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Although bradykinesia, tremor, and rigidity are the hallmark motor defects in Parkinson’s disease (PD) patients, they also experience motor learning impairments and non-motor symptoms such as depression(1). The neural circuit basis for these different PD symptoms are not well understood. While current treatments are effective for locomotion deficits in PD(2,3), therapeutic strategies targeting motor learning deficits and non-motor symptoms are lacking(4–6). We found that distinct parafascicular (PF) thalamic subpopulations project to caudate putamen (CPu), subthalamic nucleus (STN), and nucleus accumbens (NAc). While PF→CPu and PF→STN circuits are critical for locomotion and motor learning respectively, inhibition of the PF→NAc circuit induced a depression-like state. While chemogenetically manipulating CPu-projecting PF neurons led to a long-term restoration of locomotion, optogenetic long-term potentiation at PF→STN synapses restored motor learning behavior in PD model mice. Furthermore, activation of NAc-projecting PF neurons rescued depression-like PD phenotypes. Importantly, we identified nicotinic acetylcholine receptors capable of modulating PF circuits to rescue different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating motor and non-motor deficits in PD.

Primates and rodents, which descended from a common ancestor around 90 million years ago1, exhibit profound differences in behaviour and cognitive capacity; the cellular basis for these differences is unknown. Here we use single-nucleus RNA sequencing to profile RNA expression in 188,776 individual interneurons across homologous brain regions from three primates (human, macaque and marmoset), a rodent (mouse) and a weasel (ferret). Homologous interneuron types—which were readily identified by their RNA-expression patterns—varied in abundance and RNA expression among ferrets, mice and primates, but varied less among primates. Only a modest fraction of the genes identified as ‘markers’ of specific interneuron subtypes in any one species had this property in another species. In the primate neocortex, dozens of genes showed spatial expression gradients among interneurons of the same type, which suggests that regional variation in cortical contexts shapes the RNA expression patterns of adult neocortical interneurons. We found that an interneuron type that was previously associated with the mouse hippocampus—the ‘ivy cell’, which has neurogliaform characteristics—has become abundant across the neocortex of humans, macaques and marmosets but not mice or ferrets. We also found a notable subcortical innovation: an abundant striatal interneuron type in primates that had no molecularly homologous counterpart in mice or ferrets. These interneurons expressed a unique combination of genes that encode transcription factors, receptors and neuropeptides and constituted around 30% of striatal interneurons in marmosets and humans. Single-nucleus RNA-sequencing analyses of brain from humans, macaques, marmosets, mice and ferrets reveal diverse ways that interneuron populations have changed during evolution.

The four voltage-gated sodium channels SCN1/2/3/8A have been associated with heterogeneous types of developmental disorders, each presenting with disease specific temporal and cell type specific gene expression. Using single-cell RNA sequencing transcriptomic data from humans and mice, we observe that SCN1A is predominantly expressed in inhibitory neurons. In contrast, SCN2/3/8A are profoundly expressed in excitatory neurons with SCN2/3A starting prenatally, followed by SCN1/8A neonatally. In contrast to previous observations from low resolution RNA screens, we observe that all four genes are expressed in both excitatory and inhibitory neurons, however, exhibit differential expression strength. These findings provide molecular evidence, at single-cell resolution, to support the hypothesis that the excitatory/inhibitory (E/I) neuronal expression ratios of sodium channels are important regulatory mechanisms underlying brain homeostasis and neurological diseases. Modulating the E/I expression balance within cell types of sodium channels could serve as a potential strategy to develop targeted treatment for NaV-associated neuronal developmental disorders.

Fragile X syndrome (FXS) is a leading monogenic cause of intellectual disability and autism spectrum disorders, spurring decades of intense research and a multitude of mouse models. So far, these models do not recapitulate the genetic underpinning of classical FXS—CGG repeat-induced methylation of theFmr1locus—and their findings have failed to translate into the clinic. We sought to answer whether this disparity was because of low repeat length and generated a novel mouse line with 341 repeats,Fmr1hs341, which is the largest allele in mice reported to date. This repeat length is significantly longer than the 200 repeats generally required for methylation of the repeat tract and promoter region in FXS patients, which leads to silencing of theFMR1gene. Bisulfite sequencing fails to detect the robust methylation expected of FXS inFmr1hs341mice. Quantitative real-time PCR and Western blotting results also do not resemble FXS and instead produce a biochemical profile consistent with the fragile X-associated premutation disorders. These findings suggest that repeat length is unlikely to be the core determinant preventing methylation in mice, and other organisms phylogenetically closer to humans may be required to effectively model FXS.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch–seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations., An examination of motor cortex in humans, marmosets and mice reveals a generally conserved cellular makeup that is likely to extend to many mammalian species, but also differences in gene expression, DNA methylation and chromatin state that lead to species-dependent specializations.

Connecting neural circuit output to behaviour can be facilitated by the precise chemical manipulation of specific cell populations1,2. Engineered receptors exclusively activated by designer small molecules enable manipulation of specific neural pathways3,4. However, their application to studies of behaviour has thus far been hampered by a trade-off between the low temporal resolution of systemic injection versus the invasiveness of implanted cannulae or infusion pumps2. Here, we developed a remotely controlled chemomagnetic modulation—a nanomaterials-based technique that permits the pharmacological interrogation of targeted neural populations in freely moving subjects. The heat dissipated by magnetic nanoparticles (MNPs) in the presence of alternating magnetic fields (AMFs) triggers small-molecule release from thermally sensitive lipid vesicles with a 20 s latency. Coupled with the chemogenetic activation of engineered receptors, this technique permits the control of specific neurons with temporal and spatial precision. The delivery of chemomagnetic particles to the ventral tegmental area (VTA) allows the remote modulation of motivated behaviour in mice. Furthermore, this chemomagnetic approach activates endogenous circuits by enabling the regulated release of receptor ligands. Applied to an endogenous dopamine receptor D1 (DRD1) agonist in the nucleus accumbens (NAc), a brain area involved in mediating social interactions, chemomagnetic modulation increases sociability in mice. By offering a temporally precise control of specified ligand–receptor interactions in neurons, this approach may facilitate molecular neuroscience studies in behaving organisms. Controlled delivery of neuromodulators in the brain might improve the understanding of the molecular basis of behaviour. In this letter, magnetic liposomes injected in deep brain regions release small molecules under remote magnetic stimulation, activating specific neuronal circuits in freely moving mice.

Neuropsychiatric disorders are often accompanied by cognitive impairments/intellectual disability (ID). It is not clear whether there are converging mechanisms underlying these debilitating impairments. We found that many autism and schizophrenia risk genes are expressed in the anterodorsal subdivision (AD) of anterior thalamic nuclei, which has reciprocal connectivity with learning and memory structures. CRISPR-Cas9 knockdown of multiple risk genes selectively in AD thalamus led to memory deficits. While the AD is necessary for contextual memory encoding, the neighboring anteroventral subdivision (AV) regulates memory specificity. These distinct functions of AD and AV are mediated through their projections to retrosplenial cortex, using differential mechanisms. Furthermore, knockdown of autism and schizophrenia risk genes PTCHD1, YWHAG, or HERC1 from AD led to neuronal hyperexcitability, and normalization of hyperexcitability rescued memory deficits in these models. This study identifies converging cellular to circuit mechanisms underlying cognitive deficits in a subset of neuropsychiatric disease models.

