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Progress in Neuro-Psychopharmacology & Biol

Chronic nicotine and dizocilpine effects on nicotinic and NMDAglutamatergic receptor regulation: Interactions with clozapine actions and

attentional performance in rats

Amir H. Rezvani a,⁎, Yousef Tizabi b, Bruk Getachew b, Sheketha R. Hauser b, D. Patrick Caldwell a,Christopher Hunter a, Edward D. Levin a

a Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, NC 27710, USAb Department of Pharmacology, Howard University College of Medicine, Washington DC 20059, USA

Received 24 August 2007; received in revised form 4 January 2008; accepted 30 January 2008Available online 12 February 2008

Abstract

Blockade of NMDA glutamate receptors with dizocilpine (MK-801) has been shown to cause substantial cognitive deficits and has been usedto model symptoms of schizophrenia. Nicotine or nicotinic agonists, in contrast, may enhance cognitive or attentional functions and be oftherapeutic potential in schizophrenia. Nicotinic–glutamatergic interactions, therefore, may have important implications in cognitive functions andantipsychotic treatments. Clozapine, a widely used antipsychotic drug, has been shown in some studies to be effective in ameliorating thecognitive deficits associated with schizophrenia. However, there is some evidence to suggest that clozapine similar to haloperidol may impairsustained attention in rats. In this study, we sought to determine whether chronic nicotine or dizocilpine may modify the effects of acute clozapineon attentional parameters and whether the behavioral effects would correlate with nicotinic or NMDA receptor densities in discrete brain regions.Adult female rats trained on an operant visual signal detection task were given 4 weeks of nicotine (5 mg/kg/day), dizocilpine (0.15 mg/kg/day),the same doses of both nicotine and dizocilpine as a mixture, or saline by osmotic minipump. While on chronic treatment, rats received acuteinjections of various doses of clozapine (0, 0.625, 1.25, 2.5 mg/kg, sc) 10 min prior to tests on attentional tasks. The pumps were removed on day28 and 24 h later the animals were sacrificed for measurements of receptor densities in specific brain regions. The percent correct hit as a measureof sustained attention was significantly impaired by clozapine in a dose-related manner. Neither chronic nicotine nor dizocilpine affected thismeasure on their own or modified the effects of clozapine. Both nicotine and dizocilpine affected the receptor bindings in a region specific mannerand their combination further modified the effects of each other in selective regions. Attentional performance was inversely correlated with alpha-bungarotoxin binding in the frontal cortex only. In conclusion, the data suggest attentional impairments with clozapine alone and no modificationof this effect with nicotine or dizocilpine. Moreover, cortical low affinity nicotinic receptors may have a role in attentional functions.© 2008 Elsevier Inc. All rights reserved.

Keywords: Attention; MK-801; Nicotinic receptors; NMDA receptors; Schizophrenia; Animal model

1. Introduction

Patients with schizophrenia, in addition tomanifesting positiveand negative symptoms are also likely to suffer from cognitive

Abbreviations: NMDA, N-methyl-D-aspartate; PCP, Phencyclidine; BT,Bungarotoxin; 5-HT, Serotonin; D1, Dopaminergic D1 receptor.⁎ Corresponding author. Department of Psychiatry and Behavioral Sciences,

Box 3412, Bell Building, Duke University Medical Center, Durham, NC 27710,USA. Tel.: +1 919 668 1880; fax: +1 919 681 3416.

E-mail address: azadi@duke.edu (A.H. Rezvani).

0278-5846/$ - see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.pnpbp.2008.01.018

impairments. The high incidence of smoking among thesepatients and findings of cognitive enhancing effects of nicotinehas led to the self-medication hypothesis, implying that some ofthese patients may be benefiting from the nicotine intake (Adleret al., 1993; 1998; Dalack et al., 1998; Cattapan-Ludewig et al.,2005; Hughes et al., 1986; Levin and Rezvani, 2006; Levin et al.,2005a,b; Levin et al., 1996a,b; McEvoy et al., 1995; McEvoy andLindgren, 1996). There has also been the recent suggestion thatnicotine may reduce the side effects of antipsychotic medications(Barnes et al., 2006; Zhang et al., 2007). Therefore, nicotine may

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interact with antipsychotic drugs. This interaction may provecritical in final therapeutic effect of such combination, particu-larly, since some of the current antipsychotic medications withdemonstrated efficacy against positive symptoms of schizophre-nia do not appear to help the cognitive function (O'grada andDinan, 2007). In some cases the antipsychotics may evenexacerbate the cognitive impairments in these patients (Gallhoferet al., 1996; Stip, 1996; Keefe et al., 1999). In a recent study it wasshown that patients with schizophrenia who received haloperidolshowed a deficit in procedural learning as well as on a problemsolving learning task (Stip, 2006), but in another study theirperformance in Maze paradigm was enhanced (Lee et al., 2007).In general, it appears that, depending on the paradigm,antipsychotics may result in incremental improvements in someaspects of cognitive function. However, the overall treatmentoutcome in this regard remains inadequate (Gray and Roth, 2007;Keefe et al., 2007). The atypical antipsychotic clozapine has alsobeen shown to improve (Daniel et al., 1996; Galletly et al., 1997)or have no effect (Goldberg et al., 1993; Daniel et al., 1996;Lindenmayer et al., 1998; Meltzer and McGurk, 1999) oncognition in schizophrenic patients. Recently, it was shown byAmitai et al. (2007) that disruptions of five-choice serial reactiontime task induced by repeated PCP administration are preventedby chronic administration of clozapine. In our animal studies, wehave observed that an acute administration of clozapine orrisperidone significantly impaired sustained attention in rats(Rezvani et al., 2004) while an acute dose of nicotine improvedattention (Rezvani and Levin, 2003b). There is enough evidenceto confirm that nicotine improves attention in experimentalanimals (Grilly et al., 2000; Mirza and Bright, 2001; Mirza andStolerman, 1998; Stolerman et al., 2000). Recently, improvingeffects of acute and chronic nicotine administration on sustainedattention has been reported in rats (Day et al., 2007; Semenova etal., 2007). Nicotinic agonists ABT-418 and SIB-1553A have alsobeen shown to improve attention in experimental animals(McGaughy et al., 1999; Terry et al., 2002). Administration ofhaloperidol also significantly impaired choice accuracy in thevisual signal detection task in rats (Rezvani and Levin, 2004).

Glutamate is the primary excitatory neurotransmitter in the brainwith crucial roles in a variety of functions including cognition. Theglutamate N-methyl-D-aspartate (NMDA) receptor hypofunctionhas been suggested in preclinical models of schizophrenia (seerecent reviews: Lindsley et al., 2006; Morrison and Pilowsky, 2007;Tizabi, 2007). The symptoms of schizophrenia may be attributed toimpairment in NMDA glutamatergic neurotransmission which mayinclude both the dysregulated function and the actual physical loss ofNMDA synapses, particularly in the prefrontal cortex (MacDonaldand Chafee, 2006). Administration of glutamate NMDA receptorantagonists have been used to model symptoms of schizophrenia inrodents. Thus, dizocilpine (MK-801) or phencyclidine (PCP) cancause substantial working and reference memory deficits as well assensorimotor gating impairments in rats (Levin et al., 1998, 2005a,b;Rasmussen et al., 2007) and monkeys (Javitt and Zukin, 1991;Harder et al., 1998; Jentsch and Roth, 1999; Tsukada et al., 2005).Dizocilpine has also been shown to affect attentional processes byreducing signal detection accuracy in an operant visual signaldetection task (Rezvani and Levin, 2003a; Rezvani et al., 2008).

Recently, we demonstrated that the dizocilpine-induced impairmentof sustained attention in rats was significantly diminished by anacute administration of a low dose of (0.025 mg/kg) nicotine(Rezvani et al., 2008). It has been suggested that at least some of thebeneficial effects of nicotinemay bemediated through the release ofglutamate, particularly in the frontal cortical region, as schizophrenicpatients are thought to be functionally deficient in this region (Coan,1973; Freedman et al., 1995; Leonard et al., 1996; Court et al., 1997,1999; Waldo et al., 2000; Lambe et al., 2003; Wang et al., 2006).

