Bra& Research, 591 (1992) 223-238 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

BRES 18093

Distribution and relative density of p75 nerve growth factor receptors in the rat brain as a function of age and treatment with antibodies to nerve growth factor B e t h a n y A. U r s c h e l ~ a n d C l a i r e E. H u l s e b o s c h


"Department of Biomedical Sciences, McMaster Unicersity, Hamilton, Ont. (Canada) and b Department of Anatomy and Neurosciences and the Marine Biomedical Institute, Unil'ersityof Texas Medical Branch, Gah'eston, TX (USA) (Accepted 21 April 1992)

Key words: Cerebellum:, Nucleus basalis; Cochlear nucleus; Vestibular nucleus; Spinal tract of V; Medial septum; Diagonal band of Broca

it is clear that nerve growth factor (NGF) has a role in the central nervous system, in order to begin to determine the possible roles of NGF in the CNS, neonatal rats were given daily subcutaneous injections of antibodies to NGF (ANTI-NGF) beginning at birth for a period of one month. By utilizing the monoclonal antibody, 192-1gG, which recognizes the p75 NGF receptor (NGFR), and standard immunohistochemical techniques we have localized p75 NGFR in variously aged ANTI-NGF-treated animals and compared the anatomic localization and relative density of the p75 NGFR immunoreactive (p75 NGFR-I) regions to same age untreated and preimmune sera-treated littermates. We confirm previously reported Iocalizations of p75 NGFR-i in the rat brain. In addition, we demonstrate that p75 NGFR-I levels of ANTI-NGF-treated rats found in the molecular, the granular and the Purkinje cell layers of the cerebellum, the vestibular nuclei, the spinal tract of V and the cochlear nuclei remain at lower concentrations compared to same-age control animals. We also demonstrate that p75 NGFR-! levels in the basal nucleus approaches background levels after ANTI-NGF treatment. We hypothesiz,: that ANTI-NGF biologically inactivates NGF, which over a period of 30 days results in decreased p75 NGFR-I. These results are consistent with neuronal loss in these regions following ANTI-NGF tr~:atment. Furthermore, the immunological methods used to produce the specific deficits in the present study may have broader implications with respect to usefulness as a method for determining the dependency of CNS neuronal populations for a putative neurotrophic factor and as a method for the development of models of neurodegenerative diseases.

INTRODUCTION Nerve growth factor (NGF) is one of a family of proteins known collectively as neurotrophins. Included ill this family of related factors is brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and possibly other neurotrophins, such as NT-4 and NT-5 '~'2~'39'4c~'5~'c'5'8°. However, NGF is the most well characterized of the neurotrophins in terms of neurotrophic (neuron survival) and neurotropic (neurite elongation) activities both in the peripheral nervous system 27'c'°'~a and in the central nervous system '~8. Neurons that are known to be responsive to NGF are sympathetic postganglionic neurons 34'5'J'6°'6t, a subpopulation of dorsal root ganglion neurons 44'6°'6t'74't°3 and some cholinergic neurons in the C N S 54'a2'~. For example, after fimbrial-fornix lesions cholinergic neurons in

the septal nucleus will die unless exogenous NGF is administered "~°,'~'~'~'~. The acceptance of the neurotrophic activity of NGF for the septal nucleus and other basal forebrain cholinergic neurons has received widespread interest, in part because of correlations with loss of basal forebrain neurons in AIzheimer's disease in man and with memory loss in animals 6.32.37,3~.5t.98.Additionally, NGF may function as a trophic factor for several other central nervous system neuronal populations "~7. The trophic effects of NGF are thought to be mediated through a process of NGF binding to a NGF receptor complex. The current thinking is that the NGF receptor complex is composed of two proteins: a transmembrane glycoprotein with low affinity for binding NGF, p75 NGF receptor (p75 NGFR) t°° and a transmembrane tyrosine kinase protein, the gene prod-

Correspondence to: C.E. Hulsebosch, Department of Anatomy and Neurosciences, H-43, University of Texas Medical Branch, 2{}{)University Blvd., Galveston, TX 77550-2772, USA. Fax: (1) (409) 762-9382.

224 uct of the trk-I proto-oncogene (p140), which in combination with p75 is thought to confer high-affinity binding for NGF 33"4~'4'~.The NGF binds to the extracellular domain of the combined receptor complex and this binding triggers tyrosine kinase activity with subsequent induction of other gene products and neurite outgrowth and/or neuronal survival 8s'~8. The p75 protein is hypothesized to be a common component for other neurotrophins, such as BDNF and NT3, and the specificity for NG['="may be conferred by the combination of the p75 protein with the trk-1 p140 prorein 33"4~'4'~''~()''~''e'4"~.Therefore, one way to determine if a neuronal population is potentially responsive to NGF is to determine which populations express p75 NGFR mRNA and protein. The p75 NGFR mRNA has been localized to various regions in the adult brain including the medulht, cerebellum and striatum H)JT. lmmunohistochemical and autoradiographic localization of p75 NGFR protein has been demonstrated in various sensory and motor regions of the developing and adult brain, including the medulla, pens, cerebellum and diencephalon ~,..~e,.~,~,~,.~.~s.7r,.7,. H,.,i2. One way to determine the neurotrophic importance of NGF to a neuronal population is to suppress endogenous levels of NGF biologically with antibodies to NGF (ANTI-NGF). By definition, if the neurotrophic factor of a particular neuronal population is not avail. able or is biologically inactivated, that population will dic, Since NGF responsive neurons must express p75 NGFR, the loss of p75 NGFR will indicate an alter. alien in p75 NGFR expression and may indicate neuronal death. By utilizing a monoclon:tl antibody (192. leG) which recognizes the p75 NGFR ~t and standard immunohistochemical techniques, we have localized p75 NGFR in animals who have received systemic injections of ANTI.NGF and compared the anatomic localization and relative density of the p75 NGFR immunoreactive regions at various ages to those observed in untreated and preimmune sera-treated littermates. MATERIALS AND M E T H O D S Pregnant Sprague-Dawlcy rats were obtained from Harhm Sprague-Dawley. All procedures involving rats were performed in compliance with the USDA Animal Welfare Act and amendments, Ihe r~zvised Guide fi)r the Care and Use of Laboratory Animals DHEW (NIH), and were approved by the UTMB Animal Care and Use Committee. Three groups of rats were examined: (I) rats treated wilh ANTI-NGF, (2) rats treated with preimmune sera from the same rabbit~ used to generate the ANTI-NGF (PREIMM), and (3) untreated littermates (UNTR). Neonatal rats were given daily subcu. taneous injections of ANTI-NGF (3/~1 of undiluted rabbit serum per g b.wt.) or similar doses of preimmunc sera near the dorsal fat pad for a period of 14 days or I month beginning on postnatal day (PD) 0 (day of birth). The ANTI-NGF was raised in rabbits which had received sut~utaneous intx:ulations of purified B-NGF (10-20 ng/ml

for ! biological unit (bU)/ml) isolated from the 7S NGF purified from mouse submaxillary glands according to the methods described in Varon et al?:'. The classic bioassays with extirpated embryonic chick sensory ganglia 21"2~''~'1"~'~ and morphological differentiation of cultured human neuroblastoma cells 44"g2 were used and compared to NGF preparations whose specific biological activity was 10-20 ng/ml (I bU/ml), as described elsewhere% By both assays, the biological activity of the NGF and the ANTI-NGF used in this study was found to remain stable during the course of the experiments. An antiserum dilution of l : 500 blocked the effects of exogenously applied NGF at concentrations of 1 b U / m l ~l and by both assays the blocking activity of the ANTI-NGF was stable during the course of the experiments. The ANTI-NGF injections were doses of undiluted rabbit antiserum prepared against the purified/3-NGF subunit. To determine in vivo injection levels, it was determined empirically that increases in antibody concentration in the rabbit scra by ultrafiltration 43 greatly affected mortality. Consequently, the use of undiluted antisera in these experiments was deemed sufficient to block endogenous levels of NGF, which is known to be in the nanogram range. As an internal assay for the ANTI-NGF activity in vivo, wet weights were taken of the superior cervical ganglia for each of the three groups in the study. The Student's t-test was used to determine statistical differences with a level of confidence set at P < 0.05. The animals were sacrificed on PD 0, PD 14 and PD 30.

