Urocanic acid facilitates acquisition of object recognition memory in mice

Trans-urocanic acid (UCA), an isomer of cis-UCA that is mainly located in the skin, has recently been reported to have a role in short-term working memory and in the consolidation, reconsolidation and retrieval of long-term memory. However, its effect on memory acquisition remains unclear. In the present study, the effect of UCA on short-term and long-term memory acquisition in mice was investigated using novel object recognition (NOR) and object location recognition (OLR) protocols that each involved three stages: habituation, sampling and testing. UCA was intraperitoneally injected 0.5 h pre-sampling, and the discrimination index during subsequent testing was determined in NOR and OLR tasks. The results showed that 10 mg/kg UCA significantly facilitated short- term and long-term memory acquisition in both types of tasks. Furthermore, 30 mg/kg UCA significantly facilitated long-term memory acquisition in the NOR task and tended to facilitate long-term memory acquisition in the OLR tasks but did not facilitate short-term memory acquisition in either task. Additionally, the enhancing role of UCA on memory acquisition was not dependent on changes of nonspecific responses, e.g. exploratory behavior and locomotor activity. The current study suggests that UCA facilitates short-term and long-term recognition memory acquisition, which further extends the functional role of UCA in the brain function.


Introduction
Learning and memory are time-dependent, multi-step processes that include acquisition, consolidation, storage, retrieval and extinction. Of these, memory acquisition belongs to the memory encoding stage, which is the process of receiving and transforming the information from the outside world, and its storage within the brain. Multiple brain regions, but particularly the hippocampus and prefrontal cortex, play critical roles in memory acquisition [1], and impairment of this ability is a common feature of neurodegenerative disorders [2]. For example, object recognition memory is impaired in patients with Alzheimer's disease (AD) and in AD mouse models [2][3][4].
Certain types of pharmacological manipulation prior to sampling (i. e. prior to a particular experience or event) have been shown to affect memory acquisition of that experience or event. For example, RS67333, a selective agonist of 5-hydroxytryptamine (5-HT) 4 receptors, enhanced novel object recognition (NOR) acquisition in adult rats [5].
Nociceptin/Orphanin FQ injection 5 min prior to sampling significantly impaired long-term memory performance in an NOR task in mice [6]. Similarly, unilateral intracerebroventricular administration of dihydrokainic acid, an inhibitor of glutamate transporter 1 (GLT1), 30 mins prior to sampling significantly impaired short-term and long-term NOR memory acquisition in mice [7].
Urocanic acid (UCA) is a small molecular compound with two isomers, trans-UCA and cis-UCA. Trans-UCA is naturally synthesized in skin and can be isomerized to cis-UCA when exposed to UV radiation [8,9]. UCA is mainly derived from catabolism of the histidine-rich protein filaggrin. Loss-of-function mutations in the filaggrin gene cause a significantly low level of UCA in the stratum corneum [10,11]. UCA can be transformed to imidazol-propionic acid by urocanase, with further catalyzation to glutamic acid (GA) by imidazolonepropionate hydrolase [8]. Although UCA is present predominantly in skin, it is also found in the liver and the brain [8] and has been shown to have a protective role in various types of diseases [8,9]. Early studies demonstrated a cis-UCA influence on tumorigenesis, possibly via immunosuppression and activation of cell death [8]. Cis-UCA also shows protective capacity against both acute and subacute inflammation in mouse skin [12]. Additionally, cis-UCA exhibits anti-inflammatory effects in intestine, bladder and ocular tissues of rodents [8]. A recent study showed that trans-UCA can cross the blood brain barrier (BBB) and is widely distributed in the central nervous system, most abundantly in the hippocampus and the prefrontal cortex, where it can be metabolized into GA [13]. GA is one of the most abundant neurotransmitters in the central nervous system and plays an indispensable role in learning and memory [14], especially in memory acquisition [15].
Recently we have shown that UCA can facilitate spatial memory and enhance memory consolidation and reconsolidation [16,17]. In the current study, we used novel object recognition (NOR) and object location recognition (OLR) tasks, which are commonly employed in behavioral tests of learning and memory [18], to investigate the effect of UCA on short-term and long-term memory acquisition in mice.

Animals
A total of 148 male adult Institute of Cancer Research (ICR) mice (7 weeks old, weight 30±2 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd and pair-housed in a controlled animal facility with a temperature of 24±1 • C, a humidity of 50±10%, and a 12:12 h light:dark cycle (lights on at 8 a.m). All animals had ad libitum access to water and food. From one week before experimentation began, mice were individually handled for 5 min per day. Animal experiments were performed based on the Guidance for the Care and Use of Laboratory Animals, University of South China (protocol code SYXK2020-0002).

