Accessed - 1,428 times.

Umarani V1, Sudhakar M1, Ramesh A2, Lakshmi BVS1*, Rasavi S1
1Department of Pharmacology, Malla Reddy College of Pharmacy, Dhulapally (via Hakimpet), Secunderabad, TS, India.
2Department of Pharmacology, Vishnu Institute of Pharmaceutical Education and Research, Narsapur, Hyderabad, TS, India.

ORIGINAL RESEARCH ARTICLE
Volume 3, Issue 1, Page 22-28, January-April 2015.

Article history
Received: 1 April 2015
Revised: 10 April 2015
Accepted: 18 April 2015
Early view: 20 April 2015

*Author for correspondence
E-mail: adithya.neha@gmail.com

ABSTRACT
Background: Dementia is one of the age related mental problems, and a characteristic symptom of Alzheimer’s disease. Nootropic agents are clinically used in situations where there is organic disorder in learning abilities and for improving memory, mood and behavior.
Material and methods: The present work was designed to assess the potential activity of Quercetin on cognitive functions in mice using elevated plus maze model, morris water maze model, locomotor activity, rota rod test and passive avoidance paradigm and to correlate nootropic activity with acetylcholine mediated behavior. The effect of Quercetin was observed on clonidine-induced hypothermia (noradrenaline mediated behavior), haloperidol-induced catalepsy (dopamine mediated behavior). Piracetam (200 mg/kg) was used as a standard nootropic agent for comparison.
Results: The results of elevated plus maze model showed that, Quercetin treated mice at 50 mg/kg and 100 mg/kg, significantly (p<0.01) reduced the time required to find the closed arm compared to untreated mice. Further, the Quercetin treatment at 50 mg/kg and 100 mg/kg, showed significant (p<0.01) improvement in scopolamineimpaired performance with respect to acquisition and retention of memory in spatial and working memory tests, in Morris water maze model, improved step down latency in passive avoidance paradigm, increased locomotor activity and improved grip strength in Rota rod test. Conclusion: In conclusion, Quercetin appears to be a promising candidate for improving memory and it would be worthwhile to explore the potential of this compound in management of dementia and Alzheimer’s disease.

Keywords: Quercetin, Nootropic, Elevated plus maze, Morris water maze, Hypothermia, catalepsy.

INTRODUCTION

Learning is acquiring of new knowledge, behaviour, skill, values, preference or understanding and may involve synthesizing different types of information. In psychology, memory is an organism’s mental ability to store, retain and recall past information.
Dementia is a mental disorder characterized by loss of intellectual ability sufficiently severe that interfere with one?s occupational or social activities. It is of several types and invariably involves impairment of memory. The common cause of dementia is Alzheimer?s disease, which is progressive neurodegenerative disorder associated with loss of neurons in distinct areas of brain. The central cholinergic pathways play a prominent role in learning and memory process. Centrally acting anti-muscarinic drugs (e.g. Scopolamine) impairs learning and memory both in animal and human beings.
Alzheimer?s disease (AD) is the most common forms of dementia and it is associated with deposition of ß–amyloid protein in brain tissue and abnormal phosphorylation of the intracellular tau-proteins, causing abnormalities of microtubule assembly and collapse of the cytoskeleton. Pyramidal cells of the cortex and sub cortex are particularly effected (Bennet and Brown, 2003). Nootropic means “acting on the mind”, and nootropics are the substances found to have beneficial effects in the treatment of memory loss, age related memory decline and in lack of concentration. The substance piracetam was not only beneficial, but also found to have fewer side effects hence the biochemists described it “as safe as salt!”(Lanni et al., 2008, Parle and Vasudevan, 2007).
Several mediators like acetylcholine, noradrenaline, dopamine, serotonin, gamma-aminobutyric acid, glutamate, nitric oxide, and peptides influence the cognitive behaviour of the animal (Kulkarni, 2005). The animal behaviour mediated by these mediators may be a simple way of studying the neurochemical correlates of learning and memory. Therefore, in the present study we have tried to correlate the nootropic activity with the monoamine-mediated behaviour (Zarrindast et., 2012; Hasselmo, 2006; Harvey, 2003; Sigala et al., 1997).
Quercetin is a part of subclass of flavanoid called flavones, as received considerable attention because of its overwhelming presence in foods. Quercetin is believed to protect against several neurodegenerative diseases by preventing lipid peroxidation. However, the degree and method of Quercetin absorption in vivo has yet to be absolutely determined. Hence in the present study its effect on learning and memory is investigated by various models (Alexandra, 2009; Justesen and Knuthsen, 2001).

ST combined with FLX provides a potential mechanism for the anxiolytic and antidepressant properties of drugs in animal models (Santos et al., 2012). The monitoring of plasma levels of these drugs will be advantageous in the clinical or preclinical settings. Variation in pharmacodynamic responses may occur particularly at steady state, due to interaction, non-compliance, with concomitantly administered drugs or with other possible causes.
Several methods are available for the analysis of ST alone in plasma including liquid chromatographic (LC) coupled to ultra violet (UV) (Yang et al., 2003) detector gas chromatography mass spectrometric (GCMS) (Morris et al., 1993), micellar electrokinetic capillary chromatography (MEKC) (Srinivasu et al., 2002), voltammetry (Coruh and Özkan, 2006), spectrophotometry (Wang and Asgharnejad, 2000) and liquid chromatographic mass spectrometry (LCMS) (Barrett et al., 2006, Zhao et al., 2000, Jemal et al., 2000). Similarly, a few methods also have been reported for the analysis of FLX alone in plasma by gas chromatography (GC) with electron capture detection (ECD), (Dixit et al., 1991). HPLC with fluorescence detection (Kaddoumi et al., 2001, Guo et al., 2002, Gatti et al., 2003) and HPLC–UV detection (Maya et al., 2000, Thomare et al., 1992, Holladay et al., 1998, Tournel et al., 2001).
In this report, we describe the use of SPE cartridges for rapid sample extraction procedure for the determination of low level of ST, STA and FXL in rat plasma in a single run. The developed method offers a simple and fast sample preparation procedure for quantitation of ST, STA and FXL in plasma via RP-LC–UV detection.

MATERIALS AND METHODS

Drugs
Scopolamine, Piracetam, clonidine, Haloperidol was used in the study.
Experimental Animals
Swiss Albino mice (20-25g) and Wistar albino rats (180-200g) were used for the study. The rats were housed in polypropylene cages and maintained under standard conditions (12 h light and dark cycles, at 25±3º C and 35-60% humidity). Standard pelletized feed and tap water were provided ad libitum. All the pharmacological experimental protocols were approved by the Institutional Animal Ethics Committee with protocol no. (MRCP/CPCSEA/IAEC/2013-14/MPCOL/15).
Laboratory models for testing learning and memory
Twenty four Wistar Albino mice of weight 20-25g were selected for this study. Animals were divided into four groups of six animals each.
Group 1: Control group (distilled water 1ml)
Group 2: Piracetam (positive control) 200 mg/kg body weight p.o
Group 3: Quercetin 50 mg/kg body weight p.o
Group 4: Quercetin 100 mg/kg body weight p.o.
Evaluation of behavioural studies
1. Clonidine induced hypothermia model
2. Haloperidol induced catalepsy model
Thirty Wistar Albino rats of weight 180-200g were selected for this study. Animals were divided into five groups of six animals each.
Group 1: Control group (distilled water 1ml)
Group 2: Clonidine 1mg/kg body weight i.p/Haloperidol 1mg/kg body weight i.p
Group 3: Piracetam (positive control) 200 mg/kg body weight p.o
Group 4: Quercetin 50 mg/kg body weight p.o
Group 5: Quercetin 100 mg/kg body weight p.o.
Elevated plus maze model (Mrugaya et al., 2009; Gawale et al., 2001)
Mice were divided randomly into four groups of six animals. Group I comprised of saline- treated control animals; Group II animals received Piracetam 200 mg/kg, orally; and Groups III and IV were fed orally with Quercetin at doses 50 and 100 mg/kg respectively. The elevated plus maze apparatus used in the study consisted of two open and two closed arms facing each other. The maze was elevated at a height of 50 cm from the ground. The animals were placed individually 30 min after oral administration of either vehicle or test drug at the end of either of the open arms and the time taken by the animal to move from open to closed arm (transfer latency) was noted on the first day. The time elapsed between the time that the animal was placed on the open arm and the time at which all four legs were inside the enclosed arms, was noted as the transfer latency. The transfer latency was again recorded 24 hr after the first exposure.
Morris water maze model (Mrugaya et al., 2009)
The experimental groups used for spatial and working memory testing in Morris water maze model were similar to that used in elevated plus maze model described earlier. The Morris water maze consisted of a large water tank (48cm×28cm×18cm)filled with water , which was made opaque by adding milk, water provided a uniform intra maze environment, thus eliminating any olfactory interference. A 7×7 cm rectangular escape platform was constructed of water resistant material and covered with material that allows the animal to remain on top when it is submerged. The platform was 10 cm in height so that it could be submerged 2 cm below the level of water surface. The water temperature was maintained at 26°c.
Study protocol for spatial memory test
In this test, mouse was released with its head pointed towards side of the water pool. The time taken by the mouse to find a hidden platform in water pool after previous exposure to the set up, using only available external cues was determined as a measure of spatial memory. The mouse was allowed to remain on platform for 10 sec and later placed in holding cage for 15 sec. Again the mouse was released from same place and time for reaching the submerged platform was recorded. Likewise, ten trials were conducted and average time to reach the submerged platform was recorded, keeping similar experimental conditions. Drugs were administered orally and after 90 min all the groups were exposed to the training schedule. This procedure was repeated at 24 hr interval for two more days until each subject acquired minimum time interval to reach the submerged platform in the pool. On fourth day, after complete training, all groups were treated with scopolamine butyl bromide (2 mg/kg, i.p.). 30 min later, they were treated with test extract/std, and were tested for spatial memory after further 90 min. Mice with memory impairment took more time to reach the platform. The animals were also tested for spatial memory on fifth day to check the ability of test extract to restore scopolamine-induced amnesia.
Study protocol for working memory test
This test was applied after the acquisition phase of testing was completed. It is important that the mice demonstrate that they know the location of hidden platform before beginning the test. This method has been referred as the reversal test. As in spatial memory test, time for the mouse to reach the submerged platform was recorded. The submerged platform was then moved to a new location. Then, the mouse was released from the same place and time for reaching the submerged platform was recorded. In this manner, the hidden platform was changed to all four quadrants and average time to reach the submerged platform in each quadrant was recorded.
Evaluation of spatial memory and working memory tests
Latency to reach the platform in sec (mean values) was recorded on day 0, day 1, day 2, day 4 and day 5. Day 1 is the day from which animals were treated with the test extract/std. Day 4 is the day on which animals were treated with scopolamine butyl bromide 2mg/kg, i.p .
Locomotor activity in mice by using Actophotometer (Gawale et al., 2001)
Locomotor activity is an index of alertness (or) wakefulness of mental activity. The equipment was switched on and stabilized for some time and the accurate working of the photocells was checked. Each animal from their respective groups were placed in the chamber individually for 5 min. After 30 minutes all the animals were retested for activity and the results of each animal was tabulated.
Rota-rod test in mice
The Rota rod test is a classic method to assess motor coordination, balance and motor learning in mice. In the Rota rod test, a mouse is placed on a rotating rod. The speed of rotation is gradually increased and the rat?s ability to remain on the rotating rod is recorded. The mice were trained few days prior to the experiment and the “Fall of Time” for each rat within the 5 min time period was recorded.
Passive avoidance paradigm
Passive avoidance behavior based on negative reinforcement was used to examine the long-term memory. The apparatus consisted of a box (27 X 27 X 27 cm) having three walls of wood and one wall of Plexiglas, featuring a grid floor (3 mm stainless steel rods set 8 mm apart), with a wooden platform (10 X 7 X 1.7 cm) in the center of the grid floor. The box was illuminated with a 15 W bulb during the experimental period. Electric shock (20V AC) was delivered to the grid floor. Training was carried out in two similar sessions. Each mouse was gently placed on the wooden platform set in the center of the grid floor. When the mouse stepped down and placed on the wooden platform set in the center of the grid floor. When the mouse stepped down and placed all its paws on the grid floor, shocks wee delivered for 15 sec and the step-down latency (SDL) was recorded. SDL was defined as the time taken by the mouse to step down from wooden platform to grid floor with its entire paw on the grid floor. Animals showing SDL in the range (2-15 sec) during the first test were used for the second session and the retention test. The second-session was carried out 90 min after the first test. When the animals stepped down before 60 sec, electric shocks were delivered for 15 sec. During the second test, animals were removed from shock free zone if they did not step down for a period of 60 sec. Retention was tested after 24 h in a similar manner, except that the electric shocks were not applied to the grid floor. Each mouse was again placed on the platform, and the SDL was recorded, with an upper cut-off time of 300 sec (Rao et al., 2008; Hanumanthachar and Milind, 2006).
Behavioural studies
Haloperidol- induced (DA-mediated) catalepsy
Rats were divided into groups of six each. Animals received haloperidol (1mg/kg i.p.) 30 min after vehicle, Piracetam (200mg/kg), Quercetin in doses of 50 mg/kg and 100mg/kg p.o and the duration of catalepsy was noted at 5,15, 30, 60, 90 and 120 min intervals using „Bar test? . In brief, the forepaws of mouse were placed on wooden bar (0.9cm diameter) resting 2.5cm high from the table and duration of imposed posture was noted twice and greater time was chosen for calculation (Rao et al., 2008; Hanumanthachar and Milind, 2006).
Clonidine-induced (NA-mediated) hypothermia
The effect of Quercetin was studied on clonidine induced hypothermia; an animal model recommended to study the effect of drugs on the noradrenaline (NA) mediated behavior. Albino rats, divided into groups of six animals received vehicle, Piracetam (200mg/kg), Quercetin(50mg/kg) and Quercetin (100mg/kg) i.p 30 min before clonidine (0.1mg/kg) and rectal temperature was recorded at 0, 60, 120, 150 and 180 min. Due care was taken to avoid stress of inserting the probe (Rao et al., 2008; Hanumanthachar and Milind, 2006).
Estimation of brain acetyl cholinesterase (AChE) activity
Swiss mice of either sex weighing around 25 g were used. Group I (n=6), served as control and treated with distilled water. Group II (n=6), were treated with Piracetam (200 mg/kg, p.o.), Group III (n=6) with Quercetin (50 mg/kg, p.0), Group IV (n=6) were treated with (100 mg/kg, p.o.) respectively for 8 days. On the 9th day the animals were euthanized by cervical dislocation carefully to avoid any injuries to the tissue. Brain from each group was washed with saline and fixed in 10% buffered formalin for further processing of histopathological examination. The whole brain AChE activity was measured using the Ellman method (Ellman et al., 1961). This was measured on the basis of the formation of yellow color due to the reaction of thiocholine with dithiobisnitrobenzoate ions. The rate of formation of thiocholine from acetylcholine iodide in the presence of tissue cholinesterase was measured using a spectrophotometer. The sample was first treated with 5, 5?-dithionitrobenzoic acid (DTNB) and the optical density (OD) of the yellow color compound formed during the reaction at 412 nm every minute for a period of three minutes was measured (Hanumanthachar and Milind, 2006b, Ellman, 1959).
AChE activity was calculated using the following formula:
Calculations:
The enzyme activity is calculated using the following formula;
R = 5.74x 10-4 x A/CO
Where, R = Rate in moles of substrate hydrolyzed /minute / gm tissue
A = Change in absorbance / min
CO = Original concentration of the tissue (mg / ml)
Histopathological determination
For microscopic evaluation tissues were fixed in a fixative (absolute ethanol 60%, formaldehyde 30%, and glacial acetic acid 10%) and embedded in paraffin, sectioned at 4µm and subsequently stained with haematoxylin/eosin. Sections were studied under light microscope (DIALUX 20 EB) at 40 and 100 magnifications. Slides of all the treated groups were studied and photographed. A minimum 12 fields of each section were studied and approved by pathologist without saying of its treatment nature.
Statistical analysis
The values were expressed as the mean ± SEM for the 06 mice and rats in each group. Differences between groups were assessed by one-way analysis of variance (ANOVA) using the Statistical Package for Social Sciences (SPSS) software package for Windows (version 13.0). Post hoc testing was performed for intergroup comparisons using the least significant difference (LSD) test. A value corresponding to (P<0.01) was deemed to be statistically significant.

