Supplementary materials
Supplementary Methods:
Chemical Synthesis of VU0453595
Progression of Chemical synthesis: First, an NIH sponsored molecular libraries
production center network (MLPCN) high-throughput screen (~60K compounds) (Marlo
et al, 2009), and initial optimization efforts resulted in the first M1 selective probe from the
series, ML137 (Bridges et al, 2010b). In an effort to remove the isatin dicarbonyl unit the
optimized N-pyrazol-4-yl-benzyl moiety was utilized and examined within the context of
an indolinone core to give VU0448350 (Ma et al, 2009). Further examination of amide
positional isomers led to the slightly more potent isoindolinone VU0451725 (Ma et al,
2009). Unfortunately, in vitro DMPK properties of VU0451725 included high intrinsic
clearance (CLhep) in humans and rats (h,r) as well as high plasma protein binding (PPB,fu
< 1%). To address the DMPK limitations of VU0451725, additional medicinal chemistry
efforts within the isoindolinone ring system were pursued focusing on incorporation of a
pyridine ring within the western core.
These efforts provided 4-aza-isoindolinone
VU0453595, resulting in enhanced polarity and a significantly reduced calculated
octanol-water partition coefficient (VU0451725 cLogP = 2.85 vs. VU0453595 cLogP =
1.75). In addition, by incorporating the pyridine nitrogen, the tier 1 DMPK profile was
significantly enhanced for VU0453595, and the compound exhibits low predicted hepatic
clearance in both rat and human and excellent fraction unbound (fu = 4.8% rat, 6.6%
human).
General Synthetic Protocols and Methods: Analytical thin layer chromatography was
performed on Analtech silica gel GF 250 micron plates using reagent grade solvents.
Normal phase flash silica gel-based column chromatography was performed using
ready-to-connect cartridges from ISCO, on irregular silica gel, particle size 15-40 mm on
a Combi-flash Companion chromatography system from ISCO. Low resolution mass
spectra were obtained on an Agilent 1200 series 6130 mass spectrometer comprising a
binary pump with degasser, an autosampler, a column oven, a diode-array detector
(DAD) and a column as specified in the respective methods below. Flow from the
column was split to a SQ mass spectrometer and Polymer Labs ELSD. The MS detector
was configured with an ES ionization source. Nitrogen was used as the nebulizer gas.
The source temperature was maintained at 350 °C. Data acquisition was performed with
Agilent Chemstation software. Reversed phase HPLC was carried out on a Kinetex C18
column (2.6 mm, 2.1 x 30 mm) from Phenomenex, with a flow rate of 1.5 mL/min, at 45
ºC. The gradient conditions used are: 93% A (water + 0.1% TFA), 7% B (acetonitrile), to
95% B in 1.1 minutes, returning to initial conditions at 1.11 minutes. Injection volume 1
mL. Low-resolution mass spectra (single quadruple MSD detector) were acquired in
electrospray mode by scanning from 100 to 700 in 0.25 seconds, step size of 0.1 and
peak width of 0.03 minutes. The capillary needle voltage was 3.0 kV and the fragmentor
voltage was 100V. High resolution mass spectra were recorded on a Waters Q-TOF
API-US.
Preparative RP-HPLC purification was performed using a Gilson Inc.
preparative UV-based system using a Phenomenex Luna C18 column (50 x 30 mm I.D.,
5 mm) with an acetonitrile (unmodified)-water (0.1% TFA) custom gradient.
1
H NMR
spectra were recorded either on a Bruker DPX-400 or on a Bruker AV-500 spectrometer
with standard pulse sequences, operating at 400 MHz and 500 MHz respectively.
Chemical shifts (d) are reported in parts per million (ppm) downfield from
tetramethylsilane (TMS), which was used as internal standard.
