FIG. FIG.
1. Whole 2. Coronal
brain with hemisection
bilateral frontal of diencephalon
cartex suction ablations. showing rostra1 thalamic
Cats were killed electrocoagulative
7 days after lesion placement. lesion. Nissl stain. X7.
428
ARIANO
AND
ADINOLFI
FIG. 3. Intact terminal from caudate nucleus of cat with bilateral frontal pole lesion. Incubated with 0.75 mM cyclic 3’,.5’-adenosine monophosphate. A, Axon terminal. x51,000. FIG. 4. Degenerating thalamostriate terminal in caudate neuropil using 0.75 IBM cyclic 3’,5’-adenosine monophosphate as substrate. XSl,oOO.
CAUDATE
SLTBCELLULAR
PIIOSPIIODIESTERASE
429
reported the cytochemical localization of cyclic nucleotide l~liospl~odiesterase, the catalytic enzyme of cyclic 3’.5’-adenosine n~onophosphate, at postsynaptic sites in intact caudate nucleus. The lack of a suitable method for visualizing brain adenylate cyclase was noted, am1 we suggested that the demonstration of pl~ospliodiesterase activity at asymmetrical axodendritic synapses, some of which represent nigroneostriatal terminations ( 15. 16 j, supported indirectly the “dol~amine receptor” hypothesis. However, in that study we made no attenlpt to distinguish the dopaminergic nigral terminals from those which originate in the cortex or thalamus. Because all afferent fibers synapse asymmetrically on dendrites and dendritic spines in the caudate neuropil, we decided to interrupt the large thalamic and cortical inputs and determine whether or not phosphodiesterase activity would localize postsynaptic to these degenerating terminals. Following incubation of caudate slices taken from these animals with lesions, reaction product was seen postsynaptic to both intact and degenerating endings. The neurotransmitters used by the cortical and thalamic inputs are unknown (8). Thus, our present findings extend cyclic nucleotide phosphodiesterase activity to synaptic sites which are clearly not dopaminergic and demonstrate that the localization of this enzymatic activity does not occur solely at dopamine receptor sites in the caudate nucleus. MATEKIALS
AND
METHODS
Lesions of the corticostriate and thalamostriate projections were made in six adult cats. The frontal poles, including sensorimotor and orbitofrontal regions, were ablated bilaterally in two animals. Large bilateral electrocoagulative lesions destroyed rostra1 intralaminar thalamic nuclei in two animals, and combined cortical and thalamic regions were destroyed unilaterally in two animals. All animals survived for 7 clays. The cats were then anesthetized with intraperitoneal injections of sodium pentabarbitol. Brains were fixed ,in sifu by vascular perfusion with cold 2%) EM-grade glutaraldehyde (Polysciences), 2% paraformaldehyde in 0.05 M sodium cacodylate buffer containing 0.25 M dextrose at pH 7.4. Cytochemical localization of cyclic nucleotide phosphodiesterase activity is based on the sfa.fu nascendi precipitation of lead phosphate, according to the method of Florendo et al. (9), and includes the modifications reported in our previous study of intact caudate nucleus (3). The following control procedures were carried out to verify the specificity of the cytochemical localization method: 50 mM theoFIG. 5. mic lesion FIG. 6. thalamic adenosine
Degenerating terminal in caudate neuropil of cat with bilateral rostra1 thalausing 0.75 m&r cyclic 3’,5’-adenosine monophosphate as substrate. X34000. Degenerating ending within caudate nucleus following a combined unilateral with ipsilateral frontal cortical lesion. Incubated with 0.75 rnhf cyclic 3’,5’monophosphate as substrate. D, Dendrite. X58,000.
430
rZ~ImO
AND
ADIN~LFI
FIG. 7. Terminal degeneration within caudate neuropil after placement frontal cortex-ipsilateral thalamic lesion using 0.5 mAI cyclic adenosine x40.000.
of a uniIatera1 monophosphate.
CAUDATE
SVI3CELLUL.lR
I’IIOSl’IIO~IESTERhSE
431
l~hyllinc (Sigma) or 5mnl. isul~ut~liiietli~l~ai~tliiiic (Aldrich) were added to inhibit phosphodiesterase actil,ity ; q.clic 2’,3’-adenosine monophosphate, 5’adenosine monophosphate, or cJ,clic 3’,5’-guanr)sinc monophosphate were substituted as substrates ; 5’-nucleotidasc was omitted; substrate was omitted ; or substrate and 5’-nucleotidase were omitted. Substrate concentrations of 0.1 to 3.0 mh1 3’,5’-qciic atlenosine monophosphate were utilized. All tissues for electron microscqq were esamined unstained with a Hitachi HS-S electron microscope. The estent of the frontal cortical damage was assessed from whole brains (Fig. l), and the thalamic lesions were verified histologically (Fig. 2) by esamination of Nissl-stained coronal sections. RESULTS Cyclic 3’,5’-adenosine monophosphate is hydrolyzed to S’-adenosine monophosphate by the action of cyclic nucleotide phosphodiesterase. Inorganic phosphate is released by the action of 5’-nucleotidase on 5’-adenosine monophosphate, yielding adenosine and free phosphate. The activity of the membrane-bound, cyclic AMP-specific phosphodiesterase can be cytochemically visualized as an electron-dense, lead phosphate precipitate resulting from the capture of this free phosphate by a lead ion trapping agent (3,9>. Phosphodiesterase activity is visualized postsynaptically at both intact and degenerating terminals in lOO-,~m slices of caudate nucleus taken from animals with large lesions of frontal cortex and rostra1 intralaminar thalamus. Observations were made utilizing the previously determined optimum substrate concentration of 0.75 mnI cyclic 3’,5’-adenosine monophosphate (3)) as shown in Figs. 3-6. Degenerating corticostriate and thalamostriate terminations appear as electron-dense, irregular profiles which synapse asymmetrically on dendrites and dendritic spines. The lead phosphate reaction product consistently accumulates in the region of the postsynaptic density. However, at this substrate concentration background reactivity, defined as the amount of lead phosphate seenat nonsynaptic sites, was unexpectedly elevated. Reduction of the substrate concentration to 0.5 mnf cyclic 3’,5’FIG. 8. Degenerating terminalfrom caudatenucleusof cat with unilateralinterruption of thalamostriateand ipsilateral corticostriate tracts. Substrate concentration of 1.0 mlf cyclic 3’,5’-adenosine monophosphate incubated with 5 rnhz isobutylmethylxanthine was used. A, Axon terminal. X45,000. FIG. 9. Degenerating corticostria,te terminal in caudate neuropil using 0.75 mM cyclic 3’,5’-guanosine monophosphate as substrate. ~31,000. FIG. 10. Thalamostriate terminal degeneration within cat caudate nucleus, utilizing 1.0 rnsx 5’-adenosine monophosphate without .5’-nucleotidase in the eqeriment. D, Dendrite. X.50.000.
