Synaptic vesicle cycling is not impaired in a glutamatergic and a cholinergic synapse that exhibit deficits in acidification and filling

The purpose of the present work was to investigate synaptic vesicle trafficking when vesicles exhibit alterations in filling and acidification in two different synapses: a cholinergic frog neuromuscular junction and a glutamatergic ribbon-type nerve terminal in the retina. These synapses display remarkable structural and functional differences, and the mechanisms regulating synaptic vesicle cycling might also differ between them. The lipophilic styryl dye FM1-43 was used to monitor vesicle trafficking. Both preparations were exposed to pharmacological agents that collapse ΔpH (NH4Cl and methylamine) or the whole ΔµH+ (bafilomycin), a necessary situation to provide the driving force for neurotransmitter accumulation into synaptic vesicles. The results showed that FM1-43 loading and unloading in neuromuscular junctions did not differ statistically between control and experimental conditions (P > 0.05). Also, FM1-43 labeling in bipolar cell terminals proved highly similar under all conditions tested. Despite remarkable differences in both experimental models, the present findings show that acidification and filling are not required for normal vesicle trafficking in either synapse.

O objetivo do presente trabalho foi investigar o tráfego de vesículas sinápticas quando estas apresentam alterações no armazenamento de neurotransmissores e acidificação em duas distintas sinapses: a junção neuromuscular colinérgica de rãs versus o terminal nervoso glutamatérgico do tipo ribbon em céulas bipolares da retina. Essas sinapses exibem notáveis diferenças estruturais e funcionais e os mecanismos de regulação de ciclo das vesículas sinápticas podem ser diferentes entre eles. Para monitorar o tráfego de vesícula, foi utilizado o marcador lipofílico FM1-43. Ambas as preparações foram expostas a agentes farmacológicos que provocam o colapso de ΔpH (NH4Cl e metilamina) ou de todo ΔµH+ (bafilomicina), gradientes necessários para o acúmulo de neurotransmissores em vesículas sinápticas. Nossos resultados demonstram que a marcação e desmarcação de FM1-43 nas junções neuromusculares não foi estatisticamente diferente entre as diversas condições experimentais (P > 0,05). Além disso, a marcação de FM1-43 em terminais sinápticos de células bipolares foram bastante semelhantes em todas as condições testadas. Apesar das diferenças marcantes em ambos os modelos experimentais, nossos achados demonstram que a acidificação e o preenchimento de vesículas sinápticas não são necessários para o tráfico normal da vesícula nas sinapses estudadas.

Ribbon Synapses Formed at the Axon of the Calbindin-Immunoreactive ON Cone Bipolar Cell in OFF Sublamina of the Inner Plexiform Layer in the Rabbit Retina / 체질인류학회지

Some retinal neurons, including intrinsically photosensitive retinal ganglion cells have their dendrites stratified in sublamina a of the inner plexiform (IPL), the OFF sublayer, but paradoxically show light-driven ON electrophysiological responses. In order to understand the mechanism on this paradoxical response, by using immunoelectron microscopy with a specific antibody against calbindin, we examined the synaptic connections of the calbindin-immunoreactive ON cone bipolar cell of the rabbit retina, which is thought to make the ribbon synapse in sublamina a of the IPL. The ribbon synapses in sublamina a by calbindin-immunoreactive ON cone bipolar cells were mainly found at the border between the inner nuclear layer and the IPL. Interestingly, the output targets at these ribbon synapses turned out as monads, and multiple synaptic ribbons were engaged in each synapse. These findings were different from those at the conventional ribbon synapse formed by calbindin-immunoreactive ON cone bipolar axon terminals. Thus, these findings may be the characteristics of the calbindin-immunoreactive ON cone bipolar ribbon synapse in sublamina a and can be used to classify the synapse in the retinal circuit research.

Comparison of Retinal Waveform between Normal and rd/rd Mouse / 의학물리

Retinal prosthesis is regarded as the most feasible method for the blind caused by retinal diseases such as retinitis pigmentosa or age-related macular degeneration. One of the prerequisites for the success of retinal prosthesis is the optimization of the electrical stimuli applied through the prosthesis. Since electrical characteristics of degenerate retina are expected to differ from those of normal retina, we investigated differences of the retinal waveforms in normal and degenerate retina to provide a guideline for the optimization of electrical stimulation for the upcoming prosthesis. After isolation of retina, retinal patch was attached with the ganglion cell side facing the surface of microelectrode arrays (MEA). 8x8 grid layout MEA (electrode diameter: 30micrometer, electrode spacing: 200micrometer, and impedance: 50 k omega at 1 kHz) was used to record in-vitro retinal ganglion cell activity. In normal mice (C57BL/6J strain) of postnatal day 28, only short duration (<2 ms) retinal spikes were recorded. In rd/rd mice (C3H/HeJ strain), besides normal spikes, waveform with longer duration (~100 ms), the slow wave component was recorded. We attempted to understand the mechanism of this slow wave component in degenerate retina using various synaptic blockers. We suggest that stronger glutamatergic input from bipolar cell to the ganglion cell in rd/rd mouse than normal mouse contributes the most to this slow wave component. Out of many degenerative changes, we favor elimination of the inhibitory horizontal input to bipolar cells as a main contributor for a relatively stronger input from bipolar cell to ganglion cell in rd/rd mouse.

