Determining the degree of synapse formation and elimination is essential for understanding the structural basis of brain plasticity and pathology. We show that in vivo imaging of dendritic spine dynamics through an open-skull glass window, but not a thinned-skull window, is associated with high spine turnover and substantial glial activation during the first month after surgery. These findings help to explain existing discrepancies in the degree of dendritic spine plasticity observed in the mature cortex.

Highlights d Knockdown of Ptbp1 converts Mu ¨ller glia into retinal ganglion cells in mature retinas d Central projections of converted retinal ganglion cells restore visual responses d Induction of neurons with dopaminergic features in PD model mice

A fundamental feature of the mammalian neocortex is its columnar organization1. In the visual cortex, functional columns consisting of neurons with similar orientation preference have been characterized extensively2-4, but how these columns are constructed during development remains unclear5. The ‘radial unit hypothesis’6 posits that the ontogenetic columns formed by clonally related neurons migrating along the same radial glial fiber during corticogenesis7 provide the basis for functional columns in adult neocortex1. However, direct correspondence between the ontogenetic and functional columns has not been demonstrated8. Here we show that, despite the lack of discernible orientation map in mouse visual cortex4,9,10, sister neurons in the same radial clone exhibit similar orientation preference. Using a retroviral vector encoding green fluorescent protein (GFP) to label radial clones of excitatory neurons and in vivo two-photon calcium imaging to measure the neuronal response properties, we found that sister neurons preferred similar orientations, while nearby non-sisters showed no such relationship. Interestingly, disruption of gap junction coupling by viral expression of a dominant-negative mutant of Cx26 or by daily administration of a gap junction blocker carbenoxolone (CBX) during the first postnatal week greatly diminished the functional similarity between sister neurons, suggesting that the maturation of ontogenetic into functional columns requires intercellular communication through gap junctions. Together with the recent finding of preferential excitatory connections among sister neurons11, our results support the radial unit hypothesis and unify the ontogenetic and functional columns in the visual cortex.

Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.

Neuronal migration and growth-cone extension are both guided by extracellular factors in the developing brain, but whether these two forms of guidance are mechanistically linked is unclear. Application of a Slit-2 gradient in front of the leading process of cultured cerebellar granule cells led to the collapse of the growth cone and the reversal of neuronal migration, an event preceded by a propagating Ca(2+) wave from the growth cone to the soma. The Ca(2+) wave was required for the Slit-2 effect and was sufficient by itself to induce the reversal of migration. The Slit-2-induced reversal of migration required active RhoA, which was accumulated at the front of the migrating neuron, and this polarized RhoA distribution was reversed during the migration reversal induced by either the Slit-2 gradient or the Ca(2+) wave. Thus, long-range Ca(2+) signaling coordinates the Slit-2-induced changes in motility at two distant parts of migrating neurons by regulating RhoA distribution.

The autism spectrum disorders (ASDs) are a collection of human neurological disorders with heterogeneous etiologies. Hyperactivity of E3 ubiquitin (Ub) ligase UBE3A, stemming from 15q11-q13 copy number variations, accounts for 1%-3% of ASD cases worldwide, but the underlying mechanisms remain incompletely characterized. Here we report that the functionality of ALDH1A2, the rate-limiting enzyme of retinoic acid (RA) synthesis, is negatively regulated by UBE3A in a ubiquitylation-dependent manner. Excessive UBE3A dosage was found to impair RA-mediated neuronal homeostatic synaptic plasticity. ASD-like symptoms were recapitulated in mice by overexpressing UBE3A in the prefrontal cortex or by administration of an ALDH1A antagonist, whereas RA supplements significantly alleviated excessive UBE3A dosage-induced ASD-like phenotypes. By identifying reduced RA signaling as an underlying mechanism in ASD phenotypes linked to UBE3A hyperactivities, our findings introduce a new vista of ASD etiology and facilitate a mode of therapeutic development against this increasingly prevalent disease.

SUMMARY The hippocampus, as part of the cerebral cortex, is essential for memory formation and spatial navigation. Although it has been extensively studied, especially as a model system for neurophysiology, the cellular processes involved in constructing and organizing the hippocampus remain largely unclear. Here, we show that clonally related excitatory neurons in the developing hippocampus are progressively organized into discrete horizontal, but not vertical, clusters in the stratum pyramidale, as revealed by both cell type-specific retroviral labeling and mosaic analysis with double markers (MADM). Moreover, distinct from those in the neocortex, sister excitatory neurons in Cornu Ammonis 1 region of the hippocampus rarely develop electrical or chemical synapses with each other. Instead, they preferentially receive common synaptic input from nearby fast-spiking (FS), but not non-FS, interneurons and exhibit synchronous synaptic activity. These results suggest that shared inhibitory input may specify horizontally clustered sister excitatory neurons as functional units in the hippocampus.

Migration of neuronal precursor cells in the developing brain is guided by extracellular cues, but intracellular signaling processes underlying the guidance of neuronal migration are largely unknown. By examining the migration of cerebellar granule neurons along the surface of cocultured astroglial cells, we found that an extracellular gradient of Slit2, a chemorepellant for neuronal migration in vivo, caused a reversal in the direction of migration without affecting the migration speed. A Slit2 gradient elevated the intracellular concentration of Ca 2؉ , probably due to calcium release from the internal store, led to a reversal of the preexisting asymmetric intracellular Ca 2؉ distribution in the soma of migrating neurons, and this reversal was closely related with its action of reversing the migrating direction. Asymmetric Ca 2؉ distribution in the soma was both necessary and sufficient for directing neuronal migration. These results have demonstrated an important role for Ca 2؉ in mediating neuronal responses to Slit2 and suggest a general mechanism for neuronal guidance. In the developing nervous system, a large number of neuronal precursor cells migrate from the site of birth to their destination along the surface of radial glial cells (1-3). Recent studies (4-6) have shown that several secreted or cell-surface proteins, including the netrins, Slits, and the Semaphorins can function as guidance cues for directing neuronal migration. However, most studies were performed on explants of brain tissue, and intracellular mechanisms mediating neuronal responses to these guidance cues are still poorly understood. Calcium signaling is known to be involved in regulating neuronal migration. In cultured explants of cerebellar tissue, granule cell migration correlates with intracellular Ca 2ϩ fluctuations and blockade of Ca 2ϩ influx retards migration (7-9). Voltage-gated Ca 2ϩ channels are essential for postembryonic neuronal migration in Caenorhabditis elegans (10). Slit2 is a well known secreted chemorepellant for migration of many types of neurons in the CNS (11-13). In the present study, the action of Slit2 on single-neuron migration in vitro was examined, and the role of calcium signal in mediating the repulsive action of Slit2 was explored. By using a cell culture model of migration of cerebellar granule cells on cocultured radial glial cells, we found that a gradient of Slit2 in front of the migrating neuron caused a reversal of the direction of migration without significantly affecting the subsequent migratory speed. We also found that a Slit2-induced Ca 2ϩ signaling appeared to mediate the repulsive action on neuronal migration, and that Ca 2ϩ release from intracellular store through inositol trisphosphate receptor channels played a major role in the Ca 2ϩ signaling. Materials and MethodsCoculture of Cells. The methods of coculturing granular cells and astroglia cells were similar to that described by Hatten (14). In short, astroglial cells from the cerebellum of P0-P3 SD rats isolated with a step gradient (35...