Mood disorders (MDs) are caused by an interplay of genetic and environmental (GxE) risk factors. However, molecular pathways engaged by GxE risk factors are poorly understood. Using small-RNA sequencing in peripheral blood mononuclear cells (PBMCs), we show that the bipolar disorder (BD)-associated microRNA miR-708-5p is upregulated in healthy human subjects with a high genetic or environmental predisposition for MDs. miR-708-5p is further upregulated in the hippocampus of rats which underwent juvenile social isolation, a model of early life stress. Hippocampal overexpression of miR-708-5p in adult male mice is sufficient to elicit MD-associated behavioral endophenotypes. We further show that miR-708-5p directly targets Neuronatin ( Nnat ), an endoplasmic reticulum protein. Restoring Nnat expression in the hippocampus of miR-708-5p-overexpressing mice rescues miR-708-5p-dependent behavioral phenotypes. Finally, miR-708-5p is upregulated in PBMCs from patients diagnosed with MD. Peripheral miR-708-5p expression allows to differentiate male BD patients from patients suffering from major depressive disorder (MDD). In summary, we describe a potential functional role for the miR-708-5p/Nnat pathway in MD etiology and identify miR-708-5p as a potential biomarker for the differential diagnosis of MDs.

The underlying physiological and molecular mechanisms of bipolar disorder (BD) remain largely unknown. Here, by using unbiased small RNA sequencing in peripheral blood mononuclear cells (PBMCs), we found that miR-708-5p, a microRNA that was previously associated with BD, is the most strongly upregulated microRNA in peripheral blood of both healthy human subjects with a high genetic or environmental predisposition to develop mood disorders (MDs). Furthermore, miR-708-5p is strongly upregulated in patients diagnosed with BD and has potential in conjunction with the previously identified miR-499-5p to differentiate BD patients from patients suffering from major depressive disorder (MDD) and healthy controls. miR-708 is also upregulated in the hippocampus of wild type juvenile rats that underwent social isolation, as well as in juvenile rats heterozygous for the BD risk gene Cacna1c . Furthermore, ectopic overexpression of miR-708-5p in the hippocampus of adult male mice leads to BD-associated endophenotypes, such as reduced behavioral despair, enhanced compulsivity, and short-term memory impairments. miR-708-5p directly targets Neuronatin (Nnat), an endoplasmic reticulum (ER) resident protein involved in calcium homeostasis. Restoring Nnat expression in the hippocampus of miR-708-5p overexpressing mice rescues BD-associated endophenotypes. In summary, we functionally link miR-708-5p dependent regulation of Nnat to BD, with potential implications for BD diagnosis and therapy.### Competing Interest StatementThe authors have declared no competing interest.

CACNA1C encodes the α1c subunit of the L-type Ca2+ channel, Cav1.2. Ventricular myocytes from haploinsufficient Cacna1c (Cacna1c+/−) rats exhibited reduced expression of Cav1.2 but an apparently normal sarcolemmal Ca2+ influx with an impaired response to sympathetic stress. We tested the hypothesis that the altered phosphorylation of Cav1.2 might underlie the sarcolemmal Ca2+ influx phenotype in Cacna1c+/− myocytes using immunoblotting of the left ventricular (LV) tissue from Cacna1c+/− versus wildtype (WT) hearts. Activation of cAMP-dependent protein kinase A (PKA) increases L-type Ca2+ current and phosphorylates Cav1.2 at serine-1928. Using an antibody directed against this phosphorylation site, we observed elevated phosphorylation of Cav1.2 at serine-1928 in LV myocardium from Cacna1c+/− rats under basal conditions (+110% versus WT). Sympathetic stress was simulated by isoprenaline (100 nM) in Langendorff-perfused hearts. Isoprenaline increased the phosphorylation of serine-1928 in Cacna1c+/− LV myocardium by ≈410%, but the increase was significantly smaller than in WT myocardium (≈650%). In conclusion, our study reveals altered PKA-dependent phosphorylation of Cav1.2 with elevated phosphorylation of serine-1928 under basal conditions and a diminished phosphorylation reserve during β-adrenergic stimulation. These alterations in the phosphorylation of Cav1.2 may explain the apparently normal sarcolemmal Ca2+ influx in Cacna1c+/− myocytes under basal conditions as well as the impaired response to sympathetic stimulation.

