Stabilizing Immature Dendritic Spines in the Auditory Cortex: A Key Mechanism for mTORC1-Mediated Enhancement of Long-Term Fear Memories.

Mammalian target of rapamycin (mTOR) pathway has emerged as a key molecular mechanism underlying memory processes. Although mTOR inhibition is known to block memory processes, it remains elusive whether and how an enhancement of mTOR signaling may improve memory processes. Here we found in male mice that the administration of VO-OHpic, an inhibitor of the phosphatase and tensin homolog (PTEN) that negatively modulates AKT-mTOR pathway, enhanced auditory fear memory for days and weeks, while it left short-term memory unchanged. Memory enhancement was associated with a long-lasting increase in immature-type dendritic spines of pyramidal neurons into the auditory cortex. The persistence of spine remodeling over time arose by the interplay between PTEN inhibition and memory processes, as VO-OHpic induced only a transient immature spine growth in the somatosensory cortex, a region not involved in long-term auditory memory. Both the potentiation of fear memories and increase in immature spines were hampered by rapamycin, a selective inhibitor of mTORC1. These data revealed that memory can be potentiated over time by the administration of a selective PTEN inhibitor. In addition to disclosing new information on the cellular mechanisms underlying long-term memory maintenance, our study provides new insights on the molecular processes that aid enhancing memories over time.SIGNIFICANCE STATEMENT The neuronal mechanisms that may help improve the maintenance of long-term memories are still elusive. The inhibition of mammalian-target of rapamycin (mTOR) signaling shows that this pathway plays a crucial role in synaptic plasticity and memory formation. However, whether its activation may strengthen long-term memory storage is unclear. We assessed the consequences of positive modulation of AKT-mTOR pathway obtained by VO-OHpic administration, a phosphatase and tensin homolog inhibitor, on memory retention and underlying synaptic modifications. We found that mTOR activation greatly enhanced memory maintenance for weeks by producing a long-lasting increase of immature-type dendritic spines in pyramidal neurons of the auditory cortex. These results offer new insights on the cellular and molecular mechanisms that can aid enhancing memories over time.

A comprehensive atlas of perineuronal net distribution and colocalization with parvalbumin in the adult mouse brain.

Perineuronal nets (PNNs) surround specific neurons in the brain and are involved in various forms of plasticity and clinical conditions. However, our understanding of the PNN role in these phenomena is limited by the lack of highly quantitative maps of PNN distribution and association with specific cell types. Here, we present a comprehensive atlas of Wisteria floribunda agglutinin (WFA)-positive PNNs and colocalization with parvalbumin (PV) cells for over 600 regions of the adult mouse brain. Data analysis shows that PV expression is a good predictor of PNN aggregation. In the cortex, PNNs are dramatically enriched in layer 4 of all primary sensory areas in correlation with thalamocortical input density, and their distribution mirrors intracortical connectivity patterns. Gene expression analysis identifies many PNN-correlated genes. Strikingly, PNN-anticorrelated transcripts are enriched in synaptic plasticity genes, generalizing PNNs' role as circuit stability factors.

Selective Disruption of Perineuronal Nets in Mice Lacking Crtl1 is Sufficient to Make Fear Memories Susceptible to Erasure.

The ability to store, retrieve, and extinguish memories of adverse experiences is an essential skill for animals' survival. The cellular and molecular factors that underlie such processes are only partially known. Using chondroitinase ABC treatment targeting chondroitin sulfate proteoglycans (CSPGs), previous studies showed that the maturation of the extracellular matrix makes fear memory resistant to deletion. Mice lacking the cartilage link protein Crtl1 (Crtl1-KO mice) display normal CSPG levels but impaired CSPG condensation in perineuronal nets (PNNs). Thus, we asked whether the presence of PNNs in the adult brain is responsible for the appearance of persistent fear memories by investigating fear extinction in Crtl1-KO mice. We found that mutant mice displayed fear memory erasure after an extinction protocol as revealed by analysis of freezing and pupil dynamics. Fear memory erasure did not depend on passive loss of retention; moreover, we demonstrated that, after extinction training, conditioned Crtl1-KO mice display no neural activation in the amygdala (Zif268 staining) in comparison to control animals. Taken together, our findings suggest that the aggregation of CSPGs into PNNs regulates the boundaries of the critical period for fear extinction.

