Role of D2 dopamine receptors of the ventral pallidum in inhibitory avoidance learning

Behavioural Brain Research 321 (2017) 99–105

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Role of D2 dopamine receptors of the ventral pallidum in inhibitory avoidance learning László Lénárd a,b,∗ , Tamás Ollmann a , Kristóf László a , Anita Kovács a , Rita Gálosi a , Veronika Kállai a , Tóth Attila a , Erika Kertes a , Olga Zagoracz a , Zoltán Karádi a,b , László Péczely a a b

Institute of Physiology, Pécs University, Medical School, Pécs, Hungary Molecular Neuroendocrinology and Neurophysiology Research Group, Pécs University, Szentágothai Research Center, Pécs, Hungary

h i g h l i g h t s • • • • •

D2 dopamine receptor agonist quinpirole was microinjected into the ventral pallidum. The 0.1 ␮g agonist enhances memory consolidation in inhibitory avoidance learning. The same dose increases memory retention even 2 weeks after conditioning. D2 dopamine receptor antagonist sulpiride pretreatment eliminates agonist’s effect. The ventral pallidal D2 dopamine receptor activation enhances learning processes.

a r t i c l e

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Article history: Received 6 October 2016 Received in revised form 13 December 2016 Accepted 1 January 2017 Available online 3 January 2017 Keywords: Ventral pallidum Inhibitory avoidance learning Memory consolidation D2 dopamine receptors Quinpirole Sulpiride

a b s t r a c t In our present experiments, the role of D2 dopamine (DA) receptors of the ventral pallidum (VP) was investigated in one trial step-through inhibitory avoidance paradigm. Animals were shocked 3 times in the conditioning trial, with 0.5 mA current for 1 s. Subsequently bilateral microinjection of the D2 DA receptor agonist quinpirole was administered into the VP in three doses (0.1 ␮g, 1.0 ␮g or 5.0 ␮g in 0.4 ␮l saline). We also applied the D2 DA receptor antagonist sulpiride (0.4 ␮g in 0.4 ␮l saline) alone or 15 min prior to the agonist treatment to elucidate whether the agonist effect was specific for the D2 DA receptors. Control animals received saline. In a supplementary experiment, it was also investigated whether application of the same conditioning method leads to the formation of short-term memory in the experimental animals. In the experiment with the D2 DA receptor agonist, only the 0.1 ␮g quinpirole increased significantly the step-through latency during the test trials: retention was significant compared to the controls even 2 weeks after conditioning. The D2 DA receptor antagonist sulpiride pretreatment proved that the effect was due to the agonist induced activation of the D2 DA receptors of the VP. The supplementary experiment demonstrated that short-term memory is formed after conditioning in the experimental animals, supporting that the agonist enhanced memory consolidation in the first two experiments. Our results show that the activation of the D2 DA receptors in the VP facilitates memory consolidation as well as memory-retention in inhibitory avoidance paradigm. © 2017 Elsevier B.V. All rights reserved.

1. Introduction The ventral pallidum (VP), as the ventral extension of the globus pallidus was originally described by Heimer and Wilson [1]. This

∗ Corresponding author at: Institute of Physiology, Pécs University Medical School, Szigeti Str. 12, P.O. Box: 99, H−7602, Pécs, Hungary. E-mail address: [email protected] (L. Lénárd). http://dx.doi.org/10.1016/j.bbr.2017.01.005 0166-4328/© 2017 Elsevier B.V. All rights reserved.

basal forebrain structure is densely innervated by dopaminergic fibers originating predominantly from the ventral tegmental area (VTA) [2]. It has been suggested that the VP is involved in learning processes. Inactivation of the VP with lidocaine results in deficit of spatial working memory in radial arm maze test [3]. Several glycosaminoglycans, microinjected into the VP, are known to enhance memory consolidation in inhibitory avoidance learning, likely via the activation of the large cholinergic neurons of the VP [4,5]. In Pavlovian learning situation, the VP cells responded to the

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presence of the unconditioned stimulus (US), and also to the conditioned stimulus (CS) which predicted it. The US induced a stable population activity, while the response to the CS was characterized by a population activity which continuously increased in the course of the learning process, and after that activity stabilised [6]. The excytotoxic lesion of the VP prevents the acquisition of the amphetamine induced place preference, but does not influence the recall of it [7]. More information is available that the dopamine (DA) within the VP has an important role in learning processes. This is confirmed by the fact that place preference can be induced by the administration of the indirect DA agonists cocaine and amphetamine into the VP [8], and the formation of the cocaine-induced place preference is blocked by the 6-hydroxidopamine lesion of the dopaminergic nerve endings in VP [9]. Furthermore, it has been shown that the cocaine remarkably increases the DA level in the VP [9]. The DA receptors can be grouped in two major families (i.e. the D1- and the D2-subtype) [10–12], and in the VP, applying autoradiography [13,14] and mRNA immunhistochemistry [15,16] both DA receptor subtypes have been detected. It has been shown that the majority of VP neurons respond to systemic or local application of DA or its agonists [17–22]. Recently, our research group demonstrated that the activation of the D1 and D2 DA receptors of the VP facilitate memory consolidation in spatial learning [23,24], and the activation of the D1 DA receptors enhances memory consolidation and memory retention in inhibitory avoidance learning [25]. Based on our previous results [23–25] and on those of data of the literature [26] we hypothetise that the VP D2 DA receptor activation may enhance inhibitory avoidance learning, as well. Therefore, the present experiments were conducted to elucidate the potential role of the D2 DA receptors of the VP in inhibitory avoidance learning. 2. Methods 2.1. Drugs and subjects In the present experiments, the effects of bilateral microinjection of the D2 DA receptor agonist quinpirole hydrochloride (Sigma–Aldrich Co., Q102) and the D2 DA receptor antagonist (S)-(−)-sulpiride (Sigma–Aldrich Co., S7771) into the VP were investigated in inhibitory avoidance learning in male Wistar rats. We used 98 animals weighing 280–320 g at the beginning of the experiments. Rats were housed individually and cared for in accordance with institutional (BA02/2000-8/2012), national (Hungarian Government Decree, 40/2013. (II. 14.)), and international standards (European Community Council Directive, 86/609/EEC, 1986, 2010). Animals were kept in a light and temperature controlled room (12:12 h light–dark cycle with lights on at 06:00 a.m., 22 ± 2 ◦ C). Tap water and standard laboratory food pellets (CRLT/N standard rodent pellet food, Charles River Laboratories, Budapest) were available ad libitum. Food and water consumptions and body weights were measured daily. All tests were performed during the daylight period of the rats between 08:00 and 17:00 h. 2.2. Surgery Operations were carried out under anesthesia by intraperitoneal injection of a mixture of ketamine (Calypsol) and diazepam (Seduxen) mixed in a ratio of 4:1 (Calypsol, 80 mg/kg bw and Seduxen, 20 mg/kg bw, respectively; Richter Gedeon Ltd., Hungary). By means of the stereotaxic technique, 22 gauge stainless steel guide tubes were bilaterally implanted 0.5 mm above the target area (coordinates referring to the bregma: AP: −0.26 mm, ML: ±2.2 mm, DV: −7.1 mm from the surface of the dura) according to

the stereotaxic rat brain atlas of Paxinos and Watson [27]. Cannulae were fixed to the skull with self-polymerizing dental acrylic (Duracryl) anchored by 2 stainless steel screws. The guide tubes, except when being used for insertion of microinjection delivery cannula, were occluded with stainless steel obturators made of 27 gauge stainless steel wire.

2.3. Inhibitory avoidance paradigm Animals were allowed 7 days for postoperative recovery, and were frequently handled until the start of experiments. As a classic learning model for negative reinforcement, the one trial stepthrough inhibitory avoidance paradigm was used in which the rat had to learn to avoid the unpleasant stimulus of the electric shock [28,29]. The experimental apparatus consisted of a large (60 × 60 × 60 cm), square based, well illuminated chamber with light-gray walls and attached to a small box (15 × 15 × 15 cm) equipped with a removable roof, painted black and having metal-grid floor for the delivery of electrical shocks. The two compartments (the small black and the big gray) were separated by a guillotine door. A 100 W lamp (Tungsraflex) was positioned above the apparatus and provided a continuous illumination of the large chamber during the experimental period. The inhibitory avoidance procedure consisted of a habituation, a conditioning and three test trials, each lasted maximum 180 s. In the habituation trial (first day), animals were placed into the illuminated chamber and were allowed to explore the apparatus for 180 s. The conditioning session took place the following day. The rats were placed into the large illuminated chamber and after entering the dark compartment the door was closed and an unescapable weak electric foot shock (0.5 mA, 1 s) was applied through the floor grid 3 times. The latency of entering the dark box − called step-through latency- was recorded. Bilateral drug microinjections into the VP were delivered immediately after the electric shock. In the first experiment, the agonist quinpirole was administered in three different doses (0.1 ␮g, 1.0 ␮g or 5.0 ␮g, in 0.4 ␮l physiological saline, 0.98 mM, 9.77 mM and 48.89 mM, respectively). Control animals received vehicle only (physiological saline, 0.4 ␮l, also bilaterally). In the second experiment, microinjection of the D2 DA receptor antagonist sulpiride (0.4 ␮g in 0.4 ␮l physiological saline, 2.93 mM) was delivered by itself or 15 min before the administration of 0.1 ␮g of the agonist (quinpirole). All groups received two microinjections with 15 min delay: the control group, two vehicle injections; the quinpirol treated group, vehicle administration before the quinpirole treatment; the antagonist + agonist treated group, sulpiride before quinpirole treatment; the antagonist treated group received sulpiride and then vehicle. Solutions were kept in + 4 ◦C before administration. In this paper, all the doses mentioned are meant to be the dose per side values. All microinjections were applied posttrial. Solutions were microinjected for 1 min into the VP through a 27 gauge stainless steel microinjection pipette extending 0.5 mm beyond the tips of the implanted guide cannulae. A 10 ␮l Hamilton syringe (Hamilton Co., Bonaduz, Switzerland) via a polyethylene tube (PE 10) to the injector was attached. The Hamilton syringe was operated with a Cole Parmer automatic minipump (ILTC Inc. Life Sciences Instruments, USA). The microinjection pipette was left in place for an additional 60 s after infusion to allow diffusion into the VP. During the microinjections, awake, well-handled rats were gently held in hand. The same rats were tested 24 h, one week, and two weeks after conditioning (Test 1, Test 2 and Test 3, respectively) without repeated deliveries of the foot shock. In the test session, the rat was placed again into the illuminated chamber and the step-through latency was recorded again. If the animal did not enter the dark chamber within a 3 min test period, the test was terminated and the latency was recorded as 180 s. Data were recorded

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Fig. 1. Illustration of reconstructed injection sites from all experiments. Correct bilateral injection placements are indicated as circles and triangles in the VP on panel A (n = 87). Incorrect injection placements are indicated on panel B (n = 9). Brain structure diagrams of coronal sections are adapted from the stereotaxic atlas of Paxinos and Watson [27]. The numbers in the middle refer to anterior–posterior distance from the bregma in mm. Identical symbols in panel A or B indicate coherent injection sites of bilateral microinjections. Numbers beside symbols on panels A and B indicate number of animals.

