Nicotine and Clozapine Cross-Prime the Locus CoeruleusNoradrenergic System to Induce Long-Lasting

Potentiation in the Rat Hippocampus

Ramamoorthy Rajkumar,1,2,3 Sana Suri,1,2 Hong Min Deng,1,2,3 and Gavin Stewart Dawe1,2,3*

ABSTRACT: A priming-challenge schedule of nicotine treatment causeslong-lasting potentiation (LLP), a form of synaptic plasticity closely associ-ated with the norepinephrine (NE) neurotransmitter system, at the medialperforant path (MPP)-dentate gyrus (DG) synapse in the rat hippocampus.Previous reports revealed that nicotine activates the locus coeruleus (LC)noradrenergic (NAergic) system and this mechanism may underlie its beta-adrenoceptor sensitive LLP effects. Clozapine, an atypical antipsychotic, isalso known to activate the LC. Interactions between nicotine and clozapineare of interest because of the prevalence of smoking in patients with schiz-ophrenia and increasing interest in the use of nicotinic receptor ligands ascognitive enhancers. Rats were subchronically primed with nicotine, cloza-pine or saline. Twenty-one to twenty-eight days later, the effects of the nic-otine, clozapine or saline challenge on the evoked field excitatorypostsynaptic potentials (fEPSP) at the MPP-DG monosynaptic pathwaywere recorded as a measure of LLP. We confirmed the hypothesis that achallenge dose of either nicotine or clozapine induces LLP exclusively innicotine- and clozapine-primed rats, and not in saline-primed rats, thusindicating a cross-priming effect. Moreover, unilateral suppression of LCusing lidocaine abolished the LLP induced by nicotine in clozapine-primedrats. Furthermore, systemic treatment with clonidine (an a2 adrenoceptoragonist that reduces NAergic activity via autoreceptors) prior to the chal-lenge doses blocked the nicotine/clozapine-induced LLP in nicotine- andclozapine-primed rats. These findings may add to understanding of thecognitive enhancing effects of nicotine. VC 2013 Wiley Periodicals, Inc.

KEY WORDS: nicotine; clozapine; hippocampus; long-lasting poten-tiation; noradrenergic

INTRODUCTION

Nicotine has been demonstrated to bring about various neuropsycho-pharmacological changes, especially enhancement in cognitive efficiency

and performance in humans and rodents (for reviewssee Wesnes and Warburton, 1983; Grigoryan andGray, 1996; Kenney and Gould, 2008; Heishmanet al., 2010). Several decades of research on mne-monic processing has accepted and utilized hippocam-pal long-term potentiation (LTP) and long-lastingpotentiation (LLP), forms of synaptic plasticity, as ex-perimental tools in cognitive neuroscience (Bliss andLomo, 1973; Bliss and Collingridge, 1993). Amongstthe various neurotransmitter systems that have beenproposed to underlie the effect of nicotine on LLP inthe rodent hippocampus (Hamid et al., 1997; Yama-zaki et al., 2002, 2005; Tang and Dani, 2009; Zhanget al., 2010), catecholaminergic mechanisms appear tobe consistent and demand special interest. Acute nico-tine treatment (single injection) induces tyrosinehydroxylase (TH; the rate limiting enzyme in NE syn-thesis) activity in the LC, and then subsequently inthe hippocampus 21–28 days later (Mitchell et al.,1989, 1990, 1993). Nicotine directly causes NErelease in the hippocampus following both single anddual administrations (Brazell et al., 1991). Chronicnicotine treatment selectively augments TH activity inthe hippocampus and thus pretreatment with nicotineincreases the potential to synthesize and release NE,21–28 days later (Joseph et al., 1990). In addition,we had earlier demonstrated that a challenge dose ofnicotine fosters a beta-adrenoreceptor dependent LLPof fEPSPs in the granular cell layer of the hippocam-pal DG by the stimulation of MPP in nicotine-primed rats (Hamid et al., 1997).

Clozapine, an atypical antipsychotic, is prescribedto treat schizophrenia and is associated with improvedcognitive skills and diminished negative symptoms inpatients (Kane and Correll, 2010). Collective evidencesuggests that clozapine is associated with decreasedincidences of smoking in schizophrenic patients(McEvoy et al., 1995; George et al., 1995) and smok-ers have a greater therapeutic response to clozapinethan nonsmokers (McEvoy et al., 1999). The superiorclinical efficacy of clozapine has been suggested to berelated to the increase in NAergic activity by blockadeof the alpha-2C adrenergic autoreceptors (Brunelloet al., 1995; Breier et al., 1998; Kalkman andLoetscher, 2003). Chronic clozapine treatment acti-vates the LC (Souto et al., 1979; Ramirez and Wang,

1 Department of Pharmacology, Yong Loo Lin School of Medicine,National University Health System, National University of Singapore,Singapore; 2 Neurobiology and Ageing Programme, Life Sciences Insti-tute, National University of Singapore, Singapore; 3 SINAPSE, SingaporeInstitute for Neurotechnology, SingaporeGrant sponsor: Department of Pharmacology, Yong Loo Lin School ofMedicine, and Undergraduate Research Opportunities Programme inScience (UROPS), Faculty of Science, National University of Singapore.*Correspondence to: Gavin Stewart Dawe, Department of Pharmacology,Yong Loo Lin School of Medicine, National University HealthSystem, National University of Singapore, Centre for Life Sciences(CeLS), #04-04, 28 Medical Drive, Singapore 117456, Singapore. E-mail:gavindawe@nus.edu.sgAccepted 12 March 2013.DOI 10.1002/hipo.22122Published online 20 April 2013 in Wiley Online Library(wileyonlinelibrary.com).

VC 2013 WILEY PERIODICALS, INC.

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1986; Nilsson et al., 2005) and findings from our lab showincreased TH expression (Verma et al., 2007) in the LC. Clo-zapine also potentiates the synaptic responses in the DGinduced by stimulation of the rabbit perforant path (Kubotaet al., 1996, 2008).

The LC is the primary source of NE for the hippocampus andthe LC/NE - DG liaison, and plays a significant role in variousintegral aspects of memory retrieval and long term memory(Harley, 1991, 1998; Berridge and Waterhouse, 2003; Harley,2007; Lashgari et al., 2008). NE release is known to initiate abeta-adrenoceptor dependent LTP (Harley, 2007) and an MPPspecific LLP at the hippocampal DG (Neuman and Harley,1983; Dahl and Sarvey, 1989; Pelletier et al., 1994; Chaulk andHarley, 1998; Reid and Harley, 2010). The published literature,together with current understanding of the similarity in action ofnicotine and clozapine at the LC, led us to hypothesize that theseagents may cross-prime the LC NAergic system and cause anLLP at the MPP-DG synapses in the hippocampus.

MATERIALS AND METHODS

Animals and Treatment Schedule

Male Sprague-Dawley rats (180–200 g) were procured fromthe Centre for Animal Resources (CARE), Singapore, andhoused in pairs in ventilated cages at the specific pathogen freefacility at the National University of Singapore. All the rats were

given free access to food and water and they were maintained onan alternating 12 h light and dark cycle in a standard room tem-perature of 23 6 1�C. The rats were primed with either nicotine(0.8 mg/kg) or clozapine (30 mg/kg) or an equivalent volume of0.9% (w/v) NaCl solution (saline, 1 ml/kg) subcutaneously oncedaily for seven consecutive days. The drug/saline-primed ratswere subjected to electrophysiological experiments, during whichchallenge doses were administered subcutaneously, between 21and 28 days following the 7-day priming. All the procedureswere approved by the Institutional Animal Care and Use Com-mittee (IACUC), National University of Singapore.

Drugs and Chemicals

The drugs used were chloral hydrate (Sigma Alrich,Germany), (-) nicotine hydrogen tartrate (Sigma), clozapine(Tocris Bioscience, UK), clonidine (Sigma) and lidocainehydrochloride monohydrate (Sigma). Chloral hydrate (7%w:v), nicotine and clonidine were dissolved in saline. Clozapinewas prepared by dissolving in acidified (pH 5.5–6.0) hydroxy-propyl beta-cyclodextrin (20%).

Electrophysiology

Rats were anesthetized (420 mg/kg chloral hydrate, i.p.) andmaintained on a homeothermic blanket in a stereotaxic frame withmaintenance doses of anesthesia administered via the lateral tailvein. Bipolar stimulating and recording electrodes made from a pairof twisted 80% nickel: 20% chromium wires (0.125-mmdiameter), insulated except at the tips were placed in the MPP (AP:

FIGURE 1. (a) Schematic representation of rat brain regions(traced from Paxinos and Watson, 2007) showing recording site:granular cell layer of the dentate gyrus (GrDG); stimulation site:angular bundle/dorsal subiculum (AB/DS); and site of lidocaineinfusion: locus coeruleus (LC). Circles represent the sites of inter-vention. (b) Typical fEPSP waveform at the DG due to MPP stim-ulation depicting characteristic 400 ms paired pulse depression

(Scale bar: 5 mv, 20 ms). Representative micrographs of the Nisslstained brain sections with the arrows indicating the position ofthe (c) recording electrode in the GrDG, (d) stimulating electrodeat the AB/DS and (e) infusion cannula at the LC, stained with In-dian ink. 4V: 4th ventricle; Me5: mesencephalic trigeminal nu-cleus; PoDG: polymorph layer of the dentate gyrus. Scale bar: 350lm (b,d) and 400 lm (c).

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26.5 mm, ML: 64.5 mm, DV: 23 mm from the skull) andDG (AP: 23.8 mm, ML: 62 mm, DV: 23.7 mm from the skull),respectively (Fig. 1a). All coordinates were calculated relative to thebregma with the skull flat such that bregma and lambda were level.The DV coordinates were adjusted to obtain characteristic MPP-DG pair-pulse depression at a 400 ms interpulse interval (Fig. 1b)as we have previously described (Hamid et al., 1997). The stimuluswas provided by a photoelectric stimulus isolation unit (GRASS,Astro-Med Inc, USA) coupled to a digital S88X stimulator(GRASS, Astro-Med Inc). The evoked potential from the DG wasrecorded using the EX4–400 Quad Differential Amplifier (DaganCorporation, USA) connected to the bipolar recording electrode viathe head stage. The signals from the preamplifier were filtered by a50 Hz noise eliminator (Hum Bug, Quest Scientific, Canada), digi-tized (CED 1401, Cambridge Electronic Design, UK), recordedand analyzed using Signal Software, (Version 4.07). Prior to thecommencement of LLP experiments, the stimulus intensity requiredto produce a half-maximal response (ranging from 120 to 180 mA)was determined from an input–output curve.

