Blockade of the dorsal hippocampal dopamine D1 receptors inhibits the scopolamine-induced state-dependent learning in rats

Neuroscience 252 (2013) 460–467

BLOCKADE OF THE DORSAL HIPPOCAMPAL DOPAMINE D1 RECEPTORS INHIBITS THE SCOPOLAMINE-INDUCED STATE-DEPENDENT LEARNING IN RATS M. PIRI, a M. ROSTAMPOUR, b M. NASEHI c AND M. R. ZARRINDAST d,e,f,g*

phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390; 0.1 and 0.5 lg/rat, intra-CA1), resulted in apparent memory impairment, microinjection of the same doses of this agent inhibited the scopolamine-induced statedependent memory. These results indicate that the CA1 dopamine D1 receptors may potentially play an important role in scopolamine-induced amnesia as well as the scopolamine state-dependent memory. Furthermore, our results propose that dopamine D1 receptor agonist, SKF38393 reverses the scopolamine-induced amnesia via acetylcholine release and possibly through the activation of muscarinic receptors. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Biology, Faculty of Basic Sciences, Islamic Azad University, Ardabil Branch, Ardabil, Iran b Department of Biology, Islamic Azad University, Science and Research Branch, Tehran, Iran c Department of Biology, Faculty of Basic Sciences, Islamic Azad University, Garmsar Branch, Garmsar, Iran d Department of Neuroscience, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran e Department of Pharmacology, School of Medicine and Iranian National Center for Addiction Studies, Tehran University of Medical Sciences, Tehran, Iran f School of Cognitive Sciences, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran g

Key words: scopolamine, dopamine D1 receptors, dorsal hippocampus, state-dependent memory, inhibitory avoidance memory, rat.

Institute for Cognitive Sciences Studies, Tehran, Iran

Abstract—In the present study, we investigated the possible role of the dorsal hippocampal (CA1) dopamine D1 receptors on scopolamine-induced amnesia as well as scopolamine state-dependent memory in adult male Wistar rats. Animals were bilaterally implanted with chronic cannulae in the CA1 regions of the dorsal hippocampus, trained in a step-through type inhibitory avoidance task, and tested 24 h after training for their step-through latency. Results indicated that pre-training or pre-test intra-CA1 administration of scopolamine (1.5 and 3 lg/rat) dose-dependently reduced the step-through latency, showing an amnestic response. The pre-training scopolamine-induced amnesia (3 lg/rat) was reversed by the pre-test administration of scopolamine, indicating a state-dependent effect. Similarly, the pre-test administration of dopamine D1 receptor agonist, 1-phenyl-7,8-dihydroxy-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SKF38393; 1, 2 and 4 lg/rat, intra-CA1), could significantly reverse the scopolamine-induced amnesia. Interestingly, administration of an ineffective dose of scopolamine (0.25 lg/rat, intra-CA1) before different doses of SKF38393, blocked the reversal effect of SKF38393 on the pre-training scopolamine-induced amnesia. Moreover, while the pre-test intra-CA1 injection of the dopamine D1 receptor antagonist, R(+)-7-chloro-8-hydroxy-3-methyl-1-

INTRODUCTION Acetylcholine (ACh) activity seems essential to learn multiple tasks (Blokland, 1995; Robinson et al., 2011). Cumulative evidence indicates that acetylcholinesterase inhibitors, which enhance the availability of ACh in the synaptic cleft, improve performance in several cognitive models both in rodents and humans, whereas anticholinergic drugs are shown to impair learning and memory function upon a variety of tasks (Fibiger et al., 1991; Gallagher and Colombo, 1995; Power et al., 2003; Azami et al., 2010; Mahmoodi et al., 2010; Pakpour et al., 2010; Jamali-Raeufy et al., 2011). While both nicotinic and muscarinic receptors of the dorsal hippocampus are involved in learning and memory processes (Rezayof et al., 2008), the role of the muscarnic ACh receptors in learning and memory has been emphasized more (Power et al., 2003; Azami et al., 2010; Mahmoodi et al., 2010; Pakpour et al., 2010; Jamali-Raeufy et al., 2011). Similar to ACh, dopamine (DA) is known to significantly contribute to learning and memory (Jay, 2003; Wittmann et al., 2005; Nasehi et al., 2010). Several studies have suggested that the entry of information into the long-term memory in the hippocampus is affected by the neuromodulatory signals from dopaminergic projections (Gasbarri et al., 1994; Jay, 2003; Wittmann et al., 2005). A substantial body of evidence shows the complex interaction between cholinergic and dopaminergic systems (Levin and Rose, 1991; Hersi et al., 1995b;

