The Shank family of proteins interacts with NMDA receptor and metabotropic glutamate receptor complexes in the postsynaptic density (PSD). Targeted to the PSD by a PDZ-dependent mechanism, Shank promotes the maturation of dendritic spines and the enlargement of spine heads via its ability to recruit Homer to postsynaptic sites. Shank and Homer cooperate to induce accumulation of IP3 receptors in dendritic spines and formation of putative multisynapse spines. In addition, postsynaptic expression of Shank enhances presynaptic function, as measured by increased minifrequency and FM4-64 uptake. These data suggest a central role for the Shank scaffold in the structural and functional organization of the dendritic spine and synaptic junction.

Brain circuits process information through specialized neuronal subclasses interacting within a network. Revealing their interplay requires activating specific cells while monitoring others in a functioning circuit. Here we use a new platform for two-way light-based circuit interrogation in visual cortex in vivo to show the computational implications of modulating different subclasses of inhibitory neurons during sensory processing. We find that soma-targeting, parvalbumin-expressing (PV) neurons principally divide responses but preserve stimulus selectivity, whereas dendrite-targeting, somatostatin-expressing (SOM) neurons principally subtract from excitatory responses and sharpen selectivity. Visualized in vivo cell-attached recordings show that division by PV neurons alters response gain, whereas subtraction by SOM neurons shifts response levels. Finally, stimulating identified neurons while scanning many target cells reveals that single PV and SOM neurons functionally impact only specific subsets of neurons in their projection fields. These findings provide direct evidence that inhibitory neuronal subclasses have distinct and complementary roles in cortical computations.

IGF-1 ͉ plasticity ͉ autism ͉ brain ͉ synapse

A fundamental question in synaptic physiology is whether the unitary strength of a synapse can be regulated by presynaptic characteristics and, if so, what those characteristics might be. Here, we characterize a newly proposed mechanism for altering the strength of glutamatergic synapses based on the recently identified vesicular glutamate transporter VGLUT1. We provide direct evidence that filling in isolated synaptic vesicles is subject to a dynamic equilibrium that is determined by both the concentration of available glutamate and the number of vesicular transporters participating in loading. We observe that changing the number of vesicular transporters expressed at hippocampal excitatory synapses results in enhanced evoked and miniature responses and verify biophysically that these changes correspond to an increase in the amount of glutamate released per vesicle into the synaptic cleft. In addition, we find that this modulation of synaptic strength by vesicular transporter expression is endogenously regulated, both across development to coincide with a maturational increase in vesicle cycling and quantal amplitude and by excitatory and inhibitory receptor activation in mature neurons to provide an activity-dependent scaling of quantal size via a presynaptic mechanism. Together, these findings underscore that vesicular transporter expression is used endogenously to directly regulate the extent of glutamate release, providing a concise presynaptic mechanism for controlling the quantal efficacy of excitatory transmission during synaptic refinement and plasticity.

Summary Inhibitory interneurons in the cerebral cortex include a vast array of subtypes, varying in their molecular signatures, electrophysiological properties, and connectivity patterns. This diversity suggests that individual inhibitory classes have unique roles in cortical circuits; however, their characterization to date has been limited to broad classifications including many subtypes. We used the Cre/LoxP system, specifically labeling parvalbumin(PV)-expressing interneurons in visual cortex of PV-Cre mice with red fluorescent protein (RFP), followed by targeted loose-patch recordings and two-photon imaging of calcium responses in vivo to characterize the visual receptive field properties of these cells. Despite their relative molecular and morphological homogeneity, we find that PV+ neurons have a diversity of feature-specific visual responses that include sharp orientation and direction-selectivity, small receptive fields, and bandpass spatial frequency tuning. These results suggest that subsets of parvalbumin interneurons are components of specific cortical networks, and that perisomatic inhibition contributes to the generation of precise response properties.

