Isolation and partial amino acid sequence analysis of nerve growth factor from the guinea pig prostate

Nerve growth factor (NGF) has been purified from the guinea pig prostate using a modification of the Bocchini-Angeletti method for isolating 2.5S NGF from mouse submaxillary glands. As with the mouse preparation, guinea pig prostate NGF appears to migrate as a high molecular weight entity at physiological pH. Following dissociation, NGF, active in neurite proliferation assays and similar in size to mouse 2.5S NGF, can be isolated by chromatography on a column of carboxymethyl-cellulose at pH 4.8. Based on gel filtration, SDS-polyacrylamide gel analysis, and amino-terminal sequence studies, this material consists of two, noncovalently linked, identical polypeptide chains each with a molecular weight of about 13,000. The amino-terminal third of the polypeptide chain is at least 90% identical to the corresponding region of the murine molecule, confirming the homology of the guinea pig prostate protein to NGFs obtained from different tissues in other species. However, in contrast to the mouse preparation, the putative high molecular weight form of guinea pig NGF does not contain a subunit with arginine esteropeptidase activity. Although there is an abundance of this enzymatic activity in the homogenate, it does not appear to be associated with the fractions containing NGF. This apparent difference in the mouse and guinea pig material is of interest because the mouse γ subunit, possessing the arginine esteropeptidase activity, has been alleged to participate in the processing of a precursor of the β NGF polypeptide chain.     

Nerve Growth Factor Regulates Expression of the Nerve Growth Factor Receptor Gene in Adult Sensory Neurons

Sensory neurons of the adult rat dorsal root ganglion (DRG) can be maintained in culture in the absence of nerve growth factor (NGF). We have thus used dissociated cultures of these neurons to study effects of NGF on the regulation of expression of mRNA encoding the nerve growth factor receptor (NGF-R). In the absence of NGF, levels of NGF-R mRNA remained constant for 7 days in cultures of adult rat DRG neurons. In the presence of NGF, NGF-R mRNA levels rose two - three-fold after 2 days, reaching plateau levels (five - six-fold elevation) after 5 days. This NGF-induced up-regulation could be demonstrated even after prior NGF-deprivation for 3 - 4 days. NGF had no effect upon NGF-R mRNA levels in DRG non-neuronal cells. Epidermal growth factor (EGF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF) were without effect on NGF-R mRNA levels, but 8-bromo-cAMP decreased NGF-R mRNA levels by 65% after 2 days. NGF also induced a rapid (30 min) rise in expression of c-fos in DRG neurons, but not in non-neuronal cells. Our results suggest that endogenous levels of NGF may regulate the expression of NGF-R in vivo.     

Effects of autoimmune NGF deprivation in the adult rabbit and offspring

An experimental autoimmune approach to the production of nerve growth factor deprivation, which we have previously described in the rat and guinea pig, has been applied to the rabbit. This species was chosen for study because of several potential advantages. The rabbit produces large litters and has a relatively short gestation period. More importantly, rabbits generate high titers of antibody against mouse NGF and large amounts of maternal antibody are passively transferred to the developing rabbit fetus compared to most other species, particularly the rat. The sympathetic nervous system of adult rabbit immunized against mouse NGF underwent degeneration with up to an 85% decrease in neuronal numbers in the superior cervical ganglion after 10 months of immunization, thus providing further evidence that NGF is required for the survival of mature sympathetic neurons. Despite the fact that newborn rabbits born to anti-NGF producing mothers had much higher titers of anti-NGF than did rats, the effects on the developing sympathetic and sensory nervous systems were not found to be any greater than in rats. Reductions in norepinephrine levels in the heart and spleen of adult rabbits born to anti-NGF producing mothers were greater than in small intestine. Prenatal exposure to maternal anti-NGF caused reductions (up to 70%) in the number of neurons in the dorsal root ganglia. Substance-P immunoreactivity was reduced in the substantia gelatinosa of the spinal cord of rabbit exposed to maternal anti-NGF. These changes, however, were not greater than seen in the rat. We conclide that although the rabbits offers some advantage in the study of the effects of NGF deprivation in the adult animal, it appears less well suited than the rat or guinea pig to the study of the effects of NGF deprivation on development.     

