5-HT2C AND NMDA RECEPTORS, IP3, cGMP AND cAMP FUNCTIONAL REGULATION IN PILOCARPINE INDUCED TEMPORAL LOBE EPILEPSY IN RATS: NEUROPROTECTIVE ROLE OF Bacopa monnieri THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY

s Presented 1. Amee Krishnakumar, Pretty Mary Abraham and C. S. Paulose. Enhanced 5-HT2C receptor gene expression in the cerebral cortex of pilocarpine inducedepileptic rats: Neuroprotective role of Bacopa monnieri. InternationalSymposium on Regenerative Neuroscience and Annual Meeting of Societyfor Neurochemistry (India) concurrently held at National Institute of MentalHealth & Neurosciences (NIMHANS) and National Center for BiologicalSciences (NCBS), Bangalore, India from 7th-16 January, 2008 (January2008). 2. Amee Krishnakumar and C. S. Paulose. Down regulation of 5-HT2C receptorgene expression in cerebellum of pilocarpine induced epileptic rats:Therapeutic role of Bacopa monnieri (L.) Pennel. Annual Meeting of Societyfor Biotechnologist, India and National Symposium on Current Trends inStem Cell Biology. Organised by Rajiv Gandhi Centre for Biotechnology,Thiruvanathpuram, India (December 2007) 3. Amee Krishnakumar, Remya Robinson and C. S. Paulose.Acetylcholinestrase activity in the cerebral cortex and muscle of insulininduced hypoglycemic and Streptozotocin induced diabetic rats. XXVNational Symposium on Reproductive Biology and ComparativeEndocrinology. Translational endocrinology and reproductive biology.Organised by Society for Reproductive Biology and ComparativeEndocrinology and Dept. of Zoology, University of Kerala, India (January2007). 4. Amee Krishnakumar, Finla Chathu and C. S. Paulose. Acetylcholineesterase activity in the cerebral cortex and muscle of hypoxic rats: regulatoryrole of glucose, oxygen and epinephrine. National conference onBiotechnology in molecular medicine. Organised by AIMS, Amrita Instituteof Biotechnology Kochi and Society for Biotechnologists, India (January2007). 5. C. S. Paulose, Amee Krishnakumar, Anu Joseph (2006). NeurotransmitterFunctional Role in Neurodegenerative Diseases. Procedings sent to NationalConference on “Environment & Human Health” held by School ofEnvironmental Studies, Dept. of Environmental Science, Cochin University ofScience and Technology, Cochin, India (December 2006). 6. Amee Krishnakumar, Thomas Paul and C.S. Paulose. “Stem cell isolationfrom human umbilical cord blood and neurotransmitters functional role inproliferation of these cells.” Presented at the 18th Kerala Science congressheld at Centre for Earth Science Studies, Trivandrum, India (December2005). 7. Amee Krishnakumar, Rakesh R. and C.S Paulose “Collection AndProcessing Of Human Umbilical Cord Blood For Stem Cell Isolation,” inInternational Conference on Biotechnology and Neurosciences, Dec 29-31,Centre for Neuroscience, Cochin University of Science and Technology,Cochin, India (December 2004). 8. Rakesh R., Amee Krishnakumar, and C.S Paulose “Effect ofneurotransmitters on Stem Cell Proliferation.” in International Conference onBiotechnology and Neurosciences, Dec 29-31, Centre for Neuroscience,Cochin University of Science and Technology, Cochin, India (December2004). Table1Body weight (g) of control and experimental groups of rats Animal statusInitial day ofExperiment30 day ofExperiment45 day ofExperiment Control252 ± 1.10 284 ± 4.78 288 ± 1.22 Epileptic258 ± 2.03 236 ± 3.86 228 ± 3.59 Control+Bacopa monnieri 263 ± 3.27 287 ± 2.17*** 291 ± 3.14*** Epilepsy+Bacopa monnieri 260 ± 2.12 259 ± 1.32*** 265 ± 2.39*** Epileptic+Carbamazepine 262 ± 1.28 254 ± 2.70*** 260 ± 2.27*** Values are mean ± SEM of 4-6 separate experiments; n = 5-6 in each group. ANOVAfollowed by Students-Newman-Keuls Test.p<0.001when compared to Control group***p<0.001 wen compared to Epileptic group Table2Feed intake of control and experimental groups of rats Animal statusInitial day ofExperiment30 day ofExperiment45 day ofExperiment Control118 ± 2.4 128 ± 2.2 130 ± 2.4 Epileptic120 ± 1.5 101 ± 2.0 98 ± 1.7 Control+Bacopa monnieri 122 ± 1.7 131 ± 2.6*** 133 ± 3.0*** Epilepsy+Bacopa monnieri 117 ± 2.0 110 ± 1.7* 122 ± 2.1*** Epileptic+Carbamazepine 119 ± 1.6 108 ± 1.5* 117 ± 2.3*** Values are mean ± SEM of 4-6 separate experiments; n = 5-6 in each group. ANOVAfollowed by Students-Newman-Keuls Test.p<0.001when compared to Control group***p<0.001, *p<0.05 when compared to Epileptic group Table3Water consumption of control and experimental groups of rats Animal statusInitial day ofExperiment(ml)30 day ofExperiment(ml)45 day ofExperiment(ml) Control129 ± 3.3 136 ± 2.4 130 ± 3.1 Epileptic127 ± 2.8 112 ± 2.0 96 ± 2.5 Control+Bacopa monnieri 129 ± 3.5 134 ± 3.2*** 126 ± 2.8*** Epilepsy+Bacopa monnieri 130 ± 3.6 124 ± 2.2* 122 ± 2.3*** Epileptic+Carbamazepine 132 ± 3.4 122 ± 2.6* 125 ± 2.5*** Values are mean ± SEM of 4-6 separate experiments; n = 5-6 in each group. ANOVAfollowed by Students-Newman-Keuls Test.p<0.001when compared to Control group***p<0.001, *p<0.05 when compared to Epileptic group Table4Blood glucose level of control and experimental groups of rats Animal statusInitial day ofExperiment(mg/dL)30 day ofExperiment(mg/dL)45 day ofExperiment(mg/dL) Control119 ± 1.74 118 ± 2.21 120 ± 1.68 Epileptic114 ± 1.83 120 ± 2.56 116 ± 1. 85 Control+Bacopa monnieri 113 ± 1.87 122 ± 1. 77 122 ± 1.74 Epilepsy+Bacopa monnieri 117 ± 2.12 121 ± 1.84 115 ± 1.59 Epileptic+Carbamazepine 120 ± 2.23 120 ± 1.44 121 ± 1.79 Values are mean ± SEM of 4-6 separate experiments; n = 5-6 in each group. ANOVAfollowed by Students-Newman-Keuls Test. Table55-HT and 5-HIAA content in the cerebral cortex ofcontrol and experimental groups of rats Animal status5-HT(nmol/g wet wt.)5-HIAA(nmol/g wet wt.)5-HT/5-HIAA Control1.88 ± 0.08 1.88 ± 0.18 1.02 ± 0.14 Epileptic1.03 ± 0.01 2.53 ± 0.01 0.40 ± 0.19 Control+Bacopa monnieri 1.91 ± 0.03*** 1.84 ± 0.08** 1.04 ± 0.15 *** Epileptic+Bacopa monnieri 1.62 ± 0.07** 2.22 ± 0.06** 0.73 ± 0.10** Epileptic+Carbamazepine 1.34 ± 0.18* 2.35 ± 0.03* 0.57 ± 0.16 Values are mean ± SEM of 4-6 separate experiments; n = 5-6 in each group. ANOVAfollowed by Students-Newman-Keuls Test.p<0.001, p<0.01 when compared to Control group***p<0.01, **p<0.01, *p<0.05 when compared to Epileptic group Table65-HT and 5-HIAA content in the hippocampus ofcontrol and experimental groups of rats Animal status5-HT(nmol/g wet wt.)5-HIAA(nmol/g wet