Leucine5-enkephalin afferents to midbrain dopaminergic neurons: light and electron microscopic examination

The relationship between leucine5-enkephalin-containing nerve terminals and midbrain dopaminergic neurons was studied in the adult rat by light and electron microscopy. For light microscopy, alternate midbrain sections were immunostained with rabbit polyclonal antibodies against leucine5-enkephalin and tyrosine hydroxylase, by means of the peroxidase antiperoxidase technique. Leucine5-enkephalin stained fibers and terminals were observed with varying density in the retrorubral field (dopaminergic nucleus A8 region), substantia nigra pars compacta (dopaminergic nucleus A9 region), and ventral tegmental area and related nuclei (dopaminergic nucleus A10 region). For electron microscopy, midbrain sections were immunostained with a mouse monoclonal antibody against leucine5-enkephalin and a rabbit polyclonal antibody against tyrosine hydroxylase, by means of the peroxidase antiperoxidase technique and silver-intensified colloidal gold reactions, respectively. The nucleus A10 area was examined at the electron microscopic level, and there were a) both symmetric (75%) and asymmetric (25%) synapses made between leucine5-enkephalin axon terminals and dopaminergic dendrites, and also synaptic contacts with unlabeled dendrites; b) leucine5-enkephalin synaptic contacts with dopaminergic dendrites that were covered with astrocytic membranes; and c) leucine5-enkephalin appositions with unlabeled nerve terminals that made synaptic contacts with dopaminergic dendrites, suggestive of axo-axonic connections. These findings provide the structural basis for both direct and indirect control of A10 dopaminergic neurons by enkephalin-containing nerve terminals.