Ribbon synapses of the mammalian retina contain two types of synaptic bodies-ribbons and spheres

Summary The present paper reports that the synaptic bodies of the retinal ribbon synapses in rat, guinea pig, golden hamster and mouse are a heterogeneous population of organelles. In addition to the well-known synaptic ribbonssensu stricto which consist of a platelike electron-dense central structure surrounded by electron-lucent synaptic vesicles, there are what is termed synaptic spheres, in which the core is not platelike, but round to oval. In rat retinae procured at day, ribbons outnumbered spheres by a factor of 4. At night spheres were not seen in photoreceptor cells. Spheres, like ribbons, may lie some distance from the synaptic site, perhaps indicating transit from their site of origin to the synapse. At night ribbons are longer than at daytime. In addition to the previously described connecting stalks between synaptic vesicles and the electron-dense ribbons, the presence of filamentous stalks between adjacent synaptic vesicles is described. The latter stalks, depending on their presence or absence, may influence the position of the synaptic vesicles in relation to the synaptic body and/or the presynaptic membrane. It is concluded that the plasticity of retinal synapses cannot be fully appreciated unless the temporal changes of ribbons, spheres and the connecting stalks are taken into consideration.     

Distribution of synaptic ribbons in the developing organ of Corti

Summary Studies of synaptogenesis in the developing organ of Corti in the intact mouse and in culture indicate that the inner and outer hair cells contain three populations of synaptic ribbons, i.e. ribbons adjacent to nerve fibres, free intracellular ribbons and misplaced ribbons apposed to non-neuronal elements. Ribbons adjacent to nerve fibres can be further classified into: ribbons synaptically engaged, ribbons participating in formation of presynaptic complexes only and ribbons that are not engaged to the hair cell membrane. In the developing innervated cultures the ribbon distributions are similar to those in the normal animal. Inner and outer hair cells differ in distribution of the ribbons. In the inner hair cells the ribbons adjacent to the nerve fibres are dominant (over 90%) and most of them (88%) are synaptically engaged. In the outer hair cells the presynaptic ribbons dominate the population (up to 60%) during the first postnatal week when the cells acquire afferent synaptic connections. This stage is followed by a marked reduction in the number of all ribbons. In the intact animal the rapid decrease results in a relative increase of misplaced and free ribbons. These changes are presumably due to the loss of some of the afferents. In the denervated hair cells the distribution of ribbons indicated the presence of conspicuous scatter. In the areas of incomplete denervation, however, the ribbons are apposed to the preserved fibres. Despite denervation, most of the ribbons develop the entire presynaptic complex in apposition to non-neuronal structures.The different populations of synaptic ribbons appear to reflect different stages in synapse formation. Possibly, the synaptic body originates in the interior of the hair cell and subsequently migrates to the cell membrane. In any case, a nerve fibre appears critical in influencing the location of the synaptic ribbon. At the apposition of the ribbon to the hair cell membrane, presynaptic densities are formed and the ribbon appears to become anchored. Typically, the nerve fibre membrane apposed to the presynaptic complex responds with the formation of postsynaptic densities.     

Vesicle depletion and synaptic depression at a mammalian ribbon synapse

We estimated the size of the readily releasable pool (RRP) of vesicles at a ribbon synapse in the rat retina by making paired voltage-clamp recordings from presynaptic rod bipolar cells (RBCs) and postsynaptic AII amacrine cells in an in vitro retinal slice preparation. The RRP at each active zone was estimated to constitute seven vesicles, in the range of estimated RRP sizes at conventional synapses. During sustained presynaptic Ca(2+) entry, the RRP could be released with a time constant of about 4 ms. This ribbon synapse exhibited pronounced paired-pulse depression (PPD), which was attributable primarily to vesicle depletion. Recovery from PPD was slow (tau approximately 4 s) but could be accelerated by increasing the duration of the depressing stimulus. The small RRP and very high release probability likely contribute to the transient characteristics of neurotransmission at RBC synapses.     

Diurnal changes in exocytosis and the number of synaptic ribbons at active zones of an ON-type bipolar cell terminal

The number and morphology of synaptic ribbons at photoreceptor and bipolar cell terminals has been reported to change on a circadian cycle. Here we sought to determine whether this phenomenon exists at goldfish Mb-type bipolar cell terminals with the aim of exploring the role of ribbons in transmitter release. We examined the physiology and ultrastructure of this terminal around two time points: midday and midnight. Nystatin perforated-patch recordings of membrane capacitance (C(m)) revealed that synaptic vesicle exocytosis evoked by short depolarizations was reduced at night, even though Ca(2+) currents were larger. The efficiency of exocytosis (measured as the DeltaC(m) jump per total Ca(2+) charge influx) was thus significantly lower at night. The paired-pulse ratio remained unchanged, however, suggesting that release probability was not altered. Hence the decreased exocytosis likely reflects a smaller readily releasable vesicle pool at night. Electron microscopy of single sections from intact retinas averaged 65% fewer ribbons at night. Interestingly, the number of active zones did not change from day to night, only the probability of finding a ribbon at an active zone. Additionally, synaptic vesicle halos surrounding the ribbons were more completely filled at night when these on-type bipolar cells are more hyperpolarized. There was no change, however, in the physical dimensions of synaptic ribbons from day to night. These results suggest that the size of the readily releasable vesicle pool and the efficiency of exocytosis are reduced at night when fewer ribbons are present at bipolar cell terminal active zones.     

Pineal "synaptic" ribbons and spherules during the estrous cycle in rats

In previous studies pineal "synaptic" ribbons have been shown to undergo striking numerical changes under various physiological and experimental conditions and to be regulated by beta-adrenergic mechanisms. The aim of the present investigation was to study the numbers of pineal "synaptic" ribbons and spherules in Wistar rats throughout the estrous cycle and to compare them with those in males. There were no statistically significant differences in the numbers of ribbons and spherules between males and females and in the females at the different stages of the estrous cycle, indicating that the structures in question, in vivo, do not appear to be regulated by naturally occurring changes of sex steroid hormones and gonadotrophins.     

Signalling within the neurovascular unit in the mammalian retina

Neuronal activity in the central nervous system evokes localized changes in blood flow, a response termed neurovascular coupling or functional hyperaemia. Modern functional imaging methods, such as functional magnetic resonance imaging (fMRI), measure signals related to functional hyperaemia in order to determine localization of brain function and to diagnose disease. The cellular mechanisms that underlie functional hyperaemia, however, are not well understood. Glial cells have been hypothesized to be intermediaries between neurons and blood vessels in the control of neurovascular coupling, owing to their ability to release vasoactive factors in response to neuronal activity. Using an in vitro preparation of the isolated, intact rodent retina, we have investigated two likely mechanisms of glial control of the vasculature: glial K(+) siphoning and glial induction of vasoactive arachidonic acid metabolites. Potassium siphoning is a process by which a K(+) current flowing through glial cells transfers K(+) released from active neurons to blood vessels. Since slight increases in extracellular K(+) can cause vasodilatation, this mechanism was hypothesized to contribute to neurovascular coupling. Our data, however, suggest that glial K(+) siphoning does not contribute significantly to neurovascular coupling in the retina. Instead, we suggest that glial cells mediate neurovascular coupling by inducing the production of two types of arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE), which dilate and constrict vessels, respectively. We show that both light flashes and direct glial stimulation produce vasodilatation or vasoconstriction mediated by EETs and 20-HETE, respectively. Further, we show that the type of vasomotor response observed (dilatation or constriction) depends on retinal levels of nitric oxide. Our data also demonstrate that glial cells are necessary intermediaries for signalling from neurons to blood vessels, since functional hyperaemia does not occur when neuron-to-glia communication is interrupted. These results indicate that glial cells play an important role in mediating functional hyperaemia and suggest that the regulation of blood flow may involve both vasodilating and vasoconstricting components.     

Short-term synaptic depression and recovery at the mature mammalian endbulb of Held synapse in mice

The endbulb of Held synapses between the auditory nerve fibers (ANF) and cochlear nucleus bushy neurons convey fine temporal information embedded in the incoming acoustic signal. The dynamics of synaptic depression and recovery is a key in regulating synaptic transmission at the endbulb synapse. We studied short-term synaptic depression and recovery in mature (P22-38) CBA mice with stimulation rates that were comparable to sound-driven activities recorded in vivo. Synaptic depression in mature mice is less severe ( approximately 40% at 100 Hz) than reported for immature animals and the depression is predominately due to depletion of releasable vesicles. Recovery from depression depends on the rate of activity and accumulation of intracellular Ca(2+) at the presynaptic terminal. With a regular stimulus train at 100 Hz in 2 mM external [Ca(2+)], the recovery from depletion was slow (tau(slow), approximately 2 s). In contrast, a fast (tau(fast), approximately 25 ms), Ca(2+)-dependent recovery followed by a slower recovery (tau(slow), approximately 2 s) was seen when stimulus rates or external [Ca(2+)] increased. In normal [Ca(2+)], recovery from a 100-Hz Poisson-like train is rapid, suggesting that Poisson-like trains produce a higher internal [Ca(2+)] than regular trains. Moreover, the fast recovery was slowed by approximately twofold in the presence of calmidazolium, a Ca(2+)/calmodulin inhibitor. Our results suggest that endbulb synapses from high spontaneous firing rate auditory nerve fibers normally operate in a depressed state. The accelerated synaptic recovery during high rates of activity is likely to ensure that reliable synaptic transmission can be achieved at the endbulb synapse.     

Growth factor-responsive progenitors in the postnatal mammalian retina.

It is thought that the adult mammalian retina lacks the regenerative capacity of fish and amphibians retina because it does not harbor a progenitor population. However, recent observations suggest that another derivative of the optic neuroepithelium, the ciliary body, contains a mitotically quiescent population of neural progenitors that proliferate in the presence of growth factors and demonstrate properties of neural stem cells. Examination of the hypothesis that similar mitotically quiescent and growth factor-responsive progenitors may exist in the postnatal retina revealed a population of cells located in the periphery of the retina that displayed proliferative responsiveness to growth factors and possessed potential to support neurogenesis. Given their marginal position and neural properties and potential, these cells may represent a residual population of retinal progenitors, analogous to those found in the ciliary marginal zone of fish and amphibians. Their progressive decrease in proliferative potential and number in postnatal stages suggests a temporal decline in regulatory signaling that supports their maintenance during retinal neurogenesis.     

Neurotrophins and synaptic plasticity in the mammalian spinal cord

The pathway mediating the monosynaptic stretch reflex has served as an important model system for studies of plasticity in the spinal cord. Its usefulness is extended by evidence that neurotrophins, particularly neurotrophin-3 (NT-3), which has been shown to promote spinal axon elongation, can modulate the efficacy of the muscle spindle-motoneurone connection both after peripheral nerve injury and during development. The findings summarized here emphasize the potential for neurotrophins to modify function of both damaged and undamaged neurones. It is important to recognize that these effects may be functionally detrimental as well as beneficial.     

Diurnal variations in the photoreceptor synaptic terminals of the newt retina

Newt photoreceptor synaptic terminals undergo a variety of morphological changes over a 24-hr (LD 12:12) cycle. During the day, densecored synaptic vesicles were found to increase in number and accumulate near the synaptic lamellae; during the dark phase, the dense-cored vesicles decreased in number, while large clear vesicles and profiles of smooth endoplasmic reticulum increased in frequency. The most marked change in photoreceptor synaptic terminal morphology occurred after 10 hr of darkness, at 0730 hr. At this time, photoreceptor synaptic terminal cross-sectional area was found to increase dramatically. Morphometric analysis showed that the number of synaptic vesicles in these terminals remained constant throughout the day, as did the perimeter of photoreceptor terminal profiles. The observed increase in area of synaptic terminals at 0730 hr was found to be due to a decrease in the folding of the terminal plasma membrane. Qualitative observations showed endocytosis to be occurring at a rapid rate at this time as well; and since the number of synaptic vesicles and terminal perimeter did not change, exocytosis of synaptic vesicles was assumed to be occurring at an equally rapid rate. These findings support an extension to the hypothesis of Monaghan and Osborne (1975), suggesting that photoreceptor synaptic vesicles become “supercharged” with transmitter substance in the light.     

Modulation by melatonin of glutamatergic synaptic transmission in the carp retina

Melatonin is involved in a variety of physiological functions through activating specific receptors coupled to GTP-binding protein. Melatonin and its receptors are abundant in the retina. Here we show for the first time that melatonin modulates glutamatergic synaptic transmission from cones to horizontal cells (HCs) in carp retina. Immunocytochemical data revealed the expression of the MT₁ receptor on carp HCs. Whole-cell recordings further showed that melatonin of physiological concentrations potentiated glutamate-induced currents from isolated cone-driven HCs (H1 cells) in a dose-dependent manner, by increasing the efficacy and apparent affinity of the glutamate receptor. The effects of melatonin were reversed by luzindole, but not by K 185, indicating the involvement of the MT₁ receptor. Like melatonin, methylene blue (MB), a guanylate cyclase inhibitor, also potentiated the glutamate currents, but internal infusion of cGMP suppressed them. The effects of melatonin were not observed in cGMP-filled and MB-incubated HCs. These results suggest that the melatonin effects may be mediated by decreasing the intracellular concentration of cGMP. Consistent with these observations, melatonin depolarized the membrane potential of H1 cells and reduced their light responses, which could also be blocked by luzindole. These effects of melatonin persisted in the presence of the antagonists of receptors for dopamine, GABA and glycine, indicating a direct action of melatonin on H1 cells. Such modulation by melatonin of glutamatergic transmission from cones to HCs is thought to be in part responsible for circadian changes in light responsiveness of cone HCs in teleost retina.     

Suppression by zinc of AMPA receptor-mediated synaptic transmission in the retina

Zinc is strikingly co-localized with glutamate-containing vesicles in the synaptic terminals of retinal photoreceptors, and it is thought to be co-released with glutamate onto postsynaptic neurons such as horizontal cells and bipolar cells. Here we examined exogenous zinc modulation of glutamate receptors on cultured retinal horizontal cells using patch-clamp recording and endogenous zinc effect on intact horizontal cells using intracellular recording techniques. Application of 3, 30, and 300 microM zinc reduced the whole cell peak current of response to 200 microM glutamate by 2, 30, and 56%, respectively. Zinc suppression of glutamate response persisted in the presence of 10 microM cyclothiazide (CTZ). Glutamate responses of outside-out patches were completely abolished by 30 microM 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466), and the receptor desensitization was blocked by 30 microM CTZ, indicating that receptor target for the zinc action on horizontal cells is alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid (AMPA) receptors. Zinc decreased the amplitude of outside-out patch peak current without an effect on either its 10-90% rise time or the rate of receptor desensitization. Dose-response curves for glutamate show that zinc reduced the maximal current evoked by glutamate and increased EC(50) from 50 +/- 3 to 70 +/- 6 microM without changing the Hill coefficient. Chelation of endogenous zinc with 1 mM Ca-EDTA depolarized horizontal cells in the intact retina by 3 mV, consistent with relief of the partial glutamate receptor inhibition by zinc. Overall, the results describe a unimodal form of zinc modulation of AMPA-type glutamate receptor responses not previously described in native neuronal preparations and a novel role for endogenous zinc in modulating neurotransmission.     

The effects of nerve stimulation and hemicholinium on synaptic vesicles at the mammalian euromuscular junction

1. Electron micrographs of nerve terminals in rat phrenic nerve-diaphragm preparations have been studied. This has been done before and after prolonged nerve stimulation. The effectiveness of nerve stimulation has been monitored by intracellular micro-electrode recordings from the muscle cells.2. Characteristic changes in the form and distribution of the nerve terminal mitochondria were noted after nerve stimulation.3. Synaptic vesicle numbers in the region of nerve terminal less than 1800 A from the synaptic cleft were significantly greater in tissue taken 2 and 3 min after nerve stimulation, than in unstimulated preparations.4. The long and short diameters of the synaptic vesicle profiles less than 1800 A from the synaptic cleft were measured. Analysis of the distribution of the diameters indicated synaptic vesicles to be basically spherical structures. Estimates of synaptic vesicle volume were made from the measurements. Synaptic vesicle volume was significantly reduced in tissue taken 2 and 4 min following nerve stimulation.5. If hemicholinium, a compound which inhibits acetylcholine synthesis, was present during the period of nerve stimulation, much greater reductions in synaptic vesicle volume occurred. Synaptic vesicle numbers in the region of nerve terminal less than 1800 A from the synaptic cleft were also reduced, compared with unstimulated control preparations.6. These results are regarded as support for the hypothesis that the synaptic vesicles in nerve terminals at the mammalian neuromuscular junction represent stores of the transmitter substance, acetylcholine.