Pre-synaptic Neuronal Changes of AII Amacrine Cells in the Streptozotocin-induced Diabetic Rat Retina / 대한해부학회지

It has been previously reported that parvalbumin expression was downregulated in AII amacrine cells, while upregulated in a subset of cone bipolar cells electrically synapse with AII amacrine cell in the streptozotocin-induced diabetic rat retina. In the present study, we aimed to trace biochemical changes of pre-synaptic neurons to AII amacrine cells in rat retina following diabetic injury. Diabetic condition was induced by streptozotocin injection into Sprague-Dawley rats aged of 8 weeks. The experimental term of induced diabetes was set at 1, 4, 12 and 24 weeks. Changes of pre-synaptic neurons were evaluated by immunohistochemistry and Western blot analysis with anti-protein kinase C (PKC)-alpha and anti-tyrosine hydroxylase (TH) antibodies. Rod bipolar cells immunolocalized with PKC-alpha antibody extended their enlarged axon terminals into stratum 5 of the inner plexiform layer. In later diabetes, the axon was shorten and its terminals of rod bipolar cell are slightly enlarged. The protein levels of PKC-alpha were slightly increased along with the duration of diabetes. TH immunoreactive neurons are morphologically classified into two subtypes of amacrine cells in the inner nuclear layer: one (type 1) has large soma with long and primary dendrites, classified with dopaminergic, and the other (type 2) has small soma with dendritic arborization. In the outermost inner plexiform layer, ring-like structures being composed of type 1 cell processes were densely distributed. In diabetic retina, the intensity of TH immunoreactivity in type 1 neurons was reduced. In accordance with morphological changes, the protein levels of TH were reduced during diabetes. These results demonstrate that TH immunoreactive dopaminergic amacrine cells are more susceptible to diabetic injury than the rod bipolar cells in the rat retina and may suggest that downregulation of parvalbumin expression in AII amacrine cells of diabetic retina is mainly due to dysfunction of pre-synaptic dopaminergic amacrine cells.

Altered Expression of Parvalbumin in Neural Retina of the Insulin-dependent Diabetic Rat / 대한해부학회지

Calcium-binding proteins in the nervous system are functioned in Ca2+ buffering and Ca2+ transport and regulation of various enzyme systems. They potentially have a number of different effects on cells includingaltering the duration of action potentials, promoting neuronal bursting activity and protecting cells against the damaging effects of excessive calcium influx. The present study has been designed to clarify the differential responding patterns of parvalbumin immunoreactive neurons in the rat retina following diabetic injury, for better understandings of role of parvalbumin in the retinal circuitry and in calcium homeostasis. Experimental diabetes was induced by a single intravenous injection of streptozotocin in a dose of 60 mg/kg body weight. Diabetic rats showing high blood glucose levels (above 300 mg/dL) were cared for 1, 4, 8, 12 and 24 weeks, respectively. The retinas at each time point were processed for immunohistochemistry and Western blotting using antiparvalbumin antibody. In the rat retina at normal, parvalbumin immunoreactivity appeared in AII amacrine cells, amacrine cells of a widefield type and displaced amacrine cells. A few bipolar cells are also showed the reactivity. During diabetes, the intensity of parvalbumin immunoreactivity is decreased especially in the AII amacrine cells. The cell number of parvalbumin immunoreactive neurons has showed no large changes throughout the diabetes, except that of bipolar cells. That population of parv immunoreactive of bipolar cells has increased remarkably at later diabetic periods. The protein levels of parvalbumin have showed transiently a slight increase at earlier diabetic periods, and then decreased to lower than that of normal. Parvalbumin immunoreactive bipolar cells at diabetes are co-localized not with PKC-alpha or recoverin, but with glutamate transporter Glt-1b. AII amacrine cell processes were joined with each other and with axon terminals of presumed cone bipolar cells by gap junction. These results suggest that the calcium buffering activity of parvalbumin is shifted from AII amacrine cells to a certain type of cone bipolar cells, in response to diabetes. This event may be occurred through electrically coupled gap junction in between these cell processes.

