This study examined the effect of circulating oestrogen on cardiovascular parameters and on neuronal activation in the hindbrain nuclei that comprise the baroreflex circuit in response to systemic administration of Isop. The results show that EB attenuated the depressor response to Isop and the smaller decrease in MAP was accompanied by a disproportionate increase in HR. We also found that Isop increased Fos-IR, an indication of neuronal activation, in the NTS, AP, RVLM and lPBN and show for the first time that EB treatment attenuated Fos-IR in specific medullary (AP) and supramedullary (lPBN) nuclei implicated in blood pressure regulation. EB modulation of the cardiovascular and central responses to Isop was not secondary to hormonal effects on body weight because Isop-induced changes in MAP, HR, and Fos-IR in WT Loss rats were similar to those of rats treated with Oil.
Cardiovascular effects of EB
Despite the reduced depressor response to Isop in rats treated with EB, the accompanying increase in HR was of the same magnitude as that in WT Loss and Oil-treated rats. In fact, the compensatory tachycardia in EB-treated rats was similar to that in rats in which MAP decreased nearly two-fold more. This observation, in conjunction with the exaggerated ratio between change in HR and change in MAP in EB-treated rats, suggests that EB augments the HR response to Isop and may, in fact, account for the attenuated depressor response. Consistent with this idea, Ciric & Susic (1980) found that, when hypotensive responses to Isop were similar, OVX rats treated with EB had a greater increase in HR than did OVX rats without EB. Because Isop is a β-adrenergic agonist that increases heart rate and contractility, Ciric & Susic (1980) attributed the enhanced tachycardia to oestrogen modulation of β-adrenergic receptors on the heart. However, more recent studies have shown that EB blunts the stimulatory actions of Isop on heart rate and cardiac contractility in isolated rat hearts (Li et al. 2000) and prevents the up-regulation of β-adrenergic receptors on the heart that typically occurs after OVX (Thawornkaiwong et al. 2003). Thus, our finding of enhanced tachycardia in response to Isop in EB-treated rats is unlikely to be due to EB effects on β-adrenergic receptors on the heart.
Another possibility is that the weight loss associated with oestrogen replacement influenced the HR response to Isop. In conjunction with reports of enhanced parasympathetic tone after systemic administration of oestrogen (Saleh et al. 2000a,b), our observation of a mild basal bradycardia in EB-treated rats (Table 1) is consistent with increased vagal influence such as may occur as a result of weight loss (Rissanen et al. 2001). Thus, hypotension may have a greater effect on HR in EB-treated rats due to the withdrawal of elevated vagal efferent activity that is secondary to weight loss. Accordingly, we food restricted rats to produce body weight loss similar to that which occurs in EB-treated rats. Moreover, rats in the WT Loss condition exhibited basal bradycardia indicative of elevated parasympathetic tone (Rissanen et al. 2001) and comparable to that of EB-treated rats (Table 1). Nonetheless, enhanced compensatory tachycardic responses to Isop did not occur in WT Loss rats, even though the hypotension after Isop was the greatest in this condition (Table 1). Hence, weight loss alone does not affect reflex HR responses to Isop-induced hypotension, nor do slight basal bradycardia or hypotension (Table 1) related to body weight loss. Rather, given that EB augments the increase in vagal nerve activity (and thereby results in greater bradycardia) in response to hypertension (Saleh et al. 2000a,b), it is feasible that EB may contribute directly to the augmented HR response to Isop by altering baroreflex-mediated parasympathetic withdrawal.
Taken together, these observations suggest that the cardiovascular effects we observed after Isop reflect selective EB enhancement of baroreflex responses. Several studies have shown that OVX rats treated with EB had greater decreases in HR in response to phenylephrine-induced increases in blood pressure (el-Mas & Abdel-Rahman, 1998, Mohamed et al. 1999; Saleh & Connell, 2000). Although oestrogen effects on peripheral vasculature may contribute to EB-mediated changes in such compensatory HR responses, the enhancement of reflex bradycardia is likely to also involve EB modulation of central baroreflex pathways: EB treatment enhanced HR responses elicited by electrical stimulation of the cut aortic depressor nerve, but did not affect HR responses to electrical stimulation of the efferent vagus nerve (Mohamed et al. 1999). Therefore, the present findings that EB-treated rats exhibit compensatory increases in HR disproportionate to the decreased blood pressure after Isop also may be attributable to EB actions within the central baroreflex pathway.
Central effects of EB
During decreased blood pressure, the central baroreflex pathway is activated by unloading of arterial baroreceptors in the aortic arch and carotid sinus. Afferent fibres from these baroreceptors terminate on cell bodies of neurons in the NTS which integrate input from baroreceptors and, in turn, influence neural activity in the ventrolateral medulla. Hypotension decreases activity in GABAergic neurons in the CVLM that normally inhibit RVLM neurons, thereby increasing sympathetic outflow via disinhibition of excitatory projections from RVLM to sympathetic preganglionic neurons (Dampney & Horiuchi, 2003; Babic & Ciriello, 2004). Similar to other studies that examined Fos-IR after hypotension, we found that Isop increased Fos-IR in cNTS, mNTS and RVLM (Chan & Sawchenko, 1994; Dun et al. 1995; Graham et al. 1995; Curtis et al. 1999). We did not see an effect of Isop on Fos-IR in the CVLM, whereas others report increased Fos-IR in CVLM after hypotension (Graham et al. 1995; Curtis et al. 1999). Methodological differences including type of drug, route of administration, or duration of hypotension may account for the discrepancy; nonetheless, our observation is consistent with reports that hypotension reduces activity in CVLM neurons that send inhibitory projections to RVLM (Dampney & Horiuchi, 2003) and that Fos expression is not associated with decreased neuronal activity (Dragunow & Faull, 1989).
Increased Fos-IR in cNTS, mNTS, CVLM and RVLM after Isop did not differ among the groups, indicating no differences in afferent activation of the NTS or activation in CVLM and RVLM. In other words, neither EB treatment nor weight loss altered activity in the afferent terminal field or in the ‘preautonomic’ area of the hindbrain in response to Isop-induced hypotension. Although we did not record sympathetic nerve activity in this study, similar levels of Fos-IR in the RVLM after Isop suggest that sympathetic outflow also was comparable among the groups. Ultimately, then, activation in the efferent limb of the reflex pathway produced comparable reflex tachycardia in the three groups. However, the idea that typical patterns of activation in the afferent terminal field and in the sympathoexcitatory area lead to predictable and comparable changes in HR rests on the assumption that there were no differences in depressor responses (i.e. changes from baseline MAP) among the groups, which was not the case.
It is well known that hypotension increases Fos-IR in the NTS (Chan & Sawchenko, 1994; Curtis et al. 1999; Potts et al. 1999) and the effect requires intact arterial baroreceptors (Potts et al. 1999). Thus, the Fos-IR we observed in the NTS following Isop is likely to be attributable to unloading of arterial baroreceptors. We would therefore have expected that differences in depressor responses after Isop (Fig. 1) would produce different levels of Fos-IR in the NTS, as suggested from studies of hypotensive and non-hypotensive haemorrhage (Badoer et al. 1993; Buller et al. 1999). Clearly, however, there were no differences among EB, Oil, or WT Loss rats in the number of Fos-IR neurons in the NTS after Isop (Fig. 2). One possible explanation for this observation is that EB increases baroreceptor sensitivity such that afferent signals arising from peripheral baroreceptors are comparable among the groups and lead to similar activation in the NTS. An alternative possibility is that oestrogen increases the responsiveness of neurons in the NTS to the reduced baroreceptor input by classic steroid actions that lead to changes in protein expression, especially of neurotransmitter receptors (e.g. serotonin, noradrenaline). Consistent with this possibility, oestrogen receptors α (ERα) and β (ERβ) have been localized to the NTS (Schlenker & Hasen, 2006) and Fos-IR in the NTS produced by other stimuli (Flanagan-Cato et al. 1998) also is modulated by oestrogen. However, it is important to note that, without phenotypic labelling and neuroanatomical tract-tracing, we can say only that the number (Fig. 2) and general distribution (Fig. 4) of Fos-IR neurons in the NTS are comparable, not whether these are the same population of cells. It is possible that EB differentially affects subpopulations of neurons within the NTS or alters input to the NTS from other brain areas involved in cardiovascular regulation. Clearly, additional studies will be necessary to assess these possibilities, but the latter is an especially intriguing explanation for the comparable Fos-IR in the NTS given the broad distribution of oestrogen receptors throughout the central nervous system (McEwen, 2002) and the convergence of ascending and descending neural pathways at the NTS (Spyer, 1994).
