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.

Interaction between Agmatine and CRF1 Receptor in Chronic

Unpredictable Mild Stress Induced Depression in Mice.
By

Madhura P. Dixit

Guide

Co- guide

Prof. C. T. Chopde

Mr. B. G. Taksande

M. Pharm., Ph.D., F.I.C

M. Pharm.

SMT. KISHORITAI BHOYAR COLLEGE of PHARMACY,

Kamptee, Nagpur (Mh) 441002.
India.
Rashtrasant Tukodji Maharaj Nagpur University, Nagpur.
2011 2012.

Introduction
Several evidences have demonstrated link between stressful life events with an
increased vulnerability for affective and anxiety disorders. Stressful events often precede the
onset of depression and also increase the severity of the illness by several folds. The
regulation of stress response is co-ordinated through complex circuitry of several
neuropeptides, biogenic amines and brain neuromessengers at the level of hypothalamus.
Agmatine, a putative neurotransmitter is increased in response to stress. Agmatine
attenuated polysaccharide induced hyperthermia, (Aricioglu and Regunathan, 2005)
immobilization induced elevated corticosterone levels and glutamate efflux in brain nuclei
associated with modulation of stress response. (Zhu et al., 2008). Agmatine is synthesised and
expressed in different brain regions like frontal cortex, hippocampus and pituitary gland (Iyo
et al., 2006) which have abundant agmatine immunoreactivity and co-localized with
neuropeptide (Wang et al., 2002). In view of this background, it is proposed as an endogenous
modulator of stress. However the precise mechanism by which agmatine exhibit response
remains poorly understood.
Corticotropin releasing factor (CRF), 41 amino acid peptide is widely distributed
throughout the central nervous system, with highest density in hypothalamic paraventricular
nucleus (PVN). CRF system in the brain plays critical role in regulation of neuroendocrine,
autonomic and behavioural response to stress. Central administration of CRF produced
physiological and behavioural changes almost identical to those observe in response to stress
including increased heart rate and mean arterial pressure due to alterations in the autonomic
nervous system, suppression of exploratory behaviour in an unfamiliar environment,
induction of grooming behaviour, increased conflict behaviour, and decreased food intake and
sexual behaviour (Dunn and Berridge 1990, Owens and Nemeroff 1991, Koob et al., 1993).
In non-human primates, central CRF administration increases vocalizations, decreases
environmental exploration and increases huddling and lying down behaviour which are
symptoms of behavioural despair typically seen after maternal separation in infant monkeys
(Kalin 1990). The behavioural effects of centrally administered CRF can be reversed by CRF
antagonists and are independent of activation of the HPA axis. Furthermore, CRF receptor
antagonist alone attenuates many of the behavioural consequences of stress, under-scoring the

role of endogenous CRF in mediating many stress-induced behaviours. (Heinrichs et al.,

1995)
It is important to note that both agmatine and CRF are widely distributed in brain
including hypothalamus and release in response to stress. Agmatine normalised the levels of
corticosterone elevated during stress induction in rodents. However, their direct interaction
in regulation of stress response remains to be investigated.
We hypothesize that agmatine might modulate the functioning of CRF in framework
of hypothalamus to regularise the stress response and their imbalance may be associated with
neurobiological abnormality in mammals. This study designed to explore the possible
interaction between agmatine and CRF in stress induced depression in mice.
Chronic mild stress induced depression represents relatively realistic animal model
analogue of the depressed patients. Furthermore the levels of stress needed to implement the
chronic mild stress are ethically unproblematic. In addition the time course of antidepressant
action of drug in this model closely resembles to clinical time course (Willner et al, 1991).
Therefore the present study focuses on the induction of depression by applying mild stressors
chronically and its modulation by exogenous agmatine or antalarmin administration.
Material and methods
Subjects:
Adult Swiss Albino mice weighing 22-30 g will be housed in polypropylene cages in a
temperature (252 0C) relative humidity (5070%) and maintained on a 12:12-h light/dark
cycle (lights on 07:0019:00 h). Food and water will be provided ad libitum except during
specific experimental protocols. The experiments will be performed as per the protocol
approved by Institutional animal and ethical committee according to the guidelines of
CPCSEA
Drugs:
Agmatine
CRF1 antagonist

Antalarmin

Agmatine Modulators:

L Arginine

Aminoguanide

Arcaine

DMFO

D - arginine

Chronic Unpredictable Mild Stress (CUMS) procedure:

The CUMS procedure consists of following stressors:1. Overnight food and/or water deprivation (14-15 hrs)
2. Cage tilt at 30 (3 hrs)
3. Damp sawdust cage (3 hrs)
4. Exposure to continuous light (14- 15 hrs)
5. Restrain stress (3 hrs)
6. Sole housing of every animal (14-15 hrs)
7. Crowded housing (3 hrs)
8. Exposure to empty bottle following to water deprivation. (1 hr)
Everyday two or three stressors will be given to the mice randomly. During the drug
pretreatment, stress will be continued throughout. FST will be performed on 22 nd day.
(Beilajew et al., 2002; Gorkaet Z.et al., 1996).
Plan of work:
1. Standardization of CUMS to induce depression.
A group of mice will be exposed to the stress procedure for 21 days and will be
observed for FST as a model of depression on 22nd day.
The control group will be handled as frequently as CMS group but will not be
subjected to any stressor.
2. Dose dependent effect of chronic agmatine or Antalarmin (CRF1 antagonist)
administration on CUMS induced depression.
Separate groups of mice exposed to CUMS will be administered simultaneously with
different doses of agmatine (5, 10, 20, mg/kg i.p.) daily at 9 a.m. for 21 days, and will
be subjected to forced swim test on 22nd day.
3. Effect of acute agmatine or CRF1 antagonist-Antalarmin administration on
CUMS induced depression.

In a separate group of mice subjected to CUMS, agmatine or Antalarmin (5,10, 20,

mg/kg i.p.) will be administered acutely 30min before being tested in Forced Swim
Test or Open field test.
4. Effect of Antalarmin and Agmatine combination on CUMS induced depression.
Separate group of mice will be administered with sub-therapeutic dose of Antalarmin
and sub therapeutic dose of Agmatine and will be subjected to FST on 22nd day.
5. Effect of agmatine modulators on inhibitory effect of Antalarmin on CUMS
induced depression
Separate group of animals will be daily treated with agmatine precursor L- Arginine,
an ornithine decarboxylase inhibitor, DMFO, a diamine oxidase inhibitor,
Aminoguanide and an agmatininase inhibitor arcaine 15 min before Antalarmin
during the period of CUMS and will be subjected to FST.
In separate experiment, the effect of arginine decarboxylase inhibitor, D- Arginine that
block biosynthesis of agmatine will be examined on the effect of Antalarmin.
6. Effect of Agmatine on corticosterone levels in mice subjected to CUMS.
Immediately after completion of behavioral test the blood samples from CUMS and
CUMS+ Agmatine treated group will be collected and subjected to measurement of
corticosterone levels by HPLC technique as reported earlier ( Sheikh et al., 2007).
7. Determination Agmatine levels in mice subjected to CUMS.
Mice subjected for 21 days of CUMS will be checked for agmatine levels by HPLC
method as reported earlier. (Halaris et al., 1996)
Behavioral Paradigms:
Forced Swim Test:
Mice will be placed into an 800ml glass beaker (21 cm height and 12 cm internal diameter)
containing 450-500ml of water (approximately 25C). The duration of immobility will be
manually scored during last 4 min of a 6 min test. Mice will be considered immobile when
floating and only making those movements necessary to keep their head above water.
(Nielsen et al., 2004)

Open Field test:

This test will be performed to assess whether drug effect on immobility is associated with
changes in motor activity. The ambulatory behaviour will be assessed in an open-field test as
described previously (Eckeli et al., 2000; Zomkowski et al., 2002). The apparatus consists of
a wooden box measuring406050 cm high. The floor of the arena will be divided into 12
equal squares. The number of squares crossed with all paws (crossings) will be counted in a
6-min session. Animals were placed in the test field 15 or 30 min after. i.p. administration,
respectively.
Possible Outcome:
The study may provide functional evidences for possible association between agmatine and
CRF in regulation of stress associated depression and may be helpful to derive novel
pathways for treatment of stress induced neurological disorders.

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Signature of Guide

Signature of Co-guide

Prof. C.T. Chopde

Mr. B. G. Taksande

Place:

Signature of Student

Madhura P. Dixit

Date: