Journal of Neuroscience Methods, 37 (1991) 37-45
37
~,31991 Elsevier Science Publishers B.V. 0165-0270/91/$03.50 NSM01202
Immunohistochemical detection of a monoclonal antibody directed against the NGF receptor in basal forebrain neurons following intraventricular injection L. B r a n n o n T h o m a s 1, A d a m A. B o o k 2 a n d J o h n B. S c h w e i t z e r 1,2 I Department of Pathology (Division of Neuropathology) and 2 Department of Anatomy and Neurobiology University of Tennessee, Memphis, TN (U.S.A.)
(Received 11 June 1990) (Revised version received 23 October 1990) (Accepted 24 November 1990)
Ke y words: N e r v e growth factor receptor; Cholinergic basal forebrain; I m m u n o h i s t o c h e m i s t r y ; Autoradi-
ography; Monoclonal antibody; Cerebrospinal fluid It has been shown by autoradiography that, following intraventricular administration, a monoclonal antibody directed against the rat nerve growth factor (NGF) receptor is specifically accumulated bilaterally by numerous cholinergic neurons of the basal forebrain. This is consistent with the evidence that cholinergic basal forebrain neurons have NGF receptors and respond to NGF under a variety of experimental conditions. The present study demonstrates that the immunohistochemical detection of unmodified monoclonal antibody in cholinergic forebrain neurons following transport from CSF is feasible, although injection of larger amounts of the antibody is required to obtain an image equivalent to the one obtained with the autoradiographic method. The location of the immunohistochemical product clearly indicates that the antibody has been internalized, probably in an endosomal compartment.
Nerve growth factor ( N G F ) is a neurotrophic protein necessary for the survival of sympathetic and certain sensory neurons in the peripheral nervous system (Thoenen and Barde, 1980; LeviMontalcini, 1987). There is evidence that it plays a role in the maintenance of function of the magnocellular cholinergic basal forebrain (CBF) neurons, which have N G F receptors (Hefti et al., 1984; Hefti, 1986; G n a h n et al., 1983; Korsching et al., 1985; Kromer, 1987; M o b l e y et al., 1986; Whittemore et al., 1986). As part of an investigation into methods of specifically manipulating the cells comprising this system, the distribution of a
Correspondence: John B. Schweitzer, M.D., Department of
Pathology. University of Tennessee, Memphis, 800 Madison Ave., Rm 568 BMH-M, Memphis, TN 38163, U.S.A. Phone: (901)-528-5872 Fax: (901)-528-6979
monoclonal a n t i b o d y which recognizes the rat N G F receptor is being evaluated following intraventricular administration. It has been shown by a u t o r a d i o g r a p h y that radioiodinated m o n o clonal a n t i b o d y 192 (1251-192-IGG), a well-characterized a n t i b o d y of the I g G class that recognizes only the rat N G F receptor in b o t h peripheral and central nervous tissues (Chandler et al., 1984: Taniuchi and Johnson, 1985; Taniuchi et al., 1986), is accumulated by neurons in the forebrain in a highly specific fashion following intraventricular administration (Schweitzer, 1987). N e u r o n s which accumulated t251-192-IgG from the C S F failed to accumulate a radioiodinated control m o n o c l o n a l a n t i b o d y and the accumulation of 1251-192-IgG was nearly abolished by the co-administration of a 95-fold excess of unlabelled 192-IgG. A double labelling study utilizing the concurrent detection of choline acetyltransferase (CAT) by i m m u n o -
38 histochemical means and the detection of 1251-192IgG (following intraventricular administration) by autoradiographic methods showed that the neurons labelled by 1251-192-IgG are in fact cholinergic and that a high proportion of CBF neurons are labelled by limited amounts of 1251-192-IgG following intraventricular administration (Schweitzer, 1989). Other methods such as retrograde transport from defined brain areas (Seiler and Schwab, 1984), immunohistochemistry (Yan and Johnson, 1989) sometimes combined with the administration of drugs (Pioro and Cuello, 1990) and hybridization studies for the detection of N G F receptor m R N A (Koh et al., 1989) show numerous other brain regions to have N G F receptors. Although the present technique confirms the presence of N G F receptors in certain brain regions, its main interest from our point of view derives from the demonstration that a spatially diffuse brain system such as the CBF can selectively and specifically accumulate a large molecular weight substance infused into the CSF. This implies an ability to specifically manipulate this important group of neurons through the delivery of molecules into the CSF which are capable of recognizing the N G F receptor. In this study we use immunohistochemical techniques to obtain additional information about the cellular localization of injected 192-IgG and to provide a comparison with the autoradiographic method. 192-IgG and MAB 20.4, a monoelonal antibody which recognizes only the higher primate N G F receptor, were generously supplied by E.M. Johnson, Jr. They were purified from ascitic fluid using standard protein A affinity methods. Female Sprague-Dawley rats (Harlan) weighing 200-290 g were anesthetized with a mixture of ketamine (87 m g / k g ) and xylazine (13 m g / k g ) given intramuscularly and were placed into a stereotaxic apparatus (Kopf) with the incisor bar 4 - 5 mm above the interaural line. Injections were made into the right lateral ventricle using a 5/~1 syringe with a 31-gauge needle (Hamilton). The needle was placed 1.5 mm lateral to bregma and inserted to a depth of 3.3 mm. Preliminary injections with dye were made to verify the coordinates. Injections of 5 #1 were made over a 3 rain period. The needle was then slowly withdrawn over an ad-
