Introduction Ageing is the biological process characterized by the progressive and irreversible loss of physiological function accompanied by increasing mortality with advancing age. It is a complex physiological phenomenon associated with a multitude of biological changes at the molecular level, which is eventually manifested at the tissue and organism level. Understanding the molecular mechanism behind the ageing process is therefore, key to the development of therapeutics which prolong human life span, and more importantly, to reduce morbidity among the elderly. The mechanisms involved in plasticity in the nervous system are thought to support cognition, and some of these processes are affected during normal ageing. Genes that are downregulated over the age include GluRI AMP A receptor subunit, NMDA R2A receptor subwlit involved in learning, subunits of the GABA A receptor, genes involved in longterm potentiation like calmodulin 1 and CAM kinase lia, calcium signalling genes, synaptic plasticity genes, synaptic vesicle release and recycling genes. Genes that are up regulated include genes associated with stress response, DNA repair and antioxidant defense. Several studies showed that the age-related loss of a number of functions is associated with an oxidative damage in the tissues mediating those functions. In the ageing brain, neurological deficits related to ageing have been suggested to be due to a breakdown of calcium (Ca 2 +) homeostasis, and an increase in intracellular Ca 2 +. It is possible that age-related changes in Ca 2 + regulation cause some portion of the observed age-related plasticity deficits. Ageing is characterized by progressive impairment of bodily activities. Normal human ageing is associated with a progressive impairment of glucose tolerance (Davidson 1979). Total glucose stimulated insulin secretion has been described as being unchanged (Draznin et al., 1985), suppressed (Molina et al., 1985)
or increased (Curry et al., 1984) as an animal ages. Recently it was demonstrated in Wistar rats ageing is indeed associated with progressive decline in beta cell number, the pancreatic insulin content, amount of insulin secreted and insulin mrna levels (Perfetti et al., 1995). Impairment of insulin action as a function of age has also been reported. There is an impairment of insulin induced glucose disposal in old compared with young subjects (Haruo et al., 1988). Few laboratories have attributed the alterations in glucose stimulated insulin secretion with age to changes in diet rather than ageing, per se (Hara et al., 1992). The central nervous system (CNS) neurotransmitters play an important role in the regulation of glucose homeostasis. During normal ageing prominent alterations occur in various neurotransmitter systems in the brain, related to reductions in the number of neurons and to a decrease in concentration, synthesis and turnover of neurotransmitters. Reductions of the levels of transmitter substances and of the activities of enzymes involved in their synthesis have been demonstrated in the ageing brain. These neurotransmitters mediate rapid intracellular communications not only within the central nervous system but also in the peripheral tissues. They exert their function through receptors present in both neuronal and non-neuronal cell surface that trigger second messenger signaling pathways. Neurotransmitters have been reported to show significant alterations during diabetes resulting in altered functions causing neuronal degeneration. Chronic hyperglycaemia during diabetes mellitus is a major initiator of diabetic micro-vascular complications like retinopathy, neuropathy and nephropathy. Age related changes in the capacity of f3-cell for proliferation affect the insulin production and contribute to a decrease in glucose tolerance with advance in age. Acetylcholine is a neurotransmitter that has been implicated in various central neuronal degenerative disorders like Alzheimer's disease, dementia and other age
