Abstract Not Provided. / Thesis / Master of Science (MSc)

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13608号 / 論医博第2318号 / 新制||医||1073(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 村井 俊哉, 教授 高橋 淳 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The work in this thesis concerns mechanisms of excitability of neurons. Specifically, it deals with how neurons respond to input, and how their response is controlled by ion channels and other active components of the neuron. I have studied excitability in two systems of the nervous system, the hippocampus which is responsible for memory and spatial navigation, and the peripheral C–fibre which is responsible for sensing and conducting sensory information to the spinal cord. Within the work, I have studied the role of excitability mechanisms in normal function and in pathological conditions. For hippocampus the normal function includes changes in excitability linked to learning and memory. However, it also is intimately linked to pathological increases in excitability observed in epilepsy. In C–fibres, excitability controls sensitivity to responses to stimuli. When this response becomes enhanced, this can lead to pain. I have used computational modelling as a tool for studying hyperexcitability in neurons in the central nervous system in order to address mechanisms of epileptogenesis. Epilepsy is a brain disorder in which a subject has repeated seizures (convulsions) over time. Seizures are characterized by increased and highly synchronized neural activity. Therefore, mechanisms that regulate synchronized neural activity are crucial for the understanding of epileptogenesis. Such mechanisms must differentiate between synchronized and semi synchronized synaptic input. The candidate I propose for such a mechanism is the fast outward current generated by the A-type potassium channel (KA). Additionally, I have studied the propagation of action potentials in peripheral axons, denoted C–fibres. These C–fibres mediate information about harmful peripheral stimuli from limbs and organs to the central nervous system and are thereby linked to pathological pain. If a C–fibre is activated repeatedly, the excitability is altered and the mechanisms for this alteration are unknown. By computational modelling, I have proposed mechanisms which can explain this alteration in excitability. In summary, in my work I have studied roles of particular ion channels in excitability related to functions in the nervous system. Using computational modelling, I have been able to relate specific properties of ion channels to functions of the nervous system such as sensing and learning, and in particular studied the implications of mechanisms of excitability changes in diseases. / <p>QC 20102423</p>

Hypertension is associated with marked cardiac sympathetic over-activity and end organ hyper-responsiveness. The sympathetic dysfunction is caused by aberrant calcium (Ca²⁺) handling resulting in enhanced neurotransmission. However, it remains unclear whether the sympathetic neuron or the myocytes is the primary driver behind the initiation and maintenance of the autonomic phenotype. The work in this thesis characterises the Ca²⁺ dysfunction and regulation at the membrane level. Further, it employs physiologically coupled sympathetic neurons and ventricular myocytes to determine the cellular driver of cardiac dysautonomia in the pro-hypertensive state. <b>Chapter 1</b> provides a general overview of the field of autonomic hypertension with a specific focus on the sympathetic control of cardiac excitability. In particular, the role of Ca²⁺ and cyclic nucleotides in the facilitation of neurotransmission are explored. <b>Chapter 2</b> details the methods used in this thesis. It provides rationale for the approaches taken to record membrane Ca2+ currents, cyclic adenosine monophosphate (cAMP) levels and cAMP-activated protein kinase (PKA) activity, and the development and uses of a co-culture of coupled sympathetic neurons and ventricular myocytes. <b>Chapter 3</b> describes the successful development of an effective voltage clamp method to isolate whole cell Ca²⁺ currents in sympathetic neurons. It details the issue of space clamp problem when using this technique on peripheral neurons and provides experimental guidance on how to quantify and limit theses issues. <b>Chapter 4</b> identifies that the pro-hypertensive four-week old neurons from the spontaneously hypertensive rat (SHR) have significantly larger whole cell Ca²⁺ currents when compared to normotensive (Wistar Kyoto-WKY) neurons, that are largely N-type in nature. Restoring the cGMP cyclic nucleotide dysfunction seen in these cells, rescues the ion channel phenotype and bring the Ca²⁺ down to levels seen in the normotensive WKY neuron. Further, it identifies that phosphodiesterase (PDE) 2A inhibition differentially affects the currents in the WKY and SHR, further supporting the notion of PDE2A dominance. <b>Chapter 5</b> identifies the presence and functional relevance of cGMP cross-talk with the cAMP-PKA pathway in sympathetic neurons. This cross talk is significantly altered in the pro-hypertensive state, via the differential involvement of PDEs. It functionally identifies the presence of PDE3 and PDE2A and provides further evidence that these enzymes could be dysregulated in pro-hypertensive neurons. <b>Chapter 6</b> describes the use of a co-culture model of ventricular myocytes and sympathetic neurons. Physiological stimulation of the sympathetic neuron with nicotine whilst monitoring cAMP levels in the myocytes confirms that the cellular phenotypes seen in the individual cells are functionally present in the co-culture. Using cross-cultures, it identifies the neuron as the principal driver behind the cardiac sympathetic responses observed in pro-hypertension. The results provide evidence for a dominant role played by the neuron in driving the adrenergic phenotype seen in cardiovascular disease and highlights the potential of using healthy neurons to turn down the gain of neurotransmission, akin to a smart pre-synaptic &beta;-blocker. <b>Chapter 7</b> forms the concluding discussion that summarises the main findings of this thesis and attempt to place it in a clinical context, and highlights avenues of further research. In particular, the possibility of using a cell therapeutic approach to treat sympathetic hyperactivity.

