Gordon Wang

Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways

To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.

Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones

Ion channels formed by the TRP (transient receptor potential) superfamily of proteins act as sensors for temperature, osmolarity, mechanical stress and taste. The growth cones of developing axons are responsible for sensing extracellular guidance factors, many of which trigger Ca2+ influx at the growth cone; however, the identity of the ion channels involved remains to be clarified. Here, we report that TRP-like channel activity exists in the growth cones of cultured Xenopus neurons and can be modulated by exposure to netrin-1 and brain-derived neurotrophic factor, two chemoattractants for axon guidance. Whole-cell recording from growth cones showed that netrin-1 induced a membrane depolarization, part of which remained after all major voltage-dependent channels were blocked. Furthermore, the membrane depolarization was sensitive to blockers of TRP channels. Pharmacological blockade of putative TRP currents or downregulation of Xenopus TRP-1 (xTRPC1) expression with a specific morpholino oligonucleotide abolished the growth-cone turning and Ca2+ elevation induced by a netrin-1 gradient. Thus, TRPC currents reflect early events in the growth cones detection of some extracellular guidance signals, resulting in membrane depolarization and cytoplasmic Ca2+ elevation that mediates the turning of growth cones.

Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors

In the developing central nervous system (CNS), the control of synapse number and function is critical to the formation of neural circuits. We previously demonstrated that astrocyte-secreted factors powerfully induce the formation of functional excitatory synapses between CNS neurons. Astrocyte-secreted thrombospondins induce the formation of structural synapses, but these synapses are postsynaptically silent. Here we use biochemical fractionation of astrocyte-conditioned medium to identify glypican 4 (Gpc4) and glypican 6 (Gpc6) as astrocyte-secreted signals sufficient to induce functional synapses between purified retinal ganglion cell neurons, and show that depletion of these molecules from astrocyte-conditioned medium significantly reduces its ability to induce postsynaptic activity. Application of Gpc4 to purified neurons is sufficient to increase the frequency and amplitude of glutamatergic synaptic events. This is achieved by increasing the surface level and clustering, but not overall cellular protein level, of the GluA1 subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptor (AMPAR). Gpc4 and Gpc6 are expressed by astrocytes in vivo in the developing CNS, with Gpc4 expression enriched in the hippocampus and Gpc6 enriched in the cerebellum. Finally, we demonstrate that Gpc4-deficient mice have defective synapse formation, with decreased amplitude of excitatory synaptic currents in the developing hippocampus and reduced recruitment of AMPARs to synapses. These data identify glypicans as a family of novel astrocyte-derived molecules that are necessary and sufficient to promote glutamate receptor clustering and receptivity and to induce the formation of postsynaptically functioning CNS synapses.

Circadian and Homeostatic Regulation of Structural Synaptic Plasticity in Hypocretin Neurons

Neurons exhibit rhythmic activity that ultimately affects behavior such as sleep. In living zebrafish larvae, we used time-lapse two-photon imaging of the presynaptic marker synaptophysin in hypocretin/orexin (HCRT) neurons to determine the dynamics of synaptic modifications during the day and night. We observed circadian rhythmicity in synapse number in HCRT axons. This rhythm is regulated primarily by the circadian clock but is also affected by sleep deprivation. Furthermore, NPTX2, a protein implicated in AMPA receptor clustering, modulates circadian synaptic changes. In zebrafish, nptx2b is a rhythmic gene that is mostly expressed in hypothalamic and pineal gland cells. Arrhythmic transgenic nptx2b overexpression (hcrt:NPTX2b) increases synapse number and abolishes rhythmicity in HCRT axons. Finally, hcrt:NPTX2b fish are resistant to the sleep-promoting effects of melatonin. This behavioral effect is consistent with NPTX2b-mediated increased activity of HCRT circuitry. These data provide real-time in vivo evidence of circadian and homeostatic regulation of structural synaptic plasticity.

Sleep–wake regulation and hypocretin–melatonin interaction in zebrafish

In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep–wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr−/−). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr−/− pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr−/− fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr−/− fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation.

Synaptic plasticity in sleep: learning, homeostasis and disease.

Sleep is a fundamental and evolutionarily conserved aspect of animal life. Recent studies have shed light on the role of sleep in synaptic plasticity. Demonstrations of memory replay and synapse homeostasis suggest that one essential role of sleep is in the consolidation and optimization of synaptic circuits to retain salient memory traces despite the noise of daily experience. Here, we review this recent evidence and suggest that sleep creates a heightened state of plasticity, which may be essential for this optimization. Furthermore, we discuss how sleep deficits seen in diseases such as Alzheimers disease and autism spectrum disorders might not just reflect underlying circuit malfunction, but could also play a direct role in the progression of those disorders.

Asymmetric PI(3,4,5)P3 and Akt Signaling Mediates Chemotaxis of Axonal Growth Cones

The action of many extracellular guidance cues on axon pathfinding requires Ca2+ influx at the growth cone (Hong et al., 2000; Nishiyama et al., 2003; Henley and Poo, 2004), but how activation of guidance cue receptors leads to opening of plasmalemmal ion channels remains largely unknown. Analogous to the chemotaxis of amoeboid cells (Parent et al., 1998; Servant et al., 2000), we found that a gradient of chemoattractant triggered rapid asymmetric PI(3,4,5)P3 accumulation at the growth cones leading edge, as detected by the translocation of a GFP-tagged binding domain of Akt in Xenopus laevis spinal neurons. Growth cone chemoattraction required PI(3,4,5)P3 production and Akt activation, and genetic perturbation of polarized Akt activity disrupted axon pathfinding in vitro and in vivo. Furthermore, patch-clamp recording from growth cones revealed that exogenous PI(3,4,5)P3 rapidly activated TRP (transient receptor potential) channels, and asymmetrically applied PI(3,4,5)P3 was sufficient to induce chemoattractive growth cone turning in a manner that required downstream Ca2+ signaling. Thus, asymmetric PI(3,4,5)P3 elevation and Akt activation are early events in growth cone chemotaxis that link receptor activation to TRP channel opening and Ca2+ signaling. Altogether, our findings reveal that PI(3,4,5)P3 elevation polarizes to the growth cones leading edge and can serve as an early regulator during chemotactic guidance.

Enhanced phasic GABA inhibition during the repair phase of stroke: a novel therapeutic target

While tonic GABA appears to suppress brain repair after stroke, the effects of phasic (synaptic) GABA signalling are unclear. Hiu et al. reveal an increase in phasic GABA signalling during the repair phase that enhances plasticity-related recovery in mice. Increasing phasic signalling with zolpidem improves behavioural recovery, suggesting therapeutic potential.

Imaging zebrafish neural circuitry from whole brain to synapse

Recent advances in imaging tools are inspiring zebrafish researchers to tackle ever more ambitious questions in the neurosciences. Behaviorally fundamental conserved neural networks can now be potentially studied using zebrafish from a brain-wide scale to molecular resolution. In this perspective, we offer a roadmap by which a zebrafish researcher can navigate the course from collecting neural activities across the brain associated with a behavior, to unraveling molecular identities and testing the functional relevance of active neurons. In doing so, important insights will be gained as to how neural networks generate behaviors and assimilate changes in synaptic connectivity.

Fmr1 KO and Fenobam Treatment Differentially Impact Distinct Synapse Populations of Mouse Neocortex

Cognitive deficits in fragile X syndrome (FXS) are attributed to molecular abnormalities of the brains vast and heterogeneous synapse populations. Unfortunately, the density of synapses coupled with their molecular heterogeneity presents formidable challenges in understanding the specific contribution of synapse changes in FXS. We demonstrate powerful new methods for the large-scale molecular analysis of individual synapses that allow quantification of numerous specific changes in synapse populations present in the cortex of a mouse model of FXS. Analysis of nearly a million individual synapses reveals distinct, quantitative changes in synaptic proteins distributed across over 6,000 pairwise metrics. Some, but not all, of these synaptic alterations are reversed by treatment with the candidate therapeutic fenobam, an mGluR5 antagonist. These patterns of widespread, but diverse synaptic protein changes in response to global perturbation suggest that FXS and its treatment must be understood as a networked system at the synapse level.