Biomedical subjects

H Thoenen

Publications and source records attributed to H Thoenen.

At least 145 records · Page 8Linked to original sources

PubMed1985

Transganglionic regulation of central terminals of dorsal root ganglion cells by nerve growth factor (NGF).

Blockade of axonal transport or transection of the rat sciatic nerve results in transganglionic degenerative atrophy (TDA) of nerve terminals containing fluoride-resistant acid phosphatase (FRAP) in the Rolando substance of the spinal cord. Application of vinblastine (9 micrograms) in a cuff around the sciatic nerve of adult rats blocked the retrograde transport of [125I]NGF in sensory fibers; this amount of vinblastine is identical to the threshold amount that induces TDA. Conversely, application of NGF to the proximal stump of the transected sciatic nerve prevented or delayed the occurrence of TDA as reflected by the maintenance of FRAP in the upper dorsal horn, that otherwise would inevitably disappear following the peripheral nerve lesion. These results suggest that endogenous NGF transported retrogradely in peripheral sensory fibers of the adult rat under normal conditions may be responsible for the regulation of the structural and functional integrity of the central terminals of these FRAP-containing primary sensory neurons and that TDA may be the consequence of the failure of NGF to reach the perikarya of these neurons.

Acid Phosphatase↗

PubMed1985

Nerve growth factor supply for sensory neurons: site of origin and competition with the sympathetic nervous system.

Nerve growth factor (NGF) levels in the sensory nervous system were measured by a highly sensitive two-site enzyme immunoassay for NGF. Dorsal root ganglia and the adjacent spinal nerves contained 2.8 +/- 0.3 and 1.7 +/- 0.4 ng NGF/g wet wt., respectively, whereas no NGF was detectable in dorsal roots and spinal cord (less than 0.05 ng NGF/g wet wt.). It is concluded that sensory neurons are supplied with NGF exclusively from their peripheral and not from their central field of projection. Two days after treatment with 6-hydroxydopamine, which destroys sympathetic nerve terminals and thereby prevents the removal of NGF by sympathetic neurons, the NGF content of dorsal root ganglia and trigeminal ganglia increased to 285% and 161% of control, respectively. This indicates that in peripheral target tissues sensory and sympathetic neurons compete for NGF.

Animals↗

PubMed1985

Nerve growth factor.

In contrast to all other molecules which are labelled 'growth factor', NGF is not a mitogen. It is a neurotrophic molecule essential for the development and maintenance of function of specific populations of peripheral and possibly also central neurons. The availability of NGF in large quantities from exocrine glands (e.g. male mouse submandibular gland), where NGF does not play a neurotrophic role, has allowed the purification of NGF, the production of specific antibodies, the determination of its amino acid sequence and finally the molecular cloning of NGF leading to the elucidation of its precursor structure and its genomic organization. Comparison of the biological activities and the immunological properties of NGF isolated from different sources demonstrated that the active centre of the molecule has been highly conserved during evolution, whereas other parts of the molecule determining immunological properties have undergone considerable changes. After a survey of the essential biological actions of NGF, this paper concentrates on two actual questions of NGF research, namely the regulation of NGF synthesis in the target tissues of NGF-responsive neurons, and the molecular mechanism(s) of action of NGF on these neurons.

Animals↗

PubMed1985

Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation.

The levels of nerve growth factor (NGF) and its mRNA in the rat central nervous system were determined by two-site enzyme immunoassay and quantitative Northern blots, respectively. Relatively high NGF levels (0.4-1.4 ng NGF/g wet weight) were found both in the regions innervated by the magnocellular cholinergic neurons of the basal forebrain (hippocampus, olfactory bulb, neocortex) and in the regions containing the cell bodies of these neurons (septum, nucleus of the diagonal band of Broca, nucleus basalis of Meynert). Comparatively low, but significant NGF levels (0.07-0.21 ng NGF/g wet weight) were found in various other brain regions. mRNANGF was found in the hippocampus and cortex but not in the septum. This suggests that magnocellular cholinergic neurons of the basal forebrain are supplied with NGF via retrograde axonal transport from their fields of innervation. These results, taken together with those of previous studies showing that these neurons are responsive to NGF, support the concept that NGF acts as trophic factor for magnocellular cholinergic neurons.

Animals↗

PubMed1985

Progenitor cells from embryonic chick dorsal root ganglia differentiate in vitro to neurons: biochemical and electrophysiological evidence.

We have analyzed the appearance of neurons and glial cells in chick dorsal root ganglia during development. Neurons were identified by the presence of polysialogangliosides recognized by tetanus toxin (GD1b, GT1) or by the monoclonal antibody Q211 directed against polysialogangliosides containing four, five and six sialic acid residues. Glial cells were identified by the presence of 04 antigen. A population of undifferentiated cells, i.e., cells which express neither neuronal nor glial cell surface antigens, present in dorsal root ganglia until embryonic day 7, was separated from the neuronal and glial population. This cell population contains neuronal progenitor cells which differentiate to neurons within 1 day in culture. This differentiation process is characterized by the appearance of neuronal morphology, of neuron-specific gangliosides and by the appearance of voltage-dependent sodium and calcium channels.

Animals↗

PubMed1985

Nerve growth factor changes the relative levels of neuropeptides in developing sensory and sympathetic ganglia of the chick embryo.

The effects of chronic nerve growth factor administration on the development of neuropeptides in the embryonic chick peripheral nervous system were quantitated by radioimmunoassays. Starting at embryonic Day 3.5, daily doses of 20 micrograms of nerve growth factor (NGF) increased the substance P content of lumbosacral spinal sensory ganglia at all ages studied (Days 10-14), while having no effect on substance P levels of thoracic sensory ganglia. In contrast, the contents of somatostatin were increased in both thoracic and lumbosacral ganglia, but only at comparatively late time points (Day 14). Nerve growth factor administration was also found to decrease the somatostatin contents of lumbosacral paravertebral sympathetic ganglia at early time points (Day 8) while increasing levels at later stages (Day 14), thus acting to accelerate the normally occurring developmental changes in level of this peptide. These changes were shown to be specific for somatostatin by demonstrating that NGF increased tyrosine hydroxylase levels in sympathetic neurons at Day 8, and had no effect on sympathetic vasoactive intestinal polypeptide levels at Day 14. It has been concluded that exogenous NGF does not simply act to increase or prolong the expression of neuron-specific phenotypes in the chick, but rather its action is time and location dependent to accelerate development.

Animals↗

PubMed1985

Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor.

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60-70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a "central-target" (CNS) derived neurotrophic factor--BDNF, while they are differentially responsive to "peripheral-target"-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.

Animals↗

PubMed1985

Treatment with 6-hydroxydopamine and colchicine decreases nerve growth factor levels in sympathetic ganglia and increases them in the corresponding target tissues.

A two-site enzyme immunoassay was used to determine the nerve growth factor (NGF) contents of sympathetic ganglia and their corresponding target tissues in adult rats. The destruction of sympathetic nerve terminals by 6-hydroxydopamine (6-OHDA) and the blockade of axonal transport by colchicine resulted in a rapid increase in the NGF levels of sympathetically innervated organs and a rapid decrease in the sympathetic ganglia. NGF levels in heart atrium, heart ventricle, submandibular gland, and iris increased 2- to 4-fold 12 hr after injection of 6-OHDA, whereas the NGF contents of stellate and superior cervical ganglia dropped to a minimal level of 3 to 4% of control 24 hr after injection. Twelve hours after treatment with colchicine the NGF levels in sympathetically innervated organs increased 2- to 3-fold, whereas the NGF contents of sympathetic ganglia fell to one-third of control values. The half-lives of NGF in the superior cervical and stellate sympathetic ganglia were 4.5 and 4.8 hr, respectively, as determined by the decrease of NGF content after treatment with 6-OHDA. These results indicate that the synthesis of NGF is normally confined to the innervated target organs with no significant contribution of ganglionic cells. This is consistent with the concept that NGF acts as a retrograde messenger between target organs and innervating sympathetic neurons.

Animals↗

PubMed1985

Dissociated neurons regenerate into sciatic but not optic nerve explants in culture irrespective of neurotrophic factors.

Explants of adult or 10-day-old rat sciatic and optic nerves were implanted as "bridges" through a silicon grease seal in a three-compartment chamber culture system, leading from a narrow center chamber to two adjacent side chambers. Dissociated newborn rat sympathetic or sensory neurons were plated into the center chamber and grown in the presence of optimal concentrations of nerve growth factor (NGF). By light microscopy, nerve fibers were seen to grow out of the sciatic nerve explants in the side chambers after 2 to 3 weeks. Electron microscopy showed large numbers of axons present inside the sciatic nerves, irrespective of the presence and number of living Schwann cells. Besides their tendency to fasciculate, axons grew with high preference on Schwann cell membranes and the Schwann cell side of the basal lamina, a situation identical to in vivo regeneration. In contrast to the sciatic nerves, no axons could be found under any condition in the optic nerves. This result points to the existence of extremely poor, non-permissive substrate conditions in the differentiated optic nerves which cannot be overcome by the strong fiber outgrowth-promoting effects of NGF.

Animals↗

PubMed1984

Relationship between levels of nerve growth factor (NGF) and its messenger RNA in sympathetic ganglia and peripheral target tissues.

We have developed a sensitive assay for the quantification of nerve growth factor mRNA (mRNANGF) in various tissues of the mouse using in vitro transcribed RNANGF. Probes of both polarities were used to determine the specificity of the hybridization signals obtained. Comparison of NGF levels with its mRNA revealed that both were correlated with the density of sympathetic innervation. Thus, vas deferens contained high levels of both NGF and mRNANGF, whereas skeletal muscle levels were barely detectable, indicating that in peripheral tissues NGF levels are primarily regulated by the quantity of mRNANGF and not by the rate of processing of NGF precursor to NGF. However, although superior cervical ganglia contained the highest levels of NGF, its mRNA was barely detectable. Thus, the high levels of NGF in sympathetic ganglia result from retrograde axonal transport rather than local synthesis. The quantity of NGF found in the submandibular glands of female animals was three orders of magnitude higher than expected from their mRNA levels. This observation is discussed in the context of the difference between the mechanism of storage and exocytosis of exocrine glands versus the constitutive release from other tissues.

Animals↗

PubMed1984

Interchangeability of nerve growth factor and high potassium in the long-term survival of chick sympathetic neurons in serum-free culture medium.

The results of the present experiments demonstrate that embryonic sympathetic neurons initially selected in culture by nerve growth factor could be subsequently supported by raising potassium concentration of the medium to 35 mM. Alternatively, neurons initially selected by 35 mM potassium could be supported in a normal culture medium but supplemented with nerve growth factor. It is concluded that nerve growth factor and high concentrations of potassium act on the same subpopulation of chick embryonic sympathetic neurons to promote their long-term survival in a serum-free culture medium.

Animals↗

PubMed1984

The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival.

The survival of cultured chick sympathetic neurons and the outgrowth of neurites were stimulated by the basement membrane protein laminin coated onto polyornithine culture substrates. The survival-potentiating activity was dependent on the presence of nerve growth factor. Both effects of laminin could be completely inhibited by affinity-purified antibodies against laminin fragment 3, the product of a limited proteolysis that corresponds to the heparin-binding globular domain at the end of the long arm of the laminin molecule. Antibodies against other laminin fragments were inactive, including those against previously determined cell-binding domains. A large laminin fragment, E8, was produced by brief elastase digestion and shown to consist of fragment 3 and an adjacent rod-like structure. Although lacking the cell binding domains, fragment E8 potentiated both neuronal survival and neurite outgrowth, and these effects could be blocked by antibodies against fragment 3. Weak survival and neurite potentiating activity was also detected in another fragment corresponding to the short arms of laminin, but as these effects were not inhibited by any of the antibodies tested they probably arose de novo during proteolysis. The heparin-binding domain of laminin is therefore responsible for its effects on neurons.

Animals↗

PubMed1984

Regulation of nerve growth factor synthesis and release in organ cultures of rat iris.

We studied the synthesis and release of nerve growth factor (NGF) in cultured rat iris with a two-site enzyme immunoassay by measuring the time course of NGF levels remaining in the iris and relased into the medium up to 72 h. For up to 3 h, the NGF levels in the iris did not change significantly. After that, they increased to a maximal level of 350 +/- 30 pg NGF/iris at 19 h, which is 200 times higher than the in vivo content. Between 20 and 72 h in culture, the NGF level decreased to 130 +/- 10 pg NGF/iris, whereas general protein synthesis did not change during that time period. Maximal rate of NGF production (203 pg NGF/h/iris) was seen between 9 and 12 h in culture. In the medium, NGF levels were first detectable after 6 h. Levels then increased with a time course similar to that seen within the iris, reaching a maximal level of 1,180 +/- 180 pg after 19 h in vitro, and then did not significantly change for up to 48 h. The NGF production of the densely sympathetically innervated dilator was three times higher than that of the predominantly cholinergically innervated sphincter. The NGF production was blocked by inhibitors of messenger RNA synthesis (actinomycin D) and of polyadenylation (9-beta-D-arabinofuranosyladenine) as well as by inhibitors of translation (cycloheximide). Monensin, which interferes with the transport of proteins through the Golgi apparatus, decreased NGF levels to 8-12% of controls in the medium, suggesting that the Golgi apparatus is involved in the intracellular processing of NGF.

Animals↗

PubMed1984

Nerve growth factor-mediated enzyme induction in primary cultures of bovine adrenal chromaffin cells: specificity and level of regulation.

Primary cultures of bovine adrenal chromaffin cells provide large quantities of a homogeneous population of target cells for nerve growth factor (NGF) and, thus, are a suitable system for studying the molecular mechanism of action of NGF. In this study, we have shown that NGF mediates the specific induction of the key enzymes in catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT). Acetylcholinesterase (AChE), an enzyme which catalyzes the breakdown of acetylcholine, is also induced by NGF. We have compared NGF-mediated TH and AChE induction and have provided pharmacological evidence that TH induction involves a post-transcriptional, polyadenylation-dependent event (blockable by 9-beta-arabinofuranosyladenine but not by alpha-amanitin), whereas AChE induction requires transcription (blockable by alpha-amanitin). DBH and PNMT appear to be regulated via the same mechanism as TH. The time course of TH induction is such that NGF must be continuously present for at least the first 36 hr (during which time TH levels remain unchanged), and then the entire increase takes place during the subsequent 12 hr. In contrast, AChE induction proceeds linearly with time of NGF exposure. These data suggest that there may be multiple mechanisms by which NGF regulates enzyme induction. We have also compared the effects of cAMP with those of NGF. As compared to NGF, cAMP produces a different pattern of enzyme induction (in addition to TH, DBH, PNMT, and AChE, dopa decarboxylase (DDC) is also induced), it acts rapidly (a 12-hr exposure produces the full effect), and it acts only at the transcriptional level (its effects are blocked by alpha-amanitin). These data provide evidence that cAMP does not act as a second messenger for NGF with regard to enzyme induction.

Acetylcholinesterase↗

PubMed1984

Nerve growth factor-mediated induction of choline acetyltransferase in PC12 cells: evaluation of the site of action of nerve growth factor and the involvement of lysosomal degradation products of nerve growth factor.

In previous experiments it has been demonstrated that nerve growth factor (NGF), subsequent to its binding to specific membrane receptors, is internalized. Ultrastructurally, this internalized NGF is localized in membrane-confined compartments which ultimately fuse with lysosomes. The present experiments were designed, first, to evaluate whether a very small but functionally important portion of the internalized NGF might reach the free cytoplasm (and subsequently the nuclear chromatin) and might be responsible for the induction of choline acetyltransferase (ChAT) in PC12 cells. Second, we investigated whether a lysosomal proteolytic degradation product of NGF might act as a second messenger in the NGF-mediated ChAT induction. In one series of experiments, guinea pig erythrocyte ghosts, loaded with NGF (or NGF antibodies), fluorescein isothiocyanate-coupled bovine serum albumin, and/or horseradish peroxidase (HRP) were fused with PC12 pheochromocytoma cells. Electron microscopy showed that [125I]NGF and HRP reaction product were located throughout the cytoplasm and the nucleus but did not penetrate membrane compartments such as the endoplasmic reticulum, the Golgi complex, the perinuclear space, or mitochondria. Biochemically, NGF injected into the cytoplasm did not produce an induction of ChAT, whereas NGF acting via cell surface receptors resulted in a 2-fold increase in ChAT. Conversely, injection of NGF antibodies did not prevent the receptor-mediated ChAT induction. In a second series of experiments, the half-life of internalized NGF was increased from 40 min to 24 hr by the administration of leupeptin, a protease inhibitor which is accumulated in lysosomes. However, the NGF-mediated ChAT induction was not affected by this treatment. It is concluded that NGF itself does not act directly on cytoplasmic or nuclear target sites, nor is a proteolytic degradation product of NGF responsible for the NGF-mediated ChAT induction. Thus, NGF must act via a second messenger mechanism, the nature of which remains to be established.

Choline O-Acetyltransferase↗

PubMed1983

Quantitative demonstration of the retrograde axonal transport of endogenous nerve growth factor.

The level of endogenous nerve growth factor (NGF) in rat sciatic nerve was determined using a highly sensitive two-site enzyme immunoassay. After crushing this nerve NGF accumulated linearly distal to the crush during the first 12 h to reach levels 13-fold higher than in the uncrushed contralateral side. In contrast, proximal to the crush NGF levels approached or were below the detection limit of the assay. The asymmetrical distribution of NGF on the two sides of a crush is direct evidence for the retrograde axonal transport of endogenous NGF.

Animals↗

PubMed1983

Cholinergic neurons in the rat cerebral cortex demonstrated by immunohistochemical localization of choline acetyltransferase.

The presence of cholinergic neurons in rat cerebral cortex was demonstrated by immunohistochemical localization of choline acetyltransferase, the enzyme synthesizing the neurotransmitter acetylcholine. The stained neurons were found throughout the entire cortex and were present in layers II--VI. Two morphological distinct classes of cholinergic neurons have been identified and compared with those neurons containing acetylcholinesterase.

Acetylcholinesterase↗

PubMed1983

Relationship between NGF-mediated volume increase and "priming effect" in fast and slow reacting clones of PC12 pheochromocytoma cells. Role of cAMP.

Nerve Growth Factor (NGF)-mediated fiber outgrowth in pheochromocytoma PC12 cells is a slow process, developing over a period of several days. However, if these cells are pre-exposed to NGF for 7-10 days, renewed NGF treatment of the subcultured cells elicits fiber outgrowth within 24 h, comparable to the rate of response of physiological target cells to NGF. The present experiments demonstrated that this effect, previously termed "priming", was accompanied by a 60% increase in the volume of the PC12 cells, and that the dose-response curves for NGF-mediated induction of fiber outgrowth and for the increase in cell volume were very similar. Furthermore, the rates of NGF-mediated fiber outgrowth and of cell volume increase were both much slower in conventional PC12 cells (slow-reacting) compared to a newly-selected, fast-responding (FR)subclone of PC12 cells. These results suggested a possible causal relationship between the increase in cell volume and the induction of fiber outgrowth. However, when the cells were pre-exposed for 7 days to dibutyryl-cAMP (db-cAMP), the increase in cell volume was 3-fold higher than that effected by NGF. Nevertheless, db-cAMP had only a very limited ability to "prime" the cells for a subsequent response to NGF. Thus, the induction of cell volume increase and the increased availability of structural elements is not sufficient to explain the "priming" effect of NGF. The effects of db-cAMP are discussed in the context of a possible role of cAMP as a second messenger in the action of NGF.

Animals↗