Barry W. Connors, PhD - US grants

Extracellular fluids and glial cells are a significant proportion of the mammalian brain volume, and together constitute the local environment of the neurons. This environment is not static, but may undergo large alterations of size and chemical content during various stages of neuronal activity and pathology. Such changes may in turn have effects on neural activity. The anatomy and chemistry of the neuronal environment appear to change dramatically during postnatal development of the neocortex, but there is very little quantitative information regarding the extent of these changes, or the consequences for neuronal function during maturation. The proposed investigation will utilize in vitro brain slice techniques, ion-sensitive microelectrodes and intra- and extracellular recording to assess the phsiological state of the neuronal environment during the development of rat neocortex. Specifically, we will measure the volume fraction and tortuosity of the extracellular fluid and the diffusion of ions through these fluids. Since astrocytes proliferate and mature primarly during the postnatal period, we will test several predictions based on their physiological properties in the mature brain: extracellular slow potentials induced by increases in extracellular [K+] should increase with age; the diffusion of K+ relative to large impermeant cations, should increase with age; and activity- and K+ related decreases in the size of the extracellular space should become more prominent with age. Variations in extracellular K+ and Ca2+ activities will be measured under different modes of stimulation. Finally, the sensitivity of neuronal excitability to alterations in extracellular [K+] will be assessed in developing and mature neocortex. These studies will provide unique insight into the relationships between neuronal activity and the brain microenvironment, and should provide functional correlates of the anatomical and biochemical evidence already available for developing neocortex. The results will help to explain the cause and consequences of large ionic fluctuations seen during epileptic seizures, and the developmental study of glial function may have relevance to the mechanisms of cerebral edema.

The primary sensory areas of neocortex perform complex transformations of the information flowing into them. In the primary somatosensory (SmI) area of the rat, many of these operations are carried out by discrete, repeating units of interconnected neurons, each of which can be visually identified by a barrel-shaped aggregate of small cells in cortical layer IV. Under pathological conditions, such as a decrease in synaptic inhibition, the normal activity of a local neocortical circuit can be usurped by large, synchronized bursts of epileptic excitation in all of its neurons. The proposed research will investigate the morphological and physiological properties of the neurons and their synaptic connections within rat barrel cortex. A method has been devised for visualizing individual barrels in living cortical slices in vitro. Intracellular recordings plus dye injections will be used to correlate membrane properties with the soma-dendritic and axonal branching characteristics of neurons. Electrophysiological techniques and axonal labelling methods, carried out in the slices and in intact animals, will help to delineate interlaminar and interbarrel connections. A combination of these methods and immunocytochemical identification will characterize the spatial organization of local inhibitory circuits that use the neurotransmitter gamma- aminobutyric acid. The mechanisms of synchronized epileptic discharge will also be studied. Specifically, the hypothesis that a subpopulation of middle layer bursting cells initiates and disperses synchronized activity will be examined. This interdisciplinary approach should provide a uniquely detailed view of the functional properties of a local neocortical area. The information obtained will contribute to the formulation of theories of cortical information processing, as well as suggest mechanisms for the genesis and control of focal seizures.

The neocortex comprises 80% of brain volume in humans, and it is essential for normal sensation, movement and cognition. Enormous effort has been expended attempting to understand the many functions of the neocortex. Basic studies of the physiology of its neurons, synapses and local circuits have benefitted immeasurably from the development of in vitro slice preparations over the past 2 decades. However, a question that has plagued in vitro physiologists from the beginning is: how do neurons in isolated preparations differ from neurons in the intact brain? A related question is: to what extent do traditional methods of intracellular recording with sharp microelectrodes perturb and distort the neuronal properties being measured? The answers are critically important. No one would argue that a neuron in vitro behaves entirely like a neuron in vivo but precisely how they differ can so far only be guessed. The goal of this project is to study the cellular properties of neocortical neurons in their natural milieu, in vivo, using methods of whole-cell patch recording to minimize cellular trauma. This approach will clarify differences between neurons in isolated and intact brains, and allow experiments that were not feasible with traditional recording methods. Neurons will be recorded with whole-cell methods from the primary somatosensory cortex of anesthetized rats, and from the same area in isolated slices. Recordings will be made from both cell bodies and apical dendrites. Natural sensory stimuli, consisting of controlled deflections of the facial vibrissae, will be used for many of the in vivo experiments. Several hypotheses that have arisen from studies in vitro will be tested in the intact animal. There are three specific questions: l) How do the passive and active membrane properties of different classes of neurons (soma.ta and dendrites) in vivo differ from those characterized in vitro? 2) Do the intrinsic physiological properties of a neuron predict the type of synaptic input it receives during natural sensory stimulation? 3) What are the mechanisms of GABAergic inhibition onto somata and dendrites in the intact neocortex? The results should reveal important differences and similarities between neocortical neurons in vitro and in vivo. Information about the biophysical properties of neurons in their natural environment will be essential to understand how the neocortex effects normal and pathological behaviors.

DESCRIPTION The organization and mechanisms of the fast-acting neurotransmitters (GABA and glutamate) in the neocortex have been studied extensively. On the other hand, data about the modes and sites of action of the modulatory neurotransmitters in the neocortex are scant. The investigators lack knowledge about the effects of modulators on synaptic transmission. The goal of the proposed research is to examine the influence of selected modulatory transmitters on specific axonal pathways. The studies will test a general hypothesis: modulators differentially regulate specific synaptic tracts in the neocortex. Using intracellular recording techniques in thalamocortical slices from mouse brains, we will examine and characterize the effects of three different modulatory neurotransmitters (acetylcholine, norepinephrine, and the actions of GABA on GABA-B receptors) on synaptic transmission, between specific tracts and identified neuronal types: 1) thalamocortical and intracortical pathways onto single, layer 5 pyramidal cells will be selectively activated, and 2) isolated inhibitory inputs will be evoked onto single, layer 5 pyramidal cells. In addition, they will examine the influence of modulators on the frequency-sensitivity of each synaptic tract. The modulatory transmitter systems have been implicated in many functions and dysfunctions of the cerebral cortex, and are essential for normal brain development. This study will advance significantly an understanding of cortical organization and the mechanisms by which the modulatory transmitters affect activity in complex cortical circuits.

DESCRIPTION (Adapted From The Applicant's Abstract): Nicotine is the world's most widely used addictive substance. Nicotine abuse, in the form of smoking, causes many chronic diseases and almost 20% of the deaths in developed countries. The mechanisms of nicotine's addictiveness are well studied. However nicotine has behavioral effects distinct from its physically addictive qualities, and these contribute to its popularity. Nicotine is said to elevate mood and arousal, reduce pain, and improve attention, working memory, and the rapid processing of sensory information. The mechanisms for these effects of nicotine are very poorly understood. Nicotine receptor in the brain have recently been implicated in a familial form of epilepsy and other neurological and psychiatric disorders, and drugs with nicotinic activity may have potential in the treatment of Alzheimer's and Parkinson's disease. The goal of this proposal is to study the cellular mechanisms of nicotine's effects on the forebrain, in particular the sensory neocortex. Neocortex and its connections comprise 80% of human brain volume, and it is essential for normal sensation, movement, memory and cognition. Nicotine's direct action on neocortex probably plays a major role in the drug's diverse effects on behavior. Nicotine binds to neuronal nicotinic acetylcholine receptors : in neocortex neuronal nicotinic acetylcholine receptors are abundant on the input (I.e. Thalamocortical) synapses to the cortex, and on some inhibitory and pyramidal neurons. The aims of this investigation are to test the hypothesis that nicotine, and the activation of specific neuronal nicotinic acetylcholine receptors, alters the strength and dynamics of input synapsis to the neocortex, modulates synaptic inhibition and inhibitory neurons, and thus systematically changes the way the neocortex transforms sensory information from the thalamus. Experiments will be performed in vitro and in vivo in the somatosensory system of rats. A formal computational model will be used to understand how nicotine's effects on neurons and synapses lead to complex but predictable alterations of the neocortical network, and thus alter sensation.

DESCRIPTION (Applicant's Abstract): Almost 80% of adults diagnosed with polymicrogyria, a developmental abnormality of the cerebral cortex, have an associated seizure disorder (Barkovich and Kjos, 1992). Unfortunately, this form of epilepsy tends to respond poorly to surgery or medication. Now there is an animal model for microgyria that causes reproducible, focal, seizure-like activity (Dvorak and Feit, 1977; Jacobs et al., 1996). Small freeze lesions are made on the cortical surface of normal neonatal rats; these cause destruction of deep cortical layers and compression of the layers above, creating a dimple, or microgyrus once the cortex matures. The microgyrus produces epileptiform activity that can be measured in slices of lesioned neocortex in vitro. There is very little information about the synaptic and cellular mechanisms underlying chronically epileptic cortex. The goal of this proposal is to use the freeze lesion model to explore the synaptic and cellular changes that cause seizures. More specifically, we will record from identified pairs of excitatory and inhibitory neurons in layer V to look for 1) changes in the intrinsic membrane properties of individual neurons, and 2) changes in the synaptic coupling of excitatory-excitatory, excitatory-inhibitory, and inhibitory-inhibitory cell pairs. In addition, we will look for morphological changes in the axonal and dendritic branching patterns of the neurons involved in the generation of epileptiform activity. Finally, we will explore the role of a molecule known to promote dendritic arborization (cpg 15; Nedivi et al., 1993) by using in situ hybridization, and we will attempt to reverse any overarborization in the epileptic zone by viral vaccination with a truncated form. of cpg l5. In this way, we hope to elucidate some of the underlying mechanisms of hyperexcitability in microgyria, including the extent of synaptic and cellular reorganization.

DESCRIPTION (provided by applicant): Electrical synapses are specialized connections, comprising gap junctions,that allow ionic current and small organic molecules to pass from one neuron to another. Only in the past few years has it become clear that electrical synapses are a major feature of the neuronal circuitry across the mammalian central nervous system. The PI's research has shown that electrical synapses are common between inhibitory neurons of the neocortex and thalamus, and that the protein connexin36 is necessary for these connections. This investigation will characterize the functions and the regulation of electrical synapses in the thalamus and neocortex. There are three specific aims: 1) the distribution of electrical synapses in both inhibitory and excitatory neurons of the thalamocortical system will be characterized in detail using electrophysiological, anatomical, and molecular methods; 2) the hypothesis that electrical synapses can coordinate the activity of inhibitory interneuron networks, and the excitatory neurons they contact, will be tested under controlled conditions in thalamus and neocortex; 3) the hypothesis that electrical synapses can be modulated by neural activity, neurotransmitters, and intracellular signaling systems will be tested in thalamus and neocortex. This investigation will provide a deeper understanding of the properties and functions of electrical synapses, a newly recognized feature of the mammalian forebrain.

DESCRIPTION (provided by applicant): Electrical synapses are specialized connections, comprising gap junctions,that allow ionic current and small organic molecules to pass from one neuron to another. Only in the past few years has it become clear that electrical synapses are a major feature of the neuronal circuitry across the mammalian central nervous system. The PI's research has shown that electrical synapses are common between inhibitory neurons of the neocortex and thalamus, and that the protein connexin36 is necessary for these connections. This investigation will characterize the functions and the regulation of electrical synapses in the thalamus and neocortex. There are three specific aims: 1) the distribution of electrical synapses in both inhibitory and excitatory neurons of the thalamocortical system will be characterized in detail using electrophysiological, anatomical, and molecular methods;2) the hypothesis that electrical synapses can coordinate the activity of inhibitory interneuron networks, and the excitatory neurons they contact, will be tested under controlled conditions in thalamus and neocortex;3) the hypothesis that electrical synapses can be modulated by neural activity, neurotransmitters, and intracellular signaling systems will be tested in thalamus and neocortex. This investigation will provide a deeper understanding of the properties and functions of electrical synapses, a newly recognized feature of the mammalian forebrain.

ABSTRACT

Integration of Dynamic Sensing and Actuating of Neural Microcircuits
PI: Arto V. Nurmikko

The proposed EFRI program aims to develop transformative paradigms in our understanding of the complex nonlinear dynamics of brain microcircuits and their function, by developing and fusing a new generation biosensing (recording) and actuation (neurostimulation) techniques to a potent toolbox. The proposed research engages brain circuits with external photonic and microelectronic interfaces in animal models, in particular for the study of the so-called "working memory" - the brain's "random access memory". At the neuroengineering level, the proposed research integrates new set of neural sensing and actuation tools on the microscale that are applied to engage with specific sensing and planning action by the brain - in particular the dynamics of information processing in the prefrontal cortex. A key experimental driver is the development of a new micro-optical/photonic device technology that will enable precise spatio-temporal targeting through sensory pathways of cortical microcircuitry and the imaging of this circuitry in real time in specific animal models. The unique device technology elements in the sensor/actuator engineering integrate ultracompact multi-element arrays of light emitters and microelectronic chip-scale sensors for excitation and mapping of the brain microcircuitry in real-time, which has been rendered both stimulus responsive and recordable by cellular-level genetic and nanomaterial sensitizing. The goal of the development of sensing/actuation microtools with associated brain science paradigms is to pave way for microdevice interfaces for bidirectional access across a population of neurons in the brain. Bidirectionality requires that both neural recording and neural stimulation can be achieved simultaneously at cellular level for multiple neurons, and ultimately multiple brain sites, spatially and temporally. Development of a class of specific brain-interfaces probes which synergize approaches from contemporary photonics/optoelectronics for "reading" and "writing" neural information from/to brain's microcircuits is the contributing aim of this planned EFRI proposal.

In a broader context, the research aims to facilitate the implementation of a closed-loop feedback compact device technology that offers the promise of entirely new classes of neural interfaces for (i) advancing the understanding of the brain from sensing to actuation- with cellular level resolution of microcircuit dynamics, (ii) aim the application of the technology to potentially therapeutic and prosthetic applications. For example, the study of the working memory function in the brain is closely associated with neurological diseases such as schizophrenia, attention deficit disorder and has been linked to epilepsy. The team aims to leverage the research outcomes from this program in mammalian animal models (in vitro and in vivo) so that key brain science paradigms such as the fundamentally important "working memory" will find translation to human neuroscience and rehabilitative goals. By including within the team a clinical neurology interface, our proposed research is envisioned to contribute to our unraveling of neurological disease, pave way for elucidating and exploring the applicability the nature of the brain-like systems to other technologies, as well as improve U.S. competitiveness in the global economy through advanced technology development in a frontier area at the intersection of physical and life sciences. The research on these topics is also expected to create a generation of "neuroengineering" graduate students with true interdisciplinary education, as well as innovative businesses and entrepreneurs.

Project 6 will focus on the most basic neurobiological effects of DBS, i.e. how does long-lasting, high- frequency electrical stimulation of subcortical white matter affect the axons, synapses, and neurons of prefrontal regions of neocortex? To this end, we will use contemporary methods of in vitro cellular neurophysiology to make high resolution measurements from groups of specific, genetically identified cortical neurons and their synaptic connections. Several lines of GFP-expressing mice will be used. Experiments will focus on the influence of DBS on conduction properties of afferent and efferent axons, and identified types of excitatory and inhibitory synaptic connections and their local cortical circuits. We will also use a c- fos-GFP mouse line to ask how high frequency stimulation in vivo alters the physiological properties of c-fos- expressing neurons. The results of this project should illuminate the basic mechanisms of DBS in brain structures relevant to OCD. RELEVANCE (See instmctions): Obsessive Compulsive Disorder (OCD) is a chronic psychiatric illness that affects 2-3% of the worldwide population. This is disease is in the top ten dehabilitating diseases. This study will examine the neural network and mechanisms that underiie behaviors associated with OCD. These behaviors not limited to OCD, but are associated with a range of affective and addictive disorders. The collective proposed studies will generate new hypotheses of how dysfunctions within these brain networks are expressed across diseases and provide insight into the mechanisms underlying normal behavioral responses. PROJECT/

Seeinstructions): This renewal application is to support a Postdoctoral Program in Systems and Behavioral Neuroscience that will prepare trainees for independent professional careers in the brain sciences. Trainees will learn how to critically evaluate the scientific literature, to identify fruitful lines of inquiry, to design incisive, interpretable 3xperiments, and to apply cutting-edge methods. In this renewal, our Program retains its longstanding strength in modern, multidisciplinary approaches for the study of neural processes underlying behavior, ncluding perception, orientation and communication; synaptic plasticity, learning and memory; and oriented motor actions. We offer training in the full spectrum of essential, state-of-the-art techniques in the neurosciences. These include noninvasive functional MRI, robotics and neuroprosthetics, manipulations of neuronal genes, sophisticated recording and imaging methods, and functional proteomics. The breadth of our training program is enhanced by the Brown Institute for Brain Science a network that links more that 90 acuity from eleven departments and successfully integrates techniques from applied mathematics, computer science, and biomedical engineering with the biological and behavioral neurosciences. This grant supports candidates early in their postdoctoral studies who display significant promise for a successful career in research. Beyond developing excellence in bench science, the Program provides venues for trainees to develop ancillary skills important for career development, including opportunities to mentor graduate and undergraduate students in research, to craft successful fellowship and grant proposals, to attend journal clubs and courses, and to hone their presentation skills. Retreats and seminar series offer trainees other opportunities to learn about groundbreaking science and interact with leaders in the field. Career development workshops are given on topics including responsible conduct of research, time management, women in science, scientific writing and nonacademic career tracks. Funds are requested for five years of support for four postdoctoral trainees. RELEVANCE (See instructions): The training provided in this Program will contribute to our understanding and treatment of a wide range of psychiatric and neurological diseases in two direct ways. First, trainees will participate in ongoing brain and behavioral neuroscience research programs. Second, the Program will prepare trainees for their independent careers in these fields.

PROJECT SUMMARY/ABSTRACT This project will investigate the functions of electrical synapses within inhibitory circuits of the mammalian forebrain. Electrical synapses are gap junctions that interconnect neurons, and they serve as rapid, bidirectional communication pathways. A considerable amount is known about the basic biophysical properties of mammalian electrical synapses, their locations, and their dependence on the gap junction protein connexin36 (Cx36). Gap junctions can strongly influence the timing, phase, synchrony, probability, and rate of action potentials in pairs and small groups of neurons, yet we still do not know how electrical synapses contribute to larger network functions. The complexity of forebrain circuits has long been an impediment, but powerful new genetic and optical tools can now be brought to bear on these issues. Remarkably, in the mature thalamus and neocortex electrical synapses occur almost exclusively between GABAergic neurons. These junctions are quite specific; they usually interconnect inhibitory neurons of the same subtype in the cortex, and excitatory cells in the mature forebrain rarely express them. This investigation will focus on the roles of electrical synapses that interconnect inhibitory neurons of the thalamus (specifically in the somatosensory thalamic reticular nucleus, TRN), and several subtypes of interneurons in the neocortex (barrel cortex). There are three specific aims. The first is to determine the roles of electrical synapses in thalamocortical network activity, specifically slow (delta, theta) and fast (gamma) network oscillations studied in vitro and in vivo. We will use electrophysiology, selective deletion of Cx36 from subtypes of cortical and thalamic inhibitory cells, and optogenetics to control specific neurons and axonal pathways. The second aim is to test the hypothesis that electrical synapses play an important role in the powerful feedforward inhibitory circuits activated by both thalamocortical and corticothalamic pathways. The third aim is to define the spatial and cell type-specific organization of gap junction-coupled networks in somatosensory segments of the TRN and neocortex. Inhibitory circuits are universal, and essential for all sensory, motor, and cognitive functions; electrical synapses are ubiquitous components of inhibitory circuits. Abnormalities of inhibition are implicated in a wide variety of neurological, psychiatric, and developmental disorders, and mutations in gap junction genes are associated with epilepsy and other neurological dysfunctions. Our investigation will help to clarify the relevance and functions of electrical synapses in inhibitory systems of the forebrain.

Project Summary/Abstract This project will investigate how the neocortex controls sensory processing in the thalamus. The neocortex and thalamus together constitute the majority of the mammalian brain and are crucial for sensation, motor control, and cognitive function. Virtually all sensory information enters the neocortex by way of the thalamus. The neocortex also provides massive top-down input to the thalamus. ?Corticothalamic? (CT) projections outnumber ascending ?thalamocortical? projections 10:1. This suggests that the cortex has a strong influence on thalamic activity and, thereby, on its own sensory input. Indeed, altered thalamic-cortical communication has been associated with disorders such as epilepsy and schizophrenia. Despite its obvious importance, a thorough understanding of CT function has been elusive. It is generally assumed that the cortex influences thalamic throughput by modulating the excitability of thalamic relay cells. However, in previous studies the scale and even the sign of modulation have varied. The complexity of CT circuits has long been an impediment to understanding them, but powerful genetic and optical tools can now be utilized to drive major advances. The central goal of our proposal is to determine how top-down projections from the neocortex influence thalamic sensory processing at the level of cellular, synaptic, and circuit mechanisms. We address this goal in three aims by utilizing the widely studied and technically tractable somatosensory system of the mouse. Aim 1 will focus on the extensive layer 6 CT system. In awake animals performing a sensory detection task, we will test our hypothesis that the TC system bidirectionally controls thalamic sensory processing, and that the sign of control is dynamically determined by layer 6 cell spike rates, short-term synaptic plasticity, and behavioral state. Aim 2 will focus on the layer 5 CT system, which is structurally distinct. Layer 5 CT projections, unlike those of layer 6, bypass the inhibitory thalamic reticular nucleus and the primary thalamic relay nuclei and make strong driver-type synapses in higher-order thalamic nuclei. Using specific Cre-expressing mouse lines and optogenetics, we will test the prediction that the dynamic balance of thalamic excitation and inhibition caused by layer 5 CT circuitry is dramatically different from that of layer 6. We will also characterize how these two CT circuits interact to produce integrated effects. Aim 3 will examine how the CT cells are themselves controlled, focusing on effects of diverse thalamic inputs. We, and others, have shown that neurons in layers 5 and 6 are powerfully innervated by thalamus, and that distinct thalamic nuclei have unique cortical projections. Here we will test the hypothesis that feed-forward inputs from first-order and second-order thalamic nuclei exert powerful but complementary control over CT cells of layers 5 and 6. Our project will provide much-needed insight about how CT systems influence thalamic processing. Such information will be essential for understanding neurological disorders involving CT communication.