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Summary model. A , Dopamine and norepinephrine afferents synapse onto CRF-producing neurons in the BNST that in turn influence neurotransmitter release from glutamatergic afferents (Glu) onto BNST neurons projecting to the VTA. B , Close up view of proposed neurocircuitry described in A . C , D , Model of CRF modulation of glutamatergic transmission onto a VTA-projecting BNST neuron in a drug-naive state ( C ) or during acute ethanol withdrawal following CIE ( D ). Note that there are higher levels of CRF and glutamate release during withdrawal compared to the drug-naive state.
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A growing literature suggests that catecholamines and corticotropin-releasing factor (CRF) interact in a serial manner to activate the bed nucleus of the stria terminalis (BNST) to drive stress- or cue-induced drug- and alcohol-seeking behaviors. Data suggest that these behaviors are driven in part by BNST projections to the ventral tegmental area...
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... next sought to determine how exposure and withdrawal from chronic ethanol might alter the ability of CRF to modulate glu- through a CRFR1-dependent mechanism and that during acute withdrawal BNST CRFR1 may be maximally active. Stress-induced reinstatement is hypothesized to occur via increased BNST excitability, likely through catecholamine and CRF signaling interactions within BNST (Brown et al., 2009; Briand et al., 2010; Buffalari and See, 2011). Here we used a combination of strategies to elucidate a potential mechanism by which catecholamines may influence CRF activity to enhance BNST excitatory neurotransmission, and we show that this system is modulated by chronic ethanol exposure. Our data show that catecholamines can depolarize BNST CRF neurons, potentially leading to increased CRF release. In turn, CRF enhances glutamatergic transmission onto VTA-projecting BNST neurons, suggesting that elevated extracellular CRF in the BNST may modulate neurocircuitry critical to drug-seeking behavior. Importantly, CIE increases basal glutamatergic tone onto VTA- projecting BNST neurons and occludes the effect of CRF. This effect of CIE can be blocked by pretreating EtOH exposed mice with a CRFR1 antagonist, suggesting that CIE modulates BNST CRF neurocircuitry in vivo and that this neurocircuit can become hyperactive during withdrawal. Together, these findings suggest the BNST contains an ethanol-sensitive circuit wherein elevated catecholamines increase CRF neuron activity to enhance excitatory transmission onto BNST neurons projecting to the VTA (Fig. 6, summary model). Previous reports indicate that BNST neurons may be classified into distinct subtypes based on electrophysiologic properties. In the rat BNST, three different classes of neurons have been described based on membrane voltage responses to current injections (Hammack et al., 2007). In addition, BNST projection We have previously shown that DA and ISO can enhance sEPSC frequency in the BNST in an activity-dependent manner (Kash et al., 2008b; Nobis et al., 2011). This effect can be mimicked by application of CRF and blocked by pretreatment with a CRFR1 antagonist, suggesting that catecholamine effects on BNST excitatory transmission require CRF receptor activation, possibly by increasing CRF release from local sources. To record specifically from CRF neurons in the BNST, we crossed two lines of commercially available mice to create the CRF- tomato line, which expresses a red fluorescent protein specifically in CRF neurons. The expression of red fluorescent protein in these mice is most dense in brain regions that have high levels of CRF-producing neurons, like the PVN, BNST, and CeA. In recordings from these mice, we found that DA and ISO caused significant and prolonged depolarization of BNST CRF neurons. Importantly, the time course of DA and ISO depolarization of CRF neurons is similar to the time course of activity-dependent DA- and ISO-induced increases of glutamatergic transmission in the BNST (Kash et al., 2008b; Nobis et al., 2011), suggesting that catecholamine enhancement of BNST excitability may be due to increased CRF release from local BNST neurons. BNST activity is necessary for reinstatement to drug seeking (Buffalari and See, 2011). Previous findings show that stress- mediated increases in extracellular BNST NE (Pacak et al., 1995) may drive reinstatement to drug seeking (Brown et al., 2011) via activation of  -ARs (Leri et al., 2002), a system that may interact with BNST CRF circuitry (Erb and Stewart, 1999; Funk et al., 2006; Wang et al., 2006) to initiate reinstatement. The data presented here expand on this hypothesis and suggest that catecholamine–CRF neurocircuit interactions in the BNST may be an important locus for initiation of stress-induced reinstatement. While the role for BNST  -ARs in reinstatement is well characterized, a potential role of BNST DA in reinstatement is much less clear. Amygdala DA signaling has been shown to be important in cue-induced reinstatement to drug seeking (Berglind et al., 2006), but the role of BNST DA in reinstatement has not yet been tested. However, inactivation of the BNST has been shown to reduce cue-induced reinstatement of cocaine seeking (Buffalari and See, 2011), and rewarding stimuli