Glutamate transporter GLT1 inhibitor dihydrokainic acid impairs novel object recognition memory performance in mice

Accepted Manuscript Glutamate transporter GLT1 inhibitor dihydrokainic acid impairs novel object recognition memory performance in mice

Shao-Wen Tian, Xu-Dong Yu, Lian Cen, Zhi-Yong Xiao PII: DOI: Reference:

S0031-9384(18)30411-6 https://doi.org/10.1016/j.physbeh.2018.10.019 PHB 12348

To appear in:

Physiology & Behavior

Received date: Revised date: Accepted date:

24 June 2018 29 October 2018 29 October 2018

Please cite this article as: Shao-Wen Tian, Xu-Dong Yu, Lian Cen, Zhi-Yong Xiao , Glutamate transporter GLT1 inhibitor dihydrokainic acid impairs novel object recognition memory performance in mice. Phb (2018), https://doi.org/10.1016/j.physbeh.2018.10.019

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ACCEPTED MANUSCRIPT Glutamate transporter GLT1 inhibitor dihydrokainic acid impairs novel object recognition memory performance in mice Shao-Wen Tiana,1*, Xu-Dong Yua,1*, Lian Cena, Zhi-Yong Xiaob

a

Department of Physiology, Hengyang Medical College, University of South China, Hengyang, Hunan,

421001, P.R China; bDepartment of Anesthesiology, The First Affiliated Hospital, University of South

Authors contributed to the paper equally.

*

Corresponding author: Shao-Wen Tian or X-D. Yu

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China, Hengyang, Hunan, 421001, P.R China

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Address: Department of Physiology, Hengyang Medical College,, University of South China, Hengyang, Hunan, 421001, China (S-W. Tian and X-D. Yu)

+86-0734-8281389 (S-W. Tian and X-D. Yu)

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Fax:

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Phone: +86-0734-8281389 (S-W. Tian and X-D. Yu)

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E-mail: [email protected] (S-W. Tian); [email protected] (X-D. Yu)

ACCEPTED MANUSCRIPT Abstract Glutamate transporter GLT1 mediates glutamate uptake, and maintains glutamate homeostasis in the synaptic cleft. Previous studies suggest that blockade of glutamate uptake affects synaptic transmission and plasticity. However, the effect of GLT1 blockade on learning and memory still receives little attention. In the present study, we examined the effect of unilateral intracerebroventricular injection of dihydrokainic acid (DHK), a GLT-1 inhibitor, on novel object recognition (NOR) memory performance.

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The NOR task involved three sessions including habituation, sampling and test. In experiment 1, DHK injection 0.5 h pre-sampling impaired short-term NOR memory performance. In experiment 2, DHK

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injection 0.5 h pre-sampling impaired long-term NOR memory acquisition. In experiment 3, DHK

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injection immediately but not 6 h post-sampling impaired long-term NOR memory consolidation. In experiment 4, DHK injection 0.5 h pre-test impaired long-term NOR memory retrieval. Furthermore,

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DHK-induced memory performance impairment was not due to its effects on nonspecific responses such as locomotor activity and exploratory behavior. The current findings further extend previous studies on

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the effects of disruption of glutamate homeostasis on learning and memory.

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Keywords: GLT1; glutamate; astrocyte; learning; memory

1. Introduction

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Glutamate is the primary excitatory amino acid in the central nervous system, and critically participates in various physiological and pathological processes. Disruption of glutamate homeostasis [1-4]

. Excitatory amino acid

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has been indicated to contribute to a number of major neurological diseases

transporters (EAATs) play a critical role in the synaptic reuptake of glutamate. The EAAT family includes five subtypes, namely EAAT1–5 in the human, and corresponding GLAST, GLT-1, EAAC1, EAAT4 and EAAT5 in rodents [5]. GLAST (EAAT1) and GLT-1 (EAAT2) are predominantly expressed in astrocytes while EAAC1 (EAAT3), EAAT4 and EAAT5 are neuronal-specific transporters [6]. Among of them, GLT-1 is the major glutamate transporter that accounts for >90% of total glutamate uptake in the brain [5, 7]. Increasing evidence has shown that blockade of glutamate uptake leads to aberrant behavioral consequences. For example, intracerebroventricular (i.c.v) injection or microinjection of the GLT-1 inhibitor, dihydrokainic acid (DHK), into the prefrontal cortex induces anhedonia [8, 9]. Microinjection of

