D1 but not D2 dopamine receptors or adrenoceptors mediate dopamine-induced potentiation of N-methyl-d -aspartate currents in the rat prefrontal cortex

Neuroscience Letters 372 (2004) 89–93

D1 but not D2 dopamine receptors or adrenoceptors mediate dopamine-induced potentiation of N-methyl-d-aspartate currents in the rat prefrontal cortex Kerstin Wirknera,∗ , Thomas Krausea , Laszlo K¨olesa,b , Susanne Th¨ummlera , Mahmoud Al-Khrasania,b , Peter Illesa a

Rudolf–Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, H¨artelstraße 16–18, D-04107 Leipzig, Germany b Department of Pharmacology and Pharmacotherapy, Semmelweis University, Nagyvarad ter 4, Budapest H-1445, Hungary Received 30 July 2004; received in revised form 31 August 2004; accepted 8 September 2004

Abstract Dopamine–glutamate interactions in the prefrontal cortex (PFC) are associated with higher order cognitive functions, and are involved in the pathophysiology of schizophrenia and addiction. Recordings with intracellular sharp microelectrodes and patch-clamp pipettes were used to investigate these interactions in layer V pyramidal cells of brain slices obtained from the rat PFC. Dopamine (100 ␮M) potentiated Nmethyl-d-aspartate (NMDA; 10 mM)-evoked depolarizations, but did not change those elicited by ␣-amino-3-hydroxy-5-methyl-4-isoxazole4-propionic acid (AMPA; 1 mM). Dopamine (100 ␮M) increased the amplitude of the NMDA (30 ␮M)-induced currents as well, and 1-phenyl2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF 38393; 1, 10 ␮M), a D1 receptor agonist, concentration-dependently reproduced this effect. Furthermore, 7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzapine hydrochloride (SCH 23390; 10 ␮M), a D1 receptor antagonist, reversed both the dopamine- and the SKF 38393-evoked potentiation. The D2 receptor agonists lisuride and quinpirole (10 ␮M both), as well as noradrenaline (100 ␮M) failed to mimic the stimulatory effect of dopamine. Isoproterenol (1, 10 ␮M) concentrationdependently facilitated NMDA responses. However, neither this effect at 10 ␮M nor that of dopamine at 100 ␮M could be antagonized by propranolol (10 ␮M), a non-selective ␤ adrenoceptor blocker. The isoproterenol-induced facilitation of NMDA currents was abolished by SCH 23390 (10 ␮M). The results indicate that dopamine potentiates NMDA responses in layer V pyramidal cells of the PFC solely by activating D1 receptors. D2 receptors and ␣ or ␤ adrenoceptors are not involved in the dopamine–NMDA interaction. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Dopamine D1 receptor; NMDA receptor; Prefrontal cortex; Brain slice; Electrophysiology

The rich dopaminergic [5] and glutamatergic [10,23] innervation of the prefrontal cortex (PFC), and interactions between the two transmitter systems are essential for higher order cognitive functions such as attention, memory and learning [22]. Disturbances of the excitatory amino acid (EAA) [25] or dopaminergic systems [8] may impair PFC functions. Hence, the PFC is involved in the pathophysiology of schizophrenia, addiction and cognitive disorders [9]. The site of interaction between the EAA and dopaminergic systems is the close apposition of afferent fibres at the ∗

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0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.09.015

soma, dendritic shafts and synaptic spines of PFC pyramidal neurons [7,21] which express all five subtypes of dopamine receptor mRNA [12] as well as mRNA for the NMDA type EAA receptor [18]. NMDA receptors are essential for normal information processing and for proper memory function [14]. A number of in vitro electrophysiological data have pointed out the complex (excitatory or inhibitory) neuromodulatory influence of dopamine on the PFC, especially on its EAA system [1–3,6,11,16,19,24,26,28]. Although the use of structural analogues of dopamine with selectivities for the D1 receptor unequivocally indicates potentiation of NMDA responses [2,3,19,24,26,28], the application of the endogenous transmitter dopamine itself yielded less homogenous results.

