Loss of GDE2 leads to complex behavioral changes including memory impairment

Mice were anesthetized with Avertin solution and perfused transcardially with 0.1 M phosphate buffer, pH7.4 (PB), followed by 4% paraformaldehyde (PFA) in 0.1 M PB as previously described [20]. The brain tissues were harvested, post-fixed in 4% PFA overnight, and then transferred to 30% sucrose at 4C for at least 3 days. The tissues were embedded in O.C.T, flash frozen, and sectioned at 20–30 µm on Leica 3050S.

RNAscope coupled with immunohistochemistry was carried out using the RNAscope Multiplex Fluorescent v2 Assay with the Co-Detection kit according to the manufacturer’s protocol. Gde2 (Gdpd5) mRNA was visualized using the probe Mm-Gdpd5-C2 (ACDBio, Cat No. 494181-C2) and Opal-570 fluorophore (Akoya Biosciences, FP1488001KT, 1:500), and costained with NEUN (Millipore Sigma, MAB377, 1:500). Slides were mounted with ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher, P36931), coverslipped, and imaged on confocal microscope Zeiss LSM800.

All procedures involving animals were performed in accordance with the guidelines of the Johns Hopkins University Institutional Animal Care and Use Committee. Male and female Gde2KO and WT mice on a C57BL/6J strain background were used for all tests and were genotyped and maintained as previously described [18]. To obtain these mice, breeding cages were set up with 1 male and 2 female mice, all three of which were either Gde2KO or WT, so that the pups in each litter were all one genotype. This breeding scheme was utilized in order to generate a large number of animals (> 70) in a short period of time to obtain the appropriate cohort sizes. To minimize variation, all animals were of the same genetic background and generated from the same number of crosses. Two cohorts of 30–40 mice, 1.5 months apart in age, with approximately equal numbers of Gde2KO and WT mice were tested in the same sequence of behavioral experiments. Results from both cohorts were pooled together for analysis for all tests. Mice were kept in cages where up to 5 males and females were housed together separated by sex. All mice were inspected before any behavioral tests were conducted and any that displayed injuries or health problems were removed from the cohort. Mice were housed in a 12-h light/dark cycle, where mice were tested at the start of the light cycle in rooms and chambers in which the light was turned on and sound disturbances were minimized.

All tests were performed by DD and AW in isolated behavior rooms with ambient temperatures with low background noise levels. Mice were handled daily for 3 days before the start of all behavioral testing. Testing was conducted during the light phase of the circadian cycle, with experimenters blinded to genotype while testing was performed. Before every test, mice were transferred to the testing room to habituate to the new location for at least 30 min. Behavioral performances in the Open Field (OF), Y maze, plus maze, Fear Conditioning (FC), Social Motivation, and Morris Water Maze (MWM) tests were recorded by a computer-based video tracking system (ANY-maze, Stoelting Co., IL). For any tests where unexpected noises could cause startle responses and disrupt the tests, gentle lo-fi music was played to provide quiet background sound. PPI of acoustic startle reaction (ASR) was conducted in a startle soundproof chamber (model SR-LAB, San-Diego Instruments, CA). In the two tests that were performed at two different ages (OF and Y maze), conditions were altered slightly between testing ages; specifically, we utilized testing apparatuses that were constructed of different materials and changed the room in which the tests were conducted to prevent initial testing memories from interfering with the performance of the mice. Since no learning or memory deficits were observed in tests conducted at the 7-month time point and Fear Conditioning and Morris Water Maze are stressful tests for animals, we conducted these tests at a single time point (11- and 16-months, respectively). The 5-month gap was to allow the mice sufficient recovery time before embarking upon the Morris Water Maze test. For tests that were conducted at the same age point, at least 5 rest days were given to mice between different tasks to allow for sufficient recovery from previous exposure to novel environment and equipment.

Locomotor activity and anxiety measurements in Open Field (OF) experiments were assessed in two different settings depending on the age of the mice using similar equipment and protocols as previously described [23]. At the 7-month time point, mice were placed in a clear plexiglass OF arena box (40 cm by 40 cm by 40 cm) for 30 min with the lights off. At the 16-month time point, mice were placed in a different OF arena box made of white plastic (37 cm by 37 cm by 35 cm) for 45 min with the lights turned on. Before each trial, the cages were rubbed with bedding from the home cage, cleaned with a 30% ethanol solution, and dried with a paper towel. The trial began with the mouse being released near the OF wall. The total distance traveled during each block of 5 min, along with the distance and percentage of time spent in the center vs perimeter of the box as measures of anxiety-like behavior, were analyzed. The percent of time spent in the center area due to chance was 34%.

Mice were tested in a Y-shaped maze using a similar protocol as previously described [24]. Each maze arm measures 7 cm by 48 cm. For the 7-month test, a wooden Y maze was used, while the 16-month test was conducted in a different behavioral room with a metal Y maze. The different room, maze color and texture, and spatial cues were all used to prevent similarities between the tasks. Both mazes were elevated 70 cm off the ground. Before each mouse was tested, the maze was rubbed with bedding from the home cage, cleaned with 30% ethanol solution, and dried. In trial 1, mice were placed in one end of the start arm while one of the remaining two arms was blocked off. The arm selected to be blocked off alternated for each mouse tested. All mice explored the maze freely for 5 min. The number of arm entries and locomotor activity were recorded using ANY-maze software (Stoelting Co., IL). Trial 2 began 30 min after the initial exploration period. The arm block was removed, and mice were placed at the end of the start arm and allowed to explore all 3 arms for 5 min. The time spent in the novel arm compared to the previously explored arm in the second trial and the total distance traveled in both trials were analyzed using the ANY-maze software.

Mice were allowed to run freely for 10 min in an elevated plus maze to assess motor activity in this environment and evaluate how much time the mice spent in the closed arms vs. open arms as a metric of anxiety-like behavior using similar equipment and protocols as previously described by Savonenko et. al. [25]. The plus maze consisted of 2 open arms and 2 closed arms (all measuring 50 cm × 10 cm) extending from an open square platform (10 cm × 10 cm). All arms were made of wood with plastic walls (40 cm high) for the closed arms and were angled at 90 degrees to each other (see Fig. 2K). Like the Y maze, the plus maze was elevated on 70 cm tall metal poles. Using ANY-maze software, the total distance traveled by the mice and the time spent in the open and closed arms were tracked. Before starting the trials, the maze was wiped down with bedding. Before each trial started, the maze and walls were wiped down with a 30% ethanol solution and dried with disposable paper towels.

The social preferences of mice were tested in a three-chamber apparatus using similar protocols and equipment as previously described [26]. The apparatus was constructed from opaque Plexiglas (60 cm long × 40 cm wide × 35 cm high) with 2 middle dividers with cutout doors dividing the chamber into 3 equal-sized compartments (see Fig. 4A). During the 5-min habituation trial, mice were allowed to roam freely in the compartments. In both side chambers, there was one inverted empty mesh metal cup holder. After a 30-min delay in which the test mouse was removed to a waiting cage, the mouse was put back in the middle compartment for the social motivation trial and again allowed to explore for 5 min. A 2-month-old male WT mouse on a C57BL/6J background was used as the stimulus animal. This mouse came from a set of three littermates that were alternated between each test. The stimulus animal was placed under the mesh cup holder in one side chamber of the apparatus, while a blue ball toy was placed under the cup holder in the other side chamber. The distance traveled in each compartment, as well as the amount of time spent in the areas directly around the mesh enclosures was analyzed using ANY-maze. The area around the mesh was 7 cm wide circle as defined only in the AnyMaze program, no physical circle was present in the cage. This size annulus was based on the tracking of the nose and central point of the mouse body while they investigated the meshed enclosure with the social object. After each trial, all compartments and the metal cup holders were cleaned with a 30% ethanol solution and dried afterward.

Testing for the prepulse inhibition (PPI) task was conducted using similar protocols and equipment as described previously by Savonenko et. al. [25]. Briefly, mice were tested in a closed off box equipped with speakers while the lights inside were turned off (29 cm wide × 30 cm deep × 29 cm high). The mice were placed in a small cylinder in the center of the box that sat on sensors that could record the latency and maximal amplitudes of the startle response when the mice reacted to the pulse stimuli or pre-pulse (SR-LAB Startle Response System, SIC 002668). After a 5-min acclimation period, the mice were exposed to 10 startle pulses (25 ms, 120 dB) of white noise. This was used to determine the ASR (Acoustic Startle Reaction) at the beginning of the test. Mice were then given 52 trials of either prepulse-pulse stimuli, pulse stimuli, or background-only noise in a pseudorandom pattern every 30 s to measure the startle reaction as the test progressed and the PPI at different prepulse levels. Prepulse levels were 25 ms pulses of white noise, slightly higher than background noise (63 dB) levels. 6 trials each for the following prepulses were given and averaged together for analysis: 74, 78, 82, 86, and 90 dB. Prepulses were delivered 75 ms before the startle pulse was delivered. The maximum startle amplitude and latency for the pulse delivery were analyzed and used to calculate the PPI in prepulse trials and ASR in pulse-only trials (see Fig. 3G). PPI was calculated according to the following formula: % PPI = 100 × (reactivity on pulse alone trials – reactivity on prepulse trials + pulse trials)/reactivity on pulse alone trials.

The MWM task was performed using similar equipment and protocols as described previously by Savonenko et. al. [27]. Briefly, mice were trained to swim in a large water pool (130 cm diameter) with a clearly delineated swimming path. The water was kept at 21 ± 1 ℃ and colored with non-toxic washable white paint to make the platform invisible. Visual cues were placed around the edge of the maze and along the walls of the training room. All mice were pre-trained for 4 days to build stamina and learn to swim and stay on submerged platform (~ 1 cm below the surface). The purpose of the pretraining was for the mice to learn the procedural aspects of the MWM before the training phase. Mice were allowed to swim until they found the platform or until 60 s elapsed, at which point they were directed to the submerged platform and then removed to a warm, dry holding cage after sitting there stably for 5 s. The training phase to test spatial memory started in a different room with a set of big visual cues situated along the walls (distal cues) and a set of smaller visual cues situated on a rim of the pool (proximal cues). During this phase, mice were tested a total of 6 times a day for 4 days. During the first two training days, the platform was placed in quadrant 1, and the mice were started at designated positions in the other 3 quadrants in a semi-random order and varied for each trial (Fig. 5A). At the next reversal stage which lasted for 2 training days, the platform was moved to quadrant 3, and the mice were started randomly at positions in quadrants 2 and 4. During each day, two types of trials were conducted (Fig. 5A): training trial in which the platform was hidden under the water and mice were able to climb on it as soon as they reached it. The second type of trials, called probe trials, were conducted with the platform collapsed at the bottom for the duration of the probe trial. If the mice could not find the platform 20 s after the platform was raised, they were directed to the platform as previously described for training trials. The probe trials lasted 40 s after which the platform was raised to allow the animal to climb on it. To test the short- and long-term memory of the platform location, the probe trials were conducted at the end and at the beginning of a training day, respectively. Short-delay probe trials were run ~ 20 min after the last training trial, and the long-delay probe trials were conducted after the over-night delay (~ 20 h). Probe tests were given at the end of each day to assess the short-term memory of platform location from the previous 5 trials the mice had just completed that day. Probe tests at the beginning of days 2–4 were given to assess the long-term memory the mice had of the platform location from the previous day. These types of probe trials were used in numerous previous studies of cognitive deficits in different mouse and rat models of Alzheimer’s and other diseases [23, 27‐29]. The amount of time spent around the quadrant platform locations (40 cm diameter circle) was analyzed using ANY-maze software, along with swim distances and latency to find the platform for each trial.

The FC task uses classical conditioning to train mice to associate a neutral conditioned stimulus (CS, audio tone) and novel context with an aversive unconditioned stimulus (US, foot shock) and we performed this task using similar protocols and equipment as previously described [30]. After training, mice learn to associate the CS and context with the US and exhibit a freezing response, an unconditioned fear response when presented with the CS or the same context. A mouse training chamber with a metal grated bottom that delivered the foot shock (Stoelting Co., Wood Dale, IL) was used (17 cm wide × 17 cm long × 25 cm high) (Fig. 6A). With lights turned on inside the chamber, mice were released into the box and allowed to roam freely. After 120 s (intertrial interval, ITI), a 15-s long 85 dB, 600 Hz tone (CS) was delivered, followed immediately by a 2-s long 0.6 mA foot shock. This was performed 3 times, followed by one last ITI. Freezing durations of longer than 1 s were measured using ANY-maze software. The cage was cleaned with a 30% ethanol solution at the start and end of each test. 24 h after trial 1, mice were tested in the same context for 5 min with no CS or US to test for contextual fear memory (trial 2). 3 h after trial 2, the test chamber was substituted for a different shape, size, and color box (25 cm wide × 25 cm long × 38 cm high with curved white paper insert in one corner, all white walls, and solid floor) in which small amounts of bedding from the home cage were scattered. Mice were tested using the same paradigm as in trial 1 without the US (only CS). The percent time spent freezing in all contexts was measured during the ITI and during the CS.

Statistics were analyzed using Statistica 13.3 (TIBCO Software Inc., CA) with a minimum significance level of P < 0.05. Two-way ANOVA tests were used for a majority of the behavioral experiments, after which Fisher’s least significant difference post hoc tests were conducted to examine significant main effects/interactions. Genotype and sex were treated as the main effects, while effects of time intervals, area, trial type, arm type, or object type were treated as repeated measures. Interactions were orthogonal sets. Bonferroni corrections were performed on individual comparisons when the main effect was not significant. See Additional file 2: Table S1 for a detailed list of tests run. The number and sex of all animals are included in the main and supplemental figures. The means were all graphed with error bars that represent the SEM.

GDE2 is expressed in the developing and postnatal spinal cord and brain [20, 31, 32]; however, its expression in the adult brain has not been determined. To assess GDE2 expression in the adult mouse brain, we performed RNAscope for Gde2 transcripts in coronal sections from 4-month-old WT mice. We detected Gde2 expression in major brain areas including the thalamus (Th) and caudate putamen (CP) (Fig. 1A, B), with particularly strong expression in the medial habenula and the medial and anterior amygdala (Fig. 1C–F), which are areas associated with regulation of emotional and motivational aspects of behavior including fear/anxiety responses [33, 34]. Gde2 is also expressed throughout the hippocampus, a region implicated in learning and memory (Fig. 1G–J) [35, 36]. In addition, Gde2 transcripts are detected in the cortex with enriched expression in deep cortical layers 4–6 (Fig. 1K, L). Similar to previous studies using younger mice [31, 32], Gde2 expression in the cortex is mainly localized to neurons with some expression in non-neuronal cells (Fig. 1M, N). This aligns with previous expression and transcriptomic data showing Gde2 expression in neurons, mature oligodendrocytes, and endothelial cells [37, 38].

Similar to previously published work [18], Gde2KO mice were viable, fertile, and born at the expected Mendelian frequencies. Male and female Gde2KO mice were born in about equal numbers. Gde2KO mice showed no obvious differences in health or appearance from WT mice. However, upon weighing, male Gde2KO mice consistently have slightly lower body weight compared to WT mice (~ 10%, Additional file 1: Fig. S1A). This difference in body weight between genotypes decreases with age (Additional file 1: Fig. S1A). No difference in body weight was observed for female mice (Additional file 1: Fig. S1B).

Animals were first tested in the Open Field (OF) task at 7-months of age (Additional file 1: Fig. S1C). At the 7-month time point, WT and Gde2KO mice showed no significant difference in the distance traveled in the OF chamber (Fig. 2A–C, Additional file 1: Fig. S2A–C). We next tested these same mice at 16-months of age to see if any age-progressive phenotypes emerged (Additional file 1: Fig. S1C). At this time point, Gde2KO mice showed significantly increased locomotion, and this hyperactivity was the most pronounced at the beginning of testing (Fig. 2D, E). Importantly, despite increased novelty-induced activity, Gde2KO males demonstrated significant habituation as testing progressed (Additional file 1: Fig. S2D, E). Female Gde2KOs showed a trending increase in activity at both ages; however, these differences did not reach significance (Additional file 1: Fig. S2A, C, D, F).

Hyperactivity in Gde2KO mice was also evident in the two-trial Y maze at 7- and 16-months of age and in both sexes (Fig. 2F–J, Additional file 1: Fig. S2G–L). In this task, mice explore a Y-shaped maze across two 5-min trials. In the first trial, they are allowed to explore two of the arms. In the second trial, a novel third arm is opened for exploration (Fig. 2F, see “Methods”). Notably, at 7-months of age, the between-genotype differences in distance traveled were more pronounced in trial 2 than in trial 1, indicating increased hyperactivity in Gde2KOs upon the opening of the new arm in the second trial (Fig. 2G, H). At 16-months of age, increased activity of Gde2KO mice was observed in trial 1 and did not increase further in trial 2 (Fig. 2I, J).

In the plus maze task, Gde2KO animals at 7-months were tested to see if hyperactivity was novelty induced (Additional file 1: Fig. S1C). Mice were placed in a plus shaped maze with open and closed arms and were allowed to explore for 10 min (Fig. 2K; see “Methods”). Compared to WT, Gde2KO mice travel more distance over time compared with WT in both open and closed arms (Fig. 2L, M). The between-genotype difference is observed in both males and females (Additional file 1: Fig. S3A–C). Notably, we see a significant difference in distance traveled by Gde2KOs during the first minute of the test when the environment is most novel (Fig. 2M). We further analyzed the distance traveled by mice in the plus maze task in blocks of 5 min to match the block durations of the OF analysis. Gde2KOs still demonstrate hyperactivity when analyzed in 5-min blocks of time (Additional file 1: Fig. S3D). This observation is in agreement with the hyperactivity phenotypes observed in the Y maze task at 7 months.

Taken together, Gde2KO mice display hyperactivity at younger ages mainly in response to new situations/environments; however, aged Gde2KO mice show hyperactivity across all tasks measuring distance traveled regardless of novelty. Importantly, despite the hyperactivity observed in Gde2KO mice, the extent of the hyperactivity was moderate and not debilitating as the motor exploration in these mice showed preserved habituation across all tests.

Mice normally avoid open spaces such as the center area of the OF and the open arms in the plus maze. Thus, distance traveled and time spent in these areas can be used as a measure of anxiety levels [39, 40]. Gde2KO mice were tested at 7 months and 16 months in the OF test to determine if they had an age-progressive anxiety phenotype. At both time points, male and female Gde2KO mice show no difference in the percent distance traveled in the center of the OF arena (Fig. 3A–D; Additional file 1: Fig. S4A–F). Additionally, no difference in the percent time spent in the center was found between genotypes at either age (Additional file 1: Fig. S4G–K). In line with these observations, WT and Gde2KO mice spent an equivalent amount of time in the open arms of the plus maze (Fig. 3E, F) and showed no significant difference in distance traveled in the open arms whether analyzed in 1- or 5-min blocks of time (Additional file 1: Fig. S4L–N). Since Gde2KO mice in the OF task showed no increased anxiety phenotype between time points, the plus maze task was not repeated at the later age. In sum, these observations suggest that loss of GDE2 has no effect on anxiety levels.

Startle response and sensorimotor gating can be assessed using the startle reflex and pre-pulse inhibition (PPI) to acoustic stimuli. In this test, animals exhibit a startle reflex in response to a sound stimulus (pulse). Their startle reflex is typically dampened by playing a lesser prepulse ahead of the main pulse. This dampening of the startle response is indicative of sensorimotor gating [41].

To test if loss of GDE2 affects these behaviors, we placed 7-month-old mice in a startle chamber and played a series of pulses or pulses preceded by a prepulse (Fig. 3G; Additional file 1: Fig. S1C; see “Methods”). Gde2KO mice exhibit a significantly reduced startle amplitude in response to the sound stimulus (Fig. 3H). The most significant difference in startle response for male Gde2KOs was at the start of the test and was less pronounced when the startle amplitude decreased due to habituation (Fig. 3I). Gde2KO females show a significant decrease in startle response (Additional file 1: Fig. S5A) that was not modulated by habituation (Fig. 3J, trending decrease not significant after Bonferroni correction).

No overall differences in PPI were observed between WT and Gde2KO mice (Additional file 1: Fig S5B); however, when analyzed separately for each sex, there was a significant reduction in PPI for female Gde2KO mice (Fig. 3K). This reduction in PPI for females was observed as a main effect of genotype without any interaction of genotype and level of prepulse (Fig. 3M). Male Gde2KO mice do not exhibit any changes in PPI (Fig. 3K, L). We examined the correlation between startle activity and PPI to see if the difference in startle response between genotypes could be related to the levels of PPI. These phenotypes appear independent, with startle reactivity accounting for less than 36% of the variability in PPI in all cases (Additional file 1: Fig. S5C). Since a startle and PPI phenotype was evident at the 7-month time point in Gde2KO mice, we did not retest the animals in this task at a later time point. Based on these observations, we conclude that Gde2KO mice of both sexes have reduced startle reactivity, with female Gde2KOs showing additional deficits in PPI.

GDE2 is expressed at high levels in the medial habenula (Fig. 1C, D), an area associated with encoding social behaviors [33]. Accordingly, we used the 3-chamber social preference test to evaluate social motivation in the absence of GDE2 (Fig. 4A). Briefly, one chamber contained a wire enclosure with a stimulus mouse, and the other chamber contained a wire enclosure with a toy. After a habituation trial where both chambers were empty, the mouse being tested was placed in the middle chamber, and social preference was assessed based on time spent around the stimulus mouse compared to the toy (Fig. 4A; see “Methods”). Mice were tested at 11-months in the social motivation task (Additional file 1: Fig. S1C, see “Methods”). While WT mice spent significantly more time with the mouse than the toy, Gde2KOs showed no preference between the two (Fig. 4B, Additional file 1: Fig. S6A). This suggests that Gde2KO mice have reduced social preference compared to WT animals. This loss of social preference is seen in both male and female Gde2KO mice (Additional file 1: Fig. S6B–E). During the social motivation trial, Gde2KOs showed no differences in motor activity that could confound interpretation (Fig. 4C). However, during habituation, there was an increase in the activity of Gde2KO mice, particularly females, in the center compartment but not in the other two compartments (Additional file 1: Fig. S6F–J). Since Gde2KO mice exhibited a decrease in social preference at the 11-month time point, we did not repeat the task at 16 months.

We further tested the spatial working memory of Gde2KO mice using the two-trial Y maze described earlier (Fig. 2F; see “Methods”). In this paradigm, the time spent in the novel arm in trial 2 is indicative of spatial working memory. At 7-months, both WT and Gde2KO mice prefer to spend time in the novel arm rather than the old arm (Fig. 4D, Additional file 1: Fig. S1C). As is typical, both genotypes show a strong preference for the novel arm during the first minute of the test (Fig. 4E), and these effects were not modified by sex (Additional file 1: Fig. S7A–D). We tested the mice again at 16-months, since memory deficits can be an age progressive phenotype (Additional file 1: Fig. S1C). WT and Gde2KO mice still prefer the novel arm over the old arm on average (Fig. 4F). WT mice retain their preference for the novel arm throughout the task; however, Gde2KO mice lose this preference after the first 2 min (Fig. 4G). These results suggest that 16-month-old Gde2KO mice have impaired performance in this spatial working memory test compared to WT mice of the same age. These deficits were also observed when males and females were analyzed separately (Additional file 1: Fig. S7E–H). Taken together, aged Gde2KO mice show abnormal social and spatial preferences in the time scales associated with working and/or short-term memory. Accordingly, we performed further tests to assess memory and cognition in Gde2KO mice.

The Morris Water Maze (MWM) test was used to assess learning and spatial memory in Gde2KO mice [42, 43]. In this task, mice were made to swim in a pool that contained a hidden platform in one quadrant (Fig. 5A; see “Methods”). Since the MWM task is stressful for the mice and we had not seen memory deficits in Gde2KO mice at the 7-month time point in the Y maze, we tested the mice only at the 16-month time point (Additional file 1: Fig. S1C). The mice were trained over the course of two days to locate the platform in quadrant one (Fig. 5A; see “Methods”). During training trials, Gde2KO mice showed no differences in distance traveled or latency to reach the platform (Fig. 5B; Additional file 1: Fig. S8A), and there was no effect of sex on performance (Additional file 1: Fig. S8B, C). These results suggest that Gde2KO mice are capable of learning the platform location.

