Behavioural Brain Research 271 (2014) 177–183

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Nitric oxide facilitates active avoidance learning via enhancement of glutamate levels in the hippocampal dentate gyrus Shi Wang a,1 , De-Xi Pan a,b,1 , Dan Wang a , Peng Wan a , De-Lai Qiu a , Qing-Hua Jin a,∗ a Department of Physiology and Pathophysiology, Yanbian University College of Medicine and Cellular Function Research Center, Yanbian University, 977 Gongyuan Road, Yanji 133002, Jilin Province, China b China-Japan Union Hospital of Jilin University, 126 Xiantai Street, Changchun 130033, Jilin Province, China

h i g h l i g h t s • Role of NO of DG in active avoidance learning is studied in freely moving rats. • We examine the effects of l-NMMA or SNP on Glu and fEPSP in DG during avoidance learning. • NO facilitates the learning via enhancements of glutamate level and synaptic efficiency in DG.

a r t i c l e

i n f o

Article history: Received 24 January 2014 Received in revised form 3 June 2014 Accepted 6 June 2014 Available online 13 June 2014 Keywords: Nitric oxide Hippocampal dentate gyrus Active avoidance learning Glutamate level Synaptic efficiency

a b s t r a c t The hippocampus is a key structure for learning and memory in mammals, and long-term potentiation (LTP) is an important cellular mechanism responsible for learning and memory. Despite a number of studies indicating that nitric oxide (NO) is involved in the formation and maintenance of LTP as a retrograde messenger, few studies have used neurotransmitter release as a visual indicator in awake animals to explore the role of NO in learning-dependent long-term enhancement of synaptic efficiency. Therefore, in the present study, the effects of l-NMMA (a NO synthase inhibitor) and SNP (a NO donor) on extracellular glutamate (Glu) concentrations and amplitudes of field excitatory postsynaptic potential (fEPSP) were measured in the hippocampal dentate gyrus (DG) region during the acquisition and extinction of activeavoidance behavior in freely-moving conscious rats. In the control group, the extracellular concentration of Glu in the DG was significantly increased during the acquisition of active-avoidance behavior and gradually returned to baseline levels following extinction training. In the experimental group, the change in Glu concentration was significantly reduced by local microinjection of l-NMMA, as was the acquisition of the active-avoidance behavior. In contrast, the change in Glu concentration was significantly enhanced by SNP, and the acquisition of the active-avoidance behavior was significantly accelerated. Furthermore, in all groups, the changes in extracellular Glu were accompanied by corresponding changes in fEPSP amplitude and active-avoidance behavior. Our results suggest that NO in the hippocampal DG facilitates active avoidance learning via enhancements of glutamate levels and synaptic efficiency in rats. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The hippocampus is a critical site for several learning and memory processes including avoidance learning [1–3], and a number of studies have stressed the importance of long-term potentiation (LTP) in learning and memory formation [4–6]. Hippocampal LTP is a widely studied cellular model of synaptic plasticity and

∗ Corresponding author. Tel.: +86 433 2435131; fax: +86 433 2435104. E-mail addresses: [email protected], [email protected] (Q.-H. Jin). 1 These authors contributed to the work equally and should be regarded as co-first authors. http://dx.doi.org/10.1016/j.bbr.2014.06.011 0166-4328/© 2014 Elsevier B.V. All rights reserved.

is believed to underlie declarative forms of learning [7]. In the hippocampus, the induction of LTP is typically dependent on the activation of N-methyl-d-aspartate (NMDA)-type glutamate receptors, a consequent increase in Ca2+ influx into the postsynaptic cells [7,8], and a number of Ca2+ -activated biochemical processes in the postsynaptic neurons, including nitric oxide (NO) formation [9,10]. Furthermore, the induction and maintenance of LTP require that a retrograde messenger released from the postsynaptic cell acts on presynaptic terminals, where it enhances transmitter release [11]. Nitric oxide (NO), a gaseous free radical is an important neuronal messenger in the central nervous system (CNS) and is synthesized from l-arginine, molecular oxygen, and NADPH by NO synthases (NOS) [12,13]. Several lines of evidence indicate that NO is involved

