Larval Retina Research Articles

Cell-type expression and activation by light of neuropsins in the developing and mature Xenopus retina.

Photosensitive opsins detect light and perform image- or nonimage-forming tasks. Opsins such as the "classical" visual opsins and melanopsin are well studied. However, the retinal expression and functions of a novel family of neuropsins are poorly understood. We explored the developmental time-course and cell-type specificity of neuropsin (opn5, 6a, 6b, and 8) expression in Xenopus laevis by in situ hybridization and immunohistochemistry. We compared the Xenopus results with publicly available single cell RNA sequencing (scRNA-seq) data from zebrafish, chicken, and mouse. Additionally, we analyzed light-activation of neuropsin-expressing cells through induction of c-fos mRNA. opn5 and opn8 expression begins at stage 37/38 when the retinal circuits begin to be activated. Once retinal circuits connect to the brain, opn5 mRNA is distributed across multiple retinal cell types, including bipolar (~70%-75%), amacrine (~10%), and retinal ganglion (~20%) cells, with opn8 present in amacrine (~70%) and retinal ganglion (~30%) cells. opn6a and opn6b mRNAs emerge in newborn-photoreceptors (stage 35), and are colocalized in rods and cones by stage 37/38. Interestingly, in the mature larval retina (stage 43/44), opn6a and opn6b mRNAs become preferentially localized to rods and cones, respectively, while newborn photoreceptors bordering the proliferative ciliary marginal zone express both genes. In zebrafish, opn6a and opn6b are also expressed in photoreceptors, while Müller glia and amacrine cells express opn8c. Most neuropsin-expressing retinal ganglion cells display c-fos expression in response to light, as do over half of the neuropsin-expressing interneurons. This study gave a better understanding of retinal neuropsin-expressing cells, their developmental onset, and light activation.

Differential Regenerative Capacity of the Optic Tectum of Adult Medaka and Zebrafish.

Zebrafish have superior regenerative capacity in the central nervous system (CNS) compared to mammals. In contrast, medaka were shown to have low regenerative capacity in the adult heart and larval retina, despite the well-documented high tissue regenerative ability of teleosts. Nevertheless, medaka and zebrafish share similar brain structures and biological features to those of mammals. Hence, this study aimed to compare the neural stem cell (NSC) responses and regenerative capacity in the optic tectum of adult medaka and zebrafish after stab wound injury. Limited neuronal differentiation was observed in the injured medaka, though the proliferation of radial glia (RG) was induced in response to tectum injury. Moreover, the expression of the pro-regenerative transcriptional factors ascl1a and oct4 was not enhanced in the injured medaka, unlike in zebrafish, whereas expression of sox2 and stat3 was upregulated in both fish models. Of note, glial scar-like structures composed of GFAP+ radial fibers were observed in the injured area of medaka at 14 days post injury (dpi). Altogether, these findings suggest that the adult medaka brain has low regenerative capacity with limited neuronal generation and scar formation. Hence, medaka represent an attractive model for investigating and evaluating critical factors for brain regeneration.

Immunohistochemical Characterization of the Development of Long Photoreceptor Cells in the Lamprey Retina.

The adult lamprey retina has two types of photoreceptor cells, short and long photoreceptor cells, which are equivalent to rods and cones of other vertebrates. In contrast, the retina of lamprey larvae only contains a single type of photoreceptor cell, which appears to correspond to the short photoreceptor cell. However, the developmental pattern of the long photoreceptor cell is unknown. Previously, we reported that antibodies against rhodopsin and iodopsin (the chicken red cone opsin) could discriminate between the outer segments of short and long photoreceptor cells, respectively, in the retina of adult Japanese river lamprey (Lethenteron camtschaticum). Here, we immunohistochemically investigate the appearance of long photoreceptor cells in the larval and adult retinas of the Far Eastern brook lamprey (Lethenteron reissneri), which is a close relative of the Japanese river lamprey, by using anti-iodopsin antibody. We found that iodopsin immunoreactivity was localized not only in the adult retina but also in the larval retina. Moreover, we examined the immunohistochemical localization of signal transduction molecules, such as transducin and arrestin, in the iodopsin-immunoreactive cells of the larval retina. The iodopsin-immunoreactive cells also contained both transducin and arrestin, suggesting that long photoreceptor cells are already functional in the larval stage before the acquisition of visual function. Our results suggest that the iodopsin-immunoreactive cells may be related to not only cone vision in the adult but also photoreception in the larval lamprey.

