William J. Spain - US grants

The long term objectives of the research proposal are to answer the question, what are the mechanisms underlying the spread of focal seizures through normal neocortex? Specifically, what role do neurotransmitters play, what is the influence of extracellular potassium ions (K+) and are there changes in neuron membrane properties that facilitate the process? Epilepsy affects approximately 5% of the population. The mechanisms of how focal seizures spread are partially understood. A more complete unraveling of the mechanisms will lead to better methods of therapeutic intervention in the treatment of epileptic patients. Work towards answering these questions will utilize in vitro slices of mammalian pericruciate cortex. Large layer V pyramidal cells (Betz cells) will be impaled for intracellular microelectrode recording and single electrode voltage clamp (SEVC). Some studies will also use specific channel blocker, ionic substitutions and iontophoresis of neurotransmitters. Initially, the effects on firing properties of excitatory amino acids, and neuropeptides will be studied and the underlying ionic currents evaluated with SEVC. The effects of increased (K+)o on firing properties and subthreshold ionic currents and its effects on transmitter release and Betz cell response to synaptic input will likewise be evaluated. Finally, a model for focal seizures will be developed in the neocortical slices using electrical stimulation. Betz cells will be studied before, during and after seizures, specifically looking at changes in synaptic transmission, alterations of membrane properties, an the influence of (K+) on the propagation of the seizure activity through normal Betz cells.

This project investigates the membrane properties of neurons from the avian cochlear nucleus, the nucleus magnocellularis (NM). The NM contains the cells which are homologous to the bushy cells of the mammalian anteroventral cochlear nucleus. NM neurons project to nucleus laminaris neurons which underlie the spatial localization of sound by generating action potentials in response to appropriately timed inputs from the ipsilateral and contralateral NM. In order for accurate localization of sound, NM neurons must relay precise timing information about the auditory stimulus. Stimulation of the auditory nerve results in rapid volleys of EPSPs in NM neurons. Since NM neurons fire action potentials in response to AC inputs but not to DC inputs, temporal summation of the EPSPs would not produce firing of action potentials in NM neurons. We have shown that there is a K+ current in NM neurons which causes the large synaptic potentials to decay rapidly with minimal temporal summation. The NM is tonotopically organized: neurons in the posterolateral NM receive auditory nerve inputs activated by the lowest sound frequencies while those in the anteromedial NM receive inputs activated by the highest sound frequencies. An intriguing question is whether there are differences in the membrane properties that permit anteromedial NM neurons to respond best to high frequency stimuli and posterolateral NM neurons to respond best to low frequency stimuli. We have evidence that the membrane properties do indeed vary because action potential threshold is more depolarized in NM neurons from the anterior region of the nucleus than neurons from the posterior region. The aim of this proposal is to determine how transduction properties of NM neurons vary along the tonotopic axis. The hypotheses to be tested are: I. Hypothesis: NM neurons contain several different types of K+ channels and their expression varies along the tonotopic axis. 2. Hypothesis: The density and/or characteristics of Na+ channels vary along the tonotopic axis of NM. 3. Hypothesis: The tonotopic variation of NM neuron membrane properties results in response properties that differ along the tonotopic axis. The electrotonically compact NM neurons are ideal central neurons for voltage-clamp studies. The K+ and Na+ conductances will be isolated in vitro and their contribution to post-synaptic transduction will be mapped with respect to the position of the cells on the tonotopic axis. This study will provide basic information about how the auditory system works. It will also provide a foundation for future investigations about how ion channel expression is regulated over space.

Our research is aimed at elucidating how ion channels regulate the processing of information by neurons in the cerebral cortex, i.e., the diverse mechanisms neurons use to convert synaptic input into action potentials. The proposed experiments will determine basic principles of how voltage-gated potassium (Kv) channels regulate postsynaptic processing of inputs in layer 5 (L5) neocortical pyramidal neurons (PNs). PNs are the output cells of cortex and key players in learning, memory, and sensorimotor processing, as well as the targets of central nervous system diseases (e.g., epilepsy). The proposed studies go beyond the standard notion that potassium channels act as an intrinsic brake on excitability. They are designed to determine the influence Kv2 and Kv7 channels have on the types of information that L5 PNs respond to and how that information is filtered before downstream transmission. We will study mechanisms controlling firing behavior in two classes of pyramidal neurons: intratelencephalic-projecting (IT) and pyramidal tract (PT) type, represented by two genetically-identified PNs with GFP expressed in populations of L5 PNs under control of unique genes: etv1 (IT) and thy1 (PT). We will test hypotheses concerning how Kv2 and Kv7 channels regulate burst firing (Aim 1) and continuous firing (repetitive bursting and suprathreshold resonance: Aim 2). Kv channel properties and expression are dynamic. They can undergo plastic changes in response to activity or signaling pathways and thus change neuronal filtering properties. Thus, we will also study use-dependent plasticity of intrinsic excitability (Aim 3). We use transgenic mouse lines and state-of-the-art electrophysiological approaches, including somatic / dendritic paired recordings, dynamic clamp, internal pipet perfusion, nucleated patch and on-cell patch recordings, as well as whole cell and gramicidin perforated patch. We also use two-photon and charge-coupled device (CCD)-based Ca2+ imaging systems. Our stimulus protocols are designed to mimic natural synaptic activity arriving at the soma of a neuron (the common summing point for all dendrites) and will be systematically varied to simulate different levels or composition of inputs. Our findings will have major implications for cortical processing, ion channel function, understanding neural computations, and mechanisms underlying epilepsy, anesthesia, learning and memory.