An immunohistochemical marker for Wallerian degeneration of fibers in the central and peripheral nervous system

Brain Research 828 Ž1999. 41–59

Research report

An immunohistochemical marker for Wallerian degeneration of fibers in the central and peripheral nervous system Pedro Pesini b

a,1

, Jutta Kopp a , Helen Wong b , John H. Walsh b , Gunnar Grant a , Tomas Hokfelt ¨

a, )

a Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden Gastroenteric Biology Center (CURE), CHS 44-127, Department of Medicine, UniÕersity of California (UCLA), Los Angeles, CA 90024-1684, USA

Accepted 16 February 1999

Abstract This work was prompted by the accidental observation that a newly developed, affinity purified polyclonal antibody against the C-terminus of the neuropeptide tyrosine ŽNPY. Y1-receptor protein decorates degenerating fibers in the central nervous system ŽCNS.. This staining did not appear in control animals in which the antibody marked perikarya and dendrites at previously described locations wX. Zhang, L. Bao, Z.-Q. Xu, J. Kopp, U. Arvidsson, R. Elde, T. Hokfelt, Localization of neuropeptide Y Y1-receptors in the rat nervous ¨ system with special reference to somatic receptors on small dorsal root ganglion neurons, Proc. Natl. Acad. Sci. USA 91 Ž1994. 11738–11742x. Three models of experimental lesions were studied: sciatic nerve transection, spinal cord transection and parietal cortex thermocoagulation. In each model, animals were divided in groups Ž n s 2. and processed for indirect immunofluorescence at different time intervals up to 28 days post-lesion ŽPL. Žsee below.. All three experimental lesions produced a very intense immunolabeling of fibers in the projection pathways of the lesioned structures, strongly reminding of Wallerian degeneration ŽWD.. In the sciatic nerve, the staining first appeared on day 1 PL, was strongly increased on day 3 PL, then declined after 7 days and had almost completely disappeared after 14 days. In the CNS, the staining appeared later and was first observed on day 3 PL and remained for a longer period, thus showing different time courses in the brain and spinal cord as compared to the sciatic nerve. The labeling was completely abolished, both in the CNS and in the sciatic nerve, by pre-incubation of the Y1-R antibody with the immunogenic peptide at a dilution of 10y6 M. The appearance of the staining and its time course strongly suggest that the process was related to degenerating axons. Although the protein actually detected remains to be determined, it is suggested that the staining ability of this antibody could be used as a positive marker of axonal degeneration following experimental or naturally occurring lesions of the nervous system. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Brain lesion; Nerve injury; Neuropeptide Y ŽNPY.; NPY Y1-receptor; Tracing

1. Introduction Wallerian degeneration ŽWD. w61x offered an early approach to trace connections in the nervous system when visualized with the Marchi method w10x Žsee also Refs. w32,36x., which stains degenerating myelin. It has been followed by more sensitive methods which stain degenerating axons and terminals, in particular those developed by Glees w29x, Nauta w44x and by Fink and Heimer w24x Žsee also Ref. w32x.. The fundamental features of WD Žsee Refs. w23,32x., including axonal degeneration and myelin clear-

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Corresponding author. Fax: q 46-8-33-16-92; E-m ail: [email protected] 1 Present address: Departamento de Anatomıa, ´ Universidad de Santiago de Compostela, Facultad de Veterinaria, 27002 Lugo, Spain.

ance, are shared by lesioned fibers in both the peripheral and central nervous systems ŽPNS and CNS, respectively., although distinct differences exist with respect to the timing of the process. The first clearly observable morphological modification in degenerating fibers is the granular disintegration of the cytoskeleton usually considered to be slower in the CNS. Most of the events in WD have subsequently been shown to reflect changes in the antigenic characteristics of both axons and neuroglia allowing immunohistochemical techniques to be used as a powerful diagnostic tool in neurology w12,13,53x. In particular, some antibodies against non-phosphorylated epitopes of neurofilaments, which show a transient increased immunoreactivity after different kind of injuries, have shed light on the initial intraaxonal events and the fate of axonal debris in degenerating fibers w20,27,41,62x. In contrast, the staining of neurofilaments

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 2 8 3 - 4

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seen in normal rats with antibodies against phosphorylated epitopes is markedly attenuated after lesions w27,40,42x. This disappearance of labeling thus makes it possible to recognize degenerating nerves or central fiber tracts after axotomy, but this approach is not suitable to detect changes in situations of more widespread brain damage, for example after traumatic injuries or ischemia w41x. Furthermore, the anatomical information provided by staining for neurofilaments is rather complex due to the heterogeneous distribution of those epitopes in the nervous system and to the simultaneous labeling of a mesh of uninjured or regenerating fibers w17,30,39,40,51,52,55x. In this work we report, based on an initial accidental observation that a newly developed, affinity purified antibody raised against a synthetic 13 amino acid peptide from the C-terminus of the neuropeptide tyrosine ŽNPY. Y1-receptor ŽY1-R. protein decorates degenerating fibers both in the CNS and PNS. NPY is a 36-amino acid peptide isolated from bovine brain w56,57x. The first NPY receptor, the Y1-R Žsee Ref. w60x., was cloned some years ago w22,33,38x, and anti-Y1-R antibodies have been used to study its cellular localization w4,8,9,25,34,35,67x. The present affinity purified antibody also shows genuine Y1-Rlike immunoreactivity ŽLI. in perikarya and dendrites as

previously described w67x. The pattern of the labeling after lesions appears highly reproducible and distinct, and could not be detected in normal, not injured animals. We therefore decided to explore the remarkable staining ability of this antibody as a positive marker to visualize WD in the nervous system, by making lesions at three sites, the sciatic nerve, the spinal cord and the cerebral cortex. The guinea pig sciatic nerve was also analysed. We have also tried to see whether or not this antibody is actually labeling Y1-R protein in relation to the degenerating fibers using in situ hybridization.

