RESUMO
Synaptic connectivity and molecular composition provide a blueprint for information processing in neural circuits. Detailed structural analysis of neural circuits requires nanometer resolution, which can be obtained with serial-section electron microscopy. However, this technique remains challenging for reconstructing molecularly defined synapses. We used a genetically encoded synaptic marker for electron microscopy (GESEM) based on intra-vesicular generation of electron-dense labeling in axonal boutons. This approach allowed the identification of synapses from Cre recombinase-expressing or GAL4-expressing neurons in the mouse and fly with excellent preservation of ultrastructure. We applied this tool to visualize long-range connectivity of AGRP and POMC neurons in the mouse, two molecularly defined hypothalamic populations that are important for feeding behavior. Combining selective ultrastructural reconstruction of neuropil with functional and viral circuit mapping, we characterized some basic features of circuit organization for axon projections of these cell types. Our findings demonstrate that GESEM labeling enables long-range connectomics with molecularly defined cell types.
Assuntos
Conectoma/métodos , Comportamento Alimentar/fisiologia , Rede Nervosa/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Drosophila melanogaster , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , Estimulação Luminosa/métodos , Fatores de TempoRESUMO
Hunger is a complex behavioural state that elicits intense food seeking and consumption. These behaviours are rapidly recapitulated by activation of starvation-sensitive AGRP neurons, which present an entry point for reverse-engineering neural circuits for hunger. Here we mapped synaptic interactions of AGRP neurons with multiple cell populations in mice and probed the contribution of these distinct circuits to feeding behaviour using optogenetic and pharmacogenetic techniques. An inhibitory circuit with paraventricular hypothalamus (PVH) neurons substantially accounted for acute AGRP neuron-evoked eating, whereas two other prominent circuits were insufficient. Within the PVH, we found that AGRP neurons target and inhibit oxytocin neurons, a small population that is selectively lost in Prader-Willi syndrome, a condition involving insatiable hunger. By developing strategies for evaluating molecularly defined circuits, we show that AGRP neuron suppression of oxytocin neurons is critical for evoked feeding. These experiments reveal a new neural circuit that regulates hunger state and pathways associated with overeating disorders.
Assuntos
Ingestão de Alimentos/fisiologia , Comportamento Alimentar/fisiologia , Fome/fisiologia , Vias Neurais/fisiologia , Neurônios/fisiologia , Proteína Relacionada com Agouti/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Axônios/metabolismo , Feminino , Privação de Alimentos , Masculino , Camundongos , Modelos Neurológicos , Ocitocina/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Pró-Opiomelanocortina/metabolismo , Inanição , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Ionic flux mediates essential physiological and behavioral functions in defined cell populations. Cell type-specific activators of diverse ionic conductances are needed for probing these effects. We combined chemistry and protein engineering to enable the systematic creation of a toolbox of ligand-gated ion channels (LGICs) with orthogonal pharmacologic selectivity and divergent functional properties. The LGICs and their small-molecule effectors were able to activate a range of ionic conductances in genetically specified cell types. LGICs constructed for neuronal perturbation could be used to selectively manipulate neuron activity in mammalian brains in vivo. The diversity of ion channel tools accessible from this approach will be useful for examining the relationship between neuronal activity and animal behavior, as well as for cell biological and physiological applications requiring chemical control of ion conductance.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante/genética , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Neurônios/fisiologia , Engenharia de Proteínas , Animais , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , Encéfalo/citologia , Encéfalo/fisiologia , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/metabolismo , Compostos Bicíclicos com Pontes/farmacologia , Comportamento Alimentar , Feminino , Células HEK293 , Humanos , Ativação do Canal Iônico , Canais Iônicos de Abertura Ativada por Ligante/química , Ligantes , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Terciária de Proteína , Quinuclidinas/química , Quinuclidinas/metabolismo , Quinuclidinas/farmacologia , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores 5-HT3 de Serotonina/genética , Receptores 5-HT3 de Serotonina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Synaptic plasticity in response to changes in physiologic state is coordinated by hormonal signals across multiple neuronal cell types. Here, we combine cell-type-specific electrophysiological, pharmacological, and optogenetic techniques to dissect neural circuits and molecular pathways controlling synaptic plasticity onto AGRP neurons, a population that regulates feeding. We find that food deprivation elevates excitatory synaptic input, which is mediated by a presynaptic positive feedback loop involving AMP-activated protein kinase. Potentiation of glutamate release was triggered by the orexigenic hormone ghrelin and exhibited hysteresis, persisting for hours after ghrelin removal. Persistent activity was reversed by the anorexigenic hormone leptin, and optogenetic photostimulation demonstrated involvement of opioid release from POMC neurons. Based on these experiments, we propose a memory storage device for physiological state constructed from bistable synapses that are flipped between two sustained activity states by transient exposure to hormones signaling energy levels.