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Capsaicin Attenuates Amyloid-β-Induced Synapse Loss and Cognitive Impairments in Mice

Abstract

Alzheimer’s disease (AD) is the most common cause of progressive cognitive impairment in the aged. The aggregation of the amyloid β-protein (Aβ) is a hallmark of AD and is linked to synapse loss and cognitive impairment. Capsaicin, a specific agonist of the transient receptor potential vanilloid 1 (TRPV1), has been proven to ameliorate stress-induced AD-like pathological and cognitive impairments, but it is unclear whether TRPV1 activation can affect cognitive and synaptic functions in Aβ-induced mouse model of AD. In this study, we investigated the effects of TRPV1 activation on spatial memory and synaptic plasticity in mice treated with Aβ. To induce AD-like pathological and cognitive impairments, adult C57Bl/6 mice were microinjected with Aβ42 (100 μM, 2.5 μl/mouse, i.c.v.). Two weeks after Aβ42 microinjection, spatial learning and memory as well as hippocampal long-term potentiation (LTP) were examined. The results showed that Aβ42 microinjection significantly impaired spatial learning and memory in the Morris water maze and novel object recognition tests compared with controls. These behavioral changes were accompanied by synapse loss and impaired LTP in the CA1 area of hippocampus. More importantly, daily capsaicin (1 mg/kg, i.p.) treatment throughout the experiment dramatically improved spatial learning and memory and synaptic function, as reflected by enhanced hippocampal LTP and reduced synapse loss, whereas the TRPV1 antagonist capsazepine (1 mg/kg, i.p.) treatment had no effects on cognitive and synaptic function in Aβ42-treated mice. These results indicate that TRPV1 activation by capsaicin rescues cognitive deficit in the Aβ42-induced mouse model of AD both structurely and functionally.

INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurologic disease of the brain, which is characterized by progressive memory loss and other cognitive dysfunctions [1]. Currently there is no effective treatment for AD, but the pursuit of new and effective therapeutics is the objective of intense investigation. Synapse loss in the brain is a major pathological correlate of memory decline in early period of AD [2–5]. To date, despite the fact that all of the therapeutic trials for AD focused on Aβ failed, a growing body of evidence from laboratories and clinics worldwide support the concept that an imbalance between production and clearance of Aβ and related Aβ peptides is a very early, often initiating factor in AD [6–9]. In the early period, amyloid-β protein precursor (AβPP) incision dysfunction and increased Aβ are observed in the AD brain [10], among which Aβ40 and Aβ42 are the most physiologically relevant. Aβ40 is the most abundant, but Aβ42 is more neurotoxicity to form the core of Aβ plaque deposition prior to aggregation [11]. Soluble Aβ oligomers are considered to play a more important role in the early synapse loss and cognitive impairment than fibrous status observed in AD. Thus, Aβ42 injection has been reported to serve as an animal model of AD in the laboratory condition [12–14].

The transient receptor potential vanilloid 1 (TRPV1) channel, as a calcium-permeable cation channel, is expressed in different part of brain regions in both human and animals [15]. Although the TRPV1 channel is well recognized as a signal detector activated by painful stimuli [16, 17], several studies have found that the expression of TRPV1 significantly increased in the brain of patients with AD [18, 19], and a more recent study shows that a capsaicin-rich diet is able to exert favorable effects on AD blood biomarkers and cognitive function in middle-aged and elderly adults [20]. It has been well documented that TRPV1 activation is involved in certain types of synaptic plasticity, such as long-term potentiation (LTP) and term depression (LTD) [21–27]. Further genetic studies have shown that TRPV1 knockout mice displayed a dramatic reduction in hippocampal CA1 LTP compared to wild type mice [28]. It is well known that activity-dependent synaptic plasticity in the hippocampus is widely believed to be the cellular and molecular mechanism underlying certain types of learning and memory[29, 30], and hippocampal LTP is dramatically impaired, whereas LTD is significantly facilitated in animal models of AD [31–33] and AD patients [34]. Our recent study suggests that preventing LTP decay and converting it into nondecaying LTP prolongs memory retention in normal animals and reduced memory loss in the mouse model of AD [35]. However, it remains unknown whether the activation of TRPV1 by capsaicin could modulate hippocampal LTP and improve learning and memory in the Aβ42-induced mouse model of AD.

In this study, we used i.c.v. microinjection of Aβ42 to mimic the progress of AD and investigated whether the regulation of TRPV1 activation using apharmacological could affect the synaptic function, and then rescue the impairment of learning and memory in mice.

