Effects of chemotherapeutic agents 5-fluorouracil and methotrexate alone and combined in a mouse model of learning and memory

Abstract

Rationale: The concern that adjuvant cancer chemotherapy agents cause cognitive impairment in a significant number of patients has been expressed by patients and healthcare providers, but clinical studies have yielded conflicting results to date.

Objective: We directly tested two commonly used chemotherapeutic agents in a mouse model of learning and memory.

Materials and methods: In the present study, mice were conditioned to respond for a liquid reinforcer (Ensure solution) in the presence of an audible tone on day 1 as a measure of acquisition and were then required to perform the same response on day 2 as a measure of retrieval and retention. Methotrexate and 5-fluorouracil were administered prior to the day 1 session.

Results: Methotrexate (1.0-32 mg/kg) alone failed to alter mean latency acquisition, retrieval, or reinforced response rates. Similar to scopolamine, a known amnesic in this assay, 5-fluorouracil (3-75 mg/kg) failed to alter response rates or acquisition latency on day 1 but significantly altered latency to retrieve a previously learned response on day 2. In combination, 3.2 mg/kg methotrexate plus 75 mg/kg 5-fluorouracil significantly increased day 1 and day 2 acquisition and retrieval latencies without altering response rates or motivation to respond as measured by progressive ratio responding.

Conclusion: Taken together, these data demonstrate that 5-fluorouracil causes increased latencies for retrieval of previously learned behavioral responses and that combination of chemotherapeutic agents may produce greater delays than either agent alone, including when neither agent alone does so.

Fig. 1

Effects of scopolamine (upper panels) and methylscopolamine (lower panels) on autoshaped-operant responding on day 1 (open squares) and day 2 (filled squares) in male, Swiss–Webster mice. Left panels The adjusted latency (latency to the tenth reinforcer minus the latency to the first reinforcer). Previous administration of scopolamine significantly increased adjusted latency on day 2 (F_{(4, 32)}=6.7, p<0.0006) compared to saline. Middle panels Rate of nose-poke responses per second in the dipper well. Dipper response rate was slower (F_{(4, 41)}=3.6, p<0.01) on day 2 in mice treated with scopolamine compared to saline-treated mice. Right panels Rate of nose-pokes in either the right or left holes per second as a measure of general activity. Non-reinforced nose-poke rate was higher (F_{(4, 41)}=3.5, p<0.02) on day 2 after previous administration of scopolamine compared to saline. Total number of mice tested on day 1/number of mice completing ten reinforcers on day 1 and therefore included on day 2: scopolamine [1 mg/kg (6/6); 3.2 mg/kg (6/5); 5.6 mg/kg (10/6); 10 mg/kg (8/5)] and methylscopolamine [1 mg/kg (6/4); 10 mg/kg (6/5); 32 mg/kg (4/4)]. Abscissa Dose in milligram per kilogram administered i.p. Points above S are the effects in two groups of saline-treated mice tested 5 months apart and averaged into one group mean represented in Figs. 1 and 2. Vertical lines represent SEM. Asterisk Significantly different than saline control by Dunnett’s multiple comparison tests, p<0.05. Caret Significantly different than day 1 as determined by paired t tests: mean-adjusted latency, p<0.01; dipper response rates, p<0.0001; non-reinforced nose-poke rate, p<0.02

Fig. 2

Effects of the chemotherapeutic agents methotrexate (upper panels) and 5-fluorouracil (lower panels) on day 1 measures (open squares) and day 2 measures (filled squares) in male, Swiss–Webster mice. Adjusted latency on day 2 was significantly longer (F_{(3, 28)}=4.8; p<0.01) after treatment with 5-fluorouracil than after saline. Total number of mice tested on day 1/number of mice completing ten reinforcers on day 1 and therefore included on day 2 per dose: methotrexate [1 mg/kg (9/7); 3.2 mg/kg (6/6); 10 mg/kg (9/8); 32 mg/kg (11/9)] and 5-fluorouracil [3 mg/kg (12/8); 30 mg/kg (6/5); 75 mg/kg (6/5)]. Abscissa Dose in milligram per kilogram administered i.p. Asterisk Significantly different than saline control determined by Dunnett’s multiple comparison tests, p<0.05. Caret Significantly different than day 1 as determined by paired t tests: mean-adjusted latency, p<0.01; dipper response rates, p<0.0001; non-reinforced nose-poke rate, p<0.02. Other details as in Fig. 1

