Loss-of-function mutation in Mirta22/Emc10 rescues specific schizophrenia-related phenotypes in a mouse model of the 22q11.2 deletion.

Identification of protective loss-of-function (LoF) mutations holds great promise for devising novel therapeutic interventions, although it faces challenges due to the scarcity of protective LoF alleles in the human genome. Exploiting the detailed mechanistic characterization of animal models of validated disease mutations offers an alternative. Here, we provide insights into protective-variant biology based on our characterization of a model of the 22q11.2 deletion, a strong genetic risk factor for schizophrenia (SCZ). Postnatal brain up-regulation of Mirta22/Emc10, an inhibitor of neuronal maturation, represents the major transcriptional effect of the 22q11.2-associated microRNA dysregulation. Here, we demonstrate that mice in which the Df(16)A deficiency is combined with a LoF Mirta22 allele show rescue of key SCZ-related deficits, namely prepulse inhibition decrease, working memory impairment, and social memory deficits, as well as synaptic and structural plasticity abnormalities in the prefrontal cortex. Additional analysis of homozygous Mirta22 knockout mice, in which no alteration is observed in the above-mentioned SCZ-related phenotypes, highlights the deleterious effects of Mirta22 up-regulation. Our results support a causal link between dysregulation of a miRNA target and SCZ-related deficits and provide key insights into beneficial LoF mutations and potential new treatments.

A Schizophrenia-Related Deletion Leads to KCNQ2-Dependent Abnormal Dopaminergic Modulation of Prefrontal Cortical Interneuron Activity.

Altered prefrontal cortex function is implicated in schizophrenia (SCZ) pathophysiology and could arise from imbalance between excitation and inhibition (E/I) in local circuits. It remains unclear whether and how such imbalances relate to genetic etiologies. We used a mouse model of the SCZ-predisposing 22q11.2 deletion (Df(16)A+/- mice) to evaluate how this genetic lesion affects the excitability of layer V prefrontal pyramidal neurons and its modulation by dopamine (DA). Df(16)A+/- mice have normal balance between E/I at baseline but are unable to maintain it upon dopaminergic challenge. Specifically, in wild-type mice, D1 receptor (D1R) activation enhances excitability of layer V prefrontal pyramidal neurons and D2 receptor (D2R) activation reduces it. Whereas the excitatory effect upon D1R activation is enhanced in Df(16)A+/- mice, the inhibitory effect upon D2R activation is reduced. The latter is partly due to the inability of mutant mice to activate GABAergic parvalbumin (PV)+ interneurons through D2Rs. We further demonstrate that reduced KCNQ2 channel function in PV+ interneurons in Df(16)A+/- mice renders them less capable of inhibiting pyramidal neurons upon D2 modulation. Thus, DA modulation of PV+ interneurons and control of E/I are altered in Df(16)A+/- mice with a higher excitation and lower inhibition during dopaminergic modulation.

A novel model of early experiences involving neonatal learning of a T-maze using maternal contact as a reward or its denial as an event of mild emotional adversity.

We developed a novel animal model of early life experiences in which rat pups are trained during postnatal days (PND) 10-13 in a T-maze with maternal contact as a reward (RER group) or its denial (DER group) as a mildly aversive event. Both groups of animals learn the T-maze, albeit the RER do so more efficiently. Training results in activation of the basal ganglia in the RER and of the hippocampus and prefrontal cortex in the DER. Moreover, on PND10 DER training leads to increased corticosterone levels and activation of the amygdala. In adulthood, male DER animals show better mnemonic abilities in the Morris water maze while the RER exhibit enhanced fear memory. Furthermore, DER animals have a hypofunctioning serotonergic system and express depressive-like behavior and increased aggression. However, they have increased hippocampal glucocorticoid receptors, indicative of efficient hypothalamic-pituitary-adrenal axis function, and an adaptive pattern of stress-induced corticosterone response. The DER experience with its relatively negative emotional valence results in a complex behavioral phenotype, which cannot be considered simply as adaptive or maladaptive.

The abiding relevance of mouse models of rare mutations to psychiatric neuroscience and therapeutics.

Studies using powerful family-based designs aided by large scale case-control studies, have been instrumental in cracking the genetic complexity of the disease, identifying rare and highly penetrant risk mutations and providing a handle on experimentally tractable model systems. Mouse models of rare mutations, paired with analysis of homologous cognitive and sensory processing deficits and state-of-the-art neuroscience methods to manipulate and record neuronal activity have started providing unprecedented insights into pathogenic mechanisms and building the foundation of a new biological framework for understanding mental illness. A number of important principles are emerging, namely that degradation of the computational mechanisms underlying the ordered activity and plasticity of both local and long-range neuronal assemblies, the building blocks necessary for stable cognition and perception, might be the inevitable consequence and the common point of convergence of the vastly heterogeneous genetic liability, manifesting as defective internally- or stimulus-driven neuronal activation patterns and triggering the constellation of schizophrenia symptoms. Animal models of rare mutations have the unique potential to help us move from "which" (gene) to "how", "where" and "when" computational regimes of neural ensembles are affected. Linking these variables should improve our understanding of how symptoms emerge and how diagnostic boundaries are established at a circuit level. Eventually, a better understanding of pathophysiological trajectories at the level of neural circuitry in mice, aided by basic human experimental biology, should guide the development of new therapeutics targeting either altered circuitry itself or the underlying biological pathways.

Learning of a T-maze by rat pups when contact with the mother is either permitted or denied.

Mother-pup interactions constitute an important component of environmental stimulation of the offspring during the neonatal period. Employing maternal contact as either a positive reinforcer or, its denial, as a frustrative, non-rewarding stimulus, we developed a novel experimental paradigm involving learning by rat neonates of a T-maze. When trained under the reward of maternal contact during postnatal days 10-13 Wistar rat pups learned the choice leading to the mother in a T-maze. When tested 2h later, in the absence of the mother, pups showed a clear preference for the arm of the T-maze leading to the position of the mother during training. Furthermore, pups receiving the expected reward of maternal contact had higher numbers of c-Fos immunopositive cells in the dorsal striatum compared to either naïve or pups denied the expected reward. The above behavioral and cellular results indicate that pups receiving the expected reward developed a procedural-like memory. When trained under frustrative non-reward pups learned to make the correct choice in the T-maze, albeit less efficiently than pups receiving the expected reward. Following this training condition c-Fos immunohistochemistry revealed increased activation of the CA1 area of the hippocampus and the orbitofrontal cortex. Expression of the information learned by the pups denied the expected reward was contingent upon the presence of the mother in the experimental setup in exactly the same configuration as during the training.

Long-term effects of neonatal handling on mu-opioid receptor levels in the brain of the offspring.

Neonatal handling is an experimental paradigm of an early experience which permanently alters hypothalamic-pituitary-adrenal axis function resulting in increased ability to cope with stress, and decreased emotionality. In the present work we investigated the effect of neonatal handling on adult rat brain mu-opioid receptor levels, since the opioid system is known to play an important role in emotional processing, anxiety and stress responses. Neonatal handling resulted in increased levels of mu-opioid receptors in the basolateral and central amygdaloid nuclei, in the CA3 and CA4 hippocampal areas, in the ventral tegmental area, the nucleus accumbens and the prefrontal cortex. Handled animals of both sexes had lower anxiety as measured in the elevated plus maze. The increased mu receptor levels could participate in the molecular mechanisms underlying the well-documented decreased stress and anxiety responses of handled animals.

Neurocognitive and Perceptual Processing in Genetic Mouse Models of Schizophrenia: Emerging Lessons.

During the past two decades, the number of animal models of psychiatric disorders has grown exponentially. Of these, genetic animal models that are modeled after rare but highly penetrant mutations hold great promise for deciphering critical molecular, synaptic, and neurocircuitry deficits of major psychiatric disorders, such as schizophrenia. Animal models should aim to focus on core aspects rather than capture the entire human disease. In this context, animal models with strong etiological validity, where behavioral and neurophysiological phenotypes and the features of the disease being modeled are in unambiguous homology, are being used to dissect both elementary and complex cognitive and perceptual processing deficits present in psychiatric disorders at the level of neurocircuitry, shedding new light on critical disease mechanisms. Recent progress in neuroscience along with large-scale initiatives that propose a consistent approach in characterizing these deficits across different laboratories will further enhance the efficacy of these studies that will ultimately lead to identifying new biological targets for drug development.

Recapitulation and Reversal of Schizophrenia-Related Phenotypes in Setd1a-Deficient Mice.

SETD1A, a lysine-methyltransferase, is a key schizophrenia susceptibility gene. Mice carrying a heterozygous loss-of-function mutation of the orthologous gene exhibit alterations in axonal branching and cortical synaptic dynamics accompanied by working memory deficits. We show that Setd1a binds both promoters and enhancers with a striking overlap between Setd1a and Mef2 on enhancers. Setd1a targets are highly expressed in pyramidal neurons and display a complex pattern of transcriptional up- and downregulations shaped by presumed opposing functions of Setd1a on promoters and Mef2-bound enhancers. Notably, evolutionarily conserved Setd1a targets are associated with neuropsychiatric genetic risk burden. Reinstating Setd1a expression in adulthood rescues cognitive deficits. Finally, we identify LSD1 as a major counteracting demethylase for Setd1a and show that its pharmacological antagonism results in a full rescue of the behavioral and morphological deficits in Setd1a-deficient mice. Our findings advance understanding of how SETD1A mutations predispose to schizophrenia (SCZ) and point to novel therapeutic interventions.

An early experience of mild adversity involving temporary denial of maternal contact affects the serotonergic system of adult male rats and leads to a depressive-like phenotype and inability to adapt to a chronic social stress.

Adverse early life experiences can affect adaptability to chronic stressors and lead to depressive-like behaviors in animal models. We employed an early experience model in which rat pups during postnatal days 10-13 are exposed to a T-maze in which they learn the location of their mother motivated by the rewarding stimulus of maternal contact; one group of rats receives the expected reward, by being allowed contact with the mother upon finding her, while the other group is temporarily denied this contact (Denied Expected Reward, DER), thus experiencing mild adversity. The results presented herein show that the DER early life experience results in a depressive-like phenotype in adulthood, as indicated by the absence of sucrose preference -anhedonia- exhibited by these animals, in adulthood. Following exposure to a chronic social stress (CSS), DER male rats were unable to adapt, evident by reduced general locomotion and time spent in the centre of an open field which indicate anxiety and/or decreased motivation for exploration. They also exhibited increased immobility time in the forced swimming test, suggesting a passive coping strategy. The depressive-like and anxious phenotype of the DER males was accompanied by changes in the serotonergic system, such as lower serotonin levels, higher serotonin turnover and higher levels of the type 1 serotonin receptor in the hippocampus. Our results corroborate findings showing that early life adversity disturbs behavioral regulation in adulthood. They also suggest that even mild adversity, if it involves intervention in mother-offspring interactions, can be sufficient to compromise adaptability.

Impaired hippocampal place cell dynamics in a mouse model of the 22q11.2 deletion.

Hippocampal place cells represent the cellular substrate of episodic memory. Place cell ensembles reorganize to support learning but must also maintain stable representations to facilitate memory recall. Despite extensive research, the learning-related role of place cell dynamics in health and disease remains elusive. Using chronic two-photon Ca2+ imaging in hippocampal area CA1 of wild-type and Df(16)A+/- mice, an animal model of 22q11.2 deletion syndrome, one of the most common genetic risk factors for cognitive dysfunction and schizophrenia, we found that goal-oriented learning in wild-type mice was supported by stable spatial maps and robust remapping of place fields toward the goal location. Df(16)A+/- mice showed a significant learning deficit accompanied by reduced spatial map stability and the absence of goal-directed place cell reorganization. These results expand our understanding of the hippocampal ensemble dynamics supporting cognitive flexibility and demonstrate their importance in a model of 22q11.2-associated cognitive dysfunction.

Age-Dependent Specific Changes in Area CA2 of the Hippocampus and Social Memory Deficit in a Mouse Model of the 22q11.2 Deletion Syndrome.

Several neuropsychiatric disorders are associated with cognitive and social dysfunction. Postmortem studies of patients with schizophrenia have revealed specific changes in area CA2, a long-overlooked region of the hippocampus recently found to be critical for social memory formation. To examine how area CA2 is altered in psychiatric illness, we used the Df(16)A(+/-) mouse model of the 22q11.2 microdeletion, a genetic risk factor for developing several neuropsychiatric disorders, including schizophrenia. We report several age-dependent CA2 alterations: a decrease in the density of parvalbumin-expressing interneurons, a reduction in the amount of feedforward inhibition, and a change in CA2 pyramidal-neuron intrinsic properties. Furthermore, we found that area CA2 is less plastic in Df(16)A(+/-) mice, making it nearly impossible to evoke action potential firing in CA2 pyramidal neurons. Finally, we show that Df(16)A(+/-) mice display impaired social cognition, providing a potential mechanism and a neural substrate for this impairment in psychiatric disorders.

Early experience of a novel-environment in isolation primes a fearful phenotype characterized by persistent amygdala activation.

Prolonged maternal separation (MS) activates the neonate's hypothalamus-pituitary-adrenal axis causing elevated basal and stress-induced corticosterone levels that may initiate amygdala-dependent fear learning. Here we test the hypothesis that the adult fearful phenotype is programmed by the pup's stressful experience during prolonged MS rather than by prolonged maternal absence per se. For this purpose, Wistar rat pups were exposed, on postnatal-day (pnd) 3, to: (i) repeated-MS in home-environment (HOME-SEP), 8h-MS daily for three days with the pups remaining together in the home-cage; (ii) repeated-MS in a novel-environment (NOVEL-SEP), with the same separation procedure, but now the pups were individually housed in a novel-environment during the 8h dam's absence; (iii) repeated handling, which consisted of daily brief (15 min instead of 8h) MS in the home-altogether or in a novel-environment individually (HOME-HAN and NOVEL-HAN, respectively); (iv) no-separation/no-handling (NON-SEP/NON-HAN) control condition, in which pups were left undisturbed in their home-cage. Compared to HOME-SEP rats, the NOVEL-SEP rats showed one day after the last MS enhanced stress-induced amygdala c-Fos expression and ACTH-release, despite of reduced adrenal corticosterone secretion. The higher amygdala c-Fos expression, ACTH-release and reduced corticosterone output observed postnatally, persisted into adulthood of the NOVEL-SEP animals. Behaviorally, NOVEL-SEP juvenile rats displayed deficits in social play, had intact spatial memory in the peri-pubertal period and showed more contextual fear memory compared to HOME-SEP in adulthood. Finally, NOVEL-HAN, compared to HOME-HAN, displayed increased stress-induced corticosterone output, no deficits in social play and reduced contextual fear. In conclusion, programming of an adult fearful phenotype linked to amygdala priming develops if pups are repeatedly isolated from peers in a novel-environment, while away from the dam for a prolonged period of time.

Effects of an Early Experience Involving Training in a T-Maze Under either Denial or Receipt of Expected Reward through Maternal Contact.

The mother is the most salient stimulus for the developing pups and a number of early experience models employ manipulation of the mother-infant interaction. We have developed a new model which in addition to changes in maternal behavior includes a learning component on the part of the pups. More specifically, pups were trained in a T-maze and either received (RER rats) or were denied (DER) the reward of maternal contact, during postnatal days 10-13. Pups of both experimental groups learn the T-maze, but the RER do so more efficiently utilizing a procedural-type of learning and memory with activation of the dorsal basal ganglia. On the other hand, the DER experience leads to activation of the hippocampus, prefrontal cortex, and amygdala in the pups. In adulthood, male DER animals exhibit better mnemonic abilities in the Morris water maze and higher activation of the hippocampus, while they have decreased brain serotonergic activity, exhibit a depressive-like phenotype and proactive aggressive behavior in the resident-intruder test. While male RER animals assume a reactive coping style in this test, and showed increased freezing during both contextual and cued memory recall following fear conditioning.

Denial or receipt of expected reward through maternal contact during the neonatal period differentially affect the development of the rat amygdala and program its function in adulthood in a sex-dimorphic way.

Early experiences affect brain development and thus adult brain function and behavior. We employed a novel early experience model involving denial (DER) or receipt of expected reward (RER) through maternal contact in a T-maze. Exposure to the DER experience for the first time, on postnatal day 10 (PND10), was stressful for the pups, as assessed by increased corticosterone levels, and was accompanied by enhanced activation of the amygdala, as assessed by c-Fos immunohistochemistry. Re-exposure to the same experience on days 11-13 led to adaptation. Corticosterone levels of the RER pups did not differ on the first and last days of training (PND10 and 13 respectively), while on PND11 and 12 they were lower than those of the CTR. The RER experience did not lead to activation of the amygdala. Males and females exposed as neonates to the DER or RER experience, and controls were tested as adults in the open field task (OF), the elevated plus maze (EPM), and cued and contextual fear conditioning (FC). No group differences were found in the EPM, while in the OF, both male and female DER animals, showed increased rearings, compared to the controls. In the FC, the RER males had increased memory for both context and cued conditioned fear, than either the DER or CTR. On the other hand, the DER males, but not females showed an increased activation, as assessed by c-Fos expression, of the amygdala following fear conditioning. Our results show that the DER early experience programmed the function of the adult amygdala as to render it more sensitive to fearful stimuli. This programming by the DER early experience could be mediated through epigenetic modifications of histones leading to chromatin opening, as indicated by our results showing increased levels of phospho-acetyl-histone-3 in the amygdala of the DER males.

Effects of denial of reward through maternal contact in the neonatal period on adult hypothalamic-pituitary-adrenal axis function in the rat.

Emotional behavioral traits associated with stress response are well documented to be affected by early life events. In the present work, we used a novel paradigm of neonatal experience, in which pups were trained in a T-maze and either received (RER rats) or were denied (DER) the reward of maternal contact, during postnatal days 10-13. We then evaluated stress coping and key factors controlling the function of the hypothalamic-pituitary-adrenal axis in adulthood. Adult male DER rats exposed to a single session of forced swim stress (FSS) showed increased immobility, while RER rats exhibited increased escape attempts. The corticosterone response following this stressor was higher although not prolonged in the DER rats. Their CRH mRNA levels in the PVN were increased up to 2h after the forced swim. However, basal levels of these hormones did not differ among groups. In addition, the DER neonatal experience induced an increase in hippocampal GR but a decrease in CRH-R1 immunopositive cells in the CA1 area of the hippocampus and the central amygdala. Overall, these data show a distinct stress response profile in the DER male rats, characterized by passive coping during the forced swim, increased hormonal response following stress, increased inhibitory control through GR and an indirect contribution of CRH-R1, the latter two factors resulting in a modified regulation of the response termination. It thus appears that DER rats have an enhanced potential for appropriate reactivity upon an incoming challenge, while maintaining in parallel an adequate control of the duration of their stress responses.

Fear memory for cue and context: opposite and time-dependent effects of a physiological dose of corticosterone in male BALB/c and C57BL/6J mice.

Highly emotional, stress reactive BALB/c mice secrete more corticosterone in response to fear conditioning than the low stress reactive C57BL/6J mice. Fear memory to cue and context differs between the strains. We injected corticosterone at physiological concentrations (250 µg/kg i.p.) 30 min before fear conditioning. Fear memory was tested 48 and 72 h later. Although corticosterone had little effect on acquisition, it differentially affected fear memories in strain dependent manner: while BALB/c mice decreased freezing during cue and context episodes, C57BL/6J mice showed an overall increase in freezing. BALB/c mice showed extinction over days while no such extinction was seen in C57BL/6J mice. Evaluation of these data in the perspective of previous studies using the same fear conditioning paradigm with corticosterone injections 5 min before or immediately after acquisition, revealed the impact of corticosterone during conditioning on the strength of fear memories. In C57BL/6J mice the overall increase in fear memories was higher if corticosterone was injected 30 min pre acquisition than if injected 5 min pre. In contrast, BALB/c mice showed reduced fear memories when injected 30 min pre compared to that seen 5 min pre acquisition. Both strains showed decreased fear memories compared to vehicle if corticosterone was administered immediately after acquisition. We conclude that the timing of physiologically relevant, stress levels increase in corticosterone is essential for the processing of aversive events and the formation of fear memories. However, the quality of the effect depends on the genetic background. These findings contribute to the understanding of the etiology of stress-related disorders.

Denial of reward in the neonate shapes sociability and serotonergic activity in the adult rat.

BACKGROUND: Manipulations of the early environment are linked to long-lasting alterations of emotionality and social capabilities. Denial of rewarding mother-pup interactions in early life of rats could serve as model for child neglect. Negative consequences for social competence in later life, accompanied by changes in the serotonergic system would be expected. In contrast, rewarding mother-pup contact should promote adequate social abilities. METHODOLOGY/PRINCIPAL FINDINGS: Male Wistar rats trained in a T-maze during postnatal days 10-13 under denial (DER) or permission (RER) of maternal contact were tested for play behavior in adolescence and for coping with defeat in adulthood. We estimated serotonin (5-HT) levels in the brain under basal conditions and following defeat, as well as serotonin receptor 1A (5-HT1A) and serotonin transporter (SERT) expression. DER rats exhibited increased aggressive-like play behavior in adolescence (i.e. increased nape attacks, p<0.0001) and selected a proactive coping style during defeat in adulthood (higher sum of proactive behaviors: number of attacks, flights, rearings and defensive upright posture; p = 0.011, p<0.05 vs RER, non-handled-NH). In adulthood, they had lower 5-HT levels in both the prefrontal cortex (p<0.05 vs RER) and the amygdala (p<0.05 vs NH), increased 5-HT levels following defeat (PFC p<0.0001) and decreased serotonin turnover (amygdala p = 0.008). The number of 5-HT1A immunopositive cells in the CA1 hippocampal area was increased (p<0.05 DER, vs RER, NH); SERT levels in the amygdala were elevated (p<0.05 vs RER, NH), but were lower in the prefrontal cortex (p<0.05 vs NH). CONCLUSIONS/SIGNIFICANCE: Denial of expected maternal reward early in life negatively affects sociability and the serotonergic system in a complex manner. We propose that our animal model could contribute to the identification of the neurobiological correlates of early neglect effects on social behavior and coping with challenges, but also in parallel with the effects of a rewarding early-life environment.