Mice with Mutations in Trpm1, a Gene within the locus of 15q13.3 Microdeletion Syndrome, Display Pronounced Hyperactivity and Decreased Anxiety-Like Behavior.

15q13.3 microdeletion syndrome is a genetic disorder caused by a deletion of a region containing seven genes on chromosome 15, MTMR10, FAN1, TRPM1, MIR211, KLF13, OTUD7A, and CHRNA7, and characterized by a wide spectrum of psychiatric disorders. The contribution of each gene in this syndrome has been studied using mutant mouse models, but the phenotypes of these mice do not account for human phenotypes and the results are still controversial. The behavior of Trpm1 −/− mice with relation to 15q13.3 microdeletion syndrome has not been investigated due to the visual impairment in these mice, which may confound the results of behavior tests that involve vision. We have now applied a comprehensive behavioral test battery to examine the relationship of TRPM1 and 15q13.3 microdeletion syndrome by using Trpm1 null mutant mice. Our data indicate abnormal behavior of Trpm1 −/− mice which may explain some phenotypes of 15q13.3 microdeletion syndrome, including reduction of anxiety behavior, abnormality of social interaction, attenuation in fear memory, and hyperactivity, which is the most prominent phenotype of Trpm1 mutant mice. While the ON visual transduction pathway is impared in Trpm1 −/− mice, we did not detect compensatory high sensitivities for other sensory modalities. Although Trpm1 −/− mice share the same pathway for visual impairment with mGluR6 −/− mice, hyperlocomotion activity has not been reported in mGluR6 −/− mice. These data suggest that the phenotype of Trpm1 −/− mice extends beyond that expected from visual impairment alone. This is the �rst evidence to associate TRPM1 with impairment of cognitive function similar to that found in the phenotypes of 15q13.3 microdeletion syndrome.


Introduction
TRPM1, the rst member of the melanoma-related transient receptor potential (TRPM) subfamily to be discovered, is the visual transduction channel downstream of metabotropic glutamate receptor 6 (mGluR6) in retinal ON bipolar cells (BCs) [1,2].Mutations in TRPM1 are found in humans with an autosomal recessive form of complete congenital stationary night blindness (CSNB) and Trpm1 mutant mice lack a b-wave in the electroretinogram (ERG) and have no light responses in ON BCs [3].TRPM1 is located in human chromosome 15q13.3,a region that is associated with 15q13.3microdeletion syndrome, a genetic disorder caused by a deletion encompassing a region of ~ 1.5 megabase (MB) from break-point (BP) 4 to BP5, with seven genes; MTMR10; FAN1; TRPM1; MIR211; KLF13; OTUD7A; and CHRNA7 (OMIM #612001) [4].Individuals with 15q13.3microdeletion syndrome have mild to moderate intellectual disorder, mild learning delays, autism spectrum disorder, epilepsy (recurring seizures), attention de cit hyperactivity disorder (ADHD) and mental illnesses including schizophrenia and bipolar disorder [5].Deletion of CHRNA7 is thought to account for the neuropsychiatric disorder in 15q13.3microdeletion syndrome, although the phenotype of Chrna7 de cient mice is controversial [6].Otud7a mutant mice also show many features, including neurological features, in common with those in 15q13.3microdeletion syndrome, including reduced body weight, developmental delay, abnormal electroencephalogram (EEG) patterns and seizures, reduced ultrasonic vocalizations, decreased grip strength, impaired motor learning/motor coordination, and reduced acoustic startle [7].
The role of TRPM1 in behavioral disorders has not been studied, probably because of its strong relationship with vision.TRPM1 and its regulator, mGluR6, both cause CSNB.In mutant retinas, both cause ON but not OFF pathway to fail to respond to light stimuli [1,8,9].We previously reported an unexpected difference between Trpm1 −/− and mGluR6 −/− mouse retinas.Spontaneous oscillations are observed in Trpm1 −/− retinas, but not in mGluR6 −/− retinas by recording spiking in retinal ganglion cells (RGCs) using a multielectrode array (MEA) [10].We also previously reported that rod ON BC terminals were signi cantly smaller in Trpm1 −/− retinas than those of mGluR6 −/− [10].These data suggest that a de ciency of TRPM1, but not of mGluR6, in rod ON BCs may effect synaptic terminal maturation and could underlie the observed differences in oscillatory response.Spurred on by the observation of Trpm1deletion speci c RGC oscillations and a gene location in the targeted region of 15q13.3microdeletion syndrome, we searched for central and behavior changes that might attribute to a persistent, rhythmic visual out ow.
In this study, we thoroughly examined Trpm1 −/− mice with a battery of behavioral tests [11].We also examined structural and functional change in Trpm1 −/− mouse brain, which may explain the abnormal behaviors of this mutant as a model of 15q13.3microdeletion syndrome.

Animals and Experimental Design
Trpm1 −/− mice were generated as described previously [1].In this study, we analyzed Trpm1 −/− mice with their wild type (WT) littermates on the 129 Sv/Ev Taconic background.All behavioral tests were performed with male that were 11-12 weeks old at the starting of the testing (Trpm1 −/− mice, n = 24; WT littermates, n = 24).Mice were housed as 2 pairs of Trpm1 −/− and WT mice in a cage with a 12-hour light/dark cycle (light on 7:00 a.m. and off 7:00 p.m.).All mice had access to food and water ad libitum.Behavioral testing was performed between 8:30 A.M. and 6:30 P.M. unless otherwise noted.Our behavioral test battery consists of the tests listed in Table 1.After the tests, all the testing apparatus were cleaned with diluted hypochlorous solution or 70% ethanol to prevent a bias due to olfactory cues.
Brain weight measurement and monoamine quanti cation in brain tissues were performed with 129 Sv/Ev male at 4 months (Trpm1 −/− mice, n = 24; WT littermates, n = 24) or 1 months (Trpm1 −/− mice, n = 4; WT littermates, n = 5).Gene expression analysis was performed with 129 Sv/Ev male at 1 months (WT, n = 5).Mice used for monoamine quanti cation were housed as 2 pairs of Trpm1 −/− mice and WT mice in a cage with a 12-hour light/dark cycle (light on 8:00 a.m. and off 8:00 p.m.), and tissue dissection was performed at the same time point (1:00 p.m.).All mice had access to food and water ad libitum.The experimental procedures and housing conditions for animals were approved by Institutional Animal Care and Use Committee of National Institute for Physiological Sciences, Fujita Health University and Ritsumeikan University.

General health and neurological screen
A general health and neurological screen examined the body weight, rectal temperature, whisker, coat, simple re exes such as righting, whisker touch, eye blink, ear twitch re exes and reaching behavior using 11 ~ 12 weeks old, as described previously [12].Grip strength test and wire hang test were conducted to measure muscle strength.Grip strength was measured by using a grip strength meter (O'Hara & Co., Japan).In the wire hang test, the mouse was placed on a wire cage lid that was then inverted, so that the subject gripped the wire.Latency to fall onto the bedding was recorded, with a 60 sec cutoff time.

Light/dark transition test
Light/dark transition test was performed as described previously [13][14][15].The apparatus used for light/dark transition test consisted of a cage (21 × 41.5 × 25 cm) divided into two sections of equal size by a partition with a door (O'Hara & Co., Japan).One section was brightly illuminated (390 ± 20 lux), whereas the other section was dark (< 2 lux).Mice were placed into the dark side of the apparatus, and allowed to move freely between the two sections for 10 min, while the door remained open.In the same way, mice (34-35 weeks old) were placed into the light side of the apparatus allowed to move freely between the two sections for 10 min.The total number of transitions, time spent in each section, initial latency to the light section, and distance traveled were recorded automatically using Image LD software.
Social interaction test was performed as described previously [16].A pair of mice (12-13 weeks old) was placed simultaneously at opposing corners in the open eld apparatus (40 × 40 × 30 cm; O'Hara & Co., Japan), whose illumination level was 10.0 lx at the center of the oor, and allowed to explore freely for 10 min.The pair of mice had been housed in different cages.The number of active contacts, the number of contacts, mean duration per contact, total duration of contact, and total distance traveled were measured.Analysis was performed automatically using Image SI software.

