Accelerated Resensitization of the D 1 Dopamine Receptor-Mediated

The Journal of Neuroscience, October 1994, 74(10): 6260-6266

Accelerated Resensitization of the D 1 Dopamine Receptor-mediated Response in Cultured Cortical and Striatal Neurons from the Rat: Respective Role of CY1 -Adrenergic and /U-methybaspartate Receptors

Fabrice Trovero, Philippe Marin, Jean-PO1 Tassin, JoQl Premont, and Jacques Glowinski INSERM U 114, Chaire de Neuropharmacologie, College de France, 75231 Paris Cedex, France

As previously shown in vivo, noradrenergic and glutama- cortex. In the rat, bilateral electrolytic lesions of the mesence- tergic neurons can regulate the denervation supersensitivity phalic ventral tegmentalarea induce a complex and permanent of Dl dopaminergic (DA) receptors in the rat prefrontal cor- behavioral syndrome characterized by a locomotor hyperactiv- tex and striatum respectively. Therefore, the effects of meth- ity and the incapacity of the animal to focalize its attention (Le oxamine (an al-adrenergic agonist) and glutamate on the Moal et al., 1969). Some of the behavioral deficits observed in resensitization of Dl DA receptors were investigated in cul- the lesionedanimals, particularly the locomotor hyperactivity, tured cortical and striatal neurons from the embryonic rat. have beenattributed for a large part to the selectivedestruction In the presence of sulpiride and propranolol, DA stimulated of the cortical dopaminergic (DA) innervation (Tassin et al., the Dl DA receptor-mediated conversion of 3H-adenine into 1978). This locomotor hyperactivity was markedly reduced in 3H-cAMP in both intact cortical and striatal cells and these rats with 6-hydroxydopamine (6-OHDA) lesions,which destroy responses were markedly desensitized in cells preexposed not only the ascendingDA neuronsbut also the ascendingnor- for 15 min to DA (50 AM). The complete recovery of the Dl adrenergicfibers passingnear the ventral tegmentalarea (Tassin DA response was more rapid in striatal (15 min) than in et al., 1982). (Y1 -Adrenergic receptorssensitive to the (Y1 antag- cortical (80 min) neurons. Methoxamine accelerated the re- onist prazosin seemto play a prominent role in the permissive sensitization of the Dl response in cortical but not in striatal effect of cortical noradrenalineon the appearanceof behavioral neurons. The effect of the al -adrenergic agonist in cortical deficits in rats with ventral tegmental area electrolytic lesions. neurons was blocked by prazosin and chlorethylclonidine. Indeed, in these lesioned animals, the peripheral injection of In contrast, glutamate accelerated the resensitization of the prazosin immediately suppressedlocomotor hyperactivity Dl response in striatal but not in cortical neurons and the (Trovero et al., 1992a). The effect of prazosin appears to be effect observed in striatal neurons was totally blocked by mediated at the cortical level since high affinity binding sites 2-amino-5-phosphonovaleric acid, an NMDA receptor antag- for prazosin were found in the prefrontal cortex, and the cortical onist. Protein kinase C was shown to be involved in the al - application of prazosin abolished the increasedlocomotor ac- adrenergic-induced resensitization of the cortical Dl re- tivity evoked by amphetamineapplication into the nucleusac- sponse but not in the glutamate-evoked resensitiration of cumbens(Blanc et al., 1993). In addition, the denervation su- the striatal Dl response. Finally, for comparison, similar ex- persensitivity of cortical DA-sensitive adenylatecyclase observed periments were performed on 8-adrenergic receptors using in rats with electrolytic lesionsof the ventral tegmentalarea was isoproterenol (1 PM) as an agonist. Methoxamine did not prevented by the concomitant destruction of the cortical nor- modify the resensitization of the B-adrenergic response in adrenergicinnervation (Tassinet al., 1982, 1986). Similarly, the cortical neurons, but glutamate accelerated the resensiti- denervation supersensitivity of the cortical DA-sensitive ad- zation of this response in striatal neurons. enylate cyclase was not seen in rats with bilateral [Key words: cortical neurons, striatal neurons, D 1 DA re- 6-hydroxydopamine (6-OHDA) lesionsof the ventral tegmental ceptors, heterologous resensitization, a 1 -adrenergic recep- area (Tassin et al., 1982). tors, NMDA receptors] Together, thesebehavioral and biochemical observationsled us in the present study to determine whether the stimulation of Several years ago, we provided behavioral and biochemical ev- 011-adrenergic receptors could regulate the sensitivity of the D 1 idence for an interaction betweenthe mesocorticoprefrontal do- DA-induced formation of CAMP (Dl response)in intact cortical paminergic (DA) neurons and the noradrenergic neurons orig- neuronsfrom rat embryos in primary culture. For this purpose, inating from the locus coeruleus and innervating the cerebral we first investigated the effect of an (~1agonist, methoxamine, on the recovery of the D 1 responsefollowing a short-term DA- induced desensitization. For comparison, similar experiments Received Jan. 13, 1994; revised Apr. 14, 1994; accepted Apr. 21, 1994. were performed on cultured striatal neurons.Following this, the This work was supported by grants from Institut National de la Sante et de la Recherche MCdicale (INSERM) and RhBne Poulenc Rarer. We thank Robert effects of glutamate on the rate of resensitization of the cortical Williams for critical reading of the manuscript. and striatal Dl responseswere also analyzed sincethe cerebral Correspondence should be addressed to Fabrice Trovero, INSERM U 114, Chaire de Neuropharmacologie, Coll&e de France, 11 Place Marcelin Berthelot, cortex is rich in glutamatergic neurons and the corticostriatal 7523 1 Paris Cedex, France. glutamatergic innervation has been shown to contribute to the Copyright 0 1994 Society for Neuroscience 0270-6474/94/146280-09$05.00/O regulation of the denervation supersensitivity of striatal D 1 DA The Journal of Neuroscience, October 1994, f4(10) 6281

