REVIEW OF LITERATURE

Stress

Organisms survive by maintaining the dynamic equilibrium with their

environment. Organization of this homeostasis exists at molecular, cellular,

physiological and behavioural levels. Successful adaptation requires not only the

ability to respond to stress but also the ability to control the stress response

appropriately (reviewed in OConnor et al. 2000). The mammalian response to

stress exists to maximize an individuals chances of surviving a life-

threatening experience, to allow the organisms chance to

reproduce and thereby increase its contribution to the gene pool at

a later time. The stress-response is classically divided into three categories:

behavioral, autonomic, and hormonal responses. Behavioral responses govern the

conscious response to a stressor, and include fear, anxiety, heightened vigilance,

clarity of thought, anorexia, and decreased libido. Autonomic responses, mediated by

the sympathetic nervous system, mobilize certain organ systems to carry out fight or

flight activities. These include increased heart rate, cardiac output, breathing rate, and

bronchodilation, increased blood flow to brain and muscles, decreased blood flow to

skin and gut, activation of innate immunity, maintenance of blood glucose, and

increased free fatty acid generation. Hormonal responses largely provide fuel for these

activities (during a time of induced anorexia) and include maintenance of blood

glucose, increased free fatty acid generation, maintenance of blood pressure, and

restraint of the immune system (reviewed in Majzoub, 2006).

Stress is a state of threatened homeostasis provoked by psychological,

physiological or environmental stressors (Chrousos and Gold, 1992). The stimuli that
 Review of Literature

disturb homeostasis are termed as stressors. Originally, the word Stress was coined

by Selye and he proposed that in addition to specific homeostatic and local responses

caused by demands on the body, there was a single overall stereotypic stress response

common to all stressors which he referred as the general adaptation syndrome.

Selye also observed marked changes in the size of endocrine tissues subsequent to

stressor exposure (Selye, 1939; 1946). Selyes observations have been confirmed

many times over, and it is clear, that endocrine responses constitute an integral

component of the stress response (Van de Kar, 1991; Stratakis and Chrousos, 1995).

Stress can affect the hormonal control of metabolism (Weissman, 1990; Wenk, 1998),

reproduction (Moberg, 1991; Rivier and Rivest, 1991; Rivest and Rivier, 1995),

growth (Stratakis et al. 1995) and immunity (Peterson et al. 1991; Sheridan et al.

1998). Since hormone signalling plays a vital role in the maintenance of homeostasis,

virtually every endocrine system responds in some fashion to specific stressors. Stress

can acutely alter cognition, memory, cardiovascular activity and glucose, protein and

fat metabolism (reviewed in Lightman, 2008). Other investigators (Chrousos, 1998,

2000; Habib et al. 2001; Charmandari et al. 2003; Dallman, 2003) have further

clarified the concept of stress and summarized it as follows: The internal dynamic

equilibrium is constantly challenged by intrinsic or extrinsic adverse forces or

stressors.

Acute and chronic stress: Stress responses vary enormously according to the

nature of the stressor. A rapid onset of unpredictable stress event, such as a predatory

attack, will cause a short term acute stress response. The long term maintenance of

adverse environmental conditions for example, an unpredictable reduction in food

supply, will lead to chronic stress response.

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 Review of Literature

The paraventricular nucleus (PVN) is located in the hypothalamus on either

side of the third ventricle and comprises of two sets of neurons- the parvocellular and

magnocellular neurons (Fig. A). The parvocellular cells of the PVN of the

hypothalamus are the major information junction for the neuroendocrine response to

stressors. Inputs from both limbic circuits and brain stem centres ensure these cells

can be activated by both psychological and physical stressors (Herman et al. 2003)

with a rapid increase in c-fos (Ceccatelli et al. 1989) followed by increased activation

of many genes of which CRH and AVP are the most important for neuroendocrine

activation of the HPA axis (Lightman and Young, 1987; 1988).

Fig. A: Coronal brain section passing through the paraventricular nucleus (PVN) of the
hypothalamus of mouse stained with cresyl violet

During acute stress, the amplitude and synchronization of the CRH and AVP

pulsations in the hypophyseal portal system markedly increases, resulting in the

increase of ACTH and cortisol secretory episodes (Tsigos and Chrousos, 1994).

Circulating ACTH is the key regulator of glucocorticoid secretion by the adrenal

cortex. Other hormones or cytokines, either originating from the adrenal medulla or

coming from the systemic circulation, as well as neuronal information from the

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autonomic innervation of the adrenal cortex may also participate in the regulation of

cortisol secretion (Ottenweller and Meier, 1982; Hinson, 1990).

Hypothalamic-pituitary-adrenal (HPA) axis

One of the best known and consistent neuroendocrine responses to stress is

activation of the HPA axis, resulting in the secretion of steroid hormones from the

adrenal gland. This relationship between stress and adrenocortical activation was one

of the first recognised in the study of the endocrinology of stress (Selye, 1939). One

major neuroendocrine system underlying an individuals capacity to cope with stress

is the HPA axis. Early investigators concluded that the regulation of glucocorticoid

secretion from the adrenal gland depended on a linkage between hypothalamus and

the pituitary gland. Harris (1948) suggested that neurons of the hypothalamus regulate

the secretion of hormones from the anterior pituitary. Factors produced in

hypothalamic neurons did indeed regulate the secretion of ACTH from the anterior

pituitary (Guillemin and Rosenberg, 1955; Saffran et al. 1955; Porter and Jones,

1956). ACTH stimulates the synthesis and release of steroids from the adrenal cortex

by promoting the uptake of cholesterol and its enzymatic conversion to cortisol/

corticosterone, the glucocorticoid hormones.

The stressful situations cause an increase in the activity of the HPA axis, and

this has been studied in detail in mammals (Vazquez 1998; Makino et al. 2002; Tsigos

and Chrousos, 2002) as well as in birds (Harvey and Hall, 1990). The Activation of

the pituitary component of the HPA axis can be mediated by several neuroendocrine

hormones. Saffran and colleagues (1955) named the ACTH regulator as corticotropin-

releasing factor (CRF), presently this is commonly termed as corticotropin-releasing

hormone (CRH).

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In mammals, the adrenal gland comprises multiple endocrine tissues: the

adrenal cortex and the adrenal medulla. The adrenal medulla is encapsulated by the

adrenal cortex, its primary secretions are the catecholamines epinephrine,

norepinephrine and dopamine. The adrenal cortex comprises three layers: (i) zona

glomerulosa, (ii) zona fasciculata and (iii) zona reticularis. Mineralocorticoids,

predominantly aldosterone, are synthesized in the zona glomerulosa, whereas the

major products of the zona fasciculata and zona reticularis are the glucocorticoids and

androgens (Greep and Deane, 1947).

Regulation of the stress response at the level of the adrenal gland is no less

complex than regulation at the level of the hypothalamus or anterior pituitary.

Glucocorticoid secretion from the adrenal cortex is mediated primarily by endocrine

action of ACTH. Corticotropin releasing hormone may also regulate glucocorticoid

production and secretion via paracrine actions within the adrenal gland. The ability of

CRH to increase adrenal blood flow and the localization of CRH mRNA in the

adrenal suggest an intra-adrenal role for this neurohormone (Minamino et al. 1988;

Usui et al. 1988; Muglia et al. 1994). Monoamines (catecholamines and serotonin)

influence the HPA axis at many levels, including stimulating neurohormone release

from the hypothalamus (Plotsky et al. 1989), ACTH release from pituitary (Axelrod

and Reisine, 1984; Dinan, 1996) and cortisol release from the adrenal cortex (Dinan,

1996).

HPA axis activation is an important and complex adaptive response to stress.

Plotsky and co-workers (1985a, b) demonstrated that specific stressors would elicit

specific ACTH secretogogues. During haemorrhage, CRH, AVP, oxytocin and

catecholamines (all are stimulators of ACTH secretion) are released; however, during

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hypotension, CRH is the only secretogogue released. Specific stressors elicit specific

patterns of neurohormonal activation (Mason, 1974; Seggie and Brown, 1982), which

also supports the concept that activation of the HPA axis is stress specific. Yadawa

and Chaturvedi (2016) reported the activation of HPA axis of mice following water

and food deprivation of 2 and 4 days. It is now well-established that HPA axis

hormones regulate inflammation, and that the extent of this regulation varies in

response to acute and chronic psychosocial stress (Rohleder, 2012).

Hyperactivity of central neuropeptidergic circuits such as CRH and AVP

neuronal systems is thought to play a causal role in the etiology and symptomatology

of mood and anxiety disorders (Gold et al. 1984; Hokfelt et al. 2000; Holsboer, 2000).

In recent years, no. of evidences have been accumulated suggesting that normalization

of the HPA system might be the final step necessary for stable remission of the disease

(Barden et al. 1995; Zobel et al. 1999; Holsboer, 2000; Ising et al. 2005).

Corticotropin releasing hormone

Corticotropin releasing hormone (CRH), a 41 amino acid neuropeptide is

involved in the control of stress-related behaviours (Vale et al. 1981, Bale and Vale,

2004, Keck, 2006). CRH is present in the nerve cell bodies in and near the

dorsomedial parvicellular division of the paraventricular nucleus (PVN) of the

hypothalamus. These neurons send axons to the median eminence and other

hypothalamic and midbrain targets. CRH is the primary hypothalamic

hypophysiotropic hormone, which regulates both basal and stress induced release of

pituitary corticotropin (ACTH) and is the major constituent of the HPA system (see

Fig. B).

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Fig. B: Schematic diagram of HPA axis (Turnbull and Rivier, 1996)

Exposure to a stressor triggers the synthesis of CRH in the PVN of the

hypothalamus. Subsequently, CRH is released via the median eminence into the portal

blood to reach the anterior pituitary. The peptide then activates CRH receptor1 on

pituitary corticotrophs, thereby stimulating adrenocorticotropic hormone synthesis

and release into the circulatory system, which subsequently elevates the production

and secretion of glucocorticoid hormones (cortisol in humans and corticosterone in

rats and mice) from the adrenal cortex (Guillemin et al. 1959; Hiroshige et al. 1968).

CRH is required for adrenal response to different stressors (Jacobson et al.

2000) including fasting (Jeong et al. 2004). The tissue distribution of the type 1 and 2

receptors of CRH varies considerably (Van et al. 2000, Bale and Vale, 2004). CRH-R1

mediates the actions of CRH at the corticotroph as well as some aspects of the

behavior stress-response, including fear and anxiety (Liebsch et al. 1995; 1999;

Skutella et al. 1998). CRH-R2 involved in vasodilation and blood pressure control and

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recovery phase of HPA response (Coste et al. 2000). CRH secreted by postganglionic

sympathetic neurons at inflammatory sites has proinflammatory properties, one of its

key roles in the degranulation of mast cells (Chrousos et al. 1998; Elenkov et al.

1999). CRH neurons are largely controlled by serotonergic input from the amygdala

and hippocampus of the limbic system, and brainstem regions, involved in autonomic

functions. Besides PVN, CRH is also expressed in amygdala and bed nucleus of stria

terminalis and increases in response to stress (Chalmers et al. 1995).

Arginine Vasopressin (AVP)

Arginine vasopressin, also known as vasopressin, antidiuretic hormone (ADH)

or argipressin, is a Neurohypophyseal hormone found in the most mammals. AVP is a

nine amino acid neurohormone. It is synthesized within the neurons of the supraoptic

and paraventricular nuclei (SON and PVN) and certain accessory nuclei in the

anterior hypothalamus and released into the circulation from nerve endings in the

neurohypophysis. Stimulation or inhibition of release of vasopressin is controlled by

changes in blood volume or pressure and in plasma osmolality. These two control

systems will be referred to as volume control and osmotic control. Volume control is

mediated by peripheral receptors in the cardiovascular system and clearly involves a

neuroendocrine reflex (Harris, 1978). A clear relationship has been established

between plasma osmolality, plasma vasopressin concentration (Schrier et al. 1979;

Robertson, 1985) and the electrical activity of vasopressin-secreting cells in the SON

and PVN (Poulain, 1983; Leng et al. 1985).

The PVN consists of two parts with different functions. One contains

parvocellular neurons which project to the median eminence, brain stem and spinal

cord and may influence the regulation of anterior pituitary secretion and the

autonomic nervous system. The other part, which contains the magnocellular neurons

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 Review of Literature

projecting to the neurohypophysis, is divided into three subnuclei; the anterior, medial

and posterior (Swanson and Sawchenko, 1983). The SON contains only

magnocellular neurons and is divided into two parts, principal or anterior (SONa) and

retrochiasmatic or tuberal (SONt). Vasopressin is synthesized with carrier protein or

neurophysin II (NP II) as a part of precursor or pre-prohormone within the perikarya

of the magnocellular neurons.

AVP exhibits three main physiological roles: regulation of water homeostasis,

vascular constriction and control of ACTH secretion from pituitary via its different

receptor subtypes belonging to G protein coupled receptor family (Murat et al. 2012).

The physiological role of AVP is mediated by three specific receptor subtypes. The V2

receptors expressed in kidney are positively coupled to adenylyl cyclase (AC) and

mediate water reabsorption in the kidney tubules, V1a isoform induces vascular

constriction via phospholipase C (PLC) activation (Barberis et al. 1999) and the V1b

receptor, discovered last in the pituitary gland, is also coupled to the PLC pathway

and has a well-known effect on ACTH secretion. The magnocellular neurons project

through median eminence to neuronal lobe of hypophysis being involved in water

balance. The expression of isoform of AVP in birds (AVT) increased in water deprived

domestic fowl and Japanese quail. Chaturvedi and colleagues (1986) reported that

osmotic stress upregulates expression of AVT gene and increases the accumulation of

AVT mRNA in the hypothalamus of domestic fowl. During osmotic stress,

hypothalamic AVT gene expression and AVT peptide release in Japanese quail is

gender specific (Chaturvedi et al. 2000). It has been also reported that osmotic stress

not only upregulates the expression of the AVT gene in existing neurons but also

recruits many more neurons to increase the rate of AVT synthesis and secretion in

Japanese quail (Seth et al. 2004). Photoperiod (Short day and long day) also

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 Review of Literature

modulates the no. of ir-AVP neurons in water deprived chicken (Singh and

Chaturvedi, 2006).

It has been reported that AVP is a key regulator of the HPA axis and has a

strong impact on the stress related behaviours (Zelena and Jain, 2010) due to its effect

on ACTH secretion. At the pituitary level, the effects of CRH on ACTH secretion are

amplified by AVP, which, after prolonged stress, is increasingly co-expressed and co-

secreted from parvocellular hypothalamic CRH neurons located in the PVN (Antoni,

1993; Keck et al. 2000; 2002). These parvocellular neurons project to the median

eminence (ME), where CRH and AVP are released into hypophyseal portal blood

vessels to trigger ACTH release from pituitary corticotropes through the activation of

CRH receptors1 and AVP 1b receptors (V1b) (see Fig. C).

Selective afferentation

Paraventricular nucleus

Median eminence

Pituitary gland

Supraoptic nucleus
Pituitary portal vessel
ACTH

Fig. C: Schematic diagram of Hypothalamic-pituitary axis showing CRF and/or AVP

secreting neurons in the PVN and SON (Antoni, 1993). Note Co-expression of
CRF (CRH) and AVP in the parvocellular neurons of PVN. Axons of these neurons
along with the axons of magnocellular AVP neurons project and release their

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secretion in the median eminence to activate ACTH release from the anterior
pituitary.

On the other hand, magnocellular AVP neurons of PVN also project to median

eminence to stimulate ACTH secretion from anterior pituitary in addition to the

projections of its classical hypothalamic-hypophyseal tract directly in to posterior lobe

(neurohypophysis) of pituitary to secrete AVP as ADH in the peripheral circulation. In

contrast to this, magnocellular AVP neurons terminate their axons directly into the

neurohypophysis (without any projection into median eminence). Taking together the

independent description of Turnbull and Rivier (1996) as well as Antoni (1993) with

respect to HPA axis, it appears that some magnocellular neurons of PVN project their

axons in median eminence also, to regulate ACTH secretion. Moreover, parvocellular

CRH neurons may also co-express AVP which is also released into median eminence.

Recently, the involvement of hypothalamic AVP in benzodiazepine induced

HPA-system attenuation could be demonstrated (Welt et al. 2006). In clinical studies,

plasma AVP concentrations were found to be significantly correlated with anxiety-

related symptoms in healthy volunteers in response to an anxiogenic drug challenge

(Abelson et al. 2001). After prolonged stress, AVP is increasingly expressed and

released from hypothalamic neurons in both humans and rodents (Antoni, 1993).

Unlike initial concept, that AVP (along with Oxytocin-OT) synthesized in

magnocellular neurons of SON and PVN is released into the neurohypophysis via

hypothalamo-hypophyseal tract (as antidiuretic hormone), now it is also evident that it

also stimulates pituitary ACTH via its release into ME (as stress hormone), since it is

also having its receptors on pituitary corticotrophs parallel to CRH receptors.

Glucocorticoids/ Corticosterone

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Corticosterone, also known as 17-deoxycortisol and 11, 21-

dihydroprogesterone is a 21-carbon steroid hormone of the corticosteroid type

produced in adrenal cortex of rodents and other non- human animals (Hill et al. 1991).

In many species, including amphibians, reptiles, birds and rodents, corticosterone is

the main glucocorticoid involved in the regulation of energy, immune reactions and

stress responses (Sapolsky et al. 2000; Charmandari et al. 2005) unlike human where

cortisol is the major glucocorticoid. On the other hand, mineralocorticoid aldosterone

is mainly involved in ion balance (Hadley, 2000).Various deleterious effects of

chronic corticosterone elevation have been documented in many species, including

suppression of reproductive function and behavior, immune system suppression,

muscle wasting, growth suppression, and neuronal cell death (Wingfield et al. 1997;

Sapolsky et al. 2000).

Corticosterone plays a regulatory role in stress-induced HPA axis activity in

rodents (Osterlund and Spencer, 2011). Chronically elevated corticosterone levels

activate the chronic stress-response network, and as a result, affect various processes

involved in coping with stress (Dallman et al. 2003). Previous studies showed that

chronic corticosterone administration in rodents inhibits stress-induced serum

corticosterone elevation (Ago et al. 2008) and causes increase in serum insulin and

leptin, adipose-derived hormones (la Fleur et al. 2004; Warne et al. 2009, Karatsoreos

et al. 2010) that regulate food intake and body weight (Friedman, 2009). In addition,

chronic corticosterone administration normally induces depression in rodents

(Gourley and Taylor, 2009). Animal models of repeated stress exposure simulate the

presumed etiology of depression, and this stress may be produced by exogenous

corticosterone administration. Corticosterone treatment of rats (Kalynchuk et al. 2004;

Gregus et al. 2005; Johnson et al. 2006) and mice (Zhao et al. 2008) produces changes

in emotional behavior that may correspond to symptoms of clinical depression.

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Glucocorticoids are hormones that coordinate the stress response system and

the ability of an organism to cope with the stress (de Kloet et al. 1999). These

hormones easily cross the blood brain barrier and subsequently interact with their

specific intracellular receptors, i.e. glucocorticoids and mineralocorticoids receptors,

in different brain areas related to cognitive processes (Sauro et al. 2003; Nater et al.

2007). Scaccianoce et al. (2003) reported that animals with high levels of

corticosterone presented superior performance in a learning paradigm. However, a

study from Luine et al. (1993) showed that some rats submitted to corticosterone

treatment had their learning performance impaired, whereas other corticosterone-

treated rats showed no change on radial maze task performance.

Nitric oxide (NO) and nitric oxide synthase (NOS)

In mammals including human, NO is an important cellular signaling gaseous

molecule involved in many physiological and pathological processes (Hou et al.

1999). It is a powerful vasodilator with a short half-life of a few seconds in the blood.

As a consequence of its importance in neuroscience, physiology and immunology,

NO was proclaimed Molecule of the Year in 1992 (Culotta and Koshland, 1992).

Nitric oxide, known as the endothelium-derived relaxing factor, is biosynthesized

endogenously from L-arginine, oxygen, and NADPH by various nitric oxide synthase

(NOS) enzymes.

NOS
L-Arginine Nitric oxide + Citrulline
O2, NADPH

Fig.D: Schematic representation of Nitric oxide (NO) Synthesis

Reduction of inorganic nitrate may also serve to make nitric oxide.

The endothelium of blood vessels uses nitric oxide to signal the surrounding smooth

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muscle to relax, thus resulting in vasodilation and increasing blood flow. Nitric oxide

is highly reactive, yet diffuses freely across membranes. These attributes make nitric

oxide ideal for a transient paracrine and autocrine signaling molecule (Stryer, 1995).

Biological and functional properties of nitric oxide as a non-conventional

neurotransmitter in the nervous system have been reviewed extensively (Garthwaite,

2008). Nitric oxide in the CNS elicits a reduction in sympathetic nerve activity

(Zhang and Patel, 1998). It is produced by nitric oxide synthase, which has three

isoforms; neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). All the three

isoforms generate nitric oxide from L-arginine and are found in the brain, although

they do have different spatial distributions (Knowles and Moncada, 1994). Neuronal

nitric oxide synthase is present in the PVN, nucleus tractus solitaries (NTS) and

caudal medulla (Vincent and Kimura, 1992) and its activation results in the formation

of relatively large quantities of nitric oxide (Bredt, 1999; Garthwaite, 2008). Neuronal

nitric oxide synthase (nNOS) plays a major role in NO biosynthesis. In the

hypothalamus, nNOS expression can be found in parvocellular neurosecretory and

medullary-projecting preautonomic neurons (Nylen et al. 2001).

Immunohistochemical staining using an antibody against nNOS reveals many neurons

within the PVN to express nNOS, with far more neurons in the magnocellular

compared to parvocellular being immunopositive for nNOS (Stern, 2004; Watkins et

al. 2009).

As a gaseous substance, nitric oxide readily diffuses across cell membranes to

activate cGMP mechanisms to exert its biological effect (Kennedy, 2000). In the

neuroendocrine system, nitric oxide (NO) influences the secretion of many endocrine

hormones like CRH (Costa et al. 1993; Karanth et al. 1993), ACTH (Rivier and Shen.,

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1994) and vasopressin (Ota et al. 1993; Kadowaki et al. 1994). Nitric oxide mediates

the neuroendocrine function in HPA axis (Nelson et al. 1997). The magnocellular

neurosecretory neurons of the PVN constitute a major cellular source of nitric oxide

(Nylen et al. 2001; Stern, 2004). A recent hypothesis proposes that, nitric oxide

originating from the magnocellular neurons contributes to the nitric oxide mediated

modulation of autonomic outflow from the PVN. This probably is the case as the cell

bodies of the NO-containing neurons and/or dendrites have been shown to be closely

associated with spinally projecting neurons in the PVN and as such any gaseous nitric

oxide emanating from these neurons could diffuse to modulate the function of the

presympathetic neurons (Stern, 2004; Watkins et al. 2009). Neuronal NOS mRNA and

immunoreactivity have been also detected in the adrenal cortex (Tsuchiya et al. 1996).

Previous studies suggested a role for nNOS on the correct postnatal development of

vasopressinergic and oxytocinergic cells of the hypothalamus (Yuan et al. 2006).

Immobilization-induced stress also upregulates the nNOS mRNA in PVN (Calza et al.

1993).

Arginine-derived NO synthesis has been identified in mammals, fish, birds,

invertebrates, and bacteria (Liu and Gross, 1996). Nitric oxide is also reported to

modulate gonadal activity of many vertebrates (Chaturvedi and Kumar, 2007; Singh

and Chaturvedi, 2014) including sperm motility, spermatogenesis and steroidogenesis

(McCann et al. 1999). In addition to its role in modulating reproductive performance,

NO is reported to have various physiological and pathological functions (see Fig. E).

This important signaling molecule is also reported to have protective as well as

cytotoxic effects depending on its level. NO is also known to induce nitrosative stress

in the form of reactive nitrogen species (RNS).

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Cardiovascular Homeostasis
Neurotransmission

Cell proliferation Apoptosis

NO
Immune response Gonadal activity & Reproduction

Angiogenesis

Fig.E: Physiological/ pathophysiological roles of Nitric oxide

Nuclear factor-B (NF-B)

Survival of an organism is dependent on its ability to rapidly and effectively

respond to adverse changes in its environment. This is often achieved through changes

in the program and rates of gene expression, leading to the production of proteins that

exert a protective effect on the cell. NF-B exists in the cytoplasm of majority of cell

types as homo or heterodimers of a family of structurally related proteins (Baldwin,

1996; Kopp and Ghosh, 1995). NF-B was first identified as a protein bound to a

sequence in the immunoglobulin light chain enhancer in B cells stimulated with

lipopolysaccharide (Sen and Baltimore, 1986). This dimeric transcription factor is

composed of different members of the Rel family, such as p65 (RelA), p50, p52, c-Rel

and RelB (Baeuerle and Baltimore, 1996), which can activate a great variety of genes

involved in stress responses, inflammation and apoptosis.

Viral infection, bacterial lipids, parasites, UV irradiation, shear stress,

chemotherapeutic agents, oxidative stress, pro-inflammatory cytokines and DNA

damage are some agents which are capable of inducing NF-B (reviewed in Mercurio

and Manning, 1999). A growing number of highly diverse genes have been

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demonstrated to be induced by NF-B, including viral genes (HIV-1, CMV), immuno-

receptors (IL-2 receptor a-chain, T cell receptor b2), cell adhesion molecules,

cytokines and growth factors, chemokines, acute phase proteins, oxidative stress-

related enzymes and anti-apoptotic proteins (reviewed in Baeuerle and Baichwal,

1997; May and Ghosh, 1998).

These reports indicate that, different stressors are capable of inducing NF-B

which in turn can activate a variety of genes involved in stress responses, apoptosis,

inflammation etc. On the other hand, HPA axis plays a major role in stress responses

and hence also termed as stress axis. In spite of these two facts/ evidences, there is no

report relating NF-B with that of HPA axis or vice-versa during stressful conditions.

Heat Shock Protein (Hsp70)

Following a disturbance in cellular homeostasis, the expression of a specific

set of proteins, the stress proteins, is increased, and the coordinated sequence of

events resulting in this response is the stress response. The term stress proteins

primarily refers to members of the Hsp family. Because the expression of certain

members of this protein family is greatly enhanced following a variety of stressful

stimuli, they appear to be an excellent candidate as markers of neurotoxicity. Hsps are

ubiquitous proteins found in all living organisms studied to date and are so named

because of their rapid induction during periods of sudden heat stress. These proteins

were originally discovered in fruit flies (Drosophila) exposed to high temperatures

(Ritossa, 1996). In addition to heat shock, a variety of other stimuli, including

ischemia/reperfusion, seizures, heavy metals, alcohol, hormones, amino acid analogs,

and abnormal proteins, also increase the expression of Hsps (Kiang and Tsokos 1998).

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Hsp70 (also called Hsp72) is the major inducible stress protein, and its

expression is increased following exposure of cells to any stressful stimulus that

produces denatured proteins. It is hypothesized that denatured proteins in injured cells

activate transcription of hsp70 via heat shock factor activation. Increased Hsp70

protein in addition to upregulated expression of constitutive members is thought to

protect cells from noxious stimuli by binding to denatured proteins and preventing

their further denaturation. Moreover, these proteins also facilitate the restoration of

function of renatured proteins and transport the irreversibly damaged proteins to

degradative organelles (reviewed in Rajdev and Sharp, 2000).

Many drugs and agents with neurotoxic potential may cause increases in body

temperature and thus increase Hsp70 expression. Thus, their effects on Hsp70

expression are secondary to their effects on body temperature. One such example is

the psychotropic drug lysergic acid diethylamide which rapidly induces Hsp70 in

adult and foetal rabbit brain (Cosgrove and Brown, 1983; Freedman et al. 1981).

Amphetamine-induced hyperthermia has also been reported to induce Hsp70 in mouse

brain (Nowak Jr, 1988). Hsp70 chaperones increase more under stressful conditions.

Mutated proteins seem to require more attention by the Hsp70 chaperones than the

corresponding wild type protein due to the fact that along with hyperthermia other

stress factors such as oxidative and nitrosative stress, ischemia, hypoxia, viral

infection etc. denature the proteins (Meacham et al. 1999; Gaiddon et al. 2001; King

et al. 2001; Shinder et al. 2001).

At the molecular level, the effects of glucocorticoids are mediated by

glucocorticoid receptor (GR). In the cytoplasm, the inactivated GR is associated with

a chaperone complex consisting of several molecules including Hsp90 and Hsp70

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(Rajapandi et al. 2000). The main role of Hsp90 and Hsp70 is in maintaining proper

GR conformation for ligand binding, its intracellular shuttling, stabilization and

protection (Dittmar and Pratt, 1997; Rajapandi et al. 2000). Hsp70 is involved in

correct GR protein folding (Caamano et al. 2001), facilitation of receptor

translocation to the nucleus (Sanchez et al. 1990) and the interaction with

glucocorticoid-responsive elements (Srinivasan et al. 1994). Simic et al. (2012)

reported increase of the GR and Hsp70 in hypothalamic cellular compartments as a

response of naive rats to acute stress, whereas the response of chronic psychological

isolation rats to acute stress was associated with elevation of the GR in the cytosol

and decrease of Hsps in the nucleus.

*******

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