Bipolar Cell Processes Research Articles

Cell Migration: Catapulting Neurons from the Ventricular Zone?

In the developing vertebrate brain, newly born neurons migrate away from the proliferative zones. A new paper suggests that the initial phase of this migration may be propelled by the mechanical properties of the new neurons' processes rather than more conventional mechanisms of cell migration.

Immunolocalization of metabotropic glutamate receptors 1 and 5 in the synaptic layers of the chicken retina

We have examined the distribution of metabotropic glutamate receptors (mGluRs) 1 and 5 in the adult chicken retina using preembedding immuno-electronmicroscopy. Immunoreactivity for mGluRs 1 and 5 was found in both the outer plexiform layer (OPL) and the inner plexiform layer (IPL). For mGluR1, OPL labeling was observed at cone pedicles and horizontal and bipolar cell processes. In the IPL, mGluR1 labeling could be found on bipolar cell terminals, as well as postsynaptic processes, including amacrine cell processes. Neither presynaptic nor postsynaptic elements were labeled at rod synapses. For mGluR5, OPL labeling was associated with cone pedicles as well as bipolar and horizontal cell processes. As for mGluR1, rod synapses were unlabeled. In the IPL, labeling for mGluR5 was found on bipolar cell terminals and amacrine cell processes. The presynaptic expression of these receptors in the OPL was confirmed at the light level by double-labeling experiments with SV2. The distributions of mGluRs 1 and 5 indicate that they have the potential to regulate function in both synaptic layers. Furthermore, the similar expression patterns for these two receptors indicate that they might be co-expressed and thus have the potential to interact functionally.

Axons and dendrites originate from neuroepithelial-like processes of retinal bipolar cells

The cellular mechanisms underlying axogenesis and dendritogenesis are not completely understood. The axons and dendrites of retinal bipolar cells, which contact their synaptic partners within specific laminae in the inner and outer retina, provide a good system for exploring these issues. Using transgenic mice expressing enhanced green fluorescent protein (GFP) in a subset of bipolar cells, we determined that axonal and dendritic arbors of these interneurons develop directly from apical and basal processes attached to the outer and inner limiting membranes, respectively. Selective stabilization of processes contributed to stratification of axonal and dendritic arbors within the appropriate synaptic layer. This unusual mode of axogenesis and dendritogenesis from neuroepithelial-like processes may act to preserve neighbor-neighbor relationships in synaptic wiring between the outer and inner retina.

Early changes in synaptic connectivity following progressive photoreceptor degeneration in RCS rats

The Royal College of Surgeons (RCS) rat has a retinal pigment epithelial cell defect that causes progressive loss of photoreceptors. Although it is extensively used in retinal degeneration and repair studies, how photoreceptor degeneration affects retinal circuitry has not been fully explored. This study examined the changes in synaptic connectivity between photoreceptors and their target cells using immunocytochemistry and correlated these changes with retinal function using the electroretinogram (ERG). Immunostaining with bassoon and synaptophysin (as presynaptic markers) and metabotropic glutamate receptor (mGluR6, a postsynaptic marker for ON-bipolar dendrites) was already impaired at postnatal day (P) 21 and progressively lost with infrequent pairing of presynaptic and postsynaptic elements at P60. By P90 to P120, staining became increasingly patchy and was eventually restricted to sparsely and irregularly distributed foci in which the normal pairing of presynaptic and postsynaptic markers was lost. ERG results showed that mixed scotopic a-waves and b-waves were already reduced by P21 but not oscillatory potentials. While cone-driven responses (photopic b-wave) reached normal levels at P30, they were impaired by P60 but could still be recorded at P120, although with reduced amplitude; rod responses never reached normal amplitudes. Thus, only cone-driven activity attained normal levels, but declined rapidly thereafter. In conclusion, the synaptic markers associated with photoreceptors and processes of bipolar and horizontal cells show abnormalities prior to significant photoreceptor loss. These changes are paralleled with the deterioration of specific aspects of ERG responsiveness with age. Besides providing information on the effects of photoreceptor dysfunction and loss on connection patterns in the retina, the work addresses the more general issue of how disorder of input neurons affects downstream circuitry.

Microfilament and microtubule organization and dynamics in process extension by central glia-4 oligodendrocytes: evidence for a microtubule organizing center.

Microfilaments in freshly adhering CG-4 cells and differentiated CG-4 oligodendrocytes are concentrated at the tips and edges of rapidly forming processes while microtubules are concentrated in new processes and extend from a concentrated spot of alpha-tubulin staining in the cell body to the cell periphery. In motile bipolar CG-4 cells, microfilaments are heavily concentrated at the flattened end of one process and along the rim of processes and the cell body: microtubules are concentrated along main processes and splay out into process tips and the cell body. In differentiated CG-4 oligodendrocytes, microfilaments are concentrated at the many process tips, in filopodia and in fine processes, but are not obvious in main processes where separate bundles of microtubules, which diverge at process branch points, are concentrated. gamma-tubulin, involved in microtubule nucleation, is concentrated at a small discrete area in the cell body, indicative of a microtubule organizing center. Polymerization of both actin and tubulin is required for initial process elaboration. Depolymerization of microtubules, but not of microfilaments, causes complete retraction of bipolar CG-4 cell processes. This process retraction does not occur if microfilaments are depolymerized first, indicating that process extension/retraction in motile bipolar CG-4 cells may occur by a balance of motor protein-driven forces as suggested for growth cone motility. Cytoskeleton organization in CG-4 cells is very similar to that reported for oligodendrocytes. CG-4 cells are thus a useful model for investigating the signals and mechanisms regulating oligodendrocyte process dynamics.

Distribution of rod bipolar cells in the retina of the albino rat

Purpose: In the retina of albino mammals so far examined there is a deficit in the rod photoreceptor population. We reasoned that a consequence of the rod deficit might be a subsequent abnormality in the next level of the rod pathway, the rod bipolar cell. We therefore compared the distribution of rod bipolar cells in pigmented and albino rats.Methods: Rod bipolar cells were labelled in 15 µm thick sections of fixed retinas with a monoclonal antibody directed against protein kinase C, and visualized using the ABC method. Counts of the cell bodies and processes of rod bipolar cells were undertaken at various locations across the retina.Results: Qualitatively, the protein kinase C staining suggested differences between the albino and pigmented phenotypes both in the outer and inner plexiform layers and in the inner nuclear layer. Staining was denser in the pigmented retina and the bipolar cell bodies were arranged in multiple rows rather than a single row as found in the albino retina. The actual number of rod bipolar cells was reduced in the albino phenotype. At the neonatal age we examined (post‐natal day 15), there was a 14% reduction in the total number whereas in the adult retina the reduction was around 9%.Conclusions: Although the reduction in the rod bipolar population is not as great as that previously found for the rod photoreceptors (∼25%), the reduction in both populations suggests a general abnormality in the rod pathway of albinos. Further, our data support the view that melanin is crucially involved in the normal development of retinal structure.

How Müller glial cells in macaque fovea coat and isolate the synaptic terminals of cone photoreceptors.

A cone synaptic terminal in macaque fovea releases quanta of glutamate from approximately 20 active zones at a high rate in the dark. The transmitter reaches approximately 500 receptor clusters on bipolar and horizontal cell processes by diffusion laterally along the terminal's 50 microm(2) secretory face and approximately 2 microm inward. To understand what shapes transmitter flow, we investigated from electron photomicrographs of serial sections the relationship between Müller glial processes and cone terminals. We find that each Müller cell has one substantial trunk that ascends in the outer plexiform layer below the space between the "footprints" of the terminals. We find exactly equal numbers of Müller cell trunks and foveal cone terminals, which may make the fovea particularly vulnerable to Müller cell dysfunction. The processes that emerge from the single trunk do not ensheathe a single terminal. Instead, each Müller cell partially coats two to three terminals; in turn, each terminal is completely coated by two to three Müller cells. Therefore, the Müller cells that coat one terminal also partially coat the surrounding ( approximately six) terminals, creating a common environment for the cones supplying the center/surround receptive field of foveal midget bipolar and ganglion cells. Upon reaching the terminals, the trunk divides into processes that coat the terminals' sides but not their secretory faces. This glial framework minimizes glutamate transporter (EAAT1) beneath a terminal's secretory face but maximizes EAAT1 between adjacent terminals, thus permitting glutamate to diffuse locally along the secretory face and inward toward inner receptor clusters but reducing its effective spillover to neighboring terminals.