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For instance, low concentrations of dopamine (<50 ␮M) via D1 receptor stimulation have been reported to facilitate, while high concentrations of dopamine (>50 ␮M) via D2 receptor stimulation have been described to inhibit NMDA current responses [28]. In addition, dopamine at high concentrations is known to be no longer selective for its own receptortypes and appears to activate both ␣ and ␤ adrenoceptors. ␤ adrenoceptors alone or in interaction with ␣ adrenoceptors strongly modulate the excitability of hippocampal pyramidal cells [13,15] and also facilitate NMDA receptor function in cultured hippocampal neurons [17]. In view of the rather controversial data regarding the effect of dopamine in the PFC, we investigated the interaction between dopamine and EAA receptors, primarily of the NMDA-type, in layer V pyramidal cells of PFC slices, putting emphasis on the possible involvement of adrenoceptor subtypes in dopamine actions. Recordings were carried out with intracellular sharp microelectrodes or patch-clamp pipettes. 200–400 ␮m thick coronal slices containing the prelimbic portion of the medial PFC from male Wistar rats (6–8 weeks old and weighing 150–200 g for recordings with intracellular sharp microelectrodes; 10–14 days old and weighing 24–30 g for whole-cell patch-clamp recordings) were cut using a Vibratome 1000 (Plano, Marburg, Germany). Slices were transferred to a holding chamber, where they stayed for at least 1 h in oxygenated artificial cerebrospinal fluid (aCSF, pH 7.4) of the following composition (mM): NaCl 126, KCl 2.5, NaH2 PO4 1.2, CaCl2 2.4, MgCl2 1.3, NaHCO3 25 and glucose 11. Then, a single slice was placed in a recording chamber and was superfused with oxygenated aCSF at a rate of 2.5–3 ml/min. Recordings were performed at 35–36 ◦ C and 23–25 ◦ C by intracellular microelectrodes and patch-clamp pipettes, respectively. In the first series of experiments, recordings with standard sharp microelectrodes (60–120 M tip resistance; filled with 2 M KCl) were made from layer V PFC neurons. A high impedance preamplifier and a bridge circuit (Axoclamp2A; Axon Instruments, Union City, CA, USA) were used for recordings. The pyramidal neurons impaled in layer V did not fire spontaneous action potentials [27]. Only those cells were included in this study which had a stable membrane potential of <−70 mV and an input resistance of ∼60 M. They were characterized by injecting depolarizing current pulses of 300 ms duration to evoke action potentials. The majority of the cells had firing properties similar to those classified as ‘intrinsic bursting’ neurons; the residual cells were ‘regular spiking’ or ‘rhythmic oscillatory bursting’ neurons [27]. No differences in the pharmacological behaviour of all neurons investigated were observed in the present study. NMDA (10 mM) or AMPA (1 mM) were applied by pressure pulses (60 kPa, 20–650 ms) generated by a Picospritzer II (General Valve, East Hanover, NJ, USA) to evoke reproducible depolarizations of about 15 mV amplitude. EAA responses were elicited every 5 min throughout, except when superfusion with dopamine started or ceased. Dopamine was applied by changing the superfusion medium by means of three-way

