Ellen Townes-Anderson

Synapsins in the vertebrate retina: Absence from ribbon synapses and heterogeneous distribution among conventional synapses

The vertebrate retina contains two ultrastructurally distinct types of vesicle-containing synapses: conventional synapses, made predominantly by amacrine cells, and ribbon synapses, formed by photoreceptor and bipolar cells. To identify molecular differences between these synapse types, we have compared the distribution of the synapsins, a family of nerve terminal phosphoproteins, with that of synaptophysin (p38) and SV2, two intrinsic membrane proteins of synaptic vesicles. We report an absence of synapsin I and II immunoreactivity from all ribbon-containing nerve terminals. These include terminals of rod cells in developing and adult rat retina, rod and cone cells in monkey and salamander retinas, and rat bipolar cells. Furthermore, we show that synapsins I and II are differentially distributed among conventional synapses of amacrine cells. The absence of the synapsins from ribbon synapses suggests that vesicle clustering and mobilization in these terminals differ from that in conventional synapses.

A Highly Ca2+-Sensitive Pool of Vesicles Contributes to Linearity at the Rod Photoreceptor Ribbon Synapse

Studies of the properties of synaptic transmission have been carried out at only a few synapses. We analyzed exocytosis from rod photoreceptors with a combination of physiological and ultrastructural techniques. As at other ribbon synapses, we found that rods exhibited rapid kinetics of release, and the number of vesicles in the releasable pool is comparable to the number of vesicles tethered at ribbon-style active zones. However, unlike other previously studied neurons, we identified a highly Ca(2+)-sensitive pool of releasable vesicles with a relatively shallow relationship between the rate of exocytosis and [Ca(2+)](i) that is nearly linear over a presumed physiological range of intraterminal [Ca(2+)]. The low-order [Ca(2+)] dependence of release promotes a linear relationship between Ca(2+) entry and exocytosis that permits rods to relay information about small changes in illumination with high fidelity at the first synapse in vision.

L‐type calcium channels in the photoreceptor ribbon synapse: Localization and role in plasticity

Calcium (Ca2+) influx through voltage‐gated Ca2+channels stimulates a variety of neural activities, including process outgrowth, neurotransmission, and synaptic plasticity. In general, L‐type channels control Ca2+ influx into the soma and dendrites, whereas other Ca2+ channel types control presynaptic activities. Neurons that make ribbon synapses, however, are among a select group of nerve cells whose presynaptic Ca2+‐dependent secretion is linked to L‐type channels. Recently, photoreceptor ribbon synapses have been shown to be capable of dramatic structural remodeling and neuritic outgrowth. Here, we have examined 1) the distribution of dihydropyridine (DHP)‐sensitive (L‐type) Ca2+ channels in photoreceptor presynaptic structures and 2) the role of these channels in axonal plasticity and process outgrowth in culture. Using anti‐alpha1C and the fluorescent dihydropyridine, (–)‐DM‐BODIPY DHP, L‐type channels were localized in the outer plexiform layer of retinal sections and in presynaptic terminals of freshly isolated photoreceptors. In the rod terminal, dense patches of label were present; their distribution and number matched that of synaptic ribbons. After 1–7 days in vitro, punctate alpha1C staining occurred along newly formed neurites and presynaptic varicosities. Functional channels were present throughout the culture period, as determined by fura‐2 imaging. Channel blockage by nicardipine, a DHP antagonist, inhibited axonal remodeling. Specifically, it prevented axon retraction and lamellipodium formation, reduced neurite growth, and produced long, thin processes on some, primarily cone, photoreceptors. L‐type Ca2+ channel activity, therefore, not only stimulates neurotransmission but contributes to presynaptic structural plasticity at the ribbon synapse. J. Comp. Neurol. 415:1–16, 1999.

Differential expression of synapsins I and II among rat retinal synapses

The synapsins are a family of synaptic vesicle-associated phosphoproteins thought to regulate the availability of vesicles for neurotransmitter release. In order to assess variability of synapsin isoform expression, we compared the localization of synapsins Ia, Ib, IIa, and IIb in the inner plexiform layer of the rat retina. Double labeling in conjunction with confocal fluorescence and electron microscopy allowed imaging of synapsin I and II immunoreactivity within single presynaptic terminals. No qualitative differences were observed between expression of the a and b isoforms of synapsin I in individual terminals; likewise, the a and b isoforms of synapsin II were identically distributed. In contrast, marked differences were seen upon comparison of synapsin I and synapsin II expression in single terminals. Our results indicate the existence of three classes of presumed amacrine cell synaptic terminals: synapsin I+/synapsin II-, synapsin I-/synapsin II+, and synapsin I+/synapsin II+. Each class of synapse has a different distribution among five IPL sublayers, suggesting that they represent different subpopulations of amacrine cells. Double labeling with an antibody to choline acetyltransferase indicates that synapsin I-/II+ terminals may be those of cholinergic amacrine cells. Furthermore, all synapsin II+ terminals appear to be distinct from those expressing the GABA synthetic enzyme glutamic acid decarboxylase. The observed variations in synapsin content suggest the existence of presynaptic terminal heterogeneity that is not apparent from conventional morphological studies.