more than 95% of the cells were immunocytochemically defined as cortical neurons neurons according to El Etr et al. (1989). ‘H-CAMP assay 1000 r Cells cultured in 24 well culture dishes were incubated for 2 hr with 2-‘H-adenine (27 Ci/mmol, Amersham, 2 j&i per well containing 0.5 ; ; ml of culture medium). Desensitization procedure. DA (50 PM) was added to the culture medium 15 min before the end of 3H-adenine incorporation, in the presence of both propranolol(1 NM) and sulpiride (50 PM) to prevent &adrenergic and D2 DA receptor stimulation. Resensitization procedure. After removal of the desensitization medium, neurons were washed once with 0.5 ml of Krebs’ phosphate buffer (in mM: NaCl, 120; NaH,PO,, 15.6; KCI, 4.8; MgSO,, 1.2; CaCl,, 1.2; **glucose, 33.3; pH 7.4), and then incubated for the indicated time in 0.5 ml of the same medium containing appropriate agonists and/or antagonists. 3H-cAMP measurement. After removal of the resensitization medium, cells were incubated for 5 min in 0.5 ml of Krebs’ phosphate buffer containing DA (50 PM), sulpiride (5 PM), propranolol (1 PM), and the nonselective inhibitor of phosphodiesterases, 3-isobutyl- 1-methylxan- thine (IBMX: 1 mtvt). The incubation was stonned bv reolacina the medium with 0.5 mi of ice-cold trichloroacetic acid (I?%).- Cells-were 0 then scraped and 100 ~1 of medium containing 1% SDS, 50 mM Tris- HCI, 3 mM ATP, 3 mM CAMP, pH 7.4, was added to the samples. After Dopamine + + I centrifugation (10 min at 12000 x g), the labeled ATP and CAMP Sulpiride + + present in 0.5 ml of supematant were separated according to the pro- +cedure described by Salomon et al. (1974). Radioactivity was estimated Propranolol + in 10 ml of Aquasol- (New England Nuclear-Du Pont) with a beta +scintillation counter. Results, calculated as percentage of conversion of Yohimbine +)H-ATP into 3H-cAMP by (‘H-CAMP x lOO)/CH-ATP + ‘H-CAMP), SCH 23390 were expressed as a percentage of the basal values obtained without agonists. Figure 1. Pharmacological characterization of the dopamine-induced production of CAMP in cortical neurons. Cortical neurons from+ rat Inositol phospholipid turnover assay embryos were cultured for 6 din serum-free medium and then incubated for 2 hr with 3H-adenine. DA (50 PM) and antagonists (sulpiride, 5 PM; Neurons were incubated for 24 hr in culture medium supplemented propranolol, 1 PM; yohimbine, 1 PM; SCH 23390, 0.2 PM) were then with 1 &/well of myo-3H-inositol. Cells were then preincubated for added to the medium in the presence of IBMX (1 mM) and incubated 10 min in Krebs’ phosphate buffer supplemented with LiCl (10 mM). for 5 min. The 3H-cAMP that accumulated in cells was then estimated. Methoxamine (100 PM) or glutamate (100 PM) was then added for 20 Neither sulpiride nor propranolol nor yohimbine significantly modified min. The incubation was stopped by adding successively 200 pl of 0.1% the response obtained with DA alone. Results are the mean ? SEM of Triton X-100/0.1 M NaOH, 200 ~1 of 0.1% Triton X-100/0.1 M HCl, values obtained from triplicate in a typical experiment. *, significantly and 500 ~1 ofwater. Cells were then scraped, and )H-inositol phosphates different (p < 0.05, Student’s t test) from basal values; **, significantly were extracted and estimated according to the procedure of El Etr et different (p < 0.05, Student’s t test) from the DA-evoked response. Four al., (1989). other experiments performed with different cultures provided similar results. Results Desensitization of the DI DA-induced formation of CAMP in cortical neuronsby preexposureto DA receptors (Hervt et al., 1989). Finally, in order to demonstrate As illustrated in Figure 1, a 5 min exposure to DA stimulated further the specificity of the effectsobserved with methoxamine and glutamate, parallel experiments were made on the resen- the formation of 3H-cAMP in cortical neuronsprelabeled with 3H-adenine(EC,, = 1.5 * 0.2 PM, mean + SEM calculatedfrom sitization of the /3-adrenergic-inducedformation of CAMP @- three independent experiments, n = 3). Most of the responseto adrenergic response),following isoproterenol-induced desensi- DA (50 PM) wasblocked by the Dl antagonist SCH 23390 (0.2 tization of p-adrenergic receptors in cortical and striatal neurons. MM), suggestingthe involvement of D 1 receptorsin this process. The simultaneousaddition of sulpiride (5 PM) only slightly en- Materials and Methods hanced the DA-evoked response.Moreover, the addition of Primary cultures of cortical and striatal neurons propranolol (1 PM) or yohimbine (1 PM) did not alter the DA- Cortices and striata were removed from 17 d old Sprague-Dawley rat induced formation of 3H-cAMP, excluding the involvement of embryos (Iffa Credo, Lyon, France) and mechanically dissociated in /3- and/or a2-adrenergic receptors(Fig. 1). serum-free medium with a fire-narrowed Pasteur pipette. Cells were A reduction in the DA-induced formation of 3H-cAMP was plated (5. lo5 cells/ml) on 24-well (0.5 ml/well) Nunc culture dishes observed when cortical neurons were preincubatedfor 15 min previously coated successively with poly-L-omithine (1.5 rg/ml; MW, with DA (50 PM) in the presenceof both sulpiride (5 PM) and 40,000; Sigma) and culture medium containing 10% fetal calf serum. After removing the last coating solution, cells were seeded in a culture propranolol (1 FM). In fact, a comparison of the DA dose- medium composed of a 1: 1 mixture of Dulbecco’s modified Eagle’s responsecurves in untreated and DA-preincubated cells re- medium (DMEM) and Ham’s F12 nutrient (GIBCO, Paris), supple- vealed a marked decreasein the maximal response(-55%, n mented with glucose (33 mM), glutamine (2 mM), sodium bicarbonate = 10) with only a weak but not significant reduction in the (3 mM), and HEPES buffer (5 mM, pH 7.4). To this culture medium was added a mixture of hormones and salt composed of insulin (25 rcg! potency of DA (Fig. 2). ml), transferrin (100 &ml), putrescine (60 &ml), progesterone (20 The DA-induced desensitizationof the cortical Dl response nM), and sodium selenate (30 nM) (all from Sigma). After 6 d in culture, was reversible. Indeed, a slow recovery of the initial Dl DA- 6262 Trovero et al. - Resensitization of Cortical and Striatal Dl DA Receptors

