Brain Research, 523 (1990) 288-290
288
Elsevier
BRES 24162
Short Communications
Striatonigral projection neurons contain
D1
dopamine receptor-activated
c-los G.S. Robertson, S.R. Vincent and H.C. Fibiger Division of Neurological Sciences, Department of Psychiatry, Universityof British Columbia, Vancouver, B.C. (Canada) (Accepted 20 March 1990)
Key words: C-los proto-oncogene; Dopamine D~ receptor; Substantia nigra pars reticulata; Medium spiny neuron Dopamine receptor agonists which stimulate the D~ receptor have been shown to activate c-los in the striatum ipsilateral to a 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway. In the present study, striatai neurons ipsilateral to a 6-OHDA lesion of the medial forebrain bundle were retrogradely labelled by injection of the fluorescent tracer Fluoro-Gold into the substantia nigra pars reticulata. Five days later, c-los was induced in the 6-OHDA-denervated striatum by injection of the selective D 1 agonist SKF 38393. C-los-positive nuclei were frequently found in medium-sized striatal cell bodies labelled with Fluoro-Gold. These results indicate that D 1 agonists activate c-los in medium-sized neurons that project to the substantia nigra pars reticulata. The proto-oncogene c-fos can be activated in the central nervous system (CNS) by a variety of physiological and pharmacological treatments 1'3'7'1°. For example, non-noxious brushing and manipulation of the hind limb activates c-fos in layers I I - I V of the spinal cord 3 while administration of morphine I or caffeine at subconvulsive doses 7 activates c-fos in the striatum. Recently, it has been demonstrated that D1, but not D2, dopamine receptor agonists produce a massive increase in c-los immunoreactivity in nuclei of striatal neurons ipsilateral to a 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra (SN) 9. Animals with incomplete 6 - O H D A lesions failed to demonstrate an increase in c-los immunoreactivity in the ipsilateral striatum 8. The nature of the striatal neurons in which c-los is induced by a D 1 agonist in the 6-OHDAdenervated striatum has yet to be determined. The most common striatal cell type is the medium spiny neuron which accounts for approximately 95% of Golgi-impregnated cell bodies in this structure 4. Medium spiny neurons utilize the inhibitory transmitter ~,-aminobutyric acid ( G A B A ) and project to the globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata (SNR) 6. Excitatory amino acid lesions of the striatum produce a concomitant loss of medium spiny neurons, glutamic acid decarboxylase activity6, adenylate cyclase activity 2'4, DARPP-3214 and D~ antagonist binding 11 in the striatum and SNR. These results suggest that D 1 receptors are located on medium spiny neurons which project to the SNR. Consequently, c-fos immunoreactiv-
ity induced by a D 1 agonist, in the 6-OHDA-denervated striatum, may be situated in the cell bodies of medium spiny neurons that project to the SNR. The present study examined the nature of striatal neurons containing D 1 receptor-activated c-fos using the fluorescent retrograde tracer Fluoro-Gold (FG) 12. FG was injected into the SNR ipsilateral to a 6 - O H D A lesion of the medial forebrain bundle (MFB). Five days later, c-los was induced in the ipsilateral striatum by injection of the selective D 1 receptor agonist SKF 38393. Clos-positive nuclei were observed in striatal cell bodies retrogradely labelled from the SNR with FG. Male Wistar rats (275-300 g) were injected with desmethylimipramine HCI (25 mg/kg, i.p.) and 30 min later anaesthetized with sodium pentobarbital (50 mg/kg, i.p.). Unilateral lesions of the right MFB were made by injecting 8/~g (base) of 6 - O H D A HBr (Sigma) in 4/~1 of sterile saline containing 0.02% ascorbic acid; the solution was injected over a 10 min period into the right MFB at the coordinates AP 4.5, ML (right) 1.3, DV 1.7, from interaural zero. Behavioural screening was carried out after at least 2 weeks post surgery. Animals that rotated no less than 7 turns/min to an apomorphine injection (0.5 mg/kg, i.p.) were included in the study. FG (Fluorochrome Inc.) was dissolved in sterile saline (4 mg/100/~1) and injected under pentobarbital anaesthesia into the right SNR using a 5/~1 Hamilton syringe at the coordinates AP 3.2, ML (right) 2.2, DV 1.7, from interaural zero. In one of the cases a 0.5/tl volume of FG was injected while in the other 2 cases 0.1/~1 of FG was
Correspondence: G.S. Robertson, University of British Columbia, Division of Neurological Sciences, Department of Psychiatry, 2255 Wesbrook Mall, Vancouver, B.C., Canada, V6T lW5.
289
Fig. 1. The top panel (A) shows c-los-positive nuclei in a portion of the anterolateral region of the striatum ipsilateral to a 6-OHDA lesion of the MFB. The bottom panel (B) shows striatal cell bodies retrogradely labelled with FG from the same portion of the striatum illustrated in panel (A). Nuclei immunofluorescent for c-los (A) can be seen to be frequently contained in striatal cell bodies retrogradely labelled with FG (B) (arrowheads). Bar = 15 gin.
290 injected into the SNR. One week after the FG injection, subjects were placed in 20 liter rotation buckets and injected with the D 1 agonist SKF 38393 (4 mg/kg, i.p.); the number of contralateral turns completed in a 1 min period was determined every 20 min for 2 h. Two hours following the SKF 38393 injection, rats were anaesthetized with pentobarbital and were perfused with 0.02 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Freezing and thawing the tissue resulted in a loss of c-los immunoreactivity. Therefore, 20 /~m sections were cut at room temperature using a Vibratome. Sections were washed 3 times (20 min per wash) with PBS, then incubated for 48 h with sheep anti-c-los antibody diluted 1:150 Cambridge Research Biochemicals) in PBS containing 0.3% Triton X-100 and 0.02% Azide. They were then washed 3 times with PBS and incubated with Texas red-labelled rabbit anti-sheep antibody (Jackson Immunoresearch Laboratories) for 1 h. Sections were washed 3 times with PBS, mounted and coverslipped with Immersionoel 518C (Zeiss). The slides were observed using a Leitz fluorescence microscope either under ultraviolet light (340-380 nm) to examine FG, or green light (530-560 nm) to look at Texas red fluorescence. Administration of SKF 38393 (4.0 mg/kg, i.p.) resulted in a high rate of circling (12-14 turns/min) that lasted for 4 h. As reported previously 8'9, SKF 38393 produced a dramatic increase in c-fos immunoreactivity in striatal cells ipsilateral, but not contralateral (not shown), to a 6 - O H D A lesion (Fig. 1A). Injection of F G into the SNR ipsilateral to the 6 - O H D A lesion produced intense labelling of medium-sized striatal cell bodies (12-18 ktm) 4 1 Chang, S.L., Squinto, S.P. and Harlan, R.E., Morphine activation of c-fos expression in the rat brain, Biochem. Biophys. Res. Commun., 2 (1988) 698-704. 2 Gale, K., Guidotti, A. and Costa, E.C., Dopamine-sensitive adenylate cyclase: location in substantia nigra, Science, 195 0977) 503-505. 3 Hunt, S.E, Pini, A. and Evans, G., Induction of c-fos-like protein in spinal cord neurons following sensory stimulation, Nature, 328 (1987) 632-634. 4 Kemp, J.M. and Powell, T.ES., The structure of the caudate nucleus of the cat: light and electron microscopy, Phil. Trans. R. Soc. Lond. Ser. B., 262 (1971) 403-412. 5 McGeer, E.G., Innanen, V.T. and McGeer, EL., Evidence on the cellular localization of adenyi cyclase in the neostriatum, Brain Research, 118 (1976) 356-358. 6 Nagy, J.I., Carter, D.M. and Fibiger, H.C., Anterior striatal projections to the globus pallidus, entopeduncular nucleus and substantia nigra in the rat: the GABA connection, Brain Research, 158 (1978) 15-29. 7 Nakajima, T., Daval, J.-L., Morgan, P.E, Post, P.M. and Marangos, EL., Adenosinergic modulation of caffeine-induced c-los mRNA expression in mouse brain, Brain Research, 501 (1989) 307-314. 8 Robertson, G.S., Herrera, D.G., Dragunow, M. and Robertson, H.A., L-DOPA activates c-los in the striatum ipsilateral to
in the homolateral striatum (Fig. 1B). Examination of the injection site for each of the 3 cases revealed that diffusion of F G was restricted to the SN. C-los-positive nuclei were frequently located in striatal cell bodies retrogradely labelled with F G (Fig. 1A,B). The results shown in Fig. 1A,B are representative of the 3 cases examined. Hence, the D 1 agonist SKF 38393 activates c-los in medium spiny neurons which project to the SNR. Whether other types of striatal neurons (striato-pallidal, interneurones, etc.) also express D 1 receptor-activated c-los remains to be determined. The ability of a host of growth factors, neurotransmitters, drugs and physiological manipulations to activate c-fos in the CNS indicate that c-fos induction occurs as a consequence of synaptic activation m. This has led to the suggestion that c-fos immunohistochemistry might be used in a manner similar to 2-deoxyglucose to map metabolic pathways in the CNS m. Administration of the D 1 agonist SKF 38393 has been shown to increase 2-deoxyglucose utilization in the 6-OHDA-lesioned SN 13. The results of the present study support the suggestion made by Trugman and Wooten ~3 that enhanced glucose utilization in the 6-OHDA-lesioned SN represents increased activity of striatonigral efferents. Nevertheless, future studies which examine the anatomical relationships between c-los activation and other measures of neuronal activity are required to further evaluate the utility of c-los immunohistochemistry as a metabolic marker with cellular resolution. This work was supported by the Medical Research Council of Canada PG-(23). G.S.R. is an MRC Fellow, and S.R.V. an MRC scientist. The authors thank Ms. Ester Leung for excellent technical assistance. a 6-hydroxydopamine lesion of the substantia nigra, Eur. J. Pharmacol., 159 (1989) 99-100. 9 Robertson, H.A., Peterson, M.R., Murphy, K. and Robertson, G.S., Dl-dopamine receptor agonists selectively activate c-fos independent of rotational behaviour, Brain Research, 503 (1989) 346-349. 10 Sagar, S.M., Sharp, ER. and Curran, T., Expression of c-los protein in brain: metabolic mapping at the cellular level, Science, 240 (1988) 1328-1331. 11 Savasta, M., Dubois, A., Benavides, J. and Scatton, B., Different neuronal location of [3H]SCH 23390 binding in pars reticulata and pars compacta of the substantia nigra, Neurosci. Lett., 72 (1986) 265-271. 12 Schmued, L.C. and Fallon, J.H., Fluoro-Gold: a new fluorescent retrograde axonal tracer with numerous unique properties, Brain Research, 377 (1986) 147-154. 13 Trugman, J.M. and Wooten, G.E, Selective D1 and D2 dopamine agonists differentially alter basal ganglia glucose utilization in rats with unilateral 6-hydroxydopamine substantia nigra lesions, J. Neurosci., 7 (1987) 2927-2935. 14 Walaas, S.I. and Greengard, P., A dopamine- and adenosine 3":5"-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Regional and cellular distribution in the rat brain, J. Neurosci., 4 (1984) 84-98.
288
Elsevier
BRES 24162
Short Communications
Striatonigral projection neurons contain
D1
dopamine receptor-activated
c-los G.S. Robertson, S.R. Vincent and H.C. Fibiger Division of Neurological Sciences, Department of Psychiatry, Universityof British Columbia, Vancouver, B.C. (Canada) (Accepted 20 March 1990)
Key words: C-los proto-oncogene; Dopamine D~ receptor; Substantia nigra pars reticulata; Medium spiny neuron Dopamine receptor agonists which stimulate the D~ receptor have been shown to activate c-los in the striatum ipsilateral to a 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway. In the present study, striatai neurons ipsilateral to a 6-OHDA lesion of the medial forebrain bundle were retrogradely labelled by injection of the fluorescent tracer Fluoro-Gold into the substantia nigra pars reticulata. Five days later, c-los was induced in the 6-OHDA-denervated striatum by injection of the selective D 1 agonist SKF 38393. C-los-positive nuclei were frequently found in medium-sized striatal cell bodies labelled with Fluoro-Gold. These results indicate that D 1 agonists activate c-los in medium-sized neurons that project to the substantia nigra pars reticulata. The proto-oncogene c-fos can be activated in the central nervous system (CNS) by a variety of physiological and pharmacological treatments 1'3'7'1°. For example, non-noxious brushing and manipulation of the hind limb activates c-fos in layers I I - I V of the spinal cord 3 while administration of morphine I or caffeine at subconvulsive doses 7 activates c-fos in the striatum. Recently, it has been demonstrated that D1, but not D2, dopamine receptor agonists produce a massive increase in c-los immunoreactivity in nuclei of striatal neurons ipsilateral to a 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra (SN) 9. Animals with incomplete 6 - O H D A lesions failed to demonstrate an increase in c-los immunoreactivity in the ipsilateral striatum 8. The nature of the striatal neurons in which c-los is induced by a D 1 agonist in the 6-OHDAdenervated striatum has yet to be determined. The most common striatal cell type is the medium spiny neuron which accounts for approximately 95% of Golgi-impregnated cell bodies in this structure 4. Medium spiny neurons utilize the inhibitory transmitter ~,-aminobutyric acid ( G A B A ) and project to the globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata (SNR) 6. Excitatory amino acid lesions of the striatum produce a concomitant loss of medium spiny neurons, glutamic acid decarboxylase activity6, adenylate cyclase activity 2'4, DARPP-3214 and D~ antagonist binding 11 in the striatum and SNR. These results suggest that D 1 receptors are located on medium spiny neurons which project to the SNR. Consequently, c-fos immunoreactiv-
