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Cortical lesions increase reinnervation of the dorsal striatum by substantia nigra grafts
Brain Research, 446 (1988) 133-143 Elsevier
133
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Cortical lesions increase reinnervation of the dorsal striatum by substantia nigra grafts William J. Freed and H. Eleanor Cannon-Spoor Preclinical Neurosciences Section, Neuropsychiatry Branch, National Institute of Mental Health, Saint Elizabeth's Hospital, Washington, DC20032 (U.S.A.)
(Accepted 29 September 1987) Key words: Transplantation; Neurite; Fluorescence histochemistry; Cerebral cortex
Effects of aspiration lesions of the cerebral cortex on intraventricular substantia nigra grafts were investigated, increased reinnerration of the dorsal striatum was observed in animals with cortical aspirations. This reinnervation was confined to the dorsal onefourth of the striatum, immediately underneath the cortical lesions. The increased reinnervation of the striatum by substantia nigra grafts in animals with cortical lesions is suggested to be related either to secretion of neurotrophic substances by the injured brain tissue or to removal of competition between corticostriatal inputs and graft-derived neurites for sytaapticcontacts.
INTRODUCTION When embryonic substantia nigra (SN) is transplanted into the lateral cerebral ventricle, adjacent to the denervated corpus striatum, these grafts produce dopamine-containing neurites which penetrate into the host striatum for limited distances 13'15-17"35. Although substantial growth of dopaminergic neurites from these grafts is observed within 2-3 weeks 13, the distance to which these neurites grow is limited to less than 1.5 mm even more than a year later 15-17. The degree of reinnervation of the str~atum produced by grafts of SN in cavities in the cerebral cortex or of dissociated SN cells is also limited 2'8-~1. In most studies of SN transplantation, the SN grafts are implanted several months after the SN lesions are produced. By this time, the vacated postsynaptic sites in the striatum may have disappeared, either because of filling due to sprouting of adjacent inputs 6, astrocyte proliferation 36, or degeneration of dendritic spines 2~. It may be that the reinnervation of the striatum by SN grafts is limited because of a limited availability of sites for synaptic contacts. The af-
ferents to the striatum from the cerebral cortex 2°'43 contact the same dendritic spines as the afferents from the S N 3"!8 o r from SN grafts 1s'28. It is therefore conceivable that potential sites for synaptic contacts could become available for short periods of time after the induction of cortical lesions. For this reason, cortical lesions which are induced just prior to SN transplantation might increase the graft-derived striatal reinnervation. Injury to the central and peripheral nervous s','stern elicits the release of substances which support the survival of neurons in tissue culture 4"5"3°-32"u. This survival-promoting effect is time-depende~at, with maximum activity present between 10 and 16 days after the induction of injury 3°'3~. Subsequent experiments have shown that the survival of brain grafts can also be enhanced by brain injury. When embryonic tissue is transplanted into cortical injury sites, survival of the grafts is maximal when the time between the production of injury and implantation of grafts is delayed to coincide with the time at which concentrations of trophic activity are greatest 3~. Survival of brain grafts was also found to be enhanced by
Correspondence: W.J. Freed, Preclinicai Neurosciences Section, Neuropsychiatry Branch, National Institute of Mental Health. Saint Elizabeth's Hospital, Washington, DC 20032, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
134 administration of extracts from injured brain into the implant site a2. Trophic activity also appears in denerrated brain areas, when the innervating nuclei are destroyed or when pathways are severed 19'a~. The nervous system therefore, appears to have a ubiquitous tendency to produce trophic substances in response to injury. Besides playing a role in neuron survival, injuryinduced neurotrophic factors may be associated with neurite formation as well29. Laminin, which promotes neuronal process formation, is produced by astrocytes 1'23'25"26. Schonfeld et al. 3a found that hippocampal injury enhanced the accumulation of choline acetyltransferase activity in intrahippocampal iris implants, an index of cholinergic sprouting. These data suggest a possible role of injury-induced neurotrophic factors produced by neuroglia in neurite extension as well as in neuron survival. In another type of graft experiment, dopaminergic neurons were transplanted into preprepared cavities in the cortex 2,8. These cortical transplantation cavities were much like the lesion sites which have been found to promote the production of trophic substances. In the transplantation experiments, however, grafts were implanted 3.5-5 weeks after the induction of injury, at a time when trophic activity would be expected to have returned to nearly baseline levels2,8,9. Nevertheless, some of the published photographs suggest that the degree of neurite penetration into the striatum from SN grafts in cortical cavities may exceed that observed after transplantation of SN into the lateral ventricle 2. The present experiment was, therefore, undertaken to investigate the possibility that cortical injury would enhance the effects of SN grafts. By transplanting tissue into the lateral ventricle and lesioning the cerebral cortex, it may be possible to separate the effects of cortical injury from those of SN grafts. The time interval between the induction of injury and graft implantation therefore was chosen as 10 days, to coincide with the time of maximal concentrations of neurotrophic substances in injured brain tissue 3°,31,44.
were ho~.lsed in groups of 4 on a 12-h !igh~-dark cycle with continuous access to food and water. A total of 32 rats were used in the experiment. Timed pregnant rats (tissue donors) were obtained from Zivic-Miller Labs., Inc.
MATERIALSAND METHODS
Brain grafts
Animals Male Sprague-Dawlcy rats (Zivic.-Miller), weighing 150-175 g at the start of the experiment,
Surgery All surgery was performed under Chloropent (Fort Dodge Labs.) anesthesia. Animals received Flo-Cillin (Bristol Labs.) 0.4 ml/kg following each surgical procedure.
SN lesions SN lesions were performed by stereotaxic administration of 4.67/~g 6-hydroxydopamine hydrobromide (Sigma Chemicals) in a vehicle of 0.4% ascorbic acid in normal saline, in a volume of 4.0 gl over 4 rain at stereotaxic coordinates 4.4 mm posterior and 1.2 mm lateral to the bregma and 7.8 mm below the dura with the incisor bar 2.4 mm below the interaural line, according to the atlas of K6nig and Klippe122. All lesions were unilateral, on the right side. Two months after SN lesions, animals were screened for rotational behavior after administration of 0.1 mg/kg apemorphine s.c. 42. Animals that turned at least 80 rotations/h were selected for use in the experiment. Studies of rotational behavior will be reported separately.
Cortical lesions Cortical lesions were performed two and one-half months after SN lesions by aspiration of the cerebral cortex down to the white matter extending approximately laterally from 0.5 mm from the midline to the supraorbital ridge and from 2 mm posterior to the bregma to the olfactory bulb. Following aspiration, the cavity was packed with gelfoam and the skin closed with wound clips. For sham-operated animals, the bone was removed but no tissue was aspirated. For some of the animals that were sacrificed after long time intervals, the lesions were considerably more extensive medially and laterally.
Ten days after corticai or sham lesions all animals received solid SN grafts from two 17-day gestational rat embryos. The grafts were implanted into the lateral ventricle at Pellegrino et al. 34 coordi~ates 1.5
135 mm lateral, 1.5 mm anterior to the bregma, and 3.5 mm below the dura (bite-bar set at 5.0 mm above the interaural line) as previously described 14a5.
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Histology Animals were sacrificed four weeks ('short-term study,' n = 25) or one year ('long-term study,' n = 7) after transplantation. Animals received pargylene 75 mg/kg i.p. and after 4 h received an overdose of Chloropent i.p. Animals were perfused with a magnesium-sucrose solution as described by Loren et al. 27 and processed for glyoxylic acid-induced histochemical fluorescence as described by de la Torre 7. Depth of penetration of catecholamine-containing neurites into the striatum was measured using an eyepiece micrometer and stage coordinates. Because the cortical lesions were obvious, it was impossible to be blind to the status of the animals, so an observer who was naive concerning the hypothesis of the experiment was trained to perform the measurements of depth of neurite penetration. For measurements of neurite penetration, the striatum was divided into 4 dorsoventral sectors, labelled 'a', 'b', 'c' and 'd', corresponding to the 4 quarters of the ventricle as shown in Fig. la. Tyrosine hydroxylase immunocytochemistry was performed on two sham-lesioned rats using the immunoperoxidase technique of Sternberger 41. The tyrosine hydroxylase antibody was supplied by N. Weiner and W. Tank of the University of Colorado.
Statistics Statistical calculations were performed either by hand or with the aid of the StatView 512 + program (BrainPower, Inc., Calabases, CA), except for multivariate analysis of variance, for which the statistical analysis system (SAS Institute Inc., Cary, NC) general linear models procedure was used. RESULTS
General histology TH immunocytochemical staining revealed that the graft.~ contained groups of neurons with TH immunoreactivity (Fig. 2). Fluorescence histochemistry reveale¢t dense catecholaminergic innervation of parts off the grafts, often obscuring the catecholaminergic neurons. The grafts consistently reinnervated parts of the striatum, particularly areas adja-
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Fig. 1. a: illustration of the method of dividingthe caudate-putamen into 4 dorsoventral 'sectors' labelled a-d. b: illustration of the extent of a typical cortical lesion, c: pictorial representation of the depth of reinnervation produced by intraventricular SN grafts in each of the four sectors 'a' through 'd' in animals with cortical lesions as compared to sham operations (means + S.E.M.). v, ventricle; g, graft.
cent to those parts of the grafts with the most dense accumulations of catecholamines. The cortex lesions in most cases were found to be fairly small and extended laterally from about 1-4 mm from the midline ar.,J 4-6 mm in the rostral-caudal dimension. Some of the lesions partially compromised the white matter. A representative lesion is shown in Fig. lb. One o f the larger lesions that was used in the long-term study is shown in Fig. 6. In a substantial number of the rats with cortical lesions, the grafts were found to be misp l a c e d - for example, in parts of the brain other then tl-.e ventricle such as within the cortical lesion site. These animals were not used for further quantitative analysis.
Effects of cortical lesio~s Evaluation of the deepest reinnervation of the striatum (in any of the 4 sectors) for each animal from the short-term study revealed a depth of reinnerva-
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Fig. 2. Tyrosine hydroxylase immunocytochemical staining of transplanted SN neurons. Peroxidase-antiperoxidase technique, Nomarski optics, a: a graft located in the dorsal part of the lateral ventricle (bar = 100pm). b: a graft which filled the ventral part of the lateral ventricle (bar = 100pm).
tion (means + S.E.M.) of 0.77 _+ 0.08 mm for the sham-lesioned group and 1.04 + 0.11 mm for the cortical-lesioned rats. This difference did not reach statistical significance (P < 0.07, two-tailed t-test, t(14) = 2.03). Evaluations of the depths of reinnervation in each of the 4 sectors 'a' through 'd', however, showed an increase in the depth of reinnervation in sector 'a' (the most dorsal sector) in the cortex-lesioned group (Fig. lc). A multivariate analysis of variance revealed that the depth of reinnervation was increased in sector 'a' (El.15 = 10.83, P = 0.005). There was no difference in sector 'b', (P = 0.327), nor in sectors 'c' and 'd' (P > 0.5). The depth of reinnervation for each animal was also evaluated at the point where catecholaminergic
neurites were most prominently seen to oe entering the striatum (cf. Fig. 3). At the same time, depths of reinnervation were measured 0.5 mm dorsal to the entry point, 0.5 mm ventral to the entry point, and for sector 'a'. These data are shown in Fig. 4. In every one of the sham-lesioned rats, the depth of reinnervation was greater at the entry point than in sector 'a' (Fig. 4). In contrast, this was not true of the animals with cortical lesions. In 4 of the 7 cortex-lesioned rats, the reinnervation depth was greater in sector 'a' than at the entry point. In two animals (nos. I and 6) the entry point v,..~ located within sector 'a', and the depths of reinnervation were therefore equal. In only one of the 7 cortex-lesioned rats (no. 9) was the depth of reinnervation greater at the entry point than in sector 'a'. In the sham-lesioned group, the depth of
137
Fig. 3. Examples of entry of catecholaminergic neurites into host brain. In each case, the graft is on the left and the host caudate-putamen is on the right. In (a), (b) and (c), neurites enter into the brain over fairly broad areas, and in (d) and (e), the neurites enter in small bundles. Bars in ~,b = 1001~m; in c,d, and e ~- 50~m.
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Fig. 4. Depth of reinnervation in sector 'a' as compared to the depth of reinnervation perpendicular to (1) the most prominent location where catecholaminergic neurites were seen to enter the host striatum (E for 'Entry'), (2) a point 0.5 mm ventral to the entry point (V) and (3) a point 0.5 mm dorsal to the entry point (D). Data are shown for each of the 9 sham-operated animals and the 7 cortex-lesioned animals. Note that in each sham-operated animal, the reinnervation is deeper at the entry point than in sector 'a'. In 4 of the 7 cortexlesioned rats, the reinnervation in sector 'a' is deeper than that found at the entry point (E). In rat nos. 1 and 6, the entry point and sector 'a' coincided. Only rat no. 9 clearly shows a pattern similar to that of the sham-operated animals.
139 reinnervation was 40 + 9.1% less in sector 'a' then at the entry point (mean + S.E.M.), whereas in the cortex-lesioned group the depth of reinnervation was 15 + 13% greater in sector 'a' (P < 0.01, two-tailed ttest, t(14) = 3.61).
