Kuan Hong Wang

Slit Proteins Bind Robo Receptors and Have an Evolutionarily Conserved Role in Repulsive Axon Guidance

Extending axons in the developing nervous system are guided in part by repulsive cues. Genetic analysis in Drosophila, reported in a companion to this paper, identifies the Slit protein as a candidate ligand for the repulsive guidance receptor Roundabout (Robo). Here we describe the characterization of three mammalian Slit homologs and show that the Drosophila Slit protein and at least one of the mammalian Slit proteins, Slit2, are proteolytically processed and show specific, high-affinity binding to Robo proteins. Furthermore, recombinant Slit2 can repel embryonic spinal motor axons in cell culture. These results support the hypothesis that Slit proteins have an evolutionarily conserved role in axon guidance as repulsive ligands for Robo receptors.

Functional annotation of a full-length mouse cDNA collection

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

Biochemical Purification of a Mammalian Slit Protein as a Positive Regulator of Sensory Axon Elongation and Branching

Many neurons in both vertebrates and invertebrates innervate multiple targets by sprouting secondary axon collaterals (or branches) from a primary axon shaft. To begin to identify molecular regulators of axon branch initiation or extension, we studied the growth of single sensory axons in an in vitro collagen assay system and identified an activity in extracts of embryonic spinal cord and of postnatal and adult brain that promotes the elongation and formation of extensive branches by these axons. Biochemical purification of the activity from calf brain extracts led to the identification of an amino-terminal fragment of Slit2 as the main active component and to the discovery of a distinct activity that potentiates its effects. These results indicate that Slit proteins may function as positive regulators of axon collateral formation during the establishment or remodeling of neural circuits.

In Vivo Two-Photon Imaging Reveals a Role of Arc in Enhancing Orientation Specificity in Visual Cortex

Cortical representations of visual information are modified by an animals visual experience. To investigate the mechanisms in mice, we replaced the coding part of the neural activity-regulated immediate early gene Arc with a GFP gene and repeatedly monitored visual experience-induced GFP expression in adult primary visual cortex by in vivo two-photon microscopy. In Arc-positive GFP heterozygous mice, the pattern of GFP-positive cells exhibited orientation specificity. Daily presentations of the same stimulus led to the reactivation of a progressively smaller population with greater reactivation reliability. This adaptation process was not affected by the lack of Arc in GFP homozygous mice. However, the number of GFP-positive cells with low orientation specificity was greater, and the average spike tuning curve was broader in the adult homozygous compared to heterozygous or wild-type mice. These results suggest a physiological function of Arc in enhancing the overall orientation specificity of visual cortical neurons during the post-eye-opening life of an animal.

Loss of Arc renders the visual cortex impervious to the effects of sensory experience or deprivation

A myriad of mechanisms have been suggested to account for the full richness of visual cortical plasticity. We found that visual cortex lacking Arc is impervious to the effects of deprivation or experience. Using intrinsic signal imaging and chronic visually evoked potential recordings, we found that Arc−/− mice did not exhibit depression of deprived-eye responses or a shift in ocular dominance after brief monocular deprivation. Extended deprivation also failed to elicit a shift in ocular dominance or open-eye potentiation. Moreover, Arc−/− mice lacked stimulus-selective response potentiation. Although Arc−/− mice exhibited normal visual acuity, baseline ocular dominance was abnormal and resembled that observed after dark-rearing. These data suggest that Arc is required for the experience-dependent processes that normally establish and modify synaptic connections in visual cortex.

Optogenetic Inactivation Modifies Monkey Visuomotor Behavior

A critical technique for understanding how neuronal activity contributes to behavior is determining whether perturbing it changes behavior. The advent of optogenetic techniques allows the immediately reversible alteration of neuronal activity in contrast to chemical approaches lasting minutes to hours. Modification of behavior using optogenetics has had substantial success in rodents but has not been as successful in monkeys. Here, we show how optogenetic inactivation of superior colliculus neurons in awake monkeys leads to clear and repeatable behavioral deficits in the metrics of saccadic eye movements. We used our observations to evaluate principles governing the use of optogenetic techniques in the study of the neuronal bases of behavior in monkeys, particularly how experimental design must address relevant parameters, such as the application of light to subcortical structures, the spread of viral injections, and the extent of neuronal inactivation with light.

