The projection-specific signals that establish functionally segregated dopaminergic synapses.

Dopaminergic projections regulate various brain functions and are implicated in many neuropsychiatric disorders. There are two anatomically and functionally distinct dopaminergic projections connecting the midbrain to striatum: nigrostriatal, which controls movement, and mesolimbic, which regulates motivation. However, how these discrete dopaminergic synaptic connections are established is unknown. Through an unbiased search, we identify that two groups of antagonistic TGF-ß family members, bone morphogenetic protein (BMP)6/BMP2 and transforming growth factor (TGF)-ß2, regulate dopaminergic synapse development of nigrostriatal and mesolimbic neurons, respectively. Projection-preferential expression of their receptors contributes to specific synapse development. Downstream, Smad1 and Smad2 are specifically activated and required for dopaminergic synapse development and function in nigrostriatal vs. mesolimbic projections. Remarkably, Smad1 mutant mice show motor defects, whereas Smad2 mutant mice show lack of motivation. These results uncover the molecular logic underlying the proper establishment of functionally segregated dopaminergic synapses and may provide strategies to treat relevant, projection-specific disease symptoms by targeting specific BMPs/TGF-ß and/or Smads.

ASD/OCD-Linked Protocadherin-10 Regulates Synapse, But Not Axon, Development in the Amygdala and Contributes to Fear- and Anxiety-Related Behaviors.

The Protocadherin-10 (PCDH10) gene is associated with autism spectrum disorder (ASD), obsessive-compulsive disorder (OCD), and major depression (MD). The PCDH10 protein is a homophilic cell adhesion molecule that belongs to the δ2-protocadherin family. PCDH10 is highly expressed in the developing brain, especially in the basolateral nucleus of the amygdala (BLA). However, the role of PCDH10 in vivo has been debatable: one paper reported that a Pcdh10 mutant mouse line showed changes in axonal projections; however, another Pcdh10 mutant mouse line was reported to have failed to detect axonal phenotypes. Therefore, the actual roles of PCDH10 in the brain remain to be elucidated. We established a new Pcdh10 KO mouse line using the CRISPR/Cas9 system, without inserting gene cassettes to avoid nonspecific effects, examined the roles of PCDH10 in the brain, and studied the behavioral consequences of Pcdh10 inactivation. Here, we show that Pcdh10 KO mice do not show defects in axonal development. Instead, we find that Pcdh10 KO mice exhibit impaired development of excitatory synapses in the dorsal BLA. We further demonstrate that male Pcdh10 KO mice exhibit reduced anxiety-related behaviors, impaired fear conditioning, decreased stress-coping responses, and mildly impaired social recognition and communication. These results indicate that PCDH10 plays a critical role in excitatory synapse development, but not axon development, in the dorsal BLA and that PCDH10 regulates anxiety-related, fear-related, and stress-related behaviors. Our results reveal the roles of PCDH10 in the brain and its relationship to relevant psychiatric disorders such as ASD, OCD, and MD.SIGNIFICANCE STATEMENTProtocadherin-10 (PCDH10) encodes a cell adhesion molecule and is implicated in autism spectrum disorder (ASD), obsessive-compulsive disorder (OCD), and major depression (MD). PCDH10 is highly expressed in the basolateral nucleus of the amygdala (BLA). However, the phenotypes of previously published Pcdh10 mutant mice are debatable, and some are possibly because of the nonspecific effects of the LacZ/Neo cassette inserted in the mice. We have generated a new Pcdh10 mutant mouse line without the LacZ/Neo cassette. Using our new mouse line, we reveal the roles of PCDH10 for excitatory synapse development in the BLA. The mutant mice exhibit anxiety-related, fear-related, and stress-related behaviors, which are relevant to ASD, OCD, and MD, suggesting a possible treatment strategy for such psychiatric disorders.

Stimulation of lateral parabrachial (LPB) to central amygdala (CeA) pituitary adenylate cyclase-activating polypeptide (PACAP) neurons induces anxiety-like behavior and mechanical allodynia.

BACKGROUND: Anxiety disorders are the most prevalent psychiatric disorders, and they are highly comorbid with chronic pain conditions. The central nucleus of the amygdala (CeA) is known not only for its role in the regulation of anxiety but also as an important site for the negative affective dimension of pain. Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide whose terminals are abundant in the CeA, is strongly implicated in the stress response as well as in pain processing. Here, using Cre-dependent viral vectors, we explored in greater detail the role of the PACAP projection to the CeA that originates in the lateral parabrachial nucleus (LPB). METHODS: We first performed a circuit mapping experiment by injecting an anterograde Cre-dependent virus expressing a fluorescent reporter in the LPB of PACAP-Cre mice and observing their projections. Then, we used a chemogenetic approach (a Cre-dependent Designer Receptors Activated by Designer Drugs, DREADDs) to assess the effects of the direct stimulation of the PACAP LPB to CeA projection on general locomotor activity, anxiety-like behavior (using a defensive withdrawal test), and mechanical pain sensitivity (using the von Frey test). RESULTS: We found that the CeA, together with other areas, is one of the major downstream projection targets of PACAP neurons originating in the lateral parabrachial nucleus (LPB). In the DREADD experiment, we then found that the selective activation of this neuronal pathway is sufficient to increase both anxiety-like behavior and mechanical pain sensitivity in mice, without affecting general locomotor activity. CONCLUSION: In conclusion, our data suggest that the dysregulation of this circuit may contribute to a variety of anxiety disorders and chronic pain states, and that PACAP may represent an important therapeutic target for the treatment of these conditions.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) of the Bed Nucleus of the Stria Terminalis Mediates Heavy Alcohol Drinking in Mice.

