Neuron

Supplemental Information

Biased mGlu5-Positive Allosteric Modulators

Provide In Vivo Efficacy without Potentiating

mGlu5 Modulation of NMDAR Currents

Jerri M. Rook, Zixiu Xiang, Xiaohui Lv, Ayan Ghoshal, Jonathan W. Dickerson, Thomas

M. Bridges, Kari A. Johnson, Daniel J. Foster, Karen J. Gregory, Paige N. Vinson,

Analisa D. Thompson, Nellie Byun, Rebekah L. Collier, Michael Bubser, Michael T.

Nedelcovych, Robert W. Gould, Shaun R. Stauffer, J. Scott Daniels, Colleen M.

Niswender, Hilde Lavreysen, Claire Mackie, Susana Conde-Ceide, Jesus Alcazar, José

M. Bartolomé-Nebreda, Gregor J. Macdonald, John C. Talpos, Thomas Steckler, Carrie

K. Jones, Craig W. Lindsley, and P. Jeffrey Conn

Supplemental Figures & Tables

Figure S1, Related to Figure 1. Structure activity relationship and chemical structure of VU0409551, 5-[(4-Fluorophenyl)carbonyl]-2-(phenoxymethyl)-4,5,6,7-tetrahydro[1,3]oxazolo[5,4-c]pyridine.

1

Figure S2, Related to Figure 2, 4. Application of VU0409551 alone does not lead to LTD in hippocampal

SC-CA1 synapses. (A) Bath application of 10 µM VU0409551 for 30 minutes (gray circle, solid line) resulted in a transient depression of the fEPSP slope (n = 5) which readily came back to baseline levels by 10 min after the compound was washed out. Inset shows representative fEPSP traces for baseline (1); 25 minutes after VU0409551 application (2) and 10 min after compound washout (3). (B) Quantification of the change in fEPSP slope measured 25 minutes after VU0409551 application and 10 min after compound washout. 10 µM VU0409551 led to a significant transient depression during compound add. The change in fEPSP slope 10 min after compound washout was not significantly different from baseline. Error bars represent S.E.M. ** P < 0.01, when compared to baseline.

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Figure S3, Related to Figure 3. VU0409551 has no effects on DHPG-induced depolarization and increase in NMDAR-mediated response in CA1 pyramidal cells under current clamp condition and has no effects on NMDAR-fEPSPs at the SC-CA1 synapse. (A-B) Sample traces of membrane potential responses to puff application of NMDA through patch pipettes positioned near the recorded CA1 pyramidal cells in control and during application of 3 µM DHPG (A); in control, during application of 10 µM VU0409551 and co-application of 10µM VU0409551 with 3 µM DHPG. DHPG was applied for 5 min; VU0409551 was applied for 5 min followed by co-application of VU0409551 and DHPG for 5 min. (C-D) Bar graphs summarizing the effects of DHPG, VU0409551 and a combination of DHPG and VU0409551 on NMDA-induced response (C; 286.5 ± 43.0% of baseline in 3µM DHPG, n = 6, compared with 271.9 ± 49.9% of baseline in 10 µM VU0409551 and 3µM DHPG, n = 5, p > 0.5; 112.0 ± 12.9% of baseline in 10 µM VU0409551), and membrane potential (D; change in membrane potential, Vm; 8.8 ± 2.2 mV in 3µM DHPG, n = 6, compared with 10.6 ± 2.4 mV in 10 µM VU0409551 and 3µM DHPG, n = 5, p > 0.5; 0.7 ± 0.4 mV in 10 µM VU0409551 alone). (E) Resting membrane potential of cells subject to application of 3μM DHPG and those to co-application of 10µM VU0409551 and 3 µM DHPG (-63.7 ± 0.8 mV, n = 6, compared with -64.2 ± 1.4 mV, n = 5, p > 0.5). (F) Time course of normalized NMDAR-fEPSP slope in control, during application of 30 μM VU0409551 and 50 μM AP-5 (n = 5). Inset: sample traces of NMDAR-fEPSPs recorded from the stratum radiatum of the CA1 area and evoked by electrical stimulation of the Schaffer collaterals. The experiment was carried out in the presence of 20 μM

DNQX and 20 μM bicuculline in 0.1 mM Mg2+ aCSF to block AMPA and GABAA receptors and reduce the Mg2+

block of NMDAR responses. (G) Summary of the effects of 30 μM VU0409551 (measured at last 4 min of VU0409551 application) and 50 μM AP-5 on the slope of NMDAR-fEPSPs (95.1 ± 4.2% and 2.2 ± 7.0% of baseline level, respectively; * p < 0.001).

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Figure S4, Related to Figures 5-7. VU0409551 exhibits moderate clearance from plasma (CLp; 33 mL/min/kg) with a large volume of distribution at steady-state (Vss; 9.6 L/kg) and a moderate half-life (t1/2; 3.9 hr) following a single intravenous (IV) administration (2.5 mg/kg, vehicle: 20% hydroxypropyl--cyclodextrin in water) in male Sprague Dawley rat. Data represent a single determination from n = 1 animal serially sampled.

Figure S5, Related to Figures 5-7. VU0409551 exhibits favorable oral pharmacokinetics in male Sprague

Dawley rats. Following a single 3 mg/kg administration (vehicle: 0.5% methylcellulose in water), VU0409551 reached a maximum concentration in plasma (Cmax,p) of 270 nM at a time to reach Cmax,p (Tmax,p) of 1.3 hr and provided an area-under-the-curve from 0-last (AUC0-last) of 2.9 µM*hr. Data represent mean ± SEM from n = 3 animals serially sampled.

