An Experimental Study to Evaluate the Effect of Memantine in Animal Models of Anxiety in Swiss Albino Mice

The incidence of pathologic anxiety states is rapidly increasing among the community amounting to life time prevalence of 30.5% in women and 19.2% in males [1]. Hence, it is very important to address the problem of anxiety and explore safe, effective alternative medicines. The pathophysiology underlying the anxiety disorders is mostly associated with dysfunction of GABAergic neurotransmission. Although Benzodiazepines still remain the first line of anxiolytic drugs but side effects like sedation, addiction and development of tolerance may limit their chronic usage. However, it has been suggested that NMDA receptors may also contribute enormously to the neurobiological and psychological mechanisms in anxiety states [2–4].

In a previous study by Poleszak E et al., it was demonstrated that NMDA receptor activation antagonizes the NMDA antagonist-induced antianxiety effect in the elevated plus-maze test in mice [5]. This stimulated us to evaluate the anxiolytic effect of an uncompetitive NMDA antagonist, memantine which is currently used in Alzheimer’s disease. Rationale for using memantine in our study is because of improved clinical tolerability of memantine when compared to other NMDA antagonists [6,7]. Phencyclidine, Dizocilpine which are other NMDA antagonists have demonstrated the antianxiety effects in rodents [8,9].

In our study, the test drug is memantine, which is a voltage and use-dependent blocker, can become trapped in the ligand-gated channel and has rapid blocking and unblocking dissociation kinetics. In a previous study by Shuijin He et al., [10], it was evident that chronic memantine administration to rat pups hippocampal slice cultures which served as differentiation-induced epileptogenesis model, increased the GABA release and GABA_A receptor function. This also indicates the interplay of NMDA and GABA_A receptors in anxiety states.

Earlier preclinical and clinical data have revealed the antianxiety effects of other competitive NMDA receptor antagonists and these classes of drugs are well compared to benzodiazepines or barbiturates as anxiolytics [11–13]. Previous studies undertaken by Bagewadi HG et al., has conclusively showed that memantine possessed the antidepressant effects [14,15], in animal models of depression and antianxiety effect [15], in Elevated plus maze model of anxiety. To strengthen our evidence, we carried out the present study to evaluate the antianxiety effects of memantine in different experimental models of anxiety like passive avoidance test and open field test.

The animal models of anxiety used today are mainly of two types. The first involves the animal’s conditioned responses to aversive painful stimuli e.g. exposure to electric foot shock i.e. Vogel conflict test, four-plate test, passive avoidance response, defensive burying. The second is ethologically based paradigms and involves the animal’s natural reactions like flight; avoidance and freezing that do not involve pain e.g. elevated plus maze, social interaction test, open field test, hole board test, staircase test [16]. In our study, we have used one conditioned and another ethologically based unconditioned model to avoid the bias and to assess the anxiolytic effect of memantine more effectively. Both open-field and passive avoidance tests are simple, having high sensitivity and specificity and are effective screening methods for different compounds to evaluate their anxiolytic activity [17].

In earlier study by Janel M Boyce-Rustay it was evident that NMDA receptor subunit (NR2A) knockout mice showed decreased anxiety-like behaviour in open field test when compared to wild-type mice depicting the role of NMDA receptors in modulation of anxiety [18]. In the previous study done by Tomilenko RA [19], passive avoidance response test was used to evaluate the role of NMDA receptor agonist D-cycloserine and antagonist- Dizocilpine on learning and extinction of passive avoidance response in different anxiety states in mice.

The open-field and passive avoidance tests were used in earlier studies [17,20–26], to establish more conclusively the role of NMDA receptors in anxiety states. As our test drug is memantine- an uncompetitive NMDA antagonist and to make our study more appropriate, we have performed these two behavioural tests of anxiety. The BALB/C strains of swiss albino mice were used in our study. In the open field test, this strain of mice have reported with higher levels of anxiety-like behaviours when compared to C57BL/6 strain mice, which was consistent with previous findings by An XL [27].

The passive avoidance response test assesses anxiety states by training rodents to avoid electric shock stimuli by curbing their normal exploratory behaviour. In the previous study by Glotzbach et al., [28], it was evident that human participants in the Virtual reality (VR) arena, they gradually avoided the state which is associated with shock and exhibited greater amount of subjective fear when compared to participants who did not avoid the stimuli. Contextual fear conditioning predicts subsequent passive avoidance behaviour in a VR environment which is in homologous to passive avoidance response in animals. The pathological anxiety states in humans is usually associated with changes in physiological and behavioural responses to fear, painful stimuli and similar human responses were noted in animals to such stimuli. The open field and passive avoidance test simulate the human pathological anxiety state in rodents and they induce a fearful response by an aversive stimuli and preventing mice from their normal exploratory activity. This suggest the possibility of homologous, ethologically motivated defensive responses to such stimuli in animals and giving substantial face validity for both of these anxiety level assessment paradigms [29–34]. Thus, memantine was evaluated for its anti-anxiety effect in the present study.

Materials and Methods

Swiss albino mice which belonged to either of the sex and weighed between 25 to 30 g were issued from animal house of MVJ medical college and research hospital. The animals were housed in polypropylene cages and were maintained in favorable conditions with a humidity 50-55% and temperature 22±2°C. CPCSEA guidelines were followed while maintaining and handling of the animals during the experimentation on the animals. Institutional Animal Ethics Committee permission was taken in order to carry out the study.

Drugs and Chemicals

Memantine was obtained from Sun Pharma drugs Pvt.Ltd. and lorazepam from Intas Pharmaceuticals Ltd. The drugs were diluted in normal saline and freshly prepared before drug administration.

Experimental Groups

The animals were divided into four groups. Twelve mice were randomly allotted in each group. Group I - was administered normal saline 10 ml/kg by intraperitoneal route (i.p.) which served as control group. Group II - was given memantine 3 mg/kg, i.p. Group III – received lorazepam 0.5 mg/kg, i.p. Group IV - was administered memantine 3 mg/kg, i.p. + lorazepam 0.5 mg/kg, i.p. All the animals in various groups received the respective drugs by intraperitoneal route for 7 days of experimental period. The experiments were carried out on the 1^st day which was 24 hours after the 1^st exposure of the animals to the open field and passive avoidance apparatus. The findings were again recorded on the 8^th day. The anxiety behavioural assessment was performed on the days of testing i.e. on the 1^st day and on the 8^th day, 30 minutes after the drug administration. The dose of the memantine was chosen from previous studies done by Sufka KJ [35], and Fredriksson A [36], and dose of the lorazepam was chosen from earlier study by V. Sathyanathan [37].

Assessment of behavioural tests

Passive avoidance - The passive avoidance response was assessed in the apparatus with the dimension of 34 cm x 34 cm x 20 cm. In the center of the chamber a shock-free zone (SFZ) was placed. It consisted of grid floor through which 20 mV electric shock was delivered. Mouse was initially kept on the SFZ and electric shock was delivered whenever the mouse tried to come in contact with the grid floor. The animals by curbing their normal exploratory behaviour subsequently learned to avoid the aversive electric shock stimuli by remaining in the SFZ with freezing or minimal motility [38,39]. The mouse was trained for minimum of 60 seconds to stay at the SFZ. The following parameters were noted: 1.Step-down latency, the time interval during which the animal avoids electric shock and remain in the SFZ 2. Step-down error, number of times the animal tries to come back to SFZ in order to avoid painful electric stimuli. 3. Total duration of stay at Shock Zone (SZ).

Open Field test [17]- This is a standard behavioural model that assess anxiety states in rodents in which the anxious behaviour of mice to avoid open, unprotected area, preference for peripheral areas, along with periodic freezing were noted. A reduction in normal behaviours such as rearing and grooming were also considered as an index of anxiety. The apparatus consisted of floor space with dimension of 40cm x 40cm and 30cms in height. The floor space was divided into 16 squares equally. Prior to the testing, the mouse was placed at the center of the floor space and allowed to acclimatize with the surrounding area for 2 minutes. The following parameters were noted: 1) Duration for which animal stays in the central square; 2) Ambulation was indicated by the total no. of squares crossed; 3) Rearing was indicated by the total no. of times the animal stands on its rear paws.

Statistical Analysis

All the findings were expressed in terms of Mean± SEM. For comparison between the groups, one-way ANOVA followed by post-hoc Tukey’s test was utilized. The p-value ≤0.05 was considered to represent statistical significant difference.

Results

In passive avoidance test on the 1^st day the animals treated with lorazepam as a standard, when compared to the control group, showed significant decrease p<0.001 in step down latency period in shock free zone (175.8±5.98 Vs 280.4±8.17), in step down error (6.4±0.82 Vs 1.5±0.29) and total time spent in shock zone (34.2±1.63 Vs 6.7±0.86) as shown in [Table/Fig-1]. On the 8^th day, animals showed significant decrease p<0.001 in step down latency period in SFZ (161.4±2.73 Vs 278.3±5.49). The animals showed significant increase p<0.001 in step down error (8.1± 0.98 Vs 1.4±0.19) and total time spent in SZ (38.3±1.82 Vs 5.6±0.6) as shown in [Table/Fig-2].

[Table/Fig-1]:

Effect of single dose observation in passive avoidance test on 1^st day

Groups, (dose)	Step down Latency (s)	Step down Error (no.)	Time in shock zone (s)
1. Control (10 ml/kg, i.p.)	280.4±8.17	1.5± 0.29	6.7±0.86
2. Lorazepam (0.5mg/kg, i.p.)	175.8±5.98*	6.4± 0.82*	34.2±1.63*
3. Memantine (3mg/kg, i.p.)	267.2±2.51††	2.1± 0.64††	8.6±0.75††
4. Memantine + Lorazepam	152.3±3.82‡‡	9.4±0.56‡‡	42.4±1.72‡‡

(n=12), s-seconds, values expressed as mean±SEM. (*p< 0.001 vs. normal saline-control), (^†p< 0.01, ^††p< 0.001 vs. lorazepam), (^‡p< 0.01, ^‡‡p< 0.001 vs. memantine)

[Table/Fig-2]:

Effect of multiple dose observation in passive avoidance test on 8^th day.

Groups, (dose)	Step down Latency (s)	Step down Error (no.)	Time in shock zone (s)
1. Control (10 ml/kg, i.p.)	278.3±5.49	1.4±0.19	5.6±0.6
2. Lorazepam (0.5 mg/kg, i.p.)	161.4±2.73*	8.1±0.98*	38.3±1.82*
3. Memantine (3mg/kg, i.p.)	185.4±3.87*,†	6.8±0.78*	32.1±2.22*
4. Memantine + Lorazepam	155.8±1.67‡‡	9.2±0.63‡	41.8 ±1.81‡

(n=12), values expressed as mean±SEM. (*p< 0.001 vs. normal saline-control), (^†p< 0.01, ^††p< 0.001 vs. lorazepam), (^‡p< 0.01, ^‡‡p< 0.001 vs. memantine)

In passive avoidance test, on the 1^st day the animals treated with memantine when compared to control group, no statistical significant anxiolytic effect was as shown in [Table/Fig-1]. Whereas, on the 8th day the animals showed significant decrease p<0.001 in step down latency period in SFZ (185.4±3.87 Vs 278.3±5.49), significant increase p<0.001 in step down error (6.8±0.78 Vs 1.4±0.19) and in total time spent in SZ (32.1±2.22 Vs 5.6±0.6) as shown in [Table/Fig-2].

In passive avoidance test on the 1^st day the animals treated with lorazepam when compared to memantine treated group, animals showed significant decrease p<0.001 in step down latency period in SFZ (175.8±5.98 Vs 267.2±2.51), significant increase p<0.001 in step down error (6.4±0.82 Vs 2.1±0.64) and in total time spent in SZ (34.2±1.63 Vs 8.6±0.75) as shown in [Table/Fig-1]. But on the 8^th day, there was significant decrease p<0.01 in step down latency period in SFZ (161.4±2.73 Vs 185.4±3.87) as shown in [Table/Fig-2]. On the 1^st day, memantine + lorazepam treated group when compared to memantine alone treated group, animals showed significant decrease p<0.001 in step down latency period in SFZ (152.3±3.82 Vs 267.2±2.51), significant increase p<0.001 in step down error (9.4±0.56 Vs 2.1±0.64) and in total time spent in SZ (42.4±1.72 Vs 8.6±0.75) as shown in [Table/Fig-1]. But on the 8^th day, animals showed significant decrease p<0.001 in step down latency period in SFZ (155.8±1.67 Vs 185.4±3.87), significant increase p<0.01 in step down error (9.2±0.63 Vs 6.8±0.78) and total time spent in SZ (41.8±1.81 Vs 32.1±2.22) as shown in [Table/Fig-2].

In open field test on the 1^st day the animals treated with lorazepam as a standard when compared to control group, showed statistically significant anxiolytic activity as shown in [Table/Fig-3]. On the 8^th day, the animals showed significant increase p<0.001 in no. of squares crossed (126.4±2.77 Vs 83.2±2.96), time spent in central square (14.3±1.53 Vs 3.4±0.65), no. of rearings (37.5±2.42 Vs 17±1.81) and significant decrease p<0.001 in freezing time (8.53±0.59 Vs 20.2±2.29) as shown in [Table/Fig-4]. In open field test on the 1^st day the animals treated with memantine when compared to control group, no statistical significant anxiolytic effect was as shown in [Table/Fig-3]. Whereas on the 8^th day animals showed significant increase p<0.001 in no. of squares crossed (112.7±2.69 Vs 83.2±2.96), time spent in central square (11.5±1.26 Vs 3.4±0.65), no. of rearings (32.4±2.61 Vs 17±1.81) and significant decrease (p<0.001) in freezing time (15.2±1.12 Vs 20.2±2.29) as shown in [Table/Fig-4].

[Table/Fig-3]:

Effect of single dose observation in open field test on 1^st day

Groups, (dose)	Squares Crossed(no.)	Time spent in CS (s)	Rearing (no.)	Freezing time (s)
1. Control (10ml/kg, i.p)	86.3±1.08	2.7±0.14	19.3±1.6	20.6±2.36
2. Lorazepam (0.5mg/kg, i.p)	104.6±0.92*	8.4 ±0.18*	34.4±1.17*	9.6±1.35*
3. Memantine (3mg/kg, i.p.)	90.2±1.61††	3.1± 0.15††	23.5±2.39††	18.1±1.18†
4. Memantine + Lorazepam	135.6±1.6‡‡	26.32±1.1‡‡	51.2±2.06‡‡	7.3±0.86‡‡

s- seconds, CS-Central Square, (n=12), values expressed as mean±SEM. (*p< 0.001 vs. normal saline-control),(^†p< 0.01, ^††p< 0.001 vs. lorazepam), (^‡‡p< 0.001 vs. memantine)

[Table/Fig-4]:

Effect of multiple dose observation in open field test on 8^th day

Groups, (dose)	Squares Crossed(no.)	Time spent in CS (s)	Rearing (no.)	Freezing time (s)
1. Control (10ml/kg, i.p)	83.2±2.96	3.4±0.65	17±1.81	20.2±2.29
2. Lorazepam (0.5mg/kg, i.p.)	126.4±2.77*	14.3±1.53*	37.5±2.42*	8.53±0.59*
3. Memantine (3mg/kg, i.p.)	112.7±2.69*,†	11.5±1.26*	32.4±2.61*	15.2±1.12*,†
4. Memantine + Lorazepam	131.2±2.98‡‡	25.2±1.17‡‡	46.1±1.51‡‡	5.9±0.62‡‡

s- seconds, CS-Central Square, (n=12), values expressed as mean±SEM. (*p< 0.001 vs. normal saline-control), (^†p< 0.01, ^††p< 0.001 vs. lorazepam), (^‡‡p< 0.001 vs. memantine)

In open field test on 1^st day the animals treated with lorazepam when compared to memantine group, animals showed significant increase p<0.001 in no. of squares crossed (104.6±0.92 Vs 90.2±1.61), time spent in central square (8.4±0.18 Vs 3.1±0.15), no. of rearings (34.4±1.17 Vs 23.5±2.39) and significant decrease p<0.01 in freezing time (9.6±1.35 Vs 18.1±1.18) as shown in [Table/Fig-3]. But on the 8^th day there was significant increase p<0.01 in no. of squares crossed (126.4±2.77 Vs 112.7±2.69) and significant decrease p<0.01 in freezing time (8.53±0.59 Vs 15.2±1.12) as shown in [Table/Fig-4].

In open field test on 1st day the animals treated with memantine + lorazepam when compared to memantine alone group, showed statistical significant anxiolytic activity as shown in [Table/Fig-3]. On the 8^th day, animals showed significant increase p<0.001 in no. of squares crossed (131.2±2.98 Vs 112.7±2.69), time spent in central square (25.2±1.17 Vs 11.5±1.26) no. of rearings (46.1±1.51 Vs 32.4±2.61) and significant decrease (p<0.001) in freezing time (5.9±0.62 Vs 15.2±1.12) as shown in [Table/Fig-4]. Photograph shows the mouse staying at Shock Free Zone (SFZ) in passive avoidance test shown in [Table/Fig-5]. Photograph shows the mouse crossing the central square in open field test as shown in [Table/Fig-6].

[Table/Fig-5]:

Showing the mouse staying at shock free zone in passive avoidance test

[Table/Fig-6]:

Showing the mouse crossing the central square in open field test

Discussion

In the management of anxiety disorders, Benzodiazepines are mainly preferred as the first line treatment. However, chronic administration of Benzodiazepines results in side effects like sedation, amnesia, tolerance which limits their usage [40]. Mammalian brain mainly contains three types of inotropic glutamate receptors: N-methyl-D-aspartate (NMDA), 2-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid (AMPA) and kainate [41]. Among these, NMDA receptor plays a key role in modulation of anxiety disorders [42,43]. Interestingly it was found that when NMDA receptor antagonists were administered by microinjection into the dorsolateral periaqueductal gray area in rats, anxiolytic-like effects were more evident [44]. It was demonstrated from a previous study that in hippocampal neurons, conditioning with 20 μM NMDA for 20 sec caused 50% suppression of GABA responses and lorazepam potentiation reliably increased with GABA_A receptors when there was NMDA-induced suppression in plasticity of fast synaptic transmission [45].

In passive avoidance test, the mice stay at the shock free zone in order to escape from the aversive electric shock stimuli. The reduction in normal behaviour is exhibited by decrease in step down latency period and increase in number of step-down errors. Memantine showed statistically significant antianxiety effects in both the anxiety behaviour paradigms on the 8^th day when compared to 1^st day as shown in [Table/Fig-1,2,3 and 4]. Our study also demonstrated synergistic interaction between memantine and lorazepam in their antianxiety activity. Antianxiety effects of other competitive NMDA receptor antagonists were observed from previous studies [46,47].

Phencyclidine and its derivatives have produced anxiolytic effects in rodents via modulating NMDA, nicotinic acetylcholine and 5-HT receptors [46]. NMDA antagonist like phencyclidine which showed antianxiety effects in rodents causes characteristic side effects like hallucinations which limit its usage and ketamine produces profound drowsiness [47].

Further studies are needed to explore the antianxiety effect of memantine in other experimental animal models of anxiety like stair case test, vogel conflict test, social interaction test, novelty induced suppressed feeling latency test and hole board test to strengthen the evidence and address the anxiety disorders in the community in future. Recently, the over activity of hypothalamus pituitary adrenal axis (HPA) is postulated to play a significant role in anxiety disorders. The activity of the HPA axis is governed by the amygdala and hippocampus with interplay of various neuropeptides such as corticotrophin-releasing factor, substance P, vasopressin and neuropeptide Y (NPY) [48].

The hypothesis by Olney [49], suggest that over activation of NMDA receptors may cause subsequent damage of GABA neurons and further damage can be produced by disinhibited neurons e.g., glutamate, Ach, NPY. From the previous study by Wieronska JM et al., [50], it was evident that in the amygdala, the glutaminergic neurotransmission is mediated by NMDA receptors which may regulate neuropeptide Y neurons. Activity of memantine on NPY activity which might contribute to the antianxiety activity, throws light on its further exploration as antianxiety drug.

Conclusion

Memantine could be producing its antianxiety activity by blocking NMDA receptor. However, its modulating effect on NPY which might contribute to antianxiety effect cannot be ruled out. Further research is required to gain closer insights into the exact mechanism of action of memantine as antianxiety drug, which might benefit the patients of anxiety in clinical scenario.

