Alan R. Adolph

Inner plexiform circuits in the carp retina: effects of cholinergic agonists, GABA, and substance P on the ganglion cells.

Th effects on ganglion cell light responses and spontaneous activity of neurotransmitter candidates, applied by nebulizer spray and iontophoresis, were studied in the isolated carp retina. ACh, GABA, and substance P had strong effects on the ganglion cells; dopamine and the amino acids aspartate, glutamate, and glycine and only weak effects. ACh and substance P exerted their actions even when synaptic transmission was blocked by cobalt chloride, suggesting postsynaptic receptors for those agents on the ganglion cell membrane. The 3 amino acids and dopamine do not appear to act directly on the ganglion cells. The pharmacological sensitivity of ganglion cells was correlated with their physiological response type. About three-quarters of ON/OFF and half of other transiently responding ganglion cells were excited by micromolar concentrations of cholinergic agonists; most ON-center sustained ganglion cells were insensitive. The light response of some of the ACh-sensitive cells could be suppressed by cholinergic antagonists. Substance P generally excited ganglion cells with an ON-component in their light response. GABA inhibited cells of all response types, but affected least the OFF-center tonic cells. In view of these observations, and of corroborating histological evidence, we propose that ACh, GABA, and substance P are neurotransmitters that are released by amacrine cells and affect receptors located on ganglion cells.

OFF ganglion cells cannot drive the optokinetic reflex in zebrafish

Whereas the zebrafish retina has long been an important model system for developmental and genetic studies, little is known about the responses of the inner retinal neurons. Here we report single-unit ganglion cell recordings from 5- to 6-day-old zebrafish larvae. In wild-type larvae we identify at least five subtypes of ganglion cell responses to full-field illumination, with ON-OFF and ON-type cells predominating. In the nrc mutant retina, in which the photoreceptor terminals develop abnormally, we observe normal OFF responses but abnormal ON-OFF responses and no ON responses. Previously characterized as blind, these mutants lack an optokinetic reflex (OKR), but in another behavioral assay nrc mutant fish have near-normal responses to the offset of light and slow and sluggish responses to the onset of light. Pharmacological block of the ON pathway mimics most of the nrc visual defects. We conclude that the abnormal photoreceptor terminals in nrc mutants predominantly perturb the ON pathway and that the ON pathway is necessary to drive the OKR in larval zebrafish.

Ultrastructure of human retinal cell transplants with long survival times in rats

Human fetal retinas (6-12 weeks post-conception) were obtained from elective abortions, transplanted to rat retinas and examined by electron microscopy. The oldest transplants that form the basis of this report were obtained 40 and 41 total weeks post-conception. The host rats were immunosuppressed with cyclosporin A. The transplants developed according to their intrinsic, genetically determined timetable. The development was heterogeneous with some parts showing almost normal differentiation and others, little. Both rods and cones developed with inner and outer segments and synaptic terminals. In regions corresponding to the inner plexiform layer, bipolar cell processes were seen in the typical dyad arrangement. Likewise, amacrine cell processes formed typical conventional synapses. Serial synapses were seen, engaging amacrine cell synapses as well as a few reciprocal synapses at the bipolar cell dyads. Monad-type synaptic complexes, a sign of immaturity, were common in bipolar cell processes. Similarly, incompletely differentiated synapses of both the amacrine and bipolar cell types were often observed. Ganglion cell processes could not be identified with certainty. A structure with morphological characteristics similar to the inner limiting membrane was noted to form inside the transplant. Both epi-retinal and sub-retinal transplants were obtained. Transplant cells touched host photoreceptor cells or pigment epithelium without any obvious specializations. The host pigment epithelium microvilli were absent adjacent to the graft. However, graft cells did appear in the host retina, and nerve cell processes were observed to cross the membrane separating the transplant and host.

Zebrafish larvae lose vision at night.

Darkness serves as a stimulus for vertebrate photoreceptors; they are actively depolarized in the dark and hyperpolarize in the light. Here, we show that larval zebrafish essentially turn off their visual system at night when they are not active. Electroretinograms recorded from larval zebrafish show large differences between day and night; the responses are normal in amplitude throughout the day but are almost absent after several hours of darkness at night. Behavioral testing also shows that larval zebrafish become unresponsive to visual stimuli at night. This phenomenon is largely circadian driven as fish show similar dramatic changes in visual responsiveness when maintained in continuous darkness, although light exposure at night partially restores the responses. Visual responsiveness is decreased at night by at least two mechanisms: photoreceptor outer segment activity decreases and synaptic ribbons in cone pedicles disassemble.

Neuronal markers in rat retinal grafts

Rat E15 retina was grafted to the retina of adult rat hosts. After varying survival times (1 week-6 months), grafts were stained by immunohistochemistry for neurofilament 160 kDa (NF), HPC-1 (an amacrine cell marker), choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and somatostatin-28 (SS-28). The first differentiating graft amacrine cells (cholinergic and dopaminergic) could be seen 1 week after transplantation (corresponding to postnatal day 1 = P1). The inner plexiform layer of the graft started to differentiate at 2 weeks (corresponding to P8) seen by HPC-1 and GAD staining. ChAT, TH and SS-28 immunostaining revealed an abnormal lamination pattern in the graft inner plexiform layer. Also by 2 weeks, the outer plexiform layers of the graft contained NF-immunoreactive horizontal cells. No NF-stained retinal ganglion cells could be observed in the graft. Five and 7 weeks after grafting, the transplants had obtained the same staining intensity with different markers as the host retina.

Function and Structure in Retinal Transplants

Embryonic mammalian donor retina transplanted into the subretinal space of a mature host develops into a graft with wellorganized, but atypical retinal structure. We tested the effect of this organization on rabbitto-rabbit graft functional properties, isolating the graft to avoid contamination of graft responses by host retinal activity. Transient ON or ON-OFF spike-like responses and local electroretinograms (L-ERGs) were recorded simultaneously via a single electrode on the graft surface. These response components depended on stimulus diameter, sometimes in a way indicating antagonistic center-surround receptive field organization and spatial tuning (43%). Other times, the responses were an increasing function of stimulus diameter which saturated for large spots (57%). Response amplitudes were transplantation surgery is to be done with therapeutic aims.

Glutamatergic mechanisms in the outer retina of larval zebrafish: analysis of electroretinogram b- and d-waves using a novel preparation.

A new preparation is described for recording the electroretinogram (ERG) from larval zebrafish (5-8 days postfertilization) which has allowed the investigation of the pharmacology of cone photoreceptor inputs onto bipolar cells. By using a pharmacological cocktail to isolate the photoreceptors and bipolar cells from inhibitory influences, it was found that an excitatory amino acid transporter (EAAT) presumably linked to a Cl() channel mediates most of the synaptic transmission from the cone photoreceptors to the ON bipolar cells, although metabotropic glutamate receptors (presumably mGluR6) also make a small contribution. On the other hand, alpha-amino-3-hydroxy- 5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors mediate synaptic transmission from cone photoreceptors to OFF bipolar cells. The glutamatergic input mechanisms underlying bipolar cell responses in the larval zebrafish are adultlike and similar to those in other teleost species.

Lazy eyes zebrafish mutation affects Müller glial cells, compromising photoreceptor function and causing partial blindness.

A behavioral assay based on the optokinetic reflex was used to screen chemically mutagenized zebrafish larvae for deficits in visual function. A homozygous recessive mutation, lazy eyes (lze), was isolated based on the observation that 5‐day postfertilization (dpf) mutants displayed weaker and less frequent eye movements than wild‐type fish in response to moving stripes. Electroretinographic (ERG) recordings revealed that mutants had severely reduced a‐ and b‐wave amplitudes relative to wild‐type fish, indicating outer retinal dysfunction. Retinal lamination and cellular differentiation were normal in the lze retina; however, mutant photoreceptor cells had small outer segments and pyknotic nuclei were occasionally observed in the outer retina and the marginal zone of lze. Cone, rod, amacrine, bipolar, and Müller cell marker analyses indicated that the typical lze retina contained fewer rod photoreceptors and fewer Müller cells than wild‐type fish at 5 dpf. At 3 dpf, however, mutant retinas had normal numbers of rod photoreceptors and Müller cells, suggesting that the initial differentiation of these cell types occurred normally. Rod photoreceptor histology was normal at this early stage, but Müller cells were often hypertrophied, suggesting that they were unhealthy. Constant light rearing of mutant animals accelerated the Müller cell degeneration, severely worsened the visual deficit, but had no obvious affect on the photoreceptors. When ERG responses and Müller cell degeneration from the same mutant animals were analyzed, the extent of the Müller cell loss matched closely the degree to which ERG responses were reduced. In summary, the lze gene appears to be required for Müller cell viability and normal visual function. The lze mutant may be a model for the study of the involvement of Müller cells in photoreceptor development and function. J. Comp. Neurol. 463:265–280, 2003.

Monoaminergic neurons of the mudpuppy retina.

SummaryThe mudpuppy retina was investigated with the histofluorescence method of Falck and Hillarp in normal animals and in animals injected intraocularly with α-methylnoradrenaline, 5,6-dihydroxytryptamine, or a combination of the two drugs. Catecholaminergic amacrine cells were found to form a thin layer of terminals at the border between the inner nuclear and the inner plexiform layers. Catecholaminergic interplexiform cells were not found. Indoleamine-accumulating amacrine cells were also observed. They are fifteen to twenty times more numerous than the catecholaminergic cells, and their terminals occur diffusely throughout the inner plexiform layer. In a number of eyes the majority of the indoleamine-accumulating terminals were eliminated with intraocular injections of the neurotoxin, 5,7-dihydroxytryptamine, but the reproducibility of this effect was not consistent. Intravitreal injections of 5,6-dihydroxytryptamine were used to label both types of neurons for electron microscopy. They were found to make conventional type synapses on amacrine cells and, less frequently, on bipolar cells.

Ultrastructural Circuitry in Retinal Cell Transplants to Rat Retina

The development of five transplants of fetal retinal tissue to adult rat eyes was examined with the electron microscope. The transplants were of 9 to 10 weeks total age after conception in four cases and 20 weeks in one case. They were at stage E15 when transplanted. Transplants developed in both the epiretinal and subretinal spaces. The transplants were heterogeneously developed with some parts showing almost normal differentiation and others little. Subretinal transplants examined in this study were more developed than epiretinal grafts. Photoreceptor cells developed both inner and outer segments. Their synaptic terminals possessed output ribbon synapses with postsynaptic processes similar to those seen in normal retinas. In regions corresponding to the inner plexiform layer, the adult complement of synapses was seen, including advanced features such as serial synapses as well as reciprocal synapses at bipolar cell dyads. Incompletely differentiated synapses of both the amacrine and bipolar cell types were often observed, especially in the rat epiretinal transplants. Ganglion cell processes could not be identified with certainty. Although transplant cells were adjacent to host photoreceptor cells and pigment epithelium, obvious specializations or interactions were not observed. The experiments suggest that embryonic rat retinal cell transplants develop most or perhaps all of the structural components and neuronal circuitry necessary to transduce light and process some visual information.