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Published in final edited form as: J Mol Neurosci. 2021 Nov 22;72(3):451–458. doi: 10.1007/s12031-021-01946-z

Activation of Lateral Parabrachial Nucleus (LPBn) PACAP-Expressing Projection Neurons to the Bed Nucleus of the Stria Terminalis (BNST) Enhances Anxiety-like Behavior

Melissa N Boucher 1,2, Mahafuza Aktar 1,2, Karen M Braas 1,2, Victor May 1,2, Sayamwong E Hammack 1,2,*
PMCID: PMC8930475  NIHMSID: NIHMS1759116  PMID: 34811712

Abstract

Anxiety disorders are among the most common psychiatric disorders, and understanding the underlying neurocircuitry of anxiety- and stress-related behaviors may be important for treatment. The bed nucleus of the stria terminalis (BNST) has been studied for its role in many stress-related pathologies, such as anxiety, pain, depression, and addiction. Our prior work has demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) receptor activation in the BNST mediates many of the behavioral consequences of chronic stress. While the BNST contains local PACAP-expressing neurons, a major source of afferent PACAP is the lateral parabrachial nucleus (LPBn), and excitotoxic lesions of the LPBn substantially decreasess PACAP immunostaining in the BNST. Here, we first assessed Cre-dependent reporter expression by injecting AAV2-hSyn-DIO-mCherry into the LPBn of PACAP-IRES-Cre mice for circuit mapping studies and identified PACAP projections to the BNST, lateral capsular central nucleus of the amygdala (CeLC), and ventromedial hypothalamus (VMH). In a second study, we assessed the effects of chemogenetically activating LPBn PACAP afferents in the BNST by injecting AAV2-hSyn-DIO-hM3D(Gq)-mCherry into the LPBn of PACAP-IRES-Cre mice for Cre-dependent expression of excitatory designer receptors exclusively activated by designer drugs (DREADDs). Before behavioral testing, clozapine-N-oxide (CNO), the selective agonist of our DREADD, was infused directly into the BNST. We found that after specific activation of LPBn PACAP afferents in the BNST, mice had increased anxiety-like behavior compared with controls, while total locomotor activity was unaffected. These results indicate that activation of PACAPergic LPBn projections to the BNST may play an important role in producing anxiety-like behavior.

Keywords: Anxiety, Stress, PAC1 receptor, DREADD, Extended amygdala, Post-traumatic stress disorder

Introduction

Dysregulation in emotional processing represents a significant public health concern, with one third of the population being affected by an anxiety disorder in their lifetime, making them the most prevalent psychiatric disorder (Bandelow and Michaelis 2015). Stress has been linked to several of these disorders and can drive these hyper-active anxiety and fear states characteristic of disorders of emotional processing. Chronic stress can lead to physiological changes that may contribute to psychopathologies such as depression, pain, and anxiety, or fear disorders, including post-traumatic stress disorder (PTSD). The bed nucleus of the stria terminalis (BNST) has been heavily implicated in both the activation of endocrine and autonomic stress-responses, as well as anxiety-like responding to temporally distant threats (Waddell, Morris, and Bouton 2006), threats of long duration (Walker, Miles, and Davis 2009), and/or stimuli that poorly predict danger (Goode and Maren 2017). The BNST is connected to several stress- and anxiety-related areas of the brain, such as the amygdala, hypothalamus, dorsal raphe nucleus, cerebral cortex, and parabrachial nucleus (Lebow and Chen 2016; Saggu and Lundy 2008); hence, the BNST serves as a nexus integrating and coordinating complex regulatory signals for relay to mediate the diverse responses to stress.

Among many neurotransmitters and neuropeptides that signal within the BNST, pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1), and activation of its cognate G protein-coupled PAC1 receptor (Adcyap1r1) has been shown to be a critical regulator of BNST function. The BNST expresses both PACAP and PAC1 receptors, and we have shown previously that chronic variate stress (CVS) paradigms can augment their transcript expression levels in the BNST (Hammack et al. 2009). We and others have also demonstrated that BNST PACAP infusions produce many of the physiological and behavioral consequences of stressor exposure in both male and female rodents, including anxiety-like behavior. And, CVS-induced stress responses (including attenuated weight gain and anxiety-like behavior) were abrogated by the PACAP receptor antagonist PACAP(6–38). Importantly, PACAP-mediated stress-like responses could also be mimicked by the PAC1 receptor-selective agonist maxadilan but not by the related family member vasoactive intestinal polypeptide (VIP), implicating the prominent roles of PAC1 receptor function. These data complement observations that elevated blood PACAP levels and polymorphism in the ERE of the PAC1 receptor gene have been associated with PTSD symptoms in women (Ressler et al. 2011) and other data implicating PACAP dysregulation in psychopathology (see, Hammack and May 2015, for review).