Rota-rod test
Motor coordination and balance were tested with the rota-rod test old as described previously [16].The rota-rod test using an accelerating rota-rod (UGO Basile, Italy) was performed by placing a mouse on a rotating drum (3 cm diameter) and measuring the time each animal was able to maintain its balance on the rod.The speed of the rota-rod accelerated from 4 to 40 rpm over a 5-min period.
Social approach and novelty preference test Social approach and preference for social novelty were tested with the The three-chamber social approach test as described previously [14,16].The apparatus comprised a rectangular, three-chambered box and a lid with a video camera (O'Hara& Co., Japan).Each chamber was 20 cm × 40 cm × 22 cm and the dividing walls had small openings (5 cm × 3 cm) to allow exploration into each chamber.The day before testing, the mice were individually placed in the middle chamber and allowed to freely explore the entire apparatus for 10 min.During the test session, the amount of time spent in each chamber and time spent around each cage were recorded and analyzed automatically using Image CSI.

Acoustic Startle response/prepulse inhibition tests
Acoustic Startle response/prepulse inhibition tests were performed as described previously [16] (O'Hara & Co., Japan).A test session began by placing a mouse in a Plexiglas cylinder where it was left undisturbed for 10 min.The duration of white noise that was used as the startle stimulus was 40 msec for all trial types.A test session consisted of six trial types (i.e., two types for startle stimulus-only trials and four types for prepulse inhibition trials).The intensity of startle stimulus was 110 or 120 dB.The prepulse was presented 10.0 msec before the startle stimulus, and its intensity was 74 or 78 dB.Four combinations of prepulse and startle stimuli were used (74 − 110, 78 − 110, 74 − 120, and 78-120).Six blocks of the six trial types were presented in pseudorandom order such that each trial type was presented once within a block.The average intertrial interval was 15 sec (range, 10-20 sec).

Porsolt Forced Swimming test
Depression-related behavior was assessed, using the forced swimming test as described previously [12].
The apparatus consisted of Plexiglas cylinders (22 cm height x 12 cm diameter).The cylinders were lled with water (room temperature, 23 ± 2 °C), up to a height of 7.5 cm.Mice were placed into the cylinders, and their behavior was recorded over a 10-min test period.Immobility and distance traveled were measured analyzed automatically using Image PS software.

Gait analysis
The gait during walk/trot locomotion was assessed using DigiGait Imaging System (Mouse Speci cs, U.S.A.) as described previously [17].Digital video images of the underside of mice were collected at 150 frames per second.We placed the mice on a treadmill belt that moves at a speed of 24.7 cm/s.The percent of the time of stride or stance duration, stride length, stance width, step angle and paw angle were calculated.

Barnes maze
The Barnes maze test was performed as described previously [12].The circular open eld (O'Hara & Co., Japan) was elevated 97 cm from the oor.Training session were conducted one to three per day.After 24 hours after 15th training session, a probe test was conducted without the escape box, to con rm that this spatial task was acquired based on navigation by distal environmental room cues.One month after last (16th) training session, probe trial tests were conducted again to evaluate memory retention.After ve additional training sessions conducted after the memory retention test, the escape box was moved to a new position opposite to the original (reversal learning).Mice were then trained with 8 sessions to locate the new position of the escape hole using the same procedure as described above.Latency to reach the target hole, distance to reach the target hole, number of errors and time spent around each hole were recorded automatically using Image BM software.

T-maze Spontaneous Alteration
T-maze spontaneous alteration test was performed as described previously [15] using the automatic modi ed T-maze apparatus (O'Hara & Co., Japan).Mice were subjected to a the spontaneous alteration protocol for 5 sessions.One session consists of 10 choices with a 50-min cutoff time.Mice were rst placed in the start compartment of the T-Maze.Mice chose entering either the left or the right arm and could return to the start compartment.The mice was then given a 3-sec delay followed by a free choice between both T arms.A correct choice was made if the mouse entered the arm which was not visited in the previous choice.The percentage of correct response, latency (sec) to complete a session, distance traveled during the session.Data acquisition was performed automatically using Image TM software.

Tail suspension test
Depression-related behavior was assessed by the tail suspension test as described previously [17].Mice were suspended 30 cm above the oor in a visually isolated area by adhesive tape placed ,1 cm from the base of the tail, and their behavior was recorded over a 10-min test period.Data acquisition and analysis were performed automatically using Image TS software.

Contextual and cued fear conditioning
The ability of mice to learn and remember an association between environmental cues and aversive experiences was assessed by fear conditioning test as described previously [15,16].Each mouse was placed in a test chamber (26 × 34 × 33 cm, O'Hara & Co., Japan) and allowed to explore freely for 2 min.A 55 dB white noise, which served as the conditioned stimulus (CS), was presented for 30 sec.Next, a mild (2 sec, 0.3 mA) foot shock, which served as the unconditioned stimulus (US), was presented immediately after the CS.Two more CS-US pairings were presented with a 2-min interstimulus interval.Context testing was conducted 1 day after conditioning in the same chamber for 30.0 sec without CS and US presentations.
Cued testing with altered context was conducted after conditioning using a triangular box (33 × 33 × 33 cm) made of white opaque Plexiglas, which was located in a different room.Mice are allowed to explore the chamber for 360 sec.In the rst 3 min, neither a CS nor US is presented, then a CS (a 55dB white noise) is presented for the last 3 min.Freezing and distance traveled were recorded.Data acquisition, control of stimuli (i.e.tones and shocks), and data analysis were performed automatically using Image FZ software.

Twenty-four hour home cage monitoring test
The 24-hour home cage test was performed as described previously [15].The system for monitoring social interaction comprised a home cage (19 × 29 × 13 cm) and a ltered cage top with an infrared video camera (O'Hara & Co., Japan).Two mice with the same genotype that had been housed separately were placed together in a home cage.To evaluate their locomotor activity and social interaction, their behavior was monitored with a video camera for a week.Distance traveled was measured auto-matically using ImageHA software.The occurrence of social interaction was detected by counting the number of particles consisting of the mice as follows: 2 particles indicated that the mice were not in contact whereas 1 particle indicated that 2 mice were in contact.Locomotor activity of the mice was also measured.

Methylphenidate administration in the open eld
After the behavioral test battery, the behavioral response to methylphenidate (MPH) was assessed in the open led.A quarter of the area of the open eld apparatus (20 × 20 × 30 cm) was used by installing a divider.Other conditions were the same as for the open led test.The mice of each genotype were randomly divided into two groups for treatment with MPH and saline.The experiment was repeated twice with varying drug doses.Locomotor activity was recorded continuously during the 60-min habituation period and for 120 min after injection of saline or MPH (3 mg/kg or 10 mg/kg).

