Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

High efficiency two-photon uncaging coupled by the correction of spontaneous hydrolysis.

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.

[Fast three-dimensional two-photon scanning methods for studying neuronal physiology on cellular and network level]. / Háromdimenziós, gyors, kétfoton-pásztázó eljárások sejt- és hálózatszintu idegsejtvizsgálatokhoz.

Erratum to the article published on December 27th 2015 in Issue 52 of Orvosi Hetilap [Orv. Hetil., 2015, 156(52), 2120-2126, DOI: 10.1556/650.2015.30329]. The name of Dávid Mezey was not correctly typed. The corresponding author asked for the following correction to be published.

Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes.

The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution, acousto-optic two-photon microscope with continuous three-dimensional (3D) trajectory and random-access scanning modes that reaches near-cubic-millimeter scan range and can be adapted to imaging different spatial scales. We performed 3D calcium imaging of action potential backpropagation and dendritic spike forward propagation at sub-millisecond temporal resolution in mouse brain slices. We also performed volumetric random-access scanning calcium imaging of spontaneous and visual stimulation-evoked activity in hundreds of neurons of the mouse visual cortex in vivo. These experiments demonstrate the subcellular and network-scale imaging capabilities of our system.

[Fast three-dimensional two-photon scanning methods for studying neuronal physiology on cellular and network level]. / Háromdimenziós, gyors, kétfoton-pásztázó eljárások sejt- és hálózatszintu idegsejtvizsgálatokhoz.

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 Ca(2+) 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.

Seeing and Cleaving: Turn-Off Fluorophore Uncaging and Its Application in Hydrogel Photopatterning and Traceable Neurotransmitter Photocages.

The advancements in targeted drug release and experimental neuroscience have amplified the scientific interest in photolabile protecting groups (PPGs) and photouncaging. The growing need for the detection of uncaging events has led to the development of reporters with fluorescence turn-on upon uncaging. In contrast, fluorescent tags with turn-off properties have been drastically underexplored, although there are applications where they would be sought after. In this work, a rhodamine-based fluorescent tag is developed with signal turn-off following photouncaging. One-photon photolysis experiments reveal a ready loss of red fluorescence signal upon UV (365 nm) irradiation, while no significant change is observed in control experiments in the absence of PPG or with irradiation around the absorption maximum of the fluorophore (595 nm). The two-photon photolysis of the turn-off fluorescent tag is explored in hydrogel photolithography experiments. The hydrogel-bound tag enables the power-, dwell time-, and wavelength-dependent construction of intricate patterns and gradients. Finally, a prominent caged neurotransmitter (MNI-Glu) is modified with the fluorescent tag, resulting in the glutamate precursor named as GlutaTrace with fluorescence traceability and turn-off upon photouncaging. GlutaTrace is successfully applied for the visualization of glutamate precursor distribution following capillary microinjection and for the selective excitation of neurons in a mouse brain model.

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging.

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

Cortex-wide response mode of VIP-expressing inhibitory neurons by reward and punishment.

Neocortex is classically divided into distinct areas, each specializing in different function, but all could benefit from reinforcement feedback to inform and update local processing. Yet it remains elusive how global signals like reward and punishment are represented in local cortical computations. Previously, we identified a cortical neuron type, vasoactive intestinal polypeptide (VIP)-expressing interneurons, in auditory cortex that is recruited by behavioral reinforcers and mediates disinhibitory control by inhibiting other inhibitory neurons. As the same disinhibitory cortical circuit is present virtually throughout cortex, we wondered whether VIP neurons are likewise recruited by reinforcers throughout cortex. We monitored VIP neural activity in dozens of cortical regions using three-dimensional random access two-photon microscopy and fiber photometry while mice learned an auditory discrimination task. We found that reward and punishment during initial learning produce rapid, cortex-wide activation of most VIP interneurons. This global recruitment mode showed variations in temporal dynamics in individual neurons and across areas. Neither the weak sensory tuning of VIP interneurons in visual cortex nor their arousal state modulation was fully predictive of reinforcer responses. We suggest that the global response mode of cortical VIP interneurons supports a cell-type-specific circuit mechanism by which organism-level information about reinforcers regulates local circuit processing and plasticity.

Sharp-wave ripple doublets induce complex dendritic spikes in parvalbumin interneurons in vivo.

Neuronal plasticity has been shown to be causally linked to coincidence detection through dendritic spikes (dSpikes). We demonstrate the existence of SPW-R-associated, branch-specific, local dSpikes and their computational role in basal dendrites of hippocampal PV+ interneurons in awake animals. To measure the entire dendritic arbor of long thin dendrites during SPW-Rs, we used fast 3D acousto-optical imaging through an eccentric deep-brain adapter and ipsilateral local field potential recording. The regenerative calcium spike started at variable, NMDA-AMPA-dependent, hot spots and propagated in both direction with a high amplitude beyond a critical distance threshold (~150 µm) involving voltage-gated calcium channels. A supralinear dendritic summation emerged during SPW-R doublets when two successive SPW-R events coincide within a short temporal window (~150 ms), e.g., during more complex association tasks, and generated large dSpikes with an about 2.5-3-fold amplitude increase which propagated down to the soma. Our results suggest that these doublet-associated dSpikes can work as a dendritic-level temporal and spatial coincidence detector during SPW-R-related network computation in awake mice.

Theoretical Design, Synthesis, and In Vitro Neurobiological Applications of a Highly Efficient Two-Photon Caged GABA Validated on an Epileptic Case.

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.

Enhanced dendritic action potential backpropagation in parvalbumin-positive basket cells during sharp wave activity.

In this study two-photon imaging and single cell electrophysiological measurements were carried out in PV+ hippocampal interneurons to compare the dendritic calcium dynamics of somatically evoked backpropagating action potentials (BAPs) and in vitro sharp wave oscillation (SPW) activated BAPs at different distances from the soma. In the case of 300 µm thick, non-oscillating slices, the BAP-evoked Ca(2+) (BAP-Ca(2+)) influx propagated along the dendritic tree in a non-uniform manner and its amplitude gradually reduced when measured at more distal regions. In contrast to the evoked BAP-Ca(2+)s, the spontaneous SPW-induced Ca(2+) influx had only a small distance-dependent decrement. Our results suggest that similarly to nicotinic acetylcholine receptor activation, synaptic activity during hippocampal SPWs increases AP backpropagation into distant dendritic segments. Bath application of Nimodipine, a specific Ca(2+) channel blocker and tetrodotoxine decreased the amplitude of the somatically evoked Ca(2+) influx, which suggests that L-type Ca(2+) channels play an important role both during somatically evoked and SPW-induced BAPs.

Fast 3D Imaging of Spine, Dendritic, and Neuronal Assemblies in Behaving Animals.

Understanding neural computation requires methods such as 3D acousto-optical (AO) scanning that can simultaneously read out neural activity on both the somatic and dendritic scales. AO point scanning can increase measurement speed and signal-to-noise ratio (SNR) by several orders of magnitude, but high optical resolution requires long point-to-point switching time, which limits imaging capability. Here we present a novel technology, 3D DRIFT AO scanning, which can extend each scanning point to small 3D lines, surfaces, or volume elements for flexible and fast imaging of complex structures simultaneously in multiple locations. Our method was demonstrated by fast 3D recording of over 150 dendritic spines with 3D lines, over 100 somata with squares and cubes, or multiple spiny dendritic segments with surface and volume elements, including in behaving animals. Finally, a 4-fold improvement in total excitation efficiency resulted in about 500 × 500 × 650 µm scanning volume with genetically encoded calcium indicators (GECIs).

Combined two-photon imaging, electrophysiological, and anatomical investigation of the human neocortex in vitro.

Spontaneous synchronous population activity (SPA) can be detected by electrophysiological methods in cortical slices of epileptic patients, maintained in a physiological medium in vitro. In order to gain additional spatial information about the network mechanisms involved in the SPA generation, we combined electrophysiological studies with two-photon imaging. Neocortical slices prepared from postoperative tissue of epileptic and tumor patients were maintained in a dual perfusion chamber in a physiological incubation medium. SPA was recorded with a 24-channel extracellular linear microelectrode covering all neocortical layers. After identifying the electrophysiologically active regions of the slice, bolus loading of neuronal and glial markers was applied on the tissue. SPA-related [Formula: see text] transients were detected in a large population of neighboring neurons with two-photon microscopy, simultaneous with extracellular SPA and intracellular whole-cell patch-clamp recordings. The intracellularly recorded cells were filled for subsequent anatomy. The cells were reconstructed in three dimensions and examined with light- and transmission electron microscopy. Combining high spatial resolution two-photon [Formula: see text] imaging techniques and high temporal resolution extra- and intracellular electrophysiology with cellular anatomy may permit a deeper understanding of the structural and functional properties of the human neocortex.

Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves.

Sharp-wave ripples are transient oscillatory events in the hippocampus that are associated with the reactivation of neuronal ensembles within specific circuits during memory formation. Fast-spiking, parvalbumin-expressing interneurons (FS-PV INs) are thought to provide fast integration in these oscillatory circuits by suppressing regenerative activity in their dendrites. Here, using fast 3D two-photon imaging and a caged glutamate, we challenge this classical view by demonstrating that FS-PV IN dendrites can generate propagating Ca(2+) spikes during sharp-wave ripples. The spikes originate from dendritic hot spots and are mediated dominantly by L-type Ca(2+) channels. Notably, Ca(2+) spikes were associated with intrinsically generated membrane potential oscillations. These oscillations required the activation of voltage-gated Na(+) channels, had the same frequency as the field potential oscillations associated with sharp-wave ripples, and controlled the phase of action potentials. Furthermore, our results demonstrate that the smallest functional unit that can generate ripple-frequency oscillations is a segment of a dendrite.