Neuroprotective effects of quercetin on memory and anxiogenic-like behavior in diabetic rats: Role of ectonucleotidases and acetylcholinesterase activities

Biomedicine & Pharmacotherapy 84 (2016) 559–568

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Original article

Neuroprotective effects of quercetin on memory and anxiogenic-like behavior in diabetic rats: Role of ectonucleotidases and acetylcholinesterase activities Roberto M. Maciela , Fabiano B. Carvalhob,**, Ayodeji A. Olabiyib,d , Roberta Schmatzb , Jessié M. Gutierresb , Naiara Stefanellob , Daniela Zaninib , Michelle M. Rosab , Cinthia M. Andradea,b , Maribel A. Rubinb , Maria Rosa Schetingerb , Vera Maria Morschb , Cristiane C. Danesic , Sonia T.A. Lopesa,* a Programa de Pós-Graduação em Medicina Veterinária, Laboratório de Análises Clínicas Veterinária, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Santa Maria/RS 97105-900, Brazil b Programa de Pós-Graduação em Ciências Biológicas: Bioquímica Toxicológica, Departamento de Bioquímica e Biologia Molecular, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria/RS 97105-900, Brazil c Programa de Pós-Graduação em Ciências Odontológicas, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil d Department of Medical Biochemistry, Afe Babalola University, Ado Ekiti, P.M.B 5454. Ado Ekiti, Nigeria

A R T I C L E I N F O

Article history: Received 1 June 2016 Received in revised form 13 September 2016 Accepted 19 September 2016 Keywords: Diabetes Memory Anxiety Acetylcholinesterase Ectonucleotidases Quercetin

A B S T R A C T

The present study investigated the protective effect of quercetin (Querc) on memory, anxiety-like behavior and impairment of ectonucleotidases and acetylcholinesterase (AChE) activities in brain of streptozotocin-induced diabetic rats (STZ-diabetes). The type 1 diabetes mellitus was induced by an intraperitoneal injection of 70 mg/kg of streptozotocin (STZ), diluted in 0.1 M sodium-citrate buffer (pH 4.5). Querc was dissolved in 25% ethanol and administered by gavage at the doses of 5, 25 and 50 mg/kg once a day during 40 days. The animals were distributed in eight groups of ten animals as follows: vehicle, Querc 5 mg/kg, Querc 25 mg/kg, Querc 50 mg/kg, diabetes, diabetes plus Querc 5 mg/kg, diabetes plus Querc 25 mg/kg and diabetes plus Querc 50 mg/kg. Querc was able to prevent the impairment of memory and the anxiogenic-like behavior induced by STZ-diabetes. In addition, Querc prevents the decrease in the NTPDase and increase in the adenosine deaminase (ADA) activities in SN from cerebral cortex of STZdiabetes. STZ-diabetes increased the AChE activity in SN from cerebral cortex and hippocampus. Querc 50 mg/kg was more effective to prevent the increase in AChE activity in the brain of STZ-diabetes. Querc also prevented an increase in the malondialdehyde levels in all the brain structures. In conclusion, the present findings showed that Querc could prevent the impairment of the enzymes that regulate the purinergic and cholinergic extracellular signaling and improve the memory and anxiety-like behavior induced by STZ-diabetes. ã 2016 Published by Elsevier Masson SAS.

1. Introduction

* Corresponding author at: Programa de Pós
Graduação em Medicina Veterinária, Laboratório de Análises Clínicas Veterinária, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Santa Maria/RS 97105-900, Brazil. ** Corresponding author at: Programa de Pós Graduação em Ciências Biológicas: Bioquímica Toxicológica, Departamento de Bioquímica e Biologia Molecular, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria/RS 97105-900 Brazil. E-mail addresses: [email protected] (F.B. Carvalho), [email protected] (S.T.A. Lopes). http://dx.doi.org/10.1016/j.biopha.2016.09.069 0753-3322/ã 2016 Published by Elsevier Masson SAS.

