Guidance-cue control of horizontal cell morphology, lamination, and synapse formation in the mammalian outer retina

In the vertebrate retina, neuronal circuitry required for visual perception is organized within specific laminae. Photoreceptors convey external visual information to bipolar and horizontal cells at triad ribbon synapses established within the outer plexiform layer (OPL), initiating retinal visual processing. However, the molecular mechanisms that organize these three classes of neuronal processes within the OPL, thereby ensuring appropriate ribbon synapse formation, remain largely unknown. Here we show that mice with null mutations in Sema6A or PlexinA4 (PlexA4) exhibit a pronounced defect in OPL stratification of horizontal cell axons without any apparent deficits in bipolar cell dendrite or photoreceptor axon targeting. Furthermore, these mutant horizontal cells exhibit aberrant dendritic arborization and reduced dendritic self-avoidance within the OPL. Ultrastructural analysis shows that the horizontal cell contribution to rod ribbon synapse formation in PlexA4?/? retinas is disrupted. These findings define molecular components required for outer retina lamination and ribbon synapse formation.     

Modulation of cone horizontal cell activity in the teleost fish retina. I. Effects of prolonged darkness and background illumination on light responsiveness

The effects of prolonged (greater than 2 hr) darkness and background illumination on the light responsiveness of cone horizontal cells were examined in isolated, superfused white perch retinas. In retinas from fish maintained in complete darkness for more than 2 hr, cone horizontal cells had a resting membrane potential of about -18 mV, and they generated only slow, low-amplitude (3-4 mV) responses even when stimulated with bright flashes. Following the presentation of dim background light, the cone horizontal cells slowly hyperpolarized and thereafter remained at a more hyperpolarized level (about -25 mV). Concurrently, their light responses were dramatically enhanced in size, and response amplitudes to bright flashes eventually increased to about 50 mV. This was accompanied by noticeable changes in response waveforms; following light exposure, the responses became faster and showed initial on-transients. The increase in cone horizontal cell responsiveness was graded with intensity of the background light. A similar enhancement in response amplitudes of cone horizontal cells occurred after presenting bright flashes repetitively at intervals of 9 sec. This background sensitization phenomenon was observed in both L- and C-type cone horizontal cells. When light-sensitized isolated retinas were maintained in darkness for long periods (greater than 30 min), the light responsiveness of cone horizontal cells gradually decreased. The changes in membrane potential and response waveform were opposite to those that occurred when prolonged dark-adapted retinas were exposed to background lights; the cells depolarized by 5-7 mV and light-evoked responses became slower. Effects of background illumination on rod-driven horizontal cells were examined as well. Rod horizontal cells were about 2 log units more sensitive to white light than were cone horizontal cells. When recorded in prolonged dark-adapted retinas, rod horizontal cells showed large responses (approximately 50 mV), which could not be further enhanced by background illumination. Cone horizontal cell responsiveness in the carp retina was also depressed by prolonged darkness and increased by illumination in a fashion similar to that observed in the white perch retina. The change in response amplitude was only about 2-fold in carp compared with 5- to 10-fold changes observed in white perch. These results indicate that the light responsiveness of cone horizontal cells in the teleost retina is suppressed in prolonged darkness and that background lights release the cells from suppression.(ABSTRACT TRUNCATED AT 400 WORDS)     

Renewal of electrotonic synapses in teleost retinal horizontal cells

In teleost retinas, the somata of same-type cone horizontal cells are electrically coupled via extensive gap junctions, as are the axon terminals of same-type cells. This coupling persists throughout the animal's life and is modulated by dopamine and conditions of light- vs. dark-adaptation. Gap junction particle density in goldfish horizontal cell somata has also been shown to change under these conditions, indicating that these junctions are dynamic. We have used electron microscopy to examine gap junctions in bass horizontal cells with a fixation method that facilitates detection of gap junctions. Annular gap junction profiles were observed in the somatic cytoplasm of all cone horizontal cell types in both light- and dark-adapted animals. Serial sections showed that most profiles represented gap junction vesicles free within the cytoplasm; the remainder represented vesicles still attached to extensive plasma membrane gap junctions by a thin cytoplasmic neck, suggestive of an intermediate stage in endocytosis. Observations of gap junction vesicles containing fragments of gap junctional membrane and/or fused with lysosomal bodies further supported this hypothesis. Because gap junctions persist between the horizontal cells, we propose that gap junction endocytosis and lysosomal degradation are balanced by addition of new junctions. While endocytosis has been widely demonstrated to serve in programmed removal of gap junctions (without subsequent replacement), from both nonneuronal cells and developing neurons, this study indicates that it can also function in the renewal of electrical synapses in the adult teleost retina, where gap junction elimination is not the goal.     

Lack of protein-tyrosine sulfation disrupts photoreceptor outer segment morphogenesis, retinal function and retinal anatomy

To investigate the role(s) of protein‐tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1^?/?/Tpst2 ^?/?) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein‐tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance.     

A comparative analysis of glial and neuronal markers in the retina of fish: variable character of horizontal cells

The immunohistochemical localizations of the enzymes glutamine synthetase, carbonic anhydrase-C, and the intermediate filament protein GFA were examined for potential neuroglial specificity in the retinas of several types of fish. Both glutamine synthetase and carbonic anhydrase-C appear to be characteristic markers for retinal Müller cells. However, the horizontal neurons of most fish examined also possess high levels of carbonic anhydrase. Furthermore, GFA, the characteristic marker for fibrous astroglia in higher vertebrates, was found specifically localized in the horizontal neurons of several teleost fish. The identity of the GFA antigens was qualified by immunochemical as well as cytological examinations. Furthermore, specific antisera to other intermediate filament proteins, including neurofilaments, validated and contrasted with the observations made with antisera to GFA. The presence of GFA in horizontal neurons of fish is widespread but not typical of all fish. These observations indicate an evolutionary constancy of retinal Müller glial cells. However, these results again focus attention on whether horizontal cells are truly neurons or rather represent an intermediate cell type that may prove useful in studying the evolution, ontogeny, and functional significance of the neuronal-glial phenotypic dichotomy.     

Modulation of cone horizontal cell activity in the teleost fish retina. II. Role of interplexiform cells and dopamine in regulating light responsiveness

Following the destruction of the terminals of the dopaminergic interplexiform cells by intraocular injections of 6-hydroxydopamine (6-OHDA), cone horizontal cells exhibited high light responsiveness in prolonged darkness and their responses to moderate and bright full-field flashes were as large as 60 mV. Furthermore, the light responsiveness of these cells in the 6-OHDA-treated retinas was not enhanced by background illumination. The application of dopamine (50 microM) by superfusion to 6-OHDA-treated retinas resulted in a decrease in light responsiveness and changes in response waveform of the cone horizontal cells. Twenty minutes following dopamine application the responses of the cone horizontal cells closely resembled the response of cells recorded in prolonged dark-adapted retinas. Dopamine caused similar changes in cone horizontal cells recorded in light-exposed retinas, but had no obvious effects on rod horizontal cells. The selective dopamine D1 receptor antagonist, Sch 23390, enhanced cone horizontal cell responsiveness when applied to prolonged dark-adapted retinas, mimicking background illumination. The light responsiveness of cone horizontal cells recorded after application of Sch 23390 was less than that for cells in retinas that had been exposed to background lights, but light responsiveness could not be further enhanced by background illumination. Another dopamine antagonist, (+)-butaclamol, was found to have effects similar to Sch 23390 on cone horizontal cells, but (-)-butaclamol, the inactive enantiomer, did not enhance the light responsiveness of these cells. The results suggest that the dopaminergic interplexiform cells play a crucial role in the regulation of cone horizontal cell responsiveness by prolonged darkness and background illumination. These cells may release dopamine tonically in the dark, which suppresses cone horizontal cell responsiveness. Background illumination may decrease dopamine release and liberate cone horizontal cells from the suppression.     

Retinal structure in the smooth dogfish, Mustelus canis: light microscopy of photoreceptor and horizontal cells

Photoreceptor and horizontal cells in retinas of the smooth dogfish were impregnated by the rapid Golgi method. Cones as well as rods are present; there are hundreds of rods per cone. The rod synaptic endings are small spherules which send out basal processes (telodendria). The cone synaptic endings are large pedicles which also extend telodendria. Horizontal cells of three distinctive varieties are segregated in vertically separate layers. Horizontal cells of the first (external) row are thick and cuboidal. They send out processes which enter into the invaginations in rod spherules. These cells and their processes cover a circular to elliptical field measuring 75–125 by 125–200 μm. They probably contact every rod in this field. Horizontal cells of the second (intermediate) row are flattened and stellate. Their processes also enter into the rod spherules. These cells cover a circular to elliptical field slightly larger than that covered by cells of the first row. Although they appear not to contact every rod in their field, the fields of adjacent cells of this type overlap. Every rod, therefore, probably contacts one horizontal cell of the second row as well as one of the first row. Horizontal cells of the third (internal) row are stellate, with a few long cylindrical horizontal processes. Finer vertical processes go from these to contact cone pedicles. Probably each cone contacts one such process, and each third-row horizontal cell contacts on the order of ten cones. Although somewhat uncertain, the size, shape, and degree of overlap of the fields covered by third-row cells are probably not very different from those of first- and second-row cells. The vertical sequence of horizontal cells which contact rods or cones in dogfish is inverted from that in teleosts.     

Effects of GABA and glycine on the distal cells of the cyprinid retina

Two putative retinal neurotransmitters, γ-aminobutyric acid (GABA) and glycine, were applied to the isolated carp and goldfish retinas while intracellular recordings from horizontal cells and cones were made. At relatively low concentrations (0.1–1 mM), GABA consistently hyperpolarized dark-adapted H₁ or L-type cone horizontal cells and cone photoreceptors, reduced light evoked responses and suppressed the on-transients. The effects of GABA on H₁ horizontal cells were abolished when Co²⁺ was applied to the retina, indicating that GABA exerts its effects on the horizontal cells via the receptors. Thus GABA is likely to be involved in the feedback synapse between H₁ horizontal cells and the cones. Low concentrations of glycine (0.1–1 mM), hyperpolarized a number of H₂ or C-type horizontal cells and selectively abolished their dopolarizing responses to red light; thus glycine may be involved in synaptic pathways which mediate or modulate the depolarizing responses to red flashes in C-type horizontal cells.     

Horizontal cell electrical coupling in the giant danio: synaptic modulation by dopamine and synaptic maintenance by calcium

Electrical synapses, and their structural manifestation, gap junctions, are critical elements of retinal circuitry. These synapses are subject to both rapid modulation and slower structural changes by physiological signals which mediate changes in the adaptational state of the retina. The electrical synapses of fish retinal horizontal cells are an excellent preparation for in vitro studies of electrical synapses. We have examined the rapid modulation of electrical coupling by dopamine and effects on the expression and maintenance of electrical synapses by cell calcium in pairs of horizontal cells isolated from retinas of the giant danio (Danio aquipinnatus). We report that rapid modulation by dopamine reduces junctional conductance by modifying gap junction channel gating, while maintaining cells in reduced calcium medium, and lowering; intracellular calcium concentration, results in the loss of electrical coupling. The effects of calcium on synaptic maintenance may be related to structural changes observed in horizontal cell electrical synapses during light adaptation.     

Anatomy of the retina of the mink (Mustela vison)

The retina of the normal pigmented mink has been studied by light and electron microscopy. This retina resembles the typical vertebrate retina in its pastterns of lamination and synaptic interconnectivity. Rod and cone outer segments and receptor spherule and pedicle endings are found. At least two different types of horizontal cell processes are seen with the electron microscope, suggestive of rabbit A and B types. Ribbon and conventional synapses are found in both plexiform layers; conventional synapes are also present in the inner nuclear layer. Quantitative studies of the inner plexiform layer revealed amacrine:bipolar synapse ratios (3.3:1) similar to those of the cat and monkey. Other quantitative parameters also resembled those previously reported for species with retinas that predominantly contain concentric-type receptive fields.     

Dopaminergic regulation of horizontal cell gap junction particle density in goldfish retina

Light- or dark-adapted goldfish (Carassius auratus) retinas were treated with dopamine, which is believed to uncouple horizontal cells via D1 receptors, or with the dopamine antagonist haloperidol. Aldehyde-fixed retinas were freeze-fractured and the replicas examined by electron microscopy to identify horizontal gap junctions. The density (number per micron2) of intra-membrane particles of horizontal cell soma gap junctions was significantly lower in light-adapted and dopamine-treated retinas than in dark-adapted and haloperidol-treated retinas. There was no statistically significant difference between gap junction particles densities in (I) light-adapted (untreated) and in dopamine-treated (light- or dark-adapted) retinas, or between (II) dark-adapted (untreated) and haloperidol-treated (light- or dark-adapted). These results suggest that the uncoupling of horizontal cell somas by dopamine is accompanied by a decrease in gap junction particle density and that there is a greater release of dopamine during light-adaptation than dark-adaptation. Unlike horizontal cell somas, horizontal cell axon terminals did not show consistent changes in gap junction particle density with light- or dark-adaptation. Although the data suggests that there may be a reduction in axon terminal gap junction particle density with dopamine treatment, this effect is not reversible with haloperidol treatment. Our results suggest that the regulation of gap junctions may differ at two sites within the same cell.     

Dopamine release from interplexiform cells in the retina: effects of GnRH, FMRFamide, bicuculline, and enkephalin on horizontal cell activity.

In teleost fish, dopaminergic interplexiform cells provide an intraretinal centrifugal pathway from the inner to the outer plexiform layer, where they make abundant synapses on cone-related horizontal cells. The interplexiform cells receive all their input in the inner plexiform layer from centrifugal fibers and amacrine cells. In fish, centrifugal fibers contain gonadotropin hormone-releasing hormone (GnRH)-like and FMRFamide-like peptides (Munz et al., 1982; Stell et al., 1984), whereas amacrine cells contain a variety of neuroactive substances, including a number of peptides. In this study, we examined the effects of GnRH, FMRFamide, bicuculline, and enkephalin on horizontal cell activity in the white perch retina in an attempt to understand the synaptic inputs to the interplexiform cells. When the retina was superfused with Ringer's solution containing GnRH, horizontal cells depolarized (approximately 10 mV), and their responses to small spots increased, whereas their responses to full-field lights decreased. Thus, GnRH closely mimicked the effects of dopamine on horizontal cells. The GnRH antagonist [D-Phe2, Pro3, D-Phe6]-GnRH blocked the effects of GnRH, as did haloperidol. GnRH also had no effect on horizontal cells in retinas treated with 6-hydroxydopamine. The results indicate that GnRH acts by stimulating the release of dopamine from interplexiform cells. FMRFamide alone produced no changes on either the membrane potential or light responses of horizontal cells, but it did suppress the effects of GnRH on horizontal cells in some experiments. FRMFamide also reversed the effects of prolonged darkness on horizontal cell responses. When bicuculline was applied to the retina, horizontal cells also depolarized (approximately 10 mV), responses to full-field illumination decreased, and responses to small spots increased. Most of the effects of bicuculline were suppressed by haloperidol, indicating that bicuculline also stimulates the release of dopamine from interplexiform cells. Similar results were obtained when [D-Ala2]-met-enkephalinamide was applied to the retina; horizontal cells depolarized (approximately 10 mV), responses to full-field stimuli decreased, and responses to the light spots increased. On the other hand, [D-Ala2]-leuenkephalinamide and [D-Ala2, D-Leu5]-enkephalin had no effects on horizontal cells. Both haloperidol and naloxone blocked the effects of [D-Ala2]-met-enkephalinamide on horizontal cells, indicating that [D-Ala2]-met-enkephalinamide stimulates dopamine release from interplexiform cells via specific opiate receptors.     

