Effects of Typical and Atypical Antipsychotics and Receptor Selective Compounds on Acetylcholine Efflux in the Hippocampus of the Rat Sudabeh Shirazi-Southall, M.A., Dana Ellen Rodriguez, A.H.T., George G. Nomikos, M.D., Ph.D. Some atypical antipsychotic drugs appear to improve 100,907), the 5-HT2C (SB 242,084), the 5-HT6 (Ro 04-6790), ␣ cognitive function in schizophrenia and since acetylcholine the D2 (raclopride) receptors, and the 1-adrenoceptors (ACh) is of importance in cognition, we used in vivo (prazosin) modestly increased ACh by about 50%. The ϩ ␣ microdialysis to examine the effects of antipsychotics 5-HT1A agonist R-( )-8-OH-DPAT and the 2- administered acutely (SC or IP) at pharmacologically adrenoceptor antagonist yohimbine significantly increased comparable doses on ACh outflow in the hippocampus of the ACh by about 100% and 50%, respectively. Thus, olanzapine rat. The atypical antipsychotics olanzapine and clozapine and clozapine increased ACh to a greater extent than other tested produced robust increases in ACh up to 1500% and 500%, antipsychotics, explaining perhaps their purported beneficial respectively. The neuroleptics haloperidol, thioridazine, and effect in cognitive function in schizophrenia. It appears that chlorpromazine, as well as the atypical antipsychotics selective activity at each of the monoaminergic receptors studied risperidone and ziprasidone produced modest increases in is not the sole mechanism underlying the olanzapine and ACh by about 50–100%. Since most atypical antipsychotics clozapine induced increases in hippocampal ACh. affect a variety of monoaminergic receptors, we examined [Neuropsychopharmacology 26:583–594, 2002] whether selective ligands for some of these receptors affect © 2002 American College of Neuropsychopharmacology. hippocampal ACh. Antagonists for the 5-HT2A (MDL Published by Elsevier Science Inc. KEY WORDS: Antipsychotic; Schizophrenia; Olanzapine; Impairments in cognitive processes, such as deficits in Clozapine; Microdialysis; Acetylcholine executive function, verbal memory and attention, are prevalent in most patients with schizophrenia (Breier 1999). Some atypical antipsychotic drugs appear to im- prove not only the positive symptoms, but also the neg- ative symptoms and the cognitive deficits when com- From the Neuroscience Discovery Research, Lilly Research Labo- ratories, Eli Lilly and Company, Lilly Corporate Center, Indianapo- pared with classical neuroleptics (Kinon and Lieberman lis, IN 46285-0510, USA 1996; Meltzer and McGurk 1999; Remington and Kapur Address correspondence to: G.G. Nomikos, Neuroscience Dis- 2000). For example, some atypical antipsychotic drugs covery Research Eli Lilly and Company Lilly Corporate Center Indi- anapolis, IN 46285-0510, Tel.: (317) 433-2541, Fax: (317) 276-5546, have been shown to improve attention, motor and exec- E-mail: [email protected] utive function in schizophrenic patients (Meltzer and Received May 29, 2001; revised October 8, 2001; accepted October McGurk 1999). It has been well documented that all 22, 2001. Online publication: 10/24/01 at www.acnp.org/citations/ clinically effective antipsychotic drugs block dopamine Npp102401193 D2 receptors in the brain. Additionally, many preclini- NEUROPSYCHOPHARMACOLOGY 2002–VOL. 26, NO. 5 © 2002 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/02/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(01)00400-6 584 S. Shirazi-Southall et al. NEUROPSYCHOPHARMACOLOGY 2002–VOL. 26, NO. 5 cal and clinical studies have indicated that atypical effects of typical and atypical antipsychotic drugs on antipsychotics, such as clozapine, olanzapine and ris- hippocampal ACh efflux. peridone, potently inhibit serotonin 5-HT2 receptor function and affect a variety of other receptors in the brain (Meltzer et al. 1989; Leysen et al. 1992; Bymaster MATERIALS AND METHODS et al. 1996). In particular, clozapine and olanzapine show Animals and Surgery a complex pharmacological profile, being described as “multi-acting-receptor-targeted antipsychotic agents” Adult male Wistar rats (250–300 g; Harlan Sprague Daw- (MARTAs) (Bymaster et al. 2000). ley, Inc., Indianapolis, IN) were housed under 12-h Acetylcholine has been shown to be an important light/dark cycle (lights on at 7:00 A.M.) in a temperature neurotransmitter in motor function, attention and var- (23ЊC) controlled animal facility with food and water ious aspects of cognition. Cholinergic synapses are available ad libitum. Rats were anesthetized with a solu- prevalent in many areas of the brain, including the tion containing chloral hydrate (Sigma) and pentobar- striatum, the prefrontal cortex and the hippocampus. bital sodium (Bergen Brunswick) and were mounted in a Neuronal processes