Inhibition of Locus Coeruleus

In a subset of clozapine-primed animals, the LC was targetedfor suppression using a guide cannula fitted with an implantingcannula dummy or stylet (30 G PlasticsOne, USA) and positioned1 mm above the structure (AP: 29.8 mm, ML: 61.1–1.3 mmand DV: 26 mm relative to the bregma and perpendicular to theplane of the skull with the skull flat such that bregma and thelambda were level). The cannula was fixed using dental cement(Bosworth Company, USA). At the time of saline/lidocaine infu-sion the cannula dummy was replaced by the infusion cannula (33G), which was 1 mm longer than the guide cannula. A Hamiltonsyringe (Hamilton Company, USA) and pump (Micro4, WorldPrecision Instruments Inc, USA) assembly infused 0.5 ll of 40%lidocaine/saline into the LC over a period of 5 min. The infusioncannula was left in place for an extra 5 min after infusion foradequate permeation of lidocaine/saline into the brain tissue.

Brain Tissue Processing

At the end of the experiments, Indian ink was infused intothe LC using the same assembly. The rats were then sacrificedby an excess dose of sodium pentobarbital and transcardiallyperfused with ice-cold saline followed by 4% paraformaldehyde(in 0.1 M phosphate buffer). The brains were harvested andpost fixed overnight in 4% paraformaldehyde and dropped in30% sucrose solution. Once the brains were saturated insucrose, they were cryosectioned (CM3050, Leica Biosystems,Germany). Twenty micron thick cryosections were cresyl violetstained to verify the position of the electrodes and the infusioncannula (Figs. 1c–e). Rats with incorrect infusion positionswere excluded from analysis.

Statistical Analysis

The slope of the fEPSP was expressed as mean percenta-ge 6 S.E.M normalized to the baseline. Statistical analysis

(PASW Statistics, Version 18.0) was carried out using repeatedmeasures ANOVA and the level of statistical significance wasfixed at P< 0.05. Planned contrasts were performed against thebaseline. Two-tailed Student’s t-tests were used to compare theeffects of challenge treatments on the 30 min average of thefEPSP slopes normalized to baseline.

RESULTS

Nicotine and Clozapine Challenge in Nicotine-Primed Rats

A challenge dose of nicotine (0.4 mg/kg) significantly(F15,90 5 10.793, P< 0.001) increased the percentage slope ofthe fEPSP compared to the baseline in the nicotine- (0.8 mg/kg) primed rats (Fig. 2a). A significant increase in the percent-age fEPSP slope was evident from the first 10 min after thechallenge dose of nicotine and maintained until 120 min afterinjection. Nicotine challenge had no significant effect on thefEPSP slope in saline-primed rats. On comparing the effects ofthe nicotine and saline priming, it was found that nicotine sig-nificantly increased the fEPSP slope in nicotine-primed rats atthe T61–90 min (P< 0.05) and T91–120 min (P< 0.01) (Fig. 2b).Based on our hypothesis that nicotine and clozapine crossprime the locus coeruleus to cause LLP in the MPP-DG syn-apse, we expected clozapine to cause LLP in nicotine-primedrats. Indeed, clozapine (15 mg/kg) challenge significantlyincreased the fEPSP slope in nicotine-primed rats(F15,75 5 29.899, P< 0.001) compared to baseline, but not inthe saline-primed rats (Fig. 2c). Clozapine challenge in nico-tine-primed rats significantly increased the fEPSP slope com-pared to saline-primed rats (saline priming 3 nicotine primingT1–30 min: P< 0.01, T31–60 min: P< 0.05, T61–90 min: P< 0.01and T91–120 min: P< 0.01, Fig. 2d).

Nicotine and Clozapine Challenge in Clozapine-Primed Rats

Further to the observation that clozapine and nicotine chal-lenge induced an LLP in nicotine-primed rats, we investigatedwhether clozapine itself could cause such a priming effect. Thedose of clozapine was selected from previous studies (Kapuret al., 2003; Verma et al., 2007). As predicted, a challengedose of clozapine (15 mg/kg) significantly increased the fEPSPslope in the clozapine- (30 mg/kg) primed rats(F15,60 5 19.624, P< 0.001), whereas such an effect was notobserved in saline-primed rats (Fig. 3a). The significantincrease was evident from the 30th min post administration,The mean fEPSP slopes at T61–90 min and T91–120 min weresignificantly higher (P< 0.05 and P< 0.01, respectively) thanpercentages obtained in the saline-primed rats (Fig. 3b). Like-wise, nicotine challenge (0.4 mg/kg) significantly(F15,120 5 22.010, P< 0.001) increased the percentage slopefEPSP compared to the baseline in the clozapine-primed rats

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FIGURE 2. Nicotine or clozapine challenge induces LLP innicotine-primed rats. The data points in (a) and (c) representmean percentage fEPSP slope of the preceding 10 min, normalizedto the baseline. *P < 0.05, **P < 0.01 when the increase in percent-age fEPSP slope is compared to baseline (planned contrast follow-ing repeated measures ANOVA). The grey and black traces are the

representative fEPSP waveforms in pre- and post-LLP phases,respectively (Scale bar: 20 ms, 5 mv). Thirty minute epochs ofmean percentage fEPSP slope after (b) nicotine and (d) clozapinechallenge in saline- (blank) and nicotine- (black) primed rats.*P < 0.05, **P < 0.01 when compared to saline-primed rats (Two-tailed Student’s t-test). Error bars represent SEM.

FIGURE 3. Clozapine or nicotine challenge induces LLP in clo-zapine-primed rats. The data points in (a) and (c) represent meanpercentage fEPSP slope of the preceding 10 min, normalized to thebaseline. *P < 0.05, **P < 0.01 and ***P < 0.001 when the increasein percentage fEPSP slope is compared to baseline (planned con-trast following repeated measures ANOVA). The grey and black

traces are the representative fEPSP waveforms in pre- and post-LLPphases, respectively (Scale bar: 20 ms, 5 mv). Thirty minute epochsof mean percentage fEPSP slope after (b) clozapine and (d) nicotinechallenge in saline- (blank) and clozapine- (black) primed rats.*P < 0.05, **P < 0.01 when compared to saline-primed rats (Two-tailed Student’s t-test). Error bars represent SEM.