Synapse Formation Is Arrested in Retinal Photoreceptors of the ZebrafishnrcMutant

We describe the effects of a recessive mutation on visual behavior, the electroretinogram (ERG), and photoreceptor structure in zebrafish. At 6 d post-fertilization (dpf), no optokinetic reflex could be elicited in no optokinetic response c (nrc) mutant animals under any test condition. The animals exhibited ERG responses at 5-7 dpf that were markedly abnormal and could be categorized into two groups. The first showed an initial negative a-wave followed by a delayed positive b-wave of small amplitude. Often a second ERG-like response was recorded after the initial b-wave. The second group showed only a large negative a-wave; an initial b-wave was not evident. In most recordings additional oscillatory waves varying in number, amplitude, and time course were observed. Multiple responses at the cessation of long-duration flashes were also observed. Light and electron microscopy revealed that the cone photoreceptor pedicles of nrc fish were highly abnormal. Although the appropriate number of synaptic ribbons formed in these terminals, they "floated" in the terminal, unassociated with postsynaptic processes or arciform densities. The few processes invaginating the nrc pedicles resembled those of horizontal cells. Invaginating bipolar cell processes were rare, but basal contacts were observed on pedicle surfaces. The severity of the mutation did not change between 6 and 8 dpf, showing that there is neither a delay in development nor a degeneration of the terminals; rather, nrc pedicle development appears arrested. Bipolar cell terminals in the inner plexiform layer made normal ribbon synapses; thus, the mutation appears to affect only the outer retina.

Organization of the inner retina following early elimination of the retinal ganglion cell population: effects on cell numbers and stratification patterns.

The present study has examined the effects of early ganglion cell elimination upon the organization of the inner retina in the ferret. The population of retinal ganglion cells was removed by optic nerve transection on the second postnatal day, and retinas were subsequently studied in adulthood. Numbers of amacrine and bipolar cells were compared in the nerve-transected and nerve-intact retinas of operated ferrets, while stratification patterns within the inner plexiform layer were compared in these and in normal ferret retinas. Early ganglion cell elimination was found to produce a 25% reduction in the population of glycine transporter-immunoreactive amacrine cells, and 18 and 15% reductions in the populations of parvalbumin and calbindin-immunoreactive amacrine cells, respectively. GABAergic amacrine cells were also reduced by 34%. The number of calbindin-immunoreactive displaced amacrine cells, by contrast, had increased in the ganglion cell-depleted retina, being three times their normal number. Other amacrine and bipolar cell types were unaffected. Despite these changes, the stratification patterns associated with these cell types remained largely intact within the inner plexiform layer. The present results demonstrate a class-specific dependency of inner retinal neurons upon the ganglion cell population in early postnatal life, but the ganglion cells do not appear to provide any critical signals for stratification within the inner plexiform layer, at least not after birth. Since they themselves do not produce stratified dendritic arbors until well after birth, the signals for stratification of the bipolar and amacrine cell processes should arise from other sources.

The cone pedicle, a complex synapse in the retina.

Cone pedicles, the synaptic terminals of cone photoreceptors, are connected in the macaque monkey retina to several hundred postsynaptic dendrites. Using light and electron microscopy, we found underneath each cone pedicle a laminated distribution of dendritic processes of bipolar and horizontal cells. Superimposed were three strata of glutamate receptor (GluR) aggregates, including a novel layer of glutamate receptors clustered at desmosome-like junctions. They are, most likely, postsynaptic densities on horizontal cell dendrites. GABA(A) and GABA(C) receptors are aggregated on bipolar cell dendrites in a narrow band underneath the cone pedicle. Glutamate released from cone pedicles and GABA released from horizontal cell dendrites act not only through direct synaptic contacts but also (more so) through diffusion to the appropriate receptors.

Morphological analysis of peripheral nerve regenerated by means of vein grafts filled with fresh skeletal muscle.