From pupil to the brain: New insights for studying cortical plasticity through pupillometry.

Pupil size variations have been associated with changes in brain activity patterns related with specific cognitive factors, such as arousal, attention, and mental effort. The locus coeruleus (LC), a key hub in the noradrenergic system of the brain, is considered to be a key regulator of cognitive control on pupil size, with changes in pupil diameter corresponding to the release of norepinephrine (NE). Advances in eye-tracking technology and open-source software have facilitated accurate pupil size measurement in various experimental settings, leading to increased interest in using pupillometry to track the nervous system activation state and as a potential biomarker for brain disorders. This review explores pupillometry as a non-invasive and fully translational tool for studying cortical plasticity starting from recent literature suggesting that pupillometry could be a promising technique for estimating the degree of residual plasticity in human subjects. Given that NE is known to be a critical mediator of cortical plasticity and arousal, the review includes data revealing the importance of the LC-NE system in modulating brain plasticity and pupil size. Finally, we will review data suggesting that pupillometry could provide a quantitative and complementary measure of cortical plasticity also in pre-clinical studies.

Cell-specific vulnerability to metabolic failure: the crucial role of parvalbumin expressing neurons in creatine transporter deficiency.

Mutations in the solute carrier family 6-member 8 (Slc6a8) gene, encoding the protein responsible for cellular creatine (Cr) uptake, cause Creatine Transporter Deficiency (CTD), an X-linked neurometabolic disorder presenting with intellectual disability, autistic-like features, and epilepsy. The pathological determinants of CTD are still poorly understood, hindering the development of therapies. In this study, we generated an extensive transcriptomic profile of CTD showing that Cr deficiency causes perturbations of gene expression in excitatory neurons, inhibitory cells, and oligodendrocytes which result in remodeling of circuit excitability and synaptic wiring. We also identified specific alterations of parvalbumin-expressing (PV+) interneurons, exhibiting a reduction in cellular and synaptic density, and a hypofunctional electrophysiological phenotype. Mice lacking Slc6a8 only in PV+ interneurons recapitulated numerous CTD features, including cognitive deterioration, impaired cortical processing and hyperexcitability of brain circuits, demonstrating that Cr deficit in PV+ interneurons is sufficient to determine the neurological phenotype of CTD. Moreover, a pharmacological treatment targeted to restore the efficiency of PV+ synapses significantly improved cortical activity in Slc6a8 knock-out animals. Altogether, these data demonstrate that Slc6a8 is critical for the normal function of PV+ interneurons and that impairment of these cells is central in the disease pathogenesis, suggesting a novel therapeutic venue for CTD.

Convolutional neural network classifies visual stimuli from cortical response recorded with wide-field imaging in mice.

Objective.The optic nerve is a good location for a visual neuroprosthesis. It can be targeted when a subject cannot receive a retinal prosthesis and it is less invasive than a cortical implant. The effectiveness of an electrical neuroprosthesis depends on the combination of the stimulation parameters which must be optimized, and an optimization strategy might be performing closed-loop stimulation using the evoked cortical response as feedback. However, it is necessary to identify target cortical activation patterns and to associate the cortical activity with the visual stimuli present in the visual field of the subjects. Visual stimuli decoding should be performed on large areas of the visual cortex, and with a method as translational as possible to shift the study to human subjects in the future. The aim of this work is to develop an algorithm that meets these requirements and can be leveraged to automatically associate a cortical activation pattern with the visual stimulus that generated it.Approach.Three mice were presented with ten different visual stimuli, and their primary visual cortex response was recorded using wide-field calcium imaging. Our decoding algorithm relies on a convolutional neural network (CNN), trained to classify the visual stimuli from the correspondent wide-field images. Several experiments were performed to identify the best training strategy and investigate the possibility of generalization.Main results.The best classification accuracy was 75.38% ± 4.77%, obtained pre-training the CNN on the MNIST digits dataset and fine-tuning it on our dataset. Generalization was possible pre-training the CNN to classify Mouse 1 dataset and fine-tuning it on Mouse 2 and Mouse 3, with accuracies of 64.14% ± 10.81% and 51.53% ± 6.48% respectively.Significance.The combination of wide-field calcium imaging and CNNs can be used to classify the cortical responses to simple visual stimuli and might be a viable alternative to existing decoding methodologies. It also allows us to consider the cortical activation as reliable feedback in future optic nerve stimulation experiments.