*Correspondence to: M. R. Zarrindast, Department of Neuroscience, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran. Tel/fax: +98-21-66402569. E-mail address: [email protected] (M. R. Zarrindast). Abbreviations: ACh, acetylcholine; ANOVA, analysis of variance; CA1, dorsal hippocampal; cAMP/PKA, cyclic AMP/protein kinase A; DA, dopamine; intra-CA1, intra-dorsal hippocampal; SCH23390, R(+)-7chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3benzazepine hydrochloride; SKF38393, 1-phenyl-7,8-dihydroxy2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride.

0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.08.003 460

M. Piri et al. / Neuroscience 252 (2013) 460–467

Gasbarri et al., 1997). For instance, intraperitoneal administration of scopolamine decreases DA release in the hippocampus (Memo et al., 1988), whereas, the D1 receptor agonist, 1-phenyl-7,8-dihydroxy-2,3,4,5tetrahydro-1H-3-benzazepine hydrochloride (SKF38393) increases in vivo hippocampal ACh release and the D1 receptor agonist, R(+)-7-chloro-8-hydroxy-3-methyl-1phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390) inhibits ACh release in the hippocampus (Hersi et al., 1995a,b). Furthermore, the continuous intravenous infusion of the muscarinic cholinergic receptor agonist is shown to alter the cholinergic and dopaminergic receptors’ density in monkey’s brain (Hartvig et al., 2002). In addition, evidence suggests that some anti-dementia drugs such as Ginkgo biloba leaf extract enhance patients’ memory performance by means of increasing DA and ACh release in the hippocampus or other sites of the brain (Kehr et al., 2012; Zurkovsky et al., 2012; Shi et al., 2013). Administration of scopolamine, a muscarinic cholinergic receptor antagonist, is shown to impair memory performance in different models of memory assessment (Collerton, 1986; Jensen et al., 1987; Quartermain and Leo, 1988; Azami et al., 2010). Scopolamine, may be utilized as a useful pharmacological tool to produce a partial model of the aging and (Alzheimer’s) dementia (Bartus, 2000). This model is frequently employed as a simple and quick way for testing cognition-enhancing properties of new drugs (Snyder et al., 2005; Klinkenberg and Blokland, 2010). When a compound is found effective in restoring the scopolamine-induced amnesia in animals, it might possibly improve cognitive functions in healthy participants or in patients diagnosed with impaired memory function (Snyder et al., 2005; Klinkenberg and Blokland, 2010). According to some reports, memory impairment induced by the intraperitoneal injection of scopolamine can be ameliorated by the intra-ventral hippocampal injection of D2 receptor agonists (Fujishiro et al., 2005). Yet, the involvement of D1 dopaminergic receptors of the dorsal hippocampus on amnesia induced by scopolamine cannot be excluded. Given the fact that D1 receptor agonists increase the ACh release (Hersi et al., 1995a,b) and scopolamine could decrease DA release in the hippocampus (Memo et al., 1988), the present study was designed to investigate the effects of the bilateral intra-dorsal hippocampal (intra-CA1) microinjections of D1 receptor agonist and antagonist agents on scopolamine-induced amnesia as well as scopolamine state-dependent memory, using an inhibitory avoidance task.

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between 8:00 and 14:00. Experimental groups comprised eight rats and each animal was tested once. All procedures were performed in accordance with the institutional guidelines for animal care and use. Surgery Animals were anaesthetized by intraperitoneal (i.p.) injection of ketamine/xylazine mixture (100 and 10 mg/ kg, respectively) and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) with a flatskull position and the incisor bar 3.3 mm relative to the interaural line. After making a midline surgical incision, the skin and underlying periosteum were retracted. Two holes were drilled in the skull, as described in previous studies (Ishikawa et al., 1982; Packard and White, 1991), at stereotaxic coordinates of AP: 3 to 3.5 mm (depending on body weight and age) posterior to the bregma, and L: ±1.8–2 mm from midline according to the atlas of Paxinos and Watson (2006). According to the Paxinos atlas, these coordinates correspond to the length of 9 mm for bregma-lamda interval, thus, as we measured this interval in each rat’s skull, we considered a ratio to locate the actual sites for cannulae implantation. Two guide cannulae (22 gauge) were inserted into the holes. For animals receiving bilateral injections into the dorsal hippocampal (CA1) area of the hippocampus, the guide cannulae were advanced 2.8– 3 mm below the bregma through the holes drilled at the above-mentioned coordinates. The guide cannulae were anchored with a jeweler’s screw, and the incision was closed with dental cement. After surgery, stainless steel stylets (27 gauge) were inserted into the guide cannulae and left in the place until injections were made to keep them patent prior to the injections. All animals were allowed 1 week to recover from surgery and to get cleared from anesthetics’ effects. Drugs and microinfusions The drugs included scopolamine hydrobromide (Tocris, Bristol, BS11 0QL, UK), SKF38393 and SCH23390 (Sigma, St Louis, CA, USA). All drugs were dissolved in sterile saline and were injected into the CA1 region of the dorsal hippocampus. For bilateral drug infusion, the animals were gently restrained in hand; the stylets were removed from the guide cannulae and replaced by 27gauge injection needles (1 mm below the tip of the guide cannula). The injection solutions were administered at a total volume of 1 ll/rat (0.5 ll in each side) over a 60-s period. Injection needles were left in place for an additional 60 s to facilitate the diffusion of the drugs.

EXPERIMENTAL PROCEDURES Animals

Inhibitory avoidance apparatus

Adult male Wistar rats (Pasteur institute, Tehran, Iran) weighing 220–270 g at the time of surgery were used. They had free access to food and water, were housed four in a cage, and kept at (22 ± 2) °C under a 12/12 h light–dark cycle (light beginning at 7:00 a.m). All experiments were carried out during the light phase

We used a step-through inhibitory avoidance apparatus consisting of two compartments of the same size (20  20  30 cm3). In the middle of a dividing wall, there was a guillotine door (7.9 cm2) which could be lifted manually. The walls and floor of one compartment consisted of white opaque resin, while the other

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compartment was (3 mm in diameter floor of the dark shocks (50 Hz, 3 s, grid floor of the stimulator.

M. Piri et al. / Neuroscience 252 (2013) 460–467

dark in color. Stainless steel bars and 1-cm intervals) constituted the compartment. Intermittent electric 1 mA intensity) were delivered to the dark compartment by an isolated

Behavioral procedures Training was done according to our previous studies (Azami et al., 2010; Piri and Zarrindast, 2011; Zarrindast et al., 2012). All animals were allowed to habituate in the experimental room (light and sound attenuated) for at least 30 min prior to the experiments. Each animal was then gently placed in the brightly lit compartment of the apparatus. After 5 s the guillotine door was opened and the animal was allowed to enter the dark compartment. The latency after which the animal crossed into the dark compartment was recorded. Animals which waited more than 100 s to cross to the dark compartment were excluded from the experiments. Once the animal crossed with all four paws placed in the next compartment, the guillotine door was closed and the rat was immediately withdrawn from the compartment. The trial was repeated after 30 min as in the acquisition trial where after 5 s the guillotine door was opened and as soon as the animal crossed to the dark (shock) compartment the door was closed and a foot shock (50 Hz, 1 mA and 3 s) was immediately delivered to the grid floor of the dark room. After 20 s, the rat was removed from the apparatus and temporarily placed into its home cage. Two minutes later, the animal was retested in the same way as in the previous trials. If the rat did not enter the dark compartment during 120 s, a successful acquisition of inhibitory avoidance response was recorded. Otherwise, when the rat entered the dark compartment (before 120 s) for the second time, the door was closed and the animal received the shock again. Only 11 rats of 264 failed to acquire the inhibitory avoidance test in the first time and all these rats fulfilled the acquisition of inhibitory avoidance successfully, during the second session. It is important to note that there was no significant difference between the numbers of trials to acquisition. On the test day, intra-CA1 infusions were performed 5 min prior to the test. For the study of memory 24 h after the training, each animal was gently placed in the light compartment and after 5 s the door was opened, and step-through latency was measured in the absence of electric foot shocks, as indicators of inhibitory avoidance behavior. The upper cut-off time of 300 s was set. The retention test was also carried out between 8:00 a.m. and 2:00 p.m. Data analysis The data are expressed as mean ± S.E.M. The statistical analysis was performed using one- and two-way analysis of variance (ANOVA). For multiple comparisons, post hoc comparison of means was carried out with the Tukey’s test, when appropriate. The level of statistical significance was set at P < 0.05. Calculations were performed using the SPSS statistical package.