Monoamine quanti cation in brain tissues
Monoamine transmitter quanti cation was performed as described previously [18].Tissue concentrations of biogenic monoamines were analyzed after dissection in various brain regions; prefrontal cortex, hippocampus, striatum, cerebral cortex, olfactory bulb, cerebellum, midbrain, pons and medulla, thalamus, hypothalamus.The weight of the brain tissue was measured, and homogenized in 0.2 M icecold perchloric acid (including 10.0 µM EDTA 2Na) and the homogenates were cooled on ice for 30 min to deproteinize.The homogenates were centrifuged at 20,000 G for 15 min at 0 ℃.Then, the pH of the supernatant was adjusted to approximately 3.0 by adding 1 M sodium acetate.The samples were ltered through a 0. 45 mm lter (Millipore, Billerica, USA).Next, 10 µL of ltrate was loaded into a high performance liquid chromatography (HPLC) system (Eicom, Japan).The HPLC system had a ø3.0 mm x 150 mm octadecyl silane column (SC-5ODS, Eicom), and an electrochemical detector (ECD) (HTEC-50.0;Eicom, Japan) set to an applied potential of + 750 mV versus an Ag/AgCl reference analytical electrode.The change in electric current (nA) was recorded using a computer interface at 25 ℃.The mobile phase was composed of 0.1 M aceto-citric acid buffer (pH 3.5), methanol, sodium-1-octane sulfonate (0.46 M), and EDTA 2Na (0.015 mM) [830: 170: 1.9: 1].The ow rate was 0.5 mL/min.

Corticosterone measurement
Blood was collected from mice at 4 months old by Cardiac puncture immediately after cervical dislocation.A serum was separated by centrifuging at 2,000 g for 20 min, and stored at -80 ℃ until use.Corticosterone measurements were performed on serum by enzyme-linked immuno sorbent assay (ELISA) using a Corticosterone immunoassay (R&D Systems, U.S.A.) according to the manufacturer's instructions.

Data analysis
All statistical analyses were performed using Graph Pad Prism7.Statistical methods are indicated in the gure legends.Data are presented as mean ± SEM.Unpaired 2-tailed Student's t test or Welch's t test were used for 2-group comparisons.Two-way analysis of variance (ANOVA) or repeated-measures two-way ANOVA following Tukey's test and one-way ANOVA following Dunnett's test was used for multiple comparison.Unless otherwise noted, the p values are for the genotype effect.

Data repository
The raw data of the behavioral tests and the information about each mouse are accessible on the public database "Mouse Phenotype Database" (http://www.mouse-phenotype.org/).
We performed a battery of more than 20 behavioral tests (Table 1).There was almost no signi cant difference for general physical characteristics, such as body weight, body temperature, grip strength, and motor coordination between Trpm1 −/− and wild type mice (Fig. S1A-K).Trpm1 −/− did not show depression-like behaviors in Porsolt forced swim test and tail suspension test (Fig. S1L-N).Intriguingly, Trpm1 −/− mice showed signi cantly high daily locomotor activity (Fig. 1A) in contrast to mGluR6 −/− mice [19].It has been reported that the absence of vision in human and animals enhances auditory, haptic, and pain sensitivities [20][21][22][23][24][25][26][27][28][29], and causes structural changes in the visually deprived cortex and in other areas [23,30,31].We examined sensory responses in Trpm1 −/− mice, but we did not nd any signi cance difference between Trpm1 −/− mice and wild type for the hot plate test, the acoustic startle response, and prepulse inhibition (Fig. 1B-D).Although Trpm1 −/− mice lack a functional ON visual pathway and thus have limited light responses, Trpm1 −/− mice showed hyper-locomotor activity but did not show sensory hypersensitivities that was reported in other visually impaired animals.
Hyperactivity and reduction of anxiety-like behavior in Trpm1 −/− mice To assess anxiety-like behavior, we performed the light/dark transition test, the open-eld test, and the elevated plus maze test (Fig. 2).In the light/dark transition test, distance traveled in the light and dark chamber were signi cantly increased in Trpm1 −/− mice suggesting a reduction in anxiety-like behavior (Fig. 2A).However, the defect in the ON visual pathway may have affected the longer stay time in light and increased transition time and shorter latency to light for tests started at dark (Fig. 2B-D).In the open eld test, which measures voluntary locomotor activity in a novel environment, Trpm1 −/− mice exhibited a signi cant increase in total distance, vertical activity, center time and stereotypic behavior relative to WT mice (Fig. 2E-H), suggesting strong hyperactivity, which also explains longer distance traveled in the light/dark transition test.To implicate the hyperactivity in Trpm1 −/− mice with ADHD, we performed the open eld test after administration of MPH, which is often used as a rst choice for treatment of ADHD in human (Fig. 2I) [32].120 min after administration of MPH, both WT and Trpm1 −/− mice showed prominent hyperactivity, especially in mice which were injected at 10 mg/kg.As a result, MPH administration did not support the idea that the ADHD behavior displayed in Trpm1 −/− mice can be reduced by MPH administration [33].
Additionally, in the elevated plus maze test, Trpm1 −/− mice exhibited signi cantly increased numbers of entries and longer traveled distance, which are also explained by hyperactivity (Fig. 2J, M).Although visually impaired, Trpm1 −/− mice did not show differences for entries to open arms, but stayed longer time in open arms suggesting a reduction of anxiety-like behavior (Fig. 2K, L).
To examine what causes a reduction in anxiety-like behavior in Trpm1 −/− mice, we examined serum corticosterone levels in Trpm1 −/− mice by ELISA [34,35].The idea is that an authentic reduction in anxiety should be correlated with an decrease in serum corticosterone levels, while a reduction in anxiety-like behavior in the absence of a decrease may have some other cause.The serum levels of corticosterone were not signi cantly different between Trpm1 −/− mice and WT mice (Fig. S1O).This observations suggests that the hyperactivity of Trpm1 −/− mice simulates a reduction of anxiety in our tests.
Abnormal social interaction in Trpm1 −/− mice Four kinds of social interaction tests (novel environment, sociability, novelty preference, and home cage test) were performed to evaluate social behaviors in the Trpm1 −/− mice (Fig. 3).The novel environment test revealed signi cant differences between Trpm1 −/− and WT mice, including a shorter duration per contact, increased contact number, and total traveled distances, which may be explained by the hyperactivity in Trpm1 −/− mice (Fig. 3A, D, E).Although total duration of contact was shortened, but not signi cantly, active contacts were longer in Trpm1 −/− mice (Fig. 3B, C).Both Crawley's sociability and social novelty preference test and the test in the home cage did not reveal signi cant differences between WT and mutant mice (Fig. 3F-M, S1P).These results suggest that social interactions were attenuated in Trpm1 −/− mice, which may be partially explained by hyperlocomotion behavior.
Attenuation of fear and spatial memories in Trpm1 −/− mice The contextual and cued fear conditioning test is used to assess fear memory (Fig. 4).In the conditioning phase, Trpm1 −/− mice showed a lower level of freezing and traveled longer distances during sessions (Fig. 4A, B).The mutant mice traveled longer immediately after foot shock, an index of pain sensitivity (Fig. 4C).24 hours after conditioning, Trpm1 −/− mice showed decreased freezing and increased distance traveled.Similar signi cant differences were observed in tests 28 days after conditioning (Fig. 4D, E).These data suggest that attenuation of fear memory in Trpm1 −/− mice could be related to hyperactivity.
We performed the Barnes maze test to determine whether the de cit in fear memory of Trpm1 −/− contributes to hyper-locomotion or results from a de cit of memory.In both training sessions and reversal task tests, the distance to the escape box (Fig. 4F) and the number of errors to reach the escape box were signi cantly higher in Trpm1 −/− mice (Fig. 4G), but latency to rst reach the escape box was equivalent or shorter in Trpm1 −/− mice than in WT mice (Fig. 4H), which may be related to hyper-locomotion activity.The probe tests were performed 24 hours and 1 month after the nal training sessions.In these tests, Trpm1 −/− and WT mice exhibited a signi cant effect of hole location target against the rest holes: 24 hour, WT p < 0.0001, Trpm1 −/− p < 0.0001; 1 month, WT p < 0.0001, Trpm1 −/− p < 0.0001; one-way ANOVA followed by Dunnett's multiple comparison test), indicating that both genotypes were able to distinguish the location of the target.Time spent around the correct hole did not differ signi cantly between both genotypes at 24 hours after training, but was signi cantly shorter in Trpm1 −/− mice 1 month later (Fig. 4I,   J).These results suggest that Trpm1 −/− mice have a de cit in long-term memory.In the reversal probe test, although both genotypes distinguished the location of the target (WT p < 0.0001, Trpm1 −/− p < 0.0001; one-way ANOVA followed by Dunnett's multiple comparison test), there was no signi cant difference in time spent around the correct hole between both genotypes (Fig. 4K).This result indicates that there was no de cit in behavioral exibility in Trpm1 −/− mice.We also performed a T-maze test to examine the working memory of Trpm1 −/− mice.Although the shorter latency and longer distance traveled in Trpm1 −/− mice were both signi cantly different, the correct responses at each trial were not (Fig. 4L-N).Taken together, Trpm1 −/− mice showed attenuation for fear and long term memories, but no obvious deviation for exibility and working memory.
Abnormal Structural and biochemical changes in the brains of Trpm1 −/− mice We have shown differences in the behavioral phenotype in Trpm1 −/− mice relative to WT.However, Trpm1 functions predominantly as a component of the retinal ON bipolar transduction cascade and its expression is quite minor in the brain.To determine whether there are central structural changes, we compared brain regions between Trpm1 −/− and WT mice.The cerebral cortex, olfactory bulb, and pons and medulla of Trpm1 −/− mice were signi cantly heavier than in WT mice at 1 month old (Fig. 5A).In addition, the cerebral cortex, hippocampus, midbrain and cerebellum of Trpm1 −/− mice were signi cantly heavier than those of WT mice at 4 months old (Fig. 5B).
We detected a subtle expression of Trpm1 mRNA throughout the WT mouse brain with the exception of the cerebellum (Fig. 5C).We also quanti ed levels of biogenic monoamines ex vivo, including dopamine (DA), noradrenaline (NAd), serotonin (5-HT), and their major metabolites using HPLC-ECD in several adult brain regions.Levels of DA, NA, and NM were signi cantly decreased in the cerebellum (Fig. 5D-F).There was no signi cant change in the levels of the other monoamines and their metabolites in any other brain region (Fig. S2).The decreased levels of monoamines in the cerebellum of Trpm1 −/− mice could in uence hyper-locomotory activity, locomotion is regulated by the cerebellum.The lack of overlap between the Trpm1 expression pattern and the change of monoamine distribution in the brain is consistent with the idea that Trpm1 expresses in monoaminergic neurons that project to the cerebellum.

Discussion
15q13.3 microdeletion syndrome shows a spectrum of neurobehavioral phenotypes in human patients.So far, several investigations have suggested that mutants that abolish the functions of OTUD7A and CHRNA7 can partially explain the phenotypes of 15q13.3microdeletion syndrome.However, we still lack a full accounting for the microdeletion phenotypes, especially those related to hyperactivity.In this paper, we examined the behavior of Trpm1 de cient mice with a comprehensive behavioral test battery.Our data show abnormal behaviors in Trpm1 de cient mice including a reduction of anxiety behavior, an abnormality of social interaction, the attenuation of fear and spatial memories, and hyper-locomotion activity, which is the most prominent phenotype of Trpm1 mutant mice and may also underlie or contribute to other phenotypes of Trpm1 −/− mice (Fig. 1-3).Hyperactivity is one of the features of ADHD, and a patient with 15q13.3deletion, who had a relative lack of expression of genes including TRPM1, showed ADHD behavior [36][37][38][39][40][41][42][43].We examined the effect of MPH which is often used for treatment of ADHD, however, MPH signi cantly increase the locomotion activity of Trpm1 −/− mice (Fig. 2I).Intriguingly, MPH-like compounds were ineffective in approximately 35% of patients with ADHD [44,45].There are several mouse models of hyperactivity which were also insensitive to MPH.In Ndrg2 de cient mice, their ADHD-like hyperactivity was not rescued by MPH [46].Shank2 and Fmr1 mutant mice, both are the model of autism, display hyperactivity, but the injection of MPH increased their locomotion activity [47,48].Relevant to the effect of MPH in Shank2 and Fmr1 de cient mice, hyperactivity in Trpm1 de cient mice may not be related to ADHD, but instead autism which is also one of the phenotypes in 15q13.3microdeletion syndrome (Table S1) [5,49].
Trpm1 −/− mice showed prominent locomotion activities (Fig. 1A, 2E) that are not observed in mGluR6 −/− mice [19].Both mice lack of a functional ON visual transduction pathway and lack a b-wave in the ERG [3,50] as well as having no ON response [1,8,9].Additional evidence for visual impairment in Trpm1 −/− mice comes from measurements of the spatial frequency and contrast sensitivity thresholds of the optokinetic response (OKR) was repotrted.Thresholds were reduced by approximately 10% and 30%, respectively, compared to WT [51].While both mGluR6 and Trpm1 de cient mice lack ON BCs responses, spontaneous oscillatory ring in RGCs, the output cells of the retina, was only detected in Trpm1 −/− mice [10].An attractive idea is that these retinal oscillations might be communicated along the optic nerve to higher visual centers and result in hyperlocomotion in Trpm1 −/− mice.
Thus is it unlikely that the behavioral changes in Trpm1 −/− mice are secondary to changes in non-visual sensory perception.
There is another possible explanation for the behavioral phenotypes in Trpm1 −/− mice.The expression of mGluR6 is known to be restricted to retinal ON BCs, whereas TRPM1 is expressed in the retina and skin in humans [57][58][59], and a short form of TRPM1, which does not have channel function, is expressed in embryonic retinal pigment epithelial and skin in mouse [1,2].Thus, there is a precedent for the expression of TRPM1 outside of the retina, including alternate splice forms.We analyzed the expression of Trpm1 in the brain and detected a faint expression by qPCR thoughout most of the brain with the exception of the cerebellum (Fig. 5C).Thus, Trpm1 may be expressed in some parts of brain and the presence or lack of Trpm1 in a particular region may effect behavior.A link between TRPM1 and brain function was previously suggested by the demonstration that capsaicin-induced activation of TRPM1 channels contributed to the induction of long-term depression (LTD) in the lateral amygdala, which was speci cally mediated by group I mGluRs and via interactions with another member of the TRP family, TRPC5 [60].
De ciency of Trpm1 expression in the brain including the amygdala may lead to a neurochemical attenuation in brain function that may cause behavioral phenotypes in Trpm1 de cient mice similar to those demonstrated here.
In summary, our results are consistent with the idea that spontaneous oscillatory ring in the retina may be transmitted to the higher visual system through the optical nerve and more central projections during development and after, and as a result may modify the function and structure of the brain leading to the observed behavior changes.An alternate but not mutually exclusive view is that the lack of expression of Trpm1 in the brain may change the distribution of biogenic monoamines and behaviors in Trpm1 −/− mice.Irrespective of the mechanism, this is the rst report to implicate TRPM1 loss in 15q13.
Behavioral data were obtained automatically by customized applications based on a public domain ImageJ program (Image LD, Image EP, Image SI, Image CSI, Image PS, Image BM, Image TM, Image TS, Image FZ, Image HA).The ImageJ plugins, and the precompiled plugins for light/dark transition test (Image LD), elevated plus maze (Image EP), open eld test (Image OF), fear conditioning test (Image FZ), and T-maze (Image TM) are freely available on the website of "Mouse Phenotype Database" (http://www.mouse-phenotype.org/software.html) .

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Figure 1 Physical
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Figure 3 Social
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