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Central nuclei mediating estrogen-induced changes in autonomic tone and baroreceptor reflex in male rats
Brain Research 961 (2003) 190–200 www.elsevier.com / locate / brainres
Research report
Central nuclei mediating estrogen-induced changes in autonomic tone and baroreceptor reflex in male rats Tarek M. Saleh*, Barry J. Connell Department of Anatomy and Physiology, University of Prince Edward Island, 550 University Avenue, Charlottetown, Canada C1 A 4 P3 Accepted 29 October 2002
Abstract The current investigation examines the significance of estrogen in central cardiovascular regulatory nuclei in modulating autonomic tone and baroreceptor reflex function. Experiments were done in anaesthetized male Sprague-Dawley rats. Changes in autonomic tone were assessed by monitoring vagal and renal efferent nerve activities before and following bilateral injection of estrogen into select central autonomic nuclei. In the first study, selective blockade of neurotransmission through the central nucleus of the amygdala (CNA), lateral hypothalamic area (LHA) and ventral posteromedial thalamic nucleus (VPM) using the local anaesthetic lidocaine was done to determine which nuclei were involved in mediating the autonomic changes observed following bilateral injections of estrogen into the insular cortex (IC). In the second study, the role of the parabrachial nucleus (PBN) in mediating the autonomic changes observed following bilateral estrogen injections into the CNA, LHA, VPM and IC was determined by blocking neurotransmission through the PBN using lidocaine. Injections of estrogen into the IC produced a significant increase in renal sympathetic nerve activity (RSNA; from 1062 to 2464 mV/ sec; p,0.05). This estrogen-induced increase in RSNA was significantly attenuated when lidocaine was pre-injected into the LHA, CNA or PBN (5566, 3364 and 9167% decrease respectively; p,0.05) but not when injected into the VPM (1666% decrease; p.0.05). Injection of estrogen into the CNA resulted in a significant decrease in RSNA (4865%; p,0.05) whereas estrogen injection into the LHA resulted in a significant increase (2864%; p,0.05) in RSNA. Pre-injection of lidocaine into the PBN resulted in complete blockade of the autonomic changes observed following estrogen injection into the CNA but did not affect the changes observed following estrogen injection into the LHA. These results suggest that estrogen acting in forebrain and midbrain cardiovascular nuclei activated efferent pathways which synapse in the LHA, CNA and / or PBN prior to projecting to autonomic preganglionic nuclei to affect autonomic tone. These nuclei may therefore provide an added level of processing and / or integration of the autonomic response(s) following activation by local or systemic estrogen. 2002 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Insular cortex; Central nucleus of the amygdala; Lateral hypothalamic area; Ventral posteromedial thalamus; Parabrachial nucleus
1. Introduction Estrogen has become well established as a central modulator of autonomic tone and baroreceptor reflex function. Manipulation of systemic estrogen levels whether via ovariectomy or exogenous supplementation produces profound changes in sympathovagal balance. Specifically,
*Corresponding author. Tel.: 11-902-566-0819; fax: 11-902-5660832. E-mail address: [email protected] (T.M. Saleh).
elevated plasma estrogen is associated with increased parasympathetic tone and enhanced baroreflex sensitivity in male rats [27,28]. Female rats benefit further from estrogen supplementation demonstrating an additional attenuation of sympathetic tone [22,29]. The cardiac baroreflex is in turn directly affected by changes in autonomic tone. Testing of the baroreflex with phenylephrine following systemic injection of estrogen in both male and ovariectomized female rats has demonstrated a potent estrogenic enhancement of baroreflex sensitivity [22,23,28–30]. Further, these estrogen-induced cardiovascular and autonomic effects were abolished by either
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medullary or spinal (intrathecal) injections of an estrogen receptor antagonist [22,23,28–30]. Autoradiographic evidence has demonstrated a generous distribution of estrogenic receptors within the central nervous system raising the possibility that estrogen may exert effects on several biological systems. The presence of estrogen receptors and estrogen-concentrating neurons in various cardiovascular and autonomic nuclei throughout both the fore- and hindbrain of both the rat and the cat [10,14,34,35] has raised the possibility that estrogen may act within the autonomic network as it were to influence cardiovascular and autonomic responses to various environmental stimuli. Indeed, this proposition is supported by evidence that direct injection of estrogen into the insular cortex (IC), the central nucleus of the amygdala (CNA), the lateral hypothalamic area (LHA) and the parabrachial nucleus (PBN) [35], all of which are known to modulate baroreflex sensitivity primarily via efferent projections to autonomic preganglionic nuclei in the medulla [15], has been shown to alter both baroreflex sensitivity and autonomic tone [22,23,26]. The effects of estrogen within the IC are of particular interest because the IC is a forebrain area involved in the integration of sensory and visceral (including cardiovascular) information from peripheral receptors [21]. Cardiovascular and autonomic responses are coordinated via the insula’s efferent connections with various subcortical nuclei including the parabrachial nucleus (PBN), central nucleus of the amygdala (CNA), the lateral hypothalamic area (LHA) and the ventral posteromedial thalamic nucleus (VPM) [6,16,21,32]. Each of these nuclei appear to modulate baroreflex sensitivity primarily via efferent projections to autonomic preganglionic nuclei in the medulla or spinal cord [15]. Taken together, these results suggest that endogenous estradiol within this and possibly other CNS nuclei plays an important role in modulating autonomic and / or cardiovascular function in male rats and could be a potential source of therapeutic intervention in pathologies involving autonomic dysfunction. The present study examined the role of various subcortical nuclei hypothesized to be involved in mediating the estrogen-induced changes in baseline autonomic tone as well as baroreceptor reflex function in male rats. First, to determine which subcortical nuclei are involved in modulating the autonomic changes observed following estrogen injection into the IC, the reversible anesthetic, lidocaine hydrochloride, was injected into the CNA, LHA, VPM or PBN. Secondly, injections of lidocaine were made into the PBN to determine its role in mediating the estrogeninduced cardiovascular changes following estrogen injection into the CNA, LHA or VPM. Changes in autonomic tone were measured by assessing vagal and renal efferent nerve activities before and following central estrogen injection. The baroreceptor reflex was evoked using the intravenous administration of phenylephrine and the ratio of the magnitude of the reflex bradycardia to the phenylep-
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hrine-evoked pressor response before and following central injections of estrogen was used to assess changes in this reflex.
2. Materials and methods All experiments were carried out in accordance with the guidelines of the Canadian Council on Animal Care and were approved by the University of Prince Edward Island Animal Care Committee.
2.1. General surgical procedures Experiments were performed on a total of 68 male Sprague–Dawley rats (250–275 g; Charles Rivers; Montreal, QC) anesthetized with sodium thiobutabarbital (Inactin; RBI, Natick, MA; 100 mg / kg; i.p.). Polyethylene catheters were inserted into the right femoral artery (PE50; Clay Adams, Parsippany, NJ) and the right femoral vein (PE-10) to monitor blood pressure and heart rate, and to permit the intravenous administration of drugs. Arterial blood pressure was measured with a pressure transducer (Gould P23 ID; Cleveland, OH) connected to a Gould Pressure Processor. Heart rate was determined from the pulse pressure using a Gould tachograph (Biotach). Blood pressure and heart rate data were displayed using the PolyView Pro / 32 data acquisition and analysis system (Grass; Warwick, RI). An endotracheal tube was inserted and animals were paralyzed (decamethonium hydrochloride; Sigma–Aldrich, St. Louis, MO; 0.5 mg / kg; i.v.) and ventilated with room air (Harvard rodent ventilator; 65 strokes / min; 2.5 ml tidal volume).
2.2. Autonomic nerve isolation and recording All animals were instrumented to record both parasympathetic and sympathetic efferent nerve activity. To record parasympathetic efferent nerve activity, the left cervical vagus nerve was isolated through a midline cervical incision and a platinum bipolar recording electrode was secured in place using dental impression material (Basilex; Ash Temple, Bedford, NS). Vagal nerves were crushed distal to the recording electrode to allow for the recording of efferent activity only. To record sympathetic efferent nerve activity, the right kidney was exposed and a platinum bipolar recording electrode was secured to a branch of the renal nerve. The multi-unit activity from each nerve was amplified by a Grass model P55 preamplifier (Grass, Warwick, RI) with a 100 Hz to 3 kHz bandpass and 60 Hz notch filter and then displayed using the PolyView Pro / 32 data acquisition system (Grass). Raw nerve activity was sampled at a rate of 2000 / s.
