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Abnormal expression of tyrosine hydroxylase-like immunoreactivity in intraocular transplants of rat caudate nucleus
Neuroscience Letters, 96 (1989) 253 258 Elsevier Scientific Publishers Ireland Ltd.
253
NSL 05839
Abnormal expression of tyrosine hydroxylase-like immunoreactivity in intraocular transplants of rat caudate nucleus T h o m a s J. M a h a l i k l, Ingrid S t r o m b e r g 2, T h o m a s E. Finger I and Lars O l s o n 2 :Department o1'Cellular and Structural Biology, University of Colorado Health Sciences ('enter, Den ver, ( ' 0 80262 (U.S.A.) and eDepartment o/" Histology, Karolinska Institute, Stockholm (Sweden (Received 20 July 1988; Revised version received 30 August 1988: Accepted 28 September 1988) Key n'ords."
Tyrosine hydroxylase; Striatum: Transplant: Gene expression
The purpose of the present study was to examine tyrosine hydroxylase (TH)-like immunoreactivaty in single intraocular grafts of caudate nucleus, and in caudate grafts which were co-grafted with substantia nigra. Grafts of caudate obtained from fetal rats (crown rump length: 15 17 mm) were placed into the anterior chamber of eye of adult female Sprague Dawley rats, and were allowed to survive from 6 to 20 months. The host rats were then perfused with fixative and the tissue was prepared for immunocytochemistry for TH. In 7 out of 8 surviving caudate grafts, including those containing no subslantia nigra, tyrosine TH-like immunoreactive (THLI) cell bodies were present. The results of our study suggest that TH is inappropriately expressed in caudate grafts placed in the anterior chamber of the eye.
Grafts of CNS tissue can be used to examine the relative influence of intrinsic and extrinsic factors on the development of specific CNS neurons or CNS regions [17]. Fetal tissue grafted either to the anterior chamber of the eye [17], or to the CNS adult animals [3] survives to express a number of biochemical [8, 9, 20, 21], morphological (8, 9, 12], electrophysiological [I 7] and functional [3, 10] characteristics appropriate to the caudate nucleus of the adult rat. Other evidence suggests however, that grafted caudate neurons express phenotypic characteristics which are not present in adult caudate neurons. For example, grafted medium spiny neurons in 35-day-old grafts have fewer spines than medium spiny neurons in the normal adult rat [20]. Further, the density of opioid receptor binding sites is lower in intraocular caudate grafts than in the adult caudate nucleus [12]. Thus, grafted caudate neurons express a combination of normal and abnormal phenotypical traits. In the course of a preliminary ultrastructural study of TH immunoreactivity in intraocular co-grafts of caudate nucleus and substantia nigra, we noticed that some cell bodies in the caudate part of the double graft expressed TH-like immunoreactiCorre,g;~mdence." T.J. Mahalik, Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, Box Bill, CO 80262, U.S.A.
254
vity. This finding was unusual in that T H immunoreactivity is not normally expressed by neurons in the rat caudate nucleus. The purpose of the present study is to followup on these initial observations by examining TH-like immunoreactivity in single grafts of caudate nucleus, and in caudate nucleus grafts which were co-grafted with substantia nigra. Nine grafts obtained from fetal Sprague-Dawley rats were used in the study. Two host animals received unilateral single grafts of the fetal caudate nucleus; 3 other hosts received double grafts of substantia nigra and caudate nucleus. Two additional host animals received bilateral single caudate grafts in each eye yielding a total of 9 grafts. This information is summarized in Table I. lntraoeular grafting. Donor material was obtained from Sprague-Dawley rat fetuses. Fetal tissue for caudate grafts was taken from fetuses with a crown-rump length of 15-17 mm; fetal nigral tissue was obtained from rats with a crown-rump length of 19-21 mm. Uteri were removed from pregnant Sprague-Dawley rats and kept at room temperature for up to 2 h. Fetuses were removed from the uterus and decapitated; the brains were removed and placed in lactated saline. Fetal tissue was dissected and placed in sterile lactated saline. The transplantation procedure previously has been described in detail [17]. Briefly, rats were lightly anesthetized with metaphane and the recipient eye was treated topically with a 1% solution of atropine sulfate. A razor blade was used to cut a small slit in the cornea, and a fire-polished pasteur pipette was used to pick up fetal tissue in approximately 5/tl of lactated saline. Gentle pressure was used to eject pieces of tissue into the anterior chamber of the eye. For double grafts of fetal substantia nigra and caudate, the striatal tissue was transplanted first; after 6 weeks the eyes were reopened and the nigral graft was placed in contact with the piece of striatal tissue. Single and double grafts were allowed to survive from 6 to 20 months. One month prior to perfusion, adrenergic input to the host iris was eliminated by removal of the superior cervical ganglion on the side of the graft. Tissue preparation. The host rats were deeply anesthetized with an overdose of sodium pentobarbital and injected intracardially with 1 ml of a 1% solution of sodium