and evaluated by means of the Noldus Ethovision System (Noldus Information Technology, The Netherlands). 2.4. Verification of the formation of the short-term memory in inhibitory avoidance paradigm To demonstrate the formation of the short-term memory in case of the weak shock applied in our inhibitory avoidance paradigm, an additional experiment was performed. Control animals were habituated to the apparatus similar to the first two experiments. After habituation, animals were divided into two groups. For both groups, the experiment was similarly performed like in the first two experiments up to the shocking procedure. After shocking, the rats of the first group were microinjected into the VP with physiological saline (0.4 ␮l), while the animals of the second group were re-placed into the apparatus after 1 min (re-placed controls), and the step-through latencies were measured (test after 1 min). Thereafter the re-placed animals were also microinjected with physiological saline (0.4 ␮l). Twenty-four hours later both groups were tested (corresponding to the Test 1). 2.5. Histology At the end of all experiments, rats were anesthetized with i.p. urethane (20%) and perfused transcardially with physiological saline (0.15 M) followed by 10% formaldehyde solution. Brains were then removed, sliced with a freezing microtome in 40 ␮m sections and stained with Cresyl violet. Cannula tracks and location of cannula tips were reconstructed according to a stereotaxic rat brain atlas [27]. Identification of cannula tracks, tissue debris and moderate glial proliferation were used for the localization of microinjections. Only data from rats with correctly placed cannulae were analysed.

2.6. Statistical analysis Data were evaluated by one-way and two-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test using the SPSS data analysis program. In case of the third experiment oneway ANOVA and paired samples t-test were applied to analyse data. The criterion for statistical significance was set at the p < 0.05 level. All results are presented as mean ± standard error of the mean (S.E.M.).

3. Results 3.1. Histology Histological examination showed that the cannulae were precisely and symmetrically tipped in the target area in 87 of the 98 animals. Schematic illustration of cannula placements is shown in Fig. 1. The remaining eleven rats were excluded from the statistical analysis because their cannulae were not correctly positioned in the VP. Among these rats, in four cases cannula tips were symmetrically located 1 mm below the target area, so bilateral microinjections were delivered in the nucleus of the horizontal limb of the diagonal band or in the magnocellular preoptic nucleus. In one case, cannula tips were localized laterally or medially to the VP, so that these microinjections were made in the lateral preoptic area on one side and in the medial part of the interstitial nucleus of the posterior limb of the anterior commissure on the other side. In three cases, cannula tips were located above the VP, so these injections were made into the globus pallidus. In another case, the cannula tips symmetrically reached into the liquor space at the basis of the brain. Additional two rats were excluded because of their acrylate headpiece was damaged or came off.

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Fig. 2. Effect of ventral pallidal bilateral microinjection of different doses of the D2 DA receptor agonist quinpirole (D2ago) on the step-through latency in inhibitory avoidance learning paradigm. Rats were microinjected after the conditioning trial and tested without repeated drug administration 24 h (Test 1), 1 week (Test 2) and 2 weeks (Test 3) later. Columns represent mean latency time values (±S.E.M.); ’n’, the number of animals for each group; *, p < 0.05 and #, p < 0.01 indicate significant differences from the control, the 1.0 ␮g and the 5.0 ␮g agonist treated groups (one-way ANOVA within each session). For further explanation see the text.

Behavioral data of animals with the above incorrect and diverse placements were not sufficient to draw far-reaching conclusions with respect to related behavioural functions of these neighbouring structures. 3.2. Effect of D2 DA agonist quinpirole In the first experiment we investigated the effect of the D2 DA receptor agonist quinpirole administered into the VP on step-through latencies of the rats (Fig. 2). The analysis with twoway ANOVA unraveled significant difference among the trials [F (3,37) = 4.398, p < 0.01], the treatment [F (3,40) = 29.392, p < 0.001], and also the interaction of trials and the treatment proved to be significant [F (9,148) = 2.649, p < 0.01]. The Tukey’s post hoc analysis revealed that the 0.1 ␮g (n = 10) D2 DA agonist increased the stepthrough latency compared to the control (n = 9, p < 0.001), the 1.0 ␮g (n = 9, p < 0.001) and the 5.0 ␮g (n = 9, p < 0.001) agonist treated group. In addition to the two-way ANOVA analysis, the experimental data were also processed for each trial using one-way ANOVA. There were no differences among groups during the conditioning trial (Cond.). By contrast, significant differences were indicated to exist among the groups within all 3 test trials [Test1, Test2 and Test3: F (3,33) = 6.227, p < 0.005; F (3,33) = 7.434, p < 0.001; F (3,33) = 10.316, p < 0.001; respectively]. Twenty four hours after conditioning, in the first test trial (Test 1), the 0.1 ␮g agonist treatment significantly increased the step-through latency in comparison to the control, the 1.0 ␮g, as well as the 5.0 ␮g agonist treated group (based on the Tukey’s post hoc test; in all three cases: p < 0.01). Furthermore, the 0.1 ␮g dose of the agonist facilitated the retention one week (Test 2) and two weeks (Test 3) after conditioning (in both tests and comparisons made with all three groups p < 0.005). 3.3. Effect of D2 DA antagonist sulpiride The D2 DA receptor antagonist sulpiride was applied to investigate the D2 DA receptor specificity of the agonist (Fig. 3). In one group of animals only the antagonist was applied, in another group the antagonist was delivered 15 min prior to the administration of 0.1 ␮g agonist. The two-way ANOVA analysis revealed significant effect for trials [F (3,31) = 4.633, p < 0.005], for treatment [F (3,32) = 17.233, p < 0.001], and also for the interaction between tri-

als and treatment [F (9,124) = 2.137, p < 0.05]. The Tukey’s post hoc test showed that means of the 0.1 ␮g agonist treated group (n = 8) significantly differ from those of the control (n = 8), the antagonist + agonist (n = 7) and the antagonist alone (n = 8) treated group (in all three cases, p < 0.001). The one-way ANOVA analysis within the conditioning trial (Cond.) did not reveal significant differences among the groups. However, in the first (Test 1), the second (Test 2) and the third (Test 3) test trials the statistical analysis demonstrated significant group differences [Test 1, Test 2 and Test 3: F (3,27) = 9.120, p < 0.001; F (3,27) = 4.925, p < 0.01; F (3,27) = 4.279, p < 0.05; respectively]. The Tukey’s post hoc test indicated that the 0.1 ␮g dose of the D2 DA agonist increased the step-through latency in the first (Test 1, p < 0.005) and in the second test (Test 2, p < 0.05) trial compared to the control, the antagonist + agonist and the antagonist alone treated groups. In the third test trial, the 0.1 ␮g agonist facilitated retention only in comparison to the control and the antagonist + agonist treated group (Test 3, for both groups p < 0.05). The D2 DA receptor antagonist sulpiride, acting to antagonize the effect of quinpirole, eliminated the agonist effect on learning (Test 1), and on retention (Test 2 and Test 3) as well, whereas the antagonist alone in the applied dose did not have any effect.

3.4. Formation of the short-term memory In the third, supplementary experiment, we aimed to demonstrate the formation of the short-term memory whose consolidation could be enhanced by the agonist (Fig. 4). The means of data obtained in the single sessions were compared to each other within both groups. In the case of the control group (conditioned and tested only 24 h after conditioning, n = 10), the paired samples ttest did not reveal any significant difference between the means of the conditioning trial and the first test (after 24 h). In case of the re-placed control group (n = 9) one-way ANOVA (conditioned, then tested 1 min and 24 h after conditioning) showed significant difference among trials [F (2,24) = 7.494, p < 0.005], and the Tukey’s post hoc test demonstrated that 1 min after conditioning the animals entered into the dark compartment significantly later than in the conditioning trial or after 24 h, in the test trial (in both cases, p < 0.01).

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Fig. 3. Effect of ventral pallidal administration of the D2 DA receptor agonist quinpirole (D2ago), the D2 DA receptor antagonist sulpiride (D2ant), or the antagonist microinjected 15 min before agonist (D2ant + ago) on the step-through latency in inhibitory avoidance learning paradigm. Rats were microinjected immediately after conditioning and tested without repeated drug administration 24 h (Test 1), 1 week (Test 2) and 2 weeks (Test 3) later. Columns represent mean latency time values (±S.E.M.); ’n’, the number of animals in each group; *, p < 0.005 and #, p < 0.01 indicate significant differences from the control, the D2ant and the D2ant + ago groups, while †, p < 0.05 from the control and the D2ant + ago groups, respectively (one-way ANOVA for each session). For further explanation see the text.

Fig. 4. To demonstrate the short-term memory formation in inhibitory avoidance learning paradigm, two physiological saline treated groups were used: one group was re-placed into the apparatus only 24 h after the conditioning (control), while the other group was re-placed one minute and also 24 h (corresponding to the first test in case of the first two experiments) after the conditioning (re-placed control). Columns represent mean latency time values (±S.E.M.); ’n’, the number of animals in each group; *, p < 0.001 indicates significant differences (the conditioning and the test trials, one-way ANOVA, the re-placed control group). For further explanation see the text.

4. Discussion The inhibitory avoidance test is a broadly used paradigm to investigate the negative reinforcement (punishment-learning). In our experiments, the D2 DA receptor agonist quinpirole proved to be effective in 0.1 ␮g dose only, i.e. improved learning and retention, and the formed memory was stable even 2 weeks after conditioning. Pretreatment with the D2 DA antagonist sulpiride substantiated that the effect of the 0.1 ␮g dose of the agonist prevailes via the activation of the D2 DA receptors in the VP. The antagonist applied by itself did not have any effect, i.e. it did not influence the learning processes. In the supplementary experiment, two control groups were employed, one was re-placed into the apparatus 1 min after conditioning and tested 24 h after it, and the other was tested after 24 h only. The formation of short-term memory has been demonstrated in the re-placed control group. After 24 h, both groups entered into the dark compartment with a relatively short latency, reflecting to

the obvious fact that the animals have forgotten the learned information. Based on these findings, it is reasonable to suppose that also in the first two experiments, during the conditioning process all rats have learned to avoid the dark compartment from the weak shock intensity. The drugs were microinjected into the VP immediately after conditioning, which ensured the required contiguity between potential drug effect and the formation of short-term memory, so the drugs, in fact, could exert their memory-consolidating effect. However, memory consolidation occurred only in case of the 0.1 ␮g quinpirole treated group, animals of the other groups appeared to have forgotten, what they had learned during the conditioning trial. Our result clearly shows that the one-trial inhibitory avoidance test with such weak shock intensity is a very sensitive method to detect the potential memory consolidation facilitating effect of various drugs: in case of using strong shock intensities memory consolidation appear to take place in all animals, so that further facilitation can hardly be observed. This is also supported by our earlier studies and the results of other research groups [4,5,25,30–32] as well. The ineffectiveness of the antagonist when applied alone is particularly interesting, because it has been shown in our recent study that microinjection of sulpiride into the VP impairs spatial learning processes [24]. One possible plausible explanation for this apparent contradiction could be that the single conditioning trial with the low shock-intensity is not sufficient to induce the consolidation of memory, which thus, could be prevented by the sulpiride treatment. This is supported by the supplementary experiment of the present study, which demonstrated the formation of short-term memory in rats: in the control animals memory consolidation did not occur. The role of the DA and its receptors in inhibitory avoidance learning and in the related memory consolidation processes has already been proven in various brain areas. We have recently demonstrated that the activation of the D1 DA receptors of the VP facilitate memory consolidation and retention [23,25]. In the mouse, microinjection of the D1 DA receptor antagonist SCH23390 and the D2 DA receptor antagonist sulpiride in the core region of the nucleus accumbens dose-dependently prevented memory consolidation, while in the shell region of the nucleus accumbens only the sulpiride had a similar effect [26]. The memory consolidation in inhibitory avoidance learning, among others, also requires the co-activation of the DA receptors of the nucleus accumbens shell

