Mechanisms in the Bed Nucleus of the Stria Terminalis Involved in Control of Autonomic and Neuroendocrine Functions: A Review

2.1. Glutamate

Glutamate exerts its effects by interacting with metabotropic and ionotropic receptors [65]. Metabotropic glutamate receptors (mGluR) are divided into three groups: type I (mGluR 1 and 5), type II (mGluR 2, 3) and type III (mGluR 4, 6, 7 and 8) [66]. The ionotropic receptors are classified as N-Methyl-D-aspartate (NMDA) and non-NMDA receptors [67]. The non-NMDA receptors are further divided into α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor(AMPA) and kainite [67].

Functional and electrophysiological studies have suggested the presence of glutamatergic terminals in the BNST [68-72]. Source of glutamatergic innervation to the BNST is not completely established. However, available biochemical and electrophysiological results suggest that glutamatergic inputs to the BNST originate from neurons located in the ventral subiculum of the hippocampus and the infralimbic region (IL) of the MPFC [41, 73]. Based mainly on innervation of the BNST by neurons from the IL cortex and the ventral subiculum [22, 74], it has been suggested that glutamatergic neurons innervate the entire rostro-caudal extension of the BNST [48, 70].

Several studies have demonstrated that local BNST glutamatergic neurotransmission is an important neuro-chemical mechanism involved in modulation of cardiovascular function. Gelsema and Calaresu (1987) were the first to describe that the glutamatergic system present in the BNST is involved in control of cardiovascular function [75]. They demonstrated that microinjection of DL–homo-cysteate, a glutamate analog, into the BNST of urethane-anesthetized rats resulted in cardiovascular changes. The range of responses included both pressor and depressor effects on blood pressure and fall or no changes in HR, all of which depended on the site of stimulation within the BNST [75]. Indeed, results published later by Dunn & Williams (1995) gave support to the idea that glutamate action in distinct BNST regions may differently modulate cardiovascular function [42]. In urethane-anesthetized rats, they noted that glutamate microinjected into the medial portion of BNST anterior division caused increases on blood pressure and HR, stimulation of the lateral division decreased cardiovascular parameters, and both pressor and depressor response were observed when injection were centered into the BNST ventral region. Although the above results suggest a region-specific control of cardiovascular function by the BNST, glutamate microinjected into the BNST of conscious rats evoked exclusively depressor responses [46]. In fact, results suggesting that glutamate action in distinct BNST regions differently modulate cardiovascular function are controversial. Some groups have reported in anesthetized rats that stimulation of BNST with glutamate elicits depressor response regardless of region stimulated [45, 55, 76].

The mechanisms involved in depressor and bradycardiac responses to glutamate microinjection into the BNST have been extensively investigated. The largest cardiovascular responses are elicited more often when glutamate microinjection sites are centered into the anterior BNST [45, 75], consistent with the rostral division being critical in cardiovascular control. In unanesthetized rats, the fall in blood pressure mediated by anterior BNST stimulation was accompanied by an increase in the conductance of vessels supplying the hindlimb musculature without changes in conductance in renal and mesenteric beds [46]. Also, the decrease in both blood pressure and HR induced by glutamate microinjection into the BNST of chloralose-anesthetized rats were not affected by intravenous treatment with a muscarinic receptor antagonist, but were abolished after ganglion blockade [45]. Together, above results suggest that decrease of blood pressure and HR evoked by glutamate administration into the anterior BNST are mediated by an inhibition of sympathetic fibers to the vasculature supplying the hindlimb and the heart [45, 76].

The local glutamatergic mechanisms and neural pathways involved in control of cardiovascular function by BNST glutamatergic neurotransmission are not completely understood. A recent study reported that fall in blood pressure and HR evoked by glutamate microinjection into the BNST of anesthetized rats were reduced by local BNST pretreatment with either a selective NMDA or non-NMDA glutamate receptor antagonist [55]. In addition, glutamate cardiovascular responses were inhibited after nonselective synaptic ablation of the caudal ventrolateral medulla (CVLM) [76]. The CVLM projects to and inhibits sympathetic premotor neurons in the rostral ventrolateral medulla, thus decreasing sympathetic preganglionic neuronal outflow [77]. Therefore, above evidence suggest that depressor and bradycardiac responses to glutamate microinjected into the BNST are mediated by co-activation of local NMDA and non-NMDA glutamate receptors that, in turn, stimulate a pathway originating in the BNST and relaying in the CVLM.

