Chapter 9 Transmission at the photoreceptor synapse

H. Kolb, H. Ripps and S. Wu (Eds.)

progress in Brain Research, Vol. 131 © 2001 Elsevier Science B.V. All rights reserved

CHAPTER 9

Transmission at the photoreceptor synapse Paul Witkovsky l'*, Wallace Thoreson 2 and Daniel T r a n c h i n a 3 1Departments of Ophthalmology and Physiology, New York University School of Medicine, 550 First Avenue, New York, N Y 10016, USA 2Departments of Ophthalmology and Pharmacology, University of Nebraska Medical Center, 985540 Nebraska Medical Center, Omaha, NE 68198, USA 3Departments of Biology and Mathematics, New York University, 100 Washington University Square East, New York, N Y 10003, USA

Introduction The rod and cone photoreceptors of mature retinas develop from non-motile ciliated cells whose distal endings elaborate membranous disks that house the visual pigments. The biochemical transduction cascade coupling light capture by visual pigment molecules to surface membrane electrical events converges on the gating of a cGMP-dependent cation channel. Light brings about the reduction of [cGMP] and the closing of some cation channels, resulting in a hyperpolarization of the photoreceptor. Another of the photoreceptor's special features is its slow kinetics. A bright flash delivered in 1 ms initiates voltage changes in rods and cones that can take up to seconds, to complete, and in the more usual case, a constantly changing light pattern of modest intensity induces a continuous modulation of photoreceptor voltages. The unusual nature of photoreceptor electrophysiology--relative depolarization in darkness changing to a more hyperpolarized level during light--sets the parameters for the functioning of its synapse at which the photoreceptor communicates information about changing light patterns to second order retinal neurons. The synaptic signal is conveyed through a neurotransmitter whose release is a calcium-dependent process; because calcium channels have a higher * Corresponding author: Paul Witkovsky, Tel.: 212-263-6488; Fax: 212-263-7602; E-mail: [email protected]

open probability as the membrane depolarizes, there is a greater calcium current during darkness than in light. For the photoreceptor to signal time-modulated light signals effectively, its calcium channel has to be non-inactivating. The effective operating range of rod and cone photoreceptors is from - 35 to - 40 mV in darkness, grading smoothly to about - 60 mV as incident light intensity increases towards saturation, so the calcium current has to be modulated by voltage in this range. In fact, the particular calcium channels found in photoreceptors (discussed in more detail below) are only weakly activated between - 3 5 and - 6 0 mV and the slope of the relation between voltage and Ca channel activation function is shallow over that range of voltages. Corresponding measurements of intracellular calcium concentration, [Ca]i, show that it attains its highest level in darkness, when it is still less than 1 gM, according to some estimates (Krizaj and Copenhagen, 1998; Krizaj et al., 1999), falling to perhaps 0.1 ~tM in bright light. Thus photoreceptors have evolved mechanisms to control transmitter release with very low levels of calcium, compared to those which gate transmitter release at other, faster, synapses (Neher, 1998). This essay focuses on the special/features of transmitter release by rods and cones and the activation of the bipolar and horizontal cells with which photoreceptors communicate. It begins with a survey of the components that are known to participate in signal transfer between photoreceptors and their synaptic targets, or to

146 influence that process. It ends with a quantitative model of the photoreceptor synapse that utilizes some of the described components to describe the kinetics of synaptic transfer.

The photoreceptor transmitter is glutamate The neurotransmitter in question is glutamate, the most widely encountered excitatory transmitter in the brain. The evidence that both rods and cones utilize glutamate is compelling (Thoreson and Witkovsky, 1999). Immunocytochemical studies show that rods and cones, bipolar cells and ganglion cells concentrate glutamate in synaptic terminals. In contrast, retinal inhibitory interneurons, horizontal and amacrine cells, utilize gaba and glycine, and typically colocalize another monoamine or peptide transmitter. Glutamate release evoked by elevated [K + ] has been measured from individual cones (Copenhagen and Jahr, 1989) and light-gated glutamate release from the photoreceptor layer freed from the inner retina was shown by Schmitz and Witkovsky (1996). Correspondingly, the post-synaptic neurons, horizontal and bipolar cells, possess glutamate receptors in their synaptic endings. These may be ionotropic receptors, usually of the AMPA (Sasaki and Kaneko, 1996; Maple et al., 1999) or KA sub-types (deVries and Schwartz, 1999), but at least in one horizontal cell, also of the N M D A type (O'Dell and Christensen, 1986). And in addition a wide variety of metabotropic glutamate receptors is associated with the photoreceptor synapse, at both pre- and post-synaptic locations (reviewed in Brandstatter et al., 1998).

