Directionally selective calcium signals in dendrites of starburst amacrine cells
TL;DR: Dendritic calcium signals, but not somatic membrane voltage, are directionally selective for stimuli that move centrifugally from the cell soma, demonstrating that direction selectivity is computed locally in dendritic branches at a stage before ganglion cells.
Abstract: The detection of image motion is fundamental to vision. In many species, unique classes of retinal ganglion cells selectively respond to visual stimuli that move in specific directions. It is not known which retinal cell first performs the neural computations that give rise to directional selectivity in the ganglion cell. A prominent candidate has been an interneuron called the 'starburst amacrine cell'. Using two-photon optical recordings of intracellular calcium concentration, here we find that individual dendritic branches of starburst cells act as independent computation modules. Dendritic calcium signals, but not somatic membrane voltage, are directionally selective for stimuli that move centrifugally from the cell soma. This demonstrates that direction selectivity is computed locally in dendritic branches at a stage before ganglion cells.
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TL;DR: The Ca2+-signalling toolkit is used to assemble signalling systems with very different spatial and temporal dynamics and has a direct role in controlling the expression patterns of its signalling systems that are constantly being remodelled in both health and disease.
Abstract: Ca2+ is a highly versatile intracellular signal that operates over a wide temporal range to regulate many different cellular processes. An extensive Ca2+-signalling toolkit is used to assemble signalling systems with very different spatial and temporal dynamics. Rapid highly localized Ca2+ spikes regulate fast responses, whereas slower responses are controlled by repetitive global Ca2+ transients or intracellular Ca2+ waves. Ca2+ has a direct role in controlling the expression patterns of its signalling systems that are constantly being remodelled in both health and disease.
5,042 citations
Cites background from "Directionally selective calcium sig..."
...In some cases, these waves are highly localized, for example in starburst amacrine cells in the retina, in which localized dendritic signals are used to compute the direction of motio...
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TL;DR: It is demonstrated that datasets meeting these requirements can be obtained by automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope, opening the possibility of automatically obtaining the electron-microscope-level 3D datasets needed to completely reconstruct the connectivity of neuronal circuits.
Abstract: Three-dimensional (3D) structural information on many length scales is of central importance in biological research. Excellent methods exist to obtain structures of molecules at atomic, organelles at electron microscopic, and tissue at light-microscopic resolution. A gap exists, however, when 3D tissue structure needs to be reconstructed over hundreds of micrometers with a resolution sufficient to follow the thinnest cellular processes and to identify small organelles such as synaptic vesicles. Such 3D data are, however, essential to understand cellular networks that, particularly in the nervous system, need to be completely reconstructed throughout a substantial spatial volume. Here we demonstrate that datasets meeting these requirements can be obtained by automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope. Backscattering contrast is used to visualize the heavy-metal staining of tissue prepared using techniques that are routine for transmission electron microscopy. Low-vacuum (20–60 Pa H2O) conditions prevent charging of the uncoated block face. The resolution is sufficient to trace even the thinnest axons and to identify synapses. Stacks of several hundred sections, 50–70 nm thick, have been obtained at a lateral position jitter of typically under 10 nm. This opens the possibility of automatically obtaining the electron-microscope-level 3D datasets needed to completely reconstruct the connectivity of neuronal circuits.
1,506 citations
Cites background from "Directionally selective calcium sig..."
...We are particularly interested in the complete reconstruction of local neural circuits such as those that underlie the detection of motion in the retina (Barlow et al. 1964; Euler et al. 2002)....
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TL;DR: The authors' eyes send different 'images' of the outside world to the brain — an image of contours (line drawing), a colour image (watercolour painting) or a image of moving objects (movie) — and circuits that involve complex inhibitory and excitatory interactions represent filters that select 'what the eye tells the brain'.
Abstract: Our eyes send different 'images' of the outside world to the brain - an image of contours (line drawing), a colour image (watercolour painting) or an image of moving objects (movie). This is commonly referred to as parallel processing, and starts as early as the first synapse of the retina, the cone pedicle. Here, the molecular composition of the transmitter receptors of the postsynaptic neurons defines which images are transferred to the inner retina. Within the second synaptic layer - the inner plexiform layer - circuits that involve complex inhibitory and excitatory interactions represent filters that select 'what the eye tells the brain'.
1,099 citations
Cites methods from "Directionally selective calcium sig..."
...TWO-PHOTON MICROSCOPY was used to record Ca 2+ signals in the dendritic trees of cholinergic amacrine cells, and these signals showed direction-selective response...
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TL;DR: The principles of 2PE microscopy are reviewed, recent applications are highlighted, its limitations are discussed, and areas for future research and development are pointed to.
969 citations
Cites background from "Directionally selective calcium sig..."
...2PE microscopy has been exploited to dissect the mechanisms underlying direction-selectivity in the dendrites of retinal interneurons (Euler et al., 2002; Oesch et al., 2005)....
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TL;DR: Circuit motifs that emerge from the data indicate a functional mechanism for a known cellular response in a ganglion cell that detects localized motion, and predict that another ganglions cell is motion sensitive.
Abstract: 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.
929 citations
References
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TL;DR: The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation.
Abstract: Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.
8,905 citations
TL;DR: Experiments are described which show, first, that directional selectivity is not due to optical aberrations of some kind and, secondly, that it is not a simple matter of the latency of response varying systematically across the receptive field.
Abstract: Directionally selective single units have recently been found in the cerebral cortex of cats (Hubel, 1959; Hubel & Wiesel, 1959, 1962), the optic tectum of frogs and pigeons (Lettvin, Maturana, McCulloch & Pitts, 1959; Maturana & Frenk, 1963), and the retinae of rabbits (Barlow & Hill, 1963; Barlow, Hill & Levick, 1964). The term 'directionally selective' means that a unit gives a vigorous discharge of impulses when a stimulus object is moved through its receptive field in one direction (called the preferred direction), whereas motion in the reverse direction (called null) evokes little or no response. The preferred direction differs in different units, and the activity of a set of such units signals the direction of movement of objects in the visual field. In the rabbit the preferred and null directions cannot be predicted from a map of the receptive field showing the regions yielding on or off-responses to stationary spots. Furthermore, the preferred direction is unchanged by changing the stimulus; in particular, reversing the contrast of a spot or a black-white border does not reverse the preferred direction. Hubel & Wiesel (1962) thought that the directional selectivity of the cat's cortical neurons could be explained by the asymmetrical arrangement of on and off zones in the receptive field, and the simple interaction of effects summated over these zones, but the foregoing results rule out this explanation, at least in the rabbit's retina (Barlow & Hill, 1963). In the present paper we go on from this point to describe experiments which show, first, that directional selectivity is not due to optical aberrations of some kind and, secondly, that it is not a simple matter of the latency of response varying systematically across the receptive field. After these negative results we describe experiments upon the organization of directional selectivity within the receptive field, and upon its mechanism. These lead us to the conclusion that the ganglion cells responding to a
1,574 citations
TL;DR: The quantitative dependence of transmitter release on external calcium concentration has been studied at the frog neuromuscular junction, using intracellular recording and taking the amplitude of the end‐plate potential (e.p.p.) as an index of the number of packets released.
Abstract: 1. The quantitative dependence of transmitter release on external calcium concentration has been studied at the frog neuromuscular junction, using intracellular recording and taking the amplitude of the end-plate potential (e.p.p.) as an index of the number of packets released.
2. The relation between [Ca] and the e.p.p. is highly non-linear. The initial part of this relation on double logarithmic co-ordinates gives a straight line with a slope of nearly four (mean 3·78 ± 0·2 S.D. in 28 experiments). Addition of a constant amount of Mg reduces the e.p.p. without altering the slope of the log e.p.p./log Ca relation.
3. The slope of this logarithmic relation diminishes as [Ca] is raised towards the normal level.
4. The results are explained quantitatively on the hypothesis that Ca ions combine with a specific site X on the nerve terminal forming CaX, and that the number of packets of acetylcholine released is proportional to the fourth power of [CaX].
5. The analysis suggests that a co-operative action of about four calcium ions is necessary for the release of each quantal packet of transmitter by the nerve impulse.
1,277 citations
816 citations
TL;DR: The acetylcholine-synthesizing neurons of the rabbit retina were selectively stained by intraocular injection of the fluorescent dye 4, 6-diamidino-2-phenylindole (DAP1) and found to form a single morphological population.
Abstract: The acetylcholine-synthesizing neurons of the rabbit retina were selectively stained by intraocular injection of the fluorescent dye 4, 6-diamidino-2-phenylindole (DAP1). Retinas were then isolated from the eye, fixed for 10-30 min with 4% paraformaldehyde, and mounted flat on the stage of a fluorescence microscope. The acetylcholine-synthesizing cells were penetrated under visual control by microelectrodes filled with lucifer yellow CH. When the dye was electrophoretically injected into the cells, complete filling of their dendrites often occurred. Cells were successfully injected as long as one month after fixation of the tissue. Complete or nearly complete filling of 281 cells was accomplished, at retinal locations systematically covering the retinal surface. The cells stained with DAPI were found to form a single morphological population. They have two to seven primary dendrites, which branch repeatedly within a narrow plane and form a round or slightly oval dendritic tree. The branching becomes very fine for the distal one third of the dendritic tree, and the dendrites there are studded with small swellings. The distal dendritic tree lies mainly within one of the two thin strata of the inner plexiform layer where acetylcholine is present. The shape and size of the dendritic tree are continuously graded across the retina ; the dendritic tree is narrower and the branching denser in the central retina, wider and sparser in the periphery. From knowledge of the population density and the shape of the neurons, one can reconstruct the array of dendrites that exists within the inner plexiform layer. The overlap of the dendritic fields is an order of magnitude greater than of any other retinal neuron previously described. Because the cells not only overlap widely but branch quite profusely, a very dense plexus of cholinergic dendrites is created.
373 citations