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Modulation of medial prefrontal cortical D1 receptors on the excitatory firing activity of nucleus accumbens neurons elicited by (−)-Stepholidine
Life Sciences 67 (2000) 1265Ð1274
Modulation of medial prefrontal cortical D1 receptors on the excitatory Þring activity of nucleus accumbens neurons elicited by (2)-Stepholidine Zi-Tao Zhua,1, Yu Fua, Guo-Yuan Hub, Guo-Zhang Jina,* a
Department of Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China
Abstract (2)-Stepholidine (SPD), with D1 agonistic action, elicited an excitatory Þring activity of nucleus accumbens (NAc) neurons by intravenous administration, but this effect was hardly observed by iontophoresis of SPD into the NAc. The present study intends to determine whether D1 receptors in the medial prefrontal cortex (mPFC) are involved in the action of SPD on the Þring activity of NAc neurons in the chloral hydrate-anesthetized male rats. The results showed that the intra-mPFC microinjected SCH-23390 (D1 antagonist, 30 mM), but not the D2 antagonist spiperone (30 mM), signiÞcantly attenuated the enhanced Þring activity induced by intravenous injection of SPD (2 mg/kg). Similarly, the excitatory Þring of NAc neurons was also exhibited by the microinjection of either SPD or D1 agonist SKF-38393 into the mPFC. The SPD-induced excitatory effect was in a dose-dependent way from 277.8 6 51.3% (10 mM) to 1105.4 6 283.5% (30 mM) of NAc basal Þring, which was completely reversed by SCH-23390 (i.v.). Furthermore, the direct D1 agonistic action of SPD on the mPFC neuron was observed with microiontophoresis. These results indicate that SPD possesses a direct agonistic action on the mPFC D1 receptors, by which it modulates the Þring activity of NAc neurons. © 2000 Elsevier Science Inc. All rights reserved. Keywords: (2)-Stepholidine; D1 dopamine receptor; Microinfusion; Prefrontal cortex; Nucleus accumbens; Electrophysiology
Introduction Recently, considerable evidences from neuroimaging (1), receptor occupancy (2) and clinical studies (3) lend a strong support to the hypothesis that the failure of prefrontal cortical * Corresponding author. Tel.: 86-21-64311833 ext. 402; fax: 86-21-64370269. E-mail address: [email protected] (G.-Z. Jin) 1 Current address: Dept. of Physiology & Pharmacology, Oregon Health Sciences Univ., Portland, OR 97201, USA. 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 7 2 9 -3
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activation in schizophrenic patients is due to hypoactivity of the meso-cortical DA system, especially the dysfunction of D1 receptors of the PFC, which in turn leads to the hyperactivity of subcortical DA neurons, particularly in the meso-accumbens DA system (4Ð6). Hence, adjustment of D1 receptor level or activation of D1 receptors in the cortex may become an important goal of future antipsychotic drug regimes (2,7). Based on this hypothesis, the new type of antipsychotic drug should consist of D1 agonistic and D2 antagonistic characteristics. Our previous studies have demonstrated that (2)-Stepholidine (SPD), a leading compound of tetrahydroprotoberberines (8), acts as a D1 agonist - D2 antagonist in the nigro-striatal DA system (9,10). This dual action on DA receptor subtypes attracts our much great interest to know whether it is also available in the meso-cortico-limbic DA system. Since it has been shown that SPD selectively produces a depolarization inactivation (DI) of the DA neurons in the ventral tegmental area (VTA, A10), but not in the SNC (A9) DA neurons, and behaves with a low incidence of extrapyramidal side-effects (11,12), it is deÞnitely revealed that SPD possesses some atypical neuroleptic properties. More recently, authors have observed that SPD could produce a biphasic effect (decrease followed by increase) on the Þring activity of nucleus accumbens (NAc) neurons. The biphasic effect was closely related to its antagonistic action to D2 receptors and agonistic to D1 receptors respectively in the meso-accumbens DA system (13). However, the D1 agonistic effect of SPD was hardly observed directly in the NAc by iontophoresis. This implies that the D1 agonistic action of SPD on NAc neurons is more likely mediated via some indirect pathways, which may be involved in the interaction between the cortical projection neurons and meso-accumbens DA neurons. The immuno-histochemical studies have conÞrmed that D1 receptors locate on the mPFC pyramidal neurons, which project directly to the NAc with excitatory glutamate efferent (14). Nevertheless, it is still unclear whether the D1-agonistic effect of SPD on NAc Þring is related to its action on mPFC neurons. The present study, thus, aims to investigate the possibility that the mPFC D1 receptors are involved in the excitatory Þring activity of NAc neurons elicited by SPD in rat brain. Materials and methods Drugs (2)-SPD (Shanghai Institute of Materia Medica, Chinese Academy of Sciences), m.p. 161Ð1628C, [a]D-4408 in pyridine, was dissolved in a small amount of 0.1 M H2SO4, then diluted with distilled water and adjusted with 0.1 M NaOH to pH 5.5. The other drugs used in this study were (6)-SKF-38393?HCl, SCH-23390?HCl and spiperone?HCl (Research Biochemicals Incorporated, USA). Each drug is given as the weight of its salt. Animal and surgical preparation Male Sprague-Dawley rats (Shanghai Experimental Animal Center, China) were housed in a light (12:12 h light/dark cycle)-and temperature (238C)-controlled room with food and water available ad libitum. Animals (270Ð320 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic apparatus. A plastic cannula was inserted into a lateral tail vein for administration of drugs and additional anesthetic as needed. Body temperature was maintained at 36 z 388C and ECG were monitored. In preparation for intracranial
