RESUMO
Recent large-scale collaborations are generating major surveys of cell types and connections in the mouse brain, collecting large amounts of data across modalities, spatial scales, and brain areas. Successful integration of these data requires a standard 3D reference atlas. Here, we present the Allen Mouse Brain Common Coordinate Framework (CCFv3) as such a resource. We constructed an average template brain at 10 µm voxel resolution by interpolating high resolution in-plane serial two-photon tomography images with 100 µm z-sampling from 1,675 young adult C57BL/6J mice. Then, using multimodal reference data, we parcellated the entire brain directly in 3D, labeling every voxel with a brain structure spanning 43 isocortical areas and their layers, 329 subcortical gray matter structures, 81 fiber tracts, and 8 ventricular structures. CCFv3 can be used to analyze, visualize, and integrate multimodal and multiscale datasets in 3D and is openly accessible (https://atlas.brain-map.org/).
Assuntos
Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Encéfalo/fisiologia , Animais , Atlas como Assunto , Mapeamento Encefálico/métodos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Although there have been major advances in elucidating the functional biology of the human brain, relatively little is known of its cellular and molecular organization. Here we report a large-scale characterization of the expression of â¼1,000 genes important for neural functions by in situ hybridization at a cellular resolution in visual and temporal cortices of adult human brains. These data reveal diverse gene expression patterns and remarkable conservation of each individual gene's expression among individuals (95%), cortical areas (84%), and between human and mouse (79%). A small but substantial number of genes (21%) exhibited species-differential expression. Distinct molecular signatures, comprised of genes both common between species and unique to each, were identified for each major cortical cell type. The data suggest that gene expression profile changes may contribute to differential cortical function across species, and in particular, a shift from corticosubcortical to more predominant corticocortical communications in the human brain.
Assuntos
Perfilação da Expressão Gênica , Neocórtex/metabolismo , Lobo Temporal/metabolismo , Córtex Visual/metabolismo , Adulto , Animais , Regulação da Expressão Gênica , Humanos , Camundongos , Neocórtex/citologia , Neurônios/metabolismo , Especificidade da Espécie , Lobo Temporal/citologia , Córtex Visual/citologiaRESUMO
The mammalian cortex is a laminar structure containing many areas and cell types that are densely interconnected in complex ways, and for which generalizable principles of organization remain mostly unknown. Here we describe a major expansion of the Allen Mouse Brain Connectivity Atlas resource1, involving around a thousand new tracer experiments in the cortex and its main satellite structure, the thalamus. We used Cre driver lines (mice expressing Cre recombinase) to comprehensively and selectively label brain-wide connections by layer and class of projection neuron. Through observations of axon termination patterns, we have derived a set of generalized anatomical rules to describe corticocortical, thalamocortical and corticothalamic projections. We have built a model to assign connection patterns between areas as either feedforward or feedback, and generated testable predictions of hierarchical positions for individual cortical and thalamic areas and for cortical network modules. Our results show that cell-class-specific connections are organized in a shallow hierarchy within the mouse corticothalamic network.
Assuntos
Córtex Cerebral/anatomia & histologia , Córtex Cerebral/citologia , Vias Neurais/anatomia & histologia , Vias Neurais/citologia , Tálamo/anatomia & histologia , Tálamo/citologia , Animais , Axônios/fisiologia , Córtex Cerebral/fisiologia , Feminino , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vias Neurais/fisiologia , Tálamo/fisiologiaRESUMO
Fluorescent labeling allows for imaging and tracking of vesicles down to single-particle level. Among several options to introduce fluorescence, staining of lipid membranes with lipophilic dyes provides a straightforward approach without interfering with vesicle content. However, incorporating lipophilic molecules into vesicle membranes in an aqueous solution is generally not efficient because of their low water solubility. Here, we describe a simple, fast (<30 min), and highly effective procedure for fluorescent labeling of vesicles including natural extracellular vesicles. By adjusting the ionic strength of the staining buffer with NaCl, the aggregation status of DiI, a representative lipophilic tracer, can be controlled reversibly. Using cell-derived vesicles as a model system, we show that dispersion of DiI under low-salt condition improved its incorporation into vesicles by a factor of 290. In addition, increasing NaCl concentration after labeling induced free dye molecules to form aggregates, which can be filtered and thus effectively removed without ultracentrifugation. We consistently observed 6- to 85-fold increases in the labeled vesicle count across different types of dyes and vesicles. The method is expected to reduce the concern about off-target labeling resulting from the use of high concentrations of dyes.