General anatomical measurements In view of the misplacement of the grafts in some of the animals with cortical lesions, as well as the possibility of anatomical distortion induced by the cortical lesions, a number of measurements were made to compare the location of the grafts and other features of the anatomy of the lesioned animals and controls from the short-term study. The first possibility that was considered was that the lesions had altered the dorsal-ventral position of the grafts in the ventricle. The position of the center of each graft in the ventricle was evaluated for each rat, on a scale of 0-100%, where 100% represented the most dorsal limit of the ventricle. The position of the graft for the controls was at 73 + 3~1%, and 75 + 3.1% for the cortex-lesioned group (P > 0.2, t-test). In most animals the graft extended in the dorsalventral dimension over at least two sectors, often all sectc :s. A second analysis was therefore conducted. Every third section over the entire area where any substantial innervation of the striatum was seen was used. For each section, each of the 4 sectors of the ventricle ('a' through 'd') was scored as positive or negative for the presence of catecholamines in the graft. Each sector of the ventricle in each section was also scored as positive or negative for areas in which catecholaminergic neurites appeared to be entering the striatum. Each positive observation was assigned a score for 1 for present in sector 'a', 2 for 'b', 3 for 'c', and 4 for 'd'. The scores were summed and divided by the total number of positive observations to obtain a mean neurite entry score (catecholamines in striatum) and catecholamine presence score (catecholamines in the graft) for each animal. For the control animals, the catecholamine presence score was 2.3 + 0.17, vs 2.4 +_ 0.23 for the cortex-lesioned rats (P > 0.2); i.e. the predominant areas of the ventricle in which catecholamines were seen in the graft was between sectors 'b' and 'c' for both groups. The neurite entry scores were 2.4 _+_ 0.18 for the control group and 2.3 + 0.25 for the cortex-lesioned group (P > 0.2), so that neurites also were seen to be
entering the striatum predominantly between sectors 'b' and 'c' for both groups. A second possible source of artifact was anatomical distortion of the host striatum due to the removal of tissue from the lesion site. For each rat, one slide was selected from the 4 which showed the greatest degrees of reinnervation, counterstained with Cresyl violet, and measurements made with the aid of a projection microscope. The dorsal-ventral 'height' of the striatum was (means + S.E.M.) 5.2 + 0.18 mm for the controls and 5.4 + 0.30 mm for the cortex-lesioned group (P > 0.2, two-tailed t-test). The mediallateral 'width' of the striatum at the center of the graft was 2.73 + 0.14 mm for the controls and 2.72 + 0.13 mm for the cortex-lesioned rats (P > 0.2). At its widest point, the medial-lateral width of the striatum was 3.46 + 0.09 mm for the controls and 3.53 + 0.09 mm for the cortex-lesioned group (P > 0.2). Finally, measurements of the areas of the striatum were made with a mechanical planimeter. In both groups the right striatum was slightly smaller than the left striatum, to a similar degree for both groups. The area of the right striatum was 15.9 + 1.4 mm 2 for the controls and 15.0 + 0.6 mm 2 for the cortex-lesioned group (P > 0.2). The area of the left striatum (contralateral to the lesion(s) and the graft) was 17.2 + 1.2 mm 2 for the controls and 16.9 + 0.7 mm 2 for the cortex-le~i:med group (P > 0.2).
Short-term study: qualitative observations In many of the individual animals, peculiarities in the pattern of striatal reinnervation in the cortex-lesioned group were present (Fig. 5). For example, in animal no. 10, the catecholamines in the graft were largely confined to the ventral part of the ventricle, yet the striatal reinnervation was primarily in sector 'a'. In animal no. 6, the striatal reinnervation was very sparse along the walls of the ventricle, but a more dense clump of neurites had formed just underneath the medial border of the lesion. Plexuses of neurites were observed in the striatum underneath the lesion in four rats, nos. 6, 17, 23, and one (no. 3) that could not be scored because of an incomplete lesion. In the latter animal (no. 3), a bundle of catecholaminergic neurites could be seen to penetrate through the white matter and form a plexus of neurites around a small area of cortical injury (Fig. 5c,d). A similar penetration of neurites through the white
Fig. 5. Patterns of reinnervation in animals with cortical lesions, a and b: clumping of fluorescent neurites (small curved arrow in 'a') in the dorsal c~adate-putamen. The cortical lesion site can be seen in the upper right of photograph 'a'. c: a bundle of fluorescent neurites which left the reinnervated striatum and penetrated through the corpus callosum, entering into the cortex, apparently forming a plexus adjacent to a small area of brain injury. The amorphous material in the upper right is autofluorescent debris (arrows). d: higher magnification of the section shown in 'c'. The evenly-stained amorphous material in the right of the photograph (arrows) is autofluorescent debris. Bars in a and c = 200#m; in b and d = 100gm.
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matter, forming a plexus adjacent to the lesion, was also seen in rat no. 17. In two animals, nos. 10 and 15, the lateral limit of the reinnervation in sector "a~coincided with the lateral extent of the cortical lesions. Finally, in two animals with misplaced grafts (nos. 14 and 24), the grafts were located entirely in the cortex, yet the reinnervation of the striatum did not radiate equally in all directions but clearly followed the roof of the striatum just under the cortical lesion.
Long-term study In sham-lesioned animals, the density of striatal reinnervation was markedly increased, and the depth of reinneration was somewhat increased, as compared to that observed in the animals sacrificed in earlier time periods. There was a marked tendency for the preferential reinnervation of sector 'a' in t h e animals with cortical lesions (Fig. 6), as was observed io the short-term study. D I S C U S S I O N
Cortical aspiration lesions increased the reinnerration of the dorsal striatum by catecholaminergic neurites from SN grafts. The present experiment does not entirely elucidate the mechanism through
which cortical lesions produced this effect. One possibility is that the increased reinnervation of the striatum in animals with cortical lesions was due to a competition between cortical and SN inputs for synoptic contacts 24's7. In other words, cortical inputs might sprout tc fill in synaptic sites vacated following a SN lesion. A subsequent cortical lesion might remove some of this sprouted input, freeing up synaptic sites for contact by neurites from transplanted SN. This possibility is probably unlikely in view of the topography of the corticostriatal projection in the rat 43. The corticostriatal pathway is organized in a medial-lateral manner, so that medial cortical lesions like those used in the present study would be expected to produce degeneration of the cortical input along the medial striatum, extending at least into sector 'c '43. The areas of increased reinnervation in the present study corresponded to physical proximity to the lesion, more than to the topography of the corticostriatal projection. Nevertheless, the corticostriatal projection from the parts of the cortex that were lesioned would be the most dense in the dorsal striatum, so that competition cannot be ruled out entirely. The increased reinnervation of the mos~ ,Set,a! part of the striatum, directly underneath the cortical
142 lesion, suggests that a diffus[i,!e substance originating nee,r the lesion site might have influenced the growth of the graft-derived neurites. Nieto-Sampedro et al. 3°'31 have reported that brain tissue adjacent to an area of cortical injury produces a substance or substances which enhance the survival of neurons in tissue culture. Concentrations of this substance become maximal 10-16 days after lesioning 3°'31. The 10-day time interval between lesioning and transplantation in the present study was, in fact, selected so that transplantation and initial growth of the grafts would coincide with the presence of maximal concentrations of lesion-induced neurotrophic factor(s). A situation analogous to the present experiment was observed by Olson, Seiger, and their colleagues 33'39.4° following transplantation of locus coeruleus to the anterior chamber of the eye. These grafts were usually found to innervate about onethird of the iris. When the trigeminal innervation of the iris was removed, the innervation expanded to cover the entire iris 33. Although, on the face of it, this phenomenon appears to represent competition between trigeminal and graft-derived neurites for the same terminal field, subsequent studies suggested an alternative possibility, that trophic factors secreted by the denervated iris might be involved 12. The relative contributions of trophic factors and competition in this situation have not yet been entirely determined.
