatment with adrenoceptor antagonists (Tsushima et al., 1996), indicating the existence of bilateral adrenergic neural connections between supraoptic nuclei. Because the pressor response to the microinjection of carbachol into the BST was inhibited by the blockade of either the ipsilateral or the contralateral SON, it is possible that carbachol administration into the BST activates a pathway from the BST to the ipsilateral SON, in relation to BST microinjection site, which would stimulate neuron(s) that project to contralateral SON, thus suggesting that carbachol responses would depend on a bilateral SON cross-talking. Therefore, activation of vasopres- sinergic neurons in the contralateral SON in relation to BST stimulation site would mediate pressor response to carbachol administration into the BST. A schematic representation sketching the mechanism by which carbachol microinjection into the BST evokes a vasopressin-mediated pressor response is presented in Fig. 9. The pathway for the neural connection between bilateral SON is not totally understood. Moos and Richard (1989) concluded that the supraventricular gray commissura is important for interconnection of oxytocin-containing neurons in the SON, because synchronization of oxytocin-containing neurons in the bilateral SON disappeared after an inter hemisphere sectioning (including the supraventricular gray commissura and the corpus callosum), but persisted after a superficial interhemisphere sectioning that was limited to the corpus callosum. Therefore, the supraventricular gray com- missura is a possible pathway for interconnections between bilateral SON vasopressin-containing neurons. Also, other image of Fig.�7 Fig. 8 – A) Photomicrograph of a coronal brain section showing a microinjection site in the contralateral PVN, in relation to BSTmicroinjectionsite, of one representative animal. B)Diagrammatic representation,modified fromthe rat brainatlas of Paxinos andWatson (1997), indicating aCSF (open circles) and CoCl2 (filled black circles) microinjection sites in the contralateral PVN. 3V, third ventricle; IA, interaural coordinates (mm); LH, lateral hypothalamus; ox, optic chiasm; and sox, supraoptic decussation. Fig. 9 – Schematic representation illustrating the proposed mechanism by which carbachol (CBH) microinjection into the BST evokes vasopressin release. ac— anterior commissure, AVP— vasopressin, CBH— carbachol, and M2— subtype M2 of cholinergic muscarinic receptor. 39B R A I N R E S E A R C H 1 3 9 3 ( 2 0 1 1 ) 3 1 – 4 3 connections between bilateral supraoptic nuclei, through the medulla oblongata and pons, have been suggested to exist (Tsushima et al., 1996), thus indicating alternative pathways for a bilateral SON cross-talking. It has been reported that the microinjection of noradren- aline into the BST also evoked pressor response that was mediated by an acute release of vasopressin into the circulation (Crestani et al., 2007). This response was blocked by PVN pretreatment with CoCl2 and not affected by SON blockade (Crestani et al., 2009b), thus suggesting a major involvement of PVN magnocellular neurons without any significant involvement of neurons in the SON. The present study led to the interesting observation that BST noradrener- gic and cholinergic neurotransmissionsmodulate vasopressin release into the circulation through different neural pathways. This modulation of the vasopressin release by BST noradren- ergic and cholinergic neurotransmission through specific neural pathway may have a physiological importance, since BST neurons can stimulate vasopressin release through local cholinergic receptor despite of changes in pathway related to local noradrenergic neurotransmission, or vice versa. There is evidence pointing to the BST as a relay in the neural circuitry connecting limbic structures that are known to modulate neuroendocrine responses, such as the medial image of Fig.�8 image of Fig.