All procedures involving animals described in this protocol were approved by the University of Wisconsin-Madison School of Medicine and Public Health Animal Care and Use Committee.
1. Breeding mice to express fluorescent reporter protein in interneuron subpopulations
2. Performing unilateral stereotaxic injection of viral construct
3. Preparation of acute brain slices
4. Preparation of experimental artificial cerebral spinal fluid (eACSF) bags containing dissolved volatile anesthetic isoflurane
5. Preparation of hardware and software for multi-channel recordings
6. Configuration of light stimulation protocols
7. Placing multi-channel probe in ex vivo brain tissue slice
8. Patch clamping targeted neurons and obtaining whole-cell configuration
9. Layer-specific optogenetic activation of axon terminals
A timeline of steps described in the protocol is shown in Figure 1. Cortical inputs arriving from higher order cortical areas or from non-primary thalamic nuclei have partially overlapping terminal fields in layer 1 of non-primary visual cortex24. To isolate independent thalamocortical or corticocortical afferent pathways, a viral vector containing ChR2 and an eYFP fluorescent reporter into either Po or Cg was injected. Cells within the injection radius take up the viral vector and, after 2-4 weeks, express the non-specific cation channel ChR2 and the reporter in both the soma and projecting axons (Figure 2A). Coronal slices were collected. With the appropriate filter cube engaged, axons expressing the viral construct were imaged (Figure 2B). The use of ChR2 to activate axon terminals allows for activation of afferents without the prerequisite for an attached soma.
The animals used in the experiments described here were SOM-tdTomato or PV-tdTomato hybrid animals, which express the fluorescent reporter protein tdTomato in either somatostatin- (SOM+) or parvalbumin-positive (PV+) interneurons, respectively. SOM+ or PV+ interneurons in layer 2/3 were targeted for patch clamping under visual guidance with the appropriate filter cube engaged (Layer 1C). These interneurons have dendrites in layer 1 and are targets of corticocortical inputs (Figure 3A).
Addition of 125 mL of 3.0% isoflurane gas and 175 mL of 95% O2/5% CO2 to a sealed bag resulted in a pre-equilibrium concentration of gas of 1.3%. Gas dissolved into eACSF according to its partition coefficient; the predicted gas phase equilibrium concentration of isoflurane at room temperature was 0.6% (Figure 2D). This was confirmed via gas monitor.
The tissue slice was transferred to the recording chamber and the 16×1 multi-channel recording probe was placed orthogonally to the cortical laminae (Figure 2E). A 150 μm circle of 470 nm light centered over cortical layer 1 was delivered via the objective light path, while extracellular field potentials were collected using the 16 x 1 multi-channel probe and targeted whole-cell patch clamp recordings were conducted in interneurons. A schematic of the recording set-up is shown in Figure 2F.
Post-synaptic potentials (PSPs) were observed in interneurons in response to a train of four 2 ms pulses of light (10 Hz; Figure 3A). Local field potentials were also recorded (Figure 3B). Current source density (CSD; Figure 3C) and multi-unit activity (MUA; Figure 3D) were extracted from local field potentials. Ten trials at several different light intensities were used to conduct post hoc analyses. The amplitude of current sinks extracted from the CSD increased as a function of light intensity (Figure 4A). A three-parameter nonlinear logistic equation was fit to the data for comparisons across pathways. PSP amplitude also increased with current sink amplitude (Figure 4B).
Synaptic responses to thalamocortical and corticocortical inputs were measured during control, isoflurane (0.28 mM), and recovery conditions. Post-synaptic responses of somatostatin- (Figure 5A) to corticocortical stimuli were suppressed during isoflurane, as were evoked current sinks (Figure 5B).
Figure 1: A schematic outlining timeline of important steps in protocol.
Top: Describes timeline of steps necessary for breeding of transgenic animals and expression of viral vector. Bottom: Depicts steps and timeline for preparing materials and conducting experiment on the day of slice preparation. Please click here to view a larger version of this figure.
Figure 2: Injection of viral vector and preparation ex vivo coronal brain slices.