Introduction related memory disorders. An acetylcholine receptor is an integral membrane protein that responds to the binding of the neurotransmitter acetylcholine. There are two main classes of acetylcholine receptor (AChR), nicotinic acetylcholine receptors (nachr) and muscarinic acetylcholine receptors (machr). Muscarinic receptors are metabotropic and affect neurons over a longer period. They are stimulated by muscarine and acetylcholine, and blocked by atropine. Muscarinic receptors are found in both the central nervous system and the peripheral nervous system, in heart, lungs, upper GI tract and sweat glands. Acetylcholine in the central nervous system is involved in the control of motor activity, emotional behaviour, cognition and endocrine regulation. Hyperglycaemia during diabetes is reported to damage cholinergic functions. The progression of diabetes is associated with an impaired ability of the neurons in the CNS to release neurotransmitters resulting in behavioural changes. Parasympathetic activity plays an important role in insulin secretion from pancreatic p-ce11s. Cholinergic agonist carbachol increases insulin secretion from isolated rat islets (Zawalich & Zawalich, 2002). The muscarinic receptor stimulation by ACh leads to activation of phospholipase C, which, in turn, hydrolyzes phosphatidylinositol 4, 5-bisphosphate (PIP2) to produce D-myo-Inositol 1,4,5- triphosphate (IP3) and diacylglycerol (DAG) (Best & Malaisse, 1983; Zawalich et al., 1989). In pancreatic p-cells, IP3 mobilizes Ca 2 + from intracellular stores, resulting in an elevation of the intracellular concentration of Ca 2 + and allowing activation of Ca 2 +/calmodulin. DAG on the other hand, activates PKC (Nishizuka, 1995; Renstrom et al., 1996). PKC, like Ca 2 +/calmodulin, accelerates exocytosis of insulin granules (Nanko et al., 2002). The mitogenic effect of acetylcholine has been studied in different cel1 types. Muscarinic acetylcholine receptors activate many downstream signaling pathways,
some of which can lead to mitogen activated protein kinase (MAPK) phosphorylation and activation. Mitogen activated protein kinases play a role in regulating cell growth, differentiation and synaptic plasticity. Both Gi and Gq coupled muscarinic receptors have been shown to activate MAPK in various system. Muscarinic M3 receptors activate MAPK in the oligodendrocyte progenitors (Ragheb et al., 2001). The involvement of M 1 receptors has been reported in muscarinic activation of MAPK in PC12 cells (Berkeley et al., 2000). Acetylcholine analogue carbachol stimulated DNA synthesis via muscarinic receptors in primary astrocytes derived from perinatal rat brain (Ashkenazi, 1989). Carbachol is also mitogenic in certain brain derived astrocytoma and neuroblastoma, as well as in Chinese hamster ovary (CHO) cells expressing recombinant muscarinic receptors (Ashkenazi, 1989). Proliferation experiments with subtype specific antagonists in astrocytes suggest that cell proliferation is induced by the activation of muscarinic M3 receptors (Guizzetti, 1996, 2002). Regeneration is a complex interplay of several factors - growth factors, hormones and neurotransmitters. The stimulatory effect of growth honnone on insulin (INS) production and p-cell replication are well documented (Swenne et al., 1987; Nielsen, 1986, Sjoholm et al., 2000). In vitro and in vivo studies have established the role of insulin in p-cell replication (Chick et al., 1973). Insulin interacts with type 1 IGF receptor and stimulates p-cell proliferation. Somatotropin (STH), is an important anabolic honnone which exerts stimulatory effects on protein synthesis and on lipolysis. Pituitary STH release is regulated primarily by the interaction of the hypothalamic peptides, GHRH, which stimulates and somatostatin which inhibits STH production. STH (Growth hormone, GH) regulates body growth and metabolism. STH exerts its biological action by stimulating JAK2, a GH receptor (GHR)-associated tyrosine kinase. Activated JAK2 phosphorylates itself and GHR, thus initiating multiple signa ling pathways. 4
If glucose supply to the brain is not maintained, there may be a decrease in cerebral electrical activity, membrane breakdown with release of free fatty acids, and altered amino acid metabolism. Deterioration in glucose homeostasis that results from hyperglycaemia can trigger neuronal injuries; the molecular basis of this neuronal vulnerability is not yet explored. The reports so far stated did not attempt to emphasis the functional correlation of cholinergic receptors in diabetic brain as a function of age. Studies targeting the exact molecular mechanism of cholinergic receptor subtypes in diabetic rats of different age groups will be useful in improving