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. 2018 Oct:92:82-92.
doi: 10.1016/j.mcn.2018.07.003. Epub 2018 Jul 22.

Physiological signature of a novel potentiator of AMPA receptor signalling

Blanka R Szulc  1 , Stephen T Hilton  2 , Arnaud J Ruiz  3
Affiliations

Physiological signature of a novel potentiator of AMPA receptor signalling

Blanka R Szulc et al. Mol Cell Neurosci. 2018 Oct.

Abstract

We have synthesized a novel small molecule based on the pyrrolidinone-containing core structure of clausenamide, which is a candidate anti-dementia drug. The synthetic route yielded multi-gram quantities of an isomeric racemate mixture in a short number of steps. When tested in hippocampal slices from young adult rats the compound enhanced AMPA receptor-mediated signalling at mossy fibre synapses, and potentiated inward currents evoked by local application of l-glutamate onto CA3 pyramidal neurons. It facilitated the induction of mossy fibre LTP, but the magnitude of potentiation was smaller than that observed in untreated slices. The racemic mixture was separated and it was shown that only the (-) enantiomer was active. Toxicity analysis indicated that cell lines tolerated the compound at concentrations well above those enhancing synaptic transmission. Our results unveil a small molecule whose physiological signature resembles that of a potent nootropic drug.

