Behaviorally conditioned effects of psychoactive drugs in experimental animals: What we have learned from nearly a century of research and what remains to be learned.

Continuous treatment with drugs is a crucial requirement for managing various clinical conditions, including chronic pain and neuropsychiatric disorders such as depression or schizophrenia. Associative learning processes, i.e. Pavlovian conditioning, can play an important role for the effects of drugs and could open new avenues for optimizing patient treatment. In this narrative literature review, we summarize available data in experimental animals regarding the behaviorally conditioned effects of psychostimulants such as d-amphetamine and cocaine, the dopamine receptor agonist apomorphine, the dopamine receptor antagonist haloperidol, morphine and antidepressant drugs. In each section, the drug under discussion is briefly introduced, followed by a detailed examination of conditioning features, including doses and dosing regimens, characteristics of the conditioning process such as test environments or specific conditioned stimuli, testing and conditioned response characteristics, possible extinction or reconditioning or reversal training, neural mechanisms, and finally, the potential clinical relevance of the research area related to the drug. We focus on key outcomes, delve into methodical issues, identify gaps in current knowledge, and suggest future research directions.

Bipolar-associated miR-499-5p controls neuroplasticity by downregulating the Cav1.2 subunit CACNB2.

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 ß-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.

Transient reduction in dendritic spine density in brain-specific profilin1 mutant mice is associated with behavioral deficits.

Actin filaments form the backbone of dendritic spines, the postsynaptic compartment of most excitatory synapses in the brain. Spine density changes affect brain function, and postsynaptic actin defects have been implicated in various neuropathies. It is mandatory to identify the actin regulators that control spine density. Based on previous studies, we hypothesized a role for the actin regulator profilin1 in spine formation. We report reduced hippocampal spine density in juvenile profilin1 mutant mice together with impairments in memory formation and reduced ultrasonic communication during active social behavior. Our results, therefore, underline a previously suggested function of profilin1 in controlling spine formation and behavior in juvenile mice.

Advanced paternal age as a risk factor for neurodevelopmental disorders: a translational study.

Advanced paternal age (APA) is a risk factor for several neurodevelopmental disorders, including autism and schizophrenia. The potential mechanisms conferring this risk are poorly understood. Here, we show that the personality traits schizotypy and neuroticism correlated with paternal age in healthy subjects (N = 677). Paternal age was further positively associated with gray matter volume (VBM, N = 342) in the right prefrontal and the right medial temporal cortex. The integrity of fiber tracts (DTI, N = 222) connecting these two areas correlated positively with paternal age. Genome-wide methylation analysis in humans showed differential methylation in APA individuals, linking APA to epigenetic mechanisms. A corresponding phenotype was obtained in our rat model. APA rats displayed social-communication deficits and emitted fewer pro-social ultrasonic vocalizations compared to controls. They further showed repetitive and stereotyped patterns of behavior, together with higher anxiety during early development. At the neurobiological level, microRNAs miR-132 and miR-134 were both differentially regulated in rats and humans depending on APA. This study demonstrates associations between APA and social behaviors across species. They might be driven by changes in the expression of microRNAs and/or epigenetic changes regulating neuronal plasticity, leading to brain morphological changes and fronto-hippocampal connectivity, a network which has been implicated in social interaction.

Interaction of the Psychiatric Risk Gene Cacna1c With Post-weaning Social Isolation or Environmental Enrichment Does Not Affect Brain Mitochondrial Bioenergetics in Rats.

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.

A placental mammal-specific microRNA cluster acts as a natural brake for sociability in mice.

Aberrant synaptic function is thought to underlie social deficits in neurodevelopmental disorders such as autism and schizophrenia. Although microRNAs have been shown to regulate synapse development and plasticity, their potential involvement in the control of social behaviour in mammals remains unexplored. Here, we show that deletion of the placental mammal-specific miR379-410 cluster in mice leads to hypersocial behaviour, which is accompanied by increased excitatory synaptic transmission, and exaggerated expression of ionotropic glutamate receptor complexes in the hippocampus. Bioinformatic analyses further allowed us to identify five "hub" microRNAs whose deletion accounts largely for the upregulation of excitatory synaptic genes observed, including Cnih2, Dlgap3, Prr7 and Src. Thus, the miR379-410 cluster acts a natural brake for sociability, and interfering with specific members of this cluster could represent a therapeutic strategy for the treatment of social deficits in neurodevelopmental disorders.

