David A. McCormick - US grants

The proposed experiments will examine the ionic and pharmacological mechanisms of action of acetylcholine (ACh) and norepinephrine (NE) within the cerebral cortex and thalamus. These two putative neurotransmitters have been implicated in the modulation of a number of important normal (e.g. learning and memory, sleep, attention, development) and abnormal (e.g. Alzheimer's disease, epilepsy, aging related cognitive deficits) neural functions. Information as to the ionic and pharmacological characteristics of the actions of ACh and NE in the forebrain is expected to further our understanding of their involvement in these normal and abnormal brain processes as well as making progress towards a possible pharmacological therapy of such. Investigations will be carried out using the in vitro slice technique in which high quality intracellular recordings and control over the neuronal chemical environment is possible. The ionic mechanisms of actions of ACh and NE will be examined in cerebral cortical and thalamic neurons using conventional intracellular current clamp and voltage clamp techniques. The intracellular second messengers which underlie these actions within the thalamus will also be examined. The actions of ACh and NE will be examined in the cerebral cortex because of the importance of the cortex in cognitive function, while the actions of these two agents will be examined in the thalamus because of the intimate relations of the thalamus with the cerebral cortex as well as it's central role in rhythmic generation of neural activity in the forebrain. Intracellular labeling with dye injections will be used to identify subtypes of neurons recorded so that a correlation between neuronal morphological subtype and electrophysiological as well as pharmacological properties can be made. Thalamic recordings will focus on three regions: 1) the lateral geniculate nucleus will be used as a model of thalamic relay nuclei; 2) the nucleus reticularis; and 3) the intralaminar nuclei. It is expected that an increased understanding of the actions of ACh and NE in the forebrain will substantially further our understanding of not only the function of these neurotransmitter systems in the CNS, but also how ascending systems from the brainstem and basal forebrain can influence the processing of information in the cerebral cortex and thalamus.

The cerebral cortex is a highly interconnected sheet of neurons that are constantly active and interacting. This ongoing interaction allows context-dependent complex behaviors to be generated by providing a mechanism for the rapid and dynamic modulation of neuronal responsiveness and flexible coupling of cortical neurons and networks. The generation and impact of cortical-cortical interactions depends upon a precise balance of recurrent excitation and inhibition. In-addition, information processing in the cortex depends critically upon the rate and timing of action potentials. The proposed projects will reveal the functional balance of recurrent excitation and inhibition in vivo and the cellular mechanisms by which the rate and timing of action potential generation is controlled, including during natural sleep and waking. The cortex is built to generate periods of persistent activity, such as during the operation of working memory, or in relation to selective attention. The mechanisms through which persistent, but at the same time rapid, changes in neuronal activity and responsiveness are generated in cortical networks will be investigated. The h-current has proven to be a very important contributor to network activity, through the control of communication between the soma and dendrites of single neurons. The important role of the h-current in persistent activity will be investigated. Finally, the mechanisms by which neuronal network discharge, such as with epileptic seizures, spontaneously stops will be examined with recordings from synaptically connected pairs of neurons. The possibility that synaptic depression or the activation of intrinsic ionic currents contributes to the natural, but temporary, cessation of epileptiform or sleep-related cortical bursts of activity will be examined. These studies will yield valuable information into the basic mechanisms by which cortical networks operate through recurrent excitation and inhibition and how this operation is prevented from being converted into the abnormal discharges of neurological disorders such as epilepsy.

The visual cortex of the brain is a major site of activity involved in visual perception. This large network of interacting nerve cells, or neurons, analyzes the activity of neural information coming from the eyes, and generates activity patterns itself. The precise mechanisms of how this analysis is achieved have not yet been fully detailed at the level of neurons and connections between neurons. There are a variety of categories of interacting nerve cell classes, based on their morphology, connections, electrophysiological characteristics, and areas of the visual field to which they respond. This project uses electrophysiological, anatomical and computational tools to see how function and structure are correlated in this complex structure. Recent results from several laboratories have revealed a pattern of synchronization in a subset of cortical neurons, showing oscillatory electrophysiological activity at frequencies in the range of 30-60 Hz. This project is designed to reveal some of the cellular mechanisms which may underlie how these synchronized oscillations depend on the configuration of visual stimuli, and which may play a role in sensory processing. Results from this work will be important in testing a currently controversial issue of visual processing, and will be important beyond visual physiology, to help understand cortical and complex brain organization in general, and to understand mechanisms of perception of the external environment.

Development of thalamocortical networks proceeds in an activity-independent phase during which the basic axonal connections are formed, followed by an activity-dependent phase during which these axonal connections are refined to achieve the high anatomical and synaptic specificity characteristic of the adult brain. During this activity-dependent phase, athe activation of the NMDA subtype of glutamate inotropic receptor is believed to facilitate synaptic plasticity. In the mammalian visual system, retinogeniculate fibers segregate into eye specific layers prior to visual experience, and this segregation is disrupted by block of spontaneous activity. These results indicate that non-visually driven activity may play a key role in the determination of appropriate anatomical connections in the visual system. In addition, during this period of formation and definition of synaptic circuits, spontaneous activity in the forebrain, as represented by the electroencephalogram (EEG), is becoming more and more characteristic of the adult animal. This development of the EEG includes the appearance and maturation of both normal (e.g. spindle waves) and abnormal (e.g. absence seizures) synchronized oscillations in thalamocortical systems. Recently we have demonstrated that the ferret LGNd, maintained as a slice in vitro, exhibits spontaneous synchronized oscillations characteristic of the developing and adult brain in situ and that this in vitro preparation is useful for the cellular analysis of development of synaptic and network properties in thalamocortical systems. Therefore, in the present study, we will investigate the cellular mechanisms of development of non-visually related synchronized activity int he ferret LGNd, the development of GABAergic inhibition within the LGNd, the contribution of NMDA receptors to thalamocortical monosynaptic EPSPs, and the regulation of activation of NMDA receptors by intracortical GABAergic inhibitory interneurons. These studies will provide for a cellular level understanding for the development, generation, and propagation of synchronized network activity in thalamocortical systems. This increased understanding of synchronized oscillations and synaptic function will lead to a better understanding of the development of synaptic connections and function in the mammalian forebrain and possible to therapeutic options for the prevention of generalized seizures.

[unreadable] Description (provided by applicant): The basic operation of the cerebral cortex, a massively and dynamically interconnected sheet of cortical neurons and networks, depends critically on the rapid modulation of excitability of individual cells, allowing them to participate or not participate in currently active neural networks, in response to behavioral and contextual demands. This rapid gain modulation is reflected in the change in the number and timing of action potentials. Using the primary visual cortex as a model system, we will examine the synaptic and network mechanisms that control the responsiveness of cortical neurons on a moment-to-moment basis, with particular attention to naturalistic visual input (natural movies). To understand gain modulation thoroughly, we will first investigate the cellular mechanisms of the contrast response function curve, as a prototypical example of a cortical input-output relationship. These studies will examine the origins of threshold responsiveness, mid-range sensitivity, and saturation at high contrast. Once the cellular mechanisms of this prototypical relationship are understood, we will then manipulate the membrane potential, input conductance, and membrane variance of individual visual cortical neurons (as well as stimulate the non-classical receptive field) to determine how these factors affect the contrast response function curve. These gain modulation studies will be complemented with experiments designed to reveal the synaptic mechanisms that control response timing precision and variability. Visual cortical neurons respond to full field natural visual scenes (movies) with temporally precise, highly selective and unusually space action potential sequences. These responses are often not well predicted by the linear properties of cells' classical receptive fields. By recording the pattern of excitatory and inhibitory inputs arriving in visual cortical neurons during presentation of natural stimuli, we will identify the contributions of excitatory, inhibitory, and spike initiation properties to spike timing precision. By varying the size of visual stimuli (to include stimulation of the non-classical receptive field as well as the classical receptive field) while recording from V1 neurons at various membrane potentials, we will determine the relative contributions of excitatory and inhibitory events to increases in action potential selectivity and sparseness associated with responses to naturalistic stimulation. These studies will reveal fundamental mechanisms of cortical gain control, spike timing, and neuronal signaling and lead to a better and more thorough understanding of visual cortical network function and dysfunction. The cerebral cortex is the most important structure of the human brain, yet is only partially understood. We will examine the basic operating principles of the visual cortex and how it encodes information, allowing for proper behavioral function. Our studies will give fundamental information relevant to understanding not only vision, but also attention and memory. [unreadable] [unreadable] [unreadable]

In the present CCNMD we propose to conduct a new program of translational research concerned with the endogenous processes responsible for cognitive dysfunctions of the dorsolateral prefrontal cortex (DLPFC) in schizophrenia and related disorders. The central hypothesis of the proposal is based on a new set of findings pointing to deficiencies in Ca2-associated intracellular cascades in schizophrenia, among them the recent discovery of a new class of dopamine (DA) receptor interacting proteins (DRIPs) which affect calcium signaling in model systems. We propose that such deficiencies, which may arise from many causes, constitute the underlying causal mechanism in the disorder and can account for both diverse and widespread neuropathologies as well as prominent vulnerability of higher-order functions. Extensive research inspired by the DA hypothesis of schizophrenia has yet to isolate a clear disease-associated abnormality in any particular component of the DA system, but the role of dopamine in the core cognitive dysfunctions of mental diseases cannot be denied. The present CCNMD is comprised of projects at 4 different universities designed to integrate dopamine pharmacology, cell biology, circuit mechanisms and neuronal processes with behavioral endpoints in animals and patients. State-of-the-art methodologies range from the isolation of novel DRIPs and characterization of their cellular functions in cell cultures through a clinical trial in schizophrenia patients based on a broad array of preclinical research. One subproject interrogates the status of DRIPs in postmortem brains from patients and controls with molecular, neurochemical and anatomical tools. Another subproject examines DA modulation and DA mediated calcium signaling of neural interactions in identified neurons and circuits in PFC cortical slices. Studies of PKA and PKC signaling in rodents, including mice genetically engineered to express DRIPs, are carried out. Other projects will interrogate similar pharmacological as well as physiological mechanisms in nonhuman primates performing cognitive tasks under normal and chronic drug regimens designed to elucidate basic properties of cognitive function and simultaneously parallel the clinical study in Project 9. These multidimensional projects based on novel concepts and discoveries are challenging and risk-taking, but all Pl's have a long record of effective interactions and productive collaboration to justify investment in the potential of this proposal.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This study is a subcontract of a Conte Center grant based at Yale University. During the reporting period, we used immunoelectron microscopy and quantitative analyses developed in our laboratory to study the distribution of D1 family dopamine receptors (D1 and D5) and signal transduction proteins in normal monkey prefrontal cortex as well as in various interneuron subpopulations. We tested the hypothesis that differential distribution of D1 and D5, receptors that differ in their affinity for the endogenous ligand dopamine, play a role in the inverted U curve relationship between D1 family dopamine receptor stimulation and prefrontal functioning. These studies provided us with a basis for understanding how dopamine controls cognition in primates and how this control might be disordered in schizophrenia.

[unreadable] DESCRIPTION (provided by applicant): Traditionally, communication between neurons in the cerebral cortex, and, indeed, within much of the brain, is believed to be mediated largely or solely through the rate and pattern of action potentials in a pulse or digital code. Recently, however, we have shown that the average amplitude of synaptic potentials evoked by action potentials is dependent upon the membrane potential of the presynaptic neuron and that these membrane potential changes travel long distances down the axon. This means that information communication between nearby cortical neurons may use a combined pulse and graded or analog and digital code. Here we will examine the mechanisms of this possible analog code. Specifically, we will examine the mechanisms by which the membrane potential of cortical neurons can influence the amplitude of synaptic potentials that the neuron induces in nearby cells. We will perform whole cell recordings from synaptically connected pairs of neurons in prefrontal cortical slices maintained in vitro. In addition, we will examine the possible involvement of changes in presynaptic Calcium concentrations through two-photon imaging of Ca2+ levels in presynaptic boutons while their parent soma is moved to different membrane potentials. Our investigations will also examine the electrophysiological properties of intracortical axons, particularly those properties that may be involved in detecting changes in membrane potential at the soma. We are particularly interested in the properties of voltage-dependent K+ currents within intracortical axons and how these may control axonal excitability. To examine these in detail, we will perform simultaneous whole cell patch clamp recordings from the soma and axon of cortical neurons during the application of voltage clamp steps and examine the respond of the axons to the application of toxins that are specific for different types of K+ channel. Be performing whole cell recordings from intracortical axons in mice in which particular K+ channels have been knocked out, we will be able to examine the involvement of specific ionic channels and subunits in these effects. Through this combination of synaptic and axonal electrophysiological investigations, we will achieve a more detailed and integrated understanding of how information communication operates within the cerebral cortex. This information will allow us to better manage disorders of axonal and synaptic communication including multiple sclerosis and epilepsy. PUBLIC HEALTH RELEVANCE: The proper function of synaptic transmission and axons, the output of neurons, is critical to the proper functioning of the brain, particularly the cerebral cortex. Numerous neurological disorders result from disruption of synaptic and axonal function. Our study will examine important basic properties of synaptic and axonal function in the cerebral cortex. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Neurons of the cerebral cortex are embedded in a highly interconnected network, allowing them to participate in a nearly infinite variety of neural circuits. The activity of a single neuron is determined by the flow of activity within this flexibl and powerful circuit. We hypothesize that neurons receive two broad categories of synaptic inputs that determine their action potential discharge: a slow, and sometimes, maintained component that strongly influences neuronal excitability and provides the cell with context; and a more rapid, temporally precise component that determines exactly when a cell discharges. The broad component is mediated by a proportional or balanced interaction of recurrent excitatory and inhibitory interactions in the cortex. The more rapid component appears to be mediated by temporally precise interactions of specific excitatory and inhibitory networks. Who are the interneurons that contribute to these two components and how are they achieving their task? Here we will address this important question through advanced in vitro and in vivo imaging and recording techniques. Using a recently developed in vitro mouse entorhinal cortical slice preparation that robustly generates spontaneous rhythmic Up and Down states, we will address the activity of several subclasses of neuron, including two excitatory cell types (Pyramidal and Stellate) and several inhibitory neuronal subtypes (PV/FS, SOM, NPY, 5HT3a, CR, VIP). This preparation not only generates the slow oscillation between Up and Down states, but also robust gamma-frequency oscillations during the Up state. Thus, we will also utilize this preparation to investigate the interneuronal cell types robustly involved in the generation of not only the general balance of recurrent excitation and inhibition that underlies persistent activity n the cortex, but also the generation of higher frequency network oscillations and the fast synaptic events that trigger action potentials on a precise time scale. These experiments will involve whole cell patch clamp and local field/MU recordings, as well as the selective inactivation/activation of known interneuronal cell populations with light activation of virally expressed archaerhodopsin, halorhodopsin, or channelrhodopsin. We will extend these investigations in vivo by studying the activity of identified subpopulations of interneurons in the mouse somatosensory cortex through patch clamp recording in a custom-built two-photon microscope. In a separate, but related, study, we will also investigate the possibility that local electrical fields may make a contribution to the synchronization and timing of higher frequency network oscillations. These investigations will help determine and define how the cerebral cortex operates, how it achieves a relative balance of recurrent excitation and inhibition, and how deviations from this balance may be critical in the generation of precisely timed action potential generation. The balanced operation of recurrent excitation and inhibition in the cortex is essential to the normal operation of the cerebral cortex and the breakdown of this balance readily results in the generation of epileptic seizures as well as psychiatric disorders.

Summary How do rapid variations of the waking state influence central auditory responses and what is the role of noradrenergic and cholinergic systems? The central hypothesis that we wish to examine is ?Rapid modulation of the waking state result from variations in activity of the noradrenergic and cholinergic pathways and occur along a continuum from: 1) reduced responses owing to cortical/subcortical spontaneous slow rhythms/low frequency cell and network burst firing during low arousal, 2) quiescent but accurately responsive networks during intermediate alertness, and 3) reduced responsiveness during high arousal or walking owing to high levels of both excitatory and inhibitory spontaneous activity. This is a timely topic, given that modulation of auditory responses and processing is important not only to normal hearing, but also in the recovery of function following damage or disease. Here we will test our central hypothesis (stated above) by performing extracellular unit and whole cell (synaptic) recordings from auditory cortical neurons and related subcortical structures (medial geniculate, inferior colliculus) during precisely measured variations in the waking state along with monitoring and manipulating the the activity of ascending cholinergic and noradrenergic pathways. Together, these results will yield a significant increase in our understanding of the cellular and network mechanisms of both normal, and recovery, of auditory cortical and subcortical function.

What are the cellular and network cortical mechanisms of optimal neural and behavioral performance? Through what mechanisms do spontaneous and evoked changes in the waking state of the cortex influence sensory processing and behavior? The goal of my laboratory is to answer these broad and important questions at levels extending from cellular and synaptic properties, to local circuits, to thalamocortical networks, modulation, and behavior. Answering these questions are fundamental not only to understanding the normal operation of the brain, but also its operation in a variety of disorders, from schizophrenia, to ADHD, autism, and others. Our proposal is ambitious, but not unreasonable or unfocused. We have already made significant progress towards a broad outline of important pieces of the puzzle, and therefore are confident that we will make very significant progress towards a detailed clarification of these two fundamental questions during the funding period. More than a hundred years ago, two investigators, Yerkes and Dodson, noted that optimal performance on difficult detection tasks was related to arousal level in an ?inverted-U? shaped fashion. Increases from low arousal to intermediate arousal would enhance performance on difficult tasks, while further increases in arousal from intermediate to high would decrease performance. This result suggests that there is an ?optimal state? for both the brain and behavior. Surprisingly, until our recent study in behaving mice performing a difficult auditory detection task, the cortical activity or circuit representation of optimal state had not been investigated. Our investigation revealed that the optimal state for performance of a difficult auditory sensory detection task occurred at intermediate levels of arousal and was associated with the suppression of slow corticocortical and thalamocortical activity, a hyperpolarized and low variability of pyramidal cell membrane potential, and large amplitude and highly reliable evoked auditory cortical synaptic responses. In an indication of the broad nature of these effects, we observed that we could predict more than half of the variance in cortical neuronal membrane potential, action potential, and even behavioral performance simply by measuring the pupil diameter ? an easily obtained measure of rapid (second to second) fluctuations in behavioral state. By explaining a large fraction of neuronal and behavioral variance, we have demonstrated that the brain is much more precise and reliable than previously thought. Here we propose to reveal the detailed cellular, modulatory, and network mechanisms that account for these prominent effects of state variation on neural and behavioral performance. Through a combination of state-of-the-art imaging, whole cell recording, optogenetic manipulation, and high quality behavioral monitoring, we will be able to detail the contribution of multiple neuronal and neuromodulatory pathways to the determination of optimal state for neural/behavioral responses.