sign in sign up
<<
Home , Donepezil, Huperzine A

EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

Original article:

CANNABIS-INDUCED IMPAIRMENT OF LEARNING AND MEMORY: EFFECT OF DIFFERENT DRUGS

Omar M.E. Abdel-Salam1, Neveen A. Salem1, Marwa El-Sayed El-Shamarka1, Noha Al-Said Ahmed1, Jihan Seid Hussein2, Zakaria A. El-Khyat2

Departments of Toxicology and Narcotics1 and Medical Biochemistry2, National Research Centre, Cairo

* corresponding author: Omar M.E. Abdel-Salam, Department of Toxicology and Narcotics, National Research Centre, Tahrir St., Dokki, Cairo, Egypt E-mail: [email protected]; FAX: 202-33370931

ABSTRACT Cannabis sativa preparations are the most commonly used illicit drugs worldwide. The pre- sent study aimed to investigate the effect of Cannabis sativa extract in the working memory version of the Morris water maze (MWM; Morris, 1984) test and determine the effect of standard memory enhancing drugs. Cannabis sativa was given at doses of 5, 10 or 20 mg/kg (expressed as 9-) alone or co-administered with (1 mg/kg), (150 mg/ kg), (1.5 mg/kg) or (25 mg/kg) once daily sub- cutaneously (s.c.) for one month. Mice were examined three times weekly for their ability to locate a submerged platform. Mice were euthanized 30 days after starting cannabis injection when biochemical assays were carried out. Malondialdehyde (MDA), reduced (GSH), , glucose and brain monoamines were determined. Cannabis resulted in a significant increase in the time taken to locate the platform and enhanced the memory im- pairment produced by scopolamine. This effect of cannabis decreased by memory enhancing drugs with piracetam resulting in the most-shorter latency compared with the cannabis. Bio- chemically, cannabis altered the oxidative status of the brain with decreased MDA, increased GSH, but decreased nitric oxide and glucose. In cannabis-treated rats, the level of GSH in brain was increased after vinpocetine and donepezil and was markedly elevated after Ginkgo biloba. Piracetam restored the decrease in glucose and nitric oxide by cannabis. Cannabis caused dose-dependent increases of brain serotonin, noradrenaline and dopamine. After can- nabis treatment, noradrenaline is restored to its normal value by donepezil, vinpocetine or Ginkgo biloba, but increased by piracetam. The level of dopamine was significantly reduced by piracetam, vinpocetine or Ginkgo biloba. These data indicate that cannabis administration is associated with impaired memory performance which is likely to involve decreased brain glucose availability as well as alterations in brain monoamine neurotransmitter levels. Pirace- tam is more effective in ameliorating the cognitive impairments than other by alle- viating the alterations in glucose, nitric oxide and dopamine in brain.

Keywords: Cannabis sativa extract, nootropics, water maze, mice, oxidative stress, brain monoamines

INTRODUCTION commonly used illicit drugs worldwide. The cannabis preparations marijuana These are derived from the female plant of and hashish are the most popular and most Cannabis sativa L (family Cannabinaceae).

193 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

Marijuana is prepared from the dried flow- 2008). The precise nature of memory defi- ering tops and leaves; hashish consists of cits in cannabis users and their neural sub- dried cannabis resin and compressed flow- strates still require further research (Solowij ers (Ashton, 2001). Cannabinoids are a and Battisti, 2008). group of C21 terpenophenolic compounds In the treatment of memory disorders uniquely produced by Cannabis sativa whether occurring as a part of normal aging plant. The primary psychoactive constituent or due to a pathological process e.g., Alz- is 9-tetrahydrocannabinol (THC) (Mech- heimer's disease, drugs such as piracetam, oulam and Gaoni, 1967). Other plant can- vinpocetine, Ginkgo biloba or donepezil nabinoids include 8-THC, cannabinol and which improve learning and memory are cannabidiol (Adams and Martin, 1996). frequently prescribed (McDaniel et al., Memory or the retention of learned in- 2003). Piracetam was the first of the so formation is fundamental to human beings. called "nootropics", a term introduced by Cannabis use causes working and short- Giurgea (1973) to indicate this category of term memory deficits in humans as well as drugs that enhance memory, facilitate learn- in experimental animals (Solowij and Bat- ing and protect memory processes against tisti, 2008; Fadda et al., 2004). This effect conditions which tend to disrupt them. The applies to both short and long-term use of drug is a pyrrilodine derivative (2-oxo-1- cannabis (Solowij and Battisti, 2008; pyrrolidine acetamide), a cyclic derivative Solowij et al., 2011); the impairments may of gamma-aminobutyric acid, which has persist well beyond the period of intoxica- been shown to facilitate learning and pre- tion, and recovery of functions might take serve memory from disruption under differ- weeks following abstinence (Pope et al., ent experimental conditions (Gouliaev and 2001) or persist for longer time (Solowij et Senning, 1994; He et al., 2008). Piracetam al., 2002). In chronic users, cannabis might enhances recovery from post-stroke aphasia impair the ability to learn and remember (Kessler et al., 2000), improves cognitive new information. Early onset, long-term use performance in the elderly (Waegemans et and higher frequency of use are seen as risk al., 2002) and in patients undergoing bypass factors for cognitive impairments (Harvey surgery (Holinski et al., 2008). The drug et al., 2007). Cannabinoids exert their ef- possesses rheological, blood flow promot- fects by interaction with specific endoge- ing effects (Müller et al., 1999) and mito- nous cannabinoid receptors. Two G protein- chondrial membrane stabilizing properties coupled cannabinoid receptor subtypes (Keil et al., 2006; Leuner et al., 2010). have been cloned: CB1 receptor which is Vinpocetine (vinpocetine-ethyl apovin- widely distributed throughout the brain, but caminate), a synthetic derivative of the al- particularly in the , hippo- kaloid vincamine from periwinkle (Vinca campus, cerebellum, thalamus and basal minor) is phosphodiesterase (PDE) inhibi- ganglia (the brain regions involved in cog- tor, selective for PDE1 (van Staveren et al., nition, memory, reward, pain perception, 2001) and a blocker of voltage-gated Na+ and motor coordination) and CB2 receptor channels (Sitges et al., 2006) which im- which is mainly expressed on immune cells, proves learning and memory deficits in ro- but also in . These dents (DeNoble, 1987). The drug is widely receptors also respond to endogenous lig- used to improve the cognitive functions of ands, the endocannabinoids such as anan- patients with cerebrovascular disease and damide and 2-arachidonoylglycerol, which chronic cerebral hypoperfusion (Horváth, are arachidonic acid derivatives. Canna- 2001) and to decrease the risk of transient binoid CB1 receptors were identified in the ischemic attacks and strokes in patients hippocampus, the brain region most closely with chronic cerebrovascular insufficiency associated with memory (Pertwee and Ross, (Valikovics, 2007). Vinpocetine is a potent 2002; Pertwee, 2005; Svíženská et al., vasodilator at the cerebral vascular bed, in-

194 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

creasing cerebral blood flow and regional bis-induced memory alterations; (3) detect cerebral glucose uptake (Vas et al., 2002). alterations in the level of the brain neuro- Standardized extracts from the leaves of transmitters dopamine, serotonin (5-HT) Ginkgo biloba contains 24 % ginkgo-flavo- and noradrenaline; (4) assess the effect of ne glycosides and 6 % terpenoids (gink- cannabis administration on oxidative stress golides, bilobalide). Ginkgo biloba extracts markers in brain since oxidative stress has are widely used to improve cognition and been implicated in memory impairment memory in cerebral insufficiency and mild (Clausen et al., 2010). In addition, the effect cognitive impairment (Bäurle et al., 2009). of cannabis on liver oxidative stress and The extract also showed a beneficial effect liver under the different experi- in reducing the total and negative symp- mental conditions was studied. A total ex- toms of chronic when used as tract from Cannabis sativa was used based an add-on therapy to antipsychotic medica- on the fact that the effect of the whole plant tion (Singh et al., 2010). Ginkgo biloba which is abused by humans differs from prevented stress- and corticosterone- that of THC in view of its content of other induced impairments of spatial memory and cannabinoids, terpenoids and flavonoids reduced neuronal loss in hippocampus (Ta- (Russo and McPartland, 2003). kuma et al., 2007; Walesiuk and Braszko, 2009). Ginkgo biloba also showed neuro- MATERIALS AND METHODS protective and therapeutic effects in exper- Animals imental cerebral ischemia (Saleem et al., Swiss male albino mice 20-22 g of body 2008) and attenuated the toxin-induced neu- weight were used. Standard laboratory food rodegeneration of the nigrastriatal pathway and water were provided ad libitum. Ani- (Rojas et al., 2008). The extract has been mal procedures were performed in accord- shown to possess vasodilator and blood ance with the Ethics Committee of the Na- flow enhancing (Chung et al., 1999) as well tional Research Centre and followed the as antioxidant and free radical scavenging recommendations of the National Institutes properties (Rojas et al., 2008). of Health Guide for Care and Use of Labor- The main neurochemical deficit seen in atory Animals (Publication No. 85-23, re- Alzheimer's disease is reduced acetylcho- vised 1985). Equal groups of 6 mice each line content and acetyltransferase were used in all experiments. activity (the synthesizing acetyl- choline) in the nucleus basalis of Meynert Drugs and the hippocampus (Whitehouse et al., Vinpocetine (Vinporal, Amrya. Pharm. 1982). inhibitors Ind., Cairo, ARE), piracetam (Nootropil, (AChEI) e.g., donepezil are thus being used Chemical Industries Development; CID, for the symptomatic treatment of Alzhei- Cairo, ARE), Ginkgo biloba (EMA Pharm. mer's disease to enhance neuro- Co., Cairo, A.R.E) and scopolamine (Sig- transmission indirectly, by inhibiting the ma, USA) were used. All drugs were dis- enzyme which hydrolyses solved in isotonic (0.9 % NaCl) saline solu- (Musiał et al., 2007). tion immediately before use. The doses of The aims of the present study were drugs used in the study were based upon the therefore to: (1) study the behavioral altera- human dose after conversion to that of mice tions associated with long-term administra- according to Paget and Barnes conversion tion of cannabis preparations on spatial tables (1964). Cannabis sativa L. plant was learning and memory; (2) investigate the supplied by the Ministry of Justice - Egypt. effect of memory enhancing drugs pirace- tam, vinpocetine, Ginkgo biloba and the Preparation of cannabis extract acetylcholine esterase inhibitor donepezil Cannabis sativa extract was prepared for their possible modulation of the canna- from the dried flowering tops and leaves of

