RESUMEN
Fluorescent and non-fluorescent neural tract tracers enable the investigation of neural pathways in both peripheral and central nervous systems in laboratory animals demonstrating images with high resolution and great anatomic precision. Anterograde and retrograde viral tracers are important cutting-edge tools for neuroanatomical mapping. The optogenetic consists of an advanced alternative for in vivo neural tract tracing procedures, fundamentally considering the possibility to dissect and modulate pathways either exciting or inhibiting neural circuits in laboratory animals. The neurotractography by diffusion tensor imaging in vivo procedures enables the study of neural pathways in humans with reasonable accuracy. Here we describe the procedure of classical anatomic neural tract tracing and modern optogenetic technique performed in anima vili in addition to different diffusion tensor neurotractography performed in anima nobili.
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Imagen de Difusión Tensora , Optogenética , Optogenética/métodos , Animales , Imagen de Difusión Tensora/métodos , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Vías Nerviosas , Encéfalo/diagnóstico por imagen , Encéfalo/fisiología , Encéfalo/metabolismo , Trazadores del Tracto Neuronal , Humanos , RatonesRESUMEN
In the present study, we conducted a scoping review to provide an overview of the existing literature on the carbocyanine dye DiI, in human neuroanatomical tract tracing. The PubMed, Scopus, and Web of Science databases were systematically searched. We identified 61 studies published during the last three decades. While studies incorporated specimens across human life from the embryonic stage onwards, the majority of studies focused on adult human tissue. Studies that utilized peripheral nervous system (PNS) tissue were a minority, with the majority of studies focusing on the central nervous system (CNS). The most common topic of interest in previous tract tracing investigations was the connectivity of the visual pathway. DiI crystals were more commonly applied. Nevertheless, several studies utilized DiI in a paste or dissolved form. The maximum tracing distance and tracing speed achieved was, respectively, 70 mm and 1 mm/h. We identified studies that focused on optimizing tracing efficacy by varying parameters such as fixation, incubation temperature, dye re-application, or the application of electric fields. Additional studies aimed at broadening the scope of DiI use by assessing the utility of archival tissue and compatibility of tissue clearing in DiI applications. A combination of DiI tracing and immunohistochemistry in double-labeling studies have been shown to provide the means for assessing connectivity of phenotypically defined human CNS and PNS neuronal populations.
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Técnicas de Trazados de Vías Neuroanatómicas , Humanos , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Carbocianinas/química , Sistema Nervioso Central , Sistema Nervioso Periférico , Colorantes Fluorescentes/químicaRESUMEN
Purpose: After the lateral geniculate nucleus, the superior colliculus is the richest target of retinal projections in primates. Hubel et al. used tritium autoradiography to show that axon terminals emanating from one eye form irregular columns in the stratum griseum superficiale. Unlabeled gaps were thought to be filled by the other eye, but this assumption was never tested directly. Methods: Experiments were performed in two normal macaques. In monkey 1, [3H]proline was injected into the left eye and the pattern of radiolabeling was examined in serial cross-sections through the entire superior colliculus. In monkey 2, cholera toxin subunit B conjugated to Alexa 488 was injected into the right eye and cholera toxin subunit B - Alexa 594 was injected into the left eye. The two fluorescent labels were compared in a reconstruction of the superior colliculus prepared from serial sections. Results: In monkey 1, irregular columns of axon terminals were present in the superficial grey. The projection from the peripheral retina was stronger than the projection from the macula. In monkey 2, the two fluorescent Alexa tracers mainly interdigitated: a conspicuous gap in one label was usually filled by a clump of the other label. There was also partial laminar segregation of ocular inputs. In the far peripheral field representation, the contralateral eye's input generally terminated closer to the tectal surface. In the midperiphery the eyes switched, bringing the ipsilateral input nearer the surface. Conclusions: Direct retinal input to the macaque superior colliculus is segregated into alternating columns and strata, despite the fact that tectal cells respond robustly to stimulation of either eye.
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Axones/fisiología , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Neuronas Retinianas/fisiología , Colículos Superiores/anatomía & histología , Vías Visuales/anatomía & histología , Animales , Autorradiografía , Colorantes Fluorescentes/administración & dosificación , Macaca mulatta , Masculino , Prolina/administración & dosificación , Tritio/administración & dosificaciónRESUMEN
INTRODUCTION: The connections of the pedunculopontine nucleus (PPN) with motor areas of the central nervous system (CNS) are well described in the literature, in contrast relations with non-motor areas are lacking. Thus, the aim of the present study is to define the non-motor connections of the PPN in rats using the fluoro-gold (FG) tracer and compare the presence of these connections in healthy human adults using diffusion tensor tractography (DTI). MATERIALS AND METHODS: We injected FG into the PPN of 12 rats. The non-motor connections of the PPN with cortical, subcortical, and brainstem structures were documented. The non-motor connections of the rats were compared with the DTI obtained from 35 healthy adults. RESULTS: The results of the tract-tracing study in the rat showed that the PPN was connected to non-motor cortical (cingulate, somatosensory, visual, auditory, medial frontal cortices), subcortical (amygdala, hypothalamus, thalamus, habenular, and bed nucleus of stria terminalis), and brainstem (medullary reticular, trigeminal spinal, external cuneate, pontine reticular, vestibular, superior and inferior colliculus, locus ceruleus, periaqueductal gray, parabrachial, dorsal raphe, pretectal, lateral lemniscus nuclei, and the contralateral PPN) structures. The DTI obtained from healthy adults showed similar PPN non-motor connections as in rats. CONCLUSION: Understanding the connections of the PPN with non-motor cortical, subcortical, and brainstem areas of the CNS will enrich our knowledge of its contribution in various circuits and the areas that PPN activity can influence. Further, it will provide insight into the role of Parkinson's disease and related disorders and explain the non-motor complications which occur subsequent to deep brain stimulation (DBS) of the PPN.
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Vías Nerviosas/anatomía & histología , Núcleo Tegmental Pedunculopontino/anatomía & histología , Adulto , Animales , Imagen de Difusión Tensora/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Ratas , Ratas Wistar , EstilbamidinasRESUMEN
The theory holds that the anterior pituitary in mammals receives humoral regulation. Previous studies have reported that the pars distalis of the anterior pituitary of several mammalian species contains substance P-, calcitonin gene-related peptide (CGRP)-, and galanin-like immunoreactive nerve fibers, but the origins of these nerve fibers are unclear. Removal of the pituitary gland, also called hypophysectomy, involves methods that access the pituitary gland via the transauricular or parapharyngeal pathways. However, these methods are not applicable for viral tracer injection to investigate the innervation of the anterior pituitary. The transauricular technique leads to inaccuracies in locating the pituitary gland, while the parapharyngeal approach causes high mortality in animals. Here, we introduce a protocol that accesses the pituitary gland in the rat via the transsphenoidal pathway. This method imitates surgical manipulations such as endotracheal intubation and sphenoid bone drilling, which involve the use of custom-made devices. Using the transsphenoidal pathway greatly improves the survival rate of rats because no additional dissection of blood vessels and nerves is required. Moreover, the pituitary gland can be viewed clearly and directly during the operation, making it possible to accurately inject pseudorabies virus (PRV) 152-expressing enhanced green fluorescent protein (EGFP) into the anterior or posterior pituitary, respectively. After injecting PRV 152 into the anterior pituitary, we found no evidence of direct innervation of the anterior pituitary in the rat brain. However, PRV 152 injection into the posterior pituitary revealed retrograde transneuronal cell bodies in many brain areas, including the CA1 field of the hippocampus, the basolateral amygdaloid nucleus, posterior part (BLP), the arcuate hypothalamic nucleus (Arc), the dorsal portion of the dorsomedial hypothalamic nucleus (DMD), the suprachiasmatic nucleus (SCh), and the subfornical organ (SFO). In the present study, we provide a description of a possible model of hypophysectomy or pituitary injection, and identify brain regions involved in regulating the rat pituitary gland using transneuronal retrograde cell body labeling with PRV.
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Vectores Genéticos/administración & dosificación , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Neuronas/citología , Hipófisis/citología , Hipófisis/inervación , Animales , Proteínas Fluorescentes Verdes/fisiología , Herpesvirus Suido 1/fisiología , Masculino , Vías Nerviosas/citología , Ratas Sprague-Dawley , Hueso Esfenoides/cirugíaRESUMEN
Herpes simplex virus type 1 (HSV-1) has great potential to be applied as a viral tool for gene delivery or oncolysis. The broad infection tropism of HSV-1 makes it a suitable tool for targeting many different cell types, and its 150 kb double-stranded DNA genome provides great capacity for exogenous genes. Moreover, the features of neuron infection and neuron-to-neuron spread also offer special value to neuroscience. HSV-1 strain H129, with its predominant anterograde transneuronal transmission, represents one of the most promising anterograde neuronal circuit tracers to map output neuronal pathways. Decades of development have greatly expanded the H129-derived anterograde tracing toolbox, including polysynaptic and monosynaptic tracers with various fluorescent protein labeling. These tracers have been applied to neuroanatomical studies, and have contributed to revealing multiple important neuronal circuits. However, current H129-derived tracers retain intrinsic drawbacks that limit their broad application, such as yet-to-be improved labeling intensity, potential nonspecific retrograde labeling, and high toxicity. The biological complexity of HSV-1 and its insufficiently characterized virological properties have caused difficulties in its improvement and optimization as a viral tool. In this review, we focus on the current H129-derived viral tracers and highlight strategies in which future technological development can advance its use as a tool.