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.     

Multiple neurotransmitters in the tuberomammillary nucleus: comparison of rat, mouse, and guinea pig.

Tuberomammillary neurons in the posterior hypothalamus are the sole source of neuronal histamine in adult mammalian brain. In the rat, these cells are reported to contain immunoreactivity for gamma-aminobutyric acid (GABA) and several neuropeptides. We compared the presence of these substances in the tuberomammillary cells of the rat, mouse, and guinea pig. In all three species, all histamine-immunoreactive neuronal cell bodies were positive for GABA. This suggests that GABAergic transmission may be important in tuberomammillary function. No cell bodies immunoreactive for thyrotropin releasing hormone (TRH) were found in the guinea pig or mouse tuberomammillary area. In contrast, about 14% of the histamine-immunoreactive tuberomammillary cells in the rat were TRH-positive. These cells were small or medium-sized and were located only in the medial part of the tuberomammillary complex. An antibody against porcine galanin stained about 45% of the tuberomammillary cell bodies in the rat and about 28% in the mouse, but none in the guinea pig. A large proportion of the cells in the rat and mouse, but none in the guinea pig, were positive for met-enkephalin-arg-phe. In contrast, all histamine-containing tuberomammillary cells in the guinea pig, but none in the rat or mouse, were immunoreactive for met-enkephalin. This may indicate a different expression of proenkephalin-derived peptides in the tuberomammillary neurons in these species. Some substance P-immunoreactive cell bodies were located in the tuberomammillary area in all three species. However, only 3% of the histamine-immunoreactive cell bodies in the rat and mouse but none in the guinea pig were substance P-positive. The neurochemical properties of the tuberomammillary nucleus that exhibited species commonality deserve to be studied neurochemically and electrophysiologically in order to determine the functional relevance of coexisting transmitters in this nucleus.     

Regulation of substance P by nerve growth factor: disruption by capsaicin

Capsaicin depleted substance P from guinea pig dorsal root ganglia and inhibited the retrograde axoplasmic transport of nerve growth factor (NGF). Doses of capsaicin which depleted substance P also inhibited the retrograde axoplasmic transport of NGF. Inhibition of the retrograde transport of NGF by capsaicin preceded substance P depletion. Supplementation of guinea pigs with mouse NGF completely prevented capsaicin-induced substance P depletion. It is concluded that capsaicin depletes substance P from primary afferent neurons of the adult guinea pig by altering the availability of NGF. The data support a role for NGF in the normal maintenance of neuropeptide levels in some sensory neurons in the adult animal.     

Positive Feedback between Acetylcholine and the Neurotrophins Nerve Growth Factor and Brain-derived Neurotrophic Factor in the Rat Hippocampus

In the rat hippocampus, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are synthesized by neurons in an activity-dependent manner. Glutamate receptor activation increases whereas GABAergic stimulation decreases NGF and BDNF mRNA levels. Here we demonstrate that NGF and BDNF mRNA and NGF protein are up-regulated in the rat hippocampus by the activation of muscarinic receptors. Conversely, NGF and BDNF enhance the release of acetylcholine (ACh) from rat hippocampal synaptosomes containing the nerve endings of the septal cholinergic neurons. NGF also rapidly increases the high-affinity choline transport into synaptosomes. The reciprocal regulation of ACh, NGF and BDNF in the hippocampus suggests a novel molecular framework by which the neurotrophins might influence synaptic plasticity.     

