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Modification of the strength of excitatory synaptic connections is a fundamental mechanism by which neural circuits are refined during development and learning. Synapse Differentiation Induced Gene 1 (SynDIG1) has been shown to play a key role in regulating synaptic strength in vitro . Here, we investigated the role of SynDIG1 in vivo in mice with a disruption of the SynDIG1 gene rather than use an alternate loxP-flanked conditional mutant that we find retains a partial protein product. The gene-trap insertion with a reporter cassette mutant mice shows that the SynDIG1 promoter is active during embryogenesis in the retina with some activity in the brain, and postnatally in the mouse hippocampus, cortex, hindbrain, and spinal cord. Ultrastructural analysis of the hippocampal CA1 region shows a decrease in the average PSD length of synapses and a decrease in the number of synapses with a mature phenotype. Intriguingly, the total synapse number appears to be increased in SynDIG1 mutant mice. Electrophysiological analyses show a decrease in AMPA and NMDA receptor function in SynDIG1 -deficient hippocampal neurons. Glutamate stimulation of individual dendritic spines in hippocampal slices from SynDIG1-deficient mice reveals increased short-term structural plasticity. Notably, the overall levels of PSD-95 or glutamate receptors enriched in postsynaptic biochemical fractions remain unaltered; however, activity-dependent synapse development is strongly compromised upon the loss of SynDIG1, supporting its importance for excitatory synapse maturation. Together, these data are consistent with a model in which SynDIG1 regulates the maturation of excitatory synapse structure and function in the mouse hippocampus in vivo .

Background: Periventricular leukomalacia (PVL) is the leading cause of neurological disabilities including motor and cognitive deficits in premature infants. Periventricular leukomalacia is characterized by damage to the white matter in the immature brain, but the mechanisms by which damage to immature white matter results in widespread deficits of cognitive and motor function are unclear. The thalamocortical system is crucial for human consciousness and cognitive functions, and impaired development of the cortico-thalamic projections in the neonatal period is implicated to contribute importantly to abnormalities of cognitive function in children with PVL., Results: In this study, using a mouse model of PVL, we sought to test the hypothesis that PVL-like injury affects the different components of the thalamocortical circuitry that can be defined by vesicular glutamate transporters 1 and 2 (vGluT1 and vGluT2), both of which are required for glutamatergic synaptic transmission in the central nervous system. We combined immunocytochemistry and immuno-electron microscopy to investigate changes in cortico-thalamic synapses which were specifically identified by vGluT1 immunolabeling. We found that a drastic reduction in the density of vGluT1 labeled profiles in the somatosensory thalamus, with a reduction of 72-74 % in ventroposterior (VP) nucleus and a reduction of 42-82 % in thalamic reticular nucleus (RTN) in the ipsilateral side of PVL mice. We further examined these terminals at the electron microscopic level and revealed onefold-twofold decrease in the sizes of vGluT1 labeled corticothalamic terminals in VP and RTN. The present study provides anatomical and ultrastructural evidence to elucidate the cellular mechanisms underlying alteration of thalamic circuitry in a mouse model of PVL, and reveals that PVL-like injury has a direct impact on the corticothalamic projection system., Conclusions: Our findings provide the first set of evidence showing that the thalamocortical circuitry is affected and vulnerable in PVL mice, supporting a working model in which vGluT1 defined corticothalamic synapses are altered in PVL mice, and vGluT2 defined thalamocortical synapses are associated with such changes, leading to the compromised thalamocortical circuitry in the PVL mice. Our study demonstrates that the thalamocortical circuitry is highly vulnerable to hypoxia-ischemia in the PVL model, thus identifying a novel target site in PVL pathology.

The biology of cerebral white matter injury has been woefully understudied, in part because of the difficulty of reliably modeling this type of injury in rodents. Periventricular leukomalacia (PVL) is the predominant form of brain injury and the most common cause of cerebral palsy in premature infants. PVL is characterized by predominant white matter injury. No specific therapy for PVL is presently available, because the pathogenesis is not well understood. Here we report that two types of mouse PVL models have been created by hypoxia-ischemia with or without systemic coadministration of lipopolysaccharide (LPS). LPS coadministration exacerbated hypoxic-ischemic white matter injury and led to enhanced microglial activation and astrogliosis. Drug trials with the antiinflammatory agent minocycline, the antiexcitotoxic agent NBQX, and the antioxidant agent edaravone showed various degrees of protection in the two models, indicating that excitotoxic, oxidative, and inflammatory forms of injury are involved in the pathogenesis of injury to immature white matter. We then applied immunoelectron microscopy to reveal fine structural changes in the injured white matter and found that synapses between axons and oligodendroglial precursor cells (OPCs) are quickly and profoundly damaged. Hypoxia-ischemia caused a drastic decrease in the number of postsynaptic densities associated with the glutamatergic axon-OPC synapses defined by the expression of vesicular glutamate transporters, vGluT1 and vGluT2, on axon terminals that formed contacts with OPCs in the periventricular white matter, resulted in selective shrinkage of the postsynaptic OPCs contacted by vGluT2 labeled synapses, and led to excitotoxicity mediated by GluR2-lacking, Ca(2+) -permeable AMPA receptors. Overall, the present study provides novel mechanistic insights into the pathogenesis of PVL and reveals that axon-glia synapses are highly vulnerable to white matter injury in the developing brain. More broadly, the study of white matter development and injury has general implications for a variety of neurological diseases, including PVL, stroke, spinal cord injury, and multiple sclerosis., (Copyright © 2011 Wiley Periodicals, Inc.)

Major histocompatibility complex class I (MHCI) molecules modulate activity-dependent refinement and plasticity. We found that MHCI also negatively regulates the density and function of cortical synapses during their initial establishment both in vitro and in vivo. MHCI molecules are expressed on cortical neurons before and during synaptogenesis. In vitro, decreasing surface MHCI (sMHCI) on neurons increased glutamatergic and GABAergic synapse density, whereas overexpression decreased it. In vivo, synapse density was higher throughout development in β2m(-/-) mice. MHCI also negatively regulated the strength of excitatory, but not inhibitory, synapses and controlled the balance of excitation and inhibition onto cortical neurons. sMHCI levels were modulated by activity and were necessary for activity to negatively regulate glutamatergic synapse density. Finally, acute changes in sMHCI and activity altered synapse density exclusively during early postnatal development. These results identify a previously unknown function for immune proteins in the negative regulation of the initial establishment and function of cortical connections.

Throughout the nervous system, neurons restrict their connections to specific depths or "layers" of their targets to constrain the type and number of synapses they make. Despite the importance of lamina-specific synaptic connectivity, the mechanisms that give rise to this feature in mammals remain poorly understood. Here we examined the cellular events underlying the formation of lamina-specific retinal ganglion cell (RGC) axonal projections to the superior colliculus (SC) of the mouse. By combining a genetically encoded marker of a defined RGC subtype (OFF-αRGCs) with serial immunoelectron microscopy, we resolved the ultrastructure of axon terminals fated for laminar stabilization versus those fated for removal. We found that OFF-αRGCs form synapses across the full depth of the retinorecipient SC before undergoing lamina-specific arbor retraction and synapse elimination to arrive at their mature, restricted pattern of connectivity. Interestingly, we did not observe evidence of axon degeneration or glia-induced synapse engulfment during this process. These findings indicate that lamina-specific visual connections are generated through the selective stabilization of correctly targeted axon arbors and suggest that the decision to maintain or eliminate an axonal projection reflects the molecular compatibility of presynaptic and postsynaptic neurons at a given laminar depth.

Immune molecules have been discovered recently to play critical roles in the development, function, and plasticity of the cerebral cortex. MHC class I (MHCI) molecules are expressed in the central nervous system and regulate activity-dependent refinement of visual projections during late postnatal development. They have also been implicated in neurodevelopmental diseases such as schizophrenia and autism. Despite the excitement generated by these unique roles for immune proteins in the brain, little is known about how these molecules regulate cortical connections. The first step toward elucidating the mechanism is to identify the spatial and temporal distribution of MHCI proteins throughout development. Using a pan-specific antibody that recognizes many MHCI variants for biochemistry and immunohistochemistry, we found that MHCI proteins are expressed in the rat visual cortex at all ages examined-during the peak of synaptogenesis, the critical period of synaptic refinement, and adulthood. Their abundance in the cortex peaked during early postnatal development, declining during periods of plasticity and adulthood. In contrast to current assumptions, pre- and postembedding immunogold electron microscopy (EM) revealed that MHCI proteins were present both pre- and postsynaptically at all ages examined. They were often found in the postsynaptic density and were closely associated with synaptic vesicles in the presynaptic terminal. These results suggest a previously undescribed model in which MHCI molecules function on both sides of the synapse to regulate connectivity in the mammalian visual cortex before, during, and after the establishment of connections.

New neurons are continuously generated in restricted regions of the adult mammalian brain. Although these adult-born neurons have been shown to receive synaptic inputs, little is known about their synaptic outputs. Using retrovirus-mediated birth-dating and labeling in combination with serial section electron microscopic reconstruction, we report that mossy fiber en passant boutons of adult-born dentate granule cells form initial synaptic contacts with CA3 pyramidal cells within 2 weeks after their birth and reach morphologic maturity within 8 weeks in the adult hippocampus. Knockdown of Disrupted-in-Schizophrenia-1 (DISC1) in newborn granule cells leads to defects in axonal targeting and development of synaptic outputs in the adult brain. Together with previous reports of synaptic inputs, these results demonstrate that adult-born neurons are fully integrated into the existing neuronal circuitry. Our results also indicate a role for DISC1 in presynaptic development and may have implications for the etiology of schizophrenia and related mental disorders.

Adult neurogenesis occurs throughout life in discrete regions of the adult mammalian brain. Little is known about the mechanism governing the sequential developmental process that leads to integration of new neurons from adult neural stem cells into the existing circuitry. Here, we investigated roles of Disrupted-In-Schizophrenia 1 (DISC1), a schizophrenia susceptibility gene, in adult hippocampal neurogenesis. Unexpectedly, downregulation of DISC1 leads to accelerated neuronal integration, resulting in aberrant morphological development and mispositioning of new dentate granule cells in a cell-autonomous fashion. Functionally, newborn neurons with DISC1 knockdown exhibit enhanced excitability and accelerated dendritic development and synapse formation. Furthermore, DISC1 cooperates with its binding partner NDEL1 in regulating adult neurogenesis. Taken together, our study identifies DISC1 as a key regulator that orchestrates the tempo of functional neuronal integration in the adult brain and demonstrates essential roles of a susceptibility gene for major mental illness in neuronal development, including adult neurogenesis.

Differential composition of GABA(A) receptor (GABA(A)R) subunits underlies the variability of fast inhibitory synaptic transmission; alteration of specific GABA(A)R subunits in localized brain regions may contribute to abnormal brain states such as absence epilepsy. We combined immunocytochemistry and high-resolution ImmunoGold electron microscopy to study cellular and subcellular localization of GABA(A)R alpha1, alpha3, and beta2/beta3 subunits in ventral posterior nucleus (VP) and reticular nucleus (RTN) of control rats and WAG/Rij rats, a genetic model of absence epilepsy. In control rats, alpha1 subunits were prominent at inhibitory synapses in VP and much less prominent in RTN; in contrast, the alpha3 subunit was highly evident at inhibitory synapses in RTN. beta2/beta3 subunits were evenly distributed at inhibitory synapses in both VP and RTN. ImmunoGold particles representing all subunits were concentrated at postsynaptic densities with no extrasynaptic localization. Calculated mean number of particles for alpha1 subunit per postsynaptic density in nonepileptic VP was 6.1 +/- 3.7, for alpha3 subunit in RTN it was 6.6 +/- 3.4, and for beta2/beta3 subunits in VP and RTN the mean numbers were 3.7 +/- 1.3 and 3.5 +/- 1.2, respectively. In WAG/Rij rats, there was a specific loss of alpha3 subunit immunoreactivity at inhibitory synapses in RTN, without reduction in alpha3 subunit mRNA or significant change in immunostaining for other markers of RTN cell identity such as GABA or parvalbumin. alpha3 immunostaining in cortex was unchanged. Subtle, localized changes in GABA(A)R expression acting at highly specific points in the interconnected thalamocortical network lie at the heart of idiopathic generalized epilepsy.

A key step in glutamatergic synapse maturation is the replacement of developmentally expressed N-methyl-D-aspartate receptors (NMDARs) with mature forms that differ in subunit composition, electrophysiological properties and propensity to elicit synaptic plasticity. However, the mechanisms underlying the removal and replacement of synaptic NMDARs are poorly understood. Here we demonstrate that NMDARs containing the developmentally regulated NR3A subunit undergo rapid endocytosis from the dendritic plasma membrane in cultured rat hippocampal neurons. This endocytic removal is regulated by PACSIN1/syndapin1, which directly and selectively binds the carboxy-terminal domain of NR3A through its NPF motifs and assembles a complex of proteins including dynamin and clathrin. Endocytosis of NR3A by PACSIN1 is activity dependent, and disruption of PACSIN1 function causes NR3A accumulation at synaptic sites. Our results reveal a new activity-dependent mechanism involved in the regulation of NMDAR expression at synapses during development, and identify a brain-specific endocytic adaptor that confers spatiotemporal and subunit specificity to NMDAR endocytosis.

Plexin signaling is required for stereotyped pruning of long axon collaterals in the vertebrate CNS; however, a cellular basis for plexins on stereotyped pruning has not been determined. Using quantitative electron microscopy and immunocytochemistry, we found that infrapyramidal mossy fiber axon collaterals form transient synaptic complexes with basal dendrites of CA3 pyramidal cells in the early postnatal mouse hippocampus. At later postnatal ages, these synaptic complexes stop maturing and are removed before stereotyped pruning by a mechanism that does not involve axon degeneration and glial cell engulfment. In knock-out mice that lack plexin-A3 signaling, the synaptic complexes continue to mature, and, as a result, the collaterals are not pruned. Thus, our results suggest that intact plexin-A3 signaling contributes to synaptic complex elimination, which is associated with stereotyped axon pruning.

Switching of the NMDA receptor 2A (NR2A) and NR2B subunits at NMDA receptors is thought to underlie the functional changes that occur in NMDA receptor properties during the developmental epoch when neural plasticity is most pronounced. The cellular expression of NR2A and NR2B and the NR2 synaptic binding protein postsynaptic density-95 (PSD-95) was examined in the mouse somatosensory cortex and thalamus from postnatal day 2 (P2) to P15 using reverse transcription-PCR, in situ hybridization histochemistry, and immunocytochemistry. The localization of NR2A and NR2B subunits and PSD-95 was then studied at synapses in layer IV of somatosensory cortex and in the ventral posterior nucleus of the thalamus using high-resolution immunoelectron microscopy. At both cortical and thalamic synapses, a quantitative switch in the dominant synaptic subunit from NR2B to NR2A was accompanied by a similar change in the cellular expression of NR2A but not of NR2B. Synaptic PSD-95 developed independently, although both NR2A and NR2B colocalized with PSD-95. Displacement of NR2B subunits from synapses was not accompanied by an increase in an extrasynaptic pool of this subunit. Thus, the switch in synaptic NR2 subunit predominance does not occur by changes in expression or displacement from synapses and may reflect the formation of new synapses from which NR2B is lacking.