2. Material and methods 2.1. Animals Three types of experimental lesions were performed in adult male Sprague–Dawley rats Žb.wt 300 to 400 g; B & K Universal, Stockholm, Sweden.: Ži. sciatic nerve transection, Žii. spinal cord transection and Žiii. parietal cortex electrocoagulation. In addition, a unilateral sciatic nerve transection was done in two guinea pigs Žb.wt 400 g; B & K Universal.. All surgical manipulations were done

Fig. 1. Ža–f. Immunofluorescence micrographs of frontal sections of the corpus callosum 3 days after cortical electrocoagulation Ža and d. and of transverse Žb and e. and longitudinal Žc and f. sections of part of the sciatic nerve distal to the lesion 3 days after crush-injury and after incubation with antiserum a96106 Ža–c. or after pre-absorption of the antiserum with the immunogenic peptide Ž370–382. Žd–f.. Intensely fluorescent bead-like structures can be seen both in the central and peripheral tissues distal to the lesion Ža–c., but not after absorption Žd–f.. Scale bar indicates 100 mm. All micrographs have the same magnification.

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under anaesthesia with 7% chloral hydrate Ž0.5 mlr100 g b.wt.. The experiments were approved by a local ethical committee. In addition some normal rats Žwithout any lesion. were analysed. Ži. The left sciatic nerve was transected at mid-thigh level. Žii. For spinal cord transection, the animal was placed in a holder to immobilize the vertebral column. The vein leading from the dorsal hibernating gland to the azygos vein, arising from the vertebral canal between T4 and T5 was used as a reference to localize the arch of vertebra T8 which was removed w19x. The median dorsal

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artery was slightly elevated, and its branches at this level were thermocoagulated. Then a complete transection of the spinal cord was performed with pairs of microdissection scissors. Žiii. For cortical lesion, the animal was placed in a David Kopf stereotaxic frame with the skull oriented according to the atlas of Konig and Klippel w37x, and a ¨ parietal cortex electrocoagulation was made under stereotaxic guidance. The electrode was lowered 1.5 mm from the dura mater at six sites Žcoordinates from bregma: antero-posterior y0.5 and y2; medio-lateral 2, 3, and 4 mm to the right w46x.. A stimulation current of 3 mA was

Fig. 2. Ža–b. Immunofluorescence micrographs of longitudinal sections of the sciatic nerve distal to nerve crush 3 days after the lesion incubated with antiserum a96106 Ža. or after pre-absorption with the full peptide Ž370–382. Žb., the C-terminal pentapeptide fragment Ž378–382. Žc., the mid-tetrapeptide fragment Ž374–377. Žd. or the N-terminal tetrapeptide fragment Ž370–373. Že.. Ža. Strongly fluorescent beaded fibers and dots as well as a small blood vessel Žarrowhead. can be seen after incubation with antiserum a96106. Žb. After absorption with the full peptide both bead-like structures and blood vessels are unstained. Žc. Pre-absorption with the C-terminal peptide results in strong attenuation of fluorescent beaded structures, whereas blood vessels Žarrowheads. still are positive. Žd and e. In contrast, after pre-absorption with the mid- or N-terminal fragments both beaded structures and blood vessels appear as strongly fluorescent as in controls Žcf. Žd., Že. with Ža... Scale bar indicates 50 mm. All micrographs have the same magnification.

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given during 10 s at each site. After each type of experimental lesion, the animals were separated in groups Ž n s 2. and allowed to survive 1, 3, 7 or 14 days after sciatic nerve axotomy, or 1, 3, 7, 14 and 28 days after the CNS lesions. Two additional groups of animals were included. In one of them the sciatic nerve was crushed for 10 s at the same level Žmid-thigh. with a watchmaker’s forceps instead of being transected, and the animals were allowed to survive for 3 days. The other group included five animals in which post-mortem examination at 3 days Ž n s 2., 7 days Ž n s 2. and 28 days Ž n s 1. revealed that the cortical lesion also affected the superficial layers of the septal part of the hippocampus. The animals were re-anaesthetized with sodium pentobarbital ŽMebumal w , 60 mgrkg. and perfused via the ascending aorta with a solution of 4% paraformaldehyde and 0.3% picric acid in 0.1 M phosphate buffer w64x. In the animals with sciatic nerve lesions, the nerve segments proximal and distal to the cut, the L 4 and L 5 dorsal root ganglia and spinal cord segments as well as the contralat-

eral nerve were dissected out, immersed in the same, ice cold fixative for 90 min and then in 15% sucrose in 0.1 M phosphate buffer for at least 24 h. The same protocol was used for the brain and spinal cord from the animals with cortical or spinal cord lesions. All tissues were then quickly frozen, cut into 14 mm thick sections in a cryostat ŽMicrom, Heidelberg, Germany. and mounted on chrome– alum, gelatine-coated slides. 2.2. Immunofluorescence A polyclonal rabbit antibody ŽY1-R ab a96106. against the C-terminus of the NPY Y1-R Žamino acids 370–382; AFKKISMNDNEKI. was produced in a rabbit and affinity purified at CURErGastroenteric Biology Center, AntibodyrRIA Core ŽWong and Walsh, unpublished.. In addition, some other rabbit antibodies raised against the same Žantiserum a96107; unpublished. or a longer w67x C-terminal fragment were tested. Indirect immunofluorescence staining Žsee Ref. w15x. was carried out, incubating the

Fig. 3. Ža–d. Immunofluorescence micrographs of longitudinal sections of the guinea pig sciatic nerve distal Ža and b. and proximal Žc. to nerve cut 3 days after lesion, and of guinea pig dorsal horn of the spinal cord Žd. incubated with antiserum a96106. Many fluorescent beaded elongated structures are seen distal Ža and b. but not proximal Žc. to the cut. No fluorescent structures can be seen in the guinea pig dorsal horn Žd.. Scale bar indicates 50 mm Ža s d; b s c..