Glutamatergic mechanisms within the BNST play a key role in cardiac baroreflex responses. Bilateral microinjection of a selective NMDA antagonist, but not a selective non-NMDA antagonist, into the BNST increased bradycardiac responses to blood pressure increments [72]. Considering the aforementioned results showing that nonselective BNST ablation enhance reflex bradycardia [50], it is possible that the inhibitory influence of the BNST on reflex bradycardia is mediated by activation of local NMDA glutamate receptors.

Microdialysis results demonstrate absence of a glutamatergic tonic activity in the BNST [70]. Indeed, either unilateral [55] or bilateral [72] treatment of the BNST with glutamate receptor antagonists does not induce any significant change in baseline values of both arterial pressure and HR.

2.2. Gamma-aminobutyric Acid

There are two main classes of gamma-aminobutyric acid (GABA) receptors: GABA_A and GABA_B [78, 79]. GABA_A receptors are pentameric ligand-gated ion channels (chloride channels) that mediate the majority of fast inhibitory neurotransmission in the central nervous system [79]. In contrast, GABA_B receptors are metabotropic receptors that act by coupling to G(i)/G(o) protein and mediate slow GABA effects [78].

The activity of BNST neurons is regulated by GABAergic inputs from both intrinsic and extrinsic sources. The vast majority of MeA and CeA neurons are positive for GABA and glutamic acid decarboxylase (GAD), the enzyme that converts glutamate to GABA and is responsible for the majority of de novo GABA synthesis [80, 81]. Based on these results, it has been proposed that extrinsic projections of these amygdalar nuclei are predominantly GABAergic [81]. Neuroanatomical results have indicated that the CeA densely innervate the BNST anterior division, whereas MeA neurons project preferentially to the posterior division [5]. These available anatomical and neurochemical results suggest that GABAergic inputs from amygdala innervate the entire rostro-caudal extension of the BNST.

In addition to extrinsic sources, a network of internal GABAergic neurons within the BNST has been described. This idea is supported by observation that the majority of BNST neurons are GABAergic [74, 80]. Moreover, electro-physiological data suggest the presence of a functional GABAergic local circuit [69]. However, additional studies are necessary for better characterization of a possible network of local GABAergic interneurons.

A limited number of studies have investigated the role of BNST GABAergic neurotransmission in control of cardiovascular function. A recent study demonstrated that microinjection of the selective GABA_A receptor antagonist bicuculline methiodide (BMI) into the BNST significantly increased both blood pressure and HR in anesthetized rats [82]. However, microinjection of muscimol, a GABA_A receptor agonist, or the selective GABA_B receptor antagonist phaclofen did not produce any significant cardiovascular changes. The authors also observed that BMI responses were not affected by intravenous pretreatment with a muscarinic receptor blocker. In contrast, a ganglion blocker inhibited the HR response without affecting the blood pressure change, whereas a selective V₁-vasopressin receptor antagonist inhibited exclusively the pressor response [82]. These results demonstrated that BNST GABAergic neurotransmission, via activation of local GABA_A receptor, play a tonic role on maintenance of blood pressure and heart rate. The pressor and tachycardiac responses were mediated by a modulation of vasopressin release into the circulation and cardiac sympathetic activity, respectively [82]. Also, orexin-containing neurons in the medial hypothalamus seems to be part of the neural pathway involved in control of cardiorespiratory function by GABA receptors within the BNST [47]. The tonic modulation of cardiovascular function by GABAergic mechanisms in the BSNT is consistent with electrophysiological results suggesting that BNST neurons are tonically inhibited by GABA receptors [69].

The influence of chronic loss of inhibitory GABAergic tone in the BNST in panic-like responses was investigated [83]. However, the baseline cardiovascular parameters were not reported. Moreover, GABA inhibition in the BNST did not affect tachycardia, hypertension and tachypnea induced by sodium lactate infusions [83].

2.3. Noradrenaline

Adrenoceptors were initially subdivided in two subtypes: α and β [84]. Further, α-adrenoceptor was subdivided in two subtypes (α₁ and α₂) and three subtypes of β were described (β₁, β₂, and β₃) [85-87]. Currently, nine adrenoceptors subtype have been characterized, including three subtypes of α₁ (α_1A, α_1B, α_1D) and α₂ (α_2A, α_2B, α_2C) adrenoceptors [85, 87, 88].

Among the numerous neural inputs to the BNST, noradrenergic synaptic terminals are among the prominent [34, 37]. In fact, the BNST is one of the major targets of noradrenergic innervation in the brain [36, 89, 90]. Nonetheless, the distribution of noradrenergic terminals in the BNST is heterogeneous. The highest noradrenaline levels are found in anterior division of the BNST [91]. Moreover, noradrenergic terminals are mainly located in the ventral portion of the BNST [34, 37]. Noradrenergic neural terminals in the BNST originate mainly from neurons located in the A1, A2 and A5 brainstem nuclei, and in a lesser degree from the A6 [92-96]. Moreover, projections from the A1 noradrenergic nucleus to the BNST send collateral projections to the CeA and the PVN [97, 98]. Importantly, electrophysiological studies have demonstrated that noradrenaline play an inhibitory role in activity of BNST neurons [63, 99, 100].

Noradrenergic cell groups in medullary regions involved in control of cardiovascular function are important source of BNST noradrenergic innervation [93, 95]. Functional studies further indicate that local noradrenaline microinjection into the BNST evokes cardiovascular responses. Noradrenaline microinjection into the BNST of unanesthetized rats increases arterial pressure and causes bradycardia [101]. Cardiovascular changes were observed only when noradrenaline was microinjected into the anterior division of the BNST (our laboratory, data not published), consistent with results that noradrenergic innervation are mainly located in this region of the nucleus [34, 37, 91, 101]. Also, cardiovascular changes were not different when noradrenaline was microinjected into the anterolateral, anterodorsal or anteroventral regions of the anterior BNST (Fig. ​22). Cardiovascular responses to noradrenaline microinjected into the BNST were blocked by intravenous pretreatment with a selective V₁-vasopressin receptor antagonist [101], suggesting its mediation by vasopressin release into circulation. Detailed discussion of the BNST role in control of vasopressin release into the circulation is presented below (BNST and neuroendocrine function).

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Fig. (2)

Changes in mean arterial pressure (Δ MAP) and heart rate (ΔHR) in response to noradrenaline (10nmol/100nL) microinjection into the anterodorsal, anterolateral and anteroventral area in the anterion division of the BNST. Columns represent the mean and bars the SEM. Cardiovascular changes were not different when noradrenaline was microinjected into the different regions of BNST anterior division (One-way ANOVA, P>0.05).

The pressor and bradycardiac responses evoked by noradrenaline microinjected into the BNST was only partially reduced after local treatment with either a selective α₁- or α₂-adrenoceptor antagonist [102]. The response was completely abolished only when both α₁- and α₂-adrenoceptor antagonists were combined, thus indicating that co-activation of α₁- and α₂-adrenoceptor within the BNST is required for noradrenergic modulation of cardiovascular function [102]. In addition, BNST pretreatment with a selective β₁-adrenoceptor antagonist, but not a β₂-adrenoceptor antagonist, enhanced cardiovascular responses to noradrenaline [102], indicating that activation of local β₁-adrenoceptors opposes actions at the α-adrenoceptors.

Involvement of local noradrenergic neurotransmission in control of BNST baroreflex function was also demonstrated. Crestani and collaborators (2008) reported that bilateral microinjection of a selective α₁-adrenoceptor antagonist increased the gain of reflex bradycardia in response to blood pressure increases [103]. Effects on baroreflex activity were no longer observed when animals were pretreated intravenously with a cholinergic muscarinic receptor antagonist. These results provided evidence that local α₁-adrenoceptors are involved in tonic BNST regulation of the baroreflex bradycardiac response controlling cardiac parasympathetic activity. Bilateral microinjection of either selective α₂- or β-adrenoceptor antagonists into the BNST did not affect baroreflex activity [103].

