Synaptic Mechanisms of Bipolar Cell Terminals

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Vision Research 39 (1999) 2469–2476

Synaptic mechanisms of bipolar cell terminals

Gary Matthews *

Department of Neurobiology and Beha6ior, State Uni6ersity of New York, Stony Brook, NY 11794-5230, USA

Received 8 May 1998; received in revised form 24 August 1998

Abstract

Giant synaptic terminals of goldﬁsh bipolar neurons allow direct studies of presynaptic mechanisms underlying neurotransmitter release and its modulation. Calcium inﬂux via L-type calcium channels of the terminal triggers synaptic vesicle exocytosis, which can be monitored in isolated terminals by means of the associated changes in membrane capacitance. Information about the kinetics and calcium dependence of synaptic exocytosis has been obtained from capacitance measurements in these ribbon-type synaptic terminals. © 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Synaptic mechanisms; Terminals; Bipolar neurons

Studies of presynaptic mechanisms in the vertebrate enzymatic digestion of goldfish retina or in single, central nervous system (CNS) are complicated by the isolated terminals. Consistent with their central role in small size of most CNS synaptic terminals. However, neurotransmitter release, voltage-dependent calcium several notable exceptions allow direct electrical mea- channels are located predominantly in the synaptic surements to be made from single terminals, including terminal of the bipolar cell (Tachibana & Okada, 1991; giant caliciform terminals in the brainstem auditory Heidelberger & Matthews, 1992), although there is also system of the rat (Forsythe, 1994) and in the ciliary a low density of channels in the soma. The synaptic ganglion of the chick (Stanley & Atrakchi, 1990; Stan- calcium current is sensitive to dihydropyridine calcium- ley, 1993) and large, bulbous terminals of certain types channel agonists and antagonists, activates at a rela- of bipolar neuron in goldfish retina (Kaneko & tively positive voltage range, and shows sustained Tachibana, 1985; Heidelberger & Matthews, 1992). The activation during prolonged depolarization (Heidel- latter are of interest in retinal neurobiology, and in this berger & Matthews, 1992). All of these characteristics review, I will focus on our current understanding of are typical of L-type calcium channels (Fox, Nowycky presynaptic events that regulate neurotransmitter re- & Tsien, 1987). The dihydropyridine-sensitive channels lease in the giant terminals of goldfish bipolar neurons. have been demonstrated to control glutamate release from the synaptic terminal (Tachibana, Okada, Arimura, Kobayashi & Piccolino, 1993). Thus, in goldfish bipolar cells, neurotransmitter release is con- 1. Calcium current of the synaptic terminal trolled by L-type calcium channels. The large size (diameter:10 mm) of the synaptic terminal of some classes of bipolar cell in carp and goldfish (Stell, 1967; Ishida, Stell & Lightfoot, 1980; 2. Presynaptic inhibition at the bipolar-cell terminal Saito & Kujiraoka, 1982; Sherry & Yazulla, 1993) permits direct patch-clamp recording of synaptic cal- The synaptic terminals of goldfish bipolar neurons cium current, either in intact bipolar cells isolated after receive extensive feedback synapses from amacrine cells (Marc, Stell, Bok & Lam, 1978; Yazulla, Studholme & Wu, 1987), which serve to modulate the synaptic output * Fax: +1-516-632-6661. from the bipolar cells. Several functional effects of the E-mail address: [email protected] (G. Matthews) synaptic feedback have been described, mediated by

0042-6989/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S0042-6989(98)00249-1 2470 G. Matthews / Vision Research 39 (1999) 2469–2476 different neurotransmitters and neurotransmitter recep- pamine was mimicked by forskolin and by a permeant tors. Many of the amacrine cells that target bipolar-cell analog of cAMP, suggesting that dopamine acts by terminals are GABAergic (Yazulla et al., 1987), and elevating cAMP in the terminal. Taken together, the GABA acts on two distinct GABA receptors to pro- actions of GABA, dopamine, and neuropeptides duce presynaptic inhibition in the terminal. First, demonstrate that synaptic feedback onto the bipolar

GABA activates a GABAA-like receptor and increases cell terminal is capable of modulating transmitter re- chloride conductance (Tachibana & Kaneko, 1987; lease from the bipolar cell and thus the transfer of Heidelberger & Matthews, 1991; Matthews, Ayoub & information from the outer to the inner retina. Heidelberger, 1994). Secondly, GABA produces GTP- dependent inhibition of the calcium current of the bipolar-cell terminal via a GABAB-like receptor 3. Capacitance measurements of exocytosis (Heidelberger & Matthews, 1991; Matthews et al., 1994). The GABA-activated chloride current hyperpo- When a secretory granule fuses with the plasma larizes the bipolar cell and thus inhibits calcium influx membrane during exocytosis, the surface area of the into the terminal via voltage-gated calcium channels secreting cell increases. The increased surface area can (Heidelberger & Matthews, 1991; Matthews et al., be estimated by monitoring the associated change in 1994). GABA inhibits the calcium current by shifting electrical capacitance of the cell, as shown schematically its voltage dependence toward more positive potentials, in Fig. 1. Sensitive techniques for monitoring capaci- thus tending to prevent activation of the current upon tance in real time in single cells are well-established depolarization. (Lindau & Neher, 1988; Gillis, 1995) and have been Both actions of GABA in the terminal are mediated used to gather information about exocytosis in various by receptors with unusual pharmacology. The receptor types of secretory cell, including adrenal chromaffin coupled to chloride conductance is insensitive to bicu- cells (Neher & Marty, 1982), mast cells (Fernandez, culline and SR95531, which typically block GABAA Neher & Gomperts, 1984), pituitary cells (Thomas, receptors, while the action of GABA on calcium cur- Suprenant & Almers, 1990), and neuroendocrine nerve rent is neither mimicked by the GABAB agonist ba- terminals (Fidler Lim, Nowycky & Bookman, 1990; clofen nor blocked by the GABAB antagonist saclofen. Lindau, Stuenkel & Nordmann, 1992). Because of the bicuculline resistance of the GABAA-like Neurotransmitter is released at synapses by a process receptors, they are commonly referred to in retinal of exocytosis, during which transmitter-containing research as GABAC receptors (Feigenspan, Wa¨ssle & synaptic vesicles fuse with the membrane of the presy- Bormann, 1993; Qian & Dowling, 1993) to distinguish naptic terminal in response to calcium influx via them from bicuculline-sensitive isoforms of the GABAA voltage-gated calcium channels. Therefore, capacitance receptor. The bicuculline-resistant GABAA-like recep- measurements are potentially useful at synapses as an tor probably corresponds to the retina-specific GABA index of neurotransmitter release. However, a difficulty z receptor subunit, 1 (Cutting, Lu, O’Hara, Kasch, for capacitance measurements from synaptic terminals Montrose-Rafizader, Donovan, Shimada, Antonarakis, is the small size—and hence the small capacitance—of Guggino, Uhl & Kazazian, 1991; Shimada, Cutting & the synaptic vesicles at most synapses. A synaptic vesi- Uhl, 1992), which produces bicuculline-resistant cle with a diameter of about 30 nm, which would be GABA-activated chloride channels when expressed in typical for glutamatergic synapses in the CNS, would Xenopus oocytes. Studies using conformationally locked have an expected capacitance of about 3×10−17 F analogs of GABA show that the bipolar-cell GABAA- (assuming that capacitance of biological membranes is like receptor prefers the trans (extended) isomer of approximately 10−14 F/mm2). The addition of such a GABA to the cis (folded) conformation (Ayoub & small amount of capacitance is below the level of Matthews, 1991). resolution presently attainable in whole-cell recordings, In addition to GABA, other neurotransmitters affect and so capacitance measurements are limited to special- the bipolar-cell terminal. Neuropeptides that are found ized synapses at which the number of synaptic vesicles in amacrine cells, including substance P and met- fusing with the plasma membrane during a stimulus is enkephalin, produce GTP-dependent inhibition of cal- sufficiently large to allow detection of the correspond- cium current by shifting the voltage dependence, similar ing increase in capacitance. In neurons, capacitance to the action produced by GABA (Ayoub & Matthews, measurements have been used to study synaptic release 1992). Goldfish bipolar-cell terminals also receive do- in the giant synaptic terminals of goldfish bipolar cells paminergic inputs (Dowling & Ehinger, 1978; Yazulla (von Gersdorff & Matthews, 1994), in retinal photore- & Zucker, 1988), and dopamine was found to enhance ceptors (Rieke & Schwartz, 1994), and in saccular hair the increase in intracellular calcium evoked in the ter- cells (Parsons, Lenzi, Almers & Roberts, 1994). All of minal by activation of voltage-gated calcium channels these cells have distinctive presynaptic structures (Heidelberger & Matthews, 1994). This action of do- (synaptic ribbons or synaptic bodies), which are G. Matthews / Vision Research 39 (1999) 2469–2476 2471

Fig. 1. Schematic diagram of the changes in membrane capacitance that accompany exocytosis and endocytosis. Electrical access to the interior of the synaptic terminal is provided by means of a patch pipette in the whole-cell recording configuration. For simplicity, the series resistance through the patch pipette and the membrane resistance of the terminal are omitted in the equivalent circuits. (A) In the resting state, a synaptic vesicle is docked at the active zone, but there is no electrical continuity between the interior of the vesicle and the extracellular space. Therefore, the capacitance observed in the patch-clamp recording is the resting membrane capacitance of the terminal, Cm. (B) Upon depolarization, voltage-gated calcium channels open, allowing influx of calcium ions (black circles) into the synaptic terminal. Calcium binds to a calcium sensor molecule, causing opening of the putative fusion pore, which is assumed to be part of the molecular assembly of the docking complex. Release of neurotransmitter commences through the open fusion pore. At this point, the vesicle capacitance, C6, becomes electrically accessible through the conductance of the fusion pore, Gp. Because of the small size of the vesicle capacitance, virtually the entire capacitance of the vesicle is immediately observable even if the fusion pore has the conductance of a single ion channel. (C). Full fusion of the vesicle membrane with the plasma membrane is thought to occur as the docking/fusion complex disassembles. The opening of the fusion pore may also be reversible, producing so-called ‘kiss-and-run’ exocytosis. (D) The added membrane, including proteins associated with the synaptic vesicle, is retrieved via endocytosis, which may by mediated via formation of a clathrin coat (as shown) or by other mechanisms. thought to be specializations that increase the available index of the amount of exocytosis triggered by activa- pool of releasable vesicles at the synaptic output site. tion of the calcium channels, while the return of capac- Fig. 2 shows an example of the change in capacitance itance to baseline represents endocytotic retrieval of the elicited by depolarization in the goldfish bipolar-cell added membrane. terminal. The dihydropyridine-sensitive calcium current activated by the depolarization is shown in the inset. Depolarization was followed by a rapid jump in mem- 4. Depletion and replenishment of vesicle pools brane capacitance of the terminal, after which capacitance returns to baseline, typically within a few seconds In bipolar-cell terminals, the capacitance jump elic- after the stimulus. The rapid rise in capacitance is an ited by depolarization increases with the duration of the 2472 G. Matthews / Vision Research 39 (1999) 2469–2476 depolarizing pulse up to a maximum size of approximately 150 fF, which represents the maximum size of the readily releasable pool of vesicles whose release is triggered by activation of calcium channels. This pool is released within about 200 ms during depolarization that maximally activates calcium current (i.e. depolarization to about 0 mV). The 150-fF capacitance pool can be converted into a corresponding pool of 5700 synaptic vesicles from the measured average diameter of the vesicles in goldfish bipolar-cell terminals (29 nm; von Gersdorff, Vardi, Matthews & Sterling, 1996) and assuming a membrane capacitance of 10 fF/mm2.Itis interesting that reconstructions of giant terminals from electron micrographs of serial thin sections suggest that there are about 6000 vesicles tethered to synaptic ribbons in the entire terminal (von Gersdorff et al., 1996). The close agreement between the morphologically defined pool of vesicles on the ribbons and the readily releasable pool estimated from capacitance measurements suggests that the entire population of vesicles on the ribbon can be released within about 200 ms during depolarization. This implies that the average fusion rate during depolarization is therefore about 30 000 vesicles/ s for the terminal as a whole, or about 500 vesicles/sat each of the approximately 55 synaptic ribbons in the terminal. The synaptic vesicles are tethered to the ribbon of goldfish bipolar cells in about five rows on each face of the ribbon, and the vesicles in the bottom row at the base of the ribbon are in close contact with the plasma Fig. 3. Two kinetically distinct components of exocytosis in bipolar membrane of the terminal. The close proximity suggests cell synaptic terminals. (A) Changes in capacitance (upper traces) that this subgroup of vesicles might be docked and measured in response to brief depolarization given at the arrows. primed for release, and if so, the docked subgroup There was no change in access resistance (lower traces). (B) The average capacitance change evoked in five synaptic terminals by might be released more rapidly upon depolarization pulses of different durations. The solid line, fitted to the first four than the rest of the vesicles in the higher rows on the points, represents an exponential rise with a time constant of 1.5 ms ribbon. Capacitance measurements have indeed re- and an amplitude of 33 fF. The point at the longest duration deviates vealed a small pool of vesicles that are released very significantly from the line because of the slower component of exocytosis that is more readily apparent in panel C. (C). The slow component of exocytosis is revealed more clearly with longer pulse durations. Data points show average capacitance responses from eight terminals. The solid line represents the best-fitting sum of two exponential components, with time constants of 1.6 and 350 ms and amplitudes of 31 and 81 fF.

rapidly upon the opening of voltage-gated calcium channels (Mennerick & Matthews, 1996). The size of this pool (approximately 1100 vesicles) is in close agreement with the expected number of vesicles at the bottom of the ribbon (von Gersdorff et al., 1996). As illustrated in Fig. 3, the time constant for the depletion Fig. 2. Change in membrane capacitance elicited by depolarization in of the fast component was 1.5 ms, while the remainder a bipolar cell synaptic terminal. The arrow indicates the timing of the of the releasable pool was released more slowly during depolarizing pulse, and the calcium current activated by the depolar- depolarization, with a time constant of about 300 ms ization is shown on expanded time scale in the inset. The rapid jump (Mennerick & Matthews, 1996). Thus, fusion of the in capacitance represents exocytosis, and the return to baseline capacitance over the next few seconds represents endocytosis of the added docked vesicles at the base of the ribbon may occur membrane. extremely rapidly as calcium channels open, while the G. Matthews / Vision Research 39 (1999) 2469–2476 2473 vesicles in rows more distant from the plasma mem- much lower (1 mM) concentration of calcium brane achieve fusion more slowly during sustained de- achieved in the terminal as a whole during activation of polarization. Similar fast and slow components of calcium current. release have also been observed in experiments in which Although dialysis experiments give a qualitative indi- glutamate release from goldfish bipolar-cell terminals cation of the level of calcium necessary to drive exocy- was directly monitored, using electrical responses of tosis, the slowness of dialysis via a whole-cell patch closely opposed horizontal cells to detect release (Sak- pipette does not allow conclusions about the calcium aba, Tachibana, Matsui & Minami, 1997). Tachibana & dependence of the rate of exocytosis. This type of Okada, 1991 also used retinal horizontal cells as detec- information was obtained instead in experiments em- tors of glutamate release from bipolar cells and found ploying photorelease of caged calcium from calcium- that the ‘postsynaptic’ response activated with a delay loaded DM-nitrophen (Heidelberger, Heinemann, as brief as 1 ms after the onset of depolarization in the Neher & Matthews, 1994). Fig. 4 illustrates sample bipolar cell. Thus, direct measurements of glutamate results of such an experiment. With a bright flash of release independently verify the features of synaptic uncaging light, which elevated calcium concentration exocytosis revealed by capacitance measurements. to100 mM in the example of Fig. 4, capacitance As described earlier, activation of calcium current for increased rapidly after the flash. With dimmer flashes, approximately 250 ms is sufficient to completely deplete which elevated calcium to lower levels, the rate of rise the releasable pool of vesicles in bipolar-cell terminals. of the capacitance after the flash was slower, and at If a second depolarization is given soon after a deplet- calcium concentrations below about 10–20 mM, no ing stimulus, the evoked capacitance response to the capacitance change was observed. By repeating such second stimulus is strongly depressed (von Gersdorff & photolysis experiments at a variety of calcium concen- Matthews, 1997). The depression recovers with a time trations, the relation between calcium concentration constant of about 8 s, and full recovery of the response to the second stimulus requires about 20 s. This pro- and the rate of exocytosis was obtained. The relation vides an estimate of the time for functional recovery of could be modeled assuming that synaptic vesicle fusion the release-ready pool after a single, strong stimulus. was triggered by cooperative binding of calcium to four With weaker stimuli, the amount of release produced binding sites, followed by a rapid fusion step. The by a single stimulus is less, and the terminal is able to overall half-saturating calcium concentration in the m sustain exocytosis at a constant rate for up to several model was 190 M, and the maximal rate constant of seconds (von Gersdorff & Matthews, 1997). Thus, the amount of initial exocytosis, and hence the degree of depletion of the releasable pool, determines the ability of the bipolar cell to continue to release transmitter during sustained or repetitive stimuli.

5. Calcium dependence of exocytosis

The ability to monitor exocytosis by means of capacitance changes in the bipolar-cell terminal offers the opportunity for direct study of the calcium-dependence of neurotransmitter release. One method of estimating the calcium dependence of exocytosis is to monitor changes in membrane capacitance while dialyzing the cell with solutions containing buffered levels of elevated calcium concentration (Augustine & Neher, 1992). In bipolar-cell synaptic terminals, dialysis with 1.4 mM calcium (10 times the resting level) failed to elicit an increase in membrane capacitance, but when calcium concentration was increased to50 mM, capacitance did increase during dialysis (von Gersdorff & Matthews, 1994). This suggests that exocytosis is trig- Fig. 4. Capacitance change in a bipolar cell synaptic terminal evoked gered in bipolar-cell terminals by the high concentra- by photolytic release of caged calcium. The timing of the ﬂash of uncaging light is shown by the arrow. The upper trace shows the tion of calcium expected within the microdomain of capacitance response, with the initial rise shown on a faster time scale elevated calcium near open calcium channels (Chad & in the inset. The rise in calcium resulting from the release of caged Eckert, 1984; Fogelson & Zucker, 1985), and not by the calcium is shown in the lower trace on a slow time scale. 2474 G. Matthews / Vision Research 39 (1999) 2469–2476 about 3000 s−1 was achieved as calcium concentration approached several hundred mM (Heidelberger et al., 1994; Heidelberger, 1998).

6. Calcium action potentials in bipolar cells

Capacitance measurements suggest that large-terminal bipolar cells of goldfish retina are capable of very rapid release of a finite pool of neurotransmitter, at least when stimulated with depolarizations that are sufficient to maximally activate the voltage-dependent calcium channels of the synaptic terminal. However, the question arises whether such depolarizations would occur natu- rally in bipolar neurons. Intracellular recordings in intact retina suggest that they do. Saito, Kondo and Toyoda (1979) showed that light flashes elicited a rapid depolarization in on-type rod bipolar cells that peaked at a membrane potential of approximately −10 to −15 mV. This spike-like depolarization lasted about 200 ms and is therefore comparable to the stimuli used in capacitance measurements to activate rapid exocytosis. The waveform of the intracellularly recorded light response of large-terminal, on-type bipolar cells (Saito et Fig. 5. Calcium action potentials are triggered by depolarization in al., 1979; Saito & Kujiraoka, 1982) may in part reflect goldfish bipolar cells. Each set of four traces shows the voltage calcium action potentials triggered in response to synap- responses recorded in current clamp in response to the indicated tic depolarization. Large-terminal bipolar cells isolated levels of depolarizing current, injected at the time indicated by the from goldfish retina are electrically excitable and produce pulse below the upper set of traces. Depolarization above a threshold level evoked a regenerative action potential that was abolished by robust calcium action potentials (see Fig. 5) that origi- addition of 50 mM cadmium to the external solution (middle set of nate from the voltage-gated calcium channels of the traces). synaptic terminal (Zenisek & Matthews, 1998). The amplitude and duration of the calcium action potential dynamics of releasable vesicle pools and about the are similar to the light responses observed by Saito et al. calcium dependence of exocytosis at these ribbon (1979) and are sufficient to release most of the pool synapses. The bipolar-cell synaptic terminal is in a central of6000 readily releasable vesicles inferred from capac- position to determine the efficacy of synaptic transfer itance measurements. Thus, an active response to depo- between the outer and inner retina and is a prominent larization, resulting from voltage-gated calcium channels target for synaptic feedback in the retina. Several actions in the synaptic terminal, may serve to amplify the of this feedback on bipolar-cell terminals have already depolarizing response to illumination. If such calcium been identified. It seems likely that vesicle pool size, pool action potentials do occur in the intact retina, then refilling rates, and other aspects of the release machinery neurotransmitter release from the rod bipolar cell would of the synapse might be additional targets for neurotrans- be large, rapid, and transient. It is important to empha- mitters and neuromodulators. If so, capacitance mea- size, however, that action potentials, and thus rapid and surements should prove valuable in unraveling these transient synaptic signaling, might occur only under modulatory actions. restricted conditions (for example, in the highly dark- The retina contains many different kinds of bipolar adapted state). With smaller, graded depolarizations to cell, and the conclusions summarized here are based on illumination, capacitance measurements suggest that studies of a single type, selected because of its large neurotransmitter release would occur at a slower rate but synaptic terminal. Other types of bipolar neurons might would be more sustained. differ in important physiological and pharmacological ways from the rod-dominant, on-type cells on which we have focused. For instance, cone-driven bipolar cells 7. Conclusions might not require the high amplification provided by calcium action potentials and so might lack this feature. Capacitance measurements provide a direct view of Also, our results emphasize the transient nature of exocytosis in the giant synaptic terminals of goldfish transmitter release from the large-terminal bipolar cells, bipolar neurons. New information has resulted about the but the retina must possess bipolar cells that are capa- G. Matthews / Vision Research 39 (1999) 2469–2476 2475 ble of sustained transmitter release to signal steady Heidelberger, R. (1998). Adenosine triphosphate and the late steps illumination levels. Clearly, characterization of the in calcium-dependent exocytosis at a ribbon synapse. 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