432
ARIANO
AND
ADINOLFI
adenosine monophosphate virtually eliminated this background reactivity (Fig. 7). In control sections of caudate incubated with the methylxanthines (isobutylmethylxanthine and theophylline) no lead precipitate could be detected (Fig. 8). Reaction product was also absent in sections which were incubated with cyclic 2’,3’-adenosine monophosphate, cyclic 3’.5’-guanosine monophosphate (Fig. 9), without nucleotidase, without substrate of any kind, or without nucleotidase and substrate. When 1.O DIM 5’-adenosine monophosphate was substituted as a substrate, with nucleotidase in the preincubation only, reaction product was scattered throughout the 100-q tissue section. However, if 5’-nucleotidase was omitted from the experiment using 5’-adenosine monophosphate as a substrate, no reaction product was seen, indicating that no endogenous nucleotidase activity survived the aldehyde prefixation (Fig. 10). DISCUSSION This cytochemical study demonstrates cyclic nucleotide phosphodiesterase activity at sites postsynaptic to degenerating corticostriate and thalamostriate projections. Previous studies (15) reported that lesions of these pathways resulted in widespread terminal degeneration in the caudate nucleus. It was also shown that massive midbrain-nigral lesions produce sparse terminal degeneration. Those findings suggested to us that destruction of cortical and thalamic, but not nigral, inputs would produce sufficient terminal degeneration in the caudate neuropil to be compatible with the ultrastructural localization of phosphodiesterase activity. The deposition of reaction product opposite degenerating terminals precludes using phosphodiesterase localization to visualize the “dopamine receptor” (14) sites in the caudate nucleus, as we suggested earlier (3). Additionally, visualization of reaction product postsynaptic to nondegenerating terminals suggests that this cytochemical methodology localizes phosphodiesterase at synapses in the caudate nucleus regardless of the source of afferent endings or the neurotransmitters utilized by these endings. The caudate nucleus is known to contain the highest concentration of brain phosphodiesterase (4). Phosphodiesterase exists in multiple forms in brain tissue (18) and the low-K,, cyclic AMP-specific enzyme is particulate and found in synaptosomal fractions (2, 7, 10). In view of these early reports, it does not seem inconsistent to have localized phosphodiesterase activity in sitl~ at most caudate synapses. By combining terminal degeneration and cytochemistry, we were able to demonstrate phosphodiesterase activity at sites postsynaptic to two major striatal afferent pathways whose neurotransmitter content is unknown. We also found reaction product at many intact synapses in this study which may include afferents from other sites such as the substantia nigra and the intrinsic circuitry. The caudate nucleus contains the highest concentrations of brain dopamine in the nigro-
neostriatal pathway and of acetylcholine in its intrinsic connections (12). Subcellular localization of phosphodiesterase at identifiable clopatninergic and cholinergic synapses would lend the necessary support to our conclusion that this enzyme is active at all synapses in the caudate nucleus. REFERENCES 1. ANDEN, N. E., A. DRHLSTRORI, K. FCSE, AKD K. LARSWX. 1965. Further evidence for the presence of nigro-neostriatal dopamine neurons in the rat. *4~lc,r. J. .-ir~f. 116 : 329-334. 2. APPLEXAN, M. M., W. J. THOMPSOX, AND T. R. RUSSELL. 1973. Cyclic nucleotide phosphodiesterases. ,4dv. Cyclic Nuclcotidc Rcs. 3 : 65-98. 3. ARIANO, hi. A., AND A. hi. ADINOLFI. 1977. Subcellular localization of cyclic nucleotide phosphodiesterase in caudate nucleus. Exp. Nczrvol. 55 : 8494. 4. BREXENRIDGE, B. McL., AXLI R. E. JOHNSTON. 1969. Cyclic 3’,5’-nucleotide phosphodiesterase in brain. J. Histoclrrm. Cytoclrrr~. 17 : 505-511. 5. BROWN, J. H., AND M. H. MAUIAN. 1972. Stimulation of adenylate cyclase in retina1 homogenates and of adenosine 3’.5’-monophosphate formation in intact retina. Proc. Natl. Bead. Sri. USA-1 69 : 539-543. 6. CARPENTER, M. B. 1976. Anatomy of the basal ganglia and related nuclei : a review. A&. Nrnrol. 14: 7-48. 7. DEROBERTIS, E., G. RODRIWE~ DE LORES ARNI.IZ, M. ALBERICI, R. W. BUTCHER, Z\N~ E. SUTHERLAND. 1967. Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex. J. Biol. Chcuz. 242 : 3487-3493. 8. FAHN, S. 1976. Biochemistry of the basal ganglia. .-ldv. Ncwol. 14: 59-89. 9. FLORENDO, N. T., R. J. BARRNETT, ASD P. GREENCARD. 1971. Cyclic 3‘,5’-nucleotide phosphodiesterase : cytochemical localization in cerebral cortex. Scirrzce 173 : 745-747. 10. GABALLAH, S., AND C. POPOFF. 1971. Cyclic 3’,5’-nucleotide phosphodiesterase in nerve endings of developing rat brain. Braiu Rcs. 25 : 220-222. 11. GREENGARD, P., D. A. MCAFEE, AND J. W. KEBABIAN. 1972. On the mechanism of action of cyclic AMP and its role in synaptic transmission. =I&>. Cyclic Nzlclcotide Rcs. 1: 337-355. 12. HORNYIXEWIC~, 0. 1972. Dupamine and extrapyramidal motor function and dysfunction. Rcs. Pubi. =Issoc. Rcs. Ncrv. ilfcut. Dis. 50 : 390-415. 13. KEBABIAN, J. W., AND P. GREENGARD. 1971. Dopamine-sensitive adenyl cyclase: possible role in synaptic transmission. Scicrrcc 174 : 1346-1349. 14. KEBABIAN, J. W., G. L. PETZOLD, AXD P. GREENGARD. 1972. Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain, and its similarity to the “dopamine receptor.” Proc. Nat/. Acnd. Sci. USA 69: 2145-2149. 15. KEiXP, J. M., AND T. P. S. POWELI.. 1971. The site of termination of afferent fibers in the caudate nucleus. Phil. Trcrrrs. R. Sot. Lorzd. B. 262 : 413427. 16. TENNYSON, V. X., R. E. BARRETT, G. COHEX, L. COTE, R. HEII~IULA, AND C. MYTIUNEOU. 1972. The developing neostriatum of the rabbit: correlation of fluorescence histochcmistry, electron microscopy, endogenous dopamine levels, and HZ-dopamine uptake. Brlrirc Rcs. 46 : 2.5-285. 17. TENNYSON, V. M., C. MYTILINEOV, R. HEXKILA, R. E. BARRETT, G. COHEN, 1~. COTE, P. E. DuFE.~, AND L. A. MARCO. 1975. Dopamine-containing neurons of the substantia nigra and their terminals in the neostriatum. Pages 227-264 ire N. A. BUCII\V.~LD AND 51. A. B. BRAZIER, Eds., Brain filccl~ar~isms in Mmtal Rctardafio~t. Academic Press, New York. 18. THOMPSON, W. J., AND M. M. APPLE&TAN. 1971. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biocfze?fzisfry 10 : 311316.
1. Whole 2. Coronal
brain with hemisection
bilateral frontal of diencephalon
cartex suction ablations. showing rostra1 thalamic
Cats were killed electrocoagulative
7 days after lesion placement. lesion. Nissl stain. X7.
428
ARIANO
AND
ADINOLFI
FIG. 3. Intact terminal from caudate nucleus of cat with bilateral frontal pole lesion. Incubated with 0.75 mM cyclic 3’,.5’-adenosine monophosphate. A, Axon terminal. x51,000. FIG. 4. Degenerating thalamostriate terminal in caudate neuropil using 0.75 IBM cyclic 3’,5’-adenosine monophosphate as substrate. XSl,oOO.
CAUDATE
SLTBCELLULAR
PIIOSPIIODIESTERASE
429
reported the cytochemical localization of cyclic nucleotide l~liospl~odiesterase, the catalytic enzyme of cyclic 3’.5’-adenosine n~onophosphate, at postsynaptic sites in intact caudate nucleus. The lack of a suitable method for visualizing brain adenylate cyclase was noted, am1 we suggested that the demonstration of pl~ospliodiesterase activity at asymmetrical axodendritic synapses, some of which represent nigroneostriatal terminations ( 15. 16 j, supported indirectly the “dol~amine receptor” hypothesis. However, in that study we made no attenlpt to distinguish the dopaminergic nigral terminals from those which originate in the cortex or thalamus. Because all afferent fibers synapse asymmetrically on dendrites and dendritic spines in the caudate neuropil, we decided to interrupt the large thalamic and cortical inputs and determine whether or not phosphodiesterase activity would localize postsynaptic to these degenerating terminals. Following incubation of caudate slices taken from these animals with lesions, reaction product was seen postsynaptic to both intact and degenerating endings. The neurotransmitters used by the cortical and thalamic inputs are unknown (8). Thus, our present findings extend cyclic nucleotide phosphodiesterase activity to synaptic sites which are clearly not dopaminergic and demonstrate that the localization of this enzymatic activity does not occur solely at dopamine receptor sites in the caudate nucleus. MATEKIALS
AND
METHODS
