Published: October 29th, 2012
In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.
Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9′A mice 1 and α6 L9′S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.
1. Preparation of Solutions for Brain Slice Preparation and Electrophysiology
In our experiments, we routinely record from dopamine (DA)-producing neurons of the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). In voltage-clamp mode, pressure application of acetylcholine or nicotine to these cells will typically result in a rapid, inward cation current that reaches peak within 100-200 msec (Figure 1A-B). Decay of the current is largely dictated by diffusion of the drug from the site of action, and whether enzymes in the slice are present to metabolize the dru.......
The method presented in this paper is broadly useful for studying ligand-gated ion channel function in brain slice preparations. However, there are a number of factors that will significantly affect the quality and reproducibility of experimental data that result from utilizing this method. For example, evoked currents are very sensitive to the diameter of the tip of the drug-filled pipette. Small tips will cause difficulty with ejecting the drug solution, and large tips with low resistance will be more likely to disrupt.......
This work was supported by National Institutes of Health (NIH) grant DA030396. Thanks to members of the Drenan lab for helpful discussion and critique of the manuscript. Special thanks to Mi Ran Kim for technical assistance and Jonathan Thomas Ting for advice regarding adult mouse brain slices.....
|Name of the reagent
|DSK-Zero 1 Vibrating slicer
|Ted Pella, Inc.
|P-97 Flaming/Brown micropipette puller
|RC-27 Recording chamber
|TC-344B Perfusion heater controller
|SH-27B Solution heater
|C-7500 CCD Video camera
|General Valve Co.
|PA-100 Piezoelectric translator
|piezosystem jena, Inc.
|12V40 piezo amplifier
|piezosystem jena, Inc.
|Molecular Devices Corp.
|Molecular Devices Corp.
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