Structure-activity studies of C- and N-terminal fragments of cholecystokinin 26-33 in guinea pig isolated tissues

In vitro structure-activity studies with cholecystokinin (CCK)/gastrin-related peptides, including C- and N-terminal fragments of CCK 26-33, were undertaken in guinea pig gallbladder and ileum. The general order of potency in both smooth muscle preparations is CCK 26-33 greater than CCK 1-33 greater than 27-33 much much greater than nonsulfated (NS) CCK 26-33 greater than pentagastrin greater than CCK 30-33. None of the CCK fragments exhibit antagonistic properties such as in guinea pig, rat and mouse pancreatic acinar cells and hog duodenum. These observations suggest the existence of CCK receptor sub-types in peripheral tissues.     

Mutual Induction of TGFβ1 and NGF after Treatment with NGF or TGFβ1 in Grafted Chromaffin Cells of the Adrenal Medulla

Chromaffin cells have been recognized for their ability to transform into sympathetic ganglion-like cells in response to nerve growth factor (NGF) or to stimulation of other neurotrophic factors. Transforming growth factor β (TGFβ) family members have been shown to potentiate the effect of different trophic factors. The aim of this study was to investigate if TGFβ may influence NGF-induced neuronal transformation and regulation of NGF, TGFβ1, and their receptors in the adult rat chromaffin tissue after grafting. Intraocular transplantation of adult chromaffin tissue was employed and grafts were treated with TGFβ1 and/or NGF. Graft survival time was 18 days after which the grafts were processed for TGFβ luciferase detection assay, NGF enzyme immunoassay, or in situ hybridization. In grafts stimulated with NGF, increased levels of TGFβ1 and TGFβ1 mRNA were detected. When grafts instead were treated with TGFβ1, enhanced levels of NGF protein were found. Furthermore, a positive mRNA signal corresponding to the transforming growth factor II receptor (TβRII) was found in the chromaffin cells of the normal adrenal medulla as well as after grafting. No increase of TβRII mRNA levels was detected after transplantation or after TGFβ1 treatment. Instead a reduction of TβRII mRNA expression was noted after NGF treatment. NGF stimulation of grafts increased the message for NGF receptors p75 and trkA in the chromaffin transplants. Grafts processed for evaluations of neurite outgrowth were allowed to survive for 28 days and were injected weekly with NGF and/or TGFβ1. NGF treatment resulted in a robust innervation of the host irides. TGFβ1 had no additive effect on nerve fiber formation when combined with NGF. Combined treatment of NGF and anti-TGFβ1 resulted in a significantly larger area of reinnervation. In conclusion, it was found that NGF and TGFβ1 may regulate the expression of each other's protein in adult chromaffin grafts. Furthermore, TβRII mRNA was present in the adult rat chromaffin cells and became downregulated as a result of NGF stimulation. Although no synergistic effects of TGFβ1 were found on NGF-induced neurite outgrowth, it was found that TGFβ1 and NGF signaling are closely linked in the chromaffin cells of the adrenal medulla.     

Nerve growth factor mRNA-containing cells are distributed within regions of cholinergic neurons in the rat basal forebrain

It has been proposed that nerve growth factor (NGF) provides critical trophic support for the cholinergic neurons of the basal forebrain and that it becomes available to these neurons by retrograde transport from distant forebrain targets. However, neurochemical studies have detected low levels of NGF mRNA within basal forebrain areas of normal and experimental animals, thus suggesting that some NGF synthesis may actually occur within the region of the responsive cholinergic cells. In the present study with in situ hybridization and immunohistochemical techniques, the distribution of cells containing NGF mRNA within basal forebrain was compared with the distribution of cholinergic perikarya. The localization of NGF mRNA was examined by using a ³⁵S-labeled RNA probe complementary to rat preproNGF mRNA and emulsion autoradiography. Hybridization of the NGF cRNA labeled a large number of cells within the anterior olfactory nucleus and the piriform cortex as well as neurons in a continuous zone spanning the lateral aspects of both the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus. In the latter regions, large autoradiographic grain clusters labeled relatively large Nissl-pale nuclei; it did not appear that glial cells were autoradiographically labeled. Comparison of adjacent tissue sections processed for in situ hybridization to NGF mRNA and immunohistochemical localization of choline acetyltransferase (ChAT) demonstrated overlapping fields of cRNA-labeled neurons and ChAT-immunoreactive perikarya in both the horizontal limb of the diagonal band and magnocellular preoptic regions. However, no hybridization of the cRNA probe was observed in other principal cholinergic regions including the medial septum, the vertical limb of the diagonal band, or the nucleus basalis of Meynert. These results provide evidence for the synthesis of NGF mRNA by neurons within select fields of NGF-responsive cholinergic cells and suggest that the generally accepted view of “distant” target-derived neurotrophic support should be reconsidered and broadened.     