have been shown to increase extracellular DA in the BNST (Carboni et al., 2000; Park et al., 2012), which would likely increase BNST excitation based on our data. Together, these findings suggest a possible role of BNST DA activity in cue-induced reinstatement. Recent evidence suggests that DA can be released from NE terminals due to amphetamine-induced alterations in NE transporter activity, at least in the hippocampus (Smith and Greene, 2012). NE transporters in the BNST can become upregulated after chronic cocaine self-administration (Macey et al., 2003), suggesting that drug-induced alterations in BNST could also alter extracellular NE and/or DA levels through NE transporters. Our data indicate that DA can depolarize BNST CRF neurons, similar to  -AR activation. Since NE terminals in the BNST likely become activated during stress (Pacak et al., 1995), these findings suggest DA may also be released in the BNST following stress after drug exposure and further suggest a potential role of BNST DA in stress- induced reinstatement. The BNST has widespread projections that can regulate a variety of behaviors, such as stress and reward (Dong et al., 2001; Dong and Swanson, 2004; Dong and Swanson, 2006; Ulrich-Lai and Herman, 2009). However, it is not clear whether distinct populations of BNST neurons project to one specific brain region or whether a single BNST neuron may project to multiple down- stream targets simultaneously. Here, we provide initial evidence that BNST neurons projecting to the hypothalamus and VTA, two well characterized BNST targets, likely only partially overlap, suggesting that distinct pools of BNST projections neurons may independently regulate circuitry involved in reward and/or stress behavior. Supporting this hypothesis, previous reports indicate that reinstatement increases neuronal markers of activation specifically in BNST neurons that project to the VTA (Briand et al., 2010). The data presented here are in agreement with these previous findings and suggest that CRF may be an important modulator of excitatory drive onto VTA-projecting BNST neurons. We find that acute withdrawal from CIE can enhance basal glutamatergic tone onto VTA-projecting BNST neurons but occlude the effect of exogenous CRF. Additionally, the CIE enhancement of basal glutamatergic tone is blocked by pretreatment with a CRFR1 antagonist. These data are in agreement with previous work in- dicating that extracellular CRF is elevated during ethanol withdrawal (Olive et al., 2002) and suggest that CIE can modulate normal CRF signaling in the BNST in vivo . CIE can increase CRFR1 sensitivity in other components of the extended amygdala after prolonged withdrawal (Roberto et al., 2010), which might suggest that CRFR1 receptors in the BNST may also become sen- sitized following long-term withdrawal. In conjunction with the hypothesis that catecholamine–CRF interactions in the BNST may be critical for reinstatement, EtOH withdrawal-induced sen- sitization CRF circuitry in the BNST could be one mechanism for the long-term challenge of stress-induced relapse in recovering alcoholics (Sinha, 2007). Our data indicate a putative two-step reinstatement circuit centered around CRFR1 activity in the BNST, wherein catecholamines can enhance activity of BNST CRF neurons that may in turn increase the excitability of BNST neurons that project to the VTA. While more work will be needed to conclusively show the exact mode of action of this circuitry during reinstatement, we hypothesize that engagement of this circuitry following exposure to drug-associated cues or stressors likely results in increased signaling from the BNST to the VTA, thereby driving drug seeking. While the data presented here focuses on the potential role of the BNST-CRF neurocircuitry in alcoholism, recent work indicates that this neurocircuitry may also be recruited following chronic cocaine (Nobis et al., 2011) or morphine exposure (Wang et al., 2006). Together, these findings suggest that per- turbations of BNST CRF signaling may be a common mechanism for relapse regardless of the drug abused. Future research identifying specific targets within this circuitry may improve strategies for more effective long-term treatment of alcohol and drug ...
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... However, the time course for the development of increased glutamatergic input remained unknown and potential changes in GABAergic input had not been previously assessed. To address this gap with a focus on BNST neurons expressing CRF (BNST CRF ), previously implicated in both negative affect and stress-induced seeking behaviors (Silberman et al., 2013), CRF-Cre mice were crossed with an Ai14-ROSA reporter line to identify CRF-positive neurons with tdTomato expression. Female offspring were placed in the chronic drinking followed by forced abstinence (CDFA) paradigm. ...
... ;https://doi.org/10.1101https://doi.org/10. /2024 been linked to both negative affect and stress-induced seeking behaviors (Silberman et al., 2013) and are known to express Grin2d (Salimando et al., 2020). Using this approach we found that neither glutamatergic nor GABAergic transmission onto BNST CRF neurons were altered in acute withdrawal ( Figure 1B,D) but replicated enhanced glutamatergic transmission in protracted withdrawal ( Figure 1C) (Centanni et al., 2019b). ...