ACCEPTED MANUSCRIPT DHK into the amygdala central nucleus produces both depressive-like and anxiety behaviors

[10]

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Intrathecal or i.c.v administration of a non-selective glutamate transporters inhibitor L-trans-PDC induces an inhibitory effect on the micturition reflex paralleling memory training plasticity

[12]

[11]

. GLT1 complex expression levels are

, and blockade of glutamate uptake affects synaptic transmission and

[13-15]

. However, the effect of GLT1 blockade on learning and memory still receives little

attention.

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The novel object recognition (NOR) task is a learning and memory paradigm in which mice or rats show an innate preference for novelty such that they will spend more time exploring the novel object

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once they recognize the familiar one [16]. Currently, this paradigm is widely used to evaluate recognition

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memory in rodents [17]. In the present study, we firstly determined the effect of unilateral i.c.v injection of DHK on short-term NOR memory performance in mice. Then, we determine whether unilateral i.c.v

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injection of DHK affects the acquisition, consolidation and retrieval of long-term NOR memory.

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2. Material and methods 2.1. Animals

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Adult male Kunming strains of Swiss mice (SJA Laboratory Animal, Hunan, China) weighing 35-40 g with sawdust bedding (2~3cm),

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were used. The mice were housed individually in plastic cages

maintained on a 12 h light/darks cycle, with lights on at 7 A.M at a room temperature of 23 ± 1℃. The

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animals were given standard chow and free access to water. All animals were allowed to habituate to the housing conditions and experimenter for 1 week prior to the beginning of experiments. All procedures

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concerning animal care and treatment were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the animal care and use committee at University of South China.

2.2. Surgical procedures and intracerebroventricular (i.c.v) injection The procedures for surgical operation, cannulas placement and i.c.v injection were described in our previous study with some modifications

[18, 19]

. In brief, under anesthesia with sodium pentobarbital

injected intraperitoneally (65 mg/kg), a guide cannula (Mode 62203, RWD Life Science Co., Ltd, Shenzhen, China) was aseptically implanted into the right lateral ventricle of each mouse (stereotaxic coordinates: AP= -0.3 mm, ML=1.0 mm, DV=-2.5 mm ) [20]. After surgery, mice were housed individually and

ACCEPTED MANUSCRIPT allowed to recover for 5 to 7 days before experiment beginning. At the end of the experiment, cannula placement into the lateral ventricle was verified by observation of the presence of Evans Blue dye in the lateral ventricle. DHK (Abcam, Cambridge, UK) was dissolved in 0.01 M PBS (pH 7.4). The doses (6.25, 12.5, 25 nmol in 1 μl; 1μl/mouse) of DHK were derived from a previous study [8]. Drug was delivered at a speed of 0.5 μl/min, and the needle remained in place for 2 min after the injection to allow for adequate

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2.3. General procedures of novel object recognition (NOR) task

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diffusion.

