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

Abstract

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 concentrationresponse 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. Video LinkThe video component of this article can be found at http://www.jove.com/video/50034/ Protocol Preparation of Solutions for Brain Slice Preparation and Electrophysiology1. 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 comp...

Nicotine regulates activity of lateral habenula neurons via presynaptic and postsynaptic mechanisms

“…To minimize indirect effects through MHb, we locally applied ACh (30 μΜ) onto the somata of recorded LHb neurons by pressure injection from a second micropipette33. In the presence of atropine, ACh induced an inward current (25.6 ± 7.1 pA) in 6/10 neurons tested ( p  = 0.005) which was significantly depressed by MII [H9A; L15A] (to 33.0 ± 4.0% of ACh, p  = 0.016; Fig.…”

Nicotine Dependence Reveals Distinct Responses from Neurons and Their Resident Nicotinic Receptors in Medial Habenula

“…By puff applying low concentrations of ACh, a6* nAChRs can be selectively activated (Drenan et al, 2008;Engle et al, 2012Engle et al, , 2013Powers et al, 2013). This is a helpful method for determining whether a particular cell type in the brain expresses functional a6* nAChRs.…”

α6-Containing nicotinic acetylcholine receptors in midbrain dopamine neurons are poised to govern dopamine-mediated behaviors and synaptic plasticity

“…7A). AMPAR function was measured using local application of AMPA to the recorded cell using a puff-pipette as previously described and validated (Engle et al, 2012, Engle et al, 2013, Engle et al, 2015). Based on the dose-range found to be sufficient to stimulate locomotor activity in α6L9S mice (0.02 to 0.05 mg/kg; Fig.…”

“…prior to cardiac perfusion. Brain slices were prepared as previously described (Engle et al, 2012). Mice were perfused with oxygenated (95% O 2 /5% CO 2 ) 4°C N-methyl-D-glucamine (NMDG)-based recovery solution containing (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH 2 PO 4 , 30 NaHCO 3 , 20 HEPES, 25 glucose, 5 Na + ascorbate, 2 thiourea, 3 Na + pyruvate, 10 MgSO 4 ·7H 2 O, and 0.5 CaCl 2 ·2H 2 O.…”

“…Cells were held at −60 mV and AMPA receptor (AMPAR) function was analyzed as previously described (Engle et al, 2012, Engle et al, 2013, Engle et al, 2015). A micropipette filled with AMPA (100 μM) was loaded onto a single-dimension piezoelectric translator attached to a micromanipulator.…”