Original article
Involvement of D₂/D₂ dopamine antagonists upon open-arms exploratory behaviours induced by intra-nucleus accumbens shell administration of N-methyl-D-aspartate

Introduction

Nucleus accumbens (NAc) is one of the main limbic system nuclei receiving rich dopaminergic inputs hence taking an important role in the regulation of many physiological cognitive and non-cognitive behaviours [39]. Anatomical studies have determined at least two main functionally important parts in NAc, i.e. the core and the shell [8,87]. With regard to the dopaminergic system, these two parts seem different. For instance, the dopamine plexus and concentration are richer and higher in the shell than in the core, respectively [20,80]. Evidence has indicated the pivotal role of NAc dopaminergic system in the modulation of learning, memory [11,14,43], fear and/or anxiety-like behaviours [50]. Dopamine exerts its effect via two different dopamine subtypes receptors, called the D₂- and D₂-like families [55]. These two dopamine family receptors have a high expression in both parts of NAc while at different distribution patterns. For example, the D₂- and D₂-like dopamine receptor families are higher and lower in the NAc shell than in core, respectively [30]. This may partly explain the different physiological functions of the two parts of NAc [40,42]. Due to the abundant dopaminergic inputs which NAc shell receives from various brain parts and the consequent high contraction level of dopamine in the NAc shell, this part is believed to render a critical role in dopamine-mediated functions [30]. The NAc receives its dopaminergic afferents from the ventral tegmental area (VTA) and the substantia nigra (SN), and the glutamatergic inputs from several brain areas such as prefrontal cortex, amygdala and hippocampus [30].
On the other hand, glutamate is known as one of the most excitatory neurotransmitters modulating learning, memory and anxiety-like behaviours in different parts of the brain [33,94]. With respect to the preferential agonists, at least three subtypes have been identified for glutamate, including N-methyl-D-aspartate (NMDA), AMPA and kainate [57]. Investigations have revealed that the NMDA receptor plays a critical part in regulation of learning, memory formation (possibly through long-term potentiation and depression) [9,38] and anxiety-related behaviours [26,46,63,73,78]. A plethora of anatomical experiments have demonstrated that there are close relationships between NAc glutamatergic and dopaminergic systems [66,67]. For instance, it has been shown that NMDA receptors are localized on dopamine D₂ receptor-containing neurons in the NAc shell [27].
Emotional states (including, fear and aversion) can be modulated through amplification of impairment in memory formation [51]. Due to possible misinterpretations, the available animal models for learning and memory seem to have a limited ability to detect the effect of drugs on fear and anxiety. Therefore, the proposed test-retest paradigm in the elevated plus-maze (EPM) task is an attempt to concomitantly assess the effects of drugs on anxiety, learning and memory in rodents [6]. The use of EPM in testing anxiety is based on the natural tendency of animals to avoid dangerous situations when they face height and open spaces [91]. In general, animals acquire information with regard to safe and dangerous areas in the maze upon test. Animals retested in the EPM avoid exploring open spaces and displaying a clear enclosed arm preference with a low percentage of entries and time spent in the open arms [59], relative to their respective measures during the test [10,22,44,74,79]. The aversive and fear-inducing nature of the open arms represents a useful tool for the study of aversively motivated learning processes in the EPM [16]. Based on the above, learning and memory are usually studied in the EPM through avoidance to open-arms during the retest session. Given this, the purpose of the current study was to examine the possible involvement of the NAc shell D₂ and/or D₂ dopaminergic receptors on NMDA-induced effects in the aversive memory using the elevated-plus maze (EPM) task.

Material and methods

Animals

Male Wistar rats weighing approximately 250-280 γ were provided by the Central Animal House of the Institute for Cognitive Science Studies (ICSS), Tehran, Iran. Prior to the experiments, animals underwent a period of seven days habituation in groups of five in polypropylene home cages (45 × 30 × 15 cm), having access to food and water ad libitum, under a light/dark cycle of 12 h (lights on at 06:00) and the temperature ranging between 20°C and 24°C. Animal handling was restricted to the time of home cage cleaning (each 48 h), weighing and drug administration. Each experimental group comprised eight animals. All experimental procedures were conducted in compliance with the recommendations set down by the Institute’s Ethics Commission for the use of experimental animals.

