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Intranasal administration of oxytocin (OXT) might be a promising new adjunctive therapy for mental disorders characterized by social behavioral alterations such as autism and schizophrenia. Despite promising initial studies in humans, it is not yet clear the specificity of the behavioral effects induced by chronic intranasal OXT and if chronic intranasal OXT could have different effects compared with single administration. This is critical for the aforementioned chronic mental disorders that might potentially involve life-long treatments. As a first step to address these issues, here we report that chronic intranasal OXT treatment in wild-type C57BL/6J adult mice produced a selective reduction of social behaviors concomitant to a reduction of the OXT receptors throughout the brain. Conversely, acute intranasal OXT treatment produced partial increases in social behaviors towards opposite-sex novel-stimulus female mice, while on the other hand, it decreased social exploration of same-sex novel stimulus male mice, without affecting social behavior towards familiar stimulus male mice. Finally, prolonged exposure to intranasal OXT treatments did not alter, in wild-type animals, parameters of general health such as body weight, locomotor activity, olfactory and auditory functions, nor parameters of memory and sensorimotor gating abilities. These results indicate that a prolonged over-stimulation of a 'healthy' oxytocinergic brain system, with no inherent deficits in social interaction and normal endogenous levels of OXT, results in specific detrimental effects in social behaviors.

Epilepsy affects 1% of the general population and 30% of patients are resistant to antiepileptic drugs. Although optogenetics is an efficient antiepileptic strategy, the difficulty of illuminating deep brain areas poses translational challenges. Thus, the search of alternative light sources is strongly needed. Here, we develop pH-sensitive inhibitory luminopsin (pHIL), a closed-loop chemo-optogenetic nanomachine composed of a luciferase-based light generator, a fluorescent sensor of intracellular pH (E 2 GFP), and an optogenetic actuator (halorhodopsin) for silencing neuronal activity. Stimulated by coelenterazine, pHIL experiences bioluminescence resonance energy transfer between luciferase and E 2 GFP which, under conditions of acidic pH, activates halorhodopsin. In primary neurons, pHIL senses the intracellular pH drop associated with hyperactivity and optogenetically aborts paroxysmal activity elicited by the administration of convulsants. The expression of pHIL in hippocampal pyramidal neurons is effective in decreasing duration and increasing latency of pilocarpine-induced tonic-clonic seizures upon in vivo coelenterazine administration, without affecting higher brain functions. The same treatment is effective in markedly decreasing seizure manifestations in a murine model of genetic epilepsy. The results indicate that pHIL represents a potentially promising closed-loop chemo-optogenetic strategy to treat drug-refractory epilepsy. Optogenetics is a powerful antiepileptic strategy in cases of drug resistance but light delivery to deep brain structure is hard to achieve. Here, authors develop a closed-loop chemo-optogenetic tool made of a luciferase, a sensor of intracellular pH, and an optogenetic actuator for silencing neuronal hyperactivity.

The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is an epigenetic master regulator that plays a crucial role during nervous system development and maturation. REST function was originally described during development, where it determines neuronal phenotype. However, recent studies showed that REST participates in several processes in the adult brain, including neuronal plasticity and epileptogenesis. In this regard, the relationships between REST and epilepsy are still controversial and need further investigation. As forebrain excitatory neurons are the common final pathway of seizure susceptibility, we investigated the role of REST in epilepsy by inducing REST conditional knockout (REST-cKO) specifically in excitatory neurons of the hippocampus. To target the excitatory neuronal population, we cloned the calcium/calmodulin-dependent protein kinase IIα minimal promoter upstream of Cre recombinase. After assessing the specificity of the promoter's expression, the transgenes were packaged in an engineered adeno-associated virus able to cross the blood–brain and blood–cerebrospinal fluid barriers and delivered in the lateral ventricles of 2-month-old REST flox/flox mice to characterize, after 1 month, the cognitive phenotype and the seizure propensity. We show that REST-cKO mice display lower levels of anxiety in the light–dark test with respect to control mice but have unaltered motor, social, and cognitive profiles. The evaluation of the susceptibility to epileptic seizures showed that REST-cKO mice are more resistant to pentylenetetrazole-induced kindling but not to seizures induced by a single administration of the convulsant and show higher survival rates. Overall, these data suggest that the absence of REST in forebrain excitatory neurons decreases seizure susceptibility, pointing to a pro-epileptogenic role of the transcriptional repressor under conditions of pathological excitation/inhibition imbalance.