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slides overnight at 48C with the primary antiserum Ždilution 1:400. and, after rinsing for 1 h at room temperature, with a fluorescein isothiocyanate-conjugated ŽFITC. donkey anti-rabbit antibody Ž1:80; Jackson Immuno Research, West Grove, PA, USA.. Some sections were double-stained with a mixture of the Y1-R antiserum Ž1:400. and monoclonal mouse antibodies to myelin basic protein ŽMBP, 1:500; Boehringer-Mannheim, Mannheim, Germany. or glial fibrillary acidic protein ŽGFAP, 1:50; BoehringerMannheim.. The secondary incubation was made with a

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mixture of FITC-conjugated donkey anti-rabbit and lissamine rhodamine B sulfonyl chloride ŽLRSC.-conjugated donkey anti-mouse antibodies Ž1:80; Jackson Immuno Research.. Furthermore, the staining with the monoclonal antibodies was controlled by replacing them by mouse IgG at the same concentrations. The sections were examined in a Nikon Microphot-FX microscope equipped for epifluorescence and with proper filter combinations. Some sections were processed for indirect immunofluorescence staining, based on the tyramide signal amplifica-

Fig. 4. Immunofluorescence micrographs of longitudinal sections of the sciatic nerve at various time points after crush ŽŽa. 1 day; Žb and e. 3 days; Žc. 7 days. and after incubation with antiserum a96106. d shows contralateral sciatic nerve 3 days after nerve crush. Ža. One day after nerve crush fairly long, beaded fluorescent structures can be seen although in low numbers. Žb. After 3 days the stained structures are shorter, more strongly beaded and occur more frequently. Žc. After 7 days only comparatively few dot-like structures can be observed. Žd. No positive structures can be seen on the contralateral side. Že. The beaded structures are mainly seen distal to the nerve crush, but some positive structures are also present in the crush area Žarrow. and nearby proximal portion of the nerve. The distal part of the nerve is to the right. Scale bars indicate 100 mm Ža s b s c s d..

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tion ŽTSA. method w1,6x by using a commercial kit ŽTSAindirect kit, DuPont, Boston, MA, USA.. In this case antiserum a96106 was diluted 1:4000. As secondary antibody we used horseradish peroxidase ŽHRP.-conjugated swine anti-rabbit immunoglobulin Ždiluted 1:100; Dako, Glostrup, Denmark.. The deposited biotin was detected with fluorescein-labeled streptavidin Ž1:500, TSA kit.. The sections were mounted and examined as described above. For control, sections were incubated with Y1-R antiserum a96106 pre-absorbed with an excess of the tridecapeptide ŽAFKKISMNDNEKI; aa 370–382., the N-terminal tetrapeptide ŽAFKK; aa 370–373., the mid-tetrapeptide ŽISMN; aa 374–377., the C-terminal pentapeptide ŽDNEKI; aa 378–382. of the tridecapeptide, a mixture of these three short peptides or a mixture of the mid- and C-terminal short peptides, all at 10y6 or 10y5 M. The penta- and tetrapeptides were purchased from MWG-Biotech, Ebersberg, Germany. 2.3. In situ hybridization In three adult, deeply anaesthetized male Sprague– Dawley rats Žas above. the left sciatic nerve was transected at mid-thigh level. After 3 days the animals were deeply anaesthetized Žas above., and the sciatic nerve segments distal and proximal to the lesion as well as the contralateral nerve were rapidly dissected and frozen. Longitudinal sections Ž14 mm. were cut in a cryostat ŽMicrom. and thawed onto ‘Probe On’ slides ŽFisher Scientific, Pittsburgh, PA, USA. and stored in sealed boxes at y208C until hybridization. An oligonucleotide complementary to nucleotides 546– 585 of the rat NPY Y1-R mRNA w21x was purchased from Scandinavian Gene Synthesis ŽKoping, Sweden.. The ¨ oligonucleotide probe was labeled at the 3X end with a-35 S-dATP ŽNew England Nuclear, Boston, MA, USA. using terminal deoxynucleotidyl transferase ŽAmersham, Amersham, UK.. The specific activities obtained ranged from 1 = 10 6 to 4 = 10 6 cpmrng oligonucleotide. Our procedure followed previously published protocols w16x. The slides were dipped in NTB2 nuclear track emul-

sion ŽKodak., exposed, developed and analysed in a Nikon Microphot-FX microscope equipped with a darkfield condenser or stained with Toluidine blue for viewing under brightfield. Control hybridizations were carried out with an excess of cold probe Ž100-fold. together with the labeled probe.

3. Results 3.1. General and methodological aspects Every experimental lesion model gave rise to particular and highly reproducible staining patterns ŽFigs. 1–10. that were completely abolished after pre-absorption of the primary antiserum a96106 with the tridecapeptide Y1-R peptide at 10y6 M Žcf. Fig. 1a–c, Fig. 2a with Fig. 1d–f, Fig. 2b, respectively.. In Fig. 2a the staining of the sciatic nerve is shown 3 days after nerve transection, and Fig. 2b shows the effect of pre-absorption with the tridecapeptide, that is the peptide used for immunization. When fragments of this peptide were analysed, there was a strong reduction in staining intensity after pre-absorption with the five amino acid C-terminal fragment of the tridecapeptide at 10y5 M ŽFig. 2c. as well as with the mix of all three small peptides, whereas no effect of the N-terminal tetrapeptide ŽFig. 2d. or the mid-tetrapeptide ŽFig. 2e. or a combination of these two peptides was observed. None of the other two Y1-R antisera Žw67x; a96107; Wong and Walsh, unpublished. raised against the same peptide fragment as antiserum a96106 or against a longer fragment, respectively, stained degenerating fibers, although they visualized the ‘genuine’ Y1-R-positive structures also seen with Y1-R antiserum a96106, for example the interneurons in lamina II of the dorsal horn Ždata not shown. Žsee below.. After hybridization of sciatic nerve segments in situ 3 days after axotomy with the Y1-R probe, no signal above background could be observed, neither proximal nor distal to the transection, but only over blood vessels Ždata not shown.. The degeneration-related staining patterns did not appear in control Žno lesion. animals, in which the antibody

Fig. 5. Immunofluorescence micrographs of sections of the spinal cord 3 days Ža–d. and 28 days Že–h. after spinal cord transection at the T8 level and after incubation with antiserum a96106. Ža, b, e and f. Sections cranial to the lesion; Žc, d, g and h. sections caudal to the lesion; Ža and e. cervical enlargement; Žb and f. T6 , Žc. a high lumbar segment, Žg. the T10 segment, and Žd and h. a lumbar enlargement. In all sections and at all levels a strong fluorescence Žcurved arrow. can be seen in lamina II of the dorsal horn, representing genuine Y1-R-LI present in local dorsal horn neurons. With regard to this staining, no certain difference can be seen above or below the lesion. The remaining fluorescent structures represent degenerating axons with different patterns above Ža, b, e and f. and below Žc, d, g and h. transection, whereby similar distribution patterns can be observed 7 Ža–d. and 28 Že–h. days after the transection. However, the reaction is much stronger after 3 days Ža–d. as compared to 4 weeks Že–h.. Here, we focus the description on the sections 3 days after transection Ža–d.. Ža and b. Cranial to the lesion numerous positive fibers are seen in the ventral Ždouble arrowhead. and lateral Žarrowhead. funiculi, whereas the dorsal part of the lateral funiculus virtually lacks positive fibers Žbig arrow.. The fasciculus gracilis Žsmall arrow. is strongly stained at the cervical level Ža., whereas the staining is weaker and more dispersed at T6 Žb.. Žc and d. Below the lesion positive fibers are seen in the lateral and ventral funiculi including the most medial part, where the uncrossed corticospinal tract runs Žthin arrow., as well as in the dorsal part of the lateral funiculus, where the rubrospinal tract is localized. Thick arrow points to the crossed corticospinal tract in the dorsal funiculus. Note lack of fibers in the gracile fascicles Žshort arrow.. As stated above, Že. – Žh. Ž28 days post-lesion. show essentially the same results but with a weaker fluorescence. Scale bar indicates 100 mm. All micrographs have the same magnification.