REFERENCES 1 Bernstein, J.J., Getz, R., Jefferson, M. and Kelemen, M., Astrocytes secrete basal lamina after hemisection of the spinal cord, Brain Research, 327 (1985) 135-141. 2 Bj6rk!und, A., Dunnett, S.B., Stenevi, U., Lewis, M.E. and Iversen, S.D., Reinnervation of the denervated striatum by substantia nigra transplants: Functional consequences as revealed by pharmacological and sensorimotor testing, Brain Research, 199 (1980) 307-333. 3 Bouyer, J.J., Park, D.H., Joh, T.H. and Pickel, V.M., Chemical and structural analysis of the relationship between cortical inputs and tyrosine hydroxylase-containing terminals in the rat striatum, Brain Research, 302 (1984) 267-275. 4 Collins, F. and Crutcher, K., Neurotrophic activity in the adult rat hippocampal formation: regional distribution and increase after septal lesion, J. Neurosci., 5 (1985) 2809-2814. 5 Cotman, C.W. and Nieto-SamFedro, M., Cell biology of synaptic plasticity, Science, 225 (1984) 1287-1294. 6 Cotman, C.W., Nieto-Sampedro, M, and Harris, E., Synapse replacement in the nervous system of adult verte-
In summary, cerebral cortical aspiration lesions produced a highly localized augmentation of the reinnervation of the dorsal striatum by SN grafts. Several general anatomical measurements failed to support the possibility that anatomical distortion could have accounted for this increased reinnervation of the dorsal striatum. There was no distortion of the general dimensions of the striatum, of the size of the striatum, or of the position of the grafts in the ventricle. The present study did not address the time dependency of the effect of lesions of the growth of neurites from SN grafts. Nevertheless, it appears that reinnervation of the striatum by SN grafts can be substantially influenced by lesioning of the overlying cerebral cortex. ACKNOWLEDGEMENTS We thank Eleanor Krauthamer for preparing the histological materials, and the clerical, administrative, and animal care staff of the William A. White Building for invaluable assistance. We also thank Drs. Carl Cotman, Anthony Adinolfi, Daniel Weinberger, Richard Wyatt, George Jaskiw and Joel Kleinman for reading and commenting on the manuscript. Some of these data were presented at the April 1986 meeting of the New York Academy of Sciences entitled, 'Cell and Tissue Transplantation into the Adult Brain.'
brates, Physiol. Rev., 61 (1981 684-784. 7 De la Torre, J.C., An improved approach to histofluorescence using the SPG method for tissue monoamines, J. Neurosci. Methods, 3 (1980) 1-5. 8 Dunnett, S.B., Bj6rklund, A., Stenevi, U. and Iversen, S.D., Behavioral recovery following transplantation of substantia n',.'3rain rats subjected to 6-OHDA lesions of the nigrostriatal pathway. I. Unilateral lesions, Brain Research, 215 (1981a) 147-161. 9 Dunnett, S.B., Bj6rklund, A., Stenevi, U. and Iversen, S.D., Grafts of embryonic substantia nigra reinnervation the ventrolateral striatum ameliorate sensorimotor impairments and akinesia in rats with 6-OHDA lesions of the nigrostriatal pathway, Brain Research, 229 ( 1981b) 209-217. 10 Dunnett, S.B., Bj6rklund, A., Gage, F.H. and Stenevi, U., Transplantation of mesencephalic dopamine neurons to the striatum of adult rat. In A. Bj6rklund and U. Stenevi (Eds.), Neural Grafting in the Mammalian CNS, Elsevier, Amsterdam, 1985, pp. 451-469. 11 Dunnett, S.B., Bj6rklund, A., Schmidt, R.H., Stenevi, U. and Iversen, S.D., Intracerebral grafting of neuronal cell suspensions. IV. Behavioral recovery in rats with unilateral 6-OHDA lesions following implantation of nigral cell sus-
143 pensions in different brain sites, Acta Physiol. Scand., Suppl. 522 (1983 29-37. 12 Ebendal, T., Olson, L., Seiger, A. and Hedlund, K.-O., Nerve growth factors in the rat iris, Nature (Lond.), 286 (1980 25-28. 13 Freed, W.J., GI~I ganglioside does not stimulate reinnervation of the striatum by substantia nigra grafts, Brain Res. Bull., 14 (1985) 91-95. 14 Freed, W.J., T~'ansplantation of tissues to the cerebral ventricles: methodological details and rate of graft survival. In A. Bjrrklund and U. Stenevi (Eds), Neural Grafting in the Mammalian CNS, Elsevier, Amsterdam, 1985, pp. 31-40. 15 Freed, W.J., Functional brain tissue transplantation: reversal of lesion-induced rotation by intraventricular substantia nigra and adrenal medulla grafts, with a note on intracranical retinal grafts, Biol. Psychiatry, 18 (1983) 1205-1267. 16 Freed, W.J., Perlow, M.J., Karoum, F., Seiger, A., Olson, L., Hoffer, B.J. and Wyatt, R.J., Restoration of dopaminergic function by grafting of fetal rat substantia nigra to the caudate nucleus: Long-term behavioral, biochemical, and histochemical studies, Ann. Neurol., 8 (1980) 510-519. 17 Freed, W.J., Ko, G.N., Niehoff, D.L., Kuhar, M.J., Holler, B.J., Olson, L., Cannon-Spoor, H.E., Morihisa, J.M. and Wyatt, R.J., Normalization of spiroperidol binding in the denervated rat striatum by homologous grafts of substantia nigra, Science, 222 (1983) 937-939. 18 Freund, T.F., Bolam, J.P., Bj6rklund, A., Stenevi, U., Dunnett, S.B., Poqweli, J.F. and Smith A.D., Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neostriatum: A tyrosine hydroxylase immunocytochemical study, J. Neurosci., 5 (1985) 603-616. 19 Gage, F.H., Bjrrklund, A. and Stenevi, U., Denervation releases a neuronal sur,:ival factor in adult rat hippocampus, Nature (Lond.), 308 (1984) 637-639. 