Neurotrophin-3 and neurotrophin receptor immunoreactivity in peptidergic enteric neurons.

In the rat small intestine, neurotrophin-3 immunoreactivity was identified in ganglion cells and in processes mostly innervating the mucosa and occasionally the muscle layer and vasculature. The vast majority of neurotrophin-3 immunoreactive neurons contained vasoactive intestinal polypeptide (VIP), but not substance P or related tachykinin (SP/TK). Neurotrophin receptors visualized by pan-trk immunoreactivity were found in numerous ganglion cells of both plexuses and in nerve processes in the intestinal wall. Pan-trk submucosal neurons contained VIP (36%) or SP/TK-IR (47%). Pan-trk myenteric neurons contained VIP-IR (57%) or SP/TK (27%). Our data suggest that neurotrophin-3 and neurotrophin receptors may be involved in the maintenance of enteric neuronal circuits, transmission and phenotypic expression.

Brain-derived neurotrophic factor (BDNF) peptide antibodies: characterization using a Vaccinia virus expression system.

We describe and characterize a series of polyclonal antibodies, generated against amino acid sequences unique to various regions within pro- and mature brain-derived neurotrophic factor (BDNF), a member of the highly conserved nerve growth factor (NGF) family of neurotrophins. Synthetic peptides were coupled to carrier proteins in the presence of glutaraldehyde to restrict the host animals' immune response to epitopes that are compatible with aldehyde fixation. Initial screenings of the reactivity of the antisera were made on brain sections processed for immunohistochemistry after peptide injections into brain parenchyma. As a means of further characterizing these peptide antisera, we have evaluated the reactivity and specificity of the peptide antibodies in BHK cells expressing recombinant pro- and mature BDNF protein from a T7 RNA polymerase-driven Vaccinia virus system. Several of the antibodies strongly stained components of cells transfected with the BDNF gene but did not label wild-type cells nor cells containing only the expression vector. It has also been possible to detect differential compartmentalization of the BDNF protein at various stages of processing in the BHK cells, as well as in situ in cryostat sections of brain tissue, with antisera to the pro- and mature protein. We conclude that several of our antisera recognize not only the specific peptide immunogens but also what appears to be the corresponding protein native to neurons.

Rescue of basal forebrain cholinergic neurons after implantation of genetically modified cells producing recombinant NGF.

Mouse 3T3 fibroblasts were genetically modified by transfection with a mammalian expression vector containing the rat beta-nerve growth factor (NGF) gene. The transfected cell line, designated 3E, contains several hundred copies of the rat NGF gene and secretes high levels of biologically active NGF. Pieces of collagen gel containing the NGF-secreting 3E cells were grafted to the brains of unilaterally fimbria-fornix-lesioned rats. Grafts of the genetically modified NGF-producing cells rescued axotomized basal forebrain cholinergic neurons and significantly reduced cholinergic cell death in the medial septum as compared with rats treated with grafts of the parental 3T3 cells. Grafted fibroblast cells were detected, and rescue effects were noted up to 6 weeks after grafting. Local effects of NGF secreted by grafted cells were also seen at the gel-brain border in the form of sprouting acetylcholinesterase immunoreactive host cortical fibers. We suggest that implantation of genetically modified cells producing NGF may have therapeutic applications in rescuing damaged central cholinergic neurons in senile dementia of the Alzheimer type as well as in providing trophic support for chromaffin tissue grafts in Parkinson's disease.