Copper is an endogenous modulator of neural circuit spontaneous activity.

For reasons that remain insufficiently understood, the brain requires among the highest levels of metals in the body for normal function. The traditional paradigm for this organ and others is that fluxes of alkali and alkaline earth metals are required for signaling, but transition metals are maintained in static, tightly bound reservoirs for metabolism and protection against oxidative stress. Here we show that copper is an endogenous modulator of spontaneous activity, a property of functional neural circuitry. Using Copper Fluor-3 (CF3), a new fluorescent Cu(+) sensor for one- and two-photon imaging, we show that neurons and neural tissue maintain basal stores of loosely bound copper that can be attenuated by chelation, which define a labile copper pool. Targeted disruption of these labile copper stores by acute chelation or genetic knockdown of the CTR1 (copper transporter 1) copper channel alters the spatiotemporal properties of spontaneous activity in developing hippocampal and retinal circuits. The data identify an essential role for copper neuronal function and suggest broader contributions of this transition metal to cell signaling.

The Impact of Steroid Activation of TRPM3 on Spontaneous Activity in the Developing Retina.

In the central nervous system, melastatin transient receptor potential (TRPM) channels function as receptors for the neurosteroid pregnenolone sulfate (PregS). The expression and function of TRPM3 has been explored in adult retina, although its role during development is unknown. We found, during the second postnatal week in mice, TRPM3 immunofluorescence labeled distinct subsets of inner retinal neurons, including a subset of retinal ganglion cells (RGCs), similar to what has been reported in the adult. Labeling for a TRPM3 promoter-driven reporter confirmed expression of the TRPM3 gene in RGCs and revealed additional expression in nearly all Müller glial cells. Using two-photon calcium imaging, we show that PregS and the synthetic TRPM3 agonist CIM0216 (CIM) induced prolonged calcium transients in RGCs, which were mostly absent in TRPM3 knock-out (KO) mice. These prolonged calcium transients were not associated with strong membrane depolarizations but induced c-Fos expression. To elucidate the impact of PregS-activation of TRPM3 on retinal circuits we took two sets of physiological measurements. First, PregS induced a robust increase in the frequency but not amplitude of spontaneous postsynaptic currents (PSCs). This increase was absent in the TRPM3 KO mice. Second, PregS induced a small increase in cell participation and duration of retinal waves, but this modulation persisted in TRPM3 KO mice, indicating PregS was acting on wave generating circuits independent of TRPM3 channels. Though baseline frequency of retinal waves was slightly reduced in the TRPM3 KO mice, other properties of waves were indistinguishable from wildtype. Together, these results indicate that the presence of neurosteroids impact spontaneous synaptic activity and retinal waves during development via both TRPM3-dependent and independent mechanisms.

Microglial P2Y12 deficiency/inhibition protects against brain ischemia.

OBJECTIVE: Microglia are among the first immune cells to respond to ischemic insults. Triggering of this inflammatory response may involve the microglial purinergic GPCR, P2Y12, activation via extracellular release of nucleotides from injured cells. It is also the inhibitory target of the widely used antiplatelet drug, clopidogrel. Thus, inhibiting this GPCR in microglia should inhibit microglial mediated neurotoxicity following ischemic brain injury. METHODS: Experimental cerebral ischemia was induced, in vitro with oxygen-glucose deprivation (OGD), or in vivo via bilateral common carotid artery occlusion (BCCAO). Genetic knock-down in vitro via siRNA, or in vivo P2Y12 transgenic mice (P2Y12-/- or P2Y12+/-), or in vivo treatment with clopidogrel, were used to manipulate the receptor. Neuron death, microglial activation, and microglial migration were assessed. RESULTS: The addition of microglia to neuron-astrocyte cultures increases neurotoxicity following OGD, which is mitigated by microglial P2Y12 deficiency (P<0.05). Wildtype microglia form clusters around these neurons following injury, which is also prevented in P2Y12 deficient microglia (P<0.01). P2Y12 knock-out microglia migrated less than WT controls in response to OGD-conditioned neuronal supernatant. P2Y12 (+/-) or clopidogrel treated mice subjected to global cerebral ischemia suffered less neuronal injury (P<0.01, P<0.001) compared to wild-type littermates or placebo treated controls. There were also fewer microglia surrounding areas of injury, and less activation of the pro-inflammatory transcription factor, nuclear factor Kappa B (NFkB). INTERPRETATION: P2Y12 participates in ischemia related inflammation by mediating microglial migration and potentiation of neurotoxicity. These data also suggest an additional anti-inflammatory, neuroprotective benefit of clopidogrel.