TABLE I S U M M A R Y OF G R A F T S + , THLI cell present; - , no TH cells present; 0, no graft. Double =eaudate grafted with ventral mesencephalon from 19 to 20 mm (CRL) fetal donor. Crown-rump length (mm)
Type
TH cells
Crown rump length (mm)
Type
TH cells
15 16 17 17 17 17
caudate caudate caudate caudate caudate
+ 0 ~+ +
17 15-16 15 16 15 16
caudate double double double
+ + + +
255
nitrite, followed by an injection of 200 U of sodium heparin. After the rats had been perfused with 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), the grafts were removed from the eyes and placed in 4% paraformaldehyde in 0.1 M sodium bicarbonate buffer (pH 10.4) overnight at 4°C. The grafts then were washed in phosphate-buffered saline (PBS, pH 7.4) for 1 h and then cryoprotected in phosphate buffer containing increasing concentrations of sucrose and glycerol [5]. The grafts were frozen with dry-ice powder, and sectioned (40/lm) with a sliding microtome. Before exposure to the antibody solutions, the tissue sections were incubated in I% sodium borohydride in PBS for 25 min at room temperature. Following two 15 min rinses in PBS, the sections were incubated in PBS containing 2% normal goat serum to quench non-specific binding. The sections then were incubated in rabbit antiserum directed against T H (diluted 1:500 in PBS with 2% normal goat serum) for 1 6 4 8 h at 4°C. The T H antibody was a generous gift of Drs. Norman Weiner and William Tank. The immunocytochemical specificity of this antiserum has been described previously [16]. After 3 washes in PBS, the sections then were placed in fluorescein-labeled goat anti-rabbit IgG for 1 h at room temperature. After 3 wash steps, the sections were placed on gelatin-coated microscope slides and coverslipped in phosphate-buffered glycerol. Labeled material was viewed a n d p h o t o g r a p h e d on a Zeiss microscope equipped for epifluorescence (excitation = BP 450-490 nm; barrier = BP 520-560 nm). The control sections were incubated in pre-immune rabbit seruin.
Light microscopy of TH immunoreactivity. One of the grafts did not survive. The remaining 8 grafts survived from 6 to 20 months in oculo (Table I). T H immunocytochemistry revealed that TH-like immunoreactive (THLI) sympathetic innervation of the host iris was eliminated in 7 out of the 8 surviving cases. In control sections incubated with the T H pre-immune serum, no labeled cell bodies were observed. Several T H L I cell bodies were present in 4 of 5 single caudate grafts, and in the caudate of each of the 3 double grafts (Table I). T H L I cell bodies varied in size and in shape (Fig. 1). Some of the cells could not be identified on the basis of light microscopy (Fig. 1A, C); other cells however, possessed a prominent oval nucleus and a thin cytoplasm reminiscent of medium spiny neurons [4, 15]. Each 40/~m section through a striatal graft contained 2-5 THL1 cell bodies. T H L I cell bodies also were present in the nigral part of the nigrostriatal co-grafts. In each one of these cases T H L I fibers streamed across the boundary between the 2 types of graft. The presence of T H L I cell bodies in the caudate portion of the double grafts, however, made it impossible to assess the amount of nigral innervation of the striatal tissue. The present immunocytochemical study has shown that TH-like immunoreactivity is inappropriately expressed in neurons of the caudate nucleus following grafting into the anterior chamber of the eye. Of 8 surviving grafts, 7 contained T H L I cell bodies. Immunoreactive cell bodies were present in both single grafts of the caudate nucleus, and in caudate tissue which was co-grafted with substantia nigra. Some of the cell bodies within the grafted caudate resembled medium spiny neurons, the most common neuronal cell type in the caudate nucleus [4, 15]. A combined immunocytochem-
256
Fig. 1. Examples of THLI cell bodies in intraocular striatal grafts. A: graft 334-1. Note the large primary dendrite. B: several THLI cell bodies in a striatal graft. C: a THLI cell body; note round nucleus and scant cytoplasm, 2 characteristics of medium spiny neurons. D F: other THLI cell bodies in striatal grafts. The morphology of the cell in E resembles that of a medium spiny neuron.
