imaging-ca2-dynamics-cone-photoreceptor-axon-terminals-mouse

Imaging Ca<sup>2+</sup> Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

University of Tübingen

For all nervous systems, motoneurons are the main output pathway. They are involved in generating episodic motor activity as well as enduring motor rhythms. To determine whether changes in cytosolic Ca(2+) correlate with motor performance, we studied the spatiotemporal dynamics, mode of entry and role of free intracellular Ca(2+) in cricket (Gryllus bimaculatus) front leg tibial extensor and flexor motoneurons. Synaptic activation or intracellular depolarising current injection uniformly increased Ca(2+) with the same dynamics throughout the primary and secondary branches of the dendritic tree of all motoneurons. Ca(2+) rise times (mean tau(rise), 233-295 ms) were lower than decay times (mean tau(decay), 1927-1965 ms), and resulted in a Ca(2+) plateau during repetitive activation, such as during walking. The neurons therefore operate with a different Ca(2+) level during walking than during episodic leg movements. Ca(2+) enters the dendritic processes of motoneurons via a voltage-activated mechanism. Entry is driven by subthreshold excitation, and is largely independent of the neurons' spiking activity. To what extent ligand-activated mechanisms of Ca(2+) entry operate remains uncertain. We found no evidence for any prominent Ca(2+)-activated secondary currents in these motoneurons. Excitatory postsynaptic potentials evoked by extracellular stimulation of descending neurons were unaffected by the level of free intracellular Ca(2+). The activity of tibial motoneurons therefore appears to be only weakly dependent on the level of free intracellular Ca(2+) in dendrites. This is different to what has been found for many other neurons studied, and may represent an essential prerequisite for insect motoneurons to support a wide range of both episodic and rhythmic motor sequences underlying behaviour.

Dynamics of free intracellular Ca2+ during synaptic and spike activity of cricket tibial motoneurons.

Presynaptic inhibition is a widespread mechanism modulating the efficiency of synaptic transmission and in sensory pathways is coupled to primary afferent depolarizations. Axonal terminals of bush-cricket auditory afferents received 2-5 mV graded depolarizing inputs, which reduced the amplitude of invading spikes and indicated presynaptic inhibition. These inputs were linked to a picrotoxin-sensitive increase of Ca(2+) in the terminals. Electrophysiological recordings and optical imaging showed that in individual afferents the sound frequency tuning based on spike rates was different from the tuning of the graded primary afferent depolarizations. The auditory neuropil of the bush-cricket Mecopoda elongata is tonotopically organized, with low frequencies represented anteriorly and high frequencies represented posteriorly. In contrast graded depolarizing inputs were tuned to high-frequencies anteriorly and to low-frequencies posteriorly. Furthermore anterior and posterior axonal branches of individual afferents received different levels of primary afferent depolarization depending on sound frequency. The presence of primary afferent depolarization in the afferent terminals indicates that presynaptic inhibition may shape the synaptic transmission of frequency-specific activity to auditory interneurons.

Primary afferent depolarization and frequency processing in auditory afferents.

Amacrine cells constitute a diverse, yet poorly characterized, cell population in the inner retina. Here, the authors sought to characterize the morphology, molecular physiology, and electrophysiology of a subpopulation of EGFP-expressing retinal amacrine cells identified in a novel zebrafish transgenic line.

Zebrafish Tg(7.2mab21l2:EGFP)ucd2 transgenics reveal a unique population of retinal amacrine cells.

Retinal bipolar cells have been assumed to generate purely graded responses to light. To test this idea we imaged the presynaptic calcium transient in live zebrafish. We found that ON, OFF, transient and sustained bipolar cells are all capable of generating fast 'all-or-none' calcium transients modulated by visual stimulation.

In vivo evidence that retinal bipolar cells generate spikes modulated by light.

The conversion of an analog stimulus into the digital form of spikes is a fundamental step in encoding sensory information. Here, we investigate this transformation in the visual system of fish by in vivo calcium imaging and electrophysiology of retinal bipolar cells, which have been assumed to be purely graded neurons.

Spikes in retinal bipolar cells phase-lock to visual stimuli with millisecond precision.

Far from being a simple sensor, the retina actively participates in processing visual signals. One of the best understood aspects of this processing is the detection of motion direction. Direction-selective (DS) retinal circuits include several subtypes of ganglion cells (GCs) and inhibitory interneurons, such as starburst amacrine cells (SACs). Recent studies demonstrated a surprising complexity in the arrangement of synapses in the DS circuit, i.e. between SACs and DS ganglion cells. Thus, to fully understand retinal DS mechanisms, detailed knowledge of all synaptic elements involved, particularly the nature and localization of neurotransmitter receptors, is needed. Since inhibition from SACs onto DSGCs is crucial for generating retinal direction selectivity, we investigate here the nature of the GABA receptors mediating this interaction. We found that in the inner plexiform layer (IPL) of mouse and rabbit retina, GABA(A) receptor subunit α2 (GABA(A)R α2) aggregated in synaptic clusters along two bands overlapping the dendritic plexuses of both ON and OFF SACs. On distal dendrites of individually labeled SACs in rabbit, GABA(A)R α2 was aligned with the majority of varicosities, the cell's output structures, and found postsynaptically on DSGC dendrites, both in the ON and OFF portion of the IPL. In GABA(A)R α2 knock-out (KO) mice, light responses of retinal GCs recorded with two-photon calcium imaging revealed a significant impairment of DS responses compared to their wild-type littermates. We observed a dramatic drop in the proportion of cells exhibiting DS phenotype in both the ON and ON-OFF populations, which strongly supports our anatomical findings that α2-containing GABA(A)Rs are critical for mediating retinal DS inhibition. Our study reveals for the first time, to the best of our knowledge, the precise functional localization of a specific receptor subunit in the retinal DS circuit.

GABA(A) receptors containing the α2 subunit are critical for direction-selective inhibition in the retina.

In the mammalian retina, 10-12 different cone bipolar cell (BC) types decompose the photoreceptor signal into parallel channels, providing the basis for the functional diversity of retinal ganglion cells (RGCs). BCs differing in their temporal properties appear to project to different strata of the retina's inner synaptic layer, based on somatic recordings of BCs and excitatory synaptic currents measured in RGCs. However, postsynaptic currents in RGCs are influenced by dendritic morphology and receptor types, and the BC signal can be transformed at the axon terminals both through interactions with amacrine cells and through the generation of all-or-nothing spikes. Therefore, the temporal properties of the BC output have not been analyzed systematically across different types of mammalian BCs. We recorded calcium signals directly within axon terminals using two-photon imaging and show that BCs can be divided into ≥eight functional clusters. The temporal properties of the BC output were directly reflected in their anatomical organization within the retina's inner synaptic layer: faster cells stratified closer to the border between ON and OFF sublamina. Moreover, ≥three fastest groups generated clear all-or-nothing spikes. Therefore, the systematic projection pattern of BCs provides distinct temporal "building blocks" for the feature extracting circuits of the inner retina.

Spikes in mammalian bipolar cells support temporal layering of the inner retina.

Image processing begins in the retina, where neurons respond with graded voltage changes that must be converted into spikes. This conversion from 'analog' to 'digital' coding is a fundamental transformation carried out by the visual system, but the mechanisms are still not well understood. Recent work demonstrates that, in vertebrates, graded-to-spiking conversion of the visual signal begins in the axonal system of bipolar cells (BCs), which transmit visual information through ribbon-type synapses specialized for responding to graded voltage signals. Here, we explore the evidence for and against the idea that ribbon synapses also transmit digital information. We then discuss the potential costs and benefits of digitization at different stages of visual pathways in vertebrates and invertebrates.

Spikes and ribbon synapses in early vision.

Advances in neuroscience research over the last few decades have increased our understanding of how individual neurons acquire their specific properties and assemble into complex circuits, and how these circuits are affected in disease. One of the important motives driving neuroscience research is the development of new scientific techniques and interdisciplinary cooperation. Compared to developed countries, many countries on the African continent are confronted with poor facilities, lack of funding or career development programs for neuroscientists, all of which deter young scientists from taking up neuroscience as a career choice. This article highlights some steps that are being taken to promote neuroscience education and research in Africa.

Bridging the Gap: establishing the necessary infrastructure and knowledge for teaching and research in neuroscience in Africa.

For efficient coding, sensory systems need to adapt to the distribution of signals to which they are exposed. In vision, natural scenes above and below the horizon differ in the distribution of chromatic and achromatic features. Consequently, many species differentially sample light in the sky and on the ground using an asymmetric retinal arrangement of short- (S, "blue") and medium- (M, "green") wavelength-sensitive photoreceptor types. Here, we show that in mice this photoreceptor arrangement provides for near-optimal sampling of natural achromatic contrasts. Two-photon population imaging of light-driven calcium signals in the synaptic terminals of cone-photoreceptors expressing a calcium biosensor revealed that S, but not M cones, preferred dark over bright stimuli, in agreement with the predominance of dark contrasts in the sky but not on the ground. Therefore, the different cone types do not only form the basis of "color vision," but in addition represent distinct (achromatic) contrast-selective channels.

A tale of two retinal domains: near-optimal sampling of achromatic contrasts in natural scenes through asymmetric photoreceptor distribution.

A recent study provides new insights into how the very different response characteristics of the main visual pathways from the eye to the brain may directly result from the presence or absence of a 'spike trigger-zone' in retinal bipolar cells.

Early vision: where (some of) the magic happens.

Retinal bipolar cells are the first ‘projection neurons’ of the vertebrate visual system—all of the information needed for vision is relayed by this intraretinal connection. Each of the at least 13 distinct types of bipolar cells systematically transforms the photoreceptor input in a different way, thereby generating specific channels that encode stimulus properties, such as polarity, contrast, temporal profile and chromatic composition. As a result, bipolar cell output signals represent elementary ‘building blocks’ from which the microcircuits of the inner retina derive a feature-oriented description of the visual world.

Retinal bipolar cells: elementary building blocks of vision.

The visual system transmits information about fast and slow changes in light intensity through separate neural pathways. We used in vivo imaging to investigate how bipolar cells transmit these signals to the inner retina. We found that the volume of the synaptic terminal is an intrinsic property that contributes to different temporal filters. Individual cells transmit through multiple terminals varying in size, but smaller terminals generate faster and larger calcium transients to trigger vesicle release with higher initial gain, followed by more profound adaptation. Smaller terminals transmitted higher stimulus frequencies more effectively. Modeling global calcium dynamics triggering vesicle release indicated that variations in the volume of presynaptic compartments contribute directly to all these differences in response dynamics. These results indicate how one neuron can transmit different temporal components in the visual signal through synaptic terminals of varying geometries with different adaptational properties.

A synaptic mechanism for temporal filtering of visual signals.

The introduction of affordable, consumer-oriented 3-D printers is a milestone in the current "maker movement," which has been heralded as the next industrial revolution. Combined with free and open sharing of detailed design blueprints and accessible development tools, rapid prototypes of complex products can now be assembled in one's own garage--a game-changer reminiscent of the early days of personal computing. At the same time, 3-D printing has also allowed the scientific and engineering community to build the "little things" that help a lab get up and running much faster and easier than ever before.

Article	Year
Dynamics of free intracellular Ca2+ during synaptic and spike activity of cricket tibial motoneurons. The European journal of neuroscience\| PubMed ID: 19309317	2009
Primary afferent depolarization and frequency processing in auditory afferents. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 21048145	2010
Zebrafish Tg(7.2mab21l2:EGFP)ucd2 transgenics reveal a unique population of retinal amacrine cells. Investigative ophthalmology & visual science\| PubMed ID: 21051702	2011
In vivo evidence that retinal bipolar cells generate spikes modulated by light. Nature neuroscience\| PubMed ID: 21706020	2011
Spikes in retinal bipolar cells phase-lock to visual stimuli with millisecond precision. Current biology : CB\| PubMed ID: 22055291	2011
GABA(A) receptors containing the α2 subunit are critical for direction-selective inhibition in the retina. PloS one\| PubMed ID: 22506070	2012
Spikes in mammalian bipolar cells support temporal layering of the inner retina. Current biology : CB\| PubMed ID: 23246403	2013
Spikes and ribbon synapses in early vision. Trends in neurosciences\| PubMed ID: 23706152	2013
Bridging the Gap: establishing the necessary infrastructure and knowledge for teaching and research in neuroscience in Africa. Metabolic brain disease\| PubMed ID: 24166356	2014
A tale of two retinal domains: near-optimal sampling of achromatic contrasts in natural scenes through asymmetric photoreceptor distribution. Neuron\| PubMed ID: 24314730	2013
Early vision: where (some of) the magic happens. Current biology : CB\| PubMed ID: 24355785	2013
Retinal bipolar cells: elementary building blocks of vision. Nature reviews. Neuroscience\| PubMed ID: 25158357	2014
A synaptic mechanism for temporal filtering of visual signals. PLoS biology\| PubMed ID: 25333637	2014
Open Labware: 3-D printing your own lab equipment. PLoS biology\| PubMed ID: 25794301	2015

Tom Baden

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