A positively tuned voltage indicator for extended electrical recordings in the brain.

Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.

Dichotomous regulation of striatal plasticity by dynorphin.

Modulation of corticostriatal plasticity alters the information flow throughout basal ganglia circuits and represents a fundamental mechanism for motor learning, action selection, and reward. Synaptic plasticity in the striatal direct- and indirect-pathway spiny projection neurons (dSPNs and iSPNs) is regulated by two distinct networks of GPCR signaling cascades. While it is well-known that dopamine D2 and adenosine A2a receptors bi-directionally regulate iSPN plasticity, it remains unclear how D1 signaling modulation of synaptic plasticity is counteracted by dSPN-specific Gi signaling. Here, we show that striatal dynorphin selectively suppresses long-term potentiation (LTP) through Kappa Opioid Receptor (KOR) signaling in dSPNs. Both KOR antagonism and conditional deletion of dynorphin in dSPNs enhance LTP counterbalancing with different levels of D1 receptor activation. Behaviorally, mice lacking dynorphin in D1 neurons show comparable motor behavior and reward-based learning, but enhanced flexibility during reversal learning. These findings support a model in which D1R and KOR signaling bi-directionally modulate synaptic plasticity and behavior in the direct pathway.

Phase-Locking Requires Efficient Ca2+ Extrusion at the Auditory Hair Cell Ribbon Synapse.

Proper perception of sounds in the environment requires auditory signals to be encoded with extraordinary temporal precision up to tens of microseconds, but how it originates from the hearing organs in the periphery is poorly understood. In particular, sound-evoked spikes in auditory afferent fibers in vivo are phase-locked to sound frequencies up to 5 kHz, but it is not clear how hair cells can handle intracellular Ca2+ changes with such high speed and efficiency. In this study, we combined patch-clamp recording and two-photon Ca2+ imaging to examine Ca2+ dynamics in hair cell ribbon synapses in the bullfrog amphibian papilla of both sexes. We found that Ca2+ clearance from single synaptic ribbons followed a double exponential function, and the weight of the fast component, but not the two time constants, was significantly reduced for prolonged stimulation, and during inhibition of the plasma membrane Ca2+ ATPase (PMCA), the mitochondrial Ca2+ uptake (MCU), or the sarcolemma/endoplasmic reticulum Ca2+ ATPase (SERCA), but not the Na+/Ca2+ exchanger (NCX). Furthermore, we found that both the basal Ca2+ level and the Ca2+ rise during sinusoidal stimulation were significantly increased by inhibition of PMCA, MCU, or SERCA. Consistently, phase-locking of synaptic vesicle releases from hair cells was also significantly reduced by blocking PMCA, MCU, or SERCA, but not NCX. We conclude that, in addition to fast diffusion mediated by mobile Ca2+ buffer, multiple Ca2+ extrusion pumps are required for phase-locking at the auditory hair cell ribbon synapse.SIGNIFICANCE STATEMENT Hair cell synapses can transmit sound-driven signals precisely in the kHz range. However, previous studies of Ca2+ handling in auditory hair cells have often been conducted in immature hair cells, with elevated extracellular Ca2+ concentration, or through steady-state stimulation that may not be physiologically relevant. Here we examine Ca2+ clearance from hair cell synaptic ribbons in a fully mature preparation at physiological concentration of external Ca2+ and at physiological temperature. By stimulating hair cells with sinusoidal voltage commands that mimic pure sound tones, we recapitulated the phase-locking of hair cell exocytosis with an in vitro approach. This allowed us to reveal the Ca2+ extrusion mechanisms that are required for phase-locking at auditory hair cell ribbon synapses.

Multiplexed neurochemical sensing with sub-nM sensitivity across 2.25 mm2 area.

Multichannel arrays capable of real-time sensing of neuromodulators in the brain are crucial for gaining insights into new aspects of neural communication. However, measuring neurochemicals, such as dopamine, at low concentrations over large areas has proven challenging. In this research, we demonstrate a novel approach that leverages the scalability and processing power offered by microelectrode array devices integrated with a functionalized, high-density microwire bundle, enabling electrochemical sensing at an unprecedented scale and spatial resolution. The sensors demonstrate outstanding selective molecular recognition by incorporating a selective polymeric membrane. By combining cutting-edge commercial multiplexing, digitization, and data acquisition hardware with a bio-compatible and highly sensitive neurochemical interface array, we establish a powerful platform for neurochemical analysis. This multichannel array has been successfully utilized in vitro and ex vivo systems. Notably, our results show a sensing area of 2.25 mm2 with an impressive detection limit of 820 pM for dopamine. This new approach paves the way for investigating complex neurochemical processes and holds promise for advancing our understanding of brain function and neurological disorders.

Motor learning selectively strengthens cortical and striatal synapses of motor engram neurons.

Learning and consolidation of new motor skills require plasticity in the motor cortex and striatum, two key motor regions of the brain. However, how neurons undergo synaptic changes and become recruited during motor learning to form a memory engram remains unknown. Here, we train mice on a motor learning task and use a genetic approach to identify and manipulate behavior-relevant neurons selectively in the primary motor cortex (M1). We find that the degree of M1 engram neuron reactivation correlates with motor performance. We further demonstrate that learning-induced dendritic spine reorganization specifically occurs in these M1 engram neurons. In addition, we find that motor learning leads to an increase in the strength of M1 engram neuron outputs onto striatal spiny projection neurons (SPNs) and that these synapses form clusters along SPN dendrites. These results identify a highly specific synaptic plasticity during the formation of long-lasting motor memory traces in the corticostriatal circuit.

Author Correction: Spectrally filtered passive Si photodiode array for on-chip fluorescence imaging of intracellular calcium dynamics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Spectrally filtered passive Si photodiode array for on-chip fluorescence imaging of intracellular calcium dynamics.

On-chip fluorescence imaging devices are recognized for their miniaturized and implantable nature that can benefit the study of intracellular dynamics at a variety of settings. However, it is challenging to integrate a spectral filter onto such devices (to block the excitation light) that has similar performance to the state-of-the-art emission filters used in fluorescence microscopes. In this work, we report a 100%-yield, spectrally filtered passive Si photodiode array designed for on-chip fluorescence imaging of intracellular Ca2+ dynamics. Coated with a spectral filter layer that has a high extinction ratio (>103), our array features high wavelength selectivity (>102), high linearity (R2 > 0.98), and low detection limit (45.1 µW 640/30 nm light). Employing fluorescence microscopy as the reference, we demonstrate that our array can conduct on-chip Ca2+ imaging in C2C12 cells that were chemically triggered to increase their intracellular Ca2+ levels. Importantly, our array-level data qualitatively captured the static fluorescence image of the cells and the intracellular Ca2+ dynamics, both of which are correlated with the microscope-collected data. Our results suggest the possible use of the spectrally filtered array towards a miniaturized on-chip fluorescence imaging device, which may open up new opportunities in tissue-level pharmaceutical screening and fundamental studies on cell networks.