ical a n d u l t r a s t r u c t u r a l s t u d y h o w e v e r , w o u l d be r e q u i r e d to definitely e s t a b l i s h the i d e n t i t y o f T H L I cells in c a u d a t e grafts. O u r f i n d i n g s a r e s i m i l a r to the results o f o t h e r s w h o h a v e s h o w n t h a t T H - l i k e i m m u n o r e a c t i v i t y m a y be i n a p p r o p r i a t e l y e x p r e s s e d in cell t y p e s w h i c h are n o t n o r -
257 mally catecholaminergic. For example, T H immunoreactivity is transiently expressed during development in cells of the rat and mouse peripheral nervous systems [2, 13, 14, 19]. Similarly, TH immunoreactivity is transiently expressed in neurons of the inferior colliculus [1 1] and neocortex [1] during development. In these developmental studies, ~inappropriate" T H expression was limited to a 2 3 day period, A more recent study has shown that TH-like immunoreactivity and tyrosine hydroxylase enzymatic activity is transiently expressed in cultured fetal cortical neurons [7]. Further, Park et al. [18] and Herman et al. [6] have shown that neurons within grafts of neocortical tissue express TH immunoreactivity for periods of up to 10 months [6] (the longest time span examined). In our study TH-like immunoreactivity was expressed in caudate grafts which were allowed to remain in oculo for up to 20 months. This result suggests that ~inappropriate" expression of T H is a stable characteristic of grafted caudate neurons. Thus the results of this and other transplantation studies [6, 20] contrast with the findings of in vitro or developmental studies which have shown that the expression of TH in ~inappropriate' cell populations is a transient phenomenon. One explanation of our results may be that TH expression is extremely labile in some neurons, and that it depends upon the presence of appropriate inputs, as well as on the availability of appropriate tar'gets [6]. Thus, in some peripheral and central neurons, TH is expressed transiently, before normal, adult-type cell -cell contacts are established. The TH gene then may be turned off once appriopriate contacts are tk~rmed. In the case of intraocular grafts, caudate neurons may never establish normal synaptic connections because they are isolated from other CNS tissue. The expression of TH may result form the absence of normal input, or from the lack of appropriate extrinsic targets. Alternatively, the inappropriate expression of TH immunoreactivity in oculo may result from changes introduced during manipulation of the fetal tissue, or from environmental influences unique to the anterior chamber of the eye. One way of addressing this issue would be to co-graft the caudate with each ot" its major sources of input, or targets of its output, e.g., cortex or pallidum. Our experiments show that co-grafting caudate and substantia nigra does not inhibit the abnormal expression of T H L I by the caudate neurons. It should be noted that the nigro striatal system represents a relatively low percentage of the total connectivity of the caudate. Thus co-grafting of caudate and other brain areas might reveal a system of connectivity that inhibits expression of T H L I by caudate neurons. This work was supported by N I H G r a n t NS09199 to T.E.F. and Magnus Bergvalls Stiftelse, and the Loo and Hans Ostermans Foundation. 1 Bcrger, B., Verney, C., Gaspar, P. and Febvret, A., Transient expression of tyrosine hydroxylase immunoreactivityin some neurons of the rat neocortex during development, Dev. Brain Res., 25 (1985) 141 I44. 2 Cochard, P., Goldstein, M. and Black, I.B., Ontogenetic appearance and disappearance of tyrosine hydroxylaseand catecholamines in the rat embryo, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2986 2990.
258 3 Deckel, A.W., Robinson, R.G., Coyle, J.T. and Sandberg, P.R., Reversal of long-term locomotor abnormalities in the kainic acid model of Huntington's disease by day 18 fetal striatal implants, Eur. .h Pharmacol., 93 (1983) 287 288. 4 DiFiglia, M.. Pasik, P, and Pasik, T., A Golgi study of neuronal types in the neostriatum of monkeys, Brain Res., 114 (1976) 245 256. 5 Eldred, W.D., Zucker, C., Karten, H.J., and Yazulla, S.J., Comparison of fixation and penetration enhancement techniques for use in ultrastructurat immunocytochemistry, J. Histochem. Cytochem.. 31 (1983) 258 292. 6 Herman, J.P., Abrous, N., Vigny, A., Dulluc, J. and LeMoal, M., Distorted development of intracerebral grafts: long-term maintenance of tyrosine hydroxylase-containing neurons in grafts of cortical tissue, Dev. Brain Res., 40 (1988) 81 88. 7 lacovitti, L., Lee, J., Joh, T.H. and Reis, D.J., Expression of tyrosine hydroxylase neurons of cultured cerebral cortex: Evidence for phenotypic plasticity in neurons of the CNS, J. Neurosci., 7 (1987) 1264 1270. 8 lsacson, O., Dawbarn, D, Brundin, P., Gage, F.H., Emson, P.C. and Bj6rklund, A., Neural grafting in a rat model of Huntington's disease: striosomal-like organization of striatal grafts as revealed by acetylcholinesterase histochemistry, immunocytochemistry and receptor autoradiography, Neuroscience, 22 (1987) 481~497. 9 Isacson, O., Brundin, P., Gage, F.H. and Bj6rklund, A., Neural grafting in a rat model of Huntington's disease: progressive neurochemicat changes after neostriatal ibotenate lesions and striatal tissue grafting, Neuroscience, 16 (1985) 799-817. 10 Isacson. O., Brundin, P., Kelly, P.A.T., Gage, F.H.~and Bj6rklund, A., Functional neuronal replacement by grafted striatal neurons in the ibotenic acid-lesioned rat striatum, Nature (Lond.), 311 (1984) 458 460. 11 Jaeger, C.B. and Joh, T.H., Transient expression of tyrosine hydroxylase in some neurons in the developing inferior colliculus of the rat, Dev. Brain Res., 11 (1983) 128 132. 12 Johnston, J.G., Boyd, S.R. and Van der Kooy, D., Compartmentalization of the embryonic striatum after transplantation, Dev. Brain Res., 33 (1987) 311~314. 13 Jonakait, G.M., Wolf, J., Cochard, P., Goldstein, M. and Black, I.B., Selective loss of noradrenergic phenotypic characters in neuroblasts of the rat embryo, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 4683 4686. 14 Jonakait, G.M., Miller, K.A., Goldstein, M. and Black, I.B., Transient expression of selected catecholaminergic traits in cranial sensory and dorsal root ganglia of the embryonic rat, Dev. Biol., 101 (1984) 51 60. 15 Kemp, J.M. and Powell, T.P.S., The structure of the caudate nucleus of the cat: light and electron microscopy, Phil. Trans. R. Soc. Lond., 262 (1971) 383~,01. 16 Mahalik, T.J., Finger, T.E., Stromberg, I. and Olson, L., Substantia nigra transplants into the denervated striatum of the rat: ultrastructure of graft and host interactions, J. Comp. Neurol., 240 (1985) 6(~ 70. 17 Olson, L., Seiger, A., Hoffer, B. and Taylor, D., Isolated catecholaminergic projections from the substantia nigra and locus coeruleus to caudate, hippocampus and cerebral cortex formed by intraocular sequential double brain grafts, Exp. Brain Res., 35 (1979) 47 ~57. 18 Park, J.K., Joh, T.H. and Ebner, F.F., Tyrosine hydroxylase is expressed by neocortical neurons after transplantation, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7495-7498. 19 Teitelman, G., Gershon, M.D., Rothman, T.P., Joh, T.H., and Reis, D.J., Proliferation and distribution of cells that transiently express a catecholaminergic phenotype during development in mice and rats, Dev. Biol., 86 (1981) 348 355. 20 Zemanick, M.C., Walker, P.D. and McAllister, J.P., Quantitative analysis of dendrites from transplanted neostriatal neurons, Brain Res., 414 (1987) 149-152. 21 Zhou, F.C., Hull, C.D., Levine, M.S. and Buchwald, N.A., Transplantation of fetal striatal tissue to striatum and substantia nigra of adult rats, Anat. Rec. 214 (1986) 150A.