Glutamate transporters Removal of glutamate from the synaptic cleft occurs partly by diffusion, but mainly is brought about by glutamate transporters (Arriza et al., 1994). Genetic studies show that the population of glutamate transporters is diverse, while immunocytochemical studies show that they have particular locations, some in neurons and others in glial cells (Rauen et al., 1998). Glutamate transporters have certain common properties, including a high affinity for glutamate and a voltage dependence, which is in the direction

of increasing transport rate with hyperpolarization. Gaal et al. (1998) showed that the transporter of the cone cleared glutamate more rapidly when the cone was illuminated, resulting in a hyperpolarization of the horizontal cell. These data suggest a role for neuronal glutamate transporters in shaping the kinetics ofpost-synaptic responses (Roska et al., 1998). Glutamate transporters also are electrogenic, the stoichiometry varying with the particular transporter. In photoreceptors (Grant and Werblin, 1996; Eliasof and Jahr, 1996) and at least some ON-bipolar cells (Grant and Dowling, 1995), glutamate transporter activity is associated with CI flux. The pharmacology and physiological properties of glutamate-activated C1 current suggest an amalgam of a transporter and a channel, and the resultant C1 current can affect transmitter release by influencing the Ca current of rods and cones. One mechanism is a direct action whereby reduction in [C1]i inhibits Ca current (Thoreson et al., 1997). Another mechanism is through a voltage change, whose direction and amplitude are hard to assess, since Ecl is close to the membrane potential of the photoreceptor in darkness. Another complication in that connection is that cones have a prominent Ca-dependent C1 current (Maricq and Korenbrot, 1988). The glutamate transporter of the Mueller glial cell of the retina (Barbour et al., 1991; Derouiche and Rauen, 1995) works in series with those of photoreceptors, horizontal and bipolar cells. Glial processes are excluded from the synapse, but avidly take up any residual glutamate from extracellular space. In darkness, moreover, when transmitter release by rods and cones is at its highest, photoreceptors, horizontal and bipolar cells are relatively depolarized, whereas the Muller glial cell is hyperpolarized. Thus in darkness glial glutamate transport is at a relatively high rate, neuronal glutamate transport at a low rate. The relative contributions of this mix of transporter activity in setting the [glutamate] of the synaptic cleft is still unknown, as are the kinetics of the change in [glutamate] brought about by changing light levels.

Desensitization of ionotropic glutamate receptors The rate of change of glutamate concentration in the cleft controls the conductance of the receptors on

147 second-order neurons that initiate the post-synaptic potential. Receptor desensitization, which refers to a loss of conductance while the transmitter is still bound to the receptor, may play a role in shaping the post-synaptic response. B o t h AMPA receptors (AMPARs) and kainate receptors (KARs) in distal retinal neurons show desensitization (Eliasof and Jahr, 1997; deVries and Schwartz, 1999). Eliasof and Jahr (1997) studied A M P A R desensitization in horizontal cells by applying saturating concentrations of glutamate. Their experiments revealed an extremely rapid and profound desensitization. Yang et al. (1998), however, found that cyclothiazide, which blocks A M P A R desensitization, did not change the kinetics of light-evoked responses in horizontal cells. This apparent contradiction might be resolved if [glutamate] in the outer plexiform layer did not achieve the required level for desensitization. In contrast, de Vries and Schwartz (1999) noted an asymmetry in the OFF-bipolar cell currents evoked by imposing a voltage step on a cone from - 7 0 to - 3 5 mV or the reverse. In the former case [glut] at - 7 0 mV is presumed to be too low to induce desensitization, so the depolarizing step elicits a large current. In the reverse case, K A receptors are desensitized at - 35 mV by tonic [glut], resulting in a small current in response to the hyperpolarizing step. Thus KARs appear to undergo desensitization at the glutamate concentrations found in vivo and the differences between AMPARs and KARs in the outer retina in this respect may reflect different inherent affinities for glutamate. AMPARs in horizontal cells which are directly adjacent to the sites of vesicle release have a low affinity for glutamate. KARs in OFF-bipolar cells making flat contacts are farther from the release site, if it turns out, as postulated (deVries and Schwartz, 1999), that photoreceptors release glutamate only at ribbon synapses. And in this case they would be presumed to have a relatively high affinity for glutamate. In the inner plexiform layer, the kinetics of certain amacrine cell and ganglion cell responses to light are affected by cyclothiazide (Tran et al., 1999). Amacrines receive input from ON-bipolar cells whose phasic synapses release glutamate at a much higher rate than that achieved by photoreceptors (yon Gersdorff et al., 1996), which suggests that under certain conditions the glutamate concentration in the synaptic cleft will

be much higher than that at the photoreceptor synapse. Much further work is required to define the properties of these synapses and to establish the concentration(s) of glutamate which obtain in the synaptic clefts of different retinal synapses.

