The optopatcher: an electrode holder allowing the insertion of an optical fiber into a patch pipette
A picture of the optopatcher. The holder is connected to a typical intracellular headstage (HS-2A-x0.1LU, Molecular devices, Sunnyvale, CA, USA). On the right side a silicone tube is connected to the pressure port and the optical fiber is inserted inside the hollow screw on the left side. A standard glass pipette is connected to the holder (1.5mm O.D.) and the fiber within it emanating the blue light at its end. Note that the lower part of the pipette was coated (starting 2 mm away from its tip) with an opaque lacquer in order to prevent illumination aside from tip.
In order to perform simultaneous intracellular recording and light stimulation of a single neuron, two separate positioning systems are often needed (one to position the recording electrode, one to position a waveguide near the recorded neuron). More sophisticated solutions for single neuron photostimulation involve light patterning techniques which are not suited for deep in vivo recordings. Katz et al. came up with a simple and affordable solution for this problem, by designing a patch pipette holder containing an additional port for the insertion of an optical fiber into the pipette.
This device, which they called “OptoPatcher” allows whole cell patch-clamp recording simultaneously with direct projection of light from the recording pipette. The holder spares the use of an additional manipulator and, importantly, enables accurate, stable and reproducible illumination. Moreover, the presence of the bare fiber within an aqueous solution instead of the brain can prevent tissue damage due to heating of the brain. In addition, replacement of standard pipettes is done as easily as with the available commercial holders.
The OptoPatcher was used successfully in vivo for intracellular recordings from different cortical layers in the motor cortex of transgenic mice expressing channelrhodopsin-2 under the Thy1 promoter and it was also used in-vitro for NMDA uncaging using UV flash.
The properties of the optopatcher. (A) Drawing of the electrode holder. The bare optical fiber is inserted through the optic fiber port into the glass pipette which is inserted at the pipette connector. (B) Light transmission through the brain was measured by mounting a brain slice on a cover glass and advancing the electrode into the tissue. (C) Light intensity was reduced on average by about 70% at 1000 µm depth (n = 15). (D, E) Beam profile in saline (D) and cortex (E). Images are sections of a 3D map along the illumination axis trough the pipette’s tip and are normalized to the maximal intensity. Notice the decreased light transmission in the center of the beam profile in saline which results due the glass pipette (See Supplementary Fig. S4).
In-vivo optogenetic stimulation and recording of cortical neurons using the optopatcher. (A) Low magnification widefield fluorescence image of the motor cortex showing EYFP fluorescence (in green) localized to pyramidal cells of layer 5a (and b) and their apical dendrites in the supragranular layers 1-3. Arrow points to a patched and biocytin-filled cell (red). (B) Higher magnification of the same neuron (ApoTome, maximum intensity projection) shows the typical features of a small pyramidal cell. (C) A depth profile of LFP recorded by the optopatcher in response to a brief light pulse (1 ms) illuminated by the recording pipette. (D) Two traces showing the response of intracellularly L5 recorded cell to 50Hz light stimulation. The pattern of light stimulation (blue ticks) is depicted below the voltage traces. (E) Overlay of the 8 first consecutive responses shows a short delay of the highly reproducible responses. (F) Two traces recorded intracellularly from a L2 cell showing the response to 50 Hz light stimulation. Optical stimulation trace as above. (G) Overlay of the 10 first consecutive responses to the light stimulations showing the 2.4 ± 0.55 ms delay for the responses. Scale bars for D, F are similar.
This entry was posted in Journal Club
and tagged fiberoptics
. Bookmark the permalink