A dendrite-autonomous mechanism for direction selectivity in retinal starburst amacrine cells

Detection of image motion direction begins in the retina, with starburst amacrine cells (SACs) playing a major role. SACs generate larger dendritic Ca²⁺ signals when motion is from their somata towards their dendritic tips than for motion in the opposite direction. To study the mechanisms underlying the computation of direction selectivity (DS) in SAC dendrites, electrical responses to expanding and contracting circular wave visual stimuli were measured via somatic whole-cell recordings and quantified using Fourier analysis. Fundamental and, especially, harmonic frequency components were larger for expanding stimuli. This DS persists in the presence of GABA and glycine receptor antagonists, suggesting that inhibitory network interactions are not essential. The presence of harmonics indicates nonlinearity, which, as the relationship between harmonic amplitudes and holding potential indicates, is likely due to the activation of voltage-gated channels. [Ca²⁺] changes in SAC dendrites evoked by voltage steps and monitored by two-photon microscopy suggest that the distal dendrite is tonically depolarized relative to the soma, due in part to resting currents mediated by tonic glutamatergic synaptic input, and that high-voltage–activated Ca²⁺ channels are active at rest. Supported by compartmental modeling, we conclude that dendritic DS in SACs can be computed by the dendrites themselves, relying on voltage-gated channels and a dendritic voltage gradient, which provides the spatial asymmetry necessary for direction discrimination.     

Realistic retinal modeling unravels the differential role of excitation and inhibition to starburst amacrine cells in direction selectivity

Retinal direction-selectivity originates in starburst amacrine cells (SACs), which display a centrifugal preference, responding with greater depolarization to a stimulus expanding from soma to dendrites than to a collapsing stimulus. Various mechanisms were hypothesized to underlie SAC centrifugal preference, but dissociating them is experimentally challenging and the mechanisms remain debatable. To address this issue, we developed the Retinal Stimulation Modeling Environment (RSME), a multifaceted data-driven retinal model that encompasses detailed neuronal morphology and biophysical properties, retina-tailored connectivity scheme and visual input. Using a genetic algorithm, we demonstrated that spatiotemporally diverse excitatory inputs–sustained in the proximal and transient in the distal processes–are sufficient to generate experimentally validated centrifugal preference in a single SAC. Reversing these input kinetics did not produce any centrifugal-preferring SAC. We then explored the contribution of SAC-SAC inhibitory connections in establishing the centrifugal preference. SAC inhibitory network enhanced the centrifugal preference, but failed to generate it in its absence. Embedding a direction selective ganglion cell (DSGC) in a SAC network showed that the known SAC-DSGC asymmetric connectivity by itself produces direction selectivity. Still, this selectivity is sharpened in a centrifugal-preferring SAC network. Finally, we use RSME to demonstrate the contribution of SAC-SAC inhibitory connections in mediating direction selectivity and recapitulate recent experimental findings. Thus, using RSME, we obtained a mechanistic understanding of SACs’ centrifugal preference and its contribution to direction selectivity.     

A model of direction selectivity in the starburst amacrine cell network

Displaced starburst amacrine cells (SACs) are retinal interneurons that exhibit GABA _A receptor-mediated and Cl ^? cotransporter-mediated, directionally selective (DS) light responses in the rabbit retina. They depolarize to stimuli that move centrifugally through the receptive field surround and hyperpolarize to stimuli that move centripetally through the surround (Gavrikov et al, PNAS 100(26):16047–16052, 2003, PNAS 103(49):18793–18798, 2006). They also play a key role in the activity of DS ganglion cells (DS GC; Amthor et al, Vis Neurosci 19:495–509 2002; Euler et al, Nature 418:845–852, 2002; Fried et al, Nature 420:411– 414, 2002; Gavrikov et al, PNAS 100(26):16047–16052, 2003, PNAS 103(49):18793–18798, 2006; Lee and Zhou, Neuron 51:787–799 2006; Yoshida et al, Neuron 30:771–780, 2001). In this paper we present a model of strong DS behavior of SACs which relies on the GABA-mediated communication within a tightly interconnected network of these cells and on the glutamate signal that the SACs receive from bipolar cells (a presynaptic cell that receives input from cones). We describe how a moving light stimulus can produce a large, sustained depolarization of the SAC dendritic tips that point in the direction that the stimulus moves (i.e., centrifugal motion), but produce a minimal depolarization of the dendritic tips that point in the opposite direction (i.e., centripetal motion). This DS behavior, which is quantified based on the relative size and duration of the depolarizations evoked by stimulus motion at dendritic tips pointing in opposite directions, is robust to changes of many different parameter values and consistent with experimental data. In addition, the DS behavior is strengthened under the assumptions that the Cl^? cotransporters Na^?+?-K^?+?-Cl^?? and K^?+?-Cl^?? are located in different regions of the SAC dendritic tree (Gavrikov et al, PNAS 103(49):18793–18798, 2006) and that GABA evokes a long-lasting response (Gavrikov et al, PNAS 100(26):16047–16052, 2003, PNAS 103(49):18793–18798, 2006; Lee and Zhou, Neuron 51:787–799, 2006). A possible mechanism is discussed based on the generation of waves of local glutamate and GABA secretion, and their postsynaptic interplay as the waves travel between cell compartments.     