(A) Schematic representation of injection of viral vector into SOM-tdTomato or PV-tdTomato hybrid mice. (B) Coronal slices of the medial parietal association area (mPtA) were harvested, and thalamocortical (top) or corticocortical (bottom) afferent fibers were identified by their eYFP reporter in layer 1. This figure is modified with permission from24. (C) Overlay of eYFP-labeled axon terminals in layer 1 (green) and tdTomato-labelled SOM+ interneurons (red) in superficial layer 2/3. (D) Sealed bags were prepared with a 50:50 solution-to-gas mixture. (E) Placement of a 16 x 1 probe into mPtA (black outline). (F) Schematic of the recording set-up in the cortical slice. Please click here to view a larger version of this figure.
Figure 3: Simultaneous intracellular and multi-channel extracellular recordings in cortical slice.
(A) Whole-cell current clamp patch recording from the soma of a layer 2/3 PV+ interneuron. Four pulses (2 ms each, blue arrows) of blue light (2.2 mW) at 10 Hz were delivered to corticocortical axon terminals in L1. Average (red trace) of ten trials (grey traces) are shown. (B) Raw data from 16 channels of extracellular 16 x 1 probe. Channels placed in cortical tissue are shown in black, and those lying outside of cortex in grey. (C) A current source density diagram, extracted from the local field potential signal, shows synaptic current sinks (blue) in layer 1. (D) Multi-unit activity, generated by applying a high-pass filter to the local field potential signal, isolates spiking activity evoked in lower layers. Please click here to view a larger version of this figure.
Figure 4: Comparison of responses from recordings in two different slices.
Multiple light intensities were used to evoke synaptic responses in cortical layer 1. For each trial, the peak amplitude of the evoked response was extracted from the layer 1 extracellular current sink and EPSPs in layer 2/3 PV+ interneurons. (A) Extracellular response profiles of thalamocortical and corticocortical afferents are compared as a function of light intensity. (B) The relationship between current sink amplitude and EPSP amplitude is pathway dependent. Within each stimulus pathway, data from (A) and (B) were collected simultaneously. Please click here to view a larger version of this figure.
Figure 5: Bath application of isoflurane dissolved in eACSF during simultaneous recordings.
(A) Intracellular whole-cell current clamp recording from layer 2/3 SOM+ interneuron upon activation of corticocortical afferents during control, isoflurane, and wash conditions. Vertical blue lines indicate light stimuli (2 ms; 1.65 mW). (B) Current source density trace extracted from electrode in layer 1. Data were collected simultaneously with those collected in (A). Recovery of responses upon wash demonstrates depression of synaptic responses by isoflurane. Please click here to view a larger version of this figure.
Micropipette for virus injection | ||||
Glass | ID: 0.05 mm, OD: 0.11 mm | |||
Loops | 1 | |||
Heat | Pull | Vel | Time | Pressure |
Ramp + 10 | 20 | 40 | 200 | 300 |
Micropipette for whole-cell patch clamp recordings | ||||
Glass | ID: 1.1 mm, OD: 1.7 mm | |||
Loops | 4 | |||
Heat | Pull | Vel | Time | Pressure |
Ramp | 0 | 25 | 250 | 500 |
Table 1: Recommended glass and parameters for pulling micropipettes for viral injections and whole-cell patch clamp recordings. Glass used for viral injections and whole-cell patch clamp recordings is described, as well as the parameters for pulling micropipettes using the micropipette puller. Consult instruction manuals for micropipette puller for further recommendations or fine-tuning of settings.
Slicing ACSF, sACSF (in mM) | Experiment ACSF, eACSF (in mM) | |
NaCl | 111 | 111 |
NaHCO3 | 35 | 35 |
HEPES | 20 | 20 |
KCl | 1.8 | 1.8 |
CaCl2 | 1.05 | 2.1 |
MgSO4 | 2.8 | 1.4 |
KH2PO4 | 1.2 | 1.2 |
glucose | 10 | 10 |
Internal Solution | ||
K-gluconate | 140 | |
NaCl | 10 | |
HEPES | 10 | |
EGTA | 0.1 | |
MgATP | 4 | |
NaGTP | 0.3 | |
pH = 7.2 |
Table 2: Composition of artificial cerebral spinal fluid and intracellular solution. Reagents and concentrations for sACSF, eACSF, and intracellular pipette solution for patch clamp recordings are listed.
Supplementary Figure 1: Template for preparing block of tissue to collect brain slices. The template is adjusted to the appropriate size, printed, and glued to a microscope slide. A cover slip is glued over the template to prolong its use. The tissue block is placed on a piece of filter paper with the sagittal plane down, aligned to the pink background, and a vertical cut is made in the coronal plane along the black line. Please click here to download this figure.
Supplementary Figure 2: Incubation chamber for harvested brain slices. The chamber is filled with sACSF and bubbled with 95% O2/5% CO2 gas mixture via a bent needle attached to tubing. Incubation platform is made of nylon stretched over a plastic circular fitting. Please click here to download this figure.
Supplementary Figure 3: Platinum structures for slice in recording chamber. Brain slice is transferred to recording chamber via pipette and placed on top of nylon mesh, which is stretched over a horseshoe-shaped piece of flattened platinum wire and super glued in place. Platinum harp is placed over brain slice to anchor it in place during recording. Please click here to download this figure.
Supplementary Table 1: Ostwald (λ) and Bunsen (α) coefficients for other volatile anesthetics. Adapt this protocol for study of other volatile gas anesthetics, such as halothane, sevoflurane, or desflurane. Substitute the equations described in the protocol with the appropriate coefficients as listed in this table. Please click here to download this table.
2.5x broadfield objective lens | Olympus | MPLFLN2.5X | |
40x water immersion objective lens | Olympus | LUMPLFLN40XW | |
95% O2/5% CO2 mixture | Airgas | Z02OX95R2003045 | |
A16 probe | NeuroNexus | A16x1-2mm-100-177-A16 | 16-channel probe |
AAV2-hSyn-hChR2(H134R)-EYFP | Karl Deisseroth Lab, UNC Vector Core | ||
Anesthetic gas monitor (POET II) | Criticare | 602-3A | |
ATP, Magnesium Salt | Sigma Aldrich | A9187 | intracellular solution |
B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J | The Jackson Laboratory | 007914 | Cre-dependent tdTomato mouse |
B6;129P2-Pvalbtm1(cre)Arbr/J | The Jackson Laboratory | 008069 | PV-Cre mouse |
Belly Dancer Shaker | Thomas Scientific | 1210H86-TS | for equilibration of sealed gas bags |
Betadine solution | Generic brand | ||
Bleach | Generic brand | for silver chloriding patch clamp electrode | |
Bupivicaine | |||
Calcium Chloride (CaCl2) | Dot Scientific | DSC20010 | ACSF |
Capillary glass (patch clamp recordings) | King Precision Glass, Inc. | KG-33 | Borosilicate, ID: 1.1mm, OD: 1.7mm, Length: 90.0mm |
Capillary glass (viral injections) | Drummond Scientific Company | 3-000-203-G/X | 3.5" |
Control of junior micromanipulator | Luigs and Neumann | SM8 | for control of junior micromanipulator |
Control of manipulators and shifting table | Luigs and Neumann | SM7 | for control of multichannel electrode and shifting table |
Digidata 1440A + Clampex 10 | Molecular Devices | 1440A | Digitizer and software |
E-3603 tubing | Fisher Scientific | 14171208 | for delivery of 95% O2/5% CO2 gas mixture to incubation chamber + application of pressure during patch clamping |
EGTA | Dot Scientific | DSE57060 | intracellular solution |
ERP-27 EEG Reference/Patch Panel | Neuralynx | Retired | |
Filling needle | World Precision Instruments | 50821912 | for filling patch clamp pipettes |
Filter cube for imaging EYFP | Olympus | U-MRFPHQ | |
Filter paper | Fisher Scientific | 09801E | lay over slice template during preparation of tissue block |
Flaming/Brown micropipette puller | Sutter Instrument | P-1000 | 2.5×2.5 Box filament |