We next tested Gde2KO mice for their ability to remember the platform location after platform removal. During the short delay probe trial at the end of day 2 (see scheme in Fig. 5A), we assessed short-term spatial memory by analyzing the percent time that mice spent in each quadrant area. Both genotypes prefer the correct platform location (quadrant area 1) at higher than chance level; however, only WT mice could accurately distinguish between the first and second quadrants (Fig. 5C). The inability of Gde2KOs to discern the first and second quadrants suggests that they have decreased short-term recall of the platform location. This effect is mainly seen in males rather than females (Fig. 5D, E).

We assessed long-term spatial memory by measuring how well mice remembered the platform location after an overnight delay (~ 24 h). Using data collected during the long delay probe trial at the start of day 3 (see scheme in Fig. 5A), we found that similar to the short delay probe trials, WT mice spent significantly more time in the correct quadrant area (quadrant area 1) than the neighboring quadrant area 2 (Fig. 5F). In contrast, Gde2KO mice did not show a preference between these two quadrants (Fig. 5F). Thus, Gde2KO mice have less precise long-term recall of the platform location than WT mice. This pattern is significant only in males and not females (Fig. 5G, H).

We then measured the ability of animals to learn a new platform location and decrease their preference for the old location of the platform by moving the platform to quadrant 3 on the third day of the MWM test (see scheme in Fig. 5A). Over the course of days 3 and 4, both genotypes learned the new location of the platform (Additional file 1: Fig. S8D) and spent less time around the quadrant area that contained the previous platform location (Additional file 1: Fig. S8G). No sex differences were observed in this reversal portion of the task (Additional file 1: Fig. S8E, F, H, I).

These collective observations show that Gde2KO mice, particularly males, fail to recall the specific location of the platform in short- and long-term memory probe trials, while still preserving a general memory of the platform’s location. However, their ability to successfully learn new information is not affected. These findings provide evidence that Gde2KO mice have short and long-term spatial memory impairments.

GDE2 is expressed in the amygdala (Fig. 1D, E), which is implicated in fear acquisition and memory [34]. Accordingly, we assessed these behaviors using a cued and contextual fear conditioning (FC) test (Fig. 6A, see “Methods”). During the acquisition trial, mice were introduced to context 1 (see scheme in Fig. 6A), where mice received a mild shock (US) after hearing a tone (CS). This was repeated three times throughout the trial, so that mice learned to associate the CS and context with the US. Mice were tested at the 11-month time point to determine if a memory deficit developed after the 7-month time point when no memory deficit was apparent (Additional file 1: Fig. S1C). Gde2KO mice of both sexes showed no differences in contextual or cued fear acquisition in context 1 compared with WT controls (Fig. 6B, C; Additional file 1: Fig. S9A–D). We next tested animals for contextual fear memory 24 h after the acquisition trial. In this trial, mice were put back in context 1 but received no CS or US (see scheme in Fig. 6A). We found no significant difference in context 1 fear memory between WT and Gde2KO mice of either sex (Fig. 6D, E; Additional file 1: Fig. S9E, F).

We then tested cued fear memory by placing mice in context 2 and playing the CS without any shock (see scheme in Fig. 6A). Gde2KO mice, on average, had no significant difference in cued fear memory (Fig. 6F). However, male Gde2KO mice froze significantly less during the first CS (Additional file 1: Fig. S9G) indicating that the immediate recall of CS is likely impaired. Females Gde2KOs show this trend, but it is not significantly different from WT after Bonferroni correction (Additional file 1: Fig. S9H), which may reflect some sex-specific delay in fear recall in response to the CS. We also assessed freezing during the intertrial interval (ITI). Gde2KO mice of both sexes displayed less freezing as the testing progressed compared to their WT counterparts (Fig. 6G, Additional file 1: Fig. S9I, J), consistent with reduced acquisition of secondary contextual fear to context 2.

Overall, Gde2KOs showed no difference in the acquisition of cued fear memory or context 1 fear memory. However, when tested for cued fear memory, Gde2KOs, particularly males, showed impaired immediate recall of cued fear memory. Moreover, the reduced acquisition of fear response in context 2 might reflect impaired secondary contextual fear acquisition in Gde2KO mice.