Presumptive Renshaw Cells Contain Decreased Calbindin During

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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3048-3052, April 1993 Neurobiology Presumptive Renshaw cells contain decreased calbindin during recovery from sciatic nerve lesions (axotomy/botulinum toxin/calcium-binding proteins/spinal cord/regeneration) PIETRO PAOLO SANNA*, MARCO R. CELIOt, FLOYD E. BLOOM*, AND MARIO RENDEU§ *Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037; tInstitute of Histology, University of Fribourg, Fribourg, Switzerland; and *Section of Human Anatomy, Department of Experimental Medicine and Biochemical Science, University of Perugia, Perugia, Italy Contributed by Floyd E. Bloom, October 29, 1992

ABSTRACT A subpopulation of calbindin-immunoreac- MATERIALS AND METHODS tive neurons in lamnina VII ofthe spinal cord has been identified by its location as Renshaw cells, the anatomical substrate for Sciatic Nerve Lesions. For this study, six female Sprague- recurrent inhibition. The expression of calbindin (28 kDa) in Dawley rats weighing 200-220 g were anesthetized intraperi- toneally with a mixture of ketamine, acepromazine, and these calbindin-containing rat ventral horn interneurons was xylazine, and their right sciatic nerve was crushed with studied with immunocytochemistry after sciatic nerve injuries. forceps at the mid-thigh level three times for 5 sec and left in after calbindin was One week axotomy immunoreactivity place. Six other animals received a sciatic nerve cut with levels and strongly reduced on the lesioned side between L4 L6, removal ofa 2-cm segment to prevent reconnection ofthe two while calbindin-containing neurons and fibers were still nu- stumps. Six sham-operated rats were used as controls. Three merous contralateraily and cranially to the lesioned levels. further rats received an intramuscular injection of 5 ng of With the progression of regeneration, calbindin-immunoreac- botulinum toxin A (Sigma) in 5 ,u of saline in the right tibialis tive neurons reappeared, reaching a normal distribution 6-8 anterior muscle. Three control rats received an injection of weeks after the crush. Similar changes could be mimicked by saline. Four normal unperturbed rats were also used. the intramuscular adminitration of botulinum toxin. These Immunohistochemistry. Rats were deeply anesthetized and results suggest that calbindin expression in putative Ren- perfused intracardially with ice-cold heparinized 0.1 M phos- shaw cells of the spinal cord might be functionally responsive phate buffer (pH 7.4) followed by 250 ml of ice-cold 4% and that maintenance of calbindin expression may depend paraformaldehyde in phosphate buffer. on the integrity of motoneurons and neuromuscular transmis- Spinal cords were treated as described (24). Briefly, the sion. spinal cord was removed after laminectomy, anchored to a dental wax sheet to prevent curvature or distortions, and Spinal cord motoneurons give offaxon collaterals that project postfixed in 4% paraformaldehyde in phosphate buffer for 16 to small interneurons, the Renshaw cells. These neurons in hr at 4°C. The spinal cords were then rinsed in phosphate turn synapse on the somata of motoneurons (1). Renshaw buffer for 24 hr and cryoprotected in 10% and 30% sucrose in cells are mostly glycinergic (2, 3) inhibitory neurons that phosphate bufferfor 24 hreach. Prior to sectioning, the spinal cords were divided under the dissecting microscope into reduce the rate of a-motoneurons in a discharge synergistic three segments by cuts across the dorsal roots at C8 and T10; negative-feedback fashion. Renshaw cells also project to in the present study, only the caudal segment was analyzed. y-motoneurons (4), ventral spinocerebellar tract neurons (5), The dorsal root entry zone on the right side ofLi was marked Ia-inhibitory interneurons (6), and other Renshaw cells (7). with a small cut for orientation. The spinal cord segments A subpopulation of 28-kDa calbindin-immunoreactive neu- were then sectioned on a freezing microtome, and individual rons located in the ventral portion of lamina VII, medial to sections (40 ,um) were collected and stored at 4°C in Mil- the motoneuron column, has recently been proposed to be lonig's buffer until immunocytochemistry. Renshaw cells (8, 9) based on the anatomical location. Sections were incubated free floating at room temperature Calbindin (10) is a Ca2+-binding protein which, like parval- in 0.1 M Tris (pH 7.4) containing 1.4% NaCl and 10%o bumin, is thought to buffer intracellular Ca2+. Such Ca2+- (vol/vol) heat-inactivated horse serum (Tris-serum) for 30 buffering proteins tend to be expressed in discrete popula- min. The sections were then treated in Tris-serum with a