Deep brain optical measurements of cell type-specific neural activity in behaving mice

Abstract

Recent advances in genetically encoded fluorescent sensors enable the monitoring of cellular events from genetically defined groups of neurons in vivo. In this protocol, we describe how to use a time-correlated single-photon counting (TCSPC)-based fiber optics system to measure the intensity, emission spectra and lifetime of fluorescent biosensors expressed in deep brain structures in freely moving mice. When combined with Cre-dependent selective expression of genetically encoded Ca(2+) indicators (GECIs), this system can be used to measure the average neural activity from a specific population of cells in mice performing complex behavioral tasks. As an example, we used viral expression of GCaMPs in striatal projection neurons (SPNs) and recorded the fluorescence changes associated with calcium spikes from mice performing a lever-pressing operant task. The whole procedure, consisting of virus injection, behavior training and optical recording, takes 3-4 weeks to complete. With minor adaptations, this protocol can also be applied to recording cellular events from other cell types in deep brain regions, such as dopaminergic neurons in the ventral tegmental area. The simultaneously recorded fluorescence signals and behavior events can be used to explore the relationship between the neural activity of specific brain circuits and behavior.

Figure 1

General scheme for using TCSPC-based photometry to measure the fluorescence of genetically encoded biosensors in vivo. Schematic drawing of a TCSPC-based fiber optic system to measure local fluorescence signals in the dorsal striatum of a mouse performing a lever-pressing operant task. (1) Jacketed single-mode fiber for excitation. (2) Jacketed multimode fiber for photon collection. (3) Optional electrical cable for simultaneous electrophysiological recording. (4) Right lever. (5) Food magazine. (6) Left lever. The inset illustrates a hybrid fiber probe (8) lowered into the dorsal striatum and fixed in place by dental acrylic (7). This figure is adapted with permission from ref. , Nature Publishing Group.

Figure 2

In vivo measurement of GCaMP5G fluorescence using TCSPC-based photometry. (a) An example trace of GCaMP5G fluorescence intensity in the format of ΔF/F over time in a freely moving A2A–Cre mouse expressing GCaMP5G specifically in striatal indirect-pathway SPNs. Fluorescence intensity was calculated by integrating the photon count of peak GCaMP5G spectral channels (channels 6–13) in each time-resolved spectrum (b,c). Individual spectra were acquired at 20 Hz. (b,c) Examples of individual time-resolved GCaMP5G spectra at the baseline level (b) and at the peak of a fluorescence transient (c). (d,e) Normalized GCaMP5G spectra (d) and fluorescence decay curves (e) acquired at the baseline (as in b) and at the peak of a fluorescence transient (as in c). Despite the large difference in fluorescence intensity (b versus c), there is no difference between normalized spectra (d) and fluorescence lifetime (e), consistent with previous studies. All animal protocols used in this study were approved by the US National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee.

Figure 3

Potential applications of a TCSPC-based fiber optics system for ratiometric measurement in a dual-fluorophore system and lifetime measurement in FRET-based biosensors. (a–f) Examples of time-resolved spectra measured in solutions containing different volume ratios of Alexa Fluor 488 and Alexa Fluor 546 fluorescent dyes. (g) Normalized spectra of Alexa Fluor 488 (green), Alexa Fluor 546 (orange) and a mixture of Alexa Fluor 488 and Alexa Fluor 546 with the volume ratio of 1:4 (black). The dashed gray trace is a reconstructed spectrum using the equation Y = a × Y1 + b × Y2, where Y1 is the normalized spectrum of Alexa Fluor 488, Y2 is the normalized spectrum of Alexa Fluor 546, a = 0.2 and b = 0.8. (h) Normalized fluorescence decay curve of Alexa Fluor 488 and the 1:4 mixture of Alexa Fluor 488 and Alexa Fluor 546. (i) Goodness of fit to show the reconstructed spectrum (gray in g) and the measured spectrum (black in g) has a nearly perfect match. (j,k) Examples of fluorescence spectra (j) and lifetime (k) acquired from HEK cells expressing FRET-based cAMP sensor ^TEpac^VV before (black) and after (red) adding the adenylyl cyclase activator forskolin (100 µM) into the medium. (l) Continuous measurement of fluorescence lifetime of ^TEpac^VV before and after the addition of forskolin.