DA (50PM) wash DA (SO CM) cortical neurons

II \, ,I Desensitization Resensitization CAMP

IS min 0 to 80min 5 min

100

0 0 20 40 60 80 ++ -00 -7 -6 -5 -4 I 1 log [DA WI (9 Figure 2. Dose-response curves for the Dl receptor-mediated formation of ‘H-CAMP in cortical neurons pretreated with or without dopamine. Data represent the amount of ‘H-CAMP produced (v) when neurons were incubated for 5 min with increasing concentrations (s) of DA in the presence of sulpiride (5 PM) and propranolol (1 P(M), as described in Figure 1. DA-pretreated neurons were preincubated for 15 min in medium containing DA (50 PM), sulpiride (5 PM), and propranolol(1 PM). Cells were then extensively washed with Krebs’ phosphate buffer before the estimation of the dopamine-induced formation of 3H- CAMP as described above. Results are the mean + SEM of values 0’ I obtained in an experiment performed in triplicate. Curves shown are 0 5 10 15 representative of data obtained in three independent experiments made with different neuronal cultures. Inset, Eadie’s representation ofthe data. Resensitization time (min)

-- induced formation of 3H-cAMP could be observed in cortical o- - control -0 ~ methoxamine neurons after the removal of DA, the half-maximal response Figure 3. Effects of methoxamine on the resensitization of the Dl being restored within 40 min and the total recovery of the full response in cortical and striatal neurons. Neurons were preincubated for 15 min with DA (50 PM) as described for Figure 2. Following washing responsebeing achieved after 80 min (Fig. 3). (see Materials and Methods), cells were incubated for various times in the resensitization medium with or without methoxamine (100 PM). All Accelerated resensitization of the Dl DA-induced formation of incubations with methoxamine were performed in the presence of yo- CAMP by stimulation of al-adrenergic receptorsin cortical himbine (1 P(M) to prevent a potential stimulation of cY2-adrenergic neurons receptors. Values at zero time correspond to the formation of ‘H-CAMP over a 5 min period, estimated immediately after the 15 min D 1-induced The addition of methoxamine (100 FM), an al-adrenergic ag- desensitization and washing of the cells. Values are the means + SEM onist, during the resensitization period of the cortical Dl re- of data obtained in three experiments, each performed in triplicate. *, sponse,accelerated the recovery of the Dl DA-induced for- significantly different (p < 0.05, Student’s t test) when compared to mation of 3H-cAMP (Figs. 3, 4), without modifying the fully control values. restored Dl response(estimated after a 80 min resensitization period) (Fig. 3). a2-Adrenergic receptors did not contribute to also stimulatesphospholipase C activity in cortical neurons,did the effect of methoxamine sincethe (Y1 agonist-inducedresponse not modify the resensitization of the Dl response(Table 1). wasnot affected by yohimbine (1 PM) (Fig. 4). The acceleratingeffect of methoxamine on the resensitization Lack of effect of methoxamine on the resensitizationof the of the Dl responsewas suppressedby prazosin (1 PM), an crl- P-adrenergic-inducedformation of CAMP in cortical neurons adrenergicantagonist (Fig. 4). The incubation of cortical neu- A 5 min incubation of cortical neurons with isoproterenol (1 ronswith chlorethylclonidine (CEC, 100 PM, added during 30 PM) resultedin a largeformation of 3H-cAMP (seeFig. 5 legend). min before methoxamine addition), a compound that has been This ,f3-adrenergicresponse was reduced (80%) following prior shownto inactivate LY1p-adrenergic receptors irreversibly (Han exposure (15 min) of the cells to isoproterenol(1 PM) according et al., 1987) also suppressedthe effect of methoxamine (Fig. 4). to the procedure usedfor the estimation of the Dl DA-induced In the absenceof methoxamine, neither prazosin nor CEC af- desensitization. The recovery of the full /3-adrenergicresponse fected the resensitization of the Dl response(Fig. 4). Interest- was more rapid than that of the Dl responsesince 75% of the ingly, in contrast to methoxamine, glutamate (100 PM), which initial responsewas restored 15 min after the removal of iso- The Journal of Neuroscience, October 1994. f4(10) 6283

cortical neurons

D 1 response

I 0 5 10 15 Resensitization time (min) / control m 15 min res. Figure 5. Resensitization of the P-adrenergic response in cortical and @I 15 min res.+met. striatal neurons. The @adrenergic-induced formation of ‘H-CAMP was desensitized m estimated by incubating neurons with isoproterenol (1 PM) for 5 min Figure 4. Effects of oil-adrenergic antagonists on the methoxamine- (p-response). Desensitization was achieved by adding isoproterenol (1 induced acceleration of the resensitization of the D 1 response in cortical PM) for 10 min. Following washing (see Materials and Methods), cells neurons. Experiments were performed with or without methoxamine were incubated for various times in the resensitization medium. Values (100 PM, met.) as described in Figure 3. When used, yohimbine (JO., 1 at zero time correspond to the formation of ‘H-CAMP over a 5 min GM) and prazosin (prz., 1 PM) were added without or with methoxamine period, estimated immediately after the 10 min isoproterenol-induced during the 15 min resensitization period while chlorethylclonidine (CEC, desensitization and washing of the cells. Results are expressed as a 100 FM) was added 15 min before the desensitization period. Values percentage of the control P-responses (control values). &Responses rep- are the mean f SEM of data obtained in triplicate from a typical ex- resented 272 + 17 and 436 + 23% of increase in basal ‘H-CAMP periment. Results are expressed as a percentage of the control Dl re- formation in cortical and striatal neurons respectively, the basal 3H- sponse. *, significantly different (p < 0.05, Student’s t test) from the CAMP levels being 247 -t 36 and 605 ? 45 d.p.m./well. Values are the control resensitized Dl response (15 min res.).Three other experiments means + SEM of data obtained in three experiments, each performed performed with different neuronal cultures led to similar results. in triplicate.

proterenol (Fig. 5). In contrast to that observed for the Dl sulpiride and propranolol induced a marked desensitization of response, the addition of methoxamine (100 /II@ during the the Dl response (Fig. 7). Indeed, the DA-induced formation of resensitization period did not affect the recovery of the p-ad- 3H-cAMP was markedly reduced (-90% of the maximal Dl renergic response (Fig. 6). response) following a prolonged exposure of the cells to DA. Moreover, the resensitization of the striatal Dl response was Accelerated resensitization by glutamate, but not by more rapid than that observed in cortical neurons since the methoxamine, of the Dl DA-induced formation of CAMP in maximal Dl DA-induced formation of 3H-cAMP was almost striatal neurons completely restored (95%) only 15 min after the removal of DA Confirming previous results (Chneiweiss et al., 1990), prein- (Fig. 3). cubation of striatal neurons with DA (50 WM) in the presence of Interestingly, in contrast to cortical neurons, the addition of methoxamine (100 PM) during the resensitization period did not Table 1. Respective effects of methoxamine and glutamate on modify the rate of resensitization of the D 1 response in striatal phospholipase C activity and on the resensitization of the Dl response neurons (Fig. 3). However, this resensitization was accelerated in cortical neurons by glutamate (100 FM) (Fig. 7). Indeed, the full Dl response was observed as rapidly as 5 min after the beginning of the resen- Effect on Dl sitization period in the presence of the excitatory amino acid. resensitization PLC activation (in % of control NMDA receptors appear to be involved in the glutamate-in- (in % of basal resensitized duced resensitization of the D 1 response since the effect of glu- activity) response) tamate was completely abolished in the presence of 2-amino- Methoxamine 5-phosphonovaleric acid (APV) (50 PM), a competitive inhibitor (100 PM) 191 + 7* 180 * 14** of NMDA receptors (Fig. 7). In addition, the D,L-cu-amino-3- Glutamate hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) antagonist 6,7-dinitroquinoxaline-2-3-dione (DNQX; 50 PM) was not (100 w) 242 t 12* 97 + 16 effective (Fig. 7). Furthermore, 2-amino-3-phosphonopropionic The production ofaH-inositol phosphate induced by a 5 min incubation of cortical neurons in the presence of either methoxamine or glutamate is expressed as a acid (AP3; 1 mM), an antagonist of metabotropic receptors cou- percentage of the basal )H-inositol phosphate formation. Values are the mean f pled to phospholipase C was also without significant effect (Fig. SEM of data obtained in three separate experiments, each being made in triplicate. 7). The effect of methoxamine or glutamate on the resensitization of the Dl response was expressed as a percentage of the resensitized Dl response estimated under As observed with DA, the /3-adrenergic agonist isoproterenol control conditions (see Fig. 4). Values are the mean f SEM of three independent (1 PM) stimulated the formation of 3H-cAMP in striatal neurons experiments. but this effect was less pronounced than that observedin cortical * Significantly different @ -C 0.05, Student’s t test) from the basal value (342 k 14 dpmkell). neurons (see Fig. 5 legend). Following a 15 min preexposure to ** Significantly different @ i 0.05, Student’s t test) from the control resensitized isoproterenol, in order to desensitizethe P-adrenergicreceptors Dl response. on these cells, a rapid resensitization of the /3-adrenergicre- 6264 Trovero et al. - Resensitization of Cortical and Striatal Dl DA Receptors

cortical neurons striatal neurons striatal neurons 1 I3 response I3 response D 1 response L '1 * T

u control m desensitized

5 min res. m 5 min res. + met. a control 5 min res. m 5 min *es. + glu. m desensitized m 5 min res.+glu. Figure 6. Respective effects of methoxamine and glutamate on the w 5 min res.+glu.+AP3 resensitization of the P-adrenergic response in cortical and striatal neurons. The P-adrenergic-induced formation of ‘H-CAMP was estimated m 5 min res.+glu.+DNQX by incubating neurons with isoproterenol(1 FM) for 5 min (@-response). Desensitization and resensitization procedures were achieved as de- k§3 5 min res.+glu.