Identification of 5-HT(3A) and 5-HT(3B) receptor subunits in mammalian retinae: potential pre-synaptic modulators of photoreceptors.

Although serotonin (5-HT) is found in the mammalian retina only at low levels, considerable evidence suggests that it plays a role in visual processing. Pharmacological experiments indicate that numerous receptors for 5-HT are present in the mammalian retina. One of these is the ionotropic 5-HT(3) receptor. So far, two subunits for this receptor have been identified in the nervous system, 5-HT(3A) and 5-HT(3B). Co-expression of these subunits in Xenopus oocytes is sufficient to reconstitute native 5-HT(3) receptor properties. Thus, it is believed that a native neuronal 5-HT(3) receptor is multimeric similar to the related acetylcholine receptor family. To determine whether this receptor is expressed in the mammalian retina, we first performed reverse transcription polymerase chain reaction and first demonstrated the presence of transcripts for both the 5-HT(3A) and 5-HT(3B) receptor subunits. Then using a well-characterized polyclonal antiserum against the 5-HT(3A) receptor subunit, we demonstrated 5-HT(3A) receptor immunoreactivity (IR) in the rabbit, rat, and human retina. This IR was localized specifically to the rod photoreceptor terminals in all three species, suggesting that this receptor may modulate the rod signaling pathway by controlling the output at the rod terminals.

Structural analysis and mutation detection strategy for the human LAMC3 gene.

Laminins are heterotrimeric extracellular matrix molecules, present in a wide range of basement membranes within human tissues. They consist of a combination of different alpha, beta, and gamma subunits. Three different gamma subunits have been described to date. Two of them, the gamma1 and gamma2 chains are constituents of basement membrane related laminins, while the gamma3 chain was detected in skin, heart, lung, reproductive tract, brain, and in the retina. Unlike other laminins, the expression of the gamma3 chain was localized to peripheral nerves and to the apical surface of ciliated epithelial cells and in the retina. To further investigate the function and the possible pathogenic role of laminin gamma3 in human disease, we elucidated the structure of the corresponding LAMC3 gene which encodes this polypeptide. Here we report the genomic organization of the LAMC3 gene and a mutation detection strategy for use in genetic studies.

A novel member of the netrin family, beta-netrin, shares homology with the beta chain of laminin: identification, expression, and functional characterization.

The netrins are a family of laminin-related molecules. Here, we characterize a new member of the family, beta-netrin. beta-Netrin is homologous to the NH(2) terminus of laminin chain short arms; it contains a laminin-like domain VI and 3.5 laminin EGF repeats and a netrin C domain. Unlike other netrins, this new netrin is more related to the laminin beta chains, thus, its name beta-netrin. An initial analysis of the tissue distribution revealed that kidney, heart, ovary, retina, and the olfactory bulb were tissues of high expression. We have expressed the molecule in a eukaryotic cell expression system and made antibodies to the expressed product. Both in situ hybridization and immunohistochemistry were used to describe the cellular source of beta-netrin and where beta-netrin is deposited. beta-Netrin is a basement membrane component; it is present in the basement membranes of the vasculature, kidney, and ovaries. In addition, beta-netrin is expressed in a limited set of fiber tracts within the brain, including the lateral olfactory tract and the vomeronasal nerve. Functional studies were performed and show that beta-netrin promotes neurite elongation from olfactory bulb explants. Together, these data suggest that beta-netrin is important in neural, kidney, and vascular development.

5-HT(1A) and 5-HT(7) receptor expression in the mammalian retina.

Serotonin or 5-hydroxytryptamine (5-HT) is a common neurotransmitter found widely in the nervous system. Here, using RT-PCR, we have identified both the 5-HT(1A) and 5-HT(7) receptor transcripts in the rabbit retina. Furthermore, we found the same two receptors in the rat retina which was previously believed not to have a serotoninergic system. These results confirm previous reports of 5-HT(7) gene expression in retina and together with other biochemical, physiological and anatomical studies, they support the presence of multiple 5-HT receptors in the mammalian retina and suggest that the action of serotonin in the retina may be more complicated than previously believed.

Laminin expression in adult and developing retinae: evidence of two novel CNS laminins.

