Sprouting of sympathetic nerves in the absence of afferent input

One day before a medial septal lesion, the preganglionic afferents to the superior cervical ganglion were transected. We found that deafferented peripheral noradrenergic neurons appear in the hippocampal formation in response to the septal lesion in spite of loss of input from the central nervous system. This observation supports the hypothesis that sprouting is initiated by some factor derived from the target tissue.     

Expression of L1 and PSA during sprouting and regeneration in the adult hippocampal formation

The objective of the present study was to evaluate the expression of polysialic acid (PSA) and the cell adhesion molecule L1 during axonal regeneration and sprouting after injury to the adult rat brain. All animals received a complete lesion of the fimbria-fornix (FF). Grafts of nerve growth factor (NGF)- or β-galactosidase (βGal)-producing fibroblasts were placed in the FF lesion cavity and induced septohippocampal cholinergic regeneration or sympathetic tyrosine hydroxylase (TH)-positive sprouting, respectively. Cholinergic regeneration was evaluated from 2 to 8 weeks following grafting of NGF-producing fibroblasts in the FF lesion cavity. In the graft area, choline acetyltransferase (ChAT)-positive fibers expressed L1 and PSA. Once cholinergic axons reached the hippocampal formation (HF), they no longer expressed L1 or PSA. Eight weeks after a lesion of the FF and transplantation of βGal-producing fibroblasts, TH-positive fibers sprouted in the denervated HF and expressed L1 but not PSA. At the zone of reactive gliosis, PSA but not L1 expression was increased following a lesion of the FF and transplantation of NGF- or βGal-producing fibroblasts. In animals that received a graft of NGF-producing fibroblasts in the FF lesion cavity, numerous ChAT-positive axons were observed along these areas rich in PSA and reactive astrocytes. Taken together, these results suggest that the expression of PSA and L1 is upregulated on regenerating cholinergic axons during axonal elongation and downregulated upon target innervation. In contrast, TH-positive fibers that sprout in the denervated HF express and maintain their expression of L1. Finally, the expression of PSA in the area of reactive gliosis may contribute to a permissive environment for axonal regrowth. J. Comp. Neurol. 399:1–19, 1998. © 1998 Wiley-Liss, Inc.     

Effects of endogenous neurotrophins on sympathetic sprouting in the dorsal root ganglia and allodynia following spinal nerve injury

Peripheral nerve injury is often complicated by a chronic pain syndrome that is difficult to treat. In animal models of peripheral nerve injury, sympathetic nerve terminals in the dorsal root ganglia (DRG) sprout to form baskets around large diameter neurons, an anatomical change that has been implicated in the induction of neuropathic pain. In the present study, we have investigated whether neurotrophins derived from peripheral sources play any roles in sympathetic sprouting and neuropathic pain in a rat model of peripheral nerve injury. After transection of the left lumbar (L) 5 spinal nerve, antisera specific to neurotrophins were injected intraperitoneally twice a week for 2 weeks. The foot withdrawal response to von Frey hairs was examined on days 1, 3, 7, 10, and 14 postlesion. After completion of behavioral tests, sympathetic sprouting in DRG was examined by tyrosine hydroxylase (TH) immunohistochemistry. The number of TH-immunoreactive (ir) fibers and baskets around large neurons within the lesioned DRG was dramatically increased in the rats treated with control normal sheep serum. Antisera specific to nerve growth factor (NGF), neurotrophin-3 (NT3), and brain-derived neurotrophic factor (BDNF) significantly reduced the sympathetic sprouting and the formation of baskets. L5 spinal nerve lesion induced a significant increase in foot withdrawal responses to von Frey hair stimuli, which was attenuated by treatment of antisera to neurotrophins with a different time sequential. The effect of BDNF antiserum occurred earlier and lasted longer than those of NGF and NT3 antisera. These results implicate that peripherally derived neurotrophins are involved in the induction of sympathetic sprouting and neuropathic pain following peripheral nerve injury.     

TrkA immunoreactive astrocytes in dendritic fields of the hippocampal formation across estrous

Neurotrophins are important modulators of structural synaptic plasticity. (Through trophic action (Jordan. J Neurobiol 40:434-445, 1999), astrocytes serve as permissive substrates to support axonal regrowth (Ridet et al. Trends Neurosci 20:570-571, 1997), and are involved in estrogen-induced synaptic structural plasticity (Garcia-Segura et al. Cell Mol Neurobiol 16:225-237, 1996). Previously, we reported that tyrosine kinase A receptor (TrkA) immunoreactivity was present both in presynaptic neuronal processes (axons and terminals) and in select astrocytes of the male rat hippocampal formation (Barker-Gibb et al. J Comp Neurol 430:182-199, 2001). We show that the number of TrkA-immunoreactive astrocytes in female rats fluctuates 16-fold across the estrous cycle in dendritic fields of the hippocampal formation, with the greatest number at estrus after the peak plasma estradiol concentration of proestrus. Few TrkA-labeled astrocytes were found in ovariectomized animals; after estrogen replacement, this number increased by 12-fold in the hippocampal formation, indicating estrogen-mediated induction. Dual-labeling studies showed that TrkA-labeled astrocytes were also immunoreactive for vimentin, a protein expressed by reactive astrocytes. Ultrastructural analysis of the dentate gyrus molecular layer demonstrated that TrkA immunoreactive astrocytes are positioned primarily next to dendrites and unmyelinated axons. Because nerve growth factor (NGF) has been reported to stimulate astrocytes to function as substrates for axon growth (Kawaja and Gage. Neuron 7:1019-1030, 1991), these findings are consistent with the theory that TrkA immunoreactive astrocytes serve a role in structural plasticity, axon guidance, and synaptic regeneration across the estrous cycle in the hippocampal formation.     

Production in vitro of a neurotropic substance from proliferative neurolemma-like cells.

The objective of this study was to better define the biological and chemical properties of a neurotropic substance capable of mediating recognition between neurolemma-like cells and neurites during growth. Using in vitro methods, we showed that it was a macromolecular protein apparently synthesized by proliferative cells and was responsible for attracting and promoting neurite outgrowth from chick embryo dorsal root ganglia. The substance appeared to be a normal component of the cell coat during the proliferative phase, and was secreted into the tissue culture medium. Fractionation of the culture fluid by gel filtration chromatography yielded three major fractions containing the biologically active component. The fraction that had the greatest nerve-growth-stimulating activity appeared in a small molecular fraction (mol. wt., 10,000 to 20,000), and was biologically similar to the mouse salivary gland nerve growth factor (NGF). The neurotropic substance appeared to play a central role in promoting and guiding axonal growth during normal development and regeneration of the peripheral nerve.     

The localization of nerve growth factor-like immunoreactivity in the adult rat basal forebrain and hippocampal formation.

The role of nerve growth factor (NGF) as a target derived neurotrophic agent for specific cell populations in the peripheral nervous system has been well documented and much evidence suggests that NGF may serve a similar neurotrophic role in the CNS supporting the cholinergic neurons of the basal forebrain. Previous attempts to localize NGF by immunocytochemical methods, however, have not yielded evidence confirming the regional distribution expected based upon reported levels of extractable NGF. In the present study, affinity purified polyclonal antibodies to beta-NGF and a modified immunohistochemical protocol were used to demonstrate specific NGF-like immunoreactivity in the adult rat hippocampal formation and basal forebrain. In the hippocampal formation, NGF-like immunoreactivity was localized primarily within the hilus of the dentate gyrus and within stratum lucidum of the CA3 and CA2 hippocampal subfields. Staining appeared to be associated with cell processes and was similar to the reported distribution of mossy fibers suggesting that granule cells may either serve as a primary source of hippocampal NGF or that mossy fibers selectively accumulate NGF produced by other cell populations. In the basal forebrain, NGF-like immunoreactivity was localized within neuronal cell bodies of the medial septum, diagonal band, and nucleus basalis of Meynert and was further demonstrated to colocalize exclusively with LNGF-R positive neurons. These findings demonstrate the presence of an NGF-like antigen in association with cholinergic neurons of the basal forebrain and strongly support the hypothesis that NGF may serve as an endogenous trophic factor for this adult neuronal population.     

Effects of chronic paralysis with α-bungarotoxin on development of innervation

Chronic paralysis of the duck embryo with α-bungarotoxin increases the number of trochlear motor neurons that survive by decreasing the magnitude of embryonic cell death. The present study examined how such an increase in the motor neuron projection is accommodated at the peripheral target. The results indicate that the overall size of the duck superior oblique muscle was drastically reduced following chronic paralysis with α-bungarotoxin. The number of motor end-plates was reduced to about 25% of the control. Although the total number of end-plates was significantly reduced, their size was larger and they became innervated by far more nerve terminals than in the control. Thus, increased motor neuron survival occurred while the size of the periphery and the number of synaptic sites (end-plates) were greatly reduced. These observations suggest that the cause of embryonic cell death may not be related to the availability of adequate numbers of synapses with the periphery. They also suggest that the physiologic activity of the embryo may play a significant role in regulation of cell number in the developing nervous system.     

In vivo ANTI-NGF induces sprouting of sensory axons in dorsal roots

Newborn rats were given subcutaneous injections of antibodies to mouse beta -NGF (ANTI-NGF) daily for 1 month. The number of neurons in T4-T6 dorsal root ganglia (DRG) and the numbers of myelinated and unmyelinated axons in the dorsal roots of the same segments were counted in the ANTI-NGF animals and in normal littermates. The ANTI-NGF rats had 38% fewer neurons in thoracic ganglia but 17% more myelinated and 40% more unmyelinated fibers than their untreated littermates. Dorsal root ganglion cells also have a larger average size in the ANTI-NGF animals, which we interpret as a disproportionate loss of small cells. These data are interpreted as showing that some dorsal root ganglion cells, principally small ones, die when endogenous NGF is inactivated, and that the remaining cells emit more processes than normal. Thus, removal of NGF has what appears to be a paradoxical effect, a reduction in dorsal root ganglion cell numbers but an increase in dorsal root axon numbers. The relation of myelin thickness to fiber diameter is also altered, with small fibers being more thinly myelinated in the ANTI-NGF group. Thus, Schwann cell-neuronal interactions are also affected by inactivation of NGF.     

Neurotrophin-like immunoreactivity in the human hippocampal formation

The immunohistochemical occurrence and localisation of the neurotrophins nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-4, and neurotrophin-3 is described in the human post-mortem hippocampal formation from subjects aged 23 weeks of gestation to 68 years. Labeled neuronal cell bodies and processes were detectable for each neurotrophin at all examined ages with age-related changes in their distribution pattern. As a general rule, a higher number of immunoreactive perikarya was found in subjects at pre- and perinatal ages than in adults. At variance with the other neurotrophins, the BDNF antiserum labelled also extensive nerve fibre systems, whose occurrence and distribution widened with age. The results obtained provide a morphological ground in support to the concept that the neurotrophins play a functional role in the human hippocampal circuitry throughout life.     

Alkylcatechols regulate NGF gene expression in astroglial cells via both protein kinase C-and cAMP-independent mechanisms

It has been previously demonstrated that, in mouse astroglial cells and fibroblast cells in culture, alkylcatechols cause a rapid increase in the nerve growth factor (NGF) mRNA level followed by a marked increase in the amount of NGF released into the medium. To understand the mechanism of this alkylcatechol effect on NGF gene expression in astroglial cells, we examined the effects of protein kinases that influence intracellular signal transduction and of their inhibitors. The reagents to increase the intracellular content of cyclic AMP (cAMP) such as dibutyryl cyclic AMP (Bt₂cAMP), forskolin, or cholera toxin did not mimic alkylcatechol induction of NGF gene expression. Phorbol ester, a direct activator of protein kinase C (PKC), caused an increase in the NGF synthesis/secretion. The stimulatory effect of homocatechol (4-methylcatechol) on NGF synthesis was not completely inhibited by staurosporine, an inhibitor of PKC. The treatment of cells with homocatechol resulted in the translocation of PKC from cytosol to membrane-associated fractions, although the levels of the subcellular location of PKC were not correlated with the level of the induction of NGF gene expression. The concomitant administration of homocatechol (10^?4M) and PMA (10^?8?10^?6 M) evoked a drastic and prolonged increase in the NGF mRNA level, and also markedly increased the amounts of NGF secreted by the cells (approximately 150-fold). This synergism was inhibited in part by staurosporine, but the level of increase in NGF mRNA and in NGF protein was rather greater than that of activation of PKC. These results suggest that neither PKC-dependent pathway nor cAMP-dependent pathway is dominant in the stimulatory effect of alkylcatechol on NGF synthesis. © 1993 Wiley-Liss, Inc.     

Early rapid and transient increase in ornithine decarboxylase activity within sympathetic neurons after axonal injury

The activity of ornithine decarboxylase (ODC) was examined in the rat superior cervical ganglion during development and after postganglionic axotomy. During development the enzyme activity in the ganglion was transiently elevated at 19 days gestation (2 days before birth) and then declined to low adult activities by 5 days after birth. After axotomy the enzyme activity in the ganglion was rapidly increased. As early as 2 h after the injury the activity was increased to 200% and by 10 h it attained a peak of 453% above control values. The activity then rapidly declined to a lower value of about 200% above control by 24 h, where it remained as long as 5 days and then returned to control values by 10 days postoperative. The early increase in ODC activity was demonstrated by inhibitor cytochemistry to occur within neuronal cell bodies. The ODC activity was also increased after preganglionic nerve transection (denervation) (275%), end organ removal (60%), and 6-hydroxydopamine treatment (75%). We conclude that increased ODC activity is one of the earliest events in the reaction of the nerve cell body to axonal injury, and that increased ODC activity is a common feature of drastic metabolic alterations in neurons of the superior cervical ganglion.     

