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Cognitive functions depend on the time of day in various organisms. Previously, we found that 24‑h recognition memory performance of nocturnal mice changes diurnally through SCOP protein‑ dependent regulation. It remains unknown whether diurnal change and SCOP‑ dependent regulation of memory performance are conserved across species with diurnal/nocturnal habits. We tested whether the memory performance of diurnal Japanese macaques depends on the time of day. The memory association between bitter taste of drinking water and the nozzle color of the water bottle was established during the light period of the day to evaluate of memory performance for macaques. Here we found diurnal variation of declarative memory in Japanese macaques. The middle of the daytime is the most effective time for memory performance during the light period. To assess whether SCOP is involved in declarative memory performance, we interfered with SCOP expression by using lentiviral vector expressing shRNA against Scop in the hippocampus of Japanese macaques. Scop knockdown in the hippocampus abrogated the memory performance in the middle of the daytime. Our results implicate that SCOP in the hippocampus is neces‑ sary for the diurnal rhythm of the memory system and that the SCOP‑ dependent memory regulation system may be conserved in mammals. Keywords Diurnal variation, Nonhuman primates, Memory, Hippocampus, SCOP (PHLPP1) mice exhibited the highest performance of a recognition Introduction memory task during the early (subjective) night even in Daily rhythms are widely observed in a variety of physi- constant darkness . Several studies have also demon- ological functions among many organisms. In mammals, strated daily variations in spatial memory performance blood pressure , body temperature , hormone secre- [8–10] and contextual/cued fear memory performance tion [3, 4], and even higher brain functions such as mood [7, 8, 11, 12] in rodents. In humans, on the other hand, regulations  and memory performances  show the the recognition memory task was performed better in daily rhythms, which are controlled by the internal cir- the mid-afternoon than in the early morning [13, 14]. cadian clocks [5–7]. We previously found that nocturnal The hippocampus is known to contribute largely to the declarative memory formation in humans [15, 16], non- *Correspondence: human primates , and rodents [6, 18–20]. It has been Kimiko Shimizu reported that the diurnal change in synaptic transmission email@example.com Yoshitaka Fukada in the hippocampus is maximal at night in nocturnal rats firstname.lastname@example.org‑tokyo.ac.jp but during the daytime in diurnal monkeys . Given Hiroo Imai that the integrity of the hippocampal formation is criti- imai.hiroo.5m@kyoto‑u.ac.jp Full list of author information is available at the end of the article cal for cognitive behavior, nocturnal and diurnal animals © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Shimizu et al. Molecular Brain (2023) 16:31 Page 2 of 9 possibly show maximal memory performances at differ - ent times of day. SCOP (suprachiasmatic nucleus circadian oscillatory protein, later termed PHLPP1β [22, 23]) was initially identified as a protein whose expression is circadian- regulated in the rat SCN (suprachiasmatic nucleus) of the hypothalamus . In rodents, the SCOP protein is predominantly expressed in the central nervous sys- tem, particularly enriched in pyramidal neurons in the hippocampal CA1 to CA3  and other brain areas essential for memory formation [26, 27]. SCOP in the hippocampal pyramidal neurons is a critical media- tor that links the memory formation with the circadian clockwork in mice . Knockout or knockdown of Scop in the hippocampus caused a deficit in long-term recog - nition memory and blunted its circadian regulation in mice . The amino acid sequences and the domains of SCOP were highly conserved across rodents and humans , implicating a common role in mammals. In the present study, we addressed whether memory performance of a diurnal nonhuman primate, Japanese Fig. 1 Daily water consumption from regular water supply system. Several days of water consumption of two macaques in 12‑h light/ macaques, shows a time-of-day-dependent variation dur- dark condition. Blue dots are water drops the monkey consumed ing the daytime (the light period) and whether SCOP per 5 min. The monkeys are fed three times a day: ZT 1.5, ZT5.5, and regulates learning and memory performances. We estab- ZT10.0 during a daytime lished a behavioral test, the “color-taste association task,” to estimate the declarative memory performance of caged macaques. This task allows them to choose water bottles without severe aversion and, therefore, should be performance in the color-taste association task (Fig. 2a– unrelated to a reward or fear response in the brain sys- c). Six monkeys reared in individual cages were sub- tem. Because experiments under the constant light (or jected to the task at randomly-assigned either three-time dark) condition are not allowed for nonhuman primates points of the day: dawn (ZT 1.5), midday (ZT5.5), and due to the regulations of research ethics, the monkeys dusk (ZT10). (Fig. 2a, b). The task was performed for were subjected to the memory task only during the 12-h five consecutive days consisting of three parts: 3-day light period. Here we found diurnal variations in declara- “practice,” 1-day “training,” and 1-day “testing” (Fig. 2b). tive memory, indicating that SCOP plays an important In the “practice,” two bottles of bitter water and normal role in memory performance. water equipped with nozzles in different colors were presented to the monkeys to learn that colors and water tastes are associated. The animals were allowed to freely Results drink water from the bottles for 2 h with their position Diurnal variations in recognition memory exchange 1 h after the bottle setting. The bottle position Before exploring diurnal variations in recognition mem- exchange eliminates an association between water taste ory by a color-taste association task utilizing water- and bottle position, left or right. “Training” and “test- drinking behavior in Japanese macaques, we monitored ing” were performed by using a set of water bottles with their daily water consumption from a regular water sup- nozzles having a color set different from that used in the ply system in a 12-h light/dark cycle (Fig. 1). Two ran- “practice”. An association between specific nozzle color domly-selected monkeys were fed with a diet three times and water taste formed during the "training” was evalu- a day: dawn (ZT [zeitgeber time] 1.5), midday (ZT5.5), ated in the” testing”, during which the two bottles of the and dusk (ZT10.0), where ZT0 is defined as light-on same color set as was used in the “training” were both and ZT12 as light-off. We identified that these monkeys filled with normal water. Then, the monkeys were allowed drank water mainly when fed. Therefore, we decided that to freely drink water from the bottles for 30 min, during the animals should be fed at the time of the task to let which the position of the bottles was exchanged 15 min them motivate to drink water during the task. after the bottle setting. In the “testing”, we monitored We explored diurnal variations in hippocampus- which color of a nozzle the animals had first selected 24 h dependent recognition memory by examining monkey’s Shimizu et al. Molecular Brain (2023) 16:31 Page 3 of 9 after “training”, by which they had experienced an asso- reduced the endogenous SCOP protein level as compared ciation between the bitter taste and the specific nozzle to COS-7 cells treated by the control Scramble shRNA color. The memory of the association between the bitter - lentiviral vector (Fig. 3b). ness and the nozzle color during the “training” would let We randomly selected two monkeys, one for knock- the animals drink water from the color nozzle that was down (Scop shRNA) and another for its control (Scram- not bitter in the “testing.” When the animals first chose ble shRNA). The shRNA lentiviral vectors were injected the color nozzle with normal water both before and after into the hippocampus. Fourteen injections were made the bottle exchange, then 1-point was given as it was into the bilateral CA1 regions (seven injections per uni- judged that they remembered the association. This scor - lateral CA1) of each monkey to cover the whole CA1 area ing method should exclude the case of selecting the right positioned by an MRI-guided navigation system. After color nozzle by chance. The tasks were carried out 1–4 the behavioral analysis, the injection sites of the lenti- times for each time point for each monkey. The resulting viral vectors in the hippocampal CA1 region were con- points of the tasks (a total of 16 tasks for ZT1.5, 13 tasks firmed by GFP immunostaining (Fig. 4a), indicating that for ZT5.5, and 16 tasks for ZT10.0 at each time point in the vectors were transfected at the targeted CA1 region. six monkeys) were combined. The color-taste associa - The efficiency of Scop knockdown was verified by quan - tion task revealed an apparent diurnal change with the titation of Scop mRNA in the hippocampus using RT- highest level at ZT5.5 midday among the three-time qPCR, and Scop mRNA in the hippocampus was reduced points during the light period (Fig. 2c). The accuracy rate significantly to 51% in comparison with the control case at midday was significantly higher than the 0.25 chance delivered with Scramble shRNA (Fig. 4b). level (dotted line). Since the color-taste association task After the lentiviral vector injections, the animals were determined midday as the best time of day for memory allowed to recover for eight weeks, and then the color- reinforcement, we then approached the molecular mech- taste association task was performed at ZT5.5 (midday), anism to augment declarative memory performance. which was the highest performance time of day in the non-injected animals (Fig. 2c). Six trials of the color-taste Abolishment of color‑taste association by Scop knockdown association task were conducted over time for each ani- in hippocampus mal. The Scop knockdown suppressed the performance SCOP in the hippocampus is a critical molecule for diur- of the color-taste association task, contrasting with nal regulation and memory reinforcement of recognition the control animal that had been injected with the len- memory in nocturnal mice . Of interest was to ask tiviral vector expressing Scramble shRNA (Fig. 4c). The whether SCOP in the hippocampus might play a similar Scop-knocked-down monkey did not earn even 1-point role in mice and diurnal mammalian species. To investi- over six trials (Fig. 4c Scop shRNA), whereas the con- gate the role of SCOP in the hippocampus in monkeys, trol monkey correctly chose the colored nozzle before we performed knockdown experiments of SCOP by using and after three over six trials (Fig. 4c Scramble shRNA). a lentiviral vector. The lentiviral vector was designed to These results suggest that SCOP in the hippocampal CA1 express both Scop shRNA (short-hairpin RNA) driven by is indispensable for the color-taste association mem- Japanese macaque H1 promoter (mkH1) and enhanced ory in macaques, although our results from the SCOP green fluorescent protein (GFP) driven by CMV pro - knocked-down experiments are relatively premature, moter (Fig. 3a). The COS-7 monkey cell line was used to because of the small sample size. Further studies with a evaluate the knockdown efficiency of SCOP by the Scop more sample size will confirm the conclusion. shRNA lentiviral vector. Infection of the vector efficiently (See figure on next page.) Fig. 2 Diurnal change of color‑taste association task. a A Japanese macaque in a cage with two water bottles. b The method for the color ‑taste association task. The color‑taste association task is done in five consecutive days consisting of three parts, three days of “practice”, one day of “training”, and one day of “testing” at either three‑time points, ZT 1.5 (Group 1), ZT 5.5 (Group 2), or ZT 10 (Group 3). In the “practice,” two bottles of bitter water and normal water equipped with nozzles in different colors were presented to the monkeys. The “practice” let animals learn that nozzle color is associated with water taste. Monkeys were allowed to freely drink water from the bottles for 2 h with the location exchange at 1‑h after the bottle setting. “Training” and “testing” were carried out at the same time of day as the “practice,” and nozzle color sets are the same in the “training” and “testing” but different from those used in the “practice.” In the “training,” the bottles are presented for 2 h with the location exchange at 1‑h after the bottle setting. In the “testing,” the two bottles were both filled with normal water, and the animals were allowed to drink water from the bottles for 30 min, with the location exchanged at 15‑min after the bottle setting. An association between specific nozzle color and water taste formed during the "training” was evaluated in the” testing.” c The accuracy rate was examined at ZT1.5, ZT5.5, or ZT10. *p = 0.002 (ZT5.5) by Student’s t‑test (versus 0.25 chance level). Error bars, s.e.m. (n = 6 macaques, a total of 16 task trials for ZT1.5, 13 task trials for ZT5.5, 16 task trials for ZT10.0). The dotted line represents performance by chance 0.25. n.s., not statistically significant Shimizu et al. Molecular Brain (2023) 16:31 Page 4 of 9 Fig. 2 (See legend on previous page.) Shimizu et al. Molecular Brain (2023) 16:31 Page 5 of 9 Fig. 3 Evaluation for the knockdown efficiency of Scop shRNA expressing lentiviral vector. a Schematic diagram of the shRNA‑ expressing lentiviral vector. The shRNA is controlled by the macaque H1 promoter (mkH1), and the CMV promoter drives the EGFP marker gene for tracking transduced cells. 5′‑LTR, HIV ‑1 5′‑LTR; 3′ LTR, HIV‑1 self‑inactivating 3′‑LTR. b Western blot analysis of SCOP protein level after shRNA expressing lentivirus infection. Decrease in SCOP protein level in COS7 cells by infection of anti‑Scop shRNA lentivirus (shScop). Scramble shRNA lentivirus was used as a control. The sample in each lane is from a different culture dish. The displayed blot was cropped from the full‑length blot in the Additional file 1 Discussion The present study has established a color-taste asso - ciation task to investigate memory performance using macaques reared inside cages. The memory perfor - mance of this task was the best at midday during the day- time (Fig. 2c). A similar result was reported on a human declarative memory task which was performed better in the mid-afternoon than in the early morning . This similarity supports that the newly established color- taste association task is a reasonable method to verify memory ability. In our previous study, on the other hand, mice performed a recognition memory task better dur- Fig. 4 Eec ff t of Scop knockdown in the hippocampus on color‑taste ing the early night (their active phase) than during the association task. a A representative EGFP fluorescence (green) and Hoechst 33258 (Blue) image of a hippocampal section of the resting phase . Such a difference in the best timing macaque that received shRNA lentivirus. The white square in the left for declarative memory performance would be depend- photo is enlarged in the right photo. Scale bars are indicated in the ent on the temporal habitat, nocturnal vs. diurnal. Syn- photos. The white dotted area shows the hippocampal CA1 to CA4. aptic excitability that is the highest at night in nocturnal DG, dentate gyrus; LGN, lateral geniculate nucleus. b Evaluation of rats but during the daytime in diurnal monkeys  may the knockdown activity of anti‑Scop shRNA lentivirus on Scop mRNA level. Decrease in Scop mRNA level in the hippocampus by infection explain the difference in the best time of day for memory of anti‑Scop shRNA lentivirus (Scop shRNA). Scramble shRNA lentivirus formation. On the other hand, the best timing for mem- was used as a control. Error bars, SEM (n = 6 subregions). c The ory formation might depend on the task type; nocturnal accuracy rate for Scop knockdown and control macaques (one each) mice perform a fear memory task better during a daytime is shown. Memory performance at ZT 5.5 (midday) in each monkey (their inactive phase) [7, 11]. Considering these findings, that received lentivirus expressing Scop shRNA or Scramble shRNA were measured. The task was carried out six times for each animal. we can assume that the diurnal regulation for learning The number above each bar on the graph indicates the number of and memory performance may be universal, although the corrects for the number of trials Shimizu et al. Molecular Brain (2023) 16:31 Page 6 of 9 timing of the best performance varies among animal spe- contained normal water in the “practice” and the “train- cies and types of tasks . ing” (see Fig. 2b). For the discrimination of the two bot- Scop is widely conserved in vertebrates [29, 30], impli- tles, stainless steel nozzles of the bottles were treated cating its crucial role throughout vertebrate evolution. with oxidized coloration and stabilized in six different The present results and our previous work  indicate colorings, i.e., magenta, black, blue, brown, gray, and that SCOP in the hippocampus has the ability to enhance white (processed by Nakano Kagaku, Niigata, Japan). memory capability and generate a diurnal rhythm of The task was performed in five consecutive days con - memory performance across species at an appropriate sisting of three parts: three days of “practice,” 1-day time of day. In mice, we revealed SCOP-dependent diur- “training,” and 1-day “testing.” In the “practice,” two bot- nal regulation of long-term memory through a mecha- tles of bitter water and normal water equipped with noz- nism that the SCOP levels in the hippocampal membrane zles in different colors were presented simultaneously to rafts regulate the K-Ras-ERK-CREB pathway and con- the macaques (see Fig. 2a). The three days of the “prac - sequently control the CRE-mediated transcription and tice” let animals learn that nozzle color is associated with long-term memory formation . The SCOP protein water taste. Monkeys were allowed to freely drink water level in the mouse hippocampal membrane rafts is higher from the bottles for 2 h with the location exchange at during the active phase (nighttime), and, therefore, the 1-h after the bottle setting. In the “training,” bitter water amount of SCOP in the macaque hippocampal mem- and normal water were presented to the same animals by brane rafts may be higher during the daytime (their active bottles with nozzle color sets different from those used phase). Further investigations are needed to address this in the “practice.” Again, monkeys were allowed to freely issue. drink water from the bottles for 2 h with the location exchange at 1-h after the bottle setting. An association Materials and methods between specific nozzle color and water taste formed Animals during the "training” was evaluated in the” testing,” dur- Six adult Japanese macaques (Macaca fuscata, 7–10 kg, ing which the two bottles of the same color set as was 5–10 years old) of either sex (four males and two females) used in the “training” were both filled with normal water were used in this study. The monkeys were housed in and presented at the same time. Then, animals were individual cages in a 12-h light/dark cycle. Animals were allowed to freely drink water from the bottles for 30 min, fed regularly with dietary pellets and had ad libitum during which the location of the bottles was exchanged access to water by a water supply system except for the 15 min after the bottle setting. In this “testing” process, experimental periods. memory performance was assessed as the degree of cor- respondence between the nozzle color and water taste Measurement of water consumption the animals had experienced in the “training.” All the Water consumption was measured by a drinkometer “testing” process was recorded by a video camera. When (O’Hara & Co., Tokyo, Japan) placed into a regular water the monkey in the “testing” first chose the nozzle color supply system route when not conducting the memory both before and after the bottle exchange that the animal task. The drinkometer records the number of drops con - had experienced normal water in it, then 1-point was sumed per 5 min. given as it was judged that the animal remembered the association between the color and taste. The task (prac - Color‑taste association task tice, training, and testing) was carried out a maximum of The color-taste association task was conducted with four times at each time point at ZT1.5, ZT5.5, or ZT10.0 feeding/drinking limitations. In order to let the macaques (ZT10 means 10 h after the light ON) for each monkey. more motivate the drinking behavior during the behav- The total tasks at each time point in six monkeys were ioral task, the regular water supply was stopped for 3 h 16 for ZT1.5, 13 for ZT5.5, and 16 for ZT10.0. A differ - before starting the behavioral paradigm for midday ent color set was used for each task trial at a single time and dusk. For tests in the dawn, the regular water sup- points for each animal. The number of points divided by ply was stopped at a light-off time (ZT12) the day before the trials was used as the accuracy rate. The inter-task the behavioral experiment. The behavioral tests were interval was at least 1 month. performed using a two-bottle system approved by the Animal Welfare and Animal Care Committee of the Production of shRNA‑expressing lentiviral vector Primate Research Institute, Kyoto University (no. 2011- The plasmids, pENTR4-H1, CS-RfA-CG, pCMV-VSV- 093). Briefly, two filled bottles (500 mL) were set in front G-RSV-Rev and pCAG-HIVgp were provided by Dr. of the monkeys: one contained bitter water containing Hiroyuki Miyoshi, RIKEN Bioresource Center, Tsukuba, 20 mM salicin (Sigma-Aldrich, MO, USA), and the other Japan. shRNA targeting Scop was designed using Shimizu et al. Molecular Brain (2023) 16:31 Page 7 of 9 siDirect (http:// design. RNAi. jp/), and the target sequence 140 mM NaCl and 1 mM MgCl ; pH 7.4), for 2 h at room (GGATA TTGGC CATAA TCAAA CGTGT GCTGT temperature. Then the blots were incubated for 4 h at CCGTT TGATT ATGGC CAATA TCCA) was used for room temperature with anti-SCOP antibody (1:2,000, the down-regulation of macaque Scop. A control shRNA αCB in Ref. 24) diluted in the blocking solution. The sig - with a scrambled sequence (GATAT GGCAC TGATA nals were visualized by an enhanced chemiluminescence ATCAA CGTGT GCTGT CCGTT GATTA TCAGT detection system (PerkinElmer, Boston, MA, USA). GCCAT ATCA) was designed. The pairs of the comple - mentary oligonucleotides containing these sequences Injections of lentiviral vectors were synthesized (SIGMA), annealed, and cloned into the Based on the 3R principle (Replacement, Reduction, modified pENTR4-H1, in which human H1promoter was and Refinement) for animal experiments, lentivirus was replaced by Japanese macaque H1 promoter (mkH1; see administered to each macaque for Scop shRNA (one Fig. 3a). Cloning of the mkH1 promotor was performed male) and Scramble shRNA (one female) in the experi- from the genome of a Japanese macaque with reference ment. The animals were first sedated with ketamine to the homologous region of the rhesus H1 sequence and hydrochloride (5 mg/kg, i.m.) and xylazine hydrochlo- the human H1 sequence at the UCSC genome browser ride (0.5 mg/kg, i.m.) and then anesthetized with sodium (https:// genome. ucsc. edu). The H1-shRNA fragment pentobarbital (20 mg/kg, i.v.). The monkeys were kept from pENTR4 H1-shRNA was then inserted into lenti- hydrated during the surgical operation with a lactated viral vector CS-RfA-CG by the Gateway system (Invitro- Ringer’s solution (i.v.). An antibiotic (Ceftazidime; 25 mg/ gen, CA, USA) to obtain CS-H1-shRNA-CMV-GFP. kg, i.v.) and an analgesic (Meloxicam; 0.2 mg/kg, s.c.) HEK293T cells were transfected with transfer (CS- were administered at the first anesthesia. After partial H1-shRNA-CMV-GFP), envelope, and packaging removal of the skull, multiple injections of each vector (pCMV-VSV-G-RSV-Rev and pCAG-HIVgp) plasmids were performed into the hippocampal CA1 area with the by the polyethylenimine method. Eighteen hours after aid of an MRI-guided navigation system (Brainsight Pri- transfection, the medium was replaced with a fresh one, mate, Rogue Research, Montreal, QC, Canada). A total and after that, the cells were incubated for 24 h. Then, the volume of 70 µL of each vector was injected into multiple medium was harvested and filtered through a 0.22 µm sites (5 µL/site, seven sites per side, 14 sites per animal) PVDF filter (Millipore, Burlington, MA, USA). The fil - through a 10 µL Hamilton microsyringe. The injection tered medium of 32 ml was centrifuged with bottomed titer of the viral vector was 2 × 10 gc/mL. After the 20% (w/v) sucrose (5 ml) at 35,000 ×g for 2 h at 4 °C in injections were completed, the scalp incision was closed. a Beckman SW32 Ti. The pelleted viral particles were All experiments were performed in a specific laboratory resuspended in 0.001% Pluronic-F68 in phosphate-buff - (biosafety level 2) established at the Primate Research ered saline (PBS; pH 7.4) at 4 °C for 2–4 h. Institute, Kyoto University, designed for in vivo animal For measuring RNA titer, viral RNA in 50 nL of the infectious experiments. vector stock solution was isolated with a NucleoSpin RNA virus kit (Takara, Shiga, Japan), and the copy num- Immunohistochemical analysis ber of the RNA genome was determined by quantitative At the end of experiments in macaques, the animals were PCR using Taq-Man technology (Thermo Fisher Scien - deeply anesthetized with sodium pentobarbital (50 mg/ tific, Waltham, MA, USA). The viral biological titers were kg, i.p.) and perfused transcardially with PBS. The brains also determined by infection of COS-7 cells with a dilu- were cut in the coronal plane at the 3-mm thickness, and tion series and counting colonies of GFP-positive cells. the slices containing the hippocampus were immersed in 4% paraformaldehyde in PBS overnight, followed by 30% Assessment for Scop knockdown lentiviral vector in vitro sucrose in PBS for two days at 4° C and cut into serial cor- To determine the efficiency of Scop shRNA express - onal sections (20 µm) by a microtome in a cryostat (Leica, ing lentivirus, we chose monkey cell line COS-7. The Germany). The sections were washed with 0.1% Triton- COS-7 cells were infected with Scop shRNA or scram- X100 in PBS for 15 min × 3 times and then blocked in bled shRNA (Scramble shRNA) expression lentivi- 1% normal goat serum, 1% BSA, 0.1% Triton-100 in PBS 4 2 rus (2 × 10 ifu / 3.8 cm dish surface) and cultured for for 1 h at room temperature, and incubated in anti-GFP three days before western blot analysis for SCOP pro- antibody (1:1000, Invitrogen, G10362). The immunoreac - tein expression. For western blot analysis, proteins tivity was visualized with Alexa 488-conjugated goat anti- separated by SDS–PAGE were transferred to a polyvi- rabbit IgG (1:1000; Molecular Probes) and then stained nylidene difluoride membrane (Millipore, USA). The blot with 1 ng/ml Hoechst 33342 (Sigma) to visualize nuclei. was blocked in a blocking solution of 3% bovine serum The hippocampal sections were imaged on BZ-9000TS albumin in T-TBS (0.05% Tween20, 50 mM Tris–HCl, Microscope (Keyence, Osaka, Japan). Shimizu et al. Molecular Brain (2023) 16:31 Page 8 of 9 and Animal Care Committee of the Primate Research Institute, Kyoto Univer‑ RT‑q PCR analysis sity (2010‑066, 2011‑093, 2012‑047). All the experiments in this study were also Total RNA was isolated from six subregions of the performed in accordance with the guidelines of The University of Tokyo. macaque hippocampus using TRIzol reagent (Invitro- Consent for publication gen) and was subsequently purified by RNeasy Mini Not applicable. kit (Qiagen) according to the manufacturer’s protocol. RT–qPCR analysis was performed using Go Taq 2-step Competing interests The authors declare that they have no competing interests. RT–PCR system (Promega) in a Step One Plus (Applied Biosystems). Data are presented as values normalized to Author details the housekeeping gene Gapdh. PCR primers used are;for Department of Biological Sciences, School of Science, The University of Tokyo, Tokyo 113‑0033, Japan. Department of Pathological Cell Biology, Medical Scop FW 5′-CCCCA GCTGT TTGGA GTCAT-3′ and Research Institute, Tokyo Medical and Dental University, Tokyo 113‑8510, RV 5′-TCAAA CACAC CGTAG AGGGC-3′ for Gapdh Japan. Molecular Biology Section, Center for the Evolutionary Origins FW 5′-ACCGT GGTCA TGAGT CCTTC C-3′ and RV of Human Behavior, Kyoto University, Inuyama, Aichi 484‑8506, Japan. Sys‑ tems Neuroscience Section, Center for the Evolutionary Origins of Human 5′-GCACC ACCAA CTGCT TAGCA-3′. Behavior, Kyoto University, Inuyama, Aichi 484‑8506, Japan. Laborator y of Animal Resources, Graduate School of Medicine, Center for Disease Biology Supplementary Information and Integrative Medicine, The University of Tokyo, Tokyo 113‑0033, Japan. The online version contains supplementary material available at https:// doi. Received: 26 January 2023 Accepted: 22 March 2023 org/ 10. 1186/ s13041‑ 023‑ 01022‑0. 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Molecular Brain – Springer Journals
Published: Mar 25, 2023
Keywords: Diurnal variation; Nonhuman primates; Memory; Hippocampus; SCOP (PHLPP1)
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