J Neurophysiol 116: 2663–2675, 2016. First published September 21, 2016; doi:10.1152/jn.00243.2016.

␣ 7-nAChR enhances neural plasticity in the hippocampus via a GABAergic circuit

Matthew Townsend,1 Andrew Whyment,2 Jean-Sebastien Walczak,2 Ross Jeggo,2 Marco van den Top,2 Dorothy G. Flood,1 Liza Leventhal,1 Holger Patzke,1 and Gerhard Koenig1 1FORUM Pharmaceuticals, Inc., Waltham, Massachusetts; and 2Cerebrasol, Ltd., Montreal, Quebec City, Canada

Submitted 21 March 2016; accepted in final form 18 September 2016

Townsend M, Whyment A, Walczak JS, Jeggo R, van den Top thalamus of patients (Deutsch et al. 2005; Guil- ␣ M, Flood DG, Leventhal L, Patzke H, Koenig G. 7-nAChR agonist lozet-Bongaarts et al. 2014), which may contribute to cognitive enhances neural plasticity in the hippocampus via a GABAergic circuit. impairment that is not adequately treated by current therapies Downloaded from J Neurophysiol 116: 2663–2675, 2016. First published September 21, ␣ that treat positive and negative symptoms (Wallace and Porter 2016; doi:10.1152/jn.00243.2016.— of the 7-nicotinic acetyl- ␣ 2011). A genetic linkage to the ␣ -nAChR gene locus, which receptor ( 7-nAChR) have entered clinical trials as procog- 7 nitive agents for treating schizophrenia and Alzheimer’s disease. The includes CHRNA7 and CHRFAM7A, has been identified in most advanced compounds are orthosteric agonists, which occupy the families with a history of schizophrenia, suggesting that de- ligand binding site. At the molecular level, agonist activation of fects in ␣ -nAChR function may contribute to the etiology of ␣ 7 7-nAChR is reasonably well understood. However, the consequences the disease (Gault et al. 2003; Sinkus et al. 2015). http://jn.physiology.org/ ␣ of activating 7-nAChRs on neural circuits underlying cognition ␣ In AD, the loss of neurons in the basal forebrain remain elusive. Here we report that an 7-nAChR agonist (FRM- 17848) enhances long-term potentiation (LTP) in rat septo-hippocam- is an early hallmark of the disease and results in a deficit in (ACh) signaling throughout the cerebral cortex pal slices far below the cellular EC50 but at a concentration that coincides with multiple functional outcome measures as we reported and hippocampus (Bartus et al. 1982; Whitehouse 1998). in Stoiljkovic M, Leventhal L, Chen A, Chen T, Driscoll R, Flood D, Acetylcholinesterase inhibitors (AChEIs), which slow the Hodgdon H, Hurst R, Nagy D, Piser T, Tang C, Townsend M, Tu Z, clearance of ACh during neurotransmission, remain the stan- Bertrand D, Koenig G, Hajós M. Biochem Pharmacol 97: 576–589, dard of care for patients with mild to moderate AD (Anand et 2015. In this same concentration range, we observed a significant ␣

al. 2014). While a direct link between -nAChR dysfunction by 10.220.33.1 on February 15, 2017 increase in spontaneous ␥-aminobutyric acid (GABA) inhibitory post- 7 or dysregulation and AD is less well established (Neri et al. synaptic currents and a moderate suppression of excitability in whole ␣ cell recordings from rat CA1 pyramidal neurons. This modulation of 2012), selectively restoring 7-nAChR function with agonists GABAergic activity is necessary for the LTP-enhancing effects of may offer a novel, highly targeted, and well-tolerated approach ␣ FRM-17848, since inhibiting GABAA 5-subunit-containing re- to improving cognition in multiple central nervous system ␣ ceptors fully reversed the effects of the 7-nAChR agonist. These (CNS) indications including AD and schizophrenia (Toyohara ␣ data suggest that 7-nAChR agonists may increase synaptic plas- and Hashimoto 2010; Wallace and Porter 2011). ␣ ticity in hippocampal slices, at least in part, through a circuit-level The 7-nAChR is a homopentameric ligand-gated ion chan- enhancement of a specific subtype of GABAergic receptor. nel (Drisdel and Green 2000). Upon binding its endogenous ␣ ␣ ligand ACh, the ␣ -nAChR becomes selectively permeable to 7-nAChR; GABAA 5-receptor; FRM-17848; Alzheimer’s disease; 7 schizophrenia calcium and rapidly desensitizes (Albuquerque et al. 2009; ␣ Dani and Bertrand 2007). The relationships between 7- nAChR activation and desired therapeutic effects on cognitive function are not fully understood. Using Xenopus laevis ␣ NEW & NOTEWORTHY oocytes, agonists activate 7-nAChR with cellular EC50 values of Ͼ100 nM (Prickaerts et al. 2012), although these typically This article describes a potential circuit-level mechanism ␣ obtained EC50 values may be artificially elevated 10-fold by of action by which 7-nAChR agonists increase synaptic plasticity and therefore may improve cognition. ␣ -nAChR the recording conditions employed (Papke and Thinschmidt 7 ␣ agonists have shown promise in treating cognitive impair- 1998). Yet, lower concentrations of an 7-nAChR agonist (at ␣ ment in Alzheimer disease and schizophrenia, and these or below detectable 7-nAChR activation and the binding Ki) data support continued research on these compounds. can increase the response to the natural ligand ACh (Papke et al. 2011; Prickaerts et al. 2012; Quik et al. 1997; Stoiljkovic et AGONISTS OF THE ␣ -nicotinic (␣ - 7 7 al. 2015). This phenomenon, referred to as “priming,” was nAChR) are procognitive in many preclinical animal models observed and modeled at neuromuscular nAChRs (Mukhtasi- (Medeiros et al. 2014; Stoiljkovic et al. 2015). Several agonists mova et al. 2009). Cognition is improved in rodents with and positive allosteric modulators (PAMs) have advanced into free-drug concentrations of ␣ -nAChR agonist below the cel- clinical trials for treatment of cognitive impairment in disor- 7 lular EC values (but similar to the priming concentrations) ders such as schizophrenia and Alzheimer’s disease (AD) 50 (Bitner et al. 2010; Prickaerts et al. 2012; Stoiljkovic et al. (Wallace and Porter 2011). Levels of ␣ -nAChR mRNA and 7 2015; Werkheiser et al. 2011). protein are reduced in the hippocampus, cerebral cortex, and ␣ In the hippocampus, 7-nAChRs are predominantly ex- Address for reprint requests and other correspondence: M. Townsend, 200 pressed on ␥-aminobutyric acid (GABAergic) interneurons and Sidney St., Cambridge, MA 02139 (e-mail: [email protected]). to some extent on glutamatergic neurons and astrocytes (Fabi- www.jn.org 0022-3077/16 Copyright © 2016 the American Physiological Society 2663 ␣ 2664 7-nAChR AGONIST ENHANCES PLASTICITY CIRCUIT an-Fine et al. 2001; Frazier et al. 1998; Halff et al. 2014; Animals. Male Sprague-Dawley rats (5–8 wk of age) were obtained ␣ from Charles River UK (Kent, UK) and were maintained on a 12:12-h Sharma and Vijayaraghavan 2001). A selective 7-nAChR agonist (PNU-282987) increased synaptic GABAergic activity light-dark cycle with free access to food and water. All experiments in hippocampal slices (Hajos et al. 2005) by enhancing the were approved by the Cerebrasol Insitutional Animal Care and Use Committee (IACUC) and were carried out in compliance with the UK excitability of interneurons (Hurst et al. 2005). Similar results Animals (Scientific Procedures) Act, 1986, including the recent revi- ␣ were seen with another 7-nAChR agonist S24795 (Lagostena sion incorporating the European Directive 2010/63/EU on the protec- et al. 2008), and (Alkondon et al. 1997). While it is tion of animals used for scientific purposes. ␣ clear that 7-nAChRs are expressed on GABAergic interneu- Female Xenopus laevis frogs were obtained from Xenopus Express ␣ (Haute-Loire, France) and used for in vitro oocyte electrophysiology. rons and 7-nAChR agonists have a pronounced effect on GABAergic synaptic transmission in the hippocampus, the All experiments were approved by the local IACUC and were carried functional consequences on cognition remain to be established. out in compliance with the European Union regulations (Directive ␣ 2010/63/EU). To assess how 7-nAChR agonists promote cognitive func- ␣ Characterization of FRM-17848 at 7-nAChRs. Binding assays at tion at a cellular and circuit level, we have examined the ␣ rat 7-nAChR receptors were performed at Perkin-Elmer Discovery pharmacology of FRM-17848, [(R)-7-cyano-N-quinuclidin-3- Services (formerly Caliper Life Sciences/Novascreen, Hanover, MD) yl]benzo[b]thiophene-2-carboxamide, using electrophysiologi- according to their standard protocols, which follow published methods Downloaded from cal recording techniques in rat brain septo-hippocampal slice (Marks et al. 1986; Meyer et al. 1998). Briefly, rat brains were rapidly preparations. In this preparation, measures of synaptic strength removed, homogenized in buffer, and prepared for incubation with the and plasticity such as long-term potentiation (LTP) are widely radioactive ligand [125I]-␣-. The reaction was terminated considered a model for the cellular basis of learning and by diluting with buffer, followed immediately by filtration through memory (Izquierdo 1994). Previous studies have demonstrated glass fiber filters soaked in buffer containing polyethylenimine. Bind- ing of the radioactive ligand was measured using a scintillation that ␣ -nAChR agonists or PAMs can enhance LTP in the 7 counter. Nonspecific binding was determined with unlabeled ligand. http://jn.physiology.org/ hippocampus of rodents (Kroker et al. 2011; Ondrejcak et al. Each condition was measured in duplicate. The reference compound 2012). This enhancement is absent in CHRNA7 knockout mice was MLA. K values were calculated by the equation of Cheng and ␣ i (Biton et al. 2007; Lagostena et al. 2008) and 7-nAChR Prusoff (1973). antagonists such as methylcaconitine (MLA) fail to enhance Electrophysiological experiments were carried out with human ␣ LTP, indicating that agonists are activating rather than desen- 7-nAChR receptors expressed in Xenopus laevis oocytes. Oocytes ␣ ␣ sitizing 7-nAChRs (Griguoli et al. 2013). were prepared, injected with cDNA encoding for the 7-nAChR, and We recently reported that the procognitive effects of an recorded using standard procedures (Hogg et al. 2008; Stoiljkovic et ␣ al. 2015). Oocytes were grown in the presence of penicillin, and all 7-nAChR agonist could be accounted for in terms of the concentration-response function of ␣ -nAChR receptor phar- recordings were made in antibiotic-free OR2 medium containing the by 10.220.33.1 on February 15, 2017 7 following (in mmol/l): 88.5 NaCl, 2.5 KCl, 5 HEPES, 1.8 macology (Stoiljkovic et al. 2015). The bell-shaped efficacy CaCl2·2H2O, and 1 MgCl2·6H2O, pH 7.4 at 18°C. Four oocytes were dose response was shown to align with the free drug levels used in generating each set of data from at least two preparations. across assays (oocytes, brain slice LTP, in vivo theta-power, Field potential recordings for LTP. Detailed methods of the septo- and rodent cognition). In this study, we extend those findings hippocampal slice preparation and field potential recording are de- ␣ by examining how an 7-nAChR agonist enhances LTP in the scribed in Stoiljkovic et al. (2015). For all electrophysiology experi- hippocampal brain circuit at select concentrations. We demon- ments, the experimenters were blinded to the identity of the ␣ strate that the 7-nAChR-mediated enhancement of synaptic compounds. plasticity (LTP) depends on increased GABAergic neurotrans- Whole cell voltage/current clamp. Whole cell patch-clamp record- ␣ ings were performed from pyramidal neurons of the CA1 region of the mission that is mediated by GABAA 5-receptors (GABAA ␣ hippocampus at room temperature (17–21°C) using the “blind” ver- 5R). These data indicate that priming and procognitive con- ␣ sion of the patch-clamp technique and Axopatch 1D or Multiclamp centrations of 7-nAChR agonists promote synaptic plasticity, 700B amplifiers (Molecular Devices). Patch pipettes were pulled from at least in part, through a circuit-level enhancement of the thin-walled borosilicate glass (GF150TF-10; Harvard Apparatus) with activity of a specific subtype of GABAergic receptor. resistances of 3–8 M⍀ when filled with intracellular solution of the following composition (in mM): 140 potassium gluconate, 10 KCl, 1

EGTA-Na, 10 HEPES, 2 Na2ATP, and 0.3 GTP (Sigma-Aldrich). All METHODS recorded neurons were morphologically confirmed as pyramidal neu- Reagents. FRM-17848 (FRM-2 in Tang et al. 2014) was synthe- rons post-experiment via passive diffusion of Lucifer yellow (1 sized by SAI Life Sciences (Hyderabad, India) and was prepared as mg/ml) from the pipette recording solution into the recorded cell and ␮ 31.6- M stock solutions in dimethyl sulfoxide (DMSO). The GABAA fixation of the hippocampal slice tissue overnight in 4% paraformal- ␣ 5R inhibitor (hydroxypropylthio-derivative of MRK-536 and named dehyde followed by 0.1 M phosphate buffer (pH 6.8) for 24–48 h ␣ FRM-35440 herein) (Atack 2011) and the GABAA 5R PAM, SH- before visualization under a fluorescent microscope (Zeiss) (data not 053-2=F-R-CH3 (Drexler et al. 2013), were synthesized by WuXi shown). Apptech (MaShan, China). , 2,3-dioxo-6-nitro-1,2,3,4-tet- A 10-min stable baseline was established for each pyramidal rahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), DL-2-amino-5- neuron. Inhibitory postsynaptic currents (IPSCs) and inhibitory post- phosphonopentanoic acid sodium salt (D-AP5), and CGP-55845 were synaptic potentials (IPSPs) were isolated by the addition of 10 ␮M purchased from Abcam (Bristol, UK). MLA and were NBQX and 10 ␮M AP-5 to block glutamatergic receptors. FRM- purchased from Tocris Biosciences (Bristol, UK) and dissolved in 17848 (with or without FRM-35440) or donepezil was applied for 10 DMSO. All compounds were stored at Ϫ20°C and diluted to the min followed by compound washout for 20 min. In control experi- required concentrations in artificial cerebrospinal fluid (aCSF; com- ments, 50 nM MLA alone or MLA ϩ FRM-17848 was perfused over

position in mM: 127.0 NaCl, 1.6 KCl, 1.24 KH2PO4, 1.3 MgSO4, 2.4 slices after isolation of IPSCs/IPSPs. A final 5-min application of 10 ␮ CaCl2, 26.0 NaHCO3, and 10 D-glucose) immediately before use. All or 20 M bicuculline (as indicated), a GABAA , other reagents were obtained from Sigma-Aldrich. was used to demonstrate that the remaining recorded currents were

J Neurophysiol • doi:10.1152/jn.00243.2016 • www.jn.org ␣ 7-nAChR AGONIST ENHANCES PLASTICITY CIRCUIT 2665 GABAergic. Compounds were administered to the slices by bath To confirm that the enhancement of LTP by FRM-17848 ␣ perfusion from 50-ml syringes arranged in series with the main was mediated by 7-nAChR, experiments were repeated with perfusion line from the aCSF reservoir via three-way valves. Maxi- ␣ the selective 7-nAChR antagonist MLA. At a concentration of mum final DMSO concentration was 0.3%. 100 nM MLA, the effect of 3.16 nM FRM-17848 was fully Profiling FRM-35440 selectivity. The activity of FRM-35440, a inhibited (Fig. 1, D and E). This result confirmed that the ␣ GABAA 5R inhibitor, was tested at ChanTest/Charles River (Cleve- enhancement of LTP by 3.16 nM FRM-17848 was through ␣ ␣ ␣ ␣ land, OH) using HEK-293 cells expressing GABAA 1-, 2-, 3-, 4-, ␣ ␣ ␤ ␥ activation of 7-nAChRs rather than desensitization. or 5-subunits with 3/ 2-subunits using an IonWorks Barracuda ␣ 3.16 nM FRM-17848 primes 7-nAChRs. To examine the system (Molecular Devices). The intracellular solution contained the effects of 3.16 nM FRM-17848 on ␣ -receptor pharmacology, following (in mM): 50 CsCl, 90 CsF, 5 MgCl , 1 EGTA, and 10 7 2 whole cell recordings were made from Xenopus oocytes ex- HEPES, pH adjusted to 7.2 with CsOH. In preparation for a recording ␣ session, the intracellular solution was loaded into the intracellular pressing human 7-nAChRs. The addition of 3.16 nM FRM- ␣ compartment of a planar patch-clamp electrode. The extracellular 17848 to the perfusion enhanced the 7-nAChR response to the solution contained the following (in mM): 137 NaCl, 4.0 KCl, 1.8 subsequent application of 40 ␮M ACh (Fig. 2, A and B). This

CaCl2, 1 MgCl2, 10 HEPES, and 10 D-glucose, pH adjusted to 7.4 phenomenon described as priming in Stoiljkovic et al. (2015)

with NaOH. Two recordings (scans) were performed. First the test establishes a concentration-response function that is proven to Downloaded from ␣ article was added to measure agonist effects. Five minutes later, a be highly predictive of 7-nAChR agonist efficacy in LTP and second scan was recorded during the application of GABA (30 ␮M ϳ cognition (Prickaerts et al. 2012). Thus LTP in rat hippocampal Ϫ EC90) to detect antagonism. Percent inhibition was calculated as (1 slices is enhanced at a concentration of FRM-17848 that Current /Current ) ϫ 100%. FRM-35440 GABA EC90 primes ␣ -nAChRs. Analysis. Data were captured online and stored on a computer 7 running pCLAMP data acquisition software for later offline analysis. FRM-17848 hyperpolarizes hippocampal CA1 pyramidal Analysis of all data was carried out using Clampfit (Molecular neurons. To understand the effects of FRM-17848 on cellular Devices) and Microsoft Excel software. IPSP/IPSC frequencies were physiology, whole cell recordings were made from CA1 pyra- http://jn.physiology.org/ calculated from 3-min continuous recordings. LTP data are expressed midal cells in septo-hippocampal slices. For individual neu- as the mean Ϯ SE. Statistical analysis was performed using ANOVA rons, wash-in of 3.16 nM FRM-17848 did not significantly followed by Dunnett’s multiple comparisons test. For studies with change the input resistance from baseline (Fig. 3A). In contrast, sequential treatments, a repeated-measures ANOVA was applied to application 3.16 nM FRM-17848 induced a small, but highly the data followed by a Bonferroni multiple comparisons test (PRISM significant hyperpolarization of the membrane potential in Ͻ 6, GraphPad Software, San Diego, CA) with P 0.05 taken to most cells (Fig. 3B). This result was initially unexpected, indicate statistical significance. All other data are expressed as the ␣ Ϯ because in normal physiological saline, 7-nAChRs have a mean SD. ϳ ␣

reversal potential of 0 mV. Thus activation of 7-nAChRs on by 10.220.33.1 on February 15, 2017 hippocampal CA1 pyramidal neurons would be expected to be RESULTS depolarizing rather than hyperpolarizing. FRM-17848 increases GABAergic IPSCs. Based on previ- ␣ FRM-17848 enhances LTP in a bell-shaped concentration- ous work showing that 7-nAChRs are predominantly ex- response curve. In Stoiljkovic et al. (2015), it was established pressed on GABAergic interneurons in the hippocampus and ␣ ␣ that the 7-nAChR agonist FRM-17874 enhanced hippocampal our new finding that 7-nAChR activation induced a hyperpo- ␣ LTP at concentrations below the Ki for displacement of -bun- larization of pyramidal neurons, we recorded IPSCs from ϭ garotoxin. A similar compound FRM-17848 (Ki 11 nM for pyramidal neurons. Glutamatergic currents were inhibited with ␣ ϭ rat 7-nAChR, EC50 455 nM) was tested over a wider range NBQX and AP-5, leaving bicuculline-sensitive IPSCs. Addi- of concentrations for effects on LTP in rat septo-hippocampal tion of 3.16 nM FRM-17848 induced an increase in IPSC slices. As shown in Fig. 1, A and C, 3.16 nM FRM-17848 frequency within minutes (Fig. 3C). This effect was reversed induced a significant enhancement of LTP, measured during by a 20 min washout of FRM-17848 and was inhibited by the 50- to 60-min interval following theta-burst stimulation coapplication of 50 nM MLA (Fig. 3, C and D). IPSC ampli- (TBS), compared with vehicle-treated slices. The activity of tude was similarly affected in individual neurons upon wash-in this single concentration was confirmed in an additional study of 3.16 FRM-17848 (Fig. 3E), as were IPSPs (data not shown). (see Fig. 6D), and no other tested concentration significantly Thus a concentration of FRM-17848 that was found to prime ␣ enhanced LTP compared with control. This narrow efficacious the 7-nAChR and enhance LTP also increased IPSC fre- window is consistent the results in Stoiljkovic et al. (2015) that quency and amplitude in hippocampal slices. showed a similar molecule (FRM-17874) to be active only at The results from the LTP experiments identified a narrow 3.2 and 5 nM (Tang et al. 2014). concentration-response range of FRM-17848 with 3.16 nM The AChE inhibitor donepezil was used as a positive control being the only active concentration. Since 5.6 nM FRM-17848 for enhancement of LTP (Fig. 1, B and C) (Kapai et al. 2012). was inactive in LTP, this concentration was tested for its At 500 nM, donepezil showed a significant enhancement of effects on spontaneous IPSCs. As shown in Fig. 3, F and G, 5.6 LTP that was comparable to 3.16 nM FRM-17848. Neither nM FRM-17848 had no effect on the frequency (or amplitude, compound had an effect on baseline (untetanized) field excit- data not shown) of spontaneous IPSCs. Therefore, 3.16 nM atory postsynaptic potential (fEPSP) amplitude (data not FRM-17848 was found to enhance both LTP and increase shown). The 3.16-nM concentration of FRM-17848 is compa- spontaneous IPSC frequency, while 5.6 nM FRM-17848 af- ␣ rable to the free drug levels of similar 7-nAChR agonists such fected neither. as FRM-17848 and EVP-6124, which have proven benefits on Donepezil increases GABAergic IPSCs. Observing that the cognition (Keefe et al. 2015; Prickaerts et al. 2012; Stoiljkovic same concentration of FRM-17848 increased LTP and IPSC et al. 2015). frequency, we considered whether the correlation would

J Neurophysiol • doi:10.1152/jn.00243.2016 • www.jn.org ␣ 2666 7-nAChR AGONIST ENHANCES PLASTICITY CIRCUIT

A

vehicle control 3.16 nM FRM-17848 TBS fEPSP of baseline) amplitude (% B D Downloaded from

vehicle control vehicle control 3.16 nM FRM-17848 500 nM donepezil 50 nM MLA + 3.16 nM FRM-17848 TBS TBS

100 nM MLA + 3.16 nM FRM-17848 http://jn.physiology.org/ fEPSP (% amplitude of baseline) fEPSP of baseline) amplitude (% Time (min) Time (min) 200 C E # 200 180 * 180 * * 160 by 10.220.33.1 on February 15, 2017 160

140 140

120 120 post-TBS (% post-TBS (% baseline) fEPSP amplitude (% baseline) fEPSP amplitude 50-60 min 100 100

␣ ␣ Fig. 1. The 7-nicotinic acetylcholine receptor ( 7-nAChR) agonist FRM-17848 enhances long-term potentiation (LTP) in a narrow concentration range. Extracellular recordings were made of the field excitatory postsynaptic potential (fEPSP) amplitude in the CA1 region of rat septo-hippocampal slices. Theta-burst stimulation (TBS) of the Schaffer collateral afferents evoked stable LTP in all treatment groups. The average fEPSP amplitude is shown from slices treated with vehicle (control), 3.16 nM FRM-17848 (A), and 500 nM donepezil (B). The period of compound application is indicated by the black bar. C: each point in the scatterplot depicts the fEPSP amplitude averaged from 50–60 min post-TBS in brain slices treated with the indicated compounds. At 50–60 min post-TBS, both 3.16 nM FRM-17848 and 500 nM donepezil significantly increased LTP over vehicle (vehicle ϭ 129 Ϯ 4% SE, n ϭ 16; 3.16 nM FRM-17848 ϭ 148 Ϯ 4% SE, n ϭ 11; and 500 nM donepezil ϭ 145 Ϯ 6% SE, n ϭ 7; ANOVA, Dunnett’s multiple comparison test compared with vehicle, *P Ͻ 0.05). D: ␣ 7-nAChR antagonist MLA blocked the enhancement of LTP by 3.16 nM FRM-17848. Coapplication of 50 nM MLA partially blocked the enhancement of LTP by 3.16 nM FRM-17848, while 100 nM MLA fully blocked the effect. E: the scatterplot depicts the average fEPSP amplitude at 50–60 min as a percent of baseline (vehicle ϭ 129 Ϯ 4% SE, n ϭ 16; 50 nM MLA ϭ 132 Ϯ 5% SE, n ϭ 8; 3.16 nM FRM-17848 ϭ 148 Ϯ 4% SE, n ϭ 11; FRM-17848 ϩ 50 nM MLA ϭ 137 Ϯ 3% SE, n ϭ 8; FRM-17848 ϩ 100 nM MLA ϭ 131 Ϯ 7% SE, n ϭ 6; ANOVA, Dunnett’s multiple comparison test for vehicle vs. 3.16 nM FRM-17848, *P Ͻ 0.05, and 3.16 nM FRM-17848 vs. 100 nM MLA ϩ 3.16 nM FRM-17848, #P Ͻ 0.05). also apply to donepezil. To test this hypothesis, IPSC agonist) each modulate IPSC frequency at the same concen- frequency was measured in response to ascending concen- trations that enhance LTP. trations of donepezil (50–500 nM, Fig. 4A). There was a Increased GABA IPSCs modestly hyperpolarize pyramidal trend to increased IPSC frequency beginning at 50 nM neurons. To determine the consequences of increased IPSC donepezil. However, only 500 nM donepezil, the same activity on the hippocampal circuitry, current-clamp recordings concentration that significantly enhanced LTP, showed a were used to investigate the effects of FRM-17848 on the firing statistically significant increase in IPSC frequency (Fig. 4B). properties of pyramidal neurons. A current-step protocol was These data demonstrate that two distinct cognition-enhanc- applied to induce action potential firing (Fig. 5). Wash-in of ␣ ing cholinergic mechanisms (AChE inhibitor and 7-nAChR 3.16 nM FRM-17848 reduced the firing activity triggered by a

J Neurophysiol • doi:10.1152/jn.00243.2016 • www.jn.org ␣ 7-nAChR AGONIST ENHANCES PLASTICITY CIRCUIT 2667 A 2 min

␣ Fig. 2. 3.16 nM FRM-17848 can prime 7- nAChRs. Electrophysiological recordings were made from Xenopus oocytes overex- ␣ pressing human 7-nAChRs. A: sample trace from a single oocyte, shows the response to 40 ␮M ACh before and during the coappli- ␣ cation of the 7-nAChR agonist (3.16 nM FRM-17848). Cells were pulsed with 40 ␮M AChfor5sat2-or10-min intervals. B: the

histogram shows the average response from Downloaded from 4 oocytes. A stable baseline was established with 4 pulses of 40 ␮M ACh (A). Addition of 3.16 nM FRM-17848 to the bath enhanced the ACh-evoked currents recorded at 2-min intervals (3 currents, B) and then 10-min intervals (2 currents, C and D). Finally, FRM-17848 was removed from the bath and

an additional 3 ACh-evoked currents were http://jn.physiology.org/ recorded at 2-min intervals (E). The addition of FRM-17848 increased the response to ACh, enhancing the peak ACh-evoked cur- ␮ ϭ Ϯ ␮ B 3.16 nM FRM-17848 rent [40 M ACh (A) 1.0 0.06; 40 M ACh ϩ 3.16 nM FRM-17848 (B) ϭ 1.63 Ϯ 2.50 0.22; 40 ␮M ACh ϩ 3.16 nM FRM-17848 ϩ 10 min (C) ϭ 1.86 Ϯ 0.32; 40 ␮M ACh ϩ C D 3.16 nM FRM-17848 ϩ 20 min (D) ϭ Ϯ ␮ ϭ 2.00 B E 2.16 0.30; 40 M ACh washout (E) 1.74 Ϯ 0.19; repeated-measures ANOVA, by 10.220.33.1 on February 15, 2017 Bonferroni’s multiple comparison test, all treatments were significantly different from 1.50 40 ␮M ACh (A; P Ͻ 0.01)]. The priming effect persisted for a short interval after re- A moval of FRM-17848 from the bath. 1.00

0.50 Normalized AChNormalized current evoked

0.00 depolarizing current injection, and the effect could be reversed 1992). Therefore, as a procognitive agent that increases ␣ ␣ upon washout of compound. The 7-nAChR antagonist MLA GABAergic activity, 7-nAChR agonists could not simply had the opposite effect, eliciting an increase in spike activity in mimic the activity of benzodiazepines and nonspecifically response to a depolarizing current step. Importantly, 50 nM increase GABAergic activity. Previous work has demonstrated ϩ ␣ MLA 3.16 nM FRM-17848 exhibited the same effect as that selective modulators of GABAA 5Rs can be procognitive MLA alone. GABAergic inhibitory neurons are known to exert (Drexler et al. 2013; Johnstone et al. 2011; Koh et al. 2013). ␣ a tonic hyperpolarizing effect on pyramidal cells (Caraiscos et Therefore we postulated that 7-nAChR agonists enhance LTP al. 2004). These results support the conclusion that by increas- by activating a subtype of GABAergic interneuron or receptor ing IPSC activity, 3.16 nM FRM-17848 induces a modest in the neural circuit. hyperpolarization of the resting membrane potential, thereby To test this, we identified a hydroxypropylthio- variant of making pyramidal neurons less excitable. MRK-536 (herein called FRM-35440) that was reported to act ␣ ␣ ␣ GABAA 5R mediation of 7-nAChR agonist effects. We as a highly selective and potent partial agonist at GABAA 5Rs ␣ ϭ then considered whether the effects of an 7-nAChR agonist on (Ki 2 nM). As a partial agonist, FRM-35440 suppresses the GABAergic synaptic activity may be directly linked to the full activation of the receptor by GABA (Atack 2011). Mea- Ϫ ␣ enhancement of LTP. This hypothesis appears counterintuitive, suring Cl currents in HEK cells overexpressing GABAA 1–5, since FRM-17848 induced a hyperpolarization of pyramidal FRM-35440 functions as a selective antagonist of the GABAA ␣ neurons and reduced excitability. It is well known that benzo- 5R under these conditions (Fig. 6, A and B). FRM-35440 ␣ Ͼ ␮ diazepines, which are nonselective PAMs of GABAA recep- showed no binding activity at 7-nAChRs (IC50 10 M, data tors, inhibit LTP and are sedatives in humans (del Cerro et al. not shown). When tested in LTP experiments, 50 nM FRM-

J Neurophysiol • doi:10.1152/jn.00243.2016 • www.jn.org ␣ 2668 7-nAChR AGONIST ENHANCES PLASTICITY CIRCUIT 35440 alone had no effect on LTP (Fig. 6C). Thus the activity FRM-17848 on IPSC frequency. As shown in Fig. 7, A and B, ␣ of FRM-35440 at the GABAA 5R did not disrupt the basic treatment with 50 nM FRM-35440 modestly suppressed the mechanism of TBS-induced potentiation. As in Fig. 1, 3.16 nM frequency of spontaneous IPSCs recorded in CA1 pyramidal FRM-17848 was again found to enhance LTP. Strikingly, the neurons. Maintenance of the majority of IPSCs is consistent coapplication of FRM-17848 with 50 nM FRM-35440 fully with the prevalence of other GABAAR subtypes, such as ␣ inhibited the increase in LTP by 3.16 nM FRM-17848, while a GABAA 1R in this circuit, that remain active in the presence ␣ lower concentration of FRM-35440 (5 nM) partially inhibited of the GABAA 5R subtype-specific FRM-35440. Subsequent (Fig. 6, D and E). application of 3.16 nM FRM-17848, had no effect on IPSC ␣ Based on this result, we predicted that GABAA 5R inhibi- frequency. Washout of both compounds restored IPSC fre- tion by FRM-35440 should also disrupt the effect of 3.16 nM quency to baseline. FRM-35440 also fully inhibited the in-

C A Input Resistance B Membrane Potential baseline 40 5 * Downloaded from

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J Neurophysiol • doi:10.1152/jn.00243.2016 • www.jn.org ␣ 7-nAChR AGONIST ENHANCES PLASTICITY CIRCUIT 2669 crease of IPSC amplitude by 3.16 nM FRM-17848 (Fig. 7C) to that for donepezil tested at concentrations that are likely without affecting total IPSC amplitude. These data indicate to exceed the unbound concentration achievable in patients ␣ that activating 7-nAChRs at priming concentrations of FRM- (5–10 nM) (Gomolin et al. 2011; Seltzer 2005; Yang ␣ 17848 causes an enhancement of LTP and IPSC frequency and 2013a). In addition to being an agonist at the 7-nAChR, that this effect can be reversed by coapplication of a GABAA FRM-17848 is also a potent 5-HT receptor antagonist ␣ 3 5R inhibitor. (binding K ϭ 35 nM and cellular IC in oocytes ϭ 9 nM). ␣ i 50 Since FRM-35440 suppressed the effects of the 7-nAChR Since the enhancement of LTP with FRM-17848 was com- agonist FRM-17848, we tested whether a selective GABAA pletely blocked by MLA, a highly specific ␣ -nAChR an- ␣ 7 5R PAM (SH-053-2’F-R-CH3) would be sufficient to en- tagonist, it is reasonable to postulate that activation of hance LTP. A concentration of 250 nM was chosen based on ␣ -nAChRs is mediating the observed effects (Palma et al. the reported selectivity of SH-053-2’F-R-CH3 at GABA 7 A 1996). Although other classes of 5-HT antagonists can ␣ Rs (Savíc et al. 2010). As shown in Fig. 8A, a 250-nM 3 5 enhance hippocampal LTP, they also reduce GABAergic concentration of SH-053-2’F-R-CH3 significantly enhanced activity in hippocampal interneurons (Dale et al. 2014; LTP measured during the 50- to 60-min interval post-TBS. Reznic and Staubli 1997). On the contrary, 3.16 nM FRM-

This effect was concentration dependent (Fig. 8B). These data Downloaded from demonstrate that potentiating the activity of the GABA ␣ Rs 17848 increased GABAergic activity, suggesting that at this A 5 ␣ in this hippocampal circuit is sufficient to promote synaptic concentration FRM-17848 activity at 7-nAChRs predomi- plasticity. nated over activity at 5-HT3 receptors. In addition the enhancement of LTP was lost when the concentration of FRM-17848 was increased from 3.16 nM to 5.6 nM, which DISCUSSION is inconsistent with a 5-HT3 antagonism hypothesis.

Accumulating evidence suggests that selective activation of The observation that FRM-17848 causes a hyperpolariza- http://jn.physiology.org/ ␣ 7-nAChRs may be an effective therapy for enhancing cogni- tion, rather than depolarization, of CA1 pyramidal neurons tive function in patients with schizophrenia and AD. Previous led us to investigate the effects of FRM-17848 on sponta- ␣ reports have demonstrated that 7-nAChR agonists can en- neous IPSCs. IPSC frequency increased in response to hance LTP in rodent brain slices with a bell-shaped concen- FRM-17848, as might be expected based on the predomi- ␣ tration-response relationship and a narrow effective concentra- nant expression of 7-nAChRs on GABAergic interneurons tion range (Kroker et al. 2011; Lagostena et al. 2008). The (Fabian-Fine et al. 2001; Frazier et al. 1998; Freedman et al. effective concentration range for LTP was found to be similar 1993; Jones 1997; Kawai et al. 2002). These results are ␣ ␣ to the binding Ki at 7-nAChRs. consistent with previous findings that showed that 7- ␣ by 10.220.33.1 on February 15, 2017 In this report we have studied the effects of an 7-nAChR nAChR agonists increase the synaptic transmission of agonist on the basic hippocampal neuronal physiology and GABAergic interneurons at or near the binding Ki (PNU- ϭ ϭ neuroplasticity to elucidate a possible mechanism of action 282987 Ki 26 nM and FRM-17874 Ki 4.6 nM for rat ␣ ␣ underlying the procognitive effects of 7-nAChR agonists. 7-nAChRs) (Hajos et al. 2005; Stoiljkovic et al. 2015). ␣ ␣ Using concentrations of an 7-nAChR agonist (FRM- Similarly, an 7-nAChR PAM, PNU-120596, increased 17848) (FRM-2 in Tang et al. 2014), well below the agonist IPSC frequency near the EC50 for potentiating ACh-evoked EC50 (455 nM) and approximately a half-log below the responses (Hurst et al. 2005). binding Ki (11 nM), but consistent with ACh priming One potential explanation for the finding that increases in concentration (3.16 nM), we now show enhanced LTP in rat both LTP and IPSC frequency occurred at concentrations near ␣ brain slices in a narrow concentration range. The percent the binding Ki values of 7-nAChR agonists is the phenome- increase in LTP after FRM-17848 treatment was comparable non described as priming (Papke et al. 2011; Quik et al. 1997),

Fig. 3. Spontaneous inhibitory postsynaptic current (IPSC) frequency and amplitude is increased by 3.16 nM FRM-17848. A: the input resistance for an individual CA1 pyramidal neuron was measured before and after application of 3.16 nM FRM-17848. Compound application had no significant effect on input resistance [change in input resistance (⌬IR) ϭϩ5.4 Ϯ 14.9 M⍀, repeated-measures ANOVA, Bonferroni’s multiple comparison test, P ϭ 0.14, n ϭ 18]. B: the membrane ⌬ ϭϪ Ϯ potential became significantly more hyperpolarized upon application of 3.16 nM FRM-17848 [change in membrane potential ( Vm) 1.4 2.0 mV, repeated-measures ANOVA, Bonferroni’s multiple comparison test, *P Ͻ 0.01, n ϭ 38]. This hyperpolarization was not observed with 5.6 nM FRM-17848 ⌬ ϭϩ Ϯ ϭ ⌬ ϭϩ Ϯ ϭ ( Vm 0.4 3.9 mV, n 8, data not shown) or when 50 nM MLA was coapplied with 3.16 nM FRM-17848 ( Vm 0.3 4.2 mV, n 4, data not shown). C: spontaneous IPSC activity was recorded by inhibiting glutamatergic currents with 10 ␮M AP-5 and 10 ␮M NBQX. Three-minute time blocks are shown from a pyramidal neuron excerpted from a continuous recording. Addition of 3.16 nM FRM-17848 to the perfusion resulted in an increase in IPSC frequency which was prevented by 50 nM MLA. The measured currents were GABAergic, as shown by the absence of IPSCs after addition of 10 ␮M bicuculline. ϭϪ Vh 40 mV. D: the scatterplot depicts the IPSC frequency averaged over 3 min from 8 pyramidal neurons as they were subjected to sequential treatment with ␣ ϭ Ϯ compounds. The addition of the 7-nAChR agonist FRM-17848 (3.16 nM) resulted in a significant increase in IPSC frequency (baseline 0.48 Hz 0.13, n ϭ 8; 3.16 nM FRM-17848 ϭ 0.70 Hz Ϯ 0.15; wash ϭ 0.41 Hz Ϯ 0.06; repeated-measures ANOVA, Bonferroni’s multiple comparison test, *P Ͻ 0.05 vs. ␣ ϭ Ϯ baseline). Coapplication of the 7-nAChR antagonist MLA fully inhibited the effect of 3.16 nM FRM-17848 on IPSC frequency (50 nM MLA 0.47 Hz 0.08; 50 nM MLA ϩ 3.16 nM FRM-17848 ϭ 0.46 Hz Ϯ 0.09, n ϭ 3; repeated-measures ANOVA, Bonferroni’s multiple comparison test, P ϭ 0.97). Inhibitory postsynaptic potentials (IPSPs) frequency was similarly affected by FRM-17848 (baseline ϭ 0.17 Hz Ϯ 0.04; 3.16 nM FRM-17848 ϭ 0.27 Hz Ϯ 0.06; wash ϭ 0.19 Hz Ϯ 0.05, n ϭ 15, data not shown). E: as with frequency, IPSC amplitude was increased in individual neurons upon wash-in of 3.16 nM FRM-17848 (baseline ϭ 43.9 pA Ϯ 26.8, n ϭ 8; 3.16 nM FRM-17848 ϭ 95.6 pA Ϯ 67.4; wash ϭ 53.3 pA Ϯ 33.9; repeated-measures ANOVA, Bonferroni’s multiple comparison test, *P Ͻ 0.05 vs. baseline; 50 nM MLA ϭ 36.7 pA Ϯ 26.0; 50 nM MLA ϩ 3.16 nM FRM-17848 ϭ 37.6 pA Ϯ 24.6, n ϭ 3; repeated-measures ANOVA, Bonferroni’s multiple comparison test P ϭ 0.71). F: a sample recording of IPSCs from an individual neuron reveal that application of a higher concentration ϭϪ of FRM-17848 (5.6 nM) had no effect on IPSC frequency (Vh 40 mV). G: the scatterplot depicts the IPSC frequency (averaged over 3 min) of 8 cells upon addition and washout of 5.6 nM FRM-17848 (baseline ϭ 0.31 Hz Ϯ 0.14; 5.6 nM FRM-17848 ϭ 0.29 Hz Ϯ 0.14; wash ϭ 0.33 Hz Ϯ 0.13; n ϭ 8; repeated-measures ANOVA, Bonferroni’s multiple comparison test, P ϭ 0.19).

J Neurophysiol • doi:10.1152/jn.00243.2016 • www.jn.org ␣ 2670 7-nAChR AGONIST ENHANCES PLASTICITY CIRCUIT

A baseline

50 nM donepezil

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250 nM donepezil Fig. 4. Donepezil increases IPSC frequency at concentrations Downloaded from that enhanced LTP. Pyramidal neurons were voltage clamped and IPSCs were isolated with 10 ␮M NBQX and 10 ␮M AP-5. A: sequential recordings from an individual pyramidal neuron 500 nM donepezil show that step increases in donepezil concentration cause an increase in spontaneous IPSC frequency. Washout of donepe- zil restored the IPSC frequency to baseline and bicuculline eliminated the currents confirming their GABAergic origin wash

ϭϪ http://jn.physiology.org/ (Vh 50 mV). B: the scatterplot depicts the IPSC frequency averaged over 3 min for 9 cells as each was treated with ascending concentrations of donepezil. Only 500 nM donepe- zil produced a statistically significant increase in IPSC fre- quency compared with baseline (baseline ϭ 0.21 Hz Ϯ 0.11; 10 μM Bicuculline 50 nM donepezil ϭ 0.32 Hz Ϯ 0.21; 100 nM donepezil ϭ 0.34 100 pA Ϯ ϭ Ϯ Hz 0.24; 250 nM donepezil 0.33 Hz 0.19; 500 nM 10 sec donepezil ϭ 0.43 Hz Ϯ 0.29; wash ϭ 0.21 Hz Ϯ 0.13; n ϭ 9; repeated-measures ANOVA, Bonferroni’s multiple compari- son test, *P Ͻ 0.05 vs. baseline). Two of the recorded cells B * showed no change in IPSC frequency in response to any by 10.220.33.1 on February 15, 2017 concentration of donepezil.

which proposes that binding of a single molecule of an agonist function. In the canonical model of nAChR activation, at least such as FRM-17848 at an orthosteric binding site primes the two molecules of ACh are required for activation of the receptor to open in response to a second binding event of one receptor (Changeux 1992). Without rapid clearance of the ␣ molecule of an endogenous ligand such as ACh. The correlation ligand, 7-nAChRs quickly desensitize (Dani and Bertrand among priming concentrations, enhanced LTP, and increased 2007). Similarly, at higher concentrations of FRM-17848, two ␣ IPSC frequency is significant because they align with the concen- molecules could bind, activate, and desensitize the 7-nAChR trations for improving performance in cognition and memory- in the absence of ACh. We have reported that a similar related tasks in animal models (Stoiljkovic et al. 2015). compound, FRM-17874, enhances LTP at 3.16 and 5 nM but Our experiments demonstrate that FRM-17848 exhibits a not at 10 nM (Stoiljkovic et al. 2015). Thus our model would narrow, bell-shaped concentration-response function. While predict a narrow concentration-response function between this narrow efficacy concentration range for FRM-17848 may priming a sufficient number of receptors to observe efficacy ␣ be prohibitive for the development of this compound into a and desensitizing them. Designing 7-nAChR agonists that commercial drug, understanding the biological parameters that show reduced desensitization or exhibit long a half-life (T½) set the upper and lower boundaries may be useful in develop- may be desirable to maintain drug levels in an efficacious ing other molecules that widen the concentration-response concentration range.

J Neurophysiol • doi:10.1152/jn.00243.2016 • www.jn.org ␣ 7-nAChR AGONIST ENHANCES PLASTICITY CIRCUIT 2671 nAChR agonist (PHA543613) continue to increase glutamate release, yet the procognitive effect diminished (Yang 2013b), ␣ suggesting that higher concentrations of 7-nAChR agonists ␣ disrupt neuronal network functioning before 7-nAChRs be- Control come desensitized. (Vm -60 mV) However, our data do not support the latter hypothesis for this hippocampal circuit. First, we show that 500 nM donepezil exhibited a comparable effect on LTP as FRM-17848, yet 500 nM donepezil doubled the IPSC frequency (spontaneous IPSC frequency with 500 nM donepezil ϭ 204% of baseline) in FRM-17848 contrast to FRM-17848’s more modest increase (spontaneous (Vm -62 mV) IPSC frequency with 3.16 nM FRM-17848 ϭ 146% of base- line). Therefore, further increasing the IPSC frequency beyond that achieved with 3.16 nM FRM-17848 is not consistent with

suppression of plasticity in this circuit. Second, we showed that Downloaded from 5.6 nM FRM-17848 was ineffective at enhancing LTP and that Wash IPSC frequency was lower (not higher) than with 3.16 nM (Vm -60 mV) FRM-17848. The receptor-level model can account for this if ␣ 3.16 nM FRM-17848 is optimal for priming the 7-nAChR in the presence of ACh, while 5.6 nM begins to desensitize it. These data are consistent with previous studies using other

␣ http://jn.physiology.org/ 7-nAChR agonist/PAM compounds, which also showed nar- row active concentration ranges (Hajos et al. 2005; Hurst et al. MLA 2005; Kroker et al. 2011; Lagostena et al. 2008). Together, (Vm -60 mV) these studies suggest that receptor desensitization rather than overactivation of the GABAergic circuit may set the upper boundary of the concentration response curve in the hippocam- pus. Our data indicate that the modulation of GABA is directly ␣

MLA + linked to the enhanced LTP, because the GABAA 5R inhibitor by 10.220.33.1 on February 15, 2017 ␣ FRM-17848 blocked the LTP-enhancing effects of an 7-nAChR agonist (Vm -60 mV) without affecting baseline LTP. These results provide direct ␣ 50 mV pharmacological evidence that activation of 7-nAChRs en- hances the activity of a specific subtype of GABAergic syn- 250 ms apse, which in turn modulates synaptic plasticity in the hip- pocampus. Further confirmation could be established by testing Fig. 5. Pyramidal neurons are less excitable upon application of 3.16 nM ␣ ␣ FRM-17848. Whole cell current-clamp recordings were made from CA1 7-nAChR agonists in brain slices derived from GABAA 5R pyramidal neurons. Based on the salt concentrations in the electrode and knockout mice. There remains some controversy regarding the artificial cerebrospinal fluid (aCSF), the chloride reversal potential was pre- synaptic vs. extrasynaptic localization of GABA ␣ Rs in the Ϫ A 5 dicted to be 63 mV. In a sample recording, depolarizing square wave current hippocampus. Our results with FRM-35440 in Fig. 7B are injection in pyramidal neurons induced a series of accommodating action ␣ potentials. Perfusion with 3.16 nM FRM-17848 hyperpolarized the resting consistent with the existence of a synaptic pool GABAA 5Rs membrane potential (Vm) and reduced action potential firing, which was as has been shown directly by electron microscopy (Serwanski restored after a 10 min washout period (baseline ϭ 5.7 Ϯ 3.6 Hz; 3.16 nM et al. 2006). The data reported here provide a testable frame- FRM-17848 ϭ 3.9 Ϯ 3.3 Hz; wash ϭ 4.9 Ϯ 3.5 Hz; n ϭ 11; repeated- work for a “procognitive circuit” that may contribute to the measures ANOVA, Bonferroni’s multiple comparison test baseline vs. 3.16 nM FRM-17848, *P Ͻ 0.05). Addition of 50 nM MLA increased action understanding of cognitive enhancement throughout the brain. potential frequency which was unaffected by coapplication of 3.16 nM FRM- While this report has focused on the effects of FRM-17848 17848. on the GABAergic circuit and its impact on synaptic plasticity, there may be effects on glutamatergic currents as well (Gu et An alternative hypothesis could explain the bell-shaped al. 2012). In Fig. 2 we show that FRM-17848 and donepezil concentration-response function using a GABAergic circuit have no effect on baseline fEPSPs, which is consistent with a mechanism rather than a receptor-level mechanism. By this previous report (Lagostena et al. 2008). In a separate report reasoning, low concentrations of FRM-17848 would induce a (manuscript in preparation) we looked at the effects of FRM- modest increase in GABAergic activity. By moderately hyper- 17848 on evoked glutamatergic EPSCs and found no enhance- polarizing pyramidal neurons, spontaneous background activ- ment. Our data therefore support the conclusion that the pri- ity in the hippocampus would be dampened, thereby enhancing mary effect of FRM-17848 is on GABAergic rather than the contrast of burst signal-to-noise. However, at higher FRM- glutamatergic neurotransmission in this hippocampal circuit. 17848 concentrations, a further increase in GABAergic activity At first glance, it is unexpected that an increase in GABAergic would hyperpolarize pyramidal neurons to the point of disrupt- activity would be associated with enhanced LTP. Nonselective ing neurotransmission. A similar phenomenon has been shown GABAA receptor PAMs such as benzodiazepines are known to ␣ in the frontal cortex, where 7-nAChRs are localized on suppress LTP (del Cerro et al. 1992) and are associated with ␣ glutamatergic neurons, escalating concentrations of an 7- amnesia in humans (Brown and Dundee 1968; Clarke et al.

J Neurophysiol • doi:10.1152/jn.00243.2016 • www.jn.org ␣ 2672 7-nAChR AGONIST ENHANCES PLASTICITY CIRCUIT

A D 80 FRM-35440 ( 5 Agonist) FRM-35440 ( 5 Antagonist) 60

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Time (min) ␣ ␣ Fig. 6. A GABAA 5R inhibitor (FRM-35440) prevents the LTP-enhancing effect of the 7 nAChR agonist FRM-17848. A: FRM-35440 acted as a selective and ␣ ␣ ␣ ␣ potent inhibitor of GABAA 5Rs. FRM-35440 (hydroxypropylthio- derivative of MRK-536) was profiled on HEK293 cells expressing GABAA 1-, 2-, 3-, ␣ ␣ ␤ ␥ 4-, or 5-subunits with 3/ 2-subunits using an IonWorks Barracuda system. Peak currents were recorded in quadruplicate during agonist and antagonist scans. No intrinsic agonist activity of FRM-35440 was detected at 0.1–300 nM in the absence of GABA. The subsequent application of 30 ␮M GABA revealed that ␣ ϭ FRM-35440 concentration-dependently inhibited the evoked GABAA 5R current (IC50 158 nM) under these conditions. The activity of FRM-35440 may be ␣ ␣ ␮ more potent in brain slices than in recombinant cells (Farzampour et al. 2015). B: FRM-35440 was inactive on the other 1- 4 GABAA R subtypes up to 3 M. ␣ C: field potential recordings were used to measure LTP in the CA1 region of hippocampal slices. At a concentration of 50 nM, the GABAA 5R inhibitor FRM-35440 alone had no effect on TBS-induced LTP (control ϭ 134 Ϯ 4% SE, n ϭ 7, 50 nM FRM-35440 ϭ 133 Ϯ 6% SE, n ϭ 4). D: coapplication of FRM-35440 prevented the enhancement of LTP induced by 3.16 nM FRM-17848 in a concentration-dependent manner. E: each circle reports the LTP (average fEPSP amplitude from 50–60 min post-TBS) recorded from an individual slice. FRM-35440 inhibited the LTP-enhancing effects of FRM-17848 (control ϭ 134 Ϯ 4% SE, n ϭ 7, 3.16 nM FRM-17848 ϭ 151 Ϯ 6% SE, n ϭ 6; 3.16 nM FRM-17848 ϩ 5 nM FRM-35440 ϭ 143 Ϯ 3% SE, n ϭ 8; 3.16 nM FRM-17848 ϩ 50 nM FRM-35440 ϭ 132 Ϯ 4% SE, n ϭ 8; 50 nM FRM-35440 ϭ 133 Ϯ 6%, n ϭ 4; ANOVA, Dunnett’s multiple comparison test, *P Ͻ 0.05 for 3.16 nM FRM-17848 vs. vehicle, #P Ͻ 0.05 for 3.16 nM FRM-17848 vs. 3.16 nM FRM-17848 ϩ 50 nM FRM-35440).

1970). Moreover, the increase in IPSC activity with 3.16 nM in response to a depolarizing current step. Mechanistically, FRM-17848 clearly coincided with a modest hyperpolarization there are many potential circuit-based explanations such as in pyramidal neurons. We observed that the addition of bicu- feed forward inhibition (Larson and Lynch 1986) or rebound culline caused a depolarization in pyramidal neurons, indicat- after-hyperpolarization (Aizenman et al. 1998; Sah and Bek- ing that GABA contributes significantly to the hyperpolarized kers 1996) that may account for this apparent paradox. In a resting membrane potential (data not shown). We have dem- recent set of experiments it was shown in cortical circuits that onstrated that the hyperpolarization of pyramidal neurons while a nonselective GABAA receptor PAM such as diazepam caused by the increase in GABAergic activity with FRM- decreased the discharge rate of neocortical neurons during up ␣ 17848 leads to reduced firing of spontaneous action potentials states (active firing), a GABAA 5R PAM such as SH-053-

J Neurophysiol • doi:10.1152/jn.00243.2016 • www.jn.org ␣ 7-nAChR AGONIST ENHANCES PLASTICITY CIRCUIT 2673 2’F-R-CH3 had the opposite effect by increasing bursting A activity (Drexler et al. 2013). Some additional recent work by ␣ baseline Koh et al. (2013) has demonstrated that GABAA 5R PAMs are also procognitive in rats. We favor the possibility that ␣ 7-nAChR agonists may mimic this type of activation of a subset of GABAergic synapses in the circuit, thereby enhanc- 50 nM FRM-35440 ing bursting activity of glutamatergic neurons. This would have the anticipated effect of improving signal-to-noise in the hippocampal circuit, which may be relevant to cognition (Lis- 50 nM FRM-35440 + 3.16 nM FRM-17848 man 1997). While additional studies are needed to test this hypothesis, the data reported here support a convergence of the ␣ cholinergic (both 7-nAChR activation and AChE inhibition) wash 200 pA

10 sec A Downloaded from B * * http://jn.physiology.org/

vehicle control TBS 250 nM SH-053-2’F-R-CH3 fEPSP (% amplitude of baseline)

Time (min) by 10.220.33.1 on February 15, 2017 B C *

␣ Fig. 7. The GABAA 5R inhibitor (FRM-35440) prevents the increase in IPSC ␣ frequency caused by the 7-nAChR agonist FRM-17848. A: a sample trace from an individual pyramidal neuron, shows that preapplication of FRM-35440 blocked the increase in IPSC frequency induced by the subsequent addition of FRM-17848. B: quantification of the IPSC frequency in 5 pyramidal neurons demonstrates that the ␣ ␣ GABAA 5R inhibitor FRM-35440 modestly reduced IPSC frequency, and prevents Fig. 8. SH-053-2’F-R-CH3, a selective GABAA 5R PAM, enhanced LTP in the increase in IPSC frequency that occurs with 3.16 nM FRM-17848 alone. Washout septo-hippocampal brain slices. A: field potential recordings were made in CA1 of both compounds restored IPSC frequency to baseline levels (baseline ϭ 0.25 Ϯ 0.07 of rat septo-hippocampus slices. Addition of 250 nM SH-053-2’F-R-CH3 Hz; 50 nM FRM-35440 ϭ 0.20 Ϯ 0.07 Hz; 50 nM FRM-35440 ϩ 3.16 nM enhanced LTP similar to FRM-17848. B: the scatterplot depicts the fEPSP FRM-17848 ϭ 0.21 Ϯ 0.08 Hz; washout ϭ 0.25 Ϯ 0.09 Hz, n ϭ 5; repeated-measures amplitude averaged from the 50–60 min post-TBS interval from individual ANOVA, Bonferroni’s multiple comparisons test, *P Ͻ 0.05 vs. baseline). C: a similar slices. SH-053-2’F-R-CH3 exhibits a concentration-dependent enhancement of result was observed with IPSC amplitude (baseline ϭ 30.3 Ϯ 12.6 pA; 50 nM LTP (vehicle ϭ 123 Ϯ 4% SE, n ϭ 7; 50 nM SH-053-2’F-R-CH3 ϭ 121 Ϯ FRM-35440 ϩ 3.16 nM FRM-17848 ϭ 30.9 Ϯ 12.0 pA; washout ϭ 31.2 Ϯ 12.0 pA, 4% SE, n ϭ 6; 100 nM SH-053-2’F-R-CH3 ϭ 129 Ϯ 5% SE, n ϭ 6; 250 nM n ϭ 5; repeated-measures ANOVA, Bonferroni’s multiple comparisons test, P Ͼ SH-053-2’F-R-CH3 ϭ 135 Ϯ 4% SE, n ϭ 7; Dunnett’s multiple comparisons 0.05). test compared with vehicle, *P Ͻ 0.05).

J Neurophysiol • doi:10.1152/jn.00243.2016 • www.jn.org ␣ 2674 7-nAChR AGONIST ENHANCES PLASTICITY CIRCUIT and GABAergic systems on regulating neuronal plasticity in terization of a novel selective ␣7 neuronal nicotinic acetylcholine receptor the hippocampus. agonist ABT-107: preclinical considerations in Alzheimer’s disease. J Phar- In conclusion, we report that an ␣ -nAChR agonist en- macol Exp Ther 334: 875–886, 2010. 7 Biton B, Bergis OE, Galli F, Nedelec A, Lochead AW, Jegham S, Godet D, hanced LTP in the hippocampus of rat brain slices in a narrow Lanneau C, Santamaria R, Chesney F, Léonardon J, Granger P, concentration range consistent with a concentration that primes Debono MW, Bohme GA, Sgard F, Besnard F, Graham D, Coste A, the receptor. The addition of FRM-17848 stimulated an in- Oblin A, Curet O, Vigé X, Voltz C, Rouquier L, Souilhac J, Santucci V, crease in GABAergic activity among a subset of GABAergic Gueudet C, Françon D, Steinberg R, Griebel G, Oury-Donat F, George ␣ P, Avenet P, Scatton B. SSR180711, a novel selective ␣7 nicotinic receptor synapses. The coapplication of a GABAA 5R inhibitor blocked the effects of FRM-17848 on LTP and IPSC fre- partial agonist: (1) binding and functional profile. Neuropsychopharmacol- ␣ ogy 32: 1–16, 2007. quency. We conclude that the 7-nAChR agonists promote the Brown SS, Dundee JW. Clinical studies of induction agents. XXV. Diaze- activity of a subset of GABAergic synapses as part of a pam. Br J Anaesth 40: 108–112, 1968. cognition circuit in the hippocampus. These studies help to Caraiscos VB, Elliott EM, You-Ten KE, Cheng VY, Belelli D, Newell JG, elucidate the mechanism of action by which ␣ -nAChR ago- Jackson MF, Lambert JJ, Rosahl TW, Wafford KA, MacDonald JF, 7 Orser BA. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons nists produce their procognitive activity and demonstrate their is mediated by alpha5 subunit-containing gamma-aminobutyric acid type A potential utility in treating cognitive impairments in schizo- receptors. Proc Natl Acad Sci USA 101: 3662–2667, 2004. Downloaded from phrenia and AD. Changeux JP. 1992 Functional organisation of the nicotinic acetylcholine receptor. C R Acad Sci III 314: 89–94, 1992. Cheng Y, Prusoff WH. Relationship between the inhibition constant (K1) and ACKNOWLEDGMENTS the concentration of inhibitor which causes 50 percent inhibition (I50) of an We thank Raymond Hurst of FORUM Pharmaceuticals for reviewing enzymatic reaction. Biochem Pharmacol 22: 3099–3108, 1973. and discussing the data sets. We are grateful to Daniel Bertrand, Sonia Clarke PR, Eccersley PS, Frisby JP, Thornton JA. The amnesic effect of Bertrand, and Fabrice Marger of HiQScreen Sa`rl, Geneva, Switzerland for diazepam (Valium). Br J Anaesth 42: 690–697, 1970. performing the oocyte experiments, which were part of a fee-for-service Dale E, Zhang H, Leiser SC, Xiao Y, Lu D, Yang CR, Plath N, Sanchez C. http://jn.physiology.org/ contract. We also appreciate the contributions of Yuri Kuryshev of Vortioxetine disinhibits pyramidal cell function and enhances synaptic ChanTest for performing the patch recordings on HEK cells overexpressing plasticity in the rat hippocampus. J Psychopharmacol 28: 891–902, 2014. GABA receptor subtypes. Editorial assistance was provided by Hannah Dani JA, Bertrand D. Nicotinic acetylcholine receptors and nicotinic cholin- Lederman (Caudex, New York, NY). ergic mechanisms of the central nervous system. Annu Rev Pharmacol Toxicol 47: 699–729, 2007. del Cerro S, Jung M, Lynch G. Benzodiazepines block long-term potentia- GRANTS tion in slices of hippocampus and piriform cortex. Neuroscience 49: 1–6, This work was funded by FORUM Pharmaceuticals. 1992. Deutsch SI, Rosse RB, Schwartz BL, Weizman A, Chilton M, Arnold DS, ␣ Mastropaolo J. Therapeutic implications of a selective 7 nicotinic receptor DISCLOSURES abnormality in schizophrenia. Isr J Psychiatry Relat Sci 42: 33–44, 2005. by 10.220.33.1 on February 15, 2017 Drexler B, Zinser S, Huang S, Poe MM, Rudolph U, Cook JM, Antkowiak M. Townsend, D. G. Flood, L. Leventhal, H. Patzke, and G. Koenig were B. Enhancing the function of alpha5-subunit-containing GABAA receptors employees of FORUM Pharmaceuticals, a privately owned corporation. A. promotes action potential firing of neocortical neurons during up-states. Eur Whyment, J.-S. Walczak, R. Jeggo, and M. van den Top are employees of J Pharmacol 703: 18–24, 2013. 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