(nerve growth factor/retrogradetransport/substance P/sensory neurons). MICHEL ..... Hughes, A. F. & Carr, V. M. (1978) in Handbook of Sensory. Physiology, ed. ... Henry, J. L. (1976) in Substance P, eds. von Euler, U. S. & Per- now, B. (Raven ...
Proc. NatL Acad. Sci. USA Vol. 78, No. 9, pp. 5895-5898, September 1981
Neurobiology
Biological importance of the retrograde axonal transport of nerve growth factor in sensory neurons (nerve growth factor/retrograde transport/substance P/sensory neurons)
MICHEL GOEDERT*t, KLAus STOECKEL*, AND UWE OTTEN* *Department of Pharmacology, Biocentre of the University, and *F. Hoffmann-La Roche & Co., Basel, Switzerland
Communicated by Tadeus Reichstein, May 21, 1981
ABSTRACT Nerve growth factor is retrogradely transported in sympathetic and sensory neurons throughout life. Although this transport is known to be biologically significant in sympathetic neurons, such a function was not yet known in sensory ganglia. By using the neuropeptide substance P as a biochemical marker, we show that sensory ganglia from newborn and adult rats respond to nerve growth factor and that its retrograde axonal transport is biologically relevant, as indicated by an increase in substance P and in general protein content.
administration of anti-NGF antibodies leads to a decrease in the substance P content of dorsal root ganglia indicates that NGF is required for a normal postnatal maturation ofthese neurons (20). Moreover, it has been shown that the prenatal exposure of rats and guinea pigs to anti-NGF antibodies leads to a marked reduction in the number of dorsal root ganglion neurons (21) and in the substance P content (unpublished data). The use of substance P as a biochemical marker of sensory neurons allowed us to investigate the possible physiological importance of the retrograde axonal transport of NGF in sensory neurons. We report that the retrogradely transported NGF leads to a marked increase in the substance P content of sensory ganglia from adult rats. In addition, the effects of systemically administered NGF on dorsal root ganglia from newborn and adult rats were compared.
The protein nerve growth factor (NGF) purified from the male mouse submaxillary gland has profound growth-promoting effects on the peripheral sympathetic nervous system ofmammals (1). It also influences the differentiation of these neurons, as evidenced by an increase in tyrosine hydroxylase activity, the key enzyme in the synthesis of norepinephrine (2). The presence of NGF is an indispensable prerequisite for a normal development of the peripheral sympathetic nervous system, as shown by the fact that the administration of anti-NGF antibodies to newborn animals leads to an irreversible destruction of their peripheral sympathetic nervous system (3, 4). This effect is mediated by a neutralization of NGF rather than by a complement-mediated cytotoxic mechanism (5, 6). The general concept that the development of neuronal structures is critically dependent on the production of trophic substances by their respective target organs (7) received considerable support from the finding that NGF is taken up with a high selectivity by adrenergic nerve terminals and is transported retrogradely to the respective perikarya (8). The NGF reaching the cell bodies in this way is responsible to a large extent for the biological effects of the growth factor, as indicated by the fact that it leads to a selective increase in the activity of tyrosine hydroxylase (9). The original investigations on the retrograde axonal transport of NGF have shown that it occurs throughout life not only in adrenergic but also in sensory neurons, whereas it is not present in motor neurons (10, 11). However, the biological significance ofthe retrograde transport in sensory neurons has been unclear so far. Until very recently, developmental studies on sensory neurons had to rely on purely morphological grounds because there were no biochemical marker substances known. The demonstration that the undecapeptide substance P is present in sensory neurons indicated that it could be such a marker (12, 13). It is synthesized in dorsal root ganglia (14) and transported to the terminals of C-fibers located in the dorsal horn ofthe spinal cord and in the skin (15), where its release can be demonstrated (16, 17). The pre- and postnatal injection of NGF leads to a marked increase in substance P in sensory ganglia and in its central and peripheral endings (18-20). Our finding that the
MATERIALS AND METHODS Experimental Animals. Sprague-Dawley rats (Suddeutsche Versuchstierfarm, Tuttlingen, Federal Republic of Germany) were used throughout this study. They were kept at a light/ dark cycle of 12 hr, a constant temperature of 23 ± 10C and had free access to food pellets and tap water. Litter sizes were adjusted so that each litter had between 10 and 12 pups. Surgical Procedure. Two- and 30-day-old rats received a subcutaneous injection of 1 mg of NGF per kg of body weight during three consecutive days and were killed one day after the last injection. Control animals were treated with saline. The spinal cord was exposed from the cervical to the lumbar region and the spinal ganglia (Th 10-U) removed for study. One milligram of NGF dissolved in 50 ,1 ofsterile saline was injected into the right forepaw of ether-anesthesized 30-day-old rats with a 100-p1 Hamilton syringe. For the sensory denervation, the brachial plexus was exposed and transected after separation from the axillary vein. Great care was taken in order to transAct both the axillary and the musculocutaneus nerves. Control animals received a unilateral injection of equimolar concentrations of cytochrome c (Sigma), a protein with a molecular weight and an isoelectric point similar to NGF. The animals were killed 72 hr after the injection, and the dorsal root ganglia C6 and C7 ofthe injected and the noninjected side were removed. Preparation ofNGF. From submaxillary glands of adult male mice, 2.5S NGF was prepared as described by Bocchini and Angeletti (22) with the modifications described by Suda et at (23). The biological activity of NGF was determined as described by Fenton (24); it amounted to 250 biological units per ,g of protein.