Reduced Efficacy of d-Amphetamine and 3,4-Methylenedioxymethamphetamine in Inducing Hyperactivity in Mice Lacking the Postsynaptic Scaffolding Protein SHANK1.

Genetic defects in the three SH3 and multiple ankyrin repeat domains (SHANK) genes (SHANK1, SHANK2, and SHANK3) are associated with multiple major neuropsychiatric disorders, including autism spectrum disorder (ASD), schizophrenia (SCZ), and bipolar disorder (BPD). Psychostimulant-induced hyperactivity is a commonly applied paradigm to assess behavioral phenotypes related to BPD and considered to be the gold standard for modeling mania-like elevated drive in mouse models. Therefore, the goal of our present study was to test whether Shank1 plays a role in the behavioral effects of psychostimulants and whether this is associated with genotype-dependent neurochemical alterations. To this aim, male and female null mutant Shank1-/- mice were treated with d-amphetamine (AMPH; 2.5 mg/kg) and 3,4-methylenedioxymethamphetamine (MDMA, commonly known as ecstasy; 20 mg/kg), and psychostimulant-induced hyperactivity was compared to heterozygous Shank1+/- and wildtype Shank1+/+ littermate controls. Results show that Shank1-/- mice display reduced psychostimulant-induced hyperactivity, although psychostimulants robustly stimulated locomotor activity in littermate controls. Shank1 deletion effects emerged throughout development, were particularly prominent in adulthood, and seen in response to both psychostimulants, i.e., AMPH and MDMA. Specifically, while AMPH-induced hyperactivity was reduced but still detectable in Shank1-/- mice, MDMA-induced hyperactivity was robustly blocked and completely absent in Shank1-/- mice. Reduced efficacy of psychostimulants to stimulate hyperactivity in Shank1-/- mice might be associated with alterations in the neurochemical architecture in prefrontal cortex, nucleus accumbens, and hypothalamus. Our observation that psychostimulant-induced hyperactivity is reduced rather than enhanced in Shank1-/- mice clearly speaks against a behavioral phenotype with relevance to BPD. Lack of BPD-like phenotype is consistent with currently available human data linking mutations in SHANK2 and SHANK3 but not SHANK1 to BPD.

Impaired Object Recognition but Normal Social Behavior and Ultrasonic Communication in Cofilin1 Mutant Mice.

Autism spectrum disorder (ASD), schizophrenia (SCZ) and intellectual disability (ID) show a remarkable overlap in symptoms, including impairments in cognition, social behavior and communication. Human genetic studies revealed an enrichment of mutations in actin-related genes for these disorders, and some of the strongest candidate genes control actin dynamics. These findings led to the hypotheses: (i) that ASD, SCZ and ID share common disease mechanisms; and (ii) that, at least in a subgroup of affected individuals, defects in the actin cytoskeleton cause or contribute to their pathologies. Cofilin1 emerged as a key regulator of actin dynamics and we previously demonstrated its critical role for synaptic plasticity and associative learning. Notably, recent studies revealed an over-activation of cofilin1 in mutant mice displaying ASD- or SCZ-like behavioral phenotypes, suggesting that dysregulated cofilin1-dependent actin dynamics contribute to their behavioral abnormalities, such as deficits in social behavior. These findings let us hypothesize: (i) that, apart from cognitive impairments, cofilin1 mutants display additional behavioral deficits with relevance to ASD or SCZ; and (ii) that our cofilin1 mutants represent a valuable tool to study the underlying disease mechanisms. To test our hypotheses, we compared social behavior and ultrasonic communication of juvenile mutants to control littermates, and we did not obtain evidence for impaired direct reciprocal social interaction, social approach or social memory. Moreover, concomitant emission of ultrasonic vocalizations was not affected and time-locked to social activity, supporting the notion that ultrasonic vocalizations serve a pro-social communicative function as social contact calls maintaining social proximity. Finally, cofilin1 mutants did not display abnormal repetitive behaviors. Instead, they performed weaker in novel object recognition, thereby demonstrating that cofilin1 is relevant not only for associative learning, but also for "non-matching-to-sample" learning. Here we report the absence of an ASD- or a SCZ-like phenotype in cofilin1 mutants, and we conclude that cofilin1 is relevant specifically for non-social cognition.

