RESUMEN
The motivation to seek social contact may arise from either positive or negative emotional states, as social interaction can be rewarding and social isolation can be aversive. While ventral tegmental area (VTA) dopamine (DA) neurons may mediate social reward, a cellular substrate for the negative affective state of loneliness has remained elusive. Here, we identify a functional role for DA neurons in the dorsal raphe nucleus (DRN), in which we observe synaptic changes following acute social isolation. DRN DA neurons show increased activity upon social contact following isolation, revealed by in vivo calcium imaging. Optogenetic activation of DRN DA neurons increases social preference but causes place avoidance. Furthermore, these neurons are necessary for promoting rebound sociability following an acute period of isolation. Finally, the degree to which these neurons modulate behavior is predicted by social rank, together supporting a role for DRN dopamine neurons in mediating a loneliness-like state. PAPERCLIP.
Asunto(s)
Neuronas Dopaminérgicas/patología , Núcleo Dorsal del Rafe/patología , Soledad , Animales , Dopamina/metabolismo , Núcleo Dorsal del Rafe/fisiopatología , Ácido Glutámico/metabolismo , Técnicas In Vitro , Masculino , Ratones , Optogenética , Técnicas de Placa-Clamp , Recompensa , Sinapsis , Área Tegmental Ventral/fisiologíaRESUMEN
The lateral hypothalamic (LH) projection to the ventral tegmental area (VTA) has been linked to reward processing, but the computations within the LH-VTA loop that give rise to specific aspects of behavior have been difficult to isolate. We show that LH-VTA neurons encode the learned action of seeking a reward, independent of reward availability. In contrast, LH neurons downstream of VTA encode reward-predictive cues and unexpected reward omission. We show that inhibiting the LH-VTA pathway reduces "compulsive" sucrose seeking but not food consumption in hungry mice. We reveal that the LH sends excitatory and inhibitory input onto VTA dopamine (DA) and GABA neurons, and that the GABAergic projection drives feeding-related behavior. Our study overlays information about the type, function, and connectivity of LH neurons and identifies a neural circuit that selectively controls compulsive sugar consumption, without preventing feeding necessary for survival, providing a potential target for therapeutic interventions for compulsive-overeating disorder.
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Conducta Animal , Área Hipotalámica Lateral/fisiología , Área Tegmental Ventral/fisiología , Animales , Retroalimentación , Área Hipotalámica Lateral/citología , Ratones , Modelos Neurológicos , Vías Nerviosas , Neuronas/citología , Recompensa , Sacarosa , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Dopamine modulates medial prefrontal cortex (mPFC) activity to mediate diverse behavioural functions1,2; however, the precise circuit computations remain unknown. One potentially unifying model by which dopamine may underlie a diversity of functions is by modulating the signal-to-noise ratio in subpopulations of mPFC neurons3-6, where neural activity conveying sensory information (signal) is amplified relative to spontaneous firing (noise). Here we demonstrate that dopamine increases the signal-to-noise ratio of responses to aversive stimuli in mPFC neurons projecting to the dorsal periaqueductal grey (dPAG). Using an electrochemical approach, we reveal the precise time course of pinch-evoked dopamine release in the mPFC, and show that mPFC dopamine biases behavioural responses to aversive stimuli. Activation of mPFC-dPAG neurons is sufficient to drive place avoidance and defensive behaviours. mPFC-dPAG neurons display robust shock-induced excitations, as visualized by single-cell, projection-defined microendoscopic calcium imaging. Finally, photostimulation of dopamine terminals in the mPFC reveals an increase in the signal-to-noise ratio in mPFC-dPAG responses to aversive stimuli. Together, these data highlight how dopamine in the mPFC can selectively route sensory information to specific downstream circuits, representing a potential circuit mechanism for valence processing.
Asunto(s)
Reacción de Prevención/fisiología , Dopamina/metabolismo , Sustancia Gris Periacueductal/citología , Sustancia Gris Periacueductal/fisiología , Corteza Prefrontal/citología , Corteza Prefrontal/fisiología , Animales , Señalización del Calcio , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Vías Nerviosas , Ratas , Ratas Long-Evans , Relación Señal-Ruido , Análisis de la Célula Individual , Cola (estructura animal)RESUMEN
BACKGROUND: The nuclear hormone receptor RORγ regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. Our goal was to develop a RORγ specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent. RESULTS: We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. We show that the AF2 helix of RORγ is proteolytically sensitive when inverse agonist BIO399 binds. Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of RORγ. Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and ß. CONCLUSION: The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. The proteolytic sensitivity of the AF2 helix of RORγ demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism.
