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The Function of Hippocampal Opioid Peptides in the Genetically-Controlled Exploratory Responses to Novelty in Mice J.H.H.M. van Daal THE FUNCTION OF HIPPOCAMPAL OPIOID PEPTIDES IN THE GENETICALLY-CONTROLLED EXPLORATORY RESPONSES TO NOVELTY IN MICE THE FUNCTION OF HIPPOCAMPAL OPIOID PEPTIDES IN THE GENETICALLY-CONTROLLED EXPLORATORY RESPONSES TO NOVELTY IN MICE een wetenschappelijke proeve op het gebied van de natuurwetenschappen PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Katholieke Universiteit te Nijmegen, volgens besluit van het College van Decanen in het openbaar te verdedigen op woensdag 14 maart 1990 des namiddags te 1.30 precies door JAN HENRI HUBERT MARIE VAN DAAL geboren op 15 juni 1953 te Venray promotores : Prof. Dr. S.E. Wendelaar Bonga Prof. Dr. R. Nieuwenhuys co-promotores: Dr. J.H.F. van Abeelen Dr. B.G. Jenks ISBN 90-9063232-7 Omslagillustratie: een hippocampus getekend ter versiering van de eerste letter in Vestlings Syntagma anatomicum, 1647. We shall not cease from exploration And the end of all our exploring Will be to arrive where we started And know the place for the first time. Four Quartets: Little Gidding T.S. Eliot List of Abbreviations BSA : Bovine serum albumin CA : Cornu ammonis СРВ : Carboxypeptidase В DAB : Diaminobenzidine ECF : Enkephalin-containing peptide EGTA : Ethyleneglycol-bis-(|l-aminoethyl ether) Ν,Ν,Ν',Ν' tetraacetic acid) Gamma-amino-n-butync acid Goat-anti-rabbit Broad-sense hentability Narrow-sense herltability 0.1N HCl : methanol - 1 : 1 v/v Intra- and infrapyramidal mossy fibers Kilodalton Normal goat serum Peroxidase-anti-peroxidase Polyethylen glycol Additive-genetic correlation coefficient Environmental correlation coefficient Genetic correlation coefficient Phenotypical correlation coefficient Radioimmunoassay Sodium dodecyl sulphate Standard error Standard error of the mean Suprapyramidal mossy fibers Tris-buffered saline Tris(hydroxyraethyl)aminomethane Additive-genetic variance Dominance variance Environmental variance Genetic variance Phenotypical variance (within the heterozygous population) Phenotypical variance CONTENTS Chapter 1 General Introduction 1.1. Exploratory behavior 1 1.1.1. Adaptive value 1 1.1.2. Processing of novel information 2 1.2. The hippocampus and exploration 3 1.2.1. Morphology of the hippocampus 3 1.2.2. Theories of hippocampal functioning 7 1.2.3. Neurochemical mechanisms 10 1.3. The importance of the genotype 14 1.3.1. The genotype as a tool 14 1.3.1.1. Inbred strains 16 1.3.1.2. Heterogeneous lines 17 1.3.1.3. Selection lines 18 1.3.1.4. Effects of Y-chromosomal variation 18 1.3.2. Quantitative genetics 19 1.3.2.1. Heritability 20 1.3.2.2. Genetic correlations 21 1.4. Objectives of the present research 22 Chapter 2 Dynorphin В and Methionine-Enkephalin in the Mouse Hippocampus 2.1. Introduction 25 2.2. Methods 26 2.2.1. Production and characterization of antisera directed against dynorphin В and Methionine- enkephalin 26 2.2.2. The mouse strains used 27 2.2.3. Colchicine treatment 27 2.2.4. Perfusion technique and tissue staining 28 2.2.4.1. Light-microscopy 28 2.2.4.2. Electron-microscopy 29 2.3. Results 30 2.3.1. Characterization of the antisera 30 2.3.2. Localization at the light-microscopic level 31 2.3.3. Ultrastructural localization of dynorphin В 32 2.4. Discussion 34 Chapter 3 A Genetic-Correlational Study of Hippocampal Structural Variation and Variation in Exploratory Activities of Mice 3.1. Introduction 37 3.2. Methods 38 3.2.1. Strains of mice and husbandry 38 3.2.2. Exploratory behavior 39 3.2.2.1. Ethograra 39 3.2.2.2. Recording and quantification 39 3.2.3. Morphometry 40 3.2.3.1. Defining the morphometric variables 40 3.2.3.2. Selection of hippocampal level 41 3.2.3.3. Instrumentation and method of measurement .... 42 3.2.4. Tissue preparation and immunohistochemistry 43 3.2.5. Statistics 43 3.3. Results 44 3.3.1. Within- and between-strain differences 44 3.3.2. Y-chromosomal effects 46 3.3.3. Quantitative-genetic analysis 47 3.4. Discussion 55 Chapter 4 A Genotype-Dependent Hippocampal Dynorphinergic Mechanism Controls Mouse Exploration 4.1. Introduction 59 4.2. Methods 60 4.2.1. Behavioral testing 60 4.2.2. Antiserum 60 4.2.3. Injection procedures 61 4.3. Results 61 4.4. Discussion 65 Chapter S A Genetic-Correlational Study of Hippocampal Neurochemical Variation and Variation in Exploratory Activities of Mice 5.1. Introduction 67 5.2. Methods 68 5.2.1. Mouse strains used 68 5.2.2. Observation and procedure 68 5.2.3. Tissue preparation for radioimmunoassay 69 5.2.4. Radioimmunoassy of dynorphin В and Methionine- enkephalin 70 5.2.4.1. Radio-labeling 70 5.2.4.2. The technique 71 5.2.4.3. Specificity and sensitivity 72 5.2.4.4. Evaluation of the radioimmunoassay 72 5.2.5. Statistical evaluation 77 5.3. Results 77 5.3.1. Effects of exposure to novelty 77 5.3.2. Strain-dependent influences of novelty 79 5.3.3. Y-chroraosomal effects 82 5.3.4. Quantitative-genetic analysis 82 5.3.5. Correlated responses to selection 89 5.4. Discussion 90 Chapter 6 Biosynthesis and Release of Mouse Hippocampal Dynorphin and Enkephalin 6.1. Introduction 95 6.2. Methods 97 6.2.1. Animals 97 6.2.2. Preparation of hippocampal slices 98 6.2.3. The incubation medium 98 6.2.4. Labeling of hippocainpal slices with [ H]-amino acids .. 