Assuntos
Corantes Fluorescentes , Cloreto de Sódio , Corantes Fluorescentes/metabolismo , Ultracentrifugação , Coloração e RotulagemRESUMO
The posterior parietal cortex (PPC) is a major multimodal association cortex implicated in a variety of higher order cognitive functions, such as visuospatial perception, spatial attention, categorization, and decision-making. The PPC is known to receive inputs from a collection of sensory cortices as well as various subcortical areas and integrate those inputs to facilitate the execution of functions that require diverse information. Although many recent works have been performed with the mouse as a model system, a comprehensive understanding of long-range connectivity of the mouse PPC is scarce, preventing integrative interpretation of the rapidly accumulating functional data. In this study, we conducted a detailed neuroanatomic and bioinformatic analysis of the Allen Mouse Brain Connectivity Atlas data to summarize afferent and efferent connections to/from the PPC. Then, we analyzed variability between subregions of the PPC, functional/anatomical modalities, and species, and summarized the organizational principle of the mouse PPC. Finally, we confirmed key results by using additional neurotracers. A comprehensive survey of the connectivity will provide an important future reference to comprehend the function of the PPC and allow effective paths forward to various studies using mice as a model system.
Assuntos
Atenção/fisiologia , Cognição/fisiologia , Rede Nervosa/patologia , Lobo Parietal/fisiologia , Animais , Mapeamento Encefálico/métodos , Camundongos , Rede Nervosa/fisiologiaRESUMO
Cell-derived vesicles (CDVs) have been investigated as an alternative to exosomes. Here, we generated CDVs from Prokineticin receptor 1 (PROKR1) overexpressing HEK293T cells using micro-extrusion. More than 60 billion PROKR1-enriched CDV (PROKR1Tg CDVs) particles with canonical exosome properties were recovered from 107 cells. With 25⯵g/mL of PROKR1Tg CDVs, we observed delivery of PROKR1, significant reduction of apoptosis, and myotube formation in C2C12Prokr1-/- myoblasts that have lost their myogenic potential but underwent apoptosis following myogenic commitment. Expression levels of early and late myogenic marker genes and glucose uptake capacity were restored to equivalent levels with wild-type control. Furthermore, PROKR1Tg CDVs were accumulated in soleus muscle comparable to the liver without significant differences. Therefore, CDVs obtained from genetically engineered cells appear to be an effective method of PROKR1 protein delivery and offer promise as an alternative therapy for muscular dystrophy.
Assuntos
Apoptose/efeitos dos fármacos , Micropartículas Derivadas de Células/química , Desenvolvimento Muscular/efeitos dos fármacos , Receptores Acoplados a Proteínas G/química , Animais , Diferenciação Celular/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genéticaRESUMO
Comprehensive knowledge of the brain's wiring diagram is fundamental for understanding how the nervous system processes information at both local and global scales. However, with the singular exception of the C. elegans microscale connectome, there are no complete connectivity data sets in other species. Here we report a brain-wide, cellular-level, mesoscale connectome for the mouse. The Allen Mouse Brain Connectivity Atlas uses enhanced green fluorescent protein (EGFP)-expressing adeno-associated viral vectors to trace axonal projections from defined regions and cell types, and high-throughput serial two-photon tomography to image the EGFP-labelled axons throughout the brain. This systematic and standardized approach allows spatial registration of individual experiments into a common three dimensional (3D) reference space, resulting in a whole-brain connectivity matrix. A computational model yields insights into connectional strength distribution, symmetry and other network properties. Virtual tractography illustrates 3D topography among interconnected regions. Cortico-thalamic pathway analysis demonstrates segregation and integration of parallel pathways. The Allen Mouse Brain Connectivity Atlas is a freely available, foundational resource for structural and functional investigations into the neural circuits that support behavioural and cognitive processes in health and disease.
Assuntos
Encéfalo/anatomia & histologia , Encéfalo/citologia , Conectoma , Animais , Atlas como Assunto , Axônios/fisiologia , Córtex Cerebral/citologia , Corpo Estriado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Técnicas de Rastreamento Neuroanatômico , Tálamo/citologiaRESUMO
DAF-16, a forkhead transcription factor, is a key regulator of longevity, metabolism and dauer diapause in Caenorhabditis elegans. The precise mechanism by which DAF-16 regulates multiple functions, however, is poorly understood. Here, we used chromatin immunoprecipitation (ChIP) to identify direct targets of DAF-16. We cloned 103 target sequences containing consensus DAF-16 binding sites and selected 33 targets for further analysis. Expression of most of these genes is regulated in a DAF-16-dependent manner, and inactivation of more than half of these genes significantly altered DAF-16-dependent functions, including life span, fat storage and dauer formation. Our results show that the ChIP-based cloning strategy leads to greater enrichment for DAF-16 target genes than previous screening strategies. We also demonstrate that DAF-16 is recruited to multiple promoters to coordinate regulation of its downstream targets. The large number of target genes discovered provides insight into how DAF-16 controls diverse biological functions.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Longevidade/fisiologia , Fatores de Transcrição/metabolismo , Alelos , Animais , Caenorhabditis elegans/fisiologia , Imunoprecipitação da Cromatina , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica , Genes de Helmintos , FenótipoRESUMO
The cell-derived vesicles (CDVs) obtained using a proprietary extrusion process are the foundation of BioDrone platform technology. With superior productivity and versatility, this technology has garnered increasing attention in broad applications, particularly as a drug delivery vehicle. Previously, we showed that CDVs exhibited varying levels of expression for tetraspanin and organelle membrane markers while revealing no discernible differences in physical characteristics compared to naturally produced extracellular vesicles (EVs). To further understand and utilize the therapeutic potentials of CDVs, a more comprehensive study of membrane protein profiles is necessary. In addition, it is crucial to validate that the CDVs produced from extrusion are indeed intact lipid vesicles rather than other impurities. Here, we produced multiple batches of CDVs and EVs from HEK293 cells. CDVs and EVs were subjected to the same purification processes for subsequent proteome and particle analyses. The proteome analyses revealed unique proteome signatures between CDVs, EVs, and parental cells. Extensive proteome analyses identified the nine most prominent membrane markers that are abundant in CDVs compared to cells and EVs. Subsequent western blotting and nanoparticle flow cytometry analyses confirmed that CD63, lysosome-associated membrane glycoprotein 1 (LAMP1), and nicastrin (NCSTN) are highly enriched in CDVs, whereas CD81, CD9, and prostaglandin F2 receptor negative regulator (PTGFRN) are more abundant in EVs. This highlights the unique membrane composition and marker signature of CDVs that are distinct from EVs. Lastly, we demonstrated that more than 90% of the CDVs are genuine lipid vesicles by combining two different classes of vesicle labeling dyes and detergents to disrupt lipid membranes. This indicates that our proprietary extrusion technology is highly compatible with other well-characterized EV production methods. The robust CDV markers identified in this study will also facilitate the engineering of CDVs to achieve enhanced therapeutic effects or tissue-selective cargo delivery.
Assuntos
Vesículas Extracelulares , Nanopartículas , Humanos , Proteoma/metabolismo , Citometria de Fluxo/métodos , Células HEK293 , Vesículas Extracelulares/metabolismo , Organelas/metabolismo , LipídeosRESUMO
Membrane-bound vesicles such as extracellular vesicles (EVs) can function as biochemical effectors on target cells. Docking of the vesicles onto recipient plasma membranes depends on their interaction with cell-surface proteins, but a generalizable technique that can quantitatively observe these vesicle-protein interactions (VPIs) is lacking. Here, we describe a fluorescence microscopy that measures VPIs between single vesicles and cell-surface proteins, either in a surface-tethered or in a membrane-embedded state. By employing cell-derived vesicles (CDVs) and intercellular adhesion molecule-1 (ICAM-1) as a model system, we found that integrin-driven VPIs exhibit distinct modes of affinity depending on vesicle origin. Controlling the surface density of proteins also revealed a strong support from a tetraspanin protein CD9, with a critical dependence on molecular proximity. An adsorption model accounting for multiple protein molecules was developed and captured the features of density-dependent cooperativity. We expect that VPI imaging will be a useful tool to dissect the molecular mechanisms of vesicle adhesion and uptake, and to guide the development of therapeutic vesicles.