20 Goldman, P.S. and Nauta, W.J.N., An intricately patterned prefronto-caudate projection in the rhesus monkey, J. Comp. Neurol., 171 (1977) 369-386. 21 Hattori, T. and Fibiger, H.C., On the use of lesions of afferents to localize neurotransmitter receptor sites in the striatum, Brain Research, 238 (1982) 245-250. 22 Krnig, J.F.R. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas, Krieger, New York, 1963. 23 Lander, A.D., Fujii, D.K. and Reichardt, L.F., Laminin is associated with the 'neurite outgrowth-promoting factors' found in conditioned media, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 2183-2187. 24 Leong, S.K. and Luad, R.D., Anomalous bilateral corticofugal pathways in albino rats after neonatal lesions, Brain Research, 62 (1973) 218-221. 25 Liesi, P., Dahl, D. and Vaheri, A., Neurons cultured from developing rat brain attach and spread preferentially to laminin, J. Neurosci. Res., 11 (1984)241-251 26 Liesi, P., Kaakkola, S., Dahi, D. and Vaheri, A., Laminin is induced in astrocytes of adult brain by injury, EMBO J., 3 (1984) 683-686. 27 Loren, I., Bj6rklund, A., Lindvall, O. and Schmidt, R.H., Improved catecholamine histofluorescence in the developing brain based on the magnesium and aluminum (ALFA) perfusion techniques: methodology and anatom-
ical observations, Brain Res. Bull., 9 (1982) 11-26. 28 Mahalik, T.J. Finger, T.E. Stromberg, I. and Olson, L., Substantia nigra transplants into denervated striatum of the rat: ultrastructure of graft and host connections, J. Comp. Neurol., 240 (1985) 60-70. 29 Manthorpe, M., Engvali, E., Ruoslahti, E., Longo, F.M., Davis, G.E. and Varon. S., Laminin promotes neuritic regeneration from cultured peripheral and central neurons, J. Cell Biol., 97 (1983) 1882-1890. 30 Nieto-Sampedro, M., Lewis, E., Cotman, C., Manthorpe, M., Skaper, S., Barbin, G., Longo, F. and Varon, S., Brain injury causes time-dependend increase in neuronotrophic activuy at the lesion site, Science, 217 (1982) 860-861. 31 Nieto-Sampedro, M., Manthorpe, M., Barbin, G., Varon, S. and Cotman, C., Injury-induced neuronotrophic activity in adult rat brain: correlation with survival of delayed implants in the wound cavity, J. Neurosci., 3 (1983) 2219-2229. 32 Nieto-Sampedro, M., Whittemore, S.R., Needels, D.L., Larson, J. and Cotman, C.W., The survival of brain transplants is enhanced by extracts from injured brain, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 6250-6254. 33 Olson, L., Seiger, A. and Alund, M., Locus coeruleus fiber growth in oculo induced by trigeminotomy, Med. Biol., 56 (1978) 23-27. 34" Pellegrino, J., Pellegrino, A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, 2nd edn., Plenum, New York, 1979. 35 Perlow, M.J., Freed, W.J., Holler, B.J., Seiger, A., Olson, L. and Wyatt, R.J., Brain grafts reduce motor abnormalities produced by destruction of the nigrostriatal dopamine system, Science, 204 (1979) 643-647. 36 Rose, G., Lynch, G. and Cotman, C.W., Hypertrophy and redistribution of astrocytes in the deafferented dentate gyrus, Brain Res. Bull., 1 (1976) 87-92. 37 Schneider, G.E., Early lesions of superior colliculus: factors affecting the formation of abnormal retinal projections, Brain Behav. Evol., 8 (1973) 73-109. 38 Schonfeld, A.R., Heacock, A.~L and Katzman, R., Enhancement of central cholinergic sprouting by prior injury: correlation with endogenous trophic con~cnt of hippocampus, Brain Research, 321 (1984) 377-380. 39 Seiger, A. and Olson, L., Quantitation of fiber growth in transplanted central monoamine neurons, Cell Tissue Res., 179 (1977) 285-316. 40 Seiger, A. and Oison, L., Reinitiation of directed nerve fiber growth in central monoamine neurons after intraocular maturation, Exp. Brain Res., 29 (1977) 15-44. 41 Sternberger, L.A., lmmunocytochemistry, 2nd edn., Wiley, New York, 1979. 42 Urlgerstedt, U. and Arbuthnott, G.W., Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatai dopamine system, Brain Research, 24 (1970) 485-493. 43 Webster, K.E., Cortico-striatal interrelations in the albino rat, J. Anat., 95 (1961) 532-545. 44 Whittemore, S.R., Nieto-Sampedro, M., Needels, D.L. and Cotman, C.W., Neuronotrophic activity for mammalian brain neurons. Injury induction in neonatal, adult, and aged rat brain, Dev. Brain Res., 20 (1985) 169-178.
133
BRE13439
Cortical lesions increase reinnervation of the dorsal striatum by substantia nigra grafts William J. Freed and H. Eleanor Cannon-Spoor Preclinical Neurosciences Section, Neuropsychiatry Branch, National Institute of Mental Health, Saint Elizabeth's Hospital, Washington, DC20032 (U.S.A.)
(Accepted 29 September 1987) Key words: Transplantation; Neurite; Fluorescence histochemistry; Cerebral cortex
Effects of aspiration lesions of the cerebral cortex on intraventricular substantia nigra grafts were investigated, increased reinnerration of the dorsal striatum was observed in animals with cortical aspirations. This reinnervation was confined to the dorsal onefourth of the striatum, immediately underneath the cortical lesions. The increased reinnervation of the striatum by substantia nigra grafts in animals with cortical lesions is suggested to be related either to secretion of neurotrophic substances by the injured brain tissue or to removal of competition between corticostriatal inputs and graft-derived neurites for sytaapticcontacts.
INTRODUCTION When embryonic substantia nigra (SN) is transplanted into the lateral cerebral ventricle, adjacent to the denervated corpus striatum, these grafts produce dopamine-containing neurites which penetrate into the host striatum for limited distances 13'15-17"35. Although substantial growth of dopaminergic neurites from these grafts is observed within 2-3 weeks 13, the distance to which these neurites grow is limited to less than 1.5 mm even more than a year later 15-17. The degree of reinnervation of the str~atum produced by grafts of SN in cavities in the cerebral cortex or of dissociated SN cells is also limited 2'8-~1. In most studies of SN transplantation, the SN grafts are implanted several months after the SN lesions are produced. By this time, the vacated postsynaptic sites in the striatum may have disappeared, either because of filling due to sprouting of adjacent inputs 6, astrocyte proliferation 36, or degeneration of dendritic spines 2~. It may be that the reinnervation of the striatum by SN grafts is limited because of a limited availability of sites for synaptic contacts. The af-
ferents to the striatum from the cerebral cortex 2°'43 contact the same dendritic spines as the afferents from the S N 3"!8 o r from SN grafts 1s'28. It is therefore conceivable that potential sites for synaptic contacts could become available for short periods of time after the induction of cortical lesions. For this reason, cortical lesions which are induced just prior to SN transplantation might increase the graft-derived striatal reinnervation. Injury to the central and peripheral nervous s','stern elicits the release of substances which support the survival of neurons in tissue culture 4"5"3°-32"u. This survival-promoting effect is time-depende~at, with maximum activity present between 10 and 16 days after the induction of injury 3°'3~. Subsequent experiments have shown that the survival of brain grafts can also be enhanced by brain injury. When embryonic tissue is transplanted into cortical injury sites, survival of the grafts is maximal when the time between the production of injury and implantation of grafts is delayed to coincide with the time at which concentrations of trophic activity are greatest 3~. Survival of brain grafts was also found to be enhanced by
Correspondence: W.J. Freed, Preclinicai Neurosciences Section, Neuropsychiatry Branch, National Institute of Mental Health. Saint Elizabeth's Hospital, Washington, DC 20032, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
134 administration of extracts from injured brain into the implant site a2. Trophic activity also appears in denerrated brain areas, when the innervating nuclei are destroyed or when pathways are severed 19'a~. The nervous system therefore, appears to have a ubiquitous tendency to produce trophic substances in response to injury. Besides playing a role in neuron survival, injuryinduced neurotrophic factors may be associated with neurite formation as well29. Laminin, which promotes neuronal process formation, is produced by astrocytes 1'23'25"26. Schonfeld et al. 3a found that hippocampal injury enhanced the accumulation of choline acetyltransferase activity in intrahippocampal iris implants, an index of cholinergic sprouting. These data suggest a possible role of injury-induced neurotrophic factors produced by neuroglia in neurite extension as well as in neuron survival. In another type of graft experiment, dopaminergic neurons were transplanted into preprepared cavities in the cortex 2,8. These cortical transplantation cavities were much like the lesion sites which have been found to promote the production of trophic substances. In the transplantation experiments, however, grafts were implanted 3.5-5 weeks after the induction of injury, at a time when trophic activity would be expected to have returned to nearly baseline levels2,8,9. Nevertheless, some of the published photographs suggest that the degree of neurite penetration into the striatum from SN grafts in cortical cavities may exceed that observed after transplantation of SN into the lateral ventricle 2. The present experiment was, therefore, undertaken to investigate the possibility that cortical injury would enhance the effects of SN grafts. By transplanting tissue into the lateral ventricle and lesioning the cerebral cortex, it may be possible to separate the effects of cortical injury from those of SN grafts. The time interval between the induction of injury and graft implantation therefore was chosen as 10 days, to coincide with the time of maximal concentrations of neurotrophic substances in injured brain tissue 3°,31,44.
were ho~.lsed in groups of 4 on a 12-h !igh~-dark cycle with continuous access to food and water. A total of 32 rats were used in the experiment. Timed pregnant rats (tissue donors) were obtained from Zivic-Miller Labs., Inc.
MATERIALSAND METHODS
Brain grafts
Animals Male Sprague-Dawlcy rats (Zivic.-Miller), weighing 150-175 g at the start of the experiment,
Surgery All surgery was performed under Chloropent (Fort Dodge Labs.) anesthesia. Animals received Flo-Cillin (Bristol Labs.) 0.4 ml/kg following each surgical procedure.
SN lesions SN lesions were performed by stereotaxic administration of 4.67/~g 6-hydroxydopamine hydrobromide (Sigma Chemicals) in a vehicle of 0.4% ascorbic acid in normal saline, in a volume of 4.0 gl over 4 rain at stereotaxic coordinates 4.4 mm posterior and 1.2 mm lateral to the bregma and 7.8 mm below the dura with the incisor bar 2.4 mm below the interaural line, according to the atlas of K6nig and Klippe122. All lesions were unilateral, on the right side. Two months after SN lesions, animals were screened for rotational behavior after administration of 0.1 mg/kg apemorphine s.c. 42. Animals that turned at least 80 rotations/h were selected for use in the experiment. Studies of rotational behavior will be reported separately.
Cortical lesions Cortical lesions were performed two and one-half months after SN lesions by aspiration of the cerebral cortex down to the white matter extending approximately laterally from 0.5 mm from the midline to the supraorbital ridge and from 2 mm posterior to the bregma to the olfactory bulb. Following aspiration, the cavity was packed with gelfoam and the skin closed with wound clips. For sham-operated animals, the bone was removed but no tissue was aspirated. For some of the animals that were sacrificed after long time intervals, the lesions were considerably more extensive medially and laterally.
Ten days after corticai or sham lesions all animals received solid SN grafts from two 17-day gestational rat embryos. The grafts were implanted into the lateral ventricle at Pellegrino et al. 34 coordi~ates 1.5
135 mm lateral, 1.5 mm anterior to the bregma, and 3.5 mm below the dura (bite-bar set at 5.0 mm above the interaural line) as previously described 14a5.
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Histology Animals were sacrificed four weeks ('short-term study,' n = 25) or one year ('long-term study,' n = 7) after transplantation. Animals received pargylene 75 mg/kg i.p. and after 4 h received an overdose of Chloropent i.p. Animals were perfused with a magnesium-sucrose solution as described by Loren et al. 27 and processed for glyoxylic acid-induced histochemical fluorescence as described by de la Torre 7. Depth of penetration of catecholamine-containing neurites into the striatum was measured using an eyepiece micrometer and stage coordinates. Because the cortical lesions were obvious, it was impossible to be blind to the status of the animals, so an observer who was naive concerning the hypothesis of the experiment was trained to perform the measurements of depth of neurite penetration. For measurements of neurite penetration, the striatum was divided into 4 dorsoventral sectors, labelled 'a', 'b', 'c' and 'd', corresponding to the 4 quarters of the ventricle as shown in Fig. la. Tyrosine hydroxylase immunocytochemistry was performed on two sham-lesioned rats using the immunoperoxidase technique of Sternberger 41. The tyrosine hydroxylase antibody was supplied by N. Weiner and W. Tank of the University of Colorado.
Statistics Statistical calculations were performed either by hand or with the aid of the StatView 512 + program (BrainPower, Inc., Calabases, CA), except for multivariate analysis of variance, for which the statistical analysis system (SAS Institute Inc., Cary, NC) general linear models procedure was used. RESULTS
General histology TH immunocytochemical staining revealed that the graft.~ contained groups of neurons with TH immunoreactivity (Fig. 2). Fluorescence histochemistry reveale¢t dense catecholaminergic innervation of parts off the grafts, often obscuring the catecholaminergic neurons. The grafts consistently reinnervated parts of the striatum, particularly areas adja-
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cent to those parts of the grafts with the most dense accumulations of catecholamines. The cortex lesions in most cases were found to be fairly small and extended laterally from about 1-4 mm from the midline ar.,J 4-6 mm in the rostral-caudal dimension. Some of the lesions partially compromised the white matter. A representative lesion is shown in Fig. lb. One o f the larger lesions that was used in the long-term study is shown in Fig. 6. In a substantial number of the rats with cortical lesions, the grafts were found to be misp l a c e d - for example, in parts of the brain other then tl-.e ventricle such as within the cortical lesion site. These animals were not used for further quantitative analysis.
Effects of cortical lesio~s Evaluation of the deepest reinnervation of the striatum (in any of the 4 sectors) for each animal from the short-term study revealed a depth of reinnerva-
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tion (means + S.E.M.) of 0.77 _+ 0.08 mm for the sham-lesioned group and 1.04 + 0.11 mm for the cortical-lesioned rats. This difference did not reach statistical significance (P < 0.07, two-tailed t-test, t(14) = 2.03). Evaluations of the depths of reinnervation in each of the 4 sectors 'a' through 'd', however, showed an increase in the depth of reinnervation in sector 'a' (the most dorsal sector) in the cortex-lesioned group (Fig. lc). A multivariate analysis of variance revealed that the depth of reinnervation was increased in sector 'a' (El.15 = 10.83, P = 0.005). There was no difference in sector 'b', (P = 0.327), nor in sectors 'c' and 'd' (P > 0.5). The depth of reinnervation for each animal was also evaluated at the point where catecholaminergic
neurites were most prominently seen to oe entering the striatum (cf. Fig. 3). At the same time, depths of reinnervation were measured 0.5 mm dorsal to the entry point, 0.5 mm ventral to the entry point, and for sector 'a'. These data are shown in Fig. 4. In every one of the sham-lesioned rats, the depth of reinnervation was greater at the entry point than in sector 'a' (Fig. 4). In contrast, this was not true of the animals with cortical lesions. In 4 of the 7 cortex-lesioned rats, the reinnervation depth was greater in sector 'a' than at the entry point. In two animals (nos. I and 6) the entry point v,..~ located within sector 'a', and the depths of reinnervation were therefore equal. In only one of the 7 cortex-lesioned rats (no. 9) was the depth of reinnervation greater at the entry point than in sector 'a'. In the sham-lesioned group, the depth of
137
Fig. 3. Examples of entry of catecholaminergic neurites into host brain. In each case, the graft is on the left and the host caudate-putamen is on the right. In (a), (b) and (c), neurites enter into the brain over fairly broad areas, and in (d) and (e), the neurites enter in small bundles. Bars in ~,b = 1001~m; in c,d, and e ~- 50~m.
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Fig. 4. Depth of reinnervation in sector 'a' as compared to the depth of reinnervation perpendicular to (1) the most prominent location where catecholaminergic neurites were seen to enter the host striatum (E for 'Entry'), (2) a point 0.5 mm ventral to the entry point (V) and (3) a point 0.5 mm dorsal to the entry point (D). Data are shown for each of the 9 sham-operated animals and the 7 cortex-lesioned animals. Note that in each sham-operated animal, the reinnervation is deeper at the entry point than in sector 'a'. In 4 of the 7 cortexlesioned rats, the reinnervation in sector 'a' is deeper than that found at the entry point (E). In rat nos. 1 and 6, the entry point and sector 'a' coincided. Only rat no. 9 clearly shows a pattern similar to that of the sham-operated animals.
139 reinnervation was 40 + 9.1% less in sector 'a' then at the entry point (mean + S.E.M.), whereas in the cortex-lesioned group the depth of reinnervation was 15 + 13% greater in sector 'a' (P < 0.01, two-tailed ttest, t(14) = 3.61).
General anatomical measurements In view of the misplacement of the grafts in some of the animals with cortical lesions, as well as the possibility of anatomical distortion induced by the cortical lesions, a number of measurements were made to compare the location of the grafts and other features of the anatomy of the lesioned animals and controls from the short-term study. The first possibility that was considered was that the lesions had altered the dorsal-ventral position of the grafts in the ventricle. The position of the center of each graft in the ventricle was evaluated for each rat, on a scale of 0-100%, where 100% represented the most dorsal limit of the ventricle. The position of the graft for the controls was at 73 + 3~1%, and 75 + 3.1% for the cortex-lesioned group (P > 0.2, t-test). In most animals the graft extended in the dorsalventral dimension over at least two sectors, often all sectc :s. A second analysis was therefore conducted. Every third section over the entire area where any substantial innervation of the striatum was seen was used. For each section, each of the 4 sectors of the ventricle ('a' through 'd') was scored as positive or negative for the presence of catecholamines in the graft. Each sector of the ventricle in each section was also scored as positive or negative for areas in which catecholaminergic neurites appeared to be entering the striatum. Each positive observation was assigned a score for 1 for present in sector 'a', 2 for 'b', 3 for 'c', and 4 for 'd'. The scores were summed and divided by the total number of positive observations to obtain a mean neurite entry score (catecholamines in striatum) and catecholamine presence score (catecholamines in the graft) for each animal. For the control animals, the catecholamine presence score was 2.3 + 0.17, vs 2.4 +_ 0.23 for the cortex-lesioned rats (P > 0.2); i.e. the predominant areas of the ventricle in which catecholamines were seen in the graft was between sectors 'b' and 'c' for both groups. The neurite entry scores were 2.4 _+_ 0.18 for the control group and 2.3 + 0.25 for the cortex-lesioned group (P > 0.2), so that neurites also were seen to be
entering the striatum predominantly between sectors 'b' and 'c' for both groups. A second possible source of artifact was anatomical distortion of the host striatum due to the removal of tissue from the lesion site. For each rat, one slide was selected from the 4 which showed the greatest degrees of reinnervation, counterstained with Cresyl violet, and measurements made with the aid of a projection microscope. The dorsal-ventral 'height' of the striatum was (means + S.E.M.) 5.2 + 0.18 mm for the controls and 5.4 + 0.30 mm for the cortex-lesioned group (P > 0.2, two-tailed t-test). The mediallateral 'width' of the striatum at the center of the graft was 2.73 + 0.14 mm for the controls and 2.72 + 0.13 mm for the cortex-lesioned rats (P > 0.2). At its widest point, the medial-lateral width of the striatum was 3.46 + 0.09 mm for the controls and 3.53 + 0.09 mm for the cortex-lesioned group (P > 0.2). Finally, measurements of the areas of the striatum were made with a mechanical planimeter. In both groups the right striatum was slightly smaller than the left striatum, to a similar degree for both groups. The area of the right striatum was 15.9 + 1.4 mm 2 for the controls and 15.0 + 0.6 mm 2 for the cortex-lesioned group (P > 0.2). The area of the left striatum (contralateral to the lesion(s) and the graft) was 17.2 + 1.2 mm 2 for the controls and 16.9 + 0.7 mm 2 for the cortex-le~i:med group (P > 0.2).
Short-term study: qualitative observations In many of the individual animals, peculiarities in the pattern of striatal reinnervation in the cortex-lesioned group were present (Fig. 5). For example, in animal no. 10, the catecholamines in the graft were largely confined to the ventral part of the ventricle, yet the striatal reinnervation was primarily in sector 'a'. In animal no. 6, the striatal reinnervation was very sparse along the walls of the ventricle, but a more dense clump of neurites had formed just underneath the medial border of the lesion. Plexuses of neurites were observed in the striatum underneath the lesion in four rats, nos. 6, 17, 23, and one (no. 3) that could not be scored because of an incomplete lesion. In the latter animal (no. 3), a bundle of catecholaminergic neurites could be seen to penetrate through the white matter and form a plexus of neurites around a small area of cortical injury (Fig. 5c,d). A similar penetration of neurites through the white
Fig. 5. Patterns of reinnervation in animals with cortical lesions, a and b: clumping of fluorescent neurites (small curved arrow in 'a') in the dorsal c~adate-putamen. The cortical lesion site can be seen in the upper right of photograph 'a'. c: a bundle of fluorescent neurites which left the reinnervated striatum and penetrated through the corpus callosum, entering into the cortex, apparently forming a plexus adjacent to a small area of brain injury. The amorphous material in the upper right is autofluorescent debris (arrows). d: higher magnification of the section shown in 'c'. The evenly-stained amorphous material in the right of the photograph (arrows) is autofluorescent debris. Bars in a and c = 200#m; in b and d = 100gm.
141
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~
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Fig. 6. Camera lucida drawings of catecholamine fluorescence in the striatum of two animals with grafts sacrificed 12 months after transplantation, a: a sham-lesioned animal, b: an animal with a cortical lesion. Note that the lesion is more extensive than those used for the short-term animals. G, graft; L, lesion; V, ventricle; CX, cortex; CC, corpus callosum; CP, caudate-putamen.
matter, forming a plexus adjacent to the lesion, was also seen in rat no. 17. In two animals, nos. 10 and 15, the lateral limit of the reinnervation in sector "a~coincided with the lateral extent of the cortical lesions. Finally, in two animals with misplaced grafts (nos. 14 and 24), the grafts were located entirely in the cortex, yet the reinnervation of the striatum did not radiate equally in all directions but clearly followed the roof of the striatum just under the cortical lesion.
Long-term study In sham-lesioned animals, the density of striatal reinnervation was markedly increased, and the depth of reinneration was somewhat increased, as compared to that observed in the animals sacrificed in earlier time periods. There was a marked tendency for the preferential reinnervation of sector 'a' in t h e animals with cortical lesions (Fig. 6), as was observed io the short-term study. D I S C U S S I O N
Cortical aspiration lesions increased the reinnerration of the dorsal striatum by catecholaminergic neurites from SN grafts. The present experiment does not entirely elucidate the mechanism through
which cortical lesions produced this effect. One possibility is that the increased reinnervation of the striatum in animals with cortical lesions was due to a competition between cortical and SN inputs for synoptic contacts 24's7. In other words, cortical inputs might sprout tc fill in synaptic sites vacated following a SN lesion. A subsequent cortical lesion might remove some of this sprouted input, freeing up synaptic sites for contact by neurites from transplanted SN. This possibility is probably unlikely in view of the topography of the corticostriatal projection in the rat 43. The corticostriatal pathway is organized in a medial-lateral manner, so that medial cortical lesions like those used in the present study would be expected to produce degeneration of the cortical input along the medial striatum, extending at least into sector 'c '43. The areas of increased reinnervation in the present study corresponded to physical proximity to the lesion, more than to the topography of the corticostriatal projection. Nevertheless, the corticostriatal projection from the parts of the cortex that were lesioned would be the most dense in the dorsal striatum, so that competition cannot be ruled out entirely. The increased reinnervation of the mos~ ,Set,a! part of the striatum, directly underneath the cortical
142 lesion, suggests that a diffus[i,!e substance originating nee,r the lesion site might have influenced the growth of the graft-derived neurites. Nieto-Sampedro et al. 3°'31 have reported that brain tissue adjacent to an area of cortical injury produces a substance or substances which enhance the survival of neurons in tissue culture. Concentrations of this substance become maximal 10-16 days after lesioning 3°'31. The 10-day time interval between lesioning and transplantation in the present study was, in fact, selected so that transplantation and initial growth of the grafts would coincide with the presence of maximal concentrations of lesion-induced neurotrophic factor(s). A situation analogous to the present experiment was observed by Olson, Seiger, and their colleagues 33'39.4° following transplantation of locus coeruleus to the anterior chamber of the eye. These grafts were usually found to innervate about onethird of the iris. When the trigeminal innervation of the iris was removed, the innervation expanded to cover the entire iris 33. Although, on the face of it, this phenomenon appears to represent competition between trigeminal and graft-derived neurites for the same terminal field, subsequent studies suggested an alternative possibility, that trophic factors secreted by the denervated iris might be involved 12. The relative contributions of trophic factors and competition in this situation have not yet been entirely determined.
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In summary, cerebral cortical aspiration lesions produced a highly localized augmentation of the reinnervation of the dorsal striatum by SN grafts. Several general anatomical measurements failed to support the possibility that anatomical distortion could have accounted for this increased reinnervation of the dorsal striatum. There was no distortion of the general dimensions of the striatum, of the size of the striatum, or of the position of the grafts in the ventricle. The present study did not address the time dependency of the effect of lesions of the growth of neurites from SN grafts. Nevertheless, it appears that reinnervation of the striatum by SN grafts can be substantially influenced by lesioning of the overlying cerebral cortex. ACKNOWLEDGEMENTS We thank Eleanor Krauthamer for preparing the histological materials, and the clerical, administrative, and animal care staff of the William A. White Building for invaluable assistance. We also thank Drs. Carl Cotman, Anthony Adinolfi, Daniel Weinberger, Richard Wyatt, George Jaskiw and Joel Kleinman for reading and commenting on the manuscript. Some of these data were presented at the April 1986 meeting of the New York Academy of Sciences entitled, 'Cell and Tissue Transplantation into the Adult Brain.'
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