We describe automated technologies to probe the structure of neural tissue at nanometer resolution and use them to generate a saturated reconstruction of a sub-volume of mouse neocortex in which all cellular objects (axons, dendrites, and glia) and many sub-cellular components (synapses, synaptic vesicles, spines, spine apparati, postsynaptic densities, and mitochondria) are rendered and itemized in a database. We explore these data to study physical properties of brain tissue. For example, by tracing the trajectories of all excitatory axons and noting their juxtapositions, both synaptic and non-synaptic, with every dendritic spine we refute the idea that physical proximity is sufficient to predict synaptic connectivity (the so-called Peters' rule). This online minable database provides general access to the intrinsic complexity of the neocortex and enables further data-driven inquiries.

The structural dynamics of synapses probably has a crucial role in the development and plasticity of the nervous system. In the mammalian brain, the vast majority of excitatory axo-dendritic synapses occur on dendritic specializations called 'spines'. However, little is known about their long-term changes in the intact developing or adult animal. To address this question we developed a transcranial two-photon imaging technique to follow identified spines of layer-5 pyramidal neurons in the primary visual cortex of living transgenic mice expressing yellow fluorescent protein. Here we show that filopodia-like dendritic protrusions, extending and retracting over hours, are abundant in young animals but virtually absent from the adult. In young mice, within the 'critical period' for visual cortex development, approximately 73% of spines remain stable over a one-month interval; most changes are associated with spine elimination. In contrast, in adult mice, the overwhelming majority of spines (approximately 96%) remain stable over the same interval with a half-life greater than 13 months. These results indicate that spines, initially plastic during development, become remarkably stable in the adult, providing a potential structural basis for long-term information storage.

Myelin is a defining feature of the vertebrate nervous system. Variability in the thickness of the myelin envelope is a structural feature affecting the conduction of neuronal signals. Conversely, the distribution of myelinated tracts along the length of axons has been assumed to be uniform. Here, we traced high-throughput electron microscopy (EM) reconstructions of single axons of pyramidal neurons in the mouse neocortex and built high-resolution maps of myelination. We find that individual neurons have distinct longitudinal distribution of myelin. Neurons in the superficial layers displayed the most diversified profiles, including a new pattern where myelinated segments are interspersed with long, unmyelinated tracts. Our data indicate that the profile of longitudinal distribution of myelin is an integral feature of neuronal identity and may have evolved as a strategy to modulate long-distance communication in the neocortex.

Coherent network oscillations in several distinct frequency bands are seen in the hippocampus of behaving animals. To investigate how different neuronal types within this network respond to oscillatory inputs we made whole‐cell current clamp recordings from three different types of neurones in the CA1 region of rat hippocampal slices: pyramidal cells, fast‐spiking interneurones and horizontal interneurones, and recorded their response to sinusoidal inputs at physiologically relevant frequencies (1‐100 Hz). Pyramidal neurones showed firing preference to inputs at theta frequencies (range 2‐7 Hz; n= 30). They showed subthreshold resonance in the same frequency range (2‐7 Hz; mean 4.1 ± 0.4 Hz; n= 19). Interneurones differed in their firing properties. Horizontal interneurones in the stratum oriens showed firing preference to inputs at theta frequencies (range 1.5‐10 Hz; n= 10). These interneurones also showed resonance at low frequencies (range 1‐5 Hz; mean 2.4 ± 0.5 Hz; n= 7). In contrast, fast‐spiking interneurones with cell bodies in the pyramidal cell layer fired preferentially at input frequencies in the gamma band (range 30‐50 Hz; n= 10/12). These interneurones showed resonance at beta‐gamma frequencies (10‐50 Hz; mean 26 ± 5 Hz; n= 7/8). Thus, in the hippocampus, different types of neurones have distinct frequency preferences. Therefore, in the CA1 layer of the hippocampal network, a compound oscillatory input may be segregated into distinct frequency components which are processed locally by distinct types of neurones.

The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used novel staining and automated ultra-thin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell.