EXPERIMENTAL

NEUROLOGY

84-94

55,

(1977)

Subcellular Localization of Cyclic Phosphodiesterase in the Caudate

Nucleotide Nucleus’

M. A. ARIANO 3 AND A. M. ADINOLFI Dcpartmcltts Center,

of Anatomy UGmrsity

and

Psychiatry

and the Mental Retardation Research Los Angeles, California 90024

of California, Received

September

24, 1976

The subcellular localization of cyclic nucleotide phosphodiesterase activity is described in the caudate nucleus and the neocortex of the cat and rat. Reaction product is visualized postsynaptically at asymmetrical axodendritic synapses by the sfatu ~zasccxdi precipitation of lead phosphate. The effect of varying substrate concentrations and incubation times to localize enzyme activity at cortical and caudate synapses in the two species is discussed. Demonstration of phosphodiesterase activity at postsynaptic sites adds further indirect evidence suggesting a role for cyclic nucleotides in synaptic

transmission. INTRODUCTION

Direct visualization of cyclic 3’,5’-nucleotide phosphodiesterase activity is based on the statu nascmdi precipitation of lead phosphate, as described by Florendo and co-workers (10). This cytochemical method has been used with positive results in rat neocortex (lo), developing mouse occipital cortex (1)) and mouse photoreceptor outer segments (23). The present study was undertaken to localize cytochemically the subcellular distribution of cyclic nucleotide phosphodiesterase activity in cat caudate nucleus and to determine whether this enzymatic activity is concentrated at postsynaptic sites in the caudate neuropil, similar to the cyclic nucleotide phosphodiesterase localization reported previously for the superficial neocortex (1, 10). The caudate nucleus was selected because the highest levels of brain phosphodiesteraseactivity have been re1 This study was supported in part by U.S. Public and MH 7097. 2 This work was submitted by M.A.A. in partial for the degree of Doctor of Philosophy in Anatomy at Los Angeles.

Health

Service

Grants

NS

11932

fulfillment of the requirements at the University of California

84 Copyright All rights

0 1977 by Academic Press, of reproduction in any form

Inc. reserved.

ISSN

0014-

4886

PHOSPHODIESTERASE

IN

CATJDATE

85

ported for this region (5) and it has been suggested that a dopaminesensitive adenylate cyclase might mediate dopaminergic transmission in the basal ganglia ( 1s). We believe that the demonstration of phosphodiesterase activity in close proximity to asymmetrical axodendritic synapses, some of which represent nigral terminations within the caudate neuropil (20, 36), will provide the necessary preliminary evidence to further explore cytochemically whether or not the presence of this enzyme will support the suggestion that dopamine action is mediated by cyclic aclenosine monophosphate (AMP). Additionally, demonstration of cyclic nucleotide phosphodiesterase activity at synaptic sites will add to the growing body of indirect evidence suggesting that cyclic nucleotides are associated with synaptic transmission (15, 16, 21, 24, 25). In our first experiments, using cat caudate nucleus and following the previously published methods (1, lo), reaction product could not be localized due to the high amount of background reactivity in the elements of the neuropil. However, we were able to reproduce the results of Florendo ct al. (10) in rat neocortex, as well as demonstrate postsynaptic localization of phosphodiesterase activity in rat caudate nucleus. These observations suggested that some modification of the cytochemical method was needed. By a simple fourfold reduction of the substrate concentration, localization of the phosphodiesterase activity in cat caudate nucleus was achieved. MATERIALS

AND

METHODS

Ten adult cats and three rats were anesthetized with intraperitoneal injections of sodium pentobarbital. Brains were fixed in sifzb by vascular perfusion with coId 3% EM-grade glutaraldehyde (Polysciences), 2% l~araformaldehycle in 0.05 M sodium cacodylate buffer containing 0.25 M dextrose at pH 7.4. The brains were quickly removed, midsagittally sectioned, and placed for 1 hr in fresh, cold fixative. Pieces of caudate nuclei, approximately 3 mm3, were sectioned at 100 pm with a Smith-Farquhar tissue chopper. Cat frontal cortex and rat parietal cortex were also chopped at 100 pm. The slices were collected in cold 0.05 M sodium cacodylate buffer (pH 7.3) containing 0.25 I\I dextrose, and washed 30 min. They were rinsed in a Tris-maleate-sucrose buffer consisting of 60 nlM Trismaleate (Sigma), 2 mnl magnesium chloride, and 0.25 M sucrose at pH 7.4. Slices were preincubated 30 min at room temperature with excess 5’-nucleotidase (30 to 50 units enzyme activity per milliliter, Sigma), in the Tris-maleate-sucrose solution. Tissue was next incubatecl 15 to 60 min at 37°C in solutions containing 0.3 to 3.0 mM 3’,5’-cyclic AMP (Sigma), excess 5’-nucleotidase, and 2 111~ lead nitrate in Tris-maleate-sucrose buffer.

