Restoration of Visual Function by Enhancing Conduction in Regenerated Axons.

Although a number of repair strategies have been shown to promote axon outgrowth following neuronal injury in the mammalian CNS, it remains unclear whether regenerated axons establish functional synapses and support behavior. Here, in both juvenile and adult mice, we show that either PTEN and SOCS3 co-deletion, or co-overexpression of osteopontin (OPN)/insulin-like growth factor 1 (IGF1)/ciliary neurotrophic factor (CNTF), induces regrowth of retinal axons and formation of functional synapses in the superior colliculus (SC) but not significant recovery of visual function. Further analyses suggest that regenerated axons fail to conduct action potentials from the eye to the SC due to lack of myelination. Consistent with this idea, administration of voltage-gated potassium channel blockers restores conduction and results in increased visual acuity. Thus, enhancing both regeneration and conduction effectively improves function after retinal axon injury.

CDKL5 sculpts functional callosal connectivity to promote cognitive flexibility.

Functional and structural connectivity alterations in short- and long-range projections have been reported across neurodevelopmental disorders (NDD). Interhemispheric callosal projection neurons (CPN) represent one of the major long-range projections in the brain, which are particularly important for higher-order cognitive function and flexibility. However, whether a causal relationship exists between interhemispheric connectivity alterations and cognitive deficits in NDD remains elusive. Here, we focused on CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental disorder caused by mutations in the X-linked Cyclin-dependent kinase-like 5 (CDKL5) gene. We found an increase in homotopic interhemispheric connectivity and functional hyperconnectivity across higher cognitive areas in adult male and female CDKL5-deficient mice by resting-state functional MRI (rs-fMRI) analysis. This was accompanied by an increase in the number of callosal synaptic inputs but decrease in local synaptic connectivity in the cingulate cortex of juvenile CDKL5-deficient mice, suggesting an impairment in excitatory synapse development and a differential role of CDKL5 across excitatory neuron subtypes. These deficits were associated with significant cognitive impairments in CDKL5 KO mice. Selective deletion of CDKL5 in the largest subtype of CPN likewise resulted in an increase of functional callosal inputs, without however significantly altering intracortical cingulate networks. Notably, such callosal-specific changes were sufficient to cause cognitive deficits. Finally, when CDKL5 was selectively re-expressed only in this CPN subtype, in otherwise CDKL5-deficient mice, it was sufficient to prevent the cognitive impairments of CDKL5 mutants. Together, these results reveal a novel role of CDKL5 by demonstrating that it is both necessary and sufficient for proper CPN connectivity and cognitive function and flexibility, and further validates a causal relationship between CPN dysfunction and cognitive impairment in a model of NDD.

Deep learning of spontaneous arousal fluctuations detects early cholinergic defects across neurodevelopmental mouse models and patients.

Neurodevelopmental spectrum disorders like autism (ASD) are diagnosed, on average, beyond age 4 y, after multiple critical periods of brain development close and behavioral intervention becomes less effective. This raises the urgent need for quantitative, noninvasive, and translational biomarkers for their early detection and tracking. We found that both idiopathic (BTBR) and genetic (CDKL5- and MeCP2-deficient) mouse models of ASD display an early, impaired cholinergic neuromodulation as reflected in altered spontaneous pupil fluctuations. Abnormalities were already present before the onset of symptoms and were rescued by the selective expression of MeCP2 in cholinergic circuits. Hence, we trained a neural network (ConvNetACh) to recognize, with 97% accuracy, patterns of these arousal fluctuations in mice with enhanced cholinergic sensitivity (LYNX1-deficient). ConvNetACh then successfully detected impairments in all ASD mouse models tested except in MeCP2-rescued mice. By retraining only the last layers of ConvNetACh with heart rate variation data (a similar proxy of arousal) directly from Rett syndrome patients, we generated ConvNetPatients, a neural network capable of distinguishing them from typically developing subjects. Even with small cohorts of rare patients, our approach exhibited significant accuracy before (80% in the first and second year of life) and into regression (88% in stage III patients). Thus, transfer learning across species and modalities establishes spontaneous arousal fluctuations combined with deep learning as a robust noninvasive, quantitative, and sensitive translational biomarker for the rapid and early detection of neurodevelopmental disorders before major symptom onset.

Accelerated Hyper-Maturation of Parvalbumin Circuits in the Absence of MeCP2.

