Involvement of the Bed Nucleus of the Stria Terminalis in Tonic Regulation of Paraventricular Hypothalamic CRH and AVP mRNA Expression

The bed nucleus of the stria terminalis (BNST) occupies a central position in pathways regulating hypothalamo-pituitary-adrenocortical (HPA) stress regulation. The potential role of the BNST in tonic neural control of HPA function was assessed by examining effects of selective BNST lesions on expression of ACTH secretagogues in HPA-integrative neurons of the medial parvocellular paraventricular nucleus. Anterior BNST lesions (ABN) involved major portions of the anteromedial, anterolateral, ventromedial, ventrolateral, dorsolateral and juxtacapsular subnuclei. These lesions resulted in significant (30%) decreases in corticotropin-releasing hormone (CRH) mRNA expression across the rostrocaudal extent of the medial parvocellular PVN, with no accompanying changes in basal arginine vasopressin (AVP) mRNA levels. Posterior BNST (PBN) lesions involved large but subtotal damage to the posterior intermediate, posterior medial, posterior lateral and preoptic subnuclei; these lesions resulted in small but significant changes in CRH mRNA and slight increases in number of AVP mRNA-producing parvocellular neurons. PBN effects on CRH mRNA expression were most pronounced at the caudal extent of the medial parvocellular zone, suggesting a topographic input from the posterior BNST to the PVN that is only partially compromised by PBN lesions. Analysis of individual cases revealed a correlation between damage of the anterolateral BNST and decreased CRH mRNA levels, and damage of the posterior intermediate and/or posterior medial BNST and increased CRH mRNA levels. The results suggest differential BNST input into HPA regulation, perhaps reflecting the diversity of limbic input into the BNST region.     

Sexually Dimorphic Expression of Androgen Receptor Immunoreactivity by Somatostatin Neurones in Rat Hypothalamic Periventricular Nucleus and Bed Nucleus of the Stria Terminalis

Gonadal steroids exert important regulatory actions on the hypothalamic neurones regulating growth hormone secretion and are believed to play a role in generating its sexually dimorphic pattern of secretion. Recent evidence indicates that estrogen actions on one of these neural populations, the periventricular somatostatin (SOM) neurones, are likely to be indirect as they do not possess nuclear estrogen receptors in either sex although androgen receptors (ARs) have been reported within these cells in male rats. The present study has used double-labelling immunocytochemistry procedures to examine whether sex differences exist in AR expression by SOM neurones located in the periventricular nucleus and bed nucleus of the stria terminalis (BNST). Within the hypothalamus, SOM-immunoreactive neurones were found concentrated in the periventricular nucleus while both anterior and posterior divisions of the BNST contained scattered populations of SOM cells. Cells immunoreactive for the AR were detected in all of these areas. Although the intensity of AR cell nuclei staining was equivalent in males and females in regions such as the lateral septum, the intensity of AR staining in many individual cells of the periventricular nucleus and posterior BNST of the female was reduced when compared with the male. Double-labelling experiments revealed that approximately 40% of periventricular SOM neurones expressed AR immunoreactivity in the male compared with significantly (P<0.01) fewer cells in the female (～7%). In the BNST, double-labelled cells were only detected within the principle encapsulated, interfascicular and transverse nuclei of its posterior division. Approximately 60% of SOM cells in these nuclei expressed AR immunoreactivity in the male while significantly (P<0.01) fewer did so in the female (～25%). These results indicate that substantial sex differences exist in AR expression by SOM neurones in both the periventricular nucleus and BNST. Such differences in AR expression by periventricular SOM cells may contribute to their sexually dimorphic nature and, consequently, sex differences in growth hormone secretion.     