Effects of nicotine and other nicotinic agonists on learning andmemory as well as sustained attention have been studiedextensively (see Levin et al., 2006). However, interactionsbetween antipsychotics, particularly the atypical class, andnicotine in context of a hypoglutamatergic state has not beenadequately addressed. We have observed that acute administra-tion of clozapinemay have detrimental effects on some attentionalparameters (Rezvani and Levin, 2004; Rezvani et al., 2008). Wehave also reported that simultaneous infusions of nicotine anddizocilpine in rats canmodify the effects of each drug on nicotinicand NMDA receptor densities in a regionally specific manner(Levin et al., 2005b). Nicotinic receptors were assessed by usingradiolabled alpha-bungarotoxin and cytisine to detect low andhigh affinity nicotinic receptors, respectively and NMDA re-ceptors were measured using radiolabled MK-801. In this studywe sought to determine whether chronic nicotine or dizocilpinemay modify the effects of acute clozapine on attentionalparameters and whether the behavioral effects would be reflectedin nicotinic or NMDA receptor changes in discrete brain regions.

2. Materials and methods

2.1. Animals and housing

Adult female Sprague Dawley rats (N=40) (Taconic Farms,Germantown, NY, USA) were used. Rats were housed in groupsof three in plastic cages with corn cob bedding in an animalcolony room with 12 h reversed light/dark schedule (light on at7:00 p.m.). Room temperature was set at 21±1 °C and relativehumidity at 50±10%. Rats had free access to water in their homecage and were fed daily after testing in order to maintain 85% oftheir ad lib bodyweights. Rats weighed 280±3.1 g (mean±S.E.M)when the injection phase was initiated. All training and testingsessions were performed between 8:00 a.m. and 4:00 p.m. duringthe dark phase of the circadian cycle when the lights were off andanimals were in their active phase. The treatment and care of theanimals were carried out under an experimental protocol approvedby the Animal Care and Use Committee of Duke University in afacility approved by Association for Assessment and Accreditationof Laboratory Animal Care.

2.2. Experimental protocol

The following experiments were conducted in order tocharacterize the acute effects of the prototypic atypical antipsycho-tic drug clozapine on attentional performance and the interaction ofthis drug with chronic nicotine, chronic dizocilpine, and chronicnicotine plus dizocilpine in this task. A dose-function for clozapine

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was determined in rats receiving chronic nicotine, dizocilpine,combination of nicotine and dizocilpine, or placebo infusions.

2.3. Chronic nicotine, dizocilpine and the combination

After training on the signal detection taskwas complete, animalswere treated chronically for 28 days with saline, 5 mg/kg/daynicotine, 0.15 mg/kg/day dizocilpine, or a mixture of nicotine anddizocilpine (5 mg/kg nicotine+0.15 mg/kg dizocilpine), viaosmotic pump (Model 2ML4, Alzet Corporation, Palo Alto, CA).The pumpswere implanted subcutaneously into a pocketmade by ablunt instrument through an incision between the scapulaewhile therats were anesthetized with a cocktail of ketamine (0.6 mg/kg) anddormitor (0.15 mg/kg) administered IP. The incision was closedwith surgical clips after the osmotic pump was implanted. Thenicotine dose rate was chosen to match plasma levels seen in heavysmokers (Murrin et al., 1987; Lichtensteiger et al., 1988;Richardson and Tizabi, 1994; Trauth et al., 1999) and has beenshown to increase neuronal cholinergic receptor expression in adultrats (Trauth et al., 1999).

2.4. Drug preparation

Clozapinewas first dissolved in 0.1MHCl andwas thendilutedwith saline. Clozapine solution was injected subcutaneously in avolume of 2 ml/kg body weight. Dizocilpine and nicotine weredissolved in saline. For nicotine treatment, the nicotine ditartratesalt was used, and the dose was adjusted such that 5 mg/kg/day ofnicotine base was delivered via the osmotic minipump. The pH ofall solutions was adjusted to 7 by 0.1 mM NaOH. Clozapine anddizocilpine doses refer to the weight of the salt of each compound.All drugs were purchased from Sigma, St. Louis, MO, USA.