Preparation
of
6-(2-Fluoro-4-(1-methyl-1H-pyrazol-4-yl)benzyl)-6,7-dihydro-5H-
pyrrolo[3,4-b]pyridin-5-one:
Step 1. 6-(4-Bromo-2-fluorobenzyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one.
To a
flame-dried argon flushed flask containing a magnetic stir bar was added sodium hydride
(1.34 g, 55.9 mmol) and DMF (100 mL). The flask was cooled to 0 oC and 6,7-dihydro5H-pyrrolo[3,4-b]pyridin-5-one (5.0 g, 37.3 mmol) added portionwise. The mixture was
maintained at 0 °C for 20 min until foaming had subsided. The mixture was allowed to
warm to room temperature, and 4-bromo-1-(bromomethyl)-2-fluorobenzene (10.9 g, 41
mmol) was added as a solution in DMF (25 mL). After stirring at room temperature for an
additional 1.5 h, the mixture was slowly poured into ice water (400 mL). The resulting
aqueous suspension was extracted successively with EtOAc (4 x 100 mL). The organic
layers were combined, dried over MgSO4, evaporated and the residue was purified by
silica gel chromatography (0-70% EtOAc in hexanes) to afford the title compound as a
yellow solid (4.01g, 34%): LC-MS >98% (215, 254 nm), Rt = 0.905, m/z = 320.8 [M+H].
Step 2. To a round bottom flask charged with 6-(4-bromo-2-fluorobenzyl)-6,7-dihydro5H-pyrrolo[3,4-b]pyridin-5-one from step 1 (4.02 g, 12.5 mmol), 1-methyl-4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (3.12 g, 14.9 mmol), cesium carbonate
(8.14 g, 25 mmol), and PdCl2(dppf) (915 mg, 1.25 mmol) under argon was added
degassed THF (100 mL) and water (10 mL). The reaction was heated to 80 oC and
stirred for 16h. The mixture was cooled to rt and diluted with EtOAc and water. The
organic phase was washed sequentially with brine (2x), water (2x), followed by saturated
aqueous NaHCO3. The crude organic was dried over MgSO4 and concentrated under
reduced pressure. The solid was purified on silica gel with 2-10% MeOH:CH2Cl2 and
concentrated to give title compound as a yellow powder (2.21 g, 55%): LC-MS >98%
(215, 254 nm), Rt = 0.781, m/z = 323.1 [M+H]; 1H NMR (400 MHz, CDCl3, δ 8.7-8.6 (m,
1H), 8.2-8.1 (m, 1H), 7.8 (s, 1H), 7.6 (s, 1H), 7.4-7.3 (m, 2H), 7.2-7.1 (m, 2H), 4.9 (s, 2H)
4.4 (s, 2H), 4.0 (s, 3H); HRMS calculated for C18H16FN4O (M+H) m/z: 323.1308,
measured: 323.1306.
Calcium mobilization assays
Compound-evoked increases in intracellular calcium were measured using Chinese
Hamster Ovary (CHO) cells stably expressing rat M1 muscarinic receptors. Cells were
plated in 384-well, black-walled, clear-bottomed plates in 20 µL of assay medium
(DMEM supplemented with 10% dialyzed fetal bovine serum, 20 mM HEPES and 1 mM
sodium pyruvate) at a density of 30,000 cells/well and grown overnight at 37°C/5% CO2.