+ APV scribed for Figure 5. A 5 min resensitization period was chosen (5 min res.). When used, methoxamine (met., 100 PM) was added during the Figure 7. Accelerating effect of glutamate on the resensitization of the resensitization period as described in Figure 4. Similarly, when used, Dl resoonse in striatal neurons: involvement of NMDA recenters. De- glutamate (glu., 100 PM) was added during the beginning of the resen- sensitization with DA (50 PM, desensitized) and resensitization were sitization period. Values are the mean + SEM of data obtained in performed as described for Figure 2, the Dl response (5 min formation triplicate in a typical experiment. Results are expressed as a percentage of ‘H-CAMP in the presence of 50 PM DA) was estimated after a 5 min of the control @responses. Three other experiments made with different resensitization period (5 min res.),and glutamate (glu., 100 PM) was cultures led to similar results. *, significantly different (p < 0.05, Stu- added during the resensitization period. When used, AP3 (1 mM), DNQX dent’s t test) from the corresponding control value (5 min res.). (50 PM), or APV (50 PM) was added with glutamate. Data are the mean -t SEM obtained in triplicate in a typical experiment; results are expressed as a percentage of the control Dl response. *, significantly dif- sponsewas observed since the initial maximal isoproterenol- ferent (p < 0.05, Student’s t test) when compared to the corresponding induced formation of ‘H-CAMP was seenafter only 10 min of control value (5 min res.):**. significantlv different (a < 0.05. Student’s the resensitization period (Fig. 5). As demonstrated for the Dl t test) when cdmpared td ihe’cokespondkg glutamate value (5 min res. response,glutamate (100 PM) also acceleratedthe resensitization + glu.). of the /3-adrenergicresponse (Fig. 6). celeration of the resensitizationof the Dl responsewas further Selective involvement of protein kinase C in the Lul-adrenergic suggestedby the suppressionof the effect of methoxamine in acceleratedresensitization of the DI responsein cortical PMA-pretreated cortical neurons (Table 2). neurons Surprisingly, in striatal neurons,the addition of PMA (10 nM) As already indicated, (Y1 b-adrenergic receptorsappear to be in- during the resensitization period did not modify the recovery volved in the modulation of the resensitization of the cortical of the Dl response,indicating that PKC is not involved in the D 1 DA-induced formation of ‘H-CAMP. Sincecy 1 b-adrenergic acceleratingeffect of glutamate (100 PM) on the resensitization receptorsare coupled to phospholipaseC (Han et al., 1987),the of the Dl response(Fig. 8). stimulation of these receptors may result in a subsequentacti- vation of protein kinase C (PKC). This led us to examine the Discussion effect of phorbol myristate acetate (PMA; 10 nM), a PKC-acti- The prolonged exposure of cells to hormones or neurotrans- vating agent, on the resensitization rate of the Dl responsein mitters reducestheir responsivenessto further stimulation. This cortical neurons. desensitization processhas been extensively described in nu- The addition of PMA (10 nM) insteadof methoxamine during merous studiesdealing with receptorscoupled to adenylate cy- the resensitization period accelerated the recovery of the Dl clase,particularly for the /3-adrenergicreceptors (for review, see responsein cortical neurons (Fig. 8). Interestingly, when added Huganir and Greengard, 1990). Briefly, the processof desen- with DA, PMA altered neither the full DA-induced formation sitization involves a rapid and reversible uncoupling of the re- of )H-CAMP (data not shown)nor the desensitizationof the Dl ceptors from the a-subunits of G-proteins, followed by a slow response(Fig. 8). Furthermore, staurosporine (0.5 KM), a non- reversible downregulation of the receptors. Two major mech- selectiveinhibitor of PKC, markedly decreasedthe accelerating anisms have been shown to be involved in the desensitization effect of methoxamine (100 PM) on the resensitizationof the D 1 of @-adrenergicreceptors: homologous desensitization, which response(Table 2). involves a phosphorylation of the receptor by a /3-adrenergic When cells were pretreated for 40 hr with PMA (100 nM), a receptor-specific kinase (P-ARK), and heterologousdesensiti- procedureknown to downregulatethe activity of PKC, no mod- zation, in which the receptor is phosphorylated by CAMP-de- ification of the DA-induced desensitization could be observed pendent protein kinase(Clark et al., 1990; Johnsonet al., 1990; (Table 2). The involvement of PKC in the Cal-adrenergicac- for review, seeHuganir and Greengard, 1990). The Journal of Neuroscience. October 1994, 14(10) 6285

