α7-nAChR agonist enhances neural plasticity in the ......2016/03/21 · agonist (PNU-282987) increased synaptic GABAergic activity in hippocampal slices (Hajos et al. 2005) by enhancing

�7-nAChR agonist enhances neural plasticity in the hippocampus via aGABAergic 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 TopM, Flood DG, Leventhal L, Patzke H, Koenig G. �7-nAChR agonistenhances neural plasticity in the hippocampus via a GABAergic circuit.J Neurophysiol 116: 2663–2675, 2016. First published September 21,2016; doi:10.1152/jn.00243.2016.—Agonists of the �7-nicotinic acetyl-choline receptor (�7-nAChR) have entered clinical trials as procog-nitive agents for treating schizophrenia and Alzheimer’s disease. Themost advanced compounds are orthosteric agonists, which occupy theligand binding site. At the molecular level, agonist activation of�7-nAChR is reasonably well understood. However, the consequencesof activating �7-nAChRs on neural circuits underlying cognitionremain elusive. Here we report that an �7-nAChR agonist (FRM-17848) enhances long-term potentiation (LTP) in rat septo-hippocam-pal slices far below the cellular EC50 but at a concentration thatcoincides with multiple functional outcome measures as we reportedin Stoiljkovic M, Leventhal L, Chen A, Chen T, Driscoll R, Flood D,Hodgdon H, Hurst R, Nagy D, Piser T, Tang C, Townsend M, Tu Z,Bertrand D, Koenig G, Hajós M. Biochem Pharmacol 97: 576–589,2015. In this same concentration range, we observed a significantincrease in spontaneous �-aminobutyric acid (GABA) inhibitory post-synaptic currents and a moderate suppression of excitability in wholecell recordings from rat CA1 pyramidal neurons. This modulation ofGABAergic activity is necessary for the LTP-enhancing effects ofFRM-17848, since inhibiting GABAA �5-subunit-containing re-ceptors fully reversed the effects of the �7-nAChR agonist. Thesedata suggest that �7-nAChR agonists may increase synaptic plas-ticity in hippocampal slices, at least in part, through a circuit-levelenhancement of a specific subtype of GABAergic receptor.

�7-nAChR; GABAA �5-receptor; FRM-17848; Alzheimer’s disease;schizophrenia

NEW & NOTEWORTHY

This article describes a potential circuit-level mechanismof action by which �7-nAChR agonists increase synapticplasticity and therefore may improve cognition. �7-nAChRagonists have shown promise in treating cognitive impair-ment in Alzheimer disease and schizophrenia, and thesedata support continued research on these compounds.

AGONISTS OF THE �7-nicotinic acetylcholine receptor (�7-nAChR) are procognitive in many preclinical animal models(Medeiros et al. 2014; Stoiljkovic et al. 2015). Several agonistsand positive allosteric modulators (PAMs) have advanced intoclinical trials for treatment of cognitive impairment in disor-ders such as schizophrenia and Alzheimer’s disease (AD)(Wallace and Porter 2011). Levels of �7-nAChR mRNA andprotein are reduced in the hippocampus, cerebral cortex, and

thalamus of schizophrenia patients (Deutsch et al. 2005; Guil-lozet-Bongaarts et al. 2014), which may contribute to cognitiveimpairment that is not adequately treated by current therapiesthat treat positive and negative symptoms (Wallace and Porter2011). A genetic linkage to the �7-nAChR gene locus, whichincludes CHRNA7 and CHRFAM7A, has been identified infamilies with a history of schizophrenia, suggesting that de-fects in �7-nAChR function may contribute to the etiology ofthe disease (Gault et al. 2003; Sinkus et al. 2015).

In AD, the loss of cholinergic neurons in the basal forebrainis an early hallmark of the disease and results in a deficit inacetylcholine (ACh) signaling throughout the cerebral cortexand hippocampus (Bartus et al. 1982; Whitehouse 1998).Acetylcholinesterase inhibitors (AChEIs), which slow theclearance of ACh during neurotransmission, remain the stan-dard of care for patients with mild to moderate AD (Anand etal. 2014). While a direct link between �7-nAChR dysfunctionor dysregulation and AD is less well established (Neri et al.2012), selectively restoring �7-nAChR function with agonistsmay offer a novel, highly targeted, and well-tolerated approachto improving cognition in multiple central nervous system(CNS) indications including AD and schizophrenia (Toyoharaand Hashimoto 2010; Wallace and Porter 2011).

The �7-nAChR is a homopentameric ligand-gated ion chan-nel (Drisdel and Green 2000). Upon binding its endogenousligand ACh, the �7-nAChR becomes selectively permeable tocalcium and rapidly desensitizes (Albuquerque et al. 2009;Dani and Bertrand 2007). The relationships between �7-nAChR activation and desired therapeutic effects on cognitivefunction are not fully understood. Using Xenopus laevisoocytes, agonists activate �7-nAChR with cellular EC50 valuesof �100 nM (Prickaerts et al. 2012), although these typicallyobtained EC50 values may be artificially elevated 10-fold bythe recording conditions employed (Papke and Thinschmidt1998). Yet, lower concentrations of an �7-nAChR agonist (ator below detectable �7-nAChR activation and the binding Ki)can increase the response to the natural ligand ACh (Papke etal. 2011; Prickaerts et al. 2012; Quik et al. 1997; Stoiljkovic etal. 2015). This phenomenon, referred to as “priming,” wasobserved and modeled at neuromuscular nAChRs (Mukhtasi-mova et al. 2009). Cognition is improved in rodents withfree-drug concentrations of �7-nAChR agonist below the cel-lular EC50 values (but similar to the priming concentrations)(Bitner et al. 2010; Prickaerts et al. 2012; Stoiljkovic et al.2015; Werkheiser et al. 2011).

In the hippocampus, �7-nAChRs are predominantly ex-pressed on �-aminobutyric acid (GABAergic) interneurons andto some extent on glutamatergic neurons and astrocytes (Fabi-

Address for reprint requests and other correspondence: M. Townsend, 200Sidney St., Cambridge, MA 02139 (e-mail: [email protected]).

