anti-NGF Antiserum Research Articles

Nerve growth factor from Vipera lebetina venom

Nerve growth factor was isolated from the Vipera lebetina venom by a four-step procedure including gel filtration, ion exchange, heparin and hydrophobic chromatography. The purified protein is a glycosylated non-covalently bound homodimer with monomeric molecular mass of 14,380 Da. The cDNA encoding NGF is cloned and sequenced. The amino acid sequence translated from the cDNA comprises 117 or 119 amino acids depending on the N-terminus (truncated or not). The recombinant NGF (expressed in Escherichia coli) was used to prepare the anti-NGF antiserum. The antiserum interacted with the wild-type NGF and enabled to localize NGF during the purification procedure in parallel with MALDI-TOF analysis of tryptic peptides. The isolated NGF caused neurite outgrowth from PC12 cells in concentrations beginning from 2.5 ng/ml.

The role of cytokines and prostaglandin-E 2 in thymulin induced hyperalgesia

We have recently reported that intraperitoneal (i.p.) injection of thymulin at low doses (50 ng) resulted in thermal and mechanical hyperalgesia and upregulation of the level of interleukin-1β in the liver. In this study, we demonstrate that such injections of thymulin result in a significant elevation in the levels of TNF-α ( P<0.01), NGF ( P<0.01) and PGE 2 ( P<0.01) in the liver of the treated rats, in addition to the increase in the levels of IL-1β. Pretreatment with specific antagonists to each of these factors (polyclonal anti-TNF-α, anti-NGF antiserum and IL-1 receptor antagonist) did not result in the abolition of the hyperalgesia as assessed by the paw pressure, hot plate, paw immersion and tail flick tests. However, pretreatment with a combination of the above antagonist and antisera almost completely prevented thymulin-induced hyperalgesia. The cyclooxygenase inhibitor, meloxicam, reversed in a dose dependent manner (0.2, 0.4 and 2 mg/kg) thymulin effects as assessed by the different pain tests. It also abolished the thymulin-induced increase in the level of cytokines and NGF in the liver. Our results indicate that PGE 2 could be the key mediator of the hyperalgesic action of thymulin and the observed upregulation of proinflammatory cytokines and NGF.

Involvement of NGF in the induction of increased noradrenergic innervation of the ureter in neonatally capsaicin-treated rats

Neonatal denervation of primary afferents with capsaicin leads to increased sympathetic innervation of the rat ureter. In the present study the development and the immunohistochemical characterization of this sympathetic hyperinnervation as well as the specific involvement of nerve growth factor (NGF) was investigated. Noradrenaline levels were found elevated in neonatally capsaicin-treated rats by 2 weeks of age and remained at that high level into adulthood. Injections of an anti-NGF antiserum during postnatal days (PN) PN 8–14, PN 13–19 or during PN 17–23 counteracted the capsaicin effect and reduced noradrenaline towards control levels. Immunohistochemical localization of tyrosine hydroxylase (TH), a marker for sympathetic nerve fibres, revealed that the capsaicin-induced hyperinnervation was mainly represented by fibres in deeper muscle layers and to a smaller extent by fibres in the submucosa. In control animals and in rats treated with capsaicin and anti-NGF antiserum fibres were mainly distributed in the adventitia and in the outer part of the smooth muscle layer. These results show that NGF is responsible for the development of an increased noradrenergic innervation in the rat ureter after neonatal capsaicin treatment.

Involvement of Interleukin-1β, Nerve Growth Factor, and Prostaglandin-E2in the Hyperalgesia Induced by Intraplantar Injections of Low Doses of Thymulin

The effect of various doses of intraplantar thymulin injection, on nociceptive thresholds, in the hind paw of rats was assessed using different pain tests. As little as 0.5 ng thymulin resulted in localized mechanical hyperalgesia as assessed by the paw pressure test and thermal hyperalgesia as assessed by the paw immersion, hot plate, and tail flick tests. The highest dose of thymulin (10 ng) reduced both paw pressure and paw immersion latencies in the noninjected paw also. Thymulin (5 ng) also resulted in significant elevation in the levels of interleukin-1β (IL-1β) and nerve growth factor (NGF) levels in the injected paw. Both dexamethasone and indomethacin reversed thymulin-induced hyperalgesia. Also interleukin-1 receptor antagonist (IL-1ra) and a polyclonal anti-NGF antiserum significantly reduced thymulin-induced hyperalgesia. On the other hand, the tripeptide lys-D-pro-val (known to antagonize IL-1β and PGE2induced hyperalgesia) reversed the hyperalgesia due to thymulin. In conclusion, thymulin induces localized hyperalgesia which is mediated by PGE2-dependent mechanisms and this pathway could be either partially dependent on or totally independent of IL-1β mechanisms.

Cytokines, nerve growth factor and inflammatory hyperalgesia: the contribution of tumour necrosis factor alpha.

1. Peripheral inflammation is characterized by heightened pain sensitivity. This hyperalgesia is the consequence of the release of inflammatory mediators, cytokines and growth factors. A key participant is the induction of the neurotrophin nerve growth factor (NGF) by interleukin-1 beta (IL-1 beta). 2. Tumour necrosis factor alpha (TNF alpha) has been shown both to produce hyperalgesia and to upregulate IL-1 beta. We have now examined whether the induction of TNF alpha in inflammatory lesions contributes to inflammatory sensory hypersensitivity by inducing IL-1 beta and NGF. 3. The intraplantar injection of complete Freund's adjuvant (CFA) in adult rats produced a localized inflammation of the hindpaw with a rapid (3 h) reduction in withdrawal time in the hot plate test and in the mechanical threshold for eliciting the flexion withdrawal reflex. 4. The CFA-induced inflammation resulted in significant elevation in the levels of TNF alpha, IL-1 beta and NGF in the inflamed paw. In the case of TNF alpha, an elevation was detected at 3 h, rose substantially at 6 h, peaked at 24 h and remained elevated at 5 days, with similar but smaller changes in the contralateral non-inflamed hindpaw. No increase in serum TNF alpha was detected at 24 h post CFA injection. 5. Intraplantar recombinant murine TNF alpha injections produce a short-lived (3-6 h) dose-dependent (50-500 ng) increase in thermal and mechanical sensitivity which was significantly attenuated by prior administration of anti-NGF antiserum. 6. Intraplantar TNF alpha (100-500 ng) also elevated at 6 but not 48 h the levels of IL-1 beta and NGF in the hindpaw. 7. A single injection of anti-TNF alpha antiserum, 1 h before the CFA, at a dose sufficient to reduce the effects of a 100 ng intraplantar injection of TNF alpha, significantly delayed the onset of the resultant inflammatory hyperalgesia and reduced IL-1 beta but not NGF levels measured at 24 h. 8. The elevation of TNF alpha in inflammation, by virtue of its capacity to induce IL-1 beta and NGF, may contribute to the initiation of inflammatory hyperalgesia.

The progressive tactile hyperalgesia induced by peripheral inflammation is nerve growth factor dependent.

Experimental peripheral inflammation results in cutaneous mechanical hypersensitivity, and repeated low intensity mechanical stimulation of the inflamed skin induces a progressively incrementing hyperalgesia. We have now examined whether the elevation in nerve growth factor (NGF) induced by the inflammation contributes to this progressive hyperalgesia. An i.p. injection of anti-NGF antiserum (5 microliters g-1) 1 h before induction of inflammation by intraplantar complete Freund's adjuvant (CFA) injection and 24 h after, both reduced the basal inflammatory hypersensitivity and significantly attenuated the progressive increase of spontaneous activity, touch-, pinch- and A beta-afferent-evoked responses, and the progressive reduction of the mechanical threshold of biceps femoris/semitendinosus alpha motoneurones normally evoked by repeated (every 5 min) tactile stimulation of the inflamed hindpaw, in decerebrate-spinal rats. NGF contributes, therefore, to the progressive tactile hyperalgesia elicited by repeated touch stimulation of inflamed tissue.

