Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

Abstract

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas. Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=3136 (We also use this same protocol in mice.) Under deep urethane anesthesia, animals are decapitated and the brain is quickly dissected out. Then 300 μm-thick coronal brain slices containing the midrostral part of substantia nigra are cut on a Leica vibratome (VT-1200S). The slice cutting procedure is performed in an ice-cold, oxygenated high sucrose cutting solution containing (in mM): 220 sucrose, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 10 D-glucose. It is bubbled with a mixture of 95% O2/5% CO2 to keep oxygenated and maintain pH at 7.4. Keep the cutting solution ice-cold throughout the procedure. 2. The brain slices are incubated in an incubation chamber filled with an oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2.5 CaCl2, 1.3 MgCl2, 10 D-glucose. It is bubbled with a mixture of 95% O2/5% CO2 to keep oxygenated and maintain pH at 7.4. The incubation temperature is 34°C for 45 min and then at room temperature until the brain slice is transferred to the recording chamber.1. One brain slice is transferred to a patch clamp recording chamber continuously perfused with the oxygenated aCSF at 32°C. 2. Patch pipettes are pulled from autoclaved borosilicate (KG-33) glass capillary tubing (1.10 mm i.d., 1.65 mm o.d., King Precision Glass, Claremont, CA) using a PC-10 puller (Narishige, Tokyo, Japan) and filled with intracellular solution prepared with DNase-RNase-free water (Fisher Scientific) (containing 135 mM KCl, 0.5 mM EGTA, 10 mM HEPES, 1.5 mM MgCl2, pH adjusted to 7.3). The patch pipettes have resistances of 2-3 MΩ in the bath. 3. The substantia nigra is identified according to its distinct anatomical location ( Fig. 1A1-2). Under visual guidance of a video microscope (Olympus...