Nerve growth factor increases the size of intracortical cholinergic transplants

The effects of continuous intracortical mouse Nerve Growth Factor on fetal rat basal forebrain transplants in denervated adult rat neocortex were investigated. Enzyme-linked immunoassay (ELISA) was used to measure the time course of endogenous NGF protein production in neocortex, hippocampus, and basal forebrain in a cohort of animals receiving unilateral ibotenic acid (IBO) lesions of the nucleus basalis magnocellularis (nBM). A second cohort of IBO-nBM lesioned animals received transplants of fetal basal forebrain followed by two to four weeks of continuous NGF or cytochrome-C infusion into the ipsilateral frontoparietal neocortex. To study the effects of abnormally high NGF doses on transplanted and host tissue, the cumulative dose of intracortical NGF was on the order of micrograms, compared with maximum picogram levels of neocortical NGF produced following IBO-nBM lesions. A four-fold increase in transplant size, and greater cell and fiber densities were observed in NGF-treated compared with NGF-untreated transplants. No adverse histological effects of long-term, high-dose NGF treatment were observed on transplanted basal forebrain or host neocortical tissue. These data indicate that cholinergic-rich mammalian brain tissue and intrinsic host tissue can be stimulated by high doses exogenous NGF without obvious deleterious effects.     

Characterization of antibodies to synthetic nerve growth factor (NGF) and proNGF peptides

Sequence data for the mature nerve growth factor (NGF) protein and its precursor are available from molecular cloning of the NGF gene in several species, including mice, humans, rats, and chickens. Hydrophilicity analysis of the predicted rat and chicken prepro-NGF was carried out to locate putative antigenic determinants. Eight peptides were selected and synthesized based on hydrophilicity profiles. Two peptides represent sequences in the rat (and mouse) pro-NGF, one peptide (our peptide P3) represents a highly conserved region of the mature NGF protein (identical in humans, mice, rats, and chickens), two peptides are specific for the mature chicken NGF, and the remaining three peptides are specific for the mature rat NGF (each with only one amino acid substitution compared with corresponding segments of the mouse NGF). For immunization, the peptides were conjugated to keyhold limpet hemocyanin and used to produce antisera in rabbits. After bleeding, peptide-specific antibodies were purified on affinity columns prepared by coupling each of the synthetic peptides. The different peptide antisera and affinity-purified antibodies then were characterized by enzyme-linked immunoassay (ELISA) and immunohistochemistry of the male mouse submandibular gland, a rich exocrine source of NGF. ELISA analysis showed that all peptide antisera bound two to four orders of magnitude better than normal rabbit serum to a coat of their proper peptide. The higher binding was retained by the purified peptide antibodies compared with normal rabbit immunoglobulin. Specific tests, in which one peptide antiserum was checked against different peptide coats in the ELISA, also showed two to four orders of magnitude higher binding of antibodies to the proper synthetic peptide. The peptide antibodies also were tested for their ability to bind to native mouse beta NGF coated to the immunoplates. Only antibodies raised to the conserved P3 peptide recognized native NGF to an extent similar to that obtained with polyclonal anti-NGF antibodies. Conversely, P3 was well recognized by several different NGF antisera. Immunohistochemically, both peptide antisera against the pro-NGF stained the perinuclear cytoplasm in the basal part of the cells of the granulated convoluted tubules in the mouse submandibular gland.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spectra of G ratio, myelin sheath thickness, and axon and fiber diameter in the guinea pig optic nerve