ditional 3 min period. A total of 17 animals were injected with 192-IgG ranging in concentration from 0.1 to 6.0 m g / m l , and control animals were injected with 0.45 m g / m l (n = 2) or 5.0 m g / m l ( n = 2 ) MAB 20.4 or 0.9% NaC1 ( n = 2 ) . MAB 20.4 and the saline solution were used as controls in these experiments. Twenty-four hours after injection, the animals were anesthetized and perfused transcardially with 100 ml of buffered saline followed by 250 ml 4% paraformaldehyde. Following brain removal, the brains were post-fixed for at least 2 h in the same fixative. The brains were subsequently placed in a 15% buffered sucrose solution for several hours and then equilibrated in a 30% buffered sucrose solution. Sections of brain (40 /xm) were cut and washed in Tris-buffered saline (TBS) with 0.3% Triton X-100 (Sigma). An anti-mouse immunoglobulin kit (Vector Laboratories) was then used to detect the injected antibody on free floating sections by the avidin-biotinhorseradish peroxidase method according to the manufacturer's instructions, with the exception that 0.3% Triton X-100 was added to the secondary antibody solution. Biotinylated anti-mouse immunoglobulin previously adsorbed against rat immunoglobulins was found to lower the background staining and was used for some of the animals. The sections were reacted with 1 m g / m l diaminobenzidine in the presence of 4 m g / m l D-fl-glucose and 20 /~g/ml glucose oxidase. In some reactions, 0.006% CoC12was also present. The sections were mounted, dried and coverslipped. We utilized sections from the previously reported autoradiographic experiment (Schweitzer, 1987) to provide the comparison with immunohistochemical detection of the injected antibody. Briefly, 192-IgG was radioiodinated using the lactoperoxidase method and Na125I from Amersham. Specific activities of 1.0-1.5 Ci//xmol were obtained. 1.5 or 5 /~1 of 0.3 m g / m l 1251-192-IgG was injected using the same coordinates as already described. The animals were perfused transcardially at 24 h with buffered saline followed by buffered 4% paraformaldehyde, the brains were removed and fixed overnight, and then they were embedded in paraffin. Sections (6 /xm) were cut and every twentieth section mounted, dried and deparaffinized. Standard autoradiographic tech-
39
niques were then used (Rogers, 1973). Slides were coated with nuclear tracking emulsion (Kodak NTB-2), developed (Kodak D-19) 30 days later and counterstained with toluidine blue. To develop the numbers provided in Table I, one section through the medial septal nucleus (ms) (at the level of plate 15 (Paxinos and Watson, 1986)) was chosen and positive neurons located on a camera lucida drawing from each of n experimental animals. Positive neurons (defined immunohistochemically as those containing 3 or more dark black grains against a faintly positive somal outline; for autoradiography, defined as 10 or more silver grains detected over a neuron) located above a line drawn between the top of the anterior commissure on each side were counted. The animals injected with 192-IgG demonstrated varying degrees of specific (i.e., not seen with MAB 20.4-injected animals) basal forebrain neuronal staining dependent on the dose of 192IgG administered. The most intense reaction product was found in the animals injected with the highest concentrations (4.5-6.0 m g / m l ) of 192-IgG (22.5-30 #g total dose of antibody), followed in order by the lesser concentrations. Injections of 1 m g / m l solutions of 192-IgG produced images qualitatively similar to those in the 4 - 6 m g / m l range, but specific staining was much less obvious at injected concentrations less than 1 m g / m l . The number of positive neurons were few and the intensity of the staining was sufficiently
faint at 0.1 m g / m l that specific staining was almost undetectable. Although the labelling of individual neurons was more robust and easily appreciated with autoradiography and darkfield illumination than with immunohistochemical detection, it was our impression that the number of neurons labelled immunohistochemically following the intraventricular injection of 1-5 m g / m l of 192-IGG was quite similar to that seen following autoradiographic detection of 0.3 m g / m l ~251-192IgG (Fig. 2). Counts through standardized sections of the ms confirmed this impression, as well as showing that the number of positive neurons seen with either method is dependent on the dose of antibody injected (see Table I). Specific staining of neuronal perikarya was not found in brain areas other than the CBF. Animals injected with either antibody showed some diffusely positive cells around the injection site. There was also very faint, non-punctate cellular staining seen in variable subpial loci and variable staining of Purkinje cells with either antibody, which was judged nonspecific. Another region which often showed nonspecific (i.e. seen with either antibody or saline injection) staining under these conditions was the brain surrounding the lateral recess of the fourth ventricle (cochlear nuclei, flocculus of cerebellum). There was no immunohistochemical product present in CBF neurons in the control animals injected with MAB 20.4 or 0.9% NaC1. Figure 1B demonstrates the absence of specific staining ob-
TABLE I COMPARISON OF I M M U N O C Y T O C H E M I C A L A N D A U T O R A D I O G R A P H I C A L M E T H O D S OF D E T E C T I O N IN T H E MS F O L L O W I N G I N T R A V E N T R I C U L A R A D M I N I S T R A T I O N OF 192-IgG OR 1251-192-IGG, RESPECTIVELY Doses of antibody were given in a volume of 5 / d or less. Counts of positive neurons were made above a line drawn through the top of the anterior commissure at the level of plate 15 (Paxinos and Watson, 1986). Methods of deriving the counts are given in the text. One section from each of n experimental animals was evaluated. Dose (txg)
No. of neurons
n
Immunohistochemical detection 0.5 5.0 25.0
3 +_1 25.7 + 3.5 34.5 + 4.8
3 3 4
Autoradiographic detection 0.5 1.5
20.5 32.7 + 3.5
2 3
40 tained following the intraventricular administrat i o n o f 5,0 m g / m l o f M A B 20.4 a n d m a y b e compared with the specific staining of neurons s e e n at the s a m e l e v e l w i t h 1 9 2 - I g G (Fig. 1A).
~
~
~~i
i
~
Positive staining of neurons for the accumulat i o n o f the N G F r e c e p t o r a n t i b o d y w a s i n d i c a t e d b y the p r e s e n c e o f f a i n t s t a i n i n g o f t h e cell b o r d e r s accompanied by moderate numbers of punctate.
!~i
i
Fig. 1. Coronal sections through rat forebrain processed for immunohistochemical localization of antibody following intraventricular administration of 192-1gG (A,C,D,E,F) or MAB 20.4 (B). A: vdB showing labelled neurons following administration of 5/tl of 6.0 /tg//tl 192-IgG. B: section from the same level of vdB following administration of 5/tl of 6.0/tg//.tl MAB 20.4, a control antibody. Some reaction product due to endogenous peroxidase activity in blood vessels is appreciable. C: high power view of a neuron demonstrating 192-IgG irnmunoreactivity in vdB. Note the numerous punctate, black granules distributed throughout the soma. D - E : neurons showing 192-IgG immunoreactivity are seen in ms (D) nbm (E). F: high power view of a neuron in nbm demonstrating 192-IgG immunoreactivity. Bar in A,B,D = 100/~m; bar in C,F = 10/~m; bar in E = 20/tin.
41 dark granules in the cell bodies of neurons in the ms, nucleus of the diagonal band of Broca, both the vertical (vdB) and horizontal limbs and the nucleus basalis magnocellularis (nbm) (Fig. 1 A , C F). This localization is different from that seen using 192-IgG in a conventional immunohistochemical paradigm, in which there is heavy staining on the cell surfaces of perikarya, dendrites, and presumptive axons, as would be expected for a cell surface receptor (Yan and Johnson, 1989). Following intraventricular administration and a
24-h survival, the predominantly intracellular and coarsely punctate staining indicates that 192-IgG has been internalized, most likely in a lysosomal or endosomal compartment. The immunohistochemical image of non-perikaryal labelling following the intraventricular administration of 192-IgG shows specific staining in a number of regions previously demonstrated to contain N G F receptors. Moderately strong nonperikaryal staining was seen in the suprachiasmatic nucleus (Fig. 3A) and strong staining was
t
J J
e
Fig. 2. Comparison of autoradiography and immunohistochemistryin coronal sections through rat forebrain followingintraventricular administration of 1251-192-IGGor 192-IgG, respectively. A: dark-field illumination of autoradiographically positive neurons following intraventricular administration of 5 ~l of 0.31 /tg/~l 12sI-192-IgG.Bar = 100 /~m. B: brightfield illumination of positive immunoreactivity followingintraventricular administration of 5/tl of 6.0/~g//~l 192-1gG.Same magnificationas in A.
42 present in the entire region of hypothalamus comprising the regions ventral and lateral to the third ventricle (median eminence/arcuate nucleus region) (Fig. 3C,D). This included some long fiberlike staining which extended in an arc from the wall of the third ventricle to the pial surface at the base of the brain. This pattern is suggestive of staining of tanycytes, a specialized ependymal cell found in this region. A few cell bodies in the median eminence region were also stained. These could not be clearly identified as to cell type. The area postrema showed variable, but sometimes moderately strong staining of neuropil and vessels (Fig. 3E,F), while the subfornical organ did not show staining. The interpretation of hippocampus, which receives a large N G F receptor positive input from CBF, was made difficult by the fact that there generally was significant non-specific staining in this region following injection of MAB 20.4 or saline. This was apparently related in some way to the injection itself, since the staining was greater on the injected side. Individual ependymal cells in the lateral aspects of the lateral ventricles were densely stained sometimes uniformly, sometimes in a checkerboard pattern; however, there was a similar process observed with injection of MAB 20.4 and no specific pattern of staining due to 192-IgG could be discerned. Staining of the cerebellar molecular layer could be detected which showed band-like zones of faint staining alternating with interdigitated zones of no detectable reaction product (not shown), similar to what has been described in conventional immunohistochemistry (Koh et al., 1989). Although a number of sensory systems with central projections are known to be N G F receptor positive, the only white matter tract known to conduct N G F receptor positive axons which demonstrates (faint) staining under these conditions is the spinal trigeminal tract (Fig. 3B). Detectable immunohistochemical product was not observed in the tract or nucleus gracilis or cuneatus (Fig. 3E). This study demonstrates that the localization in basal forebrain neurons of 192-IgG injected into the ventricle of the rat can be accomplished using immunohistochemical methods. The localization appears specific, in that cells characteristic of the magnocellular CBF are labelled following 192-IgG
injection, but not following injection of control substances and previous studies with 1251-192-IgG have established that magnocellular CBF neurons are specifically labelled. Since the labelling is bilateral and roughly equivalent in either hemisphere and CBF projections are largely ipsilateral in nature (Meibach and Siegal, 1977: Price and Stein, 1983), dissemination in the CSF pathways with subseqent penetration into brain rather than transport from the injection tract likely explains the labelling pattern which is generated. The amount of reaction product shows a dependency on the dose of 192-IgG injected, as judged by visual inspection of the resultant sections, and by actual counts in a defined region of brain. Since the reaction product is largely intracellular and appears coarsely granular, the 192-1gG has been internalized, probably in an endosomal or lysosomal compartment. The regions of brain which are labelled following intraventricular injection of 192-IgG are a subset of the N G F receptor positive areas demonstrated by conventional immunohistochemistry using the same antibody (t92-IgG) as described in two recent reports (Yan and Johnson, 1989; Koh et al., 1989). Thus, neurons in the ventral premammillary nucleus, in the mesencephalic nucleus of the trigeminal nerve (me5), and some midbrain raphe neurons are not labelled following intraventricutar injection, but are labelled in conventional immunohistochemistry. Areas of neuropil shown to be N G F receptor positive in one or both of these reports but not labelled in the current study include the subfornical organ, the olivary pretectal nucleus, the ventral lateral geniculate nucleus, the root of the vagus, the solitary tract and nucleus, the inferior olive, the cuneate nucleus and thegracile nucleus. The olfactory bulb, infundibular stalk, and vestibulocochlear ganglion, which were described as N G F receptor positive in the above-referenced reports. were not examined in this study. The reason why some but not all N G F receptor positive areas of brain are labelled following intraventricular administration is not known. Possibilities include: (1) the method lacks the sensitivity to demonstrate N G F receptor positivity (many, but not all, of these areas are the more weakly positive areas in
43
tz
SCh C Arc 3V
n
m
E
~
Gr
\
....
CU
m
IF
Cu
SolC m
m
l
Fig. 3. Coronal sections through rat brain processed for immunohistochemistry following the intraventricular administration of 192-1gG. Brightfield illumination. A: section through level of suprachiasmatic nucleus (SCh), showing moderately strong staining of neuropil. Faint staining of ependymal layer of third ventricle (3V) is also present. B: section through the spinal tract of the trigeminal nerve (spS), demonstrating faint immunostaining of axons (arrows) in sp5 and an absence of staining in the adjacent trapezoid body (tz). C: section through the region of the caudal retrochiasmatic nucleus and rostral median eminence, showing relatively strong immunostaining of neuropil lateral and ventral to 3V and in the median eminence. The long fiber-like staining extending from the wall of 3V to the base of the brain suggests the staining of tanycytes in this region. D: section through the level of the median eminence (ME) and arcuate nucleus (Arc) of a different animal than that shown in (C). In addition to the neuropil staining, several cell bodies appear to be immunopositive in the ME. E: section through the area postrema (AP), showing strong immunostaining of neuropil in AP and no detectable staining in the gracile nucleus (Gr), cuneate nucleus (Cu), cuneate tract (cu), or commissure of the solitary nucleus (SolC). F: higher power view of AP from a different animal than the one shown in (E). The staining of neuropil in AP is weaker, but the presence of immunoproduct on vessels is more easily appreciated. Magnification bar in A,C,D,F = 100 /xm; B = 50/tin; bar in E = 200/~m.