cortical neurons striatal neurons Table 2. Effect of methoxamine on the resensitization of the Dl response in cortical neurons following long-term pretreatment with PMA D 1 response D 1 response 1 Res.+ met. Des. Res. Res.fmet. +stauro. Control corticalneurons 21.6 * 4 47.7 + 5 88.6 f 7* 42 + 9** PMA-pretreated corticalneurons 27.2 f 7 37.2 I? 5 45.4 f ll** - Cortical neurons were pretreated for 48 hr with PMA 100 nhr; then, following extensive washing, the Dl desensitization (des.) and the resensitization @es.) of the Dl response in the presence (res.+met.) or absence of methoxamine were induced and estimated as described for Figures 3 and 4, respectively. Staurospotine (0.5 PM) was added with methoxamine during the resensitization period only in control cortical neurons. Results are expressed as a percentage of the control Dl response. Values represent the mean k SEM of data obtained in triplicate in a typical experiment. Three other experiments made with different neuronal cultures j control m des. m des.+PMA led to similar results. * Significantly different (p < 0.05, Student’s t test) from the control resensitized Dl response (res.). res. ** Significantly different (p < 0.05, Student’s t test) from the corresponding value m res.+PMA of the resensitized Dl response in the presence of methoxamine (res.+met.) in Figure 8. Effect of phorbol myristateacetate (PMA) on the desensi- control cortical neurons. tization and on the resensitizationof the Dl responsein corticaland striatal neurons.Experiments with cortical and striatal neuronswere processmay result from the phosphorylation of the Dl DA performedas described for Figures4 and7, respectively.PMA (10 nM) receptorsby a specificprotein kinase as shown for /I-adrenergic wasadded during the desensitization@es. + PMA) or the resensitization receptors (Balmforth et al., 1990). period(res. + PMA). *, significantlydifferent Cp < 0.05,Student’s t test) from the resensitizedDl response(res.). Values are the meanf SEM of data obtainedin triplicate in a typical experiment.Results are ex- Role of heterologoustransmitters in the in vitro resensitization pressedas a percentageof the controlDl response.Three other exper- of the Dl responsein cortical and striatal neurons imentsmade with differentcultures led to similarresults. As already mentioned, little is known of the biochemical events underlying the resensitization of D 1 DA receptor-mediated re- Desensitization of Dl DA receptorsin cultured cortical and sponses.Our study indicatesthat transmitters (or agonists)other striatal neurons than DA can play a prominent role in the regulation of the In contrast to ,&adrenergic receptors, relatively little is known resensitization rate of the Dl DA-evoked formation of CAMP. on the biochemical mechanismsinvolved in the regulation of Indeed, both methoxamine (through (Y1 -adrenergic receptors) Dl DA receptor sensitivity. Nevertheless,studies performed on and glutamate (through NMDA receptors) can accelerate this cell cultures have demonstrated that desensitizationof Dl DA resensitization process.However, these regulations are struc- receptorsoccurs following sustainedexposure of neuronsto DA ture-specific since the al-adrenergic acceleration of the resen- (Balmforth et al., 1990; Barton and Sibley, 1990; Chneiweisset sitization of the Dl responsewas observed in cortical but not al., 1990). A similar homologousdesensitization has been ob- in striatal neuronswhile the reverse wasfound with glutamate. served in several in vivo studies in which striatal DA release Finally, sincethere is no evidence for synaptic contacts in 6-d- was enhanced by amphetamine treatment (Barnett and Ku- old neuronsin primary culture (El Etr et al., 1989),a population czenski, 1986;Robert-Lewis et al., 1986;Roseboom and Gnegy, of cortical neuronsmust possessboth D 1 DA and (Y1 -adrenergic 1989). In agreementwith theseobservations, we demonstrated receptorswhile both D 1 DA and glutamatergicreceptors should in the present study, that a 15 min preincubation with DA of be colocalized on a population of striatal neurons. This is in either cortical or striatal neurons from the embryonic rat in agreementwith electrophysiologicalstudies indicating that some primary culture resulted in a reversible decreasein the respon- cortical cells in the prefrontal cortex are sensitive to both DA sivenessof the Dl DA receptors to further DA stimulation, as and noradrenaline (Bunney and Aghajanian, 1976) and that a estimated by the 5 min formation of 3H-cAMP in intact cells. population of striatonigral neurons possessboth Dl DA and This effect was indeed mediated by Dl DA receptors sincethe glutamatergic receptors (Cepedaet al., 1992). experiments were performed in the presenceof sulpiride and propranolol to eliminate any possible interaction of DA with Acceleration of the resensitizationof the DI responsein both D2 DA and P-adrenergic receptors. In addition, the DA- cortical neuronsby the al-adrenergic agonist methoxamine induced desensitization was suppressedin the presenceof the Several observations demonstrated that the stimulation of cul- Dl antagonist SCH 23390. adrenergic receptors acceleratesthe resensitization of Dl DA The rapid and complete recovery of the Dl response,which receptorscoupled to adenylatecyclase in cortical neurons.Meth- was faster in striatal than in cortical neurons, strongly suggests oxamine acceleratedthe recovery of the full Dl responseand that this desensitization involved the uncoupling of Dl DA this was even observed in the presenceof yohimbine, which receptors from the G-protein linked to adenylate cyclaserather prevents the possible stimulation of cr2-adrenergicreceptors. than a loss of receptors from neuronal membranes. In fact, Moreover, the effect of methoxamine was prevented by both longer periodsof exposure to DA are required for the occurrence prazosin, a nonselective a!1 -adrenergic antagonist, and CEC, ofa downregulation of Dl DA receptors(Chneiweiss et al., 1990; which is known to inactivate (Y1 b-adrenoceptors irreversibly Bates et al., 1991). This rapid and reversible desensitization (Han et al., 1987). 6266 Trovero et al. - Resensitization of Cortical and Striatal Di DA Receptors

The specificity of the stimulatory effect of methoxamine on sensitization process was totally prevented by APV, a compet- the resensitization of the D 1 response was demonstrated in sev- itive antagonist of NMDA receptors, while DNQX, an AMPA eral ways. (1) The addition of methoxamine did not affect the antagonist, was without effect. In addition to ionotropic recep- full DA-induced formation of jH-CAMP (before the desensiti- tors, striatal neurons in primary culture possess metabotropic zation or after the resensitization) or the DA-induced desensi- glutamatergic receptors coupled to phospholipase C (Sladeczek tization. (2) As shown by experiments made with isoproterenol, et al., 1985). However, these receptors do not seem to be im- methoxamine did not affect the resensitization of the P-adre- plicated in the stimulatory effect of glutamate. Indeed, the phor- nergic-induced formation of 3H-cAMP in cortical neurons. (3) bol ester PMA did not accelerate the rate of recovery of the D 1 As previously indicated, methoxamine did not modify the rate response in striatal neurons, suggesting that PKC is not involved of resensitization of the striatal D 1 response. in this process. Confirming this finding, AP3, an antagonist of Further studies will be necessary in order to elucidate the metabotropic receptors coupled to phospholipase C did not sig- molecular mechanism(s) involved in the stimulatory effect of nificantly modify the stimulatory effect of glutamate. Recent methoxamine on the Dl DA receptor resensitization process. studies from our laboratory have demonstrated that a G,,r pro- Nevertheless, our results already provide some preliminary in- tein is present in striatonigral neurons possessing Dl DA re- dications. A possible modulation of phosphodiesterase activity, ceptors (He& et al., 1993) and a specific adenylate cyclase as suggested by Beavo and Reifsnyder (1990) can be excluded sensitive to DA has recently been identified in the striatum since all our experiments were performed in the presence of the (Glatt and Snyder, 1993). These observations could eventually non selective phosphodiesterase inhibitor 3-isobutyl- 1-meth- explain why distinct intracellular biochemical events are in- ylxanthine. As already indicated, LY1 b-adrenergic receptors ap- volved in the regulation of the recovery of the full Dl DA pear to be involved in the stimulatory effect of methoxamine. response in cortical and striatal neurons. These receptors are coupled to phospholipase C, leading to the Some specificity was also found in the stimulatory effect of formation of diacylglycerol and inositol phosphates. Diacylgly- glutamate on the resensitization of the striatal D 1 DA receptors. cerol and/or calcium mobilized from intracellular stores by ino- Indeed, glutamate affected neither the full DA response nor the sitol phosphates can stimulate PKC and therefore could poten- DA-induced desensitization. Moreover, glutamate did not affect tially phosphorylate a protein involved in the accelerated the resensitization rate of the cortical D 1 DA receptors although resensitization of the Dl response. The stimulatory effect of there is evidence, particularly in the prefrontal cortex, that there methoxamine persisted in both the absence of extracellular cal- are cortical cells that are sensitive to both DA and glutamate cium or the presence of a calcium chelating agent (data not (Pralong and Jones, 1993). However, glutamate also accelerated shown), suggesting that intracellular calcium could be eventually the resensitization of P-adrenergic receptors coupled to adeny- implicated in this process. Experiments performed with the late cyclase in striatal neurons, indicating that the glutamatergic phorbol ester PMA, which mimics the effect of diacylglycerol, and ,&adrenergic receptors involved are colocalized on the same support the involvement of PKC. Indeed, the direct activation population of striatal neurons. These neurons could also possess of PKC by PMA reproduced the effect of methoxamine. More- Dl receptors since we have previously shown using additivity over, the