Local adrenoceptors also mediate control of stress- and exercise-evoked cardiovascular adjustments at the BNST. It was initially demonstrated that BNST pretreatment with a selective α₁-adrenoceptor antagonist enhanced the HR increase associated with acute restraint stress without affecting pressor responses [56]. This effect was similar to that observed after nonselective blockade of BNST synapses [56], indicating an involvement of local noradrenergic neurotransmission in inhibition of restraint-related HR responses. Bilateral microinjection into the BNST of either a selective α₂- or β-adrenoceptor antagonist did not affect restraint-evoked cardiovascular responses. A completely opposite influence of BNST noradrenergic neurotransmission was observed in a conditioned stress model [53]. Indeed, BNST pretreatment with either a selective α₁- or β₁-adrenoceptor reduced both freezing and autonomic responses induced by an aversive context, observed in the contextual fear-conditioning model [53]. These results corroborate data obtained after nonselective ablation,indicating that BNST differentially modulates cardiovascular responses to unconditioned and conditioned stress [56, 64], and reinforce the idea that the role of the BNST in neurobiological mechanisms of emotional stress depends on the type of stress.

The role of BNST noradrenergic neurotransmission in autonomic control during exercise was suggested by a recent study from our group demonstrating that local α₁- and α₂-adrenoceptors differentially modulate cardiovascular responses to a protocol of dynamic exercise [49]. Bilateral microinjection of a selective α₁-adrenoceptor antagonist into the BNST enhanced the HR increase evoked by running on a rodent treadmill, without affecting the blood pressure increase.In contrast, BNST pretreatment with a selective α₂-adrenoceptor antagonist reduced exercise-evoked pressor response without changing the tachycardiac response [49]. These results brought the first direct evidence for the involvement of brain adrenoceptors in cardiovascular responses observed during dynamic exercise. Considering that blockade of α₂-adrenoceptor and the nonselective synaptic ablation within the BNST produced similar effects on exercise-evoked pressor response [51], it is possible that local α₂-adrenoceptors mediate, at least in part, a BNST influence on blood pressure during exercise. The neurotransmitter(s) in the BNST that are involved in its influence on tachycardiac response to exercise remain to be elicited.However, the facilitation of exercise-evoked tachycardiac response following the blockade of α₁-adrenoceptor within the BNST indicates that activation of these receptors may have a physiological role in counteracting excessive cardiac activation during dynamic exercise [49].

In vivo microdialysis results demonstrated a noradrenergic tonic activity in the BNST [104]. However, either unilateral [102] or bilateral [49, 53, 56, 103] treatment of the BNST with adrenoceptor antagonists did not induce any significant change in baseline values of either blood pressure or HR. Therefore, apart of a key physiological role played by BNST adrenoceptors in control of baroreflex activity and cardiovascular responses to stress and physical exercise [49, 53, 56, 103], BNST noradrenergic neurotransmission does not play a role in tonic maintenance of cardiovascular function.

2.4. Acetylcholine

There are two main classes of acetylcholine receptors: muscarinic and nicotinic receptors [105-107]. The muscarinic receptors are metabotropic receptors [105], and mediate physiological effects via five muscarinic receptor subtypes(M_1, M₂, M₃, M₄ and M₅) [105, 106]. Nicotinic receptors are ionotropic composed of five subunits arranged symmetrically around a central ion-conducting pore [107].

The activity of neurons within the BNST is modulated by local acetylcholine release. Indeed, cholinergic terminals were identified in the BNST [38]. Moreover, autoradiographic ligand-binding studies identified the presence of both muscarinic and nicotinic cholinergic receptors in the BNST [108, 109]. Neuroanatomical and electrophysiological evidence have suggested the presence of cholinergic terminals throughout the BNST [38, 63, 110]. Moreover, acetylcholine seems to play an excitatory role in activity of BNST neurons [63, 110]. However, although some data indicate that cholinergic innervation to BNST is supplied by the latero-dorsal tegmental nucleus [111], the source of these inputs is not completely established.

