i PLACENTAL MORPHOLOGY AND THE CELLULAR BRAIN IN MAMMALIAN EVOLUTION by Eric Lewitus A Dissertation submitted in part fulfillment of the degree of Doctorate of Philosophy of Biological Anthropology University College London ii I, Eric Lewitus confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. iii – Harpo iv A major focus of evolutionary neurobiology has been on whether different regions of the eutherian brain evolve in concert, and how free the brain is to evolve independently of body plans. Since the eutherian brain is loosely modularized, such that one region is rarely isolated for specialization at the expense of others, but the design of modularization itself can be adapted by tweaking developmental programs, the degree to which brain regions must evolve in concert and can evolve independently may carry a deep phylogenetic signal. Using data collected from preserved brain tissue of 37 primate, 21 carnivore, and 15 other eutherian species (spanning 11 orders), I examined the phylogenetic level at which the proliferation of neurons and glia in the primary visual cortex and hippocampus proper, as well as granular layer volumes of the dentate gyrus and cerebellum, may be constrained by conserved developmental programs. In doing so, I was able to test for cellular signatures of (1) evolutionary changes in metabolic activity, (2) phylogenetic divergences, (3) specializations in behavior, and (4) developmental constraints. The degree to which disparate brain regions evolve in concert is shown to be generally conserved in Eutheria, although a derived ability to evolve regions independently is observed along the primate lineage. Using a separate dataset on placental and life-histroy character states, a comprehensive comparative phylogenetic approach was used to resolve relationships among five aspects of placental structure and to identify syndromes of placental morphology with life-history variables. My results support two discrete biological phenotypes of placental morphology and life- history, which are shown to have an evolutionary affect on allocortical, but not neocortical, brain organization. I have provided a new perspective on exploring how developmental constraints – acting both within and without the brain – may affect brain organization at the cellular level, and the extent to which those constraints have been adapted along certain eutherian lineages. v TABLE OF CONTENTS INTRODUCTION 1 CHAPTER 1: CELLULAR PROCESSES (HOW THE BRAIN EVOLVES) 4 Neurogenesis and cortical expansion 4 Glia 5 Brain metabolism 10 CHAPTER 2: THE COMPARATIVE METHOD IN NEUROSCIENCE 13 Brain scaling 13 Environment, behavior, and placentation 14 CHAPTER 3: HOW THE BRAIN EVOLVED 17 Homology 17 Mammalian brain 20 Total brain size 23 Regional brain size 26 Primate brain 30 Carnivore brain 35 CHAPTER 4: METHODOLOGY (BRAIN DATA) 37 Fractionator principle 37 Optical fractionator 38 Cavalieri volume estimator 41 Coefficient of error 41 Materials 43 Artifactual error 45 The effect of phylogeny on scaling 45 Data analysis 46 CHAPTER 5: PRIMARY VISUAL CORTEX 53 Anatomy 53 Evolution 55 Analysis I 65 Aim 65 vi Materials 65 Demarcation 65 Cell counting 65 Results I 68 Hypothesis and predictions 68 Cellular scaling patterns among taxonomic groups 68 Cellular scaling patterns within primates 69 Cellular scaling patterns among placental groups 72 Discussion I 74 Constraints on cellular densities in a limited cortical regions 74 Placentatation and the neocortex in eutheria 76 Specializations in primate motion-processing pathways show glia-neuron signatures 77 Body mass is a poor parameter for intelligence 80 CHAPTER 6: HIPPOCAMPUS 82 Structure 82 Circuitry 85 Evolution 85 Shared and derived functions 89 Analysis II 93 Aim 93 Materials 93 Demarcation of CA1-3 93 Demarcation of the dentate gyrus 93 Cell counting in CA1-3 94 Volumetric estimates of the dentate gyrus 95 Results II 96 Hypotheses and predictions 96 Scaling of the CA subfields in mammals 96 The CA subfields and the dentate gyrus 102 Human predictions for the cellular hippocampus based on primates and other mammals 103 Mode of placentation and the hippocampus 104 Discussion II 107 Fetal development regulates evolution of the hippocampus 107 vii CHAPTER 7: DEVELOPMENTAL CONSTRAINTS 110 Cerebellar function and evolution 111 Analysis III 116 Volumetric estimates of the cerebellum 116 Results III 117 Hypotheses and predictions 117 Regressors of the cerebellum 118 V1 and the hippocampal formation 119 Discussion III 121 Divorcing the cerebrum and the cerebellum 121 Constraints on cellular reorganization in diverse regions of the brain 123 CHAPTER 8: LIFE-HISTORY CORRELATES OF PLACENTAL EVOLUTION 125 Materials and methods 125 Definitions 130 Results IV 136 Ancestral reconstructions 136 Mutational mapping 138 Character associations 138 Discussion IV 141 Yolk sac and placental shape 142 Diffusion and exchange 143 Viviparity-driven and maternal-fetal conflicts 147 CONCLUSIONS 150 REFERENCES 152 APPENDIX TABLES A1 SUPPLEMENTAL FIGURES S1 viii LIST OF TABLES Table 1: List of species by taxonomy 47 Table 2: List of species by placental group 48 Table 3: Slope estimates and correlation coefficients in taxonomic groups for V1 70 Table 4: Hominoid predictions based on OLS regressions of Old and New World monkeys 71 Table 5: Human predictions based on OLS regressions of hominoids 72 Table 6: Slope estimates and correlation coefficients in taxonomic groups for the hippocampus 97 Table 7: Slope estimates and correlation coefficients in placental groups for the hippocampus 104 Table 8: Slope estimates and correlation coefficients in taxonomic groups between cerebellar and cerebral variables 118 Table 8: Slope estimates and correlation coefficients in taxonomic groups between V1 and the hippocampus 118 Table 10: Definitions of placental and life-history variables 126 Table 11: Ancestral reconstructions of placental character states 136 Table 12: Mutational mapping of placental character states 137 Table 13: Association statistics of placental character states 137 Table 14: Constellations of placental and life-history variables 140 APPENDIX TABLES Table A1a: Stereological results for carnivores and AS A2 Table A1b: Anatomical variables for carnivores and AS A3 Table A1c: Stereological results for primates A4 Table A1d: Anatomical variables for primates A5 Table A2: Slope estimates and correlation coefficients in placental groups for V1 A6 Table A3: Stepwise AIC regression models for CA1-3 A7 Table A4a: Stepwise AIC regression models for all brain regions in AS A8 Table A4b: Stepwise AIC regression models for all brain regions in carnivores A9 Table A4c: Stepwise AIC regression models for all brain regions in primates A10 Table A6a: Placental data matrix A11 Table A6b: Categories for variables in placental matrix A12 Table A7: Categories and ranges for discrete life-history variables A12 Table A8: Life-history data matrix A13 ix Table A9a: Definitions of life-history variables A14 Table A9b: Residuals of life-history variables after size correction A15 Table A10a: Association statistics for uterus A16 Table A10b: Association statistics for placental shape A17 Table A10c: Association statistics for maternofetal interdigitation A18 Table A10d: Association statistics for interhemal barrier A19 Table A10e: Association statistics for yolk sac A20 x LIST OF FIGURES Figure 1: Supragranular layers of the subventricular zone 6 Figure 2: Myelin sheathing of axons 9 Figure 3: Derived features of the mammalian brain 21 Figure 4: Three-dimensional disector 39 Figure 5: Optical fractionator 40 Figure 6: Point-grid system for Cavalieri method 44 Figure 7: Photograph of mounted slides 46 Figure 8: Phylogeny of species sample for brain data 49 Figure 9: Subdivisions of the visual cortex 55 Figure 10: Layers of the primary visual cortex 56 Figure 11: Schematic of honeycomb model of layer IVa organization 61 Figure 12: Evolution of layer IVa in Haplorrhini 62 Figure 13: Boundaries of the primary visual cortex 66 Figure 14: Identification of neurons and glia in the primary visual cortex 67 Figure 15: Bar graph of cellular densities in V1 69 Figure 16: Scaling of glia on neuron density and glia-neuron ratio on brain mass in V1 for taxonomic groups 73 Figure 17: Position, structure, and composition of the hippocampal formation 83 Figure 18: Layers of the hippocampus proper 84 Figure 19a: Relative importance metrics and recursive trees for glia-neuron ratio in CA1-3 for AS 98 Figure 19b: Relative importance metrics and recursive trees for glia-neuron ratio in CA1-3 for carnivores 99 Figure 19c: Relative importance metrics and recursive trees for glia-neuron ratio in CA1-3 for primates 100 Figure 20: Bar graph of hippocampal variables 101 Figure 21: Scaling of dentate gyrus as a function of brain mass in taxonomic groups 102 Figure 22: Scaling of glia on neuron density in CA1-3 for primates 103 Figure 23: Scaling of glia on neuron density and glia-neuron ratio on brain mass in CA1-3 for taxonomic groups 105 Figure 24: Scaling of dentate gyrus as a function of brain mass in placental groups 106 Figure 25: Y-intercepts of glia on neuron density for primates and other mammals 108 Figure 26: Gross anatomy of the cerebellum 112 Figure 27: Demarcation of the granule cell layer of the cerebellum 113 xi Figure 28: Scaling of dentate gyrus with cerebellum in taxonomic groups 119 Figure 29: Scaling of glia density, neuron density, and glia-neuron