The spectra of fiber and axon diameter, myelin sheath thickness, fiber density, and g ratio of the optic nerve were analyzed for the strain-13 guinea pig, an animal extensively utilized in the investigation of experimental disorders of demyelination. Our detailed analytical study of the normal guinea pig optic nerve provides the basis for comparison to disease states and the morphology of other species. As in the rat, mouse, and chipmunk, fiber diameters in the guinea pig were unimodal, but dissimilar to the trimodal fiber spectra of the cat and primate. The predominance of medium-sized fibers (0.80-2.00 microns), common to most species, contributed to the larger mean fiber diameter (1.45 microns) of the guinea pig optic nerve, in which small fibers (0.50 microns or less) were infrequent and fibers larger than 5.00 microns in diameter, seen in the cat and primate, were absent. While myelin sheath thickness increased with axon diameter in the guinea pig, as in other species, a g ratio of 0.81 in the guinea pig was greater than in most mammals. Since conduction velocity is dependent on axon size, as well as myelin properties, the relatively larger mean axon diameter of the guinea pig optic nerve (1.18 microns) may compensate for the decrease in its myelination.     

Prolonged alkylcatechol-induced expression of c-jun proto-oncogene followed by elevation of NGF mRNA in cultured astroglial cells

We have already shown that alkylcatechol markedly enhances synthesis/secretion of nerve growth factor (NGF) in cultured mouse fibroblasts and astroglial cells through immediate accumulation of NGF mRNA and that the stimulatory effect of alkylcatechol on NGF synthesis/secretion is synergistically enhanced by the coadministration of phorbol 12-myristate 13-acetate (PMA). The stimulatory effect on NGF mRNA expression of astroglial cells in culture by 4-methylcatechol (MC), an alkylcatechol, and/or PMA was blocked by treatment of the cells with cycloheximide, suggesting de novo synthesis of some cellular protein(s) is essential for the observed increase in the NGF mRNA level. The exposure to MC and/or PMA caused a rapid increase in c-fos mRNA content, which was immediately followed by an increase in c-jun mRNA, prior to NGF mRNA elevation. The expression of c-fos mRNA was transiently enhanced in all cases of the treatment with MC and/or PMA. The c-jun mRNA expression was also observed transiently when the cells were treated with PMA alone, while the expression of c-jun mRNA was pronounced and long-lasting after the treatment with MC, which was much further enhanced by the coadministration of PMA. The result that the profile of the change in c-jun mRNA expression resembled that in NGF mRNA expression suggests that the increase in c-jun mRNA is responsible for the subsequent increase in NGF mRNA after MC treatment. The cotransfection of mouse astroglial cells with expression plasmids of c-fos and/or c-jun and NGF promoter gene showed that simultaneous expression of both c-fos and c-jun genes was necessary to enhance NGF promoter activity. These results suggest that alkylcatechol induces NGF mRNA by means of transient induction of c-fos mRNA and long-lasting induction of c-jun mRNA. © 1994 Wiley-Liss, Inc.     

Glucocorticoids modulate the NGF mRNA response in the rat hippocampus after traumatic brain injury

Nerve growth factor (NGF) expression in the rat hippocampus is increased after experimental traumatic brain injury (TBI) and is neuroprotective. Glucocorticoids are regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the expression of NGF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury and in situ hybridisation to evaluate the expression of NGF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomised rats (with or without CORT replacement). TBI increased expression of NGF mRNA in sham-ADX rats, but not in ADX rats. Furthermore, CORT replacement in ADX rats restored the increase in NGF mRNA induced by TBI. These findings suggest that glucocorticoids have an important role in the induction of hippocampal NGF mRNA after TBI.     

Differential regulation of the nerve growth factor and brain-derived neurotrophic factor genes in L929 mouse fibroblasts.

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are structurally related survival and differentiation factors for distinct sets of peripheral and central neurons. The regulation of NGF gene expression has been extensively studied in L929 mouse fibroblasts. L929 cells also express the BDNF gene. Northern blot hybridization analysis revealed 4 discrete BDNF mRNA species in L929 cells and rat hippocampus after induction of seizures with kainic acid. Serum as well as 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated NGF and all 4 BDNF mRNAs in L929 cells. Treatment with both agents induced NGF mRNA to a much larger extent than the BDNF mRNAs. The induction of the BDNF mRNAs was rapid, with nearly maximal levels by 1 hr. In contrast, NGF mRNA induction occurred later and peaked at 4-6 hr. Both NGF and BDNF mRNA induction were inhibited by actinomycin D. Cycloheximide, on the other hand, inhibited only NGF but not BDNF mRNA induction. Corticosterone rapidly decreased NGF mRNA but not the BDNF mRNAs, and had no effect on seizure-induced NGF or BDNF mRNAs. Forskolin did not stimulate NGF or BDNF mRNAs. In contrast to NGF mRNA, forskolin did not interfere with the serum induction of BDNF mRNAs. These results demonstrate that 2 genes which encode closely related neurotrophic factors are differentially regulated in L929 cells. The molecular mechanisms which bring about this differential regulation remain to be elucidated.     

Inflammation causes expression of NGF in epithelial cells of the rat colon

Nerve growth factor (NGF) is a neurotrophin implicated in intestinal pathophysiology, such as impaired barrier function, altered motility and a lowered threshold to noxious stimuli in colitis. We evaluated the cellular source of NGF and determined the effect of inflammation on its expression in TNBS-induced colitis in the rat. Receptors for NGF were studied by immunocytochemistry, showing that submucosal neurons expressed both trkA and p75(NTR). NGF presence and activity was assessed by bioassay, ELISA, western blotting and immunocytochemistry. Bioassay of colonic mucosa using the PC12 cell line showed low levels in control tissue but a marked increase in NGF activity with inflammation. Western blotting showed the appearance of 13 kDa NGF in inflamed mucosa by 6 h, declining over time to become similar to control by 35 days. Semi-quantitative PCR showed minimal mRNA for NGF in control mucosa that increased sharply by 6 h post-TNBS. Laser-capture microdissection was used to collect colonic epithelial cells, where mRNA for NGF was markedly increased by 6 h post-TNBS. While the epithelium of the inflamed colon was positive for NGF by immunocytochemistry, other cell types remained negative. A potential precursor form of NGF, but not 13 kDa NGF itself, was detected in several epithelial cell lines and a mucosal mast cell line. We conclude that NGF is principally synthesized by epithelial cells in the inflamed colon, where the presence of specific receptors suggests the potential for wide-spread action.     

Long-Term Estrogen Replacement Coordinately Decreases trkA and β-PPT mRNA Levels in Dorsal Root Ganglion Neurons

A population of adult dorsal root ganglion (DRG) neurons bind NGF with high affinity and express the trkA gene. In these cells, NGF regulates gene expression and function. Recently, a number of laboratories reported the presence of estrogen receptors in DRG neurons and profound effects of estrogen on DRG gene expression. Our laboratory, for example, has reported a significant and coordinate decrease in DRG trkA and beta-preprotachykinin (beta-PPT) mRNA levels following 90 days of daily estrogen injections to ovariectomized (OVX) rats. These data suggest, as has been suggested for medial septal cholinergic neurons, that estrogen may collaborate with NGF in the regulation of DRG neuronal gene expression and function. The current study examined further this potential collaboration in the DRG by determining the effect of short-term estrogen replacement in OVX rats on DRG trkA mRNA levels following sciatic nerve transection and the resulting removal of a vital source of NGF for those cells. In OVX rats, about 40% of lumbar DRG neurons contained trkA mRNA. Short-term estrogen replacement had no effect on the percentage of neurons containing trkA mRNA, but increased the mean trkA mRNA level in uninjured DRGs of OVX rats by 23%. Axotomy in OVX rats reduced the mean trkA mRNA level by 55% but did not significantly decrease the percentage of neurons containing the mRNA. Estrogen replacement, 7 days after axotomy, partially and significantly restored the mean trkA mRNA level. It was 49% greater than that of the untreated axotomized DRGs. It did not, however, significantly increase the percentage of DRG neurons containing trkA in axotomized DRGs. These observations show that short-term estrogen has an opposite effect on DRG neuronal trkA mRNA levels as compared to that of long-term estrogen demonstrated in our previous study. Moreover, the current data show that estrogen regulates trkA mRNA levels in the absence of target-derived NGF. These data suggest that estrogen may collaborate with NGF in the maintenance of normal adult DRG gene expression and function. Furthermore, these data suggest that loss of estrogen, such as that associated with menopause, may contribute to a decline in DRG neuronal function and an exacerbation of ongoing neuropathic processes.     