Few studies have investigated the involvement of BNST cholinergic neurotransmission on cardiovascular modulation. We have observed that microinjection of carbachol, a nonselective muscarinic receptor agonist, into the BNST of unanesthetized rats caused increase in blood pressure that was followed by bradycardia [112]. These responses were abolished after local pretreatment with a selective M₂ receptor antagonist as well as after intravenous treatment with a selective V₁-vasopressin receptor antagonist [112]. Therefore, these results suggested that cardiovascular effects of carbachol were mediated by activation of local M₂ receptors that, in turn, evokes acute release of vasopressin into the systemic circulation. Detailed discussion of the BNST role in control of vasopressin release into the circulation is presented below (BNST and neuroendocrine function).

A recent study demonstrated that microinjection into the BNST of the endogenous neurotransmitter acetylcholine also caused pressor responses, but without changing HR in urethane-anesthetized rats, and effects were blocked by local pretreatment with a nonselective muscarinic receptor antagonist [54]. Urethane decreases the baroreflex control of HR [62]. Although authors reported reflex responses in response to intravenous administration of phenylephrine, it remains possible that anesthesia can account for the absence of bradycardia following acetylcholine microinjection into the BNST of anesthetized animals. Other interesting difference between cardiovascular responses following local administration of carbachol or the endogenous neurotransmitter acetylcholine was that although acetylcholine microinjection into the BNST evokes pressor response in anesthetized animals, urethane-anesthesia inhibited carbachol cardiovascular responses [112]. The reasons for the discrepancy are not clear. Therefore, further studies are necessary to clarify whether cardiovascular responses to carbachol microinjection into the BNST mimic responses evoked by local treatment with the endogenous neurotransmitter.

Nasimi & Hatam (2011) also investigated a possible influence of BNST cholinergic neurotransmission in baroreflex control of the HR [54]. They observed that although bilateral treatment of the BNST with a muscarinic receptor antagonist did not affect reflex bradycardia, local acetylcholine microinjection significantly reduced bradycardiac baroreflex response in urethane-anesthetized rats [54].

2.5. Nitric Oxide

Nitric oxide (NO) is a small/short-lived molecule that modulates numerous physiological functions [113]. NO was initially described as the endothelium-derived relaxing factor (EDRF) [114, 115], been previously described by Furchgott and Zawadzki (1980) [116]. However, a set of key findings led to the identification of NO as a signalling molecule in the brain and other tissues (for review see [117]).

NO is synthesized from l-arginine by three cell-specific nitric oxide synthases (NOS) isoforms. Neuronal nitric oxide synthase (nNOS or Type I) and endothelial nitric oxide synthase (eNOS or Type III) are constitutively expressed enzymes, whose activities are stimulated by increases in intracellular Ca⁺², whereas inducible nitric oxide synthase (iNOS or Type II) is calcium-independent and mediates immune functions of NO [118]. Although other mechanisms have been described (e.g., nitration of protein thiol groups or S-nitration and inhibition of mitochondrial cytochrome c oxidase) [117], the majority of NO effects are mediated by activation of soluble guanylyl cyclase(sGC) and consequent cyclic guanosine monophosphate(cGMP) formation [117, 119].

The nNOS has been demonstrated as the major isoform involved on NO synthesis in the central nervous system [120]. Garthwaite and collaborators demonstrated that activation of NMDA glutamate receptors present in postsynaptic neurons in the central nervous system results in the formation of NO, through a mechanism dependent on Ca⁺² [121]. Therefore, it has been proposed that the nNOS enzyme in the brain is activated in response to Ca⁺² influx following the activation of NMDA receptors by glutamate [117, 122]. NO has been demonstrated to play an important role in several cerebral functions and/or dysfunctions, including regulation of neuronal excitability, synaptic plasticity (e.g., long-term potentiation and depression), neurotoxicity, neuroprotection, modulation of several behaviors and, importantly, control of cardiovascular function [117, 119].

NOS-positive neurons were identified in numerous regions associated with control of cardiovascular function, including the BNST [40]. However, a participation of BNST nitrergic mechanisms in control of cardiovascular function has not been extensively investigated. To address this issue, a recent study from our group demonstrated that bilateral microinjection of either the nonselective NOS inhibitor L-NG-Nitroarginine methyl ester (L-NAME) or a selective nNOS inhibitor into the BNST increased bradycardiac baroreflex response [72]. Also, BNST treatment with a NO scavenger caused an effect similar to that observed after NOS blockade [72]. Interestingly, changes on baroreflex activity following local NOS blockade or NO scavenger was similar to that observed after BNST treatment with a selective NMDA receptor antagonist, suggesting a NMDA receptor–NO interaction [72]. This idea was further reinforced by observation that changes on reflex bradycardia induced by local NMDA receptors were reverted by BNST treatment with a NO-donor [72].

