In situ hybridization with complementary DNA probes for nerve growth factor (NGF) was used to identify cells containing NGF messenger RNA in rat and mouse brain. The most intense labeling occurred in hippocampus, where hybridizing neurons were found in the dentate gyrus and the pyramidal cell layer. The neuronal identity of NGF mRNA-containing cells was further assessed by a loss of NGF-hybridizing mRNA in hippocampal areas where neurons had been destroyed by kainic acid or colchicine. RNA blot analysis also revealed a considerable decrease in the level of NGF mRNA in rat dentate gyrus after a lesion was produced by colchicine. This lesion also caused a decrease in the level of Thy-1 mRNA and an increase in the level of glial fibrillary acidic protein mRNA. Neuronal death was thus associated with the disappearance of NGF mRNA. These results suggest a synthesis of NGF by neurons in the brain and imply that, in hippocampus, NGF influences NGF-sensitive neurons through neuron-to-neuron interactions.

In situ hybridization using a-nerve growth factor (NGF) DNA probes was used to demonstrate NGF mRNA in spermatocytes and early spermatids of adult mouse. NGF mRNA-containing cells were also identified in the epithelium of convoluted ducts in mouse corpus epididymidis. Blot-hybridization analysis of RNA prepared from mouse testis and epididymis as well as from rat epididymis confirmed the presence of a 1.3-kilobase (kb) NGF mRNA in these tissues. In the rat testis, however, only a 1.5-kb NGF mRNA was found, corresponding in size to a minor NGF mRNA detected in the rat brain, heart, and epididymis. By using affinity-purified anti-NGF antibodies, NGF-like immunoreactivity was observed in germ cells of rat and mouse testis and in the lumen of epididymis. Extracts of both mouse epididymis and testis stimulated fiber outgrowth in cultured sympathetic ganglia, and the effect was blocked by antibodies to mouse NGF. A two-site enzyme immunoassay showed the presence of 10 and 70 ng of NGF per g of tissue in the mouse testis and epididymis, respectively. Furthermore, RNA blot analysis showed the presence of mRNA for the NGF receptor in mouse testis. These results suggest a nonneurotrophic role for NGF in the male reproductive system, possibly in survival maturation and/or motility of spermatozoa.P-Nerve growth factor (NGF) is a protein essential for the development and maintenance of sensory and sympathetic neurons in the peripheral nervous system (1, 2). Recently RNA blot analysis, enzyme-linked immunoassays, and immunohistoche'mistry have been used to demonstrate NGF mRNA and protein in the mammalian brain (3-6), where NGF has been suggested to support basal forebrain cholinergic neurons (7-10). The levels of NGF mRNA in most peripheral tissues as well as in the brain appear to correlate with their degree of innervation by NGF-sensitive fibers either sympathetic in the periphery or cholinergic in the central nervous system (3-6, 11, 12). In the male mouse submandibular gland, which constitutes an extremely rich source of NGF mRNA and protein, the level of NGF synthesis does not correlate, however, with the innervation by NGF-sensitive fibers (1). Also, relatively high levels of NGF protein have been found in the snake venom gland (1), in guinea pig and rabbit prostate (13-16), and in bull semen and seminal vesicle (17,18). Furthermore, in the mouse testis, NGF-like immunoreactivity was recently shown to be present in germ cells, and a possible role for NGF in sperm maturation or motility was hypothesized (19).In the study reported here, we used a mouse NGF cDNA probe for in situ hybridization and RNA blot analysis to reveal the identity and distribution of cells synthesizing NGF in rodent male genital organs. The presence of NGF protein in the same tissues was studied by immunohistochemistry, immunoassays, and biological tests of activity. Our results show that male mouse germ cells synthesize both NGF mRNA and protein. In addition, NGF mRNA-containing cells were found in the epithelium of convoluted ducts of cor...

Substances which regulate expression of nerve growth factor (NGF) were examined in embryonic rat hippocampal cultures containing both neurons and glial cells. Both cell types expressed NGF mRNA when cultivated in vitro. Lipopolysaccharide, an activator of macrophages, elicited a significant increase in NGF mRNA. Interleukin-1 beta evoked a similar increase in NGF mRNA which was accompanied by a rise in NGF protein. The Il-1-induced increase was partially blocked by indomethacin, suggesting that prostaglandins might mediate this effect. Treatment of the cultures directly with prostaglandin E2 resulted in elevated levels of both NGF mRNA and protein. Thus, agents which promote inflammatory activity appear to increase NGF expression. Moreover, a suppressor of inflammation, dexamethasone, decreased NGF expression. Our observations indicate that a variety of immunomodulators regulate NGF expression in the hippocampus.

A cDNA clone encoding rat basic fibroblast growth factor (bFGF) was used as a hybridization probe to determine the level of bFGF mRNA during rat brain development as well as in different adult rat brain regions. In the rat brain, a 3.7kb bFGF mRNA was detected together with lower levels of two minor bFGF mRNA species of 1.8kb and 1.5kb, respectively. The 3.7kb bFGF mRNA was detected in the rat brain already at embryonic day 16, the earliest time point tested. The embryonic brain contained 1.5 to 2 times higher levels of the 3.7kb bFGF mRNA than the adult brain. The amount of the 3.7kb bFGF mRNA in the adult rat brain was approximately 50 times higher than the level of beta-nerve growth factor mRNA in the rat brain. bFGF mRNA was found in all 12 brain regions tested in the adult rat brain with the highest level in colliculi, cerebral cortex, thalamus, and olfactory bulb. The lowest levels were found in pons and medulla oblongata. All three bFGF mRNA species showed the same regional distribution in the brain. In contrast to nerve growth factor mRNA, the level of bFGF mRNA in the neonatal hippocampus was slightly decreased 10 days after a cholinergic denervation by transection of the fimbria-fornix.

A series of polyclonal affinity-purified antibodies against mouse submandibular-gland nerve growth factor (NGF) are described. Using the submandibular gland of the male mouse and indirect immunofluorescence, the specificity and sensitivity of affinity-purified immunoglobulins and various other fractions from the immunized animals have been tested. It will be shown that affinity-purification schemes, including pre-purification of protein A-fractionated immunoglobulins to remove antibodies that bind to unrelated hydrophilic and hydrophobic proteins, significantly enhance the signal-to-noise ratio and specificity of the antibodies. The antibodies effectively detect NGF-like immunoreactivity in both fresh and fixed glandular tissue. Optimal fixation procedures are described. Fluorescence intensities are linearly correlated to log antibody concentration. By use of the best antibody fractions and optimal fixation protocols, the distribution of NGF-like immunoreactivity is described in eight different salivary glands (rat and mouse, male and female, submandibular and sublingual glands). In addition to the well-known large numbers of immunoreactive cells in the submandibular gland of the male mouse, immunoreactive cells were found in the sublingual gland of male mice and in the submandibular and sublingual glands of female mice. One antibody revealed a weak specific fluorescence also in the submandibular gland of the male mouse. In a survey of genital organs of male mice, one antibody revealed fluorescence in the germ cell line. We conclude that several polyclonal affinity-purified antibodies have been characterized that show a strong NGF-dependent binding to the secretory granules of tubular cells in the submandibular gland of male mice. These antibodies should make it possible to locate endogenous and perturbed NGF levels immunocytochemically, e.g., in the peripheral and central nervous system, where NGF concentrations may be several orders of magnitude lower than in the salivary glands.