MATERIAL AND METHODS

Animals

Male C57Bl/6 mice (7-8 weeks, 22–25 g body weight at the beginning of the experiment) were obtained from Charles River (Beijing Office, China) and maintained at the Children’s Hospital of Chongqing Medical University Animal Care Centre. Mice were housed under 12 h light and 12 h dark cycle (lights on from 7:00 a.m. to 7:00 p.m.) with free access to food and water in a temperature and humidity controlled room. All procedures were performed in accordance with Chongqing Science and Technology Commission guidelines for animal research and approved by the Chongqing Medical University Animal Care Committee, and every effort was made to minimize both the animal suffering and the number of animals used.

Drugs preparation and injection

All drugs including Aβ42 peptide, hexafluoroisopropanol (HFIP), dimethyl sulfoxide (DMSO), capsaicin (Cap), and capsazepine (Cpz) were obtained from Sigma Chemicals (St. Louis, MO). Aβ42 peptide was incubated as previously described [12]. Briefly, the peptides were dissolved in HFIP, and then HFIP was removed by evaporation with traces removed under vacuum. The Aβ peptide was then dissolved in DMSO to 5 mM, which was then added to phosphate buffer saline (PBS) to a final concentration of 100 μM. This solution was incubated at 4°C for 48 h to enhance oligomer formation. Cap and Cpz were dissolved in a 1:1:8 mixture of Tween80: ethanol: saline as previously described [36].

Mice were deeply anesthetized with sodium pentobarbital at a dose of 60 mg/kg (i.p.) within its home cage. Scalp skin was shaved with clippers and disinfected using iodine before the mouse was mounted on a stereotaxic instrument (Stoelting Co., USA). The core temperature was monitored and kept at 36.5±0.5°C. After opening the scalp skin and exposing the skull, the incubated Aβ42 peptides (100 μM, 2.5 μl/mouse) or isopyknic PBS were injected in left lateral ventricle (brain coordinates are expressed as mm from bregma: 0.6 posterior, 1.1 lateral, and 2 ventral) with a 10 μl syringe at a rate of 1 μl/min, handled by a microsyringe pump (Harvard Apparatus, Holliston, MA) as described previously [37]. Then mice were randomly divided into four groups: PBS+vehicle (Ctl), Aβ+vehicle (Aβ), Aβ+Cap, and Aβ+Cpz. For the Ctl and Aβ group, animals received vehicle (5 ml/kg, i.p.) treatment, which is a 1:1:8 mixture of Tween 80: ethanol: saline. Mice in Aβ+Cap and Aβ+Cpz group were subjected to capsaicin (1 mg/kg, i.p.) and capsazepine (1 mg/kg, i.p.) treatment for two weeks before behavioral tests(Fig. 1).

Fig.1

Experimental design. Mice received an Aβ42 treatment (100 μM, 2.5 μl/mouse, i.c.v.) or the same volume of vehicle on day 1, and capsaicin (1 mg/kg, i.p.) or capsazepine (1 mg/kg, i.p.) was administrated daily throughout the experiment. Two weeks after Aβ42 treatment, behavioral tests were performed, and brain tissues were collected immediately after behavioral tests.

Experimental design. Mice received an Aβ42 treatment (100 μM, 2.5 μl/mouse, i.c.v.) or the same volume of vehicle on day 1, and capsaicin (1 mg/kg, i.p.) or capsazepine (1 mg/kg, i.p.) was administrated daily throughout the experiment. Two weeks after Aβ42 treatment, behavioral tests were performed, and brain tissues were collected immediately after behavioral tests.

Morris water maze test

As previously described [35], the animals completed the Morris water maze test in a circular stainless steel pool (150 cm in diameter) filled with water (25±1°C) made opaque with nontoxic white paint. The pool was surrounded by light blue curtains, and 3 distal visual cues were fixed to the curtains. A CCD camera suspended above the pool center recorded the swim paths of the animals, and video output was digitized by an Any-maze tracking system (Stoelting). The pool was divided into four equally quadrants: NE, NW, SW, and SE. The water maze test includes spatial training and a probe test. Twenty-four hours before spatial training, the animals were allowed to adapt to the maze for a 120-s free swim. The animals were then trained in the spatial learning task for 4 trials per day for 5 consecutive days. In each trial, mice were placed into water from 4 starting positions (NE, NW, SW, and SE), facing the pool wall. They were then required to swim to find the hidden platform (13 cm in diameter, located in the SW quadrant), which was submerged 1 cm under water. During each trial, mice were allowed to swim until they found the hidden platform where they remained for 20 s before being returned to a holding cage. Mice that failed to find the hidden platform in 120 s were guided to the platform where they remained for 20 s. Twenty-four hours after the final training trial, a probe test was conducted. Mice were returned to the pool from a novel drop point with the hidden platform absent for 120 s, and their swim path wasrecorded.

