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Dopamine D1 autoreceptor function: possible expression in developing rat prefrontal cortex and striatum
229
Developmental Brain Research, 63 (1991) 229-235 Elsevier ADONIS 016538069151359X BRESD 51359
D o p a m i n e D 1 autoreceptor function: possible expression in
developing rat prefrontal cortex and striatum Martin H. Teicher 1, Amelia L. Gallitano 1, Harris A. Gelbard 2., Henriette K. Evans 1, Elda R. Marsh 1, Raymond G. Booth 1'4 and Ross J. Baldessarini 1'3 Departments of 1Psychiatry, 2Neurology and 3Neuroscience Program, Harvard Medical School Mailman Laboratories for Psychiatric Research, McLean Hospital Belmont, MA 02178 (U.S.A.) and 4Department of Medicinal Chemistry, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7360 (U.S.A.) (Accepted 23 July 1991)
Key words: Dopamine; Presynaptic receptor; Autoreceptor; Dopamine 1 receptor; Prefrontal cortex; Corpus striatum; Dopamine agonist
Synthesis-modulating dopamine (DA) autoreceptor function was studied in vivo using y-butyrolactone (GBL) to block propagation along DA axons. DA synthesis was measured by the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) after inhibition of aromatic L-amino acid decarboxylase. GBL treatment markedly increased DOPA accumulation in both the striatum and prefrontal cortex of developing rats. The selective DA partial D 1 agonist SKF-38393 inhibited this GBL-induced rise in DA synthesis in both the striatum and prefrontal cortex of 15- and 22-day-old rats, but not in adults. The effects of SKF-38393 in developing rats were mimicked by the non-catechol D1 partial agonist CY-208-243, and were blocked by the D 1 antagonist SCH-23390, suggesting receptor mediation. The mixed D2/D3 agonist quinpirole attenuated DA synthesis in striatum of both two-week-old and adult rats, but failed to inhibit the GBL-induced increase in DA synthesis in the developing prefrontal cortex. These findings suggest that synthesis-modulating Dl-like receptor function may emerge transiently in the developing mammalian forebrain. In the adult striatum these functions appear to be subsumed by D2-1ike receptors, whereas all synthesismodulating DA receptor function in prefrontal cortex appears to be essentially lost with maturation. INTRODUCTION Dopamine ( D A ) receptors in the central nervous system can be subdivided by their anatomical localization as well as their physiochemical properties, pharmacology and molecular structure. The majority of D A receptors lie across the synaptic cleft on n o n - D A neurons, and are referred to as postsynaptic receptors. Neuropharmacological, electrophysiological, behavioral and histochemical data also suggest that D A receptors exist in D A cell bodies and dendrites in midbrain, and on terminals in some forebrain regions 13'34. The latter 'autoreceptors' function to modulate neuronal activity and the synthesis and release of D A 34. Until recently, synthesis modulating D A autoreceptors appeared to be exclusively of the D 2 type 7'32. Recently, a D 3 receptor has been cloned and characterized, and may function as both a postsynaptic receptor and an autoreceptor 26. Although a D2-1ike D 4 receptor 31, and a Dl-like D 5 receptor 27 have also been cloned and partially characterized, their physiological roles remain to be elucided. Attempts to detect synthesis-modulating D 1 autoreceptors in the adult rat striatum
have failed to elicit any compelling evidence for their existence 7'33. However, the density of D 1 receptors in forebrain appears to change markedly with development. In both rats 12 and man 23, the density of D1 receptors enigmatically rises and then falls several-fold during the postnatal period. We hypothesized that the early postnatal surge in DI receptor density might include some D1 autoreceptors, followed by selective loss of this class of autoreceptors with further maturation. We now report the interesting possibility that synthesis-modulating D~ receptors may arise and function transiently in both the striatum and prefrontal cortex between postnatal days 15 and 22, but cease to function by day 60. The present findings suggest, more generally, that certain receptor classes may be gained and lost during development and encourages speculation that certain neuropsychiatric disorders may stem from the failure of these receptor types to arise or recede at the appropriate time. MATERIALS AND METHODS Lactating female Sprague-Dawley rats were housed with their litters on a 12 h light-dark cycle (lights on: 07.00-19.00 h) with food
* Present address: Division of Pediatric Neurology, Box 631, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, U.S.A. Correspondence: M.H. Teicher, McLean Hospital, 115 Mill Street, Belmont, MA 02178, U.S.A. Fax: (1) (617) 855-3299.
230 and water provided ad libitum. Litters were culled to 10 pups of either sex on postnatal day 1 and, in long-term experiments, were weaned at day 28. Pups were sacrificed on postnatal days 15, 22 and 60 in some experiments, and at 16 days and adulthood (ca. 275 g body weight) in others. Their brains were rapidly removed, chilled, and dissected on ice. Corpus striatum and mesial prefrontal cerebral cortex (PFCTX) were individually dissected as previously described 3°, and stored at -70 °C. For HPLC determination of DOPA (L-3,4-dihydroxyphenylalanine) concentrations, individual tissue samples were homogenized in 1.0 ml of 0.1 N perchloric acid, 0.2 mM EDTA, and 0.4 mM sodium metabisulfite, Homogenates were extracted and assayed by slight modification of the methods of Baldessarini et al. 4. Sample aliquots (100/d) were separated by high pressure liquid chromatography (HPLC) on a C-18, reverse-phase, 5 /,m resin particle column measuring 4.6 mm × 25 cm, and analytes were detected using a Bioanalytical System electrochemical detector (+0.55 V vs Ag/AgCI reference electrode). The mobile phase consisted of 0.05 M NaOAc.3H20, 0.1 mM EDTA, 1.5% acetonitrile (vols.), and 4.2 mM 1-heptanesulfonic acid (pH 3.5). The recovery of authentic catechols consistently exceeded 85%, The presence of synthesis-modulating autoreceptors were explored using the y-butyrolactone (GBL) technique of Waiters and Roth 33. In this paradigm, tyrosine hydroxylase activity was measured as accumulation of DOPA for 30 min following administration of the decarboxylase inhibitor, m-hydroxyphenylhydrazine (NSD-1015). GBL was administered to block propagation along DA axons, thus isolating DA terminals from long-loop feedback effects that can accompany postsynaptic receptor stimulation. We hypothesized that, if synthesis-modulating D ~ autoreceptors were present, then the selective D~ agonist SKF-38393 would antagonize DOPA accumulation induced by GBL and this effect, in turn, would be blocked by administration of the selective D ~ antagonist SCH-23390. The sequence of treatments, given by intraperitoneal (i.p.) injections, are summarized in Table I; group sizes are indicated in the accompanying figures. A total of 300 rats were used in these studies. In the first study, the effects of SKF-38393 and SCH-23390 on DOPA accumulation in striatum and PFCTX were explored in 15-, 22- and 60-day-old rats using the GBL technique. In the second study, the age-dependent effects of the D l agonist CY-208-243 were compared to the effects of SKF-38393. This comparison was made because SKF-38393 is a catechol and may produce direct chemical inhibition of tyrosine hydroxylase 2,7,2o, possibly confounding the interpretation of its action as an autoreceptor agonist. CY208-243 is a non-catechol D t receptor agonist, that does not directly inhibit tyrosine hydroxylase activity L 16.20.21. Although there is some concern that the monohydroxyergoline CY-208-243 has a relatively high affinity for D 2 as well as D~ binding sites 2"2°, it lacks any discernable intrinsic D 2 agonist effects (e.g., can not induce stereotypic behavior, nor induce emesis in dogs, and has no effect on plasma prolactin levels2°). In contrast, CY-208-243 has clear intrinsic partial D z agonist effects. It produces characteristic grooming behavior 2°, stimulates DA-sensitive adenylate cyclase 2°, and in-
duccs rotation in the unilateral 6-hydroxydopaminc model that can be antagonized by SCH-23390 but not by the D~ antagonist sulpride 2°. Several other D~ agonists arc known, including some with greater intrinsic activity and selectivity in ligand binding studies, however all these agents arc catechols 29, and thus can not serve as adequate controls for the potential catechol effect of SKF-38393. In the third study, we explored the effects of lhe probably mixed D2/D 3 agonist quinpirole (LY-171555) on DOPA accumulation in the striatum of 15-day-old and adult rats. The affinity of quinpirole for the D 3 receptor is over 100-fold greater than its affinity for the D e receptor 26, and it is the most selective known D+ agonist. Since relatively large doses were used here (1 mg/kg), it is likely that both receptor types were affected. Quinpirole was also paired with spiperone, a potent antagonist of both D e and D~ rcceptorsZC'~
Statistical analysis Effects of age, drug treatment, and their interaction were determined using unweighted ANOVA for unequal group sizetS. Individual planned comparisons were made using Student's t-test based on predicted effects. Materials SCH-23390, SKF-38393, and spiperone were obtained from Research Biochemicals Incorporated (RBI, Natick, MA). Generous donations of drugs included quinpirole by Eli Lilly and Sons Laboratories (Indianapolis, IN), and CY-208-243 by Dr. Paul Herrling of the Sandoz-Wander Research Institute (Berne, Switzerland). GBL, NSD-1015, and the various HPLC reagents were obtained from Sigma Chemical Company (St. Louis, MO) in the highest available purity.