The next day, medium was removed and the cells were incubated with 20 µl/well of 1 µM
Fluo-4AM (Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in dimethyl sulfoxide
(DMSO) and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in
calcium assay buffer (Hank’s Balanced Salt Solution (HBSS; Invitrogen, Carlsbad, CA)
supplemented with 20 mM HEPES and 2.5 mM probenecid, pH 7.4) for 50 minutes at 37
°C. Dye was removed and replaced with 20 µl/well of assay buffer and further incubated
at 37 °C for 10 minutes. For PAM potency curves, VU0453595 was diluted in calcium
assay buffer and added to the cells followed by the addition of an EC20 concentration of
acetylcholine 140 seconds later, and then an EC80 concentration of acetylcholine 120
seconds later. For fold shift experiments, multiple fixed concentrations (50 nM - 30 µM)
of VU0453595 or vehicle were added followed by the addition of a concentrationresponse curve of acetylcholine (1fM - 10µM) 140 seconds later. Calcium flux was
measured over time as an increase in fluorescence using a Functional Drug Screening
System 6000 or 7000 (FDSS 6000/FDSS 7000, Hamamatsu, Japan). The change in
relative fluorescence over basal was calculated before normalization to the maximal
response to acetylcholine. As described previously, shifts of acetylcholine concentration-
response curves were globally fitted to an operational model of allosterism (Leach et al,
2007).
To assess the selectivity of VU0453595 calcium mobilization assays were
performed using CHO cells stably expressing rat M2-M5 muscarinic receptors (M2 and M4
cells were co-transfected with Gqi5) as described above for the potency assay.
Radioligand binding
Radioligand binding studies were performed based on previous studies(Shirey et al,
2009). Cell membranes were prepared from CHO cells expressing rat M1. Cells were
harvested and pelleted by centrifugation and re-suspended in ice-cold homogenization
buffer (50 mM Tris-HCl, 10 mM EDTA, 0.9% NaCl, pH7.4), and homogenized by 3 x 10
second bursts. Cell fractions were separated by centrifugation and the resulting pellet resuspended in ice-cold assay buffer (50 mM Tris-HCl, 0.9% NaCl, pH7.4). For inhibition
binding experiments, membranes (10 µg/well) were incubated with 150 pM [3H]Nmethylscopolamine (NMS), a fixed concentration of VU0453595 (3µM – 30 µM) or
vehicle and a range of concentrations of acetylcholine (1 nM – 1 mM) for 2 hours at
room temperature with shaking in assay buffer. Non-specific binding was determined
using 10 µM atropine. Assays were terminated by rapid filtration using a Brandel 96-well
plate Harvester, and washed three times with ice-cold assay buffer. The next day,
MicroScint20 was added and radioactivity was counted.
Brain homogenate binding:
The brain homogenate binding of VU0453595 was determined in brain homogenates via
equilibrium dialysis as previously described (Noetzel et al, 2012). Brain homogenate
binding samples were analyzed using LC-MS/MS techniques on a Thermo Electron TSQ
Quantum Ultra triple quad detector via electrospray ionization with two Themo Electron
Accella pumps (San Jose, CA), and a Leap Technologies CTC PAL autosampler.
In Vivo Pharmacokinetic Analysis
Test compound was formulated as 10% Tween 80 in sterile water at the concentration of
3.33 mg/mL and administered intraperitoneally to male C57/bl6 mice weighing (Harlan,
Indianapolis, IN) at a dose of 10 mg/kg. Mice blood (cardiac puncture) and brain were
collected at 0.25, 0.5, 1, 3, and 6 h. Animals were euthanized and decapitated, and the
brains were removed, thoroughly washed in ice-cold (4 °C) phosphate-buffered saline,
and immediately frozen on dry ice. Brain samples were processed and concentrations of
compound were determined via electrospray ionization on an AB Sciex API-4000 (Foster
City, CA) triple-quadrupole instrument that was coupled with Shimadzu LC-10AD pumps
(Columbia, MD) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC) as
previously described(Noetzel et al, 2012). All data were analyzed using AB Sciex
Analyst 1.5.1 software. Compound exposures, in the form of area-under-the-curve were
calculated by trapezoidal method employing PRISM software (GraphPad, La Jolla, CA).
