The Ginkgo biloba extract EGb 761® and its main constituent flavonoids and ginkgolides increase extracellular dopamine levels in the rat prefrontal cortex

Abstract

Background and purpose:

Experimental and clinical data suggest that extracts of Ginkgo biloba improve cognitive function. However, the neurochemical correlates of these effects are not yet fully clarified. The purpose of this study was to examine the effects of acute and repeated oral administration of the standardized extract EGb 761® on extracellular levels of dopamine, noradrenaline and serotonin (5-HT), and the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the prefrontal cortex (PFC) and striatum of conscious rats.

Experimental approach:

Monoamines and their metabolites were monitored by the use of microdialysis sampling and HPLC with electrochemical or fluorescence detection.

Key results:

A single oral dose of EGb 761 (100 mg·kg⁻¹) had no effect on monoamine levels. However, following chronic (100 mg·kg⁻¹/14 days/once daily) treatment, the same dose significantly increased extracellular dopamine and noradrenaline levels, while 5-HT levels were unaffected. Chronic treatment with EGb 761 showed dose-dependent increases in frontocortical dopamine levels and, to a lesser extent, in the striatum. The extracellular levels of HVA and DOPAC were not affected by either acute or repeated doses. Treatment with the main constituents of EGb 761 revealed that the increase in dopamine levels was mostly caused by the flavonol glycosides and ginkgolide fractions, whereas bilobalide treatment was without effect.

Conclusions and implications:

The present results demonstrate that chronic but not acute treatment with EGb 761 increased dopaminergic transmission in the PFC. This finding may be one of the mechanisms underlying the reported effects of G. biloba in improving cognitive function.

Introduction

Extracts of Ginkgo biloba (EGb 761®) have been shown to exert beneficial effects as cognitive enhancers in ageing, anti-stress agents and in therapy of age-related neurological disorders such as Alzheimer's disease (see DeFeudis and Drieu, 2000; DeFeudis, 2003; DeKosky and Furberg, 2008). A number of clinical studies have demonstrated that extracts of G. biloba, and particularly the standardized extract EGb 761, could ameliorate cognitive defects associated with mild to moderate Alzheimer's disease (Andrieu et al., 2003; Kanowski and Hoerr, 2003; Mazza et al., 2006; Napryeyenko et al., 2007; Scripnikov et al., 2007; Dodge et al., 2008). However, a recent Cochrane review on published randomized, double-blind, clinical trials has found that the use of G. biloba extracts was safe but the evidence for a significant benefit in people with dementia or cognitive impairment was inconsistent and unconvincing (Birks and Grimley Evans, 2007). In addition, a randomized, double-blind, placebo-controlled clinical study on 3069 volunteers aged 75 years or older receiving EGb 761 at 120 mg twice a day did not confirm the therapeutic efficacy of EGb 761 in reducing the overall incidence rate of dementia or Alzheimer's disease in elderly individuals with normal cognition or those with mild cognitive impairment (DeKosky et al., 2008). A similar long-lasting clinical study initiated in Europe is still in progress (Vellas et al., 2006).

In animal experiments, G. biloba extracts were shown to improve spatial memory deficits in aged rats (Wang et al., 2006; Blecharz-Klin et al., 2009) and a transgenic mouse model of Alzheimer's disease (Stackman et al., 2003) as well as to improve acquisition of working memory in rats (Satvat and Mallet, 2009). However, the precise neurochemical correlates of these behavioural effects of G. biloba are not known. Extracts of G. biloba exhibit potent antioxidant activity, scavenging various reactive oxygen species, including superoxide, peroxy and hydroxyl radicals (see DeFeudis and Drieu, 2000; Ahlemeyer and Krieglstein, 2003). G. biloba has been reported to enhance the activities of superoxide dismutase and catalase, and to decrease lipid peroxidation in the striatum, substantia nigra and hippocampus. The neuroprotective effects of G. biloba were demonstrated in animal models of Parkinson's disease, both in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice (Wu and Zhu, 1999; Rojas et al., 2008) and 6-hydroxydopamine-lesioned rats (Ahmad et al., 2005). In addition, the extracts of G. biloba were shown to reduce the activity of both forms of MAO-A and MAO-B in the rat (White et al., 1996), in the aged mice (Pardon et al., 2000) and mouse brain in vitro (Wu and Zhu, 1999). The activity of MAO is closely associated with mitochondrial function and production of hydrogen peroxide as a by-product of oxidation of monoamines. Accordingly, the proposed neuroprotective effects of G. biloba in Parkinson's and Alzheimer's diseases were attributed to the ability of Ginkgo extracts to stabilize mitochondrial function (Abdel-Kader et al., 2007).

Acute administration of G. biloba extracts was reported to reduce stress-induced increases in whole brain levels of catecholamines and serotonin (5-HT) in the rat (Shah et al., 2003). Similarly, chronic administration of EGb 761 enhanced copulatory behaviour and reduced serum prolactin levels in male rats, suggesting involvement of the dopaminergic system in the effects of G. biloba (Yeh et al., 2008). Based on these reports, we have hypothesized that there could be a causal link between the central protective effects of G. biloba and monoaminergic neurotransmission. Strong evidence exists for the role of prefrontal cortical dopamine and its receptors in modulating prefrontal cortical neurons, which are commonly viewed as a basis for cognitive operations (for review, see Goldman-Rakic et al., 2000; Mehta and Riedel, 2006; Phillips et al., 2008). Indeed, several studies have shown enhanced dopamine release in the rat prefrontal cortex (PFC) during various cognitive tasks (Rossetti and Carboni, 2005; Phillips et al., 2008). However, there are no data available demonstrating possible in vivo effects of G. biloba extracts on monoamine neurotransmitters in the brain areas implicated in regulating cognitive function, motivation and mood.

The purpose of the present study was to examine whether the acute and sub-chronic (14 days) daily administration of G. biloba extract and its main constituents, flavonol glycosides, ginkgolides and bilobalide, could affect basal extracellular levels of monoamines dopamine, noradrenaline and 5-HT monitored by microdialysis in the PFC and striatum of awake rats. Some preliminary data of this study have been reported at Gesellschaft für Arzneipflanzenforschung 2006 – International Congress and 54th Annual Meeting of the Society for Medicinal Plant Research, 29 August–2 September 2006, Helsinki, Finland.

Methods

Animals

All animal care and experimental procedures were approved by the local ethical committee following the directives of the ‘Principles of Laboratory Animal Care’ (National Institute of Health publication no. 8023) and the Council of the European Communities (86/809/EEC). Male Sprague-Dawley rats (weighing 250–350 g, total number of 75 rats) were used in the study. The rats (three animals/cage) were maintained on a 12 h light–dark cycle (light at 7 am), room temperature of 23 ± 2°C and humidity of 55–65%. All efforts were made to minimize animal suffering and the amount of animals used for the study.

Surgery and microdialysis experiments

The microdialysis experiments were carried out on conscious rats following the protocol described elsewhere (Osborne et al., 1990; Kehr, 1999; Kehr and Yoshitake, 2006). Fourteen days before the microdialysis experiment, the rats were anaesthetized with isoflurane (Forene®, Abbott Laboratories, Abbott Park, IL, USA) using a Univentor 400 anaesthesia unit (AgnThos, Lidingö, Sweden) and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) in a flat skull position with the incisor bar set to −3.2 mm. The body temperature of the rat was controlled by a rectal thermometer and maintained at +37°C using a CMA/105 temperature controller (CMA/Microdialysis, Stockholm, Sweden). A mid-scalp incision of 2–3 cm was made, and the flaps were kept open with haemostat forceps. After exposing the skull, a hole for a probe and two holes for the fixing screws were made using a fine trephine drill. The guide cannula for a microdialysis probe (Eicom Corp., Kyoto, Japan) was implanted into the PFC (AP +3.2 mm, L −0.5 mm, V −1.1 mm; from the bregma and the dural surface, according to the stereotaxic atlas of Paxinos and Watson, 1997). In a separate group of rats, the guide cannula was implanted into the striatum at the following coordinates: AP +0.2 mm, L −3.0 mm, V −3.5 mm. The guide cannula was fixed firmly to the skull surface using dental cement. Following 14 days of recovery and repeated daily administration of the Ginkgo extract or carboxymethylcellulose (CMC) suspension alone, a microdialysis probe (Eicom A-I; 0.22 mm o.d., 2 mm membrane length with 50 000 Da cut-off) was inserted into the guide cannula of the conscious rat. A typical placement of the guide cannula and the microdialysis probe in the PFC is illustrated in Figure 1. The probe was perfused with Ringer's solution (NaCl, 147 mM; KCl, 4 mM; CaCl₂, 2.3 mM) at a flow rate of 1 µL·min⁻¹. After the initial stabilization period of 2–3 h, the microdialysis samples were collected in 20 min intervals. The first four samples were used for estimation of basal levels of dopamine, noradrenaline, 5-HT, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Then, one dose of EGb 761 suspension in 0.3% w/v CMC or only CMC (vehicle-treated group) was given (p.o.), and the fractions were collected for a further 180 min.

Figure 1.

