Functional topography of cardiovascular ... - Wiley Online Library

Clinical and Experimental Pharmacology and Physiology (2016) 43, 484–493

doi: 10.1111/1440-1681.12542

ORIGINAL ARTICLE

Functional topography of cardiovascular regulation along the rostrocaudal axis of the rat posterior insular cortex Fernanda Ribeiro Marins,* Marcelo Limborcßo-Filho,* Carlos Henrique Xavier,* Vinicia C Biancardi,‡ Gisele C Vaz,* Javier E. Stern,‡ Stephen M Oppenheimer† and Marco Antonio Peliky Fontes* *Department of Physiology and Biophysics, INCT, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil, †Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, Maryland and ‡Department of Physiology, Georgia Regents University, Augusta, GA, USA

SUMMARY Cardiovascular (CV) representation has been identified within the insular cortex (IC) and a lateralization of function previously suggested. In order to further understand the role of IC on cardiovascular control, the present study compared the CV responses evoked by stimulation of N-metil-D-aspartate (NMDA) receptors in the right and left posterior IC at different rostrocaudal levels. Intracortical microinjections of NMDA were performed into the IC of male Wistar rats anaesthetized with urethane (1.4 g/kg) prepared for blood pressure, heart rate and renal sympathetic nerve activity. Gene expression of NMDA receptor subunits NR2A and NR2B in the IC was confirmed by RT-PCR. Immunofluorescence for the NMDA receptor NR1 subunit was demonstrated in the IC (coordinates anteroposterior (AP) +1.5, 0.0 and 1.5 mm). A cardiac sympathoinhibitory site was identified, more rostrally located than identified in previous studies. A site of sympathoexcitatory cardiac control was identified more caudal to this region in agreement with earlier work. Under the experimental conditions, no lateralization of cardiovascular function was identified with chemical stimulation eliciting the same responses from either left or right insular cortices. No tonic role of the insula on cardiovascular control was identified with the use of the NMDA antagonist, AP-5. Periinsular microinjection of NMDA was without cardiovascular effect indicating the specificity of the insula as a cardiovascular regulatory site. The current study reveals a functional topography for autonomic cardiovascular control along the rostrocaudal axis of the posterior IC. Key words: autonomic nervous system, cardiovascular control, insular cortex, N-metil-D-aspartate (NMDA) .

Correspondence: Marco Antonio Peliky Fontes, Laboratorio de Hipertens~ao, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, Pampulha, UFMG - ICB - Bl: B4/Sala: 244, Brasil. Email: [email protected] Received 19 June 2015; revision 16 December 2015; accepted 4 January 2016. © 2016 John Wiley & Sons Australia, Ltd

INTRODUCTION Various cortical regions are involved in cardiovascular control including the medial prefrontal cortex,1 anterior cingulate cortex2 and the insular cortex (IC).3,4 In the IC, rostrocaudal separation of viscerotopic representation has been demonstrated.4 Within the cardiovascular region itself, there is evidence for a separation of regulatory zones, with pressor and tachycardia sites more rostrally situated to those from which depressor and bradycardia effects are elicitable on both electrical and chemical stimulation.5,6 These responses are robust and not anaesthesia dependent.6 Additionally, lateralization of cardiovascular response has been demonstrated in the human,7 rat8 and monkey insula9 in electrical stimulation, extracellular recording and lesion studies. To investigate lateralization of cardiovascular regulatory function more specifically, the use of excitatory amino acid (EAA) receptor agonists, which stimulate cell bodies and dendritic processes of neurons within the injection site,10 could provide further insight into the role of IC in cardiovascular control. The main objective of this work was to gather in a single study a re-evaluation of the cardiovascular responses along the rat posterior IC using EAA receptor stimulation. For this purpose, the autonomic and cardiovascular effects resulting from stimulation of N-metil-D-aspartate (NMDA) glutamate receptors along the rostrocaudal axis of the posterior IC were determined. Based on previous studies,5,6 three different subregions were chosen within the IC (anteroposterior (AP) +1.5, 0.0 and 1.5 mm from bregma, respectively). Using the same approach, the study also investigated a possible functional asymmetry for autonomic cardiovascular responses between the left and right sides of posterior IC. Given the data from clinical studies indicating that the gravity of cardiovascular and autonomic derangement after IC stroke in humans is hemisphere dependent,11–13 the current data may shed further light on this important and controversial area.

RESULTS Table 1 shows the baseline values of cardiovascular parameters of all groups before any peripheral injection or central microinjection. No differences between the groups were observed.

397 11 82 8 392 8 97 6

NMDA microinjection at AP +1.5 mm

396 3 95 2

Unilateral microinjection of NMDA into the right or left IC evoked decreases in heart rate (DHR, R = 38 5 vs L = 35 4 bpm, P = 0.0001 and P < 0.0001, respectively vs baseline) and renal sympathetic nerve activity (DRSNA, R = 25 6 vs L = 23 2%, P = 0.0015 and P = 0.0002 respectively vs baseline) associated with increases in mean arterial pressure (DMAP, R = +15 2 vs L = +17 3 mmHg, P < 0.0001 and P = 0.0001 respectively vs baseline). The bradycardia lasted 9 min, the fall in RSNA lasted 11 min and the pressor response lasted 8 min (Fig. 1a,b). There were no differences in the magnitude or latency of the cardiovascular response dependent on right or left insular injection (Fig. 2a–c). Microinjection of vehicle control into R-IC at this level did not change cardiovascular parameters.

394 3 81 3 369 10 79 5 364 14 89 6

NMDA microinjection at AP 0.0 mm

IC, insular cortex; HR, heart rate; MAP, mean arterial pressure; NMDA, N-metil-D-aspartate.

396 2 94 2 405 3 89 4 338 14 73 3 400 3 85 7 392 8 87 4 389 5 85 4 399 5 91 2 HR (bpm) MAP (mmHg)

485

Activation of NMDA receptors at different levels of the IC on heart rate, mean arterial pressure and renal sympathetic nerve activity

383 6 79 2

NMDA R-IC NMDA+ AP-5 NMDA L-IC NMDA + MA NMDA+ PZ NMDA+AP5 NMDA L-IC NMDA R-IC Control R-IC

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Coordinates anteroposterior(bregma) Protocols

Table 1 Baseline cardiovascular values of all experimental groups before any injections of drugs

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Cardiovascular control in rat insular cortex

Unilateral microinjection of NMDA into either R-IC or L-IC evoked increases in HR (DHR, R-IC = +36 7 vs L-IC = +40 8 bpm, P = 0.0041 and P = 0.0038, respectively vs baseline), small changes in MAP (DMAP, R-IC = +7 2 vs L-IC = +5 1 mmHg, P = 0.0106 and P = 0.0059, respectively vs baseline) and increased RSNA (DRSNA, R-IC = +31 5 vs L-IC = +28 4%, P = 0.001 and P = 0.0009, respectively vs baseline). The tachycardia lasted 14 min and it was accompanied by a gradual increase in blood pressure. The increases in RSNA lasted 12 min (Fig. 1c,d). No differences were found in the magnitude of the responses when comparing R-IC and L-IC (Fig. 2d–f). Microinjection of vehicle control into this coordinate in R-IC did not change the parameters evaluated. NMDA microinjection at AP 1.5 mm Microinjection of NMDA at this site did not change cardiovascular parameters (DHR, R-IC = +1 2 vs L-IC = +2 3 bpm; DMAP, R-IC = +2 2 vs L-IC = +3 2 mmHg; and DRSNA, R-IC = +1 5 vs L-IC = +5 5%). Likewise, microinjection of vehicle control into this coordinate did not change cardiovascular parameters (DHR = 2 1 bpm, DMAP = +3 1 mmHg, and DRSNA = +1 3%). NMDA microinjection at peri-insular sites Microinjection of NMDA outside of the IC anatomical limits (negative control) did not evoke significant changes in cardiovascular parameters (mean maximum changes: DHR, +2 2 bpm; DMAP, +2 1 mmHg; DRSNA, +2 1%). Examples of injection sites outside of the IC limits are shown in the histological analysis. Effect of blockade of NMDA receptors on MAP, HR and renal sympathetic nerve activity responses evoked by NMDA stimulation Since the magnitudes and profile of the responses evoked by NMDA microinjection into R-IC or L-IC were similar, all subsequent protocols were evaluated only in the R-IC.

© 2016 John Wiley & Sons Australia, Ltd

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FR Marins et al. Effect of blockade of muscarinic cholinergic receptors in the bradycardia evoked by stimulation of NMDA receptors into +1.5 mm AP level of the IC To investigate whether the bradycardic effect evoked by injection of NMDA at the +1.5 mm coordinate was mediated by vagal activity, we tested the effect of NMDA in this coordinate in the presence of the muscarinic cholinergic antagonist, methylatropine. Intravenous injection of methylatropine increased baseline HR (D= +55 10 bpm, P = 0.0149 vs baseline) without major changes in MAP (D= 0 4 mmHg, P = 0.0587 vs baseline) and in RSNA (D= +11 7%, P = 0.2041 vs baseline). Methylatropine attenuated the bradycardia evoked by NMDA microinjection into R-IC at AP +1.5 mm (DNMDA = 38 6 bpm vs DNMDA after methylatropine = 19 1 bpm, P = 0.0285). The increases in MAP (D= +11 2 mmHg, P = 0.2427 vs NMDA) and the decreases in RSNA (D= 23 7%, P = 0.8070 vs NMDA) evoked by microinjection of NMDA were not changed by methylatropine (Fig. 2a–c). Effect of blockade of alpha 1-adrenergic receptors on the bradycardia evoked by stimulation of NMDA receptors into +1.5 mm AP level of the IC

Fig. 1 Representative tracings showing effects on baseline cardiovascular and autonomic parameters (HR, heart rate; MAP, mean arterial pressure; RSNA, renal sympathetic nerve activity) elicited by NMDA (0.2 mm) microinjection into the (a) R-IC or (b) L-IC in +1.5 mm level and into the (c) R-IC or (d) L-IC in 0.0 mm level of rostrocaudal axis (n = 6 each side). The numbers near the traces are the mean values for the predrug and mean maximum value for postdrug conditions for the respective group. Statistical analysis: Paired t test, P < 0.05, * versus predrug values.

To confirm the specificity of the NMDA effects, the response evoked by NMDA was tested in the presence of its selective antagonist, AP-5. Microinjection of AP-5 at AP +1.5 mm or AP 0.0 mm coordinates did not evoke significant changes in baseline cardiovascular and autonomic parameters (IC +1.5 mm: DHR, 1 3 bpm; DMAP, +1 2 mmHg; DRSNA, +2 5%; IC 0.0 mm: DHR, +3 1 bpm; DMAP, +2 7 mmHg; DRSNA, +2 5%). However, AP-5 blocked the response to NMDA microinjection at both coordinates (AP +1.5 mm: DHR = 3 1 bpm; DMAP = 1 2 mmHg; DRSNA = 5 4%; AP 0.0 mm: DHR = 3 2 bpm; DMAP = 2 1 mmHg; DRSNA = 4 3%; Fig. 2a–f).

To investigate whether the bradycardic effect elicited by NMDA injection at the +1.5 mm site was elicited by a baroreceptor reflex response, we tested the effect of NMDA in this coordinate in presence of the a-adrenergic antagonist, prazosin. Intravenous injection of prazosin decreased HR (DHR = 15 7 bpm, P = 0.0071 vs baseline) and MAP (DMAP = 33 3 mmHg, P < 0.0001 vs baseline) without major changes in RSNA (DRSNA = +5 4%, P = 0.3323 vs baseline). The subsequent microinjection of a1-adrenergic agonist phenylephrine in three rats did not significantly change the cardiovascular parameters (DHR, 7 4 bpm, P = 0.1845 vs baseline; DMAP, +4 1 mmHg, P = 0.0741 vs baseline; DRSNA, +4 8%, P = 0.5896 vs baseline) indicating complete blockade of a1- receptors. The pressor response and the decrease in RSNA evoked by NMDA microinjection at +1.5 mm were abolished when prazosin was previously injected (DMAP = +2 1 mmHg, P < 0.0001 vs NMDA, and DRSNA = 7 4%, P = 0.0263 vs NMDA). Prazosin also attenuated the bradycardic effect (DNMDA = 38 6 bpm vs DNMDA after prazosin = 17 3 bpm, P = 0.0263) evoked by NMDA microinjection into R-IC at AP +1.5 mm level (Fig. 2a–c). Effect of blockade of b1 adrenergic receptors on the tachycardia evoked by stimulation of NMDA receptors into 0.0 mm AP level of the IC To investigate whether the tachycardia evoked by NMDA injection at the 0.0 mm coordinate was sympathetic mediated, we tested the effect of NMDA at this coordinate in presence of the b1 adrenergic receptor antagonist, atenolol. Intravenous injection of atenolol decreased baseline HR (DHR = 65 6 bpm, P = 0.0007 vs baseline) without significant changes in MAP (DMAP = +5 4 mmHg P = 0.1412 vs baseline) or in RSNA (DRSNA = +7 4% P = 0.1479 vs baseline). Atenolol completely blocked the tachycardic and pressor responses evoked


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Fig. 2 Changes in heart rate (HR), mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) elicited by NMDA microinjection into the right insula (R-IC) or left insula (L-IC) into rostrocaudal coordinates: (a–c) +1.5 mm or (d–f) 0.0 mm. NMDA was injected alone (n = 6 each side) or after: AP-5 (n = 4), methylatropine (n = 4), prazosin (n = 6) or atenolol (n = 4). Saline (n = 4) was used as volume control. Statistical analysis: ANOVA one-way with Newman-Keuls post test, P < 0.05, * all groups versus saline, # all groups versus NMDA.

by subsequent NMDA microinjection into R-IC at AP 0.0 mm level (DHR, NMDA = +36 7 bpm vs NMDA after atenolol = +2 4 bpm, P = 0.0076; and DMAP, NMDA = +7 2 mmHg vs NMDA after atenolol = 2 3 mmHg, P = 0.0356). The increase in RSNA evoked by microinjection of NMDA into this coordinate was not altered by atenolol (DRSNA, NMDA = +31 5% vs NMDA after atenolol = +25 4%, P = 0.4257; Fig. 2d–f). Expression of NMDA receptor subunits NR2A and NR2B and immunofluorescence for NMDA receptor subunit NR1 into posterior IC In order to further confirm the functional findings evoked by NMDA microinjection, we evaluated the NMDA receptor expression and the specific presence of NR1 subunit in the posterior IC. Supporting the functional findings, RT-PCR analysis showed two products of 402 bp and 449 bp, indicating the expression of NR2A and NR2B subunits, respectively, in the posterior IC (Fig. 3a). Immunofluorescence of confocal photomicrographs from the sections of the three regions of the IC — coordinates AP +1.5 mm (Fig. 3b,e), 0.0 mm (Fig. 3c,f) and 1.5 mm (Fig. 3d,g) — reveals expression of NR1 subunit highly concentrated in the same region where NMDA injection elicited cardiovascular effects, mainly on the granular and dysgranular IC. Histological analysis NMDA cardiovascular and sympathetic responses were mainly elicited in the deeper layers of the IC (layer V and layer VI) and

were primarily located in the granular and dysgranular areas (Fig. 4a–i). An illustration representing the main findings is shown in Fig. 4j. Anatomical experiments: descending anatomical projections from rostral (+1.5 mm) and intermediate (0.0 mm) IC regions Anatomical experiments were performed with the aim of understanding the origin of the differential cardiovascular responses between rostral and intermediate regions of IC. Two possible downstream synaptic relays were targeted, the caudal portion of the ventrolateral medulla (CVLM) and the rostral portion of the ventrolateral medulla (RVLM). These regions were chosen considering that CVLM and RVLM play a sympathoinhibitory and sympathoexcitatory roles in cardiovascular control, respectively.14 Low power photomicrograph depicting the retrograde tracer injection site at CVLM (Fig. 5a,b) or RVLM (Fig. 5c,d). Two images (white light and fluorescence light) were taken from the same section to show the precise location of the rhodamine retrobeads injection (red area, arrow; Fig. 5a and c, respectively CVLM and RVLM). Neurons retrogradely labelled from CVLM were located in the IC coordinate AP +1.5 mm (Fig. 5e,f, respectively right and left rostral IC region) and coordinate AP 0.0 mm (Fig. 5i,j, respectively right and left intermediate IC region). Interestingly, neurons retrogradely labelled from RVLM were found just at coordinate AP 0.0 mm. (Fig. 5k,l, respectively right and left intermediate IC region). No neuronal labelling was found at coordinate AP +1.5 mm (Fig. 5g,h, respectively right and left rostral IC region). Note that the projections from cortical regions to CVLM or RVLM were bilateral in all cases.


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Fig. 3 (a) Agarose gel electrophoresis of NMDA receptor RT-PCR products amplified from posterior IC. The analysis showed two products of 402 bp (lanes 1, 3 and 5) and 449 bp (lanes 2, 4 and 6), indicating the presence of subunits NR2A and NR2B, respectively. Lane M contains a molecular DNA marker with fragment sizes (bp) of 1000 to 100 (n = 3). Immunofluorescence of representative confocal photomicrographs from the sections of the three regions of the IC (coordinates AP +1.5 mm (b and e), 0.0 mm (c and f) and 1.5 mm (d and g)) stained for NMDA NR1 subunit. Left side 10x and right side 25x magnification (n = 3).

DISCUSSION The present study found that activation of NMDA receptors in the IC produces autonomic and cardiovascular responses with different profiles along the rostrocaudal axis of IC (summarized in Fig. 4j). These results extend previous investigations by compar-

ing the response of right and left insular neurons to NMDA microinjection, as opposed to investigation of one side alone. Using this approach, no clear evidence of functional asymmetry for autonomic cardiovascular control between the left and right sides of the posterior IC was found. The choice of these three different regions (coordinates) aimed to cover the posterior IC subregions involved in cardiovascular control as previously described in the literature.5,8,15,16 In the rostral portion of posterior IC (coordinate AP +1.5 mm) pressor and bradycardic effects can be elicited with chemical stimulation. Results obtained with NMDA in this region after peripheral blockade of a-adrenergic or muscarinic cholinergic receptors indicate that this region of IC is mainly involved in controlling a non-renal sympathoexcitatory vasomotor component. The pressor response is abolished by prazosin which largely attenuates (but does not abolish) the HR response. This suggests that the bradycardic response elicited at this site may in part be mediated by reflex baroreceptor response to the pressor effect evoked by the NMDA injection, but also the response may be due to a direct centrally-mediated effect on HR. Since the bradycardic response was only partially abolished by methylatropine without a significant effect on blood pressure this would suggest that either the residual bradycardia was mediated by a withdrawal of sympathetic tone. We speculate therefore that the bradycardic effect seen on NMDA injection at +1.5 mm is mediated by two mechanisms: in the presence of a pressor response, by a reflex baroreceptor-mediated bradycardia and chiefly a parasympathetic effect and, secondly, when the pressor response is blocked, by activation of a cardiac sympathoinhibitory pathway. Conversely, activation of NMDA receptors at the intermediate region of IC (coordinate AP 0.0 mm) resulted in a significant increase in MAP that was accompanied by tachycardia and large increases in RSNA. Similar results were described in previous studies after chemical or electrical stimulation at equivalent coordinates.6,17,18 The tachycardic and pressor responses, but not the increases in RSNA, evoked by NMDA at coordinate AP 0.0 mm were completely abolished by atenolol. This confirm and extends previous findings suggesting that the tachycardia is sympathetically mediated,5 and further suggests that the pressor response evoked from this IC region results primarily from increases in cardiac activity. Taken together, these findings suggest that the intermediate (AP 0.0 mm) region of posterior IC controls coordinated cardiac and renal sympathoexcitatory responses. Activation of NMDA receptors into coordinate AP 1.5 mm did not promote a significant change in cardiovascular parameters. The current data using NMDA are in agreement with the findings of Yasui and colleagues suggesting that this area is outside of the cardiovascular regulatory zone in the rat IC6 and with a previous study by Butcher and Cechetto, which, targeting a similar IC coordinate, demonstrated that injections of D,L-homocysteic acid, a glutamatergic non-specific agonist, did not change HR, MAP and RSNA.17 The cardiovascular effects evoked by NMDA at coordinates AP +1.5 mm and AP 0.0 mm were completely abolished by AP5. This finding added to the results showing gene expression of NMDAR subunits (NR2A and NR2B) and the presence of NR1 in the IC, confirms the specificity of NMDA effects. Furthermore, confirming the data, the NR1 subunit is highly concentrated in


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Fig. 4 From rostral to caudal the figure shows schematic coronal sections of the rat brain corresponding to the atlas of Paxinos and Watson39 illustrating sites of injections into the IC. Each level is accompanied by a representative photomicrograph example of a microinjection site into (a, d and g) right or (c, f and i) left sides: (a–c) at AP level +1.5 mm; (d–f) at AP level 0.0 mm and (g–i) at AP level 1.5 mm. (j) Schematic drawing of rat brain (sagittal plane, redrawing from Paxinos and Watson:39 the scheme summarizes the main findings of the current manuscript showing the topographical organization of the rat insular cortex on the cardiovascular and autonomic control. CL, claustrum; IC, insular cortex; AHC, anterior hypothalamic area, central part; ac, anterior commissure; cc, corpus callosum; LV, lateral ventricle; 3V, 3rd ventricle; CPu, caudate putamen (striatum); och, optic chiasm; gcc, genu of the corpus callosum; aca, anterior commissure, anterior part.


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Fig. 5 Distribution of neurons within the insular cortex rostral, +1.5 mm and intermediate, 0.0 mm that are retrogradely labelled from ventrolateral medulla (n = 2, each region). Representative example of a rhodamine beads injection site in (a,b) the CVLM (AP: –14.6 mm) or (c,d) RVLM (AP: – 11.8 mm), in white light and fluorescence light, respectively. The precise location of the rhodamine retrobeads injection is demonstrated by the red area (arrow) for (a) CVLM and (c) RVLM. Neurons retrogradely labelled (excited by helium-neon laser at 543 nm) from CVLM were located in the (e,f) rostral (respectively right and left IC AP +1.5 mm) and (i,j) intermediate (respectively right and left IC AP 0.0 mm) IC regions. Neurons retrogradely labelled from RVLM were found just at intermediate IC (k,l; respectively right and left IC AP 0.0 mm). No neuronal labelling was found at rostral IC (g,h; respectively right and left IC AP +1.5 mm). Note that the projections from cortical regions to CVLM or RVLM were bilateral in all cases. (a–d) at 5x of magnification and (e-l) at 25x of magnification, 30 lm z-stack. Scale bars 100 lm in (a–d) and 50 lm in (e–l). CVLM, Caudal ventrolateral medulla; RVLM, rostral ventrolateral medulla; Py, pyramidal tract; IO, inferior olive; NA, nucleus ambiguous; NTS, nucleus of the solitary tract; CC, central canal; CL, claustrum; IC, insular cortex; ac, anterior commissure; cc, corpus callosum; LV, lateral ventricle; CPu, caudate putamen (striatum); och, optic chiasm; gcc, genu of the corpus callosum; aca, anterior commissure, anterior part.

the same region where NMDA injection elicited cardiovascular effects, mainly on the layers V and VI of the granular and dysgranular IC. The selective competitive NMDA antagonist, AP-5, has high affinity for NR1, NR2A and NR2B subunits19,20 and blocks the effects of NMDA in a dose dependent way.21–23 It is worth noting that both NMDA receptor subunits evaluated are critical to determine the biophysical, pharmacological and functional properties of NMDA receptor.24 Injection of NMDA into peri-insular zones did not result in any cardiovascular responses, again emphasizing the specificity and localization of this effect within the insula. The current data showing that injections of AP5 did not affect resting HR or blood pressure, indicates that NMDA receptors in the IC may not be involved in any tonic inhibitory activity on cardiovascular function at least under these experimental conditions. A recent study by Alves and colleagues showed that bilateral injection of the competitive NMDAR antagonist, LY235959, into the IC has no effect on baseline HR or

MAP.25 Altogether, these findings indicate that NMDAR in the IC are not tonically involved in the control of autonomic output to the cardiovascular system. Aiming to understand the differential responses evoked by NMDA microinjection at rostral (+1.5 mm) and intermediate (0.0 mm) IC, this study re-evaluated the descending projections from these two specific subregions of the IC. We targeted the ventrolateral medulla (VLM) subregions, the rostral portion of the VLM (RVLM) and the caudal portion of the VLM (CVLM), since these regions play a sympathoexcitatory and sympathoinhibitory role in cardiovascular control, respectively.14 Interestingly, we found that both regions of the IC (+1.5 mm and 0.0 mm) send efferent projections to the CVLM. However, only the intermediate region of IC (IC 0.0 mm) sends projections to the RVLM. RVLM neurons provide sympathoexcitatory drive to the heart, kidney and vascular resistance.26,27 Therefore, the selective projection to the RVLM can explain the sympathoexci-