mGluR5 PAMs rescue cortical and behavioural defects in a mouse model of CDKL5 deficiency disorder.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a devastating rare neurodevelopmental disease without a cure, caused by mutations of the serine/threonine kinase CDKL5 highly expressed in the forebrain. CDD is characterized by early-onset seizures, severe intellectual disabilities, autistic-like traits, sensorimotor and cortical visual impairments (CVI). The lack of an effective therapeutic strategy for CDD urgently demands the identification of novel druggable targets potentially relevant for CDD pathophysiology. To this aim, we studied Class I metabotropic glutamate receptors 5 (mGluR5) because of their important role in the neuropathological signs produced by the lack of CDKL5 in-vivo, such as defective synaptogenesis, dendritic spines formation/maturation, synaptic transmission and plasticity. Importantly, mGluR5 function strictly depends on the correct expression of the postsynaptic protein Homer1bc that we previously found atypical in the cerebral cortex of Cdkl5-/y mice. In this study, we reveal that CDKL5 loss tampers with (i) the binding strength of Homer1bc-mGluR5 complexes, (ii) the synaptic localization of mGluR5 and (iii) the mGluR5-mediated enhancement of NMDA-induced neuronal responses. Importantly, we showed that the stimulation of mGluR5 activity by administering in mice specific positive-allosteric-modulators (PAMs), i.e., 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) or RO6807794, corrected the synaptic, functional and behavioral defects shown by Cdkl5-/y mice. Notably, in the visual cortex of 2 CDD patients we found changes in synaptic organization that recapitulate those of mutant CDKL5 mice, including the reduced expression of mGluR5, suggesting that these receptors represent a promising therapeutic target for CDD.

Modeling RTT Syndrome by iPSC-Derived Neurons from Male and Female Patients with Heterogeneously Severe Hot-Spot MECP2 Variants.

Rett syndrome caused by MECP2 variants is characterized by a heterogenous clinical spectrum accounted for in 60% of cases by hot-spot variants. Focusing on the most frequent variants, we generated in vitro iPSC-neurons from the blood of RTT girls with p.Arg133Cys and p.Arg255*, associated to mild and severe phenotype, respectively, and of an RTT male harboring the close to p.Arg255*, p.Gly252Argfs*7 variant. Truncated MeCP2 proteins were revealed by Western blot and immunofluorescence analysis. We compared the mutant versus control neurons at 42 days for morphological parameters and at 120 days for electrophysiology recordings, including girls' isogenic clones. A precocious reduced morphological complexity was evident in neurons with truncating variants, while in p.Arg133Cys neurons any significant differences were observed in comparison with the isogenic wild-type clones. Reduced nuclear size and branch number show up as the most robust biomarkers. Patch clamp recordings on mature neurons allowed the assessment of cell biophysical properties, V-gated currents, and spiking pattern in the mutant and control cells. Immature spiking, altered cell capacitance, and membrane resistance of RTT neurons, were particularly pronounced in the Arg255* and Gly252Argfs*7 mutants. The overall results indicate that the specific markers of in vitro cellular phenotype mirror the clinical severity and may be amenable to drug testing for translational purposes.

Expression of a Secretable, Cell-Penetrating CDKL5 Protein Enhances the Efficacy of Gene Therapy for CDKL5 Deficiency Disorder.

Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases.

Behavioral impulsivity is associated with pupillary alterations and hyperactivity in CDKL5 mutant mice.

