1C), we trained TMZ/saline-treated rats in VLD eyeblink conditioning, a task that is also dependent on an intact hippocampus. We then trained the same rats in trace eyeblink conditioning, to examine whether learning VLD conditioning would facilitate learning this more complex hippocampus-dependent task. In the last experiment (Fig. 1D), rats were trained in

trace eyeblink conditioning until they acquired a robust conditioned response, and then tested for the memory of the conditioned response 3 weeks later. This experiment was conducted to control RG7204 ic50 for the effects of acute, non-specific side effects of TMZ and to further assess the effects of chemotherapy on retention of trace memories. Each rat undergoing eyeblink conditioning was acclimated to

the conditioning chamber by being placed inside for 1 h with the headstage secured. On the next day, training was begun by giving 10 presentations of the white noise (83 dB, 250 ms) to determine whether the rats showed any sensitised responses to the noise. Eyeblink conditioning was then started. White noise was used as a CS, and a 100-ms periorbital shock (0.65 mA) as a US. A trace conditioning trial consisted of a 250-ms CS followed by a 500-ms stimulus-free time interval that separated the CS from the presentation of the US. A delay conditioning trial consisted of an 850-ms CS that overlapped and coterminated with the US. Finally, a VLD conditioning trial consisted of a 1500-ms CS that overlapped and coterminated with the US. Trials were presented with an intertrial interval of 25 ± 5 s. The number of trials per day and the number Regorafenib of days of training for each variation of eyeblink Phospholipase D1 conditioning were determined on the basis of the difficulty of the task evaluated in light of previous experience in our laboratory. Trace conditioning is harder to learn than VLD conditioning (Nokia et al.,

2012), whereas VLD conditioning is harder to learn than delay conditioning. Thus, for trace conditioning, 200 trials/day for up to 6 days were given, for VLD conditioning, 200 trials/day for 4 days were given, and for delay conditioning, 100 trials/day for 4 days were given (Fig. 1). During training, electromyographic (EMG) signals from the upper eyelid and local-field potentials from the hippocampus were recorded. The EMG signal was bandpass filtered between 300 and 500 Hz (1700 Differential AC amplifier; A-M Systems). The local-field potentials were filtered between 1 and 500 Hz (PGA16; MultiChannel Systems, Reutlingen, Germany). All signals were sampled at a rate of 2000 Hz and recorded continuously (Digidata1440 and AxoScope; Molecular Devices, Sunnyvale, CA, USA). Matlab (MathWorks, Natick, MA, USA) was used for data analyses. To determine learned responding from the EMG signals, the signal amplitude was derived with Hilbert transformation. Next the mean and the standard deviation (SD) of the signal during a 250-ms period immediately preceding the onset of the CS were obtained.