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and not in saline-primed rats (Fig. 3c). The effect of nicotinechallenge was significant in clozapine-primed rats compared tosaline-primed rats at all time points following the challengedose (saline priming 3 clozapine priming, T1–30 min, T31–60

min, T61–90 min, and T91–120 min : P< 0.05, Fig. 3d).

Lidocaine-Induced Inhibition of the LC

The results obtained from the above experiments indicatethat nicotine and clozapine can cross-prime each other. To findthe locus of action, which is likely to be the LC, the nicotine-induced LLP in the clozapine-primed rats was examined byreversibly inhibiting the LC using local application of lidocaine(Lashgari et al., 2008). Lidocaine (4%) infusion at the LCabolished the nicotine challenge-induced increase in the fEPSPslope in clozapine-primed rats (Fig. 4a) and the percentagefEPSP slope did not significantly differ from the baseline.Saline infusion at the LC did not affect the significant increasein percentage slope fEPSP induced by nicotine challenge(F15,45 5 14.092, P< 0.001). Two-tailed Student’s t-testcomparison between the saline and lidocaine infused groupsindicated that the differences in the percentage fEPSP weresignificant at all the time points calculated (saline infusion 3

lidocaine infusion, T1–30 min: P< 0.05; T31–60 min, T61–90 min

and T91–120 min: P< 0.01, Fig. 4b)

Systemic Clonidine Pretreatment

Clonidine, an alpha-2 adrenergic receptor agonist, suppressesLC/NE neurotransmission (Grant and Redmond, 1981; Agha-janian and Vandermaelen, 1982; Washburn and Moises, 1989).The literature on nicotine-induced LLP, specifically in theMPP-DG synapse, has notably indicated the role of NE. Clo-nidine (0.4 mg/kg) pretreatment significantly reduced(F18,90 5 7.988, P< 0.001) the nicotine challenge-inducedincrease in the fEPSP slope in the nicotine-primed rats (Fig.5a), whereas the saline pretreatment had no influence. Oncomparing the effects of saline and clonidine pretreatment(Fig. 5b), clonidine-treated rats had significantly lower fEPSP

slope compared to the saline pretreated group at all the timepoints recorded (saline 3 clonidine, T1–30 min: P< 0.05,T31–60 min: P< 0.01, T61–90 min: P< 0.001,T91–120 min:P< 0.01 and T121–150 min: P< 0.01). Similarly, clonidine pre-vented the effects of clozapine challenge in nicotine-primedrats (saline time effect: F18,54 5 19.230, P< 0.001; clonidinetime effect: F18,90 5 5.107, P< 0.001; saline 3 clonidine,T1–30 min: P< 0.05, T31–60 min: P< 0.001, T61–90 min:P< 0.01, T91–120 min: P< 0.001 and T121–150 min: P< 0.001,Figs. 5c,d). In addition, clonidine pretreatment prevented theclozapine-induced LLP in clozapine-primed rats (saline timeeffect: F18,54 5 55.988, P< 0.001; clonidine time effect:F18,90 5 0.511, NS; saline 3 clonidine, T61–90 min: P< 0.01,T91–120 min: P< 0.001 and T121–150 min: P< 0.001, Figs.6a,b) and nicotine-induced LLP in clozapine-primed rats(saline time effect: F18,54 5 19.115, P< 0.001; clonidinetime effect: F18,90 5 5.286, P< 0.001; saline 3 clonidine,T31–60 min : P< 0.01, T61–90 min : P< 0.01, T91–120 min:P< 0.01 and T121–150 min: P< 0.01, Figs. 6c,d).

DISCUSSION

This study clearly demonstrates the cross-priming effect ofnicotine and clozapine on the LC/NE neurotransmitter systemin inducing an LLP at MPP-DG synapse in the rat hippocam-pus. The cross-priming effect and abolition of LLP due to lido-caine-induced suppression of LC and systemic clonidinepretreatment substantiate the influence of the LC in the inter-action between nicotine and clozapine. In vitro and in vivostudies point to a critical involvement of the LC-NE system inthe initiation of LTP in the hippocampus (Segal and Bloom,1976; Brown et al., 2005). An in vitro (rat brain slice) studyshowed that NE causes a LLP in the DG, which is specific toMPP but not lateral perforant path stimulation (Dahl andSarvey, 1989). More specifically, recent research has shownthat LC activation initiates a beta-adrenoceptor dependent

FIGURE 4. Lidocaine-induced reversible inhibition of locuscoeruleus prevents nicotine challenge-induced LLP in clozapine-primed rats. (a) The data points represent mean percentagefEPSP slope of the preceding 10 min normalized to the baseline.*P < 0.05, **P < 0.01 when the increase in percentage fEPSP slopeis compared to baseline (planned contrasts following repeatedmeasures ANOVA). (b) The grey and black traces are the

representative fEPSP waveforms in pre- and post-LLP phases,respectively. (Scale bar: 20 ms, 5 mv). (c) Thirty minute epochs ofmean percentage fEPSP slope after the nicotine challenge inlidocaine- (blank) and saline- (black) infused (at LC) clozapine-primed rats. *P < 0.05, **P < 0.01 when compared to saline-primedrats (Two-tailed Student’s t-test). Error bars represent SEM.

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FIGURE 5. Clonidine pretreatment prevents nicotine and clo-zapine challenge-induced LLP in nicotine-primed rats. The datapoints in (a) and (c) represent mean percentage fEPSP slope ofthe preceding 10 min, normalized to the baseline. *P < 0.05,**P < 0.01, when the increase in percentage fEPSP slope is com-pared to baseline (planned contrasts following repeated measuresANOVA). The grey and black traces are the representative fEPSP

waveforms in pre- and post-LLP phases, respectively. (Scale bar:20 ms, 5 mv). Thirty minute epochs of mean percentage fEPSPslope after (a) nicotine and (c) clozapine challenge in nicotine-primed rats that received saline (blank) or clonidine (blank) pre-treatment before the challenge dose. *P < 0.05, **P < 0.01 and ***P < 0.001 when compared to saline-primed rats (Two-tailed Stu-dent’s t-test). Error bars represent SEM.

FIGURE 6. Clonidine pretreatment prevents clozapine andnicotine challenge-induced LLP in clozapine-primed rats. The datapoints in (a) and (c) represent mean percentage fEPSP slope ofthe preceding 10 min, normalized to the baseline. *P < 0.05,**P < 0.01, when the increase in percentage fEPSP slope is com-pared to baseline (planned contrasts following repeated measuresANOVA). The grey and black traces are the representative fEPSP

waveforms in pre- and post-LLP phases, respectively. (Scale bar:20 ms, 5 mv). Thirty minute epochs of mean percentage fEPSPslope after (a) clozapine and (c) nicotine challenge in clozapine-primed rats that received saline (blank) or clonidine (blank) pre-treatment before the challenge dose . *P < 0.05, **P < 0.01 and***P < 0.001 when compared to saline-primed rats (Two-tailed Stu-dent’s t-test). Error bars represent SEM.

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long-lasting increase in the synaptic strength of concurrentlyactivated perforant path inputs to the DG (Brown et al., 2005;Reid and Harley, 2010).

Nicotine and Clozapine Induce LLP

A challenge dose of nicotine induced an LLP of fEPSPs atthe MPP-DG synapse in the hippocampus of anesthetizedrats, primed 4 weeks earlier with seven daily injections ofnicotine, but not saline. This data is consistent with earlierfindings (Hamid et al., 1997), which suggest a beta-adreno-ceptor basis to this observation as the effect was blocked bypropranolol. A plausible mechanism can be deduced fromprevious reports, which showed that a single subcutaneousinjection of nicotine increases TH mRNA in the locuscoeruleus, resulting in enhanced TH activity in the hippo-campus 21–28 days later. Nicotine priming-associatedenhanced TH activity results in an increased NE release inthe hippocampus (Smith et al., 1991; Mitchell et al., 1989,1990, 1993). The delay in the augmented NE release isattributed to axonal transport of TH from the locus coeru-leus to the hippocampus. Subchronic administration of nico-tine (seven consecutive days), as was done in this study,results in maximal TH activity and NE release (Smith et al.,1991). Another study proposed dual mechanisms for nico-tine mediated NE release in the hippocampus, which are (1)activation of nicotinic acetylcholine receptors of the LC and(2) via GABA release (Leslie et al., 2002). The nicotine-induced LLP may represent its procognitive effects, whichhave been evidenced in smokers with or without a historyof schizophrenia (McEvoy et al., 1999; Harris et al., 2004;Barr et al., 2008) and in preclinical behavioral investigations(Socci et al., 1995; Abdulla et al., 1996; Levin et al., 1998;Bernal et al., 1999; Rezvani et al., 2006).

Preclinical studies have implicated NAergic (direct and indi-rect) mechanisms behind the superior efficacy of clozapine inthe management of cognitive dysfunction in schizophrenicpatients (Nilsson et al., 2005; Verma et al., 2006, 2007;Masana et al., 2011). A challenge dose of clozapine adminis-tered 21–28 days after priming rats with nicotine induced LLPat the MPP-DG synapse in the hippocampus, which suggeststhat the nicotine-induced enhanced NE neurotransmission isutilized by clozapine in inducing the LLP. This phenomenoncould relate to the clinical findings of McEvoy et al. (1999)demonstrating enhanced cognitive functions observed inpatients with schizophrenia who smoke.

On comparing the pattern of effects following nicotine andclozapine challenge in the nicotine-primed rats, it is apparentthat nicotine caused an almost immediate increment in thefEPSP slope, whereas clozapine showed a gradual and delayedincrease. This could be explained by the striking difference inthe log P values of nicotine and clozapine (0.95 and 3.5,respectively), which influences the release of the drugs from thesite of administration (in this case, the subcutaneous tissue),and subsequent transport across the blood brain barrier(Ghosheh et al., 2001; Liao et al., 1999).

Nicotine- and Clozapine-Induced LLP is LC-NASystem Dependent

Atypical antipsychotics can induce a de novo expression ofTH in the LC (Verma et al., 2006, 2007). The newly synthe-sized TH takes several weeks to be transported along LC pro-jections to the mPFC, thereby causing a delayed increase inTH levels in the mPFC (Verma et al., 2006), an observationsimilar to that of nicotine-induced delayed TH increase in thehippocampus (Mitchell et al., 1989, 1990; Smith et al., 1991;Mitchell, 1993). It has been established that acute administra-tion of atypical antipsychotics, including olanzapine and cloza-pine, increases the firing of NAergic LC neurons and NErelease (Souto et al. 1979; Ramirez and Wang 1986; Daweet al., 2001). This increased activity of the LC upon adminis-tration of atypical antipsychotics could contribute to the poten-tiation observed in clozapine-primed rats. The role of the LCin clozapine/nicotine priming-challenge regimen-induced LLPis clearly established in this investigation by suppression of theLC by infusion of 4% lidocaine, which abolished the nicotine-induced LLP in clozapine-primed rats.

Nicotine, like clozapine, activates the LC. Increases in firingrate, membrane depolarization, and inward current have beenobserved in vitro in response to nicotine treatment (Egan andNorth, 1986). Application of nicotine proximal to LC causedincreased extracellular levels of NE in the hippocampus (Mitch-ell et al., 1993). Hamid et al. (1997) demonstrated that nicotineinduced a beta-adrenoceptor-mediated LLP in nicotine-primedrats. In order to verify whether the clozapine-induced LLP alsofollowed a similar adrenergic mechanism, clonidine, a selectivealpha-2 adrenergic agonist, was used. Clonidine stimulatesalpha-2 adrenergic receptors and so inhibits firing of LC neuronsand the release of NE at LC axon terminals. When clonidinewas administered to clozapine- or nicotine-primed rats 30 minprior to nicotine or clozapine challenge, the potentiation wasblocked. This is consistent with the blockade of the LLP byinfusion of lidocaine into the LC and confirms the involvementof NAergic mechanisms in the hippocampal LLP induced.

Nicotine and Clozapine can Cross-Prime

To the best of our knowledge, this is the first study showingthat both clozapine and nicotine induce an LLP at the MPP-DG synapse in clozapine- or nicotine-primed rats. Althoughclozapine would acutely antagonize the effects of nicotine atalpha-7-nicotinic receptors (Grinevich et al., 2009) in the hip-pocampus, the results show a similarity in LLP priming-chal-lenge effects among these agents. It appears that subchronicpriming with clozapine increases LC TH expression, which isconsistent with our earlier findings (Verma et al., 2007), andsubsequent release of NE on acute challenge with either cloza-pine or nicotine. The antagonistic actions of clozapine at nico-tinic receptors are not evident because the clozapine andnicotine were not simultaneously administered.

In conclusion, our findings indicate that both clozapine andnicotine can prime the induction of LLP in the hippocampus

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to subsequent challenge with clozapine or nicotine by a mecha-nism involving the LC NAergic system. This has potentialimplications for understanding enhanced cognitive function inpatients with schizophrenia on atypical antipsychotic therapywho smoke and the use of nicotinic ligands as cognitiveenhancers. Our findings also emphasize the importance ofNAergic mechanisms in regulation of synaptic plasticity in theDG of the hippocampus.

REFERENCES

Abdulla FA, Bradbury E, Calaminici MR, Lippiello PM, WonnacottS, Gray JA, Sinden JD. 1996. Relationship between up-regulationof nicotine binding sites in rat brain and delayed cognitiveenhancement observed after chronic or acute nicotinic receptorstimulation. Psychopharmacology (Berl) 124:323–331.

Aghajanian GK, Vandermaelen CP. 1982. Alpha 2-adrenoceptor-medi-ated hyperpolarization of locus coeruleus neurons: Intracellularstudies in vivo. Science 215:1394–1396.

Barr RS, Culhane MA, Jubelt LE, Mufti RS, Dyer MA, Weiss AP,Deckersbach T, Kelly JF, Freudenreich O, Goff DC, Evins AE.2008. The effects of transdermal nicotine on cognition in non-smokers with schizophrenia and nonpsychiatric controls. Neuropsy-chopharmacology 33:480–490.

Bernal MC, Vicens P, Carrasco MC, Redolat R. 1999. Effects of nico-tine on spatial learning in C57BL mice. Behav Pharmacol 10:333–336.

Berridge CW, Waterhouse BD. 2003. The locus coeruleus noradrener-gic system: Modulation of behavioral state and state-dependentcognitive processes. Brain Res Rev 42:33–84.

Bliss TVP, Collingridge GL. 1993. A synaptic model of memory:Long-term potentiation in the hippocampus. Nature 36:31–39.

Bliss TVP, Lomo T. 1973. Long-lasting potentiation of synaptic trans-mission in the dentate area of the anaesthetized rabbit followingstimulation of the perforant path. J Physiol 232:331–356.

Brazell MP, Mitchell SN, Gray JA. 1991. Effect of acute administra-tion of nicotine on in vivo release of noradrenaline in the hippo-campus of freely moving rats: A dose-response and antagoniststudy. Neuropharmacology 30:823–833.

Breier A, Elman I, Goldstein DS. 1998. Norepinephrine and schizo-phrenia: A new hypothesis for antipsychotic drug action. Adv Phar-macol 42:785–788.

Brown RA, Walling SG, Milway JS, Harley CW. 2005. Locus coeru-leus activation suppresses feedforward interneurons and reducesbeta-gamma electroencephalogram frequencies while it enhancestheta frequencies in rat dentate gyrus. J Neurosci 25:1985–1991.

Brunello N, Masotto C, Steardo L, Markstein R, Racagni G. 1995.New insights into the biology of schizophrenia through the mecha-nism of action of clozapine. Neuropsychopharmacology 13:177–213.

Chaulk PC, Harley CW. 1998. Intracerebroventricular norepinephrinepotentiation of the perforant path-evoked potential in dentategyrus of anesthetized and awake rats: A role for both alpha- andbeta-adrenoceptor activation. Brain Res 787:59–70.

Dahl D, Sarvey JM. 1989. Norepinephrine induces pathway-specificlong-lasting potentiation and depression in the hippocampal den-tate gyrus. Proc Nat Acad Sci USA 86:4776–4780.

Dawe GS, Huff KD, Vandergriff JL, Sharp T, O’Neill MJ, RasmussenK. 2001. Olanzapine activates the rat locus coeruleus: In vivo elec-trophysiology and c-Fos immunoreactivity. Biol Psychiatry 50:510–520.

Egan TM, North RA. 1986. Actions of acetylcholine and nicotine onrat locus coeruleus neurons in vitro. Neuroscience 19:565–571.

George TP, Sernyak MJ, Ziedonis DM, Woods SW. 1995. Effects ofclozapine on smoking in chronic schizophrenic outpatients. J ClinPsychiatry 56:344–346.

Ghosheh OA, Dwoskin LP, Miller DK, Crooks PA. 2001. Accumula-tion of nicotine and its metabolites in rat brain after intermittentor continuous peripheral administration of [20-(14)C]nicotine.Drug Metab Dispos 29:645–651.

Grant SJ, Redmond DE Jr. 1981. The neuroanatomy and pharmacol-ogy of the nucleus locus coeruleus. Prog Clin Biol Res 71:5–27.

Grigoryan GA, Gray JA. 1996. A single dose of nicotine proactivelyenhances the partial reinforcement extinction effect in the rat. Psy-chobiology 24:136–146.

Grinevich VP, Papke RL, Lippiello PM, Bencherif M. 2009. Atypicalantipsychotics as noncompetitive inhibitors of alpha4beta2 andalpha7 neuronal nicotinic receptors. Neuropharmacology 57:183–191.

Hamid S, Dawe GS, Gray JA, Stephenson JD. 1997. Nicotine induceslong-lasting potentiation in the dentate gyrus of nicotine-primedrats. Neurosci Res 29:81–85.

Harley CW. 1991. Noradrenergic and locus coeruleus modulation ofthe perforant path evoked potential in rat dentate gyrus supports arole for the locus coeruleus in attentional and memorial processes.Prog Brain Res 88:307–321.

Harley CW. 1998. Noradrenergic long-term potentiation in the den-tate gyrus. Adv Pharmacol 42:952–956.

Harley CW. 2007. Norepinephrine and the dentate gyrus. Prog BrainRes 163:299–318.

Harris JG, Kongs S, Allensworth D, Martin L, Tregellas J, Sullivan B,Zerbe G, Freedman R. 2004. Effects of nicotine on cognitive defi-cits in schizophrenia. Neuropsychopharmacology 29:1378–1385.

Heishman SJ, Kleykamp BA, Singleton EG. 2010. Meta-analysis ofthe acute effects of nicotine and smoking on human performance.Psychopharmacology (Berl) 210:453–469.

Joseph MH, Peters SL, Prior A, Mitchell SN, Brazel MP Gray JA.1990. Chronic nicotine administration increases tyrosine hydroxy-lase selectively in the rat hippocampus. Neurochem Int 16:269–273.

Kalkman HO, Loetscher E. 2003. alpha2C-Adrenoceptor blockade byclozapine and other antipsychotic drugs. Eur J Pharmacol 462:33–40.

Kane JM, Correll CU. 2010. Past and present progress in the pharma-cologic treatment of schizophrenia. J Clin Psychiatry 71:1115–1124.

Kapur S, VanderSpek SC, Brownlee BA, Nobrega JN. 2003. Antipsy-chotic dosing in preclinical models is often unrepresentative of theclinical condition: A suggested solution based on in vivo occu-pancy. J Pharmacol Exp Ther 305: 625–631.

Kenney JW, Gould TJ. 2008. Modulation of hippocampus-dependentlearning and synaptic plasticity by nicotine. Mol Neurobiol38:101–121.

Kubota T, Jibiki I, Ishikawa A, Kawamura T, Kurokawa S, Wang M.2008. Increase in extracellular dopamine levels during clozapine-induced potentiation in the hippocampal dentate gyrus of chroni-cally prepared rabbits. Can J Physiol Pharmacol 86:249–256.

Kubota T, Jibiki I, Kishizawa S, Kurokawa K. 1996. Clozapine-induced potentiation of synaptic responses in the perforant path-dentate gyrus pathway in chronically prepared rabbits. NeurosciLett 211:21–24.

Lashgari R, Khakpour-Taleghani B, Motamedi F, Shahidi S. 2008.Effects of reversible inactivation of locus coeruleus on long-termpotentiation in perforant path-DG synapses in rats. NeurobiolLearn Mem 90:309–316.

Leslie FM, Gallardo KA, Park MK. 2002. Nicotinic acetylcholine re-ceptor-mediated release of [3H]norepinephrine from developing

NICOTINE AND CLOZAPINE CROSS-PRIME NAERGIC HIPPOCAMPAL LLP 623

Hippocampus

and adult rat hippocampus: Direct and indirect mechanisms. Neu-ropharmacology 42:653–661.

Levin ED, Bettegowda C, Weaver T, Christopher NC. 1998. Nico-tine-dizocilpine interactions and working and reference memoryperformance of rats in the radial-arm maze. Pharmacol BiochemBehav 61:335–340.

Liao Y, Venhuis BJ, Rodenhuis N, Timmerman W, Wikstr€om H,Meier E, Bartoszyk GD, B€ottcher H, Seyfried CA, Sundell S.1999. New (sulfonyloxy)piperazinyldibenzazepines as potentialatypical antipsychotics: Chemistry and pharmacological evaluation.J Med Chem 42:2235–2244.