Components of the extracellular matrix exert myriad effects on tissues throughout the body. In particular, the laminins, a family of heterotrimeric extracellular glycoproteins, have been shown to affect tissue development and integrity in such diverse organs as the kidney, lung, skin, and nervous system. Of these, we have focused on the roles that laminins play in the differentiation and maintenance of the nervous system. Here, we examine the expression of all known laminin chains within one component of the CNS, the retina. We find seven laminin chains-alpha3, alpha4, alpha5, beta2, beta3, gamma2, and gamma3-outside the retinal basement membranes. Anatomically, these chains are coexpressed in one or both of two locations: the matrix surrounding photoreceptors and the first synaptic layer where photoreceptors synapse with retinal interneurons. Biochemically, four of these chains are coisolated from retinal extracts in two independent complexes, confirming that two novel heterotrimers-alpha4beta2gamma3 and alpha5beta2gamma3-are present in the retinal matrix. During development, all four of these chains, along with components of laminin 5 (the alpha3, beta3, and gamma2 chains) are also expressed at sites at which they could exert important effects on photoreceptor development. Together, these data suggest the existence of two novel laminin heterotrimers in the CNS, which we term here laminin 14 (composed of the alpha4, beta2, and gamma3 chains) and laminin 15 (composed of the alpha5, beta2, and gamma3 chains), and lead us to hypothesize that these laminins, along with laminin 5, may play roles in photoreceptor production, stability, and synaptic organization.

Disruption of laminin beta2 chain production causes alterations in morphology and function in the CNS.

From the elegant studies of Ramon y Cajal (1909) to the current advances in molecular cloning (e.g., Farber and Danciger, 1997), the retina has served as an ideal model for the entire CNS. We have taken advantage of the well described anatomy, physiology, and molecular biology of the retina to begin to examine the role of the laminins, one component of the extracellular matrix, on the processes of neuronal differentiation and synapse formation in the CNS. We have examined the effect of the deletion of one laminin chain, the beta2 chain, on retinal development. The gross development of retinas from laminin beta2 chain-deficient animals appears normal, and photoreceptors are formed. However, these retinas exhibit several pathologies: laminin beta2 chain-deficient mice display abnormal outer segment elongation, abnormal electroretinograms, and abnormal rod photoreceptor synapses. Morphologically, the outer segments are reduced by 50% in length; the outer plexiform layer of mutant animals is disrupted specifically, because only 7% of observed rod invaginating synapses appear normal, whereas the inner plexiform layer is undisturbed; finally, the rate of apoptosis in the mutant photoreceptor layer is twice that of control mice. Physiologically, the electroretinogram is altered; the amplitude of the b-wave and the slope of the b-wave intensity-response function are both decreased, consistent with synaptic disruption in the outer retina. Together, these results emphasize the prominence of the extracellular matrix and, in particular, the laminins in the development and maintenance of synaptic function and morphogenesis in the CNS.

5-HT2a receptors in the rabbit retina: potential presynaptic modulators.

Three 5-HT receptors have been implicated in retinal processing but positive identification of the receptors and the localization of receptor subtypes in the retina have not been achieved. In this study, molecular techniques were used to identify one class of 5-HT receptor--5-HT2a--in the retina, and immunohistochemical techniques were used to localize the receptor in the retinal network. Reverse transcription polymerase chain reaction (RT-PCR) techniques were used to identify a segment of the rabbit 5-HT2a gene; a 422 base fragment was identified, cloned, and sequenced. The fragment shows a high degree (ca. 90%) of nucleotide sequence identity with the 5-HT2a receptor gene from other mammals. 5-HT2a immunoreactivity was seen in both the inner and outer plexiform (synaptic) layers of the retina. Using cell-type-specific markers, the 5-HT2a immunoreactivity was shown to be on the terminals of photoreceptor and rod bipolar cells. This association of 5-HT2a receptors with these two synapses suggests that serotonin may be a modulator of synaptic function in the retina.

Characterization and expression of the laminin gamma3 chain: a novel, non-basement membrane-associated, laminin chain.

Laminins are heterotrimeric molecules composed of an alpha, a beta, and a gamma chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel laminin, composed of known alpha and beta chains but containing a novel gamma chain, gamma3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that gamma3 contains all the expected domains of a gamma chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that gamma3-containing laminins are likely to exist in a stable matrix. Studies of the tissue distribution of gamma3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, gamma3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The gamma3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, ductus deferens, and seminiferous tubules. The distribution of gamma3-containing laminins on the apical surfaces of a variety of epithelial tissues is novel and suggests that they are not found within ultrastructurally defined basement membranes. It seems likely that these apical laminins are important in the morphogenesis and structural stability of the ciliated processes of these cells.

Serotonin receptors modulate rod signals: a neuropharmacological comparison of light- and dark-adapted retinas.

