Dual-polarity voltage imaging of the concurrent dynamics of multiple neuron types.

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between targeted cell populations. However, current indicators lack the requisite sensitivity for in vivo multipopulation imaging. We describe next-generation green and red voltage sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants pAce and pAceR. Our indicators enable 0.4- to 1-kilohertz voltage recordings from >50 spiking neurons per field of view in awake mice and ~30-minute continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered brain state-dependent antagonism between neocortical somatostatin-expressing (SST+) and vasoactive intestinal peptide-expressing (VIP+) interneurons and contributions to hippocampal field potentials from cell ensembles with distinct axonal projections. By combining three mutually compatible indicators, we performed simultaneous triple-population imaging. These approaches will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution.

Optimizing Strategies for Developing Genetically Encoded Voltage Indicators.

Genetically encoded optical indicators of neuronal activity enable unambiguous recordings of input-output activity patterns from identified cells in intact circuits. Among them, genetically encoded voltage indicators (GEVIs) offer additional advantages over calcium indicators as they are direct sensors of membrane potential and can adeptly report subthreshold events and hyperpolarization. Here, we outline the major GEVI designs and give an account of properties that need to be carefully optimized during indicator engineering. While designing the ideal GEVI, one should keep in mind aspects such as membrane localization, signal size, signal-to-noise ratio, kinetics and voltage dependence of optical responses. Using ArcLight and derivatives as prototypes, we delineate how a probe should be optimized for the former properties and developed along other areas in a need-based manner. Finally, we present an overview of the GEVI engineering process and lend an insight into their discovery, delivery and diagnosis.

Fast, in vivo voltage imaging using a red fluorescent indicator.

Genetically encoded voltage indicators (GEVIs) are emerging optical tools for acquiring brain-wide cell-type-specific functional data at unparalleled temporal resolution. To broaden the application of GEVIs in high-speed multispectral imaging, we used a high-throughput strategy to develop voltage-activated red neuronal activity monitor (VARNAM), a fusion of the fast Acetabularia opsin and the bright red fluorophore mRuby3. Imageable under the modest illumination intensities required by bright green probes (<50 mW mm-2), VARNAM is readily usable in vivo. VARNAM can be combined with blue-shifted optical tools to enable cell-type-specific all-optical electrophysiology and dual-color spike imaging in acute brain slices and live Drosophila. With enhanced sensitivity to subthreshold voltages, VARNAM resolves postsynaptic potentials in slices and cortical and hippocampal rhythms in freely behaving mice. Together, VARNAM lends a new hue to the optical toolbox, opening the door to high-speed in vivo multispectral functional imaging.

Visual Deprivation During the Critical Period Enhances Layer 2/3 GABAergic Inhibition in Mouse V1.

UNLABELLED: The role of GABAergic signaling in establishing a critical period for experience in visual cortex is well understood. However, the effects of early experience on GABAergic synapses themselves are less clear. Here, we show that monocular deprivation (MD) during the adolescent critical period produces marked enhancement of GABAergic signaling in layer 2/3 of mouse monocular visual cortex. This enhancement coincides with a weakening of glutamatergic inputs, resulting in a significant reduction in the ratio of excitation to inhibition. The potentiation of GABAergic transmission arises from both an increased number of inhibitory synapses and an enhancement of presynaptic GABA release from parvalbumin- and somatostatin-expressing interneurons. Our results suggest that augmented GABAergic inhibition contributes to the experience-dependent regulation of visual function. SIGNIFICANCE STATEMENT: Visual experience shapes the synaptic organization of cortical circuits in the mouse brain. Here, we show that monocular visual deprivation enhances GABAergic synaptic inhibition in primary visual cortex. This enhancement is mediated by an increase in both the number of postsynaptic GABAergic synapses and the probability of presynaptic GABA release. Our results suggest a contributing mechanism to altered visual responses after deprivation.

Loss of the neuron-specific F-box protein FBXO41 models an ataxia-like phenotype in mice with neuronal migration defects and degeneration in the cerebellum.

