Developmental and age-related changes in D1-dopamine receptors and dopamine content in the rat striatum

Developmental Brain Research, 35 (1987) 283-290

283

Elsevier BRD 50604

Developmental and age-related changes in Dl-dopamine receptors and dopamine content in the rat striatum O. Giorgi 1, G. De Montis 2, M.L. Porceddu 1, S. Mele 1, G. Calderini 3, G. Toffano 3 and G. Biggio 1 1Department of Experimental Biology, Chairof Pharmacology and 2Instituteof Biochemical Pharmacology, University of Cagliari (Italy) and 3FIDIA Research Laboratories, Abano Terme (Italy) (Accepted 24 February 1987)

Key words: D rdopamine receptors; [3H]SCH 23390; Adenylate cyclase; Dopamine; Development; Aging; Striatum The relationship between the postnatal development of dopaminergic (DAergic) nerve endings and the maturation of D l DA receptots in the rat striatum was analyzed by measuring the content of DA and dihydroxyphenylacetic acid (DOPAC), two biochemical markers of DAergic nerve terminal proliferation, and the ontogenetic changes in [3H]SCH 23390 binding sites. DA-stimulated adenylate cyclase (AC) activity was also measured in order to characterize the coupling of [~H]SCH 23390 binding sites to the responses mediated by the activation of D 1 DA receptors. Striatal levels of DA and DOPAC, as well as the density and affinity of [3H]SCH 23390 binding sites and DAostimulated AC activity were also measured in senescent rats. The striatal content of DA increased slowly after birth, reaching adult levels by postnatal day 60 and remaining constant through adulthood and senescence (up to 20 months of age). The density of [3H]SCH 23390 binding sites increased 14-fold from birth to postnatal day 35, when a peak value was reached, whereas a significant decrease was observed in the striatum of aged rats. In contrast, the affinity of D 1 DA receptors for [3H]SCH 23390 remained unchanged from birth through senescence. The stimulation of cyclic AMP formation induced by 100/~M DA increased 4-fold from birth to postnatal day 14, when the maximal responsiveness to DA was observed and then returned to adult levels. No significant alterations were observed in the K mvalues during development, whereas the stimulatory effect of 100/~M DA on AC activity was significantly decreased in senescent rats. These findings are consistent with the view that the maturation of D 1DA receptors does not depend upon the proliferation of DAergic nerve endings in the rat striatum. Our results also indicate that the loss of striatal D 1DA receptots precedes the alterations in DA content observed in senescent rats. INTRODUCTION The establishment of neurotransmission in the developing nervous system requires outgrowth of functional presynaptic terminals as well as proliferation of n e u r o t r a n s m i t t e r receptors c o u p l e d to their response systems. It is unclear, however, w h e t h e r maturation of the different synaptic c o m p o n e n t s is a c o o r d i n a t e d process or if p r e s y n a p t i c nerve terminal proliferation p r e c e d e s and controls the d e v e l o p m e n t of postsynaptic r e c e p t o r s and r e c e p t o r - m e d i a t e d cellular events o r vice versa. Brain d o p a m i n e ( D A ) receptors have b e e n divided into two subtypes: D~ D A receptors, that increase cyclic A M P ( c A M P ) synthesis u p o n stimulation, and D2 D A receptors, that either decrease or have no el-

fect on c A M P synthesis 16,44. T h e r e is e x p e r i m e n t a l evidence that the develo p m e n t of D2 D A receptors, and the responses that they m e d i a t e , are d e p e n d e n t u p o n the m a t u r a t i o n of their corresponding presynaptic nerve terminals 6,35, although o t h e r reports do not s u p p o r t this view 5,3°. Similarly, it remains to be d e t e r m i n e d w h e t h e r the d e v e l o p m e n t of D : D A receptors linked to a d e n y l a t e cyclase ( A C ) is influenced by the presence of D A ergic terminals. Thus, the m a t u r a t i o n of D A - s t i m u lated A C activity in the striatum and o t h e r brain areas 43'45 precedes that of the presynaptic D A e r g i c neuron m a r k e r s 4,12.17. The aim of this study was to characterize the relationship b e t w e e n the p o s t n a t a l d e v e l o p m e n t of D A ergic nerve endings and the m a t u r a t i o n of D 1 D A re-

Correspondence: 0. Giorgi, Dept. of Experimental Biology, Chair of Pharmacology, University of Cagliari, Via Palabanda, 12, 1-09123 Cagliari, Italy. 0165-3806/87/$03.50 ~ 1987 Elsevier Science Publishers B.V. (Biomedical Division)

284 ceptors in the rat striatum. Hence, we examined the ontogeny of DA-stimulated AC activity. Moreover, in order to analyze the coupling of receptor binding sites to receptor-mediated responses, we studied the development of D 1 DA receptors labelled with [3H]SCH 23390, the most selective ligand for these receptors available to date 2'13'14. Striatal contents of DA and of its major metabolite dihydroxyphenylacetic acid (DOPAC) were measured by highpressure liquid chromatography (HPLC) to provide indices of DAergic nerve terminal proliferation. Different neurotransmitter systems of the mammalian brain, including the nigrostriatal DAergic pathway, undergo age-related changes (for a review see ref. 7). Thus, alterations in DA synthesis and metabolism 22 and in DA uptake 15'2°have been described in the brain of aged rats. Although several reports indicate that DA-stimulated AC activity is decreased in the striatum of senescent rats 1°'41, different patterns of age-related changes in D1 DA receptors have been described in different animal species. Thus, it has been shown that D1 DA receptors increase in the human caudate nucleus and putamen during normal aging 26, but decrease 27 or remain unchanged 29 in the rat striatum. In these studies D 1 DA receptors were labelled with antagonist ligands, like [3H]piflutixol and [3H]fluphenazine, which bind with similar affinities to D 2 and D 1 DA receptors 2. Therefore we used [3H]SCH 23390 as a selective probe to study age-related alterations in the density and/or affinity of D t DA receptors in the rat striatum. Age-related changes in DA-stimulated AC activity as well as in DA and DOPAC concentrations were also investigated. Our results indicate that [3H]SCH 23390 binding sites and DA-stimulated AC activity have quite similar developmental patterns and precede maturation of DAergic innervation in the rat striatum. Moreover, we provide evidence that age-related loss of striatal D~ DA receptors and DA-stimulated AC activity occurs well in advance of the reductions in D A content. MATERIALS AND METHODS

Animals Rat pups and adults were the offspring of Sprague-Dawley (Charles River, Como, Italy) rats bred

in our animal care unit. After mating, dams were singly housed in plexiglass cages with food and water freely available. Rats were weaned at 30 days of age and housed with same-sexed littermates until time of testing. Aged rats (14, 20 and 26 months old) and their respective 3-month-old controls were provided by the Italian Study Group on Brain Aging. Rats were decapitated at different times after birth (1 day to 26 months) and their brains were rapidly removed and placed on an ice-cold glass surface. Striata were dissected out and used for [3H]SCH 23390 binding assays, DA-sensitive and AC activity assays and for the determination of DA and DOPAC content by HPLC. Tissue from 90-day-old rats was included in each [3H]SCH 23390 binding assay, AC assay and catecholamine determination in striata obtained from rats of different ages. The results from some experiments are shown as percent of the values corresponding to the 90-day-old group assayed simultaneously with tissues from rats of different ages.

[3H]SCH 23390 binding Membrane preparation. Striata were homogenized with a Polytron PT 10 for 20 s in 100 volumes (w:v) of ice-cold 50 mM Tris-HC1 buffer, pH 7.4 and centrifuged for 10 min at 48.000 g. The pellet was washed once by resuspension and recentrifugation in 100 volumes of the same buffer. The final pellet was resuspended in 200 volumes of 50 mM Tris-HCl buffer, pH 7.4, containing 120 mM NaCi, 5 mM KC1, 2 mM CaC12 and 1 mM MgC12. Assay. 400/A of membrane suspension (approximately 120/~g protein) was added to ice-cold glass tubes containing 50 ~1 of 50 mM Tris-HCl buffer, pH 7.4 and incubated with [3H]SCH 23390 (spec. act. 85 Ci/mmol, Amersham) in concentrations ranging from 0.098 to 6 nM, in a final volume of 0.5 ml. Nonspecific binding was determined in the presence of 10-5 M cis-flupentixol. After 20 min incubation at 37 °C, the reaction was stopped by adding 3,5 ml of ice-cold Tris-HC1 buffer, pH 7.4, followed by filtration under vacuum through Whatman GF/B filters. Filters were washed twice with 3.5 ml of ice-cold 50 mM Tris-HC1 buffer, pH 7.4, and placed into plastic minivials containing 3.5 ml of scintillation fluid (Atomlight, New England Nuclear). The radioactivity was measured in a scintillation spectrophotometer

285 with an efficiency of 40%. Data are presented as specific binding calculated by subtracting binding in the presence of 10 -5 M c/sflupentixol from total binding determined in the absence of the unlabelled ligand. The maximum number of binding sites (Bmax) and the apparent dissociation constant (K•) were calculated by linear regression analysis of Scatchard plots obtained from [3H]SCH 23390 specific binding isotherms. Protein was measured according to Lowry et al., using bovine serum albumin as standard 19.