44
the brain (Koh et al., 1989)); (2) the areas would be positive at some other time point; a n d / o r (3) the pharmacokinetics of the antibody in CSF a n d / o r the anatomy of some of the NGF receptor positive regions favor the labelling of some neurons and areas over others. The last consideration is likely to be an important element in the restricted labelling which is observed, as antibodies are known to penetrate for only relatively short distances into brain tissue from CSF (Klatzo et al., 1964). Support for the idea that 192-IgG labels CBF neurons by retrograde intra-axonal transport is provided by the fact that the neurons of me5, which are strongly NGF receptor positive but whose axons project outside the CNS. are not labelled under these conditions. The sensitivity of autoradiography using 1251192-IgG appears superior to the detection of comparable m o u n t s of unmodified 192-IgG using the immunohistochemical method, but similar results can be achieved with immunohistochemistry, if higher doses of 192-IgG can be injected into the animal. Immunohistochemical detection has the advantage of being a more rapid procedure that does not require a dark room. We would anticipate that the data concerning the localization of both 192-IgG and 12sI-192-IgG following intraventricular administration would indicate the feasibility of delivering a cytotoxin attached to 192-IgG such as has been synthesized (DiStefano et al., 1985) to create a highly specific CBF lesioning technique and further that the localization of the antibody portion of the toxin could be achieved using this technique. The authors gratefully acknowledge the support and constructive criticism of E.M. Johnson, Jr. and the skillful technical assistance of Ellen B. Looney and Lisa C. Wathen. This research has been supported by grants from the National Institute of Neurological Disorders and Stroke NS25122 and NS01230 as well as the National Institute of Aging AHR 1 PSO AG05681. References Chandler, C.E., L.M. Parsons, M. Hosang and E.M. Shooter (1984) A monoclonal antibody modulates the interaction of
nerve growth factor with PC12 cells. J. Biol. (;hem., 259: 6882-6889. DiStefano, P.S., J.B. Schweitzer, M. Taniuchi and E.M. Johnson, Jr. (1985) Selective destruction of nerve growth factor receptor-bearing cells in vitro using a hybrid toxin composed of ricin A chain and a monoclonal antibody against the nerve growth factor receptor. J. Celt Biol., 10l: 11071114. G n a h n , H., F. Hefti, R. H e u m a n n , M.E. Schwab and H. T h o e n e n (1983) N G F - m e d i a t e d increase of choline acetyltransferase (CHAT) in the neonatal rat forebrain: Evidence for a physiological role of N G F in the brain. Dev. Brain Res., 9: 45-52. Hefti. F.. A. Dravid and J. Hartikka (1984) Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septo-hippocampal lesions. Brain Res.. 293: 305-311. Hefti. F (1986) Nerve growth factor promotes survival of septal cholinergm neurons after fimbrial transections. J. Neurosci.. 6: 2155-2162. Klatzo. I.. J. Miquel. P.J. Ferris. J.D. Prokop and D.E. Smith (1964) Observations on the passage of the fluoroscem labeled serum proteins (FLSP) from the cerebrospinal fluid. J. Neuropath. Exp. Neurol., 23: 18-35. Koh. S., G. A. Oyler and G.A. H i g h s (1989) Localization of nerve growth factor receptor messenger R N A a n d protein in the adult rat brain. Exp. Neurol.. 106: 209-221. Korsching, S., G. Auburger, R H e u m a n n . J. Scott and H. Thoenen (1985) Levels of nerve growth factor and its m R N A in the central nervous system of the rat correlate with cholinergic innervation. E M B O J., 4: 1389-1393. Kromer, L.F. (1987) Nerve growth factor treatment after brain injury prevents neuronal death. Science, 235: 214-216. Levi-Montalcini, R. (1987) The nerve growth factor 35 years later. Science, 237: 1154-1162. Meibach, R. and A. Siegel. (1977) Efferent connections of the septal area in the rat: an analysis using retrograde and anterograde transport methods. Brain Res., 119: 1-20. Mobley, W.C., J.L. Rutkowski, G.I. Termekoon, J. Gemski, K. Buchanan and M.V. Johnston (1986) Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons. Mol. Brain Res., 1: 53-62. Paxinos, G. and C. Watson (1986) The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney. Pioro, E.P. and A.C. Cuello (1990) Distribution of nerve growth factor receptor-like immunoreactivity in the adult rat central nervous system. Effect of colchicine and correlation with the cholinergic system. I. Forebrain. Neuroscience, 34: 5 7 87. Price, J.L. and R. Stein (1983) Individual cells m the nucleus basahs-diagonal band complex have restricted axonal projections to the cerebral cortex in the rat. Brain Res., 269: 352-356. Rogers, A.W. (1973) Techniques of Autoradiography. Elsevier Science Publishing Co., Amsterdam, pp. 309-310.
45 Schweitzer, J.B. (1987) Nerve growth factor receptor-mediated transport from cerebrospinal fluid to basal forebrain neurons. Brain Res., 423: 309-317. Schweitzer, J.B. (1989) Nerve growth factor receptor-mediated transport from CSF labels cholinergic neurons: direct demonstration by a double-labelling study. Brain Res., 490: 390-396. Seiler, M. and M.E. Schwab (1984) Specific retrograde transport of nerve growth factor (NGF) from neocortex to nucleus basalis in the rat. Brain Res., 300: 33-39. Taniuchi, M. and E.M. Johnson (1985) Characterization of the binding properties and retrograde axonal transport of a monoclonal antibody directed against the rat nerve growth receptor. J. Cell Biol., 101: 1100-1106.
Taniuchi, M., J.B. Schweitzer and E.M. Johnson (1986) Nerve growth factor receptor molecules in rat brain. Proc. Natl. Acad. Sci. USA, 83: 1950-1954. Thoenen, H. and Y.A. Barde (1980) Physiology of nerve growth factor. Physiol. Rev., 60: 1284-1335. Whittemore, S.R., T. Ebendal, L. Larkfors, L. Olson, A. Seiger, I. Stromberg and H. Persson (1986) Developmental and regional expression of/~ nerve growth messenger R N A and protein in the rat central nervous system. Proc. Natl. Acad. Sci. USA, 83: 817-821. Yan, Q. and E.M. Johnson, Jr. (1989) Immunohistochemical Localization and Biochemical Characterization of Nerve Growth Factor Receptor in Adult Rat Brain. J. Comp. Neurol., 290: 585-598.
