Plasmids
Vectors encoding EGFP- and mCherrry-PLIN2 were described previously [5]. In brief, human cDNAs encoding PLIN2 was amplified from HeLa cells by PCR with specific primers and were then subcloned into pEGFP or pmCherrry (Clontech).
Reagents
LysoTracker Red was from Thermo Fisher Scientific. Filipin complex from Streptomyces filipinensis was from Sigma-Aldrich.
Cell culture and transfection
PC12 cells were cultured under a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM, Wako) supplemented with 10% fetal bovine serum (FBS, Nichirei) and on plates coated with poly-L-lysine (150–300 kDa, Sigma) before exposure to mouse submaxillary gland nerve growth factor (Merck Millipore) at 100 ng/ml in RPMI 1640 supplemented with 1% horse serum (Thermo Fisher Scientific). They were transfected with siRNAs or expression vectors with the use of a Nucleofector system (Lonza).
RNA interference
Stealth siRNAs targeted to rat PDZD8 mRNA were obtained from Invitrogen–Life Technologies. The sequences of the siRNAs were 5'-GGGCCGGCTTAAAGTTACATTGCTA-3' (#1), 5'-CAGTCCCAAACGTACTCCAACAACA-3' (#2), and 5'-GAGGTGGCTTTAGGATGCCTAGCTA-3' (#3). The siRNAs were introduced into PC12 cells with the use of a Nucleofector instrument (Lonza), and the cells were then cultured for 72 h before experiments. As for both human and rat siRNAs, results are shown for siRNA #1, but similar findings were obtained with the other two siRNAs.
Fluorescence imaging of live cells
Cells that had been transfected with plasmids encoding fluorescently tagged proteins or metabolically labeled with fluorescent probes were observed with an LSM800 confocal microscope (Zeiss), and the images were processed for calculation of fluorescence intensity with ZEN imaging software (Zeiss).
TEM
Mouse brain was fixed with 2% glutaraldehyde (Nisshin EM) in 0.05 M cacodylate buffer containing 5 mM CaCl2. The fixed tissue was washed in cacodylate buffer with CaCl2, exposed to buffered 1% OsO4 plus 0.8% K4[Fe(CN)6]•3H2O for 1 to 2 h, dehydrated with acetone or ethanol, and embedded in Epon-Araldite or Spurr’s medium (Nisshin EM). Thin sections were stained with uranyl acetate and lead citrate and observed with a JEM-1400 Plus instrument (JEOL).
Mutant mice
Generation of PDZD8-deficient mice was described previously [10]. In brief, Cas9 nickase mRNA (IDT 1074181, Alt-R S.p. Cas9 Nuclease 3NLS; IDT, San Jose, CA), Generic tracrRNA (IDT 1072532, Alt-R CRISPR-Cas9 tracrRNA), and target-specific crRNA (IDT, Custom Alt-R CRISPR crRNA) were mixed and injected into C57BL/6 J mouse zygotes with the use of a Super Electroporator NEPA21 Type II (Nepagene, Tokyo, Japan). The 5' and 3' single guide RNAs with 20-nucleotide sequences targeted to exon 1 (which contains the start codon) of the PDZD8 gene (GenBank accession number NM_001033222) and containing protospacer-adjacent motif sequences were 5'-GCAGGCCGAGGGTTGCGGCGGGG-3' and 5'-GCAGATTCCCAGCACGACCCTGG-3', respectively. Potential predicted off-target sites were checked for at http://crispr.mit.edu. The resultant mutant mice were backcrossed with C57BL/6 J mice before experiments. Animals were genotyped by PCR with the primers 5'-CTCAACAGACCCAGGAGAGG-3', 5'-AGCCCGACTTATCCAGGTCT-3', and 5'-CGCTGGAGACCTGCTACTTC-3' and with MightyAmp DNA polymerase (Takara, Shiga, Japan). All mouse experiments were approved by the animal ethics committee of Nagoya City University.
General protocol for behavioral tests
WT and PDZD8-KO mice were group-housed (three or four animals per cage) in a room with a 12-h-light, 12-h-dark cycle (lights on at 7 a.m. and off at 7 p.m.) and with access to food and water ad libitum. Behavioral tests were performed between 9 a.m. and 6 p.m. with male mice at 10 to 20 weeks of age as described previously [36,37,38,39,40], unless indicated otherwise. Each apparatus was cleaned with hypochlorite solution before testing of each animal in order to prevent bias due to olfactory cues.
Neurological screen
Mice were subjected to physical assessment, including measurement of rectal temperature and body weight. A wire-hang test apparatus (O’Hara & Co., Tokyo, Japan) was used to assess muscular strength. The apparatus consists of a box (21.5 by 22 by 23 cm) with a wire-mesh grid (10 by 10 cm) on top that can be inverted. Mice were placed on the wire mesh, which was then inverted, causing the animal to grip the wire. The latency to falling was recorded, with a 60-s cutoff time. For assessment of forelimb grip strength, a mouse was held by its tail and lifted so that its forepaws could grasp the wire grid of a grip-strength meter (O’Hara & Co.). It was then gently pulled backward by the tail until it released the grid. The peak force applied by the forelimbs was recorded. Each mouse was tested three times, and the highest value was used for statistical analysis.
Hot-plate test
Sensitivity to a painful stimulus was assessed with the hot-plate test. Mice were placed on a hot plate maintained at 55.0° ± 0.2 °C and with a black anodized aluminum surface (Columbus Instruments, Columbus, OH). The latency to the first paw response (foot shake or paw lick) was recorded, with a cutoff time of 15 s.
Rotarod test
Motor coordination and balance were evaluated with the rotarod test. Each mouse was placed on a rotating rod (Accelerating Rotarod; UGO Basile, Varese, Italy), and the latency to falling off the rod (the time that each animal was able to maintain its balance on the rod) during its acceleration from 5 to 40 rpm over 5 min was measured in three trials per day over two consecutive days. The mice were subjected to this test without any pretest training.
Open-field test
Each mouse was placed in the corner of an open-field apparatus that consisted of a transparent Plexiglas chamber (40 by 40 by 30 cm) with a white plastic floor (Accuscan Instruments, Columbus, OH) and which was illuminated at 100 lx. The total distance traveled, vertical activity (rearing, measured by counting the number of photobeam interruptions), time spent in the central area (20 by 20 cm), and beam-break counts for stereotyped behavior were recorded over 120 min.
Light–dark transition test
The apparatus for the light–dark transition test consisted of a cage with a white floor made of PVC (21 by 42 by 25 cm) that was divided into two sections of equal size by a partition with a door (O’Hara & Co.). The walls and roof of one chamber were made of white plastic and the chamber was brightly illuminated (390 lx), whereas the walls and roof of the other chamber were made of black plastic and it was maintained dark (2 lx). Mice were placed in the dark chamber and allowed to move freely between the two rooms with the door open for 10 min. The distance traveled in both chambers, the number of transitions between the two chambers, the time spent in each chamber, and the latency to entry into the light chamber were recorded with the use of ImageLD software.
Elevated plus-maze test
The apparatus (O’Hara & Co.) consisted of two open arms (25 by 5 cm) and two enclosed arms of the same size with 15-cm-high transparent walls, and the arms were connected by a central square (5 by 5 cm). The arms and central square were made of white plastic plates and were elevated to a height of 55 cm above the floor. The likelihood of animals falling from the apparatus was minimized by the attachment of 3-mm-high plastic ledges to the open arms. Arms of the same type were arranged on opposite sides of the apparatus. Each mouse was placed in the central square of the maze facing one of the closed arms, and its behavior was recorded over 10 min. The total distance traveled, total number of arm entries, percentage of entries into the open arms, and percentage of time spent in the open arms were measured with the use of ImageEP software. Data for one mutant mouse that fell from the maze were excluded from analysis.
Contextual and cued fear conditioning test
For assessment of fear-related learning and memory, a mouse was placed in and allowed to explore freely for 2 min a plastic chamber consisting of white lateral and transparent front, rear, and top surfaces (33 by 25 by 28 cm) and with a stainless-steel grid floor (bars 0.2 cm in diameter, spaced 0.5 cm apart) (O’Hara & Co.). A conditioned stimulus (CS) of 55-dB white noise was presented for 30 s and was followed by a mild foot shock (0.3 mA), which was administered during the last 2 s of the CS and served as the unconditioned stimulus (US). Two more CS-US pairings were presented with an interval of 2 min between each pair. A context test was conducted in the same chamber for 5 min both 1 and 28 days after conditioning. A cued test with an altered context was also performed on these days for 6 min in a triangular chamber (33 by 29 by 32 cm) that had walls and a floor made of white plastic and was located in a different sound-attenuating room. In the cued test, the mouse was allowed to move freely for 3 min and was then exposed to the auditory stimulus (55-dB white noise) for 3 min. The test chambers were equipped with a ceiling-mounted video camera connected to a computer for monitoring of mouse behavior. Images were captured at a rate of one frame per second. For each successive frame, the amount of area (pixels) within which the mouse moved was measured. When this area was below a certain threshold, the behavior was judged as “freezing.” When the area equaled or exceeded the threshold, the behavior was judged as “nonfreezing.” The optimal threshold (number of pixels) for judgment of freezing was determined by adjustment based on the degree of freezing measured by human observation. In each test, the percent freezing time and distance traveled were calculated automatically with the use of ImageFZ software. After each conditioning and context test, the plastic surface and grid floor of the chamber were wiped with hypochlorite solution and 65% ethanol, respectively. After each cued test, the walls and floor of the chamber were cleaned with hypochlorite solution.