Extracellular Field Potential Recordings
8-9 week old male C57BL6/J mice (Jackson Laboratories) or B6.Cg-Tg(ChatCOP4*H134R/EYFP,Slc18a3)5Gfng/J mice were anesthetized with isofluorane, and the
brains were removed and submerged in ice-cold cutting solution (in mM: 230 sucrose,
2.5 KCl, 8 MgSO4, 0.5 CaCl2, 1.25 NaH2PO4, 10 D-glucose, 26 NaHCO3). Coronal
slices containing the prelimbic prefrontal cortex were cut at 400 µm using a
compresstome (Precisionary Instruments, Greenville, NC). Slices were transferred to a
holding chamber containing NMDG-HEPES recovery solution (in mM: 93 NMDG, 2.5
KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 D-glucose, 5 sodium ascorbate, 2
thiourea, 3 sodium pyruvate, 10 MgSO4, 0.5 CaCl2, 12 N-acetyl-L-cysteine, pH 7.3, <310
mOsm) for 15 minutes at 32 ºC. Slices were then transferred to a room temperature
holding chamber for at least 1 hour containing ACSF (in mM: 126 NaCl, 1.25 NaH2PO4,
2.5 KCl, 10 D-glucose, 26 NaHCO3, 2 CaCl2, 1 MgSO4) supplemented with 600 µM
sodium ascorbate for slice viability. All buffers were continuously bubbled with 95%
O2/5% CO2. Subsequently, slices were transferred to a 32 ºC submersion recording
chamber where they were perfused with ACSF at a rate of 2 mL/min. Borosilicate glass
electrodes were pulled using a Flaming/Brown micropipette puller (Sutter Instruments)
and had a resistance of 2-4 MΩ when filled with ACSF. Sampled data was analyzed
offline using Clampfit 10.2 (Molecular Devices, Sunnyvale, CA). The slopes from three
sequential sweeps were averaged.
All slopes calculated were normalized to the
average slope calculated during the pre-drug period (percent of baseline). Data were
digitized using a Multiclamp 700B, Digidata 1322A, and pClamp 10 software (Molecular
Devices). Input-output curves were generated to determine the stimulation intensity that
produced 50-60% of the maximum response before each experiment, which was used
as the baseline stimulation. A minimum of 10 min stable baseline was recorded before
application of any drugs. 50 and 10 µM carbachol was applied for 10 min to induce
saturated and threshold form of muscarinic LTD in the prefrontal cortex. M1 PAM
VU0453595 and M1 orthosteric antagonists were treated alone for 10 min immediately
followed by co-application with carbachol. Carbachol (Sigma, St. Louis, MO) stocks were
prepared in water and all other compound stocks were prepared in DMSO. All drugs
were diluted in ACSF (0.1% DMSO final conc.) and bath applied.
For studies involving optical stimulation, blue light (470 nm) was delivered using
a High Power LED (Thorlabs Inc. Newton, NJ) which was mounted to the epi-illunimaton
port of an Olympus BX50WI upright microscope (Olympus). Blue light was shined onto
slices through the objective lens for 15 min at a frequency of 2 Hz to stimulate release of
endogenous acetylcholine from cholinergic fibers terminating in the prefrontal cortex.
Electrical stimulations continued during the 15 min optical stimulation.
Whole-Cell Patch-Clamp Recordings
Whole-cell patch-clamp recordings were performed using coronal slices prepared from
8-9 week old male C57BL6/J mice (Jackson Laboratories). Animals were anesthetized
using a mixture of ketamine and xylazine (100mg/kg and 10mg/kg, respectively,
intraperitoneal injection) and then transcardially perfused with ice-cold cutting solution.