Figure 4

Surgical procedures for intra-striatal virus injection and optical fiber implantation. (a–d) The first surgery for virus injection and creating a dental cement base. (a) A virus injection kit attached to the vertical arm of a stereotaxis. The kit consists of a 1-µl (or 2-µl) Hamilton syringe, connection polyethylene tubing (filled with saline) and a 30-gauge needle. (b) Picture showing the virus injection needle placed above the burr hole targeting the left striatum. Three anchoring screws have been driven into the skull. (c) Dental cement is applied with a paintbrush. (d) Picture showing the final step of the first surgery. A dummy cannula (marking the position of the burr hole) is fixed in place by dental cement. A thin dental cement base is created to cover the skull surface. Animals are allowed at least 2 weeks of recovery time before proceeding to next steps. (e,f) The second surgery for fiber implantation on the day of optical measurement. (e) The dummy cannula has been removed. The burr hole made during the first surgery has been re-exposed for lowering the fiber probe into the brain. (f) The final step of the second surgery. The fiber probe is fixed in place with a generous amount of dental cement. (g) Available options for connection between the implanted fibers and the fibers to the laser and the detector. (h) A detachable ferrule-sleeve connection for single-mode fibers. Panels g and h are courtesy of L. Bergann at Becker & Hickl. All animal protocols used in this study were approved by the US National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee.

Figure 5

Procedures to make a hybrid optical fiber probe. (a) A fiber optic stripper is used to remove the jacket and acrylic coating of a single-mode fiber. (b) A single pull of the fiber stripper can remove both the jacket and coating. (c) The stripped single-mode fiber is placed on a high-precision fiber cleaver to create a flat-cleaved end. (d) A perfectly round bright spot is formed by the blue light coming out of a single-mode fiber that has been stripped and cleaved through the steps shown in a–c. (e) The multimode and single-mode fibers are glued together. (f) A metal tube is added to reinforce the probe.

Figure 6

A series of snapshots of time-resolved spectra acquired during the process of lowering the fiber probe into the striatum. (a) Detected photons are mostly from the ambient light when the fiber probe is placed above the brain surface. (b) The photon count decreases after the tip of the probe is submerged below the brain surface. (c) The laser-excited GCaMP5G spectrum starts to appear while the probe is going down through the cortex. (d) The GCaMP5G spectrum becomes dominant when the tip of the probe is in the dorsal striatum. (e) The intensity of GCaMP5G (measured at the same place as in d) is markedly increased after the animal wakes up from anesthesia (note the peak values highlighted by red dashed ovals). In all time-resolved spectrum plots, the x axis (‘Rout Chan X’) depicts the wavelength channels for detecting the fluorescence spectrum; the y axis depicts the time channels (in ns) for measuring fluorescence decay time; and the z axis is the photon count. All animal protocols used in this study were approved by the US National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee.

Figure 7

Examples of fluorescence changes in freely moving mice expressing GCaMPs. (a) Comparison between GCaMP3 and GCaMP5G fluorescence transients recorded in freely moving mice expressing GCaMP3 or GCaMP5G in indirect-pathway SPNs. (b) The GCaMP5G transients (expressed in indirect-pathway SPNs) are abolished by isoflurane anesthesia and come back after the mouse wakes up. (c) Fluorescence transients detected in the ventral tegmental area (VTA) in TH-Cre mice after viral injection of AAV-FLEX-GCaMP6s. (d,e) GCaMP3 (expressed in direct-pathway SPNs) fluorescence aligned to the first food magazine entry made by the mouse after left lever presses (d), and its reversal behavior: the first left lever press after food magazine entries (e). Top, individual trials with color-coded fluorescence intensity. Black squares indicate left lever presses. White dots indicate food magazine entries. Bottom, averaged response from all the trials expressed as mean (black trace) ± s.e.m. (gray traces). *** and ### indicate P < 0.0001, paired t test between the fluorescence at 0 s and the peak (in d) or trough (in e). The peak (d) and trough (e) are calculated by averaging the fluorescence between −1 s and −0.8 s. Two vertical lines are drawn to highlight the area of interest in d and e. All animal protocols used in this study were approved by the US National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee.