RESUMEN

The Claustrum/dorsal endopiriform cortex complex (CLA) is an enigmatic brain region with extensive glutamatergic projections to multiple cortical areas. The transcription factor Nurr1 is highly expressed in the CLA, but its role in this region is not understood. By using conditional gene-targeted mice, we show that Nurr1 is a crucial regulator of CLA neuron identity. Although CLA neurons remain intact in the absence of Nurr1, the distinctive gene expression pattern in the CLA is abolished. CLA has been hypothesized to control hallucinations, but little is known of how the CLA responds to hallucinogens. After the deletion of Nurr1 in the CLA, both hallucinogen receptor expression and signaling are lost. Furthermore, functional ultrasound and Neuropixel electrophysiological recordings revealed that the hallucinogenic-receptor agonists' effects on functional connectivity between prefrontal and sensorimotor cortices are altered in Nurr1-ablated mice. Our findings suggest that Nurr1-targeted strategies provide additional avenues for functional studies of the CLA.

Asunto(s)

Claustro , Alucinógenos , Neuronas , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Animales , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Ratones , Alucinógenos/farmacología , Claustro/metabolismo , Neuronas/metabolismo , Masculino , Ratones Noqueados , Ratones Endogámicos C57BL , Corteza Prefrontal/metabolismo , Corteza Prefrontal/fisiología , Corteza Sensoriomotora/metabolismo , Corteza Sensoriomotora/fisiología

RESUMEN

The ability to represent one's own position in relation to cues, goals, or threats is crucial to successful goal-directed behavior. Using optotagging in knock-in rats expressing Cre recombinase in parvalbumin (PV) neurons (PV-Cre rats), we demonstrate cell-type-specific encoding of spatial and movement variables in the medial prefrontal cortex (mPFC) during goal-directed reward seeking. Single neurons encoded the conjunction of the animal's spatial position and the run direction, referred to as the spatial context. The spatial context was most prominently represented by the inhibitory PV interneurons. Movement toward the reward was signified by increased local field potential (LFP) oscillations in the gamma band but this LFP signature was not related to the spatial information in the neuronal firing. The results highlight how spatial information is incorporated into cognitive operations in the mPFC. The presented PV-Cre line opens the door for expanded research approaches in rats.

RESUMEN

Excitatory projections from the lateral hypothalamic area (LHA) to the lateral habenula (LHb) drive aversive responses. We used patch-sequencing (Patch-seq) guided multimodal classification to define the structural and functional heterogeneity of the LHA-LHb pathway. Our classification identified six glutamatergic neuron types with unique electrophysiological properties, molecular profiles and projection patterns. We found that genetically defined LHA-LHb neurons signal distinct aspects of emotional or naturalistic behaviors, such as estrogen receptor 1-expressing (Esr1+) LHA-LHb neurons induce aversion, whereas neuropeptide Y-expressing (Npy+) LHA-LHb neurons control rearing behavior. Repeated optogenetic drive of Esr1+ LHA-LHb neurons induces a behaviorally persistent aversive state, and large-scale recordings showed a region-specific neural representation of the aversive signals in the prelimbic region of the prefrontal cortex. We further found that exposure to unpredictable mild shocks induced a sex-specific sensitivity to develop a stress state in female mice, which was associated with a specific shift in the intrinsic properties of bursting-type Esr1+ LHA-LHb neurons. In summary, we describe the diversity of LHA-LHb neuron types and provide evidence for the role of Esr1+ neurons in aversion and sexually dimorphic stress sensitivity.

Asunto(s)

Habénula , Femenino , Ratones , Animales , Habénula/fisiología , Hipotálamo/fisiología , Área Hipotalámica Lateral , Neuronas/fisiología , Afecto , Vías Nerviosas/fisiología

RESUMEN

Stem cell therapies for Parkinson's disease (PD) have entered first-in-human clinical trials using a set of technically related methods to produce mesencephalic dopamine (mDA) neurons from human pluripotent stem cells (hPSCs). Here, we outline an approach for high-yield derivation of mDA neurons that principally differs from alternative technologies by utilizing retinoic acid (RA) signaling, instead of WNT and FGF8 signaling, to specify mesencephalic fate. Unlike most morphogen signals, where precise concentration determines cell fate, it is the duration of RA exposure that is the key-parameter for mesencephalic specification. This concentration-insensitive patterning approach provides robustness and reduces the need for protocol-adjustments between hPSC-lines. RA-specified progenitors promptly differentiate into functional mDA neurons in vitro, and successfully engraft and relieve motor deficits after transplantation in a rat PD model. Our study provides a potential alternative route for cell therapy and disease modelling that due to its robustness could be particularly expedient when use of autologous- or immunologically matched cells is considered.

