2009 — 2012 |
Barnea, Gilad |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A Molecular Method to Selectively Record Activation of Dopamine Receptor Subtypes
Neurons communicate with one another by secreting chemical signals called neurotransmitters. The neurotransmitters secreted from one neuron bind specific receptors on the membranes of other cells and elicit a cascade of responses in these cells. Dopamine is a neurotransmitter that regulates a diverse array of biological processes including cognition and emotion, motivation and reward, locomotion, and the release of certain hormones. Imbalances in the dopamine system have been implicated in disorders as diverse as schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, Tourette's syndrome, addiction, Parkinson's disease, and hypertension. The mammalian genome encodes five different receptors for dopamine that can be grouped into two classes based on their cellular signaling and sequence homology. The various types of receptors are thought to mediate different biological functions and are implicated in different disorders. The two classes of dopamine receptors can also be distinguished pharmacologically, but this discrimination is not absolute. Furthermore, it is much more difficult to distinguish pharmacologically between receptors within the same class. A given drug often acts on multiple receptors, producing unwanted side effects. Thus, having highly specific drugs for the various dopamine receptors is critical for the successful treatment of a disorder that involves a particular dopamine receptor type with minimal side effects. Since many neurons express multiple types of dopamine receptors, it is currently impossible to attribute the effects of a particular drug to a specific receptor. Clinically, this gap of knowledge translates into an inability to predict and address the side effects of a given drug. Here we present a novel molecular method to selectively record activation of a particular dopamine receptor subtype in the murine brain. Since our system is extremely selective, it can be used to unequivocally determine which receptor subtype has been activated in a particular neuron in response to a given drug. This is accomplished regardless of the presence of other kinds of dopamine receptors in this neuron. The animal models that we will generate will enable the development and testing of specific drugs with fewer side effects. Moreover, our technology can be used to identify changes that occur in particular circuits in mouse models for human diseases such as schizophrenia and Parkinson's disease, providing clues regarding the mechanisms underlying the progression of these diseases. Finally, the current inability to monitor the activation of a particular receptor subtype also applies to other families of receptors. Since our system is modular, it can be readily adapted to study other receptors. A method to selectively monitor activation of specific receptors in an animal model will thus have a major impact on a very broad segment of the biomedical research community. We are proposing to generate a novel molecular method to selectively record the activation of a particular dopamine receptor subtype in an animal model. The dopamine system has been implicated in multiple disorders such as schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, Tourette's syndrome, addiction, Parkinson's disease and hypertension. The availability of such animal models will enable the development and testing of much more specific dopamine receptor agonists and antagonists with fewer side effects.
|
1 |
2013 — 2017 |
Barnea, Gilad Lomvardas, Stavros [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Controlling Epigenetic States and Nuclear Architecture in the Brain @ Columbia University Health Sciences
DESCRIPTION (provided by applicant): The realization that epigenetic control of gene expression can override regulatory information encoded in DNA provides the exciting opportunity to stably alter gene expression programs in vivo with the use of epigenetic modifiers. However, this aspiration is challenged by limitations in our ability to alter the epigenetic state of specific target genes in restricted cell types in a temporally regulated fashio. For this reason, we propose to combine novel genetic approaches that afford tight spatiotemporal control in vivo with innovative biochemical tools that allow the targeting of specific genomic loci in a sequence-specific manner. We will modify an assay that we previously designed for the inducible labeling of specific neuronal populations, named Tango, towards the controlled expression of synthetic TALE (Transcription Activator Like Effectors)-fusion proteins that will bind to target genomic loci and alter their epigenetic properties. As a model for these proof-of-principle experiments we will use the genetically, epigenetically and biochemically tractable mouse olfactory system. As we previously showed, the monogenic and monoallelic expression of olfactory receptor (OR) genes in olfactory sensory neurons (OSNs) is epigenetically regulated, both at the level of post-translational histone modifications and at the level of nuclear organization and distribution of active and silent OR alleles. Therefore, we propose to express TALE-fusion proteins with specificity for OR genes and their regulating enhancers in an inducible fashion in specific OSN subpopulations using variations of the TANGO system. This way we will alter the epigenetic state of active or silent ORs, and induce their re-positioning to distinct nuclear territories with the goal of stably altering their expresson pattern. This strategy of chemically or optically controlled epigenetic manipulations will be directly applicable to any other cell type in the mouse, and compatible with viral delivery methods that will make our approach applicable to future therapeutic interventions for human disease.
|
0.966 |
2014 — 2018 |
Barnea, Gilad |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
An Olfactory Subsystem That Mediates Innate Behaviors
DESCRIPTION (provided by applicant): The mammalian olfactory system responds both to neutral odors, whose significance for the organism is assigned by learning, and to odors that elicit innate behaviors. A subset of olfactory sensory neurons expresses trace amine associated receptors (TAARs) that respond to volatile amines that are mostly aversive to rodents. We recently described the projection patterns of TAAR-expressing neurons (TRNs) to the olfactory bulb and examined the molecular mechanisms that control the expression of a single TAAR per TRN. Based on these findings, our overall hypothesis is that the TRNs constitute a distinct olfactory subsystem and that they integrate into hard-wired circuits that enable them to extract specific environmental cues and drive robust innate behavioral responses. Several predictions stem from this hypothesis: 1. TRNs are molecularly distinct from ORNs and they use specific mechanisms to ensure expression of a single receptor per neuron; 2. Activation of the TRNs is sufficient to elicit innate behavioral responses and, 3. The projections from the TAAR glomeruli are stereotyped and target central neural structures appropriate to aversive behavior responses. To test these predictions, we will conduct an interdisciplinary, multi-tiered approach that spans the molecular, neuroanatomical, neurophysiological and behavioral levels. At the molecular level, we will use unbiased approaches to unequivocally determine whether TRNs express only TAARs or co-express a subset of ORs. Further, we will definitively determine whether TRNs are molecularly committed to exclusively express TAARs, or can switch to express ORs upon choosing a deleted Taar gene. We will also analyze sorted populations of TRNs to identify repressive epigenetic marks and activating enhancer sequences that control expression of a single TAAR per TRN. Together, these studies will provide the molecular evidence for defining the TRNs as a distinct olfactory subsystem. At the anatomical level, we will map the projections from the TAAR glomeruli in the bulb to higher olfactory centers in the brain. We predict that these projections will be stereotyped and these glomeruli may project predominantly to the amygdala. At the systems/behavioral level, we will determine the behavioral consequences of selective optogenetic activation of TRNs, following systematic characterization of the optimal stimuli for driving bulb responses. These experiments will test whether TAAR circuits are hard wired to induce innate behavioral responses such as sniffing, and hard wired to assign aversive valence. Our studies are the first to examine innate responses to aversive olfactory stimuli in the context of known receptors and ligands. They will shed light on the molecular and anatomical substrate of innate responses to aversive stimuli, and provide foundation for direct links between molecular and anatomical organization and behavioral outcomes. Understanding the neural mechanisms mediating the conversion of sensory information to eliciting innate behaviors may provide new insight into the underpinnings of several psychiatric conditions.
