Intrinsic Membrane Properties And Firing Response Dynamics Of Rat Medial Vestibular Nucleus Neurons By Infrared Visual Patch Clamp Method

Part 1: Modified Preparing Methods of Brainstem Slices for Visual Patch Clamp TechniquesObjective: To approach a modified method of preparing brainstem slices, and to discuss the factors affecting slices health. Methods: Brainstem slices with medial vestibular nuclei were made on the basis of reported methods, and the quality of slices was judged by naked eye observation, light microscope observation and firing activities recordings. The effects of rats age, physiological fluids and temperature upon slices health were observed. Results: A simple and reliable preparation procedure of brain slices was established. The medial vestibular nucleus neurons were visualized by infrared differential interference contrast technique and readily-patched at a depth of 50-100μm underneath the surface of slices. Brain slices were easy to obtain from juvenile animals, while performing intracardiac perfusion with cold artificial cerebrospinal fluid, using high-sucrose solution and pre-incubating slices at a warmer temperature were useful when prepared slices from adult rats. Within definite temperature rage, the excitability of neurons improved with the increasing of temperature during incubation and recording, while the survival time shortened. Conclusions: Modified preparing methods could supply sufficient healthy medial vestibular nucleus neurons for electrophysiological recordings. The preparation procedures and the controlling of physiological surroundings should be adjusted according to rats age. Part 2: Spontaneous Firing Activities and Basic Membrane Properties of Rat Medial Vestibular Nucleus Neurons in Brain SlicesObjective: To study the basic membrane properties of rat medial vestibular nucleus(MVN) neurons and their firing activities at resting membrane potential, and to discuss how the intrinsic membrane properties contribute to physiological functions in central vestibular system. Methods: By using infrared differential interference contrast technique, whole-cell recordings were made from rat MVN neurons. Firing activities were recorded by current clamp mode in artificial cerebrospinal fluid (ACSF) and low Ca2+-high Mg2+ ACSF. On the basis of their averaged action potential shapes, the MVN neurons were classified. The differences of intrinsic membrane properties and firing mode were observed between two types. Results: Regular discharge activities were recorded in MVN neurons in ACSF. In low Ca2+-high Mg2+ ACSF, Neurons showed more irregular and depressive firing activities. MVN neurons were classified as type A (n=17, 33%) characterized by a single deep after-hyperpolarization (AHP) and A-like rectification, or type B (n=32, 63%) characterized by double AHP, and another two neurons (4%) with all or none of the characters. The passive membrane properties were not significantly different between type A and type B neurons, while part of active membrane properties was significantly different. Conclusions: The discharge activities of MVN neurons were initiated by their intrinsic membrane properties, and calcium mechanism contributed to firing mode. Most MVN neurons were classified as type A and B, while several showed unrepresentative firing properties. The differences of membrane properties between neurons determined their different physiological functions. Part 3: Firing Responses of Rat Medial Vestibular Nucleus Neurons to Simulated Input SignalsObjective: To observe the firing response dynamics of rat medial vestibular nucleu(sMVN) neurons to simulated input signals, and to discuss how the firing dynamics contribute to physiological functions in central vestibular system. Methods: By using infrared differential interference contrast technique, whole-cell recordings were made from rat MVN neurons under direct observation. Firing activities were recorded in current clamp mode. Hyperpolarizing currents were injected into MVN neurons to simulated inhibitory postsynaptic currents (IPSC). Depolarizing currents and sinusoidal currents were injected into neurons to simulate the input signals from peripheric vestibular organs when head in linear accelerated motion and uniform rotation motion. Firing dynamics of MVN neurons to stimulus currents were recorded. Results: After hyperpolarizing currents injected into neurons, the after-hyperpolarization amplitude of action potentials increased and post-inhibitory rebound was recorded. Neurons firing rate increased with the depolarized current. Compared with type A neurons, type B neurons showed the weaker adaptation and the stronger overshot phenomenon to same depolarizing currents. Firing rate of neurons resonantly oscillated with sinusoidal currents, and phase lead was observed. Conclusions: When head made linear accelerated motion and uniform rotation motion, the MVN neurons responded actively to the input signals from peripheric vestibular organs. Differences of firing dynamics between type A and B neurons determined their different physiological functions.