Roles for Pituitary Adenylate Cyclase-Activating Peptide (PACAP) Expression and Signaling in the Bed Nucleus of the Stria Terminalis (BNST) in Mediating the Behavioral Consequences of Chronic Stress

Anxiety disorders are frequently long-lasting and debilitating for more than 40 million American adults. Although stressor exposure plays an important role in the etiology of some anxiety disorders, the mechanisms by which exposure to stressful stimuli alters central circuits that mediate anxiety-like emotional behavior are still unknown. Substantial evidence has implicated regions of the central extended amygdala, including the bed nucleus of the stria terminalis (BNST) and the central nucleus of the amygdala as critical structures mediating fear- and anxiety-like behavior in both humans and animals. These areas organize coordinated fear- and anxiety-like behavioral responses as well as peripheral stress responding to threats via direct and indirect projections to the paraventricular nucleus of the hypothalamus and brainstem regions (Walker et al. Eur J Pharmacol 463:199–216, 2003, Prog Neuropsychopharmacol Biol Psychiatry 33(8):1291–1308, 2009; Ulrich-Lai and Herman Nat Rev Neurosci 10:397–409, 2009). In particular, the BNST has been argued to mediate these central and peripheral responses when the perceived threat is of long duration (Waddell et al. Behav Neurosci 120:324–336, 2006) and/or when the anxiety-like response is sustained (Walker and Davis Brain Struct Funct 213:29–42, 2008); hence, the BNST may mediate pathological anxiety-like states that result from exposure to chronic stress. Indeed, chronic stress paradigms result in enhanced BNST neuroplasticity that has been associated with pathological anxiety-like states (Vyas et al. Brain Res 965:290–294, 2003; Pego et al. Eur J Neurosci 27:1503–1516, 2008). Here we review evidence that suggests that pituitary adenylate cyclase-activating polypeptide (PACAP) and corticotropin-releasing hormone (CRH) work together to modulate BNST function and increase anxiety-like behavior. Moreover, we have shown that BNST PACAP as well as its cognate PAC1 receptor is substantially upregulated following chronic stress, particularly in the BNST oval nucleus where PACAP-containing neurons closely interact with CRH-containing neurons (Kozicz et al. Brain Res 767:109–119, 1997; Hammack et al. Psychoneuroendocrinology 34:833–843, 2009). We describe how interactions between PACAP and CRH in the BNST may mediate stress-associated behaviors, including anorexia and anxiety-like behavior. These studies have the potential to define specific mechanisms underlying anxiety disorders, and may provide important therapeutic strategies for stress and anxiety management.     

Exogenous Corticosterone Induces the Expression of the Clock Protein,PERIOD2, in the Oval Nucleus of the Bed Nucleus of the Stria Terminalis and the Central Nucleus of the Amygdala of Adrenalectomized and Intact Rats

The cyclical expression of the clock protein PERIOD2 (PER2) in select regions of the limbic forebrain is contingent upon the rhythmic secretion of the adrenal glucocorticoid, corticosterone. Daily rhythmic PER2 expression in the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and the central nucleus of the amygdala (CEA) is abolished with the removal of the adrenal glands but restored with rhythmic hormone replacement via the drinking water at a time corresponding to the endogenous peak of circulating glucocorticoids. Here, we investigated the effects of serial or acute systemic injections of corticosterone on the expression of PER2 in the BNSTov and CEA of both adrenalectomized (ADX) and intact rats. We sought to determine whether there is a temporal window of corticosterone sensitivity by delivering the hormone at a time corresponding to trough levels of circulating glucocorticoids, at lights on. We found that daily morning injections of corticosterone induced PER2 expression in the BNSTov and CEA of ADX rats, with levels peaking 1 h after injection. In intact rats, daily morning injections significantly upregulated the expression of PER2 in the BNSTov and CEA 1 h after injection and dampened the evening peak, while a single injection abolished the rhythm of PER2 expression in the CEA but had no effect on PER2 in the BNSTov. Our findings suggest that despite the potential masking effect of signals from the light-entrained master clock, daytime chronic and acute corticosterone administration can alter the rhythmic expression of PER2 in the BNSTov and CEA, and that the response is region-specific and dependent on the duration of treatment.     

Cocaine Augments Dopamine-Mediated Inhibition of Neuronal Activity in the Dorsal Bed Nucleus of the Stria Terminalis