selective cognitive processes and insulin function with increasing chronological age. In the present study, the alterations of muscarinic M 1 and M3 receptor subtypes in the brain regions of streptozotocin induced diabetic rats were carried out in different experimental age groups ofrats. Also, the second messengers, IP3 and cgmp, in brain regions of diabetic rats were studied as a function of age. Calcium imaging studies were carried out to observe the role of muscarinic receptor antagonists in intracellular Ca 2 + release from pancreatic islets of young and old rats in vitro. The functional role of long term low dose STH and INS treatment in the regulation of neurotransmitter levels in the ageing brain was studied. Also, the possible linkage between the STH and INS induced changes in second messengers like IP3 and cgmp has been elucidated as a function of age. Electroencepalogram studies were performed to analyse the brain wave signaling pattern in young and old rats. This will have immense therapeutic application in improving cognition, learning and memory and rejuvenating brain functions during ageing. 5
OBJECTIVES OF THE PRESENT STUDY 1. To study acetylcholine esterase (AChE) activity in the brain regions; cerebral cortex (CC), brainstem (BS) & corpus striatum (CS) of 7 weeks (young) and 90 weeks (old) control, diabetic and insulin treated diabetic rats. 2. To study AChE activity in the pancreas of 7 weeks (young) and 90 weeks (old) control, diabetic and insulin treated diabetic rats. 3. To study acetylcholine receptor changes in the cerebral cortex of 7 weeks and 90 weeks old control, diabetic and insulin treated diabetic rats. 4. To study muscarinic M I and M3 receptors changes in the brain regions of 7 weeks and 90 weeks old control, diabetic and insulin treated diabetic rats. 5. To study muscarinic MI and M3 receptor gene expression in the brain regions of 7 weeks and 90 weeks old control, diabetic and insulin treated diabetic rats. 6. To study muscarinic M3 receptors changes in the pancreas of 7 weeks and 90 weeks old control, diabetic and insulin treated diabetic rats. 7. To study muscarinic M3 receptor gene expression in the pancreas of 7 weeks and 90 weeks old control, diabetic and insulin treated diabetic rats. 8. To study glutamate NMDARI, mglu-5, a2a, P2, GABA Aal and GABA B, DAD2 and 5-HT 2e receptors gene expression in the cerebral cortex of 7 weeks and 90 weeks old control, diabetic and insulin treated diabetic rats. 6
9. To quantify IP3 and cgmp content in the brain regions and pancreas of 7 weeks and 90 weeks old control, diabetic and insulin treated diabetic rats. 10. To study the role of carbachol and muscarinic MI, M3 receptors antagonists on glucose induced insulin secretion by pancreatic islets of young and old rats in vitro. 11. To study the role of carbachol and muscarinic M 1, M3 receptors antagonists on IP3 and cgmp release by pancreatic islets of young and old rats in vitro. 12. To study the role of carbachol and dopamine on IP3 and cgmp release by pancreatic islets of young and old rats in vitro. 13. To quantify the triiodothyronine (T3) content in the serum of 7 weeks and 90 weeks old control, diabetic and insulin treated diabetic rats. 14. To quantify epinephrine (EPI), norepinephrine (NE), dopamine (DA) and serotonin (5-HT) content in the brain regions CC, CS, BS & Hypothalamus (Hypo) of long term low dose somatotropin and insulin treated young and old rats using HPLC. 15. To study AChE activity In the cerebral cortex of long term low dose somatotropin and insulin treated young and old rats. 16. To study muscanmc MI, M3, glutamate NMDARI, mglu-5, U2A. P2, GABA Aaj and GABAs, DAD2 and 5-HT 2c receptors gene expression in the 7
cerebral cortex of long tenn low dose somatotropin and insulin treated young and old rats. 17. To quantify the IP3 and cgmp content in the brain regions of long tenn low dose somatotropin and insulin treated young and old rats. 18. To quantify the triiodothyronine (T3) content in the serum of long tenn low dose somatotropin and insulin treated young and old rats. 19. To study the effect of carbachol and muscarinic Ml, M3 receptor antagonists on intracellular calcium release in pancreatic islets of young and old rats in vitro. 20. To perfonn neurophysiologic analysis of the electrical activity of the brain using electroencephalogram of experimental rats. 8