Stereotaxic surgery and microinjections

Animals were anaesthetized intraperitoneally using ketamine hydrochloride (50 mg/kg) and xylazine (4 mg/kg) then placed in a Stoelting stereotaxic instrument (Wood Dale, IL, USA). Two stainless steel guide cannulae (22 gauge) were implanted in the right and left of the NAc shell regions according to the atlas of Paxinos and Watson [58]. The stereotaxic coordinates for the NAc regions were as follows: +1.4 mm posterior to bregma, ±0.8 mm lateral to the Midline and –5.5 mm ventral to the dorsal surface of the skull. The cannulae were fixed to the skull using acrylic dental cement. Rats were allowed 5 days before the test to recover from surgery. The left and right of the NAc areas were infused by means of an internal cannula (27 gauge), terminating 2 mm below the tip of the guides and connected by polyethylene tubing to a 2 µL Hamilton syringe (Bonaduz, GR, Switzerland). A volume of 0.3 µL solution was injected over a 60-second period, in each side. The inner cannulae were left in place for an additional 60 seconds to allow diffusion of the solution and to reduce the possibility of reflux. Intra-NAc shell injections were made just five minutes before testing. Control groups and drug infused groups were surgeried and all of the animals were anaesthetized using a ketamine solution, therefore all of groups were under the same condition and under the same effect of ketamine anaesthesia.

Elevated plus-maze (EPM) apparatus

This plexiglas, plus-shaped apparatus, was set at 50 cm height from the floor. This apparatus was composed of two 50 × 10-cm open arms and two 50 × 10 × 40-cm enclosed arms, each with an open roof. The junction area of the four arms (central platform) measured 10 × 10 cm. The maze was placed at the centre of a quiet and dimly lit room [91,92].

Behavioural testing

Rats were placed in the experimental room at least 1 h before testing. All experiments were done during the light phase of the light/dark cycle between 11 a.m. and 2 p.m. Animals were randomly assigned to treatment conditions and tested in a counter-balanced order. Animals’ behaviours were tracked and recorded by an observer who quietly sat 1 m behind one of the closed arms of the maze, using a chronometer. During the five-minute post-drug treatment, rats were individually placed at the centre of the plus maze facing one of the open arms and allowed for 5 min free exploration in EPM (test session) then were taken back to their home cages. In 24 hours, rats were returned to the test room and placed again in the EPM for a new exploration period of 5 min (retest session). The observer measured: 1) time spent in open arms, 2) time spent in closed arms, 3) number of entries into open arms and 4) number of entries into closed arms during the five-minute period both upon test and retest. An entry was defined as ‘all four paws in the arm’. Between EPM sessions and after each rat the maze was cleaned with distilled water. The obtained data were used to calculate: a) % OAT (the ratio of time spent in open arms to the time spent in all arms ×100); b) % OAE (the ratio of entries into open arms to total entries ×100) [55,97,98], and c) the total closed and open arm entries were considered as a relatively pure index for the locomotor activity [93,95].
Drugs The drugs used in the present study were ketamine and xylazine (Alfasan Chemical Co, Woerden, Holland) for animal anaesthesia, NMDA (N-methyl-D-aspartic acid as NMDA receptor agonist, Tocris Cookson, Bristol, UK), SCH 23390 (as dopamine D₂ receptor antagonist) and sulpiride (as dopamine D₂ receptor antagonist). NMDA and SCH 23390 were dissolved in sterile 0.9% saline while sulpiride was dissolved in vehicle (the vehicle was one drop of glacial acetic acid from Hamilton microsyringe, made up to a volume of 5 ml with sterile 0.9% saline, then diluted to the required volume) just before the experiment. Control animals received either saline or vehicle. NMDA and SCH were administered into the shell of the nucleus accumbens at a volume of 0.3 µL/rat.

Drug treatments

Experiment 1: Effects of NMDA administration on open arms exploratory-like behaviours in the presence or absence of SCH 23390

To substantiate whether the microinjection of drugs involved in anxiety, the drug infusion took place before EPM testing. In this experiment, 12 groups of animals were examined. These were as follows: 1) animals which received intra-NAc shell saline (0.3 µL/rat) or NMDA (0.125, 0.25, and 0.5 µg/rat), 5 min after saline (0.3 µL/rat); 2) animals which received intra-NAc shell saline (0.3 µL/rat) or SCH (0.125, 0.25 and 0.5 µg/rat), 5 min before saline (0.3 µL/rat) and 3) animals which received intra-NAc shell saline (0.3 µL/rat) or the subthreshold dose of SCH (0.0125 µg/rat), 5 min before different doses of NMDA (0.125, 0.25, and 0.5 µg/rat).
To investigate the possible drug carryover effects of aversive learning during test day to aversive memory upon retest, treated groups were retested undrugged in EPM 24 h later.

Experiment 2: Effects of NMDA administration on open-arms exploratory-like behaviours in the presence or absence of sulpiride

To substantiate whether the microinjection of drugs involved in anxiety, the drug infusions were done before EPM testing. In this experiment, 12 groups of animals were examined. These included: 1) animals which received intra-NAc shell saline (0.3 µL/rat) or NMDA (0.125, 0.25, and 0.5 µg/rat), 5 min after saline (0.3 µL/rat); 2) animals which received intra-NAc shell vehicle (0.3 µL/rat) or sulpiride (0.125, 0.25 and 0.5 µg/rat), 5 min before saline (0.3 µL/rat) into NAc shell, and 3) animals which received intra-NAc shell vehicle (0.3 µL/rat) or the subthreshold dose of sulpiride (0.0125 µg/rat), 5 min before different doses of NMDA (0.125, 0.25, and 0.5 µg/rat).
To investigate the possible drug carryover effects of aversive learning during test day to aversive memory upon retest, treated groups were retested undrugged in EPM 24 h later.

Histology

Following the completion of behavioural testing, animals were euthanized using an overdose of chloroform. Ink (0.3 µL of 1% aquatic methylene blue solution) was injected into the guide cannulae using 27-gauge injection cannulae. Brains were then removed and fixed in a 10% formalin solution for 48 hours before sectioning. The brains were sliced using the vibro-slice apparatus in transverse planes (40 µm). Cannula placements were verified based on the corresponding map of Paxinos and Watson atlas of rodents’ brain [58]. Data from the animals in which injection sites were located outside the NAc shell were not included in the analyses.

Statistical analysis

Data were expressed as mean ± SEM and analyzed using the repeated measure protocol during test and retest days. In addition to the analysis made to compare test to test or retest to retest, the two-way analysis of variance (ANOVA) was also applied. Where F-value was significant, one-way ANOVA and post-hoc analysis (Tukey-test) were performed. Between-groups differences with p < 0.05 were considered statistically significant.

Results

Histology

Data from the animals in which injection sites were located outside the NAc shell were not included in the analyses and in the present study we used only data from animals that were cannulated clearly within-NAc-shell. Cannulae were implanted into the NAc shell of a total of 208 rats, however only the data from 192 animals with correct cannulae implants were included in statistical analyses.

Experiment 1 results

Effects of NMDA administration on open arms exploratory-like behaviours

Repeated measure and post hoc analyses demonstrated that NMDA increases the %OAT (at 0.25 and 0.5 µg/rat, as seen in Fig. 1; panel 1A and Fig. 2; panel 1A, respectively), %OAE (at 0.25 and 0.5 µg/rat as seen in Fig. 1; panel 1B and Fig. 2; panel 1B, respectively) and decreases the locomotor activity (non-significantly and significantly as seen in Fig. 1; panel 1C and Fig. 2; panel 1C, respectively), indicating an anxiolytic-like response to NMDA.
Adding to the above, data showed that NMDA increases the %OAT (at 0.25 and 0.5 µg/rat, as shown in Fig. 1; panel 2A and at 0.125 and 0.5 µg/rat in Fig. 2; panel 2A) and %OAE (at 0.25 and 0.5 µg/rat as shown in Fig. 1; panel 2B and at 0.125 and 0.5 µg/rat in Fig. 2; panel 2B). However, this did not alter the locomotor activity (Fig. 1, panel 2C and Fig. 2, panel 2C) upon retest as compared to the control group, indicating an NMDA-induced impairment of the aversive memory acquisition.
According to the above data, NMDA induced anxiolytic-like behaviours. Furthermore, the retest data suggested that the NMDA anxiolytic-like effect may also be linked to the impairment in further avoidance acquisition. The corresponding repeated measure results have been demonstrated in Table I and Table II.