Animal treatment with trans-UCA
Trans-UCA, purchased from Merck Life Science UK Limited (CAS number 104-98-3, 99% purity), was dissolved in sterile saline (vehicle). The doses of UCA (10, 20, 30 mg/kg) were chosen based on our previous studies [16,17] and administered by intraperitoneal (i.p.) injection. To assess the effect of trans-UCA on short-term NOR memory, 40 mice were habituated for 24 h then randomly divided into four groups (10 mice per group), with each group being administered either saline (vehicle control), 10 mg/kg, 20 mg/kg or 30 mg/kg trans-UCA 30 min prior to sampling; to assess the effect of trans-UCA on short-term OLR memory, 32 mice were habituated for 24 h then randomly divided into four groups (8 mice per group), with each group administered either saline (vehicle control), 10 mg/kg, 20 mg/kg or 30 mg/kg trans-UCA 30 min prior to sampling; to assess the effect of trans-UCA on long-term NOR memory, 40 mice were habituated for 24 h then randomly divided into four groups (10 mice per group), with each group administered either saline (vehicle control), 10 mg/kg, 20 mg/kg or 30 mg/kg trans-UCA 30 min prior to sampling; to assess the effect of trans-UCA on long-term OLR memory, 36 mice were habituated for 24 h then randomly divided into four groups (9 mice per group), with each group administered either saline (vehicle control), 10 mg/kg, 20 mg/kg or 30 mg/kg trans-UCA 30 min prior to sampling.

Behavioral testing
For the NOR task, mice were placed in an empty box (30 cm × 30 cm × 60 cm) for 10 min habituation. 24 h later, the mice were placed into the same box, now containing two identical objects, for a sampling period of 3 min (short-term memory task) or 5 min (long-term memory task). After 2 h (short-term memory task) or 24 h (long-term memory task), one of the two objects in the box was replaced with a novel one, and the mice were returned to the box for a test period of 5 min. During the sampling and testing periods, the distance traveled and the exploration time for each object were analyzed. For the OLR task, the procedure was identical to the NOR task except that, instead of being replaced by a novel object during the test period, one of the original objects was moved to a new location within the box. Travelled distance was analyzed by behavioral tracking software (Anymaze 6.16), while the exploration time for each object was determined by an expert observer who was blind to the treatment. Exploratory behavior was judged to be taking place when the mouse's nose was directed towards the object (<2 cm), accompanied by active sniffing or vibrissal sweeping. Discrimination index (DI) was calculated as the difference between the time spent exploring the novel object or location (N) and the familiar object or location (F) divided by the total time exploring both objects or

Statistical analysis
Data was statistically analyzed by SigmaPlot 12.5 software and presented as mean ±SEM. Statistical significance of total exploration time, travel distance and discriminant index was determined by one-way analysis of variance (ANOVA) test with post hoc Tukey test. Before the ANOVA test, normality was tested. p < 0.05 is statistically significant.

UCA facilitated short-term memory performance in the NOR task
In order to evaluate the effect of UCA on memory acquisition, we first determined whether UCA injection 0.5 h pre-sampling could alter shortterm memory performance in the NOR task. Mice were injected with vehicle or UCA at various doses (10, 20, 30 mg/kg body weight) 0.5 h pre-sampling, with the test performed 2 h after the 3 min sampling period. The schematic of the short-term NOR task is illustrated in Fig. 1A. In the short-term memory NOR task, during the sampling the mice in each of the four groups had equal traveling distances ( Fig. 1B; p > 0.05) and total object exploration times ( Fig. 1C; p > 0.05). During the test period, none of the doses of UCA had an effect on travelling distance ( Fig. 1D; p > 0.05) or total exploration time ( Fig. 1E; p > 0.05). However, a 10 mg/kg dose of UCA significantly increased the discrimination index of the mice (Fig. 1F; p < 0.05).

UCA facilitated short-term memory performance in the OLR task
We then determined the effect of UCA injection 0.5 h pre-sampling on short-term memory performance in the OLR task. Mice were injected with vehicle or UCA at various doses (10, 20, 30 mg/kg body weight) 0.5 h pre-sampling, with the test performed 2 h after the 3 min sampling period. The schematic of the short-term OLR task is illustrated in Fig. 2A.
In the short-term memory OLR task, during the sampling the mice in each of the four groups had equal traveling distances ( Fig. 2B; p > 0.05) and total object exploration times ( Fig. 2C; p > 0.05). During the test period, none of the doses of UCA affected the traveling distance ( Fig. 2D; p > 0.05) or total exploration time ( Fig. 2E; p > 0.05). However, a 10 mg/kg dose of UCA significantly increased the discrimination index of the mice (Fig. 2F; p < 0.05).

UCA facilitated long-term memory performance in the NOR task
Next, we aimed to determine the effect of UCA injection 0.5 h presampling on long-term memory performance in the NOR task. Mice were injected with vehicle or UCA at various doses (10, 20, 30 mg/kg body weight) 0.5 h pre-sampling, with the test performed 24 h after the 5 min sampling period. The schematic of the long-term NOR task is illustrated in Fig. 3A. In the long-term memory NOR task, during the sampling the mice in each of the four groups had equal travelling distances ( Fig. 3B; p > 0.05) and total object exploration times ( Fig. 3C; p > 0.05). During the test period, none of the doses of UCA affected the travelling distance ( Fig. 3D; p > 0.05) or total exploration time ( Fig. 3E; p > 0.05). However, 10 and 30 mg/kg doses of UCA significantly increased the discrimination index of the mice (Fig. 3F; both p < 0.05).

UCA facilitated long-term memory performance in the OLR task
We next determined the effect of UCA injection 0.5 h pre-sampling on long-term memory performance in the OLR task. Mice were injected with vehicle or UCA at various doses (10, 20, 30 mg/kg body weight) 0.5 h pre-sampling, with the test performed 24 h after the 5 min sampling period. The schematic of the long-term memory OLR task is illustrated in Fig. 4A. In the long-term memory OLR task, during the sampling the mice in each of the four groups had equal traveling distances ( Fig. 4B; p > 0.05) and total object exploration times ( Fig. 4C; p > 0.05). During the test period, none of the doses of UCA affected the distance traveled ( Fig. 4D; p > 0.05) or total exploration time ( Fig. 4E; p > 0.05). However, a 10 mg/kg dose of UCA significantly increased the discrimination index of the mice (Fig. 4F; p < 0.05).

Discussion
The present study investigated the effect of UCA on short-term and long-term memory acquisition in NOR and OLR tasks in mice. We observed that UCA injection 0.5 h pre-sampling enhanced not only short-term memory performance but also long-term memory performance in NOR and OLR tasks. In both protocols, the UCA-treated mice displayed behavior that indicated an enhanced ability to discriminate between previously-encountered and newly-encountered objects and locations, thus demonstrating that UCA facilitated their ability to remember familiar situations.
Growing evidence strongly suggests that UCA plays a critical role in the memory process [13,16,17]. While a previous study showed that UCA at 25 mg/kg did not influence short-term memory in NOR tasks [13], results from our own earlier work showed that UCA at 10 mg/kg Fig. 1. The effect of UCA (10, 20, 30 mg/kg) injection 0.5 h pre-sampling on short-term memory performance in the NOR task. (A) The timeline of short-term NOR task procedure and UCA injection. (B) Total exploration time and (C) distance traveled during the sampling period. (D) Total exploration time, (E) distance traveled and (F) the discrimination index during the test period. *p < 0.05 VS control group. All data are presented as mean ± SEM. NOR, novel object recognition memory; UCA, urocanic acid; n = 10. enhanced short-term memory in Y-maze tasks [16]. Consistently, our present study showed that mice treated with UCA at a dose of 10 mg/kg but not 20 or 30 mg/kg 0.5 h pre-sampling significantly enhanced memory acquisition in short-term memory tasks.
In addition to its effect on short-term memory, UCA has been shown to influence long-term memory. Our previous studies reported that UCA at 10 mg/kg enhanced long-term memory consolidation and reconsolidation in NOR tasks [17] and enhanced long-term memory consolidation and retrieval in OLR tasks [16]. The present study further confirmed that administration of 10 mg/kg of UCA 0.5 h pre-sampling enhanced long-term memory acquisition in both NOR and OLR tasks.
The mechanisms underlying UCA facilitation of short-term and longterm memory acquisition remain unclear. Previous studies have shown that peripheral injection of UCA resulted in increased UCA levels in the hippocampus and prefrontal cortex (two brain regions that are crucial for learning and memory), promoted the production of glutamate through the UCA-glutamate metabolic pathway, and enhanced glutamatergic transmission, which facilitates memory retrieval in mice [13]. It is reasonable to assume that elevated glutamate level and increased glutamatergic synaptic transmission via glutamate receptors, such as N-methyl-D-aspartate) ionotropic glutamate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ionotropic glutamate receptors (AMPARs) [19], are associated with the potentiating effect of UCA on memory acquisition. In fact, both NMDARs and AMPARs have been reported to mediate memory acquisition [19,20]. Desensitization of glutamate receptors plays a critical role in synaptic transmission and neural function. The degree of desensitization of these receptors depends on the glutamate concentration [21,22]. Whether trans-UCA (dose 10 mg/kg or 30 mg/kg) has a potential effect on desensitization of glutamate receptors, thus mediating short-term and long-term memory acquisition, requires further investigation.
It is also known that trans-UCA can cross the blood-brain barrier and is then converted to its isomer, cis-UCA, which has a chemical structure similar to serotonin (5-hydroxytryptamine, 5-HT), with a relatively  All data are presented as mean ± SEM. OLR, object location recognition memory; UCA, urocanic acid; n = 9.
higher binding affinity to 5-HT 2A receptor and is thus regarded as an agonist of 5-HT 2A receptor [23]. 5-TH 2A receptors are widely expressed in the central nervous system, but are particularly enriched in the amygdala and the hippocampus, which are associated with memory functions [24,25]. Previous work has shown that 5-TH 2A receptor mediates retrieval, consolidation and reconsolidation of object recognition memory in rodents [26][27][28], but it is currently unknown if the promoting effects of UCA on object recognition memory is mediated by 5-HT 2A receptor.
In summary, the current study demonstrates that UCA facilitates short-term and long-term memory acquisition in NOR and OLR tasks. However, the underlying molecular mechanism by which UCA promotes memory acquisition requires further elucidation.

Declaration of Competing Interest
None