(n=12), s-seconds, values expressed as mean±SEM. (*p< 0.001 vs. normal saline-control), (^†p< 0.01, ^††p< 0.001 vs. lorazepam), (^‡p< 0.01, ^‡‡p< 0.001 vs. memantine)(n=12), values expressed as mean±SEM. (*p< 0.001 vs. normal saline-control), (^†p< 0.01, ^††p< 0.001 vs. lorazepam), (^‡p< 0.01, ^‡‡p< 0.001 vs. memantine)s- seconds, CS-Central Square, (n=12), values expressed as mean±SEM. (*p< 0.001 vs. normal saline-control),(^†p< 0.01, ^††p< 0.001 vs. lorazepam), (^‡‡p< 0.001 vs. memantine)s- seconds, CS-Central Square, (n=12), values expressed as mean±SEM. (*p< 0.001 vs. normal saline-control), (^†p< 0.01, ^††p< 0.001 vs. lorazepam), (^‡‡p< 0.001 vs. memantine)

[1]. Kessler RC, McGonagle KA, Zhao S, Nelson CB, Hughes M, Eshleman S, Lifetime and 12-month prevalence of DSM-III—R psychiatric disorders in the United States: Results from the National Comorbidity Survey Archives of General Psychiatry 1994 51(1):8-19.  [Google Scholar]

[2]. Simon AB, Gorman JM, Advances in the treatment of anxiety: targeting glutamate Neuro Rx 2006 3(1):57-68.  [Google Scholar]

[3]. Barkus C, McHugh SB, Sprengel R, Seeburg PH, Rawins JN, Bannerman DM, Hippocampal NMDA receptors and anxiety: at the interface between cognition and emotion Eur J Pharmacol 2010 626(1):49-56.  [Google Scholar]

[4]. Jessa M, Nazar M, Bidzinski A, Plaznik A, The effects of repeated administration of diazepam, MK-801 and CGP 37849 on rat behaviour in two models of anxiety Eur Neuropsychopharmacol 1996 6(1):55-61.  [Google Scholar]

[5]. Poleszak E, Serefko A, Szopa A, Wosko S, Dudka J, Wrobel A, NMDA receptor activation antagonizes the NMDA antagonist-induced antianxiety effect in the elevated plus-maze test in mice Pharmacol Rep 2013 65(5):1124-31.  [Google Scholar]

[6]. Lipton SA, Chen HS, Paradigm shift in neuroprotective drug development: clinically tolerated NMDA receptor inhibition by memantine Cell Death Differ 2004 11:18-20.  [Google Scholar]

[7]. Lipton SA, Failures and successes of NMDA receptor antagonists: molecular basis for the use of open-channel blockers like memantine in the treatment of acute and chronic neurologic insults Neuro Rx 2004 1(1):101-10.  [Google Scholar]

[8]. Martin P, Carlsson M, Systemic PCP treatment elevates brain extracellular 5-HT: a micro dialysis study in awake rats Neuroreport 1998 9(13):2985  [Google Scholar]

[9]. Sharma AC, Kulkarni SK, MK-801 produces antianxiety effect in elevated plus-maze in mice Drug development research 1991 22(3):251-58.  [Google Scholar]

[10]. Shuijin He, Bausch SB, Synaptic plasticity in glutamatergic and GABAergic neurotransmission following chronic memantine treatment in anin vitro model of limbic epileptogenesis Neuropharmacology 2014 77:379-86.  [Google Scholar]

[11]. Molchanov ML, Guimaraes FS, Anxiolytic-like effects of AP7 injected into the dorsolateral or ventrolateral columns of the periaqueductal gray of rats Psychopharmacology (Berl) 2002 160:30-38.  [Google Scholar]

[12]. Criswell HE, Knapp DJ, Overstreet DH, Breese GR, Effects of ethanol, chlordiazepoxide, and MK-801 on performance in the elevated-plus maze and on locomotor activity Alcohol Clin Exp Res 1994 18:596-601.  [Google Scholar]

[13]. Kotlinska J, Liljequist S, A characterization of anxiolytic-like actions induced by the novel NMDA/glycine site antagonist, L-701,324 Psychopharmacology (Berl) 1998 135(2):175-81.  [Google Scholar]

[14]. Bagewadi HG, Nayaka SR, Venkatadri TV, Effect of memantine in experimental models of depression in swiss albino mice J Chem Pharm Res 2014 6(12):880-84.  [Google Scholar]

[15]. Bagewadi HG, Venkatadri TV, Rajeshwari B, To investigate the role of memantine as anxiolytic in elevated plus maze test and as antidepressant in tail suspension test in Swiss albino mice Int J Basic Clin Pharmacol 2015 4(2):213-88.  [Google Scholar]

[16]. Rodgers RJ, Animal models of anxiety. Where next? Behav Pharmacol 1997 8:477-96.  [Google Scholar]

