Alison Barth - US grants

DESCRIPTION (provided by applicant): Identifying the cells and circuits that underlie perception, behavior, and learning is a central goal of contemporary neuroscience. Although techniques such as lesion analysis, fMRI, 2-deoxyglucose studies, and induction of gene expression have been helpful in determining the brain areas responsible for particular functions, these methods are technically limited. Currently there is no method that allows the identification of individual neurons that are associated with a particular function in living tissue. We have developed a strain of transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the promoter of the activity-dependent gene c-fos. Cortical and subcortical fosGFP expression could be induced in a regionally restricted fashion following specific activation of neuronal ensembles. This proposal seeks to further characterize the fosGFP transgenic mouse and validate this tool as a method for anatomical and electrophysiological analysis of neuronal subsets activated by in vivo experience. Single whisker stimulation induces c-fos and fosGFP expression in a cortical whisker barrel during experience-dependent plasticity. In order to better understand how neuronal gene expression in activated subsets of neurons leads to expansion of receptive fields for spared sensory inputs, fosGFP fluorescent neurons will be targeted for whole-cell recording. The fosGFP mice will be used to understand the anatomical and synaptic changes that underlie experience-dependent plasticity. These mice enable an in vivo or ex vivo characterization of the cells and synapses activated by particular pharmacological and behavioral manipulations. This method will enhance our ability to study the way neuronal networks are activated and changed by both experience and pharmacological manipulations.

DESCRIPTION (provided by applicant): The immediate early gene c-fos couples neural activity to gene expression and has been widely used as a marker to identify stimulus-specific neural ensembles. Here we propose to create and characterize transgenic mice that use the c-fos promoter to drive expression of the light-activated cation channel, channelrhodopsin (ChR), coupled to the yellow-fluorescent protein YFP. This proposal presents a fusion of cutting-edge technologies (direct visualization of gene expression and use of light-activated ion channels for circuit reactivation) that facilitate investigation of novel questions in systems neuroscience. Fos-ChR transgenic mice will allow us to 1) mark the identity of activated cells and 2) subsequently drive activity in these neurons to test the role of specific neurons in perception and behavior. Controlled reactivation of neuronal subsets will answer important questions about how specific neuronal subsets encode perception and behavior, and may facilitate active retraining of maladaptive neural circuits that have been altered by drug exposure or disease. PUBLIC HEALTH RELEVANCE: The mammalian brain contains millions to trillions of neurons that drive a wide array of complex behaviors. Understanding how specific neuronal ensembles are engaged by and encode perception and behavior requires identification and analysis of these neural subsets. Expression of the immediate-early gene c-fos can "mark" populations of task- or stimulus-specific neurons, providing a functional criterion to define neural subpopulations. Using fos gene promoter sequences to drive expression of the light-activated cation channel, channelrhodopsin, (ChR) we will create and characterize fos-ChR transgenic mice. These animals will enable us to identify and then reactivate functionally-defined neural ensembles to understand how the controlled reactivation of small subsets of neurons can drive perception and behavior. In addition, neural reactivation may allow retraining of specific circuits to eliminate maladaptive behaviors in addiction or psychiatric disorders.

DESCRIPTION (provided by applicant): We will investigate the factors regulating the subcellular distribution of the high conductance, calcium- and voltage-gated BK channel, a critical ion channel that regulates neuronal firing output in health and disease. Despite its powerful role in modulating excitability, experimental evidence indicates that it is sparsely distributed at the plasma membrane, a phenomenon that is regulated via interactions with the brain-specific accessory subunit, ?4. Conventional methods to study membrane protein localization have relied heavily upon overexpression of tagged proteins, a method that can significantly alter protein distribution by changing the stoichiometry of the target with its regulatory factors. To accurately determine how BK channels are distributed across the cell, it is important to be able to determine the location of individual molecules at endogenous expression levels to preserve critical concentration-dependent interactions with regulatory partners. We have developed a novel protein/dye tag with high-fluorescence emission that enables single-molecule detection, for both high- and low-abundance proteins. To preserve normal channel expression levels, we will generate a transgenic mouse where this tag has been inserted into the endogenous BK channel gene. The localization of this channel in primary neurons derived from these animals will be evaluated, and its accessory subunit and activity-regulated surface distribution will be determined.

DESCRIPTION (provided by applicant): Abnormal temperature and pain sensation is a common symptom of patients after stroke, and in some cases can lead to central pain syndrome, a debilitating disorder. Plasticity in neocortical circuits may play an important role in this disease. Here we will use sophisticated molecular tools and state-of-the-art recording techniques to identify the primary neocortical area representing pain and temperature stimuli in mice. Experiments will focus on cold sensation mediated by the TrpM8 receptor. TrpM8 is the sole receptor required for thermal sensation from ~10-24o C, and animals genetically lacking TrpM8 have a lack of temperature sensation in this range. In addition to cold temperature, TrpM8-expressing neurons are excited by the chemical ligand menthol, offering complementary routes for specific receptor activation. Our preliminary data indicate that TrpM8 stimulation specifically activates neurons in the posterior insula and that this activation is absent in TrpM8 receptor knock-out mice. We will use cold- or menthol stimulation in fosGFP transgenic mice followed by 2-photon targeted in vivo recordings of fosGFP+ neurons to determine the receptive field properties of cold-activated neurons. These experiments will lay a critical foundation for understanding how pain and temperature circuitry in the neocortex can be modified by experience and disease.

