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Auditory brainstem neurons as components of a biohybrid system [Elektronische Ressource] / vorgelegt von Thomas Künzel

85 pages
Auditory br ainstem ne urons a scomponents of  a  bi ohybrid s ystemVon de r F akultät für M athematik, Inf ormatik und N aturwissenschaften de r Rhe inisch­Westfälischen T echnischen H ochschule A achen z ur E rlangung de s a kademischen G radeseines D oktors de r N aturwissenschaften g enehmigte D issertationvorgelegt v onDiplom­Bi ologe T homas K ünzelaus M arlBerichter:  Universitätsprofessor D r. H arald L ukschUniversitätsprofessor D r. H ermann W agnerTag de r m ündlichen P rüfung: 25. S eptember 200 7Diese D issertation i st a uf de n Int ernetseiten de r H ochschulbibliothek onl ine v erfügbar.Contents1 Introduction 51.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.1.1 Functional neuronal circuits in vitro and biohybrid systems . . . . 51.1.2 Form and function of the auditory brainstem of birds . . . . . . . . 71.1.3 Development of the auditory brainstem of birds . . . . . . . . . . . 111.2 Aims of the thesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 Materials and methods 162.1 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.1.1 Culture Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.1.2 Primary culture preparation of the E6.5 auditory brainstem . . . . 172.1.3 Prelabeling of NM/NL neurons with fluorescent tracers. . . . . . . 182.1.4 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.
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Auditory brainstem neurons as components of a biohybrid system
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-WestfäHochschule Aachen zur Erlangung des akademischen Gradeslischen Technischen eines Doktors der Naturwissenschaften genehmigte Dissertation
Berichter:
vorgelegt von
Diplom-Biologe Thomas Künzel aus Marl
Universitätsprofessor Dr. Harald Luksch Universitätsprofessor Dr. Hermann Wagner
Tag der mündlichen Prüfung: 25. September 2007
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
Contents
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Introduction 1.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 Functional neuronal circuits in vitro and biohybrid systems 1.1.2 Form and function of the auditory brainstem of birds . . . . 1.1.3 Development of the auditory brainstem of birds . . . . . . . 1.2 Aims of the thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Materials and methods 2.1 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 Culture Substrates . . . . . . . . . . . . . . . . . . . . . . . 2.1.2 Primary culture preparation of the E6.5 auditory brainstem 2.1.3 Prelabeling of NM/NL neurons with fluorescent tracers . . . 2.1.4 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Staining and microscopy techniques . . . . . . . . . . . . . . . . . . 2.2.1 Phase-contrast and morphometry . . . . . . . . . . . . . . . 2.2.2 Immunocytochemistry . . . . . . . . . . . . . . . . . . . . . 2.2.3 FM1-43fx dye loading of synaptic vesicles . . . . . . . . . . 2.2.4 DiI labeling of live and fixed cells . . . . . . . . . . . . . . . 2.3 Electrophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1 Setup and media . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 Current clamp experiments . . . . . . . . . . . . . . . . . . 2.3.3 Voltage clamp experiments . . . . . . . . . . . . . . . . . . 2.3.4 Extracellular stimulation . . . . . . . . . . . . . . . . . . . .
Results 3.1 Cell survival and outgrowth . . . . . . . . . . . . . . . . . . . . . 3.2 Identity of cells in the culture system . . . . . . . . . . . . . . . . 3.3 Electrophysiological characterisation . . . . . . . . . . . . . . . . 3.3.1 Current clamp experiments . . . . . . . . . . . . . . . . . 3.3.2 Voltage clamp experiments . . . . . . . . . . . . . . . . . 3.4 Synaptogenesis in the culture system . . . . . . . . . . . . . . . . 3.4.1 Morphological evidence . . . . . . . . . . . . . . . . . . . 3.4.2 FM dye loading of synaptic vesicles . . . . . . . . . . . . . 3.4.3 Electrophysiological evidence . . . . . . . . . . . . . . . . 3.5 Assigning identity: evidence from identified NM/NL neurons . . . 3.6 From generalist to specialist – experiments with barn owl embryos
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First application of the culture system in a biohybrid context . . . . . . . 3.7.1 Guiding neurite outgrowth: micro-contact printing of ECM proteins 3.7.2 Auditory brainstem neurons on iridiumoxide surfaces . . . . . . . .
Discussion 4.1 Identity of the neurons in the culture system . . . . . . . . . . . . . . . . 4.2 Reduction of neuron numbers in vitro . . . . . . . . . . . . . . . . . . . . 4.3 Autonomous neuronal differentiation in vitro . . . . . . . . . . . . . . . . 4.4 Incomplete specialised development in the culture . . . . . . . . . . . . . . 4.5 Comparison with other culture systems of the auditory brainstem . . . . . 4.6 Usefulness of the auditory brainstem culture as a biological component in biohybrid systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Summary
Zusammenfassung
Bibliography
Appendix 8.1 Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Curriculum Vitae von Thomas Künzel . . . . . . . . . . . . . . . . . . . .
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Culture phases and cell survival . . . . . . . . . . . . Morphometrical analysis . . . . . . . . . . . . . . . . Neuronal identity of cells in the culture system . . . Expression of calretinin . . . . . . . . . . . . . . . . Expression of Kv channels . . . . . . . . . . . . . . . AP fire types . . . . . . . . . . . . . . . . . . . . . . Mean age and membrane parameters by firetype . . AP parameters by AP firetype . . . . . . . . . . . . AP parameters by AP firetype(cont.). . . . . . . . Development of basic membrane parameters . . . . . Development of AP parameters . . . . . . . . . . . . Development of AP parameters(cont.). . . . . . . . Exemplary voltage-clamp recordings . . . . . . . . . mean voltage-current relationship curves . . . . . . . Development of membrane currents . . . . . . . . . . Cytochemical stainings against synaptic markers . . Visualising vesicle uptake with FM1-43fx . . . . . . . Elicited postsynaptic activity . . . . . . . . . . . . . Recordings of spontaneous activity . . . . . . . . . . Recordings from identified NM neurons in vitro . . . Neurons from the auditory brainstem of the barn-owl Neurons on micro-contact printed surfaces . . . . . . Auditory brainstem neurons on iridiumoxide surfaces
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Excision of NM/NL at E 6.5 . . . . Injection sites and labeled neurons
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NM-NL circuit as biohybrid system The Jeffress model . . . . . . . . .
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1.1 1.2
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1.1
Introduction
Background
1.1.1 Functional neuronal circuits in vitro and biohybrid systems
Many of the established cell culture models of functional neuronal circuits suffer from a fundamental flaw. Several approaches have shown, that recurring activity patterns emerge in long-term neuronal cultures (van Ooyen et al., 1992; Segev et al., 2002; van Pelt et al., 2004a,b, 2005; Berdondini et al., 2006; Wagenaar et al., 2006a,b). These circuits are well balanced between excitation and inhibition, undergo maturation and refinement and even show complex network responses to pharmacological agents. It has also been tried to interpret the correlation of electrical external input into the in vitro networks with reproduceable activity pattern changes as conditional output of the system (DeMarse et al., 2001; Wagenaar et al., 2005; Potter et al., 2005). Thus a biohybrid signal processing system is generated. These biohybrid systems, incorporating a neuronal sub-system into silicon circuitry, allow interesting insights into biological as well as technical questions. However, at this point a conceptual problem arises – how can the function of the artificial system be interpreted in neurobiological terms? Does the emergence of activity and (in an experimental system) signal processing give meaningful information of how functional circuitry is formed in the brain? These questions are difficult to answer, because no "biological function" per se against which hypotheses could be tested can be attributed to a diffuse network of dissociated neurons in culture. To address this conceptual shortcoming a neuronal circuit, of which the biological func-tion in the brain is known, should be reconstructed in culture. The anatomy, physiology and function of the in vivo circuit taken as a model forms the ideal endpoint of the in vitro development. Ideally, the output of the reconstructed circuit should be identical to the output of the in vivo circuit, if the same input is presented to both. Only un-der these conditions can differences between the in vivo and the in vitro output provide meaningful information about the system, thus allowing more precise technical as well as neurobiological conclusions.
What are the properties of a good candidate circuit for this function-guided reconstruc-tion? Since cultivation of neuronal cells implies many methodological difficulties on its own, the circuit should be simple, i.e. not be composed of many different neuron types. Its function should already be well defined. To allow for precise comparisons between the in vivo and in vitro situation, it should have been studied on many different levels of complexity in respect to both anatomy and physiology.
Introduction
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One neural circuit was chosen to be reconstructed as a biohybrid system within the framework of a collaborative effort of several groups1of which this thesis work was part. It is the sound localisation circuit from the auditory brainstem of birds.
Briefly (since I will describe the in vivo nuclei in the following sections), the circuit consists of two neuron types. Two populations of the first neuron type (Nucleus mag-nocellularis neurons, NM) on both sides of the brain project onto the population of the second neuron type (Nucleus laminaris neurons, NL). The nature of the projection and the coincidence detector properties of the NL neurons enables this simple circuit to per-form a complex task: the measurement of very small timing differences between excitation in NM neurons and the representation of these time differences as a map formed by the NL neurons. See figure 1.1 for a depiction of the biohybrid system whose construction in vitro is the ultimate goal of the project. Eventually, the NL neurons are situated on recording electrodes (or field-effect transistors to allow recording of sub-threshold events) and the two populations of NM neurons can be individually or collectively stimulated. In this reconstruction the detailed physiology and developmental dynamics of such a coincidence detection circuit can be studied.
Figure 1.1:Depiction of the avian auditory brainstem coincidence detector circuitry as a biohybrid system in vitro. Red and green: NM neurons. Yellow: NL neuron. Lightblue: micro-contact printed growth lanes. Grey: Stimulation and recording electrodes integrated in the microchip substrate
1virtuelles Institut für Biohybridtechnologie, vIBHT; sponsored by the Helmholtz Gesellschaft
Introduction
1.1.2
Form and function of the auditory brainstem of birds
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The Jeffress modelIn 1948 Loyd A. Jeffress proposed a model of "a place theory of sound localization based on the discrimination of small time intervals" (Jeffress, 1948), which included delay lines and coincidence detectors as key components. The delay lines compensate the interaural time difference in the sensory information that is due to spa-tial difference between the two ears, if the sound source that is perceived is not directly in front (or behind). The second key component of the Jeffress-model are the coinci-dence detectors, at that time hypothetical neurons that are only active when excitation reaches them from contra- and ipsilateral ear at exactly the same time. Thus, "we have a difference of time represented as a difference in place" (Jeffress, 1948) and therefore a continuous map of horizontal auditory space formed by the population of coincidence detector units. In the classical paper, based on the cat auditory system, Jeffress speculated that "we
Figure 1.2:Original drawing of Jeffress’ model of sound localisation utilising interaural time differences.
must look for the center either in the inferior colliculus or in the medial geniculate body" (Jeffress, 1948). Later it became clear that the Jeffress model in its pure form is not found in the mammalian auditory system. But a circuit that very closely resembles the delay line and coincidence detector scheme was found in the avian auditory brainstem (Kon-ishi et al., 1988; Overholt et al., 1992; Joseph & Hyson, 1993; Carr & Boudreau, 1993; Pena et al., 2001). Here the axons of the Nucleus magnocellularis (NM) neurons, which probably are homologous to the bushy cells in the mammalian anteroventral cochlear nucleus, take on the role of the delay lines. The Nucleus laminaris (NL) neurons, which are bilaterally innervated by NM, represent the coincidence detectors and constitute the
Introduction
place map – the first representation of interaural time differences.
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Anatomy of the auditory brainstem and morphological specialisations of NM/NL In the auditory brainstem of birds the morphological and physiological specialisations can be connected to the function of the neurons – the fast and faithful transmission of auditory information. Auditory nerve fibres (the axons of the cochlear ganglion neurons) project onto neurons in the two cochlear nuclei, Nucl. magnocellularis (NM) and Nucl. angularis (NA). The tonotopical order, that results from the frequency analysis performed by the cochlea, is conserved in this projection (Parks & Rubel, 1978). NM neurons project bilaterally onto the Nucl. laminaris and also maintain the tonotopy (Parks & Rubel, 1975). The ipsilateral NM axons innervate the NL from the dorsal side, the contralateral axons cross the midline and innervate the NL from the ventral side. The NM terminals cover a large proportion of the somatic (42%) and proximal dendritic (63%) membrane area (Parks et al., 1983). These two synaptic steps, from CN to NM to NL, form the time domain of the auditory brainstem pathway, i.e. the information processed in this part of the audi-tory pathway is processed according to its timing information content (Adolphs, 1993). Both NA and NL project to the superior olivary nucleus (SON), which is situated in the ventral brainstem. The neurons in this nucleus provide reciprocal GABAergic projections back onto NA and NL and also to NM (Code et al., 1989; Burger et al., 2005a). The NL neurons (and the NA neurons) are the output neurons of the auditory brainstem (Adolphs, 1993) and project into higher order parts of the auditory pathway – namely the nuclei of the lateral lemniscus (nucleus ventralis lemnisci lateralis, pars anterior and nucleus lemnisci lateralis pars ventralis) and the nucl. mesencephalicus lateralis pars dor-salis (or torus semicircularis), the avian homologue of the mammalian inferior colliculus.
The NM is the most caudal of the auditory nuclei (Carr & Boudreau, 1991). In the adult (or post-hatch) chicken it contains two neuron types: most of the NM neurons are large, round neurons that have few or no short dendrites and a large axon (Jhaveri & Morest, 1982b). They receive input from the auditory nerve via a large synaptic ending, the endbulbs of Held (Jhaveri & Morest, 1982a). This calyx-shaped giant synaptic ending covers a large amount of somatic membrane space. The other neuron type is found in the most caudolateral low-frequency region of NM and has longer dendrites. These cells do not receive input via endbulb synapses but via bouton-like synaptic endings (Jhaveri & Morest, 1982b). The GABAergic terminals from the SON neurons can be found on somata and dendrites of the NM neurons, as opposed to the strictly axo-somatic synapses of the auditory nerve fibres (Carr et al., 1989; Code et al., 1989). The NL lies slightly anteromedial and ventral in respect to the NM. In the chicken it contains a single plane of evenly spaced, fairly large bitufted neurons that receive input from contra- and ipsilateral NM via bouton-like synapses (Parks & Rubel, 1975; Jhaveri & Morest, 1982a; Pettigrew et al., 1988). The neurons have several dendrites positioned on opposing poles of the cells, with each dendritic tuft reaching into a cell free area dorsal or ventral of the nucleus (Parks & Rubel, 1975). Along the axis of tonotopy, which runs
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