Histology After the testing sessions, each rat was deeply anesthetized and 1 ll of a 4% methylene-blue solution was bilaterally infused into the CA1 (0.5 ll/side), as described in the drug section. Animals were then decapitated and their brain removed and placed in formaldehyde (10%). After several days, the brains were sliced and the sites of injections were verified according to Paxinos and Watson (2006). Only the data from animals with correct cannulae implants were included in the statistical analyses. Experimental design Eight animals were used in each experimental group. In experiments where animals received one or two injections, the control groups identically received saline. The intervals of drug administration were based on the previous studies. The experimental design has been summarized in Table 1. Experiment 1: effect of scopolamine on inhibitory avoidance memory. In this experiment, the effect of pretraining and pre-test administration of scopolamine on inhibitory avoidance response was examined. Thirteen groups of animals were used in this experiment (n = 8 rats/group). Five groups of animals received saline or different doses of scopolamine (0.25, 0.75, 1.5 and 3 lg/rat, intra-CA1), 5 min prior to the training phase (pre-training). On the test day, animals received saline (1 ll/rat, intra-CA1), 5 min before the test. The other eight groups of animals received saline (1 ll/rat, intraCA1) or scopolamine (3 lg/rat, intra-CA1), 5 min before the training and pre-test injection of the different doses of scopolamine (0.25, 0.75, 1.5 and 3 lg/rat, intra-CA1). Experiment 2: effects of pre-test administration of SKF38393 on memory retrieval in the presence or absence of scopolamine. Twelve groups of animals were used here (n = 8 rats/group). On the training day, four groups of animals received saline (1 ll/rat, intraCA1) 5 min before the training (pre-training). On the test day, the animals received pre-test intra-CA1 administration of SKF38393 (0, 1, 2 and 4 lg/rat) 2 min after saline injection (1 ll/rat, intra CA1; Fig. 2, left panel). Another eight groups received scopolamine (3 lg/rat, intra-CA1) 5 min before the training (pretraining). On the test day, four groups of these animals received SKF38393 (0, 1, 2 and 4 lg/rat, intra-CA1), 2 min after saline (1 ll/rat, intra CA1; Fig. 2, middle panel). The other four groups of rats received SKF38393 (0, 1, 2 and 4 lg/rat, intra-CA1) 2 min after scopolamine injection (0.25 lg/rat, intra-CA1; right panel). The step-through latency was measured 5 min after the last injection. Experiment 3: effects of the pre-test administration of SCH23390 on memory retrieval in the presence or absence of scopolamine. This experiment dealt with eight groups of animals (n = 8 rats/group). On the training day, all groups of animals received pre-training

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M. Piri et al. / Neuroscience 252 (2013) 460–467 Table 1. Summary of experimental design Figure

1

2

3

Left panel Middle panel Right panel

Number of groups and number of animals per groups

Pre-training treatment (5 min prior training) Intra-CA1 microinjection (lg/rat or 1 ll/rat)

Intra-CA1 microinjection (lg/rat or 1 ll/rat)

Saline (1 ll/ rat)

Scopolamine

Saline (1 ll/ rat)

(0, 0.25, 0.75,1.5, 3)

1

5 (n = 8 rats/group) 4 (n = 8 rats/group)

1

4 (n = 8 rats/group)

Left panel Middle panel Right panel

4 (n = 8 rats/group)

Left panel Right panel

4 (n = 8 rats/group)

1 3

4 (n = 8 rats/group)

3 1

Scopolamine

1

(0, 1, 2, 4)

1

(0, 1, 2, 4) 0.25

1 3

administration of saline (1 ll/rat, intra-CA1; left panel) or scopolamine (3 lg/rat, intra-CA1; right panel). On the test day, the animals received pre-test intra-CA1 administration of SCH23390 (0, 0.02, 0.1 and 0.5 lg/rat, intra-CA1), 2 min before saline (Fig. 3, left panel) or scopolamine injection (3 lg/rat) (Fig. 3, right panel). Likewise, the step-through latency was measured 5 min after the last injection.