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The NOR task was run in a black Plexiglas training box (30 cm × 30 cm × 30 cm) that was placed in a sound-attenuating cabinet. The objects used in the experiments were plastic cuboids with yellow color (3

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cm long × 3cm wide × 6 cm high) and round glasses with white color (3cm diameter × 6 cm high).The objects were fixed to the floor of the training box, with a 10 cm distance from the wall. Mice showed no

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a spontaneous preference for one of the objects in a pilot experiment (data no shown). The objects and their locations were counterbalanced during the period of behavior assays. Additionally, the floor of the

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training box was divided into twenty five isometric squares. The numbers of complete crossings from

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one square to another were calculated to assess locomotive activity when appropriate. The NOR task involved three sessions including habituation, sampling and test. During the

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habituation session (on day 1), the mice were transported from the housing room to the test room, and introduced into the training box without object for 10 min to habituate them to the training box. During

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the sampling session (on day 2), the mice were introduced into the training box, and permitted to freely explore two identical objects for 5 min (short-term NOR memory) or 10 min (long-term NOR memory). An expert observer recorded the total exploring of the two objects. The test session was conducted 2 h (short-term NOR memory) or 24 h (long-term NOR memory) after the sampling session, respectively. The mice were introduced into the training box in which one of the objects was randomly replaced for a novel object. Mice were permitted to freely explore two objects for 5 min. An expert observer recorded the time spent on exploring each object and the total exploring of the two objects. For assessing NOR memory performance, the discrimination index during the test session was calculated as a percentage of difference in time exploring the novel and familiar object, over the total time spent exploring of the two objects.

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2.4. Experiment designs Experiment 1 was designed to determine the effect of DHK on short-term NOR memory performance. The NOR task was conducted as described above. The mice received unilateral i.c.v injection of vehicle or DHK (6.25, 12.5, 25 nmol) 0.5 h pre-sampling, and were subjected to NOR memory test 2 h after sampling. At the same time, the numbers of complete crossings from one square to another were

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calculated to assess locomotor activity during the sampling and test sessions. The number of samples was N=8 per group. In experiment 1, we found that DHK at the doses of 12.5 and 25 but not 6.25 nmol

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decreased significantly the locomotive activity during the sampling session. Thus, a low dose of DHK

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(6.25 nmol) was chosen in the following experiments.

Experiment 2 was designed to determine the effect of DHK on long-term NOR memory acquisition.

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The NOR task was conducted as described above. The mice received unilateral i.c.v injection of vehicle or DHK (6.25 nmol) 0.5 h pre-sampling, and were subjected to NOR memory test 24 h after sampling.

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Additionally, the numbers of complete crossings from one square to another were calculated to assess locomotor activity during the test session. The number of samples was N=8 per group.

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Experiment 3 was designed to determine the effect of DHK on long-term NOR memory consolidation.

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The NOR task was conducted as described above. The mice received unilateral i.c.v injection of vehicle or DHK (6.25 nmol) immediately or 6 h after sampling, and were subjected to NOR memory test 24 h

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after sampling. The 6 hr time point was selected on the basis of a previous study in which the protein synthesis inhibitor anisomycin administrated immediately but not 6 h after sampling impairs NOR

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memory consolidation [21]. The number of samples was N=9 per group. Experiment 4 was designed to determine the effect of DHK on long-term NOR memory retrieval. The NOR task was conducted as described above. The mice were subjected to NOR memory test 24 h after sampling, and received unilateral i.c.v injection of vehicle or DHK (6.25 nmol) 0.5 h pre-test. The number of samples was N=8 per group.

2.5. Statistical analyses Statistical analyses were finished utilizing Sigma Stat 3.1. One-way analysis of variance (ANOVA) was run to analyse the total exploration time, numbers of crossings and discrimination index. Multiple comparisons were run with the Tukey HSD method. In addition, we ran a one-sample t-test to analyse

ACCEPTED MANUSCRIPT the discrimination index of the mice to chance performance (discrimination index = 0%) when appropriate. Significance level was set at P < 0.05. The data were presented as mean ± SEM.