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Figure S6, Related to Figures 6. VU0409551 demonstrates antipsychotic-like activity and precognitive

function in rodent behavioral models. (A-B) VU0409551 dose-dependently (3 – 100 mg/kg, p.o., β-cyclodextrin in water) reverses amphetamine-induced hyperlocomotion in rats with an ED50 of 5.9 mg/kg and maximum reversal of 78.1% at 100 mg/kg. Data are expressed as the mean total number of beam breaks per 5-min intervals ± S.E.M. (n = 7–8). Area under the curve from time interval t = 60 to 120 min was quantified for each treatment group and expressed as total ambulations. Percent reversal was calculated after normalizing to the vehicle + amphetamine treatment group. Comparisons of group effects relative to the vehicle + amphetamine group were completed across all treatment groups, p < 0.0001, Dunnett's test. (C) VU0409551 reverses amphetamine-induced disruptions in prepulse inhibition of the startle reflex (PPI) in rats. Amphetamine (3 mg/kg, s.c., 15 min) resulted in a significant disruption in PPI. This disruption was reversed by 56.6 and 100 mg/kg, p.o., β-cyclodextrin in water, 30 min pretreatment of VU0409551. Data are expressed as the mean percent PPI ± S.E.M. (n = 8-10). Comparisons of group effects relative to the vehicle + amphetamine group were completed across all treatment groups, *** p < 0.001, ** p < 0.01, * p < 0.05, Dunnett's test. Statistical analysis was completed using a one way analysis of variance. If significant (p < 0.05), comparison of group effects relative to the vehicle group was completed using a Dunnett’s test, *** p < 0.001, ** p < 0.01 and * p < 0.05. (D) VU0409551 (3 – 60 mg/kg, p.o., 20% cyclodextrin/Tween 80 in water) does not affect the number of total trials initiated in the delayed non-matching to position task. Data are expressed as the mean total number of completed trials ± S.E.M.

5

Figure S7, Related to Figure 7. VU0409551 provides similar maximum concentration in plasma (Cmax,p) and exposure (area-under-the-curve from 0-last [AUC0-last]) on the final day (day four; Cmax,p: 7.5 µM; AUC0-last: 95 µM*hr) of chronic once daily (QD) administration (120 mg/kg, p.o., vehicle: 75% PEG400 in water) and the first day (day one; Cmax,p: 7.0 µM; AUC0-last: 96 µM*hr) in male Sprague Dawley rats. Data represent mean ± SEM from n = 4-6 animals non-serially sampled.

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Table S1, Related to Figure 1.

Target Species %

Inhibition Target Species

% Inhibition

Adenosine A1 human 25 Histamine H2 human -9 Adenosine A2A human -10 Histamine H3 human 16 Adenosine A3 human 2 Interleukin IL-1 mouse 13 Adrenergic 1A rat 1 Leukotriene, Cysteinyl CysLT1 human 3 Adrenergic 1B rat 1 Melatonin MT1 human -6 Adrenergic 1D human -4 Muscarinic M1 human 2 Adrenergic A human 27 Muscarinic M2 human -5 Adrenergic human 6 Muscarinic M3 human -1 Adrenergic human -1 Neuropeptide Y Y1 human -5

Androgen (Testosterone) AR rat 0 Neuropeptide Y Y2 human 5 Bradykinin B1 human 9 Nicotinic Acetylcholine human -5

Bradykinin B2 human 8 Nicotinic Acetylcholine 1, Bungarotoxin

human -2

Calcium Channel L-Type, Benzothiazepine

rat 23 Opiate (OP1, DOP) human -2

Calcium Channel L-Type, Dihydropyridine

rat -2 Opiate (OP2, KOP) human 2

Calcium Channel N-Type rat -6 Opiate (OP3, MOP) human 3 Cannabinoid CB1 human 9 Phorbol Ester mouse -4

Dopamine D1 human 1 Platelet Activating Factor (PAF) human 18 Dopamine D2S human 3 Potassium Channel [KATP] human 2 Dopamine D3 human -3 Potassium Channel hERG human 2 Dopamine D4.2 human 8 Prostanoid EP4 human -2 Endothelin ETA human -2 Purinergic P2X rabbit 16 Endothelin ETB human 5 Purinergic P2Y rat 2

Epidermal Growth Factor (EGF) human -5 Rolipram rat 11

Estrogen ER human 8 Serotonin (5- Hydroxytryptamine) 5-HT1A

human 0

GABAA, Flunitrazepam, Central rat -24 Serotonin (5- Hydroxytryptamine) 5-HT2B

human 22

GABAA, Muscimol, Central rat 3 Serotonin (5- Hydroxytryptamine) 5-HT3

human -1

GABAB1A human -2 Sigma 1 human -2 Glucocorticoid human -1 Tachykinin NK1 human -10

Glutamate, Kainate rat 12 Thyroid Hormone rat 12 Glutamate, NMDA, Agonism rat 1 Transporter, Dopamine (DAT) human 6 Glutamate, NMDA, Glycine rat 5 Transporter, GABA rat 11

Glutamate, NMDA, Phencyclidine

rat 7 Transporter, Norepinephrine (NET)

human 6

Histamine H1 human 5 Transporter, Serotonin (5- Hydroxytryptamine) (SERT)

human 2

Ricerca Biosciences, LLC Lead Profiling Selectivity Screen. Radioliganding Binding assay at 10 M (n=2). No significant binding noted.

7

Table S2, Related to Figures 5-7.