within the hippocampus, in par- stereotaxic apparatus (Stoelting) with the incisor bar 3 ticular, mediate declarative memory, and participate mm under the horizontal plane passing through the in- in cognitive mapping in both experimental animals teraural line. Animals were implanted with a guide can- and humans (Eichenbaum et al. 1999). It appears likely nula (Bioanalytical Systems, Inc.) in the hippocampus therefore that a dysfunction in cholinergic neurotrans- (coordinates relative to bregma: AP ϭ Ϫ5.2, ML ϭ ϩ5.0, mission within the hippocampus may be responsible DV ϭ Ϫ3.8) according to the atlas of Paxinos and Watson for the cognitive impairments often encountered in (1986). While the animal was still anesthetized, a com- schizophrenia. In turn, the beneficial effects of some mercially available microdialysis probe (Bioanalytical atypical antipsychotic drugs in the treatment of cogni- Systems, Inc.), with an active membrane surface of 4.0 tive deficits in schizophrenia may be related to their mm, was inserted through the guide cannula and se- selective action on cholinergic function in the hippo- cured in place. After surgery, rats were housed individu- campus, as a consequence of their multireceptor phar- ally in plexiglass cages. Principles of laboratory animal macological profile. care (Guide for the Care and Use of Laboratory Animals, Na- Earlier in vivo microdialysis experiments have re- tional Academy Press 1996) were followed, and all pro- vealed a selective action of clozapine on ACh outflow tocols were approved by the Animal Care and Use Com- in limbocortical, dopaminergic regions of the brain, mittee of Eli Lilly and Company. such as the prefrontal cortex, as compared with classi- cal neuroleptics (Parada et al. 1997; Moore et al. 1999). Microdialysis Experiments and In general, antipsychotic drugs appear to either in- Biochemical Analysis crease or have no effect on ACh efflux within the stria- tum, a dopaminergic region of the brain related to mo- The day prior to microdialysis experiments, rats were tor function (Imperato et al. 1993; Ueda et al. 1995; transported from their home cage to the microdialysis DeBoer and Abercrombie 1996; Ikarashi et al. 1997; testing room for habituation. All experiments were per- Parada et al. 1997; Moore et al. 1999). To this end, we formed approximately 48 h postsurgery in awake, freely sought to examine the effects the atypical antipsy- moving animals during the light period. Microdialysis chotic drugs, olanzapine, clozapine, risperidone and experiments and the subsequent biochemical analysis ziprasidone, vis-à-vis the effects of the typical antipsy- were performed using an automated on-line injection chotic drugs, haloperidol, thioridazine and chlorpro- system. On the day of the experiment, microdialysis mazine, on ACh efflux in the hippocampus of the rat probes were perfused with a modified Ringer’s solution by means of in vivo microdialysis. In order to be able comprised of: 1.3 mM CaCl2 (Sigma), 1.0 mM MgCl2 to differentiate between the effects of typical and atyp- (Sigma), 3.0 mM KCl (Sigma), 147.0 mM NaCl (Sigma), ϫ ical antipsychotics on ACh outflow, pharmacologically 1.0 mM Na2HPO4 7H20 (Malinckrodt), 0.2 mM ϫ ␮ comparable doses of the tested antipsychotics were used, NaH2PO4 2H2O (Malinckrodt) and 0.3 M neostig- ␮ primarily based on ex vivo occupancy data for the D2 mine bromide (Sigma) (pH 7.2) at a rate of 2.4 l/min and/or the 5-HT2 receptors, i.e. doses that achieve consid- set by a microinfusion pump (Scipro). The dialysate erable occupancy for these receptors (Schotte et al. 1996; was loaded directly into a 100 ␮l sample loop of the in- Zhang and Bymaster 1999). We also examined the ACh jector (Valco Instruments Co.) and automatically in- responses to selective ligands for some monoaminergic jected into the analytical system (ESA, Inc.) every 15 ␣ ␣ receptors (D2, 5-HT2A, 5-HT2C, 5-HT6, 5-HT1A, 1- and 2- min. The loading and injecting modes of the injector adrenoceptors) that are targeted by the studied atypical were computer driven (EZ Chrom software, Scientific antipsychotic drugs. Thus, we aimed at unraveling the Software). Dialysate concentrations of acetylcholine receptor system(s) involved in the tentative differential were determined by high-performance liquid chroma- NEUROPSYCHOPHARMACOLOGY 2002–VOL. 26, NO. 5 Antipsychotics and Hippocampal Acetylcholine 585 tography with electrochemical detection (HPLC-ED), lution (RBI). SB 242,084 (3mg/kg, SC)-LRL was dis- with a 150 ϫ 3mm ACH-3 column (ESA, Inc.) main- solved in a 5% 2-hydroxypropyl-␤-cyclodextrin solu- tained at 35ЊC. The mobile phase was comprised of 100 tion (RBI)/10% cremophor EL (Sigma) solution with 10 mM di-Sodium hydrogen phosphate (Fluka), 2.0mM ␮l