Alcohol use disorder (AUD) is a complex psychiatric disease characterized by periods of heavy drinking and periods of withdrawal. Chronic exposure to ethanol causes profound neuroadaptations in the extended amygdala, which cause allostatic changes promoting excessive drinking. The bed nucleus of the stria terminalis (BNST), a brain region involved in both excessive drinking and anxiety-like behavior, shows particularly high levels of pituitary adenylate cyclase-activating polypeptide (PACAP), a key mediator of the stress response. Recently, a role for PACAP in withdrawal-induced alcohol drinking and anxiety-like behavior in alcohol-dependent rats has been proposed; whether the PACAP system of the BNST is also recruited in other models of alcohol addiction and whether it is of local or nonlocal origin is currently unknown. Here, we show that PACAP immunoreactivity is increased selectively in the BNST of C57BL/6J mice exposed to a chronic, intermittent access to ethanol. While pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor-expressing cells were unchanged by chronic alcohol, the levels of a peptide closely related to PACAP, the calcitonin gene-related neuropeptide, were found to also be increased in the BNST. Finally, using a retrograde chemogenetic approach in PACAP-ires-Cre mice, we found that the inhibition of PACAP neuronal afferents to the BNST reduced heavy ethanol drinking. Our data suggest that the PACAP system of the BNST is recruited by chronic, voluntary alcohol drinking in mice and that nonlocally originating PACAP projections to the BNST regulate heavy alcohol intake, indicating that this system may represent a promising target for novel AUD therapies.

The Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) System of the Central Amygdala Mediates the Detrimental Effects of Chronic Social Defeat Stress in Rats.

Many psychiatric diseases stem from an inability to cope with stressful events, as chronic stressors can precipitate or exacerbate psychopathologies. The neurobiological mechanisms underlying the response to chronic stress and the resulting anxiety states remain poorly understood. Stress neuropeptides in the extended amygdala circuitry mediate the behavioral response to stress, and hyperactivity of these systems has been hypothesized to be responsible for the emergence of persistent negative outcomes and for the pathogenesis of anxiety-related and trauma-related disorders. Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor PAC1R are highly expressed within the central amygdala (CeA) and play a key role in stress regulation. Here, we used chronic social defeat stress (CSDS), a clinically relevant model of psychosocial stress that produces robust maladaptive behaviors in rodents. We found that 10 days of CSDS cause a significant increase in PACAP levels selectively in the CeA of rats, as well as an increase in PAC1R mRNA. Using a viral vector strategy, we found that PAC1R knock-down in the CeA attenuates the CSDS-induced body weight loss and prevents the CSDS-induced increase in anxiety-like behavior. Notably, CSDS animals display reduced basal corticosterone (CORT) levels and PAC1R knock-down in CeA further reduce them. Finally, the CeA PAC1R knock-down blocks the increase in corticotropin-releasing factor (CRF) immunoreactivity induced by CSDS in CeA. Our findings support the notion that the persistent activation of the PACAP-PAC1R system in the CeA mediates the behavioral outcomes of chronic psychosocial stress independently of the hypothalamic-pituitary-adrenal axis, perhaps via the recruitment of the CRF system.

Role of the PACAP system of the extended amygdala in the acoustic startle response in rats.

Anxiety-related disorders are the most prevalent mental disorders in the world and they are characterized by abnormal responses to stressors. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide highly expressed in the extended amygdala, a brain macrostructure involved in the response to threat that includes the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). The aim of this series of experiments was to systematically elucidate the role of the PACAP system of the CeA and BNST under both control, unstressed conditions and after the presentation of a stressor in rats. For this purpose, we used the acoustic startle response (ASR), an unconscious response to sudden acoustic stimuli sensitive to changes in stress which can be used as an operationalization of the hypervigilance present in anxiety- and trauma-related disorders. We found that infusion of PACAP, but not the related peptide vasoactive intestinal peptide (VIP), into either the CeA or the BNST causes a dose-dependent increase in ASR. In addition, while infusion of the antagonist PACAP(6-38) into either the CeA or the BNST does not affect ASR in non-stressed conditions, it prevents the sensitization of ASR induced by an acute footshock stress. Finally, we found that footshock stress induces a significant increase in PACAP, but not VIP, levels in both of these brain areas. Altogether, these data show that the PACAP system of the extended amygdala contributes to stress-induced hyperarousal and suggest it as a potential novel target for the treatment of stress-related disorders.