2.5. Acute clozapine injections

Starting four days after osmotic pump implantation, the ratswere injected acutely SCwith a dose of clozapine (0.625, 1.25 and2.5 mg/kg) or saline during weeks 1–4 of chronic infusion. Eachdose was injected twice, once during the first 2–weeks of chronictreatment and once during the last 2-weeks of the chronictreatment. Thus, all animals on chronic treatment received a totalof 8 acute clozapine (0, 0.625, 1.25, and 2.5 mg/kg, sc) injections.Tenmin after each injection, animals were transferred into operantchambers for testing. Rats in each group received all treatmentsassigned to that group following a counter-balanced design withrandom assignment. The interval between injections was at least48 h. In order to maintain performance, animals were testedwithout treatment during the 2-day inter-session intervals.

2.6. Visual signal detection task

Operant visual signal detection tasks have been used extensivelyto study the effects of different compounds on sustained attention inrats. In this sensitive task, both sensory and sustained attention canbe simultaneously assessed. Animals are required to discriminatebetween visual signals and non-signals (Bushnell et al., 1997;Bushnell, 1998; Rezvani et al., 2004, 2005, 2006, 2008). The

operant chambers of 29×25×29 cm (HWD) were equipped with asignal light; a house light; two retractable levers 13 cm apart, 2.5 cmabove the floor of the chamber, and inserted horizontally 2.5 cminto the chamber; a food cup in the center of the front panel of thechamber 2.2 cm above the floor (Coulbourn Instruments, LehighValley, PA, USA); and a white noise amplifier (Med AssociatesInc., Georgia, VT, USA)mounted above the signal lever generatingbackground white noise of about 65 dB. The signal light, alsoreferred to as the cue light, was located above the food cup at thecenter of the front panel 28 cm above the floor of the chamberduring the final stages of training and during the actual test. Theduration of the signal light (illumination) was set at 500ms. Signalswere generated using Windows based Med Associates Inc.software running on a Pentium computer processor.

The taskwas conducted daily in 240-trial sessions (for details seeBushnell, 1998; Rezvani et al., 2002; Rezvani and Levin, 2003a,b;Rezvani et al., 2004). Two trial types, “signal” and “blank” werepresented an equal number of times per session in groups of four(2 signal and 2 blank, in random order). Only one signal intensitywas used for these experiments. Each signal trial included a pre-signal interval, the signal (cue light), and a post-signal interval. Thepre-signal intervals were selected randomly from 12 different valuesranging from0.3 to 24.4 s. Following the presentation of the signal, apost-signal interval of 2, 3, or 4 s (selected randomly) occurred.Afterthe post-signal interval, both levers were inserted into the chambersimultaneously. These temporal parameters yield a trial presentationrate of five trials/min. Blank trials were presented in an identicalmanner to the signal trials but without a signal light.

Rats were trained for several months to perform the visualsignal detection task (Bushnell et al., 1997; McGaughy andSarter, 1998; McGaughy et al., 1999; Sarter et al., 2001). A trialbegan with both levers retracted from the chamber. At the end ofthe post-signal interval, both levers were inserted into thechamber simultaneously. The levers were both retracted whenone was pressed or if 5 s elapsed without a response. If the ratfailed to press a lever, a response failure was recorded and thetrial was not repeated. Every correct response (i.e. a press on thesignal lever in a signal trial or a press on the blank lever in a blanktrial) was followed by the illumination of the food cup and thedelivery of one 20-mg food pellet. After each incorrect response(i.e. a press on the signal lever in a blank trial or a press on theblank lever in a signal trial) or response failure, the rat received a2 s period of darkness (time out). The left lever was defined as thesignal lever for half of the rats and the right lever as the blanklever; the opposite assignment was made for the remaining rats(Rezvani and Levin, 2003a,b). Rats were trained for approxi-mately 3 months. A performance level of 60% or higher wasrequired before the initiation of pharmacological studies.

