The proper connectivity between neurons is essential for the implementation of the algorithms used in neural computations, such as the detection of directed motion by the retina. The analysis of ...neuronal connectivity is possible with electron microscopy, but technological limitations have impeded the acquisition of high-resolution data on a large enough scale. Here we show, using serial block-face electron microscopy and two-photon calcium imaging, that the dendrites of mouse starburst amacrine cells make highly specific synapses with direction-selective ganglion cells depending on the ganglion cell's preferred direction. Our findings indicate that a structural (wiring) asymmetry contributes to the computation of direction selectivity. The nature of this asymmetry supports some models of direction selectivity and rules out others. It also puts constraints on the developmental mechanisms behind the formation of synaptic connections. Our study demonstrates how otherwise intractable neurobiological questions can be addressed by combining functional imaging with the analysis of neuronal connectivity using large-scale electron microscopy.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Comprehensive high-resolution structural maps are central to functional exploration and understanding in biology. For the nervous system, in which high resolution and large spatial extent are both ...needed, such maps are scarce as they challenge data acquisition and analysis capabilities. Here we present for the mouse inner plexiform layer--the main computational neuropil region in the mammalian retina--the dense reconstruction of 950 neurons and their mutual contacts. This was achieved by applying a combination of crowd-sourced manual annotation and machine-learning-based volume segmentation to serial block-face electron microscopy data. We characterize a new type of retinal bipolar interneuron and show that we can subdivide a known type based on connectivity. Circuit motifs that emerge from our data indicate a functional mechanism for a known cellular response in a ganglion cell that detects localized motion, and predict that another ganglion cell is motion sensitive.
Neuroanatomic analysis depends on the reconstruction of complete cell shapes. High-throughput reconstruction of neural circuits, or connectomics, using volume electron microscopy requires dense ...staining of all cells, which leads even experts to make annotation errors. Currently, reconstruction speed rather than acquisition speed limits the determination of neural wiring diagrams. We developed a method for fast and reliable reconstruction of densely labeled data sets. Our approach, based on manually skeletonizing each neurite redundantly (multiple times) with a visualization-annotation software tool called KNOSSOS, is ∼50-fold faster than volume labeling. Errors are detected and eliminated by a redundant-skeleton consensus procedure (RESCOP), which uses a statistical model of how true neurite connectivity is transformed into annotation decisions. RESCOP also estimates the reliability of consensus skeletons. Focused reannotation of difficult locations promises a rather steep increase of reliability as a function of the average skeleton redundancy and thus the nearly error-free analysis of large neuroanatomical datasets.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
High-resolution, comprehensive structural information is often the final arbiter between competing mechanistic models of biological processes, and can serve as inspiration for new hypotheses. In ...molecular biology, definitive structural data at atomic resolution are available for many macromolecules; however, information about the structure of the brain is much less complete, both in scope and resolution. Several technical developments over the past decade, such as serial block-face electron microscopy and trans-synaptic viral tracing, have made the structural biology of neural circuits conceivable: we may be able to obtain the structural information needed to reconstruct the network of cellular connections for large parts of, or even an entire, mouse brain within a decade or so. Given that the brain's algorithms are ultimately encoded by this network, knowing where all of these connections are should, at the very least, provide the data needed to distinguish between models of neural computation.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Directionally tuned signalling in starburst amacrine cell (SAC) dendrites lies at the heart of the circuit that detects the direction of moving stimuli in the mammalian retina. The relative ...contributions of intrinsic cellular properties and network connectivity to SAC direction selectivity remain unclear. Here we present a detailed connectomic reconstruction of SAC circuitry in mouse retina and describe two previously unknown features of synapse distributions along SAC dendrites: input and output synapses are segregated, with inputs restricted to proximal dendrites; and the distribution of inhibitory inputs is fundamentally different from that observed in rabbit retina. An anatomically constrained SAC network model suggests that SAC–SAC wiring differences between mouse and rabbit retina underlie distinct contributions of synaptic inhibition to velocity and contrast tuning and receptive field structure. In particular, the model indicates that mouse connectivity enables SACs to encode lower linear velocities that account for smaller eye diameter, thereby conserving angular velocity tuning. These predictions are confirmed with calcium imaging of mouse SAC dendrites responding to directional stimuli.
The detection of visual motion is a fundamental function of the visual system. How motion speed and direction are computed together at the cellular level, however, remains largely unknown. Here, we ...suggest a circuit mechanism by which excitatory inputs to direction-selective ganglion cells in the mouse retina become sensitive to the motion speed and direction of image motion. Electrophysiological, imaging, and connectomic analyses provide evidence that the dendrites of ON direction-selective cells receive spatially offset and asymmetrically filtered glutamatergic inputs along motion-preference axis from asymmetrically wired bipolar and amacrine cell types with distinct release dynamics. A computational model shows that, with this spatiotemporal structure, the input amplitude becomes sensitive to speed and direction by a preferred direction enhancement mechanism. Our results highlight the role of an excitatory mechanism in retinal motion computation by which feature selectivity emerges from non-selective inputs.
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•Space-time wiring between ON DS cells and bipolar cell types was identified•Presynaptic inhibition and NaV channels set the glutamate release dynamics•Preferred direction enhancement supports the tuning to direction and slow speed•Global dendritic summation is involved in computing image motion
Matsumoto et al. find the space-time wiring between the dendrites of ON direction-selective cells and bipolar cell types in the mouse retina. Temporally diverse glutamatergic inputs, which are asymmetrically organized across the dendrites, are globally summated by a preferred direction enhancement mechanism for computing motion speed and direction.
Highlights ► The dense analysis of neuronal circuits requires volume electron microscopy. ► We review new EM methods that provide increases in z-resolution and throughput. ► Tradeoffs between ...resolution, acquisition speed, and reliability are discussed. ► We argue wiring diagrams are necessary but not sufficient to understand circuits.
Local and global forms of inhibition controlling directionally selective ganglion cells (DSGCs) in the mammalian retina are well documented. It is established that local inhibition arising from ...GABAergic starburst amacrine cells (SACs) strongly contributes to direction selectivity. Here, we demonstrate that increasing ambient illumination leads to the recruitment of GABAergic wide-field amacrine cells (WACs) endowing the DS circuit with an additional feature: size selectivity. Using a combination of electrophysiology, pharmacology, and light/electron microscopy, we show that WACs predominantly contact presynaptic bipolar cells, which drive direct excitation and feedforward inhibition (through SACs) to DSGCs, thus maintaining the appropriate balance of inhibition/excitation required for generating DS. This circuit arrangement permits high-fidelity direction coding over a range of ambient light levels, over which size selectivity is adjusted. Together, these results provide novel insights into the anatomical and functional arrangement of multiple inhibitory interneurons within a single computational module in the retina.
•Wide-field, but not local GABAergic inhibition is modulated by ambient illumination•Spiking WACs co-modulate inhibitory and excitatory inputs to DSGCs•WACs confer spatial tuning without altering directional preferences•Electron microscopy confirms WAC synapses at bipolar cell axon terminals
Understanding the roles of diverse GABAergic interneurons in a given neural circuit is a challenge. Hoggarth et al., describe how distinct GABAergic amacrine cells allow multiplexing of spatial and directional information in the same output neuron with little mutual interference.
Optimal epoxy resin embedding is crucial for obtaining consistent serial sections from large tissue samples, especially for block faces spanning >1 mm
. We report a method to quantify non-uniformity ...in resin curing using block hardness measurements from block faces. We identify conditions that lead to non-uniform curing as well as a procedure to monitor the hardness of blocks for a wide range of common epoxy resins used for volume electron microscopy. We also assess cutting repeatability and uniformity by quantifying the transverse and sectional cutting forces during ultrathin sectioning using a sample-mounted force sensor. Our findings indicate that screening and optimizing resin formulations is required to achieve the best repeatability in terms of section thickness. Finally, we explore the encapsulation of irregularly shaped tissue samples in a gelatin matrix prior to epoxy resin embedding to yield more uniform sections.
Danionella cerebrum (DC) is a promising vertebrate animal model for systems neuroscience due to its small adult brain volume and inherent optical transparency, but the scope of their cognitive ...abilities remains an area of active research. In this work, we established a behavioral paradigm to study visual spatial navigation in DC and investigate their navigational capabilities and strategies. We initially observed that adult DC exhibit strong negative phototaxis in groups but less so as individuals. Using their dark preference as a motivator, we designed a spatial navigation task inspired by the Morris water maze. Through a series of environmental cue manipulations, we found that DC utilize visual cues to anticipate a reward location and found evidence for landmark-based navigational strategies wherein DC could use both proximal and distal visual cues. When subsets of proximal visual cues were occluded, DC were capable of using distant contextual visual information to solve the task, providing evidence for allocentric spatial navigation. Without proximal visual cues, DC tended to seek out a direct line of sight with at least one distal visual cue while maintaining a positional bias toward the reward location. In total, our behavioral results suggest that DC can be used to study the neural mechanisms underlying spatial navigation with cellular resolution imaging across an adult vertebrate brain.
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