Pharmacological Reports 66 (2014) 10–14
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
The effect of intra-ventral hippocampus administration of TRPV1 agonist and antagonist on spatial learning and memory in male rats Sedigheh Amiresmaili c, Ali Shamsizadeh c, Mohammad Allahtavakoli c, Ali Asghar Pourshanazari b,c, Ali Roohbakhsh a,c,* a b c
Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Department of Physiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Physiology-Pharmacology Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
A R T I C L E I N F O
Article history: Received 19 December 2012 Received in revised form 28 May 2013 Accepted 13 June 2013 Available online 30 January 2014 Keywords: Spatial learning OLDA AMG9810 TRPV1 Morris water maze
A B S T R A C T
Background: The role of transient receptor potential vanilloid 1 (TRPV1) in peripheral nervous system has been studied well but its role in the central nervous system remains to be studied in detail. The expression of TRPV1 receptors in hippocampus is suggesting that they may play an important role in higher cognitive functions such as learning and memory. Methods: In the present study, the role of TRPV1 receptors in acquisition and retrieval of spatial memory of male Wistar rats was evaluated by intra-ventral hippocampus administration of TRPV1 selective agonist (OLDA) and antagonist (AMG9810) using Morris water maze. Results: The results demonstrated that administration of either OLDA (0.001, 0.01 and 0.1 mg/rat) or AMG9810 (0.003, 0.03 and 0.3 mg/rat) did not influence memory acquisition or retrieval. Conclusions: These data suggest that ventral-hippocampal TRPV1 receptors possibly are not involved in spatial learning and memory. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Learning and memory are made possible via changes at neuronal synapses in the brain, a process known as synaptic plasticity. Synaptic plasticity consists of both long-term potentiation (LTP) and long-term depression (LTD) [2]. Memory is often considered to be a process that has different stages, including acquisition, consolidation and retrieval [1]. Acquisition is the stage during which an animal learns a task, consolidation is the stage during which the memory is stabilized and the retrieval brings back the learned task. Hippocampus plays an important role in learning and memory processes [7]. Its key role in acquisition, consolidation and retrieval of spatial memory has clearly been established [3]. N-methyl-Daspartate receptors have a key role in both LTD and LTP but recently transient receptor potential vanilloid 1 (TRPV1) has been shown to be involved in some types of synaptic plasticity in the hippocampus [2,6]. TRPV1 channel is a nonselective cation channel with high Ca2+ permeability that belongs to the TRP family of
* Corresponding author. E-mail address: [email protected] (A. Roohbakhsh).
proteins and is activated by capsaicin, the pungent ingredient found in the hot chilli pepper, noxious heat (>43 8C) and low pH [21]. In the central nervous system (CNS), TRPV1 is expressed in brain regions including cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus, locus ceruleus, periaqueductal gray, amygdala, striatum, mesencephalon and olfactory bulb [11]. The function of TRPV1 channel in the CNS is less well known compared to the peripheral nervous system since these receptors are unlikely to be exposed to low pH or noxious heat directly. They tonically stimulate glutamate release [14] and appear to participate in pain perception, regulation of body temperature, bladder function, itching, pulmonary diseases [16], and control of locomotion [4] and anxiety [19]. It was demonstrated that TRPV1 knock-out (TRPV1KO) mice have reduced CA1 LTP and anxiety-like behaviors compared to wild type (TRPV1-WT) controls [15]. Previous reports have shown that anadamine, N-arachidonoyldopamine and 12hydroperoxyeicosatetraenoic (12-HPETE) are endogenous TRPV1 agonists [21]. Gibson et al. [6] in their study showed that 12-HPETE is involved in TRPV1-induced LTD in hippocampal interneurons. In addition, it was identified that capsaicin by activation of TRPV1 enhanced NMDA-receptor dependent CA1 LTP and decreased NMDA-receptor dependent LTD [12]. Based on the above evidence, using pharmacological tools, we evaluated the role of TRPV1
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.06.002
S. Amiresmaili et al. / Pharmacological Reports 66 (2014) 10–14
receptors of the ventral hippocampus in acquisition and retrieval of memory in Morris water maze.
11
Behavioral procedure
The drugs used in the present study were AMG9810 (selective TRPV1 antagonist) and N-oleoyldopamine (OLDA, selective TRPV1 agonist) and were purchased from Tocris Bioscience (Bristol, UK). The drugs were dissolved in dimethyl sulfoxide and sterile 0.9% saline (up to 10%, v/v) and Tween 80.
The Morris water maze task was based on methods described by Haj-ali et al. [8]. In a single training protocol each rat accomplished three blocks separated by a 30-min resting period. Each animal was exposed to four trials per block. On each trial, rats were randomly released into the water from one of the four quadrants of the maze with its face toward the wall of the quadrant where it was released. Each rat had 4 different releasing points. During acquisition, the location of the platform remained constant and rats were allowed to swim to the hidden escape platform. After the animal found the platform, it was allowed to remain there for 20 s and then located in animal cage to wait 30 s before the start of the next trial. While a rat failed to find the platform in 60 s, the experimenter guided it toward the platform and allowed to rest there for 20 s. A single probe trial was performed 24 h after the last training trial to test the spatial memory in the water maze. In this trial the platform was removed and the rat was allowed to swim for 90 s. The time and distance spent in the target quadrant were analyzed as a measure of spatial memory retrieval. After the probe trial, rats completed a visible platform test to determine any possibility of interference of tested drugs with sensory and motor coordination. In this session, the animals’ ability to escape to a visible platform (platform was raised 2 cm above the water level and became visible with aluminum foil) was calculated.
Surgery
Histology
Rats were implanted with bilateral cannula aimed at the ventral hippocampus. Before surgery, animals were anesthetized with intraperitoneal injections of ketamine (60 mg/kg) and xylazine (5 mg/kg). The animals were mounted into a stereotaxic frame to position the 22-gauge stainless steel guide cannula in the right and left ventral hippocampus according to rat brain atlas [18] (AP: 4.40 mm; ML: 5 mm; DV: 7.5 mm). The cannula was anchored to the skull using acrylic cement.
After the completion of behavioral training, animals were sacrificed and the brains were removed and stored in 10% formalin. After one week, the brains sectioned and cannula placements were examined for verification of needle tip locations.
Materials and methods Animals Adult male Wistar rats (200–250 g) were used in the present study. The animal house temperature was maintained at 23 2.0 8C with a 12:12 h light/dark cycle. Food and water were provided ad libitum except during behavioral testing in Morris water maze (MWM). The method of this study was approved by local ethics committee (Ethics and Animal Care Committee of Rafsanjan University of Medical Sciences). Animals were handled in accordance with criteria outlined in the Guide for Care and Use of Laboratory Animals (NIH publication, 7th edition) [10]. Drugs
Microinjection procedure The animals were allowed 6 days to recover before the test. Intra-ventral hippocampus (intra-VH) injections were done using a microinfusion pump (KD Scientific, Holliston, MA, USA). Injector (27-gauge) was glued to polyethylene tubing filled with distilled water. The tubing was connected to a 10-ml microsyringe (Hamilton, Reno, NV, USA) mounted on the microinfusion pump. Rats were hand-held as the experimenter inserted the injector. The injector projected 1 mm beyond the guide cannula and 0.5 ml solution was infused in the right and left ventral regions of the hippocampus over 60 s. It was left in place for an additional 60 s. OLDA and AMG9810 were administered 15 min before training blocks or before test probe. The doses of the drugs were selected as followed in previous studies [9,20]. Morris water maze (MWM) The MWM consisted of a black circular pool, 160 cm diameter, 60 cm height, and was filled with water maintained at 22 1 8C to a depth of 25 cm. The pool was divided into four quadrants of equal size and starting points were designated at each quadrant as N, S, E, and W. A square platform (10 cm diameter) submerged 1.5 cm beneath the surface of the water. Fixed, extra maze visual cues were present at various locations around the maze (e.g., circles, squares or triangles). The experiments were carried out in a dimly illuminated room. Animal’s performances such as latency to find the platform and traveled distance were recorded by a video tracking system (Noldus Ethovision1 system, version 5, USA).
Data analysis The time spent and distance traveled to find the platform during training sessions were analyzed using repeated measures analysis of variance (ANOVA). Comparisons among the groups for the data collected in the MWM were analyzed with one-way ANOVA followed by Dunnett’s post hoc test. Data are expressed as means SEM of eight animals per group. p < 0.05 was considered statistically significant. Results The effect of bilateral intra-ventral hippocampus administration of OLDA and AMG9810 on spatial learning acquisition During acquisition (learning phase), animals in vehicle-treated group learned to find the hidden platform as showed by the decrease in distance traveled and escape latency with subsequent blocks of training (p = 0.016 and p = 0.005 respectively; Fig. 1A and B). The results demonstrated that administration of OLDA (0.001, 0.01 and 0.1 mg/rat) influenced neither the distance traveled nor escape latency when compared with vehicle-treated rats at the same block (p > 0.05; Fig. 1A and B). Similar to OLDA, administration of AMG9810 (0.003, 0.03 and 0.3 mg/rat) did not change distance traveled and escape latency significantly compared with vehicle-treated rats (p > 0.05; Fig. 2A and B). The effect of bilateral intra-ventral hippocampus administration of OLDA and AMG9810 on memory retrieval One-way ANOVA for distance traveled and time spent in target quadrant revealed no significant difference between rats treated
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S. Amiresmaili et al. / Pharmacological Reports 66 (2014) 10–14
Fig. 1. (A) The effect of different doses of OLDA on the distance traveled. Each block represents the average distance traveled of four consecutive trials. *p < 0.05 indicating the difference between blocks 1, 2 and 3 in vehicle-treated rats. Treatment with OLDA (0.001, 0.01 and 0.1 mg/rat) did not influence distance traveled when compared with vehicle-treated group at the same block (p > 0.05). (B) The effect of different doses of OLDA on the escape latency to find the hidden platform. *p < 0.05 and **p < 0.01 indicating the difference between blocks 1, 2 and 3 in vehicle-treated rats. Data are presented as means SEM; n = 8. OLDA treatments (0.001, 0.01 and 0.1 mg/rat) did not influence escape latency when compared with vehicle-treated group at the same block (p > 0.05).
Fig. 2. (A) The effect of different doses of AMG9810 on the distance traveled. Each block represents the average distance traveled of four consecutive trials. *p < 0.05 indicating the difference between blocks 1, 2 and 3 in vehicle-treated rats. Treatment with AMG9810 (0.003, 0.03 and 0.3 mg/rat) did not influence distance traveled when compared with vehicle-treated group at the same block (p > 0.05). (B) The effect of different doses of AMG9810 on the escape latency to find the hidden platform. *p < 0.05 and **p < 0.01 indicating the difference between blocks 1, 2 and 3 in vehicle-treated rats. Data are presented as means SEM; n = 8. AMG9810 treatments (0.003, 0.03 and 0.3 mg/rat) did not influence escape latency when compared with vehicle-treated group at the same block (p > 0.05).
with different doses of OLDA (0.001, 0.01 and 0.1 mg/rat) compared with vehicle-treated rats (p > 0.05; Fig. 3A and B). Similarly, administration of AMG9810 (0.003, 0.03 and 0.3 mg/rat) before probe test also did not affect distance traveled or time spent in target quadrant significantly compared to vehicle-treated group (p > 0.05; Fig. 4A and B).
spatial memory. The present results may be linked to the doses of the drugs that we used but it should be noted that at the same doses OLDA and AMG9810 had anxiogenic- and anxiolytic-like effects respectively [9]. Furthermore, these doses are higher than their EC50 (36 nM) and IC50 (17 nM) respectively. There are a wide range of well-developed models that are available to study and analyze spatial memory. The most commonly used model is the Morris water maze in which an animal’s capacity to remember spatial cues is required to locate a hidden platform underwater [17]. Up to now, most of the previous studies have focused on the role of TRPV1 receptors in LTD and LTP besides, but only a limited number of behavioral studies dealing with the role of TRPV1 receptors in memory have been published so far. In line with the present results, it has been reported that administration of capsaicin (TRPV1 receptor agonist) into the dorsal hippocampus of the rats had no significant effect on retrieval of spatial memory in Morris water maze [12]. In another study, Bennion et al. [2] investigated the possibility that TRPV1 is endogenously activated and involved normally in CA1 LTP induction. They found that
The effect of OLDA and AMG9810 on visible platform performance The results of the visual test showed no difference in escape latency (p > 0.05) to find the visible platform compared to the vehicle group in both OLDA and AMG9810-treated groups (Fig. 5). Discussion The main finding of the present study is that administration of both TRPV1 receptor selective agonist and antagonist into the ventral hippocampus failed to show any significant effect on memory acquisition and retrieval in Morris water maze test of
S. Amiresmaili et al. / Pharmacological Reports 66 (2014) 10–14
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Fig. 3. The effect of different doses of OLDA on memory retrieval. (A) The distance traveled in target quadrant. (B) Time spent in target quadrant. Data are presented as means SEM; n = 8.
Fig. 4. The effect of different doses of AMG9810 on memory retrieval. (A) The distance traveled in target quadrant. (B) Time spent in target quadrant. Data are presented as means SEM; n = 8.
capsazepine, as a selective TRPV1 receptor antagonist, had no effect on LTP induced by theta burst stimulation. Their finding suggests that, at least in an in vitro preparation, TRPV1 is not involved in CA1 LTP induction. Based on these reports it may be suggested that TRPV1 receptors at their baseline activity are not involved in learning and memory processes. In agreement with this hypothesis, it was reported that the involvement of these receptors in memory may be dependent on the intensity of peripheral stimulus [15]. The authors evaluated the role of TRPV1 in nonassociative memory processes. In their study, both TRPV1-KO and TRPV1-WT were exposed to one or three inescapable and unsignaled footshocks and exposed to a tone one and seven days later. After sensitization with a single footshock, there was no difference in the freezing response to the tone between TRPV1-KO and TRPV1-WT. After sensitization with three footshocks, TRPV1KO consistently showed less freezing to the tone than TRPV1-WT from day 1 to day 7. The authors concluded that TRPV1 seems to be implicated in non-associative memory processes after strong sensitization procedures. Furthermore, Li et al. [12] showed that capsaicin by itself had no significant effect on spatial memory when administered into the dorsal hippocampus but it prevented stress-induced impairment in retrieval of spatial memory. We further suggest that the effect of capsaicin on stress-induced memory impairment possibly is not due to its anxiolytic effect as its anxiogenic effect following intra-CA1 administration has been reported elsewhere [9]. Moreover, a recent study using passive avoidance test and novel object recognition test showed that TRPV1-KO mice had long-term memories similar to WT mice [23]. In the same study, TRPV1-KO mice had deficits in short-term memory (1.5 h) in novel object recognition test with reduced expression of NR2A subunit of NMDA receptors indicating the
involvement of NMDA receptors in TRPV1-mediated deficits in short-term memory. However, in the present study the rats that received TRPV1 antagonist had memory acquisition similar to vehicle-treated rats. Previous studies represent a connection between the age of animals and the biological actions of TRPV1 receptors. For example, Zavitsanou et al. [24] showed that administration of capsaicin significantly increased 5-HT2A and M2/M4 receptor expression in the brain of two days old rats compared with 15–16 weeks old rats. Another study showed that long-term depression in the rodent superior colliculus can be induced by TRPV1 receptors during development but not in aged mice [13]. Therefore, the involvement of TRPV1 receptors in the
Fig. 5. The effect of OLDA and AMG9810 on escape latency during visible platform test. Data are presented as means SEM.