Behavioral phenotypes and neurobiological mechanisms in the Shank1 mouse model for autism spectrum disorder: A translational perspective.

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders, characterized by early-onset deficits in social behavior and communication across multiple contexts, together with restricted, repetitive patterns of behavior, interests, or activities. ASD is among the most heritable neuropsychiatric conditions with heritability estimates higher than 80%, and while available evidence points to a complex set of genetic factors, the SHANK (also known as ProSAP) gene family has emerged as one of the most promising candidates. Several genetic Shank mouse models for ASD were generated, including Shank1 knockout mice. Behavioral studies focusing on the Shank1 knockout mouse model for ASD included assays for detecting ASD-relevant behavioral phenotypes in the following domains: (I) social behavior, (II) communication, and (III) repetitive and stereotyped patterns of behavior. In addition, assays for detecting behavioral phenotypes with relevance to comorbidities in ASD were performed, including but not limited to (IV) cognitive functioning. Here, we summarize and discuss behavioral and neuronal findings obtained in the Shank1 knockout mouse model for ASD. We identify open research questions by comparing such findings with the symptoms present in humans diagnosed with ASD and carrying SHANK1 deletions. We conclude by discussing the implications of the behavioral and neuronal phenotypes displayed by the Shank1 knockout mouse model for the development of future pharmacological interventions in ASD.

Aberrant cognitive phenotypes and altered hippocampal BDNF expression related to epigenetic modifications in mice lacking the post-synaptic scaffolding protein SHANK1: Implications for autism spectrum disorder.

Autism spectrum disorder (ASD) is a class of neurodevelopmental disorders characterized by persistent deficits in social communication/interaction, together with restricted/repetitive patterns of behavior. ASD is among the most heritable neuropsychiatric conditions, and while available evidence points to a complex set of genetic factors, the SHANK gene family has emerged as one of the most promising candidates. Here, we assessed ASD-related phenotypes with particular emphasis on social behavior and cognition in Shank1 mouse mutants in comparison to heterozygous and wildtype littermate controls across development in both sexes. While social approach behavior was evident in all experimental conditions and social recognition was only mildly affected by genotype, Shank1-/- null mutant mice were severely impaired in object recognition memory. This effect was particularly prominent in juveniles, not due to impairments in object discrimination, and replicated in independent mouse cohorts. At the neurobiological level, object recognition deficits were paralleled by increased brain-derived neurotrophic factor (BDNF) protein expression in the hippocampus of Shank1-/- mice; yet BDNF levels did not differ under baseline conditions. We therefore investigated changes in the epigenetic regulation of hippocampal BDNF expression and detected an enrichment of histone H3 acetylation at the Bdnf promoter1 in Shank1-/- mice, consistent with increased learning-associated BDNF. Together, our findings indicate that Shank1 deletions lead to an aberrant cognitive phenotype characterized by severe impairments in object recognition memory and increased hippocampal BDNF levels, possibly due to epigenetic modifications. This result supports the link between ASD and intellectual disability, and suggests epigenetic regulation as a potential therapeutic target.

Acoustic Communication in Rats: Effects of Social Experiences on Ultrasonic Vocalizations as Socio-affective Signals.