Cyclin-dependent kinase-like 5 (Cdkl5) deficiency disorder (CDD) is a severe neurodevelopmental condition caused by mutations in the X-linked Cdkl5 gene. CDD is characterized by early-onset seizures in the first month of life, intellectual disability, motor and social impairment. No effective treatment is currently available and medical management is only symptomatic and supportive. Recently, mouse models of Cdkl5 disorder have demonstrated that mice lacking Cdkl5 exhibit autism-like phenotypes, hyperactivity and dysregulations of the arousal system, suggesting the possibility to use these features as translational biomarkers. In this study, we tested Cdkl5 male and female mutant mice in an appetitive operant conditioning chamber to assess cognitive and motor abilities, and performed pupillometry to assess the integrity of the arousal system. Then, we evaluated the performance of artificial intelligence models to classify the genotype of the animals from the behavioral and physiological phenotype. The behavioral results show that CDD mice display impulsivity, together with low levels of cognitive flexibility and perseverative behaviors. We assessed arousal levels by simultaneously recording pupil size and locomotor activity. Pupillometry reveals in CDD mice a smaller pupil size and an impaired response to unexpected stimuli associated with hyperlocomotion, demonstrating a global defect in arousal modulation. Finally, machine learning reveals that both behavioral and pupillometry parameters can be considered good predictors of CDD. Since early diagnosis is essential to evaluate treatment outcomes and pupillary measures can be performed easily, we proposed the monitoring of pupil size as a promising biomarker for CDD.

Learning to count biological structures with raters' uncertainty.

Exploiting well-labeled training sets has led deep learning models to astonishing results for counting biological structures in microscopy images. However, dealing with weak multi-rater annotations, i.e., when multiple human raters disagree due to non-trivial patterns, remains a relatively unexplored problem. More reliable labels can be obtained by aggregating and averaging the decisions given by several raters to the same data. Still, the scale of the counting task and the limited budget for labeling prohibit this. As a result, making the most with small quantities of multi-rater data is crucial. To this end, we propose a two-stage counting strategy in a weakly labeled data scenario. First, we detect and count the biological structures; then, in the second step, we refine the predictions, increasing the correlation between the scores assigned to the samples and the raters' agreement on the annotations. We assess our methodology on a novel dataset comprising fluorescence microscopy images of mice brains containing extracellular matrix aggregates named perineuronal nets. We demonstrate that we significantly enhance counting performance, improving confidence calibration by taking advantage of the redundant information characterizing the small sets of available multi-rater data.

The gut microbiota of environmentally enriched mice regulates visual cortical plasticity.

Exposing animals to an enriched environment (EE) has dramatic effects on brain structure, function, and plasticity. The poorly known "EE-derived signals'' mediating the EE effects are thought to be generated within the central nervous system. Here, we shift the focus to the body periphery, revealing that gut microbiota signals are crucial for EE-driven plasticity. Developmental analysis reveals striking differences in intestinal bacteria composition between EE and standard rearing (ST) mice, as well as enhanced levels of short-chain fatty acids (SCFA) in EE mice. Depleting the microbiota of EE mice with antibiotics strongly decreases SCFA and prevents activation of adult ocular dominance plasticity, spine dynamics, and microglia rearrangement. SCFA treatment in ST mice mimics EE induction of ocular dominance plasticity and microglial remodeling. Remarkably, transferring the microbiota of EE mice to ST recipients activates adult ocular dominance plasticity. Thus, experience-dependent changes in gut microbiota regulate brain plasticity.

Combining Optogenetic Stimulation and Motor Training Improves Functional Recovery and Perilesional Cortical Activity.

Background. An ischemic stroke is followed by the remapping of motor representation and extensive changes in cortical excitability involving both hemispheres. Although stimulation of the ipsilesional motor cortex, especially when paired with motor training, facilitates plasticity and functional restoration, the remapping of motor representation of the single and combined treatments is largely unexplored. Objective. We investigated if spatio-temporal features of motor-related cortical activity and the new motor representations are related to the rehabilitative treatment or if they can be specifically associated to functional recovery. Methods. We designed a novel rehabilitative treatment that combines neuro-plasticizing intervention with motor training. In detail, optogenetic stimulation of peri-infarct excitatory neurons expressing Channelrhodopsin 2 was associated with daily motor training on a robotic device. The effectiveness of the combined therapy was compared with spontaneous recovery and with the single treatments (ie optogenetic stimulation or motor training). Results. We found that the extension and localization of the new motor representations are specific to the treatment, where most treatments promote segregation of the motor representation to the peri-infarct region. Interestingly, only the combined therapy promotes both the recovery of forelimb functionality and the rescue of spatio-temporal features of motor-related activity. Functional recovery results from a new excitatory/inhibitory balance between hemispheres as revealed by the augmented motor response flanked by the increased expression of parvalbumin positive neurons in the peri-infarct area. Conclusions. Our findings highlight that functional recovery and restoration of motor-related neuronal activity are not necessarily coupled during post-stroke recovery. Indeed the reestablishment of cortical activation features of calcium transient is distinctive of the most effective therapeutic approach, the combined therapy.

Creatine deficiency and heart failure.

Impaired cardiac energy metabolism has been proposed as a mechanism common to different heart failure aetiologies. The energy-depletion hypothesis was pursued by several researchers, and is still a topic of considerable interest. Unlike most organs, in the heart, the creatine kinase system represents a major component of the metabolic machinery, as it functions as an energy shuttle between mitochondria and cytosol. In heart failure, the decrease in creatine level anticipates the reduction in adenosine triphosphate, and the degree of myocardial phosphocreatine/adenosine triphosphate ratio reduction correlates with disease severity, contractile dysfunction, and myocardial structural remodelling. However, it remains to be elucidated whether an impairment of phosphocreatine buffer activity contributes to the pathophysiology of heart failure and whether correcting this energy deficit might prove beneficial. The effects of creatine deficiency and the potential utility of creatine supplementation have been investigated in experimental and clinical models, showing controversial findings. The goal of this article is to provide a comprehensive overview on the role of creatine in cardiac energy metabolism, the assessment and clinical value of creatine deficiency in heart failure, and the possible options for the specific metabolic therapy.

MEYE: Web App for Translational and Real-Time Pupillometry.

Pupil dynamics alterations have been found in patients affected by a variety of neuropsychiatric conditions, including autism. Studies in mouse models have used pupillometry for phenotypic assessment and as a proxy for arousal. Both in mice and humans, pupillometry is noninvasive and allows for longitudinal experiments supporting temporal specificity; however, its measure requires dedicated setups. Here, we introduce a convolutional neural network that performs online pupillometry in both mice and humans in a web app format. This solution dramatically simplifies the usage of the tool for the nonspecialist and nontechnical operators. Because a modern web browser is the only software requirement, this choice is of great interest given its easy deployment and setup time reduction. The tested model performances indicate that the tool is sensitive enough to detect both locomotor-induced and stimulus-evoked pupillary changes, and its output is comparable to state-of-the-art commercial devices.

The Role of Preclinical Models in Creatine Transporter Deficiency: Neurobiological Mechanisms, Biomarkers and Therapeutic Development.

Creatine (Cr) Transporter Deficiency (CTD) is an X-linked metabolic disorder, mostly caused by missense mutations in the SLC6A8 gene and presenting with intellectual disability, autistic behavior, and epilepsy. There is no effective treatment for CTD and patients need lifelong assistance. Thus, the research of novel intervention strategies is a major scientific challenge. Animal models are an excellent tool to dissect the disease pathogenetic mechanisms and drive the preclinical development of therapeutics. This review illustrates the current knowledge about Cr metabolism and CTD clinical aspects, with a focus on mainstay diagnostic and therapeutic options. Then, we discuss the rodent models of CTD characterized in the last decade, comparing the phenotypes expressed within clinically relevant domains and the timeline of symptom development. This analysis highlights that animals with the ubiquitous deletion/mutation of SLC6A8 genes well recapitulate the early onset and the complex pathological phenotype of the human condition. Thus, they should represent the preferred model for preclinical efficacy studies. On the other hand, brain- and cell-specific conditional mutants are ideal for understanding the basis of CTD at a cellular and molecular level. Finally, we explain how CTD models might provide novel insight about the pathogenesis of other disorders, including cancer.

Chondroitin 6-sulphate is required for neuroplasticity and memory in ageing.

Perineuronal nets (PNNs) are chondroitin sulphate proteoglycan-containing structures on the neuronal surface that have been implicated in the control of neuroplasticity and memory. Age-related reduction of chondroitin 6-sulphates (C6S) leads to PNNs becoming more inhibitory. Here, we investigated whether manipulation of the chondroitin sulphate (CS) composition of the PNNs could restore neuroplasticity and alleviate memory deficits in aged mice. We first confirmed that aged mice (20-months) showed memory and plasticity deficits. They were able to retain or regain their cognitive ability when CSs were digested or PNNs were attenuated. We then explored the role of C6S in memory and neuroplasticity. Transgenic deletion of chondroitin 6-sulfotransferase (chst3) led to a reduction of permissive C6S, simulating aged brains. These animals showed very early memory loss at 11 weeks old. Importantly, restoring C6S levels in aged animals rescued the memory deficits and restored cortical long-term potentiation, suggesting a strategy to improve age-related memory impairment.

Age-Related Cognitive and Motor Decline in a Mouse Model of CDKL5 Deficiency Disorder is Associated with Increased Neuronal Senescence and Death.

CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental disease caused by mutations in the X-linked CDKL5 gene. Children affected by CDD display a clinical phenotype characterized by early-onset epilepsy, intellectual disability, motor impairment, and autistic-like features. Although the clinical aspects associated with CDKL5 mutations are well described in children, adults with CDD are still under-characterized. Similarly, most animal research has been carried out on young adult Cdkl5 knockout (KO) mice only. Since age represents a risk factor for the worsening of symptoms in many neurodevelopmental disorders, understanding age differences in the development of behavioral deficits is crucial in order to optimize the impact of therapeutic interventions. Here, we compared young adult Cdkl5 KO mice with middle-aged Cdkl5 KO mice, at a behavioral, neuroanatomical, and molecular level. We found an age-dependent decline in motor, cognitive, and social behaviors in Cdkl5 KO mice, as well as in breathing and sleep patterns. The behavioral decline in older Cdkl5 KO mice was not associated with a worsening of neuroanatomical alterations, such as decreased dendritic arborization or spine density, but was paralleled by decreased neuronal survival in different brain regions such as the hippocampus, cortex, and basal ganglia. Interestingly, we found increased ß-galactosidase activity and DNA repair protein levels, γH2AX and XRCC5, in the brains of older Cdkl5 KO mice, which suggests that an absence of Cdkl5 accelerates neuronal senescence/death by triggering irreparable DNA damage. In summary, this work provides evidence that CDKL5 may play a fundamental role in neuronal survival during brain aging and suggests a possible worsening with age of the clinical picture in CDD patients.

MiR-29 coordinates age-dependent plasticity brakes in the adult visual cortex.

Visual cortical circuits show profound plasticity during early life and are later stabilized by molecular "brakes" limiting excessive rewiring beyond a critical period. The mechanisms coordinating the expression of these factors during the transition from development to adulthood remain unknown. We found that miR-29a expression in the visual cortex dramatically increases with age, but it is not experience-dependent. Precocious high levels of miR-29a blocked ocular dominance plasticity and caused an early appearance of perineuronal nets. Conversely, inhibition of miR-29a in adult mice using LNA antagomirs activated ocular dominance plasticity, reduced perineuronal nets, and restored their juvenile chemical composition. Activated adult plasticity had the typical functional and proteomic signature of critical period plasticity. Transcriptomic and proteomic studies indicated that miR-29a manipulation regulates the expression of plasticity brakes in specific cortical circuits. These data indicate that miR-29a is a regulator of the plasticity brakes promoting age-dependent stabilization of visual cortical connections.