The distributions of mRNAs for the protooncogene trk and the low- affinity NGF receptor (LNGFR) were studied by hybridization with oligonucleotide probes on sections of adult rat primary sensory and sympathetic ganglia. For comparison with high-affinity binding sites, adjacent sections were processed for NGF receptor radioautography. Among neurons in lumbar dorsal root ganglia and trigeminal ganglia, trk mRNA and NGF-binding sites were closely colocalized; this finding together with previous direct evidence in other cell types is taken to indicate that trk protein is an essential component of the high- affinity NGF receptor in adult sensory neurons. In lumbar dorsal root ganglia and trigeminal ganglia, abundant LNGFR mRNA was found in all neurons with strong 125I-NGF labeling and on additional neurons lacking high-affinity NGF-binding sites. The presence of abundant LNGFR in neurons with high-affinity receptors could be the cause and/or consequence of their ability to respond to NGF. Neurons with abundant LNGFR mRNA but few high-affinity NGF-binding sites may have receptors for other members of the neurotrophin family. In nodose ganglia, neurons with high concentrations of LNGFR mRNA greatly outnumbered the small percentage with abundant trk mRNA. Following intrathecal infusion of NGF to otherwise normal dorsal root ganglia, the concentrations of LNGFR mRNA but not those of trk mRNA and NGF-binding sites were increased in NGF-responsive neurons. The usual single normal pattern of frequency histograms of LNGFR labeling indices became bimodal in response to NGF. Concentrations of NGF-binding sites, LNGFR mRNA, and trk mRNA were all decreased by peripheral nerve transection and restored by exogenous NGF, the restoration being complete for LNGFR mRNA and partial for trk mRNA and NGF-binding sites. The data indicate that NGF can regulate both LNGFR and trk mRNAs but do not clarify the possible contribution of the LNGFR protein to high-affinity binding sites.

To survey the distribution of neuronal receptors for NGF, sections of the rat brain, spinal cord, and peripheral ganglia were incubated in vitro with radioiodinated NGF and examined by autoradiography. NGF binds selectively with high affinity to most sympathetic neurons and many primary sensory neurons together with their intraspinal or intramedullary axons. In autoradiographs of the brain, labeled neuronal perikarya are seen in the basal forebrain, the caudate-putamen, the medulla oblongata, the ventral cochlear nucleus, and the dorsal nucleus of the lateral lemniscus. The distribution of neurons binding NGF resembles the distribution of cholinergic neurons in the forebrain, but these 2 systems overlap very little in the brain stem. In extracts of the brain or spinal cord enriched for plasma membranes, avid binding sites are regionally manifest with properties similar to those of fetal peripheral neurons. The localization of neurons expressing the high-affinity receptor for NGF defies simple correlation with neurotransmitter function or embryogenesis.

The density and binding properties of receptors for nerve growth factor (NGF) in normal and injured sensory neurons have been analyzed by quantitative radioautography following incubation of tissue sections with radioiodinated NGF. The technique is designed to study binding sites that are half-maximally saturated by picomolar concentrations of NGF: Additional sites of lower affinity have not been emphasized. In normal adult rats, approximately half of lumbar sensory neurons have high-affinity receptors for NGF. One month after the sciatic nerve is cut, the mean number of high-affinity sites on heavily labeled neurons in the fifth lumbar dorsal root ganglion falls to less than 20% of normal values because of reduced receptor density and cell volume. Neurons with high-affinity receptors are more liable to atrophy after injury than those lacking such receptors. Receptors are lost not only in the cell bodies of sensory neurons but also on their peripheral and central processes. Delayed administration of NGF to the sciatic nerve 3 weeks after it is cut restores the receptor density to normal values and partially restores the neuronal cell volume. As part of the response to axonal injury and possibly because the cell body is deprived of NGF, fewer high-affinity receptors are displayed by sensory neurons. For at least 3 weeks after nerve transection, neurons that are atrophic and depleted of NGF receptors can be resuscitated by exogenous NGF.

To investigate the distribution of nerve growth factor (NGF) receptors on peripheral and central axons, [125I]NGF was injected into the sciatic nerve or spinal cord of adult rats. Accumulation of [125I]NGF in lumbar dorsal root ganglia was monitored by gamma emission counting and radioautography. [125I]NGF, injected endoneurially in small quantities, was taken into sensory axons by a saturable process and was transported retrogradely to their cell bodies at a maximal rate of 2.5 to 7.5 mm/hr. Because very little [125I]NGF reached peripheral terminals, the results were interpreted to indicate that receptors for NGF are present on nonterminal segments of sensory axons. The specificity and high affinity of NGF uptake were illustrated by observations that negligible amounts of gamma activity accumulated in lumbar dorsal root ganglia after comparable intraneural injection of [125I] cytochrome C or [125I]oxidized NGF. Similar techniques were used to demonstrate avid internalization and retrograde transport of [125I]NGF by intraspinal axons arising from dorsal root ganglia. Following injection of [125I]NGF into lumbar or cervical regions of the spinal cord, neuronal perikarya were clearly labeled in radioautographs of lumbar dorsal root ganglia. Sites for NGF uptake on primary sensory neurons in the adult rat are not restricted to peripheral axon terminals but are extensively distributed along both peripheral and central axons. Receptors on axons provide a mechanism whereby NGF supplied by glia could influence neuronal maintenance or axonal regeneration.

No abstract

Kindling, an animal model of epilepsy wherein seizures are induced by subcortical electrical stimulation, results in the upregulation of neurotrophin mRNA and protein in the adult rat forebrain and causes mossy fiber sprouting in the hippocampus. Intraventricular infusion of a synthetic peptide mimic of a nerve growth factor domain that interferes with the binding of neurotrophins to their receptors resulted in significant retardation of kindling and inhibition of mossy fiber sprouting. These rmdings suggest a critical role for neurotrophins in both kindling and kindling-induced synaptic reorganization.

Using a dissociated primary sensory neuron culture system, it is observed that some naturally occurring porphyrins produce dose-dependent neurotoxicity as measured by neuron death and by inhibition of the neurite outgrowth induced by Nerve Growth Factor (NGF). However, the porphyrin precursor delta-aminolevulinic acid (delta-ALA) is not toxic up to millimolar concentrations within a 30-h time period. Two synthetic porphyrins, tetraphenylporphine sulfonate (TPPS) and hematoporphyrin derivative (HPD), have also been shown to be toxic in vitro. Cultures and cocultures of isolated populations of neurons and glia of central and peripheral nervous system origin may prove advantageous in the study of porphyrin influences on the intact nervous system. This in vitro assay system can complement in vivo paradigms and may be useful for rapid quantitative screening for neurotoxicity of radiation sensitizers including the synthetic porphyrins and other chemotherapeutic agents.

Dissociated neuronal cultures have become a popular tool to address a number of questions in neurobiology. These culture systems may provide useful paradigms to approach problems related to in vivo neurotoxicity of xenobiotics. The clinical spectra of vinca neurotoxicity and alcohol teratogenesis is reviewed, and some observations on the use of dissociated cultures to study neuronal toxicity of vinca alkaloids and alcohol are presented.