Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Podsumowanie

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.

Streszczenie

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.

Protokół

1. Preparation of Solutions for Brain Slice Preparation and Electrophysiology

1. Solutions for preparation of brain slices were previously described ^{3, 4}. Prepare N-methyl D-glucamine (NMDG)-based cutting and recovery solution of the following composition (in mM): 93 n-methyl D-glucamine, 2.5 KCl, 1.2 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 25 glucose, 5 Na⁺ ascorbate, 2 thiourea, 3 Na⁺ pyruvate, 10 MgSO₄•7H₂O, 0.5 CaCl₂•2H₂O. Adjust to 300-310 mOsm with NMDG. Adjust pH to 7.3-7.4 with 10 N HCl.
   1. Dissolve MgSO₄•7H₂O and CaCl₂•2H₂O in Millipore water to make 2 M stock solutions of each.
   2. Weigh out other components in the order listed and add to a volumetric flask partially filled with Millipore water while stirring to dissolve. Once everything is added, fill volumetric flask with Millipore water.
   3. Measure the pH and adjust with 10 N HCl.
   4. Measure the osmolarity and adjust with NMDG if necessary.
2. Prepare HEPES holding artificial cerebrospinal fluid (aCSF) of the following composition (in mM): 92 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 25 glucose, 5 Na⁺ ascorbate, 2 thiourea, 3 Na⁺ pyruvate, 2 MgSO₄•7H₂O, 2 CaCl₂•2H₂O. Adjust to 300-310 mOsm with sucrose. Adjust pH to 7.3-7.4 with 1 N HCl or NaOH.
   1. Weigh out components in the order listed and add to a volumetric flask partially filled with Millipore water while stirring to dissolve. Once everything is added, fill volumetric flask with Millipore water.
   2. Measure the pH and adjust with NaOH or HCl if needed.
   3. Measure the osmolarity and adjust with sucrose if necessary.
3. Prepare standard aCSF recording solution of the following composition (in mM): 124 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 24 NaHCO₃, 12.5 glucose, 2 MgSO₄•7H₂O, 2 CaCl₂•2H₂O. Adjust to 300-310 mOsm with sucrose. Adjust pH to 7.3-7.4 with 1 N HCl or NaOH.

2. Preparation of Acute Brain Slices

1. Brain slice cutting and recovery are done as previously described ^{3, 4}. Bubble two crystal dishes filled with NMDG recovery solution with 95% oxygen/5% CO₂ gas (carbogen), one dish on ice, the other at 33 °C.
2. Bubble one crystal dish filled with HEPES holding aCSF with carbogen at room temperature.
3. Anesthetize adult mouse (aged 2 to 12 months) with Na⁺ pentobarbital (200 mg/kg, i.p.). Proceed with procedure when the animal has no response to a toe-pinch.
4. If necessary, cut a tail tissue sample for later genotyping of the animal.
5. Open the chest cavity, expose the heart, and clamp the descending aorta. Lacerate the right atrium.
6. Transcardially perfuse animal with 5-10 ml of 0-4 °C NMDG-recovery solution.
7. Decapitate animal, remove the brain and place it in 4 °C NMDG-recovery solution for 1 min.
8. On an ice-chilled metal plate, trim the brain in the desired orientation (coronal, parasagittal, horizontal, etc.) to include the area of interest.
9. Affix the brain block with superglue to the dry stage surface of a vibrating slicer (DSK-Zero 1; Dosaka), submerge the brain block in 4 °C NMDG-recovery solution, and continuously bubble the solution with carbogen.
10. Cut slices of the brain area of interest. Slice thickness is 200-400 μm, depending on the specific brain area of interest, animal age, and experiment to be performed.
11. Immediately after cutting, incubate desired slices in carbogenated, 33 °C NMDG-recovery solution for exactly 12 min, then transfer to carbogenated, room temperature HEPES holding solution for an initial period of 60 min. After this 60 min incubation, slices can be used for recording. Slices are maintained in HEPES holding solution throughout the day until they are used for recording.

3. Patch Clamp Recording from Neurons in Brain Slices

1. Pull a standard patch micropipette with a programmable Flaming-Brown puller (Sutter P-97 or equivalent vertical puller). Pipettes that are optimal for giga-ohm seal formation typically have a resistance of 4-6 MΩ
2. Transfer one slice to the recording chamber (RC-27L; Warner Instruments) on the stage of an upright microscope (Nikon FN-1). Continuously superfuse (1.5 - 2.0 ml/min) the slice with carbogenated, standard recording aCSF heated and maintained at 32 °C. Alternatively, slices can be maintained at room temperature.
3. Locate and center the brain area of interest using a 10X air objective (Nikon Plan Fluor 10X; NA 0.3; WD: 16 mm).
4. Visualize individual neurons using a 40X near-infrared (IR) water immersion objective (Nikon APO 40XW; NA: 0.80; WD: 3.5 mm) connected to IR-differential interference contrast (DIC) optics and a high-speed near infrared charged coupled device (CCD) video camera (Hamamatsu C-7500). Under IR-DIC observation, healthy neurons exhibit a smooth, light-grey plasma membrane.
5. Fill a micropipette with a suitable intracellular recording solution. For most applications, we use the following solution (in mM): 135 potassium gluconate, 5 EGTA, 0.5 CaCl₂, 2 MgCl₂, 10 HEPES, 2 Mg-ATP, and 0.1 GTP (pH adjusted to 7.25 with Tris base, osmolarity adjusted to 290 mOsm with sucrose). Establish a whole-cell recording from a visualized neuron.