Fig. 3

Effects of 5-fluorouracil (5-FU) alone (open squares and closed squares) and in combination with methotrexate (MTX) (open and closed circles) on autoshaped responses on day 1 (upper panels) and day 2 (lower panels). Combined administration of 3.2 mg/kg methotrexate and with 5-fluorouracil altered day-1-adjusted latency (F_{(3, 23)}=4.4, p<0.02) relative to the effects of 3.2 mg/kg methotrexate alone and produced a decrease in day 1 dipper response rates (F_{(3, 23)}=3.3, p<0.04). On day 2, adjusted latency was increased by combinations of 3.2 mg/kg (F_{(3, 21)}=50, p<0.0001) and 32 mg/kg methotrexate (F_{(2, 18)}=8.2, p<0.004) with 5-fluorouracil relative to methotrexate alone. In addition, 3.2 mg/kg methotrexate and 75 mg/kg 5-fluorouracil produced longer adjusted latencies than 75 mg/kg 5-fluorouracil alone (p<0.01). Day 2 dipper response rates were significantly different than methotrexate alone for 3.2 mg/kg (F_{(3, 23)}=3.3, p<0.04) and 10 mg/kg (F_{(3, 26)}=5.2, p<0.007) methotrexate in combination with 5-fluorouracil. Total number of mice tested on day 1/number of mice completing ten reinforcers on day 1 and therefore included in day 2 per dose: [3.2 mg/kg methotrexate+3 mg/kg 5-fluorouracil (6/6), +30 mg/kg 5-fluorouracil (6/6), +75 mg/kg 5-fluorouracil (6/4); 10 mg/kg methotrexate+3 mg/kg 5-fluorouracil (6/6), +30 mg/kg 5-fluorouracil (6/5), +75 mg/kg 5-fluorouracil (6/6); 32 mg/kg methotrexate+30 mg/kg 5-fluorouracil (6/4), +75 mg/kg 5-fluorouracil (6/5)]. Points above MTX are the effects of 3.2, 10, or 32 mg/kg methotrexate alone tested 3–6 months apart in two separate groups of mice per dose (Fig. 2). Asterisk Significantly different than methotrexate alone, p<0.05. Caret Significantly different than 75 mg/kg 5-fluorouracil alone, p<0.05 (lower left panel), p<0.0004 (lower middle panel). Other details as in Fig. 1

Fig. 4

Effects of drugs on the motivation to respond for Ensure solution in mice. Doses of 10 mg/kg scopolamine (N=7), 75 mg/kg 5-fluorouracil alone (N=9) and in combination with 3.2 mg/kg methotrexate (N=7), or 3.2 mg/kg mCPP (N=12) administered i.p. prior to the session (day 1) or 24 h prior to the session (day 2). Scopolamine significantly increased breakpoint (F_{(2, 20)}=3.9; p<0.05), whereas mCPP decreased breakpoint (F_{(2, 35)}=17.6; p<0.0001). Ordinate Final number of ratios completed (left axis) and total number of responses emitted (right axis) on the log PR schedule. Abscissa Saline injection on the day prior to testing (baseline responding), drug injection 30 min prior to testing day 1, saline injection 30 min prior to testing on day 2 and 24 h after drug injection on day 1. Star Significantly different (p<0.05, p<0.01, respectively) than baseline responding. Vertical bars represent SEM