To investigate the physiology and plasticity of mixed rod–cone ON-bipolar cells (Mb) in the goldfish retina, we established a slice preparation which allows us to optically monitor the synaptic activity of bipolar cell axon terminals. We used the styryl dye FM1-43 which is incorporated into active axon terminals due to synaptic vesicle cycling and thus reflects synaptic activity. Different activity states of the axon terminals were revealed when slices prepared from light-adapted retinae were incubated in the presence of FM1-43 under various conditions. Depolarizing high K+ Ringer (50 mM) and the gamma-butyric acid (GABA) antagonist bicuculline (100 μM) resulted in more than two-fold increase in the number of stained terminals compared to slices stained in normal Ringer. In contrast, GABA treatment (0.5 mM) reduced the frequency of stained terminals. Thus, in light-adapted retinal slices the synaptic activity of Mb axon terminals can be modulated towards higher and lower activity states. The fact that the GABA antagonist bicuculline had similar effects as stimulatory high K+ Ringer suggests that inhibitory control is an important component in the regulation of synaptic activity and transmitter release in Mb terminals.

Previous research in adults has demonstrated the utility of the visual evoked potential (VEP) to measure the integrity of the chromatic and achromatic visual pathways. The VEP has also been shown to be a valuable indicator of maturation of these pathways in infants up to 1 year of age. The present manuscript reports changes in the visual pathways from 2 years to adulthood as measured by the spatio-chromatic VEP. The responses to achromatic reversal stimuli designed to preferentially activate the low spatial-frequency achromatic (luminance) pathways appear adult-like by 1 year of age. The responses to low spatial-frequency isoluminant onset stimuli designed to preferentially activate the chromatic pathway do not appear as they do in the adult until after 12–13 years of age. The shapes of the chromatic VEP waveforms shift from a positive–negative complex to a negative–positive complex. These changes can be modeled by a decrease in the latency of a large negative component between the ages of 1 year and adulthood. The results suggest that for low spatial-frequency stimuli, there are long-term changes in the development of the chromatic pathways that are not observed in the low spatial-frequency achromatic pathways. The changes in the chromatic VEP waveforms with age may be a physiological correlate of reported behavioral changes.

The activity of the endogenous retinal dopamine (DA) pathway has been examined in the pigmented rat using retinas obtained from normal adult (∼3 months) and senile adults (∼24 months) using an in vitro electrophysiological approach. By comparing the pharmacological sensitivity of the horizontal cells (HCs) to exogenous DA, a D1 receptor antagonist (SCH 23390) and a DA-transport inhibitor (nomifensine), it is suggested that there is a functional deficit in the endogenous DA activity in the senile retina. Cells recorded from retinae obtained from senile animals are more sensitive to exogenous DA, whilst the senile retina is insensitive to SCH 23390. In addition, nomifensine was effective in potentiating subthreshold DA applications, but only in the normal adult retina. The data may suggest that endogenous DA release upon the HCs and selective re-uptake are suppressed in these retinae. These functional deficits also appear to be associated with changes in the receptive fields of the HCs, suggesting there is a corresponding deficit in spatial processing at the outer plexiform layer (OPL) of the senile rat.

The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin α subunits (Gαt) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of Gαt. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander Gαt-1 has 91.2–93.7% amino acid sequence identity to mammalian rod Gαt subunits and 79.7–80.9% to mammalian cone Gαts. The salamander Gαt-2 has 86.2–87.9% sequence identity to mammalian cone Gαts and 78.9–80.9% to mammalian rod Gαts at the amino acid level. The Gαt-1 cDNA encodes 350 amino acids while the Gαt-2 cDNA encodes 354 residues, which is typical for rod and cone Gαts, respectively, and we thus identified the Gαt-1 as rod and Gαt-2 as cone Gαt. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone Gαt subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod Gαt and 2.6 kb for cone Gαt. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod Gαt is localized to rods, predominantly in the outer segments; similarly, cone Gαt is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.