RE-1 Silencing Transcription factor (REST) controls several steps in neural development by modulating the expression of a wide range of neural genes. Alterations in REST expression have been associated with the onset of epilepsy; however, whether such alterations are deleterious or represent a protective homeostatic response remains elusive. To study the impact of REST modulation on seizure propensity, we developed a tool for its negative modulation . The tool is composed of the paired-amphipathic helix 1 (PAH1) domain, a competitive inhibitor of REST activation by mSin3, fused to the light-oxygen-voltage sensing 2 (LOV2) domain of phototropin 1, a molecular switch to alternatively hide or expose the PAH1 inhibitor. We employed the C450A and I539E light-independent AsLOV2 variants to mimic the closed (inactive) and open (active) states of LOV2-PAH1, respectively. Recombinant AAV1/2 viral particles (rAAVs) allowed LOV2-PAH1 expression in HEK293T cells and primary neurons, and efficiently transduced hippocampal neurons . mRNA expression analysis revealed an increased expression of several neuronal genes in the hippocampi of mice expressing the open probe. AAV-transduced mice received a single dose of kainic acid (KA), a treatment known to induce a transient increase of REST levels in the hippocampus. Remarkably, mice expressing the active variant displayed a reduced number of KA-induced seizures, which were less severe compared to mice carrying the inactive probe. These data support the validity of our tool to modulate REST activity and the potential impact of REST modulation on epileptogenesis.

The mechanisms underlying the neurodevelopmental deficits associated with CHARGE syndrome, which include cerebellar hypoplasia, developmental delay, coordination problems, and autistic features, have not been identified. CHARGE syndrome has been associated with mutations in the gene encoding the ATP-dependent chromatin remodeler CHD7. CHD7 is expressed in neural stem and progenitor cells, but its role in neurogenesis during brain development remains unknown. Here we have shown that deletion of Chd7 from cerebellar granule cell progenitors (GCps) results in reduced GCp proliferation, cerebellar hypoplasia, developmental delay, and motor deficits in mice. Genome-wide expression profiling revealed downregulated expression of the gene encoding the glycoprotein reelin (Reln) in Chd7-deficient GCps. Recessive RELN mutations have been associated with severe cerebellar hypoplasia in humans. We found molecular and genetic evidence that reductions in Reln expression contribute to GCp proliferative defects and cerebellar hypoplasia in GCp-specific Chd7 mouse mutants. Finally, we showed that CHD7 is necessary for maintaining an open, accessible chromatin state at the Reln locus. Taken together, this study shows that Reln gene expression is regulated by chromatin remodeling, identifies CHD7 as a previously unrecognized upstream regulator of Reln, and provides direct in vivo evidence that a mammalian CHD protein can control brain development by modulating chromatin accessibility in neuronal progenitors.

Reelin is a large secreted extracellular matrix glycoprotein playing an important role in early neurodevelopment. Several genetic studies found an association between RELN gene and increased risk of autism suggesting that reelin deficiency may be a vulnerability factor in its etiology. Moreover, a reduced reelin expression has been observed in several brain regions of subjects with Autism Spectrum Disorders. Since a number of reports have documented presence of vocal and neuromotor abnormalities in patients with autism and suggested that these dysfunctions predate the onset of the syndrome, we performed a fine-grain characterization of the neonatal vocal and motor repertoire in reelin mutant mice to explore the developmental precursors of the disorder. Our findings evidence a general delay in motor and vocal development in heterozygous (50% reduced reelin) and reeler (lacking reelin gene) mutant mice. As a whole, an increased number of calls characterized heterozygous pup's emission. Furthermore, the typical ontogenetic peak in the number of calls characterizing wild-type pups on postnatal day 4 appeared slightly delayed in heterozygous pups (to day 6) and was quite absent in reeler littermates, which exhibited a flat profile during development. We also detected a preferential use of a specific call category (two-components) by heterozygous and reeler mice at postnatal days 6 and 8 as compared to their wild-type littermates. With regard to the analysis of spontaneous movements, a differential profile emerged early in development among the three genotypes. While only slight coordination difficulties are exhibited by heterozygous pups, all indices of motor development appear delayed in reeler mice. Overall, our results evidence a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reelin mutant pups.

Background CHD8 haploinsufficiency causes autism and macrocephaly with high penetrance in the human population. Chd8 heterozygous mice exhibit relatively subtle brain overgrowth and little gene expression changes in the embryonic neocortex. The purpose of this study was to generate new, sub-haploinsufficient Chd8 mouse models to allow us to identify and study the functions of CHD8 during embryonic cortical development. Methods To examine the possibility that certain phenotypes may only appear at sub-heterozygous Chd8 levels in the mouse, we created an allelic series of Chd8 -deficient mice to reduce CHD8 protein levels to approximately 35% (mild hypomorph), 10% (severe hypomorph) and 0% (neural-specific conditional knockout) of wildtype levels. We used RNA sequencing to compare transcriptional dysregulation, structural MRI and brain weight to investigate effects on brain size, and cell proliferation, differentiation and apoptosis markers in immunostaining assays to quantify changes in neural progenitor fate. Results Mild Chd8 hypomorphs displayed significant postnatal lethality, with surviving animals exhibiting more pronounced brain hyperplasia than heterozygotes. Over 2000 genes were dysregulated in mild hypomorphs, including autism-associated neurodevelopmental and cell cycle genes. We identify increased proliferation of non-ventricular zone TBR2+ intermediate progenitors as one potential cause of brain hyperplasia in these mutants. Severe Chd8 hypomorphs displayed even greater transcriptional dysregulation, including evidence for p53 pathway upregulation. In contrast to mild hypomorphs, these mice displayed reduced brain size and increased apoptosis in the embryonic neocortex. Homozygous, conditional deletion of Chd8 in early neuronal progenitors resulted in pronounced brain hypoplasia, partly caused by p53 target gene derepression and apoptosis in the embryonic neocortex. Limitations Our findings identify an important role for the autism-associated factor CHD8 in controlling the proliferation of intermediate progenitors in the mouse neocortex. We propose that CHD8 has a similar function in human brain development, but studies on human cells are required to confirm this. Because many of our mouse mutants with reduced CHD8 function die shortly after birth, it is not possible to fully determine to what extent reduced CHD8 function results in autism-associated behaviours in mice. Conclusions Together, these findings identify important, dosage-sensitive functions for CHD8 in p53 pathway repression, neurodevelopmental gene expression and neural progenitor fate in the embryonic neocortex. We conclude that brain development is acutely sensitive to reduced CHD8 expression and that the varying sensitivities of different progenitor populations and cellular processes to CHD8 dosage result in non-linear effects on gene transcription and brain growth. Shaun Hurley, Conor Mohan and Philipp Suetterlin have contributed equally to this work.