Route [Dose] Property Unit Value

IV [2.5 mg/kg] CLp mL/min/kg 33

Vss L/kg 9.6

Elimination t1/2 hr 3.9

PO [3 mg/kg]

F % 63

AUC0-last µM*hr 2.9

Cmax,p µM 0.27

Tmax,p hr 1.3

PO [3-100 mg/kg] Brain:Plasma Kpa - 2.3a

Brain:Plasma Kp,uua - 1.3a

aconcentrations obtained 1.5 hr after administration; values represents mean from multiple doses in rat AHL studies; unbound concentrations based on fuplasma (0.07) and fubrain (0.04) determined in vitro

8

Supplemental Experimental Procedures

Materials

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and antibiotics were

purchased from Invitrogen (Carlsbad, CA). Astrocytes were obtained from Lonza (Basel,

Switzerland). Phosphorylated extracellular signal-regulated kinase (pERK) was detected

utilizing the ALPHAScreen-based ERK Surefire kit from Perkin-Elmer, TGR Biosciences (South

Australia, Australia). 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and dihydroxyphenylglycine

(DHPG) were obtained from Ascent Scientific (Bristol, UK) and Tocris (Ellisville, Missouri),

respectively. FluoroJade C was purchased from Millipore (Billerica, MA). 5PAM523 (Parmentier-

Batteur et al., 2013) and VU0424465 (Williams et al., 2011) were synthesized as described

previously. VU0409551 was synthesized as described in Supplemental Experimental

Procedures. Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich

(St. Louis, MO) and were either analytical or HPLC grade.

VU0409551 Synthesis

Synthetic Scheme

9

Step 1. 3-Hydroxy-4-(2-phenoxy-acetylamino)-piperidine-1-carboxylic acid ethyl ester (1).

Phenoxyacetyl chloride (19.3 mL, 139.5 mmol) was added dropwise to a stirred solution of cis-

ethyl (3S,4R)-4-amino-3-hydroxypiperidine-1-carboxylate (25 g, 132.8 mmol) and TEA (22.1 mL,

159.4 mmol) in dichloromethane (660 mL) at 0 ºC. The reaction mixture was stirred at room

temperature for 1 h and then a saturated solution of Na2CO3 was added. The organic layer was

separated, dried (Na2SO4), filtered and the solvent evaporated in vacuo. The product was

purified by flash column chromatography (7 M solution of ammonia in MeOH in DCM 0/100 to

7/93). The desired fractions were collected and evaporated in vacuo to yield 3-hydroxy-4-(2-

phenoxy-acetylamino)-piperidine-1-carboxylic acid ethyl ester (1, 38 g, 89%) as a white solid.

Step 2. 3-Oxo-4-(2-phenoxy-acetylamino)-piperidine-1-carboxylic acid ethyl ester (2).

Dess-Martin Periodinane (57.5 g, 135.5 mmol) was added to a stirred solution of 3-hydroxy-4-

(2-phenoxy-acetylamino)-piperidine-1-carboxylic acid ethyl ester (1, 38 g, 117.9 mmol) in DCM

(590 mL). The mixture was stirred at room temperature for 16 hr and the solvent was

evaporated in vacuo. The product was purified by flash column chromatography (EtOAc in DCM

0/100 to 100/0). The desired fractions were collected and evaporated in vacuo to yield 4-oxo-3-

[(phenoxyacetyl)amino]-1-piperidinecarboxylic acid 1,1-dimethylethyl ester (2, 30.6 g, 81%).

Step 3. 2-Phenoxymethyl-6,7-dihydro-4H-oxazolo[5,4-c]pyridine-5-carboxylic acid ethyl

ester (3). Phosphorus oxychloride (9.3 mL, 99.9 mmol) was added to a stirred solution of 2

(30.3 g, 90.8 mmol) in 1,4-dioxane (454 mL). The mixture was stirred at 100 ºC for 2 hr. The

mixture was cooled at 0 ºC, treated with water and extracted with EtOAc. The organic layer was

separated, dried (Na2SO4), filtered and the solvents evaporated in vacuo. The product was

purified by flash column chromatography (EtOAc in DCM 0/100 to 30/70). The desired fractions

were collected and evaporated in vacuo to yield 2-phenoxymethyl-6,7-dihydro-4H-oxazolo[5,4-

c]pyridine-5-carboxylic acid ethyl ester (3, 23.2 g, 84 %) as a colourless oil.

Step 4. 4,5,6,7-Tetrahydro-2-(phenoxymethyl)-oxazolo[5,4-c]pyridine (4).

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Lithium hydroxide (0.66 g, 27.7 mmol) was added to a stirred solution of 3 (1.77 g, 5.55 mmol)

in a mixture of H2O (5 mL) and 1,4-dioxane (15 mL) under N2. The mixture was stirred at 170 ºC

for 40 min under microwave irradiation, diluted with H2O and extracted with DCM. The organic

layer was separated, dried (Na2SO4), filtered and the solvents evaporated in vacuo to yield

4,5,6,7-tetrahydro-2-(phenoxymethyl)-oxazolo[5,4-c]pyridine (4, 0.58 g, 45%) as a purple oil that

was used in the next step without further purification: LCMS tR 1.07, m/z 231 [M+H].

Step 5. 5-[(4-fluorophenyl)carbonyl]-2-(phenoxymethyl)-4,5,6,7-tetrahydro[1,3]oxazolo

[5,4-c]pyridine (5).

