Visual processing: The Devil is in the details

AbstractGanglion cells convey information from the retina back to the brain. Recent experiments have examined how ganglion cell receptive fields are assembled from many incoming signals

The transfer characteristics of hair cells encoding mechanical stimuli in the lateral line of zebrafish

Hair cells transmit mechanical information by converting deflection of the hair bundle into synaptic release of glutamate. We have investigated this process in the lateral line of larval zebrafish (male and female) to understand how mechanical stimuli are encoded within a neuromast. Using multiphoton microscopy in vivo, we imaged synaptic release of glutamate using the reporter iGluSnFR and deflections of the cupula. We found that the neuromast is composed of a functionally diverse population of hair cells. Half the hair cells signalled cupula motion in both directions from rest, either by increasing glutamate release in response to a deflection in the positive direction or by reducing release in the negative direction. The relationship between cupula deflection and glutamate release demonstrated maximum sensitivity at displacements of just ~40 nm in the positive direction. The remaining hair cells only signalled motion in one direction and were less sensitive, extending the operating range of the neuromast beyond 1 μm. Adaptation of the synaptic output was also heterogeneous, with some hair cells generating sustained glutamate release in response to a steady deflection of the cupula and others generating transient outputs. Finally, a distinct signal encoded a return of the cupula to rest: a large and transient burst of glutamate release from hair cells unresponsive to the initial stimulus. A population of hair cells with these different sensitivities, operating ranges and adaptive properties will allow the neuromast to encode weak stimuli while maintaining the dynamic range to signal the amplitude and duration of stronger deflections

The mouth of a dense-core vesicle opens and closes in a concerted action regulated by calcium and amphiphysin

Secretion of hormones and peptides by neuroendocrine cells occurs through fast and slow modes of vesicle fusion but the mechanics of these processes are not understood. We used interference reflection microscopy to monitor deformations of the membrane surface and found that both modes of fusion involve the tightly coupled dilation and constriction of the vesicle. The rate of opening is calcium dependent and occurs rapidly at concentrations <5 μM. The fast mode of fusion is blocked selectively by a truncation mutant of amphiphysin. Vesicles do not collapse when fusion is triggered by strontium, rather they remain locked open and membrane scission is blocked. In contrast, constriction of the vesicle opening continues when endocytosis is blocked by inhibiting the function of dynamin. Thus, fast and slow modes of fusion involve similar membrane deformations and vesicle closure can be uncoupled from membrane scission. Regulation of these processes by calcium and amphiphysin may provide a mechanism for controlling the release of vesicle contents

The mouth of a dense-core vesicle opens and closes in a concerted action regulated by calcium and amphiphysin

Secretion of hormones and peptides by neuroendocrine cells occurs through fast and slow modes of vesicle fusion but the mechanics of these processes are not understood. We used interference reflection microscopy to monitor deformations of the membrane surface and found that both modes of fusion involve the tightly coupled dilation and constriction of the vesicle. The rate of opening is calcium dependent and occurs rapidly at concentrations <5 muM [corrected] The fast mode of fusion is blocked selectively by a truncation mutant of amphiphysin. Vesicles do not collapse when fusion is triggered by strontium, rather they remain locked open and membrane scission is blocked. In contrast, constriction of the vesicle opening continues when endocytosis is blocked by inhibiting the function of dynamin. Thus, fast and slow modes of fusion involve similar membrane deformations and vesicle closure can be uncoupled from membrane scission. Regulation of these processes by calcium and amphiphysin may provide a mechanism for controlling the release of vesicle contents

Exocytosis at the ribbon synapse of retinal bipolar cells studied in patches of presynaptic membrane

The distribution of exocytic sites and ion channels in the synaptic terminal of retinal bipolar cells was investigated by measuring capacitance and conductance changes in cell-attached patches of presynaptic membrane. Patch depolarization evoked capacitance and conductance increases that were inhibited by blocking Ca(2+) influx or loading the terminal with EGTA. The increase in capacitance declined as the depolarization approached the reversal potential for Ca(2+), indicating that it was a result of Ca(2+)-dependent exocytosis. The conductance increase was caused by K(Ca) channels that were also activated by Ca(2+) influx. Two observations indicated that sites of exocytosis and endocytosis colocalized with clusters of Ca(2+) channels and K(Ca) channels; the initial rate of exocytosis was correlated with the activation of K(Ca) channels, and exocytosis did not occur in the 41% of patches lacking this conductance. Electron microscopy demonstrated that there were approximately 16 vesicles docked to the plasma membrane at each active zone marked by a ribbon, but vesicles were also attached to the rest of the membrane at a density of 1.5/microm(2). The density of ribbons was 0.10 +/- 0.02/microm(2), predicting that approximately 43% of cell-attached patches would lack an active zone. The density of Ca(2+) channel clusters assayed by capacitance and conductance responses was therefore similar to the density of ribbons. These results are consistent with the idea that Ca(2+) channel clusters were colocalized with ribbons but do not exclude the possibility that calcium channels also occurred at other sites. The wide distribution of vesicles docked to the plasma membrane suggests that exocytosis might also be triggered by the spread of Ca(2+) from Ca(2+) channel clusters