RESULTS

Effect of Quercetin on transfer latency of mice in elevated plus maze model
Quercetin treatment at doses of 50mg/kg and 100 mg/kg, showed significant (p<0.1) decrease in transfer latency to find closed arm on day two, compared to vehicle treated mice (Figure 1A). Effect of Quercetin on scopolamine –induced working memory impairment.
Quercetin at both the doses (50mg/kg and 100mg/kg) significantly (p<0.01) improved scopolamine –impaired performance in all four quadrants on days four and five, compared to vehicle control group (Table 2). Effect of Quercetin on locomotor activity of mice in actophotometer.
In the present study, Quercetin showed significant change in the locomotor function of animals (score30.5±7.96 and 45.25±9.24) as compared to control group (score 29.75±3.57) when tested using a photoactometer (Figure 1B).
Effect of Quercetin on grip strength using Rota rod apparatus
In the present study treatment with Quercetin at 50mg/kg and 100 mg/kg, increased the grip strength in mice when compared to control group (Figure 1C).
Effect of Quercetin on scopolamine–induced spatial memory impairment
Scopolamine treated mice without any treatment, took longer time to reach the platform due to memory impairment on day four and five. However, Quercetin at 50 mg/kg and 100mg/kg significantly (P<0.01) reduced the time required by mouse to reach the platform after scopolamine treatment on day four and five compared to normal control (Table 1). Effect of Quercetin on step-down-latency (SDL) using passive-avoidance apparatus
In the present study Quercetin 50 mg/kg and Quercetin 100mg/kg profoundly increased SDL significantly as compared to control group on the second day indicating improvement in memory of mice. Further, this dose of Quercetin reversed scopolamine-induced amnesia (Figure 1D).
Effect of Quercetin on AchE activity in mice
AchE activity was decreased significantly in piracetam, Quercetin 50mg/kg and 100mg/kg treated animals, when compared to control group (Figure 1 E).
Effect of Quercetin on Clonidine-induced (NA mediated) hypothermia in rats.
The rectal temperature decreased from 35±0.01°c to 31.5±0.28°c 90 min after clonidine administration. Piracetam, Quercetin 50mg/kg and 100mg/kg showed significant effect till 120 min. Treatment with the respective drugs counteracted the decrease in rectal temperature (Table 4).

Figure 1. Effect of Quercetin on Various models.
Click here to view full image

Table 1. Effect of Quercetin on scopolamine induced spatial memory impairment in Morris water maze.
Click here to view full image

Table 2. Effect of Quercetin on scopolamine induced working memory impairment in Morris water maze.
Click here to view full image

Table 3. Effect of Quercetin on Haloperidol-induced (DA mediated) catalepsy in rats.
Click here to view full image

Table 4. Effect of Quercetin on Clonidine-induced (NA mediated) hypothermia in rats.
Click here to view full image

DISCUSSION

Nootropics are the drugs better known as smart drugs, memory cognitive enhancers, supplements, functional foods or nutraceuticals that improve mental functions such as cognitive attention, concentration, motivation, intelligence.
The elevated plus maze is used to measure the anxiety state in animals, however transfer latency i.e. the time elapsed between the movement of the animal from an open to an enclosed arm is markedly shortened, if the animal has previously experienced entering open and close arms, and this shortened transfer latency has been shown to be related with memory processes (Jaiswal and Bhattacharya, 1992). In elevated plus maze model, acquisition (learning) can be considered as transfer latency on first day trials and the retention (memory) is examined 24 hr later . The animals treated with Quercetin at 50 mg/kg and 100 mg/kg, showed significant (p<0.1) decrease in transfer latency on day two compared to vehicle control group, indicating cognitive enhancing effect of the extract in rodents. Morris water maze model has been extensively used to study the neurological mechanisms that underlie spatial navigation to influence special cognitive processes. The same task can also be used to test working memory by changing the hidden platform from one quadrant to another quadrant. This model throws light on ability of the agent in improving cognitive functions impaired by scopolamine (centrally acting acetylcholine blocker), thereby emphasizing its utility in cognitive disorders involving dementia. The Quercetin treatment at 50 mg/kg and 100 mg/kg, showed significant (p<0.01) improvement in scopolamine-impaired performance with respect to acquisition and retention of memory, in both spatial and working memory tests, thereby reinforcing the nootropic potential of Quercetin. The improved spatial and working memory observed in the treatment groups, suggests that Quercetin might act on the central cholinergic system for reversing the scopolamine- impaired memory in Morris water maze test. In the present study, Quercetin increases the locomotor function of animals (p<0.05) as compared to control group when tested using an actophotometer. In the present study, Quercetin increases the step- down latency (p<0.001) as compared to control group. In Clonidine induced hypothermia different doses of Quercetin (50 and 100 mg/kg) have reversed the clonidine induced hypothermia in rats. The rectal temperature decreased from 35±0.01°c to 31.5±0.28°c 90 min after clonidine. Piracetam, Quercetin 50mg/kg and 100mg/kg showed significant effect till 120 min. Treatment with the respective drugs counteracted the decrease in rectal temperature. Haloperidol produced peak catalepsy 30 min after its administration. Piracetam inhibited catalepsy, lower dose of Quercetin had little effect on haloperidol – induced catalepsy and higher dose of Quercetin significantly inhibited catalepsy (Ferre et al., 1990). Quercetin demonstrated significant nootropic activity by inhibiting AchE activity and increasing global/regional blood flow and improves oxygen supply and brain energy and nerve growth stimulation and brain cell protection.