Spatial summation and degree of center-surround antagonism were examined in the receptive field of nonlagged cells in the dorsal lateral geniculate nucleus (dLGN). We recorded responses to stationary light or dark circular spots that were stepwise varied in width. The spots were centered on the receptive field. For a sample of nonlagged X-cells, we made simultaneous recordings of action potentials and S-potentials, and could thereby compare spatial summation in the dLGN cell and in the retinal input to the cell. Plots of response versus spot diameter showed that the response for a dLGN cell was consistently below the response in the retinal input at all spot sizes. There was a marked increase of antagonism at the retinogeniculate relay. The difference between the retinal input and dLGN cell response suggested that the direct retinal input to a relay cell is counteracted in dLGN by an inhibitory field that has an antagonistic center-surround organization. The inhibitory field seems to have the same center sign (ON- or OFF-center), but a wider receptive-field center than the direct retinal input to the relay cell. The broader center of the inhibitory field can explain the increased center-surround antagonism at the retinogeniculate relay. The ratio between the response of a dLGN cell and its retinal input (transfer ratio) varied with spot width. This variation did not necessarily reflect a nonlinearity at the retinogeniculate relay. Plots of dLGN cell response against retinal input were piecewise linear, suggesting that both excitatory and inhibitory transmission in dLGN are close to linear. The variation in transfer ratio could be explained by sustained suppression evoked by the background stimulation, because such suppression has relatively stronger effect on the response to a spot evoking weak response than to a spot evoking a strong response. A simple model for the spatial receptive-field organization of nonlagged X-cells, that is consistent with our findings, is presented.

Spatial receptive fields of relay cells in dorsal lateral geniculate nucleus (dLGN) have commonly been modeled as a difference of two Gaussian functions. We present alternative models for dLGN cells which take known physiological couplings between retina and dLGN and within dLGN into account. The models include excitatory input from a single retinal ganglion cell and feedforward inhibition via intrageniculate interneurons. Mathematical formulas describing the receptive field and response to circular spot stimuli are found both for models with a finite and an infinite number of ganglion-cell inputs to dLGN neurons. The advantage of these models compared to the common difference-of-Gaussians model is that they, in addition to providing mathematical descriptions of the receptive fields of dLGN neurons, also make explicit contributions from the geniculate circuit. Moreover, the model parameters have direct physiological relevance and can be manipulated and measured experimentally. The discrete model is applied to recently published data (Ruksenas et al., 2000) on response versus spot-diameter curves for dLGN cells and for the retinal input to the cell (S-potentials). The models are found to account well for the results for the X-cells in these experiments. Moreover, predictions from the discrete model regarding receptive-field sizes of interneurons, the amount of center-surround antagonism for interneurons compared to relay cells, and distance between neighboring retinal ganglion cells providing input to interneurons, are all compatible with data available in the literature.

In retinal rods, light exposure decreases the total outer segment content of both cGMP and cAMP by about 50%. The functional role of the light-evoked change in cAMP is not known. It is postulated to trigger changes in the phosphorylation state of phosducin, a phosphoprotein that is phosphorylated in the dark by cAMP-dependent protein kinase (PKA) and dephosphorylated by basal phosphatase activity when PKA is inhibited by the light-evoked drop in cAMP. In biochemical studies, dephosphorylated phosducin binds to free βγ dimer of transducin (Tβγ) and prevents the regeneration of heterotrimeric transducin by blocking the re-association of the βγ and α subunits. Phosducin's interaction with Tβγ is blocked when it is phosphorylated on a single residue by PKA. To evaluate the effect of the light-evoked fall in cAMP, functionally intact isolated lizard rod outer segments were dialyzed in whole-cell voltage clamp with a standard internal solution and electrical light responses were recorded with and without adding cAMP to the dialysis solution. Since the total outer segment content of cAMP in darkness is ∼5 μM, internal dialysis with solution containing a much higher concentration (100 μM) of cAMP (or 8-bromo-cAMP) will overcome the effects of a light-evoked decrease in its concentration by keeping cAMP-dependent processes fully activated. Neither cyclic nucleotide had any influence on the generation, light sensitivity, recovery, or background adaptation of the flash response. These results also argue against the participation of phosducin in the sequence of events that are responsible for these aspects of rod function. This does not exclude the possibility of phosducin being involved in adaptation caused by higher light levels than used in the present study, that is, bleaching adaptation, or in light-dependent processes other than phototransduction.

The cat's lateralis medialis-suprageniculate nuclear complex (LM-Sg) in the thalamus receives input from various brain regions such as the superior colliculus, brain stem, and spinal cord, as well as from visual association cortex. In a previous study, we demonstrated that LM-Sg receives cholinergic fibers from the pedunculopontine tegmental nucleus (PPT) and that cholinergic terminals make synaptic contacts with the dendrites of glutamatergic projection neurons and of GABAergic interneurons (Hoshino et al., 1997). In this study, we investigate the distribution and the organization of PPT terminals by means of a combined anterograde tracer (biotinylated dextran amine, BDA) and immunohistochemical methods. When stained by acetylcholinesterase (AChE), the LM-Sg is not uniformly immunoreactive, but rather is patchily labeled and shows a streaming type of reactivity. The tissue content appears high in enzyme activity in AChE-positive zones and is much lighter in activity in AChE-negative zones. We compared the synaptic organization between AChE-positive and AChE-negative portions of the LM-Sg in separate groups of electron-microscopic material: four types of vesicle containing profiles (RS, RL, F1, and PSD) as well as synaptic glomeruli were observed in this nucleus. Among these, the PSD profiles were observed more frequently in AChE-positive portions than in AChE-negative zones. Furthermore, the number of glomeruli was significantly higher in AChE-positive than in AChE-negative zones. Following the injection of BDA into PPT, labeled terminals within LM-Sg were rather more concentrated in the AChE-positive portion. Although the majority of PPT terminals made synaptic contacts with dendrites in the neuropil, a few terminals were involved in the synaptic glomeruli. The present results show that the synaptic organization is distinctly different between the AChE-positive and AChE-negative portions of LM-Sg. These results suggest that the AChE-positive portions of LM-Sg are relatively more involved in integrating information arising from a diverse set of inputs and processing that information within glomeruli in a complex manner than occurs in the AChE-negative portion of LM-Sg.

The avian retinotectofugal pathway has been suggested to be homologous to the mammalian retinotectofugal pathway. The projection of the nucleus rotundus upon the ectostriatum is equivalent to that of the pulvinar nucleus upon the extrastriate cortex in mammals. In this system, the optic tectum relays retinal input to the nucleus rotundus, which then ascends to the ectostriatum of the telencephalon. Given the fact that the chick retinotectofugal system becomes mature early during development, the present study attempted to investigate the timecourse of neuronal connections of the embryonic rotundoectostriatal pathway. We used multiple injections of cholera toxin B subunit (CTb) in the ectostriatum of chick embryos to retrogradely trace projections to the nucleus rotundus. We found CTb-labeled neurons in the nucleus rotundus at embryonic day 7.5–8. By embryonic day 8–8.5, increased numbers of CTb-labeled neurons were seen in the nucleus rotundus. It was noted that the time of this initial connection between the nucleus rotundus and the ectostriatum is nearly synchronous with that of the retinotectal and tectorotundal pathways, respectively (Crossland et al., 1975; Thanos & Bonhoeffer, 1987; Wu et al., 2000). These findings, combined with the present study, suggest that the retinotectofugal system becomes established, at least at a structural level, by embryonic day E8.

Recently, it was reported that the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA) and linolenic acid (LNA), activate the light-sensitive channels in Drosophila photoreceptors (Chyb et al. 1999). We have examined whether these PUFAs activate the light-sensitive channels in Limulus ventral photoreceptors. We find that, whether applied from the outside or injected into a Limulus ventral photoreceptor, either AA or LNA fails to activate the light-sensitive channels. Moreover, the synthetic diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, also fails to activate the light-sensitive channels. We suggest that these findings require us to rethink our view about the generality of the process of phototransduction in invertebrate microvillar photoreceptors. We propose that the photoreceptors of Drosophila and Limulus evolved to utilize different branches of the phosphoinositide pathway for phototransduction: those of Limulus evolved to utilize IP3-mediated calcium release while those of Drosophila evolved to utilize diacylglycerol and it's downstream products.

Light responses of R/G chromaticity-type horizontal cells (R/G HCs) and luminosity-type horizontal cells (LHCs) were intracellularly recorded in isolated superfused carp retina, and the response dynamics analyzed. The results revealed that (1) No significant difference in delay was detected between R/G HC red and green responses; (2) The rising speed was quicker for R/G HC depolarizing red responses compared to that of its hyperpolarizing green responses; and (3) Dynamic characteristics of R/G HC red responses and its changes caused by green background illumination did not follow that of LHC red response. All these results suggest that the depolarizing response of the R/G HCs cannot be entirely mediated by the negative feedback pathway from LHCs onto cones. A direct inhibitory input from red cones to R/G HCs may exist.

Gamma aminobutyric acid (GABA) has been established as an important developmental signal in a number of regions of the central nervous system (CNS), including retina. Our previous studies have shown that GABAergic horizontal cells act as the initial synaptic target for developing cone photoreceptors in neonatal rabbit retina. Since intraocular injections of the GABAA receptor antagonists, picrotoxin or bicuculline, disrupt cone synaptogenesis in vivo, GABA released from horizontal cells may provide a necessary signal for cone axon growth and/or synapse formation. In the current report, we have used cultured retinal explants to examine the effects of GABAA receptor antagonists on other aspects of developing cones. These include the distribution pattern of cone cell bodies across the outer surface of the retina and the expression of GABAA receptors within both cone cell bodies and axonal processes. Peanut agglutinin (PNA), a plant lectin that specifically labels cone plasma membrane and extracellular matrix, was used to monitor cone development, and a GABAA receptor antibody against the β2/3 subunits of the protein was used to label GABAA receptors. Results showed that cones maintained in the explant culture express GABAA receptors in a temporal and spatial pattern similar to that observed in vivo, namely a low expression of receptors on cone cell bodies at postnatal day 1 (P1), peaking around P3 and diminishing by P7. Neonatal retinal explants exposed to the GABAA receptor antagonists, bicuculline (10 μM) or SR95531 (5 μM), for 24 h in culture showed disruption of the normal distribution of cone cell bodies. When GABA (100 μM) was added along with either antagonist, cone cell bodies appeared normal. Neither bicuculline nor SR95531 alone had any effect on the general morphology of other retinal layers, suggesting that these GABAA receptor antagonists at the concentrations used were not acting as nonspecific disruption agents. The effects of GABA antagonists were confined to the first week after birth with no disruption seen in P9 or adult explant cultures. These data provide a direct demonstration of the necessity for GABAergic input to cones during active synaptogenesis. As we have previously shown, GABAA receptor activation causes a substantial increase in intracellular calcium concentrations in cones and thereby could provide a mechanism by which GABA regulates cone maturation.

The retinae of insectivores have been rarely studied, and their photoreceptor arrangements and expression patterns of visual pigments are largely unknown. We have determined the presence and distribution of cones in three species of shrews (common shrew Sorex araneus, greater white-toothed shrew Crocidura russula, dark forest shrew Crocidura poensis; Soricidae) and in the lesser hedgehog tenrec Echinops telfairi (Tenrecidae). Spectral cone types were identified and quantified in flattened whole retinae by antisera/antibodies recognizing the middle-to-long-wavelength-sensitive (M/L-)cone opsin and the short-wavelength-sensitive (S-)cone opsin, respectively. A combination of immunocytochemistry with conventional histology was used to assess rod densities and cone/rod ratios. In all four species the rods dominate at densities of about 230,000–260,000/mm2. M/L- and S-cones are present, comprising between 2% of the photoreceptors in the nocturnal Echinops telfairi and 13% in Sorex araneus that has equal diurnal and nocturnal activity phases. This suggests dichromatic color vision like in many other mammals. A striking feature in all four species are dramatically higher S-cone proportions in ventral than in dorsal retina (0.5% vs. 2.5–12% in Sorex, 5–15% vs. 30–45% in Crocidura poensis, 3–12% vs. 20–50% in Crocidura russula, 10–30% vs. 40–70% in Echinops). The functional and comparative aspects of these structural findings are discussed.

This study examined the question of which features of a complex grouping discrimination make it vulnerable to permanent elimination by V4 lesions. We first verified that the line element grouping discrimination, which we previously reported to be devastated by V4 lesions, was similarly affected in the monkeys of this study. The permanence of the deficit was established by mapping its visual field distribution and then testing this discrimination for an extended period at a locus on the border of the deficit. Also, a staircase procedure was used to provide the monkey with within session instruction in the grouping discrimination, but this did not improve V4 lesion performance. Grouping was then compared with several discriminations that shared some features with it, but which were found not to be permanently eliminated by V4 lesions. This comparison suggested that grouping (rather than segmentation or response to a single element) was one feature that made the discrimination vulnerable, a second was the similarity in shape of the texture elements to be grouped. Finally, we tested visual crowding, a property of peripheral vision that is thought to reflect neuronal interactions early in visual cortex, possibly in area V1, and found no effect of V4 lesions. A control experiment with human observers tested whether the elimination of grouping by V4 lesions might be due to an alteration of attention, but found no evidence to support this hypothesis. These results show that severe disruption of texture discriminations by V4 lesions depends on both the nature of the discrimination and the type of texture elements involved, but does not necessarily involve the disruption of attention.

In five subjects, we measured visual evoked potentials (VEPs) elicited by Vernier targets in which the contrast of the two components of the stimuli were modulated by sinusoids at distinct frequencies f1 and f2. This approach allows for the extraction of VEP signatures of spatial interactions, namely, responses at intermodulation frequencies n1f1 + n2f2, without the need to introduce motion into the stimulus. The most prominent interactions were at the sum frequency f1 + f2, and, for frequency pairs that were sufficiently separated, the difference frequency f1 − f2. These responses had a systematic dependence on the temporal parameters of the stimulus, corresponding to an effective latency of 145 to 165 ms. Fourth-order interactions were also detected, particularly at the frequencies 2f1 ± 2f2. These VEP signatures of interaction were similar to interactions seen for colinear line segments separated by a gap. Thus, for Vernier stimuli devoid of motion, VEP signatures of interaction are readily detected but are not specific to hyperacuity displacements. The distribution of interactions across harmonic orders is consistent with local rectification preceding the spatial interactions. Their effective latencies and dependence on spatial parameters are consistent with interactions within V1 receptive fields or mediated by horizontal connections between cells with a similar orientation tuning within V1.