taps. Depolarization by NMDA or AMPA was evoked three times before the application of dopamine; the last control response was compared with the agonist responses after 5, 10 and 15 min superfusion with dopamine. In the second series of experiments, pyramidal cells were visualized with an upright interference contrast microscope (Axioscope FS; Carl Zeiss, Oberkochen, Germany) and membrane currents of layer V pyramidal cells in the PFC were recorded by means of the whole-cell patch-clamp method, similar to that described by Edwards et al. [4]. Patch pipettes (tip resistance, 5–7 M) were filled with intracellular solution of the following composition (mM): K-gluconate 140, NaCl 10, MgCl2 1, HEPES [N-(2-hydroxyethyl)piperazineN -(2-ethanesulphonic acid)] 10, EGTA [ethyleneglycol-bis(␤-aminoethylether)-N,N,N ,N ,-tetraacetic acid] 11, MgATP 1.5 and GTP 0.3; pH 7.3 adjusted with KOH. The membrane potential of pyramidal cells, measured in the current-clamp mode of the patch-clamp amplifier (List EPC-7, Darmstadt, Germany), immediately after establishing whole-cell access, was −65 to −85 mV and remained stable during the whole experiment. The system was left for 5–10 min to allow for the settling of a diffusion equilibrium between the patch pipette and the cell interior, before current responses to NMDA were recorded in the voltage-clamp mode. Data were filtered at 3–10 kHz with the inbuilt filter of the EPC-7, digitized at 0.15 kHz (Model 1401; Cambridge Electronic Devices, Cambridge, UK). Data analysis was made by a patch-clamp software (Cambridge Electronic Devices). Drugs were applied by changing the superfusion medium by means of three-way taps. NMDA (30 ␮M) was applied three times for 1.5 min each (T1 , T2 and T3 ) being separated by superfusion periods of 10 min with drug-free aCSF. Dopamine (10, 100 ␮M), SKF 38393 (1, 10 ␮M), lisuride (10 ␮M), quinpirole (10 ␮M), noradrenaline (100 ␮M) or isoproterenol (1, 10 ␮M), were present in the bath 5 min before and during T3 . SCH 23390 (10 ␮M) or propranolol (10 ␮M) were present in the superfusion medium throughout the whole experiment, except when their own effects on NMDA responses were investigated. In the latter case, these antagonists were present in the bath 5 min before and during T3 . Only one cell was used from each brain slice. Since the amplitudes of NMDA-induced currents showed large variabilities, the effects of all drugs applied before and during T3 were normalized with respect to the response measured at T2 , and the effects at T3 were represented as a percentage change compared with the response at T2 . All compounds were obtained from Sigma/RBI (Taufkirchen, Germany). Means ± S.E.M. are given throughout. Comparisons with the control value were performed either by the parametric Student’s t test or the non-parametric Mann–Whitney test, as appropriate. A probability level of 0.05 or less was considered to be statistically significant. The version 2.0 of the package SigmaStat (Jandel Scientific, Erkrath, Germany) was used for all statistical evaluations. Stable intracellular recordings were obtained from pyramidal cells located in layer V of the PFC. The pressure-

K. Wirkner et al. / Neuroscience Letters 372 (2004) 89–93

Fig. 1. Effect of dopamine on the NMDA- or AMPA-induced depolarization in layer V pyramidal neurons of the rat PFC. NMDA (10 mM) or AMPA (1 mM) was pressure-applied every 5 min throughout, except when superfusion with dopamine started or ceased. Recordings were made with standard sharp intracellular microelectrodes. (A) Representative responses to pressure-applied NMDA or AMPA. (Aa) Dopamine (DA; 100 ␮M) potentiates NMDA evoked responses (n = 5). (Ab) Dopamine (100 ␮M) fails to influence AMPA-induced depolarizations (n = 7). (B) Potentiation of the second response to NMDA or AMPA measured in the presence of dopamine in comparison with the third control response to the respective excitatory amino acid. Means ± S.E.M. of n experiments similar to those shown in (A). * P < 0.05; significant difference from the third control response to NMDA.