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2.3. Central microinjections Following nerve isolations, animals were placed in a David Kopf (Tujunga, CA) stereotaxic frame and holes (see Table 1 for coordinates; [19]) were drilled bilaterally through the temporal-occipital bones to permit the stereotaxic insertion of a 30-gauge stainless steel 1 ml Hamilton microsyringe. Each group consisted of four animals. Every animal received bilateral injections into two separate central nuclei (i.e. lidocaine or saline injected into the first nucleus followed by estrogen or saline injected into the second nucleus). In the first experiment, lidocaine hydrochloride (5%, 100 nl per side; Vetoquinol Canada, Joliette, PQ) or saline vehicle (0.9%; 100 nl / side) was injected bilaterally into either the central nucleus of the amygdala (CNA; n54 / drug), lateral hypothalamus (LHA; n54 / drug), parabrachial nucleus (PBN; n54 / drug) or ventral posteromedial thalamus (VPM; n54 / drug) 10 min prior to the bilateral injection of estrogen (17b-estradiol; Sigma– Aldrich; 0.5 mM; 100 nl / side) into the insular cortex (IC; n528). This concentration of estrogen was determined as the optimal dose for local microinjections based on a dose–response relationship preformed in a previous studies in our laboratory [23,31]. In the second experiment, saline or lidocaine was injected bilaterally into the PBN (n524) 10 min prior to estrogen injections into either the CNA (n54), LHA (n54) or VPM (n54). Finally, to determine the effect of lidocaine on cardiovascular and autonomic function, lidocaine was injected into the PBN, CNA, LHA or VPM (n54 / nucleus). Central injection of lidocaine, at a similar volume and concentration as that used in the present study has previously been shown to suppress neuronal activity in a region approximately 1 mm in diameter from the tip of the injector [9,25]. The effects on neurotransmission recovered within 90 min [25].
central injection and then 5, 30, 60, 90 and 120 min following the final central injection. The ratio of the peak changes in the magnitude of the reflex bradycardia to the magnitude of the phenylephrine-induced pressor response (DHR /DMAP beats / min per mmHg) was calculated and used as an index of the sensitivity of the baroreceptor reflex. Changes in sympathovagal balance were also assessed at each time point by measuring baseline levels and phenylephrine-induced changes in vagal and renal efferent nerve activity.
2.5. Data analysis All data are presented as mean6standard error of the mean (S.E.M). Changes from baseline in mean arterial blood pressure, heart rate, baroreceptor sensitivity, sympathetic and parasympathetic nerve activities were analyzed with one-way analysis of variance (ANOVA) for repeated measures, and when necessary, followed by a Student– Newman–Keuls post hoc analysis. Differences between groups at identical time points were determined using unpaired student t-tests with a Bonferroni correction for multiple comparisons. In all cases, differences were considered significant if P#0.05.
2.6. Histology At the end of each experiment animals were perfused transcardially with 0.9% saline followed by 10% formalin. The brains were removed and stored in 10% formalin until the location of the microsyringe tracks could be verified histologically from thionin-stained coronal sections (100 mm).
3. Results
2.4. Baroreflex testing and autonomic tone measurements In all animals, an index of baroreflex sensitivity was measured following the intravenous administration of phenylephrine hydrochloride (PE; 0.1 mg / kg). All phenylephrine injections were made 5 min prior to each Table 1 Stereotaxic coordinates used to locate central autonomic nuclei for the bilateral injection of drugs
IC CNA LHA VPM PBN
Anterior–posterior
Medial–lateral
Dorsal–ventral
0.8 21.8 22.2 22.7 29
65.3 64.1 61.8 62.3 62.0
25 27.3 28 26.2 26.4
IC, insular cortex; CNA, central nucleus of the amygdala; LHA, lateral hypothalamic nucleus; VPM, ventral posteromedial thalamus; PBN, parabrachial nucleus (modified coordinates obtained from Ref. [19]).
Prior to estrogen injection, mean arterial pressure (MAP) and heart rate (HR) were 110611 mmHg and 325621 beats / min, respectively (n568). Baseline values for vagal parasympathetic nerve activity (VPNA) and renal sympathetic nerve activity (RSNA) were 962 mV/ s (n5 68) and 1163 mV/ s (n568), respectively. Control injections of saline into forebrain nuclei had no effect on baseline MAP, HR or nerve activities, nor on the phenylephrine-evoked changes in these parameters. For all animals, injection of phenylephrine prior to central injection of estrogen produced an increase in MAP (2364 mmHg; n568) which was accompanied by a reflexive decrease in HR (21162 beats / min; n568; see Fig. 1 at 25 min). The index of baroreflex sensitivity at this time point was 0.4860.05 beats / min per mmHg (n568). During baroreflex testing with phenylephrine, VPNA was increased 45611% (n568) and RSNA was decreased 55612% (n5 68; see Fig. 1 at 25 min) relative to baseline levels. No significant changes in baseline MAP, HR, VPNA or
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Fig. 1. Cardiovascular and autonomic responses to bilateral estrogen injection into the central nucleus of the amygdala (CNA). (A) Representative physiograph tracings demonstrating that baseline blood pressure (25 min) as well as the pressor response to phenylephrine injection (0.1 mg / kg; ↑) are significantly decreased at 30 min post-estrogen injection (0.5 mM; 100 nl / side). (B) Representative samples of renal sympathetic nerve activity (RSNA) and vagal parasympathetic nerve activity (VPNA) indicating basal autonomic tone before (25 min) and following estrogen injection into the CNA. Changes in nerve activities measured during baroreflex testing before (25 min) and following estrogen injection into CNA originate at the arrow (↑) and were measured for 1 min immediately following the PE injection (time scale bar51 min). Both baseline and evoked nerve activities returned to pre-injection values at 60 min post-estrogen injection.
RSNA could be measured at any time point following the injection of lidocaine into the VPM, CNA, LHA or PBN (n54 / nucleus; data not shown; P.0.05). However, following lidocaine injection into the PBN, a significant increase in baroreflex sensitivity was measured at 30 min (from 0.4860.06 to 0.9860.15 beats / min per mmHg; P,0.05). This increase in baroreflex sensitivity measured at 30 min was accompanied by a significant enhancement in both the phenylephrine-evoked reflex bradycardia (22365 beats / min) and VPNA (73611%; n54; P,0.05) compared to pre-lidocaine values (21162 beats / min and 45611%, respectively). These changes in cardiovascular and autonomic reflex function following lidocaine injection into the PBN recovered when measured at 90 min postinjection (P.0.05).
3.1. The role of subcortical nuclei in mediating the autonomic response to estrogen injection into IC Bilateral injection of estrogen into IC produced a significant increase in baseline RSNA within 30 min of
injection (from 1062 to 2664 mV/ s; n54; Fig. 2; P, 0.05). Changes in baseline RSNA returned to pre-injection values by 60 min post-estrogen injection. The phenylephrine-evoked pressor response (2062 mmHg) and reflex bradycardia (21062 beats / min) were not significantly different at all time points following estrogen injection into the IC (P.0.05). As a result, baroreflex sensitivity did not differ from pre-injection values throughout the time course of the experiment (0.5560.06 beats / min per mmHg; P. 0.05). Similarly, reflex changes in RSNA and VPNA during baroreflex testing were not significantly different from pre-injection values (1063 mV/ s and 1364 mV/ s, respectively; P.0.05). Bilateral injection of lidocaine into the PBN resulted in almost complete blockade (91.466% decrease; n54; Fig. 2; P,0.05) of the increase in baseline sympathetic tone observed following estrogen injection into the IC as described above. Although not as dramatic, the sympathoexcitatory response to estrogen injection into the IC was also significantly attenuated when lidocaine was pre-injected into either the CNA (3364% decrease; n54; P,
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Fig. 2. Effects of bilateral estrogen injection (0.5 mM; 100 nl / side) into the insular cortex (IC; filled circle) on renal sympathetic nerve activity (RSNA). Also shown is the change in the magnitude of this sympathoexcitatory response when lidocaine (1lidocaine) was pre-injected into either the central nucleus of the amygdala (CNA; open circle), lateral hypothalamic area (LHA; closed triangle), ventral posteromedial thalamus (VPM; open triangle) or parabrachial nucleus (PBN; closed square). In all cases, * indicates significance from the pre-estrogen injection value (25 min), and ** indicates significance from the post-estrogen injection into the IC value at 30 min (P,0.05; ANOVA).
0.05) or LHA (5566% decrease; n54; Fig. 2; P,0.05). In contrast to the above three nuclei, following lidocaine injection into the VPM, the sympathoexcitatory response to estrogen injection into the IC was not significantly different (1666% decrease; n54; Fig. 2; P.0.05).
3.2. The role of the PBN in mediating the autonomic and cardiovascular responses to estrogen injection into the CNA Bilateral injection of estrogen into CNA significantly decreased baseline MAP (to 7263 mmHg) and RSNA (to 5.361 mV/ s) within 30 min of injection (n54; P,0.05; Fig. 3A,C) but did not alter baseline HR or VPNA (P. 0.05; Fig. 3B,D). The changes in MAP and RSNA returned to pre-injection values by 60 min post-estrogen injection (P.0.05; Fig. 3A,C). Also at 30 min postestrogen injection, the phenylephrine-evoked pressor response was significantly attenuated (from 2464 to 1064 mmHg; P,0.05; Fig. 1). In contrast, the reflexive bradycardia (21063 beats / min) evoked by phenylephrine injection was at no time significantly different from the phenylephrine-evoked reflex bradycardia observed prior to estrogen injection (21162 beats / min; P.0.05; Fig. 1). The value obtained for the index of baroreflex sensitivity at 60 min post-estrogen injection was significantly enhanced (1.160.05 beats / min per mmHg; P,0.05) relative to the pre-injection value (0.4860.05 beats / min per mmHg). Following estrogen injection into the CNA, the magnitude
of the reflex decrease in RSNA was significantly attenuated when compared to that obtained prior to estrogen injection (a decrease of 2568% from baseline vs. 55612% prior to estrogen injection; P,0.05). This effect was evident at 30 min post-estrogen injection only. Phenylephrine-evoked changes in VPNA during baroreflex testing were not different at any time point throughout the course of the experiment (P.0.05). The decrease in baseline MAP and RSNA described above following estrogen injection into the CNA, were all significantly attenuated (greater than 90% for each parameters; P,0.05) following the prior bilateral injection of lidocaine into the PBN (Fig. 3A,C). As a result, the estrogen-induced enhanced baroreflex sensitivity was blocked and remained not different from baseline values (P.0.05). Also, the observed decrease in the magnitude of the RSNA following phenylephrine injection in the presence of estrogen in the CNA, was blocked when lidocaine was pre-injected into the PBN (P.0.05).
3.3. The role of the PBN in mediating the autonomic and cardiovascular responses to estrogen injection into the LHA Bilateral injection of estrogen into LHA resulted in a significant increase in baseline RSNA within 30 min of injection (from 960.7 to 11.261 mV/ s; n54; P,0.05; Fig. 4C) but did not affect baseline MAP, heart rate or
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Fig. 3. Effects of bilateral estrogen injection into the central nucleus of the amygdala (CNA) on the baseline cardiovascular and autonomic responses in the presence of either saline or lidocaine pre-injected into the parabrachial nucleus (PBN). Average baseline values of mean arterial pressure (MAP; A), heart rate (HR; B), renal sympathetic nerve activity (RSNA; C) and vagal parasympathetic nerve activity (VPNA; D) before (25 min) and following bilateral injection of estrogen (0.5 mM; 100 nl / side) into CNA are shown. In all cases, * indicates significance (P,0.05; ANOVA) from pre-estrogen injection value (25 min).
VPNA (P.0.05; Fig. 4A,B,D). The change in RSNA returned to pre-injection values by 60 min post-estrogen injection (P.0.05; Fig. 4C). The phenylephrine-evoked pressor response or reflex bradycardia were not affected following estrogen injection into the LHA compared to pre-injection values (2064 mmHg and 1063 beats / min, respectively; P.0.05). The value obtained for the index of baroreflex sensitivity was also unchanged post-estrogen injection into the LHA relative to the pre-injection value (0.4760.04 beats / min per mmHg; P.0.05). No changes in the reflex decrease in RSNA or increase in VPNA were observed following estrogen injection into the LHA (P. 0.05). The increase in baseline RSNA described above following estrogen injection into the LHA, was not significantly attenuated following the prior bilateral injection of lidocaine into the PBN (11.061 mV/ s; n54; P.0.05; Fig. 4C).
3.4. The role of the PBN in mediating the autonomic and cardiovascular responses to estrogen injection into the VPM Bilateral injection of estrogen into VPM did not affect any cardiovascular or autonomic baseline parameter measured (n54; P.0.05; not shown). Also, no changes were observed in the phenylephrine-evoked pressor response (2164 mmHg), reflex bradycardia (21062 beats / min) or reflex decrease in RSNA (49610%) and reflex increase in VPNA (4369%) following estrogen injection into the VPM (P.0.05). Therefore, when the index of baroreflex sensitivity was calculated, no significant change was observed post-estrogen injection into the VPM (0.4860.05; P.0.05). Since estrogen injection into the VPM resulted in no significant change in any of the baseline or reflex cardiovascular or autonomic measures, we did not repeat the study in the presence of lidocaine in the PBN.
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Fig. 4. Effects of bilateral estrogen injection into the lateral hypothalamic area (LHA) on the baseline cardiovascular and autonomic responses in the presence of either saline or lidocaine pre-injected into the parabrachial nucleus (PBN). Average baseline values of mean arterial pressure (MAP; A), heart rate (HR; B), renal sympathetic nerve activity (RSNA; C) and vagal parasympathetic nerve activity (VPNA; D) before (25 min) and following bilateral injection of estrogen (0.5 mM; 100 nl / side) into LHA are shown. In all cases, * indicates significance (P,0.05; ANOVA) from pre-estrogen injection value (25 min).
3.5. Histological verification of cannulae placement Data from animals in which both cannulae were not localized to the appropriate nuclei were not included in this study. Bilateral injections of estrogen observed to be unilateral or completely outside the intended region produced no significant effects on baseline parameters nor on phenylephrine-evoked changes in the same parameters (data not shown). For all animals receiving bilateral injections of estrogen (n54 / nucleus), saline (n54 / nucleus), or lidocaine (n54 / nucleus) into the IC, VPM, CNA, LHA or PBN, the sites (n564 in total) were confirmed as previously described [22,23]. Briefly, in the IC, cannulae tracks were predominantly located in the dysgranular IC between bregma 20.26 and 11.2 [19]. In the VPM, cannulae tracks were found predominantly in the parvocellular region of the VPM (VPMpc) just dorsal to the medial lemniscus be-
tween bregma 22.3 and 23.8. In the CNA, sites were located between bregma 22.12 and 22.8 evenly distributed throughout the medial and lateral subdivisions. In the LHA, sites were located between bregma 21.8 and 22.8 between the zona incerta and internal capsule at the level of the fornix and the optic tract. In the PBN, sites were predominantly located in the lateral subnucleus distributed within the dorsal, central and external and internal lateral subdivisions between bregma 29.0 and 29.7.
4. Discussion The present study demonstrated that in male rats, direct injections of estrogen into various central nuclei resulted in significant changes in cardiovascular and autonomic parameters. Specifically, estrogen injected into the IC enhanced sympathetic tone while in the in the LHA, estrogen
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injections increased both sympathetic tone and mean arterial pressure. Estrogen injections into the CNA attenuated sympathetic tone and mean arterial pressure in addition to enhancing baroreceptor reflex function. When neurotransmission through the PBN was blocked with the reversible anesthetic, lidocaine hydrochloride, the sympathoexcitatory effect of estrogen in the IC was completely blocked, whereas the cardiovascular and autonomic changes observed following estrogen injection into the CNA were only partially blocked. In contrast, the effects of estrogen in the LHA were not altered in the presence of lidocaine in the PBN. Prior injection of lidocaine into the CNA or LHA also resulted in a significant attenuation of the sympathoexcitatory effect of estrogen into the IC. Estrogen injected into the VPM did not alter any of the cardiovascular or autonomic baseline or reflex parameters measured. These results suggest that estrogen activates a population of neurons in the IC which project directly or synapse in the CNA and / or LHA prior to projecting to the PBN before synapsing on presympathetic neurons in the brainstem and spinal cord (Fig. 5). The same seems to be true for estrogen-sensitive neurons in the CNA as there appears to be a synapse in the PBN prior to influencing autonomic preganglionic neurons. The estrogen-sensitive neurons originating in the LHA, on the other hand, seem to bypass the PBN and possibly project directly to autonomic
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preganglionic nuclei in the brainstem and spinal cord (Fig. 5). A technical consideration should be taken into account with respect to the use of lidocaine to block synaptic transmission in select nuclei. Lidocaine may also inhibit fibres of passage traveling through the nucleus or in close proximity (approx. 1 mm in diameter from the tip of the injection cannulae). Previous studies using lidocaine to block synaptic transmission in the PBN and LHA [9,25] have shown that lidocaine is an effective tool in that it is reversible and thus allows for the investigation of the effect of the blockade and the recovery of responses in a reasonable amount time (minutes to hours). It is for this advantage that lidocaine was the drug of choice in this investigation. The effects of estrogen injection in all nuclei (except the VPM) seemed to have an effect within 30 min of injection. This rapid (non-genomic?) change in membrane excitability in response to estrogen may likely be mediated by the yet uncharacterized cell surface receptor for estrogen. Previous work in our laboratory has shown similar rapid actions of estrogen in central nuclei on autonomic tone which were completely antagonized by the potent estrogen receptor antagonist, ICI 182,780 [23,28,30]. Since this antagonist was not used in this study, we cannot rule out the possibility that the estrogen-induced changes in neuro-
Fig. 5. Schematic diagram illustrating the neuronal pathway suggested to be activated by estrogen-sensitive neurons in the forebrain. The diagram summarizes the results of the present investigation which demonstrates which midbrain and pontine nuclei are involved in mediating the autonomic and cardiovascular effects of estrogen in the forebrain. Solid line, suggested pathway based on results presented here; dashed lined, subsequent pathway activated in order to activate autonomic preganglionic cells in the brainstem and spinal cord. CNA, central nucleus of the amygdala; IC, insular cortex; LHA, lateral hypothalamic area; PBN, parabrachial nucleus.
