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.

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Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.energy metabolism | phototransduction A significant energy distribution problem can arise from the relative locations of mitochondria, ion pumps, and synapses in neurons. In photoreceptors, ion pumps occupy the intervening space between the centrally located mitochondria and the synaptic terminal. Ion pumping in dark-adapted photoreceptors consumes ∼20× more energy than neurotransmission (1). Therefore, the pumps could intercept all the metabolic energy made by the mitochondria before it can reach the synaptic terminal. In the vascularized retinas of mice, rats, and humans (2-4) this problem is solved by the presence of additional mitochondria in the terminal. However, in the avascular retinas of zebrafish, salamanders, rabbits, and guinea pigs there are no mitochondria in the terminals (2, 4, 5), which creates a need to partition some of the energy made by the central mitochondria into a protected form that can bypass the ion pumps to support the essential energy demands of the synaptic terminal.Energy consumption within retinal photoreceptors is compartmentalized and light-dependent. During illumination, phototransduction and light adaptation consume energy in the outer segment (OS). In darkness, energy is consumed by ion pumps in the inner segment and by glutamate release at the synaptic terminal (1). Energy demands and O 2 consumption are far greater in darkness than in light (1, 6-8).Metabolic energy is distributed in most cells as either ATP or phosphocreatine (PCr). There are 2 isoforms of creatine kinase (CK) in neurons, ubiquitous mitochondrial creatine kinase (uMtCK), and brain-type cytoplasmic creatine kinase (CK-B). uMtCK creates PCr from ATP at mitochondria (9), and CK-B can recreate ATP from PCr at sites of energy demand. In this way uMtCK and CK-B can collaborate to transfer metabolic energy between neuronal compartments (10, 11). This paper descr...

To test the effects of isolation on adult neurons, we investigated the fine structure and synaptic activity of rod cells dissociated from the mature salamander retina and maintained in vitro. First, freshly isolated rod cells appeared remarkably similar to their counterparts in the intact retina: the outer segment retained its stack of membranous disks and the inner segment contained its normal complements of organelles. Some reorganization of the cell surface, however, was observed: (a) radial fins, present at the level of the cell body, were lost; and (b) the apical and distal surfaces of the inner and outer segments, respectively became broadly fused. Second, the synaptic endings or pedicles retained their presynaptic active zones: reconstruction of serially sectioned pedicles by using three-dimensional computer graphics revealed that 73% of the synaptic ribbons remained attached to the plasmalemma either at the cell surface or along its invaginations. Finally, tracer experiments that used horseradish peroxidase demonstrated that dissociated rod cells recycled synaptic vesicle membrane in the dark and thus probably released transmitter by exocytosis. Under optimal conditions, a maximum of 40% of the synaptic vesicles within the pedicle were labeled. As in the intact retina, uptake of horseradish peroxidase was suppressed by light. Thus, freshly dissociated receptor neurons retained many of their adult morphological and physiological characteristics. In long-term culture, the photoreceptors tended to round up; however, active zones were present even 2 wk after removal of the postsynaptic processes.Rod cells from the tiger salamander are ideally suited for the study of the cell biology of isolated adult neurons: they survive intact after dissociation ( 1 ); they can be maintained in culture for long periods of time (2); and they give normal hyperpolarizing responses to light (1). Although the electrical responses of these cells have been studied in some detail (3-5), a number of crucial properties are still unknown; it seems especially important to establish whether solitary rod cells, maintained in vitro, preserve their morphologically differentiated state and whether their synaptic endings retain functional active zones capable of releasing transmitter by exocytosis in the dark.In this paper, the fine structure of salamander rod cells was compared in the intact retina and after dissociation. The geometry of the active zones in the synaptic terminals of solitary rod cells was analyzed by three-dimensional computer graphic reconstruction of serial sections. Finally, the functional state of their endings was tested by observing the uptake of the extracellular tracer, horseradish peroxidase, into synaptic vesicles. Portions of this work have appeared in preliminary form (2, 6, 7). MATERIALS AND METHODSAnimals: Aquatic-phase salamanders (Ambystoma tigrinum) measuring from 18 to 25 cm in total length were used. Animals of this size, although in the aquatic or larval phase, are considered adult inasmuch as ...

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.