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Crocins, the active constituents of Crocus sativus L., counteracted apomorphine-induced performance deficits in the novel object recognition task, but not novel object location task, in rats
Neuroscience Letters 644 (2017) 37–42
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Crocins, the active constituents of Crocus sativus L., counteracted apomorphine-induced performance deficits in the novel object recognition task, but not novel object location task, in rats Nikolaos Pitsikas a,∗ , Petros A. Tarantilis b a
Department of Pharmacology, School of Medicine, Faculty of Health Sciences, University of Thessaly, Larissa, Greece Laboratory of Chemistry, Department of Food Science and Human Nutrition, School of Food Biotechnology and Development, Agricultural University of Athens, Athens, Greece b
h i g h l i g h t s • Apomorphine disrupted recognition memory. • Crocins reversed the recognition memory deficits described above. • An interaction between crocins and dopamine relevant to schizophrenia is evidenced.
a r t i c l e
i n f o
Article history: Received 15 December 2016 Received in revised form 15 February 2017 Accepted 16 February 2017 Available online 17 February 2017 Keywords: Crocins Apomorphine Recognition memory Locomotor activity Schizophrenia Rat
a b s t r a c t Schizophrenia is a chronic mental disease that affects nearly 1% of the population worldwide. Several lines of evidence suggest that the dopaminergic (DAergic) system might be compromised in schizophrenia. Specifically, the mixed dopamine (DA) D1 /D2 receptor agonist apomorphine induces schizophrenia-like symptoms in rodents, including disruption of memory abilities. Crocins are among the active components of saffron (dried stigmas of Crocus sativus L. plant) and their implication in cognition is well documented. The present study investigated whether crocins counteract non-spatial and spatial recognition memory deficits induced by apomorphine in rats. For this purpose, the novel object recognition task (NORT) and the novel object location task (NOLT) were used. The effects of compounds on mobility in a locomotor activity chamber were also investigated in rats. Post-training peripheral administration of crocins (15 and 30 mg/kg) counteracted apomorphine (1 mg/kg)-induced performance deficits in the NORT. Conversely, crocins did not attenuate spatial recognition memory deficits produced by apomorphine in the NOLT. The present data show that crocins reversed non-spatial recognition memory impairments produced by dysfunction of the DAergic system and modulate different aspects of memory components (storage and/or retrieval). The effects of compounds on recognition memory cannot be attributed to changes in locomotor activity. Further, our findings illustrate a functional interaction between crocins and the DAergic system that may be of relevance for schizophrenia-like behavioral deficits. Therefore, the utilization of crocins as an adjunctive agent, for the treatment of cognitive deficits observed in schizophrenic patients should be further investigated. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Schizophrenia is a severe psychiatric disorder that affects up to 1% of the population in the world [16]. Several lines of evi-
∗ Corresponding author at: Department of Pharmacology, School of Medicine, Faculty of Health Sciences, University of Thessaly, Biopolis, Panepistimiou 3, 415-00, Larissa, Greece. E-mail address: [email protected] (N. Pitsikas). http://dx.doi.org/10.1016/j.neulet.2017.02.042 0304-3940/© 2017 Elsevier B.V. All rights reserved.
dence suggest that the dopaminergic (DAergic) system might be compromised in schizophrenia. Deficits in performance in various cognitive tasks have been described in this disorder, linked to abnormal dopamine (DA) functioning [24]. Experimental evidence suggests that either too little or too much stimulation of DA impairs cognitive performance [37]. Traditional neuroleptics have demonstrated a certain efficacy in the treatment of the positive symptoms of schizophrenia; however their efficacy is weak in the treatment of cognitive deficits of this disease [16].
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Crocus sativus L. (C. sativus) is a plant cultivated in many countries such as Iran, India, Italy, Spain and Greece. Their dried stigmas are the well-known spice called saffron. The main substances of saffron are crocins, picrocrocin and safranal. Crocins, glucosyl esters of crocetin, are water-soluble carotenoids and their implication in neurodegenerative and psychiatric disorders has been proposed [34]. In addition, it has recently been demonstrated that crocins counteracted behavioral deficits induced by the N-methyl-d-aspartate (NMDA) receptor antagonist ketamine, including recognition memory impairments in rats [19]. At present, it is not clear if and how crocins are able to reverse cognitive deficits produced by dysfunction of the DAergic system in animals. Based on the above described findings, the purpose of the current work was to investigate the efficacy of crocins in counteracting apomorphine-induced recognition memory deficits. Recognition memory is a type of memory that is impaired in schizophrenic patients [7,11] and disrupted by apomorphine, a mixed DA D1 /D2 agonist, in healthy volunteers [28] and rats [20]. For this purpose, the novel object recognition task (NORT) [12] and the novel object location task (NOLT) [14] were used. These procedures assess nonspatial and spatial recognition memory, respectively, in rodents. Locomotor activity was also assessed as an independent measure of the potential motoric effects of the compound that could influenced rats’ performance in the NORT and NOLT respectively. 2. Material and methods 2.1. Animals Independent groups of naive male (3-month-old) Wistar rats (Hellenic Pasteur Institute, Athens, Greece) weighing 250–300 g were used. The animals were housed in Makrolon cages (47.5 cm length × 20.5 cm height × 27 cm width), three per cage, in a regulated environment (21 ± 1 ◦ C; 50–55% relative humidity; 12-h/12-h light/dark cycle, lights on at 07.00 h) with free access to food and water. All animal care and procedures were in accordance with international guidelines and national (Animal Act, P.D. 160/91) and international laws and policies (EEC Council Directive 86/609, JL 358, 1, December 12, 1987; NIH Guide for Care and Use of Laboratory Animals, NIH publication no. 85-23, 1985). 2.2. Novel object recognition task (NORT) The test apparatus consisted of a dark open box made of Plexiglas (80 cm length × 50 cm height × 60 cm width) that was illuminated by a 60-W light suspended 60 cm above the box. The light intensity was equal in the different parts of the apparatus. The objects to be discriminated (in triplicate) made of glass, plastic, or metal, were in three different shapes: metallic cubes, glass pyramids and plastic cylinders 7 cm high; and could not be displaced by rats. NORT was performed as described previously [19]. Briefly, during the week before the test, the animals were handled twice a day for 3 consecutive days. Before testing, the rats were allowed to explore the apparatus for 2 min for 3 consecutive days. During testing, a session that consisted of two 2-min trials was conducted. During the “sample” trial (T1), two identical samples (objects) were placed in two opposite corners of the apparatus in a random fashion, 10 cm from the side walls. A rat was placed in the middle of the apparatus and allowed to explore the two identical objects. After T1, the rat returned to its home cage and an intertrial interval (ITI) followed. Subsequently, the “choice” trial (T2) was performed. During T2, a novel object replaced one of the objects presented during T1. Accordingly, the rats were re-exposed to two objects: a copy of the
familiar (F) object and the novel (N) object. All combinations and locations of the objects were counterbalanced to reduce potential bias caused by preference for particular locations or objects. Exploration was defined as follows: directing the nose toward the object at a distance of 2 cm or less and/or touching the object with the nose. Turning around or sitting on the object was not considered exploratory behavior. The time spent by the rats exploring each object during T1 and T2 was manually recorded with a stopwatch. Based on this measure, a series of variables was then calculated: the total time spent exploring the two identical objects in T1 and the time spent exploring the two different objects, (F) and (N) in T2. The discrimination between (F) and the (N) during T2 was measured by comparing the time spent exploring the familiar object with the time spent exploring the novel object. Because this time may be biased by differences in the overall level of exploration [8], we used a discrimination index (D) to represent the preference for novel as opposed to familiar object calculated as follows; D = N-F/N + F [8]. 2.3. Novel object location task (NOLT) The test apparatus was the same apparatus as the one used in the object recognition task. The test arena was located in a large observation room with external cues (large and distinctive objects) that surrounded the experimental box to help rats complete the spatial memory task. These cues were kept in a constant location throughout the testing period. The objects were the same objects as in the NORT. The NOLT was performed as described elsewhere [32]. Briefly, during the week before the test, the animals were handled twice daily for 3 consecutive days. Before testing, the rats were allowed to explore the apparatus for 2 min for 3 consecutive days. During testing, a session that consisted of two 2-min trials was conducted. During the “sample” trial (T1), two identical samples (objects) were placed in two opposite corners of the apparatus in a random fashion; 10 cm form the side wall. A rat was placed in the middle of the apparatus and allowed to explore these two identical objects. After T1, the rat was returned to its home cage, and an intertrial interval (ITI) followed. Subsequently, the “choice” trial (T2) was performed. During T2, one of the two similar objects was moved to a different location (new location [NL]) while the other object remained in the same position (familiar location [FL]) as in T1. Thus, the two objects were now in diagonal corners. All combinations and locations of the objects were counterbalanced to reduce potential bias caused by preferences for particular locations. The definition of exploration is provided above in the context of describing the NORT protocol. The time spent by the rats exploring each object during T1 and T2 was manually recorded with a stopwatch. Based on this measure, a series of variables was then calculated: the total time spent exploring the two identical objects in T1 and the time spent exploring the two objects in the two different locations in T2. The discrimination between the FL and NL during T2 was measured by comparing the time spent in exploring the object in the FL with the time spent exploring the object in the NL. Because this time may be biased by differences in the overall level of exploration [8], we used a discrimination index (D) to represent the preference for novel, as opposed to familiar location that was calculated as D = NL − FL/NL + FL [8]. 2.4. Spontaneous locomotor activity test Spontaneous locomotor activity was assessed in an activity cage (Ugo Basile, Varese, Italy). The apparatus consisted of a box made of Plexiglas (41 cm length × 33 cm height × 41 cm width). Every movement of the animal produced a signal caused by variations in the inductance and capacitance of resonance circuitry of the apparatus. The signals were then automatically converted into numbers
N. Pitsikas, P.A. Tarantilis / Neuroscience Letters 644 (2017) 37–42
Crocins used in the current experimentation were derived from the same batch of plant material (saffron) and the same purification procedure, extraction and separation. Our plant material was kindly offered by the Cooperative of Saffron, Krokos, Kozani, Greece. Crocins were isolated from the red dried stigmas (saffron) of C. sativus as described previously [25]. The purity of crocins was 81%. The compound was dissolved in saline (NaCl 0.9%). Apomorphine hydrochloride (Sigma, St. Louis, MO, USA) was dissolved in saline that contained 0.1% ascorbic acid to prevent oxidation. All solutions were freshly prepared on the day of testing and were administered intraperitoneally (i.p.) in a volume of 1 ml/kg. Control animals received isovolumetric amounts of the specific vehicle solution.
Vehicle+vehicle Vehicle+crocins (15 mg/kg) Vehicle+crocins (30 mg/kg) Apomorphine (1 mg/kg)+vehicle Apomorphine+crocins (15 mg/kg) Apomorphine+crocins (30 mg/kg)
0,6 0,5
Discrimination index D
2.5. Chemicals
A
0,4 0,3 0,2 0,1
*
0,0
B 16 14
Exploration time (s.)
that reflected activity counts. Changes in activity counts represent a standard behavioral assay for testing the motoric effects of drugs. The test was performed as described previously [21]. Each animal was placed into the locomotor activity arena and spontaneous locomotion was recorded for 5 min. To avoid the presence of olfactory cues, all the apparatuses (NORT, NOLT and motor activity cage) were thoroughly cleaned with 20% ethanol and then wiped with dry paper after each trial.
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Vehicle+vehicle Vehicle+crocins (15 mg/kg) Vehicle+crocins (30 mg/kg) Apomorphine (1 mg/kg)+vehicle Apomorphine+crocins (15 mg/kg) Apomorphine+crocins (30 mg/kg)
12 10 8 6 4
2.6. Experimental protocol 2
Separate cohorts of naive rats were used for the NORT, NOLT, and the locomotor activity experiments. Experiments were conducted between 10.00 am and 14.00 pm in a room where only these animals were housed. Animals’ behavior was video-recorded. An experimenter who was unaware of the pharmacological treatment of each subject evaluated the data.
2.6.1. Experiment 1: effects of crocins and apomorphine on rats’ performance in the NORT To examine the effects of crocins, apomorphine and their combination on non-spatial recognition memory we administered the vehicle or apomorphine immediately after T1. Crocins were administered 5–10 s after vehicle or apomorphine. For this study, the 3-h ITI has been used since at this delay condition recognition memory is still intact in the vehicle-treated rat [2], while impairments associated with apomorphine administration (hyperactivity) were not observed at this time point [20]. Doses of crocins were selected based on a previous study [19]. The dose of apomorphine (1 mg/kg) was selected based on a prior report in which it was found to impair rats’ performance in the NORT without producing side effects [20]. Rats were randomly divided into six experimental groups (10 rats per group) as follows: vehicle (saline) + vehicle (saline + 0.1% ascorbic acid); vehicle (saline) + crocins 15 mg/kg; vehicle (saline) + crocins 30 mg/kg; vehicle (saline + 0.1% ascorbic acid) + apomorphine 1 mg/kg; apomorphine 1 mg/kg + crocins 15 mg/kg; apomorphine 1 mg/kg + crocins 30 mg/kg.
2.6.2. Experiment 2: effects of crocins and apomorphine on rats’ performance in the NOLT To examine the effects of crocins, apomorphine and their combination on spatial recognition memory we administered the vehicle or apomorphine immediately after T1. Crocins were administered 5–10 s after vehicle or apomorphine. For this study, the ITI used and the dose range of compounds was that used in experiment 1. Six experimental groups (8 rats per group) were used in experiment 2 and were the same groups of experiment 1.
0 Fig. 1. Novel object recognition task. Histograms represent the mean ± S.E.M. of 10 rats in each group. The 3-h ITI was used. (A) Discrimination index D performance expressed by different groups of rats during T2. * p < 0.05 vs. all the other groups. (B) Total exploration time in the different groups of rats during T2.
2.6.3. Experiment 3: effects of crocins and apomorphine on rats’ performance in the spontaneous locomotor activity test To study the effects of compounds on rats’ locomotor activity we first administered the vehicle or apomorphine. Crocins were administered 5–10 s after vehicle or apomorphine and 3 h later the spontaneous locomotor activity test was performed. Six experimental groups (7 rats per group) were used in experiment 3 and were the same groups of experiment 1. 2.7. Statistical analysis The data were expressed as mean ± S.E.M. Data were analyzed using the two-way analysis of variance (ANOVA) test. Post hoc comparisons between treatment means were made using the Tukey’s test. Values of p < 0.05 were considered statistically significant [26]. 3. Results 3.1. Experiment 1: effects of crocins and apomorphine on rats’ performance in the NORT Crocins counteracted apomorphine-induced performance deficits in the NORT (Fig. 1A). Specifically, index D data analysis evidenced a statistically significant interaction between crocins and apomorphine (F(2,59) = 4.7, p < 0.05), a statistically significant main effect of crocins (F(2,59) = 7.1, p < 0.01) and of apomorphine (F(1,59) = 36, p < 0.01). Subsequent post hoc comparisons revealed that the vehicle + apomorphine-treated rats exhibited a lower discrimination index D compared to vehicle + vehicle-treated rats (p < 0.05) and that the index D displayed by apomorphine + crocins (15 mg/kg) and apomorphine + crocins (30 mg/kg)-treated rats
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A Vehicle+vehicle Vehicle+crocins (15 mg/kg) Vehicle+crocins (30 mg/kg) Apomorphine (1 mg/kg)+vehicle Apomorphine+crocins (15/mg/kg) Apomorphine+crocins (30 mg/kg)
0,6
0,4 0,3 0,2 0,1
***
0,0
B
16
Exploration time (s.)
14 12
800
Motor activity (counts/5 min)
Discrimination index D
0,5
Vehicle+vehicle Vehicle+crocins (15 mg/kg) Vehicle+crocins (30 mg/kg) Apomorphine (1 mg/kg)+vehicle Apomorphine+crocins (15 mg/kg) Apomorphine+crocins (30 mg/kg)
10 8 6
Vehicle+vehicle Vehicle+crocins (15 mg/kg) Vehicle+crocins (30 mg/kg) Apomorphine (1 mg/kg)+vehicle Apomorphine+crocins (15 mg/kg) Apomophine+crocins (30 mg/kg)
600
400
200
0 Fig. 3. Spontaneous locomotor activity test. Histograms represent the mean ± S.E.M. of 7 rats in each group.
(F(2,41) = 0.1, p > 0.05) or a significant interaction between apomoprhine and crocins (F(2,41) = 0.7, p > 0.05) (Fig. 3). 4. Discussion
4 2 0
Fig. 2. Novel object location task. Histograms represent the mean ± S.E.M. of 8 rats per in each group. The 3-h ITI was used. (A) Discrimination index D performance expressed by different groups of rats during T2. * p < 0.05 vs. all the other groups. (B) Total exploration time in the different groups of rats during T2.
was higher than that of the group of rats that received vehicle + apomorphine (both p < 0.05, Fig. 1A). Analysis of the total exploration times during T2 did not show a statistically significant apomorphine x crocins interaction (F(2,59) = 0.7, p > 0.05) neither a main effect of crocins (F(2,59) = 0.6, p > 0.05) or of apomorphine (F(1,59) = 2.5, p > 0.05) (Fig. 1B). 3.2. Experiment 2: effects of crocins and apomorphine on rats’ performance in the NOLT Crocins did not counteract apomorphine-induced performance deficits in the NOLT (Fig. 2A). The analysis of the D index data showed a significant main effect of apomorphine (F(1,47) = 162.9, p < 0.01), but no main effect of crocins (F(2,47) = 0.007, p > 0.05) and no apomorphine x crocins interaction (F(2,47) = 0.2, p > 0.05). Preplanned comparisons revealed that all animals treated with apomorphine had a significantly lower D index compared with their respective control groups (p < 0.05; Fig. 2A). Total exploration times were not different among the various experimental groups (Fig. 2B). Analysis of the total exploration times during T2 did not show a statistically significant apomorphine x crocins interaction (F(2,47) = 0.69, p > 0.05), neither a main effect of crocins (F(2,47) = 2, p > 0.05) or of apomorphine (F(1,47) = 0.08, p > 0.05) (Fig. 2B). 3.3. Experiment 3: effects of crocins and apomorphine on rats’ performance in the locomotor activity test Total motor activity was not different among the various experimental groups. Overall analysis did not show a significant main effect either of apomorphine (F(1,41) = 0.1, p > 0.05) or of crocins
The NORT evaluates non-spatial recognition memory in rodents. It is a non-rewarded paradigm that it is based on spontaneous exploratory behavior in rodents [12]. The NOLT is a version of the NORT that evaluates spatial recognition memory. This task assesses the ability of rodents to discriminate the novelty of the object locations but not the objects itself because the behavioral testing arena is already familiar to the animals [14]. Both of these recognition memory tasks do not involve explicit reward or punishment but rely on the natural curiosity of rodents and preference for novelty which do not appear to be influenced by reinforcement/response contingencies [9]. These paradigms are quite similar to procedures used in humans and should have a significant level of construct and predictive validity [12]. The current findings are in line with prior studies, in which post-training administration of apomorphine disrupted rats’ performance in either of the two recognition memory tasks [20,27]. These detrimental effects of apomorphine on memory seem to be dependent on its action on hippocampus and prefrontal cortex (PFC). Specifically, local infusion of apomorphine into CA1 but not CA3 subregion of hippocampus produced non-spatial and spatial memory impairments [35,36]. Alterations in the DA activity in the dorsolateral PFC have also been suggested to underlie the effects of apomorphine on cognition [17,18]. Crocins, at any doses tested reversed apomorphine-induced performance deficits in the NORT. As in the present study, the retention delay interval was no longer than 3 h; the effects of compounds were limited to short-term memory. The effects of crocins and apomorphine on performance during the retention phase reflect the modulation of post-training mnemonic processes (storage and/or retrieval of information) [23]. In contrast, crocins at all doses tested did not counteract the performance deficits produced by apomorphine in the NOLT. Several reasons might underlie this apparent discrepancy. The lack of a protective effect of crocins in the NOLT may originate from the fact that spatial memory is more susceptible to hippocampal dysfunction than non-spatial memory (NORT procedure). Thus, performance in the spatial object location task is easier to disrupt than performance in the non-spatial object recognition task, making performance in this task less susceptible to crocins administration [6]. In this context, it has been reported that object location is more vulnerable
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than object recognition because it is based on less redundant information: the various features of the objects can be encoded from several dimensions and attributes, which are not all required during recognition, while a location offers fewer cues [13,15]. Collectively, tests of spatial memory typically tax the ability of the animal to discriminate between two or more highly familiar locations, often on the basis of which the animal has last visited, set within a familiar environment while non-spatial tasks often involve stimuli that are relatively unfamiliar or completely novel. Thus, this difference in stimuli intensity (higher in non-spatial tasks as compared to spatial tasks) might underlie the longer preservation of memory in the object recognition with respect to the object location [10]. The NOLT study results are inconsistent with previous findings of ours in which crocins attenuated spatial memory deficits produced by the muscarinic receptor antagonist scopolamine in rats [33]. These controversial findings with regards the effects of crocins on spatial memory may be attributable to differences in experimental settings. Specifically, the radial water maze task used in our previous study [33] is a negatively reinforced spatial memory procedure, whereas the NOLT used in the present experiment is a non-rewarded spatial memory paradigm. Moreover, in the present experimentation compounds were acutely administered, while in the above described study a sub-chronic treatment protocol was used. Finally, apomorphine and scopolamine act on different neurotransmitter systems since apomorphine supresses DA neurotransmission, while scopolamine disrupts the integrity of the cholinergic system. These considerations probably offer a plausible explanation as to why crocins did not reverse the apomorphineinduced impairments in the NOLT. It seems unlikely that non-specific factors, as attentional or sensorimotor deficits, may have influenced rats’ cognitive performance assessed in the NORT since exploration levels were not different among the various experimental groups during the choice trial T2. Further, the motor activity cage results indicate that the effects of compounds on recognition memory cannot be attributed to changes in locomotor activity. In addition, the effects of apomorphine on memory cannot be ascribed to the residual presence of the drug during testing since this drug has a very short half-life (10–15 min) [4]. Thus, this pattern of results indicates that nonspecific factors did not affect animals’ performance in either of the two memory tasks. The mechanism(s) by which crocins exert their effects on apomorphine-induced non-spatial recognition memory deficits are not yet clarified. In this context, it has been previously demonstrated that crocins reduced schizophrenia-like effects, including recognition memory deficits, in rodents due to excessive glutamatergic activity [3,19,22]. An explanation of the current findings might be based on the well-documented antioxidant properties of crocins. The antioxidant properties of crocins have been revealed in different preclinical models of oxidative stress [29], brain ischemia [38], Parkinson’s disease [1] and Alzheimer’s disease [30]. Although the pathogenesis of schizophrenia remains unknown, a link between oxidative stress and this pathology has been suggested [5] and the disrupting effect of apomorphine on memory seem to be dependent on its pro-oxidative stress properties [31]. Additional research, however, is required aiming to clarify this issue. The current results are in line and extend previous our findings in which the efficacy of crocins to counteract psychotomimetic effects, including recognition memory deficits, caused by dysfunction of the glutamatergic system has been revealed [19]. Collectively, these findings suggest a functional interaction between crocins and both the glutamatergic and DAergic systems and support a potential therapeutic role of crocins in the treatment of some behaviors that may be altered in schizophrenia. Specifically, the utilization of crocins as an adjunctive agent, for the
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[32] N. Pitsikas, Effects of scopolamine and L-NAME on rats’ performance in the object location test, Behav. Brain Res. 179 (2007) 294–298. [33] N. Pitsikas, S. Zisopoulou, P.A. Tarantilis, C.D. Kanakis, M.G. Polissiou, N. Sakellaridis, Effects of the active constituents of Crocus sativus L., crocins on recognition and spatial rats’ memory, Behav. Brain Res. 183 (2007) 141–146. [34] N. Pitsikas, The effects of Crocus sativus L. and its constituents on memory: basic studies and clinical applications, Evid. Based Complement. Alternat. Med. (2015) 926284. [35] D.R. Vago, A. Bevan, R.P. Kessner, The role of the direct perforant path input to the CA1 subregion of the dorsal hippocampus in memory retention and retrieval, Hippocampus 17 (2007) 977–987. [36] D.R. Vago, R.P. Kessner, Disruption of the direct perforant path input to the CA1 subregion of the dorsal hippocampus interferes with spatial working memory and novelty detection, Behav. Brain Res. 189 (2008) 273–283. [37] S. Vijayraghavan, M. Wang, S.G. Birnbaum, G.V. Williams, A.F. Arnsten, Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory, Nat. Neurosci. 10 (2007) 376–384. [38] Y.Q. Zheng, J.X. Liu, J.N. Wang, L. Xu, Effects of crocin on reperfusion induced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia, Brain Res. 1138 (2007) 86–94.
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Research article
Crocins, the active constituents of Crocus sativus L., counteracted apomorphine-induced performance deficits in the novel object recognition task, but not novel object location task, in rats Nikolaos Pitsikas a,∗ , Petros A. Tarantilis b a
Department of Pharmacology, School of Medicine, Faculty of Health Sciences, University of Thessaly, Larissa, Greece Laboratory of Chemistry, Department of Food Science and Human Nutrition, School of Food Biotechnology and Development, Agricultural University of Athens, Athens, Greece b
h i g h l i g h t s • Apomorphine disrupted recognition memory. • Crocins reversed the recognition memory deficits described above. • An interaction between crocins and dopamine relevant to schizophrenia is evidenced.
a r t i c l e
i n f o
Article history: Received 15 December 2016 Received in revised form 15 February 2017 Accepted 16 February 2017 Available online 17 February 2017 Keywords: Crocins Apomorphine Recognition memory Locomotor activity Schizophrenia Rat
a b s t r a c t Schizophrenia is a chronic mental disease that affects nearly 1% of the population worldwide. Several lines of evidence suggest that the dopaminergic (DAergic) system might be compromised in schizophrenia. Specifically, the mixed dopamine (DA) D1 /D2 receptor agonist apomorphine induces schizophrenia-like symptoms in rodents, including disruption of memory abilities. Crocins are among the active components of saffron (dried stigmas of Crocus sativus L. plant) and their implication in cognition is well documented. The present study investigated whether crocins counteract non-spatial and spatial recognition memory deficits induced by apomorphine in rats. For this purpose, the novel object recognition task (NORT) and the novel object location task (NOLT) were used. The effects of compounds on mobility in a locomotor activity chamber were also investigated in rats. Post-training peripheral administration of crocins (15 and 30 mg/kg) counteracted apomorphine (1 mg/kg)-induced performance deficits in the NORT. Conversely, crocins did not attenuate spatial recognition memory deficits produced by apomorphine in the NOLT. The present data show that crocins reversed non-spatial recognition memory impairments produced by dysfunction of the DAergic system and modulate different aspects of memory components (storage and/or retrieval). The effects of compounds on recognition memory cannot be attributed to changes in locomotor activity. Further, our findings illustrate a functional interaction between crocins and the DAergic system that may be of relevance for schizophrenia-like behavioral deficits. Therefore, the utilization of crocins as an adjunctive agent, for the treatment of cognitive deficits observed in schizophrenic patients should be further investigated. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Schizophrenia is a severe psychiatric disorder that affects up to 1% of the population in the world [16]. Several lines of evi-
∗ Corresponding author at: Department of Pharmacology, School of Medicine, Faculty of Health Sciences, University of Thessaly, Biopolis, Panepistimiou 3, 415-00, Larissa, Greece. E-mail address: [email protected] (N. Pitsikas). http://dx.doi.org/10.1016/j.neulet.2017.02.042 0304-3940/© 2017 Elsevier B.V. All rights reserved.
dence suggest that the dopaminergic (DAergic) system might be compromised in schizophrenia. Deficits in performance in various cognitive tasks have been described in this disorder, linked to abnormal dopamine (DA) functioning [24]. Experimental evidence suggests that either too little or too much stimulation of DA impairs cognitive performance [37]. Traditional neuroleptics have demonstrated a certain efficacy in the treatment of the positive symptoms of schizophrenia; however their efficacy is weak in the treatment of cognitive deficits of this disease [16].
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Crocus sativus L. (C. sativus) is a plant cultivated in many countries such as Iran, India, Italy, Spain and Greece. Their dried stigmas are the well-known spice called saffron. The main substances of saffron are crocins, picrocrocin and safranal. Crocins, glucosyl esters of crocetin, are water-soluble carotenoids and their implication in neurodegenerative and psychiatric disorders has been proposed [34]. In addition, it has recently been demonstrated that crocins counteracted behavioral deficits induced by the N-methyl-d-aspartate (NMDA) receptor antagonist ketamine, including recognition memory impairments in rats [19]. At present, it is not clear if and how crocins are able to reverse cognitive deficits produced by dysfunction of the DAergic system in animals. Based on the above described findings, the purpose of the current work was to investigate the efficacy of crocins in counteracting apomorphine-induced recognition memory deficits. Recognition memory is a type of memory that is impaired in schizophrenic patients [7,11] and disrupted by apomorphine, a mixed DA D1 /D2 agonist, in healthy volunteers [28] and rats [20]. For this purpose, the novel object recognition task (NORT) [12] and the novel object location task (NOLT) [14] were used. These procedures assess nonspatial and spatial recognition memory, respectively, in rodents. Locomotor activity was also assessed as an independent measure of the potential motoric effects of the compound that could influenced rats’ performance in the NORT and NOLT respectively. 2. Material and methods 2.1. Animals Independent groups of naive male (3-month-old) Wistar rats (Hellenic Pasteur Institute, Athens, Greece) weighing 250–300 g were used. The animals were housed in Makrolon cages (47.5 cm length × 20.5 cm height × 27 cm width), three per cage, in a regulated environment (21 ± 1 ◦ C; 50–55% relative humidity; 12-h/12-h light/dark cycle, lights on at 07.00 h) with free access to food and water. All animal care and procedures were in accordance with international guidelines and national (Animal Act, P.D. 160/91) and international laws and policies (EEC Council Directive 86/609, JL 358, 1, December 12, 1987; NIH Guide for Care and Use of Laboratory Animals, NIH publication no. 85-23, 1985). 2.2. Novel object recognition task (NORT) The test apparatus consisted of a dark open box made of Plexiglas (80 cm length × 50 cm height × 60 cm width) that was illuminated by a 60-W light suspended 60 cm above the box. The light intensity was equal in the different parts of the apparatus. The objects to be discriminated (in triplicate) made of glass, plastic, or metal, were in three different shapes: metallic cubes, glass pyramids and plastic cylinders 7 cm high; and could not be displaced by rats. NORT was performed as described previously [19]. Briefly, during the week before the test, the animals were handled twice a day for 3 consecutive days. Before testing, the rats were allowed to explore the apparatus for 2 min for 3 consecutive days. During testing, a session that consisted of two 2-min trials was conducted. During the “sample” trial (T1), two identical samples (objects) were placed in two opposite corners of the apparatus in a random fashion, 10 cm from the side walls. A rat was placed in the middle of the apparatus and allowed to explore the two identical objects. After T1, the rat returned to its home cage and an intertrial interval (ITI) followed. Subsequently, the “choice” trial (T2) was performed. During T2, a novel object replaced one of the objects presented during T1. Accordingly, the rats were re-exposed to two objects: a copy of the
familiar (F) object and the novel (N) object. All combinations and locations of the objects were counterbalanced to reduce potential bias caused by preference for particular locations or objects. Exploration was defined as follows: directing the nose toward the object at a distance of 2 cm or less and/or touching the object with the nose. Turning around or sitting on the object was not considered exploratory behavior. The time spent by the rats exploring each object during T1 and T2 was manually recorded with a stopwatch. Based on this measure, a series of variables was then calculated: the total time spent exploring the two identical objects in T1 and the time spent exploring the two different objects, (F) and (N) in T2. The discrimination between (F) and the (N) during T2 was measured by comparing the time spent exploring the familiar object with the time spent exploring the novel object. Because this time may be biased by differences in the overall level of exploration [8], we used a discrimination index (D) to represent the preference for novel as opposed to familiar object calculated as follows; D = N-F/N + F [8]. 2.3. Novel object location task (NOLT) The test apparatus was the same apparatus as the one used in the object recognition task. The test arena was located in a large observation room with external cues (large and distinctive objects) that surrounded the experimental box to help rats complete the spatial memory task. These cues were kept in a constant location throughout the testing period. The objects were the same objects as in the NORT. The NOLT was performed as described elsewhere [32]. Briefly, during the week before the test, the animals were handled twice daily for 3 consecutive days. Before testing, the rats were allowed to explore the apparatus for 2 min for 3 consecutive days. During testing, a session that consisted of two 2-min trials was conducted. During the “sample” trial (T1), two identical samples (objects) were placed in two opposite corners of the apparatus in a random fashion; 10 cm form the side wall. A rat was placed in the middle of the apparatus and allowed to explore these two identical objects. After T1, the rat was returned to its home cage, and an intertrial interval (ITI) followed. Subsequently, the “choice” trial (T2) was performed. During T2, one of the two similar objects was moved to a different location (new location [NL]) while the other object remained in the same position (familiar location [FL]) as in T1. Thus, the two objects were now in diagonal corners. All combinations and locations of the objects were counterbalanced to reduce potential bias caused by preferences for particular locations. The definition of exploration is provided above in the context of describing the NORT protocol. The time spent by the rats exploring each object during T1 and T2 was manually recorded with a stopwatch. Based on this measure, a series of variables was then calculated: the total time spent exploring the two identical objects in T1 and the time spent exploring the two objects in the two different locations in T2. The discrimination between the FL and NL during T2 was measured by comparing the time spent in exploring the object in the FL with the time spent exploring the object in the NL. Because this time may be biased by differences in the overall level of exploration [8], we used a discrimination index (D) to represent the preference for novel, as opposed to familiar location that was calculated as D = NL − FL/NL + FL [8]. 2.4. Spontaneous locomotor activity test Spontaneous locomotor activity was assessed in an activity cage (Ugo Basile, Varese, Italy). The apparatus consisted of a box made of Plexiglas (41 cm length × 33 cm height × 41 cm width). Every movement of the animal produced a signal caused by variations in the inductance and capacitance of resonance circuitry of the apparatus. The signals were then automatically converted into numbers
N. Pitsikas, P.A. Tarantilis / Neuroscience Letters 644 (2017) 37–42
Crocins used in the current experimentation were derived from the same batch of plant material (saffron) and the same purification procedure, extraction and separation. Our plant material was kindly offered by the Cooperative of Saffron, Krokos, Kozani, Greece. Crocins were isolated from the red dried stigmas (saffron) of C. sativus as described previously [25]. The purity of crocins was 81%. The compound was dissolved in saline (NaCl 0.9%). Apomorphine hydrochloride (Sigma, St. Louis, MO, USA) was dissolved in saline that contained 0.1% ascorbic acid to prevent oxidation. All solutions were freshly prepared on the day of testing and were administered intraperitoneally (i.p.) in a volume of 1 ml/kg. Control animals received isovolumetric amounts of the specific vehicle solution.
Vehicle+vehicle Vehicle+crocins (15 mg/kg) Vehicle+crocins (30 mg/kg) Apomorphine (1 mg/kg)+vehicle Apomorphine+crocins (15 mg/kg) Apomorphine+crocins (30 mg/kg)
0,6 0,5
Discrimination index D
2.5. Chemicals
A
0,4 0,3 0,2 0,1
*
0,0
B 16 14
Exploration time (s.)
that reflected activity counts. Changes in activity counts represent a standard behavioral assay for testing the motoric effects of drugs. The test was performed as described previously [21]. Each animal was placed into the locomotor activity arena and spontaneous locomotion was recorded for 5 min. To avoid the presence of olfactory cues, all the apparatuses (NORT, NOLT and motor activity cage) were thoroughly cleaned with 20% ethanol and then wiped with dry paper after each trial.
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Vehicle+vehicle Vehicle+crocins (15 mg/kg) Vehicle+crocins (30 mg/kg) Apomorphine (1 mg/kg)+vehicle Apomorphine+crocins (15 mg/kg) Apomorphine+crocins (30 mg/kg)
12 10 8 6 4
2.6. Experimental protocol 2
Separate cohorts of naive rats were used for the NORT, NOLT, and the locomotor activity experiments. Experiments were conducted between 10.00 am and 14.00 pm in a room where only these animals were housed. Animals’ behavior was video-recorded. An experimenter who was unaware of the pharmacological treatment of each subject evaluated the data.
2.6.1. Experiment 1: effects of crocins and apomorphine on rats’ performance in the NORT To examine the effects of crocins, apomorphine and their combination on non-spatial recognition memory we administered the vehicle or apomorphine immediately after T1. Crocins were administered 5–10 s after vehicle or apomorphine. For this study, the 3-h ITI has been used since at this delay condition recognition memory is still intact in the vehicle-treated rat [2], while impairments associated with apomorphine administration (hyperactivity) were not observed at this time point [20]. Doses of crocins were selected based on a previous study [19]. The dose of apomorphine (1 mg/kg) was selected based on a prior report in which it was found to impair rats’ performance in the NORT without producing side effects [20]. Rats were randomly divided into six experimental groups (10 rats per group) as follows: vehicle (saline) + vehicle (saline + 0.1% ascorbic acid); vehicle (saline) + crocins 15 mg/kg; vehicle (saline) + crocins 30 mg/kg; vehicle (saline + 0.1% ascorbic acid) + apomorphine 1 mg/kg; apomorphine 1 mg/kg + crocins 15 mg/kg; apomorphine 1 mg/kg + crocins 30 mg/kg.
2.6.2. Experiment 2: effects of crocins and apomorphine on rats’ performance in the NOLT To examine the effects of crocins, apomorphine and their combination on spatial recognition memory we administered the vehicle or apomorphine immediately after T1. Crocins were administered 5–10 s after vehicle or apomorphine. For this study, the ITI used and the dose range of compounds was that used in experiment 1. Six experimental groups (8 rats per group) were used in experiment 2 and were the same groups of experiment 1.
0 Fig. 1. Novel object recognition task. Histograms represent the mean ± S.E.M. of 10 rats in each group. The 3-h ITI was used. (A) Discrimination index D performance expressed by different groups of rats during T2. * p < 0.05 vs. all the other groups. (B) Total exploration time in the different groups of rats during T2.
2.6.3. Experiment 3: effects of crocins and apomorphine on rats’ performance in the spontaneous locomotor activity test To study the effects of compounds on rats’ locomotor activity we first administered the vehicle or apomorphine. Crocins were administered 5–10 s after vehicle or apomorphine and 3 h later the spontaneous locomotor activity test was performed. Six experimental groups (7 rats per group) were used in experiment 3 and were the same groups of experiment 1. 2.7. Statistical analysis The data were expressed as mean ± S.E.M. Data were analyzed using the two-way analysis of variance (ANOVA) test. Post hoc comparisons between treatment means were made using the Tukey’s test. Values of p < 0.05 were considered statistically significant [26]. 3. Results 3.1. Experiment 1: effects of crocins and apomorphine on rats’ performance in the NORT Crocins counteracted apomorphine-induced performance deficits in the NORT (Fig. 1A). Specifically, index D data analysis evidenced a statistically significant interaction between crocins and apomorphine (F(2,59) = 4.7, p < 0.05), a statistically significant main effect of crocins (F(2,59) = 7.1, p < 0.01) and of apomorphine (F(1,59) = 36, p < 0.01). Subsequent post hoc comparisons revealed that the vehicle + apomorphine-treated rats exhibited a lower discrimination index D compared to vehicle + vehicle-treated rats (p < 0.05) and that the index D displayed by apomorphine + crocins (15 mg/kg) and apomorphine + crocins (30 mg/kg)-treated rats
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A Vehicle+vehicle Vehicle+crocins (15 mg/kg) Vehicle+crocins (30 mg/kg) Apomorphine (1 mg/kg)+vehicle Apomorphine+crocins (15/mg/kg) Apomorphine+crocins (30 mg/kg)
0,6
0,4 0,3 0,2 0,1
***
0,0
B
16
Exploration time (s.)
14 12
800
Motor activity (counts/5 min)
Discrimination index D
0,5
Vehicle+vehicle Vehicle+crocins (15 mg/kg) Vehicle+crocins (30 mg/kg) Apomorphine (1 mg/kg)+vehicle Apomorphine+crocins (15 mg/kg) Apomorphine+crocins (30 mg/kg)
10 8 6
Vehicle+vehicle Vehicle+crocins (15 mg/kg) Vehicle+crocins (30 mg/kg) Apomorphine (1 mg/kg)+vehicle Apomorphine+crocins (15 mg/kg) Apomophine+crocins (30 mg/kg)
600
400
200
0 Fig. 3. Spontaneous locomotor activity test. Histograms represent the mean ± S.E.M. of 7 rats in each group.
(F(2,41) = 0.1, p > 0.05) or a significant interaction between apomoprhine and crocins (F(2,41) = 0.7, p > 0.05) (Fig. 3). 4. Discussion
4 2 0
Fig. 2. Novel object location task. Histograms represent the mean ± S.E.M. of 8 rats per in each group. The 3-h ITI was used. (A) Discrimination index D performance expressed by different groups of rats during T2. * p < 0.05 vs. all the other groups. (B) Total exploration time in the different groups of rats during T2.
was higher than that of the group of rats that received vehicle + apomorphine (both p < 0.05, Fig. 1A). Analysis of the total exploration times during T2 did not show a statistically significant apomorphine x crocins interaction (F(2,59) = 0.7, p > 0.05) neither a main effect of crocins (F(2,59) = 0.6, p > 0.05) or of apomorphine (F(1,59) = 2.5, p > 0.05) (Fig. 1B). 3.2. Experiment 2: effects of crocins and apomorphine on rats’ performance in the NOLT Crocins did not counteract apomorphine-induced performance deficits in the NOLT (Fig. 2A). The analysis of the D index data showed a significant main effect of apomorphine (F(1,47) = 162.9, p < 0.01), but no main effect of crocins (F(2,47) = 0.007, p > 0.05) and no apomorphine x crocins interaction (F(2,47) = 0.2, p > 0.05). Preplanned comparisons revealed that all animals treated with apomorphine had a significantly lower D index compared with their respective control groups (p < 0.05; Fig. 2A). Total exploration times were not different among the various experimental groups (Fig. 2B). Analysis of the total exploration times during T2 did not show a statistically significant apomorphine x crocins interaction (F(2,47) = 0.69, p > 0.05), neither a main effect of crocins (F(2,47) = 2, p > 0.05) or of apomorphine (F(1,47) = 0.08, p > 0.05) (Fig. 2B). 3.3. Experiment 3: effects of crocins and apomorphine on rats’ performance in the locomotor activity test Total motor activity was not different among the various experimental groups. Overall analysis did not show a significant main effect either of apomorphine (F(1,41) = 0.1, p > 0.05) or of crocins
The NORT evaluates non-spatial recognition memory in rodents. It is a non-rewarded paradigm that it is based on spontaneous exploratory behavior in rodents [12]. The NOLT is a version of the NORT that evaluates spatial recognition memory. This task assesses the ability of rodents to discriminate the novelty of the object locations but not the objects itself because the behavioral testing arena is already familiar to the animals [14]. Both of these recognition memory tasks do not involve explicit reward or punishment but rely on the natural curiosity of rodents and preference for novelty which do not appear to be influenced by reinforcement/response contingencies [9]. These paradigms are quite similar to procedures used in humans and should have a significant level of construct and predictive validity [12]. The current findings are in line with prior studies, in which post-training administration of apomorphine disrupted rats’ performance in either of the two recognition memory tasks [20,27]. These detrimental effects of apomorphine on memory seem to be dependent on its action on hippocampus and prefrontal cortex (PFC). Specifically, local infusion of apomorphine into CA1 but not CA3 subregion of hippocampus produced non-spatial and spatial memory impairments [35,36]. Alterations in the DA activity in the dorsolateral PFC have also been suggested to underlie the effects of apomorphine on cognition [17,18]. Crocins, at any doses tested reversed apomorphine-induced performance deficits in the NORT. As in the present study, the retention delay interval was no longer than 3 h; the effects of compounds were limited to short-term memory. The effects of crocins and apomorphine on performance during the retention phase reflect the modulation of post-training mnemonic processes (storage and/or retrieval of information) [23]. In contrast, crocins at all doses tested did not counteract the performance deficits produced by apomorphine in the NOLT. Several reasons might underlie this apparent discrepancy. The lack of a protective effect of crocins in the NOLT may originate from the fact that spatial memory is more susceptible to hippocampal dysfunction than non-spatial memory (NORT procedure). Thus, performance in the spatial object location task is easier to disrupt than performance in the non-spatial object recognition task, making performance in this task less susceptible to crocins administration [6]. In this context, it has been reported that object location is more vulnerable
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than object recognition because it is based on less redundant information: the various features of the objects can be encoded from several dimensions and attributes, which are not all required during recognition, while a location offers fewer cues [13,15]. Collectively, tests of spatial memory typically tax the ability of the animal to discriminate between two or more highly familiar locations, often on the basis of which the animal has last visited, set within a familiar environment while non-spatial tasks often involve stimuli that are relatively unfamiliar or completely novel. Thus, this difference in stimuli intensity (higher in non-spatial tasks as compared to spatial tasks) might underlie the longer preservation of memory in the object recognition with respect to the object location [10]. The NOLT study results are inconsistent with previous findings of ours in which crocins attenuated spatial memory deficits produced by the muscarinic receptor antagonist scopolamine in rats [33]. These controversial findings with regards the effects of crocins on spatial memory may be attributable to differences in experimental settings. Specifically, the radial water maze task used in our previous study [33] is a negatively reinforced spatial memory procedure, whereas the NOLT used in the present experiment is a non-rewarded spatial memory paradigm. Moreover, in the present experimentation compounds were acutely administered, while in the above described study a sub-chronic treatment protocol was used. Finally, apomorphine and scopolamine act on different neurotransmitter systems since apomorphine supresses DA neurotransmission, while scopolamine disrupts the integrity of the cholinergic system. These considerations probably offer a plausible explanation as to why crocins did not reverse the apomorphineinduced impairments in the NOLT. It seems unlikely that non-specific factors, as attentional or sensorimotor deficits, may have