Weekly weighing indicated no weight loss in TMZ-treated rats (Fig. S1). Bromodeoxyuridine (BrdU; Sigma) was injected intraperitoneally at a dose of 200 mg/kg (concentration, 15 mg/mL) to mark the dividing cells in the dentate gyrus. In the first experiment selleck chemical (Fig. 1A), the overall effect of TMZ on adult hippocampal neurogenesis was examined in naïve adult rats. To evaluate the effect of chemotherapy on a larger population of cells generated

during and surviving past the drug treatment, we injected BrdU multiple times during the first treatment cycle (three daily injections) – BrdU was injected first, and this was followed by a TMZ injection at least 2 h later. Each BrdU injection labeled the population of cells that were in S-phase during the 2 h for which BrdU remains systemic. In all further experiments, BrdU was injected only once, to enable more straightforward determination of the age of the labeled cell population. In the next two experiments Small molecule library clinical trial (Fig. 1B and C), BrdU was injected at different time points with regard to both drug treatment and learning/training, to verify the expected reduction in the number of BrdU-labeled cells caused by TMZ, and to examine possible changes in this reduction. The rats in the first three experiments (Fig. 1A–C) were all euthanised 21 days after the (last) BrdU injection. In the last experiment (Fig. 1D), we assessed the effects of long-term chemotherapy on the size of the proliferating cell population. For

this, BrdU was injected after a total of four cycles of drug treatment, and rats were euthanised 7 days later. It is acknowledged that the repeated injections might act as a stressor, and thus affect the outcome of the experiments. However, the number Cytidine deaminase of injections was the same for rats treated with saline and for those treated with TMZ. In addition, in male rats, stress facilitates rather than impairs learning (Maeng et al., 2010). To assess learning and memory, we used different variations of classical eyeblink conditioning, a type of learning for which the neural basis is well known, and learning does not require

physical activity or exploration. In eyeblink conditioning, a neutral conditioned stimulus (CS) is repeatedly paired with aversive stimulation of the eyelid [unconditioned stimulus (US)]. As a result, the subject learns to blink the eyelid shut in response to the CS. In the trace variant of this task, the CS precedes the US, but the two stimuli do not overlap. In the VLD and delay variants, the CS onset precedes the US, and the two stimuli overlap and coterminate. To study the effects of chemotherapy on hippocampus-dependent associative learning, we trained TMZ/saline-treated rats in trace eyeblink conditioning (Fig. 1B). The same rats were then trained in standard delay eyeblink conditioning, a hippocampus-independent task, to ensure that possible learning deficits observed during trace conditioning were not caused by an overall inability to learn an eyeblink conditioned response.