INTRODUCTION

Uncaging means activation of biochemically masked (’caged’) molecules via photolysis that mimics physiological release of bioactive compounds. This technique is widely used in the neuroscience where the caged molecule is usually neurotransmitter. Using two-photon excitation, precise release can be elicited in miniature volumes and uncaging is a powerful opportunity to investigate the distribution of receptors on the neurons or activate dendrites or spines.

PHOTOSTIMULATION AND IMAGING

Photolysis of caged glutamate can be performed with 720 nm ultrafast pulsed laser light. This stimulating laser beam is coupled to the existing light path with a dichroic mirror, and it is used simultaneously by the imaging IR laser. The light path is optimized for both wavelengths thanks for the full optical engineering. The stimulation and imaging can be performed along various patterns, which flexibility is established by galvanometer based scanner (see below). The imaging is interleaved with uncaging periods when the scanner jumps to the locations. The microsecond-scale switching time between the stimulation and imaging is established by using of Pockels cell.

Figure: Photostimulation of the MNI-Glu-TFA close to the dendritic segment of a hippocampal interneuron. The near simultaneous stimulation and the imaging are accomplished by point and line scanning, respectively. The stimulation is performed in the distributed points, and the evoked changes is followed along a line covering the dendrite. Ca2+ responses along the selected dendritic segment are analyzed as the ∆F/F ratio of OGB-1 fluorescence.

MICROSCOPE SOLUTION FOR UNCAGING STUDIES

FemtoS-Galvo equipped with Multiple beam module
Uncaging in well-defined points of a specimen

The accuracy of the excitation point and the high flexible scanning patterns enables the FemtoS-Galvo equipped by Multiple beam module to be the best choice for performing uncaging experiments. Multiple point scanning performed by galvo scanner allows stimulation in points around dendrites or even around spines, multiple line scanning along the dendrite allows following the evoked changes with high speed and high SNR. (Femto2D-Galvo equipped with multiple beam module is still an existing opportunity.)

The accuracy of the uncaging and imaging is established by the followings:
  • the stimulating laser is focused on femtoliter excitation volume
  • the galvanometric scanner ensures microsecond-precision
  • MES control software allows flexible spatiotemporal scanning

Stimulus mapping and analysis software module


Specialized functions for photostimulation mapping

This module of MES control software allows performing photostimulation mapping experiments at a range of locations and shapes. The locations can be picked manually one-by-one, along a line or in a raster, and various stimulation patterns can be selected like point, spiral, x, zigzag. It forms datasets quickly by evaluating fluorescence changes images at defined time intervals. Analysis tool for stimulation mapping can create (multichannel) map images formed by the elicited responses after a mapping experiment.

SYNTHESIZED DERIVATIVES OF NEUROTRANSMITTER GLUTAMATE

DNI-Glu-TFA

The DNI-Glu-TFA is a dinitro-indoline masked form of the glutamate patented by Femtonics which releases the bioactive glutamate rapidly than other commercially available compound. It was developed for high quantum yield so it need less irradiation for releasing and its effective concentration is lower than other caging scaffolds.

Features
  • photostimulation at 740 nm with femtosecond IR laser
  • high quantum yield
  • low effective concentration
  • exists as trifluoroacetic acid salted form
Benefits
  • seven-fold higher quantum yield than other caged form
  • high excitatory postsynaptic potential (EPSP) and high calcium transient as a response of the photorelease
  • lighter illumination sufficient to elicit the same response as with alternative compounds
  • eliciting large transients or regenerative activity
  • multiple illumination of the same structure
  • receptor mapping experiments
  • primarily used for in vitro experiments
  • TFA salt of compound ensures good solubility, stability and low hygroscopicity of the complex
Figure: Photoactivation of DNI-Glu close to a dendritic segment of a hippocampal interneuron. The elicited calcium responses were measured by line scannings along the dendrite. The stimulation and the imaging was performed by Femto2D-Galvo.

Femtonics has aimed to design and develop new caged-neurotransmitters for the frontier neuroscience research for custom order. Feel free to contact us if you have an unmatched need or want to test pre-release compounds. See more.