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microinjection and single-unit recording, two burr holes (2 mm each) were drilled in the skull overlying the mPFC and NAc, and the dura was retracted. All surgical procedures were performed in accordance with the Guiding Principles in Care and Use of Animals approved by the Council of American Physiological Society. Cannulation and intracranial microinjection A 22-gauge stainless-steel guide cannula was implanted at a point which is 1 mm above the injection site in the mPFC and Þxed to the skull by dental cement and screws. The site is ipsilateral to the recording (coordinates: AP 13.2 mm, ML 0.8 mm, DV 23.2 z 3.5 mm, related to bregma and midline according to the atlas) (15). For intracranial injection, SPD and D1 antagonist SCH-23390 or D2 antagonist spiperone (dissolved in NN9-Dimethyl formamide solution) were freshly made before use. The solutions (pH 5.5) or vehicle were injected at a constant rate of 1 ml over 2 min via a stainless steel internal cannula, which was connected to a syringe infusion pump (B. Braun Melsungen AG, Germany) through a length of polyethylene tubing. Single-unit recording and microiontophoresis Single-barrel glass electrode was pulled and then Þlled with a 2 M NaCl solution containing 2% Pontamine Sky Blue dye. The in vitro impedance of the electrode was measured 4Ð9 MV at 60 Hz. The recording microelectrode was passed through a small burr hole drilled in the skull to the NAc at the coordinates AP 11.4 z 2.2 mm, ML 0.8 z 1.5 mm, DV 25.5 z 7.5 mm with a pulse-motor microdriver (Narishige, Japan). Electrical signals were ampliÞed and displayed on a storage oscilloscope (Nicolet 2090-1, USA), then led into a window discriminator and an audio monitor. A computer automatically collected the Þring activity and simultaneously constructed the Þring rate histogram on-line. The electrical signals were also stored in a tape recorder (TEAC-R61, Japan). The Þring activity of NAc neurons was identiÞed by their electrophysiological characteristics (White and Wang, 1986). The basal Þring (the Þring change was less than about 20% of average rate) was recorded at least 3Ð5 min before the onset of intracranial drug administration. Some experiments were carried out by means of microiontophoresis of drugs into the mPFC, while the spontaneous Þring activity was recorded from the mPFC neurons (AP 12.5 z 3.2 mm, ML 0.9 z 1.4 mm, DV 23 z 4 mm from cortical surface). Five-barrel micropipettes were pulled and broken mechanically to an overall tip diameter of 10Ð18 mm. A single-barrel recording electrode (as mentioned above) was glued adjacent to this Þve-barrel iontophoretic micropipette. The recording pipette tip extended 10 z 20 mm beyond the drug pipette tip. Barrels of the micropipette were Þlled with either 10 mM SKF-38393, 30 mM SPD, 20 mM SCH-23390 or 2 M NaCl solutions (all at pH 4). The drugs were iontophoretically applied using a Þve-channel Neuro Phore System (Medical Systems Corp., USA; Model BH-2). The impedance of the iontophoretic barrels was between 20 and 70 MV. Histology At the end of each experiment, the recording and injection sites were marked by passing a cathodal current (30 mA, 20 z 30 min) and 2% Pontamine Sky Blue dye solution (1 ml, 1 min), respectively. The rats were then perfused transcardially with 0.9% NaCl followed by
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10% formalin. The brains were removed and stored in formalin for at least 48 h until serial frozen sections of 40 mm thickness could be made and stained with cresyl violet and neutral red. The dye spots of both electrode and cannula were veriÞed with a light microscopy. The position of the recording electrode in the NAc (A) and the site of injection cannula in the mPFC (B) were examined (Fig. 1). Statistics The statistical signiÞcance of the effect of each drug was compared with the change of Þring rates (raw data, i.e., spikes/sec) before and after drug injection using an unpaired t test. All numerical data were expressed as mean 6 SEM. Results Excitatory Þring activity of NAc neurons induced by SPD (i.v.) and reversed by D1 antagonist microinjected into the mPFC In general, intravenous administration of SPD produced a consistent biphasic response, i.e., a reduction followed by an increase of NAc neuron Þring during the cumulative doses (0.02 z 2 mg/kg), and the excitatory activity induced by a high dose of SPD could last for 10 z 30 min after the cease of drug injection (n 5 8). In order to verify whether D1 receptors in the mPFC were involved in the SPD (i.v.)-induced excitatory Þring on NAc neurons, the
Fig. 1. Photomicrograph illustrating the sites of recording electrode (arrow) within the NAc (A), and ejecting cannula within the mPFC (B). Abbreviations: CPu, caudate putamen; ac, anterior commissura; LV, lateral ventricle; fmi, forceps minor corpus callosum.
