Thesis (Ph. D. in Neuroscience)--Vanderbilt University, Aug. 2008. / Title from title screen. Includes bibliographical references.

M.A. (Philosophy) / Philosophy of mind has begun to rely on input from neurobiology and neuroscience to answer questions concerning consciousness, representation and the subjective character of experience. Some philosophers believe that through studies done on the brain, neuroscience will help us answer the hard-problems of consciousness. The first chapter of this paper is concerned with the kind of contributions neurobiology can make to certain debates in philosophy of mind and proceeds to explain that even though neurobiology is mostly a positive contributor to philosophy of mind, it still fails to answer some of the more pressing issues in philosophy of mind. In the second chapter of this paper, I focus on Frank Jackson’s Knowledge Argument. Jackson’s Knowledge Argument is perhaps one of the most challenged arguments concerning experience in philosophy of mind and responses to this argument will possibly tell us more about the mind. Jackson argues that we gain a new kind of knowledge when we experience something, for example when seeing the colour red. He argues that these new learnt facts are non-physical. David Lewis argues that a person learns no new facts upon experiencing red, but rather abilities such as the ability to recognize, imagine and remember. In this chapter I also examine some of the counterarguments to Jackson’s Knowledge Argument and conclude that these philosophers have approached the Knowledge Argument incorrectly. I suggest a different physicalist response to the Knowledge Argument. In the third chapter of this paper, I propose a new physicalist account of experience I call ‘bifactualism’. The Knowledge Argument is an argument for dualism that claims that there are both physical and non-physical facts which can be learnt through experience. I reject the Knowledge Argument and suggest bifactualism. Bifactualism is a new physicalist account essentially comprising two elements. First, it distinguishes between two kinds of physical facts: general and particular. The second element is the claim that this distinction explains facts about consciousness, without resorting to dualism. I disagree with the dualist claims made in the Knowledge Argument and show that the Knowledge Argument neither supports dualist claims nor does it refute bifactualist claims. Most contributions made with regards to the Knowledge Argument focus on what Mary is able to learn once outside the black and white room. Bifactualism is interested in what she is able to learn in the black and white room which makes this a novel approach to the Knowledge Argument. In the fourth chapter of this paper I propose bifactualist responses to several issues that have been highlighted throughout this paper. In this chapter, I primarily focus on Nagel’s What it is Like to be a Bat? Nagel claims that we cannot know what it is like to be a bat subjective character of experience. This chapter argues that it is as difficult to know the feeling of what it is like, or WIL (Prinz, 2012), to be a bat which has a feeling of WIL, as it is to know the feeling of what it is like to be a book (which has no feeling of what it is like: non- WIL). I argue that this is not because of two different ways of knowing two different properties, but rather that there are two different physical facts about both WIL and non-WIL properties. I show that with a bifactualist account, there are particular physical facts that can be known about WIL and non-WIL properties alike that are not expressible in the language of physics.

The evolution, development and function of the embryonic and larval peripheral nervous system (PNS) were investigated in a number of dipteran species, including Aedes aegypti, Hermetia pallescens, Lucilia cuprina, Bactrocera tryoni and Drosophila melanogaster. Comparative immuno-cytochemical data was generated for developmental proteins in the embryonic PNS and degenerative PCR was employed to identify homologous proneural genes amongst the species. Immunocytochemistry revealed aspects of sense-organ evolution amongst the Diptera, particularly an increase in sense-cell number and number of sensilla comprising proprioceptive organs in the crawling versus swimming larvae. The function of putative proprioceptive cells was investigated via mutant analysis and laser ablation. Results indicate that the multiple-dendrite (md) and bipolar-dendrite (bd) neurons contribute in a cumulative and roughly equal fashion to maintain peristaltic waves in crawling larvae. This work highlights the usefulness of the dipteran PNS as a model of adaptive evolution that can be investigated via developmental mechanisms.

