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Evaluation of alpha7 nicotinic receptor agonist on gabaergic synaptic activity

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NEUROSERVICE
NEUROSERVICE

This document summarizes an experiment evaluating the effects of an alpha7 nicotinic acetylcholine receptor agonist on GABAergic synaptic transmission in rat hippocampal slices. Acute hippocampal slices were prepared from rats and patch clamp recordings were obtained from CA1 pyramidal neurons. The alpha7 nAChR agonist PNU282987 was bath applied at different concentrations. PNU282987 increased the frequency of spontaneous inhibitory postsynaptic currents at concentrations of 30nM and 300nM, but decreased it at 1000nM, possibly due to desensitization of alpha7 nAChRs. The effects were blocked by the GABA-A receptor antagonist bicuculline, confirming the GABA

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www.neuroservice.com Evaluation of an ?7 nicotinic receptor agonist on GABAergic spontaneous synaptic transmission in the hippocampus
www.neuroservice.com SUMMARY Objectives Materials & Methods Preparation of acute rats hippocampal slices Slice perfusion and neurons visualization Patch-clamp recordings and analysis Results
www.neuroservice.com OBJECTIVES To document the pharmacological activity of a selective a7 nAChR agonist in an acute hippocampal slice To illustrate the pre-synaptic modulation of a7 nAChRs on spontaneous GABAergic synpatic transmission
www.neuroservice.com MATERIALS & METHODS Preparation of acute rats hippocampal slices Experiments are carried out with Sprague-Dawley between 3 and 4 week of age Acute horizontal brain slices (400 ?m thickness) are cut with a Leica VT1200S in a ice-cold oxygenated sucrose solution (in mM: sucrose 249, KCl 2.5, MgSO4 2, NaH2PO4 1.2, NaHCO3 26, D-glucose 11 and kynurenic acid 1). Then 4-5 slices containing the hippocampus are incubated at room temperature for at least 1h into an incubation chamber containing ACSF (in mM: NaCl 119, KCl 2.5, CaCl2 2.5, MgCl2 1.3, NaHCO3 25, NaH2PO4 1.2, D-glucose 11). All solutions are continuously bubbled with carbogen gas (95% CO2, 5% O2). Slice perfusion and neurons visualization For experiments, the slices are transferred into a recording chamber containing about 1 ml of the above ACSF and continuously perfused with ACSF at 1.5 ml/min. Neurons are visualized using infrared differential interference contrast (IR-DIC) microscopy and a 60x objective. Pyramidal neurons of the CA1 region are visually identified for recordings.
www.neuroservice.com Patch-clamp recordings and analysis Whole-cell patch clamp experiments are performed in voltage-clamp mode using a software- controlled MultiClamp 700B amplifier in combination with a Digidata 1440A digitizer. Data were low-pass filtered at 2 kHz before being sampled at 10 kHz using Clampex 10 (all from Molecular Devices). The pipette solution contains (in mM): KCl 110, Kgluconate 5, NaCl 5, CaCl2 1, MgCl2 2, EGTA 10, HEPES 10, Na2ATP 2, Na3GTP 0.3, pH 7.2. Cells are held at -60 mV. In order to block postsynaptic spiking activities, 5 mM QX-314(-Cl) are added to the pipette solution. With a holding potential of -60 mV, the GABA-A-evoked currents are inward. Spontaneous inhibitory postsynaptic currents (sIPSCs) are pharmacologically isolated by adding 30 ?M of the NMDA receptor antagonist D-AP5 and 10 ?M of the AMPA/kainate receptor antagonist CNQX to the bath solution. GABAergic spontaneous IPSCs (sIPSCs) are recorded from pyramidal cells of the CA1 area of hippocampus for 10 minutes (control) after observing a stable activity for a 5 minute period. The compound (PNU282987) is then wash-in for 20 minutes and, an additional 10 minutes slot is recorded under the compound. To further verify if the sIPSCs are GABAA-mediated currents, 10 ?M bicuculline are perfused at the end of each experiment into the bath solution to block all the spontaneous events. The frequency of the sIPSCs are analyzed with a proprietary script developed by NEUROSERVICE: automatic sIPSCs detection is performed with margin of errors less than 5% in detection of the overall events. MATERIALS & METHODS
www.neuroservice.com RESULTS Representative traces of recording cells under different conditions: Freq=4,5 Hz Freq=7,2 Hz (57% increase relative to control) 400pA 2s 400pA 2s 400pA 2s Addition of Bicuculline at 10?M block all activity, confirming that synaptic events where GABAergic spontaneous IPSCs ACSF ACSF + PNU282987 300nM Control VS PNU282987 ACSF + PNU282987 300nM + Bicuculline 10?M
www.neuroservice.com RESULTS Statistical analysis of the effect of PNU282987 on the frequency of sIPSCs Cumulative plot of inter-event interval showing that PNU282987 at 300nM increase the frequency of sIPSCs (n=6) Change in the frequency of spontaneous activity relative to baseline and PNU282987 at 30, 300 and 1000 nM (n=6 per concentration). PNU282987 (nM)
www.neuroservice.com CONCLUSIONS The frequency of the GABAergic sIPSCs recorded from pyramidal cells in the CA1 area of the hippocampus changes in the presence of PNU282987 (30, 300, 1000nM) indicating an ?7 nAChRs-mediated modulation of GABAergic minis sIPSCs. As previously shown (Hajos M et al., 2004), concentrations of 30 and 300 nM increase the frequency of sIPSCs whereas 1000 nM decreases it, possibly reflecting the desensitization of ?7 nAChRs. Bath application of bicuculline block all the sIPSC in pyramidal cells, confirming the GABAergic nature of spontaneous IPSCs.
Domaine de St Hilaire 595, rue Pierre CS 30531 13 593 Aix-en-Provence Cedex 3 FRANCE Tel : +33 (0)442 991 220 contact@neuroservice.com www.neuroservice.com www.neuroservice.com