The cerebellum is crucial for sensorimotor coordination. The cerebellar architecture not only requires proper development but also long-term integrity to ensure accurate functioning. Developmental defects such as impaired neuronal migration or neurodegeneration are thus detrimental to the cerebellum and can result in movement disorders including ataxias. In this study, we identify FBXO41 as a novel CNS-specific F-box protein that localizes to the centrosome and the cytoplasm of neurons and demonstrate that cytoplasmic FBXO41 promotes neuronal migration. Interestingly, deletion of the FBXO41 gene results in a severely ataxic gait in mice, which show delayed neuronal migration of granule neurons in the developing cerebellum in addition to deformities and degeneration of the mature cerebellum. We show that FBXO41 is a critical factor, not only for neuronal migration in the cerebellum, but also for its long-term integrity.

p250GAP is a novel player in the Cdh1-APC/Smurf1 pathway of axon growth regulation.

Axon growth is an essential process during brain development. The E3 ubiquitin ligase Cdh1-APC has emerged as a critical regulator of intrinsic axon growth control. Here, we identified the RhoGAP p250GAP as a novel interactor of the E3 ubiquitin ligase Cdh1-APC and found that p250GAP promotes axon growth downstream of Cdh1-APC. We also report that p250GAP undergoes non-proteolytic ubiquitination and associates with the Cdh1 substrate Smurf1 to synergistically regulate axon growth. Finally, we found that in vivo knockdown of p250GAP in the developing cerebellar cortex results in impaired migration and axonal growth. Taken together, our data indicate that Cdh1-APC together with the RhoA regulators p250GAP and Smurf1 controls axon growth in the mammalian brain.

The E3 ligase Cdh1-anaphase promoting complex operates upstream of the E3 ligase Smurf1 in the control of axon growth.

Axon growth is an essential event during brain development and is extremely limited due to extrinsic and intrinsic inhibition in the adult brain. The E3 ubiquitin ligase Cdh1-anaphase promoting complex (APC) has emerged as an important intrinsic suppressor of axon growth. In this study, we identify in rodents the E3 ligase Smurf1 as a novel substrate of Cdh1-APC and that Cdh1 targets Smurf1 for degradation in a destruction box-dependent manner. We find that Smurf1 acts downstream of Cdh1-APC in axon growth and that the turnover of RhoA by Smurf1 is important in this process. In addition, we demonstrate that acute knockdown of Smurf1 in vivo in the developing cerebellar cortex results in impaired axonal growth and migration. Finally, we show that a stabilized form of Smurf1 overrides the inhibition of axon growth by myelin. Taken together, we uncovered a Cdh1-APC/Smurf1/RhoA pathway that mediates axonal growth suppression in the developing mammalian brain.

Mevastatin accelerates loss of synaptic proteins and neurite degeneration in aging cortical neurons in a heme-independent manner.

The therapeutic use of statins in reducing cholesterol requires careful assessment of potential neuroprotective and/or neurotoxic mechanisms. Chronic treatment with mevastatin (MV) exerts effects on cortical neuron morphology, protein expression and synaptic function in primary culture. MV impaired expression of synaptic proteins, reduced N-methyl-d-aspartate receptor (NMDAR) currents and accelerated neurodegeneration associated with aging. The down-regulating effect of MV on neuronal protein expression was additive with aging-associated decline in culture. Induction of Heme oxygenase-1 (HMOX1) by MV was superimposed on age-related up-regulation. Comparison of MV-treated and heme-deficient neurons showed that inhibition of heme synthesis (by succinyl acetone) had similar damaging effect on neurite integrity and MNDAR expression and function but not on expression of the receptor for neuropeptide Y1 (NPY1R). Replacement of heme in heme-deficient cultures restored protein expression but had no effect in those cultures co-treated with MV. Despite the dramatic induction of HMOX1, intracellular heme remained sufficient in MV-treated cultures, consistent with a heme-independent mechanism of MV-induced neurotoxicity and this was confirmed by analysing neurons with lentiviral over-expression of HMOX1. We conclude that MV exerts a neurotoxic effect in cultured neurons in a heme-independent manner.