Uptake and transepithelial transport of nerve growth factor in suckling rat ileum.

Nerve growth factor (NGF) is necessary for the development of sympathetic and some sensory neurons. Milk may be a source of NGF for suckling young, but sites of intestinal absorption of the protein have not been identified. To determine whether NGF is transported across the absorptive epithelium of suckling rat ileum, we assessed binding, uptake, and transport of 125I-NGF by light microscopy and EM autoradiography. Blood and tissue extracts were analyzed by biochemical and immunological methods to determine whether NGF was taken up structurally intact. NGF binding sites were identified on microvilli and apical invaginations of ileal absorptive cells in vitro. Injected into ileal loops in vivo, NGF radioactivity retained by fixation was evident after 20 min in apical regions of absorptive cells, in endocytic tubules (which mediate the uptake of membrane-bound ligands), in vesicles (which mediate nonspecific endocytosis), and in the supranuclear lysosomal vacuole. At 1 and 2 h, radiolabel in these compartments increased and silver grains were evident at the basal cell surface, and in cells, matrix, and vessels of the lamina propria. In blood and liver, radiolabeled molecules that were immunologically and electrophoretically indistinguishable from NGF and that co-eluted with NGF on gel filtration columns were detected, confirming that some NGF was transported across the epithelium structurally intact. Thus, absorptive cells of suckling rat ileum can take up NGF by both receptor-mediated and nonspecific endocytosis, and direct NGF either to the lysosome for degradation, or into a transepithelial transport pathway.

Nerve growth factor and epidermal growth factor in mouse submandibular glands: identical diurnal changes and rates of secretagogue-induced synthesis.

The granular convoluted tubule cells of the mouse submandibular gland contain high concentrations of nerve growth factor (NGF) and epidermal growth factor (EGF). Levels of the two growth factors increase in parallel during development and after stimulation by thyroid hormone, androgens, and glucocorticoids, suggesting that similar controls regulate the production of both proteins. At the same time, however, the granular convoluted tubules hypertrophy, with resulting increases in glandular size and protein content. In this study, we have monitored changes in salivary concentrations of NGF and EGF in the absence of detectable changes in submandibular size or protein concentration. Both growth factors exhibited identical diurnal changes, peaking between 1200-2000 h. In addition, after secretagogue-stimulated release of the proteins into saliva, reaccumulation began after 2 h, with normal levels of both molecules attained 6 h posttreatment. These data indicate that submandibular NGF and EGF can be controlled by similar cellular or molecular mechanisms independent of the regulation of generalized growth responses.

Steroids and triiodothyronine reduce nerve growth factor concentrations in medium conditioned by L-929 fibroblasts.

Thyroid hormone, testosterone, and glucocorticoids stimulate the production of nerve growth factor (NGF) in mouse submandibular glands, but little is known about the influences of these hormones on other sources of NGF. In this study we tested the effects of thyroid hormone and steroids on NGF levels in medium conditioned by L-929 fibroblasts (L-cells), a mouse cell line that secretes NGF in culture. NGF concentrations in conditioned medium were measured by RIA and were normalized for cell number. In the absence of hormones, NGF levels in medium averaged 5.57 ng/ml. T3, cortisone, and aldosterone decreased NGF levels below the sensitivity of the RIA (less than 0.37 ng/ml), with respective ID50 values of 1 X 10(-5), 1 X 10(-8), and 5 X 10(-8) M. Testosterone decreased NGF levels by 66%, with an ID50 of 9 X 10(-9) M, and 17 beta-estradiol decreased it by up to 50%, although the hormone's effects had not plateaued at 10(-6) M. NGF concentrations were unaffected by progesterone at doses as high as 10(-6) M. Cell viability was unaffected by hormone treatment, and the total amount of secreted protein in medium decreased only in cultures treated with high doses of cortisone and aldosterone. Qualitatively similar results were obtained by bioassay; steroids markedly reduced, but did not eliminate, the nerve growth-promoting effects of L-cells on embryonic chick dorsal root ganglia in cocultures. These data indicate that hormones that stimulate NGF production in salivary glands inhibit NGF production by L-cells in culture.

Effects of postnatal anti-nerve growth factor serum exposure on development of apical nerves of the rat molar.

The present study was undertaken to test the hypothesis that development of the pulpal innervation is dependent on nerve growth factor (NGF). Newborn rats were given subcutaneous injections of a rabbit anti-mouse NGF serum on alternate days for the first 24 days postnatally. Control animals were untreated and normal rabbit serum-treated litter mates. The animals were deeply anesthetized on postnatal day 26, perfused with fixative and the first mandibular molars were processed for transmission electron microscopy to obtain a complete census of axons entering the four roots. The composition of the mental nerve was also examined. Compared to control animals, the apical innervation of molars from anti-NGF-treated rats had only 62% as many myelinated fibers and 41% as many unmyelinated axons. Those myelinated fibers present in antiserum-treated animals were slightly, but significantly, smaller in average diameter than controls. In teeth of control animals, about 20% of all unmyelinated axons were located in fibers coursing outside of nerve fascicles; these isolated fibers were disproportionately rare after antiserum exposure. The average number of unmyelinated axons per Schwann cell unit was also significantly lower. Postnatal exposure to anti-NGF had milder effects on mental nerve composition compared to the tooth innervation. Numbers of myelinated fibers were 83% of controls, unmyelinated axons were 74% of controls and there was no change in the average number of unmyelinated axons per Schwann cell unit. We conclude that development of dental innervation is highly susceptible to postnatal NGF deprivation. This may be a consequence of the mostly nociceptive composition of dental nerves and their late development.

Ultrastructural changes in the masseter muscle of Macaca fascicularis resulting from intramuscular injections of botulinum toxin type A.

Intramuscular injections of botulinum toxin type A (Oculinum) is used to treat strabismus and focal dystonias affecting orofacial muscles. However, the toxin-induced morphological changes that underlie the therapeutic alterations of tone in the muscles of mastication have not been described. In this study, paired intramuscular injections of botulinum toxin (10 units) were made in three adult monkeys (Macaca fascicularis) allowed to survive 14, 28 and 63 days. Another monkey received multiple injection-pairs over 84 days. Animals were killed by deep pentobarbital anaesthesia before transcardiac perfusion-fixation. Tissue sampled from comparable regions of the injected masseter, the uninjected masseter and an uninjected animal was processed for ultrastructural analysis. Few changes were found 14 days post-injection. However, muscle fibres showed myofibrillar dissolution, aberrations in the Z-line, and enlarged mitochondria in the region of the I-band by 28 days. In the 63-day and 84-day animals, the injected muscle was considerably smaller than the uninjected, contralateral muscle. Regions of the injected muscle contained fibres with markedly reduced cross-sectional area. Internalization of myonuclei, loss of myofibrillar organization, and helical complexes were common. Toxin-induced changes, though similar to those that follow denervation by axotomy, were not accompanied by degeneration of neuromuscular junctions. Instead, morphological evidence for axonal sprouting in the region of the neuromuscular junction, possibly contributing to functional recovery, was seen as early as 14 days in toxin-treated muscles.

Quantitative study of the apical nerve fibers of adult and juvenile rat molars.

The rat molar has become an important model for studies of interactions between nerves and the pulp-dentin complex, yet there is only limited quantitative information on the number and size distribution of axons entering the roots of this tooth. This study was undertaken to provide such a detailed characterization of the apical innervation of the rat molar. An additional objective was to compare the apical nerve composition of young, recently erupted rat molars with that of mature teeth in order to determine whether there is ongoing maturation of the innervation after the teeth have attained functional occlusion. A complete census was made of the nerve fibers entering the roots of both mature and recently erupted juvenile mandibular first molars in Sprague-Dawley rats. Each of the four roots of the first molars was processed for electron microscopy of thin sections near the apex. The majority of intradental nerve fibers entered the molar via the two larger (mesial and distal) roots. Within the apical root pulp, most, but not all, axons occurred within well-defined fascicles associated with blood vessels. Molars from adult animals (age 4 months) had a mean total of 232 (S.D. = 49, N = 7 teeth) myelinated fibers and 806 (S.D. = 143) unmyelinated axons entering the four roots. Fibers exceeding the A delta size range (circumference > or = 19 microns) accounted for only 4% of the myelinated axons at the apex. Molars from juvenile animals (age 4 weeks) had fewer myelinated fibers (mean 176, S.D. 18, N = 8), but more unmyelinated axons (mean 1,174, S.D. 160) than adults. The mean ratio of unmyelinated axons to myelinated axons was 6.6:1 for juveniles compared to 3.5:1 for adults. Juvenile teeth contained no myelinated fibers that exceeded 19 microns in circumference. These results indicate that the innervation of the rat molar resembles that of teeth of non-rodent mammals in that (1) innervation density is high, (2) there is a high ratio of unmyelinated axons, and (3) most of the myelinated fibers are of thin caliber. Furthermore, it appears that after the molar erupts, maturation of the nerve fiber composition continues with processes that include both a marked decrease in the number of unmyelinated axons and an increase in the number and size heterogeneity of myelinated fibers.

Acetylcholinesterase in the developing ferret retina.

The function of acetylcholinesterase (AChE) is to terminate the action of acetylcholine at the cholinergic synapse. Recent evidence suggests additional roles for acetylcholinesterase as a peptidase and/or a protease which is expressed by growing neurites as part of their invasion of developing neural structures. We report the localization of acetylcholinesterase in developing ferret retina. AChE histochemical staining is seen in the developing inner plexiform layer (IPL) of ferret retina at birth (post-natal day zero, PO), the earliest developmental stage examined. Transient expression is seen at the border between the ganglion cell layer and the nerve fiber layer at P14 and P21. A small amount of transient expression is seen in the outer plexiform layer (OPL) at this age as well. By P28, the transient expression in the OPL is at its peak, and is found at photoreceptor terminals and associated with apparent horizontal cell axons. Labeling is also seen intracellularly in the inner nuclear layer (INL), at the OPL/INL border, suggesting that horizontal cells are the source of the transient AChE expression in the OPL. Overt synaptic profiles also appear in the inner plexiform layer (IPL) at P21 and P28. About 2 days layer, the eyes open and the photoreceptor outer segments are fully developed. By 2 weeks later, at P42, the AChE staining pattern in the retina has taken on its adult appearance: no reaction product in the outer retina; intracellular reaction product in the Golgi apparatus of a subset of amacrine and displaced amacrine cells which manufacture AChE; and extracellular reaction product at both synaptic and non-synaptic sites in the IPL. These data are consistent with a role for AChE as a peptidase early in development, and as an enzyme essential in the termination of synaptic action at mature synapses.

Course and composition of the nerves that supply the mandibular teeth of the rat.

The rodent dentition has become an important model for investigations of interactions between dental tissues and peripheral neurons. Although experimental nerve injury has been widely used for such studies, there is uncertainty about the courses of nerve fibers supplying the mandibular teeth. In order to clarify this, we used a mixture of monoclonal antibodies against neurofilament proteins to enhance demonstration of nerve fibers so that small nerves could be readily traced in serial frozen sections of mandibles of Sprague Dawley rats ranging in age from embryonic day (E) 18 to postnatal day (P) 90. The 1st molar and anterior portion of the 2nd molar were innervated by small nerves that emerged as distinct branches of the IAN trunk at or near the mandibular foramen. In contrast, the nerve supply to the 3rd molar and posterior part of the 2nd molar was a branch of the lingual nerve that bypassed the mandibular canal altogether. The IAN trunk split into the mental nerve and a large branch to the incisor about 2 mm anterior to the mandibular foramen. Thick branches of the incisor nerve descended into the incisor socket to form a dense plexus of nerve fiber bundles extending along the length of the incisor periodontium. The sparse pulpal innervation of the incisor was provided by a few thin fascicles that emerged from the caudal portion of the periodontal plexus to enter the incisor apex. The dental branches of the IAN and lingual nerve seen in the adult were well established and readily identifiable at age E18 even though their targets were limited to the follicles of the developing teeth. These studies show that the trigeminal branches that supply the mandibular teeth can be identified at a wide range of ages as distinct nerves at a considerable distance proximal to their targets. This detailed information on the courses taken by the dental nerves can provide an anatomical basis for increased precision in characterization and perturbation of neural pathways from the molars and incisor.