CACNA1C encodes the pore-forming α1C subunit of the L-type Ca2+ channel, Cav1.2. Mutations and polymorphisms of the gene are associated with neuropsychiatric and cardiac disease. Haploinsufficient Cacna1c+/− rats represent a recently developed model with a behavioral phenotype, but its cardiac phenotype is unknown. Here, we unraveled the cardiac phenotype of Cacna1c+/− rats with a main focus on cellular Ca2+ handling mechanisms. Under basal conditions, isolated ventricular Cacna1c+/− myocytes exhibited unaltered L-type Ca2+ current, Ca2+ transients (CaTs), sarcoplasmic reticulum (SR) Ca2+ load, fractional release, and sarcomere shortenings. However, immunoblotting of left ventricular (LV) tissue revealed reduced expression of Cav1.2, increased expression of SERCA2a and NCX, and augmented phosphorylation of RyR2 (at S2808) in Cacna1c+/− rats. The β-adrenergic agonist isoprenaline increased amplitude and accelerated decay of CaTs and sarcomere shortenings in both Cacna1c+/− and WT myocytes. However, the isoprenaline effect on CaT amplitude and fractional shortening (but not CaT decay) was impaired in Cacna1c+/− myocytes exhibiting both reduced potency and efficacy. Moreover, sarcolemmal Ca2+ influx and fractional SR Ca2+ release after treatment with isoprenaline were smaller in Cacna1c+/− than in WT myocytes. In Langendorff-perfused hearts, the isoprenaline-induced increase in RyR2 phosphorylation at S2808 and S2814 was attenuated in Cacna1c+/− compared to WT hearts. Despite unaltered CaTs and sarcomere shortenings, Cacna1c+/− myocytes display remodeling of Ca2+ handling proteins under basal conditions. Mimicking sympathetic stress with isoprenaline unmasks an impaired ability to stimulate Ca2+ influx, SR Ca2+ release, and CaTs caused, in part, by reduced phosphorylation reserve of RyR2 in Cacna1c+/− cardiomyocytes.

BackgroundHealthy brain development depends on early social practices and experiences. The risk gene CACNA1C is implicated in numerous neuropsychiatric disorders, in which key characteristics include deficits in social functioning and communication. Recently, we reported sex-dependent impairments in social behavior and ultrasonic vocalizations (USV) in juvenile heterozygous Cacna1c+/− (HET) rats. Specifically, HET females displayed increases in rough-and-tumble play that eliminated the typically observed sex difference between male and female rats. Interestingly, female wild-type Cacna1c+/+ (WT) pairs also showed a similar increase in social play when housed with HET females, suggesting their behavior may be influenced by HET cage mates. This indicates that the genetic makeup of the social environment related to Cacna1c can influence social play, yet systematic studies are lacking.MethodsIn the present study, we housed juvenile females in MIXED- or SAME-genotype cages and tested them in a social play paradigm with a same- and opposite-genotype partner.ResultsThe results show that the early social environment and the genotype of the play partner influence social play and 50-kHz USV emission. Experience with a WT play partner appears necessary for HET females to show comparable levels of play and 50-kHz USV emission. Same-genotype HET pairs played less and emitted fewer 50-kHz USV than same-genotype WT or opposite-genotype pairs; however, we found that the decrease in social play and 50-kHz USV in HET pairs can be rescued by playing with a WT partner. The effect was particularly prominent when the first play partner was WT, as we found it increased play and 50-kHz USV emission in all subsequent interactions with ensuing partners.ConclusionThese findings suggest that the genetic makeup related to the social environment and/or social peers influences social play in Cacna1c+/− haploinsufficient rats. Specifically, our results show that WT peers can rescue behavior and communication alterations in Cacna1c female rats. Our findings have important implications because they show that the genetic makeup of the social environment can divulge phenotypic changes in genetic rat models of neuropsychiatric disorders.