RESULTS
D O P A accumulation in striatum and prefrontal cerebral cortex after SKF-38393 A s s h o w n in Fig. 1, D O P A a c c u m u l a t i o n in s t r i a t u m was m a r k e d l y a f f e c t e d by age (F2,9o = 51.1, P < 0.001), d r u g c o n d i t i o n (Fs.9o = 42.2, P < 0 . 0 0 t ) , and their int e r a c t i o n (F6.9o = 3.9, P < 0.01). G B L (750 m g / k g ) inc r e a s e d D O P A a c c u m u l a t i o n by a b o u t 1 0 0 % at e a c h age (all P < 0.0005). T h e G B L - i n d u c e d rise in D O P A
ac-
c u m u l a t i o n was a n t a g o n i z e d by 56% with 10 m g / k g S K F 38393 at 15 days ( P < 0.0001), and b y 33% at 22 days ( P < 0.05), but was n o t a n t a g o n i z e d at 60 days, S C H 23390 at 10 m g / k g fully a t t e n u a t e d t h e effects o f S K F 38393 at 15 ( P < 0.005) and 22 days ( P < 0.05). A s s h o w n in Fig. 2, D O P A a c c u m u l a t i o n in P F C T X ,
TABLE I
Sequence of treatments CY, CY-208-243 (10 mg/kg); GBL, y-butyrolactone (750 mg/kg); NSD, NSD-1015 (100 mg/kg); QNP, quinpirole (LY-171,555, 1 mg/kg)~ sal, 0.9% (w/v) NaCI (saline); SCH, SCH-23390 (10 mg/kg); SKF, SKF-38393 (10 mg/kg); SPI, spiperone (1 mg/kg). Rats were sacrificed at 0 min; test agents were all administered intraperitoneally (i.p.).
Time (rain) Control
GBL
SKF
CY
QNP
SCH
SP1
SCH+SKF
SCH+CY
SPI+QNP
-45 -~ -35 -30
sal sal GBL NSD
sal SKF GBL NSD
sal CY GBL NSD
sal QNP GBL NSD
SCH sal GBL NSD
SPI sal GBL NSD
SCH SKF GBL NSD
SCH CY GBL NSD
SPI QNP GBL NSD
sal sal sal NSD
231 STRIA TUM
S TRIA TUM
4
[] • [] •
l
SAUSAWSAL SAUSAUGBL I SAWSKF/GBL I SCH/SKF/GBLI
3
16-DAYS
ADULT
AGE GROUP
~
O, = 22
15
60
AGE (d)
Fig. 1. Effects of 7-butyrolactone (GBL) and SKF-38393 (SKF), alone or with SCH-23390 (SCH), on accumulation of L-3,4,-dihydroxyphenylalanine (DOPA) in corpus striatum of rats aged 15, 22, or 60 days after pretreatment with NSD-1015. Doses of agents (given i.p.) are summarized in Table I. Data are means of stated numbers of subjects (at the base of each bar) -+ S.E.M. too, was markedly affected by age (/'2,70 = 24.3, P < 0.001), drug treatment (/'3,7o = 10.4, P < 0.001), and their interaction (F6,7o = 3.2, P < 0.01). G B L produced a 100% increase in D O P A levels at 15 days (P < 0.005), a 34% increase at 22 days (P < 0.02), and only a 23% increase at 60 days (P < 0.05). As in striatum, the effects of G B L on D O P A accumulation were fully reversed by SKF-38393 at 15 days (P < 0.005) and 22 days (P < 0.02) and, in turn, SKF-38393 was antagonized by SCH-23390 at these young ages (both P < 0.05), but neither SKF-38393 alone nor in combination with SCH23390 had a significant effect at 60 days.
PREFRONTAL CORTEX
0.3 ~
/
'~
~, 8
[
T
|
i [I SAL~AUGBL.~
•
I
! llm SO:I,,S,.:~,G.1 l
n
/
) / . S, SKF,GB' /
o.~
0.0
15
22 AGE (d)
60
Fig. 2. Effects of GBL, SKF and SCH on accumulation of DOPA in prefrontal cerebral cortex of rats aged 15, 22, or 60 days, as described for Fig. 1 and in Table I.
Fig. 3. Effects of GBL, SKF or CY-208-243 (CY), alone or after SCH, on accumulation of DOPA in corpus striatum of rats aged 16 days or adults, as described for Fig. 1 and in Table I.
DOPA accumulation in striatum after CY-208-243 In this replication experiment (Fig. 3) there were again strong effects of age (F1,87 = 28.8, P < 0.001) and drug (F5,87 = 22.7, P < 0.001), and a significant age by drug interaction (F5,87 = 2.35, P < 0.05). G B L increased D O P A accumulation by about 3-fold in both 16-day-old and adult rats (both P < 0.0005). In 16-day-olds, D O P A accumulation was inhibited by about 50% by 10 mg/kg of either CY-208-243 or SKF-38393 (both P < 0.01). The effects of SKF-38393 were fully inhibited by 10 mg/kg SCH-23390 (P < 0.01). Inexplicably, the combination of CY-208-243 and SCH-23390 proved lethal to 16-day-olds in the G B L paradigm (n = 6), so that data on the antagonism of CY-208-243 by SCH-23390 were not obtained for young rats. In adult striatum (Fig. 3) there was no significant effect of the same dose of SKF-38393 on D O P A accumulation. CY-208-243 produced a slight, but significant, 24% decrease in D O P A accumulation in the adult striatum (P < 0.05), that was not antagonized by the D 1 antagonist SCH-23390 (Fig. 3). Thus, CY-208-243 may have some pharmacological or toxic effects that are not mediated by DI receptors. In 16-day-olds, SCH-23390 alone (10 mg/kg) produced a 23% increase in striatal D O P A accumulation over that induced by G B L alone and, in adults, this D 1 antagonist produced a 13% increase. While these observations may suggest some degree of tonic D 1 inhibition, particularly in the preweanling rat, neither effect attained statistical significance (P > 0.10; Fig. 3).
DOPA accumulation in striatum after quinpirole Overall, there were main effects of age (F1,48 = 13.6, P < 0.001), and drug (F3,49 = 33.0, P < 0.001), but no significance interaction (F3,49 = 1.92, ns), indicating that
232 STRIA TUM
==
=¢ 0
0 ¢3
15-DAYS
ADULTS
AGE GROUP
Fig. 4. Effects of GBL and quinpirole (QNP) alone or with spiperone (SPI) on accumulation of DOPA in corpus striatum of 15day-old or adult rats, as described for Fig. 1 and in Table I.
the pattern of response was comparable at the two ages tested (Fig. 4). GBL increased DOPA accumulation by 120% in the striatum of 16-day-old rats (P < 0.0001), and by 60% in adults (P < 0.005). Quinpirole (1 mg/kg) fully antagonized the effects of GBL in 16-day-olds (P < 0.0001). In adults, this agent not only reversed the effects of GBL (P < 0.0005), but reduced DOPA accumulation below the level obtained in the saline control group (P < 0.0005). Spiroperidol (1 mg/kg) fully antagonized the effects of quinpirole in both 16-day-olds (P < 0.0001) and adults (P < 0.005). Combined treatment of 16-day-olds with SKF-38393 and quinpirole produced only a 7% greater inhibitory effect than quinpirole alone, which though statistically significant (P < 0.05; data not shown), probably has little biological meaning.
DOPA accumulation in prefrontal cortex of 16-day-old rats after quinpirole In this experiment there were main effects of treatment (F2,15 = 5.85, P < 0.02) with GBL (n = 7), which increased D O P A accumulation by 50% relative to saline treated controls (n = 6; P < 0.005). However, quinpirole (n = 5) failed to antagonize the effects of GBL in this brain region, producing only a statistically insignificant 7% reduction (data not shown). DISCUSSION
Two major observations emerge from these experiments. First, important age-related differences in D O P A accumulation were found in prefrontal cortex. GBL induced a prominent increase in tyrosine hydroxylation in 15-day-old rats, but only a modest increase at 22 days, and a trivial increase at 60 days (Fig. 2). This observa-
tion suggests that synthesis-modulating DA receptors may be present in the PFCTX during early development, but may cease to exist, or at least not function, later in development. It is also interesting that the partial I) 1 agonist SKF-38393 fully antagonized the GBL effect in the developing PFCTX, and that SCH-23390 blocked these agonist effects (Fig. 2), whereas the mixed D2/D 3 agonist quinpirole failed to attenuate DOPA accumulation in PFCTX. These observations suggest that DA synthesis-modulating receptors may appear transiently in the prefrontal cerebral cortex, and that they may belong to the D 1 class. Second, the D 1 agonists SKF-38393 and CY-208-243 antagonized, by about 50%, the GBL-induced increase in accumulation of DOPA in the corpus striatum of 11516-day-old rats (Figs. 1 and 3). The effects of SKF-38393 were fully blocked by the D~ antagonist SCH-23390, suggesting again that these effects were mediated by D~ receptors. On the other hand, SKF-38393 exerted little or no effect on DOPA accumulation of 60-day-old or adult rats (Figs. 1 and 3). Thus, synthesis-modulating D 1 receptors may be present in the striatum of developing rats, but cease to function as the animal matures. On the other hand, the mixed D2/D 3 agonist quinpirole exerted a consistent and powerful effect on striatal DOPA accumulation across age (Fig. 4). Thus, there may be different populations of synthesis-modulating DA receptors in the striatum, and they may have a dissimilar developmental time course. The possibility that synthesis-modulating D~ receptors might arise transiently during development, and particularly in PFCTX, is interesting and unexpected. In general, the emergence of a function followed by its loss is consistent with many observations on the synaptogenesis of the developing nervous system, in many brain regions there is a marked increase in the density of synaptic contacts during early postnatal development, followed by prominent pruning during adolescence 15. Such a time course seems to be followed by D 1 receptors in the rat striatum ~2. On the other hand, the concept of a Dt autoreceptor is somewhat perplexing, as DA has a much weaker (i.e.,/~M) apparent affinity for the D 1 receptor, that it does for D 2 (nM), D 3 o r D4 (sub-nM) receptors 26"31. Some models of presynaptic autoreceptor function hypothesize that terminal autoreceptors are highly sensitive and responsive to released transmitter, and provide exquisite feedback control over the range of physiological activity2s. Other theoretical models, however, suggest that some autoreceptors are relatively insensitive to released transmitter, and only come into play during periods of excessive activation 3, possibly to prevent the system from entering into a pathological range of activity ~7. It is possible that the developing nervous
233 system transiently requires a low sensitivity feedback control mechanism on DA synthesis in the prefrontal cortex, and may utilize this process as a redundant emergency backup system in the striatum. The concept of a D1 autoreceptor may appear untenable if one posits that the majority of synthesis-modulating autoreceptors are D 2 receptors that are coupled by an inhibitory G-protein to adenylate cyclase 6'8, and presume that a D 1 autoreceptor would have to exert opposite effects through a stimulatory G-protein. It is now clear that there is sufficient heterogeneity in the coupling mechanism of D1 and D2 receptor families 8'22'26 to encompass the possibility of a subclass of synthesis-modulating D1 presynaptic receptors. The strongest additional experimental support for the existence of synthesis-modulating D 1 receptors derives from the work of Berry and Gudelsky 5. They found that both SKF-38393 and CY-208-243 attenuated D O P A accumulation (after NSD-1015) in tuberoinfundibular DA neurons of adult rat hypothalamus after activation of DA turnover by haloperidol, reserpine, or neurotensin. However, these D 1 agonists failed to exert any effect on the basal rate of DA synthesis. Thus, D1 receptor stimulation appears to have a strong modulatory role on the rate of D A synthesis when such neurons are in a stimulated state. There are, however, a number of caveats that are relevant to the interpretation of the present findings. First, apparent age-related differences in autoreceptor function could be an artifact of ontogenic differences in the action of GBL. However, one study of developmental differences in response to 7-hydroxybutyrate (GHB, the active metabolic product of GBL), found that the doseresponse for accumulation of DA following GHB was nearly identical at 14 days and 4 weeks of age 19, indicating that a developmental change in the potency of this agent, and presumably GBL, is unlikely to be a significant factor in the present study. It should also be acknowledged that the GBL paradigm, while experimentally convenient, provides only an indirect means of exploring autoreceptor function. Although this technique largely isolates DA terminals from long-loop, multi-synaptic, feedback regulation via DA axons, it does not suppress local feedback from interneurons which contain postsynaptic D 1 receptors, and which may reciprocally communicate with DA terminal heteroreceptors that can modulate tyrosine hydroxylase activity 6. In this regard, we have not been able to extend the present D~-like autoreceptor effects to isolated minces or synaptosomal preparations from the developing rat striatum. SKF-38393 inhibited tyrosine hydroxylase activity, but this effect was not reversed by SCH-23390 (R. Booth and R.J. Baldessarini, unpublished observations, 1990).
However, such in vitro preparations may be limited in their ability to model autoreceptor function 7"11, and the operation of the autoreceptor may depend on the integrity of a cascade of molecular events in the terminal that may not be preserved in isolated tissue, or may require stimulation or interactions with other transmitter systems that are not readily duplicated in vitro 5. Thus, this initial study leaves unresolved the question as to whether these apparent transient synthesis-modulating D l-like receptors are located on dopamine terminals, and can rightfully be called 'autoreceptors', or whether they are postsynaptic receptors, and act on DA terminals indirectly through heteroreceptors. We have chosen to refer to the effects of these receptors on dopamine synthesis as 'autoreceptor function', aware that these receptors may not be presynaptic. This caveat applies to all studies that utilize the GBL technique or similar procedures. Relatively little attention has been given to the ontogeny of DA autoreceptor function in mammalian brain. Behavioral studies utilizing low doses of DA agonists (apomorphine or 3-[3-hydroxyphenyl]-N-n-propylpiperidine [3PPP]) to probe for autoreceptor-induced behavioral suppression generally have failed to elicit evidence for DA autoreceptor function until 28-35 days of age 14, 25. We have found however, that 8-day-old rats, when sufficiently aroused by maternal deprivation, can display marked low-dose behavioral suppression with R(-) N-npropylnorapomorphine 29. To our knowledge, there are only two reports on the ontogeny of synthesis-modulating DA autoreceptors using the GBL technique. Shalaby et al. 25 studied the accumulation of DA in striatum and olfactory tubercle in 7- to 90-day-old rats after GBL and apomorphine (no decarboxylase inhibitor was used). GBL significantly increased D A accumulation in the striatum even at day 7, but was not reversed by apomorphine until day 14. In the olfactory tubercle, GBL failed to produce a significant increase in DA until day 21, and effects of apomorphine did not clearly emerge until day 35. Hedner and Lundborg 14 reported that (---)3PPP inhibited DOPA accumulation after GBL and NSD-1015 in striatum of 28-day-old rats, but did not do so in 4-dayolds. In conclusion, the present study adds to our understanding of the development of dopaminergic autoreceptor function, suggesting that both Dl-like and D2-1ike synthesis-modulating receptors may be present in the striatum of two-week-old rats, and that synthesis-modulating autoreceptors of the D 1 type may also be present in the prefrontal cortex temporarily at this age. With further maturation there is an apparent loss of synthesis-modulating receptor function (and GBL effect) in prefrontal cortex, and a loss of the synthesis-modulating effects of D1 receptor stimulation in striatum. Further
234 studies are needed to ascertain whether synthesis-modulating D 1 (or D5) receptors exist at earlier ages, and to ascertain whether probes for such receptors or their gene messages can be localized in D A cell bodies or terminals at relevant ages.
Acknowledgements. Supported in part by USPHS (N1HM) Grants MH-43743, MH-34006, MH-31154 and MH-47370, as well as awards from the Charles Dana Foundation, Scottish Rite Schizophrenia Research Program, Hall Mercer Foundation, Snider Family, the Bruce J. Anderson Foundation, Milton Foundation, and Marion Ireland Benton Trust.