No description available.

Although the processes used for temporarily storing and manipulating neural information have been extensively studied at the synaptic level far less attention has been given to the underlying cellular and molecular mechanisms that contribute to change in the intrinsic excitability of neurons. More importantly, how do these mechanisms of plasticity integrate with ongoing mechanisms of regulation of neural intrinsic excitability and, in turn, homeostasis of entire neural circuits? In this dissertation I describe the underlying mechanisms that contribute to persistent neural activity and, more globally, sensorimotor adaptation using weakly electric fish as my model system. Weakly electric fish have evolved a behavior adaptation known as the jamming avoidance response (JAR), and it is this adaptation that allows the organism to elevate its own electrical discharge in response to intraspecific interactions and subsequent distortions of the animal’s electric field. The elevation operates over a wide range and in vivo can last tens of hours upon cessation of a jamming stimulus. I demonstrate that the underlying mechanisms of the adaptation are mediated by calcium-dependent signaling in the pacemaker nucleus and that calcium-mediated phosphorylation plays an important role in the regulation of the long-term frequency elevation (LTFE). I demonstrate using an in vitro brain slice preparation from the weakly electric fish, Apteronotus leptorhynchus that the engram of memory formation depends on the cooperativity of calcium-dependent protein kinases and protein phosphatases. In addition, I show that the memory formation (in the form of LTFE) does not depend on the continued flux of calcium, but rather the phosphorylation events downstream of NMDA receptor activation. Moreover, I describe the differences in the expression of protein phosphatases and protein kinases as they relate to species-specific differences in sensorimotor adaptation. It is important to note that this is the first time that the cooperativity between different isoforms of protein kinase C (PKC) have been shown to play a role in graded long-term change in neuronal activity and, in turn, providing the neural basis of species-specific behavior. The neural adaptation of the electromotor system in weakly electric fish provides an excellent model system to study the underlying cellular and molecular events of vertebrate memory formation. / text