Growth and synapse formation among major classes of adult salamander retinal neurons in vitro

Adult neurons, isolated from the salamander retina, were maintained in low-density cell culture and examined for synapse formation by electrophysiological and electron microscopic techniques. Morphologically identifiable rod, cone, horizontal, bipolar, and amacrine/ganglion cells survived for many months, grew processes, and formed numerous cell contacts. Intracellular recordings showed the presence of a variety of voltage- and time-dependent conductances and both electrical and chemical transmission among these cells. At the ultrastructural level, gap junctions, monad ribbon synapses, and conventional synapses, like those present in the intact retina, were observed in sibling cultures. Thus, all major classes of adult retinal neurons, in addition to ganglion cells, are able to regenerate processes and reform synapses. The regenerated synaptic contacts are functional and structurally diverse.

The Nitric Oxide-cGMP Signaling Pathway Differentially Regulates Presynaptic Structural Plasticity in Cone and Rod Cells

Although abundant structural plasticity in the form of axonal retraction, neurite extension, and formation of presynaptic varicosities is displayed by photoreceptors after retinal detachment and during genetic and age-related retinal degeneration, the mechanisms involved are mostly unknown. We demonstrated recently that Ca2+ influx through cGMP-gated channels in cones and voltage-gated L-type channels in rods is required for neurite extension in vitro (Zhang and Townes-Anderson, 2002). Here, we report that the nitric oxide (NO)-cGMP signaling pathway is active in photoreceptors and that its manipulation differentially regulates the structural plasticity of cone and rod cells. The NO receptor soluble guanylyl cyclase (sGC) was detected immunocytochemically in both cone and rod cells. Stimulation of sGC increased cGMP production in retinal cultures. In cone cells, quantitative analysis showed that NO or cGMP stimulated neuritic sprouting; this stimulatory effect was dependent on both Ca2+ influx through cGMP-gated channels and phosphorylation by protein kinase G (PKG). At the highest levels of cGMP, however, cone outgrowth was no longer increased. In rod photoreceptors, NO or cGMP consistently inhibited neuritic growth in a dose-dependent manner; this inhibitory effect required PKG. When NO-cGMP signaling was inhibited, changes in the neuritic development of cone and rod cells were also observed but in the opposite direction. These results expand the role of cGMP in axonal activity to adult neuritogenesis and suggest an explanation for the neurite sprouting observed in an autosomal recessive form of retinitis pigmentosa that is characterized by high cGMP levels in photoreceptor layers.

Injury-induced remodelling and regeneration of the ribbon presynaptic terminal in vitro.

SummaryThe neuronal response to axonal injury may relate to the type of insult incurred. Recently, neuritic and presynaptic varicosity regeneration by isolated adult salamander photoreceptors was demonstrated. We have used this system to compare the rod photoreceptor response to two types of injury:denervation/detargeting, the removal of pre-and postsynaptic partners from the axon terminal, andaxotomy, the removal of the axon terminal itself. Cells were followed with time-lapse video microscopy for 24–48h in culture and immunolabelled for SV2 or synaptophysin to identify synaptic vesicle-containing varicosities. Although all injured cells responded with regenerative growth, denervated/detargeted photoreceptors (i.e. neurons which retain their axon terminal) grew 80% more processes and fourfold more presynaptic varicosities than axotomized neurons. In cells which retained their original axon and terminal, varicosity formation generally began with axon retraction. Retraction was followed by elaboration of a lamellipodium and, by 48 h, development of varicosity-bearing neurites from the lamellipodium. Synaptic vesicle protein localization in denervated/detargeted cells paralleled axon terminal reorganization. Axotomized cells, in contrast, lacked synaptic vesicle protein immunoreactivity during this period. To detect synaptic protein synthesis, photoreceptors were examined for colocalization of synaptic vesicle protein with rab6, a Golgi marker, by confocal microscopy. As expected, synaptic vesicle protein staining was present in the Golgi complex during regeneration; however, in cells with an axon, new synaptic vesicle protein-labelled varicosities were found at early stages, prior to the appearance of immunolabel in the Golgi complex. The data demonstrate remarkable plasticity in the ribbon synapse, and suggest that in adult rod cells with an intact axon terminal, synaptic vesicle protein synthesis is not a prerequisite for the formation of new presynaptic-like terminals. We propose that preexisting axonal components are reutilized to expedite presynaptic renewal as an early response to denervation/detargeting.

RhoA and its role in synaptic structural plasticity of isolated salamander photoreceptors.

PURPOSE Adult salamander photoreceptors retract existing axons and extend new neuritic processes in vitro. In mammalian retina, similar forms of structural plasticity occur in injury and disease. The authors asked whether RhoA is present in photoreceptor axon terminals and whether activity in the RhoA-ROCK pathway contributes to the structural plasticity observed in rod and cone cells. METHODS Antibodies against RhoA were used to immunolabel Western blots sections and isolated neurons from salamander retina. Isolated photoreceptors were treated with lysophosphatidic acid (LPA; a RhoA activator) or Y27632 (an inhibitor of RhoA effector ROCK) for the first 24 hours, the first 3 days, or the last 24 hours of culture. Growth and retraction were assessed with time-lapse and image analyses. RESULTS RhoA protein was found throughout the retina, including in rod and cone synaptic terminals. When treated with LPA, photoreceptors significantly reduced the growth of new neuritic processes and presynaptic varicosities and retracted growth at the highest LPA concentrations. When treated with Y27632, rod cells significantly increased the number of varicosities, whereas cone cells increased process growth. Treatment with Y27632 also dramatically reduced retraction of the existing axon, which occurs spontaneously in rod cells during the first 24 hours of culture. CONCLUSIONS Thus, RhoA-ROCK activity reduces and retracts neuritic growth, but inhibition of activity increases neuritic development and blocks retraction. The results suggest that RhoA activation contributes to axon retraction by rod cells after retinal detachment, whereas inhibition of RhoA contributes to the neuritic sprouting seen in reattached and degenerating retina.

Adenosine A2a receptor‐mediated inhibition of rod opsin mRNA expression in tiger salamander

The neuromodulator adenosine mediates dark‐adaptive changes in retinal photoreceptors through A2a receptors. In cold‐blooded vertebrates, opsin mRNA expression is lower at night than during the day. In the present study, we tested whether adenosine could inhibit opsin mRNA expression in cultured rod cells and if endogenous adenosine acts to suppress opsin mRNA in the intact retina at night. Semi‐quantitative in situ hybridization showed that treatment with 100 nm of the A2a/A2b agonist N 6‐[2‐(3,5‐dimethoxyphenyl)‐2‐(2‐methylphenyl)‐ethyl]adenosine (DPMA) reduced opsin mRNA 41% in cultured rod cells. The effect of DPMA was blocked by 10 µm of the A2a antagonist 8‐(3‐chlorostyryl)caffeine (CSC) but not by 10 µm of the A2b antagonist alloxazine. One micromolar adenosine alone had no effect on opsin mRNA. However, in the presence of the adenosine deaminase inhibitor erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine hydrochloride (EHNA), 1 µm adenosine reduced opsin mRNA 61%. EHNA alone reduced opsin mRNA by 26%. Consistent with an A2a receptor mechanism, 100 nm forskolin (adenylate cyclase agonist) decreased opsin mRNA 34%. Finally, northern blots showed that intravitreal injection of 10 µm CSC at night increased opsin I mRNA 38%. Thus, endogenous adenosine suppresses rod opsin I mRNA expression at night; in vitro results indicate this reduction occurs through A2a‐like receptor binding and stimulation of adenylate cyclase activity.

Dopamine D4 receptor-mediated regulation of rod opsin mRNA expression in tiger salamander

Light stimulates dopamine release in the retina and has been shown to rapidly up‐regulate rod opsin mRNA. In the present study, we tested the effect of dopamine on rod opsin mRNA expression and examined the hypothesis that dopamine can mediate a light‐evoked increase in opsin gene expression. Northern blots showed that a 30‐min light‐exposure increased rod opsin mRNA expression 27%. In situ hybridization on isolated rods showed that 500 nm dopamine and 1 µm quinpirole (dopamine D2/D3/D4 agonist) increased opsin mRNA 45% and 26%, respectively. The effect of quinpirole was selectively blocked by the D4 antagonist, L750,667 (20 µm). In very low density cultures, quinpirole increased opsin expression 46%, suggesting a direct effect on rod photoreceptors. Consistent with a dopamine D4 receptor mechanism, 1 µm H‐89 (protein kinase A inhibitor) increased opsin mRNA 39%. Finally, intravitreal injection of quinpirole increased opsin mRNA 21% whereas injection of L750,667 (10 µm) blocked the light‐evoked increase in opsin expression. These data show that rod opsin mRNA is up‐regulated by dopamine binding a D4‐like receptor on rods, possibly through inhibition of protein kinase A, and that endogenous dopamine can mediate the light‐evoked increase in opsin mRNA expression.