Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.
This protocol can be used to investigate whether volatile anesthetics disrupts some synopsis more than others, to determine if their effects are cell type or as pathway specific, and to determine what effects occur at the population level. The main advantage of this technique is the specificity with which we can assess the effects of volatile anesthetics. We can target specific pathways and cell types in simultaneously assay population activity.
Begin by preparing the tissue block for sectioning. Gently lift the brain from the skull cavity and place it on the filter paper. Make a cut along the sagittal plane of the brain parallel to the midline and place the tissue block on this sagittal plane.
Guide the filter paper over the blocking template, and align the brain with the underlying template outline. Make a parallel cut in the coronal plane as indicated by the lines on the template, adding a small drop of slicing aCSF to keep the filter paper wet if necessary. Place the tissue block in four degrees Celsius slicing aCSF and apply a small amount of glue to the ice-cold specimen stage.
Lift the tissue block from the slicing aCSF and wick away excess solution with a corner of an absorbent towel. Glue the posterior coronal plane of the tissue block to the specimen stage with the dorsal surface of the brain facing the sapphire blade. Collect 500 micrometer thick coronal brain slices and place them on a nylon mesh in 34 degrees Celsius slicing aCSF.
Allow the container to reach room temperature. To prepare experimental aCSF bags with dissolved isoflurane, add 600 milliliters of experimental aCSF and 600 milliliters of 95%oxygen and 5%carbon dioxide gas mixture to an empty polytetrafluoroethylene gas bag. Label this bag control.
Add 300 milliliters of experimental aCSF to another polytetrafluoroethylene gas bag, and label this bag isoflurane. Proceed with preparing the isoflurane bag according to the manuscript directions. Configure the light simulation protocols by randomly assigning wave forms to each profile.
Each profile with its assigned wave form corresponds to one trigger pulse from a digital TTL input or one trial. When finished, save the profile sequence. To place the multi-channel probe in the ex vivo brain tissue slide, perfuse bubbled experimental aCSF at three to six milliliters per minute, and transfer the brain slice containing the area of interest onto the mesh grid in the microscope perfusion chamber.
Anchor the brain slice with a platinum harp. Rotate the mesh grid so that the line of electrode contacts on the distal end of the multi-channel probe is approximately perpendicular to the peel surface. Under broad field illumination and fine control of the micromanipulator, lower the multichannel probe toward the surface of the slice.
Engage the appropriate filter cube for visualization of the fluorescent reporter protein expressed in axon terminals of cortical afferents. If necessary, rotate the slice to more precisely align the probe with the peel surface. Position the probe just above the plane of the slice 200 micrometers short of the final target position along the x-axis, leaving at least one channel outside of the area of tissue being recorded.
Slowly insert the probe into the slice along its longitudinal axis. To minimize damage to the tissue, only advance the probe to the extent that the sharp tips are just visible below the tissue surface. Switch experimental aCSF source to bagged control solution and identify the fluorescently-labeled cell for targeted patch clamp recording.
Restrict the aperture to the smallest diameter and engage the high power water immersion objective, taking care to avoid contact between the multichannel probe and objective lens. Bring the tissue into focus. Center the light over an area of tissue adjacent to but not overlapping the multi-channel probe.
Engage the appropriate filter set to allow imaging of cells expressing the Cre-dependent fluorescent marker. Raise the objective lens to create ample space for lowering a patch pipette. Load a patch pipette with internal solution and mount it on the electrode holder.
Use a one milliliter syringe to apply positive pressure corresponding to approximately 0.1 milliliter air. Lower the patch pipette into the solution bringing the tip into focus under visual guidance, and obtain whole cell recording from the targeted cell. The animals used in this protocol expressed fluorescent reporter protein tdTomato in either somatostatin or parvalbumin-positive interneurons.
These interneurons were targeted for patch clamping under visual guidance with the appropriate filter cube engaged. Postsynaptic potentials were observed in interneurons in response to a train of four two millisecond pulses of light. Local field potentials were also recorded.
Current source density and multi-unit activity were extracted from local field potentials. The amplitude of current sinks extracted from the current source density increased as a function of light intensity. A three parameter non-linear logistic equation was fit to the data for comparisons across pathways.
Postsynaptic potential amplitude also increased with current sink amplitude. Synaptic responses to the thalamocortical and cortico-cortical inputs were measured during control, isoflurane, and recovery conditions. Post synaptic responses of somatostatin and cortico-cortical stimuli and the evoked current sinks were suppressed in isoflurane conditions.
When preparing the brains slices, maintaining the health of the tissue is paramount. It's important to keep the tissue cool throughout the slicing procedure, working quickly without damaging the tissue. Multichannel extracellular recordings may be post-processed in a variety of ways.
For example, high pass filtering of the wideband signal isolates population spiking activity while current source density calculations highlight synaptic activity. Together, these techniques have allowed us to explore anesthetic effects on specific isolated components of cortical circuits, providing insight into the mechanisms underlying loss of consciousness in vivo.
