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Exp Neurobiol 2019; 28(4): 485-494
Published online August 31, 2019
© The Korean Society for Brain and Neural Sciences
Min Jung Kim1†, Ro Un Lee1†, Jihae Oh1, Ja Eun Choi1, Hyopil Kim1, Kyungmin Lee2, Su-Kyeong Hwang3, Jae-Hyung Lee4, Jin-A Lee5, Bong-Kiun Kaang1, Chae-Seok Lim6* and Yong-Seok Lee7*
1Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, 2Behavioral Neural Circuitry and Physiology Laboratory, Department of Anatomy, Brain Science & Engineering Institute, Kyungpook National University Graduate School of Medicine, Daegu 41944, 3Department of Pediatrics, Kyungpook National University Hospital, Daegu 41944, 4Department of Life and Nanopharmaceutical Sciences, Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul 02447, 5Department of Biotechnology and Biological Sciences, Hannam University, Daejeon 34430, 6Department of Pharmacology, Wonkwang University School of Medicine, Iksan 54538, 7Department of Physiology, Biomedical Sciences, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea
Correspondence to: *To whom correspondence should be addressed.
Chae-Seok Lim, TEL: 82-63-850-6765, FAX: 82-63-850-7262
Yong-Seok Lee, TEL: 82-2-740-8225, FAX: 82-2-763-9667
†These authors equally contributed to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License(http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, andreproduction in any medium, provided the original work is properly cited.
Vacuolar protein sorting-associated protein 13B (VPS13B), also known as COH1, is one of the VPS13 family members which is involved in transmembrane transport, Golgi integrity, and neuritogenesis. Mutations in the
Keywords: Vps13b, Cohen syndrome, Learning disability
VPS13B (also known as COH1) is one of the VPS13 protein family members which consist of four mammalian members; VPS13A, VPS13B, VPS13C, and VPS13D . Each of
In this study, we generated the first rodent model of
RNA isolation method was previously described [18, 19]. Briefly, RNA was extracted from hippocampus of wild type mice,
RT-PCR was performed as previously described . Briefly, cDNA was synthesized by using SuperScriptIII and random hexamers following manufacturer’s instructions (Invitrogen). Quantitative real-time (qRT)-PCR was conducted with SYBR Premix Ex TaqII (Cat. # RR820A, Takara) and 7300 Real-Time PCR system (Applied Biosystems). Primers for
We used 2~5 month-old male (25~33 g) and female (18~22 g) mice for the behavioral experiments. Since we did not find any significant difference between male and female, we merged the results from both sexes. Behavioral analyses were performed by a test battery in a single cohort as the following order. Mice were tested in open field, elevated zero maze, light-dark box and Y-maze on consecutive days. After Y-maze, mice were handled for 5 days and trained in Morris water maze. Three days after finishing the water maze task, mice were trained for fear conditioning test. Three chamber test was performed 19 days after the fear conditioning test. Rotarod test was independently performed in a different cohort. The genotypes of the mice were blinded to the experimenters.
Procedure for open field test was previously described . Mice were placed in the center of open field box (40×40×40 cm) under dim light. Free movement of the mice were recorded for 30 min and analyzed by tracking software, EthoVision 9.0 (Noldus).
Elevated zero maze test was performed as described . The maze (round-shaped, inner diameter: 50 cm, outer diameter: 60 cm, height of two walls: 20 cm) includes two close arms and two open arms, elevated to a height of 65 cm above the floor. Mice were placed in the center of the one closed arm, and allowed to move all arms freely for 10 min under bright light. The time spent in each arms was analyzed by EthoVision 9.0 (Noldus).
Specific experimental procedures were previously described [19, 23]. Briefly, light-dark box apparatus includes both light and dark chambers adhered to each other, and the mice could move freely from one another through the gate. The illumination of light chamber (20×30×20 cm, a large compartment) was 404 – 408 lux, while dark chamber (20×13×20 cm, a small apartment) was fully blocked from illuminator. Mice were first introduced to the dark chamber, and free movement between two chambers was recorded for 10 min. The time spent in each chamber was manually and blindly counted.
We followed the protocols previously described . Mice encountered to the center of the Y-maze with three identical arms at a 120o angle from each other under dim light. The movement was recorded for 8 min. Each arm was 30 cm long, 6 cm wide, and 15 cm height. The sequence of entries and the total number of each arm entries were recorded, and manually and blindly counted.
Test was performed as previously described [22, 24–27]. Briefly, mice were handled for 3 min for 5 successive days. Mice were trained in a swimming pool (140 cm diameter, 100 cm height) filled with a soluble white paint with multiple spatial cues on the walls of the test room. The temperature of the water was 21~22 degree of Celsius. Mice were released from four edges of the maze respectively, and allowed to find the hidden platform (round-shaped, 10 cm diameter), which was placed at the center of a target quadrant (TQ), within 60 s. Mice were trained with 4 trials (start swimming from four different edges of the maze) per day. Probe tests were conducted at day 7 and 11 with an absence of the platform, and training was not performed on the probe test days. The visual platform test was performed by placing a black flag on the platform after finishing hidden platform version of water maze test. The time spent in each quadrant and mean velocity were scored by EthoVision 9.0 (Noldus).
Mice were trained and tested as formerly described [20, 25]. For conditioning, mice were located in the conditioning chamber (Coulbourn Instruments) for 3 min, and an electrical foot shock (3 s, 0.8 mA) was given through the floor grid. After the conditioning, mice went back to the home cages. After 24 h later, mice were disclosed to the same chamber for 3 min without a shock to recognize whether mice remember electric shock (fear memory) or not. Freezing level was automatically quantified by a software (Freeze Frame, Actimetrics).
Sociability and social novelty recognition tests were conducted as previously described . An apparatus we used had three sequential chambers and each chamber was connected with gates. For sociability test, a mouse (stranger 1) and an object were placed in a wired-cup sitting in two end chamber of the apparatus. A test mouse was placed into the center chamber and allowed to move whole apparatus for 10 min. For social novelty recognition test, the object was replaced to another mouse (stranger 2), with which test mouse had not met ever, and the test mouse was introduced and allowed to explore between stranger 1 and 2. The time spent exploring object, stranger 1, or stranger 2 was manually counted.
Detailed experimental procedures were previously described . Mice were habituated to the rotarod apparatus (LE8200, Harvard apparatus) slowly accelerated from 4 to 20 rpm over 11 min for two days. On the test day, the rod was accelerated from 4 to 40 rpm over 5 min and the latency to fall off the rod was recorded. The test was replayed for 3 days and the average time to fall was analyzed.
Data were analyzed followed procedures previously defined . Statistical comparisons were made by using unpaired
We investigated the behavioral phenotypes of the
We examined the
Next, we assessed hippocampus-dependent spatial learning and memory by testing the mice in the Morris water maze test. In the hidden platform version of this task, mice were trained to learn spatial cues around the maze to find a platform hidden under the water during training sessions (Fig. 3C). Hippocampus-dependent memory can be assessed in probe tests in which the platform is removed. We trained the mice for 10 days and preformed probe tests at day 7 and day 11 (Fig. 3D and 3E).
We have generated the first mouse model of
Authors thank Macrogen for technical service generating
Table 1 Primer sequences for RT-PCR
|Sense primer (5′→3′)||Antisense primer (5′→3′)||Product size (bp)|
|Exon 1–4||1:TCCAGCCTCTCTGCCTACTC||5:CGGGGTTTTATGGATGATTTT||424 bp (intact)|
|Exon 2–3||2:CGAGTTAAAGTTGGACGTTCTGG||3:AGGTTCGGAGCCCAATTTTGT||115 bp|
|Exon 3–4||4:AGTGGCCATATCCATGAACTG||5:CGGGGTTTTATGGATGATTTT||194 bp|
|Exon 22–23||6:CAGTAAAGAGTCTCACGCTACAG||7:TGTTCCAGGGATGTCACCAGA||110 bp|