Bipolar disorder (BD) is a chronic mood disorder characterized by manic and depressive episodes. Dysregulation of neuroplasticity and calcium homeostasis are frequently observed in BD patients, but the underlying molecular mechanisms are largely unknown. Here, we show that miR‐499‐5p regulates dendritogenesis and cognitive function by downregulating the BD risk gene CACNB2. miR‐499‐5p expression is increased in peripheral blood of BD patients, as well as in the hippocampus of rats which underwent juvenile social isolation. In rat hippocampal neurons, miR‐499‐5p impairs dendritogenesis and reduces surface expression and activity of the L‐type calcium channel Cav1.2. We further identified CACNB2, which encodes a regulatory β‐subunit of Cav1.2, as a direct functional target of miR‐499‐5p in neurons. miR‐499‐5p overexpression in the hippocampus in vivo induces short‐term memory impairments selectively in rats haploinsufficient for the Cav1.2 pore forming subunit Cacna1c. In humans, miR‐499‐5p expression is negatively associated with gray matter volumes of the left superior temporal gyrus, a region implicated in auditory and emotional processing. We propose that stress‐induced miR‐499‐5p overexpression contributes to dendritic impairments, deregulated calcium homeostasis, and neurocognitive dysfunction in BD.

ABSTRACTBipolar disorder (BD) is a chronic mood disorder characterized by alternating manic and depressive episodes, often in conjunction with cognitive deficits. Dysregulation of neuroplasticity and calcium homeostasis as a result of complex genetic environment interactions are frequently observed in BD patients, but the underlying molecular mechanisms are largely unknown. Here, we show that a BD-associated microRNA, miR-499-5p, regulates neuronal dendrite development and cognitive function by downregulating the BD risk gene CACNB2. miR-499-5p expression is increased in peripheral blood of BD patients and healthy subjects at risk of developing the disorder due to a history of childhood maltreatment. This up-regulation is paralleled in the hippocampus of rats which underwent juvenile social isolation. Elevating miR-499-5p levels in rat hippocampal pyramidal neurons impairs dendritogenesis and reduces surface expression and activity of the voltage-gated L-type calcium channel Cav1.2. We further identified CACNB2, which encodes a regulatory β-subunit of Cav1.2, as a direct target of miR-499-5p in neurons. CACNB2 downregulation is required for the miR-499-5p dependent impairment of dendritogenesis, suggesting that CACNB2 is an important downstream target of miR-499-5p in the regulation of neuroplasticity. Finally, elevating miR-499-5p in the hippocampus in vivo is sufficient to induce short-term memory impairments in rats haploinsufficient for the Cav1.2 pore forming subunit Cacna1c. Taken together, we propose that stress-induced upregulation of miR-499-5p contributes to dendritic impairments and deregulated calcium homeostasis in BD, with specific implications for the neurocognitive dysfunction frequently observed in BD patients.

The top-ranked cross-disorder risk gene CACNA1C is strongly associated with multiple neuropsychiatric dysfunctions. In a recent series of studies, we applied a genomically informed approach and contributed extensively to the behavioral characterization of a genetic rat model haploinsufficient for the cross-disorder risk gene Cacna1c. Because deficits in processing social signals are associated with reduced social functioning as commonly seen in neuropsychiatric disorders, we focused on socio-affective communication through 22-kHz and 50-kHz ultrasonic vocalizations (USV). Specifically, we applied a reciprocal approach for studying socio-affective communication in sender and receiver by including rough-and-tumble play and playback of 22-kHz and 50-kHz USV. Here, we review the findings obtained in this recent series of studies and link them to the key features of 50-kHz USV emission during rough-and-tumble play and social approach behavior evoked by playback of 22-kHz and 50-kHz USV. We conclude that Cacna1c haploinsufficiency in rats leads to robust deficits in socio-affective communication through 22-kHz and 50-kHz USV and associated alterations in social behavior, such as rough-and-tumble play behavior.

The cross-disorder risk gene CACNA1C is strongly involved in the etiology of all major neuropsychiatric disorders, with women often being more affected by CACNA1C mutations than men. Human neuroimaging studies provided evidence that CACNA1C variants are associated with anatomical and functional brain alterations, such as decreased prefrontal volumes, microstructural changes in the hippocampus, and reduced hippocampal activity during memory tasks. In mouse models, Cacna1c alterations were repeatedly linked to disorder-like behavioral phenotypes and reduced adult hippocampal neurogenesis, which has been implicated in the pathology of neuropsychiatric disorders. Here, we applied a recently developed rat model and conducted two studies to investigate the effects of partial Cacna1c depletion on adult hippocampal neurogenesis and volumetric properties of the hippocampus and the prefrontal cortex in adult female constitutive heterozygous (Cacna1c+/-) rats and wildtype (Cacna1c+/+) littermate controls. In study 1, we analyzed proliferation versus survival of adult-born hippocampal cells based on a 5-bromodeoxyuridine assay ensuring neuronal cell-type specificity through applying an immunofluorescent multiple staining approach. In study 2, we performed a detailed volumetric analysis with high structural resolution of the dorsal hippocampus and the medial prefrontal cortex, including their major substructures. Our results indicate comparable levels of cell proliferation and neuronal survival in Cacna1c+/- rats and Cacna1c+/+ controls. Additionally, we found similar volumes of the dorsal hippocampus and the medial prefrontal cortex across major substructures irrespective of genotype, indicating that Cacna1c haploinsufficiency has no prominent effects on these brain features in female rats.

Sexual dimorphisms are widespread in the animal kingdom. At the behavioral level, there is evidence for sex differences in social play behavior. In rats, males typically engage more in rough-and-tumble play than females. One prominent component of the rough-and-tumble play repertoire in rats is the emission of 50-kHz ultrasonic vocalizations (USV). Such 50-kHz USV reflect the rewarding nature of play and serve as socioaffective signals. Here, we provide evidence for sexual dimorphisms within rough-and-tumble play-induced 50-kHz USV in juvenile rats. Specifically, females displayed reduced 50-kHz USV emission during playful interactions. This reduction was associated with changes in 50-kHz USV emission rates and subtype profiles during specific rough-and-tumble components, i.e., pinning, wrestling, and chasing, as well as differences in acoustic parameters. Interestingly, sex differences were modulated by Cacna1c, a gene strongly implicated in major neuropsychiatric disorders, often characterized by prominent sex biases, most notably autism. Specifically, Cacna1c haploinsufficiency affected the emission of 50-kHz USV during rough-and-tumble play in female rats and we provide evidence supporting the notion that such effects of Cacna1c haploinsufficiency are driven by male-typical features of 50-kHz USV emission. This is in line with the hypermasculinized social play repertoire previously observed in juvenile Cacna1c haploinsufficient females.

Deficits in processing social signals leads to reduced social functioning and is typically associated with neuropsychiatric disorders, including autism spectrum disorder, schizophrenia, and major depressive disorder. The cross-disorder risk gene CACNA1C is implicated in the etiology of all of these disorders and single-nucleotide polymorphisms within CACNA1C are ranked among the best replicated and most robust genetic findings from genome-wide association studies in psychiatry. Rats are highly social, live in large social groups, and communicate through ultrasonic vocalizations (USV), with low-frequency 22-kHz USV emitted in dangerous and often life-threating situations, such as predator exposure, serving an alarming function. In the present study, we applied an alarm 22-kHz USV playback paradigm to investigate the role of Cacna1c in socio-affective information processing in rats. Specifically, we assessed behavioral inhibition evoked by 22-kHz USV in constitutive heterozygous Cacna1c(+/+) females and males, as compared to wildtype Cacna1c(+/+) littermate controls. To probe specificity, two sets of alarm 22-kHz USV were presented, i.e. 22-kHz USV elicited by predator urine exposure and 22-kHz USV emitted during a retention test on learned fear, together with acoustic control stimuli. Our results show that behavioral inhibition evoked by playback of alarm 22-kHz USV is robust and occurs in response to both sets, yet is modulated by Cacna1c in a sex-dependent manner. In male but not female rats, Cacna1c haploinsufficiency led to less pronounced and less specific behavioral inhibition, supporting the idea that Cacna1c haploinsufficiency results in a lower motivation and/or diminished capability to display appropriate responses to important socio-affective communication signals.