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nal excitability were receptor mediated or due to nonspecific alterations in ionic conductance or membrane resistance. Both estrogen receptor subtypes (ERa and ERb) are found in all nuclei under investigation in the current study in approximately equal density [2,14,34,35]. Therefore, we cannot conclude that differential activation of one receptor subtype results in a specific autonomic change (i.e. ERa activation resulting in sympathoinhibition). Opposing effects of estrogen (i.e. sympathoexcitation in the IC and LHA versus sympathoinhibition in the CNA) may be rationalized by an altered binding affinity of estrogen to a particular receptor subtype or accessibility to the nucleus may be differentially regulated.
4.1. Subcortical sites mediating sympathetic responses from the IC The insular cortex, particularly the caudal granular insula, is a cortical area known to participate in autonomic and cardiovascular regulation [18]. Bilateral injections of estrogen into the IC of male rats increased sympathetic tone without altering baseline MAP, HR, VPNA or baroreflex sensitivity. Our laboratory has previously demonstrated that in estrogen-replaced ovariectomized female rats, bilateral injections of estrogen into the IC significantly depressed RSNA, whereas bilateral injections of estrogen into the IC of non-estrogen replaced ovariectomized female rats had no effect [23]. Comparison of the present results with our previous findings suggests that circulating estrogen levels may influence the density and / or distribution of estrogen-sensitive neurons or estrogen receptors within the IC of male versus female rats. The difference in estrogen-induced sympathetic responses from the IC of male versus female rats may be due to a gender difference in estrogen receptor density and / or sensitivity. The IC receives a variety of visceral afferent projections and has efferent connectivity with several subcortical sites, including those involved in autonomic control [6,16,21]. In particular, the IC possesses reciprocal connections with the LHA [32,36], the CNA [5,37], the VPM [5] and the PBN [32]. The interconnectivity of these nuclei would suggest that these pathways are involved in the integration of sensory and visceral information and would thus be responsible for modulating autonomic efferent tone. The estrogen-induced sympathoexcitation following injection into the IC appeared to be mediated by a synapse in the PBN since prior injection of lidocaine into this nucleus decreased the enhanced level of RSNA by 91.4%. In addition, the estrogen-induced increase in RSNA appeared to activate pathways passing through both the LHA and CNA as the sympathetic response was decreased by 55% and 33%, respectively, as a result of synaptic blockade of these nuclei. The presence of sympathoexcitatory neurons within the IC has been previously described [38], and it has been suggested that following electrical stimulation of the IC,
the LHA is a mandatory synaptic site for the transmission of subsequent neural activity resulting in autonomic responses [4]. Consistent with these results, we have demonstrated that estrogen-sensitive neurons in the IC activate a pathway to the LHA leading to sympathoexcitation. In addition, our results also demonstrated the importance of the PBN and the CNA for transmitting estrogen-induced sympathoexcitatory commands originating in the IC.
4.2. Role of the PBN in mediating cardiovascular and autonomic responses from the CNA, LHA and VPM Estrogen injections into the CNA resulted in a significant decrease in RSNA accompanied by a dramatic hypotension independent of a reflexive decrease in heart rate (or increase in VPNA). Fig. 3D suggests that VPNA was elevated at this time point, but not significantly compared to baseline (pre-injection) values and thus heart rate remained the same. It is possible that the neurons activated by estrogen in the CNA not only resulted in sympathoexcitation, but also may have been involved in simultaneous inhibition of parasympathetic tone (at least to some extent) due to the fact that, following the baroreceptor response to hypotension, VPNA did not increase and heart rate remained the same. The PBN has topographically organized afferent and efferent connections to a number of forebrain sites, including the CNA and LHA [1,5,11,17,33]. Lidocaine injections into the PBN completely blocked all estrogen-induced autonomic and cardiovascular changes from the CNA, demonstrating the importance of the PBN synapse prior to projecting to autonomic preganglionic nuclei in the brainstem. The CNA can also influence autonomic preganglionic neurons directly since efferent pathways have also been demonstrated to the nucleus tractus solitarius and the rostral ventrolateral medulla [20]. However, our results seem to suggest that the estrogen-sensitive neurons in the CNA may not directly modulate the activity of these brainstem autonomic nuclei since the effects of estrogen injections into the CNA were completely abolished following lidocaine injections into the PBN. This suggested mandatory synapse in the PBN for the CNA projection to autonomic preganglionic neurons may not be the case for all neurons in the CNA, only the ones that were activated by estrogen using our preparation and experimental paradigm. The CNA participates in the regulation of autonomic responses relative to behavioral and neuroendocrine responses to environmental stimuli. One possible physiologically relevant role for estrogen-induced cardiovascular changes within the CNA may be in modulating the response to pain. Both the PBN and CNA have been shown to receive noxious inputs [3,13]. This is particularly relevant in light of the recent literature suggesting that elevations in circulating estrogen during late gestation is associated with a significant increase in nociceptive response threshold and that this is likely due to an action
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within the dorsal horn of the spinal cord [2,12]. Along the same line of evidence, a similar role for estrogen in IC could be suggested due to the convergence of nociceptive and baroreceptive inputs previously shown in the rat [5,6,37,38] and monkey [16]. The findings reported here may suggest a novel supraspinal role for estrogen in the modulation of nociceptive inputs to the forebrain. Estrogen injections into the LHA significantly enhanced RSNA without affecting MAP, HR or VPNA. Since the prior injection of lidocaine in the PBN did not alter the enhanced sympathetic tone observed following estrogen injections into the LHA, the efferent pathways mediating this effect do not synapse within the PBN. The lateral hypothalamus is well known to be involved in the modulation of autonomic and neuroendocrine responses relative to feeding and reproductive behavior. The LHA has been demonstrated to have efferent connections to the rostral ventrolateral medulla and electrical stimulation of the LHA has been shown to result in sympathoexcitation [7,8]. Perhaps it is this estrogen-sensitive pathway originating within the LHA that is activated in order to produce the observed increase in sympathetic tone. The bilateral injection of estrogen into the VPM did not alter any of the autonomic or cardiovascular parameters measured. The PBN has reciprocal connections to the NTS [11] and is an obligatory synapse between the NTS and the VPM in the relay of visceral information destined for higher autonomic regulatory centers such as the IC [24]. Also, visceral afferent activation of the VPM can be modulated by estrogen injections into the PBN [31]. However, it appears that the VPM does not play a role in mediating estrogen-induced efferent changes in autonomic tone or baroreceptor sensitivity.
4.3. Conclusions The results of this experiment are the first to propose a functional anatomical pathway at various levels throughout the neuraxis activated by estrogen and the specific nuclei involved in mediating the autonomic and cardiovascular changes observed. Each of the forebrain nuclei examined in this study are involved in the integration of environmental stimuli with internal sensory information in order to coordinate an appropriate autonomic, endocrine and behavioral response to these stimuli. This study suggests that these nuclei are sensitive to changes in the levels of hormones which can activate the same pathways involved in mediating the autonomic and cardiovascular response in an attempt to restore homeostasis.