Although we have observed evidence for intrinsic BNST PACAP neurons, our studies have also demonstrated that a substantial source of BNST PACAP may originate from the lateral parabrachial nucleus (LPBn; Missig et al. 2014). Nearly 60% of LPBn PACAP immunoreactivity can be colocalized with calcitonin gene-related peptide (CGRP), and LPBn excitotoxic lesions removed more than 70% of the PACAP immunoreactive fibers in the BNST. While we have observed an anxiogenic response to BNST PACAP infusion, it remains unclear whether direct activation of the LPBn PACAP afferents in the BNST is anxiogenic. Here, using chemogenetic techniques, we demonstrate that activation of LPBn PACAP neuronal terminals in the BNST produces anxiety-like behavior in mice.

Methods

Animals

PACAP-IRES-Cre mice (generous gift from Bradford Lowell, Harvard Medical School), harboring the internal ribosomal entry sequence (IRES) tethered to Cre-recombinase downstream of the Adcyap1 gene stop codon (for gene driven expression), were bred in-house and housed with food and water ad libitum. The colony room was maintained on a 12-h light/dark cycle, and all the experiments were carried out during the light phase. All the procedures were approved by the University of Vermont Institutional Animal Care and Use Committee and carried out with accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

Stereotaxic Surgery

For anatomical characterizations, adult male PACAP-IRES-Cre mice were anesthetized with isoflurane (4% induction, 1.5–2% maintenance) and placed in the stereotaxic device (Stoelting, Wood Dale, IL). Nanoliters of 250–500 of AAV2-hSyn-DIO-mCherry (Addgene, Watertown, MA), a viral vector leading to Cre-dependent mCherry expression in PACAP-expressing neurons, was injected bilaterally into the LPBn (AP: −5.3, ML: +/− 1.3, DV: −3.5 mm) over the course of 5 min with an additional 5-min wait to allow for diffusion. Following infusions, the wound was sutured, and mice were given subcutaneous injections of 5 mg/kg Carprofen at the end of surgery and 24 h after for analgesia. Following stereotaxic surgery and post-operative care, mice were held in their home cages for 14 days prior to transcardial perfusion and histology (see below).

For chemogenetic activation of LPBn PACAP afferents in the BNST studies, adult male PACAP-IRES-Cre mice were anesthetized with isoflurane (4% induction, 1.5–2% maintenance) and placed in the stereotaxic device (Stoelting, Wood Dale, IL). Nanoliters of 250–500 of either AAV2-hSyn-DIO-hM3D(Gq)-mCherry, a viral vector leading to Cre-dependent expression of an excitatory DREADD receptor in PACAP-expressing neurons, or the control virus AAV2-hSyn-DIO-mCherry (Addgene, Watertown, MA) were injected bilaterally into the LPBn (AP: −5.3, ML: +/− 1.3, DV: −3.5 mm) over the course of 5 min with an additional 5-min wait to allow for diffusion. A 1-uL Hamilton syringe and Harvard Apparatus infusion pump were used for injection of viral vectors. Following viral injection, cannula (P1 Technologies, Roanoke, VA) were placed in the BNST (AP: +0.3, ML: +/− 2.6, DV: −4.6, 20°), and a headcap was fashioned using glue (Loctite 454, Locktite, Westlake, OH) and a glue hardening accelerator (Loctite 7542). Mice were given subcutaneous injections of 5 mg/kg Carprofen at the end of surgery and 24 h after for analgesia. Following stereotaxic surgery and post-operative care, mice were handled daily for 1 week in order to reduce stress the day of behavioral testing.