37
~,31991 Elsevier Science Publishers B.V. 0165-0270/91/$03.50 NSM01202
Immunohistochemical detection of a monoclonal antibody directed against the NGF receptor in basal forebrain neurons following intraventricular injection L. B r a n n o n T h o m a s 1, A d a m A. B o o k 2 a n d J o h n B. S c h w e i t z e r 1,2 I Department of Pathology (Division of Neuropathology) and 2 Department of Anatomy and Neurobiology University of Tennessee, Memphis, TN (U.S.A.)
(Received 11 June 1990) (Revised version received 23 October 1990) (Accepted 24 November 1990)
Ke y words: N e r v e growth factor receptor; Cholinergic basal forebrain; I m m u n o h i s t o c h e m i s t r y ; Autoradi-
ography; Monoclonal antibody; Cerebrospinal fluid It has been shown by autoradiography that, following intraventricular administration, a monoclonal antibody directed against the rat nerve growth factor (NGF) receptor is specifically accumulated bilaterally by numerous cholinergic neurons of the basal forebrain. This is consistent with the evidence that cholinergic basal forebrain neurons have NGF receptors and respond to NGF under a variety of experimental conditions. The present study demonstrates that the immunohistochemical detection of unmodified monoclonal antibody in cholinergic forebrain neurons following transport from CSF is feasible, although injection of larger amounts of the antibody is required to obtain an image equivalent to the one obtained with the autoradiographic method. The location of the immunohistochemical product clearly indicates that the antibody has been internalized, probably in an endosomal compartment.
Nerve growth factor ( N G F ) is a neurotrophic protein necessary for the survival of sympathetic and certain sensory neurons in the peripheral nervous system (Thoenen and Barde, 1980; LeviMontalcini, 1987). There is evidence that it plays a role in the maintenance of function of the magnocellular cholinergic basal forebrain (CBF) neurons, which have N G F receptors (Hefti et al., 1984; Hefti, 1986; G n a h n et al., 1983; Korsching et al., 1985; Kromer, 1987; M o b l e y et al., 1986; Whittemore et al., 1986). As part of an investigation into methods of specifically manipulating the cells comprising this system, the distribution of a
Correspondence: John B. Schweitzer, M.D., Department of
Pathology. University of Tennessee, Memphis, 800 Madison Ave., Rm 568 BMH-M, Memphis, TN 38163, U.S.A. Phone: (901)-528-5872 Fax: (901)-528-6979
monoclonal a n t i b o d y which recognizes the rat N G F receptor is being evaluated following intraventricular administration. It has been shown by a u t o r a d i o g r a p h y that radioiodinated m o n o clonal a n t i b o d y 192 (1251-192-IGG), a well-characterized a n t i b o d y of the I g G class that recognizes only the rat N G F receptor in b o t h peripheral and central nervous tissues (Chandler et al., 1984: Taniuchi and Johnson, 1985; Taniuchi et al., 1986), is accumulated by neurons in the forebrain in a highly specific fashion following intraventricular administration (Schweitzer, 1987). N e u r o n s which accumulated t251-192-IgG from the C S F failed to accumulate a radioiodinated control m o n o c l o n a l a n t i b o d y and the accumulation of 1251-192-IgG was nearly abolished by the co-administration of a 95-fold excess of unlabelled 192-IgG. A double labelling study utilizing the concurrent detection of choline acetyltransferase (CAT) by i m m u n o -
38 histochemical means and the detection of 1251-192IgG (following intraventricular administration) by autoradiographic methods showed that the neurons labelled by 1251-192-IgG are in fact cholinergic and that a high proportion of CBF neurons are labelled by limited amounts of 1251-192-IgG following intraventricular administration (Schweitzer, 1989). Other methods such as retrograde transport from defined brain areas (Seiler and Schwab, 1984), immunohistochemistry (Yan and Johnson, 1989) sometimes combined with the administration of drugs (Pioro and Cuello, 1990) and hybridization studies for the detection of N G F receptor m R N A (Koh et al., 1989) show numerous other brain regions to have N G F receptors. Although the present technique confirms the presence of N G F receptors in certain brain regions, its main interest from our point of view derives from the demonstration that a spatially diffuse brain system such as the CBF can selectively and specifically accumulate a large molecular weight substance infused into the CSF. This implies an ability to specifically manipulate this important group of neurons through the delivery of molecules into the CSF which are capable of recognizing the N G F receptor. In this study we use immunohistochemical techniques to obtain additional information about the cellular localization of injected 192-IgG and to provide a comparison with the autoradiographic method. 192-IgG and MAB 20.4, a monoelonal antibody which recognizes only the higher primate N G F receptor, were generously supplied by E.M. Johnson, Jr. They were purified from ascitic fluid using standard protein A affinity methods. Female Sprague-Dawley rats (Harlan) weighing 200-290 g were anesthetized with a mixture of ketamine (87 m g / k g ) and xylazine (13 m g / k g ) given intramuscularly and were placed into a stereotaxic apparatus (Kopf) with the incisor bar 4 - 5 mm above the interaural line. Injections were made into the right lateral ventricle using a 5/~1 syringe with a 31-gauge needle (Hamilton). The needle was placed 1.5 mm lateral to bregma and inserted to a depth of 3.3 mm. Preliminary injections with dye were made to verify the coordinates. Injections of 5 #1 were made over a 3 rain period. The needle was then slowly withdrawn over an ad-
