Single-cell characterization of endothelin system gene expression in the cerebellum in situ

To evaluate the expression of components of the endothelin (ET) system in single Purkinje neurons and Bergmann glial cells in situ, patch-clamp recording was combined with a multiplex RT-PCR approach. Cerebellar slices were rapidly isolated from 20- to 28-day-old mice. Cells were characterized morphologically and electrophysiologically and cell contents were aspirated and immediately reverse-transcribed. The cDNA was used as a template in a multiplex PCR reaction containing primers specific for ET-1, ET-2, and ET-3, ET-converting enzyme 1 (ECE-1) and ECE-2, and ETA and ETB receptors. The resulting PCR products were used as templates in a second PCR reaction containing only one pair of nested primers. Specific single bands were obtained from positive cells, which was confirmed by DNA sequencing of the PCR products. Of the 25 Purkinje neurons assayed, 84% were positive for ECE-1 mRNA and 68% for ECE-2 mRNA. No ET and ETA receptor mRNAs were detected, and only one cell was positive for ETB receptor mRNA. In Bergmann glial cells, ETB receptor mRNA was predominant. A total of 68% of the 25 cells assayed were positive. Sixteen percent were positive for ETA receptor mRNA, 8% for ECE-1 mRNA, and 12% for ECE-2 mRNA. Again, no ET mRNAs were detected. These results confirm the role of the ETB receptor in Bergmann glial cells and provide evidence for expression of ECE-1 and ECE-2 in Purkinje neurons.