influenced rats’ cognitive performance assessed in the NORT since exploration levels were not different among the various experimental groups during the choice trial T2. Further, the motor activity cage results indicate that the effects of compounds on recognition memory cannot be attributed to changes in locomotor activity. In addition, the effects of apomorphine on memory cannot be ascribed to the residual presence of the drug during testing since this drug has a very short half-life (10–15 min) [4]. Thus, this pattern of results indicates that nonspecific factors did not affect animals’ performance in either of the two memory tasks. The mechanism(s) by which crocins exert their effects on apomorphine-induced non-spatial recognition memory deficits are not yet clarified. In this context, it has been previously demonstrated that crocins reduced schizophrenia-like effects, including recognition memory deficits, in rodents due to excessive glutamatergic activity [3,19,22]. An explanation of the current findings might be based on the well-documented antioxidant properties of crocins. The antioxidant properties of crocins have been revealed in different preclinical models of oxidative stress [29], brain ischemia [38], Parkinson’s disease [1] and Alzheimer’s disease [30]. Although the pathogenesis of schizophrenia remains unknown, a link between oxidative stress and this pathology has been suggested [5] and the disrupting effect of apomorphine on memory seem to be dependent on its pro-oxidative stress properties [31]. Additional research, however, is required aiming to clarify this issue. The current results are in line and extend previous our findings in which the efficacy of crocins to counteract psychotomimetic effects, including recognition memory deficits, caused by dysfunction of the glutamatergic system has been revealed [19]. Collectively, these findings suggest a functional interaction between crocins and both the glutamatergic and DAergic systems and support a potential therapeutic role of crocins in the treatment of some behaviors that may be altered in schizophrenia. Specifically, the utilization of crocins as an adjunctive agent, for the
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treatment of cognitive deficits observed in schizophrenic patients should be further investigated. In this context, it is important to take into account the good safety profile of saffron which was revealed in different clinical studies [34]. Conflict of interest statement The authors declare no potential conflicts of interest with respect to authorship and/or publication of this article. Acknowledgements The present study was supported by a grant of the Research Committee of the University of Thessaly (no. 3219) to N.P. References [1] A.S. Ahmad, M.A. Ansari, M. Ahmad, S. Saleem, S. Yousuf, M.N. Hoda, F. Islam, Neuroprotection by crocetin in a hemi-parkinsonian rat model, Pharmacol. Biochem. Behav. 81 (2005) 805–813. [2] L. Bartolini, F. Casamenti, G. Pepeu, Aniracetam restores object recognition impaired by age, scopolamine and nucleus basalis lesions, Pharmacol. Biochem. Behav. 53 (1996) 277–283. [3] F. Berger, A. Hensel, K. Nieber, Saffron extracts and trans-crocetin inhibit glutamatergic synaptic transmission in rat cortical brain slices, Neuroscience 180 (2011) 238–247. [4] G. Bianchi, M. Landi, S. Garattini, Disposition of apomorphine in rat brain areas: relationship to stereotypy, Eur. J. Pharmacol. 131 (1986) 229–236. [5] B.K. Bitanihirwe, T.U. Woo, Oxidative stress in schizophrenia: an integrated approach, Neurosci. Biobehav. Rev. 35 (2011) 878–893. [6] N.J. Broadbent, L.R. Squire, R.E. Clark, Spatial memory, recognition memory, and the hippocampus, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 14515–14520. [7] A. Calev, P.H. Venables, A.F. Monk, Evidence for distinct verbal memory pathologies in severely and mildly disturbed schizophrenics, Schizophr. Bull. 9 (1983) 247–264. [8] A. Cavoy, J. Delacour, Spatial but not object recognition is impaired by aging in rats, Physiol. Behav. 53 (1993) 527–530. [9] E. Dere, J.P. Huston, M.A. De Souza Silva, The pharmacology, neuroanatomy and neurogenetics of one-trial object recognition in rodents, Neurosci. Biobehav. Rev. 31 (2007) 673–704. [10] S.L. Dix, J.P. Aggleton, Extending the spontaneous preference test of recognition: evidence of object location and object-context recognition, Behav. Brain Res. 99 (1999) 191–200. [11] J. Edwards, H.J. Jackson, P.E. Pattison, Emotion recognition via facial expression and affective prosody in schizophrenia: a methodological review, Clin. Psychol. Rev. 22 (2002) 789–832. [12] A. Ennaceur, J. Delacour, A new one-trial test for neurobiological studies of memory in rats. 1. Behavioral data, Behav. Brain Res. 31 (1988) 47–59. [13] A. Ennaceur, K. Meliani, A new-trial for neurobiological studies of memory in rats. III. Spatial vs. Non-spatial working memory, Behav. Brain Res. 51 (1992) 83–92. [14] A. Ennaceur, N. Neave, J.P. Aggleton, Spontaneous object recognition and object location memory in rats: the effects of lesions in the cingulated cortices, the medial prefrontal cortex, the cingulum bundle and the fornix, Exp. Brain Res. 113 (1997) 509–519. [15] A. Ennaceur, S. Michalikova, A. Bradford, S. Ahmed, Detailed analysis of behaviour of Lister and Wistar rats in anxiety, object recognition and object location tasks, Behav. Brain Res. 159 (2005) 247–266. [16] J.R. Field, A.G. Walker, P.J. Conn, Targeting glutamate synapses in schizophrenia, Trends Mol. Med. 17 (2011) 689–698. [17] P.C. Fletcher, C.D. Frith, P.M. Grasby, K.J. Friston, R.J. Dolan, Local and distributed effects of apomorphine on fronto-temporal function in acute unmedicated schizophrenia, J. Neurosci. 16 (1996) 7055–7062. [18] K.J. Friston, P.M. Grasby, C.J. Bench, C.D. Frith, P.J. Cowen, P.F. Liddle, R.S.J. Frackowiak, R. Dolan, Measuring the neuromodulatory effects of drugs in men with positron emission tomography, Neurosci. Lett. 141 (1992) 106–110. [19] G. Georgiadou, V. Grivas, P.A. Tarantilis, N. Pitsikas, Crocins, the active constituents of Crocus Sativus L., counteracted ketamine-induced behavioural deficits in rats, Psychopharmacology 231 (2014) 717–726. [20] I. Gourgiotis, N.G. Kampouri, V. Koulouri, I.G. Lempesis, M.D. Prasinou, G. Georgiadou, N. Pitsikas, Nitric oxide modulates apomorphine-induced recognition memory deficits in rats, Pharmacol. Biochem. Behav. 102 (2012) 507–514. [21] V. Grivas, A. Markou, N. Pitsikas, The metabotropic glutamate 2/3 receptor agonist LY379268 induces anxiety-like behavior at the highest dose tested in two rat models of anxiety, Eur. J. Pharmacol. 715 (2013) 105–110. [22] H. Hosseinzadeh, H.R. Sadeghnia, A. Rahimi, Effects of safranal on extracellular hippocampal levels of glutamate and aspartate during kainic Acid treatment in anesthetized rats, Planta Med. 74 (2008) 1441–1445. [23] B. Hunter, S.F. Zornetzer, M.E. Jarvik, J.L. McGaugh, Modulation of learning and memory: effects of drugs influencing neurotransmitters, in: L.L. Iversen, S.D.
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