Effects of intra-NAc shell microinjection of SCH 23390 on open arms exploratory-like behaviours

In this experiment two-way ANOVA analysis was done to assess the NMDA treated group dose-response as compared to controls as well as the interaction with SCH 23390 or sulpiride treatment. According to the repeated measure and Post hoc analyses, SCH 23390 increased the %OAT (at 0.25 µg/rat, Fig. 1; panel 3A), %OAE (at 0.25 and 0.5 µg/rat, Fig. 1, panel 3B) and decreased the locomotor activity (at 0.5 µg/rat, Fig. 1; panel 3C) upon test, indicating that SCH 23390 may induce an anxiolytic-like response.
Moreover, the data revealed that SCH 23390 increases the %OAT (at 0.5 µg/rat, Fig. 1; panel 4A), and %OAE (at 0.25 and 0.5 µg/rat, Fig. 1; panel 4B), while does not alter the locomotor activity (Fig. 1; panel 4C) on retest day as compared to the control group, indicating that SCH 23390 possibly impairs the aversive memory retrieval.
In conclusion, the data revealed that SCH 23390 induces an anxiolytic-like response. Besides, the increased %OAT upon retest indicates that SCH 23390-treated rats had their aversive memory to open-arm exploration negatively affected as compared to the control group. The corresponding repeated measure results have been summarized in Table I.

Effects of NAc shell microinjection of SCH 23390 prior to NMDA on open arms exploratory-like behaviours

Two-way ANOVA and post hoc analyses revealed that intra-NAc microinjection of SCH 23390 prior to NMDA causes a significant decrease in %OAT (at 0.125, 0.25 and 0.5 µg/rat, Fig. 1; panel 5A) while exerts no significant change in the %OAE and the locomotor activity (Fig. 1; panel 5B, 5C) on test day as compared to NMDA-treated groups (Fig. 1; panel 1A, 1B and 1C), indicating that SCH 23390 potentially reverses the anxiolytic-like response induced by the intra-NAc shell microinjection of NMDA.
On the other hand, this intervention resulted in a significant decrease in the %OAT (at 0.125, 0.25 and 0.5 µg/rat, Fig. 1; panel 6A) while leading to no significant change in %OAE and locomotor activity (Fig. 1; panel 6B, 6C) on retest day as compared to NMDA-treated groups (Fig. 1; panel 2A, 2B and 2C). The above indicates that SCH 23390 potentially restores the aversive memory impairment already induced by the intra-NAc shell microinjection of NMDA.
Having mentioned these, we may conclude that the intra-NAc shell injection of the subthreshold dose of SCH 23390 decreases the anxiolytic-like behaviours induced by the intra-NAc shell injection of NMDA, and meanwhile improves the aversive memory impairment. The two-way ANOVA results have been outlined in Table I.

Experiment 2 results

Effects of NAc shell microinjection of sulpiride on open arms exploratory-like behaviours

According to the repeated measure and post hoc analyses, the data showed that sulpiride does not alter the %OAT (Fig. 1; panel 3A) while increases the %OAE (at 0.25 and 0.5 µg/rat, Fig. 2, panel 3B) and decreases the locomotor activity (at 0.25 and 0.5 µg/rat, Fig. 2; panel 3C) upon test, indicating that sulpiride may induce an anxiolytic-like response. Moreover, the data revealed that sulpiride increases the %OAT (at 0.5 µg/rat, Fig. 2; panel 4A), %OAE (at 0.5 µg/rat, Fig. 2; panel 4B) whereas decreases the locomotor activity (at 0.5 µg/rat, Fig. 2; panel 4C) on retest day as compared to the control group, indicating that sulpiride possibly impairs the aversive memory retrieval.
In conclusion, the data revealed that sulpiride induces an anxiolytic-like response. Besides, the increased %OAT upon retest indicates that sulpiride-treated rats had their aversive memory to open-arm exploration negatively affected as compared to the control group. The corresponding repeated measure results have been summarized in Table II.

