RESUMEN
Brain circuits in the neocortex develop from diverse types of neurons that migrate and form synapses. Here we quantify the circuit patterns of synaptogenesis for inhibitory interneurons in the developing mouse somatosensory cortex. We studied synaptic innervation of cell bodies, apical dendrites, and axon initial segments using three-dimensional electron microscopy focusing on the first 4 weeks postnatally (postnatal days P5 to P28). We found that innervation of apical dendrites occurs early and specifically: Target preference is already almost at adult levels at P5. Axons innervating cell bodies, on the other hand, gradually acquire specificity from P5 to P9, likely via synaptic overabundance followed by antispecific synapse removal. Chandelier axons show first target preference by P14 but develop full target specificity almost completely by P28, which is consistent with a combination of axon outgrowth and off-target synapse removal. This connectomic developmental profile reveals how inhibitory axons in the mouse cortex establish brain circuitry during development.
Asunto(s)
Conectoma , Neuronas GABAérgicas/fisiología , Interneuronas/fisiología , Red Nerviosa/crecimiento & desarrollo , Corteza Somatosensorial/crecimiento & desarrollo , Sinapsis/fisiología , Animales , Axones/ultraestructura , Conjuntos de Datos como Asunto , Dendritas/ultraestructura , Neuronas GABAérgicas/ultraestructura , Imagenología Tridimensional/métodos , Interneuronas/ultraestructura , Ratones , Microscopía Electrónica/métodos , Red Nerviosa/ultraestructura , Corteza Somatosensorial/ultraestructura , Sinapsis/ultraestructuraRESUMEN
Automated tape-collecting ultramicrotomy in conjunction with scanning electron microscopy (SEM) is a powerful approach for volume electron microscopy and three-dimensional neuronal circuit analysis. Current tapes are limited by section wrinkle formation, surface scratches and sample charging during imaging. Here we show that a plasma-hydrophilized carbon nanotube (CNT)-coated polyethylene terephthalate (PET) tape effectively resolves these issues and produces SEM images of comparable quality to those from transmission electron microscopy. CNT tape can withstand multiple rounds of imaging, offer low surface resistance across the entire tape length and generate no wrinkles during the collection of ultrathin sections. When combined with an enhanced en bloc staining protocol, CNT tape-processed brain sections reveal detailed synaptic ultrastructure. In addition, CNT tape is compatible with post-embedding immunostaining for light and electron microscopy. We conclude that CNT tape can enable high-resolution volume electron microscopy for brain ultrastructure analysis.
Asunto(s)
Encéfalo/ultraestructura , Microscopía Electrónica de Rastreo/instrumentación , Microscopía Electrónica de Rastreo/métodos , Nanotubos de Carbono , Animales , Masculino , Ratones , Microtomía , Tereftalatos Polietilenos , Ratas WistarRESUMEN
Research on neuronal connectivity in the cerebral cortex has focused on the existence and strength of synapses between neurons, and their location on the cell bodies and dendrites of postsynaptic neurons. The synaptic architecture of individual presynaptic axonal trees, however, remains largely unknown. Here we used dense reconstructions from three-dimensional electron microscopy in rats to study the synaptic organization of local presynaptic axons in layer 2 of the medial entorhinal cortex, the site of grid-like spatial representations. We observe path-length-dependent axonal synapse sorting, such that axons of excitatory neurons sequentially target inhibitory neurons followed by excitatory neurons. Connectivity analysis revealed a cellular feedforward inhibition circuit involving wide, myelinated inhibitory axons and dendritic synapse clustering. Simulations show that this high-precision circuit can control the propagation of synchronized activity in the medial entorhinal cortex, which is known for temporally precise discharges.
Asunto(s)
Axones/fisiología , Corteza Entorrinal/citología , Corteza Entorrinal/fisiología , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Sinapsis/fisiología , Animales , Axones/ultraestructura , Conectoma , Dendritas/fisiología , Dendritas/ultraestructura , Corteza Entorrinal/ultraestructura , Potenciales Postsinápticos Excitadores , Imagenología Tridimensional , Interneuronas/fisiología , Masculino , Microscopía Electrónica , Modelos Neurológicos , Inhibición Neural/fisiología , Vías Nerviosas/ultraestructura , Ratas , Sinapsis/ultraestructuraRESUMEN
Nerve tissue contains a high density of chemical synapses, about 1 per µm3 in the mammalian cerebral cortex. Thus, even for small blocks of nerve tissue, dense connectomic mapping requires the identification of millions to billions of synapses. While the focus of connectomic data analysis has been on neurite reconstruction, synapse detection becomes limiting when datasets grow in size and dense mapping is required. Here, we report SynEM, a method for automated detection of synapses from conventionally en-bloc stained 3D electron microscopy image stacks. The approach is based on a segmentation of the image data and focuses on classifying borders between neuronal processes as synaptic or non-synaptic. SynEM yields 97% precision and recall in binary cortical connectomes with no user interaction. It scales to large volumes of cortical neuropil, plausibly even whole-brain datasets. SynEM removes the burden of manual synapse annotation for large densely mapped connectomes.
Asunto(s)
Automatización de Laboratorios/métodos , Conectoma/métodos , Imagenología Tridimensional/métodos , Microscopía Electrónica/métodos , Corteza Somatosensorial/anatomía & histología , Sinapsis/ultraestructura , Animales , RatonesRESUMEN
The 28th annual Barrels meeting was held prior to the Society for Neuroscience meeting in October 2015 at the Northwestern University School of Law in Chicago, Illinois. The meeting brought together researchers focused on the rodent sensorimotor system. The meeting focused on modern techniques to decipher cortical circuits, social interactions among rodents, and decision-making. The meeting allowed investigators to share their work via short talks, poster presentations, and a data blitz.