Information Processing at the Calyx of Held Synapse Under Natural Conditions Joachim Hermann Zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegte Dissertation der Fakultät für Biologie der Ludwig–Maximilians–Universität München von Joachim Hermann aus Münster / Westfalen München, 17.01.2008 Erstgutachter: . Univ.-Prof. Dr. Benedikt Grothe Zweitgutachter: . Univ.-Prof. Dr. Christian Leibold Tag der mündlichen Prüfung:. .11.03.2008 2 “No job too dirty for a fucking scientist” – William S. Burroughs 3 4 Contents List of Figures9 Abbreviations 11 Summary 13 Zusammenfassung 15 1 Introduction 19 1.1 The auditory brainstem.......................... 20 1.2 The calyx of Held.............................. 23 1.3 Short-term plasticity............................ 24 1.4 Objective of this work........................... 26 2 Materials and Methods 29 2.1 In vivo recordings.............................. 29 2.1.1 Surgical procedures......................... 29 2.1.2 Recordings of neural activity.................... 30 2.1.3 Stimulus presentation and recording protocols.......... 32 2.2 In vitro recordings............................. 32 2.2.1 Slice preparation.......................... 33 2.2.2 Whole-cell recordings........................ 33 2.2.3 Stimulation of synaptic inputs................... 34 2.2.4 Conductance clamp experiments.................. 34 2.2.5 Miniature EPSC analysis...................... 35 2.3 Vesicle release model............................ 35 2.3.1 Model variants........................... 36 2.3.2 Model predictions.......................... 37 2.4 Statistical analysis............................. 38 5 6 Contents 3 Synaptic Transmission at the Calyx of Held 39 3.1 Introduction................................. 39 3.2 Results.................................... 40 3.2.1 Spontaneous firing rates...................... 40 3.2.2 Introducing spontaneous rates................... 42 3.2.3 Effects of ‘spontaneous’ firing................... 43 3.2.4 Effects of simulated tone-bursts.................. 45 3.2.5 Recovery of firing.......................... 48 3.2.6 Recovery from depression..................... 50 3.2.7 Reduced synaptic reliability.................... 53 3.3 Discussion.................................. 59 3.3.1 Background firing in MNTB neurons............... 59 3.3.2 Prolonged spontaneous spiking.................. 61 3.3.3 Reliability of synaptic transmission................ 62 4 Modeling Short-Term Synaptic Plasticity 65 4.1 Introduction................................. 65 4.2 Results.................................... 66 4.2.1 Poisson distributed test trains................... 67 4.2.2 Model variants........................... 71 4.2.3 Stimulation with trains consisting of regularly spaced intervals. 74 4.2.4 Natural stimuli........................... 78 4.3 Discussion.................................. 80 5 Transition Between Unconditioned and Conditioned State 83 5.1 Introduction................................. 83 5.2 Results.................................... 84 5.2.1 Beginning of conditioning period................. 84 5.2.2 Vesicle pool recovery........................ 87 5.2.3 Miniature EPSCs.......................... 92 5.3 Discussion.................................. 94 6 General Discussion 97 6.1 The functional role of the MNTB..................... 97 6.2 Additional input to the MNTB...................... 100 Contents 7 6.3 Implications for other nuclei........................ 101 Bibliography 105 Contributions to the Manuscript 117 Curriculum Vitæ 119 Danksagung 121 Ehrenwörtliche Versicherung 123 8 List of Figures 1.1 A schematic drawing of the auditory brainstem.............. 21 1.2 An artist’s rendering of a MNTB neuron.................. 24 2.1 Spike waveforms and raster displays of two single MNTB neurons... 31 3.1 Illustration of in vivo activity in the MNTB............... 41 3.2 Spontaneous firing rates.......................... 42 3.3 EPSC amplitudes in response to different conditioning pulse trains... 44 3.4 Response of neurons to high frequency stimulation............ 47 3.5 PSTHs of a single MNTB neuron’s in vivo response........... 49 3.6 Recovery from depression.......................... 51 3.7 Reliability of synaptic transmission in spontaneously active synapses.. 55 4.1 Poisson distributed test trains....................... 69 4.2 Model variants............................... 72 4.3 Stimulation with trains consisting of regularly spaced intervals..... 75 4.4 Summary graphs showing the differences between experimental data and model prediction............................ 77 4.5 Natural stimuli............................... 79 5.1 Development of EPSC amplitudes during conditioning......... 85 5.2 Development of model parameters over time............... 86 5.3 Vesicle pool recovery............................ 88 5.4 Comparison of recovery between unconditioned and conditioned state. 91 5.5 Development of miniature EPSCs..................... 93 9 10 Abbreviations AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid BF best frequency CV coefficient of variation CN cochlear nucleus ECS extracellular solution EPSC excitatory postsynaptic current EPSG excitatory postsynaptic conductance EPSP excitatory postsynaptic potential IID interaural intensity difference IPSP inhibitory postsynaptic potential ISI inter spike interval ITD interaural time difference LSO lateral superior olive LTD long term depression LTP long term potentiation MNTB medial nucleus of the trapezoid body MSO medial superior olive NMDA N-methyl-D-aspartate PSTH peri stimulus time histogram PTP post tetanic potentiation 11 12 Summary This study investigates the role of the medial nucleus of the trapezoid body (MNTB) in sound processing. The experimental part focuses on in vitro experiments in acute brain slices of Mongolian gerbils, in parallel a theoretical approach explains the experimental results in the context of a mathematical vesicle-release model. One of the hallmarks of auditory neurons in vivo is spontaneous activity that occurs even in the absence of any sensory stimuli. Sound evoked bursts of discharges are thus embedded within this background of random firing. The calyx of Held synapse has been characterized in vitro as a fast relay that reliably fires at high stimulus frequencies (up to 800 Hz). However, inherently due to the preparation method, spontaneous activity is absent in studies using brain slices. This study deals with the question how this ongoing activity is influencing synaptic transmission. The answer is divided into three parts. In the first part a phenomenological description of the effects of spontaneous activity on synaptic transmission is given. Therefore in vivo spontaneous firing rates were determined and then reintroduced as random firing patterns to in vitro brain stem synapses. After conditioning synapses for two minutes at Poisson averaged rates of 20, 40, and 60 Hz, a number of differences in synaptic transmission were observed. Accordingly, current-clamp, dynamic-clamp, and loose-patch recordings revealed a number of failures at the postsynaptic cell, although the initial onset of evoked activity was still transmitted with higher fidelity. The conclusion of these observations is that in vivo auditory synapses are in a tonic state of reduced EPSCs as a consequence of spontaneous spiking. In the second part the conditioned state of calyx of Held synapse is closer inves- tigated by modeling the short-term dynamics with a biophysically motivated vesicle release model. The mechanisms regulating short-term plasticity can be demonstrated in physiological studies as well as computer models aimed at testing the functional role of them. In the case of the calyx of Held synapse, considerable progress has 13 14 Summary been made in understanding the dynamics of transmission both on a physiological and modeling level. Nevertheless, little is known about the processing of complex, long lasting stimulation patterns mimicking the input typically present in the intact brain. Furthermore, calyx of Held synapses are chronically active emphin vivo due to sponta- neous activity in the auditory brainstem. Here we test synaptic responses to complex stimulation protocols mimicking periods of low and high activity, as well as protocols derived from natural sound clips. Additionally, all stimuli were embedded in chronic background activity attempting to imitate the naturally occurring spontaneous activ- ity. We measured synaptic responses to these stimulus trains and then used the data to test how well several vesicle-release models could capture the dynamics observed physiologically. Already the most basic model variant produced very good results with correlation coefficients between the experimental data and the model prediction of more than 90%. None of the more complex model variants, which incorporated additional physiological effects, could improve this prediction accuracy significantly. The conclusion of these results is that the functional state of chronically active ca- lyces differs from the functional state of silent calyces, and that this chronically active functional state can be described in simpler terms. Finally the third part focuses on the transition phase between completely rested synapses and synapses conditioned with simulated spontaneous activity. Modeling the transition phase at the beginning of the conditioning period reveals significant changes in the model parameters thus suggesting changes in the underlying biophys- ical parameters including the vesicle