REFERENCES I Abbott, B., Starr, B.S. and Starr, M., CY-208-243 behaves as a typical D-1 agonist in the reserpine-treated mouse, Pharmacol. Biochem. Behav., 38 (1991) 259-263. 2 Andersen, P.H. and Jansen, J.A., Dopamine receptor agonists selectivity and dopamine D1 receptor efficacy, Eur. J. Pharmacol. Mol. Pharmacol. Sect., 188 (1990) 335-347. 3 Angus, J.A., Korner, P.I., Jackman, G.P., Bobik, A. and Kopin, I.J., Role of autoinhibitory feedback in cardiac sympathetic transmission, Clin. Exp. Hypertens, A6 (1984) 371-385. 4 Baldessarini, R.J., Marsh, E.R., Kula, N.S., Zong, R., Gao, Y. and Neumeyer, J.L., Effects of isomers of hydroxyaporphines on dopamine metabolism in rat brain regions, Biochem. Pharmacol., 40 (1990) 417-423. 5 Berry, S.A. and Gudelsky, G.A., D x receptors function to inhibit the activation of tuberoinfundibulardopamine neurons, J. Pharmacol. Exp. Ther., 254 (1990) 677-682. 6 Booth, R.G. and Baldessarini, R.J., Adenosine A 2 stimulation of tyrosine hydroxylase in rat striatal minces is reversed by dopamine D2 autoreceptor activation, Eur. J. Pharmacol., 185 (1990) 217-221. 7 Booth, R.G., Baldessarini, R.J., Kula, N.S., Gao, Y., Zong, R. and Neumeyer, J.L., Presynaptic inhibition of dopamine synthesis in rat striatal tissue by enantiomeric mono- and dihydroxyaporphines, Mol. Pharmacol., 38 (1990) 92-101. 8 Bowyer, J.G. and Weiner, N., K+ channel and adenylate cyclase involvement in regulation of Ca2+-evoked release of [3H]dopamine from synaptosomes, J. Pharmacol. Exp. Ther., 248 (1989) 514-519. 9 Brewster, W.K., Nichols, D.E., Riggs, R.M., Mottola, D.M., Lovenberg, T.W., Lewis, M,H. and Mailman, R.B., trans-lO,11Dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]-phenanthridine: a highly potent selective dopamine D a full agonist, J. Med. Chem., 33 (1990) 1756--1764. 10 Faedda, G.L., Kula, N.S. and Baldessarini, R.J., Pharmacology of binding of 3H-SCH-23390, a ligand selective for D1 dopamine receptor sites, in rat brain tissue, Biochem. Pharmacol., 38 (1989) 473-480. 11 Fowler, C.J., ThoreU, G., Andersson, M. and Magnusson, O., Is inhibition of striatal synaptosomal tyrosine hydroxylation by dopamine agonists a measure of dopamine autoreceptor function?, Naunyn-Schmiedebergs Arch. Pharmacol., 331 (1985) 1219. 12 Gelbard, H.A., Teicher, M.H., Faedda, G. and Baldessarini, R.J., Postnatal development of dopamine Da and D 2 receptor sites in rat striatum, Dev. Brain Res., 49 (1989) 123-130. 13 Goldstein, M., Harada, K., Meller, E., Schalling, M. and H6kfelt, T., Dopamine autoreceptors: biochemical, pharmacological and morphological studies, Ann. N.Y. Acad. Sci., 604 (1990) 169-175. 14 Hedner, T. and Lundborg, P., Development of dopamine autoreceptors in the postnatal rat brain, J. Neural. Transm., 62 (1985) 53-63. 15 Huttenlocher, P.R. and de Courten, C., The development of synapses in striate cortex of man, Hum. Neurobiol., 6 (1987) 1-9. 16 Imperato, A. and Di Chiara, G., Effects of locally applied D-1 and D-2 receptor agonists and antagonists studied with brain dialysis, Eur. J. Pharmacol., 156 (1988) 385-393. 17 Kato, E., Koketsu, K., Kuba, K. and Dumamoto, E., The
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31
32
mechanism of the inhibitory action of adrenaline on transmitter release in bullfrog sympathetic ganglia: independence of cyclic AMP and calcium ions, Br. J. Pharmacol., 84 (1985) 435-443. Keppel, G., Design and Analysis: A Researcher's Handbook. Prentice-Hall, Englewood Cliffs, 1973, pp. 355-360. Lundborg, P., Hedner, T. and Engel, J., Catecholamine concentration in the developing rat brain after y-hydroxybutyric acid, J. Neurochem., 35 (1980) 425-429. Markstein, R., Seller, M.P., Vigouret, J.M., Urwyler, S., Enz, A. and Dixon, K., Pharmacologic properties of CY-208-243, a novel D-1 agonist. In M. Sandier, A. Dahlstrom and R.H. Belmaker (Eds.), Progress in Catecholamine Research, Part B: Central Aspects, Alan Liss, New York, 1988, pp. 59-64. Markstein, R., Vigouret, J.M., Enz, A., Coward, D., Jaton, A. and Briner, V., Dopaminergic ergots. In P.M. Beact, G~N. Woodruff and D.M. Jackson (Eds.), Pharmacology and Functional Regulations of Dopaminergic Neuronsl Macmillan, London, 1988, pp. 22-29. Murray, A.M. and Waddington, J.L., The interaction of clozapine with dopamine D-1 vs. dopamine D-2 receptor mediated functions: behavioral indices, Eur. J. Pharmacol., 186 (1990) 79-86. Petitto, J.M., Cook, L,L., Lewis, M.H., Nichols, D.E. and Mailman, R.B., Multiple Dl-like dopamine receptors not linked to adenylate cyclase exhibit different affinities for SCH-23390, Eur. J. Pharmacol., in press. Seeman, P., Bzowej, N.H., Guan, H.C., Bergeron, C., Becker, L.E., Reynolds, G.P., Bird, E.D., Riederer, P., JeUinger, K., Watanabe, S. and Tourtellone, W.W., Human brain dopamine receptors in children and aging adults, Synapse, 1 (1987) 399404. Shalaby, I.A., Dendel, P.S. and Spear, L.P., Differential functional ontogeny of dopamine presynaptic receptor regulation, Dev. Brain Res., 1 (198i) 434-439. Shalaby, I.A. and Spear, L.P., Psychopharmacological effects of low and high doses of apomorphine during ontogeny, Eur. J. Pharmacol., 67 (1980) 451-459. Sokoloff, P., Giros, B., Martres, M-P., Bouthenet, M-L. and Schwartz, J-C., Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics, Nature, 347 (1990) 146-151. Starke, K., Regulation of noradrenaline release by presynaptie receptor systems, Rev. Physiol. Biochem PharmacoL, 77 (1977) 1-124. Sunahara, R.K., Guan, H.-C., O'Dowd, B.F., Seeman, P.. Laurier, L.G., Ng, G., George, S.R., Torchia, J., Van Tol, H.H.M. and Niznik, H.B., Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D 1, Nature, 350 (1991) 614-619. Teieher, M.H. and Baldessarini, R.J., Developmental pharmacodynamics. In. C. Popper (Ed.), Psychiatric Pharmacosciences of Children and Adolescents, APA, Washington DC, 1987, pp. 47-80. Teicher, M.H., Barber, N.I., Reichheld, J.H., Baldessarini, R.J. and Finklestein, S.P., Selective depletion of cerebral norepinephfine with 6-hydroxydopamine and GBR-t2909 in neonatal rats, Dev. Brain Res., 30 (1986) 124-128. Van Tol, H.H.M., Bunzow, J.R., Guan, H.-C., Sunahara, R.K., Seeman, P., Niznik, H.B. and Civelli, O., Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine, Nature, 350 (1991) 610-614.
235 33 Waiters, J.R. and Roth, R.H., Dopaminergic neurons: an in vivo system for measuring drug interactions with presynaptic receptors, Naunyn-Schmiedeberg 's Arch. Pharmacol., 296 (1976) 5-14. 34 Wanatabe, H., Suda, H., Sekihara, S. and Nomura, Y., D t type of dopamine autoreceptors are not involved in the regulation of
dopamine synthesis in the striatum, Jpn. J. Pharmacol., 43 (1987) 327-330. 35 Wolf, M.E. and Roth, R.H., Dopamine autoreceptors. In I. Creese and C.M. Fraser (Eds.), Dopamine Receptors, Alan Liss, New York, 1987, pp. 45-96.
Developmental Brain Research, 63 (1991) 229-235 Elsevier ADONIS 016538069151359X BRESD 51359
D o p a m i n e D 1 autoreceptor function: possible expression in
developing rat prefrontal cortex and striatum Martin H. Teicher 1, Amelia L. Gallitano 1, Harris A. Gelbard 2., Henriette K. Evans 1, Elda R. Marsh 1, Raymond G. Booth 1'4 and Ross J. Baldessarini 1'3 Departments of 1Psychiatry, 2Neurology and 3Neuroscience Program, Harvard Medical School Mailman Laboratories for Psychiatric Research, McLean Hospital Belmont, MA 02178 (U.S.A.) and 4Department of Medicinal Chemistry, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7360 (U.S.A.) (Accepted 23 July 1991)
Key words: Dopamine; Presynaptic receptor; Autoreceptor; Dopamine 1 receptor; Prefrontal cortex; Corpus striatum; Dopamine agonist
Synthesis-modulating dopamine (DA) autoreceptor function was studied in vivo using y-butyrolactone (GBL) to block propagation along DA axons. DA synthesis was measured by the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) after inhibition of aromatic L-amino acid decarboxylase. GBL treatment markedly increased DOPA accumulation in both the striatum and prefrontal cortex of developing rats. The selective DA partial D 1 agonist SKF-38393 inhibited this GBL-induced rise in DA synthesis in both the striatum and prefrontal cortex of 15- and 22-day-old rats, but not in adults. The effects of SKF-38393 in developing rats were mimicked by the non-catechol D1 partial agonist CY-208-243, and were blocked by the D 1 antagonist SCH-23390, suggesting receptor mediation. The mixed D2/D3 agonist quinpirole attenuated DA synthesis in striatum of both two-week-old and adult rats, but failed to inhibit the GBL-induced increase in DA synthesis in the developing prefrontal cortex. These findings suggest that synthesis-modulating Dl-like receptor function may emerge transiently in the developing mammalian forebrain. In the adult striatum these functions appear to be subsumed by D2-1ike receptors, whereas all synthesismodulating DA receptor function in prefrontal cortex appears to be essentially lost with maturation. INTRODUCTION Dopamine ( D A ) receptors in the central nervous system can be subdivided by their anatomical localization as well as their physiochemical properties, pharmacology and molecular structure. The majority of D A receptors lie across the synaptic cleft on n o n - D A neurons, and are referred to as postsynaptic receptors. Neuropharmacological, electrophysiological, behavioral and histochemical data also suggest that D A receptors exist in D A cell bodies and dendrites in midbrain, and on terminals in some forebrain regions 13'34. The latter 'autoreceptors' function to modulate neuronal activity and the synthesis and release of D A 34. Until recently, synthesis modulating D A autoreceptors appeared to be exclusively of the D 2 type 7'32. Recently, a D 3 receptor has been cloned and characterized, and may function as both a postsynaptic receptor and an autoreceptor 26. Although a D2-1ike D 4 receptor 31, and a Dl-like D 5 receptor 27 have also been cloned and partially characterized, their physiological roles remain to be elucided. Attempts to detect synthesis-modulating D 1 autoreceptors in the adult rat striatum
have failed to elicit any compelling evidence for their existence 7'33. However, the density of D 1 receptors in forebrain appears to change markedly with development. In both rats 12 and man 23, the density of D1 receptors enigmatically rises and then falls several-fold during the postnatal period. We hypothesized that the early postnatal surge in DI receptor density might include some D1 autoreceptors, followed by selective loss of this class of autoreceptors with further maturation. We now report the interesting possibility that synthesis-modulating D~ receptors may arise and function transiently in both the striatum and prefrontal cortex between postnatal days 15 and 22, but cease to function by day 60. The present findings suggest, more generally, that certain receptor classes may be gained and lost during development and encourages speculation that certain neuropsychiatric disorders may stem from the failure of these receptor types to arise or recede at the appropriate time. MATERIALS AND METHODS Lactating female Sprague-Dawley rats were housed with their litters on a 12 h light-dark cycle (lights on: 07.00-19.00 h) with food
* Present address: Division of Pediatric Neurology, Box 631, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, U.S.A. Correspondence: M.H. Teicher, McLean Hospital, 115 Mill Street, Belmont, MA 02178, U.S.A. Fax: (1) (617) 855-3299.
230 and water provided ad libitum. Litters were culled to 10 pups of either sex on postnatal day 1 and, in long-term experiments, were weaned at day 28. Pups were sacrificed on postnatal days 15, 22 and 60 in some experiments, and at 16 days and adulthood (ca. 275 g body weight) in others. Their brains were rapidly removed, chilled, and dissected on ice. Corpus striatum and mesial prefrontal cerebral cortex (PFCTX) were individually dissected as previously described 3°, and stored at -70 °C. For HPLC determination of DOPA (L-3,4-dihydroxyphenylalanine) concentrations, individual tissue samples were homogenized in 1.0 ml of 0.1 N perchloric acid, 0.2 mM EDTA, and 0.4 mM sodium metabisulfite, Homogenates were extracted and assayed by slight modification of the methods of Baldessarini et al. 4. Sample aliquots (100/d) were separated by high pressure liquid chromatography (HPLC) on a C-18, reverse-phase, 5 /,m resin particle column measuring 4.6 mm × 25 cm, and analytes were detected using a Bioanalytical System electrochemical detector (+0.55 V vs Ag/AgCI reference electrode). The mobile phase consisted of 0.05 M NaOAc.3H20, 0.1 mM EDTA, 1.5% acetonitrile (vols.), and 4.2 mM 1-heptanesulfonic acid (pH 3.5). The recovery of authentic catechols consistently exceeded 85%, The presence of synthesis-modulating autoreceptors were explored using the y-butyrolactone (GBL) technique of Waiters and Roth 33. In this paradigm, tyrosine hydroxylase activity was measured as accumulation of DOPA for 30 min following administration of the decarboxylase inhibitor, m-hydroxyphenylhydrazine (NSD-1015). GBL was administered to block propagation along DA axons, thus isolating DA terminals from long-loop feedback effects that can accompany postsynaptic receptor stimulation. We hypothesized that, if synthesis-modulating D ~ autoreceptors were present, then the selective D~ agonist SKF-38393 would antagonize DOPA accumulation induced by GBL and this effect, in turn, would be blocked by administration of the selective D ~ antagonist SCH-23390. The sequence of treatments, given by intraperitoneal (i.p.) injections, are summarized in Table I; group sizes are indicated in the accompanying figures. A total of 300 rats were used in these studies. In the first study, the effects of SKF-38393 and SCH-23390 on DOPA accumulation in striatum and PFCTX were explored in 15-, 22- and 60-day-old rats using the GBL technique. In the second study, the age-dependent effects of the D l agonist CY-208-243 were compared to the effects of SKF-38393. This comparison was made because SKF-38393 is a catechol and may produce direct chemical inhibition of tyrosine hydroxylase 2,7,2o, possibly confounding the interpretation of its action as an autoreceptor agonist. CY208-243 is a non-catechol D t receptor agonist, that does not directly inhibit tyrosine hydroxylase activity L 16.20.21. Although there is some concern that the monohydroxyergoline CY-208-243 has a relatively high affinity for D 2 as well as D~ binding sites 2"2°, it lacks any discernable intrinsic D 2 agonist effects (e.g., can not induce stereotypic behavior, nor induce emesis in dogs, and has no effect on plasma prolactin levels2°). In contrast, CY-208-243 has clear intrinsic partial D z agonist effects. It produces characteristic grooming behavior 2°, stimulates DA-sensitive adenylate cyclase 2°, and in-
duccs rotation in the unilateral 6-hydroxydopaminc model that can be antagonized by SCH-23390 but not by the D~ antagonist sulpride 2°. Several other D~ agonists arc known, including some with greater intrinsic activity and selectivity in ligand binding studies, however all these agents arc catechols 29, and thus can not serve as adequate controls for the potential catechol effect of SKF-38393. In the third study, we explored the effects of lhe probably mixed D2/D 3 agonist quinpirole (LY-171555) on DOPA accumulation in the striatum of 15-day-old and adult rats. The affinity of quinpirole for the D 3 receptor is over 100-fold greater than its affinity for the D e receptor 26, and it is the most selective known D+ agonist. Since relatively large doses were used here (1 mg/kg), it is likely that both receptor types were affected. Quinpirole was also paired with spiperone, a potent antagonist of both D e and D~ rcceptorsZC'~
Statistical analysis Effects of age, drug treatment, and their interaction were determined using unweighted ANOVA for unequal group sizetS. Individual planned comparisons were made using Student's t-test based on predicted effects. Materials SCH-23390, SKF-38393, and spiperone were obtained from Research Biochemicals Incorporated (RBI, Natick, MA). Generous donations of drugs included quinpirole by Eli Lilly and Sons Laboratories (Indianapolis, IN), and CY-208-243 by Dr. Paul Herrling of the Sandoz-Wander Research Institute (Berne, Switzerland). GBL, NSD-1015, and the various HPLC reagents were obtained from Sigma Chemical Company (St. Louis, MO) in the highest available purity.
RESULTS
D O P A accumulation in striatum and prefrontal cerebral cortex after SKF-38393 A s s h o w n in Fig. 1, D O P A a c c u m u l a t i o n in s t r i a t u m was m a r k e d l y a f f e c t e d by age (F2,9o = 51.1, P < 0.001), d r u g c o n d i t i o n (Fs.9o = 42.2, P < 0 . 0 0 t ) , and their int e r a c t i o n (F6.9o = 3.9, P < 0.01). G B L (750 m g / k g ) inc r e a s e d D O P A a c c u m u l a t i o n by a b o u t 1 0 0 % at e a c h age (all P < 0.0005). T h e G B L - i n d u c e d rise in D O P A
ac-
c u m u l a t i o n was a n t a g o n i z e d by 56% with 10 m g / k g S K F 38393 at 15 days ( P < 0.0001), and b y 33% at 22 days ( P < 0.05), but was n o t a n t a g o n i z e d at 60 days, S C H 23390 at 10 m g / k g fully a t t e n u a t e d t h e effects o f S K F 38393 at 15 ( P < 0.005) and 22 days ( P < 0.05). A s s h o w n in Fig. 2, D O P A a c c u m u l a t i o n in P F C T X ,
TABLE I
Sequence of treatments CY, CY-208-243 (10 mg/kg); GBL, y-butyrolactone (750 mg/kg); NSD, NSD-1015 (100 mg/kg); QNP, quinpirole (LY-171,555, 1 mg/kg)~ sal, 0.9% (w/v) NaCI (saline); SCH, SCH-23390 (10 mg/kg); SKF, SKF-38393 (10 mg/kg); SPI, spiperone (1 mg/kg). Rats were sacrificed at 0 min; test agents were all administered intraperitoneally (i.p.).
Time (rain) Control
GBL
SKF
CY
QNP
SCH
SP1
SCH+SKF
SCH+CY
SPI+QNP
-45 -~ -35 -30
sal sal GBL NSD
sal SKF GBL NSD
sal CY GBL NSD
sal QNP GBL NSD
SCH sal GBL NSD
SPI sal GBL NSD
SCH SKF GBL NSD
SCH CY GBL NSD
SPI QNP GBL NSD
sal sal sal NSD
231 STRIA TUM
S TRIA TUM
4
[] • [] •
l
SAUSAWSAL SAUSAUGBL I SAWSKF/GBL I SCH/SKF/GBLI
3
16-DAYS
ADULT
AGE GROUP
~
O, = 22
15
60
AGE (d)
Fig. 1. Effects of 7-butyrolactone (GBL) and SKF-38393 (SKF), alone or with SCH-23390 (SCH), on accumulation of L-3,4,-dihydroxyphenylalanine (DOPA) in corpus striatum of rats aged 15, 22, or 60 days after pretreatment with NSD-1015. Doses of agents (given i.p.) are summarized in Table I. Data are means of stated numbers of subjects (at the base of each bar) -+ S.E.M. too, was markedly affected by age (/'2,70 = 24.3, P < 0.001), drug treatment (/'3,7o = 10.4, P < 0.001), and their interaction (F6,7o = 3.2, P < 0.01). G B L produced a 100% increase in D O P A levels at 15 days (P < 0.005), a 34% increase at 22 days (P < 0.02), and only a 23% increase at 60 days (P < 0.05). As in striatum, the effects of G B L on D O P A accumulation were fully reversed by SKF-38393 at 15 days (P < 0.005) and 22 days (P < 0.02) and, in turn, SKF-38393 was antagonized by SCH-23390 at these young ages (both P < 0.05), but neither SKF-38393 alone nor in combination with SCH23390 had a significant effect at 60 days.
PREFRONTAL CORTEX
0.3 ~
/
'~
~, 8
[
T
|
i [I SAL~AUGBL.~
•
I
! llm SO:I,,S,.:~,G.1 l
n
/
) / . S, SKF,GB' /
o.~
0.0
15
22 AGE (d)
60
Fig. 2. Effects of GBL, SKF and SCH on accumulation of DOPA in prefrontal cerebral cortex of rats aged 15, 22, or 60 days, as described for Fig. 1 and in Table I.
Fig. 3. Effects of GBL, SKF or CY-208-243 (CY), alone or after SCH, on accumulation of DOPA in corpus striatum of rats aged 16 days or adults, as described for Fig. 1 and in Table I.
DOPA accumulation in striatum after CY-208-243 In this replication experiment (Fig. 3) there were again strong effects of age (F1,87 = 28.8, P < 0.001) and drug (F5,87 = 22.7, P < 0.001), and a significant age by drug interaction (F5,87 = 2.35, P < 0.05). G B L increased D O P A accumulation by about 3-fold in both 16-day-old and adult rats (both P < 0.0005). In 16-day-olds, D O P A accumulation was inhibited by about 50% by 10 mg/kg of either CY-208-243 or SKF-38393 (both P < 0.01). The effects of SKF-38393 were fully inhibited by 10 mg/kg SCH-23390 (P < 0.01). Inexplicably, the combination of CY-208-243 and SCH-23390 proved lethal to 16-day-olds in the G B L paradigm (n = 6), so that data on the antagonism of CY-208-243 by SCH-23390 were not obtained for young rats. In adult striatum (Fig. 3) there was no significant effect of the same dose of SKF-38393 on D O P A accumulation. CY-208-243 produced a slight, but significant, 24% decrease in D O P A accumulation in the adult striatum (P < 0.05), that was not antagonized by the D 1 antagonist SCH-23390 (Fig. 3). Thus, CY-208-243 may have some pharmacological or toxic effects that are not mediated by DI receptors. In 16-day-olds, SCH-23390 alone (10 mg/kg) produced a 23% increase in striatal D O P A accumulation over that induced by G B L alone and, in adults, this D 1 antagonist produced a 13% increase. While these observations may suggest some degree of tonic D 1 inhibition, particularly in the preweanling rat, neither effect attained statistical significance (P > 0.10; Fig. 3).
DOPA accumulation in striatum after quinpirole Overall, there were main effects of age (F1,48 = 13.6, P < 0.001), and drug (F3,49 = 33.0, P < 0.001), but no significance interaction (F3,49 = 1.92, ns), indicating that
232 STRIA TUM
==
=¢ 0
0 ¢3
15-DAYS
ADULTS
AGE GROUP
Fig. 4. Effects of GBL and quinpirole (QNP) alone or with spiperone (SPI) on accumulation of DOPA in corpus striatum of 15day-old or adult rats, as described for Fig. 1 and in Table I.
the pattern of response was comparable at the two ages tested (Fig. 4). GBL increased DOPA accumulation by 120% in the striatum of 16-day-old rats (P < 0.0001), and by 60% in adults (P < 0.005). Quinpirole (1 mg/kg) fully antagonized the effects of GBL in 16-day-olds (P < 0.0001). In adults, this agent not only reversed the effects of GBL (P < 0.0005), but reduced DOPA accumulation below the level obtained in the saline control group (P < 0.0005). Spiroperidol (1 mg/kg) fully antagonized the effects of quinpirole in both 16-day-olds (P < 0.0001) and adults (P < 0.005). Combined treatment of 16-day-olds with SKF-38393 and quinpirole produced only a 7% greater inhibitory effect than quinpirole alone, which though statistically significant (P < 0.05; data not shown), probably has little biological meaning.
DOPA accumulation in prefrontal cortex of 16-day-old rats after quinpirole In this experiment there were main effects of treatment (F2,15 = 5.85, P < 0.02) with GBL (n = 7), which increased D O P A accumulation by 50% relative to saline treated controls (n = 6; P < 0.005). However, quinpirole (n = 5) failed to antagonize the effects of GBL in this brain region, producing only a statistically insignificant 7% reduction (data not shown). DISCUSSION
Two major observations emerge from these experiments. First, important age-related differences in D O P A accumulation were found in prefrontal cortex. GBL induced a prominent increase in tyrosine hydroxylation in 15-day-old rats, but only a modest increase at 22 days, and a trivial increase at 60 days (Fig. 2). This observa-
tion suggests that synthesis-modulating DA receptors may be present in the PFCTX during early development, but may cease to exist, or at least not function, later in development. It is also interesting that the partial I) 1 agonist SKF-38393 fully antagonized the GBL effect in the developing PFCTX, and that SCH-23390 blocked these agonist effects (Fig. 2), whereas the mixed D2/D 3 agonist quinpirole failed to attenuate DOPA accumulation in PFCTX. These observations suggest that DA synthesis-modulating receptors may appear transiently in the prefrontal cerebral cortex, and that they may belong to the D 1 class. Second, the D 1 agonists SKF-38393 and CY-208-243 antagonized, by about 50%, the GBL-induced increase in accumulation of DOPA in the corpus striatum of 11516-day-old rats (Figs. 1 and 3). The effects of SKF-38393 were fully blocked by the D~ antagonist SCH-23390, suggesting again that these effects were mediated by D~ receptors. On the other hand, SKF-38393 exerted little or no effect on DOPA accumulation of 60-day-old or adult rats (Figs. 1 and 3). Thus, synthesis-modulating D 1 receptors may be present in the striatum of developing rats, but cease to function as the animal matures. On the other hand, the mixed D2/D 3 agonist quinpirole exerted a consistent and powerful effect on striatal DOPA accumulation across age (Fig. 4). Thus, there may be different populations of synthesis-modulating DA receptors in the striatum, and they may have a dissimilar developmental time course. The possibility that synthesis-modulating D~ receptors might arise transiently during development, and particularly in PFCTX, is interesting and unexpected. In general, the emergence of a function followed by its loss is consistent with many observations on the synaptogenesis of the developing nervous system, in many brain regions there is a marked increase in the density of synaptic contacts during early postnatal development, followed by prominent pruning during adolescence 15. Such a time course seems to be followed by D 1 receptors in the rat striatum ~2. On the other hand, the concept of a Dt autoreceptor is somewhat perplexing, as DA has a much weaker (i.e.,/~M) apparent affinity for the D 1 receptor, that it does for D 2 (nM), D 3 o r D4 (sub-nM) receptors 26"31. Some models of presynaptic autoreceptor function hypothesize that terminal autoreceptors are highly sensitive and responsive to released transmitter, and provide exquisite feedback control over the range of physiological activity2s. Other theoretical models, however, suggest that some autoreceptors are relatively insensitive to released transmitter, and only come into play during periods of excessive activation 3, possibly to prevent the system from entering into a pathological range of activity ~7. It is possible that the developing nervous
233 system transiently requires a low sensitivity feedback control mechanism on DA synthesis in the prefrontal cortex, and may utilize this process as a redundant emergency backup system in the striatum. The concept of a D1 autoreceptor may appear untenable if one posits that the majority of synthesis-modulating autoreceptors are D 2 receptors that are coupled by an inhibitory G-protein to adenylate cyclase 6'8, and presume that a D 1 autoreceptor would have to exert opposite effects through a stimulatory G-protein. It is now clear that there is sufficient heterogeneity in the coupling mechanism of D1 and D2 receptor families 8'22'26 to encompass the possibility of a subclass of synthesis-modulating D1 presynaptic receptors. The strongest additional experimental support for the existence of synthesis-modulating D 1 receptors derives from the work of Berry and Gudelsky 5. They found that both SKF-38393 and CY-208-243 attenuated D O P A accumulation (after NSD-1015) in tuberoinfundibular DA neurons of adult rat hypothalamus after activation of DA turnover by haloperidol, reserpine, or neurotensin. However, these D 1 agonists failed to exert any effect on the basal rate of DA synthesis. Thus, D1 receptor stimulation appears to have a strong modulatory role on the rate of D A synthesis when such neurons are in a stimulated state. There are, however, a number of caveats that are relevant to the interpretation of the present findings. First, apparent age-related differences in autoreceptor function could be an artifact of ontogenic differences in the action of GBL. However, one study of developmental differences in response to 7-hydroxybutyrate (GHB, the active metabolic product of GBL), found that the doseresponse for accumulation of DA following GHB was nearly identical at 14 days and 4 weeks of age 19, indicating that a developmental change in the potency of this agent, and presumably GBL, is unlikely to be a significant factor in the present study. It should also be acknowledged that the GBL paradigm, while experimentally convenient, provides only an indirect means of exploring autoreceptor function. Although this technique largely isolates DA terminals from long-loop, multi-synaptic, feedback regulation via DA axons, it does not suppress local feedback from interneurons which contain postsynaptic D 1 receptors, and which may reciprocally communicate with DA terminal heteroreceptors that can modulate tyrosine hydroxylase activity 6. In this regard, we have not been able to extend the present D~-like autoreceptor effects to isolated minces or synaptosomal preparations from the developing rat striatum. SKF-38393 inhibited tyrosine hydroxylase activity, but this effect was not reversed by SCH-23390 (R. Booth and R.J. Baldessarini, unpublished observations, 1990).
However, such in vitro preparations may be limited in their ability to model autoreceptor function 7"11, and the operation of the autoreceptor may depend on the integrity of a cascade of molecular events in the terminal that may not be preserved in isolated tissue, or may require stimulation or interactions with other transmitter systems that are not readily duplicated in vitro 5. Thus, this initial study leaves unresolved the question as to whether these apparent transient synthesis-modulating D l-like receptors are located on dopamine terminals, and can rightfully be called 'autoreceptors', or whether they are postsynaptic receptors, and act on DA terminals indirectly through heteroreceptors. We have chosen to refer to the effects of these receptors on dopamine synthesis as 'autoreceptor function', aware that these receptors may not be presynaptic. This caveat applies to all studies that utilize the GBL technique or similar procedures. Relatively little attention has been given to the ontogeny of DA autoreceptor function in mammalian brain. Behavioral studies utilizing low doses of DA agonists (apomorphine or 3-[3-hydroxyphenyl]-N-n-propylpiperidine [3PPP]) to probe for autoreceptor-induced behavioral suppression generally have failed to elicit evidence for DA autoreceptor function until 28-35 days of age 14, 25. We have found however, that 8-day-old rats, when sufficiently aroused by maternal deprivation, can display marked low-dose behavioral suppression with R(-) N-npropylnorapomorphine 29. To our knowledge, there are only two reports on the ontogeny of synthesis-modulating DA autoreceptors using the GBL technique. Shalaby et al. 25 studied the accumulation of DA in striatum and olfactory tubercle in 7- to 90-day-old rats after GBL and apomorphine (no decarboxylase inhibitor was used). GBL significantly increased D A accumulation in the striatum even at day 7, but was not reversed by apomorphine until day 14. In the olfactory tubercle, GBL failed to produce a significant increase in DA until day 21, and effects of apomorphine did not clearly emerge until day 35. Hedner and Lundborg 14 reported that (---)3PPP inhibited DOPA accumulation after GBL and NSD-1015 in striatum of 28-day-old rats, but did not do so in 4-dayolds. In conclusion, the present study adds to our understanding of the development of dopaminergic autoreceptor function, suggesting that both Dl-like and D2-1ike synthesis-modulating receptors may be present in the striatum of two-week-old rats, and that synthesis-modulating autoreceptors of the D 1 type may also be present in the prefrontal cortex temporarily at this age. With further maturation there is an apparent loss of synthesis-modulating receptor function (and GBL effect) in prefrontal cortex, and a loss of the synthesis-modulating effects of D1 receptor stimulation in striatum. Further
234 studies are needed to ascertain whether synthesis-modulating D 1 (or D5) receptors exist at earlier ages, and to ascertain whether probes for such receptors or their gene messages can be localized in D A cell bodies or terminals at relevant ages.
Acknowledgements. Supported in part by USPHS (N1HM) Grants MH-43743, MH-34006, MH-31154 and MH-47370, as well as awards from the Charles Dana Foundation, Scottish Rite Schizophrenia Research Program, Hall Mercer Foundation, Snider Family, the Bruce J. Anderson Foundation, Milton Foundation, and Marion Ireland Benton Trust.
