Transient Impairment in Microglial Function Causes Sex-Specific Deficits in Synaptic and Hippocampal Function in Mice Exposed to Early Adversity.

Abnormal development and function of the hippocampus are two of the most consistent findings in humans and rodents exposed to early life adversity, with males often being more affected than females. Using the limited bedding (LB) paradigm as a rodent model of early life adversity, we found that male adolescent mice that had been exposed to LB exhibit significant deficits in contextual fear conditioning and synaptic connectivity in the hippocampus, which are not observed in females. This is linked to altered developmental refinement of connectivity, with LB severely impairing microglial-mediated synaptic pruning in the hippocampus of male and female pups on postnatal day 17 (P17), but not in adolescent P33 mice when levels of synaptic engulfment by microglia are substantially lower. Since the hippocampus undergoes intense synaptic pruning during the second and third weeks of life, we investigated whether microglia are required for the synaptic and behavioral aberrations observed in adolescent LB mice. Indeed, transient ablation of microglia from P13-21, in normally developing mice caused sex-specific behavioral and synaptic abnormalities similar to those observed in adolescent LB mice. Furthermore, chemogenetic activation of microglia during the same period reversed the microglial-mediated phagocytic deficits at P17 and restored normal contextual fear conditioning and synaptic connectivity in adolescent LB male mice. Our data support an additional contribution of astrocytes in the sex-specific effects of LB, with increased expression of the membrane receptor MEGF10 and enhanced synaptic engulfment in hippocampal astrocytes of 17-day-old LB females, but not in LB male littermates. This finding suggests a potential compensatory mechanism that may explain the relative resilience of LB females. Collectively, these studies highlight a novel role for glial cells in mediating sex-specific hippocampal deficits in a mouse model of early-life adversity.

A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell-cell contacts.

Junctions between the ER and the plasma membrane (ER/PM junctions) are implicated in calcium homeostasis, non-vesicular lipid transfer and other cellular functions. Two ER proteins that function both as membrane tethers to the PM via a polybasic motif in their C-terminus and as phospholipid transporters are brain-enriched TMEM24 (C2CD2L) and its paralog C2CD2. Based on an unbiased proximity ligation analysis, we found that both proteins can also form a complex with band 4.1 family members, which in turn can bind a variety of plasma membrane proteins including cell adhesion molecules such as SynCAM 1. This complex results in the enrichment of TMEM24 and C2CD2 containing ER/PM junctions at sites of cell contacts. Dynamic properties of TMEM24-dependent ER/PM contacts are impacted when in complex as TMEM24 present at cell adjacent junctions is not shed by calcium rise, unlike TMEM24 at non-cell adjacent junctions. These findings suggest that cell-contact interactions control ER/PM junctions via TMEM24 complexes involving band 4.1 proteins.

The human milk component myo-inositol promotes neuronal connectivity.

Effects of micronutrients on brain connectivity are incompletely understood. Analyzing human milk samples across global populations, we identified the carbocyclic sugar myo-inositol as a component that promotes brain development. We determined that it is most abundant in human milk during early lactation when neuronal connections rapidly form in the infant brain. Myo-inositol promoted synapse abundance in human excitatory neurons as well as cultured rat neurons and acted in a dose-dependent manner. Mechanistically, myo-inositol enhanced the ability of neurons to respond to transsynaptic interactions that induce synapses. Effects of myo-inositol in the developing brain were tested in mice, and its dietary supplementation enlarged excitatory postsynaptic sites in the maturing cortex. Utilizing an organotypic slice culture system, we additionally determined that myo-inositol is bioactive in mature brain tissue, and treatment of organotypic slices with this carbocyclic sugar increased the number and size of postsynaptic specializations and excitatory synapse density. This study advances our understanding of the impact of human milk on the infant brain and identifies myo-inositol as a breast milk component that promotes the formation of neuronal connections.

CADM2 is implicated in impulsive personality and numerous other traits by genome- and phenome-wide association studies in humans and mice.

Impulsivity is a multidimensional heritable phenotype that broadly refers to the tendency to act prematurely and is associated with multiple forms of psychopathology, including substance use disorders. We performed genome-wide association studies (GWAS) of eight impulsive personality traits from the Barratt Impulsiveness Scale and the short UPPS-P Impulsive Personality Scale (N = 123,509-133,517 23andMe research participants of European ancestry), and a measure of Drug Experimentation (N = 130,684). Because these GWAS implicated the gene CADM2, we next performed single-SNP phenome-wide studies (PheWAS) of several of the implicated variants in CADM2 in a multi-ancestral 23andMe cohort (N = 3,229,317, European; N = 579,623, Latin American; N = 199,663, African American). Finally, we produced Cadm2 mutant mice and used them to perform a Mouse-PheWAS ("MouseWAS") by testing them with a battery of relevant behavioral tasks. In humans, impulsive personality traits showed modest chip-heritability (~6-11%), and moderate genetic correlations (rg = 0.20-0.50) with other personality traits, and various psychiatric and medical traits. We identified significant associations proximal to genes such as TCF4 and PTPRF, and also identified nominal associations proximal to DRD2 and CRHR1. PheWAS for CADM2 variants identified associations with 378 traits in European participants, and 47 traits in Latin American participants, replicating associations with risky behaviors, cognition and BMI, and revealing novel associations including allergies, anxiety, irritable bowel syndrome, and migraine. Our MouseWAS recapitulated some of the associations found in humans, including impulsivity, cognition, and BMI. Our results further delineate the role of CADM2 in impulsivity and numerous other psychiatric and somatic traits across ancestries and species.

Concerted roles of LRRTM1 and SynCAM 1 in organizing prefrontal cortex synapses and cognitive functions.

Multiple trans-synaptic complexes organize synapse development, yet their roles in the mature brain and cooperation remain unclear. We analyzed the postsynaptic adhesion protein LRRTM1 in the prefrontal cortex (PFC), a region relevant to cognition and disorders. LRRTM1 knockout (KO) mice had fewer synapses, and we asked whether other synapse organizers counteract further loss. This determined that the immunoglobulin family member SynCAM 1 controls synapse number in PFC and was upregulated upon LRRTM1 loss. Combined LRRTM1 and SynCAM 1 deletion substantially lowered dendritic spine number in PFC, but not hippocampus, more than the sum of single KO impairments. Their cooperation extended presynaptically, and puncta of Neurexins, LRRTM1 partners, were less abundant in double KO (DKO) PFC. Electrophysiology and fMRI demonstrated aberrant neuronal activity in DKO mice. Further, DKO mice were impaired in social interactions and cognitive tasks. Our results reveal concerted roles of LRRTM1 and SynCAM 1 across synaptic, network, and behavioral domains.

A synaptomic analysis reveals dopamine hub synapses in the mouse striatum.

Dopamine transmission is involved in reward processing and motor control, and its impairment plays a central role in numerous neurological disorders. Despite its strong pathophysiological relevance, the molecular and structural organization of the dopaminergic synapse remains to be established. Here, we used targeted labelling and fluorescence activated sorting to purify striatal dopaminergic synaptosomes. We provide the proteome of dopaminergic synapses with 57 proteins specifically enriched. Beyond canonical markers of dopamine neurotransmission such as dopamine biosynthetic enzymes and cognate receptors, we validated 6 proteins not previously described as enriched. Moreover, our data reveal the adhesion of dopaminergic synapses to glutamatergic, GABAergic or cholinergic synapses in structures we named "dopamine hub synapses". At glutamatergic synapses, pre- and postsynaptic markers are significantly increased upon association with dopamine synapses. Dopamine hub synapses may thus support local dopaminergic signalling, complementing volume transmission thought to be the major mechanism by which monoamines modulate network activity.

Subsynaptic positioning of AMPARs by LRRTM2 controls synaptic strength.

Recent evidence suggests that nano-organization of proteins within synapses may control the strength of communication between neurons in the brain. The unique subsynaptic distribution of glutamate receptors, which cluster in nanoalignment with presynaptic sites of glutamate release, supports this hypothesis. However, testing it has been difficult because mechanisms controlling subsynaptic organization remain unknown. Reasoning that transcellular interactions could position AMPA receptors (AMPARs), we targeted a key transsynaptic adhesion molecule implicated in controlling AMPAR number, LRRTM2, using engineered, rapid proteolysis. Severing the LRRTM2 extracellular domain led quickly to nanoscale declustering of AMPARs away from release sites, not prompting their escape from synapses until much later. This rapid remodeling of AMPAR position produced significant deficits in evoked, but not spontaneous, postsynaptic receptor activation. These results dissociate receptor numbers from their nanopositioning in determination of synaptic function and support the novel concept that adhesion molecules acutely position receptors to dynamically control synaptic strength.

Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution.

Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.

Synaptic recognition molecules in development and disease.

Synaptic connectivity patterns underlie brain functions. How recognition molecules control where and when neurons form synapses with each other, therefore, is a fundamental question of cellular neuroscience. This chapter delineates adhesion and signaling complexes as well as secreted factors that contribute to synaptic partner recognition in the vertebrate brain. The sections follow a developmental perspective and discuss how recognition molecules (1) guide initial synaptic wiring, (2) provide for the rejection of incorrect partner choices, (3) contribute to synapse specification, and (4) support the removal of inappropriate synapses once formed. These processes involve a rich repertoire of molecular players and key protein families are described, notably the Cadherin and immunoglobulin superfamilies, Semaphorins/Plexins, Leucine-rich repeat containing proteins, and Neurexins and their binding partners. Molecular themes that diversify these recognition systems are defined and highlighted throughout the text, including the neuron-type specific expression and combinatorial action of recognition factors, alternative splicing, and post-translational modifications. Methodological innovations advancing the field such as proteomic approaches and single cell expression studies are additionally described. Further, the chapter highlights the importance of choosing an appropriate brain region to analyze synaptic recognition factors and the advantages offered by laminated structures like the hippocampus or retina. In a concluding section, the profound disease relevance of aberrant synaptic recognition for neurodevelopmental and psychiatric disorders is discussed. Based on the current progress, an outlook is presented on research goals that can further advance insights into how recognition molecules provide for the astounding precision and diversity of synaptic connections.

FARP-1 deletion is associated with lack of response to autism treatment by early start denver model in a multiplex family.

BACKGROUND: Children with autism spectrum disorder (ASD) display impressive clinical heterogeneity, also involving treatment response. Genetic variants can contribute to explain this large interindividual phenotypic variability. METHODS: Array-CGH (a-CGH) and whole genome sequencing (WGS) were performed on a multiplex family with two small children diagnosed with ASD at 17 and 18 months of age. Both brothers received the same naturalistic intervention for one year according to the Early Start Denver Model (ESDM), applied by the same therapists, yielding dramatically different treatment outcomes. RESULTS: The older sibling came out of the autism spectrum, while the younger sibling displayed very little, in any, improvement. This boy was subsequently treated applying a structured Early Intensive Behavioral Intervention paired with Augmentative Alternative Communication, which yielded a partial response within another year. The ESDM nonresponsive child carries a novel maternally inherited 65 Kb deletion at chr. 13q32.2 spanning FARP1. Farp1 is a synaptic scaffolding protein, which plays a significant role in neural plasticity. CONCLUSION: These results represent a paradigmatic example of the heuristic potential of genetic markers in predicting treatment response and possibly in supporting the targeted prescription of specific early intervention approaches.

Synaptic Connectivity and Cortical Maturation Are Promoted by the ω-3 Fatty Acid Docosahexaenoic Acid.

Brain development is likely impacted by micronutrients. This is supported by the effects of the ω-3 fatty acid docosahexaenoic acid (DHA) during early neuronal differentiation, when it increases neurite growth. Aiming to delineate DHA roles in postnatal stages, we selected the visual cortex due to its stereotypic maturation. Immunohistochemistry showed that young mice that received dietary DHA from birth exhibited more abundant presynaptic and postsynaptic specializations. DHA also increased density and size of synapses in a dose-dependent manner in cultured neurons. In addition, dendritic arbors of neurons treated with DHA were more complex. In agreement with improved connectivity, DHA enhanced physiological parameters of network maturation in vitro, including bursting strength and oscillatory behavior. Aiming to analyze functional maturation of the cortex, we performed in vivo electrophysiological recordings from awake mice to measure responses to patterned visual inputs. Dietary DHA robustly promoted the developmental increase in visual acuity, without altering light sensitivity. The visual acuity of DHA-supplemented animals continued to improve even after their cortex had matured and DHA abolished the acuity plateau. Our findings show that the ω-3 fatty acid DHA promotes synaptic connectivity and cortical processing. These results provide evidence that micronutrients can support the maturation of neuronal networks.

Emerging Roles of Synapse Organizers in the Regulation of Critical Periods.

Experience remodels cortical connectivity during developmental windows called critical periods. Experience-dependent regulation of synaptic strength during these periods establishes circuit functions that are stabilized as critical period plasticity wanes. These processes have been extensively studied in the developing visual cortex, where critical period opening and closure are orchestrated by the assembly, maturation, and strengthening of distinct synapse types. The synaptic specificity of these processes points towards the involvement of distinct molecular pathways. Attractive candidates are pre- and postsynaptic transmembrane proteins that form adhesive complexes across the synaptic cleft. These synapse-organizing proteins control synapse development and maintenance and modulate structural and functional properties of synapses. Recent evidence suggests that they have pivotal roles in the onset and closure of the critical period for vision. In this review, we describe roles of synapse-organizing adhesion molecules in the regulation of visual critical period plasticity and we discuss the potential they offer to restore circuit functions in amblyopia and other neurodevelopmental disorders.

Synapse-Selective Control of Cortical Maturation and Plasticity by Parvalbumin-Autonomous Action of SynCAM 1.

Cortical plasticity peaks early in life and tapers in adulthood, as exemplified in the primary visual cortex (V1), wherein brief loss of vision in one eye reduces cortical responses to inputs from that eye during the critical period but not in adulthood. The synaptic locus of cortical plasticity and the cell-autonomous synaptic factors determining critical periods remain unclear. We here demonstrate that the immunoglobulin protein Synaptic Cell Adhesion Molecule 1 (SynCAM 1/Cadm1) is regulated by visual experience and limits V1 plasticity. Loss of SynCAM 1 selectively reduces the number of thalamocortical inputs onto parvalbumin (PV+) interneurons, impairing the maturation of feedforward inhibition in V1. SynCAM 1 acts in PV+ interneurons to actively restrict cortical plasticity, and brief PV+-specific knockdown of SynCAM 1 in adult visual cortex restores juvenile-like plasticity. These results identify a synapse-specific, cell-autonomous mechanism for thalamocortical visual circuit maturation and closure of the visual critical period.

Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins.

Synapses are specialized neuronal cell-cell contacts that underlie network communication in the mammalian brain. Across neuronal populations and circuits, a diverse set of synapses is utilized, and they differ in their molecular composition to enable heterogenous connectivity patterns and functions. In addition to pre- and post-synaptic specializations, the synaptic cleft is now understood to be an integral compartment of synapses that contributes to their structural and functional organization. Aiming to map the cleft proteome, this study applied a peroxidase-mediated proximity labeling approach and used the excitatory synaptic cell adhesion protein SynCAM 1 fused to horseradish peroxidase (HRP) as a reporter in cultured cortical neurons. This reporter marked excitatory synapses as measured by confocal microcopy and was targeted to the edge zone of the synaptic cleft as determined using 3D dSTORM super-resolution imaging. Proximity labeling with a membrane-impermeant biotin-phenol compound restricted labeling to the cell surface, and Label-Free Quantitation (LFQ) mass spectrometry combined with ratiometric HRP tagging of membrane vs. synaptic surface proteins was used to identify the proteomic content of excitatory clefts. Novel cleft candidates were identified, and Receptor-type tyrosine-protein phosphatase zeta was selected and successfully validated. This study supports the robust applicability of peroxidase-mediated proximity labeling for synaptic cleft proteomics and its potential for understanding synapse heterogeneity in health and changes in diseases such as psychiatric disorders and addiction.

Social Stimulus Causes Aberrant Activation of the Medial Prefrontal Cortex in a Mouse Model With Autism-Like Behaviors.

Autism spectrum disorder (ASD) is a highly prevalent and genetically heterogeneous brain disorder. Developing effective therapeutic interventions requires knowledge of the brain regions that malfunction and how they malfunction during ASD-relevant behaviors. Our study provides insights into brain regions activated by a novel social stimulus and how the activation pattern differs between mice that display autism-like disabilities and control littermates. Adenomatous polyposis coli (APC) conditional knockout (cKO) mice display reduced social interest, increased repetitive behaviors and dysfunction of the ß-catenin pathway, a convergent target of numerous ASD-linked human genes. Here, we exposed the mice to a novel social vs. non-social stimulus and measured neuronal activation by immunostaining for the protein c-Fos. We analyzed three brain regions known to play a role in social behavior. Compared with control littermates, APC cKOs display excessive activation, as evidenced by an increased number of excitatory pyramidal neurons stained for c-Fos in the medial prefrontal cortex (mPFC), selectively in the infralimbic sub-region. In contrast, two other social brain regions, the medial amygdala and piriform cortex show normal levels of neuron activation. Additionally, APC cKOs exhibit increased frequency of miniature excitatory postsynaptic currents (mEPSCs) in layer 5 pyramidal neurons of the infralimbic sub-region. Further, immunostaining is reduced for the inhibitory interneuron markers parvalbumin (PV) and somatostatin (SST) in the APC cKO mPFC. Our findings suggest aberrant excitatory-inhibitory balance and activation patterns. As ß-catenin is a core pathway in ASD, we identify the infralimbic sub-region of the mPFC as a critical brain region for autism-relevant social behavior.

Structural analyses of FERM domain-mediated membrane localization of FARP1.

FARP1 is a multi-domain protein that is involved in regulating neuronal development through interacting with cell surface proteins such as class A Plexins and SynCAM 1. The N-terminal FERM domain in FARP1 is known to both promote membrane localization and mediate these protein interactions, for which the underlying molecular mechanisms remain unclear. Here we determined the crystal structures of the FERM domain of FARP1 from zebrafish, and those of FARP2 (a close homolog of FARP1) from mouse and zebrafish. These FERM domains adopt the three-leaved clover fold that is typical of all FERM domains. Our structures reveal a positively charged surface patch that is highly conserved in the FERM domain of FARP1 and FARP2. In vitro lipid-binding experiments showed that the FARP1 FERM domain binds specifically to several types of phospholipid, which is dependent on the positively charged surface patch. We further determined through cell-based analyses that this surface patch on the FERM domain underlies the localization of FARP1 to the plasma membrane, and that FERM domain interactions recruit it to postsynaptic sites in neurons.

Open Up to Make New Contacts: Caldendrin Senses Postsynaptic Calcium Influx to Dynamically Organize Dendritic Spines.

In this issue of Neuron, Mikhaylova et al. (2018) report how the Ca2+ sensor caldendrin interacts in dendritic spines with cortactin to control actin remodeling. Combining molecular and functional approaches, this work gains insights into postsynaptic dynamics relevant for synaptic plasticity.

Transcellular Nanoalignment of Synaptic Function.

At each of the brain's vast number of synapses, the presynaptic nerve terminal, synaptic cleft, and postsynaptic specialization form a transcellular unit to enable efficient transmission of information between neurons. While we know much about the molecular machinery within each compartment, we are only beginning to understand how these compartments are structurally registered and functionally integrated with one another. This review will describe the organization of each compartment and then discuss their alignment across pre- and postsynaptic cells at a nanometer scale. We propose that this architecture may allow for precise synaptic information exchange and may be modulated to contribute to the remarkable plasticity of brain function.

Reduced Insulin/Insulin-Like Growth Factor Receptor Signaling Mitigates Defective Dendrite Morphogenesis in Mutants of the ER Stress Sensor IRE-1.

