Transverse hippocampal slices were obtained from P21–P30 Chrna2-cre/R26tom and wild type littermate mice, Chrna2-cre/Viaatlx mice, and 1–2 month-old hChR2 carrying mice of either sex (see Virus injection) as previously described and according to the rules of Animal Experimentation of the Uppsala University. Slices were maintained in artificial CSF (in mmol: 124 NaCl, 3.5 KCl, 1.25 NaH2PO4, 1.5 MgCl2, 1.5 CaCl2, 24 NaHCO3, 10 glucose), constantly bubbled with 95% O2 and 5% CO2. Borosilicate glass electrodes (resistance = 4–8MΩ for somatic recordings; 12–18MΩ for dendritic recordings) were filled with either K-gluconate or CsCl-based internal solution49 (link). Current/voltage clamp recordings were obtained from using either a Dagan BVC700 (Dagan), Axopatch 200B or a Multiclamp 700B (Molecular Devices) amplifiers; data was acquired by National Instruments DAQ cards and winWCP (Dr John Dempster, Strathclyde University, UK). No differences between firing and passive membrane properties and morphology of CA1 OLM cells were found between Chrna2-cre and WT littermates (n=153 cells); therefore, data is presented only from mice carrying Cre recombinase. Postsynaptic currents were obtained in voltage clamp at a holding potential of −60mV using a CsCl-based internal solution (Cl− reversal potential = 0mV).
Extracellular field EPSP (fEPSP) recordings (LTP experiments) were obtained by placing a concentric stimulation electrode (FHC) either at SR or SLM (for SC or TA stimulation, respectively) as previously described14 (link). A borosilicate glass pipette (4–8MΩ) filled with ACSF was used to record SC or TA fEPSPs at the CA1 region 200–400 µm away from the stimulation electrode. Stimulation strength was adjusted to obtain 50–60% of the maximum fEPSP amplitude followed by 20min recordings (200ms pulses delivered every 20s) to obtain a stable fEPSP baseline. Stimulus-response curves were obtained from fEPSPs slopes and synaptic potentiation was induced by weak theta burst stimulation (wTBS; two bursts of four pulses at 100 Hz spaced by 200 ms). The following drugs were bath applied to brain slices: tetrodotoxin (1µM), methyllycaconitine citrate (MLA, Tocris, 10nM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, Sigma, 10uM), d-(−)-2-Amino-5-phosphonopentanoic acid (dAP5, Sigma, 30µM), picrotoxin (PTX, Sigma, 10uM), mecamylamine hydrochloride (MEC, Sigma, 25µM), and (−)-Nicotine ditartrate (Nic, Tocris, 1µM).
Extracellular field EPSP (fEPSP) recordings (LTP experiments) were obtained by placing a concentric stimulation electrode (FHC) either at SR or SLM (for SC or TA stimulation, respectively) as previously described14 (link). A borosilicate glass pipette (4–8MΩ) filled with ACSF was used to record SC or TA fEPSPs at the CA1 region 200–400 µm away from the stimulation electrode. Stimulation strength was adjusted to obtain 50–60% of the maximum fEPSP amplitude followed by 20min recordings (200ms pulses delivered every 20s) to obtain a stable fEPSP baseline. Stimulus-response curves were obtained from fEPSPs slopes and synaptic potentiation was induced by weak theta burst stimulation (wTBS; two bursts of four pulses at 100 Hz spaced by 200 ms). The following drugs were bath applied to brain slices: tetrodotoxin (1µM), methyllycaconitine citrate (MLA, Tocris, 10nM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, Sigma, 10uM), d-(−)-2-Amino-5-phosphonopentanoic acid (dAP5, Sigma, 30µM), picrotoxin (PTX, Sigma, 10uM), mecamylamine hydrochloride (MEC, Sigma, 25µM), and (−)-Nicotine ditartrate (Nic, Tocris, 1µM).
