SiRNA-mediated serotonin transporter knockdown in the dorsal raphe nucleus rescues single prolonged stress-induced hippocampal autophagy in rats

Journal of the Neurological Sciences 360 (2016) 133–140

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SiRNA-mediated serotonin transporter knockdown in the dorsal raphe nucleus rescues single prolonged stress-induced hippocampal autophagy in rats Zhong-Min Wu a,d, Chun-Hua Zheng b, Zhen-Hua Zhu c, Feng-Tian Wu e, Gui-Lian Ni d, Yong Liang a,⁎ a

Department of anatomy, Medical College of Taizhou University, Taizhou 318000, China Outpatient Office, Taizhou Hospital, Taizhou 317000, China c Department of Pediatrics, Taizhou Central Hospital, Taizhou 318000, China d Department of Neurology, First People's Hospital of Linhai City, Linhai 317000, China e City Colloege of Zhejiang University, Hanzhou 310031, China b

a r t i c l e

i n f o

Article history: Received 28 October 2015 Received in revised form 26 November 2015 Accepted 30 November 2015 Available online 2 December 2015 Keywords: Small interfering RNA Hippocampal autophagy Dorsal raphe nucleus Serotonin transporter Post-traumatic stress disorder

a b s t r a c t The neurobiological mechanisms underlying the development of post-traumatic stress disorder (PTSD) remain elusive. One of the hypotheses is the dysfunction of serotonin (5-HT) neurotransmission, which is critically regulated by serotonin transporter (SERT). Therefore, we hypothesized that attenuation of SERT gene expression in the hippocampus could prevent hippocampal autophagy and the development of PTSD-like behavior. To this end, we infused SLC6A4 siRNAs into the dorsal raphe nucleus (DRN) to knockdown SERT gene expression after a single prolonged stress (SPS) treatment in rats. Then, we evaluated the effects of SERT gene knockdown on anxietyrelated behaviors and extinction of contextual fear memory. We also examined the histological changes and the expression of Beclin-1, LC3-I, and LC3-II in the hippocampus. We found that SPS treatment did not alter anxiety-related behaviors but prolonged the extinction of contextual fear memory, and such a behavioral phenomenon was correlated with increased hippocampal autophagy, decreased 5-HT level, and increased expression of Beclin-1 and LC3-II/LC3-I ratio in the hippocampus. Furthermore, intra-DRN infusion of SLC6A4 siRNAs promoted the extinction of contextual fear memory, prevented hippocampal autophagy, increased 5-HT level, and decreased expression of Beclin-1 and LC3-II/LC3-I ratio. These results indicated that SERT may play a critical role in the pathogenesis of hippocampal autophagy, and is likely involved in the development of PTSD. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Post-traumatic stress disorder (PTSD) refers to long-lasting symptoms of mental disorders due to the experience of life-threatening events or severe trauma [1–4]. The clinical manifestations of reexperience the trauma is characterized and accompanied by emotional irritability and avoidance behavior [1,2,5]. Nightmare-ridden patients usually experience hallucinations and exhibit severe anxiety, fear, and trauma related environmental escape, which seriously affect the quality of life of patients [1,2]. While great effort has been spent on studying PTSD, it is still not clear about the neurobiological mechanisms underlying the development of PTSD [6].

Abbreviations: PTSD, post-traumatic stress disorder; SERT, serotonin transporter; DRN, dorsal raphe nucleus; SPS, single prolonged stress; 5-HT, serotonin; LC3, light chain 3. ⁎ Corresponding author at: Department of Anatomy, Medical College of Taizhou University, 1139 Taizhou City Government Avenue, Taizhou 318000, China. E-mail address: [email protected] (Y. Liang).

http://dx.doi.org/10.1016/j.jns.2015.11.056 0022-510X/© 2015 Elsevier B.V. All rights reserved.

One of the many hypotheses about the pathogenesis of PTSD is the dysfunction of serotonin (5-HT) neurotransmission system [4,6–9]. Previous studies in humans have demonstrated that low levels of 5-HT in the brains are correlated with unadjusted alertness, such as increased frightened, increased awareness of new stimuli, and pain [10,11]. Additionally, it has also been shown that low levels of 5-HT is associated with impulsive and/or aggressive behavior in humans, which are associated with PTSD symptoms [8,10,11]. Furthermore, animal studies have illustrated that 5-HT1A receptor gene knockout can illicit significant anxiety-like behavior, and over-expression of 5-HT1A receptor can inhibit the occurrence of anxiety-like behavior [12]. Finally, studies in PTSD patients and animals have confirmed that selective 5-HT reuptake inhibitors (SSRI), such as fluoxetine, have a clear therapeutic effect on PTSD symptoms [9,10,13,14]. Therefore, it is likely that 5-HT reuptake dysfunction is an important factor in the development of PTSD. 5-HT reuptake is mediated by 5-HT transporter (5-hydroxytryptamine transporter; 5-HTT; SERT). In the brain, 5-HT neurons are mainly located in the brainstem raphe nuclei, including dorsal raphe nucleus (DRN) and raphe nucleus, which sends serotoninergic projections to