A schematic illustration of the placement of the guide cannula and the microdialysis probe in the rat prefrontal cortex. The membrane of the probe was placed in the cingulate cortex, area 1 (Cg1) and prelimbic cortex (PrL); the other marked areas are secondary motor cortex (M2) and infralimbic cortex (IL). Adapted from Paxinos and Watson (1997).

At the end of the experiment, the animals were killed by CO₂ inhalation and dislocation of the neck. The brains were removed and examined for correct placement of the probe (the probe track) in the rat brain.

HPLC determination of monoamines and acidic metabolites

A highly sensitive liquid chromatographic method, based on precolumn derivatization and fluorescence detection, allowing simultaneous determination of 5-HT, noradrenaline and dopamine in brain microdialysis samples, was used following the method described elsewhere (Yoshitake et al., 2004a,b;) Briefly, 5-HT, noradrenaline and dopamine were derivatized with benzylamine and 1,2-diphenylethylenediamine in the presence of potassium hexacyanoferrate(III) and glycine, which yielded highly fluorescent and stable benzoxazoles. The derivatized monoamines were separated on a microbore column (150 × 1.0 mm i.d., packed with C18 silica, 5 µm) within 60 min. The mobile phase consisted of a mixture (32:68, v/v) of acetonitrile and Britton-Robinson buffer (composition: 25 mM boric acid, 25 mM acetic acid, 25 mM phosphoric acid and pH adjusted to 7.2 with 125 mM NaOH) containing 5 mM Na₂EDTA and 5 mM octanesulfonic acid sodium salt. The detection limits (signal-to-noise ratio of 3) for 5-HT, noradrenaline and dopamine were 76, 42 and 95 amol·10 µL^–1 injected on-column respectively.

Dopamine and its metabolites DOPAC and HVA were determined by ion-pair reversed-phase column liquid chromatography with electrochemical detection as described elsewhere (Kehr and Yoshitake, 2006). The HPLC system included a pump, a degasser and an amperometric detector (HTEC-500, Eicom Corp.). Dopamine and metabolites were separated on a 150 × 2.1 i.d. mm column (CA-5-ODS, Eicom Corp.). The mobile phase consisted of 0.1 M citric acid buffer at pH 3.5, 0.13 mM Na₂EDTA, 2.3 mM sodium-1-octanesulfonate and 20% (v/v) methanol. The detection limit (signal-to-noise ratio = 3) for dopamine was 5·10⁻¹¹ M, that is, 0.5 fmol in 10 µL injected onto the column respectively.

Data presentation and statistical analysis

The basal concentrations of monoamines and metabolites were expressed as mean ± standard error of the mean (SEM) of four fractions collected at −60 to 0 min from the rats in each treated group. These mean values were taken as 100%, and all the following values were related to these averaged levels. The overall effects of EGb 761 on dopamine levels were also expressed as the relative area under the curves (AUC) calculated as the mean percentage increase in extracellular dopamine levels over the entire 180 min post-treatment sampling period (nine samples) and compared with the corresponding value of the vehicle-treated group. Statistical analysis was performed using Prism 5 (GraphPad Software, San Diego, CA, USA) statistical software. Mean basal levels were compared by the use of one-way ANOVA followed by Newman–Keuls multiple comparison test. Differences between the groups and treatments were analysed by repeated-measures two-way ANOVA followed by Bonferroni's test.

Materials

The monoamines noradrenaline, 5-HT and dopamine, and the metabolites DOPAC and HVA, as well as all chemicals for mobile phase and reagent preparations, were purchased from Sigma-Aldrich (St. Louis, MO, USA), Wako Pure Chemical Co. (Osaka, Japan) or from Kisida Chemical Co. (Tokyo, Japan). Deionized and distilled water, purified with a Barnstead EASYpure RF (Hansen Co., Hyogo, Japan) system, was used for all aqueous solutions. Benzylamine hydrochloride was obtained from Tokyo Kasei Kogyo Co. (Tokyo, Japan) and was used after purification by recrystallization with absolute ethanol. 1,2-Diphenylethylenediamine was purchased from Tosoh (Tokyo, Japan). Standard solutions of monoamines were prepared in water and kept frozen (−20°C) in amber-coloured test tubes.

The standardized extract of G. biloba (EGb 761®) and its main constituents, flavonol glycosides, ginkgolides and bilobalide, were kindly provided by Willmar-Schwabe (Karlsruhe, Germany). The extract contains 24% flavonol glycosides, 6% terpene lactones and trace amounts of other substances including proanthocyanidins and organic acids (DeFeudis and Drieu, 2000; DeFeudis, 2003; Chan et al., 2007). The flavonol constituents are essentially flavonol-O-glycosides quercetin, kaempferol or isorhamnetin conjugated to D-glucose, L-rhamnose or glucorhamnose. The terpene trilactones are characteristic of G. biloba. Of those ginkgolides, A, B, C and J account for 3.1%, and bilobalide for 2.9% of the total extract. The dried extract or the constituents were resuspended in 0.3% w/v CMC (Sigma-Aldrich). Ginkgo suspension (EGb 761) was given orally (p.o.) via a gastro-oesophageal gavage, once daily for 14 days at doses of 30, 100 or 300 mg·kg⁻¹. A separate group of rats received only the CMC solution (control group). The dose of each constituent fraction given p.o., once daily for 14 days, corresponded to its content in 300 mg·kg⁻¹ of the EGb 761 extract: flavonol glycosides 72 mg·kg⁻¹, ginkgolides 9.3 mg·kg⁻¹ and bilobalide 8.7 mg·kg⁻¹.

The nomenclature of drugs and molecular targets conforms to the ‘Guide to Receptors and Channels’ (Alexander et al., 2008).

Results

Basal extracellular levels of dopamine, noradrenaline, 5-HT, and metabolites DOPAC and HVA in the rat PFC

The basal concentrations (calculated in 10⁻¹⁰ M, i.e. fmol in 10 µL injected onto the column and expressed as mean ± SEM, n= 5) of monoamines, as well as the dopamine metabolites, DOPAC and HVA, in the dialysates from the PFC of conscious rats in all treated groups are summarized in Table 1. The basal levels of monoamines and metabolites did not significantly differ within the respective treated groups and analytical methods used.

Table 1.

Basal concentrations of dopamine (DA), noradrenaline (NA), 5-HT, and the metabolites DOPAC and HVA in the prefrontal cortex of vehicle- and EGb 761-treated rats

1.Analytical method: HPLC with electrochemical detection
Treatment (EGb 761)		Basal concentrations (fmol in 10 µL, i.e. 10−10 M)
Dose (mg·kg−1)	Dosing schedule	DA	DOPAC	HVA
Vehicle	Once	6.19 ± 0.39	987.7 ± 69.9	1489.2 ± 92.4
100	Once	5.92 ± 0.60	1091.3 ± 79.7	1527.3 ± 105.5
Vehicle	Daily, 14 days	5.84 ± 0.47	1022.2 ± 59.1	1662.3 ± 86.2
30	Daily, 14 days	5.63 ± 0.50	–	–
100	Daily, 14 days	6.55 ± 0.48	–	–
300	Daily, 14 days	7.61 ± 0.59	1280.0 ± 96.9	1784.4 ± 102.1
Treatment (constituents)
Vehicle	Daily, 14 days	4.54 ± 0.53	–	–
Ginkgolides	Daily, 14 days	4.33 ± 0.41	–	–
Bilobalide	Daily, 14 days	4.71 ± 0.53	–	–
Flavonoids	Daily, 14 days	4.34 ± 0.59	–	–
2.Analytical method: HPLC with fluorescence detection
Treatment (EGb 761)		DA	NA	5-HT
Vehicle	Daily, 14 days	4.24 ± 0.42	3.41 ± 0.51	3.24 ± 0.63
100	Daily, 14 days	5.13 ± 0.46	4.01 ± 0.54	3.41 ± 0.62

The concentrations are expressed as mean ± SEM, n= 5 rats, where the basal value for each rat was calculated as the mean of four microdialysis samples collected at −60 to 0 min before the drug or vehicle administration. The basal levels of monoamines and metabolites did not significantly differ between the respective treated groups and analytical methods used.

DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid.

Effects of a single dose of EGb 761 on dopamine and metabolites in the rat PFC

A single dose of EGb 761 at a dose of 100 mg·kg⁻¹ p.o. had no significant effect on basal extracellular levels of dopamine, DOPAC and HVA during the next 180 min, as shown in Figure 2. Similarly, the basal levels of noradrenaline and 5-HT were not affected by a single does of EGb 761 (data not shown).

Figure 2.

Effect of single administration of EGb 761 (100 mg·kg⁻¹ p.o.) on the extracellular levels of dopamine (DA) and the metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the prefrontal cortex (PFC) of conscious rats. Administration of EGb 761 had no effect on the levels of dopamine and its metabolites.

Effects of sub-chronic administration of EGb 761 on monoamines and metabolites in the rat PFC

In the first part of this experiment, the monoamines dopamine, noradrenaline and 5-HT were determined by HPLC with fluorescence detection following precolumn derivatization. The basal concentrations of monoamines were not significantly different between the vehicle- and EGb 761-treated groups, as listed in Table 1. A single dose of EGb 761 (100 mg·kg⁻¹ p.o.) following 2 weeks daily pretreatment with EGb 761 (100 mg·kg⁻¹ p.o.) now significantly increased extracellular dopamine levels, within 40–180 min, reaching a maximal value at 140 min after the dose (Figure 3). The noradrenaline levels increased to a lesser extent within 80–180 min, with maximal value reached at 120 min, whereas EGb 761 showed no significant effect on concentrations of extracellular 5-HT. There was a significant difference between the groups both for the treatment [F_(5,288)= 48.80; P < 0.0001] and the time points [F_(12,288)= 21.42; P < 0.0001]. The time courses for each monoamine following chronic EGb 761 administration are shown in Figure 3.

Figure 3.

Effects of sub-chronic administration of EGb 761 (100 mg·kg⁻¹ p.o. for 14 days, once daily) on extracellular levels of dopamine (DA), noradrenaline (NA) and 5-HT in the rat prefrontal cortex (PFC). Administration of a single dose of EGb 761 (100 mg·kg⁻¹ p.o.) following 2 weeks daily pretreatment with EGb 761 caused a significant increase in extracellular dopamine levels within 40–180 min (***P < 0.001), whereas the NA levels increased to a lesser extent (*P < 0.05; ***P < 0.001) within 80–180 min after the single dose of EGb 761.

In the next experiment, separate groups of rats were treated for 14 days with EGb 761 at three doses (30, 100 and 300 mg·kg⁻¹ p.o. once daily), and following the last EGb challenge on day 14, the dopamine, DOPAC and HVA levels in the microdialysates were determined by the use of HPLC with electrochemical detection. Basal dopamine levels in this group did not significantly differ from those receiving only single vehicle, or EGb 761 doses, whether measured by HPLC with electrochemical detection or HPLC with fluorescence detection (Table 1). Further, this experiment confirmed the previous results showing the significant [F_(3,192)= 50.43; P < 0.0001] increase in extracellular dopamine levels that lasted for at least 3 h after the EGb 761 dose (Figure 4A). Interestingly, in spite of increased dopamine levels, the concentrations of metabolites DOPAC and HVA were unchanged even following sub-chronic treatment with EGb 761 at the highest dose (data not shown). The low dose (30 mg·kg⁻¹) of EGb 761 extract had no effect on basal extracellular levels of dopamine, whereas both 100 mg·kg⁻¹ and 300 mg·kg⁻¹ EGb 761 doses caused marked and rapid increases in extracellular levels of dopamine from 40 and 60 min respectively. Following the 100 mg·kg⁻¹ treatment, the dopamine levels gradually increased to a maximal value at 160 min, whereas the dose of 300 mg·kg⁻¹ caused a faster and sharper increase to a maximal value at 100 min. The overall effect expressed as relative AUC_{(0–180 min)} values for dopamine following vehicle, acute and sub-chronic treatments with three doses of EGb 761 is shown in Figure 4B. As seen, chronic EGb 761 caused dose-dependent increases in AUCs for the 100 mg·kg⁻¹ and 300 mg·kg⁻¹ EGb 761 doses.

Figure 4.

Sub-chronic (14 days) treatment with EGb 761 at three different doses caused dose-dependent and long-lasting increases in the extracellular dopamine (DA) levels in the rat prefrontal cortex as shown in (A) the upper panel for the time courses (**P < 0.01; ***P < 0.001) and (B) the lower panel summarizing the relative (rel.) area under the curve (AUC) values. The AUCs were significantly higher (***P < 0.001) for the 100 and 300 mg·kg⁻¹ doses compared with the vehicle-treated group.

Effects of sub-chronic administration of EGb 761 on dopamine in the rat striatum

The basal concentration (expressed in 10⁻¹⁰ M) of dopamine in the dialysates from the striatum of conscious rats was 45.8 ± 5.5 (mean ± SEM, n= 5; vehicle-treated group). Following repeated (14 days) oral administration of 100 mg·kg⁻¹ and 300 mg·kg⁻¹ of EGb 761, the corresponding basal levels of dopamine were 47.9 ± 6.3 and 48.8 ± 7.1 respectively. A single dose of 100 mg·kg⁻¹ given to rats treated with this dose for 14 days caused only a minor transient increase in dopamine levels at 80 min and at 100 min (P < 0.01), whereas a single dose of 300 mg·kg⁻¹ following 14 days administration of EGb 761 at this dose caused a significant [F_(2,156)= 15.27; P < 0.001] increase in extracellular dopamine levels, reaching a maximal value at 120 min after administration (Figure 5). The corresponding AUC_{(0–180 min)} values for the lower and higher doses were 107.4 ± 2.4% and 119.6 ± 3.6% (P < 0.001 compared with the vehicle-treated group, and P < 0.01 compared with the 100 mg·kg⁻¹-treated group), respectively, suggesting a dose-dependent effect.

Figure 5.

Sub-chronic (14 days) treatment with EGb 761 at doses of 100 and 300 mg·kg⁻¹ p.o., respectively, caused only minor, but yet significant, (*P < 0.05; **P < 0.01; ***P < 0.001) increases in extracellular dopamine (DA) levels in the striatum of awake rats.

Effects of sub-chronic administration of EGb 761 constituents on dopamine levels in the rat PFC

The basal concentrations of dopamine in the dialysates from the PFC of conscious rats treated with EGb 761 constituents are shown in Table 1. Repeated (14 days) administration of flavonol glycosides (flavanoids) caused a significant [F_(3,192)= 49.17; P < 0.001] increase in extracellular levels of dopamine starting at 40 min and increasing to a maximal value at 120 min compared with the vehicle-treated group (Figure 6). Correspondingly, repeated administration of ginkgolides caused only a moderate but significant increase in dopamine levels within 100–180 min, reaching a maximal value at 140 min, and treatment with bilobalide extract had no effect on extracellular levels of dopamine. The calculated AUC_{(0–180 min)} values for bilobalide, ginkgolide and flavonoid groups compared with the vehicle-treaded group were 102.8 ± 0.7%, 110.1 ± 1.0% and 127.3 ± 3.0% respectively. Thus, the relative AUC_{(0–180 min)} calculated as the sum of the increments of all three constituents was 40.2% above the AUC of the vehicle-treated group, which can be compared with the corresponding value of 52.9% observed for the treatment with 300 mg·kg⁻¹ of the EGb 761 extract (shown in Figure 4B).

Figure 6.

Effects of sub-chronic administration of flavonol glycosides, ginkgolides or bilobalide, the major constituents of the EGb 761 extract, on extracellular levels of dopamine (DA) in the rat prefrontal cortex. Administration of flavonol glycosides (flavanoids; 72 mg·kg⁻¹ p.o.) following 2 weeks daily treatment caused a significant increase in extracellular DA levels within 40–180 min (***P < 0.001), whereas ginkgolides increased the DA levels to a lesser extent (*P < 0.05; **P < 0.01; ***P < 0.001) within 100–180 min after the administration.

Discussion

The purpose of the present study was to examine the effects of acute and chronic treatment with the extract of G. biloba (EGb 761) on the extracellular levels of the monoamines dopamine, noradrenaline and 5-HT in the PFC of conscious rats. In addition, the three main constituents of the extract, flavonol glycosides, ginkgolides and bilobalide, were tested in order to evaluate their role in mediating the effects of repeated administration of the EGb 761 extract on dopamine signalling in the rat PFC. The rationale for monitoring the levels of monoamines was based on the earlier studies suggesting that G. biloba reduced the activity of both MAO-A and MAO-B forms in the rat (White et al., 1996) and mouse (Wu and Zhu, 1999; Pardon et al., 2000) brains. We have applied the microdialysis technique in combination with highly sensitive liquid chromatographic methods for determination of the extracellular levels of dopamine, noradrenaline and 5-HT, as well as the acidic metabolites DOPAC and HVA, in the PFC of conscious rats. A single oral administration of EGb 761 had no effect on the levels of basal extracellular monoamines and metabolites. Likewise, an acute dose of G. biloba extract had no effect on brain tissue levels of catecholamines or 5-HT (Shah et al., 2003). However, the present data show that a single dose of EGb 761, following a sub-chronic daily treatment with the same dose for 14 days, caused a marked elevation of extracellular levels of dopamine and a moderate increase in noradrenaline levels, but had no effect on extracellular 5-HT values in the rat PFC. Interestingly, the levels of DOPAC and HVA were not affected by any dose and treatment. In the subsequent studies, the sub-chronic administration of EGb 761 at three different doses revealed a dose-dependent and long-lasting (>3 h) effect on increasing the dopamine levels in the rat PFC and, to a lesser extent, in the striatum. The finding that striatal dopamine levels increased only marginally is in good agreement with our behavioural observations (Yoshitake et al., 2004a), where no significant increases in locomotor activity were recorded. The EGb 761 doses of 100 and 300 mg·kg⁻¹ used in this study are considered to be relevant to the currently recommended clinical doses of 240 mg·kg⁻¹ daily (Schwabe, Tebonin konzent, Fachinformation, 2008).

A major finding of the study is that the sub-chronic treatment with EGb 761 preferentially increased extracellular dopamine in the PFC of conscious rats. In addition, the same treatment with the three main constituents of EGb 761, at the doses corresponding to their respective contents in the EGb 761 extract, revealed that flavonol glycosides and, to a lesser extent, the ginkgolide fractions were the main components contributing to the observed effects of the EGb 761 extract on the dopamine levels. The sum of the overall effects of the three major components on the increase of dopamine levels was comparable with that of chronic treatment with the corresponding dose of the whole EGb 761 extract. This finding indicates that the effects of the EGb 761 extract, at least those observed for the dopamine levels in the PFC, are simply additive, that is, they reflect a sum of contributions of active constituents, rather than a synergistic potentiation effect, triggered by the individual components, or a ‘polyvalent’ action as often proposed for the mechanisms of action of herbal extracts, including EGb 761 (DeFeudis and Drieu, 2000).

Several studies have indicated that G. biloba extracts may affect the brain dopamine system and dopamine-mediated functions (Shah et al., 2003; Szasz et al., 2008; Yeh et al., 2008; Fehske et al., 2009). However, this is the first report linking the effects of EGb 761 to the PFC, that is, the brain area that is neuroanatomically relevant to learning and memory processing. Previous reports have suggested that G. biloba extracts could increase monoaminergic function via inhibition of MAO activity (White et al., 1996; Wu and Zhu, 1999; Pardon et al., 2000). However, chronic (14 days) daily treatment with EGb 761 (100 mg·kg⁻¹, p.o.) had no effect on MAO-A or MAO-B activities in the homogenates of mice brains (Fehske et al., 2009). Our findings on extracellular levels of monoamines and metabolites following repeated treatment with EGb 761 support the conclusions of the latter study.

Thus, chronic, but not acute, administration of EGb 761 increased dopamine levels. However, the concentrations of noradrenaline were increased only marginally, while the levels of 5-HT remained unaltered, and there was no effect on the acidic metabolites DOPAC and HVA. This differs from the effects of MAO inhibitors, which increase extracellular levels of all three monoamines and decrease the levels of their respective metabolites, even after a single injection. For example, in our previous study (Yoshitake et al., 2004a), we have shown that systemic administration of MAO-A/B inhibitor phenelzine (5 mg·kg⁻¹ i.p.) caused a gradual increase in extracellular dopamine, noradrenaline and 5-HT levels, which at 120 min reached peak values of 171, 121 and 140% of the pre-drug levels respectively. The corresponding extracellular levels of DOPAC and 5-hydroxyindoleacetic acid (5-HIAA) decreased to 41 and 83% respectively (Yoshitake et al., 2004a). These findings are in agreement with an earlier study showing that chronic treatment with the selective MAO-A inhibitor, clorgyline, (1 mg·kg⁻¹) or MAO-B inhibitor [(-)-selegiline, 10 mg·kg⁻¹] produced sustained elevations of concentrations of dopamine and 5-HT, and decreased their deaminated metabolites in the rat forebrain tissue (Yeghiayan et al., 1997). Thus, we postulate that the EGb 761 extract affects brain dopaminergic systems through other more specific and treatment time-dependent mechanisms than inhibiting the MAO or COMT activities.

A possible explanation for the effects exerted by chronic EGb 761 administration could involve desensitization or down-regulation of receptors modulating dopamine and noradrenaline release in the mesocortical and mesolimbic structures. For example, it was shown that chronic but not acute treatment with sertraline caused an increase in the noradrenaline levels in the frontal cortex but not in the hippocampus of rats, most likely as a consequence of desensitization of cortical α₂-adrenoceptors (Thomas et al., 1998). In addition, blockade of α₂-adrenoceptors with the α₂-antagonist idazoxan was shown to preferentially increase extracellular dopamine levels in the PFC (Hertel et al., 1999) and potentiate the effects of venlafaxine on dopamine and noradrenaline levels (Weikop et al., 2004). In addition, modulation of other receptors such as 5-HT or muscarinic acetylcholine receptors by chronic EGb 761 treatment may also account for the increased extracellular dopamine levels in the rat PFC. There is evidence that exists for the role of 5-HT_2A/2C antagonists or inverse agonists, 5-HT₆ or 5-HT₇ receptor antagonists (see Meltzer and Huang, 2008), 5-HT_1A and muscarinic M₁ receptor agonists (Li et al., 2009) in enhancing cortical dopamine transmission, particularly when given in combination with typical antipsychotic drugs such as haloperidol, as well as atypical antipsychotic drugs, which possess intrinsic affinities for these and other receptor subtypes. Some data are available on the effects of EGb 761 and its main constituents on binding affinity to central neurotransmitter receptors. Thus, the G. biloba extract reduced [³H]ketanserin binding to 5-HT_2A receptors in the frontal cortex of MAO-A knockout mice (Shin et al., 2000). Chronic treatment with EGb 761 increased binding (B_max values) to muscarinic acetylcholine receptors (Taylor, 1986) and α₂-adrenoceptors (Huguet and Tarrade, 1992) in the hippocampal membranes, and 5-HT_1A receptors (Huguet et al., 1994) in cortical membranes of aged, but not young, rats. Administration of EGb 761 to adult rats increased noradrenaline turnover in the cerebral cortex only after sub-chronic treatment (Brunello et al., 1985). More recent findings demonstrate that ginkgolides, active constituents of G. biloba, are effective blockers of the glycine receptor pore (Chatterjee et al., 2003; Heads et al., 2008) and recombinant human GABA_A receptors (Huang et al., 2004). However, it is difficult to predict the functional outcome of these effects on behaviour as blockers of glycine and GABA_A receptors are typically proconvulsant. Taken together, although several reports indicate that EGb 761 and its constituents interact with seven-transmembrane receptors and transmitter-gated ion channel receptors, these data are inconclusive and support only a tentative explanation for the findings of this study.

The monoamines noradrenaline, 5-HT and, possibly, dopamine are traditionally implicated in the aetiology of depression and related mood disorders, whereas the progressive impairment of learning and memory functions, observed in Alzheimer's disease, is strongly associated with the atrophy and loss of cholinergic neurons in the basal forebrain (Bartus et al., 1982; Coyle et al., 1983; Terry and Buccafusco, 2003). However, an increasing body of evidence exists for the role of the non-cholinergic neurotransmitter systems in Alzheimer's disease pathology (Terry, 1994; Francis, 1996; Dringenberg, 2000). Thus, the dysfunction of cortical and hippocampal glutamatergic and GABAergic networks, and the major afferent systems such as 5-hydroxytryptaminergic, noradrenergic and histaminergic systems has been implicated in the neuropathology of Alzheimer's disease and appears to affect the majority of patients with this condition (Cross et al., 1981; Bowen and Davison, 1986; Rossor and Lversen, 1986; Baker and Reynolds, 1989; Cross, 1990). A body of evidence exists for the role of prefrontal cortical dopamine and dopamine receptors in modulating the neurons essential for working memory (Goldman-Rakic, 1995; Goldman-Rakic et al., 2000; Castner and Goldman-Rakic, 2004; Paspalas and Goldman-Rakic, 2005). Thus, cognitive deficits have been observed experimentally in rhesus monkeys following dopamine depletion in the PFC (Brozoski et al., 1979; Williams and Goldman-Rakic, 1995), as well as following modulation of dopamine D₁ and D₂ receptors in humans (Müller et al., 1998). In addition, a microdialysis study in freely moving rats during a spatial working memory task provided direct evidence that both noradrenaline and dopamine were increased in the PFC (Rossetti and Carboni, 2005). The dopamine increase was primarily attributed to reward expectancy in tasks involving the memory-guided search for food, whereas the noradrenaline increase was more likely associated with the attention necessary for behavioural activation during the task.

These reports together with the recent findings on the stimulatory effects of EGb 761 on dopamine and noradrenaline transmission in the rat PFC may provide a working hypothesis for the mechanism of action of EGb 761 in relation to improved cognitive performance in aged and healthy people. Recently, a placebo-controlled, double-blind study conducted on 177 test persons between the ages of 45 years and 60 years, and receiving a daily dose of 240 mg Ginkgo extract for 6 weeks revealed a significant twofold improvement in so-called implied learning (the ability needed in everyday situations to quickly reach a correct solution from the new information and to learn from this). The retention capacity (e.g. remembering dates after a 45 min diversion) increased in the EGb-treated group by 26%, but not in the placebo group (Kaschel, 2007). However, the therapeutic efficacy of G. biloba extracts in reducing the incidence of cognitive decline was not confirmed in a recent clinical study (DeKosky et al., 2008).

In summary, the present data demonstrate for the first time that repeated administration of G. biloba extract EGb 761 results in increased dopaminergic and noradrenergic transmission in the frontocortical brain areas, which may be one of the underlying mechanisms behind the clinically observed effects of G. biloba on improved cognitive function. In addition, G. biloba extracts, besides their reported neuroprotective effects and improved memory function, may possess mood/motivation-enhancing properties in disorders associated with abnormal monoaminergic and, in particular, dopaminergic function.

Glossary

Abbreviations:

CMC

carboxymethylcellulose

DOPAC

3,4-dihydroxyphenylacetic acid

HPLC

high-performance liquid chromatography

HVA

homovanillic acid

MAO

monoamine oxidase

PFC

prefrontal cortex

Conflict of interest

The authors declare no conflict of interest.