Localized infusions of the partial alpha 7 nicotinic receptor agonist SSR180711 evoke rapid and transient increases in prefrontal glutamate release

Neuroscience 255 (2013) 55–67

LOCALIZED INFUSIONS OF THE PARTIAL ALPHA 7 NICOTINIC RECEPTOR AGONIST SSR180711 EVOKE RAPID AND TRANSIENT INCREASES IN PREFRONTAL GLUTAMATE RELEASE D. M. BORTZ, a J. D. MIKKELSEN c AND J. P. BRUNO a,b*

of a7 nAChR-positive modulators in treating cognitionimpairing disorders in humans. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Department of Psychology, The Ohio State University, Columbus, OH, United States b Department of Neuroscience, The Ohio State University, Columbus, OH, United States

Key words: rat, schizophrenia.

c

Neurobiology Research Unit, Copenhagen University Righospitalet, Copenhagen, Denmark

acetylcholine,

glutamate,

cognition,

INTRODUCTION

Abstract—The ability of local infusions of the alpha 7 nicotinic acetycholine receptor (a7 nAChR) partial agonist SSR180711 to evoke glutamate release in prefrontal cortex was determined in awake rats using a microelectrode array. Infusions of SSR180711 produced dose-dependent increases in glutamate levels. The lower dose (1.0 lg in 0.4 lL) evoked a rapid rise (1.0 s) in glutamate (1.41 ± 0.30 lM above baseline). The higher dose (5.0 lg) produced a similarly rapid, yet larger increase (3.51 ± 0.36 lM above baseline). After each dose, the glutamate signal was cleared to basal levels within 7–18 s. SSR180711-evoked glutamate was mediated by the a7 nAChR as co-infusion of the selective a7 nAChR antagonist a-bungarotoxin (10.0 lM) + SSR1808711 (5.0 lg) reduced the effect of 5.0 lg alone by 87% (2.62 vs. 0.35 lM). Finally, the clearance of the SSR180711 (5.0 lg)-evoked glutamate was bidirectionally affected by drugs that inhibited (threobeta-benzyl-oxy-aspartate (TbOA), 100.0 lM) or facilitated (ceftriaxalone, 200 mg/kg, i.p.) excitatory amino acid transporters. TbOA slowed both the clearance (s) and rate of clearance (lM/s) by 10-fold, particularly at the mid-late stages of the return to baseline. Ceftriaxone reduced the magnitude of the SSR180711-evoked increase by 65%. These results demonstrate that pharmacological stimulation of a7 nAChRs within the prefrontal cortex is sufficient to evoke rapid yet transient increases in glutamate levels. Such increases may underlie the cognition-enhancing effects of the drug in animals; further justifying studies on the use

Dysregulated neurotransmission within the prefrontal cortex (PFC) is thought to underlie the deficits in executive function, such as working memory (Perlstein et al., 2001; Abi-Dargham et al., 2002) attention (Nuechterlein et al., 2004; Demeter et al., 2008; Luck and Gold, 2008) and cognitive flexibility (Everett et al., 2001; Thoma et al., 2007) seen in patients with schizophrenia (SZ). In addition, the severity of impairments in cognitive control represents the best predictor of long-term outcome for persons afflicted with SZ (Green et al., 2004; Chen et al., 2005; Prouteau et al., 2005) yet are only modestly attenuated by current antipsychotic regimens (Bowie and Harvey, 2006). Thus, there is a need to develop novel or adjunctive medications to ameliorate these cognitive symptoms through experiments on drugs acting to promote/ normalize chemo-transmission within task-related brain regions. One group of drugs that has received considerable attention as potential cognition-enhancing adjunctive medications is the nicotinic acetylcholine receptor (nAChR) agonists and their positive allosteric modulators (Thomsen et al., 2010; Kucinski et al., 2011; Jones et al., 2012). Clinically, this focus is based, in part, upon the relationship between expression of the nAChR and the development of SZ (Guan et al., 1999; Severance and Yolken, 2008). In addition, the vast majority of patients within the SZ population smoke (70–90%) (Hughes et al., 1986; Goff et al., 1992; de Leon and Diaz, 2005), and the speculation that this behavior represents a self-medication strategy is supported by findings that nicotine has positive effects on attention and learning in both humans and rodents (Levin and Simon, 1998; Levin and Rezvani, 2002; Sacco et al., 2005). Moreover, nicotine withdrawal impairs cognitive performance in patients with SZ and such withdrawal induces greater cognitive deficits in patients than in normal individuals (Weinberger et al., 2007; AhnAllen et al., 2008). Finally, task performance

*Correspondence to: J. P. Bruno, Department of Psychology, 1835 Neil Avenue, The Ohio State University, Columbus, OH 43016, United States. Tel: +1-614-292-1770; fax: +1-614-688-4733. E-mail address: [email protected] (J. P. Bruno). Abbreviations: a7, alpha 7; aBGT, a-bungarotoxin; AA, L-ascorbic acid; ACh, acetylcholine; ANOVA, analysis of variance; BSA, bovine serum albumin; CEF, ceftriaxone; DA, dopamine (3-hydroxytyramine); EAAT, excitatory amino acid transporter; Gluox, glutamate oxidase; MEA, microelectrode array; MK-801, (+)-5-methyl-10,11-dihydro-5Hdibenzo [a, d] cyclohepten-5,10-imine maleate; m-PD, m-Phenylenediamine dihydrochloride; nAChR, nicotinic acetycholine receptor; PFC, prefrontal cortex; SZ, schizophrenia; Tcx, the clearance rate during x time period; Tx, the time to clear x% of the signal from the maximum amplitude; TbOA, threo-beta-benzyl-oxy-aspartate.

0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.09.047 55

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has been shown to rebound following the reinstatement of nicotine (Barr et al., 2008). However, nicotine’s clinical utility, as a non-selective nAChR agonist, is limited because it can rapidly desensitize the receptor (James et al., 1994; Quick and Lester, 2002) and also causes a number of unwanted side effects (Benowitz, 1988; Benowitz and Gourlay, 1997). Therefore, subsequent research has attempted to avoid these issues by focusing on the pro-cognitive effects of agonists and positive allosteric modulators that are selective for specific subtypes of the nAChR. The alpha 7 nAChR (a7 nAChR) subtype has received considerable attention due to its reported link to the development of SZ. Furthermore, there are several lines of evidence that have attracted interest for this receptor in SZ. Several polymorphisms in the a7 nAChR gene, CHRNA7, have been linked to P50 auditory gating deficits in SZ (Freedman et al., 1997; Stassen et al., 2000; Leonard et al., 2002); and such deficits predict the severity of cognitive deficits in SZ patients (Erwin et al., 1998). In addition, post-mortem histochemical and binding studies have revealed reduced a7 nAChR expression in critical brain regions in SZ, such as the hippocampus, thalamic reticular nucleus, and the cingulate cortex (Freedman et al., 1995; Court et al., 1999; Guan et al., 1999; Marutle et al., 2001). While the number of clinical studies testing the cognition-enhancing effects of a7 nAChRs is relatively small, there have been promising results for several a7 agonists, partial agonists, and positive modulators (Koike et al., 2005; Olincy et al., 2006). The present paper focuses on the neurochemical effects of intracortical administration of SSR180711, a selective a7 nAChR partial agonist for rat and human receptors that has no significant binding at non-a7 nAChRs, including the a4b2 (Biton et al., 2007). Administration of SSR180711 has been shown to promote performance in several cognitive tasks in intact animals as well as in models of SZ (Pichat et al., 2007; Barak et al., 2009; Brooks et al., 2012). Despite these promising results, the mechanisms by which SSR180711 exerts its cognition-enhancing effects remain to be fully understood, although several effects that might contribute to attentional processing and working memory have been reported. SSR180711 induces immediate early gene expression, c-Fos and Arc, in both the medial PFC and the nucleus accumbens of rats (Hansen et al., 2007; Kristensen et al., 2007; Thomsen et al., 2008). It also increases extracellular dopamine levels in the PFC, and stimulates acetylcholine (ACh) efflux in the hippocampus (Biton et al., 2007; Pichat et al., 2007). Given the critical roles for prefrontal cholinergic and glutamatergic transmission in the normal expression of selective attention (Sarter and Bruno, 1997; Kozak et al., 2006; Parikh et al., 2008), working memory (Lewis and Moghaddam, 2006; Timofeeva and Levin, 2011), and cognitive flexibility (Stefani et al., 2003; Stefani and Moghaddam, 2005; Alexander et al., 2012), we hypothesized that SSR180711’s cognition-enhancing properties are based on the drug’s ability to locally

elevate prefrontal glutamate levels via its activation of the a7 nAChR within the PFC. First, we determined, using a glutamate-sensitive biosensor and amperometry (Burmeister and Gerhardt, 2001; Konradsson-Geuken et al., 2009), whether the effects of SSR180711 on glutamate levels were dose-dependent (1.0 vs. 5.0 lg). Second, we confirmed the a7 nAChR mediation of SSR180711’s effects by measuring the impact of a wellestablished concentration (10.0 lM; (Van der Kloot, 1993; Utkin et al., 2001; Konradsson-Geuken et al., 2009; Momi et al., 2013) of the selective antagonist a-bungarotoxin (aBGT) on the drug’s ability to elevate glutamate levels. Finally, we provided additional evidence that our electrochemical signal was indeed glutamate by determining the bidirectional effects on amplitude and clearance of a standard concentration of the excitatory amino acid transport inhibitor threo-betabenzyl-oxy-aspartate (TbOA; 100 lM; (Shimamoto et al., 1998; Hascup et al., 2010; Medrano et al., 2013) and the glutamate transport promoter ceftriaxone (CEF; (Thone-Reineke et al., 2008; Wei et al., 2012)) on the SSR180711 response.

EXPERIMENTAL PROCEDURES Subjects Male Wistar rats, weighing 280–420 g, were used as subjects in these experiments. Animals were maintained in a temperature and humidity controlled room on a 12:12-h light:dark cycle (lights on at 06:00 a.m.) and individually housed in plastic cages lined with corn cob bedding (Harlan Teklad, Madison, WI, USA). Animals had access to food and water ad libitum. One group of rats (N = 7) was used for the initial SSR180711 dose– response study. A second group (N = 5) was used for determining the effects of aBGT and TbOA on druginduced glutamate levels. A final group (N = 4) was used to test the effects of CEF on SSR180711-evoked glutamate levels and clearance. All procedures involving animals were approved by The Ohio State University Institutional Animal Care and Use Committee in accordance with the NIH Guide for the Care and Use of Laboratory Animals. As such, all efforts were made to minimize animal suffering, to reduce the number of animals used, and to consider alternatives to in vivo techniques. Drugs All solutions used for intracranial infusion were prepared in 0.9% saline and adjusted to a pH of 7.1–7.4. These solutions were made with the following drugs: SSR180711 (4-bromophenyl 1,4diazabicyclo(3.2.2) nonane-4-carboxylate, monohydrochloride), synthesized at the Department of Medicinal Chemistry, NeuroSearch A/S (Copenhagen, Denmark), aBGT, purchased from Tocris Bioscience (Ellisville, MO, USA), CEF, purchased from Sigma–Aldrich Corp. (St. Louis, MO, USA), and TbOA, purchased from Tocris Cookson (Bristol, UK). All solutions used in preparation and calibration of the Gluox microelectrode array (MEA) were prepared using

D. M. Bortz et al. / Neuroscience 255 (2013) 55–67

distilled, deionized water. These include the following solutions: m-Phenylenediamine dihydrochloride (m-PD), purchased by Acros Organics (New Jersey, USA), L-ascorbic acid (AA), 3-hydroxytyramine (DA), L-glutamate monosodium salt, glutaraldehyde [25% (w/w) in water], bovine serum albumin (BSA), H2O2, obtained from Sigma–Aldrich Corp. (St. Louis, MO, USA), and L-glutamate oxidase (Gluox), purchased from Seikaghaku America, Inc (East Falmouth, MA, USA). Detection of glutamate-generated signals Glutamate levels were detected using the MEA recording system (Quanteon, LLC, Nicholasville, KY, USA) described in detail in Rutherford et al. (2007). Briefly, the MEA is a ceramic electrode, bearing four 15  333 lm platinum recording sites. Each pair of recording sites was designated to be either Gluox or not (background or sentinel). The Gluox channels were coated with the glutamate oxidase (2%, 1 unit/1 lL, 100 nL), BSA (1%), and glutaraldehyde (0.125%) whereas the sentinel channels were coated with just BSA and glutaraldehyde. MEA’s were electro-plated with m-PD to block the oxidation of larger endogenous molecules (i.e. ascorbic acid, dopamine, norepinephrine, serotonin) that would also oxidize at the applied potential (0.7 V). In this detection scheme, glutamate is oxidized by Gluox, generating a-ketoglutarate and H2O2. Because the MEA is maintained at a constant potential of +0.7 V vs. an Ag/AgCl reference electrode, the H2O2 reporting molecule is oxidized, yielding two electrons. The differential coating scheme allows for the isolation of current generated solely by the oxidation of glutamate by self-referencing the sentinel channel activity from the Gluox channel activity. The resulting current is then amplified and recorded by the FAST-16 mkII recording system (Quanteon, LLC, Nicholasville, KY, USA). Extracellular glutamate reaches the platinum surface of the sentinel channels, but in the absence of Gluox, any oxidation current detected reflects electroactive molecules other than glutamate. Such non-glutamate derived current is presumed to be similar across each of the recording sites (Burmeister and Gerhardt, 2001; Day et al., 2006; Rutherford et al., 2007). In vitro calibration of microelectrodes Microelectrodes were calibrated in vitro just prior to implantation. Calibrations were performed in a stirred solution of PBS (0.05 M, 40 ml; pH 7.4; 37 °C; +0.7 V). After a stable baseline was established, AA (250 lM), glutamate (3  20 lM), DA (2 mM), and H2O2 (8.8 lM), were sequentially added to the calibration beaker in 40 lL aliquots and amperometric signals were acquired at a rate of 1.0 Hz. Fig. 1 depicts a representative in vitro calibration. The tracings represent change in current detected at each of the channels following the addition of the chemicals listed above (noted by the arrows). To simplify the figure, only two of the four channels are illustrated. The top tracing is from the Gluox channel, whereas

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the bottom tracing represents the background channel (sentinel). The addition of AA produces little to no change in current on either channel. Successive additions of glutamate produce large and consistent increases in current on only the Gluox channel. The increase in current is linear as the beaker concentration of glutamate becomes progressively higher. The addition of DA produces no change in current indicating the effectiveness of the m-PD barrier layer. Finally, the addition of H202 produces equal increases in current between the two channels, indicating that both recording sites are similarly sensitive to the reporting molecule. From these recordings, the slope (sensitivity, nA/lM glutamate), limit of detection (L.O.D, lM glutamate), selectivity (ratio of glutamate over ascorbic acid), and linearity (R2) were calculated. In order to qualify for use during in vivo recordings, the MEAs had to conform to the following calibration criteria: (i) similar background current (i.e., no greater than a 20-pA difference) between the Gluox and sentinel channels, (ii) linear response to increasing concentrations of glutamate (R2 > 0.998), (iii) a minimum slope of 0.003 nA/lM glutamate, (iv) a minimum limit of detection of <0.5 lM, (v) a high selectivity for glutamate over either AA or DA (i.e., >50:1), and (vi) similar sensitivity to the reporting molecule H2O2 on all four channels (>80% similarity for each channel pair). Implantation of microelectrodes and infusion cannulae Animals were anesthetized using isofluorane (2%, 0.6 L/min) and implanted with the MEAs unilaterally in the PFC (in mm from bregma: AP (anterior, posterior) + 2.7, ML (medial, lateral) ± 0.65, DV (dorsal, ventral) 4.0; hemispheres counterbalanced). Stainless steel guide cannulae (Plastics One, Roanoke, VA, USA), used for intracortical infusions of various drugs, were implanted anterior (20 degrees) to the MEA such that the tip of the infusion cannula was positioned approximately 50 lm from the upper portion of the more ventral pair of recording sites. A dummy cannula, was inserted in the guide cannula and extended 0.7 mm beyond the tip of the guide. The Ag/AgCl reference electrode was implanted in the contralateral side at a site distant from the recording area. All coordinates were determined from the atlas of Paxinos and Watson (1998). In vivo recordings and intracortical infusions Electrochemical activity was recorded in freely moving rats placed inside a wooden, Faraday-screened test chamber (dimensions: H 57.2 cm; W 341.9 cm; L 317.0 cm). On post-surgery day 1 and 2, rats were habituated to the test environment by placing them inside the chamber without connecting them to the preamplifier. On the third day after implantation, recordings of cortical glutamate began. At the onset of each testing day, animals were placed in the recording box and connected to the preamplifier. Animals were then given approximately 3 h to establish a stable

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Fig. 1. A representative in vitro calibration of the MEA conducted immediately before implantation into the prefrontal cortex. Two of the four recording channels are shown for clarity of presentation. The top tracing represents the glutamate-sensitive (Gluox) recording channel and the bottom tracing the sentinel background channel. Arrows correspond to the addition of various substances into the stirred calibration beaker. Current (nAmp) is depicted along the vertical axis and time (s) along the horizontal axis. The successive additions of glutamate (20 lM/aliquots) produced a linear increase in current (due to the oxidation of H2O2 generated from glutamate) on the Gluox channel. Expectedly, there were no changes in glutamate-related current on the sentinel channel. Important for self-referencing, the calibration also reveals equivalently high sensitivities on both channels to the reporting molecule H2O2. The effectiveness of the m-PD barrier is evident by the lack of increase in current depicted following the addition of large concentrations of potential in vivo interferents, AA and DA, to the beaker.