Alcohol use disorder (AUD) is a chronic, relapsing disease, highly comorbid with anxiety and depression. The bed nucleus of the stria terminalis (BNST), and Crh+ neurons in this region are thought to play a key role in chronic ethanol-induced increases in volitional ethanol intake. This role has been hypothesized to be driven by emergent BNST-dependent negative affective behaviors. Indeed, we report here that in female mice undergoing a home cage chronic drinking forced abstinence model (CDFA), excitatory transmission undergoes time-dependent upregulation in BNST Crh+ cells. Excitatory NMDA receptors (NMDARs) are a major target of ethanol, and chronic ethanol exposure has been shown to regulate NMDAR function and expression. GluN2D subunit-containing NMDARs have emerged as a target of interest due to their limited distribution and potential roles in affective behavior. We find that knockdown of dorsal BNST (dBNST) GluN2D expression significantly decreases ethanol intake in female, but not male, mice. While BNST Grin2b expression was significantly increased in protracted abstinence following CDFA, no differences in Grin2d expression were observed in dBNST or specifically in dBNST Crh+ neurons. Finally, to determine the impact of GluN2D expression on negative affective behaviors, open field, elevated zero maze, and forced swim tasks were used to measure anxiety- and depressive-like behaviors in constitutive and conditional BNST GluN2D knockout mice. Surprisingly, we find that deletion of GluN2D fails to alter negative affect in ethanol-naive female mice. Together, these data suggest a role for BNST GluN2D-containing NMDARs in ethanol drinking behaviors but not abstinence from ethanol, highlighting potential sex differences and behavioral specificity in the context of AUD behaviors. Overall, these data further suggest roles for BNST synaptic signaling in volitional ethanol intake that are partially independent of actions on affective behavior.
... As such, human neuroimaging studies have shown that the BNST is a hub region in the dysregulated neural circuitry found in both AUD and anxiety (O'Daly et al., 2012;Avery et al., 2014;Avery et al., 2016) that may play a causal role in the expression of alcohol drinking and anxiety behaviors. Work from our group and others has found that the BNST is a critical site for the regulation of excessive alcohol consumption in rodents (Eiler et al., 2003;Eiler and June, 2007;Pina et al., 2015;Pleil et al., 2015b;Rinker et al., 2016); and, many studies have described plasticity in the excitability and synaptic transmission of BNST neurons following chronic alcohol exposure related to the maintenance of excessive alcohol consumption and withdrawal-induced anxiety and negative affect (Olive et al., 2002;Kash et al., 2009;McElligott and Winder, 2009;Kash, 2012;McElligott et al., 2013;Silberman et al., 2013;Kash et al., 2015;Pleil et al., 2015a). ...
... BNST neurons in rodents have previously been categorized into multiple types based on voltage responses to hyperpolarizing and depolarizing current injection steps, including inward rectification and voltage sag, the latter of which has been used as a proxy measure for HCN channel-mediated Ih, and action potential firing pattern (Type I: large voltage sag, little inward rectification, persistent firing, and no It; Type II: little/no voltage sag or inward rectification, large It, burst firing, and/or rebound firing; Type III: large inward rectification, little/no voltage sag or It) (Hammack et al., 2007;Daniel et al., 2017). While these categories were determined in male rats and segregate BNST neurons into equal thirds in that subject population, both male mouse and male monkey BNST neurons have been shown to be less distinguishable based on the combination of these measures (Silberman et al., 2013;Daniel et al., 2017;Miura et al., 2022). This is, in part, due to the presence of additional features in these cells, such as irregular firing and a lack of voltage sag in most neurons (which we also found here). ...
... Further, in male mice, the use of these categories has not proven to be useful in distinguishing specific cell types of BNST projection populations. For example, both the CRF neuron and VTAprojecting neuron populations in the BNST of male mice have been shown to have all three Types, as well as a large proportion of neurons that fall into an "Other" category because they do not fit into the three Types (Silberman et al., 2013;Miura et al., 2022). Here, the use of voltage clamp recordings (in the same cells in which we performed current clamp recordings) to more directly assess I h showed than many cells in CON M monkeys displayed a large Ih in voltage clamps even though they displayed almost no voltage sag in current clamp. ...
