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

Summary

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

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.

Protocol

1. Brain slice preparation

1. Young (15-40 days old) male and female Sprague-Dawley rats are used. (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% O_2....

Discussion

The combination of patch clamp recording in brain slice with scRT-PCR we demonstrated here provide an excellent method to investigate the mRNA expression profiles for ion channels, receptors, and key enzymes for neurotransmitter synthesis in individually characterized neurons. This is particularly useful when the protein in question can not be detected and localized using other methods such as immunohistochemistry due to low expression level and/or lack of suitable antibody (Surmeier et al. 1996; Zhou et al. 2009). The h.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Table 1. Key reagents and equipment
Table 2. Primer pairs for rat neuronal Kv3 channel, tyrosine hydroxylase (TH) and glutamic acid decarboxylase 1 (GAD1) mRNAs for scRT-PCR