The dorsal region of the bed nucleus of the stria terminalis (dBNST) receives substantial dopaminergic input which overlaps with norepinephrine input implicated in stress responses. Using ex vivo fast scan cyclic voltammetry in male C57BL6 mouse brain slices, we demonstrate that electrically stimulated dBNST catecholamine signals are of substantially lower magnitude and have slower uptake rates compared with caudate signals. Dopamine terminal autoreceptor activation inhibited roughly half of the catecholamine transient, and noradrenergic autoreceptor activation produced an ∼30% inhibition. Dopamine transporter blockade with either cocaine or {"type":"entrez-protein","attrs":{"text":"GBR12909","term_id":"2281680821","term_text":"GBR12909"}}GBR12909 significantly augmented catecholamine signal duration. We optogenetically targeted dopamine terminals in the dBNST of transgenic (TH:Cre) mice of either sex and, using ex vivo whole-cell electrophysiology, we demonstrate that optically stimulated dopamine release induces slow outward membrane currents and an associated hyperpolarization response in a subset of dBNST neurons. These cellular responses had a similar temporal profile to dopamine release, were significantly reduced by the D2/D3 receptor antagonist raclopride, and were potentiated by cocaine. Using in vivo fiber photometry in male C57BL/6 mice during training sessions for cocaine conditioned place preference, we show that acute cocaine administration results in a significant inhibition of calcium transient activity in dBNST neurons compared with saline administration. These data provide evidence for a mechanism of dopamine-mediated cellular inhibition in the dBNST and demonstrate that cocaine augments this inhibition while also decreasing net activity in the dBNST in a drug reinforcement paradigm.SIGNIFICANCE STATEMENT The dorsal bed nucleus of the stria terminalis (dBNST) is a region highly implicated in mediating stress responses; however, the dBNST also receives dopaminergic inputs from classically defined drug reward pathways. Here we used various techniques to demonstrate that dopamine signaling within the dBNST region has inhibitory effects on population activity. We show that cocaine, an abused psychostimulant, augments both catecholamine release and dopamine-mediated cellular inhibition in this region. We also demonstrate that cocaine administration reduces population activity in the dBNST, in vivo. Together, these data support a mechanism of dopamine-mediated inhibition within the dBNST, providing a means by which drug-induced elevations in dopamine signaling may inhibit dBNST activity to promote drug reward.     

Somatostatin Neurons of the Bed Nucleus of Stria Terminalis Enhance Associative Fear Memory Consolidation in Mice

Excessive fear learning and generalized, extinction-resistant fear memories are core symptoms of anxiety and trauma-related disorders. Despite significant evidence from clinical studies reporting hyperactivity of the bed nucleus of stria terminalis (BNST) under these conditions, the role of BNST in fear learning and expression is still not clarified. Here, we tested how BNST modulates fear learning in male mice using a chemogenetic approach. Activation of GABAergic neurons of BNST during fear conditioning or memory consolidation resulted in enhanced cue-related fear recall. Importantly, BNST activation had no acute impact on fear expression during conditioning or recalls, but it enhanced cue-related fear recall subsequently, potentially via altered activity of downstream regions. Enhanced fear memory consolidation could be replicated by selectively activating somatostatin (SOM), but not corticotropin-releasing factor (CRF), neurons of the BNST, which was accompanied by increased fear generalization. Our findings suggest the significant modulation of fear memory strength by specific circuits of the BNST.SIGNIFICANCE STATEMENT The bed nucleus of stria terminalis (BNST) mediates different defensive behaviors, and its connections implicate its integrative modulatory role in fear memory formation; however, the involvement of BNST in fear learning has yet to be elucidated in detail. Our data highlight that BNST stimulation enhances fear memory formation without direct effects on fear expression. Our study identified somatostatin (SOM) cells within the extended amygdala as specific neurons promoting fear memory formation. These data underline the importance of anxiety circuits in maladaptive fear memory formation, indicating elevated BNST activity as a potential vulnerability factor to anxiety and trauma-related disorders.     

Oxytocin Receptors in the Anteromedial Bed Nucleus of the Stria Terminalis Promote Stress-Induced Social Avoidance in Female California Mice

Background

The neuropeptide oxytocin (OT) is a key regulator of social and emotional behaviors. The effects of OT are context dependent, and it has been proposed that OT increases the salience of both positive and negative social cues. Here we tested whether the bed nucleus of the stria terminalis (BNST) mediates anxiogenic effects of OT.