Suitable light intensity during the ontogenetic development of white trevally, Pseudocaranx dentex (Bloch and Schneider, 1801), larvae

Three experiments were conducted to elucidate the most suitable rearing light intensity during the ontogenetic changes in white trevally (Pseudocaranx dentex) larvae. In Experiment 1, larvae were reared under 1000, 3000 or 9000 lx. At 10 days post hatching (dph), larval growth was significantly higher in the 1000 lx treatment than in the other treatments. In Experiment 2, larvae were reared under 30, 100, 300, 1000, 3000, and 9000 lx, and 300 lx was revealed to be the most suitable light intensity for larval growth and survival until 10 dph. However, light intensities higher than 1000 lx may promote larval growth after 10 dph when the larval retina has developed prior to the metamorphic stage. In Experiment 3, larvae were exposed to increasing light intensities (i.e. from 300 lx to 1000, 3000 and 9000 lx) from 10 to 18 dph. Our results indicated that the most suitable rearing light intensity was 300 lx during the first week of development and at the commencement of feeding, and between 1000 and 3000 lx when the larvae approached the metamorphic stage. The rearing light intensity should, therefore, be adapted throughout the ontogenetic development of white trevally to promote larval growth and production efficiency.

Marine larval vision suffers in low oxygen

Human brains don't do well without oxygen. When mountaineer Thomas Hornbein ascended Everest in 1963, he described his vision dimming when his oxygen regulator jarred loose. Fortunately, few of us experience hypoxia in such dramatic situations, but Lillian McCormick, from the Scripps Institution of Oceanography, explains that many aquatic creatures routinely encounter potentially threatening oxygen levels. ‘In nearshore areas of the Pacific Ocean off southern California, organisms can be exposed to reduced oxygen even in 0–50 m depth,’ she says. And when she reviewed the literature, it was clear that species with complex eyes would be most at risk of visual impairment. However, she and her colleagues Lisa Levin and Nicholas Oesch noticed that little was known about the ill effects of hypoxia on the larvae of many of these species.To rectify the situation, McCormick went trawling for plankton. ‘I hand-sorted each tow to set aside the larvae that we were interested in’, says McCormick, who decided to test the vision of larvae of the two spot octopus (Octopus bimaculatus), the tuna crab (Pleuroncodes planipes) and the graceful rock crab (Metacarcinus gracilis). However, she had to wait for the first and new moons of each month, when the market squid (Doryteuthis opalescens) spawn, to collect freshly laid egg sacs, which she took back to the lab to wait for the youngsters to hatch. Then, she painstakingly inserted a minute electrode into the retina of each tiny larva to record the eye's responses to light. Gradually decreasing and then increasing the concentration of oxygen dissolved in the water, McCormick recorded the response of each larva's retina to 1 s flashes of light. And when the oxygen was at ∼21, ∼10 and ∼3%, she shone a series of increasingly dimmer 1 s flashes into their eyes. In addition, McCormick shone a flickering light that gradually speeded up until the flashes blurred into one into the eyes of the squid and tuna crab larvae to find out how well the tiny animals could see fast-moving objects as the oxygen dwindled.Shockingly, all of the larvae lost some or all of their vision as the oxygen declined, with the market squid and graceful rock crab larvae unexpectedly beginning to suffer vision loss as soon as the oxygen began dropping. In contrast, the light response of the two spot octopus and tuna crab larval retinas held out reasonably well until the oxygen fell to ∼13%. By the time the oxygen had dipped to ∼3%, most of the squid, octopus and graceful rock crab larvae had completely lost their vision, although the retinal response of the tuna crab larvae only dipped to 60%. And while the speed of the tuna crab larvae's vision barely faltered at the lower oxygen levels, the response time of the squid larvae's retina reduced significantly, suggesting that they may really struggle to see the fast-moving copepods, upon which they dine, when oxygen is scarce.‘We calculate that the decline in retinal function due to decreased oxygen availability in larvae that move from 0 to 30 m depth in spring in southern California would be 15–59%’, says McCormick; which means that these minute creatures may already be experiencing visual loss in their natural environment. And she is concerned that additional declines in oxygen availability could place the larvae at further risk. ‘If larvae are unable to detect small changes in light intensity, or the swimming movements of their prey, they may not survive’, she warns.

Connectivity of cone photoreceptor telodendria in the zebrafish retina.

The connectivity amongst photoreceptors is critical to their function, as it underpins lateral inhibition and effective translation of stimuli into neural signals. Despite much work characterizing second-order interneurons in the outer retina, the synapses directly connecting photoreceptors have often been overlooked. Telodendria are fine processes that connect photoreceptor pedicles. They have been observed in diverse vertebrate groups, yet their roles in vision remain speculative. Here, we visualize telodendria via fluorescent protein expression in photoreceptor subtypes. We characterized short wavelength cone telodendria in adult and larval zebrafish retina. Additionally, in the larval retina, we investigated rod telodendria and UV cone telodendria in mutant and transgenic retinas with altered complements of cone types. In the adult retina, telodendria are twice as abundant and branch almost twice as often on blue cones compared to UV cones. Pedicles of neighboring UV and blue cones typically converge into contiguous pairs, despite the regular spacing of their cell bodies. In contrast to adults, larval UV cone telodendria are more numerous (1.3 times) than blue cone telodendria. UV cone telodendria are not detectably affected by ablation of blue cones, and are reduced twofold in mutant larval retina with few UV cones. We thus saw no evidence that telodendria increase in number in the absence of their typical cellular neighbors. We also found that larval rod telodendria are less abundant than short wavelength cone telodendria. In summary, we describe the development and morphology of zebrafish photoreceptor synaptic connectivity toward appreciating the function of telodendria in visual signal processing.

The lens regenerative competency of limbal vs. central regions of mature Xenopus cornea epithelium

The frog, Xenopus laevis, is capable of completely regenerating a lens from the cornea epithelium. Because this ability appears to be limited to the larval stages of Xenopus, virtually all the work to understand the mechanisms regulating this process has been limited to pre-metamorphic tadpoles. It has been reported that the post-metamorphic cornea is competent to regenerate under experimental conditions, despite the fact that the in vivo capacity to regenerate is lost; however, that work didn't examine the regenerative potential of different regions of the cornea. A new model suggests that cornea-lens regeneration in Xenopus may be driven by oligopotent stem cells, and not by transdifferentiation of mature cornea cells. We investigated the regenerative potential of the limbal region in post-metamorphic cornea, where the stem cells of the cornea are thought to reside. Using EdU (5-Ethynyl-2’-deoxyuridine), we identified long-term label retaining cells in the basal cells of peripheral post-metamorphic Xenopus cornea, consistent with slow-cycling stem cells of the limbus that have been described in other vertebrates. Using this data to identify putative stem cells of the limbal region in Xenopus, we tested the regenerative competency of limbal regions and central cornea. These regions showed a similarly high ability for the cells of the basal epithelium to express lens proteins when cultured in proximity to larval retina. Thus, the regenerative competency in the post-metamorphic cornea is not restricted to stem cells of the limbal region, but also occurs in the transit amplifying cells throughout the basal layer of the cornea epithelium.

Electroretinogram analysis of the visual response in zebrafish larvae.

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

High-throughput RNA sequencing reveals the effects of 2,2',4,4' -tetrabromodiphenyl ether on retina and bone development of zebrafish larvae.