4-Fluorobenzoyl chloride (0.25 mL, 2.12 mmol) was added dropwise to a stirred solution of 4

(0.37 g, 1.63 mmol) and TEA (0.34 mL, 2.44 mmol) in DCM (8.15 mL) at 0 ºC. The reaction

mixture was stirred at room temperature for 15 min and then diluted with a saturated solution of

NaHCO3. The organic layer was separated, dried (Na2SO4), filtered and the solvent evaporated

in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in

DCM 0/100 to 30/70). The desired fractions were collected and the solvents evaporated in

vacuo. The crude product was triturated with heptane to yield 5-(4-fluorobenzoyl)-4,5,6,7-

tetrahydro-2-(phenoxymethyl)-oxazolo[5,4-c]pyridine (5, 0.38 g, 67%) as a white solid: LCMS

m/z 353 [M+H]; 1H NMR (400 MHz, CDCl3) 2.73 (br. s., 2 H), 3.69 (br. s., 1.4 H), 3.99 (br. s.,

0.6 H), 4.57 (br. s., 0.6 H), 4.78 (br. s., 1.4 H), 5.12 (br. s., 2 H), 7.02 (d, J=7.4 Hz, 3 H), 7.14 (t,

J=8.6 Hz, 2 H), 7.31 (t, J=7.7 Hz, 2 H), 7.43 - 7.51 (m, 2 H).

In Vitro Assays

Selectivity Screening

mGlu1

HEK293 cells stably expressing rat mGlu1 were plated in black-walled, clear-bottomed, poly-D-

lysine coated 384-well plates (Greiner Bio-One, Monroe, NC) in assay medium at a density of

20,000 cells/well the day prior to assaying. Calcium flux was measured using the Functional

11

Drug Screening System 6000 (FDSS6000, Hamamatsu, Japan); vehicle or a fixed concentration

of test compound (10 µM) was added followed by a concentration response curve of glutamate

2.5 min later. The change in relative fluorescence over basal was calculated before

normalization to the maximal response to glutamate (Hammond et al., 2010; Rodriguez, 2010).

Group II and Group III mGlu’s

Compound activity at the rat group II and III mGlus was assessed using thallium flux through

GIRK channels as previously described in detail (Hammond et al., 2010; Niswender et al.,

2008). Briefly, HEK-293-GIRK cells expressing mGlu subtype 2, 3, 4, 6, 7 or 8 were plated into

384-well, black-walled, clear-bottom poly-D-lysine coated plates at a density of 15,000 cells/well

in assay medium the day prior to the assay. On the day of the assay, the medium was aspirated

and replaced with assay buffer (Hank’s Balanced Salt Solution, 20 mM HEPES, pH7.4)

supplemented with 0.16 µM Fluozin2-AM (Invitrogen, Carlsbad, CA). For these assays, vehicle

or fixed concentration of test compound (10 µM) was added followed by a concentration

response curve to glutamate (or L-AP4 in the case of mGlu7) diluted in thallium buffer (125 mM

NaHCO3, 1 mM MgSO4, 1.8 mM CaSO4, 5 mM glucose, 12 mM thallium sulfate, 10 mM

HEPES) and fluorescence measured using a FDSS 6000. Data were analyzed as described

previously (Niswender et al., 2008).

ERK Phosphorylation Assay

HEK293A cells stably expressing rat mGlu5 were seeded at ~40,000/well in assay medium. Six

hr prior to assay, medium was replaced with serum-free medium (DMEM + HEPES) and cells

returned to 37 ºC incubator. Media was exchanged at 30 min, and cells were exposed to

VU0409551 1 min prior to stimulation with glutamate for 7 min. At t = 0, ligand-containing media

was aspirated and 50 L of 1X Lysis buffer added. Plates were shaken for 10 min and lysates

frozen. pERK1/2 levels were detected using the ALPHAScreen-based ERK Surefire kit (Perkin-

Elmer, TGR Biosciences) as per manufacturer’s instructions. Data were normalized to the

12

maximal response elicited by glutamate and transformed and fitted using GraphPad Prism 5.0

(Graph-Pad Software, San Diego, California).

Astrocytes

Primary rat cortical astrocytes were stored in liquid nitrogen until use. Astrocytes were thawed

following the protocol provided by Lonza and plated on CD Falcon Primaria dishes in assay

growth media (AGM; assay basal media supplemented with AGM Singlequots from Lonza,).

Four hr after initial plating, the media was removed and replaced with fresh media. Every 3 days

until the astrocytes reached confluency, half of the media volume was removed and replaced

with the same volume of fresh media. When confluent, astrocytes were plated in 384-well poly-

D-lysine coated, black-walled, clear-bottomed plates (BD Falcon) in a 20 µL volume of AGM.

Each dish of astrocytes yielded 1 plate. The next day, astrocytes were supplemented with G5

diluted 1:100 in AGM.

Ca+2 mobilization assay protocol. One day after G5 supplementation, the growth media in the

astrocyte plate was exchanged with assay buffer using an ELX405 cell plate washer. A Ca2+-

sensitive fluorescent dye (Fluo4-AM, Invitrogen Corp) was added at a final concentration of 1

µM inthe presence of 2.5 mM probenecid. The cells were allowed to take up the dye during an

incubation period of 45 min at 37 °C/5% CO2. A second exchange with fresh assay buffer was

performed to remove dye remaining extracellularly and, after an incubation period of 10 min at

room temperature, the assay was initiated. All data were collected at a frequency of 1 frame/sec

for 350 sec monitoring fluorescence of the Ca2+-bound dye at an excitation wavelength of 480

nm and emission at 540 nm using a Functional Drug Screening System (FDSS, Hamamatsu

Corp). First, compound alone was added at 3 sec to detect any agonist activity. Next, a low sub-

maximal concentration (nominally 20% of the maximum response, EC20) of glutamate was

added at 143 sec to detect any potentiation of the glutamate response by the test compounds. A

range of 1.1 – 1.4 µM glutamate was used for the EC20 concentration. Lastly, a high sub-

13

maximal concentration of 8 µM (nominally 80% of the maximum response, EC80) was added at

240 sec to detect any antagonism of the glutamate response by the test compounds. A

glutamate concentration (60 µM) resulting in a maximal response was also added in the third

addition to wells not receiving compound or previous glutamate additions. The resulting Emax

response was used to normalize test responses during data analysis.

Data Analysis. Data files containing raw values from the FDSS were inserted into a Microsoft

Excel analysis template using Excel formulas for data reduction and IDBS XLfit (Guildford, UK)

for curve fitting. Each point was normalized to the initial value for that well (static ratio). For each

window (agonist, potentiator, antagonist), the peak static ratio response was determined and

corrected by subtraction of the minimum response at the beginning of the data collection. Each

corrected response was then expressed as a percent of the average baseline corrected static

ratio of the Emax. For the agonist, potentiator, and antagonist windows, each corresponding

%Emax response was plotted versus the log of the molar concentration of test compound. The

data were fit to a 4 parameter logistic equation to determine the minimum response, maximum

response (%Emax), the log concentration giving the half-maximal response (log EC50), and the

slope factor of the curve.

In Vivo Studies

Brain Slice Electrophysiology

Whole-cell voltage and current clamp recordings. Transverse hippocampal slices were

prepared from male SD rats (age 17–28 days) (Charles River, Wilmington, MA). In brief, after

anesthetized with isoflurane, rats were decapitated and the brains were rapidly removed from

the skull and submerged in ice-cold cutting solution (in mM: 210 sucrose, 2.5 KCl, 0.5 CaCl2, 8

MgSO4, 1.25 NaH2PO4, 26 NaHCO3, and 10 D-glucose) oxygenated with 95% O2 /5% CO2.

Transverse hippocampal slices (300 μm) were cut using a Leica VT1200S Microtome (Leica

Microsystems Inc, Buffalo Grove IL), and then incubated in oxygenated aCSF (in mM, 126

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NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, and 10 D-glucose) at 31-32°C

for 30 min and maintained at room temperature afterward until transferred individually to a

submersion recording chamber, which was continuously perfused with oxygenated aCSF at 30-

32°C.

Whole-cell recordings were made from visually identified hippocampal CA1 pyramidal

neuron soma under an Olympus BX50WI upright microscope (Olympus, Lake Success). A low-

power objective (4x) was used to identify CA1 region of the hippocampus, and a 40x water

immersion objective coupled with Hoffman optics and video system was used to visualize

individual pyramidal cells. Patch pipettes (3-5 MΩ) were prepared from borosilicate glass (World

Precision Instrument, Sarasota, FL) using a Narashige vertical patch pipette puller (Narashige,

Japan) or Flaming/Brown micropipette puller (Sutter Instrument Company, Novato, CA). For

voltage clamp studies, patch pipettes were filled with the intracellular solution containing (in mM)

130 Cs-MeSO3, 5 NaCl, 10 TEA-Cl, 5 QX-314, 10 HEPES, 0.2 EGTA, 4 Mg-ATP, and 0.4 Na-

GTP. The pH of the pipette solution was adjusted to 7.3 with 1 M CsOH, and osmolarity was

adjusted to ~295 mOsm. For current clamp studies, patch pipettes were filled with the solution

containing (in mM) 120 K-MeSO3, 1 MgCl2, 0.1 CaCl2, 10 HEPES, 1 EGTA, 12

phosphocreatine, 0.4 Na-GTP and 2 Mg-ATP. The pH of the pipette solution was adjusted to 7.3

with 1 M KOH, and osmolarity was adjusted to 285-295 mOsm. NMDA receptor mediated

responses (current or voltage changes) were recorded under voltage or current clamp condition

and induced by pressure ejection of 0.5-1 mM NMDA to the dendritic field near the soma of the

recorded cell every 30 sec through a patch pipette using a Picospritzer II (General Valve,

Fairfield, NJ). In voltage clamp experiments, the cell was typically clamped at -65 to -70 mV.

The current clamp experiments were performed at the resting membrane potential. These

experiments were carried out in the presence of tetrodotoxin (1 μM) to block voltage-gated

sodium channels. AMPAR and NMDAR mediated excitatory postsynaptic currents (EPSCsAMPA

and EPSCsNMDA) were evoked by electrical stimulation of Schaffer collaterals every 30 sec and

15

recorded at holding potential of -60 mV and +40mV, respectively, in the presence of 20 μM

bicuculline to block GABAA receptor mediated inhibitory currents. The electrophysiological

signals were acquired using a MultiClamp 700B amplifier (Molecular Devices), DigitData 1440A

and pClamp 10.2 software (Molecular Devices). All drugs were bath applied. Data were

analyzed using Clampfit 10.2, Origin 6 (OriginLab, Northampton, MA) and GraphPad Prism 5.0

(GraphPad Software, La Jolla, CA), and presented as percentage of the baseline value or

percentage potentiation. The ratio of EPSCNMDA and EPSCAMPA was calculated by dividing

EPSCNMDA amplitude measured at 100 ms after the stimulation artifact by EPSCAMPA peak

amplitude. The percentage potentiation was defined by [I(max)/I(baseline)-1]x100, where

I(baseline) was the average amplitude of NMDA receptor currents of 4 trials immediately before

application of DHPG or mGlu5 PAM and I(max) is the maximum current amplitude during

application of the compound(s).