The effects of intra-NAc shell microinjection of sulpiride prior to NMDA on open arms exploratory-like behaviours

Two-way ANOVA and post hoc analyses demonstrated that the intra-NAc microinjection of sulpiride prior to NMDA causes a significant decrease in %OAT (at 0.25 and 0.5 µg/rat, Fig. 2; panel 5A) and %OAE (at 0.5 µg/rat, Fig. 2; panel 5B) while exerts no significant change in locomotor activity (Fig. 2; panel 5C) on test day as compared to NMDA-treated groups (Fig. 2; panel 1A, 1B and 1C). This indicates that sulpiride potentially reverses the anxiolytic-like response induced by intra-NAc shell microinjection of NMDA.
On the other hand, this intervention resulted in a significant decrease in %OAT (at 0.125, 0.25 and 0.5 µg/rat, Fig. 2; panel 6A) while leading to no significant change in %OAE and locomotor activity (Fig. 2; panel 6B, 6C) on retest day as compared to NMDA-treated groups (Fig. 2; panel 2A, 2B and 2C). The above indicates that sulpiride potentially restores the aversive memory impairment already induced by the intra-NAc shell microinjection of NMDA.
Based on these, we may conclude that the intra-NAc shell microinjection of the subthreshold dose sulpiride decreases the anxiolytic-like behaviours induced by the intra-NAc shell injection of NMDA, and meanwhile improves the aversive memory impairment. The corresponding two-way ANOVA results are outlined in Table II.

Discussion

In our study, animals were given pretest intracerebral drugs injection followed by no injection upon retest 24 h later. Based on this, drug effects on anxiety-like behaviours and aversive learning with subsequent long-term effects on memory in 24 h were tested. It has been reported that the prior experience of an undrugged EPM testing session may alter the behavioural responses in an undrugged retest session [13,74]. The injury caused by the injection might change the expression of messenger RNAs and proteins. But there is a study that investigated changes in the expression of messenger RNAs for trkA, trkB and trkC in the brain following an injury caused by insertion of a 30-gauge needle into adult rat hippocampus or neocortex. The increased levels of mRNA after the injury returned to control levels a few hours after the injury. Pretreatment of the animals with the ketamine completely prevented the changes of trkB and trkC messenger RNAs, suggesting that the brain injury caused a release of glutamate with subsequent activation of NMDA receptor [54]. In the present study, rats were allowed 5 days before the test to recover from surgery so the changes of mRNA levels after the injury may be returned to control levels after the recovery days.

Effects of intra-NAc shell NMDA administration on anxiolytic-like behaviours and aversive memory formation

Our results indicated that the intra-NAc shell infusion of NMDA receptor agonist at applied doses induces an anxiolytic-like response in EPM. This NMDA-induced anxiolytic-like effect emerges into the retest day. Current findings suggest that NMDA treatment induces impairment in the aversive memory acquisition upon test. There is a body of evidence supporting that NAc shell is an essential brain site regulating emotion, motor activity [17], motivation-related learning, memory [12,29], and anxiety-like behaviours [48,49,61]. On the other hand, the NMDA receptor (as an ionotropic glutamate receptor) plays a critical role in the regulation of glutamate-induced behaviours such as learning and memory formation (possibly through long-term potentiation and depression) [9,38], and anxiety-related behaviours [26,63].
Our results are in agreement with the previous investigation showing that NMDA agonist releases behavioural and anxiolytic-like behaviours indicating the role of NMDA receptors in modulation of anxiety-related behaviours [26,73]. Moreover, there is also an investigation showing that the activation of NMDA receptor induces anxiogenic-like effects in EPM and social interaction tasks [21].
Evidence has suggested the critical role of NAc in regulation of several learning functions which require a flexible use of sensory information [47,64, 65]. It has been postulated that NAc manipulations induce spatial memory deficit in the Morris water maze [64,68] and radial maze [24,70]. In agreement with our results, evidence has demonstrated that the systemic administration of NMDA leads to an impaired dark-avoidance learning in rats [89]. On the other hand, some investigations have postulated that the deactivation of the NAc glutamatergic ionotropic receptors disrupt the working memory [7,35, 41] and spatial responses [71] while other contradictory studies have shown that the NMDA receptor blockade in NAc shell does not alter the spatial learning [47,71]. Furthermore, it appears that the NAc shell is more involved in the regulation of spatial learning and memory as compared to the NAc core since the NAc shell (but not the core) lesions disrupt the spatial learning [35]. The NAc sends a dense GABA projection to the ventral pallidum (VP), and stimulation of either the NAc or its glutamatergic afferents can inhibit VP neuronal firing [23]. The VP can influence DA neural activity via a direct projection to the ventral tegmental area (VTA) [23]. Dopaminergic neurons projection of the VTA sends back to the NAc. Dopaminergic terminals arising from this area make synaptic contacts with NAc GABAergic neurons. According to the present results, stimulation of the NAc by injection of NMDA in this area would have increased firing of GABAergic projection neurons of the NAc causing a decrease in VP the GABAergic neural activity. The decrease in VP activity would then be expected to cause a reduction of the GABAergic inhibition over the VTA and this could alter an increased release of dopamine in NAc. The present data suggested that maybe glutamate exerts its function by affecting the release of dopamine. Several studies have indicated that dopaminergic system modulates the neuronal activities involved in fear or anxiety-like behaviours [25,60]. Several investigations have substantiated that the mesocortical DA system produces a robust and specific response to stressors [28]. Some investigations have suggested that these high levels of DA released under stress are above the optimal range for working memory and therefore impair this cognitive function [5,88].