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Figures

Fig. 1
Fig. 1
Application of BRS–015 in stratum lucidum potentiates dentate–evoked fEPSPs in CA3. In red is the fEPSP amplitude plotted against time showing a rapid and reversible increase following the pressure application of BRS–015 (113.1 ± 22.8%, n = 5, P = 0.007). In black, no significant increase in fEPSP amplitude after local application of ACSF. Data are from 5 slices. Sample traces show fEPSPs from one experiment (stimulus artifacts removed for clarity). Picrotoxin (100 μM), CGP–52432 (5 μM) and d–AP5 (50 μM) are present in the bathing solution. Arrow indicates the onset of BRS–015 or ACSF puffs. Circles represent the mean fEPSP amplitude. Error bars: SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Dose–dependent effect of BRS–015 at mossy fibre synapses. A, Plot of normalized EPSC amplitude against time showing a reversible increase (63.5 ± 12.5%, P = 0.002) in the presence of BRS–015 (100 μM). Consecutive application of the mGluRII agonist DCG–IV (1 μM) depresses EPSCs by 70.7 ± 29.3% (P = 0.01). Data pooled from 5 neurons. Representative current traces for each condition are shown on top. B, No effect of BRS–015 (100 μM) on holding current (∆Iholding: 10.9 ± 7.4 pA, P = 0.21). Horizontal bar: mean. Error bars: SEM. C, BRS–015 (100 μM) does not alter the PPR (control: 1.89 ± 0.09 versus BRS–015: 1.78 ± 0.07, P = 0.14). Sample traces show consecutive paired EPSCs in control condition (black) and in the presence of BRS–015 (red). D, The EPSC τdecay is not affected by BRS–015 (control: 46.8 ± 3.7 ms versus BRS–015: 43.9 ± 2.5 ms, P = 0.45). Example traces show peak–scaled EPSCs from one cell, in control condition (black) and in the presence of BRS–015 (red). Each paired circle represents data from one experiment. E, in red is the mean fractional change in CV−2 plotted against the mean fractional change in EPSC amplitude (BRS–015/baseline ratio of CV−2 = 1.1 ± 0.1, n = 5; P = 0.9). Error bars: SEM. Vectors represent fractional changes in CV−2 and amplitude in individual cells. Responses on the horizontal (y = 1) line depict changes in EPSC amplitude without changes in variance and therefore represent changes in quantal size. The dashed grey line is the 45° identity line. F, Concentration–facilitation relation and fitting with a non–linear logistic function. BRS–015 (1 μM) produces a non–significant increase in EPSC amplitude (7.1 ± 6.5%, n = 4, P = 0.3). BRS–015 (10 μM) increases it by 35.2 ± 18.4% (n = 4, P = 0.15) and BRS–015 (1 mM) by 87.5 ± 5.2% (n = 4; P = 4.E−4). Error bars: SEM. Vertical dashed line indicates the EC50. Picrotoxin (100 μM), CGP–52432 (5 μM) and d–AP5 (50 μM) are continuously present in the bathing solution. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
BRS–015 does not affect kainate and NMDA receptor–mediated EPSCs. A, Kainate receptor–mediated EPSCs isolated by adding picrotoxin (100 μM), CGP–52432 (5 μM), d–AP5 (50 μM), and GYKI–53655 (50 μM) to the bathing solution. Superfusion of slices with BRS–015 (100 μM) has little effect on EPSC amplitude (13.8 ± 7.7%, n = 5, P = 0.17). Sample current traces are shown on top (averages of 5 consecutive trials). B, Superfusion of BRS–015 (100 μM) does not affect NMDA receptor–mediated EPSCs (n = 9; P = 0.75), whereas application of DCG–IV (1 μM) depresses them by 92.2 ± 0.5% (n = 6; P = 0.01). Each point represents the mean current amplitude. Error bars: SEM. NBQX (20 μM), picrotoxin (100 μM) and CGP–52432 (5 μM) are present in the bathing solution.
Fig. 4
Fig. 4
BRS–015 does not affect the electrical membrane properties of CA3 pyramidal neurons and dentate granule cells. A, Voltage deflections recorded in a CA3 pyramidal neuron in response to hyperpolarizing and depolarizing current steps (−20–50 pA, 1 s), in control condition (black) and in the presence of BRS–015 (100 μM, red). There are no significant changes in membrane potential, Rinput, rheobase current, and firing. B, Summary data for membrane potential (control: −67.7 ± 3.6 mV versus BRS–015: −62.9 ± 3.9 mV, P = 0.2); Rinput (control: 206.7 ± 19.5 MΩ versus BRS–015: 171.7 ± 17.3 MΩ, P = 0.053); rheobase current (control: 35.7 ± 5.7 pA versus BRS–015: 35.7 ± 4.8 pA); mean firing frequency (control: 7.4 ± 0.8 Hz versus BRS–015: 7.1 ± 0.7 pA); maximum firing frequency (control: 12.2 ± 1.5 Hz versus BRS–015: 13.5 ± 2.1 Hz, P = 0.21). Data pooled from 7 neurons. C, Voltage deflections recorded in a dentate granule cell in response to hyperpolarizing and depolarizing current steps (−20–50 pA, 1 s), in control condition (black), and in the presence of BRS–015 (100 μM, red). Note the presence of a “sag” at hyperpolarized potential (asterisk). D, Summary data for membrane potential (control: −81.8 ± 1.1 mV versus BRS–015: −78.8 ± 2.1 mV, P = 0.2); Rinput (control: 246.4 ± 18.9 MΩ versus BRS–015: 212.8 ± 27.6 MΩ, P = 0.15); sag ratio (control: 0.99 ± 0.01 versus BRS–015: 0.99 ± 0.03 pA, P = 0.83); maximum firing frequency (control: 102.2 ± 2.1 Hz versus BRS–015: 101.6 ± 1.5 Hz, P = 0.66). Data pooled from 5 granule cells. Each circle represents the data from one experiment. Horizontal bar: mean. Error bars: SEM. The bathing solution contains picrotoxin (100 μM), CGP–52432 (5 μM) and D–AP5 (50 μM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
