Three of them are molecules used in several drug preparations and drug testing for medical purposes (fluoxetine, verapamil and kainic acid) and two of them (permethrin and deltamethrin) are from the most commonly used and best described pesticides (pyrethroids, respectively, of types I and II). The compounds used were: 1. R-(()-Fluoxetine hydrochloride1 (FLU, Sigma–Aldrich – F1678), CAS: 114247-09-5. FLU is a serotonin reuptake inhibitor. In both vertebrates and invertebrates, serotonin functions as a neuromodulator to
either Selleckchem Cyclopamine facilitate or inhibit synaptic activity mediated by neurotransmitters (Fink and Göthert, 2007). Mention of trade names or commercial products does not constitute endorsement or recommendation for use. 60-electrode MEA chips have been employed with 30 μm diameter electrodes, 200 μm inter-electrode spacing with an integrated reference electrode (Multichannel Systems GmbH, Reutlingen, Germany). Prior to plating the cells, the MEA chip was sterilized (2 h in oven at 122 °C) and afterwards, to promote cell adhesion and neurite outgrowth,
it was Selleck Doramapimod coated with laminin (Sigma L2020) and poly-d-Lysin (Sigma P6407). Neuronal activity was recorded by the MEA120-2-System from Multi Channel Systems (MCS GmbH, Ruetlingen, Germany, http://www.multichannelsystems.com). The MEA chip was fed into the MEA Amplifier (Gain 1000×) and data were recorded by MC_Rack software at a sampling rate of 10 kHz. A band pass digital filter (60–4000 Hz)
was also applied. The system also includes a heating system connected to a temperature controller (TC02, MCS GmbH) that keeps the MEA chamber at 37 °C. Spikes were detected when the amplitude of the neuronal electrical activity overcame a threshold set at (6.5 times the standard deviation of the mean square root noise; the threshold was VAV2 set at a negative value since the action potentials had a negative voltage peak (see Fig. 1). The recorded signals were then processed to extract parameters related to the spontaneous electrophysiology at both spike and burst level as previously described (Chiappalone et al., 2005). Neuronal cultures were recorded for spike activity from the 3rd to the 5th week in vitro. The experiments were performed on different days using cultures from a minimum of two different isolations. At the beginning of the experimental session a medium change (50%) is performed to establish the “reference activity” and the spontaneous activity which was recorded for 40 min. The medium volume during the experiment is 1000 μl. The experimental protocol is an “accumulative treatment”, and it consists of the administration of 5–8 serial concentrations of each compound or mixture (see Table 1).