Binding pattern of alpha-bungarotoxin on horizontal cells of a marine teleost retina

A conjugate of alpha-bungarotoxin and a fluorescent marker (fluorescein isothiocyanate) has been used to localize "nicotinic" acetylcholine receptors on neurons in the outer plexiform layer of marine teleost retina. Toxin binding was confined to bipolar cell dendrites and to intermediate horizontal cells. The arrangement of labeled horizontal cells appears irregular in the whole retina, with a peak density in the ventral and dorsal quandrants. Alpha-bungarotoxin receptors on horizontal cells differ from those on bipolar cells and from those on dendrites in the inner plexiform layer in their sensitivity to agonists and antagonists such as d-tubocurarine and nicotine. They constitute a different type of "nicotinic" receptor that probably has a different function.     

Centrifugal innervation of the lamprey retina. Light- and electron microscopic and electrophysiological investigations

Centrifugal fibers and their synaptic connections were studied in retinas of the lamprey Lampetra fluviatilis. The morphological analysis of retinofugal and retinopetal elements was performed after their horseradish peroxidase (HRP) filling through either the cut optic nerve in isolated retina preparations or after intracerebral HRP injections. In flat-mounted retinas, labeled ganglion cell bodies with their dendritic arborizations as well as centrifugal axons were found. The topography of labeled ganglion cell bodies and fibers in semi-thin plastic sections is described. The electron microscopic analysis revealed that the centrifugal terminals synapse either upon unlabeled somata or profiles containing synaptic vesicles (PCSVs). In more rare cases these boutons seem to establish synaptic contacts on ganglion cell dendrites. The target cell bodies were located within the inner part of the inner nuclear layer, whereas postsynaptic dendrites and PCSVs were mainly observed in the outer portion of the internal synaptic layer. Stimulation of the optic nerve in isolated retinas produced antidromic responses in 23 neurons and in 9 of these cells, an antidromic spike was followed by a postsynaptic potential (PSP). Ten cells yielded no antidromic response, but showed PSPs sometimes associated with spikes. The morphological and physiological evidence obtained indicate that these PSP-generating cells were activated synaptically by centrifugal fibers and that in the lamprey retina, these fibers make contacts either with dendrites or somata of amacrine cells and probably with ganglion cell dendrites.     

A new type of wide-field horizontal cell, presumably linked to blue cones, in rabbit retina

Horizontal cells of vertebrate retina play an important role in the formation of visual receptive field surrounds of bipolar cells, and hence in the centre-surround receptive field organization of retinal ganglion cells. In some retinas, horizontal cells also play a major functional role in the first stage of colour-coding of visual stimuli. We have identified a new type of horizontal cell, called 'type C', in rabbit retina, which unlike type A and type B horizontal cells, contacts only a small fraction of cone photoreceptors, possibly blue cones. The multiple, sparsely branched axons of type C cells are well-positioned to contact bipolar dendrites in a feedforward manner. In summary, we propose (1) that the presence in rabbit retina of 3 types of cone horizontal cell, A, B, and C, may represent a more common pattern in mammalian retinae, shared with many non-mammalian retinas which contain colour-coded neurons, (2) that type C cells connect principally to blue cones, and (3) that type C cells are more common in retinas, such as those of squirrels, and to a lesser extent rabbits, in which blue cones play a major role in the colour coding of visual signals.     

Localization of L-glutamic acid decarboxylase mRNA in cat retinal horizontal cells by in situ hybridization

Retinal horizontal cells receive synaptic input from photoreceptors and provide a pathway for lateral interactions in the vertebrate retina. In nonmammalian retinas, the H1 horizontal cells appear to use gamma-amino butyric acid (GABA) as their neurotransmitter. The transmitter used by mammalian horizontal cells, however, remains to be identified. In the present study, we have employed in situ hybridization to examine whether cat retinal horizontal cells contain L-glutamic acid decarboxylase (GAD) mRNA and hence might use GABA as their transmitter. In the cat retina, labeled cell bodies were found in the inner nuclear layer and the ganglion cell layer. No labeled cells were found in the photoreceptor layer. In the inner nuclear layer, labeled somata were present at two locations. The majority of them (approximately 72%) were located in the vitread side of the inner nuclear layer bordering the inner nuclear layer/inner plexiform layer boundary. A second class of labeled cells in the inner nuclear layer (approximately 20%) had larger somata and were present at the inner nuclear layer/outer plexiform layer boundary. Double labeling experiments with antisera to parvalbumin, a horizontal cell marker, showed that these perikarya belonged to horizontal cells. RNA blot analysis showed that cat retina contains a single species of GAD mRNA that is about 4 kb in size. These data show that in addition to GABAergic amacrine, displaced amacrine, and interplexiform cells described previously, horizontal cells contain GAD mRNA and may use GABA as their neurotransmitter. Hence, GABA may be a transmitter that is involved in lateral inhibition in both nonmammalian and mammalian retinas.     

Postnatal development of 3H-GABA-accumulating cells in rabbit retina

Light and electron microscopic autoradiography demonstrates that 3H-GABA is accumulated by horizontal cells in neonatal rabbit retina but not in the adult. A specific population of horizontal cells appears to be mature at birth and they avidly accumulate 3H-GABA during a 15-minute incubation period in vitro. Uptake into horizontal cells is not observed after the fifth postnatal day; 3H-GABA-accumulating horizontal cell bodies and their processes are the first identifiable components that clearly mark the future location of the outer plexiform layer at birth and as such, may be considered pioneering elements. Our observations raise the interesting possibility that the pioneering horizontal cell may provide structural and/or chemical factors necessary for the subsequent development of the outer plexiform layer of the retina. Labeling patterns of other retinal cells also show varying degrees of change during development. A population of amacrine cells accumulate 3H-GABA at birth. These cells show little change in their morphological or 3H-GABA uptake properties from birth to adulthood. Müller cells show weak accumulation of 3H-GABA at birth. Subsequent to this time, labeling of Müller cells is significantly more robust, resulting in Müller cell domination of retinal autoradiographic patterns in more mature retinas. Every cell body in the ganglion cell layer accumulates 3H-GABA at birth. The number of labeled cells declines during postnatal development, resulting in a very limited adult population. We conclude that the ability of retinal cells to accumulate 3H-GABA does not remain constant during postnatal development; rather each cell population displays a unique maturation sequence that results in a dramatic developmental shift in the number and types of GABA-accumulating cells present in the retina.     

Protein content and cAMP-dependent phosphorylation of fractionated white perch retina

In the retinas of teleost fish dopamine, released from interplexiform cells, modulates synaptic transmission at both the chemical and electrical synapses of retinal horizontal cells. This modulation is due to activation of adenylate cyclase and phosphorylation by protein kinase A, perhaps of the synaptic ion channel proteins themselves. In this study we have fractionated the white perch retina by Percoll density gradient centrifugation in order to identify proteins which coenrich with horizontal cells. In addition we have tested retinal fractions for phosphorylation by native cAMP-dependent kinase. Our findings indicate that there are at least 3 proteins of molecular weights 28, 43/44 and 50 kDa which coenrich with horizontal cells and 3 proteins of 30/31 kDa, 35 kDa (putative rhodopsin) and 48 kDa (putative arrestin) which coenrich with photoreceptor fractions. The 43/44 kDa phosphoprotein is a target for cAMP-dependent protein phosphorylation and thus is apparently an element of the dopaminergic modulatory pathway in perch horizontal cells.