Cardiovascular control in rat insular cortex tatory profile of the responses evoked by NMDA injection into the 0.0 mm coordinate of the IC. On the other hand, the cardiac and renal inhibitory effects evoked by NMDA injection into the +1.5 mm coordinate can be mediated by descending projections via the CVLM. Activation of CVLM neurons inhibits the activity of RVLM neurons diminishing the sympathoexcitatory drive to the cardiovascular system.27 Therefore, this anatomical finding supports our conclusion that at least part of the bradycardia evoked by NMDA injection into the +1.5 mm coordinate was mediated by a withdrawal of sympathetic tone. Previous studies have demonstrated lateralized differences in insular representation of cardiovascular control. Cortical lateralization of cardiovascular control has been demonstrated in both animal and human studies8,28 in general indicates a predominance of parasympathetic cardioregulatory sites in the left insula, and of sympathetic cardiovascular sites in the right insula. The present findings do not support a left-parasympathetic, right-sympathetic division of insular function in regard to NMDA stimulation. Possible reasons include the difference between the stimulation tool used in this compared to the other studies, that can affect fibres of passage which could be recruited in electrical stimulation studies,5,7,8 or destroyed in lesion studies.28–30 In humans, IC stroke causes imbalance of the autonomic control. In left IC (L-IC) cases, there is predominance of increase in sympathetic output, whereas right IC (R-IC) stroke seems to drive parasympathetic-mediated responses. These changes in autonomic outflow could contribute to the generation of cardiovascular dysfunctions11,12,31,32 including arrhythmias.13,33 Additionally, intraoperative electrical stimulation demonstrated functional asymmetry in the IC for cardiovascular control,34 suggesting that pathophysiological activation of the insular cortex by stroke, epileptic seizure, or under conditions of severe emotional stress could predispose autonomic and cardiovascular disarrangement.11,12,31 It is important to mention that, in contrast to what occurs in humans after IC stroke, the NMDA microinjection in the current study reached a more delimited region/population of neurons and stimulates specific subunits of NMDA receptors. On the other hand, stroke distribution territories are not as well delimited and may result in neuronal silencing and death, with additional release of different toxic factors to surrounding neurons and glial cells.35–37 The topographical functional arrangement for the autonomic cardiovascular regulation described in the current study allows us to speculate that the cardiac arrhythmias resulting from stroke can result from damage to intermingled IC regions. Perspectives The current study confirms and extends previous findings demonstrating the presence of a significant functional topography for autonomic cardiovascular control along the posterior IC rostrocaudal axis. This topographical organization allows us to understand the cardiovascular and autonomic derangement resulting from unilateral IC damage in humans as a result of concomitant damage to functionally different IC sub regions controlling the autonomic output. Future experiments using focal experimental stroke along the rat IC will also be valuable to unravel the mechanisms involved in generation of cardiac arrhythmias resulting from damage to IC in humans.

491 METHODS

Animals All experiments were performed on male Wistar rats (300– 320 g), bred at the animal facilities of the Biological Sciences Institute (CEBIO, UFMG) or were purchased from Harlan Laboratories (Indianapolis, IN, USA) conducted in accordance with the guidelines established by CETEA/UFMG (protocol 11 412/ 2012), and in strict compliance with NIH guidelines and carried out in agreement with Georgia Regents University Institutional Animal Care and Use Committee Guidelines. Experimental procedures In all functional experiments rats were anesthetized with urethane (1.4 g/kg intraperitoneally (i.p.), and the trachea was cannulated to maintain open airways. Body temperature was kept in the range of 37–37.5°C with a heating pad (Physitemp TCAT-2DF Controller). The animals were positioned in a stereotaxic frame (Lab Standard with18 Degree Ear bars, Stoelting, IL, USA), and a small unilateral craniectomy was made to allow the insertion of a glass pipette (Sigma-Aldrich, St Louis, MO, USA) into the IC. Catheters were placed into the femoral artery to record MAP and HR, and into the vein for the injection of drugs. Using a retroperitoneal approach, the left renal nerve was isolated and prepared for recording as previously reported.38 After all surgical procedures, a minimum period of 20 min was held for cardiovascular parameters stabilization. Drugs Drugs employed in the experiments were: (i) NMDA (0.2 mm); (ii) NMDA antagonist, D()-2-Amino-5-phosphonopentanoic acid, AP-5 (5 mm); (iii) vehicle (NaCl 0.9%); (iv) muscarinic antagonist, methylatropine (3 mg/kg, intravenous, i.v.); (v) alpha 1-adrenergic antagonist, prazosin hydrochloride (1 mg/kg, i.v.); (vi) alpha 1-adrenergic agonist, phenylephrine hydrochloride (50 lg/mL, i.v.); (vii) b1 adrenergic antagonist, atenolol (2 mg/ kg, i.v.). The volume for central microinjections was 100 nL, and for peripheral injections 0.1 mL. Drugs were purchased from Sigma Chemical, St. Louis, MO, USA. Experimental design Using data from previous studies indicating the position of the cardiovascular zone within the insula,5,6 NMDA (0.2 mm/ 100 nL) microinjections were made at three different sites rostrocaudally in either the right or left insula: AP +1.5, 0.0 or 1.5 mm from the bregma, 5.8 mm lateral and 7.0 mm ventral39 and cardiovascular and autonomic responses recorded. Only one site was investigated per animal and six rats were used per insular coordinate site. As a control for non-specific effects of the microinjection, saline (NaCl 0.9%) was injected into the right IC (n = 4 for each coordinate). NMDA microinjection outside the insular cortex (n = 6 in each coordinate) was included as a negative control group. Since the nature and magnitude of the NMDA evoked responses were similar for right and left IC and in order to minimize the number of animals, the following protocols were performed only into right IC but in separated groups of rats: AP-


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5 followed by NMDA; methylatropine followed by NMDA; prazosin followed by NMDA; atenolol followed by NMDA. At the end of each experiment histological analysis was performed as previously described.1 Sites of microinjection were confirmed using the atlas of Paxinos and Watson as a reference.39 Expression of NMDA receptor subunits NR2A and NR2B and immunofluorescence for NMDA receptor subunit NR1 into posterior insular cortex To evaluate the gene expression of NMDA receptor subunits NR2A and NR2B from tissue samples corresponding to the IC (punch from AP coordinate +1.5 to 1.5 mm), reverse transcription polymerase chain reaction (RT-PCR) was performed. Forward and reverse primers for each subunit (NR2A - Forward: 50 -T TCATCTGGGAGCACCTCTT-30 ; Reverse: 50 -GGCCACAAAT GTTTGGAGTT-30 / NR2B - Forward: 50 -GGCATTGCTATCC AAAAGGA-30 ; Reverse: 50 -GACAGGTTGGCCATGTTCTT-30 ), as previously described.40 An electrophoresis was conducted for visualization of the bands corresponding to the gene expression of NR2A and NR2B subunits in a 1% agarose gel. Immunofluorescence in insular cortex tissue: animals were transcardially perfused with 0.01 mol/L phosphate-buffered saline (PBS; 150 mL) and 4% paraformaldehyde (PFA; 350 mL). Brains were dissected and post fixed overnight in 4% PFA followed by cryoprotection in PBS containing 30% sucrose for 3 days at 4°C. Sections of 50 lm, containing the evaluated IC regions were collected, following a pre-incubation in 5% horse blocking serum for 1 h, primary antibody incubation, NR1 (goat anti-NMDA zeta 1-polyclonal, diluted 1 : 100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), for 48 h41 and revealed by secondary reaction with anti-goat FITC (1 : 250) (Jackson Immunoresearch, West Grove, PA, USA) as previously described.42 The specificity of each antibody was tested by the omission of the primary antibody. Immunofluorescence from insular cortex sections was examined with an upright Zeiss LSM510 confocal microscope as previously described.42 Anatomical experiments: descending anatomical projections from rostral (+1.5 mm) and intermediate (0.0 mm) IC regions Rats were anaesthetized with isoflurane 4%, and the animal head was placed in a stereotaxic frame. For microinjections into the CVLM or the rostral portion of the ventrolateral medulla (RVLM), a 4 mm burr hole was made in the skull and undiluted rhodamine-labelled microspheres (Lumaflor, Naples, FL, USA) were pressure injected unilaterally (100 nL) using the bregma as reference. The coordinates for injections were: 14.6 mm posterior and 2.0 mm lateral at a depth of 8.0 mm below the dura for CVLM and 11.96 mm posterior, 2.2 mm lateral at a depth of 8.0 mm for RVLM, as determined by the atlas of Paxinos and Watson.39 Buprenorphine (Bruprenex C3 0.3 mg/kg; Butler Schein/NLS, Dublin, OH) was given in a single subcutaneous injection immediately after surgery to minimize postsurgical pain. Animals were allowed to recover and 7 days after surgery were killed by deep anesthetization and an intraperitoneal injection of sodium pentobarbitone (50 mg/kg⁻¹) and transcardially perfused. The brain was dissected and maintained in 4% PFA for 4 h and

then transferred to a 30% sucrose solution for 48 h. Insular cortex sections from rostral (+1.5 mm) and intermediate (0.0 mm) regions were collected and then processed for analysis. Acquired stained sections were examined with a Leica TCS SL confocal microscope system (Leica Microsystems, Mannheim, Germany), using a helium-neon laser to excite rhodamine fluorochrome at 543 nm. All images were acquired and digitized using identical acquisition settings. Statistical analysis Analyses were performed by Student’s t test for paired and unpaired data or one-way ANOVA followed by Newman-Keuls post test. Significance was taken at P < 0.05. Data are expressed as mean standard error of the mean (SEM).

ACKNOWLEDGEMENTS The authors thank the financial support from Brazilian agencies, CNPq (PQ 306 000/2013-0) and Fundacß~ao de Amparo a Pesquisa do Estado de Minas Gerais (Edital Universal 2013 Fapemig, CBB-APQ-00 353-13). Marins FR received a Masters PostGraduation Program fellowship from CAPES / Brazil Current address for CHX is Dept. of Physiological Sciences, Federal University of Goias, Goiânia, Brazil.

DISCLOSURE The authors declare that there are no conflicts of interest.

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