Oxytocin in the Bed Nucleus of the Stria Terminalis and Lateral Septum Facilitates Bursting of Hypothalamic Oxytocin Neurons in Suckled Rats

Several regions of the forebrain possess high densities of oxytocin (OT)-binding sites including the bed nucleus of the stria terminalis (BST) and lateral septum (LS). In order to examine whether these regions participate in the central facilitation of the milk ejection reflex by OT, microinjections of OT (1 ng in 100 nl containing Janus Green dye) were made into the BST (13 tests) or LS (9 tests) of anaesthetized, suckled rats, while recording the electrical activity of OT neurons in the contralateral supraoptic nucleus. Histological localization of injection sites using Janus Green demonstrated that all BST injections were close to the anterior commissure, and LS injections were all located in the ventral division of the LS. Film autoradiographic visualization of OT-binding sites (in 7 tests using [¹²⁵I]OT antagonist) confirmed that the BST and LS injections were located within regions of high OT binding. Injections into both regions facilitated the milk ejection reflex by increasing either the frequency and/or amplitude of OT neuron bursts, or by triggering bursts in animals that previously had shown no milk ejection responses; the mean number of milk ejections in the 30 min before and after injection increasing from 1.6·0.5 to 3.6·0.5 for BST and from 1.5·0.6 to 3.9·0.4 for LS. The OT microinjections had a more variable effect on background activity of OT neurons, increasing firing in some cases and not in others. This facilitatory effect was similar to that induced by microinjections into the lateral ventricle, but was smaller and delayed compared to that induced by injection into the third ventricle (9 tests), possibly due to unilateral activation of target sites. The facilitatory effect was unlikely to have been due to diffusion of OT into the ventricle, since injections into control sites (striatum and thalamus) at similar distances from the ventricle (9 tests) had no facilitatory effect (number of bursts during 30 min before and after injection; 2.2·0.5 and 1.8·0.5, respectively). These data suggest that limbic structures (BST and LS) participate in the action of central OT on the pattern of milk ejections in the suckled rat.     

Maternal Behaviour is Associated with Vasopressin Release in the Medial Preoptic Area and Bed Nucleus of the Stria Terminalis in the Rat

The neuropeptide arginine vasopressin was recently shown to be an important regulator of female social behaviour, including maternal care and aggression. A key brain site for vasopressin‐ as well as oxytocin‐mediated maternal care is the medial preoptic area (MPOA). Together with the adjacent bed nucleus of the stria terminalis (BNST), these brain regions are considered to form a ‘super‐region’ for maternal behaviour. In the present study, we investigated the vasopressin and oxytocin systems within the MPOA and the BNST during maternal care in lactating rats in more detail. Binding to V1a and oxytocin receptors in the BNST and to oxytocin receptors in the MPOA was increased in lactation. Furthermore, microdialysis revealed that vasopressin release significantly increased (MPOA) or tended to increase (BNST) during different phases of maternal care (i.e. with or without suckling stimulus). In support, manipulations of V1a receptors in the MPOA are known to alter maternal care. We now show that local injection of a selective V1a receptor antagonist bilaterally into the BNST did not affect maternal care, but reduced maternal aggression and tended to lower anxiety‐related behaviour. The release of oxytocin did not change in any of the brain regions during maternal care. The results obtained indicate that locally‐released vasopressin within the MPOA and the BNST is important for the maintenance of complex maternal behaviours, including maternal care and aggression, respectively.     

Plasticity in the Stress-Regulating Circuit: Decreased Input from the Bed Nucleus of the Stria Terminalis to the Hypothalamic Paraventricular Nucleus in Wistar Rats following Adrenalectomy

The bed nucleus of the stria terminalis is involved in the stress-regulating circuit by funnelling limbic information to the hypothalamic paraventricular nucleus. Since adrenalectomy influences both limbic structures (by inducing cell death in the hippocampus) and the hypothalamic paraventricular nucleus (by increased corticotrophin-releasing hormone synthesis), we investigated whether the bed nucleus of the stria terminalis is also influenced by adrenalectomy. For this purpose, we analysed and compared the projections from the bed nucleus of the stria terminalis to the hypothalamic paraventricular nucleus in normal and adrenalectomized rats by anterograde tracer injections in the bed nucleus of the stria terminalis. Quantitative analysis of the fibre pattern in the hypothalamic paraventricular nucleus of normal rats revealed a homogeneous distribution of fibres of the bed nucleus of the stria terminalis over the different subdivisions of the hypothalamic paraventricular nucleus. In adrenalectomized rats, the absolute fibre density was significantly lower in the whole hypothalamic paraventricular nucleus (1.17 ± 0.27 10^?3μm/μm³ in adrenalectomized rats versus 2.59 ± 0.24 10^?3μm/μm³ in normal rats; P < 0.01) and all its subdivisions. The largest decrease of fibre density was found in the corticotrophin-releasing hormone-rich part of the hypothalamic paraventricular nucleus (relative fibre density; adrenalectomized rats: 0.602 ± 0.106, versus 1.095 ± 0.019 in normal rats, P < 0.01). These results show a loss of input from the bed nucleus of the stria terminalis to the hypothalamic paraventricular nucleus, and particularly to the corticotrophin-releasing hormone neurons, following adrenalectomy. The data suggest that this pathway within the stress-regulating circuit is functionally affected by corticosteroids in adult rats and may imply that human disorders associated with corticosteroid imbalance are allied to a changed circuitry in the brain.     

