Immunocytochemical and in situ hybridization evidence for the coexistence of GABA and tyrosine hydroxylase in the rat locus ceruleus.

We have demonstrated the coexistence of GABA-like and tyrosine hydroxylase-like immunoreactivities (GABA-LI and TH-LI, respectively) in the same neurons of the rat locus ceruleus (LC). The profiles of these cells were labeled by alternately immunostaining adjacent sections for GABA-LI or TH-LI by the avidin-biotin-peroxidase complex method or the peroxidase-anti-peroxidase method after perfusion (either Zamboni's fixative or PPG), and observation at light and electron microscopic levels. For light microscopy, pairs of adjacent sections of more than 590 (Zamboni's) and 260 (PPG), and for electron microscopy, 40 ultrathin sections cut from adjacent semithin plastic sections (Zamboni's), were examined. GABA-LI was found in 80% (1,309/1,642 in total) of small and medium-sized neurons, uniformly scattered throughout the LC. Observations unequivocally show that the majority of GABA-ergic neurons are also noradrenergic. Several neurons are neither noradrenergic nor GABA-ergic, while other noradrenergic neurons do not show GABA-LI. It is shown that astrocytes, but not oligodendrocytes, contain GABA. In situ hybridization using a probe DNA fragment of the glutamic acid decarboxylase (GAD) cDNA, amplified by the polymerase chain reaction, detected GAD mRNA signals in many neurons throughout the LC, supporting the presence of a GAD/GABA system in the LC. Multiple "classical" transmitters, including GABA, serotonin, and noradrenaline, coexist in many LC neurons and may contribute to its widely diverging projections throughout the entire CNS.