ity induced by a D 1 agonist, in the 6-OHDA-denervated striatum, may be situated in the cell bodies of medium spiny neurons that project to the SNR. The present study examined the nature of striatal neurons containing D 1 receptor-activated c-fos using the fluorescent retrograde tracer Fluoro-Gold (FG) 12. FG was injected into the SNR ipsilateral to a 6 - O H D A lesion of the medial forebrain bundle (MFB). Five days later, c-los was induced in the ipsilateral striatum by injection of the selective D 1 receptor agonist SKF 38393. Clos-positive nuclei were observed in striatal cell bodies retrogradely labelled from the SNR with FG. Male Wistar rats (275-300 g) were injected with desmethylimipramine HCI (25 mg/kg, i.p.) and 30 min later anaesthetized with sodium pentobarbital (50 mg/kg, i.p.). Unilateral lesions of the right MFB were made by injecting 8/~g (base) of 6 - O H D A HBr (Sigma) in 4/~1 of sterile saline containing 0.02% ascorbic acid; the solution was injected over a 10 min period into the right MFB at the coordinates AP 4.5, ML (right) 1.3, DV 1.7, from interaural zero. Behavioural screening was carried out after at least 2 weeks post surgery. Animals that rotated no less than 7 turns/min to an apomorphine injection (0.5 mg/kg, i.p.) were included in the study. FG (Fluorochrome Inc.) was dissolved in sterile saline (4 mg/100/~1) and injected under pentobarbital anaesthesia into the right SNR using a 5/~1 Hamilton syringe at the coordinates AP 3.2, ML (right) 2.2, DV 1.7, from interaural zero. In one of the cases a 0.5/tl volume of FG was injected while in the other 2 cases 0.1/~1 of FG was
Correspondence: G.S. Robertson, University of British Columbia, Division of Neurological Sciences, Department of Psychiatry, 2255 Wesbrook Mall, Vancouver, B.C., Canada, V6T lW5.
289
Fig. 1. The top panel (A) shows c-los-positive nuclei in a portion of the anterolateral region of the striatum ipsilateral to a 6-OHDA lesion of the MFB. The bottom panel (B) shows striatal cell bodies retrogradely labelled with FG from the same portion of the striatum illustrated in panel (A). Nuclei immunofluorescent for c-los (A) can be seen to be frequently contained in striatal cell bodies retrogradely labelled with FG (B) (arrowheads). Bar = 15 gin.
290 injected into the SNR. One week after the FG injection, subjects were placed in 20 liter rotation buckets and injected with the D 1 agonist SKF 38393 (4 mg/kg, i.p.); the number of contralateral turns completed in a 1 min period was determined every 20 min for 2 h. Two hours following the SKF 38393 injection, rats were anaesthetized with pentobarbital and were perfused with 0.02 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Freezing and thawing the tissue resulted in a loss of c-los immunoreactivity. Therefore, 20 /~m sections were cut at room temperature using a Vibratome. Sections were washed 3 times (20 min per wash) with PBS, then incubated for 48 h with sheep anti-c-los antibody diluted 1:150 Cambridge Research Biochemicals) in PBS containing 0.3% Triton X-100 and 0.02% Azide. They were then washed 3 times with PBS and incubated with Texas red-labelled rabbit anti-sheep antibody (Jackson Immunoresearch Laboratories) for 1 h. Sections were washed 3 times with PBS, mounted and coverslipped with Immersionoel 518C (Zeiss). The slides were observed using a Leitz fluorescence microscope either under ultraviolet light (340-380 nm) to examine FG, or green light (530-560 nm) to look at Texas red fluorescence. Administration of SKF 38393 (4.0 mg/kg, i.p.) resulted in a high rate of circling (12-14 turns/min) that lasted for 4 h. As reported previously 8'9, SKF 38393 produced a dramatic increase in c-fos immunoreactivity in striatal cells ipsilateral, but not contralateral (not shown), to a 6 - O H D A lesion (Fig. 1A). Injection of F G into the SNR ipsilateral to the 6 - O H D A lesion produced intense labelling of medium-sized striatal cell bodies (12-18 ktm) 4 1 Chang, S.L., Squinto, S.P. and Harlan, R.E., Morphine activation of c-fos expression in the rat brain, Biochem. Biophys. Res. Commun., 2 (1988) 698-704. 