Optical Imaging as a Link Between Cellular Neurophysiology and Circuit Modeling

The relatively simple and highly modular circuitry of the cerebellum raised expectations decades ago that a realistic computational model of cerebellar circuit operations would be feasible, and prove insightful for unraveling cerebellar information processing. To this end, the biophysical properties of most cerebellar cell types and their synaptic connections have been well characterized and integrated into realistic single cell models. Furthermore, large scale models of cerebellar circuits that extrapolate from single cell properties to circuit dynamics have been constructed. While the development of single cell models have been constrained by microelectrode recordings, guidance and validation as these models are scaled up to study network interactions requires an experimental methodology capable of monitoring cerebellar dynamics at the population level. Here we review the potential of optical imaging techniques to serve this purpose

Engineering of a Genetically Encodable Fluorescent Voltage Sensor Exploiting Fast Ci-VSP Voltage-Sensing Movements

Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement (‘gating’ current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs

Engineering and Characterization of an Enhanced Fluorescent Protein Voltage Sensor

BACKGROUND: Fluorescent proteins have been used to generate a variety of biosensors to optically monitor biological phenomena in living cells. Among this class of genetically encoded biosensors, reporters for membrane potential have been a particular challenge. The use of presently known voltage sensor proteins is limited by incorrect subcellular localization and small or absent voltage responses in mammalian cells. RESULTS: Here we report on a fluorescent protein voltage sensor with superior targeting to the mammalian plasma membrane and high responsiveness to membrane potential signaling in excitable cells. CONCLUSIONS AND SIGNIFICANCE: This biosensor, which we termed VSFP2.1, is likely to lead to new methods of monitoring electrically active cells with cell type specificity, non-invasively and in large numbers, simultaneously

The role of the calcium transporter protein plasma membrane calcium ATPase PMCA2 in cerebellar Purkinje neuron function

Genetic deletion of the plasma membrane calcium ATPase type 2 (PMCA2), a calcium transporter protein, is associated with an overtly ataxic phenotype in mice. PMCA2 is expressed at high levels in cerebellar Purkinje neurons (PNs) where functional integrity is essential for normal cerebellar function. Indeed, loss of PN function accompanies cerebellar ataxia in humans and mouse models. In the ataxic PMCA2 knockout (PMCA2-/-) mouse the ability of the PNs to control their cytosolic calcium levels was severely impaired; basal calcium levels were high and calcium recovery kinetics slow. Whole cell patch clamp recordings from PMCA2-/- PNs revealed that they possessed hyperpolarised membrane potentials, reduced frequency and increased irregularity of spontaneous action potential firing, curtailed complex spikes and sustained calcium-dependent outward K+ currents. We propose that these alterations limit pathological excursions in PN cytosolic calcium as an aid to survival but that they are insufficient to prevent loss of functional cerebellar outpu

Photoinduced intramolecular charge transfer in push-pull polyenes: Effects of solvation, electron-donor group, and polyenic chain length

International audienceSubpicosecond absorption spectroscopy is used to characterize the primary photoinduced processes in a class of push−pull polyenes bearing a julolidine end group as the electron donor and a diethylthiobarbituric acid end group as the electron acceptor. The excited-state decay time and relaxation pathway have been studied for four polyenes of increasing chain length (n = 2−5 double bonds) in aprotic solvents of different solvation time, polarity, and viscosity. Intramolecular charge transfer (ICT) leading to a transient state of cyanine-like structure (fully conjugated with no bond length alternation) is observed in all polar solvents at a solvent dependent rate, but the reaction is not observed in cyclohexane, a nonpolar solvent. In polar solvents, the reaction time increases with the average solvation time but remains slightly larger, except in the viscous solvent triacetin. These facts are interpreted as an indication that both solvent reorganization and internal restructuring are involved in the ICT-state formation. The observed photodynamics resemble those we previously found for another class of polyenes bearing a dibutylaniline group as the donor, including a similar charge-transfer rate in spite of the larger electron donor character of the julolidine group. This observation brings further support to the proposal that an intramolecular coordinate is involved in the charge-transfer reaction, possibly a torsional motion of the donor end group. On the other hand, relaxation of the ICT state leads to cis−trans isomerization or crossing to the triplet state, depending on the length of the polyenic chain. In dioxane, tetrahydrofuran, and triacetin, the ICT state of the shorter chains (n = 2, 3) relaxes to the isomer with a viscosity-dependent rate, while that of the longer ones (n = 4, 5) leads to the triplet state with a viscosity-independent rate, as expected. In acetonitrile, the ICT-state lifetime is generally much shorter. A change from photoisomerization to intersystem crossing at n = 4 is also proposed in this solvent, but the formation of a photoproduct at n = 2 is not clear. In cyclohexane, where the ICT state is not formed, the relaxation pathway of the initially excited state is found to lead to an isomer for n = 2. As in polar solvents, a change to intersystem crossing at n = 4 is proposed. The direct relaxation to the ground state found at n = 3 for the series bearing a dibutylaniline group is not observed with the julolidine group. The results clearly illustrate that photoinduced reaction trajectories in push−pull polyenes are controlled by the static and dynamic properties of the solvent, the chemical nature and size of the end groups, and the conjugated-chain length and flexibility

Fast spatial beam shaping by acousto-optic diffraction for 3D non-linear microscopy

International audienceAcousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial structure of light beams, most notably their phase. AOD light modulation of ultra-short laser pulses, however, is not straightforward to implement because of intrinsic chromatic dispersion and non-stationarity of acousto-optic diffraction. While schemes exist to compensate chromatic dispersion, non-stationarity remains an obstacle. In this work we demonstrate an efficient AOD light modulator for stable phase modulation using time-locked generation of frequency-modulated acoustic waves at the full repetition rate of a high power laser pulse amplifier of 80 kHz. We establish the non-local relationship between the optical phase and the generating acoustic frequency function and verify the system for temporal stability, phase accuracy and generation of non-linear twodimensional phase functions