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Benzoxazinas/química , Agonismo Inverso de Drogas , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Benzoxazinas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Ligandos , Simulación de Dinámica Molecular , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
RATIONALE: The basolateral amygdala (BLA) and medial geniculate nucleus of the thalamus (MGN) have both been shown to be necessary for the formation of associative learning. While the role that the BLA plays in this process has long been emphasized, the MGN has been less well-studied and surrounded by debate regarding whether the relay of sensory information is active or passive. OBJECTIVES: We seek to understand the role the MGN has within the thalamoamgydala circuit in the formation of associative learning. METHODS: Here, we use optogenetics and in vivo electrophysiological recordings to dissect the MGN-BLA circuit and explore the specific subpopulations for evidence of learning and synthesis of information that could impact downstream BLA encoding. We employ various machine learning techniques to investigate function within neural subpopulations. We introduce a novel method to investigate tonic changes across trial-by-trial structure, which offers an alternative approach to traditional trial-averaging techniques. RESULTS: We find that the MGN appears to encode arousal but not valence, unlike the BLA which encodes for both. We find that the MGN and the BLA appear to react differently to expected and unexpected outcomes; the BLA biased responses toward reward prediction error and the MGN focused on anticipated punishment. We uncover evidence of tonic changes by visualizing changes across trials during inter-trial intervals (baseline epochs) for a subset of cells. CONCLUSION: We conclude that the MGN-BLA projector population acts as both filter and transferer of information by relaying information about the salience of cues to the amygdala, but these signals are not valence-specified.
Asunto(s)
Amígdala del Cerebelo , Complejo Nuclear Basolateral , Amígdala del Cerebelo/fisiología , Tálamo , Complejo Nuclear Basolateral/fisiología , Condicionamiento Clásico/fisiología , Nivel de AlertaRESUMEN
NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys²66 and Cys³°9 with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.
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Disulfuros/metabolismo , Proteínas de la Mielina/química , Ingeniería de Proteínas/métodos , Receptores de Superficie Celular/química , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Animales , Conducta Animal/efectos de los fármacos , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Humanos , Masculino , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Proteínas de la Mielina/genética , Receptor Nogo 1 , Estabilidad Proteica , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Raíces Nerviosas Espinales/lesionesRESUMEN
The basolateral amygdala (BLA) mediates associative learning for both fear and reward. Accumulating evidence supports the notion that different BLA projections distinctly alter motivated behavior, including projections to the nucleus accumbens (NAc), medial aspect of the central amygdala (CeM), and ventral hippocampus (vHPC). Although there is consensus regarding the existence of distinct subsets of BLA neurons encoding positive or negative valence, controversy remains regarding the anatomical arrangement of these populations. First, we map the location of more than 1,000 neurons distributed across the BLA and recorded during a Pavlovian discrimination task. Next, we determine the location of projection-defined neurons labeled with retrograde tracers and use CLARITY to reveal the axonal path in 3-dimensional space. Finally, we examine the local influence of each projection-defined populations within the BLA. Understanding the functional and topographical organization of circuits underlying valence assignment could reveal fundamental principles about emotional processing.
Asunto(s)
Amígdala del Cerebelo/crecimiento & desarrollo , Complejo Nuclear Basolateral/crecimiento & desarrollo , Neuronas/metabolismo , Animales , Masculino , RatonesRESUMEN
The membrane protein interacting with kinase C1 (PICK1) plays a trafficking role in the internalization of neuron receptors such as the amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor. Reduction of surface AMPA type receptors on neurons reduces synaptic communication leading to cognitive impairment in progressive neurodegenerative diseases such as Alzheimer disease. The internalization of AMPA receptors is mediated by the PDZ domain of PICK1 which binds to the GluA2 subunit of AMPA receptors and targets the receptor for internalization through endocytosis, reducing synaptic communication. We planned to block the PICK1-GluA2 protein-protein interaction with a small molecule inhibitor to stabilize surface AMPA receptors as a therapeutic possibility for neurodegenerative diseases. Using a fluorescence polarization assay, we identified compound BIO124 as a modest inhibitor of the PICK1-GluA2 interaction. We further tried to improve the binding affinity of BIO124 using structure-aided drug design but were unsuccessful in producing a co-crystal structure using previously reported crystallography methods for PICK1. Here, we present a novel method through which we generated a co-crystal structure of the PDZ domain of PICK1 bound to BIO124.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Sitios de Unión/efectos de los fármacos , Cristalografía , Diseño de Fármacos , Humanos , Modelos Moleculares , Conformación Molecular , Dominios PDZ , Unión Proteica/efectos de los fármacos , Receptores AMPA/metabolismo , Relación Estructura-ActividadRESUMEN
Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.
