Peter B. Detwiler

Directionally selective calcium signals in dendrites of starburst amacrine cells

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.

Molecular cloning and characterization of retinal photoreceptor guanylyl cyclase-activating protein

Guanylyl cyclase-activating protein (GCAP) is thought to mediate Ca(2+)-sensitive regulation of guanylyl cyclase (GC), a key event in recovery of the dark state of rod photoreceptors following light exposure. Here, we characterize GCAP from several vertebrate species by molecular cloning and provide evidence that GCAP contains a heterogeneously acylated N-terminal region that interacts with GC. Vertebrate GCAPs consist of 201-205 amino acids, and sequence analysis indicates the presence fo three EF hand Ca(2+)-binding motifs. These results establish that GCAP is a novel photoreceptor-specific member of a large family of Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-sensitive activation of GC.

The calcium feedback signal in the phototransduction cascade of vertebrate rods

Intracellular free Ca (Cai) was measured in functionally intact rod outer segments in darkness and during light responses using the fluorescent Ca indicator Indo-dextran. In darkness, Cai was 554 +/- 25 nM (n = 28) for -85 +/- 2 pA of circulating dark current (Id) and declined in saturating light to a minimum value of approximately 50 nM with a time course that paralleled the fall in Na:Ca,K exchange current. During a subsaturating flash response that reduced Id by 70%, Cai fell to a minimum of approximately 325 nM and recovered incompletely to a plateau of approximately 450 nM that lasted approximately 15 s after full recovery of Id. During a 60 s step that caused approximately 7-fold reduction in sensitivity of superimposed flash responses, Cai reached a steady-state level of approximately 252 nM.

The effect of recoverin-like calcium-binding proteins on the photoresponse of retinal rods.

The rod photoresponse is triggered by an enzyme cascade that stimulates cGMP hydrolysis. The resulting fall in cGMP leads to a decrease in Ca2+, which promotes photoresponse recovery by activating guanylate cyclase, causing cGMP resynthesis. In vitro biochemical studies suggest that Ca2+ activation of guanylate cyclase is medicated by recoverin, a 26 kd Ca(2+)-binding protein. To evaluate this, exogenous bovine recoverin and two other homologous Ca(2+)-binding proteins from chicken and Gecko retina were dialyzed into functionally intact Gecko rods using whole-cell recording. All three proteins prolonged the rising phase of the photoresponse without affecting the kinetics of response recovery. These results suggest that recoverin-like proteins affect termination of the transduction cascade, rather than mediate Ca(2+)-sensitive activation of guanylate cyclase.

Functional Stability of Retinal Ganglion Cells after Degeneration-induced Changes in Synaptic Input

Glutamate released from photoreceptors controls the activity and output of parallel pathways in the retina. When photoreceptors die because of degenerative diseases, surviving retinal networks are left without their major source of input, but little is known about how photoreceptor loss affects ongoing synaptic activity and retinal output. Here, we use patch-clamp recording and two-photon microscopy to investigate morphological and physiological properties of identified types of ON and OFF retinal ganglion cells (RGCs) in the adult (36–210 d old) retinal degeneration rd-1/rd-1 mouse. We find that strong rhythmic synaptic input drives ongoing oscillatory spike activity in both ON and OFF RGCs at a fundamental “beating” frequency of ∼10 Hz. Despite this aberrant activity, ON and OFF cells maintain their characteristic dendritic stratification, intrinsic firing properties, including rebound firing in OFF cells, balance of synaptic excitation and inhibition, and dendritic calcium signaling. Thus, RGCs are inherently stable during degeneration-induced retinal activity.

The influence of arrestin (48K protein) and rhodopsin kinase on visual transduction

The shutoff of the phototransduction cascade in retinal rods requires the inactivation of light-activated rhodopsin. The underlying mechanisms were studied in functionally intact detached rod outer segments by testing the effect of either sangivamycin, an inhibitor of rhodopsin kinase, or phytic acid, an inhibitor of 48K protein binding to phosphorylated rhodopsin, on light responses recorded in whole-cell voltage clamp. The results suggest that isomerized rhodopsin is inactivated fully by multiple phosphorylation and that the binding of 48K protein accelerates recovery by quenching partially phosphorylated rhodopsin. Higher concentrations of sangivamycin cause changes in the light response that cannot be explained by selective inhibition of rhodopsin kinase and suggest that other protein kinases are needed for normal rod function.

Different mechanisms generate maintained activity in ON and OFF retinal ganglion cells.

Neuronal discharge is driven by either synaptic input or cell-autonomous intrinsic pacemaker activity. It is commonly assumed that the resting spike activity of retinal ganglion cells (RGCs), the output cells of the retina, is driven synaptically, because retinal photoreceptors and second-order cells tonically release neurotransmitter. Here we show that ON and OFF RGCs generate maintained activity through different mechanisms: ON cells depend on tonic excitatory input to drive resting activity, whereas OFF cells continue to fire in the absence of synaptic input. In addition to spontaneous activity, OFF cells exhibit other properties of pacemaker neurons, including subthreshold oscillations, burst firing, and rebound excitation. Thus, variable weighting of synaptic mechanisms and intrinsic properties underlies differences in the generation of maintained activity in these parallel retinal pathways.

Deletion of PrBP/δ impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments

The mouse Pde6d gene encodes a ubiquitous prenyl binding protein, termed PrBP/δ, of largely unknown physiological function. PrBP/δ was originally identified as a putative rod cGMP phosphodiesterase (PDE6) subunit in the retina, where it is relatively abundant. To investigate the consequences of Pde6d deletion in retina, we generated a Pde6d−/− mouse by targeted recombination. Although manifesting reduced body weight, the Pde6d−/− mouse was viable and fertile and its retina developed normally. Immunocytochemistry showed that farnesylated rhodopsin kinase (GRK1) and prenylated rod PDE6 catalytic subunits partially mislocalized in Pde6d−/− rods, whereas rhodopsin was unaffected. In Pde6d−/− rod single-cell recordings, sensitivity to single photons was increased and saturating flash responses were prolonged. Pde6d−/− scotopic paired-flash electroretinograms indicated a delay in recovery of the dark state, likely due to reduced levels of GRK1 in rod outer segments. In Pde6d−/− cone outer segments, GRK1 and cone PDE6α′ were present at very low levels and the photopic b-wave amplitudes were reduced by 70%. Thus the absence of PrBP/δ in retina impairs transport of prenylated proteins, particularly GRK1 and cone PDE, to rod and cone outer segments, resulting in altered photoreceptor physiology and a phenotype of a slowly progressing rod/cone dystrophy.

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.

Visual transduction in dialysed detached rod outer segments from lizard retina

1. Properties of a new preparation for studying the physiology and biochemistry of phototransduction in retinal rods are described. Whole‐cell voltage clamp was used to record the generation, maintenance and light‐sensitivity of dark current in rod outer segments that had been isolated from the rest of the receptor cell by detachment at the connecting cilium. 2. Detached outer segments dialysed with standard internal solution supplemented with physiological amounts of ATP (5 mM) and GTP (1 mM) developed a standing inward dark current that was the sum of three components: approximately 91% light‐sensitive current, approximately 6% Na(+)‐Ca2+,K+ exchange current and approximately 3% leakage current. Light‐sensitive dark current (mean amplitude approximately ‐63 pA) was suppressed transiently by brief flashes in an intensity‐dependent manner. Light responses had the same kinetics, sensitivity and intensity‐response relationship as those recorded from intact rods. 3. Dialysed outer segments differed from intact rods in that intense flashes evoked saturating responses that recovered incompletely to a plateau of reduced dark current caused by incomplete inactivation of the transduction cascade. Light sensitivity was reduced for a short time following an intense flash and then recovered despite persistent reduction of dark current. This suggests that there is no fixed relationship between dark current amplitude and light sensitivity. 4. Light‐sensitive dark current faded rapidly when outer segments were not supplied with nucleotides. Outer segments dialysed with solution that contained cyclic GMP, but no ATP or GTP, supported dark current at a level that increased with [cyclic GMP]. When basal phosphodiesterase (PDE) activity is inhibited, 8 microM cyclic GMP supports a dark current of approximately 70 pA. 5. Light sensitivity decreased during recordings made with solution that contained only cyclic GMP, consistent with the inhibition of G protein activation by loss of GTP. After thorough nucleoside triphosphate depletion, however, intense illumination evoked a transient increase rather than a decrease in dark current, i.e. an inverted light response. This result suggests that isomerized rhodopsin may generate a signal that causes either inhibition of basal PDE activity or release of bound cyclic GMP. 6. Sustained Na(+)‐Ca2+,K+ exchange current was recorded during steady illumination when Ca2+, but not when Mg2+, was added to the dialysis solution. Exchange current increased with the amount of added Ca2+ and saturated at approximately 18 pA when the dialysis solution contained > or = 10 mM Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)