application of both NMDA (10 mM) and AMPA (1 mM) caused reproducible depolarizations of PFC pyramidal cells. Superfusion with dopamine (100 ␮M) for 15 min increased the NMDA-induced depolarization. The maximum effect was reached within 5 min and did not alter afterwards (10 min incubation; potentiation by 58.3 ± 15.1%; n = 7; P < 0.05; Fig. 1Aa and B). The facilitation by dopamine was reversed by washout. In analogous experiments, dopamine (100 ␮M) failed to influence the effect of AMPA both after a superfusion time of 10 min (−1.2 ± 8.4% change; Fig. 1Ab and B) and 15 min (1.6 ± 4.9% change; Fig. 1Ab; n = 5 and P > 0.05 each). NMDA (30 ␮M) evoked reproducible current amplitudes at T2 and T3 in whole-cell patch-clamp experiments. (T3 /T2 = 0.99 ± 0.01; n = 6). Dopamine at a low concentration (10 ␮M) did not alter the NMDA currents, however, at a higher concentration (100 ␮M) potentiated the effect of NMDA (Fig. 2Aa and B). SKF 38393 concentration-dependently (1, 10 ␮M) increased the NMDA currents (Fig. 2B). SCH 23390 (10 ␮M) prevented both the dopamine (100 ␮M) and SKF 38393 (10 ␮M)-induced potentiation of NMDA currents (Fig. 2Ab and B). Both lisuride (10 ␮M) and quinpirole (10 ␮M) failed

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Fig. 2. Effects of dopamine as well as dopamine receptor agonists and antagonists on NMDA-induced currents. Whole-cell patch-clamp recordings were made at a holding potential of −80 mV. NMDA (30 ␮M) was applied three times (T1 , T2 , T3 ) for 1.5 min with a 10-min interval between applications. Current responses were reproducible at T2 and T3 under these conditions. Dopamine, SKF 38393, lisuride and quinpirole, were applied 5 min before and during T3 either in the absence or presence of SCH 23390. Superfusion with SCH 23390 started 20 min before T1 and lasted throughout the whole experiment. (A) Representative tracings of the second and third (T2 , T3 ) NMDA responses. (Aa) Potentiation by dopamine of the current response to NMDA. (Ab) Antagonism by SCH 23390 of the dopamine-induced potentiation. (B) Potentiation of NMDA current amplitudes by dopamine itself as well as by dopamine receptor agonists. Means ± S.E.M. of n experiments. Effects of dopamine (10, 100 ␮M; n = 8 each), SKF 38393 (1, 10 ␮M; n = 5 and 8, respectively), lisuride (10 ␮M; n = 7) and quinpirole (10 ␮M; n = 8). Antagonism of the dopamine (100 ␮M; n = 7)- and SKF 38393 (10 ␮M; n = 8)-induced potentiation by SCH 23390 (10 ␮M). No effect of SCH 23390 (10 ␮M; n = 6), when given alone. The names of antagonists and their concentrations are written with faint letters. * P < 0.05; significant difference from the controls at T2 (0%). # P < 0.05; significant difference from the effects of agonists when given in the absence of antagonists.

to mimic the stimulatory effect of dopamine on NMDA responses (Fig. 2B). Whereas noradrenaline (100 ␮M) did not influence the NMDA-induced currents in layer V pyramidal cells of the PFC, isoproterenol concentration-dependently increased the NMDA conductance (1, 10 ␮M) (Fig. 3Aa and B). Surprisingly, SCH 23390 (10 ␮M) but not propranolol (10 ␮M) reversed the stimulatory effect of isoproterenol; propranolol did not alter the potentiation caused by dopamine (Fig. 3Ab and

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Fig. 3. Effects of noradrenaline and isoproterenol as well as propranolol and SCH 23390 on NMDA-induced currents. Whole-cell patch-clamp recordings were made as described in the legend to Fig. 2. Noradrenaline and isoproterenol were applied 5 min before and during T3 either in the absence or presence of propranolol or SCH 23390. Superfusion with propranolol or SCH 23390 started 20 min before T1 and lasted throughout the whole experiment. (A) Representative tracings of the second and third (T2 , T3 ) NMDA responses. (Aa) Potentiation by isoproterenol of the current response to NMDA. (Ab) Antagonism by SCH 23390 of the isoproterenol-induced potentiation. (B) Potentiation of NMDA current amplitudes by isoproterenol but not by noradrenaline. Means ± S.E.M. of n experiments. Effects of noradrenaline (100 ␮M; n = 11) and isoproterenol (1, 10 ␮M; n = 7 and 8, respectively). No antagonism of the isoproterenol (10 ␮M; n = 7)- or dopamine (100 ␮M; n = 6)-induced potentiation by propranolol (10 ␮M). No effect of propranolol (10 ␮M; n = 7), when given alone. Antagonism of the isoproterenol (10 ␮M; n = 8)-induced potentiation by SCH 23390 (10 ␮M). The names of antagonists and their concentrations are written with faint letters. * P < 0.05; significant difference from the controls at T (0%). # P < 0.05; sig2 nificant difference from the effects of agonists when given in the absence of antagonists.

B). When given alone, neither SCH 23390 (10 ␮M, Fig. 2B) nor propranolol (10 ␮M, Fig. 3B) influenced the NMDA responses. Our data unequivocally show that, in layer V pyramidal cells of the rat PFC, the dopamine-induced potentiation of NMDA responses is mediated via the activation of D1 dopamine receptors. This suggestion is supported by threefold evidence. Firstly, the endogenous agonist dopamine facilitated NMDA currents at 100, but not at 10 ␮M, and the effect of the higher concentration of dopamine was abolished by the D1 receptor antagonist SCH 23390. Secondly,

SKF 38393, a D1 receptor agonist, mimicked the dopamineinduced facilitation; this effect was also reversed by SCH 23390. Thirdly, the D2 receptor agonists lisuride and quinpirole did not alter NMDA currents. The present data conflict with a recent report demonstrating an inhibitory D2 –NMDA interaction in the PFC [24] and also disagree with the previously proposed dual action of dopamine on NMDA responses [28]. It has been suggested that dopamine at low concentrations (<50 ␮M) enhances NMDA currents by activating D1 receptors, while at higher concentrations dopamine inhibited NMDA currents by stimulating D2 receptors. However, in our experiments, there was absolutely no indication for a depression by dopamine of responses to NMDA, in perfect agreement with most scientific reports generated in the PFC by either membrane potential or membrane current measurements [2,3,19,26]. In addition, our data revealed that adrenoceptors are not involved in the dopamine-induced potentiation of NMDA responses in the PFC. The ␣ and ␤1 adrenoceptor selective endogenous agonist noradrenaline did not influence NMDA currents. Furthermore, the non-selective ␤ adrenoceptor antagonist propranolol failed to reverse the dopamineinduced stimulatory effect. Although the mixed ␤1 and ␤2 receptor agonist isoproterenol concentration-dependently facilitated the NMDA-induced inward current, isoproterenol apparently activated D1 rather than ␤ adrenoceptors, since its effect was abolished by SCH 23390 but not by propranolol. On the basis of these results it could be excluded that dopamine acts via a new type of dopamine-receptor or by non-specific mechanisms in PFC pyramidal cells [20]. In accordance with previous findings, in the present experiments only NMDA but not AMPA responses were altered by dopamine [28]. It is noteworthy that some authors have reported that dopamine negatively interacts with AMPA [11], supposedly by activating D2 receptors [24]; in contrast, D1 receptor-activation did not influence AMPA-induced postsynaptic responses [2,19]. Since the primary purpose of the present study was to clarify the interaction between dopamine and NMDA receptors, it was not attempted to search for the possible reasons of discrepancy between our data and those of a few other authors. In summary, we demonstrated both by measuring membrane potential changes and membrane currents that ␣ or ␤ adrenoceptors are not involved in the dopamine-induced potentiation of NMDA responses in layer V pyramidal cells of the rat PFC. These effects appear to be mediated via D1 but not D2 receptor activation. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (IL 20/9-2). We are grateful to Dr. P. Scheibler and Mr. J.M. Guzman for methodological support.

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