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D1-selective antagonist SCH-23390 (30 mM, 1 ml/2 min) was microinjected into mPFC following the injection of SPD (2 mg/kg, i.v.). It was clearly shown that the increased Þring activities (385.8 6 100.0% of baseline, n 5 6) of NAc neurons elicited by SPD were completely reversed by SCH-23390 (Fig. 2A and B). The reversing rate was 102.7 6 14.6% of baseline (p , 0.05 vs pre-SCH, Fig. 2B). But the D2 antagonist spiperone (30 mM, 1 ml) could not reverse the increased Þring of NAc elicited by SPD (p 5 0.779, n 5 5, Fig. 3). These results indicate that the excitatory effect of SPD on NAc neurons is resulted from the activation of D1 receptors in the mPFC. Excitatory Þring activity of NAc neurons elicited by SPD-microjected into the mPFC and reversed by D1 antagonist (i.v.) In eleven tested rats, the microinjected SPD (10 or 30 mM, 1 ml/2 min) directly into the mPFC immediately increased the Þring rate of NAc neurons (Fig. 4). This enhancing effect, lasting for 5Ð10 minutes after the cessation of drug injection, was in a dose-dependent way from 277.8 6 51.3% (10 mM, n 5 6) to 1105.4 6 283.5% (30 mM, n 5 5) vs baseline (p , 0.05 & 0.01 vs vehicle, respectively; Fig. 4B). Similar effect was also observed with the mi-
Fig. 2. A. Histogram showing the reversal of SPD-elicited Þring activity in the NAc by D1 antagonist SCH-23390 (30 mM) in the mPFc. B. The enhancing effect of NAc neurons elicited by SPD (2 mg/kg, i.v.) was completely reversed by subsequent microinjection of SCH (30 mM, n 5 6) in the mPFC.
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Fig. 3. D2 antagonist spiperone (30 mM) microinjected into the mPFC had no antagonism to the excitatory Þring activity of NAc neurons elicited by SPD (2 mg/kg, i.v.; n 5 5).
croinjection of D1 agonist SKF-38393 (10 mM, n 5 5; Fig. 4B). However, the intravenously administrated D1 antagonist SCH-23390 (1 mg/kg, n 5 5) was capable of reversing the increased Þring activity elicited by SPD (116.5 6 17.5%; p , 0.01 vs 30 mM SPD; Fig. 4A and B). These results were just consistent to that of SPD applied with intravenous injection, and further supported the standpoint that the SPD-induced excitatory Þring of NAc neurons is subjected to an indirect action mediated via the D1 receptors on the mPFC neurons. D1 agonist action of iontophoresis of SPD on the mPFC neuron To further ascertain the D1 agonistic action of SPD directly on the mPFC neurons, the spontaneous Þring activity of mPFC neurons was investigated by microiontophoresis of SPD. The Þring of mPFC neurons (8/10) were inhibited by iontophoretically applied SPD (30 mM). This inhibition (46.3 6 5.2% vs baseline) of SPD was similar to that of D1 agonist SKF38393 (10 mM, n 5 11; Fig. 5A). Moreover, the inhibitory effect of SPD on the Þring of mPFC neurons (4/5) was completely blocked by D1 antagonist SCH-23390 (20 mM; Fig. 5B). Thus, it is indicated that SPD possesses D1 agonistic action on the Þring of mPFC neurons. Discussion In our previous studies, it had been clearly shown that the excitatory Þring activity of NAc neurons induced by intravenous administration of SPD was completely reversed by D1 antagonist SCH-23390 (13). Obviously, the D1 agonistic action of SPD could facilitate the Þring activity of NAc neurons. However, this D1 agonistic action to the NAc neurons could not be
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Fig. 4. A. Histogram showing the reversed effect of D1 antagonist SCH-23390 (i.v.) on the excitatory Þring activity of NAc neuron elicited by microinjection of SPD (30 mM) into the mPFC (i.c., intra-cortical injection). B. Effects of both D1 agonist SKF-38393 (10 mM, n 5 5) and SPD (10, 30 mM, n 5 5) microinjected into the mPFc on the Þring activity of NAc neurons, and SCH-23390 (i.v.) completely reversed the enhancing effect induced by SPD (30 mM). * P , 0.05 and ** P , 0.01 vs vehicle; # P , 0.05 vs pre-SCH.
directly observed with iontophoresis of SPD. These evidences raise a presumption that the D1 agonistic effect of SPD (i.v.) on NAc neurons is an indirect action in the meso-cortico-limbic DA system. One of the possible indirect mechanisms of SPD (i.v.) on NAc neurons is the modulation of mPFC neurons involved in. Particularly, glutamatergic efferent neurons would enhance the
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Fig. 5. A. Histogram showing that the microiontophoresis of both D1 agonist SKF-38393 and SPD inhibited the spontaneous Þring of mPFC neurons. B. Histogram illustrating that the inhibition of SPD (30 mM) on the mPFC neuron Þring was blocked by D1 antagonist SCH-23390 (20 mM).
NAc neuron activity via some pathways between the mPFC and the NAc (14). The present work ascertained that the microinfusion of either SPD or D1 agonist SKF-38393 into the mPFC could really produce a striking excitatory effect on NAc neuron Þring, which was also reversed by the D1 antagonist SCH-23390 (i.v.). Moreover, it was shown that intra-mPFC injected SCH-23390, but not the D2 antagonist spiperone, deÞnitely attenuated the enhanced Þring activity of NAc neurons induced by intravenous administration of SPD. Furthermore, the fact that the iontophoresis of SPD activated the D1 receptors in the mPFC neurons, but not in the NAc neurons (13), strongly imply that in normal conditions, the D1 agonistic action of SPD is more sensitive to mPFC D1 receptors than that of the NAc. Our present results showed that iontophoresis of either D1 agonist SKF-38393 or SPD could produce a marked decrease on the Þring of PFC neurons, which is consistent to the report in vivo by ParÞtt, et al. (21), though some works showed that SKF-38393 failed to inhibit the Þring of PFC neurons or even enhanced the membrane excitability in response to depolarizing current. It may be due to the different sensitivity of DA subtype receptors to DA agonistic agents between the superÞcial and deep layer neurons in PFC area (22).
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Based on the recent evidence (17,23) which has revealed that the excitation of PFC glutamatergic output neurons could lead to the hyperactivity of VTA DA neurons and increased the DA release from the subcortex, such as the nucleus accumbens, the inhibitiory effect of D1 agonistic action on the Þring of PFC neurons may cause a signiÞcant decrease of DA efßux from the NAc, which might give rise to a disinhibition of DA transmitter acting on the subcortical NAc neurons or instead of exciting the accumbens neurons (24). These results not only verify the D1 agonistic character of SPD originated from the mPFC, but also support the hypothesis that the mPFC D1 receptors are involved in the modulatory mechanism of corticoaccumbens neurons (5). Recently, some evidence indicates that the D1 receptors in the mPFC are implicated in the important physiological functions. Electrophysiological studies show that the activation of D1 receptors in the deep layer PFC, but not D2 receptors, enhances the membrane excitability in response to depolarizing current pulses in PFC output neurons, including those that project to the NAc (16). It has been reported that D1 receptor agonist SKF-38393 in the mPFC tends to decrease the NAc DA stress response (17); likewise, SCH-23390 injected into the mPFC signiÞcantly increased the locomotion induced by intra-NAc SKF-38393 (18). More attractive, considerable results have proposed that the hypofunction in the D1 receptor activity in the mPFC is involved in schizophrenia: (a) the D1 receptors are implicated in the control of working memory, and its dysfunction resulted in the prominent feature of schizophrenic patients (19); (b) D1 receptors are reduced in the mPFC of schizophrenia, and this reduction is related to the severity of the negative symptoms (1); (c) the D1 antagonist would worsen the status of schizophrenics (7); (d) D1-speciÞc drugs have already revealed their promising beneÞcial effects on the negative symptoms of schizophrenia (20). It has, thus, suggested that future antipsychotic drugs should be designed with D1 agonistic-D2 antagonistic dual action to DA receptors, i.e. to optimize stimulation of cortical D1 sites as well as to antagonize D2 receptors in subcortex and/or cortex for the amelioration of positive and negative symptoms (2,5). Based on the previous studies and present work, the pharmacological characteristics of SPD have been established with a dual action, i.e. antagonistic to D2 receptors and agonistic to D1 receptors in the meso-mPFC-NAc DA system. Thus, SPD is well correspondent with the current opinion for new antipsychotic drugs, and it has been attempted to try in clinic. In conclusion, SPD possesses agonist actions on D1 receptors in the mPFC, by which it exerts an excitatory inßuence on the Þring activity of NAc neurons. Acknowledgments This work was supported by the foundation of National Nature Sciences of China (Project No. 39670829) and the grant (No. K-016) of State Key Laboratory of Drug Research in Shanghai Institute of Materia Medica. References 1. Y. OKUBO, T. SUHARA, K. SUZUKI, Nature 385 634Ð636 (1997). 2. M.S. LIDOW, G.V. WILLIAM, P.S. GOLDMAN-RAKIC, Trends Pharmacol. Sci. 19 136Ð140 (1998). 3. W.T. Jr. CARPENTER, Biol. Psychiatry 29 735Ð737 (1991).
1274 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
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M. GOLDSTEIN, A.Y. DEUTCH, FASEB J. 6 2413Ð2421 (1992). A.Y. DEUTCH, J. Neural. Transm. (suppl) 36 61Ð89 (1992). E.J. NESTLER, Nature 385 578Ð579 (1997). G. SEDVALL, L. FRADE, Lancet. 346 743Ð749 (1995). G.Z. JIN, Trends Pharmacol. Sci. 8 81Ð82 (1987). K.X. HUANG, B.C. SUN, G.Z. JIN, Acta Pharmacol. Sin. 13 17Ð22 (1992). Z.J. DONG, X. GUO, L.J. CHEN, Y.F. HAN, G.Z. JIN, Life Sci. 61 465Ð472 (1997). X.X. ZHANG, B.C. SUN, G.Z. JIN, Sci. China [C] 40 531Ð538 (1997). B.C. SUN, G.Z. JIN, Acta Pharmaccol. Sin. 13 395Ð399 (1992). Z.T. ZHU, Y. FU, G.Y. HU, G.Z. JIN, Acta Pharmacol. Sin. (1999) (in press). H.W. BERENDSE, Y.G. GRAAF, H.J. GROENEWEGEN, J. Comp. Neurol. 316 314Ð347 (1992). G. PAXINOS, C. WATSON, The Rat Brain in Stereotaxic Coordinates. (1986). C.R. YANG, J.K. SEAMANS, J. Neurosci. 16 1922Ð1935 (1996). M.D. DOHERTY, A. GRATTON, Brain Res. 715 86Ð97 (1996). P. VEZINA, G. BLANC, J. GLOWINSKI, J.P. TASSIN, Synapse 16 104Ð112 (1994) J. ZAHRT, J.R. TAYLOR, R.G. MATHE, A.F. ARNSTEN, J. Neurosci. 17 8528Ð8535 (1997). M. DAVIDSON, P.D. HARVEY, P.L. BERGMAN, Arch. Gen. Psychiatry 47 190Ð191 (1990). K.D. PARFFIT, A. GRATTON, P.C. BICKFORD-WIMER, J. Parmacol. Exp. Ther. 254 539Ð545 (1990). B.S. BUNNEY, G.K. AGHAJANIAN, Life Sci. 19 783Ð1792 (1976). S. MURASE, J. GRENHOFF, G. CHOUVET, F.G. GONON, T.H. SVENSSON, Neurosci. Lett. 157 53Ð56 (1993). 24. Z.T. ZHU, W.R. WU, Y. FU, G.Z. JIN, Acta Pharmacol. Sin. In press (2000).
Modulation of medial prefrontal cortical D1 receptors on the excitatory Þring activity of nucleus accumbens neurons elicited by (2)-Stepholidine Zi-Tao Zhua,1, Yu Fua, Guo-Yuan Hub, Guo-Zhang Jina,* a
Department of Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China
Abstract (2)-Stepholidine (SPD), with D1 agonistic action, elicited an excitatory Þring activity of nucleus accumbens (NAc) neurons by intravenous administration, but this effect was hardly observed by iontophoresis of SPD into the NAc. The present study intends to determine whether D1 receptors in the medial prefrontal cortex (mPFC) are involved in the action of SPD on the Þring activity of NAc neurons in the chloral hydrate-anesthetized male rats. The results showed that the intra-mPFC microinjected SCH-23390 (D1 antagonist, 30 mM), but not the D2 antagonist spiperone (30 mM), signiÞcantly attenuated the enhanced Þring activity induced by intravenous injection of SPD (2 mg/kg). Similarly, the excitatory Þring of NAc neurons was also exhibited by the microinjection of either SPD or D1 agonist SKF-38393 into the mPFC. The SPD-induced excitatory effect was in a dose-dependent way from 277.8 6 51.3% (10 mM) to 1105.4 6 283.5% (30 mM) of NAc basal Þring, which was completely reversed by SCH-23390 (i.v.). Furthermore, the direct D1 agonistic action of SPD on the mPFC neuron was observed with microiontophoresis. These results indicate that SPD possesses a direct agonistic action on the mPFC D1 receptors, by which it modulates the Þring activity of NAc neurons. © 2000 Elsevier Science Inc. All rights reserved. Keywords: (2)-Stepholidine; D1 dopamine receptor; Microinfusion; Prefrontal cortex; Nucleus accumbens; Electrophysiology
Introduction Recently, considerable evidences from neuroimaging (1), receptor occupancy (2) and clinical studies (3) lend a strong support to the hypothesis that the failure of prefrontal cortical * Corresponding author. Tel.: 86-21-64311833 ext. 402; fax: 86-21-64370269. E-mail address: [email protected] (G.-Z. Jin) 1 Current address: Dept. of Physiology & Pharmacology, Oregon Health Sciences Univ., Portland, OR 97201, USA. 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 7 2 9 -3
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activation in schizophrenic patients is due to hypoactivity of the meso-cortical DA system, especially the dysfunction of D1 receptors of the PFC, which in turn leads to the hyperactivity of subcortical DA neurons, particularly in the meso-accumbens DA system (4Ð6). Hence, adjustment of D1 receptor level or activation of D1 receptors in the cortex may become an important goal of future antipsychotic drug regimes (2,7). Based on this hypothesis, the new type of antipsychotic drug should consist of D1 agonistic and D2 antagonistic characteristics. Our previous studies have demonstrated that (2)-Stepholidine (SPD), a leading compound of tetrahydroprotoberberines (8), acts as a D1 agonist - D2 antagonist in the nigro-striatal DA system (9,10). This dual action on DA receptor subtypes attracts our much great interest to know whether it is also available in the meso-cortico-limbic DA system. Since it has been shown that SPD selectively produces a depolarization inactivation (DI) of the DA neurons in the ventral tegmental area (VTA, A10), but not in the SNC (A9) DA neurons, and behaves with a low incidence of extrapyramidal side-effects (11,12), it is deÞnitely revealed that SPD possesses some atypical neuroleptic properties. More recently, authors have observed that SPD could produce a biphasic effect (decrease followed by increase) on the Þring activity of nucleus accumbens (NAc) neurons. The biphasic effect was closely related to its antagonistic action to D2 receptors and agonistic to D1 receptors respectively in the meso-accumbens DA system (13). However, the D1 agonistic effect of SPD was hardly observed directly in the NAc by iontophoresis. This implies that the D1 agonistic action of SPD on NAc neurons is more likely mediated via some indirect pathways, which may be involved in the interaction between the cortical projection neurons and meso-accumbens DA neurons. The immuno-histochemical studies have conÞrmed that D1 receptors locate on the mPFC pyramidal neurons, which project directly to the NAc with excitatory glutamate efferent (14). Nevertheless, it is still unclear whether the D1-agonistic effect of SPD on NAc Þring is related to its action on mPFC neurons. The present study, thus, aims to investigate the possibility that the mPFC D1 receptors are involved in the excitatory Þring activity of NAc neurons elicited by SPD in rat brain. Materials and methods Drugs (2)-SPD (Shanghai Institute of Materia Medica, Chinese Academy of Sciences), m.p. 161Ð1628C, [a]D-4408 in pyridine, was dissolved in a small amount of 0.1 M H2SO4, then diluted with distilled water and adjusted with 0.1 M NaOH to pH 5.5. The other drugs used in this study were (6)-SKF-38393?HCl, SCH-23390?HCl and spiperone?HCl (Research Biochemicals Incorporated, USA). Each drug is given as the weight of its salt. Animal and surgical preparation Male Sprague-Dawley rats (Shanghai Experimental Animal Center, China) were housed in a light (12:12 h light/dark cycle)-and temperature (238C)-controlled room with food and water available ad libitum. Animals (270Ð320 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic apparatus. A plastic cannula was inserted into a lateral tail vein for administration of drugs and additional anesthetic as needed. Body temperature was maintained at 36 z 388C and ECG were monitored. In preparation for intracranial
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microinjection and single-unit recording, two burr holes (2 mm each) were drilled in the skull overlying the mPFC and NAc, and the dura was retracted. All surgical procedures were performed in accordance with the Guiding Principles in Care and Use of Animals approved by the Council of American Physiological Society. Cannulation and intracranial microinjection A 22-gauge stainless-steel guide cannula was implanted at a point which is 1 mm above the injection site in the mPFC and Þxed to the skull by dental cement and screws. The site is ipsilateral to the recording (coordinates: AP 13.2 mm, ML 0.8 mm, DV 23.2 z 3.5 mm, related to bregma and midline according to the atlas) (15). For intracranial injection, SPD and D1 antagonist SCH-23390 or D2 antagonist spiperone (dissolved in NN9-Dimethyl formamide solution) were freshly made before use. The solutions (pH 5.5) or vehicle were injected at a constant rate of 1 ml over 2 min via a stainless steel internal cannula, which was connected to a syringe infusion pump (B. Braun Melsungen AG, Germany) through a length of polyethylene tubing. Single-unit recording and microiontophoresis Single-barrel glass electrode was pulled and then Þlled with a 2 M NaCl solution containing 2% Pontamine Sky Blue dye. The in vitro impedance of the electrode was measured 4Ð9 MV at 60 Hz. The recording microelectrode was passed through a small burr hole drilled in the skull to the NAc at the coordinates AP 11.4 z 2.2 mm, ML 0.8 z 1.5 mm, DV 25.5 z 7.5 mm with a pulse-motor microdriver (Narishige, Japan). Electrical signals were ampliÞed and displayed on a storage oscilloscope (Nicolet 2090-1, USA), then led into a window discriminator and an audio monitor. A computer automatically collected the Þring activity and simultaneously constructed the Þring rate histogram on-line. The electrical signals were also stored in a tape recorder (TEAC-R61, Japan). The Þring activity of NAc neurons was identiÞed by their electrophysiological characteristics (White and Wang, 1986). The basal Þring (the Þring change was less than about 20% of average rate) was recorded at least 3Ð5 min before the onset of intracranial drug administration. Some experiments were carried out by means of microiontophoresis of drugs into the mPFC, while the spontaneous Þring activity was recorded from the mPFC neurons (AP 12.5 z 3.2 mm, ML 0.9 z 1.4 mm, DV 23 z 4 mm from cortical surface). Five-barrel micropipettes were pulled and broken mechanically to an overall tip diameter of 10Ð18 mm. A single-barrel recording electrode (as mentioned above) was glued adjacent to this Þve-barrel iontophoretic micropipette. The recording pipette tip extended 10 z 20 mm beyond the drug pipette tip. Barrels of the micropipette were Þlled with either 10 mM SKF-38393, 30 mM SPD, 20 mM SCH-23390 or 2 M NaCl solutions (all at pH 4). The drugs were iontophoretically applied using a Þve-channel Neuro Phore System (Medical Systems Corp., USA; Model BH-2). The impedance of the iontophoretic barrels was between 20 and 70 MV. Histology At the end of each experiment, the recording and injection sites were marked by passing a cathodal current (30 mA, 20 z 30 min) and 2% Pontamine Sky Blue dye solution (1 ml, 1 min), respectively. The rats were then perfused transcardially with 0.9% NaCl followed by
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10% formalin. The brains were removed and stored in formalin for at least 48 h until serial frozen sections of 40 mm thickness could be made and stained with cresyl violet and neutral red. The dye spots of both electrode and cannula were veriÞed with a light microscopy. The position of the recording electrode in the NAc (A) and the site of injection cannula in the mPFC (B) were examined (Fig. 1). Statistics The statistical signiÞcance of the effect of each drug was compared with the change of Þring rates (raw data, i.e., spikes/sec) before and after drug injection using an unpaired t test. All numerical data were expressed as mean 6 SEM. Results Excitatory Þring activity of NAc neurons induced by SPD (i.v.) and reversed by D1 antagonist microinjected into the mPFC In general, intravenous administration of SPD produced a consistent biphasic response, i.e., a reduction followed by an increase of NAc neuron Þring during the cumulative doses (0.02 z 2 mg/kg), and the excitatory activity induced by a high dose of SPD could last for 10 z 30 min after the cease of drug injection (n 5 8). In order to verify whether D1 receptors in the mPFC were involved in the SPD (i.v.)-induced excitatory Þring on NAc neurons, the
Fig. 1. Photomicrograph illustrating the sites of recording electrode (arrow) within the NAc (A), and ejecting cannula within the mPFC (B). Abbreviations: CPu, caudate putamen; ac, anterior commissura; LV, lateral ventricle; fmi, forceps minor corpus callosum.
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D1-selective antagonist SCH-23390 (30 mM, 1 ml/2 min) was microinjected into mPFC following the injection of SPD (2 mg/kg, i.v.). It was clearly shown that the increased Þring activities (385.8 6 100.0% of baseline, n 5 6) of NAc neurons elicited by SPD were completely reversed by SCH-23390 (Fig. 2A and B). The reversing rate was 102.7 6 14.6% of baseline (p , 0.05 vs pre-SCH, Fig. 2B). But the D2 antagonist spiperone (30 mM, 1 ml) could not reverse the increased Þring of NAc elicited by SPD (p 5 0.779, n 5 5, Fig. 3). These results indicate that the excitatory effect of SPD on NAc neurons is resulted from the activation of D1 receptors in the mPFC. Excitatory Þring activity of NAc neurons elicited by SPD-microjected into the mPFC and reversed by D1 antagonist (i.v.) In eleven tested rats, the microinjected SPD (10 or 30 mM, 1 ml/2 min) directly into the mPFC immediately increased the Þring rate of NAc neurons (Fig. 4). This enhancing effect, lasting for 5Ð10 minutes after the cessation of drug injection, was in a dose-dependent way from 277.8 6 51.3% (10 mM, n 5 6) to 1105.4 6 283.5% (30 mM, n 5 5) vs baseline (p , 0.05 & 0.01 vs vehicle, respectively; Fig. 4B). Similar effect was also observed with the mi-
Fig. 2. A. Histogram showing the reversal of SPD-elicited Þring activity in the NAc by D1 antagonist SCH-23390 (30 mM) in the mPFc. B. The enhancing effect of NAc neurons elicited by SPD (2 mg/kg, i.v.) was completely reversed by subsequent microinjection of SCH (30 mM, n 5 6) in the mPFC.
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Fig. 3. D2 antagonist spiperone (30 mM) microinjected into the mPFC had no antagonism to the excitatory Þring activity of NAc neurons elicited by SPD (2 mg/kg, i.v.; n 5 5).
croinjection of D1 agonist SKF-38393 (10 mM, n 5 5; Fig. 4B). However, the intravenously administrated D1 antagonist SCH-23390 (1 mg/kg, n 5 5) was capable of reversing the increased Þring activity elicited by SPD (116.5 6 17.5%; p , 0.01 vs 30 mM SPD; Fig. 4A and B). These results were just consistent to that of SPD applied with intravenous injection, and further supported the standpoint that the SPD-induced excitatory Þring of NAc neurons is subjected to an indirect action mediated via the D1 receptors on the mPFC neurons. D1 agonist action of iontophoresis of SPD on the mPFC neuron To further ascertain the D1 agonistic action of SPD directly on the mPFC neurons, the spontaneous Þring activity of mPFC neurons was investigated by microiontophoresis of SPD. The Þring of mPFC neurons (8/10) were inhibited by iontophoretically applied SPD (30 mM). This inhibition (46.3 6 5.2% vs baseline) of SPD was similar to that of D1 agonist SKF38393 (10 mM, n 5 11; Fig. 5A). Moreover, the inhibitory effect of SPD on the Þring of mPFC neurons (4/5) was completely blocked by D1 antagonist SCH-23390 (20 mM; Fig. 5B). Thus, it is indicated that SPD possesses D1 agonistic action on the Þring of mPFC neurons. Discussion In our previous studies, it had been clearly shown that the excitatory Þring activity of NAc neurons induced by intravenous administration of SPD was completely reversed by D1 antagonist SCH-23390 (13). Obviously, the D1 agonistic action of SPD could facilitate the Þring activity of NAc neurons. However, this D1 agonistic action to the NAc neurons could not be
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Fig. 4. A. Histogram showing the reversed effect of D1 antagonist SCH-23390 (i.v.) on the excitatory Þring activity of NAc neuron elicited by microinjection of SPD (30 mM) into the mPFC (i.c., intra-cortical injection). B. Effects of both D1 agonist SKF-38393 (10 mM, n 5 5) and SPD (10, 30 mM, n 5 5) microinjected into the mPFc on the Þring activity of NAc neurons, and SCH-23390 (i.v.) completely reversed the enhancing effect induced by SPD (30 mM). * P , 0.05 and ** P , 0.01 vs vehicle; # P , 0.05 vs pre-SCH.
directly observed with iontophoresis of SPD. These evidences raise a presumption that the D1 agonistic effect of SPD (i.v.) on NAc neurons is an indirect action in the meso-cortico-limbic DA system. One of the possible indirect mechanisms of SPD (i.v.) on NAc neurons is the modulation of mPFC neurons involved in. Particularly, glutamatergic efferent neurons would enhance the
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Fig. 5. A. Histogram showing that the microiontophoresis of both D1 agonist SKF-38393 and SPD inhibited the spontaneous Þring of mPFC neurons. B. Histogram illustrating that the inhibition of SPD (30 mM) on the mPFC neuron Þring was blocked by D1 antagonist SCH-23390 (20 mM).
NAc neuron activity via some pathways between the mPFC and the NAc (14). The present work ascertained that the microinfusion of either SPD or D1 agonist SKF-38393 into the mPFC could really produce a striking excitatory effect on NAc neuron Þring, which was also reversed by the D1 antagonist SCH-23390 (i.v.). Moreover, it was shown that intra-mPFC injected SCH-23390, but not the D2 antagonist spiperone, deÞnitely attenuated the enhanced Þring activity of NAc neurons induced by intravenous administration of SPD. Furthermore, the fact that the iontophoresis of SPD activated the D1 receptors in the mPFC neurons, but not in the NAc neurons (13), strongly imply that in normal conditions, the D1 agonistic action of SPD is more sensitive to mPFC D1 receptors than that of the NAc. Our present results showed that iontophoresis of either D1 agonist SKF-38393 or SPD could produce a marked decrease on the Þring of PFC neurons, which is consistent to the report in vivo by ParÞtt, et al. (21), though some works showed that SKF-38393 failed to inhibit the Þring of PFC neurons or even enhanced the membrane excitability in response to depolarizing current. It may be due to the different sensitivity of DA subtype receptors to DA agonistic agents between the superÞcial and deep layer neurons in PFC area (22).
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Based on the recent evidence (17,23) which has revealed that the excitation of PFC glutamatergic output neurons could lead to the hyperactivity of VTA DA neurons and increased the DA release from the subcortex, such as the nucleus accumbens, the inhibitiory effect of D1 agonistic action on the Þring of PFC neurons may cause a signiÞcant decrease of DA efßux from the NAc, which might give rise to a disinhibition of DA transmitter acting on the subcortical NAc neurons or instead of exciting the accumbens neurons (24). These results not only verify the D1 agonistic character of SPD originated from the mPFC, but also support the hypothesis that the mPFC D1 receptors are involved in the modulatory mechanism of corticoaccumbens neurons (5). Recently, some evidence indicates that the D1 receptors in the mPFC are implicated in the important physiological functions. Electrophysiological studies show that the activation of D1 receptors in the deep layer PFC, but not D2 receptors, enhances the membrane excitability in response to depolarizing current pulses in PFC output neurons, including those that project to the NAc (16). It has been reported that D1 receptor agonist SKF-38393 in the mPFC tends to decrease the NAc DA stress response (17); likewise, SCH-23390 injected into the mPFC signiÞcantly increased the locomotion induced by intra-NAc SKF-38393 (18). More attractive, considerable results have proposed that the hypofunction in the D1 receptor activity in the mPFC is involved in schizophrenia: (a) the D1 receptors are implicated in the control of working memory, and its dysfunction resulted in the prominent feature of schizophrenic patients (19); (b) D1 receptors are reduced in the mPFC of schizophrenia, and this reduction is related to the severity of the negative symptoms (1); (c) the D1 antagonist would worsen the status of schizophrenics (7); (d) D1-speciÞc drugs have already revealed their promising beneÞcial effects on the negative symptoms of schizophrenia (20). It has, thus, suggested that future antipsychotic drugs should be designed with D1 agonistic-D2 antagonistic dual action to DA receptors, i.e. to optimize stimulation of cortical D1 sites as well as to antagonize D2 receptors in subcortex and/or cortex for the amelioration of positive and negative symptoms (2,5). Based on the previous studies and present work, the pharmacological characteristics of SPD have been established with a dual action, i.e. antagonistic to D2 receptors and agonistic to D1 receptors in the meso-mPFC-NAc DA system. Thus, SPD is well correspondent with the current opinion for new antipsychotic drugs, and it has been attempted to try in clinic. In conclusion, SPD possesses agonist actions on D1 receptors in the mPFC, by which it exerts an excitatory inßuence on the Þring activity of NAc neurons. Acknowledgments This work was supported by the foundation of National Nature Sciences of China (Project No. 39670829) and the grant (No. K-016) of State Key Laboratory of Drug Research in Shanghai Institute of Materia Medica. References 1. Y. OKUBO, T. SUHARA, K. SUZUKI, Nature 385 634Ð636 (1997). 2. M.S. LIDOW, G.V. WILLIAM, P.S. GOLDMAN-RAKIC, Trends Pharmacol. Sci. 19 136Ð140 (1998). 3. W.T. Jr. CARPENTER, Biol. Psychiatry 29 735Ð737 (1991).
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