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The effect of hippocampal NMDA receptor blockade by MK-801 on cued fear extinction
Accepted Manuscript Title: The effect of hippocampal NMDA receptor blockade by MK-801 on cued fear extinction Authors: Bo Zhang, Chuan-Yu Li, Xiu-Song Wang PII: DOI: Reference:
S0166-4328(16)30953-6 http://dx.doi.org/doi:10.1016/j.bbr.2017.05.067 BBR 10917
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
28-10-2016 24-5-2017 30-5-2017
Please cite this article as: Zhang Bo, Li Chuan-Yu, Wang Xiu-Song.The effect of hippocampal NMDA receptor blockade by MK-801 on cued fear extinction.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2017.05.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The effect of hippocampal NMDA receptor blockade by MK-801 on cued fear extinction Running Title： Hippocampal NMDA receptor blockade in extinction Bo Zhang1^*, Chuan-Yu Li1^, Xiu-Song Wang2* 1 Kunming University of Science and Technology, Kunming, Yunnan 650500, China 2 Key Laboratory of Animal Resistance of Shandong Province, Department of Anatomy, Physiology and Development，College of Life Science, Shandong Normal University, Jinan 250000, China ^
The two authors contribute equally to this paper work
*Correspondence: [email protected]; [email protected]
1
Abstract Extinction of conditioned fear has been suggested to be a new form of learning instead of erasure of what was originally learned, and the process is NMDA (Nmethyl D-aspartate) receptor (NMDAR) dependent. Most of studies have so far revealed the important roles of NMDARs in the amygdala and medial prefrontal cortex (mPFC) in cued fear extinction. Although the ventral hippocampus has intimately reciprocal connections with the amygdala and mPFC, the role of its NMDARs in cued fear extinction remains unclear. The present experiment explored the issue by bilateral pre-extinction microinjection of the noncompetitive NMDAR antagonist MK-801 into the ventral hippocampus. Four groups of rats were given habituation, tone cued fear conditioning, fear extinction training and extinction test. Prior to extinction training, rats received bilateral infusions of either MK-801 (1.5, 3, or 6 μg/0.5 μl) or saline. Our results showed that MK-801 reduced freezing on the first trial of extinction training with no impact on within-session acquisition of extinction, and that the lower doses of MK-801 resulted in increased freezing on the extinction retrieval test. These findings suggest that ventral hippocampal NMDARs are necessary for the consolidation of tone cued fear extinction.
Key words: NMDA receptors; ventral hippocampus; consolidation; cued fear extinction
2
Pavlovian fear conditioning and extinction have been widely used as an animal model to investigate neurobiological mechanisms underlying anxiety and stressrelated disorders (e.g. post-traumatic stress disorder) in humans. In the conditioning paradigm, a conditioned stimulus (CS; e.g., tone or the context) is paired with an aversive unconditioned stimulus (US; e.g., foot shock), and after repeated training pairs, CS alone causes a conditioned response (CR; e.g., freezing). The fear extinction process involves reduction of CR by repeated presentation of CS without US, which is considered as a new learning process that inhibits initial fear memory [1]. However, it has been suggested that the mediating networks and possible neural mechanisms underlying fear conditioning remain largely distinct from those behind fear extinction [1, 2]. It is well known that the NMDA (N-methyl D-aspartate) receptors (NMDARs) are required for learning and memory [3], and moreover, studies with systemic injections of NMDA agonists or antagonists suggest its essential role in fear extinction [4, 5]. However, the precise process (acquisition, consolidation or retrieval) of fear extinction learning involving NMDARs remains unclear. While some reports suggest its involvement in acquisition process [6, 7], others have shown its importance in consolidation process [4]. Furthermore, most of later studies with local brain microinjections reveal the requirement of NMDARs in the lateral amygdala (LA) for extinction acquisition, and those in medial prefrontal cortex (mPFC) for consolidation process [7, 8]. As well known, the ventral hippocampus has intimately reciprocal connections with amygdala [9], which plays a central role in fear memory [10]. Also, there is the evidence indicating the involvement of the ventral hippocampus in cued fear conditioning [11], however, NMDAR blockade in the ventral hippocampal failed to impair cued fear conditioning [12]. Regarding the hippocampal NMDAR role in cued fear extinction, to our knowledge, only one related study showed that activation of NMDARs in the dorsal hippocampus facilitated acquisition and retrieval of extinction memory [6]. However, the effect of ventral hippocampal NMDAR blockade on cued fear extinction remains unclear. Therefore, the present study aimed to investigate this issue by bilateral microinjection of three doses of MK-801 (1.5, 3, or 6 μg/0.5 μl), a noncompetitive NMDAR antagonist, or saline into the ventral hippocampus of rats prior to cued fear extinction. Sprague-Dawley male rats (Kunming Medical College Animal Center, Kunming, China) weighing 280-320 g upon arrival were individually housed in transparent 3
polyethylene cages within a temperature- and humidity-controlled environment under a 12 h light/dark cycle (lights on from 7:00 AM to 7:00 PM). Four groups of animal (n=8 for each group) were used. Food and water were available ad libitum throughout the experiment. Experimental and animal care procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). After application of the local anesthetic lidocaine, the scalp was incised to expose the skull, and the bregma and lambda were aligned in the same horizontal plane. Stainless steel guide cannulas (length, 10 mm; diameter, 0.6 mm) were implanted bilaterally through small holes (diameter, 0.8 mm; coordinates: 5.2 mm posterior and 5 mm lateral to the bregma) drilled on each side of the skull. The tips of the guide cannulas were aimed above the ventral hippocampus at the following coordinates, 5.2 mm posterior and 5 mm lateral to the bregma, and 5 mm ventral to the dura, according to the brain atlas [13]. The guide cannulas were fixed with dental cement, and three small stainless steel screws used as anchors were screwed into the skull previously. Stainless steel stylets (diameter, 0.3 mm) that extended 0.5 mm beyond the tips of the guide cannulas were placed inside the guide cannulas to prevent occlusion. MK-801 (dizocilpine; Sigma, St. Louis, MO, USA) infusion solutions were prepared freshly on the day of injection, by been dissolved in isotonic 0.9% saline to a final concentration of 12 mg/ml. Three doses (1.5, 3, or 6 μg/0.5 μl) were adopted based on previous reports [12]. For microinjection procedure, firstly the stylets were removed from the guide cannula with rats manually restrained. Then, infusion cannulas (diameter 0.3 mm) which were connected to a 1 μl microsyringes (Shanghai Medical Laser Instrument Factory, Shanghai, China) mounted on a microinfusion pump (WZ-50C6, Medical Instrument Corporation of Zhejiang University, Hangzhou, Zhejiang, China), were inserted into the guide cannulas with its tip protruded 1.0 mm beyond the tip of guide cannulas. Rats were then bilaterally infused with 0.5 μl of MK-801 or saline with the speed 0.2 μl/min, and the infusion cannulas were left inside the guide cannulas for an additional 2 min after infusion for absorption of the drug, and then replaced by the stylets. Histological verification of cannula placement was performed after behavioral testing. Rats were anesthetized with ethyl carbamate (20% solution, 0.6 ml/100 g of body weight) and then perfused across the heart with physiological saline, followed by 10% formalin solution. Brains were postfixed in 10% formalin solution and then 30% sucrose solution, and subsequently sections (10 4
μm) were cut on a cryostat (Leica CM1850 UV, Leica Biosystems, Nussloch, BadenWürttemberg, Germany). After histological HE-staining, sections were examined on a light microscope for visualization of cannula tracts and injector tip localizations. One observation chamber without ceiling (40 cm × 40 cm × 50 cm) was placed in a sound-attenuating cabinet (50 cm × 50 cm × 60 cm), which was located in a brightly lit and isolated room. Walls of the chamber were constructed of black opaque plexiglas with white round spheres (4 cm in diameter) attached inside and spaced 3.5 cm apart. An infrared digital camera was attached to the roof and connected to a computer, to monitor and record rat behavior for behavioral scoring later. The US foot shocks (0.5 mA) were delivery by 25 stainless steel rods (diameter, 5 mm) of the floor, and the CS tones (5 kHz, 80 dB, 20 s) were delivered by a buzzer in the cabinet, which presentations were controlled by a computer program. The chamber was washed with 50% alcohol solution before the introduction of each subject. For behavioral procedures, after 1 week of recovery from surgery, rats were submitted to four experimental phases carried out over four consecutive days: habituation on day 0, fear conditioning on day 1, extinction training on day 2 and extinction test on day 3. Rats were put inside the chamber for 30 min with no stimuli presented during habituation. For fear conditioning, three minutes after being transported from their home cages and placed in the chamber, rats received six CS-US pairings with US foot shock presented 0.6 s before the ending and co-terminated with the CS tone, and with averaged inter-trial interval of 90 s (ranging from 60 to 120 s). Thirty seconds after the final shock, rats were returned to their home cages. During fear extinction training, firstly rats received drug infusion and were placed in the chamber 20 min later. Three minutes after rats being placed in the chamber, extinction training began, and 12 tones were delivered in the absence of US with the same inter-trial interval as training. On the following day, the extinction test was given with the similar procedure to that of extinction training, with one exception of 10 tones delivered. Fear response to the CS was assessed by measuring freezing behavior, which was defined as the absence of all movement with the exception of movement related to respiration. Freezing was scored offline from the videos using a digital stopwatch by an experimenter who was blind to the treatment conditions, and quantified by computing the percentage of the time spent for freezing during each tone presentation. Immobility was the baseline freezing behavior during three minutes before onset of first tone on day 2 and day 3 and quantified by computing the percentage of the time spent for being immobile. 5
Statistical analyses were conducted using the SPSS software (SPSS Inc, Chicago, Illinois, USA). The trial-by-trial data were analyzed using two-way repeated analysis of variance (ANOVA), with the drug treatment being the between-subjects factor and the trial being the within-subjects repeated measures factor. Locomotion time, percentage of immobility and percentage of freezing measures presented in histograms were analyzed using one-way analysis of variance (ANOVA). Post hoc comparison was performed using the Turkey’s method and all data were represented as means ± SEM. Differences were considered significant at P < 0.05.
Fig. 1
Fig. 2 Localization of injector tips was shown in Fig. 1. The trial-by-trial data for freezing behavior of subjects during fear conditioning, extinction training and extinction test were shown in Fig. 2A. A two-way repeated ANOVA revealed no significant difference between groups during conditioning on day 1 (F(3,27) = 0.57, P = 0.64). A one-way ANOVA revealed no significant difference in freezing response between groups during the last trial (F(3,27) = 0.733, P = 0.542), suggesting reaching the same conditioning level in all groups. However, there was a significant difference between trials (F(5,135) = 165.71, P < 0.001), suggesting the fast establishment of fear conditioning. During extinction training on day 2, a two-way repeated ANOVA revealed significant differences between trials (F(11,308) = 6.777, P < 0.001), but not between groups (F(3,28) = 2.042, P = 0.131), and also revealed significant trial × treatment interaction (F(33,308) = 2.317, P < 0.001), suggesting a differential treatment effect between groups across trials (Fig. 2A, also see Fig.2C). In order to evaluate the effects of MK-801 in each trial, a one-way ANOVA was used. During the first trial, a oneway ANOVA revealed significant differences between groups (F(3,28) = 12.514, P < 0.001). Further post hoc comparison showed significant differences between saline and all drug groups (all Ps < 0.01), but no significant differences among drug groups (all Ps > 0.05) (also see Fig. 2B). Similarly, ANOVA also revealed significant differences between groups during the following two trials (all Ps < 0.05), and further post hoc comparison showed significant differences between saline and the high dose group (all Ps < 0.05). For the remaining trials, there were no significant differences 6
between groups (all Ps > 0.05), possibly suggesting that MK-801 had no effects on fear extinction acquisition. There were two possible explanations for the results of first three trials. One possibility was that MK-801 could impair retrieval of cued fear conditioning memory. However, an alternative possibility was MK-801 induced hyperlocomotion as indicated in one previous report [12]. To clarify these two possibilities, the activity data during three minutes before the onset of first tone on day 2 were analyzed. A one-way ANOVA revealed significant drug effects on locomotion activity (F(3,27) = 3.317, P = 0.035), and further post hoc comparison only revealed significantly higher level of locomotion in high dose group compared with saline group (P = 0.03) (Fig. 3 A). The result indicated that MK-801 induced hyperlocomotion in the high dose group. This drug effect contributed to the significant low immobility in high dose group as shown in Fig. 3 B (F(3,27) = 3.317, P = 0.035). Taken together, for both lower dose groups, the first possibility could mainly contribute to our results, but for the high dose of group, two possibilities could coexist. During extinction test, a two-way repeated ANOVA revealed significant differences between groups (F(3,28) = 3.174, P = 0.04), trials (F(9,252) = 10.091, P < 0.001), but no significant trial × treatment interaction (F(27,252) = 0.739, P = 0.825) (Fig. 2A). Further post hoc comparison only revealed significant differences between saline and two low dose groups (all Ps < 0.05). Results indicated impairment of extinction consolidation by those two doses, reflected by significantly higher freezing response of subjects in those groups compared with saline group (also see Fig. 2D). This result was consistent with another previous report adopting systemic drug administration [4], which together supports the critical role of NMDARs in consolidation of fear extinction. Moreover, together with another hippocampal NMDAR activation study [6], these results indicate the important role of hippocampal NMDARs in cued fear extinction process. Considering the central role of mPFC in extinction consolidation and bidirectional connections between hippocampus and PFC [14], hippocampal NMDAR might take effect through its cooperation with PFC. However, it remains unclear why the subjects in the high dose group showed no impairment of extinction memory. One possibility could be that the MK-801 induced hyperlocomotion interfered with its potential influence on extinction memory consolidation. Further studies are required to clarify or test this issue. Moreover, in order to rule out contextual fear as a potential confound, due to the fact that the same chamber was used during conditioning and extinction in the present study, immobility 7
data during three minutes before the onset of the first tone on day 3 were analyzed. A one-way ANOVA revealed no significant differences between groups (F(3,28) = 0.807, P = 0.501) and subjects in all groups showed low immobility, suggesting no significant contextual influence on conditioned fear response (Fig. 3C). Taken together, the present study only showed impairment of extinction consolidation but not within-session acquisition. The present result was consistent with the idea suggesting a shift from NMDAR independent short-term memory formation to NMDAR dependent consolidation. Moreover, the underlying possible mechanism has been also suggested previously [15, 16]. MK-801 could take effect by its influence on long-term potentiation (LTP) and synaptic plasticity, which plays central roles in learning and memory [17]. For example, a previous study showed MK-801 blocked the induction of LTP in rat hippocampal slices [18]. Additionally, it has been shown that administration of MK-801 increased acetylcholine (Ach) release in hippocampus [19], and that elevated levels of the hippocampal Ach disrupted cued fear conditioning [20]. Therefore, MK-801 might also take effect indirectly through its influence on Ach release in hippocampus. Key brain regions involved in fear extinction have been elucidated, indicating the central role of amygdala in acquisition while the PFC in consolidation process [21]. Furthermore, fear extinction involves interactions between cortical and subcortical brain areas, for example, PFC could regulate the expression of fear extinction by inhibiting the amygdala [22]. Additionally, studies reveal strong functional connections between hippocampus and mPFC, which has been suggested to be important in extinction [14, 23]. In Maren’s model of fear extinction, hippocampus sends inhibitory projections to PFC, while PFC sends inhibitory projections to amygdala [24]. According to this model, hippocampus could indirectly modulate the function of amygdala via the mPFC during fear extinction. Hippocampus may also modulate the function of amygdala directly, as there is a direct projection from the hippocampus to the amygdala [25]. Therefore, despite the central role of hippocampus in contextual fear memory, it could also play an important role in extinction process of cued fear memory as revealed in the present study, together with other studies, possibly via its influence on mPFC and amygdala directly or indirectly.