Asunto(s)

Enfermedad de Parkinson , Células Madre Pluripotentes , Animales , Diferenciación Celular , Neuronas Dopaminérgicas , Humanos , Mesencéfalo , Enfermedad de Parkinson/terapia , Ratas , Tretinoina/farmacología

RESUMEN

Maps of the nervous system inspire experiments and theories in neuroscience. Advances in molecular biology over the past decades have revolutionized the definition of cell and tissue identity. Spatial transcriptomics has opened up a new era in neuroanatomy, where the unsupervised and unbiased exploration of the molecular signatures of tissue organization will give rise to a new generation of brain maps. We propose that the molecular classification of brain regions on the basis of their gene expression profile can circumvent subjective neuroanatomical definitions and produce common reference frameworks that can incorporate cell types, connectivity, activity, and other modalities. Here we review the technological and conceptual advances made possible by spatial transcriptomics in the context of advancing neuroanatomy and discuss how molecular neuroanatomy can redefine mapping of the nervous system.

Asunto(s)

Neurociencias , Transcriptoma , Animales , Encéfalo , Mapeo Encefálico , Neuroanatomía

RESUMEN

The prefrontal cortex (PFC) is considered to constitute the highest stage of neural integration and to be devoted to representation and production of actions. Studies in primates have laid the foundation for theories regarding the principles of prefrontal function and provided mechanistic insights. The recent surge of studies of the PFC in mice holds promise for evolvement of present theories and development of novel concepts, particularly regarding principles shared across mammals. Here we review recent empirical work on the mouse PFC capitalizing on the experimental toolbox currently privileged to studies in this species. We conclude that this line of research has revealed cellular and structural distinctions of the PFC and neuronal activity with direct relevance to theories regarding the functions of the PFC. We foresee that data-rich mouse studies will be key to shed light on the general prefrontal architecture and mechanisms underlying cognitive aspects of organized actions.

Asunto(s)

Cognición/fisiología , Ratones , Modelos Animales , Vías Nerviosas/fisiología , Neuronas/fisiología , Corteza Prefrontal/fisiología , Animales , Fenómenos Electrofisiológicos , Perfilación de la Expresión Génica , Corteza Prefrontal/anatomía & histología , Corteza Prefrontal/metabolismo , Transcriptoma

RESUMEN

The mouse prefrontal cortex (PFC) encompasses a collection of agranual brain regions in the rostral neocortex and is considered to be critically involved in the neuronal computations underlying intentional behaviors. Flexible behavioral responses demand coordinated integration of sensory inputs with state, goal and memory information in brain-wide neuronal networks. Neuronal oscillations are proposed to provide a temporal scaffold for coordination of neuronal network activity and routing of information. In the present book chapter, we review findings on the role neuronal oscillations in prefrontal functioning, with a specific focus on research in mice. We discuss discoveries pertaining to local prefrontal processing, as well to interactions with other brain regions. We also discuss how the recent discovery of brain-wide respiration-entrained rhythms (RR) warrant re-evaluation of certain findings on slow oscillations (<10Hz) in prefrontal functioning.

Asunto(s)

Neuronas , Corteza Prefrontal , Animales , Ratones , Neuronas/fisiología , Corteza Prefrontal/fisiología

RESUMEN

Inhibitory interneurons expressing parvalbumin (PV) are central to cortical network dynamics, generation of γ oscillations, and cognition. Dysfunction of PV interneurons disrupts cortical information processing and cognitive behavior. Brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase B (trkB) signaling regulates the maturation of cortical PV interneurons but is also implicated in their adult multidimensional functions. Using a novel viral strategy for cell-type-specific and spatially restricted expression of a dominant-negative trkB (trkB.DN), we show that BDNF/trkB signaling is essential to the integrity and maintenance of prefrontal PV interneurons in adult male and female mice. Reduced BDNF/trkB signaling in PV interneurons in the medial prefrontal cortex (mPFC) resulted in deficient PV inhibition and increased baseline local field potential (LFP) activity in a broad frequency band. The altered network activity was particularly pronounced during increased activation of the prefrontal network and was associated with changed dynamics of local excitatory neurons, as well as decreased modulation of the LFP, abnormalities that appeared to generalize across stimuli and brain states. In addition, our findings link reduced BDNF/trkB signaling in prefrontal PV interneurons to increased aggression. Together our investigations demonstrate that BDNF/trkB signaling in PV interneurons in the adult mPFC is essential to local network dynamics and cognitive behavior. Our data provide direct support for the suggested association between decreased trkB signaling, deficient PV inhibition, and altered prefrontal circuitry.SIGNIFICANCE STATEMENT Brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase B (trkB) signaling promotes the maturation of inhibitory parvalbumin (PV) interneurons, neurons central to local cortical dynamics, γ rhythms, and cognition. Here, we used a novel viral approach for reduced BDNF/trkB signaling in PV interneurons in the medial prefrontal cortex (mPFC) to establish the role of BDNF/trkB signaling in adult prefrontal network activities. Reduced BDNF/trkB signaling caused pronounced morphologic alterations, reduced PV inhibition, and deficient prefrontal network dynamics. The altered network activity appeared to manifest across stimuli and brain states and was associated with aberrant local field potential (LFP) activities and increased aggression. The results demonstrate that adult BDNF/trkB signaling is essential to PV inhibition and prefrontal circuit function and directly links BDNF/trkB signaling to network integrity in the adult brain.

Asunto(s)

Interneuronas/metabolismo , Glicoproteínas de Membrana/metabolismo , Red Nerviosa/metabolismo , Parvalbúminas/metabolismo , Corteza Prefrontal/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Factores de Edad , Animales , Femenino , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Parvalbúminas/genética , Proteínas Tirosina Quinasas/genética

RESUMEN

Synchronous activity of cortical inhibitory interneurons expressing parvalbumin (PV) underlies expression of cortical γ rhythms. Paradoxically, deficient PV inhibition is associated with increased broadband γ power in the local field potential. Increased baseline broadband γ is also a prominent characteristic in schizophrenia and a hallmark of network alterations induced by NMDAR antagonists, such as ketamine. Whether enhanced broadband γ is a true rhythm, and if so, whether rhythmic PV inhibition is involved or not, is debated. Asynchronous and increased firing activities are thought to contribute to broadband power increases spanning the γ band. Using male and female mice lacking NMDAR activity specifically in PV neurons to model deficient PV inhibition, we here show that neuronal activity with decreased synchronicity is associated with increased prefrontal broadband γ power. Specifically, reduced spike time precision and spectral leakage of spiking activity because of higher firing rates (spike "contamination") affect the broadband γ band. Desynchronization was evident at multiple time scales, with reduced spike entrainment to the local field potential, reduced cross-frequency coupling, and fragmentation of brain states. Local application of S(+)-ketamine in (control) mice with intact NMDAR activity in PV neurons triggered network desynchronization and enhanced broadband γ power. However, our investigations suggest that disparate mechanisms underlie increased broadband γ power caused by genetic alteration of PV interneurons and ketamine-induced power increases in broadband γ. Our study confirms that enhanced broadband γ power can arise from asynchronous activities and demonstrates that long-term deficiency of PV inhibition can be a contributor.SIGNIFICANCE STATEMENT Brain oscillations are fundamental to the coordination of neuronal activity across neurons and structures. γ oscillations (30-80 Hz) have received particular attention through their association with perceptual and cognitive processes. Synchronous activity of inhibitory parvalbumin (PV) interneurons generates cortical γ oscillation, but, paradoxically, PV neuron deficiency is associated with increases in γ oscillations. We here reconcile this conundrum and show how deficient PV inhibition can lead to increased and asynchronous excitatory firing, contaminating the local field potential and manifesting as increased γ power. Thus, increased γ power does not always reflect a genuine rhythm. Further, we show that ketamine-induced γ increases are caused by separate network mechanisms.

Asunto(s)

Potenciales de Acción/fisiología , Encéfalo/metabolismo , Ritmo Gamma/fisiología , Interneuronas/metabolismo , Red Nerviosa/metabolismo , Animales , Química Encefálica/fisiología , Femenino , Interneuronas/química , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Red Nerviosa/química , Parvalbúminas/análisis , Parvalbúminas/metabolismo , Receptores de N-Metil-D-Aspartato/análisis , Receptores de N-Metil-D-Aspartato/metabolismo

RESUMEN

Injuries to the central nervous system (CNS) are inefficiently repaired. Resident neural stem cells manifest a limited contribution to cell replacement. We have uncovered a latent potential in neural stem cells to replace large numbers of lost oligodendrocytes in the injured mouse spinal cord. Integrating multimodal single-cell analysis, we found that neural stem cells are in a permissive chromatin state that enables the unfolding of a normally latent gene expression program for oligodendrogenesis after injury. Ectopic expression of the transcription factor OLIG2 unveiled abundant stem cell-derived oligodendrogenesis, which followed the natural progression of oligodendrocyte differentiation, contributed to axon remyelination, and stimulated functional recovery of axon conduction. Recruitment of resident stem cells may thus serve as an alternative to cell transplantation after CNS injury.

Asunto(s)

Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Oligodendroglía/fisiología , Regeneración de la Medula Espinal/fisiología , Animales , Astrocitos/fisiología , Axones/fisiología , Linaje de la Célula , Epéndimo/citología , Epéndimo/metabolismo , Ratones , Ratones Endogámicos C57BL , Neurogénesis/genética , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Oligodendroglía/citología , Recuperación de la Función/genética , Recuperación de la Función/fisiología , Remielinización/genética , Remielinización/fisiología , Análisis de la Célula Individual , Traumatismos de la Médula Espinal/fisiopatología , Regeneración de la Medula Espinal/genética

RESUMEN

Electrophysiological recording and optogenetic control of neuronal activity in behaving animals have been integral to the elucidation of how neurons and circuits modulate network activity in the encoding and causation of behavior. However, most current electrophysiological methods require substantial economical investments and prior expertise. Further, the inclusion of optogenetics with electrophysiological recordings in freely moving animals adds complexity to the experimental design. Expansion of the technological repertoire across laboratories, research institutes, and countries, demands open access to high-quality devices that can be built with little prior expertise from easily accessible parts of low cost. We here present an affordable, truly easy-to-assemble micro-drive for electrophysiology in combination with optogenetics in freely moving rodents. The DMCdrive is particularly suited for reliable recordings of neurons and network activities over the course of weeks, and simplify optical tagging and manipulation of neurons in the recorded brain region. The highly functional and practical drive design has been optimized for accurate tetrode movement in brain tissue, and remarkably reduced build time. We provide a complete overview of the drive design, its assembly and use, and proof-of-principle demonstration of recordings paired with cell-type-specific optogenetic manipulations in the prefrontal cortex (PFC) of freely moving transgenic mice and rats.

Asunto(s)

Potenciales de Acción/fisiología , Diseño de Equipo , Neuronas/fisiología , Optogenética/instrumentación , Corteza Prefrontal/fisiología , Animales , Conducta Animal/fisiología , Dependovirus/genética , Dependovirus/metabolismo , Electrodos Implantados , Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neuronas/citología , Optogenética/métodos , Corteza Prefrontal/citología , Impresión Tridimensional , Ratas , Ratas Transgénicas , Técnicas Estereotáxicas , Proteína Fluorescente Roja

RESUMEN

The Supplementary Information is available in the online version of this Publisher Correction.

RESUMEN

The local and long-range connectivity of cortical neurons are considered instrumental to the functional repertoire of the cortical region in which they reside. In cortical networks, distinct cell types build local circuit structures enabling computational operations. Computations in the medial prefrontal cortex (mPFC) are thought to be central to cognitive operation, including decision-making and memory. We used a retrograde trans-synaptic rabies virus system to generate brain-wide maps of the input to excitatory neurons as well as three inhibitory interneuron subtypes in the mPFC. On the global scale the input patterns were found to be mainly cell type independent, with quantitative differences in key brain regions, including the basal forebrain. Mapping of the local mPFC network revealed high connectivity between the different subtypes of interneurons. The connectivity mapping gives insight into the information that the mPFC processes and the structural architecture underlying the mPFC's unique functions.

Asunto(s)

Encéfalo/citología , Interneuronas/citología , Corteza Prefrontal/citología , Animales , Atlas como Asunto , Neuronas Colinérgicas/citología , Femenino , Vectores Genéticos , Masculino , Ratones Endogámicos C57BL , Vías Nerviosas/citología , Técnicas de Trazados de Vías Neuroanatómicas , Virus de la Rabia/genética

RESUMEN

Encoding and predicting aversive events are critical functions of circuits that support survival and emotional well-being. Maladaptive circuit changes in emotional valence processing can underlie the pathophysiology of affective disorders. The lateral habenula (LHb) has been linked to aversion and mood regulation through modulation of the dopamine and serotonin systems. We have defined the identity and function of glutamatergic (Vglut2) control of the LHb, comparing the role of inputs originating in the globus pallidus internal segment (GPi), and lateral hypothalamic area (LHA), respectively. We found that LHb-projecting LHA neurons, and not the proposed GABA/glutamate co-releasing GPi neurons, are responsible for encoding negative value. Monosynaptic rabies tracing of the presynaptic organization revealed a predominantly limbic input onto LHA Vglut2 neurons, while sensorimotor inputs were more prominent onto GABA/glutamate co-releasing GPi neurons. We further recorded the activity of LHA Vglut2 neurons, by imaging calcium dynamics in response to appetitive versus aversive events in conditioning paradigms. LHA Vglut2 neurons formed activity clusters representing distinct reward or aversion signals, including a population that responded to mild foot shocks and predicted aversive events. We found that the LHb-projecting LHA Vglut2 neurons encode negative valence and rapidly develop a prediction signal for negative events. These findings establish the glutamatergic LHA-LHb circuit as a critical node in value processing.

Asunto(s)

Reacción de Prevención/fisiología , Habénula/fisiología , Hipotálamo/fisiología , Afecto/fisiología , Animales , Dopamina/metabolismo , Fármacos actuantes sobre Aminoácidos Excitadores/metabolismo , Globo Pálido/fisiología , Ácido Glutámico/metabolismo , Habénula/metabolismo , Área Hipotalámica Lateral/fisiología , Hipotálamo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vías Nerviosas/fisiología , Neuronas/fisiología , Recompensa

RESUMEN

CNS injury often severs axons. Scar tissue that forms locally at the lesion site is thought to block axonal regeneration, resulting in permanent functional deficits. We report that inhibiting the generation of progeny by a subclass of pericytes led to decreased fibrosis and extracellular matrix deposition after spinal cord injury in mice. Regeneration of raphespinal and corticospinal tract axons was enhanced and sensorimotor function recovery improved following spinal cord injury in animals with attenuated pericyte-derived scarring. Using optogenetic stimulation, we demonstrate that regenerated corticospinal tract axons integrated into the local spinal cord circuitry below the lesion site. The number of regenerated axons correlated with improved sensorimotor function recovery. In conclusion, attenuation of pericyte-derived fibrosis represents a promising therapeutic approach to facilitate recovery following CNS injury.

Asunto(s)

Cicatriz/patología , Traumatismos de la Médula Espinal/patología , Animales , Axones/fisiología , Axones/efectos de la radiación , Modelos Animales de Enfermedad , Potenciales Evocados/efectos de la radiación , Matriz Extracelular/metabolismo , Fibrosis , Luz , Ratones , Ratones Transgénicos , Pericitos/citología , Pericitos/metabolismo , Estimulación Luminosa , Tractos Piramidales/fisiología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Recuperación de la Función , Regeneración , Corteza Sensoriomotora/fisiología , Traumatismos de la Médula Espinal/fisiopatología

RESUMEN

To deconstruct the architecture and function of brain circuits, it is necessary to generate maps of neuronal connectivity and activity on a whole-brain scale. New methods now enable large-scale mapping of the mouse brain at cellular and subcellular resolution. We developed a framework to automatically annotate, analyze, visualize and easily share whole-brain data at cellular resolution, based on a scale-invariant, interactive mouse brain atlas. This framework enables connectivity and mapping projects in individual laboratories and across imaging platforms, as well as multiplexed quantitative information on the molecular identity of single neurons. As a proof of concept, we generated a comparative connectivity map of five major neuron types in the corticostriatal circuit, as well as an activity-based map to identify hubs mediating the behavioral effects of cocaine. Thus, this computational framework provides the necessary tools to generate brain maps that integrate data from connectivity, neuron identity and function.

Asunto(s)

Mapeo Encefálico , Encéfalo/citología , Red Nerviosa/fisiología , Vías Nerviosas/fisiología , Neuronas/fisiología , Animales , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Genes Inmediatos-Precoces/fisiología , Glutamato Descarboxilasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Masculino , Ratones Transgénicos , Actividad Motora , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuropéptido Y/metabolismo , Parvalbúminas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

In the version of this article initially published online, Daniel Fürth was not listed as a corresponding author. The error has been corrected in the print, PDF and HTML versions of this article.

RESUMEN

During evolution, the prefrontal region grew in size relative to the rest of the cortex. It reached its largest extent in the human brain, where it constitutes 30% of the total cortical area. This growth was accompanied by phylogenetic differentiation of the cortical areas. It has been argued that the human brain holds prefrontal regions that are both qualitatively and functionally unique. Present-day neuroscientists studying the prefrontal cortex increasingly use mice. An important goal is to reveal how the prefrontal cortex enables complex behavior. However, the prefrontal cortex still lacks a conclusive definition. The structure and function of this brain area across species remain unresolved. This state of affairs is often overlooked, warranting renewed focus on what the prefrontal cortex is and does.

Asunto(s)

Corteza Prefrontal/anatomía & histología , Animales , Evolución Biológica , Humanos , Ratones , Tamaño de los Órganos , Ratas

RESUMEN

While signatures of attention have been extensively studied in sensory systems, the neural sources and computations responsible for top-down control of attention are largely unknown. Using chronic recordings in mice, we found that fast-spiking parvalbumin (FS-PV) interneurons in medial prefrontal cortex (mPFC) uniformly show increased and sustained firing during goal-driven attentional processing, correlating to the level of attention. Elevated activity of FS-PV neurons on the timescale of seconds predicted successful execution of behavior. Successful allocation of attention was characterized by strong synchronization of FS-PV neurons, increased gamma oscillations, and phase locking of pyramidal firing. Phase-locked pyramidal neurons showed gamma-phase-dependent rate modulation during successful attentional processing. Optogenetic silencing of FS-PV neurons deteriorated attentional processing, while optogenetic synchronization of FS-PV neurons at gamma frequencies had pro-cognitive effects and improved goal-directed behavior. FS-PV neurons thus act as a functional unit coordinating the activity in the local mPFC circuit during goal-driven attentional processing.

Asunto(s)

Atención , Neuronas/citología , Corteza Prefrontal/citología , Animales , Conducta Animal , Cognición , Ritmo Gamma , Ratones , Optogenética , Parvalbúminas/metabolismo , Corteza Prefrontal/fisiología

RESUMEN

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼ 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure-function relationships of the light-gated pore.

Asunto(s)

Reacción de Prevención/fisiología , Cloruros/metabolismo , Activación del Canal Iónico/fisiología , Optogenética , Rodopsina/química , Potenciales de Acción , Secuencia de Aminoácidos , Animales , Arginina/química , Reacción de Prevención/efectos de la radiación , Complejo Nuclear Basolateral/fisiología , Complejo Nuclear Basolateral/efectos de la radiación , Células Cultivadas , Dependovirus/genética , Electrochoque , Miedo , Tecnología de Fibra Óptica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Células HEK293 , Hipocampo/citología , Histidina/química , Humanos , Concentración de Iones de Hidrógeno , Activación del Canal Iónico/efectos de la radiación , Masculino , Memoria/fisiología , Memoria/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neuronas/fisiología , Conformación Proteica , Ratas , Ratas Sprague-Dawley , Rodopsina/metabolismo , Rodopsina/efectos de la radiación , Alineación de Secuencia , Área Tegmental Ventral/fisiología