Increased neurite outgrowth of cultured rat dorsal root ganglion cells following transection or inhibition of axonal transport of the sciatic nerve

Dissociated dorsal root ganglion cells (DRGs), taken from rats 2 weeks after sciatic nerve transection, have an increase in the percentage of cells with neurites compared to DRGs taken from normal animals. This study examines the possible factors that may contribute to the nerve injury-induced increase in neuritogenesis. Topical application of the local anaesthetic, bupivicaine, either to the nerve trunk prior to transection or to the proximal nerve stump for 2 weeks had no effect on the increased neurite outgrowth induced by nerve transection. Neurite outgrowth was also not influenced by administration of either nerve growth factor (NGF) via the femoral artery into normal rats or anti-NGF antiserum to the proximal nerve stump. Inhibition of axonal transport by topical application of vinblastine, however, induced a significant increase in neurite outgrowth compared to untreated controls. In addition, vinblastine-treated animals also develop hyperalgesia to mechanical stimulation and transganglionic labelling of sensory neurons with choleragenoid-horseradish peroxidase shows that the area of termination of myelinated sensory neurons in the spinal cord expands into lamina II. The results suggest that nerve injury-induced increase in neurite outgrowth is not dependent on NGF nor nerve impulses generated at the site of injury and supports the view that the absence of an inhibitory factor(s), that in normal animals may regulate neuronal outgrowth.

Nerve growth factor alleviates a painful peripheral neuropathy in rats

Nerve growth factor (NGF) reverses some effects of axotomy and prevents toxic neuropathy in adult rodents. We tested the effect of NGF on behavioral hyperalgesia resulting from a chronic constriction injury (CCI) of the sciatic nerve in the rat [5]. CCI rats exhibit thermal hyperalgesia as demonstrated by a reduction of paw withdrawal latency to a noxious thermal stimulus applied to the paw on the side of injury. The mechanical sensitivity of the ipsilateral hindpaw, assessed with von Frey filaments, was also significantly increased. There were no significant changes in nociceptive thresholds on the contralateral side. When NGF was infused directly on the ligated nerve via an osmotic pump (0.5 μg/μl/h for 7 days) immediately after the ligation, thermal hyperalgesia was abolished from postoperative days 5 up to at least two weeks. The CCI-induced decrease in mechanical threshold was also abolished by NGF. However, NGF had only a minor effect on the abnormally long response duration, a second measure of mechanical sensitivity, to the mechanical stimulus. Delayed infusion of NGF four days after the ligation failed to block hyperalgesia. Infusion of NGF on the sciatic nerve of rats that had no CCI had no significant effect on paw withdrawal latency. Infusion of anti-NGF antiserum did not enhance hyperalgesia in CCI rats. These results suggest that alterations in neurotrophic factor(s) contribute to the development of behavioral hyperalgesia in an animal model of neuropathy and that NGF may have therapeutic value in the treatment of neuropathic pain in humans.

Anti-NGF treatment blocks the upregulation of NGF receptor mRNA expression associated with collateral sprouting of rat dorsal root ganglion neurons

The collateral sprouting of intact cutaneous sensory neurons has been shown to be dependent upon the presence of nerve growth factor (NGF). We have examined NGF receptor (NGFR) mRNA expression in DRG neurons undergoing sprouting and in dorsal root ganglion (DRG) neurons whose sprouting had been prevented through exposure to anti-NGF antiserum during the course of the experiment. The results indicate that the low affinity p75 NGFR mRNA is increased by 4 days post-operatively in DRGs from control serum-treated animals, and that this increase is most pronounced in smaller neurons. In contrast, the expression of p75 mRNA was maintained at control levels in DRGs from anti-NGF-treated animals. Results for trkA expression indicate an increase in expression at days 4–6 post-op in both ;groups of animals, with the anti-NGF treatment having a delayed influence on mRNA levels. Examination of GAP-43 mRNA levels revealed an increased expression in sprouting DRG neurons, whereas this increase was not observed in DRGs from anti-NGF treated animals. Taken together, these results provide further evidence of NGF's role in the collateral sprouting of nociceptive neurons.

Localization of nerve growth factor (NGF) and low‐affinity NGF receptors in touch domes and quantification of NGF mRNA in keratinocytes of adult rats