Methyl-CpG-binding protein 2 (MeCP2) mutations are the primary cause of Rett syndrome, a severe neurodevelopmental disorder. Cortical parvalbumin GABAergic interneurons (PV) make exuberant somatic connections onto pyramidal cells in the visual cortex of Mecp2-deficient mice, which contributes to silencing neuronal cortical circuits. This phenotype can be rescued independently of Mecp2 by environmental, pharmacological, and genetic manipulation. It remains unknown how Mecp2 mutation can result in abnormal inhibitory circuit refinement. In the present manuscript, we examined the development of GABAergic circuits in the primary visual cortex of Mecp2-deficient mice. We identified that PV circuits were the only GABAergic interneurons to be upregulated, while other interneurons were downregulated. Acceleration of PV cell maturation was accompanied by increased PV cells engulfment by perineuronal nets (PNNs) and by an increase of PV cellular and PNN structural complexity. Interestingly, selective deletion of Mecp2 from PV cells was sufficient to drive increased structure complexity of PNN. Moreover, the accelerated PV and PNN maturation was recapitulated in organotypic cultures. Our results identify a specific timeline of disruption of GABAergic circuits in the absence of Mecp2, indicating a possible cell-autonomous role of MeCP2 in the formation of PV cellular arbors and PNN structures in the visual cortex.

NMDA 2A receptors in parvalbumin cells mediate sex-specific rapid ketamine response on cortical activity.

Ketamine has emerged as a widespread treatment for a variety of psychiatric disorders when used at sub-anesthetic doses, but the neural mechanisms underlying its acute action remain unclear. Here, we identified NMDA receptors containing the 2A subunit (GluN2A) on parvalbumin (PV)-expressing inhibitory interneurons as a pivotal target of low-dose ketamine. Genetically deleting GluN2A receptors globally or selectively from PV interneurons abolished the rapid enhancement of visual cortical responses and gamma-band oscillations by ketamine. Moreover, during the follicular phase of the estrous cycle in female mice, the ketamine response was transiently attenuated along with a concomitant decrease of grin2A mRNA expression within PV interneurons. Thus, GluN2A receptors on PV interneurons mediate the immediate actions of low-dose ketamine treatment, and fluctuations in receptor expression across the estrous cycle may underlie sex-differences in drug efficacy.

Behavioral analyses of animal models of intellectual and developmental disabilities.

Intellectual and developmental disabilities (IDDs) are a common group of disorders that frequently share overlapping symptoms, including cognitive deficits, altered attention, seizures, impaired social interactions, and anxiety. The causes of these disorders are varied ranging from early prenatal/postnatal insults to genetic variants that either cause or are associated with an increased likelihood of an IDD. As many of the symptoms observed in individuals with IDDs are a manifestation of altered nervous system function resulting in altered behaviors, it should not be surprising that the field is very dependent upon in vivo model systems. This special issue of Neurobiology of Learning and Memory is focused on the methods and approaches that are being used to model and understand these disorders in mammals. While surveys by the Pew Foundation continue to find a high degree of confidence/trust in scientists by the public, several recent studies have documented issues with reproducibility in scientific publications. This special issue includes both primary research articles and review articles in which careful attention has been made to transparently report methods and use rigorous approaches to ensure reproducibility. Although there have been and will continue to be remarkable advances for treatment of subset of IDDs, it is clear that this field is still in its early stages. There is no doubt that the strategies being used to model IDDs will continue to evolve. We hope this special issue will support this evolution so that we can maintain the trust of the public and elected officials, and continue developing evidence-based approaches to new therapeutics.

Rigor and reproducibility in rodent behavioral research.

Behavioral neuroscience research incorporates the identical high level of meticulous methodologies and exacting attention to detail as all other scientific disciplines. To achieve maximal rigor and reproducibility of findings, well-trained investigators employ a variety of established best practices. Here we explicate some of the requirements for rigorous experimental design and accurate data analysis in conducting mouse and rat behavioral tests. Novel object recognition is used as an example of a cognitive assay which has been conducted successfully with a range of methods, all based on common principles of appropriate procedures, controls, and statistics. Directors of Rodent Core facilities within Intellectual and Developmental Disabilities Research Centers contribute key aspects of their own novel object recognition protocols, offering insights into essential similarities and less-critical differences. Literature cited in this review article will lead the interested reader to source papers that provide step-by-step protocols which illustrate optimized methods for many standard rodent behavioral assays. Adhering to best practices in behavioral neuroscience will enhance the value of animal models for the multiple goals of understanding biological mechanisms, evaluating consequences of genetic mutations, and discovering efficacious therapeutics.

Visual evoked potentials detect cortical processing deficits in Rett syndrome.

OBJECTIVE: Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutation of the X-linked MECP2 gene and characterized by developmental regression during the first few years of life. The objective of this study was to investigate if the visual evoked potential (VEP) could be used as an unbiased, quantitative biomarker to monitor brain function in RTT. METHODS: We recorded pattern-reversal VEPs in Mecp2 heterozygous female mice and 34 girls with RTT. The amplitudes and latencies of VEP waveform components were quantified, and were related to disease stage, clinical severity, and MECP2 mutation type in patients. Visual acuity was also assessed in both mice and patients by modulating the spatial frequency of the stimuli. RESULTS: Mecp2 heterozygous female mice and RTT patients exhibited a similar decrease in VEP amplitude that was most striking in the later stages of the disorder. RTT patients also displayed a slower recovery from the principal peak of the VEP response that was impacted by MECP2 mutation type. When the spatial frequency of the stimulus was increased, both patients and mice displayed a deficit in discriminating smaller patterns, indicating lower visual spatial acuity in RTT. INTERPRETATION: VEP is a method that can be used to assess brain function across species and in children with severe disabilities like RTT. Our findings support the introduction of standardized VEP analysis in clinical and research settings to probe the neurobiological mechanism underlying functional impairment and to longitudinally monitor progression of the disorder and response to treatment.

Aberrant development and plasticity of excitatory visual cortical networks in the absence of cpg15.

During development, experience plays a crucial role in sculpting neuronal connections. Patterned neural activity guides formation of functional neural circuits through the selective stabilization of some synapses and the pruning of others. Activity-regulated factors are fundamental to this process, but their roles in synapse stabilization and maturation is still poorly understood. CPG15, encoded by the activity-regulated gene candidate plasticity gene 15, is a small, glycosylphosphatidylinositol (GPI)-linked, extracellular protein that promotes synapse stabilization. Here we show that global knock-out of cpg15 results in abnormal postnatal development of the excitatory network in visual cortex and an associated disruption in development of visual receptive field properties. In addition, whereas repeated stimulation induced potentiation and depression in wild-type mice, the depression was slower in cpg15 knock-out mice, suggesting impairment in short-term depression-like mechanisms. These findings establish the requirement for cpg15 in activity-dependent development of the visual system and demonstrate the importance of timely excitatory network development for normal visual function.

Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors.

The mature optic nerve cannot regenerate when injured, leaving victims of traumatic nerve damage or diseases such as glaucoma with irreversible visual losses. Recent studies have identified ways to stimulate retinal ganglion cells to regenerate axons part-way through the optic nerve, but it remains unknown whether mature axons can reenter the brain, navigate to appropriate target areas, or restore vision. We show here that with adequate stimulation, retinal ganglion cells are able to regenerate axons the full length of the visual pathway and on into the lateral geniculate nucleus, superior colliculus, and other visual centers. Regeneration partially restores the optomotor response, depth perception, and circadian photoentrainment, demonstrating the feasibility of reconstructing central circuitry for vision after optic nerve damage in mature mammals.

Visual acuity development and plasticity in the absence of sensory experience.

Visual circuits mature and are refined by sensory experience. However, significant gaps remain in our understanding how deprivation influences the development of visual acuity in mice. Here, we perform a longitudinal study assessing the effects of chronic deprivation on the development of the mouse subcortical and cortical visual circuits using a combination of behavioral optomotor testing, in vivo visual evoked responses (VEP) and single-unit cortical recordings. As previously reported, orientation tuning was degraded and onset of ocular dominance plasticity was delayed and remained open in chronically deprived mice. Surprisingly, we found that the development of optomotor threshold and VEP acuity can occur in an experience-independent manner, although at a significantly slower rate. Moreover, monocular deprivation elicited amblyopia only during a discrete period of development in the dark. The rate of recovery of optomotor threshold upon exposure of deprived mice to light confirmed a maturational transition regardless of visual input. Together our results revealed a dissociable developmental trajectory for visual receptive-field properties in dark-reared mice suggesting a differential role for spontaneous activity within thalamocortical and intracortical circuits.

CAGE-defined promoter regions of the genes implicated in Rett Syndrome.

