RESUMEN
Studies in several important areas of neuroscience, including analysis of single neurons as well as neural networks, continue to be limited by currently available experimental tools. By combining molecular probes of cellular function, such as voltage-sensitive or calcium-sensitive dyes, with advanced microscopy techniques such as multiphoton microscopy, experimental neurophysiologists have been able to partially reduce this limitation. These approaches usually provide the needed spatial resolution along with convenient optical sectioning capabilities for isolating regions of interest. However, they often fall short in providing the necessary temporal resolution, primarily due to their restrained laser scanning mechanisms. In this regard, we review a method of laser scanning for multiphoton microscopy that overcomes the temporal limitations of pervious approaches and allows for what is known as 3D Random Access Multiphoton (3D RAMP) microscopy, an imaging technique that supports full three dimensional recording of many sites of interest on physiologically relevant time scales.
Asunto(s)
Imagenología Tridimensional/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Neuronas/fisiología , Imagen Óptica/métodos , Imagen de Colorante Sensible al Voltaje/métodos , Animales , Calcio/metabolismo , Colorantes Fluorescentes/química , Hipocampo/fisiología , Hipocampo/ultraestructura , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional/instrumentación , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Red Nerviosa/fisiología , Red Nerviosa/ultraestructura , Neuronas/ultraestructura , Imagen Óptica/instrumentación , Factores de Tiempo , Imagen de Colorante Sensible al Voltaje/instrumentaciónRESUMEN
Clones of excitatory neurons derived from a common progenitor have been proposed to serve as elementary information processing modules in the neocortex. To characterize the cell types and circuit diagram of clonally related excitatory neurons, we performed multi-cell patch clamp recordings and Patch-seq on neurons derived from Nestin-positive progenitors labeled by tamoxifen induction at embryonic day 10.5. The resulting clones are derived from two radial glia on average, span cortical layers 2-6, and are composed of a random sampling of transcriptomic cell types. We find an interaction between shared lineage and connection type: related neurons are more likely to be connected vertically across cortical layers, but not laterally within the same layer. These findings challenge the view that related neurons show uniformly increased connectivity and suggest that integration of vertical intra-clonal input with lateral inter-clonal input may represent a developmentally programmed connectivity motif supporting the emergence of functional circuits.