The AP is a hindbrain circumventricular organ that, by virtue of reciprocal connections with the NTS, is among the brain areas that may influence activity in the NTS. Importantly, the AP itself receives baroreceptor input (Papas & Ferguson, 1991) and, due to the incomplete blood–brain barrier that is characteristic of circumventricular organs, also receives humoral signals pertaining to blood pressure and blood volume from circulating hormones such as AngII. The AP expresses angiotensin type 1 (AT1) receptors (Lenkei et al. 1998), which bind circulating AngII, thereby activating neurons that modify baroreflex responses (Hasser et al. 2000; Xue et al. 2003). In addition to receptors for AngII, ERα and ERβ have been localized to the AP (Shughrue et al. 1997), raising the possibility of interactions between AngII and EB. In fact, Pamidimukkala & Hay (2003a) recently reported that EB inhibited AngII activation of cultured AP neurons, suggesting that EB also inhibits AngII activation of AP neurons in vivo. These observations are of particular importance because Isop is a potent activator of the renin–angiotensin system (RAS) and leads to large increases in levels of circulating AngII (Stocker et al. 2000). Although our overall ANOVA did not reveal an interaction between group and drug, our planned comparisons revealed an effect of EB treatment on Fos-IR in the AP after Isop. Thus, the attenuation of Isop-induced Fos-IR in the AP of EB-treated rats observed in the present study may be attributable to EB inhibition of AP neural responses to AngII.
The present finding of attenuated Fos-IR in a circumventricular organ is similar to that in our previous study (Krause et al. 2006), in which EB treatment attenuated Isop-induced Fos-IR in a forebrain circumventricular organ, the subfornical organ (SFO). The decreased neural activation in that study was associated with EB reduction of a behavioural response, water intake. The central neural pathways that mediate AngII-stimulated water intake have not been defined as well as the baroreflex pathway, so it has been difficult to elaborate on the previous observation. On the other hand, many years of research on the neurochemistry, electrophysiology and neuroanatomy of the baroreflex pathway (Spyer, 1994) allow speculation about the functional implications of attenuated Fos-IR in the AP. AngII binds to and activates AP neurons that modulate activity of barosensitive neurons in the NTS (Cai et al. 1994), in part, via inhibitory GABAergic projections from the AP to the NTS (Pickel et al. 1989). Thus, EB attenuation of AngII-induced activity in the AP may reduce GABAergic inhibiton of the NTS and thereby contribute to normal baroreceptor activation of NTS after Isop despite the reduced depressor response in EB-treated rats.
As an additional parallel between attenuated AngII-induced activation in forebrain and hindbrain circumventricular organs, it is interesting that Fos-IR in the SFO after Isop and Isop-induced water intake were reduced by both EB treatment and weight loss. In contrast, EB treatment, but not weight loss, reduced Fos-IR in the AP after Isop and increased baroreflex HR responses to Isop. These observations raise the possibility of regional differences in the effects of EB (or weight loss) on AngII-mediated activation of circumventricular organs and in the consequences of such activation. But is there also a hindbrain–forebrain difference in the mechanisms by which such effects occur? Our previous study showed that both EB treatment and weight loss reduced AT1 receptor mRNA in the SFO (Krause et al. 2006). Similar analyses of AngII receptors in the AP remain to be done; nonetheless, these findings suggest that EB treatment (but not weight loss) reduces AT1 receptor mRNA in the AP which underlies the attenuation of Isop-induced Fos-IR in the AP and the concomitant enhancement of baroreflex HR responses.
We have focused on the role of AngII in modulating baroreflex responses largely because of the ability of Isop to stimulate RAS and produce large increases in circulating AngII (Stocker et al. 2000). However, hypotension also increases circulating levels of other hormones, including vasopressin. Vasopressin promotes important compensatory responses to decreased blood pressure such as fluid retention and peripheral vasoconstriction. In the present context, it is equally important that vasopressin augments baroreflex responses (Hasser et al. 2000), particularly since oestrogen increases both circulating levels of vasopressin (Peysner & Forsling, 1990) and vasopressin release stimulated by haemorrhage (Hartley et al. 2004). Vasopressin receptors have been localized to the AP and application of vasopressin to the AP alters activity in AP neurons that, in turn, influence neurons in the NTS that also receive baroreceptor input (Nakayama et al. 1997). Thus, alternative explanations for the present findings are that the augmented baroreflex HR response to Isop in EB-treated rats is attributable to oestrogen enhancement of vasopressin release or of AP responsiveness to vasopressin. However, several observations argue against these explanations. First, our previous study (Krause et al. 2006) showed no effect of EB treatment on Fos-IR in neurosecretory neurons in the hypothalamus. Admittedly, this measure is not equivalent to measuring plasma levels of vasopressin, but we are aware of no studies that have assessed the effect of hypotension on circulating vasopressin in EB-treated rats. Second, vasopressin augmentation of baroreflex responses is eliminated by lesions of the AP (Hasser et al. 1997). If the blunted activation in AP after Isop indicated by attenuated Fos-IR signifies decreased responsiveness of AP neurons to vasopressin, augmented baroreflex HR responses would not be expected. However, it should be noted that responses of AP neurons to vasopressin include both excitation and inhibition, as do subsequent responses in NTS barosensitive neurons (Cai et al. 1994). In this regard, vasopression has been reported to attenuate baroreflex-mediated excitatory HR responses in male rabbits (Luk et al. 1993); thus, additional studies may be necessary to further examine this issue.
For purposes of this analysis, we have concentrated primarily on neural activation in the hindbrain baroreflex pathway and interpreted our results assuming essentially linear, unidirectional information flow. In point of fact, the baroreflex circuit is considerably more complex: reciprocal connections among many of these areas allow feed-forward and feed-back modulation within the hindbrain; in addition, activity at several levels within the hindbrain circuit is modulated by descending input from forebrain areas which, in turn, receive ascending input from these same hindbrain areas (Spyer, 1994). In our previous study (Krause et al. 2006), EB treatment did not affect Isop-induced Fos-IR in the hypothalamic supraoptic (SON) or paraventricular nuclei (PVN) which, on the surface, might imply that differences in neural activation related to baroreflex responses are specific to the hindbrain baroreflex pathway. Interestingly, however, EB treatment also attenuated Fos-IR in the pontine lPBN, one of the major relays for cardiovascular information (Spyer, 1994). It is possible that this effect is attributable to indirect projections from AP to the forebrain via the lPBN (Leslie & Gwyn, 1984). In other words, the attenuated activity in AP after Isop may underlie the attenuation in the lPBN with as yet undetermined consequences for activity in forebrain areas other than the SON and PVN. Alternatively, the attenuated Fos-IR in the lPBN may have implications for activation in the hindbrain baroreflex circuit via descending projections from the lPBN to the NTS (Spyer, 1994). Stimulation of the lPBN decreases baroreflex HR responses (Len & Chan, 2001) and inhibits responses of barosensitive NTS neurons (Hamilton et al. 1981; Felder & Mifflin, 1988; Len & Chan, 1999) and, interestingly, Len & Chan (2001) recently reported that stimulation of lPBN elevates extracellular GABA in the commissural NTS. Thus, similar to the AP, EB attenuation of activity in the lPBN may disinhibit NTS neurons by reducing GABAergic input, thus contributing to normal baroreceptor activation of NTS after Isop despite the reduced depressor response in EB-treated rats. Work by Saleh & Connell (2000, 2003) also has pointed to oestrogen inhibition of neural activity in the PBN as an important factor in oestrogen effects on autonomic functions and, in conjunction with the localization of oestrogen receptors to the lPBN (McEwen, 2002), our findings are consistent with this idea. At present, however, the mechanism(s) underlying attenuated activity in the lPBN remain to be determined, as do the complex interactions among central areas that mediate baroreflex responses to hypotension.
