An Lmmunohistochemical Study of the Nerve Growth Factor Receptor in Developing Rats
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The Journal of Neuroscience, September 1988, 8(9): 34813498 An lmmunohistochemical Study of the Nerve Growth Factor Receptor in Developing Rats Qiao Yan and Eugene M. Johnson, Jr. Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 Nerve growth factor (NGF) receptor expression was studied Nerve growth factor (NGF) is essentialfor the development and in rats between embryonic day 11 (El 1) to postnatal day maintenance of the function of peripheral sympathetic neurons 10 (PNDlO) by using a monoclonal antibody, 1924gG, that and neural crest-derived sensoryneurons (Levi-Montalcini and specifically recognizes rat NGF receptor. Sympathetic gan- Angeletti, 1968; Johnsonet al., 1980). Recent experiments show glia were lightly stained by 1924gG for NGF receptor im- that NGF also haseffects on central cholinergic neuronsin basal munoreactivity (NGFRI) (E 11 -PNDlO). Neural crest-derived forebrain (Gnahn et al., 1983)and striatum (Mobley et al., 1985). sensory ganglia were moderately to densely stained (El l- Although the precisebiochemical mechanism(s)by which the PNDl 0). Areas in CNS innervated by the central processes effects of NGF are mediated is unknown, NGF binding with of these ganglia were also stained. Parasympathetic ciliary the surfacereceptor on NGF-responsive cellsis an essentialstep ganglion showed some detectable staining (E16-PND6). for NGF to produce its biological effects.The expressionofNGF Placode-derived sensory ganglia were stained more densely receptor is regarded as a prerequisite for a tissue to be NGF than that of neural crest-derived sensory ganglia. The most responsive.It has been shown that the expressionof NGF bind- densely stained tissue for NGFRI was found in all peripheral ing sites is developmentally regulated in a broad range of tis- nerves. Basal forebrain cholinergic neurons were NGFRI suesin chicken embryo by autoradiographic studies(Raivich et positive from El5 throughout the period examined. Moto- al., 1985, 1987) and in rats by a quantitative immunoprecipi- neurons in both spinal cord and brain stem were positive for tation assay (Yan and Johnson, 1987). It is clear from these NGFRI between E 15 and PND 10. NGFRI staining was seen studies that NGF receptor is expressedvery early in develop- in a variety of sensory pathways and related structures, such ment. In most tissuesthe NGF receptor level is decreasedduring as olfactory tract and glomerular layer of olfactory bulb; ret- development (Yan and Johnson, 1987). The studies in chick ina, optic nerve and tract, lateral geniculate nucleus, medial embryo (Raivich et al., 1985, 1987) show a wide distribution terminal nucleus of the accessory optic tract, and olivary of specific NGF binding sites, but so far there has been no pretectal nucleus; ventral cochlear nucleus and to a lesser systematicmorphological study of NGF receptor in mammalian degree in dorsal cochlear nucleus, superior olive, and nu- development. The temporal changeof NGF dependenceof em- cleus of lateral lemniscus; solitary tract; cuneate nucleus, bryonic avian dorsal root ganglion (DRG) is somewhatdisparate gracile nucleus, and ventroposterior thalamic nucleus. The from that of mammalian DRG (Herrup and Shooter, 1975; specific staining was also found in some other CNS struc- Stockel et al., 1975; Barde et al., 1980; Johnson et al., 1980) tures, including brain-stem reticular formation; amygdala; which suggestedthat the expression of NGF receptor in these medial nucleus of inferior olive but not the rest of inferior different speciesmight be different. Here we useda specific anti- olive, external granule cell layer and Purkinje’s cells of cer- rat NGF receptor monoclonal antibody, 192-IgG (Chandler et ebellum, and deep cerebellar nuclei. Some non-neuronal tis- al., 1984; Taniuchi and Johnson, 1985), for immunohistochem- sues such as meninges and dental tissue showed very dis- ical study to localize NGF receptor in developing rats. The NGF tinctive staining. Limb buds and somites were NGFRI positive receptor immunoreactivities (NGFRI) were found in all the starting at El 1, and the staining on muscle tissue became classicalNGF targets such as peripheral sympathetic and sen- very dense at El 5-El6 and largely disappeared around sory neurons, aswell asbasal forebrain choline& neurons.The PNDlO. Embryonic thymus was positive for NGFRI. The ad- data also show many other, unexpected NGF receptor-immu- ventitia surrounding blood vessels was very densely stained. nopositive sites. The changes in NGFRI staining seen in this study suggest that NGF may have broader effects during development than Materials and Methods previously thought. Materials. Mouse anti-rat NGF receptormonoclonal antibody (192- IgG) (Chandleret al., 1984)was affinity-purified as previously described (Yan and Johnson,1987). Anti-mouse antibody ABC-peroxidasekits Received Nov. 5, 1987; accepted Jan. 12, 1988. werepurchased from Vector Lab (Burlingame,CA). NalZSI(100 mCi/ We wish to thank Dr. Thomas A. Woolsey for the use of his laboratory equip- ml) waspurchased from Amersham(Chicago). Other chemicalswere ment and helpful discussion of results. We also wish to thank Drs. Lawrence F. Kramer, Joseph L. Price, David Menton, and Barbara Bohne for valuable dis- obtainedfrom Sigma(St. Louis). cussion of results and Mr. David Martin, Ms. Pat Osborne, and Dr. Ian Ferguson Animals and tissue sectioning. Sprague-Dawleyrats (ChappelBreed- for reading the manuscript, and Jenny Colombo for general laboratory assistance. ers,St. Louis)were used. Vaginal plug positive is recordedas embryonic This work was supported by NIH Grants NS 1807 1 and NS24679. day zero (EO).Parturition is betweenE21 to E22, taken as postnatal Correspondence should be addressed to Eugene M. Johnson, Jr., Ph.D., De- day zero (PNDO).Timed-pregnant rats were killed by decapitationunder partment of Pharmacology, Box 8 103, Washington University School ofMedicine, light ether anesthesia,and embryoswere quickly removedfrom the 660 South Euclid Avenue, St. Louis, MO 63 110. uterus.Embryos younger than E 18were immersion-fixed. El 8 andolder Copyright 0 1988 Society for Neuroscience 0270-6474/88/093481-18$02.00/O animalswere perfused through the left ventricle of the heartwith PBS, The Journal of Neuroscience, September 1988, 8(9) 3483 pH 7.4, followed by fixative. Both 1 and 4% paraformaldehyde in 0.1 crest-derived sensoryganglia, NGFRI was found in the trigem- M sodium phosphate buffer, pH 7.4, were used in initial experiments. inal ganglion (Fig. lA), rostra1DRG, and potential caudal DRG The immunoreactivity was decreased only slightly at the higher con- centration. Most data in this report came from 4% paraformaldehyde- region (lateral to spinal cord) as early as El 1 (Fig. 2, A and B). fixed tissues. Tissues were postfixed overnight (prolonged fixation would At this stage, some caudal DRG were not discrete structures greatly decrease the immunoreactivity), equilibrated with 30% sucrose, and might still have been in the processof DRG formation and and frozen in an isobutane/dry ice bath. Fourteen- to twenty-micron neural crest cell migration (Fig. 2B); this suggeststhat the mi- sections were cut with a cryostat (-2o”C), mounted on chrome-alum- coated slides, and air-dried. grating neural crest cells might be NGFRI positive. At E 13 all Immunohistochemistrywith 192-IgG. The indirect immunohisto- the DRG were distinct structures that showed relatively low chemistry was carried out by using the avidin-biotin-peroxidase com- NGFRI staining levels (Fig. 2C). The staining density in DRG plex (ABC) method (Vectastain. Vector Laboratories) as described be- increasedand becamequite densebetween El 8 to PNDO (Figs. low. After’ preincubation with 0.003% hydrogen peroxide in absolute 2E, 5D). The staining ofjugular ganglion (Fig. 5C), also a neural methanol for 1 hr at room temperature (RT) to inhibit endogenous peroxidase, sections were incubated overnight at 4°C with 2 r&ml of crest-derived sensory ganglion, was basically the same as that 192-IgG or any of several control antibodies in PBS/IO% horse serum, of DRG. Trigeminal ganglia showeda similar, but slightly more pH 7.4. The sections were washed 3 times in PBS, 10 min each at RT intense, temporal pattern of staining. The staining density in all (same wash procedure was performed between each of the following theseneural crest-derived sensoryganglia was reduced by PND6 steps), and incubated overnight at 4°C with biotinylated secondary anti- mouse antibodies, dilution 1:300 in PBS/IO% horse serum, pH 7.4. The and further declined in PND 10 DRG (Table i), consistentwith sections were incubated for 3 hr at RT with gentle agitation with a our previous quantitative biochemical data (Yan and Johnson, solution containing a preformed avidin/biotin-peroxidase complex, at 1987). a dilution of 1:250 in PBS, pH 7.4. The sections were then incubated Placode-derived sensoryganglia showeda surprisingly intense with 3,3’-diaminobenzidine tetrahydrochloride (DAB) reaction solution NGFRI stainingwith 192-IgG (Table 1). Otic vesiclewas NGFRI (0.05% DAB/O.0 17% cobalt chloride/O.01 36% nickel ammonium sul- fate/0.1 M phosphate buffer, pH 7.6) for 10 min, and then 0.0066% positive at El I-E13. The geniculate ganglion of seventh nerve, hydrogen peroxide was added to the DAB solution for another 5-10 the spiral cochlear ganglion and vestibular ganglion of eighth min. The reaction took place at RT with agitation. The sections were nerve, and nodoseganglion (Fig. 1D) of tenth nerve were mod- then dehydrated and coverslips applied. erately stained at El 5-E16. The staining became very intense Quantitativeimmunoprecipitation assay for NGFreceptor.Mouse NGF (2.5s) was purified from male mouse submaxillary glands by the method between El8 to PND6 (Figs. 1, B, C, E; 5, B, C, and Table 1). of Bocchini and Angeletti (1969) and iodinated (Marchalonis, 1969) to The density of staining in placode-derived sensoryganglia was obtain a specific activity of 2200 cpm/fmol. NGF receptors in SCG and somewhatgreater than that in neural crest-derived sensorygan- nodose ganglia were quantitated in duplicates of tissue homogenates by glia and much higher than that in sympathetic ganglia.