Neurons receive excitatory or sensory inputs through their dendrites, which often branch extensively to form unique neuron-specific structures. How neurons regulate the formation of their particular arbor is only partially understood. In genetic screens using the multidendritic arbor of PVD somatosensory neurons in the nematode Caenorhabditis elegans, we identified a mutation in the ER stress sensor IRE-1/Ire1 (inositol requiring enzyme 1) as crucial for proper PVD dendrite arborization in vivo. We further found that regulation of dendrite growth in cultured rat hippocampal neurons depends on Ire1 function, showing an evolutionarily conserved role for IRE-1/Ire1 in dendrite patterning. PVD neurons of nematodes lacking ire-1 display reduced arbor complexity, whereas mutations in genes encoding other ER stress sensors displayed normal PVD dendrites, specifying IRE-1 as a selective ER stress sensor that is essential for PVD dendrite morphogenesis. Although structure function analyses indicated that IRE-1's nuclease activity is necessary for its role in dendrite morphogenesis, mutations in xbp-1, the best-known target of non-canonical splicing by IRE-1/Ire1, do not exhibit PVD phenotypes. We further determined that secretion and distal localization to dendrites of the DMA-1/leucine rich transmembrane receptor (DMA-1/LRR-TM) is defective in ire-1 but not xbp-1 mutants, suggesting a block in the secretory pathway. Interestingly, reducing Insulin/IGF1 signaling can bypass the secretory block and restore normal targeting of DMA-1, and consequently normal PVD arborization even in the complete absence of functional IRE-1. This bypass of ire-1 requires the DAF-16/FOXO transcription factor. In sum, our work identifies a conserved role for ire-1 in neuronal branching, which is independent of xbp-1, and suggests that arborization defects associated with neuronal pathologies may be overcome by reducing Insulin/IGF signaling and improving ER homeostasis and function.

Excitatory Synaptic Drive and Feedforward Inhibition in the Hippocampal CA3 Circuit Are Regulated by SynCAM 1.

UNLABELLED: Select adhesion proteins control the development of synapses and modulate their structural and functional properties. Despite these important roles, the extent to which different synapse-organizing mechanisms act across brain regions to establish connectivity and regulate network properties is incompletely understood. Further, their functional roles in different neuronal populations remain to be defined. Here, we applied diffusion tensor imaging (DTI), a modality of magnetic resonance imaging (MRI), to map connectivity changes in knock-out (KO) mice lacking the synaptogenic cell adhesion protein SynCAM 1. This identified reduced fractional anisotropy in the hippocampal CA3 area in absence of SynCAM 1. In agreement, mossy fiber refinement in CA3 was impaired in SynCAM 1 KO mice. Mossy fibers make excitatory inputs onto postsynaptic specializations of CA3 pyramidal neurons termed thorny excrescences and these structures were smaller in the absence of SynCAM 1. However, the most prevalent targets of mossy fibers are GABAergic interneurons and SynCAM 1 loss unexpectedly reduced the number of excitatory terminals onto parvalbumin (PV)-positive interneurons in CA3. SynCAM 1 KO mice additionally exhibited lower postsynaptic GluA1 expression in these PV-positive interneurons. These synaptic imbalances in SynCAM 1 KO mice resulted in CA3 disinhibition, in agreement with reduced feedforward inhibition in this network in the absence of SynCAM 1-dependent excitatory drive onto interneurons. In turn, mice lacking SynCAM 1 were impaired in memory tasks involving CA3. Our results support that SynCAM 1 modulates excitatory mossy fiber inputs onto both interneurons and principal neurons in the hippocampal CA3 area to balance network excitability. SIGNIFICANCE STATEMENT: This study advances our understanding of synapse-organizing mechanisms on two levels. First, the data support that synaptogenic proteins guide connectivity and can function in distinct brain regions even if they are expressed broadly. Second, the results demonstrate that a synaptogenic process that controls excitatory inputs to both pyramidal neurons and interneurons can balance excitation and inhibition. Specifically, the study reveals that hippocampal CA3 connectivity is modulated by the synapse-organizing adhesion protein SynCAM 1 and identifies a novel, SynCAM 1-dependent mechanism that controls excitatory inputs onto parvalbumin-positive interneurons. This enables SynCAM 1 to regulate feedforward inhibition and set network excitability. Further, we show that diffusion tensor imaging is sensitive to these cellular refinements affecting neuronal connectivity.