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Evaluation of alpha7 nicotinic receptor agonist on gabaergic synaptic activity

  • 1. www.neuroservice.com Evaluation of an ?7 nicotinic receptor agonist on GABAergic spontaneous synaptic transmission in the hippocampus
  • 2. www.neuroservice.com SUMMARY Objectives Materials & Methods Preparation of acute rats hippocampal slices Slice perfusion and neurons visualization Patch-clamp recordings and analysis Results
  • 3. www.neuroservice.com OBJECTIVES To document the pharmacological activity of a selective a7 nAChR agonist in an acute hippocampal slice To illustrate the pre-synaptic modulation of a7 nAChRs on spontaneous GABAergic synpatic transmission
  • 4. www.neuroservice.com MATERIALS & METHODS Preparation of acute rats hippocampal slices Experiments are carried out with Sprague-Dawley between 3 and 4 week of age Acute horizontal brain slices (400 ?m thickness) are cut with a Leica VT1200S in a ice-cold oxygenated sucrose solution (in mM: sucrose 249, KCl 2.5, MgSO4 2, NaH2PO4 1.2, NaHCO3 26, D-glucose 11 and kynurenic acid 1). Then 4-5 slices containing the hippocampus are incubated at room temperature for at least 1h into an incubation chamber containing ACSF (in mM: NaCl 119, KCl 2.5, CaCl2 2.5, MgCl2 1.3, NaHCO3 25, NaH2PO4 1.2, D-glucose 11). All solutions are continuously bubbled with carbogen gas (95% CO2, 5% O2). Slice perfusion and neurons visualization For experiments, the slices are transferred into a recording chamber containing about 1 ml of the above ACSF and continuously perfused with ACSF at 1.5 ml/min. Neurons are visualized using infrared differential interference contrast (IR-DIC) microscopy and a 60x objective. Pyramidal neurons of the CA1 region are visually identified for recordings.
  • 5. www.neuroservice.com Patch-clamp recordings and analysis Whole-cell patch clamp experiments are performed in voltage-clamp mode using a software- controlled MultiClamp 700B amplifier in combination with a Digidata 1440A digitizer. Data were low-pass filtered at 2 kHz before being sampled at 10 kHz using Clampex 10 (all from Molecular Devices). The pipette solution contains (in mM): KCl 110, Kgluconate 5, NaCl 5, CaCl2 1, MgCl2 2, EGTA 10, HEPES 10, Na2ATP 2, Na3GTP 0.3, pH 7.2. Cells are held at -60 mV. In order to block postsynaptic spiking activities, 5 mM QX-314(-Cl) are added to the pipette solution. With a holding potential of -60 mV, the GABA-A-evoked currents are inward. Spontaneous inhibitory postsynaptic currents (sIPSCs) are pharmacologically isolated by adding 30 ?M of the NMDA receptor antagonist D-AP5 and 10 ?M of the AMPA/kainate receptor antagonist CNQX to the bath solution. GABAergic spontaneous IPSCs (sIPSCs) are recorded from pyramidal cells of the CA1 area of hippocampus for 10 minutes (control) after observing a stable activity for a 5 minute period. The compound (PNU282987) is then wash-in for 20 minutes and, an additional 10 minutes slot is recorded under the compound. To further verify if the sIPSCs are GABAA-mediated currents, 10 ?M bicuculline are perfused at the end of each experiment into the bath solution to block all the spontaneous events. The frequency of the sIPSCs are analyzed with a proprietary script developed by NEUROSERVICE: automatic sIPSCs detection is performed with margin of errors less than 5% in detection of the overall events. MATERIALS & METHODS
  • 6. www.neuroservice.com RESULTS Representative traces of recording cells under different conditions: Freq=4,5 Hz Freq=7,2 Hz (57% increase relative to control) 400pA 2s 400pA 2s 400pA 2s Addition of Bicuculline at 10?M block all activity, confirming that synaptic events where GABAergic spontaneous IPSCs ACSF ACSF + PNU282987 300nM Control VS PNU282987 ACSF + PNU282987 300nM + Bicuculline 10?M
  • 7. www.neuroservice.com RESULTS Statistical analysis of the effect of PNU282987 on the frequency of sIPSCs Cumulative plot of inter-event interval showing that PNU282987 at 300nM increase the frequency of sIPSCs (n=6) Change in the frequency of spontaneous activity relative to baseline and PNU282987 at 30, 300 and 1000 nM (n=6 per concentration). PNU282987 (nM)
  • 8. www.neuroservice.com CONCLUSIONS The frequency of the GABAergic sIPSCs recorded from pyramidal cells in the CA1 area of the hippocampus changes in the presence of PNU282987 (30, 300, 1000nM) indicating an ?7 nAChRs-mediated modulation of GABAergic minis sIPSCs. As previously shown (Hajos M et al., 2004), concentrations of 30 and 300 nM increase the frequency of sIPSCs whereas 1000 nM decreases it, possibly reflecting the desensitization of ?7 nAChRs. Bath application of bicuculline block all the sIPSC in pyramidal cells, confirming the GABAergic nature of spontaneous IPSCs.
  • 9. Domaine de St Hilaire 595, rue Pierre CS 30531 13 593 Aix-en-Provence Cedex 3 FRANCE Tel : +33 (0)442 991 220 contact@neuroservice.com www.neuroservice.com www.neuroservice.com