Long-term effect of uncomplicated Plasmodium berghei ANKA malaria on memory and anxiety-like behaviour in C57BL/6 mice

Abstract

Background: Cerebral malaria, the main complication of Plasmodium falciparum infection in humans, is associated with persistent neurocognitive sequels both in human disease and the murine experimental model. In recent years, cognitive deficits related to uncomplicated (non-cerebral) malaria have also been reported in chronically exposed residents of endemic areas, but not in some murine experimental models of non-cerebral malaria. This study aimed at evaluating the influence of uncomplicated malaria on different behavioural paradigms associated with memory and anxiety-like parameters in a murine model that has the ability to develop cerebral malaria.

Methods: Plasmodium berghei ANKA-infected and non-infected C57BL/6 mice were used. Development of cerebral malaria was prevented by chloroquine treatment starting on the fourth day of infection. The control group (non-infected mice) were treated with PBS. The effect of uncomplicated malaria infection on locomotor habituation, short and long-term memory and anxious-like behaviour was evaluated 64 days after parasite clearance in assays including open field, object recognition, Y-maze and light/dark tasks.

Results: Plasmodium berghei ANKA-infected mice showed significant long-lasting disturbances reflected by a long-term memory-related behaviour on open field and object recognition tasks, accompanied by an anxious-like phenotype availed on open field and light-dark tasks.

Conclusions: Long-term neurocognitive sequels may follow an uncomplicated malaria episode in an experimental model prone to develop cerebral malaria, even if the infection is treated before the appearance of clinical signs of cerebral impairment.

Keywords: Anxiety; Behavioural impairment; C57BL/6 mice; Memory; Plasmodium berghei; Uncomplicated malaria.

Fig. 1

Experiment flowchart. Plasmodium berghei ANKA-infected C57BL/6 mice received chloroquine treatment for 7 days, starting on the day 4 of infection. Mice were assessed for performance regarding the indicated behavioural tasks on memory and anxiety-like evaluation 64 days after treatment (resting time)

Fig. 2

Effect of chloroquine (CQ) administration for full treatment of PbA infection in C57BL/6 mice. CQ was given by gavage at a concentration of 25 mg/kg daily for 7 days (from day 4 to day 10 of infection, n = 10, combining 2 experiments). This therapeutic scheme does not allow recrudescence of parasitaemia for up to 4 months of follow-up

Fig. 3

Influence of chloroquine (CQ) administration on recognition memory and anxiety-like parameters in non-infected animals. CQ was used by gavage at a concentration of 25 mg/kg daily for 7 days (from day 4 to day 10 of infection, n = 10, combining 2 experiments). This therapeutic scheme does not affect the recognition memory (training a; test b) and the anxiety-like parameters (c and d; n = 24, combining 2 experiments) performed 64 days after the administration of CQ on the NO Recognition and Light/Dark task. Control: PBS treated mice. Abbreviations: FO, familiar object; NO, novel object

Fig. 4

PbA-infected mice show long-lasting impairment in memory-related task performance. Effect of a treated PbA 4 day-infection on the total distance travelled in open field training and test sessions (a) and on the Δ between the first and the third minute distance travelled (b); effect of PbA 4 day-infection on the time spent in each object in the training (c) and test (d) sessions on the novel object recognition task; and effect of treated PbA 4 day-infection on time spent in each arm in the training (e) and test (f) sessions in the Y-Maze task. Data were analysed by two-way ANOVA, followed by Tukey’s multiple range post-hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001, comparing the training (OFT1) and test (OFT2) sessions; the familiar (FO) and the novel (NO) object recognition task; and the different arms in the Y-maze task (n = 22 animals/group, combining 2 experiments). See Methods for behavioural task description. Arm 1 (closed and open on training and test sessions, respectively), arms 2 and 3 (open). Abbreviations: Non-inf mice, uninfected and PBS treated mice; PbA-inf mice, infected and chloroquine treated mice

Fig. 5

PbA-infected mice presented long-lasting increase in anxious-related behaviour. The distance travelled (a) in the centre zone was evaluated in the open field task training session (n = 22, combining 2 experiments). The time spent in the light compartment (b) was evaluated in the light/dark task (n = 12). Data are reported as the mean ± SEM and were analysed by unpaired Student’s t-test. *P < 0.05 compared to the non-infected animals. See Methods for behavioural task description. Abbreviations: Non-inf mice, uninfected and PBS treated mice; PbA-inf mice, infected and chloroquine treated mice