2. Amplifiers: Axopatch 200B (Axon Instruments, Foster City CA) for ... both the Axopatch 200B and Iso-Flex to allow for alternating stimulation/voltage.
6 Visualizing Cannabinoid Effects Using Brain Slice Imaging and Electrophysiological Approaches Alexander F. Hoffman and Carl R. Lupica
Summary The use of electrophysiological recordings in brain slices is now routinely used to assess the actions of cannabinoid ligands within various central nervous system nuclei. In this chapter we describe common protocols involving both intracellular and extracellular recording techniques in the hippocampus, where the presynaptic modulatory effects of cannabinoid receptor activation have been studied in detail. In addition to describing the basic electrophysiological setup needed for these recordings, we will address common technical problems and limitations involved in working with highly lipophilic compounds, such as the cannabinoid ligands, in brain slices. Key Words: Electrophysiology; brain slice; marijuana; drugs of abuse; intracellular; extracellular; hippocampus; synaptic transmission.
1. Introduction The brain slice preparation has long enhanced our ability to study the fundamental synaptic targets for a variety of drugs (1), including those abused by humans. Intracellular and extracellular recording techniques, in combination with appropriate pharmacological tools, have elucidated the cellular substrates of many drugs of abuse, including nicotine (2), opiates (3,4), alcohol (5), and cocaine (6,7). Recently, the availability of selective ligands has allowed for the application of these traditional approaches to the study of cannabinoid receptors. In this chapter, we discuss the methods routinely used in our laboratory to study the synaptic effects of cannabinoid receptor activation in the hippocampus (8,9). We will first describe preparation and storage of hippocampal brain slices, and then separately describe intracellular and extracellular recording setups, including preparation of cannabinoid ligands routinely used in these studies. From: Methods in Molecular Medicine: Marijuana and Cannabinoid Research: Methods and Protocols Edited by: E. S. Onaivi © Humana Press Inc., Totowa, NJ
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2. Materials 1. Artificial cerebrospinal fluid (aCSF): 126 mM NaCl, 3 mM KCl, 1.5 mM MgCl2, 2.4 mM CaCl2, 1.2 mM NaH2PO4, 11 mM glucose, 26 mM NaHCO3. This can be prepared as a stock (10X) solution and diluted fresh daily as needed. 2. 1% Tween-80, 2% dimethylsulfoxide (DMSO), dissolved in physiological saline. 3. DMSO (for preparation of stock solutions of cannabinoid ligands). 4. “Injector-style” stainless steel razor blades (Ted Pella Inc.) 5. Wecprep single-edge razor blades. 6. Whatman no. 3 qualitative filters. 7. Loctite tissue adhesive (Ted Pella Inc., Redding CA). 8. 10-cc syringe, 23 G needle. 9. Polyethylene PE-50 tubing. 10. 1 mL glass Pasteur pipets + rubber bulbs. 11. Intracellular electrode filling solution (composition varies according to experiment). 12. Borosilicate glass electrodes (1.5 mm O.D., 0.86 mm I.D.; Sutter Instrument Corp., Novato, CA).
3. Methods 3.1. Preparation of Hippocampal Slices 1. Rats are rapidly decapitated, and the brains are removed within 1 min and placed into a chilled aCSF solution. After 1–2 min, the brain is transferred to the filter paper that is kept on a chilled petri dish and is blocked using a single-edge Wecprep razor blade. One blocking cut is made just anterior to the cerebellum, and the other block is made approx 3 mm posterior to the anterior tip of the brain. A small piece of filter paper is then used to transfer the brain, anterior side up (e.g., the filter paper adheres to the anterior surface), to the cutting chamber. A drop of Loctite glue is placed on the stage of the chamber, and the brain is gently placed on the glue. Applying gentle downward pressure on the brain with an index finger, ice-cold (0–4°C) aCSF is then poured into the chamber to completely immerse the brain in solution. Sections are then taken at 300–400 µm thickness (thinner sections are preferred for visualized whole-cell recordings; slightly thicker sections are appropriate for “blind patch” or extracellular recordings). 2. Slices are transferred to a holding chamber using a broken-back Pasteur pipet (the tip is broken off and a bulb is placed over this end). Numerous holding chambers are commercially available through suppliers, including Warner Instruments (Hamden, CT). However, we have found that homemade Gibb chambers (10) work well for maintaining the tissue. The holding chamber may be maintained at room temperature (~22°C) with no detrimental effect on slice viability. However, continuous saturation of the solution with a 95% O2/5% CO2 mixture is absolutely critical. 3. Slices are incubated for at least 90 min prior to being transferred to the recording chamber. This allows for equilibration of the tissue, and recovery of synaptic activity, following the cutting procedure.
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3.2. Preparation of Cannabinoid Solutions 1. Stock solutions of WIN55,212-2, AM251, SR141716A, and most other ligands are prepared as 10 mM in DMSO. These stocks should be stable for many weeks when stored cold in a lab refrigerator or freezer. 2. Drugs are diluted to 100X the desired final bath concentration in a solvent consisting of 1% Tween-80, 2% DMSO, and 97% physiological saline. Thus, for a 1-µM bath concentration, 100 µL of stock would be dissolved in 9.9 mL of solvent to produce a 100-µM solution. 3. Drugs are added to a 10-cc syringe. A 23 G needle is cut roughly in half using a Dremel tool, and the ends filed to allow for PE50 tubing to cleanly fit over the blunt ends. The tubing will thus lead from the nonsharp (Luer-fitting) end attached to the syringe to the sharp (beveled) end, which will be inserted into a rubber septum near the superfusion inlet of the slice chamber. 4. Following experiments, drug syringes may be kept cold in a lab refrigerator for 24–72 h. Thereafter, old drug solutions should be discarded, the syringes and tubing rinsed several times in EtOH and distilled H2O, and fresh drug solutions prepared from stocks.
