Systemic L-Kynurenine sulfate administration disrupts object recognition memory, alters open field behavior and decreases c-Fos immunopositivity in C57Bl/6 mice

Abstract

L-Kynurenine (L-KYN) is a central metabolite of tryptophan degradation through the kynurenine pathway (KP). The systemic administration of L-KYN sulfate (L-KYNs) leads to a rapid elevation of the neuroactive KP metabolite kynurenic acid (KYNA). An elevated level of KYNA may have multiple effects on the synaptic transmission, resulting in complex behavioral changes, such as hypoactivity or spatial working memory deficits. These results emerged from studies that focused on rats, after low-dose L-KYNs treatment. However, in several studies neuroprotection was achieved through the administration of high-dose L-KYNs. In the present study, our aim was to investigate whether the systemic administration of a high dose of L-KYNs (300 mg/bwkg; i.p.) would produce alterations in behavioral tasks (open field or object recognition) in C57Bl/6j mice. To evaluate the changes in neuronal activity after L-KYNs treatment, in a separate group of animals we estimated c-Fos expression levels in the corresponding subcortical brain areas. The L-KYNs treatment did not affect the general ambulatory activity of C57Bl/6j mice, whereas it altered their moving patterns, elevating the movement velocity and resting time. Additionally, it seemed to increase anxiety-like behavior, as peripheral zone preference of the open field arena emerged and the rearing activity was attenuated. The treatment also completely abolished the formation of object recognition memory and resulted in decreases in the number of c-Fos-immunopositive-cells in the dorsal part of the striatum and in the CA1 pyramidal cell layer of the hippocampus. We conclude that a single exposure to L-KYNs leads to behavioral disturbances, which might be related to the altered basal c-Fos protein expression in C57Bl/6j mice.

Keywords: C57Bl/6 mice; L-Kynurenine; anxiety; c-Fos; hippocampus; novel object recognition; open field; striatum.

Figure 1

Effects of L-KYNs treatment on the ambulatory activity of C57Bl/6j mice. (A) Total distance moved (cm). No difference was found between the control and treated groups. (B) Percentage of resting time: treatment elevated the percentage of resting time. Data are shown as means ± SEM (mANOVA; ^*p ≤ 0.05; n = 19 animals).

Figure 2

Effects of L-KYNs treatment on the movement velocity of C57Bl/6j mice. (A) Average speed (cm/s). Treatment significantly accelerated the average speed of the animals. (B) Maximal speed (cm/s). Treatment significantly elevated the maximal speed of the animals. (C) Proportion of high-speed movement (>22.5 cm/s). Treatment significantly elevated the percentage of fast moving. (D) Proportion of low-speed movement (<22.5 cm/s). Treatment significantly diminished the percentage of slow moving. Data are shown as means ± SEM (mANOVA; ^*p ≤ 0.05; ^***p ≤ 0.001; n = 19 animals).

Figure 3

Effects of L-KYNs treatment on the anxiety-related behaviors of C57Bl/6j mice. (A) Percentage of time spent in the central area of the OF arena. Treatment significantly diminished the preference for the central zone. (B) Number of rearings. Treatment significantly diminished the rearing activity of the animals. (C) Time (s) spent expressing stereotypy, e.g., grooming and freezing. Treatment significantly elevated the time of expressing stereotypy behavior. Data are shown as means ± SEM (mANOVA or Mann-Whitney U-test ^**p ≤ 0.01; ^***p ≤ 0.001; n = 19 animals).

Figure 4

Effects of L-KYNs treatment on object recognition memory performance, expressed as discrimination index (DI). The DI of the control group during the choice phase was significantly elevated as compared with the sample phase. However, there was no such difference in the treated group. The DI of the choice phase for treated group was significantly lower than that for the controls. Data are shown as means ± SEM (paired t-test; ^*p ≤ 0.05; independent t-test; ^*p ≤ 0.05; n = 20 animals). [DI: novel × 100/(novel + familiar)].

Figure 5

Effects of L-KYNs treatment on the number of c-Fos⁺ cells in the hippocampal CA1 area of C57Bl/6j mice. (A) Schematic illustration of the hippocampus. The red box (500 × 140 μm) indicates the captured and analyzed subregion of CA1. (B) Representative photomicrographs of c-Fos immunostaining in the CA1. There were a lower number of c-Fos⁺ cells in the L-KYNs-treated group (bottom panel) in comparison with the vehicle-treated group (top panel). Scale bars represent 50 μm. (C) Number of c-Fos⁺ cells in the CA1 area. The number of c-Fos⁺ cells was significantly reduced following L-KYNs administration. Data are shown as median, interquartile ranges ± minimum/maximum values (GLMM, ^*p ≤ 0.05; n = 20 animals).

Figure 6

Effects of L-KYNs treatment on the number of c-Fos⁺ cells in the dorsal striatum of C57Bl/6j mice. (A) Schematic illustration of the striatum. The red line indicates the captured and analyzed subregion of the dorsal striatum. (B) Representative photomicrographs of c-Fos immunostaining in the dorsal striatum. There were a lower number of c-Fos⁺ cells in the L-KYNs-treated group (bottom panel) in comparison with the vehicle-treated group (top panel). Scale bars represent 200 μm. (C) Number of c-Fos⁺ cells in the dorsal part of the striatum. The number of c-Fos⁺ cells was significantly reduced following L-KYNs administration. Data are shown as median, interquartile ranges ± minimum/maximum values (GLMM; ^***p ≤ 0.001; n = 20 animals).