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Research report
Central nuclei mediating estrogen-induced changes in autonomic tone and baroreceptor reflex in male rats Tarek M. Saleh*, Barry J. Connell Department of Anatomy and Physiology, University of Prince Edward Island, 550 University Avenue, Charlottetown, Canada C1 A 4 P3 Accepted 29 October 2002
Abstract The current investigation examines the significance of estrogen in central cardiovascular regulatory nuclei in modulating autonomic tone and baroreceptor reflex function. Experiments were done in anaesthetized male Sprague-Dawley rats. Changes in autonomic tone were assessed by monitoring vagal and renal efferent nerve activities before and following bilateral injection of estrogen into select central autonomic nuclei. In the first study, selective blockade of neurotransmission through the central nucleus of the amygdala (CNA), lateral hypothalamic area (LHA) and ventral posteromedial thalamic nucleus (VPM) using the local anaesthetic lidocaine was done to determine which nuclei were involved in mediating the autonomic changes observed following bilateral injections of estrogen into the insular cortex (IC). In the second study, the role of the parabrachial nucleus (PBN) in mediating the autonomic changes observed following bilateral estrogen injections into the CNA, LHA, VPM and IC was determined by blocking neurotransmission through the PBN using lidocaine. Injections of estrogen into the IC produced a significant increase in renal sympathetic nerve activity (RSNA; from 1062 to 2464 mV/ sec; p,0.05). This estrogen-induced increase in RSNA was significantly attenuated when lidocaine was pre-injected into the LHA, CNA or PBN (5566, 3364 and 9167% decrease respectively; p,0.05) but not when injected into the VPM (1666% decrease; p.0.05). Injection of estrogen into the CNA resulted in a significant decrease in RSNA (4865%; p,0.05) whereas estrogen injection into the LHA resulted in a significant increase (2864%; p,0.05) in RSNA. Pre-injection of lidocaine into the PBN resulted in complete blockade of the autonomic changes observed following estrogen injection into the CNA but did not affect the changes observed following estrogen injection into the LHA. These results suggest that estrogen acting in forebrain and midbrain cardiovascular nuclei activated efferent pathways which synapse in the LHA, CNA and / or PBN prior to projecting to autonomic preganglionic nuclei to affect autonomic tone. These nuclei may therefore provide an added level of processing and / or integration of the autonomic response(s) following activation by local or systemic estrogen. 2002 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Insular cortex; Central nucleus of the amygdala; Lateral hypothalamic area; Ventral posteromedial thalamus; Parabrachial nucleus
1. Introduction Estrogen has become well established as a central modulator of autonomic tone and baroreceptor reflex function. Manipulation of systemic estrogen levels whether via ovariectomy or exogenous supplementation produces profound changes in sympathovagal balance. Specifically,
*Corresponding author. Tel.: 11-902-566-0819; fax: 11-902-5660832. E-mail address: [email protected] (T.M. Saleh).
elevated plasma estrogen is associated with increased parasympathetic tone and enhanced baroreflex sensitivity in male rats [27,28]. Female rats benefit further from estrogen supplementation demonstrating an additional attenuation of sympathetic tone [22,29]. The cardiac baroreflex is in turn directly affected by changes in autonomic tone. Testing of the baroreflex with phenylephrine following systemic injection of estrogen in both male and ovariectomized female rats has demonstrated a potent estrogenic enhancement of baroreflex sensitivity [22,23,28–30]. Further, these estrogen-induced cardiovascular and autonomic effects were abolished by either
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03928-8
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medullary or spinal (intrathecal) injections of an estrogen receptor antagonist [22,23,28–30]. Autoradiographic evidence has demonstrated a generous distribution of estrogenic receptors within the central nervous system raising the possibility that estrogen may exert effects on several biological systems. The presence of estrogen receptors and estrogen-concentrating neurons in various cardiovascular and autonomic nuclei throughout both the fore- and hindbrain of both the rat and the cat [10,14,34,35] has raised the possibility that estrogen may act within the autonomic network as it were to influence cardiovascular and autonomic responses to various environmental stimuli. Indeed, this proposition is supported by evidence that direct injection of estrogen into the insular cortex (IC), the central nucleus of the amygdala (CNA), the lateral hypothalamic area (LHA) and the parabrachial nucleus (PBN) [35], all of which are known to modulate baroreflex sensitivity primarily via efferent projections to autonomic preganglionic nuclei in the medulla [15], has been shown to alter both baroreflex sensitivity and autonomic tone [22,23,26]. The effects of estrogen within the IC are of particular interest because the IC is a forebrain area involved in the integration of sensory and visceral (including cardiovascular) information from peripheral receptors [21]. Cardiovascular and autonomic responses are coordinated via the insula’s efferent connections with various subcortical nuclei including the parabrachial nucleus (PBN), central nucleus of the amygdala (CNA), the lateral hypothalamic area (LHA) and the ventral posteromedial thalamic nucleus (VPM) [6,16,21,32]. Each of these nuclei appear to modulate baroreflex sensitivity primarily via efferent projections to autonomic preganglionic nuclei in the medulla or spinal cord [15]. Taken together, these results suggest that endogenous estradiol within this and possibly other CNS nuclei plays an important role in modulating autonomic and / or cardiovascular function in male rats and could be a potential source of therapeutic intervention in pathologies involving autonomic dysfunction. The present study examined the role of various subcortical nuclei hypothesized to be involved in mediating the estrogen-induced changes in baseline autonomic tone as well as baroreceptor reflex function in male rats. First, to determine which subcortical nuclei are involved in modulating the autonomic changes observed following estrogen injection into the IC, the reversible anesthetic, lidocaine hydrochloride, was injected into the CNA, LHA, VPM or PBN. Secondly, injections of lidocaine were made into the PBN to determine its role in mediating the estrogeninduced cardiovascular changes following estrogen injection into the CNA, LHA or VPM. Changes in autonomic tone were measured by assessing vagal and renal efferent nerve activities before and following central estrogen injection. The baroreceptor reflex was evoked using the intravenous administration of phenylephrine and the ratio of the magnitude of the reflex bradycardia to the phenylep-
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hrine-evoked pressor response before and following central injections of estrogen was used to assess changes in this reflex.
2. Materials and methods All experiments were carried out in accordance with the guidelines of the Canadian Council on Animal Care and were approved by the University of Prince Edward Island Animal Care Committee.
2.1. General surgical procedures Experiments were performed on a total of 68 male Sprague–Dawley rats (250–275 g; Charles Rivers; Montreal, QC) anesthetized with sodium thiobutabarbital (Inactin; RBI, Natick, MA; 100 mg / kg; i.p.). Polyethylene catheters were inserted into the right femoral artery (PE50; Clay Adams, Parsippany, NJ) and the right femoral vein (PE-10) to monitor blood pressure and heart rate, and to permit the intravenous administration of drugs. Arterial blood pressure was measured with a pressure transducer (Gould P23 ID; Cleveland, OH) connected to a Gould Pressure Processor. Heart rate was determined from the pulse pressure using a Gould tachograph (Biotach). Blood pressure and heart rate data were displayed using the PolyView Pro / 32 data acquisition and analysis system (Grass; Warwick, RI). An endotracheal tube was inserted and animals were paralyzed (decamethonium hydrochloride; Sigma–Aldrich, St. Louis, MO; 0.5 mg / kg; i.v.) and ventilated with room air (Harvard rodent ventilator; 65 strokes / min; 2.5 ml tidal volume).
2.2. Autonomic nerve isolation and recording All animals were instrumented to record both parasympathetic and sympathetic efferent nerve activity. To record parasympathetic efferent nerve activity, the left cervical vagus nerve was isolated through a midline cervical incision and a platinum bipolar recording electrode was secured in place using dental impression material (Basilex; Ash Temple, Bedford, NS). Vagal nerves were crushed distal to the recording electrode to allow for the recording of efferent activity only. To record sympathetic efferent nerve activity, the right kidney was exposed and a platinum bipolar recording electrode was secured to a branch of the renal nerve. The multi-unit activity from each nerve was amplified by a Grass model P55 preamplifier (Grass, Warwick, RI) with a 100 Hz to 3 kHz bandpass and 60 Hz notch filter and then displayed using the PolyView Pro / 32 data acquisition system (Grass). Raw nerve activity was sampled at a rate of 2000 / s.