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Herpesvirus Humano 1/metabolismo , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Animales , Ingeniería Genética/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Herpesvirus Humano 1/genética , Neuronas/metabolismo , Sinapsis/metabolismoRESUMEN
Viral tracers are important tools for neuroanatomical mapping and genetic payload delivery. Genetically modified viruses allow for cell-type-specific targeting and overcome many limitations of non-viral tracers. Here, we summarize the viruses that have been developed for neural circuit mapping, and we provide a primer on currently applied anterograde and retrograde viral tracers with practical guidance on experimental uses. We also discuss and highlight key technical and conceptual considerations for developing new safer and more effective anterograde trans-synaptic viral vectors for neural circuit analysis in multiple species.
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Conectoma/métodos , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Sinapsis/fisiología , Virus/genética , Animales , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Sinapsis/metabolismo , Virus/metabolismoRESUMEN
The cerebral cortex and cerebellum both play important roles in sensorimotor processing, however, precise connections between these major brain structures remain elusive. Using anterograde mono-trans-synaptic tracing, we elucidate cerebrocerebellar pathways originating from primary motor, sensory, and association cortex. We confirm a highly organized topography of corticopontine projections in mice; however, we found no corticopontine projections originating from primary auditory cortex and detail several potential extra-pontine cerebrocerebellar pathways. The cerebellar hemispheres were the major target of resulting disynaptic mossy fiber terminals, but we also found at least sparse cerebrocerebellar projections to every lobule of the cerebellum. Notably, projections originating from association cortex resulted in less laterality than primary sensory/motor cortices. Within molecularly defined cerebellar modules we found spatial overlap of mossy fiber terminals, originating from functionally distinct cortical areas, within crus I, paraflocculus, and vermal regions IV/V and VI - highlighting these regions as potential hubs for multimodal cortical influence.
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Cerebelo/anatomía & histología , Cerebro/anatomía & histología , Vías Nerviosas/anatomía & histología , Animales , Tronco Encefálico/anatomía & histología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Puente/anatomía & histologíaRESUMEN
Neural activity in the corticothalamic network is crucial for sensation, memory, decision, and action. Nevertheless, a systematic characterization of corticothalamic functional connectivity has not been achieved. Here, we developed a high throughput method to systematically map functional connections from the dorsal cortex to the thalamus in awake mice by combing optogenetic inactivation with multi-channel recording. Cortical inactivation resulted in a rapid reduction of thalamic activity, revealing topographically organized corticothalamic excitatory inputs. Cluster analysis showed that groups of neurons within individual thalamic nuclei exhibited distinct dynamics. The effects of inactivation evolved with time and were modulated by behavioral states. Furthermore, we found that a subset of thalamic neurons received convergent inputs from widespread cortical regions. Our results present a framework for collecting, analyzing, and presenting large electrophysiological datasets with region-specific optogenetic perturbations and serve as a foundation for further investigation of information processing in the corticothalamic pathway.
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Conectoma , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Optogenética/métodos , Corteza Somatosensorial/citología , Tálamo/citología , Animales , Femenino , Masculino , Ratones , Conducción Nerviosa , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Neuronas/citología , Neuronas/metabolismo , Neuronas/fisiología , Corteza Somatosensorial/fisiología , Tálamo/fisiologíaRESUMEN
We describe our connectomics pipeline for processing anterograde tracer injection data for the brain of the common marmoset (Callithrix jacchus). Brain sections were imaged using a batch slide scanner (NanoZoomer 2.0-HT) and we used artificial intelligence to precisely segment the tracer signal from the background in the fluorescence images. The shape of each brain was reconstructed by reference to a block-face and all data were mapped into a common 3D brain space with atlas and 2D cortical flat map. To overcome the effect of using a single template atlas to specify cortical boundaries, brains were cyto- and myelo-architectonically annotated to create individual 3D atlases. Registration between the individual and common brain cortical boundaries in the flat map space was done to absorb the variation of each brain and precisely map all tracer injection data into one cortical brain space. We describe the methodology of our pipeline and analyze the accuracy of our tracer segmentation and brain registration approaches. Results show our pipeline can successfully process and normalize tracer injection experiments into a common space, making it suitable for large-scale connectomics studies with a focus on the cerebral cortex.
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Inteligencia Artificial , Encéfalo/citología , Conectoma/métodos , Imagen por Resonancia Magnética , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Neuronas/citología , Animales , Atlas como Asunto , Callithrix , Vías Nerviosas/citologíaRESUMEN
OBJECTIVE: The dual-pathway model has been proposed to explain the heterogeneity in symptoms of attention deficit hyperactivity disorder (ADHD) by two independent psychological pathways based on distinct brain circuits. The authors sought to test whether the hypothesized cognitive and motivational pathways have separable neural correlates. METHODS: In a longitudinal community-based cohort of 1,963 adolescents, the neuroanatomical correlates of ADHD were identified by a voxel-wise association analysis and then validated using an independent clinical sample (99 never-medicated patients with ADHD, 56 medicated patients with ADHD, and 267 healthy control subjects). The cognitive and motivational pathways were assessed by neuropsychological tests of working memory, intrasubject variability, stop-signal reaction time, and delay discounting. The associations were tested between the identified neuroanatomical correlates and both ADHD symptoms 2 years later and the polygenic risk score for ADHD. RESULTS: Gray matter volumes of both a prefrontal cluster and a posterior occipital cluster were negatively associated with inattention. Compared with healthy control subjects, never-medicated patients, but not medicated patients, had significantly lower gray matter volumes in these two clusters. Working memory and intrasubject variability were associated with the posterior occipital cluster, and delay discounting was independently associated with both clusters. The baseline gray matter volume of the posterior occipital cluster predicted the inattention symptoms in a 2-year follow-up and was associated with the genetic risk for ADHD. CONCLUSIONS: The dual-pathway model has both shared and separable neuroanatomical correlates, and the shared correlate in the occipital cortex has the potential to serve as an imaging trait marker of ADHD, especially the inattention symptom domain.
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Trastorno por Déficit de Atención con Hiperactividad , Síntomas Conductuales , Cognición/fisiología , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Lóbulo Occipital , Corteza Prefrontal , Adolescente , Trastorno por Déficit de Atención con Hiperactividad/diagnóstico , Trastorno por Déficit de Atención con Hiperactividad/fisiopatología , Trastorno por Déficit de Atención con Hiperactividad/psicología , Síntomas Conductuales/diagnóstico , Síntomas Conductuales/fisiopatología , Ciencias Bioconductuales , Femenino , Sustancia Gris/diagnóstico por imagen , Sustancia Gris/patología , Humanos , Estudios Longitudinales , Masculino , Motivación/fisiología , Neuroimagen/métodos , Pruebas Neuropsicológicas , Lóbulo Occipital/diagnóstico por imagen , Lóbulo Occipital/patología , Tamaño de los Órganos , Corteza Prefrontal/diagnóstico por imagen , Corteza Prefrontal/patología , Medición de Riesgo/métodosRESUMEN
Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.
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Clonación Molecular , Proteínas Luminiscentes/genética , Optogenética/métodos , Optogenética/tendencias , Transducción de Señal/genética , Animales , Técnicas Biosensibles , Rastreo Celular/métodos , Rastreo Celular/tendencias , Clonación Molecular/métodos , Humanos , Proteínas Luminiscentes/metabolismo , Imagen Molecular/métodos , Imagen Molecular/tendencias , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Técnicas de Trazados de Vías Neuroanatómicas/tendenciasRESUMEN
Neurotropic viral transsynaptic tracing is an increasingly powerful technique for dissecting the structure and function of neural circuits. Herpes simplex virus type 1 strain H129 has been widely used as an anterograde tracer. However, HSV tracers still have several shortcomings, including high toxicity, low sensitivity and non-specific retrograde labeling. Here, we aimed to construct high-brightness HSV anterograde tracers by increasing the expression of exogenous genes carried by H129 viruses. Using a Trojan horse-like strategy, a HSV/AAV (adeno-associated virus) chimaera termed H8 was generated to enhance the expression of a fluorescent marker. In vitro and in vivo assays showed that the exogenous gene was efficiently replicated and amplified by the synergism of the HSV vector and introduced AAV replication system. H8 reporting fluorescence was brighter than that of currently available H129 tracers, and H8 could be used for fast and effective anterograde tracing without additional immunostaining. These results indicated that foreign gene expression in HSV tracers could be enhanced by integrating HSV with AAV replication system. This approach may be useful as a general enhanced expression strategy for HSV-based tracing tools or gene delivery vectors.
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Transporte Axonal/fisiología , Encéfalo/citología , Virus Defectuosos/fisiología , Dependovirus/fisiología , Proteínas Fluorescentes Verdes/análisis , Virus Helper/fisiología , Herpesvirus Humano 1/fisiología , Vías Nerviosas/ultraestructura , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Trazadores del Tracto Neuronal/análisis , Neuronas/ultraestructura , Virus Reordenados/fisiología , Animales , Línea Celular , Núcleo Celular/virología , Virus Defectuosos/genética , Dependovirus/genética , Genes Reporteros , Genes Sintéticos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Virus Helper/genética , Herpesvirus Humano 1/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/virología , Virus Reordenados/genética , Proteinas del Complejo de Replicasa Viral/genética , Replicación ViralRESUMEN
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated 18F positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping.
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Drogas de Diseño , Radioisótopos de Flúor/análisis , Trazadores del Tracto Neuronal/análisis , Animales , Encéfalo , Clozapina/análogos & derivados , Clozapina/química , Células HEK293 , Haplorrinos , Humanos , Ligandos , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Trazadores del Tracto Neuronal/química , Tomografía de Emisión de Positrones/métodos , RoedoresRESUMEN
BACKGROUND: Movement performance depends on the synaptic interactions generated by coherent parallel sensorimotor cortical outputs to different downstream targets. The major outputs of the neocortex to subcortical structures are driven by pyramidal tract neurons (PTNs) located in layer 5B. One of the main targets of PTNs is the spinal cord through the corticospinal (CS) system, which is formed by a complex collection of distinct CS circuits. However, little is known about intracortical synaptic interactions that originate CS commands and how different populations of CS neurons are functionally organized. To further understand the functional organization of the CS system, we analyzed the activity of unambiguously identified CS neurons projecting to different zones of the same spinal cord segment using two-photon calcium imaging and retrograde neuronal tracers. RESULTS: Sensorimotor cortex slices obtained from transgenic mice expressing GCaMP6 funder the Thy1 promoter were used to analyze the spontaneous calcium transients in layer 5 pyramidal neurons. Distinct subgroups of CS neurons projecting to dorsal horn and ventral areas of the same segment show more synchronous activity between them than with other subgroups. CONCLUSIONS: The results indicate that CS neurons projecting to different spinal cord zones segregated into functional ensembles depending on their hodology, suggesting that a modular organization of CS outputs controls sensorimotor behaviors in a coordinated manner.
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Conectoma , Tractos Piramidales/fisiología , Médula Espinal/fisiología , Animales , Calcio/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Ratones , Ratones Transgénicos , Corteza Motora/metabolismo , Corteza Motora/fisiología , Vías Nerviosas/metabolismo , Vías Nerviosas/fisiología , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Neuronas/fisiología , Tractos Piramidales/metabolismo , Médula Espinal/metabolismoRESUMEN
Monosynaptic tracing using deletion-mutant rabies virus allows whole-brain mapping of neurons that are directly presynaptic to a targeted population of neurons. The most common and robust way of implementing it is to use Cre mouse lines in combination with Cre-dependent adeno-associated viral vectors for expression of the required genes in the targeted neurons before subsequent injection of the rabies virus. Here we present a step-by-step protocol for performing such experiments using first-generation (ΔG) rabies viral vectors.
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Encéfalo/metabolismo , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Trazadores del Tracto Neuronal , Neuronas/metabolismo , Virus de la RabiaRESUMEN
Reorganization of residual descending motor circuits underlies poststroke recovery. We previously clarified a causal relationship between the cortico-rubral tract and intensive limb use-induced functional recovery after internal capsule hemorrhage (ICH). However, other descending tracts, such as the cortico-reticular tract, might also be involved in rehabilitation-induced compensation. To investigate whether rehabilitation-induced recovery after ICH involves a shift in the compensatory circuit from the cortico-rubral tract to the cortico-reticular tract, we established loss of function of the cortico-rubral tract or/and cortico-reticular tract using two sets of viral vectors comprising the Tet-on system and designer receptors exclusively activated by the designer drug system. We used an ICH model that destroyed almost 60% of the corticofugal fibers. Anterograde tracing in rehabilitated rats revealed abundant sprouting of axons from the motor cortex in the red nucleus but not in the medullary reticular formation during the early phase of recovery. This primary contribution of the cortico-rubral tract was demonstrated by its selective blockade, whereas selective cortico-reticular tract silencing had little effect. Interestingly, cortico-rubral tract blockade from the start of rehabilitation induced an obvious increase of axon sprouting in the reticular formation with substantial functional recovery. Additional cortico-reticular tract silencing under the cortico-rubral tract blockade significantly worsened the recovered forelimb function. Furthermore, the alternative recruitment of the cortico-reticular tract was gradually induced by intensive limb use under cortico-rubral tract blockade, in which cortico-reticular tract silencing caused an apparent motor deficit. These findings indicate that individual cortico-brainstem pathways have dynamic compensatory potency to support rehabilitative functional recovery after ICH.SIGNIFICANCE STATEMENT This study aimed to clarify the interaction between the cortico-rubral and the cortico-reticular tract during intensive rehabilitation and functional recovery after capsular stroke. Pathway-selective disturbance by two sets of viral vectors revealed that the cortico-rubral tract was involved in rehabilitation-induced recovery of forelimb function from an early phase after internal capsule hemorrhage, but that the cortico-reticular tract was not. The sequential disturbance of both tracts revealed that the cortico-reticular tract was recruited and involved in rehabilitation-induced recovery when the cortico-rubral tract failed to function. Our data demonstrate a dynamic compensatory action of individual cortico-brainstem pathways for recovery through poststroke rehabilitation.
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Tronco Encefálico/fisiología , Corteza Motora/fisiología , Tractos Piramidales/fisiología , Recuperación de la Función/fisiología , Núcleo Rojo/fisiología , Accidente Cerebrovascular/fisiopatología , Animales , Tronco Encefálico/química , Tronco Encefálico/patología , Masculino , Corteza Motora/química , Corteza Motora/patología , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Tractos Piramidales/química , Tractos Piramidales/patología , Ratas , Ratas Wistar , Núcleo Rojo/química , Núcleo Rojo/patología , Accidente Cerebrovascular/patologíaRESUMEN
An understanding of how the brain processes information requires knowledge of its underlying wiring diagrams, as well as insights into the relationship between circuit architecture and physiological function. Notably, rabies virus based single-cell genetic manipulations that can facilitate an experimental link between physiology and genetics have recently advanced the field of systems neuroscience. It allows capturing the synaptic and the anatomical receptive fields of individual neurons. Recently, the methodological portfolio has been upgraded by two novel approaches, single cell electroporation with genetically encoded Ca2+ sensors allowing for functionalized transsynaptic tracing and single cell targeted virus stamping. Especially the development of virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types with different viruses at once, both in vitro and in vivo. Here we will summarize the latest developments in this rapidly moving field and provide a perspective for automated, quantitative analysis of single cell initiated connectomes.
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Red Nerviosa , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Neuronas , Neurociencias/métodos , Virus de la Rabia , AnimalesRESUMEN
Herpes simplex virus type 1 H129 strain has been widely used as a useful anterograde neuronal circuit tracing tool. However, whether H129 is a rigorous anterograde tracer and undergoes anterograde-only spreading are questions of significant interest. In the present study, we evaluated the retrograde labeling efficiency of H129 using a TK and ICP34.5 dual deleted H129 recombinant (named as H306) which was replication-deficient in non-dividing postmitotic neurons. The novel tracer was tested in vitro and in vivo for evaluating its invasion properties and tracing capacities. The results demonstrated that H306 could efficiently label the neurons following intracerebral injection. Notably, H306 could also efficiently infect upstream innervating neurons through axon terminal uptake and displayed obvious retrograde labeling phenotype, regardless of 3 days or 10 days of tracing. The data implied that replication-competent, trans-multisynaptic H129 tracing results might be a mixed neural networks from two types of starter cells, because the retrogradely infected neurons would also replicate H129 and spread virus anterogradely through their axon collaterals (ectopic starter sites), as the local infected neurons in the injection site (true starter site). Therefore, the interpretation of the anterogradely tracing neural networks by current H129 tools at longer post-inoculation intervals need to be cautious, and effective modification strategies are needed to avoid or block the axon terminal invasion process of H129, which is important for rigorous anterograde H129 tracer.