Diabetes mellitus (DM) consists a group of metabolic dysfunction characterized by hyperglycemia resulting from a defect in insulin secretion or insulin action [1]. Hyperglycemia persistent is indeed the causal link in the evolution of neuropathy and uncontrolled diabetes [2]. It has been shown that persistent hyperglycemia increases production of reactive oxygen species (ROS) for all tissues across of glucose auto-oxidation and protein glycosylation.

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The cholinergic systems provide diffuse innervations to practically all brain [3]. The broad cholinergic innervation acting via nicotinic acetylcholine receptors has been found to influence arousal, attention, sleep, fatigue, anxiety, central processing of pain, and a number of cognitive functions [4–6]. The enzyme acetylcholinesterase (AChE) is found in the cholinergic terminal and is the most efficient enzyme that rapidly hydrolyze the neurotransmitter acetylcholine (ACh) at cholinergic synapses as well as the neuromuscular junction [7]. In addition, the hippocampus and cortical regions of the brain are main sites for cholinergic transmission to monitor learning and memory processing, and seem to be more sensitive to oxidative damage [8] which contributes neuronal damage and establishing of cognitive deficit [9]. The extracellular nucleotide ATP and its nucleoside derivative adenosine are important signaling molecules involved in numerous physiological and pathological functions [10]. The ATP and extracellular adenosine levels are regulated by a cascade of cellsurface-bound enzymes named ectonucleotidases. The NTPDase is an enzyme that hydrolyses ATP and ADP into AMP, which is subsequently converted to adenosine by the enzyme 50 -nucleotidase [11,12]. Moreover, adenosine is cleaved by enzyme adenosine deaminase to inosine in the synaptic cleft [13,14]. Together, these enzymes constitute an organized enzymatic cascade for the regulation of nucleotide-mediated signaling, controlling rate, degradation, and nucleoside formation [15,16]. It has been described also the ectonucleotidases involvement on learning and memory process in rats [17–19]. The various purinergic receptor subtypes are said to be widely distributed throughout the central nervous system (CNS) and they control local network behaviors by regulating the balance between the release and effects of ATP and adenosine as well as ectonucleotidases activities on synaptic transmission [18,20–25]. Furthermore, the extracellular ATP and adenosine levels have been related to processes of learning and memory formation, since various evidences point to LTP and LTD and synaptic plasticity as a neural basis for cognitive processes [26–28]. Furthermore, adenosine receptor antagonists, such as caffeine has been shown to improve memory and induce anxiety in rats [29–33]. Antioxidants, antihyperglycemics and insulin sensitizing agents are reported to reduce cognitive dysfunction in diabetic condition [34–36]. However, at present, no specific treatments are available for the management and/or prevention of cognitive dysfunction in DM. Quercetin (Querc) is a flavonoid that possesses free radical scavenging properties and can protect from oxidative injury by its ability to modulate intracellular signals and promote cellular survival [37]. It has been shown that Querc is able to protect the memory loss in diabetic rats however studies involving the ectonucleotidases and AChE enzymes have not been described. Therefore, this study seeks to investigate the neuroprotective effects of quercetin on memory and anxiogenic-like behavior and ectonucleotidases and acetylcholinesterase activities in STZ induced diabetic rats.

2.2. Animals Male Wistar rats (100 animals with 70–90 days old; 200–250 g) from the Central Animal House of the Federal University of Santa Maria were used in this study. The animals were maintained at a constant temperature (23  1  C), on a 12 h dark/light cycle with free access to food and water. All procedures were approved by the Animal Ethics Committee from the Federal University of Santa Maria (protocol number: 57/2010). 2.3. Experimental protocol All animals were acclimatized for the period of 15 days, at five rats per cage, before the initiation of the experimental protocols as previously described [38–42]. The rats were randomly divided into eight groups: Vehicle, Querc 5 mg/kg, Querc 25 mg/kg, Querc 50 mg/kg, diabetic, diabetic plus Querc 5 mg/kg, diabetic plus Querc 25 mg/kg and diabetic plus Querc 50 mg/kg. Diabetes was induced by a single intraperitoneal injection of 70 mg/kg streptozotocin (STZ) diluted in 0.1 M sodium-citrate buffer (pH 4.5). STZ-treated rats received 5% of glucose instead of water for 24 h after diabetes induction in order to reduce death due to hypoglycemic shock. Blood samples were taken from the tail vein 48 h after STZ induction to measure glucose levels. Only animals with fasting glycaemia over 250 mg/dL were considered diabetic and used for the study. Ten days after diabetes induction, the treatments with vehicle or Querc were initiated. Querc was freshly prepared in 25% ethanol (vehicle) and was administered at between 3 and 4 p.m. once a day during 40 days, by gavage. The Querc doses were based on previous experimental doses used by our research group which reported beneficial effects [43–46] and was adjusted weekly based on individual weight. During the experiment the blood glucose levels were verified six times (15 days before, on the first day, 10, 20, 30, and 40 days after the beginning of treatment). A saline group was performed in control and STZ-diabetic rats to exclude possible effects of ethanol vehicle. The saline groups were not included in this study since there were not significant differences between vehicles saline and ethanol. 2.4. Behavioral parameters 2.4.1. Inhibitory avoidance task After treatment with Querc (40 days), animals were subjected to training in a step-down inhibitory avoidance task as previously describe [17,47]. Twenty four hours later, the animals were subjected to the test in a step-down inhibitory avoidance task and recorded. Briefly, the inhibitory avoidance apparatus consisted of a 25  25  35 cm box with a grid floor whose left portion was covered by a 7  25 cm platform, 2.5 cm high was used. The rat was placed gently on the platform facing the rear left corner, and when the rat stepped down with all four paws on the grid, a 3-s 0.4 mA shock was applied to the grid. Retention test took place in the same apparatus 24 h later. Test step-down latency was taken as a measure of retention, and a cut-off time of 300 s was established.

2. Materials and methods 2.1. Chemical reagents Quercetin (Querc, >95% purity, Q4951), 5,50 -dithio-bis-2nitrobenzoic acid (DTNB), streptozotocin, acetylthiocholine chloride, malondialdehyde tetrabutylammonium salt (MDA), 2thiobarbituric acid (TBA), trizma base, nucleotides and Percoll reagent were obtained from Sigma Chemical Co (St. Louis, MO, USA). All the other chemicals used in this experiment were of highest purity.

2.4.2. Open field Immediately after the inhibitory avoidance test session, the animals were transferred to an open-field measuring 56  40  30 cm, with the floor divided into 12 squares measuring 12  12 cm each. The open field session lasted for 5 min and during this time, an observer, who was not aware of the pharmacological treatments, recorded the number of crossing responses and rearing responses manually. This test was carried out to identify motor disabilities, which might influence inhibitory avoidance performance at testing.

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2.4.3. Elevated plus maze task Anxiolytic-like behavior was conducted after open-field and was evaluated using the elevated plus maze task as previously described [48,49]. The apparatus consists of a wooden structure raised to 50 cm from the floor. This apparatus is composed of 4 arms of the same size, with two closed-arms (walls 40 cm) and two open-arms. Initially, the animals were placed on the central platform of the maze in front an open arm. The animal had 5 min to explore the apparatus, and the time spent and the number of entries in open and closed-arms were recorded. The apparatus was thoroughly cleaned with 30% ethanol between each session. 2.5. Biochemical assays 2.5.1. Brain tissue preparation After behavioral tests, the animals were anesthetized under halothane atmosphere. The brains were removed and separated into the cerebral cortex, hippocampus and striatum. The brain structures were placed in a solution of Tris–HCl 10 mM, pH 7.4, on ice and gently homogenized in a glass potter. After, the homogenate was centrifuged 800 g during 15 min at 4  C. Aliquots of supernatant were stored at
80  C until the acetylcholinesterase assay and malondialdehyde quantification. Protein was determined previously for each structure: cerebral cortex (0.7 mg/mL), hippocampus (0.8 mg/mL) and striatum (0.4 mg/mL), as determined by the Coomassie blue method described [50], using bovine serum albumin as standard solution. 2.5.2. Cerebral cortex synaptosomes Synaptosomes (SN) were obtained from cerebral cortex and isolated as previously described [51], using a discontinuous Percoll gradient. The cerebral cortex was gently homogenized in a glass potter 10 vol of an ice-cold medium (medium I) containing 320 mM sucrose, 0.1 mM EDTA and 5 mM HEPES, pH 7.5 and then centrifuged at 1000 g for 10 min. An aliquot of 0.5 mL of the crude mitochondrial pellet was mixed with 4.0 mL of an 8.5% Percoll solution and layered into an isosmotic discontinuous Percoll/ sucrose gradient (10%/16%). The SN that banded at the 10/16% Percoll interface were collected with a wide-tip disposable plastic transfer pipette. The SN were washed twice with an isosmotic solution consisting of 320 mM sucrose, 5.0 mM HEPES, pH 7.5, and 0.1 mM EDTA by centrifugation at 15,000 g to remove the contaminating Percoll. The pellet of the second centrifugation was resuspended in an isosmotic solution to a final protein concentration of 0.9 mg/mL. SN were prepared fresh daily and maintained at 4  C throughout the procedure and used for acetylcholinesterase and ectonucleotidases assays. 2.5.3. Lactate dehydrogenase assay The integrity of the SN was confirmed by determining the lactate dehydrogenase (LDH) activity which was obtained after SN lysis with 0.1% Triton X-100 and comparing it with an intact preparation, using the Labtest kit (Labtest, Lagoa Santa, MG, Brasil). 2.5.4. NTPDase and 50 -nucleotidase activities The NTPDase enzymatic assay of the SN was carried out in plates with a reaction medium containing 5 mM KCl, 1.5 mM CaCl2, 0.1 mM EDTA, 10 mM glucose, 225 mM sucrose and 45 mM Tris– HCl buffer, pH 8.0, in a final volume of 200 mL as described in a previous work from our laboratory [52]. The 50 -nucleotidase activity was determined [53] in a reaction medium containing 10 mM MgSO4 and 100 mM Tris–HCl buffer, pH 7.5, in a final volume of 200 mL. In SN, 20 mL of enzyme preparation (8–12 mg of protein) was added to the reaction mixture and pre-incubated at 37  C for 10 min. The reaction was initiated by the addition of ATP or ADP to obtain a final concentration of 1.0 mM and incubation

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proceeded for 20 min. For AMP hydrolysis, the 50 -nucleotidase activity was carried out as previously described [53] and the final concentration of the nucleotide AMP added was 2 mM. The reactions were stopped by the addition of 200 mL of 10% trichloroacetic acid (TCA) to provide a final concentration of 5%. Subsequently, the tubes were chilled on ice for 10 min. Released inorganic phosphate (Pi) was assayed using malachite green as the colorimetric reagent and KH2PO4 as standard [54]. All samples were in triplicate readings. Enzyme specific activities are reported as hmol Pi/min/mg of protein. 2.5.5. Adenosine deaminase (ADA) assay ADA activity was determined in SN from cerebral cortex as previously described [55] with minor modifications [19]. The assay is based on the direct measurements of the formation of ammonia, produced when ADA acts in excess of adenosine. The samples were added to the reaction mixture containing 21 mM of adenosine, pH 6.5, and were incubated at 37  C for 60 min. The reaction was stopped by adding the samples on a 106/0.16 mM phenol–nitroprusside/mL. The reaction mixtures were immediately mixed with 125/11 mM alkaline-hypochlorite (sodium hypochlorite). 75 mM ammonium sulphate was used as ammonium standard. The protein content used for this experiment was adjusted to 0.4–0.6 mg/mL. Results were expressed as unit of specific enzyme activity/mg protein (U/mg of protein for ADA). One unit of ADA is defined as the amount of enzyme required to release 1 mmol of ammonia/min from adenosine at standard assay condition. 2.5.6. Acetylcholinesterase (AChE) assay The AChE enzymatic assay was determined according to the method of Ellman et al. [56] with some modifications of the spectrophotometric method [57]. The reaction mixture (2 mL final volume) contained 100 mM K+-phosphate buffer, pH 7.5 and 1 mM 5,50 -dithiobisnitrobenzoic acid (DTNB). The method is based on the formation of the yellow anion, 5, 50 - dithio-bis-acidnitrobenzoic, measured by absorbance at 412 nm during 2 min incubation at 25  C. The enzyme (40–50 mg of protein) was preincubated for 2 min. The reaction was initiated by adding 0.8 mM acetylthiocholine iodide (AcSCh). All samples were in triplicate readings and the enzyme activities were expressed in mmol AcSCh/h/mg of protein. 2.5.7. Brain thiobarbituric acid reactive substances (TBARS) level measurement Brain malondialdehyde (MDA) levels were determined by the thiobarbituric acid reactive substances measurement method as previously described [58], with a few modifications [59]. In short, the reaction mixture contained 200 mL of cerebral cortex, hippocampus or striatum homogenates or standard (MDAmalondialdehyde 0.03 mM), 200 mL of 8.1% sodium dodecylsulfate (SDS), 750 mL of acetic acid solution (2.5 M HCl, pH 3.5) and 750 mL of 0.8% TBA. The mixtures were heated at 95  C for 90 min. After centrifugation at 1700 g for 5 min, the absorbance was measured at 532 nm. MDA tissue levels were expressed as hmol MDA/mg of protein. 2.5.8. Protein determination Protein was measured by the Coomassie Blue method of Bradford [50], using bovine serum albumin as standard. 2.6. Statistical analysis Statistical analysis of latency test was carried by Kruskal– Wallis test (a non-parametric extension of One-way ANOVA). Training latency, elevated plus maze task, open field results,