Kinase D interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a transmembrane scaffold protein that participates in fundamental aspects of neuronal physiology including cell survival, differentiation, and synaptic plasticity. The Kidins220 constitutive knockout line displays developmental defects in the nervous and cardiovascular systems that lead to embryonic lethality, which has so far precluded the study of this protein in the adult. Moreover, Kidins220 mRNA is tightly regulated by alternative splicing, whose impact on nervous system physiology has not yet been addressed in vivo. Here, we have asked to what extent the absence of Kidins220 splicing and the selective knockout of Kidins220 impact on adult brain homeostasis. To answer this question, we used a floxed line that expresses only the full-length, non-spliced Kidins220 mRNA, and a forebrain-specific, CaMKII-Cre driven Kidins220 conditional knockout (cKO) line. Kidins220 cKO brains are characterized by enlarged ventricles in the absence of cell death, and by deficient dendritic arborization in several cortical regions. The deletion of Kidins220 leads to behavioral changes, such as reduced anxiety-like traits linked to alterations in TrkB-BDNF signaling and sex-dependent alterations of hippocampal-dependent spatial memory. Kidins220 floxed mice present similarly enlarged brain ventricles and increased associative memory. Thus, both the absolute levels of Kidins220 expression and its splicing pattern are required for the correct brain development and related expression of behavioral phenotypes. These findings are relevant in light of the increasing evidence linking mutations in the human KIDINS220 gene to the onset of severe neurodevelopmental disorders.

Dietary restriction, such as low glycemic index diet (LGID), have been successfully used to treat drug-resistant epilepsy. However, if such diet could also counteract antiepileptogenesis is still unclear. Here, we investigated whether the administration of LGID during the latent pre-epileptic period, prevents or delays the appearance of the overt epileptic phenotype. To this aim, we used the Synapsin II knockout (SynIIKO) mouse, a model of temporal lobe epilepsy in which seizures manifest 2–3 months after birth, offering a temporal window in which LGID may affect epileptogenesis. Pregnant SynIIKO mice were fed with either LGID or standard diet during gestation and lactation. Both diets were maintained in weaned mice up to 5 months of age. LGID delayed the seizure onset and induced a reduction of seizures severity only in female SynIIKO mice. In parallel with the epileptic phenotype, high-density multielectrode array recordings revealed a reduction of frequency, amplitude, duration, velocity of propagation and spread of interictal events by LGID in the hippocampus of SynIIKO females, but not mutant males, confirming the gender-specific effect. ELISA-based analysis revealed that LGID increased cortico-hippocampal allopregnanolone (ALLO) levels only in females, while it was unable to affect ALLO plasma concentrations in either sex. The results indicate that the gender-specific interference of LGID with the epileptogenic process can be ascribed to a gender-specific increase in cortical ALLO, a neurosteroid known to strengthen GABAergic transmission. The study highlights the possibility of developing a personalized gender-based therapy for temporal lobe epilepsy.

Kinase D-interacting substrate of 220 kDa (Kidins220) is a transmembrane protein that participates in neural cell survival, maturation, and plasticity. Mutations in the human KIDINS220 gene are associated with a neurodevelopmental disorder (‘SINO’ syndrome) characterized by spastic paraplegia, intellectual disability, and in some cases, autism spectrum disorder. To better understand the pathophysiology of KIDINS220-linked pathologies, in this study, we assessed the sensory processing and social behavior of transgenic mouse lines with reduced Kidins220 expression: the CaMKII-driven conditional knockout (cKO) line, lacking Kidins220 in adult forebrain excitatory neurons, and the Kidins220floxed line, expressing constitutively lower protein levels. We show that alterations in Kidins220 expression levels and its splicing pattern cause impaired response to both auditory and olfactory stimuli. Both transgenic lines show impaired startle response to high intensity sounds, with preserved pre-pulsed inhibition, and strongly reduced social odor recognition. In the Kidins220floxed line, olfactory alterations are associated with deficits in social memory and increased aggressive behavior. Our results broaden our knowledge of the SINO syndrome; understanding sensory information processing and its deviations under neuropathological conditions is crucial for devising future therapeutic strategies to enhance the quality of life of affected individuals.