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Haloperidol induces Fos and related molecules in intrastriatal grafts derived from fetal striatal primordia
Brain Research, 530 (1990) 309-311 Elsevier BRES 24328
309
Short Communications
Haloperidol induces Fos and related molecules in intrastriatal grafts derived from fetal striatal primordia M. Dragunow 1, M.
Williams 2 and R.L.M. Faull 2
Departments of 1Pharmacology and 2Anatomy, School of Medicine, The University of Auckland, Auckland (New Zealand) (Accepted 3 July 1990)
Key words: Haloperidol; Fos; Striatal graft; Fetal tissue
Haloperidol induces Fos and related molecules (Fos-related antigens, FRAs) in adult striatal neurons. We tested whether a similar induction of Fos and FRAs by haloperidoi injection would occur in fetal striatal neurons transplanted into adult quinolinic acid-lesioned striatum. We found that Fos and FRAs were induced in striatal neurons after haloperidol. This induction had a time course in transplanted neurons that was identical to the time course of induction in normal adult striatum. Furthermore, the relative numbers of Fos- and FRA-immunopositive neurons induced by haloperidol were identical for both transplanted and normal striatal neurons. These results provide the first demonstration that neurons in intrastriatal grafts derived from fetal striatal primordia have similar intracellular biochemical characteristics to normal adult striatal neurons. The successful transplantation of fetal neurons into the adult brain has raised the possibility that neural transplants may have the potential of ameliorating the symptoms of various neurodegenerative neurological disorders such as Parkinson's, Alzheimer's, and Huntington's disease 1. However, the fundamental question in all transplantation studies is whether the transplanted neurons develop identical biochemical and physiological properties to normal neurons in the host brain. Much effort has been directed at this problem using neurophysiological, neurochemical and behavioural techniques. For example, intrastriatal grafts derived from fetal striatal primordia express many of the chemical markers (e.g. acetylcholinesterase, substance P, enkephalin, somatostatin, cholecytstokinin, neuropeptide Y, calbindin) and neurotransmitter receptors (e.g. muscarinic, dopamine, opiate) found in the normal striatum 5'7'8, and lead to functional recovery 6'9. Furthermore, afferents and efferents are also to some extent reestablished ~°. However, it is not known if transplanted striatal neurons transduce extracellular signals in the same way as normal striatal neurons. We have recently shown that haloperidol induces the transcription modulators (third messengers) Fos and Fos-related antigens (FRAs) in normal striatal neurons 3. The number and kinetics of Fos and F R A induction differs, with F R A s being induced in approximately twice as many striatal neurons as Fos itself 3. Furthermore, Fos levels are back to baseline after
24 h whereas F R A s return to baseline with a longer time course. Also, F R A s , but not Fos, are expressed basally in striatal neurons 3. Because Fos and F R A s are intracellular messengers we investigated whether haloperidol would also induce Fos and F R A s in transplanted striatal neurons to test the hypothesis that the transplanted neurons have similar intracellular biochemical properties to normal striatal cells. Male Wistar rats were anaesthetized with sodium pentobarbital and received a unilateral intrastriatal injection of quinolinic acid (30 nmol/15 nl) which destroys a population of striatal neurons. At least 4 weeks after lesion rats were again anaesthetized with sodium pentobarbital and received intrastriatal grafts of fetal striatal primordia using a modification of the technique described by Schmidt et al. it. Some rats were not injected with striatal primordia and served as controls for the haloperidol studies. The striatal transplantation technique involved the careful removal of the striatal ridge from the brains of 17-day-old rat fetuses using a dissecting microscope; the cells were then incubated for 20 min at 37 °C in glucose saline and trypsin and then dissociated in D N A a s e in glucose saline to form a cell suspension which was immediately injected through a glass pipette into the lesioned striatum of adult rats. Rats were left for at least 6 months, and then transplanted and control (lesion but no transplant) rats were injected with either haloperidol (2 mg/kg, i.p.) or
Correspondence: M. Dragunow, Department of Pharmacology and Clinical Pharmacology, School of Medicine, The University of Auckland, Private Bag, Auckland, New Zealand. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
310 saline (equivalent volume). They were then deeply anaesthetized with sodium p e n t o b a r b i t a l at various times after haloperidol or saline injection (10 rain, 2 h, or 24 h) and perfused transcardially with saline followed by 4% p a r a f o r m a l d e h d y d e in 0.1 M phosphate buffer (pH 7.4).
Their brains were then cut coronally on a vibroslice machine (50 ~ m sections). Sections were then processed for the immunocytochemical detection of Fos and F R A s using our standard immunocytochemical p r o c e d u r e s z. A l t e r n a t e sections were processed with an antibody
Fig. 1. Photomicrographs showing rat striatum immunostained for the detection of Fos (a-c) and Fos-related antigens (d-f) from the striatum of a normal rat (a,d), a quinolinic acid-lesioned rat (b,e), and a rat which received a fetal striatal implant in the quinolinic acid-lesioned region (c,f). Bar = 180/~m. Note that all rats received an injection of 2 mg/kg haloperidol, i.p., 2 h before perfusion. Arrows indicate examples of Fos- and FRA-positive striatal neurons.
311 specific for Fos (Medac, O P A 08/112) and an antibody that recognizes Fos as well as F R A s (generously provided by Tom Curran4). O u r striatal transplants had a m o r p h o l o g y similar to that r e p o r t e d by others 7. In saline o r haloperidol injected control lesioned rats (i.e. quinolinic acid lesion but no transplant) there were no Fos- or F R A - i m m u n o p o s i t i v e striatal cells in the lesioned region. In saline injected t r a n s p l a n t e d rats there were no Fos-immunopositive striatal neurons in the n o r m a l or transplanted striatum, as m e a s u r e d with the Fos-specific antiserum. H o w e v e r , there were F R A - i m m u n o p o s i t i v e striatal neurons in n o r m a l and transplanted striatum for saline-injected rats, as m e a s u r e d with the non-specific antiserum. In haloperidol-injected t r a n s p l a n t e d rats there was a large induction of Fos- and F R A - i m m u n o p o s i t i v e neurons in the normal and t r a n s p l a n t e d striatum but not in lesioned striatum (Fig. 1). T h e r e were m o r e than twice as m a n y F R A immunopositive than Fos-immunopositive neurons in the n o r m a l and transplanted striatum (Fig. 1). The time course of Fos and F R A induction after haloperidol (i.e. Fos back to baseline after 24 h, but F R A s still elevated) was identical in n o r m a l and transplanted striatum. These results clearly d e m o n s t r a t e that transplanted
striatal neurons express Fos and F R A s after haloperidoi injection in the same p r o p o r t i o n and with the same time course as normal striatal neurons. F u r t h e r m o r e , similar to normal striatal neurons t r a n s p l a n t e d striatal neurons express F R A s , but not Fos itself, constitutively (in one rat there was evidence of increased expression of F R A ) . These results suggest that t r a n s p l a n t e d striatal neurons transduce extraceUular signals (i.e. h a l o p e r i d o l ) and generate third messengers (i.e. Fos and F R A s ) in the same way as n o r m a l striatal neurons. Thus, when these results are t a k e n t o g e t h e r with the results of other studies showing that t r a n s p l a n t e d fetal striatal neurons establish c o m p a r a b l e fibre connections and express a similar range of n e u r o t r a n s m i t t e r s and their receptors to host striatal neurons, it appears that transplanted fetal striatal neurons have the potential to replace and function in a similar fashion to differentiated adult striatal neurons. These collective scientific data from animal studies indicate that fetal neural transplants offer a real possibility for t r e a t m e n t of neurological disorders in the h u m a n brain.
1 Bj6rklund, A., Lindvall, O., Isacson, O., Brundin, P., Wictorin, K., Strecker, R.E., Clarke, D,J. and Dunnett, S.B., Mechanisms of action of intracerebral neural implants: studies on nigral and striatal grafts to the lesioned striatum, Trends Neurosci., 12 (1987) 509-516. 2 Dragunow, M. and Robertson, H.A., Localization and induction of c-los protein-like immunoreactive material in the nuclei of adult mammalian neurons, Brain Research, 440 (1988) 252-260. 3 Dragunow, M., Robertson, G.S., Faull, R.L.M., Robertson, H.A. and Jansen, K., D2 Dopamine receptor antagonists induce los and related proteins in rat striatal neurons, Neuroscience, in press. 4 Franza, B.R., Sambucetti, L.C., Cohen, D.R. and Curran, T., Analysis of Fos protein complexes and Fos-related antigens by high-resolution two-dimensional gel electrophoresis, Oncogene, 1 (1987) 213-221. 5 Graybiel, A.M., Liu, F.-C. and Dunnett, S.B., Intrastriatal grafts derived from fetal striatal primordia phenotypy and modular organization, J. Neurosci., in press. 6 Isacson, O., Brundin, P., Kelly, P.A.T., Gage, EH. and Bj6rklund, A., Functional neuronal replacement by grafted striatal neurones in the ibotenic acid-lesioned rat striatum, Nature, 311 (19--) 458. 7 Isacson, O., Dawbarn, D., Brundin, P., Gage, EH., Emson,
P.C. and Bj6rklund, A., Neural grafting in a rat model of Huntinton's disease: striosomal-like organization of striatal grafts as revealed by acetylcholinesterase histochemistry, immunocytochemistry and receptor autoradiography, Neuroscience, 22 (1987) 481-497. Liu, E-C., Graybiel, A.M., Dunnett, S.B. and Baughman, R.W., Intrastriatal grafts derived from fetal striatal primordia. II. Reconstitution of cholinergic and dopaminergic systems, J. Comp. Neurol., 295 (1990) 1-14. Norman, A.B., Calderon, S.F., Giordano, M. and Sanberg, ER., Striatal tissue transplants attenuate apomorphine-induced rotational behavior in rats with unilateral kainic acid lesions, Neuropharmacology, 27 (1988) 333-336. Pritzel, M., [sacson, O., Brundin, P., Wiklund, L. and Bjfrklund, A., Afferent and efferent connections of striatal grafts implanted into the ibotenic acid lesioned neostriatum in adult rats, Exp. Brain Res., 65 (1986) 112-126. Schmidt, R.H., Bj6rklund, A. and Stenevi, U., Intracerebral grafting of dissociated CNS tissue suspensions: a new approach for neuronal transplantation to deep brain sites, Brain Research, 218 (1981) 347-356. Verrier, B., Muller, D., Bravo, R. and Muller, R., Wounding a fibroblast monolayer results in the rapid induction of the c-fos proto-oncogene, EMBO J., 5 (1986) 913-917.
Supported by grants from the New Zealand Neurological Foundation, the New Zealand Medical Research Council, MRC, and the Auckland Medical Research Foundation.
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309
Short Communications
Haloperidol induces Fos and related molecules in intrastriatal grafts derived from fetal striatal primordia M. Dragunow 1, M.
Williams 2 and R.L.M. Faull 2
Departments of 1Pharmacology and 2Anatomy, School of Medicine, The University of Auckland, Auckland (New Zealand) (Accepted 3 July 1990)
Key words: Haloperidol; Fos; Striatal graft; Fetal tissue
Haloperidol induces Fos and related molecules (Fos-related antigens, FRAs) in adult striatal neurons. We tested whether a similar induction of Fos and FRAs by haloperidoi injection would occur in fetal striatal neurons transplanted into adult quinolinic acid-lesioned striatum. We found that Fos and FRAs were induced in striatal neurons after haloperidol. This induction had a time course in transplanted neurons that was identical to the time course of induction in normal adult striatum. Furthermore, the relative numbers of Fos- and FRA-immunopositive neurons induced by haloperidol were identical for both transplanted and normal striatal neurons. These results provide the first demonstration that neurons in intrastriatal grafts derived from fetal striatal primordia have similar intracellular biochemical characteristics to normal adult striatal neurons. The successful transplantation of fetal neurons into the adult brain has raised the possibility that neural transplants may have the potential of ameliorating the symptoms of various neurodegenerative neurological disorders such as Parkinson's, Alzheimer's, and Huntington's disease 1. However, the fundamental question in all transplantation studies is whether the transplanted neurons develop identical biochemical and physiological properties to normal neurons in the host brain. Much effort has been directed at this problem using neurophysiological, neurochemical and behavioural techniques. For example, intrastriatal grafts derived from fetal striatal primordia express many of the chemical markers (e.g. acetylcholinesterase, substance P, enkephalin, somatostatin, cholecytstokinin, neuropeptide Y, calbindin) and neurotransmitter receptors (e.g. muscarinic, dopamine, opiate) found in the normal striatum 5'7'8, and lead to functional recovery 6'9. Furthermore, afferents and efferents are also to some extent reestablished ~°. However, it is not known if transplanted striatal neurons transduce extracellular signals in the same way as normal striatal neurons. We have recently shown that haloperidol induces the transcription modulators (third messengers) Fos and Fos-related antigens (FRAs) in normal striatal neurons 3. The number and kinetics of Fos and F R A induction differs, with F R A s being induced in approximately twice as many striatal neurons as Fos itself 3. Furthermore, Fos levels are back to baseline after