REFERENCES I Abbott, B., Starr, B.S. and Starr, M., CY-208-243 behaves as a typical D-1 agonist in the reserpine-treated mouse, Pharmacol. Biochem. Behav., 38 (1991) 259-263. 2 Andersen, P.H. and Jansen, J.A., Dopamine receptor agonists selectivity and dopamine D1 receptor efficacy, Eur. J. Pharmacol. Mol. Pharmacol. Sect., 188 (1990) 335-347. 3 Angus, J.A., Korner, P.I., Jackman, G.P., Bobik, A. and Kopin, I.J., Role of autoinhibitory feedback in cardiac sympathetic transmission, Clin. Exp. Hypertens, A6 (1984) 371-385. 4 Baldessarini, R.J., Marsh, E.R., Kula, N.S., Zong, R., Gao, Y. and Neumeyer, J.L., Effects of isomers of hydroxyaporphines on dopamine metabolism in rat brain regions, Biochem. Pharmacol., 40 (1990) 417-423. 5 Berry, S.A. and Gudelsky, G.A., D x receptors function to inhibit the activation of tuberoinfundibulardopamine neurons, J. Pharmacol. Exp. Ther., 254 (1990) 677-682. 6 Booth, R.G. and Baldessarini, R.J., Adenosine A 2 stimulation of tyrosine hydroxylase in rat striatal minces is reversed by dopamine D2 autoreceptor activation, Eur. J. Pharmacol., 185 (1990) 217-221. 7 Booth, R.G., Baldessarini, R.J., Kula, N.S., Gao, Y., Zong, R. and Neumeyer, J.L., Presynaptic inhibition of dopamine synthesis in rat striatal tissue by enantiomeric mono- and dihydroxyaporphines, Mol. Pharmacol., 38 (1990) 92-101. 8 Bowyer, J.G. and Weiner, N., K+ channel and adenylate cyclase involvement in regulation of Ca2+-evoked release of [3H]dopamine from synaptosomes, J. Pharmacol. Exp. Ther., 248 (1989) 514-519. 9 Brewster, W.K., Nichols, D.E., Riggs, R.M., Mottola, D.M., Lovenberg, T.W., Lewis, M,H. and Mailman, R.B., trans-lO,11Dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]-phenanthridine: a highly potent selective dopamine D a full agonist, J. Med. Chem., 33 (1990) 1756--1764. 10 Faedda, G.L., Kula, N.S. and Baldessarini, R.J., Pharmacology of binding of 3H-SCH-23390, a ligand selective for D1 dopamine receptor sites, in rat brain tissue, Biochem. Pharmacol., 38 (1989) 473-480. 11 Fowler, C.J., ThoreU, G., Andersson, M. and Magnusson, O., Is inhibition of striatal synaptosomal tyrosine hydroxylation by dopamine agonists a measure of dopamine autoreceptor function?, Naunyn-Schmiedebergs Arch. Pharmacol., 331 (1985) 1219. 12 Gelbard, H.A., Teicher, M.H., Faedda, G. and Baldessarini, R.J., Postnatal development of dopamine Da and D 2 receptor sites in rat striatum, Dev. Brain Res., 49 (1989) 123-130. 13 Goldstein, M., Harada, K., Meller, E., Schalling, M. and H6kfelt, T., Dopamine autoreceptors: biochemical, pharmacological and morphological studies, Ann. N.Y. Acad. Sci., 604 (1990) 169-175. 14 Hedner, T. and Lundborg, P., Development of dopamine autoreceptors in the postnatal rat brain, J. Neural. Transm., 62 (1985) 53-63. 15 Huttenlocher, P.R. and de Courten, C., The development of synapses in striate cortex of man, Hum. Neurobiol., 6 (1987) 1-9. 16 Imperato, A. and Di Chiara, G., Effects of locally applied D-1 and D-2 receptor agonists and antagonists studied with brain dialysis, Eur. J. Pharmacol., 156 (1988) 385-393. 17 Kato, E., Koketsu, K., Kuba, K. and Dumamoto, E., The
18 19
20
21
22
23
24
25 26
27
28 29
30
31
32
mechanism of the inhibitory action of adrenaline on transmitter release in bullfrog sympathetic ganglia: independence of cyclic AMP and calcium ions, Br. J. Pharmacol., 84 (1985) 435-443. Keppel, G., Design and Analysis: A Researcher's Handbook. Prentice-Hall, Englewood Cliffs, 1973, pp. 355-360. Lundborg, P., Hedner, T. and Engel, J., Catecholamine concentration in the developing rat brain after y-hydroxybutyric acid, J. Neurochem., 35 (1980) 425-429. Markstein, R., Seller, M.P., Vigouret, J.M., Urwyler, S., Enz, A. and Dixon, K., Pharmacologic properties of CY-208-243, a novel D-1 agonist. In M. Sandier, A. Dahlstrom and R.H. Belmaker (Eds.), Progress in Catecholamine Research, Part B: Central Aspects, Alan Liss, New York, 1988, pp. 59-64. Markstein, R., Vigouret, J.M., Enz, A., Coward, D., Jaton, A. and Briner, V., Dopaminergic ergots. In P.M. Beact, G~N. Woodruff and D.M. Jackson (Eds.), Pharmacology and Functional Regulations of Dopaminergic Neuronsl Macmillan, London, 1988, pp. 22-29. Murray, A.M. and Waddington, J.L., The interaction of clozapine with dopamine D-1 vs. dopamine D-2 receptor mediated functions: behavioral indices, Eur. J. Pharmacol., 186 (1990) 79-86. Petitto, J.M., Cook, L,L., Lewis, M.H., Nichols, D.E. and Mailman, R.B., Multiple Dl-like dopamine receptors not linked to adenylate cyclase exhibit different affinities for SCH-23390, Eur. J. Pharmacol., in press. Seeman, P., Bzowej, N.H., Guan, H.C., Bergeron, C., Becker, L.E., Reynolds, G.P., Bird, E.D., Riederer, P., JeUinger, K., Watanabe, S. and Tourtellone, W.W., Human brain dopamine receptors in children and aging adults, Synapse, 1 (1987) 399404. Shalaby, I.A., Dendel, P.S. and Spear, L.P., Differential functional ontogeny of dopamine presynaptic receptor regulation, Dev. Brain Res., 1 (198i) 434-439. Shalaby, I.A. and Spear, L.P., Psychopharmacological effects of low and high doses of apomorphine during ontogeny, Eur. J. Pharmacol., 67 (1980) 451-459. Sokoloff, P., Giros, B., Martres, M-P., Bouthenet, M-L. and Schwartz, J-C., Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics, Nature, 347 (1990) 146-151. Starke, K., Regulation of noradrenaline release by presynaptie receptor systems, Rev. Physiol. Biochem PharmacoL, 77 (1977) 1-124. Sunahara, R.K., Guan, H.-C., O'Dowd, B.F., Seeman, P.. Laurier, L.G., Ng, G., George, S.R., Torchia, J., Van Tol, H.H.M. and Niznik, H.B., Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D 1, Nature, 350 (1991) 614-619. Teieher, M.H. and Baldessarini, R.J., Developmental pharmacodynamics. In. C. Popper (Ed.), Psychiatric Pharmacosciences of Children and Adolescents, APA, Washington DC, 1987, pp. 47-80. Teicher, M.H., Barber, N.I., Reichheld, J.H., Baldessarini, R.J. and Finklestein, S.P., Selective depletion of cerebral norepinephfine with 6-hydroxydopamine and GBR-t2909 in neonatal rats, Dev. Brain Res., 30 (1986) 124-128. Van Tol, H.H.M., Bunzow, J.R., Guan, H.-C., Sunahara, R.K., Seeman, P., Niznik, H.B. and Civelli, O., Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine, Nature, 350 (1991) 610-614.
235 33 Waiters, J.R. and Roth, R.H., Dopaminergic neurons: an in vivo system for measuring drug interactions with presynaptic receptors, Naunyn-Schmiedeberg 's Arch. Pharmacol., 296 (1976) 5-14. 34 Wanatabe, H., Suda, H., Sekihara, S. and Nomura, Y., D t type of dopamine autoreceptors are not involved in the regulation of
dopamine synthesis in the striatum, Jpn. J. Pharmacol., 43 (1987) 327-330. 35 Wolf, M.E. and Roth, R.H., Dopamine autoreceptors. In I. Creese and C.M. Fraser (Eds.), Dopamine Receptors, Alan Liss, New York, 1987, pp. 45-96.