? DESCRIPTION (provided by applicant): Neurons in the cerebral cortex communicate with each other using highly specified, hierarchical rules of connectivity. There are more than 30 cell types in the neocortex, and these cell types can be differentiated by their developmental lineage, projection target, or expression of marker genes. Previous studies have attempted to reveal the logic of neural circuits by low-throughput anatomical or electrophysiological methods. Here we propose to develop and employ a novel trans-synaptic fluorescent complex formation strategy to chemically tag synapses defined by pre- and post- synaptic cell identity. Cell contacts made between genetically specified pre- and post- synaptic neurons will bring together a fluorescence-activating protein and one of a pair of covalently anchored fluorogenic dyes to trigger a 20,000-fold increase in fluorescence, easily detectable over background signal. The outstanding signal-to-noise and spectral properties of the dye will enable quantitative and in vivo analysis of cell-type specific synapses in the mammalian neocortex. We will use sequential labeling in different colors to differentially label newly formed synapses, allowing single endpoin measurement of synaptic density changes in response to experience. Applying these tools in the context of seizure models will reveal the cellular and molecular mechanism underlying changes in inhibition in cortex that result in increased seizure risk. The long- term goal of this proposal is to develop chemical biology tools for a complete index of cell-type specific synaptic contacts in order to establish how these contacts change in health and disease states.

? DESCRIPTION (provided by applicant): How synapses function in their native environment, during sensation and perception and in the context of network activity, are critical to understanding how they can transmit information and be modified by experience. Traditionally, electrophysiological studies have been carried out under experimental conditions that are profoundly different from the intact brain, where one of the most critical differences is the near absence of background activity in acute brain slices. Thus, the functional connectivity between neurons in active cortical circuits, across different brain states, remains unknown. We propose to investigate the properties of excitatory synapses, not in isolation, but as they function embedded in a dynamic neural network, using both in vitro and in vivo recordings. Analysis will focus on individual synaptic connections between pairs of pyramidal neurons in superficial layers of the rodent somatosensory cortex, a well-characterized exemplar of the mammalian neocortex. Our preliminary data indicates that under conditions of high network activity, excitatory synaptic connections onto multiple neuronal cell types can be effectively silenced by GABAb activation. We will identify the cellular source of GABA responsible for this suppression and examine how GABAb signaling is regulated in vivo. Understanding both the static and dynamic patterns of synaptic connectivity in the neocortex will be essential for understanding circuit function and plasticity in health and disease.

PROJECT DESCRIPTION Synapses are formed, broken and reformed dynamically both during development, normal function and in response to activity. Although this general principle is well-established, the way in which this is manifested in specific subtypes of neurons across a complex network, and how altered patterns of synaptic input will determine network function, have not been quantitatively investigated. Here we propose to develop molecular genetic tools for defining synaptic organization and connectivity in the mouse brain using fluorogen activating proteins (FAPs), a robust and modular system that enables multiplexed fluorescence identification of synapses and cell-specific connectivity. Our preliminary data indicate that we can target FAPs to synapses for quantitative analysis, as well as import 3D fluorescence image data for automated synapse detection using the image processing platform Imaris. Here we will create and validate pre- and postsynaptic targeting of fluorescent and FAP proteins respectively, acheiveing trans-synaptic FRET signal with high signal-to-background sensitized emission, allowing selective detection of synaptic connections formed between two genetically selected cell populations. These constructs, and the associated imaging and analysis approach, establish a pipeline for high- throughput data acquisition and analysis for assignment of cell-type specific contacts. As a test- bed for this technology, we will employ it to determine the synaptic input map for an important subset of cortical interneurons, somatostatin-expressing GABAergic cells, in the mouse neocortex.

ABSTRACT We will use our expertise in somatosensory organization and plasticity to develop novel and automated solutions for cell identification based upon neural activity, in order to decode the algorithms neural circuits use for information processing. Extracellular recordings in sensory cortex have been thought to primarily represent excitatory neuron activity, since these cells comprise ~80% of the total cell population. However, targeted cell recordings in S1 reveal that firing activity is dominated by inhibitory neurons, and that excitatory neurons can show 10-100 fold lower firing rates depending on cortical layer. Furthermore, new findings that reveal the complex relationship between different subtypes of inhibitory neurons make it difficult to relate blindly-recorded firing activity to local- or network-level computations. Clearly, cell-types matter, and massively parallel extracellular recordings that do not enable the simultaneous identification of multiple cell types will be limited in identifying principles for information transmission and encoding. Based on our preliminary findings, we hypothesize that the complex spontaneous and evoked spike trains from molecularly-identified neurons will provide unique and cell-type specific signatures that will enable cell identification from in vivo extracellular recordings. In collaboration with computer scientists at Carnegie Mellon, we will develop machine-learning algorithms for cell classification, using data collected from in vitro and in vivo recordings.

ABSTRACT Synapse addition and loss have been linked to learning throughout the brain. This has been well-documented in the neocortex using anatomical analysis of axonal boutons or dendritic spines in both fixed tissue and in vivo, using longitudinal imaging. However, it has been difficult to extrapolate the consequences of these synaptic changes without understanding how they lie within a defined network of cell-type specific contacts, requiring identification of the pre- and postsynaptic partners of the synapse. Here we will use in vivo imaging and multicolor fluorescence labeling of molecularly-defined pre- and postsynaptic neurons to monitor input- specific synapse addition and loss during sensory learning in a whisker-dependent task in mice. Synaptic contacts will be validated using post-hoc expansion microscopy and immunohistochemistry for nanoscale resolution. Analysis will be focused on learning-dependent reorganization of thalamic inputs from a higher-order thalamic nucleus, the posterior medial nucleus (POm) onto the dendrites of layer 5 (L5) pyramidal neurons that have been specifically implicated in experience-dependent plasticity. Quantitative, multicolor, in vivo imaging across different stages of learning will provide insight into how cortical circuits encode and are changed by salient sensory information.