Effect of intra-NAc shell dopamine D₂ and D₂ receptors antagonists injection on anxiolytic-like behaviours and aversive memory formation

It has been made clear that the NAc shell plays a critical part in modulation of dopamine-mediated functions as it contains the largest amount of dopaminergic terminals and thus the highest concentration of dopamine [20,27,80]. Therefore, the NAc dopaminergic system has a pivotal role in modulation of learning and memory [11,14,43], fear and/or anxiety [50]. The idea of investigating the role of NAc shell dopamine D₂ and D₂ receptors in NMDA-induced effects using the elevated plus-maze test, appealed to our interest.
Present results indicated that the intra-NAc shell microinjection of SCH 23390 (a dopamine D₂ receptor antagonist) increases both %OAT and %OAE whereas decreases the locomotor activity. On the other hand, inhibition of the dopamine D₂ receptor in NAc shell by sulpiride (a dopamine D₂ receptor antagonist) did not affect the %OAT, however; increased %OAE and decreased the locomotor activity. Several investigations have substantiated that the extensive D₂- and D₂-like receptors are on the presynaptic varicosities of medium-spiny neurons of nucleus accumbens [84]. Similar to the response seen with SCH 23390 and sulpiride on anxiolytic-like behaviours there is a study showing that dopamine D₂ and D₂ receptors blockade in the basolateral amygdala exerts anxiolytic-like behaviours [96]. Intra-NAc injection of SCH 23390 or sulpiride induced anxiolytic- and did not alter anxiety-like behaviours, respectively [90]. Furthermore, our present data indicated that beside the increased %OAT on the test day, the increased %OAT upon retest in both SCH 23390 and sulpiride show that these drug-induced anxiolytic-like effects emerge on the retest day. Current findings suggest that SCH 23390 and sulpiride treatments impair the aversive memory function upon retest. Moreover, evidence has reemphasized the crucial role of dopaminergic system in the regulation of several neural activities which are involved in learning and memory (see [34], for a review). For instance, it has been shown that the activation or deactivation of dopamine receptors provides a capability to learn and store information [1]. Other studies have demonstrated that the immediate post-training blockade of D₂ and D₂ receptors located within the NAc impairs the performance of spatial learning tasks [52]. Based on some other reports, the intra-dorsal hippocampal [62] and peripheral [2,15] administration of the antagonists impair the one-trial passive avoidance and spatial or non-spatial memories in mice, respectively. However, other investigations indicate that pre-test single administration of SCH 23390 or sulpiride causes no significant change in the step-down latency [56]. One study demonstrated that inhibition of the dopamine exocytosis from pre-synaptic neuron via Ca2+-channel blockade by SKF96365 decreases anxiolytic-like behaviours induced by sulpiride in the NAc shell region indicating the involvement of the pre-synaptic dopamine D₂ receptors in sulpiride induced anxiolytic-like behaviours [3]. However, blockade of pre-synaptic dopamine D₂ receptors increases the presynaptic release of dopamine, which in turn induced anxiolytic-like behaviours and aversive memory acquisition impairment. The anxiolytic-like behaviours and aversive memory acquisition impairment that induced by SCH may be related to its effects on pre-synaptic dopamine D₂ receptors in shell on NAc which will cause the increase in dopamine release.

Effect of intra-NAc shell microinjection of D₂ and D₂ receptors antagonists on anxiolytic-like behaviours and aversive memory deficits induced by the intra-NAc shell NMDA

Data indicated that the intra-NAc shell administration of the subthreshold dose (0.125 µg/rat) of SCH 23390 or sulpiride together with different doses of NMDA, reduce the anxiolytic-like response and improve the aversive memory impairment already induced by the intra-NAc shell infusion of NMDA. These results may suggest the involvement of the dopamine transmission through D₂ and D₂ receptors of the NAc. Several investigations have suggested the possible dopaminergic and glutamatergic systems interaction in the NAc [66,67] based on which the NAc glutamate transmission is modulated by the dopamine system [37,53,82]. With respect to the interaction of these systems, there is a study showing that the NMDA receptors are localized on the NAc shell neurons which abundantly contain dopamine D₂ receptors [81]. Some evidence has also indicated the modulation of dopamine function through NMDA and AMPA receptors [30]. In an interesting report, Kalivas et al. declared that SCH 23390 and sulpiride restore the amphetamine-induced glutamate level decrease in NAc suggesting the involvement of presynaptic dopamine receptors in this phenomenon [36]. Furthermore, Dalia et al. indicated that dopamine increases the extracellular glutamate levels in the NAc [18]. Meanwhile, activation of glutamate receptor increases the dopamine release in the NAc [31]. We also know from the literature that NAc dopamine and glutamate signalling interactions are crucially required in behavioural reinforcement and habit formation and dopamine can modulate excitatory glutamatergic projection from the PFC [82]. The elevation of extracellular dopamine and glutamate levels in the striatum might disrupt Ca2+ homeostasis leading to the endoplasmic reticulum (ER) stress response [4,72]. The stimulation of dopamine D₂ receptors in the mouse neostriatum activates the cAMP/protein kinase A (PKA) pathway [76]. PKA activated increases in the levels of nitric oxide (NO) in the dorsal striatum [45]. NO in turn produces peroxynitrite which can regulate poly (ADPribose) polymerase-1 (PARP-1) activation [86]. Activation of three subtypes of glutamatergic ionotropic receptors leads to calcium influx, nitric oxide (NO) and reactive oxygen species (ROS) generation. Superoxide can combine with NO forming peroxynitrite (ONOO–). Excessive production of peroxynitrite and other free radicals induces chromosomal DNA nicks and breaks resulting in PARP-1 activation [19,83]. This enzyme is the nuclear target for different types of stress and signalling pathways. PARP-1 plays a crucial role in regulation of many transcription factors and nuclear proteins. Under physiological conditions, this enzyme is involved in memory formation and it should not be inhibited [32]. However, under massive stress and other pathological conditions it could be over-activated and involved in cell death by different mechanisms including activation of pro-inflammatory gene expression or modulation of NMDA and cholinergic receptor signalling [75,77]. Thus, there are three possible mechanisms for PARP-1 activation: first, dopamine D₂ receptor-dependent cAMP/PKA pathway is able to activate PARP-1; second, group I mGluRs and NMDA receptors interact with each other to increase PAPR-1 activation; and third, crosstalk between dopamine and glutamate receptors may provide another means of interaction [85]. Another study demonstrated that systemic inflammation evoked by an intraperitoneal injection of lipopolysaccharide induces morphological and biochemical changes in the brain including alterations of PARP-1 activity and expression of several genes [32]. PARP-1 inhibitor protects against LPS-evoked recognition impairment and significantly improves spatial memory in LPS-treated mice. PARP-1 inhibitor in control mice decreased memory function because under physiological conditions this enzyme is involved in memory formation and it should not be inhibited [32].
In conclusion, the stimulation of NMDA receptors in the shell of NAc and the effects of dopamine antagonists on presynaptic receptors might cause the increase in dopamine levels in the striatum and disrupt Ca2+ homeostasis leading to the endoplasmic reticulum (ER) stress response [4,72]. In addition, stimulating dopamine and glutamate receptors increases nitric oxide efflux which activates PARP-1 in the central nervous system [69,86]. Under physiological conditions, this enzyme is involved in memory formation and it should not be inhibited [32]. Activation of PARP-1 by elevation of extracellular dopamine and glutamate levels in the striatum might disrupt memory function and induce anxiolytic-like behaviours. Finally, our results suggest a modulatory role of the NAc shell dopaminergic system on NMDA-induced effects in the aversive memory.

Disclosure

Authors report no conflict of interest.

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