BRS–015 potentiates glutamate–evoked currents in CA3 pyramidal neurons. Amplitude of puff–evoked glutamatergic currents plotted against time showing a reversible increase (57.6 ± 15.2%, n = 4, P = 0.03) in the presence of BRS–015 (100 μM). Application of NBQX (20 μM) at the end of the experiment strongly depresses the currents, leaving a small residual component. Traces on top show glutamatergic currents (single trials) from one neuron, in control condition and in the presence of BRS–015 (100 μM). Glutamate puff: 5–20 psi, 10–50 ms, every 60 s (grey circle). Error bars: SEM.
Fig. 6
Fig. 6
(−) BRS–015 is more potent than piracetam at low concentration. A, Plot of EPSP amplitude against time showing an increase (51.8 ± 15.1%, n = 6, P = 0.02) in the presence of (−) BRS–015 (100 μM). The consecutive application of DCG–IV (1 μM) depresses fEPSPs by 91.1 ± 7.2% (n = 5; P = 2E−4). Example voltage traces from one experiment are shown on top for each condition (stimulation artifacts truncated for clarity). B, Superfusion of (+) BRS–015 (100 μM) has no significant effect on fEPSP amplitude. DCG–IV (1 μM) depresses fEPSPs by 83.9 ± 11.3% (n = 5; P = 0.04). Sample traces show fEPSPs from one experiment (stimulus artifact removed for clarity). C, Summary data for (+/−) BRS–015 (100 μM): 49 ± 18.9%, n = 5, P = 0.04; DMSO 0.1%: 1.7 ± 2.2%, n = 2; piracetam (100 μM): 2.3 ± 6.7%, n = 3; piracetam (500 μM): 26.5 ± 10.1%, n = 3, P = 0.11; (+) BRS–015 (100 μM): 0.2 ± 3.2%, n = 5, and (−) BRS–015 (100 μM): 51.8 ± 15.1%, n = 6, P = 0.02. Error bars: SEM. *, P < 0.05, paired t–test.
Fig. 7
Fig. 7
BRS–015 induced modulation of mossy fibre LTP. A, Time course of normalized fEPSP amplitude in slices continuously superfused with BRS–015 (100 μM, red) and in untreated slices (black), and depression by the mGluR II agonist DCG–IV (1 μM). There is a reduced mossy fibre LTP in the presence of BRS–015 (100 μM). B, Cumulative probability distribution of mossy fibre LTP measured as the percentage of fEPSP potentiation 15–20 min after tetanus compared with control period. LTP in slices treated with BRS–015 (111.51 ± 16.81%, n = 6) is smaller than that elicited in untreated slices (182.9 ± 20.4%, n = 8, P = 0.02). *, unpaired t–test. C, Time course of normalized fEPSP amplitude showing a non–significant increase (17.9 ± 13.5%, n = 4, P = 0.2) when BRS–015 (100 μM) is applied during mossy fibre LTP. fEPSPs elicited by stratum radiatum stimulation remain largely unaffected (13.3 ± 14.9% reduction, n = 4, P = 0.19). Addition of DCG–IV (1 μM) depresses fEPSPs evoked by stratum granulosum (s. g.) stimulation (70.9 ± 7.4%, n = 4, P = 0.03) but has no effect on stratum radiatum (s. rad) evoked responses. Arrow indicates the time of tetanic stimulation. D, Example traces from a single experiment depicted in panel C. E, Normalized fEPSP amplitude plotted against time showing PTP of fEPSPs (57.9 ± 12.7%, n = 11) after a stimulus burst is delivered in stratum granulosum (HFS1, 100 stimuli in 1 s). Subsequent superfusion with BRS–015 (100 μm) increases the amplitude of fEPSPs by 48.4 ± 9.6% (n = 11, P = 0.05). It has no effect on stratum radiatum evoked responses (6.2 ± 7.6%, n = 5, P = 0.3). Blue arrow indicates the time of a stimulus intensity reset at the dentate electrode when the enhancing effect of BRS–015 reaches a plateau. A second stimulus burst (HFS2, identical to HFS1) delivered after 20 min of application of BRS–015 leads to early LTP (174.3 ± 15.1%, n = 6, P = 0.04). Final application of DCG–IV (1 μM) depresses stratum granulosum evoked fEPSPs by 82.8 ± 3.8% (n = 4, P = 0.008) and has no effect on those elicited by stratum radiatum stimulation. Each point represents the mean. Error bars: SEM. Picrotoxin (100 μM), CGP–52432 (5 μM) and d–AP5 (50 μM) are included in the perfusion solution. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Supplementary Scheme 1
Supplementary Scheme 1
Synthesis of BRS–015. N–Boc–phenylalanine methyl ester was N–methylated and deprotected to generate N–methylphenylalanine methyl ester (2) in 79% yield over two steps. Dehydration using tert–butyl hypochlorite and acylation with diacetoxyacetyl chloride (5) followed by cyclisation in neat boron trifluoride diethyl etherate gave BRS–015 in 72% yield.
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