RESULTS Effects of scopolamine on inhibitory avoidance memory Fig. 1 shows the effects of pre-training (left panel) or pretest (middle panel) intra-CA1 administration of scopolamine on step-through latency. One-way ANOVA revealed that pre-training [F(4,35) = 6.71, P < 0.001] or pre-test [F(4,35) = 7.77, P < 0.001] scopolamine (1.5 and 3 lg/rat, intra-CA1), dose-dependently reduced the step-through latency in the inhibitory avoidance task, indicating a scopolamine-induced amnesia. In animals which demonstrated pre-training scopolamine-induced amnesia, pre-test administration of scopolamine (0.75, 1.5 and 3 lg/rat, intra-CA1) restored the memory to the control level (scopolamine state of memory), (Fig. 1, right panel) [F(4,35) = 10.06, P < 0.001]. These results indicated that scopolamine induced amnesia as well as state-dependent learning. Effects of pre-test administration of SKF38393 on memory retrieval in the presence or absence of scopolamine Fig. 2 indicates the effects of pre-test intra-CA1 injection of SKF38393 in the presence or absence of

SKF38393 (2 min after Saline or Scopolamine)

SCH23390 (2 min before Saline or scopolamine)

(0, 0.25, 0.75,1.5, 3) (0, 0.25, 0.75,1.5, 3)

3

4 (n = 8 rats/group)

4 (n = 8 rats/group)

Pre-test treatment

(0, 1, 2, 4) (0,0.02, 0.1, 0.5)

3

(0,0.02, 0.1, 0.5)

scopolamine on memory retrieval. Two-way ANOVA indicated an interaction between the groups of animals which received pre-training saline (1 ll/rat) and pre-test SKF38393 (0, 1, 2 and 4 lg/rat, intra-CA1) and those which received pre-training scopolamine (3 lg/rat, intraCA1) and pre-test SKF38393 [for Treatment, F(1,56) = 18.32, P < 0.001; Dose, F(3,56) = 5.12, P < 0.01; and Treatment  Dose interaction, F(3,56) = 3.61, P < 0.05] on inhibitory avoidance memory. Two-way ANOVA also revealed a significant effect of treatment [F(1,56) = 84.12, P < 0.00] between the groups of animals which received pre-training saline (1 ll/rat) and pre-test SKF38393 and those with pretraining scopolamine (3 lg/rat, intra-CA1), followed by pre-test SKF38393 plus a lower dose of scopolamine (0.25 lg/rat, intra-CA1) on inhibitory avoidance memory. The results indicated neither a significant effect for dose [F(3,56) = 0.94, P > 0.05] nor the treatment  dose interaction [F(3,56) = 0.47, P > 0.05] on inhibitory avoidance memory. These results indicated that D1 receptors agonist influences on scopolamine amnesia as well as the scopolamine state-dependent learning. Furthermore, two-way ANOVA revealed a significant difference between the groups of animals which received pre-training scopolamine (3 lg/rat, intra-CA1) and pre-test SKF38393 and those which received pretraining scopolamine (3 lg/rat, intra-CA1), followed by pre-test SKF38393 plus a lower dose of scopolamine (0.25 lg/rat, intra-CA1) [for Treatment, F(1,56) = 7.99, P < 0.01; Dose, F(3,56) = 6.28, P < 0.001; and Treatment  Dose interaction, F(3,56) = 3.52, P < 0.05] on inhibitory avoidance memory. These results indicated that an ineffective dose of scopolamine administered on the testing day could block reversing the effect of SKF38393 on scopolamine-induced