P. Pesini et al.r Brain Research 828 (1999) 41–59

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Fig. 6. Ža–g. Immunofluorescence micrographs of transverse Ža–f. and longitudinal Žg. sections of the dorsal spinal cord 3 Ža, d and g., 7 Žb and e. and 28 Žc and f. days after transection, above Ža, b, c and g. and below Žd–f; note that Žf. is more caudal than Žd. and Že.. the transection and after incubation with antiserum a96106. Ža–f. The immunoreactivity both in the fasciculus gracilis above the transection Ža–c., as well as in the crossed corticospinal tract below the transection Žd–f. is strongest 3 days Ža, d and g. after lesion and is then attenuated with time. However, the decrease in intensity and number of positive structures is more marked in the corticospinal tract than in the fasciculus gracilis. Scale bar indicates 100 mm Ža s b s c; d s e s f..

only marked perikarya and dendrites in locations described elsewhere w67x as well as blood vessels. Examples of such genuine Y1-R staining observed in the present study are

seen in blood vessels in the sciatic nerve ŽFig. 2a,c,d,e. and in the spinal cord, where a strong staining was found in lamina II of the dorsal horn ŽFig. 5a–h. as described

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Fig. 7. Immunofluorescence micrographs of transverse sections of the lower medulla oblongata Ža–c., cerebellum Žd and f. and pons Že. 3 days Ža, b, c, e and f. and 28 days Žd. after spinal cord transection and after incubation with antiserum a96106. Note presumably ‘genuine’ Y1-R1-LI in lamina II of the spinal trigeminal nucleus Žcurved arrow in Žc.. and in the parabrachial nucleus Žbig asterisk in Že.; diffuse fluorescence extending dorso-medially.. A strong immunoreactivity is seen in the fasciculus gracilis Žarrows in Ža. and Žc.. and in fibers ascending along the ventrolateral surface of the medulla oblongata ŽŽb. arrowheads in Žc.., presumably representing spinocerebellar fibers. Spinocerebellar fibers Žarrowheads. can also be seen in the ventral spinocerebellar tract in Že. and entering the cerebellum Žbig arrowhead in Žf... Note the weakly fluorescent fibersrbeads terminating in the granular layer Žsmall arrowheads in Žd. and Žf... Small asterisk in Že. shows superior cerebellar peduncle, big asterisk the lateral parabrachial nucleus. Small asterisk in Žf. indicates the molecular layer and large asterisk the granular layer in cerebellum. Scale bars indicate 100 mm Žb s e..

previously w67x. The blood vessel staining was not absorbed with any of the three peptide fragments ŽFig. 2c–e., but was completely abolished by the full peptide ŽFig. 2b.. Stained degenerating nerve fibers with a similar appearance were also observed in guinea pig sciatic nerve distal to a nerve cut made 3 days previously ŽFig. 3a,b.. No such

fibers were seen contralaterally or proximal to the cut ŽFig. 3c., nor was any staining seen in blood vessels or in the dorsal horn of the spinal cord ŽFig. 3d.. In this study we mainly used the routine indirect immunofluorescence method originally described by Coons and collaborators Žsee Ref. w15x., since this approach pro-

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duced a strong staining of the degenerating axons vs. a weak staining of ‘genuine’ Y1-R-positive neurons. Such a favourable situation was not observed when using the, in general terms, ‘more sensitive’ TSA method, since in

some regions the ‘genuine’ Y1-R staining obscured the ‘Y1-R’-decorated, degenerating axons Ždata not shown; but see Kopp et al., in preparation.. Two possible areas, where ‘genuine’ Y1-R-LI may overlap with ‘degeneration’

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staining are the dorsal thalamus and the parabrachial nucleus. Here a diffuse, probably ‘genuine’ Y1-R-LI partly mixed with dot-like fibers could be seen Žsee below.. No labeling was seen when the sections were looked at with other filter combinations, and thus no evidence for a contribution of autofluorescence Že.g., lipids droplets. within the degenerating fibers was obtained. As a rule, the immunoreactive material observed after any of the experimental lesions was composed of intensely fluorescent dots, and of elongated structures of variable length. When the nerve or tracts were cut longitudinally, these dots were aligned in rows, often forming beaded fibers during the first week post-lesion, for example in the cingulate cortex and in the spinal cord gray matter Žsee below.. A conspicuous exception to this general rule was the uniformly small, evenly distributed dust-like material found in the thalamus after ipsilateral cortical lesions, but this may represent ‘genuine’ Y1-R-LI Žsee above and below.. Double-staining of the sciatic nerve with MBP antiserum revealed that many ‘Y1-R’-positive fragments were surrounded by myelin sheaths 3 days after axotomy Ždata not shown.. 3.2. Sciatic nerÕe lesions Stained degenerating nerve fibers were only seen in the segment distal to the cut. One day after axotomy fairly long segments of a small proportion of the nerve fibers were labeled ŽFig. 4a.. By day 3 many nerve fibers were stained but now appeared more fragmented, forming short segments or rows of dots localized along the neurites inside the myelin sheath showing different degrees of segmentation ŽFig. 4b.. By the end of the first week the staining was strongly reduced with a few intensely fluorescent dots scattered along the distal segment of the nerve ŽFig. 4c.. The number of these dots further diminished with time, and by day 14 almost all the labeling had disappeared and only a very few isolated dots were found Žnot shown.. In the crushed nerves the labeling of the nerve was less dense on day 3 Žcf. Fig. 4e,b., and as seen both in transverse and longitudinal sections of the most distal branches, many fibers were not labeled Žsee also Fig. 1b,c.. No labeled degenerating fibers were observed in the contralateral sciatic nerve at any time interval studied ŽFig. 4d., nor was any staining seen in the L 4 –L 5 dorsal root ganglia or spinal cord segments Ždata not shown..

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3.3. Spinal cord transection The distribution of the labeling within the spinal cord white matter was different when comparing segments rostral and caudal to the transection. The highest density and most extensive distribution of the staining was found 3 days after lesion. We will first describe the pattern of the staining at this time point ŽFig. 5a–d. and then consider the aspects related to its time course. At all levels, without any certain difference when compared to normal spinal cords, a strong Y1-R-LI was observed in lamina II of the dorsal horn ŽFig. 5a–h., which at higher magnification could be resolved into small cell bodies and numerous processes Žnot shown.. These cells and processes are known to represent ‘genuine’ Y1-R-positive structures w67x. Rostral to the transection transversely cut, stained fibers were found peripherally in the white matter of the ventral and lateral funiculi ŽFig. 5a,b.. The density decreased towards the deeper parts of the white matter, but close to the lesion ŽT6 . there was a substantial number of positive fibers also more deeply. The most dorsal part of the lateral funiculus lacked positive fibers ŽFig. 5a,b.. Immunoreactive fibers were also seen in the fasciculus gracilis of the dorsal columns ŽFig. 5a,b. with a more dense and distinct staining at some distance from the transection Žcervical level; Fig. 5a., as compared to close to it ŽT6 ; Fig. 5b.. The cuneate fasciculus was devoid of positive fibers. Below the transection the lateral and ventral funiculi were strongly labeled but with a higher density more close to the lesion Žhigh lumbar level; Fig. 5c. than in the most caudal segments Žlumbar level; Fig. 5d.. In these segments, the staining continued medially to the ventral median fissure to the area occupied by the uncrossed fibers of the corticospinal tract ŽFig. 5c,d.. The dorsal part of the lateral funiculus, close to the neck of the dorsal horn where the rubrospinal tract is localized, was also labeled in the lumbar segments ŽFig. 5c,d.. The most striking difference in the distribution of labeled fibers occurred in the dorsal funiculus, when compared to the segments situated rostral to the transection. Thus, within the low thoracic and lumbar segments only the corticospinal tract fibers were stained in this funiculus ŽFig. 5c,d.. Essentially the same staining patterns were observed 28 days after the lesion, but much fewer fluorescent fibers could be detected ŽFig. 5e–h.. In particular, the staining of the deep dorsal columns had almost disappeared below the

Fig. 8. Immunofluorescence micrographs of transverse sections of corpus callosum Ža–d., pons Že–g., and of cervical Žh–i. and lumbar Žj and k. spinal cord 7 Ža, c, f, h and j. and 28 Žb, d, g, i and k. days after electrocoagulation of the right cortex and after incubation with antiserum a96106. Že. Contralateral pons from the same animal and level as Žf.. Ža–d. Note fluorescent beads Žarrowheads in c. both 7 Ža and c. and 28 Žb and d. days after lesion. Že. No fluorescent structures can be seen contralaterally. Žf–k. The degenerating fibers can be followed on the ipsilateral side in the cerebral peduncle Žf and g., as well as in the contralateral corticospinal tract in the deep dorsal columns Žh–k.. Note that the immunoreactivity is approximately of the same strength 7 and 28 days in the corpus callosum Žcf. Žc. with Žd.., whereas there is a marked attenuation over time at the spinal level Žcf. Ži., Žk. with Žh., Žj... Scale bars indicate 100 mm Ža s b; c s d; e s f s g; h s i s k..

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Fig. 9. Ža–d. Immunofluorescence micrographs of frontal sections of cortex Ža and d. and thalamus Žb and c. 7 days Ža, b and d. and 28 days Žc. after cortical lesion Žto the right in a. and after incubation with antiserum a96106. Ža and d. Fluorescent degenerating fibers can be seen in corpus callosum Žasterisk in Ža.. traversing the midline to the contralateral side Žarrowheads in Ža... Note fluorescent fibers entering the cingulate cortex Ždouble-arrowhead in Ža.. with fine fibers running to the superficial layers Žarrowheads in Žd... Žb and c. Fluorescent, degenerating, dot-like fibers are also seen in the internal capsule and along the ventral border of the stria terminalis Žbig asterisk in Žb.. in the dorsal thalamus. In addition, diffuse, presumably ‘genuine’ Y1-R LI can be seen in the reticular, the ventral anterior–lateral Žarrowheads in Žc.., the lateral dorsal Žarrow in Žc.. and anteroventral thalamic nuclei Žb and c., as well as in caudate putamen Žsmall asterisk in b. Žcf. Kopp et al., in preparation.. Curved arrows in Žb. indicate the sharp border formed by fluorescent degenerating fibers along the lateral border of the anterodorsal thalamic nucleus Žb.. Note spongy appearance of the tissue in some labeled thalamic areas Žasterisk in Žc... Scale bars indicates 100 mm.

lesion ŽFig. 5g,h., and the staining was in general weaker in the more distal segments ŽFig. 5e,h..

In Fig. 6, the staining patterns of the dorsal columns are detailed at a higher magnification showing the decrease in

P. Pesini et al.r Brain Research 828 (1999) 41–59

staining with time, 3 ŽFig. 6a., 7 ŽFig. 6b. and 28 ŽFig. 6c. days after transection, as well as the strong bilateral staining in the deep dorsal column Žcorticospinal tract. below the transection 3 ŽFig. 6d., 7 ŽFig. 6e. and 28 ŽFig. 6f. days after lesion with a dramatic decrease at the latest time interval. Fig. 6g shows a horizontal section through the deep dorsal columns below the lesion with dot-like staining patterns following the myelinated axons 3 days after the lesion. At the leÕel of the caudal medulla oblongata labeled fibers were found in the nucleus gracilis ŽFig. 7a,c. and in the ventrolateral part ŽFig. 7c.. Positive fibers were mostly localized between the lateral reticular nucleus and the medullary surface but also extending into the reticular formation ŽFig. 7b,c.. Again, the ‘genuine’ Y1-R staining was found in lamina II of the spinal trigeminal nucleus ŽFig. 7c.. More rostrally, labeled fibers appeared in both the dorsal and ventral spinocerebellar tracts that could be followed entering the cerebellum through the ventral spinocerebellar tract ŽFig. 7e,f., with very thin, weakly fluorescent fibers in the cerebellar white matter terminating within the granular layer in vermis of the anterior cerebellar lobe ŽFig. 7d,f.. Note that in the dorsal parabrachial nucleus a diffuse, presumably ‘genuine’ Y1R-Li could be seen ŽFig. 7e.. In summary, the staining patterns described above were maintained during the whole period studied, although in general a substantial reduction in the density of immunoreactive material was observed after 14 days and a further decrease after 28 days. This clearly was the case in the lateral and ventral funiculi, where the most substantial reduction of the labeling occurred caudal to the transection in the ventral part of the lateral funiculus and rostral to the transection in the ventral funiculus. Both these parts were almost devoid of labeled fibers 28 days after the spinal cord transection. Within the dorsal funiculus, the corticospinal tract followed a similar time course and by day 28 contained only small amounts of immunoreactive dots. In contrast, the labeling in the fasciculus gracilis, as well as in the above mentioned areas of the medulla oblongata and cerebellum, was strongest after 3 days, decreased after 7 days but was then maintained with a similar intensity throughout the period of time studied. 3.4. Parietal cortex lesion The electrocoagulation procedure yielded a lesion of the cortex which was approximately 4 mm wide, and extended from bregma q0.5 mm to y3.0 mm in the rostro-caudal direction. This area included most of the hindlimb and forelimb cortical regions as well as part of the parietal cortical area 1 and the caudo-lateral portion of the frontal cortex. One day after surgery changes could not be seen in the projection pathways of the parietal cortex, nor in their terminal areas Žcf. below.. After 3 days, the pattern of

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staining appeared fully developed, and no further modifications in the topographic distribution of the labeling could be observed throughout the time period studied. A description of the most distinct features of this pattern will follow. A wide band of densely packed immunoreactive dots of variable size was found within the ventral aspects of the corpus callosum from approximately bregma y0.5 mm to its caudal end ŽFig. 8a–d, Fig. 9a.. This pattern of labeling, though with a more loose appearance, could be followed along the dorsal half of both the contralateral and ipsilateral external capsule beyond the lateral border of the lesion. On the ipsilateral side, however, the highest density of labeled fibers was found entering the internal capsule where they coursed ventrally ŽFig. 9b, Fig. 10a.. At middiencephalic level, a dense band of immunoreactive material branched from the internal capsule, bent medially under the stria terminalis and penetrated into the thalamus ŽFig. 9b,c, Fig. 10a,c,d.. This band terminated medially in rather sharply delineated areas containing fine immunoreactive dots within the laterodorsal and ventroposterior nuclear groups ŽFig. 9b,c, Fig. 10a,c,d.. These areas with a high density of fluorescent dots had varying intensities, and in addition to the dot-like structures, a more diffuse background fluorescence was observed ŽFig. 9c, Fig. 10a,c,d.. Eventually, the remaining labeled fibers in the internal capsule bent caudally and appeared in a relatively well circumscribed patch within the ipsilateral cerebral peduncle along the entire brain stem ŽFig. 8f–g.. Caudal to the pyramidal decussation, the contralateral corticospinal tract in the deep dorsal columns contained high amounts of immunoreactive material that could be observed down to the lumbar segments of the spinal cord ŽFig. 8h–k.. In addition, a conspicuous group of densely packed immunoreactive dots appeared on the dorsomedial aspect of the ipsilateral cingulum in parallel to the rostrocaudal extension of the lesion ŽFig. 9a.. From there, a small amount of immunoreactive dots and beaded fibers spread towards layer I of the cingulate cortex, where a low density of stained fibers was found ŽFig. 9d.. Sparse labeling was also found in the deepest layers of the ipsilateral frontal, retrosplenial and occipital cortical regions as well as in the contralateral parietal and cingulate cortex ŽFig. 9a.. In the group of animals, in which the superficial layers of the septal hippocampus were damaged together with the cortical areas, the pattern of staining described above was supplemented by the appearance of labeled fibers in the ipsilateral fornix ŽFig. 10e. along its course from the rostral hippocampus to the mammillary body. Moreover, in these animals a wide band of densely packed immunoreactive material occupied the dorso-caudal part of the alveus ŽFig. 10b., and a sparse labeling appeared in the dorsal part of the ipsilateral subiculum and pre-subiculum. As mentioned above, the intensity of the staining in brain areas remained without significant changes throughout the period of time studied ŽFig. 8a–g, Fig. 10a,c,d..

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However, within the spinal cord, a substantial decay of the labeling was observed in the corticospinal tract of the

animals sacrificed 28 days post-surgery ŽFig. 8h–k.. In addition, 14 days post-surgery the tissue in some of the

P. Pesini et al.r Brain Research 828 (1999) 41–59

labeled areas of the thalamus acquired a spongy appearance ŽFig. 9c, Fig. 10c,d.. Double-staining with the antibody against GFAP showed an increase in reactive astrocytes in these areas Žnot shown..

4. Discussion The main finding of this work is that a recently raised, affinity purified NPY Y1-R antiserum ŽY1-R ab a96106. decorates anterogradely degenerating fibers both in the CNS and PNS. The pattern of staining was very distinct, highly reproducible, and the ‘Y1-R’ double-staining with MBP antiserum revealed that the immunoreactivity was confined to the neurite process and not associated with the myelin sheath. Moreover, the pattern and time course were dependent of the site of the lesion. It could in almost all cases be easily distinguished from the ‘genuine’ Y1-R staining patterns seen both in normal and injured animals w67x; see the work of Kopp et al., to be published.. Briefly, it has been found that ‘genuine’ Y1-R-LI can be observed in numerous brain regions, with a particularly strong staining in, for example, the arcuate nucleus, the dorsal horn of the spinal cord, and in some peripheral tissues such as dorsal root ganglia w67x. These studies suggest that the Y1-R at least mainly is localized to cell bodies andror dendrites, in agreement with the classification by Wahlestedt et al. w60x of Y1-Rs as post-synaptic Žand Y2-receptors as pre-synaptic.. With two exceptions the lesion did not seem to ‘interfere’ with this ‘genuine’ Y1-R staining, such as the Y1-R in the dorsal horn of the spinal cord and in the arcuate nucleus, when the tissues were processed according to the routine, indirect immunofluorescence methodology of Coons Žsee Ref. w15x.. In fact, all micrographs in this study are from this material. The exceptions were found in some areas of the dorsal, lateral aspects of the thalamus, where a diffuse Y1-R-LI overlapped with the dot-like degenerating fibers, and possibly in the parabrachial nucleus. In controls immunoreactivity could normally not be observed in these areas. However, in the extensive mapping of Y1-R systems now under way, we employ the TSA method w1,6x which reveals a much more

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extensive distribution of the Y1-Rs ŽKopp et al., to be published., including dorsal lateral thalamic areas and the parabrachial nucleus. It is thus possible that the lesion has in an unknown way induced an increase in Y1-R levels in these regions, alternatively that in these particular sections the Coons method was sensitive enough to detect genuine Y1-R-LI. The localization, appearance and time course of the stain closely followed the sequence seen in WD w61x. In particular, our results after axotomy or crush of the sciatic nerve were essentially identical to those reported, when silver impregnation or antisera against neurofilaments were used w7x. With regard to the CNS, the labeling appeared primarily within the fiber tracts passing through or coming from the lesioned area. This could be clearly seen after transection of the spinal cord at the T8 level. Caudal to that point, the bulk of the immunoreactive material was found overlapping the areas of the descending corticospinal and rubrospinal tracts, including the uncrossed component of the corticospinal tract in the ventral funiculus w2,11,65x as well as bulbospinal systems Žcf. Refs. w5,14,18x.. The former tracts were both unlabeled at upper thoracic and cervical levels, that is rostral to the lesion. Here, instead, the staining appeared concentrated to the ascending gracilis tract and in the dorsal and ventral spinocerebellar tracts extending up to their respective termination sites in the gracile nucleus and the granular layer of the rostral vermis w58,63x. Most of the remaining labeling appearing in the lateral and ventral funiculi within the immediate vicinity of the transection area could be accounted for by the staining of propriospinal fibers andror fibers arising from the grey matter at these levels and traversing the lateral and ventral funiculi towards their final destination in one of the principal ascending or descending pathways w58x. From our studies of the spinal cord it could not be determined if other ascending tracts, mainly the spinothalamic and spinoreticular tracts, were labeled. This is because their fibers widely overlap with those of the spinocerebellar tracts w28,43,63,66x, but in contrast to the degeneration seen in cerebellum, no degenerative changes were observed in the terminal fields or in the brain pathways of the

Fig. 10. Ža–e. Immunofluorescence micrographs of frontal sections of thalamus 7 Ža., 14 Žc., and 28 Žd. days following parietal cortex lesion Ža, c and d., and of the most dorsal aspects of hippocampusrcorpus callosumrbasal cortex Žb. and the anterior fornix Že. 28 Žb. and 7 Že. days, respectively, following cortical lesion also including the superficial layers of the septal hippocampus Žb and e. and after incubation with antiserum a96106. Ža, c and d. Fluorescent, degenerating, dot-like fibers are seen at 7 Ža., 14 Žc., and 28 Žd. days following cortical lesion. Degenerating fibers are seen in the internal capsule Žarrowheads in Ža.. and along the ventral surface of the stria terminalis Ždouble-arrowheads in Ža.. as well as in dorsolateral aspects of the thalamus Žasterisks in Ža., Žc. and Žd... These nuclei also exhibit presumably ‘genuine’, diffuse Y1-R LI Žarrow in Ža.; arrowhead in Žd.., being strongest in the lateral dorsal nucleus of the thalamus Žarrowhead in Žc.. Žcf. Kopp et al., in preparation.. Note the sharp border between weak ‘genuine’, diffuse Y1-R LI and immunofluorescent degenerating fibers in the thalamic lateral dorsal and lateral posterior nuclei towards the central lateral thalamic nucleus Žarrows in Žd... Aslo note the spongy appearance of the tissue in some thalamic areas Žasterisk in Žc. and Žd... Žb and e. Densely packed fluorescent, degenerating fibers Žarrowheads in Žb.. are seen in the alveus but not in the external capsule Žbig asterisk. 28 days following a cortical lesion affecting the superficial hippocampus Žb.. Scattered immunoreactive dots also appear in the deepest layers of the occipital cortex Žb.. Note some weakly stained immunoreactive cell bodies with processes Ždouble-arrowheads in Žb.. in the hippocampus representing ‘genuine’ Y1-R LI Žcf. Kopp et al. in preparation. Žsmall asterisk b.. Že. Scattered fluorescent degenerating fibers are seen in the ipsilateral anterior fornix Žarrowheads in Že.., whereas no such fibers were observed contralaterally Žasterisk in Že... Scale bars indicate 50 mm.

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spinothalamic and spinoreticular tracts. The lack of labeling in the dorsal part of the lateral funiculus, which is a site for ascending spinal axons of lamina I neurons Žsee Ref. w58x., could be a further indication that fibers ascending to the midbrain and medullary reticular formation, maybe also to the thalamus, are not labeled by the antibody. Thus, our results suggest that degenerating axons in these systems did not express the protein recognized by Y1-R ab a96106 or at least not at levels detectable by our immunohistochemical procedure. Variability in the staining of degenerating axons has been observed ever since the introduction of the silver based methods for identifying degenerating fibers, and it was thought that they could be associated with fine structural differences w32x. More recently, the use of a wide panel of monoclonal antibodies against different neurofilament epitopes has shown a considerable heterogeneity in the degree of phosphorylation of this important component of the cytoskeleton, even between the perikaryon and its axon or along a single axon w17,39,45,49,51,52x. Furthermore, in some cases at least, these differences in the neurofilament lattice network and phosphorylation state seem to occur as a function of the cell type and are independent of the length or caliber of the axons w55x. Thus, the absence of labeling that we found in the spinothalamic and spinoreticular tracts might be signalling a metabolic or allosteric peculiarity in the chemoarchitecture of these axons. Unfortunately, detailed studies on the cytoskeleton of the spinothalamic or spinoreticular axons that could allow us to go further in the discussion of this point are not available from the literature and the present explanation should be considered hypothetical. The electrocoagulation of the cortex yielded immunoreactivity primarily in the corpus callosum and in the ipsilateral corticothalamic and corticospinal projections. Caudal to the pyramidal decussation the contralateral corticospinal tract was labeled down to lumbar levels, and scattered immunoreactive dots were also seen in the ipsilateral ventral funiculus, close to the wall of the median sulcus, where the uncrossed fibers of the ventral corticospinal tract are localized w11,14x. In general terms, these finding agree with the distribution of fronto-parietal cortex projection pathways w68x. On the other hand, the labeling extending from the cingulum to the superficial layer of cingulate cortex as well as the sparse immunoreactive material found in the ipsilateral retrosplenial occipital and frontal cortex, could be explained by a possible affection of thalamo-cortical fibers passing through the area of the lesion. The topography of this labeling was coincident with the projection areas of the anterior thalamic nuclei w48,50x. In addition, the presence of immunoreactive dots along the ipsilateral fornix and in Žlimited. areas of the alveus in those animals in which the lesion affected the dorsal part of the septal hippocampus, further supports an anatomical correspondence between the distribution of stained fibers and the lesioned structures.