24 h whereas F R A s return to baseline with a longer time course. Also, F R A s , but not Fos, are expressed basally in striatal neurons 3. Because Fos and F R A s are intracellular messengers we investigated whether haloperidol would also induce Fos and F R A s in transplanted striatal neurons to test the hypothesis that the transplanted neurons have similar intracellular biochemical properties to normal striatal cells. Male Wistar rats were anaesthetized with sodium pentobarbital and received a unilateral intrastriatal injection of quinolinic acid (30 nmol/15 nl) which destroys a population of striatal neurons. At least 4 weeks after lesion rats were again anaesthetized with sodium pentobarbital and received intrastriatal grafts of fetal striatal primordia using a modification of the technique described by Schmidt et al. it. Some rats were not injected with striatal primordia and served as controls for the haloperidol studies. The striatal transplantation technique involved the careful removal of the striatal ridge from the brains of 17-day-old rat fetuses using a dissecting microscope; the cells were then incubated for 20 min at 37 °C in glucose saline and trypsin and then dissociated in D N A a s e in glucose saline to form a cell suspension which was immediately injected through a glass pipette into the lesioned striatum of adult rats. Rats were left for at least 6 months, and then transplanted and control (lesion but no transplant) rats were injected with either haloperidol (2 mg/kg, i.p.) or
Correspondence: M. Dragunow, Department of Pharmacology and Clinical Pharmacology, School of Medicine, The University of Auckland, Private Bag, Auckland, New Zealand. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
310 saline (equivalent volume). They were then deeply anaesthetized with sodium p e n t o b a r b i t a l at various times after haloperidol or saline injection (10 rain, 2 h, or 24 h) and perfused transcardially with saline followed by 4% p a r a f o r m a l d e h d y d e in 0.1 M phosphate buffer (pH 7.4).
Their brains were then cut coronally on a vibroslice machine (50 ~ m sections). Sections were then processed for the immunocytochemical detection of Fos and F R A s using our standard immunocytochemical p r o c e d u r e s z. A l t e r n a t e sections were processed with an antibody
Fig. 1. Photomicrographs showing rat striatum immunostained for the detection of Fos (a-c) and Fos-related antigens (d-f) from the striatum of a normal rat (a,d), a quinolinic acid-lesioned rat (b,e), and a rat which received a fetal striatal implant in the quinolinic acid-lesioned region (c,f). Bar = 180/~m. Note that all rats received an injection of 2 mg/kg haloperidol, i.p., 2 h before perfusion. Arrows indicate examples of Fos- and FRA-positive striatal neurons.
311 specific for Fos (Medac, O P A 08/112) and an antibody that recognizes Fos as well as F R A s (generously provided by Tom Curran4). O u r striatal transplants had a m o r p h o l o g y similar to that r e p o r t e d by others 7. In saline o r haloperidol injected control lesioned rats (i.e. quinolinic acid lesion but no transplant) there were no Fos- or F R A - i m m u n o p o s i t i v e striatal cells in the lesioned region. In saline injected t r a n s p l a n t e d rats there were no Fos-immunopositive striatal neurons in the n o r m a l or transplanted striatum, as m e a s u r e d with the Fos-specific antiserum. H o w e v e r , there were F R A - i m m u n o p o s i t i v e striatal neurons in n o r m a l and transplanted striatum for saline-injected rats, as m e a s u r e d with the non-specific antiserum. In haloperidol-injected t r a n s p l a n t e d rats there was a large induction of Fos- and F R A - i m m u n o p o s i t i v e neurons in the normal and t r a n s p l a n t e d striatum but not in lesioned striatum (Fig. 1). T h e r e were m o r e than twice as m a n y F R A immunopositive than Fos-immunopositive neurons in the n o r m a l and transplanted striatum (Fig. 1). The time course of Fos and F R A induction after haloperidol (i.e. Fos back to baseline after 24 h, but F R A s still elevated) was identical in n o r m a l and transplanted striatum. These results clearly d e m o n s t r a t e that transplanted
striatal neurons express Fos and F R A s after haloperidoi injection in the same p r o p o r t i o n and with the same time course as normal striatal neurons. F u r t h e r m o r e , similar to normal striatal neurons t r a n s p l a n t e d striatal neurons express F R A s , but not Fos itself, constitutively (in one rat there was evidence of increased expression of F R A ) . These results suggest that t r a n s p l a n t e d striatal neurons transduce extraceUular signals (i.e. h a l o p e r i d o l ) and generate third messengers (i.e. Fos and F R A s ) in the same way as n o r m a l striatal neurons. Thus, when these results are t a k e n t o g e t h e r with the results of other studies showing that t r a n s p l a n t e d fetal striatal neurons establish c o m p a r a b l e fibre connections and express a similar range of n e u r o t r a n s m i t t e r s and their receptors to host striatal neurons, it appears that transplanted fetal striatal neurons have the potential to replace and function in a similar fashion to differentiated adult striatal neurons. These collective scientific data from animal studies indicate that fetal neural transplants offer a real possibility for t r e a t m e n t of neurological disorders in the h u m a n brain.