�9 40 B R A I N R E S E A R C H 1 3 9 3 ( 2 0 1 1 ) 3 1 – 4 3 and central amygdala, hippocampus and medial prefrontal cortex (Choi et al., 2007; Feldman et al., 1995; Herman and Cullinan, 1997; Herman et al., 2005; Ulrich-Lai and Herman, 2009). This idea is reinforced by results showing that BST lesions inhibit amygdala and hippocampus-related neuroen- docrine responses (Feldman et al., 1990; Zhu et al., 2001). Based on this, the present results suggest that BST cholinergic neurotransmission could be an important system in the neural circuitry of neuroendocrine regulation involved in the integration to vasopressin systemic release by SON. In summary, the present results indicate that cardiovas- cular responses following carbachol microinjection into the BST are mediated by SON magnocellular neurons, without significant involvement of those in the PVN. The results also indicate that responses to the carbachol microinjection into the BST are mediated by a pathway involving a bilateral SON cross-talking, possibly through ipsilateral projections from the BST to the SON and activation of vasopressinergic neurons in the contralateral SON. 4. Experimental procedures 4.1. Animals Sixty-four male Wistar rats weighing 230–270 g were used. Animals were kept in the Animal Care Unit of the Department of Pharmacology of the School of Medicine of Ribeirão Preto, University of São Paulo. Rats were kept under a 12 h:12 h light– dark cycle (lights on between 06:00 am and 6:00 pm) and had free access to water and standard laboratory food. Housing conditions and experimental procedures were approved by the University of São Paulo Animal Ethical Committee, which complies with the Guiding Principles of Research Involving Animals and Human Beings of the American Physiological Society. 4.2. Surgical preparation Four days before the experiment rats were anesthetized with tribromoethanol (250 mg⁄kg, i.p.). After scalp anesthesia with 2% lidocaine, the skull was exposed and stainless steel guide cannulae (26 G) were implanted in the BST and in the SON or PVN, using a stereotaxic apparatus (Stoelting, Wood Dale, Illinois, USA). Cannulae were positioned 1 mm above injection sites. Stereotaxic coordinates for cannula implantation in the BST, PVN or SON were selected according to the rat brain atlas of Paxinos and Watson (1997). Cannula was implanted unilaterally in the BST and stereotaxic coordinates were: anteroposterior: +8.6 mm from the interaural, lateral: 4.0 mm from the medial suture, ventral: −5.8 mm from the skull, with a lateral inclination of 23°. Cannulae were implanted in the ipsilateral or contralateral PVN, in relation to BST cannula, and stereotaxic coordinates were: anteroposterior: +7.2 mm from the interaural, lateral: 2 mm from the medial suture, ventral: −6.9 mm from the skull, with a lateral inclination of 12°. Cannulae were implanted in the ipsilateral or contralateral SON, in relation to BST cannula, and stereotaxic coordinates were: anteroposterior: +6.9 mm from the interaural, lateral: 1.8 mm from the medial suture, ventral: −8.1 mm from the skull. Cannulaewere fixed to the skull with dental cement and one metal screw. After surgery, the animals received a poly- antibiotic veterinarian preparation of streptomycins and penicillins (i.m., 0.27 mg/kg, Pentabiotico®; Fort Dodge, Cam- pinas, SP, Brazil), to prevent infection, and the nonsteroidal anti-inflammatory flunixine meglumine (i.m., 0.025 mg/kg, Banamine®; Schering Plough, Cotia, SP, Brazil), for post- operative analgesia. One day before the experiment, animals were anesthetized with tribromoethanol (250 mg⁄kg, i.p.) and a catheter was inserted into the abdominal aorta through the femoral artery for arterial pressure andHR recording. Catheters consisted of a 4 cm piece of PE-10 heat-bound to a 13 cm piece of PE-50 (Clay Adams, Parsippany, NJ, USA). The catheters were tunneled under the skin and exteriorized on the animal's dorsum. After surgery, animals were kept in individual cages, which were later used for transport to the experimental room. The nonsteroidal anti-inflammatory flunixine meglumine (i.m., 0.025 mg⁄kg, Banamine®; Schering Plough, Cotia, SP, Brazil) was administered for postoperative analgesia. 4.3. Measurement of cardiovascular responses On the day of the experiment, the arterial cannulas were connected to a pressure transducer. The pulsatile arterial pressure (PAP) of freely moving animals was recorded using an HP-7754A amplifier (Hewlett Packard, Palo Alto, CA, USA) and an acquisition board (MP100A; Biopac Systems Inc., Goleta, CA,USA) connected to a computer. Mean arterial pressure (MAP) and HR values were derived from PAP recordings and processed on-line. 4.4. Drug microinjection in the central nervous system The needles (33 G; Small Parts, Miami Lakes, FL, USA) used for microinjection into the BST, SON and PVN were 1 mm longer than the guide cannulas and were connected to a 2 μL syringe (7002 KH; Hamilton, Reno, NV, USA) through PE-10 tubing. The needle was carefully introduced into the guide cannula without touching or restraining the animal and drugs were injected in a final volume of 100 nL. After a 20 s period, the needle was removed. 