3. Results 3.1. DHK impairs short-term NOR memory performance in experiment 1 Fig.1 shows the effects of unilateral i.c.v injection of DHK 0.5 h pre-sampling on short-term NOR

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memory performance in experiment 1. During the 5-min sampling session, DHK dose-dependently decreased both the total object exploration time (F (3, 28) = 8.428, P < 0.001; Fig. 1A) and the numbers

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of crossings (F (3, 28) = 16.098, P < 0.001; Fig. 1B). Further multiple comparisons revealed that mice

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treated with DHK at the doses of 12.5 and 25 nmol showed a significant reduction in the total object exploration time (both, P < 0.01) and numbers of crossings (both, P < 0.001) than those of

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vehicle-treated mice. DHK at the dose of 6.25 nmol did not affect the total object exploration time and numbers of crossings (both, P > 0.05). The results indicate that DHK at the doses of 12.5 and 25 nmol

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leads to the exploring behavior and locomotor activity impairments in mice. During the 5-min test session 2 h after sampling, DHK did not influence both the total object

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exploration time (F (3, 28) = 0.815, P > 0.05; Fig. 1C) and the numbers of crossings (F (3, 28) = 1.232,

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P > 0.05; Fig. 1D), but significantly decreased the discrimination index (F (3, 28) = 6.168, P < 0.01; Fig. 1E). Further multiple comparisons revealed that mice treated with DHK at the doses of 6.25, 12.5 and 25

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nmol showed a significant reduction in the discrimination index than that of vehicle-treated mice (P < 0.05, P < 0.01 and P < 0.05, respectively). Additionally, one-sample t-test indicated that mice treated

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with vehicle performed the NOR task well above chance (P < 0.001), and mice treated with DHK at the doses of 6.25, 12.5 and 25 nmol performed the NOR task at chance (all, P > 0.05). As DHK at the doses of 12.5 and 25 nmol affects the exploring behavior and locomotor activity, these observations suggest that DHK at the dose of 6.25 nmol impairs short-term NOR memory performance in mice. Thus, a low dose of DHK (6.25 nmol) was chosen in the following experiments.

3.2. DHK impairs long-term NOR memory acquisition in experiment 2 Fig.2 shows the effects of unilateral i.c.v injection of DHK 0.5 h pre-sampling on long-term NOR memory performance in experiment 2. During the 10-min sampling session, the two groups presented comparable level of the total object exploration time (F (1, 14) = 0.987, P > 0.05; Fig.2A). During the

ACCEPTED MANUSCRIPT 5-min test session 24 h after sampling, DHK did not influence the total object exploration time (F (1, 14) = 0.812, P > 0.05; Fig. 2B) and the numbers of crossings (F (1, 14) = 1.338, P > 0.05; Fig. 2C), but significantly decreased the discrimination index (F (1, 14) = 8.540, P < 0.05; Fig. 2D). Additionally, one-sample t-test indicated that mice treated with vehicle performed the NOR task well above chance (P < 0.001), and mice treated with DHK performed the NOR task at chance (P > 0.05). These observations

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suggest that DHK injection 0.5 h pre-sampling impairs long-term NOR memory acquisition in mice.

3.3. DHK time-dependently impairs long-term NOR memory consolidation in experiment 3

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Fig.3 shows the effects of unilateral i.c.v injection of DHK immediately or 6 h post-sampling on

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long-term NOR memory performance in experiment 3. During the 10-min sampling session, the three groups presented comparable level of the total object exploration time (F (2, 24) = 0.590, P > 0.05; Fig.

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3A). During the 5-min test session 24 h after sampling, DHK did not influence the total object exploration time (F (2, 24) = 2.145, P > 0.05; Fig. 3B), but time-dependently decreased the

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discrimination index (F (2, 24) = 4.981, P < 0.05; Fig. 3C). Further multiple comparisons revealed that mice treated with DHK immediately but not 6 h post-sampling showed a significant reduction in the

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discrimination index than that of vehicle-treated mice (P < 0.05 and P > 0.05). There was no significant

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difference in the discrimination index between mice-treated with DHK 6 h post-sampling and mice treated with vehicle (P > 0.05). Additionally, one-sample t-test indicated that both mice treated with

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vehicle and DHK 6 h post-sampling performed the NOR task well above chance (P < 0.001 and P < 0.01), and mice treated with DHK immediately post-sampling performed the NOR task at chance (P >

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0.05). These observations suggest that DHK injection immediately but not 6 h post-sampling impairs long-term NOR memory consolidation in mice.