Extracellular field potential recordings. Transverse hippocampal slices were prepared from

young adult (age 29–36 days) male SD rats (Charles River, Wilmington, MA) with use of

standard techniques and buffers as previously described (Ayala et al., 2009; Noetzel, 2012). In

brief, rats were anesthetized with isoflurane and decapitated, and the brains were quickly

removed and submerged into ice-cold cutting solution (110 mM sucrose, 60 mM NaCl, 3 mM

KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 5 mM D-glucose, 0.6 mM (+)-sodium-L-ascorbate,

0.5 mM CaCl2, and 7 mM MgCl2) continuously bubbled with 95% O2/5% CO2. 400 μm thick

transverse slices were made using a Compresstome (Precisionary Instruments, Greenville,

North Carolina). Individual hippocampi were microdissected from the slice and transferred to a

room temperature mixture containing equal volumes of cutting solution and artificial

cerebrospinal fluid (aCSF) (125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 15

mM glucose, 2 mM CaCl2, and 1 mM MgCl2), where they were allowed to equilibrate for 30 min.

The hippocampi were then transferred to room temperature aCSF continuously bubbled with

95% O2/5% CO2 for an additional hour. Slices were transferred to a submersion recording

16

chamber and allowed to equilibrate for 5 to 10 min at 30–32 °C with a flow rate of 1.5-2 ml/min.

A bipolar-stimulating electrode was placed in the stratum radiatum near the CA3-CA1 border to

stimulate the Schaffer collaterals. Recording electrodes (3-5 MΩ) were pulled with a

Flaming/Brown micropipette puller (Sutter Instrument Company, Novato, CA), filled with aCSF,

and placed in the stratum radiatum of area CA1. Field potential recordings were acquired using

a MultiClamp 700B amplifier (Molecular Devices) and pClamp 10.2 software (Molecular

Devices). A stimulus intensity that produced 50–60% [long-term depression (LTD)] and 40–50%

[long-term potentiation (LTP)] of the maximum fEPSP slope was set before each experiment.

mGlu5 compounds were diluted to the appropriate concentrations in dimethylsulfoxide (0.1%

final) in aCSF and applied to the bath for 10-20 min with use of a perfusion system. Chemically

induced mGlu LTD was initiated by the application of DHPG in aCSF (25 or 75 µM) for 10 min.

Threshold LTP was induced by one train of theta burst stimulation (TBS; nine bursts of four

pluses at 100 Hz, 230-millisecond interburst interval). Saturated LTP was induced by four trains

of 10 Hz TBS (nine bursts of four pulses at 100 Hz, 100-millisecond interburst interval). For the

NMDAR-fEPSP experiment, DNQX (20 μM) and bicuculline (20 μM) were included in aCSF to

block AMPA and GABAA receptors, and MgCl2 was decreased to 0.1 mM in aCSF to reduce

Mg2+ block of NMDAR currents. Data were analyzed using Clampfit 10.2 and GraphPad Prism

5.0 as described previously (Noetzel, 2012). Between group statistics were performed using

one-way analysis of variance (ANOVA) and for within group analysis repeated measures

ANOVA was performed with a critical p of 0.05. Multiple comparisons were performed using

Dunnett’s multiple comparison test for comparing with a control group with a critical p of 0.05.

Novel object recognition. Effects of VU0409551 on recognition memory were evaluated in rats

(275-300 g). Rats were habituated in an empty novel object recognition (NOR) arena (consisting

of dark colored plexiglass box (32 x 32 x 40 inches) for 10 min for 2 consecutive days prior to

testing. Approximately 24 hr following habituation, rats were administered vehicle, 1, 3, or 10

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mg/kg of VU0409551 and placed back into their home cage for 30 min. Rats were then placed

back into the NOR arena containing 2 identical objects for 10 min. Following the exposure

period, rats were placed back into their home cages, and 24 hr later returned to the arena in

which one of the previously exposed (familiar) objects was replaced by a novel object. The rats

were video recorded for 10 min while they explored the objects. Time spent exploring each

object was scored by an observer blinded to the experimental conditions and the recognition

index was calculated as [(Time spent exploring novel object) – (Time spent exploring familiar

object)] / Total time exploring objects.

Sleep-wake electroencephalography studies

Male SD rats (250-275 g) were surgically implanted with a transmitter (4-ET, Data Sciences

International, St. Paul, MN) for recording electroencephalography (EEG), electromyography

(EMG), temperature and motor activity via telemetric recording. Briefly, under isoflurane

anesthesia (4% induction; 1.5-2.5% maintenance), transmitters were implanted subcutaneously

just off the midline of the dorsal flank of each animal. Transmitter leads were tunneled

subcutaneously to the skull. Holes were drilled in the skull and exposed wires were placed in

contact with the dura and secured in place via dental cement (Butler Schein, USA). Three sets

of leads were placed, each bilaterally to record from cortical regions corresponding with the

frontal, parietal and occipital cortices (+3.0 mm, -3 mm and -6 mm from Bregma, respectively

and +/- 2 mm lateral to the midline). An additional set of leads were placed bilaterally in the

nuchal muscle for EMG recording. Rats received 5 mg/kg ketoprofen (s.c.) just prior to surgery

and once daily for 3 days following surgery. A prophylactic antibiotic regimen (5 mg/kg Baytril

and Enrofloxacin, s.c.) was administered for 7 days following surgery. Animals were individually

housed following surgery and allowed to recover and acclimate to the recording room for a

minimum of 10 days prior to recording.