Repeated alcohol drinking contributes to a number of neuropsychiatric diseases, including alcohol use disorder and co-expressed anxiety and mood disorders. Women are more susceptible to the development and expression of these diseases with the same history of alcohol exposure as men, suggesting they may be more sensitive to alcohol-induced plasticity in limbic brain regions controlling alcohol drinking, stress responsivity, and reward processing, among other behaviors. Using a translational model of alcohol drinking in rhesus monkeys, we examined sex differences in the basal function and plasticity of neurons in the bed nucleus of the stria terminalis (BNST), a brain region in the extended amygdala shown to be a hub circuit node dysregulated in individuals with anxiety and alcohol use disorder. We performed slice electrophysiology recordings from BNST neurons in male and female monkeys following daily open access (22 hr/day) to 4% ethanol and water for more than one year or control conditions. We found that BNST neurons from control females had reduced overall current density, hyperpolarization-activated depolarizing current (Ih), and inward rectification, as well as higher membrane resistance and greater synaptic glutamatergic release and excitatory drive, than those from control males, suggesting that female BNST neurons are more basally excited than those from males. Chronic alcohol drinking produced a shift in these measures in both sexes, decreasing current density, Ih, and inward rectification and increasing synaptic excitation. In addition, network activity-dependent synaptic inhibition was basally higher in BNST neurons of males than females, and alcohol exposure increased this in both sexes, a putative homeostatic mechanism to counter hyperexcitability. Altogether, these results suggest that the rhesus BNST is more basally excited in females than males and chronic alcohol drinking produces an overall increase in excitability and synaptic excitation. These results shed light on the mechanisms contributing to the female-biased susceptibility to neuropsychiatric diseases including co-expressed anxiety and alcohol use disorder.
... 2 plasticity in the excitability and synaptic transmission of BNST neurons following chronic alcohol exposure related to the maintenance of excessive alcohol consumption and withdrawal-induced anxiety and negative affect (Olive et al., 2002;Kash et al., 2009Kash et al., , 2015McElligott and Winder, 2009;Kash, 2012;McElligott et al., 2013;Silberman et al., 2013;Pleil et al., 2015a). ...
... voltage sag, the latter of which has been used as a proxy measure for HCN channel-mediated I h , and action potential firing pattern (Type I: large voltage sag, little inward rectification, persistent firing, and no I t ; Type II: little/no voltage sag or inward rectification, large I t , burst firing, and/or rebound firing; Type III: large inward rectification, little/no voltage sag or I t ) (Hammack et al., 2007;Daniel et al., 2017). While these categories were determined in male rats and segregate BNST neurons into equal thirds in that subject population, both male mouse and male monkey BNST neurons have been shown to be less distinguishable based on the combination of these measures (Silberman et al., 2013;Daniel et al., 2017;Miura et al., 2022). This is, in part, due to the presence of additional features in these cells, such as irregular firing and Fig. 6. ...
... Further, in male mice, the use of these categories has not proven to be useful in distinguishing specific cell types of BNST projection populations. For example, both the CRF neuron and VTA-projecting neuron populations in the BNST of male mice have been shown to have all three Types, as well as a large proportion of neurons that fall into an "Other" category because they do not fit into the three Types (Silberman et al., 2013;Miura et al., 2022). Here, the use of voltage clamp recordings (in the same cells in which we performed current clamp recordings) to more directly assess I h showed than many cells in CON M monkeys displayed a large I h in voltage clamps even though they displayed almost no voltage sag in current clamp. ...
Repeated alcohol drinking contributes to a number of neuropsychiatric diseases, including alcohol use disorder and co-expressed anxiety and mood disorders. Women are more susceptible to the development and expression of these diseases with the same history of alcohol exposure as men, suggesting they may be more sensitive to alcohol-induced plasticity in limbic brain regions controlling alcohol drinking, stress responsivity, and reward processing, among other behaviors. Using a translational model of alcohol drinking in rhesus monkeys, we examined sex differences in the basal function and plasticity of neurons in the bed nucleus of the stria terminalis (BNST), a brain region in the extended amygdala shown to be a hub circuit node dysregulated in individuals with anxiety and alcohol use disorder. We performed slice electrophysiology recordings from BNST neurons in male and female monkeys following daily “open access” (22 h/day) to 4% ethanol and water for more than one year or control conditions. We found that BNST neurons from control females had reduced overall current density, hyperpolarization-activated depolarizing current (Ih), and inward rectification, as well as higher membrane resistance and greater synaptic glutamatergic release and excitatory drive, than those from control males, suggesting that female BNST neurons are more basally excited than those from males. Chronic alcohol drinking produced a shift in these measures in both sexes, decreasing current density, Ih, and inward rectification and increasing synaptic excitation. In addition, network activity-dependent synaptic inhibition was basally higher in BNST neurons of males than females, and alcohol exposure increased this in both sexes, a putative homeostatic mechanism to counter hyperexcitability. Altogether, these results suggest that the rhesus BNST is more basally excited in females than males and chronic alcohol drinking produces an overall increase in excitability and synaptic excitation. These results shed light on the mechanisms contributing to the female-biased susceptibility to neuropsychiatric diseases including co-expressed anxiety and alcohol use disorder.