Key Points• Recessive mutations in the thrombopoietin gene are a novel cause of aplastic anemia.• Such patients may benefit from treatment with eltrombopag or romiplostim.We recently identified 2 siblings afflicted with idiopathic, autosomal recessive aplastic anemia. Whole-exome sequencing identified a novel homozygous missense mutation in thrombopoietin (THPO, c.112C>T) in both affected siblings. This mutation encodes an arginine to cysteine substitution at residue 38 or residue 17 excluding the 21-amino acid signal peptide of THPO receptor binding domain (RBD). THPO has 4 conserved cysteines in its RBD that form 2 disulfide bonds. Our in silico modeling predicts that introduction of a fifth cysteine may disrupt normal disulfide bonding to cause poor receptor binding. In functional assays, the mutant-THPO-containing media shows two-to threefold reduced ability to sustain UT7-TPO cells, which require THPO for proliferation. Both parents and a sibling with heterozygous R17C change have reduced platelet counts, whereas a sibling with wild-type sequence has normal platelet count. Thus, the R17C partial loss-of-function allele results in aplastic anemia in the homozygous state and mild thrombocytopenia in the heterozygous state in our family. Together with the recent identification of THPO receptor (MPL) mutations and the effects of THPO agonists in aplastic anemia, our results have clinical implications in the diagnosis and treatment of patients with aplastic anemia and highlight a role for the THPO-MPL pathway in hematopoiesis in vivo.

Neurons in plastic regions of the brain undergo fundamental changes in the number of cells connecting to them as a result of development, plasticity and disease. Across these same time periods, functional changes in cellular and synaptic physiology are known to occur and are often characterized as developmental features of these periods. However, it remains possible that many such changes are direct consequences of the modified degree of partnering, and that neurons intrinsically scale their physiological parameters with network size. To systematically vary a recurrent network's number of neurons while measuring its synaptic properties, we used microfabricated extracellular matrix adhesive islands created with soft lithography to culture neuronal clusters of precise sizes, and assessed their intrinsic connectivity using intracellular recordings and confocal microscopy. Both large and small clusters supported constant densities of excitatory and inhibitory neurons. However, neurons that were provided with more potential partners (larger clusters) formed more connections per cell via an expanded dendritic surface than cocultured smaller clusters. Electrophysiologically, firing rate was preserved across clusters even as size and synapse number increased, due in part to synapses in larger networks having reduced unitary strengths, and sparser paired connectivity. Larger networks also featured a particular increase in the number of excitatory connections onto inhibitory dendrites. We suggest that these specific homeostatic mechanisms, which match the number, strength, and architecture of connections to the number of total available cellular partners in the network, could account for several known phenomena implicated in the formation, organization and degeneration of neuronal circuits.

Cranial neural crest cells (CNCCs) delaminate from embryonic neural folds and migrate to pharyngeal arches, which give rise to most mid-facial structures. CNCC dysfunction plays a prominent role in the etiology of orofacial clefts, a frequent birth malformation. Heterozygous mutations in SPECC1L have been identified in patients with atypical and syndromic clefts. Here, we report that in SPECC1L-knockdown cultured cells, staining of canonical adherens junction (AJ) components, β-catenin and E-cadherin, was increased, and electron micrographs revealed an apico-basal diffusion of AJs. To understand the role of SPECC1L in craniofacial morphogenesis, we generated a mouse model of Specc1l deficiency. Homozygous mutants were embryonic lethal and showed impaired neural tube closure and CNCC delamination. Staining of AJ proteins was increased in the mutant neural folds. This AJ defect is consistent with impaired CNCC delamination, which requires AJ dissolution. Further, PI3K-AKT signaling was reduced and apoptosis was increased in Specc1l mutants. In vitro, moderate inhibition of PI3K-AKT signaling in wildtype cells was sufficient to cause AJ alterations. Importantly, AJ changes induced by SPECC1L-knockdown were rescued by activating the PI3K-AKT pathway. Together, these data indicate SPECC1L as a novel modulator of PI3K-AKT signaling and AJ biology, required for neural tube closure and CNCC delamination.