2.7. Brain dissection and determination of receptor densities

Rats were sacrificed by decapitation for receptor bindinganalysis one day after pump removal and two days after the lastinjection. The brains were immediately frozen on dry ice afterremoval. For dissection, brains were thawed and maintained on anice-cold plate. Eight areas of the brain were dissected under amagnifying lens and stored at −80 °C before preparation for

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binding assays: hypothalamus, frontal cortex (up to the genu ofcorpus callosum and excluding the olfactory bulb and tubercle),cerebral cortex (both hemispheres of the remaining cortex),striatum (bilateral), hippocampus (bilateral), midbrain (containingthe thalamus), colliculi (superior and inferior), and cerebellum(Tizabi et al., 1999). The inclusion of all these areas was todetermine whether the effects of drugs on receptor binding and/orfunction are selective or non-discriminative. Moreover, includingsuch areas of the brain have the potential for identifying novelareas involved in attentional functions.

Tissue was homogenized in ice-cold 50 mM Tris–HCl buffer(pH 7.0 at room temperature). The tissue homogenate wascentrifuged at 38,000 ×g for 12 min at 4 °C. The pellet waswashed twice by suspension in fresh buffer and centrifuged again.Aliquots of homogenate, equivalent to approximately 10mg tissue,were divided into three sets of tubes for determination of [3H]cytisine, [125I]alpha-bungarotoxin, and [3H]MK-801 binding.These ligands are commonly used to quantify the high affinityα4–β2 nicotinic receptor, the low affinity α7 nicotinic receptor,and the glutamatergic NMDA receptor, respectively (Pabreza et al.,1991; Flores et al., 1992; Berger, 2000). Samples were run intriplicate for both total and non-specific binding. Specific bindingwas determined as the difference between total and non-specificbinding and expressed in femto-mole per milligram protein (fmol/mg protein). Protein assay was performed using the Pierce BCA kit(Pierce Chemical Company, Rockford, IL).

For total [3H]cytisine binding, approximately 4 nM [3H]cytisine (35.2 Ci/mmol, PerkinElmer, Billerica, MA) wasincubated in a final volume of 0.25 ml at 2 °C for 75 min.Non-specific binding was obtained in the presence of 100 μM(−)-nicotine ditartrate (Pabreza et al., 1991).

For total [125I]alpha-bungarotoxin binding, approximately2 nM [125I]alpha-bungarotoxin (113 Ci/mmol, PerkinElmer,Billerica, MA) was incubated in a final volume of 0.25 ml at37 °C for 2 h. Non-specific binding was obtained in the presenceof 200 μM (−)-nicotine ditartrate (Pabreza et al., 1991).

Fig. 1. Effects of acute clozapine on attentional performance of rats chronically tredizocilpine. Values are mean±S.E.M. with N=10 per group.

Saturation studies were carried out in the cortex andcerebellum for α-BT and cytisine binding. The selection of theareas was based on adequacy of tissue to generate the saturationcurve. For this purpose, 8 concentrations of [3H]cytisine (0.3–12 nM) or 8 concentrations of [125I]α-BT (0.1–10 nM) wereutilized. Scatchard plots (for determination ofBmax andKd)weregenerated by Radioligand Binding Analysis Program.

For total [3H]MK-801 binding, approximately 5 μM [3H]MK-801 (24.2 Ci/mmol, PerkinElmer, Billerica, MA) was incubated inthe presence of 10 μM glutamate and 10 μM glycine in a finalvolume of 0.25ml at room temperature for 2 h (Berger, 2000). Non-specific bindingwas obtained in the presence of 100μMdizocilpine.

In all cases, membrane-bound ligand was separated from freeligand by filtration using Brandel GF/C filter paper (soaked in0.5% polyethylenimine to reduce non-specific binding) and aBrandel cell harvester.

2.8. Behavioral measures and statistical analysis of data

The following dependent variables were measured in eachexperiment: percent hit, percent correct rejection, and responseomissions. The threshold for significance was pb0.05. Receptorbinding data were analyzed by one-way analysis of variancefollowed by planned comparisons of the treated groups withcontrols. The Superanova/Statview computer program (SAS;Cary, NC) was used for the statistical analysis of data. Significantinteractions were followed-up by tests of the simple main effects.

3. Results

3.1. Attentional performance

Percent hit was significantly impaired by clozapine in a dose-related manner (Fig. 1). The main effect of clozapine treatmentwas significant (F(3, 105)=11.62, pb0.0001). Hit choiceaccuracy declined in a significant linear fashion (F(1,105)=

ated with control vehicle, dizocilpine, nicotine or combination of nicotine and

Fig. 2. A–H. Effects of chronic nicotine, chronic dizocilpine and their combination on α-bungarotoxin binding to low affinity nicotinic receptors in different areas ofthe brain. Values are mean±S.E.M. with N=8–10 per group.

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33.96, pb0.0001) from 65.1% with no clozapine to 59.6% with0.625 mg/kg clozapine, to 57.5% with 1.25 mg/kg and finally53.1% with the highest dose of 2.5 mg/kg of clozapine. Plannedcomparisons of each clozapine dose back to control showedthat each of the clozapine doses significantly (0.625 mg/kg(F(1,105)=6.95, pb0.01), 1.25 mg/kg (F(1,105)=13.21,pb0.0005), and 2.5 mg/kg (F(1,105)=33.75, pb0.0001))decreased hit choice accuracy relative to no clozapine. Therewere no significant main effects of chronic dizocilpine ornicotine seen with percent hit performance. Also, there wereno significant interactions of clozapine with chronic nicotineor dizocilpine. In contrast to percent hit performance, percentcorrect rejection was not found to be significantly affected byclozapine (data not shown). Similar to percent hit perfor-mance, no effects of chronic nicotine, dizocilpine, or theirinteractions with clozapine were seen on percent correctrejection. Neither hit nor correct rejection showed significantinteractions of drug treatment over weeks.

Response omissions were not significantly altered as tested bythemain effects of nicotine and dizocilpine. Therewas a significant

Fig. 3. A–H. Effects of chronic nicotine, chronic dizocilpine and their combination onValues are mean±S.E.M. with N=8–10 per group.

(F(3, 108)=6.70, pb0.0005) main effect of clozapine, with thehigher (2.5 mg/kg) clozapine dose causing a significant increasein response omissions. There was a significant interaction ofdizocilpine across weeks (F(1,36)=5.17, pb0.025), but tests of thesimple main effects of dizocilpine during weeks 1 and 2 vs. weeks3 and 4 did not show any significant effects of dizocilpine duringeither time period.

3.2. Receptor binding

3.2.1. Bungarotoxin (BT) bindingExcept for the cerebellum, no significant effect of chronic

nicotine, dizocilpine or their combination on BT bindingwas detected (Fig. 2A–H). In the cerebellum, chronic ni-cotine significantly (F(1,31)=14.85, pb0.001) increased BTbinding relative to controls. Interestingly, co-administration ofdizocilpine significantly (F(1,31)=5.63, pb0.05) attenuatedthis effect of nicotine (Fig. 2C). In the frontal cortex,combination of nicotine and dizocilpine showed a trendtoward increase in BT binding, which did not achieve statistical

cytisine binding to high affinity nicotinic receptors in different areas of the brain.

Fig. 3 (continued ).

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significance (Fig. 2A). There were no significant differences inKd values for either cortex or cerebellum between the groups(data not shown).

3.2.2. Cytisine bindingAs has been reported in a variety of studies, chronic nicotine

increased high affinity nicotinic receptor binding in almost allareas. In the current study the increase was statistically significantin the frontal cortex (F(1,31)=7.61, pb0.01), posterior cortex(F(1,28)=29.22, pb0.0005), midbrain (F(1,29)=8.62, pb0.01),striatum (F(1,35)=13.63, pb0.001), colliculi (F(1,28)=11.59,pb0.005), hypothalamus (F(1,26)=9.08, pb0.01), and hippo-campus (F(1,30)=29.41, pb0.0005), but not in the cerebellum(Fig. 3A–H). No significant differences in Kd values for eithercortex or cerebellum between the groups were noted (data notshown).

Chronic administration of dizocilpine alone did not have anyeffect on cytisine bindings in the areas examined (Fig. 3A–H).However, dizocilpine significantly attenuated nicotine-inducedincreases in cytisine binding in the midbrain (F(1,29)=4.24,pb0.05) and hippocampus (F(1,30)=13.54, pb0.001) (Fig. 3Dand H).

3.2.3. Dizocilpine bindingChronic nicotine administration significantly decreased

dizocilpine bindings in the cerebellum (F(1,30)=4.41, pb0.05)(Fig. 4C) and the colliculi (F(1,30)=5.55, pb0.05) (Fig. 4F).NMDA binding in other areas was not affected by chronicadministration of nicotine (Fig. 4A–H). Chronic dizocilpinesignificantly (F(1,26)=5.98, pb0.025) increased NMDA bind-ing in the hippocampus only (Fig. 4H). Combination of nicotineand dizocilpine did not modify the effect of either drug in anyarea except in the striatum where the combination resulted in asignificant (F(1,31)=4.83, pb0.05) reduction in dizocilpinebinding compared to the control (Fig. 4E).

3.3. Correlation analysis

Attentional performance averaged over the four weeks ofchronic nicotine and dizocilpine administration when noclozapine was given, as assessed by percent hit was significantly(pb0.05, r2 =0.124) correlated in an inverse fashion with alpha-bungarotoxin binding in the frontal cortex (Fig. 5). This was aselective effect in that no correlation with alpha-bungarotoxinreceptor binding in any of the other areas assessed was observed.

Fig. 4. A–H. Effects of chronic nicotine, chronic dizocilpine and their combination on dizocilpine binding to NMDA glutamate receptors in different areas of the brain.Values are mean±S.E.M. with N=8–10 per group.

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Fig. 5. Correlation between α-bungarotoxin in the frontal cortex and percent hitin all chronic treatment groups without clozapine as an index of correctperformance in the visual signal detection attentional task (pb0.05).

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Also, there were no significant correlations between percent hitand receptor binding of cytisine or dizocilpine in any of the brainregions assessed.

4. Discussion

The results of this study suggest that acute administration ofclozapine, a widely used atypical antipsychotic drug, may haveunfavorable effects on some attentional parameters. Thesefindings, while confirming our previous reports (Rezvani et al.,2004, 2008), further suggest that chronic administration of anNMDA antagonist or nicotine may not exacerbate the effects ofclozapine. Clozapine has been shown to interact with variousreceptors. For instance, in addition to its well characterizedinteractions with D2 receptors, it has been shown to have lowaffinity antagonism for D1 and moderate antagonism for the 5-HT2a/2c receptors (Keefe et al., 1999). Since antagonism of thesereceptors may worsen aspects of cognitive function (Keefe etal., 2007), it is likely that some of the detrimental effects ofclozapine observed in this study may be related to its interactionwith these receptors. Recently, it was shown that the principalmetabolite of clozapine, but not clozapine itself, is a potent andefficacious muscarinic receptor agonist (Weiner et al., 2004).Since muscarinic agonists may posses an antipsychotic-likeprofile (Stanhope et al., 2001), it would be of interest todetermine whether this metabolite contributes to the clinicalefficacy of clozapine.

The results of the current study may be of relevance toantipsychotic treatments in patientswith schizophrenia that smoke.Therefore, addition of a nicotinic agonist in a schizophrenic patientundergoing treatment with atypical antipsychotics may benefit andnot be of further detriment. Obviously, the experimental paradigmutilized in this study might have significant limitations inextrapolation to human cognitive functions. Thus, possible thera-peutic potentials of nicotine or nicotinic agonists in overallsymptoms of schizophrenia, in general, and cognitive functions, inparticular, warrant further studies.