Structure of the photoreceptor synapse Photoreceptor synaptic transmission occurs at sites with a stereotyped but complex geometry (Dowling, 1987). During retinal development, outgrowing neurites of horizontal and bipolar cells approach the rod and cone synaptic terminals. Some neurites penetrate more or less deeply into the rod-cone bases; these are the invaginating contacts. Or, in the case of flat bipolar cell contacts with cones, they array themselves in shallow indentations along the surface of the synaptic terminal. Photoreceptors themselves extend telodendritic processes which form electrical synapses with neighboring photoreceptors, or, more rarely, become one of the post-synaptic processes at a ribbon synapse of a neighboring photoreceptor (Lasansky, 1973). The processes arrayed in the invaginations have a specific geometry in relation to the pre-synaptic specializations associated with the positioning of transmitter-filled vesicles for release. These include a synaptic ribbon, arciform density and paramembranous proteins which form the complex required for vesicle exocytosis. The calcium channels which gate the calcium influx that triggers exocytosis are distributed near the docking sites (Taylor and Morgans, 1998). The positioning of post-synaptic processes with respect to sites of glutamate release presumably is important in relation to whether their glutamate receptors have high or low affinity for glutamate, because as mentioned above, glutamate released from vesicles is actively taken up by neural and glial transporters. This consideration may be particularly important for flat bipolar contacts on cones in view of the postulate that glutamate is released not adjacent to the site of flat contact, but rather from a more distant ribbon synapse. The presently available evidence about the composition of naturally occurring gluRs and their locations within post-synaptic terminals is far from complete. In fact, the full subunit composition of any

148 naturally occurring GluR in a retinal neuron is unknown. EM-immunocytochemistry (Vardi et al., 1998; Brandstatter et al., 1998) suggests the presence of certain glutamate receptor (GluR) subunits within terminal dendrites. A number of studies show that immunoreactivity for GluR1, GluR2/3 and GluR4 are found among bipolar and horizontal cell processes in the outer plexiform layer. These are the subunits of AMPA receptors (Hollman and Heinemann, 1994) but there are the inevitable species differences which complicate reaching general conclusions about the make up of AMPA receptors. Immunocytochemical studies provide evidence for the presence of K A R subunits (GluR 5 7, KA1,2) in outer retinal synapses. EM-immunocytochemistry also reveals some unexpected findings, such as the presence of ionotropic gluR subunits in ON-bipolar cells which are known to respond to light through a metabotropic receptor, mGluR6. Brandstatter et al., (1996) have drawn attention to an intriguing finding, which is that only one of the two post-synaptic processes emanating from the same cell type contains a particular mGluR. GluRs typically are clustered with the aid of scaffolding proteins, of which a variety have been identified, and been shown to be specific to the different classes of GluR, including ionotropic and metabotropic types. Recent evidence shows that such scaffolding proteins are present in retinal synapses (Koulen et al., 1998) where they may serve to link GluRs to certain intracellular enzymes, e.g. soluble guanylate kinases, as has been found in brain (Ziff, 1997).

The calcium currents of rods and cones The advent of voltage clamp methods that could be applied to small cells (Hamill et al., 1981) made it possible to study calcium currents in photoreceptors. The literature is consistent in identifying L-type voltage-gated Ca channels in rods and cones (Bader et al., 1982; Corey et al., 1984; Lasater and Witkovsky, 1991; Wilkinson and Barnes, 1996). L-Ca channels are characterized by sensitivity to dihydropyridines, half-maximum of the Boltzmann activation function at relatively depolarized voltages ( - 3 0 to - 15 mV), lack of inactivation in the face of a sustained depolarizing step, and relatively slow

kinetics. An L-Ca current has been shown to underlie glutamate release by rods and cones (Rieke and Schwartz, 1996; Schmitz and Witkovsky, 1997), retinal bipolar cells (Tachibana et al., 1993) and to contribute to catecholamine release from chromaffin cells (Artalejo et al., 1991), whereas N, P, Q and R calcium channels gate transmitter release at fast synapses (Olivera et al., 1994). It has been reported that a cGMP-dependent, Ca current in cones contributes to the control of transmitter release (Rieke and Schwartz, 1994).

Intracellular regulation of calcium in photoreceptors Photoreceptors experience a steady influx of calcium at both ends, which is greater in darkness than in light. In the outer segment, the plasma membrane is abundantly provided with cGMP-gated channels that have a high relatively permeability to divalent cations, such that ca. 15% of the inward current is carried by Ca 2 +. In the inner segment and synaptic terminal, the L-Ca channels have a relatively high open probability at the membrane potentials of rods and cones in darkness, permitting a steady calcium influx. Light reduces Ca 2 + permeability at both ends of the photoreceptor. In the outer segment a Na/KCa exchanger restores the low [Ca] of the cytoplasm (McCarthy et al., 1994), whereas in the inner segment, a Ca-ATPase is largely responsible for maintaining cytosolic [Ca] levels (Krizaj and Copenhagen, 1998; Morgans et al., 1998). Influx of calcium through voltage-gated Ca channels is only one route whereby rods and cones regulate [Ca]/. The intracellular cisternae found in photoreceptor inner segments are calcium stores that can be triggered either to release Ca 2 + or to sequester it. Ca release from the endoplasmic reticulum is under the control of two types of receptor. The inositol trisphosphate (IP? ~ receptor is activated through a metabotropic, G protein-linked cascade in which the enzyme phospholipase C is activated to breakdown a membrane lipid, phosphatidyl inositol trisphosphate, into diacylglycerol (DAG) and IP3. D A G remains in the membrane, where it activates PKC, while IP3 diffuses to its receptor, causing a release of Ca and an up-regulation of PKC. Protein

149 kinase C has been found to up-regulate transmitter release by retinal bipolar cells (Minami et al., 1998). The ryanodine receptor, whose structure resembles that of the IP3 receptor, is activated by Ca influx, a phenomenon called calcium induced calcium release (CICR). Besides these receptors, a host of transporter and pumps act both at intracellular sites and at the surface membrane to maintain calcium homeostasis. Ca imaging experiments, using fluorescent Ca sensors, indicate that [Ca] is kept in the range 0.1-1.0 micromolar as photoreceptors respond to light/darkness. Krizaj et al. (1999) examined whether Ca released from intracellular stores affected transmitter release by rods. Caffeine was used to stimulate release and changes in [Ca]/ were monitored by fluorescence. Puffs of caffeine evoked a transient 1 2 s rise in [Ca] followed by a more sustained decay. Parallel measures of glutamate release by the photoreceptor layer showed a caffeine-induced depression. The transient rise in [Ca]/could not be resolved by the glutamate measures which were averaged over several minutes. However when horizontal cell membrane potential was monitored during exposure of the eyecup to caffeine, a brief depolarization followed by a sustained hyperpolarization was observed, on the time scale which roughly paralleled the changes in [Ca]. These data raise an important question: does the exocytotic machinery of rod photoreceptors respond to the general cytoplasmic level of [Ca], or does it sample [Ca] in a microzone that is part of the local region where exocytosis occurs?

Modulation of photoreceptor calcium currents Voltage-gated calcium channels in rods and cones are subject to modulation by numerous neuroactive substances and also show a substantial degree of regulation by calcium itself and by pH. Barnes et al. (1993) found that an alkaline shift of pH increased L-channel current. Since retinal pH changes as a function of its metabolic state, which in turn is influenced by light and darkness, the Ca current will be influenced by the metabolic activity of photoreceptors, which is known to be very high (Shichi, 1983). Recently, Verweij et al. (1996) proposed that the feedback influence which horizontal cells exert upon cones is mediated through a

leftward shift of the Ca activation function along the voltage axis, an effect which works against the direct hyperpolarizing action of light on cones, hence a "negative" feedback. A similar result was reported for somatostatin (Akopian et al., 2000), which, acting through an sst-2a receptor, increases the delayed rectifying current of rods and cones. However this same peptide had a differential action on Ca currents, increasing that of cones, while diminishing that of rods. The increase results from a leftward shift of the Ca activation function. Koulen et al. (1999) recently identified a metabotropic glutamate receptor (mGluR8) which is coupled to Ca entry. The location of mGluR8 is on the photoreceptor terminal, where it functions as an autoreceptor.

Photoreceptor coupling Photoreceptor coupling is well documented in the retinas of lower vertebrates and it has been inferred for mammalian retinas based on the transmission of rod signals through the retinal network (Nelson, 1977). In amphibian and turtle eyes, rods are coupled to other rods (Copenhagen and Owen, 1976; Gold and Dowling, 1979), which results in noise reduction and spatial integration that may be important for perception of very dim lights. Studies of dim light responses in primate photoreceptors, however (Schneeweis and Schnapf, 1995) led to the conclusion that the rods were uncoupled from other rods, but were coupled to cones. Rod-cone coupling also is well documented in amphibian retinas. Physiological evidence suggests that a small fraction of rods are very tightly coupled to cones; such rods have markedly altered kinetics reflecting cone input. Because both rod-cone and rod-rod coupling exist, the cone signal is transmitted with progressive attenuation through the rod network, resulting in inhomogeneity in light-evoked response kinetics among the rod population. R o d cone coupling is increased by dopamine through a D2 receptor (Krizaj et al., 1998), consistent with the general role of dopamine in promoting light adaptive changes in retinal function (reviewed in Witkovsky and Dearry, 1991). Presumably rod-cone coupling will contribute to the dynamics of transmitter release by rods, but this requires experimental confirmation.

150

Replenishment of glutamate released at photoreceptor synapses In their classical study, Heuser and Reese (1973) showed that exocytosing vesicles added vesicular membrane to the synaptic terminal, but that exocytosis was balanced by recovery of membrane (endocytosis) at an adjacent region. There membrane was taken up by coated vesicles and added to intracellular cisternae from which new vesicles were derived. Photoreceptors were shown to conform to this pattern of renewal (Ripps et al., 1976). The source of the glutamate which is used to fill the vesicles is still not fully determined. An important cycle involves glutamine and the Muller glial cell. Glial cells take up glutamate, converting it to glutamine with an enzyme, glutamine synthetase which is confined to glial cells (Linser and Moscona, 1979). Glutamine is moved out of glia and into photoreceptors by transporters that need to be better characterized. Glutamine is converted to glutamate by glutaminase, which has been shown to be present in rods and cones. Although there is evidence that this system operates for the photoreceptor, Winkler et al. (1999) showed that blocking glutamine synthetase had almost no effect on generation of the electroretinographic b-wave, which is a measure of ON-bipolar cell activity (Stockton and Slaughter, 1989) and hence an indirect monitor of the photoreceptor synapse. On the other hand, blocking a glutamate transporter rapidly eliminated the b-wave.

Glutamate in the synaptic cleft The concentration and kinetics of extracellular glutamate at the photoreceptor synapse are still subjects of active investigation. The function of other components of the synapse depend on the rise and fall of glutamate in a very direct way. For example, the proximity of the GluR to th¢ release site will have to match its affinity for glutamate and the degree of desensitization and recovery from desensitization of a given GluR will depend directly on the kinetics and magnitude of glutamate concentration changes. Gaal et al. (1998) studied cone to horizontal transfer in the salamander retina. They found that when photoreceptor transmission was blocked with

20 mM Mg 2+, horizontal cells hyperpolarized and lost light responsiveness. In this state exogenous glutamate depolarized the HC. A superimposed light flash, to which photoreceptors would still be responsive, induced a horizontal cell hyperpolarization, suggesting that a cone glutamate transporter was more active when hyperpolarized. Supporting evidence came from the findings that the hyperpolarizing response was blocked by DHKA, and that substituting kainate, which is not transported, for glutamate, blocked the phenomenon. Gaal et al. (1998) also found that the plot of horizontal cell depolarization against [glutamate], when the transporter and synaptic transmission were blocked, was fit by a logistic function with a Hill coefficient near 2 and half-activation at 32 gm. This data suggest that [glutamate] varies in the low micromolar range over the functional range of cone light responsiveness.

Modeling transmission at the photoreceptor synapse The preceding sections outline some of the basic membrane properties of rods and cones that bear on the way they release their transmitter, glutamate. Neither rods, cones, nor the cells with which they communicate produce action potentials, so synaptic transfer is the process whereby slow light-induced potential changes in photoreceptors induce similar slow potentials in bipolar and horizontal cells. When one takes into account that in addition to multiple photoreceptor classes, there exist a variety of postsynaptic receptors for glutamate which are arrayed on various dendritic terminals of horizontal ar~l bipolar cells, it is clear that there are multiple photoreceptor synapses. For example, in photoreceptor to horizontal and OFF-bipolar cell transmission the polarity of the potential changes is the same, i.e. the synapse is sign-conserving. In photoreceptor to ON-bipolar cell transmission, the synapse is sign-inverting. The evidence is very strong that sign inversion results from a metabotropic receptor (mGluR6; Nakajima et al., 1993) on the ON-bipolar cell. Its activation by glutamate leads to a second messenger cascade whose details are still not entirely worked out, but whose end result is to decrease a cyclic nucleotide-dependent cation current

151 (Nawy and Jahr, 1991). Thus when light hyperpolarizes the photoreceptor, its release of glutamate is reduced, resulting in an opening of cation channels in the ON-bipolar cell and a resulting depolarization. As a beginning step towards understanding the kinetic properties of synaptic transfer from photoreceptors to second-order retinal neurons, the present authors have developed a quantitative model for transmission at a sign-conserving synapse, i.e. through ionotropic gluRs between rods or cones and horizontal or OFF-bipolar neurons. Although, as discussed above, the glutamate receptors of horizontal vs. OFF-bipolar cells may be different, a fundamental similarity is that glutamate opens a nonspecific cation channel, resulting in depolarization of the post-synaptic neuron. Our model has a completely physiological focus in that its components and their behavior have been described in the literature. The point of generating a model is to see whether the interactions of its components that we postulate provide a realistic description of the kinetics of postsynaptic responses. When that is accomplished, the consequences of changing the parameters of model components or of introducing new variables can be evaluated. At sign-conserving photoreceptor synapses, the sequence of events resulting in synaptic transmission is: (a) Light activates a transduction cascade in the photoreceptor outer segment resulting in closure of some of its cGMP-dependent channels and a resulting hyperpolarization. Other intrinsic channels help to shape the complex waveform of the photoreceptor's light-evoked voltage. (b) The photoreceptor's voltage controls Ca influx through L-type channels. The relation between voltage and Ca influx is given by a Boltzmann relation with appropriate slope factor and voltage of half-activation. (c) Ca influx determines the rate of glutamate release according to a relation described in the next section. (d) Glutamate diffuses to its post-synaptic targets, but its concentration in the synaptic cleft is lowered by transporters located in both neurons and glia. (e) Glutamate activates post-synaptic receptors to initiate the conductance changes that mediate

post-synaptic currents. The relation between [glutamate] and conductance change is determined by a Michaelis-Menten function with a Hill coefficient near 2. The following section provides a quantitative description of (b) (e) above, using data of Corey et al., 1984; Rieke and Schwartz, 1996; Schmitz and Witkovsky, 1996, 1997; Witkovsky et al., 1997 and Thoreson et al., work in progress. Although other workers have dealt with elements of the model outlined above (Attwell et al., 1987; Wu, 1988; Belgum and Copenhagen, 1988), those previous studies did not attempt to account for the kinetics of post-synaptic responses and/or they dealt only with rod transmission.

Glutamate release A preparation consisting of an isolated layer of photoreceptors developed by Cahill and Besharse (1992) to study circadian rhythms was adapted by Schmitz and Witkovsky (1996) to study glutamate release. The method utilized an enzyme cascade (Fosse et al., 1986) terminating in bacterial luciferase by which photons were generated in proportion to glutamate degradation. The main findings were that glutamate release was higher in darkness than in light by a factor of when glutamate uptake was inhibited with dihydrokainate. The process was calciumdependent and was inhibited by blockers of L-type Ca channels, but not blockers of N- or P-type calcium channels (Schmitz and Witkovsky, 1997). Spectral sensitivity measures showed that only glutamate release from rods was being monitored. Light steps of graded intensity induced steady-state plateau voltages in rods and a progressive attenuation of glutamate release. When [ight was factored out and glutamate release plotted against rod plateau voltage, the data were well fit by a Boltzmann function for the L-Ca current of amphibian rods (Corey et al., 1984 and cf. Fig. 1).

Relation of Ca influx to glutamate release The goodness of fit illustrated in Fig. 1 implies that glutamate release is a linear function of Ca current.

152

3.5 (D

3 ~O

2.5 a

'-~ 1.5 -60

j -55

-50

-45

--40

membrane potential (mV) Fig. 1. Relation of rod voltage and glutamate release to calcium current. The data points ±SEM were obtained from plots of rod voltage vs. intensity and glutamate release vs. intensity, then factoring out intensity to yield rod voltage vs. glutamate release (cf. Witkovsky et al., 1997 for more details). The line through the points is the Boltzmann function for the L-type Ca current, with values for half-saturation and slope factor taken from Corey et al. (1984). Reproduced from Witkovsky et al., 1997 with permission of the J. Neuroscience.

Similarly, Rieke and Schwartz (1996) determined for salamander rod terminals that changes in capacitance, which are a measure o f net [ e x o c y t o s i ~ endocytosis] changed in a linear manner with [Ca]i, which was monitored with a fluorescent calcium indicator.

Input-output relation between photoreceptors and second-order neurons We established conditions that permitted examination o f pure rod or pure cone input to either horizontal or O F F - b i p o l a r cells. F o r rod input, animals were fully d a r k - a d a p t e d and the retinal slices prepared under infra-red illumination. A D2 dopamine antagonist, spiperone, was a d d e d to the bath to prevent rod~cone coupling (Krizaj et al., 1998). F o r cone input, the light-sensitivity o f rods was suppressed by a blue b a c k g r o u n d light:. The same b a c k g r o u n d illumination reduced cone sensitivity only slightly, but did make cone kinetics more transient. The light-evoked currents o f second-order neurons were recorded with perforated patch electrodes under voltage clamp. Vhold was - - 4 0 mV, i.e. close to the membrane potential o f the neuron in darkness.

Fig. 2. Intracellular dual recordings from a rod-horizontal cell pair under scotopic conditions. The upper record is the rod response. The stimulus marker below the traces indicates the timing of a 200 ms flash of 567 nm light delivering 12.3 log quanta incident cm - 2 s - 1 . The arrowheads indicate plateau responses of the rod and the horizontal cell.

Our model was developed in two stages. The first stage (Witkovsky et al., 1997) concerns the "steadystate" gain between rods and horizontal cells. That is, when a step of light is imposed, rods reach a maintained membrane potential v a l u e - - t h e plateau o f the rod's light-evoked r e s p o n s e - - a n d horizontal cells do the same (Fig. 2). The model attempts to account for the membrane potential, u, of the HC, as foUows:

u=

(GsEs + G,.Er) +

'

(1)

where Gs is the synaptic conductance (glutamategated), Es is the equilibrium potential for current through this conductance, Gr is the combined conductance o f other currents (mainly K), and Er is the net reversal potential of those currents. The post-synaptic conductance G~, is defined by a Hill function: [glutamate] ~ G~ = G~m~x K~[ut + [glutamate]n,

(2)

where n is close to 2 and Kglut is the concentration required to half-saturate the receptor population. G l u t a m a t e release from the photoreceptor gives rise to a concentration o f glutamate in the synaptic

153 cleft, governed by [glutamate] = c~r(v),

(3)

where c~ is a constant, r is the rate of release and v is the rod potential. The rate of release of synaptic transmitter is given by

r(v) =

C[,~ 1 + exp

+ r0

(4)

where C is a constant and r0 is the baseline release rate. A and B are, respectively, the voltage for half activation of the Ca current and the slope factor in the Boltzmann relation. It follows that our model takes glutamate release to be a linear function of [Ca]i The justification for this assumption is that, in our study (Witkovsky et al., 1997) we found that the relation between rod voltage and glutamate release was matched perfectly by the activation function for the L-type Ca current of the rod (Fig. 1). If we define a normalized glutamate conductance, g,= Gs/Gr, we can reconfigure Eq. (1) above as: u - (gsEr + er) ( g s + 1)

(5)

This equation provides a very good fit to the data points on a plot of rod voltage vs. horizontal cell voltage (Fig. 3). -4(

,

-4~t

0

o_50 O

-r"

-55

-61~

o o

-55

-50 rod voltage

-45

= g(v)

-

y,

(6)

where v = p h o t o r e c e p t o r voltage, and the function g(v) is the fractional activation of the voltage-gated Ca current, normalized by its value in darkness. A Boltzmann relation governs g(v)= (l+exp[-A--~darkl)

( 1 + e x p [@~1) -1

(7)

We define z = [ g l u t ] / [ g l U t ] d a r k , the normalized glutamate concentration in the synaptic cleft. It increases in direct proportion to [Ca 2+ ] (the term, y, in Eq. 7) and decreases at a rate determined by diffusion and activate re-uptake. If we ignore the voltagedependence of re-uptake and make the simplifying assumption that the rate of glutamate removal is proportional to [Glut], then we have:

o o

>

dy 3-Ca d t

x

,

t33

In the second stage of the model we extend the above formulation to the generation of post-synaptic currents. We chose to look at post-synaptic currents rather than voltages to avoid the complications of the voltage-dependent conductances present in secondorder retinal neurons which contribute to the different kinetics of the fight-evoked responses of photoreceptors compared to those of horizontal and bipolar cells (cf. Fig. 2). For this determination we need to consider the dynamics of calcium change, since that governs the rate of glutamate release. We define y = [Ca2+]/[Ca2+]dark, the normalized free calcium concentration at any point in the cytoplasm. The calcium concentration reflects a balance between calcium influx through voltagegated Ca channels and efflux which is governed by a Ca transporter (exchange pump). In Eq. (6) below it is assumed that the rate of the transporter is proportional to the free calcium concentration.

dz

-40

Fig. 3. Steady state model of rod vs. horizontal cell lightinduced potential changes. The data points are taken from simultaneous intracellular recordings of rods and horizontal cells. The line through the data is the best fit of Eq. (5) in the text. Reproduced from Witkovsky et al., 1997 with permission of the J. Neuroscience.

glut

27 =

y

-

z

(8)

The term, z, determines the fraction of bound glutamate receptors, f, according to: Zn

f -- -

-

Z n Jr-

kglut

(9)

154 In this equation kglut is the [glutamate] sufficient to bind half the receptors, and n is the Hill coefficient for cooperativity, whose value is near 2. The glutamate conductance, G, is proportional to the fraction of bound receptors, f Thus we have:

(Gaal et al., 1998) will affect the fit of the model to data. Our hope and expectation is that our model will contribute to understanding how photoreceptors faithfully convey information about light stimuli to the retinal network.

Z// = amax Zn +

kglut

-- Gmax f .

~,

(lO)

To this point, receptor desensitization has not been taken into account. Since we find that including desensitization does not improve the fit of the model to data, it is omitted here. The fits to the data were obtained for matched sets of photoreceptor voltages and second-order cell currents generated by a series of 1 s light flashes of different intensities. Equations (6) and (8) were integrated; any choice of free parameters in those equations gave rise to a family of postsynaptic conductance responses. The values of the free parameters were varied and the best-fit values determined by a least squares method, using an iterative computer program. Figure 4 shows how the model matches data from four photoreceptor synapses: rod to HC, rod to OFF-BC, cone to HC, and cone to OFF BC. The essential finding was that for the four pairs (rod to HC; cone to HC; rod to OFF BC; cone to OFF BC), the relation between peak light-evoked current in the second-order neuron to peak photoreceptor voltage was fit by a Michaelis-Menten function with a slope very close to 1.0. This fixed relation implies a constancy of synaptic transfer over a wide range of illumination levels. Accordingly, we take the photoreceptor voltage change to be the controlling element for a change in calcium current at the photoreceptor synaptic terminal. The strength of the model is that, with relatively simple assumptions and few free parameters, it captures the essence of post-synaptic kinetics for both rod and cone synaptic transfers mediated through ionotropic glutamate receptors. Another strength is that the components of the model are physiological in nature and accessible to experimentation. Undoubtedly the model is incomplete in the sense that additional components need to be included to achieve a more perfect fit of predicted and real responses. For example, we are currently exploring how making glutamate transport voltage-dependent

Conclusions and future perspectives The retina arises embryologically from the central nervous system and its cells share many of the properties of neurons in the brain. Yet the retina has a unique role in providing information to the brain about a complex visual world, and certain of its special features have evolved in relation to that task. One of the most compelling is its ceaseless activity: even in complete darkness photoreceptors drip glutamate at a relatively brisk rate, while many ganglion cells fire spikes at a more or less steady pace. Moreover, there can be no "timeout" while the retina reorganizes itself for daytime or nighttime vision. Instead an interconnected set of neuromodulatory mechanisms accomplishes this task on the run. In this context the photoreceptor and ON-bipolar cell output synapses have become model systems for investigating the properties of tonic neurotransmission. At no other known synapses do noninactivating, high voltage-activated (HVA) calcium channels play such a dominant role in triggering vesicle release. The HVA calcium currents are subject to multiple controls, including direct neuromodulation of the channels by, e.g. neuropeptides (Akopian et al., 2000), dopamine (Stella and Thoreson, 2000) and by nitric oxide (Kurenny et al., 1994). Many other elements contribute to the regulation of [Ca]i, including intracellular stores (Krizaj et al., 1999), calcium pumps and transporters (Krizaj and Copenhagen, 1998; Morgans et al., 1998) and calcium-dependent currents which contribute to the regulation of membrane voltage and so have an impact on the calcium current (Maricq and Korenbrot, 1988). The link between Ca entry and exocytosis in photoreceptors and ON-bipolar cells also is poorly understood from a molecular biological perspective. In conventional presynaptic terminals a host of intracellular proteins organize vesicle transport, docking, priming and release (Chapman et al., 1995). There is some indication that photoreceptors may

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Fig. 4. Dynamic model of photoreceptor to second-order retinal neuron synaptic transfer. Each set of two panels shows light-evoked photoreceptor responses to a set of ls stimuli o f different intensity in the upper panel, whose light-evoked voltages are the input to the model. Stimuli were 580 nm for rods (panels A, B) and 680 nm for cones (C, D). The lower panel illustrates the best fit of the model to photocurrents recorded from horizontal cells (A, C) and OFF-bipolar cells (B, D). All recordings were made using the perforated patch technique. Horizontal and bipolar cells were held at - 40 inV. See text for presentation of the equations underlying the model.

156 lack certain of these proteins (Mandell et al., 1990) and in any case we lack basic information about their function. In relation to the apparent linear relation between calcium current and release (cf. Fig. 1) we need to know the properties of the calcium sensor. Is it synaptotagmin, which is found at photoreceptor ribbon synapses (Koontz and Hendrickson, 1993) as has been proposed for spiking synapses (Augustine et al., 1994)? Synaptotaginin is thought to require 4 C a 2+ to initiate vesicle exocytosis: were this the case for the photoreceptor, then the linear calcium-release relation probably would reflect the independence of calcium microdomains (Augustine et al., 1991). On the post-synaptic side several crucial question have emerged recently: (1) to what extent does the glutamate transporter control [glutamate] in the synaptic cleft? (2) what is the true extracellular concentration of glutamate and does it cause desensitization of post-synaptic ionotropic glutamate receptors? (3) what is the subunit composition of igluRs in retinal cells? How many different igluRs reside in a given post-synaptic cell and is their relative number fixed or subject to regulation, as has been shown for hippocampal neurons (Hayashi et al., 2000)? Our intuition, based on a survey of the literature, is that every aspect of synaptic communication in the retina is subject to modulation. Given that the retina has to transmit a coherent statement to the brain about the visual world, either in dim light or in bright light, it is unthinkable that each retinal synapse be subject to modulation without some overarching control, i.e. we suppose that a hierarchical framework of neuromodulatory mechanisms exists. In that context, a primary task for future studies is to examine the interrelations among the more than fifty putative neuromodulators which have been identified in retinal neurons.

A personal note One of the authors (P.W.) takes this opportunity to pay a personal tribute to John Dowling. Long ago, as a recent PhD and having just finished a post-doctoral year with Gunnar Svaetichin in Caracas, Venezuela, I met John Dowling for the first time at Johns Hopkins University. Together with Brian Boycott he was working out the central principles of the retinal

wiring diagram, based on a combination of light and electron microscopy. The retina they studied in greatest detail was that of the primate, but at the time (mid 1960s) the mammalian eye seemed largely inaccessible to microelectrode recording, whereas the fish retina that I and others worked on was more suitable for functional anatomy, i.e. drawing inferences about the responses of individual retinal cells from their synaptic connections. John, who has always shown remarkable prescience about which were the important new directions of research, suggested that I come to Baltimore to study the synaptic organization of the carp retina. Naively, I imagined that I would carry out neurophysiological studies, while John would do the electron microscopy. But he had another idea: I should learn electron microscopy and related techniques and do the work myself, under his guidance. So with his considerable help, both on questions of technique and of interpretation, I was introduced to the field of retinal synaptic connectivity. We published one paper together in 1969 on the synaptic organization of the carp retina which was well received, and the orientation I received from that brief period in his laboratory has influenced my way of looking at the nervous system ever since. In my view, the papers of John Dowling on the synaptic organization of the retina have played a seminal role in guiding our interpretations of retinal function. Many other scientists also contributed importantly to our understanding of retinal synapses, among them Helga Kolb, who was in the DoMing laboratory during the period I worked there and Brian Boycott, who was a frequent visitor. Still others, who have gone on to become the leaders in our field, were his students (e.g. Frank Werblin) and collaborators at this same time. John Dowling's ability to attract people to his laboratory and his charismatic persuasiveness as a teacher are two more of his many talents. It is a pleasure to acknowledge John Dowling's pivotal role in bringing retinal neurobiology to the forefront of modern investigations of nervous system function.

Acknowledgments Supported by the National Eye Institute (EY 03570 and EY 10542), Research to Prevent Blindness, Inc.,

157 the N e b r a s k a

Lions Foundation

and the Hoffritz

Foundation.

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