Further studies are required for full characterization of cardiovascular control by BNST NMDA receptor-NO pathway. Moreover, the expression of iNOS and eNOS has been documented in brain cells [123, 124], suggesting a role of other NOS isoforms in control of NO function in the central nervous system. In addition, signaling from cerebral vascular beds to neurons by endothelial production of NO has been proposed [117]. Therefore, investigation of mechanisms other than those involving nNOS is also necessary.

2.6. Cannabinoids

The neuronal endocannabinoid system is classically described as being composed of two best-characterized endocannabinoids: anandamide and 2-arachidonoylglycerol [125]. Endocannabinoid signalling is mediated by the cannabinoid receptors type-1 (CB1) and type-2 (CB2), as well as by proteins responsible for endocannabinoid synthesis, transport and degradation [125, 126].

The endocannabinoid system plays an important regulatory role in several brain functions. The CB1 receptor is highly expressed throughout the central nervous system and modulates synaptic transmission and plasticity in many brain regions, including the BNST [35, 127]. Activation of these receptors on axon terminals regulates ion channel activity, inhibiting neurotransmitter release [125, 128]. The CB1 receptor is present in glutamatergic and GABAergic terminals in the BNST [127, 129, 130]. Moreover, electro-physiological studies have demonstrated that activation of CB1 receptors inhibits inhibitory and excitatory transmission in BNST slices [73, 127], which could be important for the regulation of behavioral and physiological responses in this area. Despite these pieces of evidence, no study so far has investigated the effects of direct injections into the BNST of CB1 receptor agonist or antagonist on cardiovascular function.

A recent study from our group have shown that systemic treatment with cannabidiol, a non-psychotomimetic component of Cannabis sativa that has shown anxiolytic properties, was able to reduce c-Fos expression in the BNST following re-exposure to an aversive context previously paired with footshocks [131]. These results provide evidence that the BNST is involved in antiaversive effects of cannabidiol. We have also shown that direct injection of cannabidiol into the BNST attenuated the behavioral and cardiovascular changes induced by contextual fear conditioning [132]. In addition, BNST treatment with cannabidiol enhanced tachycardiac response evoked by acute exposure to restraint stress, without affecting blood pressure changes [133]. However, cannabidiol into the BNST did not induce any significant change in baseline values of arterial pressure and HR. Therefore, it is unlikely that the attenuation of the cardiovascular responses to aversive context depends on direct cardiovascular effects, but rather on an attenuation of the emotional response. In agreement with this proposal, intra-BNST administration of cannabidiol has been shown to induce anxiolytic-like effects in animal submitted to elevated plus-maze and Vogel conflict test [134]. However, a direct influence of cannabidiol in cardiovascular function could not be ruled out. Accordingly, we have recently documented that cannabidiol microinjected into the BNST facilitates bradycardiac baroreflex response [135].

The mechanisms associated with effects of cannabidiol are still poorly understood. Although cannabidiol has low affinity for cannabinoids receptors [136], it can block the reuptake of the endogenous cannabinoid anandamide and its metabolism by the enzyme fatty acid amide hydrolase [137, 138]. Also, cannabidiol can activate TRPV₁ receptors [137], enhance adenosine signaling through inhibition of uptake [139], and modulate serotoninergic neurotransmission by allosterically inhibiting 5-HT₃ receptor [140] or acting as agonist at the 5-HT_1A receptor [141].

The BNST receives dense serotoninergic innervation from caudal regions of the dorsal raphe nucleus [142, 143]. Moreover, BNST neurons express a variety of serotonin receptors, and 5-HT_1A receptors is one the most prevalent receptor subtypes expressed [144]. In this way, serotonin elicits inhibitory responses, evoking membrane hyper-polarization, in the majority of BNST neurons by activation of postsynaptic 5-HT_1A receptors [145, 146]. Interestingly, all effects following CBD microinjection into the BNST were inhibited after local pretreatment with a 5-HT_1A antagonist [132, 133, 135]. Therefore, activation of local 5-HT_1A seems to be a key mechanism involved in CBD action in the BNST.

