- Email: [email protected]
Restoration of dopamine transmission in graft reinnervated striatum. Evidence for regulation of dopamine D2 receptor function in regions lacking dopamine
L. E Agnati, K: Fuxe, C. Nicholson and E. Sykovfi (Eds.) Progress in Brain Research,Vol 125 © 2000 Elsevier Science B'~ All rights reserved.
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Restoration of dopamine transmission in graft reinnervated striatum. Evidence for regulation of dopamine D2 receptor function in regions lacking dopamine Ingrid Strfmberg*, Jan Kehr and Kjell Fuxe Department of Neuroscience, Karolinska Institute, S-17177 Stockholm, Sweden
Introduction to grafting as a tool to restore dopamine deficiency
Grafting of catecholamine-rich tissue into dopamine depleted striatum to restore the loss of dopaminergic input into the striatum has become a useful tool (Bjfrklund and Stenevi, 1979; Perlow et al., 1979; Freed et al., 1981). The first clinical attempts were performed by the grafting of adrenal medullary tissue into the caudate nucleus of patients suffering from Parkinson's disease (Backlund et al., 1985). Although the chromaffin cells store and release adrenaline and noradrenaline (NA) rather than dopamine (DA), it has been shown in animal experiments that implantation of adrenal chromaffin cells to the DA depleted striatum affects drug-induced rotational behavior (Freed et al., 1981, 1986; Strfmberg et al., 1985). Studies documenting effects on rotational behavior induced acutely after implantation of the tissue showed that rotations were induced by release of catecholamines from the grafts (Herrera-Marschitz et al., 1984; Strfmberg et al., 1984), and consequently, it was suggested that the graft exerted its action via diffusion or volume transmission rather than over synaptic contacts, since no outgrowth was found in this case. *Corresponding author: Tel.: 46-8-728 7087; Fax: 46-8-728 7437; e-mail: [email protected]
Later, grafting of fetal ventral mesencephalic tissue was shown to be more powerful to use than chromaffin cells. Robust documentation of graft survival, graft-derived dopaminergic reinnervation of host brain, spontaneous activity of dopaminergic neurons within the transplants, and DA released from the graft-derived outgrowth were shown (Wuerthele et al., 1981; Arbuthnott et al., 1985; Brundin et al., 1985; Rose et al., 1985; Strecker et al., 1987; Zetterstrfm et al., 1986). Furthermore, functional evidence of DA transmission was shown when local application of phencyclidine affected the striatal neuronal activity in graft-reinnervated striatum (Strfmberg et al., 1985). It was suggested that DA transmission in the stfiatum after reinnervation occurred via synaptic contacts, since reciprocal contacts between graft and host had been found, showing membrane specializations of the same type that had been characterized for a dopaminergic synaptic input to the striatal neurons (Freund et al., 1985; Jaeger, 1985; Mahalik et al., 1985; Bolam et al., 1987). Furthermore, evidence for functional dopaminergic input to the graft-reinnervated striatum was obtained based on studies showing that the increased D2 receptor binding found after a chronic DA depletion (Creese et al., 1977) was normalized after grafting (Freed et al., 1983). Later, it was shown that the D2 receptor binding became normalized in the total volume of dorsal striatum,
310
although the graft-derived dopaminergic reinnervation of dorsal striatum is limited (Dawson et al., 1991; Gagnon et al., 1991; Blunt et al., 1992; Freed et al., 1983). In fact, outgrowth from fetal ventral mesencephalon terminates already approximately 2 weeks after implantation (Barker et al., 1996), and at this time point only 1/3 to 1/2 of the volume of dorsal striatum has become reinnervated.
Conditions of dopamine transmission via volume transmission? The mismatch between striatal reinnervated areas and normalization of D2 receptor binding raised the question whether the dopamine transmission in graft-reinnervated striatum occurs via volume transmission in addition to synaptic transmission. Indeed it has been shown that clearance time for extracellular dopamine is prolonged distal compared to proximal to the graft (Strtmberg et al., 1991). Dopamine reuptake sites are present in THpositive nerve fibers after grafting (Kordower et al., 1996). Hence, the explanation for the longer clearance time was suggested to be due to the distribution of DA nerve fibers with less density distal than proximal to the graft, resulting in a more efficient DA reuptake close to the graft with higher DA nerve fiber densities and DA reuptake sites than that found distally. The conclusion was drawn that the graft may act over a larger volume of the host brain than that becomes reinnervated, probably via diffusion in the extracellular space (long-distance volume transmission).
Functional dopamine D2 receptors in dopamine reinnervated versus denervated striatum To further explore the possibility of DA transmission via volume transmission in non-DAinnervated areas of graft-reinnervated striatum, extracellular recordings of graft reinnervated striatum were employed. Recordings were performed in the DA depleted striatum reinnervated by a fetal ventral mesencephalic graft implanted into the lateral ventricle. The host striatum becomes dopaminergically reinnervated as visualized by tyrosine hydroxylase (TH)-positive nerve fibers in a zone close to the ventricle, and thus, reinnervated and
non-innervated areas can be localized to medial and lateral striatum respectively (Fig. 1) (Strtmberg and Bickford, 1996). Extracellular recordings in the striatum including local applications of the D2-1ike agonist quinpirole revealed a dose-dependent reduction in striatal neuronal firing rates. When recording in graftreinnervated areas of the striatum there was no shift in sensitivity to quinpirole when compared to the control side. Furthermore, there was no difference in sensitivity to quinpirole when recording in noninnervated versus reinnervated regions of graftreinnervated striatum (Fig. 2). However, a significant supersensitivity was found when recording in 6-hydroxydopamine (6-OHDA) lesioned control animals (Fig. 2). Thus, the D2 receptor supersensitivity seen after an almost complete DA denervation may be functionally removed in graft reinnervated striatum, not only within reinnervated areas, but also lateral to the DA reinnervation. Hence, the normalization of D2 receptor binding in incomplete graft-reinnervated striatum had physiological relevance.
Striatal spontaneous neuronal activity The striatal neuronal firing rates are significantly upregulated in DA depleted striatum (Siggins et al., 1974; Schultz and Ungerstedt, 1978; Strtmberg et al., 1985). After graft-reinnervation the spontaneous discharge rate becomes normalized, but the normalization is found only in areas that are reinnervated (Fig. 3) (Strtmberg et al., 1985; Fisher et al., 1991). Since the D2 receptors were shown to be functionally normalized, the upregulated neuronal spontaneous activity might be due to malfunction at the D1 receptor level. It has been suggested that the D1 receptor sensitivity shows either decrease, increase or no change after a 6-OHDA lesion (Buonamici et al., 1986; Marshall et al., 1989; Dawson et al., 1991; Gagnon et al., 1991; Robertson et al., 1991; Blunt et al., 1992; Savasta et al., 1992). Although the reports show divergent results, it has been found that the D1 receptor levels are normalized after grafting and some studies have shown reduced D1 receptor levels (Blunt et al., 1992; Strtmberg et al., 1995). Nevertheless, electrophysiological recordings in
311 DA depleted striatum indicate no significant change of the dose-response curve to the D1 agonist N0437 (Str6mberg and Bickford-Wimer, 1991). However, inactivation of the D2 receptor results in a downregulation of the sensitivity to a D1 agonist, indicating that there is a kind of synergistic action between D1 and D2 receptors in the 6-OHDA lesioned striatum (Str6mberg and Bickford-Wimer, 1991). Extracellular recordings using local applications of the D1 agonist SKF 81297 in DA reinnervated striatum revealed no physiological differences of striatal neuronal responses to the D1 agonist in reinnervated versus noninnervated areas of grafted striatum (StrtSmberg et al., 1999). Thus, the upregulated striatal neuronal discharge rate in non-innervated areas did not seem to be due to a malfunction of the D 1 receptor.
Cortical excitatory input to the striatum after grafting The results showing that the dopaminergic reinnervation normalized the D2 receptor function but not the spontaneous neuronal activity in noninnervated areas of grafted striatum turned our interest to the excitatory input to the striatum, i.e. the cortical glutamatergic innervation of the striatum. It has been shown that a DA depletion enhance extracellular levels of glutamate (Yamamoto and Davy, 1992; Meshul et al., 1999), and accordingly this might be the explanation for the upregulated striatal neuronal activity in noninnervated areas of the graft reinnervated striatum. However, there is a loss of asymmetric synapses after a DA depletion (Ingham et al., 1998; Meshul et al., 1999), and since
Fig. 1. TH-immunohistochemistryof a fetal ventral mesencephalicgraft transplanted to the lateral ventricleof a unilaterally 6-OHDA lesioned rat. Graft outgrowthinto the host brain is limitedto a zone close to the ventricle.Extracellularelectrophysiologicalrecordings were performed in host striatum within the zone of TH-positive nerve fibers and in dopamine denervated regions lateral to THimmunoreactiveareas. Scale bar: 200 ~tm.
312
asymmetric synapses are related to the glutamatergic input to striatal dentritic spines (Kemp and Powell, 1971; Somogyi et al., 1981), enhanced glutamate levels are accompanied with loss of glutamate synaptic input. Microdialysis using the dual-probe approach (Morari et al., 1996) and recording of extracellular levels of glutamate after DA reinnervation showed no differences in glutamate overflow in reinner-
vated compared to non-innervated areas of graft-derived DA reinnervated striatum. Extracellular levels of potassium-induced glutamate overflow showed approximately 200% increase of baseline levels in all areas measured. Thus, it is not likely that the upregulated neuronal activity in noninnervated areas of graft-reinnervated striatum is due to an increase in extracellular glutamate in these areas.
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Fig. 2. Dose-response to locally applied quinpirole expressed as percentage change from baseline. Recordings were performed in 6-OHDA lesioned (a) and in graft DA reinnervated striatum (b). In DA depleted striatum there was a shift in dose-response (p < 0.001 ) to quinpirole and the supersensitivity to the D2 agonist was shown, while in graft reinnervated striatum there was no difference in dose response when recording in DA reinnervated vs. noninnervated regions compared to control side. From Str6mberg et al., 2000.
313
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Fig. 3. Extracellularrecordings performed at 2.2 and 3.3 mm lateral to bregma in 6-OHDAlesioned rats and in DA-depleted animals followed by grafting of fetal ventral mesencepahlic tissue to the lateral ventricle. Striatal neuronal discharge rates were significantly increased in DA depleted regions compared to normal firing rate. In DA reinnervated regions of grafted striatum (2.2 mm grafted side) the spontaneous discharge rate was normal. ***p< 0.0001.
Conclusion The results indicate that the functional normalization of the D2 receptors within as well as distal to reinnervated areas of the striatum is regulated by diffusion of DA from graft-derived reinnervated areas to denervated areas. Binding studies have shown a normalization of D2 receptor levels in total volume of dorsal striatum, and it has not been correlated to the exact location of the graft or the reinnervated areas. However, the present data indicate that the distance the volume transmitted DA diffuses to functionally normalize the D2 receptor levels in non-innervated regions lateral to the DA reinnervated areas is rather at the 500 txm levels than at smaller distances, since recordings were performed at a minimum of 500 txm lateral to the zone of DA reinnervation. Trials to measure extracellular levels of DA at 500 ~xm distance from the reinnervated areas have not been successful, but
since the D2 receptors require more than a 90% DA depletion to become upregulated (Heikkila et al., 1981), the DA levels needed to normalize the D2 receptors in denervated areas might be below detection limit. Thus, these data provides evidence that DA action in graft-reinnervated striatum may occur via long distance volume transmission in DA denervated areas.
Acknowledgements This study was supported by the Swedish Medical Research Council, grant No. 09917, 13233, Tore Nilsson's, Loo and Hans Osterman's, and Karolinska Institutet's foundations.
References Arbuthnott, G., Dunnett, S. and MacLeod, N. (1985) Electrophysiological properties of single units in dopamine-rich mesencephalic transplants in rat brain. Neurosci. Lett., 57: 205-210.
314 Backlund, E.-O., Granberg, P.O., Hamberger, B., Knutsson, E., M~rtensson, A., Sedvall, G., Seiger, ~. and Olson, L. (1985) Transplantation of adrenal medullary tissue to striatum in parkinsonism. First clinical trials. J. Neurosurg., 62: 169-173. Barker, R.A., Dunnett, S.B., Faissner, A. and Fawcett, J.W. (1996) The time course of loss of dopaminergic neurons and the gliotic reaction surrounding grafts of embryonic mesenceohalon to the striatum. Exp. Neurol., 141: 79-93. Bjrrklund, A. and Stenevi, U. (1979) Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants. Brain Res., 177: 555-560. Blunt, S.B., Jenner, P. and Marsden, C.D. (1992) Autoradiographic study of striatal Dt and D2 dopamine receptors in 6-OHDA-lesioned rats receiving foetal ventral mesencephalic grafts and chronic treatment with L-DOPA and carbidopa. Brain Res., 582:299-311. Bolam, J.P., Freund, T.F., Bjrrklund, A., Dunnett, S.B. and Smith, A.D. (1987) Synaptic input and local output of dopaminergic neurons in grafts that functionally reinnervate the host striatum. Exp. Brain Res., 68: 131-146. Brundin, P., Isacson, O. and Bjtirklund, A. (1985) Monitoring of cell viability in suspensions of embryonic CNS tissue and its use as a criterion for intracerebral graft survival. Brain Res., 331: 251-259. Buonamici, M., Caccia, M., Carpenteri, M., Pegrassi, L., Rossi, A.C. and Di Chiara, G. (1986) D-1 receptor supersensitivity in the rat striatum after unilateral 6-hydroxydopamine lesions. Eur. J. Pharmacol., 126: 347-348. Creese, I., Burt, D.R. and Snyder, S.H. (1977) Dopamine receptor binding enhancement accompanies lesion-induced behavioral sensitivity. Science, 17: 596-598. Dawson, T.M., Dawson, V.L., Gage, EH., Fisher, L.J., Hunt, M.A. and Wamsley, J.K. (1991) Functional recovery of supersensitive dopamine receptors after intrastriatal grafts of fetal substantia nigra. Exp. Neurol., 111: 282-292. Fisher, L.J., Young, S.J., Tepper, J.M., Groves, P.M. and Gage, EH. (1991) Electrophysiological characteristics of cells within mesencephalon suspension grafts. Neuroscience, 40: 109-122. Freed, W., Morihisa, J., Spoor, E., Hoffer, B., Olson, L., Seiger, A. and Wyatt, R. (1981) Transplanted adrenal chromaffin ceils in rat brain reduce lesion-induced rotational behaviour. Nature, 292:351-352. Freed, W.J., Cannon-Spoor, H.E. and Krauthamer, E. (1986) Intrastriatal adrenal medulla grafts in rats. Long-term survival and behavioral effects. J. Neurosurg., 65: 664-670. Freed, W.J., Neihoff, D.L., Kuhar, M.J., Hoffer, B.J., Olson, L., Cannon-Spoor, H.E., Morihisa, J.M. and Wyatt, R.J. (1983) Normalization of spiroperidol binding in the denervated rat striatum by homologous grafts of substantia nigra. Science, 222: 937-939. Freund, T.E, Bolam, J.P., Bjrrklund, A., Stenevi, U., Dunnett, S.B., Powell, J.F. and Smith, A.D. (1985) Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neostriatum: a tyrosine hydroxylase immunocytochemical study. J. Neurosci., 5: 603~i16.
Gagnon, C., Brdard, EJ., Rioux, L., Gaudin, D., Martinoli, M.G., Pelletier, G. and Di Paolo, T. (1991) Regional changes of striatal dopamine receptors following denervation by 6-hydroxydopamine and fetal mesencephalic grafts in the rat. Brain Res., 558: 251-263. Heikkila, R.D., Shapiro, B.S. and Duvoisin, R.C. (1981) The relationship between loss of dopamine nerve terminals, striatal 3H-spiroperidol binding and rotational behavior in unilaterally 6-OHDA lesioned rats. Brain Res., 211: 285-292. Herrera-Marschitz, M., Strrmberg, I., Olsson, D., Olson, L. and Ungerstedt, U. (1984) Adrenal medullary implants in the dopamine-denervated rat striatum. II. Rotational behavior during the first seven hours as a function of graft amount and location and its modulation by neuroleptics. Brain Res., 297: 53-61. Ingham, C.A., Hood, S.H., Taggart, P. and Arbuthnott, G.W. (1998) Plasticity of synapses in the rat neostriatum after unilateral lesion of the nigrostriatal dopaminergic pathway. J. Neurosci., 18: 4732-4743. Jaeger, C.B. (1985) Cytoarchitectonics of substantia nigra grafts: a light- and electron-microscopic study of immunocytochemically identified dopaminergic neurons and fibrous astrocytes. J. Comp. Neurol., 231: 121-135. Kemp JM, Powell TPS (1971) The synaptic organization of the caudate nucleus. Philos. Trans.R. Soc. (Lond.) B. Biol. Sci., 262: 403-412. Kordower, J.H., Rosenstein, J.M., Collier, T.J., Burke, M.A., Chen, E.-Y., Li, J.M., Martel, L., Levey, A.E., Mufson, E.J., Freeman, T.B. and Olanow, C.W. (1996) Functional fetal nigral grafts in a patient with Parkinson's disease: chemoanatomic, ultrastructural, and metabolic studies. J. Comp. Neurol., 370: 203-230. Mahalik, T.J., Finger, T.E., Strrmberg, I. and Olson, L. (1985) Substantia nigra transplants into denervated striatum of the rat: Ultrastructure of graft and host interconnections. J. Comp. Neurol., 240: 60-70. Marshall, J.E, Navarrete, R. and Joyce, J.N. (1989) Decreased striatal D1 binding density following mesetelencephalic 6-hydroxydopamine injections: an autoradiographic analysis. Brain Res., 493: 247-257. Meshul, C.K., Emre, N., Nakamura, C.M., Allen, C., Donohue, M.K. and Buckman, J.E (1999) Time-dependent changes in striatal glutatmate synapses following 6-hydroxydopamine lesion. Neuroscience, 88: 1-16. Morari, M., O'Connor, W.T., Darvelid, M., Ungerstedt, U., Bianchi, C. and Fuxe, K. (1996) Functional neuroanatomy of the nigrostriatal and the striatonigral pathways as studied with dual probe microdialysis in the awake rat. I. Effects of perfusion with tetrodotoxin and low calcium medium. Neuroscience, 72: 79-87. Perlow, M.J., Freed, W.J., Hoffer, B.J., Seiger, ~., Olson, L. and Wyatt, R.J. (1979) Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science, 204: 643~547.
315 Robertson, G.S., Fine, A. and Robertson, H.A. (1991) Dopaminergic grafts in the striatum reduce D1 but not D2 receptor-mediated rotation in 6-OHDA-lesioned rats. Brain Res., 539: 304-311. Rose, G., Gerhardt, G., Str6mberg, I., Olson, L. and Hoffer, B. (1985) Monoamine release from dopamine-depleted rat caudate nucleus reinnervated by substantia nigra transplants: an in vivo electrochemical study. Brain Res., 341: 92-100. Savasta, M., Mennicken, E, Chritin, M., Abrous, D.N., Feuerstein, C., Le Moal, M. and Herman, J. (1992) Intrastriatal dopamine-rich implants reverse the changes in dopamine D2 receptor densities caused by 6-hydroxydopaine lesion of the nigrostriatal pathway in rats: an autoradiographic study. Neuroscience, 46: 729-738. Schultz, W. and Ungerstedt, U. (1978) Short-term increase and long-term reversion of striatal cell activity after degeneration of the nigrostriatal dopamine system. Exp. Brain Res., 33: 159-171. Siggins, G., Hoffer, B. and Ungerstedt, U. (1974) Electrophysiological evidence for the involvement of cyclic adenosine monophosphate in dopamine responses of caudate neurons. Life Sci., 15: 779-792. Somogyi, P., Bolam, J.P. and Smith, A.D. (1981) Monosynaptic cortical input and local axon collaterals of identified striatonigral neurons, a light and electron microscopic study using the Golgi-peroxidase transport-degeneration procedure. J. Comp. Neurol., 195: 567-584. Strecker, R.E., Sharp, T., Brundin, P., Zetterstr6m, T., Ungerstedt, U. and Bj6rklund, A. (1987) Autoregulation of dopamine release and metabolism by intrastriatal nigral grafts as revealed by intracerebral dialysis. Neuroscience, 22: 169-178. Str6mberg, I., Adams, C., Bygdeman, M., Hoffer, B., Boyson, S. and Humpel, C. (1995) Long-term effects of human-to-rat mesencephalic xenografts on rotational behavior, striatal dopamine receptor binding, and mRNA levels. Brain Res. Bull., 38: 221-233. Str6mberg, I. and Bickford, P. (1996) Reduced aging effects of striatal neuronal discharge rate by aged ventral mesencephalic grafts. NeuroReport, 7: 693-696.
Str6mberg, I. and Bickford-Wimer, E (1991) Effects of locally applied D~ and D 2 agonists on striatal neurons with 6-OHDA and pertussis toxin lesions. Brain Res., 564: 279-285. Str6mberg, I., Herrera-Marschitz, M., Hultgren, L., Ungerstedt, U. and Olson, L. (1984) Adrenal medullary implants in the dopamine-denervated rat striatum. I. Acute catecholamine levels in grafts and host candate as determined by HPLCelectrochemistry and fluorescence histochemical image analysis. Brain Res., 297: 41-51. Str6mberg, I., Herrera-Marschitz, M., Ungerstedt, U., Ebendal, T. and Olson, L. (1985) Chronic implants of chromaffin tissue into the dopamine-denervated striatum. Effects of NGF on graft survival, fiber growth and rotational behavior. Exp. Brain Res., 60: 335-349. Str/3mberg, I., Johnson, S., Hoffer, B. and Olson, L. (1985) Reinnervation of dopamine-denervated striatum by substantia nigra transplants. Immunocytochemical and electrophysiological correlates. Neuroscience, 14:981-998. Str0mberg, I., Kehr, J., Andbjer, B. and Fuxe, K. (2000) Fetal ventral mecencephalic grafts functionally reduce the dopamine D2 receptor supersensitivity in partially dopamine reinnervated host striatum. Exp. Neurol., 1648. StrOmberg, I., van Home, C., Bygdeman, M., Weiner, N. and Gerhardt, G. (1991) Function of intraventricular human mesencephalic xenograft in immunosuppressed rats: an electrophysiological and neurochemical analysis. Exp. Neurol., 112: 140-152. Wuerthele, S., Freed, W., Olson, L., Morihisa, J., Spoor, L., Wyatt, R. and Hoffer, B. (1981) Effects of dopamine agonists and antagonists on the electrical activity of substantia nigra neurons transplanted into the lateral ventricle of the rat. Exp. Brain Res., 44: 1-10. Yamamoto, B.K. and Davy, S. (1992) Dopaminergic modulation of glutamate release in striatum as measured by microdialysis. J. Neurochem., 58: 1736-1742. Zetterstr6m, T., Brundin, E, Gage, EG., Sharper, T., Isacson, O., Dunnett, S.B., Ungerstedt, U. and BjOrklund, A. (1986) In vivo measurement of spontaneous release and metabolism of dopamine from intrastriatal nigral grafts using intracerebral dialysis. Brain Res., 362: 344-349.
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18
Restoration of dopamine transmission in graft reinnervated striatum. Evidence for regulation of dopamine D2 receptor function in regions lacking dopamine Ingrid Strfmberg*, Jan Kehr and Kjell Fuxe Department of Neuroscience, Karolinska Institute, S-17177 Stockholm, Sweden
Introduction to grafting as a tool to restore dopamine deficiency
Grafting of catecholamine-rich tissue into dopamine depleted striatum to restore the loss of dopaminergic input into the striatum has become a useful tool (Bjfrklund and Stenevi, 1979; Perlow et al., 1979; Freed et al., 1981). The first clinical attempts were performed by the grafting of adrenal medullary tissue into the caudate nucleus of patients suffering from Parkinson's disease (Backlund et al., 1985). Although the chromaffin cells store and release adrenaline and noradrenaline (NA) rather than dopamine (DA), it has been shown in animal experiments that implantation of adrenal chromaffin cells to the DA depleted striatum affects drug-induced rotational behavior (Freed et al., 1981, 1986; Strfmberg et al., 1985). Studies documenting effects on rotational behavior induced acutely after implantation of the tissue showed that rotations were induced by release of catecholamines from the grafts (Herrera-Marschitz et al., 1984; Strfmberg et al., 1984), and consequently, it was suggested that the graft exerted its action via diffusion or volume transmission rather than over synaptic contacts, since no outgrowth was found in this case. *Corresponding author: Tel.: 46-8-728 7087; Fax: 46-8-728 7437; e-mail: [email protected]
Later, grafting of fetal ventral mesencephalic tissue was shown to be more powerful to use than chromaffin cells. Robust documentation of graft survival, graft-derived dopaminergic reinnervation of host brain, spontaneous activity of dopaminergic neurons within the transplants, and DA released from the graft-derived outgrowth were shown (Wuerthele et al., 1981; Arbuthnott et al., 1985; Brundin et al., 1985; Rose et al., 1985; Strecker et al., 1987; Zetterstrfm et al., 1986). Furthermore, functional evidence of DA transmission was shown when local application of phencyclidine affected the striatal neuronal activity in graft-reinnervated striatum (Strfmberg et al., 1985). It was suggested that DA transmission in the stfiatum after reinnervation occurred via synaptic contacts, since reciprocal contacts between graft and host had been found, showing membrane specializations of the same type that had been characterized for a dopaminergic synaptic input to the striatal neurons (Freund et al., 1985; Jaeger, 1985; Mahalik et al., 1985; Bolam et al., 1987). Furthermore, evidence for functional dopaminergic input to the graft-reinnervated striatum was obtained based on studies showing that the increased D2 receptor binding found after a chronic DA depletion (Creese et al., 1977) was normalized after grafting (Freed et al., 1983). Later, it was shown that the D2 receptor binding became normalized in the total volume of dorsal striatum,
310
although the graft-derived dopaminergic reinnervation of dorsal striatum is limited (Dawson et al., 1991; Gagnon et al., 1991; Blunt et al., 1992; Freed et al., 1983). In fact, outgrowth from fetal ventral mesencephalon terminates already approximately 2 weeks after implantation (Barker et al., 1996), and at this time point only 1/3 to 1/2 of the volume of dorsal striatum has become reinnervated.
