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.

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.

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.

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.

Elasmobranch oculomotor organization: anatomical and theoretical aspects of the phylogenetic development of vestibulo-oculomotor connectivity.

The oculomotor organization of two elasmobranch species, smooth dogfish (Mustelus canis) and little skate (Raja erinacea), was studied by investigating the extraocular muscle apparatus and the oculomotor motoneuron distribution. The macroscopic appearance of the eye muscles was similar to any lateral-eyed vertebrate species (e.g., goldfish, rabbit). The size of extraocular muscles was expressed by counting single muscle fibers and comparing cross-sectional areas of the extraocular muscles. There were significant differences in the number of fibers in the six extraocular muscles in dogfish, but not in skate. Fiber sizes varied considerably; thus, the number of fibers did not relate to cross-sectional areas. In the dogfish, no one pair of agonist-antagonist extraocular muscles was larger than the others, suggesting that there was no preference for eye movements in a particular plane of space. However, the lateral rectus was more than twice the size of most of the other muscles. In the skate, cross-sectional areas of the horizontal eye muscles were smaller than those of the vertical eye movers. This may indicate a reduced utilization of horizontal eye muscles, which may reflect the bottom-dwelling habitat and mode of locomotion of the skate. The distribution of the extraocular motoneurons was determined by injecting horseradish peroxidase (HRP) into single eye muscles. Medial rectus, superior rectus, and superior oblique motoneuron populations were located contralateral to their respective muscles. Lateral rectus, inferior rectus, and inferior oblique motoneurons were located ipsilateral to their muscles. This distribution is in contrast to almost all other vertebrates studied thus far, where medial rectus motoneurons are located ipsilateral to the muscle which they innervate. The oculomotor arrangement in elasmobranchs is likely to have consequences for the circuitry responsible for the production of conjugate compensatory eye movements in the horizontal plane. We hypothesize that, in contrast to other vertebrates, the basic elasmobranch vestibulo-ocular reflex pathway consists of three identically structured three-neuron-arcs connecting the three semicircular canals to their respective extraocular muscles. This innervation pattern may constitute a special feature of the elasmobranch brain or a phylogenetically older arrangement of eye movement pathways.

Retinal neurochemistry of three elasmobranch species: an immunohistochemical approach.

We surveyed retinas of Raja erinacea, Mustelus canis, and Squalus acanthias for neurotransmitter substances by using antisera directed against the substances themselves or against their synthesizing enzymes. Both the peroxidase-antiperoxidase (PAP) and indirect fluorescent techniques were employed to visualize the primary antisera. In all three species positive results were obtained with antisera directed against tyrosine hydroxylase (TOH), glutamic acid decarboxylase (GAD), serotonin (5-HT), and leucine enkephalin (Lenk). Antisera directed against glucagon, neurotensin, beta-endorphin, vasoactive intestinal peptide, or bombesin failed to show any specific staining. Immunoreactivity was located in amacrine, interplexiform, and horizontal cells as well as in axons of the optic fiber layer. The four antisera labelled different amacrine cell classes, distinguished on the bases of perikaryal morphology and the distribution of cell processes in the inner plexiform layer (IPL). Amacrine cells that labelled with the same marker were seen to have different morphologies in the species studied. Thus, TOH-like immunoreactivity was distributed in layers 1, 3, and 5 of the IPL in Mustelus but only in layers 1 and 3 in Raja retina. GAD-like immunoreactivity was found diffusely over all layers of the IPL in Raja, but in Mustelus it was confined primarily to layers 1, 3, and 5 of the IPL. Lenk- and 5-HT-like immunoreactivities showed similar species variations. Two neurochemical classes of interplexiform cell were identified in this study. In Mustelus GAD-like and Lenk-like immunoreactive interplexiform cells were seen whereas in Raja only GAD-positive interplexiform cells were detected. In squalus no unequivocal demonstration of any interplexiform cell was made with these antisera. The GAD antiserum also labelled a subset of the horizontal cells in the dorsal retina of Raja. TOH and 5-HT-antisera labelled axons in the optic fiber layer of all three species but reactive ganglion cell perikarya were not identified.

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.

5-HT2 antagonists reduce ON responses in the rabbit retina.