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region and those of the basolateral amygdala [33]. In inhibitory avoidance learning paradigm, blocking the D1 DA receptors in the anterolateral prefrontal and the entorhinal cortices causes longterm memory impairment [34]. The timing of the microinjections appears to be important, which is reflected by the experiments with SCH23390 microinjection into the anterior medial or posterior medial precentral area of the prefrontal cortex [35]. Both interventions had amnesic effect, but in the former case the memory consolidation was blocked by the antagonist when given 0, 1.5 or 3 h after conditioning, while in the latter case the antagonist was effective only when given 1.5 h after it [35]. The significance of timing of the microinjections is also underlined by the findings demonstrating that the activation of the D1 DA receptors in the CA1 region of the hippocampus facilitates, whereas the blocking of these receptors impairs memory consolidation related to avoidance learning in the time range of 3–6 h after conditioning [36]. In the present experiments only the 0.1 ␮g dose of the agonist was effective, but not the 1.0 or 5.0 ␮g doses. In connection with the VP D1 DA receptor activation in spatial learning, we have already observed a similar phenomenon, namely, only the 0.1 and 1.0 ␮g dose of the D1 DA agonist enhanced consolidation of spatial memory, while the 5.0 ␮g dose was ineffective [23]. Supposedly, a theoretical minimal effective dose exists, in the present study and in the experiment with the D1 DA agonist [23], the dose-response curves of the agonists remind us to the inverse Ushaped dose-response relationship. The linear − or asymptotic − dose-response curve does not require any particular explanation, in case of the experiments with DA agonist or antagonist generally this type of curve can often be observed independently from the paradigm [26,37–39]. Nevertheless, the inverse U-shaped doseresponse curve is not unprecedented in the literature [35,40]. It should be taken into consideration that D2 DA receptors can be found in the VP both presynaptically on the accumbal GABA-ergic fibers, and postsynaptically on the local neurons [41]. It is plausible to think that the pre- and postsynaptic effects could interfere with each other when the higher dose of the agonist was applied. Another possibility is related to the receptorial inhomogeneity of the VP. It has been demonstrated that in certain cases blocking the D3 DA receptors does not impair, but facilitates memory consolidation [42,43]. The VP contains both D2 and D3 DA receptors in a similar proportion [44], however, a potential inhomogeneity can still be present in the distribution of these receptors within the structure. Accordingly, it is reasonable to suppose that the lower dose of the agonist in the microinjection site affected the D2 DA receptors resulting in the enhancement of memory consolidation, while in case of microinjections with the higher doses could affect other, farther parts of the VP (for instance the anterior VP), activating predominantly the D3 DA receptors, which could lead to the experienced ineffectiveness of the higher doses. Although our results did not reveal any significant difference in the effects of microinjections with different injection sites within the VP, we cannot exclude the possibility of such topographical influence, therefore, to clarify this issue further experiments are required. In summary, the present study provided evidence for that activation of the VP D2 DA receptors enhances memory consolidation and the long-term stability of the formed memory in inhibitory avoidance learning.

Acknowledgements The authors express their thanks to Katalin Oláhné Várady for her efforts in the experiments, furthermore to Erika Szabó, Eniko˝ Károly and Erzsébet Korona for technical contribution to this work. This study was supported by SROP-4.2.2/B-10/1-2010-

0029, SROP-4.2.1.B-10/2/KONV-2010-0002, the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/ 2-11/1-2012-0001 ‘National Excellence Program’, Pécs University, Medical School (PTE-AOK-KA 2013/34039/1), Pécs University, Medical School (PTE AOK KA-201515), Pécs University, Medical School (PTE AOK PD-2016-06), the ÚNKP-16-4-I New National Excellence Program of the Ministry of Human Capacities and the ÚNKP-16-3-III New National Excellence Program of the Ministry of Human Capacities.

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