tions of neurons as well as in selected nonneuronal cell types mouse anti-calbindin monoclonal antibody (25) used at a (11-15). dilution of 1:6000 overnight or with the mouse anti-LNGFR After peripheral nerve lesions, motoneurons undergo com- monoclonal antibody (26) 192-IgG used at 5 ug/ml for 30 hr. plex morphological (16), physiological (17-19), and biochem- LNGFR expression was used to visualize regenerating a-mo- ical (20-23) changes. We have tested whether the presump- toneurons as reported (22, 24). Immunoreactivity was re- tive Renshaw neurons might respond to peripheral nerve vealed with the avidin-biotin complex method (ABC-elite; injuries by modulating their calbindin-expression as a result Vector Laboratories) (27) in Tris without serum. When of the reduced motoneuronal activity and the consequent double labeling for calbindin and LNGFR was performed on reduced need for Ca2+-buffering power. the same section sequentially, calbindin immunoperoxidase Low-affinity nerve growth factor receptor (LNGFR) im- was revealed with diaminobenzidine (DAB; Sigma) as a munoreactivity was used as marker for peripherally lesioned chromogen, while aminoethylcarbazole (AEC, Vector Lab- a-motoneurons, since previous studies showed that mo- oratories) was used for LNGFR. The DAB-peroxidase re- toneurons express LNGFR during regeneration after sciatic nerve injuries (22, 24). Abbreviations: LNGFR, low-affinity nerve growth factor receptor; DAB, diaminobenzidine; AEC, aminoethylcarbazole. §To whom reprint requests should be addressed at: Department of The publication costs of this article were defrayed in part by page charge Experimental Medicine and Biochemical Science, Section of Hu- payment. This article must therefore be hereby marked "advertisement" man Anatomy, University of Perugia, via del Giochetto, Perugia, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Italy 06100.

3048 Downloaded by guest on September 30, 2021 Neurobiology: Sanna et al. Proc. Natl. Acad. Sci. USA 90 (1993) 3049 action was performed by incubation of the samples in 0.04% bation in 0.025% AEC/0.03% H202/0.1 M Tris, pH 7.4, for DAB/0.06% NiCl2/0.02% H202/0.1 M Tris, pH 7.4, for 10 20 min. Nonspecific binding in the second staining was min. The AEC-peroxidase reaction was performed by incu- prevented by incubating the section sequentially with avidin

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FIG. 1. Comparison of calbindin immunoreactivity as expressed in a normal spinal cord (A-C), after sciatic nerve crush (D-F), or after intramuscular administration of botulinum toxin (G-I). (B, E, and H) Magnification of a segment of the upper (undamaged) side of A, D, and G. (C, F, and H) Magnification of a segment of the lower (damaged) side of A, D, and G. (D-F) One week after sciatic nerve crush, calbindin-immunoreactive cells on the lesioned side (lower side) were fewer and calbindin-immunoreactive fibers had disappeared; a striking difference can be seen between the undamaged contralateral side (E) and the damaged side (F), where calbindin immunoreactivity was strongly reduced and staining of calbindin-immunoreactive neurons tended to be nuclear. (G-I) Similarly, 1 week after intramuscular administration of botulinum toxin, calbindin immunoreactivity was strongly reduced on the lesioned (lower) side (G and I). (Bar = 0.5 mm in A, D, and G and 250 ,um in B, C, E, F, H, and I.) Downloaded by guest on September 30, 2021 3050 Neurobiology: Sanna et al. Proc. Natl. Acad. Sci. USA 90 (1993)

FIG. 2. Double labeling for calbindin and LNGFR in a rat ventral horn 1 week after sciatic nerve crush, showing the lesioned side levels L3-L6. Calbindin-immunoreactive neurons appear black (DAB-labeled immunoperoxidase), and LNGFR-immunoreactive neurons appear red (AEC-labeled immunoperoxidase). (A) Numerous calbindin-immunoreactive neurons (arrowheads) were present at lumbar levels cranial to the lesion at levels L3 and LU; a rich plexus ofcalbindin-immunoreactive fibers was also visible. (B) Calbindin-immunoreactive neurons were fewer at levels L5 and L6, where LNGFR-immunoreactive neurons (white asterisks) became more and more numerous. At these lumbar levels, calbindin-immunoreactive neurons had virtually disappeared. (C and D) However, in the area of transition (L4-L4/5), few calbindin- immunoreactive neurons (arrowhead with asterisk) coexisted with LNGFR-immunoreactive a-motoneurons (asterisks), and calbindin- Downloaded by guest on September 30, 2021 Neurobiology: Sanna et al. Proc. Natl. Acad. Sci. USA 90 (1993) 3051 D and biotin blocking solutions (Vector Laboratories) prior to B 0. w;t . :; the incubation with the anti-LNGFR antibody. Sections were mounted with water-soluble medium (Cristalmount) to pre- serve AEC staining.

RESULTS ' .A :.:.E 0: 0: E u-Zw s-/ s st .. l-e- In the ventral horn of normal unperturbed rats, calbindin immunoreactivity identified a population of small neurons mostly distributed in lamina VII and particularly abundant at ti5.pt,f' . ' ' : t .....-.^5:.: the level ofthe cervical and lumbar enlargements (Fig. 1 A-C) C W D o that have been proposed to represent Renshaw cells (8, 9). ... Also corroborating such identification was their fusate or .. .R *j multipolar cellular morphology (Fig. 2F), strongly reminis- _ cent of electrophysiologically identified Renshaw neurons (28). Immunohistochemistry revealed calbindin immunore- .- .. activity of the cell bodies, dendrites, and axons (Fig. 1 A-C _?i ...... : and 2 E and F). The number ofcalbindin-immunoreactive cell _ . .w.... bodies and fibers and the intensity of their staining showed a FIG. 3. Calbindin-immunoreactive cells displayed a range of certain degree ofinteranimal variation. One week after sciatic cellular staining patterns. (A) In the lumbar and cervical enlarge- nerve crush, on the lesioned side of the cord between levels ments, calbindin-immunoreactive neurons usually displayed a Golgi- L4 and L6, calbindin immunoreactivity was strongly re- like pattern of staining. (B-D) However, at the lesioned levels after duced, and the number ofcalbindin-immunoreactive neurons axotomy, cells with predominantly or exclusively nuclear staining had drastically declined (Figs. 1 D-F and 2B). While calbin- were often encountered. Cells with similar morphology were often din-immunoreactive neurons in the normal lumbar enlarge- seen at thoracic levels of normal unperturbed spinal cords and may ments usually displayed a Golgi-like pattern of staining (Figs. represent functional stages characterized by reduced need for Ca2+- 1 A-C, 2F, and 3A), in the lesioned levels the calbindin- buffering power. (Bar = 20 ,um.) immunoreactive neurons still visible had few or no calbindin- reinnervation of muscular targets, LNGFR-immunoreactive immunoreactive fibers, and their staining was often predom- a-motoneurons disappeared in a cranio-caudal manner as inantly or exclusively nuclear (Figs. 1 D-F, 2B, and 3). Cells described (24). with a similar pattern of staining were not infrequently seen in the ventral horns of normal unperturbed spinal cords especially at thoracic levels. DISCUSSION At levels cranial to those affected by the lesions (L1-L3; The development and maintenance of many neuronal char- Fig. 2A), calbindin-immunoreactive neurons could still be the observed, and a robust plexus of calbindin-immunoreactive acteristics depends both on the trophic action exerted by neurites spanning from Li to L3/4 was clearly evident (Fig. target (29) and on neuronal activity (30-32). Peripheral nerve 2A). A similar morphological pattern was present on the lesions are known to induce several morphological (16), contralateral side, where calbindin-immunoreactive neurons biochemical (20-23), and physiological (17-19) changes in were still numerous at any lumbar level (Figs. 1 D and E and a-motoneurons. The alterations in a-motoneuron physiolog- 2 E and F). Comparable effects were consistently observed ical properties seen after sciatic nerve lesions are known to in all rats perfused at 7 days after sciatic nerve crush. A be reversible upon target reinnervation (33-35) and have similar pattern of reduced calbindin immunoreactivity on the been ascribed primarily to the loss offunctional contact with lesioned side and a reduced number of calbindin-immunore- the muscular targets, since they can be reproduced by the active neurons was observed 1 week after cut. A similar intramuscular administration of botulinum toxin (36) and are reduction ofcalbindin immunoreactivity