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in various forms of synaptic plasticity, including hippocampal LTP, as an intercellular retrograde messenger [10,14–16]. However, because these experiments were conducted in in vitro hippocampal slices, in which neuronal connectivity is decreased and the extracellular milieu is significantly altered compared to in vivo preparations, the results may not faithfully reflect the natural functioning of the organism. Behavioral studies have revealed that NO has modulatory effects on learning and memory processes [17–19]. For instance, hippocampal NO is involved in spatial learning and inhibitory avoidance tasks in rats [3,20,21]. This raises the possibility that NO is involved in learning-dependent longterm enhancement of synaptic efficiency (LD-LTE) as a retrograde messenger. However, these studies did not consider the possibility of using both neurotransmitter release and synaptic efficiency as read-outs to elucidate the role of NO in conscious, freely moving animals. Although chronic in vivo recordings in freely moving animals are more difficult and time-consuming than in vitro recordings, the results obtained may provide the most accurate and holistic view of the functioning brain. The dentate gyrus (DG), a hippocampal subregion, plays a critical role in learning and memory, and both computational modeling and physiological evidence indicate that the DG is important for encoding memory processes [22–25]. The perforant path (PP) is the main excitatory afferent pathway to the hippocampus. The response of DG granule cells to PP stimulation and the expression of LTP in this pathway vary with the behavioral state [26,27]. Although it has been demonstrated that nNOS is highly concentrated within the hippocampal DG region [12], the role of NO within the DG in the active avoidance learning is rarely studied. Therefore, in the present study, we microinjected NG -methyl-l-arginine acetate salt (l-NMMA, an inhibitor of NO synthase) or sodium nitroprusside (SNP, a NO donor) directly into the DG region, then measured both glutamate (Glu) release – using an in vivo brain microdialysis technique – and synaptic efficiency in the DG, during the acquisition and extinction of active-avoidance behavior in conscious, freelymoving rats. 2. Materials and methods 2.1. Animals Male Wistar rats weighing 180–220 g (Vital River Laboratories, Beijing, China) were used. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the animals’ suffering, and the minimum number of animals was used. 2.2. Drugs l-NMMA and SNP (both from Sigma, USA) were dissolved in modified Ringer’s solution (147 mM NaCl, 4 mM KCl, 2.3 mM CaCl2 ; pH 6.5) at concentrations of 1 mM and 0.1 mM, respectively, and stored at 4 ◦ C. 2.3. Surgical procedures Rats were anesthetized with 10% chloral hydrate (300 mg/kg, i.p.) and placed on a stereotaxic frame (David Kopf, USA). A guide cannula (0.90 mm OD) affixed a 20-gauge stainless steel tubing (Terumo, Japan) was stereotaxically implanted 1.5 mm above the DG region according to the atlas of Paxinos and Watson [28], and sealed with a dummy cannula after implantation. The stereotaxic coordinates were 3.4 mm posterior to bregma, 2.2 mm lateral to the midline, and 2.5 mm ventral to the dural surface. A bipolar stimulating electrode (made of epoxylite-coated stainless steel; A-M

Systems, Inc., USA) was lowered into the ipsilateral perforant path (PP; 6.8 mm posterior to bregma, 4.0 mm lateral to the midline, 5.0 mm ventral to the dural surface), and the guide cannula and stimulating electrode were fixed to the skull by dental cement. The animals were individually housed with access to food and water and allowed to recover from surgery for 3 days. On the day before the experiment, the animal was anesthetized with isoflurane (2.5–3.0% in 100% oxygen) and the dummy cannula was replaced with a microdialysis probe bonded to microinjection tubing. To reach the DG region, the tip of the microdialysis probe, covered with a 1.5-mm long hollow fibers (200 ␮m OD, acetate cellulose membrane, cut-off 5.0 × 104 mol wt; Eicom, Japan), was set to extend 1.5 mm beyond the guide shaft. A monopolar recording electrode (stainless steel pin; A-M Systems, Inc, USA) inserted into the 20-gauge stainless steel tubing reached the DG region, where extracellular field potentials evoked by stimulation of the PP were recorded. Ground and reference electrodes consisting of uninsulated stainless steel machine screws were positioned contralaterally on the skull surface at locations corresponding to the parietal cortex. The recording electrode was lowered until the maximal evoked response was visually confirmed, and then fixed by dental cement.

2.4. Experimental procedure The animals were divided into the following groups: (1) nonbehavioral test groups: 1 ␮L of the drug solution (modified Ringer solution; l-NMMA; SNP) was microinjected into the DG region, and the extracellular concentrations of Glu and amplitudes of fEPSP were examined; (2) l-NMMA groups: on every session of behavioral test, 1 ␮L of the drug solution (modified Ringer solution; l-NMMA) was microinjected into the DG region 10 min before the test, and the Glu concentrations and fEPSP amplitudes were examined 10 min after behavioral test; (3) SNP groups: on every day of behavioral test, 1 ␮L of the drug solution (modified Ringer solution; SNP) was microinjected into the DG region 30 min before the test, and the Glu concentrations and fEPSP amplitudes were examined 10 min after behavioral test. On the day of experiments, the collection of dialysates for Glu and measurement of fEPSP amplitudes were carried out under freely-moving conditions. The microdialysis probe was perfused with modified Ringer’s solution at a constant rate of 1.5 ␮L/min, and the dialysate from the DG region was automatically collected by a fraction collector (EFC-82, Eicom, Japan) at 4 ◦ C every 10 min. Three consecutive dialysate samples were collected to measure the Glu concentration. The measurement of fEPSP amplitudes was performed simultaneously: the PP was stimulated 10 times by single-phase square wave pulses (0.1 ms/phase, intensity was chosen to elicit 50% of the maximal fEPSP, interval was 30 s) generated with The Flexible Stimulus Isolator (ISO-Flex, A.M.P.I., Israel). Evoked responses were filtered (0.5–2.0 kHz) and amplified (1000×) by an AC amplifier (Neurolog, Digitimer, UK), digitized (Micro3, CED, UK), and analyzed on a computer with Spike2 software (CED, UK). 10 fEPSP traces were averaged to obtain the mean amplitude. Glu levels were measured using high-performance liquid chromatography with electrochemical detection (HTEC-500, Eicom, Japan), as described previously [29]. Briefly, an o-phthalaldehyde (OPA) solution (40 mM) was made by adding 13.5 mg of OPA and 10 ␮L of 2-mercaptoethanol to 2.5 mL of 0.1 M K2 CO3 buffer (pH = 9.5) with 10% ethanol. The solution was then stored at −4 ◦ C and diluted in 0.1 M K2 CO3 to yield a 4 mM OPA solution just before detection. The dialysate (12 ␮L) was mixed with 3 ␮L of the 4 mM OPA solution and allowed to react for 2.5 min at 25 ◦ C incubation. After completing the reaction, 10 ␮L of the reaction mixture was

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Table 1 Basal levels of Glu concentration in the hippocampal DG. Groups Non-behavioral test Control l-NMMA SNP Behavioral test Control of l-NMMA l-NMMA Control of SNP SNP

n

Glu (␮M)

7 7 7

2.06 ± 0.09 2.21 ± 0.19 1.99 ± 0.17

7 7 7 7

2.11 2.33 2.17 2.25

± ± ± ±

0.19 0.47 0.21 0.23

applied to the HTEC-500. The elution buffer for Glu consisted of 0.1 M phosphate buffer, 30% methanol, and 0.5 mM EDTA (pH 6.5). At the end of each experiment, rats were killed via an overdose injection of chloral hydrate and brains were fixed in 10% neutral buffer formalin. The implantation site of the dialysis probe and electrode were verified histologically in 40 ␮m coronal sections after cresyl violet staining. 2.5. Behavioral tests The active avoidance learning was measured using a shuttle box (RD1106-SB, YiShu, China) that consisted of two compartments. In order to avoid an electric foot shock, the animal had to move from one compartment to the other when a buzzer sounded. A session for the acquisition of active avoidance conditioning was performed using the following parameters: 5 s buzzer, 10 s foot shock (shock intensity was set to a level at which all rats were startled), 20 s intervals, 20 trials each day. When the rate of avoidance reached the acquisition criterion (upward of 70%), the experimental extinction session was started using the same parameters in the absence of a foot shock. The extinction criterion for this task was that the avoidance rate was reduced to 30%. 2.6. Data analysis All data are expressed as mean ± SEM, and statistical analysis was performed with SPSS 17.0 software. A comparison of baseline Glu values between the experimental groups was carried out using a one-way ANONA followed by an LSD t-test; other data were analyzed using a one-way ANOVA followed by the Dunnett test. A value of P < 0.05 was considered statistically significant. 3. Results In the present study, the ‘basal level’ is the average of Glu concentrations across three consecutive dialysate samples before the treatment. The basal levels are shown in Table 1 and are not significantly different between the groups. 3.1. Effects of l-NMMA and SNP in non-behavioral test groups The effects of local microinjection of l-NMMA or SNP on Glu levels in the hippocampal DG region are summarized in Fig. 1A. In control group, microinjection of modified Ringer’s solution did not affect Glu levels in the DG region. In contrast, Glu levels in the DG region showed an immediate decrease upon the local microinjection of l-NMMA, reaching 35.11 ± 12.30% of the basal level (P < 0.05) before gradually returning to basal levels. In addition, the microinjection of SNP elicited a significant increase in Glu levels at 30 min post-injection, reaching 353.84 ± 100.09% of the basal level (P < 0.05) before rapidly returning to basal levels. To examine the effects of l-NMMA and SNP on synaptic efficacy in the DG region, the fEPSP amplitude was measured synchronously.

Fig. 1. Effects of local microinjection of l-NMMA or SNP on Glu level (A) and fEPSP amplitude (B) in the hippocampal DG in non-behavioral test groups. Glu levels and fEPSP amplitudes are expressed as percentages of values obtained before microinjection. Arrowhead shows the onset of microinjection. Inset: solid line, before microinjection; dotted line, 30 min after microinjection (left: l-NMMA, right: SNP). Data are mean ± SEM. * P < 0.05 compared with before microinjection, # P < 0.05 compared with control group.

The fEPSP amplitude in the DG region was decreased by microinjection of l-NMMA, whereas local administration of SNP enhanced (Fig. 1B). Interestingly, the changes of Glu in the DG were paralleled with changes in fEPSP amplitude. Based on these results, we decided the time point of drug’s administration in the behavioral test groups including l-NMMA and SNP groups. 3.2. Effects of l-NMMA in behavioral test groups Due to the limitation of the in vivo microdialysis technology, in our experiment, the maximal measure period of the Glu is 10 days. In order to complete the acquisition and extinction of activeavoidance behavior within 10 days, the period of acquisition is a maximum of 7 days. In our pre-experiment, the active avoidance conditioning was difficult to establish within 7 d of l-NMMA injection. Therefore, we carried out the behavioral tests twice a day (interval of 8 h) in l-NMMA groups and measured Glu level and fEPSP amplitude in the DG region during the second session of behavioral testing on each day. In the control of l-NMMA group, rats reached the acquisition criterion on the third day (the rate of avoidance increased to 83.89 ± 8.19%), after which the extinction procedure was carried out immediately. Rats reached the criterion of extinction on the fifth day (the rate of avoidance returned to 29.89 ± 15.20%) (Fig. 2A). Moreover, in this group, extracellular levels of Glu (Fig. 2B) and fEPSP amplitudes (Fig. 2C) in the DG region were significantly

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Fig. 2. Effects of local microinjection of l-NMMA on active avoidance learning (A), Indicates the Glu level (B) and fEPSP amplitude (C) in the hippocampal DG. time at which the avoidance rate reached the criterion of acquisition, and indicates the time at which the avoidance rate reached the criterion of extinction. Glu levels and fEPSP amplitudes are expressed as percentages of values obtained on the first day. Data are mean ± SEM. * P < 0.05 compared with first day, # P < 0.05 compared with control group.

increased (P < 0.05, respectively) during the acquisition of the active avoidance behavior and gradually returned following extinction training. These results show that changes of Glu in the DG were accompanied by changes in fEPSP, and that both changes correspond roughly to the avoidance rate. In l-NMMA group, rats did not acquire the conditioned reflex within 5 days, although the maximal rate of avoidance reached 21.39 ± 2.44% (Fig. 2A). In this group, Glu levels were significantly lower on the third (P < 0.05) and fourth (P < 0.05) days as compared to the control group, and returned to baseline levels on the fifth day (Fig. 2B). Similarly, fEPSP amplitude was reduced on the third day but returned to baseline levels on the fourth day (Fig. 2C).

Fig. 3. Effects of local microinjection of SNP on active avoidance learning (A), Glu level (B) and fEPSP amplitude (C) in the hippocampal DG. Indicates the time indicates at which the avoidance rate reached the criterion of acquisition, and the time at which the avoidance rate reached the criterion of extinction. Glu levels and fEPSP amplitudes are expressed as percentages of values obtained on the first day. Data are mean ± SEM. * P < 0.05 compared with first day.

3.3. Effects of SNP in behavioral test groups The effects of SNP microinjection on Glu levels and fEPSP amplitudes within the hippocampal DG during the active avoidance learning are shown in Fig. 3. In control of SNP group, rats reached the acquisition criterion on the sixth day (the rate of avoidance increased to 80.83 ± 1.54%) and the extinction criterion was reached on the eighth day (the rate of avoidance returned to 17.50 ± 3.10%); in contrast, rats in SNP group reached the acquisition criterion on the fifth day (the rate of avoidance increased to 85.00 ± 4.47%) and the extinction criterion on the eighth day (the rate of avoidance returned to 10.00 ± 3.10%) (Fig. 3A). In addition,

S. Wang et al. / Behavioural Brain Research 271 (2014) 177–183 Table 2 The number of training trial during the acquisition and extinction of activeavoidance behavior. Groups

Acquisition

Extinction

Control of SNP SNP

125.76 ± 3.30 96.70 ± 5.76*

33.34 ± 2.65 51.66 ± 5.81*

*

P < 0.05 compared with control group.

compared with control group, the number of training trial required to reach the acquisition criterion was decreased, and the number required to reach the extinction criterion was increased in SNP group (Table 2). These results demonstrate that SNP in the DG region promotes the acquisition and suppresses the extinction of the active avoidance behavior. In both groups, extracellular Glu levels and fEPSP amplitudes in the DG region were significantly increased (P < 0.05, respectively) during the acquisition of the active avoidance behavior and gradually returned following extinction training. The data also revealed that changes of Glu concentration were accompanied by fEPSP amplitude, and that both changes roughly correspond to the avoidance rate. However, the maximal changes in Glu level, fEPSP amplitude, and avoidance rate during the conditioned reflex were not significantly different between the two groups (Table 3). 4. Discussion The results of the present study demonstrated that microinjection of the non-selective NOS inhibitor l-NMMA into the hippocampal DG region inhibited the acquisition of activeavoidance behavior and simultaneously reduced both local Glu release and fEPSP amplitude during avoidance training. Additionally, microinjection of SNP (a NO donor) in the DG region facilitated the acquisition and suppressed the extinction of active-avoidance behavior. Furthermore, these behavioral changes were associated with alterations in Glu levels that paralleled with LD-LTE. Since the first demonstration in cerebellar granule cells that NO could act as a neuronal messenger [30], NO has been reported to be involved in various physiological activities as a non-conventional neurotransmitter, and considerable evidence has shown that NO in the hippocampus plays an important role in LTP and consequent learning and memory [15,31–33]. Several studies have reported that systemic administration of NOS inhibitors impairs spatial learning and reference memory formation in rats [34–36]. Moreover, it has been reported that local administration of NOS inhibitors in the hippocampus significantly disrupts memory processes including inhibitory avoidance learning, spatial learning and object recognition task [3,20,21,37]. Previous studies have shown that the hippocampal DG region is an important site for several learning and memory processes. For instance, the survival of new neurons in the DG region is enhanced by exposure to an enriched environment as well as by some forms of hippocampus-dependent learning [38]. In addition, the perforant path input to the DG is known to play a critical role in spatial pattern separation during spatial memory tasks [25]. In agreement with these studies, our results exhibited that microinjection of l-NMMA (a NOS inhibitor) into the hippocampal DG region inhibited the establishment of Table 3 The maximal changes of the experimental parameters during active-avoidance behavior. Groups

Rate of avoidance (%)

Control of SNP SNP

80.83 ± 1.54 85.00 ± 4.47

% of values obtained on the first day Glu levels

fEPSP amplitudes

265.65 ± 58.42 312.50 ± 59.52

266.29 ± 25.67 321.35 ± 56.22

181

a conditioned reflex, whereas SNP (a NO donor) facilitated its establishment and suppressed its extinction. Therefore, our results indicate that NO in the hippocampal DG region might have a facilitative effect on active avoidance learning. LTP is an activity-dependent modification of synaptic efficacy whose induction requires the activation of post synaptic neurons; this can be achieved via high frequency afferent stimulation [7]. Hippocampal LTP is a model system for investigating the mechanisms of long-term synaptic plasticity within the brain and its relevance in learning and memory [39]. Several studies have shown that hippocampal LTP appears during the establishment of a conditioned reflex and disappears upon extinction training. Moreover, both the maximal level of LTP and its complete extinction preceded the conditioned behavior, suggesting that the LTP was learningdependent [5,40,41]. In the present study, the Glu levels and fEPSP amplitudes in the DG did not change when the rats were placed in the shuttle box without avoidance test (data not shown), furthermore, the in vivo induction of long-term enhancement on synaptic efficiency correlated with the rats’ ability to perform a shuttle-box avoidance task, and, therefore, such long-term enhancement can be considered as learning-dependent. A wealth of evidence indicates that the expression of LTP requires both pre-synaptic [42,43] and post-synaptic [44,45] changes. For example, LTP induction in hippocampal CA1 region is initiated by the activation of postsynaptic AMPA and NMDA glutamate receptors, leading to increases of [Ca2+ ]i , and the activation of Ca2+ /CaMKII. Subsequently, NOS activity is increased, and NO is diffused retrogradely across the synaptic cleft, where it can augment transmission in the presynaptic cell [39,46–48]. Similarly, it has been shown that LTP in the hippocampal DG region also requires the activation of NMDA receptors [49,50], and that stimulation of PP results in an increase in the number of nNOSimmunoreactive neurons in the DG [51]. The general scheme for NO action in hippocampal LTP is that Ca2+ influx via NMDA receptors activates NOS, which synthesizes NO from l-arginine. Once synthesized, NO diffuses out of the postsynaptic cell and acts on soluble guanylyl cyclase in the presynaptic neuron, thereby switching on cGMP-dependent protein kinases and increasing the release of neurotransmitters necessary for the maintenance of LTP [52]. While a number of in vitro studies have reported that NO acts as a retrograde messenger during LTP induction [14–16,47,53,54], it remains unclear whether NO acts retrogradely in hippocampal LTP. This is because the absence of important afferents (such as inputs from the medial septum and the dorsal and median raphe) in hippocampal slices in in vitro experiments may cause a difference in results from in vivo and in vitro experiments [55]. Although several in vivo studies suggest that NO in hippocampus may be critical for the learning performance and learning-dependent LTP [41,56], this is the first study in which the effects of NO on both neurotransmitter release and LD-LTE have been directly measured in the same region. In our study, inhibition of NOS in the hippocampal DG region reduced local release of Glu and simultaneously inhibited the induction of LD-LTE. Furthermore, application of a NO donor to the DG region facilitated the induction and maintenance of LDLTE, and the local release of Glu was correlated with the LD-LTE. In addition, microinjection of l-NMMA decreased the Glu levels and synaptic efficacy in the hippocampal DG region, whereas local administration of SNP elicited increases in these parameters. These results indicate that NO in the hippocampal DG region is involved in LD-LTE as an intercellular retrograde messenger, where it may facilitate the presynaptic release of Glu. It is thought that the l-arginine-NO pathway is directly triggered by the activation of the NMDA but not AMPA receptors [57], and that the glutamate-NMDA receptor-NOS system-NOcGMP system plays an essential role in modulating learning-related synaptic plasticity such as learning-dependent LTP [58–60].