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ectonucleotidases and AChE activities and TBARS levels were analyzed by One or Two-Way ANOVA, followed by Student Newman-Keuls post hoc test. P < 0.05 was considered to represent a significant difference in all experiments. Latency test was expressed as median  interquartile range. All the other data were expressed as mean  SEM. 3. Results 3.1. Body weight and blood glucose Diabetic rats from this study presented clinical disorders commonly observed in this condition, such as, polydipsia, polyuria, lethargy, polyphagia, and weight loss. The values of body weight and blood glucose levels have been previously published [43]. 3.2. Querc prevents the impairment of memory in STZ-diabetic rats The Fig. 1 shows the training latency (A), test latency (B), number of crossing (C) and rearing (D) of diabetic rats treated with Querc in the inhibitory avoidance and open-field tasks. No significant difference between groups for the training latency (A), number of crossing (C) and rearing (D) were observed. It can be seen in the graph B that diabetic rats showed a reduction in the latency to step-down from platform compared to the control group. In addition, Querc 25 and 50 were able to prevent the reduction in the step-down latency induced by diabetes.

3.3. Querc prevents the anxiogenic-like behavior in STZ-diabetic rats Fig. 2 shows the number of entries in the open (A) and closed arms (C), time spent in the open (B) and closed arms (D), time in center (E) and number of entries in arms (F) from diabetic rats treated with Querc in the elevated plus maze task. Diabetic rats showed a decreased in the number of entries into the open arms and Querc 5, 25 and 50 mg/kg prevented this effect (A). Querc 50 mg/kg showed a per se effect on the time spent in the open arms (B). However, no significant differences in the number of entries into open arms (C) and number of entries into the arms (F) were observed. Querc 50 mg/kg also showed a per se effect by reducing the time spent in the closed arms (D). Furthermore, diabetic rats showed an increase in the time spent to closed arms (D). However, Querc 5, 25 and 50 mg/kg prevented the increase in the time spent to closed arms induced by STZ-diabetes (D). Diabetic rats showed a reduction in the time spent in the center and all Querc doses reversed this effect. 3.4. Querc prevents the impairment of ectonucleotidases activities in the SN from cerebral cortex of STZ-diabetic rats Fig. 3 shows the NTPDase (A and B), 50 -nucleotidase (C) and adenosine deaminase (D) activities in the SN from cerebral cortex of diabetic rats treated with Querc. Diabetes reduced the NTPDase activity using ATP (A) and ADP (B) as substrate. Querc showed a protective effect on the reduction in the NTPDase activity but no

Fig. 1. Quercetin (Querc) prevents the impairment of memory in STZ-diabetic rats. Training latency (a), test latency (B), number of crossing (C) and rearing (D) of diabetic rats treated with Querc (5, 25 and 50 mg/kg) in the inhibitory avoidance task and open-field. * denotes a significant difference from the vehicle group. # denotes a significant difference compared with the diabetes group (Test latency was carried by Kruskal–Wallis test. All the other date were carried by ANOVA one-way or two followed by post-hoc SNK, n = 10).

