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.

Biphasic Cholinergic Modulation of Reverberatory Activity in Neuronal Networks.

Acetylcholine (ACh) is an important neuromodulator in various cognitive functions. However, it is unclear how ACh influences neural circuit dynamics by altering cellular properties. Here, we investigated how ACh influences reverberatory activity in cultured neuronal networks. We found that ACh suppressed the occurrence of evoked reverberation at low to moderate doses, but to a much lesser extent at high doses. Moreover, high doses of ACh caused a longer duration of evoked reverberation, and a higher occurrence of spontaneous activity. With whole-cell recording from single neurons, we found that ACh inhibited excitatory postsynaptic currents (EPSCs) while elevating neuronal firing in a dose-dependent manner. Furthermore, all ACh-induced cellular and network changes were blocked by muscarinic, but not nicotinic receptor antagonists. With computational modeling, we found that simulated changes in EPSCs and the excitability of single cells mimicking the effects of ACh indeed modulated the evoked network reverberation similar to experimental observations. Thus, ACh modulates network dynamics in a biphasic fashion, probably by inhibiting excitatory synaptic transmission and facilitating neuronal excitability through muscarinic signaling pathways.

Mesophasic organization of GABAA receptors in hippocampal inhibitory synapses.

Information processing in the brain depends on specialized organization of neurotransmitter receptors and scaffolding proteins within the postsynaptic density. However, how these molecules are organized in situ remains largely unknown. In this study, template-free classification of oversampled sub-tomograms was used to analyze cryo-electron tomograms of hippocampal synapses. We identified type-A GABA receptors (GABAARs) in inhibitory synapses and determined their in situ structure at 19-Å resolution. These receptors are organized hierarchically: from GABAAR super-complexes with a preferred inter-receptor distance of 11 nm but variable relative angles, through semi-ordered, two-dimensional receptor networks with reduced Voronoi entropy, to mesophasic assembly with a sharp phase boundary. These assemblies likely form via interactions among postsynaptic scaffolding proteins and receptors and align with putative presynaptic vesicle release sites. Such mesophasic self-organization might allow synapses to achieve a 'Goldilocks' state, striking a balance between stability and flexibility and enabling plasticity in information processing.

Excitation wavelength optimization improves photostability of ASAP-family GEVIs.

Recent interest in high-throughput recording of neuronal activity has motivated rapid improvements in genetically encoded calcium or voltage indicators (GECIs or GEVIs) for all-optical electrophysiology. Among these probes, the ASAPs, a series of voltage indicators based on a variant of circularly permuted green fluorescent protein (cpGFP) and a conjugated voltage sensitive domain (VSD), are capable of detecting both action potentials and subthreshold neuronal activities. Here we show that the ASAPs, when excited by blue light, undergo reversible photobleaching. We find that this fluorescence loss induced by excitation with 470-nm light can be substantially reversed by low-intensity 405-nm light. We demonstrate that 405-nm and 470-nm co-illumination significantly improved brightness and thereby signal-to-noise ratios during voltage imaging compared to 470-nm illumination alone. Illumination with a single wavelength of 440-nm light also produced similar improvements. We hypothesize that reversible photobleaching is related to cis-trans isomerization and protonation of the GFP chromophore of ASAP proteins. Amino acids that influence chromophore isomerization are potential targets of point mutations for future improvements.

[Study on simulation of clarification for Vegetative Filter Strips by VFSMOD Model].

The functions and mechanism of Vegetative Filter Strips Model (VFSMOD) had been introduced in this paper, and the applicability and performance ability of this model have been tested by plot experiment data. The results show that the relative deviations between simulated values and measured values of outflow quantity are within +/- 15%, while with the concentration of SS, the relative deviations are within +/- 20%, and the determination coefficients between simulated values and measured values for outflow and SS are 0.995 and 0.889 respectively. Therefore, the simulation precision of this model is satisfactory, the model can be used as a tool for the design of VFS.