Influence of nerve growth factor on neurofilament gene expression in mature primary sensory neurons (original) (raw)
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Role of nerve growth factor in the adult dorsal root ganglia neuron and its response to injury
The Journal of Comparative Neurology, 1984
The response of dorsal root ganglia (DRG) neurons to NGF deprivation and to axotomy was examined in adult guinea pigs. The success of NGF deprivation by means of an autoimmune approach was monitored by the measurement of serum antibody titer levels against guinea pig NGF with the standard bioassay for NGF activity. That the antibody produced NGF deprivation was confirmed by histologic evidence of neuronal atrophy and apparent cell loss in sections of the superior cervical ganglia (SCG) and by marked decreases (65-80%) of SCG neurotransmitter-synthesizing enzyme activity levels. By using the autoimmune approach a new source of guinea pigs was found which consistently produced high titers of cross-reacting anti-NGF antibodies. Experiments were designed to examine the response of the sensory neuron to injury while chronically deprived of NGF. Total neuronal counts in the sixth lumbar DRG 98 days after sciatic nerve crush showed no difference between NGF-deprived and control ganglia. Measurement of the size spectrum of DRG neurons showed evidence of atrophy of the NGFdeprived neurons in both the uninjured and axotomized side compared to respective controls. The mean volume of uninjured sensory neurons measured in the NGF-deprived guinea pigs was decreased 27.7% (P < .05) compared with that of control guinea pigs. The degree of regeneration 6 days following a nerve crush was the same in NGF-deprived sensory neurons and in controls when measured by the "pinch test" and by isotope-labeled axonal transport studies. The long-term regenerative response was analyzed with various histologic and morphometric techniques studying the sural nerve 98 days after sciatic nerve crush. No differences between control and NGF-deprived guinea pig sural nerves were noted at the light microscopic and ultrastructural levels. These studies demonstrated an unimpaired regenerative capability of sensory neurons deprived of NGF compared with controls.
Neurofilament Phosphorylation in Peripheral Nerve: Changes with Axonal Length and Growth State
Journal of Neurochemistry, 1990
We have previously reported that the intrinsic ability of motor axons to sprout can vary considerably from nerve to nerve with an inverse correlation to axonal length. In this study we asked whether this variation might be associated with differences in one axonal component, neurofilaments, near the site of outgrowth. The phosphorylation of epitopes on the heavy subunit of neurofilaments (NF-H) was compared in normal and regenerating axons from long and short nerves in the rat. Quantitative determination of phosphorylation states on NF-H epitopes was made by measuring immunoreactivity to monoclonal antibodies using an enzyme-linked immunosorbent assay system. Our results showed a much higher degree of phosphorylation of epitopes on NF-H in terminal portions of short compared to long axons. There was a significant inverse correlation between phosphorylation of NF-H epitopes and axonal length. In newly formed sprouts NF-H phosphorylation was reduced compared to normal. However, the absolute levels were related to the degree of NF-H phosphorylation in the parent axons. The ability to phosphorylate axonal proteins near the site of outgrowth may correlate with the potential for plastic changes in the axon such as sprouting.
Brain Research, 2000
In previous studies, we found that a 2-week in vivo intracerebroventricular infusion of nerve growth factor (NGF) elicited a sprouting response by sympathetic perivascular axons associated with the intradural segment of the internal carotid artery. We hypothesized that NGF infused into the ventricular system would be internalized by responsive sympathetic cerebrovascular axons, retrogradely transported to parent cell bodies in the superior cervical ganglion (SCG), and subsequently released into the local ganglionic environment. Because fibers exhibiting immunoreactivity for calcitonin gene related peptide (CGRP) have been localized in the SCG, we used immunohistochemical methods to investigate whether a response by CGRP-immunoreactive axons in the SCG occurred following the proposed transport to and release of exogenous NGF in the ganglion. In consecutive tissue sections of the SCG stained for either CGRP or NGF, we found CGRP pericellular 'baskets' surrounding identified NGF-immunoreactive cell bodies. Nerve growth factor infusion resulted in a significant increase both in the number of CGRP pericellular baskets and in NGF-immunoreactive cell bodies. A significant positive correlation (r50.95, P,0.05) between the pericellular baskets and NGF-immunoreactive cell bodies was observed, suggesting that intracranial projection neurons in the SCG released infused NGF (or possibly a converted signal) into the local ganglionic environment to elicit remodeling of CGRP fibers to form pericellular baskets. These findings were confirmed in sections double labeled for NGF and CGRP immunoreactivity. This remodeling suggests that exogenous NGF may mediate retrograde transneuronal plasticity, allowing for future in vivo examinations of the mechanisms involved in neurotrophin transport and release.
Morphological and biochemical changes of neurofilaments in aged rat sciatic nerve axons
Journal of Neurochemistry, 2004
We have made a detailed comparison of neurofilaments (NFs) in the axons of the sciatic nerves between young and aged rats. In young rats, NF density was similar between proximal and distal sciatic nerve, but it became higher in the proximal region of sciatic nerve of aged rats. In accordance with this morphological change, NF protein content decreased dramatically in the middle region of the sciatic nerves of aged rats. The ratio of NF-M to NF-H in aged rats was lower than that in young rats at the proximal region of sciatic nerves and further decreased in the distal region of sciatic nerve. We analyzed transcription and axonal transport of NF proteins in motor neurons in spinal cord which are the major constituents of sciatic nerve axons. Of the transcripts of the NF subunits, NF-M mRNA was particularly reduced in aged rats. Examination of slow axonal transport revealed that the transport rate for NF-M was slightly faster than that for NF-H in young rats, but slightly slower in aged rats. A decrease in both the synthesis and transport rate of NF-M with aging may contribute to the relative reduction in NF-M in the aged rat sciatic nerve. Although the relationship between NF packing and reduced NF-M is not clear at present, these changes in NFs may be associated with age-dependent axonal degeneration diseases. Abbreviations used: DRG, dorsal root ganglion; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; NFs, neurofilaments; NF-L, low molecular weight subunit of neurofilament; NF-M, middle molecular weight subunit of neurofilament; NF-H, high molecular weight subunit of neurofilament; RT-PCR, reverse transcription-polymerase chain reaction.
Brain Research, 2000
Neurofilament proteins (NFs) are made by co-polymerization of three intermediate filament proteins, NF-L, NF-M and NF-H and constitute the most abundant cytoskeletal element in large myelinated axons. NFs have a well-established role as intrinsic determinants of axon caliber with all the functional implications, but the role of each individual NF subunit is much less clear. The aim of our study was to examine functional properties of large myelinated axons with altered morphology from mice bearing a targeted disruption of each NF genes (NF-L 2 / 2, NF-M2 / 2 and NF-H 2 / 2 mice). Membrane properties, action potentials and single axon refractory period were measured in isolated sciatic nerves in vitro, using intra-axonal microelectrode recording in conjunction with current-clamp technique. Some results were obtained from whole nerves by sucrose-gap recording. The NF-knockout mice showed several deficits in physiological properties of low-threshold fibers. In keeping with smaller axon diameter, the conduction velocity was significantly decreased in NF-L 2 / 2 and NF-M 2 / 2 transgenic animals (control, 39.961.8 m / s, NF-M 2 / 2; 23.561.4 m / s, and NF-L2 / 2; 12.060.7 m / s, mean6S.E.M.; intra-axonal recording; similar ratios obtained by sucrose-gap recording; 22-268C). However, in spite of their preserved caliber, large myelinated axons in NF-H 2 / 2 mice also showed a significant decrease in conduction velocity (22.861.0 m / s, mean6S.E.M.). Although action potential amplitudes, duration and shape did not differ between control axons and transgenic animals, the refractory period was prolonged in NF-H 2 / 2 and NF-M 2 / 2 animals. Intracellular injections of 200 ms depolarizing and hyperpolarizing currents revealed outward and inward rectification in all animal groups. In comparison to control animals, NF-H 2 / 2 mice expressed a significant decrease in outward rectification. Potassium channel blockers (4AP and TEA) and cesium ions were able to block outward and inward rectification in all myelinated axons in qualitatively the same manner. These results suggest that NF-H may have a specific role in modulating ion channel functions in large myelinated fibers.
Ultrastructural localization of brain-derived neurotrophic factor in rat primary sensory neurons
Neuroscience Research, 2001
In a previous study we have shown that brain-derived neurotrophic factor (BDNF) is present in a subpopulation of small-to medium-sized sensory neurons in the dorsal root ganglia (DRG) and is anterogradely transported in both the peripheral and central processes. Within the spinal cord, BDNF is localized to varicosities of sensory nerve terminals in laminae I and II of the dorsal horn. This study raised the question of whether BDNF is localized in synaptic vesicles of the afferent nerve terminals. Using immunohistochemical and immunocytochemical techniques we have now investigated the ultrastructural localization of BDNF in the spinal cord of the rat. In addition, its colocalization with the low affinity neurotrophin receptor, p75, and calcitonin gene related peptide (CGRP) was also investigated. In lamina II of the spinal cord, BDNF immunoreactivity was restricted to nerve terminals. The reaction product appeared associated with dense-cored and clear vesicles of terminals superficial laminae. Double labelling experiments at the light microscopic level showed that 55% of BDNF immunoreactive neurons in DRG are colocalized with CGRP and many nerve terminals in laminae I and II of the spinal cord contained both BDNF and CGRP immunoreactivities. The results of double labelling at the ultrastructural level showed that most BDNF-ir (immunoreactive) nerve terminals contained CGRP or the low affinity neurotrophin receptor, p75, but not vice versa. These results point to the conclusion that BDNF may be released in parallel with neurotransmitters from nerve terminals in the spinal cord from a subpopulation of nociceptive primary afferents.
2000
Genetic transfer of growth-promoting molecules was proposed as a potential strategy to modify the nonpermissive nature of the adult CNS to induce axonal regeneration. To evaluate whether overexpression of neurotrophins or cellular adhesion molecules would effect axonal plasticity, adenoviruses encoding fibroblast growth factor-2 (FGF-2/Adts), nerve growth factor (NGF/Adts), neurotrophin-3, and the cell adhesion molecules N-cadherin and L1 were injected into the dorsal horn of the adult spinal cord. Transgene expression was primarily localized to astrocytes in the dorsal horn and motor neurons within the ventral horn. Overexpression of these factors, with the exception of NGF/Adts, failed to increase axonal sprouting. Eight days after NGF/Adts injections, axonal sprouting within the dorsal horn was apparent, and after 4 weeks, extensive spouting was observed throughout the entire dorsal horn, extending into the ventral horn and the white matter of the lateral funicu-lus. These axons were identified primarily as a subpopulation of nociceptive fibers expressing calcitonin gene-related peptide and substance-P. Behavioral analysis revealed thermal hyperalgesia and perturbation of accurate paw placement on gridwalking tasks for both FGF-2-and NGF-treated animals. These results indicate that the administration of growth-promoting molecules can induce robust axonal plasticity of normal adult primary sensory neurons into areas of transgene expression, causing significant alterations in behavioral responses. This observation also indicates that gene transfer protocols that aim to reconstruct diseased or injured pathways should also be designed to prevent the sprouting of the normal circuitry from adjacent unaffected neurons.
Neuroscience, 2001
AbstractöSeveral types of changes have been reported to occur in dorsal root ganglia following peripheral nerve injury, including loss of neurons and increases and decreases in peptide expression. However, with regard to loss of neurons, results have not been consistent, presumably due to di¡erent quantitative methodologies employed and species analyzed. So far, most studies have been conducted on rats; however, with the fast development of the transgenic techniques, the mouse has become a standard model animal in primary sensory research. Therefore we used stereological methods to determine the number of neurons, as well as the expression of galanin message-associated peptide, a marker for galaninexpressing neurons, neuropeptide Y, and calcitonin gene-related peptide in lumbar 5 dorsal root ganglia of both control C57 BL/6J mice and in mice subjected to a`mid-thigh' sciatic nerve transection (axotomy).
Annals of the New York Academy of Sciences, 1991
In newborn rats the levels of nerve growth fa tor (NGF) mRNA (mRNANGF) and NGF receptor mRNA ( A') in the sciatic nerve were 10 and 120 times higher, r pectively, than in adult animals. mRNAL levels decreased st adily from birth, approaching adult levels by the third stnatal week, whereas mRNANUF levels decreased only after th first postnatal week, although also reaching adult levels by the third week. Transection of the adult sciatic nerve resulted in a marked biphasic increase in mRNANGF with time. On the p oximal side of the cut, this increase was confined to the area immediately adjacent to the cut; peripherally, a similar biphasic increase was present in all segments. mRNAL levels were al markedly elevated distal to the transection site, in agreem nt with previous results obtained by immunological methods aniuchi, M., Clark, H. B. & Johnson, E. M., Jr. (1986) P oc. Nal. Acad. Sci. USA 83, 4094-4098]. Following a crush le ion (allowing regeneration), the mRNA1 levels were rapidly