4. Local Application of Drugs to Neurons in Slices

1. Pull a standard patch clamp micropipette as described above. Using a pipette puller such as a Sutter P-97 that produces two identical "sister" tips from the same piece of glass is advantageous when multiple drug solutions are to be used on the same cell. Tip sizes that would have a resistance of ~5 MΩ if they were filled with the intracellular solution described above are optimal.
2. Backfill the micropipette with a solution of drug diluted in carbogenated, standard recording aCSF. Point the tip downward, and flick the drug-filled micropipette to remove any air bubbles trapped in the column of solution.
3. Mount the drug-filled micropipette in a pipette holder connected with tubing to a suitable pressure ejection system, such as a Picospritzer III (General Valve Co.). The pipette holder should be mounted onto a micromanipulator with the same resolution as manipulators commonly used for patch clamp recording (for example, Sutter MP-285). Additionally, the manipulator should allow for diagonal movement into and out of the slice. Manually eject pressure 3 to 4 times into the micropipette in order to build up adequate pressure behind the column of fluid.
4. Using the micromanipulator controls, lower the drug pipette into the slice while visualizing the tip under IR-DIC optics. Ideally, one should visually identify and center a neuron to be recorded from, followed by positioning of the drug pipette near the cell prior to lowering the recording micropipette into the slice. Positioning the drug pipette can cause movement of tissue in the vicinity of the recorded cell that could disrupt the recording if the drug pipette is moved into position after establishing a gigaohm seal.
5. Using IR video, position the drug micropipette at the top surface of the tissue slice. Execute one pressure ejection and monitor 1) the vicinity of the tip for movement of debris related to the ejection, and 2) the micropipette tip for any signs that the tip is clogged or blocked. If the tip is blocked/clogged, retract the pipette and replace it with a new one.
6. Approach the recorded cell diagonally from the top of the slice such that when in final position, the tip of the drug-filled pipette is 1) in the same focal plane (or slightly below) as the recorded cell and the tip of the recording electrode, and 2) 10 to 40 μm from the recorded cell. If the drug-filled pipette tip is above the recorded cell, the force from the pressure ejection could disrupt the gigaohm seal.
7. Record the desired cellular response while holding the cell in voltage clamp mode or current clamp mode. We routinely record acetylcholine and/or nicotine-elicited cellular currents while holding neurons in voltage clamp mode. Although pressure ejection can be triggered manually when using a Picospritzer III, for more reproducible results it is advisable to trigger the pressure ejection using the acquisition system (Axon Digidata 1440 and pClamp 10.3) with a digital TTL pulse.

5. Controlling the Drug-filled Micropipette with a Piezoelectric Translator

1. If possible, mount the drug-filled pipette holder to a single-dimension piezoelectric translator (e.g. Piezojena PA-100 or Burleigh PZM-150) that is capable of accepting an analog voltage input (again, from the acquisition system), which is then mounted onto the high-precision micromanipulator (Sutter MP-285). A piezoelectric translator is useful because the movement of the drug-filled pipette into and out of the slice, including the timing and speed of entry/exit, can be more easily controlled and reproduced from experiment to experiment. When studying nAChRs, the desensitizing effects of nicotine leakage from the drug-filled pipette, should they ever occur, are minimized when the pipette is only moved near the cell with a piezoelectric translator for the moment of pressure ejection.
2. Using the manually-controlled potentiometer on the piezoelectric voltage control amplifier, dial the voltage to its maximal value.
3. Using the micromanipulator, position the drug-filled pipette in the slice where the pipette will eject its contents onto the recorded cell, and retract the pipette by manually reducing the voltage to zero on the piezoelectric voltage control amplifier.
4. Using an analog output signal from the recording acquisition system, move the drug-filled pipette into the slice over a period of 250 msec starting 300 msec before the pressure ejection TTL pulse occurs. Retract the pipette over a period of 250 msec, starting 50 msec after the TTL pulse ends.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
N-Methyl D-glucamine	Sigma	M2004
KCl	Sigma	P3911
NaH2PO4	Sigma	S9638
NaHCO3	Sigma	S6014
HEPES	Sigma	H3375
glucose	Sigma	G5767
Na+ ascorbate	Sigma	A4034
thiourea	Sigma	T8656
Na+ pyruvate	Sigma	P2256
MgSO4∙7H2O	Sigma	230391
CaCl2∙2H20	Sigma	223506
NaCl	Sigma	S9625
Na+ pentobarbital	Vortech Pharmaceuticals	76351315
potassium gluconate	Sigma	G4500
EGTA	Sigma	E3889
Mg-ATP	Sigma	A9187
GTP	Sigma	G8877
DSK-Zero 1 Vibrating slicer	Ted Pella, Inc.
P-97 Flaming/Brown micropipette puller	Sutter
RC-27 Recording chamber	Warner
TC-344B Perfusion heater controller	Warner	640101
SH-27B Solution heater	Warner	640102
Nikon FN-1	Nikon
C-7500 CCD Video camera	Hamamatsu
Picospritzer III	General Valve Co.
MP-285 Micromanipulator	Sutter
PA-100 Piez–lectric translator	piezosystem jena, Inc.
12V40 piezo amplifier	piezosystem jena, Inc.
Axopatch 200B	Molecular Devices Corp.
Digidata 1440A	Molecular Devices Corp.