In situ hybridization detection of trka mRNA in brain: Distribution,colocalization with p75NGFR and up-regulation by nerve growth factor

In situ hybridization techniques were used to examine the distribution and the nerve growth factor (NGF) regulation of trkA mRNA in the adult rat brain in order to identify neurons in discrete regions of the brain that may be NGF responsive. In agreement with previous studies, trkA mRNA was detected within cells located in the medial septum (MS), diagonal band of Broca (DBB), and caudate. trkA mRNA was also detected in many other regions of the brain, including the nucleus basalis of Meynert, substantia innominata, paraventricular nucleus of the thalamus, interpeduncular nucleus, prepositus hypoglossal nucleus, vestibular nudei raphe obscuris, cochlear nucleus, sensory trigeminal nuclei, and gigantocellular as well as perigigantocellular neurons in the medullary reticular formation. By combining in situ hybridization detection of trkA mRNA with immunocytochemical detection ofp75^NGFR, it was determined that the vast majority (> 90%) of the trkA mRNA-containing cells detected in the MS and DBB also express p75^NGFR. Likewise, the vast majority of p75^NGFR-IR cells detected in the MS and DBB expressed trkA mRNA. Intracerebroventricular infusions of NGF into the third ventricle adjacent to the preoptic area resulted in a 58% increase in relative cellular levels of trkA mRNA in the horizontal limb of the DBB. These data provide evidence that both p75^NGFR and trkA are expressed by NGF-responsive neurons in the MS and DBB. In addition, we note that areas that contained trkA mRNA and that also have been reported to contain p75^NGFR are areas where high-affinity NGF binding sites have been observed autoradio-graphically, whereas areas that contain either trkA or p75^NGFR alone are areas where no high-affinity NGF binding has been reported. Together, these findings suggest that both trkA and p75^NGFR play an important role in the formation of high-affinity NGF receptors in brain and, furthermore, suggest that NGF may have physiological effects within many regions of the brain outside of the basal forebrain.     

Expression of the nerve growth factor gene is controlled by the microtubule network

Colchicine, nocodazol, and vinblastine, three microtubule-disrupting drugs were shown to increase the levels of both nerve growth factor (NGF) mRNA and cell-secreted NGF protein in L929 cells, with levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or amyloid precursor protein (APP) mRNAs remaining unaffected. Northern blot analysis demonstrated that colchicine also increased NGF mRNA levels in rat primary astrocytes and mouse skin fibroblasts. The specificity of the effects observed was assessed by the fact that the microtubulestabilizing agent Taxotere®, a semisynthetic compound structurally related to taxol, suppressed the effects of colchicine, whereas lumicolchicine, a colchicine derivative that has no action on the microtubule network, had no influence on NGF expression. Likewise, the disruption of the microfilament network by cytochalasin B did not increase NGF mRNA levels in L929 cells. Furthermore, the increase in NGF gene expression observed following microtubule disruption depended on a cascade of events involving at least one protein kinase, which is not down-regulated by phorbol ester, and on a pertussis toxin sensitive step. These results support the concept that tubulin and/or the microtubule cytoskeleton play an active role in the regulation of the NGF gene. © 1995 Wiley-Liss, Inc.