neuropeptide-y and 3-3--5-5--tetramethylbenzidine

The morphological support of interactions between enkephalins and three systems--beta-endorphin (beta-END), tyrosine hydroxylase (TH), or neuropeptide Y (NPY)--well represented in the arcuate nucleus, was examined by using an electron microscopic double immunostaining combining two sensitive chromogens, diaminobenzidine (DAB) and tetramethylbenzidine (TMB). The first step consisted of visualizing Metenkephalinergic terminals with DAB reaction product, and the second one involved detecting the antigens TH, beta-END, and NPY in their respective neurons with TMB reaction product. Ultrastructural analysis revealed enkephalinergic terminals presynaptic to TH-immunopositive cells and dendrites, principally in the dorsal portion of the arcuate nucleus. Enkephalinergic nerve terminals also contacted synaptically ventrolaterally located beta-END-immunoreactive cells. In the ventromedial arcuate nucleus, few synaptic contacts were observed between enkephalinergic boutons and NPY neurons, which were principally in close apposition with glial processes. Enkephalin-immunoreactive synapses were more frequently seen on TH-immunopositive neurons. This TH neuronal group is known to correspond to the dopaminergic tuberoinfundibular neurons implicated in the control of reproductive functions. The pattern of distribution of the different synapses within the arcuate nucleus (TH dorsal, beta-END ventrolaterally; NPY ventromedially) suggests that enkephalins may play a role in the neuroendocrine regulation of gonadotropin and prolactin secretion. The results provide evidence that enkephalins, in the arcuate nucleus, exert a postsynaptic action on the beta-END cells in addition to the presynaptic regulation previously demonstrated in the mediobasal hypothalamus, related to beta-END release. Moreover, the arcuate nucleus is a site of intercellular relationships between enkephalins and dopamine and between enkephalins and other peptides such as NPY.

Topics: 3,3'-Diaminobenzidine; Animals; Arcuate Nucleus of Hypothalamus; Benzidines; beta-Endorphin; Chromogenic Compounds; Enkephalin, Methionine; Enkephalins; Male; Microscopy, Immunoelectron; Neurons; Neuropeptide Y; Presynaptic Terminals; Rats; Rats, Wistar; Tyrosine 3-Monooxygenase

Techniques for correlative double-label immunocytochemistry (ICC) at light and electron microscopic (EM) level are useful for determining the neurotransmitter phenotype of inputs onto immunocytochemically identified neurons. Tetramethylbenzidine (TMB) has been used as a chromogen at the EM level for horseradish peroxidase tract tracing. We have found that TMB, in combination with diaminobenzidine (DAB), can be used in a double-label immunocytochemical protocol to examine neuropeptide Y inputs onto luteinizing hormone-releasing hormone cells in the sheep preoptic area. At both light and EM levels, TMB reaction product is visibly distinct from DAB reaction product. The ultrastructural preservation we have been able to obtain with our technique is better than that obtained with techniques that use TMB at a lower pH. Furthermore, this technique allows the demonstration of synaptic contacts between neurochemically identified terminals and cells with different neurotransmitter phenotypes.

Topics: 3,3'-Diaminobenzidine; Animals; Benzidines; Chromogenic Compounds; Female; Gonadotropin-Releasing Hormone; Immunoenzyme Techniques; Microscopy, Electron; Neuropeptide Y; Preoptic Area; Sheep