Lesions of the corticostriate and thalamostriate projections were made in six adult cats. The frontal poles, including sensorimotor and orbitofrontal regions, were ablated bilaterally in two animals. Large bilateral electrocoagulative lesions destroyed rostra1 intralaminar thalamic nuclei in two animals, and combined cortical and thalamic regions were destroyed unilaterally in two animals. All animals survived for 7 clays. The cats were then anesthetized with intraperitoneal injections of sodium pentabarbitol. Brains were fixed ,in sifu by vascular perfusion with cold 2%) EM-grade glutaraldehyde (Polysciences), 2% paraformaldehyde in 0.05 M sodium cacodylate buffer containing 0.25 M dextrose at pH 7.4. Cytochemical localization of cyclic nucleotide phosphodiesterase activity is based on the sfa.fu nascendi precipitation of lead phosphate, according to the method of Florendo et al. (9), and includes the modifications reported in our previous study of intact caudate nucleus (3). The following control procedures were carried out to verify the specificity of the cytochemical localization method: 50 mM theoFIG. 5. mic lesion FIG. 6. thalamic adenosine
Degenerating terminal in caudate neuropil of cat with bilateral rostra1 thalausing 0.75 m&r cyclic 3’,5’-adenosine monophosphate as substrate. X34000. Degenerating ending within caudate nucleus following a combined unilateral with ipsilateral frontal cortical lesion. Incubated with 0.75 rnhf cyclic 3’,5’monophosphate as substrate. D, Dendrite. X58,000.
430
rZ~ImO
AND
ADIN~LFI
FIG. 7. Terminal degeneration within caudate neuropil after placement frontal cortex-ipsilateral thalamic lesion using 0.5 mAI cyclic adenosine x40.000.
of a uniIatera1 monophosphate.
CAUDATE
SVI3CELLUL.lR
I’IIOSl’IIO~IESTERhSE
431
l~hyllinc (Sigma) or 5mnl. isul~ut~liiietli~l~ai~tliiiic (Aldrich) were added to inhibit phosphodiesterase actil,ity ; q.clic 2’,3’-adenosine monophosphate, 5’adenosine monophosphate, or cJ,clic 3’,5’-guanr)sinc monophosphate were substituted as substrates ; 5’-nucleotidasc was omitted; substrate was omitted ; or substrate and 5’-nucleotidase were omitted. Substrate concentrations of 0.1 to 3.0 mh1 3’,5’-qciic atlenosine monophosphate were utilized. All tissues for electron microscqq were esamined unstained with a Hitachi HS-S electron microscope. The estent of the frontal cortical damage was assessed from whole brains (Fig. l), and the thalamic lesions were verified histologically (Fig. 2) by esamination of Nissl-stained coronal sections. RESULTS Cyclic 3’,5’-adenosine monophosphate is hydrolyzed to S’-adenosine monophosphate by the action of cyclic nucleotide phosphodiesterase. Inorganic phosphate is released by the action of 5’-nucleotidase on 5’-adenosine monophosphate, yielding adenosine and free phosphate. The activity of the membrane-bound, cyclic AMP-specific phosphodiesterase can be cytochemically visualized as an electron-dense, lead phosphate precipitate resulting from the capture of this free phosphate by a lead ion trapping agent (3,9>. Phosphodiesterase activity is visualized postsynaptically at both intact and degenerating terminals in lOO-,~m slices of caudate nucleus taken from animals with large lesions of frontal cortex and rostra1 intralaminar thalamus. Observations were made utilizing the previously determined optimum substrate concentration of 0.75 mnI cyclic 3’,5’-adenosine monophosphate (3)) as shown in Figs. 3-6. Degenerating corticostriate and thalamostriate terminations appear as electron-dense, irregular profiles which synapse asymmetrically on dendrites and dendritic spines. The lead phosphate reaction product consistently accumulates in the region of the postsynaptic density. However, at this substrate concentration background reactivity, defined as the amount of lead phosphate seenat nonsynaptic sites, was unexpectedly elevated. Reduction of the substrate concentration to 0.5 mnf cyclic 3’,5’FIG. 8. Degenerating terminalfrom caudatenucleusof cat with unilateralinterruption of thalamostriateand ipsilateral corticostriate tracts. Substrate concentration of 1.0 mlf cyclic 3’,5’-adenosine monophosphate incubated with 5 rnhz isobutylmethylxanthine was used. A, Axon terminal. X45,000. FIG. 9. Degenerating corticostria,te terminal in caudate neuropil using 0.75 mM cyclic 3’,5’-guanosine monophosphate as substrate. ~31,000. FIG. 10. Thalamostriate terminal degeneration within cat caudate nucleus, utilizing 1.0 rnsx 5’-adenosine monophosphate without .5’-nucleotidase in the eqeriment. D, Dendrite. X.50.000.
432
ARIANO
AND
ADINOLFI
adenosine monophosphate virtually eliminated this background reactivity (Fig. 7). In control sections of caudate incubated with the methylxanthines (isobutylmethylxanthine and theophylline) no lead precipitate could be detected (Fig. 8). Reaction product was also absent in sections which were incubated with cyclic 2’,3’-adenosine monophosphate, cyclic 3’.5’-guanosine monophosphate (Fig. 9), without nucleotidase, without substrate of any kind, or without nucleotidase and substrate. When 1.O DIM 5’-adenosine monophosphate was substituted as a substrate, with nucleotidase in the preincubation only, reaction product was scattered throughout the 100-q tissue section. However, if 5’-nucleotidase was omitted from the experiment using 5’-adenosine monophosphate as a substrate, no reaction product was seen, indicating that no endogenous nucleotidase activity survived the aldehyde prefixation (Fig. 10). DISCUSSION This cytochemical study demonstrates cyclic nucleotide phosphodiesterase activity at sites postsynaptic to degenerating corticostriate and thalamostriate projections. Previous studies (15) reported that lesions of these pathways resulted in widespread terminal degeneration in the caudate nucleus. It was also shown that massive midbrain-nigral lesions produce sparse terminal degeneration. Those findings suggested to us that destruction of cortical and thalamic, but not nigral, inputs would produce sufficient terminal degeneration in the caudate neuropil to be compatible with the ultrastructural localization of phosphodiesterase activity. The deposition of reaction product opposite degenerating terminals precludes using phosphodiesterase localization to visualize the “dopamine receptor” (14) sites in the caudate nucleus, as we suggested earlier (3). Additionally, visualization of reaction product postsynaptic to nondegenerating terminals suggests that this cytochemical methodology localizes phosphodiesterase at synapses in the caudate nucleus regardless of the source of afferent endings or the neurotransmitters utilized by these endings. The caudate nucleus is known to contain the highest concentration of brain phosphodiesterase (4). Phosphodiesterase exists in multiple forms in brain tissue (18) and the low-K,, cyclic AMP-specific enzyme is particulate and found in synaptosomal fractions (2, 7, 10). In view of these early reports, it does not seem inconsistent to have localized phosphodiesterase activity in sitl~ at most caudate synapses. By combining terminal degeneration and cytochemistry, we were able to demonstrate phosphodiesterase activity at sites postsynaptic to two major striatal afferent pathways whose neurotransmitter content is unknown. We also found reaction product at many intact synapses in this study which may include afferents from other sites such as the substantia nigra and the intrinsic circuitry. The caudate nucleus contains the highest concentrations of brain dopamine in the nigro-
neostriatal pathway and of acetylcholine in its intrinsic connections (12). Subcellular localization of phosphodiesterase at identifiable clopatninergic and cholinergic synapses would lend the necessary support to our conclusion that this enzyme is active at all synapses in the caudate nucleus. REFERENCES 1. ANDEN, N. E., A. DRHLSTRORI, K. FCSE, AKD K. LARSWX. 1965. Further evidence for the presence of nigro-neostriatal dopamine neurons in the rat. *4~lc,r. J. .-ir~f. 116 : 329-334. 2. APPLEXAN, M. M., W. J. THOMPSOX, AND T. R. RUSSELL. 1973. Cyclic nucleotide phosphodiesterases. ,4dv. Cyclic Nuclcotidc Rcs. 3 : 65-98. 3. ARIANO, hi. A., AND A. hi. ADINOLFI. 1977. Subcellular localization of cyclic nucleotide phosphodiesterase in caudate nucleus. Exp. Nczrvol. 55 : 8494. 4. BREXENRIDGE, B. McL., AXLI R. E. JOHNSTON. 1969. Cyclic 3’,5’-nucleotide phosphodiesterase in brain. J. Histoclrrm. Cytoclrrr~. 17 : 505-511. 5. BROWN, J. H., AND M. H. MAUIAN. 1972. Stimulation of adenylate cyclase in retina1 homogenates and of adenosine 3’.5’-monophosphate formation in intact retina. Proc. Natl. Bead. Sri. USA-1 69 : 539-543. 6. CARPENTER, M. B. 1976. Anatomy of the basal ganglia and related nuclei : a review. A&. Nrnrol. 14: 7-48. 7. DEROBERTIS, E., G. RODRIWE~ DE LORES ARNI.IZ, M. ALBERICI, R. W. BUTCHER, Z\N~ E. SUTHERLAND. 1967. Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex. J. Biol. Chcuz. 242 : 3487-3493. 8. FAHN, S. 1976. Biochemistry of the basal ganglia. .-ldv. Ncwol. 14: 59-89. 9. FLORENDO, N. T., R. J. BARRNETT, ASD P. GREENCARD. 1971. Cyclic 3‘,5’-nucleotide phosphodiesterase : cytochemical localization in cerebral cortex. Scirrzce 173 : 745-747. 10. GABALLAH, S., AND C. POPOFF. 1971. Cyclic 3’,5’-nucleotide phosphodiesterase in nerve endings of developing rat brain. Braiu Rcs. 25 : 220-222. 11. GREENGARD, P., D. A. MCAFEE, AND J. W. KEBABIAN. 1972. On the mechanism of action of cyclic AMP and its role in synaptic transmission. =I&>. Cyclic Nzlclcotide Rcs. 1: 337-355. 12. HORNYIXEWIC~, 0. 1972. Dupamine and extrapyramidal motor function and dysfunction. Rcs. Pubi. =Issoc. Rcs. Ncrv. ilfcut. Dis. 50 : 390-415. 13. KEBABIAN, J. W., AND P. GREENGARD. 1971. Dopamine-sensitive adenyl cyclase: possible role in synaptic transmission. Scicrrcc 174 : 1346-1349. 14. KEBABIAN, J. W., G. L. PETZOLD, AXD P. GREENGARD. 1972. Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain, and its similarity to the “dopamine receptor.” Proc. Nat/. Acnd. Sci. USA 69: 2145-2149. 15. KEiXP, J. M., AND T. P. S. POWELI.. 1971. The site of termination of afferent fibers in the caudate nucleus. Phil. Trcrrrs. R. Sot. Lorzd. B. 262 : 413427. 16. TENNYSON, V. X., R. E. BARRETT, G. COHEX, L. COTE, R. HEII~IULA, AND C. MYTIUNEOU. 1972. The developing neostriatum of the rabbit: correlation of fluorescence histochcmistry, electron microscopy, endogenous dopamine levels, and HZ-dopamine uptake. Brlrirc Rcs. 46 : 2.5-285. 17. TENNYSON, V. M., C. MYTILINEOV, R. HEXKILA, R. E. BARRETT, G. COHEN, 1~. COTE, P. E. DuFE.~, AND L. A. MARCO. 1975. Dopamine-containing neurons of the substantia nigra and their terminals in the neostriatum. Pages 227-264 ire N. A. BUCII\V.~LD AND 51. A. B. BRAZIER, Eds., Brain filccl~ar~isms in Mmtal Rctardafio~t. Academic Press, New York. 18. THOMPSON, W. J., AND M. M. APPLE&TAN. 1971. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biocfze?fzisfry 10 : 311316.