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

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an-Fine et al. 2001; Frazier et al. 1998; Halff et al. 2014;Sharma and Vijayaraghavan 2001). A selective �7-nAChRagonist (PNU-282987) increased synaptic GABAergic activityin hippocampal slices (Hajos et al. 2005) by enhancing theexcitability of interneurons (Hurst et al. 2005). Similar resultswere seen with another �7-nAChR agonist S24795 (Lagostenaet al. 2008), and nicotine (Alkondon et al. 1997). While it isclear that �7-nAChRs are expressed on GABAergic interneu-rons and �7-nAChR agonists have a pronounced effect onGABAergic synaptic transmission in the hippocampus, thefunctional consequences on cognition remain to be established.

To assess how �7-nAChR agonists promote cognitive func-tion at a cellular and circuit level, we have examined thepharmacology of FRM-17848, [(R)-7-cyano-N-quinuclidin-3-yl]benzo[b]thiophene-2-carboxamide, using electrophysiologi-cal recording techniques in rat brain septo-hippocampal slicepreparations. In this preparation, measures of synaptic strengthand plasticity such as long-term potentiation (LTP) are widelyconsidered a model for the cellular basis of learning andmemory (Izquierdo 1994). Previous studies have demonstratedthat �7-nAChR agonists or PAMs can enhance LTP in thehippocampus of rodents (Kroker et al. 2011; Ondrejcak et al.2012). This enhancement is absent in CHRNA7 knockout mice(Biton et al. 2007; Lagostena et al. 2008) and �7-nAChRantagonists such as methylcaconitine (MLA) fail to enhanceLTP, indicating that agonists are activating rather than desen-sitizing �7-nAChRs (Griguoli et al. 2013).

We recently reported that the procognitive effects of an�7-nAChR agonist could be accounted for in terms of theconcentration-response function of �7-nAChR receptor phar-macology (Stoiljkovic et al. 2015). The bell-shaped efficacydose response was shown to align with the free drug levelsacross assays (oocytes, brain slice LTP, in vivo theta-power,and rodent cognition). In this study, we extend those findingsby examining how an �7-nAChR agonist enhances LTP in thehippocampal brain circuit at select concentrations. We demon-strate that the �7-nAChR-mediated enhancement of synapticplasticity (LTP) depends on increased GABAergic neurotrans-mission that is mediated by GABAA �5-receptors (GABAA�5R). These data indicate that priming and procognitive con-centrations of �7-nAChR agonists promote synaptic plasticity,at least in part, through a circuit-level enhancement of theactivity of a specific subtype of GABAergic receptor.

METHODS

Reagents. FRM-17848 (FRM-2 in Tang et al. 2014) was synthe-sized by SAI Life Sciences (Hyderabad, India) and was prepared as31.6-�M stock solutions in dimethyl sulfoxide (DMSO). The GABAA

�5R inhibitor (hydroxypropylthio-derivative of MRK-536 and namedFRM-35440 herein) (Atack 2011) and the GABAA �5R PAM, SH-053-2=F-R-CH3 (Drexler et al. 2013), were synthesized by WuXiApptech (MaShan, China). Bicuculline, 2,3-dioxo-6-nitro-1,2,3,4-tet-rahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), DL-2-amino-5-phosphonopentanoic acid sodium salt (D-AP5), and CGP-55845 werepurchased from Abcam (Bristol, UK). MLA and donepezil werepurchased from Tocris Biosciences (Bristol, UK) and dissolved inDMSO. All compounds were stored at �20°C and diluted to therequired concentrations in artificial cerebrospinal fluid (aCSF; com-position in mM: 127.0 NaCl, 1.6 KCl, 1.24 KH2PO4, 1.3 MgSO4, 2.4CaCl2, 26.0 NaHCO3, and 10 D-glucose) immediately before use. Allother reagents were obtained from Sigma-Aldrich.

Animals. Male Sprague-Dawley rats (5–8 wk of age) were obtainedfrom Charles River UK (Kent, UK) and were maintained on a 12:12-hlight-dark cycle with free access to food and water. All experimentswere approved by the Cerebrasol Insitutional Animal Care and UseCommittee (IACUC) and were carried out in compliance with the UKAnimals (Scientific Procedures) Act, 1986, including the recent revi-sion incorporating the European Directive 2010/63/EU on the protec-tion of animals used for scientific purposes.

Female Xenopus laevis frogs were obtained from Xenopus Express(Haute-Loire, France) and used for in vitro oocyte electrophysiology.All experiments were approved by the local IACUC and were carriedout in compliance with the European Union regulations (Directive2010/63/EU).

Characterization of FRM-17848 at �7-nAChRs. Binding assays atrat �7-nAChR receptors were performed at Perkin-Elmer DiscoveryServices (formerly Caliper Life Sciences/Novascreen, Hanover, MD)according to their standard protocols, which follow published methods(Marks et al. 1986; Meyer et al. 1998). Briefly, rat brains were rapidlyremoved, homogenized in buffer, and prepared for incubation with theradioactive ligand [125I]-�-bungarotoxin. The reaction was terminatedby diluting with buffer, followed immediately by filtration throughglass fiber filters soaked in buffer containing polyethylenimine. Bind-ing of the radioactive ligand was measured using a scintillationcounter. Nonspecific binding was determined with unlabeled ligand.Each condition was measured in duplicate. The reference compoundwas MLA. Ki values were calculated by the equation of Cheng andPrusoff (1973).

Electrophysiological experiments were carried out with human�7-nAChR receptors expressed in Xenopus laevis oocytes. Oocyteswere prepared, injected with cDNA encoding for the �7-nAChR, andrecorded using standard procedures (Hogg et al. 2008; Stoiljkovic etal. 2015). Oocytes were grown in the presence of penicillin, and allrecordings were made in antibiotic-free OR2 medium containing thefollowing (in mmol/l): 88.5 NaCl, 2.5 KCl, 5 HEPES, 1.8CaCl2·2H2O, and 1 MgCl2·6H2O, pH 7.4 at 18°C. Four oocytes wereused in generating each set of data from at least two preparations.

Field potential recordings for LTP. Detailed methods of the septo-hippocampal slice preparation and field potential recording are de-scribed in Stoiljkovic et al. (2015). For all electrophysiology experi-ments, the experimenters were blinded to the identity of thecompounds.