Mice were then decapitated and the brains were removed and submerged in ice-cold
cutting solution as described above. Coronal slices containing the prelimbic prefrontal
cortex were cut at 300 µm using a Compresstome (Precisionary Instruments). Slices
were then transferred to a holding chamber containing a modified NMDG-HEPES
recovery solution and recovered as described above. After the initial recovery period,
slices were transferred to a holding chamber containing modified ACSF for at least 30
minutes (in mM: 126 NaCl, 1.25 NaH2PO4, 2.5 KCl, 10 D-glucose, 26 NaHCO3, 2 CaCl2,
1 MgSO4) supplemented with 600 µM sodium ascorbate for slice viability. Slices were
perfused with 2 ml/min ACSF at 32ºC in a submersion recording chamber. Neurons were
visualized with a 40X water-immersion lens with Hoffman modulation contrast optics
coupled with an Olympus BX50WI upright microscope (Olympus). Borosilicate glass
pipette electrodes were pulled as described above and had a resistance of 3-6 MΩ when
filled with an intracellular solution containing K-Gluconate (in mM: 123 K-gluconate, 7
KCl, 1 MgCl2, 0.025 CaCl2, 10 HEPES, 0.1 EGTA, 2 ATP, 0.2 GTP) at pH 7.3 and
osmolarity of 290-295. Whole-cell recordings were made from visualized prelimbic
prefrontal pyramidal neurons. Pyramidal neurons were further identified by their regular
spiking pattern following depolarizing current injections in current clamp mode.
Spontaneous EPSCs were recorded at a holding potential of -70 mV. The voltage clamp
signal was low pass filtered at 5 kHz and digitized at 10 kHz and acquired using
clamplex 10.2/Digidata1332A system (Molecular Devices). After a stable baseline was
recorded for 5-10 min, the effect of 100 µM CCh on baseline sEPSC frequency was
examined. Inward currents were recorded in the presence of 0.5-1 µM tetrodotoxin, a
concentration which completely blocked action potential firing upon depolarizing current
injections in current-clamp mode. EPSCs were detected and analyzed using the Mini
Analysis Program (Synaptosoft, Decatur, GA). The inter-event intervals of sEPSCs from
2 min episodes during baseline and drug application were used to generate cumulative
probability plots. The inter-event intervals from each experiment were then expressed as
frequency and the mean values from the 2 min episodes were grouped and compared.
Inward current data analysis was performed using Clampfit software where the peak
amplitude of the inward current was measured (v10.2, Molecular Devices). Carbachol
(Sigma, St. Louis, MO) stocks were prepared in water and all other compound stocks
were prepared in DMSO. All drugs were diluted in ACSF (0.1% DMSO final conc.) and
bath applied.
Supplementary Results:
Characterization of the novel selective M1 PAM VU0453595
We and others recently reported the discovery of multiple highly selective M1
PAMs that act at allosteric sites on the receptor to increase the affinity and/or efficacy of
ACh (Bridges et al, 2010a; Bridges et al, 2010b; Bridges et al, 2010c; Digby et al, 2012;
Ma et al, 2009; Marlo et al, 2009; Melancon et al, 2013; Mistry et al, 2013; Shirey et al,
2009; Uslaner et al, 2013). While earlier M1 PAMs provide high selectivity and excellent
in vitro properties for assessing M1 function, previous compounds suffer from poor CNS
exposure after systemic administration and adverse effects in mice that limit their utility
for in vivo studies(Kuduk et al, 2010a; Kuduk et al, 2011; Kuduk et al, 2010b; Shirey et
al, 2009; Thomsen et al, 2012).
Based on this, we performed extensive chemical
optimization studies to develop an M1 PAM with improved brain penetration and
pharmacokinetic properties when administered systemically to mice.
This effort
culminated in the discovery of a novel and selective M1 PAM, 6-(2-Fluoro-4-(1-methyl-Hpyrazol-4-yl)benzyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one
(VU0453595).
VU0453595 belongs to a series of isoindolinone M1-selective PAMs, which evolved from
extensive small molecule lead optimization efforts that resulted in the first M1 selective
probe, ML137 (Supplementary Figure S1a; Supp. methods). Evaluation of effects of
VU0453595 in CHO cells expressing M1 receptors revealed that this compound does not
possess intrinsic allosteric agonist activity but potentiates the response to an EC20
concentration of ACh in a concentration-dependent manner with a mean EC50 of 3.2 µM
and potentiation to 75% of ACh maximum response (n=6; Supplementary Figure S1b).
Increasing concentrations of VU0453595 also led to a progressive leftward shift in the
glutamate concentration-response curve (n=3; Supplementary Figure S1c) resulting in
the following fold shifts: 1.3 (50 nM), 1.1 ± 0.05 (100 nM), 1.2 ± 0.06 (500 nM), 1.5 ±
0.14 (1 μM), 5.6 ± 1.13 (10 μM) and 15.6 ± 2.81 (30 μM). VU0453595 exhibited a
predicted affinity (logKb) of -2.76 ± 0.06 (1738 μM) and a cooperativity factor (log β) of
1.16 (cooperativity ~ 14.6; value constrained to the average maximum fold shift). To
characterize the interaction of VU0453595 with the M1 receptor, we determined the
effects of multiple concentrations of VU0453595 on ACh-induced displacement of [3H] Nmethylscopolamine ([3H]-NMS) binding (150 pM) to M1 in membranes from M1expressing cells. VU0453595 readily shifted the ACh competition curve leftward (n=3;
log α = 1.81 ± 0.35; 65 fold; Supplementary Figure S1d), further providing evidence for
its function as a PAM that acts primarily via increasing the affinity of ACh at M1.
VU0453595 was also highly selective for M1 and had no detectable activity at M2 - M5
(Supplementary Figure S1e), with no significant off-target activity at other G-proteincoupled receptors, ion channels or transporters (Eurofins Inc. screening; Supplementary
Table S3). VU0453595 demonstrated excellent brain penetration when dosed
systemically, with a greatly improved exposure profile relative to previously reported
PAMs (Supplementary Figure S1f; Supplementary Table S2). In addition, VU0453595
also showed an excellent fraction unbound in brain and plasma and reduced hepatic
clearance from brain as compared to previous M1 PAMs including
BQCA
(Supplementary Figure S1a) (Kuduk et al, 2010a; Kuduk et al, 2011; Kuduk et al, 2010b;
Shirey et al, 2009). Finally, while BQCA can be used in rats without inducing serious
adverse effects, it induces adverse effects (Thomsen et al, 2012) and fully generalized
convulsive seizures in mice (Supplementary Figure S2) that compromise its utility for use
in behavioral studies in this species. In contrast, VU0453595 has no adverse effect
liabilities in mice (Supplementary Table S4), including the absence of seizure activity at
doses as high as 100 mg/kg (Supplementary Figure S2).
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activation of the M1 muscarinic acetylcholine receptor achieved by allosteric
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Supplementary Figure Legends:
Figure S1: Synthesis and characterization of a novel M1 selective PAM VU0453595.
a. Progression steps for the chemical synthesis of the M1 PAM VU0453595 (Detailed
description in supplementary methods). CLhep (h,r): Hepatic clearance in humans and
rats; PPB fu (h,r): Plasma protein binding- fraction unbound in humans and rats. b. A
VU0453595 concentration response curve (CRC) derived from Ca2+ mobilization assays
performed in CHO cells stably expressing M1 receptors is displayed. The CRC shows
that VU0453595 potentiated the response to a submaximal (EC20) concentration of ACh.
Data were normalized to the maximal response to 10 µM ACh. c. The ability of
VU0453595 to potentiate the response of the M1 receptor to ACh is also shown by a
concentration-dependent leftward shift of the ACh CRC. Data were normalized as a
percentage of maximal response to 10 μM ACh d. [3H]-NMS competition binding,
showing that increasing fixed concentrations of VU0453595 (3 – 30 μM) were able to
increase the affinity of ACh binding to M1 receptors. Experiments were performed using
membranes prepared from M1 CHO cells. Data in (C and D) represent the mean ±
S.E.M. from at least three independent determinations. e. Calcium mobilization assays
were performed using CHO cells stably expressing M2-M5 muscarinic receptors (M2 and
M4 containing cells were co-transfected with Gqi5) to assess selectivity of VU0453595. In
contrast to concentration-dependent potentiation of M1 (B), VU0453595 does not exhibit
any functional activity at the other rat muscarinic receptor subtypes. f. VU0453595
exhibits favorable pharmacokinetics in mice, when dosed intraperitoneally (i.p.) at a 10
mg/kg dose (vehicle: 10% Tween 80). Data represent mean ± S.E.M. from n = 3 animals
per timepoint.
