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In this paper, we present an additional, new cage-GABA compound, called 4-amino-1-(4′-dimethylaminoisopropoxy-5′,7′-dinitro-2′,3′-dihydro-indol-1-yl)-1-oxobutane-γ-aminobutyric acid (iDMPO-DNI-GABA), and currently, this compound is the only photoreagent, which can be applied for GABA uncaging without experimental compromises. By a systematic theoretical design and successful synthesis of several compounds, the best reagent exhibits a high two-photon efficiency within the 700–760 nm range with excellent pharmacological behavior, which proved to be suitable for a complex epileptic study. Quantum chemical design showed that the optimal length of the cationic side chain enhances the two-photon absorption by 1 order of magnitude due to the cooperating internal hydrogen bonding to the extra nitro group on the core. This feature increased solubility while suppressing membrane permeability. The efficiency was demonstrated in a systematic, wide range of in vitro single-cell neurophysiological experiments by electrophysiological as well as calcium imaging techniques. Scalable inhibitory ion currents were elicited by iDMPO-DNI-GABA with appropriate spatial–temporal precision, blocking both spontaneous and evoked cell activity with excellent efficiency. Additionally, to demonstrate its applicability in a real neurobiological study, we could smoothly and selectively modulate neuronal activities during artificial epileptic rhythms first time in a neural network of GCaMP6f transgenic mouse brain slices.

Objective Multimodal neuroimaging approaches are beneficial to discover brain functionalities at high spatial and temporal resolution. In our work, a novel material composition of a microECoG device relying on Parylene HT and indium-tin-oxide (ITO) is presented, which facilitates two-photon imaging of Ca2+ signals and concurrent recording of cortical EEG. Approach Long-term stability of the interfaces of the transparent microdevice is confirmed in vitro by electrochemical and mechanical tests. The outstanding optical properties, like high transmittance and low auto-fluorescent are proven by fluorimetric measurements. Spatial resolution of fluorescent two-photon imaging through the microECoG device is presented in transgenic hippocampal slices, while concurrent recording of Ca2+ signals and cortical EEG is demonstrated in vivo. Photoartefacts and photodegradation of the materials are also investigated in detail to provide safety guidelines for further use in two-photon in vivo imaging schemes. Main results Two-photon imaging of Ca signals can be safely performed through the proposed transparent ECoG device, without significant distortion in the dimensions of detected neuronal structures or in the temporal signaling. In chronic use, we demonstrated that fluorescent Ca signals of individual neurons can be clearly recorded even after 51 d. Significance Our results give a firm indication that highly transparent microECoG electrode arrays made of Parylene HT/ITO/Parylene HT multilayer are excellent candidates for synergetic recording of optical signals and EEG from intact brains with high resolution and are free of electrical and optical artefacts.

Two-photon (TP) uncaging of neurotransmitter molecules is the method of choice to mimic and study the subtleties of neuronal communication either in the intact brain or in slice preparations. However, the currently available caged materials are just at the limit of their usability and have several drawbacks. The local and focal nature of their use may for example be jeopardized by a high spontaneous hydrolysis rate of the commercially available compounds with increased photochemical release rate. Here, using quantum chemical modelling we show the mechanisms of hydrolysis and two-photon activation, and synthesized more effective caged compounds. Furthermore, we have developed a new enzymatic elimination method removing neurotransmitters inadvertently escaping from their compound during experiment. This method, usable both in one and two-photon experiments, allows for the use of materials with an increased rate of photochemical release. The efficiency of the new compound and the enzymatic method and of the new compound are demonstrated in neurophysiological experiments.

Introduction: Two-photon microscopy is the ideal tool to study how signals are processed in the functional brain tissue. However, early raster scanning strategies were inadequate to record fast 3D events like action potentials. Aim: The aim of the authors was to record various neuronal activity patterns with high signal-to-noise ratio in an optical manner. Method: Authors developed new data acquisition methods and microscope hardware. Results: Multiple Line Scanning enables the experimenter to select multiple regions of interests, doing this not just increases repetition speed, but also the signal-to-noise ratio of the fluorescence transients. On the same principle, an acousto-optical deflector based 3D scanning microscope has been developed with a sub-millisecond temporal resolution and a millimeter z-scanning range. Its usability is demonstrated by obtaining 3D optical recordings of action potential backpropagation in several hundred micrometers long neuronal processes of single neurons and by 3D random-access scanning of Ca2+ transients in hundreds of neurons in the mouse visual cortex. Conclusions: Region of interest scanning enables high signal-to-noise ratio and repetition speed, while keeping good depth penetration of the two-photon microscopes. Orv. Hetil., 2015, 156(52), 2120–2126.

Two-photon microscopy is the ideal tool to study how signals are processed in the functional brain tissue. However, early raster scanning strategies were inadequate to record fast 3D events like action potentials.The aim of the authors was to record various neuronal activity patterns with high signal-to-noise ratio in an optical manner.Authors developed new data acquisition methods and microscope hardware.Multiple Line Scanning enables the experimenter to select multiple regions of interests, doing this not just increases repetition speed, but also the signal-to-noise ratio of the fluorescence transients. On the same principle, an acousto-optical deflector based 3D scanning microscope has been developed with a sub-millisecond temporal resolution and a millimeter z-scanning range. Its usability is demonstrated by obtaining 3D optical recordings of action potential backpropagation in several hundred micrometers long neuronal processes of single neurons and by 3D random-access scanning of Ca(2+) transients in hundreds of neurons in the mouse visual cortex.Region of interest scanning enables high signal-to-noise ratio and repetition speed, while keeping good depth penetration of the two-photon microscopes.