253
NSL 05839
Abnormal expression of tyrosine hydroxylase-like immunoreactivity in intraocular transplants of rat caudate nucleus T h o m a s J. M a h a l i k l, Ingrid S t r o m b e r g 2, T h o m a s E. Finger I and Lars O l s o n 2 :Department o1'Cellular and Structural Biology, University of Colorado Health Sciences ('enter, Den ver, ( ' 0 80262 (U.S.A.) and eDepartment o/" Histology, Karolinska Institute, Stockholm (Sweden (Received 20 July 1988; Revised version received 30 August 1988: Accepted 28 September 1988) Key n'ords."
Tyrosine hydroxylase; Striatum: Transplant: Gene expression
The purpose of the present study was to examine tyrosine hydroxylase (TH)-like immunoreactivaty in single intraocular grafts of caudate nucleus, and in caudate grafts which were co-grafted with substantia nigra. Grafts of caudate obtained from fetal rats (crown rump length: 15 17 mm) were placed into the anterior chamber of eye of adult female Sprague Dawley rats, and were allowed to survive from 6 to 20 months. The host rats were then perfused with fixative and the tissue was prepared for immunocytochemistry for TH. In 7 out of 8 surviving caudate grafts, including those containing no subslantia nigra, tyrosine TH-like immunoreactive (THLI) cell bodies were present. The results of our study suggest that TH is inappropriately expressed in caudate grafts placed in the anterior chamber of the eye.
Grafts of CNS tissue can be used to examine the relative influence of intrinsic and extrinsic factors on the development of specific CNS neurons or CNS regions [17]. Fetal tissue grafted either to the anterior chamber of the eye [17], or to the CNS adult animals [3] survives to express a number of biochemical [8, 9, 20, 21], morphological (8, 9, 12], electrophysiological [I 7] and functional [3, 10] characteristics appropriate to the caudate nucleus of the adult rat. Other evidence suggests however, that grafted caudate neurons express phenotypic characteristics which are not present in adult caudate neurons. For example, grafted medium spiny neurons in 35-day-old grafts have fewer spines than medium spiny neurons in the normal adult rat [20]. Further, the density of opioid receptor binding sites is lower in intraocular caudate grafts than in the adult caudate nucleus [12]. Thus, grafted caudate neurons express a combination of normal and abnormal phenotypical traits. In the course of a preliminary ultrastructural study of TH immunoreactivity in intraocular co-grafts of caudate nucleus and substantia nigra, we noticed that some cell bodies in the caudate part of the double graft expressed TH-like immunoreactiCorre,g;~mdence." T.J. Mahalik, Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, Box Bill, CO 80262, U.S.A.
254
vity. This finding was unusual in that T H immunoreactivity is not normally expressed by neurons in the rat caudate nucleus. The purpose of the present study is to followup on these initial observations by examining TH-like immunoreactivity in single grafts of caudate nucleus, and in caudate nucleus grafts which were co-grafted with substantia nigra. Nine grafts obtained from fetal Sprague-Dawley rats were used in the study. Two host animals received unilateral single grafts of the fetal caudate nucleus; 3 other hosts received double grafts of substantia nigra and caudate nucleus. Two additional host animals received bilateral single caudate grafts in each eye yielding a total of 9 grafts. This information is summarized in Table I. lntraoeular grafting. Donor material was obtained from Sprague-Dawley rat fetuses. Fetal tissue for caudate grafts was taken from fetuses with a crown-rump length of 15-17 mm; fetal nigral tissue was obtained from rats with a crown-rump length of 19-21 mm. Uteri were removed from pregnant Sprague-Dawley rats and kept at room temperature for up to 2 h. Fetuses were removed from the uterus and decapitated; the brains were removed and placed in lactated saline. Fetal tissue was dissected and placed in sterile lactated saline. The transplantation procedure previously has been described in detail [17]. Briefly, rats were lightly anesthetized with metaphane and the recipient eye was treated topically with a 1% solution of atropine sulfate. A razor blade was used to cut a small slit in the cornea, and a fire-polished pasteur pipette was used to pick up fetal tissue in approximately 5/tl of lactated saline. Gentle pressure was used to eject pieces of tissue into the anterior chamber of the eye. For double grafts of fetal substantia nigra and caudate, the striatal tissue was transplanted first; after 6 weeks the eyes were reopened and the nigral graft was placed in contact with the piece of striatal tissue. Single and double grafts were allowed to survive from 6 to 20 months. One month prior to perfusion, adrenergic input to the host iris was eliminated by removal of the superior cervical ganglion on the side of the graft. Tissue preparation. The host rats were deeply anesthetized with an overdose of sodium pentobarbital and injected intracardially with 1 ml of a 1% solution of sodium
TABLE I S U M M A R Y OF G R A F T S + , THLI cell present; - , no TH cells present; 0, no graft. Double =eaudate grafted with ventral mesencephalon from 19 to 20 mm (CRL) fetal donor. Crown-rump length (mm)
Type
TH cells
Crown rump length (mm)
Type
TH cells
15 16 17 17 17 17
caudate caudate caudate caudate caudate
+ 0 ~+ +
17 15-16 15 16 15 16
caudate double double double
+ + + +
255
nitrite, followed by an injection of 200 U of sodium heparin. After the rats had been perfused with 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), the grafts were removed from the eyes and placed in 4% paraformaldehyde in 0.1 M sodium bicarbonate buffer (pH 10.4) overnight at 4°C. The grafts then were washed in phosphate-buffered saline (PBS, pH 7.4) for 1 h and then cryoprotected in phosphate buffer containing increasing concentrations of sucrose and glycerol [5]. The grafts were frozen with dry-ice powder, and sectioned (40/lm) with a sliding microtome. Before exposure to the antibody solutions, the tissue sections were incubated in I% sodium borohydride in PBS for 25 min at room temperature. Following two 15 min rinses in PBS, the sections were incubated in PBS containing 2% normal goat serum to quench non-specific binding. The sections then were incubated in rabbit antiserum directed against T H (diluted 1:500 in PBS with 2% normal goat serum) for 1 6 4 8 h at 4°C. The T H antibody was a generous gift of Drs. Norman Weiner and William Tank. The immunocytochemical specificity of this antiserum has been described previously [16]. After 3 washes in PBS, the sections then were placed in fluorescein-labeled goat anti-rabbit IgG for 1 h at room temperature. After 3 wash steps, the sections were placed on gelatin-coated microscope slides and coverslipped in phosphate-buffered glycerol. Labeled material was viewed a n d p h o t o g r a p h e d on a Zeiss microscope equipped for epifluorescence (excitation = BP 450-490 nm; barrier = BP 520-560 nm). The control sections were incubated in pre-immune rabbit seruin.
Light microscopy of TH immunoreactivity. One of the grafts did not survive. The remaining 8 grafts survived from 6 to 20 months in oculo (Table I). T H immunocytochemistry revealed that TH-like immunoreactive (THLI) sympathetic innervation of the host iris was eliminated in 7 out of the 8 surviving cases. In control sections incubated with the T H pre-immune serum, no labeled cell bodies were observed. Several T H L I cell bodies were present in 4 of 5 single caudate grafts, and in the caudate of each of the 3 double grafts (Table I). T H L I cell bodies varied in size and in shape (Fig. 1). Some of the cells could not be identified on the basis of light microscopy (Fig. 1A, C); other cells however, possessed a prominent oval nucleus and a thin cytoplasm reminiscent of medium spiny neurons [4, 15]. Each 40/~m section through a striatal graft contained 2-5 THL1 cell bodies. T H L I cell bodies also were present in the nigral part of the nigrostriatal co-grafts. In each one of these cases T H L I fibers streamed across the boundary between the 2 types of graft. The presence of T H L I cell bodies in the caudate portion of the double grafts, however, made it impossible to assess the amount of nigral innervation of the striatal tissue. The present immunocytochemical study has shown that TH-like immunoreactivity is inappropriately expressed in neurons of the caudate nucleus following grafting into the anterior chamber of the eye. Of 8 surviving grafts, 7 contained T H L I cell bodies. Immunoreactive cell bodies were present in both single grafts of the caudate nucleus, and in caudate tissue which was co-grafted with substantia nigra. Some of the cell bodies within the grafted caudate resembled medium spiny neurons, the most common neuronal cell type in the caudate nucleus [4, 15]. A combined immunocytochem-
256
Fig. 1. Examples of THLI cell bodies in intraocular striatal grafts. A: graft 334-1. Note the large primary dendrite. B: several THLI cell bodies in a striatal graft. C: a THLI cell body; note round nucleus and scant cytoplasm, 2 characteristics of medium spiny neurons. D F: other THLI cell bodies in striatal grafts. The morphology of the cell in E resembles that of a medium spiny neuron.