Acoustic startle response and prepulse inhibition test
A startle-reflex measurement system (O’Hara & Co.) was used to measure an acoustic startle response (ASR) elicited by a loud stimulus as well as prepulse inhibition of the startle response (PPI). Mice were placed in a PVC plastic cylinder. They were left undisturbed for 10 min and then subjected to test trials consisting of six trial types: two types of startle stimulus–only trial and four types of PPI trial. White noise of 110 or 120 dB (duration of 40 ms) was used as the startle stimulus for all trial types. A prepulse stimulus with an intensity of 74 or 78 dB (duration of 20 ms) was presented 100 ms before the onset of the startle stimulus. The four combinations of prepulse and startle stimuli were thus 74 and 110, 78 and 110, 74 and 120, and 78 and 120 dB. Mice were subjected to six blocks of the six trial types in a pseudorandom order such that each trial type was presented once within each block. The average intertrial interval was 15 s (range, 10–20 s). The startle response was recorded for 400 ms beginning with the onset of the startle stimulus. The peak startle amplitude was used as a dependent variable. The background noise level was 70 dB during all the test sessions. Percent PPI was calculated for each mouse according to the following formula: PPI (%) = 100 × {1 – [(ASR amplitude in prepulse + startle trial)/(ASR amplitude in startle stimulus–only trial)]}.
Social interaction test in a novel environment
Two mice of the same genotype that had been housed in different cages were placed together in a box (40 by 40 by 30 cm) (O’Hara & Co.) and allowed to explore freely for 10 min. Analysis was performed automatically with the use of ImageSI software. Images were captured at a rate of three frames per second, and the distance traveled between two successive frames was determined for each mouse. If the two mice contacted each other and the distance traveled by them was > 10 cm, then the behavior was considered an active contact. The total duration of contacts, total number of contacts, total duration of active contacts, mean duration per contact, and total distance traveled were measured.
Social interaction test in the home cage
The home cage–monitoring system consisted of a home cage (25 by 15 by 23.5 cm, interior dimensions) and a cage top equipped with an infrared video camera (O’Hara & Co.). Two mice of the same genotype that had been housed separately were placed together in the home cage. Images from the cage were captured at a rate of one frame per second. Social interaction was measured by counting the number of particles detected in each frame (two particles indicated that the mice were not in contact with each other, and one particle indicated contact between the two mice). The activity level of the mice was also measured by quantifying the number of pixels that changed between each pair of successive frames. The mean number of particles and mean activity level were calculated over 1-h intervals for 7 days, with the values for the first 3 days and the last 3 days being averaged for analysis of social behavior in a familiar environment. Analysis was performed automatically with the use of ImageHC software.
Three-chamber tests of sociability and social novelty preference
The testing apparatus consisted of a rectangular, three-chambered box with a lid fitted with a video camera (O’Hara & Co.). The dividing walls of the box were made of transparent plastic, with small square openings (5 by 3 cm) allowing access to each chamber (20 by 40 by 47 cm). A small round wire cage (9 cm in diameter and 11 cm in height, with vertical bars 0.5 cm apart) was located in a corner of the left and right chambers. The test mice were first placed in the middle chamber and allowed to explore the entire apparatus for 10 min. It was then transferred to a clean holding cage, while a male C57BL/6 J mouse (stranger 1) that had had no prior contact with the test mouse was enclosed in one of the wire cages. The location of stranger 1 in the left versus right side chamber was systematically alternated between trials. The test mouse was then returned to the middle chamber of the test box and allowed to explore for 10 min (sociability test). After the test session, the test mouse was again placed in the holding cage, and a second unfamiliar male C57BL/6 J mouse (stranger 2) was enclosed in the remaining empty wire cage. The test mouse was returned to the middle chamber of the test box and now had a choice to explore either the already-investigated unfamiliar mouse or the novel unfamiliar mouse for 10 min (social novelty preference test). The time spent in each chamber and the time spent around each cage were automatically measured from images with the use of ImageCSI software.
