M1-M5 MUSCARINIC RECEPTOR DISTRIBUTION IN RAT CNS BY RT-PCR AND HPLC

Five muscarinic receptor genes (m1-m5) that encode distinct muscarinic receptor subtypes have been cloned. Because of their structural homol ogy and pharmacological similarity, ligand binding probes currently av ailable do not clearly distinguish among the subtypes. To obtain a cle ar distribution within the CNS of molecularly defined muscarinic recep tor subtypes, seven brain regions were examined for the expression of the respective mRNAs. The most sensitive method for detecting mRNA is through amplification of the respective cDNAs. Brain regions were obta ined from male Wistar rats, and total RNA was isolated. The isolates w ere extensively treated with RNase-free DNase to remove any residual g enomic DNA. Total RNA (1 mu g) was reverse-transcribed using random pr imers and reverse transcriptase. The resulting cDNA was amplified usin g a thermal cycler, and the polymerase chain reaction (PCR)-amplified products were analyzed by gel electrophoresis containing ethidium brom ide and visualized with fluorescent illumination. PCR-amplified sample s were also injected directly onto an HPLC anion exchange column and q uantified by UV detection. Each of the five muscarinic subtypes was fo und in every brain region examined. The m1 subtype was most abundant i n cortex and gradually declined in content caudally to the spinal cord . The m2 subtype was most abundant in thalamus-hypothalamus and ponsme dulla. The m4 subtype was found in greatest amount in the striatum, wh ereas m3 and m5 were expressed consistently throughout the CNS. The co mbination of RT-PCR and HPLC provides a rapid and sensitive method for quantifying the expression of mRNA coding for all five muscarinic rec eptor subtypes derived from the CNS.