Behavioural Brain Research 274 (2014) 211–218

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Effects of ventral pallidal D1 dopamine receptor activation on memory consolidation in morris water maze test László Péczely a , Tamás Ollmann a , Kristóf László a , Anita Kovács a , Rita Gálosi a , Ádám Szabó a , Zoltán Karádi a,b , László Lénárd a,b,∗ a b

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

h i g h l i g h t s • • • •

D1 dopamine receptor agonist SKF38393 was microinjected into the ventral pallidum. SKF38393 in lower doses enhances memory consolidation in spatial learning. SKF38393 in lower doses increases stability of the memory trace against extinction. D1 dopamine receptor antagonist SCH23390 pretreatment eliminates SKF38393 effects.

a r t i c l e

i n f o

Article history: Received 15 April 2014 Received in revised form 11 July 2014 Accepted 21 July 2014 Available online 16 August 2014 Keywords: Ventral pallidum Morris water maze test Memory consolidation SKF38393 SCH23390 D1 dopamine receptor

a b s t r a c t In the present experiments, in adult male Wistar rats, the effect of microinjection of the D1 dopamine receptor agonist SKF38393 into the ventral pallidum on memory consolidation, as well as on resistance of the resulting memory trace against extinction were investigated in Morris water maze test. SKF38393 was applied in three doses (0.1, 1.0 or 5.0 ␮g in 0.4 ␮l physiological saline, respectively). To clarify whether the effect of the agonist was specific, in a separate group of animals, the D1 dopamine receptor antagonist SCH23390 (5.0 ␮g in 0.4 ␮l physiological saline) was administered 15 min prior to 1.0 ␮g agonist treatment. In another group of animals, the same dose of antagonist was applied by itself. The two lower doses (0.1 and 1.0 ␮g) of the agonist accelerated memory consolidation relative to controls and increased the stability of the consolidated memory trace against extinction. Antagonist pretreatment eliminated the effects of the agonist, thus confirming that the effect was selectively specific to D1 dopamine receptors. Our findings indicate that the ventral pallidal D1 dopamine receptors are intimately involved in the control of the consolidation processes of spatial memory. © 2014 Elsevier B.V. All rights reserved.

1. Introduction It is well known that the mesolimbic dopaminergic system (MLDS) arising from the ventral tegmental area (VTA) plays important role in motivation, reinforcement, learning and memory processes [1,2]. Dopamine released from the endings of the MLDS modulates synaptic plasticity [3,4]. The ventral pallidum (VP), receiving terminal elements of the MLDS, has reciprocal connections with several important brain regions essential for reinforcement, learning and memory [5–7].

∗ Corresponding author at: Institute of Physiology, Pécs University Medical School, Szigeti str. 12, P.O. Box 99, H-7602 Pécs, Hungary. Tel.: +36 72 536243; fax: +36 72 536244. E-mail address: [email protected] (L. Lénárd). http://dx.doi.org/10.1016/j.bbr.2014.07.031 0166-4328/© 2014 Elsevier B.V. All rights reserved.

The VP is the main target area of projection fibers from the nucleus accumbens (these fibers are predominantly GABAergic ones), and the VP also receives strong glutamatergic innervations from the prefrontal cortex and amygdala [8,9]. Although the VP has no direct connection to the hippocampus, this latter can influence ventral pallidal activity via the nucleus accumbens [10]. The VP is innervated by the dopaminergic fibers of the VTA, and in turn, the VP sends GABAergic fibers to the originating areas of the MLDS [5,11–13]. It has been shown that in the VP both D1 and D2 dopamine receptor subtypes are found, however, receptor density of the former is much higher compared to the latter [14–17]. VP neurons respond to both local and systemic application of dopamine and its agonists [18–22]. Local application of the D1 dopamine receptor agonist SKF38393 and the D2 dopamine receptor agonist quinpirole evoke, in most cases, opposite changes in the firing rate of VP neurons [18]. Systemically administered SKF38393