Molecular mechanisms underlying neural circuit formation

The functions of the nervous system are mediated by neural circuits that are formed during development and modulated by experiences. Central to the assembly of neural circuits is the regulation of synaptic connectivity by synaptic molecules and neuronal activity. Extensive studies have focused on identifying molecules involved in synapse formation. Besides factors promoting synaptogenesis, several antisynaptogenic factors have been discovered. These factors act in concert to ensure the timing and specificity of circuit formation. Moreover, progress has been made in revealing how neuronal activity governs the balance of excitatory and inhibitory synapses. Intriguingly, several transcription factors, as well as activity-dependent transcription of BDNF through promoter IV, have been shown to selectively regulate cortical inhibitory circuits by promoting GABAergic synapse formation.

Motor Learning Consolidates Arc-Expressing Neuronal Ensembles in Secondary Motor Cortex

Motor behaviors recruit task-specific neuronal ensembles in motor cortices, which are consolidated over subsequent learning. However, little is known about the molecules that can identify the participating neurons and predict the outcomes of the consolidation process. Using a mouse rotarod-learning task, we showed that lesion or inactivation of the secondary motor (M2) cortex disrupts learning of skilled movements. We tracked the endogenous promoter activity of the neuronal activity-regulated gene Arc in individual M2 neurons during rotarod learning by in vivo two-photon imaging of a knockin reporter. We found that task training initially recruits Arc-promoter-activated neurons and then consolidates them into a specific ensemble exhibiting persistent reactivation of Arc-promoter. The intensity of a neurons initial Arc-promoter activation predicts its reactivation probability and neurons with weak initial Arc-promoter activation are dismissed from the ensemble during subsequent training. Our findings demonstrate a task-specific Arc-dependent cellular consolidation process in M2 cortex during motor learning.

Dopamine, cognitive function, and gamma oscillations: role of D4 receptors

Cognitive deficits in individuals with schizophrenia (SCZ) are considered core symptoms of this disorder, and can manifest at the prodromal stage. Antipsychotics ameliorate positive symptoms but only modestly improve cognitive symptoms. The lack of treatments that improve cognitive abilities currently represents a major obstacle in developing more effective therapeutic strategies for this debilitating disorder. While D4 receptor (D4R)-specific antagonists are ineffective in the treatment of positive symptoms, animal studies suggest that D4R drugs can improve cognitive deficits. Moreover, recent work from our group suggests that D4Rs synergize with the neuregulin/ErbB4 signaling pathway, genetically identified as risk factors for SCZ, in parvalbumin (PV)-expressing interneurons to modulate gamma oscillations. These high-frequency network oscillations correlate with attention and increase during cognitive tasks in healthy subjects, and this correlation is attenuated in affected individuals. This finding, along with other observations indicating impaired GABAergic function, has led to the idea that abnormal neural activity in the prefrontal cortex (PFC) in individuals with SCZ reflects a perturbation in the balance of excitation and inhibition. Here we review the current state of knowledge of D4R functions in the PFC and hippocampus, two major brain areas implicated in SCZ. Special emphasis is given to studies focusing on the potential role of D4Rs in modulating GABAergic transmission and to an emerging concept of a close synergistic relationship between dopamine/D4R and neuregulin/ErbB4 signaling pathways that tunes the activity of PV interneurons to regulate gamma frequency network oscillations and potentially cognitive processes.

Genetic Disruption of Arc/Arg3.1 in Mice Causes Alterations in Dopamine and Neurobehavioral Phenotypes Related to Schizophrenia

Human genetic studies have recently suggested that the postsynaptic activity-regulated cytoskeleton-associated protein (Arc) complex is a convergence signal for several genes implicated in schizophrenia. However, the functional significance of Arc in schizophrenia-related neurobehavioral phenotypes and brain circuits is unclear. Here, we find that, consistent with schizophrenia-related phenotypes, disruption of Arc in mice produces deficits in sensorimotor gating, cognitive functions, social behaviors, and amphetamine-induced psychomotor responses. Furthermore, genetic disruption of Arc leads to concomitant hypoactive mesocortical and hyperactive mesostriatal dopamine pathways. Application of a D1 agonist to the prefrontal cortex or a D2 antagonist in the ventral striatum rescues Arc-dependent cognitive or psychomotor abnormalities, respectively. Our findings demonstrate a role for Arc in the regulation of dopaminergic neurotransmission and related behaviors. The results also provide initial biological support implicating Arc in dopaminergic and behavioral abnormalities related to schizophrenia.