in presumptive Ren- not prevented by chronic electric stimulation (37). The major shaw neurons was also seen ipsilaterally after intramuscular functional changes in a-motoneurons after peripheral nerve administration of botulinum toxin (Fig. 1 G-1). The distribu- lesions include reduced action-potential amplitude, de- tion ofcalbindin-immunoreactive neurons returned to normal creased after-hyperpolarization duration, decreased axonal- within 6-8 weeks after sciatic nerve crush. A certain degree conduction velocity (17, 19), and reduction ofintramedullary of interanimal variation was observed in the timing of the axon collaterals originating from the axotomized motoneu- reappearance of calbindin immunoreactivity. The specific rons (38). In the present report we have analyzed the distri- temporal aspects of the recovery of calbindin expression and bution of presumptive calbindin-immunoreactive Renshaw its relationship to functional recovery will be reported sep- neurons in the ventral horn after peripheral nerve lesions and arately. after intramuscular administration of botulinum toxin. Cal- The double staining with LNGFR antiserum showed im- bindin immunoreactivity was severely reduced in the le- munoreactive perikarya on the lesioned side at spinal levels sioned segments after these perturbations, suggesting that the L4-L6 within the column of the sciatic nerve motoneurons. maintenance of calbindin expression in presumptive Ren- At spinal levels L4-L4/5 on the lesioned side, few calbindin- shaw cells might depend on the integrity of a-motoneurons immunoreactive neurons coexisted with LNGFR-immunore- and their capacity for neuromuscular transmission. The active neurons, and calbindin-immunoreactive neurites oc- slower conduction velocity of a-motoneurons, their reduced casionally could be observed spreading to the vicinity of activity pattern, and the reduction of their input on Renshaw LNGFR-immunoreactive a-motoneurons (Fig. 2 C and D). cells would be expected to lead to reduced activity of these With the progression of sciatic nerve regeneration and the latter neurons. As a result, these neurons should have a

immunoreactive processes (arrowheads) were occasionally observed spreading to the vicinity of the red LNGFR-IR a-motoneurons. (E) Contralateral unaffected side (L4 and L5), in which arrowheads indicate calbindin-immunoreactive neurons. (F) Detail ofD showing the cellular morphology of calbindin-immunoreactive neurons in the anterior horn. These cells displayed the fusiform (arrowheads with asterisks) or multipolar (arrowheads) morphology typical of electrophysiologically identified Renshaw cells (28). (Bar = 200 Am in A, B, and E and 50 Am in C, D, and F.) Downloaded by guest on September 30, 2021 3052 Neurobiology: Sanna et al. Proc. Natl. Acad. Sci. USA 90 (1993) reduced need for Ca2+-buffering power, leading to reduced 11. Celio, M. R. & Heizmann, C. W. (1982) Nature (London) 297, expression of calbindin. The range of cellular staining pat- 504-506. terns that we have observed (Fig. 3) both at lesioned levels 12. Berchtold, M. W., Celio, M. R. & Heizmann, C. W. (1984) J. Biol. Chem. 259, 5189-5196. and in the normal cord, especially at thoracic levels, may 13. Celio, M. R. (1986) Science 231, 995-997. represent a range of functional stages characterized by dif- 14. Celio, M. R. (1990) Neuroscience 35, 375-475. ferent needs for Ca2+-buffering power. This would be con- 15. Sanna, P. P., Keyser, K. T., Battenberg, E. & Bloom, F. E. sistent with a plastic, activity-responsive regulation of cal- (1990) Neurosci. Lett. 118, 136-139. bindin expression in the normal spinal cord as well as in the 16. Clark, P., Jones, K. J. & LaVella, A. (1991) J. Comp. Neurol. lesioned one. A functional plasticity in the expression of 312, 132-144. parvalbumin, another Ca2+-buffering, Ca2+-binding protein, 17. Gustafsson, B. (1979) J. Physiol. (London) 293, 197-215. has been previously shown in striated muscle where parval- 18. Gustafsson, B. & Pinter, M. J. (1984) J. Physiol. (London) 356, 433-442. bumin-expression is normally restricted to fast myotubules 19. Kuno, M., Myata, Y. & Mufioz-Martinez, E. J. (1974) J. (11). However, if fast myotubules are made to become slow Physiol. (London) 240, 725-739. by replacing the fast nerve fibers innervating them with slow 20. Miller, F. D., Tetzlaff, W., Bisby, M. A., Fawcett, J. W. & fibers, they stop expressing parvalbumin (39). Milner, R. J. (1989) J. Neurosci. 9, 1452-1463. Calbindin expression in putative Renshaw cells of the 21. Scott, J. N. & Parhard, I. M. (1991) Mol. Brain Res. 10, anterior horn thus appeared to be functionally responsive and 315-325. was strongly reduced when peripheral nerves were injured or 22. Emfors, P., Henschen, A., Olson, L. & Persson, H. (1989) when neuromuscular transmission was disrupted by intra- Neuron 2, 1605-1613. of botulinum toxin. These results 23. Jenkins, R. & Hunt, S. P. (1991) Neurosci. Lett. 129, 107-110. muscular administration 24. Rende, M., Hagg, T., Manthorpe, M. & Varon, S. (1992) J. suggest that the maintenance of calbindin expression in this Comp. Neurol. 319, 285-298. neuronal population might depend on the activity and integ- 25. Celio, M. R., Baier, W., Sharer, L., Gregersen, H. J., de rity of motoneurons. Viragh, P. A. & Norman, A. W. (1990) Cell Calcium 11, 599-602. This study was partially supported by Grant NS 22347 from 26. Sutter, A., Riopelle, R. J., Harris-Warrick, R. M. & Shooter, National Institute ofNeurological Disorders and Stroke, a grant from E. M. (1979) J. Biol. Chem. 254, 5972-5980. the Swiss National Foundation to M.R.C., and a grant from Tele- 27. Hsu, S. M., Raine, L. & Fanger, H. (1981) J. Histochem. thon-Italia to M.R. This is manuscript number NP 7627 of The Cytochem. 29, 577-588. Scripps Research Institute. 28. Fyffe, R. E. W. (1990) Brain Res. 536, 301-304. 29. Purves, D. & Lichtman, J. W. (1985) Principles of Neural 1. Renshaw, B. (1941) J. Neurophysiol. 4, 167-183. Development (Sinauer, Sunderland, MA), pp. 290-328. 2. Eccles, J. C., Fatt, P. & Koketsu, K. (1954) J. Physiol. 30. Benoit, P. & Changeux, J. P. (1975) Brain Res. 99, 354-358. (London) 159, 479-499. 31. Benoit, P. & Changeux, J. P. (1978) Brain Res. 149, 89-96. 3. Cullheim, S. & Kellerth, J.-O. (1981) J. Physiol. (London) 312, 32. Laufer, R. & Changeux, J. P. (1989) Mol. Neurobiol. 3, 1-53. 209-224. 33. Vanden Noven, S. & Pinter, M. J. (1989) J. Neurophysiol. 62, 4. Ellaway, P. H. (1971) J. Physiol. (London) 216, 524-562. 325-333. 5. Lindstrom, D. & Schomburg, E. D. (1973) Acta Physiol. 34. Kuno, M., Myata, Y. & Munioz-Martinez, E. J. (1974) J. Scand. 88, 505-515. Physiol. (London) 242, 273-288. 6. Hultborn, H. (1972) Acta Physiol. Scand. Suppl. 375, 1-42. 35. Foehring, R. C., Sypert, G. W. & Munson, B. J. (1986) J. 7. Ryall, R. W. (1970) J. Neurophysiol. 32, 257-270. Neurophysiol. 55, 947-965. 8. Antal, M., Freund, T. F. & Polgar, E. (1990) J. Comp. Neurol. 36. Pinter, M. J., Vanden Noven, S., Muccio, D. & Wallace, N. 295, 467-484. (1991) J. Neurosci. 11, 657-666. 9. Arvidsson, U., Ulfhake, B., Cullheim, S., Ramirez, V., Shu- 37. Gordon, T., Gillespie, J., Orozco, R. & Davis, L. (1991) J. pliakov, 0. & Hokfelt, T. (1992) J. Neurosci. 12, 718-728. Neurosci. 11, 2157-2169. 10. Wassermann, R. H. & Taylor, A. N. (1966) Science 152, 791- 38. Havton, L. & Kellerth, J. 0. (1989) Exp. Brain Res. 79, 65-74. 793. 39. Leberer, E. & Pette, D. (1986) Biochem. J. 235, 67-73. Downloaded by guest on September 30, 2021