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increases firing rate of the cells in the VP, the quinpirole, however, was demonstrated to suppress firing rate [20]. Nevertheless, it is important to note that most VP neurons do not respond to quinpirole, suggesting a low density of D2 receptors in this forebrain region [18]. Increasing amount of information is available about the role of VP dopamine and its receptors mainly in motor behavior, and, to a more limited extent, in motivation and reward/positive reinforcement processes. Dopamine administration at low doses into the VP increases locomotor activity [23]. It has been shown that microinjection of the D1 dopamine receptor agonist SKF38393 into the VP generally increased, while that of the D2 dopamine receptor agonist quinpirole rather decreased locomotion [24]. Thus, it is reasonable to suppose, that the above locomotor effects of dopamine are exerted predominantly via D1 dopamine receptors of the VP. It has also been demonstrated that psychostimulants such as amphetamine and cocaine injected into the VP induce place preference [25]. Acquisition of place preference evoked by the dopamine reuptake inhibitor cocaine can be blocked by 6-hydroxydopamine [26]. The D1 and D2 dopamine receptor antagonists increased the threshold of intracranial self-stimulation and decreased the maximal rate of it [27]. Putting it all together, VP dopamine appears to be fundamental in the organization of motivated behaviors, but little is known yet about its exact role in learning and memory processes. Our recent results demonstrate that the D1 dopamine receptor agonist SKF38393 dose-dependently improves memory consolidation and retention in inhibitory avoidance learning [28]. Considering the data above, the present experiments, by using the Morris water maze paradigm, were designed to investigate the role of D1 dopamine receptor subtype of the VP in the consolidation of spatial memory. 2. Methods 2.1. Drugs and subjects In the present experiments, effect of bilateral microinjection of the D1 dopamine receptor agonist (R)-(+)-SKF-38393 hydrochloride (Sigma–Aldrich Co., S101) and the D1 dopamine receptor antagonist R(+)-SCH-23390 hydrochloride (Sigma–Aldrich Co., D054) into the VP was investigated in Morris water maze test (MWM) in male Wistar rats. We used 78 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 food pellet, Charles River Laboratories, Budapest) were available ad libitum. Food and water consumption and body weight 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 [29]. 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. Morris water maze test Experiments were carried out in a circular pool with a diameter of 1.5 m and filled with water (temperature: 23 ± 1 ◦ C) [30]. The pool was divided into four quadrants. One of these was chosen to place a square (10 cm × 10 cm) plexiglass platform in it. The location of the platform was fixed during the experiments, except in the habituation and extinction trials when the animals swam without the presence of the platform. Surface of the water was kept 2 cm above the platform and the water was colored to make the water opaque, and the platform hidden for the animals. The pool was surrounded with external cues, which helped the orientation of the rats. These cues were kept in constant position throughout the whole experiment. The behavior of animals was recorded by a video camera and registered by a specific software (EthoVision; Noldus Information Technology, The Netherlands). One day before the start of training, rats were habituated to the pool by allowing them to perform swimming for 90 s without platform. In the morning of the first day, two trials for spatial learning (detailed below) were performed, the two trials were separated by 1 min interval. This short intertrial interval ensured the possibility to observe in the second trial the short term memory trace formed during the first trial. On the second day, 24 h later, the schedule of the first day was repeated to investigate the possible further consolidation of memory. In these trials, the latency to finding the safe platform was measured. On the third day, 24 h later, in the morning, an extinction trial was performed: the platform was removed, and the latency to the first crossing of the removed platform’s place was measured. In the extinction trial, in addition to the first crossing, three other parameters were measured: number of crossings at the place of the removed hidden platform, number of the entrances into the target quadrant (where the platform was previously placed) and the time spent in the target quadrant. The same day, in the afternoon, the test trial was carried out: the platform was replaced and the latency to finding the safe platform was measured again. The first four trials were conducted as follows: rats were placed into the water maze at randomly assigned but predetermined locations to avoid the egocentric orientation. The task required animals to swim to the hidden platform guided by external spatial cues. After finding the platform, the rats were allowed to remain there for 60 s. Animals failing to find the platform in 180 s were placed on the platform and were allowed to rest for 60 s. During the experiments, in each trial the mean swimming velocities of the animals were measured. 2.4. Microinjection protocol The first and the second day, the two series of trials were immediately followed by the bilateral microinjection of D1 dopamine receptor agonist into the VP, that is, one experimental day one microinjection was performed. The agonist was applied in three different doses (SKF38393, 0.1 ␮g, 1.0 ␮g or 5.0 ␮g, in 0.4 ␮l physiological saline, 0.85 mM, 8.56 mM and 42.84 mM, respectively). Control animals received only vehicle (physiological saline) in all cases (0.4 ␮l, bilaterally). In a second experiment, D1 dopamine receptor antagonist (SCH23390, 5.0 ␮g in 0.4 ␮l physiological saline, 38.55 mM) was microinjected by itself or 15 min before the administration of 1.0 ␮g agonist. Solutions were kept in +4 ◦ C

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before application. In this paper, all the doses mentioned are meant to be the dose per side values. Drugs or vehicles were bilaterally microinjected through a 27 gauge stainless steel injection tube extending 0.5 mm below the tips of the implanted guide cannulae. The microinjection pipette was attached to a 10 ␮l Hamilton microsyringe via polyethylene tubing (PE-10) (Hamilton Co., Bonaduz, Switzerland). All microinjections were delivered by a syringe pump in volume of 0.4 ␮l (Cole Parmer, IITC, Life Sci. Instruments, California) over a 60 s interval. After finishing the microinjection, the pipette was left in place for an additional 60 s to allow diffusion into the surrounding tissue. During the microinjections, awake, well-handled rats were gently held in hand. 2.5. Histology At the end of the experiments animals were anesthetized with i.p. urethane (20%) and perfused transcardially with saline (0.15 M) followed by 10% formaldehyde solution. Brains were 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 [29]. Identification of cannula tracks with tissue debris and moderate glial proliferation were used for the localization of microinjections. Only data from rats with correctly placed cannulae were analyzed. 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. The statistical significance criterion was set at p < 0.05 level. All results are presented as mean ± standard error of the mean (S.E.M.).

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3. Results 3.1. Histology Histological examination showed that the cannulae were precisely and symmetrically tipped in the target area in 70 of the altogether 78 rats. Schematic illustration of cannula placements is shown in Fig. 1. The remaining eight (8/78) rats were excluded from the statistical analysis since their cannulae were not correctly positioned in the VP. Among these rats, in three cases, cannula tips symmetrically located about 1 mm below the target area, so in these animals bilateral microinjections were made in the nucleus of the horizontal limb of the diagonal band or in the magnocellular preoptic nucleus. In two cases, cannula tips were localized above the VP, so that these microinjections were in fact delivered in the caudateputamen or in the globus pallidus. In another case, cannula tips were localized below the VP, so these microinjections were made in the islands of Calleja. Additional two rats were excluded because of their acrylate “headpiece” was damaged or came off. Behavioral data of animals with these incorrect and diverse placements were not enough to draw far-reaching conclusions. 3.2. Effect of D1 agonist SKF38393 In the first experiment, the effect of microinjection of different doses of the D1 agonist SKF38393 into the VP was investigated on the escape latency (Fig. 2). The two-way ANOVA revealed that there was a significant effect for both trials [F (5,37) = 22.694, p < 0.001], and treatment [F (3,60) = 6.816, p < 0.001], but the interaction of treatment and trials proved not be significant [F (15,222) = 0.994, p > 0.05]. Tukey’s post hoc test indicated that the 0.1 and 1.0 ␮g doses (n = 10, n = 9, respectively) of the D1 receptor agonist SKF38393 significantly decreased the escape latency compared to the control group (n = 10, p < 0.005, p < 0.001; respectively). To reveal the potential short-term memory formation (acquisition)

Fig. 1. Illustration of reconstructed injection sites from all experiments. Correct bilateral injection placements are indicated as circles (open and closed in different shades of gray) and black triangles in the VP in panel A (n = 70). Incorrect injection placements are indicated in panel B (n = 6). Brain structure diagrams of coronal sections are adapted from the stereotaxic atlas of Paxinos and Watson [29]. Numbers in the middle refer to anterior–posterior distance from bregma in mm. Identical symbols in panels A and B within each brain section indicate appropriate, well corresponding placements of the bilateral injections. Numbers above symbols in panels A and B indicate numbers of animals having these injection sites.

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Fig. 2. Effect of ventral pallidal bilateral microinjection of different doses of D1 dopamine receptor agonist SKF38393 (D1ago) on the latency of finding hidden platform in a spatial learning test paradigm. On the first day two trials (1st trial and 2nd trial) were performed with 1 min inter-trial interval. The 2nd trial was immediately followed by microinjection of the agonist. On the second day, the schedule of the first day was repeated (3rd trial and 4th trial). In the morning of the third day, the hidden platform was removed (extinction), while in the afternoon, in the test trial, the platform was placed back (re-placed platf.). In case of the 1st–4th and test trials, columns represent the mean latencies for finding the hidden platform (±S.E.M.), or in case of the extinction trial, the mean latencies for finding the place of the removed hidden platform (±S.E.M.). In the latter case not the latencies to find the platform were measured as suggested by the label of the vertical axis, but the latencies to the site where the platform was originally placed in the 1st–4th and test trials. ‘n’ is the number of animals in each group. *p < 0.05 and # p < 0.05 indicate significant differences compared to controls and 5.0 ␮g D1ago treated group, respectively (Tukey’s post hoc test within each trial). For further explanation see the text.

and the consolidation of the formed short-term memory trace, we compared the latencies of the six trials within each group with one-way ANOVA. Significant differences were found among trials within all groups [0.1 ␮g, 1.0 ␮g, 5.0 ␮g, control; F (5,54) = 13.219, p < 0.001; F (5,48) = 9.004, p < 0.001; F (5,42) = 5.308, p < 0.001; F (5,54) = 3.846, p < 0.005; respectively]. Tukey’s post hoc test revealed that in every group the animals learned the place of the platform in the first trial, because 1 min after, in the second trial the animals found the platform significantly faster (0.1 ␮g, 1.0 ␮g, 5.0 ␮g, control; p < 0.001, p < 0.001, p < 0.05, p < 0.05; respectively). The second trial was immediately followed by the microinjection of the drugs. After 24 h, in the third trial, the latencies of the controls and the 5.0 ␮g agonist treated group almost totally returned to the level of the first trial, while the latencies of the 0.1 and 1.0 ␮g treated groups were significantly shorter relative to the first trial (in both cases p < 0.001), but not compared to the latencies of the second trial. Like the first day, 1 min after the third trial, in the fourth trial, all groups found the platform significantly faster compared to the first trial (0.1 ␮g, 1.0 ␮g, 5.0 ␮g, control; p < 0.001, p < 0.001, p < 0.005, p < 0.05; respectively). After 24 h, in the extinction trial, all groups displayed short latency to find the place of the platform relative to the latencies of the first trial (0.1 ␮g, 1.0 ␮g, 5.0 ␮g, control; p < 0.001, p < 0.001, p < 0.005, p < 0.05; respectively). After the extinction trial, in the test trial when the platform was placed back, the escape latencies of the 0.1 ␮g and 1.0 ␮g treated groups (in both cases p < 0.001), but not latencies of the controls or the 5.0 ␮g treated group remained significantly shorter relative to the first trial. We have also investigated the possible statistical differences among groups within each trial with one-way ANOVA. Significant difference could not be observed among the groups within the first (1st trial), the second (2nd trial), the fourth (4th trial) trials and the extinction trial (extinction). However, within the third (3rd trial) and the test trial (re-placed platf.), one-way ANOVA revealed a significant effect of treatment [F (3,33) = 5.033, p < 0.01; F (3,33) = 4.175, p < 0.05; respectively]. Tukey’s post hoc test showed that in the third trial the 0.1 ␮g and 1.0 ␮g doses of the D1 receptor agonist SKF38393 significantly decreased the escape latency relative to the control group (in both cases p < 0.05) and that of the 1.0 ␮g dose compared to the 5.0 ␮g agonist-treated group (p < 0.05). In the test trial, when the platform was placed back, Tukey’s post hoc test revealed that the mean latency of the 0.1 ␮g and 1.0 ␮g

agonist treated groups was significantly smaller than the mean of the control group (in both cases p < 0.05). In the extinction trial, the number of entrances into the target quadrant (where the platform was previously placed), the time spent in the target quadrant and the number of crossings at the place of the removed hidden platform were measured (see Table 1). The mean of the groups was compared with one-way ANOVA for each parameter. Significant differences were not revealed by oneway ANOVA among groups in any parameter. In all trials the mean swimming velocities of the animals were measured, and the means of the groups were compared with oneway ANOVA within each trial (data are not shown). Statistical difference was not found among groups within any trial. 3.3. Effect of the D1 antagonist SCH23390 To clarify that the previous results are specifically induced by the activation of D1 dopamine receptors, experiments with the D1 dopamine receptor antagonist SCH23390 were performed (Fig. 3). The antagonist was administered by itself or 15 min before 1.0 ␮g agonist, and the effects were compared to those of the control and 1.0 ␮g agonist treatments. The two-way ANOVA revealed that there was a significant effect for trials [F (5,33) = 26.322, p < 0.001] and also a significant effect for treatment [F (3,54) = 5.559, p < 0.001], but the interaction of treatment and trials proved not to be significant [F (15,198) = 1.191, p > 0.05]. Subsequent comparisons (Tukey’s post hoc test) between group means indicated that the 1.0 ␮g (n = 9) dose Table 1 Effect of ventral pallidal bilateral microinjection of different doses of D1 dopamine receptor agonist SKF38393 (D1ago) in the extinction trial on the number of entrances into the target quadrant (where the platform was previously placed) [Entrances (No.)], on the time spent in the target quadrant [Time spent (s)] and on the number of crossings at the place of the removed hidden platform [Crossings (No.)]. Numbers are mean values (±S.E.M.) during extinction trial. The groups and the number of the animals in each group are identical with those demonstrated in Fig. 2. Regarding each parameter, there was no significant difference among groups. Entrances (No.) Control 0.1 ␮g D1ago 1.0 ␮g D1ago 5.0 ␮g D1ago

18.8 19.6 17.6 15.4

± ± ± ±

1.6 1.4 2.3 1.1

Time spent (s) 54.5 53.8 48.4 45.2

± ± ± ±

4.5 4.7 4.4 2.2

Crossings (No.) 3.0 4.1 2.9 4.1

± ± ± ±

0.7 0.9 0.6 1.0

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Fig. 3. Effect of ventral pallidal administration of D1 dopamine receptor agonist SKF38393 (D1ago), D1 dopamine receptor antagonist SCH23390 (D1ant), or antagonist microinjected 15 min before the agonist (D1ant + ago) on the platform finding latency (escape latency). On the first day two trials (1st trial and 2nd trial) were performed with 1 min inter-trial interval. The 2nd trial was immediately followed by the microinjection of the agonist. On the second day, the schedule of the first day was repeated (3rd trial and 4th trial). In the morning of the third day, the hidden platform was removed (extinction), while in the afternoon, in the test trial, the platform was placed back (re-placed platf.). In case of the 1st–4th and test trials, columns represent the mean latencies for finding the hidden platform (±S.E.M.), or in case of the extinction trial, the mean latencies for finding the place of the removed hidden platform (±S.E.M.). In the latter case not the latencies to find the platform were measured as suggested by the label of the vertical axis, but the latencies to the site where the platform was placed in the 1st–4th and test trials. *p < 0.05 indicates significant differences from controls and D1ant + ago treated group (Tukey’s post hoc test within each trial). For further explanation see the text.

of the D1 receptor agonist SKF38393 decreased the platform finding latency, i.e., enhanced learning compared to the controls (n = 8, p < 0.01) and to the antagonist-agonist (n = 8, p < 0.001) treated groups. Similarly to the experiments with the agonist, we compared the latencies of the six trials within each group with one-way ANOVA. Significant differences were found among trials within all groups [control, 1.0 ␮g agonist, antagonist-agonist, antagonist; F (5,42) = 6.862, p < 0.001; F (5,48) = 12.707, p < 0.001; F (5,42) = 6.595, p < 0.001; F (5,42) = 5.462, p < 0.001; respectively]. Tukey’s post hoc test revealed that 1 min after the first trial, in the second trial the animals found the platform significantly faster compared to the first trial (control,1.0 ␮g agonist, antagonist–agonist, antagonist; p < 0.005, p < 0.001, p < 0.005, p < 0.005; respectively). After 24 h, in the third trial, the latencies of the controls, the antagonist and the antagonist-agonist treated groups decreased compared to the first trial, but these results were not statistically significant. However, the latencies of the 1.0 ␮g D1 agonist treated group were significantly shorter relative to the first trial (p < 0.001), but not compared to the latencies of the second trial. In the fourth trial, all groups found the platform significantly faster compared to the first trial (control, 1.0 ␮g agonist, antagonist-agonist, antagonist; p < 0.005, p < 0.001, p < 0.005, p < 0.001; respectively). After 24 h, in the extinction trial latencies of all groups were significantly shorter compared to the latencies of the first trial (in all cases p < 0.005). After the extinction trial, in the test trial when the platform was placed back, the escape latencies of the 1.0 ␮g agonist and the antagonist treated groups, but not latencies of the controls or the antagonist–agonist treated group remained significantly shorter relative to the first trial (1.0 ␮g agonist, antagonist; p < 0.001, p < 0.05; respectively). Differences among groups were also investigated with one-way ANOVA within each trial. Significant difference was not found during the trials of the first day (1st trial and 2nd trial) among the groups. On the second day of the experiment, at the first swimming of the day (3rd trial), the ANOVA revealed a significant difference for treatment [F (3,29) = 4.710, p < 0.01]. Tukey’s post hoc test showed that the mean of the escape latencies of the 1.0 ␮g agonist-treated group was significantly shorter compared to that of the controls and of the antagonist-agonist treated group (p < 0.05 in both cases). In the second swimming of the second day (4th trial), there was no statistical difference among groups. In the morning of the third day, in the extinction trial (extinction), there was also no statistical difference among groups. In the afternoon of the third day, in the test

trial (re-placed platf.) statistically significant difference for treatment was revealed by one-way ANOVA [F (3,29) = 3.925, p < 0.05]. Tukey’s post hoc test indicated that bilateral administration of the 1.0 ␮g D1 dopamine receptor agonist significantly decreased the escape latency relative to the controls and to the antagonist-agonist treated group (p < 0.05 in both cases). In the extinction trial, the number of entrances into the target quadrant (where the platform was previously placed), the time spent in the target quadrant and the number of crossings at the place of the removed hidden platform were measured (see Table 2). The means of the groups were compared with one-way ANOVA concerning each parameter. Significant differences were found in case of all parameters: the number of entrances into the target quadrant [F (3,29) = 3.449, p < 0.05], the time spent in the target quadrant [F (3,29) = 5.953, p < 0.005] and the number of crossings [F (3,29) = 3.482, p < 0.05]. Tukey’s post hoc test revealed significant differences in all three parameters between the 1.0 ␮g agonist and the antagonist treated groups (Entrances, Time spent and Crossings; p < 0.05, p < 0.005, p < 0.05; respectively) and in the time spent in the target quadrant between the control and the antagonist treated group (p < 0.05). In all trials the mean swimming velocities of the animals were measured, and the means of the groups were compared with oneway ANOVA within each trial. Statistical difference was not found among groups within any trial. Table 2 Effect of ventral pallidal administration of D1 dopamine receptor agonist SKF38393 (D1ago), D1 dopamine receptor antagonist SCH23390 (D1ant), or antagonist microinjected 15 min before agonist (D1ant + ago) in the extinction trial on the number of entrances into the target quadrant (where the platform was previously placed) [Entrances (No.)], on the time spent in the target quadrant [Time spent (s)] and on the number of crossings at the place of the removed hidden platform [Crossings (No.)]. Numbers are mean values (±S.E.M.) during extinction trial. The groups and the number of the animals in each group are identical with those demonstrated in Fig. 3. *p < 0.05 and † p < 0.005 indicate significant differences from 1.0 ␮g D1ago treated group while # p < 0.05 compared to vehicle treated controls (Tukey’s post hoc test). Entrances (No.) Control 1.0 ␮g D1ago D1ant + ago D1ant

17.1 18.7 16.1 13.1

± ± ± ±

1.3 1.5 1.2 1.0*

Time spent (s) 52.3 54.9 46.6 34.9

± ± ± ±

4.4 3.3 4.7 3.1†,#

Crossings (No.) 3.9 4.3 4.2 1.6

± ± ± ±

0.9 0.8 0.5 0.3*

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4. Discussion In the present experiments effect of D1 dopamine receptors on memory consolidation and on stability of the consolidated memory trace against extinction were investigated in Morris water maze test. In our interpretation, the results of the second trial reflect to the fact that in case of all groups short-term memory have been formed in the first trial, while the results of the third trial indicate that the two lower doses (0.1 and the 1.0 ␮g) of the D1 dopamine receptor agonist SKF38393 enhanced memory consolidation relative to the controls. In the latter group, however, a tendency could also be observed. Interestingly, further consolidation processes were not influenced by a second microinjection of the drugs (see the extinction trial), that is, it can be said that the agonist accelerates memory consolidation compared to the controls. D1 selective dopamine receptor antagonist SCH23390 pretreatment confirmed that the effect of the agonist on the consolidation processes was specific to the D1 dopamine receptors of the VP. The role of the D1 dopamine receptor in learning processes is supported by recent electrophysiological and behavioral findings. The long-term potentiation (LTP) is the main electrophysiological correlate of memory consolidation [31]. It has been demonstrated that D1 dopamine receptor activation is critical for development of LTP in the hippocampus [32–34], and these receptors in the striatum and the prefrontal cortex modulate LTP [35,36]. In D1 dopamine receptor knockout mice impaired spatial learning was observed [32,37]. In rats, 6OH-dopamine lesion of the hippocampus resulted in impairment of spatial memory [38]. The microinjection of the D1 dopamine receptor antagonist SCH23390 into the nucleus accumbens, the main input structure of the VP, caused memory consolidation deficit in inhibitory avoidance learning [39]. The D1 dopamine receptor antagonist SCH23390 microinjection into the nucleus accumbens impairs consolidation of spatial memory [40]. This latter finding shows that D1 dopamine receptor activation in the accumbens is a necessary condition of spatial memory consolidation. Accordingly, we suppose that when memory consolidation was induced by the first microinjection of the D1 agonist in the VP, the D1 dopamine receptors in the accumbens should have been activated as well. This activation might be dependent on the VP since this latter is known to influence the activity of the VTA [41,42], the main source of dopamine in the nucleus accumbens. Several reports suggest that also the hippocampus – another key structure of spatial learning processes – can regulate the activity of the VTA through a hippocampus-NAC-VP-VTA loop [10,43]. Based on the above and on the LTP inducing effect of D1 dopamine receptors [3,4,32–36], it is reasonable to suppose that the activation of these receptors in the VP can lead to synaptic changes locally and also – via the VTA – in other brain regions. Additional important finding of the present experiments is that after extinction trial, when the platform was re-placed (test trial), the 0.1 ␮g and the 1.0 ␮g agonist treated groups found significantly faster the re-placed platform than the controls. Furthermore, this effect was specific to the D1 dopamine receptors, because it could be eliminated by the SCH23390 pretreatment. In our hypothesis this shortening in the latencies is due to the increased stability of the consolidated memory against extinction. However, another possible explanation for the observed phenomenon is that animals treated with 0.1 ␮g or 1.0 ␮g agonist perseverate to visit the original platform location, while the controls search elsewhere the escape possibility. It is well known that activation of dopamine receptors in certain brain areas induces perseveration. D2 receptor stimulation in the NAC causes impaired behavioral flexibility [44]. The D2 dopamine agonist quinpirole, but not the D1 dopamine agonist SKF38393 injected into the dorsomedial striatum increased perseverative responding [45]. In delayed tasks, injections of higher

concentration of D1 agonists into the medial PFC of well-trained rats tended to cause animals to perseverate to revisit the previously rewarded locations [46,47]. Consequently, it cannot be excluded that dopamine receptors of the VP are playing a role in the regulation of behavioral flexibility. To clarify, whether the results of the test trial indicate merely the perseveration-inducing effect of the agonist, we measured and analyzed more parameters in the extinction trial: the number of entrances into the target quadrant, the time spent in the target quadrant and the number of crossings at the place of the removed hidden platform (see Tables 1 and 2). The results of all groups – except the antagonist treated group – were very similar and did not confirm perseverative behavior in any group. The antagonist applied by itself likely induced a faster extinction, but to prove this latter, further experiments are required. In the extinction trial the controls and the agonist treated groups returned to the place of the platform approximately the same number of times, therefore it is reasonable to suppose that being re-placed into the apparatus, in the test trial, also the control animals searched the platform. However, in the control animals the uncertainty of the memory caused by the extinction made it more difficult to find the platform. This is supported by the fact that the results of the controls in the test trial were very similar to those measured in the third trial. Based on these findings we suppose that our results indicate that the 0.1 ␮g and the 1.0 ␮g agonist treatment enhanced the stability of the memory trace against extinction compared to the control group. It has been shown by Gong and Neil that the 0.3 ␮g and the 1.0 ␮g SKF38393 injected into the VP just before placing into the apparatus evoke an increased locomotor activity in open field [24]. Although we applied post-trial microinjections, it cannot be excluded that the effects of the agonist – the effective doses were very similar to those applied by Gong and Neil – could be attributed to the longrun locomotion enhancing effect of the agonist. The analysis of the mean velocity of the animals within each trial indicated that the agonist did not have any effect on the movement of the rats and so our original hypotheses can be maintained. The last important issue which has to be discussed is that the results of microinjection of the D1 dopamine receptor agonist SKF38393 into the VP shows an inverted U-shaped-like dose–response curve in spatial learning, since the two lower doses improved memory consolidation, while the highest dose was ineffective. It is generally accepted that the neuropeptides can be characterized by an inverted U-shaped dose–response curve, having an intermediate dose with a maximum effectiveness [48–50]. Only limited data are available in the literature about the inverted U-shaped dose–response curve of the D1 dopamine receptor agonists. The prefrontal D1 dopamine receptor stimulation by lower doses of the agonist can improve, while overstimulation can impair working memory performance in rodents [47], and cognitive functions in monkeys [51]. It has been shown that this inverted U-shaped dose–response curve is likely to be caused by the characteristic effect of the D1 dopamine receptors on the cAMP level in the prefrontal neurons: an optimal receptor stimulation can lead to an optimal cAMP level which is necessary for the appropriate functioning of working memory [52]. A similar attribute of the VP D1 dopamine receptors could explain our findings, nevertheless, there are some other possible mechanisms as well. Another potential reason for ineffectiveness of the 5.0 ␮g dose of the agonist can be the different location and density of the D1 dopamine receptors within the VP. The D1 dopamine receptors in the VP can be found both presynaptically, on the GABAergic fibers arising from the NAC, and postsynaptically, on the intrinsic VP cells [18,53–55]. The pre- and the postsynaptic effect of the D1 dopamine receptor activation can interfere with each other in case of higher doses of the agonist. A further possibility to explain the phenomenon is the potential functional inhomogenity of the VP. This is supported by the finding that

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