Rapid mapping of visual receptive fields by filtered back-projection: application to multi-neuronal electrophysiology and imaging

Neurons in the visual system vary widely in the spatiotemporal properties of their receptive fields (RFs), and understanding these variations is key to elucidating how visual information is processed. We present a new approach for mapping RFs based on the filtered back projection (FBP), an algorithm used for tomographic reconstructions. To estimate RFs, a series of bars were flashed across the retina at pseudo‐random positions and at a minimum of five orientations. We apply this method to retinal neurons and show that it can accurately recover the spatial RF and impulse response of ganglion cells recorded on a multi‐electrode array. We also demonstrate its utility for in vivo imaging by mapping the RFs of an array of bipolar cell synapses expressing a genetically encoded Ca2+ indicator. We find that FBP offers several advantages over the commonly used spike‐triggered average (STA): (i) ON and OFF components of a RF can be separated; (ii) the impulse response can be reconstructed at sample rates of 125 Hz, rather than the refresh rate of a monitor; (iii) FBP reveals the response properties of neurons that are not evident using STA, including those that display orientation selectivity, or fire at low mean spike rates; and (iv) the FBP method is fast, allowing the RFs of all the bipolar cell synaptic terminals in a field of view to be reconstructed in under 4 min. Use of the FBP will benefit investigations of the visual system that employ electrophysiology or optical reporters to measure activity across populations of neurons

A novel tool to measure extracellular glutamate in the Zebrafish nervous system in vivo

Glutamate is the major excitatory neurotransmitter in the brain. Its release and eventual recycling are key to rapid sustained neural activity. We have paired the gfap promoter region with the glutamate reporter molecule, iGluSnFR, to drive expression in glial cells throughout the nervous system. Tg(gfap:iGluSnFR) is expressed on the glial membrane of Müller glia cells in the retina, which rapidly respond to stimulation and the release of extracellular glutamate. As glial cells are associated with most, if not all, synapses, Tg(gfap:iGluSnFR) is a novel and exciting tool to measure neuronal activity and extracellular glutamate dynamics in many regions of the nervous system. Glutamate is the major excitatory neurotransmitter in the brain. Its release and eventual recycling are key to rapid sustained neural activity.1 Glial cells play a key role in the uptake and recycling of glutamate from the synaptic cleft. iGluSnFR has been used to study synaptic activity by measuring glutamate dynamics in the zebrafish nervous system.2,3 Previous work has also used iGluSnFR in glial cells; however, this was done transiently in the mouse using viral vectors.2,4 As such, we designed a transgene to stably express iGluSnFR in the glial cells of the zebrafish nervous system. We report a novel transgenic zebrafish, Tg(gfap:iGluSnFR), that displays the glutamate-sensitive fluorescent reporter iGluSnFR specifically on the membrane of glial cells (Figure 1A–C). This molecule is expressed on the glial membrane in many brain regions and rapidly responds to stimulation and the release of extracellular glutamate (Figure 1D–F, Supplementary Data; Supplementary Data are available online at www.liebertpub.com/zeb). Thus, pairing the sensitivity of iGluSnFR and optical transparency of the zebrafish provides a powerful tool for understanding glutamate dynamics in neural tissues in vivo

An amplitude code transmits information at a visual synapse

Most neurons transmit information digitally using spikes that trigger the release of synaptic vesicles with low probability. The first stages of vision and hearing are distinct in that they operate with analog signals, but it is unclear how these are recoded for synaptic transmission. By imaging the release of glutamate in live zebrafish, we demonstrate that ribbon synapses of retinal bipolar cells encode contrast through changes in both the frequency and amplitude of release events. Higher contrasts caused multiple vesicles to be released within an event, and such coding by amplitude often continued after the rate code had reached a maximum frequency. Glutamate packets equivalent to five vesicles transmitted four times as many bits of information per vesicle compared with those released individually. By discretizing analog signals into sequences of numbers up to about 11, ribbon synapses can increase the dynamic range, temporal precision and efficiency with which visual information is transmitted

A retinal circuit generating a dynamic predictive code for oriented features

Sensory systems must reduce the transmission of redundant information to function efficiently. One strategy is to continuously adjust the sensitivity of neurons to suppress responses to common features of the input while enhancing responses to new ones. Here we image the excitatory synaptic inputs and outputs of retinal ganglion cells to understand how such dynamic predictive coding is implemented in the analysis of spatial patterns. Synapses of bipolar cells become tuned to orientation through presynaptic inhibition generating lateral antagonism in the orientation domain. Individual ganglion cells receive excitatory synapses tuned to different orientations but feedforward inhibition generates a high-pass filter that only transmits the initial activation of these inputs, thereby removing redundancy. These results demonstrate how a dynamic predictive code can be implemented by circuit motifs common to many parts of the brain