S. Amiresmaili et al. / Pharmacological Reports 66 (2014) 10–14
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hippocampal plasticity in the study of Bennion et al. [2] and the ineffective action of TRPV1 agonist and antagonist on the spatial memory in the present study may be attributed to the difference in animal age of these two studies (2–4 weeks old and adult rats, respectively). Based on the present and previous reports especially the study of Li et al. [12], it looks like that TRPV1 receptors of the hippocampus, despite their modulatory effects on the cellular mechanism of learning and memory in forms of both LTP and LTD, do not have an apparent effect on learning and memory processes related to spatial memory while they may have a protective role against stress-induced memory impairment. It should be noted that TRPV1 receptors other than dorsal and ventral hippocampus are not involved in this statement. Besides, as both dorsal and ventral hippocampus are involved in spatial learning, simultaneous activation or inhibition of TRPV1 receptors in dorsal and ventral regions of the hippocampus would make a more conclusive experiment for elucidation the role of TRPV1 receptors in spatial learning and memory. At present, both TRPV1 receptor selective agonists and antagonists are under development as analgesics [22]. The present finding possibly means that the impairment effect of TRPV1-based drugs on spatial learning and memory is lower compared with opioid analgesics [5]. However, we do not reject the need for more investigation regarding the exact role of TRPV1 receptors in learning and memory. This can be further investigated using other animal models of learning and memory or in brain regions other than the hippocampus. In conclusion, in the present study, administration of both TRPV1 receptor agonist and antagonist into the ventral hippocampus of the rats did not elicit any significant effect on both memory acquisition and retrieval in Morris water maze. Conflict of interest No conflict of interest. Funding This project was supported by a grant from the Rafsanjan University of Medical Sciences. References [1] Abel T, Lattal KM. Molecular mechanisms of memory acquisition, consolidation and retrieval. Curr Opin Neurobiol 2001;11:180–7. [2] Bennion D, Jensen T, Walther C, Hamblin J, Wallmann A, Couch J, et al. Transient receptor potential vanilloid 1 agonists modulate hippocampal CA1 LTP via the GABAergic system. Neuropharmacology 2011;61:730–8. [3] Bird CM, Burgess N. The hippocampus and memory: insights from spatial processing. Nat Rev Neurosci 2008;9:182–94.
[4] Di Marzo V, Bisogno T, De Petrocellis L, Brandi I, Jefferson RG, Winckler RL, et al. Highly selective CB(1) cannabinoid receptor ligands and novel CB(1)/VR(1) vanilloid receptor ‘‘hybrid’’ ligands. Biochem Biophys Res Commun 2001;281: 444–51. [5] Farahmandfar M, Karimian SM, Naghdi N, Zarrindast MR, Kadivar M. Morphine-induced impairment of spatial memory acquisition reversed by morphine sensitization in rats. Behav Brain Res 2010;211:156–63. [6] Gibson HE, Edwards JG, Page RS, Van Hook MJ, Kauer JA. TRPV1 channels mediate long-term depression at synapses on hippocampal interneurons. Neuron 2008;57:746–59. [7] Goodrich-Hunsaker NJ, Hopkins RO. Spatial memory deficits in a virtual radial arm maze in amnesic participants with hippocampal damage. Behav Neurosci 2010;124:405–13. [8] Haj-ali V, Mohaddes G, Babri SH. Intracerebroventricular insulin improves spatial learning and memory in male Wistar rats. Behav Neurosci 2009; 123:1309–14. [9] Hakimizadeh E, Oryan S, Hajizadeh Moghaddam A, Shamsizadeh A, Roohbakhsh A. Endocannabinoid system and TRPV1 receptors in the dorsal hippocampus of the rats modulate anxiety-like behaviors. Iran J Basic Med Sci 2012;15:795–802. [10] Institute of Laboratory Animal Resources. Guide for the care and use of laboratory animals. 7th ed. Washington: Academy Press; 1996. [11] Kauer JA, Gibson HE. Hot flash: TRPV channels in the brain. Trends Neurosci 2009;32:215–24. [12] Li HB, Mao RR, Zhang JC, Yang Y, Cao J, Xu L. Antistress effect of TRPV1 channel on synaptic plasticity and spatial memory. Biol Psychiatry 2008; 64:286–92. [13] Maione S, Cristino L, Migliozzi AL, Georgiou AL, Starowicz K, Salt TE, et al. TRPV1 channels control synaptic plasticity in the developing superior colliculus. J Physiol 2009;1:2521–35. [14] Marinelli S, Pascucci T, Bernardi G, Puglisi-Allegra S, Mercuri NB. Activation of TRPV1 in the VTA excites dopaminergic neurons and increases chemical- and noxious-induced dopamine release in the nucleus accumbens. Neuropsychopharmacology 2005;30:864–70. [15] Marsch R, Foeller E, Rammes G, Bunck M, Ko¨ssl M, Holsboer F, et al. Reduced anxiety, conditioned fear, and hippocampal long-term potentiation in transient receptor potential vanilloid type 1 receptor-deficient mice. J Neurosci 2007;27:832–9. [16] Moran MM, McAlexander MA, Bı´ro´ T, Szallasi A. Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov 2011;10:601–20. [17] Morris RGM. Spatial localization does not require the presence of local cues. Learn Motiv 1981;12:239–60. [18] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 4th ed. San Diego: Academic Press; 1998. [19] Rubino T, Realini N, Castiglioni C, Guidali C, Vigano´ D, Marras E, et al. Role in anxiety behavior of the endocannabinoid system in the prefrontal cortex. Cereb Cortex 2008;18:1292–301. [20] Spicarova D, Palecek J. The role of the TRPV1 endogenous agonist N-oleoyldopamine in modulation of nociceptive signaling at the spinal cord level. J Neurophysiol 2009;102:234–43. [21] Vriens J, Appendino G, Nilius B. Pharmacology of vanilloid transient receptor potential cation channels. Mol Pharmacol 2009;75:1262–79. [22] Wong GY, Gavva NR. Therapeutic potential of vanilloid receptor TRPV1 agonists and antagonists as analgesics: recent advances and setbacks. Brain Res Rev 2009;60:267–77. [23] You IJ, Jung YH, Kim MJ, Kwon SH, Hong SI, Lee SY, et al. Alterations in the emotional and memory behavioral phenotypes of transient receptor potential vanilloid type 1-deficient mice are mediated by changes in expression of 5-HT1A, GABA(A), and NMDA receptors. Neuropharmacology 2012;62: 1034–43. [24] Zavitsanou K, Dalton VS, Wang H, Newson P, Chahl LA. Receptor changes in brain tissue of rats treated as neonates with capsaicin. J Chem Neuroanat 2010;39:248–55.
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
The effect of intra-ventral hippocampus administration of TRPV1 agonist and antagonist on spatial learning and memory in male rats Sedigheh Amiresmaili c, Ali Shamsizadeh c, Mohammad Allahtavakoli c, Ali Asghar Pourshanazari b,c, Ali Roohbakhsh a,c,* a b c
Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Department of Physiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Physiology-Pharmacology Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
A R T I C L E I N F O
Article history: Received 19 December 2012 Received in revised form 28 May 2013 Accepted 13 June 2013 Available online 30 January 2014 Keywords: Spatial learning OLDA AMG9810 TRPV1 Morris water maze
A B S T R A C T
Background: The role of transient receptor potential vanilloid 1 (TRPV1) in peripheral nervous system has been studied well but its role in the central nervous system remains to be studied in detail. The expression of TRPV1 receptors in hippocampus is suggesting that they may play an important role in higher cognitive functions such as learning and memory. Methods: In the present study, the role of TRPV1 receptors in acquisition and retrieval of spatial memory of male Wistar rats was evaluated by intra-ventral hippocampus administration of TRPV1 selective agonist (OLDA) and antagonist (AMG9810) using Morris water maze. Results: The results demonstrated that administration of either OLDA (0.001, 0.01 and 0.1 mg/rat) or AMG9810 (0.003, 0.03 and 0.3 mg/rat) did not influence memory acquisition or retrieval. Conclusions: These data suggest that ventral-hippocampal TRPV1 receptors possibly are not involved in spatial learning and memory. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Learning and memory are made possible via changes at neuronal synapses in the brain, a process known as synaptic plasticity. Synaptic plasticity consists of both long-term potentiation (LTP) and long-term depression (LTD) [2]. Memory is often considered to be a process that has different stages, including acquisition, consolidation and retrieval [1]. Acquisition is the stage during which an animal learns a task, consolidation is the stage during which the memory is stabilized and the retrieval brings back the learned task. Hippocampus plays an important role in learning and memory processes [7]. Its key role in acquisition, consolidation and retrieval of spatial memory has clearly been established [3]. N-methyl-Daspartate receptors have a key role in both LTD and LTP but recently transient receptor potential vanilloid 1 (TRPV1) has been shown to be involved in some types of synaptic plasticity in the hippocampus [2,6]. TRPV1 channel is a nonselective cation channel with high Ca2+ permeability that belongs to the TRP family of
* Corresponding author. E-mail address: [email protected] (A. Roohbakhsh).