Ultrasonic vocalizations (USV) serve important communicative functions as socio-affective signals in rats. In aversive situations, such as inter-male aggression and predator exposure, 22-kHz USV are emitted. They likely function as appeasement signals during fighting and/or as alarm calls to warn conspecifics. In appetitive situations, 50-kHz USV are uttered, most notably during social interactions, such as rough-and-tumble play and mating. It is believed that they fulfill an affiliative function as social contact calls. Social experiences or their lack, such as social isolation, can have profound impact on the emission of 22- and 50-kHz USV by the sender in later life, albeit direction and strength of observed effects vary, with time point of occurrence and duration being critical determinants. Little, however, is known about how social experiences affect the behavioral responses evoked by 22- and 50-kHz USV in the recipient. By means of our 50-kHz USV radial maze playback paradigm, we recently showed that the behavioral response elicited in the recipient is affected by post-weaning social isolation. Rats exposed to four weeks of isolation during the rough-and-tumble play period did not display social approach behavior toward 50-kHz USV but some signs of social avoidance. We further found that physical environmental enrichment providing minimal opportunities for social interactions has similar detrimental effects. Together, this indicates that social experiences can affect socio-affective communication in rodents, both at the level of sender and recipient. Deficits seen following post-weaning social isolation or physical environmental enrichment might be useful to model aspects of neurodevelopmental disorders characterized by social and communication deficits, such as autism and schizophrenia.

Early communication deficits in the Shank1 knockout mouse model for autism spectrum disorder: Developmental aspects and effects of social context.

Alterations in SHANK genes were repeatedly reported in autism spectrum disorder (ASD). ASD is a group of neurodevelopmental disorders diagnosed by persistent deficits in social communication/interaction across multiple contexts, with restricted/repetitive patterns of behavior. To date, diagnostic criteria for ASD are purely behaviorally defined and reliable biomarkers have still not been identified. The validity of mouse models for ASD therefore strongly relies on their behavioral phenotype. Here, we studied communication by means of isolation-induced pup ultrasonic vocalizations (USV) in the Shank1 mouse model for ASD by comparing Shank1(-/-) null mutant, Shank1(+/-) heterozygous, and Shank1(+/+) wildtype littermate controls. The first aim of the present study was to evaluate the effects of Shank1 deletions on developmental aspects of communication in order to see whether ASD-related communication deficits are due to general impairment or delay in development. Second, we focused on social context effects on USV production. We show that Shank1(-/-) pups vocalized less and displayed a delay in the typical inverted U-shaped developmental USV emission pattern with USV rates peaking on postnatal day (PND) 9, resulting in a prominent genotype difference on PND6. Moreover, testing under social conditions revealed even more prominently genotype-dependent deficits regardless of the familiarity of the social context. As communication by definition serves a social function, introducing a social component to the typically nonsocial test environment could therefore help to reveal communication deficits in mouse models for ASD. Together, these results indicate that SHANK1 is involved in acoustic communication across species, with genetic alterations in SHANK1 resulting in social communication/interaction deficits. Autism Res 2016, 9: 696-709. © 2015 International Society for Autism Research, Wiley Periodicals, Inc.

Repetitive behaviors in the Shank1 knockout mouse model for autism spectrum disorder: developmental aspects and effects of social context.

BACKGROUND: Autism spectrum disorder (ASD) is characterized by persistent deficits in social behavior and communication, together with restricted and repetitive patterns of behavior. Several ASD candidate genes have been identified, including the SHANK gene family with its three family members SHANK1, SHANK2, and SHANK3. METHODS: Typically, repetitive behavior in mouse models for ASD is assessed by measuring self-grooming behavior. The first aim of the current study was to assess repetitive behaviors in Shank1(-/-) null mutant, Shank1(+/-) heterozygous, and Shank1(+/+) wildtype littermate control mice by means of a comprehensive approach, including the assessment of self-grooming, digging behavior, and marble burying. The second aim was to establish a test paradigm that allows for assessing the effects of social context on the occurrence of repetitive behaviors in a genotype-dependent manner. To this aim, repetitive behaviors were repeatedly tested on three consecutive days in distinct social contexts, namely in presence or absence of social odors. RESULTS: Shank1(+/-) heterozygous and to a lesser extent Shank1(-/-) null mutant mice displayed slightly elevated levels of self-grooming behavior as adults, but not as juveniles, with genotype differences being most prominent in the social context. In contrast to elevated self-grooming behavior, marble burying was strongly reduced in adult Shank1(+/-) heterozygous and Shank1(-/-) null mutant mice across social contexts, as compared to adult Shank1(+/+) wildtype littermate controls. CONCLUSION: The opposite effects of the Shank1 deletion on the two types of repetitive behaviors are in line with a number of studies on repetitive behaviors in other genetic Shank models.