The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: NGF, nerve growth factor. tPresent address: MRC Neurochemical Pharmacology Unit, MRC Centre, Medical School, Hills Road, Cambridge, England.
5895
Neurobiology: Goedert et alPProc. Nad. Acad. Sci. USA 78 (1981)
5896
Radioimmunoassay for Substance P. Spinal ganglia were rapidly dissected, freed of dorsal roots and of attached connective tissue, and put in polystyrene tubes containing 1 ml of 2 M ice-cold acetic acid. The extraction was started by heating the ganglia twice in a microwave oven (power, 1.5 kW) for three seconds to inactivate peptidases. Thereafter, the ganglia were homogenized by an ultrasonicator (Sonifer B12, Branson Sonic Power, Danbury, CT) used at 40-50 W. The samples were allowed to stand on ice for 30 min to ensure complete extraction. They were then centrifuged and washed twice, and the combined supernatants were freeze-dried. The lyophilized samples were resuspended in 50 mM sodium barbital buffer (pH 8.6) containing 5% heat-inactivated human plasma and were centrifuged to eliminate insoluble material. The iodination of substance P with 1 mCi (1 Ci = 3.7 X 1010 becquerels) of Na1"I (Radiochemical Centre, Amersham, England) was done by the chloramine-T method of Hunter and Greenwood (25). The reaction product was purified by gel-filtration column chromatography packed on Sephadex G-10 200 .
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(Pharmacia). Elution was carried out with 0.1 M acetic acid containing 1 mg of bovine serum albumin and 0.1 mg of dithiothreitol per ml. Substance P immunoreactivity was measured with a carboxyterminal antiserum as described by Mroz et aL (26). The separation between bound and free iodine was done with dextrancoated charcoal. By using this assay, the lowest detectable quantity of substance P was 12 pg, and the levels measured were linear with the amount of extracted tissue used. The recovery of synthetic substance P was more than 90%. Protein Determination. Protein concentrations were determined by the method of Lowry et aL (27) with bovine serum albumin as the standard. Sensory ganglia were homogenized in 50 mM Tris HCl buffer, pH 7.4/0.1% Triton X-100, and the proteins were measured in the ganglion homogenate. RESULTS Ontogenetic Development of Substance P and Protein. Sensory ganglia from the lower thoracic and the upper lumbar level were used to define the normal ontogenetic development of substance P and protein. Substance P increased from a value of 45 pg per ganglion at birth to about 150 pg at day 30. Thereafter, the values remained stable. The protein values increased from 30 pug per ganglion at birth to about 100 ug per ganglion at day 60 (Fig. 1). The specific activity of substance P rose from 1.5 ng/mg of protein at birth to 2.5 ng/mg ofprotein at day 30. Effects of NGF Treatment. We compared the effects of a subcutaneous injection of NGF (1 mg/kg) during three consecutive days on substance P and the protein content of sensory ganglia (Th 10-L2) in newborn and adult rats. The total substance P content per ganglion increased to 168% of the salinetreated control animals in 2-day-old animals, whereas in 30-dayold animals it showed an increase to 135% of the control values (Fig. 2). Conversely, the total protein values per ganglion increased to about 125% of control values both in newborn and in adult rats (Table 1). Thus, the administration of 1 mg of NGF per kg of body weight during 3 consecutive days led to an increase in specific activity of substance P from 1.57 to 2.14 ng/ mg of protein in newborn and from 2.33 to 2.66 ng/mg of protein in 30-day-old animals. Effects of the Retrograde Axonal Transport of NGF. Rats (30 days old) were injected with 1 mg of NGF in the right fore**
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Age, days FIG. 1. (Upper) Postnatal ontogenetic development of substance P (SP). (Bottom) Total protein in sensory ganglia from the spinal cord level Th 10-L2 of the rat. Each value is the mean + SEM for eight assays.
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400 FIG. 2. Effects of NGF (i) on substance P (SP) content in sensory ganglia (Th 10-L2) from newborn and adult rats. Rats (2- and 30-dayold) received a subcutaneous injection of NGF (1 mg/kg) on three subsequent days and were killed 1 day after the last injection. Each value represents the mean ± SEM for eight assays. **P,