Background2,2′,4,4′-Tetrabromodiphenyl ether (BDE47) is a prevalent environmental pollutant and has been demonstrated to be a serious toxicant in both humans and animals, but little is known about the molecular mechanism underlying its toxic effect on the early development of vertebrates. BDE47-treated zebrafish larvae were found to present the light-related locomotion reduction in our previous study, therefore, we aimed to use high throughput sequencing to investigate the possible reasons from a transcriptomic perspective.ResultsBy exposing zebrafish embryos/larvae to 5 μg/l and 500 μg/l BDE47, we measured the influence of BDE47 on the mRNA expression profiles of zebrafish larvae until 6 days post-fertilization, using Illumina HiSeq 2000 sequencing. Differential expression analysis and gene enrichment analysis respectively revealed that a great number of genes, and gene sets based on two popular terminologies, were affected by the treatment of 500 μg/l BDE47. Among them, BDE47 caused changes in the retinal metabolism and related biological processes involving eye morphogenesis and visual perception, as confirmed by disordered photoreceptor arrangement and thickened bipolar cell layer of larval retina from histological observations. Other altered genes such as pth1a and collaborative cathepsin family exhibited disrupted bone development, which was consistent with the body curvature phenotype. The transcriptome of larvae was not significantly affected by the treatment of 5 μg/l BDE47, as well as the treatment of DMSO vehicle.ConclusionsOur results suggest that high BDE47 concentrations disrupt the eye and bone development of zebrafish larvae based on both transcriptomic and morphological evidences. The abnormal visual perception may result in the alteration of dark adaption, which was probably responsible for the abnormal larval locomotion. Body curvature arose from enhanced bone resorption because of the intensive up-regulation of related genes. We also proposed the larval retina as a novel potential target tissue for BDE47 exposure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-014-1194-5) contains supplementary material, which is available to authorized users.

Zebrafish Guanylate Cyclase Type 3 Signaling in Cone Photoreceptors

The zebrafish guanylate cyclase type 3 (zGC3) is specifically expressed in cone cells. A specifc antibody directed against zGC3 revealed expression at the protein level at 3.5 dpf in outer and inner retinal layers, which increased in intensity between 3.5 and 7 dpf. This expression pattern differed from sections of the adult retina showing strong immunostaining in outer segments of double cones and short single cones, less intense immunoreactivity in long single cones, but no staining in the inner retina. Although transcription and protein expression levels of zGC3 are similar to that of the cyclase regulator guanylate cyclase-activating protein 3 (zGCAP3), we surprisingly found that zGCAP3 is present in a 28-fold molar excess over zGC3 in zebrafish retinae. Further, zGCAP3 was an efficient regulator of guanylate cyclases activity in native zebrafish retinal membrane preparations. Therefore, we investigated the physiological function of zGCAP3 by two different behavioral assays. Using the morpholino antisense technique, we knocked down expression of zGCAP3 and recorded the optokinetic and optomotor responses of morphants, control morphants, and wild type fish at 5–6 dpf. No significant differences in behavioral responses among wild type, morphants and control morphants were found, indicating that a loss of zGCAP3 has no consequences in primary visual processing in the larval retina despite its prominent expression pattern. Its physiological function is therefore compensated by other zGCAP isoforms.

Temporal and tissue specific gene expression patterns of the zebrafish kinesin-1 heavy chain family, kif5s, during development

Homo- and heterodimers of Kif5 proteins form the motor domain of Kinesin-1, a major plus-end directed microtubule motor. Kif5s have been implicated in the intracellular transport of organelles, vesicles, proteins, and RNAs in many cell types. There are three mammalian KIF5s. KIF5A and KIF5C proteins are strictly neural in mouse whereas, KIF5B is ubiquitously expressed. Mouse knockouts indicate crucial roles for KIF5 in development and human mutations in KIF5A lead to the neurodegenerative disease Hereditary Spastic Paraplegia. However, the developmental functions and the extent to which individual kif5 functions overlap have not been elucidated. Zebrafish possess five kif5 genes: kif5Aa, kif5Ab, kif5Ba, kif5Bb, and kif5C. Here we report their tissue specific expression patterns in embryonic and larval stages. Specifically, we find that kif5As are strictly zygotic and exhibit neural-specific expression. In contrast, kif5Bs exhibit strong maternal contribution and are ubiquitously expressed. Lastly, kif5C exhibits weak maternal expression followed by enrichment in neural populations. In addition, kif5s show distinct expression domains in the larval retina.

