Robert C. Foehring - US grants

The objective of this study is to understand the mechanisms by which extrathalamic transmitters (norepinephrine: NE;acetylcholine: ACH; and serotonin: 5HT) modulate the firing behavior of cat neocortical neurons during ontogeny. The focus of the proposed study is at the level of the neuron as an integrative unit rather than on the kinetics of individuals ion channels. The specific neurons studied are those which give rise to the primary motor output of cerebral cortex (layer V pyramidal neurons). The proposed experiments will use isolated neocortical brain slices and acutely dissociated neurons. Neuronal firing behavior and electrical properties will be studied using standard intracellular recording techniques. Singles electrode voltage clamp (SEVC), ionic substitution and pharmacological manipulations will provide qualitative information concerning the ionic basis for the observed cell behavior. The use of pharmacological blockers and current-clamp recording will then be used to test hypothesis derived from SEVC experiments. Whole cell patch-clamp of acutely dissociated cells will characterized Ca2+ and Ca-dependent K+ currents, their modulation by NE and 5HT, and the second messengers involved. Prerequisite to the study of modulation of firing behavior during development, one must know: (i) the effects extrathalamic transmitters exert on adult neurons, and (ii) the substrate for modulation: the development of firing behavior and its ionic basis.Completed studies have illustrated the effects of NE and muscarine on neocortical cells from adult animals. Those experiments also revealed that norepinephrine and muscarine decrease the same two K+ currents in neocortical cells by two apparently different second messenger systems. The proposed experiments will examine: (i) the effects of 5HT upon firing behavior in adult cats, (ii) the ontogeny of repetitive firing behavior and its modulation by extrathalamic transmitters, and (iii) the second messengers involved in modulation of Ca2+ and Ca-dependent K+ currents. These studies will answer fundamental questions concerning synaptic communication. In particular, the proposed experiments will test the hypothesis concerning the actions of neuromodulators during critical periods for cortical plasticity. The results of these experiments will facilitate formulation of hypotheses about epileptic mechanisms in neocortex and will contribute to the understanding of diseases such as Alzheimer's disease.

During the early postnatal period, the mammalian cortex undergoes rapid changes in cellular properties, laminar structure, and synaptic connectivity. Proper maturation of these properties is essential to normal brain function, and several diseases of the brain may reflect abnormal patterns of development (e.g. schizophrenia, epilepsy, depression). Many of these changes are sensitive to patterns of synaptic input and the degree of cellular activity. Increases in intracellular Ca2+ levels are thought to be an important mechanism by which firing behavior and the pattern of synaptic inputs are translated into changes in synaptic strength or connectivity. In adult cortical pyramidal cells, several ionic conductances interact to shape the activity of the cell. This interplay is incompletely understood in adult cells and even less examined in immature neurons. Neuromodulators such as norepinephrine (NE) and serotonin (5HT) activate G-proteins and second messenger systems to alter ionic conductances and firing behavior in adult neurons. These transmitter systems mature over the same time period as the intrinsic cellular properties of pyramidal cells, and may influence cortical plasticity. We have designed experiments to investigate the properties of Ca2+ and Ca-dependent K+ currents, and the effects of NE and 5HT at various postnatal ages in rat sensorimotor cortical pyramidal neurons. The central hypothesis driving this work is that NE and 5HT have multiple convergent and divergent effects on voltage-gated Ca2+ currents in immature and adult neocortical pyramidal cells. Furthermore, these effects may be age-dependent due to developmental changes in the expression of Ca2+ and K+ channels and receptors for NE and 5HT, as well as in the maturation of Ca2+ regulatory mechanisms. The Specific Aims of this proposal are (1) To investigate the ontogeny of different Ca2+ currents in neocortical neurons. (2) To determine the signalling pathways involved in the modulation of Ca2+ currents by NE and 5HT. (3) To determine the functional roles of different Ca2+ currents in eliciting Ca-dependent K+ currents and after hyperpolarizations. We will employ intracellular recordings in a brain slice preparation, whole cell patch clamp recordings from acutely dissociated neurons, pharmacological agents, and single cell mRNA amplification techniques. Data derived from these experiments are expected to help in the understanding of (1) the development of ion channels, and (2) the actions of NE and 5HT in modulating Ca2+ currents and cellular integration in cortical pyramidal cells. These mechanisms are likely to be important in the development of normal cortical function, as well as in disease processes.

DESCRIPTION: (Applicant's Abstract) Neocortical pyramidal neurons are a model system for study of the mechanisms and significance of transduction of synaptic inputs into spike trains. Several aspects of this transduction process are Ca2+-dependent, including regulation of the interspike interval (ISI), spike frequency adaptation (SFA), spike timing and afterhyperpolarizations (AHPs). Pyramidal cells express at least five different high voltage-activated Ca2+ channels. We hypothesize that Ca2+ entry has different consequences for these cells, depending upon which calcium channel subtypes are involved. An example of such partitioning of function is generation of AHPs: N-, P-, and Q-type channels couple to the sAHP and P-type to the mAHP. Two critical negative feedback systems controlling Ca2+ entry are Ca2+-dependent inactivation of Ca2+ channels and activation of Ca2+-dependent K+ channels. Both processes are potential regulators of pyramidal cell firing behavior. We will use whole cell electrical recordings and fura-2 Ca2+ imaging techniques on mature pyramidal cells in both acutely dissociated and brain slice preparations to test hypotheses about the roles of Ca2+-dependent inactivation and the relationships between action potentials, [Ca2+]i, and AHP currents (due to Ca2+-dependent K+ channels). Aim 1 addresses the importance of Ca2+-dependent inactivation of Ca2+ channel subtypes and which channel subtypes are involved. Aim 2 characterizes the relationships between firing frequency, IAHP, and [Ca2+]i in mature pyramidal cells. These data are important for understanding how pyramidal cells integrate synaptic inputs, and how this process is influenced by transmitters and ontogeny. (In neocortical pyramidal neurons, both Ca2+ and Ca2+-dep K+ channels are developmentally regulated and are targets for several neuromodulators). Neuronal activity and its modulation regulate cortical function and the use-dependent plasticity of cortical connections. These studies will contribute to understanding essential cortical functions such as attention, learning, and memory as well as basic mechanisms of diseases such as epilepsy, anxiety and depression.

DESCRIPTION: (Applicant's Abstract) Neocortical pyramidal neurons are a model system for study of the mechanisms and significance of transduction of synaptic inputs into spike trains. Several aspects of this transduction process are Ca2+-dependent, including regulation of the interspike interval (ISI), spike frequency adaptation (SFA), spike timing and afterhyperpolarizations (AHPs). Pyramidal cells express at least five different high voltage-activated Ca2+ channels. We hypothesize that Ca2+ entry has different consequences for these cells, depending upon which calcium channel subtypes are involved. An example of such partitioning of function is generation of AHPs: N-, P-, and Q-type channels couple to the sAHP and P-type to the mAHP. Two critical negative feedback systems controlling Ca2+ entry are Ca2+-dependent inactivation of Ca2+ channels and activation of Ca2+-dependent K+ channels. Both processes are potential regulators of pyramidal cell firing behavior. We will use whole cell electrical recordings and fura-2 Ca2+ imaging techniques on mature pyramidal cells in both acutely dissociated and brain slice preparations to test hypotheses about the roles of Ca2+-dependent inactivation and the relationships between action potentials, [Ca2+]i, and AHP currents (due to Ca2+-dependent K+ channels). Aim 1 addresses the importance of Ca2+-dependent inactivation of Ca2+ channel subtypes and which channel subtypes are involved. Aim 2 characterizes the relationships between firing frequency, IAHP, and [Ca2+]i in mature pyramidal cells. These data are important for understanding how pyramidal cells integrate synaptic inputs, and how this process is influenced by transmitters and ontogeny. (In neocortical pyramidal neurons, both Ca2+ and Ca2+-dep K+ channels are developmentally regulated and are targets for several neuromodulators). Neuronal activity and its modulation regulate cortical function and the use-dependent plasticity of cortical connections. These studies will contribute to understanding essential cortical functions such as attention, learning, and memory as well as basic mechanisms of diseases such as epilepsy, anxiety and depression.

DESCRIPTION (provided by applicant): Voltage-gated potassium currents play a crucial role in controlling neuronal excitability and sculpting patterns of neuronal activity. Consistent with these roles, K+ channels are exceptionally diverse. This diversity comes in part, from multiple genes, post-translational mechanisms, and heteromeric co-assembly of subunits. Recent molecular work has documented the diversity of subunits and has revealed some of the rules governing the association of subunit types. Studies in expression systems have demonstrated the biophysical and pharmacological properties of defined channel types. Relatively little is known about the composition of K+ channels in native membranes. The division of labor between the various K+ channel types in individual cells is also incompletely understood. Firing of regular-spiking (RS) pyramidal neurons in neocortex is characterized by relatively broad spikes, modest fAHPs, complex subthreshold integration, and rhythmic, repetitive firing with spike-frequency adaptation (SFA). In vivo studies indicate that the characteristic firing pattern of RS cells is integral to their functions in local circuit processing. Previous work by others and ourselves indicate that neocortical pyramidal cells express several K+ currents which regulate excitability. In particular, there is a diversity of slowly inactivating currents. This proposal is to (1) characterize the slowly-inactivating voltage-gated K+ currents and channel subunits in layer II/III pyramidal neurons from rat somatosensory cortex, (2) determine the relationship between particular channel subunits and macroscopic K+ currents, and (3) determine the mechanisms by which voltage-gated K+ currents regulate the RS firing pattern. These data are essential for understanding how pyramidal cells integrate synaptic inputs into spike trains, a process underlying cortical output. This work will also provide insights into abnormal cortical excitability and disease processes, such as epilepsy, as well as provide knowledge of the substrate for modulation by transmitters.

[unreadable] DESCRIPTION (provided by applicant): Fragile X syndrome (FXS) is the most common form of inherited human mental retardation. Neuroanatomical studies of the brains of fragile X patients and of a mouse model of the disease (Fmr1 knock-out) showed a significantly altered morphology of neurons in the neocortex, cerebellum, as well as other parts of the brain. The key anatomical finding is that dendritic spines are abnormally thin and long, as well as increased in number. In healthy brains dendritic spines contain the postsynaptic terminals of excitatory synapses, suggesting that excitatory transmission may be altered in affected parts of FXS brains. In vitro studies have shown abnormal synaptic plasticity (increased LTD in hippocampus and decreased LTD in the neocortex). The cognitive, sensory, and behavioral deficits in human fragile X strongly implicate neocortical dysfunction. [unreadable] [unreadable] Based on the FXS-associated changes in spine morphology of cortical neurons, hypersensitivity to sensory input, and increased probability of seizures, the investigators hypothesize that cortical function in FXS patients is impaired due to increased excitability of the neocortical network. It is unclear whether the primary cause of these symptoms is increased excitability of pyramidal neurons, a reduced effectiveness of inhibitory interneurons, or a combination of these. Here the investigators propose to combine in vivo and in vitro electrophysiological experiments to determine how FXS changes the function of the cortical network in awake, behaving animals and how these network changes relate to alterations in synaptic transmission or excitability in different types of cortical neurons. This project will focus particularly on the effects of FXS on inhibition. The investigators will compare normal and Fmr1 knock-out mice using the whisker-barrel cortex as a model for neocortical function. The rodent whisker barrel cortex has two major advantages: 1) its normal function has been thoroughly investigated and documented, and 2) neurons in the barrel cortex express the typical anatomical abnormalities of fragile X brains. [unreadable] [unreadable] The proposed project has two aims: Specific Aim 1 will determine the effects of fragile X syndrome on (1) the function of the awake neocortical network, and (2) intracortical inhibition using the Frm1 knock-out mouse barrel cortex as a model. The investigators will use multiple electrode extracellular recording techniques to compare spontaneous and task-related neuronal activity in the barrel cortex of awake behaving wild-type and Fmr1 null mice. They will also determine the role of inhibition in shaping size and response properties of whisker barrel receptive fields. Specific Aim 2 will determine the effects of fragile X syndrome on excitability and synaptic transmission in fast spiking interneurons. The investigators in addition will address the potential cellular underpinning for network effects in Aim 1. They will also test whether defects in Frm1 null mice are restricted to neurons with spines or also include sparsely or aspiny interneurons. Immunocytochemistry will be used to test for FMRP expression in GABAergic interneurons and whole cell recordings for changes in intrinsic excitability, excitatory drive to interneurons, and the balance of excitation/inhibition on to layer V pyramidal neurons. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Voltage-gated potassium channels control neuronal excitability and sculpt patterns of neuronal activity. Molecular studies have documented the diversity of subunits and revealed some of the rules governing the association of subunit types. Studies in expression systems have demonstrated the biophysical and pharmacological properties of defined channel types. In contrast, relatively little is known about the composition or functional division of labor of potassium channels in native membranes. We concentrate on the influence of somatic and dendritic Kv channels on computations made by neocortical pyramidal cells to convert synaptic inputs into spike trains. The average rate and timing of action potentials are integral to the functions of neocortical pyramidal cells. In particular, temporal selectivity of pyramidal cells facilitates rhythmic and synchronous activity in cortical circuits, which in turn is important in attentional and perceptual processes in vivo and underlies spread of seizures in epileptic cortex. Most synaptic inputs to pyramidal cells are to dendrites, thus dendritic ion channels are interposed between inputs and the site of spike initiation. Nonlinearities due to activation of a dendritic Ca2+dependent spike initiation zone can lead to intrinsic burst firing in pyramidal cells, which makes synaptic transmission more reliable and facilitates oscillatory behavior. Our previous work indicates that neocortical pyramidal cells express several slowly-inactivating potassium currents. We will concentrate on Kv1 and Kv2 subunits and characterize single channel properties, test functional hypotheses concerning dendritic vs. somatic distribution of channel subunits, and test for a role of Kv2 channels in filtering responses to noisy inputs (to mimic background synaptic inputs). These data are essential for understanding how pyramidal cells process synaptic inputs in health and disease. Abnormalities of Kv1 channels have been implicated in epilepsy and ataxia. Kv2 channels are targets of anesthetic agents, regulators of excitability in many neuronal and nonneuronal cell types, and mediators of apoptosis in cortical neurons. In addition, the distribution and properties of Kv2.1 channels are altered by seizures and ischemia. PUBLIC HEALTH RELEVANCE: These basic studies test how specific potassium channel subunits influence dendritic and somatic computations made by neocortical pyramidal cells to convert inputs into spike trains. These data are essential for understanding how pyramidal cells process synaptic inputs in health and disease.

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.