Overexpression of nerve growth factor by murine smooth muscle cells: Role of the p75 neurotrophin receptor on sympathetic and sensory sprouting

Elevating levels of nerve growth factor (NGF) can have pronounced effects on the survival and maintenance of distinct populations of neurons. We have generated a line of transgenic mice in which NGF is expressed under the control of the smooth muscle α‐actin promoter. These transgenic mice have augmented levels of NGF protein in the descending colon and urinary bladder, so these tissues display increased densities of NGF‐sensitive sympathetic efferents and sensory afferents. Here we provide a thorough examination of sympathetic and sensory axonal densities in the descending colon and urinary bladder of NGF transgenic mice with and without the expression of the p75 neurotrophin receptor (p75NTR). In response to elevated NGF levels, sympathetic axons (immunostained for tyrosine hydroxylase) undergo robust collateral sprouting in the descending colon and urinary bladder of adult transgenic mice (i.e., those tissues having smooth muscle cells); this sprouting is not augmented in the absence of p75NTR expression. As for sensory axons (immunostained for calcitonin gene‐related peptide) in the urinary bladders of transgenic mice, fibers undergo sprouting that is further increased in the absence of p75NTR expression. Sympathetic axons are also seen invading the sensory ganglia of transgenic mice; these fibers form perineuronal plexi around a subpopulation of sensory somata. Our results reveal that elevated levels of NGF in target tissues stimulate sympathetic and sensory axonal sprouting and that an absence of p75NTR by sensory afferents (but not by sympathetic efferents) leads to a further increase of terminal arborization in certain NGF‐rich peripheral tissues. J. Comp. Neurol. 521:2621–2643, 2013. © 2013 Wiley Periodicals, Inc.     

Glial reaction to nerve growth factor in the regenerating optic nerve of the newt (Triturus viridescens)

Nerve growth factor (NGF), administered as a single intraocular injection at the time of nerve transection, elicited cellular hyperplasia and hypertrophy in the astrocyte-like glial cell population of the regenerating newt (Triturus viridescens) optic nerve at 14 days postlesion. More specifically, there was a significant dose-dependent increase in the number of glial cells in response to various NGF concentrations [2 to 2000 biologic units (BU)], which correlated directly with the dose-dependent rise in the neuronal (regenerating axons) area of 14-day regenerating nerve cross sections. We refer to this phenomenon (i.e., cell hyperplasia) as a NGF dose-dependent glial cell response. Quantitation of the astrocyte-like cell perikaryal area and nuclear area, and calculation of cell: nuclear ratios indicated that cell hypertrophy was elicited by the 2000-BU dose of NGF, but not by lower NGF concentrations (i.e., 2 to 200 BU). Corroborative ultrastructural observations were even more revealing. Not only did electron micrographs verify cellular hypertrophy in the astrocyte-like glial cells, but also they revealed hypertrophy of cell processes and a massive increase in the number of microfilaments in response to the 2000-BU NGF treatment. We speculate that these phenomena may represent an NGF dose-independent glial cell response.     

Regenerative repair in the severed optic nerve of the newt (Triturus viridescens): Effect of nerve growth factor antiserum

At 14 days after transection those regenerating newt (Triturus viridescens) optic nerves receiving anti-nerve growth factor treatment were easily distinguished from regenerating controls. Quantitative analysis revealed that antiserum treatment significantly reduced nerve diameter and cross-sectional areas compared to the control groups. Quantitation from electron microscope montages of nerve cross sections revealed similar results. In addition, antiserum treatment significantly reduced the area of regenerating axon fascicles per nerve cross section compared to the control groups. Most significantly, the mean number of regenerating axons per nerve decreased more than 50% in the antiserum-treated group. Regenerating axons appeared normal in all three groups. Axons were filled with clear cytoplasm containing neurofilaments, neurotubules, and an occasional mitochondrion. Axon density was not significantly different among the three groups and axon diameters were similar from 0.1 to 0.8 μm. Distention of glial cell processes surrounding fascicles of axons and increased intra- and extracellular debris may indicate an alteration of glial cell activity in the antiserum group. Many of the 14-day antiserum-treated nerves have the appearance of an untreated transected optic nerve 6 to 10 days after lesion.     

Effect of applied electrical fields on sprouting of intact saphenous nerve in adult rat

Saphenous nerve sprouting was measured behaviorally and histologically after chronic sciatic denervation in the adult rat. The effect of electrical stimulation (either weak DC fields, or stronger AC fields) on the rate of sprouting of the intact saphenous was studied. Sprouting was enhanced by DC fields (1 μA) if the cathode was placed distal to the growth tips, but was unaffected by anode stimulation. Sprouting was also enhanced by AC fields (1000 μA per pulse) given at 20 Hz and 0.1 ms duration. In the discussion we postulate that separate mechanisms might mediate the AC and DC results. The DC effects are the first demonstration in mammals of results previously observed in lower vertebrates.     

Adrènal medulla grafts survice and exhibit catecholamine-specific fluorescence in the primate brain

Parkinson's disease most consistently involves pathologic changes in the substantia nigra, which is the major source of dopamine to the striatum. It has been shown that either fetal substantia nigra or adrenal medulla tissue implanted into the rat brain survives, produces dopamine, and improves behavioral abnormalities induced by deprivation of the caudate nucleus of its dopaminergic innervation. Thus, catecholaminecontaining grafts could be potential replacements for destroyed or damaged dopaminergic neurons in patients with Parkinson's disease. To explore the potential of this therapeutic approach, fetal substantia nigra or host adrenal medulla were grafted to the denervated caudate nucleus of the rhesus monkey. Under the specific conditions of our experiment, fetal substantia nigra did not survive in either of two animals tested. On the other hand, some tissue from adrenal medulla grafts survived in all four animals tested. These grafted cells contained catecholamines, as indicated by the presence of specific glyoxylic acid-induced catecholamine fluorescence. In two of the four animals, however, the grafts contained fewer than 10 surviving cells, and in the other two animals, about 190 and 300 cells were found, respectively. Despite the small numbers of cells, this is the first demonstration that peripheral tissue autografts can survive implantation into the nonhuman primate central nervous system.     

Histochemical studies of sympathetic sprouting: Fluorescence morphology of noradrenergic axons

The importance of distinguishing between central and peripheral noradrenergic axons is evident from recent observations that sympathetic fibers will invade the central nervous system following specific lesions. The present paper reviews the normal histofluorescence appearance of peripheral and central NE fibers in several species as well as their appearance following experimental manipulations. The most striking differences between these two types of NE neurons is their axonal fluorescence morphology which is apparently determined by the target tissue, and their responsiveness to nerve growth factor (NGF). The latter may account for the remarkable growth of sympathetic axons into regions of the central nervous system denervated of cholinergic fibers. The use of glyoxylic acid in studying such sprouting is also discussed.     

Purification and culture of adult rat dorsal root ganglia neurons

To study the trophic requirements of adult rat dorsal root ganglia neurons (DRG) in vitro, we developed a purification procedure that yields highly enriched neuronal cultures. Forty to fifty ganglia are dissected from the spinal column of an adult rat. After enzymatic and mechanical dissociation of the ganglia, myelin debris are eliminated by centrifugation on a Percoll gradient. The resulting cell suspension is layered onto a nylon mesh with a pore size of 10 microns. Most of the neurons, the diameter of which ranged from 17 microns to greater than 100 microns, are retained on the upper surface of the sieve; most of the non-neuronal cells with a caliber of less than 10 microns after trypsinization go through it. Recovery of neurons is achieved by reversing the mesh onto a Petri dish containing culture medium. Neurons to non-neurons ratio is 1 to 10 in the initial cell suspension and 1 to 1 after separation. When these purified neurons are seeded at a density of 3,000 neurons/cm2 in 6 mm polyornithine-laminin (PORN-LAM) coated wells, neuronal survival (assessed by the ability to extend neurites), measured after 48 hr of culture, is very low (from 0 to 16%). Addition of nerve growth factor (NGF) does not improve neuronal survival. However, when neurons are cultured in the presence of medium conditioned (CM) by astrocytes or Schwann cells, 60-80% of the seeded, dye-excluding neurons survive. So, purified adult DRG neurons require for their short-term survival and regeneration in culture, a trophic support that is present in conditioned medium from PNS or CNS glia.(ABSTRACT TRUNCATED AT 250 WORDS)