Ketamine alleviates fear memory and spatial cognition deficits in a PTSD rat model via the BDNF signaling pathway of the hippocampus and amygdala

doi:10.21203/rs.3.rs-3116158/v1

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Ketamine alleviates fear memory and spatial cognition deficits in a PTSD rat model via the BDNF signaling pathway of the hippocampus and amygdala

https://doi.org/10.21203/rs.3.rs-3116158/v1

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Background

Post-traumatic stress disorder (PTSD) is associated with traumatic stress experiences. This condition can be accompanied by learning and cognitive deficits. Studies have demonstrated that ketamine can rapidly and significantly alleviate symptoms in patients with chronic PTSD. Nonetheless, the effects of ketamine on neurocognitive impairment and its mechanism of action in PTSD remain unclear.

Methods

In this study, different concentrations of ketamine (5, 10, 15, and 20 mg/kg, i.p.) were evaluated in rat models of single prolonged stress and electropodic shock (SPS&S). Expression levels of brain-derived neurotrophic factor (BDNF) and post-synaptic density-95 (PSD-95) in the hippocampus (HIP) and amygdala (AMG) were determined by Western blot analysis, immunohistochemistry.

Results

The data showed that rats subjected to SPS&S exhibited significant PTSD-like cognitive impairment. The effect of ketamine on SPS&S-induced neurocognitive function showed a U-shaped dose effect in rats. A single administration of ketamine at a dosage of 10–15mg/kg resulted in significant changes in behavioral outcomes. These manifestations of improvements in cognitive function and molecular changes were reversed at high doses (15–20mg/kg).

Conclusion

Overall, ketamine reversed SPS&S-induced fear and spatial memory impairment and the down-regulation of BDNF and BDNF-related PSD-95 signaling in the HIP and AMG. A dose equal to 15 mg/kg rapidly reversed the behavioral and molecular changes, and promoted the amelioration of cognitive dysfunction. The enhanced association of BDNF signaling with PSD-95 effects could be involved in the therapeutic efficiency of ketamine for PTSD.

post-traumatic stress disorder

ketamine

neurocognitive function

brain-derived neurotrophic factor

postsynaptic density protein 95

1. Post-traumatic stress disorder rats had learning and cognitive impairments..

2. Ketamine provides rapid and significant relief in patients with chronic PTSD.

3. Brain-derived neurotrophic factor and postsynaptic dense region protein 95 may be involved in the treatment of PTSD with ketamine.

Post-traumatic stress disorder (PTSD) is a delayed onset and long-term mental disorder caused by abnormally threatening or catastrophic psychological trauma, which is characterized by continuous reexperience of traumatic memories, hyperarousal state, and cognitive deficit[1, 2]. Its lifetime prevalence is approximately 1.0–2.6% in the general population[3]. The disruption of neurocognitive function is a major symptom of PTSD[4]. Therefore, when individuals who have experienced traumatic events are re-exposed to traumatic situations, negative cognitive changes may occur, including fear memories and impaired mental memories[5]. However, treatment of cognitive dysfunction caused by PTSD is rarely reported.

Traditional antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) and norepinephrine reuptake inhibitors, are among the medications licensed for the treatment of PTSD (NSRIs)[6]. However, the effectiveness of SSRIs and SNRIs is relatively marginal, and they have the potential to cause side effects. The combination of some antidepressants with extinction therapy has been found to reduce PTSD symptoms in humans and fear in rodents[7]. However, the underlying mechanisms of such treatments remain largely unknown. Therefore, new targets and fast-acting therapeutic drugs need to be identified to improve cognitive impairment in PTSD-like psychiatric symptoms, including fear memory and spatial memory deficits.

Notably, a subanesthetic dose of ketamine may produce antidepressant, analgesic, and propsychotic effects for animals[8]. Recent have studies demonstrated that intravenous (IV) subanesthetic infusion of ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, quickly and essentially lessens psychiatric distress in PTSD[9]. In addition, recent data suggest that ketamine can alleviate PTSD symptoms in patients with failed conventional pharmacotherapy; in recent years, ketamine has been considered a possible prophylactic agent to prevent the emergence of PTSD-like symptoms[10]. Ketamine doses of 5,10 and 15mg /kg have been found to improve depression-like behavior without adverse effects[11]. Moreover, a single treatment with ketamine (15 mg/kg) significantly inhibited these symptoms[12]. However propsychotic effects when the dose is higher than 20 mg/kg[13]. However, the details of these effects, such as whether different concentrations ketamine treatment can improve the symptoms of single prolonged stress (SPS)-induced fear memory and spatial memory deficits in rats, or other neurocognitive diseases of PTSD, are poorly understood.

BDNF is a neurotrophic factor that is considered a key factor in mediating synaptic plasticity associated with learning and memory. Previous studies have reported that BDNF expression in the amygdala and hippocampus was likely associated with stress-related events and PTSD symptoms[14]. In cognitive activities such as learning loss and fear expression, BDNF exerts different effects on the neural plasticity of hippocampal (HIP) and amygdala (AMG) networks[15, 16]. For example, the acquisition and retrieval of contextual fear memory also requires coordinated neural activity, which is presented as encoded information in HIP and then transmitted to AMG to achieve contextual fear conditioning[17, 18]. And research indicates that BDNF signaling and changes in its concentrations affect memory performance and europsychiatric-related cognitive dysfunctions[19]. In the amygdala, BDNF transcription increased in the consolidation period 2 h after fear conditioning, and inhibition of BDNF signals in the amygdala impaired the acquisition and consolidation of fear conditioning and the consolidation of regression[20]. In accordance with these findings, a recent meta-analysis of 20 studies has revealed an increase in the serum BDNF levels of PTSD patients relative to those of controls[21]. Meanwhile, other studies that have found significantly lower serum or plasma BDNF levels in individuals with PTSD, together with impaired memory and learning[22]. In animal models of chronic stress and post-traumatic stress disorder, BDNF concentrations have been found to be lower in the plasma and hippocampus of stressed animals than those of non-stressed controls. For example, in the social isolation paradigm, BDNF levels in the hippocampus of male rats (except female rats) decrease[23].

Chronic, persistent PTSD could be attributed to the accumulation of adverse effects on synaptic structures. Growing evidence shows that altered synaptic plasticity can cause impaired spatial cognition and fear extinction. Postsynaptic density protein 95 (PSD-95) is a synaptic protein regulating glutamate receptor anchoring, synaptic stability, and certain types of memory[24]. Its transport is controlled by the BDNF-TrkB pathway, which regulates the synaptic transmission of PSD-95[25]. The contribution of PSD-95 to memory has been demonstrated behaviorally by studies showing that PSD-95 deletion or knockdown impairs spatial learning, conditioned taste aversion, and simple operant associative learning [26, 27], in addition to impaired fear memory stability[28]. PSD-95 expression was found to be regulated by the activation of BDNF and its receptor TrKB in a cellular model of genetic intervention[29]. Thus, ketamine could increase BDNF protein levels in the HIP and AMG, upregulating BDNF signal transduction and activating PSD-95 expression, which could be associated with the alleviation of PTSD symptoms. Accordingly, the PTSD model was induced with single prolonged stress and electric foot shock (SPS&S) to evaluate the effect of ketamine on fear memory and spatial cognition of SPS&S-induced rats, as well as to elucidate changes in the BDNF-related PSD-95 signaling pathway in HIP and AMG after ketamine administration. In addition, we focused on the “does gradient” of ketamine (5, 10, 15, 20 mg/kg) and studied different behavior phenotypes and the mechanism underlying the different effects of sub-anesthetic and anesthetic ketamine, to systematically evaluate the effects of ketamine at different subanesthetic concentrations on PTSD-like neurocognitive symptoms.

SPS&S model effects

The rats in the SPS&S group showed more freezing time after 24 h (t = 5.162, p = 0.000, Fig. 1B). When tested in the WMT, reductions in escape latencies and the number of platform crossings provided an index of spatial memory. The significant effects of the SPS&S model were observed in the time spent in escape latencies, and the SPS&S (46.60 ± 12.14) rats showed robust spatial memory deficits (Fig. 1C). Compared with the CON rats (17.28 ± 6.24), the SPS&S group showed a significant increase in escape latency on Days 3, 4, and 5 of the place navigation test (MANOVA: F (4, 24) = 9.168, p = 0.000; Day 3: p = 0.014; Day 4: p = 0.000; Day 5: p = 0.001. Figure 1D).

The results of Western blot analysis (Figs. 2A, 2B, 2C, and 2D) revealed that PSD-95 expression levels in the HIP (t = 4.967, p = 0.038) and AMG (t = 5.168; p = 0.036) were lower in the SPS&S group than in the CON group. Meanwhile, BDNF levels were significantly decreased in the HIP (t = 5.145, p = 0.036) of the SPS&S group, whereas no differences were detected in the AMG (t = 1.101, p = 0.386). The results of IHC data analysis indicated that the BDNF-positive cell levels in the HIP (t = 6.704, p = 0.003) and AMG (t = 5.365, p = 0.002) of the SPS-induced rats were significantly decreased relative to those in the control group; and the difference in PSD-95-positive cell levels between the two groups was only observed in the HIP (t = 6.582, p = 0.001. Figures 2E, 2F, 2G, and 2H).

Effects of ketamine on SPS&S-induced fear memory and spatial cognition behaviors

After ketamine administration, a significant difference in freezing time was observed in each group (F (4, 25 = 11.66, p < 0.001). Treatment with ketamine at 15 mg/kg (t = 6.597, p < 0.001) and 20 mg/kg (t = 3.167, p = 0.006) reversed the prolonged freezing time in the PTSD-sal group. However, no such significant difference was found between the PTSD-ket-5, PTSD-ket-10, and PTSD-sal groups (Fig. 3A). According to the polynomial fitting results, the freezing time in each group was significantly correlated with ketamine concentration (R² = 0.477, P = 0.001) (Fig. 3D). On the basis of the curve trend, treatment with ketamine at 15 and 20 mg/kg could reduce the freezing time in the PTSD group; however, with a decrease or increase in ketamine concentration, freezing time may increase. The WMT results for the number of platform crossing showed statistically significant differences. Ketamine exerted significant effects on the number of platform crossing in the PTSD-ket-15 group relative to that in the PTSD-sal, as determined from an increase in the number of platform crossings (t = 8.562, p = 0.005) (Fig. 3B). According to the polynomial fitting results, it was found that the number of platform crossing of each group were significantly correlated with ketamine concentration(R² = 0.487, p = 0.001) (Fig. 3E). The PTSD- ket-15 group had the highest number of platform crossings.

The WMT results for the escape latency time(s) of the rats to locate the platform showed statistically significant differences between the PTSD-ket-10 (t = 7.435, p = 0.028) or PTSD-ket-15 groups (t = 13.45, p = 0.043) and the PTSD-sal group (Fig. 3C). The PTSD-ket-10 (39.97 ± 7.84), and PTSD-ket-15 (32.04 ± 9.43) groups spent less time finding the platform, and the PTSD-sal (52.41 ± 10.25) showed robust spatial memory deficits, as determined from their longer escape latency. No significant differences in the results were found between the PTSD-ket-10 and PTSD-ket-15 groups. Effects of ketamine on corticosteroid levels in the SPS&S-induced group, the CORT levels in the PTSD-ket-15 group significantly decreased relative to that in the PTSD-sal group (446.21 ± 34.7) (t = 13.472, p = 0.045). The increase in CORT levels in the PTSD-ket-15 group (315.7 ± 20.13) was significantly reduced with the ketamine treatment; meanwhile, the CORT levels in the PTSD-ket-5, PTSD-ket-10, and PTSD-ket-20 groups showed no significant difference from that in the PTSD-sal group(Fig. 3F).

Effects of ketamine on protein expression of BDNF and PSD-95 under SPS&S-induced treatment

The results of Western blot analysis showed that the BDNF levels in the HIP and AMG of the rats in the PTSD-ket-15 group increased relative to that in the PTSD-sal group (t = 4.453, p = 0.048). The tendency of BDNF expression to change with the amount of ketamine injection was reversed in the PTSD-ket-20 group (0.69 ± 0.17) (Figs. 4A, 4D). The intensity of PSD-95 was relatively enhanced by ketamine infusion and was maximized in the PTSD-ket-15 group (1.50 ± 0.21) (Figs. 4B, 4E). BDNF protein was positively correlated with PSD-95 protein in the HIP (r² = 0.813, P = 0.000) and AMG (r² = 0.896, P = 0.000) (Figs. 4C, 4F).

Effects of ketamine on positive cell expression of BDNF and PSD-95 under SPS&S-induced treatment

The IHC result revealed an upregulation in the expression of BDNF-positive and PSD-95-positive cells in the AMG and HIP of SPS&S-induced rats after ketamine administration, with the highest expression levels found in the PTSD-ket-15 group (BDNF: 472.5 ± 42.57; PSD-95: 283.3 ± 27.45. Figures 5A, 5B, 5C, and 5D). However, no significant differences were noted between the PTSD-ket-10 (1.63 ± 0.27) and PTSD-ket-15 groups (t = 4.375, P = 0.025), although protein and the number of positive cells exhibited an increasing tendency with ketamine dose. Further analysis indicated a positive correlation between the BDNF level of positive cells and the expression of PTD95-positive cells in the HIP and AMG (r² = 0.771, p = 0.001, r² = 0.773. p = 0.001) (Figs. 5E and 5F). The result of linear regression analysis (Table 1) showed that BDNF protein effectively predicted contextual fear conditioning in the SPS&S-induced rats, and changes in spatial memory in the rats exposed to SPS&S might be related to the effect of PSD-95.

Table 1

Relationship between expression levels of BDNF and PSD95 proteins and various behaviors
Independent variable	Dependent variable	R	R²	F	β	T
BDNF	Freezing time	0.708	0.501	18.088	-0.623	-4.253***
PSD-95	Number of crossing time	0.786	0.618	29.14	0.705	5.398***
BDNF: brain-derived neurotrophic factor, PSD95: postsynaptic density 95
***p < 0.001
***p < 0.001 indicates statistical significance

This present study was designed to evaluate the acute pharmacological profile of ketamine to alleviate PTSD-like symptoms in an SPS&S model. Behavioral results implied that with SPS&S, the rats developed PTSD-like behaviors; however, a single treatment with ketamine (10 and 15 mg/kg) could lead to a significant remission of these symptoms, and a u-shaped dose–response relationship was observed. Further, ketamine could upregulate the expression of BDNF and PSD-95 in the HIP and AMG of SPS&S rats. Meanwhile, BDNF expression was positively correlated with PSD-95 expression, and regression analysis results indicated that increases in BDNF and PSD-95 expression levels in the HIP and AMG were closely related to the improvement in fear memory and spatial cognition deficits in PTSD, which could be attributed to the PSD-95 expression by regulating the BDNF expression of the rats exposed to SPS&S.

The model for post-traumatic stress disorder has been successfully created based on earlier laboratory investigations[12]. The current study revealed that the SPS&S model could produce more PTSD symptoms, including increases in freezing time and time to find the platform location, and a reduced number of platform crossings. These changes were accompanied by increased levels of plasma corticosterone and reduced BDNF protein levels and PSD-95 activity in the HIP and AMG. On the basis of these observations, SPS&S effectively induced PTSD-like behaviors and was established successfully in this study. The increases in freezing time, increase in time to find the platform location, and decrease in the number of platform crossings in WMT could be interpreted as typical symptoms associated with PTSD-like behavior [36]. These changes are consistent with previous findings indicating that PTSD-associated freezing and anxiogenic activities were induced by SPS&S[37].

The SPS&S procedure has led to contextual conditioning fear, and the freezing behavior may serve as a good factor for assessing the severity of anxiety due to regional brain dysfunction[38]. Studies indicate that the HIP is involved in encoding context representations; meanwhile, in conditioning models, the amygdala is critical for encoding, storing, and retrieving direct associations between contexts or cues and aversive stimuli[39]. Impaired spatial memory is another important symptom of PTSD. The WMT hidden platform is a well-accepted measure of HIP-dependent spatial learning in rats[40]. Neurons in the HIP respond vigorously to various stimuli, particularly to spatial locations. In the present study, ketamine (15 and 20 mg/kg) significantly reduced the freezing time 24 h after administration. Specifically, 15 mg/kg of ketamine could significantly shorten the time of regression and improve the number of crossings in the WMT 48 h after administration, which could be interpreted as a remission of fear and spatial memory deficits of PTSD symptoms in these ketamine-treated rats. These findings contradicted previous reports that PTSD associated with multiple foot shocks did not affect spatial memory. The fear memory caused by multiple foot shocks is separate from the spatial memory in the HIP, which may be due to the division of neurons in different hippocampal subregions [41]. In addition, the ventral HIP is associated with the fear acquisition; meanwhile, lesions of the dorsal but not the ventral HIP disrupt performance in spatial tasks[42]. Therefore, on the basis of the present data, the ventral HIP could serve as a conduit for the transfer of contextual information between the dorsal HIP and the AMG, and this conduit was blocked in the rats exposed to SPS&S.

Information acquisition and renewal are important processes of learning and memory, and ketamine induced significant changes in the expression of glutamate receptors in brain areas relevant for renewal and contextual memory[43]. These findings may explain the result that ketamine can significantly reduce the freezing time, as well as improve the spatial memory and learning ability of rats exposed to SPS&S. Subanesthetic doses of ketamine (5, 10, and 15 mg/kg) were known to improve depression-like behaviors without psychomotor adverse reactions[44], However, ketamine exceeding 20 mg/kg could exert pro-psychotic effects, including negative cognitive symptoms and deficits [45]. Another study reported that a large dose of ketamine or an infusion rate greater than 25 µg/kg/min induced hallucination and impairment of cognitive functioning[46]. These effects could be the compensatory consequence of increased glutamate release induced by high-dose ketamine treatment administered to rats 24 h earlier. Higher doses of ketamine in the brain exert a negative effect in potentiating oxidative stress. Our preclinical study shows similar results, ketamine (10 and 15 mg/kg) can significantly reduce the freezing time and increased the number of platform crossings, as well as improve the spatial memory and learning ability of rats exposed to SPS&S without psychomotor adverse reactions. However, the effect on the cognitive function of SPS rats was reversed when the ketamine dose reaches 20mg/kg. There was a U-shaped trend in behavioral results with different doses.

Stress is known to impair various endocrine, physiological, and neuronal functions. Moreover, it is often associated with higher vulnerability to psychiatric disorders. Corticosterone has become an important indicator of stress in the animal model[47]. In the present study, ketamine (15 mg/kg) produced behavioral alterations and decreased corticosterone levels relative to those in the SPS&S group. Ketamine administration also restored the corticosterone levels in the PTSD model, which was consistent with previous studies showing the normalization of reduced corticosterone levels in PTSD in the presence of ketamine and selective serotonin reuptake inhibitors (SSRIs)[48]. However, excessive ketamine caused a stress reaction in rats[49], which was confirmed by the significant increase in corticosterone levels in the PTSD-ket-20 group relative to those in the PTSD-ket-15 group. Different doses of ketamine induce different behavior and BDNF and PSD-95 level. Some studies reported that chronic ketamine administration induced negative syndrome and decreased the BDNF levels, but acute treatment with ketamine (15 mg/kg) increased the BDNF level in the hippocampus and strengthened the antidepressant efficacy by a pro-cognitive mechanism[50]. The decreased levels of BNDF protein expression were reversed by ketamine (10 and 15 mg/kg) after PTSD training, which was consistent with a previous finding that ketamine increased pro-BDNF expression after a single dose[51].

Another study found that extinction in animals receiving ketamine increased the phosphorylated and activated forms of BDNF in the HIP, as well as the levels of downstream kinases, which could mediate synaptic plasticity and maintain the stability of synaptic connections. These activated forms triggered the synaptic delivery of PSD-95[52]. In the current study, ketamine at 10 and 15 mg/kg could increase the number of PSD-95-positive cells, and protein expression levels were significantly increased in PSD-95 protein in the HIP and AMG. Therefore, PSD-95 itself and its interaction with BDNF signaling might be implicated in the retrieval of fear memories, and reduced PSD-95 expression might be the key factor in spatial cognitive dysfunction. These combined findings supported the hypothesis that ketamine enhanced extinction because of increased synaptic function in the HIP and AMG. Regression analysis results verified this idea, indicating that the increases in BDNF and PSD-95 in the HIP and AMG were closely related to the improvement of fear and spatial cognition symptoms in PTSD, which could be attributed to the effects of BDNF on PSD-95. Furthermore, different doses of ketamine selectively changed the BDNF and PSD-95 expression in both AMG and HIP at 24 h after administration. These effects could be the compensatory consequence of increased glutamate release induced by high-dose ketamine treatment administered to rats 24 h earlier. Higher doses of ketamine in the brain exert a negative effect in potentiating oxidative stress[53].

The effects of ketamine on cognitive function vary. Obtaining information is important in the cognitive process of learning and memory[54]. Endocytosis of the AMPA receptor was potentially necessary for memory remodeling after retrieval[55], while NMDAR activation was controlled by AMPA receptor endocytosis during hippocampal long-term depression(LTD)[56]. When PSD-95 is recruited into the synapse, NMDA receptors may migrate and aggregate, affecting the morphology, function, plasticity of synapses, and endocytosis of AMPA receptors[57]. Moreover, the BDNF and PSD-95 results showed a positive correlation in expression levels in the HIP and AMG, indicating that ketamine upregulated the PSD-95 protein and triggered the synaptic delivery of PSD-95 by promoting BDNF signaling to alleviate the PTSD-like effects.

However, the cause of these changes and the mechanism of cytokine between the HIP and AMG have yet to be clarified. Future studies should examine (i) whether the BDNF- and HIP-dependency of extinction is related only to the context specificity of the extinction memory as suggested by the data described earlier or (ii) whether HIP neural plasticity exerts a more general effect on fear inhibition.

Overall, treatment with 10 and 15 mg/kg ketamine was used in upregulating the expression of BDNF and PSD-95 in HIP and AMG to alleviate PTSD-like behaviors in a rat model exposed to SPS&S. This finding unravels a new role for BDNF-related PSD-95 signaling in the treatment of PTSD. Ketamine might be a useful alternative for treating fear memory and spatial cognition deficits in PTSD, further demonstrating the existence of a yet unexplored window of opportunity for the treatment of PTSD.

Animals

A total of 56 outbred adult male Sprague–Dawley rats (aged 8 weeks, weighing 275–300 g from the animal center of Weifang Medical University) were used for this experiment. All rats were housed in a limited-access rodent facility with 4 rats per polycarbonate cage in a room with a 12 h light/12 h dark cycle (lights on from 7:00 a.m. to 7:00 p.m.). All behavioral experiments were performed between 9:00 a.m. and 2:00 p.m. during the light cycle (see Fig. 1a for the treatment timeline). Ad libitum access to sterilized drinking water and standard food was provided. The rats were habituated to these conditions for 7 d. Procedures involving animal use were conducted in accordance with the National Institutes of Health Guidelines (Use of Laboratory Animals) and approved by the Weifang Medical University Animal Care and Use Committees.

Single prolonged stress and electric foot shock

The SPS&S procedure was conducted as previously described [12]. The rats in the experimental group (n = 48) were restrained for 2 h by placing them inside a polyethylene cone barrel with only the tails protruding. After immobilization, they were individually placed in a clear acrylic cylinder (diameter, 240 mm; height, 500 mm), filled two-thirds from the bottom with water (22°C–24°C), and forced to swim for 20 min. After recuperating for 15 min, the rats were exposed to diethyl ether until loss of consciousness. The unconscious rats were placed in a warm place until recovery, and four foot shocks (4 s, 0.8 mA) were delivered in a conditioning chamber (325 mm × 280 mm × 500 mm) by a shock generator-scrambler (PSS-1: Jiliang, Shanghai, China) through a stainless-steel grid floor. Two foot shocks were delivered with a intertrial interval of 30 s. Subsequently, the rats remained in the chamber for an additional period of 10 min before they were returned to their home cages. After their exposure to SPS&S stressors, the rats were left undisturbed in their home cages for 14 d. By contrast, none of these procedures were conducted on the control group (n = 8).

Testing the validity of the PTSD model

After the PTSD model was established, behavioral testing was conducted to evaluate replicability. Eight rats were randomly selected from 48 stressed rats and then tested for PTSD-like symptoms. The method conformed to the principle of placing a less stressful test in front to reduce the impact on subsequent tests. The following behavioral tests were conducted in the following sequence: (1) freezing behavior test and (2) water maze test. Biochemical assays were performed, including the plasma cortisol level test and changes in the expression levels of BDNF and PSD-95 proteins in the HIP and AMG.

Experimental design and pharmacological treatment

After the evaluation of the PTSD model, the other SPS&S-induced rats were randomly assigned into one of the five groups: the PTSD-sal group(n = 8)(saline via intraperitoneal (IP) injection, n = 8); PTSD-ket 5 group (5 mg/kg ketamine via IP injection, n = 8); PTSD-ket 10 group (10 mg/kg ketamine via IP injection, n = 8); PTSD-ket-15 group (15 mg/kg ketamine via IP injection, n = 8); and PTSD-ket 20 group ( 20 mg/kg ketamine via IP injection, n = 8). Ketamine hydrochloride (KH141202: Hengrui, Jiangsu, China) was prepared in 10% saline, and all injections were administered IP in volumes of 10 mL/kg of body weight.

