Consequences of VGluT3 deficiency on learning and memory in mice

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Consequences of VGluT3 deficiency on learning and memory in mice ´ ´ ´ Horvath ´ , Csilla Lea Fazekas , Diana Balazsfi , Hanga Reka ´ ´ Zoltan Balogh , Mano Aliczki , Agnesa Puhova , Lucia Balagova , ´ Magdalena Chmelova , Daniela Jezova , Jozsef Haller , ´ Zelena Dora PII: DOI: Reference:

S0031-9384(19)30670-5 https://doi.org/10.1016/j.physbeh.2019.112688 PHB 112688

To appear in:

Physiology & Behavior

Received date: Revised date: Accepted date:

28 June 2019 22 September 2019 22 September 2019

´ ´ ´ Horvath ´ , Please cite this article as: Csilla Lea Fazekas , Diana Balazsfi , Hanga Reka ´ Balogh , Zoltan Mano´ Aliczki , Agnesa Puhova , Lucia Balagova , Magdalena Chmelova , ´ ´ Zelena , Consequences of VGluT3 deficiency on learning and Daniela Jezova , Jozsef Haller , Dora memory in mice, Physiology & Behavior (2019), doi: https://doi.org/10.1016/j.physbeh.2019.112688

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Highlights 

Vesicular glutamate transporter 3 knockout mice exhibited the capability to learn.



They showed mild disturbances in working memory, cognitive flexibility.



It is unlikely that their anxious phenotype confounded the results.

Consequences of VGluT3 deficiency on learning and memory in mice Csilla Lea Fazekas1*, Diána Balázsfi1,2*, Hanga Réka Horváth1, Zoltán Balogh1,2, Manó Aliczki1, Agnesa Puhova3, Lucia Balagova3, Magdalena Chmelova3, Daniela Jezova3, József Haller1, Dóra Zelena1,4 1

Hungarian Academy of Sciences, Institute of Experimental Medicine, Budapest, Hungary

2

János Szentágothai School of Neurosciences, Semmelweis University, Budapest, Hungary

3

Slovak Academy of Sciences, Biomedical Research Center, Institute of Experimental

Endocrinology, Bratislava, Slovakia 4

Centre for Neuroscience, Szentágothai Research Centre, Institute of Physiology, Medical

School, University of Pécs, Pécs, Hungary *contributed equally

Abbreviated title: VGluT3 in learning and memory

Corresponding author: Dóra Zelena 1083 Budapest Szigony 43 Hungary Tel. +36-1-2109400/290 Fax. +36-1-2109951 e-mail: [email protected]

Abstract The aim of the present study was to test the hypothesis that vesicular glutamate transporter 3 (VGluT3) deficiency is associated with cognitive impairments. Male VGluT3 knockout (KO) and wild type (WT) mice were exposed to a behavioral test battery covering paradigms based on spontaneous exploratory behavior and reinforcement-based learning tests. Reversal learning was examined to test the cognitive flexibility. The VGluT3 KO mice clearly exhibited the ability to learn. The social recognition memory of KO mice was intact. The ymaze test revealed weaker working memory of VGluT3 KO mice. No significant learning impairments were noticed in operant conditioning or holeboard discrimination paradigm. In avoidance-based learning tests (Morris water maze and active avoidance), KO mice exhibited slightly slower learning process compared to WT mice, but not a complete learning impairment. In tests based on simple associations (operant conditioning, avoidance learning) an attenuation of cognitive flexibility was observed in KO mice. In conclusion, knocking out VGluT3 results in mild disturbances in working memory and learning flexibility. Apparently, this glutamate transporter is not a major player in learning and memory formation in general. Based on previous characteristics of VGluT3 KO mice we would have expected a stronger deficit. The observed hypolocomotion did not contribute to the mild cognitive disturbances herein reported, either. Keywords: y-maze, operant conditioning, active avoidance, holeboard discrimination, Morris water maze

1. Introduction Memory and learning impairments are major societal problems not only because of the aging society and the growing prevalence of Alzheimer’s disorder [1, 2], but they are also core symptoms of many other neuropsychiatric disorders (e.g. schizophrenia, depression, posttraumatic stress disorder) [3]. The processes of cognition, learning and memory formation are complex and not yet fully understood and many neurotransmitters were implicated in it (e.g. acetylcholine [4], GABA [5], serotonin [6]). However, the neurotransmitter glutamate and its receptors seem to be the key components [7, 8], as they play an important role in the mechanisms of synaptic plasticity, in long-term potentiation and in long-term depression, which are believed to form the cellular basis of learning and memory [9]. The transport of glutamate to the synaptic cleft is helped by the family of vesicular glutamate transporters (VGluTs). Their role is to load the synaptic vesicles with glutamate. Their contribution to the memory formation is therefore very likely. Three VGluTs have been identified in the central nervous system so far, namely VGluT1, 2 and 3 [10, 11]. VGluT3 is frequently colocalizing with other neurotransmitter transporters in serotoninergic (5-HT), cholinergic (ACh) or GABAergic neurons [12-14]. The VGluT3 transporter was found in the dendrites of particular neurons, indicating its potential for retrograde synaptic signaling [15]. Moreover, VGluT3 is the only VGluT present in the cholinergic cells of the basal forebrain and striatum, an area known to be deeply implicated in Alzheimer’s dementia [16, 17]. An efficient approach to study the physiological role of regulatory proteins, such as neurotransmitter transporters, is knocking out the particular gene. VGluT1 and 2 homozygous knock-out is lethal, the former dying before reaching mature adulthood unless properly taken care of, while the latter causes death right after the birth due to fatal respiratory impairments [18-20]. In contrast, VGluT3 knockout (KO) mice are viable and live relatively normally without extra care [10]. The general phenotype of the VGluT3 KO mice is already known.

Their motor coordination, exploratory behavior, touch-, thermal- and electroshock-induced pain sensation are normal compared to the wild type (WT) littermates [21]. They do not show depression-like behavior or altered aggression, but they are anxious [21, 22]. Non-convulsive electrographic seizures were recorded in VGluT3 KO mice by electroencephalography (EEG), which occurred once a day without influencing the exploratory behavior of the animal [23]. Since the inner ear also contains VGluT3, it is not surprising that the KO animals have auditory impairments [23]. In spite of the essential role of glutamate in learning and memory processes, the information on cognitive phenotyping of VGluT3 KO mice is lacking. The aim of the present study was to test the hypothesis that VGluT3 deficiency is associated with cognitive impairments. To do so, we examined the learning skills of VGluT3 KO mice. We used a test battery of behavioral examinations covering a wide range of learning and memory processes, linked to different brain regions.

2. Methods 2.1. Animals VGluT3 KO and WT mice (C57BL/6J background) were obtained from heterozygous mating pairs from the Institute of Experimental Medicine, Budapest, Hungary. The genotype was determined by PCR from a small tail sample collected from 2-3-day-old animals. Adult male mice (13-25-week-old) were housed individually in Macrolon cages (40 cm x 25 cm x 26 cm) under a standard 12h light–dark cycle (lights on at 6 a.m., 21±1°C, 50-60% humidity), with food (standard mice chow, Charles River, Hungary) and water available ad libitum. To enhance motivation to explore the environment during operant conditioning and holeboard discrimination tests, mice participating in these tests were kept on restricted diet to maintain 80% of their bodyweight beginning 3 days prior to the first experimental day. Bodyweight was regularly measured, and in case it dropped below the target bodyweight the diet was

adjusted. Individual housing was chosen because the restricted diet could be controlled better and the extra stress of manipulating the cage-mates could be avoided. For comparability of data, single-housing was applied for all other tests. All tests were approved by the local committee of animal health and care (PEI/001/334/2013) and performed according to the European Communities Council Directive recommendations for the care and use of laboratory animals (2010/63/EU). 2.2. Behavioral testing Tests were carried out between 9-13h in a separate room under similar lighting condition as in the animal facility and measured automatically by the equipment for operant chamber and active avoidance or recorded by ceiling-mounted camera (Samsung SNB 7000) for social memory, y-maze, holeboard discrimination and Morris water maze (MWM) tests. The experiments were performed blinded to genotype and with randomization of the animals. Data were analyzed later by computer-based event recorders H77, Budapest, Hungary; Noldus EthoVision or Solomon Coder (https://solomoncoder.com/). Each test apparatus was cleaned with 20% ethanol and water and dried prior the next animal was introduced. VGluT3 KO mice are anxious [22], which made prolonged habituation to the test situations necessary [24, 25]. Moreover, to overcome the confounding effect of locomotion, we used derived parameters (e.g. spontaneous alternation, preference, discrimination index) or the differences in the slope of the learning curve (calculated by interaction) was take into consideration. The test battery (Table 1.) included two types of tests, namely tests based on spontaneous exploratory behavior and reinforcement-based learning paradigms.

LEARNING TYPE

TEST

SOCIAL MEMORY TEST LEARNING BASED ON SPONTANEOUS (recognition memory) EXPLORATORY BEHAVIOR Y-MAZE (working memory) OPERANT REWARD CONDITIONING BASED SIMPLE (implicit memory) ASSOCIATION AVOIDANCE ACTIVE AVOIDANCE MOTIVATION BASED (implicit memory) BASED HOLEBOARD REWARD LEARNING DISCRIMINATION BASED COMPLEX (spatial memory) ASSOCIATION AVOIDANCE MWM BASED (spatial memory)

KO vs WT  ↓ ↓ COGNITIVE FLEXIBILITY

↓ COGNITIVE FLEXIBILITY

 

Table 1. Summary of the tests and the main results WT: wild type; KO: vesicular glutamate transporter 3 knock out; MWM: Morris Water Maze; : no difference; ↓: worse compared to WT

2.2.1. Paradigms based on spontaneous exploratory behavior These experiments were conducted without pre-test learning of a rule or reinforcement [26]. Social discrimination reflects recognition memory, which is the ability to judge a previously encountered item as familiar [27] and linked to areas of the prefrontal cortex (PFC) and the hippocampus [28-31] as well as visual [32] and olfactory processes [33]. Spontaneous alternation on y-maze measures working memory [34] with the contribution of ventral hippocampus and PFC [35]. 2.2.1.1. Social memory test We used a three chamber sociability apparatus (clear Plexiglas box; three 40 cm × 20 cm × 25 cm identical chambers) placing the test animals in the center chamber [37]. On day 1-2 (habituation days) animals were put into the empty apparatus for 10 min. On day 3-4 mice were replaced into the same apparatus for 10 min, where in one side chamber a stimulus animal (WT, 13-16-week-old virgin females, reused between experimental animals; subsequently called ‘old’ mouse) under a barred cage was placed, while the other chamber

contained only an empty cage (learning phase). On day 4, 4 hours after familiarization of the learning trials (intermediate memory), another stimulus animal (subsequently called ‘new1’ mouse) was placed under the other cage along with the ‘old’ mouse for 10 min. The last trial took place 24 hours following the first session of day 4 with ‘new1’ mouse and a third stimulus animal (subsequently called ‘new2’ mouse). The stimulus animals and their locations in the arena (left – right) were randomized to avoid place preference (Fig. 1A). The stimulus animals were not familiar with the test animals or each other beforehand. We evaluated a Sociability Index (SI; may vary between -1 and +1) for day 3-4 𝑆𝐼 =

𝑡𝑖𝑚𝑒 𝑖𝑛 𝑐ℎ𝑎𝑚𝑏𝑒𝑟 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑖𝑛𝑔 𝑎 𝑐𝑜𝑛𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 − 𝑒𝑚𝑝𝑡𝑦 𝑐ℎ𝑎𝑚𝑏𝑒𝑟 𝑡𝑖𝑚𝑒 𝑖𝑛 𝑙𝑒𝑓𝑡 + 𝑟𝑖𝑔ℎ𝑡 𝑐𝑜𝑚𝑝𝑎𝑟𝑡𝑚𝑒𝑛𝑡𝑠

and identically a Discrimination Index (DI; may vary between -1 and +1) for day 4 (4h) and day 5 (24h) 𝐷𝐼 =

𝑡𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑠𝑛𝑖𝑓𝑓𝑖𝑛𝑔 𝑛𝑒𝑤 − 𝑜𝑙𝑑 𝑚𝑜𝑢𝑠𝑒 𝑡𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑠𝑛𝑖𝑓𝑓𝑖𝑛𝑔 𝑛𝑒𝑤 + 𝑜𝑙𝑑 𝑚𝑜𝑢𝑠𝑒

where a value greater than 0 represent a preference toward a social stimulus or an intact memory, respectively [38]. Distance moved was also measured. N=10WT/10KO 2.2.1.2. Y-maze The arms of the apparatus were 25 cm long, 5 cm wide and 21 cm high. Mice were placed at the end of one arm and were allowed to explore the maze freely for 5 min. Total number of entries reflects overall locomotion, while spontaneous alternation was given as percentage (%) of ‘correct’ alternation/total entries. ‘Correct’ alternation means entry into all three arms on consecutive choices (i.e. ABC, BCA, or CAB, but not CAC, BAB, or ABA) (Fig. 2A) [39, 40]. N=9WT/9KO 2.2.2. Reinforcement-based learning paradigms

First simple associations were used. These tasks required the involvement of neuronal networks instead of a single brain area or neuron group [41]. During operant conditioning the driving force is a reward [42], while active avoidance (shuttle-box) test is based on learning to avoid harmful stimuli with deep amygdalar involvement [43]. More complex tasks testing (mostly) hippocampus based spatial memory [44] in combination with rewards (holeboard discrimination test) [45] or punishment (MWM) were also performed. Cognitive flexibility was also examined in operant conditioning, active avoidance, holeboard discrimination and MWM tests [46]. 2.2.2.1. Operant conditioning The test was performed as described previously in an automated operant chamber (Med Associates, St. Albans, VT, USA) using 45 mg food pellets (Bio-Serv Dustless Precision Rodent Pellet, Bilaney Consultants GmbH, Germany) as reward [42]. Animals were placed inside a test chamber with two nose holes for 30 min and were allowed to freely explore the environment. A nose poke into one of the nose holes was immediately associated with a reward followed by a 25 sec long timeout with the chamber light switched on (time-in period), while the other nose hole was not baited (incorrect). During the time-out period, responses were not rewarded, but were registered [47]. The test was divided into three phases: habituation (day 1-7), learning (day 8-25) and cognitive flexibility (day 26-39) (Fig. 3A). The position of the baited nose hole was changed between phases. Reward preference (ratio of responses on the rewarded nose hole) were calculated during the time-in period (when the chamber light was switched on) as follows: 𝑅𝑒𝑤𝑎𝑟𝑑 𝑝𝑟𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 =

𝑐𝑜𝑟𝑟𝑒𝑐𝑡 𝑛𝑜𝑠𝑒 𝑝𝑜𝑘𝑒 × 100 𝑖𝑛𝑐𝑜𝑟𝑟𝑒𝑐𝑡 + 𝑐𝑜𝑟𝑟𝑒𝑐𝑡 𝑛𝑜𝑠𝑒 𝑝𝑜𝑘𝑒𝑠

and the total number of responses (correct+incorrect) was also recorded. The criterion of learning was that the preference of baited hole exceeded the chance level of 50%. N=12WT/12KO 2.2.2.2. Active avoidance (shuttle-box) test Automated shuttle-box apparatus was used (Med Associates, St. Albans, VT, USA) consisting of two identical compartments with photobeam sensors, stimulus light, tone generator, stainless steel grid floor and a guillotiner door. The chambers were placed inside sound-attenuating cubicles and the shuttle-boxes were interfaced with a computer running Med-PC IV software (Med Associates, St. Albans, VT, USA) [48]. Mice were placed in the left or right compartment of the apparatus for 5 days (day 1-5) (Fig. 4A). After 1 min of habituation to the chamber without any special stimulus the 40 trials (each 30 sec long) were started. In each trial after 20 sec the light turned on and a tone was played, meanwhile the guillotine door opened (conditioning stimuli). During the last 5 sec of one trial an electric footshock (0.15 mA) was applied to the grid floor (unconditioned stimulus). At the end of the trial all stimuli were switched off, the guillotine door closed and 5 sec intertrial interval (ITI) started, then the subsequent trial was conducted. The learning phase was immediately followed by 5 days of cognitive flexibility phase (day 6-10), when the animals received the shock if they entered to the other chamber. An avoidance response was recorded when the animal avoided the electric shock by entering the other compartment during the conditioned stimuli (escape during stimulus – EDST, avoidance) or during the footshock (escape during footshock – EDFS, escape). N=12WT/12KO 2.2.2.3. Holeboard discrimination

Sixteen holes with centers 9.5 cm apart were drilled into the bottom of white, nontransparent chambers (40 cm x 36 cm x 19 cm) and bottle caps (3 cm in diameter) were glued to them and served as reward holders. Pellets were used as reinforcement. To exclude any olfactory cues, pellets were also placed under the boxes and extra-maze cues were placed on the ledges of the boxes [45]. The testing included four phases (Fig. 5A). The first phase was 15 min habituation (day 1-2) with all of the holes baited. During the learning phase four randomly chosen holes were baited (day 3-7). There were 6 trials per day, approximately with 1 h ITI (when the animals were returned to their home cages). Each was 3 min long or lasted until the animals found all four pellets. The third phase was a cognitive flexibility test (day 8-9), when four, previously not baited holes were rewarded with pellets. The fourth section (day 10, one 3 min trial) was conducted in darkness under infrared illumination (RM-25-120, 850nm, Raytec, UK) in order to assess whether the extra-maze cues were used by the mice to locate the rewards, and the animals had to find the later rewarded four holes. Number of reference memory errors (entering a non-baited hole) and error of omission (missing baited holes) were measured. N=9WT/9KO 2.2.2.4. Morris water maze (MWM) A circular pool made of bright gray plastic (90 cm in diameter and 40 cm in height) was filled with tap water (24 ± 2 °C), made opaque by white wall paint, and the level of the water was 1 cm higher than the platform (6 cm in diameter) except for learning day 1, when the platform was above the water. The apparatus was divided into 4 quadrants and the platform was installed in the middle of one quadrant (Fig. 6A). Mice were released into the water from different points across trials and were allowed to swim freely for 1 min to find the platform. If they did not find the platform in time, they were helped to find it and allowed to stay there for 10 sec. The learning phase (day 1-6)

consisted of 4 trials with 30 min ITI (when the animals were returned to their home cages) and was repeated for 6 consecutive days. On day 7 (test day 1) the platform was removed from the water and the mice had 1 min to search for the missing platform. From day 8 the platform was put to another quadrant and the animals were tested using the same protocol (cognitive flexibility, day 8-10). On the last day (day 11, test day 2) the platform was once again removed [49]. Latency to reach the platform, distance moved and time spent in as well as frequency to enter the different zones were calculated. N=8WT/8KO 2.3. Statistical analysis Data were analyzed with StatSoft 13.0 (Tulsa, USA) utilizing single sample t-test (SI, DI in comparison to 0 and y-maze, operant conditioning in comparison to 50%), one-way analysis of variance (ANOVA) (y-maze), ANCOVA with locomotor activity as covariate, repeated measure ANOVA (social memory, operant conditioning, active avoidance, holeboard discrimination, MWM) or General linear module (repeated measures on trials and days) (holeboard discrimination, MWM) followed by Newman Keuls posthoc comparison. Data are expressed as mean±SEM and p<0.05 was considered statistically significant. All marks on figures represent the results of post hoc comparison, while main ANOVA effects are described as texts, where appropriate.

3. Results 3.1. Social memory test The KO mice showed decreased locomotion during the testing (Fig. 1B; Table 2.). The distance moved by KO mice was significantly less than that of WT mice (genotype: F(1,18)=11.624, p=0.003) and decreased day by day in both groups (day: F(5,90)=30.208, p<0.001) reaching a nadir on day 3.

There was no chamber preference between the right and left compartments during the habituation phase on day 1 and 2 (Table 2.). On day 3 the SI of the WT group - but not that of the KO animals - was significantly higher than 0 (chance level) (WT: t(9)=6.050, p<0.001). When corrected to locomotion, the genotype effect on day 3 became almost significant (F(1,17)=5.901, p=0.053; covariant effect (distance moved): F(1,17)= 4.687, p=0.045). On day 4 the SI was significantly higher than 0 also in the KO group (WT: t(9)=2.685, p=0.025; KO: t(9)=2.667, p=0.026). When corrected to locomotion, the genotype difference of SI on day 4 became significant (F(1,17)=5.901, p=0.027, covariant effect (distance moved): F(1,17)=3.577, p=0.076), suggesting that changes in locomotion masked the genotype difference in the social interest.

Habituation

Day 1

Acquisition

Day 2

Day 3

Day 4

Tests

Day 4, 4h Day 5, 24h

WT

KO

Distance moved (cm)

4403.8±202.1

3410.4±303.5 **

Time spent in right chamber (%)

34.9±1.9

40.3±7.0

Time spent in left chamber (%)

36.6±3.0

34.3±7.2

Distance moved (cm)

3322.5±190.5 ++

2356.6±216.4 * ++

Time in right chamber (%)

35.2±3.3

45.2±6.9

Time in left chamber (%)

36.9±4.5

38.3±7.1

Distance moved (cm)

3156.3±150.8 ++

2228.1±171.8 * ++

SI (%)

0.353±0.058 $$

0.220±0.173

Distance moved (cm)

3558.1±92.7 ++

2655.8±195.2 * ++

SI (%)

0.129±0.048 $

0.336±0.126 $

Distance moved (cm)

3162.5±123.7 ++

2579.4±152.6 ++

DI (%)

0.171±0.052 $$

0.173±0.074 $

Distance moved (cm)

3356.6±112.3 ++

2856.2±222.1 ++

DI (%)

0.040±0.065

-0.019±0.058

Table 2. Details of the social memory test Mean±SEM; WT: wild type, KO: vesicular glutamate transporter 3 knockout; * p<0.05, ** p<0.01 compared to WT; ++ p<0.01 compared to previous day; $ p<0.05, $$ p<0.01 single sample t-test to 50%

Both genotypes had intact intermediate (4h) social memory on day 4 (Fig. 1C). Thus, mice spent more time observing the new animal than the old one (F(1,18)=15.505, p<0.001) irrespective of the genotype, even when we used locomotion as covariance, excluding the possibility that differences in the distance travelled confounded the results. The discrimination index of both groups was significantly higher than 0 (Table 2., DI; WT: t(9)=3.302, p=0.009; KO: t(9)=2.332, p=0.045).

In the long-term (24h) social memory test, there were no differences with respect to the social partner or genotype (Fig. 1D., Table 2., DI), even when we used locomotion as covariance.

Fig. 1 Results of the Social memory test (Experiment 1) A: Experimental design of the Social memory test with the timeline of the phases. The location of the animals was randomised to avoid place preference. B: During habituation phase the KO mice showed hypolocomotion. C: Both WT and KO animals had intact intermediate-term (4h) memory of the ‘old’ mouse indicated by the increased time spent with ‘new1’ compared to ‘old’ mouse. D: Neither group had long-term (24h) memory as there were no differences between the times spent with animals. Data are expressed as mean±SEM. N=10/group. WT: wild type; KO: vesicular glutamate transporter 3 knock out; Non-social: chamber without mouse; Social: chamber with ‘old’ mouse; Center: center chamber; ** p<0.01 compared to WT; × p<0.05 compared to ‘old’ mouse

3.2. Y-maze

There were no differences between the genotypes in the total number of arm entries (Fig. 2B). A significant difference was found in their spontaneous alternation, which was disturbed in KO (only WT mice were above the chance level of 50%; t(8)=4.257; p=0.002) (Fig. 2C).

Fig. 2 Results of the Y-maze test (Experiment 2) A: Experimental design of the Y-maze test. The animals freely explored the maze for 5 min. B: Mobility was measured by the total number of entries into the arms. The two genotypes showed no differences. C: Spontaneous alternation was counted as percentage (%) of ‘correct’ alternation/total entries. ‘Correct’ alternation means entry into all three arms on consecutive choices (i.e. ABC, BCA, or CAB, but not CAC, BAB, or ABA). Only the alternation of the WT mice was significantly higher than the random 50%. Data are expressed as mean±SEM. N=9/group. WT: wild type; KO: vesicular glutamate transporter 3 knock out; $$ p<0.01 compared to random 50%

3.3. Operant conditioning During the habituation and learning phases there were no significant differences between the two genotypes. Total number of responses increased across the days of habituation (day: F(6,126)=7.832, p<0.001), indicating that the animals understood the paradigm (Fig. 3B). The absence of genotype difference suggest that the locomotion did not confounded this performance. Both groups needed 16 days (7 days of habituation + 9 days of learning) to learn the task (preference of baited nose hole exceeded the chance level of 50% on day 10 of learning on figure: day 17 of the experiment) (WT: t(11)=3.740, p=0.003; KO: t(11)=2.353, p=0.038)

(Fig. 3C). However, there was no significant difference between the genotypes during the learning phase and the genotype x time interaction did not reach the level of significance,

Fig. 3 Results of the Operant conditioning test (Experiment 3) A: Experimental design of the operant conditioning test with the timeline of the phases. Between the different phases the baited nose hole was interchanged. In all of the figures the horizontal red line represents the random chance level of 50%. B: The total number of responses significantly increased after day 4, indicating successful habituation for both genotypes. C: Reward preference was calculated from total rewarded responses/(total rewarded responses + total non-rewarded responses). Reward preference during acquisition exceeded the random 50% on day 17 for both groups. There was no difference between the genotypes. D: On day 26 (first day of reversal learning) the performance of both groups dropped. On day 27-30 the performance of KO mice was significantly worse than that of WT mice. Data are expressed as mean±SEM. N=12/group. WT: wild type; KO: vesicular glutamate transporter 3 knock out; ** p<0.01 compared to WT

either. On day 26, at the beginning of the test of cognitive flexibility, the successful responses dropped for both groups, as expected (Fig. 3D). On days 26-30 the performance of the KO mice was significantly worse than that of WT mice, the KOs showing smaller preference for the rewarded nose hole (genotype: F(1,22)=14.762, p<0.001). The WT exceeded the chance level of 50% on day 2 of cognitive flexibility (day 27 of the experiment; t(11)=4.171, p=0.001), while the KO only on day 5 (experimental day 30; t(11)=2.66, p=0.026). There was no difference between the genotypes in time-out responses (data not shown). 3.4. Active avoidance In both groups the number of EDST increased (day: F(4,88)=31.066, p<0.001) (Fig. 4B). Complementary to the this, EDFS decreased gradually during the learning days (days 15) (day: F(4,88)=32.772, p<0.001) (data provided). The latency of escape during the first 10 trials of the first session (day 1) showed significant difference between the genotypes (genotype: F(1,22)=4.345, p=0.049) (Fig. 4D). The latencies of escape of KO mice were longer than those of WT mice. During the cognitive flexibility phase (days 6-10) the number of EDST decreased across the days (day: F(4,88)=25.395, p<0.001) (Fig. 4C). At the beginning of testing cognitive flexibility (day 6), the latencies of escapes increased across the first 10 trials (trial: F(9,198)=9.345, p<0.001; maximum: 10 sec), suggesting that the animals learned to stay in order to avoid the shock rather fast (Fig. 4E). Moreover, a significant difference between the genotypes was observed (genotype: F(1,22)=4.891, p=0.038).

Fig. 4 Results of the Active avoidance test (Experiment 4) A: Experimental design of the active avoidance test. Both the learning and cognitive flexibility phase lasted for 5 days, respectively. Each day consisted of 40 trials starting with 1 min habituation. B: Both groups were able to learn the task after 5 days as the improvement in the number of EDST increased between the days, but there was no significant difference between the KO and WT mice. C: The number EDST decreased across the days during cognitive flexibility phase, indicating successful learning of the new task. D: The latency to escape across the first 10 trials on day 1 were significantly higher for KO animals than that of the WT mice. E: During cognitive flexibility phase the escape latencies of the KO group peaked sooner (trial 5) than that of the WT group. Data are expressed as mean±SEM. N=12/group. WT: wild type; KO: vesicular glutamate transporter 3 knock out; EDST: escape during stimulus

3.5. Holeboard discrimination There was a significant decrease of reference memory errors across the days of the learning phase (day 3-7) (trial: F(5,80)= 3.253, p=0.010; day: F(4,64)= 4.893, p=0.002) (Fig. 5B),

indicating that the animals learned the task. There was a difference between the genotypes with less errors made by the KO mice compared to WT animals (genotype: F(1,16)= 4.961, p=0.041). No interaction was detected between factors genotype and day. As expected, the number of errors increased on the first day of testing cognitive flexibility (day 7, trial 6 as last trial of learning vs day 8, trial 1, as first trial of cognitive flexibility: F(1,16)=13.490, p=0.002) with significant genotype difference (F(1,16)=4.733, p=0.045). No genotype differences were observed during the reversal learning phase (day 8-9). On the last day (day 10), when the test was conducted in darkness, the errors of both groups significantly increased compared to day 9 (day: F(1,16)= 27.427, p<0.001). A significant decrease of errors of omission across the trials (F(5,80)=16.920, p<0.001) and days (F(4,64)=9.329, p<0.001) were found during the learning (day 3-7) (Fig. 5C). The error of omission in mice of the two genotypes did not differ. As expected, there was a difference between the last day of the learning task and the first day of testing cognitive flexibility (day 7-8: F(1,16)=11.765, p=0.003) with significant day x genotype interaction (F(1,16)=7.529, p=0.014). During reversal learning there was only a tendency for decreased error of omission between day 8 and 9 (day: F(1,16)=4.174, p=0.058) without significant genotype difference, while on day 10 no further changes were detected. The analysis by ANCOVA with locomotor behavior as a covariate did not show any significant influence of the locomotion suggesting that genotypic differences in the locomotion did not confound the results.

Fig. 5 Results of Holeboard test (Experiment 5) A: Experimental design of the holeboard test with the timeline of the phases. During habituation all holes, while during learning phase four randomly chosen holes were baited. During cognitive flexibility four, previously not baited holes were rewarded. In the dark test the baited hole configuration was the same as during cognitive flexibility phase. B: Reference-memory errors were the visits of non-baited holes. The decrease of errors during day 3-7 (learning phase) and their increase on day 8 (cognitive flexibility) suggested that both groups learnt the task. The significant increase of errors on day 10 (dark test) meant that the strain used extra-maze cues. C: Errors of omission meant missing a baited hole during the 3 min trials. Their decrease across the trials and days for both groups indicated learning. There was an interaction between the day and the genotype between day 7 and 8 at the point of testing cognitive flexibility. Data are expressed as mean±SEM. N=9/group. WT: wild type; KO: vesicular glutamate transporter 3 knock out

3.6. MWM During the 6 days of learning, the distance moved until reaching the platform decreased (day: F(5,65)=7.105, p<0.001) with only a tendency for a genotype difference (genotype: F(1,13)=3.716, p=0.076) (data provided). The latencies to reach the platform showed a significant improvement during the learning phase (day 1-6) in both genotypes (trial: F(3,39)=3.810, p=0.017; day: F(5,65)=8.715, p<0.001) (Fig. 6B). The repeated measure ANOVA for the data obtained on day 6 and day 7 (test 1) revealed a significant interaction between day x genotype (day 6 vs 7: F(1,14)=6.417, p=0.024), with a decrease in the latency in the WT and an increase in the KO mice. There was no genotype difference in the time spent in the learned quadrant during retention on day 7 (data provided). During the test of cognitive flexibility (day 8-10) there was a significant improvement in performance across the trials (F(3,42)=6.042, p=0.002) and days (F(2,28)=12.540, p<0.001) for both genotypes in distance moved (data provided). The latencies to find the platform showed a significant decrease across the trials (F(3,42)=7.540, p<0.001) and days (F(2,28)=16.579, p<0.001) during the cognitive flexibility phase (day 8-10) (Fig. 6C). The WT performed better from the third trial of the first day, while the KO only from the second trial of the second day (data provided). On day 11 (test 2) there was no difference in latencies between the genotypes. There was no genotype difference also in the time spent in the learned quadrant during retention on day 11 (data provided). As a sign of long-term memory, the number of entries into the ‘old’ platform zone was high at the beginning of testing cognitive flexibility, but decreased during the subsequent trials (F(3,42)=9.385, p<0.001) and days (F(2,28)=12.985, p<0.001) (Fig. 6D). The WT animals showed improvement from the third trial of day 8, while KO mice only from the second trial

of day 9. On day 11 (test day 2), only the KO animals entered significantly more times to the "old" zone than on the day 10 (data provided). The analysis by ANCOVA with locomotor behavior as a covariate did not show any significant influence of the locomotion excluding the confounding effect of locomotion.

Fig. 6 Results of Morris water maze test (Experiment 6) A: Experimental design of the MWM test with the timeline of the phases. Full, gray circle represents the platform to be reached. The dashed, gray circle represents the place that the test mice had to find. B: The latencies to reach the platform during learning was improved during days for both groups. There was an interaction between the genotype and days between day 6 and 7, showing a smaller latency to previous day for the WT animals, while a bigger one for the KO mice. C: The latencies to platforms during cognitive flexibility phase decreased across trials for both groups. D: The frequencies of old zone entries showed a decline across days for both genotypes. Data are expressed as mean±SEM. N=8/group. WT: wild type; KO: vesicular glutamate transporter 3 knock out

4. Discussion The VGluT3 KO mice clearly exhibited the ability to learn. We found only few signs of their impaired memory (y-maze) and reduced cognitive flexibility (operant conditioning, avoidance learning). An overview of the results is given in Table 1. Although locomotion is required for sampling the information during learning, and the anxious phenotype of KO mice [21, 22] - presented here by reduced distance moved (Fig. 1B) and higher escape latencies (Fig. 4D) during early habituation phases of tests - might influence this process [21], the repeated habituation was aimed to normalize the exploratory behavior (Fig. 1B). In case it was not successful (Table 2.), we used locomotion as covariance and also conducted analysis on derived parameters (e.g. SI, DI) corrected for movement. It was important to conduct ANCOVA as the loss of the main effect of genotype after ANCOVA may indicate that the genotypic differences in locomotion contribute as much to the variance as genotypic differences in the given measure. We did not detect any loss of the main effect genotype after using distance travelled as a covariate suggesting that the observed effects were not confounded by anxiety-induced hypolocomotion. Moreover, hypolocomotion per se might mimic learning and memory impairement, thus, could have increased the genotype difference. The lack of substantial genotype difference speaks agains the confounding effect of anxiety-related hypolocomotion. Indeed, we failed to observe any differences between the intermediate-term social memory in VGluT3 KO and WT mice. Social skills are heavily dependent on smelling. It has been demonstrated that certain neuron populations of the olfactory bulb expresses VGluT3 [50, 51], controlling the output of sensory information to the cortex [51]. As on day 3 KO animals did not show social preference (Table 2.), we may assume that VGluT3 could have

some role in social communication. Indeed, 8-day-old KO pups emitted more ultrasound after a short maternal separation, than WT [22, 52]. However, previous resident-intruder test revealing territorial aggression failed to find any genotype difference [22], therefore further tests of social behavior seem to be needed. The present studies revealed an impaired working memory of VGluT3 KO mice in the y-maze.

Y-maze

performance

is

PFC

and

hippocampus

dependent

[35].

Immunohistochemical studies have demonstrated that both brain areas contain VGluT3, either in axon terminals coming from other areas or in local cell bodies [53, 54]. Thus, present finding on reduced working memory of KO mice is understandable, but further studies are required to reveal the specific role of PFC and/or hippocampus and their innervation. The VGluT3 KO mice did not show any impairment in reward-based learning (operant conditioning or holeboard discrimination) paradigms. Reward is commonly associated with dopamine and its effects in the nucleus accumbens (NAc) [55]. It has been demonstrated that VGluT3 positive neurons project to the dopaminergic neurons of the ventral tegmental area (VTA) [56], the latter sending afferents to the NAc. The perikarya and dendrites of the NAc neurons are also VGluT3 positive [53]. Moreover, chronic consumption of sucrose, a reward, increased VGluT3 expression in the NAc [57]. Cocaine self-administration was increased in VGluT3 KO mice in connection with enhanced glutamatergic transmission in their NAc [58]. The present results are therefore somewhat surprising. In addition, we were not able to detect an overall impairment in the learning ability during avoidance learning in the shuttle-box, in which NAc also plays a role [55]. The diminished initial active avoidance and holeboard discrimination performance observed in the present study can be explained by a higher innate fear of VGluT3 KO mice leading to enhanced freezing during footshock and reduced locomotion [21]. One might also expect that hearing impairment [23] confound the results of the avoidance test as KO mice may depend

only on other cues (door opening, light). Indeed, this might explain the early genotype difference in escape latencies during the first few trials of testing day 1 (Fig. 4D). Previous findings showed that the protein expression of VGluT1, VGluT2, but not VGluT3 in aging mice correlated positively with the capacity of learning and memory in the shuttle-box [59], supporting the idea that VGluT3 is not involved in active avoidance learning. VGluT3 KO mice exhibited slower learning process compared to WT mice, but not a general learning impairment in the MWM. MWM measures spatial memory in which the hippocampus and its glutamatergic neurons are highly implicated [60]. However, the present results are in accordance with the report that heterozygous VGluT1 mice, having impaired VGluT1 expression, did not show any deficit in MWM [61]. We have observed impairments in cognitive flexibility in VGluT3 KO mice in several cognitive tests (mainly in those based on simple associations). With respect to the brain areas involved, distinct frontocortical regions are dominant in the control of behavioral flexibility [62]. As acetylcholine [63, 64] and dopamine [65] are known to play an important role in suppression of a previously learnt strategy, the disturbances in their neurotransmission – due to missing VGluT3 - might have contributed to the observed deficiency [66, 67]. 5. Conclusion Apparently, VGluT3 is not a major player in learning and memory formation in general, but the mild cognitive impairment of VGluT3 KO mice observed here suggest deficit in frontocortical function and/or PFC-hippocampal interaction. We cannot entirely exclude sex dependent alterations, therefore females should be also tested. Since cell-type specific deletion of VGluT3 in acetylcholinergic and serotoninergic neurons has not fully recapitulated some behaviors observed in full VGluT3 KO mice [66, 68], there is a possibility that VGluT3 on specific cells has deeper impact on learning and memory. Conflict of interest

We declare that there is no conflict of interest in the conduct and reporting of research. The agencies had no further role in study design, in the collection, analysis, or interpretation of the data. Author contribution DB, DJ, JH and DZ conceptualized the work, CsLF, DB, HHR, ZB, MA, AP, LB, MC and DZ conducted the investigations, CsLF, DB, HHR, ZB, MA and DZ conducted the formal analysis, CsLF, DB, DJ and DZ wrote the original draft, while all other authors contributed to the review and editing process, CsLF, DB and DZ prepared the visualization, while DJ and JH conducted the funding acquisition. Grant information This work was supported by the European Reserch Council Advanced Research grant ERC2011-ADG-294313 (SERACO), NKFIK K101645, K112907, K120311, HAS NKM-32/2016 and bilateral projects between Hungarian and Slovak Academy of Sciences. We declare that there is no conflict of interest in the conduct and reporting of research. The agencies had no further role in study design, in the collection, analysis or interpretation of the data. References [1] Soriano, J. B., Rojas-Rueda, D., Alonso, J., Anto, J. M., Cardona, P. J., Fernandez, E., et al. The burden of disease in Spain: Results from the Global Burden of Disease 2016. Med Clin (Barc). 2018. [2] Barnes, L. L., Bennett, D. A. Alzheimer's disease in African Americans: risk factors and challenges for the future. Health Aff (Millwood). 2014,33:580-6. [3] Dere, E., Pause, B. M., Pietrowsky, R. Emotion and episodic memory in neuropsychiatric disorders. Behav Brain Res. 2010,215:162-71. [4] Haam, J., Yakel, J. L. Cholinergic modulation of the hippocampal region and memory function. J Neurochem. 2017,142 Suppl 2:111-21. [5] Lucas, E. K., Clem, R. L. GABAergic interneurons: The orchestra or the conductor in fear learning and memory? Brain Res Bull. 2018,141:13-9. [6] Seyedabadi, M., Fakhfouri, G., Ramezani, V., Mehr, S. E., Rahimian, R. The role of serotonin in memory: interactions with neurotransmitters and downstream signaling. Exp Brain Res. 2014,232:723-38. [7] Riedel, G., Reymann, K. G. Metabotropic glutamate receptors in hippocampal long-term potentiation and learning and memory. Acta physiologica Scandinavica. 1996,157:1-19. [8] Riedel, G., Platt, B., Micheau, J. Glutamate receptor function in learning and memory. Behav Brain Res. 2003,140:1-47.

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