Finally, we chose to examine the effect of EB on activity within the central baroreflex pathway using Fos immunocytochemistry for several important reasons. First, Fos immunolabelling has been extensively employed in studies of cardiovascular function (though primarily in male rats, e.g. Chan & Sawchenko, 1994; Dun et al. 1995; Graham et al. 1995; Potts et al. 1999); as a consequence, much is known about neural activation and patterns of Fos-IR in response to cardiovascular challenges. Second, this technique allows examination of multiple central areas in the same animal, a distinct advantage in analysing activity in a circuit in which a change in one area may, in turn, alter ‘down-stream’ parts of the pathway. Nonetheless, there are limitations to this technique. For example, Fos is not expressed during neuronal inhibition, which may be important in central baroreflex pathways. In fact, not all neurons express Fos; thus, the absence of Fos-IR must be interpreted cautiously. The advantages and limitations of Fos immunolabelling for understanding central pathways in cardiovascular function have been thoughtfully and thoroughly reviewed (e.g. Dampney & Horiuchi, 2003; Dragunow & Faull, 1989) and will not be exhaustively discussed here. However, a more general consideration merits further mention. Specifically, the classic steroidal effects of oestrogen suggest the possibility that the observed differences in central activation might be attributable to oestrogen-mediated changes in gene transcription. We saw no differences in baseline Fos-IR, which would argue against the effects being specific to oestrogen and independent of the hypotension; however, the more telling observation is that EB did not globally alter Fos-IR after Isop. In fact, the EB effect on Fos-IR was specific to two areas within the baroreflex pathway – the AP and the lPBN. Whether these selective differences are attributable to the genomic effects of oestrogen on gene transcription or to non-genomic effects on neuronal excitability remain to be determined.
In summary, the present study shows that EB reduces the depressor response to Isop in OVX rats, and that the reduction is accompanied by a compensatory increase in HR similar to that of rats in which MAP decreased nearly two-fold more. Although peripheral effects of EB also could contribute to this effect, the actions of EB to modify activation in the central baroreflex pathway may be important in the observed EB enhancement of baroreflex responses. We show for the first time that EB treatment selectively attenuates Fos-IR, an indication of neuronal activation, in the AP and lPBN after Isop. In contrast, Isop increased Fos-IR in the NTS and RVLM (see also Chan & Sawchenko, 1994; Dun et al. 1995; Graham et al. 1995; Curtis et al. 1999), areas associated with baroreceptor afferent input and sympathoexcitation, respectively, and the increase was not altered by EB. We also show for the first time that the body weight loss associated with EB treatment does not underlie these effects, as OVX rats that were food restricted to produce weight loss similar to that which occurs with EB treatment, did not differ from OVX rats that were not given EB.
Within the central nervous system, oestrogen acts at specific membrane-bound receptors to produce rapidly occurring nongenomic effects, in addition to the classic steroid effects to alter gene transcription via actions at intranuclear receptors. We cannot distinguish between these mechanisms based on the present observations; however, it seems clear that oestrogen effects on compensatory responses to cardiovascular challenges involve differences in neuronal activation in specific areas within central baroreflex pathways. We therefore propose that EB-mediated decrease in neural activation in AP and/or lPBN after Isop alters activation in the baroreflex circuit which ultimately results in enhanced compensatory tachycardia, notwithstanding reduced depressor responses. More specifically, we propose that EB attenuation of neural responses in the AP which, based on our previous studies showing that EB reduces AT1 receptor mRNA in a forebrain circumventricular organ, may be attributable to decreased responsiveness to circulating AngII, or in the lPBN which may be attributable to alterations in neural signals relayed to the lPBN, reduces GABAergic inhibition of the NTS. This disinhibition allows activation in the NTS despite reduced baroreceptor input and, in turn, leads to activation of the RVLM and thus to increased HR.