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2.3. Central microinjections Following nerve isolations, animals were placed in a David Kopf (Tujunga, CA) stereotaxic frame and holes (see Table 1 for coordinates; [19]) were drilled bilaterally through the temporal-occipital bones to permit the stereotaxic insertion of a 30-gauge stainless steel 1 ml Hamilton microsyringe. Each group consisted of four animals. Every animal received bilateral injections into two separate central nuclei (i.e. lidocaine or saline injected into the first nucleus followed by estrogen or saline injected into the second nucleus). In the first experiment, lidocaine hydrochloride (5%, 100 nl per side; Vetoquinol Canada, Joliette, PQ) or saline vehicle (0.9%; 100 nl / side) was injected bilaterally into either the central nucleus of the amygdala (CNA; n54 / drug), lateral hypothalamus (LHA; n54 / drug), parabrachial nucleus (PBN; n54 / drug) or ventral posteromedial thalamus (VPM; n54 / drug) 10 min prior to the bilateral injection of estrogen (17b-estradiol; Sigma– Aldrich; 0.5 mM; 100 nl / side) into the insular cortex (IC; n528). This concentration of estrogen was determined as the optimal dose for local microinjections based on a dose–response relationship preformed in a previous studies in our laboratory [23,31]. In the second experiment, saline or lidocaine was injected bilaterally into the PBN (n524) 10 min prior to estrogen injections into either the CNA (n54), LHA (n54) or VPM (n54). Finally, to determine the effect of lidocaine on cardiovascular and autonomic function, lidocaine was injected into the PBN, CNA, LHA or VPM (n54 / nucleus). Central injection of lidocaine, at a similar volume and concentration as that used in the present study has previously been shown to suppress neuronal activity in a region approximately 1 mm in diameter from the tip of the injector [9,25]. The effects on neurotransmission recovered within 90 min [25].
central injection and then 5, 30, 60, 90 and 120 min following the final central injection. The ratio of the peak changes in the magnitude of the reflex bradycardia to the magnitude of the phenylephrine-induced pressor response (DHR /DMAP beats / min per mmHg) was calculated and used as an index of the sensitivity of the baroreceptor reflex. Changes in sympathovagal balance were also assessed at each time point by measuring baseline levels and phenylephrine-induced changes in vagal and renal efferent nerve activity.
2.5. Data analysis All data are presented as mean6standard error of the mean (S.E.M). Changes from baseline in mean arterial blood pressure, heart rate, baroreceptor sensitivity, sympathetic and parasympathetic nerve activities were analyzed with one-way analysis of variance (ANOVA) for repeated measures, and when necessary, followed by a Student– Newman–Keuls post hoc analysis. Differences between groups at identical time points were determined using unpaired student t-tests with a Bonferroni correction for multiple comparisons. In all cases, differences were considered significant if P#0.05.
2.6. Histology At the end of each experiment animals were perfused transcardially with 0.9% saline followed by 10% formalin. The brains were removed and stored in 10% formalin until the location of the microsyringe tracks could be verified histologically from thionin-stained coronal sections (100 mm).
3. Results
2.4. Baroreflex testing and autonomic tone measurements In all animals, an index of baroreflex sensitivity was measured following the intravenous administration of phenylephrine hydrochloride (PE; 0.1 mg / kg). All phenylephrine injections were made 5 min prior to each Table 1 Stereotaxic coordinates used to locate central autonomic nuclei for the bilateral injection of drugs
IC CNA LHA VPM PBN
Anterior–posterior
Medial–lateral
Dorsal–ventral
0.8 21.8 22.2 22.7 29
65.3 64.1 61.8 62.3 62.0
25 27.3 28 26.2 26.4
IC, insular cortex; CNA, central nucleus of the amygdala; LHA, lateral hypothalamic nucleus; VPM, ventral posteromedial thalamus; PBN, parabrachial nucleus (modified coordinates obtained from Ref. [19]).
Prior to estrogen injection, mean arterial pressure (MAP) and heart rate (HR) were 110611 mmHg and 325621 beats / min, respectively (n568). Baseline values for vagal parasympathetic nerve activity (VPNA) and renal sympathetic nerve activity (RSNA) were 962 mV/ s (n5 68) and 1163 mV/ s (n568), respectively. Control injections of saline into forebrain nuclei had no effect on baseline MAP, HR or nerve activities, nor on the phenylephrine-evoked changes in these parameters. For all animals, injection of phenylephrine prior to central injection of estrogen produced an increase in MAP (2364 mmHg; n568) which was accompanied by a reflexive decrease in HR (21162 beats / min; n568; see Fig. 1 at 25 min). The index of baroreflex sensitivity at this time point was 0.4860.05 beats / min per mmHg (n568). During baroreflex testing with phenylephrine, VPNA was increased 45611% (n568) and RSNA was decreased 55612% (n5 68; see Fig. 1 at 25 min) relative to baseline levels. No significant changes in baseline MAP, HR, VPNA or
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Fig. 1. Cardiovascular and autonomic responses to bilateral estrogen injection into the central nucleus of the amygdala (CNA). (A) Representative physiograph tracings demonstrating that baseline blood pressure (25 min) as well as the pressor response to phenylephrine injection (0.1 mg / kg; ↑) are significantly decreased at 30 min post-estrogen injection (0.5 mM; 100 nl / side). (B) Representative samples of renal sympathetic nerve activity (RSNA) and vagal parasympathetic nerve activity (VPNA) indicating basal autonomic tone before (25 min) and following estrogen injection into the CNA. Changes in nerve activities measured during baroreflex testing before (25 min) and following estrogen injection into CNA originate at the arrow (↑) and were measured for 1 min immediately following the PE injection (time scale bar51 min). Both baseline and evoked nerve activities returned to pre-injection values at 60 min post-estrogen injection.
RSNA could be measured at any time point following the injection of lidocaine into the VPM, CNA, LHA or PBN (n54 / nucleus; data not shown; P.0.05). However, following lidocaine injection into the PBN, a significant increase in baroreflex sensitivity was measured at 30 min (from 0.4860.06 to 0.9860.15 beats / min per mmHg; P,0.05). This increase in baroreflex sensitivity measured at 30 min was accompanied by a significant enhancement in both the phenylephrine-evoked reflex bradycardia (22365 beats / min) and VPNA (73611%; n54; P,0.05) compared to pre-lidocaine values (21162 beats / min and 45611%, respectively). These changes in cardiovascular and autonomic reflex function following lidocaine injection into the PBN recovered when measured at 90 min postinjection (P.0.05).
3.1. The role of subcortical nuclei in mediating the autonomic response to estrogen injection into IC Bilateral injection of estrogen into IC produced a significant increase in baseline RSNA within 30 min of
injection (from 1062 to 2664 mV/ s; n54; Fig. 2; P, 0.05). Changes in baseline RSNA returned to pre-injection values by 60 min post-estrogen injection. The phenylephrine-evoked pressor response (2062 mmHg) and reflex bradycardia (21062 beats / min) were not significantly different at all time points following estrogen injection into the IC (P.0.05). As a result, baroreflex sensitivity did not differ from pre-injection values throughout the time course of the experiment (0.5560.06 beats / min per mmHg; P. 0.05). Similarly, reflex changes in RSNA and VPNA during baroreflex testing were not significantly different from pre-injection values (1063 mV/ s and 1364 mV/ s, respectively; P.0.05). Bilateral injection of lidocaine into the PBN resulted in almost complete blockade (91.466% decrease; n54; Fig. 2; P,0.05) of the increase in baseline sympathetic tone observed following estrogen injection into the IC as described above. Although not as dramatic, the sympathoexcitatory response to estrogen injection into the IC was also significantly attenuated when lidocaine was pre-injected into either the CNA (3364% decrease; n54; P,
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Fig. 2. Effects of bilateral estrogen injection (0.5 mM; 100 nl / side) into the insular cortex (IC; filled circle) on renal sympathetic nerve activity (RSNA). Also shown is the change in the magnitude of this sympathoexcitatory response when lidocaine (1lidocaine) was pre-injected into either the central nucleus of the amygdala (CNA; open circle), lateral hypothalamic area (LHA; closed triangle), ventral posteromedial thalamus (VPM; open triangle) or parabrachial nucleus (PBN; closed square). In all cases, * indicates significance from the pre-estrogen injection value (25 min), and ** indicates significance from the post-estrogen injection into the IC value at 30 min (P,0.05; ANOVA).
0.05) or LHA (5566% decrease; n54; Fig. 2; P,0.05). In contrast to the above three nuclei, following lidocaine injection into the VPM, the sympathoexcitatory response to estrogen injection into the IC was not significantly different (1666% decrease; n54; Fig. 2; P.0.05).