195 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

the plant. The method of extraction fol- Biochemical studies lowed that described by Turner and Mahl- At the end of the study, mice were eu- berg (1984) with modification as described thanized by decapitation, brains and livers elsewhere (Abdel-Salam et al., 2012). Tet- were then removed, washed with ice-cold rahydrocannabinol (THC) content was saline solution (0.9 % NaCl), weighed and quantified using GC mass. The 9-THC stored at -80 ºC for the biochemical anal- content of the extract was 10 %. The extract yses. The tissues were homogenized with was injected intraperitoneally at doses of 5, 0.1 M phosphate buffer saline at pH 7.4, to 10 or 20 mg/kg (expressed as 9-THC). The give a final concentration of 10 % w/v for injection volume was 0.2 ml/mice. the biochemical assays. For the determina- tion of monoamine neurotransmitters, fro- Cognitive testing zen brain samples were homogenized in The maze consisted of a glass tank, nar- cold 0.1 N-perchloric acid. rowed to 20 cm wide, 40 cm in height, 70 cm in length, filled to a depth of 21 cm Determination of lipid peroxidation with water maintained at 25 °C. The escape Lipid peroxidation was assayed in brain glass platform was hidden from sight, sub- and liver homogenates by measuring the merged 1 cm below the surface of the water level of malondialdehyde (MDA). at the end of the tank (Dunnett et al., 2003). Malondialdehyde was determined by meas- The effect of cannabis extract was studied uring thiobarbituric reactive species using in normal mice and in mice treated with the method of Ruiz-Larrea et al. (1994) in scopolamine (1 mg/kg, i.p.) to induce cog- which the thiobarbituric acid reactive sub- nitive impairment (Smith et al., 2002). Mice stances react with thiobarbituric acid to were treated with scopolamine alone or in produce a red colored complex having peak combination with cannabis (20 mg/kg, s.c.) absorbance at 532 nm. (n = 6/group) 30 min prior to testing. In ad- dition, the effect of cannabis extract Determination of reduced glutathione (20 mg/kg, s.c.) was studied in mice treated Reduced glutathione (GSH) was deter- with memory enhancing drugs piracetam mined by Ellman's method (1959). The pro- (150 mg/kg, s.c.), vinpocetine (1.5 mg/kg, cedure is based on the reduction of s.c.), Ginkgo biloba (25 mg/kg, s.c.) and the Ellman´s reagent by –SH groups of GSH to acetylcholinesterase inhibitor donepezil form 2-nitro-s-mercaptobenzoic acid, the (1 mg/kg, s.c.) for their possible modulation nitromercaptobenzoic acid anion has an in- of the Cannabis sativa-induced memory tense yellow color which can be determined alterations. Treatments were given once spectrophotometrically. daily for 30 days. Each test consisted of three trials involving placing the mouse in Determination of nitric oxide the water maze with the platform hidden Nitric oxide measured as nitrite was de- until it finds the platform, leaving it on the termined by using Griess reagent, according platform for 15 s, and then retesting the to the method of Moshage et al. (1995), mouse from the same start position 3 min where nitrite, stable end product of nitric later (trial 1; reference memory or acquisi- oxide radical, is mostly used as indicator tion trial; trials 2 & 3; working memory or for the production of nitric oxide. retrieval trials). This was done 3 times a week for 4 weeks. At the end of each trial, Determination of liver enzymes the mouse was towel dried, returned to its Aspartate aminotransferase (AST) and home cage (where a heat lamp was availa- aminotransferase (ALT) activities ble). The latency to find the platform (s) is in liver were measured according to Reit- assessed with a stopwatch. man-Frankel colorimetric transaminase

196 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

procedure (Crowley, 1967) using commer- RESULTS cially available kits (BioMérieux, France). Behavioral testing

Determination of brain glucose Spatial memory Brain tissue glucose content was deter- Cannabis substantially impaired water mined according to the method of Trinder maze performance. The time taken to find (1969). Glucose in the presence of glucose the escape platform (latency) was signifi- oxidase is converted to peroxide and glu- cantly delayed by cannabis in a dose- conic acid. The produced perox- dependent manner, compared with the sa- ide reacts with phenol and 4-amino- line-treated control group (Figure 1). The antipyrine in the presence of peroxidase to average mean latency and standard error of yield a colored quinonemine, which is the mean over 4 weeks for the saline, can- measured spectrophotometrically. nabis 5 mg/kg, cannabis 10 mg/kg and can- nabis 20 mg/kg was 3.19 ± 0.07, 4.78 ± Determination of brain monoamines 0.23, 5.73 ± 0.42 and 7.62 ± 0.52 sec, re- Determination of brain serotonin, nor- spectively. There was a significant main adrenaline and dopamine was carried out drug effect (F = 36.63, p = 0.001), a signifi- using high performance liquid chromatog- cant main effect of days (F = 8.82, p = raphy (HPLC) system, Agilent technologies 0.001) but no significant main effect for 1100 series, equipped with a quaternary trials (F = 1.62, p = 0.207). There was a pump (Quat pump, G131A model). Separa- significant drug x days interaction (F = tion was achieved on ODS reversed phase 1.76, p = 0.006) but no significant drug x column (C18, 25 x 0.46 cm i.d. 5 µm). The trial (F = 0.33, p = 0.92) or trial x days in- mobile phase consisted of potassium phos- teraction (F = 1.25, p = 0.20). phate buffer/methanol 97/3 (v/v) and was Saline Cannabis sativa 5 mg/kg delivered at a flow rate of 1 ml/min. UV Cannabis sativa 10 mg/kg 18 Cannabis sativa 20 mg/kg detection was performed at 270 nm and the * 16 injection volume was 20 µl. The concentra- 14 * tion of both catecholamines and serotonin 12 were determined by external standard 10 * * method using peak areas. Serial dilutions of * 8 * * * * * standards were injected and their peak areas * 6 * * * * *

Escape latency (sec) latency Escape * were determined. A linear standard curve * * * * * was constructed by plotting peak areas ver- 4 sus the corresponding concentrations. The 2 0 concentration in samples was obtained from 024681012 the curve. Days Figure 1: Effect of cannabis extract on the la- tency to find hidden platform in the MWM test. Statistical analysis Cannabis was administered daily via subcuta- Data are expressed as mean ± SEM. The neous route for one month and observations data were analyzed by one way ANOVA were done three times weekly. Asterisks indi- and by repeated measures ANOVA, fol- cate significant change from the saline control lowed by Duncan’s multiple range test, us- group. ing SPSS software (SAS Institute Inc., Cary, NC). A probability value of less than Donepezil, piracetam, vinpocetine or 0.05 was considered statistically significant. Ginkgo biloba co-administered with canna- bis (20 mg/kg) resulted in significantly shorter latencies compared with the canna- bis (20 mg/kg) only-treated group, which indicated improved learning and memory (Figure 2). The average mean latency and 197 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

standard error of the mean over 4 weeks for * the cannabis 20 mg/kg plus donepezil, pi- * racetam, vinpocetine or Ginkgo biloba was 9 * 8 * 4.87 ± 0.21, 4.42 ± 0.19, 5.18 ± 0.28 and * 7 + 4.65 ± 0.23 sec, respectively compared to * 7.62 ± 0.52 sec for the cannabis 20 mg/kg 6 + + * + # # group (Figure 3). Compared with the can- 5 + + nabis only group, the escape latency de- 4 + creased by 36.1 %, 42 %, 32 % and 39 % 3 2 by donepezil, piracetam, vinpocetine or Escape latency (sec) Ginkgo biloba, respectively. Piracetam re- 1 sulted in significantly shorter latency com- 0 g zil o /kg e g g/k cetin k pared with vinpocetine by 14.7 %. There m np cetam o Control o a Gin 0 mg 0 ir np 1 2 P i was a significant main drug effect (F = is is V b b + nabis 5 mg/kga a g/kg + n g/kg + d kg 14.45, p = 0.001), a significant main effect nn nn / m Ca m g/kg + 0 Ca Ca 0 m 2 2 0 mg 0 of days (F = 5.27, p = 0.001), no significant s 2 2 is ab is n b n main effect of trials (F = 3.42, p = 0.038). nnabi a Ca Ca nn There was a significant drug x days interac- CannabisCa tion (F = 3.19, p = 0.001) but no significant drug x trial (F = 0.38, p = 0.93) or trial x Figure 3: The average mean latency to locate a days interaction (F = 1.4, p = 0.11). submerged platform in the MWM test over four weeks. Mice received daily injections of saline

Cannabis 20 mg/kg or cannabis extract (5, 10 or 20 mg/kg) alone or Cannabis 20 mg/kg + donepezil Cannabis 20 mg/kg + piracetam combined with donepezil, piracetam, vinpoce- Cannabis 20 mg/kg + vinpocetine Cannabis 20 mg/kg + ginkgo biloba tine or Ginkgo biloba and were tested three 18 times weekly. Asterisks indicate significant

16 change from the saline control group and be-

14 tween different groups as shown in the figure. The plus sign indicates significant change from 12 the cannabis 20 mg/kg group. The # sign indi- 10 cates significant change from the cannabis 8 10 mg/kg group. Other statistical comparisons * * between different treated groups are also 6 * *

Escape latency (sec) * * shown and are indicated by asterisks. 4 * 2 In the first trial, the time taken to find 0 024681012 the escape platform was significantly de- Days layed by Cannabis sativa (Figure 7A). Figure 2: Effect of cannabis extract combined There was a significant main effect of drug with donepezil, piracetam, vinpocetine or (F = 18.61, p = 0.001), a significant main Ginkgo biloba on the latency to find hidden effect of days (F = 2.18, p = 0.017) but no platform in the MWM test. Cannabis and test significant drug x days interaction (F = drugs were administered daily via subcutane- 0.86, p = 0.69). Mice treated with cannabis ous route for one month and observations were done three times weekly. Asterisks indicate sig- 20 mg/kg in combination with donepezil, nificant change from the cannabis only treated piracetam, vinpocetine or Ginkgo biloba group. exhibited significantly shorter escape laten- cies compared with cannabis (20 mg/kg) only-treated group, suggesting improve- ment of the cognitive impairing effects of cannabis by these drugs. There was a sig- nificant main effect of drug (F = 5.0, p = 0.004), a significant main effect of days (F = 2.09, p = 0.021) but no significant drug x

198 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

days interaction (F = 0.97, p = 0.54). The 0.28, 4.22 ± 0.29, 5.09 ± 0.32 and 4.98 ± average mean for the saline, cannabis 0.21 sec, respectively. Compared with the 5 mg/kg, cannabis 10 mg/kg and cannabis cannabis only group, the escape latency de- 20 mg/kg was 3.33 ± 0.14, 5.24 ± 0.47, 6.02 creased by 42 %, 44.2 %, 32.8 % and ± 0.45, 8.27 ± 0.44 sec, respectively. The 34.2 % by donepezil, piracetam, vinpoce- average mean latency and standard error of tine or Ginkgo biloba, respectively. Pirace- the mean over 4 weeks for the cannabis tam resulted in significantly shorter latency 20 mg/kg plus donepezil, piracetam, compared with vinpocetine by 17.1 %. vinpocetine or Ginkgo biloba was 5.64 ± In the third trial, mice that received 0.46, 5.11 ± 0.47, 5.54 ± 0.42 and 5.08 ± cannabis took significantly more time to 0.43 sec, respectively (Figure 4A). Com- find the escape platform compared with the pared with the cannabis only group, the es- saline-treated group (Figure 4C). There was cape latency decreased by 31.8 %, 38.2 %, a significant main effect of drug (F = 7.36, 33 % and 38.6 % by donepezil, piracetam, p = 0.002), a significant main effect of days vinpocetine or Ginkgo biloba, respectively. (F = 5.12, p = 0.001) but no significant drug No significant differences were observed x days interaction (F = 0.96, P = 0.53). between the different drugs in trial 1. Donepezil, piracetam, vinpocetine or Gink- In the second trial, mice that received go biloba co-administered with cannabis 10 or 20 mg/kg of cannabis were also de- (20 mg/kg) resulted in significantly shorter layed compared with the saline-treated latencies compared with cannabis (20 mg/ group in escaping from the water maze kg) only-treated group (Figure 4C). There (Figure 4B). There was a significant main was a significant main effect of drug (F = effect of drug (F = 14.89, p = 0.001), a sig- 5.60, P = 0.002), a significant main effect nificant main effect of days (F = 3.86, P= of days (F = 3.65, p = 0.0001) and a signif- 0.001) but no significant drug x days inter- icant drug x days interaction (F = 2.0, p = action (F = 1.13, p = 0.29). Donepezil, pi- 0.001). The average mean for the saline, racetam, vinpocetine or Ginkgo biloba co- cannabis 5 mg/ kg, cannabis 10 mg/kg and administered with cannabis (20 mg/kg) im- cannabis 20 mg/ kg was 3.06 ± 0.1, 4.71 ± proved performance resulting in significant- 0.36, 5.85 ± 0.41 and 7.02 ± 0.60 sec, re- ly shorter latencies to find the hidden plat- spectively. The average mean latency and form than the cannabis (20 mg/kg) only- standard error of the mean over 4 weeks for treated group (Figure 4B). There was a sig- the cannabis 20 mg/ kg plus donepezil, pi- nificant main effect of drug (F = 4.73, P = racetam, vinpocetine or Ginkgo biloba was 0.006), a significant main effect of days (F 4.95 ± 0.31, 3.94 ± 0.20, 4.91 ± 0.26 and = 2.76, p = 0.002) and a significant drug x 3.89 ± 0.19 sec, respectively. Compared days interaction (F = 1.95, p = 0.001). The with the cannabis only group, the escape average mean for the saline, cannabis latency decreased by 29.4 %, 43.9 %, 5 mg/kg, cannabis 10 mg/kg and cannabis 30.1 % and 44.6 % by donepezil, piracetam, 20 mg/kg was 3.15 ± 0.11, 4.38 ± 0.36, 5.33 vinpocetine or ginkgo biloba, respectively. ± 0.43 and 7.57 ± 0.50 sec, respectively. Piracetam or Ginkgo biloba resulted in sig- The average mean latency and standard er- nificantly shorter latency compared with ror of the mean over 4 weeks for the canna- vinpocetine by 19.7 % and 20.1 % and bis 20 mg/kg plus donepezil, piracetam, compared with donepezil by 20.4 %, vinpocetine or Ginkgo biloba was 4.39 ± 21.4 %, respectively.

199 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

A B

10.0 9 * * 8 * 7.5 7 * *+ + + * + 6 *+ * +* *+ + *+ 5.0 5 + + + + 4 + 2.5 3

Escape latency (sec)Escape 2 Escape latency (sec) Escape 1 0.0 il 0 /kg am tin g t e kgo l saline n e zi tin m onpez lin /kg e 0 mg/kg 0 d race p trol 1 2 sa n n + pi + inpoc l mg oce g + 0 o p k 1 in + ginkgo Co g /kg tro + d v g nnabis 5 nabismg/kg nabis k g n is a mg/ o b g + C C a /k Can Can 0 mg/ 20 mg/kg + gi n g 2 s n /kg is 20 Cannabisa 5 mg/kg m is abi C Cannabis0 20 mg/kg mg n 0 nnab nab is 2 2 Can b is Ca a b Cannabis 20 m n a Can n n a n Cannabis 20 mg/k C a Cannabis 20 mg/kg + piracetam C C Figure 4A-C: The average mean latency (in 8 * seconds) ± SEM of first (A); second (B) and * * third (C) trial to locate a submerged platform 7 * in the MWM test over four weeks. Mice re- 6 + ceived daily injections of saline or cannabis + * 5 * * + extract (5, 10 or 20 mg/kg) alone or combined + # + # 4 with donepezil, piracetam, vinpocetine or + Ginkgo biloba and were tested three times 3 weekly. Asterisks indicate significant change latency (sec) 2 from the saline control group. The plus sign 1 indicates significant change from the canna- bis 20 mg/ kg group. The # sign indicates 0 significant change from the cannabis 10 mg/ g kg m in g/ g/kg cet m npezil kg group. o ginkgo 0 m iraceta inpo + is 5 mg/ks 1 p ab bi bis 20 Control saline a /kg + d na g g/kg + g/kg + v Cann Can Cann m m 20 m s 20 s 20 bi nnabis abi a na Cannabis 20 mg/kg C ann C Can

Scopolamine (1 mg/kg, i.p.) substantial- = 0.09). There was no significant effect of ly impaired cognitive performance leading trials (F = 1.71, p = 0.19) and no significant to higher latencies to locate the submerged main effect of days (F = 0.89, p = 0.55). platform compared with the saline-treated There was a significant drug x days interac- group. Mice treated with scopolamine and tion (F = 1.69, P = 0.01) but no significant cannabis (10 or 20 mg/kg, sc) needed sig- drug x trial (F = 0.37, p = 0.89) or trial x nificantly more time to locate the hidden days interaction (F = 0.83, p = 0.69). The platform than the scopolamine only-treated average mean latency and standard error of group, suggesting enhancement of the cog- the mean over 4 weeks for the saline, sco- nitive impairing effects of scopolamine by polamine 1 mg/kg and the scopolamine plus cannabis (Figures 5, 6) (2-way repeated cannabis at 5, 10 or 20 mg/kg was 3.13 ± measures ANOVA; drug effect: F = 2.19, p 0.06, 6.92 ± 0.35, 6.32 ± 0.23, 8.07 ± 0.47