86

ARIANO

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After incubation, the tissue was washed in the buffer, postfixed in 1% osmium tetroxide in 0.05 M sodium cacodylate-0.25 M dextrose buffer (pH 7.4), dehydrated in a graded ethanol series, and embedded in Araldite 502. Thin sections were cut on a Sorval MT-2B ultramicrotome and examined, unstained, with a Hitachi HU-11A electron microscope. The following controls were performed to check the validity of the experimental cytochemical localization : (i) 50 mM theophylline (Sigma) or 5 mM isobutyhnethylxanthine (Aldrich) were added to the incubation to inhibit phosphodiesterase activity, (ii) 5’-AMP (Sigma) was substituted as a substrate, (iii) 2’,3’-cyclic AMP (Sigma) was substituted as a substrate, (iv) 5’-nucleotidase was omitted, (v) substrate was omitted, (vi) 5’-nucleotidase and substrate were omitted, and (vii) the tissue was heated 20 min at 80°C prior to any incubation. The localization of reaction product was considered specific when 3’,5’cyclic AMP was required as a substrate, 50 mm theophylline or 5 mM isobutylmethylxanthine inhibited the reaction, and localization could not be attributed to endogenous 5’-nucleotidase activity (1, 5, 10). RESULTS Cyclic nucleotide phosphodiesterase hydrolyzes 3’,5’-cyclic AMP to 5’-AMP. The activity is visualized cytochemically as an electron-dense, finely granular reaction product which results when inorganic phosphate, formed by the action of 5’-nucleotidase on 5’-AMP, is precipitated with lead ions. In our initial studies using the caudate nucleus and neocortex of rats, this reaction product was localized primarily at postsynaptic sites when the tissues were incubated 30 min with 1.5 or 3.0 mM 3’,5’-cyclic AMP. These studies replicated the localization of cyclic nucleotide phosphodiesterase activity in neocortex by using a 3.0 DlM substrate concentration, as first reported by Florendo et al. (10). We further demonstrated similar sites of enzymatic activity in caudate neuropil, but better localization was obtained with 1.5 mM cyclic AMP (Fig. 1). When sections of the caudate nucleus and neocortex of cats were incubated with 3.0 mM 3’,5’-cyclic AMP 15, 30, or 60 min, the reaction product was concentrated near postsynaptic junctional densities but the background reactivity was high. Background reactivity is defined as the amount of dense precipitate scattered throughout the neuropil. The distribution is random and appears to be greatest close to the edges of the sections. Although this background reactivity varies with the substrate concentrations and times of incubation, the consistent accumulation of lead phosphate precipitate at postsynaptic sites supports the validity of this cytochemical method. This random distribution of visible reaction product might result from movement of released inorganic phosphate during the time needed for

PHOSPHODIESTERASE

FIG.

1. Rat

caudate

nucleus,

incubated

IN

with

87

CAUDATE

1.5 mx

3’,5’-cyclic

AMP.

D-dendrite.

x 71,000. diffusion of the capture reagent and the precipitation of the lead phosphate, as discussed in an earlier study (1). In addition, reaction product accumulates within capillary endothelial cells. This occurs consistently, and the significance of such localization is not known. At 3’,5’-cyclic AMP concentrations of 1.5 m&f incubated 30 min, reaction product was localized at postsynaptic densities throughout the slices of cat caudate nucleus and neocortex (Fig. 2). Background activity was low to moderate, with some mitochondria and capillary endothelium exhibiting reaction product. Concentrations of 0.75 1llM cyclic AMP incubated 30 min gave the most specific localization of cyclic nucleotide phosphodiesterase activity in cat caudate nucleus (Figs. 3, 4) and cat neocortex (Figs. 5, 6). Background activity was extremely low with the reaction product seen almost entirely at postjunctional densities of asymmetrical axodendritic synapses. At substrate concentrations of 0.3 m&r 3’,5’-cyclic AMP, incubated 30 min, localization of the reaction product at postjunctional densities was evident but restricted to a narrow band just below the edges of the sections. Very little reaction product was found beyond this narrow band. Thus, the most reliable results were obtained at 0.75 nlti~ cyclic AMP concentrations. Cyclic nucleotide phosphodiesterase localization in cat caudate nucleus and neocortex using substrate concen-

88

ARIANO

FIG. 2. Cat caudate terminal. X 49,000.

nucleus,

incubated

FIG. 3. Cat caudate terminal. X 51.800.

nucleus,

incubated

AND

ADINOLFI

with

with

1.5 rnhl

0.75

mM

3’,5’-cyclic

AMP.

A-axon

3’,5’-cyclic

AMP.

A-axon

PHOSPHODIESTERASE

FIG.

5. Cat cortex,

incubated

with

0.75 m&f

IN

3’,5’-cyclic

CAUDATE

AMP.

89

D-dendrite.

X 86,300.

90

ARIANO

AND

ADINOLFI

FIG. 6. Cat cortex, incubated with 0.75 mM 3’,5’-cyclic x 86,000.

FIG. 7. Cat cortex, incubated with 0.75 mM 2’,3’-cyclic is visible at synapses. A-axon terminal. X 23,800.

AMP.

AMP.

A-axon

terminal.

No reaction product

PHOSPIIODIESTERASE

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CAUDATE

91

trations of 0.3 and 0.75 m>I suggests that the particulate enzyme in these regions has a high affinity for the cyclic nucleotide. In control sections incubated with the nietli!-lxanthines (theophylline and isobutyhnethylxanthine), no reaction product was observed. No reaction product was seen when the tissue was heated 20 min at SO”C or with -,?’ 3’-cyclic adenosine monophosphate (Fig. 7), without substrate, without 5’-nucleotidase, or without substrate and nucleotidase. When S/-AMP was substituted as a substrate, lead precipitate was seen throughout the sections. It was felt that the addition of 2% paraformaldehyde to the fixative fluid improved the morphology and comparisons with tissue fixed with glutaraldehyde alone showed no differences in localization of cyclic nucleotide phosphodiesterase activity. Use was made of EK-grade glutaraldehyde to avoid possible contaminants, such as phosphate, contained in the 235-nm peak of commercial glutaraldehyde (9). DISCUSSION This study demonstrates the postsynaptic sites of cyclic nucleotide phosphodiesterase activity in the caudate nucleus and neocortex of the cat and rat. Biochemical studies on the subcellular distribution of kinetically distinct phosphodiesterase enzymatic activities have demonstrated that, in brain, the enzyme with a high substrate affinity is found in the particulate fraction, whereas the enzyme with a lower affinity for 3’,5’-cyclic AMP is soluble. The particulate activity is associated with synaptosomal fractions (4, 8, 11, 27). The cyclic nucleotide phosphodiesterase activity that can be visualized cytochemically at postjunctional densities in these and earlier (1, 10) studies of the central nervous system probably represents a portion of the high-affinity enzyme. This suggestion is supported by the selective postsynaptic localization achieved by reducing the substrate concentrations. At low substrate concentrations, it is presumed that hydrolysis by the particulate enzyme with a low K, yields the visible reaction product. However, when substrate concentrations are reduced beyond the apparent optimum (0.$5 m&f) concentration to 0.3 mM, visualization of the reaction product becomes difficult and is restricted to a narrow band close to the surface. The limits of this cytochemical method do not permit direct correlation of the enzyme activity seen postsynaptitally at asymmetrical axodendritic synapses in caudate nucleus and neocortex with the high-affinity, low molecular weight enzyme determined biochemically (4, 27). Substrate concentrations used in routine biochemical assays vary from 1 m&l (total phosphodiesterase) to less than 0.1 PM. In this study, the lowest substrate concentrations were 300 to 750 p>l. However, despite these high substrate concentrations, it seems

92

ARIANO

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ADINOLFI

unlikely that any activity other than that of the me~~~l)rane-l~ound, highaffinity enzyme (S, 11) could be consistently localized in tissue s&ions. The cyclic nucleotide phosphodiesterase activity seen at postsynaptic sites in the caudate nucleus supports to some extent the concept of a “dopamine receptor” in this region. Approximately 80% of the total brain

dopanline

is contained

within

the nigroneostriatal

pathway

(13).

Dopamine in the substantia nigra is concentrated in cell bodies, and dopamine in the neostriatum is present in very fine, varicose nerve fibers throughout the neuropil (2, 3). Both nigral stimulation and microiontophoretic application of dopamine evoke excitatory and inhibitory responses in caudate units, indicating that dopaminergic mechanisms are an integral part of neuronal firing patterns within the caudate nucleus (7, 14). However, the precise mode of action of the nigral amine at the receptor level is unknown. Recently, a dopamine-sensitive adenylate cyclase has been identified in the superior cervical ganglion (17)) in the inner retina (6)) in the caudate nucleus (IS), and in the substantia nigra (19). These studies suggest that adenylate cyclase might act as a “dopamine receptor” and that cyclic nucleotides, generated by this enzyme activity, might mediate catecholamine-induced changes in postsynaptic neurons (12, 21, 22). Although no reliable method for the ultrastructural localization of brain adenylate cyclase activity has been reported, the cytochemical visualization of cyclic nucleotide phosphodiesterase activity at axodendritic synapses supports the suggestion of a “dopamine receptor” within the caudate nucleus. However, a definitive demonstration of the involvement of cyclic nucleotide phosphodiesterase awaits a methodology that will selectively mark dopaminergic terminals in Z&W and is compatible with the in z&o localization of phosphodiesteraseactivity. This is important because there are major projections from the thalamus and the cerebral cortex which also form asymmetrical axodendritic synapses on caudate neurons (20). The neurotransmitters used by these inputs are unknown and we cannot eliminate the possibility that cyclic nucleotides might also mediate their actions. REFERENCES 1. ADINOLFI,

A. M.,

AND

S. Y.

1974. Cyto:hemicaf

SCHMIDT.

localization

nucleotidephosphodiesterase activity at developingsynapses.Brain 21-31. 2. ANDEN, N. E., K. LARSSON. mine neurons.

3. ANDEN,

N. GERSTEDT.

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1966.Ascendingmonoamineneuronsto cephalon.Acta Physiol. Stand. 67: 313-326.

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4. APPLEMAN, M. M., 1%‘. J. T~~onrrsos, ;\m T. R. RUSSELL. 1973. Cyclic nucleotide phosphodiesterases. Pages 65-98 ill P. GREENGATIDAXD G. ~1. R~BIS~X, Eds., &wrwrs ix Cyclist A:lrc-htidr ~~rscwc~h. I’ol. 3. Raven Press, New York. 5. BRECKENRIDGE, B. McL., AND R. E. JOHNSTON. 1969. Cyclic 3’,5’-nucleotide phosphodiesterase in brain. J. Hisforkcnc. Cytochcw. 17 : SOS-51 1. 6. BROI~N, J. H., AXD M. H. MAKMAN. 1972. Stimulation of adenylate cyclase in retinal homogenates and of xdenosine-3’,5’-monophosphate formation in intact retina. Proc. Not. Amd. Sri. liS&-i 69: 539-W. 7. CONNER, J. D. 1972. The nigro-neostriatal pathway: The effects produced by iontophoretic dopamine. Xcs. I’trb. .-fssor.. iZrs. Ncrrl. Afrrrf. LG. 50 : 193-206. 8. DEROBERTIS, E., G. RODRI~XEZ DE LORES ARX Arz, hI. ~%I.BEI~I, R. W. BUTCHER, AKD E. SUTIIERLAXD. 1967. Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex. J. Viol. Chrw. 242: 3487-3493. 9. ESSNER, E. 1973. Phosphatases. Pages 4476 ilr M. A. HAYAT, Ed., Electron ~Vicrasc-opll af EPICS. I-o/. 1. Van Nostrand Reinhold, New York. 10. FLORENDO, N. T., R. J. BARRXFXTT, AND P. GREESGARD. 1971. Cyclic 3’,5’-nucleotide phosphodiesterase : Cyto:hemical 1o:alization in cerebral cortex. S’ricwe 173 : 745-747. 11. GARAI.I.AIT, S., AND C. POPOFF. 1971. Cyclic 3’,5’-nucieotide p!losphodiesterase in nerve endings of developing rat brain. nrairt Res. 25: 220-222. 12. GREENCARD, P., D. .4. MCAFEE, ASD J. ‘cz’. KEBABIAN. 1972. On the mechanism of action of cyclic AMP and its role in synaptic transmission. Pages 337-355 iit P. GREEXC,ARDAND G. A. ROBI~OX, Eds., ~~~W~KCS iI1 c~dic NttcLotidc Rcscarrk, T-01. 1. Raven Press, New York. 13. HORNYICIE\VICZ, 0. 1972. Dopamine and extrapyramidal motor function and dysfunction. Rcs. Pub. z4ssoz. Rrs. Ncru. Alrnt. Dis. 50: 390-415. 14. Hur.r., C. D., G. BEL~SAI~DI, AXD N. A. BCCH\VALD. 1970. Intracellular responses of caudate neurons to brainstem stimulation. LIrain Rss. 22: 163-179. 1.5. KAKIUCHI, S., T. W. RAI,L, ASD H. MCIL\~AIX. 1969. The effect of electrical stimulation upon the accumulation of adenosine 3’,5’-phosphate in isolated cerebral tissue. J. NC~ITOT~E~. 16 : 485-491. 16. KEBABIAN, J. W., F. E. BLOOM, A. L. STEINER, AND P. GREENGARD.1975. Neurotransmitters increase cyclic nucleotides in postganglionic neurons : Immunocytochemical demonstration. Srirwc 190 : 157-159. 17. KEBABIAN, J. W., AXD P. GREENGARD. 1971. Dopamine-sensitive adenyl cyclase: Possible role in synaptic transmission. Srimrc 174 : 1346-1349. 18. KEBABIAN, J. W., G. L. PETZOLLI, AND P. GREENGAKD. 1972. Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain, and its similarity to the “dopamine receptor.” Pror. h’at. -4cad. Sri. US24 69: 2145-2149. 19. KEBABIAN, J. W., AND J. M. SAAVEDRA. 1976. Dopamine sensitive adenylate cyclase occurs in a region of substantia nigra containing dopaminergic dendrites. Scic>~cc 193 : 683-685. 20. KE&lP, J. M., AND T. P. S. POWELL. 1971. The site of termination of afferent fibers in the caudate nucleus. Philos. Tmrrs. R. Sot. Lo& (Biol.) 262: 413427. 21. MCAFEE, D. A., hI. SCHORDERET,AXD P. GREENCARD. 1971. Adenosine 3’,5’monophosphate in nervous tissue, increase associated with synaptic transmission. S&we 173 : 1156-1158. 2.2. RALJ,, T. W., AND A. G. GILMAh: 1970. The role of cyclic AMP in the nervous system. Neurosci. Rcs. Prog. Bull. 8: 220-323.

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23. R~BB, R. M. 1974. Histochemical evidence of cyclic nucleotide phosphodiesterase in photoreceptor outer segments. Imjc~t. Oplztltahriol. 13 : 740-747. 24. SIGGINS, G. R., B. J. HOFFER, AND U. UNGERSTEDT. 1974. Electrophysiological evidence for involvement of cyclic adenosine monophosphate in dopamine responses of caudate neurons. Life Sci. 15: 779-792. 25. SIGGINS, G. R., A. P. OLIVER, B. J. HOFFER, AND F. E. BLOOM. 1971. Cyclic adenosine monophosphate and norepinephrine : Effects on transmembrane properties of cerebellar Purkinje cells. Science 171: 192. 26. TENNYSON, V. M., R. E. BARRETT, G. COHEN, L. COTE, R. HEIKKILA, AND C. MYTILINEOU. 1972. The developing neostriatum of the rabbit: Correlation of fluorescence histochemistry, electron microscopy, endogenous dopamine levels, and (3H) -dopamine uptake. Bruin Res. 46 : 251-285. 27. THOMPSON, W. J., AND M. M. APPLEMAN. 1971. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochenzistry 10 : 311-316.