Descending Octopaminergic Neurons in the Stick Insect: Their Inputs and their Output Effects on the Locomotor System

The neural networks controlling locomotion (walking) must exhibit a high degree of flexibility for task-specific adaptation of behavior to environmental influences and changes in internal state. Neuromodulatory influences are very important for this flexibility, as they can regulate the activity of all neurons in the walking system and the strengths of their synaptic connections. To fully understand the neural control of walking, it is crucial to identify the neurons that release neuromodulators and to determine their activity patterns during behavior and analyze the properties of their output effects. Octopamine, one such neuromodulator, is considered the invertebrate homolog to the vertebrate noradrenaline. It is a significant modulator in insect locomotor systems, both acting in the peripheral and central nervous systems. Octopamine modulates muscle metabolism, neuromuscular transmission, sensory sensitivity, excitability of motor neurons, and activity in the central pattern generating networks that control locomotion. The neural source of octopamine acting in the central nervous system of insect thoracic segments has not yet been identified. Thus, it is unknown to what extent effects of application of octopamine to thoracic ganglia in previous studies reflect the physiological role of octopamine. In the current thesis, I hypothesized that dorsal unpaired median neurons with bilaterally descending axons (desDUM neurons) are a source of octopaminergic modulation of activity in thoracic neural networks for the control of walking in the stick insect Carausius morosus. I revealed the morphology of desDUM neurons in the gnathal ganglion by intracellular staining. Employing the newly developed method of direct MALDI-TOF mass spectrometry, I could show that stick insect desDUM neurons are octopaminergic. Using semi-intact preparations and intracellular recordings of desDUM neurons, I found that they are phasically activated during six-legged walking and single-leg stepping. The phasic excitatory input to desDUM neurons during walking does not arise from coupling to activity of mesothoracic central pattern generating networks, but most likely from activation of mechanosensory organs of all six legs. Passive leg movement and stimulation of mesothoracic campaniform sensilla excited desDUM neurons. Furthermore, stimulation of the mesothoracic femoral chordotonal organ (fCO) had a weak excitatory influence on their activity. Further, I investigated the output effects of desDUM neurons on reflex-evoked, spontaneous, and centrally generated activity of mesothoracic motor neurons with activation of single desDUM neurons. I could show that distinct desDUM neurons mediate differential effects. Some neurons induce a decrease and others an increase, in the magnitude of reflex-induced motor neuron activity. The neurons which mediated an excitatory influence additionally increased the frequency of reversal of an fCO-induced postural reflex. Some desDUM neurons mediated an increase in the frequency of centrally generated rhythmic motor neuron activity. Collectively, the results of the current thesis provide a comprehensive characterization of desDUM neurons and their complex roles in the stick insect locomotor system. The identity of direct neural target structures for the modulatory action of desDUM neurons, as well as the net output effects of the entire population of desDUM neurons during walking remain open questions. In future experiments, genetic access to desDUM neurons could aid in the activation, silencing, or visualization of their activity, which would collectively contribute to comprehensive answers to the open questions

Investigations of effective connectivity in small and large scale neural networks

The correct signal processing of neuronal signals requires coordination of different groups of neurons. To achieve this there has to be a connection between those neurons. This connection and especially the strength of the connection is not known a priori and can only be measured directly in rare cases. In this thesis I present three publications (Rosjat et al., 2014; Tóth et al., 2015; Popovych et al., under review) and the results from two additional studies focussing on the analysis of couplings in experimental measured neuronal activities. The publications can be divided into investigations of intrinsic, as well as extrinsic intra- and intersegmental connections in the stick insect Carausius morosus and into analysis and mathematical modeling of couplings from EEG-measurements of the human brain while subjects were performing different tasks. In both parts I made use of mathematical models to build hypotheses about so far unknown coupling mechanisms. The first study deals with connectivity changes in the thalamo-cortical loop caused by schizophrenia (Rosjat et al., 2014). To build a mathematical model consisting of neural populations representing the thalamus and the auditory cortex we made use of published EEG-data, which were collected while subjects performed a double-click paradigm. The individual populations comprised a large number of phase oscillators with continuously distributed natural frequencies. Applying reduction methods by Pikovsky and Rosenblum, Ott and Antonsen together with the reduction method by Watanabe and Strogatz we investigated the influences of the bidirectional connections between the brain areas on the synchronization of the neuronal populations. The model was able to replicate the experimental data adequately. We observed that the coupling strength from the thalamic region to the cortical region mainly affected the duration of synchrony while the feedback to the thalamic region had a bigger effect on the strength of synchrony. This led to the hypothesis that the back coupling to the thalamic region might be reduced in schizophrenia patients. The second study will show an analysis of intersegmental couplings in the protractorretractor system of the pro- and mesothoracic ganglion of the stick insect Carausius morosus using mathematical models based on experimental data (Tóth et al., 2015). We made use of phase-response curves that were calculated experimentally on the one hand and simulated by mathematical models on the other hand to determine the nature and the strength of their connection. We showed that connections on both sides from the prothoracic to the mesothoracic network were necessary to achieve a good agreement with the experimental phase-response curves. Additionally, it was found that the strength of the excitatory connection played a key role, while the strength of the inhibitory connection did not have a big influence on the shape of the phase-response curves. The third study deals with the identification of a neuronal marker of movement execution (Popovych et al., under review). In this work we investigated the influence of internally and externally triggered movement on the phase synchronization in the motor system. We tested the signals, that were recorded from electrodes lying above the motor cortex, in the phase space including the major frequency bands (delta-, theta-, alpha-, beta- and low gamma-frequencies) for inter-trial phase synchrony. The study revealed a strong lateralized phase synchronization in the lower frequency bands (delta and theta) in the electrodes above the contralateral primary motor cortex independent of the hand performing and the cue triggering the movement. The results suggest that this phase synchronization could serve as an electrophysiological marker of movement execution additionally to the well established event-related desynchronization and event-related synchronization that are based on the amplitude changes in alpha- and beta- frequency bands