Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 168 The Impact of Growth Hormone and Gamma- Hydroxybutyrate (GHB) on Systems Related to Cognition JENNY JOHANSSON ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6192 ISBN 978-91-554-8552-8 UPPSALA urn:nbn:se:uu:diva-185631 2012 Dissertation presented at Uppsala University to be publicly examined in B21, BMC, Husargatan 3, Uppsala, Friday, January 18, 2013 at 09:15 for the degree of Doctor of Philosophy (Faculty of Pharmacy). The examination will be conducted in Swedish. Abstract Johansson, J. 2012. The Impact of Growth Hormone and Gamma-Hydroxybutyrate (GHB) on Systems Related to Cognition. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 168. 73 pp. Uppsala. ISBN 978-91-554-8552-8. Drug dependence is a serious and increasing problem in our society, especially among adolescents. The use of the large variety of substances available can result in a range of physiological and psychological adverse effects on individuals and negative consequences on the society overall. Several different types of drugs induce neurotoxicological damages, which in turn can generate impairment in for example the reward system and affect cognitive parameters. The drug gamma-hydroxybutyrate (GHB) is usually considered a harmless compound among abusers, but has now shown to be highly addictive. Furthermore, GHB can cause memory impairments in both humans and animals. On the contrary, growth hormone (GH) and its main mediator insulin-like growth factor 1 (IGF-1) have recently been suggested to improve memory and learning in several studies. The hormones exhibit certain neuroprotective capabilities and have also previously been demonstrated to reverse opioid induced apoptosis in hippocampal cells. These effects and the fact that GHB is shown to increase GH secretion, which attracted considerable attention among body builders, led us to initiate studies on GHB and its impact on relevant systems in the central nervous system (CNS). Thus, the main purpose of the present investigation was to elucidate some of the underlying mechanisms that could account for the effects exerted by GH and GHB in the CNS. We found that a) GH affects the density and functionality of GABAB-receptors and opioid receptors in the male rat brain, b) GHB induces cognitive deficits and down-regulates GABAB- receptors, c) GHB treatment creates an imbalance between the endogenous opioids Met- enkaphalin-Arg6Phe7 (MEAP) and dynorphin B and increases the levels of MEAP in regions of the brain that are associated with drug dependence, and d) GHB affects the expression of IGF-1 receptors but not the plasma levels of IGF-1. In conclusion, the present work demonstrates that GH interacts with both opioid and GABAB-receptors in the male rat CNS and that GHB has an impact on brain regions associated with cognition and the development of dependence. These observations may be of relevance in many aspects related to addiction and might be translated into humans. Keywords: Growth Hormone, Gamma-Hydroxybutyrate, GABAB, Opioids, Insulin-like growth factor 1, Rats, Central Nervous System, Autoradiography, Radioimmunoassay, ELISA, Water Maze, Open Field Jenny Johansson, Uppsala University, Department of Pharmaceutical Biosciences, Box 591, SE-751 24 Uppsala, Sweden. © Jenny Johansson 2012 ISSN 1651-6192 ISBN 978-91-554-8552-8 urn:nbn:se:uu:diva-185631 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-185631) Supervisor Associate professor Mathias Hallberg Co-supervisor Professor Fred Nyberg Faculty opponent Associate professor Linda Fryklund Members of the examining board Professor Lennart Dencker Associate professor Åsa Mackenzie Associate professor Anna Skottner List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Grönbladh, A., Johansson, J., Nyberg, F., Hallberg, M. (2012) Recombinant human growth hormone affects the density and func- tionallity of GABAB receptors in the male rat brain. Neuroendocri- nology, Epub ahead of press. II Johansson, J., Grönbladh, A., Nyberg, F., Hallberg, M. (2013) Application of in vitro [35S]GTPγS autoradiography in studies of growth hormone effects on opioid receptors in the male rat brain. Brain Research Bulletin, 90, 100-106. III Johansson, J., Grönbladh, A., Nyberg F., Hallberg, M. (2012) Gamma-hydroxybutyrate (GHB) induces cognitive deficits and af- fects GABAB receptors and IGF-1 receptors in male rats. In manu- script. IV Johansson, J., Brolin, E., Grönbladh, A., Nyberg, F., Hallberg, M. (2012) Gamma-hydroxybutyrate (GHB) elevates Met-enkephalin- Arg6Phe7 (MEAP) levels in the frontal cortex of the male rat brain. In manuscript. Paper I and II are published by kind permission of S. Karger AG, Basel, Switzerland and Elsevier, Oxford, United Kingdom. Contents Introduction ................................................................................................... 11 Gamma-hydroxybutyrate (GHB) .............................................................. 11 History .................................................................................................. 11 Physiological and psychological effects of GHB ................................ 11 Synthesis and metabolism of GHB ...................................................... 12 Mechanisms of action .......................................................................... 14 Growth Hormone (GH) and Insulin-like Growth factor-1 (IGF-1) .......... 15 History .................................................................................................. 15 The somatotrophic system ................................................................... 16 Pharmacodynamic aspects of GH and IGF-1 ....................................... 18 Physiological and psychological relevance of GH and IGF-1 ............. 20 The brain reward system ........................................................................... 21 The opioid system ..................................................................................... 22 Opioid peptides and receptors .............................................................. 22 Regulation of the reward system by opioid peptides ........................... 23 The GABAergic system ....................................................................... 24 Cognition; learning and memory ......................................................... 25 The aims of the thesis .................................................................................... 27 Methods ......................................................................................................... 28 Animal handling and drug treatment ........................................................ 28 Behavioural methods ................................................................................ 29 The water maze test (WM) .................................................................. 29 The open field (OF) ............................................................................. 30 Tissue and blood collection ...................................................................... 31 Autoradiographic methods ....................................................................... 31 35 GABAB, mu and delta opioid receptor stimulated [ S]GTPγS autoradiography ................................................................................... 32 GABAB receptor autoradiography ....................................................... 33 IGF-1 receptor autoradiography .......................................................... 33 IGF -1 Enzyme-linked immunosorbent assay (ELISA) ........................... 34 Peptide extraction and separation ............................................................. 34 Radioimmunoassay (RIA) ........................................................................ 35 Statistical analysis ..................................................................................... 35 Results and discussion ................................................................................... 37 GABAB receptors ...................................................................................... 37 Mu and delta opioid receptors .................................................................. 40 Insulin-like growth factor-1 receptor ........................................................ 41 Insulin-like growth factor-1 in plasma ..................................................... 42 Opioid peptides .................................................................................... 43 The water maze ......................................................................................... 44 The Open field .......................................................................................... 46 Summarizing conclusion ............................................................................... 48 Populärvetenskaplig sammanfattning ........................................................... 49 Acknowledgements ....................................................................................... 50 References ..................................................................................................... 52 List of abbreviations 1,4-BD 1,4-butanediol CNS Central nervous system DAMGO Tyr-D-Ala-Gly-NMe-Phe-Gly-ol DOP Delta opioid peptide DPDPE [D-Pen2-D-Pen5]-enkephalin Dyn B Dynorphin B ELISA Enzyme-linked immunosorbent assay GABA Gamma-aminobutyric acid GBL