Modern humans, archaic humans and great apes are genetically closely related and share many behavioral and anatomical similarities. However, modern humans differ from the others by a fast development of complex culture and technologies, that rely on complex cognitive abilities. Cognition is directly linked to brain structure and neuronal function. In this thesis, I study neuronal differences between humans and chimpanzees and bonobos as well as morphological differences of “ancestralized” and “modernized” human neurons, that could possibly contribute to cognitive differences among the different hominid species. The comparison of human and ape induced pluripotent stem cell-derived neurons (iNeurons) revealed that the human neurons mature transcriptionally, morphologically and functionally slower than their ape counterparts. By injecting the mRNA of 16 genes that are relevant for neuronal function and carry amino acid substitutions on the modern human lineage, I could show that the 16 proteins are able to increase the total neurite length and suggest a potential slower development of neurons injected with the modern human variants. In a single gene approach, I investigated the effect of modern and ancestral human SSH2 variants on neurite outgrowth and found differences in neurite length and branching pattern, making SSH2 a promising candidate for being involved in human neuron-specific morphology. I showed that iNeurons can serve as a model system for evolutionary neurobiology. I gained insights into features of neurons that are unique to modern humans in comparison to their closest relatives, the great apes and archaic humans.:BIBLIOGRAPHISCHE DARSTELLUNG 2 TABLE OF CONTENTS 3 1. THESIS SUMMARY 6 COMPARISON OF HUMAN AND APE INDUCED NEURONS 7 MICROINJECTION AS A TOOL TO STUDY RECENT HUMAN HISTORY 8 MODELING THE EFFECT OF A SINGLE GENE BY USING TRANSFECTION OF PRIMARY NEURONS 9 CONCLUSION 10 2. ZUSAMMENFASSUNG 11 VERGLEICH VON NERVENZELLEN VON MENSCHEN UND MENSCHENAFFEN 12 MIKROINJEKTION ZUR UNTERSUCHUNG DER JÜNGSTEN MENSCHLICHEN VERGANGENHEIT 14 MODELLIERUNG DES EFFEKTS EINES EINZELNEN GENS AUF DAS NEURITEN WACHSTUM VON PRIMÄREN NEURONEN 15 FAZIT 16 3. INTRODUCTION 17 3.1 THE HOMINID FAMILY 17 3.2 THE HOMINID BRAIN 19 3.3 THE INEURON MODEL SYSTEM 23 3.4 MICROINJECTION OF INS: A TOOL TO STUDY ANCIENT AND MODERN HUMAN NEURONS 24 4. MATERIAL AND METHODS 27 4.1 METHODS 27 4.1.1 GENERATION OF RTTA/NGN2-POSITIVE PLURIPOTENT STEM CELL LINES 27 4.1.2 CULTURING OF PLURIPOTENT STEM CELL LINES 27 4.1.3 CRYOPRESERVATION OF PLURIPOTENT STEM CELLS 28 4.1.4 DIFFERENTIATION OF RTTA/NGN2-POSITIVE PLURIPOTENT STEM CELLS TO INEURONS 28 4.1.5 SINGLE CELL TRANSCRIPTOMIC ANALYSIS 29 4.1.5.1 SINGLE CELL RNA-SEQ DATA GENERATION 29 4.1.5.2 DATA PROCESSING 30 4.1.5.3 IDENTIFICATION