Genetic intervention is increasingly being explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies rely upon expressing a transgene in affected cells while minimizing off-target expression. Here we show organ-specific targeting of adeno-associated virus (AAV) capsids after intravenous delivery, which we achieved by employing a Cre-transgenic-based screening platform and sequential engineering of AAV-PHP.eB between the surface-exposed AA452 and AA460 of VP3. From this selection, we identified capsid variants that were enriched in the brain and targeted away from the liver in C57BL/6J mice. This tropism extends to marmoset (Callithrix jacchus), enabling robust, non-invasive gene delivery to the marmoset brain after intravenous administration. Notably, the capsids identified result in distinct transgene expression profiles within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood–brain barrier with neuronal specificity in rodents and non-human primates enables new avenues for basic research and therapeutic possibilities unattainable with naturally occurring serotypes.

CACNA1I, a schizophrenia risk gene, encodes a subtype of voltage-gated T-type calcium channel CaV3.3. We previously reported that a patient-derived missense de novo mutation (R1346H) of CACNA1I impaired CaV3.3 channel function. Here, we generated CaV3.3-RH knock-in animals, along with mice lacking CaV3.3, to investigate the biological impact of R1346H (RH) variation. We found that RH mutation altered cellular excitability in the thalamic reticular nucleus (TRN), where CaV3.3 is abundantly expressed. Moreover, RH mutation produced marked deficits in sleep spindle occurrence and morphology throughout non-rapid eye movement (NREM) sleep, while CaV3.3 haploinsufficiency gave rise to largely normal spindles. Therefore, mice harboring the RH mutation provide a patient derived genetic model not only to dissect the spindle biology but also to evaluate the effects of pharmacological reagents in normalizing sleep spindle deficits. Importantly, our analyses highlighted the significance of characterizing individual spindles and strengthen the inferences we can make across species over sleep spindles. In conclusion, this study established a translational link between a genetic allele and spindle deficits during NREM observed in schizophrenia patients, representing a key step toward testing the hypothesis that normalizing spindles may be beneficial for schizophrenia patients.

Background Deep brain stimulation (DBS) is an effective treatment for patients with obsessive-compulsive disorder (OCD) that do not respond to conventional therapies. Although the precise mechanism of action of DBS remains unknown, modulation of activity in corticofugal fibers originating in the prefrontal cortex is thought to underlie its beneficial effects in OCD. Methods To gain more mechanistic insight into DBS in OCD, we used Sapap3 mutant mice. These mice display excessive self-grooming and increased anxiety, both of which are responsive to therapeutic drugs used in OCD patients. We selected two clinically relevant DBS targets through which activity in prefronto-corticofugal fibers may be modulated: the internal capsule (IC) and the dorsal part of the ventral striatum (dVS). Results IC-DBS robustly decreased excessive grooming, whereas dVS-DBS was on average less effective. Grooming was reduced rapidly after IC-DBS onset and reinstated upon DBS offset. Only IC-DBS was associated with increased locomotion. DBS in both targets induced c-Fos expression around the electrode tip and in different regions of the prefrontal cortex. This prefronto-cortical activation was more extensive after IC-DBS, but not associated with behavioral effects. Furthermore, we found that the decline in grooming cannot be attributed to altered locomotor activity and that anxiety, measured on the elevated plus maze, was not affected by DBS. Conclusions DBS in both the IC and dVS reduces compulsive grooming in Sapap3 mutant mice. However, IC stimulation was more effective, but also produced motor activation, even though both DBS targets modulated activity in a similar set of prefrontal cortical fibers.

Recent success in identifying gene-regulatory elements in the context of recombinant adeno-associated virus vectors has enabled cell-type-restricted gene expression. However, within the cerebral cortex these tools are largely limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple new enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we discovered enhancers selective for parvalbumin (PV) and vasoactive intestinal peptide-expressing interneurons. Demonstrating the functional utility of these elements, we show that the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across vertebrate species, including humans. Finally, we demonstrate that our selection method is generalizable and characterizes additional PV-specific enhancers with exquisite specificity within distinct brain regions. Altogether, these viral tools can be used for cell-type-specific circuit manipulation and hold considerable promise for use in therapeutic interventions.

© 2019 Elsevier Inc. Autism spectrum disorder (ASD) is associated with noise hypersensitivity, the suboptimal extraction of meaningful signals in noisy environments. Because sensory filtering can involve distinct automatic and executive circuit mechanisms, however, developing circuit-specific therapeutic strategies for ASD noise hypersensitivity can be challenging. Here, we find that both of these processes are individually perturbed in one monogenic form of ASD, Ptchd1 deletion. Although Ptchd1 is preferentially expressed in the thalamic reticular nucleus during development, pharmacological rescue of thalamic perturbations in knockout (KO) mice only normalized automatic sensory filtering. By discovering a separate prefrontal perturbation in these animals and adopting a combinatorial pharmacological approach that also rescued its associated goal-directed noise filtering deficit, we achieved full normalization of noise hypersensitivity in this model. Overall, our work highlights the importance of identifying large-scale functional circuit architectures and utilizing them as access points for behavioral disease correction.

Somatosensory over-reactivity is common among patients with autism spectrum disorders (ASDs) and is hypothesized to contribute to core ASD behaviors. However, effective treatments for sensory over-reactivity and ASDs are lacking. We found distinct somatosensory neuron pathophysiological mechanisms underlie tactile abnormalities in different ASD mouse models and contribute to some ASD-related behaviors. Developmental loss of ASD-associated genes Shank3 or Mecp2 in peripheral mechanosensory neurons leads to region-specific brain abnormalities, revealing links between developmental somatosensory over-reactivity and the genesis of aberrant behaviors. Moreover, acute treatment with a peripherally restricted GABAA receptor agonist that acts directly on mechanosensory neurons reduced tactile over-reactivity in six distinct ASD models. Chronic treatment of Mecp2 and Shank3 mutant mice improved body condition, some brain abnormalities, anxiety-like behaviors, and some social impairments but not memory impairments, motor deficits, or overgrooming. Our findings reveal a potential therapeutic strategy targeting peripheral mechanosensory neurons to treat tactile over-reactivity and select ASD-related behaviors. Treatment with a peripherally restricted GABAA receptor agonist in multiple distinct autism spectrum disorder mouse models reveals a potential therapeutic strategy for select ASD-related behaviors., National Institutes of Health (U.S.) (Grants F31 MH098641A, R01 MH097104), National Institute of Mental Health (U.S.) (Conte Center Grant P50 MH094271)

Dopaminergic and serotonergic neurons modulate and control processes ranging from reward signaling to regulation of motor outputs. Further, dysfunction of these neurons is involved in both degenerative and psychiatric disorders. Elucidating the roles of these neurons has been greatly facilitated by bacterial artificial chromosome (BAC) transgenic mouse lines expressing channelrhodopsin to readily enable cell-type specific activation. However, corresponding lines to silence these monoaminergic neurons have been lacking. We have generated two BAC transgenic mouse lines expressing the outward proton pump, enhanced ArchT3.0 (eArchT3.0), and GFP under control of the regulatory elements of either the dopamine transporter (DAT; Jax# 031663) or the tryptophan hydroxylase 2 (TPH2; Jax# 031662) gene locus. We demonstrate highly faithful and specific expression of these lines in dopaminergic and serotonergic neurons respectively. Additionally we validate effective and sensitive eArchT3.0-mediated silencing of these neurons using slice electrophysiology as well as with a well-established behavioral assay. These new transgenic tools will help expedite the study of dopaminergic and serotonergic system function in normal behavior and disease.