The Expression of SRp38 Gene in Mouse Retina and It's Regulation of Alternative Splicing of GluR-B Minigene / 生物化学与生物物理进展

SR proteins play important roles in regulating alternative pre-mRNA splicing. As a newly discovered neural and reproductive tissue specific SR protein, SRp38 regulates the alternative splicing of several genes important for neural function, such as GluR-B, Trk-C and NCAML1. It also acts as a splicing inhibitor during mitosis or stress response in order to prevent wrong splicing. The expression of SRp38 in mouse retina was investigated by Western blot and immunohistochemistry (IHC) analyses. The result shows that the expression of SRp38 proteins in mouse retina is region-specific, with extensive distribution in the outer and inner plexiform layers, inner nuclear layer and ganglion cell layer, but no expression in outer nuclear layer. Double staining of isolated retina cells with anti-SRp38 and anti-Trk-C antibodies showed that SRp38 is localized in the dendrites, somata and axon terminals of rod-bipolar cells. By transient co-transmission of over-expressed SRp38 plasmid and RT-PCR analyses, the further results showed that overexpressed SRp38 could promote the splicing of the Flip isoform of GluR-B minegene in R28 cells. The result suggests that SRp38 may play important roles in the retinal function, possibly via regulating the neural-specific alternative splicing of genes as GluR-B.

Immunocytochemical Study on the Development of the Rod Pathway in the Rat Retina / 대한해부학회지

Rod bipolar cells constitute the second-order neuron in the rod pathway. Previous investigations of the rat retina have evaluated the development of other components of the rod pathway namely the AII amacrine cell and GABAergic amacrine cell populations. To gain further insights into the maturation of this retinal circuitry, we studied the development of rod bipolar cells, immunocytochemistry with antibodies directed to the protein kinase C (PKC), in the rat retina. PKC immunoreactivity first appeared in postnatal day 9 (P9), faint PKC immunoreactivity was observed in the cell bodies located at the distal inner nuclear layer (INL), dendrites in the outer plexiform layer (OPL) and immunoreactive bands in the proximal inner plexiform layer (IPL). PKC immunoreactive cells and terminal bulbs at P10 show stronger immunostaining. At P15, the time of eye opening, PKC immunoreactive cells display stronger immunostaining than those of P10 and more mature characteristics like in the adult retina. Double fluorescence immunocytochemistry using an antiserum against parvalbumin, a marker for the AII amacrine cells, or GABA revealed that PKC immunoreactive rod bipolar cell terminals make contact with AII amacrine cells and GABAergic neurons in the proximal IPL from P9. Given these results, the different components of the rod pathway follow a similar pattern of maturation, presumably allowing the rod pathway to function at the early developmental stage of retina.

Differentiation of Recoverin Immunoreactive Cone Bipolar Cells and Their Timing of Synaptic Formation with GABAergic Amacrine Cells in the Rat Retina / 대한해부학회지

Recoverin is a member of the large family of EF-hand calcium binding proteins (Baimbridge et al., 1992), and it is thought to be involved in the regulation of phosphodiesterase in photoreceptors and in the phosphorylation of activated rhodopsin (Polans et al., 1996). Although the functional significance of recoverin in cone bipolar cells is not fully understood, the antiserum against recoverin has been widely used to identify a certain population of cone bipolar cells (Milam et al., 1993; Sasso's Pognetto et al., 1994; Euler & W sle, 1995). GABA is well known to act as major neurotransmitters in the mammalian central nervous system including retina. This study was conducted to identify the development process of recoverin-labeled cone bipolar cells, and the timing points of synaptic formation of the labeled bipolar cells and GABAergic amacrine cells in the rat retina. The results were as follows; In the adult rat retina, recoverin-labeled cone bipolar cells were subdivided into twotypes; type 2 cells with axon terminal stratified in sublamina a of the inner plexiform layer (IPL), and type 8 cells with axon terminals stratified in sublamina b of the IPL. Recoverin-labeled cone bipolar cells began to appear from postnatal day 5. The axon terminals of recoverin-labeled type 2 cone bipolar cells stratified at postnatal day 10, while those of type 8 cone bipolar cells stratified at postnatal day 13. The axon terminals of type 2 cone bipolar cells made ribbon synapses onto GABAergic amacrine cells in the IPL at postnatal day 10. These results demonstrate that recoverin-labeled type 2 cone bipolar cells differentiate earlier than recoverin-labeled type 8 cone bipolar cells, and suggest that GABAergic amacrine cells may play important roles in visual processing of recoverin-labeled type 2 cone bipolar cells by making synapse onto these cells at early stage. Synapses between type 2 cone bipolar cells and GABAergic amacrine cells are formed about the time of postnatal day 10 for visual processing.