200 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

and 9.04 ± 0.63 sec, respectively. The time 10.0 taken to find the escape platform was sig- * nificantly delayed by scopolamine alone or + in combination with cannabis compared 7.5 * + with the saline treated group in the first, *+ * second and third trial (2-way repeated 5.0 measure ANOVA: drug effect: trial 1: F = + 1.11, p = 0.37; trial 2: F = 0.5, p = 0.69; tri- 2.5 al 3: F = 1.22, p = 0.33). There was no sig- Escape latency (sec) nificant main effect of days on the escape 0.0 g g g line /k latency and no significant drug x days in- a g/kg S mg m 5 0 mg/k 0 teraction in all trials (data not shown). In 1 2 bis is a bis b a the second trial, mice treated with scopola- nn na a nn n C a copolamine 1 +mg/k C mine + 20 mg/kg cannabis showed signifi- S + + Ca cantly higher latencies to find the hidden platform compared with scopolamine + Figure 6: The average mean latency to locate a submerged platform in the MWM test over four 5 mg/kg cannabis-treated group. In the third weeks. Mice received daily injections of scopol- trial mice that received scopolamine + amine (1 mg/kg, s.c.) or cannabis extract (5, 10 20 mg/kg cannabis showed significantly or 20 mg/kg) and were tested three times week- higher latencies to find the submerged plat- ly. Asterisks indicate significant change from form compared with other groups (Figure the saline control group. The plus sign indicates significant change from the scopolamine + can- 7A-C). nabis 20 mg/kg group. 12 A 10 10.0 * + * + 8 * 7.5 * 6 * * * *

4 5.0

Escape latency(sec) 2 2.5

0 Escape latency(sec) g k /kg /kg g g mg/ m 0.0 5 0 is 2 e ine 1m b is m a b lin g/kg n a n n Sa m n opola Ca is 5 mg/kg 10 mg/kg 20 + Ca b is is Sc + Cannabis 10 mg/kg b + a annab ann + Canna C C Figure 5: Effect of cannabis extract on the la- Scopolamine 1 mg/kg + + tency to find hidden platform in the MWM test in mice treated with scopolamine (1 mg/kg, s.c.). Figure 7A The columns represent the first, second and third trail, respectively for each treatment group. Asterisks indicate significant change from trial 1.

201 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

B group (26.71 ± 0.96 . 33.47 ± 1.34 12.5 * nmol/g). Brain GSH showed significant in- * crease by 24.2 % after cannabis at 20 mg/kg 10.0 * * (4.1 ± 0.20 vs. 3.3 ± 0.13 µmol/g, p<0.05). 7.5 Meanwhile, the administration of cannabis at 20 mg/kg resulted in a significant de- 5.0 crease in brain nitrite by 32.8 % (34.48 ± 3.3 vs. 51.3 ± 3.1 µmol/g, p<0.05) and in 2.5 Escape latency (sec) brain glucose by 30.2 % (35.1 ± 1.9 vs. 50.3 0.0 ± 3.5 µg/g, p<0.05) (Figure 8 A-D). g k aline Cannabis in combination with memory en- S mg/kg mg/kg 5 mg/ 10 20 is hancing drugs nabis an nnab nnabis The level of MDA in brain increased C a a + C C + + significantly by 23.8 % after the administra- Scopolamine 1 mg/kg C * + tion of piracetam (33.08 ± 0.76 vs. 26.71 ± 10.0 * 0.96 nmol/g, p<0.05). Meanwhile, brain nitrite and glucose which decreased by can- * 7.5 * nabis were restored to near normal values by piracetam, registering 29.1% (44.5 ± 2.9 5.0 vs. 34.48 ± 3.3 µmol/g, p<0.05) and 36.2 % (47.8 ± 2.0 vs. 35.1 ± 1.9 µg/g, p<0.05) in- 2.5 crements compared with the 20 mg/kg can- Escape latency(sec) nabis only-treated group. On the other 0.0 hand, the administration of donepezil, e in /kg /kg kg kg g g vinpocetine or Ginkgo biloba resulted in Sal m m mg/ mg/ 1 5 0 0 significant increase in brain GSH by e is b is 1 is 2 in a b b m n a a la n n n 36.6 %, 41.5 % and 117.1 %, respectively o n n p Ca + Ca Ca (Figure 8A-D) Sco + +

Figure 7 A-C: The average mean latency of Cannabis in combination with scopolamine first (A); second (B) and third (C) trial to locate a The administration of scopolamine at submerged platform in the MWM test over four the dose of 1 mg/kg did not change brain weeks. Mice received daily injections of saline, MDA or glucose level. However, the level scopolamine (1 mg/kg, s.c.) or scopolamine of nitric oxide increased by 15.7 % and plus cannabis (5, 10 or 20 mg/kg) and were tested three times weekly. Asterisks indicate GSH significantly decreased by 27.3 % by significant change from the saline control group. scopolamine vs. saline control value (2.4 ± The plus sign indicates significant change from 0.12 vs. 3.3 ± 0.13 µmol/g, p<0.05). In sco- the scopolamine only or scopolamine + canna- polamine-treated mice, the administration bis 5 mg/kg group. of cannabis extract decreased MDA in a dose-dependent manner. A significant de- BIOCHEMICAL RESULTS crease in MDA level by 26.2 % and 31.7 % was observed after the administration of Effect of cannabis on markers of oxidative cannabis extract 10 or 20 mg/kg, respec- stress and glucose in brain tively (27.87 ± 1.04 and 25.76 ± 0.98 vs. Cannabis alone 37.74 ± 1.23 nmol/g, p<0.05). The level of The administration of cannabis altered glutathione was significantly increased by the redox status in brain with the effect be- 29.2 % (p<0.05) and 62.5 % (p<0.05) after ing significant with the high dose of the ex- the administration of cannabis extract at the tract which resulted in 20.2 % decrease in doses of 10 and 20 mg/kg, respectively, MDA compared with the saline-treated compared with the scopolamine only-

202 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

treated group (3.1 ± 0.0.16 and 3.9 ± 0.26 vs. 2.4 ± 0.12 µmol/g, p<0.05). The level of C nitrite significantly decreased by 26.3 % 60 + + after cannabis at 20 mg/kg, while glucose 50 + + level significantly decreased by 16.7 % and # 40 30.6 % after cannabis at 10 and 20 mg/kg, tissue) mol/g. *  respectively vs. scopolamine only-treated 30 group (Figure 8 A-D). 20

A 10

45 0

+ + ( oxide nitric Brain e g kg m g kg 40 lin / ta / e pezil Sa mg c e mg + a pocetin 35 0 mg/k on 5 mg/k 1 20 in d 10 pir v + bis # bis + + bis 30 * * * * abis g g g na * # n na /k n na * * n a n a C a 25 Can C mg C + Cannabis 5 mg/kg0 mg/k0 mg/k 0 + + + 2 2 2 Scopolamine+ 1 +mg/kg Cannabis 20 mg/kg 20 s s i i bis ab ab a 15 n n Cannabisnn 20 mg/kg + ginkgo a C 10 Can Can 5 Brain MDA (nmol/g. tissue) (nmol/g. MDA Brain 0 D e m o n /kg /kg g ezil /kg /kg k 60 Sali mg ceta ep mg on 5 mg d is bis 5 mg + ine 1 mg/kg + a bis 10 g/kg + gin ab bis 10 50 na kg + pira na + + g/ m g/kg mg/kg + vinpocetin + Cann 0 m + Cann 40 * * #* + Can+ Cannabis 20 20m mg/kg Scopolam+ Can+ Cannabis 20 mg/kg *

abis 20 g/g. tissue) is n # ab abis 2  * Can nn 30 a CannCannabis 20C 20 B * 10 Brain glucose ( * * 0 e g o g kg m g kg kg lin / a k / / g/k in g/k Sa m g m 5 0 mg 10.0 5 20 mg + 1 mg + is g donepezile is 2 * b is + b is b + vinpocetin/k b na g min na n /kg la n g/k 0 mg g Ca m po Ca 7.5 + Canna mg/kg + piracet 2 o + Canna + Cannabis+ 10 mg/kg0 is + Cannabis+ 10 mg/kg + 20 b Sc * * + * is na bis 20 m * abis 2 n a n a nnab C mol/g. tissue) mol/g. a 5.0 * Cann  * # Can C + * + +* 2.5 * Figure 8 A-D: Effect of cannabis extract alone or in combination with piracetam, vinpocetine, Brain GSH ( GSH Brain 0.0 Ginkgo biloba, donepezil or scopolamine on n o l g g g g tam /k /k /k /k brain malondialdehyde (MDA), reduced gluta- e g g g g Saline thione (GSH), nitric oxide and glucose in mice. + ginkg 5 m e 1 ms + donepeziin bi is 10 ism 20 m *: p< 0.05 vs corresponding saline control val- g/kg m a b b m la nn na na g/kg o a n n ue. +: p< 0.05 vs. cannabis (20 mg/kg) only- mg/kg + pirac m C + Cannabis 5 mg/kg mg/kgs 20 + vinpoceti + + Cannabis+ Cannabis 10 mg/kg 20 mg/kg0 i Scop + Ca+ Ca treated group. #: p< 0.05 vs scopolamine only- ab s 20 n i ab treated group Can n annabis 2 Can CannabisC 20 Effect of cannabis on brain monoamines In saline-treated mice, a dose-dependent elevation of serotonin, noradrenaline and dopamine was observed after treatment with cannabis extract alone at tested doses

203 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

of 5, 10 and 20 mg/kg. In mice given Effect of cannabis on markers of oxidative 20 mg/kg of cannabis, the increment in ser- stress in liver otonin was ameliorated by the memory en- The effect of cannabis on oxidative hancing drugs donepezil, piracetam, markers in the liver was also examined. The vinpocetine or Ginkgo biloba. Noradrena- administration of cannabis extract (5, 10 or line was restored to its normal value by 20 mg/kg) did not alter the level of MDA, donepezil, vinpocetine or ginkgo biloba, but GSH or nitrite in hepatic tissue. In mice increased by piracetam. The level of dopa- given 20 mg/kg cannabis extract, the ad- mine was significantly reduced by pirace- ministration of Ginkgo biloba significantly tam, vinpocetine or Ginkgo biloba, but not decreased nitrite level (85.0 ± 5.1 vs. 102.4 by donepezil (Table 1). In mice treated with ± 6.1 µmol/g, p<0.05). Meanwhile, GSH scopolamine, a significant increase in sero- showed further increase by 17.2% in mice tonin and dopamine by cannabis at 20 mg/ treated with Ginkgo biloba (12.9 ± 1.1 vs. kg was observed. 11.01 ± 3.1 µmol/g, p<0.05). Liver alanine aminotransferase and aspartate aminotrans- ferase were not altered in mice given 20 mg/kg cannabis alone or with memory en- hancing drugs (Table 2).