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Fig. 2. Quercetin (Querc) prevents the anxiogenic-like behavior in STZ-diabetic rats. Number of entries in the open (A) and closed arms (C), time spent in the open (B) and closed arms (D), time in center (E) and number of entries in arms (F) from diabetic rats treated with Querc (5, 25 and 50 mg/kg) in the elevated plus maze task. * denotes a significant difference from the vehicle group. # denotes a significant difference compared with the diabetes group (ANOVA one-way or two followed by post-hoc SNK, n = 10).

significant differences for 50 -nucleotidase activity (C) was observed. In addition, diabetes increased the ADA activity (D) however, Querc 25 and 50 mg/kg were able to prevent this effect. 3.5. Querc prevents the increase in the acetylcholinesterase activity in the brain of diabetic rats Fig. 4 shows the AChE activity in SN obtained from the cerebral cortex (A), and in the cerebral cortex (B), hippocampus (C) and

striatum (D) supernatants of diabetic rats treated with Querc. Diabetic rats showed an increase in AChE activity in SN from the cerebral cortex. Nevertheless, Querc 50 mg/kg prevented the increase in AChE activity induced by diabetes. A similar effect was observed in the supernatant from cerebral cortex and hippocampus of rats. Querc 50 mg/kg was able to prevent the increase in AChE activity induced by diabetes in the cerebral cortex and hippocampus but no significant difference was observed in relation to the striatum.

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Fig. 3. Quercetin (Querc) prevents the impairment of ectonucleotidases activities in the synaptosomes (SN) from cerebral cortex of STZ-diabetic rats. NTPDase (A and B), 50 nucleotidase (C) and adenosine deaminase (D) activities in the SN from cerebral cortex of diabetic rats treated with Querc (5, 25 and 50 mg/kg). * denotes a significant difference from the vehicle group. # denotes a significant difference compared with the diabetes group (ANOVA one-way or two followed by post-hoc SNK, n = 10).

3.6. Querc prevents the increase in TBARS levels in the brain of STZdiabetic rats The Fig. 5 shows the TBARS (MDA) levels in the cerebral cortex (A), hippocampus (B) and striatum (C) from diabetic rats treated with Querc. Diabetic rats showed an increased in the MDA levels in cerebral cortex, hippocampus and striatum. Querc 25 and 50 mg/kg prevented the increase in MDA levels induced by diabetes in the cerebral cortex while Querc 5, 25 and 50 mg/kg prevented the increase in the MDA levels in the hippocampus and striatum from diabetic rats. 4. Discussion Extensive evidence supports the notion that diets rich in polyphenols and/or supplementation with specific compounds provide beneficial health effects. In particular, polyphenols have been shown to exert protective actions in several pathological conditions such as cardiovascular disease, metabolic disorders, obesity, diabetes, infections, cancer, and neurotoxic/neurodegenerative processes [60,61]. Several studies show that quercetin can exert neuroprotection and antagonize oxidative stress when administered in vivo. For example, oral quercetin was shown to protect rodents from oxidative stress and neurotoxicity induced by various neurotoxic insults [62]. Among metals, quercetin has been shown to provide protection against the neurotoxicity of lead, methylmercury, and tungsten [63–65]. The present study investigated the protective effects of Querc on behavioral and biochemical alterations in the brain of STZ-diabetic rats. Recently,