REFERENCES

Roller Coaster Scanning reveals spontaneous triggering of dendritic spikes in CA1 interneurons, G. Katona, A. Kaszas, G. F. Turi, N. Hajos, G. Tamas, E.S. Vizi, B. Rozsa, PNAS (2011)

Nonlinear signal integration in interneuron dendrites through dendritic NMDA spikes. A) Maximum intensity image stack projection (top), single scan images showing the maximal 22 locations used for two-photon glutamate uncaging for the clustered (middle) and distributed input patterns (bottom). B) Representative uncaging-evoked 3D Ca2+ responses when the maximum number of inputs was activated for clustered (top) and distributed input patterns (upper middle) decreased to noise level by the NMDA receptor antagonist AP5 (Lower Middle). C) Spatial distribution of the peak 3D Ca2+ responses in (B).
Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves. B Chiovini, G F Turi, G Katona, A Kaszas, D Palfi, P Maak, G Szalay, M F Szabo, Z Szadai, Sz Kali and B Rozsa, Neuron (2014)

Effect of VGCC blockers on uncaging-evoked Ca2+ responses in an FS-PV IN. A) Maximum intensity z projection image of a distal dendritic segment. Average uncaging-evoked Ca2+ responses in control conditions (middle) and in the presence of a cocktail of VGCC blockers (bottom). White points are active input locations used for DNI-Glu,TFA uncaging (top). B) Spatial distribution of the peak dendritic Ca2+ response measured along the white line in (A) under control conditions (black) and in the presence of VGCC blockers (red). Inset: mean Ca2+ transients derived from the hot spot (green) and lateral dendritic (magenta) regions before (solid line) and after (dashed line) application of the VGCC cocktail.
Electrical behaviour of dendritic spines as revealed by voltage imaging. Marko A. Popovic, Nicholas Carnevale, Balazs Rozsa; Dejan Zecevic, Nature Communications (2015)

Comparison of eEPSPspine (red) and eEPSPdendrite (green) evoked by two-photon uncaging of glutamate. Lower black traces: somatic patch electrode recordings and timing of uncaging pulse. Left panels: upper shows z-stack of confocal images, lower is a single frame image of a spine in recording position, red dot: uncaging location.

Correlated Synaptic Inputs Drive Dendritic Calcium Amplification and Cooperative Plasticity during Clustered Synapse Development. Kevin F.H. Lee, Cary Soares, Jean-Philippe Thivierge, Jean-Claude Béïque, Neuron (2016)


Local Postsynaptic Voltage-Gated Sodium Channel Activation in Dendritic Spines of Olfactory Bulb Granule Cells Wolfgang G. Bywalez, Dinu Patirniche, Vanessa Rupprecht, Martin Stemmler, Andreas;V.M. Herz, Denes Palfi, Balazs Rozsa, Veronica Egger, Neuron (2015)


Quantitation of various indolinyl caged glutamates as their o-phthalaldehyde derivatives by high performance liquid chromatography coupled with tandem spectroscopic detections: derivatization, stoichiometry and stability studies.Vasanits-Zsigrai A, Majercsik O, Toth G, Csampai A, Haveland-Lukacs C, Palfi D, Szadai Z, Rozsa B, Molnar-Perl I, J Chromatogr A. (2015)


Molecular Tattoo: Subcellular Confinement of Drug Effects Miklos Kepiro, Boglarka H. Varkuti, Anna A. Rauscher, Miklos S.Z. Kellermayer, Mate Varga, Andras Malnasi-Csizmadia, CellPress (2015)


Combined two-photon imaging, electrophysiological, and anatomical investigation of the human neocortex in vitro. Balint Peter Kerekes, Kinga Toth, Attila Kaszas, Balazs Chiovini, Zoltan Szadai, Gergely Szalay, Denes Palfi, Attila Bago, Klaudia Spitzer, Balazs Rozsa, Istvan Ulbert, Lucia Wittner, Neurophotonics (2014)


Spine neck plasticity regulates compartmentalization of synapses J Tønnesen, G Katona, B Rozsa, U V Nägerl, Nature Neuroscience (2014)