Novel object recognition test

The novel object recognition test was performed as described previously with some modifications [38]. Briefly, mice were placed into the 40×40 cm2 open box for 5 min adaption before the test. On the next day, mice were placed in the box to explore two identical objects for 5 min, and then returned to their home cage. To evaluate long-term memory, mice were returned to the box at the same time the next day. In the box, they were exposed to two different objects, one identical to the one previously encountered and the other a novel object. The animals were allowed to explore both objects for 5 min. After the end of each test, the box and objects were cleaned with 70% ethanol. Extraordinarily, the object should not be too large or small to reject the animal standing on or turn over the objects. The two objects were fixed to the bottom and positions of each object were counterbalanced between the animals, at least 10 cm away from the wall of box. A mouse was considered to explore the objects when it was looking toward the object with its nose within 2 cm of the object. The number and time each animal spent actively exploring the objects was manually scored and the recognition index (RI) was calculated. The RI was calculated by using the equation: RI = number or time spent on novel object / total number or time spent on both objects×100%.

Western immunoblotting

After behavioral tests, half of the mice were euthanized with an overdose of urethane (3 g/kg, i.p.). The hippocampus from each mouse was collected and stored at –80°C tissue bank for preparation of synaptosomal marker detection. The western immunoblotting was performed as in our previous works [35, 39]. The brain tissues were grated and lysed on ice in the lysis buffer containing a cocktail protease inhibitors (Complete, Roche), and then the solution was centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was collected, and protein concentration was determined by BCA protein assay kit (Pierce). Equal amount of protein samples (30 μg) was mixed with 4×sample buffer, boiled at 95°C for 5 min. The samples were then separated on 10% SDS-PAGE gels and transferred onto polyvinylidenedifluoride membranes (Bio-Rad). To block nonspecific background, the membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then incubated overnight at 4°C with primary antibody. After washing 5 min for 3 times in TBST, membranes were incubated with corresponding secondary antibody for 1 h at room temperature. After another washing 5 min for 3 times with TBST, protein was developed in the Bio-Rad Imager using ECL Western blotting substrate (Pierce). Immunoblotting with anti-β-actin was used to control equal loading and protein quality. The band intensity of each protein was quantified by the Bio-Rad Quantity One software. Anti-synaptophysin antibody (1:1000) and anti-β-actin antibody (1:3000) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-PSD95 antibody (1:500) was obtained from Abcam. Anti-TRPV1 antibody (1:1000) was obtained from Alomone lab.

Transmission electron microscopy (TEM)

For TEM detection, 3 mice from each group were sacrificed with an overdose of urethane (3 g/kg, i.p.), and then transcardially perfused with 2.5% glutaraldehyde in 0.01 M PBS (pH 7.4). The brain was rapidly stripped on ice and 1 mm tissue sample from the hippocampal CA1 area of each mouse was cut out. The tissue blocks were then fixed in 2.5% glutaraldehyde for more than one day. Then samples were embedded with Epon812 epoxy resin. Tissue blocks were cut into 1-μm-thick thin sections. After uranyl acetate/lead citrate double-staining, neurons and ultrastructures were observed with a Philips EM208S transmission electron microscope (Philips, Amsterdam, Netherlands). Synapses were identified by the existence of at least 3 vesicles in the presence of a PSD and the presynaptic bouton. Using randomly obtained synapse images, the number of synapse, PSD thickness, and synaptic cleft width were measured using the Image-Pro Plus 6.0 image analysis system, following the approach used before [40, 41]. All measures were conducted by a double-blinded way.

Electrophysiology

Mice from the behavioral test were deeply anesthetized using urethane (1.5 g/kg, i.p.) and transcardially perfused with N-methyl-D-glucamine (NMDG) artificial cerebral spinal fluid (ACSF) (in mM: NaCl 124, KCl 2.8, NaH2PO4.H2O 1.25, CaCl2 2.0, MgSO4 1.2, Na-vitamin C 0.4, NaHCO3 26, Na-lactate 2.0, Na-pyruvate 2.0 and D-glucose 10.0, pH = 7.4) prior to decapitation as described previously [42]. Next, acute coronal hippocampal slices were sectioned (400 μm thick) with a vibratome (VT1000 S, Leica Microsystems) in ice-cold NMDG ACSF bubbled with 95% O2 and 5% CO2. The slices were then incubated in oxygenated HEPES ACSF for 1 h at 30°C. Subsequently, the slices were gently transferred into a recording chamber filled with normal ACSF. The field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the Schaffer collateral/commissural pathways were recorded in the hippocampus using pipettes (1–2MΩ) filled with ACSF. Test fEPSPs were evoked at a frequency of 0.033 Hz and at a stimulus intensity that was adjusted to approximately 50% of the intensity that elicited the maximal response. After a 30-min stable baseline, theta burst stimulation (TBS) was given to induce LTP. TBS consisted of 2 trains of stimuli (at 20 s interval), with each train composed of 5 bursts (4 pulses at 100 Hz in each burst) at an inter-burst interval of 200 ms. Data acquisition (filtered at 3 kHz and digitized at 10 kHz) was performed with the PatchMaster v2.73 software (HEKA Electronic, Lambrecht/Pfalz, Germany).

Statistical analyses

For the behavioral experiments, all the data are presented as the mean±SEM. The spatial training of water maze data was analyzed with a two-way between/within-subjects factorial ANOVA with drug treatment as the between-subjects factor and session (day) as the within-subjects factor. All significant main effects and interactions were further analyzed by using Turkey’s comparisons. The probe test data and novel object recognition test were analyzed with a one-way between-subjects factorial ANOVA with drug treatment as the between-subjects factor. The significance level was set at p < 0.05.

For the western immunoblotting and TEM detection, the data are presented as the mean±SEM, analyzed with one-way ANOVAs followed by post hoc Turkey’s tests where appropriate with drugtreatment as the between-subjects factor. The significance level was set at p < 0.05.

For the electrophysiological experiments, all the data are expressed as the average percent change from baseline±SEM, and the data were analyzed with one-way ANOVAs followed by post hoc Turkey’s tests where appropriate with drug treatment as the between-subjects factor. The significance level was set at p < 0.05.

RESULTS

Capsaicin rescues memory decline in Aβ42-induced mouse model of AD

We first examined the effects of activation of TRPV1 on spatial learning and memory in an Aβ42-induced mouse model of AD by using the Morris water maze task. The results showed that there was no significant difference on escape latency during the first spatial training day. However, the escape latency in the Aβ42-treated group (Aβ, n = 17) was much longer than the control group (Ctl, n = 20) from day 2 to 5 (day 2:62.3±7.7 s for Ctl, 94.5±3.8 s for Aβ, p < 0.01 versus Ctl; day 3:52.3±7.8 s for Ctl, 90.0±6.3 s for Aβ, p < 0.01 versus Ctl; day 4:35.8±5.5 s for Ctl, 82.2±7.1 s for Aβ, p < 0.01 versus Ctl; day 5:24.7±3.5 s for Ctl, 70.3±6.3 s for Aβ, p < 0.01 versus Ctl; Fig. 2B). Capsaicin (Aβ+Cap, n = 17) treatment significantly shortened escape latency for searching for the hidden platform on training day 4 (57.6±6.2 s, p < 0.05 versus Ctl, p < 0.05 versus Aβ; Fig. 2B) and day 5 (48.7±5.7 s, p < 0.05 versus Ctl, p < 0.05 versus Aβ; Fig. 2B) compared to those of the Aβ group, whereas capsazepine (Aβ+Cpz, n = 19) treatment displayed no difference with the Aβ group. Twenty-four hours after the last training trial, a probe test with the platform removed was performed to examine long-term spatial memory retrieval. The results revealed that spatial memory retrieval dramatically impaired in mice-treated with Aβ42 since they spent much less time in the targetquadrant in which the platform was previously located (Ctl: 54.1±3.6 s; Aβ: 25.2±5.7 s, p < 0.01 versus Ctl; Fig. 2C) and reduced the number of entries into the platform zone (Ctl: 5.2±0.8; Aβ: 0.9±0.3, p < 0.01 versus Ctl; Fig. 2D). As expected, capsaicin treatment significantly increased the time spend in target quadrant (Aβ+Cap: 44.2±5.7 s, p > 0.05 versus Ctl, p < 0.05 versus Aβ; Fig. 2C) and the number of entries into the platform zone (3.8±0.8; p > 0.05 versus Ctl, p < 0.05 versus Aβ; Fig. 2D) compared with the Aβ group, whereas capsazepine treatment displayed no difference with the Aβ group.