Visual Abstract, In the central and peripheral nervous systems, the myelin sheath promotes neuronal signal transduction. The thickness of the myelin sheath changes during development and in disease conditions like multiple sclerosis. Such changes are routinely detected using electron microscopy through g-ratio quantification. While g-ratio is one of the most critical measurements in myelin studies, a major drawback is that g-ratio quantification is extremely laborious and time-consuming. Here, we report the development and validation of MyelTracer, an installable, stand-alone software for semi-automated g-ratio quantification based on the Open Computer Vision Library (OpenCV). Compared with manual g-ratio quantification, using MyelTracer produces consistent results across multiple tissues and animal ages, as well as in remyelination after optic nerve crush, and reduces total quantification time by 40–60%. With g-ratio measurements via MyelTracer, a known hypomyelination phenotype can be detected in a Williams syndrome mouse model. MyelTracer is easy to use and freely available for Windows and Mac OS X (https://github.com/HarrisonAllen/MyelTracer).

Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Cavα2δ1 subunit (Cavα2δ1), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Cavα2δ1 in the spinal cord in vivo and that TSP4/Cavα2δ1-dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/Cavα2δ1-dependent processes lead to increased frequency of miniature and amplitude of evoked excitatory post-synaptic currents in second-order neurons as well as increased VGlut2- and PSD95-positive puncta, indicative of increased excitatory synapses. Blockade of TSP4/Cavα2δ1-dependent processes with Cavα2δ1 ligand gabapentin or genetic Cavα2δ1 knockdown blocks TSP4 induced nociception and its pathological correlates. Conversely, TSP4 antibodies or genetic ablation blocks nociception and changes in synaptic transmission in mice overexpressing Cavα2δ1. Importantly, TSP4/Cavα2δ1-dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Cavα2δ1-dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated presynaptic excitatory input and evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development.

© 2020 Elsevier Inc. Optogenetics is among the most widely employed techniques to manipulate neuronal activity. However, a major drawback is the need for invasive implantation of optical fibers. To develop a minimally invasive optogenetic method that overcomes this challenge, we engineered a new step-function opsin with ultra-high light sensitivity (SOUL). We show that SOUL can activate neurons located in deep mouse brain regions via transcranial optical stimulation and elicit behavioral changes in SOUL knock-in mice. Moreover, SOUL can be used to modulate neuronal spiking and induce oscillations reversibly in macaque cortex via optical stimulation from outside the dura. By enabling external light delivery, our new opsin offers a minimally invasive tool for manipulating neuronal activity in rodent and primate models with fewer limitations on the depth and size of target brain regions and may further facilitate the development of minimally invasive optogenetic tools for the treatment of neurological disorders.

Background: Obsessive–compulsive disorder (OCD) is a common psychiatric disorder that affects about 2% of the population, but the underlying neuropathophysiology of OCD is not well understood. Although increasing lines of evidence implicate dysfunction of the orbitofrontal cortex (OFC) in OCD, a detailed understanding of the functional alterations in different neuronal types in the OFC is still elusive. Methods: We investigated detailed activity pattern changes in putative pyramidal neurons and interneurons, as well as local field potential oscillations, in the lateral OFC underlying OCD-relevant phenotypes. We applied in vivo multichannel recording in an awake OCD mouse model that carried a deletion of the Sapap3 gene, and in wild type littermates. Results: Compared with wild type mice, the lateral OFC of Sapap3 knockout mice exhibited network dysfunction, demonstrated by decreased power of local field potential oscillations. The activity of inhibitory and excitatory neurons in the lateral OFC showed distinct perturbations in Sapap3 knockout mice: putative interneurons exhibited increased activity; putative pyramidal neurons exhibited enhanced bursting activity; and both putative pyramidal neurons and interneurons exhibited enhanced discharge variability and altered synchronization. Limitations To exclude motor activity confounders, this study examined functional alterations in lateral OFC neurons only when the mice were stationary. Conclusion: We provide, to our knowledge, the first direct in vivo electrophysiological evidence of detailed functional alterations in different neuronal types in the lateral OFC of an OCD mouse model. These findings may help in understanding the underlying neuropathophysiology and circuitry mechanisms for phenotypes relevant to OCD, and may help generate and refine hypotheses about potential biomarkers for further investigation.

Innate defensive responses are essential for animal survival and are conserved across species. The ventral tegmental area (VTA) plays important roles in learned appetitive and aversive behaviors, but whether it plays a role in mediating or modulating innate defensive responses is currently unknown. We report that VTAGABA+ neurons respond to a looming stimulus. Inhibition of VTAGABA+ neurons reduced looming-evoked defensive flight behavior, and photoactivation of these neurons resulted in defense-like flight behavior. Using viral tracing and electrophysiological recordings, we show that VTAGABA+ neurons receive direct excitatory inputs from the superior colliculus (SC). Furthermore, we show that glutamatergic SC-VTA projections synapse onto VTAGABA+ neurons that project to the central nucleus of the amygdala (CeA) and that the CeA is involved in mediating the defensive behavior. Our findings demonstrate that aerial threat-related visual information is relayed to VTAGABA+ neurons mediating innate behavioral responses, suggesting a more general role of the VTA. Keywords: Ventral tegmental area; GABAergic neurons; innate fear; superior collic-55ulus; looming; defensive responses.

Hyper-reactivity to sensory input is a common and debilitating symptom in individuals with autism spectrum disorders (ASD), but the neural basis underlying sensory abnormality is not completely understood. Here we examined the neural representations of sensory perception in the neocortex of a Shank3B−/− mouse model of ASD. Male and female Shank3B−/− mice were more sensitive to relatively weak tactile stimulation in a vibrissa motion detection task. In vivo population calcium imaging in vibrissa primary somatosensory cortex (vS1) revealed increased spontaneous and stimulus-evoked firing in pyramidal neurons but reduced activity in interneurons. Preferential deletion of Shank3 in vS1 inhibitory interneurons led to pyramidal neuron hyperactivity and increased stimulus sensitivity in the vibrissa motion detection task. These findings provide evidence that cortical GABAergic interneuron dysfunction plays a key role in sensory hyper-reactivity in a Shank3 mouse model of ASD and identify a potential cellular target for exploring therapeutic interventions., National Institutes of Health (Grant R01MH097104), NIMH (Grants P50MH094271, F32MH100749 and R01NS045130)

The dystrophin glycoprotein complex (DGC) is critical for muscle stability, and mutations in DGC proteins lead to muscular dystrophy. The DGC also contributes to the maturation and maintenance of the neuromuscular junction (NMJ). The gene encoding the DGC protein alpha-dystrobrevin undergoes alternative splicing to produce at least five known isoforms. Isoform-specific antibody staining and reverse transcription PCR in mutant mice with a deletion of exon 3 of the alpha-dystrobrevin gene suggested the existence of a remaining synaptic isoform, which might be compensating for alpha-dystrobrevin function. To test this possibility and to more completely understand the synaptic function of alpha-dystrobrevin, we used a two-step homologous recombination strategy combined with in vivo Cre-mediated excision to generate mice with a large deletion of the alpha-dystrobrevin gene to disrupt all isoforms. However, these mice did not exhibit a more severe NMJ phenotype than that observed in the exon 3-deleted mice. Nonetheless, these mice not only eliminate possible compensation by remaining isoforms of alpha-dystrobrevin, but also offer a conditional allele that could be used to identify tissue-specific and developmental functions of alpha-dystrobrevin. This work also demonstrates a successful strategy to achieve deletion of a large genomic sequence, which can be a valuable tool for functional studies of genes encoding multiple isoforms that span a large genomic region.

Social deficit is a core clinical feature of autism spectrum disorder (ASD) but the underlying neural mechanisms remain largely unclear. We demonstrate that structural and functional impairments occur in glutamatergic synapses in the pyramidal neurons of the anterior cingulate cortex (ACC) in mice with a mutation in Shank3, a high-confidence candidate ASD gene. Conditional knockout of Shank3 in the ACC was sufficient to generate excitatory synaptic dysfunction and social interaction deficits, whereas selective enhancement of ACC activity, restoration of SHANK3 expression in the ACC, or systemic administration of an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-positive modulator improved social behavior in Shank3 mutant mice. Our findings provide direct evidence for the notion that the ACC has a role in the regulation of social behavior in mice and indicate that ACC dysfunction may be involved in social impairments in ASD.

Numerous risk genes have recently been implicated in susceptibility to autism and schizophrenia. Translating such genetic findings into disease-relevant neurobiological mechanisms is challenging due to the lack of throughput assays that can be used to assess their functions on an appropriate scale. To address this issue, we explored the feasibility of using a micro-electrode array (MEA) as a potentially scalable assay to identify the electrical network phenotypes associated with risk genes. We first characterized local and global network firing in cortical neurons with MEAs, and then developed methods to analyze the alternation between the network active period (NAP) and the network inactive period (NIP), each of which lasts tens of seconds. We then evaluated the electric phenotypes of neurons derived from Shank3 knockout (KO) mice. Cortical neurons cultured on MEAs displayed a rich repertoire of spontaneous firing, and Shank3 deletion led to reduced firing activity. Enhancing excitation with CX546 rescued the deficit in the spike rate in the Shank3 KO network. In addition, the Shank3 KO network produced a shorter NIP, and this altered network firing pattern was normalized by clonazepam, a positive modulator of the GABAA receptor. MEA recordings revealed electric phenotypes that displayed altered excitation and inhibition in the network lacking Shank3. Thus, our study highlights MEAs as an experimental framework for measuring multiple robust neurobiological end points in dynamic networks and as an assay system that could be used to identify electric phenotypes in cultured neuronal networks and to analyze additional risk genes identified in psychiatric genetics.

Summary: The central mechanisms controlling glucose and lipid homeostasis are inadequately understood. We show that α2δ-1 is an essential regulator of glucose and lipid balance, acting in steroidogenic factor-1 (SF1) neurons of the ventromedial hypothalamus (VMH). These effects are body weight independent and involve regulation of SF1+ neuronal activity and sympathetic output to metabolic tissues. Accordingly, mice with α2δ-1 deletion in SF1 neurons exhibit glucose intolerance, altered lipolysis, and decreased cholesterol content in adipose tissue despite normal energy balance regulation. Profound reductions in the firing rate of SF1 neurons, decreased sympathetic output, and elevated circulating levels of serotonin are associated with these alterations. Normal calcium currents but reduced excitatory postsynaptic currents in mutant SF1 neurons implicate α2δ-1 in the promotion of excitatory synaptogenesis separate from its canonical role as a calcium channel subunit. Collectively, these findings identify an essential mechanism that regulates VMH neuronal activity and glycemic and lipid control and may be a target for tackling metabolic disease. : Felsted et al. show a required role of the calcium channel subunit and thrombospondin receptor α2δ-1 in regulating glucose and lipid homeostasis in the ventromedial hypothalamus (VMH). These effects are caused by regulation of SF1+ neuronal activity in the VMH through non-canonical mechanisms and concomitant influences on sympathetic output. Keywords: diabetes, VMH, hypothalamus, glucose, norepinephrine, serotonin, excitability, lipid, SF1

Mutation in the SHANK3 human gene leads to different neuropsychiatric diseases including Autism Spectrum Disorder (ASD), intellectual disabilities and Phelan-McDermid syndrome. Shank3 disruption in mice leads to dysfunction of synaptic transmission, behavior, and development. Protein S-nitrosylation, the nitric oxide (NO•)-mediated posttranslational modification (PTM) of cysteine thiols (SNO), modulates the activity of proteins that regulate key signaling pathways. We tested the hypothesis that Shank3 mutation would generate downstream effects on PTM of critical proteins that lead to modification of synaptic functions. SNO-proteins in two ASD-related brain regions, cortex and striatum of young and adult InsG3680(+/+) mice (a human mutation-based Shank3 mouse model), were identified by an innovative mass spectrometric method, SNOTRAP. We found changes of the SNO-proteome in the mutant compared to WT in both ages. Pathway analysis showed enrichment of processes affected in ASD. SNO-Calcineurin in mutant led to a significant increase of phosphorylated Synapsin1 and CREB, which affect synaptic vesicle mobilization and gene transcription, respectively. A significant increase of 3-nitrotyrosine was found in the cortical regions of the adult mutant, signaling both oxidative and nitrosative stress. Neuronal NO• Synthase (nNOS) was examined for levels and localization in neurons and no significant difference was found in WT vs. mutant. S-nitrosoglutathione concentrations were higher in mutant mice compared to WT. This is the first study on NO•-related molecular changes and SNO-signaling in the brain of an ASD mouse model that allows the characterization and identification of key proteins, cellular pathways, and neurobiological mechanisms that might be affected in ASD., Army Research Office Institute for Collaborative Biotechnologies (Grant W911NF-09-0001)

Autism spectrum disorder (ASD) is a heterogeneous disease, but genetically defined models can provide an entry point to studying the molecular underpinnings of this disorder. We generated germline mutant mice with loss-of-function mutations in Chd8, a de novo mutation strongly associated with ASD, and demonstrate that these mice display hallmark ASD behaviors, macrocephaly, and craniofacial abnormalities similar to patient phenotypes. Chd8[superscript +/–] mice display a broad, brain-region-specific dysregulation of major regulatory and cellular processes, most notably histone and chromatin modification, mRNA and protein processing, Wnt signaling, and cell-cycle regulation. We also find altered synaptic physiology in medium spiny neurons of the nucleus accumbens. Perturbation of Chd8 in adult mice recapitulates improved acquired motor learning behavior found in Chd8[superscript +/–] animals, suggesting a role for CHD8 in adult striatal circuits. These results support a mechanism linking chromatin modification to striatal dysfunction and the molecular pathology of ASD., National Science Foundation (U.S.) (1122374), National Science Foundation (U.S.) (2013169249), National Institute of Mental Health (U.S.) (F31-MH111157), Howard Hughes Medical Institute (NS046789), Simons Foundation Autism Research Initiative (306063), Simons Foundation Autism Research Initiative (6927482), National Institute of Mental Health (U.S.) (5DP1-MH100706), National Institute of Mental Health (U.S.) (1R01-MH110049), Nancy Lurie Marks Family Foundation (6928117)

We used high-speed optogenetic mapping technology to examine the spatial organization of local inhibitory circuits formed by cerebellar interneurons. Transgenic mice expressing channelrhodopsin-2 exclusively in molecular layer interneurons allowed us to focally photostimulate these neurons, while measuring resulting responses in postsynaptic Purkinje cells. This approach revealed that interneurons converge upon Purkinje cells over a broad area and that at least seven interneurons form functional synapses with a single Purkinje cell. The number of converging interneurons was reduced by treatment with gap junction blockers, revealing that electrical synapses between interneurons contribute substantially to the spatial convergence. Remarkably, gap junction blockers affected convergence in sagittal slices, but not in coronal slices, indicating a sagittal bias in electrical coupling between interneurons. We conclude that electrical synapse networks spatially coordinate interneurons in the cerebellum and may also serve this function in other brain regions., Singapore. National Research Foundation (CRP Grant), Korea (South). Ministry of Education, Science and Technology (National Research Foundation of Korea Grant WCI 2009-003)

The basal forebrain provides the primary source of cholinergic input to the cortex, and it has a crucial function in promoting wakefulness and arousal. However, whether rapid changes in basal forebrain neuron spiking in awake animals can dynamically influence sensory perception is unclear. Here we show that basal forebrain cholinergic neurons rapidly regulate cortical activity and visual perception in awake, behaving mice. Optogenetic activation of the cholinergic neurons or their V1 axon terminals improved performance of a visual discrimination task on a trial-by-trial basis. In V1, basal forebrain activation enhanced visual responses and desynchronized neuronal spiking; these changes could partly account for the behavioral improvement. Conversely, optogenetic basal forebrain inactivation decreased behavioral performance, synchronized cortical activity and impaired visual responses, indicating the importance of cholinergic activity in normal visual processing. These results underscore the causal role of basal forebrain cholinergic neurons in fast, bidirectional modulation of cortical processing and sensory perception.

Molecular genetic tools have had a profound impact on neuroscience, but until recently their application has largely been confined to a few model species, most notably mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. With the development of new genome engineering technologies such as CRISPR, it is becoming increasingly feasible to apply these molecular tools in a wider range of species, including nonhuman primates. This will lead to many opportunities for brain research, but it will also pose challenges. Here we identify some of these opportunities and challenges in light of recent and foreseeable technological advances and offer some suggestions. Our main focus is on the creation of new primate disease models for understanding the pathological mechanisms of brain disorders and for developing new approaches to effective treatment. However, we also emphasize that primate genetic models have great potential to address many fundamental questions about brain function, providing an essential foundation for future progress in disease research.

Multiple hypothalamic neuronal populations that regulate energy balance have been identified. Although hypothalamic glia exist in abundance and form intimate structural connections with neurons, their roles in energy homeostasis are less known. Here we show that selective Ca[superscript 2+] activation of glia in the mouse arcuate nucleus (ARC) reversibly induces increased food intake while disruption of Ca[superscript 2+] signaling pathway in ARC glia reduces food intake. The specific activation of ARC glia enhances the activity of agouti-related protein/neuropeptide Y (AgRP/NPY)-expressing neurons but induces no net response in pro-opiomelanocortin (POMC)-expressing neurons. ARC glial activation non-specifically depolarizes both AgRP/NPY and POMC neurons but a strong inhibitory input to POMC neurons balances the excitation. When AgRP/NPY neurons are inactivated, ARC glial activation fails to evoke any significant changes in food intake. Collectively, these results reveal an important role of ARC glia in the regulation of energy homeostasis through its interaction with distinct neuronal subtype-specific pathways., Massachusetts Institute of Technology. Poitras Center for Affective Disorders Research, Singapore. Agency for Science, Technology and Research, National Institutes of Health (U.S.) (R01EY007023), National Institutes of Health (U.S.) (R01EY018648), National Institutes of Health (U.S.) (U01NS090473), National Science Foundation (U.S.) (EF1451125), Simons Foundation, Massachusetts Institute of Technology. Simons Center for the Social Brain, Autism Science Foundation

Alzheimer's disease (AD) is the most common cause of dementia among older people. There are no effective medications currently available to prevent and treat AD and halt disease progression. Monoacylglycerol lipase (MAGL) is the primary enzyme metabolizing the endocannabinoid 2-arachidonoylglycerol in the brain. We show here that inactivation of MAGL robustly suppressed production and accumulation of β-amyloid (Aβ) associated with reduced expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) in a mouse model of AD. MAGL inhibition also prevented neuroinflammation, decreased neurodegeneration, maintained integrity of hippocampal synaptic structure and function, and improved long-term synaptic plasticity, spatial learning, and memory in AD animals. Although the molecular mechanisms underlying the beneficial effects produced by MAGL inhibition remain to be determined, our results suggest that MAGL, which regulates endocannabinoid and prostaglandin signaling, contributes to pathogenesis and neuropathology of AD, and thus is a promising therapeutic target for the prevention and treatment of AD., National Institutes of Health (U.S.) (NIH grant R01NS076815), National Institutes of Health (U.S.) (NIH Grant R01NS054886), National Institutes of Health (U.S.) (NIH Grant R21AG039669)

The precise causal factors for obsessive–compulsive disorder (OCD) are not known, although, decades of research have honed in on the cortico-striatal-thalamo-cortical (CSTC) circuitry in the brain as a critical pathway involved in obsessions and the intimately linked compulsive–repetitive behaviors. Recent progress in human and mouse genetics have led to the identification of novel candidate susceptibility genes, which in turn have facilitated a more focused approach to unraveling the nature of circuitry dysfunction in OCD. The ability to perform invasive techniques in genetic animal models of OCD will be crucial for rapid advances in this field, and as such we review the most recent developments and highlight the importance of searching out common circuitry defects underlying compulsive–repetitive behaviors., National Institutes of Health (U.S.) (Grant NIMH R01MH081201), Hartwell Foundation, Simons Foundation. Autism Research Initiative, Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard. SPARC Program, Brain & Behavior Research Foundation (Young Investigator Award), National Institute of Mental Health (U.S.) (Ruth L. Kirschstein National Research Service Award F32MH084460)

A formidable challenge in neural repair in the adult central nervous system (CNS) is the long distances that regenerating axons often need to travel in order to reconnect with their targets. Thus, a sustained capacity for axon regeneration is critical for achieving functional restoration. Although deletion of either Phosphatase and tensin homolog (PTEN), a negative regulator of mammalian target of rapamycin (mTOR), or suppressor of cytokine signaling 3 (SOCS3), a negative regulator of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, in adult retinal ganglion cells (RGCs) individually promoted significant optic nerve regeneration, such regrowth tapered off around two weeks after the crush injury1,2. Remarkably, we now find that simultaneous deletion of both PTEN and SOCS3 enables robust and sustained axon regeneration. We further show that PTEN and SOCS3 regulate two independent pathways that act synergistically to promote enhanced axon regeneration. Gene expression analyses suggest that double deletion not only results in the induction of many growth-related genes, but also allows RGCs to maintain the expression of a repertoire of genes at the physiological level after injury. Our results reveal concurrent activation of mTOR and STAT3 pathways as a key for sustaining long-distance axon regeneration in adult CNS, a crucial step toward functional recovery.

Autism spectrum disorders (ASDs) comprise a range of disorders that share a core of neurobehavioural deficits characterized by widespread abnormalities in social interactions, deficits in communication as well as restricted interests and repetitive behaviours. The neurological basis and circuitry mechanisms underlying these abnormal behaviours are poorly understood. SHANK3 is a postsynaptic protein, whose disruption at the genetic level is thought to be responsible for the development of 22q13 deletion syndrome (Phelan–McDermid syndrome) and other non-syndromic ASDs. Here we show that mice with Shank3 gene deletions exhibit self-injurious repetitive grooming and deficits in social interaction. Cellular, electrophysiological and biochemical analyses uncovered defects at striatal synapses and cortico-striatal circuits in Shank3 mutant mice. Our findings demonstrate a critical role for SHANK3 in the normal development of neuronal connectivity and establish causality between a disruption in the Shank3 gene and the genesis of autistic-like behaviours in mice., National Institute of Mental Health (U.S.) (NIMH/NIH (R01MH081201)), Hartwell Foundation (Hartwell Individual Biomedical Research Award), Simons Foundation (Autism Research Initiative (SFARI) grant Award), Brain and Behavior Research Foundation (NARSAD Young Investigator Award), National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award (F32MH084460)), National Institutes of Health (U.S.) (NIH grant (R03MH085224)), Fundação para a Ciência e a Tecnologia (SFRH/BD/15231/2004), Fundação para a Ciência e a Tecnologia (SFRH/BD/15855/2005), Instituto Gulbenkian de Ciência (“Programa Gulbenkian de Doutoramento em Biomedicina” (PGDB, Oeiras, Portugal)), University of Coimbra. Center for Neuroscience and Cell Biology (“Programa Doutoral em Biologia Experimental e Biomedicina” (CNC, Coimbra, Portugal))

SummaryAcetylcholine is an important neurotransmitter, and the habenulo-interpeduncular projection is a major cholinergic pathway in the brain. To study the physiological properties of cholinergic transmission in the interpeduncular nucleus (IPN), we used a transgenic mouse line in which the light-gated cation channel ChannelRhodopsin-2 is selectively expressed in cholinergic neurons. Cholinergic axonal terminals were activated by light pulses, and postsynaptic responses were recorded from IPN neurons. Surprisingly, brief photostimulation produces fast excitatory postsynaptic currents that are mediated by ionotropic glutamate receptors, suggesting wired transmission of glutamate. By contrast, tetanic photostimulation generates slow inward currents that are largely mediated by nicotinic acetylcholine receptors, suggesting volume transmission of acetylcholine. Finally, vesicular transporters for glutamate and acetylcholine are coexpressed on the same axonal terminals in the IPN. These results strongly suggest that adult brain “cholinergic” neurons can corelease glutamate and acetylcholine, but these two neurotransmitters activate postsynaptic neurons via different transmission modes.

Summary Neuroligins (NLs) are critical for synapse formation and function. NL3 R451C is an autism-associated mutation. NL3 R451C knockin (KI) mice exhibit autistic behavioral abnormalities, including social novelty deficits. However, neither the brain regions involved in social novelty nor the underlying mechanisms are clearly understood. Here, we found decreased excitability of fast-spiking interneurons and dysfunction of gamma oscillation in the medial prefrontal cortex (mPFC), which contributed to the social novelty deficit in the KI mice. Neuronal firing rates and phase-coding abnormalities were also detected in the KI mice during social interactions. Interestingly, optogenetic stimulation of parvalbumin interneurons in the mPFC at 40 Hz nested at 8 Hz positively modulated the social behaviors of mice and rescued the social novelty deficit in the KI mice. Our findings suggest that gamma oscillation dysfunction in the mPFC leads to social deficits in autism, and manipulating mPFC PV interneurons may reverse the deficits in adulthood.

In many parts of the vertebrate nervous system, synaptic connections are remodeled during early postnatal life. Neural activity plays an important role in regulating one such rearrangement, synapse elimination, in the developing neuromuscular system, but there is little direct evidence on roles of pre- or postsynaptic activity in regulating synapse elimination in the developing brain. To address this issue, we expressed a chloride channel-yellow fluorescent protein fusion in cerebellar Purkinje cells (PCs) of transgenic mice to decrease their excitability. We then assessed elimination of supernumerary climbing fiber inputs to PCs. Individual PCs are innervated by multiple climbing fibers at birth; all but one are eliminated during the first three postnatal weeks in wild-type mice, but multiple innervation persists for at least three months in the transgenic mice. The normal redistribution of climbing fiber synapses from PC somata to proximal dendrites was also blunted in transgenics. These results show that normal electrical activity of the postsynaptic cell is required for it to attain a mature innervation pattern.

The ability to control and manipulate neuronal activity within an intact mammalian brain is of key importance for mapping functional connectivity and for dissecting the neural circuitry underlying behaviors. We have previously generated transgenic mice that express channelrhodopsin-2 for light-induced activation of neurons and mapping of neural circuits. Here we describe transgenic mice that express halorhodopsin (NpHR), a light-driven chloride pump, that can be used to silence neuronal activity via light. Using the Thy-1 promoter to target NpHR expression to neurons, we found that neurons in these mice expressed high levels of NpHR-YFP and that illumination of cortical pyramidal neurons expressing NpHR-YFP led to rapid, reversible photoinhibition of action potential firing in these cells. However, NpHR-YFP expression led to the formation of numerous intracellular blebs, which may disrupt neuronal function. Labeling of various subcellular markers indicated that the blebs arise from retention of NpHR-YFP in the endoplasmic reticulum. By improving the signal peptide sequence and adding an ER export signal to NpHR-YFP, we eliminated the formation of blebs and dramatically increased the membrane expression of NpHR-YFP. Thus, the improved version of NpHR should serve as an excellent tool for neuronal silencing in vitro and in vivo.