BACKGROUND: Mutations in three functionally diverse genes cause Rett Syndrome. Although the functions of Forkhead box G1 (FOXG1), Methyl CpG binding protein 2 (MECP2) and Cyclin-dependent kinase-like 5 (CDKL5) have been studied individually, not much is known about their relation to each other with respect to expression levels and regulatory regions. Here we analyzed data from hundreds of mouse and human samples included in the FANTOM5 project, to identify transcript initiation sites, expression levels, expression correlations and regulatory regions of the three genes. RESULTS: Our investigations reveal the predominantly used transcription start sites (TSSs) for each gene including novel transcription start sites for FOXG1. We show that FOXG1 expression is poorly correlated with the expression of MECP2 and CDKL5. We identify promoter shapes for each TSS, the predicted location of enhancers for each gene and the common transcription factors likely to regulate the three genes. Our data imply Polycomb Repressive Complex 2 (PRC2) mediated silencing of Foxg1 in cerebellum. CONCLUSIONS: Our analyses provide a comprehensive picture of the regulatory regions of the three genes involved in Rett Syndrome.

Phenotypic characterization of Cdkl5-knockdown neurons establishes elongated cilia as a functional assay for CDKL5 Deficiency Disorder.

CDKL5 Deficiency Disorder (CDD) is a severe encephalopathy characterized by intractable epilepsy, infantile spasms, and cognitive disabilities. The detrimental CNS manifestations and lack of therapeutic interventions represent unmet needs, necessitating identification of CDD-dependent phenotypes for in vitro disease modeling and therapeutic testing. Here, we optimized a high-content assay to quantify cilia in CDKL5-deficient neurons. Our work shows that Cdkl5-knockdown neurons have elongated cilia and uncovers cilium lengthening in hippocampi of Cdkl5 knockout mice. Collectively, our findings identify cilia length alterations under CDKL5 activity loss in vitro and in vivo and reveal elongated cilia as a robust functional phenotype for CDD.

Optimization of somatic inhibition at critical period onset in mouse visual cortex.

Local GABAergic circuits trigger visual cortical plasticity in early postnatal life. How these diverse connections contribute to critical period onset was investigated by nonstationary fluctuation analysis following laser photo-uncaging of GABA onto discrete sites upon individual pyramidal cells in slices of mouse visual cortex. The GABA(A) receptor number decreased on the soma-proximal dendrite (SPD), but not at the axon initial segment, with age and sensory deprivation. Benzodiazepine sensitivity was also higher on the immature SPD. Too many or too few SPD receptors in immature or dark-reared mice, respectively, were adjusted to critical period levels by benzodiazepine treatment in vivo, which engages ocular dominance plasticity in these animal models. Combining GAD65 deletion with dark rearing from birth confirmed that an intermediate number of SPD receptors enable plasticity. Site-specific optimization of perisomatic GABA response may thus trigger experience-dependent development in visual cortex.

Autism: a "critical period" disorder?

Cortical circuits in the brain are refined by experience during critical periods early in postnatal life. Critical periods are regulated by the balance of excitatory and inhibitory (E/I) neurotransmission in the brain during development. There is now increasing evidence of E/I imbalance in autism, a complex genetic neurodevelopmental disorder diagnosed by abnormal socialization, impaired communication, and repetitive behaviors or restricted interests. The underlying cause is still largely unknown and there is no fully effective treatment or cure. We propose that alteration of the expression and/or timing of critical period circuit refinement in primary sensory brain areas may significantly contribute to autistic phenotypes, including cognitive and behavioral impairments. Dissection of the cellular and molecular mechanisms governing well-established critical periods represents a powerful tool to identify new potential therapeutic targets to restore normal plasticity and function in affected neuronal circuits.

Dynamical Characteristics of Wild-Type Mouse Spontaneous Pupillary Fluctuations.

Spontaneous pupil size fluctuations in humans and mouse models are noninvasively measured data that can be used for early detection of neurodevelopmental spectrum disorders. While highly valuable in such applied studies, pupillometry dynamics and dynamical characteristics have not been fully investigated, although their understanding may potentially lead to the discovery of new information, which cannot be readily uncovered by conventional methods. Properties of pupillometry dynamics, such as determinism, were previously investigated for healthy human subjects; however, the dynamical characteristics of pupillometry data in mouse models, and whether they are similar to those of human subjects, remain largely unknown. Therefore, it is necessary to establish a thorough understanding of the dynamical properties of mouse pupillometry dynamics and to clarify whether it is similar to that of humans. In this study, dynamical pupillometry characteristics from 115 wild-type mouse datasets were investigated by methods of nonlinear time series analysis. Results clearly demonstrated a strong underlying determinism in the investigated data. Additionally, the data's trajectory divergence rate and predictability were estimated.