ical a n d u l t r a s t r u c t u r a l s t u d y h o w e v e r , w o u l d be r e q u i r e d to definitely e s t a b l i s h the i d e n t i t y o f T H L I cells in c a u d a t e grafts. O u r f i n d i n g s a r e s i m i l a r to the results o f o t h e r s w h o h a v e s h o w n t h a t T H - l i k e i m m u n o r e a c t i v i t y m a y be i n a p p r o p r i a t e l y e x p r e s s e d in cell t y p e s w h i c h are n o t n o r -
257 mally catecholaminergic. For example, T H immunoreactivity is transiently expressed during development in cells of the rat and mouse peripheral nervous systems [2, 13, 14, 19]. Similarly, TH immunoreactivity is transiently expressed in neurons of the inferior colliculus [1 1] and neocortex [1] during development. In these developmental studies, ~inappropriate" T H expression was limited to a 2 3 day period, A more recent study has shown that TH-like immunoreactivity and tyrosine hydroxylase enzymatic activity is transiently expressed in cultured fetal cortical neurons [7]. Further, Park et al. [18] and Herman et al. [6] have shown that neurons within grafts of neocortical tissue express TH immunoreactivity for periods of up to 10 months [6] (the longest time span examined). In our study TH-like immunoreactivity was expressed in caudate grafts which were allowed to remain in oculo for up to 20 months. This result suggests that ~inappropriate" expression of T H is a stable characteristic of grafted caudate neurons. Thus the results of this and other transplantation studies [6, 20] contrast with the findings of in vitro or developmental studies which have shown that the expression of TH in ~inappropriate' cell populations is a transient phenomenon. One explanation of our results may be that TH expression is extremely labile in some neurons, and that it depends upon the presence of appropriate inputs, as well as on the availability of appropriate tar'gets [6]. Thus, in some peripheral and central neurons, TH is expressed transiently, before normal, adult-type cell -cell contacts are established. The TH gene then may be turned off once appriopriate contacts are tk~rmed. In the case of intraocular grafts, caudate neurons may never establish normal synaptic connections because they are isolated from other CNS tissue. The expression of TH may result form the absence of normal input, or from the lack of appropriate extrinsic targets. Alternatively, the inappropriate expression of TH immunoreactivity in oculo may result from changes introduced during manipulation of the fetal tissue, or from environmental influences unique to the anterior chamber of the eye. One way of addressing this issue would be to co-graft the caudate with each ot" its major sources of input, or targets of its output, e.g., cortex or pallidum. Our experiments show that co-grafting caudate and substantia nigra does not inhibit the abnormal expression of T H L I by the caudate neurons. It should be noted that the nigro striatal system represents a relatively low percentage of the total connectivity of the caudate. Thus co-grafting of caudate and other brain areas might reveal a system of connectivity that inhibits expression of T H L I by caudate neurons. This work was supported by N I H G r a n t NS09199 to T.E.F. and Magnus Bergvalls Stiftelse, and the Loo and Hans Ostermans Foundation. 1 Bcrger, B., Verney, C., Gaspar, P. and Febvret, A., Transient expression of tyrosine hydroxylase immunoreactivityin some neurons of the rat neocortex during development, Dev. Brain Res., 25 (1985) 141 I44. 2 Cochard, P., Goldstein, M. and Black, I.B., Ontogenetic appearance and disappearance of tyrosine hydroxylaseand catecholamines in the rat embryo, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2986 2990.
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