... BNSTc (Walter et al., 1991) BNSTl Walter et al., 1991) BNSTlj BNSTm (Walter et al., 1991) Input (Dabrowska et al., 2016) Prelimbic cortex (Dabrowska et al., 2016) - Behavioural function and dysfunction -Stress induced nociception (Tran et al., 2012) (Acute) pain related behaviors (Myers et al., 2014;Fang et al., 2023) -Anxiety related responses (freezing behaviour) (Kalin et al., 2005) -Sustained anxiety/threat anticipa tion/threat processing (Davis et al., 2010;Alvarez et al., 2011;Grupe et al., 2012;Somerville et al., 2013;Herrmann et al., 2016;Brinkmann et al., 2018;Camilo et al., 2022) (Continued) (Huang et al., 2010) -Arousal (Ju et al., 1989) -Conditioned anxiety/ contextual fear/ sustained anxiety (Sullivan et al., 2004;Vantrease et al., 2022) -Sustained anxiety/threat anticipation/ threat processing (Fellinger et al., 2021) -Pavlovian fear and reward behaviour/learning (Bruzsik et al., 2021;Kaouane et al., 2021) -Regulation of food intake (Wang et al., 2019) -Alcohol-and drug-motivated behaviour (Silberman et al., 2013) -Threat anticipation/evaluation of threatening stimuli (Hoffman et al., 2007) -Behavioural inhibition (Fox et al., 2010) -Social/affiliative behaviour: aggression, mating behaviour, maternal care, social interaction (Jacobs et al., 2023) -Phasic threat processing (Gungor and Paré, 2016;Shackman and Fox, 2016;Klumpers et al., 2017;Brinkmann et al., 2018;Koller et al., 2019) -Anxious temperament (Fox et al., 2018) -PTSD (Ahearn et al., 2007;Brinkmann et al., 2017;Awasthi et al., 2020;Feola et al., 2023) -Generalized Anxiety disorder (Avery et al., 2016;Buff et al., 2017;Brinkmann et al., 2018) -Social Anxiety (Avery et al., 2016;Clauss, 2019;Clauss et al., 2019) -Affective disorders (Fox et al., 2010) thought to regulate BNST activity in relation to affective behavior (Luchsinger et al., 2021;Glangetas, 2022). Interesting is this regard is the presence of so-called Von Economo or spindle cells which are found in high numbers in insular cortex and other anterior limbic areas in humans but not in many other primates or species, potentially providing a unique input and regulation of BNST in humans only (Seeley et al., 2012; Table 1). ...
... The electrophysiological heterogeneity of BNST neurons has been extensively studied in rodents by multiple research groups (Hammack et al., 2007;Szücs et al., 2010;Hazra et al., 2011;Dabrowska et al., 2013;Rodríguez-Sierra et al., 2013;Silberman et al., 2013;Nagano et al., 2015;Daniel et al., 2017;Yamauchi et al., 2018). Historically defined in the BNST ALG of adult male rats by Hammack and others, three GABAergic neuronal cell types (Type I, II, and III) have been described based on their responses to hyperpolarizing and depolarizing current injections. ...
The bed nucleus of the stria terminalis (BNST), as part of the extended amygdala, has become a region of increasing interest regarding its role in numerous human stress-related psychiatric diseases, including post-traumatic stress disorder and generalized anxiety disorder amongst others. The BNST is a sexually dimorphic and highly complex structure as already evident by its anatomy consisting of 11 to 18 distinct sub-nuclei in rodents. Located in the ventral forebrain, the BNST is anatomically and functionally connected to many other limbic structures, including the amygdala, hypothalamic nuclei, basal ganglia, and hippocampus. Given this extensive connectivity, the BNST is thought to play a central and critical role in the integration of information on hedonic-valence, mood, arousal states, processing emotional information, and in general shape motivated and stress/anxiety-related behavior. Regarding its role in regulating stress and anxiety behavior the anterolateral group of the BNST (BNST ALG ) has been extensively studied and contains a wide variety of neurons that differ in their electrophysiological properties, morphology, spatial organization, neuropeptidergic content and input and output synaptic organization which shape their activity and function. In addition to this great diversity, further species-specific differences are evident on multiple levels. For example, classic studies performed in adult rat brain identified three distinct neuron types (Type I-III) based on their electrophysiological properties and ion channel expression. Whilst similar neurons have been identified in other animal species, such as mice and non-human primates such as macaques, cross-species comparisons have revealed intriguing differences such as their comparative prevalence in the BNST ALG as well as their electrophysiological and morphological properties, amongst other differences. Given this tremendous complexity on multiple levels, the comprehensive elucidation of the BNST ALG circuitry and its role in regulating stress/anxiety-related behavior is a major challenge. In the present Review we bring together and highlight the key differences in BNST ALG structure, functional connectivity, the electrophysiological and morphological properties, and neuropeptidergic profiles of BNST ALG neurons between species with the aim to facilitate future studies of this important nucleus in relation to human disease.