We have investigated the effects of serotonin (5-HT2) antagonists in the rabbit retina. These antagonists reduce the ON responses of ON-center cells as well as the surround (ON) responses of OFF-center cells, and enhance the center (OFF) responses of the latter cells. The result is consistent with the anatomy of the indoleamine-accumulating cells in the rabbit retina, which ramify in sublamina b (ON) of the inner plexiform layer and contact primarily bipolar cells that are depolarizing in the rabbit. This suggests that at least part of the surround (ON) responses to OFF-center cells is generated in the inner plexiform layer.

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.

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.

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.

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.

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 agonists and antagonists on the response properties of complex ganglion cells in the rabbit's retina.

Selective agonists and antagonists were employed to determine the role of indoleaminergic amacrine cells in the generation of the light-evoked responses and spontaneous activity of direction and orientation selective cells. Perfusion with 5-HT2 antagonists reduced the spontaneous activity and both the leading and trailing edge responses of ON/OFF direction selective cells. 5-HT1a agonists had a similar effect on this class of cell, namely, a reduction of light-evoked and spontaneous activity. Results from ON-center and OFF-center orientation selective cells were consistent with those obtained from direction selective cells in that no disruption of direction or orientation selectivity was observed during perfusion of these drugs. These data suggest that the indoleaminergic cells are not directly involved in the generation of the trigger features of complex ganglion cells, but may be facilitating synaptic transmission in the inner retina. This function is discussed relative to the connectivity of the rod bipolar cells and the putative indoleaminergic amacrine cells. The similarity of the effects of 5-HT1a agonists and 5-HT2 antagonists supports the hypothesis, developed during our prior studies of brisk ganglion cells, that these two receptor classes mediate antagonistic processes in the target neurons.

The actions of serotonergic agonists and antagonists on the activity of brisk ganglion cells in the rabbit retina.

Extracellular electrophysiological recordings were obtained from rabbit retinal ganglion cells in either a superfused eyecup or an in vivo preparation. Selective antagonists or agonists of serotonin at the 5-HT2 or 5-HT1A receptors were applied, and the changes in light-evoked and spontaneous activity were studied. Both 5-HT1A agonists and 5-HT2 antagonists reduced the ON-components of the light-evoked responses of all classes of brisk ganglion cell; spontaneous activity was reduced in these cells as well. These results suggest that the indoleamine-accumulating amacrine cells of the rabbit retina serve to facilitate the output of the depolarizing rod bipolar cell and thereby increase the efficacy of transmission between this and other cells in the rabbit retina, and that this process is mediated by 5-HT2 receptors. On the basis of the similarity of the actions of the 2 classes of drug studied, we hypothesize further that 5-HT1A receptors mediate an inhibitory process that serves to terminate the indoleamine-induced facilitation. This process may be located either in the bipolar terminal or presynaptic to it in the terminal of the putative indoleaminergic cells.

Neuropharmacological analysis of the role of indoleamine-accumulating amacrine cells in the rabbit retina.

In order to elucidate the role of putative indoleaminergic amacrine cells in visual processing, we have employed pharmacological agents specific for the two classes of serotonin receptor, 5-HT2 and 5-HT1, which have been identified in both the retina and brain. Perfusion of the rabbit retina with 5-HT2 selective antagonists decreases the ON-excitation of all classes of ganglion cell as well as the spontaneous activity of these cells. The effect on OFF-responses depends on the cell type: OFF-excitation of center-surround brisk and sluggish cells is increased or not affected by these drugs, but OFF excitation of ON/OFF direction selective cells is reduced. No disruption of the trigger features of direction selective or orientation selective cells was discovered, suggesting that indoleaminergic amacrine cells do not play a role in the generation of the complex properties of these cells. 5-HT1 receptors are heterogeneous and classified as a, b, or c subtypes. Since no selective antagonists are available for these sites, we have employed specific agonists. The most specific of these are for the 5-HT1A receptor. Perfusion with these agonists had physiological effects similar to those with perfusion of 5-HT2 antagonists. Thus, we have suggested that these two classes of serotonin receptors mediate opponent processes in the neural pathway. Since indoleaminergic cells make reciprocal synaptic connections with rod bipolar cell terminals, which are depolarizing in the rabbit retina, we hypothesize that 5-HT2 receptors facilitate the synaptic transmission from the depolarizing rod bipolar cell thus facilitating ON-excitation in the retinal network while 5-HT1A receptors mediate an inhibitory process. Similar self-opponent processing appears to take place in the hypothalamic and hippocampal serotonergic systems as well as in the dopaminergic retinal system. Thus, it is likely that this organization is a general feature of monoamine signal transmission in the central nervous system.