Muscarinic M1, M3, Nicotinic,GABAA and GABAB Receptor ...

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DETERMINATION OF BLOOD GLUCOSE The diabetic state of animals was assessed by measuring blood glucose concentrations at 72 hours after streptozotocin treatment. The rats with a blood sugar level above 250 mg/dl were selected as diabetic rats. SACRIFICE AND TISSUE PREPARATION The animals were then sacrificed on 15 th day by decapitation. The cerebral cortex, cerebellum, brain stem, corpus striatum and hippocampus were dissected out quickly over ice according to the procedure of Glowinski & Iversen, (1966) and the pancreas was dissected quickly over ice. Hippocampus was dissected according to the procedure of Heffner et al., (1980). The blood samples were collected and plasma was separated by centrifugation. The tissue samples and plasma were kept at -80° C until assay. All animal care and procedures were in accordance with Institutional and National Institute of Health guidelines. ESTIMATION OF BLOOD GLUCOSE Blood glucose was estimated using Glucose estimation kit (Merck). The spectrophotometric method using glucose oxidase-peroxidase reactions is as follows: Principle: Glucose oxidase (GOD) catalyses the oxidation of glucose in accordance with the following equation: Glucose + O2 + H2O (GOD) Gluconic acid + H2O2. H2O2 + Phenol 4-aminoantipyrene (Peroxidase) Coloured complex + H2O The hydrogen peroxide formed reacts with 4-aminoantipyrine and 4hydroxybenzoic acid in the presence of peroxidase (POD) to form N-(-4- 47
antipyryl)-p-benzo quinoneimine. The addition of mutarotase accelerates the reactions. The amount of dye formed is proportional to the glucose concentration. The absorbance was read at 510nm in spectrophotometer (Shimadzu UV-1700 pharmaspec). ESTIMATION OF CIRCULATING INSULIN BY RADIOIMMUNOASSAY Principle of the assay The assay was done according to the procedure of BARC radioimmunoassay kit. The radioimmunoassay method is based on the competition of unlabelled insulin in the standard or samples and [ 125 I] insulin for the limited binding sites on a specific antibody. At the end of incubation, the antibody bound and free insulin were separated by the second antibodypolyethylene glycol (PEG) aided separation method. Measuring the radioactivity associated with bound fraction of sample and standards quantitates insulin concentration of samples. Assay Protocol Standards, ranging from 0 to 200 μU/ml, insulin free serum and insulin antiserum (50μl each) were added together and the volume was made up to 250μl with assay buffer. Samples of appropriate concentration from experiments were used for the assay. They were incubated overnight at 2°C. Then [ 125 I] insulin (50μl) was added and incubated at room temperature for 3 hours. The second antibody was added (50μl) along with 500μl of PEG. The tubes were then vortexed and incubated for 20 minutes and they were centrifuged at 1500 x g for 20 minutes. The supernatant was aspirated out and the radioactivity in the pellet was determined in a gamma counter. 48
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MUSCARINIC M1, M3, NICOTINIC, GABAA
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ACKNOWLEDGEMENT To the Almighty God
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Seema Chandran, Ms. Neema Ani Manga
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ABBREVIATIONS 5-HIAA 5 Hydroxy indo
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PFC Prefrontal cortex PLC Phospholi
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GABAC Receptors 34 Glutamic Acid De
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Behavioural response of control and
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the cerebral cortex of control and
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Muscarinic M1 receptor analysis Sca
Page 21 and 22: REAL TIME-PCR ANALYSIS 81 Real Time
Page 23 and 24: Real Time PCR analysis of GABAAα1
Page 25 and 26: pancreas of control and experimenta
Page 27 and 28: utilization is decreased in the bra
Page 29 and 30: impairment, coma or seizure (Cryer,
Page 31 and 32: achieving optimal blood sugar contr
Page 33 and 34: OBJECTIVES OF THE PRESENT STUDY 1.
Page 35 and 36: Literature Review Diabetes mellitus
Page 37 and 38: 12 Literature Review from the adren
Page 39 and 40: 14 Literature Review Hypoglycemic c
Page 41 and 42: 16 Literature Review respectively)
Page 43 and 44: 18 Literature Review in extracellul
Page 45 and 46: 20 Literature Review substrates in
Page 47 and 48: 22 Literature Review nucleus, altho
Page 49 and 50: 24 Literature Review al., 1990; Lev
Page 51 and 52: 26 Literature Review muscarinic M2
Page 53 and 54: Signal transduction by muscarinic a
Page 55 and 56: 30 Literature Review nicotinic acet
Page 57 and 58: GABAA Receptors 32 Literature Revie
Page 59 and 60: 34 Literature Review GABAB-mediated
Page 61 and 62: 36 Literature Review (Erlander et a
Page 63 and 64: 38 Literature Review which could be
Page 65 and 66: 40 Literature Review administration
Page 67 and 68: 42 Literature Review the hippocampu
Page 69 and 70: 44 Literature Review understanding
Page 71: Confocal Dyes Rat primary antibody
Page 75 and 76: was analyzed. Data was expressed as
Page 77 and 78: ANALYSIS OF THE RECEPTOR BINDING DA
Page 79 and 80: Taqman probes of muscarinic M1, M3,
Page 81 and 82: Taylor, 1968). The islets were isol
Page 83 and 84: Results BODY WEIGHT AND BLOOD GLUCO
Page 85 and 86: 60 Results IIH and C + IIH rats sho
Page 87 and 88: Real Time-PCR analysis of muscarini
Page 89 and 90: Real Time PCR analysis of SOD 64 Re
Page 91 and 92: 66 Results GABAAα1 receptor antibo
Page 93 and 94: 68 Results compared to diabetic gro
Page 95 and 96: 70 Results compared to diabetic rat
Page 97 and 98: 72 Results Muscarinic M3 receptor a
Page 99 and 100: Muscarinic M3 receptor analysis 74
Page 101 and 102: 76 Results group, α7 nAChR mRNA si
Page 103 and 104: 78 Results diabetic rats. In C + II
Page 105 and 106: Muscarinic M1 receptor analysis 80
Page 107 and 108: Real Time-PCR analysis of α7 nAChR
Page 109 and 110: Real Time PCR analysis of phospholi
Page 111 and 112: HIPPOCAMPUS Total muscarinic recept
Page 113 and 114: 88 Results IIH and C + IIH group sh
Page 115 and 116: 90 Results group, GLUT3 mRNA signif
Page 117 and 118: 92 Results α7 nACh receptor antibo
Page 119 and 120: GABA receptor analysis 94 Results S
Page 121 and 122: 96 Results control. D + IIH and C +
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Discussion Glucose is the principal
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100 Discussion The ability to sense
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102 Discussion The Y-maze test is a
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104 Discussion CHOLINERGIC ENZYME A
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106 Discussion Pancreas Insulin sec
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108 Discussion 2003). Cholinergic m
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110 Discussion brain for learning,
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112 Discussion hypoglycemia on brai
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114 Discussion shows impaired expre
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116 Discussion glycemic control is
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118 Discussion release (Ilcol et al
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120 Discussion al., 1992), it has t
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122 Discussion metabolic cycles are
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124 Discussion al., 1988). Low bloo
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126 Discussion modulate the second
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128 Discussion between excitation a
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130 Discussion hypoglycemic seizure
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132 Discussion Insulin secretion fr
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134 Discussion hyper and hypoglycem
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136 Discussion receptors in glucose
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138 Discussion exacerbated by recur
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140 Discussion Haces et al., 2010).
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142 Discussion hypoglycemic and dia
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144 Discussion transcription factor
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Summary 1. Insulin induced hypoglyc
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148 Summary specific antibodies con
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150 Summary 16. Transcription facto
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References Abdelmalik PA, Shannon P
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Akwa Y, Ladurelle N, Covey F, Bauli
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Cryer PE. (2002b). The pathophysiol
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Salkovic M, Lackovic Z. (1992). Bra
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(2002). Sulfonylurea receptor type
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pentabromodiphenyl ether (PBDE 99).
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Weiner DM, Levey AI, Brann MR. (199
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6. Anju T R, Pretty Mary Abraham, S
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Body weight (gm) 300 250 200 150 10
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Figure-3 Blood glucose levels at di
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Figure-5 Behavioural response of co
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Figure-7 Scatchard analysis of [ 3
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Figure-11 Real Time PCR amplificati
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C D + IIH Figure--25 Muscarinic M1
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Figure--27 α 7 nACh receptor expre
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Figure--47 Muscarinic M1 receptor e
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Figure--49 α7 nicotinic acetylchol
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Figure--71 α7 nicotinic acetylchol
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C Figure--93 GABAAα1 receptor expr
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Figure-101 Real Time PCR amplificat
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Figure—113 Muscarinic M3 receptor
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Figure--115 GABAAα1 receptor expre
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Figure-117 Scatchard analysis of [
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Figure-119 Scatchard analysis of [
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Figure-131 Muscarinic M1 receptor e
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C D + IIH Figure-133 GABAAα1 recep
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