CONCLUSION

In our study it was found that Quercetin which is one of the strongest known natural antioxidant found in a large quantity in plants and foods, such as red wine, onions, green tea, apples and berries, being a flavonoid may be responsible for the observed nootropic activity in different experimental animal models used in the study..

ACKNOWLEDGEMENTS

The authors are thankful to the authorities of Malla Reddy College of Pharmacy, Secunderabad, for providing support to this study.

CONFLICT OF INTEREST
None declared.

REFERENCES

Alexandra B. Bent. A Review of Quercetin: Chemistry, Antioxidant Properties, and Bioavailability. 1, 9, 2009.
Bennet PN, Brown MJ. Clinical pharmacology. 10th ed. Churchil Livingstone, New York, 406-408, 2003.
Ellman Georg L. Tissue sulfhydryl group: Archives of Biochemistry and Biophysics. 82 (1), 70-77, 1959.
Ferre S, Guix T, Part G, Jane F. Is experimental catalepsy properly measured? Pharmacology Biochemistry and Behavior. 35, 753-757, 1950.
Gawale NS, Pal SC, Kasture VS, Kasture SB. Effect of Butea monosperma on memory and behaviour mediated via monoamine neurotransmitters in laboratory animals. Journal of Natural Remedies. 1(1), 33-41, 2001.
Hanumanthachar Joshi, Milind Parle. Evaluation of Nootropic potential of Ocimum sanctum Linn. in mice. Indian Journal of Experimental Biology. 44, 133-136, 2006.
Hanumanthachar Joshi, Milind Parle. Zingiber officinale: Evaluation of its Nootropic effect in mice. African Journal of Traditional Medicine. CAM 3 (1), 64-74, 2006.
Harvey JA. Role of the Serotonin 5-HT2A receptor in learning. Learning and Memory. 10, 355-362, 2003.
Hasselmo ME. The role of acetylcholine in learning and memory. Current Opinion in Pharmacology. 16, 710-715, 2006.
Jaiswal AK, Bhattacharya SK. Effects of Shilajit on memory, anxiety and brain monoamines in rats. Indian Journal of Pharamacology. 24, 12-17, 1992.
Justesen U, Knuthsen P. Composition of flavonoids in fresh herbs and calculation of flavonoid intake by use of herbs in traditional Danish dishes. Food Chemistry. 73 (2), 245–50, 2001.
Kulkarni SK. Handbook of experimental pharmacology. 3rd ed.Vallabh Prakashan, Delhi, 44-45, 2005.
Lanni C, Lenzken SC, Pascale A, et al., Cognition enhancers between treating and doping the mind. Pharmacology. 57 (3), 196–213, 2008.
Mrugaya P Kulkarni, Archana R Juvekar. Studies on nootropic activity of roots of Tylophora indica in mice. Journal of Natural Remedies. 9, 62-67, 2009.
Parle M, Vasudevan M. Memory enhancing activity of Abana®: An Indian ayurvedic poly-herbal formulation. Journal of Healing Science. 53(1), 43-52, 2007.
Rao NV et al., Nootropic activity of tuber extract of Pueraria Tuberosa. Indian Journal of Experimental Biology. 41, 78-85, 2008.
Sigala S, Missale C, Spano P. Opposite effects of dopamine D2 and D3 receptors on learning and memory in the rat. European Journal of Pharmacology. 336, 107-112, 1997.
Vervliet B. Learning and memory in conditioned fear extinction: effects of D-cycloserine. Acta Psychologica. 27(3), 601-613, 2008.
Zarrindast MR, Lahiji P, Shafaghi B, Sadegh M. Effects of GABAergic drugs on Physostigmine-induced improvement in memory acquisition of passive avoidance learning in mice. General Pharmacology. 31 (1), 81-86, 2012.