proteins and is activated by capsaicin, the pungent ingredient found in the hot chilli pepper, noxious heat (>43 8C) and low pH [21]. In the central nervous system (CNS), TRPV1 is expressed in brain regions including cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus, locus ceruleus, periaqueductal gray, amygdala, striatum, mesencephalon and olfactory bulb [11]. The function of TRPV1 channel in the CNS is less well known compared to the peripheral nervous system since these receptors are unlikely to be exposed to low pH or noxious heat directly. They tonically stimulate glutamate release [14] and appear to participate in pain perception, regulation of body temperature, bladder function, itching, pulmonary diseases [16], and control of locomotion [4] and anxiety [19]. It was demonstrated that TRPV1 knock-out (TRPV1KO) mice have reduced CA1 LTP and anxiety-like behaviors compared to wild type (TRPV1-WT) controls [15]. Previous reports have shown that anadamine, N-arachidonoyldopamine and 12hydroperoxyeicosatetraenoic (12-HPETE) are endogenous TRPV1 agonists [21]. Gibson et al. [6] in their study showed that 12-HPETE is involved in TRPV1-induced LTD in hippocampal interneurons. In addition, it was identified that capsaicin by activation of TRPV1 enhanced NMDA-receptor dependent CA1 LTP and decreased NMDA-receptor dependent LTD [12]. Based on the above evidence, using pharmacological tools, we evaluated the role of TRPV1
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.06.002
S. Amiresmaili et al. / Pharmacological Reports 66 (2014) 10–14
receptors of the ventral hippocampus in acquisition and retrieval of memory in Morris water maze.
11
Behavioral procedure
The drugs used in the present study were AMG9810 (selective TRPV1 antagonist) and N-oleoyldopamine (OLDA, selective TRPV1 agonist) and were purchased from Tocris Bioscience (Bristol, UK). The drugs were dissolved in dimethyl sulfoxide and sterile 0.9% saline (up to 10%, v/v) and Tween 80.
The Morris water maze task was based on methods described by Haj-ali et al. [8]. In a single training protocol each rat accomplished three blocks separated by a 30-min resting period. Each animal was exposed to four trials per block. On each trial, rats were randomly released into the water from one of the four quadrants of the maze with its face toward the wall of the quadrant where it was released. Each rat had 4 different releasing points. During acquisition, the location of the platform remained constant and rats were allowed to swim to the hidden escape platform. After the animal found the platform, it was allowed to remain there for 20 s and then located in animal cage to wait 30 s before the start of the next trial. While a rat failed to find the platform in 60 s, the experimenter guided it toward the platform and allowed to rest there for 20 s. A single probe trial was performed 24 h after the last training trial to test the spatial memory in the water maze. In this trial the platform was removed and the rat was allowed to swim for 90 s. The time and distance spent in the target quadrant were analyzed as a measure of spatial memory retrieval. After the probe trial, rats completed a visible platform test to determine any possibility of interference of tested drugs with sensory and motor coordination. In this session, the animals’ ability to escape to a visible platform (platform was raised 2 cm above the water level and became visible with aluminum foil) was calculated.
Surgery
Histology
Rats were implanted with bilateral cannula aimed at the ventral hippocampus. Before surgery, animals were anesthetized with intraperitoneal injections of ketamine (60 mg/kg) and xylazine (5 mg/kg). The animals were mounted into a stereotaxic frame to position the 22-gauge stainless steel guide cannula in the right and left ventral hippocampus according to rat brain atlas [18] (AP: 4.40 mm; ML: 5 mm; DV: 7.5 mm). The cannula was anchored to the skull using acrylic cement.
After the completion of behavioral training, animals were sacrificed and the brains were removed and stored in 10% formalin. After one week, the brains sectioned and cannula placements were examined for verification of needle tip locations.
Materials and methods Animals Adult male Wistar rats (200–250 g) were used in the present study. The animal house temperature was maintained at 23 2.0 8C with a 12:12 h light/dark cycle. Food and water were provided ad libitum except during behavioral testing in Morris water maze (MWM). The method of this study was approved by local ethics committee (Ethics and Animal Care Committee of Rafsanjan University of Medical Sciences). Animals were handled in accordance with criteria outlined in the Guide for Care and Use of Laboratory Animals (NIH publication, 7th edition) [10]. Drugs
Microinjection procedure The animals were allowed 6 days to recover before the test. Intra-ventral hippocampus (intra-VH) injections were done using a microinfusion pump (KD Scientific, Holliston, MA, USA). Injector (27-gauge) was glued to polyethylene tubing filled with distilled water. The tubing was connected to a 10-ml microsyringe (Hamilton, Reno, NV, USA) mounted on the microinfusion pump. Rats were hand-held as the experimenter inserted the injector. The injector projected 1 mm beyond the guide cannula and 0.5 ml solution was infused in the right and left ventral regions of the hippocampus over 60 s. It was left in place for an additional 60 s. OLDA and AMG9810 were administered 15 min before training blocks or before test probe. The doses of the drugs were selected as followed in previous studies [9,20]. Morris water maze (MWM) The MWM consisted of a black circular pool, 160 cm diameter, 60 cm height, and was filled with water maintained at 22 1 8C to a depth of 25 cm. The pool was divided into four quadrants of equal size and starting points were designated at each quadrant as N, S, E, and W. A square platform (10 cm diameter) submerged 1.5 cm beneath the surface of the water. Fixed, extra maze visual cues were present at various locations around the maze (e.g., circles, squares or triangles). The experiments were carried out in a dimly illuminated room. Animal’s performances such as latency to find the platform and traveled distance were recorded by a video tracking system (Noldus Ethovision1 system, version 5, USA).