The synaptic mechanism of direction selectivity in distal processes of starburst amacrine cells

Patch-clamp recordings revealed that distal processes of starburst amacrine cells (SACs) received largely excitatory synaptic input from the receptive field center and nearly purely inhibitory inputs from the surround during both stationary and moving light stimulations. The direct surround inhibition was mediated mainly by reciprocal GABA(A) synapses between opposing SACs, which provided leading and prolonged inhibition during centripetal stimulus motion. Simultaneous Ca(2+) imaging and current-clamp recording during apparent-motion stimulation further demonstrated the contributions of both centrifugal excitation and GABA(A/C)-receptor-mediated centripetal inhibition to the direction-selective Ca(2+) responses in SAC distal processes. Thus, by placing GABA release sites in electrotonically semi-isolated distal processes and endowing these sites with reciprocal GABA(A) synapses, SACs use a radial-symmetric center-surround receptive field structure to build a polar-asymmetric circuitry. This circuitry may integrate at least three levels of interactions--center excitation, surround inhibition, and reciprocal inhibitions that amplify the center--surround antagonism-to generate robust direction selectivity in the distal processes.     

Modulation of synaptic function in retinal amacrine cells

Amacrine cells are interneurons that have diverse functionsin retinal signal processing. In order to study signaling andmodulation in retinal amacrine cells, we employ a simplifiedculture system containing identifiable GABAergic amacrine cells.Immunocytochemistry experiments indicate that GABAergic amacrinecells express metabotropic glutamate receptor 5 (mGluR5), agroup I mGluR usually linked to the IP3 signaling pathway. Ca²⁺imaging experiments using an mGluR5-specific agonist indicatethat these receptors are functional and when activated, canstimulate temporally diverse Ca²⁺ elevations. To begin to establishthe role of these receptors in modulating amacrine cell function,we have used electrophysiological methods to ask whether ionchannels are the targets of mGluR5-dependent modulation. Herewe discuss our results indicating that activation of mGluR5leads to enhancement of currents through GABA_A receptors. Thisenhancement is dependent upon elevations in cytosolic Ca²⁺ andactivation of protein kinase C (PKC). To explore the consequencesof Ca²⁺ elevations in another context, we have used nitric oxide(NO) donors to mimic the effects of activating the Ca²⁺-dependentsynthetic enzyme for NO, neuronal nitric oxide synthase. Wefind that exposure to NO donors also enhances the amplitudeof currents through GABA_A receptors. Together, these resultsindicate that glutamate from presynaptic bipolar cells has thepotential to work through multiple mechanisms to regulate thefunction of amacrine-to-amacrine cell GABAergic synapses.     

Modelling the electrotonic structure of starburst amacrine cells in the rabbit retina: A functional interpretation of dendritic morphology

A detailed morphometric analysis of a Lucifer yellow-filled Cb amacrine cell was undertaken to provide raw data for the construction of a neuronal cable model. The cable model was employed to determine whether distal input-output regions of dendrites were electrically isolated from the soma and each other. Calculations of steady state electrotonic current spread suggested reasonable electrical communication between cell body and dendrites. In particular, the centripetal voltage attenuation revealed that a synaptic signal introduced at the distal end of the equivalent dendrite could spread passively along the dendrite and reach the soma with little loss in amplitude. A functional interpretation of this results could favour a postsynaptic rather than a presynaptic scheme for the operation of directional selectivity in the rabbit retina. On the other hand, dendrites of starburst amacrine cells process information electrotonically with a bias towards the centrifugal direction and for a restricted range of membrane resistance values the voltage attenuation in the centripetal direction suggests that the action of these dendrites can be confined locally. A functional interpretation of this result favours a presynaptic version of Vaney's cotransmission model which attempts to explain how the neural network of starburst amacrine cells might account for directionally selective responses observed in the rabbit retina.     

Metabotropic glutamate receptor 5 and calcium signaling in retinal amacrine cells

To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)-dependent Ca(2+) elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both G(q) and G(s). Selective activation of mGluR5 stimulated Ca(2+) elevations that varied in waveform from cell to cell. Experiments using high external K(+) revealed that the mGluR5-dependent Ca(2+) elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca(2+) -free external solution demonstrated that the variability in the time course of mGluR5-dependent Ca(2+) elevations is largely due to the influx of extracellular Ca(2+). The sensitivity of the initial phase of the Ca(2+) elevation to thapsigargin indicates that this phase of the response is due to the release of Ca(2+) from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5-mediated Ca(2+) elevations are dependent upon the activation of phospholipase C. We rule out a role for L-type Ca(2+) channels and cAMP-gated channels as pathways for Ca(2+) entry, but provide evidence of transient receptor potential (TRP) channel-like immunoreactivity, suggesting that Ca(2+) influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C-dependent Ca(2+) release and Ca(2+) influx, possibly through TRP channels.     

Antibodies against neurofilament subunits label retinal ganglion cells but not displaced amacrine cells of hamsters.

Although neurofilament (NF) antibodies have been used to visualize ganglion cells and their axons in the retina, it is not known, however, how many ganglion cells contain NF, and how the various NF subunits are distributed in the ganglion cells. Moreover, it is not known whether displaced amacrine cells in the ganglion cell layer are also labelled. In order to see whether NF antibodies can be used as a specific marker for ganglion cells, antibodies raised against the low (NF-L), middle (NF-M) and high (NF-H) molecular weight subunits of NF were employed to stain retinal whole-mounts of adult hamsters after pre-labelling the ganglion cells with Granular Blue. It was found that NF-L and NF-H antibodies labelled 38,777 and 17,750 cells in the ganglion cell layer respectively. By co-localization with GB-labelled cells, 88% of NF-L positive cells and 91% of NF-H positive cells were found to be ganglion cells. In contrast, the NF-M antibody labelled only very few ganglion cells (418 per retina) although robust staining of axonal bundles was observed. Thus, NF antibodies may prove useful in studying this population of ganglion cells.     

Asymmetric temporal properties in the receptive field of retinal transient amacrine cells

The speed of signal conduction is a factor determining the temporal properties of individual neurons and neuronal networks. We observed very different conduction velocities within the receptive field of fast-type On-Off transient amacrine cells in carp retina cells, which are tightly coupled to each other via gap junctions. The fastest speeds were found in the dorsal area of the receptive fields, on average five times faster than those detected within the ventral area. The asymmetry was similar in the On- and Off-part of the responses, thus being independent of the pathway, pointing to the existence of a functional mechanism within the recorded cells themselves. Nonetheless, the spatial decay of the graded-voltage photoresponse within the receptive field was found to be symmetrical, with the amplitude center of the receptive field being displaced to the faster side from the minimum-latency location. A sample of the orientation of varicosity-laden polyaxons in neurobiotin-injected cells supported the model, revealing that approximately 75% of these processes were directed dorsally from the origin cells. Based on these results, we modeled the velocity asymmetry and the displacement of amplitude center by adding a contribution of an asymmetric polyaxonal inhibition to the network. Due to the asymmetry in the conduction velocity, the time delay of a light response is proposed to depend on the origin of the photostimulus movement, a potentially important mechanism underlying direction selectivity within the inner retina.     

Essential role of phosphoinositide 3-kinase delta in neutrophil directional movement

Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3K delta in neutrophil chemotaxis, we have developed a potent and selective PI3K delta inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3K delta activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3K delta, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3K delta can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.     

Neurocalcin, a novel calcium binding protein with three EF-hand domains, expressed in retinal amacrine cells and ganglion cells.

Neurocalcin (molecular weight 23,000 and 24,000) is a newly identified Ca2+ binding protein with three EF-hand domains and has a strong amino acid sequence homology with visinin and recoverin (Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. J. Biol. Chem. In press). We produced antibody against neurocalcin. Immunoblotting showed the presence of neurocalcin in bovine retina as well as brain, suggesting that neurocalcin was a neuron specific Ca2+ binding protein. Immunohistochemistry revealed the expression of neurocalcin in retinal amacrine cells and ganglion cells but not in the photoreceptor layer. This distribution of neurocalcin was quite different from that of visinin and recoverin. Our results suggest that neurocalcin may play an important role in a Ca2+ signal pathway of the nervous system.     

Mechanism of generation of spontaneous miniature outward currents (SMOCs) in retinal amacrine cells

A subtype of retinal amacrine cells displayed a distinctive array of K(+) currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of -60 to -40 mV. Depolarizations above approximately -40 mV were associated with the disappearance of SMOCs and the appearance of transient (I(to)) and sustained (I(so)) outward K(+) currents. I(to) appeared at about -40 mV and its apparent magnitude was biphasic with voltage, whereas I(so) appeared near -30 mV and increased linearly. SMOCs, I(to), and a component of I(so) were Ca(2+) dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of -20 mV and above. Their mean amplitude was Nernstian with respect to [K(+)](ext) and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca(2+)-free solutions, indicative of BK-type Ca(2+)-activated K(+) currents. Dihydropyridine Ca(2+) channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca(2+) permeation through the kainic acid receptor had no effect. Blockade of organelle Ca(2+) channels by ryanodine, or intracellular Ca(2+) store depletion with caffeine, eradicated SMOCs. Internal Ca(2+) chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca(2+) influx through L-type Ca(2+) channels and its subsequent amplification by Ca(2+)-induced Ca(2+) release via the ryanodine receptor leads to a localized elevation of internal Ca(2+). This amplified Ca(2+) signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs.     

Calcium-induced transitions between the spontaneous miniature outward and the transient outward currents in retinal amacrine cells

Spontaneous miniature outward currents (SMOCs) occur in a subset of retinal amacrine cells at membrane potentials between -60 and -40 mV. At more depolarized potentials, a transient outward current (I(to)) appears and SMOCs disappear. Both SMOCs and the I(to) are K(+) currents carried by BK channels. They both arise from Ca(2+) influx through high voltage-activated (HVA) Ca(2+) channels, which stimulates release of internal Ca(2+) from caffeine- and ryanodine-sensitive stores. An increase in Ca(2+) influx resulted in an increase in SMOC frequency, but also led to a decline in SMOC mean amplitude. This reduction showed a temporal dependence: the effect being greater in the latter part of a voltage step. Thus, Ca(2+) influx, although required to generate SMOCs, also produced a negative modulation of their amplitudes. Increasing Ca(2+) influx also led to a decline in the first latency to SMOC occurrence. A combination of these effects resulted in the disappearance of SMOCs, along with the concomitant appearance of the I(to) at high levels of Ca(2+) influx. Therefore, low levels of Ca(2+) influx, arising from low levels of activation of the HVA Ca(2+) channels, produce randomly occurring SMOCs within the range of -60 to -40 mV. Further depolarization leads to greater activation of the HVA Ca(2+) channels, larger Ca(2+) influx, and the disappearance of discontinuous SMOCs, along with the appearance of the I(to). Based on their characteristics, SMOCs in retinal neurons may function as synaptic noise suppressors at quiescent glutamatergic synapses.     

Spatial heterogeneity and function of voltage- and ligand-gated ion channels in retinal amacrine neurons.

The spatial distribution of ion channels within amacrine cells of the tiger salamander retina was studied using patch recording in the retinal slice preparation. By focally puffing kainate, GABA and glycine at amacrine cell processes in the inner plexiform layer, it was determined that the cell's glutamate receptors were located in a confined region of the processes near the soma, while glycine and GABA receptors were located throughout the processes. Likewise, similar techniques in conjunction with voltage steps demonstrated that voltage-gated sodium channels were located throughout the cell and were shown to generate sodium-dependent spikes, while only the processes contained voltage-gated calcium channels. These results suggest that this form of transient amacrine cell collects its excitatory synaptic inputs in a region confined to a central annular region near the soma, that the signal is actively propagated throughout its processes by voltage-gated sodium channels and that calcium-dependent neurotransmitter release of glycine from this neuron can occur throughout its processes. Thus, excitatory signals are collected in the processes near the soma, inhibitory signals throughout the processes and excitation is probably propagated throughout the processes of the amacrine cell.     

Center-surround,orientation, and directional properties of turtle retinal horizontal cells

The spatial transfer functions (STF) of L-type horizontal cells (HC) in turtle retina were measured using drifting sine wave grating stimuli. Two classes of STF were identified: low-pass and band-pass. A low-pass STF corresponds to a linespread function (LSF) having an excitatory center that attenuates montonically with distance; a band-pass STF corresponds to a LSF with an excitatory center and an inhibitory surround. Two models of the surround inhibitory mechanism, based on retinal outer plexiform layer (OPL) anatomy, are tested experimentally: surround mediated lateral inhibition and surround modulated self-inhibition. In both types, sign inverting pathways are based on GABA feedback synapses, and sign conserving pathways are based on excitatory synapses and gap junctions. Temperature variation was used to modify synaptic properties and study their effect on STF. The low frequency limb of band-pass STF was most sensitive to temperature changes; its slope increased with decreasing temperature. Synaptic properties were also manipulated pharmacologically. Cutoff frequency of low-pass STF decreased from 0.5 to 0.4 cpmm during exogenous GABA. Picrotoxin (PTX) increases upper cutoff frequency and decreases low frequency limb slope in band-pass STF. Band-pass STF of a ganglion cell (GC) has higher upper and lower cutoff frequencies than a HC in the same retinal region, which corresponds to strong spatial convergence from HC to GC. Orientation sensitivity and directional selectivity were found in some HC. Differences between major and minor response axes in orientation sensitive HC were small, ca. 2 dB; orientation differences in directionally selective HC were also small (ca. 1–2 dB) but directional asymmetry was large (ca. 10–12 dB).     

Numerical study of the effect of head and eye movement on progression of retinal detachment

Rhegmatogenous retinal detachment (RD) is a sight threatening condition. In this type of RD a break in the retina allows retrohyaloid fluid to enter the subretinal space. The prognosis concerning the patients’ visual acuity is better if the RD has not progressed to the macula. The patient is given a posturing advice of bed rest and semi-supine positioning (with the RD as low as possible) to allow the utilisation of gravity and immobilisation in preventing progression of the RD. It is, however, unknown what external loads on the eye contribute the most to the progression of a RD. The goal of this exploratory study is to elucidate the role of eye movements caused by head movements and saccades on the progression of an RD. A finite element model is produced and evaluated in this study. The model is based on geometric and material properties reported in the literature. The model shows that a mild head movement and a severe eye movement produce similar traction loads on the retina. This implies that head movements—and not eye movements—are able to cause loads that can trigger and progress an RD. These preliminary results suggest that head movements have a larger effect on the progression of an RD than saccadic eye movements. This study is the first to use numerical analysis to investigate the development and progression of RD and shows promise for future work.     

Group I metabotropic glutamate receptors are expressed in the chicken retina and by cultured retinal amacrine cells

Glutamate is well established as an excitatory neurotransmitter in the vertebrate retina. Its role as a modulator of retinal function, however, is poorly understood. We used immunocytochemistry and calcium imaging techniques to investigate whether metabotropic glutamate receptors are expressed in the chicken retina and by identified GABAergic amacrine cells in culture. Antibody labeling for both metabotropic glutamate receptors 1 and 5 in the retina was consistent with their expression by amacrine cells as well as by other retinal cell types. In double-labeling experiments, most metabotropic glutamate receptor 1-positive cell bodies in the inner nuclear layer also label with anti-GABA antibodies. GABAergic amacrine cells in culture were also labeled by metabotropic glutamate receptor 1 and 5 antibodies. Metabotropic glutamate receptor agonists elicited Ca(2+) elevations in cultured amacrine cells, indicating that these receptors were functionally expressed. Cytosolic Ca(2+) elevations were enhanced by metabotropic glutamate receptor 1-selective antagonists, suggesting that metabotropic glutamate receptor 1 activity might normally inhibit the Ca(2+) signaling activity of metabotropic glutamate receptor 5. These results demonstrate expression of group I metabotropic glutamate receptors in the avian retina and suggest that glutamate released from bipolar cells onto amacrine cells might act to modulate the function of these cells.