Gas dispersion tube | Sigma Aldrich | CLS3953312C | |
Glass syringe (100 mL) | Sigma Aldrich | Z314390 | for filling gas-sealed bags |
Gluconic Acid, Potassium Salt (K-gluconate) | Dot Scientific | DSG37020 | intracellular solution |
Glucose | Dot Scientific | DSG32040 | ACSF |
GTP, Sodium Salt | Sigma Aldrich | G8877 | intracellular solution |
Headstage-probe adaptor | NeuroNexus | A16-OM16 | adaptor to connect 16-channel probe to headstage input |
Hemostatic Forceps | VWR International | 76192-096 | |
HEPES | Dot Scientific | DSH75030 | ACSF,intracellular solution |
HS-16 Headstage | Neuralynx | Retired | |
Isoflurane | Patterson Veterinary | 07-893-1389 | |
Isopropyl alcohol (70%) | VWR International | 101223-746 | |
Junior micromanipulator | Luigs and Neumann | 210-100 000 0090-R | for manipulation of patch clamp electrode |
LED Light Source Control Module | Mightex | BLS-PL02_US | optogenetic light source control |
Lidocaine | |||
Lynx-8 Amplifier | Neuralynx | Retired | |
Lynx-8 Power Supply | Neuralynx | Retired | |
Magnesium Sulfate (MgSO4) | Dot Scientific | DSM24300 | ACSF |
mCherry, Texas Red filter cube | Chroma | 49008 | for imaging tdTomato fluorescent reporter |
Meloxicam | |||
Micropipette holder | Fisher Scientific | NC9044962 | |
Microsyringe pump | World Precision Instruments | UMP3-4 | |
Mineral oil | Generic brand | ||
MultiClamp 700A | Molecular Devices/Axon Instruments | 700A | Amplifier |
Nitrogen (for air table) | Airgas | NI200 | |
Nylon mesh | Fisher Scientific | 501460083 | stretched over horseshoe of flattened platinum wire, slice rest on top of this during recordings |
Nylon, cut from pantyhose | Generic brand | small piece to create slice platform in incubation chamber, single fibers to create platinum harp | |
Ophthalmic ointment | Fisher Scientific | NC1697520 | |
Pipette | Dot Scientific | 307 | For transferring tissue to rig |
Platinum wire | VWR International | BT124000 | 2 cm, flattened, to make platinum harp |
Polygon400 | Mightex | DSI-E-0470-0617-000 | optogenetic light delivery system, comes with PolyScan2 software |
Potassium Chloride (KCl) | Dot Scientific | DSP41000 | ACSF |
Potassium Phosphate (KH2PO4) | Dot Scientific | DSP41200 | ACSF |
Razor blade | Fisher Scientific | 12-640 | |
Sapphire blade (for vibratome) | VWR International | 100492-502 | |
Scalpel blade | Santa Cruz Biotechnology, Inc. | sc-361445 | |
Sealed gas bag | Fisher Scientific | 109236 | |
Shifting table for microscope | Luigs and Neumann | 380FMU | |
Sodium Bicarbonate (HCO3-) | Dot Scientific | DSS22060 | ACSF |
Sodium Chloride (NaCl) | Dot Scientific | DSS23020 | ACSF, intracellular solution |
Ssttm2.1(cre)Zjh/J (SOM-IRES-Cre) | The Jackson Laboratory | 013044 | SOM-Cre mouse |
Stereotaxic instrument | Kopf | Model 902 | Dual Small Animal |
Super glue | Staples | 886833 | to fix tissue block to specimen stage during slice preparation |
Surgical drill | RAM Products Inc. | DIGITALMICROTORQUE | Microtorque II |
Syringe (1 mL) with LuerLock tip | Fisher Scientific | 309628 | for application of pressure during patch clamping |
Syringe (1 mL) with slip tip | WW Grainger, Inc. | 19G384 | for filling patch clamp pipettes |
Syringe Filters | VWR International | 66064-414 | |
Upright microscope | Olympus | BX51 | |
Vibrating microtome | Leica Biosystems | VT1000S | |
Wypall towels | Fisher Scientific | 19-042-427 |
Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.
Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.
Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.