Figure S2: BQCA, but not VU0453595, produces generalized convulsive seizures
in WT mice. BQCA (100 mg/kg; intraperitoneal) produces time-dependent increases in
behavioral convulsions with a modified Racine score of 4, which lasts for at least 6 hours
following BQCA administration. In contrast, a similar dose of VU0453595 (100 mg/kg;
intraperitoneal) failed to produce any signs of behavioral convulsions in mice.
Figure S3: Effect of VU0453595 treatment alone in drug naïve and PCP-treated
mice. Bath application of 10 µM VU0453595 alone for 20 min (solid black line) led to an
initial depression but did not induce any form mLTD in drug naïve (a) as well as PCPtreated mice (b). Representative sample traces are included in the inset (red trace:
during mLTD and black trace: during baseline). c. The magnitude of mLTD was
calculated as the average response 65-70 min following the addition of VU0453595
(shaded gray area) and plotted as a bar graph, indicating no depression of fEPSP
responses during this time period.
Figure S4: VU0453595 can potentiate optically induced endogenous ACh release
dependent mLTD in an M1 dependent manner in ChAT-ChR2-YFP transgenic mice.
a. mLTD following endogenous ACh release in presence or absence of the M1 PAM,
VU0453595 and M1 antagonist VU0255035. Continuous blue light optostimulation (2Hz)
for 15 min (blue shaded area) led to negligible LTD in the PFC of Chat-ChR2-YFP BAC
transgenic mice (blue circles), but pretreatment of 10 µM VU0453595 for 5 min alone
and then in combination with the optical stimulation (red dashed line) potentiated the
optostimulation induced response and led to significantly more mLTD (red circles).
Moreover, treatment of the M1 antagonist VU0255035 (10 µM) simultaneously with the
M1 PAM VU0453595 (gray dashed line) completely blocked the ability of VU0453595 to
potentiate the endogenous ACh release mediated mLTD (gray circles). Sample traces
are shown above. b. Quantification of mLTD following endogenous ACh release in
presence (n = 4; red bar) or absence of VU0453595 (n = 4; blue bar) and in presence of
both VU0255035 and VU0453595 (n = 4; gray bar). mLTD observed in slices with
combined VU0453595 and optical stimulation is significantly more than that observed
with optical stimulation alone or when combined with the M1 antagonist VU0255035.
**denotes < 0.01 Error bars denote S.E.M.
Figure S5: Glutamatergic LTD in mouse PFC is spared following repeated PCP
treatment. a. Application of the Group II metabotropic glutamate receptor agonist
LY379268 (100 nM) for 10 min (solid black line) led to a robust LTD (51.68 ± 8.62%
baseline fEPSP) in mouse (drug naïve) layer V PFC that lasted at least 60 min (gray
sample trace) after application of the compound. b. Interestingly, this form of LTD was
unaffected after repeated PCP treatment as bath application of 100 nM LY379268 (solid
black line) also led to a robust LTD (48.66 ± 3.16% baseline fEPSP), similar in
magnitude and kinetics (gray sample trace), to responses observed in the absence of
PCP treatment. c. Average normalized fEPSP slope during LTD (55-60 min following
drug application; shaded gray area in a and b), is plotted as a bar-graph. Statistical
comparison between the 2 groups revealed no significant differences between them
(unpaired t test; p = 0.71), indicating that this form of glutamatergic LTD is intact in the
PFC of PCP-treated mice.