Masana M, Bortolozzi A, Artigas F. 2011. Selective enhancement ofmesocortical dopaminergic transmission by noradrenergic drugs:Therapeutic opportunities in schizophrenia. Int J Neuropsycho-pharmacol 14:53–68.

McEvoy J, Freudenreich O, McGee M, VanderZwaag C, Levin E,Rose J. 1995. Clozapine decreases smoking in patients withchronic schizophrenia. Biol Psychiatry 37:550–552.

McEvoy JP, Freudenreich O, Wilson WH. 1999. Smoking and thera-peutic response to clozapine in patients with schizophrenia. BiolPsychiatry 46:125–129.

Mitchell SN, Braze MP, Schugens MM, Gray JA. 1990. Nicotine-induced catecholamine synthesis after lesions to the dorsal or ven-tral noradrenergic bundle. Eur J Pharmacol 179:383–391.

Mitchell SN, Brazell MP, Joseph MH, Alivijeh M, Gray JA. 1989.Regionally specific effects of acute and chronic nicotine on rates ofcatecholamine and 5-hydroxytryptamine synthesis in rat brain. EurJ Pharmacol 167:311–322.

Mitchell SN, Smith KM, Joseph MH, Gray JA. 1993. Increases in ty-rosine hydroxylase messenger RNA in the locus coeruleus after asingle dose of nicotine are followed by time-dependent increases inenzyme activity and noradrenaline release. Neuroscience 56:989–997.

Mitchell SN. 1993. Role of the locus coeruleus in the noradrenergicresponse to a systemic administration of nicotine. Neuropharma-cology 32:937–949.

Neuman RS, Harley CW. 1983. Long-lasting potentiation of the den-tate gyrus population spike by norepinephrine. Brain Res273:162–165.

Nilsson LK, Schwieler L, Engberg G, Linderholm KR, Erhardt S.2005. Activation of noradrenergic locus coeruleus neurons by clo-zapine and haloperidol: Involvement of glutamatergic mechanisms.Int J Neuropsychopharmacol 8:329–339.

Paxinos G, Watson C. 2007. The Rat Brain in Stereotaxic Coordi-nates, 6th ed. Massachusetts: Elsevier Inc.

Pelletier MR, Kirkby RD, Jones SJ, Corcoran ME. 1994. Pathwayspecificity of noradrenergic plasticity in the dentate gyrus. Hippo-campus 4:181–188.

Ramirez OA, Wang RY. 1986. Locus coeruleus norepinephrine-con-taining neurons: Effects produced by acute and subchronic treat-ment with antipsychotic drugs and amphetamine. Brain Res362:165–170.

Reid AT, Harley CW. 2010. An associativity requirement for locuscoeruleus-induced long-term potentiation in the dentate gyrus ofthe urethane-anesthetized rat. Exp Brain Res 200:151–159.

Rezvani AH, Caldwell DP, Levin ED. 2006. Chronic nicotine interac-tions with clozapine and risperidone and attentional function inrats. Prog Neuropsychopharmacol Biol Psychiatry 30:190–197.

Segal M, Bloom FE. 1976. The action of norepinephrine in the rathippocampus. IV. The effects of locus coeruleus stimulation onevoked hippocampal unit activity. Brain Res 107:513–525.

Smith KM, Mitchell SN, Joseph MH. 1991. Effects of chronic andsubchronic nicotine on tyrosine hydroxylase activity in noradrener-gic and dopaminergic neurones in the rat brain. J Neurochem57:1750–1756.

Socci DJ, Sanberg PR, Arendash GW. 1995. Nicotine enhances Mor-ris water maze performance of young and aged rats. NeurobiolAging 16:857–860.

Souto M, Monti JM, Altier H.1979. Effects of clozapine on the activ-ity of central dopaminergic and noradrenergic neurons. PharmacolBiochem Behav 10:5–9.

Tang J, Dani JA. 2009. Dopamine enables in vivo synaptic plasticityassociated with the addictive drug nicotine. Neuron 63:673–682.

Verma V, Lim EP, Han SP, Nagarajah R, Dawe GS. 2007. Chronichigh-dose haloperidol has qualitatively similar effects to risperidoneand clozapine on immediate-early gene and tyrosine hydroxylaseexpression in the rat locus coeruleus but not medial prefrontal cor-tex. Neurosci Res 57:17–28.

Verma V, Rasmussen K, Dawe GS. 2006. Effects of short-term andchronic olanzapine treatment on immediate early gene protein andtyrosine hydroxylase immunoreactivity in the rat locus coeruleusand medial prefrontal cortex. Neuroscience 143:573–585.

Washburn M, Moises HC. 1989. Electrophysiological correlates ofpresynaptic alpha2-receptor-mediated inhibition of norepinephrinerelease at locus coeruleus synapses in dentate gyrus. J Neurosci9:2131–2140.

Wesnes K, Warburton DM. 1983. Smoking, nicotine and human per-formance. Pharmacol Ther 21:189–208.

Yamazaki Y, Hamaue N, Sumikawa K. 2002. Nicotine compensatesfor the loss of cholinergic function to enhance long-term potentia-tion induction. Brain Res 946:148–152.

Yamazaki Y, Jia Y, Hamaue N, Sumikawa K. 2005. Nicotine-inducedswitch in the nicotinic cholinergic mechanisms of facilitation oflong-term potentiation induction. Eur J Neurosci 22:845–860.

Zhang TA, Tang J, Pidoplichko VI, Dani JA. 2010. Addictive nicotinealters local circuit inhibition during the induction of in vivo hip-pocampal synaptic potentiation. J Neurosci 30:6443–6453.

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