Absence of the alpha and gamma subunits of 7S nerve growth factor in denervated rodent iris: immunocytochemical studies.

Immunocytochemical studies were performed to determine if denervated rodent iris produces nerve growth factor (NGF) in a form chemically similar to that of the 7S NGF complex in mouse submandibular glands. Antisera to the alpha, beta, and gamma subunits of 7S NGF were raised in rabbits and characterized on immunoblots of SDS-containing polyacrylamide gels. Antisera were applied to stretch preparations of rat and mouse irides that were cultured for periods of 2 to 6 days or sympathetically denervated by superior cervical ganglionectomy and left in situ 4 days. Antibody binding was visualized by indirect immunofluorescence. In control studies done on plastic sections of mouse submandibular glands, antisera co-localized the three subunits of 7S NGF within secretory granules of granular tubule cells. In denervated rat iris, beta NGF immunoreactivity was evident in a cellular plexus that resembled in distribution and morphology nerve fibers in the normal iris, in agreement with a previous study (R.A. Rush (1984). Nature (London) 312, 364-367). Identical staining patterns were observed in mouse iris. In neither rat or mouse, however, did the nerve-like processes stain with antibodies suggests that the NGF-like protein in denervated rodent iris is not synthesized as part of the 7S NGF complex. Iris also did not react with antibodies to epidermal growth factor, a protein co-localized with NGF in mouse submandibular glands and in guinea pig prostate.

Transforming growth factor alpha inhibits secretion of gastric acid.

Transforming growth factor alpha (TGF-alpha), a protein secreted by transformed cells and related to epidermal growth factor (EGF), was tested for its effects on gastric acid secretion. Guinea pig gastric mucosae were mounted in Ussing chambers and the rate of acid release was monitored by the pH-stat method. When administered prior to the secretagogue, TGF-alpha prevented the histamine-induced increase in the rate of acid secretion. Similarly, TGF-alpha caused a decrease in the rate of acid release in tissues that had already been stimulated with histamine. These data show that TGF-alpha inhibits gastric acid secretion in a manner similar to EGF and that the two growth factors share at least one physiological action unrelated to their mitogenic properties.

Heterogeneity of channel catfish CTL with respect to target recognition and cytotoxic mechanisms employed.

Two types of catfish alloantigen-dependent cytotoxic T cells were cloned from PBL from a fish immunized in vivo and stimulated in vitro with the allogeneic B cell line 3B11. Because these are the first clonal cytotoxic T cell lines derived from an ectothermic vertebrate, studies were undertaken to characterize their recognition and cytotoxic mechanisms. The first type of CTL (group I) shows strict alloantigen specificity, i.e., they specifically kill and proliferate only in response to 3B11 cells. The second type (group II) shows broad allogeneic specificity, i.e., they kill and proliferate in response to several different allogeneic cells in addition to 3B11. "Cold" target-inhibition studies suggest that group II CTL recognize their targets via a single receptor, because the killing of one allotarget can be inhibited by a different allotarget. Both types of catfish CTL form conjugates with and kill targets by apoptosis. Killing by Ag-specific cytotoxic T cells (group I) was completely inhibited by treatment with EGTA or concanamycin A, and this killing is sensitive to PMSF inhibition, suggesting that killing was mediated exclusively by the secretory perforin/granzyme mechanism. In contrast, killing by the broadly specific T cytotoxic cells (group II) was only partially inhibited by either EGTA or concanamycin A, suggesting that these cells use a cytotoxic mechanism in addition to that involving perforin/granzyme. Consistent with the presumed use of a secretory pathway, both groups of CTL possess putative lytic granules. These results suggest that catfish CTL show heterogeneity with respect to target recognition and cytotoxic mechanisms.