Elevated Plus Maze

Animals were allowed to briefly acclimate to the lab bench in order to reduce sudden movements while unscrewing dummy cannula and placement of internal cannula. Once the dummy cannula was removed, an internal cannula was connected via 22-gauge tubing to a 10-uL Hamilton syringe and inserted into the guide cannula. Mice that had received prior AAV2-hSyn-DIO-hM3D(Gq)-mCherry into the LPBn as well as mice that received prior control virus were both administered 300 nL of 1-mM clozapine-n-oxide (CNO) into the BNST using a Harvard Apparatus infusion pump over 1 min; CNO was allowed to diffuse for an additional 1 min before removal of the internal cannula. This process was repeated bilaterally and upon cessation, the animal was returned to its home cage. After 3–4 min, the mouse was placed on the elevated plus maze (Med Associates, Fairfax, VT) under red light and was given 5 min to freely explore. Behavior was recorded using a Logitech camera and Quicktime Player. All the videos were scored by observers blind to treatment condition and data were analyzed using unpaired t-tests in GraphPad Prism 9.

Histology

Mice were deeply anesthetized with isoflurane and transcardially perfused with an ice-cold heparinized phosphate buffered solution (PBS), followed by 4% paraformaldehyde. Brains were extracted and allowed to fix in 4% paraformaldehyde for 24 h and then were placed in 30% sucrose for cryoprotection. Once brains were cryoprotected, they were frozen in optimal cutting temperature solution, sliced at 40 μm and stored in 4 °C in wells containing PBS. Brains were then mounted on SuperFrostPlus Slides and cover-slipped with Citiflour Antifadent mounting media. An Olympus fluorescent microscope (Olympus Corporation of the Americas, Center Valley, PA, USA) was used to image mCherry expression in LPBn neurons and terminals and confirm proper BNST cannula placement.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Results

LPBn PACAP Neurons Have Long Distance Axonal Projections to the BNST, Amygdala, and Hypothalamus

From anterograde tracing and excitotoxic lesion studies, we have shown previously that LPBn PACAP neurons project to the lateral capsular division of the central nucleus of the amygdala (CeLC) and BNST (Missig et al. 2014). With currently available resources, we have now augmented with studies using PACAP-IRES-Cre mice for neural circuit mapping. Injections of Cre-inducible reporter vectors into the LPBn of PACAP-IRES-Cre mice resulted in high levels of reporter expression in a large fraction of neurons just dorsal to the superior cerebellar peduncle (Figures 1 and 2). As the reporter strongly labeled the soma and fibers, the forebrain was serially sectioned to trace and identify the distal axonal targets. Congruent with earlier work, high densities of the reporter-labeled axonal terminal puncta were identified in the CeLC and BNST (Figures 1 and 2). Notably, high densities of LPBn PACAP neuronal terminals were also found in the ventromedial hypothalamus (VMH; Figure 2), particularly in the dorsomedial aspect of the nucleus. Hence, these circuit mapping studies suggest that LPBn PACAP signaling may have the potential of coordinating complex emotional, behavioral, neuroendocrine, and autonomic responses to sensory inputs.

Figure 1.

Figure 1

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LPBn PACAP neuron reporter expression in soma and axonal projections. A AAV2-hSyn-DIO-mCherry vector infusion in to LPBn of PACAP-IRES-Cre mice resulted in reporter expression in a large population of multipolar neurons (B, high magnification). C Inspection of anterior forebrain tissue sections showed that the mCherry LPBn neurons have axonal projections to several distal target site such as the CeLC; D high magnification of the CeLC revealed high density of reporter axonal terminal puncta. Scale bar, high magnification 20 μm

Figure 2.

Figure 2

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LPBn PACAP neurons project to multiple distal emotional/behavioral target sites. Right panels: AAV2-hSyn-DIO-mCherry vector was infused into the LPBn of PACAP-IRES-Cre mice (right panel D), and reporter terminal expression was observed in the BNST, CeLC, and VMH (right panels A–C, respectively). Left and middle panels, schematic illustration of mouse brain with corresponding coronal sections of LPBn (reporter infusion site, coronal section D), and axonal terminal projections to the BNST, CeLC, and VMH (coronal sections A–C, respectively). Micrographs of mCherry reporter expression correspond to locations denoted by the colored dots on the coronal sections. ac, anterior commissure; 3v, third ventricle; scp, superior cerebellar peduncle

Chemogenetic Stimulation of LPBn PACAP Neuronal Terminals in the BNST Increases Anxiety-like Behavior