Clinical data have shown that a vein segment filled with fresh skeletal muscle can be considered a good autologous grafting conduit for the repair of peripheral nerve lesions. In this study, the long-term morphological organization of rat sciatic nerve fibers regenerated along a muscle-vein-combined graft conduit is further analysed by light and electron microscopy. Regenerated nerve fibers were organized into fascicles of various sizes that were clearly delimited by perineurial-like shells made by long and thin cytoplasmic processes of perineurial-like bipolar cells and by densely packed collagen fibrils. Grafted skeletal muscle fibers were still detectable among nerve fiber fascicles. However, in spite of the persistence of skeletal muscle along the graft, regenerated nerve fibers showed a good morphological pattern of regeneration, providing further evidence that the muscle-vein-combined grafting technique represents an effective surgical alternative to the classical fresh nerve autograft for the repair of peripheral nerve defects.

Electron microscopic observation of pituitary adenylate cyclase-activating polypeptide (PACAP)-containing neurons in the rat retina☆

The distribution and localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the rat retina were studied by immunocytochemistry with both light and electron microscopy. PACAP-like immunoreactivity (PACAP-LI) was detected in the amacrine and horizontal cells as well as in the inner plexiform layer, the ganglion cell layer and the nerve fiber layer. PACAP-LI seemed to be concentrated predominantly in the neuronal perikarya and their processes, but not in other cells in the retina. At the ultrastructural level, PACAP-LI was visible in the plasma membranes, rough endoplasmic reticulum, and cytoplasmic matrix in the PACAP-positive neurons in the inner nuclear layer. In the inner plexiform layer, PACAP-positive amacrine cell processes made synaptic contact with immunonegative amacrine cell processes, bipolar cell processes, and ganglion cell terminals. These findings suggest that PACAP may function as a neurotransmitter and/or neuromodulator.

Transient synaptic ribbons in the mammalian retina at unusual sites.

In the vertebrate retina the presence of synaptic ribbons (SRs) is well documented in two sites only, viz., in photoreceptor axon terminals in the outer plexiform layer and in bipolar cell axons in the inner plexiform layer. The present paper reports the presence of non-photoreceptor SRs in the outer plexiform layer of cattle and mouse, where they were seen in small numbers in thin cell processes near cone pedicles of light-adapted animals. They were never seen near rod spherules. Quantitative data obtained in mice killed at different time-points revealed that the SRs under consideration increased in number during day time and were absent during the dark phase. Moreover, under high light intensity of 10000 lux they were more frequent in number compared to 100-lux-exposed animals. It is concluded that the cell processes revealing the temporary presence of SRs are processes of flat bipolar cells which may provide a feedback to cones during the light phase.

Immunocytochemical localization of the glutamate transporter GLT-1 in goldfish (Carassius auratus) retina.

Glutamate is the major excitatory neurotransmitter in the retina of vertebrates. Electrophysiological experiments in goldfish and salamander have shown that neuronal glutamate transporters play an important role in the clearance of glutamate from cone synaptic clefts. In this study, the localization of the glutamate transporter GLT-1 has been investigated immunocytochemically at the light and electron microscopical levels in the goldfish retina using a GLT-1-specific antibody. GLT immunoreactivity (IR) was observed at the light microscopical level in Müller cells, bipolar cells, the outer plexiform layer (OPL), and the inner plexiform layer (IPL). At the electron microscopical level, membrane-bound and cytoplasmic GLT-IR in the OPL was located in finger-like protrusions of the cone terminal located near the invaginating postsynaptic processes of bipolar and horizontal cells. GLT-IR was not observed in the vicinity of synaptic ribbons. This location of GLT-1 allows modulation of the glutamate concentration in the synaptic cleft, thereby shaping the dynamics of synaptic transmission between cones and second-order neurons. In the inner IPL, GLT-IR was observed in the cytoplasm and was membrane bound in mixed rod/cone bipolar cell terminals and cone bipolar cell terminals. The membrane-bound GLT-1 was generally observed at some distance from the synaptic ribbon. The morphology of the bipolar cell terminal together with the localization of GLT-1 suggests that at least these glutamate transporters are not primarily involved in rapid uptake of glutamate release by the bipolar cells. The GLT-IR in the cytoplasm of Müller cells was located throughout the entire goldfish retina from the outer limiting membrane to the inner limiting membrane. The location of GLT-1 in Müller cells is consistent with the role of Müller cells in converting glutamate to glutamine.

Rods and cones project to the inner plexiform layer during development

Mature rod and cone photoreceptor cells extend terminals to the outer plexiform layer (OPL), where they form characteristic spherules or pedicles, synapsing with the second-order neurons of the inner nuclear layer (INL). The present study demonstrates that, prior to the formation of this connectivity, immature rods and cones in the ferret extend processes beyond the level of the horizontal cells and future OPL, reaching the inner plexiform layer (IPL). The number of processes extending to the IPL increases steadily as the population of photoreceptor cells expands postnatally, reaching a maximum 2 weeks after birth. These processes are immunopositive for synaptophysin, and they terminate in two strata occupied by the dendrites of amacrine cells and ganglion cells. The frequency of these processes declines rapidly during the third postnatal week, and they are no longer detectable by the fourth postnatal week. Their loss is neither a consequence of photoreceptor cell death nor is it due to selective protein trafficking mechanisms that render them immunonegative. Rather, these processes retract to the level of the OPL during this period, coincident with the maturation of bipolar and horizontal cell processes. These results demonstrate that, despite the clear presence of environmental signals presaging the formation of the OPL, photoreceptor terminals initially ignore them to grow beyond this level of the retina. Rather, they detect and respond to signals within the IPL during this period, terminating in proximity to the processes of other cells in the inner retina, where they may contribute to transient retinal circuitry during early development.

Fine structure of the retina of black bass, Micropterus salmoides (Centrarchidae, Teleostei).

The structure of light- and dark-adapted retina of the black bass, Micropterus salmoides has been studied by light and electron microscopy. This retina lacks blood vessels at all levels. The optic fiber layer is divided into fascicles by the processes of Müller cells and the ganglion cell layer is represented by a single row of voluminous cells. The inner nuclear layer consists of two layers of horizontal cells and bipolar, amacrine and interplexiform cells. In the outer plexiform layer we observed the synaptic terminals of photoreceptor cells, rod spherules and cone pedicles and terminal processes of bipolar and horizontal cells. The spherules have a single synaptic ribbon and the pedicles possess multiple synaptic ribbons. Morphologically, we have identified three types of photoreceptors: rods, single cones and equal double cones which undergo retinomotor movements in response to changes in light conditions. The cones are arranged in a square mosaic whereas the rods are dispersed between the cones.

Localization of nNOS in photoreceptor, bipolar and horizontal cells in turtle and rat retinas.

Neuronal nitric oxide synthase (nNOS), an enzyme that synthesizes NO, has been found in the outer retina using light microscopic immunocytochemistry, but its subcellular localization is unknown. We used electron immunocytochemistry to examine nNOS-like immunoreactivity (nNOS-LI) in the outer plexiform layer of turtle and rat retinas. In turtle, nNOS-LI was present in some bipolar and horizontal cell processes at photoreceptor ribbon synapses and at basal junctions between photoreceptors. In rat, nNOS-LI was present in some rod bipolar and B-type horizontal cell axon terminals at rod ribbon synapses. These results indicate that in vertebrates, NO is produced by all of the major nerve cell types in the outer retina at specific synaptic contacts.

Neurochemistry of the mammalian cone 'synaptic complex'.

The cone 'synaptic complex' is a unique structure in which a single presynaptic axon secretes glutamate onto processes of bipolar cells (both ON and OFF) and horizontal cells. In turn, the horizontal cell processes antagonize cone and bipolar responses to glutamate (probably by GABA). What still remains largely unknown is the molecular identity of the postsynaptic receptors and their exact locations. We identified several subunits of the glutamate receptor and the GABAA receptor expressed at the cone synaptic complex and localized them ultrastructurally. Glutamate receptors: (i) Invaginating (probably ON) bipolar dendrites in the monkey and rat express the metabotropic glutamate receptor, mGluR6. The stain is intense on the dendritic membrane where it first enters the invagination, and weak at the tip nearest to the ribbon. The cone membrane is electron-dense where it apposes the intense stain for mGluR6. Surprisingly, invaginating bipolar dendrites in the cat also express the AMPA receptor subunits, GluR2/3 and GluR4. (ii) Dendrites forming basal contacts in the cat (probably OFF) express the AMPA subunits GluR2/3, GluR4, and also the kainate subunit, GluR6/7. The stain is especially intense at the dendritic tips in apposition to electron-dense regions of cone membrane. (iii) Horizontal cells in the cat express the AMPA subunits GluR2/3, GluR4 and the kainate subunit, GluR6/7. The stain is strongest in the cytosol of somas and primary dendrites, but is also present in the invaginating terminals where it localizes to the membrane subjacent to the ribbon. GABAA receptors: (i) ON and OFF bipolar dendrites in the monkey express the alpha 1 and beta 2/3 subunits. The stain is localized to the bipolar cell membrane in apposition to horizontal cell processes. (ii) Cones did not express the GABAA subunits tested by immunocytochemistry, but beta 3 mRNA was amplified by RT-PCR from rat photoreceptors. (i) mGluR6 receptors concentrate on dendrites at the base of the invagination rather than at the apex. This implies that receptors at both 'invaginating' and 'basal' contacts lie roughly equidistant from the release sites and should therefore receive similar spatiotemporal concentrations of glutamate. (ii) The 'cone' membrane is electron-dense opposite to the receptor sites on both ON and OFF bipolar cells. This suggests a special role for this region in synaptic transmission. Possibly, these densities signify a transporter that would regulate glutamate concentration at sites remote (> 200 nm) from the locus of vesicle release.

Visualization of interstitial cells of Cajal in the mouse colon by vital staining.

Interstitial cells of Cajal (ICCs) are believed to be a major element in generating the spontaneous rhythm of the gastrointestinal tract. A prominent problem in the study of these cells has been the difficulty in observing them in intact tissues. We used the lipophilic dye DiI to stain ICCs in the submucosal-circular muscle border of freshly dissected mouse colon. The placement of small DiI crystals in this area resulted in the labeling of ICC-like cells. Two main morphological cell types, viz., bipolar and multipolar, were noted. Bipolar cells had two primary processes emerging from the poles of an elongated soma. The mean length of these processes was 78.7 microm. These cells constituted 42.3% of the sample (n=105). Multipolar cells (54.3% of total) had a less elongated soma and extended 3-6 main processes whose mean length was 56.3 microm. These processes showed no preferred direction. The length of the primary processes of bipolar cells was 40% greater than that of multipolar cells (P<0.02). Three cells (2.9%) had only one primary process. The DiI stain could be converted into a stable electron-opaque product. Electron-microscopic observations showed that these cells had the typical appearance of ICCs reported in previous studies. This staining method should be useful for physiological investigations of ICCs in gastrointestinal tissues.

Differential distribution of beta-dystroglycan in rabbit and rat retina.

The distribution of the dystrophin-associated glycoprotein complex was investigated in rabbit and rat retina by using the monoclonal antibody 43DAG/8D5, which specifically recognizes beta-dystroglycan, a central component of the complex. In cryostat sections of retinae from both species, the authors observed staining of blood vessels, continuous labeling around the vitreal border, and strong immunoreactivity in the outer plexiform layer (OPL). Electron microscopy showed that the immunoreactivity associated with the vitreal border of the retina was the result of a subcellular concentration of beta-dystroglycan in the endfeet of Müller glial cells. A similar concentration was observed in endfeet of perivascular astrocytes in the region of contact with the capillary basal lamina. In the OPL, beta-dystroglycan was associated with the terminals of both rods and cones. The label was almost exclusively found outside the synaptic area and was particularly strong in the extensions of the photoreceptor terminals protruding into the OPL. In the OPL of the rabbit retina, the authors found additional immunoreactivity associated with the tips of postsynaptic horizontal and bipolar cell processes. These results show that the dystrophin-associated glycoprotein complex is subcellularly concentrated in photoreceptor terminals and glial cell endfeet, and that the rabbit retina differs from the rat retina by the additional expression of this complex in bipolar and horizontal cells.