ONTOGENESIS OF DOPAMINERGIC-CHOLINERGTC INTERACTIONS IN THE RAT STRIATUM: A NEUROCHEMICAL STUDY J. T. CovLr and P. CAMPOCHIARO Departments of Pharmacologj and Experimental Therapeutics and Psychiatry and the Behavioral Sciences. The Johns Hopkins University School of Medicine, Baltimore. MD 21 205, U.S.A. (Rrcrived 23 October. 1975. Accepted 25 Fubruary 1976)
Abstract-In the striaturn of the newborn rat. the activity of tjrosine hydroxylase. the concentration of dopamine and the activity of the synaptosomal high-affinity uptake process for doparnine is IOU;, of that of the adult; there is a linear and closely associated increase in all three parameters during maturation. achieving 7-59; of adult levels by 4 weeks after birth. In contrast. the specific activity of choline acetyltransferase exhibits a more delayed developmental rise commencing 1 week after birth: the concentration of acetylcholine is disproportionately high in the neonatal striatum and precedes the developmental increase in the activity of choline acetyltransferase. At birth, the specific activit) of dopamine-sensitiveadenylate cyclase is 20% of that of the adult striaturn and achieves adult activity by 4 weeks after birth. Pretrcatment with the neuroleptic, fluphenazine. does not reduce the striatal content of acetylcholine until 8 days after birth. It is postulated that dopaminergic influences on cholinegic neuronal activity appear when the cholinergic neurons in the striatum cease dividing and start differentiating.
THEDOPAMINERGIC neurons, the cell bodies of which (MCGEERP t ul., 1971; BUTCHER& BLITHER,1974). are localized in the substantia nigra, provide an ex- Based on ultramicroscopic characteristics. it has been tremely dense innervation to the striatum estimated suggested that certain small interneurons that poputo be 15% of all nerve terminals in the region (UNGER- late the striatum are cholinergic (BAK et d.,1975). STEDT, 1971 ; HOKFELT rt al.. 1970). Electro-physioloOf the many types of striatal neurons on which the gic studies utilizing extracellular recordings indicate dopaminergic terminals synapse, there is strong evithat the dopaminergic innervation acts predomi- dence that the dopaminergic neurons modulate the nantly as an inhibitory influence on the firing rate activity of the striatal cholinergic neurons. The sysof striatal neurons (CONNOR,1970; MCLENNAN& temic administration of neuroleptics results in deYORK, 1967). A dopamine-sensitive adenylate cyclase creases in the levels of endogenous acetylcholine and has been demonstrated in the corpus striatum and increases in its turnover whereas dopamine receptor in other regions of the CNS that are innervated by agonists cause variable increases in the level of acetylcholine and decreases in the rate of turnover of the dopaminergic neurons (KEBABIAN rt al., 1972). This cyclase possesses many properties that would be neurotransmitter (SETHY & VAN WOERT, 1974; er ul., 1975a; TRABUCCHI et ul.. 1971. 1975). expected for the ‘dopamine receptor’ as characterized GUYENET Because of the modulatory influence the extrinsic by previous pharmacologic and physiologic studies; i t is responsive to low concentrations of dopamine dopaminergic neurons exert on the cholinergic and is inhibited by neuroleptic drugs including neurons intrinsic to the striatum, this neuronal relaphenothiazines. butyrophenes and thioxanthines tionship would be an excellent model system for exa(KEBABIAN rt nl., 1972; MILLER P t nl., 1974; CLEMENT- mining the biochemical aspects of the development CORMIER rt d., 1975). The recent demonstration that of neuronal innervation in the central nervous system. iontophoretically applied adenosine 3’5’-cyclicmono- In this study we have examined the ontogenesis of phosphate (cyclic AMP) produces electrophysiologic several parameters associated with the dopaminergic effects similar to dopamine in the striatum terminals and the cholinergic neurons in the striatum. strengthens the inference that dopamine-sensitive In addition, the development of dopamine-sensitive adenylate cyclase mediates the effect of dopamine on adenylate cyclase is compared to the onset of the inhibitory control of the dopaminergic neurons on the rr al., 1975). the post-synaptic neurons (SICGINS Since lesioning of the major afferents to the stria- cholinergic neurons. tum has negligible effects on the activity of choline METHODS acetyltransferase [EC 2.3.1.6; Acetyl-CoA-choline 0-acetyltransferase] in the region, the cholinergic Prepnmtioit of tissues. Sprague-Dawley rats. spermneurons appear to be intrinsic to the striatum positive on a specific date. were obtained from Hormone 673
674
J. T. COYLFand P. CA~IPO(.HI.AKO
Assay Laboratories (Chicago1 and a c r e housed in separate cages after I6 days of gestation. A t birth. the litters were reduced to 8; and runts Nere discarded. For all assays except the determination of the levels of acetjlcholine. the rats were killed hq decapitation: and broin tissues ivere dissected on a n aluminum block resting on ice. For the measurement of hasal levels of acet!lcholine. the rats were killed hy microwave irradiation focused on the skull ( 13W W: 2450 MHz) in an oven adapted b) Medical Enginecring Consultants (Lexington. MA): optimal exposure varied for each age. The striatum is defined as the tissue that lies anterior to an imaginar) line drawn perpendicular to the optic chiasm and intersecting the fornix: the lateral limits are the corpus callosum: the septum w a s removed tnediall) as previously described b) G L O W I U K &I IVEKSLS (19661. 7 j m s i r i u hpfrozyltrsc~rrssu!.. The activit) of tjrosine h!droxylase [EC 1.14.16.2: L-tlrosinc. tetrati!dropteridine: oxygen oxidoreductase (3-h!drox)lating)] was measured in 50 ktl portions of striatum homogenized in 20 vol. ( n t \@I.) of 0.05 M-Tris-HCI. pH 7.3. containing 0.2"" ( v v ) Triton X-100 (Packard Instrument Co.. Downers Grove. I L ) at a final concentration of 0.1 niwtyrosine according to thc method of CouLt (1972). Cholirir crcet!~lt~ari.~li.~u-usr usstry. The acti\ it) of choline acetyltransfersse was measured in 5 to 15 j t l portions of striatum homogenized i n 20 \ol. (at vol.) of the 0.05 TrisHC1-Triton buffer according to the method of Bi LL & ODERFELD-NOWAK ( I97 I ). Doyarriirrc.