3.2. The role of the PBN in mediating the autonomic and cardiovascular responses to estrogen injection into the CNA Bilateral injection of estrogen into CNA significantly decreased baseline MAP (to 7263 mmHg) and RSNA (to 5.361 mV/ s) within 30 min of injection (n54; P,0.05; Fig. 3A,C) but did not alter baseline HR or VPNA (P. 0.05; Fig. 3B,D). The changes in MAP and RSNA returned to pre-injection values by 60 min post-estrogen injection (P.0.05; Fig. 3A,C). Also at 30 min postestrogen injection, the phenylephrine-evoked pressor response was significantly attenuated (from 2464 to 1064 mmHg; P,0.05; Fig. 1). In contrast, the reflexive bradycardia (21063 beats / min) evoked by phenylephrine injection was at no time significantly different from the phenylephrine-evoked reflex bradycardia observed prior to estrogen injection (21162 beats / min; P.0.05; Fig. 1). The value obtained for the index of baroreflex sensitivity at 60 min post-estrogen injection was significantly enhanced (1.160.05 beats / min per mmHg; P,0.05) relative to the pre-injection value (0.4860.05 beats / min per mmHg). Following estrogen injection into the CNA, the magnitude
of the reflex decrease in RSNA was significantly attenuated when compared to that obtained prior to estrogen injection (a decrease of 2568% from baseline vs. 55612% prior to estrogen injection; P,0.05). This effect was evident at 30 min post-estrogen injection only. Phenylephrine-evoked changes in VPNA during baroreflex testing were not different at any time point throughout the course of the experiment (P.0.05). The decrease in baseline MAP and RSNA described above following estrogen injection into the CNA, were all significantly attenuated (greater than 90% for each parameters; P,0.05) following the prior bilateral injection of lidocaine into the PBN (Fig. 3A,C). As a result, the estrogen-induced enhanced baroreflex sensitivity was blocked and remained not different from baseline values (P.0.05). Also, the observed decrease in the magnitude of the RSNA following phenylephrine injection in the presence of estrogen in the CNA, was blocked when lidocaine was pre-injected into the PBN (P.0.05).
3.3. The role of the PBN in mediating the autonomic and cardiovascular responses to estrogen injection into the LHA Bilateral injection of estrogen into LHA resulted in a significant increase in baseline RSNA within 30 min of injection (from 960.7 to 11.261 mV/ s; n54; P,0.05; Fig. 4C) but did not affect baseline MAP, heart rate or
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Fig. 3. Effects of bilateral estrogen injection into the central nucleus of the amygdala (CNA) on the baseline cardiovascular and autonomic responses in the presence of either saline or lidocaine pre-injected into the parabrachial nucleus (PBN). Average baseline values of mean arterial pressure (MAP; A), heart rate (HR; B), renal sympathetic nerve activity (RSNA; C) and vagal parasympathetic nerve activity (VPNA; D) before (25 min) and following bilateral injection of estrogen (0.5 mM; 100 nl / side) into CNA are shown. In all cases, * indicates significance (P,0.05; ANOVA) from pre-estrogen injection value (25 min).
VPNA (P.0.05; Fig. 4A,B,D). The change in RSNA returned to pre-injection values by 60 min post-estrogen injection (P.0.05; Fig. 4C). The phenylephrine-evoked pressor response or reflex bradycardia were not affected following estrogen injection into the LHA compared to pre-injection values (2064 mmHg and 1063 beats / min, respectively; P.0.05). The value obtained for the index of baroreflex sensitivity was also unchanged post-estrogen injection into the LHA relative to the pre-injection value (0.4760.04 beats / min per mmHg; P.0.05). No changes in the reflex decrease in RSNA or increase in VPNA were observed following estrogen injection into the LHA (P. 0.05). The increase in baseline RSNA described above following estrogen injection into the LHA, was not significantly attenuated following the prior bilateral injection of lidocaine into the PBN (11.061 mV/ s; n54; P.0.05; Fig. 4C).
3.4. The role of the PBN in mediating the autonomic and cardiovascular responses to estrogen injection into the VPM Bilateral injection of estrogen into VPM did not affect any cardiovascular or autonomic baseline parameter measured (n54; P.0.05; not shown). Also, no changes were observed in the phenylephrine-evoked pressor response (2164 mmHg), reflex bradycardia (21062 beats / min) or reflex decrease in RSNA (49610%) and reflex increase in VPNA (4369%) following estrogen injection into the VPM (P.0.05). Therefore, when the index of baroreflex sensitivity was calculated, no significant change was observed post-estrogen injection into the VPM (0.4860.05; P.0.05). Since estrogen injection into the VPM resulted in no significant change in any of the baseline or reflex cardiovascular or autonomic measures, we did not repeat the study in the presence of lidocaine in the PBN.
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Fig. 4. Effects of bilateral estrogen injection into the lateral hypothalamic area (LHA) on the baseline cardiovascular and autonomic responses in the presence of either saline or lidocaine pre-injected into the parabrachial nucleus (PBN). Average baseline values of mean arterial pressure (MAP; A), heart rate (HR; B), renal sympathetic nerve activity (RSNA; C) and vagal parasympathetic nerve activity (VPNA; D) before (25 min) and following bilateral injection of estrogen (0.5 mM; 100 nl / side) into LHA are shown. In all cases, * indicates significance (P,0.05; ANOVA) from pre-estrogen injection value (25 min).
3.5. Histological verification of cannulae placement Data from animals in which both cannulae were not localized to the appropriate nuclei were not included in this study. Bilateral injections of estrogen observed to be unilateral or completely outside the intended region produced no significant effects on baseline parameters nor on phenylephrine-evoked changes in the same parameters (data not shown). For all animals receiving bilateral injections of estrogen (n54 / nucleus), saline (n54 / nucleus), or lidocaine (n54 / nucleus) into the IC, VPM, CNA, LHA or PBN, the sites (n564 in total) were confirmed as previously described [22,23]. Briefly, in the IC, cannulae tracks were predominantly located in the dysgranular IC between bregma 20.26 and 11.2 [19]. In the VPM, cannulae tracks were found predominantly in the parvocellular region of the VPM (VPMpc) just dorsal to the medial lemniscus be-
tween bregma 22.3 and 23.8. In the CNA, sites were located between bregma 22.12 and 22.8 evenly distributed throughout the medial and lateral subdivisions. In the LHA, sites were located between bregma 21.8 and 22.8 between the zona incerta and internal capsule at the level of the fornix and the optic tract. In the PBN, sites were predominantly located in the lateral subnucleus distributed within the dorsal, central and external and internal lateral subdivisions between bregma 29.0 and 29.7.
4. Discussion The present study demonstrated that in male rats, direct injections of estrogen into various central nuclei resulted in significant changes in cardiovascular and autonomic parameters. Specifically, estrogen injected into the IC enhanced sympathetic tone while in the in the LHA, estrogen
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injections increased both sympathetic tone and mean arterial pressure. Estrogen injections into the CNA attenuated sympathetic tone and mean arterial pressure in addition to enhancing baroreceptor reflex function. When neurotransmission through the PBN was blocked with the reversible anesthetic, lidocaine hydrochloride, the sympathoexcitatory effect of estrogen in the IC was completely blocked, whereas the cardiovascular and autonomic changes observed following estrogen injection into the CNA were only partially blocked. In contrast, the effects of estrogen in the LHA were not altered in the presence of lidocaine in the PBN. Prior injection of lidocaine into the CNA or LHA also resulted in a significant attenuation of the sympathoexcitatory effect of estrogen into the IC. Estrogen injected into the VPM did not alter any of the cardiovascular or autonomic baseline or reflex parameters measured. These results suggest that estrogen activates a population of neurons in the IC which project directly or synapse in the CNA and / or LHA prior to projecting to the PBN before synapsing on presympathetic neurons in the brainstem and spinal cord (Fig. 5). The same seems to be true for estrogen-sensitive neurons in the CNA as there appears to be a synapse in the PBN prior to influencing autonomic preganglionic neurons. The estrogen-sensitive neurons originating in the LHA, on the other hand, seem to bypass the PBN and possibly project directly to autonomic
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preganglionic nuclei in the brainstem and spinal cord (Fig. 5). A technical consideration should be taken into account with respect to the use of lidocaine to block synaptic transmission in select nuclei. Lidocaine may also inhibit fibres of passage traveling through the nucleus or in close proximity (approx. 1 mm in diameter from the tip of the injection cannulae). Previous studies using lidocaine to block synaptic transmission in the PBN and LHA [9,25] have shown that lidocaine is an effective tool in that it is reversible and thus allows for the investigation of the effect of the blockade and the recovery of responses in a reasonable amount time (minutes to hours). It is for this advantage that lidocaine was the drug of choice in this investigation. The effects of estrogen injection in all nuclei (except the VPM) seemed to have an effect within 30 min of injection. This rapid (non-genomic?) change in membrane excitability in response to estrogen may likely be mediated by the yet uncharacterized cell surface receptor for estrogen. Previous work in our laboratory has shown similar rapid actions of estrogen in central nuclei on autonomic tone which were completely antagonized by the potent estrogen receptor antagonist, ICI 182,780 [23,28,30]. Since this antagonist was not used in this study, we cannot rule out the possibility that the estrogen-induced changes in neuro-
Fig. 5. Schematic diagram illustrating the neuronal pathway suggested to be activated by estrogen-sensitive neurons in the forebrain. The diagram summarizes the results of the present investigation which demonstrates which midbrain and pontine nuclei are involved in mediating the autonomic and cardiovascular effects of estrogen in the forebrain. Solid line, suggested pathway based on results presented here; dashed lined, subsequent pathway activated in order to activate autonomic preganglionic cells in the brainstem and spinal cord. CNA, central nucleus of the amygdala; IC, insular cortex; LHA, lateral hypothalamic area; PBN, parabrachial nucleus.