It was reported previously that simultaneous infusion ofnicotine and dizocilpine in rats canmodify the effects of each drug

on nicotinic and NMDA receptor densities in a regionally specificmanner (Levin et al., 2005b). In the current study, administrationof a lower dose of dizocilpine (0.15 vs. 0.3 mg/kg in previousstudy) did not achieve the same effects on receptor densities.Thus, the effects of dizocilpine on low affinity nicotinic receptorswere not evident in the hypothalamus, colliculi, or hippocampus,whereas an increase was noted before (Levin et al., 2005b).Similarly, the increase in NMDA receptors in the hippocampus,the only area that was affected by dizocilpine in both studies, wasnot as high as in the previous study.Moreover, the combination ofthe lower dose of dizocilpine with nicotine in this study did notresult in an increase in low affinity nicotinic receptors in thefrontal cortex as noted previously (Levin et al., 2005b). Finally,the combination of dizocilpine and nicotine previously resulted ina decrease in high affinity nicotinic receptors in the frontal cortexand the striatum which was not evident in the current study.Interestingly, the neurochemical effects of dizocilpine seem toparallel the behavioral observations in that a higher dizocilpinedose appears to be necessary to bring about the changes in thecortical receptor densities as well as impairment in sustainedattention which was observed previously (Rezvani et al., 2003a).

It is important to note that in this study the animals alsoreceived clozapine treatment. Thus, the possible interactiveeffects of clozapine on receptor bindings must also be considered.Hence, some of the effects observed in this study, such as adecrease in cerebellar NMDA receptors following administra-tions of nicotine or combination of nicotine and dizocilpine,which was absent in the previous study, might be due tointeractive effects of clozapine with nicotine and dizocilpine. Inthis regard, it would be of interest to investigate the effects ofclozapine alone on nicotinic and NMDA receptors.

Attentional performance, when no clozapine was given, wasinversely correlated (r2 =0.124) with alpha-bungarotoxin bind-ing in the frontal cortex. This correlation, although rather weak,was selective and unique in that no such relationship betweenthe behavior and any of the receptors in any other region wasobserved. This finding may specifically implicate frontalcortical low affinity alpha7 nicotinic receptors in attentionalfunctions. A role for nicotinic receptors in cognitive functions issupported by a number of animal as well as human studies.Thus, there is enough evidence to suggest that nicotine mayimprove learning and memory (Rezvani and Levin, 2001; Levinand Rezvani, 2002; Hernandez and Terry, 2005; Levin et al.,2006; Weiss et al., 2007). There is also some evidence thatstimulation of nicotinic receptors by nicotine may provideimprovement of attentional functions both in animals andhumans (Day et al., 2007; Levin et al., 2006; Rezvani et al.,2004, 2005; Semenova et al., 2007). Treatment with a nicotinicanalog may also improve attention. Terry et al. (2002) foundthat the nicotinic agonist SIB-1553A significantly improvesperformance of rats on a 5-choice attentional task but only whenaccuracy was reduced behaviorally with a distracting stimulusor pharmacologically by injecting the rats with dizocilpine.Evidence of the involvement of nicotinic receptors in humancognition is also suggested by the loss of cortical nicotiniccholinergic receptors found during postmortem studies ofpatients with Alzheimere's disease (Nordberg, 2001; Nordberg

1039A.H. Rezvani et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 1030–1040

et al., 1992). Thus, further investigation of therapeutic potentialof nicotine in cognitive disorders, in general, and schizophrenia-related cognitive impairment, in particular, is necessary.

5. Conclusion

In summary, partial attentional impairments induced by acuteadministration of clozapine may not be exacerbated by chronicadministration of nicotine and/or low dose of dizocilpine.Moreover, cortical low affinity nicotinic receptors may beimplicated in attentional functions. Chronic administration ofnicotine (5 mg/kg/day) also did not improve the attentionalimpairments induced by acute administration of clozapine,probably because of inadequate dose of nicotine.

Acknowledgments

This research was supported by the National Instituteof Mental Health Grant MH64494 and NIH/NIGMS (2 SO6GM08016-37). We thank Mrs. Marty C. Cauley of DukeUniversity Medical Center for her excellent editorial skills.

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