ditional 3 min period. A total of 17 animals were injected with 192-IgG ranging in concentration from 0.1 to 6.0 m g / m l , and control animals were injected with 0.45 m g / m l (n = 2) or 5.0 m g / m l ( n = 2 ) MAB 20.4 or 0.9% NaC1 ( n = 2 ) . MAB 20.4 and the saline solution were used as controls in these experiments. Twenty-four hours after injection, the animals were anesthetized and perfused transcardially with 100 ml of buffered saline followed by 250 ml 4% paraformaldehyde. Following brain removal, the brains were post-fixed for at least 2 h in the same fixative. The brains were subsequently placed in a 15% buffered sucrose solution for several hours and then equilibrated in a 30% buffered sucrose solution. Sections of brain (40 /xm) were cut and washed in Tris-buffered saline (TBS) with 0.3% Triton X-100 (Sigma). An anti-mouse immunoglobulin kit (Vector Laboratories) was then used to detect the injected antibody on free floating sections by the avidin-biotinhorseradish peroxidase method according to the manufacturer's instructions, with the exception that 0.3% Triton X-100 was added to the secondary antibody solution. Biotinylated anti-mouse immunoglobulin previously adsorbed against rat immunoglobulins was found to lower the background staining and was used for some of the animals. The sections were reacted with 1 m g / m l diaminobenzidine in the presence of 4 m g / m l D-fl-glucose and 20 /~g/ml glucose oxidase. In some reactions, 0.006% CoC12was also present. The sections were mounted, dried and coverslipped. We utilized sections from the previously reported autoradiographic experiment (Schweitzer, 1987) to provide the comparison with immunohistochemical detection of the injected antibody. Briefly, 192-IgG was radioiodinated using the lactoperoxidase method and Na125I from Amersham. Specific activities of 1.0-1.5 Ci//xmol were obtained. 1.5 or 5 /~1 of 0.3 m g / m l 1251-192-IgG was injected using the same coordinates as already described. The animals were perfused transcardially at 24 h with buffered saline followed by buffered 4% paraformaldehyde, the brains were removed and fixed overnight, and then they were embedded in paraffin. Sections (6 /xm) were cut and every twentieth section mounted, dried and deparaffinized. Standard autoradiographic tech-
39
niques were then used (Rogers, 1973). Slides were coated with nuclear tracking emulsion (Kodak NTB-2), developed (Kodak D-19) 30 days later and counterstained with toluidine blue. To develop the numbers provided in Table I, one section through the medial septal nucleus (ms) (at the level of plate 15 (Paxinos and Watson, 1986)) was chosen and positive neurons located on a camera lucida drawing from each of n experimental animals. Positive neurons (defined immunohistochemically as those containing 3 or more dark black grains against a faintly positive somal outline; for autoradiography, defined as 10 or more silver grains detected over a neuron) located above a line drawn between the top of the anterior commissure on each side were counted. The animals injected with 192-IgG demonstrated varying degrees of specific (i.e., not seen with MAB 20.4-injected animals) basal forebrain neuronal staining dependent on the dose of 192IgG administered. The most intense reaction product was found in the animals injected with the highest concentrations (4.5-6.0 m g / m l ) of 192-IgG (22.5-30 #g total dose of antibody), followed in order by the lesser concentrations. Injections of 1 m g / m l solutions of 192-IgG produced images qualitatively similar to those in the 4 - 6 m g / m l range, but specific staining was much less obvious at injected concentrations less than 1 m g / m l . The number of positive neurons were few and the intensity of the staining was sufficiently
faint at 0.1 m g / m l that specific staining was almost undetectable. Although the labelling of individual neurons was more robust and easily appreciated with autoradiography and darkfield illumination than with immunohistochemical detection, it was our impression that the number of neurons labelled immunohistochemically following the intraventricular injection of 1-5 m g / m l of 192-IGG was quite similar to that seen following autoradiographic detection of 0.3 m g / m l ~251-192IgG (Fig. 2). Counts through standardized sections of the ms confirmed this impression, as well as showing that the number of positive neurons seen with either method is dependent on the dose of antibody injected (see Table I). Specific staining of neuronal perikarya was not found in brain areas other than the CBF. Animals injected with either antibody showed some diffusely positive cells around the injection site. There was also very faint, non-punctate cellular staining seen in variable subpial loci and variable staining of Purkinje cells with either antibody, which was judged nonspecific. Another region which often showed nonspecific (i.e. seen with either antibody or saline injection) staining under these conditions was the brain surrounding the lateral recess of the fourth ventricle (cochlear nuclei, flocculus of cerebellum). There was no immunohistochemical product present in CBF neurons in the control animals injected with MAB 20.4 or 0.9% NaC1. Figure 1B demonstrates the absence of specific staining ob-
TABLE I COMPARISON OF I M M U N O C Y T O C H E M I C A L A N D A U T O R A D I O G R A P H I C A L M E T H O D S OF D E T E C T I O N IN T H E MS F O L L O W I N G I N T R A V E N T R I C U L A R A D M I N I S T R A T I O N OF 192-IgG OR 1251-192-IGG, RESPECTIVELY Doses of antibody were given in a volume of 5 / d or less. Counts of positive neurons were made above a line drawn through the top of the anterior commissure at the level of plate 15 (Paxinos and Watson, 1986). Methods of deriving the counts are given in the text. One section from each of n experimental animals was evaluated. Dose (txg)
No. of neurons
n
Immunohistochemical detection 0.5 5.0 25.0
3 +_1 25.7 + 3.5 34.5 + 4.8
3 3 4
Autoradiographic detection 0.5 1.5
20.5 32.7 + 3.5
2 3
40 tained following the intraventricular administrat i o n o f 5,0 m g / m l o f M A B 20.4 a n d m a y b e compared with the specific staining of neurons s e e n at the s a m e l e v e l w i t h 1 9 2 - I g G (Fig. 1A).
~
~
~~i
i
~
Positive staining of neurons for the accumulat i o n o f the N G F r e c e p t o r a n t i b o d y w a s i n d i c a t e d b y the p r e s e n c e o f f a i n t s t a i n i n g o f t h e cell b o r d e r s accompanied by moderate numbers of punctate.
!~i
i
Fig. 1. Coronal sections through rat forebrain processed for immunohistochemical localization of antibody following intraventricular administration of 192-1gG (A,C,D,E,F) or MAB 20.4 (B). A: vdB showing labelled neurons following administration of 5/tl of 6.0 /tg//tl 192-IgG. B: section from the same level of vdB following administration of 5/tl of 6.0/tg//.tl MAB 20.4, a control antibody. Some reaction product due to endogenous peroxidase activity in blood vessels is appreciable. C: high power view of a neuron demonstrating 192-IgG irnmunoreactivity in vdB. Note the numerous punctate, black granules distributed throughout the soma. D - E : neurons showing 192-IgG immunoreactivity are seen in ms (D) nbm (E). F: high power view of a neuron in nbm demonstrating 192-IgG immunoreactivity. Bar in A,B,D = 100/~m; bar in C,F = 10/~m; bar in E = 20/tin.