Whole cell voltage/current clamp. Whole cell patch-clamp record-ings were performed from pyramidal neurons of the CA1 region of thehippocampus at room temperature (17–21°C) using the “blind” ver-sion of the patch-clamp technique and Axopatch 1D or Multiclamp700B amplifiers (Molecular Devices). Patch pipettes were pulled fromthin-walled borosilicate glass (GF150TF-10; Harvard Apparatus) withresistances of 3–8 M� when filled with intracellular solution of thefollowing composition (in mM): 140 potassium gluconate, 10 KCl, 1EGTA-Na, 10 HEPES, 2 Na2ATP, and 0.3 GTP (Sigma-Aldrich). Allrecorded neurons were morphologically confirmed as pyramidal neu-rons post-experiment via passive diffusion of Lucifer yellow (1mg/ml) from the pipette recording solution into the recorded cell andfixation of the hippocampal slice tissue overnight in 4% paraformal-dehyde followed by 0.1 M phosphate buffer (pH 6.8) for 24–48 hbefore visualization under a fluorescent microscope (Zeiss) (data notshown).

A 10-min stable baseline was established for each pyramidalneuron. Inhibitory postsynaptic currents (IPSCs) and inhibitory post-synaptic potentials (IPSPs) were isolated by the addition of 10 �MNBQX and 10 �M AP-5 to block glutamatergic receptors. FRM-17848 (with or without FRM-35440) or donepezil was applied for 10min followed by compound washout for 20 min. In control experi-ments, 50 nM MLA alone or MLA � FRM-17848 was perfused overslices after isolation of IPSCs/IPSPs. A final 5-min application of 10or 20 �M bicuculline (as indicated), a GABAA receptor antagonist,was used to demonstrate that the remaining recorded currents were

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GABAergic. Compounds were administered to the slices by bathperfusion from 50-ml syringes arranged in series with the mainperfusion line from the aCSF reservoir via three-way valves. Maxi-mum final DMSO concentration was 0.3%.

Profiling FRM-35440 selectivity. The activity of FRM-35440, aGABAA �5R inhibitor, was tested at ChanTest/Charles River (Cleve-land, OH) using HEK-293 cells expressing GABAA �1-, �2-, �3-, �4-,or �5-subunits with �3/�2-subunits using an IonWorks Barracudasystem (Molecular Devices). The intracellular solution contained thefollowing (in mM): 50 CsCl, 90 CsF, 5 MgCl2, 1 EGTA, and 10HEPES, pH adjusted to 7.2 with CsOH. In preparation for a recordingsession, the intracellular solution was loaded into the intracellularcompartment of a planar patch-clamp electrode. The extracellularsolution contained the following (in mM): 137 NaCl, 4.0 KCl, 1.8CaCl2, 1 MgCl2, 10 HEPES, and 10 D-glucose, pH adjusted to 7.4with NaOH. Two recordings (scans) were performed. First the testarticle was added to measure agonist effects. Five minutes later, asecond scan was recorded during the application of GABA (30 �M �EC90) to detect antagonism. Percent inhibition was calculated as (1 �CurrentFRM-35440/CurrentGABA EC90) � 100%.

Analysis. Data were captured online and stored on a computerrunning pCLAMP data acquisition software for later offline analysis.Analysis of all data was carried out using Clampfit (MolecularDevices) and Microsoft Excel software. IPSP/IPSC frequencies werecalculated from 3-min continuous recordings. LTP data are expressedas the mean � SE. Statistical analysis was performed using ANOVAfollowed by Dunnett’s multiple comparisons test. For studies withsequential treatments, a repeated-measures ANOVA was applied tothe data followed by a Bonferroni multiple comparisons test (PRISM6, GraphPad Software, San Diego, CA) with P 0.05 taken toindicate statistical significance. All other data are expressed as themean � SD.

RESULTS

FRM-17848 enhances LTP in a bell-shaped concentration-response curve. In Stoiljkovic et al. (2015), it was establishedthat the �7-nAChR agonist FRM-17874 enhanced hippocampalLTP at concentrations below the Ki for displacement of �-bun-garotoxin. A similar compound FRM-17848 (Ki 11 nM forrat �7-nAChR, EC50 455 nM) was tested over a wider rangeof concentrations for effects on LTP in rat septo-hippocampalslices. As shown in Fig. 1, A and C, 3.16 nM FRM-17848induced a significant enhancement of LTP, measured duringthe 50- to 60-min interval following theta-burst stimulation(TBS), compared with vehicle-treated slices. The activity ofthis single concentration was confirmed in an additional study(see Fig. 6D), and no other tested concentration significantlyenhanced LTP compared with control. This narrow efficaciouswindow is consistent the results in Stoiljkovic et al. (2015) thatshowed a similar molecule (FRM-17874) to be active only at3.2 and 5 nM (Tang et al. 2014).

The AChE inhibitor donepezil was used as a positive controlfor enhancement of LTP (Fig. 1, B and C) (Kapai et al. 2012).At 500 nM, donepezil showed a significant enhancement ofLTP that was comparable to 3.16 nM FRM-17848. Neithercompound had an effect on baseline (untetanized) field excit-atory postsynaptic potential (fEPSP) amplitude (data notshown). The 3.16-nM concentration of FRM-17848 is compa-rable to the free drug levels of similar �7-nAChR agonists suchas FRM-17848 and EVP-6124, which have proven benefits oncognition (Keefe et al. 2015; Prickaerts et al. 2012; Stoiljkovicet al. 2015).

To confirm that the enhancement of LTP by FRM-17848was mediated by �7-nAChR, experiments were repeated withthe selective �7-nAChR antagonist MLA. At a concentration of100 nM MLA, the effect of 3.16 nM FRM-17848 was fullyinhibited (Fig. 1, D and E). This result confirmed that theenhancement of LTP by 3.16 nM FRM-17848 was throughactivation of �7-nAChRs rather than desensitization.

3.16 nM FRM-17848 primes �7-nAChRs. To examine theeffects of 3.16 nM FRM-17848 on �7-receptor pharmacology,whole cell recordings were made from Xenopus oocytes ex-pressing human �7-nAChRs. The addition of 3.16 nM FRM-17848 to the perfusion enhanced the �7-nAChR response to thesubsequent application of 40 �M ACh (Fig. 2, A and B). Thisphenomenon described as priming in Stoiljkovic et al. (2015)establishes a concentration-response function that is proven tobe highly predictive of �7-nAChR agonist efficacy in LTP andcognition (Prickaerts et al. 2012). Thus LTP in rat hippocampalslices is enhanced at a concentration of FRM-17848 thatprimes �7-nAChRs.