OF NEURONAL CELLS AND DIFFERENTIALLY EXPRESSED GENES 30 4.1.5.4 GENE ONTOLOGY ENRICHMENT ANALYSIS 31 4.1.6 ELECTROPHYSIOLOGY 32 4.1.6.1 RECORDINGS 33 4.1.6.2 ANALYSIS 33 4.1.7 LIPOFECTION OF INEURONS 34 4.1.8 IMMUNOSTAINING OF INEURONS 34 4.1.8.1 PREPARATION OF PARAFORMALDEHYDE FIXATIVE 34 4.1.8.2 FIXATION OF GFP-LABELLED INEURONS 34 4.1.8.3 QUENCHING AND IMMUNOSTAINING OF GFP-LABELLED INEURONS 35 4.1.9 IMAGE ACQUISITION 35 4.1.10 IMAGE QUANTIFICATION 35 4.1.10.1 QUANTIFICATION OF NEURONAL MORPHOLOGY. 35 4.1.10.2 QUANTIFICATION OF TUJI SIGNAL. 36 4.1.11 ASSIGNMENT OF CELL IDENTITY. 36 4.1.12 MICROINJECTION 36 4.13 TRANSFECTION OF PRIMARY NEURONS WITH SSH2 PLASMIDS 40 4.1.3.1 CELL CULTURE 40 4.1.3.2 TRASFECTION 40 4.2 MATERIALS 41 5. RESULTS 48 5.1 COMPARISON OF INDUCED NEURONS REVEALS A SLOWER STRUCTURAL AND FUNCTIONAL MATURATION IN HUMANS THAN IN APES 48 5.1.1 ABSTRACT 49 5.1.2 INTRODUCTION 49 5.1.3 RESULTS 51 5.1.3.1 MATURATION OF HUMAN AND APE INDUCED NEURONS IN VITRO 51 5.1.3.2 MORPHOLOGICAL HETEROGENEITY IN IN POPULATIONS 53 5.1.3.3 MORPHOLOGICAL MATURATION OF APE AND HUMAN INS 55 5.1.3.4 SCRNASEQ REVEALED THAT NGN2 INDUCES CORTICAL AND SENSORY NEURON FATES 56 5.1.3.5 NGN2 ALSO INDUCES CORTICAL SENSORY NEURON FATE 59 5.1.3.6 TRANSCRIPTIONAL MATURATION OF HUMAN AND CHIMPANZEE INS 60 5.1.3.7 INTRINSIC PASSIVE ELECTROPHYSIOLOGICAL PROPERTIES OF HUMAN AND APE INS 62 5.1.3.8 ACTIVE ELECTROPHYSIOLOGICAL PROPERTIES OF APE AND HUMAN INS 63 5.1.4 DISCUSSION 65 5.1.4.1 NGN2 INDUCES HETEROGENEOUS NEURONAL FATES 65 5.1.4.2 EVOLUTIONARY ASPECTS OF NEURONAL MATURATION 65 5.5. SUPPLEMENTARY INFORMATION 68 5.5.1 SUPPLEMENTARY FIGURES 68 5.5.2 SUPPLEMENTARY TABLES 81 5.2 MRNA MICROINJECTION AS A TOOL TO STUDY RECENT HUMAN BRAIN EVOLUTION 88 5.2.1 ABSTRACT 89 5.2.2 INTRODUCTION 89 5.2.3 RESULTS 91 5.2.3.1 NEURONAL GENES CARRYING AMINO ACID SUBSTITUTIONS BETWEEN MODERN AND ARCHAIC HUMANS 91 5.2.3.2 SCREEN OF DISTINCT TRANSCRIPTOME DATASETS FOR EXPRESSION ANALYSES OF THE 16 NEURONAL GENES 94 5.2.3.3 TRANSCRIPTIONAL ANALYSES OF THE 16 NEURONAL GENES IN INEURONS 97 5.2.3.4 MICROINJECTION OF THE 16 NEURONAL GENES INTO INEURONS 99 5.2.3.5 EFFECT OF SSH2 GENE VARIANTS ON HUMAN PRIMARY NEURONS 101 5.2.4. DISCUSSION 103 6. DISCUSSION 108 6.1 COMPARISON OF HUMAN AND APE INDUCED NEURONS 108 6.2 EXPERIMENTAL SYSTEMS TO MODEL RECENT HUMAN HISTORY 109 6.3 MRNA MICROINJECTION TO MODEL MULTIGENIC HUMAN TRAITS 110 6.4 MODELING THE EFFECT OF A SINGLE GENE BY USING TRANSFECTION OF PRIMARY NEURONS 111 6.5 CONCLUDING REMARKS 111 INDEX OF FIGURES 113 SUPPLEMENTARY FIGURES 113 INDEX OF TABLES 114 SUPPLEMENTARY TABLES 114 REFERENCES 115 SOFTWARE AND SCRIPTS 128 ACKNOWLEDGEMENTS 129 CURRICULUM VITAE 130 PUBLICATIONS 133 SELECTED TALKS 134 POSTER PRESENTATIONS 134 SELBSTÄNDIGKEITSERKLÄRUNG 135