Porsolt forced swim test
A Plexiglas cylinder (20 by 10 cm) (O’Hara & Co.) filled with hypochlorite solution at 21° to 23 °C up to a height of 7.5 cm was placed in a white plastic chamber (44 by 32 by 49 cm, inside dimensions) (O’Hara & Co.). Each mouse was placed in the cylinder, and its immobility time was recorded over a 10-min test period. Images were captured at a rate of two frames per second with a video camera. For each pair of successive frames, the area (number of pixels) within which the mouse moved was measured. When this area was below a certain threshold, the mouse was judged to be immobile. When the area equaled or exceeded the threshold, the mouse was considered to be moving. The optimal threshold was determined by adjustment based on the degree of immobility measured by human observation. Immobility lasting < 2 s was not included in the analysis. Data acquisition and analysis were performed automatically with the use of ImageTS/PS software.
Barnes maze test
The Barnes maze test was performed on “dry land,” a white circular surface with a diameter of 100 cm and with 12 holes equally spaced around the perimeter (O’Hara & Co.). The circular open field was elevated 75 cm from the floor. The apparatus was illuminated by fluorescent lights mounted on the ceiling of a sound-attenuating room, with an illumination level of ~ 850 lx in the center of the field. A variety of fixed extramaze clues surrounded the apparatus. A black Plexiglas escape box (17 by 13 by 7 cm) was located under one of the holes, designated the target hole, which is analogous to the hidden platform in the Morris water-maze task. The location of the target was consistent for a given mouse but was randomized across mice. In a training session, the mouse was placed in the center of the field and allowed to explore the maze freely. After it had entered the target hole, the mouse was left undisturbed in the escape box for 30 s. The training session was conducted in two trials each day for nine consecutive days. The maze was rotated daily, with the spatial location of the target remaining unchanged with respect to the visual room cues in order to prevent bias based on olfactory or proximal cues within the maze. The latency to reach the target hole, number of errors before reaching the target hole, distance traveled to reach the target hole, and number of omission errors (defined by a visit to the target hole without subsequent entry into the escape box) were automatically recorded with the use of ImageBM software. One and 30 days after the last training session, probe trials were conducted without the escape box for 180 s in order to assess spatial reference memory. In the probe trials, the time spent around each hole was measured.
T-maze test
The spontaneous alternation task was conducted with an automatic T-maze apparatus (O’Hara & Co.) constructed of white plastic runways with 25-cm-high walls. The maze is partitioned into six areas by sliding doors that can be opened downward: stem of the T, straight runway, left and right arms, and connecting passageways from the arms to the stem of the T. Each mouse was subjected to sessions consisting of 10 trials per day (cutoff time, 50 min) for 3 days. Each trial consisted of a forced choice followed by a free choice. In the forced-choice run, the mouse was forced to enter either the left or right arm of the T-maze and was kept in the arm for 10 s. A free-choice run in which the mouse was allowed to choose one of the arms was then performed after a delay of 3, 10, 30, or 60 s. Choice by the mouse of the arm opposite that selected during the forced-choice run was considered a correct response, and the percentage of correct responses was calculated automatically with the use of ImageTM software.
Data analysis
The applications for analysis of behavioral data (ImageLD, ImageEP, and ImageFZ, ImageSI, ImageHC, ImageCSI, ImageTS/PS, ImageTM) were based on ImageJ (http://rsb.info.nih.gov/ij) and developed by T. Miyakawa[41].
Statistical analysis
Statistical analysis was performed with the use of BellCurve for Excel software (Social Survey Research Information, Tokyo, Japan). Normality of data was first assessed with the Shapiro–Wilk test, and homogeneity of variance between genotypes was examined with the F-test for each behavioral measure. If the normality assumption was not met, the Mann–Whitney U test was applied for comparisons between genotypes. If data were normally distributed and variance was homogeneous between genotypes, comparisons were performed with Student’s t test. If homogeneity of variance was not assumed, Welch’s t test was applied instead of Student’s t test. Two-way repeated-measures analysis of variance (ANOVA) was also conducted. All statistical analysis values, including ANOVA results, are included in Additional file 2: Table S2.