3.3. Electrophysiology Configuration/Setup 1. Acquisition software/hardware: We have found that Dr. John Dempster’s WCP and EDR programs, freely available for download at http://spider.science.strath.ac.uk/ PhysPharm, are highly versatile programs that are designed to work with a number of different A/D boards, amplifiers, etc. National Instruments (Austin, TX) provides low-cost boards and accessories (e.g., 6024E board and BNC-2090 interface) that may be used to acquire data to a Windows-based PC. 2. Amplifiers: Axopatch 200B (Axon Instruments, Foster City CA) for intracellular recordings; Model 1700 Differential A-C amplifier (A-M Systems, Carlsborg, WA) for extracellular recordings. 3. Stimulus isolation units: Iso-Flex (A.M.P.I., Jerusalem, Israel) provides either constant current or constant voltage stimulation. 4. Timer/pulse delivery: Master-8 (A.M.P.I., Jerusalem, Israel) readily interfaces with both the Axopatch 200B and Iso-Flex to allow for alternating stimulation/voltage step protocols. 5. Perfusion chambers: Warner Instruments (Hamden, CT) Series 20 platforms and chambers are ideally suited to maintaining slice superfusion in a relatively low bath volume (150–200 µL). Slices are perfused at 2 mL/min. A flow-meter (Fisher Scientific) can be used to calibrate solution flow rate, and an in-line solution heater (Warner, SH-27B) can be used to maintain slices at 30–32°C during recordings. 6. Microscopes: A low-power stereomicroscope is sufficient for visualizing tissue and electrode placement for extracellular recordings. A Zeiss Axioscope or other similar microscope equipped with differential interference contrast/infrared (DIC-IR) optics is necessary for visualized whole-cell recordings.
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Fig. 1. DIC-IR image of a stratum radiatum interneuron in a hippocampal brain slice. The slice was cut at 300 µm, and the depth of the recorded cell was approx 175 µm from the surface of the slice. (A) The recording electrode is visible on the left as it approaches the cell. (B) Note the slight dimpling of the cell as the recording electrode makes contact. 7. Razel infusion pumps (Model A-99, Razel Scientific, Stamford, CT) provide an easy means to apply drugs via a 10 cc syringe, as described in Subheading 3.2.
3.4. Intracellular Recordings 1. Internal solution composition will vary according to the desired experiment (see Note 2). Osmolarity should be approx 270–290 mOsm/L and pH should be adjusted to 7.2–7.4. Addition of QX-314, a lidocaine derivative, to the internal solution (1–2 mg/mL) will prevent direct activation of Na+ channels in the postsynaptic cell during electrical stimulation of the slice. 2. Electrodes are pulled on a Sutter P-97 Flaming/Brown micropipet puller. Tips are generally 2–3 µm in diameter, and resistances are 3–6 MΩ. 3. Stimulating electrodes can be purchased from a variety of sources, including Frederick Haer Co. (FHC, Bowdoinham, ME). Alternatively, electrodes can be fabricated by running two strands of formvar-insulated nichrome wire into a 22G spinal needle (or other suitable cannula), then twisting the strands together tightly with a hemostat. 4. Recording electrodes are slowly lowered into the tissue, while applying slight positive pressure to keep the tip free of debris. The targeted cell should dimple slightly as the electrode encounters the membrane. At this point, pressure is released and slight suction can be used to facilitate sealing. Figure 1 shows a hippocampal interneuron prior to, and following, impalement with the electrode. 5. A hyperpolarizing step (10–20 mV) is given every 30 s (alternating with electrical stimulation of the tissue) in order to ensure that whole-cell access is stable. 6. Stimulus intensity is set to elicit a response that is 30–40% of the maximum response amplitude.
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Fig. 2. Inhibition of GABAergic transmission in a stratum radiatum interneuron by WIN55,212-2. IPSCs were evoked by paired (100 ms) stimuli every 30 s. Under control conditions, the response showed paired-pulse facilitation (IPSC2 > IPSC1). After application of 500 nM WIN55,212-2, the first response of the pair showed slightly greater inhibition than the second, resulting in an enhancement of facilitation. This is highlighted by the scaled trace on the right, where the first responses have been scaled to match. The increase in paired-pulse facilitation is one indication of the presynaptic inhibition of GABA release by CB1 receptor activation. Note that the response does not spontaneously recover or reverse following the application of the agonist.
7. After a 10-min period to allow for stabilization of the response, cannabinoid agonists are added to the bath using the Razel pump described in Subheading 3.3. Pumps are intially set to deliver drugs at 20 µL/min, a 1:100 dilution into the flowing perfusate. This will reduce the solvent to 0.01% Tween-80 and 0.02% DMSO. Increasing the pump speed will result in a linear increase in drug concentration to the bath, but this will also increase the solvent concentration. Always run vehicle controls when higher concentrations are used to ensure the stability of the response in the presence of higher solvent concentrations (see Note 4). 8. Peak drug effects are generally observed within 10–12 min of drug application, although longer (20–25 min) time periods may be required for lower concentrations (