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M. Piri et al. / Neuroscience 252 (2013) 460–467 Pre- training treatment

Pre-training treatment

Step-Through Latency (sec)

0 0.25 0.75 1.5

Saline ( 1 µl/rat)

Scopolamine ( 3 µg/rat)

+++

200

+ 150

**

**

***

***

50

(1 µl/rat) Saline

Scopolamine (3 µg/rat)

++ 250

0.25 0.75 1.5

3

0.25 0.75 1.5

3

Scopolamine (µg/rat)

Pre- test treatment Fig. 1. The effects of scopolamine on inhibitory avoidance memory. Five groups of animals received pre-training saline (1 ll/rat), or different doses of scopolamine (0.25, 0.75, 1.5 and 3 lg/rat, intraCA1). On the test day, the animals received saline (1 ll/rat, intraCA1) 5 min before the test. Other four groups of animals received pre-training injection of saline (1 ll/rat) and pre-testing injections of different doses of scopolamine (0.25, 0.75, 1.5 and 3 lg/rat, intraCA1). In the remaining four groups, animals received the pre-training administration of a high dose of scopolamine (3 lg/rat, intra-CA1) and the pre-testing injections of different doses of scopolamine (0.25, 0.75, 1.5 and 3 lg/rat, intra-CA1). Data are expressed as mean ± S.E.M. of eight animals per group. ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 different from the pre-training saline/pre-test saline group. +P < 0.05, +++ P < 0.001 different from the pre-training scopolamine (3 lg/ rat)/pre-test saline group.

amnesia. In addition, post hoc analysis revealed that in the animals which received saline before training and were tested following the intra-CA1 administration of SKF38393 (Fig. 2, left panel), no significant change was observed in the retrieval latencies [F(3,28) = 0.45, P > 0.05] as compared to the saline/saline control group. It turned out that, animals which received the pre-training administration of scopolamine (3 lg/rat, intra-CA1) with impaired inhibitory avoidance memory, had their memory impairment significantly recovered following the administration of SKF38393 (1, 2 and 4 lg/rat), on the test day [F(3,28) = 6.32, P < 0.01] reversed memory impairment (Fig. 2, middle panel). Moreover, co-administration of SKF38393 (1, 2 and 4 lg/rat, intra-CA1) with the lower dose of scopolamine (0.25 lg/rat, intra-CA1) on the test day, altered the response induced by the pre-test injection of SKF38393 [F(3,28) = 1.035, P > 0.05]. Post-hoc analysis showed that lower dose of scopolamine (0.25 lg/rat, intra-CA1) prevented the reversal effect of SKF38393 (1, 2 and 4 lg/rat, intra-CA1) on scopolamine-induced memory impairment (Fig. 2, right panel). Effects of pre-test administration of SCH23390 on memory retrieval in the presence or absence of scopolamine Two-way ANOVA showed no significant difference between the effects of SCH23390 (0, 0.02, 0.1 and

+

+

200

150

100

*** ***

50

0 0

Saline (1 µl/rat )

+++

3

250

100

300

Step-Through Latency (sec)

300

Scopolamine (µg/rat)

0

1

2

4

0

1

2

4

0

1

2

4

SKF 38393 (µg/rat) Saline (1 µl/rat )

Scopolamine(0.25 µg/rat)

Pre-test treatment Fig. 2. Effects of the pre-test administration of SKF38393 with or without scopolamine on the step-through latencies. In one series, all animals received the pre-training administration of saline (1 ll/rat, intra CA1), and the pre-testing administration of SKF38393 (0, 1, 2 and 4 lg/rat, intra-CA1), 2 min after saline (1 ll/rat, intra CA1). In the remaining groups, the animals received pre-training administration of scopolamine (3 lg/rat, intra-CA1) and pre-test injection of SKF38393 (0, 1, 2 and 4 lg/rat, intra-CA1), 2 min after the administration of saline (1 ll/rat, intra CA1) or scopolamine (0.25 lg/rat, intra-CA1). Data are expressed as mean ± S.E.M. of eight animals per group. ⁄⁄⁄ P < 0.001 different from saline/saline group. +P < 0.05, ++ P < 0.01 different from scopolamine (3 lg/rat)/saline group.

