EXPERIMENTAL

NEUROLOGY

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The Inferior Colliculus: Calbindin and Parvalbumin lmmunoreactivity in Neural Grafts J. R. COLEMAN,*,? Departments

of *Psychology, and $Laboratoire

A. J. MCDONALD,*

iUft?riOr

COlliCUhS.

0 1992

Academic

Press,

MATERIALS

AND

METHODS

Long-Evans (Harlan) rats 60 to 90 days of age served as normal and graft host subjects and were maintained on ad libitum food and water in an AAALAC approved vivarium. The caudal two-thirds of each tectum was dissected from E17-18 fetuses individually removed from anesthetized (ketamine:xylazine 5O:lO mg/kg body wt, ip) pregnant rats after cesarian section. Overlying meninges and blood vessels were removed from tecta which were transferred to ice-cold nutrient mixture (HAM F-10; Sigma). Anesthetized host animals (N = 28) received unilateral stereotaxically placed IC lesions using direct current passed through a tungsten electrode. Grafts were implanted immediately to 3 weeks after lesion using a glass pipette (0.8 mm o.d.) attached to a 50-~1 Hamilton syringe and overlying burr holes filled with Gelfoam (Upjohn) and bone wax, and skin was sutured. Other methodological details have been described previously (20). Following 2 to 9 months grafted and control animals were anesthetized with sodium pentobarbital (100 mg/ kg body wt) and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After 24 h in perfusate at 4°C 50-pm sections were cut in frontal plane on a Vibratome or freezing microtome. Following blocking of nonspecific binding sites, tissue was incubated in a primary antibody (anti-calbindin or anti-parvalbumin; obtained courtesy of Dr. Kenneth Bairnbridge) in a solu-

Inc.

Few studies have observed the effects of direct placement of neural grafts into structures of the brain stem. One series of studies has relied upon placement of graft material onto the dorsal surface of the midbrain superior colliculus (9, 11). Recent work has demonstrated that transplantation of embryonic caudal tectum into intact or lesioned inferior colliculus (IC) provides a viable model for the study of graft-host interactions in the auditory brain stem. In particular, collicular grafts show excellent survival and display anatomical features similar to the normal IC (18,20). Features such as predominance of discoid type neurons comparable to the collicular central nucleus suggest a possible laminar or organized substrate in graft material. Furthermore, there is evidence for both afferent and efferent connections between collicular grafts and host tissues (19,20), as well as clearly defined metabolic activation of grafts by acoustic stimulation (19, 21). 142 Inc. reserved.

29208;

The question arises as to whether neurochemical specificity characteristic of normal inferior colliculus develops in tectal grafts. Typical intrinsic or extrinsic sources of neurotransmitters or other neuroactive cell constituents have been reported in grafts of other systems including hippocampus (e.g., 7, 13), basal ganglia (8,17), and spinal cord (16). We have recently identified distinct distributions of the calcium binding proteins calbindin and parvalbumin in neurons of the rat inferior colliculus (5). In the present study these calcium binding proteins are identified in neural populations of grafts of caudal tectum.

INTRODUCTION

0014-4886/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

AND M. C. ZRULL*

tPhysiology, and @Anatomy, University of South Carolina, Columbia, South Carolina de Neuropsychologie, Ecole des Hautes Etudes en Sciences Socials, Marseille, France

The ,inferior colliculus was selected as a brain stem site for study of neural grafting and identification of calcium binding proteins. Unilateral ablation sites of eight midbrain inferior colliculus in adult Long-Evans rats were implanted with E17-18 caudal tectum. After 2 to 9 months animals were sacrificed and sections reacted using antibodies for calbindin and parvalbumin. The central nucleus of normal inferior colliculus shows high density of neuronal and fiber staining for parvalbumin. Typical graft cores had similar staining distributions including discoid and stellate neuron populations. Graft cores showed low densities of reactivity for calbindin comparable to central nucleus. In surrounding graft regions there was substantiveneuronal and fiber labeling for calbindin and parvalbumin including stellate neuron populations normally found in the dorsal and lateral nuclei of inferior colliculus. These results demonstrate that the expression of calcium binding proteins in tectal grafts resembles that Of

