Influence of Metformin on learning and memory in experimental Amnesia model in Mice

Background: Metformin belongs to the antidiabetic drug but it has been shown some benefi cial effects towards Central Nervous System (CNS) disorders and found to be neuroprotective by inhibiting apoptosis in neuronal cortical cells in various animal models apart from its anti-diabetic potential as per the available reports. Aim and objectives: The present study was aimed to screen the infl uence of Metformin (MET) against experimentally induced amnesia using scopolamine (SCOP) in mice. Materials and methods: Twenty adult albino mice were used for the current study and all the animals were divided into 4 groups of fi ve animals each and treated for about three weeks with MET (10 mg/kg.p.o) whereas amnesia was induced by using SCOP (3 mg/kg ip). The various cognitive skills were assessed by using conventional apparatus like an elevated plus maze (EPM), Morris water maze (MWM), motor coordination by rota rod test and neurotoxicity by chimney test. Results: Results from EPM and MWM indicated the rise in transfer latency and escape latency respectively were due to amnesic effect by administration of SCOP when compared to control animals received normal saline. Administration of standard drug Piracetam (PTM) and test drug MET showed a signifi cant reduction of (p<0.05) in the transfer latency (TL) period were noticed. Thus, the administration of MET signifi cantly ameliorated SCOP-induced amnesia in EPM as indicated by an increase in infl ection ratio and reduction in TL. In the MWM test, MET administration showed a benefi cial reduction of escape latency (EL) period (p<0.05) when compared to SCOP induced amnesia animals. There were no signifi cant changes in motor coordination and no neurotoxic effects were observed as per the results. Conclusion: From the results, the administration of SCOP resulted in signifi cant alterations in the cognitive skills of animals particularly impaired learning and memory skills whereas acute administration of MET for about three weeks, resulted in signifi cant amelioration of scopolamine-induced amnesia. This was noticed as indicated by signifi cant reduction (p<0.05) in transfer latency in the EPM test and signifi cant reduction of (p<0.05) escape latency in the MWM test which refl ects the benefi cial effects of MET against scopolamine-induced memory and behavioral defi cit in mice. Research Article Infl uence of Metformin on learning and memory in experimental Amnesia model in Mice Jayaraman Rajangam1*, Shvetank Bhatt2, Navaneetha Krishnan1, Mounika Sammeta1 and Lagumsani Joshna1 1Department of Pharmacology, Sree Vidyanikethan College of Pharmacy – Tirupati, 517 501. Andhra Pradesh, India 2Department of Pharmacology, Amity Institute of Pharmacy, Amity University, Madhya Pradesh,Gwalior– 474005, India 3Department of Pharmaceutics, Sree Vidyanikethan College of Pharmacy – Tirupati, 517 501. Andhra Pradesh, India Received: 02 February, 2020 Accepted: 11 May, 2020 Published: 12 May, 2020 *Corresponding author: Dr. Jayaraman Rajangam, Professor & Head, Department of Pharmacology, Sree Vidyanikethan College of Pharmacy, Tirupati, 517501. Andhra Pradesh, India, E-mail:


Introduction
Cognition is one of the vital complex functions of the brain of every living species including humans comprising of perception, registration, consolidation, storage, recollection throughout the life span of every individual. Any impairment in memory called amnesia aff ects not only the individual's quality of life and it is also considered as a most important CNS disorder due to the loss of memory due to declining of neuronal population because of the aging process, any neurodegenerative disorders, head injury, brain infections, genetic abnormalities, etc. In elderly patients, dementia is associated with various co-morbid Citation: Rajangam  conditions like diabetes mellitus, hypertension, dyslipidemia, cardiovascular diseases, etc [1]. Due to an increase in life expectancy and lifestyle modifi cations, more incidence rates of memory loss or dementia are being reported and its burden is rising day by day globally. As per the records, in India around 3.7 million people with dementia in 2010 while the incidence rates are expected to be doubled by 2030 and over 100 million by 2050 [2].
As per the current scenario, only limited cognitive treatment options like the use of Cholinesterase inhibitors and few other therapeutics medicaments were used to minimize dementiarelated cognitive dysfunction symptomatically [3] and memory-related problems to a certain extent. Hence there are lots of potentials to develop therapeutic agents used for the management or prevention of early stages of dementia due to aging and other pathological complications and that kind of drug still need to be explored. Very recently, the focus has been directed towards statins and other drugs like metformin [4].
Apart from the anti-diabetic eff ect, Metformin has shown diverse therapeutic eff ects like neuroprotection on Central nervous system disorders like Alzheimer's and Parkinson's diseases in various animal models. Both are considered as major neurodegenerative disorders and metformin treatment found to be neuroprotective by inhibiting apoptosis in neuronal cortical cells in animals as per the reports of performed studies [5,6].
In continuation, There are many reports to support the benefi cial eff ect of metformin on neurogenesis along with improvement in spatial memory and signifi cant increase of health span and lifetime in various experimental animal models [7,8]. Moreover, Metformin prevented the free radical related oxidative stress which in turn prevented pathological consequences of Alzheimer's in animal models [9]. However, the potential of metformin in amnesia is remaining to be explored and deserves further investigation. Hence, we aimed to proceed to screen the infl uence of metformin on cognition against the experimental amnesia model in mice.

Experimental animals
Adult Albino male mice of 20-25 g, aged between 3-4 months were used for the present study. After the adaptation period of about one week, all the animals were randomly assigned to

Drugs and chemicals
Scopolamine was procured from Sigma Chemicals (St. Louis, USA) and dissolved in 0.9% Normal saline to inject intraperitoneally to induce amnesia to the experimental animals. Metformin and Piracetam were received as a gift sample and suspended in 1% carboxymethylcellulose (CMC) to administer orally to respective groups. Piracetam is a nootropic agent, used as a standard in the present study and the dose of Piracetam, Metformin has been taken from the earlier reported studies [10][11][12].

Experimental protocol
A total of twenty male albino mice (20-25gm) were randomly assigned to diff erent groups of fi ve animals in each. The duration of the treatment was 21 days.Exactly 30 min before the screening of behavioral parameters, except control animals, all other groups were received scopolamine injection to induce amnesia whereas Piracetam was used as a standard drug and cognitive, memory defi cit was assessed using standard laboratory tests [13]. Cognitive and behavioral assessment was performed every week.

Assessment of neurotoxicity by chimney test
Chimney test was used to assess the neurotoxic property of test drug MET on animals. This procedure was carried out by following the standard protocol for about 45 min subjected to suffi cient trials. Briefl y, mice were placed to a 25 cm long and 3cm diameter wide glass tube and made a mark at a point at 20cm from its base. As soon as the mice moved to another end of the tube, the glass tube was kept in a vertical position, and immediately the mouse will try to climb backward. The ability of animals to leave the tube and climbing backward towards moving out from the tube within 1min was taken as an endpoint to consider the lack of neurotoxic properties of Metformin [14].

Assessment of transfer latency by EPM
Mice were placed individually in the central part of the plus-maze apparatus in such a way that, head of animals facing towards the open end. The time taken for each animal to reach out to the closed platform was noted as transfer latency (TL) and observed for a cutoff period of about 90 sec. Before the beginning of the experiment adequate free trials were given and animals were allowed to move freely in the apparatus to minimize the bias [15].

Assessment of escape latency by MWM
A large circular pool (Diameter of 150 cm and height of 45 cm ) fi lled with water to a depth of 30 cm at 28±1°C was used.
To this pinch of titanium dioxide powder was added to make opaque of the circular pool. The whole pool was then equally divided into four equal quadrants and named as Q1, Q2, Q3, and Q4 and the hidden platform was placed at the center of the pool.
Animals were allowed to move freely and suffi cient trial was allowed before the experiments to avoid bias. Time is taken by Citation: Rajangam  each animal to identify the hidden platform was noted as escape latency (EL) and continued to a Cut off period of about 120sec [16].

Assessment of muscle grip strength by rotarod Test
Rotarod is considered an important experimental tool used to record the muscle coordination and relaxant property of the given drug. In the present study in the rotarod test, latency to fall animals is automatically recorded. Animals were subjected to prior exposure to the rotating bar and selected upon the suitability and ability to hold the rotating bar in a specifi ed time limit [17].

Statistical analysis
The statistical analysis was carried out by one-way analysis

Results and Discussion
Morris water maze (MWM), elevated plus maze tests were commonly used to measure learning and memory parameters in diff erent animal models and particularly behavioral manipulations in rodents models among the diff erent models available [18]. In the present study, Transfer latency was considered as one of the important parameters to assess the learning and memory process in the experimental animals The infl uence of MET on EL was depicted in Table 1  In the present study, the protective eff ect of MET on experimental amnesia model using SCOP was screened using diff erent exteroceptive behavioral models like MWM, EPM tests and rotarod test, etc. Among many behavioral screening models, both EPM and MWM models were used extensively to screen learning and memory impairments in diff erent animal models particularly, behavioral manipulations in rodents [19][20]. On the other hand, PTM is a nootropic agent, used as a standard in the present study and the dose of PTM has been taken from the earlier studies [21].
As per the results of the present investigation, SCOP administration to experimental animals refl ected signifi cant prolonged TL in the EPM task and prolonged EL in MWM task than the control animals received normal saline. In contrast, acute administration of MET showed signifi cant protection of these parameters, and the diminishment of both TL and EL were noticed.
The key fi nding of this present investigation is that MET attenuated the SCOP-induced learning and memory impairments to a signifi cant extent were refl ected in EPM and MWM models in mice and this may be due to protective eff ect by MET treatment, probably MET attenuated SCOP-induced neuronal damage in the brain. Because there were no observable signifi cant changes that were seen in motor activity and or motor coordination in the rotarod test. Hence, results show that MET treatment improves spatial learning and memory in SCOP received animals, and they were able to fi nd the hidden platform as soon as quickly and thereby reduced escape latency was noticed.
Results from chimney test reveal that all the mice were able to leave the tube within 1 min which indicates that there were no neurotoxic eff ects were observed with MET (10 mg/kg p.o) and it has no impact on motor coordination, muscle grip strength in the rotarod test against SCOP-induced amnesia model. Also, it has been shown that in earlier studies, MET can act as a neuroprotectant against apoptotic cell death [22] and it can attenuate tau phosphorylation, A generation, and increases antioxidant protection, can improve cognitive function [23].
Following that, our result also demonstrates that MET can improve learning and memory function of mice against scopolamine-induced amnesia.

Conclusion
In conclusion, the administration of SCOP resulted in