Acknowledgments: This study was supported by National Science Foundation of China (No. 81501149), Hainan special fund project for science and technology (No. 8
KJHZ2015-20) and the Natural Science Foundation of Shandong Province (No. ZR2015HQ004).
REFERENCES [1] K.M. Myers, M. Davis, Mechanisms of fear extinction, Mol Psychiatry 12(2) (2007) 120-50. [2] J.J. Kim, M.W. Jung, Neural circuits and mechanisms involved in Pavlovian fear conditioning: a critical review, Neurosci Biobehav Rev 30(2) (2006) 188-202. [3] R.G. Morris, NMDA receptors and memory encoding, Neuropharmacology 74 (2013) 32-40. [4] J.M. Langton, J.H. Kim, J. Nicholas, R. Richardson, The effect of the NMDA receptor antagonist MK-801 on the acquisition and extinction of learned fear in the developing rat, Learn Mem 14(10) (2007) 665-8. [5] E. Santini, R.U. Muller, G.J. Quirk, Consolidation of extinction learning involves transfer from NMDA-independent to NMDA-dependent memory, J Neurosci 21(22) (2001) 9009-17. [6] J. Ren, X. Li, X. Zhang, M. Li, Y. Wang, Y. Ma, The effects of intra-hippocampal microinfusion of D-cycloserine on fear extinction, and the expression of NMDA receptor subunit NR2B and neurogenesis in the hippocampus in rats, Prog Neuropsychopharmacol Biol Psychiatry 44 (2013) 25764. [7] F. Sotres-Bayon, D.E. Bush, J.E. LeDoux, Acquisition of fear extinction requires activation of NR2B-containing NMDA receptors in the lateral amygdala, Neuropsychopharmacology 32(9) (2007) 1929-40. [8] V. Laurent, R.F. Westbrook, Distinct contributions of the basolateral amygdala and the medial prefrontal cortex to learning and relearning extinction of context conditioned fear, Learn Mem 15(9) (2008) 657-66. [9] A. Pitkanen, M. Pikkarainen, N. Nurminen, A. Ylinen, Reciprocal connections between the amygdala and the hippocampal formation, perirhinal cortex, and postrhinal cortex in rat. A review, Ann N Y Acad Sci 911 (2000) 369-91. [10] M. Barad, P.W. Gean, B. Lutz, The role of the amygdala in the extinction of conditioned fear, Biol Psychiatry 60(4) (2006) 322-8. [11] S. Maren, W.G. Holt, Hippocampus and Pavlovian fear conditioning in rats: muscimol infusions into the ventral, but not dorsal, hippocampus impair the acquisition of conditional freezing to an auditory conditional stimulus, Behav Neurosci 118(1) (2004) 97-110. [12] W.N. Zhang, T. Bast, J. Feldon, The ventral hippocampus and fear conditioning in rats: different anterograde amnesias of fear after infusion of N-methyl-D-aspartate or its noncompetitive antagonist MK-801 into the ventral hippocampus, Behav Brain Res 126(1-2) (2001) 159-74. [13] George Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates.4TH.EDITION, Academic Press, Inc, San Diego, 1998. [14] M. Takita, S.E. Fujiwara, Y. Izaki, Functional structure of the intermediate and ventral hippocampo-prefrontal pathway in the prefrontal convergent system, J Physiol Paris 107(6) (2013) 441-7. [15] K.M. Myers, M. Davis, Behavioral and neural analysis of extinction, Neuron 36(4) (2002) 567-84. [16] A. Fischer, M. Radulovic, C. Schrick, F. Sananbenesi, J. Godovac-Zimmermann, J. Radulovic, Hippocampal Mek/Erk signaling mediates extinction of contextual freezing behavior, Neurobiol Learn Mem 87(1) (2007) 149-58. [17] G. Riedel, B. Platt, J. Micheau, Glutamate receptor function in learning and memory, Behav Brain Res 140(1-2) (2003) 1-47. [18] T. Frankiewicz, B. Potier, Z.I. Bashir, G.L. Collingridge, C.G. Parsons, Effects of memantine and MK-801 on NMDA-induced currents in cultured neurones and on synaptic transmission and LTP in area CA1 of rat hippocampal slices, Br J Pharmacol 117(4) (1996) 689-97. [19] P.H. Hutson, J.E. Hogg, Effects of and interactions between antagonists for different sites on the NMDA receptor complex on hippocampal and striatal acetylcholine efflux in vivo, Eur J Pharmacol 295(1) (1996) 45-52. [20] L. Calandreau, P. Trifilieff, N. Mons, L. Costes, M. Marien, A. Marighetto, J. Micheau, R. Jaffard, A. Desmedt, Extracellular hippocampal acetylcholine level controls amygdala function and promotes adaptive conditioned emotional response, J Neurosci 26(52) (2006) 13556-66. [21] S. Maren, G.J. Quirk, Neuronal signalling of fear memory, Nat Rev Neurosci 5(11) (2004) 844-52. [22] G.J. Quirk, E. Likhtik, J.G. Pelletier, D. Pare, Stimulation of medial prefrontal cortex decreases
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the responsiveness of central amygdala output neurons, J Neurosci 23(25) (2003) 8800-7. [23] K.A. Corcoran, G.J. Quirk, Recalling safety: cooperative functions of the ventromedial prefrontal cortex and the hippocampus in extinction, CNS Spectr 12(3) (2007) 200-6. [24] S. Maren, Building and burying fear memories in the brain, Neuroscientist 11(1) (2005) 89-99. [25] S. Maren, M.S. Fanselow, Synaptic plasticity in the basolateral amygdala induced by hippocampal formation stimulation in vivo, J Neurosci 15(11) (1995) 7548-64.
Figures
Fig 1
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Fig 2
Fig 3
11
Figure legends Fig. 1 Coronal drawings indicate the localization of injector tips (top to bottom: -4.8, 5.2, and -5.6 mm relative to bregma). Open circles: saline group; filled circles: 1.5 μg/0.5 μl group; open squares: 3 μg/0.5 μl group; filled squares: 6 μg/0.5 μl group.
Fig 2 (A) Trial-by-trial freezing (mean ± SEM) of rats during the experiment. Six CSUS pairings were given on day 1; MK-801 was infused 20 min before fear extinction training phase (12 CS-alone trials) as indicated by the arrow on day 2; Extinction test (10 CS-alone trials) on day 3. (B) Comparisons of freezing (mean ± SEM) between groups during the first trial of extinction training. (C) Comparisons of freezing (mean ± SEM) between groups during extinction training without the first trial. (D) Comparisons of freezing (mean ± SEM) between groups during extinction test. (* P < 0.05, ** P < 0.01, *** P < 0.001)
Fig 3 (A) Comparison of activity (mean ± SEM) between groups during 3 min before onset of the first tone on day 2. (B) Comparison of immobility (mean ± SEM) between groups during 3 min before onset of the first tone on day 2. (C) Comparison of immobility (mean ± SEM) between groups during 3 mins before onset of the first tone on day 3. (* P < 0.05)
12
S0166-4328(16)30953-6 http://dx.doi.org/doi:10.1016/j.bbr.2017.05.067 BBR 10917
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
28-10-2016 24-5-2017 30-5-2017
Please cite this article as: Zhang Bo, Li Chuan-Yu, Wang Xiu-Song.The effect of hippocampal NMDA receptor blockade by MK-801 on cued fear extinction.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2017.05.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The effect of hippocampal NMDA receptor blockade by MK-801 on cued fear extinction Running Title： Hippocampal NMDA receptor blockade in extinction Bo Zhang1^*, Chuan-Yu Li1^, Xiu-Song Wang2* 1 Kunming University of Science and Technology, Kunming, Yunnan 650500, China 2 Key Laboratory of Animal Resistance of Shandong Province, Department of Anatomy, Physiology and Development，College of Life Science, Shandong Normal University, Jinan 250000, China ^
The two authors contribute equally to this paper work
*Correspondence: [email protected]; [email protected]
1
Abstract Extinction of conditioned fear has been suggested to be a new form of learning instead of erasure of what was originally learned, and the process is NMDA (Nmethyl D-aspartate) receptor (NMDAR) dependent. Most of studies have so far revealed the important roles of NMDARs in the amygdala and medial prefrontal cortex (mPFC) in cued fear extinction. Although the ventral hippocampus has intimately reciprocal connections with the amygdala and mPFC, the role of its NMDARs in cued fear extinction remains unclear. The present experiment explored the issue by bilateral pre-extinction microinjection of the noncompetitive NMDAR antagonist MK-801 into the ventral hippocampus. Four groups of rats were given habituation, tone cued fear conditioning, fear extinction training and extinction test. Prior to extinction training, rats received bilateral infusions of either MK-801 (1.5, 3, or 6 μg/0.5 μl) or saline. Our results showed that MK-801 reduced freezing on the first trial of extinction training with no impact on within-session acquisition of extinction, and that the lower doses of MK-801 resulted in increased freezing on the extinction retrieval test. These findings suggest that ventral hippocampal NMDARs are necessary for the consolidation of tone cued fear extinction.
Key words: NMDA receptors; ventral hippocampus; consolidation; cued fear extinction
2
Pavlovian fear conditioning and extinction have been widely used as an animal model to investigate neurobiological mechanisms underlying anxiety and stressrelated disorders (e.g. post-traumatic stress disorder) in humans. In the conditioning paradigm, a conditioned stimulus (CS; e.g., tone or the context) is paired with an aversive unconditioned stimulus (US; e.g., foot shock), and after repeated training pairs, CS alone causes a conditioned response (CR; e.g., freezing). The fear extinction process involves reduction of CR by repeated presentation of CS without US, which is considered as a new learning process that inhibits initial fear memory [1]. However, it has been suggested that the mediating networks and possible neural mechanisms underlying fear conditioning remain largely distinct from those behind fear extinction [1, 2]. It is well known that the NMDA (N-methyl D-aspartate) receptors (NMDARs) are required for learning and memory [3], and moreover, studies with systemic injections of NMDA agonists or antagonists suggest its essential role in fear extinction [4, 5]. However, the precise process (acquisition, consolidation or retrieval) of fear extinction learning involving NMDARs remains unclear. While some reports suggest its involvement in acquisition process [6, 7], others have shown its importance in consolidation process [4]. Furthermore, most of later studies with local brain microinjections reveal the requirement of NMDARs in the lateral amygdala (LA) for extinction acquisition, and those in medial prefrontal cortex (mPFC) for consolidation process [7, 8]. As well known, the ventral hippocampus has intimately reciprocal connections with amygdala [9], which plays a central role in fear memory [10]. Also, there is the evidence indicating the involvement of the ventral hippocampus in cued fear conditioning [11], however, NMDAR blockade in the ventral hippocampal failed to impair cued fear conditioning [12]. Regarding the hippocampal NMDAR role in cued fear extinction, to our knowledge, only one related study showed that activation of NMDARs in the dorsal hippocampus facilitated acquisition and retrieval of extinction memory [6]. However, the effect of ventral hippocampal NMDAR blockade on cued fear extinction remains unclear. Therefore, the present study aimed to investigate this issue by bilateral microinjection of three doses of MK-801 (1.5, 3, or 6 μg/0.5 μl), a noncompetitive NMDAR antagonist, or saline into the ventral hippocampus of rats prior to cued fear extinction. Sprague-Dawley male rats (Kunming Medical College Animal Center, Kunming, China) weighing 280-320 g upon arrival were individually housed in transparent 3
polyethylene cages within a temperature- and humidity-controlled environment under a 12 h light/dark cycle (lights on from 7:00 AM to 7:00 PM). Four groups of animal (n=8 for each group) were used. Food and water were available ad libitum throughout the experiment. Experimental and animal care procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). After application of the local anesthetic lidocaine, the scalp was incised to expose the skull, and the bregma and lambda were aligned in the same horizontal plane. Stainless steel guide cannulas (length, 10 mm; diameter, 0.6 mm) were implanted bilaterally through small holes (diameter, 0.8 mm; coordinates: 5.2 mm posterior and 5 mm lateral to the bregma) drilled on each side of the skull. The tips of the guide cannulas were aimed above the ventral hippocampus at the following coordinates, 5.2 mm posterior and 5 mm lateral to the bregma, and 5 mm ventral to the dura, according to the brain atlas [13]. The guide cannulas were fixed with dental cement, and three small stainless steel screws used as anchors were screwed into the skull previously. Stainless steel stylets (diameter, 0.3 mm) that extended 0.5 mm beyond the tips of the guide cannulas were placed inside the guide cannulas to prevent occlusion. MK-801 (dizocilpine; Sigma, St. Louis, MO, USA) infusion solutions were prepared freshly on the day of injection, by been dissolved in isotonic 0.9% saline to a final concentration of 12 mg/ml. Three doses (1.5, 3, or 6 μg/0.5 μl) were adopted based on previous reports [12]. For microinjection procedure, firstly the stylets were removed from the guide cannula with rats manually restrained. Then, infusion cannulas (diameter 0.3 mm) which were connected to a 1 μl microsyringes (Shanghai Medical Laser Instrument Factory, Shanghai, China) mounted on a microinfusion pump (WZ-50C6, Medical Instrument Corporation of Zhejiang University, Hangzhou, Zhejiang, China), were inserted into the guide cannulas with its tip protruded 1.0 mm beyond the tip of guide cannulas. Rats were then bilaterally infused with 0.5 μl of MK-801 or saline with the speed 0.2 μl/min, and the infusion cannulas were left inside the guide cannulas for an additional 2 min after infusion for absorption of the drug, and then replaced by the stylets. Histological verification of cannula placement was performed after behavioral testing. Rats were anesthetized with ethyl carbamate (20% solution, 0.6 ml/100 g of body weight) and then perfused across the heart with physiological saline, followed by 10% formalin solution. Brains were postfixed in 10% formalin solution and then 30% sucrose solution, and subsequently sections (10 4
μm) were cut on a cryostat (Leica CM1850 UV, Leica Biosystems, Nussloch, BadenWürttemberg, Germany). After histological HE-staining, sections were examined on a light microscope for visualization of cannula tracts and injector tip localizations. One observation chamber without ceiling (40 cm × 40 cm × 50 cm) was placed in a sound-attenuating cabinet (50 cm × 50 cm × 60 cm), which was located in a brightly lit and isolated room. Walls of the chamber were constructed of black opaque plexiglas with white round spheres (4 cm in diameter) attached inside and spaced 3.5 cm apart. An infrared digital camera was attached to the roof and connected to a computer, to monitor and record rat behavior for behavioral scoring later. The US foot shocks (0.5 mA) were delivery by 25 stainless steel rods (diameter, 5 mm) of the floor, and the CS tones (5 kHz, 80 dB, 20 s) were delivered by a buzzer in the cabinet, which presentations were controlled by a computer program. The chamber was washed with 50% alcohol solution before the introduction of each subject. For behavioral procedures, after 1 week of recovery from surgery, rats were submitted to four experimental phases carried out over four consecutive days: habituation on day 0, fear conditioning on day 1, extinction training on day 2 and extinction test on day 3. Rats were put inside the chamber for 30 min with no stimuli presented during habituation. For fear conditioning, three minutes after being transported from their home cages and placed in the chamber, rats received six CS-US pairings with US foot shock presented 0.6 s before the ending and co-terminated with the CS tone, and with averaged inter-trial interval of 90 s (ranging from 60 to 120 s). Thirty seconds after the final shock, rats were returned to their home cages. During fear extinction training, firstly rats received drug infusion and were placed in the chamber 20 min later. Three minutes after rats being placed in the chamber, extinction training began, and 12 tones were delivered in the absence of US with the same inter-trial interval as training. On the following day, the extinction test was given with the similar procedure to that of extinction training, with one exception of 10 tones delivered. Fear response to the CS was assessed by measuring freezing behavior, which was defined as the absence of all movement with the exception of movement related to respiration. Freezing was scored offline from the videos using a digital stopwatch by an experimenter who was blind to the treatment conditions, and quantified by computing the percentage of the time spent for freezing during each tone presentation. Immobility was the baseline freezing behavior during three minutes before onset of first tone on day 2 and day 3 and quantified by computing the percentage of the time spent for being immobile. 5
Statistical analyses were conducted using the SPSS software (SPSS Inc, Chicago, Illinois, USA). The trial-by-trial data were analyzed using two-way repeated analysis of variance (ANOVA), with the drug treatment being the between-subjects factor and the trial being the within-subjects repeated measures factor. Locomotion time, percentage of immobility and percentage of freezing measures presented in histograms were analyzed using one-way analysis of variance (ANOVA). Post hoc comparison was performed using the Turkey’s method and all data were represented as means ± SEM. Differences were considered significant at P < 0.05.
Fig. 1
Fig. 2 Localization of injector tips was shown in Fig. 1. The trial-by-trial data for freezing behavior of subjects during fear conditioning, extinction training and extinction test were shown in Fig. 2A. A two-way repeated ANOVA revealed no significant difference between groups during conditioning on day 1 (F(3,27) = 0.57, P = 0.64). A one-way ANOVA revealed no significant difference in freezing response between groups during the last trial (F(3,27) = 0.733, P = 0.542), suggesting reaching the same conditioning level in all groups. However, there was a significant difference between trials (F(5,135) = 165.71, P < 0.001), suggesting the fast establishment of fear conditioning. During extinction training on day 2, a two-way repeated ANOVA revealed significant differences between trials (F(11,308) = 6.777, P < 0.001), but not between groups (F(3,28) = 2.042, P = 0.131), and also revealed significant trial × treatment interaction (F(33,308) = 2.317, P < 0.001), suggesting a differential treatment effect between groups across trials (Fig. 2A, also see Fig.2C). In order to evaluate the effects of MK-801 in each trial, a one-way ANOVA was used. During the first trial, a oneway ANOVA revealed significant differences between groups (F(3,28) = 12.514, P < 0.001). Further post hoc comparison showed significant differences between saline and all drug groups (all Ps < 0.01), but no significant differences among drug groups (all Ps > 0.05) (also see Fig. 2B). Similarly, ANOVA also revealed significant differences between groups during the following two trials (all Ps < 0.05), and further post hoc comparison showed significant differences between saline and the high dose group (all Ps < 0.05). For the remaining trials, there were no significant differences 6
between groups (all Ps > 0.05), possibly suggesting that MK-801 had no effects on fear extinction acquisition. There were two possible explanations for the results of first three trials. One possibility was that MK-801 could impair retrieval of cued fear conditioning memory. However, an alternative possibility was MK-801 induced hyperlocomotion as indicated in one previous report [12]. To clarify these two possibilities, the activity data during three minutes before the onset of first tone on day 2 were analyzed. A one-way ANOVA revealed significant drug effects on locomotion activity (F(3,27) = 3.317, P = 0.035), and further post hoc comparison only revealed significantly higher level of locomotion in high dose group compared with saline group (P = 0.03) (Fig. 3 A). The result indicated that MK-801 induced hyperlocomotion in the high dose group. This drug effect contributed to the significant low immobility in high dose group as shown in Fig. 3 B (F(3,27) = 3.317, P = 0.035). Taken together, for both lower dose groups, the first possibility could mainly contribute to our results, but for the high dose of group, two possibilities could coexist. During extinction test, a two-way repeated ANOVA revealed significant differences between groups (F(3,28) = 3.174, P = 0.04), trials (F(9,252) = 10.091, P < 0.001), but no significant trial × treatment interaction (F(27,252) = 0.739, P = 0.825) (Fig. 2A). Further post hoc comparison only revealed significant differences between saline and two low dose groups (all Ps < 0.05). Results indicated impairment of extinction consolidation by those two doses, reflected by significantly higher freezing response of subjects in those groups compared with saline group (also see Fig. 2D). This result was consistent with another previous report adopting systemic drug administration [4], which together supports the critical role of NMDARs in consolidation of fear extinction. Moreover, together with another hippocampal NMDAR activation study [6], these results indicate the important role of hippocampal NMDARs in cued fear extinction process. Considering the central role of mPFC in extinction consolidation and bidirectional connections between hippocampus and PFC [14], hippocampal NMDAR might take effect through its cooperation with PFC. However, it remains unclear why the subjects in the high dose group showed no impairment of extinction memory. One possibility could be that the MK-801 induced hyperlocomotion interfered with its potential influence on extinction memory consolidation. Further studies are required to clarify or test this issue. Moreover, in order to rule out contextual fear as a potential confound, due to the fact that the same chamber was used during conditioning and extinction in the present study, immobility 7
data during three minutes before the onset of the first tone on day 3 were analyzed. A one-way ANOVA revealed no significant differences between groups (F(3,28) = 0.807, P = 0.501) and subjects in all groups showed low immobility, suggesting no significant contextual influence on conditioned fear response (Fig. 3C). Taken together, the present study only showed impairment of extinction consolidation but not within-session acquisition. The present result was consistent with the idea suggesting a shift from NMDAR independent short-term memory formation to NMDAR dependent consolidation. Moreover, the underlying possible mechanism has been also suggested previously [15, 16]. MK-801 could take effect by its influence on long-term potentiation (LTP) and synaptic plasticity, which plays central roles in learning and memory [17]. For example, a previous study showed MK-801 blocked the induction of LTP in rat hippocampal slices [18]. Additionally, it has been shown that administration of MK-801 increased acetylcholine (Ach) release in hippocampus [19], and that elevated levels of the hippocampal Ach disrupted cued fear conditioning [20]. Therefore, MK-801 might also take effect indirectly through its influence on Ach release in hippocampus. Key brain regions involved in fear extinction have been elucidated, indicating the central role of amygdala in acquisition while the PFC in consolidation process [21]. Furthermore, fear extinction involves interactions between cortical and subcortical brain areas, for example, PFC could regulate the expression of fear extinction by inhibiting the amygdala [22]. Additionally, studies reveal strong functional connections between hippocampus and mPFC, which has been suggested to be important in extinction [14, 23]. In Maren’s model of fear extinction, hippocampus sends inhibitory projections to PFC, while PFC sends inhibitory projections to amygdala [24]. According to this model, hippocampus could indirectly modulate the function of amygdala via the mPFC during fear extinction. Hippocampus may also modulate the function of amygdala directly, as there is a direct projection from the hippocampus to the amygdala [25]. Therefore, despite the central role of hippocampus in contextual fear memory, it could also play an important role in extinction process of cued fear memory as revealed in the present study, together with other studies, possibly via its influence on mPFC and amygdala directly or indirectly.
Acknowledgments: This study was supported by National Science Foundation of China (No. 81501149), Hainan special fund project for science and technology (No. 8
KJHZ2015-20) and the Natural Science Foundation of Shandong Province (No. ZR2015HQ004).
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Figure legends Fig. 1 Coronal drawings indicate the localization of injector tips (top to bottom: -4.8, 5.2, and -5.6 mm relative to bregma). Open circles: saline group; filled circles: 1.5 μg/0.5 μl group; open squares: 3 μg/0.5 μl group; filled squares: 6 μg/0.5 μl group.
Fig 2 (A) Trial-by-trial freezing (mean ± SEM) of rats during the experiment. Six CSUS pairings were given on day 1; MK-801 was infused 20 min before fear extinction training phase (12 CS-alone trials) as indicated by the arrow on day 2; Extinction test (10 CS-alone trials) on day 3. (B) Comparisons of freezing (mean ± SEM) between groups during the first trial of extinction training. (C) Comparisons of freezing (mean ± SEM) between groups during extinction training without the first trial. (D) Comparisons of freezing (mean ± SEM) between groups during extinction test. (* P < 0.05, ** P < 0.01, *** P < 0.001)
Fig 3 (A) Comparison of activity (mean ± SEM) between groups during 3 min before onset of the first tone on day 2. (B) Comparison of immobility (mean ± SEM) between groups during 3 min before onset of the first tone on day 2. (C) Comparison of immobility (mean ± SEM) between groups during 3 mins before onset of the first tone on day 3. (* P < 0.05)
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