baseline before any drugs were delivered. All drugs were delivered over about 2 s (pH 7.1–7.4, 0.4 lL solutions) using an infusion cannula (extended 1.0 mm beyond the guide) attached to a Hamilton PB600-1 manual dispenser (Hamilton Company, Reno, NV, USA). In the first experiment, once the three hour baseline period had concluded, the dummy cannula was removed and either 1.0 or 5.0 lg of SSR180711 was delivered locally within the PFC. On the subsequent day, the dose not given on the first day was delivered using the same procedure. Dose order was counterbalanced among animals. In the second experiment, the receptor mediation of the SSR180711 effect was determined. After a stable baseline was reached (3 h), a 5.0-lg infusion of SSR180711 was delivered to the PFC. Then, following a two hour break, a second infusion made up of a combination of 5.0 lg SSR + 10.0 lM aBGT was delivered. A final experiment was conducted to corroborate the assumption that the electrochemical signal referred to as glutamate was indeed glutamate. This was accomplished by revealing, its sensitivity to a glutamate transport blocker (TbOA) as well as a drug that promoted glutamate transport (CEF). In the TbOA experiment, the effects of a 5.0-lg infusion of SSR180711 were determined following the acquisition of a stable baseline (3 h). Then, 2 h later, a solution consisting of 5.0 lg SSR180711 + 100.0 lM TbOA was infused. In the CEF experiment, the drug was administered at the end of a recording session in which the effects of 5.0 lg SSR180711 were determined. Forty-eight hours later (Wei et al., 2012) the effects of

CEF (200 mg/kg, i.p.) on evoked release were determined by measuring glutamate levels in animals infused with 5.0 lg SSR180711. Given the propensity of a7 nAChRs to desensitize (Bertrand et al., 1992; Dani et al., 2000), drug infusion order was counterbalanced whenever possible (i.e. the dose–response for SSR180711 and the effects of TbOA). Histology At the conclusion of each experiment, animals were given a sub-lethal dose of Euthasol and trans-cardially perfused with 0.9% heparinized saline followed by 10% formalin. Brains were removed and stored in formalin (10%) for at least 2 days, and then transferred to a sucrose solution (30%) for cryoprotection for at least 2 days. Brains were sectioned using a cryostat; coronal and sagittal sections (50 lm) were mounted on gelatin-coated slides, stained using Cresyl Violet, and examined under a light microscope for verification of microelectrode and cannula placement. Data analysis Measurements derived from the MEA recording and the FAST-16 data file included: (i) basal glutamate levels, (ii) maximum peak concentration (lM) of glutamate beyond basal levels, (iii) the latency (s) of onset of the effect from the time of drug infusion, (iv) T80, the time in seconds from maximum peak amplitude to 80% decay of signal (a measure of glutamate clearance), and (v)

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various rates of clearance (i.e. Tc40–80, indicates the clearance rate during which 40–80% of the glutamate signal was being cleared). The glutamate signal, initially measured in pA, was transformed to a concentration equivalent (lM) on the basis of each sensor’s individual calibration slopes generated immediately before surgery. The signal derived exclusively by the oxidation of glutamate was isolated using a self-referencing procedure between the Gluox recording channel and the adjacent background sentinel channel as we have described elsewhere (Burmeister and Gerhardt, 2001; Day et al., 2006; Rutherford et al., 2007; KonradssonGeuken et al., 2009). In terms of group comparisons, the effects of SSR180711, SSR180711 + aBGT, SSR180711 + CEF, and SSR180711 + TbOA-induced glutamate release on each of the measures described above were analyzed by analysis of variance (ANOVA) using the SPSS 18 statistics program (V19, IBM Corporations, Armonk, NY, USA). In all ANOVAs, the Huynh-Feldt correction was utilized to reduce Type I errors associated with repeated measure ANOVAs (Vasey and Thayer, 1987). When appropriate, a minimum number of post hoc comparisons were conducted using t-tests. Statistical significance was defined as P < 0.05.

RESULTS Intracortical placement of microelectrode and infusion cannula Fig. 2 depicts a representative sagittal section revealing the placement of the MEA and infusion cannula in the

Fig. 2. Photomicrograph illustrating a representative placement of the MEA and infusion cannula within the PFC. Inset (A): This high magnification image is of the MEA and infusion cannula, and demonstrates their relative positions as they are lowered into the cortex. The distance between the MEA’s platinum sites and the infusion cannula tip is maintained at 45–65 lm relative to the upper half of the more ventral channel pair. Photomicrograph (B): This sagittal brain section highlights the actual position of the ventral tip of the MEA (double asterisk) and the proximity of its infusion cannula (single asterisk). The tip of the MEA can be clearly seen at the border between the prelimbic and infralimbic cortices. Note the close proximity of the end of the infusion cannula and the recording channels of the MEA and that this implant results in only modest tissue disruption.

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PFC. The inset (Fig. 2A) indicates that the positioning of the cannula was in close proximity (50 lM) to the platinum recording sites of the MEA. The tips of the MEA and cannula (as indicated by the double and single asterisks, respectively) terminated in the prelimbic/infralimbic border of the medial PFC. This representative image also illustrates the minimal tissue damage produced by the MEA/ cannula assemblage in the brain. Only rats with MEA and properly positioned cannulas terminating in this region of the PFC were used for these analyses. Locally infused SSR180711 increases glutamate release in the PFC In order to determine whether local infusion of the partial agonist SSR180711 increases prefrontal glutamate levels, the drug was administered, in counterbalanced order, at two different doses (1.0 and 5.0 lg in 0.4 lL). Fig. 3 displays representative tracings of the MEA’s electrochemical signal in the same animal following infusion of these two doses, which are separated by 24 h (depicted by the break in the horizontal axis). Baselines for the two doses were aligned for ease of comparison. In this figure, the top tracing represents the signal generated by the Gluox channel, and the middle tracing represents the signal generated from the sentinel channel, which is sensitive to the same array of electroactive compounds as is the Gluox site except for glutamate. The bottom tracing represents the resulting signal generated from subtracting the background channel from the Gluox channel. Thus, this bottom tracing indicates the self-referenced signal or the signal generated exclusively by the oxidation of glutamate (all subsequent data shown are presented as self-referenced tracings). Inspection of the selfreferenced tracing in Fig. 3 reveals the stability of the baseline both before and after each infusion. The lower dose of SSR180711 (1.0 lg) produced a peak that had a very short onset time (1 s) and a rapid rise to maximum evoked amplitude of glutamate. The signal reached a maximum amplitude of 0.80 lM before rapidly returning to baseline (5 s). Following the higher dose of SSR180711 (5.0 lg) the peak onset and rise to max amplitude were also very rapid (2 s) and the peak cleared within 3 s. However, the resulting evoked glutamate (4.07 lM) was significantly greater than that seen after the 1.0 lg infusion. Overall, the group data (n = 7; Fig. 4) were consistent with the individual data depicted in Fig. 3. Peak amplitudes were dose-dependent with glutamate levels (mean ± SEM) after 1.0 lg SSR180711 (1.41 ± 0.30 lM) being significantly less than after 5.0 lg SSR180711 (3.51 ± 0.36 lM; F1,12 = 19.96, P = 0.001). These effects were seen regardless of the order in which the doses were presented (e.g. 5.0 lg first session = 3.48 lm; 5 lg second session = 3.54 lm). Basal glutamate levels did not differ significantly before or after any drug infusion (1.0 lg: 0.75 ± 0.21 lM, 5.0 lg: 0.79 ± 0.18 lM; P > 0.5), indicating that basal glutamate did not affect the magnitude of stimulated glutamate levels. Also, the time to peak onset did not differ between drug

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Fig. 3. Representative MEA tracings taken from the PFC following the infusion of two different doses of the a7 nAChR partial agonist, SSR180711 (1.0 lg and 5.0 lg). The top tracing represents the signal from the glutamate-sensitive channel (Gluox), whereas the middle tracing represents the signal from its adjacent sentinel or background channel. The bottom tracing (Self Ref) represents the signal derived from subtracting the second channel from the first, thereby isolating the signal obtained exclusively from the oxidation of extracellular glutamate. Changes in the vertical axis are transformed (using the regression lines from the calibration) from current to concentration (lM), whereas the horizontal axis reflects the passage of time (s). The break in the time axis represents different, counterbalanced recording sessions separated by 24 h. Baselines were aligned on either side of the time break to allow for ease of comparison (see the results section for actual basal glutamate values). Arrows represent the time at which infusions were delivered. The local infusion of the lower dose of SSR180711 (1.0 lg) produced a robust and immediate (1 s) increase in prefrontal glutamate release, which persisted for 5 s before returning to a stable baseline. This effect, seen only on the Gluox channel, reaches a maximum increase in peak amplitude of 0.80 lM after self-referencing. A local infusion of the higher dose of SSR180711 (5.0 lg), delivered during a separate recording session, also produces a rapid (2 s to peak) and transient (3 s to baseline) increase in prefrontal glutamate, but one that is larger in magnitude (4.07 lM) than following the 1.0 lg infusion.

doses (1.0 lg: 1.29 ± 0.49 s, 5.0 lg: 2.00 ± 0.37 s; P > 0.05). Finally, peak clearance, as measured by T80 (time in seconds to clear 80% of the maximum glutamate peak) was also not significantly affected by dose (1.0 lg: 7.80 ± 2.28 s, 5.0 lg: 18.40 ± 5.17 s; P > 0.05). SSR180711-induced PFC glutamate release attenuated with aBGT

Fig. 4. Group data representing the maximum peak amplitude (mean ± SEM) of prefrontal glutamate release (lM), relative to basal values, following the infusion of two different doses of SSR180711 (1.0 lg and 5.0 lg, in 0.4 lL). Each rat (n = 7) received both doses in separate, counterbalanced recording sessions and maximum amplitudes were taken after self-referencing.  = 5.0 lg drug infusions resulted in significantly higher prefrontal glutamate levels (3.51 ± 0.36 lM) than seen following 1.0 lg infusions (1.41 ± 0.30 lM; P = 0.001).

In order to determine if the SSR180711-induced increase in PFC glutamate levels was mediated via stimulation of a7 nAChRs, SSR180711 (5.0 lg) was delivered locally into the PFC alone (in order to replicate the effects seen in Figs. 3 and 4) and then again in combination with a well-established dose of the selective a7 nAChR antagonist aBGT(10.0 lM). The top tracing in Fig. 5 displays a representative self-referenced glutamate signal generated by these two infusions, delivered in this order and separated by 2 h (as depicted by the break in the axis). Baselines for the two infusions were aligned for ease of comparison. Again, the selfreferenced tracing reveals the stability of the baseline both before and after each infusion (5.0 lg, basal = 0.59 lM, 5.0 lg + 10.0 lM aBGT, basal = 0.77 lM). SSR180711 (5.0 lg) alone produced a peak similar in amplitude (4.0 lM) to that of the previous experiment and a separate group of animals.

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Fig. 5. Representative MEA tracing and group data taken from the PFC following the infusion of SSR180711 (5.0 lg) alone or SSR180711 (5.0 lg) co-administered with the selective a7 antagonist aBGT (10.0 lM). (top) This tracing represents the self-referenced glutamate derived current. The break in the time axis represents a separation of drug infusions (2 h), with both infusions occurring within the same testing day. Arrows represent the time at which infusions were delivered. As in the first experiment, a local infusion of SSR180711 (5.0 lg) produced a robust and immediate (2 s) increase in prefrontal glutamate release (4.00 lM), which persisted for 17 s before returning to a stable baseline. This glutamate peak was undetectable when the SSR180711 (5.0 lg) was co-administered with the a7 nAChR antagonist aBGT (10.0 lM) as shown in the second portion of the representative tracing. (bottom) Group data comparing the maximum amplitude (mean ± SEM) of prefrontal glutamate levels following the two conditions depicted in the top panel. Each rat (N = 5) received both drug conditions in the same experiment session and in the order shown. ⁄⁄⁄ SSR180711 (5.0 lg) + aBGT (10.0 lM) drug infusions resulted in a significantly smaller increase in prefrontal glutamate levels (0.35 ± 0.10 lM) than the infusion of SSR180711 (5.0 lg; 2.62 ± 0.36 lM; P < 0.001).

As seen in the MEA tracing, the peak had a very short onset time (2 s) and a rapid rise to maximum evoked amplitude of glutamate before returning to baseline within 17 s. However, following the co-administration of SSR180711 (5.0 lg) with aBGT (10.0 lM), the SSR180711-induced prefrontal glutamate release is dramatically reduced and, in this particular tracing, was virtually eliminated, not reaching the minimum peak criterion of 3 the average amplitude of noise. Overall, the group data (n = 5) shown at the bottom of Fig. 5 revealed peak amplitude of glutamate levels (mean ± SEM) after 5.0 lg SSR180711 (2.62 ± 0.36 lM) that were markedly reduced following the combination of 5.0 lg SSR180711 + 10.0 lM aBGT (0.35 ± 0.10 lM; F1,8 = 35.848, P < 0.001). Basal glutamate levels did not differ before or after any drug infusion (5.0 lg, basal = 0.70 lM; 5.0 lg + 10.0 lM aBGT, basal = 0.79 lM; P > 0.5). Also, the time to peak onset (as calculated only from peaks that met the 3 the amplitude of noise criteria) did not differ between drug combinations (5.0 lg: 1.5 s; 5.0 lg + 10.0 lM aBGT: 4.00 s; P > 0.1). Peak clearance, as measured by T80 (time in seconds to clear 80% of the maximum glutamate peak) was also unaffected by drug

combination (5.0 lg: 17.67 s; 5.0 lg + 10.0 lM aBGT: 11.00 s; P > 0.1). Bidirectional effects on clearance of SSR180711induced glutamate signals following drugs that facilitate or block excitatory amino acid transporter (EAAT) transporters The administration of CEF, a compound shown to facilitate the clearance of extracellular glutamate, markedly reduced the SSR180711-induced electrochemical signal. Animals that received an intraPFC infusion of SSR180711 along with a vehicle saline injection exhibited a rapid increase (1 s or less) in selfreferenced signal in PFC. The self-referenced tracing from a representative animal receiving on session 1 SSR180711 and on session 2 SSR180711 + CEF (200 mg/kg, i.p.48 h earlier) are illustrated in Fig. 6A. Fig. 6A depicts a representative self-referenced tracing of the MEA’s electrochemical signal following these two infusions in the same animal. Baselines for the two infusions were aligned for ease of comparison. Again, the self-referenced tracing reveals the stability of the basal glutamate levels both before and after each

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Fig. 6. Representative MEA tracing of the self-referenced glutamate-derived current in PFC following administration of drugs that facilitate (A, ceftriaxone) or inhibit (B, TbOA) transporters of extracellular glutamate. Arrows represent the time at which infusions were delivered. (A) As in the dose–response experiment, a local infusion of SSR180711 (5.0 lg) produced a robust and immediate (1.5 s) increase in prefrontal glutamate release (4.58 lM), which persisted for 26 s before returning to a stable baseline. Administration of the glutamate transport facilitator ceftriaxone (200 mg/kg, i.p.; 48 h earlier) markedly reduced the SSR180711-evoked glutamate signal. (B) Again, a local infusion of SSR180711 (5.0 lg) produced a robust and immediate (1.5 s) increase in prefrontal glutamate release (3.44 lM), which persisted for 5.5 s before returning to a stable baseline. Co-administration of the glutamate transport blocker TbOA (100.0 lM) with SSR180711 (5.0 lg) dramatically slowed the clearance of the glutamate peak as shown in the second half of the tracing. The infusion produced a comparably rapid and significant rise in the glutamate signal (3.73 lM) however the clearance was markedly affected by the presence of TbOA. The initial phase of clearance was only moderately slowed by the transport inhibitor, clearing approximately 40% of the maximal amplitude. Thereafter, glutamate clearance was considerably slowed, reaching the pre-infusion baseline in 70 s as opposed to the 5.5 s in the absence of TbOA.

infusion (5.0 lg: 2.40 lM; 5.0 lg + 200 mg/kg CEF: 1.89 lM). SSR180711 (5.0 lg) alone produced a peak similar to that of the previous experiments (Figs. 3–5). However, when SSR180711 (5.0 lg) was delivered 48 h after CEF (200 mg/kg), there was both a definitive decrease in the amount of glutamate released as well as a shortening of the clearance time of the glutamate peak (26 vs. 8 s, respectively). In terms of group data, the rise from baseline in the amplitude of the signal corresponded to an increase of 5.18 ± 0.24 lM (mean ± SEM; n = 4 rats, data not shown). The average T80 value for the clearance of the signal was 38.25 ± 14.4 s. In these same animals, administration of CEF (200 mg/kg, i.p.; 48 h prior to the local infusion of SSR180711) significantly reduced the maximal amplitude increase by 65% (1.82 ± 0.73 lM; F1,7 = 19.04. P = 0.005; data not shown). There was a trend toward a more rapid clearance (shortened T80; 22.0 ± 6.9 s) that may have contributed to the reduced maximal concentration of extracellular glutamate. In order to further corroborate that the self-referenced electrochemical signal was indeed glutamate, we determined the effects of an EAAT blocker on the clearance of the signal. SSR180711 (5.0 lg) was delivered locally into the PFC both alone and then, 2 h later, in combination with a standard concentration of the EAAT glutamate transport blocker, TbOA (100.0 lM). Fig. 6B depicts a representative selfreferenced tracing of the MEA’s electrochemical signal

following these two infusions in the same animal. Baselines for the two infusions were aligned for ease of comparison. Again, the self-referenced tracing reveals the stability of the basal glutamate levels both before and after each infusion (5.0 lg: 3.25 lM; 5.0 lg + 100.0 lM TbOA: 2.18 lM). SSR180711 (5.0 lg) alone produced a peak similar to that of the previous experiments (Figs. 3–5). However, when SSR180711 (5.0 lg) was co-administered with TbOA (100.0 lM), there was, in contrast to the effects of CEF (Fig. 6A), a definitive lengthening of the peak waveform indicating a marked slowing of glutamate clearance (up to 70 s). An inspection of the effects of TbOA reveals that the initial portion of signal clearance, after the apex of the amplitude, looks quite similar to that seen following SSR180711 (5.0 lg) delivered alone. It is not until the later portion of the signal that the clearance appears to be most protracted and thus affected by the TbOA. An analysis of the group data reveals that basal glutamate levels did not differ significantly before either of the two drug infusions (5.0 lg: 3.87 ± 1.95 lM, 5.0 lg + 10.0 lM TbOA: 3.26 ± 1.56 lM; P > 0.5). Also, the time to peak onset did not differ between drug combinations (5.0 lg: 1.5 ± 0.24 s, 5.0 lg + 100.0 lM TbOA: 2.00 ± 0.35 s; P > 0.1). As previously mentioned, the group data also indicate no difference in glutamate peak amplitude (5.0 lg: 3.29 ± 0.61 lM, 5.0 lg + 100.0 lM TbOA: 3.08 ± 0.40 lM; P > 0.5).

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SSR + TbOA = 0.02 ± 0.01; P < 0.05) stages of the waveform. Finally, clearance rates were normalized by peak amplitude in the bottom panel in order to determine if the clearance rate was affected by the amount of extracellular glutamate (i.e. a potential saturation of the transporter following high concentrations being released). The data reveal that TbOA continued to slow clearance rates (P < 0.03) regardless of maximal amplitude although the error bars for the normalized rates are slightly smaller, indicating that some of the variance in the rate calculation is derived from minor amplitude differences.

DISCUSSION

Fig. 7. (top panel) Group data comparing the clearance of glutamate (mean ± SEM) at several different time points (T20–T80; e.g. T20 refers to the time in s it takes to clear 20% of the maximal glutamate level increase) after two different drug infusions (5.0 lg SSR180711 and 5.0 lg SSR180711 + 100.0 lM TbOA). Each rat (n = 5) received both doses in the same recording session, and dose order was counterbalanced. Glutamate levels evoked by the combined drug infusion took significantly longer to clear at later time points (T60, T80: ⁄P < 0.05), but not earlier time points (T20, T40: P > 0.1) when compared to clearance times following an infusion of SSR180711 (5.0 lg) alone. (bottom panel): Group data comparing the clearance rates of glutamate (Tc = lM/s; mean ± SEM) at two time ranges (Tc20–60 and Tc40–80). Tc20–60 refers to the rate of clearance in lM/s during which 20–60% of the maximal glutamate level is cleared. Both clearance ranges are also displayed after being normalized for glutamate peak amplitude (Tc/max amp). This was done in order to guard against the possibility that clearance rates interacted with the maximal amplitude of glutamate (as might be the case if the transporter molecules were saturated). Regardless of how the rate of clearance is expressed, the addition of TbOA (100.0 lM) significantly slowed clearance rate during both the middle (Tc20–60) and late (Tc40–80) stages of the SSR-evoked release. ⁄P < 0.05.

Overall, the group clearance data (n = 5) shown in Fig. 7 were consistent with the individual tracing in Fig. 6B. The presence of TbOA led to significantly lengthened clearance times (top panel; mean ± SEM s) and these effects were seen whether the TbOA was administered during the first or second recording session. Sensitivity to TbOA appeared at the later time points (T60: SSR = 6.8 ± 2.3; SSR + TbOA = 47.0 ± 16.1 and T80: SSR = 11.1 ± 3.9; SSR + TbOA = 152.0 ± 50.1; P < 0.05), but not the earlier time points (T20: SSR = 2.5 ± 0.7; SSR + TbOA = 2.8 ± 1.0 and T40: SSR = 4.3 ± 1.2; SSR + TbOA = 7.2 ± 3.4; P > 0.4). TbOA also affected glutamate clearance rates (bottom panel; mean ± SEM lM/s) but, unlike the clearance time, the presence of TbOA led to significantly slower clearance rates during both the early (Tc20–60: SSR = 0.71 ± 0.28; SSR + TbOA = 0.04 ± 0.01; P < 0.05) and later (Tc40–80: SSR = 0.36 ± 0.14;

The present experiments led to three important observations. First, local administration of the partial a7 nAChR agonist, SSR180711, produced a rapid, dosedependent increase in extracellular glutamate levels that was cleared from the area surrounding the MEA in less than 20 s. Second, the SSR180711-induced increases in glutamate levels were mediated via the a7 nAChR as co-administration of SSR180711 with the selective a7 antagonist aBGT virtually eliminated the increase in evoked glutamate in PFC. Finally, the identity of the self-referenced tracing as glutamate was corroborated by bidirectional changes in either signal clearance (i.e. T80 or TC) and/or peak amplitude (i.e. concentration) following co-administration of drugs that are known to either facilitate or block excitatory amino acid transport proteins. The discussion below will focus on the following topics; (a) the identity and source of the glutamate signal; (b) mechanisms by which SSR180711 might evoke prefrontal glutamate release; and (c) the cognition-enhancing effects of SSR180711 administration in animal models and the potential for a7 nAChR agonists as adjuncts in patients with SZ. Identity and source of the glutamate signal Numerous studies have provided converging evidence that the electrochemical self-referenced signals illustrated here are of currents generated by the oxidation of glutamate at the surface of the MEA. First, control infusions of exogenous glutamate evoke rapid increases and then decreases in amplitude at the Gluox site but not at the sentinel – yielding a self-referenced wave form that resembles those presented in Figs. 3, 5 and 6 above (Burmeister and Gerhardt, 2001; Pomerleau et al., 2003; Konradsson-Geuken et al., 2009). Second, recordings made at a potential of +0.25 V, a value that is lower than the oxidation potential of the glutamate reporting molecule H2O2, reveal a wave form that is completely eliminated following self-referencing. This indicates that the same non-glutamate analyte(s) profile and amount were recorded on both the Gluox and sentinel site (and selfreferenced to zero) due to the fact that glutamate oxidation could no longer be detected (Day et al., 2006; Rutherford et al., 2007). Finally, administration of CEF, a facilitator of the glutamate transporter protein GLT-1 (Rothstein et al., 2005) and TbOA, an inhibitor of these

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transporters (Shimamoto et al., 1998) markedly decreased the signal amplitude and increased the time for clearance of the glutamate signal (Figs. 6 and 7; see also Day et al., 2006), respectively. The source(s) of the extracellular glutamate being measured in the present experiments remains to be determined. Two major glutamatergic afferents to PFC are from the mediodorsal thalamus (Leonard, 1969; Guldin et al., 1981; Groenewegen, 1988; Hoover and Vertes, 2007) and the ventral hippocampus (Ferino et al., 1987; Jay and Witter, 1991; Jay et al., 1992; Hoover and Vertes, 2007). Release from both inputs is modulated by presynaptic nACh receptors (Gray et al., 1996; Gioanni et al., 1999; Lambe et al., 2003). We are currently studying the impact of selective lesions of these input regions on basal and evoked glutamate levels. Such studies will also reveal the extent to which SSR180711 acts directly on glutamatergic nerve terminals in the PFC, as opposed to post-synaptic sites on GABAergic interneurons or pyramidal cells. SSR180711-mediated elevations of glutamate levels SSR180711 is a selective a7 nAChR partial agonist (Ki = 22 ± 4 and 14 ± 1 nM for rat and human receptors, respectively), with no significant binding at human b2 or b4 nAChRs (Biton et al., 2007; Pichat et al., 2007). Expression studies reveal intrinsic activities of 39–50% relative to ACh, with weak desensitization properties (Biton et al., 2007). Systemic administration of SSR180711 potentiates the amplitude of electrically evoked EPSPs in CA1 hippocampal neurons as well as increases ACh efflux in hippocampus and PFC in awake rats (Biton et al., 2007). SSR180711 has also been shown to increase c-Fos and Arc expression in PFC in a mechanism dependent upon activation of a7 nAChRs in basal forebrain (Thomsen et al., 2010). In terms of local regulation of prefrontal glutamate release, there is evidence supporting an a7 nAChRbased modulation of glutamate levels in PFC (Rousseau et al., 2005; Dickinson et al., 2008; Konradsson-Geuken et al., 2009). However, the ability of SSR180711 infusions to stimulate prefrontal glutamate release has not yet been reported. The increase in glutamate, following either dose of SSR180711, returned to basal values in <20 s. The rapid clearance of the signal is due to an initial passive diffusion of glutamate away from the surface of the MEA coupled with highly efficient inactivation mechanisms associated with glial and neuronal transporters (see Danbolt, 2001 for review). An acute administration of CEF, a drug shown to enhance the clearance of extracellular glutamate, through activation of GLT-1 transporter systems (Rothstein et al., 2005), markedly reduced the concentration of SSR180711-evoked glutamate sampled by the MEA. Conversely, inhibition of these transport proteins with co-infusion of TbOA had little effect on the evoked concentration of glutamate or the initial phase (T20, T40) of the clearance profile (the bases of these observations are presently unclear) but markedly slowed the clearance process in the later

stages (T60, T80; see summary in Fig. 7). The transporter proteins were unlikely saturated within the range of extracellular glutamate levels observed as similar effects were seen even after the clearance rate was normalized by the maximum levels of glutamate detected. Cognition-enhancing effects of SSR180711 A number of studies have reported cognition-enhancing effects of systemic administration of SSR180711 in normal animals and in various models of SZ. Pichat et al. (2007), in their initial characterization of the compound, reported that SSR180711 facilitated episodic memory and object recognition in rodents. These effects were blocked by the selective a7 nAChR antagonist methylycaconitine (MLA) and in a7 nAChR knock-out mice. SSR180711 reversed MK-801 and PCP-induced deficits in object recognition as well as MK-801-induced spatial working memory deficits (Pichat et al., 2007). Acute (Thomsen et al., 2009) or chronic (Hashimoto et al., 2008) administration of SSR180711 prevented the cognitive deficits associated with repeated injections of phencyclidine (PCP). Another study revealed that SSR180711 alleviated deficits in latent inhibition produced by both an acute MK-801 challenge and in a developmental model of SZ [neonatal nitrous oxide synthase (NOS) blockade; (Barak et al., 2009)]. Brooks et al. (2012) demonstrated that SSR180711 reversed set-shifting deficits in adults following transient inactivation of the ventral hippocampus during the first week of development. Deficits in set-shifting have been linked to insufficient glutamate release in PFC (Stefani et al., 2003; Stefani and Moghaddam, 2005; Alexander et al., 2012); so the present demonstration of SSR180711-induced increases in glutamate levels could provide a mechanism for these pro-cognitive effects. Finally, it should be noted that the pro-cognitive effects of SSR180711 cited above have been observed with other a7 nAChR agonists and positive allosteric modulators (PAMs) (Thomsen et al., 2010; Werkheiser et al., 2011). The studies cited above contributed to the rationale for testing the cognition-enhancing effects of agonists and positive modulators of the a7 nAChR in patients with SZ. The partial agonist tropisetron improved deficits in the auditory P50 profile in patients (Koike et al., 2005). Another partial agonist, GTS-21 (DMXB-A), elevated the total scale score of the Repeatable Battery for the Assessment of Neuropsychology as well as demonstrated improvement in P50 auditory evoked potential inhibition in a proof-of-concept study performed with non-smoking participants that were taking antipsychotics (Olincy et al., 2006; but see Freedman et al., 2008). Most recently, an exploratory trial of the effects of the selective partial a7 nAChR agonist TC-5619 on symptoms in patients with SZ was reported (Lieberman et al., 2013). The drug improved performance in a test of executive function and decreased negative symptom scores. In closing, SSR180711 and other drugs that stimulate a7 nAChRs have been shown to enhance cognitive

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performance in both animal models and in patients with SZ. Given the role of cortical glutamate transmission in the expression of executive functions (Stefani et al., 2003; Stefani and Moghaddam, 2005; Lewis and Moghaddam, 2006; Alexander et al., 2012) and the dysregulation of prefrontal glutamatergic transmission in SZ (see Goff and Coyle, 2001, for review), the present demonstration that local SSR180711 elevates glutamate levels in PFC provides a mechanism by which administration of the drug improves performance in several cognitive tasks.

DISCLOSURE Listing for each author, detailing the names of organizations, institutions, companies, and individuals, including intermediaries such as sub-contractors or conference organizers, from whom they have received compensation for professional services in any of the previous 3 years, or from whom they anticipate receiving such compensation in the near future, whether or not these affiliations appear to have any relevance to the topic covered in the submission: David Bortz and John P. Bruno have nothing to report. Jens Mikkelsen received research grants from the Neurobiology Research Unit and the Danish Research Council. Acknowledgments—This research was supported by a grant from the Danish Council for Strategic Research (COGNITO), the NOVO Nordisk Foundation, and the Lundbeck Foundation. The authors acknowledge Dr. Dan Peters for supplying the SSR180711.

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(Accepted 20 September 2013) (Available online 1 October 2013)