It will therefore be interesting

to investigate the autoi

It will therefore be interesting

to investigate the autoinhibitory effects of psychotropic drugs accumulated in synaptic vesicles on specific network activity profiles within cortical (Goto et al., 2010) and subcortical (Kellendonk, 2009) pathways as well as various neurotransmitter systems (Lisman et al., 2008), especially DA signaling, and differential effects of other classes of psychotropic drugs (Sulzer, 2011). All animal work was approved by the Kollegiales Leitungsgremium of the Franz-Penzoldt Zentrum, Erlangen and was conducted in conformity with the Animal Protection Law of the Federal Republic of Germany and the guidelines of the State of Bavaria. Hippocampal Raf phosphorylation neuronal cultures were prepared from 1- to 4-day-old Wistar rats. Briefly, newborn rats were sacrificed by decapitation. The hippocampus was removed from each brain and was transferred into ice-cold Hank’s salt solution, and the dentate gyrus was cut away. After digestion with trypsin (5 mg ml−1), cells were triturated mechanically and plated in MEM, DAPT mw supplemented with 10% fetal calf serum and 2% B27 Supplement (all from Invitrogen, Taufkirchen, Germany). If required, neurons were transfected on DIV3 (days in vitro) with spH (Sankaranarayanan et al., 2000)

under the control of a synapsin promoter with a modified calcium phosphate method by Threadgill et al. (1997). Experiments were performed between DIV25 and DIV30. N1E115 neuroblastoma cells were maintained at 37°C in 5% CO2 in DMEM (Invitrogen) with 5 g/l glucose and were supplemented with 10% fetal bovine serum (Biochrom, Berlin) Urease and 1% penicillin/streptomycin solution (Biochrom). Cells were trypsinized and plated in 3.5 cm dishes (Corning, Lowell, MA, USA). For recordings from NaV1.6r, N1E115 cells were transfected on the next day with 1 μg of cDNA for each dish and 0.5 μg of pEGFP-C1 (Mountain View, CA, USA) with Nanofectin (PAA, Pasching, Austria) according to the manufacturers’ protocol.

The mNaV1.6 was a gift from E. Leipold and S. Heinemann (Leipold et al., 2006). The TTX-resistant variant mNaV1.6r Y371S was constructed with the use of the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). Experiments were conducted at room temperature on a Nikon TI-Eclipse inverted microscope (60×, 1.2 NA water-immersion objective; Perfect Focus System). Fluorescence was excited by a Nikon Intensilight C-HGFI through excitation filters centered at 482, 561, and 628 nm using dichroic long-pass mirrors (cutoff wavelength 500, 570, and 660 nm, respectively). The emitted light passed emission band-pass filters ranging from 500 to 550 nm, 570 to 640 nm, and 660 to 730 nm (Semrock, Rochester, NY, USA) and was projected onto a cooled EM-CCD camera (iXonEM DU-885 or iXonEM DU-897; Andor). Coverslips were placed into a perfusion chamber.

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