gB,ß′-iminodipropionitrile administrations

Brain Research, 563 (1991) 151-162 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391171158

151

BRES 17115

Regulation of aberrant neurofilament phosphorylation in neuronal perikarya. III. Alterations following single and continuous fl,fl'-iminodipropionitrile administrations Bruce G. Gold and Daniel R. Austin Center for Research on Occupational and Environmental Toxicology, Oregon Health Sciences University, Portland, OR 97201-3098 (U.S.A.)

(Accepted 11 June 1991) Key words: Axon reaction; Axotomy; fl,fl'-Iminodipropionitrile; Dorsal root ganglia; Neurofilament epitope; Non-phosphorylated

neurofilament; Phosphorylated neurofilament

fl,fl'-Iminodipropionitrile (IDPN) administration produces giant neurofilament-filled axonal swellings in the first proximal internodes of large myelinated sensory and motor fibers without any accompanying axonal degeneration. In the present study, we asked whether proximal giant axonal swellings are sufficient to elicit aberrant neurofilament (NF) phosphorylation in neuronal perikarya. Rats were given a single intraperitoneal (i.p.) injection of IDPN (2 g/kg) followed by IDPN (0.1%) in the drinking water (continuous IDPN exposure) or tap water (single IDPN exposure) for two days to 7 weeks. Immunoreactivity to phosphorylated NF (pNF) epitopes (using monoclonal antibodies 6-17 and 7-05) was observed in L4 and L5 dorsal root ganglia (DRG) neurons beginning between one and 5 days, corresponding to the development of proximal giant axonal swellings. Quantitation of DRG neurons demonstrated maximal numbers of immunoreactive cell bodies to pNF epitopes (46-51%) by one week. The number of immunostained DRG cells was maintained in animals given continuous IDPN exposure, but declined significantly (P < 0.001) in rats given a single injection of IDPN to 26 --. 0.80% and 6 +- 0.04% at 3 and 5 weeks, respectively. Ventral and dorsal root fibers, which undergo axonal atrophy distal to axonal swellings, showed intense immunoreactivity to pNF epitopes and a marked reduction or a complete lack of immunostaining to antibody 2-135 (directed against non-phosphorylated NF epitopes); pretreatment with alkaline phosphatase reversed this staining pattern. In a separate study, a similar alkaline phosphatase-sensitive lack of staining to antibody 2-135 was also observed in atrophic motor fibers in the DRG 4 weeks following nerve crush. It is suggested that aberrant NF phosphorylation in DRG neuronal cell bodies from IDPN-treated rats arises secondarily to an alteration in a retrogradely transported 'trophic' signal(s) to the neuron due to the presence of giant axonal swellings, Furthermore, pNFs in atrophic axons may correspond to stationary or slowly moving NFs in the axoplasm. INTRODUCTION Neurofilaments (NFs) are extensively phosphorylated in axons but are normally poorly phosphorylated in neuronal perikarya, as revealed by monoclonal antibodies 39' 56. This regional heterogeneity in the distribution of phosphorylated neurofilament (pNF) epitopes is altered in a number of human and experimental disorders including Alzheimer's disease, where they are found in neurofibrillary tangles 9,13,30,38,45,48,57, progressive supranuclear palsy t, m o t o r neuron disease 44'55, axotomy 2,22,54, and intoxication by the neurotoxic chemicals acrylamide 21'36, aluminum 3'45'59, and organophosphate 58. The significance of this alteration in any of these situations remains unclear. In motor neuron disease (amyotrophic lateral sclerosis; ALS), aberrant NF phosphorylation in neuronal perikarya 44'55 is associated with a massive accumulation of

NFs in the proximal axon 5-7'37. These giant axonal swellings are also rich in pNF epitopes 44,55 and production of neurofilamentous axonal swellings has been suggested 44'55 to result from an increase in N F phosphorylation in the axon. Whether the altered phosphorylation of axonal NFs is related to the aberrant expression of pNF epitopes in neuronal perikarya is unknown (see ref. 13). The relationship between neurofilamentous axonal swelling formation and abnormal N F phosphorylation in neuronal perikarya was examined using the model produced by fl,fl'-iminodipropionitrile (IDPN) intoxication. I D P N produces giant neurofilamentous axonal swellings in the first proximal internodes of large sensory and motor nerve fibers 29. The swellings are first noted 48 h following a single injection, achieve giant proportions by one week, and migrate distally along the axon with time 26'z9. In contrast, the swellings are maintained in the

Correspondence: B.G. Gold, Center for Research on Occupational and Environmental Toxicology, L606, The Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201-3098, U.S.A. Fax: (1) (503) 494-4278.