Assuntos
Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Comunicação Celular , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Introduction: Salivary gland (SG) damage is commonly caused by aging, irradiation, and some medications, and currently, no damage modifying agent is available. However, cell therapy based on mesenchymal stem cells (MSCs) has been proposed as a therapeutic modality for irradiated SGs. Therefore, we administered cell-derived vesicles (CDVs) of adipose-derived mesenchymal stem cells (ADMSCs) to irradiated SG cells to investigate their radioprotective effects in vitro. Methods: The artificial CDVs were obtained from ADMSC by tangential flow filtration (TFF) purification and ultracentrifugation. Cultured human SG epithelial cells were exposed to 2, 5 or 15 Gy of 4 MV X-rays produced by a linear accelerator. The effects of ADMSC-CDVs on SG epithelial cells damaged by irradiation were tested by proliferation activity, transepithelial electrical resistance (TEER), and amylase activity. Results: Exposure to penetrating radiation inhibited the proliferation of SG epithelial cells, but the radiation intensity required to reduce the proliferation of human submandibular gland epithelial cells (hSMGECs) was greater than required for other SG cells. ADMSC-CDVs restored the proliferative ability of SG epithelial cells reduced by irradiation, and the proliferation capacities of irradiated human parotid gland epithelial cells (hPGECs) and human sublingual gland epithelial cells (hSLGECs) were increased by administering ADMSC-CDVs to non-irradiated SG epithelial cells. Furthermore, amylase activity in irradiated hPGECs, hSMGECs, and hSLGECs was lower than in non-irradiated controls. However, amylase ability was restored in all by ADMSC-CDV treatment. Also, TEER was diminished by irradiation in hPGECs, hSMGECs, and hSLGECs and restored by ADMSC-CDV administration. Conclusion: Overall, our findings demonstrate that ADMSC-CDVs have potent radioprotective effects on irradiated SG cells.
RESUMO
Extracellular vesicles (EVs) released by a variety of cell types have been shown to act as a natural delivery system for bioactive molecules such as RNAs and proteins. EV therapy holds great promise as a safe and cell-free therapy for many immunological and degenerative diseases. However, translation to clinical application is limited by several factors, including insufficient large-scale manufacturing technologies and low yield. We have developed a novel drug delivery platform technology, BioDrone™, based on cell-derived vesicles (CDVs) produced from diverse cell sources by using a proprietary extrusion process. This extrusion technology generates nanosized vesicles in far greater numbers than naturally obtained EVs. We demonstrate that the CDVs are surrounded by a lipid bilayer membrane with a correct membrane topology. Physical, biochemical and functional characterisation results demonstrate the potential of CDVs to act as effective therapeutics. Umbilical cord mesenchymal stem cell (UCMSC)-derived CDVs exhibit a biological activity that is similar to UCMSCs or UCMSC-derived EVs. Lastly, we present the establishment of a GMP-compliant process to allow the production of a large number of UCMSC-CDVs in a reproducible manner. GMP-compliant manufacturing of CDVs will facilitate the preclinical and clinical evaluation of these emerging therapeutics in anti-inflammatory or regenerative medicine. This study also represents a crucial step in the development of this novel drug delivery platform based on CDVs.
RESUMO
The connections between the claustrum and the cortex in mouse are systematically investigated with adeno-associated virus (AAV), an anterograde viral tracer. We first define the boundary and the three-dimensional structure of the claustrum based on a variety of molecular and anatomical data. From AAV injections into 42 neocortical and allocortical areas, we conclude that most cortical areas send bilateral projections to the claustrum, the majority being denser on the ipsilateral side. This includes prelimbic, infralimbic, medial, ventrolateral and lateral orbital, ventral retrosplenial, dorsal and posterior agranular insular, visceral, temporal association, dorsal and ventral auditory, ectorhinal, perirhinal, lateral entorhinal, and anteromedial, posteromedial, lateroposterior, laterointermediate, and postrhinal visual areas. In contrast, the cingulate and the secondary motor areas send denser projections to the contralateral claustrum than to the ipsilateral one. The gustatory, primary auditory, primary visual, rostrolateral visual, and medial entorhinal cortices send projections only to the ipsilateral claustrum. Primary motor, primary somatosensory and subicular areas barely send projections to either ipsi- or contralateral claustrum. Corticoclaustral projections are organized in a rough topographic manner, with variable projection strengths. We find that the claustrum, in turn, sends widespread projections preferentially to ipsilateral cortical areas with different projection strengths and laminar distribution patterns and to certain contralateral cortical areas. Our quantitative results show that the claustrum has strong reciprocal and bilateral connections with prefrontal and cingulate areas as well as strong reciprocal connections with the ipsilateral temporal and retrohippocampal areas, suggesting that it may play a crucial role in a variety of cognitive processes. J. Comp. Neurol. 525:1317-1346, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Gânglios da Base/anatomia & histologia , Córtex Cerebral/anatomia & histologia , Vias Neurais/anatomia & histologia , Animais , Imageamento Tridimensional , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BLRESUMO
14-3-3 proteins are evolutionarily conserved and ubiquitous proteins that function in a wide variety of biological processes. Here we define a new role for C. elegans 14-3-3 proteins in life span regulation. We identify two C. elegans 14-3-3 proteins as interacting proteins of a major life span regulator, the C. elegans SIR2 ortholog, SIR-2.1. Similar to sir-2.1, we find that overexpression of either 14-3-3 protein (PAR-5 or FTT-2) extends life span and that this is dependent on DAF-16, a forkhead transcription factor (FOXO), another important life span regulator in the insulin/IGF-1 signaling pathway. Furthermore, we show that both 14-3-3 proteins are co-expressed with DAF-16 and SIR-2.1 in the tissues critical for life span regulation. Finally, we show that DAF-16/FOXO also physically interacts with the 14-3-3 proteins. These results suggest that C. elegans 14-3-3 proteins can regulate longevity by cooperating with both SIR-2.1 and DAF-16/FOXO.
Assuntos
Proteínas 14-3-3/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Sirtuínas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Forkhead , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Longevidade , Modelos Animais , Organismos Geneticamente Modificados , Interferência de RNARESUMO
In Caenorhabditis elegans, the insulin/IGF-1 signaling pathway controls many biological processes such as life span, fat storage, dauer diapause, reproduction and stress response . This pathway is comprised of many genes including the insulin/IGF-1 receptor (DAF-2) that signals through a conserved PI 3-kinase/AKT pathway and ultimately down-regulates DAF-16, a forkhead transcription factor (FOXO). DAF-16 also receives input from several other pathways that regulate life span such as the germline and the JNK pathway [Hsin, H., Kenyon, C., 1999. Signals from the reproductive system regulate the lifespan of C. elegans. Nature 399, 362-366; Oh, S.W., Mukhopadhyay, A., Svrzikapa, N., Jiang, F., Davis, R.J., Tissenbaum, H.A., 2005. JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16. Proc. Natl. Acad. Sci. USA 102, 4494-4499]. Therefore, DAF-16 integrates signals from multiple pathways and regulates its downstream target genes to control diverse processes. Here, we discuss the signals to and from DAF-16, with a focus on life span regulation.
Assuntos
Envelhecimento/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição/genética , Animais , Restrição Calórica , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas/fisiologia , Insulina/genética , Longevidade/genética , Modelos Genéticos , Receptor IGF Tipo 2/genética , Receptor de Insulina/genética , Transdução de Sinais/genética , Sirtuínas/genéticaRESUMO
There have been reports of regional differences in the activation of mitogen activated protein kinases (MAPKs) and in the induction of immediate early genes after electroconvulsive shock (ECS) in the rat brain. This study was performed to determine whether ECS induce the region-specific phosphorylation of MAPK-downstream transcription factors, ATF-2, Elk-1, c-Jun, in rat hippocampus and cerebellum. Following ECS, the phosphorylation of ATF-2 was highly increased in the hippocampus but slightly in the cerebellum. The phosphorylation of Elk-1 was increased in the cerebellum but not in the hippocampus. In contrast, the phosphorylation of c-Jun was increased only in the hippocampus. These results indicate that ECS can induce the region-specific phosphorylation of MAPK-downstream transcription factors in rat hippocampus and cerebellum.
Assuntos
Cerebelo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA , Eletrochoque , Hipocampo/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Fator 2 Ativador da Transcrição , Animais , Genes Precoces/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Fosforilação , Ratos , Ratos Sprague-Dawley , Proteínas Elk-1 do Domínio etsRESUMO
Significant advances in circuit-level analyses of the brain require tools that allow for labeling, modulation of gene expression, and monitoring and manipulation of cellular activity in specific cell types and/or anatomical regions. Large-scale projects and individual laboratories have produced hundreds of gene-specific promoter-driven Cre mouse lines invaluable for enabling genetic access to subpopulations of cells in the brain. However, the potential utility of each line may not be fully realized without systematic whole brain characterization of transgene expression patterns. We established a high-throughput in situ hybridization (ISH), imaging and data processing pipeline to describe whole brain gene expression patterns in Cre driver mice. Currently, anatomical data from over 100 Cre driver lines are publicly available via the Allen Institute's Transgenic Characterization database, which can be used to assist researchers in choosing the appropriate Cre drivers for functional, molecular, or connectional studies of different regions and/or cell types in the brain.
Assuntos
Encéfalo/anatomia & histologia , Regulação da Expressão Gênica/fisiologia , Integrases/metabolismo , Neurônios/metabolismo , Recombinação Genética , Animais , Encéfalo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Integrases/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/classificação , Tamoxifeno/farmacologia , Trimetoprima/farmacologiaRESUMO
As an anterograde neuronal tracer, recombinant adeno-associated virus (AAV) has distinct advantages over the widely used biotinylated dextran amine (BDA). However, the sensitivity and selectivity of AAV remain uncharacterized for many brain regions and species. To validate this tracing method further, AAV (serotype 1) was systematically compared with BDA as an anterograde tracer by injecting both tracers into three cortical and 15 subcortical regions in C57BL/6J mice. Identical parameters were used for our sequential iontophoretic injections, producing injections of AAV that were more robust in size and in density of neurons infected compared with those of BDA. However, these differences did not preclude further comparison between the tracers, because the pairs of injections were suitably colocalized and contained some percentage of double-labeled neurons. A qualitative analysis of projection patterns showed that the two tracers behave very similarly when injection sites are well matched. Additionally, a quantitative analysis of relative projection intensity for cases targeting primary motor cortex (MOp), primary somatosensory cortex (SSp), and caudoputamen (CP) showed strong agreement in the ranked order of projection intensities between the two tracers. A detailed analysis of the projections of two brain regions (SSp and MOp) revealed many targets that have not previously been described in the mouse or rat. Minor retrograde labeling of neurons was observed in all cases examined, for both AAV and BDA. Our results show that AAV has actions equivalent to those of BDA as an anterograde tracer and is suitable for analysis of neural circuitry throughout the mouse brain.
Assuntos
Biotina/análogos & derivados , Encéfalo/anatomia & histologia , Dependovirus , Dextranos , Corantes Fluorescentes , Técnicas de Rastreamento Neuroanatômico , Marcadores do Trato Nervoso , Animais , Contagem de Células , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência , Vias Neurais/anatomia & histologia , Neurônios/citologia , Fotomicrografia , Sensibilidade e EspecificidadeRESUMO
Harnessing the natural ability of viruses to infect post-mitotic cells such as neurons has provided an explosion of new methods to manipulate and reconstruct neural circuits in vivo. Here we describe the use of recombinant adeno-associated viral vectors (rAAV) for axonal tract tracing in nontransgenic and transgenic Cre driver mice. Two protocols are presented for stereotactic-guided placement of rAAV vectors into the live mouse brain using iontophoretic or nanoliter pressure injections. The methods discussed here will result in expression of fluorescent proteins in cell bodies, dendrites, and axons in targeted neurons, and can be easily adapted to address various experimental questions.