a positive correlation of DM with cognitive impairment or dementia has been demonstrated [66,67]. Specifically in the general population, DM was associated with abnormal performance in the domains of memory, attention and psychomotor speed [68,69]. In addition, polyphenolic compounds have received considerable attention since they have been shown to protect neurons against a variety of experimental neurodegenerative conditions including cognitive deficit associated with diabetes [70]. In this context, the present study shown that diabetic rats exhibited pronounced impairment on the memory formation in the inhibitory avoidance task and that 25 and 50 mg/ kg of Querc prevented this effect. Furthermore, motor disabilities were not observed in the open field arena, indicating absence of motor problem that would compromise the findings obtained in the inhibitory avoidance task. These data were in agreement with previous studies from our group that reported impairment in memory of diabetic rats in the inhibitory avoidance task and fear conditioning [41,42,71]. Another behavioral aspect commonly found in diabetic changes is anxiety [41,72,73]. The elevated plus maze task revealed that diabetic rats showed an increase in anxiety-like behavior. The anxiety in these animals reflected in the increase of time spent in the closed arms compared to the open arms. It is important to note that diabetic animals that received treatment with Querc in all concentrations were similar to non-diabetic control. Moreover, non-diabetic rats that received 50 mg/kg showed significant increase in the time spent in the open arms and reduction of time at enclosed arms. Besides benzodiazepine derivatives, flavonoid compounds have shown anxiolytic effects

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Fig. 4. Quercetin (Querc) prevents the increase in the acetylcholinesterase (AchE) activity in the brain of STZ-diabetic rats. AChE activity in SN obtained from the cerebral cortex (A) and in the supernatant of the cerebral cortex (B), hippocampus (C) and striatum (D) of diabetic rats treated with Querc (5, 25 and 50 mg/kg). * denotes a significant difference from the vehicle group. # denotes a significant difference compared with the diabetes group (ANOVA one-way or two followed by post-hoc SNK, n = 10).

in experimental models associated with increase in the anxiety behavior [17,48]. In addition, the anxiolytic-like behavior of Querc has been recently reported in a literature [45], and was reported to reduce the anxiogenic-state induced by cadmium intoxication. In fact, from the pharmacological targets of flavonoids involving their ability to interact with the GABAA receptors, key-receptors in the anxiety regulation [74–78]. The diabetic encephalopathy is a recognized complication of untreated diabetes resulting in a progressive cognitive impairment accompanied by modification of brain function. The purinergic system is a promising novel target to control diabetic encephalopathy since it control the cerebral synaptic plasticity and glucose handling [79,80]. In addition, the ATP regulates key physiological functions such as neurotransmitter release [81], synaptic plasticity phenomena [82], glucose homeostasis and modulation of insulin secretion [83]. Besides these beneficial effects previously described, the extracellular ATP can lead to deleterious effects on brain since it is also a danger signal, which promotes influx of intracellular calcium leading to the activation of apoptotic pathways, production of reactive species, excitotoxicity and neurodegeneration [84–87]. Firstly, Duarte and colleagues found a decrease in the cerebrospinal fluid levels of ATP in diabetic rats, together with a decrease of the evoked release of ATP in hippocampal nerve terminals and ATP metabolism [88]. Similar results were obtained in the ATP and ADP hydrolysis in SN from cerebral cortex of diabetic rats in our findings. Alterations on purinergic signaling could lead

to impairment in the modulation of neurotransmission and gliotransmission, which may contribute to progressive cognitive impairment induced by diabetes, although the direct impact on both neuronal and glial functions. In contrast to our findings, Schmatz and colleagues reported an increase in the hydrolysis of ATP and ADP in the brain of STZ-diabetic rats, and this effect was enhanced by the effect of resveratrol [40]. However, our study investigated a more prolonged effect of diabetes for 40 days which can be linked to the difference in the hydrolysis profile of these nucleotides. In addition, our results demonstrated that ADA activity was increased in SN of diabetic rats. Corroborating these results, Rutkiewicz and Górski (1990) also found a significant elevation in ADA activity in tissues of diabetic rats induced with STZ [89]. Additionally, study has suggested that insulin is involved in the regulation of ADA activity in diabetes, and insulin administration is able of decrease the activity of this enzyme in skeletal muscle, heart and liver. However the mechanism involved in this phenomenon is unclear [89]. Thus, we can suggest that the increase in ADA activity in diabetes could be explained, in part, by inefficient production and action of insulin. Interestingly, ADA activity increased in SN from cerebral cortex of the diabetic rats, which may be related with insulin deficits. In this way, a rapid deamination of adenosine by ADA causes a decrease in the adenosine level in the brain and can be associated with the development of neuronal damage observed in the diabetic state. Adenosine is a molecule that has a role in suppressing effector cells,

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being directly involved in motor processes, cognitive and memory. The reestablishment of AChE activity by Querc can contribute to the maintenance of cholinergic signaling, and then recovery the memory loss in diabetic rats. Diabetic animals untreated showed a significant MDA content elevation indicating a lipid peroxidation in the cerebral cortex, hippocampus and striatum. In addition, the Querc treatment reduced the MDA levels in the brain of rats. These results confirmed the antioxidant properties of Querc, whose degree of protection varied depending on brain structure and concentration employed. In the brain, the high content of polyunsaturated fatty acids and the high utilization of oxygen account for the susceptibility to free radical damage. Oxidative stress is said to have an important role in aging and in the development of a number of neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease and Multiple Sclerosis [90–93]. Studies have also shown that enzymes anchored to biological membranes are sensitive a ROS generation [18,46,93–97]. The peroxidation of biological membranes can compromise the optimal conformation of enzymes and interferer in its catalytic activity [93]. In this context, the protection of biological membranes from lipid peroxidation induced by diabetes may contribute to maintaining the enzyme activity of the cholinergic and purinergic system to act effectively in control of extracellular neurotransmitters [17,19,46]. Taking into consideration the above-mentioned findings, it seems that hyperglycemia in the STZ-diabetic rats’ caused changes both in memory formation and anxiety. In contrast, it is plausible that Querc acts not only as a powerful antioxidant but also compound with nootropic and anxiolytic properties, and most notably, has a close interaction with the up-regulation of AChE and ectonucleotidases activities, which can be one of the target mechanisms to protect cognitive deterioration and anxiogenic-like behavior in STZ-diabetic rats. Conflict of interest statement There are no conflicts of interest. Author contributions

Fig. 5. Quercetin (Querc) prevents the increase in the MDA levels in different brain structures of STZ-diabetic rats. Cerebral cortex (A), hippocampus (B) and striatum (C) from diabetic rats treated with Querc (5, 25 and 50 mg/kg). * denotes a significant difference from the vehicle group. # denotes a significant difference compared with the diabetes group (ANOVA one-way or two followed by post-hoc SNK, n = 10).

through A2A receptor-mediated signaling, thus a low availability of this nucleoside may contribute to the inflammatory process that occurs due to high glucose-exposure. Because ATP and adenosine signaling and regulation are a continuous and physiological phenomenon, another important neurotransmitter system involved in learning and memory is the cholinergic system. Although we do not quantify the levels of acetylcholine and choline acetyltransferase activity, it was possible to observe that the activity of AChE in STZ-diabetes rats was significantly increased in the SN, homogenate of cerebral cortex and hippocampus. These findings are consistent with previous studies from our group [41,42]. Acetylcholine (ACh) is the main neurotransmitter of nervous stimuli from one neuron to another,

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the ultimate version to be published. Study conception and design: Roberto Maciel, Roberta Schmatz. Diabetes induction and treatment: Roberto Maciel, Roberta Schmatz, Naiara Stefanello. Euthanasia and synaptosomes isolation: Jessie Gutierres, Cinthia Andrade. Determination of enzyme activities: Roberta Schmatz, Naiara Stefanello, Daniela Zanini, Cristiani Danesi. Behavioral parameters: Fabiano Carvalho, Michelle Rosa, Maribel Rubin. Data analysis and elaboration of the article: Roberto Maciel, Fabiano Carvalho, Ayodeji A Olabiyi. Project Financiers: Cinthia Andrade, Vera Morsch, Maria Rosa Schetinger, Sonia T Lopes. Acknowledgments We thank the Programa de Pós-Graduação em Medicina Veterinária da UFSM, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS). References [1] P.A. Maher, D.R. Schubert, Metabolic links between diabetes and Alzheimer’s disease, Exp. Rev. Neurother. 9 (5) (2009) 617–630.

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