Table 1: Effect of cannabis extract, scopolamine or cannabis + scopolamine on brain monoamines Serotonin Dopamine Noradrenaline (µg/g tissue) (µg/g tissue) (µg/g tissue) Saline 2.83  0.20 2.94  0.05 1.95  0.15 + Cannabis 5 mg/kg 5.23  0.26* 4.65  0.51* 2.10  0.08 + Cannabis 10 mg/kg 5.19  0.23* 7.88  0.11* 2.22  0.12* + Cannabis 20 mg/kg 5.56  0.47* 11.4  0.65* 2.55  0.11* Cannabis 20 mg/kg + donepezil 1.45  0.05*+ 14.62  1.61*+ 1.58  0.08+ 1 mg/kg Cannabis 20 mg/kg + piracetam 1.95  0.07*+ 4.65  0.51*+ 3.85  0.23*+ 150 mg/kg Cannabis 20 mg/kg + vinpocetin 1.06  0.09*+ 5.71  0.33*+ 1.95  0.17+ 3 mg/kg Cannabis 20 mg/kg + Ginkgo 2.01  0.18*+ 4.64  0.52*+ 3.44  0.43*+ biloba 25 mg/kg Scopolamine 1 mg/kg 1.84  0.21* 5.68  0.41* 1.84  0.21 + Cannabis 5 mg/kg 1.91  0.12 5.43  0.34* 1.77  0.12 + Cannabis 10 mg/kg 1.96  0.13 5.49  0.24* 1.91  0.15 + Cannabis 20 mg/kg 2.82  0.22+ 7.92  0.63*+ 1.96  0.13

Results are mean ± S.E. Six mice were used per each group. Data were analyzed by one way ANOVA and means of different groups were compared by Duncan’s multiple range test. P<0.05 was considered statistically significant. *: P<0.05 vs. saline control group. +: P<0.05 vs. the scopolamine control group

Table 2: Effect of cannabis extract given with donepezil, piracetam, vinpocetine or Ginkgo biloba on liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) Saline Cannabis 20 mg/kg Cannabis 20 mg/kg + Cannabis Cannabis + donepezil piracetam 150 mg/kg 20 mg/kg + 20 mg/kg + 1 mg/kg vinpocetine Ginkgo biloba 3 mg/kg 25 mg/kg AST 66.8 ± 4.2 67.9 ±1.9 66.1 ± 2.1 65.3 ± 2.0 55.5 ± 5.3 ALT 31.1 ± 1.9 35.2 ± 2.1 32.5 ± 1.4 34.8 ± 2.0 30.9 ± 1.1 Results are mean ± S.E. Six mice were used per each group. Data were analyzed by one way ANOVA and means of different groups were compared by Duncan’s multiple range test.

204 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

The administration of scopolamine did C not alter liver malondialdehyde or nitric ox- 45 ide level. However, the level of GSH was 40 significantly decreased by 31.7 % (p<0.05) 35 + by scopolamine vs. saline control value 30 * mol/g. tissue) mol/g. (7.65 ± 1.3 vs. 11.2 ± 2.4 µmol/g, p<0.05).  25 In scopolamine-treated mice, the admin- 20 istration of cannabis at 5, 10 or 20 mg/kg 15 increased liver GSH in a dose-dependent 10 manner (33.3 %, 38.6 % and 50.3 % in- 5 0 crease vs. scopolamine only-treated group) Liver nitric oxide ( g n l ne ti e ali (Figure 9 A-C). S mg/kg 1 s 5 mg/kg inpoc 10 mg/kg A s 20 mg/kg piracetam v donepezine g + nnabi nnabi annabis 10 mg/k annabis + Ca C copolami+ Cannabis C 5 mg/kg + + Ca + + Cannabis 20 mg/kg 20 mg/kg +20 mg/kg S+ 70 Cannabis 20 mg/kg + ginkgo annabis annabis 60 CannabisC 20 mg/kC 50 Figure 9 A-C: Effects of cannabis extract 40 alone, cannabis + memory enhancing drugs or 30 cannabis + scopolamine on liver malondialde- 20 hyde (MDA), reduced glutathione (GSH) and nitric oxide in mice. *: p< 0.05 vs corresponding 10 vehicle control value. +: p< 0.05 vs. cannabis

Liver MDALiver (nmol/g. tissue) 0 (20 mg/kg) only-treated group l g m i g /kg /k a z /kg /k /kg g g nkgope g g g Salinem cet ocetini m m a p ne 5 0 mg/kg 1 0 is 1 pir vin do 10 m2 kg + g ne s is DISCUSSION ab bis 20 m / i bi b a nn /kg + kg + lam na g/kg g+ 0 mg Ca annabis mg/ anna C m m 2 C In the present study the effect of canna- + 0 0 is 0 + Cannabis 5 mg/kg + + Cann b 2 Scopo + Can+ 9 s 2 is bi bis extract with known Δ -THC content on anna ab nnabisnna 2 C nn a a working memory and on oxidative stress in Ca C C brain and liver of mice was investigated. B The repeated daily administration of the extract at doses corresponding to 5, 10 and * 20 mg Δ9-THC/kg was associated with im- 15 paired learning and memory when tested in * the water maze test. Mice given cannabis # # # spent more time to locate the hidden plat- 10 form than their untreated counterparts. The *

mol/g. tissue) mol/g. effect was most evident with the initial ad-  ministration of the extract and maintained 5 throughout the study. There was a clear dose-related response during the three trials

Liver GSH ( Liver GSH of the test. Scopolamine, an 0 e kg kg m zil kg kg kg kg drug, impaired memory performance in the lin / / ta e / / / g g e p g m c e Sa mg/kg m a n mg/ m water maze test which is in accordance with 5 0 inpocetin o 5 0 1 pir v 1 is + d ne 1 mg is b + + i b other studies (Smith et al., 2002). Mice a g a n /kg /k /kg n annabisn g g annabisn C C treated with scopolamine and cannabis Ca mg m Ca + 0 is 20 mg/kg + ginkgo+ + + Cannabis 2 2020 mb Scopolam+ + Cannabis 20 mg a is 20 needed significantly more time to locate the is n b b a a n n Can n n a hidden platform than the scopolamine only- Ca Cannabis C treated group, suggesting exacerbation of

205 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

the cognitive impairing effects of scopola- depolarization-induced suppression of ex- mine by cannabis. Studies have shown that citatory mechanism in the CA1 area of hip- cannabis preparations or their primary psy- pocampus (Ebrahimpour et al., 2010). choactive constituent Δ9-THC affect Of the important findings in the present memory processing in humans and in ex- studies is the effect of cannabis on neuro- perimental animals. Prose recall and source transmitter levels in brain. It is evident that memory were poorer in daily users with the repeated administration of cannabis ex- high THC levels in hair (Morgan et al., tract increased brain levels of serotonin, 2011). In healthy volunteers, Δ9-THC (2.5, noradrenaline and dopamine. Other studies and 5 mg) given intravenously disrupted have shown increased dopamine and nora- immediate and delayed word recall, im- drenaline release in rodent brain by THC in paired performance tests of distractibility, several regions of the brain, including stria- verbal fluency and working memory tal, nucleus accumbens and prefrontal areas (D'Souza et al., 2004). Similarly, orally (Muntoni et al., 2006; Robledo et al., 2007). given Δ9-THC led to acute impairment of Moreover, increased release of norepineph- attentional functioning and working rine in the rat frontal cortex was observed memory (Roser et al., 2008). Impaired after systemic administration of the synthet- memory performance has been reported in ic cannabinoid WIN 55,212-2 mice after the oral administration of hashish (Oropeza et al., 2005). In healthy human extract in dosages corresponding to 1, 5, subjects, THC induced dopamine release in and 10 mg Δ9-THC/kg (Frischknecht et al., the human striatum (Bossong et al., 2009). 1985) and after inhalation of marijuana As for 5HT, 9-THC attenuated methylene- smoke (Niyuhire et al., 2007) and in rats dioxymethamphetamine (MDMA)-induced following intraperitoneal injection of Δ9- decreases in 5-HT levels and in serotonin THC-rich extracts (2 or 5 mg/kg) (Fadda et transporter (SERT) binding in the frontal al., 2004). Cannabidiol, another constituent cortex, parietal cortex, and striatum (Shen of the plant Cannabis sativa, that does not et al., 2011) while stimulating CB-1 de- possess psychoactive properties, appeared creased the effect of on increas- to antagonize the memory impairing effect ing serotonin levels in the prefrontal cortex of Δ9-THC in the same extract (Fadda et al., (Kleijn et al., 2011). In contrast, rats inject- 2004), though not the working memory def- ed with the synthetic cannabinoid HU210 icits induced by scopolamine and dizocil- exhibited increased 5HT1A receptor densi- pine (Fadda et al., 2006). The cannabis in- ty and mRNA expression in the CA1 region duced memory alterations are likely to be of the hippocampus (Zavitsanou et al., mediated by 9-THC-inducecd activation of 2010). These effects of cannabis on brain cannabinoid CB1 receptor in brain. Delta monoamines could account for the cogni- (9)-THC-rich extracts impaired memory tive and attention deficits and anxiety reac- performance in rats (Fadda et al., 2004), tions seen in cannabis users. Cannabis sati- while 9-THC-induced spatial memory im- va, however, contains many constituents pairment was reversed by cannabinoid including over 70 different cannabinoids CB(1) receptor antagonist, suggesting that (Hollister, 1988). Thus cannabis can result the effect of 9-THC is mediated through in different effects from those of 9-THC cannabinoid CB(1) receptors (Egashira et alone. For example, cannabigerol a non- al., 2012). Studies also suggested the in- psychoactive constituent behaved as a po- volvement of prefrontal dopamine receptors tent alpha (2) adrenoceptor agonist and a (Rodrigues et al., 2011) as well as μ- and κ- 5HT(1A) antagonist (Cascio et al., 2010). opioid receptors (Egashira et al., 2012) in Meanwhile, cannabidiol, another non- 9-THC-induced disruption of spatial work- psychoactive increased extracellular dopa- ing memory. Cannabis effect on learning mine levels in nucleus accumbens (Murillo- and memory processes probably involves Rodríguez et al., 2011). While 9-THC acts