Activity-Induced Long-Term Potentiation of Excitatory Synapses in Developing Zebrafish Retina In Vivo

Neural activity-induced long-term potentiation (LTP) of synaptic transmission is believed to be one of the cellular mechanisms underlying experience-dependent developmental refinement of neural circuits. Although it is well established that visual experience and neural activity are critical for the refinement of retinal circuits, whether and how LTP occurs in the retina remain unknown. Using in vivo perforated whole-cell recording and two-photon calcium imaging, we find that both repeated electrical and visual stimulations can induce LTP at excitatory synapses formed by bipolar cells on retinal ganglion cells in larval but not juvenile zebrafish. LTP induction requires the activation of postsynaptic N-methyl-D-aspartate receptors, and its expression involves arachidonic acid-dependent presynaptic changes in calcium dynamics and neurotransmitter release. Physiologically, both electrical and visual stimulation-induced LTP can enhance visual responses of retinal ganglion cells. Thus, LTP exists in developing retinae with a presynaptic locus and may serve for visual experience-dependent refinement of retinal circuits.

The role of polarized light in prey capture in an aquatic predator

The role of polarized light in prey capture in an aquatic predator

Retinotopy of visual projections to the optic tectum and pretectum in larval sea lamprey

The sea lamprey has a complex life cycle with very different larval and adult stages. The eyes of larvae are subcutaneous, lack a differentiated lens and probably work only as an ocellus-like photoreceptor organ, while the well-developed adult eyes are capable of forming images. The larval retina differs greatly from the adult retina and presents a central region with differentiated photoreceptors and a lateral, largely undifferentiated part that grows in the second half of larval life. In the present study, we examined the retinotopy of projections from larval ganglion cells to the optic tectum and pretectum in sea lamprey by using retrograde tract-tracing techniques. In most regions of the tectum, application of the tracer neurobiotin (NB) resulted in labelled ganglion cells in the lateral retina, mostly in the contralateral eye. Ganglion cells of the lateral retina showed a very simple dendritic tree, possibly because of the lack of differentiation of most retinal layers in this region. The retinotectal projection is already retinotopically organized in larvae and follows a pattern similar to that observed in adult lampreys and other vertebrates. Application of NB to the central region of the tectum also led to labelling of a few ganglion cells in the central retina, which were clearly more complex than those in the lateral region, as they had dendrites that branched both in the outer and inner plexiform layers. Application of NB to the medial pretectum led to labelling of ganglion cells in the contralateral central retina. Occasional cells were also labelled in the lateral retina. The differential organization of larval retinal projections to the pretectum and tectum suggests a different role for these projections, which is consistent with the different involvement of these centres in visual behaviour, as determined in adult lampreys. The observations in larvae also reveal very different developmental timetables for these putative functions.

Cellular expression of midkine‐a and midkine‐b during retinal development and photoreceptor regeneration in zebrafish

AbstractIn the retina of adult teleosts, stem cells are sustained in two specialized niches: the ciliary marginal zone (CMZ) and the microenvironment surrounding adult Müller glia. Recently, Müller glia were identified as the regenerative stem cells in the teleost retina. Secreted signaling molecules that regulate neuronal regeneration in the retina are largely unknown. In a microarray screen to discover such factors, we identified midkine‐b (mdkb). Midkine is a highly conserved heparin‐binding growth factor with numerous biological functions. The zebrafish genome encodes two distinct midkine genes: mdka and mdkb. Here we describe the cellular expression of mdka and mdkb during retinal development and the initial, proliferative phase of photoreceptor regeneration. The results show that in the embryonic and larval retina mdka and mdkb are expressed in stem cells, retinal progenitors, and neurons in distinct patterns that suggest different functions for the two molecules. Following the selective death of photoreceptors in the adult, mdka and mdkb are coexpressed in horizontal cells and proliferating Müller glia and their neurogenic progeny. These data reveal that Mdka and Mdkb are signaling factors present in the retinal stem cell niches in both embryonic and mature retinas, and that their cellular expression is actively modulated during retinal development and regeneration. J. Comp. Neurol. 514:1–10, 2009. © 2009 Wiley‐Liss, Inc.

Cellular expression of Midkine‐a and Midkine‐b during retinal development and photoreceptor regeneration in zebrafish

In the retina of adult teleosts, stem cells are sustained in two specialized niches: the ciliary marginal zone (CMZ) and the microenvironment surrounding adult Müller glia. Recently, Müller glia were identified as the regenerative stem cells in the teleost retina. Secreted signaling molecules that regulate neuronal regeneration in the retina are largely unknown. In a microarray screen to discover such factors, we identified midkine-b (mdkb). Midkine is a highly conserved heparin-binding growth factor with numerous biological functions. The zebrafish genome encodes two distinct midkine genes: mdka and mdkb. Here we describe the cellular expression of mdka and mdkb during retinal development and the initial, proliferative phase of photoreceptor regeneration. The results show that in the embryonic and larval retina mdka and mdkb are expressed in stem cells, retinal progenitors, and neurons in distinct patterns that suggest different functions for the two molecules. Following the selective death of photoreceptors in the adult, mdka and mdkb are coexpressed in horizontal cells and proliferating Müller glia and their neurogenic progeny. These data reveal that Mdka and Mdkb are signaling factors present in the retinal stem cell niches in both embryonic and mature retinas, and that their cellular expression is actively modulated during retinal development and regeneration.

Cellular expression of midkine‐a and midkine‐b during retinal development and photoreceptor regeneration in zebrafish

AbstractIn the retina of adult teleosts, stem cells are sustained in two specialized niches: the ciliary marginal zone (CMZ) and the microenvironment surrounding adult Müller glia. Recently, Müller glia were identified as the regenerative stem cells in the teleost retina. Secreted signaling molecules that regulate neuronal regeneration in the retina are largely unknown. In a microarray screen to discover such factors, we identified midkine‐b (mdkb). Midkine is a highly conserved heparin‐binding growth factor with numerous biological functions. The zebrafish genome encodes two distinct midkine genes: mdka and mdkb. Here we describe the cellular expression of mdka and mdkb during retinal development and the initial, proliferative phase of photoreceptor regeneration. The results show that in the embryonic and larval retina mdka and mdkb are expressed in stem cells, retinal progenitors, and neurons in distinct patterns that suggest different functions for the two molecules. Following the selective death of photoreceptors in the adult, mdka and mdkb are coexpressed in horizontal cells and proliferating Müller glia and their neurogenic progeny. These data reveal that Mdka and Mdkb are signaling factors present in the retinal stem cell niches in both embryonic and mature retinas, and that their cellular expression is actively modulated during retinal development and regeneration. J. Comp. Neurol. 514:1–10, 2009. © 2009 Wiley‐Liss, Inc.

Neurochemical differentiation of horizontal and amacrine cells during transformation of the sea lamprey retina

The sea lamprey is a modern representative of the earliest vertebrates (the agnathans) in which development of the eye and retina shows unique patterns. In larval stages the retina is poorly developed, and although a small central region has developed glutamatergic vertical pathways, there is no evidence of chemical differentiation of amacrine and horizontal cells in the central or lateral larval retina [Villar-Cerviño, V., Abalo, X.M., Villar-Cheda, B., Meléndez-Ferro, M., Pérez-Costas, E., Holstein, G.R., Martinelli, G.P., Rodicio, M.C., Anadón, R., 2006. Presence of glutamate, glycine, and γ-aminobutyric acid in the retina of the larval sea lamprey: comparative immunohistochemical study of classical neurotransmitters in larval and postmetamorphic retinas. J. Comp. Neurol. 499, 810–827.]. However, in adults all the retina was differentiated and both amacrine and horizontal cells are well developed. Present immunocytochemical results show that the horizontal and amacrine cells of the retina begin their neurochemical differentiation during metamorphosis, when they start to express GABA, glycine, serotonin and dopamine; this occurs several years after the onset of development. Immunoreactivity for GABA, glycine and serotonin was found at early metamorphic stages, while expression of the markers of catecholaminergic amacrine cells, dopamine and tyrosine hydroxylase, was found to be delayed until intermediate metamorphic stages. GABA, which is found in some amacrine and horizontal cells of adults, was first observed in amacrine cells during early stages of transformation and then in horizontal cells during middle stages. All cells immunoreactive to serotonin or tyrosine hydroxylase/dopamine were amacrine cells. Interestingly, all these markers began expression before the appearance of opsin-immunoreactive photoreceptors in the lateral retina. The pattern of chemical differentiation of amacrine and horizontal cells was compared with that of other vertebrates and their significance was discussed.