Fig.2

Capsaicin rescues the Aβ42-induced deficit of spatial learning and memory in the Morris water maze test. The spatial training was carried out for 5 days (4 trials per day), and a probe test was performed on day 6. A) Representative track plot in training day 5 and probe test. B) The average escape latency to the hidden platform location is plotted for each spatial learning day. Two-way ANOVA: F (12,345)  = 1.994, p < 0.05. C, D) Bar graphs show the time spent in the hidden platform-located quadrant (C) and the number of entries into the island zone (D) during the probe test with absence of the hidden platform, which is conducted 24 h after the last learning trial. One-way ANOVA: F (3,69)  = 10.472, p < 0.001 for the time spent in the hidden platform-located quadrant and F (3,69)  = 10.218, p < 0.001 for the number of entries into the island zone. Values are expressed as mean±SEM (n = 17–20), **p < 0.01, *p < 0.05.

Capsaicin rescues the Aβ42-induced deficit of spatial learning and memory in the Morris water maze test. The spatial training was carried out for 5 days (4 trials per day), and a probe test was performed on day 6. A) Representative track plot in training day 5 and probe test. B) The average escape latency to the hidden platform location is plotted for each spatial learning day. Two-way ANOVA: F (12,345)  = 1.994, p < 0.05. C, D) Bar graphs show the time spent in the hidden platform-located quadrant (C) and the number of entries into the island zone (D) during the probe test with absence of the hidden platform, which is conducted 24 h after the last learning trial. One-way ANOVA: F (3,69)  = 10.472, p < 0.001 for the time spent in the hidden platform-located quadrant and F (3,69)  = 10.218, p < 0.001 for the number of entries into the island zone. Values are expressed as mean±SEM (n = 17–20), **p < 0.01, *p < 0.05.

To further evaluate the effects of activation of TRPV1 on learning and memory in an Aβ42-induced mouse model of AD, we introduced another behavior test, the novel object recognition task. The novel object recognition test is based on the spontaneous tendency of rodents toward novelty. Specifically, when they are first exposed to two identical objects, rodents will explore both of the objects. Next time when they are exposed to two disparate object (one familiar and one novel object), they explore the novel object for a longer time than the familiar object. In the novel object recognition test, mice from the Aβ group failed to distinguish familiar and novel objects, as reflected by similar exploration number (Ctl: n = 17, RI = 59.4±2.3 %; Aβ: n = 16, 46.4±2.8%, p < 0.01 versus Ctl; Fig. 3A) and time (Ctl: RI = 63.1±2.63 %; Aβ: 48.3±2.7 %, p < 0.01 versus Ctl; Fig. 3B) of the novel object, compared with Ctl group. Similar to water maze testing, capsaicin treatment (Aβ+Cap, n = 16) significantly increased the number (RI = 61.8±3.1 %, p > 0.05 versus Ctl, p < 0.01 versus Aβ; Fig. 3A) and time (RI = 63.2±4.0 %, p > 0.05 versus Ctl, p  <  0.01 versus Aβ; Fig. 3B) to explore the novel object, whereas capsazepine treatment (Aβ+Cpz, n = 15) had no influence on novel object recognition compared with Aβ group.

Fig.3

Capsaicin rescues the Aβ42-induced deficit of memory in the novel object recognition test. The novel object recognition test was performed 24 h later after training. Both number (A) and time (B) of recognition index (RI) were calculated. One-way ANOVA: F (3,69)  = 6.099, p < 0.01 for the number RI and F (3,69)  = 4.286, p < 0.05 for the Time RI. Values are expressed as mean±SEM (n = 15–17), **p < 0.01, *p < 0.05.

Capsaicin rescues the Aβ42-induced deficit of memory in the novel object recognition test. The novel object recognition test was performed 24 h later after training. Both number (A) and time (B) of recognition index (RI) were calculated. One-way ANOVA: F (3,69)  = 6.099, p < 0.01 for the number RI and F (3,69)  = 4.286, p < 0.05 for the Time RI. Values are expressed as mean±SEM (n = 15–17), **p < 0.01, *p < 0.05.

Taken together, these findings indicated an impaired learning and memory ability after Aβ42 i.c.v microinjection. Administration of capsaicin, but not capsazepine, significantly rescued the memory decline in the Aβ42-induced mouse model of AD.