99 6.2.5. Analysis of incorporated [ HJ-araino acids by SDS gel electrophoresis 99 6.2.6. Release of hippocainpal opioids by superfusion 100 6.2.7. High-affinity re-uptake of [3H]-GABA and superfusion in the presence of methionine-enkephalin 101 6.2.8. Separation of ECP's on Sephadex gel 101 6.2.9. Enzyme-treatment 102 6.2.10.Analysis of released ECP's from hippocampal slices .... 102 6.3. Results 103 6.3.1. Viability of hippocampal tissue in vitro 103 6.3.2. Release of hippocampal methionine-enkephalin and dynorphin В , 105 6.3.3. Release of [ HJ-GABA in the presence of methionine- enkephalin 106 6.3.4. Enzymatic generation of enkephalins IOS 6.4. Discussion 110 Chapter 7 General Discussion 113 Summary 117 Samenvatting 121 Appendix I : Quantitative-genetic method 125 Appendix II: Basic data 131 References 135 List of publications 147 Dankbetuiging 149 Curriculum vltae 151 Aan mijn moeder en in aandenken aan mijn vader Chapter 1 GENERAL INTRODUCTION 1.1. Exploratory Behavior 1.1.1. Adaptive Value ANIMALS, WHEN CONFRONTED with changes in their surroundings, or when entering new environments, often display a behavioral pattern termed exploration. In this behavior, visual, acoustic, olfactory, and tactile components can be distinguished. The concept of exploration is closely linked with that of novelty (Barnett & Cowan, 1976). The latter can only be defined in connection with an animal's previous experience. Therefore, a distinction has been made between absolute novelty, a confrontation with stimuli that the animal has never experienced before, and relative novelty, in which familiar items have been arranged in an unfamiliar way (Hennessy & Levine, 1979). The former situation can readily be achieved in the laboratory but is seldom encountered by animals in their natural environment. Novelty evokes either a seemingly spontaneous approach towards a new stimulus, termed neophilia, or avoidance, termed neophobia. The latter tendency is manifest in species that are heavily predated upon, for example the commensal rat with man as its predator. These rodents, even after a number of generations in the laboratory, avoid objects and incentives offered to them outside their home cage (Barnett 6 Cowan, 1976). Even food presented to them in an unfamiliar way is rejected (ibid.). This tendency can be considered to be a protective mechanism that guards the animal from dangers which an unknown environment may conceal. Neophilia, on the other hand, can easily be elicited in highly domesticated animals, such as mice and rats bred in the laboratory. Genetic selection can probably shift the balance in favor of either avoidance or approach behavior (Cowan, 1983). - 1 - Surprisingly, domesticated rodents' often explore without any reinforcement. Mice and rats placed in a plus-maze choose arms alternately, independent of the presentation of food (Barnett 6 Cowan, 1976). This tendency is sometimes referred to with the anthropomorphism 'curiosity'. I.e., novelty in itself is rewarding, and neophilia is regarded as an indicator of intelligent behavior {cf. Archer & Birke, 1963). It seems better, however, to use the concept of latent learning here, which means that what is learnt may not be observable at the time. For example, exploratory movements of animals, often observed after they have fed, increase their chances of finding new sources of food in their surroundings (Barnett ь Cowan, 1976). Exploration has therefore a survival value: in collecting information about an unknown environment it promotes the chances of finding necessities such as food, shelter, escape routes, and conspecifics, thereby increasing an individual's reproductive success (fitness). Also, exploration influences the dispersal of individuals from their native population so that gene flow is facilitated and genetic variability is maintained (Gaines & McClenaghan, 1980). In terms of evolution, a well-balanced exploratory behavioral system will facilitate the adaptation to new habitats and, in this way, enhance the survival of the species. 1.1.2.