immunohistochemistry Animals were anesthetized by hypothermia on PD 0 (n = 7) and by an intraperitoneal injection of sodium pentobarbital (40 mg/kg) on PD 14 (UNTR, n -- 7; PRE|MM, n -- 2; ANTI-NGF, n = 3) and PD 30 (UNTR, n = 4; PREIMM, n = 2; ANTI-NGF, n = 4), and when anesthesia was deep, as determined by reflex testing, the animals were perfused transcardially with phosphate buffered saline (PBS) followed by a fixative of buffered 4% paraformaldehyde with 0.2% picric acid. The brain and attached spinal cord to C2 were removed and placed in 30% sucrose in the fixative solution for 48 h at 4°C. The tissue was then divided into the following regions: medulla, pens and overlying cerebellum, midbrain to optic chiasm, and optic chiasm to olfactory bulb and embedded in O,C.T. Sections, 40/~m thick, were cut on a cryostat and collected as free-floating sections in PBS. All sections from a particular region were processed in partitioned carrier devices with screened bottoms (up to 136 partitions to one carrier), which were dipped in lucite boxes filled with the appropriate solutions, Thus, till sections from the same anatomical region for all individuals from all groups at all sacrifice time points were exposed to the same solutions. The sectkms were incubated in 4% normal horse serum (Vector) with 0,02% Triton X-100 for 20 rain and in a I : 1,000 to 1:2,000 dilution of purified mouse monoclonal antibody 192 with 0,02% Triton X-100 for 48 h at 4°C, The 192-1gG recognizes the p75 NGFR. The sections were exposed to a i% HzO ~ solution for 5 rain and were placed in a ! : 500 d;tution of affinity-purified, biotinylated horse anti.mouse leg (Vector) overnight at 4°C, Following a 3 h incubation in an avidinbiotin-horseradish peroxidase complex (Vector), the tissues were pre-incubated for 10 rain in a solution containing 0.025% 3,3'-diaminobenzidine (DAB), 0.0002% cobalt chloride and 0,00016% nickel ammonium sulfate, The sections were then incubated for 2-4 rain in the DAB solution with 0,009% H 20., added. Negative controls were run without the 192-1gG and were found to be at background levels of immunoreactivity, The sections were mounted on slides, without counterstaining, and coverslipped. The sections for all groups were standardized for section thickness by through-focusing, and sections < 35 p,m or > 40 p,m in thickness were eliminated. The anatomic localization of the p75 NGFR immunoreaction product for each region for each of the three groups was recorded. Light level quantification of the immunoreaction product was done using an Amersham Image Analysis System coupled to a Nikon light microscope under consistent lighting conditions. The window of signal level was set from 0-255. A standard square area was used to measure the optical density for each anatomic region. The histological sections were sampled three times within each immunoreactive area and three times within an unreacted area in the same section from 5 random sections of each animal. The mean optical density of each

225 sampled region was divided by the average background optical density. All measurements were done in a double-blind format. The data were analyzed for significance by ANOVA and Student's t-test with P < 0.05 chosen as the criterion for the level of confidence. RESULTS

The superior cervical ganglia from the ANTI-NGF rats weighed significantly less than the untreated (P 0.000001) or preimmune-treated (P ~ 0.0002) animals at PD 14, and was significantly less than the untreated (P < 0.000001) and pre-immune treated (P ~ 0.000007) animals at PD 30 (Table I). The immunoreaction product was observed in structures such as the solitary tract, prepositus hypogiossal nucleus, nucleus gracilis and cuneatus, ventral posterior nucleus of the thalamus, medial septum, diagonal band of Broca, medial terminal nucleus of the accessory optic tract, area postrema, cerebellum, nucleus basalis, medial vestibular nuclei, spinal tract of V, and lateral cochlear nuclei, corroborating results of others 16'52"53"63"75"7°'78'1°1'102. Only the areas which demonstrated a difference in the relative density of the


Superior cert,tcal ganglia Wet weights of the superior cervical ganglia from the three treatment groups of animals used in the present study. Statistical analyses were done using the Student's t-test by comparing two groups with the same superscript. The levels of significance are shown for all comparisons. Note that the ANTI-NGF treatment group demonstrated statistically significant decreases when compared to the two other treatment groups for both time points in this study. Measurements are shown in mg as means + S.E.M. PD 14, animals sacrificed on postnatal day 14; PD 30, animals sacrificed on postnatal day 30; UNTR, untreated animals; PREIMM, animals treated with preimmune sera; ANTI-NGF, animals treated with antibodies to/3-NGF.


Wet weights


0.42:1:0.02 ,i 0.40 + 0.00 b 0.08 + 0.01 a,b


1.29 + 0.04 c 1.28 + 0.02 d 0.46 + 0.02 c,0

Levels of significance: Student's t-test a p _

Distribution and relative density of p75 nerve growth factor receptors in the rat brain as a function of age and treatment with antibodies to nerve growth factor.

It is clear that nerve growth factor (NGF) has a role in the central nervous system. In order to begin to determine the possible roles of NGF in the C...
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