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Data recording began at the start of the light cycle. VU0409551 was then administered

(vehicle, 10, or 60 mg/kg) at the 2nd hour of the light period. Sleep polygraphic variables were

measured for a period of 22 hr after compound administration in rats. A crossover study design

was utilized in which all rats received each of the 3 treatments. All EEG recordings took place in

a designated recording room shielded from extraneous noise and traffic. EEG and EMG

waveform data were collected using Dataquest A.R.T. 4.3 software (DSI, Minneapolis, MN)

using a continuous sampling method. Telemetric data were sampled at a rate of 500 Hz and

transmitted via a receiver (RPC-2, DSI, MN) placed below the cage of each rat. Each receiver

was connected to a data exchange matrix (DSI, MN) which transferred EEG data to a computer

for off-line analysis.

A blinded observer manually scored each 10-second epoch using Neuroscore 3.0

software (Data Sciences International, St. Paul, MN) to determine changes in sleep-wake

stages following dosing based upon accepted characteristic oscillatory patterns. The awake

stage was defined as mixed-frequency EEG and high-voltage EMG activity; NREM sleep was

defined ashigh-amplitude, low-frequency EEG and low-EMG amplitude; and REM sleep was

defined as low-amplitude, low-frequency EEG and low-EMG with muscle atonia and occasional

muscle twitches. Sleep-wake stages were scored in 10 sec epochs then NREM and REM sleep

time was combined into 60-min bins to examine the percent of time spent in total sleep across

the first 22 hr cycle following compound administration.

Delayed Non-matching to Position

Effects of VU0409551 on working memory and executive function were evaluated in the delayed

non-matching to position (DNMTP) task using male Lister-hooded rats weighing 350-450 g.

Rats underwent extensive training prior to testing to ensure that a high level of stable and

accurate performance had been obtained (approximately 3 months). However, animals were

drug naïve during the testing phase. Rats were administered vehicle or VU0409551 (10 – 60

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mg/kg, p.o., 20% BCD + Tween) 60 min prior to testing. Four delay lengths (1, 10, 20 and 30

sec) were used in this study with 15 trials occurring at each delay (60 trials total). Rats were

allowed a maximum of 45 min to complete these trials. Detailed methods can be found in the

Supplemental Materials section. The percent correct responses and total trials completed were

quantified. Repeated measures ANOVA were used for statistical analysis with delay, treatment

with JNJ-46778212 and its interaction used as the independent variables. Scheffé adjusted

tests were used for post-hoc analysis with all comparisons being made against the appropriate

vehicle condition.

Studies were performed in a MedAssociates operant chambers [32 cm x 25 cm x 25 cm

(Georgia, Vermont)], consisting of one wall equipped with two retractable response levers with a

pair of signal lights above the levers. The opposite wall contained the reward magazine,

equipped with a reward light and an infra-red sensor. Above this was a small “house” light and

speaker. Forty five mg dustless precision pellets (Bio Serv, Flemington, New Jersey) were used

as reward. The DNMTP task consisted of two stages. In the first of these (sample), a lever was

presented and the rat was required to press it. Once the lever was pressed, it was retracted

followed by a short variable delay (1 - 30 sec), and the food magazine light was illuminated to

encourage a nosepoke behavior. The first poke after the delay initiated the choice phase of the

trial. In the choice phase, the original lever and a second lever not yet seen during the

particular trial were presented. Selection of the second lever resulted in presentation of a food

reward.

A DNMTP trial began with the illumination of the magazine receptacle. This signaled to

the rat that it must poke at the receptacle to start the sample phase, turning off the light within

the receptacle. Once this response was made, the sample phase began. One of two levers

was extended in a pseudo random fashion, and the rat had 20 sec to respond at the lever. A

response caused the lever to be retracted and marked the beginning of the delay phase

(variable 1 - 30 sec). Once the delay phase began, the food magazine was illuminated

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encouraging the rat to nosepoke at it. The first poke after the delay started the choice phase

(extension of levers and de-activation of magazine light). However, if no response occurred

within 10 sec, the trial was considered an omission. Rats were given 10 sec to respond to the

levers presented. A response at the correct lever (opposite from that of the sample) caused the

levers to retract, the magazine light to illuminate, and delivery of a food pellet along with a short

reward tone. Once the pellet was collected, the magazine light turned off and the inter trial

interval (ITI) began (5 sec). An incorrect response resulted in the retraction of the response

levers, the de-activation of the house light, and a short “punishment” period (10 sec). During this

interval, the animal was required to wait in the dark before it could initiate the next trial. When

this punishment period passed, the house light turned on and the ITI began (10 sec s). Once

the ITI passed, the magazine receptacle was again illuminated signaling the beginning of the

next trial. Animals we required to respond to progress a trial. At the sample phase they were

given 20 sec to make a response and 10 sec at both the choice initiation and choice selection

phase. A lack of response at any of these time points was counted as an omitted trial, and was

otherwise treated as an incorrect trial.

Fluoro-Jade C staining

Twenty-four hours after the last dose of VU0409551 or VU0424465, male SD rats were deeply

anesthetized using isoflurane and transcardially perfused with 250 ml of cold phosphate

buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer.

Brains were removed and post-fixed overnight, then placed in a cryoprotectant solution of 30%

sucrose in 0.1 M phosphate buffer (pH 7.4) at 4°C until sectioning. Using a freezing microtome

(Leica Microsystems; Buffalo Grove, IL), 50 µm coronal sections through the hippocampus,

including the auditory cortex, were cut and processed for Fluoro-Jade C staining as previously

described (Schmued et al., 2005). Briefly, one out of every five sections were mounted from

0.15% gelatin onto Superfrost plus slides (Fisher Scientific; Pittsburgh, PA) and allowed to dry

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overnight. The slide-mounted sections were first treated with 80% ethanol containing 1%

sodium hydroxide for 5 min followed by a 2 min rinse in 70% ethanol and then a 2 min rinse in

water. Sections were incubated in a 0.06% potassium permanganate solution for 10 min

followed by a 2 min rinse in water. The slides were then protected from light and immersed into

a 0.0001% solution of Fluoro-Jade C (dissolved in 0.1 % acetic acid) (Millipore; Billerica, MA) for

10 min. Excess Fluoro-Jade C was washed off with 1 min rinses in water repeated 3 times.