The ubiquitin–proteasome pathway has been implicated in synaptic development and plasticity. However, mechanisms by which ubiquitination contributes to precise and dynamic control of synaptic development and plasticity are poorly understood. We have identified a PDZ domain containing RING finger 3 (PDZRN3) as a synapse-associated E3 ubiquitin ligase and have demonstrated that it regulates the surface expression of muscle-specific receptor tyrosine kinase (MuSK), the key organizer of postsynaptic development at the mammalian neuromuscular junction. PDZRN3 binds to MuSK and promotes its ubiquitination. Regulation of cell surface levels of MuSK by PDZRN3 requires the ubiquitin ligase domain and is mediated by accelerated endocytosis. Gain- and loss-of-function studies in cultured myotubes show that regulation of MuSK by PDZRN3 plays an important role in MuSK-mediated nicotinic acetylcholine receptor clustering. Furthermore, overexpression of PDZRN3 in skeletal muscle of transgenic mice perturbs the growth and maturation of the neuromuscular junction. These results identify a synapse-associated E3 ubiquitin ligase as an important regulator of MuSK signaling.

To permit rapid optical control of brain activity, we have engineered multiple lines of transgenic mice that express the light-activated cation channel Channelrhodopsin-2 (ChR2) in subsets of neurons. Illumination of ChR2-positive neurons in brain slices produced photocurrents that generated action potentials within milliseconds and with precisely timed latencies. The number of light-evoked action potentials could be controlled by varying either the amplitude or duration of illumination. Furthermore, the frequency of light-evoked action potentials could be precisely controlled up to 30 Hz. Photostimulation also could evoke synaptic transmission between neurons, and, by scanning with a small laser light spot, we were able to map the spatial distribution of synaptic circuits connecting neurons within living cerebral cortex. We conclude that ChR2 is a genetically based photostimulation technology that permits analysis of neural circuits with high spatial and temporal resolution in transgenic mammals.

Tonic inhibition was imaged in cerebellar granule cells of transgenic mice expressing the optogenetic chloride indicator, Clomeleon. Blockade of GABA A receptors substantially reduced chloride concentration in granule cells due to block of tonic inhibition. This indicates that tonic inhibition is a significant contributor to the resting chloride concentration of these cells. Tonic inhibition was observed not only in granule cell bodies, but also in their axons, the parallel fibers (PFs). This presynaptic tonic inhibition could be observed in slices both at room and physiological temperatures, as well as in vivo, and has many of the same properties as tonic inhibition measured in granule cell bodies. GABA application revealed that PFs possess at least two types of GABA A receptor: one high-affinity receptor that is activated by ambient GABA and causes a chloride influx that mediates tonic inhibition, and a second with a low affinity for GABA that causes a chloride efflux that excites PFs. Presynaptic tonic inhibition regulates glutamate release from PFs because GABA A receptor blockade enhanced both the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs at the PF-Purkinje cell synapse. We conclude that tonic inhibition of PFs could play an important role in regulating information flow though cerebellar synaptic circuits. Such cross talk between phasic and tonic signaling could be a general mechanism for fine tuning of synaptic circuits., National Science Foundation (U.S.) (Grant 1512826), National Science Foundation (U.S.) (Grant MH106013)

Temporal lobe epilepsy (TLE) is one of the most common drug-resistant forms of epilepsy in adults and usually originates in the hippocampal formations. However, both the network mechanisms that support the seizure spread and the exact directions of ictal propagation remain largely unknown. Here we report the dissection of ictal propagation in the hippocampal–entorhinal cortex (HP–EC) structures using optogenetic methods in multiple brain regions of a kainic acid-induced model of TLE in VGAT-ChR2 transgenic mice. We perform highly temporally precise cross-area analyses of epileptic neuronal networks and find a feed-forward propagation pathway of ictal discharges from the dentate gyrus/hilus (DGH) to the medial entorhinal cortex, instead of a re-entrant loop. We also demonstrate that activating DGH GABAergic interneurons can significantly inhibit the spread of ictal seizures and largely rescue behavioural deficits in kainate-exposed animals. These findings may shed light on future therapeutic treatments of TLE., The network mechanism supporting seizure spread in temporal lobe epilepsy (TLE) is only partially understood. Using optogenetic methods, Lu et al. identify a feed-forward propagation pathway of ictal discharges from the dentate gyrus/hilus to the medial entorhinal cortex in a mouse model of TLE.

Developmental disabilities, including attention-deficit hyperactivity disorder (ADHD), intellectual disability (ID), and autism spectrum disorders (ASD), affect one in six children in the USA. Recently, gene mutations in patched domain containing 1 (PTCHD1) have been found in ∼1% of patients with ID and ASD. Individuals with PTCHD1 deletion show symptoms of ADHD, sleep disruption, hypotonia, aggression, ASD, and ID. Although PTCHD1 is probably critical for normal development, the connection between its deletion and the ensuing behavioural defects is poorly understood. Here we report that during early post-natal development, mouse Ptchd1 is selectively expressed in the thalamic reticular nucleus (TRN), a group of GABAergic neurons that regulate thalamocortical transmission, sleep rhythms, and attention. Ptchd1 deletion attenuates TRN activity through mechanisms involving small conductance calcium-dependent potassium currents (SK). TRN-restricted deletion of Ptchd1 leads to attention deficits and hyperactivity, both of which are rescued by pharmacological augmentation of SK channel activity. Global Ptchd1 deletion recapitulates learning impairment, hyper-aggression, and motor defects, all of which are insensitive to SK pharmacological targeting and not found in the TRN-restricted deletion mouse. This study maps clinically relevant behavioural phenotypes onto TRN dysfunction in a human disease model, while also identifying molecular and circuit targets for intervention., Simons Foundation Autism Research Initiative (Grant 307913), National Institutes of Health (U.S.) (Grant R01MH097104), National Institute of Mental Health (U.S.) (Grant R01MH097104), National Institutes of Health (U.S.) (Grant R01MH107680)

One of the great strengths of the mouse model is the wide array of genetic tools that have been developed. Striking examples include methods for directed modification of the genome, and for regulated expression or inactivation of genes. Within neuroscience, it is now routine to express reporter genes, neuronal activity indicators, and opsins in specific neuronal types in the mouse. However, there are considerable anatomical, physiological, cognitive, and behavioral differences between the mouse and the human that, in some areas of inquiry, limit the degree to which insights derived from the mouse can be applied to understanding human neurobiology. Several recent advances have now brought into reach the goal of applying these tools to understanding the primate brain. Here we describe these advances, consider their potential to advance our understanding of the human brain and brain disorders, discuss bioethical considerations, and describe what will be needed to move forward., Poitras Center for Affective Disorders Research, Stanley Center for Psychiatric Research, Brain Research Foundation (Science Innovation Award)

Because autism spectrum disorders are neurodevelopmental disorders and patients typically display symptoms before the age of three, one of the key questions in autism research is whether the pathology is reversible in adults. Here we investigate the developmental requirement of Shank3 in mice, a prominent monogenic autism gene that is estimated to contribute to approximately 1% of all autism spectrum disorder cases. SHANK3 is a postsynaptic scaffold protein that regulates synaptic development, function and plasticity by orchestrating the assembly of post synaptic density macromolecular signalling complex. Disruptions of the Shank3 gene in mouse models have resulted in synaptic defects and autistic-like behaviours including anxiety, social interaction deficits, and repetitive behaviour. We generated a novel Shank3 conditional knock-in mouse model, and show that re-expression of the Shank3 gene in adult mice led to improvements in synaptic protein composition, spine density and neural function in the striatum. We also provide behavioural evidence that certain behavioural abnormalities including social interaction deficit and repetitive grooming behaviour could be rescued, while anxiety and motor coordination deficit could not be recovered in adulthood. Together, these results reveal the profound effect of post-developmental activation of Shank3 expression on neural function, and demonstrate a certain degree of continued plasticity in the adult diseased brain., National Institutes of Health (U.S.) (Grant R01MH097104)

We describe here a molecular genetic approach for imaging synaptic inhibition. The thy-1 promoter was used to express high levels of Clomeleon, a ratiometric fluorescent indicator for chloride ions, in discrete populations of neurons in the brains of transgenic mice. Clomeleon was functional after chronic expression and provided non-invasive readouts of intracellular chloride concentration ([Cl(-)](i)) in brain slices, allowing us to quantify age-dependent declines in resting [Cl(-)](i) during neuronal development. Activation of hippocampal interneurons caused [Cl(-)](i) to rise transiently in individual postsynaptic pyramidal neurons. [Cl(-)](i) increased in direct proportion to the amount of inhibitory transmission, with peak changes as large as 4 mM. Integrating responses over populations of pyramidal neurons allowed sensitive detection of synaptic inhibition. Thus, Clomeleon imaging permits non-invasive, spatiotemporally resolved recordings of [Cl(-)](i) in a large variety of neurons, opening up new opportunities for imaging synaptic inhibition and other forms of chloride signaling.

Mechanisms of chronic pain, including neuropathic pain, are poorly understood. Upregulation of voltage-gated calcium channel (VGCC) alpha2delta1 subunit (Ca(v)alpha2delta1) in sensory neurons and dorsal spinal cord by peripheral nerve injury has been suggested to contribute to neuropathic pain. To investigate the mechanisms without the influence of other injury factors, we have created transgenic mice that constitutively overexpress Ca(v)alpha2delta1 in neuronal tissues. Ca(v)alpha2delta1 overexpression resulted in enhanced currents, altered kinetics and voltage-dependence of VGCC activation in sensory neurons; exaggerated and prolonged dorsal horn neuronal responses to mechanical and thermal stimulations at the periphery; and pain behaviors. However, the transgenic mice showed normal dorsal horn neuronal responses to windup stimulation, and behavioral responses to tissue-injury/inflammatory stimuli. The pain behaviors in the transgenic mice had a pharmacological profile suggesting a selective contribution of elevated Ca(v)alpha2delta1 to the abnormal sensations, at least at the spinal cord level. In addition, gabapentin blocked VGCC currents concentration-dependently in transgenic, but not wild-type, sensory neurons. Thus, elevated neuronal Ca(v)alpha2delta1 contributes to specific pain states through a mechanism mediated at least partially by enhanced VGCC activity in sensory neurons and hyperexcitability in dorsal horn neurons in response to peripheral stimulation. Modulation of enhanced VGCC activity by gabapentin may underlie at least partially its antihyperalgesic actions.

Summary A somatodendritic gradient of Cl − concentration ([Cl − ] i ) has been postulated to generate GABA-evoked responses of different polarity in retinal bipolar cells, hyperpolarizing in OFF cells with low dendritic [Cl − ] i , and depolarizing in ON cells with high dendritic [Cl − ] i . As glutamate released by the photoreceptors depolarizes OFF cells and hyperpolarizes ON cells, the bipolars' antagonistic receptive field (RF) could be computed by simply integrating glutamatergic inputs from the RF center and GABAergic inputs from horizontal cells in the RF surround. Using ratiometric two-photon imaging of Clomeleon, a Cl − indicator transgenically expressed in ON bipolar cells, we found that dendritic [Cl − ] i exceeds somatic [Cl − ] i by up to 20 mM and that GABA application can lead to Cl − efflux (depolarization) in these dendrites. Blockers of Cl − transporters reduced the somatodendritic [Cl − ] i gradient. Hence, our results support the idea that ON bipolar cells employ a somatodendritic [Cl − ] i gradient to invert GABAergic horizontal cell input.

Synaptopodin is an actin-associated molecule found in a subset of telencephalic spines. It is an essential component of the spine apparatus, a Ca(2+)-storing organelle and has been implicated in synaptic plasticity (Deller et al. [2003] Proc Natl Acad Sci U S A 100:10494-10499). In the rodent hippocampus, Synaptopodin is distributed in a characteristic region- and lamina-specific manner. To learn more about the cellular basis underlying this distribution, the regional, laminar, and cellular localization of Synaptopodin and its mRNA were analyzed in mouse hippocampus. First, Synaptopodin puncta densities were quantified after immunofluorescent labeling using confocal microscopy. Second, the dendritic distribution of Synaptopodin-positive puncta was studied using three-dimensional confocal reconstructions of Synaptopodin-immunostained and enhanced green fluorescence protein (EGFP)-labeled principal neurons. Synaptopodin puncta located within dendrites of principal neurons were primarily found in spines (>95%). Analysis of dendritic segments located in different layers revealed lamina-specific differences in the percentage of Synaptopodin-positive spines. Densities ranged between 37% (outer molecular layer) and 14% (stratum oriens; CA1). Finally, synaptopodin mRNA expression was studied using in situ hybridization, laser microdissection, and quantitative reverse transcriptase-polymerase chain reaction. Expression levels were comparable between all regions. These data demonstrate a lamina-specific distribution of Synaptopodin within dendritic segments of identified neurons. Within dendrites, the majority of Synaptopodin-positive puncta were located in spines where they represent spine apparatuses. We conclude, that this organelle is distributed in a region- and layer-specific manner in the mouse hippocampus and suggest that differences in the activity of afferent fiber systems could determine its distribution.

We show that the neural cell recognition molecule Close Homolog of L1 (CHL1) is required for neuronal positioning and dendritic growth of pyramidal neurons in the posterior region of the developing mouse neocortex. CHL1 was expressed in pyramidal neurons in a high-caudal to low-rostral gradient within the developing cortex. Deep layer pyramidal neurons of CHL1-minus mice were shifted to lower laminar positions in the visual and somatosensory cortex and developed misoriented, often inverted apical dendrites. Impaired migration of CHL1-minus cortical neurons was suggested by strikingly slower rates of radial migration in cortical slices, failure to potentiate integrin-dependent haptotactic cell migration in vitro, and accumulation of migratory cells in the intermediate and ventricular/subventricular zones in vivo. The restriction of CHL1 expression and effects of its deletion in posterior neocortical areas suggests that CHL1 may regulate area-specific neuronal connectivity and, by extension, function in the visual and somatosensory cortex.