RIN1

Enhanced apoptosis and decreased ampa receptors are involved in deficit in fear memory in rin1 knockout rats

Linchuan Ma, Xinzhao Chen, Beiying Zhao, YuXiu Shi, Fang Han⁎
PTSD laboratory, Department of Histology and Embryology, Basic Medical College, China Medical University, Shenyang 110122, China

A R T I C L E I N F O

Keywords: Rin1 Ampar Apoptosis
Cytoskeleton Fear memory

A B S T R A C T

Background: Ras and Rab interactor 1 (Rin1) is predominantly expressed in memory-related brain regions, and has been reported to play an important role in fear memory. Increased expression of Rin1 in an animal model of posttraumatic stress disorder (PTSD) has been associated with enhanced acquisition of fear memories, but the exact mechanism of Rin1 in memory regulation are not clear.

Methods: Here, we used Rin1-knockout rats to examine the effect of Rin1 on fear memories by fear conditional test and the molecular mechanisms that regulate these effects by immunofluorescence, western blotting and TUNEL.

Results: Our results show that Rin1-knockout rats have a deficit in formation and extinction of Auditory fear memories. Lack of Rin1 results in enhanced apoptosis in the hippocampus through a pathway related to the mitochondria rather than the endoplasmic reticulum-related pathway. Importantly, the lack of Rin1 induces a decrease in α-amino-3‑hydroXy-5-methyl-4-isoXazolepropionic acid receptors (AMPAR) found in the cytoplasm, but not in those found in the membrane. EXpression of CaMKII (which is important for insertion of cytoplasmic AMPAR into the membrane) and stargazin (which is important for immobilization of AMPAR in the membrane) was not changed. The lack of Rin1 also induced changes in AMPAR distribution, from diffuse spread in the cells to clusters around the edge of the cell. Additionally, clustered
AMPAR distribution showed a high degree of overlap with actin distribution.

Conclusion: These findings indicate that Rin1 affects not only apoptosis, but also the concentration and dis- tribution pattern of AMPAR, which are important in the formation and extinction of fear memory.

1. Introduction
Ras and Rab interactor 1 (Rin1)—a downstream effector molecule of RAS—plays a critical role in the development of many types of tumors in humans, such as gastric, breast, and liver cancer (BiaoXue et al., 2016; Shan et al., 2012). There are two main molecules in the signaling pathway downstream of Rin1: Rab5, which controls receptor en- docytosis and trafficking, and Ab1, which controls cytoskeletal re- modeling. Rab5 mediates clathrin-dependent endocytosis in hippo- campal neurons (de Hoop et al., 1994) and participates in the(for reviews, see Koleske, 2006; Colicelli, 2010; also see Perez de Arce et al., 2010; Warren et al., 2012). Rin1 is distributed in mature tele- ncephalic neurons—mainly in the amygdala, the hippocampus, and cortex (Bliss et al., 2010) and shows continuously increasing expression in the prenatal brain, reaching a peak in the postnatal brain (Han et al., 1997), with lower expression in the adult brain. In the subcellular structures, Rin1 is localized in the neuronal soma and dendrites, with high expression in postsynaptic elements (Deininger et al., 2008). Stu- dies published since 2003 have demonstrated that Rin1 plays a key role in fear regulation. Rin1-/-knockout (KO) mice showed normal devel- regulation of α-amino-3‑hydroXy-5-methyl-4-isoXazolepropionic acid opment and enhanced learning of conditioned fear, enhanced acquisi-receptors (AMPAR) in hippocampal excitatory synapses. Ab1 is ex- pressed at hippocampal excitatory synapses, where it controls synaptic functions and regulates synaptic plasticity (Moresco et al., 2003). The modulation of actin cytoskeletal remodeling, binding to β1 integrin, and synaptic clustering of postsynaptic density protein-95 have been all implicated in Abl kinase-mediated activity-dependent synaptic efficacy tion of aversive memories, and elevated long-term potentiation of the amygdala. Therefore, Rin1 may be involved in acquisition and retention of fear memory. These characteristics make Rin1-/- KO mice a potential model to study posttraumatic stress disorder (PTSD), although the molecular mechanisms underlying Rin1 function in PTSD remain un- clear.

⁎ Corresponding author: dr. Fang Han, No. 77, PuHe Road, ShenBei New District, ShenYang City, China.
E-mail addresses: [email protected] (L. Ma), [email protected] (X. Chen), [email protected] (B. Zhao), [email protected] (F. Han).
https://doi.org/10.1016/j.jad.2020.02.040
Received 28 November 2019; Received in revised form 20 February 2020; Accepted 26 February 2020
Availableonline27February2020
0165-0327/©2020ElsevierB.V.Allrightsreserved.

We previously reported increased Rin1 expression in the hippo- campus and amygdala, and enhanced fear memory in rats that under- went single prolonged stress (SPS) (Han et al., 2017). This finding ar- gues against direct negative regulation of enhanced acquisition of fear memory by Rin1 as previously reported (Colicelli et al., 2010). Over the last few years, the SPS model has become the most frequently used model for PTSD. Increased freezing in conditional fear tests after SPS is consistent with other studies (Iwamoto et al., 2007). We also found increased Rab5 and Ab1 expression in the hippocampus and amygdala in SPS rats (Han et al., 2017). These findings prompted us to further explore the role and molecular mechanisms of Rin1 in fear memory, including acquisition and extinction. It should be noted the roles of Rin1 in rats may differ from those in mice.

The Cell line study has been reported that knockdown of Rin1 ex-
pression in malignant melanoma cells suppresses cell proliferation and induced apoptosis (Fang et al., 2012). A number of studies have re- ported that enhanced apoptosis occurs in neurological conditions that affect memory. PTSD patients experience decreased volume of the hippocampus, which is an important brain region for memory (Etkin and Wager, 2007; Morey et al., 2009; Murrough et al., 2011; Xiong et al., 2013). Evidence from animal studies have shown enhanced apoptosis in the hippocampus of PTSD (Han et al., 2013; Li et al., 2010). Despite the growing evidence that Rin1 may be a crucial regulator of memory in humans, its effect on apoptosis in neuronal cells remains to be clarified.

Here, we use Rin1-/- KO rats to explore the role of Rin1 on fear
memory. We aimed to find direct evidence of correlation between Rin1 expression and fear regulation in Rin1-/- KO rats. The second aim of this study was to explore possible molecular mechanisms of Rin1 function on regulation of fear memory, for example, the effect on Rin1 on apoptosis or synapse formation. The results of this study should provide useful clues for further exploration of the mechanisms of memory, pa- thogenesis of PTSD, and even treatment of PTSD.

2. Materials and methods
2.1. Animals
In this experiment, 30 wild-type Sprague Dawley (SD) rats (male, 10 weeks old) and 30 SD Rin1-/- KO rats (male,10 weeks old) were used to explore changes in behaviors and molecular levels associated with knockout of Rin1. Twenty rats per group were used in behavioral tests (10 rats per group for fear conditioning test, 10 rats per group for open- field test), four rats per group were used in western blotting, three rats per group were used in immunofluorescence experiments, and three rats per group were used in the terminal deoXynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay.All experimental animals were group-housed on a 12 h: 12 h light/ dark cycle at a room temperature of 19–21 °C. The animals had ad libitum access to food and water. All experimental procedures were approved by the ethics committee of China Medical University and conducted in accordance with the Guidelines and Principles on Animal EXperimentations for Laboratory Animal Science set by China Medical University.

2.2. Generation of Rin1-/- KO rats
2.2.1. Transcription activator-like effector nuclease construction
Four transcription activator-like effector nucleases (TALENs) tar- geting the Rin1 gene were chosen using a web-based tool (http://asia. ensembl.org) to precisely bind opposing DNA target sequences, which were separated by the spacer, in order to search for potential targets (Fig. 1A). The TALENs were 16–18 base pairs in length. TALENs were assembled using the TALEN Toolkit (ViewSolid Biotech, Beijing, China). Finally, four pairs of TALEN sequences were designed using the pCS7-eTALEN-T plasmid (Fig. 1B). The activity of the four TALENs was
measured using a luciferase single-strand annealing kit (ViewSolid Biotech, Beijing, China), and the TALEN with the strongest activity was used for embryo microinjection (Fig. 1C). The plasmids were extracted using the E.Z.N.A® Plasmid Midi Kit (Omega Bio-tek, Norcross, GA, USA), according to the manufacturer’s instructions and transcript to mRNA in vitro. TALEN mRNA was stored at −80 °C until it was used for embryo injection.

2.2.2. Embryo microinjection
20 Female embryo donors (6 weeks old) were superovulated with
25 IU of pregnant mare serum gonadotropin (MilliporeSigma, Burlington, MA, USA), followed by 25 IU of human chorionic gonado- tropin (MilliporeSigma, Burlington, MA, USA) after 24 h, then caged individually with a male rat. The following day, donors were sacrificed and embryos were collected from the oviducts and incubated in M16 (MilliporeSigma, Burlington, MA, USA) at 37 °C in air containing 5% CO2. Fertilized one-cell embryos were transferred to medium for mi- croinjection and the mRNAs of the TALEN plasmids with the strongest activity were miXed and injected into the cytoplasm. Embryos that survived the injection procedure were surgically transferred to the oviducts of female SD rats.

2.2.3. Mutation analysis
Offspring from successfully injected embryos were obtained and examined for mutation analysis of the Rin1 gene. The tails were cut and DNA was extracted using a DNA extraction kit; then, the extracted DNA was analyzed by PCR. The primer sequences for peptidyl-prolyl iso- merase (PIN1) were as follows: sense, CTAGGAGGAGCTGGCAAGGAC; antisense, TTCCGCACCAGGAAGGTCTGTG. The PCRs were incubated according to the following protocol: initial denaturation at 98 °C for 3 min; 35 cycles of 98 °C for 10 s, 60 °C for 20 s, and 68 °C for 40 s; final extension at 68 °C for 2 min; and hold at 4 °C.
After mutation analysis, rats with the Rin1 mutation (F0) were mated with wild-type SD rats. Genetic analysis of their offspring (F1) was performed at two weeks after birth. Rats from the F1 generation that carried the Rin1 mutation were mated with each other to produce offspring (F2). Two weeks after birth, genetic mutations in the F2 generation were analyzed. Male homozygous Rin1 knockout (Rin1-/- KO) rats were used in our experiment.

2.3. Behavioral tests
2.3.1. Auditory fear conditioning
The rats from wild type group and Rin1-/- KO group were placed in the conditioning chamber (23 cm × 23 cm × 35 cm) and allowed to freely roam for 5 min. The degree of freezing during 5 min was con- sidered baseline freezing. After 5 min of exploration, an auditory cue (1000 Hz, 75 dB; conditioned stimulus [CS]) was presented for 30 s, and an electrical foot shock (2 s, 1.5 mA; unconditioned stimulus [US]) was delivered continuously during the last 2 s of the auditory cue. We repeated the CS-US protocol three times per session with 90 s intervals between each repeat. Following the final foot shock, the rats were re- turned to their home cages. The rats were placed in a novel chamber 2 h and 48 h after initial training, and were trained again and tested for freezing to the auditory cue. After 2 min of habituation to the novel chamber (pre-CS), the freezing time was measured immediately after the tone stimulation (CS, without foot shock) within 120 s.
For the fear conditioning test, the freezing activity was recorded and measured using Packwin 2.0 software (Panlab, Barcelona, Spain). Freezing times were used as an index of fear conditioning. Freezing was defined as immobility, excluding respiratory movements, with a freezing posture. For example, rats remained still, sluggish, curled, or crouched whilst breathing, and had a slight rocking motion.

2.3.2. Auditory fear extinction training and testing
Half of rats were used to carry out extinction training. Each training. Transcription activator-like effector nuclease (TALEN) construction, design, and activity (A) The sequence of the four constructed TALENs. (B) The design of the TALEN plasmid. (C) The activity of the four TALENs was measured, and the most efficient one was chosen. The Rin1-T3 plasmid was the most efficient, and was chosen for knockout. * P < 0.05.protocol began 5 min after rats were placed in the novel conditioning chamber. The extinction training protocol included 30 repetitions of 30 s of the auditory cue used in fear conditioning training, with 1 min rest between each repetition. EXtinction testing (30 repetitions of 30 s of the auditory cue, with 1 min rest between each repetition) was performed at one day, three days and seven days after extinction training (Dhaka et al., 2003). During the extinction testing, the freezing time was measured. 2.3.3. Open-field test The open-field test was used to study locomotor activity and an- Xiety-related behaviors. The procedure was done as described in Han et al. (Han et al., 2017). The apparatus was surrounded by black walls (40 cm in height) and the floor (100 cm × 100 cm). The floor was formed by 25 squares (20 cm × 20 cm each) and divided to center region (50 cm X 50 cm) and border region. During the experiment, each rat was put in the center of the center region, and behavior was re- corded for 5 min by an automatic analyzing system (Smart 3.0, Panlab, Barcelona, Spain). Total distance and time in center region were re- corded. The percentage of time in the center (time in the center region/ total time) were calculated. The apparatus was cleaned with 70% ethanol using a wet sponge and a paper towel before the introduction of each rat. 2.3.4. Immunofluorescence Three rats per group were anesthetized. The brains were removed from the skull, quickly frozen using powdered dry ice, and cut into 25 m thick frontal sections on a cryostat (CM3050; Leica Biosystems, Wetzlar, Germany). The sections were treated with a solution of 2% bovine serum albumin and 0.3% Triton X-100 in phosphate-buffered saline (PBS) for 2 h at room temperature to block nonspecific reactions. The sections were incubated with primary antibodies overnight at 4 °C. For single labeling immunofluorescence, sections were incubated with a rabbit polyclonal anti-Rin1 antibody (1:1000, Santa Cruz, USA), a mouse monoclonal anti-AMPAR antibody (1:1000, Invitrogen, USA), a mouse monoclonal anti-Caspase 7 antibody (1:500,Santa Cruz, USA) or a mouse monoclonal anti-Caspase 9 antibody (1:500, Santa Cruz, USA). After 3 time wash, sections were incubated at 37 °C for 30 min with goat anti-rabbit IgG (1:500, Boster, China) or rabbit anti-mouse IgG (1:500, Boster, China). After 3 times PBS wash, then sections were in- cubated with 4′, 6-diamidino-2-phenylindole (DAPI, 1:200, KeyGen Biotech, Nanjing, China) staining solution. For double immuno- fluorescence labeling of actin and AMPAR, sections were incubated with a mouse monoclonal anti-AMPAR antibody (1:1000, Invitrogen, USA) at 4 °C overnight. Then sections were incubated by rabbit anti- mouse IgG (1:500, Boster, China). After incubation with the secondary antibody, sections were incubated with DAPI (1:200, KeyGen Biotech, Nanjing, China) for 5 min, and then incubated with Actin-Tracker Green (1:200, KeyGen Biotech, Nanjing China) for 20 min. We ex- amined these sections under a confocal laser scanning microscope (TI- PS100W; Nikon, Tokyo, Japan). 2.3.5. TUNEL assay The In Situ Cell Death Detection Kit POD (Roche, Reinach, Switzerland) was used to detect cell apoptosis in the hippocampus ac- cording to the manufacturer's instructions. Briefly, frozen sections from three rats per group were washed for 30 min with PBS and then blocked with 3% hydrogen peroXide in methanol for 10 min. After washing with PBS, the sections were permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate for 10 min. The TUNEL reaction miXture (50 µL that was previously miXed) included 5 µL of enzyme solution and 45 µL of la- beling solution; this reaction miXture was added to the sections for 60 min in a dark and humidified chamber. Next, the sections were washed with PBS and counterstained with DAPI. Sections were washed and sealed with a glycerol and PBS miXture under cover slips. The Pannoramic MIDI Digital Slide Scanner (3DHISTECH, Budapest, Hungary) was used to scan the slides and digitize the fluorescent sig- nals, enabling us to zoom in or zoom out when examining the slides. 2.3.6. Western blotting Four rats per group were sacrificed, and the brains were im- mediately removed and cooled on ice. The whole hippocampus was then dissected from the brain tissue. The total protein of the dissected hippocampi was extracted using the Minute™ Total Protein EXtraction Kit (Invent Biotechnologies, Plymouth, MN, USA). The cytoplasm and membrane fractions of the hippocampi were fractionated using the Minute TM Plasma membrane proteins and cellular component stein expression. 2.4. Statistical analysis All data were analyzed using SPSS software, version 23.0 (IBM, Armonk, NY, USA). All data are expressed as mean ± standard devia- tion (SD). Data from the fear conditioning experiments (including ac- quisition and extinction) were analyzed by two-way analysis of var- iance (ANOVA), Tukey's range test were used as post-hoc. Other data were analyzed by t-test. P < 0.05 was considered statistically sig- nificant. 3. Results 3.1. Expression of Rin1-/- KO rats in the fear condition test and open-field test To assess short-term memory and long-term memory, fear. EXpression of Rin1-/- KO rats in the fear condition test and Open field test Upper panel shows expression of wild-type rats (n = 10 rats) and Rin1-/- KO rats (n = 10 rats) (A) 2 h after training (short-term memory), and (B) 48 h after training (long-term memory). Middle panel shows expression of wild-type rats and Rin1-/- KO rats (C) one day, (D) four days, and (E) seven days after extinction training. Bottom panel (F) shows result of open-filed test (n = 10 rats per group): no significant difference in total distance or time in center region between wild-type rats and Rin1-/- KO rats. #P < 0.01; * P < 0.05. EXpression of Rin1 in the hippocampus and the amygdala of Rin1-/- KO rats (A) Western blot showing expression of Rin1 proteins (n = 4 rats per group). (B) EXpression of Rin1 immunoreactivity in the CA1 region of the hippocampus and the amygdala. The magnification show cells in the white boX. The arrows show Rin1 immunoreactivity in the cytoplasm of the hippocampus of Rin1-/- KO rats (n = 3 rats per group). conditioning with an auditory cue was measured 2 h (short-term memory) and 48 h (long-term memory) after training. We used two- way ANOVA (independent variables were foot shock and knockout) to analyze the results. In short-term memory, the ANOVA showed that the interaction between Rin1-/- KO and foot shock were significant (F [1, 18] = 58.64, P < 0.05). The main effect of Rin1-/- KO on freezing time was significant (F [1, 18] = 26.71, P < 0.05), but the main effect of foot shock on freezing time was not significant (F [1, 18] = 19.67, P > 0.05).

The post hoc Tukey’s range test showed that Rin1-/- KO rats had a significant decrease in freezing time in comparison with wild-type rats, not only in the pre-CS period (Fig. 2A, P < 0.01), but also during the CS (Fig. 2A, P < 0.01). Similarly, for long-term memory, ANOVA showed that the interaction between Rin1-/- KO and foot shock were significant (F [1, 18] =123.75, P < 0.05). The main effect of KO on freezing time was significant (F [1, 18] = 172.23, P < 0.05), but the main effect of foot shock on freezing time was not significant (F [1, 18] = 36.94, P > 0.05), the post hoc Tukey’s range test showed that Rin1-/- KO rats had a significant decrease in freezing time in comparison with wild-type rats both in the pre-CS period (Fig. 2B, P < 0.01) and during the CS (Fig. 2B, P < 0.01). After the test, rats were then exposed to an extinction protocol with repeated presentations of CS alone. Levels of freezing at one day, four days, and seven days after extinction training were examined. Results were analyzed by two-way ANOVA (independent variables were ex- tinction test and KO). At day one, the interaction between Rin1-/- KO and foot shock were significant (F [1, 18] =18.83, P < 0.05). The main effect of KO on freezing time was significant (F [1, 18] = 26.74, P < 0.05), although the main effect of the extinction test was not significant (F [1, 18], P > 0.05). the post hoc Tukey’s range test did not show a significant difference in freezing time between rats that underwent the extinction test and rats that did not (Fig. 2C). At day four, the inter- action and two main effects were not significant according to the ANOVA (F [1, 18], P > 0.05; Fig. 2D). Additionally, the post hoc Tu- key’s range test did not show a significant difference in time freezing between rats who underwent extinction test and rats who did not in wild-type or Rin1-/- KO rats. At seven days, the ANOVA showed that the interaction and the two main effects were not significant (F [1, 18], P > 0.05). However, the post hoc Tukey’s range test showed a significant difference in freezing time between wild-type rats who underwent ex- tinction training and those who did not, and no difference in Rin1-/- KO rats (Fig. 2E, P < 0.05). The results of the open-field test did not show a significant differ- ence in total distance and percentage of time in center between wild type rats and Rin1-/- KO rats (Fig. 2F, P>0.05).

3.2. Expression of rin1 in the hippocampus and the amygdala of Rin1-/- KO rats
Using western blotting, Rin1 showed a band at 82KD in the hip- pocampus of control rats, but no band in Rin1-/- KO rats (Fig. 3A). Using immunofluorescence, we also found intense signal and distribu- tion of Rin1 in the neuronal cytoplasm of wild-type rats in the hippo- campus. Rin1-/- KO rats did not show Rin1 immunoreactivity in the neuronal cytoplasm of the hippocampus (Fig. 3B). In the other brain regions of Rin1-/- KO rats, such as the amygdala (Fig. 3B), the cortex (data not shown) and hypothalamus (data not shown), no Rin1 positive staining was found.

3.3. Rin1-/- KO rats expressed enhanced apoptosis in the hippocampus
We used the TUNEL method to examine apoptosis in the hippo- campus (Fig. 4): the nuclei of neurons fluoresced blue, while apoptotic cells fluoresced green (Fig. 4A). The total number of cells in the hip- pocampi of Rin1-/- KO rats was significantly less than in those of wild-
EXpression of apoptosis in the hippocampus of Rin1-/- KO rats (A) Upper panel: Cells identified by terminal deoXynucleotidyl transferase dUTP nick end labeling (TUNEL) as having undergone apoptosis in whole hippocampus. Bottom panel: magnification in the hippocampus shows cells identified by TUNEL as having undergone apoptosis with green fluorescence, and DAPI nuclear staining with blue fluorescence. (B) Analysis of cells in the hippocampus identified by TUNEL as having undergone apoptosis (n = 3 rats per group). (C) EXpression of caspase 7 and caspase 9 in the hippocampus using immunofluorescence staining (n = 3 rats per group). (D) EXpression of caspase 7, caspase 9, and caspase 12 using western blotting. (E) Analysis of western blotting. 7 and caspase 9 showed significant increases in Rin1-/- KO rats in comparison with that in wild-type rats, whereas there was no difference in caspase 12 between wild-type and Rin1-/- KO rats (n = 4 rats per group). * P < 0.05 vs wild type; # P < 0.01 vs wild type. 3.4. Rin1-/- KO rats expressed decreased ampar expression in the hippocampus cluster from a Rin1-/- KO rat (the brightest cluster, termed the “b” cluster) by positioning specific coordinates (intersection of two white lines; Fig 5A). AMPAR-ir showed a similar peak of intensity in clusters “a” and “b” (almost reaching 250). Additionally, the overall intensity of the immunoreactive AMPAR cluster in the wild-type rat was higher than that of the Rin1-/- KO rat (Fig. 5B). Optical density analysis also showed that the intensity of AMPAR-ir was significantly lower in Rin1-/-KO rats compared with that in the wild type (Fig. 5C, P < 0.01). Therefore, these results suggest that Rin1-/- KO induces a decrease in cytoplasmic AMPAR but not in membrane AMPAR. To confirm these immunofluorescence results, we used western blotting to examine the expression of AMPAR in the whole cell, cytoplasm, and membrane in the hippocampus (Fig. 5D). AMPAR was significantly decreased in the whole cell (p<0.01) and cytoplasm (p<0.001) in the Rin1-/- KO rats compared with that in the wild-type rats, but there was no significant difference between the groups for AMPAR in the membrane. Stargazin is essential for immobilizing AMPAR in the postsynaptic membrane. Compared with that in wild-type rats, there was no change in hippocampal stargazin expression in Rin1-/- KO rats (Fig. 6, P<0.05). Ca2+/calmodulin-dependent protein kinase (CaMKII), disEXcitatory AMPAR, which are distributed in the postsynaptic tributed in postsynaptic elements, is also important for cytoplasmic membrane, convey fast synaptic transmission and play an important role in synaptic plasticity. In the wild-type rats, AMPAR im- munoreactivity (AMPAR-ir) was diffusely expressed at cell. In Rin1-/- KO rats, AMPAR-ir were expressed in a clustered distribution at the edge of cells, and not in the cytoplasm (Fig. 5A, arrow). AMPAR loca- lized to the edge of cell are thought to be distributed in the postsynaptic membrane. To compare the intensity of AMPAR-ir from clusters in Rin1-/- KO rats and the diffuse distribution in wild-type rats, we se- lected a cluster from a wild-type rat (termed the “a” cluster) and a AMPAR because it drives insertion of cytoplasmic AMPAR directly into the perisynaptic membrane. EXpression of hippocampal CaMKII did not differ significantly between wild-type rats and Rin1-/- KO rats, sug- gesting that the lack of Rin1 does not influence the expression of CaMKII, and, therefore, decreased AMPAR in the cytoplasm is not the result of changes in CaMKII expression (Fig. 6). overlap. 4. Discussion Rin1-/- KO rats that underwent fear conditioning tests showed lower rates of freezing than the control group of wild-type rats in terms of auditory cue memory. Rin1-/- KO rats also appeared to have a reduced ability to establish short-term or long-term memories to new fear con- ditioning situations. This is in contrast to reports of Rin1-/- KO mice that showed enhanced acquisition of aversive memories (Dhaka et al., 2003; Deininger et al., 2008). Rin1-/- KO rats showed a deficit in fearextinction, which is consistent with the results from Rin1-/- KO mice, although there was a difference in extinction time (Bliss et al., 2010). However, our previous studies with a rat model of PSTD found in- creased expression of Rin1 and enhanced contextual test. But no sig- nificant difference in the freezing level was seen between the SPS and the control rats in the auditory cued fear test (Han et al., 2017). Therefore, results from our Rin1-/- KO rats confirm that Rin1 plays a positive role in the regulation of fear memory, including acquisition and extinction. We propose that the roles of Rin1 in rats may differ from those in mice. In open-field test, rat from two groups did not show significant difference in total distance and time in center region, sug- gesting Rin1-/- KO does not affect locomotor activity and anxiety-re- lated behavior. This is consistent with other researches (Dhaka et al., 2003; Deininger et al., 2008). We found that Rin1-/- KO induced enhanced apoptosis in the hip- pocampus of rats. Enhanced apoptosis also led to a decreased number of neuronal cells in Rin1-/- KO rats. Moreover, increased expression of caspase 7 and caspase 9, and unchanged expression of caspase 12, in- dicated that KO of Rin1 induced apoptosis by the mitochondrial pathway. However, we could not completely exclude the possibility of endoplasmic reticulum-mediated apoptosis, because the endoplasmic reticulum pathway may be involved in young rats or even at an earlier stage in their life cycle. To our knowledge, no study has reported a relationship between Rin1 and apoptosis in neurons: only a few studies in cancer (mainly in melanoma) have reported a correlation (Kumar et al., 2016). Knock down of RIN1 expression in the melanoma cell line A375 suppressed cell proliferation and induced apoptosis through caspase 3 activation and poly (adenosine diphosphate-ribose) polymerase cleavage, which is consistent with our findings (Zhang Role of funding source This work was supported by two grants from the China National Natural Science Foundation (No. 81,571,324) and the Department of science and technology in LiaoNing (No.2017225011). Professor Fan Han received these two grants. The funding source had no influence on study design; the collection, analysis, and interpretation of data; the writing of the report; or the decision to submit the manuscript for publication. Other authors has not received any financial support in regard to the research presented in this manuscript. Contributors Mr. Ma were involved in most animal experiments, conducting statistical analyses, interpretation of the data, and drafting of the manuscript. Mr. Chen and Mrs. Zhao were involved in TUNEL and western blotting.Professor YuXiu Shi were involved in experimental design, securing funding and decision to submit the article for pub- lication. Professor Fan Han were involved in generating hypotheses, some experiments, data collection, securing funding and editing of the manuscript. Declaration of Competing Interest There are no conflicts of interest to declare. Acknowledgments The authors are grateful to all staff members of the China Medical University EXperiment Center for their technical support. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jad.2020.02.040. References neurons: differential attachment of nmda versus ampa receptors. J. Neurosci. 18, 2423–2436. Anton, F., Fres, J.M., Schauss, A., Pinson, B., Praefcke, G.J., Langer, T., Escobar- Henriques, M., 2011. 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