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other brain regions such as the hippocampus, amygdala, and prefrontal cortex [15]. Importantly, SERT is located on 5-HT neuron cell bodies and the presynaptic area of the projecting fibers, and can dynamically alter the extracellular concentrations of 5-HT [10,13]. Thus, SERT likely plays a key role in the 5-HT neurotransmission, which are critical for the development of PTSD and other mental illness [8,15]. One important pathological phenomenon associated with PTSD is the atrophy of hippocampal neurons. Neuronal loss, apoptosis, and synaptic changes are commonly associated with hippocampal atrophy [16]. Human studies have shown that patients with recent or chronic PTSD have reduced hippocampal volume and hippocampal function disorder [16–20]. Importantly, SSRIs treatment can increase neurogenesis in the hippocampus [21–23], suggesting that inactivation of SERT may reverse the pathogenesis of PTSD. Furthermore, recent studies have suggested that serotonin transporter (SERT) genetic polymorphisms contribute to an individual's response to a traumatic event [24,25]. In consistent with human research, animal research have demonstrated that when rats received repeated restraint stress, hippocampal cell proliferation was inhibited [26,27], and rats exhibited the emergence of anxietylike behavior [27]. Furthermore, 5-HT1A receptor activation can promote adult rat hippocampal cell proliferation, while blocking the 5HT1A receptors result in the loss of hippocampal neurons [28]. These studies suggested that activation of 5-HT neurotransmission in the hippocampus may rescue the hippocampal neuron autophagy in animals. Previous studies have demonstrated that microtubule-associated protein light chain 3 (LC3) and Beclin-1 are mammalian autophagyrelated genes in mammalian cells and are critical for the process of autophagy [29]. LC3 has type I and type II isoforms, and LC3-II is a marker of autophagy [29,30]. Therefore, based on the above information, we hypothesized in the present study that attenuation of SERT gene expression in the hippocampus could prevent the development of PTSD-like behavior in animals, and prevent the hippocampal autophagy. To test this hypothesis, we used siRNA-mediated SERT gene knockdown in the DRN after a single prolonged stress treatment in rats. Twenty one days later, we evaluated the effects of siRNA-mediated SERT gene knockdown in the DRN on general anxiety behaviors and the extinction of contextual fear memory. We also examined the histological changes in the hippocampus by assessing the neuronal autophagy. Additionally, using Western blot, we investigated the expression of Beclin-1, LC3-I, and LC3-II in the hippocampus. 2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (n = 64) that weighed 220–250 g at the time of surgery were obtained from the Experimental Animal Science Center of Taizhou University, and were housed individually under 12 h light/dark cycle (lights on/off at 7 am/7 pm) and had free access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee of Taizhou University. The housing and treatment of the rats followed the guidelines of the “Guide for the Care and Use of Laboratory Rats” (Institute of Laboratory Animal Resources, Commission on Life Sciences 2011). 2.2. Stereotaxic surgery Animals were anesthetized with 10% chloral hydrate intraperitoneal injection (3.5 ml/kg) and were placed in a stereotaxic frame (David Kopf, Tujunga, CA, USA). A stainless steel guide cannula with a dummy probe (10 mm; Plastic One, Roanoke, VA, USA) was implanted into the dorsal raphe nucleus (DRN) at a 28° angle with the sagittal plane, 3.2 mm lateral to lambda, 2.5 mm below the skull surface according to Paxinos & Watson rat brain atlas. Following the surgery, rats were housed individually for additional 5 days for recovery before the following experiments.

2.3. Single prolonged stress treatment The single prolonged stress (SPS) treatment was modified based on literature. Briefly, rats were exposed to restraint for 2 h, immediately followed by 20 min of forced swimming, which occurred in a plastic tub (55 cm in diameter and 45 cm in height) filled with warm water (20–24 °C). Twenty minutes after the forced swim, rats were then exposed to ether (75 ml) in a glass dessicator until they were fully anesthetized without toe or tail pinch response. Thirty minutes later, rats were placed in an electric shock box (40 × 30 × 25 cm) and 15 cycles of the foot shock (0.8 mA current, 10 s duration, 10 s interval) were administered. After electric shock, rats were returned to their home cages and were individually house until next experiment. 2.4. Intracranial microinjection Intracranial microinjections were started the next day after the SPS treatment, a 33-gauge injector (Plastics One, Roanoke, VA, USA) attached to a PE50 tubing was inserted through the guide, so that it protruded 1 mm from the cannula guide tip. The other end of the tubing was connected to a 10 μl Hamilton syringe which was attached to a Kopf microinjection unit (Model 5000, Kopf, Tunja, CA, USA). A total volume of 0.5 μl SLC6A4 siRNA or negative control siRNA was infused over a period of 2 min, and the injector was remained in place for additional 2 min after infusion was completed. To achieve effective gene knockdown, the once daily microinjections repeated for 3 days after SPS treatment. Rats were then individual housed for additional 18 days after microinjection procedure was completed. The SLC6A4 siRNA oligo was designed using Invitrogren BLOCK-iT™ RNAi Designer based on the accession number of rat slc6a4 gene (NM_013034.3) with the target sequences of 3′-TGGGCAACATCTGGCGGTTTCCTTA-5′,3′-ACCAGTGTGG TGAACTGCATGACAA-5′, and 3′-GGGACACTTAAGGAGCGCATTATTA-5′. 2.5. Light–dark box test Light–dark box behavior was measured in a testing chamber made of Plexiglas with a black floor (72 × 30 × 34 cm; Coulbourn Instruments, Allentown, PA, USA). The testing chamber was divided into two sides by a black wall with an arched opening (11 × 12 cm) to allow the rat to cross between sides. The testing chamber consisted of a light zone (30 × 40 cm) with an open ceiling and white walls and a dark zone (30 × 30 cm) with a closed ceiling (lid) and black walls. Rats were first placed next to the wall of the light zone facing the archway and behavior was recorded for 10 min. The percentage of time spent in the light zone and latency to first entry into the dark zone were measured to assess unconditioned anxiety. The total number of transitions of light and dark zones was measured to indicate the monitor activity. 2.6. Elevated plus maze test During the elevated plus maze (EPM) test, rats were placed in the center of a maze (Coulbourn Instruments, Allentown, PA, USA), which was 52 cm above the floor, and were facing at an enclosed arm. The duration of time spent in (as a percent of total time and compared to closed arm + center zone time), and the number of entries into (with all 4 paws) both the open and enclosed arms (45 × 12 cm) were recorded for 5 min. 2.7. Contextual fear extinction During the contextual fear conditioning, rats were exposed to novel context paired with a foot electric shock (0.8 mA, 4 s) through a stainless steel grid floor (Med Associates Inc., USA). After foot shock, rats were remained in the chamber for an additional 1 min before being returned to their home cages. One day, two days, three days later, each rat was placed in the conditioning chamber where it had

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previously been foot shocked. The contextual fear response was evaluated by measuring the percentage of the duration of freezing behavior during 5 min exposure. 2.8. Central neurochemical analysis Serum corticosterone levels and hippocampal serotonin (5-HT) levels were measured using respective enzyme-linked immunosorbent assay (ELISA) kits (BlueGene Biotech Co., Ltd., Shanghai, China) according to the instructions. Serum was separated from blood samples using centrifugation at 3500 rpm for 10 min and was then used for ELISA assay. Hippocampal tissues were first homogenized in phosphatebuffered saline (PBS) with a glass homogenizer on ice and the suspensions were then subjected to two freeze-thaw cycles in order to break the cell membranes. Next, the homogenates were centrifuged at 5000 rpm for 5 min and the resulting supernatants were then used for ELISA assay. The microtiter plate in each ELISA kit has been pre-coated with an antibody specific to corticosterone or 5-HT. Standards or samples were added to the appropriate plate wells and incubated for 2 h at 37 °C. After removing the solution from each well and washing, a biotin-conjugated antibody was added to the wells for incubation. After removal of excess detection antibody, a horseradish peroxidase (HRP) conjugate streptavidin was added into the wells. After incubation and washing to remove the excess HRP conjugate, TMB substrate solution was added and the enzyme-substrate reaction was terminated by the addition of sulphuric acid solution and the color changes were measured spectrophotometrically at a wavelength of 450 nm. The concentration of corticosterone or 5-HT in the samples was then determined by comparing the O.D. value of the samples to the standard curve. 2.9. Western blot Rats were sacrificed by rapid decapitations, and the brains were rapidly removed. The whole hippocampal formation was dissected in 4 °C phosphate buffered saline and was flash frozen in liquid nitrogen, and stored at −80 °C. The total proteins were extracted and concentrations were determined using Biorad DC Protein™ Assay kit. Equal amount of protein samples (25 μg protein) were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 120 V for 1 h and transferred onto nitrocellulose membranes for 1 h at 100 V.

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Membranes were then incubated with primary antibodies against serotonin transporter (SERT; ab44520, Abcam, USA; 1:1000), Beclin-1 (ab62557, Abcam, USA; 1:1000), LC3-I and II (#4108, Cell signaling, USA, 1:1000) overnight in 5% skim milk solution at 4 °C. After being rinsed 3 times in 0.05% Tween-Tris buffered solution (TTBS), membranes were then incubated in donkey anti-rabbit polyclonal secondary antibody (1:10,000, GE Healthcare, Piscataway, NJ), conjugated to horseradish peroxidase (HRP) for 1 h followed by development with an enhanced chemiluminescence (ECL) system (Pierce Biotech, Rockford, IL, USA). β-actin (Santa Cruz, CA, USA; 1:1000) was used as loading control. SERT, Beclin-1, LC3-I and LC3-II, and β-actin protein levels were quantified by densitometry using of NIH image J (NIH, Bethesda, MD, USA). SERT, Beclin-1, LC3-I and LC3-II levels were normalized to the levels of the loading control, β-actin, and to those of naive control group. 2.10. Hematoxylin and eosin staining Immediately after the last day extinction test of fear memory, rats were transcardially perfused, and the brains were isolated and fixed in PBS-buffered 4% paraformaldehyde, paraffin embedded, and processed. Serial 4-μm sections were obtained through the hippocampus. Representative hematoxylin and eosin (H & E) staining sections were obtained every 12 sections. Photomicroscopy was taken under a light microscope (Axioskop 2-Plus, Zeiss, Shanghai, China) using Zeiss AxioVision 4.5 software. 2.11. Statistical analysis Data were expressed as mean ± SEM. Data were analyzed using one way or mixed-factorial analyses of variance (ANOVAs), where appropriate. Significant ANOVA main and interaction effects were further investigated using Tukey post hoc tests, when appropriate. Alpha was set at 0.05. 3. Results 3.1. Histology Schematics illustrating cannula placement are included in Fig. 1. The most ventral point of the infusion cannula tracks was located within the

Fig. 1. Schematics illustrating cannula placement within the dorsal raphe nucleus region. Solid circles represent the rats (n = 32) that received SLC6A4 siRNA infusions, and open triangles represent the rats (n = 32) that received negative control siRNA infusions. Numbers indicate the distance from bregma in millimeters.

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dorsal raphe nucleus region. Furthermore, high power microscopy did not reveal any evidence of abnormal tissue damage (i.e., extensive cell loss or gliosis) at the infusion site. The sample sizes are reported in the figure captions. 3.2. Effects of siRNA-mediated SERT knockdown in the DRN on anxietyrelated behaviors and extinction of fear memory Generally, 21 days after the SPS treatment, rats in either group did not exhibit altered anxiety-related behaviors as compared with rats in control group (i.e., naïve). Specifically, one-way ANOVA of animal behavior in the light–dark box test indicated that all groups of rats spent similar time in the light zone of the box (F(3,28) = 0.74, p N 0.05; Fig. 2B), similar latency to enter the dark zone (F(3,28) = 0.68, p N 0.05; Fig. 2C), and had similar total number of transitions between the light and dark zones (F(3,28) = 0.49, p N 0.05; Fig. 2D). Additionally, one-way ANOVA of animal behavior in the elevated plus maze test indicated that all groups of rats spent similar time in the open arms (F(3,28) = 0.42, p N 0.05; Fig. 2E) and had similar open arm entries (F(3,28) = 0.91, p N 0.05; Fig. 2F). However, 21 days after the SPS treatment, SPS treatment prolonged the extinction of fear memory in rats (treatment main and interaction effects, F(3–9, 28–84) = 9.24–26.38, p = 0.001–0.01, Tukey test, p b 0.05; Fig. 2G). Specifically, rats in all groups exhibited similar freezing behavior when re-exposed to fear context during the first and second days of extinction. However, during the third and fourth days of extinction, rats that received SPS treatment only or SPS treatment and negative control siRNA infusions, but not those received SPS treatment

and SLC6A4 siRNA infusions, exhibited enhanced freezing behavior as compared with naïve control group (Tukey test, p b 0.05; Fig. 2G). 3.3. Effects of siRNA-mediated SERT knockdown in the DRN on hippocampal autophagy To investigate possible neuropathological changes in the hippocampus after SPS treatment and examine the effects of siRNA-mediated SERT knockdown in the DRN on hippocampal autophagy, histological examinations of rat brains were conducted on a representative group of rats (n = 3–4/group) after all the behavioral tests were completed. In general, we found increased spongiform changes in the CA1-3 and dentate gyrus of the hippocampus of rats that received SPS treatment only (Fig. 3B, E) or SPS treatment and negative control siRNA infusions (Fig. 3C, E), as compared with naïve control rats (Fig. 3A, E). Importantly, pongiform changes in the same subregions of the hippocampus of rats that received SPS treatment and SLC6A4 siRNA infusions were robustly reduced (Fig. 3D, E). It should also be noted that no apparent inflammatory or hemorrhagic changes or microgliosis were observed surrounding the spongiform changes, neuronal cytoplasmic vacuolation, and neuronal loss. 3.4. Effects of siRNA-mediated SERT knockdown in the DRN on the levels of serum corticosterone and hippocampal 5-HT To explore the putative mechanisms of the effects of siRNAmediated SERT knockdown in the DRN on the extinction of fear memory and hippocampal autophagy, we collected serum samples and

Fig. 2. Effects of siRNA-mediated SERT knockdown in the DRN on anxiety-related behaviors and extinction of fear memory (n = 8/group). (A) Experimental design and time line. Behavioral experiment results of (B, C, D) light–dark box test (LD) (E, F), elevated plus maze (EPM), and (G) fear conditioning (FC) and extinction of contextual fear memory (FE). Asterisks represent the treatment simple main effect, Tukey test, p b 0.05.

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Fig. 3. Representative oil-immersion differential interference contrast imaging (×100) of H & E sections of hippocampal CA1 regions shows neuronal cytoplasmic vacuolation, nuclear dysmorphology, and neuronal loss in (B) rats receive SPS treatment only and (C) in rats received SPS treatment and negative siRNA infusions in contrast to (A) naïve control rat and (D) rats received intra-DRN infusions of SLC6A4 siRNA. (E) Quantification of progenitor subgranular zone cells. n = 3–4/group. The black scale bars represent 10 μm.

hippocampal tissues in rats 21 days after SPS treatment. Generally, rats in all groups exhibited similar levels of serum corticosterone (F(3,28) = 0.73, p N 0.05; Fig. 4B). In contrast, 21 days after SPS treatment, SPS treatment altered 5-HT levels in the hippocampus of rats (F(3,28) = 13.49, p = 0.01; Fig. 4C). Specifically, rats that received SPS treatment only or SPS treatment and negative control siRNA infusions, but not those received SPS treatment and SLC6A4 siRNA infusions, exhibited decreased 5-HT level in the hippocampus as compared with naïve control group (Tukey test, p b 0.05; Fig. 4C).

3.5. Effects of siRNA-mediated SERT knockdown in the DRN on hippocampal expression of SERT, Beclin-1, LC3-I, and LC3-II To verify the effects of siRNA-mediated SERT knockdown, we evaluated the expression of SERT in the hippocampus. Generally, SPS treatment did not alter protein expression of SERT in the hippocampus 21 days after SPS treatment, as compared with naïve control group. Intra-DRN infusions of SLC6A4 siRNA but not negative control siRNA reduced the expression of SERT in the hippocampus (F(3,28) = 15.13, p = 0.01, Tukey test, p b 0.05; Fig. 5A).

Fig. 4. Effects of siRNA-mediated SERT knockdown in the DRN on the levels of (B) serum corticosterone and (C) hippocampal 5-HT. (A) Experimental design and time line. Asterisks represent the significant effects as compared with naïve control group. Pond represents the significant effect as compared with SPS treatment group. n = 8/group.

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Fig. 5. Effects of siRNA-mediated SERT knockdown in the DRN on hippocampal expression of (A) SERT, (B) Beclin-1, and (C) LC3-I, and LC3-II. n = 8/group. Asterisks represent the significant effects as compared with naïve control group. Pond represents the significant effect as compared with SPS treatment group.

Importantly, SPS treatment did not alter protein expression of Beclin-1, LC3-I, and LC3-II in the hippocampus 21 days after SPS treatment. Specifically, rats that received SPS treatment only or SPS treatment and negative control siRNA infusions, but not those received SPS treatment and SLC6A4 siRNA infusions, exhibited increased expression of Beclin-1 and LC3-II/LC3-I ratio in the hippocampus as compared with naïve control group (F(3,28) = 12.83–17.02, p = 0.01–0.02, Tukey test, p b 0.05; Fig. 5B–C). 4. Discussion The present study was designed to examine the role of SERT in the development of PTSD in an animal model. Specifically, we found that SPS treatment did not alter anxiety-related behaviors but prolonged the extinction of contextual fear memory 21 days after the SPS treatment. Importantly, the SPS-induced impairment of extinction of contextual fear memory was correlated with increased hippocampal autophagy, decreased hippocampal 5-HT level, and increased expression of Beclin-1 and LC3-II/LC3-I ratio in the hippocampus. These results suggested that SPS treatment may produce long term effects on the morphology and function of the hippocampus, which are critical for the persistence of PTSD psychopathology. Furthermore, intra-DRN infusions of SLC6A4 siRNAs decreased the expression of SERT in the hippocampus, increased 5-HT level, and decreased the expression of Beclin-1 and LC3-II/LC3-I ratio in the hippocampus. Importantly, intra-DRN infusions of SLC6A4 siRNAs prevented hippocampal autophagy and promoted the extinction of contextual fear memory. These results indicated that SERT in the hippocampus may play a critical role in the pathogenesis of hippocampal autophagy, and is likely involved in the development of PTSD. The present study was designed to evaluate the effects of neurobiological manipulation (i.e., SERT gene knockdown) after the occurrence of traumatic stress exposure using our animal model. In reality, most of the patients seek the treatment after exposure to the traumatic events. Thus, the results of our study may help the development of treatment strategies after the exposure of traumatic events. Furthermore, while this study was not aimed to assess the effects of time window of SERT gene knockdown on the development of PTSD in rats, it will be important to assess whether pre-deposited SERT gene polymorphisms can contribute to the vulnerability to development of PTSD. To support this, recent studies have suggested that serotonin transporter (SERT) genetic polymorphisms contribute to an individual's response to a traumatic event [24,25]. Therefore, one of the possible animal experiments in the future will be to study whether the SERT knockout animals are resistant to SPS-treatment induced hippocampal autophagy and depression-like behavior.

It should be noted that SERT gene knockdown in the DRN will result in the decreased expression of SERT in other brain regions besides the hippocampus in the present study. For example, the amygdala is a major forebrain target of 5-HT neurons arising from the DRN and 5-HT signaling within the amygdala regulates normal fear and threat responsiveness [31–34]. Therefore, it is likely that abnormal SERT function within the amygdala is an important mechanism underlying the pathophysiology of PTSD. In support of this hypothesis, it has been shown that genetic variants causing differential expression of SERT genes are associated with differences in the acquisition of a conditioned fear response and altered startle response in humans [35,36]. Furthermore, previous studies have demonstrated that reduced amygdala SERT binding in PTSD is correlated with higher anxiety and depression symptoms in PTSD patients [37]. However, the exact role of SERT in amygdala in the development of PTSD has not been well studied. Future studies will be necessary to examine the SERT expression and function in the amygdala in the pathogenesis of PTSD using the animal model of the present study. Given that the connectivity of amygdala and hippocampus is essential for modulating hippocampal function [38,39], this line of research would be helpful to expand our understandings in the involvement of neural substrates and neurocircuits in the PTSD. The present study found that SPS treatment did not alter unconditioned anxiety-related behavior after a long period of incubation time (i.e., 21 days). While these results seem consistent with some previous studies [40–44], other studies have demonstrated that SPS treatment can increase anxiety-related behaviors [40–47]. Such a discrepancy may be due to the specific procedure of SPS treatment. For example, re-exposure to the context of forced swim during the previous SPS can enhance anxiety-related behavior in rats in the EPM test [40]. Thus, it is likely that previous trauma-related cues can promote the exhibition of anxiety-related behaviors. Furthermore, previous studies using a modified SPS protocol which included a 30 min foot shock after exposure to SPS stressors, similar to the protocol used in the present study, have shown that SPS treatment could increase anxiety-related behavior up to 14 days later [42,44]. Additionally, it has been shown that foot shock could enhance anxiety-like behavior [48]. Thus, it is likely that the incubation time after the SPS treatment may alter the exhibition of anxiety-related behaviors. Our results are consistent with previous reports that SPS treatment could impair the extinction of contextual fear memory [49]. Several studies have shown that the neural function of the hippocampus is critical for the extinction of fear memory [50–52]. Specifically, muscimol inactivation of the dorsal hippocampus reduces the rate of extinction and prevents the context dependency of extinction [50,53]. Additionally, actin rearrangement within the dorsal hippocampus is necessary for the extinction of contextual fear memory [51]. Furthermore, repeated

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administration of D-cycloserine, a selective partial agonist of the Nmethyl-D-aspartic acid (NMDA) glutamatergic receptors alleviated impaired fear extinction in SPS rats [49]. Adding to the literature, the present study indicated that SERT in the hippocampus may be critical for the SPS-induced neuroadaptations, which are critical for the maintenance of the contextual fear memory. To support it, previous studies have shown enhanced contextual fear memory in central serotonindeficient mice [54]. The present study demonstrated that SPS treatment attenuated 5HT levels in the hippocampus after 21 days of incubation, even though the expression levels of SERT in the hippocampus were not altered after 21 days of incubation. These results suggested that SPS treatment may have produced a long lasting impairment of 5-HT storage pool, which is regulated by release, reuptake, and biosynthesis. While a few studies have indicated that stress can increase the biosynthesis of 5HT in response to counterbalance neuronal depletion of 5-HT due to release [55,56], it is possible that the increase in biosynthesis could not make up the loss of 5-HT from the storage pool due to the depletion. Importantly, the present study also demonstrated that intra-DRN infusions of SLC6A4 siRNA during the early incubation after SPS treatment could prevent subsequent reduction of 5-HT levels in the hippocampus. These results suggested that increase the extracellular level of 5-HT during the early incubation after SPS treatment may stimulate the biosynthesis of 5-HT, and maintain the 5-HT storage pool in the long run. However, the mechanisms underlying the putative increase in 5-HT biosynthesis are still not clear. Future studies will be necessary to explore the effects of SPS treatment on the activity of critical enzymes involved in 5-HT biosynthesis. One important finding in the present study was that SPS treatment could induce hippocampal autophagy, which was associated with increased expression of Beclin-1 and LC3-II/LC3-I ratio, which are well known autophagy markers. The formation of large autophagic vacuoles is the typical morphological feature of autophagy, which is one of the major mechanisms for the degradation of cytoplasmic constituents, including proteins and various organelles, within lysosomes/vacuoles [57]. It has been shown that Beclin-1 plays a critical role in autophagy and is involved in the recruitment of membranes to form autophagosomes, which undergo several microtubule-dependent maturation processes before fusion with lysosomes [58,59]. Additionally, heterozygous disruption of Beclin-1 expression would reduce autophagic activity in mice [59–61]. Indeed, many studies have suggested that Beclin-1 mediated autophagy is likely important for elimination of deleterious components that have accumulated within the cytoplasm, such as misfolded protein and damaged organelles in order to maintain neuronal survival [29,57]. Therefore, it is likely that SPS treatment-induced autophagy might be protective at an early stage, but became deleterious later in the present study. To support this speculation, previous studies have shown that inhibition of autophagy 24 h before ischemia triggered a higher neuronal death rate, whereas inhibition of autophagy after reperfusion reduced neuronal death [62]. While the neurobiological mechanisms underlying the effects of SPS treatment on hippocampal autophagy are still not clear, the present study suggested that attenuation of SERT expression in the hippocampus during the early incubation period after SPS treatment was able to rescue the development of hippocampal autophagy. It should be noted that the attenuation of SERT expression in the hippocampus was associated with normalized 5-HT levels in the hippocampus. While few studies have been conducted to investigate the role of 5-HT in the central nervous system in neuronal autophagy, previous studies have shown that serotonin reduced starvation-induced autophagy of hepatocellular carcinoma cells [63]. Hence, 5-HT in the hippocampus is likely important for prevent excessive autophagy. However, when 5-HT level is reduced as seen in the present study after SPS treatment, autophagy process will lose the inhibitory control, thus leading deleterious neuronal death. Therefore, our findings in the present study indicated that SERT in the hippocampus may play a critical role in the pathogenesis

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of hippocampal autophagy, and is likely involved in the development of PTSD. Conflict of interest The authors have no conflict of interest to disclose. Acknowledgments This work was supported by the Zhejiang Provincial Science and Technology Department of Public Welfare Technology Application Project (2014C37026). We thank Professor Marong Fang for his valuable suggestions on this paper. References [1] D.S. Charney, A.Y. Deutch, J.H. Krystal, S.M. Southwick, M. Davis, Psychobiologic mechanisms of posttraumatic stress disorder, Arch. Gen. Psychiatry 50 (1993) 295–305. [2] J.R. Howlett, M.B. Stein, Prevention of trauma and stressor-related disorders: a review, Neuropsychopharmacology (2015), http://dx.doi.org/10.1038/npp.2015.261. [3] J. Ipser, S. Seedat, D.J. Stein, Pharmacotherapy for post-traumatic stress disorder — a systematic review and meta-analysis, S. Afr. Med. J. 96 (2006) 1088–1096. [4] M. Hoskins, J. Pearce, A. Bethell, L. Dankova, C. Barbui, W.A. Tol, et al., Pharmacotherapy for post-traumatic stress disorder: systematic review and meta-analysis, Br. J. Psychiatry 206 (2015) 93–100. [5] N. Brunello, J.R. Davidson, M. Deahl, R.C. Kessler, J. Mendlewicz, G. Racagni, et al., Posttraumatic stress disorder: diagnosis and epidemiology, comorbidity and social consequences, biology and treatment, Neuropsychobiology 43 (2001) 150–162. [6] V. Michopoulos, S.D. Norrholm, T. Jovanovic, Diagnostic biomarkers for posttraumatic stress disorder: promising horizons from translational neuroscience research, Biol. Psychiatry 78 (2015) 344–353. [7] A. Meneses, 5-HT systems: emergent targets for memory formation and memory alterations, Rev. Neurosci. 24 (2013) 629–664. [8] L.L. Davis, A. Suris, M.T. Lambert, C. Heimberg, F. Petty, Post-traumatic stress disorder and serotonin: new directions for research and treatment, J. Psychiatry Neurosci. 22 (1997) 318–326. [9] I. Hageman, H.S. Andersen, M.B. Jorgensen, Post-traumatic stress disorder: a review of psychobiology and pharmacotherapy, Acta Psychiatr. Scand. 104 (2001) 411–422. [10] L.A. Jans, W.J. Riedel, C.R. Markus, A. Blokland, Serotonergic vulnerability and depression: assumptions, experimental evidence and implications, Mol. Psychiatry 12 (2007) 522–543. [11] A.Y. Shalev, What is posttraumatic stress disorder? J. Clin. Psychiatry. 62 (Suppl. 17) (2001) 4–10. [12] H. Kusserow, B. Davies, H. Hortnagl, I. Voigt, T. Stroh, B. Bert, et al., Reduced anxietyrelated behaviour in transgenic mice overexpressing serotonin 1A receptors, Brain Res. Mol. Brain Res. 129 (2004) 104–116. [13] Z. Lin, J.J. Canales, T. Bjorgvinsson, M. Thomsen, H. Qu, Q.R. Liu, et al., Monoamine transporters: vulnerable and vital doorkeepers, Prog. Mol. Biol. Transl. Sci. 98 (2011) 1–46. [14] M.E. Thase, T. Denko, Pharmacotherapy of mood disorders, Annu. Rev. Clin. Psychol. 4 (2008) 53–91. [15] S. Kohler, K. Cierpinsky, G. Kronenberg, M. Adli, The serotonergic system in the neurobiology of depression: relevance for novel antidepressants, J. Psychopharmacol. (2015). [16] N. Kitayama, V. Vaccarino, M. Kutner, P. Weiss, J.D. Bremner, Magnetic resonance imaging (MRI) measurement of hippocampal volume in posttraumatic stress disorder: a meta-analysis, J. Affect. Disord. 88 (2005) 79–86. [17] D.W. Hedges, S. Allen, D.F. Tate, G.W. Thatcher, M.J. Miller, S.A. Rice, et al., Reduced hippocampal volume in alcohol and substance naive Vietnam combat veterans with posttraumatic stress disorder, Cogn. Behav. Neurol. 16 (2003) 219–224. [18] G. Villarreal, H. Petropoulos, D.A. Hamilton, L.M. Rowland, W.P. Horan, J.A. Griego, et al., Proton magnetic resonance spectroscopy of the hippocampus and occipital white matter in PTSD: preliminary results, Can. J. Psychiatr. 47 (2002) 666–670. [19] R.J. Lindauer, E.J. Vlieger, M. Jalink, M. Olff, I.V. Carlier, C.B. Majoie, et al., Effects of psychotherapy on hippocampal volume in out-patients with post-traumatic stress disorder: a MRI investigation, Psychol. Med. 35 (2005) 1421–1431. [20] E.L. Wignall, J.M. Dickson, P. Vaughan, T.F. Farrow, I.D. Wilkinson, M.D. Hunter, et al., Smaller hippocampal volume in patients with recent-onset posttraumatic stress disorder, Biol. Psychiatry 56 (2004) 832–836. [21] Y. Golub, S.F. Kaltwasser, C.P. Mauch, L. Herrmann, U. Schmidt, F. Holsboer, et al., Reduced hippocampus volume in the mouse model of posttraumatic stress disorder, J. Psychiatr. Res. 45 (2011) 650–659. [22] H.C. Yan, X. Cao, T.M. Gao, X.H. Zhu, Promoting adult hippocampal neurogenesis: a novel strategy for antidepressant drug screening, Curr. Med. Chem. 18 (2011) 4359–4367. [23] C. Schmahl, K. Berne, A. Krause, N. Kleindienst, G. Valerius, E. Vermetten, et al., Hippocampus and amygdala volumes in patients with borderline personality disorder with or without posttraumatic stress disorder, J. Psychiatry Neurosci. 34 (2009) 289–295.

140

Z.-M. Wu et al. / Journal of the Neurological Sciences 360 (2016) 133–140

[24] A. Caspi, K. Sugden, T.E. Moffitt, A. Taylor, I.W. Craig, H. Harrington, et al., Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene, Science 301 (2003) 386–389. [25] H.J. Lee, M.S. Lee, R.H. Kang, H. Kim, S.D. Kim, B.S. Kee, et al., Influence of the serotonin transporter promoter gene polymorphism on susceptibility to posttraumatic stress disorder, Depress. Anxiety 21 (2005) 135–139. [26] K. Pham, J. Nacher, P.R. Hof, B.S. McEwen, Repeated restraint stress suppresses neurogenesis and induces biphasic PSA-NCAM expression in the adult rat dentate gyrus, Eur. J. Neurosci. 17 (2003) 879–886. [27] E. Castilla-Ortega, C. Hoyo-Becerra, C. Pedraza, J. Chun, F. Rodriguez De Fonseca, G. Estivill-Torrus, et al., Aggravation of chronic stress effects on hippocampal neurogenesis and spatial memory in LPA(1) receptor knockout mice, PLoS ONE 6 (2011), e25522. [28] M. Banasr, M. Hery, R. Printemps, A. Daszuta, Serotonin-induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5-HT receptor subtypes in the dentate gyrus and the subventricular zone, Neuropsychopharmacology 29 (2004) 450–460. [29] H.J. Wu, J.L. Pu, P.R. Krafft, J.M. Zhang, S. Chen, The molecular mechanisms between autophagy and apoptosis: potential role in central nervous system disorders, Cell. Mol. Neurobiol. 35 (2015) 85–99. [30] S. Romao, C. Munz, LC3-associated phagocytosis, Autophagy 10 (2014) 526–528. [31] B.L. Jacobs, E.C. Azmitia, Structure and function of the brain serotonin system, Physiol. Rev. 72 (1992) 165–229. [32] C.L. Wellman, A. Izquierdo, J.E. Garrett, K.P. Martin, J. Carroll, R. Millstein, et al., Impaired stress-coping and fear extinction and abnormal corticolimbic morphology in serotonin transporter knock-out mice, J. Neurosci. 27 (2007) 684–691. [33] J.M. Muller, E. Morelli, M. Ansorge, J.A. Gingrich, Serotonin transporter deficient mice are vulnerable to escape deficits following inescapable shocks, Genes Brain Behav. 10 (2011) 166–175. [34] J.M. Zanoveli, M.C. Carvalho, J.M. Cunha, M.L. Brandao, Extracellular serotonin level in the basolateral nucleus of the amygdala and dorsal periaqueductal gray under unconditioned and conditioned fear states: an in vivo microdialysis study, Brain Res. 1294 (2009) 106–115. [35] T.B. Lonsdorf, A.I. Weike, P. Nikamo, M. Schalling, A.O. Hamm, A. Ohman, Genetic gating of human fear learning and extinction: possible implications for geneenvironment interaction in anxiety disorder, Psychol. Sci. 20 (2009) 198–206. [36] B. Brocke, D. Armbruster, J. Muller, T. Hensch, C.P. Jacob, K.P. Lesch, et al., Serotonin transporter gene variation impacts innate fear processing: acoustic startle response and emotional startle, Mol. Psychiatry 11 (2006) 1106–1112. [37] J.W. Murrough, Y. Huang, J. Hu, S. Henry, W. Williams, J.D. Gallezot, et al., Reduced amygdala serotonin transporter binding in posttraumatic stress disorder, Biol. Psychiatry 70 (2011) 1033–1038. [38] E.M. Blessing, F. Beissner, A. Schumann, F. Brunner, K.J. Bar, A data-driven approach to mapping cortical and subcortical intrinsic functional connectivity along the longitudinal hippocampal axis, Hum. Brain Mapp. (2015), http://dx.doi.org/10.1002/ hbm.23042. [39] Y. Fan, K. Pestke, M. Feeser, S. Aust, J.C. Pruessner, H. Boker, et al., Amygdala–hippocampal connectivity changes during acute psychosocial stress: joint effect of early life stress and oxytocin, Neuropsychopharmacology 40 (2015) 2736–2744. [40] B.H. Harvey, L. Brand, Z. Jeeva, D.J. Stein, Cortical/hippocampal monoamines, HPAaxis changes and aversive behavior following stress and restress in an animal model of post-traumatic stress disorder, Physiol. Behav. 87 (2006) 881–890. [41] Y. Peng, S.F. Feng, Q. Wang, H.N. Wang, W.G. Hou, L. Xiong, et al., Hyperbaric oxygen preconditioning ameliorates anxiety-like behavior and cognitive impairments via upregulation of thioredoxin reductases in stressed rats, Prog. NeuroPsychopharmacol. Biol. Psychiatry 34 (2010) 1018–1025. [42] H.N. Wang, Y. Peng, Q.R. Tan, Y.C. Chen, R.G. Zhang, Y.T. Qiao, et al., Quetiapine ameliorates anxiety-like behavior and cognitive impairments in stressed rats:

[43]

[44]

[45]

[46] [47] [48] [49]

[50]

[51]

[52]

[53] [54]

[55] [56] [57] [58]

[59]

[60]

[61] [62] [63]

implications for the treatment of posttraumatic stress disorder, Physiol. Res. 59 (2010) 263–271. H.T. Wang, F. Han, J.L. Gao, Y.X. Shi, Increased phosphorylation of extracellular signal-regulated kinase in the medial prefrontal cortex of the single-prolonged stress rats, Cell. Mol. Neurobiol. 30 (2010) 437–444. W. Wang, Y. Liu, H. Zheng, H.N. Wang, X. Jin, Y.C. Chen, et al., A modified singleprolonged stress model for post-traumatic stress disorder, Neurosci. Lett. 441 (2008) 237–241. A. Imanaka, S. Morinobu, S. Toki, S. Yamawaki, Importance of early environment in the development of post-traumatic stress disorder-like behaviors, Behav. Brain Res. 173 (2006) 129–137. S. Khan, I. Liberzon, Topiramate attenuates exaggerated acoustic startle in an animal model of PTSD, Psychopharmacology 172 (2004) 225–229. I. Liberzon, M. Krstov, E.A. Young, Stress–restress: effects on ACTH and fast feedback, Psychoneuroendocrinology 22 (1997) 443–453. S.M. Korte, S.F. De Boer, B. Bohus, Fear-potentiation in the elevated plus-maze test depends on stressor controllability and fear conditioning, Stress 3 (1999) 27–40. S. Yamamoto, S. Morinobu, M. Fuchikami, A. Kurata, T. Kozuru, S. Yamawaki, Effects of single prolonged stress and D-cycloserine on contextual fear extinction and hippocampal NMDA receptor expression in a rat model of PTSD, Neuropsychopharmacology 33 (2008) 2108–2116. K.A. Corcoran, T.J. Desmond, K.A. Frey, S. Maren, Hippocampal inactivation disrupts the acquisition and contextual encoding of fear extinction, J. Neurosci. 25 (2005) 8978–8987. A. Fischer, F. Sananbenesi, C. Schrick, J. Spiess, J. Radulovic, Distinct roles of hippocampal de novo protein synthesis and actin rearrangement in extinction of contextual fear, J. Neurosci. 24 (2004) 1962–1966. S.A. Heldt, L. Stanek, J.P. Chhatwal, K.J. Ressler, Hippocampus-specific deletion of BDNF in adult mice impairs spatial memory and extinction of aversive memories, Mol. Psychiatry 12 (2007) 656–670. K.A. Corcoran, S. Maren, Hippocampal inactivation disrupts contextual retrieval of fear memory after extinction, J. Neurosci. 21 (2001) 1720–1726. J.X. Dai, H.L. Han, M. Tian, J. Cao, J.B. Xiu, N.N. Song, et al., Enhanced contextual fear memory in central serotonin-deficient mice, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 11981–11986. H. Anisman, K. Matheson, Stress, depression, and anhedonia: caveats concerning animal models, Neurosci. Biobehav. Rev. 29 (2005) 525–546. F. Chaouloff, Physiopharmacological interactions between stress hormones and central serotonergic systems, Brain Res. Brain Res. Rev. 18 (1993) 1–32. D.J. Klionsky, S.D. Emr, Autophagy as a regulated pathway of cellular degradation, Science 290 (2000) 1717–1721. X. Qu, J. Yu, G. Bhagat, N. Furuya, H. Hibshoosh, A. Troxel, et al., Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene, J. Clin. Invest. 112 (2003) 1809–1820. Z. Yue, S. Jin, C. Yang, A.J. Levine, N. Heintz, Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 15077–15082. Z. Yue, A. Horton, M. Bravin, P.L. DeJager, F. Selimi, N. Heintz, A novel protein complex linking the delta 2 glutamate receptor and autophagy: implications for neurodegeneration in lurcher mice, Neuron 35 (2002) 921–933. A. Kihara, Y. Kabeya, Y. Ohsumi, T. Yoshimori, Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network, EMBO Rep. 2 (2001) 330–335. R. Shi, J. Weng, L. Zhao, X.M. Li, T.M. Gao, J. Kong, Excessive autophagy contributes to neuron death in cerebral ischemia, CNS Neurosci. Ther. 18 (2012) 250–260. C. Soll, J.H. Jang, M.O. Riener, W. Moritz, P.J. Wild, R. Graf, et al., Serotonin promotes tumor growth in human hepatocellular cancer, Hepatology 51 (2010) 1244–1254.