B. PINEK,§

CALCIUM

FIG. 1. (A) Distribution of individual midpoint. Note sparse labeling of central the dorsal and lateral nuclei (DN, LN). density of parvalbumin-reactive neurons

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neurons immunolabeled for calbindin in frontal section of the L-E rat inferior colliculus near rostra1 nucleus (CN) and ventrolateral nucleus (VL). More neurons immunoreactive for calbindin appear in (B) Distribution of individual neurons immunolabeled for parvalbumin. The CN shows the highest in the rat inferior colliculus. Bar, 500 pm.

tion of 1% goat normal goat serum and 0.3% Triton X-100 in phosphate buffer. Tissue was transferred to goat anti-rabbit solution, incubated in ABC solution (Vector Labs.), and then reacted with DAB using a glucose oxidase solution. Sections were mounted onto gelcoated slides, air-dried, and immersed in a series of alcohols and xylenes, and then slides were coverslipped. Adjacent sections not immunoreacted were stained with thionin. Following histological procedures material was observed under brightfield and phase microscopy at 10 to 1000X and subdivisions of normal IC were defined as described previously (6, 12, 15). RESULTS

The IC of the normal L-E rat, like the S-D rat (6), includes three main subdivisions: central nucleus (CN), dorsal nucleus (DN), and lateral nucleus (LN). CN is dominated by discoid type neurons (12, 15), which, along with stellate cell populations, are also found in graft material (20). Various large and small stellate cell populations permeate layers of the dorsal and lateral nuclei (12). Small spindle or fusiform cells are also common in these pericentral nuclei, particularly in superficial layers. The CN normally contains relatively few CB staining neurons (Fig. 1A) including scattered discoid and stellate cells. In contrast DN and LN react more heavily for CB, especially the superficial layers of these nuclei (Fig. 1A). The neuropil of DN and LN are considerably stained, neurons and fibers of the ventrolateral nucleus (VL) are also reactive. Therefore, the paucity of CN staining for CB and the substantive localization of CB in shell nuclei make CB labeling distribution a marker for subdivisions of the inferior colliculus. In grafted material a core or major portion of the tissue typically contains relatively few CB-labeled neurons (Figs. 2 and 3A). These include scattered discoid-type or stellate neurons similar to the normal CN. The adjacent

graft tissue often shows more extensive numbers of CBreactive neurons. These immunoreactive cells constitute a shell of tissue (Fig. 3A) or form a crescent (Fig. 2) juxtaposing the relatively unstained graft core. Common among reactive neurons are stellate and spindle cells similar to those observed in the normal shell nuclei (Fig. 3A). These findings in graft cases suggest that CB staining patterns in core and superficial zones of grafts parallel those of the normal IC. However, identification of laminar ~stributions in the circumscribed portions of graft material are not always observed in the frontal plane since such patterns may be partially obscured by variant orientations of developing grafts. Immunostaining for parvalbumin reveals remarkably different patterns in the central nucleus of rat IC. The CN has the highest density of PV staining cells in the

FIG. 2. Collicular (center) shows Iittle (under dark pointers) of the right side graft

graft immunostained for calbindin. Core area somal reactivity, while the adjacent region shows several labeled cells. Ventromedial edge appears at lower left (light pointers).

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FIG. 3. (A) Drawings of neurons immunoreactive for calbindin in graft (G) and remaining host (H) inferior colliculus in frontal section of Case 9016. The core of both graft and host tissues are relatively devoid of labeled neurons while the shell regions have many reactive stellate and spindle cells. (B) Drawings of neurons in adjacent section to that in A reacted for parvalbumin. The core of both host and graft regions show heavy cell labeling with many neurons identifiable as discoid or stellate neurons.

