SYNOPSIS

For approval of topic of the thesis to be submitted in the partial fulfillment of the
requirement for the degree of Master of Pharmacy in Pharmacology.

Role of agmatine on impaired hippocampal memory processing

insulin resistant diabetic rats

By

Manish M. Aglawe
B. Pharm

Guide

B G.Taksande
M. Pharm

SMT. KISHORITAI BHOYAR COLLEGE OF PHARMACY,

NEW KAMPTEE, NAGPUR, MAHARASHTRA, 441 002 (INDIA)

2010
Rashtrasant Tukdoji Maharaj Nagpur University, Nagpur-14.

Introduction:
The presence of insulin and insulin receptors in the brain suggests that the brain is a target organ
for insulin. In addition to its central role in hypothalamus (arcuate nucleus) in food intake and weight
control, a direct role of insulin & CNS insulin receptor signaling in cognitive functions, including
learning and memory, and the association of insulin receptor deterioration with brain degenerative
dementia (e.g.,Alzheimers disease) have attracted increasing interest. Efforts in behavioral,
electrophysiological and biochemical studies are running to uncover the cellular and molecular basis
for the CNS insulin/insulin receptor action on learning and memory. Insulin in hippocampus have
shown physiological role in memory processing. Acute delivery of physiological doses of insulin, but
not IGF-1, directly to the hippocampus specifically enhances spatial working memory via a PI3K
dependent mechanism, and increases local glucose removal from interstitial fluid. whereas blockade of
endogenous hippocampal insulin either with PI-3-kinase antagonists or small anti-insulin antibody-like
peptides impairs cognitive performance below baseline (Ewan C. McNay et al,2010). Spatial memory
training has been reported to alter hippocampal expression of insulin receptors (Zhao et al., 1999;
Zhao et al., 2004).Importantly direct specific blockade of endogenous intrahippocampal insulin
produces marked cognitive deficits, supporting a role for insulin in physiological hippocampal
memory processes (Ewan C. McNay et al,2010).
A potential role for insulin signallings in enhancement of cognitive function is of particular
interest in the context of type 2 diabetes mellitus . Diet-induced T2DM reduces baseline
hippocampally mediated cognitive function and impairs the ability of insulin to enhance memory
performance and/or increase hippocampal fuel metabolism. Animal models of T2DM show impaired
hippocampal translocation of GluT4 (Reagan, 2005; Winocur et al., 2005), reduced hippocampal
synaptic plasticity (Mielke et al., 2005). The abnormal reduction in hippocampal glucose use seen in
the hippocampus of AD patients is accompanied by severe impairment to hippocampal insulin signal
transduction (Frolich et al., 1998; Hoyer, 2004; Steen et al., 2005). Neuronal insulin resistance has
been suggested to be directly linked to the development of neurodegenerative disease (Schubert et al.,
2004). Central insulin resistance may be an important contributor to the cognitive deficits associated
with T2DM, age-related cognitive decline and dementia(Ewan C. McNay et al,2010). In addition, it
appears that chronic hyperinsulinemia is directly linked to abnormal brain amyloid accumulation (Ho

et al., 2004),a defining marker for AD. Hence drugs which improve sensitivity to insulin are currently
being tested in human trials for treatment of AD (Alzheimers disease); indeed, AD has been described
as Type 3 diabetes (Steen et al., 2005).
Agmatine an endogenous amine that exist in mammalian brain ,binds to several target receptors
such as imidazoline, N-methyl-D-aspartate (NMDA), nicotinic cholinergic (NIC), 2-adrenergic,
seretonin receptors and inhibits nirtic oxide synthase and has been proposed as a novel
neurotransmitter/neuromodulator. The distribution of agmatine-containing neurons is concentrated in
regions of the brain that subserve visceral and neuroendocrine control, processing of emotions, pain
perception and cognition and memory. Agmatine has been shown to be densly located in hippocampal
pyramidal cells of rat (Reis, D. J. and Regunathan, S), 1998. Functionally, imidazoline receptors are
introduced to mediate the smooth muscle contraction, insulin secretion and renal metabolism (W.
Raasch, U.,et al,2002). An activation of central I2-imidazoline receptors in brain by agmatine (i.c.v)
lowers plasma glucose of STZ-diabetic rats (Shuo-Bin Jou,et al,2004).Also the activation of peripheral
I(2)-imidazoline receptors by agmatine ameliorate insulin resistance in fructose-rich chow-fed rats i.e
in T2DM(Wen Ching Ko,et al,2008, Chin-Hui Su,et al,2009). The binding sites for I2 imidazoline
receptors are located in cortex, hippocampus, cerebellum and hypothalamus (Nicholas MacInnes &
Sheila L. Handley,2005) .Thus the effect of agmatine on activation of hippocampal I2 imidazoline
receptor on insulin sensitivity in insulin resistance may influence the memory processing & hence is
of interest in AD and age related cognitive functions.

Objective :
1. To study the effect of agmatine on hippocampal memory process in insulin resistance rat.
2. To study the role of imidazoline I2 receptor in the agmatine mediated effect.

Plan of work :
1.

The rats will undergo surgery for implantation of guide cannula in order to facilitate the intrahippocampal drug administration .The 8 day post-surgery recovery period will be given, during

2.

which they will be handled to adapt experimental conditions.

The induction of insulin resistance (T2DM) will be carried out in rats by giving high fat diet

3.

followed by sub effective dose of streptozotocin and will be evaluated for same.
Animals will then be trained for spatial learning in morris water maze, which includes
familiarization on 1st day followed by training on 2nd ,3rd & 4th day, and on 5th day retrieval will
be checked. All the three phases i.e. pretraining, post-training and retrieval phase will be

4.

evaluated for insulin treatment .

Depending upon this effect ,the either phase will be selected for administration of insulin and

5.

agmatine for assessment of insulin sensitivity.

Further the role of imidazoline I2 receptor will be evaluated by using I2 antagonist along with
agmatine.

Material and method :

Animals :
Male sprauge-dawley rats weighing 160-180 g will be used. . They will be housed in a
temperature- (22+1 C) and humidity-controlled (50+5%) environment and will have free access to
food and water. The animals in which T2DM is to be induced will be given high fat diet(HFD) for 2
weeks followed by low dose of streptozotocin (35mg/kg), after one week of which the drug testing can
be carried out. The animals will be treated in accordance with the CPCSEA guidelines for the care and
use of laboratory animals and in agreement with the institutional animal ethical committee. The
number of animals used and their suffering will minimized in all experiments designed.

Drugs:

The drugs used will be Insulin, Agmatine sulphate and BU224(I2-antagonist).The artificial
cerebrospinal fluid (aCSF) will be prepared in the laboratory.
Drug solutions and administration:
The aCSF will be used as vehicle for all drugs.The composition of aCSF is (composition: 125
mM NaCl, 10 mM glucose, 1.25 mM Na2HPO4 , 2.5 mM CaCl2 , 1.5 mM MgSO4 , 26 mM NaHCO2
treated for 30 min with 5% CO : 95% O, pH 7.4) . Microinjections will given to maze-tested animals
over 2 min in a total volume of 0.5 l, into the left hippocampus, 10 min prior to testing.

Intra-hippocampal Cannula Implantation:

The animal will be anaesthetized with pentobarbital (50 mg/kg i.p.) and the skull will be shaved
with hair remover. Iodine solution will be applied as an antiseptic to exposed skin. The animal will be
placed in a stereotaxic frame with flat skull position fixed in stereotaxic apparatus for implantation of
guide cannula. A midline incision will be made and skin and underlined periostium will be retracted.
Animals received either a microinjection guide cannula. into the left hippocampus as described
previously (McNay et al., 2000) with coordinates relative to bregma +3.8 mm AP, +5.0 mm lateral,
-4.5 mm from dura, nosebar at 5.0 mm above interaural line. After surgery, the animal will be placed
in individual cage for not less than 8 days, during which the animal will be handled to adapt the future
experimental conditions. The animal will be treated prophylactically with oxytetracyclin (25 mg/kg
i.p.) and Neosporin to avoid infection.

Apparatus:
Morris water maze

Morris water maze is the circular tank with 110 cm diameter, about 60 cm high filled with
water up to 30 cm maintained at 2510C. Water will be made opaque using water soluble, non-irritant
white color. The maze will be equipped with platform (9 cm diameter) 0.5 cm above or below water
level as per day of trial. The tank will be divided into four quadrants and marked accordingly, all
quadrants will be equipped with cues. The Morris water maze will be prepared and standardized in our
laboratory conditions.
Training:
It consisted of different phases such as:
1) Day 1: Familiarization
Animal will be kept on the platform for 60 seconds, which will be 0.5 cm above water
level. After 60 seconds it will be returned to its home cage. (During this period, sometimes
animal used to jump in water and explore through the maze so that it should be familiarized with
the water maze and cues inside it).
2) Day 2,3,4: Training
It consists of four sessions of three trials each with inter session delay of 15-20 minutes. All
conditions will be same as on day 1 except platform submerged 0.5 cm below water level. In each
trial animal will be introduced from starting point in the tank and allowed to locate and get on the
platform. The animal not finding platform within 60 seconds will be guided by stick to reach the
platform. Maximum latency period of 60 seconds will be recorded. Those animals not finding
platform within 60 seconds on day 3 and 4 will be excluded from the study. In each session,
starting point will be randomly changed. When animal will found platform or after 60 seconds, it
will be removed from the maze and kept under warm light to prevent hypothermia, which may
affect the next training trial. After this animal will be shifted back to its home cage.
3) Day 5: Probe test/ Retrieval
It will start exactly 24 hours after last trial on day 4. Here platform will be removed and animal
will be introduced from the starting point exactly opposite to the platform quadrant with its face
towards wall of the tank. Different treatments will be given starting from 30 minutes prior to
retrieval.

Mice will be evaluated during probe trail for the following parameters:
1) Time spent in quadrant in which platform will be placed.
2

2) Escape latency, i.e. time taken by animal to reach the position of platform.
3) Number of entries in platform quadrant.
4) Number of crossing over the position of platform.

Treatment groups:
A) Effect of insulin on different phases of memory
1)Pre-training administration
a) aCSF
b) insulin(100U,1mU) in normally fed animal
2)Post-training administration
a) aCSF
b) insulin(100U,1mU) in normally fed animal
3)Pre-testing administration
a) aCSF
b) insulin(100U, 1mU) in normally fed animal
B) Effect of agmatine on insulin sensitivity
Depending on the observed effect of insulin on different phases of memory, the agmatine
and insulin will be given in either of the three phases in HFD (high fat diet) animal.
1) Insulin (effective dose) in normally fed animal
2) Insulin (effective dose) in HFD animal
3) Agmatine (100,50 mol/kg) + insulin in HFD animal

C) Role of imidazoline I2 receptor in agmatine mediated effect

To check the effect mediated by agmatine is through imidazoline receptor or not,the I2imidazoline antagonist is given before agmatine.
1) BU224( 10mg/kg,i.p)+ Agmatine + insulin

Verification of cannulae placements:

After completion of experimental sessions, each animal will be sacrificed with an overdose of
pentobarbital sodium. Mice received intra-hippocampal injection of ink (1% methylene blue, 0.5
l/side). Brain will be removed and sections will be examined to determine the location of cannulae.
The cannula placement will be verified using the atlas of Paxinos and Franklin, (2004). Only data form
animals with correct cannulae placement will be considered.

Data analysis:
To observe the spatial learning, comparison between control and agmatine treated groups
will be done by unpaired t-test. For analysis of other data, two way analysis of variance (ANNOVA)
followed by by post hoc Bonferroni multiple comparison test will be used. A nonparametric test will
be used because of cut off time of 60 seconds. The data is expressed as a mean SEM (standard error
mean) and a value of P < 0.05 will be considered to be statistically significant.
Possible outcomes:
Agmatine may increase the insulin sensitivity in hippocampus and thereby facilitate memory
processing in T2DM.

Reference
Babri, A., Badie, H., Khamenei, S., & Seyedlar, M. (2007). Intrahippocampal insulin
improves memory in a passive-avoidance task in male wistar rats. Brain and Cognition, 64, 8691.
Frolich, L., Blum-Degen, D., Bernstein, H.-G., Engelsberger, S., Humrich, J., Laufer, S., et al. (1998).
Brain insulin and insulin receptors in aging and sporadic Alzheimers disease. Journal of Neural
Transmission, 105, 423438.

Ho, L., Qin, W., Pompl, P., Xiang, Z., Wang, J., Zhao, Z., et al. (2004). Diet-induced insulin resistance
promotes amyloidosis in a transgenic animal model of Alzheimers disease. FASEB Journal, 18, 902
904.
Hoyer, S. (2004). Glucose metabolism and insulin receptor signal transduction in Alzheimer disease.
European Journal of Pharmacology, 115125.
Joua,S.B. , Liub I.M., Cheng,J.T., (2004), Activation of imidazoline receptor by agmatine to lower
plasma glucose in streptozotocin-induced diabetic rats, Neuroscience Letters ,358, 111114
Koa,W.C., Liub ,I.M., Chung ,H.H., Cheng, J.T., (2008), Activation of I2-imidazoline receptors may
ameliorate insulin resistance in fructose-rich chow-fed rats, Neuroscience Letters, 448, 9093
MacInnes N., Handley, S. L., (2005), Autoradiographic localisation of [3H]2-BFI imidazoline I2
binding sites in animal brain ,European Journal of Pharmacology ,516 , 139 144
McNay, E. C., Ong, C. T., McCrimmon, R .J., Bogan, J. S., Sherwin, R.S., (2010), Hippocampal
memory processes are modulated by insulin and high-fat-induced insulin resistance, Neurobiology of
Learning and Memory,93,546-553.

Mielke, J., Taghibiglou, C., Liu, L., Zhang, Y., Jia, Z., Adeli, K., et al. (2005). A biochemical and
functional characterization of diet-induced brain insulin resistance. Journal of Neurochemistry, 93,
15681578.

Moosavi, M., Naghdi, N., & Choopani, S. (2007). Intra CA1 insulin microinjection improves memory
consolidation and retrieval. Peptides, 28, 10291034.
Reagan, L. (2005). Neuronal insulin signal transduction mechanisms in diabetes
phenotypes. Neurobiology of Aging, 26S, S5659.

Srinivasan K., Viswanad B., Asrat L., Kaul C.L., Ramarao P., (2005), Combination of high-fat diet-fed
and low-dose streptozotocin-treated rat: A model for type 2 diabetes and pharmacological screening,
Pharmacological Research 52 313320
Schubert, M., Gautam, D., Surjo, D., Ueki, K., Baudler, S., Schubert, D., et al. (2004). Role for
neuronal insulin resistance in neurodegenerative diseases. PNAS, 101, 31003105.
Steen, E., Terry, B., Rivera, E., Cannon, J., Neely, T., Tavares, R., et al. (2005). Impaired insulin and
insulin-like growth factor expression and signaling mechanisms in Alzheimers disease: Is this type 3
diabetes? Journal of Alzheimers Disease, 7, 6380.

Sua, C.H., Liub ,I.M., Chung ,H.H., Cheng, J.T , (2009),Activation of I2-imidazoline receptors by
agmatine improved insulin sensitivity through two mechanisms in type-2 diabetic rats, Neuroscience
Letters 457 , 125128
Winocur, G., Greenwood, C., Pirolli, G., Grillo, C., Reznikov, L., Reagan, L., et al. (2005). Memory
impairment in obese zucker rats: An investigation of cognitive function in an animal model of insulin
resistance and obesity. Behavioral Neuroscience, 119, 13891395.
Zhao, W-Q., Chen, H., Xu, H., Moore, E., Meiri, N., Quon, M., et al. (1999). Brain insulin receptors
and spatial memory: Correlated changes in gene expression, tyrosine phosphorylation, and signalling
molecules in the hippocampus of water maze trained rats. Journal of Biological Chemistry,274,
3489334902.
Zhao, W-Q., Chen, H., Quon, M., & Alkon, D. (2004). Insulin and the insulin receptor in experimental
models of learning and memory. European Journal of Pharmacology, 490, 7181.

Signature of guide

B G. Taksande

Date:
Place:

Signature of candidate

Manish M. Aglawe