The risk gene CACNA1C is strongly implicated in the etiology of all major psychiatric disorders, such as depressive disorder, bipolar disorder, autism spectrum disorder, and schizophrenia. These disorders feature high levels of comorbidity and share an overlap of symptoms; in particular, deficits in social functioning are common. Intriguingly, sex-dependent effects of CACNA1C single nucleotide polymorphisms on prevalence, health outcomes, and psychological traits have been reported, typically suggesting that women are more affected by CACNA1C mutations than men. In rodents, genetic modifications specifically targeting Cacna1c have repeatedly been linked to deficits in social behavior in male mice and rats but many studies neglect the sex-dependent effects observed in humans. Our study focused on the role of Cacna1c in regulating social behavior and communication in adult female rats. We compared social and non-social behavior together with concomitant emission of pro-social 50-kHz ultrasonic vocalizations (USV) associated with positive affect in constitutive heterozygous (Cacna1c(+/-)) rats to wildtype (Cacna1c(+/+)) littermate controls. Our results indicate that partial Cacna1c depletion leads to strongly reduced emission of 50-kHz USV and mild social deficits during female direct reciprocal social interaction. Detailed temporal analyses revealed most prominent reductions of 50-kHz USV during non-social behavior, suggesting that reduced positive affect occurs in a social context in Cacna1c(+/-) rats but is not specifically linked to social behavior. Finally, we observed increased self-grooming behavior in Cacna1c(+/-) rats, consistent with an autism-like phenotype. Our findings in rats thus support a role of Cacna1c in regulating behavioral phenotypes with relevance for several neuropsychiatric disorders.

As cross-disorder risk gene, CACNA1C is implicated in the etiology of all major neuropsychiatric disorders characterized by deficits in social behavior and communication and there is evidence for sex-dependent influences of single-nucleotide polymorphisms within CACNA1C on diagnosis, course, and recovery in humans. In this study, we aimed, therefore, at further exploring the role of Cacna1c in regulating behavioral phenotypes, focusing on sex-specific differences in social behavior and communication during the critical developmental period of adolescence in rats. Specifically, we compared rough-and-tumble play, concomitant emission of pro-social 50-kHz ultrasonic vocalizations, and social approach behavior in response to playback of 50-kHz ultrasonic vocalizations between constitutive heterozygous Cacna1c +/- females and wildtype Cacna1c +/+ littermate controls, and contrasted present female findings to data previously reported in males. Our results show for the first time that partial depletion of Cacna1c leads to sex-dependent alterations in social behavior and communication in rats. In females, Cacna1c haploinsufficiency led to hypermasculinization, with rough-and-tumble play behavior, in general, and pinning behavior, in particular, being even higher than in males without affecting concomitant 50-kHz ultrasonic vocalizations. In males, in contrast, rough-and-tumble play behavior was not altered, yet emission of 50-kHz ultrasonic vocalizations was diminished following partial Cacna1c depletion. The behavioral responses elicited by playback of 50-kHz ultrasonic vocalizations were reduced upon partial Cacna1c depletion in both sexes. It thus can be concluded that Cacna1c plays a prominent sex-dependent role in regulating juvenile rat social play behavior and pro-social 50-kHz ultrasonic communication with relevance to sex-specific effects seen in neuropsychiatric disorders.

Genetic and environmental influences are thought to interact in their contribution to the etiology of major neuropsychiatric disorders. One of the best replicated findings obtained in genome-wide association studies are genetic variants in the CACNA1C gene. Here, we used our constitutive heterozygous Cacna1c rat model in combination with a 4-week exposure to either post-weaning social isolation, standard housing or social and physical environmental enrichment during the critical juvenile developmental period to observe their long-term interactive effects with Cacna1c haploinsufficiency. Our study provides evidence for a gene × environment interaction, i.e. an interplay between Cacna1c haploinsufficiency and environment during juvenile development, on object recognition, spatial memory and reversal learning capabilities. Social and physical enrichment had a positive influence on Cacna1c+/- rats and Cacna1c+/+ littermate controls on spatial and reversal learning, while post-weaning social isolation negatively affected novel object recognition in both genotypes. Despite intact spatial learning and re-learning abilities in all groups, slight but consistent deficits were evident in Cacna1c+/- rats previously housed under standard conditions particularly during reversal learning but not Cacna1c+/- rats previously exposed to social and physical enrichment. Together, this supports the notion that Cacna1c interacts with the environment to shape disease vulnerability and associated alterations in cognitive functioning.

The pathophysiology of neuropsychiatric disorders involves complex interactions between genetic and environmental risk factors. Confirmed by several genome-wide association studies, Cacna1c represents one of the most robustly replicated psychiatric risk genes. Besides genetic predispositions, environmental stress such as childhood maltreatment also contributes to enhanced disease vulnerability. Both, Cacna1c gene variants and stressful life events are associated with morphological alterations in the prefrontal cortex and the hippocampus. Emerging evidence suggests impaired mitochondrial bioenergetics as a possible underlying mechanism of these regional brain abnormalities. In the present study, we simulated the interaction of psychiatric disease-relevant genetic and environmental factors in rodents to investigate their potential effect on brain mitochondrial function using a constitutive heterozygous Cacna1c rat model in combination with a four-week exposure to either post-weaning social isolation, standard housing, or social and physical environmental enrichment. Mitochondria were isolated from the prefrontal cortex and the hippocampus to evaluate their bioenergetics, membrane potential, reactive oxygen species production, and respiratory chain complex protein levels. None of these parameters were considerably affected in this particular gene-environment setting. These negative results were very robust in all tested conditions demonstrating that Cacna1c depletion did not significantly translate into altered bioenergetic characteristics. Thus, further investigations are required to determine the disease-related effects on brain mitochondria.

Play fighting in juvenile rats is associated with a high occurrence of 50 kHz vocalizations. These calls are varied in form, ranging from long, flat calls to short, frequency-modulated ones. We hypothesize that at least some types of calls serve as play signals to facilitate play. In the present study, pairs of juvenile male rats that were unfamiliar with one another were paired in a neutral test enclosure to which they had been habituated. Video and audio records were made of the encounters. Pairs were of two types: both pairs could vocalize or only one partner could do so. There were some differences between the play of pairs containing a devocalized partner, but overall, the pattern of play, the frequency and types of calls were similar between the two types of pairs. We used a Monte Carlo shuffling technique to analyse the correlations between the playful actions performed and the types and frequencies of various 50 kHz calls that were produced. The analyses revealed that there were strong associations between types of calls and types of social contact: an approach followed by playful nape contact was associated with calls, but an approach followed by nonplayful contact (e.g. anogenital sniffing) was not. Similarly, different calls were associated with different actions, such as nape contact, evade and wrestling, with most of these calls being uttered by the initiator of the action, not the recipient. However, coordinating calls reciprocally with complementary calls uttered by participants as they engaged in complementary actions (e.g. attacking, being attacked) appeared to be a way in which calls could potentially be used as play signals to influence the ongoing cooperation needed to sustain play fights. (C) 2018 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.