Data analysis The time spent and distance traveled to find the platform during training sessions were analyzed using repeated measures analysis of variance (ANOVA). Comparisons among the groups for the data collected in the MWM were analyzed with one-way ANOVA followed by Dunnett’s post hoc test. Data are expressed as means SEM of eight animals per group. p < 0.05 was considered statistically significant. Results The effect of bilateral intra-ventral hippocampus administration of OLDA and AMG9810 on spatial learning acquisition During acquisition (learning phase), animals in vehicle-treated group learned to find the hidden platform as showed by the decrease in distance traveled and escape latency with subsequent blocks of training (p = 0.016 and p = 0.005 respectively; Fig. 1A and B). The results demonstrated that administration of OLDA (0.001, 0.01 and 0.1 mg/rat) influenced neither the distance traveled nor escape latency when compared with vehicle-treated rats at the same block (p > 0.05; Fig. 1A and B). Similar to OLDA, administration of AMG9810 (0.003, 0.03 and 0.3 mg/rat) did not change distance traveled and escape latency significantly compared with vehicle-treated rats (p > 0.05; Fig. 2A and B). The effect of bilateral intra-ventral hippocampus administration of OLDA and AMG9810 on memory retrieval One-way ANOVA for distance traveled and time spent in target quadrant revealed no significant difference between rats treated
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Fig. 1. (A) The effect of different doses of OLDA on the distance traveled. Each block represents the average distance traveled of four consecutive trials. *p < 0.05 indicating the difference between blocks 1, 2 and 3 in vehicle-treated rats. Treatment with OLDA (0.001, 0.01 and 0.1 mg/rat) did not influence distance traveled when compared with vehicle-treated group at the same block (p > 0.05). (B) The effect of different doses of OLDA on the escape latency to find the hidden platform. *p < 0.05 and **p < 0.01 indicating the difference between blocks 1, 2 and 3 in vehicle-treated rats. Data are presented as means SEM; n = 8. OLDA treatments (0.001, 0.01 and 0.1 mg/rat) did not influence escape latency when compared with vehicle-treated group at the same block (p > 0.05).
Fig. 2. (A) The effect of different doses of AMG9810 on the distance traveled. Each block represents the average distance traveled of four consecutive trials. *p < 0.05 indicating the difference between blocks 1, 2 and 3 in vehicle-treated rats. Treatment with AMG9810 (0.003, 0.03 and 0.3 mg/rat) did not influence distance traveled when compared with vehicle-treated group at the same block (p > 0.05). (B) The effect of different doses of AMG9810 on the escape latency to find the hidden platform. *p < 0.05 and **p < 0.01 indicating the difference between blocks 1, 2 and 3 in vehicle-treated rats. Data are presented as means SEM; n = 8. AMG9810 treatments (0.003, 0.03 and 0.3 mg/rat) did not influence escape latency when compared with vehicle-treated group at the same block (p > 0.05).
with different doses of OLDA (0.001, 0.01 and 0.1 mg/rat) compared with vehicle-treated rats (p > 0.05; Fig. 3A and B). Similarly, administration of AMG9810 (0.003, 0.03 and 0.3 mg/rat) before probe test also did not affect distance traveled or time spent in target quadrant significantly compared to vehicle-treated group (p > 0.05; Fig. 4A and B).
spatial memory. The present results may be linked to the doses of the drugs that we used but it should be noted that at the same doses OLDA and AMG9810 had anxiogenic- and anxiolytic-like effects respectively [9]. Furthermore, these doses are higher than their EC50 (36 nM) and IC50 (17 nM) respectively. There are a wide range of well-developed models that are available to study and analyze spatial memory. The most commonly used model is the Morris water maze in which an animal’s capacity to remember spatial cues is required to locate a hidden platform underwater [17]. Up to now, most of the previous studies have focused on the role of TRPV1 receptors in LTD and LTP besides, but only a limited number of behavioral studies dealing with the role of TRPV1 receptors in memory have been published so far. In line with the present results, it has been reported that administration of capsaicin (TRPV1 receptor agonist) into the dorsal hippocampus of the rats had no significant effect on retrieval of spatial memory in Morris water maze [12]. In another study, Bennion et al. [2] investigated the possibility that TRPV1 is endogenously activated and involved normally in CA1 LTP induction. They found that
The effect of OLDA and AMG9810 on visible platform performance The results of the visual test showed no difference in escape latency (p > 0.05) to find the visible platform compared to the vehicle group in both OLDA and AMG9810-treated groups (Fig. 5). Discussion The main finding of the present study is that administration of both TRPV1 receptor selective agonist and antagonist into the ventral hippocampus failed to show any significant effect on memory acquisition and retrieval in Morris water maze test of
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Fig. 3. The effect of different doses of OLDA on memory retrieval. (A) The distance traveled in target quadrant. (B) Time spent in target quadrant. Data are presented as means SEM; n = 8.
Fig. 4. The effect of different doses of AMG9810 on memory retrieval. (A) The distance traveled in target quadrant. (B) Time spent in target quadrant. Data are presented as means SEM; n = 8.
capsazepine, as a selective TRPV1 receptor antagonist, had no effect on LTP induced by theta burst stimulation. Their finding suggests that, at least in an in vitro preparation, TRPV1 is not involved in CA1 LTP induction. Based on these reports it may be suggested that TRPV1 receptors at their baseline activity are not involved in learning and memory processes. In agreement with this hypothesis, it was reported that the involvement of these receptors in memory may be dependent on the intensity of peripheral stimulus [15]. The authors evaluated the role of TRPV1 in nonassociative memory processes. In their study, both TRPV1-KO and TRPV1-WT were exposed to one or three inescapable and unsignaled footshocks and exposed to a tone one and seven days later. After sensitization with a single footshock, there was no difference in the freezing response to the tone between TRPV1-KO and TRPV1-WT. After sensitization with three footshocks, TRPV1KO consistently showed less freezing to the tone than TRPV1-WT from day 1 to day 7. The authors concluded that TRPV1 seems to be implicated in non-associative memory processes after strong sensitization procedures. Furthermore, Li et al. [12] showed that capsaicin by itself had no significant effect on spatial memory when administered into the dorsal hippocampus but it prevented stress-induced impairment in retrieval of spatial memory. We further suggest that the effect of capsaicin on stress-induced memory impairment possibly is not due to its anxiolytic effect as its anxiogenic effect following intra-CA1 administration has been reported elsewhere [9]. Moreover, a recent study using passive avoidance test and novel object recognition test showed that TRPV1-KO mice had long-term memories similar to WT mice [23]. In the same study, TRPV1-KO mice had deficits in short-term memory (1.5 h) in novel object recognition test with reduced expression of NR2A subunit of NMDA receptors indicating the
involvement of NMDA receptors in TRPV1-mediated deficits in short-term memory. However, in the present study the rats that received TRPV1 antagonist had memory acquisition similar to vehicle-treated rats. Previous studies represent a connection between the age of animals and the biological actions of TRPV1 receptors. For example, Zavitsanou et al. [24] showed that administration of capsaicin significantly increased 5-HT2A and M2/M4 receptor expression in the brain of two days old rats compared with 15–16 weeks old rats. Another study showed that long-term depression in the rodent superior colliculus can be induced by TRPV1 receptors during development but not in aged mice [13]. Therefore, the involvement of TRPV1 receptors in the
Fig. 5. The effect of OLDA and AMG9810 on escape latency during visible platform test. Data are presented as means SEM.
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hippocampal plasticity in the study of Bennion et al. [2] and the ineffective action of TRPV1 agonist and antagonist on the spatial memory in the present study may be attributed to the difference in animal age of these two studies (2–4 weeks old and adult rats, respectively). Based on the present and previous reports especially the study of Li et al. [12], it looks like that TRPV1 receptors of the hippocampus, despite their modulatory effects on the cellular mechanism of learning and memory in forms of both LTP and LTD, do not have an apparent effect on learning and memory processes related to spatial memory while they may have a protective role against stress-induced memory impairment. It should be noted that TRPV1 receptors other than dorsal and ventral hippocampus are not involved in this statement. Besides, as both dorsal and ventral hippocampus are involved in spatial learning, simultaneous activation or inhibition of TRPV1 receptors in dorsal and ventral regions of the hippocampus would make a more conclusive experiment for elucidation the role of TRPV1 receptors in spatial learning and memory. At present, both TRPV1 receptor selective agonists and antagonists are under development as analgesics [22]. The present finding possibly means that the impairment effect of TRPV1-based drugs on spatial learning and memory is lower compared with opioid analgesics [5]. However, we do not reject the need for more investigation regarding the exact role of TRPV1 receptors in learning and memory. This can be further investigated using other animal models of learning and memory or in brain regions other than the hippocampus. In conclusion, in the present study, administration of both TRPV1 receptor agonist and antagonist into the ventral hippocampus of the rats did not elicit any significant effect on both memory acquisition and retrieval in Morris water maze. Conflict of interest No conflict of interest. Funding This project was supported by a grant from the Rafsanjan University of Medical Sciences. References [1] Abel T, Lattal KM. Molecular mechanisms of memory acquisition, consolidation and retrieval. Curr Opin Neurobiol 2001;11:180–7. [2] Bennion D, Jensen T, Walther C, Hamblin J, Wallmann A, Couch J, et al. Transient receptor potential vanilloid 1 agonists modulate hippocampal CA1 LTP via the GABAergic system. Neuropharmacology 2011;61:730–8. [3] Bird CM, Burgess N. The hippocampus and memory: insights from spatial processing. Nat Rev Neurosci 2008;9:182–94.
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