3.1. BNST and Hypothalamus-pituitary-adrenal Axis

The BNST plays a major role in regulation of HPA responses to stress. Several of the subregions comprising the BNST, including anterolateral and posteromedial subdivisions, send direct projections to corticotropin releasing-hormone (CRH) containing parvocellular regions of the PVN [13, 15, 174], providing a mechanism for direct actions on HPA axis output. Anatomical studies indicate that the vast majority of BNST input to the parvocellular PVN is GABAergic [74], suggesting that BNST provides largely inhibitory input to HPA axis output neurons. Innervation by BNST GABA neurons may contribute to the pronounced inhibitory tone on parvocellular PVN neurons [16]. Indeed, lesions of some BNST subregions, most notably the posterior medial zone, increase basal CRH and parvocellular arginine vasopressin (AVP) mRNA expression, suggesting release of inhibition on ACTH secretagogue gene expression [175]. In addition, lesions of the posterior BNST result in elevated corticosterone release and increased parvocellular PVN Fos activation following acute restraint [48], indicating that this cell group participates in inhibition of responses to psychogenic stressors.

However, some regions of the BNST, most notably the fusiform nucleus, contain substantial populations of PVN-projecting CRH neurons [13]. Local PVN or icv injections of CRH cause parvocellular PVN Fos activation and induce corticosterone release, suggesting that at least some BNST neurons may participate in activation of the HPA axis. This possibility is supported by data demonstrating decreased stress-induced ACTH, corticosterone and PVN c-fos mRNA induction following lesions of the anteroventral (but not anterodorsal) CRH cell groups of the BNST [48].

The effects of the BNST on stress responsiveness are dependent upon the duration of exposure. For example, lesions of the anteroventral BNST reduce HPA axis response to acute stress, but markedly potentiate HPA stress responses following chronic unpredictable stress exposure [176], suggesting that different cell populations may be recruited by acute vs. chronic stress.Notably, lesions of the posterior medial BNST potentiate stress responses after chronic unpredictable stress [177], consistent with a role in limiting sensitization of the HPA axis after prolonged stress.

The anteroventral region of the BST expresses both GABA and CRH [13]. Immunolesioning experiments demonstrate that there are mutually exclusive populations of GABA and CRH neurons [17]. Moreover, selective damage of anteroventral BNST GABAergic (but not CRH) cells causes increases in stress-induced HPA axis activation and PVN Fos expression [17], suggesting functional segregation among intermingled BNST neurons in this region. Anteroventral BNST CRH neurons are activated by stimuli that cause HPA axis activation (e.g., interleukin 1-beta) [178], suggesting that activation of the HPA axis may be mediated, at least in part, by BNST CRH neurons.

The BNST receives rich innervation from limbic forebrain structures such as the ventral subiculum, medial amygdala and central amygdala [16], as well as more limited input from the medial prefrontal cortex [17, 18]. Neurons in the ventral subiculum, prelimbic cortex and medial amygdala project to PVN-projecting neurons in the posteromedial BNST, suggesting that the BNST serves as a relay between limbic processing of emotional information with eventual elaboration of an HPA axis stress response. Indeed, there is evidence for co-innervation of PVN-projecting BNST neurons by the ventral subiculum and prelimbic cortex [179], indicating that limbic information may be integrated at the level of the individual BNST neuron.

GABAergic neurons comprise the principle target of the primary amygdalar regions innervating the BNST (medial and central amygdaloid nuclei). Moreover, the vast majority of BNST neurons are GABAergic in phenotype (>90%) [74]. Consequently, activation of the HPA axis may be mediated in part by disinhibition of PVN-projecting BNST neurons (i.e., inhibition of an inhibitory neuron) [16]. To test this hypothesis, our group performed injections of muscimol, a GABA-A receptor agonist, into the posterior-medial BNST. Muscimol causes hyperpolarization of GABA-A receptor-bearing neurons and results in a net inactivation of neurons in the area of injection. Our data indicate that local inactivation of the posterior BNST enhances HPA responses to acute restraint stress (Fig. ​33), consistent with the hypothesized disinhibitory mechanism of action. These data are supported by immunolesion studies, which demonstrate that destruction of ventral BNST GABA neurons enhance HPA axis responses to acute stress [17].

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Fig. (3)

Plasma ACTH (P<0.05) and corticosterone (cort)(P<0.05) response to an acute 30 min restraint stress challenge was enhanced in animals that received the GABA-A receptor agonist muscimol into the posterior-medial BNST. Muscimol microinjection into the posterior-medial BNST also increased c-fos expression in the PVN of the hypothalamus(P<0.05). Data are shown as mean±SEM.*P<0.05 versus saline.

The BNST receives substantial input from brainstem noradrenergic neurons, with particularly dense projections to the anterolateral and anteroventral regions [70, 180]. The relationship between BNST noradrenergic innervation and motivated behaviors, e.g., addiction and relapse, is well characterized, and numerous studies indicate that stress exposure causes release of noradrenaline in the BNST. In general, BNST noradrenaline release causes inhibition of HPA axis responses to stress [70]. Similarly, recent data from our group indicate that microinjection of a cocktail of α₁- and α₂-adrenoceptor antagonist(WB4101 and RX821002) into the anterolateral BNST (but not the β-adrenoceptor antagonist propranolol) increases corticosterone release following acute restraint stress (Fig. ​44), further suggesting an inhibitory influence of BNST noradrenergic neuro-transmission on PVN neural activity.However, presynaptic facilitation of norepinephrine release by the α₂-adrenoceptor antagonist yohimbine increases PVN Fos activation, an effect that can be attenuated by DBH-DSAP lesions of the BNST [181]. Noradrenergic effects appear to be mediated by a combination of presynaptic inhibition of noradrenaline release and inhibition of glutamate neurotransmission [182, 183].

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Fig. (4)

Plasma corticosterone response to an acute 30 min restraint stress challenge in animals that received bilateral microinjection into the anterolateral BNST of vehicle (100nL), the β-adrenoceptor antagonist propranolol (10nmol/100nL) or a cocktail containing the selective α₁-adrenoceptor antagonist WB4101 (10nmol/100nL) and the selective α₂-adrenoceptor antagonist RX821002 (10nmol/100nL) (WB+RX). The response in animals treated with the cocktail of WB+RX was enhanced (P<0.05), whereas propranolol microinjection did not affect plasma corticosterone response (P>0.05). Circles represent the mean and bars the SEM; n=12-14. *P<0.05 versus vehicle group, two-way ANOVA followed by Bonferroni’s post hoc test.

Both estrogen (ERalpha, ERbeta) and androgen (AR) receptors are abundantly expressed in the BNST, particularly its posterior-medial subdivisions [184, 185]. A substantial number of AR neurons project to the PVN [186]. Recent data suggest that dihydrotestosterone implants into the posterior BNST enhance stress-induced PVN Fos activation and increase PVN AVP mRNA, suggesting that BNST AR activation may promote HPA axis activation [187]. The role of BST ERs remains to be evaluated.

The BNST contains numerous other peptidergic terminal fields and cell groups, expressing proenkephalin, nociceptin/orphanin FQ (N/OFQ), cholecystokinin, galanin, substance P, and pituitary adenylate cyclase-activating peptide (PACAP), as well as others [12, 188-190]. While not extensively studied, some data exist to suggest these at least some of these peptidergic neurons are important in HPA axis regulation. For example, local injection of N/OFQ increases HPA axis responses to novelty [191]. Electrophysiological data suggest that N/OFQ inhibits the majority of BNST neurons [192], suggesting that N/OFNQ activates the PVN via inhibition of inhibitory BNST output neurons. Microinjection of a galanin antagonist into the BNST increases stress-induced anxiety and ACTH responses [193], suggesting that BNST galanin receptors increases activation of the HPA axis in the context of stress. Other peptides resident in the BNST, such as PACAP, can directly activate CRH neurons [194], suggesting possible excitatory peptidergic projections to the PVN. Notably, BNST PACAP is up-regulated by chronic stress, a condition that causes hyperactivity of the HPA axis [195].

The authors wish to thank financial support from FAPESP (2010/16192-8), CNPq (474177/2010-6), PADC-UNESP and FUNDUNESP.