[17]. Prut L, Belzung C, The open field as a paradigm to measure the effects of drugs on anxiety-like behaviours: A review European Journal of Pharmacology 2003 463(1–3):3-33.  [Google Scholar]

[18]. Boyce-Rustay JM, Holmes A, Genetic Inactivation of the NMDA Receptor NR2A Subunit has Anxiolytic- and Antidepressant-Like Effects in Mice Neuropsychopharmacology 2006 31(11):2405-14.  [Google Scholar]

[19]. Tomilenko RA, Dubrovina NI, Effects of activation and blockade of NMDA receptors on the extinction of a conditioned passive avoidance response in mice with different levels of anxiety Neurosci Behav Physiol 2007 37(5):509-15.  [Google Scholar]

[20]. Grzeda E, Wisniewska RJ, Wiśniewski K, Effect of an NMDA receptor agonist on T-maze and passive avoidance test in 12-week streptozotocin-induced diabetic rats Pharmacol Rep 2007 59(6):656-63.  [Google Scholar]

[21]. Zajaczkowski W, Frankiewicz T, Parsons CG, Danysz W, Uncompetitive NMDA receptor antagonists attenuate NMDA-induced impairment of passive avoidance learning and LTP Neuropharmacology 1997 36(7):961-71.  [Google Scholar]

[22]. Venable N, Kelly PH, Effects of NMDA receptor antagonists on passive avoidance learning and retrieval in rats and mice Psychopharmacology (Berl) 1990 100(2):215-21.  [Google Scholar]

[23]. Bullock S, Rose SP, Pearce B, Potter J, Training chicks on a passive avoidance task modulates glutamate-stimulated inositol phosphate accumulation Eur J Neurosci 1993 5(1):43-48.  [Google Scholar]

[24]. Wiśniewski K, Fedosiewicz-Wasiluk M, Hoły ZZ, Car H, Grzeda E, Influence of NMDA, a potent agonist of glutamate receptors, on behavioural activity in 4-week streptozotocin-induced diabetic rats Pol J Pharmacol 2003 55(3):345-51.  [Google Scholar]

[25]. Plaznik A, Palejko W, Nazar M, Jessa M, Effects of antagonists at the NMDA receptor complex in two models of anxiety Eur Neuropsychopharmacol 1994 4(4):503-12.  [Google Scholar]

[26]. Salunke BP, Umathe SN, Chavan Jagatpalsingh G, Involvement of NMDA receptor in low-frequency magnetic field-induced anxiety in mice Electromagnetic Biology and Medicine 2014 33(4):312-26.  [Google Scholar]

[27]. An XL, Tai FD, Effects of estradiol and clomiphene citrate on behaviour of BALB/cJ mice Journal of Shaanxi Normal University: Natural Science Edition 2010 38(5):82-86.  [Google Scholar]

[28]. Glotzbach E, Ewald H, Andreatta M, Pauli P, Muhlberger A, Contextual fear conditioning predicts subsequent avoidance behaviour in a virtual reality environment Cognition and Emotion 2012 26(7):1256-72.  [Google Scholar]

[29]. Blanchard RJ, Blanchard DC, Attack and defense in rodents as ethoexperimental models for the study of emotion Progress in Neuro-Psychopharmacology & Biological Psychiatry 1989 13:S3-14.  [Google Scholar]

[30]. Blanchard RJ, Griebel G, Henrie JA, Blanchard DC, Differentiation of anxiolytic and panicolytic drugs by effects on rat and mouse defense test batteries Neurosci Biobehav Rev 1997 21(6):783-89.  [Google Scholar]

[31]. Blanchard DC, Griebel G, Blanchard RJ, The mouse defensive test battery: Pharmacological and behavioural assays for anxiety and panic Eur J Pharmacol 2003 463(1-3):97-116.  [Google Scholar]

[32]. Cryan JF, Holmes A, The ascent of mouse: Advances in modeling human depression and anxiety Nature Reviews: Drug Discovery 2005 4(9):775-90.  [Google Scholar]

[33]. Hall CS, Emotional behaviour in the rat. I. Defecation and urination as measures of individual differences in emotionality Journal of Comparative Psychology 1934 18(3):385-403.  [Google Scholar]

[34]. Rodgers RJ, Cao BJ, Dalvi A, Holmes A, Animal models of anxiety: An ethological perspective Brazilian Journal of Medical and Biological Research 1997 30(3):289-304.  [Google Scholar]

[35]. Sufka KJ, Warnick JE, Pulaski CN, Slauson SR, Kim YB, Rimoldi JM, Antidepressant efficacy screening of novel targets in the chick anxiety-depression model Behav Pharmacol 2009 20(2):146-54.  [Google Scholar]

[36]. Fredriksson A, Danysz W, Quack G, Archer T, Co-administration of memantine and amantadine with sub/suprathreshold doses of L-Dopa restores motor behaviour of MPTP-treated mice J Neural Transm 2001 108(2):167-87.  [Google Scholar]

[37]. Sathyanathan V, Eswar Kumar A, Babu M Suresh, Rizwana I, Fatima F, Sedative-hypnotic activity on whole plant extract of Vernonia cinerea (linn.) less Int J of Res in Pharmacology & Pharmacotherapeutics 2012 1(2):169-71.  [Google Scholar]

[38]. Sharma AC, Kulkarni SK, Evidence for GABA-BZ receptor modulation in short term memory passive avoidance task paradigm in mice Met Find Exp Clin Pharmacol 1990 12(3):175-80.  [Google Scholar]

[39]. Sethi A, Das BP, Bajaj BK, The Anxiolytic Activity of Gabapentin in Mice The Journal of Applied Research 2005 5(3):415-22.  [Google Scholar]

[40]. Mattila-Evenden M, Bergman U, Franck J, A study of benzodiazepine users claiming drug-induced psychiatric morbidity Nord J Psychiatry 2001 55(4):271-78.  [Google Scholar]

[41]. Erreger K, Chen PE, Wyllie DJ, Traynelis SF, Glutamate receptor gating Crit Rev Neurobiol 2004 16(3):187-224.  [Google Scholar]

[42]. Barkus C, McHugh SB, Sprengel R, Seeburg PH, Rawins JN, Bannerman DM, Hippocampal NMDA receptors and anxiety: at the interface between cognition and emotion Eur J Pharmacol 2010 626(1):49-56.  [Google Scholar]

[43]. Martinez G, Ropero C, Funes A, Flores E, Blotta C, Landa AI, Gargiulo PA, Effects of selective NMDA and non-NMDA blockade in the nucleus accumbens on the plus-maze test Physiol Behav 2002 76:219-224.  [Google Scholar]

[44]. Guimaraes FS, Carobrez AP, De Aguiar JC, Graeff FG, Anxiolytic effect in the elevated plus-maze of the NMDA receptor antagonist AP7 microinjected into the dorsal periaqueductal grey Psychopharmacology (Berl) 1991 103(1):91-94.  [Google Scholar]

[45]. Chisari M, Zorumski CF, Mennerick S, Cross talk between synaptic receptors mediates NMDA-induced suppression of inhibition J Neurophysiol 2012 107(9):2532-40.  [Google Scholar]

[46]. Fryer JD, Lukas RJ, Noncompetitive functional inhibition at diverse, human nicotinic acetylcholine receptor subtypes by Bupropion, Phencyclidine and Ibogaine J Pharmacol Exp Ther 1999 288(1):88-92.  [Google Scholar]

[47]. Wiley JL, Cristello AF, Balster RL, Effects of site-selective NMDA receptor antagonists in an elevated plus-maze model of anxiety in mice Eur J Pharmacol 1995 294:101-107.  [Google Scholar]

[48]. Chen HS, Pellegrini JW, Aggarwal SK, Lei SZ, Warach S, Jensen FE, Open channel block of N-methyl-D-aspartate (NMDA) responses by memantine: therapeutic advantage against NMDA receptor-mediated neurotoxicity J Neurosci 1992 12(11):4427-36.  [Google Scholar]

[49]. Olney JW, Wozniak DF, Faber NB, Glutamate receptor dysfunction and Alzheimer’s disease Restorative Neurology and Neuroscience 1998 13(1-2):75-83.  [Google Scholar]

[50]. Wieronska JM, Branski P, Pałvcha A, Smiałowska M, The effect of competitive and non- competitive NMDA receptor antagonists, ACPC and MK-801 on NPY and CRF-like immune reactivity in the rat brain amygdala Neuropeptides 2001 35(5-6):219-26.  [Google Scholar]