Behavioral tests

The behavioral tests were conducted 24 h after a single injection of ketamine or saline. The operators performed minimal movements and made no noise during the procedure and collection of behavioral data. The Smart-3.0 video tracking system (Panlab SL, Barcelona, Spain) was used to evaluate all observations and record data for ethology. Experimenters were blind to the experimental group during the experiment. All animals were tested in the same order and in accordance with the principle of placing a less stressful test in front to reduce the effect on subsequent tests: (1) the freezing behavior test and 2) the water maze test.

Freezing behavior test

The freezing response upon re-exposure to the traumatic context was used as the measure of conditioned associative fear memory as a symptom of PTSD[30]. Each SPS&S-induced rat was placed in the conditioning chamber where the rat received footshocks during SPS&S and was allowed to explore the chamber for 180 s. The rats were then exposed to a neutral tone (80 dB, 9 kHz) for 3 min. They then remained in the chamber for an additional period of 1 min before they were returned to their home cages. Behavior was monitored via overhead cameras and freezing time was observed manually by experimenters who were blinded to conditions. Freezing is defined as the absence of any observable active movement of any part of the body (except for those related to respiration), and all other behaviors were assessed as active[31]. The chamber was cleaned with ethanol after each animal.

Morris water maze test

Spatial learning and memory were assessed using the Morris water maze test (WMT) and across repeated trials (place navigation test) as described previously[32]. Preference for the platform region in the absence of the platform (the spatial probe test) determines the reference memory[33]. All groups of rats were trained for 5 d, with random variations in the start position for each trial. The platform was hidden 1 cm below the water surface at the center of one quadrant of the pool. The rats were guided to the platform unless they were unable to locate the position in 90 s. Once the platform was reached, the rat was left for 15 s on the platform. The rats were dried with a soft towel after each trial and placed into a heating plate to keep them warm and then underwent three consecutive trials each day with an intertrial interval of 20 min to provide space between acquisition trials and prevent the effects of hypothermia. Escape latency time and the number of platform crossings were recorded. The experimental flow is shown in Fig. 1A.

Biochemical tests

Radioimmunoassay

Blood samples drawn via the tail vein were used for plasma corticosterone (CORT) analyses were collected on Day 2 post-SPS&S, approximately 2 h after the onset of the dark cycle. Blood samples were collected in chilled tubes lined with a heparin solution. The tubes were then centrifuged at 12000 r/min for 10 min. Plasma was collected and then stored at -80°C until analysis via radioimmunoassay. Plasma corticosterone levels were assessed by spectrofluorometry. The fluorescence was assessed at the emission wavelength of 525 nm on a spectrofluorometer.

Western blot assay

Western blot assay was conducted as previously described[34]. The rats were sacrificed, and the brains were removed and immediately placed in an iced dish. The HIP and AMG were dissected in accordance with the atlas of brain anatomy. The amygdala was extracted by placing the rat brain dorsal side up on a cryo-operating table, It was then gently removed using forceps because of its almond-like shape and its location in the dorsal medial part of the anterior temporal lobe; slightly in front of the hippocampus and the inferior horn of the lateral ventricle. Fresh tissue samples of rats were homogenized with a sample buffer, denatured by boiling for 3 min, loaded on a 10% SDS–polyacrylamide gel, and electroblotted onto a PVDF membrane (Millipore Corp. Bedford, MA) from the gel by using a wet transfer membrane apparatus (Bio-Rad Laboratories, Inc, Hercules, CA). The blotted membrane was then blocked with 5% skim milk for 2 h and then incubated overnight (18 h) at 4°C with primary antibodies, including mouse monoclonal antibodies against actin (1:500, ab8244, Abcam), rabbit polyclonal antibody against BDNF (1:2000, ab108319, Abcam), and rabbit polyclonal antibody against PSD-95 (1:500, ab18258, Abcam) at 4°C for 24 h. Blots were washed three times with TBST and then incubated with a second antibody (1:5000, anti-mouse, or anti-rabbit IgG-HRP; Abcam) for 2 h at room temperature. After incubation, blots were washed three times with TBST before visualization by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). The protein levels of β-actin, BDNF, and PSD-95 were determined by calculating the optical density (OD) ratio. The OD results were analyzed on the Image J analysis system.

Immunohistochemistry

An immunohistochemistry assay was performed as described in previous reports [35]. Under 8% chloral hydrate (400 mg/kg, IP) anesthesia, the rats were transcranially perfused with 200 mL of normal saline, followed by 200–300 mL of 4% paraformaldehyde in 0.1M phosphate buffer. The brains were removed and post-fixed with 4% paraformaldehyde for 6 h. They were then immersed in 30% sucrose in 0.1M PBS until they sank. Cryotome sections were washed 3 times (5 min each) with 0.01 M PBS and then treated with 2% BSA in PBS for 2 h at room temperature for blocking nonspecific reactions. The sections were treated with mouse monoclonal BDNF and a PSD-95 antibody (diluted to 1:200, Abcam, USA) in a PBS solution for 24 h at 4°C. The sections were washed 3 times with PBS and then incubated using an IHC detection reagent (PV6001, Company of Zhongshan Golden Bridge, Beijing, China) at 37°C for 30 min. A brown color appeared in the slices after 3, 3-diaminobenzidine colorization. Slices were then dehydrated and mounted with neutral gum. The sections were observed using the Olympus DP80 microscope system (Olympus Corporation, Japan), and five slides were randomly selected from each rat. For each slide, 5 randomly selected visual fields of the brains were chosen (×20/400 magnification). We recorded the number of positive cells in each field to evaluate the rate of expression of the target protein. The number of immune-positive cells was analyzed using the Image J analysis system.

Statistical analysis

Pass 11 (NCSS, USA) was used to estimate the sample size. In accordance with the preliminary experimental results, the pilot experiment was conducted using SPSS version 22.0 (SPSS, Inc. Chicago, IL, USA). Data were presented as mean ± standard deviation. The normality and variance of data distribution between the two groups were analyzed using the Kolmogorov–Smirnov test and Levene’s test, respectively. The data between the control and SPS&S groups were analyzed using Student’s t-test. Repeated measures of two-way ANOVA followed by posthoc Bonferroni multiple comparison tests were used to determine the WMT parameters. Biochemical data analyses were performed using one-way ANOVA, followed by LSD comparison posthoc tests. A single comparison between the two groups was using Student’s t-test. Pearson’s correlation coefficient r was calculated to determine the specific relationship between the expression levels of BDNF and PSD-95 proteins in the HIP and AMG. The relationship between protein levels and different behavioral tests was determined by linear regression analysis. p < 0.05 was considered statistically significant.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki and approved by the Weifang Medical University Animal Care and Use Committees.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

The authors affirm that all study participants gave informed consent.

Data Availability

Not applicable.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

This work was supported by the National Natural Science Foundation of China (NSFC) (82101588; 82071517; 31771217); Surface project of Natural Science Foundation of Shandong Province (ZR2020MC218); Institute of Psychology, Chinese Academy of Sciences(GJ202002); Medical Education Research project of Chinese Medical Association(2020A-N12063 ); The Education and Teaching Reform Project of the Psychology and Education Reference Committee of the Ministry of Education(20221013); Science and Technology Development Program of Traditional Chinese Medicine in Shandong Province(202002010572); Science and Technology Development Program in Weifang City(2021YX042).

Author Contributors

All authors contributed to the study conception and design. Designed the study, Writing-Original Draft and data analysis by [JiaYiao Niu] and [Yue Teng]; Investigation by [Yang Liu] and [Han Wang]; Writing-Review & Editing by [JinHong Chen]; Visualization by [YuJia Kong]; Funding acquisition by [Ling Wang], [Bo Lian] and [HongWei Sun]; Writing-Review & Editing by [WeiWen Wang]; Project administration, Funding acquisition and Writing-Review & Editing by [KuiTao Yue] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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No competing interests reported.

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Ketamine alleviates fear memory and spatial cognition deficits in a PTSD rat model via the BDNF signaling pathway of the hippocampus and amygdala

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Abstract

Background

Methods

Results

Conclusion

Figures

Highlights

Introduction

Results

SPS&S model effects

Effects of ketamine on SPS&S-induced fear memory and spatial cognition behaviors

Effects of ketamine on protein expression of BDNF and PSD-95 under SPS&S-induced treatment

Effects of ketamine on positive cell expression of BDNF and PSD-95 under SPS&S-induced treatment

Discussion

Conclusion

Materials and Methods

Animals

Single prolonged stress and electric foot shock

Testing the validity of the PTSD model

Experimental design and pharmacological treatment

Behavioral tests

Freezing behavior test

Morris water maze test

Biochemical tests

Radioimmunoassay

Western blot assay

Immunohistochemistry

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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