Androgenic and Oestrogenic Influences on Tyrosine Hydroxylase‐Immunoreactive Cells of the Prairie Vole Medial Amygdala and Bed Nucleus of the Stria Terminalis

The posterodorsal medial amygdala (MeApd) and principal nucleus of the bed nucleus of the stria terminalis (pBST) are densely interconnected sites integrating steroid hormone and olfactory information necessary for sociosexual behaviours in many rodents. Our laboratory recently reported sexually dimorphic populations of cells containing tyrosine hydroxylase (TH) located in the MeApd and pBST of prairie voles (Microtus ochrogaster), with males having many more TH‐immunoreactive (‐ir) cells in these sites than do females. Gonadal hormones circulating during adulthood were showm to regulate this sex difference because it was eliminated by castrating adult males or implanting females with testosterone‐filled capsules. In the present study, we demonstrate that many (25–65%) TH‐ir cells in the MeApd and pBST of adult virgin male and female prairie voles also contain immunoreactivity for either the androgen receptor or oestrogen receptor α. Subcutaneous implants of oestradiol benzoate mimicked the effects of testosterone and maintained high numbers of TH‐ir cells in these sites in castrated males. However, implants of dihydrotestosterone (DHT) did not, and these males had low numbers of TH‐ir cells similar to castrated males given empty capsules. A similar effect was found in females, where testosterone or oestradiol benzoate greatly increased the number of TH‐ir cells in these sites compared to intact or ovariectomised controls, but DHT did not. DHT implants did, however, maintain high seminal vesicle weights in males. Thus, many of the TH‐ir cells in the prairie vole MeApd and pBST are potentially sensitive to androgens and oestrogens, although maintaining immunocytochemically detectable levels of TH in these cells may depend more on an oestrogen‐mediated mechanism in both sexes. These data have implications for understanding how gonadal hormone release across the reproductive cycle modulates these species‐specific groups of catecholaminergic cells and socially monogamous behaviours in prairie voles.     

Projection Sites of Medial Preoptic Area and Ventral Bed Nucleus of the Stria Terminalis Neurons that Express Fos during Maternal Behavior in Female Rats

Medial preoptic area (MPOA) and ventral bed nucleus of the stria terminalis (VBST) neurons are involved in maternal behavior, but the neural sites to which the maternally relevant neurons project have not been determined. Since MPOA and VBST neurons express Fos during maternal behavior, we used a double-labeling immunocytochemical procedure to detect both Fos and a retrograde tracer, wheat germ agglutinin (WGA), in order to determine where these Fos neurons project. On Day 4 postpartum, fully maternal females were separated from their litters. On Day 5, WGA was iontophoretically injected into one of the following regions known to receive MPOA and/or VBST input: Lateral septum, medial hypothalamus at the level of the ventromedial nucleus, lateral habenula, ventral tegmental area, retrorubral field, or periaqueductal gray. On Day 7, females received a 2-h test with either pups or candy, after which they were perfused and their brains were processed for the detection of Fos and WGA. As expected, females tested with pups had more Fos-containing neurons in the MPOA and VBST than did females tested with candy. After WGA injections into several brain sites, the number of double-labeled cells observed in the MPOA and VBST was greater for the maternal females when compared to the non-maternal females. Therefore, these results pinpointed neural circuits that were activated during maternal behavior. For the maternal females, Fos-containing neurons in the MPOA projected most strongly to the medial hypothalamus at the level of the ventromedial nucleus and to the lateral septum, while Fos-containing neurons in the VBST projected most strongly to the retrorubral field, ventral tegmental area, and medial hypothalamus. Although relatively few MPOA and VBST neurons which expressed Fos during maternal behavior projected to the periaqueductal gray, these Fos-expressing neurons made up a relatively large proportion of the MPOA and VBST projection to the periaqueductal gray. This study suggests that MPOA and VBST efferents project to a variety of regions to promote full maternal responsiveness.     

Targeting Corticotropin‐Releasing Factor Projections from the Oval Nucleus of the Bed Nucleus of the Stria Terminalis Using Cell‐Type Specific Neuronal Tracing Studies in Mouse and Rat Brain

The bed nucleus of the stria terminalis (BNST) is known to play a critical role in mediating the behavioural and autonomic responses to stressors. The oval nucleus of the BNST (BNSTov) contains cell bodies that synthesise the stress hormone corticotropin‐releasing factor (CRF). Although afferent fibres originating from the BNSTov have been shown to innervate several key structures of the neuroendocrine and central autonomic system, the question remains as to whether some of these fibres are CRF‐positive. To directly address this question, we injected a ‘floxed’ anterograde tracer (rAAV5/EF1a‐DIO‐mCherry) into the BNSTov of CRFp3.0Cre^GFP transgenic mice, which express a green fluorescent protein (GFP) under the control of the CRF promoter. Serial sections were then analysed for the presence of double‐labelled fibres in potential projection sites. To determine whether CRF neurons in the rat BNSTov send comparable projections, we infused rat BNSTov with an adeno‐associated viral vector (AAV) in which the human synapsin promoter drives enhanced GFP expression. We then used CRF immunoreactivity to examine double‐labelled fluorescent fibres and axon terminals in projection sites from brain sections of the AAV‐infused rats. We have observed several terminal fields in the mouse and rat brain with double‐labelled fibres in the Dorsal raphe nucleus (DRD), the paraventricular nucleus of the hypothalamus and, to a lesser extent, in the ventral tegmental area. We found double‐labelled terminal boutons in the nucleus accumbens shell, prelimbic cortex and posterior basolateral nucleus of the amygdala. The most intense double‐labelling was found in midbrain, including substantia nigra pars compacta, red nucleus, periaqueductal grey and pontine nuclei, as well as DRD. The results of the present study indicate that CRF neurons are the output neurons of the BNSTov and they send projections not only to the centres of neuroendocrine and autonomic regulation, but also regions modulating reward and motivation, vigilance and motor function, as well as affective behaviour.     

Autoradiographic Detection of Oxytocin- and Vasopressin-Binding Sites in Various Subnuclei of the Bed Nucleus of the Stria Terminalis in the Rat. Effects of Functional and Experimental Sexual Steroid Variations

Oxytocin- and vasopressin-binding sites were detected by autoradiography on films and on emulsion-coated sections of rat brains using highly selective [¹²⁵|]-labelled oxytocin and vasopressin antagonists. Two distinct areas with high concentrations of oxytocin-binding sites were detected in the bed nucleus of the stria terminalis: 1) the principal encapsulated nucleus and the associated cell-sparse zone in the posterior medial part, and 2) the oval nucleus in the anterior lateral part. A weak diffuse labelling was, in addition, detected around the oval nucleus in the anterior lateral and anterior dorsal areas. The vasopressin-binding sites were restricted to the anterior lateral part of the bed nucleus of the stria terminalis where they were highly concentrated in the juxtacapsular nucleus and present with lower density in a discrete cell group dorsal to the oval nucleus. Autoradiographic analyses of the bed nucleus of the stria terminalis from pregnant, lactating and ovariectomized rats (oestradiol treated or not) indicated that only the oxytocin-binding sites in the principal encapsulated nucleus and the associated cell-sparse zone were oestrogen-dependent. These observations are in agreement with earlier data suggesting that the two major divisions of the bed nucleus of the stria terminalis are involved in distinct regulations. The anterior lateral part, including the oval nucleus in which oxytocin receptors are not oestrogen-dependent, is, rather, involved in central autonomie regulations. The posterior medial part, where oestrogen-dependent oxytocin receptors are concentrated in the principal encapsulated nucleus and the associated cell-sparse zone, is implicated in neuroendocrine regulations and in reproductive behaviour.