Touch domes are clearly delineated mechanoreceptors that are visible on the depilated skin of mammals. These structures consist of a sharply circumscribed disk of thickened epithelium surmounting a group of Merkel cells that are innervated by type I sensory neurons. These characteristic cutaneous structures provide an ideal opportunity for investigating whether the localization of nerve growth factor (NGF) in the skin is related to sites of sensory axon termination. For these reasons, we have used immunocytochemistry to study the distribution of NGF and the low-affinity NGF receptor (p75NGFR) in the touch domes of adult rat skin. Intense NGF-like immunoreactivity was sharply restricted to keratinocytes (excluding the stratum corneum) of the thickened epidermis of touch domes. The epidermis immediately surrounding touch domes and the epidermis of the tylotrich hair follicle associated with touch domes were not stained by anti-NGF antiserum. Merkel cells of the basal epidermis of touch domes were immunonegative for NGF but were immunopositive for p75NGFR as were the type I nerve endings innervating these cells. Quantitative Northern blotting revealed that the level of NGF mRNA was substantially higher in keratinocytes isolated from the stratum granulosum and stratum spinosum than in keratinocytes isolated from the stratum germinativum. These findings indicate that NGF synthesis in mature skin has a highly restricted regional distribution that is primarily associated with the innervation of a specialized touch receptor.

Early nerve growth factor-induced events in developing rat septal neurons

A culture system enriched for nerve growth factor (NGF) receptor bearing cells was developed to investigate signal transduction events activated by NGF in postmitotic central nervous system neurons. Cells from the septal region of embryonic rats at 16 days of gestation were grown on glass coverslips above a glial cell layer established from postnatal rat cortex. The separation of glial and neuronal planes in this “bilaminar” system permits the diffusion of glial-derived factors required by septal neurons for survival yet allows the investigation of NGF responses in a pure neuronal population. Approximately 15% of the neurons in this culture system were immunoreactive for the low affinity NGF receptor. NGF rapidly increased MAP kinase activity (2–5 min) and transiently induced expression of c- fos in septal neurons. NGF treatment also increased choline acetyltransferase activity, while the number of cholinergic neurons remained constant. Septal neuron survival depended on the presence of glial cells, but neuronal viability in the bilaminar system was unaffected by anti-NGF antiserum, indicating that glial-derived neurotrophic support is not mediated by NGF alone. These data suggest that the bilaminar culture system is a useful system for the study of early events in NGF-activated signal transduction and the nature of glial-derived trophic support of developing basal forebrain neurons.

Insulin and insulin-like growth factor I enhance regeneration in cultured adult rat sensory neurones

Insulin and the insulin-like growth factors (IGFs) may directly affect the growth, development, and maintenance of the vertebrate nervous system. Previous in vitro studies have focused on embrynic nervous tissue. In this study the effects of insulin, IGF-I, IGF-II and nerve growth factor (NGF) on regeneration and neuronal survival were studied in cultured adult rat sensory neurones in a cell culture environment that limited non-neuronal cell mediated effects. Regeneration, as assessed by neurite outgrowth, was significantly enhanced by insulin and IGF-I in a dose-dependent manner. The half-maximally effective concentrations, ED 50's, were approximately 1 nM and 0.1 nM for insulin and IGF-I, respectively. Concentrations of IGF-I as low as 10pM were active. There wassome evidence that IGF-II stimulated regeneration, although this failed to reach statistical significance. NGF also promoted regeneration, confirming previous studies, exhibiting an ED 50 of approximately 0.3 ng/ml and inducing a maximal response 2-fold greater than that observed with insulin or IGF-I. Combined treatment with NGF and insulin had an additive effect. Specific anti-NGF antiserum inhibited the regenerative response to NGF but failed to block the response to IGF-I, supporting the view that IGF-I was acting directly on sensory neurones rather than stimulating NGF production by non-neuronal cells. Insulin, IGF-I and NGF had no effect on neuronal survival in this cultural system. These results show that adult sensory neurons can respond with enhanced regenerative growth to insulin and IGF-I, in addition to NGF although the response to IGF-II was less clear.

NGF deprivation and neuronal degeneration trigger altered β-amyloid precursor protein gene expression in the rat superior cervical ganglia in vivo and in vitro

In order to study the expression of β-amyloid precursor protein (APP) isoforms during neuronal degeneration we have used the rat superior cervical ganglia (SCG) as an experimental model. In the neonate these sympathetic ganglia are nerve growth factor (NGF) dependent and in vivo administration of anti-NGF antiserum results in exaggerated neuronal degeneration. Analysis of APP mRNA transcripts in the SCG, following NGF deprivation, revealed a coincident decrease in APP 695 and augmentation of APP 751/770. These changes were specific to the SCG and were not seen in sensory ganglia. Subsequent in vitro studies, using primary dissociated cultures of sympathetic or cortical neurones, confirmed these changes in APP gene expression during neuronal degeneration. These observations may have important implications for the generation of β-amyloid in Alzheimer's disease.