Adenylate cyclase Membrane preparation. Striata were homogenized (1:10, w:v) in 5 mM Tris-HCl buffer (pH 7.4) containing I mM dithiothreitol, 1 mM E G T A ( T D E buffer) and 10% sucrose with a motor-driven Teflonglass tissue grinder (clearance: 0,25 mm, 400 rpm). The homogenate was then centrifuged at 900 g for 10 min. The supernatant (S0 was centrifuged at 9000 g for 20 min. The resuspended P2 fraction was immediately used for the adenylate cyclase activity assay. Assay. The reaction mixture contained the following reagents: 75 mM Tris-HCl, pH 7.4, 0.33 mM E G T A , 2 mM MgCI2, 0.5 mM IBMX, 1 mM cAMP, 0.1 mM a-[32p]ATP (spec. act. 30 Ci/mmol, New England Nuclear), 5 mM phosphocreatine, 5 units of creatine phosphokinase, 2/~M GTP, 50/~g bovine serum albumin, 0.33 mM dithiothreitol and 1-100/~M D A in a final volume of 150/~1. The reaction was started by adding 50/~1 of membrane preparation (30-40/~g protein) and continued for 5 min at 37 °C. The incubation was stopped by adding 200/d of a solution containing 2% sodium dodecylsulphate (w:v), 45 mM ATP and 1.3 mM cAMP, p H 7.5. Following the addition of [3H]cAMP (spec. act. 33 Ci/mmol, New England Nuclear) to monitor cyclic AMP recovery, the samples were placed in a boiling water bath for 3 min and cAMP was then isolated according to Salomon et al.39. Results are expressed as pmol of cAMP formed per mg of protein per rain or as a percentage of values from the 90-day-old group. The concentration of D A required to induce a half-maximal activation of adenylate cyclase activity (Kin) was calculated by linear regression analysis of E a d i e - H o f s t e e plots obtained from concentration-response curves for DA.

Determination of DA and DO PA C Sample preparation. Striata were homogenized in cold 0.1 N perchloric acid (PCA) containing 100 nM dihydroxybenzylamine ( D H B A ) as internal standard and centrifuged for 20 min at 18.000 g. Aliquots (250 /~1) of the supernatant fluid were adjusted to pH 8.3 with 0.5 M Tris buffer, containing 1% E D T A and 0.5% sodium metabisulphite and then poured into conical test tubes containing 20 mg of activated alumina. After mixing thoroughly for 10 min and centrifuging, the supernatant was discarded and the alumina washed twice with 0.005 M Tris-HC1 buffer, pH 8.3. The catechols were then eluted from alumina with 250/~1 of 0.1 M PCA by shaking the mixture for

A

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DA-stimulated AC activity in the striatum of developing and senescent rats. A: DA and DOPAC content are expressed as percent of the mean value observed in the 90-day-old group: DA = 471 + 19 pmol/mg protein (n = 4); DOPAC = 138 + 17 pmol/mg protein (n = 4). Shown are the means + S.E.M. of 4 separate experiments. B: the density of Dt DA receptors labelled with [3H]SCH23390 and the increase in cAMP formation above basal levels induced by 100 #M DA are indicated as percent of the respective mean values from 90-day-old rats (see legends to Table I and Table II). Shown are the mean + S.E.M. of 3-9 separate experiments. *P < 0.05 vs the respective control group (i.e. 90-day-oldrats).

286 10 min and, after centrifugation, the clean extract was transferred to a n o t h e r set of tubes. A l i q u o t s ( 2 5 50/d) were injected into the H P L C system.

propriate ( F value greater than 95% confidence level) posthoc comparison of the means was carried out using a two-tailed Student's t-test.

Chromatography. Samples were injected into the H P L C system through a R h e o d y n e 7125 valve ( R h e o d y n e , Cotati C A , U . S . A . ) , fitted with a 50-/A loop. The eluant (0.23 M sodium acetate/0.02 M citric acid, p H 5.20, containing 100 mg/l of E D T A ) was p u m p e d at 1.2 ml/min through a Varian m o d e l 500 p u m p (Varian, Palo A l t o , C A , U . S . A . ) connected to a Bio-Sil ODS-5S p r e c o l u m n (Bio R a d , Richmond, C A , U . S . A . ) and a M i c r o - P a k M C H - 5 - N Cap, 5-k~ analytical column (150 m m × 4 m m , Varian). D A and D O P A C were m e a s u r e d by electrochemical detection using an LC-4B a m p e r o m e t r i c controller (Bioanalytical Systems, West Lafayette, IN, U . S . A . ) . The flow cell ( T L - 5 A , Bioanalytical Systems) was e q u i p p e d with a glassy carbon electrode. The applied potential was set at +0.72 V vs an Ag/AgC1 reference electrode. Signals were r e c o r d e d on a Varian 4270 integrator. C o n c e n t r a t i o n s of D A and D O P A C were calculated from the ratio of their detector response relative to that of the internal standard introduced into the calibration curve equation obtained by linear regression analysis. Statistical analysis. M e a n s were c o m p a r e d by oneway analysis of variance ( A N O V A ) and, where ap-

RESULTS A t the first day after birth, the rat striatum contained only 12 and 24% of the adult (i.e. 90-day-old) level of D A and D O P A C , respectively. The striatal D A content increased somewhat slowly thereafter, reaching adult levels by postnatal day 60, and remaining constant through a d u l t h o o d and senescence (Fig. 1A). In contrast, D O P A C levels increased more rapidly, attaining adult values by 20 days after birth, when D A levels were only 50% of adult, and remaining unchanged through senescence (Fig. 1A). Basal adenylate cyclase activity increased 6-fold from birth to postnatal day 45 (Table I). Concentrat i o n - r e s p o n s e curves for D A - s t i m u l a t e d A C activity indicated that 100 # M D A induced a maximal stimulation of c A M P . The effect of this concentration of D A increased 4-fold from birth to postnatal day 14, when the maximal responsiveness to D A was observed, and rapidly r e t u r n e d to adult (90-day-old) levels thereafter (Fig. 1B, Table I). The concentration of D A necessary to induce a half-maximal activation of the enzyme (Km) did not change as a function of d e v e l o p m e n t (Table I),

TABLE 1

Specific [3H]SCH 23390 binding and adenylate cyclase activity in striatal membranes from developing rats The Bmax and KD values from specific [3H]SCH 23390 binding were calculated as described in Materials and Methods. Shown are the basal AC activity and the maximal increase in cAMP formation above the basal level in the presence of 100 ktM DA (A, t0 -4 M DA). The concentration of DA that induces a half-maximal stimulation of AC activity (Kin), was determined from Eadie-Hofstee plots of the increase in cAMP formation as a function of DA concentration (1-100gM). Kmvalues are the mean of two separate experiments, each run in triplicate. All the other values are the mean + S.E.M. of 3-9 independent experiments.

Age (days)

1 7 10 14 21 30 35 45 60 90

Specific [3H]SCH binding

Adenylate cyclase activity

Bmox (pmol/mg protein)

KD (nM)

Basal A (10 -4 M DA) (pmol cAMP/min/mg protein)

Km (I~M)

0.12 + 0.01" 0.41 + 0.06* n.d. 0.97 -+ 0.08 1.46 + 0.15 n.d. 1.63 + 0.10" n.d. 1.14 + 0.12 1.20 + 0.13

0.78 + 0.16 0.62 _+0.13 n.d. 0.82 + 0.19 0.54 + 0.14 n.d. 0.71 _+0.07 n.d. 0.61 + 0.15 0.67 + 0.10

21 + 5* 21 + 2* 50 + 6* 71 + 4* n.d. 70 + 6* n.d. 126 _+8 n.d. 121 + 6

1.4 2.4 1.8 2.3 n.d. 2.5 n.d. 2.2 n.d. 2.6

19 + 38 + 74 + 85 + n.d. 74 + n.d. 77 + n.d. 61 +

7* 4* 11 5* 9 6 5

* P < 0.05 when compared with the 90 day old group (ANOVA followed by the two-tailed Student's t-test); n.d., not determined.

287 TABLE II

Specific [3H]SCH23390 binding and adenylate cyclaseactivity in the striatum of senescent rats The Bmu and K o values for specific [3H]SCH 23390 binding were calculated as described in Materials and Methods. Shown are the basal AC activity and the maximal increase in cAMP synthesis induced by 100/~M DA (A, 10-4 M DA). Results are presented as the mean + S.E.M. of 3-6 experiments, each run in triplicate.