The mammalian central nervous system maintains a delicate balance between neuronal excitation and inhibition. Conventional synaptic inhibition is mediated through the transient activity of postsynaptic γ-aminobutyric acid (GABA) at type A GABA (GABAA) receptors. A subset of GABAA receptors is also located outside of inhibitory synapses. These extrasynaptic receptors generate a tonic inhibitory conductance in response to low concentrations of extracellular GABA. Tonic inhibition broadly suppresses neuronal activity and regulates many vital processes such as sleep, consciousness and memory formation. This thesis examines the physiological effects of tonic inhibition at the cellular level and in the behaving animal. This thesis also explores whether gabapentin, a commonly used sedative, anxiolytic, and analgesic drug, enhances tonic GABAergic inhibition. I hypothesize that: (1) tonic GABAA receptor activity reduces the intrinsic excitability of neurons; (2) the activity of tonically active GABAA receptors in spinal pain pathways attenuates nociception; and (3) tonic inhibition can be upregulated by gabapentin. The results show that a tonic inhibitory current generated by α5 subunit-containing GABAA (α5GABAA) receptors reduces the excitability of hippocampal pyramidal neurons excitability by increasing the rheobase, but does not change the gain of action potential firing. A similar shunting inhibition is present in spinal cord lamina II neurons that is generated by δ subunit-containing GABAA receptors. The activity of these receptors in spinal nociceptive pathways reduces acute thermal nociception and may constrain central sensitization in a behavioural model of persistent pain. Finally, gabapentin increases a tonic inhibitory current in cultured hippocampal neurons independent from changes in the expression of α5GABAA receptors or in the concentration of GABAA receptor ligands. The results of this thesis demonstrate that tonically active GABAA receptors play an important role in the regulation of neuronal activity and nociception, and that tonic inhibition represents a novel target of therapeutic drugs.

The abundance of microtubules in receptive dendrites suggests they may function in sensory transduction. Responses of frog muscle spindle receptors and joint receptors is inhibited within 25 minutes by 50 mM colchicine, a microtubuledisrupting agent. The inhibition is reversible upon removal of colchicine, and the time course of recovery is comparable to that of inhibition. Frog olfactory responses are briefly inhibited by washing the olfactory mucosa with perfusion fluid. Colchicine accentuates the inhibition and substantially retards the rate of recovery in a dose-dependent fashion. Colchicine does not affect axonal conduction, nor the oxygen uptake of isolated crab or frog leg nerves. The inhibitory action of colchicine is therefore an effect on the electrical excitability of the receptive dendrites or soma, and not an effect on axonal conduction.

Title: Motoneuron excitability depending on the level of muscle stretch Aim: The aim of this thesis is to evaluate if passive muscle lenght change may influence parameters of H - reflex of soleus muscle Method: Tibial nerve stimulation in the fossa poplitea area was performed on 6 persons while passive stretching or lengthening of the muscle. Action potentials were captured on soleus muscle. Stimulation was performed in three different time periods - 4s, 2s, 1s. Each period was performed twice. First in passive muscle stretching then the passive shortening. The Hmax and Mmax values during passive muscle stretching and shortening were evaluated and compared. Results: The measurment results showed that there was a significant decrease in the Hmax values during passive muscle stretching. Mmax values were evaluated as passive muscle length change independent. Keywords: EMG, recruitment curve, H-reflex, M-wave, m.soleus, excitability

Title of diploma thesis: Tactile discrimination and excitability of alpha motoneurons Objectives: The aim of this thesis is to detect whether tactile discrimination tasks affect the excitability of the alpha motoneurons. Methods: Seven volunteers aged between 20 and 26 years participated in this study. The H reflex, (M wave) were recorded during three control and three experimental conditions. The control conditions preceded each experimental condition. By stimulating the tibialis nerve in the popliteal fossa the H reflex was elicited and its amplitude and latency measured at rest (control) and during tactile discrimination tasks (experimental). As tactile discrimination tasks, three separate tasks were chosen-tactile stimulation, escape reaction to tactile stimulation, and two-point discrimination. We used an EMG stimulator with a constant voltage output and monophasic squared pulses, with a 0,5 ms interval. The stimulation was switched on manually every 3-5 seconds. To detect the electrical potential of the soleus muscle, we used a surface EMG device, a GrassTelefactor, with galvanic isolation complying with EU standards. The parameters measured were the latency and amplitude of the H reflex and M wave during the tactile discrimination tasks and these were then compared to the values at rest. The...