Capsaicin rescues synaptic loss in the Aβ42-induced mouse model of AD

Decreases in the density of dendritic spines and alterations in their morphology, which are related to learning and memory decline, occur in AD patient [2, 43–45] and in animals models of AD [46, 47].Thus, we next wanted to determine the effects of TRPV1 activation on synapse changes. We first detected the expression of TRPV1 level and found that there was no difference among these group (Fig. 4A, B). However, the postsynaptic marker PSD95 significantly decreased in mice treated with Aβ42 (Aβ: 76.3±5.2 % relative to Ctl, p < 0.01 versus Ctl; Fig. 4A, C) compared to the Ctl group. More importantly, capsaicin treatment restored the expression level of PSD95 (Aβ+Cap: 94.4±3.9 % relative to Ctl, p > 0.05 versus Ctl, p < 0.05 versus Aβ; Fig. 4A, C), whereas capsazepine treatment had no effect on the PSD95 expression (Aβ+Cpz: 81.8±5.8% relative to Ctl, p < 0.05 versus Ctl, p > 0.05 versus Aβ; Fig. 4A, C). Notably, the pre-synaptic marker synaptophysin remained unchanged among all groups (Fig. 4A, D).

Fig.4

Capsaicin rescues the Aβ42-induced decrease in PSD95 expression in the hippocampus. A) Sequential immunoblotting of total tissue lysates of hippocampal tissues collected from animals after behavioral tests. The relative protein levels of TRPV1 (B), PSD95 (C), and synaptophysin (D) are normalized by the control (Ctl). One-way ANOVA: F (3,16)  = 0.062, p > 0.05 for TRPV1; F (3,24)  = 6.331, p < 0.01 for PSD95; F (3,12)  = 1.076, p > 0.05 for synaptophysin. Values are expressed as mean±SEM (n = 7), **p < 0.01, *p < 0.05.

Capsaicin rescues the Aβ42-induced decrease in PSD95 expression in the hippocampus. A) Sequential immunoblotting of total tissue lysates of hippocampal tissues collected from animals after behavioral tests. The relative protein levels of TRPV1 (B), PSD95 (C), and synaptophysin (D) are normalized by the control (Ctl). One-way ANOVA: F (3,16)  = 0.062, p > 0.05 for TRPV1; F (3,24)  = 6.331, p < 0.01 for PSD95; F (3,12)  = 1.076, p > 0.05 for synaptophysin. Values are expressed as mean±SEM (n = 7), **p < 0.01, *p < 0.05.

Further TEM analysis showed that the total number of synapses in the CA1 of hippocampus dramatically reduced in the Aβ group (n = 30, 8.7±0.6, p < 0.01 versus Ctl; Fig. 5B, E) compared with the Ctl group (n = 30, 13.7±0.3; Fig. 5A, E). Moreover, the thickness of PSD also decreased in Aβ mice brain (Aβ: n = 30, 29.2±1.5 nm, p < 0.01 versus Ctl; Fig. 5B, F) compared with the Ctl group (Ctl: n = 30, 47.6±2.6 nm; Fig. 5A, F). Similar to western blotting assay, after treatment with capsaicin (synapse number: n = 30, 14.5±0.6, p > 0.05 versus Ctl, p < 0.01 versus Aβ; PSD thickness: 49.0±2.3 nm, p > 0.05 versus Ctl, p < 0.01 versus Aβ; Fig. 5C, E, F), but not capsazepine (synapse number: n = 30, 9.2±0.6, p < 0.01 versus Ctl, p > 0.05 versus Aβ; PSD thickness: 30.7±1.6 nm, p < 0.01 versus Ctl, p > 0.05 versus Aβ; Fig. 5C, E, F), the number of synapses and thickness of PSD markedly increased compared to the Aβ group. Notably, there was no significant difference in the width of the synaptic cleft among these groups (Fig. 5G).

Fig.5

Capsaicin rescues the Aβ42-induced synapse loss in the hippocampus. A-D) Electronmicrographs showing the ultrastructural features of synapses in the hippocampal CA1 area of mice. Scale bar: 1 μm. Bar graphs summarize the number of synapses (E), the thickness of postsynaptic density (F), and the width of the synaptic cleft (G) in the different groups. One-way ANOVA: F (3,116)  = 23.778, p < 0.001 for the number of synapses; F (3,116)  = 27.494, p < 0.001 for the thickness of postsynaptic density; F (3,116)  = 0.641, p > 0.05 for the width of the synaptic cleft. Values are expressed as mean±SEM (n = 30), **p < 0.01, *p < 0.05.

Capsaicin rescues the Aβ42-induced synapse loss in the hippocampus. A-D) Electronmicrographs showing the ultrastructural features of synapses in the hippocampal CA1 area of mice. Scale bar: 1 μm. Bar graphs summarize the number of synapses (E), the thickness of postsynaptic density (F), and the width of the synaptic cleft (G) in the different groups. One-way ANOVA: F (3,116)  = 23.778, p < 0.001 for the number of synapses; F (3,116)  = 27.494, p < 0.001 for the thickness of postsynaptic density; F (3,116)  = 0.641, p > 0.05 for the width of the synaptic cleft. Values are expressed as mean±SEM (n = 30), **p < 0.01, *p < 0.05.

These results indicate that Aβ treatment resulted in synapse loss and synaptic morphology change. Capsaicin, but not capsazepine, significantly rescued these abnormalities in the Aβ42-induced mouse model of AD.

Capsaicin rescues hippocampal CA1 LTP in the Aβ42-induced mouse model of AD

Because hippocampal LTP has been considered to be a cellular mechanism underlying learning and memory, we next wanted to detect the influence of TRPV1 activation on hippocampal LTP in the Aβ42-induced mouse model of AD. As shown in Fig. 5, TBS was able to reliably induce LTP in thecontrol slices (Ctl: n = 7, 172.3±9.4 % baseline, p < 0.01 versus baseline; Fig. 6). However, the LTP was significantly impaired in the slices from Aβ-treated mice (Aβ: n = 7, 122.6±6.9 % baseline, p < 0.01 versus baseline, p < 0.01 versus Ctl; Fig. 6). As expected, administration of capsaicin rescued Aβ-induced LTP impairment (Aβ+Cap: n = 8, 149.8±9.1 % baseline, p < 0.01 versus baseline, p > 0.05 versus Ctl, p < 0.05 versus Aβ; Fig. 6), whereas capsazepine had no effect on LTP induction (Aβ+Cpz: n = 9, 118.3±5.4 % baseline, p < 0.01 baseline, p < 0.01 versus Ctl, p > 0.05 versus Aβ, Fig. 6) compared with the Aβ group. These results indicate that Aβ treatment impaired hippocampal CA1 LTP, and capsaicin, but not capsazepine, significantly rescued the impairment.

Fig.6

Capsaicin rescues the Aβ42-induced impairment of LTP in the CA1 area of hippocampus. Representative fEPSP traces (A) and plots of the normalized slopes (B) of the fEPSP 5 min before and 55 min after TBS delivery. C) Bar graphs of the average percentage changes in the fEPSP slope 55–60 min after TBS delivery. One-way ANOVA: F (3,27)  = 12.002, p < 0.001. Data are expressed as means±SEM % baseline (n = 7–9 slices from 4-5 different mice), **p < 0.01, *p < 0.05.

Capsaicin rescues the Aβ42-induced impairment of LTP in the CA1 area of hippocampus. Representative fEPSP traces (A) and plots of the normalized slopes (B) of the fEPSP 5 min before and 55 min after TBS delivery. C) Bar graphs of the average percentage changes in the fEPSP slope 55–60 min after TBS delivery. One-way ANOVA: F (3,27)  = 12.002, p < 0.001. Data are expressed as means±SEM % baseline (n = 7–9 slices from 4-5 different mice), **p < 0.01, *p < 0.05.

DISCUSSION

In the present study, we confirm that i.c.v microinjection of Aβ42 induces a deficit of learning and memory in mice, and demonstrate that administration of TRPV1 agonist capsaicin, an active component of hot peppers, significantly ameliorates the learning and memory impaired by Aβ42. In addition, capsaicin treatment prevents synapse loss and improves LTP in the CA1 region of hippocampus. We have therefore provided evidence that activation of TRPV1 by capsaicin could reduce Aβ42-induced hippocampal synaptic abnormalities structurally and functionally, and subsequently improve cognitive functions in the Aβ42 mouse model of AD.

Challenged with aging population, tremendous efforts have been made to find an efficient way to treat AD. Previous studies have demonstrated that TRPV1 activation could prevent AD-like pathological and cognitive impairments in different animal models, including cold water stress [48], type 2 diabetes [49], and coapplication of streptozotocin and AlCl3 + D-galactose [50]. However, no direct evidence shows that TRPV1 agonist capsaicin can improve synaptic and cognitive functions in animals subjected to soluble Aβ oligomers treatment. Consistent with previous reports [12–14], we found here that direct Aβ42 treatment led to significant deficits in learning and memory in both the Morris water maze (Fig. 2) and novel object recognition (Fig. 3) tests, further suggesting that i.c.v. microinjection of Aβ42 is a useful mouse model of AD.

More importantly, we for the first time reported that capsaicin treatment improved the Aβ42-induced synapse loss in the hippocampus, as reflected by a significant increase in PSD95 expression (Fig. 4), synapse number, and postsynaptic density thickness (Fig. 5). It has been well documented that AD is primarily a disorder of synaptic failure [3]. Synapses in the hippocampus, a vital brain region for the encoding, consolidation, and retrieval phases of associative memory, begin to decline in patients with AD and mild cognitive impairment [2, 43–45] and in animals models of AD [46, 47]. In the early period of AD, there is a reduction of about 25% in the presynaptic vesicle protein synaptophysin [51]. With the development of the disease, synapses are disproportionately lost relative to neurons, which particularly affects the hippocampus [52], and this loss is the best correlate with dementia [4, 5, 53]. Thus, activation of TRPV1 by capsaicin reduces synapse loss in the hippocampus, and this morphological alteration may subsequently contribute to the improvements of learning and memory in Aβ42-induced mouse model of AD.

Beside structural plasticity, functional plasticity such as synaptic plasticity in the hippocampus is also known to be related to learning and memory. In particular, hippocampal CA1 LTP, an increase in the efficacy of synaptic transmission in excitatory synapses, is thought to be one of the basic mechanisms underlying certain types of learning and memory [29, 30]. Previous studies have reported that hippocampal LTP is dramatically impaired whereas LTD is significantly facilitated in animal models of AD [31–33] and AD patients [34]. More recent studies have further demonstrated that synthetic Aβ oligomers or oligomeric Aβ extracted from the cerebral cortex of AD patients can disrupt hippocampal LTP in vitro [33, 54, 55] and in vivo [56–58]. Consistent with these reports, we here found that the LTP was markedly inhibited in the hippocampal slices of mice treated with Aβ42 (Fig. 6). Many studies have shown that a brief application of capsaicin produced a prolonged Ca2 + influx via TRPV1 at the nerve terminals, which results in a prolonged increase in neurotransmitter release and LTP [21, 59, 60].Accordingly, we here reported that capsaicin treatment significantly improved the Aβ42-induced impairment of hippocampal CA1 LTP (Fig. 6), which may subsequently contribute to the improvements of learning and memory in an Aβ42-induced mouse model of AD. Nonetheless, contradictory results challenge these findings. For example, capsaicin treatment reduces the evoked excitatory postsynaptic currents in CA1 pyramidal cells [61] and LTP in the lateral amygdala [62] in a TRPV1-mediated manner. Thus, future experiments examining the exact cause of these discrepancies will help to be determine whether capsaicin’s therapeutic effect on AD can be attributed to the alteration of synaptic plasticity.

Alternatively, TRPV1 activation have been demonstrated to inhibit oxidative stress by reducing the activation of reactive oxygen species generation [63], and reduce inflammation by modulating cytokine production [64]. For instance, the activation of TRPV1 channels by vanillin has shown a significant neuroprotective effect, which may be possibly due to decreasing the level of lipid peroxidation and NO2, and elevates the activity of antioxidative enzymes along with an enhancement in the levels of brain glutathione [63, 65]. Accumulating evidence has indicated that TRPV1 plays a suppressive role in the systemic inflammatory response by inhibiting the production of tumor necrosis factor [66]. Thus, activation of TRPV1 by capsaicin may inhibit Aβ-induced oxidative stress and inflammatory response in the brain, and subsequently contribute to the improvements of synaptic and cognitive functions in the present study.

In summary, our study demonstrates that TRPV1 activation by capsaicin can ameliorate the deficits of cognitive and synaptic functions in Aβ42-induced mouse model of AD, suggesting that the TRPV1 channel may be a potential target for AD treatment. However, the beneficial effects of capsaicin on cognitive functions in different animal model of AD such as transgenic mice, and the underlying molecular mechanisms need to be further investigated in the future.

ACKNOWLEDGMENTS

This work was supported by grants from the Natural Science Foundation of China (No. 81622015 and 81571042 to ZD, 81501143 to HH), the Natural Science Foundation of Chongqing (No. cstc2015jcyjA00037 to HH), 973 Program of the Ministry of Science and Technology of China (No. 2014CB548100 to ZD) and Graduate student Innovation Project of Chongqing (No. CYS15117 to LC).

Authors’ disclosures available online (http://j-alz.com/manuscript-disclosures/17-0337r1).

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