An intriguing result was the labeling found in several ipsilateral thalamic nuclei after cortical lesion. These areas were coated with evenly distributed and uniform small dust-like dots, perhaps representing terminal fields. However, its localization presented some mismatch with the expected projections from the injured cortex, since for example a high density of these dots was found in the laterodorsal nuclei which do not receive afferents from the parietal cortex w48x. Rather surprisingly, this labeling reminded of the fine silver grain deposits described by Fink and Heimer w24x in the thalamus after cingular ipsilateral decortication. This phenomenon was attributed to extensive retrograde cell reaction and death, including the precipitation of silver grain in the degenerating dendritic trees of axotomized neurones w31,32x Žcf. Ref. w47x. or possibly of collaterals. Our results were congruent with this explanation, since the areas coated with the immunoreactive dust showed an increased spongy appearance 14 days post-lesion, and double-staining of Y1-R ab a96106 with GFAP revealed the presence of reactive astrocytes within or around these same areas. As discussed above, it cannot be excluded that in these areas there is an overlap with, possibly upregulated, ‘genuine’ Y1-R-LI. The maintenance of the labeling in the CNS throughout the whole span of time studied in this work was in agreement with the well established fact that the elimination of degenerating fibers in the CNS is a slow process that lasts for several months w7,54x. The discussion of the possible causes of this remarkable difference between the CNS and the PNS, where the axonal debris almost completely disappeared in a few weeks, as well as its implications with respect to the capability of axonal regeneration are beyond the scope of this work. However, our results showed a more rapid elimination of the immunoreactive material in the spinal cord than in the brain. This differentiated decay of the staining could be appreciated both in the animals submitted to spinal cord transection and those to cortical electrocoagulation. Moreover, the transection of the spinal cord revealed regional differences in the pace at which debris was eliminated, particularly between the gracilis and the corticospinal tracts. Though detailed studies have compared the time course of WD between the CNS and PNS w3,7,26,27x, similar systematic comparisons between different pathways within the CNS seem to be lacking in the literature. At the present moment we cannot determine if this variable pace of elimination of the debris labeled by Y1-R ab a96106 is due to intrinsic characteristics of the fibers themselves or is related to differences in the local response of the microgliarmacrophages. The epitope protein actually detected by Y1-R ab a96106 remains to be determined. The possibility that it labels Y1-R protein within the degenerating fibers is highly improbable. The Y1-R is considered to be exclusively post-synaptic, and its presence in axons has never been described w59,67x. Moreover, in situ hybridization experiments including control and transected sciatic nerves dis-

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sected 3 or 7 days post-lesion did not detect Y1-R mRNA within cells either in the neuroma or in the nerve itself, other than along blood vessel walls, where Y1-R is expressed under normal conditions w4x Žas always with immunohistochemistry, it is possible, however, that the signal is too weak to be detected with our in situ hybridization technique.. Also, whereas the C-terminal pentapeptide strongly reduced the degeneration-related staining, it did not seem to affect the specific Y1-R staining of blood vessel walls. Thus it presumably sees something different than the Y1-R. This is also supported by the analysis of the guinea pig sciatic nerve and spinal cord. Thus, fluorescent elongated, beaded structures were seen to the same extent in this species as in the rat distal, but not proximal to the lesion. Since no staining could be observed in the guinea pig dorsal horn it is unlikely that this antibody sees the Y1-R in guinea pig tissue, further supporting the view that the degeneration staining recognizes an epitope completely unrelated to the Y1-R. In conclusion, although the protein actually detected by Y1-R ab a96106 remains to be determined, its staining ability could be used as a positive marker of axonal degeneration in experimental or naturally occurring lesions of the nervous system.

Acknowledgements

˚ We thank Katarina Aman for her excellent technical work, and Johan Widenfalk for his advise on the surgery and maintenance of spinal cord transected animals. This work was supported by grants from the Swedish MRC Ž04X-2887., the EC ŽBMH4-CT95-0172., Marianne och Marcus Wallenbergs Foundation, Knut and Alice Wallenbergs Foundation as well as an Unrestricted Grant for Neuroscience Research from the Bristol-Myers Squibb Foundation. Pedro Pesini was supported by a grant from the Spanish Direccion ´ General de Investigacion ´ Cientifica y Ensenanza ˜ Superior ŽRef. PR95-470.. References w1x J.C. Adams, Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains, J. Histochem. Cytochem. 40 Ž1992. 1457–1463. w2x M. Antal, G.N.S. Antal, A.K. Moschovakis, J. Storm-Mathisen, C.W. Heizmann, W. Hunziker, The termination pattern and postsynaptic targets of rubrospinal fibers in the rat spinal cord: a light and electron microscopy study, J. Comp. Neurol. 325 Ž1992. 22–37. w3x A.M. Avellino, D. Hart, A.T. Dailey, M. MacKinnon, D. Elelgala, M. Kliot, Differential macrophage responses in the peripheral and central nervous system during Wallerian degeneration of axons, Exp. Neurol. 136 Ž1995. 183–198. w4x L. Bao, J. Kopp, X. Zhang, Z.-Q. Xu, L.-F. Zhang, H. Wong, J.H. Walsh, T. Hokfelt, Localization of neuropeptide Y Y1-receptor-like ¨ immunoreactivity in cerebral arteries, Proc. Natl. Acad. Sci. USA 11 Ž1997. 12661–12666.

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