The Stage of the Estrus Cycle Is Critical for Interpretation of Female Mouse Social Interaction Behavior.

Female animals in biomedical research have traditionally been excluded from research studies due to the perceived added complexity caused by the estrus cycle. However, given the importance of sex differences in a variety of neurological disorders, testing female mice is critical to identifying sex-linked effects in diseases. To determine the susceptibility of simple behaviors to hormonal fluctuations in the estrus cycle, we studied the effects of sex and the estrus cycle on a variety of behavioral tasks commonly used in mouse phenotyping laboratories. Male and female C57BL/6J mice were tested in a small battery of short duration tests and, immediately on completion of each test, females were classified using cytology of vaginal lavages as sexually-receptive (proestrus and estrus) or non-receptive (NR; metestrus and diestrus). We showed that there was a significant difference in 3-chamber social interaction (SI) between female mice at different stages of their estrus cycle, with sexually-receptive mice showing no preferential interest in a novel female mouse compared with an empty chamber. NR female mice showed the same level of preference for a novel female mouse as male mice did for a novel male mouse. No differences between or within sexes were found for tests of anxiety elevated plus maze (EPM; Hole board), working memory [Novel object recognition (NOR)], and motor learning (repeated tests on rotarod). We conclude that the stage of the estrus cycle may impact SI between same-sex conspecifics, and does not impact performance in the elevated plus-maze, hole board, NOR, and rotarod.

Intellectual and Developmental Disabilities Research Centers: A Multidisciplinary Approach to Understand the Pathogenesis of Methyl-CpG Binding Protein 2-related Disorders.

Disruptions in the gene encoding methyl-CpG binding protein 2 (MECP2) underlie complex neurodevelopmental disorders including Rett Syndrome (RTT), MECP2 duplication disorder, intellectual disabilities, and autism. Significant progress has been made on the molecular and cellular basis of MECP2-related disorders providing a new framework for understanding how altered epigenetic landscape can derail the formation and refinement of neuronal circuits in early postnatal life and proper neurological function. This review will summarize selected major findings from the past years and particularly highlight the integrated and multidisciplinary work done at eight NIH-funded Intellectual and Developmental Disabilities Research Centers (IDDRC) across the US. Finally, we will outline a path forward with identification of reliable biomarkers and outcome measures, longitudinal preclinical and clinical studies, reproducibility of results across centers as a synergistic effort to decode and treat the pathogenesis of the complex MeCP2 disorders.

MeCP2: an epigenetic regulator of critical periods.

Complex adult behaviors arise from the integration of sequential and often overlapping critical periods (CPs) early in life and adolescence. These processes rely on a subtle interplay between the set of genes inherited from the parents, the surrounding environment and epigenetic regulation. Methyl-CpG-binding protein 2 (MeCP2) has been shown to recognize epigenetic states and regulate gene expression by reading methylated DNA. Here, we will review the recent findings revealing the role of MeCP2 during postnatal CPs of development using mouse models of Rett (RTT) syndrome.

Transparent, Flexible, Penetrating Microelectrode Arrays with Capabilities of Single-Unit Electrophysiology.

Accurately mapping neuronal activity across brain networks is critical to understand behaviors, yet it is very challenging due to the need of tools with both high spatial and temporal resolutions. Here, penetrating arrays of flexible microelectrodes made of low-impedance nanomeshes are presented, which are capable of recording single-unit electrophysiological neuronal activity and at the same time, transparent, allowing to bridge electrical and optical brain mapping modalities. These 32 transparent penetrating electrodes with site area, 225 µm2 , have a low impedance of ≈149 kΩ at 1 kHz, an adequate charge injection limit of ≈0.76 mC cm-2 , and up to 100% yield. Mechanical bending tests reveal that the array is robust up to 1000 bending cycles, and its high transmittance of 67% at 550 nm makes it suitable for combining with various optical methods. A temporary stiffening using polyethylene glycol allows the penetrating nanomesh arrays to be inserted into the brain minimally invasively, with in vivo validation of recordings of spontaneous and evoked single-unit activity of neurons across layers of the mouse visual cortex. Together, these results establish a novel neurotechnology-transparent, flexible, penetrating microelectrode arrays-which possesses great potential for brain research.