41 dark granules in the cell bodies of neurons in the ms, nucleus of the diagonal band of Broca, both the vertical (vdB) and horizontal limbs and the nucleus basalis magnocellularis (nbm) (Fig. 1 A , C F). This localization is different from that seen using 192-IgG in a conventional immunohistochemical paradigm, in which there is heavy staining on the cell surfaces of perikarya, dendrites, and presumptive axons, as would be expected for a cell surface receptor (Yan and Johnson, 1989). Following intraventricular administration and a
24-h survival, the predominantly intracellular and coarsely punctate staining indicates that 192-IgG has been internalized, most likely in a lysosomal or endosomal compartment. The immunohistochemical image of non-perikaryal labelling following the intraventricular administration of 192-IgG shows specific staining in a number of regions previously demonstrated to contain N G F receptors. Moderately strong nonperikaryal staining was seen in the suprachiasmatic nucleus (Fig. 3A) and strong staining was
t
J J
e
Fig. 2. Comparison of autoradiography and immunohistochemistryin coronal sections through rat forebrain followingintraventricular administration of 1251-192-IGGor 192-IgG, respectively. A: dark-field illumination of autoradiographically positive neurons following intraventricular administration of 5 ~l of 0.31 /tg/~l 12sI-192-IgG.Bar = 100 /~m. B: brightfield illumination of positive immunoreactivity followingintraventricular administration of 5/tl of 6.0/~g//~l 192-1gG.Same magnificationas in A.
42 present in the entire region of hypothalamus comprising the regions ventral and lateral to the third ventricle (median eminence/arcuate nucleus region) (Fig. 3C,D). This included some long fiberlike staining which extended in an arc from the wall of the third ventricle to the pial surface at the base of the brain. This pattern is suggestive of staining of tanycytes, a specialized ependymal cell found in this region. A few cell bodies in the median eminence region were also stained. These could not be clearly identified as to cell type. The area postrema showed variable, but sometimes moderately strong staining of neuropil and vessels (Fig. 3E,F), while the subfornical organ did not show staining. The interpretation of hippocampus, which receives a large N G F receptor positive input from CBF, was made difficult by the fact that there generally was significant non-specific staining in this region following injection of MAB 20.4 or saline. This was apparently related in some way to the injection itself, since the staining was greater on the injected side. Individual ependymal cells in the lateral aspects of the lateral ventricles were densely stained sometimes uniformly, sometimes in a checkerboard pattern; however, there was a similar process observed with injection of MAB 20.4 and no specific pattern of staining due to 192-IgG could be discerned. Staining of the cerebellar molecular layer could be detected which showed band-like zones of faint staining alternating with interdigitated zones of no detectable reaction product (not shown), similar to what has been described in conventional immunohistochemistry (Koh et al., 1989). Although a number of sensory systems with central projections are known to be N G F receptor positive, the only white matter tract known to conduct N G F receptor positive axons which demonstrates (faint) staining under these conditions is the spinal trigeminal tract (Fig. 3B). Detectable immunohistochemical product was not observed in the tract or nucleus gracilis or cuneatus (Fig. 3E). This study demonstrates that the localization in basal forebrain neurons of 192-IgG injected into the ventricle of the rat can be accomplished using immunohistochemical methods. The localization appears specific, in that cells characteristic of the magnocellular CBF are labelled following 192-IgG
injection, but not following injection of control substances and previous studies with 1251-192-IgG have established that magnocellular CBF neurons are specifically labelled. Since the labelling is bilateral and roughly equivalent in either hemisphere and CBF projections are largely ipsilateral in nature (Meibach and Siegal, 1977: Price and Stein, 1983), dissemination in the CSF pathways with subseqent penetration into brain rather than transport from the injection tract likely explains the labelling pattern which is generated. The amount of reaction product shows a dependency on the dose of 192-IgG injected, as judged by visual inspection of the resultant sections, and by actual counts in a defined region of brain. Since the reaction product is largely intracellular and appears coarsely granular, the 192-1gG has been internalized, probably in an endosomal or lysosomal compartment. The regions of brain which are labelled following intraventricular injection of 192-IgG are a subset of the N G F receptor positive areas demonstrated by conventional immunohistochemistry using the same antibody (t92-IgG) as described in two recent reports (Yan and Johnson, 1989; Koh et al., 1989). Thus, neurons in the ventral premammillary nucleus, in the mesencephalic nucleus of the trigeminal nerve (me5), and some midbrain raphe neurons are not labelled following intraventricutar injection, but are labelled in conventional immunohistochemistry. Areas of neuropil shown to be N G F receptor positive in one or both of these reports but not labelled in the current study include the subfornical organ, the olivary pretectal nucleus, the ventral lateral geniculate nucleus, the root of the vagus, the solitary tract and nucleus, the inferior olive, the cuneate nucleus and thegracile nucleus. The olfactory bulb, infundibular stalk, and vestibulocochlear ganglion, which were described as N G F receptor positive in the above-referenced reports. were not examined in this study. The reason why some but not all N G F receptor positive areas of brain are labelled following intraventricular administration is not known. Possibilities include: (1) the method lacks the sensitivity to demonstrate N G F receptor positivity (many, but not all, of these areas are the more weakly positive areas in
43
tz
SCh C Arc 3V
n
m
E
~
Gr
\
....
CU
m
IF
Cu
SolC m
m
l
Fig. 3. Coronal sections through rat brain processed for immunohistochemistry following the intraventricular administration of 192-1gG. Brightfield illumination. A: section through level of suprachiasmatic nucleus (SCh), showing moderately strong staining of neuropil. Faint staining of ependymal layer of third ventricle (3V) is also present. B: section through the spinal tract of the trigeminal nerve (spS), demonstrating faint immunostaining of axons (arrows) in sp5 and an absence of staining in the adjacent trapezoid body (tz). C: section through the region of the caudal retrochiasmatic nucleus and rostral median eminence, showing relatively strong immunostaining of neuropil lateral and ventral to 3V and in the median eminence. The long fiber-like staining extending from the wall of 3V to the base of the brain suggests the staining of tanycytes in this region. D: section through the level of the median eminence (ME) and arcuate nucleus (Arc) of a different animal than that shown in (C). In addition to the neuropil staining, several cell bodies appear to be immunopositive in the ME. E: section through the area postrema (AP), showing strong immunostaining of neuropil in AP and no detectable staining in the gracile nucleus (Gr), cuneate nucleus (Cu), cuneate tract (cu), or commissure of the solitary nucleus (SolC). F: higher power view of AP from a different animal than the one shown in (E). The staining of neuropil in AP is weaker, but the presence of immunoproduct on vessels is more easily appreciated. Magnification bar in A,C,D,F = 100 /xm; B = 50/tin; bar in E = 200/~m.
44
the brain (Koh et al., 1989)); (2) the areas would be positive at some other time point; a n d / o r (3) the pharmacokinetics of the antibody in CSF a n d / o r the anatomy of some of the NGF receptor positive regions favor the labelling of some neurons and areas over others. The last consideration is likely to be an important element in the restricted labelling which is observed, as antibodies are known to penetrate for only relatively short distances into brain tissue from CSF (Klatzo et al., 1964). Support for the idea that 192-IgG labels CBF neurons by retrograde intra-axonal transport is provided by the fact that the neurons of me5, which are strongly NGF receptor positive but whose axons project outside the CNS. are not labelled under these conditions. The sensitivity of autoradiography using 1251192-IgG appears superior to the detection of comparable m o u n t s of unmodified 192-IgG using the immunohistochemical method, but similar results can be achieved with immunohistochemistry, if higher doses of 192-IgG can be injected into the animal. Immunohistochemical detection has the advantage of being a more rapid procedure that does not require a dark room. We would anticipate that the data concerning the localization of both 192-IgG and 12sI-192-IgG following intraventricular administration would indicate the feasibility of delivering a cytotoxin attached to 192-IgG such as has been synthesized (DiStefano et al., 1985) to create a highly specific CBF lesioning technique and further that the localization of the antibody portion of the toxin could be achieved using this technique. The authors gratefully acknowledge the support and constructive criticism of E.M. Johnson, Jr. and the skillful technical assistance of Ellen B. Looney and Lisa C. Wathen. This research has been supported by grants from the National Institute of Neurological Disorders and Stroke NS25122 and NS01230 as well as the National Institute of Aging AHR 1 PSO AG05681. References Chandler, C.E., L.M. Parsons, M. Hosang and E.M. Shooter (1984) A monoclonal antibody modulates the interaction of
nerve growth factor with PC12 cells. J. Biol. (;hem., 259: 6882-6889. DiStefano, P.S., J.B. Schweitzer, M. Taniuchi and E.M. Johnson, Jr. (1985) Selective destruction of nerve growth factor receptor-bearing cells in vitro using a hybrid toxin composed of ricin A chain and a monoclonal antibody against the nerve growth factor receptor. J. Celt Biol., 10l: 11071114. G n a h n , H., F. Hefti, R. H e u m a n n , M.E. Schwab and H. T h o e n e n (1983) N G F - m e d i a t e d increase of choline acetyltransferase (CHAT) in the neonatal rat forebrain: Evidence for a physiological role of N G F in the brain. Dev. Brain Res., 9: 45-52. Hefti. F.. A. Dravid and J. Hartikka (1984) Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septo-hippocampal lesions. Brain Res.. 293: 305-311. Hefti. F (1986) Nerve growth factor promotes survival of septal cholinergm neurons after fimbrial transections. J. Neurosci.. 6: 2155-2162. Klatzo. I.. J. Miquel. P.J. Ferris. J.D. Prokop and D.E. Smith (1964) Observations on the passage of the fluoroscem labeled serum proteins (FLSP) from the cerebrospinal fluid. J. Neuropath. Exp. Neurol., 23: 18-35. Koh. S., G. A. Oyler and G.A. H i g h s (1989) Localization of nerve growth factor receptor messenger R N A a n d protein in the adult rat brain. Exp. Neurol.. 106: 209-221. Korsching, S., G. Auburger, R H e u m a n n . J. Scott and H. Thoenen (1985) Levels of nerve growth factor and its m R N A in the central nervous system of the rat correlate with cholinergic innervation. E M B O J., 4: 1389-1393. Kromer, L.F. (1987) Nerve growth factor treatment after brain injury prevents neuronal death. Science, 235: 214-216. Levi-Montalcini, R. (1987) The nerve growth factor 35 years later. Science, 237: 1154-1162. Meibach, R. and A. Siegel. (1977) Efferent connections of the septal area in the rat: an analysis using retrograde and anterograde transport methods. Brain Res., 119: 1-20. Mobley, W.C., J.L. Rutkowski, G.I. Termekoon, J. Gemski, K. Buchanan and M.V. Johnston (1986) Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons. Mol. Brain Res., 1: 53-62. Paxinos, G. and C. Watson (1986) The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney. Pioro, E.P. and A.C. Cuello (1990) Distribution of nerve growth factor receptor-like immunoreactivity in the adult rat central nervous system. Effect of colchicine and correlation with the cholinergic system. I. Forebrain. Neuroscience, 34: 5 7 87. Price, J.L. and R. Stein (1983) Individual cells m the nucleus basahs-diagonal band complex have restricted axonal projections to the cerebral cortex in the rat. Brain Res., 269: 352-356. Rogers, A.W. (1973) Techniques of Autoradiography. Elsevier Science Publishing Co., Amsterdam, pp. 309-310.
45 Schweitzer, J.B. (1987) Nerve growth factor receptor-mediated transport from cerebrospinal fluid to basal forebrain neurons. Brain Res., 423: 309-317. Schweitzer, J.B. (1989) Nerve growth factor receptor-mediated transport from CSF labels cholinergic neurons: direct demonstration by a double-labelling study. Brain Res., 490: 390-396. Seiler, M. and M.E. Schwab (1984) Specific retrograde transport of nerve growth factor (NGF) from neocortex to nucleus basalis in the rat. Brain Res., 300: 33-39. Taniuchi, M. and E.M. Johnson (1985) Characterization of the binding properties and retrograde axonal transport of a monoclonal antibody directed against the rat nerve growth receptor. J. Cell Biol., 101: 1100-1106.
Taniuchi, M., J.B. Schweitzer and E.M. Johnson (1986) Nerve growth factor receptor molecules in rat brain. Proc. Natl. Acad. Sci. USA, 83: 1950-1954. Thoenen, H. and Y.A. Barde (1980) Physiology of nerve growth factor. Physiol. Rev., 60: 1284-1335. Whittemore, S.R., T. Ebendal, L. Larkfors, L. Olson, A. Seiger, I. Stromberg and H. Persson (1986) Developmental and regional expression of/~ nerve growth messenger R N A and protein in the rat central nervous system. Proc. Natl. Acad. Sci. USA, 83: 817-821. Yan, Q. and E.M. Johnson, Jr. (1989) Immunohistochemical Localization and Biochemical Characterization of Nerve Growth Factor Receptor in Adult Rat Brain. J. Comp. Neurol., 290: 585-598.