-serisiti~rudtvi i - h t c c~yc/u.sc~ t t ~ . v r I ~Fresh11 . dissected striata were homogeniLed in 25 or 50 \ol. of 3 m w Tris-acetic acid buffer. pH 7.3. containing 3 mu-eth! lene glycol his (P-aminocth!l) .Y..Y'-tetraacetic acid. Fift! pl of the whole homogenate was assabed in a total vol. of 300 pI according to the method of KEBABLA\: L'r ul.. (1972). After 2.5 min incubation at 30 C. the reaction w a s terminated by heating the assaj mix for 5 min in a water bath a t 95.C. The adenosinc 3'5'-c)clic monophosphatc ( q c l i c AMP) formed was measured b! the competitive binding assay of BKOWY c't u/.. (1971). Basal acti\it!. activit) in the presence of dopamine (50 pv).and activit! in the presence of dopamine (50 p ~ plus ) Huphenazinc (5 p i ) \\ere measured: all assaqs ~ v e r eperformed in triplicate. Eritfoqiwoirs dopcrriiirit,. The concentration of dopamine was measured i n striata homogenized in 100 vol. ( u t vol.) of 0.1 x-perchloric acid according to the radiometric enzymatic assay of COYLE& H F N R(1973). ~ Eridoyrrious r r ~ ~ e r ~ . / ~ / r o l i rThe r e , . conct'iitra tion of acet)lcholine in formic acid-acetone extracts of striatum a-as measured b! the enzjmatic radiometric assa! of Goi.rink~c; & MCCAMAN (1973). Uptakr of' [3H]dopu~m~~t. Fresh striata here Iiomogenized in 20 vol. ( a t . vol.) of 0.32 wsucrosc: a n d P I fractions were prepared according to thc method of WHITTAKER (1965). The P, fraction was resuspended in the original honiogenizin_e \ olume of sucroce. Preparation for assaqs of the striatal homogenates for all ages were performed with the u m e homogenizer and pestle and assaJed together on the same da! to insure kalidit) of comparisons. Portions of the homogenate (25 or 5 0 pl) were incubated i n a total volume of 1 ml of Krebs-Ringer buffer containing 5 x It)-' M ['H]dopainine (10 Ci mmol: Sen- England Nuclear Corp.: Boston. MA) for 5 min as dexribed by H0i.i & COYLE(1974). Synaptosomal uptake \\as tcrminated by immersion of tubes in an ice.\\ater bath and centrifugation at 5000 gd, x 10 min a t 5 C to sediment the sqnaptosomes. The pellet was superficiall> rinsed rvith
4 ml of ice-cold Krehs-Ringer buffer and then soluhilized with I nil of Protosol (New England Nuclear Corp.); and radioactivity was measured by liquid scintillation spectrometr!. Under these conditions. >95",, of the tritium accumulated i n the P , fraction co-chromatographs with dopamine (HOLZ8r Coi Lf . 1974).
RESIJLTS
Deidoprricvir
of' doptruiiiiergic
irinercution
r o the
srritrrrrrii
TJ rosine hydroxylase, the initial and rate-limiting enzJnie in the synthesis pathway for dopamine, is present in the neonatal striatum with a specific activity of IO",, of that of the adult (Fig. 1). It exhibits a relatively linrar increase in specific activity during the 1 weeks after birth. attaining 75';" of the adult specific activity. The end product of the synthetic pathwa! . dopamine. is readily detectable in the neonatal striatuin with a concentration of 12% of that of the adult. Similar to tyrosine hydroxylase. the concentration o f dopamine increases in a linear fashion. attaining nearl) 70",, of that of the adult level by 4 necks after birth. Prey ious studies have shown that [ Hldo paminc taken u p by particulate material in sucrose homogenates of brain is highly localized t o synaptosoinal fractions (COYLL& SNYDEK.1969); therefore, we essmined the uptake or [3H]dopamine into resuspcnded P2 Itactions. The initial uptake of ['Hldopamine into qnaptosomes is lo",, of that of the adult ;tt hirth. The relative activity of the uptake process increases in a linear fashion attaining 75"/,, of that of the adult by 1 weeks after birth. Kinetic studies of ['Hldopamine uptake into homogenates prepared from neonatril and adult striatum indicate that the
W
0
5
I. 1 /'
I
7
14
21
28
Adu
Days (Post-Parturn) FIG. I . De\elopment of dopaminergic innervation in the striatum. The actnitq of tqrosine hydroxylase, the concentration of dopamine and the activity of the synaptosomnl uptakc process for dopamine were measured in striatum as described in the Methods. Results are expressed in tcrms 0 1 nig n s t \vt. or tissue. Each point is the mean or four or more separate preparations: and S.F M. are C- 10"~;.
Ontogeny of DA-Ach interaction in striatum
615
increase in the specific activity of choline acetyltransferase. Development of dopamine-serisitive adenylate cyclase in striaturn
Days ( Pos1- Part urn)
FIG. 2. Development of cholinergic neurons in the striatum. The activity of choline acetyltransferase and concentration of acetylcholine in the striatum were measured as described in the Methods. Results are expressed in terms of mg wet wt of tissue. Each point is the mean of five are I 10%. or more separate preparations; and S.E.M.
increase in uptake during development reflects an increase in V,,, whereas the K , does not change significantly (not shown). Dei~e!opmeizta/changes in cholinv acetyltransjerase and acetylcholine in the striatum The specific activity of choline acetyltransferase is 25; of that of the adult in the neonatal striatum; and during the first 7 days after birth, it increases only another 2% (Fig. 2). Between 1 and 4 weeks after birth, there is a linear, 20-fold increase in the specific activity of choline acetyltransferase such that 70% of the adult activity is attained by the end of this period. The concentration of acetylcholine in the neonatal striatum is quite high, being 23% of that of the adult. Like the transferase, there is only a modest 2% increase in the concentration of the neurotransmitter during the first week after birth. However, subsequent to 1 week after birth, there is a linear increase in the concentration of acetylcholine, which attains adult levels by 4 weeks after birth. Notably, the developmental rise in acetylcholine precedes but parallels the
In cell-free homogenates prepared from the neonatal striatum, dopamine produces a significant stimulation of the formation of cyclic AMP that is completely reversed by fluphenazine (Table 1). The absolute amount of cyclic AMP formed is 20% of that measured in the adult, although the stimulation by dopamine over basal activity is twice as high in the neonate as compared to the adult striatum. The specific activity of the dopamine-sensitive adenylatc cyclase increases rapidly, attaining adult activity by 3 4 weeks after birth. Whereas the dopamine-sensitive cyclase increases 5-fold in activity from birth to adulthood, the basal activity increases 10-fold. It is noteworthy that the developmental rise in the activity of dopamirie-sensitive adenylate cyclase parallels but clearly precedes the developmental increase in the three presynaptic markers for dopaminergic neurons: tyrosine hydroxylase, endogenous dopamine and synaptosomal uptake of C3H]dopamine. Effects uf‘ ,fluphenazine on concentration of’ acetylcholine in developirtg striatuni
To determine when dopaminergic afferents to the striatum develop a functional control over the cholinergic neurons, the effects of the neuroleptic, fluphenazine, on the levels of acetylcholine in the striatum of rats of various ages were examined. Rats were administered fluphenazine ( 5 mg/kg) subcutaneously or an equivalent volume of vehicle, killed 30 min after injection and the concentration of acetylcholine in their striatum was measured. In the case of the adult, treatment with fluphenazine results in a 38% decrement in the concentration of acetylcholine in the striatum (Table 2). In contrast, fluphenazine has no significant effect on the levels of acetylcholine in the striatum of the newborn or 5-day-old rat. Eight days after birth is the first time that treatment with fluphenazine results in a significant decrement in the levels of acetylcholine. The fall in the levels of acetylcholine is 26% at this stage of development and increases to 36”/, by 17 days after birth.
TABLE1. DEVELOPMENT OF DOPAMINE-SENSITIVE Age Newborn ( 3 ) 8 Days ( 3 ) 15 Days (3) 21 Days ( I ) 28 Days (3) Adult (3)
+ fluphenazine 1.0 + 0.4
Dopamine
2.0 k 0.2 3.8 k 0.2 4.5 6.6 & 0.6 10.2 k 1.9
ADENYLATE CYCLASE IN THE STRIATUM
Dopamine
A
3.0 f 0.3 6.4 k 0.8 8.6 0.2 14.1 18.8 k 1.2 20.2 2.3
2.0 0.2 4.4 k 0.8 5.1 k 0.9 9.6 12.2 & 0.6 10.0 & 1.1
*
+
*
‘?A
Stimulation
200 220 126 213 185
98
Cell free homogenates prepared from striata of rats of various ages were assayed for dopamine-sensitive adenylate cyclase as described in the Methods; the concentrations of dopamine and of fluphenazine are 50 pM and 5 p ~ respect, ively. Each preparation was assayed in triplicate; the number of separate preparations is in brackets. Results are expressed in terms of pmol cyclic AMP formed per mg tissue during 150 s incubation S.E.M.
*
676
J. T. COYLL and P. CASIPOCHIAKO
The development of the presynaptic neurochemical markers for the GABAcrgic neurons in the striatum, which appear to be predominantly local circuit Age Control Fluphenazint: neurons like the cholinerpic neurons, also exhibit a disproportiotiality between the levels of neurotratistoo 9 101 4 Newborn ( 5 ) loo* h 119 9 5 days (10) mitter and the activity of its biosynthetic enzyme 7 1 3* 8 Days ( 5 ) 100 1 early in development (COYLE & ENNA, 1975). Since 61 6* 17 Days ( 5 ) I00 9 in the striatum peaks cell division of the interneurons I00 s 62 lr Adult (5) at 21 dajs of gestation of the rat and continues for 1974; DAS & ALTRats of various ages were administcrcd fluphenazine ( 5 several days after birth (CREEPS. mgjkg) or equivalent volume of vehicle b> subcutaneous XIAS. 1970; SCHCLTZE ct al.. 1974). the rise in activity injection; to preclude hgpothermia. immature rats here of choline acctyltransferase may be ii more reliable maintained at 35 C. Thirtl min after treatment. the rats index of the ratc of differentiation of the cholinergic were killed and the content of acetylcholinc i n the striiita was measured. Results are expressed in terms of percent neurons in contrast to the levels of acetylcholine. Tisof vehicle treated controls S.E.V. The number of rats i n sue culture studies on clones of neuroblastoma supeach experimental group is in brackets. port this hypothesis in that the activity of the enzymes * P 5 0.01. responsible for neurotransmitter synthesis rise dramatically when the neuroblastoma cells cease dividing DISCUSSION and commence differentiating (AIVIANOet ( I / . . 1971; Since the cell bodies of the dopaminergic neurons RICHELSON. 1973). The dopamine-sensitivc adetiylate cyclase has a are extrinsic to the striatum (UNGERSTEDT. 1971). the appearance and ontogenetic changes in the levels of predominantly. if not exclusively, post-synaptic localineurochemical markers for the dopamincrgic neurons zation (MISHKAor d.. 1974). Although indirect eviin the striatum would reflect the ingrowth and pro- dence suggests that the cholinergic neurons possess dopamine receptors. the diffuse innervation by the liferation of their terminals. At birth of the rat. the levels of dopamine and the activitj of tyrosine 11)- dopaminergic neurons on ultrastructurally different droxylase and of the synaptosomal uptake process for neuronal tqpes implies that other neiirons in the striatum possess receptors for dopamine (BAKet d., 1975). dopamine are ICI"~, of that of the adult. During the subsequent 4 weeks of postnatal maturation. all three In the neonatal striatum. the specific activity of the parameters increase in remarkably close association dopamine-sensitive adenylate cyclase is 2000 of that achieving approx 750, of that of the adult. A similar of the adult and increases progressively to adult levels close temporal association i n the development of pre- b j 3-4 weeks after birth. The rise in the activity of synaptic markers for the noradrenergic neurons in the dopamine-sensitive adenylate cyclase precedes but central nervous system has been previously denion- parallels the developmental rise in the prcsynaptic strated (CoYLt & AXELROD. 1971. 1972u.h: Coirt & neurochemical markers for the dopaminergic terH t w Y . 1973). These neurochemical observations are minals. Thus. the development of the dopamine recepin close agreement with the semiquantitative results tor precedes the ingrowth of dopaminergic terminals. from histofluorescent microscopic studies on the de- This observation concerning the appearance and invelopment of dopaminergic innervation to the rat crease in receptor sites prior to the development of striatum. At birth. patches of fluorescent terminals are presynaptic input is in accordance with neurophysiopresent in the striatum that become progressivelj logic observations on the development of nicotinic confluent and achieve an intensit) of fuorescence cholinergic receptors on myoblasts (FAMBROUGH, near that of the adult by 4 weeks after birth (LOIZOL. 1974). the p receptor on cerebellar Purkinje cells 1972; OLSON et d.. 1972). cr d.. 1971) and neurochemical studies (WOODWARD In contrast to the dopaminergic neurons. the cho- on the development of the GABAergic receptor in linergic neurons appear to be small interneurons with the rat cerebellum ( C o Y L t & ENNA,1975) and muscell bodies and terminals completely limited to the carinic cholinergic receptor in the striatum (Coyle & striatum (BAK e r d.. 1975). Thc specific activity of Yamamura. in preparation). Similarly, in the chick choline acetyltransferase is quite low at birth and retina. dopamine-sensitive adenylate cyclase appears remains depressed during the first postnatal week: it several days before the dopaminergic neurons begin then increases rapidly in activitj during the sub- to differentiate (Coyle gi Schwarcz, in preparation). sequent 3 weeks, attaining 70"" of the adult activity. Extracellular recordings demonstrate that the ionThere is a marked disparity between the Icvcls of ace- tophoretic application of dopamine on to neurons in tylcholine and the activity of choline acetlltransferase the striatum causes inhibition of firing of a tnajority in the neonatal striatuni with the concentration of of them; and it is significant that, in light of the eviacetylcholine 25", of that of the adult whereas the dence that dopamine-sensitive adenyla te cyclase may specific activity of choline acetyltransferase is only 3",, be the dopamine receptor, that iontophorrtic appliof adult at this time. The developmental incrcases in cation of cyclic AMP also inhibits the firing of these the concentration of acetylcholine parallel but pre- neurons ( CONNOR.1970: SIGGINS clt d . . 1975). Concede the rise in activity of choline acetyltransferase. versely. blockade of dopamine receptors by systemic TABLF2 EFFECT OF
FLLPHFNAZN o\ A C E T I L C H O L I ~ E LF\. t L S I\ THF STKIATL \I
* ** *
** ** *
Ontogeny of DA-Ach intrraction in striatiim administration of neuroleptic drugs results in increased rates of firing (CONNOR. 1970). Of the many types of striatal neurons on which the dopaminergic terminals synapse, there is strong evidence that the dopaininergic terminals modulate the activity of the striatal cholinergic interneurons. The systemic administration of dopamine-receptor blockers results in decreased levels of endogenous acetylcholine and increases in the turnover of acetylcholine in the striaet al.. tum (SETHY& VAN WOERT.1974; GUYENET 1975a: TIUBUCCHI et al., 1974). The effect of neuroleptics on the levels of acetylcholine is presumably due to blockade of dopamine receptors resulting in disinhibition from the tonic dopaminergic input. In our studies, pretreatment with fluphenazine results in a 38",, decrement in the concentration of acetylcholine in the striatum of the adult rat. In contrast, fluphenazine administration has no significant effect on the levels of acetylcholine in the striatum of newborn or 5-day-old rats. Eight days after birth is the first time that iluphenazine treatment results in a significant effect on the concentration of acetylcholine in the striatum; and this effect is apparent at later stages of development. These results are in close agreement with those of GUYENLT et al.. (l975b) on the effects of other neuroleptics on the levels of acetylcholine in the striatum of the developing rat. These studies indicate that there is a significant disparity between the development of dopaminergic innervation and dopamine-sensitive adenylate cyclase in the striatum and the appearance of sensitivity of cholinergic neurons to blockade of dopamine receptors. The lack of response of striatal cholinergic neurons to fluphenazine until 8 days after birth does not appear to be due to insufficient activity of the dopaminergic afferents to the striatum s'ince KELLERet al., (1973) have shown that the turnover of dopamine is quite high in the neonatal striatum and responds appropriately to stimulation or blockade of dopamine receptors. This change in response of the cholinergic neurons in the striatum to blockade of dopamine receptors may indicate that there is a delay between the time of appearance of the dopamine receptor on the neuron and the time when it assumes a role in regulating the electrical activity of the cholinergic neurons. An alternative and more parsimonious explanation is that the dopamine-sensitive adenylate cyclase activity mcasured in the striatum during the first week after birth is localized on other neuronal and non-neuronal cells; and that the cyclase appears on the cholinergic neurons when they cease dividing and start differentiating at 8 days after birth. Thus, the appearance of filnctional control of the dopaminergic input over the striatal cholinergic neurons coincides with the time when the activity of choline acetyltraiisferase begins to increase at a precipitous rate.
677
RHODESfor their secretarial assistance. P. CAMPOis a Year 111 Medical Student at Johns Hopkins School of Medicine. This research was supported by
VrKiE
CHIAKO
USPHS Grant DA 00266. REFERENCES AMANOT., RICHELSON E. & NIRENBERG M. W. (1972) Proc. iiatri. Acnd. Sci., U.S.A. 69. 258-263. BAK I. J., CHOIW. €3.. HASSLER R., USUNOFFK. G. & WAGNEK A. (1975) in Advatices iii Neurologjy (CALNE D. B.. CHASE, T. N. & BARBEAUA.. eds.) Vol. 9. pp. 25-41. Raven Press, New York. BKOWNB. L., ALBANO. J. D. M., ELKINSR. P.. SCHERZI A. M. & -fAMl'lON w. (1971) Biochem J . 121. 561-561. B L ~ LG. L & ODERFELD-NOWAK B. (1971)J . Neurocheiri. 19. 935-947. S. G. & BUTCHERL. L. (1974) Bruin Rrs. 71. BUTCHER 1 67-1 7 I .
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22. 147-151. HOLZR. W. & COVLEJ. T. (1971) Molec. Phurri~uc. 10. 74C758. K F B A H I A N J. w., PtTZOLD G. L. & GREENGARD P. (1972) Proc. iiatii. Acad. Sci., U.S..-L. 69. 2145-2149. KELLER H. H., BARTHOLINIG. & PLETSCHER A. (1973) Braiii Res. 64. 371-378. Loizou L. A. (1972)Bruin Res. 40. 395-418. H. C. & WICKSON MCGEER P. L., M c C t t ~E. G., FIBIGEK V. (1971) Brtriii Res. 35. 308-314. MCLENNAN H. & YORK D. H. (1967) J . Pl~ysiol.,Lorid. 189. 393-402.
R. J., HORNA. S. & IVERSEN L. L. (1974) Molrc. Ach.fio\\.I,,dgL.iiients~The authors wish to thank R. ZACZEK MILLEK for his excellent technical assistance and NANCYHIATT and Pliaritiac. 10, 759-766.
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MISHRAR. K., GARDNER E. L.. KATZMANR. & MAKMAN TRABLCCHI M.. CHENEY D. L.. RACACNIG. & COSTAE. M. H. (1974) Proc. nutn. Acad. Sci., U.S.A. 71. 388r3887. ( 1 974) Nuitrye. Loid.. 249. 664-666. OLSONL., SEIGEKA. & FUXEK. (1972) Bruin Rrs. 44. TRALILCCHI M., CHENEY D. L.. RACAGNIG. & COSTAE. 283-288. (197.5) Brairi Rex 85. 13@-1?4. RICHELSON E. (1973) J . Nrurochrrti. 21. 1139-1 145. WHITTAREK V. P. (1965) Prog. Biophps. Molec. Biol. 15. 4 1-96. SCHULTZE B., NOWAKB. & MAURER W. (1974) J . conip. WOODWAKU D. J., HOFFER B. J., SIGGINS G. R. & BLOOM Nrurol. 158. 207-218. SETHYV. H. & VAN WOEKTM. H. (1974) Rrs. Cottitti. F. E. (1974) Bruiri Rus. 34. 73-97. Chetn. Path. Pharrnuc. 8. 13-28. . T (1971) Acto phvsiol. .sctrnd. ( S u p p l . ) 367. U N G ~ K S T ~UD SIGGINS G . R.. HOFFERB. J. & UNGEKST~IIT U. (197.5) LIT? 1-172. Sci. 15. 719-792.
Abstract-In the striaturn of the newborn rat. the activity of tjrosine hydroxylase. the concentration of dopamine and the activity of the synaptosomal high-affinity uptake process for doparnine is IOU;, of that of the adult; there is a linear and closely associated increase in all three parameters during maturation. achieving 7-59; of adult levels by 4 weeks after birth. In contrast. the specific activity of choline acetyltransferase exhibits a more delayed developmental rise commencing 1 week after birth: the concentration of acetylcholine is disproportionately high in the neonatal striatum and precedes the developmental increase in the activity of choline acetyltransferase. At birth, the specific activit) of dopamine-sensitiveadenylate cyclase is 20% of that of the adult striaturn and achieves adult activity by 4 weeks after birth. Pretrcatment with the neuroleptic, fluphenazine. does not reduce the striatal content of acetylcholine until 8 days after birth. It is postulated that dopaminergic influences on cholinegic neuronal activity appear when the cholinergic neurons in the striatum cease dividing and start differentiating.
THEDOPAMINERGIC neurons, the cell bodies of which (MCGEERP t ul., 1971; BUTCHER& BLITHER,1974). are localized in the substantia nigra, provide an ex- Based on ultramicroscopic characteristics. it has been tremely dense innervation to the striatum estimated suggested that certain small interneurons that poputo be 15% of all nerve terminals in the region (UNGER- late the striatum are cholinergic (BAK et d.,1975). STEDT, 1971 ; HOKFELT rt al.. 1970). Electro-physioloOf the many types of striatal neurons on which the gic studies utilizing extracellular recordings indicate dopaminergic terminals synapse, there is strong evithat the dopaminergic innervation acts predomi- dence that the dopaminergic neurons modulate the nantly as an inhibitory influence on the firing rate activity of the striatal cholinergic neurons. The sysof striatal neurons (CONNOR,1970; MCLENNAN& temic administration of neuroleptics results in deYORK, 1967). A dopamine-sensitive adenylate cyclase creases in the levels of endogenous acetylcholine and has been demonstrated in the corpus striatum and increases in its turnover whereas dopamine receptor in other regions of the CNS that are innervated by agonists cause variable increases in the level of acetylcholine and decreases in the rate of turnover of the dopaminergic neurons (KEBABIAN rt al., 1972). This cyclase possesses many properties that would be neurotransmitter (SETHY & VAN WOERT, 1974; er ul., 1975a; TRABUCCHI et ul.. 1971. 1975). expected for the ‘dopamine receptor’ as characterized GUYENET Because of the modulatory influence the extrinsic by previous pharmacologic and physiologic studies; i t is responsive to low concentrations of dopamine dopaminergic neurons exert on the cholinergic and is inhibited by neuroleptic drugs including neurons intrinsic to the striatum, this neuronal relaphenothiazines. butyrophenes and thioxanthines tionship would be an excellent model system for exa(KEBABIAN rt nl., 1972; MILLER P t nl., 1974; CLEMENT- mining the biochemical aspects of the development CORMIER rt d., 1975). The recent demonstration that of neuronal innervation in the central nervous system. iontophoretically applied adenosine 3’5’-cyclicmono- In this study we have examined the ontogenesis of phosphate (cyclic AMP) produces electrophysiologic several parameters associated with the dopaminergic effects similar to dopamine in the striatum terminals and the cholinergic neurons in the striatum. strengthens the inference that dopamine-sensitive In addition, the development of dopamine-sensitive adenylate cyclase mediates the effect of dopamine on adenylate cyclase is compared to the onset of the inhibitory control of the dopaminergic neurons on the rr al., 1975). the post-synaptic neurons (SICGINS Since lesioning of the major afferents to the stria- cholinergic neurons. tum has negligible effects on the activity of choline METHODS acetyltransferase [EC 2.3.1.6; Acetyl-CoA-choline 0-acetyltransferase] in the region, the cholinergic Prepnmtioit of tissues. Sprague-Dawley rats. spermneurons appear to be intrinsic to the striatum positive on a specific date. were obtained from Hormone 673
674
J. T. COYLFand P. CA~IPO(.HI.AKO
Assay Laboratories (Chicago1 and a c r e housed in separate cages after I6 days of gestation. A t birth. the litters were reduced to 8; and runts Nere discarded. For all assays except the determination of the levels of acetjlcholine. the rats were killed hq decapitation: and broin tissues ivere dissected on a n aluminum block resting on ice. For the measurement of hasal levels of acet!lcholine. the rats were killed hy microwave irradiation focused on the skull ( 13W W: 2450 MHz) in an oven adapted b) Medical Enginecring Consultants (Lexington. MA): optimal exposure varied for each age. The striatum is defined as the tissue that lies anterior to an imaginar) line drawn perpendicular to the optic chiasm and intersecting the fornix: the lateral limits are the corpus callosum: the septum w a s removed tnediall) as previously described b) G L O W I U K &I IVEKSLS (19661. 7 j m s i r i u hpfrozyltrsc~rrssu!.. The activit) of tjrosine h!droxylase [EC 1.14.16.2: L-tlrosinc. tetrati!dropteridine: oxygen oxidoreductase (3-h!drox)lating)] was measured in 50 ktl portions of striatum homogenized in 20 vol. ( n t \@I.) of 0.05 M-Tris-HCI. pH 7.3. containing 0.2"" ( v v ) Triton X-100 (Packard Instrument Co.. Downers Grove. I L ) at a final concentration of 0.1 niwtyrosine according to thc method of CouLt (1972). Cholirir crcet!~lt~ari.~li.~u-usr usstry. The acti\ it) of choline acetyltransfersse was measured in 5 to 15 j t l portions of striatum homogenized i n 20 \ol. (at vol.) of the 0.05 TrisHC1-Triton buffer according to the method of Bi LL & ODERFELD-NOWAK ( I97 I ). Doyarriirrc.-serisiti~rudtvi i - h t c c~yc/u.sc~ t t ~ . v r I ~Fresh11 . dissected striata were homogeniLed in 25 or 50 \ol. of 3 m w Tris-acetic acid buffer. pH 7.3. containing 3 mu-eth! lene glycol his (P-aminocth!l) .Y..Y'-tetraacetic acid. Fift! pl of the whole homogenate was assabed in a total vol. of 300 pI according to the method of KEBABLA\: L'r ul.. (1972). After 2.5 min incubation at 30 C. the reaction w a s terminated by heating the assaj mix for 5 min in a water bath a t 95.C. The adenosinc 3'5'-c)clic monophosphatc ( q c l i c AMP) formed was measured b! the competitive binding assay of BKOWY c't u/.. (1971). Basal acti\it!. activit) in the presence of dopamine (50 pv).and activit! in the presence of dopamine (50 p ~ plus ) Huphenazinc (5 p i ) \\ere measured: all assaqs ~ v e r eperformed in triplicate. Eritfoqiwoirs dopcrriiirit,. The concentration of dopamine was measured i n striata homogenized in 100 vol. ( u t vol.) of 0.1 x-perchloric acid according to the radiometric enzymatic assay of COYLE& H F N R(1973). ~ Eridoyrrious r r ~ ~ e r ~ . / ~ / r o l i rThe r e , . conct'iitra tion of acet)lcholine in formic acid-acetone extracts of striatum a-as measured b! the enzjmatic radiometric assa! of Goi.rink~c; & MCCAMAN (1973). Uptakr of' [3H]dopu~m~~t. Fresh striata here Iiomogenized in 20 vol. ( a t . vol.) of 0.32 wsucrosc: a n d P I fractions were prepared according to thc method of WHITTAKER (1965). The P, fraction was resuspended in the original honiogenizin_e \ olume of sucroce. Preparation for assaqs of the striatal homogenates for all ages were performed with the u m e homogenizer and pestle and assaJed together on the same da! to insure kalidit) of comparisons. Portions of the homogenate (25 or 5 0 pl) were incubated i n a total volume of 1 ml of Krebs-Ringer buffer containing 5 x It)-' M ['H]dopainine (10 Ci mmol: Sen- England Nuclear Corp.: Boston. MA) for 5 min as dexribed by H0i.i & COYLE(1974). Synaptosomal uptake \\as tcrminated by immersion of tubes in an ice.\\ater bath and centrifugation at 5000 gd, x 10 min a t 5 C to sediment the sqnaptosomes. The pellet was superficiall> rinsed rvith
4 ml of ice-cold Krehs-Ringer buffer and then soluhilized with I nil of Protosol (New England Nuclear Corp.); and radioactivity was measured by liquid scintillation spectrometr!. Under these conditions. >95",, of the tritium accumulated i n the P , fraction co-chromatographs with dopamine (HOLZ8r Coi Lf . 1974).
RESIJLTS
Deidoprricvir
of' doptruiiiiergic
irinercution
r o the
srritrrrrrii
TJ rosine hydroxylase, the initial and rate-limiting enzJnie in the synthesis pathway for dopamine, is present in the neonatal striatum with a specific activity of IO",, of that of the adult (Fig. 1). It exhibits a relatively linrar increase in specific activity during the 1 weeks after birth. attaining 75';" of the adult specific activity. The end product of the synthetic pathwa! . dopamine. is readily detectable in the neonatal striatuin with a concentration of 12% of that of the adult. Similar to tyrosine hydroxylase. the concentration o f dopamine increases in a linear fashion. attaining nearl) 70",, of that of the adult level by 4 necks after birth. Prey ious studies have shown that [ Hldo paminc taken u p by particulate material in sucrose homogenates of brain is highly localized t o synaptosoinal fractions (COYLL& SNYDEK.1969); therefore, we essmined the uptake or [3H]dopamine into resuspcnded P2 Itactions. The initial uptake of ['Hldopamine into qnaptosomes is lo",, of that of the adult ;tt hirth. The relative activity of the uptake process increases in a linear fashion attaining 75"/,, of that of the adult by 1 weeks after birth. Kinetic studies of ['Hldopamine uptake into homogenates prepared from neonatril and adult striatum indicate that the
W
0
5
I. 1 /'
I
7
14
21
28
Adu
Days (Post-Parturn) FIG. I . De\elopment of dopaminergic innervation in the striatum. The actnitq of tqrosine hydroxylase, the concentration of dopamine and the activity of the synaptosomnl uptakc process for dopamine were measured in striatum as described in the Methods. Results are expressed in tcrms 0 1 nig n s t \vt. or tissue. Each point is the mean or four or more separate preparations: and S.F M. are C- 10"~;.
Ontogeny of DA-Ach interaction in striatum
615
increase in the specific activity of choline acetyltransferase. Development of dopamine-serisitive adenylate cyclase in striaturn
Days ( Pos1- Part urn)
FIG. 2. Development of cholinergic neurons in the striatum. The activity of choline acetyltransferase and concentration of acetylcholine in the striatum were measured as described in the Methods. Results are expressed in terms of mg wet wt of tissue. Each point is the mean of five are I 10%. or more separate preparations; and S.E.M.
increase in uptake during development reflects an increase in V,,, whereas the K , does not change significantly (not shown). Dei~e!opmeizta/changes in cholinv acetyltransjerase and acetylcholine in the striatum The specific activity of choline acetyltransferase is 25; of that of the adult in the neonatal striatum; and during the first 7 days after birth, it increases only another 2% (Fig. 2). Between 1 and 4 weeks after birth, there is a linear, 20-fold increase in the specific activity of choline acetyltransferase such that 70% of the adult activity is attained by the end of this period. The concentration of acetylcholine in the neonatal striatum is quite high, being 23% of that of the adult. Like the transferase, there is only a modest 2% increase in the concentration of the neurotransmitter during the first week after birth. However, subsequent to 1 week after birth, there is a linear increase in the concentration of acetylcholine, which attains adult levels by 4 weeks after birth. Notably, the developmental rise in acetylcholine precedes but parallels the
In cell-free homogenates prepared from the neonatal striatum, dopamine produces a significant stimulation of the formation of cyclic AMP that is completely reversed by fluphenazine (Table 1). The absolute amount of cyclic AMP formed is 20% of that measured in the adult, although the stimulation by dopamine over basal activity is twice as high in the neonate as compared to the adult striatum. The specific activity of the dopamine-sensitive adenylatc cyclase increases rapidly, attaining adult activity by 3 4 weeks after birth. Whereas the dopamine-sensitive cyclase increases 5-fold in activity from birth to adulthood, the basal activity increases 10-fold. It is noteworthy that the developmental rise in the activity of dopamirie-sensitive adenylate cyclase parallels but clearly precedes the developmental increase in the three presynaptic markers for dopaminergic neurons: tyrosine hydroxylase, endogenous dopamine and synaptosomal uptake of C3H]dopamine. Effects uf‘ ,fluphenazine on concentration of’ acetylcholine in developirtg striatuni
To determine when dopaminergic afferents to the striatum develop a functional control over the cholinergic neurons, the effects of the neuroleptic, fluphenazine, on the levels of acetylcholine in the striatum of rats of various ages were examined. Rats were administered fluphenazine ( 5 mg/kg) subcutaneously or an equivalent volume of vehicle, killed 30 min after injection and the concentration of acetylcholine in their striatum was measured. In the case of the adult, treatment with fluphenazine results in a 38% decrement in the concentration of acetylcholine in the striatum (Table 2). In contrast, fluphenazine has no significant effect on the levels of acetylcholine in the striatum of the newborn or 5-day-old rat. Eight days after birth is the first time that treatment with fluphenazine results in a significant decrement in the levels of acetylcholine. The fall in the levels of acetylcholine is 26% at this stage of development and increases to 36”/, by 17 days after birth.
TABLE1. DEVELOPMENT OF DOPAMINE-SENSITIVE Age Newborn ( 3 ) 8 Days ( 3 ) 15 Days (3) 21 Days ( I ) 28 Days (3) Adult (3)
+ fluphenazine 1.0 + 0.4
Dopamine
2.0 k 0.2 3.8 k 0.2 4.5 6.6 & 0.6 10.2 k 1.9
ADENYLATE CYCLASE IN THE STRIATUM
Dopamine
A
3.0 f 0.3 6.4 k 0.8 8.6 0.2 14.1 18.8 k 1.2 20.2 2.3
2.0 0.2 4.4 k 0.8 5.1 k 0.9 9.6 12.2 & 0.6 10.0 & 1.1
*
+
*
‘?A
Stimulation
200 220 126 213 185
98
Cell free homogenates prepared from striata of rats of various ages were assayed for dopamine-sensitive adenylate cyclase as described in the Methods; the concentrations of dopamine and of fluphenazine are 50 pM and 5 p ~ respect, ively. Each preparation was assayed in triplicate; the number of separate preparations is in brackets. Results are expressed in terms of pmol cyclic AMP formed per mg tissue during 150 s incubation S.E.M.
*
676
J. T. COYLL and P. CASIPOCHIAKO
The development of the presynaptic neurochemical markers for the GABAcrgic neurons in the striatum, which appear to be predominantly local circuit Age Control Fluphenazint: neurons like the cholinerpic neurons, also exhibit a disproportiotiality between the levels of neurotratistoo 9 101 4 Newborn ( 5 ) loo* h 119 9 5 days (10) mitter and the activity of its biosynthetic enzyme 7 1 3* 8 Days ( 5 ) 100 1 early in development (COYLE & ENNA, 1975). Since 61 6* 17 Days ( 5 ) I00 9 in the striatum peaks cell division of the interneurons I00 s 62 lr Adult (5) at 21 dajs of gestation of the rat and continues for 1974; DAS & ALTRats of various ages were administcrcd fluphenazine ( 5 several days after birth (CREEPS. mgjkg) or equivalent volume of vehicle b> subcutaneous XIAS. 1970; SCHCLTZE ct al.. 1974). the rise in activity injection; to preclude hgpothermia. immature rats here of choline acctyltransferase may be ii more reliable maintained at 35 C. Thirtl min after treatment. the rats index of the ratc of differentiation of the cholinergic were killed and the content of acetylcholinc i n the striiita was measured. Results are expressed in terms of percent neurons in contrast to the levels of acetylcholine. Tisof vehicle treated controls S.E.V. The number of rats i n sue culture studies on clones of neuroblastoma supeach experimental group is in brackets. port this hypothesis in that the activity of the enzymes * P 5 0.01. responsible for neurotransmitter synthesis rise dramatically when the neuroblastoma cells cease dividing DISCUSSION and commence differentiating (AIVIANOet ( I / . . 1971; Since the cell bodies of the dopaminergic neurons RICHELSON. 1973). The dopamine-sensitivc adetiylate cyclase has a are extrinsic to the striatum (UNGERSTEDT. 1971). the appearance and ontogenetic changes in the levels of predominantly. if not exclusively, post-synaptic localineurochemical markers for the dopamincrgic neurons zation (MISHKAor d.. 1974). Although indirect eviin the striatum would reflect the ingrowth and pro- dence suggests that the cholinergic neurons possess dopamine receptors. the diffuse innervation by the liferation of their terminals. At birth of the rat. the levels of dopamine and the activitj of tyrosine 11)- dopaminergic neurons on ultrastructurally different droxylase and of the synaptosomal uptake process for neuronal tqpes implies that other neiirons in the striatum possess receptors for dopamine (BAKet d., 1975). dopamine are ICI"~, of that of the adult. During the subsequent 4 weeks of postnatal maturation. all three In the neonatal striatum. the specific activity of the parameters increase in remarkably close association dopamine-sensitive adenylate cyclase is 2000 of that achieving approx 750, of that of the adult. A similar of the adult and increases progressively to adult levels close temporal association i n the development of pre- b j 3-4 weeks after birth. The rise in the activity of synaptic markers for the noradrenergic neurons in the dopamine-sensitive adenylate cyclase precedes but central nervous system has been previously denion- parallels the developmental rise in the prcsynaptic strated (CoYLt & AXELROD. 1971. 1972u.h: Coirt & neurochemical markers for the dopaminergic terH t w Y . 1973). These neurochemical observations are minals. Thus. the development of the dopamine recepin close agreement with the semiquantitative results tor precedes the ingrowth of dopaminergic terminals. from histofluorescent microscopic studies on the de- This observation concerning the appearance and invelopment of dopaminergic innervation to the rat crease in receptor sites prior to the development of striatum. At birth. patches of fluorescent terminals are presynaptic input is in accordance with neurophysiopresent in the striatum that become progressivelj logic observations on the development of nicotinic confluent and achieve an intensit) of fuorescence cholinergic receptors on myoblasts (FAMBROUGH, near that of the adult by 4 weeks after birth (LOIZOL. 1974). the p receptor on cerebellar Purkinje cells 1972; OLSON et d.. 1972). cr d.. 1971) and neurochemical studies (WOODWARD In contrast to the dopaminergic neurons. the cho- on the development of the GABAergic receptor in linergic neurons appear to be small interneurons with the rat cerebellum ( C o Y L t & ENNA,1975) and muscell bodies and terminals completely limited to the carinic cholinergic receptor in the striatum (Coyle & striatum (BAK e r d.. 1975). Thc specific activity of Yamamura. in preparation). Similarly, in the chick choline acetyltransferase is quite low at birth and retina. dopamine-sensitive adenylate cyclase appears remains depressed during the first postnatal week: it several days before the dopaminergic neurons begin then increases rapidly in activitj during the sub- to differentiate (Coyle gi Schwarcz, in preparation). sequent 3 weeks, attaining 70"" of the adult activity. Extracellular recordings demonstrate that the ionThere is a marked disparity between the Icvcls of ace- tophoretic application of dopamine on to neurons in tylcholine and the activity of choline acetlltransferase the striatum causes inhibition of firing of a tnajority in the neonatal striatuni with the concentration of of them; and it is significant that, in light of the eviacetylcholine 25", of that of the adult whereas the dence that dopamine-sensitive adenyla te cyclase may specific activity of choline acetyltransferase is only 3",, be the dopamine receptor, that iontophorrtic appliof adult at this time. The developmental incrcases in cation of cyclic AMP also inhibits the firing of these the concentration of acetylcholine parallel but pre- neurons ( CONNOR.1970: SIGGINS clt d . . 1975). Concede the rise in activity of choline acetyltransferase. versely. blockade of dopamine receptors by systemic TABLF2 EFFECT OF
FLLPHFNAZN o\ A C E T I L C H O L I ~ E LF\. t L S I\ THF STKIATL \I
* ** *
** ** *
Ontogeny of DA-Ach intrraction in striatiim administration of neuroleptic drugs results in increased rates of firing (CONNOR. 1970). Of the many types of striatal neurons on which the dopaminergic terminals synapse, there is strong evidence that the dopaininergic terminals modulate the activity of the striatal cholinergic interneurons. The systemic administration of dopamine-receptor blockers results in decreased levels of endogenous acetylcholine and increases in the turnover of acetylcholine in the striaet al.. tum (SETHY& VAN WOERT.1974; GUYENET 1975a: TIUBUCCHI et al., 1974). The effect of neuroleptics on the levels of acetylcholine is presumably due to blockade of dopamine receptors resulting in disinhibition from the tonic dopaminergic input. In our studies, pretreatment with fluphenazine results in a 38",, decrement in the concentration of acetylcholine in the striatum of the adult rat. In contrast, fluphenazine administration has no significant effect on the levels of acetylcholine in the striatum of newborn or 5-day-old rats. Eight days after birth is the first time that iluphenazine treatment results in a significant effect on the concentration of acetylcholine in the striatum; and this effect is apparent at later stages of development. These results are in close agreement with those of GUYENLT et al.. (l975b) on the effects of other neuroleptics on the levels of acetylcholine in the striatum of the developing rat. These studies indicate that there is a significant disparity between the development of dopaminergic innervation and dopamine-sensitive adenylate cyclase in the striatum and the appearance of sensitivity of cholinergic neurons to blockade of dopamine receptors. The lack of response of striatal cholinergic neurons to fluphenazine until 8 days after birth does not appear to be due to insufficient activity of the dopaminergic afferents to the striatum s'ince KELLERet al., (1973) have shown that the turnover of dopamine is quite high in the neonatal striatum and responds appropriately to stimulation or blockade of dopamine receptors. This change in response of the cholinergic neurons in the striatum to blockade of dopamine receptors may indicate that there is a delay between the time of appearance of the dopamine receptor on the neuron and the time when it assumes a role in regulating the electrical activity of the cholinergic neurons. An alternative and more parsimonious explanation is that the dopamine-sensitive adenylate cyclase activity mcasured in the striatum during the first week after birth is localized on other neuronal and non-neuronal cells; and that the cyclase appears on the cholinergic neurons when they cease dividing and start differentiating at 8 days after birth. Thus, the appearance of filnctional control of the dopaminergic input over the striatal cholinergic neurons coincides with the time when the activity of choline acetyltraiisferase begins to increase at a precipitous rate.
677
RHODESfor their secretarial assistance. P. CAMPOis a Year 111 Medical Student at Johns Hopkins School of Medicine. This research was supported by
VrKiE
CHIAKO
USPHS Grant DA 00266. REFERENCES AMANOT., RICHELSON E. & NIRENBERG M. W. (1972) Proc. iiatri. Acnd. Sci., U.S.A. 69. 258-263. BAK I. J., CHOIW. €3.. HASSLER R., USUNOFFK. G. & WAGNEK A. (1975) in Advatices iii Neurologjy (CALNE D. B.. CHASE, T. N. & BARBEAUA.. eds.) Vol. 9. pp. 25-41. Raven Press, New York. BKOWNB. L., ALBANO. J. D. M., ELKINSR. P.. SCHERZI A. M. & -fAMl'lON w. (1971) Biochem J . 121. 561-561. B L ~ LG. L & ODERFELD-NOWAK B. (1971)J . Neurocheiri. 19. 935-947. S. G. & BUTCHERL. L. (1974) Bruin Rrs. 71. BUTCHER 1 67-1 7 I .
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22. 147-151. HOLZR. W. & COVLEJ. T. (1971) Molec. Phurri~uc. 10. 74C758. K F B A H I A N J. w., PtTZOLD G. L. & GREENGARD P. (1972) Proc. iiatii. Acad. Sci., U.S..-L. 69. 2145-2149. KELLER H. H., BARTHOLINIG. & PLETSCHER A. (1973) Braiii Res. 64. 371-378. Loizou L. A. (1972)Bruin Res. 40. 395-418. H. C. & WICKSON MCGEER P. L., M c C t t ~E. G., FIBIGEK V. (1971) Brtriii Res. 35. 308-314. MCLENNAN H. & YORK D. H. (1967) J . Pl~ysiol.,Lorid. 189. 393-402.
R. J., HORNA. S. & IVERSEN L. L. (1974) Molrc. Ach.fio\\.I,,dgL.iiients~The authors wish to thank R. ZACZEK MILLEK for his excellent technical assistance and NANCYHIATT and Pliaritiac. 10, 759-766.
678
J. T. COYLE and P. CAMPCCHIARO
MISHRAR. K., GARDNER E. L.. KATZMANR. & MAKMAN TRABLCCHI M.. CHENEY D. L.. RACACNIG. & COSTAE. M. H. (1974) Proc. nutn. Acad. Sci., U.S.A. 71. 388r3887. ( 1 974) Nuitrye. Loid.. 249. 664-666. OLSONL., SEIGEKA. & FUXEK. (1972) Bruin Rrs. 44. TRALILCCHI M., CHENEY D. L.. RACAGNIG. & COSTAE. 283-288. (197.5) Brairi Rex 85. 13@-1?4. RICHELSON E. (1973) J . Nrurochrrti. 21. 1139-1 145. WHITTAREK V. P. (1965) Prog. Biophps. Molec. Biol. 15. 4 1-96. SCHULTZE B., NOWAKB. & MAURER W. (1974) J . conip. WOODWAKU D. J., HOFFER B. J., SIGGINS G. R. & BLOOM Nrurol. 158. 207-218. SETHYV. H. & VAN WOEKTM. H. (1974) Rrs. Cottitti. F. E. (1974) Bruiri Rus. 34. 73-97. Chetn. Path. Pharrnuc. 8. 13-28. . T (1971) Acto phvsiol. .sctrnd. ( S u p p l . ) 367. U N G ~ K S T ~UD SIGGINS G . R.. HOFFERB. J. & UNGEKST~IIT U. (197.5) LIT? 1-172. Sci. 15. 719-792.