FRM-17848 hyperpolarizes hippocampal CA1 pyramidalneurons. To understand the effects of FRM-17848 on cellularphysiology, whole cell recordings were made from CA1 pyra-midal cells in septo-hippocampal slices. For individual neu-rons, wash-in of 3.16 nM FRM-17848 did not significantlychange the input resistance from baseline (Fig. 3A). In contrast,application 3.16 nM FRM-17848 induced a small, but highlysignificant hyperpolarization of the membrane potential inmost cells (Fig. 3B). This result was initially unexpected,because in normal physiological saline, �7-nAChRs have areversal potential of �0 mV. Thus activation of �7-nAChRs onhippocampal CA1 pyramidal neurons would be expected to bedepolarizing rather than hyperpolarizing.

FRM-17848 increases GABAergic IPSCs. Based on previ-ous work showing that �7-nAChRs are predominantly ex-pressed on GABAergic interneurons in the hippocampus andour new finding that �7-nAChR activation induced a hyperpo-larization of pyramidal neurons, we recorded IPSCs frompyramidal neurons. Glutamatergic currents were inhibited withNBQX and AP-5, leaving bicuculline-sensitive IPSCs. Addi-tion of 3.16 nM FRM-17848 induced an increase in IPSCfrequency within minutes (Fig. 3C). This effect was reversedby a 20 min washout of FRM-17848 and was inhibited bycoapplication of 50 nM MLA (Fig. 3, C and D). IPSC ampli-tude was similarly affected in individual neurons upon wash-inof 3.16 FRM-17848 (Fig. 3E), as were IPSPs (data not shown).Thus a concentration of FRM-17848 that was found to primethe �7-nAChR and enhance LTP also increased IPSC fre-quency and amplitude in hippocampal slices.

The results from the LTP experiments identified a narrowconcentration-response range of FRM-17848 with 3.16 nMbeing the only active concentration. Since 5.6 nM FRM-17848was inactive in LTP, this concentration was tested for itseffects on spontaneous IPSCs. As shown in Fig. 3, F and G, 5.6nM FRM-17848 had no effect on the frequency (or amplitude,data not shown) of spontaneous IPSCs. Therefore, 3.16 nMFRM-17848 was found to enhance both LTP and increasespontaneous IPSC frequency, while 5.6 nM FRM-17848 af-fected neither.

Donepezil increases GABAergic IPSCs. Observing that thesame concentration of FRM-17848 increased LTP and IPSCfrequency, we considered whether the correlation would

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also apply to donepezil. To test this hypothesis, IPSCfrequency was measured in response to ascending concen-trations of donepezil (50 –500 nM, Fig. 4A). There was atrend to increased IPSC frequency beginning at 50 nMdonepezil. However, only 500 nM donepezil, the sameconcentration that significantly enhanced LTP, showed astatistically significant increase in IPSC frequency (Fig. 4B).These data demonstrate that two distinct cognition-enhanc-ing cholinergic mechanisms (AChE inhibitor and �7-nAChR

agonist) each modulate IPSC frequency at the same concen-trations that enhance LTP.

Increased GABA IPSCs modestly hyperpolarize pyramidalneurons. To determine the consequences of increased IPSCactivity on the hippocampal circuitry, current-clamp recordingswere used to investigate the effects of FRM-17848 on the firingproperties of pyramidal neurons. A current-step protocol wasapplied to induce action potential firing (Fig. 5). Wash-in of3.16 nM FRM-17848 reduced the firing activity triggered by a

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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 slicestreated 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: eachpoint 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 minpost-TBS, both 3.16 nM FRM-17848 and 500 nM donepezil significantly increased LTP over vehicle (vehicle 129 � 4% SE, n 16; 3.16 nM FRM-17848 148 � 4% SE, n 11; and 500 nM donepezil 145 � 6% SE, n 7; ANOVA, Dunnett’s multiple comparison test compared with vehicle, *P 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 LTPby 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 ofbaseline (vehicle 129 � 4% SE, n 16; 50 nM MLA 132 � 5% SE, n 8; 3.16 nM FRM-17848 148 � 4% SE, n 11; FRM-17848 � 50 nMMLA 137 � 3% SE, n 8; FRM-17848 � 100 nM MLA 131 � 7% SE, n 6; ANOVA, Dunnett’s multiple comparison test for vehicle vs. 3.16 nMFRM-17848, *P 0.05, and 3.16 nM FRM-17848 vs. 100 nM MLA � 3.16 nM FRM-17848, #P 0.05).



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depolarizing current injection, and the effect could be reversedupon washout of compound. The �7-nAChR antagonist MLAhad the opposite effect, eliciting an increase in spike activity inresponse to a depolarizing current step. Importantly, 50 nMMLA � 3.16 nM FRM-17848 exhibited the same effect asMLA alone. GABAergic inhibitory neurons are known to exerta tonic hyperpolarizing effect on pyramidal cells (Caraiscos etal. 2004). These results support the conclusion that by increas-ing IPSC activity, 3.16 nM FRM-17848 induces a modesthyperpolarization of the resting membrane potential, therebymaking pyramidal neurons less excitable.

GABAA �5R mediation of �7-nAChR agonist effects. Wethen considered whether the effects of an �7-nAChR agonist onGABAergic synaptic activity may be directly linked to theenhancement of LTP. This hypothesis appears counterintuitive,since FRM-17848 induced a hyperpolarization of pyramidalneurons and reduced excitability. It is well known that benzo-diazepines, which are nonselective PAMs of GABAA recep-tors, inhibit LTP and are sedatives in humans (del Cerro et al.

1992). Therefore, as a procognitive agent that increasesGABAergic activity, �7-nAChR agonists could not simplymimic the activity of benzodiazepines and nonspecificallyincrease GABAergic activity. Previous work has demonstratedthat selective modulators of GABAA �5Rs can be procognitive(Drexler et al. 2013; Johnstone et al. 2011; Koh et al. 2013).Therefore we postulated that �7-nAChR agonists enhance LTPby activating a subtype of GABAergic interneuron or receptorin the neural circuit.

To test this, we identified a hydroxypropylthio- variant ofMRK-536 (herein called FRM-35440) that was reported to actas a highly selective and potent partial agonist at GABAA �5Rs(Ki 2 nM). As a partial agonist, FRM-35440 suppresses thefull activation of the receptor by GABA (Atack 2011). Mea-suring Cl� currents in HEK cells overexpressing GABAA �1–5,FRM-35440 functions as a selective antagonist of the GABAA�5R under these conditions (Fig. 6, A and B). FRM-35440showed no binding activity at �7-nAChRs (IC50 � 10 �M, datanot shown). When tested in LTP experiments, 50 nM FRM-

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Fig. 2. 3.16 nM FRM-17848 can prime �7-nAChRs. Electrophysiological recordingswere made from Xenopus oocytes overex-pressing human �7-nAChRs. A: sample tracefrom a single oocyte, shows the response to40 �M ACh before and during the coappli-cation of the �7-nAChR agonist (3.16 nMFRM-17848). Cells were pulsed with 40 �MACh for 5 s at 2- or 10-min intervals. B: thehistogram shows the average response from4 oocytes. A stable baseline was establishedwith 4 pulses of 40 �M ACh (A). Addition of3.16 nM FRM-17848 to the bath enhancedthe ACh-evoked currents recorded at 2-minintervals (3 currents, B) and then 10-minintervals (2 currents, C and D). Finally,FRM-17848 was removed from the bath andan additional 3 ACh-evoked currents wererecorded at 2-min intervals (E). The additionof FRM-17848 increased the response toACh, enhancing the peak ACh-evoked cur-rent [40 �M ACh (A) 1.0 � 0.06; 40 �MACh � 3.16 nM FRM-17848 (B) 1.63 �0.22; 40 �M ACh � 3.16 nM FRM-17848 �10 min (C) 1.86 � 0.32; 40 �M ACh �3.16 nM FRM-17848 � 20 min (D) 2.16 � 0.30; 40 �M ACh washout (E) 1.74 � 0.19; repeated-measures ANOVA,Bonferroni’s multiple comparison test, alltreatments were significantly different from40 �M ACh (A; P 0.01)]. The primingeffect persisted for a short interval after re-moval of FRM-17848 from the bath.



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35440 alone had no effect on LTP (Fig. 6C). Thus the activityof FRM-35440 at the GABAA �5R did not disrupt the basicmechanism of TBS-induced potentiation. As in Fig. 1, 3.16 nMFRM-17848 was again found to enhance LTP. Strikingly, thecoapplication of FRM-17848 with 50 nM FRM-35440 fullyinhibited the increase in LTP by 3.16 nM FRM-17848, while alower concentration of FRM-35440 (5 nM) partially inhibited(Fig. 6, D and E).

Based on this result, we predicted that GABAA �5R inhibi-tion by FRM-35440 should also disrupt the effect of 3.16 nM

FRM-17848 on IPSC frequency. As shown in Fig. 7, A and B,treatment with 50 nM FRM-35440 modestly suppressed thefrequency of spontaneous IPSCs recorded in CA1 pyramidalneurons. Maintenance of the majority of IPSCs is consistentwith the prevalence of other GABAAR subtypes, such asGABAA �1R in this circuit, that remain active in the presenceof the GABAA �5R subtype-specific FRM-35440. Subsequentapplication of 3.16 nM FRM-17848, had no effect on IPSCfrequency. Washout of both compounds restored IPSC fre-quency to baseline. FRM-35440 also fully inhibited the in-

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crease of IPSC amplitude by 3.16 nM FRM-17848 (Fig. 7C)without affecting total IPSC amplitude. These data indicatethat activating �7-nAChRs at priming concentrations of FRM-17848 causes an enhancement of LTP and IPSC frequency andthat this effect can be reversed by coapplication of a GABAA�5R inhibitor.

Since FRM-35440 suppressed the effects of the �7-nAChRagonist FRM-17848, we tested whether a selective GABAA�5R PAM (SH-053-2’F-R-CH3) would be sufficient to en-hance LTP. A concentration of 250 nM was chosen based onthe reported selectivity of SH-053-2’F-R-CH3 at GABAA�5Rs (Savíc et al. 2010). As shown in Fig. 8A, a 250-nMconcentration of SH-053-2’F-R-CH3 significantly enhancedLTP measured during the 50- to 60-min interval post-TBS.This effect was concentration dependent (Fig. 8B). These datademonstrate that potentiating the activity of the GABAA �5Rsin this hippocampal circuit is sufficient to promote synapticplasticity.

DISCUSSION

Accumulating evidence suggests that selective activation of�7-nAChRs may be an effective therapy for enhancing cogni-tive function in patients with schizophrenia and AD. Previousreports have demonstrated that �7-nAChR agonists can en-hance LTP in rodent brain slices with a bell-shaped concen-tration-response relationship and a narrow effective concentra-tion range (Kroker et al. 2011; Lagostena et al. 2008). Theeffective concentration range for LTP was found to be similarto the binding Ki at �7-nAChRs.

In this report we have studied the effects of an �7-nAChRagonist on the basic hippocampal neuronal physiology andneuroplasticity to elucidate a possible mechanism of actionunderlying the procognitive effects of �7-nAChR agonists.Using concentrations of an �7-nAChR agonist (FRM-17848) (FRM-2 in Tang et al. 2014), well below the agonistEC50 (455 nM) and approximately a half-log below thebinding Ki (11 nM), but consistent with ACh primingconcentration (3.16 nM), we now show enhanced LTP in ratbrain slices in a narrow concentration range. The percentincrease in LTP after FRM-17848 treatment was comparable

to that for donepezil tested at concentrations that are likelyto exceed the unbound concentration achievable in patients(5–10 nM) (Gomolin et al. 2011; Seltzer 2005; Yang2013a). In addition to being an agonist at the �7-nAChR,FRM-17848 is also a potent 5-HT3 receptor antagonist(binding Ki 35 nM and cellular IC50 in oocytes 9 nM).Since the enhancement of LTP with FRM-17848 was com-pletely blocked by MLA, a highly specific �7-nAChR an-tagonist, it is reasonable to postulate that activation of�7-nAChRs is mediating the observed effects (Palma et al.1996). Although other classes of 5-HT3 antagonists canenhance hippocampal LTP, they also reduce GABAergicactivity in hippocampal interneurons (Dale et al. 2014;Reznic and Staubli 1997). On the contrary, 3.16 nM FRM-17848 increased GABAergic activity, suggesting that at thisconcentration FRM-17848 activity at �7-nAChRs predomi-nated over activity at 5-HT3 receptors. In addition theenhancement of LTP was lost when the concentration ofFRM-17848 was increased from 3.16 nM to 5.6 nM, whichis inconsistent with a 5-HT3 antagonism hypothesis.

The observation that FRM-17848 causes a hyperpolariza-tion, rather than depolarization, of CA1 pyramidal neuronsled us to investigate the effects of FRM-17848 on sponta-neous IPSCs. IPSC frequency increased in response toFRM-17848, as might be expected based on the predomi-nant expression of �7-nAChRs on GABAergic interneurons(Fabian-Fine et al. 2001; Frazier et al. 1998; Freedman et al.1993; Jones 1997; Kawai et al. 2002). These results areconsistent with previous findings that showed that �7-nAChR agonists increase the synaptic transmission ofGABAergic interneurons at or near the binding Ki (PNU-282987 Ki 26 nM and FRM-17874 Ki 4.6 nM for rat�7-nAChRs) (Hajos et al. 2005; Stoiljkovic et al. 2015).Similarly, an �7-nAChR PAM, PNU-120596, increasedIPSC frequency near the EC50 for potentiating ACh-evokedresponses (Hurst et al. 2005).

One potential explanation for the finding that increases inboth LTP and IPSC frequency occurred at concentrations nearthe binding Ki values of �7-nAChR agonists is the phenome-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 individualCA1 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) �5.4 � 14.9 M�, repeated-measures ANOVA, Bonferroni’s multiple comparison test, P 0.14, n 18]. B: the membranepotential 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 0.01, n 38]. This hyperpolarization was not observed with 5.6 nM FRM-17848(�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 notshown). C: spontaneous IPSC activity was recorded by inhibiting glutamatergic currents with 10 �M AP-5 and 10 �M NBQX. Three-minute time blocks areshown from a pyramidal neuron excerpted from a continuous recording. Addition of 3.16 nM FRM-17848 to the perfusion resulted in an increase in IPSCfrequency 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.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 withcompounds. 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 8; 3.16 nM FRM-17848 0.70 Hz � 0.15; wash 0.41 Hz � 0.06; repeated-measures ANOVA, Bonferroni’s multiple comparison test, *P 0.05 vs.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 � 3.16 nM FRM-17848 0.46 Hz � 0.09, n 3; repeated-measures ANOVA, Bonferroni’s multiple comparison test, P 0.97). Inhibitorypostsynaptic potentials (IPSPs) frequency was similarly affected by FRM-17848 (baseline 0.17 Hz � 0.04; 3.16 nM FRM-17848 0.27 Hz � 0.06; wash 0.19Hz � 0.05, n 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 43.9 pA � 26.8, n 8; 3.16 nM FRM-17848 95.6 pA � 67.4; wash 53.3 pA � 33.9; repeated-measures ANOVA, Bonferroni’s multiple comparison test,*P 0.05 vs. baseline; 50 nM MLA 36.7 pA � 26.0; 50 nM MLA � 3.16 nM FRM-17848 37.6 pA � 24.6, n 3; repeated-measures ANOVA,Bonferroni’s multiple comparison test P 0.71). F: a sample recording of IPSCs from an individual neuron reveal that application of a higher concentrationof 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 uponaddition and washout of 5.6 nM FRM-17848 (baseline 0.31 Hz � 0.14; 5.6 nM FRM-17848 0.29 Hz � 0.14; wash 0.33 Hz � 0.13; n 8;repeated-measures ANOVA, Bonferroni’s multiple comparison test, P 0.19).



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which proposes that binding of a single molecule of an agonistsuch as FRM-17848 at an orthosteric binding site primes thereceptor to open in response to a second binding event of onemolecule of an endogenous ligand such as ACh. The correlationamong priming concentrations, enhanced LTP, and increasedIPSC frequency is significant because they align with the concen-trations for improving performance in cognition and memory-related tasks in animal models (Stoiljkovic et al. 2015).

Our experiments demonstrate that FRM-17848 exhibits anarrow, bell-shaped concentration-response function. Whilethis narrow efficacy concentration range for FRM-17848 maybe prohibitive for the development of this compound into acommercial drug, understanding the biological parameters thatset the upper and lower boundaries may be useful in develop-ing other molecules that widen the concentration-response

function. In the canonical model of nAChR activation, at leasttwo molecules of ACh are required for activation of thereceptor (Changeux 1992). Without rapid clearance of theligand, �7-nAChRs quickly desensitize (Dani and Bertrand2007). Similarly, at higher concentrations of FRM-17848, twomolecules could bind, activate, and desensitize the �7-nAChRin the absence of ACh. We have reported that a similarcompound, FRM-17874, enhances LTP at 3.16 and 5 nM butnot at 10 nM (Stoiljkovic et al. 2015). Thus our model wouldpredict a narrow concentration-response function betweenpriming a sufficient number of receptors to observe efficacyand desensitizing them. Designing �7-nAChR agonists thatshow reduced desensitization or exhibit long a half-life (T½)may be desirable to maintain drug levels in an efficaciousconcentration range.

B

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Fig. 4. Donepezil increases IPSC frequency at concentrationsthat enhanced LTP. Pyramidal neurons were voltage clampedand IPSCs were isolated with 10 �M NBQX and 10 �M AP-5.A: sequential recordings from an individual pyramidal neuronshow that step increases in donepezil concentration cause anincrease in spontaneous IPSC frequency. Washout of donepe-zil restored the IPSC frequency to baseline and bicucullineeliminated the currents confirming their GABAergic origin(Vh �50 mV). B: the scatterplot depicts the IPSC frequencyaveraged over 3 min for 9 cells as each was treated withascending concentrations of donepezil. Only 500 nM donepe-zil produced a statistically significant increase in IPSC fre-quency compared with baseline (baseline 0.21 Hz � 0.11;50 nM donepezil 0.32 Hz � 0.21; 100 nM donepezil 0.34Hz � 0.24; 250 nM donepezil 0.33 Hz � 0.19; 500 nMdonepezil 0.43 Hz � 0.29; wash 0.21 Hz � 0.13; n 9;repeated-measures ANOVA, Bonferroni’s multiple compari-son test, *P 0.05 vs. baseline). Two of the recorded cellsshowed no change in IPSC frequency in response to anyconcentration of donepezil.



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An alternative hypothesis could explain the bell-shapedconcentration-response function using a GABAergic circuitmechanism rather than a receptor-level mechanism. By thisreasoning, low concentrations of FRM-17848 would induce amodest increase in GABAergic activity. By moderately hyper-polarizing pyramidal neurons, spontaneous background activ-ity in the hippocampus would be dampened, thereby enhancingthe contrast of burst signal-to-noise. However, at higher FRM-17848 concentrations, a further increase in GABAergic activitywould hyperpolarize pyramidal neurons to the point of disrupt-ing neurotransmission. A similar phenomenon has been shownin the frontal cortex, where �7-nAChRs are localized onglutamatergic neurons, escalating concentrations of an �7-

nAChR agonist (PHA543613) continue to increase glutamaterelease, yet the procognitive effect diminished (Yang 2013b),suggesting that higher concentrations of �7-nAChR agonistsdisrupt neuronal network functioning before �7-nAChRs be-come desensitized.

However, our data do not support the latter hypothesis forthis hippocampal circuit. First, we show that 500 nM donepezilexhibited a comparable effect on LTP as FRM-17848, yet 500nM donepezil doubled the IPSC frequency (spontaneous IPSCfrequency with 500 nM donepezil 204% of baseline) incontrast to FRM-17848’s more modest increase (spontaneousIPSC frequency with 3.16 nM FRM-17848 146% of base-line). Therefore, further increasing the IPSC frequency beyondthat achieved with 3.16 nM FRM-17848 is not consistent withsuppression of plasticity in this circuit. Second, we showed that5.6 nM FRM-17848 was ineffective at enhancing LTP and thatIPSC frequency was lower (not higher) than with 3.16 nMFRM-17848. The receptor-level model can account for this if3.16 nM FRM-17848 is optimal for priming the �7-nAChR inthe presence of ACh, while 5.6 nM begins to desensitize it.These data are consistent with previous studies using other�7-nAChR agonist/PAM compounds, which also showed nar-row active concentration ranges (Hajos et al. 2005; Hurst et al.2005; Kroker et al. 2011; Lagostena et al. 2008). Together,these studies suggest that receptor desensitization rather thanoveractivation of the GABAergic circuit may set the upperboundary of the concentration response curve in the hippocam-pus.

Our data indicate that the modulation of GABA is directlylinked to the enhanced LTP, because the GABAA �5R inhibitorblocked the LTP-enhancing effects of an �7-nAChR agonistwithout affecting baseline LTP. These results provide directpharmacological evidence that activation of �7-nAChRs en-hances the activity of a specific subtype of GABAergic syn-apse, which in turn modulates synaptic plasticity in the hip-pocampus. Further confirmation could be established by testing�7-nAChR agonists in brain slices derived from GABAA �5Rknockout mice. There remains some controversy regarding thesynaptic vs. extrasynaptic localization of GABAA �5Rs in thehippocampus. Our results with FRM-35440 in Fig. 7B areconsistent with the existence of a synaptic pool GABAA �5Rsas has been shown directly by electron microscopy (Serwanskiet al. 2006). The data reported here provide a testable frame-work for a “procognitive circuit” that may contribute to theunderstanding of cognitive enhancement throughout the brain.

While this report has focused on the effects of FRM-17848on the GABAergic circuit and its impact on synaptic plasticity,there may be effects on glutamatergic currents as well (Gu etal. 2012). In Fig. 2 we show that FRM-17848 and donepezilhave no effect on baseline fEPSPs, which is consistent with aprevious report (Lagostena et al. 2008). In a separate report(manuscript in preparation) we looked at the effects of FRM-17848 on evoked glutamatergic EPSCs and found no enhance-ment. Our data therefore support the conclusion that the pri-mary effect of FRM-17848 is on GABAergic rather thanglutamatergic neurotransmission in this hippocampal circuit.

At first glance, it is unexpected that an increase in GABAergicactivity would be associated with enhanced LTP. NonselectiveGABAA receptor PAMs such as benzodiazepines are known tosuppress LTP (del Cerro et al. 1992) and are associated withamnesia in humans (Brown and Dundee 1968; Clarke et al.

50 mV

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Fig. 5. Pyramidal neurons are less excitable upon application of 3.16 nMFRM-17848. Whole cell current-clamp recordings were made from CA1pyramidal neurons. Based on the salt concentrations in the electrode andartificial cerebrospinal fluid (aCSF), the chloride reversal potential was pre-dicted to be �63 mV. In a sample recording, depolarizing square wave currentinjection in pyramidal neurons induced a series of accommodating actionpotentials. Perfusion with 3.16 nM FRM-17848 hyperpolarized the restingmembrane potential (Vm) and reduced action potential firing, which wasrestored after a 10 min washout period (baseline 5.7 � 3.6 Hz; 3.16 nMFRM-17848 3.9 � 3.3 Hz; wash 4.9 � 3.5 Hz; n 11; repeated-measures ANOVA, Bonferroni’s multiple comparison test baseline vs. 3.16nM FRM-17848, *P 0.05). Addition of 50 nM MLA increased actionpotential frequency which was unaffected by coapplication of 3.16 nM FRM-17848.



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1970). Moreover, the increase in IPSC activity with 3.16 nMFRM-17848 clearly coincided with a modest hyperpolarizationin pyramidal neurons. We observed that the addition of bicu-culline caused a depolarization in pyramidal neurons, indicat-ing that GABA contributes significantly to the hyperpolarizedresting membrane potential (data not shown). We have dem-onstrated that the hyperpolarization of pyramidal neuronscaused by the increase in GABAergic activity with FRM-17848 leads to reduced firing of spontaneous action potentials

in response to a depolarizing current step. Mechanistically,there are many potential circuit-based explanations such asfeed forward inhibition (Larson and Lynch 1986) or reboundafter-hyperpolarization (Aizenman et al. 1998; Sah and Bek-kers 1996) that may account for this apparent paradox. In arecent set of experiments it was shown in cortical circuits thatwhile a nonselective GABAA receptor PAM such as diazepamdecreased the discharge rate of neocortical neurons during upstates (active firing), a GABAA �5R PAM such as SH-053-

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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 andpotent 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 thatFRM-35440 concentration-dependently inhibited the evoked GABAA �5R current (IC50 158 nM) under these conditions. The activity of FRM-35440 may bemore 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 inhibitorFRM-35440 alone had no effect on TBS-induced LTP (control 134 � 4% SE, n 7, 50 nM FRM-35440 133 � 6% SE, n 4). D: coapplication ofFRM-35440 prevented the enhancement of LTP induced by 3.16 nM FRM-17848 in a concentration-dependent manner. E: each circle reports the LTP (averagefEPSP amplitude from 50–60 min post-TBS) recorded from an individual slice. FRM-35440 inhibited the LTP-enhancing effects of FRM-17848 (control 134 �4% SE, n 7, 3.16 nM FRM-17848 151 � 6% SE, n 6; 3.16 nM FRM-17848 � 5 nM FRM-35440 143 � 3% SE, n 8; 3.16 nM FRM-17848 � 50 nMFRM-35440 132 � 4% SE, n 8; 50 nM FRM-35440 133 � 6%, n 4; ANOVA, Dunnett’s multiple comparison test, *P 0.05 for 3.16 nM FRM-17848vs. vehicle, #P 0.05 for 3.16 nM FRM-17848 vs. 3.16 nM FRM-17848 � 50 nM FRM-35440).



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2’F-R-CH3 had the opposite effect by increasing burstingactivity (Drexler et al. 2013). Some additional recent work byKoh et al. (2013) has demonstrated that GABAA �5R PAMsare also procognitive in rats. We favor the possibility that�7-nAChR agonists may mimic this type of activation of asubset of GABAergic synapses in the circuit, thereby enhanc-ing bursting activity of glutamatergic neurons. This wouldhave the anticipated effect of improving signal-to-noise in thehippocampal circuit, which may be relevant to cognition (Lis-man 1997). While additional studies are needed to test thishypothesis, the data reported here support a convergence of thecholinergic (both �7-nAChR activation and AChE inhibition)

*

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Fig. 8. SH-053-2’F-R-CH3, a selective GABAA �5R PAM, enhanced LTP insepto-hippocampal brain slices. A: field potential recordings were made in CA1of rat septo-hippocampus slices. Addition of 250 nM SH-053-2’F-R-CH3enhanced LTP similar to FRM-17848. B: the scatterplot depicts the fEPSPamplitude averaged from the 50–60 min post-TBS interval from individualslices. SH-053-2’F-R-CH3 exhibits a concentration-dependent enhancement ofLTP (vehicle 123 � 4% SE, n 7; 50 nM SH-053-2’F-R-CH3 121 �4% SE, n 6; 100 nM SH-053-2’F-R-CH3 129 � 5% SE, n 6; 250 nMSH-053-2’F-R-CH3 135 � 4% SE, n 7; Dunnett’s multiple comparisonstest compared with vehicle, *P 0.05).

50 nM FRM-35440

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Fig. 7. The GABAA �5R inhibitor (FRM-35440) prevents the increase in IPSCfrequency caused by the �7-nAChR agonist FRM-17848. A: a sample trace from anindividual pyramidal neuron, shows that preapplication of FRM-35440 blocked theincrease in IPSC frequency induced by the subsequent addition of FRM-17848. B:quantification of the IPSC frequency in 5 pyramidal neurons demonstrates that theGABAA �5R inhibitor FRM-35440 modestly reduced IPSC frequency, and preventsthe increase in IPSC frequency that occurs with 3.16 nM FRM-17848 alone. Washoutof both compounds restored IPSC frequency to baseline levels (baseline 0.25 � 0.07Hz; 50 nM FRM-35440 0.20 � 0.07 Hz; 50 nM FRM-35440 � 3.16 nMFRM-17848 0.21 � 0.08 Hz; washout 0.25 � 0.09 Hz, n 5; repeated-measuresANOVA, Bonferroni’s multiple comparisons test, *P 0.05 vs. baseline). C: a similarresult was observed with IPSC amplitude (baseline 30.3 � 12.6 pA; 50 nMFRM-35440 � 3.16 nM FRM-17848 30.9 � 12.0 pA; washout 31.2 � 12.0 pA,n 5; repeated-measures ANOVA, Bonferroni’s multiple comparisons test, P �0.05).



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and GABAergic systems on regulating neuronal plasticity inthe hippocampus.

In conclusion, we report that an �7-nAChR agonist en-hanced LTP in the hippocampus of rat brain slices in a narrowconcentration range consistent with a concentration that primesthe receptor. The addition of FRM-17848 stimulated an in-crease in GABAergic activity among a subset of GABAergicsynapses. The coapplication of a GABAA �5R inhibitorblocked the effects of FRM-17848 on LTP and IPSC fre-quency. We conclude that the �7-nAChR agonists promote theactivity of a subset of GABAergic synapses as part of acognition circuit in the hippocampus. These studies help toelucidate the mechanism of action by which �7-nAChR ago-nists produce their procognitive activity and demonstrate theirpotential utility in treating cognitive impairments in schizo-phrenia and AD.

ACKNOWLEDGMENTS

We thank Raymond Hurst of FORUM Pharmaceuticals for reviewingand discussing the data sets. We are grateful to Daniel Bertrand, SoniaBertrand, and Fabrice Marger of HiQScreen Sarl, Geneva, Switzerland forperforming the oocyte experiments, which were part of a fee-for-servicecontract. We also appreciate the contributions of Yuri Kuryshev ofChanTest for performing the patch recordings on HEK cells overexpressingGABA receptor subtypes. Editorial assistance was provided by HannahLederman (Caudex, New York, NY).

GRANTS

This work was funded by FORUM Pharmaceuticals.

DISCLOSURES

M. Townsend, D. G. Flood, L. Leventhal, H. Patzke, and G. Koenig wereemployees of FORUM Pharmaceuticals, a privately owned corporation. A.Whyment, J.-S. Walczak, R. Jeggo, and M. van den Top are employees ofCerebrasol, which was contracted to perform electrophysiology experiments.

AUTHOR CONTRIBUTIONS

M.T., L.L., and G.K. conception and design of research; M.T., A.W., andR.J. analyzed data; M.T., D.G.F., H.P., and G.K. interpreted results of exper-iments; M.T. prepared figures; M.T. and D.G.F. drafted manuscript; M.T.,D.G.F., H.P., and G.K. edited and revised manuscript; M.T., A.W., J.-S.W.,R.J., M.v.d.T., D.G.F., L.L., H.P., and G.K. approved final version of manu-script; A.W., J.-S.W., and M.v.d.T. performed experiments.

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α7-nAChR agonist enhances neural plasticity in the ......2016/03/21 · agonist (PNU-282987) increased synaptic GABAergic activity in hippocampal slices (Hajos et al. 2005) by enhancing