2 Gale, K., Guidotti, A. and Costa, E.C., Dopamine-sensitive adenylate cyclase: location in substantia nigra, Science, 195 0977) 503-505. 3 Hunt, S.E, Pini, A. and Evans, G., Induction of c-fos-like protein in spinal cord neurons following sensory stimulation, Nature, 328 (1987) 632-634. 4 Kemp, J.M. and Powell, T.ES., The structure of the caudate nucleus of the cat: light and electron microscopy, Phil. Trans. R. Soc. Lond. Ser. B., 262 (1971) 403-412. 5 McGeer, E.G., Innanen, V.T. and McGeer, EL., Evidence on the cellular localization of adenyi cyclase in the neostriatum, Brain Research, 118 (1976) 356-358. 6 Nagy, J.I., Carter, D.M. and Fibiger, H.C., Anterior striatal projections to the globus pallidus, entopeduncular nucleus and substantia nigra in the rat: the GABA connection, Brain Research, 158 (1978) 15-29. 7 Nakajima, T., Daval, J.-L., Morgan, P.E, Post, P.M. and Marangos, EL., Adenosinergic modulation of caffeine-induced c-los mRNA expression in mouse brain, Brain Research, 501 (1989) 307-314. 8 Robertson, G.S., Herrera, D.G., Dragunow, M. and Robertson, H.A., L-DOPA activates c-los in the striatum ipsilateral to
in the homolateral striatum (Fig. 1B). Examination of the injection site for each of the 3 cases revealed that diffusion of F G was restricted to the SN. C-los-positive nuclei were frequently located in striatal cell bodies retrogradely labelled with F G (Fig. 1A,B). The results shown in Fig. 1A,B are representative of the 3 cases examined. Hence, the D 1 agonist SKF 38393 activates c-los in medium spiny neurons which project to the SNR. Whether other types of striatal neurons (striato-pallidal, interneurones, etc.) also express D 1 receptor-activated c-los remains to be determined. The ability of a host of growth factors, neurotransmitters, drugs and physiological manipulations to activate c-fos in the CNS indicate that c-fos induction occurs as a consequence of synaptic activation m. This has led to the suggestion that c-fos immunohistochemistry might be used in a manner similar to 2-deoxyglucose to map metabolic pathways in the CNS m. Administration of the D 1 agonist SKF 38393 has been shown to increase 2-deoxyglucose utilization in the 6-OHDA-lesioned SN 13. The results of the present study support the suggestion made by Trugman and Wooten ~3 that enhanced glucose utilization in the 6-OHDA-lesioned SN represents increased activity of striatonigral efferents. Nevertheless, future studies which examine the anatomical relationships between c-los activation and other measures of neuronal activity are required to further evaluate the utility of c-los immunohistochemistry as a metabolic marker with cellular resolution. This work was supported by the Medical Research Council of Canada PG-(23). G.S.R. is an MRC Fellow, and S.R.V. an MRC scientist. The authors thank Ms. Ester Leung for excellent technical assistance. a 6-hydroxydopamine lesion of the substantia nigra, Eur. J. Pharmacol., 159 (1989) 99-100. 9 Robertson, H.A., Peterson, M.R., Murphy, K. and Robertson, G.S., Dl-dopamine receptor agonists selectively activate c-fos independent of rotational behaviour, Brain Research, 503 (1989) 346-349. 10 Sagar, S.M., Sharp, ER. and Curran, T., Expression of c-los protein in brain: metabolic mapping at the cellular level, Science, 240 (1988) 1328-1331. 11 Savasta, M., Dubois, A., Benavides, J. and Scatton, B., Different neuronal location of [3H]SCH 23390 binding in pars reticulata and pars compacta of the substantia nigra, Neurosci. Lett., 72 (1986) 265-271. 12 Schmued, L.C. and Fallon, J.H., Fluoro-Gold: a new fluorescent retrograde axonal tracer with numerous unique properties, Brain Research, 377 (1986) 147-154. 13 Trugman, J.M. and Wooten, G.E, Selective D1 and D2 dopamine agonists differentially alter basal ganglia glucose utilization in rats with unilateral 6-hydroxydopamine substantia nigra lesions, J. Neurosci., 7 (1987) 2927-2935. 14 Walaas, S.I. and Greengard, P., A dopamine- and adenosine 3":5"-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Regional and cellular distribution in the rat brain, J. Neurosci., 4 (1984) 84-98.