1 Bj6rklund, A., Lindvall, O., Isacson, O., Brundin, P., Wictorin, K., Strecker, R.E., Clarke, D,J. and Dunnett, S.B., Mechanisms of action of intracerebral neural implants: studies on nigral and striatal grafts to the lesioned striatum, Trends Neurosci., 12 (1987) 509-516. 2 Dragunow, M. and Robertson, H.A., Localization and induction of c-los protein-like immunoreactive material in the nuclei of adult mammalian neurons, Brain Research, 440 (1988) 252-260. 3 Dragunow, M., Robertson, G.S., Faull, R.L.M., Robertson, H.A. and Jansen, K., D2 Dopamine receptor antagonists induce los and related proteins in rat striatal neurons, Neuroscience, in press. 4 Franza, B.R., Sambucetti, L.C., Cohen, D.R. and Curran, T., Analysis of Fos protein complexes and Fos-related antigens by high-resolution two-dimensional gel electrophoresis, Oncogene, 1 (1987) 213-221. 5 Graybiel, A.M., Liu, F.-C. and Dunnett, S.B., Intrastriatal grafts derived from fetal striatal primordia phenotypy and modular organization, J. Neurosci., in press. 6 Isacson, O., Brundin, P., Kelly, P.A.T., Gage, EH. and Bj6rklund, A., Functional neuronal replacement by grafted striatal neurones in the ibotenic acid-lesioned rat striatum, Nature, 311 (19--) 458. 7 Isacson, O., Dawbarn, D., Brundin, P., Gage, EH., Emson,
P.C. and Bj6rklund, A., Neural grafting in a rat model of Huntinton's disease: striosomal-like organization of striatal grafts as revealed by acetylcholinesterase histochemistry, immunocytochemistry and receptor autoradiography, Neuroscience, 22 (1987) 481-497. Liu, E-C., Graybiel, A.M., Dunnett, S.B. and Baughman, R.W., Intrastriatal grafts derived from fetal striatal primordia. II. Reconstitution of cholinergic and dopaminergic systems, J. Comp. Neurol., 295 (1990) 1-14. Norman, A.B., Calderon, S.F., Giordano, M. and Sanberg, ER., Striatal tissue transplants attenuate apomorphine-induced rotational behavior in rats with unilateral kainic acid lesions, Neuropharmacology, 27 (1988) 333-336. Pritzel, M., [sacson, O., Brundin, P., Wiklund, L. and Bjfrklund, A., Afferent and efferent connections of striatal grafts implanted into the ibotenic acid lesioned neostriatum in adult rats, Exp. Brain Res., 65 (1986) 112-126. Schmidt, R.H., Bj6rklund, A. and Stenevi, U., Intracerebral grafting of dissociated CNS tissue suspensions: a new approach for neuronal transplantation to deep brain sites, Brain Research, 218 (1981) 347-356. Verrier, B., Muller, D., Bravo, R. and Muller, R., Wounding a fibroblast monolayer results in the rapid induction of the c-fos proto-oncogene, EMBO J., 5 (1986) 913-917.
Supported by grants from the New Zealand Neurological Foundation, the New Zealand Medical Research Council, MRC, and the Auckland Medical Research Foundation.
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