Selective dependence of mammalian dorsal root ganglion neurons on nerve growth factor during embryonic development

We have investigated the NGF dependence of dorsal root ganglion (DRG) neurons in mammals using a paradigm of multiple in utero injections of a high titer anti-NGF antiserum. We have determined the specificity of our antiserum in relation to other members of the NGF neurotrophin family and found no cross-reactivity with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). To identify various classes of DRG neurons, we have stained their characteristic central projections with Dill. We show here that the NGF dependence of DRG neurons is strikingly selective. Although a majority of DRG neurons are lost after NG F deprivation during embryonic life, these are almost exclusively small diameter neurons that project to laminae I and II of the dorsal horn and presumably subserve nociception and thermoreception. Larger neurons that project to more ventral spinal laminae and subserve other sensory modalities do not require NGF for survival. These NGF-independent DRG neurons likely require one of the more recently identified neurotrophins, BDNF or NT-3.

Trophic regulation of nodose ganglion cell development: Evidence for an expanded role of nerve growth factor during embryogenesis in the rat

Peripheral sensory neurons are derived from two distinct embryonic tissues, the neural crest and epibranchial placodes. Studies in the chick suggest that embryonic lineage and trophic dependence are interrelated, such that many crest-derived cells depend on NGF for survival during development, whereas placodal derivates, including nodose ganglion neurons, do not (30). It remains controversial, however, whether or not a similar dichotomy exists in mammalian species, in which trophic requirements during early development of placodal ganglia have not been defined. To approach this issue, the present study examined the effects of nerve growth factor (NGF) on neuronal survival in embryonic rat nodose ganglion cultures. Treatment of E13.5–14.5 nodose explants with 20 ng/ml NGF resulted in a four-fold increase in neuronal survival that was blocked by anti-NGF antiserum. Increased neuronal survival and neurite outgrowth were also observed in neuronenriched dissociated cell cultures; these effects were seen within 12 h of plating, indicating that NGF-responsive neurons or neuroblasts were already present in the ganglion at the time of explantation. This was further supported by immunocytochemical staining of nodose cell bodies in situ with the monoclonal antibody 192-IgG against the NGF receptor (12). These findings indicate that NGF may be important in regulating nodose development during early gangliogenesis in mammals and suggest that NGF plays a more widespread role in peripheral nervous system ontogeny than previously recognized.

Development of cholinergic retinal neurons from embryonic chicken in monolayer cultures: stimulation by glial cell-derived factors

In recent years evidence has indicated that, like the PNS, the development of the CNS is influenced by neuronotrophic polypeptide factors. In the present study, cultures of dissociated retinal neurons from 8-d-old chicken embryos were used to investigate the role of neuronotrophic factors (NTF) in the development of the neural retina. CAT, which in vivo is located in amacrine cells of the retina, served as a marker for studying the in vitro development of cholinergic retinal neurons. Differentiation of cholinergic cells under control conditions was indicated by a 10-fold increase of enzyme activity during a 7-d culture period. Addition of media conditioned by high-density retinal cultures resulted in a further stimulation of CAT activity by 100-400%. The CAT-stimulating activity was associated with a high-molecular-weight component of the retina conditioned medium (RCM) and was sensitive to protease treatment, but was not affected by other hydrolytic enzymes. The putative cholinergic factor was secreted by retinal cultures virtually free of neurons, suggesting that it is mainly produced by Müller cells. CAT-stimulating activity was also present in extracts from embryonic chicken retinae and medium conditioned by rat retinal cultures. NGF, anti-NGF antiserum, extracts from chicken brain tissues, and a number of other extracts and conditioned media, all known to contain neuronotrophic activities, were found to have no influence on cholinergic development in chicken retinal cultures. An extract from non-retinal eye tissue containing ciliary neuronotrophic factor (CNTF) stimulated CAT activity to the same extent as did RCM.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation.