0.5 lg/rat, intra-CA1) in the presence or absence of effective dose of scopolamine (3 lg/rat, intra-CA1) [for Treatment, F(1,56) = 0.10, P > 0.05; Dose, F(3,56) = 13.06, P < 0.001; and Treatment  Dose interaction, F(3,56) = 0.48, P > 0.05]. In addition, post hoc analysis revealed that pre-test injection of SCH23390 reduced the step-through latency in the inhibitory avoidance task [F(3,28) = 7.85, P < 0.01]. Upon pre-training and pre-test administration of scopolamine (3 lg/rat, intra-CA1), pre-test intra-CA1 administration of SCH23390 was shown to decrease the improvement of memory retrieval induced by the pretest scopolamine (3 lg/rat, intra-CA1) treatment [F(3,28) = 5.37, P < 0.01]. These results indicated that blockade of the D1 receptors of the dorsal hippocampus on test day, impaired the inhibitory avoidance memory and hampered the scopolamine state-dependent learning.

DISCUSSION There are many reports highlighting the role of ACh as a crucial mediator for learning and memory (Blokland, 1995). In agreement with other investigations, our data showed that pre-training or pre-test intra-CA1 administration of scopolamine by itself impaired the inhibitory avoidance memory, which is thought to be directly attributed to a decrease in central cholinergic functions (Fibiger et al., 1991; Gallagher and Colombo, 1995). Furthermore, the present data showed that

M. Piri et al. / Neuroscience 252 (2013) 460–467

Pre - training treatment Saline (1 µl/rat )

Step-Through Latency (sec)

300

Scopolamine (3 µg/rat)

250

200

*

150

+ ++

***

100

50

0

0

0.02

0.1

0.5

0

0.02

0.1

0.5

SCH23390 (µg/rat) Saline (1 µl/rat )

Scopolamine (3 µg/rat)

Pre -test treatment Fig. 3. Effects of the pre-test administration of SCH23390 with or without scopolamine on the step-through latencies. All animals received pre-training administration of saline (1 ll/rat, intra CA1) or scopolamine (3 lg/rat, intra-CA1) and pre-test administration of different doses of SCH23390 (0, 0.02, 0.1 and 0.5 lg/rat, intraCA1), 2 min before the administration of saline (1 ll/rat, intra CA1) or scopolamine (3 lg/rat, intra-CA1). Data are expressed as mean ± S.E.M. of eight animals per group. ⁄P < 0.05, ⁄⁄⁄P < 0.001 different from saline/saline group. +P < 0.05, ++P < 0.01 different from scopolamine (3 lg/rat)/scopolamine (3 lg/rat) group.

amnesia induced by pre-training scopolamine (3 lg/rat, intra-CA1) was completely reversed by injecting the same dose of scopolamine before testing. Such effects of scopolamine which are referred to as statedependent learning have been previously demonstrated (Azami et al., 2010; Jamali-Raeufy et al., 2011). Statedependent memory is a phenomenon in which the retrieval of newly acquired information is possible only if the subject is in the same sensory context and physiological state as the encoding phase (Overton, 1966; Izquierdo and Dias, 1983; Azami et al., 2010; Jamali-Raeufy et al., 2011). In our investigations, we also evaluated the possible role of the CA1 D1 receptors on inhibitory avoidance memory and scopolamine-induced amnesia. Based on our findings, the pre-test microinjection of the different doses of SKF38393 into the CA1 region of dorsal hippocampus by itself does not significantly alter the inhibitory avoidance memory. It however can reverse the memory impairment already induced by the pretraining administration of scopolamine. As such, the activation of CA1 D1 receptors appeared to abrogate the scopolamine-induced amnesia. This is in agreement with previous studies showing that D1 receptor agonists can reverse memory impairment in aged rats via facilitating the cholinergic function in the hippocampus (Hersi et al., 1995b). Our data may also be endorsed by the previous investigations showing that SKF38393 significantly alleviates the scopolamine-induced choice accuracy deficit in a radial arm maze test (Levin and Rose, 1991). Since cyclic AMP/protein kinase A (cAMP/