Age (months)

3 14 20 26

Specific [3H]SCH23390 binding

A denylate cyclaseactivity

Bmax (pmol/mgprotein)

KD (nM)

Basal A (10-4MDA) (pmol cAMP/min/mgprotein)

1.05 0.85 0.67 0.57

0.49 0.48 0.42 0.63

+ + + +

0.09 0.03 0.02* 0.06*

+ + + +

0.04 0.09 0.10 0.07

121 + 6 93 + 5* 82 + 7* n.d.

61 + 5 40 + 7* 29 + 4* n.d.

* P < 0.05 as compared with the 3-month-old group (ANOVA followed by the Student's t-test); n.d., not determined.

whereas a significant decrease in the stimulatory effect of 100 gM DA on AC activity as well as a reduced basal activity of the enzyme were observed in senescent rats (Fig. 1B and Table II). The density of D 1 DA receptors labelled with [3H]SCH 23390 was only 9% of the adult value at birth, and increased very rapidly thereafter, so that by postnatal day 35 a peak value was reached (Fig. 1B and Table I). Thus, there was a lag of about 15

days between the peak of DA-induced maximal stimulation of AC activity and the peak of maximal density of [3H]SCH 23390 binding sites in the rat striaturn. It is worth noting that the development of striatal [3H]SCH 23390 binding sites and DA-stimulated AC activity proceeded in advance of the time course for DA nerve terminal ontogeny as reflected by the postnatal increase in DA content in the striatum (Fig.

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BOUND (pmoles/mg protein) Fig. 2. Scatchard analysis of [3H]SCH 23390 binding to striatal membranes from developing and senescent rats. A: developing rats. Scatehard plots from 3 representative experiments are shown. Bmu values (pmol/mg protein): 7 day = 0.60; 35 day = 1.75; 90 day = 1.33. KD values (nM): 7 day = 0.79; 35 day = 0.80; 90 day = 0.71. B: senescent rats. Shown are Scatchard plots from 3 representative experiments. Bm~x values (pmol/mg protein): 3 month = 1.2; 14 month = 0.89; 26 month = 0.57. K D values (nM): 3 month = 0.54; 14 month --- 0.51; 26 month = 0.63.

288 1A,B). Moreover, endogenous D A increased between postnatal day 28 and adulthood, whereas the density of [3H]SCH 23390 binding sites and the activity of DA-stimulated AC decreased slightly over the same time interval (Fig. 1A,B). Finally, the density of [3H]SCH 23390 binding sites decreased significantly with age, whereas Scatchard analysis of [3H]SCH 23390 binding isotherms revealed that the affinity of D 1 D A receptors for this ligand remained unchanged from birth through senescence (Fig. 2A,B; Table I and Table II). DISCUSSION The results of the present study indicate that the maturation of striatal D1 D A receptors labelled with [3H]SCH 23390 and DA-stimulated AC activity proceeds in advance of the time course for DAergic nerve terminal ontogeny. Moreover, D A content increased during the second month after birth, whilst the density of [3H]SCH 23390 binding sites and DA-stimulated AC activity were found to decrease slightly over the same period. Accordingly, previous studies have shown that, in the neocortex as well as in the brainstem 45 and in the hypothalamus of the rat 12'17, DA-stimulated AC maturation precedes that of the presynaptic marker. Taken together, these findings are consistent with the view that the development of striatal D~ D A receptors and DA-stimulated AC activity does not depend upon DAergic nerve terminal outgrowth. It is worth noting, however, that striatal D A content reached 50% of the adult value by postnatal day 35, when the density of D1 D A receptors peaked. Hence, our results do not exclude the possibility that the maturation of striatal D 1 D A receptors is influenced by the development of presynaptic processes. In order to test this hypothesis, future experiments will be aimed at characterizing the ontogeny of [3H]SCH 23390 binding sites and DA-stimulated AC activity in the striatum of rats neonatally treated with 6-OH-DA to destroy presynaptic DAergic nerve terminals. In this context, it has been recently reported that adult rats neonatally treated with 6-OH-DA have functionally supersensitive D1 D A receptors which are critical for self-mutilation behaviour and contribute to other behavioural responses induced by L-DOPA 3. The latter findings indicate that, far from