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Consistent with this, our previous results indicated that microinjection of MK-801 (an antagonist of NMDA receptor) into the hippocampal DG region decreased the local Glu release and synaptic efficacy, whereas CNQX (an antagonist of AMPA receptor) reduced only synaptic efficacy without influencing Glu levels [61,62]. In addition to Glu, several other neurotransmitter systems have been investigated with regard to the mechanisms underlying the functions of NO. Because an enhancement of the cholinergic tone antagonized memory impairment induced by a NOS inhibitor during inhibitory avoidance task in mice, it was suggested that this memory impairment was mediated by a reduction of the activity of central muscarinic cholinergic mechanisms [63]. Majlessi et al. have shown that serotonin depletion in rat hippocampus attenuated l-NAME-induced spatial learning deficits, suggesting a dependence of spatial memory formation on an interaction between NO and serotonin (5-HT) in the hippocampus [64]. Moreover, NO is well known for its ability to modulate the release of neurotransmitters such as acetylcholine, dopamine, GABA, 5-HT [65–67], thereby affecting synaptic plasticity [68]. Although our study demonstrates that NO in the hippocampal DG region may acts as a retrograde messenger in LD-LTE, alternative mechanisms through which NO participates in LD-LTE remain to be explored. In conclusion, the present study suggests that NO promotes the acquisition and suppresses the extinction of the active avoidance behavior via enhancements of glutamate levels and synaptic efficiency in the hippocampal DG. Acknowledgment This work was supported by grant 30760064 and 31160211 from the National Natural Science Foundation of China. References [1] Grecksch G, Bernstein HG, Becker A, Höllt V, Bogerts B. Disruption of latent inhibition in rats with postnatal hippocampal lesions. Neuropsychopharmacology 1999;20:525–32. [2] Lehmann H, Lacanilao S, Sutherland RJ. Complete or partial hippocampal damage produces equivalent retrograde amnesia for remote contextual fear memories. Eur J Neurosci 2007;25(5):1278–86. [3] Harooni HE, Naghdi N, Sepehri H, Rohani AH. The role of hippocampal nitric oxide (NO) on learning and immediate, short- and long-term memory retrieval in inhibitory avoidance task in male adult rats. Behav Brain Res 2009;201(1):166–72. [4] Manabe T. Molecular mechanism of hippocampal synaptic transmission, plasticity and memory. Mol Med 2004;14(9):1095–101. [5] Whitlock JR, Heynen AJ, Shuler MG, Bear MF. Learning induces long-term potentiation in the hippocampus. Science 2006;313:1093–7. [6] Fedulov V, Rex CS, Simmons DA, Palmer L, Gall CM, Lynch G. Evidence that long-term potentiation occurs within individual hippocampal synapses during learning. J Neurosci 2007;27(30):8031–9. [7] Bliss TVP, Collingridye GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 1993;361:31–9. [8] Bashir ZI, Alford S, Davies SN, Randall AD, Collingridge GL. Long-term potentiation of NMDA receptor-mediated synaptic transmission in the hippocampus. Nature 1991;349:156–8. [9] Malenka RC, Kauer JA, Perkel DJ, Mauk MD, Kelly PT, Nicoll RA, Waxham MN. An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation. Nature 1989;340(6234):554–7. [10] O’dell TJ, Hawkins RD, Kandel ER, Arancio O. Tests of the roles of two diffusible substances in long-term potentiation: evidence for nitric oxide as a possible early retrograde messenger. Proc Nat Acad Sci 1991;88:11285–9. [11] Bekkers JM, Stevens CF. Presynaptic mechanism for long-term potentiation in the hippocampus. Nature 1990;346(6286):724–9. [12] Bredt DS, Glatt CE, Hwang PM, Fotuhi M, Dawson TM, Snyder SH. Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron 1991;7(4):615–24. [13] Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43(2):109–42. [14] Arancio O, Lev-Ram V, Tsien RY, Kandel ER, Hawkins RD. Nitric oxide acts as a retrograde messenger during long-term potentiation in cultured hippocampal neurons. J Physiol (Paris) 1996;90:321–2. [15] Bon CLM, Garthwaite J. On the role of nitric oxide in hippocampal long-term potentiation. J Neurosci 2003;23:1941–8.

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