The trafficking of glutamate receptors in neurons is of the utmost importance for synapse formation and synaptic plasticity. Recently, we demonstrated that both NMDA and AMPA receptors reside in mobile transport packets that are recruited rapidly and independently to nascent synapses. Here, we show that a large proportion of the glutamate receptor clusters in young cortical neurons are present on the surface of dendrites before synapses are formed and these surface-exposed transport packets are mobile. Exocytosis of glutamate receptors to the dendritic surface occurs via a SNARE [soluble n-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor]-dependent SNAP-23-mediated mechanism. Endocytosis occurs rapidly after surface exposure; >50% of surface-labeled NMDA receptors (NMDARs) are endocytosed within 5 min. NMDARs are transported along microtubules on large tubulovesicular organelles, as indicated by immunoelectron microscopy, and are associated with EEA1 (early endosomal antigen 1) and SAP102 (synapse-associated protein 102), as indicated by immunocytochemistry. Most surprisingly, a large proportion of these transport packets cycle through the dendritic plasma membrane before synapse formation. These results suggest a novel model in which NMDARs cycle with the plasma membrane during pauses of movement along microtubules while trafficking.

Mouse hippocampus retains the capacity for neurogenesis throughout lifetime, but such plasticity decreases with age. Adult hippocampal neurogenesis (AHN) involves the birth, maturation, and synaptic integration of newborn granule cells (GCs) into preexisting hippocampal circuitry. While functional integration onto adult-born GCs has been extensively studied, maturation of efferent projections onto CA3 pyramidal cells is less understood, particularly in aged brain. Here, using combined light and reconstructive electron microscopy (EM), we describe the maturation of mossy fiber bouton (MFB) connectivity with CA3 pyramidal cells in young adult and aged mouse brain. We found mature synaptic contacts of newborn GCs were formed in both young and aged brains. However, the dynamics of their spatiotemporal development and the cellular process by which these cells functionally integrated over time were different. In young brain newborn GCs either formed independent nascent MFB synaptic contacts or replaced preexisting MFBs, but these contacts were pruned over time to a mature state. In aged brain only replacement of preexisting MFBs was observed and new contacts were without evidence of pruning. These data illustrate that functional synaptic integration of AHN occurs in young adult and aged brain, but with distinct dynamics. They suggest elimination of preexisting connectivity is required for the integration of adult-born GCs in aged brain.

UnlabelledArginase 1 deficiency is a urea cycle disorder associated with hyperargininemia, spastic diplegia, loss of ambulation, intellectual disability, and seizures. To gain insight on how loss of arginase expression affects the excitability and synaptic connectivity of the cortical neurons in the developing brain, we used anatomical, ultrastructural, and electrophysiological techniques to determine how single-copy and double-copy arginase deletion affects cortical circuits in mice. We find that the loss of arginase 1 expression results in decreased dendritic complexity, decreased excitatory and inhibitory synapse numbers, decreased intrinsic excitability, and altered synaptic transmission in layer 5 motor cortical neurons. Hepatic arginase 1 gene therapy using adeno-associated virus rescued nearly all these abnormalities when administered to neonatal homozygous knock-out animals. Therefore, gene therapeutic strategies can reverse physiological and anatomical markers of arginase 1 deficiency and therefore may be of therapeutic benefit for the neurological disabilities in this syndrome.Significance statementThese studies are one of the few investigations to try to understand the underlying neurological dysfunction that occurs in urea cycle disorders and the only to examine arginase deficiency. We have demonstrated by multiple modalities that, in murine layer 5 cortical neurons, a gradation of abnormalities exists based on the functional copy number of arginase: intrinsic excitability is altered, there is decreased density in asymmetrical and perisomatic synapses, and analysis of the dendritic complexity is lowest in the homozygous knock-out. With neonatal administration of adeno-associated virus expressing arginase, there is near-total recovery of the abnormalities in neurons and cortical circuits, supporting the concept that neonatal gene therapy may prevent the functional abnormalities that occur in arginase deficiency.