We have developed an experimental paradigm to study the mechanism by which nerve growth factor (NGF) allows the survival of sympathetic neurons. Dissociated sympathetic neurons from embryonic day-21 rats were grown in vitro for 7 d in the presence of NGF. Neurons were then deprived of trophic support by adding anti-NGF antiserum, causing them to die between 24 and 48 h later. Ultrastructural changes included disruption of neurites, followed by cell body changes characterized by an accumulation of lipid droplets, changes in the nuclear membrane, and dilation of the rough endoplasmic reticulum. No primary alterations of mitochondria or lysosomes were observed. The death of NGF-deprived neurons was characterized biochemically by assessing [35S]methionine incorporation into TCA precipitable protein and by measuring the release of the cytosolic enzyme adenylate kinase into the culture medium. Methionine incorporation began to decrease approximately 18 h post-deprivation and was maximally depressed by 36 h. Adenylate kinase began to appear in the culture medium approximately 30 h after deprivation, reaching a maximum by 54 h. The death of NGF-deprived neurons was entirely prevented by inhibiting protein or RNA synthesis. Cycloheximide, puromycin, anisomycin, actinomycin-D, and dichlorobenzimidazole riboside all prevented neuronal death subsequent to NGF deprivation as assessed by the above morphologic and biochemical criteria. The fact that sympathetic neurons must synthesize protein and RNA to die when deprived of NGF indicates that NGF, and presumably other neurotrophic factors, maintains neuronal survival by suppressing an endogenous, active death program.

Nerve growth factor selectively increases cholinergic markers but not neuropeptides in rat basal forebrain in culture

We have previously used organotypic cultures to study mechanisms regulating phenotypic expression of neurotransmitter characters in the brain. Our previous work indicated that nerve growth factor (NGF) specifically increased the activity of choline acetyltransferase (CAT) in striatal cholinergic interneurons. In the present study we examined the effect of NGF on neurons of fetal rat basal forebrain-medial septal area (BF-MS) maintained in organotypic culture. Treatment with 200 biological units/ml of NGF resulted in a 3-to 6-fold increase in the specific activity of CAT. This effect was specifically blocked by anti-NGF antiserum, whereas treatment with antiserum alone did not alter the cholinergic enzyme. NGF also elicited a marked increase in CAT staining intensity, using a monoclonal antibody directed against the enzyme. Further, the number of CAT-positive neurons appeared to increase in the NGF-treated cultures. Exposure to NGF also increased the activity of another cholinergic marker, the catabolic enzyme, acetylcholinesterase. The effect of NGF appeared to be highly selective, since substance P and somatostatin levels were unchanged by NGF treatment.

Nerve growth factor levels in mouse serum: variations due to stress.

The presence of nerve growth factor (NGF) in the serum of adult male mice was assayed using the chick embryo dorsal root ganglion (DRG) bioassay technique in a serum free N1 supplemented medium. Wide variations in the serum-induced nerve fiber outgrowth response were observed when serum was obtained from animals maintained four per cage. Of 64 mice tested, sera of 7 animals induced a profound nerve fiber outgrowth response while the sera of 57 mice failed to show a similar response. In animals kept in isolation for 7 days prior to the start of the experiment, aggression provoked a marked increase in serum NGF levels. In contrast to the sera of aggression-unprovoked mice, the sera of all aggression-provoked mice stimulated a dense nerve fiber outgrowth. The sera of both groups of mice stimulated an intense proliferation and migration of nonneuronal cells. The neurite outgrowth responses elicited by sera from aggression-provoked and unprovoked mice were completely inhibited by the rabbit anti-NGF antiserum. In conclusion, both crowded housing and aggression in mice may provoke an elevation in the serum NGF levels that can be confirmed by the ganglion bioassay technique.

Nerve growth factor promotes development of the rat septo-hippocampal cholinergic projection in vitro.

There is now widespread evidence indicating that nerve growth factor (NGF) is involved in the function of central cholinergic neurons. Its possible role in the establishment of cholinergic fiber connectivities was studied in co-cultures of rat septum and hippocampus. Application of 100 ng/ml NGF greatly increased the number of acetylcholinesterase (AChE)-positive fibers which invaded the hippocampal slices, an effect which was accompanied by a more than 6-fold elevation of the two major cholinergic enzymes, choline acetyltransferase and AChE. In contrast, application of anti-NGF antiserum, but not a non-immune serum, reduced the number of AChE-positive fibers which grew into and remained within the hippocampal slices. Since no diffuse outgrowth of AChE-positive fibers from the septum was observed following application of NGF, these results suggest that NGF plays a role in the stabilization and long-term maintenance of the cholinergic septo-hippocampal projection in vitro.