Presence of glutamate, glycine, and γ‐aminobutyric acid in the retina of the larval sea lamprey: Comparative immunohistochemical study of classical neurotransmitters in larval and postmetamorphic retinas

The neurochemistry of the retina of the larval and postmetamorphic sea lamprey was studied via immunocytochemistry using antibodies directed against the major candidate neurotransmitters [glutamate, glycine, gamma-aminobutyric acid (GABA), aspartate, dopamine, serotonin] and the neurotransmitter-synthesizing enzyme tyrosine hydroxylase. Immunoreactivity to rod opsin and calretinin was also used to distinguish some retinal cells. Two retinal regions are present in larvae: the central retina, with opsin-immunoreactive photoreceptors, and the lateral retina, which lacks photoreceptors and is mainly neuroblastic. We observed calretinin-immunostained ganglion cells in both retinal regions; immunolabeled bipolar cells were detected in the central retina only. Glutamate immunoreactivity was present in photoreceptors, ganglion cells, and bipolar cells. Faint to moderate glycine immunostaining was observed in photoreceptors and some cells of the ganglion cell/inner plexiform layer. No GABA-immunolabeled perikarya were observed. GABA-immunoreactive centrifugal fibers were present in the central and lateral retina. These centrifugal fibers contacted glutamate-immunostained ganglion cells. No aspartate, serotonin, dopamine, or TH immunoreactivity was observed in larvae, whereas these molecules, as well as GABA, glycine, and glutamate, were detected in neurons of the retina of recently transformed lamprey. Immunoreactivity to GABA was observed in outer horizontal cells, some bipolar cells, and numerous amacrine cells, whereas immunoreactivity to glycine was found in amacrine cells and interplexiform cells. Dopamine and serotonin immunoreactivity was found in scattered amacrine cells. Amacrine and horizontal cells did not express classical neurotransmitters (with the possible exception of glycine) during larval life, so transmitter-expressing cells of the larval retina appear to participate only in the vertical processing pathway.

Choline acetyltransferase-immunoreactive neurons in the retina of adult and developing lampreys

The presence of choline acetyltransferase-immunoreactive (ChATir) amacrine cells is reported for the first time in the retinas of three species of lamprey ( Lampetra fluviatilis, Ichthyomyzon unicuspis, and Petromyzon marinus). In the three species, the ChATir cells were mainly distributed in the inner plexiform layer (IPL), which in lampreys extends from the inner nuclear layer (INL) to the inner limiting membrane. These cells had a bipolar, triangular or stellate appearance, and gave rise to processes coursing in the inner plexiform layer. In transforming lampreys, ChATir processes formed two asymmetrical inner and outer subplexuses in the inner plexiform layer, which is reminiscent of the distribution of processes of ChATir cells in the On and Off sublaminae reported in jawed vertebrates. The larval retina lacked ChAT immunoreactivity, and ChATir cells and processes appeared at early metamorphosis throughout the retina, exhibiting in late transforming stages an organization similar to that of adults. This first report of ChATir cells in the lamprey retina indicates that the appearance of cholinergic circuits in the retina of vertebrates occurred before the divergence of the agnathan and gnathostome lines.