Whereas I only worked with Bill for three years when he supervised my PhD at the University of Stirling in the mid 1970s, his influence on me has lasted a lifetime. This piece, rather than summarizing the details of his considerable contributions to science, is a personal tribute to one of the most significant people in my life.

The retina mainly contains ganglion, bipolar and photoreceptor cells which are distributed in the ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL), respectively. Whether there is an age-related loss of these retinal cells remains not well understood. Cell density and the total number of cells were two commonly used measures to evaluate such age-related changes in most previous studies and provided controversial conclusions. The use of density measures as decisive data is problematic because the total area of the retina was expanded in aging, whereas the application of the total number of cells was limited for assessing ganglion cells. In this study, thus, we wanted to test whether there is an age-related cell loss in the GCL, INL and ONL and if so, whether such a loss is correlated to the convergence ratio of these cells. We used stereological procedures to quantify the total number of cells in the three retinal nuclear layers in six young and six aged Long-Evans rats. We found that during aging, the total volume of the retina remained unchanged, but the retina became thinner. There was no cell loss in each individual nuclear layer, and the ratio of the ONL to INL to GCL was preserved.

Retinal ganglion cells (RGCs) display the phenomenon of rebound excitation, which is observed as rebound sodium action potential firing initiated at the termination of a sustained hyperpolarization below the resting membrane potential (RMP). Rebound impulse firing, in contrast to corresponding firing elicited from rest, displayed a lower net voltage threshold, shorter latency and was invariably observed as a phasic burst-like doublet of spikes. The preceding hyperpolarization leads to the recruitment of a Tetrodotoxin-insensitive depolarizing voltage overshoot, termed as the net depolarizing overshoot (NDO). Based on pharmacological sensitivities, we provide evidence that the NDO is composed of two independent but interacting components, including (1) a regenerative low threshold calcium spike (LTCS) and (2) a non-regenerative overshoot (NRO). Using voltage and current clamp recordings, we demonstrate that amphibian RGCs possess the hyperpolarization activated mixed cation channels/current, Ih, and low voltage activated (LVA) calcium channels, which underlie the generation of the NRO and LTCS respectively. At the RMP, the Ih channels are closed and the LVA calcium channels are inactivated. A hyperpolarization of sufficient magnitude and duration activates Ih and removes the inactivation of the LVA calcium channels. On termination of the hyperpolarizing influence, Ih adds an immediate depolarizing influence that boosts the generation of the LTCS. The concerted action of both conductances results in a larger amplitude and shorter latency NDO than either mechanism could achieve on its own. The NDO boosts the generation of conventional sodium spikes which are triggered on its upstroke and crest, thus eliciting rebound excitation.

GABAergic amacrine cells, cultured from embryonic chick retina, display spontaneous mini frequencies ranging from 0–4.6 Hz as a result of the release of quanta of transmitter from both synapses and autapses. We show here that at least part of this variation originates from differences in the degree to which endocannabinoids, endogenously generated within the culture, are present at terminals presynaptic to individual cells. Though all cells examined scored positive for cannabinoid receptor type I (CB1R), only those showing a low initial rate of spontaneous minis responded to CB1R agonists with an increase in mini frequency, caused by a Gi/o-mediated reduction in [cAMP]. Cells displaying a high initial rate of spontaneous minis, on the other hand, were unaffected by CB1R agonists, but they did show a rate decrease with CB1R antagonists. Such a regulation of spontaneous transmitter release by endocannabinoids might be important in network maintenance in amacrine cells and other inhibitory interneurons.