152 proximal area d u r i n g c o n t i n u o u s e x p o s u r e s . P r o d u c t i o n of these giant a x o n a l swellings by I D P N intoxication does n o t result in either a x o n a l d e g e n e r a t i o n n o r neuronal perikaryal loss. T h u s , although clearly n o t a m o d e l of A L S (see ref. 15), I D P N provides a m e a n s to directly address the role of swelling f o r m a t i o n in the p a t h o g e n esis of a b e r r a n t expression of p N F epitopes in n e u r o n a l perikarya. In the p r e s e n t study, we e x a m i n e d the distrib u t i o n of p N F epitopes in n e u r o n a l p e r i k a r y a a n d proximal axons, a n d c o m p a r e d the time course for any alteration in levels of N F p h o s p h o r y l a t i o n following single a n d c o n t i n u o u s m o d e l s of I D P N intoxication. MATERIALS AND METHODS

Animals and intoxication protocols Thirty-four 3-week-old male Sprague-Dawley rats were given a single intraperitoneal (i.p.) injection of IDPN (2 g/kg) and divided into two groups. One group was given 0.1% IDPN in the drinking water (continuous IDPN exposure group) for one (n = 2), 5 (n = 4), 7 (n = 4), 14 (n = 3), 21 (n = 4), or 49 (n = 4) days following i.p. injection. The other group was placed on tap water (single IDPN exposure group) for 7 (n = 2), 14 (n = 3), 21 (n = 3), 35 (n = 3) or 49 (n = 2) days. Four 3-week-old rats were injected with an equivalent volume of saline and studied at 35 days (i.e. 8 weeks of age). In a second study, 3 rats were given IDPN for two weeks, as above. At 5 weeks of age, the animals were anesthetized with chloral hydrate (400 mg/kg, i.p.) and the sciatic nerve crushed twice (for 30 s) on one side using a fine watchmaker's forceps. These animals were placed on IDPN in the drinking water for an additional week and studied one week following crush (i.e. 3 weeks continuous IDPN exposure). In a third study, six 3-week-old rats underwent a unilateral sciatic nerve crush, as above. These animals were studied at two (n = 2) and 4 (n = 4) weeks following crush. Tissue fixation and preparation At the time points given above, the animals were heparinized, anesthetized with chloral hydrate (400 mg/kg, i.p.) and perfused through the ascending aorta with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.6). The dorsal root ganglia (DRG), spinal cord, and spinal roots at the fourth and fifth lumbar levels (L4 and L 5, respectively), and sciatic nerve were dissected following overnight fixation in situ (4 °C). The tissues were dehydrated in graded series of alcohols and embedded in paraffin. Immunocytochemistry The immunocytochemical procedures using the present series of antibodies has been described previouslyel, Briefly, 10/~m sections were mounted on chrom-alum-subbed slides, deparaffinized, incubated for 1 h in 3% normal goat serum, and incubated overnight with one of the following primary antibodies (1:1000 dilution in 1% normal goat serum): antibody 2-135 (directed against a non-phosphorylated epitope of the 200 kilodalton (kDa) NF polypeptide); and antibodies 06-17 and 07-05 (directed against phosphorylated epitopes shared by the 200 and 145 kDa polypeptides)23'49's6. Sections were incubated for 1 h in goat-anti-mouse secondary antibody (1:20), washed, incubated for 1 h in mouse peroxidase-antiperoxidiase (1:200), and the immunoreactivity visualized with 0.05% diaminobenzidine tetrahydrochloride/0.01% hydrogen peroxidase (8 min). Alkaline phosphatase treatment of tissue sections Selected tissue sections from rats given continuous IDPN exposure for one (n = 1), 3 (n = 2), or 7 (n = 2) weeks were treated

with alkaline phosphatase (to remove phosphate groups) prior t,~ incubation with the primary antibody. Sections were incubated with alkaline phosphatase (400/~g/ml) type VII-N (Sigma) and 0.01 M phenylmethylsulfonylfluoride (Sigma) in 0.1 M Tris buffer (pH 8.0) for 16-18 h, as described by Sternberger and Sternberger56.

Assessment of immunostaining Immunoreactivity was quantitated as previously described ~e54. Immunoreactivity was quantitated by counting all cells containing immunoreactivity above background; background staining was minimal in all sections. The proportion of cells demonstrating pNF epitopes (% phosphorylated) was determined for each DRG by counting the total number of neuronal perikarya stained with antibody 7-05 and dividing this value by the total number of neuronal perikarya in the DRG (determined by counting the total number of neuronal perikarya stained with antibody 2-135 and lightly counterstained with Cresyl violet). Statistical analysis A two-way analysis of variance (ANOVA) was performed followed by Scheffe's post-hoc analysis to test for differences between individual treatment groups (i.e. continuous and single IDPN exposure groups) and time (days following the initial IDPN injection) in numbers of DRG cells demonstrating pNF epitopes. Student's t-test was used to test for differences between IDPN and IDPN/ crushed groups. All values are mean - S.E.M. RESULTS

Controls I n a g r e e m e n t with previous studies 16'21'22'54, i n t e n s e i m m u n o r e a c t i v i t y was p r e s e n t in n e u r o n a l p e r i k a r y a a n d axons using the a n t i b o d y against n o n - p h o s p h o r y l a t e d N F epitopes (2-135) (Fig. 1A). I n contrast, a n t i b o d i e s directed against p N F epitopes ( 6 - 1 7 a n d 7 - 0 5 ) stained only axons in L 4 a n d L 5 D R G (Fig. 1B).

I D P N studies: neuronal perikaryal staining N e u r o n a l p e r i k a r y a in the L 4 a n d L 5 D R G from I D P N - t r e a t e d a n i m a l s d e m o n s t r a t e d m o d e s t to i n t e n s e i m m u n o s t a i n i n g with a n t i b o d i e s 6 - 1 7 (not shown) a n d 7 - 0 5 b e g i n n i n g at 5 days (Fig. 1D; Table I); q u a n t i t a t i v e analysis r e v e a l e d i m m u n o r e a c t i v i t y to p N F epitopes in 36 --- 4.2% of D R G n e u r o n s . T h e n u m b e r of i m m u n o reactive n e u r o n s i n c r e a s e d to m a x i m a l levels ( 4 6 - 5 1 % ) at o n e w e e k in b o t h rats given a single dose of I D P N (Figs. 2 A a n d 3; Table I) a n d in those m a i n t a i n e d o n I D P N in their d r i n k i n g w a t e r (Figs. 2D a n d 3; Table I). H o w e v e r , m a r k e d differences b e t w e e n t r e a t m e n t groups were p r e s e n t at later time-points. T h e i n c i d e n c e of imm u n o s t a i n e d D R G n e u r o n s to p N F epitopes was m a i n t a i n e d at the p e a k level for at least 7 weeks in animals c o n t i n u o u s l y e x p o s e d to I D P N (Figs. 2B, C, a n d 3; Table I). In contrast, the percentage of D R G n e u r o n s expressing p N F epitopes declined significantly ( P < 0.001), beginning at 3 weeks, in animals not given I D P N in their drinking water (Figs. 2E, F, a n d 3; Table I). I m m u n o r e a c tivity to a n t i b o d i e s directed against p N F epitopes was n o t o b s e r v e d , h o w e v e r , in m o t o r n e u r o n s in the L 4 a n d

153

Fig. 1. Peroxidase-antiperoxidase staining of L 4 DRG from a control animal (A, B) and a 3-week-old rat given a single i.p. injection of IDPN (2 g/kg) followed by IDPN (0.1%) in the drinking water (continuous IDPN exposure) for 5 days (C, D). A, C: antibody 2-135, against non-phosphorylated NF epitopes, shows staining of neuronal perikarya and axons in the dorsal root (DR) and axons in the ventral root (VR). Insets: higher power views of motor fibers in the ventral root. Fewer motor axons are stained from the IDPN-treated rat (inset in C), compared to those from the control animal (inset in A). Regions devoid of immunoreactivity in cell bodies correspond to perikaryal nuclei. B, D: antibody 7-05, against pNF epitopes, shows intense axonal staining in D R G from both animals. Neuronal perikarya from the control animal are not stained (B), but many (see Table I) show modest to intense immunoreactivity from the IDPN-treated rat (D). x 110; insets: ×230.

154 L 5 spinal cords at any of the above time-points following either single or continuous IDPN administration (Fig. 4B, D, F). Immunoreactivity to antibody 2-135 in both sensory (Fig. 1C) and motor (Fig. 4C, E) neuronal perikarya from IDPN-treated rats was indistinguishable from controls (Figs. 1A and 4A, respectively). I D P N studies: axonal staining Giant axonal swellings in D R G (not shown) and anterior horns of the spinal cord showed intense immunoreactivity to pNF epitopes (Fig. 4D) and non-phosphorylated NF epitopes (Fig. 4C), as previously reported 4. In spinal cord, intense immunostaining to pNF epitopes was observed in swollen axons, whereas neighboring anterior horn cells lacked immunoreactivity (Fig. 4D); in some sections, direct continuity was observed between an axonal swelling and the cell body of a motor neuron (Fig. 4C, inset). A similar pattern of staining to nonphosphorylated NF (Fig. 4E) and pNF (Fig. 4F) epitopes was present in axonal swellings which had migrated 26 into the ventral root exit zone and proximal ventral root two weeks following a single injection of IDPN. The most striking alteration involving axons was a marked reduction in immunoreactivity to non-phospho-

TABLE I Comparison of the incidence of phosphorylated NF epitopes in DRG neuronal perikarya following continuous and single IDPN exposures

Percentage (mean ± S.E.M.) of neuronal perikarya stained with antibody 7-05 directed against phosphorylated NF epitopes (see Materials and Methods). n, number of animals; N/A, not available. % Phosphorylated Time (Days)

1 5 7 14 21 35 49

Continuous exposure

Singleexposure

0.3 ± 0.03 (,, = 2) 36 ± 2.1" (n = 4) 48 -+ 1.8 (n = 4) 45 ± 0.9

N/A N/A 50 ± 1.1 (n = 2) 43 -+ 0.6

(n = 3)

(n = 3)

50 ± 2.1 (n = 4) N/A

26 -+ 0.8* (n = 3) 6 ± 0.04* (n = 3) 7 + 0.04* (n = 2)

51 ± 1.2 (n = 4)

*P < 0.0001 compared to continuous exposure at 1, 7, 14, 21 and 49 days (two-way analysis of variance); *, P < 0.0001 compared to continuous exposure at 21 and 49 days and single exposure at 7-49 days (two-way analysis of variance); $, P < 0.0001 compared to continuous exposure at 21 and 49 days and single exposure at 7-21 days (two-way analysis of variance).

rylated NF epitopes and an intense immunostaining m antibodies directed against pNF epitopes in sensory and motor fibers in the L 4 and L5 spinal roots from animals given either single or continuous IDPN administration. This was best visualized in the L 4 and L 5 D R G where, in contrast to sensory fibers, motor fibers are located distal to the giant axonal swellings. At this level of the ventral root, the reduction in axonal staining of motor fibers to non-phosphorylated NF epitopes was first noted at 5 days (Fig. 1C). A complete lack of immunoreactivity was observed in many motor axons at the level of the D R G at all later time-points (i.e. 1-7 weeks) in animals continuously exposed to IDPN (Fig. 5A). In contrast, these fibers exhibited intense immunostaining to antibodies 6-17 (not shown) and 7-05 (Figs. ID and 5B). However, a return of the normal pattern of immunostaining was observed in motor axons by 5 weeks in animals not given IDPN in their drinking water (Fig. 5C, D); immunoreactivity to antibody 2-135 was again present in fibers in the ventral root (compare Fig. 5C with Fig. 1A, C). In contrast, dorsal root fibers in the D R G (Fig. 5 A - D ) and fibers in the sciatic nerve (not shown) from both IDPN exposure groups demonstrated intense immunoreactivity to antibodies directed against non-phosphorylated NF and pNF epitopes at all timepoints examined. The reduction in immunoreactivity to non-phosphorylated NF epitopes in the spinal root fibers from IDPNtreated animals could be due to masking of the antigenic sites by phosphate groups 39'56. To test this hypothesis, D R G in which motor axons demonstrated a reduction or a lack of immunoreactivity to antibody 2-135 (Fig. 6A) and intense immunostaining to antibody 7-05 (Fig. 6B) were selected and adjacent sections were pretreated with alkaline phosphatase (to remove phosphate groups) prior to incubation with the primary antibodies (see Materials and Methods). The pattern of immunostaining with these antibodies was reversed following pretreatment with alkaline phosphatase (Fig. 6C, D); intense immunoreactivity was now present to antibody 2-135 (Fig. 6C), but immunostaining was abolished to antibody 7-05 (Fig. 6D). Identical results were found using D R G from 3 additional IDPN-treated rats. Crush studies: neuronal perikaryal staining Aberrant expression of pNF epitopes was observed in approximately 45-50% of neuronal perikarya in L 4 and L5 D R G at two and 4 weeks following sciatic nerve crush, as previously described x6"54. In agreement with findings reported by other investigators ~1'24, immunostaining to pNF epitopes was not present in lumbar motor neurons following axotomy of the sciatic nerve. The number of immunoreactive D R G neurons to pNF

155 epitopes was not altered (P < 0.05) by axotomy in

addition, the intensity of staining in D R G n e u r o n a l

IDPN-treated rats. Immunoreactivity to antibody 7-05

perikarya from axotomized neurons was similar to that

in rats given continuous I D P N exposure for 3 weeks was present in 46 +-- 1.3% and 50 --- 1.6% of D R G n e u r o n s from crushed and non-crushed nerves, respectively. In

observed o n the contralateral non-crushed side from IDPN-treated animals (not shown).

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Fig. 2. Peroxidase-antiperoxidase staining of L 5 DRG to antibody 7-05 from rats given continuous IDPN exposure (for details, see legend in Fig. 1) (A-C) and rats given a single i.p. injection of IDPN only (D-F). Animals were studied at one (A, D), 3 (B, E), and 7 (C, F) weeks after IDPN injection. Immunoreactivity to pNF epitopes is observed in many neuronal perikarya from both treatment groups at one week (A, D) and from the continuous IDPN group at 3 (B) and 7 (C) weeks. However, the number of immunostained cell bodies from the single IDPN group appears to be reduced at 3 weeks (E) and is present in only a few neuronal perikarya at 7 weeks (F). For quantitation, see Fig. 3 and Table I. x130.

156

Crush studies: axonal staining A reduction or a complete lack of immunoreactivity to antibody 2-135 and intense staining to antibodies directed against pNF epitopes (6-17 and 7-05) was observed in ventral root fibers in the L 4 and L 5 DRG at 4 weeks, but not at two weeks, following axotomy alone (not shown). This pattern of staining was reversed by pretreatment of the sections with alkaline phosphatase (not shown), as observed above in tissue sections from IDPN-treated animals (see Fig. 6). DISCUSSION

The present study demonstrates that IDPN administration results in an increase in NF phosphorylation in peripheral neurons. Aberrant expression of pNF epitopes in neuronal perikarya is present in D R G neurons but not in anterior horn cells of the lumbar spinal cord. Furthermore, this abnormal NF phosphorylation in D R G neuronal perikarya is reversible following discontinuation of IDPN administration, but persists (for up to 7 weeks) in animals given continuous IDPN exposure. In addition, an increase in the level of NF phosphorylation is present in both sensory and motor axons, leading to an inability to detect (by immunocytochemistry) non-phosphorylated NF epitopes in the spinal roots distal to giant axonal swellings. The significance of these findings is discussed separately below.

Perikaryal neurofilament phosphorylation Development of aberrant expression of pNF epitopes in neuronal perikarya following a x o t o m y 2'22'54 suggests that this type of staining pattern (i.e., homogenous throughout the neuronal cytoplasm) represents a component of the cell body response to injury (axon reaction) in DRG neurons. Furthermore, our previous studies using colchicine 16 demonstrated that structural interruption of nerve and its target is not necessary for production of aberrant expression of pNF epitopes in D R G cells. The present study supports and extends this

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Fig. 3. Comparison of the number of immunostained neuronal perikarya in L4 and L~ DRG to pNF epitopes (% phosphorylated) from continuous (solid line) and single (dotted line) IDPN exposure groups. Cell body staining is maintained at the one-week level

in animals given continuous IDPN exposure, but falls off precipitously, beginning at 3 weeks, in rats given a single IDPN injection only (see Table I); bars indicate S.E.M. * P < 0.0001 (compared to continuous exposure at 21 and 49 days). initial observation by showing that aberrant NF phosphorylation can be induced in intact neurons containing IDPN-induced giant neurofilamentous axonal swellings. Evidence from previous electrophysiological studies in IDPN-intoxicated cats T M indicates that IDPB administration also recapitulates many of the axotomy-induced changes in electrical properties of the soma-dendritic membrane of motor neurons. Taken together, these findings suggest that IDPN neuropathy produces an axotomy-like response in the neuronal perikaryon despite a lack of axonal degeneration 15. The mechanism by which these axotomy-like alterations arise in IDPN-intoxicated animals without the presence of frank degeneration is unknown. At least 3 possibilities can be suggested. First, IDPN may have a direct effect on the neuronal perikaryon. Although this cannot be ruled out, such a mechanism seems unlikely based upon the in vivo metabolism of IDPN during the initial 48 h following a single 2 g/kg injection of the

Fig. 4. Peroxidase-antiperoxidase staining of L4 spinal cord from a control animal (A, B), from a rat given continuous IDPN exposure (for details, see legend in Fig. 1) for 7 weeks (C, D) and from a rat given a single i.p. injection of IDPN and studied at two weeks (E, F). A, B: antibody 2-135 shows staining of neuronal perikarya and neuronal processes (A) and antibody 7-05 shows staining of neuronal processes but not neuronal perikarya (asterisks) (B) in the anterior horn from a control animal. Insets: higher power views of stained (A) and nonstained (B) cell bodies. C-F: neurofilamentous swellings are present in the anterior horn of the spinal cord in the rat given continuous IDPN exposure (C, D) and have migrated into the ventral root exit zone (VREZ) and proximal ventral root by two weeks in the animal given only a single IDPN injection (E, F). C, E: antibody 2-135 shows staining of neuronal perikarya and giant axonal swellings (arrowheads) in the anterior horn following continuous IDPN exposure (C), and of axons in the VREZ (arrow) and proximal ventral root (VR) following a single IDPN injection (E). Inset (in C): higher power view of boxed region in C showing immunoreactive giant axonal swelling connected to an anterior horn cell. Inset (in E): higher power view of immunostained axonal swellings in the VREZ and proximal VR. D, F: antibody 6-17 shows lack of immunoreactivity in anterior horn ceils (asterisks); staining is present in giant axonal swellings in the anterior horn (D), and in axonal swellings in the VREZ and proximal VR (F). Insets: higher power views of immunoreactive giant swelling (D) and swollen axons (F). x80; Insets: x160 (A, B); ×285 (C, D); ×150 (E, F).

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Fig. 5. Peroxidase-antiperoxidase staining of L 5 D R G from a rat given continuous IDPN exposure (for details, see legend in Fig. 1) ff)r 7 weeks (A, B) and from a rat given a single i.p. injection of IDPN only (C, D) and studied at 5 weeks. A, C: antibody 2-135 shows staining of neuronal perikarya and axons in the dorsal root (DR); a reduction or a lack of immunoreactivity is apparent in motor axons in the ventral root (VR) following continuous exposure for up to 7 weeks (A), whereas a return to a normal (compare with Fig. 1A) intensity of immunoreactivity in motor axons is present 5 weeks after a single injection (C). B, D: antibody 7-05 shows staining of neuronal perikarya and axons in the D R and axons in the VR following both treatments. The extent of expression of pNF epitopes in cell bodies (see Table I) anti intensity of staining in motor axons appears greater following continuous exposure (C) compared to a single IDPN injection (D) at 5 weeks. xllO.

159

Fig. 6. Effect of alkaline phosphatase treatment on immunoreactivity to non-phosphorylated (A, C) and pNF (B, D) epitopes in L4 ventral root fibers at the level of the DRG in a rat given continuous IDPN exposure for 3 weeks. A, B: peroxidase-antiperoxidase staining without alkaline phosphatase pretreatment shows reduced or absence of axonal staining to antibody 2-135 (A) and intense immunoreactivity to antibody 7-05 (B). C, D: peroxidase-antiperoxidase staining of adjacent sections pretreated with alkaline phosphatase shows intense staining of motor axons to antibody 2-135 (C) and lack of immunoreactivity, as expected, to pNF epitopes (D). Identical results were obtained in DRG from 4 additional IDPN-treated rats. x440. toxin 63. In this context, it may be relevant that aberrant expression of pNF epitopes was still present, albeit in 0nly a few D R G neurons, up to 7 weeks following a single IDPN injection. Second, production of axotomy-like changes may arise from an alteration in current flow 12' ~9,4~ due to the presence of paranodally demyelinated giant axonal swellings adjacent to the initial segment of the a x o n 8'12'15'18'27'29'41. A major pathological difference between single vs continuous modes of I D P N exposure is that the giant axonal swellings are maintained in the first proximal internodes (adjacent to the initial segment) during continuous exposure a'27, but migrate down the axon (becoming smaller in size) following a single injection of the agent 26'29. Thus, migration of the swellings away from the initial segment 26 may explain the reduction over time in aberrant expression of pNF epitopes following a single IDPN injection. Third, axotomy-like alterations in IDPN-intoxicated animals may arise from an impairment in retrograde axonal transport through

the axonal swellings 14 of a target-derived 'trophic' signal (e.g. NGF) which regulates perikaryal function 1°A7,2°. This hypothesis is consistent with our previous finding of aberrant NF phosphorylation in D R G neurons following blockade of fast axonal transport using colchicine 16. Studies are in progress to examine whether the inhibition of retrograde axonal transport is reversible following discontinuation of IDPN administration; i.e., transport block is maintained only during continuous exposure. The present study also demonstrates that the number of D R G neurons expressing aberrant pNF epitopes in IDPN-treated animals is not increased by axotomy; previous studies using these antibodies 16'54 showed that aberrant NF phosphorylation is present in a maximum of 45-50% of D R G neurons following nerve crush. Taken together, these findings indicate that aberrant expression of pNF epitopes is an inherent property of a subpopulation of D R G cells.

160 In contrast, motor neurons, which also develop giant axonal swellings, did not demonstrate aberrant NF phosphorylation. Since immunoreactivity is also not observed in motor neurons of the rat following axotomy of the sciatic nerve 11'22, this may represent a difference in the response of rat motor and sensory neurons to injury. Alternatively, the demonstration that aberrant NF phosphorylation can be induced in motor neurons of the rat by a very proximal (ventral root) transection 42'53 leading to neuronal perikaryal degeneration 53 suggests that development of this phenomenon may represent a marker of cell death in dying anterior horn cells. Thus, the significance of the aberrant NF phosphorylation in motor neurons from ALS patients 44'55 remains unclear.

Axonal neurofilament phosphorylation An additional finding in the present study was a marked reduction or an absence of immunodetectable levels of non-phosphorylated NFs in motor and sensory fibers distal to giant axonal swellings in IDPN-treated rats. Our findings provide morphological support for a previous study 6x demonstrating an increase in NF phosphorylation in dorsal root fibers distal to IDPN-induced axonal swellings using quantitative biochemical (immunoassay) methods. This increased level of NF phosphorylation appears to coincide with the development of axonal atrophy distal to the swellingsS'6X; the similar changes in sensory and motor fibers despite the lack of aberrant expression of pNF epitopes in anterior horn cells (see above) argues against a direct relationship between the expression of pNF epitopes in D R G neurons and increased NF phosphorylation in sensory axons. Since axonal atrophy in IDPN-treated animals results from a reduction in delivery of NFs to the axon distal to the swelling 8, we asked whether a similar alteration in the pattern of immunoreactivity to these antibodies arises in a situation where axonal atrophy results from a different mechanism, i.e., axotomy. Axotomy also leads to a marked decrease in axonal caliber in the proximal portion of the axon 33'51. In contrast to IDPN neuropathy 5°, axotomy-induced axonal atrophy results from a reduction in NF synthesis in the neuronal perikarya 24'31'64 and a consequent decreased delivery of NFs to the proximal axon35; the atrophy advances in a somatofugal fashion (somatofugal axonal atrophy) down the nerve 33. Despite differences in levels of NF synthesis between these two models 5°, in both situations axonal atrophy develops due to a decreased delivery of NFs to the axon. It is therefore interesting that axotomy leads to an identical pattern of increased NF phosphorylation in motor fibers at the level of the D R G at a time corresponding to when somatofugal axonal atrophy would have reached this level of the ventral root33;

the significant increase observed at 4 weeks (as opposed to two weeks) in the present study appears to be supported by a recent quantitative (immunoassay) study ~'° showing a tendency towards increased NF phosphorylation in ventral root fibers at 3 weeks following axotomy. Thus, the present findings support 61 a correlation between increased NF phosphorylation in the axon and the development of axonal atrophy. The significance of the increase in NF phosphorylation in atrophic nerve fibers from either IDPN-treated animals and/or axotomized nerves is unknown. However, an increase in NF phosphorylation is also observed in IDPN-induced swellings 4 (present study). Thus, the alteration in NF phosphorylation does not appear to be due to axonal atrophy per se. Alternatively, increased NF phosphorylation may be related to the retardation in the rate of NF transport 6° observed in IDPN-treated animals 25'28. Furthermore, the increase in NF phosphorylation in dorsal and ventral root fibers from IDPN-treated animals parallels that observed during axonal maturation 61, another situation where there is a correlative slowing in NF transport 32'6e. However, the lack of a similar alteration in immunoreactivity to non-phosphorylated NF epitopes in the distal sciatic nerve (present study), despite a retardation in NF transport along the length of the nerve 28'65, indicates that a slowing in NF transport cannot completely explain the absence of immunodetectable levels of non-phosphorylated NFs in these axons. One feature common to both situations (i.e. IDPN-induced and axotomy-induced axonal atrophy) is a decreased delivery of newly synthesized NFs to the atrophic internodes; this arises due to a blockade in NF transport 25~28 during continuous IDPN administration where the swellings are maintained in the proximal internodes s or due to a reduction in NF synthesis following axotomy24'31'64. ThUS, the NFs visualized by phosphorylationdependent antibodies would correspond to those remaining in the internodes of atrophic nerve fibers. Taken together, our findings suggest that highly phosphorylated NFs correspond to stationary 46'47 or more slowly moving 61 NFs in the axoplasm. Moreover, a recent demonstration of reduced levels of pNF epitopes at nodes of Ranvier 43, where proportionately (compared to the adjacent internodes) fewer number of NFs would be expected to exist in the stationary NF phase 34, appears to support a correlation between the degree of NF phosphorylation and a retardation in the movement of these cytoskeletal structures 4°'56'6°. Thus, we propose that the increase in NF phosphorylation distal to IDPN-induced axonal swellings and in axotomized nerves is a reflection of the somatofugal progression of a reduced delivery of moving (poorly phosphorylated) NFs to the adjacent internode.

161 Acknowledgements. Supported by funds from the U.S. Public Health Service Grant NIH NS26265. The authors thank Drs. Nancy Sternberger and Ludwig Sternberger, University of Maryland, Bal-

timore, MD, USA, for theft generous gift of monoclonal antibodies, Ms. Toni Storm-Dickerson for preparation of Fig. 3, and Ms. Marcia I-Iindman for expert secretarial assistance.

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