Conditions of dopamine transmission via volume transmission? The mismatch between striatal reinnervated areas and normalization of D2 receptor binding raised the question whether the dopamine transmission in graft-reinnervated striatum occurs via volume transmission in addition to synaptic transmission. Indeed it has been shown that clearance time for extracellular dopamine is prolonged distal compared to proximal to the graft (Strtmberg et al., 1991). Dopamine reuptake sites are present in THpositive nerve fibers after grafting (Kordower et al., 1996). Hence, the explanation for the longer clearance time was suggested to be due to the distribution of DA nerve fibers with less density distal than proximal to the graft, resulting in a more efficient DA reuptake close to the graft with higher DA nerve fiber densities and DA reuptake sites than that found distally. The conclusion was drawn that the graft may act over a larger volume of the host brain than that becomes reinnervated, probably via diffusion in the extracellular space (long-distance volume transmission).
Functional dopamine D2 receptors in dopamine reinnervated versus denervated striatum To further explore the possibility of DA transmission via volume transmission in non-DAinnervated areas of graft-reinnervated striatum, extracellular recordings of graft reinnervated striatum were employed. Recordings were performed in the DA depleted striatum reinnervated by a fetal ventral mesencephalic graft implanted into the lateral ventricle. The host striatum becomes dopaminergically reinnervated as visualized by tyrosine hydroxylase (TH)-positive nerve fibers in a zone close to the ventricle, and thus, reinnervated and
non-innervated areas can be localized to medial and lateral striatum respectively (Fig. 1) (Strtmberg and Bickford, 1996). Extracellular recordings in the striatum including local applications of the D2-1ike agonist quinpirole revealed a dose-dependent reduction in striatal neuronal firing rates. When recording in graftreinnervated areas of the striatum there was no shift in sensitivity to quinpirole when compared to the control side. Furthermore, there was no difference in sensitivity to quinpirole when recording in noninnervated versus reinnervated regions of graftreinnervated striatum (Fig. 2). However, a significant supersensitivity was found when recording in 6-hydroxydopamine (6-OHDA) lesioned control animals (Fig. 2). Thus, the D2 receptor supersensitivity seen after an almost complete DA denervation may be functionally removed in graft reinnervated striatum, not only within reinnervated areas, but also lateral to the DA reinnervation. Hence, the normalization of D2 receptor binding in incomplete graft-reinnervated striatum had physiological relevance.
Striatal spontaneous neuronal activity The striatal neuronal firing rates are significantly upregulated in DA depleted striatum (Siggins et al., 1974; Schultz and Ungerstedt, 1978; Strtmberg et al., 1985). After graft-reinnervation the spontaneous discharge rate becomes normalized, but the normalization is found only in areas that are reinnervated (Fig. 3) (Strtmberg et al., 1985; Fisher et al., 1991). Since the D2 receptors were shown to be functionally normalized, the upregulated neuronal spontaneous activity might be due to malfunction at the D1 receptor level. It has been suggested that the D1 receptor sensitivity shows either decrease, increase or no change after a 6-OHDA lesion (Buonamici et al., 1986; Marshall et al., 1989; Dawson et al., 1991; Gagnon et al., 1991; Robertson et al., 1991; Blunt et al., 1992; Savasta et al., 1992). Although the reports show divergent results, it has been found that the D1 receptor levels are normalized after grafting and some studies have shown reduced D1 receptor levels (Blunt et al., 1992; Strtmberg et al., 1995). Nevertheless, electrophysiological recordings in
311 DA depleted striatum indicate no significant change of the dose-response curve to the D1 agonist N0437 (Str6mberg and Bickford-Wimer, 1991). However, inactivation of the D2 receptor results in a downregulation of the sensitivity to a D1 agonist, indicating that there is a kind of synergistic action between D1 and D2 receptors in the 6-OHDA lesioned striatum (Str6mberg and Bickford-Wimer, 1991). Extracellular recordings using local applications of the D1 agonist SKF 81297 in DA reinnervated striatum revealed no physiological differences of striatal neuronal responses to the D1 agonist in reinnervated versus noninnervated areas of grafted striatum (StrtSmberg et al., 1999). Thus, the upregulated striatal neuronal discharge rate in non-innervated areas did not seem to be due to a malfunction of the D 1 receptor.
