Modeling Brain Circuitry over a Wide Range of Scales

If we are ever to unravel the mysteries of brain function at its most fundamental level, we will need a precise understanding of how its component neurons connect to each other. Electron Microscopes (EM) can now provide the nanometer resolution that is needed to image synapses, and therefore connections, while Light Microscopes (LM) see at the micrometer resolution required to model the 3D structure of the dendritic network. Since both the topology and the connection strength are integral parts of the brain's wiring diagram, being able to combine these two modalities is critically important. In fact, these microscopes now routinely produce high-resolution imagery in such large quantities that the bottleneck becomes automated processing and interpretation, which is needed for such data to be exploited to its full potential. In this paper, we briefly review the Computer Vision techniques we have developed at EPFL to address this need. They include delineating dendritic arbors from LM imagery, segmenting organelles from EM, and combining the two into a consistent representation

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack

Formation of Dendritic Spines with GABAergic Synapses Induced by Whisker Stimulation in Adult Mice

AbstractDuring development, alterations in sensory experience modify the structure of cortical neurons, particularly at the level of the dendritic spine. Are similar adaptations involved in plasticity of the adult cortex? Here we show that a 24 hr period of single whisker stimulation in freely moving adult mice increases, by 36%, the total synaptic density in the corresponding cortical barrel. This is due to an increase in both excitatory and inhibitory synapses found on spines. Four days after stimulation, the inhibitory inputs to the spines remain despite total synaptic density returning to pre-stimulation levels. Functional analysis of layer IV cells demonstrated altered response properties, immediately after stimulation, as well as four days later. These results indicate activity-dependent alterations in synaptic circuitry in adulthood, modifying the flow of sensory information into the cerebral cortex

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ2 domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA–mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS–PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons

Glial Glutamate Transporters and Maturation of the Mouse Somatosensory Cortex

In the adult nervous system, glutamatergic neurotransmission is tightly controlled by neuron-glia interactions through glial glutamate reuptake by the specific transporters GLT-1 and GLAST. Here, we have explored the role of these transporters in the structural and functional maturation of the somatosensory cortex of the mouse. We provide evidence that GLT-1 and GLAST are early and selectively expressed in barrels from P5 to P10. Confocal and electron microscopy confirm that the expression is restricted to the astroglial membrane. By P12, and despite an increased global expression as observed by immunoblotting, the barrel pattern of GLAST and GLT-1 staining is no longer evident. In P10 GLT-1 −/− and GLAST −/− mice, the cytoarchitectural segregation of the barrels is preserved. However, at P9-10, the functional response to whisker stimulation, measured by deoxyglucose uptake, is markedly decreased in GLT-1 −/− and GLAST −/− mice. The role of GLAST is transient since the metabolic response is already restored at P11-12 in GLAST −/− mice and remains unchanged in adulthood. However, deletion of GLT-1 seems to impair the functional metabolic response until adulthood. Our data suggest that astrocyte-neuron interactions via the glial glutamate transporters are involved in the functional maturation of the whisker representation in the somatosensory corte

Pansynaptic Enlargement at Adult Cortical Connections Strengthened by Experience

Behavioral experience alters the strength of neuronal connections in adult neocortex. These changes in synaptic strength are thought to be central to experience-dependent plasticity, learning, and memory. However, it is not known how changes in synaptic transmission between neurons become persistent, thereby enabling the storage of previous experience. A long-standing hypothesis is that altered synaptic strength is maintained by structural modifications to synapses. However, the extent of synaptic modifications and the changes in neurotransmission that the modifications support remain unclear. To address these questions, we recorded from pairs of synaptically connected layer 2/3 pyramidal neurons in the barrel cortex and imaged their contacts with high-resolution confocal microscopy after altering sensory experience by whisker trimming. Excitatory connections strengthened by experience exhibited larger axonal varicosities, dendritic spines, and interposed contact zones. Electron microscopy showed that contact zone size was strongly correlated with postsynaptic density area. Therefore, our findings indicate that whole synapses are larger at strengthened connections. Synaptic transmission was both stronger and more reliable following experience-dependent synapse enlargement. Hence, sensory experience modified both presynaptic and postsynaptic function. Our findings suggest that the enlargement of synaptic contacts is an integral part of long-lasting strengthening of cortical connections and, hence, of information storage in the neocorte

FOXO3 determines the accumulation of α-synuclein and controls the fate of dopaminergic neurons in the substantia nigra

Parkinson's disease (PD) is characterized by the selective degeneration of neuronal populations presumably due to pathogenic interactions between aging and predisposing factors such as increased levels of α-synuclein. Here, we genetically modulate the activity of the transcription factor Forkhead box protein O3 (FOXO3) in adult nigral dopaminergic neurons using viral vectors and explore how this determinant of longevity impacts on neuronal fate in normal and diseased conditions. We find that dopaminergic neurons are particularly vulnerable to changes in FOXO3 activity in the substantia nigra. While constitutive activation has proapoptotic effects leading to neuronal loss, inhibition of FOXO-mediated transcription by a dominant-negative competitor causes oxidative damage and is detrimental at high vector dose. To address the role of FOXO3 in PD, we modulate its activity in dopaminergic neurons overexpressing human α-synuclein. In this pathogenic condition, we find that FOXO inhibition has protective effects, suggesting that this transcription factor ultimately contributes to neuronal cell death. Nevertheless, mild FOXO3 activity also protects nigral neurons against the accumulation of human α-synuclein, albeit to a lesser extent. FOXO3 reduces the amount of α-synuclein present in the soluble protein fraction and promotes the coalescence of dense proteinase K-resistant aggregates, with an accumulation of autophagic vacuoles containing lipofuscin. Consistent with these in vivo observations, we find that FOXO3 controls autophagic flux in neuronal cells. Altogether, these results point to FOXO3 as an important determinant of neuronal survival in the substantia nigra, which may oppose α-synuclein accumulation and proteotoxicit

Diversity of cortico-descending projections: histological and diffusion MRI characterization in the monkey

The axonal composition of cortical projections originating in premotor, supplementary motor (SMA), primary motor (a4), somatosensory and parietal areas and descending towards the brain stem and spinal cord was characterized in the monkey with histological tract tracing, electron microscopy (EM) and diffusion MRI (dMRI). These 3 approaches provided complementary information. Histology provided accurate assessment of axonal diameters and size of synaptic boutons. dMRI revealed the topography of the projections (tractography), notably in the internal capsule. From measurements of axon diameters axonal conduction velocities were computed. Each area communicates with different diameter axons and this generates a hierarchy of conduction delays in this order: a4 (the shortest), SMA, premotor (F7), parietal, somatosensory, premotor F4 (the longest). We provide new interpretations for i) the well-known different anatomical and electrophysiological estimates of conduction velocity; ii) why conduction delays are probably an essential component of the cortical motor command; and iii) how histological and dMRI tractography can be integrated