4.5. Radioimmunoassay Blood samples were collected in chilled plastic tubes contain- ing heparin (10 μL of heparin per mL of collected blood). Samples were centrifuged at 4 °C, 3000 rpm for 20 min. Plasma vasopressin levels were measured by specific radioimmuno- assay after previous extraction fromplasmausing acetone and petroleum ether (Glick and Kagan, 1979; Elias et al., 1997). The recovery rateswere greater than87%. Theassay sensitivity and intra- and inter-assay coefficients of variation were 0.9 pg/mL, 4.6 and 18.6%, respectively. All samples from a single experiment were assayed in duplicate in the same assay. 4.6. Drugs Carbachol (Sigma, St Louis, MO, USA) and CoCl2 (Sigma) were dissolved in artificial cerebrospinal fluid (aCSF), with the following composition: 100 mM NaCl, 2 mM Na3PO4, 2.5 mM 41B R A I N R E S E A R C H 1 3 9 3 ( 2 0 1 1 ) 3 1 – 4 3 KCl, 1.0 mM MgCl2, 27 mM NaHCO3 and 2.5 mM CaCl2 (pH 7.4). Tribomoethanol (Sigma) and urethane (Sigma) were dissolved in saline (0.9% NaCl). Flunixine meglumine (Banamine®, Schering Plough, Brazil) and poly-antibiotic preparation of streptomycins and penicillins (Pentabiotico®, Fort Dodge, Brazil) were used as provided. 4.7. Experimental protocols On the day of the experiment, animals were transported to the experimental room and were allowed 60 min period to adapt to the experimental room conditions, such as sound and illumination, before starting arterial pressure and HR record- ing. The experimental room was acoustically isolated and a constant background noise was generated by an air exhauster to minimize sound interference within the experimental room. 4.7.1. Effect of aCSF or carbachol microinjection into the bed nucleus of the stria terminalis of unanesthetized rats in plasma vasopressin levels This experiment aimed to study the effect of carbachol microinjection into the BST of unanesthetized rats on plasma vasopressin levels. For this, two different groups of animals received vehicle (aCSF, 100 nL, n=6) or carbachol (1 nmol/ 100 nL, n=6) injected into the BST (Alves et al., 2007). Five minutes after BST treatment, animals were decapitated and blood samples were collected to determine of plasma vaso- pressin levels. 4.7.2. Effect of supraoptic nucleus pretreatment with aCSF or CoCl2 on cardiovascular responses to carbachol microinjection into the bed nucleus of the stria terminalis These experiments aimed to study the involvement of the SON in cardiovascular responses to carbachol microinjection into the BST of unanesthetized rats. For this, animals were divided into two groups, ipsilateral and contralateral SON groups. In the ipsilateral SON group, rats had cannulas implanted unilaterally in the BST and in the ipsilateral SON, in relation to BST cannula, and were subdivided into vehicle (aCSF, 100 nL, n=7) and CoCl2 (1 mM/100 nL, n=7) groups (Busnardo et al., 2007; Crestani et al., 2009a, 2009b; Scopinho et al., 2008). In the contralateral SON group, rats had cannulas implanted unilaterally in the BST and in the contralateral SON and were subdivided into vehicle (aCSF, 100 nL, n=6) and CoCl2 (1 mM/100 nL, n=6) groups (Alves et al., 2007; Busnardo et al., 2007; Crestani et al., 2009a, 2009b). Carbachol (1 nmol/ 100 nL) was microinjected into the BST on the first day and again 24 h later, at 10 min after aCSF or CoCl2 microinjection into the SON (Alves et al., 2007). Different set of animals received aCSF or CoCl2 into the SON in either ipsilateral or contralateral SON groups. 4.7.3. Effect of paraventricular nucleus pretreatment with aCSF or CoCl2 in cardiovascular responses to carbachol microinjection into the bed nucleus of the stria terminalis These experiments aimed to study the involvement of the PVN in cardiovascular responses following carbachol microinjection into the BST of unanesthetized rats. For this, animals were also divided in two groups, ipsilateral and contralateral PVN groups. In the ipsilateral PVN group, rats had cannulas implanted unilaterally in the BST and in the ipsilateral PVN, in relation to BST cannula, andwere subdivided in vehicle (aCSF, 100 nL, n=7) andCoCl2 (1 mM/100 nL, n=7) groups (Alves et al., 2007; Crestani et al., 2009a, 2009b; Scopinho et al., 2008). In the contralateral PVN group, rats had cannulas implanted unilaterally in the BST and in the contralateral PVN and were further subdivided in vehicle (aCSF, 100 nL, n=6) and CoCl2 (1 mM/100 nL, n=6) group (Alves et al., 2007; Crestani et al., 2009a, 2009b; Scopinho et al., 2008). Carbachol (1 nmol/100 nL)wasmicroinjected into theBST on the firstdayandagain24 h later, at 10 minafter aCSForCoCl2 microinjection into the PVN (Alves et al., 2007). Different set of animals received aCSF or CoCl2 into the PVN in either ipsilateral PVN or contralateral PVN groups. 4.8. Histological determination of microinjection sites At the end of the experiment, animals were anesthetized with urethane (1.25 g⁄ kg i.p.) and 100 nL of 1% Evan's Blue dye was injected into the BST, SON and PVN as an injection site marker. Animals were submitted to intracardiac perfusion with saline (0.9% NaCl) followed by 10% formalin. Brains were removed and post fixed for 24 h at 4 °C and 40 μm sections were cut with a cryostat (CM 1900; Leica, Wetzlar, Germany). Brain sections were stained with 1% neutral red for light microscopy analysis. The actual placement of the microinjec- tion needleswas determined according to the rat brain atlas of Paxinos and Watson (1997). 4.9. Statistical analysis Data are presented asmean±SEM. ThemaximumMAP andHR responses to carbachol microinjection into the BST, MAP and HR basal values and the effect of BST treatment with aCSF or carbachol in plasma vasopressin levels were compared using paired Student's t-test. Time-course curves of the MAP and HR changes caused by carbachol microinjection into the BST before and after SON or PVN pharmacological manipulation were compared using two-way ANOVA for repeated measure- ments (treatment vs. time) with repeated measures on the second factor. Significance was set at P<0.05. Acknowledgments The authors wish to thank Ivanilda Fortunato, Simone Guilhaume and Milene M. Lopes for technical help. Alves and Busnardo is supported by FAPESP post-doctoral fellowship (2010/09462-9) and (09/05308-8) respectively. Gomes is sup- ported by FAPESP PhD fellowship (2010/17343-0). The present researchwas supported by grants from the CNPq (306381⁄2003- 6, 505394⁄2003-0 and 504321/2009-9), FAPESP and FAEPA. R E F E R E N C E S Altura, B.M., Altura, B.T., 1984. Actions of vasopressin, oxytocin, and synthetic analogs on vascular smooth muscle. Fed. 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Distribution of muscarinic cholinergic high and low affinity agonist binding sites: a light microscopic autoradiographic study. Brain Res. Bull. 12, 233–243. Zhu, W., Umegaki, H., Suzuki, Y., Miura, H., Iguchi, A., 2001. Involvement of the bed nucleus of the stria terminalis in hippocampal cholinergic system-mediated activation of the hypothalamo-pituitary–adrenocortical axis in rats. Brain Res. 916, 101–106. Hypothalamic supraoptic but not paraventricular nucleus is involved in cardiovascular responses to carbachol microinjected ... Introduction Results Cardiovascular responses to aCSF or carbachol microinjection into the bed nucleus of the stria terminalis of unanesthetized... Effect of aCSF or carbachol microinjection into the bed nucleus of the stria terminalis in plasma vasopressin levels Effect of supraoptic nucleus pretreatment with aCSF or CoCl2 in cardiovascular responses to carbachol microinjection into t... Ipsilateral supraoptic nucleus Contralateral supraoptic nucleus Effect of paraventricular nucleus pretreatment with aCSF or CoCl2 in cardiovascular responses to carbachol microinjection i... Ipsilateral paraventricular nucleus Contralateral paraventricular nucleus Discussion Experimental procedures Animals Surgical preparation Measurement of cardiovascular responses Drug microinjection in the central nervous system Radioimmunoassay Drugs Experimental protocols Effect of aCSF or carbachol microinjection into the bed nucleus of the stria terminalis of unanesthetized rats in plasma va... Effect of supraoptic nucleus pretreatment with aCSF or CoCl2 on cardiovascular responses to carbachol microinjection into t... Effect of paraventricular nucleus pretreatment with aCSF or CoCl2 in cardiovascular responses to carbachol microinjection i... Histological determination of microinjection sites Statistical analysis Acknowledgments References