IC, although all nuclei of the IC contain neurons immunoreactive for PV (Fig. 3B). Reactive neurons of various sizes in the CN include both stellate and discoid classes which are embedded in a matrix of fiber staining that sometimes suggests a laminar arrangement of the neuropil. Intrinsic cellular labeling in CN is dominated by somal staining, while punctate structures of the neuropi1 are also labeled. The pericentral nuclei especially contain different classes of PV-stained stellate neurons in deeper layers, and there is considerable reactivity in VL. Graft regions reacted for parvalbumin show staining patterns which are similar to normal IC, but are largely complementary to the calbindin-reactive distribution of neurons in both graft and normal IC (Fig. 3). Discoid and stellate neurons, as well as neuropil with puncta, are immunoreactive for PV in the graft core. Organized cell/fiber patterns reminiscent of the normal CN are sometimes, but not always, observed in the graft core. Adjacent rind- or crescent-shaped regions of the graft display substantive populations of PV-reactive stellate cells; some large and all other stained neurons resemble those of the normal DN and LN. DISCUSSION

The present results show that two calcium binding proteins, calbindin and parvalbumin, are differentially

ET

AL.

concentrated in neurons of subnuclei of the rat inferior colliculus. It is clear that most neurons of the central nucleus which contain parvalbumin do not also produce calbindin since relatively few neurons of the CN stain for CB. Which populations of PV staining discoid and stellate neurons of the CN have projections to the target medial geniculate body in rat (4) remain to be determined. In the dorsal and lateral nuclei of the IC there is some overlap in the distribution of CB and PV, although it appears that CB is more evident in the superficial layers. Double labeling studies are required to identify stellate and spindle cells which contain both CB and PV. In several other brain regions as in cerebral cortex (9) separate neuron classes contain these calcium binding proteins, although CB and PV can coexist in the same neurons of brain regions such as in Purkinje cells of cerebellum (2). Calbindin, and more often parvalbumin, may be colocalized with GABA (2,14). GABA containing neurons have been identified in the central nucleus of rat IC (l), but have not been colocalized with calcium binding proteins. The distribution and morphology of neurons in the normal inferior colliculus staining for calbindin and parvalbumin provide a useful set of reference markers for identification of neurons developing from grafts of the caudal tectum. In effect, normative material establishes a set of criteria for examination of expression of calcium binding proteins in grafted material. The present work shows that the core of tectal grafts is relatively unstained for calbindin as is the normal central nucleus of IC. In contrast the graft core of adjacent sections are heavily reactive for parvalbumin which is typical of CN. Furthermore, surrounding graft tissue labels for both calbindin and parvalbumin as is the case for pericentral nuclei. The morphologies of stained neurons in core and periphery of graft tissue are also often comparable to those of the central and rind nuclei of IC. Of special note are neurons classified as discoid and stellate types of the central nucleus which can be readily identified in the graft core. Previous work using Golgi and Nissl methods has demonstrated that relatively typically occurring cells of these classes are regularly observed in grafts of caudal tectum (18-20). These works combined suggest that normal biochemical and structural features can develop in the graft core which corresponds to a region that is uniquely organized for processing of tonotopic information and other domains (3). The appearance of large populations of stellate neurons along with other cell types in the shell-like portions of grafts which stain for CB or PV also suggests that biochemical features of corresponding morphological classes of DC and LN can be expressed. The precise nature of graft lamination features should be illuminated by more extensive examination of tissues in other planes of section. Additional information is also required on the synaptic integrity of

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graft neurons and the presence and distribution of neuroactive substances and neurotransmitters (e.g., GABA) within borders of collicular grafts. ACKNOWLEDGMENTS We thank Dr. Kenneth Bairnbridge for providing antibodies to calcium binding proteins. This research was supported by NIH BRSG SO7 RR07160, the Deafness Research Foundation, and the Fyssen Foundation.

REFERENCES 1. CASPARY, D. M., A. RAZA, B. A. LAWHORN ARMOUR, J. PIPPIN, AND S. P. ARNERIC. 1990. Immunocytochemical andneurochemical evidence for age-related loss of GABA in the inferior colliculus: Implications for neural presbycusis. J. Neurosci. 10: 23632372. 2. CELIO, M. R. 1990. Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35: 375-475. 3. CLERICI, W. J., AND J. R. COLEMAN. 1987. Resting and pure tone evoked metabolic responses in the inferior colliculus of young adult and senescent rats. Neurobiol. Aging 8: 171-178. 4. CLERICI, W. J., AND J. R. COLEMAN. 1990. Anatomy of the rat medial geniculate body. I. Cytoarchitecture, myeloarchitecture and neonatal connectivity. J. Comp. Neural. 297: 14-31. 5. COLEMAN, J. R., AND W. J. CLERICI. 1987. Sources of projections to subdivisions of inferior colliculus in rat. J. Comp. Neural. 262: 215-226. 6. COLEMAN,J.R.,B.PINEK,M.C.ZRVLL, A. J.MCDONALD,AND K. G. BAIMBRIDGE. 1990. Calbindin and parvalbumin reactivity in grafted and intact inferior colliculus. Sot. Neurosci. Abstr. 16: 722. 7. DASZUTA, A., R. E. STRECKER, P. BRUNDIN, AND A. BJ~~RKLUND. 1988. Serotonin neurons grafted to the adult rat hippocampus. I. Time course of growth as studied by immunohistochemistry and biochemistry. Bruin Res. 458: 1-19. 8. DOUCET, G., Y. MVRATA, P. BRUNDIN, 0. BOSLER, N. MONS, M. GEFFARD, C. C. OVIMET, AND A. BJ~~RKLVND. 1989. Host afferents into instrastriatal transplants of fetal ventral mesencephalon. Exp. Neural. 106: 1-19.

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9. HARVEY, A. R., AND S. S. WARTON. 1986. The morphology of neurons in rat tectal transplants as revealed by Golgi-Cox impregnation. Anat. Embryol. 174: 361-367. 10. HENDRY, S. H. C., E. G. JONES, P. C. EMSON, D. E. M. LAWSON, C. W. HEIZMANN, AND P. STREIT. 1989. Two classes of cortical GABA neurons defined by differential calcium binding protein immunoreactivities. Exp. Brain Res. 76: 467-472. 11. LUND, R. D., AND A. R. HARVEY. 1981. Transplantation of tectal tissue in rats. I. Organization of transplants and pattern of distribution of host afferents within them. J. Comp. Neurol. 201: 191-209. 12. MOREST, D. K., AND D. L. OLIVER. 1984. The neuronal architecture of the inferior colliculus in the cat: defining the functional anatomy of the auditory midbrain. J. Comp. Neural. 222: 209236. 13. MLJRATA, Y., T. CHIBA, P. BRUNDIN, A. BJBRKLVND, AND 0. LINDVALL. 1990. Formation of synaptic graft-host connections by noradrenergic locus coeruleus neurons transplanted into the adult rat hippocampus. Exp. Neurol. 110: 258-267. 14. NITSCH, R., E. SORIANO, AND M. FROTSCHER. 1990. The parvalbumin-containing nonpyramidal neurons in the rat hippocampus. Anat. Embryol. 181: 413-425. 15. OLIVER, D. L., AND K. K. MOREST. 1984. The central nucleus of the inferior colliculus in the cat. J. Comp. Neural. 222: 237-264. 16. TESSLER, A., B. T. HIMES, J. HOVLE, AND P. J. REIER. 1988. Regeneration of adult dorsal root axons into transplants of embryonic spinal cord. J. Comp. Neurol. 270: 537-548. 17. WICTORIN, K., AND A. BJBRKLUND. 1989. Connectivity of striatal grafts implanted into the ibotenic acid-lesioned striatum. II. Cortical afferents. Neuroscience 80: 297-311. 18. ZRULL, M. C., AND J. R. COLEMAN. 1990. Fetal tectum grafted as a cell suspension into the adult rat inferior colliculus. Hear. Res. 45: 237-246. 19. ZRULL, M. C., AND J. R. COLEMAN. 1990. Properties of fetal tecturn grafted to adult and neonatal rat inferior colliculus. Sot. Neurosci. Abstr. 16: 722. 20. ZRULL, M. C., AND J. R. COLEMAN. 1991. Structural features of neurons in whole grafts of the rat inferior colliculus. Hear. Res. 55: 117-132. 21. ZRULL, M. C., T. M. WESSINGER, AND J. R. COLEMAN. 1991. Differential sound-evoked metabolic activity in fetal tectum grafted into damaged adult rat inferior colliculus. Submitted for publication.

The inferior colliculus: calbindin and parvalbumin immunoreactivity in neural grafts.

The inferior colliculus was selected as a brain stem site for study of neural grafting and identification of calcium binding proteins. Unilateral abla...
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