Slides were dried overnight while being protected from light and subsequently cleared in xylene

for 2 min and coverslipped using DPX (Electron Microscopy Sciences; Hatfield, PA) mounting

media. Fluoro-Jade C labeled sections were visualized using a Leica DM IRB (Leica

Microsystems; Buffalo Grove, IL) microscope equipped with a Nikon DXM 1200C (Nikon

Instruments; Melville, NY) camera. Images were acquired using NIS-Elements software (Nikon

Instruments; Melville, NY). Fluoro-jade C positive neurons within the auditory cortex were

counted under 20x magnification. A total of five sections per animal was quantified and the data

is presented as mean number of cells per section.

Amphetamine-induced hyperlocomotion

Effects of VU0409551 on amphetamine-induced hyperlocomotion were determined as

previously described (Rodriguez, 2010). Briefly, male SD rats were placed in an open-field

chamber (KinderScientific, San Diego, CA) for 30 min. At t = 30, rats were administered vehicle

or VU0409551 (3 – 100 mg/kg, p.o., 20% β-cyclodextrin in water, 10 ml/kg, n = 8) followed by

amphetamine (1 mg/kg, s.c., saline, 1 ml/kg) at t = 60 min. Locomotor activity was measured for

an additional 60 min. Changes in locomotor activity were measured as the total number of

photobeam breaks per 5-min bins. Data are expressed as mean ± SEM and analyzed using

one-way ANOVA (60 – 120 min) with a Dunnett’s post hoc test comparing all dosing groups to

vehicle + amphetamine-treated controls. Statistical significance was determined as p < 0.05.

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Amphetamine-induced disruptions in prepulse inhibition of the acoustic startle reflex

Studies were conducted using San Diego Instruments startle chambers (SR-LAB; San Diego

Instruments, San Diego, CA) as previously described (Jones et al., 2005). Following a 20-min

pretreatment with vehicle or VU0409551 (10 - 100 mg/kg, p.o., 20% β-cyclodextrin in water, 10

ml/kg volume, n = 8), male SD rats were injected with amphetamine (3 mg/kg, s.c., saline, 1

ml/kg volume). After an additional 10 min, they were placed into individual startle chambers,

allowed to habituate for 5 min and subsequently tested following the paradigm described

previously (Gregory et al., 2013). Percent prepulse inhibition was calculated as 100 x (mean

acoustic startle reflex [ASR] - mean ASR in prepulse plus pulse trials) / mean ASR in startle

pulse trials. Data are expressed as mean ± SEM and analyzed using one-way ANOVA with a

Dunnett’s post hoc test comparing all dosing groups to vehicle + amphetamine-treated control

group. Statistical significance was determined as p < 0.05.

Statistical Analysis

For electrophysiology studies, data are expressed as percentage of the baseline value or

percentage potentiation. Between group statistics were performed using one-way analysis of

variance (ANOVA) and for within group analysis repeated measures ANOVA was performed.

Multiple comparisons were performed using Dunnett’s multiple comparison test for comparing

with a control group. EEG dose-response studies were analyzed using a two-way ANOVA to

examine effects of time and dose for each of the above described measures, followed by

Bonferroni post hoc test. Locomotor time course data were analyzed using one-way ANOVA (60

– 120 min) with a Dunnett’s post hoc test comparing all dosing groups to vehicle +

amphetamine-treated controls. Fear conditioning and novel object recognition data were

analyzed using a one-way ANOVA and all dose groups were compared with the vehicle-treated

group using a Dunnett’s post hoc test. The percent correct responses and total trials initiated

were analyzed in the DNMTP studies using a repeated measures ANOVA for statistical analysis

23

with delay and treatment with VU0409551 and its interaction used as the independent variables.

Scheffé adjusted tests were used for post-hoc analysis with all comparisons being made against

the appropriate vehicle condition. All statistical analyses used a critical p value < 0.05.

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Z., Zhang, Y., Jones, P.J., et al. (2009). mGluR5 positive allosteric modulators facilitate both

hippocampal LTP and LTD and enhance spatial learning. Neuropsychopharmacology 34, 2057-

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J.T., Jadhav, S., Bridges, T.M., Weaver, C.D., et al. (2013). N-aryl piperazine metabotropic

glutamate receptor 5 positive allosteric modulators possess efficacy in preclinical models of

NMDA hypofunction and cognitive enhancement. J Pharmacol Exp Ther 347, 438-457.

Hammond, A.S., Rodriguez, A.L., Townsend, S.D., Niswender, C.M., Gregory, K.J., Lindsley,

C.W., and Conn, P.J. (2010). Discovery of a Novel Chemical Class of mGlu(5) Allosteric

Ligands with Distinct Modes of Pharmacology. ACS Chem Neurosci 1, 702-716.

Jones, C.K., Eberle, E.L., Shaw, D.B., McKinzie, D.L., and Shannon, H.E. (2005).

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Lavreysen, H., Stauffer, S. R., Niswender, C. M., Xiang, Z., Daniels, J. S., Jones, C. K.,

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