206 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

as a CB1 and CB2 receptor partial agonist, Zhou et al., 2008; Halliwell, 2006). The 9-tetrahydrocannabivarin, behaves either present study suggests that cannabis extract as a CB1 antagonist or, at higher doses, as a alters the oxidative balance in the brain. CB1 receptor agonist (Pertwee, 2008). The This however, appears to be in favor of re- present study has also provided evidence ducing lipid peroxidation. Malondialdehyde that the nootropic drugs piracetam, an index of lipid peroxidation activity (Gut- vinpocetine, Ginkgo biloba or donepezil teridge, 1995), is decreased and there was were capable of modulating neurotransmit- significant increase of GSH, an important ter levels in mice treated with cannabis. antioxidant defense system, especially with Serotonin was decreased by all drugs. Nor- the highest dose of cannabis examined. Ni- adrenaline was normalized by vinpocetine tric oxide in brain is also decreased by can- and donepezil though increased by pirace- nabis administration. The significance of tam and Ginkgo biloba. Meanwhile, pirace- this finding is yet to be determined. Nitric tam, vinpocetine, Ginkgo biloba decreased oxide is an important signaling molecule the cannabis-induced increments in dopa- involved in neurotransmission and in main- mine. Studies have shown that most taining vascular tone via its vasodilator nootropics influencing cognitive mecha- properties. Nitric oxide can be also detri- nisms affect neurotransmitters levels in mental to neural tissue if generated in ex- brain. Vinpocetine through selective block- cess by inflammatory cytokines due to the ade of voltage-sensitive presynaptic Na+ action of inducible nitric oxide synthase channels inhibits the transporter-mediated (Moncada et al., 1991). In mice treated with release of monoamine neurotransmitters. the anticholinergic drug scopolamine so as The drug impairs the vesicular storage of to produce memory deficits, lipid peroxida- dopamine as well (Trejo et al., 2001). tion, though not significantly increased by Ginkgo biloba has been shown to increase scopolamine is decreased to normal value central dopamine, noradrenaline (Yoshitake by the highest dose of cannabis. Reduced et al., 2010) and 5-HT (Blecharz-Klin et al., glutathione which is decreased by scopola- 2009) levels. Piracetam increases dopamine mine is restored by cannabis administration. in cortex and striatum (Wustmann et al., The extract also lessened the increase in 1990; Stancheva and Alova, 1994). In one brain nitric oxide by scopolamine. Still study, piracetam abolished the amnestic ef- brain glucose decreased after cannabis ad- fect of 6-hydroxydopamine and restored to ministration under these conditions. These control values the noradrenaline level in the findings confirm observations in the saline- frontal cortex and hippocampus (Gouliaev treated mice. These results are intriguing in and Senning, 1994). Donepezil administra- view of the studies reporting neuroprotec- tion was associated with a significantly in- tive effects for certain cannabinoids under creased release of dopamine (Liang and experimental substances (Pazos et al., Tang, 2006), noradrenaline (Shearman et 2012). These antioxidant properties of the al., 2006) in cortex or hippocampus, but cannabis extract can be explained by the decreased extracellular serotonin levels fact that cannabis extract is not merely 9- (Shearman et al., 2006). THC, but rather a mixture of over 600 dif- The effect of cannabis on brain oxida- ferent chemical compounds. Cannabinoids, tive stress, an imbalance between free radi- a group of C21 terpenophenolic compounds cals generation and antioxidant defense uniquely produced by Cannabis sativa plant mechanisms is important in view of the ev- are considered to be the main biologically idence linking cellular damage arising from active constituents of the Cannabis sativa increased oxidative stress to neuronal de- plant, of which currently at least 70 are generation and decline in cognitive function known (Brenneisen, 2006). Other canna- associated with normal aging or caused by binoids such as cannabidiol (CBD), canna- different pathological states (Dröge, 2003; binol (CBN) and tetrahydrocannabivarin

207 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

(THCV) can result in different effects from compared with the cannabis. Piracetam re- those of 9-THC alone (Pertwee, 2008). sulted in significantly shorter latency com- Some cannabinoids were found to exert pared with vinpocetine over the 4 weeks of neuroprotective effects (Pazos et al., 2012). the study. The effect of piracetam was more The beneficial effects of cannabis extract evident during the second and third trials of on brain lipid peroxidation can also be as- the test. Moreover, only piracetam signifi- cribed to the flavonoids it contains. One cantly increased brain glucose compared important finding, however, was the de- with the cannabis-treated group. The drug creased brain glucose by cannabis admin- has been reported to reverse regional de- istration, thereby, impairing cerebral energy pressions in glucose in the rat metabolism. This impairment of brain ener- hippocampus after scopolamine treatment getics, can explain the effect of the extract (Piercey et al., 1987). When given to pa- on memory function. The oxidative status tients with Alzheimer's disease, piracetam of hepatic tissue was also examined. In con- significantly improved regional glucose trast to the effect of repeated cannabis ex- metabolism in most cortical areas (Heiss et tract on oxidative stress in brain, the extract al., 1988). Interestingly, GSH is increased did no affect lipid peroxidation or reduced by donepezil, vinpocetine and Ginkgo bi- glutathione levels in the liver tissue. loba. In other studies, vinpocetine and pi- The current study investigated the effect racetam increased GSH in different brain of piracetam, vinpocetine, Ginkgo biloba areas (Abdel-Salam et al., 2011). In vitro, extract or donepezil on the memory im- vinpocetine displayed scavenging activity pairment induced by cannabis extract in the at human therapeutic serum concentration water maze test. These drugs are widely (Horvath et al., 2002). Studies indicated an prescribed to enhance memory function due antioxidant effect for Ginkgo biloba ex- to mild cognitive impairment or Alzhei- tracts. In mice, Ginkgo biloba attenuated 1- mer's disease (McDaniel et al., 2003). Pi- methyl-4-phenyl-1,2,3,6-tetrahydropyridine racetam, vinpocetine, Ginkgo biloba or (MPTP)-induced neurodegeneration of the donepezil co-administered with cannabis nigrastriatal pathway, most likely due to an resulted in significantly shorter latencies inhibitory effect against oxidative stress compared with mice treated with only can- (Rojas et al., 2008). In vitro, Ginkgo biloba nabis, which indicated improved learning was able to block A (1–42)-induced cell and memory or in other words improve- apoptosis, reactive oxygen species accumu- ment of the cognitive impairing effects of lation and mitochondrial dysfunction (Shi et Cannabis sativa by these drugs. This effect al., 2009). of cannabis decreased by memory enhanc- In summary, this study examined can- ing drugs with piracetam resulting in the nabis induced memory deficits in the Mor- most-shorter latency compared with the ris water maze, using mice. Cannabis was cannabis. Biochemically, cannabis altered found to impair performance as measured the oxidative status of the brain with de- by the time taken to locate a submerged creased MDA, increased GSH, but de- platform compared to controls. The positive creased nitric oxide and glucose. In canna- control scopolamine also slowed perfor- bis-treated rats, the level of GSH in brain mance and added to the cannabis deficit showed further increase after vinpocetine when the two agents were tested together. and donepezil and was markedly elevated The impaired memory performance is likely after Ginkgo biloba. Piracetam restored the to involve decreased brain glucose availa- decrease in glucose and nitric oxide by can- bility as well as alterations in brain mono- nabis. Of the memory enhancing drugs used amine neurotransmitter levels. The cannabis in an attempt to counteract the effect of induced performance deficit was attenuated cannabis on memory performance, pirace- by the memory enhancing drugs, with pi- tam resulted in the most-shorter latency racetam resulting in the most-shorter laten-

208 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

cy compared with the cannabis and restor- Brenneisen R. Chemistry and analysis of ing the cannabis-induced decrease in brain phytocannabinoids and other cannabis con- glucose and nitric oxide. stituents. In: ElSohly MA (ed). Forensic science and medicine: marijuana and the cannabinoids (pp 17-49). Totowa, NJ: Hu- REFERENCES mana Press Inc., 2006. Abdel-Salam OME, Khadrawy YA, Salem NA, Sleem AA. Oxidative stress in a model Cascio MG, Gauson LA, Stevenson LA, of toxic demyelination in rat brain: The ef- Ross RA, Pertwee RG. Evidence that the fect of piracetam and vinpocetine. Neuro- plant cannabinoid cannabigerol is a highly chemical Res 2011;36:1062-72. potent alpha2-adrenoceptor agonist and moderately potent 5HT1A receptor antago- Abdel-Salam OME, El-Shamarka ME-S, nist. Br J Pharmacol 2010;159:129-41. Salem NA, Gaafar AE-DM. Effects of Cannabis sativa extract on - Chung HS, Harris A, Kristinsson JK, Ciulla induced catalepsy and oxidative stress in TA, Kagemann C, Ritch R. Ginkgo biloba the mice. EXCLI J 2012;11:45-58. extract increases ocular blood flow veloci- ty. J Ocul Pharmacol Ther 1999;15:233-40. Adams IB, Martin BR. Cannabis: pharma- cology and toxicology in animals and hu- Clausen A, Doctrow S, Baudry M. Preven- mans. Addiction 1996;91:1585-614. tion of cognitive deficits and brain oxida- tive stress with superoxide dismutase/cata- Ashton CH. Pharmacology and effects of lase mimetics in aged mice. Neurobiol Ag- cannabis: A brief review. Br J Psychiat ing 2010;31:425-33. 2001;178:101-6. Crowley LV. The Reitman-Frankel colori- Bäurle P, Suter A, Wormstall H. Safety and metric transaminase procedure in suspected effectiveness of a traditional ginkgo fresh myocardial infarction. Clin Chem 1967;13: plant extract - results from a clinical trial. 482–7. Forsch Komplementmed 2009;16:156-61. DeNoble VJ. Vinpocetine enhances retriev- Blecharz-Klin K, Piechal A, Joniec I, Pyr- al of a step-through passive avoidance re- zanowska J, Widy-Tyszkiewicz E. Pharma- sponse in rats. Pharmacol Biochem Behav cological and biochemical effects of Gink- 1987;26:183-6. go biloba extract on learning, memory con- solidation and motor activity in old rats. Dröge W. Oxidative stress and aging. Adv Acta Neurobiol Exp (Wars) 2009;69:217- Exp Med Biol 2003;543:191-200. 31. D'Souza DC, Perry E, MacDougall L, Am- Bossong MG, van Berckel BN, Boellaard merman Y, Cooper T, Wu YT et al. The R, Zuurman L, Schuit RC, Windhorst AD psychotomimetic effects of intravenous del- et al. Delta 9-tetrahydrocannabinol induces ta-9-tetrahydrocannabinol in healthy indi- dopamine release in the human striatum. viduals: implications for psychosis. Neuro- Neuropsychopharmacology 2009;34:759- psychopharmacology 2004;29:1558-72. 66.

209 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

Dunnett SB, Bensadoun J-C, Pask T, Gutteridge JMC. Lipid peroxidation and Brooks S. Assessment of motor impair- antioxidants as biomarkers of tissue dam- ments in transgenic mice. In: Crawley JN age. Clin Chem 1995;41:1819-28. (ed). Mouse behavioral phenotyping (pp 1- 13). Washington, DC: Society for Neuro- Halliwell B. Oxidative stress and neuro- science, 2003. degeneration: where are we now? J Neuro- chem 2006;97:1634-58. Ebrahimpour S, Tehranipour M, Behnam M, Kafaee M. Evaluating the effect of Harvey MA, Sellman JD, Porter RJ, Framp- aquatic extraction of Cannabis sativa seed ton CM. The relationship between non- on spatial memory consolidation in rats. acute adolescent cannabis use and cogni- Ann Gen Psychiatry 2010;9(Suppl 1): tion. Drug Rev 2007;26:309-19. S139. He Z, Liao Y, Zheng M, Zeng FD, Guo LJ. Egashira N, Manome N, Mishima K, Iwa- Piracetam improves cognitive deficits saki K, Oishi R, Fujiwara M. Involvement caused by chronic cerebral hypoperfusion of opioid system in cognitive deficits in- in rats. Cell Mol Neurobiol 2008;28:613- duced by ∆⁹-tetrahydrocannabinol in rats. 27. Psychopharmacology (Berlin) 2012;219: 1111-8. Heiss WD, Hebold I, Klinkhammer P, Zif- fling P, Szelies B, Pawlik G et al. Effect of Ellman GL. Tissue sulfhydryl groups. Arch piracetam on cerebral glucose metabolism Biochem 1959;82:70–7. in Alzheimer's disease as measured by posi- tron emission tomography. J Cereb Blood Fadda P, Robinson L, Fratta W, Pertwee Flow Metab 1988;8:613-7. RG, Riedel G. Differential effects of THC- or CBD-rich cannabis extracts on working Holinski S, Claus B, Alaaraj N, Dohmen memory in rats. Neuropharmacology 2004; PM, Kirilova K, Neumann K et al. Cerebro- 47:1170-9. protective effect of piracetam in patients undergoing coronary bypass surgery. Med Fadda P, Robinson L, Fratta W, Pertwee Sci Monit 2008;14:PI53-7. RG, Riedel G. Scopolamine and MK801- induced working memory deficits in rats Hollister LE. Cannabis. Acta Psychiatr are not reversed by CBD-rich cannabis ex- Scand (Suppl) 1988;345:108–18. tracts. Behav Brain Res 2006;168:307-11. Horváth S. The use of vinpocetine in chron- Frischknecht HR, Siegfried B, Schiller M, ic disorders caused by cerebral hypoperfu- Waser PG. Hashish extract impairs reten- sion. [Article in Hungarian]. Orv Hetil tion of defeat-induced submissive behavior 2001;42:383-9. in mice. Psychopharmacology (Berl) 1985; 86:270-3. Horvath B, Marton Z, Halmosi R, Alexy T, Szapary L, Vekasi J et al. In vitro antioxi- Giurgea C. The “nootropic” approach to the dant properties of pentoxifylline, piracetam, pharmacology of the integrative activity of and vinpocetine. Clin Neuropharmacol the brain. Cond Reflex 1973;8:108–15. 2002;25:37-42.

Gouliaev AH, Senning A. Piracetam and other structurally related nootropics. Brain Res Brain Res Rev 1994;19:180-222.

210 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

Keil U, Scherping I, Hauptmann S, Schues- Morgan CJ, Gardener C, Schafer G, Swan sel K, Eckert A, Müller WE. Piracetam im- S, Demarchi C, Freeman TP et al. Sub- proves mitochondrial dysfunction following chronic impact of cannabinoids in street oxidative stress. Br J Pharmacol 2006;147: cannabis on cognition, psychotic-like symp- 199-208. toms and psychological well-being. Psychol Med 2011, epub ehead of print. Kessler J, Thiel A, Karbe H, Heiss WD. Piracetam improves activated blood flow Morris R. Developments of a water-maze and facilitates rehabilitation of poststroke procedure for studying spatial learning in aphasic patients. Stroke 2000;31:2112-6. the rat. J Neurosci Methods 1984;11:47–60.

Kleijn J, Cremers TI, Hofland CM, Wester- Moshage H, Kok B, Huizenga JR. Nitrite ink BH. CB-1 receptors modulate the effect and nitrate determination in plasma: A crit- of the selective serotonin reuptake inhibitor, ical evaluation. Clin Chem 1995;41:892-6. citalopram on extracellular serotonin levels in the rat prefrontal cortex. Neurosci Res Müller WE, Eckert GP, Eckert A. Pirace- 2011;70:334-7. tam: novelty in a unique mode of action. Pharmacopsychiatry 1999;32(Suppl 1):2-9. Leuner K, Kurz C, Guidetti G, Orgogozo JM, Müller WE. Improved mitochondrial Muntoni AL, Pillolla G, Melis M, Perra S, function in brain aging and Alzheimer dis- Gessa GL, Pistis M. Cannabinoids modu- ease - the new mechanism of action of the late spontaneous neuronal activity and old metabolic enhancer piracetam. Front evoked inhibition of locus coeruleus nora- Neurosci 2010;4:pii:44. drenergic neurons. Eur J Neurosci 2006;23: 2385-94. Liang YQ, Tang XC. Comparative studies of , donepezil, and Murillo-Rodríguez E, Palomero-Rivero M, on brain acetylcholine, dopamine, norepi- Millán-Aldaco D, Mechoulam R, Drucker- nephrine, and 5-hydroxytryptamine levels Colín R. Effects on sleep and dopamine in freely-moving rats. Acta Pharmacol Sin levels of microdialysis perfusion of canna- 2006;27i:1127-36. bidiol into the lateral hypothalamus of rats. Life Sci 2011;88:504-11. McDaniel MA, Maier SF, Einstein GO. 'Brain-specific' nutrients: a memory cure? Musiał A, Bajda M, Malawska B. Recent Nutrition 2003;19:957-75. developments in cholinesterases inhibitors for Alzheimer's disease treatment. Curr Mechoulam R, Gaoni Y. The absolute con- Med Chem 2007;14:2654-79. figuration of delta-1-tetrahydrocannabinol, the major active constituent of hashish. Tet- Niyuhire F, Varvel SA, Martin BR, Licht- rahedron Lett 1967;12:1109-11. man AH. Exposure to marijuana smoke im- pairs memory retrieval in mice. J Pharma- Moncada S, Palmer RMJ, Higgs EA. Nitric col Exp Ther 2007;322:1067–75. oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43: Oropeza VC, Page ME, Van Bockstaele EJ. 109-42. Systemic administration of WIN 55,212-2 increases norepinephrine release in the rat frontal cortex. Brain Res 2005;1046:45-54.