Pituitary adenylate cyclase-activating polypeptide induces a depressive-like phenotype in rats.

BACKGROUND: Major depressive disorder (MDD) is a chronic, life-threatening psychiatric condition characterized by depressed mood, psychomotor alterations, and a markedly diminished interest or pleasure in most activities known as anhedonia. Available pharmacotherapies have limited success and the need for new strategies is clear. Recent studies attribute a major role to the pituitary adenylate cyclase-activating polypeptide (PACAP) system in mediating the response to stress. PACAP knockout mice display profound alterations in depressive-like behaviors, and genetic association studies have demonstrated that genetic variants of the PACAP gene are associated with MDD. However, the effects of PACAP administration on depressive-like behaviors in rodents have not yet been systematically examined. OBJECTIVES: The present study investigated the effects of central administration of PACAP in rats on depressive-like behaviors, using well-established animal models that represent some of the endophenotypes of depression. METHODS: We used intracranial self-stimulation (ICSS) to assess the brain reward function, saccharin preference test to assess anhedonia, social interaction to assess social withdrawal, and forced swim test (FST) to assess behavioral despair. RESULTS: PACAP raised the current threshold for ICSS, elevation blocked by the PACAP antagonist PACAP(6-38). PACAP reduced the preference for a sweet saccharin solution and reduced the time the rats spent interacting with a novel animal. Interestingly, PACAP administration did not affect immobility in the FST. CONCLUSIONS: Our results demonstrate a role for the central PACAP/PAC1R system in the regulation of depressive-like behaviors and suggest that hyperactivity of the PACAP/PAC1R system may contribute to the pathophysiology of depression, particularly the associated anhedonic symptomatology and social dysfunction.

Olanzapine, but not fluoxetine, treatment increases survival in activity-based anorexia in mice.

Anorexia nervosa (AN) is an eating disorder characterized by extreme hypophagia, hyperactivity, and fear of weight gain. No approved pharmacological treatments exist for AN despite high mortality rates. The activity-based anorexia (ABA) phenomenon models aspects of AN in rodents, including progressive weight loss, reduced food intake, and hyperactivity. First, we optimized the ABA paradigm for mice. We compared mouse strains (Balb/cJ, A/J) for susceptibility with ABA, and evaluated the effects of different food access durations (2, 4, 6, 8, and 10 h) on ABA parameters. Balb/cJ mice exhibited significantly shorter survival time (days until 25% bodyweight loss) in the ABA paradigm compared with A/J mice. Furthermore, 6 h of food access reduced survival in mice housed with wheels without reducing survival in mice housed without wheels. We then evaluated the effects of chronic treatment with fluoxetine (4 weeks) or subchronic treatment with olanzapine (OLZ) (1 week) on ABA in BALB/cJ mice. OLZ (12 mg/kg/day) significantly increased survival and reduced food anticipatory activity (FAA). However, OLZ did not alter food intake or running wheel activity during ad-lib feeding (baseline) or restriction conditions, or in mice housed without wheels. Fluoxetine (18 mg/kg/day) increased food intake and reduced FAA, but did not alter survival. Here, we report for the first time that OLZ, but not fluoxetine, reduces ABA in mice. Our findings indicate further need for clinical investigations into the effects of OLZ, but not selective serotonin reuptake inhibitors, on core features of AN.

Antidepressant response to chronic citalopram treatment in eight inbred mouse strains.

RATIONALE: The antidepressant response exhibits a characteristic delay. BALB/cJ mice respond to chronic, but not subchronic, treatment with selective serotonin reuptake inhibitors (SSRIs), providing a model of antidepressant onset. Identification of other mouse strains exhibiting this phenotype will provide additional tools for studying mechanisms of the antidepressant response. OBJECTIVES: We aimed to identify inbred mouse strains that respond to chronic, but not subchronic, SSRI treatment in the forced swim test (FST). We also assessed whether response correlated with genotype at the functional C1473G polymorphism in tryptophan hydroxylase-2 (Tph2). METHODS: BALB/cJ, three closely related strains (BALB/cByJ, SEA/GnJ, A/J), and four distantly related strains (C57BL/6J, C57BL/10J, CAST/EiJ, SM/J) received the SSRI citalopram (0-30 mg/kg/day in drinking water) for ~4 weeks and were assessed for locomotion and FST behavior. Citalopram-responsive strains were assessed identically following ~1 week of treatment. C1473G genotypes were determined. RESULTS: BALB/cJ and related strains carried the 1473G allele and responded to chronic citalopram treatment in the FST. BALB/cJ, BALB/cByJ, and SEA/GnJ mice showed either no response or an attenuated response to subchronic treatment. Distantly related strains carried the 1473C allele and showed no response to citalopram. No relationship was found between the antidepressant response and baseline immobility or locomotion. CONCLUSIONS: BALB/cJ and related strains exhibit an antidepressant response to chronic SSRI treatment that emerges over time and is likely a heritable trait. This antidepressant response is associated with carrying the 1473G allele in Tph2. In conclusion, BALB/cJ and related strains provide valuable models for studying the therapeutic mechanisms of SSRIs.