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nal excitability were receptor mediated or due to nonspecific alterations in ionic conductance or membrane resistance. Both estrogen receptor subtypes (ERa and ERb) are found in all nuclei under investigation in the current study in approximately equal density [2,14,34,35]. Therefore, we cannot conclude that differential activation of one receptor subtype results in a specific autonomic change (i.e. ERa activation resulting in sympathoinhibition). Opposing effects of estrogen (i.e. sympathoexcitation in the IC and LHA versus sympathoinhibition in the CNA) may be rationalized by an altered binding affinity of estrogen to a particular receptor subtype or accessibility to the nucleus may be differentially regulated.
4.1. Subcortical sites mediating sympathetic responses from the IC The insular cortex, particularly the caudal granular insula, is a cortical area known to participate in autonomic and cardiovascular regulation [18]. Bilateral injections of estrogen into the IC of male rats increased sympathetic tone without altering baseline MAP, HR, VPNA or baroreflex sensitivity. Our laboratory has previously demonstrated that in estrogen-replaced ovariectomized female rats, bilateral injections of estrogen into the IC significantly depressed RSNA, whereas bilateral injections of estrogen into the IC of non-estrogen replaced ovariectomized female rats had no effect [23]. Comparison of the present results with our previous findings suggests that circulating estrogen levels may influence the density and / or distribution of estrogen-sensitive neurons or estrogen receptors within the IC of male versus female rats. The difference in estrogen-induced sympathetic responses from the IC of male versus female rats may be due to a gender difference in estrogen receptor density and / or sensitivity. The IC receives a variety of visceral afferent projections and has efferent connectivity with several subcortical sites, including those involved in autonomic control [6,16,21]. In particular, the IC possesses reciprocal connections with the LHA [32,36], the CNA [5,37], the VPM [5] and the PBN [32]. The interconnectivity of these nuclei would suggest that these pathways are involved in the integration of sensory and visceral information and would thus be responsible for modulating autonomic efferent tone. The estrogen-induced sympathoexcitation following injection into the IC appeared to be mediated by a synapse in the PBN since prior injection of lidocaine into this nucleus decreased the enhanced level of RSNA by 91.4%. In addition, the estrogen-induced increase in RSNA appeared to activate pathways passing through both the LHA and CNA as the sympathetic response was decreased by 55% and 33%, respectively, as a result of synaptic blockade of these nuclei. The presence of sympathoexcitatory neurons within the IC has been previously described [38], and it has been suggested that following electrical stimulation of the IC,
the LHA is a mandatory synaptic site for the transmission of subsequent neural activity resulting in autonomic responses [4]. Consistent with these results, we have demonstrated that estrogen-sensitive neurons in the IC activate a pathway to the LHA leading to sympathoexcitation. In addition, our results also demonstrated the importance of the PBN and the CNA for transmitting estrogen-induced sympathoexcitatory commands originating in the IC.
4.2. Role of the PBN in mediating cardiovascular and autonomic responses from the CNA, LHA and VPM Estrogen injections into the CNA resulted in a significant decrease in RSNA accompanied by a dramatic hypotension independent of a reflexive decrease in heart rate (or increase in VPNA). Fig. 3D suggests that VPNA was elevated at this time point, but not significantly compared to baseline (pre-injection) values and thus heart rate remained the same. It is possible that the neurons activated by estrogen in the CNA not only resulted in sympathoexcitation, but also may have been involved in simultaneous inhibition of parasympathetic tone (at least to some extent) due to the fact that, following the baroreceptor response to hypotension, VPNA did not increase and heart rate remained the same. The PBN has topographically organized afferent and efferent connections to a number of forebrain sites, including the CNA and LHA [1,5,11,17,33]. Lidocaine injections into the PBN completely blocked all estrogen-induced autonomic and cardiovascular changes from the CNA, demonstrating the importance of the PBN synapse prior to projecting to autonomic preganglionic nuclei in the brainstem. The CNA can also influence autonomic preganglionic neurons directly since efferent pathways have also been demonstrated to the nucleus tractus solitarius and the rostral ventrolateral medulla [20]. However, our results seem to suggest that the estrogen-sensitive neurons in the CNA may not directly modulate the activity of these brainstem autonomic nuclei since the effects of estrogen injections into the CNA were completely abolished following lidocaine injections into the PBN. This suggested mandatory synapse in the PBN for the CNA projection to autonomic preganglionic neurons may not be the case for all neurons in the CNA, only the ones that were activated by estrogen using our preparation and experimental paradigm. The CNA participates in the regulation of autonomic responses relative to behavioral and neuroendocrine responses to environmental stimuli. One possible physiologically relevant role for estrogen-induced cardiovascular changes within the CNA may be in modulating the response to pain. Both the PBN and CNA have been shown to receive noxious inputs [3,13]. This is particularly relevant in light of the recent literature suggesting that elevations in circulating estrogen during late gestation is associated with a significant increase in nociceptive response threshold and that this is likely due to an action
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within the dorsal horn of the spinal cord [2,12]. Along the same line of evidence, a similar role for estrogen in IC could be suggested due to the convergence of nociceptive and baroreceptive inputs previously shown in the rat [5,6,37,38] and monkey [16]. The findings reported here may suggest a novel supraspinal role for estrogen in the modulation of nociceptive inputs to the forebrain. Estrogen injections into the LHA significantly enhanced RSNA without affecting MAP, HR or VPNA. Since the prior injection of lidocaine in the PBN did not alter the enhanced sympathetic tone observed following estrogen injections into the LHA, the efferent pathways mediating this effect do not synapse within the PBN. The lateral hypothalamus is well known to be involved in the modulation of autonomic and neuroendocrine responses relative to feeding and reproductive behavior. The LHA has been demonstrated to have efferent connections to the rostral ventrolateral medulla and electrical stimulation of the LHA has been shown to result in sympathoexcitation [7,8]. Perhaps it is this estrogen-sensitive pathway originating within the LHA that is activated in order to produce the observed increase in sympathetic tone. The bilateral injection of estrogen into the VPM did not alter any of the autonomic or cardiovascular parameters measured. The PBN has reciprocal connections to the NTS [11] and is an obligatory synapse between the NTS and the VPM in the relay of visceral information destined for higher autonomic regulatory centers such as the IC [24]. Also, visceral afferent activation of the VPM can be modulated by estrogen injections into the PBN [31]. However, it appears that the VPM does not play a role in mediating estrogen-induced efferent changes in autonomic tone or baroreceptor sensitivity.
4.3. Conclusions The results of this experiment are the first to propose a functional anatomical pathway at various levels throughout the neuraxis activated by estrogen and the specific nuclei involved in mediating the autonomic and cardiovascular changes observed. Each of the forebrain nuclei examined in this study are involved in the integration of environmental stimuli with internal sensory information in order to coordinate an appropriate autonomic, endocrine and behavioral response to these stimuli. This study suggests that these nuclei are sensitive to changes in the levels of hormones which can activate the same pathways involved in mediating the autonomic and cardiovascular response in an attempt to restore homeostasis.
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