211 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

Paget GE, Barnes JM. Toxicity tests. In: Rodrigues LC, Conti CL, Nakamura- Laurence DR, Bacharach AL (eds). Evalua- Palacios EM. Clozapine and SCH 23390 tion of drug activities. Pharmacometrics prevent the spatial working memory disrup- (p 161). London: Academic Press, 1964. tion induced by Δ(9)-THC administration into the medial prefrontal cortex. Brain Res Pazos MR, Cinquina V, Gómez A, Layunta 2011;1382:230-7. R, Santos M, Fernández-Ruiz J et al. Can- nabidiol administration after hypoxia- Rojas P, Serrano-García N, Mares-Sámano ischemia to newborn rats reduces long-term JJ, Medina-Campos ON, Pedraza-Chaverri brain injury and restores neurobehavioral J, Ogren SO. EGb761 protects against ni- function. Neuropharmacology 2012;63:776- grostriatal dopaminergic neurotoxicity in 1- 83. methyl-4-phenyl-1,2,3,6-tetrahydropyridine- induced Parkinsonism in mice: role of oxi- Pertwee RG. Pharmacological actions of dative stress. Eur J Neurosci 2008;28:41- cannabinoids. Handb Exp Pharmacol 2005; 50. 168:1-51. Roser P, Juckel G, Rentzsch J, Nadulski T, Pertwee RG. The diverse CB1 and CB2 re- Gallinat J, Stadelmann AM. Effects of ceptor pharmacology of three plant canna- acute oral Delta9-tetrahydrocannabinol and binoids: delta9-tetrahydrocannabinol, can- standardized cannabis extract on the audito- nabidiol and delta9-tetrahydrocannabivarin. ry P300 event-related potential in healthy Br J Pharmacol 2008;153:199-215. volunteers. Eur Neuropsychopharmacol 2008;18:569-77. Pertwee RG, Ross RA. Cannabinoid recep- tors and their ligands. Prostaglandins Leu- Ruiz-Larrea MB, Leal AM, Liza M, Lacort kot Essent Fatty Acids 2002;66:101-21. M, de Groot H. Antioxidant effects of es- tradiol and 2-hydroxyestradiol on iron- Piercey MF, Vogelsang GD, Franklin SR, induced lipid peroxidation of rat liver mi- Tang AH. Reversal of scopolamine-induced crosomes. Steroids 1994;59:383-8. amnesia and alterations in energy metabo- lism by the nootropic piracetam: implica- Russo EB, McPartland JM. Cannabis is tions regarding identification of brain struc- more than simply D9-tetrahydrocannabinol. tures involved in consolidation of memory Psychopharmacology 2003;165:431-2. traces. Brain Res 1987;424:1-9. Saleem S, Zhuang H, Biswal S, Christen Y, Pope HG Jr, Gruber AJ, Hudson JI, Huestis Doré S. Ginkgo biloba extract neuroprotec- MA, Yurgelun-Todd D. Neuropsychologi- tive action is dependent on heme oxygenase cal performance in long-term cannabis us- 1 in ischemic reperfusion brain injury. ers. Arch Gen Psychiatry 2001;58:909-15. Stroke 2008;39:3389-96.

Robledo P, Trigo JM, Panayi F, de la Torre Shearman E, Rossi S, Szasz B, Juranyi Z, R, Maldonado R. Behavioural and neuro- Fallon S, Pomara N et al. Changes in cere- chemical effects of combined MDMA and bral neurotransmitters and metabolites in- THC administration in mice. Psychophar- duced by acute donepezil and macology (Berl) 2007;195:255-64. administrations: a microdialysis study. Brain Res Bull 2006;69:204-13.

212 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

Shen EY, Ali SF, Meyer JS. Chronic ad- Stancheva SL, Alova LG. Biogenic mono- ministration of THC prevents the behavior- amine uptake by rat brain synaptosomes al effects of intermittent adolescent MDMA during aging. Effects of nootropic drugs. administration and attenuates MDMA- Gen Pharmacol 1994;25:981-7. induced hyperthermia and neurotoxicity in rats. Neuropharmacology 2011; 61:1183- Svíženská I, Dubový P, Šulcová A. Canna- 92. binoid receptors 1 and 2 (CB1 and CB2), their distribution, ligands and. Pharmacolo- Shi C, Zhao L, Zhu B, Li Q, Yew DT, Yao gy, Biochem Behav 2008;90:501–11. Z et al. Protective effects of Ginkgo biloba extract (EGb761) and its constituents quer- Takuma K, Hoshina Y, Arai S, Himeno Y, cetin and ginkgolide B against beta-amyloid Matsuo A, Funatsu Y et al. Ginkgo biloba peptide-induced toxicity in SH-SY5Y cells. extract EGb 761 attenuates hippocampal Chem Biol Interact 2009;181:115-23. neuronal loss and cognitive dysfunction re- sulting from chronic restraint stress in ovar- Singh V, Singh SP, Chan K. Review and iectomized rats. Neuroscience 2007;149: meta-analysis of usage of ginkgo as an ad- 256-62. junct therapy in chronic schizophrenia. Int J Neuropsychopharmacol 2010;13:257-71. Trejo F, Nekrassov V, Sitges M. Character- ization of vinpocetine effects on DA and Sitges M, Chiu LM, Nekrassov V. Single DOPAC release in striatal isolated nerve and combined effects of carbamazepine and endings. Brain Res 2001;909:59-67. vinpocetine on depolarization-induced changes in Na+, Ca2+ and glutamate re- Trinder P. Determination of glucose in lease in hippocampal isolated nerve end- blood using glucose oxidase with an alter- ings.Neurochem Int 2006;49:55-61. native oxygen acceptor. Ann Clin Biochem 1969;6:24-5. Smith JW, Evans T, Costall B, Smythe JW. Thyroid hormones, brain function and cog- Turner JC, Mahlberg PG. Separation of ac- nition. A brief review. Neurosci Biobehav id and neutral cannabinoids in Cannabis Rev 2002;26:45–60. sativa L. using HPLC. In: Agurell S, Dew- ey WL, Willette RE (eds). The canna- Solowij N, Battisti R. The chronic effects of binoids: chemical, pharmacologic and ther- cannabis on memory in humans: a review. apeutic aspects (pp 79-88). Orlando, FL: Curr Drug Abuse Rev 2008;1:81-98. Academic Press, 1984.

Solowij N, Stephens RS, Roffman RA, Ba- Valikovics A. Investigation of the effect of bor T, Kadden R, Miller M et al. Cognitive vinpocetine on cerebral blood flow and functioning of long-term heavy cannabis cognitive functions. [Article in Hungarian]. users seeking treatment. JAMA 2002;287: Ideggyogy Sz 2007;60:301-10. 1123-31. van Staveren WC, Markerink-van Ittersum Solowij N, Jones KA, Rozman ME, Davis M, Steinbusch HW, de Vente J. The effects SM, Ciarrochi J, Heaven PC et al. Verbal of phosphodiesterase inhibition on cyclic learning and memory in adolescent canna- GMP and cyclic AMP accumulation in the bis users, alcohol users and non-users. Psy- hippocampus of the rat. Brain Res 2001; chopharmacology (Berl) 2011;216:131-44. 888:275-86.

213 EXCLI Journal 2013;12:193-214 – ISSN 1611-2156 Received: February 13, 2013, accepted: February 27, 2013, published: March 12, 2013

Vas A, Gulyás B, Szabó Z, Bönöczk P, Wustmann C, Blaschke M, Rudolph E, Csiba L, Kiss B et al. Clinical and non- Fischer HD, Schmidt J. Influence of nootro- clinical investigations using positron emis- pic drugs on the age-dependent potassium- sion tomography, near infrared spectrosco- coupling of transmitter release. Biomed py and transcranial Doppler methods on the Biochim Acta 1990;49:619-24. neuroprotective drug vinpocetine: a sum- mary of evidences. J Neurol Sci 2002;203- Yoshitake T, Yoshitake S, Kehr J. The 4:259-62. Ginkgo biloba extract EGb 761(R) and its main constituent flavonoids and ginkgo- Waegemans T, Wilsher CR, Danniau A, lides increase extracellular dopamine levels Ferris SH, Kurz A, Winblad B. Clinical ef- in the rat prefrontal cortex. Br J Pharmacol ficacy of piracetam in cognitive impair- 2010;159:659-68. ment: a meta-analysis. Dement Geriatr Cogn Disord 2002;13:217-24. Zavitsanou K, Wang H, Dalton VS, Nguyen V. Cannabinoid administration increases Walesiuk A, Braszko JJ. Preventive action 5HT1A receptor binding and mRNA ex- of Ginkgo biloba in stress- and corti- pression in the hippocampus of adult but costerone-induced impairment of spatial not adolescent rats. Neuroscience 2010;169: memory in rats. Phytomedicine 2009;16:40- 315-24. 6. Zhou C, Huang Y, Przedborski S. Oxidative Whitehouse PJY, Price DL, Struble RG, stress in Parkinson's disease: a mechanism Clark AW, Coyle JT, DeLong MR. Alz- of pathogenic and therapeutic significance. heimer’s disease and senile : loss Ann N Y Acad Sci 2008;1147:93-104. of neurons in the basal forebrain. Science 1982;215:1237-9.

214