Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics

Abstract

The development of the living acute brain slice preparation for analyzing synaptic function roughly a half century ago was a pivotal achievement that greatly influenced the landscape of modern neuroscience. Indeed, many neuroscientists regard brain slices as the gold-standard model system for detailed cellular, molecular, and circuitry level analysis and perturbation of neuronal function. A critical limitation of this model system is the difficulty in preparing slices from adult and aging animals, and over the past several decades few substantial methodological improvements have emerged to facilitate patch clamp analysis in the mature adult stage. In this chapter we describe a robust and practical protocol for preparing brain slices from mature adult mice that are suitable for patch clamp analysis. This method reduces swelling and damage in superficial layers of the slices and improves the success rate for targeted patch clamp recordings, including recordings from fluorescently labeled populations in slices derived from transgenic mice. This adult brain slice method is suitable for diverse experimental applications, including both monitoring and manipulating neuronal activity with genetically encoded calcium indicators and optogenetic actuators, respectively. We describe the application of this adult brain slice platform and associated methods for screening kinetic properties of Channelrhodopsin (ChR) variants expressed in genetically defined neuronal subtypes.

Figure 1

Equipment for preparation of brain slices.(A) Compresstome VF-200 slicing machine with major components labeled as follows: 1-slicing chamber, 2-blade arm, 3-micrometer, 4-controller box.(B) Alternate view of the blade arm with lipstick inserted into the receiver of the slicing platform.(C) Brain Slice Keeper-4 apparatus.(D) Disassembled BSK-4 with major components labeled as follows: 1-lid, 2-four chambers with netting, 3-gas diffuser stone, 4-outer container.

Figure 2

The protective recovery method yields superior neuronal preservation for acute brain slice preparation from mature adult animals. (A) Comparison of procedural steps in the protective cutting versus protective recovery methods. (B) Rapid neuronal swelling and subsequent shriveling in acute brain slices prepared from 5 month old adult mice with the sucrose aCSF protective cutting method.(C) Reduced swelling and improved neuronal preservation in acute brain slices prepared from 5 month old adult littermate mice with the protective recovery method.

Figure 3

GCaMP3 calcium imaging to assess functional integrity of mature adult brain slices. (A) Transient bath application of high K⁺ solution (15 mM) evokes weak fluorescence increase in hippocampal dentate granule cells of brain slices prepared from a 5 month old Thy1-GCaMP3 transgenic mouse using the standard sucrose aCSF protective cutting method. Arrows mark examples of weakly responding neurons.(B) The same high K⁺ (15 mM) perfusion evokes a robust increase in fluorescence throughout the entire granule cell layer in brain slices prepared from an age matched littermate animal using the NMDG protective recovery method.(C) Example raw traces of fluorescence intensity measured over time for selected regions of interest (ROIs) demonstrating the effect of high K⁺ bath perfusion. Scale bars: 50%, 50 s. Summary data comparing ΔF/F for the two slice preparation methods over a range of extracellular K⁺ concentrations.

Figure 4

Positioning of laser-coupled optical fibers for focalized laser stimulation in brain slices.(A) Example positioning of a 200 μm core optic fiber directly over a recorded striatal neuron (asterisk).(B) Each laser pulse illuminates an ellipse surrounding the target neuron.(C) Estimated area of illumination for determining power output as a function of area (irradiance).

Figure 5

Comparative analysis of ChR kinetic variants in distinct cell types. Measurement of the kinetics of channel closure (Tau_off) following a brief 2-ms light stimulation was performed using whole-cell voltage clamp (−70 mV) as a sensitive assay for screening novel ChR kinetic variants.(A) Experiment #1: comparison of Tau_off measured in cortical pyramidal neurons with transgenic expression of ChR2_R or VChR1. Scale bars: 200 pA, 100 ms. Experiment #2: comparison of Tau_off measured in cortical pyramidal neurons expressing the novel variants ChETA_ARC or oChIEF_AC. Scale bars: 50 pA, 100 ms.(B) Combined data for pyramidal neurons.(C) Comparison of Tau_off measured in cortical fast spiking interneurons with transgenic expression of ChR2_R (VGAT-ChR2_R-EYFP line 8) or ChETA_TR (R26-2XChETA_TR/Pvalb-IRES-Cre). Scale bars: 200pA, 10 ms.(D) Summary data comparing Tau_off measured from either cortical fast-spiking interneurons or cortical pyramidal neurons both expressing ChR2_R. Intrinsic cell type differences influence measured kinetic properties and thus preclude comparative analysis across cell-types.

Figure 6

Improved visualization of ChR2-expressing neurons for targeted patch clamp recordings in brain slices using viral P2A linkers.(A) The challenge of identifying ChR2-EYFP expressing neurons is examined in acute striatal brain slices from Ai32/D1-Cre mice. This line has strong expression of the ChR2-EYFP transgene in roughly half of all striatal medium spiny neurons. The ChR2-EYFP gene fusion is localized to the cell membrane and produces a dense fluorescent neuropil with little signal from cell bodies. A recorded neuron is shown (asterisk) along with an inset of recorded photocurrent, thus confirming the identity as a D1+ MSN.(B) A recorded MSN in a nearby region had no photocurrent and was presumed D1 negative (D1−/D2+ MSNs account for the other half of the MSN population). The recorded neurons were indistinguishable on the basis of morphology or live EYFP fluorescence.(C) Analysis of native tdTomato fluorescence together with double immunostaining with anti-2A (indicating localization of ChR2) and DARPP32 (indicating all MSNs in the striatum region) demonstrates the unambiguous identification of ChR2 expressing neurons with the opsin-2A-XFP expression strategy. The example shown here is from our Cre-inducible ChETA-P2A-tdTomato reporter line crossed to the RGS9-Cre driver mice for labeling a subset of striatal MSNs.(D) The use of the viral P2A linker (green sequence) allows for physical uncoupling of opsin and fluorophore, and the 2A epitope tag can then be used to track the localization of the membrane-targeted opsin protein. The cytosolic fluorophore, in this case tdTomato, fills the entire cell body. Scale bars: 20 μm in panels A and B, 50 μm in panel C.

Figure 7

Avoiding photoactivation while searching for ChR2-expressing cells.(A–C) The excitation/emission spectra of various optical filter sets are plotted together with the activation spectrum for ChETA. Considerable overlap with ChETA activation is observed for the excitation range using EYFP (A) and Texas red (B) filter sets but not with a custom tdTomato filter set (C). (D) Raw traces of maximal photocurrents evoked by 1-s epifluorescent illumination (blue line) using the various filter sets for a ChETA-P2A-tdTomato expressing neuron recorded in whole-cell voltage clamp.(E) Cell-attached recordings demonstrating light-evoked spiking with the various filter sets at full and reduced light intensity. Complete elimination of spiking was only achieved with the custom tdTomato filter set at reduced light intensity.