wt.)5-HT/5-HIAA Control1.49 ± 0.10 2.20 ± 0.09 0.54 ± 0.04 Epileptic0.90 ± 0.09 3.24 ± 0.05 0.27 ± 0.05 Control+Bacopa monnieri 1.37 ± 0.04** 2.37 ± 0.15** 0.57 ± 0.08 Epileptic+Bacopa monnieri 1.28 ± 0.05** 2.68 ± 0.08** 0.48 ± 0.06 Epileptic+Carbamazepine 1.18 ± 0.06* 3.18 ± 0.12 0.30 ± 0.04 Values are mean ± SEM of 4-6 separate experiments; n = 5-6 in each group. ANOVAfollowed by Students-Newman-Keuls Test.p<0.001, p<0.01 when compared to Control group***p<0.01, **p<0.01, *p<0.05 when compared to Epileptic group Table75-HT and 5-HIAA content in the cerebellum ofcontrol and experimental groups of rats Animal status5-HT(nmol/g wetwt.)5-HIAA(nmol/g wet wt.)5-HT/5-HIAA Control2.76 ± 0.12 2.35 ± 0.05 1.18 ± 0.06 Epileptic1.52 ± 0.18 2.82 ± 0.04 0.53 ± 0.04 Control+Bacopa monnieri 2.81 ± 0.14*** 2.21 ± 0.05*** 1.27 ± 0.03*** Epileptic+Bacopa monnieri 2.32 ± 0.05** 2.52 ± 0.07** 0.92 ± 0.08** Epileptic+Carbamazepine 2.10 ± 0.09** 3.02 ± 0.09 0.68 ± 0.05 Values are mean ± SEM of 4-6 separate experiments; n = 5-6 in each group. ANOVAfollowed by Students-Newman-Keuls Test.p<0.001, p<0.01, p<0.01 when compared to Control group***p<0.01, **p<0.01, *p<0.05 when compared to Epileptic group Table85-HT and 5-HIAA content in the brainstem ofcontrol and experimental groups of rats Animal status5-HT(nmol/g wet wt.)5-HIAA(nmol/g wet wt.)5-HT/5-HIAA Control2.95 ± 0.04 3.15 ± 0.12 0.94 ± 0.04 Epileptic2.30 ± 0.06 3.70 ± 0.06 0.62 ± 0.09 Control+Bacopa monnieri 2.87 ± 0.12** 3.27 ± 0.12** 0.87 ± 0.03 Epileptic+Bacopa monnieri 2.56 ± 0.05* 3.56 ± 0.05* 0.72 ± 0.05 Epileptic+Carbamazepine 2.78 ± 0.07** 3.68 ± 0.03 0.75 ± 0.06 Values are mean ± SEM of 4-6 separate experiments; n = 5-6 in each group. ANOVAfollowed by Students-Newman-Keuls Test.p<0.001, p<0.05 when compared to Control group**p<0.01, *p<0.05 when compared to Epileptic group Bound(fmoles/mg protein)0 25 50 75 100 125 150 175 200Bound/free(fmoles/mgprotein/nM) 050100150200250300350 ControlEpilepticC+BE+BE+CFigure 1Scatchard analysis of [H]mesulergine binding against mesulergine incerebral cortex of control and experimental rats. C Control, E Epileptic, C + B Control + Bacopa monnieri,E + B Epileptic + Bacopamonnieri, E + C Epileptic + Carbamazepine Table9Scatchard analysis of [H]mesulergine binding against mesulergine incerebral cortex of control and experimental rats. Animal statusBmax(fmoles/mg protein)Kd(nM)Control65 ± 040.38 ± 0.04Epileptic176 ± 110.65 ± 0.02Control+Bacopa monnieri62 ± 080.37 ± 0.05Epileptic+Bacopa monnieri117 ± 03***0.42 ± 0.03**Epileptic+Carbamazepine132 ± 15***0.43 ± 0.03** Values are mean ± SEM of 4-6 separate experiments; n = 5-6 in each group. ANOVAfollowed by Students-Newman-Keuls Test.p<0.001, p<0.01 when compared to Control group***p<0.001, **p<0.01 when compared to Epileptic group Figure 2Real Time amplification of 5-HT2C receptor mRNA from thecerebral cortex of control and experimental rats C Control, E Epileptic, C + B Control + Bacopa monnieri, E + B Epileptic + Bacopamonnieri, E + C Epileptic + Carbamazepine Table10Real Time amplification of 5-HT2C receptor mRNA from thecerebral cortex of control and experimental rats Animal statusLog RQ value