Previous physiological studies have shown that serotonin (5-HT) reciprocally modulates ON and OFF channels in the mammalian retina. This study was undertaken to determine if the serotoninergic system is exclusively associated with the rod pathway. We tested drugs specific to 5-HT3 receptor, a serotonin-gated ion channel, in both dark- and light-adapted retina. Consistent with previous studies, we demonstrated that 5-HT3 receptors modulate the light-evoked responses of ganglion cells in the dark-adapted state. Moreover, we have extended these prior studies and shown that activation of the 5-HT3 receptor is capable of completely blocking the light-evoked response of OFF-center cells whereas inactivation of the 5-HT3 receptor is capable of completely blocking the light-evoked responses of ON-center cells. In contrast, in light-adapted retinae, serotonin agents failed to have any effect on retinal processing. These data suggest that the serotoninergic system in retina is (1) specifically associated with rod-related pathways; and (2) exerts a powerful modulatory force over information transfer in the retina. Together these observations suggests that serotonin plays an important physiological role in modulating retinal processing.

Beta 2 laminins modulate neuronal phenotype in the rat retina.

The production of cell types in the vertebrate retina follows a stereotyped time course. We have focused on a component of the extracellular matrix that may guide this schedule: the laminin beta 2 chain. Here, we have asked directly whether heterotrimeric laminins containing the laminin beta 2 chain can promote the production of presumptive rod photoreceptors ("rods") and have correlated changes in rod production with changes in the production of other cell types. In cultures in which few rods, but many Müller and bipolar cells, are produced, the production of rods can be enhanced sixfold and that of bipolar cells can be reduced by 66%, by exposing cells to a laminin beta 2-rich matrix. Substitution of a laminin beta 2-depleted matrix (created with antisense RNA) returns the density of rods and bipolar cells to control levels. These linked alterations in phenotype expression suggest that laminins may control the choice between rod photoreceptor and rod bipolar cell fates.

Identification of the cellular source of laminin beta2 in adult and developing vertebrate retinae.

The interphotoreceptor matrix (IPM) is a specialized extracellular matrix that surrounds the inner and outer segments of photoreceptors. This matrix contains molecules that may be important in directing photoreceptor differentiation and survival. For example, one molecule that we have previously identified as a component of the IPM, laminin beta2 (formerly known as s-laminin), is implicated in the differentiation of rod photoreceptor cells. Developmentally, laminin beta2 is present before rod birth in a position that is consistent with a role in directing rod differentiation; it is found, in both the rat and skate, in the ventricular space that ultimately becomes the IPM. In this study, we identify the source of laminin beta2 in the adult and developing retina. Both immunohistochemistry in the adult skate retina and in situ hybridizations in the adult rat retina reveal that laminin beta2 is produced by Müller cells. In addition, in the skate but not the rat retina, retinal pigment epithelial cells may be an alternative source of laminin beta2. During development, however, laminin beta2 is present before the birth of Müller glial cells; at this stage of development, laminin beta2 RNA is present within the neuroepithelial layer in a pattern that is consistent with its production by neuroepithelial cells.

Developmental expression of laminin beta 2 in rat retina. Further support for a role in rod morphogenesis.

PURPOSE: The authors previously hypothesized that laminin beta 2 (S-laminin) plays a role in directing photoreceptor development. The aim of this study was to examine the temporal and spatial expression pattern of beta 2 laminins in rat retina to test this hypothesis. METHODS: Retinas from Sprague-Dawley rats were harvested on embryonic days (E) 14, 16, and 21, as well as on postnatal days (P) 2, 5, and 10. Cryostat sections were probed with antibodies directed against beta 2 laminin, laminin-1 (alpha 1-beta 1-gamma 1), and von Willebrand factor. RESULTS: At the onset of rod photoreceptor birth (E14), laminin beta 2 surrounds the cells of the retinal pigmented epithelium (RPE) and is present on the apical surface of the retinal neuroepithelium. At E16, laminin beta 2 persists on the apical surface of the neuroepithelium and the subjacent apical surface of the RPE. At birth, laminin beta 2 fills the matrix between the juxtaposed surfaces of the RPE and neuroepithelium; moreover, laminin beta 2 immunoreactivity penetrates the neural retina. Throughout postnatal development, laminin beta 2 immunoreactivity surrounds maturing inner and outer segments. Laminin beta 2 also is found in association with blood vessels in the neural retina itself, as well as with choroidal blood vessels; in both places, it is co-localized with an endothelial marker, von Willebrand factor, and laminin-1. CONCLUSIONS: The spatial and temporal expression of laminin beta 2 is consistent with its hypothesized role in rod development. Laminin beta 2 is in a unique position to interact with mitotically active cells (in early retinal development), uncommitted progenitors (in late embryonic development), developing rods (in early postnatal development), and mature outer segments (throughout adulthood). Together with our earlier functional data, these data support our hypothesis that this molecule is an important component of the interphotoreceptor matrix.

A differential effect of APB on ON- and OFF-center ganglion cells in the dark adapted rabbit retina.

The glutamate analog, 2-amino-4-phosphonobutyric acid (APB) is a proven tool in exploring the retinal circuit; it has been shown to interfere specifically with the transmission from photoreceptor to depolarizing bipolar cell. Consequently, in photopic retinae, the application of APB disrupts the ON-channel leaving the OFF-channel undisturbed; on the other hand, in the scotopic state, APB application blocks all ganglion cell responses. In this paper, we will show that the ON- and OFF-channels have a differential sensitivity to application of APB. That is to say, APB blocks center responses in ON-ganglion cells at mean concentration of 22 +/- 5.1 microM (mean +/- standard error of the mean; n = 15) and in OFF-ganglion cells at mean concentration of 91 +/- 15.5 microM (n = 16). Since considerable data rule out direct effects of APB on ganglion cells, we hypothesize that this effect is due to a difference in the synaptic gain of ON and OFF pathways in the inner retina.

A role for 5HT3 receptors in visual processing in the mammalian retina.

We investigated the role of 5HT3 receptors in the mammalian retina using electrophysiological techniques to monitor ganglion cell activity. Activation of 5HT3 receptors with the selective agonist 1-phenylbiguanide (PBG) increased the ON responses of ON-center ganglion cells, while decreasing the OFF responses of OFF-center cells. The application of a selective 5HT3 antagonist had a reciprocal effect, namely it reduced the center response in ON-center cells and concomitantly increased the center responses in OFF-center cells. Since putative serotoninergic amacrine cells in the retina are connected specifically to the rod bipolar cell, these agents most likely affect the rod bipolar terminal. These data, together with previous studies, suggest that both 5HT2 and 5HT3 receptors mediate an excitatory influence which serves to facilitate the output from rod bipolar cells, the former via a phosphatidyl inositol second-messenger system, and the latter via a direct ion channel.

The effects of serotonin drugs on horizontal and ganglion cells in the rabbit retina.

We have investigated the effects of a serotonin 5-HT2 antagonist and a 5-HT1A agonist on horizontal and ganglion cells in the rabbit retina. Simultaneous intracellular horizontal cell and extracellular ganglion cell recordings were obtained from a superfused in vitro rabbit eyecup preparation and the effects of bath applied drugs on these cells' light responses observed. Sinusoidally modulated current was also injected into horizontal cells while the extracellular spike activity of nearby, single-unit ganglion cells was monitored. Although the ON components of the light-evoked responses of ganglion cells were reduced by the 5-HT2 antagonist or the 5-HT1A agonist, the membrane potential and the light responses of horizontal cells and the b-wave of the ERG were simultaneously unaffected. However, the drugs blocked current-driven ganglion cell spike activity induced by current injections into nearby horizontal cells. These results are discussed with respect to the site of action of these serotonin drugs and with respect to the circuitry of serotonergic neurons.

S-laminin expression in adult and developing retinae: a potential cue for photoreceptor morphogenesis.

The development of the neural retina follows a stereotyped time course that begins with an undifferentiated neuroepithelium populated by multipotential progenitor cells and ends with a highly differentiated tissue containing diverse cell types. The identities of the factors that guide this differentiation have remained elusive; a likely location for such factors, however, is the extracellular environment. Here, we show that the extracellular matrix component s-laminin is present in the neural retina, that s-laminin expression parallels the differentiation of rod photoreceptors, that photoreceptors interact with s-laminin in vitro, and that antibodies to s-laminin profoundly reduce the appearance of cells that express rhodopsin in vitro. These data suggest that s-laminin plays a role in the differentiation of the neural retina and provide evidence that the composition of the extracellular matrix may be an important determinant of retinal differentiation.

Rod pathways in mammalian retinae.

A variety of recent experiments has resolved the way in which signals are transmitted from rod photoreceptors to ganglion cells in the mammalian retina. Rods connect to a single class of rod bipolar cell, which depolarize in response to light. Rod bipolar cells are not connected directly to ganglion cells: they synapse onto rod amacrine cells, which excite ON-centre ganglion cells via gap junctions, and inhibit OFF-centre ganglion cells via inhibitory glycine synapses. Monoamines have particular influences on the rod system, through synapses with rod amacrine and rod bipolar cells, and a function for dopamine and indoleamines within this system can be hypothesized from recent experiments. There is evidence to suggest that dopaminergic amacrine cells bring the surround response into the rod system through synapses with the rod amacrine cell, and that an indoleamine, probably serotonin, increases the signal in the ON pathway through a feedback synapse onto the rod bipolar terminal.