|
1 |
2014 — 2017 |
Barnea, Gilad |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Functional Mapping of Mammalian Neural Circuits
DESCRIPTION (provided by applicant): Neural circuits are the basic computational units in the nervous system. Thus, our inability to anatomically label specific circuits and to manipulate their function is a major deficiency in our ability to study the brain. Imaging techniques, which have revolutionized our understanding of the functions of large brain structures, lack the resolution and precision needed for studying particular circuits. Molecular techniques, which have been extremely powerful in studying individual neurons, are currently limited to the confines of single cells. The development of a technology for circuit tracing and manipulation by selectively bridging across the synapses that connect neurons within a given circuit is still an unmet challenge. Here we propose to combine molecular biology and genetics to develop such a technology. At the core of our approach is a synthetic signaling pathway that is introduced into all neurons. Selective activation of this pathway within a particular circuit will be used to label the circuit, or to functionally manipulate it. To achieve this, we will selectively activate specifc neurons by optogenetic techniques. Glutamate release into the synapses of these neurons will activate the signaling pathway in post-synaptic neurons leading to expression of a marker protein that will label their projections. Since our system is modular, its use will be readily expanded to various neural circuits. Furthermore, our system can be configured for use in other model organisms including primates. Our technology for selective labeling and manipulation of circuits in vivo will therefore open new research avenues. In addition, it will be easily adapted t experiments in which the properties of particular circuits will be modified and the functional consequences will be studied. The type of studies of neural circuits afforded by our technology will lead to a deeper understanding of how the brain functions. Our technology will also be used in various mouse models for human diseases and help identify specific changes that occur in particular circuits in these model animals. Without a sensitive method for specific mapping and manipulation of particular circuits, the accessibility of these questions is rather limited. Our approach to circuit tracing and manipulation is totally different than any method attempted thus far, as molecular genetics has only been used to study neurons that express a given genetic marker. The system presented here will expand the use of molecular approaches to all the cells with which the neurons that express the genetic marker communicate. Thus, our technique will expand the utility of molecular biological approaches beyond the confines of a single neuron. Harnessing the enormous power of molecular genetics to study circuits will undoubtedly broaden our understanding of the brain and therefore have a major impact on the neuroscience community.
|
1 |
2015 — 2016 |
Barnea, Gilad |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Trans-Synaptic Tracing and Manipulation of Olfactory Circuits in Flies
DESCRIPTION (provided by applicant): Most behavioral responses to odors require experience and learning before an odor stimulus acquires its value for the organism. There are however some odors that elicit innate and stereotyped behavioral responses. Some of these odors are pheromones while others are environmental odors with special selective importance for the survival of the organism. It is believed that innate behaviors are governed by genetically programmed, hard-wired neural circuits. The existence of several layers within the circuit allows for modulation of the response to reflect the internal state of the organism. Due to lack of tools for specific and sensitive trans- synaptic labeling of neurons within a circuit, little is known abut the circuit level of the brain, including hard- wired circuits. We have combined molecular biology and genetics to develop a new technique for circuit mapping in fruit flies. At the core of our system is a synthetic signaling pathway that is introduced into all neurons. Selective activation of this pathway within a particular circuit will be used to trace projections within the circuit orto alter its function. To achieve this, we will genetically modify pre-synaptic neurons, for which there is a genetic marker, such that they will express in their synapses a membrane-bound ligand that will activate the signaling pathway in post-synaptic partners. Here we propose to optimize this technique for tracing projections of second and third order neurons within the olfactory circuits. Since our system is modular, its use will be readily expanded to multiple neural circuits in the fly. Furthermore, it will be also easily adapted to experiments in which the properties of particular circuits will be modified and the functional consequences will be studied. Once we optimize our trans-synaptic tracing technique, we intend to use it to trace circuits that mediate olfactory-governed innate aversion and attraction in flies. We also intend to follow this proof of concept in flies by establishing an equivalent technique for labeling circuits in mice.
|
1 |
2018 — 2021 |
Barnea, Gilad |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular and Cellular Analysis of Accessory Olfactory Circuits in Mice
In mice, the vomeronasal organ (VNO) is a main sensory organ for detecting pheromones, chemicals that affect social behaviors including: territoriality, sexual recognition and maternal care. The VNO can be divided into apical and basal layers that differ in their properties. VNO sensory neurons (VSNs) in the apical layer express members of the Vmn1r family of G protein coupled receptors (GPCRs), while basal VSNs express members of the Vmn2r family of GPCRs. Vmn2rs belong to the same family of GPCRs that includes the metabotropic glutamate, GABAB, and taste receptors. Basal VSNs do not obey the one receptor per neuron rule that is operative in all other sensory neurons in the olfactory system, with the exception of neurons of the newly identified necklace subsystem that do not express GPCRs. The Vmn2r family consists of four classes designated A, B, C and D. Members of classes A, B and D are more closely related to one another than to the seven members of class C (Vmn2r1-7). Each basal VSN expresses one Vmn2rC and one Vmn2rABD. Consequently, The seven class C receptors are broadly expressed in the VNO while class ABD receptors are sparsely expressed, like the rest of the olfactory receptors. The functional significance of the coexpression of Vmn2rs is not well understood. Based on our knowledge regarding the other members of the GPCR family to which Vmn2rs belong, we hypothesize that Vmn2rCs and Vmn2rABDs form heterodimers and that this interaction alters the functional properties of the basal VSNs. Vmn2rCs might affect the subcellular localization of Vmn2rABDs, their ligand binding and signaling properties. To examine these hypotheses, we have devised a multipronged strategy encompassing molecular and biochemical studies, neuroanatomical examination, and behavioral analysis. We have generated a mouse line carrying a deletion of the Vmn2r1-7 gene cluster using CRISPR/Cas9-mediated chromosome engineering followed by Cre recombination. Further, we generated a battery of specific antibodies against all Vmn2rCs, against specific class C receptors, and against a clade of Vmn2rAs. With these new reagents, we are poised to address these hypotheses. Our studies will reveal the mechanisms underlying the function of basal VSNs. More broadly, these experiments will shed light on how the olfactory system mediates social behaviors that are critical for the survival of the species. Thus far, only a handful of Vmn2r ligands have been identified. Since we predict that the deletion of the Vmn2rC cluster will affect the ability of the animals to properly respond to Vmn2rABD ligands, the cluster knockout line that we have generated will be invaluable for evaluating new Vmn2r ligands and their behavioral importance, as these ligands are identified. In this manner, our studies will deepen our understanding of the control of social behavior in multiple ways. Finally, our experiments will provide additional insight into the molecular mechanisms underlying the function of the GPCR family to which Vmn2rs belong. Some of these receptors have relevance to human disease.
|
1 |
2018 — 2021 |
Barnea, Gilad |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Neural Circuits Underlying Gustatory Perception in Flies
Animals use the sense of taste to make decisions regarding potential food; substances with high nutritional value are ingested, while toxins and harmful substances are rejected. Interestingly, these behaviors are common across many species. Flies respond to sweet and bitter tastants with different stereotyped behaviors: sweet substances, often calorie rich, are appetitive and accepted, while bitter compounds, usually harmful, are rejected and avoided. The linkage between stimulus quality and behavioral response suggests that sweet and bitter tastants are represented differently in the brain. Mice process information regarding sweet and bitter substances in parallel through labeled lines. By contrast, in moths, a distributed combinatorial code for individual tastants was described, suggesting that the neural circuits are convergent. It is currently unknown which of these distinct models is operative in flies. Addressing this question will require a comprehensive analysis of the gustatory circuits layer by layer. While our understanding of the first-order level within the bitter and sweet circuits is rather advanced, little is known about neurons in the second-order level of the gustatory system. Most of the second-order neurons that have been characterized thus far have been identified by genetic screens. Due to the distributive nature of the first-order gustatory projections, one cannot identify the second-order neurons by the location of their dendrites, as has been done successfully in the olfactory circuits. In addition, flies have gustatory neurons in various parts of their body, and we hypothesize that a somatotopic gustatory map exists in the brain. All of these important gaps of knowledge would benefit from a robust genetic system for transsynaptic labeling of neural circuits. We have recently developed a new method for transsynaptic tracing and manipulation of neural circuits termed trans-Tango. We have validated trans-Tango in the olfactory system of flies and established it in the gustatory circuits that process information regarding sweet compounds. Our analysis revealed that second- order neurons in the sweet circuits project to neuromodulatory areas in the brain, some of which are known to be involved in controlling feeding behavior. Here we propose to implement trans-Tango to identify second- order projections in the bitter circuits. Our preliminary data suggest that the second-order projections in the bitter circuits are very similar to the second-order sweet projections. We propose a multipronged strategy that involves anatomical, functional and behavioral analyses aimed at characterizing in detail the second- and third- order projections within the sweet and bitter circuits. For our analysis, we will establish new versions of trans- Tango that incorporate new modules for functional analysis of circuits via calcium imaging and optogenetics, for intersectional connectivity studies, and for multicolor projection analysis. Thus, our studies will deepen our understanding of gustatory information processing in flies, a topic of high importance for human health in view of the relevance of the sense of taste for the role of insects as major vectors of many insect-born diseases.
|
1 |