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Calcio/metabolismo , Imagen Molecular/métodos , Neuronas/metabolismo , Optogenética/métodos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Ingeniería Genética , Ratones , Neuronas/química , Neuronas/citología , RatasRESUMEN
Although the basolateral amygdala (BLA) is known to play a critical role in the formation of memories of both positive and negative valence, the coding and routing of valence-related information is poorly understood. Here, we recorded BLA neurons during the retrieval of associative memories and used optogenetic-mediated phototagging to identify populations of neurons that synapse in the nucleus accumbens (NAc), the central amygdala (CeA), or ventral hippocampus (vHPC). We found that despite heterogeneous neural responses within each population, the proportions of BLA-NAc neurons excited by reward predictive cues and of BLA-CeA neurons excited by aversion predictive cues were higher than within the entire BLA. Although the BLA-vHPC projection is known to drive behaviors of innate negative valence, these neurons did not preferentially code for learned negative valence. Together, these findings suggest that valence encoding in the BLA is at least partially mediated via divergent activity of anatomically defined neural populations.
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Complejo Nuclear Basolateral/fisiología , Núcleo Amigdalino Central/fisiología , Hipocampo/fisiología , Recuerdo Mental/fisiología , Núcleo Accumbens/fisiología , Animales , Señales (Psicología) , Masculino , Ratones , Vías Nerviosas/fisiologíaRESUMEN
The basolateral amygdala (BLA) and ventral hippocampus (vHPC) have both been implicated in mediating anxiety-related behaviors, but the functional contribution of BLA inputs to the vHPC has never been directly investigated. Here we show that activation of BLA-vHPC synapses acutely and robustly increased anxiety-related behaviors, while inhibition of BLA-vHPC synapses decreased anxiety-related behaviors. We combined optogenetic approaches with in vivo pharmacological manipulations and ex vivo whole-cell patch-clamp recordings to dissect the local circuit mechanisms, demonstrating that activation of BLA terminals in the vHPC provided monosynaptic, glutamatergic inputs to vHPC pyramidal neurons. Furthermore, BLA inputs exerted polysynaptic, inhibitory effects mediated by local interneurons in the vHPC that may serve to balance the circuit locally. These data establish a role for BLA-vHPC synapses in bidirectionally controlling anxiety-related behaviors in an immediate, yet reversible, manner and a model for the local circuit mechanism of BLA inputs in the vHPC.
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Amígdala del Cerebelo/fisiopatología , Ansiedad/patología , Hipocampo/fisiopatología , Vías Nerviosas/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Ansiedad/genética , Proteínas Bacterianas/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Channelrhodopsins , Modelos Animales de Enfermedad , Antagonistas de Aminoácidos Excitadores/farmacología , Conducta Exploratoria , Halorrodopsinas/genética , Halorrodopsinas/metabolismo , Hipocampo/citología , Técnicas In Vitro , Proteínas Luminiscentes/genética , Masculino , Aprendizaje por Laberinto , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/fisiología , Bloqueadores de los Canales de Sodio/farmacología , Sinapsis/fisiología , Tetrodotoxina/farmacología , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 A resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 A resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.
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Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Agammaglobulinemia Tirosina Quinasa , Secuencia de Aminoácidos , Cristalografía por Rayos X , Dasatinib , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/química , Pirroles/química , Tiazoles/químicaRESUMEN
Nogo-66 receptor (NgR1) is a leucine-rich repeat (LRR) protein that forms part of a signaling complex modulating axon regeneration. Previous studies have shown that the entire LRR region of NgR1, including the C-terminal cap of the LRR, LRRCT, is needed for ligand binding, and that the adjacent C-terminal region (CT stalk) of the NgR1 contributes to interaction with its coreceptors. To provide structure-based information for these interactions, we analyzed the disulfide structure of full-length NgR1. Our analysis revealed a novel disulfide structure in the C-terminal region of the NgR1, wherein the two Cys residues, Cys-335 and Cys-336, in the CT stalk are disulfide-linked to Cys-266 and Cys-309 in the LRRCT region: Cys-266 is linked to Cys-335, and Cys-309 to Cys-336. The other two Cys residues, Cys-264 and Cys-287, in the LRRCT region are disulfide-linked to each other. The analysis also showed that Cys-419 and Cys-429, in the CT stalk region, are linked to each other by a disulfide bond. Although published crystal structures of a recombinant fragment of NgR1 had revealed a disulfide linkage between Cys-266 and Cys-309 in the LRRCT region and we verified its presence in the corresponding fragment, this is artificially caused by the truncation of the protein, since this linkage was not detected in intact NgR1 or a slightly larger fragment containing Cys-335 and Cys-336. A structural model of the LRRCT with extended residues 311-344 from the CT stalk region is proposed, and its function in coreceptor binding is discussed.