prolonged (40 hr) preexposure of cortical neurons to experiments that D 1 DA and P-adrenergic receptors are in part PMA, a procedure that desensitizes PKC (Hepler et al., 1988), colocalized in a population of striatal neurons (Chneiweiss et prevented the accelerating effect of methoxamine on the resen- al., 1985). sitization of the Dl response in cortical neurons. Providing further evidence for the involvement of distinct regulatory Relevance of the stimulatory effects of methoxamine and mechanisms in the desensitization and resensitization processes, glutamate on the resensitization processes of cortical and the inactivation of PKC induced by long term pretreatment of striatal Dl receptors to in vivo findings cortical neurons with PMA did not affect the desensitization of Although the questions raised in the present study were sug- Dl DA receptors. Experiments are in progress to identify the gested by in vivo findings, the interpretation of in vivo events in protein that is phosphorylated by PKC following exposure to adult rats based on data obtained from cultured embryonic neu- methoxamine and is responsible for the accelerated recovery of rons can be criticized. For instance, the long term development the full Dl DA response in cortical neurons. of the denervation supersensitivity of the cortical or striatal Dl DA receptors may involve additional or different mechanisms Acceleration by glutamate of the resensitization of the DI than those implicated in the rapid resensitization processes de- response in striatal neurons scribed in the present study. In fact, it has been reported in Through CAMP formation and protein kinase A activation, the several binding studies that following denervation, changes in stimulation of D 1 DA receptors in the striatum leads via CAMP the sensitivity of receptors coupled to adenylate cyclase are in to the phosphorylation of DARPP 32, an inhibitor of phospha- most cases very likely to be associated with the appearance of tase I (Hemmings et al., 1984). As elegantly shown, by acting new receptors on the postsynaptic neuronal membrane (Spom on NMDA receptors, glutamate activates calcineurine, which et al., 1976). As already discussed, this is certainly not the case dephosphorylates DARPP 32 and thus counteracts some of the in our in vitro experimental paradigm. However, the amplitude effects of DA (Halpain et al., 1990). Interestingly, our study of the full DA responses in cortical and striatal embryonic neu- demonstrates a new regulatory process of DA transmission by rons, when compared to those observed in tissues of normal glutamate. Indeed, glutamate was found to accelerate the resen- adult rats, suggests that Dl DA receptors in neuronal cultures sitization of the Dl DA-induced formation of 3H-cAMP in are likely to be in an hypersensitive state. Although not strictly cultured striatal neurons. comparable, the hypersensitive Dl DA receptors in cultured NMDA receptors appear to be involved in the stimulatory neurons and in adult rats with denervation of DA neurons may effect of glutamate on the resensitization rate of striatal Dl DA thus have some common properties. receptors. Indeed, the glutamate-induced facilitation of this re- According to our in vivo data, the presence of the cortical The Journal of Neuroscience, October 1994, 14(10) 6287 noradrenergic innervation plays a prominent permissive role in Bates MD, Caron MG, Raymond JR (199 1) Desensitization of DA1 the appearance of the denervation supersensitivity of the pre- dopamine receptors coupled to adenylate cyclase in opossum kidney cells. Am J Physiol 260:F937-F945. frontocortical D 1 DA receptors (Tassin et al., 1986). Moreover, Beavo JA, Reifsnyder DH (1990) Primary sequence of cyclic nucle- arl-adrenergic receptors sensitive to prazosin were shown to be otide phosphodiesterase isoenzymes and the design of selective in- involved in this heterologous receptor regulation (Trovero et hibitors. Trends Pharmacol Sci 11: 150-l 55. al., 1992b). This is in agreement with our present results. 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