hindering the development of D~ DA receptors, the neonatal destruction of DAergic neurons induces a dramatic increase in the sensitivity to the behavioural effects induced by selective agonists for these receptors. A lag of about 15 days was observed between the peak of DA-induced maximal stimulation of AC activity and the peak of maximal density of [3H]SCH 23390 binding sites. Different mechanisms, including changes in AC availability or regulation may underlie this time course dissociation. Further studies are required to clarify this issue. In contrast with our present results, it has been reported that the concentration-response curve for DA-stimulated AC activity in newborn (3-day-old) rats is virtually superimposed on the corresponding curve for adults 43, whereas in agreement with our data, different studies on the developmental variations of the AC activity in the neocortex and brainstem 45 as well as in the hypothalamus ~2 also reported highest activity around day 20 after birth. The time course for the maturation of striatal O a DA receptors recently reported by Zeng et al. 46 is quite similar to that observed using [3H]SCH 23390 as a selective ligand for D 1 DA receptors. Confirming previous reports 1°'41 we observed a progressive decline in basal and DA-stimulated AC activity in the striatum of aging rats. We also found a concomitant decrease in the density of [3H]SCH 23390 binding sites, indicating that D 1 D A receptors undergo age-related changes. However, contradictory alterations in the density of striatal D1 D A receptors have been reported to occur with advancing age. Thus, in agreement with our results, Norman et al. 27 observed a decrease in the density of D 1 D A receptors in the striatum of aging rats. In contrast, it has been reported that D1 D A receptors labelled with [3H]piflutixol are unchanged in the striatum of s e n e s c e n t rats 29. In this context, it is worth noting that the Hill coefficient for displacement of [3H]piflutixol by SCH 23390 is less than unity 28, whereas the Hill coefficients for displacement of [3H]SCH 23390 by various D A receptor antagonists are indistinguishable from unity It. Hence, the results of binding studies using [3H]SCH 23390 or [3H]piflutixol are not necessarily equivalent. The large increment in the density of D~ D A receptors observed in the caudate nucleus and putamen of the ag-

289 ing human brain 26 indicates that these receptors may undergo opposite changes in different species, albeit the cause of this species-related difference is still unclear. Our finding that D A and D O P A C content in the striatum does not change with age is in line with previous reports indicating that, in rodents, the loss of striatal DA-sensitive AC may precede by 6 to 12 months smaller reductions (no greater than 25%) of striatal D A levels and turnover 8. Therefore, the loss of nigral DAergic neurons with age is probably not a major factor in the decrease in D 1 D A receptors and DA-stimulated A C activity in the striatum of senescent rats. In addition, several lines of experimental evidence indicate that D: D A receptors lost during aging are located on the cell bodies of striatal neurons rather than in the nerve endings of afferent fibers. Thus, in adult rats, 6-OH-DA and surgically induced lesions of the nigrostriatal DAergic pathway or the chronic administration of D A receptor blockers increase the density of O 1 D A receptors 3:'33 as well as the activity of DA-stimulated AC 23, whereas opposite changes are observed in the rat striatum during normal aging (refs. 10, 27, 41 and this study). Moreover, the nearly complete loss of the DA-stimulated AC activity42 and [3H]SCH 23390 binding sites 32 following striatal kainate lesions indicates that D 1 D A receptors are largely confined to the cell bodies of striatal neurons. Finally, surgical lesions of the corticostriatal fibers fail to affect the activity of DA-stimulated AC 9'41 and the density of [3H]SCH

23390 binding sites in the rat striatum (O. Giorgi, unpublished results). The above findings are consistent with the view that the decline of striatal D 1 D A receptors with age can result from a loss of striatal neurons or from a decreased number of receptors per cell. Another possible locus in the loss of binding sites is the dendrospinous process of striatal neurons, since alterations of dendritic spines have been observed in the striatum of different animal species 18'21'4°. The decrease in [3H]SCH 23390 binding sites and DA-stimulated AC activity with advancing age may be relevant in view of the behavioural and biochemical effects mediated by D 1 D A receptors that have been evidenced only recently using selective agonists and antagonists of the D 1 and D 2 D A receptors. Thus, it has been shown that not only D 2 D A receptor stimulation, but also the selective stimulation of D 1 D A receptors elicits rotation in rats with 6-OHDA-induced unilateral lesions of the nigrostriatal pathway 1. It has also been reported that certain forms of stereotypy and grooming are mediated by D 1D A receptors 24'25'36. In addition, evidence is accumulating that a significant functional interaction exists between D1 and D 2 D A receptors both in vivo and in vitro 37,38. Hence, it is tempting to speculate that neuronal systems dependent on D1 D A receptor-mediated mechanisms become disfunctional below a critical extent of loss, thereby contributing to some of the multiple alterations in DAergic functions observed in aged rodents 8,34.

REFERENCES

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