3.4. DHK impairs long-term NOR memory retrieval in experiment 4 Fig.4 shows the effects of unilateral i.c.v injection of DHK 0.5 h pre-test on long-term NOR memory retrieval in experiment 4. During the 10-min sampling session, the two groups presented comparable level of the total object exploration time (F (1, 14) = 0.0086, P > 0.05; Fig. 4A). During the 5-min test session 24 h after sampling, DHK did not influence the total object exploration time (F (1, 14) = 1.711, P > 0.05; Fig. 4B), but significantly decreased the discrimination index (F (1, 14) = 5.758, P < 0.05; Fig. 4C). Additionally, one-sample t-test indicated that mice treated with vehicle performed the NOR task

ACCEPTED MANUSCRIPT well above chance (P < 0.001), and mice treated with DHK performed the NOR task at chance (P > 0.05). These observations suggest that DHK injection 0.5 h pre-test impairs long-term NOR memory retrieval in mice.

4. Discussion In the present study, we explored the effects of unilateral i.c.v injection of DHK on NOR memory

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performance, a hippocampal-dependent task[17], in mice. We found that DHK at 6.25 nmol impaired both short-term NOR memory performance in experiment 1, and the acquisition, consolidation and

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retrieval of long-term NOR memory in experiment 2, 3 and 4, respectively. Importantly, DHK at 6.25

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nmol did not affect the numbers of crossings and total object exploratory time 0.5, 2 and 24.5 h after drug injection, suggesting that DHK-induced NOR memory performance impairment is not due to its

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effects on nonspecific responses such as exploratory behavior and locomotor activity. In experiment 1, DHK at higher doses (12.5 and 25 nmol) significantly decreased the numbers of

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crossings and total exploratory time during the sampling but not test sessions, suggesting that DHK at higher doses produces an acute impairment in locomotor activity and exploratory behavior. This impairment might be due to acute excitotoxicity induced by DHK as selective inhibition of GLT1 using

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chronic antisense oligonucleotide administration leads to excitotoxicity and a progressive paralysis[22]. A previous study reported that i.c.v injection of DHK at the doses of 25 and 50 nmol did not affect [8]

. A difference

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locomotor activity during a 30-min period immediately after drug administration in rats in animal species (mouse versus rat) may account for the inconsistent data.

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A large number of studies suggests that these exits a critical time window, usually 1-6 h post-training, only during which pharmacological manipulation or sleep deprivation is proved to be effective on memory consolidation

[21,23]

. In experiment 3, we observed that DHK injection immediately but not 6 h

post-sampling impaired long-term NOR memory performance, suggesting a time-dependent effect of DHK on NOR memory consolidation. This differential effect is consistent with a previous finding that the protein synthesis inhibitor anisomycin administrated immediately but not 6 h after sampling impairs NOR memory consolidation

[21]

. Additionally, DHK injection 6 h post-sampling did not impaired NOR

memory performance, further suggesting that DHK-induced NOR memory performance impairment is not the result of nonspecific responses. Accumulated data suggest that multiple brain regions such as the hippocampus and prefrontal cortex

ACCEPTED MANUSCRIPT are critically involved in NOR memory[24,25]. GLT1 is highly expressed in the hippocampus and pivotal for maintaining glutamate homeostasis

[12, 22]

. Disruption of glutamate homeostasis is usually

associated with hippocampal-dependent learning and memory impairments under normal physiological condition. For example, pharmacological upregulation of GLT-1 impairs hippocampal synaptic transmission and plasticity, and learning in rats

[26-28]

. Pharmacological inhibition of GLT-1 via DHK

disrupts hippocampal-dependent spatial memory in the Morris water maze task in rats[8]. The current

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results indicate that DHK impairs NOR memory performance in mice. Presumably, a dysfunction of glutamate uptake in the hippocampus might be related to NOR memory performance impairment

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induced by DHK. Furthermore, previous studies have shown that i.c.v injection or infusion of DHK into

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the prefrontal cortex or the amygdala produces anhedonia and impairs spatial memory [29-31]. These observations imply that i.c.v injection of DHK might result in a cumulative global disruption of

This issue needs to be clarified in future studies.

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glutamate uptake, which possibly affect non-memory circuits and impair memory performance overall.

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The neurobiological mechanisms underling DHK-induced NOR learning and memory impairments remain unclear. Accumulated evidence shows that astrocyte-neuron lactate shuttle is of crucial important in synaptic plasticity and memory formation, and regulation of neuronal gene expression related to

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synaptic plasticity and memory formation [32-34]. Astrocytic glutamate uptake can trigger glucose uptake and processing via glycolysis, which results in astrocytic lactate production and release

[35]

. GLT1

synaptic transmission

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maintains low glutamate concentrations in the synaptic cleft and ensures a high signal-to-noise ratio in [6,36]

. Inhibition of GLT1 impairs the temporal contingency that is required for

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Hebbian learning[15]. Mutant mice lacking GLT-1 shows impaired long-term potentiation in the hippocampal CA1 region[14]. Possibly, blockade of astrocytic glutamate uptake disrupts neuron–glia metabolic coupling, synaptic plasticity and its underlying molecules, which might underlies DHK-induced NOR learning and memory impairments. In conclusion, we demonstrate that GLT1 blockade by DHK impairs NOR memory performance in mice. The current findings further extend previous studies on the effects of disruption of glutamate homeostasis on learning and memory. Further studies are needed to determine the neurobiological mechanisms underlying DHK-induced NOR memory performance impairment.

Acknowledgements

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Role of funding source This work was supported by the National Natural Science Foundation of China (81771170) and the Hunan Provincial Natural Science Foundation of China (2018JJ3465; 2018JJ2359). The funding sources

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had no further role.

Conflict of interest

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None.

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ACCEPTED MANUSCRIPT Figure captions Fig.1. DHK injection 0.5 h pre-sampling impairs short-term novel object recognition memory performance in experiment 1. The test session was conducted 2 h after sampling session. (A) The total object exploration time during the sampling session; (B) The numbers of crossings during the sampling session; (C) The total object exploration time during the test session; (D) The numbers of crossings during the test session; (E) The discrimination index during the test session. * P < 0.05 and

P < 0.01

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versus mice treated with vehicle.

**

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Fig.2. DHK injection 0.5 h pre-sampling impairs long-term novel object recognition memory acquisition

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in experiment 2. The test session was conducted 24 h after sampling session. (A) The total object exploration time during the sampling session; (B) The total object exploration time during the test

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session; (C) The numbers of crossings during the test session; (D) The discrimination index during the

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test session. * P < 0.05 versus mice treated with vehicle.

Fig.3. DHK injection immediately but not 6 h post-sampling impairs long-term novel object recognition

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memory consolidation in experiment 3. The test session was conducted 24 h after sampling session. (A)

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The total object exploration time during the sampling session; (B) The total object exploration time during the test session; (C) The discrimination index during the test session.

P < 0.05 versus mice

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treated with vehicle.

*

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Fig.4. DHK injection 0.5 h pre-test impairs long-term novel object recognition memory retrieval in experiment 4. The test session was conducted 24 h after sampling session. (A) The total object exploration time during the sampling session; (B) The total object exploration time during the test session; (C) The discrimination index during the test session. * P < 0.05 versus mice treated with vehicle.

ACCEPTED MANUSCRIPT Highlights

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 DHK 0.5 h pre-sampling impairs short-term NOR memory performance  DHK injection 0.5 h pre-sampling impairs long-term NOR memory acquisition  DHK injection immediately but not 6 h post-sampling impairs long-term NOR memory consolidation  DHK injection 0.5 h pre-test impairs long-term NOR memory retrieval

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