... Neural circuitry and behavioral evidence suggest CRF+ aBNST neurons are also involved in stress effects on motivation behaviors. CRF+ aBNST neurons project to [27][28][29] and inhibit [17,30] ventral tegmental area (VTA) and nucleus accumbens (NAc) shell. Stress-induced neuroplasticity increases the inhibitory actions of CRF in VTA and impairs reward responsivity [31][32][33]. ...
... Stress-induced neuroplasticity increases the inhibitory actions of CRF in VTA and impairs reward responsivity [31][32][33]. CRF + aBNST neurons also promote aversive effects of withdrawal [34], drug-seeking [35,36], and cocaine-induced dopamine release [30]. In a two-choice progressive ratio task for sucrose or optogenetic photostimulation of CRF + BNST neurons paired with sucrose, rats lever press less for paired sucrose and photostimulation [37]. ...
... aBNST CRF+ neurons are involved in stressinduced avoidance [16,18,20,21,39], but it was unknown whether they also play a role in effortful responding. A potential mechanism is that stress drives drug-seeking [31] and activates CRF + BNST projections that inhibit dopaminergic reward-related regions, including the VTA and NAc [30,33], thereby reducing motivated behavior. Here, we tested whether highly specific chemogenetic activation of CRF+ aBNST neurons mimics stress effects on willingness to exert effort for a high value reward when a low value reward is freely available in both effort-related choice and concurrent choice. ...
Corticotropin-releasing factor (CRF) in the anterior bed nucleus of the stria terminalis (aBNST) is associated with chronic stress and avoidance behavior. However, CRF + BNST neurons project to reward- and motivation-related brain regions, suggesting a potential role in motivated behavior. We used chemogenetics to selectively activate CRF+ aBNST neurons in male and female CRF-ires-Cre mice during an effort-related choice task and a concurrent choice task. In both tasks, mice were given the option either to exert effort for high value rewards or to choose freely available low value rewards. Acute chemogenetic activation of CRF+ aBNST neurons reduced barrier climbing for a high value reward in the effort-related choice task in both males and females. Furthermore, acute chemogenetic activation of CRF+ aBNST neurons also reduced effortful lever pressing in high-performing males in the concurrent choice task. These data suggest a novel role for CRF+ aBNST neurons in effort-based decision and motivation behaviors.
... Dopamine projections to the BNST are concentrated in the dBNST and synapse specifically onto the CRFergic neurons (Meloni et al., 2006;Phelix et al., 1994). Molecular and electrophysiology studies suggest that dopamine increases local CRF release in the dBNST and drug-induced synaptic plasticity in the dBNST requires both dopamine and CRF (Day et al., 2002;Kash et al., 2008;Silberman et al., 2013a). These anatomical and ex vivo physiology studies suggest dopamine and CRF are critically interacting to drive reward and stress-related behaviors. ...
Midbrain and striatal dopamine signals have been extremely well characterized over the past several decades, yet novel dopamine signals and functions in reward learning and motivation continue to emerge. A similar characterization of real-time sub-second dopamine signals in areas outside of the striatum has been limited. Recent advances in fluorescent sensor technology and fiber photometry permit the measurement of dopamine binding correlates, which can divulge basic functions of dopamine signaling in non-striatal dopamine terminal regions, like the dorsal bed nucleus of the stria terminalis (dBNST). Here, we record GRABDA signals in the dBNST during a Pavlovian lever autoshaping task. We observe greater Pavlovian cue-evoked dBNST GRABDA signals in sign-tracking (ST) compared to goal-tracking/intermediate (GT/INT) rats and the magnitude of cue-evoked dBNST GRABDA signals decreases immediately following reinforcer-specific satiety. When we deliver unexpected rewards or omit expected rewards, we find that dBNST dopamine signals encode bidirectional reward prediction errors in GT/INT rats, but only positive prediction errors in ST rats. Since sign- and goal-tracking approach strategies are associated with distinct drug relapse vulnerabilities, we examined the effects of experimenter-administered fentanyl on dBNST dopamine associative encoding. Systemic fentanyl injections do not disrupt cue discrimination but generally potentiate dBNST dopamine signals. These results reveal multiple dBNST dopamine correlates of learning and motivation that depend on the Pavlovian approach strategy employed.
... The bed nucleus of the stria terminalis (BNST) is a key structure in the limbic system, playing a well-established role in anxiety and stress-reward interaction. The projection from the BNST to the ventral tegmental area (VTA) is a critical neural pathway involved in reward processing (Jennings et al., 2013), and is extensively studied in regards to drug and alcohol addiction (Dumont et al., 2008;Briand et al., 2010;Sartor and Aston-Jones, 2012) and anxiety (Jennings et al., 2013;Kim et al., 2013). Specifically, GABA inputs from the BNST onto the VTA have been found to be necessary to promote binge-like ethanol intake (Companion and Thiele, 2018), which can be strengthened by stress (Ostroumov et al., 2016). ...
... Hyperpolarizing current injection induces a voltage sag in Type I, rebound spiking exclusively in Type II, but neither in Type III neurons (Hammack et al., 2007;Yamauchi et al., 2018). Type "Others" is identified by firing with a single spike (Rodríguez-Sierra et al., 2013;Silberman et al., 2013;Daniel et al., 2017;Ch'ng et al., 2019). ...
... Similarly, it is unknown if the cells maintain similar functional differences as described in rats. Research in rats found that 80% of VTA-projecting BNST neurons are Type III (Yamauchi et al., 2018) while one study in mice showed that there are no Type III neurons in the same projection (Silberman et al., 2013). Moreover, multiple studies have relied on visual discrimination to categorize the various neuronal types potentially biasing the classifications. ...
The bed nucleus of the stria terminalis (BNST) is a highly heterogeneous limbic forebrain structure that serves as a relay connecting autonomic, neuroendocrine and behavioral function. It can be divided into over 16 individual subregions with distinct neuronal subpopulations based on receptors, transmitters, and neuropeptides. Specifically, the BNST projection to the ventral tegmental area (VTA), the dopamine hub of the brain, has been shown to have a crucial role in the stress response. However, in mice there is a lack of unbiased data on the functional diversity of this sub-population which serves as an upstream input to the VTA. The dopaminergic neurons in the VTA modify their ion channel activity and intrinsic membrane properties to adapt to stress in part from inputs from BNST projections. Therefore, we aimed to perform a multi-component characterization of the functional diversity of the BNST-VTA pathway. We studied the passive and active electrophysiological properties of virally identified population of BNST neurons that project to the VTA. We used a comprehensive series of in vitro recordings of electrophysiological variables and performed hierarchical clustering to determine the functional diversity of the projection neurons in the BNST-VTA pathway. Our study revealed four subpopulations in the BNST-VTA pathway, all of which differ in their activation profiles and likely have distinct inputs and function in the VTA. Our results will help resolve the discord in interpretation of the various roles of this electrophysiologically diverse projection and builds a foundation for understanding how the different neuronal types integrate signals.
... These results are consistent with prior literature supporting a general hyperactive state of the BNST in acute withdrawal following CIE. For example, work from our group and others indicates withdrawal from CIE increases neuronal excitability (Kash et al., 2009;Pati et al., 2020), excitatory drive (Silberman et al., 2013;Pleil et al., 2015), and long-term potentiation of glutamatergic synapses in the BNST (Wills et al., 2012). ...
The bed nucleus of the stria terminalis (BNST) is a component of the extended amygdala that regulates motivated behavior and affective states and plays an integral role in the development of alcohol-use disorder (AUD). The dorsal subdivision of the BNST receives dense dopaminergic input from the ventrolateral periaqueductal gray (vlPAG)/dorsal raphe (DR). To date, no studies have examined the effects of chronic alcohol on this circuit. Here, we used chronic intermittent ethanol exposure (CIE), a well-established rodent model of AUD, to functionally interrogate the vlPAG/DR-BNST dopamine circuit during acute withdrawal. We selectively targeted vlPAG/DR DA neurons in tyrosine hydroxylase-expressing transgenic adult male mice. Using ex vivo electrophysiology, we found hyperexcitability of vlPAG/DR DA neurons in CIE-treated mice. Further, using optogenetic approaches to target vlPAG/DR DA terminals in the dBNST, we revealed a CIE-mediated shift in the vlPAG/DR-driven excitatory-inhibitory ratio to a hyperexcitable state in dBNST. Additionally, to quantify the effect of CIE on endogenous DA signaling, we coupled optogenetics with fast-scan cyclic voltammetry to measure pathway-specific DA release in dBNST. CIE-treated mice had significantly reduced signal half-life, suggestive of faster clearance of DA signaling. CIE treatment also altered the ratio of vlPAG/DR DA -driven cellular inhibition and excitation of a subset of dBNST neurons. Overall, our findings suggest a dysregulation of vlPAG/DR to BNST dopamine circuit, which may contribute to pathophysiological phenotypes associated with AUD.
SIGNIFICANCE STATEMENT:
The dorsal bed nucleus of stria terminalis (dBNST) is highly implicated in the pathophysiology of alcohol use disorder and receives dopaminergic inputs from ventrolateral periaqueductal gray/dorsal raphe regions (vlPAG/DR). The present study highlights the plasticity within the vlPAG/DR to dBNST dopamine (DA) circuit during acute withdrawal from chronic ethanol exposure. More specifically, our data reveal that chronic ethanol strengthens vlPAG/DR-dBNST glutamatergic transmission while altering both DA transmission and dopamine-mediated cellular inhibition of dBNST neurons. The net result is a shift toward a hyperexcitable state in dBNST activity. Together, our findings suggest chronic ethanol may promote withdrawal-related plasticity by dysregulating the vlPAG/DR-dBNST DA circuit.
... In mice, systemic clenbuterol administration also increases CRF mRNA levels in the BNST [47], while intra-BNST administration of the CRF-R1 receptor antagonist, antalarmin, prevents reinstatement of cocaine seeking in response to intra-BNST administration of clenbuterol [83]. A series of studies by Winder and colleagues have demonstrated that beta adrenergic receptors promote excitatory regulation of key BNST output pathways involved in cocaine seeking via a mechanism that likely requires CRF release from a local population of neurons intrinsic to the BNST and BNST CRF-R1 receptor activation [84,85]. These pathways include CRF-positive neurons that innervate the VTA [85][86][87], where CRF receptor activation is required for stressor-induced cocaine seeking ( [88,89]; see below). ...
... A series of studies by Winder and colleagues have demonstrated that beta adrenergic receptors promote excitatory regulation of key BNST output pathways involved in cocaine seeking via a mechanism that likely requires CRF release from a local population of neurons intrinsic to the BNST and BNST CRF-R1 receptor activation [84,85]. These pathways include CRF-positive neurons that innervate the VTA [85][86][87], where CRF receptor activation is required for stressor-induced cocaine seeking ( [88,89]; see below). Indeed, bath application of the non-selective beta-adrenergic receptor agonist, isoproterenol, promotes excitatory synaptic regulation of retro-labeled VTA-projecting CRFpositive neurons via CRF-R1-dependent glutamate release in the BNST [85]. ...
... These pathways include CRF-positive neurons that innervate the VTA [85][86][87], where CRF receptor activation is required for stressor-induced cocaine seeking ( [88,89]; see below). Indeed, bath application of the non-selective beta-adrenergic receptor agonist, isoproterenol, promotes excitatory synaptic regulation of retro-labeled VTA-projecting CRFpositive neurons via CRF-R1-dependent glutamate release in the BNST [85]. The BNST-VTA projection is comprised of both GABAergic and glutamatergic neurons [90]. ...
The neuropeptide, corticotropin releasing factor (CRF), has been an enigmatic target for the development of medications aimed at treating stress-related disorders. Despite a large body of evidence from preclinical studies in rodents demonstrating that CRF receptor antagonists prevent stressor-induced drug seeking, medications targeting the CRF-R1 have failed in clinical trials. Here, we provide an overview of the abundant findings from preclinical rodent studies suggesting that CRF signaling is involved in stressor-induced relapse. The scientific literature that has defined the receptors, mechanisms and neurocircuits through which CRF contributes to stressor-induced reinstatement of drug seeking following self-administration and conditioned place preference in rodents is reviewed. Evidence that CRF signaling is recruited with repeated drug use in a manner that heightens susceptibility to stressor-induced drug seeking in rodents is presented. Factors that may determine the influence of CRF signaling in substance use disorders, including developmental windows, biological sex, and genetics are examined. Finally, we discuss the translational failure of medications targeting CRF signaling as interventions for substance use disorders and other stress-related conditions. We conclude that new perspectives and research directions are needed to unravel the mysterious role of CRF in substance use disorders.
... Both chronic stress and alcohol exposure disrupt norepinephrine signaling at CRF neurons in the BNST (Snyder et al., 2019). Changes at the synaptic level, such as reduced activity in CRF+ VTA-projecting BNST neurons (Silberman et al., 2013) may also lead to long-lasting mood-related symptoms of withdrawal known to drive excessive alcohol intake. ...
The sexually dimorphic bed nucleus of the stria terminalis (BNST) is comprised of several distinct regions, some of which act as a hub for stress-induced changes in neural circuitry and behavior. In rodents, the anterodorsal BNST is especially affected by chronic exposure to stress, which results in alterations to the corticotropin-releasing factor (CRF)-signaling pathway, including CRF receptors and upstream regulators. Stress increases cellular excitability in BNST CRF+ neurons by potentiating miniature excitatory postsynaptic current (mEPSC) amplitude, altering the resting membrane potential, and diminishing M-currents (a voltage-gated K+ current that stabilizes membrane potential). Rodent anterodorsal and anterolateral BNST neurons are also critical regulators of behavior, including avoidance of aversive contexts and fear learning (especially that of sustained threats). These rodent behaviors are historically associated with anxiety. Furthermore, BNST is implicated in stress-related mood disorders, including anxiety and Post-Traumatic Stress Disorders in humans, and may be linked to sex differences found in mood disorders.