Cortical excitatory input to the striatum after grafting The results showing that the dopaminergic reinnervation normalized the D2 receptor function but not the spontaneous neuronal activity in noninnervated areas of grafted striatum turned our interest to the excitatory input to the striatum, i.e. the cortical glutamatergic innervation of the striatum. It has been shown that a DA depletion enhance extracellular levels of glutamate (Yamamoto and Davy, 1992; Meshul et al., 1999), and accordingly this might be the explanation for the upregulated striatal neuronal activity in noninnervated areas of the graft reinnervated striatum. However, there is a loss of asymmetric synapses after a DA depletion (Ingham et al., 1998; Meshul et al., 1999), and since
Fig. 1. TH-immunohistochemistryof a fetal ventral mesencephalicgraft transplanted to the lateral ventricleof a unilaterally 6-OHDA lesioned rat. Graft outgrowthinto the host brain is limitedto a zone close to the ventricle.Extracellularelectrophysiologicalrecordings were performed in host striatum within the zone of TH-positive nerve fibers and in dopamine denervated regions lateral to THimmunoreactiveareas. Scale bar: 200 ~tm.
312
asymmetric synapses are related to the glutamatergic input to striatal dentritic spines (Kemp and Powell, 1971; Somogyi et al., 1981), enhanced glutamate levels are accompanied with loss of glutamate synaptic input. Microdialysis using the dual-probe approach (Morari et al., 1996) and recording of extracellular levels of glutamate after DA reinnervation showed no differences in glutamate overflow in reinner-
vated compared to non-innervated areas of graft-derived DA reinnervated striatum. Extracellular levels of potassium-induced glutamate overflow showed approximately 200% increase of baseline levels in all areas measured. Thus, it is not likely that the upregulated neuronal activity in noninnervated areas of graft-reinnervated striatum is due to an increase in extracellular glutamate in these areas.
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Fig. 2. Dose-response to locally applied quinpirole expressed as percentage change from baseline. Recordings were performed in 6-OHDA lesioned (a) and in graft DA reinnervated striatum (b). In DA depleted striatum there was a shift in dose-response (p < 0.001 ) to quinpirole and the supersensitivity to the D2 agonist was shown, while in graft reinnervated striatum there was no difference in dose response when recording in DA reinnervated vs. noninnervated regions compared to control side. From Str6mberg et al., 2000.
313
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Fig. 3. Extracellularrecordings performed at 2.2 and 3.3 mm lateral to bregma in 6-OHDAlesioned rats and in DA-depleted animals followed by grafting of fetal ventral mesencepahlic tissue to the lateral ventricle. Striatal neuronal discharge rates were significantly increased in DA depleted regions compared to normal firing rate. In DA reinnervated regions of grafted striatum (2.2 mm grafted side) the spontaneous discharge rate was normal. ***p< 0.0001.
Conclusion The results indicate that the functional normalization of the D2 receptors within as well as distal to reinnervated areas of the striatum is regulated by diffusion of DA from graft-derived reinnervated areas to denervated areas. Binding studies have shown a normalization of D2 receptor levels in total volume of dorsal striatum, and it has not been correlated to the exact location of the graft or the reinnervated areas. However, the present data indicate that the distance the volume transmitted DA diffuses to functionally normalize the D2 receptor levels in non-innervated regions lateral to the DA reinnervated areas is rather at the 500 txm levels than at smaller distances, since recordings were performed at a minimum of 500 txm lateral to the zone of DA reinnervation. Trials to measure extracellular levels of DA at 500 ~xm distance from the reinnervated areas have not been successful, but
since the D2 receptors require more than a 90% DA depletion to become upregulated (Heikkila et al., 1981), the DA levels needed to normalize the D2 receptors in denervated areas might be below detection limit. Thus, these data provides evidence that DA action in graft-reinnervated striatum may occur via long distance volume transmission in DA denervated areas.
Acknowledgements This study was supported by the Swedish Medical Research Council, grant No. 09917, 13233, Tore Nilsson's, Loo and Hans Osterman's, and Karolinska Institutet's foundations.
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