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We investigated how transmission of hunger- and satiety-promoting neuropeptides, NPY and αMSH, is integrated at the level of intracellular signaling to control feeding. Receptors for these peptides use the second messenger cAMP. How cAMP integrates opposing peptide signals to regulate energy balance, and the in vivo spatiotemporal dynamics of endogenous peptidergic signaling, remain largely unknown. We show that AgRP axon stimulation in the paraventricular hypothalamus evokes probabilistic NPY release that triggers stochastic cAMP decrements in downstream MC4R-expressing neurons (PVHMC4R). Meanwhile, POMC axon stimulation triggers stochastic, αMSH-dependent cAMP increments. Release of either peptide impacts a ~100 µm diameter region, and when these peptide signals overlap, they compete to control cAMP. The competition is reflected by hunger-state-dependent differences in the amplitude and persistence of cAMP transients: hunger peptides are more efficacious in the fasted state, satiety peptides in the fed state. Feeding resolves the competition by simultaneously elevating αMSH release and suppressing NPY release, thereby sustaining elevated cAMP in PVHMC4R neurons. In turn, cAMP potentiates feeding-related excitatory inputs and promotes satiation across minutes. Our findings highlight how biochemical integration of opposing, quantal peptide signals during energy intake orchestrates a gradual transition between stable states of hunger and satiety.
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We developed a platform that utilizes a calcium-dependent luciferase to convert neuronal activity into activation of light sensing domains within the same cell. The platform is based on a Gaussia luciferase variant with high light emission split by calmodulin-M13 sequences that depends on influx of calcium ions (Ca2+) for functional reconstitution. In the presence of its luciferin, coelenterazine (CTZ), Ca2+ influx results in light emission that drives activation of photoreceptors, including optogenetic channels and LOV domains. Critical features of the converter luciferase are light emission low enough to not activate photoreceptors under baseline condition and high enough to activate photosensing elements in the presence of Ca2+ and luciferin. We demonstrate performance of this activity-dependent sensor and integrator for changing membrane potential and driving transcription in individual and populations of neurons in vitro and in vivo.
RESUMO
We investigated how transmission of hunger- and satiety-promoting neuropeptides, NPY and αMSH, is integrated at the level of intracellular signaling to control feeding. Receptors for these peptides use the second messenger cAMP, but the messenger's spatiotemporal dynamics and role in energy balance are controversial. We show that AgRP axon stimulation in the paraventricular hypothalamus evokes probabilistic and spatially restricted NPY release that triggers stochastic cAMP decrements in downstream MC4R-expressing neurons (PVH MC4R ). Meanwhile, POMC axon stimulation triggers stochastic, αMSH-dependent cAMP increments. NPY and αMSH competitively control cAMP, as reflected by hunger-state-dependent differences in the amplitude and persistence of cAMP transients evoked by each peptide. During feeding bouts, elevated αMSH release and suppressed NPY release cooperatively sustain elevated cAMP in PVH MC4R neurons, thereby potentiating feeding-related excitatory inputs and promoting satiation across minutes. Our findings highlight how state-dependent integration of opposing, quantal peptidergic events by a common biochemical target calibrates energy intake.
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Understanding percepts, engrams and actions requires methods for selectively modulating synaptic communication between specific subsets of interconnected cells. Here, we develop an approach to control synaptically connected elements using bioluminescent light: Luciferase-generated light, originating from a presynaptic axon terminal, modulates an opsin in its postsynaptic target. Vesicular-localized luciferase is released into the synaptic cleft in response to presynaptic activity, creating a real-time Optical Synapse. Light production is under experimenter-control by introduction of the small molecule luciferin. Signal transmission across this optical synapse is temporally defined by the presence of both the luciferin and presynaptic activity. We validate synaptic Interluminescence by multi-electrode recording in cultured neurons and in mice in vivo. Interluminescence represents a powerful approach to achieve synapse-specific and activity-dependent circuit control in vivo.
Assuntos
Neurônios/metabolismo , Optogenética/métodos , Sinapses/metabolismo , Animais , Encéfalo/citologia , Células Cultivadas , Luciferases/genética , Luciferases/metabolismo , Luciferinas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , RatosRESUMO
In genetic and pharmacological models of neurodevelopmental disorders, and human data, neural activity is altered within the developing neocortical network. This commonality begs the question of whether early enhancement in excitation might be a common driver, across etiologies, of characteristic behaviors. We tested this concept by chemogenetically driving cortical pyramidal neurons during postnatal days 4-14. Hyperexcitation of Emx1-, but not dopamine transporter-, parvalbumin-, or Dlx5/6-expressing neurons, led to decreased social interaction and increased grooming activity in adult animals. In vivo optogenetic interrogation in adults revealed decreased baseline but increased stimulus-evoked firing rates of pyramidal neurons and impaired recruitment of inhibitory neurons. Slice recordings in adults from prefrontal cortex layer 5 pyramidal neurons revealed decreased intrinsic excitability and increased synaptic E/I ratio. Together these results support the prediction that enhanced pyramidal firing during development, in otherwise normal cortex, can selectively drive altered adult circuit function and maladaptive changes in behavior.
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The ability to manipulate specific neuronal populations of the spinal cord following spinal cord injury (SCI) could prove highly beneficial for rehabilitation in patients through maintaining and strengthening still existing neuronal connections and/or facilitating the formation of new connections. A non-invasive and highly specific approach to neuronal stimulation is bioluminescent-optogenetics (BL-OG), where genetically expressed light emitting luciferases are tethered to light sensitive channelrhodopsins (luminopsins, LMO); neurons are activated by the addition of the luciferase substrate coelenterazine (CTZ). This approach utilizes ion channels for current conduction while activating the channels through the application of a small chemical compound, thus allowing non-invasive stimulation and recruitment of all targeted neurons. Rats were transduced in the lumbar spinal cord with AAV2/9 to express the excitatory LMO3 under control of a pan-neuronal or motor neuron-specific promoter. A day after contusion injury of the thoracic spine, rats received either CTZ or vehicle every other day for 2 weeks. Activation of either neuron population below the level of injury significantly improved locomotor recovery lasting beyond the treatment window. Utilizing histological and gene expression methods we identified neuronal plasticity as a likely mechanism underlying the functional recovery. These findings provide a foundation for a rational approach to spinal cord injury rehabilitation, thereby advancing approaches for functional recovery after SCI. SUMMARY: Bioluminescent optogenetic activation of spinal neurons results in accelerated and enhanced locomotor recovery after spinal cord injury in rats.
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Neurons and glia are functionally organized into circuits and higher-order structures that allow the precise information processing required for complex behaviors. To better understand the structure and function of the brain, we must understand synaptic connectivity, action potential generation and propagation, as well as well-orchestrated molecular signaling. Recently, dramatically improved sensors for voltage, intracellular calcium, and neurotransmitters/modulators, combined with advanced microscopy provide new opportunities for in vivo dissection of cellular and circuit activity in awake, behaving animals. This review focuses on the current trends in genetically encoded sensors for molecules and cellular events and their potential applicability to the study of nervous system in health and disease.
Assuntos
Técnicas Biossensoriais/métodos , Química Encefálica , Encéfalo/fisiologia , Proteínas Luminescentes/análise , Imagens com Corantes Sensíveis à Voltagem/métodos , Potenciais de Ação , Animais , Encéfalo/citologia , Cálcio/análise , Cálcio/metabolismo , Humanos , Proteínas Luminescentes/genética , Microscopia de Fluorescência/métodos , Neurônios/citologia , Neurônios/fisiologia , Neurotransmissores/análise , Neurotransmissores/metabolismoRESUMO
Previous work has demonstrated that fusion of a luciferase to an opsin, to create a luminescent opsin or luminopsin, provides a genetically encoded means of manipulating neuronal activity via both chemogenetic and optogenetic approaches. Here we have expanded and refined the versatility of luminopsin tools by fusing an alternative luciferase variant with high light emission, Gaussia luciferase mutant GLucM23, to depolarizing and hyperpolarizing channelrhodopsins with increased light sensitivity. The combination of GLucM23 with Volvox channelrhodopsin-1 produced LMO4, while combining GLucM23 with the anion channelrhodopsin iChloC yielded iLMO4. We found efficient activation of these channelrhodopsins in the presence of the luciferase substrate, as indicated by responses measured in both single neurons and in neuronal populations of mice and rats, as well as by changes in male rat behavior during amphetamine-induced rotations. We conclude that these new luminopsins will be useful for bimodal opto- and chemogenetic analyses of brain function.
Assuntos
Channelrhodopsins , Luciferases , Neurônios/fisiologia , Optogenética/métodos , Potenciais de Ação , Adenoviridae/fisiologia , Animais , Channelrhodopsins/genética , Channelrhodopsins/fisiologia , Feminino , Vetores Genéticos , Células HEK293 , Hipocampo/fisiologia , Humanos , Luciferases/genética , Luciferases/fisiologia , Masculino , Camundongos , Cultura Primária de Células , Ratos Sprague-Dawley , Volvox/genéticaRESUMO
Mice socially isolated during adolescence exhibit behaviors of anxiety, depression and impaired social interaction. Although these behaviors are well documented, very little is known about the associated neurobiological changes that accompany these behaviors. It has been hypothesized that social isolation during adolescence alters the development of the prefrontal cortex, based on similar behavioral abnormalities observed in isolated mice and those with disruption of this structure. To establish relationships between behavior and underlying neurobiological changes in the prefrontal cortex, Thy-1-GFP mice were isolated from weaning until adulthood and compared to group-housed littermates regarding behavior, electrophysiological activity and dendritic morphology. Results indicate an immaturity of dendritic spines in single housed animals, with dendritic spines appearing smaller and thinner. Single housed mice additionally show impaired plasticity through measures of long-term potentiation. Together these findings suggest an altered development and impairment of the prefrontal cortex of these animals underlying their behavioral characteristics.