To demonstrate directly that activation of LPBn PACAP neurons can facilitate stress-related behaviors, we used the PACAP-IRES-Cre mice in chemogenetic experiments. For these studies we infused the LPBn bilaterally with an AAV2-hSyn-DIO-hM3D(Gq)-mCherry, a Cre-dependent construct containing an excitatory DREADD coupled to Gq, to allow for specific activation of PACAP neurons. Following the infusion of the excitatory DREADD (or control virus) into the LPBn, and after 14 days to allow for DREADD expression and distribution throughout LPBn PACAP neurons (Figure 3), CNO was infused into the BNST to stimulate the LPBn PACAP projection terminals (Figure 3). Histological analyses confirmed that all viral infusions were in the region of the LPBn, and CNO infusions were made into the region of the BNST (data not shown). Stimulation of LPBn PACAP afferents in the BNST significantly reduced open-arm exploration on the elevated-plus maze as compared to controls t(15) = 2.0973, p < 0.05. Strikingly, open arm times exploration following LBPn PACAP stimulation in the BNST was decreased more than 50% compared to controls; on the other hand, total locomotor activity as measured by total crossing between the arms of the plus-maze was not altered t(15) = 0.043, p > 0.05. The responses were robust and demonstrated unequivocally that LPBn signaling from PACAP-expressing neurons can be contributory to stress-associated behaviors.

Figure 3.

Figure 3

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Activation of LPBn PACAP neuronal terminals in BNST enhances anxiety-like responses. Following injections of AAV2-hSyn-DIO-hM3D(Gq)-mCherry or control virus into the LPBn of PACAP-IRES-Cre mice, CNO was infused directly into the BNST prior to behavioral testing on the elevated plus maze. A Schematic diagram illustrating LPBn virus injections and BNST CNO infusions. B DREADD or control virus injections were localized to the LPBn. C After CNO activation of LPBn PACAP afferents in the BNST, mice exhibited increased anxiety-like behavior compared with controls, exploring the open arms of the elevated-plus maze significantly less (top panel). Total locomotor activity on the elevated plus maze was unaffected (bottom panel). scp, superior cerebellar peduncle shown in dotted line

Discussion

Here, we have demonstrated that LPBn PACAP-expressing neurons project to several areas of the brain involved in stress, emotional behavior, and neuroendocrine responses. Infusion of a viral vector containing a Cre-dependent mCherry reporter into the LPBn of PACAP-IRES-Cre mice revealed axon-terminals in the CelC, BNST, and dorsomedial VMH. Furthermore, chemogenetic activation of LPBn PACAPergic terminals in the BNST-produced anxiety-like behavior as compared with mice that received a control virus, while locomotor activity was not affected. Combined with our prior work, in which excitotoxic lesioning of the LPBn reduced PACAP immunostaining in both the BNST and CelC, these results demonstrate that a LPBn to BNST circuit may be critical for anxiety-like behavior, and further implicate PACAP as a critical peptide within this circuit.

The lateral parabrachial nucleus (LPBn) is among several in a complex located in a thin cellular layer just dorsal to the pontine superior cerebellar peduncle. The LPBn receives afferent input from the nucleus tractus solitarius and the dorsal laminae of the spinal cord, as well as from several forebrain regions, including the thalamus, cortex, central nucleus of the amygdala (CeA), hypothalamus, and the BNST (see Palmiter 2018, for review). Broadly, this brain region is involved in many physiological and behavioral functions, including feeding (i.e., anorexia), food palatability and satiety, ascending sensory processing, visceral malaise, body temperature regulation, and arousal (especially to hypercapnia) (Palmiter 2018; Chiang et al. 2019). Moreover, there has been an increasing interest in the role of the LPBn in affective and anxiety (Jaramillo, Brown, and Winder 2021). From these attributes, the parabrachial nucleus has been considered to encode and transmit aversive and alarm or danger signals for coordinated responses (Palmiter 2018; Campos et al. 2018; Chiang et al. 2020). In alignment with other work, our previous anterograde and excitotoxic lesion studies demonstrated LPBn PACAP neuronal projections to the CeLC and BNST. PACAP expression in the LPBn neurons could be induced in a sciatic nerve chronic constriction injury (CCI) pain model which was reflected by increased fiber density and terminals at the target sites (Missig et al. 2017). Our current data corroborated these observations and identified major targets in the ventromedial hypothalamus (VMH). The LPBn PACAP projections to the CeLC have been implicated to mediate the emotional component of chronic pain (Missig et al. 2017); a similar function may be present in the BNST (Tran, Wiskur, and Greenwood-Van Meerveld 2012). The VMH participates in a variety of homeostatic responses including feeding, metabolism, and weight gain (Ribeiro et al. 2009; Bray 2000; Balagura and Devenport 1970). PACAP has been well studied as an anorexic peptide (Hawke et al. 2009; Kocho-Schellenberg et al. 2014; Iemolo et al. 2015; Matsuda et al. 2005), and the LPBn PACAP projections to the VMH may well mediate chronic pain/stress-associated alterations in feeding behavior and weight change.

The BNST has been heavily implicated in both stress responding and behavioral phenotypes associated with anxiety (Hammack, Braas, and May 2021; Gungor and Pare 2016). Here, we have demonstrated that LPBn to BNST projections can be an important component driving the overall stress response, and in particular, we show that chemogenetic activation of LPBn PACAPergic terminals in the BNST can produce anxiety-like behavior, compared with mice that received a control virus, without affecting locomotor activity. These data complement several other reports implicating a LPBn-BNST circuit in defensive behavior. For example, Chiang et al. (2020) examined the differential roles of LPBn projections to several regions including the BNST, CeA, VMH, and lateral periaqueductal gray, and found that LPBn activity is necessary for both escape behavior and aversive learning. Flavin and others (2014) have shown that the LPBn provides glutamatergic input to the BNST and that LPBn neurons are sensitive to the α2A-adrenergic receptor agonist guanfacine. Further research demonstrated that some PBn afferents synapse specifically onto BNST CRF cells, a BNST subpopulation that has been heavily implicated in stress- and anxiety-responding (Fetterly et al. 2019). Using a transsynaptic anterograde AAV, Jamarillo et al. (2020) showed that activation of BNST cells that were innervated by the PBn increased the latency to consume food in the novelty-suppressed feeding task in female mice. The data presented in this report suggest that LPBn PACAP neurons may play important roles in the LPBn-to-BNST circuit.

While LPBn PACAP terminals in the BNST were activated in the current study, the critical transmitter/neuropeptides responsible for enhancing the anxiety-like responses are under investigation. LPBn afferents to the BNST may contain multiple transmitters, including glutamate (Flavin et al. 2014; Ye and Veinante 2019), CGRP (Missig et al. 2014; Carter et al. 2013; Chen et al. 2018) and PACAP (Missig et al. 2014), and one or a combination of regulators may drive the response. In addressing comparable questions mediating hypothalamic feeding behavior, the primary driver was shown to be PACAP rather than the glutamate co-transmitter (Krashes et al. 2014). CGRP and PACAP are often co-expressed and can elicit comparable responses; we have shown previously that PACAP and CGRP can be co-expressed in a large fraction of LPBn neurons. Our current data complement our prior work to suggest that PACAP release could play an important function in these circuits. Interestingly, like many peptides, PACAP release in stress circuits may be promoted by high frequency stimulation. Hence, PACAP release could work in conjunction with glutamate signaling to augment LPBn-to-BNST synaptic strength and promote anxiety-like responding during periods of high or chronic stress. Moreover, we and others have shown that PACAP receptor activation enhances action potential frequency following depolarization. Again, combined with depolarization following glutamate release, such an action could enhance the salience of LPBn input.

The current study investigated the consequences of LPBn afferent stimulation in the BNST in male mice. Given the sexually dimorphic nature of the BNST (Laflamme et al. 1998; Allen and Gorski 1990) and sex differences in both the PACAP and stress systems (see King, Toufexis, and Hammack 2017, for review), investigations into female mice are also critical. Our prior work has demonstrated that BNST PACAP infusion also mimics many of the consequences of stressor exposure in females, and these effects sometimes interact with estradiol, such that stronger BNST PACAP effects are often observed when estradiol levels are high (King et al. 2017). In summary, the present data support other work implicating the LPBn-to-BNST circuit as critical for some anxiety-like states and suggest that LPBn PACAP signaling may be important in these processes.

Acknowledgements

We thank Galen Missig and Matt McCabe for the preliminary viral injection studies.

Funding

This work was supported in part by the National Institutes of Health (NIH) grant and the National Institute of Mental Health (NIMH) MH097988 (SEH and VM).

Footnotes

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Ethics Approval and Consent to Participate

All the animal procedures were approved by the University of Vermont Institutional Animal Care and Use Committee and carried out with accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. No human studies were performed in this article.

Competing Interests

The authors declare no competing interests.

Availability of Data and Materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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