General Anesthetic Conditions Induce Network Synchrony and Disrupt Sensory Processing in the Cortex

General anesthetics are commonly used in animal models to study how sensory signals are represented in the brain. Here, we used two-photon (2P) calcium activity imaging with cellular resolution to investigate how neuronal activity in layer 2/3 of the mouse barrel cortex is modified under the influence of different concentrations of chemically distinct general anesthetics. Our results show that a high isoflurane dose induces synchrony in local neuronal networks and these cortical activity patterns closely resemble those observed in EEG recordings under deep anesthesia. Moreover, ketamine and urethane also induced similar activity patterns. While investigating the effects of deep isoflurane anesthesia on whisker and auditory evoked responses in the barrel cortex, we found that dedicated spatial regions for sensory signal processing become disrupted. We propose that our isoflurane-2P imaging paradigm can serve as an attractive model system to dissect cellular and molecular mechanisms that induce the anesthetic state, and it might also provide important insight into sleep-like brain states and consciousness

Quantitative Comparison of Genetically Encoded Ca2+ Indicators in Cortical Pyramidal Cells and Cerebellar Purkinje Cells

Genetically encoded Ca2+ indicators (GECIs) are promising tools for cell type-specific and chronic recording of neuronal activity. In the mammalian central nervous system, however, GECIs have been tested almost exclusively in cortical and hippocampal pyramidal cells, and the usefulness of recently developed GECIs has not been systematically examined in other cell types. Here we expressed the latest series of GECIs, yellow cameleon (YC) 2.60, YC3.60, YC-Nano15, and GCaMP3, in mouse cortical pyramidal cells as well as cerebellar Purkinje cells using in utero injection of recombinant adenoviral vectors. We characterized the performance of the GECIs by simultaneous two-photon imaging and whole-cell patch-clamp recording in acute brain slices at 33 ± 2°C. The fluorescent responses of GECIs to action potentials (APs) evoked by somatic current injection or to synaptic stimulation were examined using rapid dendritic imaging. In cortical pyramidal cells, YC2.60 showed the largest responses to single APs, but its decay kinetics were slower than YC3.60 and GCaMP3, while GCaMP3 showed the largest responses to 20 APs evoked at 20 Hz. In cerebellar Purkinje cells, only YC2.60 and YC-Nano15 could reliably report single complex spikes (CSs), and neither showed signal saturation over the entire stimulus range tested (1–10 CSs at 10 Hz). The expression and response of YC2.60 in Purkinje cells remained detectable and comparable for at least over 100 days. These results provide useful information for selecting an optimal GECI depending on the experimental requirements: in cortical pyramidal cells, YC2.60 is suitable for detecting sparse firing of APs, whereas GCaMP3 is suitable for detecting burst firing of APs; in cerebellar Purkinje cells, YC2.60 as well as YC-Nano15 is suitable for detecting CSs

Genetically encoded Ca2+ indicators; expanded affinity range, color hue and compatibility with optogenetics

Fluorescent protein-based indicators are invaluable tools for functional imaging of living cells and organisms. Genetically encoded calcium indicators (GECIs) such as derivatives of yellow cameleons (YCs) and GCaMPs/pericams (Miyawaki et al., 1997; Nakai et al., 2001; Nagai et al., 2001) are a highly advanced class of indicators. Continued efforts for improvement of the performance of GECIs have resulted in brighter indicators with better photo-stability and expanded dynamic range, thus improving the sensitivity of detection. Fine-tuning of other properties, including Ca2+ affinity and Hill constant, have also contributed to increase the detectability of Ca2+ dynamics. Emerging optogenetic technology has forced the spectrally compatible GECI color variants. In this opinion, we highlight the recent development of GECIs including photo-switchable Ca2+ indicators and bioluminescence-based Ca2+ indicator, mainly invented in our group, focusing especially on the parameters determining their performance in order to provide a guideline for the selection of appropriate GECI for a given experiment