The number of L cones in the retina normally exceeds that of the M cones. Because normal color vision does not depend on the ratio of L- and M-photoreceptors, their signals must undergo an alteration in gain before being analyzed in the cortex. Previous studies have shown that this gain must take place before the cortex, but after the bipolar/amacrine cell layer of the retina. The aim of this study was to obtain topographical information about L- and M-cone activity at the ganglion cell layer using multifocal pattern electroretinography (mfPERG). A standard (black and white) stimulus was used, as well as stimuli modulating only the long wavelength-sensitive (L) or only the middle wavelength-sensitive (M) cones. The L:M ratio was calculated from the amplitude of the L-cone isolating mfPERG to that of the M-cone isolating mfPERG of 10 trichromats. Both the positive and negative components of the waveform were analyzed. Additional recordings of single cone modulated mfERGs were obtained from nine of the 10 subjects. We also recorded from one protanope and one deuteranope. The L:M cone amplitude ratios for both deflections of the mfPERG in the trichromats were around unity (medians 1.18 and 1.16, respectively) for the central 8° of retina. In the peripheral retina between 12.8° and 26°, this ratio increased to 1.42 for the positive component, and 1.37 for the negative component. The median L:M cone amplitude ratios for the mfPERG were higher and ranged between 1.00–2.78 in the central 8° and 1.29–2.78 in the periphery. The results indicate that a major gain adjustment of the retinal signals takes place at the ganglion cell level, and that the ratio is higher at eccentric locations than in the central retinal area.

Horizontal cells mediate inhibitory feed-forward and feedback communication in the outer retina; however, mechanisms that underlie transmitter release from mammalian horizontal cells are poorly understood. Toward determining whether the molecular machinery for exocytosis is present in horizontal cells, we investigated the localization of syntaxin-4, a SNARE protein involved in targeting vesicles to the plasma membrane, in mouse, rat, and rabbit retinae using immunocytochemistry. We report robust expression of syntaxin-4 in the outer plexiform layer of all three species. Syntaxin-4 occurred in processes and tips of horizontal cells, with regularly spaced, thicker sandwich-like structures along the processes. Double labeling with syntaxin-4 and calbindin antibodies, a horizontal cell marker, demonstrated syntaxin-4 localization to horizontal cell processes; whereas, double labeling with PKC antibodies, a rod bipolar cell (RBC) marker, showed a lack of co-localization, with syntaxin-4 immunolabeling occurring just distal to RBC dendritic tips. Syntaxin-4 immunolabeling occurred within VGLUT-1-immunoreactive photoreceptor terminals and underneath synaptic ribbons, labeled by CtBP2/RIBEYE antibodies, consistent with localization in invaginating horizontal cell tips at photoreceptor triad synapses. Vertical sections of retina immunostained for syntaxin-4 and peanut agglutinin (PNA) established that the prominent patches of syntaxin-4 immunoreactivity were adjacent to the base of cone pedicles. Horizontal sections through the OPL indicate a one-to-one co-localization of syntaxin-4 densities at likely all cone pedicles, with syntaxin-4 immunoreactivity interdigitating with PNA labeling. Pre-embedding immuno-electron microscopy confirmed the subcellular localization of syntaxin-4 labeling to lateral elements at both rod and cone triad synapses. Finally, co-localization with SNAP-25, a possible binding partner of syntaxin-4, indicated co-expression of these SNARE proteins in the same subcellular compartment of the horizontal cell. Taken together, the strong expression of these two SNARE proteins in the processes and endings of horizontal cells at rod and cone terminals suggests that horizontal cell axons and dendrites are likely sites of exocytotic activity.

We examined the functional properties of a low-voltage-activated (LVA) calcium current in ganglion cells of the neotenous tiger salamander (Ambystoma tigrinum) retina. Our analysis was based on whole-cell recordings from acutely dissociated ganglion cell bodies identified by retrograde dye injections. Using a continuously perfused cell preparation, the LVA current was isolated with the use of potassium channel blocking agents added to the bathing medium and the pipette solution, while tetrodotoxin was added to the bathing medium to block Na+ channels. Approximately 70% of ganglion cells had an easily identified LVA current. The LVA current activated at membrane potentials more positive than −90 mV, and inactivated rapidly. It was relatively insensitive to nickel (IC50 > 500 μM) and amiloride (IC50 > 750 μM). Voltage- and current-clamp studies allowed us to generate a model of this current using the NEURON simulation program. Studies were also carried out to measure the LVA Ca2+ current in ganglion cells with dendrites to confirm that it had a significant dendritic representation. Physiological mechanisms that may depend on LVA Ca2+ currents are discussed with an emphasis on the role that dendrites play in ganglion cell function.

The isolated retina of the goldfish has proven a valuable resource for studying the variability of firing of retinal ganglion cells. Three major areas of study are considered here: the variability of maintained discharges, the correlated firing of neighboring ganglion cells, and the variability of responses to light. The sources of variability, its relationship to retinal processing, and its possible functional role in perception are examined through these three aspects of variability. The results are related to similar studies in mammals (mainly cats). This retrospective is biased toward my studies over 30 years.

Light-dependent translocation of invertebrate visual guanine-nucleotide binding protein, iGqα, from rhabdomeric membranes to the cytoplasm is one of many mechanisms that contribute to light adaptation in the invertebrate eye. We have previously cloned iGqα from a Loligo pealei photoreceptor cDNA library and shown that when expressed in HEK 293T cells it is palmitoylated. In this study we compared the activation, cytoplasmic translocation, and turnover of iGqα with that of a non-palmitoylated mutant, iGqα(C3,4A). In the HEK 293T cells, muscarinic M1 receptors coupled equally well to iGqα and iGqα(C3,4A) to activate phospholipase C. Activation of iGqα(C3,4A), but not iGqα, induced translocation of the α subunit from the membrane to cytosol with rapid degradation of the soluble protein resulting in a decreased half-life for iGqα(C3,4A) of 10 hours compared to 20 hours for iGqα. Degradation of iGqα(C3,4A) was inhibited by proteasomal inhibitors but not by inhibitors of lysosomal proteases or calpain. The presence of the proteasomal inhibitor led to the accumulation of polyubiquitinated species of either iGqα or iGqα(C3,4A). Our results suggest that palmitoylation of iGqα is required to maintain membrane association of the protein in its active conformation, and whereas membrane-bound and soluble iGqα can be polyubiquitinated, membrane association protects the protein from rapid degradation by the proteasomal pathway.

The role of intracellular organelles in Ca2+ homeostasis was studied in salamander rod and cone photoreceptors under conditions that simulate photoreceptor activation by darkness and light. Sustained depolarization evoked a Ca2+ gradient between the cell body and ellipsoid regions of the inner segment (IS). The standing pattern of calcium fluxes was created by interactions between the plasma membrane, endoplasmic reticulum (ER), and mitochondria. Pharmacological experiments suggested that mitochondria modulate both baseline [Ca2+]i in hyperpolarized cells as well as kinetics of Ca2+ entry via L type Ca2+ channels in cell bodies and ellipsoids of depolarized rods and cones. Inhibition of mitochondrial Ca2+ sequestration by antimycin/oligomycin caused a three-fold reduction in the amount of Ca2+ accumulated into intracellular organelles in both cell bodies and ellipsoids. A further 50% decrease in intracellular Ca2+ content within cell bodies, but not ellipsoids, was observed after suppression of SERCA-mediated Ca2+ uptake into the ER. Inhibition of Ca2+ sequestration into the endoplasmic reticulum by thapsigargin or cyclopiazonic acid decreased the magnitude and kinetics of depolarization-evoked Ca2+ signals in cell bodies of rods and cones and decreased the amount of Ca2+ accumulated into internal stores. These results suggest that steady-state [Ca2+]i in photoreceptors is regulated in a region-specific manner, with the ER contribution predominant in the cell body and mitochondrial buffering [Ca2+] the ellipsoid. Local [Ca2+]i levels are set by interactions between the plasma membrane Ca2+ channels and transporters, ER and mitochondria. Mitochondria are likely to play an essential role in temporal and spatial buffering of photoreceptor Ca2+.

Alpha 2 adrenergic agonists are used for controlling intraocular pressure (IOP) in the treatment of glaucoma. They have also been shown to be neuroprotective to retinal cells in a variety of injury models. Despite this significance, the localization of the three known alpha 2 adrenergic receptors has not been unequivocally established. The aim of this study was to determine the location of the three alpha 2 adrenergic receptors in ocular tissues using immunohistochemical techniques. New antibodies were generated and their specificity was determined using Western blotting and preadsorption. In the anterior segment of the eye alpha 2A immunoreactivity was located in the nonpigmented ciliary epithelium, corneal, and conjunctival epithelia. Alpha 2B staining was not apparent in these tissues. Alpha 2C immunostaining was present in the membrane of pigmented ciliary epithelium and corneal and conjunctival epithelial cells. In the rat retina, all three receptor subtypes were present but were differentially localized. Alpha 2A was present in the somata of ganglion cell layer and inner nuclear layer somas, alpha 2B was located in the dendrites and axons of most of the neurons as well as glia, while alpha 2C was present in the somata and inner segment of the photoreceptors. In human and monkey retinas, similar pattern of labeling for alpha 2A and 2B receptors were observed, while alpha 2B was additionally present in the membranes of many cell somata in addition to dendrites and axons. Alpha 2C labeling was much weaker but exhibited similar pattern to that observed in the rat. These data provide additional information on the location of the alpha 2 receptors in the anterior portion of the eye and present new information on their specific location in the retina. This offers insights into possible targets for adrenergic agonists in a therapeutic context.

In the cat, the analysis of visual motion cues has generally been attributed to the posteromedial lateral suprasylvian cortex (PMLS) (Toyama et al., 1985; Rauschecker et al., 1987; Rauschecker, 1988; Kim et al., 1997). The responses of neurons in this area are not critically dependent on inputs from the primary visual cortex (VC), as lesions of VC leave neuronal response properties in PMLS relatively unchanged (Spear & Baumann, 1979; Spear, 1988; Guido et al., 1990b). However, previous studies have used a limited range of visual stimuli. In this study, we assessed whether neurons in PMLS cortex remained direction-selective to complex motion stimuli following a lesion of VC, particularly to complex random dot kinematograms (RDKs). Unilateral aspiration of VC was performed on post-natal days 7–9. Single unit extracellular recordings were performed one year later in the ipsilateral PMLS cortex. As in previous studies, a reduction in the percentage of direction selective neurons was observed with drifting sinewave gratings. We report a previously unobserved phenomenon with sinewave gratings, in which there is a greater modulation of firing rate at the temporal frequency of the stimulus in animals with a lesion of VC, suggesting an increased segregation of ON and OFF sub-regions. A significant portion of neurons in PMLS cortex were direction selective to simple (16/18) and complex (11/16) RDKs. However, the strength of direction selectivity to both stimuli was reduced as compared to normals. The data suggest that complex motion processing is still present, albeit reduced, in PMLS cortex despite the removal of VC input. The complex RDK motion selectivity is consistent with both geniculo-cortical and extra-geniculate thalamo-cortical pathways in residual direction encoding.

To determine whether regenerating neural pathways can support visual behavior, adult goldfish (Carassius auratus) were injected intraocularly with ouabain and tested for the presence of reflexive visual behaviors (dorsal light reflex and optokinetic nystagmus) and the ability to respond to visual stimuli in a classical conditioning paradigm. All visual behaviors were absent or greatly diminished until 8 to 10 weeks, when retinal layering had returned. At 10 weeks post-ouabain, reflexive behaviors to supra-threshold stimuli were near normal; however the ability to detect supra-threshold stimuli in the conditioning paradigm did not recover until 13 weeks. Absolute dark-adapted threshold and light-adapted spectral sensitivity measured at 13 to 17 weeks were abnormal: Dark-adapted threshold was elevated by 1.5 log units and light-adapted spectral sensitivity was markedly narrower than normal. No responses to 50% contrast sinusoidal gratings could be obtained through ouabain-treated eyes using the classical conditioning technique, even though responses through the untreated eye remained. Results demonstrate that: (a) visually mediated behaviors return in goldfish with ouabain-treated retinas; (b) the time course of recovery of reflexive responses in luminance and spatial domains parallels return of ERG function and of tectal activity; and (c) visual function that is mediated by regenerating retina appears not to be as sensitive as vision via normally developed retinal pathways.

Visual spatial attention has been described as a process that favors the processing of sensory information that falls into the “spotlight of attention.” Recent studies have provided support for an ability to split this attentional focus to selectively process spatially separate locations. Using functional magnetic resonance imaging, the signature for the presence of multiple spotlights is the presence of multiple retinotopically specific foci of activation in striate and extrastriate visual areas. We used this approach to investigate the presence of such separable activations as a function of the eccentricity of the spatial foci of attention. Visual stimuli consisted of letters and digits displayed in rapid serial visual presentation (RSVP). Five RSVP streams were presented simultaneously, one in the center of the visual field and one in each visual field quadrant. Subjects had to deploy their attention either to a single peripheral location or two non-contiguous regions performing a match-mismatch judgment. The results show that dividing attention leads to multiple spotlights of attention for central as well as more peripheral locations of the visual field. However, depending on the exact location and width of the attentional spotlights, resulting activation maps might reveal merged activation patterns even in the presence of distinct attentional spotlights.

In metacontrast a masking stimulus reduces the visibility of an adjacent target stimulus. This effect has been interpreted in terms of magno-/parvocellular interactions. It has also been found that a second masking stimulus, which precedes the primary mask by about 90 ms reduces the masking effect. This reduction, which is termed “target recovery,” has been hypothesized to reflect parvocellular inhibition of the magnocellular system. However, this is problematic because the time course of this effect is much larger than would be expected from magno-/parvocellular interactions. For this and other reasons, it is difficult to understand metacontrast in terms of magno- and parvocellular mechanisms.

Strychnine is considered a selective competitive antagonist of glycine gated Cl− channels (Saitoh et al., 1994) and studies have used strychnine at low micromolar concentrations to study the role of glycine in rabbit retina (Linn, 1998; Protti et al., 2005). However, other studies have shown that strychnine, in the concentrations commonly used, is also a potent competitive antagonist of α7 nicotinic acetylcholine receptors (nAChRs; Matsubayashi et al., 1998). We tested the effects of low micromolar concentrations of strychnine and 3-[2′-phosphonomethyl[1,1′-biphenyl]-3-yl] alanine (PMBA), a specific glycine receptor blocker (Saitoh et al., 1994; Hosie et al., 1999) on the activation of both α7 nAChRs on retinal ganglion cells and on ganglion cell responses to a light flash. Extracellular recordings were obtained from ganglion cells in an isolated retina/choroid preparation and 500 μM choline was used as an α7 agonist (Alkondon et al., 1997). We recorded from brisk sustained and brisk transient OFF cells, many of which have been previously shown to have α7 receptors (Strang et al., 2005). Further, we tested the effect of strychnine, PMBA and α-bungarotoxin on the binding of tetramethylrhodamine α-bungarotoxin in the inner plexiform layer. Our data indicates that strychnine, at doses as low as 1.0 μM, can inhibit the α7 nAChR-mediated response to choline, but PMBA at concentrations as high as 0.4 μM does not. Binding studies show strychnine and α-bungarotoxin inhibit binding of labeled α-bungarotoxin in the IPL. Thus, the effects of strychnine application may be to inhibit glycine receptors expressed by ganglion cell or to inhibit amacrine cell α7 nAChRs, both of which would result in an increase in the ganglion cell responses. Further research will be required to disentangle the effects of strychnine previously believed to be caused by a single mechanism of glycine receptor inhibition.

We measured the latency of 35 cells from V1 in two rhesus monkeys, to dynamic random dot stimuli monocular and binocularly presented. Mean latencies after non-dominant eye stimulation (97.9 ms) were longer than those for dominant eye (78.2 ms) and binocular (70.7 ms) stimulation. Differences between latencies for dominant eye and binocular stimulation were not statistically significant. For dominant eye, there was a significant statistical correlation between dominance strength and latency (R = −0.36; p = 0.03). We failed to find significant statistical differences between latencies for cells with temporal and nasal dominant receptive-field. We conclude that, in V1, the response latency is largely determined by the dominant eye, whereas interocular interactions do not seem to play a relevant role regarding response latency.

Neighboring retinal ganglion cells often spike synchronously, but the possible function and mechanism of this synchrony is unclear. Recently, the strength of the fast correlation between ON-OFF directionally selective cells of the rabbit retina was shown to be stimulus dependent. Here, we extend that study, investigating stimulus-dependent correlation among multiple ganglion-cell classes, using multi-electrode recordings. Our results generalized those for directionally selective cells. All cell pairs exhibiting significant spike synchrony did it for an extended edge but rarely for full-field stimuli. The strength of this synchrony did not depend on the amplitude of the response and correlations could be present even when the cells' receptive fields did not overlap. In addition, correlations tended to be orientation selective in a manner predictable by the relative positions of the receptive fields. Finally, extended edges and full-field stimuli produced significantly greater and smaller correlations than predicted by chance respectively. We propose an amacrine-network model for the enhancement and depression of correlation. Such an apparently purposeful control of correlation adds evidence for retinal synchrony playing a functional role in vision.

GABA receptor antagonists produce an unexpectedly significant inhibition of native glycine receptors in retina and in α1 or α2 homomeric glycine receptors (GlyRs) expressed in HEK 293 cells. In this study we evaluate this phenomenon in heteromeric glycine receptors, formed by mixing α1, α2, and β subunits. Picrotoxinin, picrotin, SR95531, and bicuculline are all more effective antagonists at GlyRs containing α2 subunits than α1 subunits. Inclusion of β subunits reduces the inhibitory potency of picrotoxinin and picrotin but increases the potency of SR95531 and bicuculline. As a result of these two factors, bicuculline is particularly poor at discriminating GABA and glycine receptors. Picrotin, which has been reported to be inactive at GABA receptors, blocks glycine currents in retina and in HEK293 cells, suggesting its utility as a selective glycine antagonist. However, picrotin is a more potent inhibitor of GABA than glycine in retinal neurons. We also tested if GABA and glycine receptor subunits can combine to form functional receptors. If GABAAR γ2S subunits are co-expressed with GlyR α subunits, the mixed receptor is glycine-sensitive and GABA-insensitive. But the mixed receptor exhibits a non-competitive picrotoxinin inhibition that is not observed in the homomeric GlyRs. This suggests that glycine and GABA subunits can co-assemble to form functional glycine receptors.

Evidence suggests that patients with schizophrenia show impaired performances on tests assessing the magnocellular (M) visual pathway. The aim of this study was to investigate M pathway functioning in persons at high-risk of psychosis. Sixteen high-risk persons at the prodromal phase of psychosis and 20 healthy controls participated. Two types of contrast sensitivity measurements were used, during which participants were asked to detect a briefly presented target Gabor patch. In the pulsed-pedestal paradigm, the luminance of the background field was decreased to saturate M pathways and to bias information processing to parvocellular (P) pathways. In the steady-pedestal paradigm, the luminance of the background field was constant and briefly presented targets were processed by the M pathway. Anomalous perceptual experiences were assessed using the Structured Interview for Assessing Perceptual Anomalies (SIAPA). Results revealed that the high-risk persons showed elevated contrast sensitivity during the M pathway test, and normal sensitivity during the P pathway test. The visual SIAPA scores showed significant positive correlations with the M pathway sensitivity values. These results suggest that the high-risk mental state is associated with hyper-reactive M pathways, which may be responsible for some anomalous perceptual experiences, including abnormal intensity of environmental stimuli, feelings of being flooded and inundated, and inability to focus attention to relevant details.