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PKA) signaling has an important role in learning and memory (Drain et al., 1991), one may expect that intraCA1 injections of SKF38393 may influence inhibitory avoidance memory via activation of cAMP/PKA pathway. On the other hand, there is also a report showing that local administrations of SKF38393 into the hippocampus increase the ACh release in this site (Imperato et al., 1993; Hersi et al., 1995a,b). Referring to the proven ACh’s memory-improving properties in the inhibitory avoidance task (Fibiger, 1991; Blokland, 1995; Mahmoodi et al., 2010), one may expect that the intraCA1 injections of SKF38393 may influence inhibitory avoidance memory via modulating ACh release in the dorsal hippocampus. In the present study, we examined this hypothesis. Our result indicated that when ineffective dose of scopolamine was injected before SKF38393-induced D1 blockade, it appeared to improve the effect of SKF38393 on the pre-training scopolamineinduced amnesia. Our results obtained by pre-test scopolamine injection, 2 min before SKF38393, may further support the hypothesis that the intra-CA1 injections of D1 receptor agonist, SKF38393, may influence the inhibitory avoidance memory via increasing ACh release in the dorsal hippocampus and activation of muscarinic receptors. The idea may be supported by previous investigations showing that the activation of D1 receptors increase the production of ACh via the activation of cAMP/PKA pathway (Berrard et al., 1995; Berse and Blusztajn, 1995; Inoue et al., 1995; Hersi et al., 1995b). Later in our studies, the effect of D1 receptor antagonist on inhibitory avoidance memory and scopolamine state-dependent learning in the dorsal hippocampus was investigated. Our results revealed impairment in inhibitory avoidance performance after the pre-test intra-CA1 administration of SCH23390. This result is in agreement with that of a previous study showing that the dopaminergic system (Hefco et al., 2003) or even dopamine D1 receptors (Seamans et al., 1998) have an important role in the modulation of memory function. Since the D1 antagonist, SCH23390, inhibited the in vivo hippocampal ACh release (Hersi et al., 1995b), one may expect that intra-CA1 injection of SCH23390 may influence the inhibitory avoidance memory via the inhibition of ACh release in the dorsal hippocampus. Another explanation is that the effects were observed following D1 receptors inhibition by SCH23390 administration could be due to the inhibition of the cAMP/PKA pathway. Based on the present data, the pre-test intra-CA1 injection of SCH23390 inhibited the scopolamine statedependent memory, which may indicate that the scopolamine response in the dorsal hippocampus is at least partly mediated through the D1 receptor system. The results are in agreement with others who found that pre-test injection of the D1 receptor antagonist inhibits morphine (Azizbeigi et al., 2011), cannabinoid (Zarrindast et al., 2010) and ethanol (Rezayof et al., 2007) state-dependent memory. Since, state-dependent memory is considered as a reward-related kind of learning; there may be a possibility that SCH23390

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inhibits the state-dependent learning through suppression of the reward-related learning mechanisms. The results of the present and previous investigations (Rezayof et al., 2007; Zarrindast et al., 2010; Azizbeigi et al., 2011) may substantiate that the activation of the D1 receptor subtype in the CA1 regions is necessary for statedependent learning, and may support the hypothesis that the restoration of scopolamine amnesia by SKF38393 is possibly mediated through D1 receptors. To demonstrate the involvement of the D1 receptors for the observed results, the effects of SCH23390 on the restoration of scopolamine-induced amnesia by SKF38393 was examined in the dorsal hippocampus. Based on our findings, the intra-CA1 injection of different doses of SCH23390 before administration of the effective dose of SKF38393 blocked the reversal effect of SKF38393 on pre-training scopolamine-induced amnesia (data not shown). These results indicate that D1 receptors of the dorsal hippocampus (the CA1 region) may play a definitive role in scopolamineinduced amnesia as well as the scopolamine statedependent memory. In summary, the activation of D1 receptors of the dorsal hippocampus reversed the scopolamine-induced amnesia, while the inhibition of D1 receptors of the dorsal hippocampus blocked the scopolamine statedependent learning. Moreover, the activation of D1 receptors via ACh release and finally through the activation of the muscarinic receptors was shown to reverse the scopolamine-induced amnesia.

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(Accepted 1 August 2013) (Available online 9 August 2013)