299
Brain Research, 584 (1992) 299-304 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25248
The spatial relationship between MOiler cell processes and the photoreceptor output synapse Monique Sarantis and Peter Mobbs Department of Physiology, University College London, London (UK) (Accepted 31 March 1992)
Key words: Photoreceptor; Ribbon synapse; Miiller cell; Electron microscopy; Horseradish peroxidase; Glutamate; Uptake
Glutamate is the neurotransmitter released by photoreceptors in the retina. The postsynaptic action of glutamate is terminated partly by uptake into glial (Miiller) cells. The anatomical distribution of Miiller cell processes around the synaptic terminals of photoreceptors was investigated electron microscopically in the tiger salamander retina. Miiller cells wrap around the synaptic terminals of both rods and cones and come within 1-3/~m of the sites of glutamate release, close enough to contribute to terminating the synaptic action of glutamate.
Glutamate is a major neurotransmitter in the brain and is thought to be the transmitter released from photoreceptors 3'4'17. Since there are no extracellular enzymes present to inactivate it, the action of glutamate must be terminated either by diffusion or by glutamate being taken up into cells. Even if diffusion of glutamate is initially the predominant mechanism, this is only made possible by glutamate uptake maintaining a low extracellular concentration around the synapse. Despite the importance of uptake for terminating glutamate's synaptic action, there have been no studies of the spatial localization of the uptake process near glutamate-releasing synapses. Studies of the uptake of radiolabelled glutamate in the retina have shown that it is taken up mainly into Miiller cells9A3,24, but also into photoreceptors 5,16. Miiller cells of the salamander 6 and rabbit retina 2° possess a .high-affinity electrogenic uptake mechanism for glutamate. It has been calculated, assuming a spatially uniform extracellular glutamate concentration, that the electrogenic uptake mechanism in salamander Miiller cells can lower the concentration of glutamate in the extracellular space with a time constant of 32 ms t. However, in reality the extracellular glutamate concentration will not be spatially uniform, and it is not known how close MiJller cell processes, that pre-
sumably contain the uptake carriers, come to the sites of glutamate release at the photoreceptor output synapse. Consequently, it is difficult to assess the importance of glutamate uptake by Miiller cells for terminating synaptic action. Copenhagen et al. 7 have calculated (by deconvolving the pre- and postsynaptic light responses to dim flashes) the impulse response function (the calculated form of the voltage response in the postsynaptic horizontal cell for a brief change in voltage in the photoreceptor 22) for transmission from rods and cones. In the turtle retina the decaying phase of this function, which is probably determined by the rate of removal of glutamate from the extracellular space, is 7 times slower for transmission from rods than for transmission from cones to horizontal cells7. This could, in principle, reflect slower removal of glutamate from the rod output synapse, due to a greater diffusion distance to nearby Miiller cell processes. To provide further information on the role of Miiller cells in terminating transmitter action at the photoreceptor output synapse, we have studied the anatomical arrangement of Miiller cell processes near the photoreceptor ribbon synapse, in the outer plexiform layer of the salamander retina. Ribbon synapses are the sites of chemical synaptic contact between photoreceptors and
Correspondence: M. Sarantis, Department of Physiology, University College London, Gower Street, London, WC1E 6BT, London, UK.
300 postsynaptic horizontal and bipolar ceils 15. They offer the methodological advantage that their precise site of glutamate release is easily visible in the electron microscope (Fig. 1B). Salamanders (Ambystoma tigrinum) were decapitated and their eyes removed. The retina was removed and cut into quarters u n d e r Ringer's solution containing (in raM) NaCl 104, KC1 2.5, CaC12 3, MgCI 2 0.5, glucose 15, H E P E S 5, N a O H 3 (pH 7.3). The pieces of retina were stuck, vitreal surface uppermost, over a window of coverslip-glass inserted into holes cut in a Millipore filter TM. For intracellular recording and injection, microelectrodes (resistance = 200 M J2 in Ringer's) were filled with 1 ~1 5% H R P (horseradish peroxidase, Sigma P-8375) in 0.1 M T r i s / 0 . 2 M KC1 at p H 7.6 (ref. 23), and backfilled with 3 M potassium acetate. Miiller cells were impaled with electrodes from the retina's vitreal surface TM. The identity of cells was confirmed by the m o r e negative resting potential found in Miiller cells (between - 8 0 and - 9 0 mV), than in retinal neurons. H R P was injected into the cell by passing 1 - 2 n A depolarizing current pulses (50% duty cycle) for 1-3 min. T h e H R P was later found to be distributed t h r o u g h o u t the whole of the Miiller cell cytoplasm (as observed in the electron microscope), including the processes near the photoreceptor output synapse (100 ~ m away through the retina from the site of H R P injection). Following injection, the recording c h a m b e r was flooded with primary fixative (1% freshly p r e p a r e d p a r a f o r m a l d e h y d e and 2.5% glutaraldehyde with 3% sucrose in 0.066 M sodium phosphate (S6rensen) buffer at p H 7.4). Tissue was fixed at r o o m t e m p e r a t u r e for 30 min followed by 85 min at 5°C. Tissue which was only going to be examined in the light microscope was fixed in buffered 2.5% glutaraldehyde (Fig. 1A). Tissue which did not contain an H R P - i n j e c t e d cell but which was processed for electron microscopy was fixed in 2.5% g l u t a r a l d e h y d e / l % O s O 4 (ref. 12) in buffer (Fig. 1B). H R P was visualized using a histochemical procedure using H a n k e r - Y a t e s reagent ( p - p h e n y l e n e d i a mine-pyrocatechol (PPD-PC)) 11 in cacodylate buffer. T h e tissue was then processed for examination in the electron microscope by post-fixation in 1% O s O 4, dehydration in graded ethanol solutions and embedding in Epon. E p o n blocks were cut into 30-txm sections and the sections that contained HRP-filled Miiller cells
A
IPL["
GCLE
Fig. 1. A: light micrograph of a 50-tzm transverse section through a tiger salamander retina containing an HRP-injected Miiller cell. The retinal layers are indicated: ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL) and the outer nuclear layer (ONL). Miiller cell processes project out laterally from the stalk into the IPL and project around the cell bodies in the INL and ONL. B: electron micrograph of a rod synaptic pedicle (rp). The pedicle contains several ribbon synapses each surrounded by an array of synaptic vesicles (arrows). Arrow head indicates the arciform density. Postsynaptic processes from horizontal and bipolar cells invaginate the synaptic pedicle of the photoreceptor in the outer plexiform layer (OPL) resulting in a 'dyadic' (d) or 'triadic' (t) arrangement, where a synaptic ribbon releases transmitter to two or three postsynaptic processes (Lasansky, 1973). A cone cell body (cn) and pedicle (cp) are indicated.
were r e e m b e d d e d . T h e HRP-filled Miiller cells were serially sectioned onto slot grids that were coated with formvar and evaporated carbon. Sections were stained with uranyl acetate and Reynolds lead stain.
Fig. 2. Miiller cell processes in the outer plexiform layer. A: cone nucleus (cn) with synaptic pedicle (cp) containing ribbon synapses (arrows). HRP-filled Miiller cell process is indicated by Mc. The cone cell body borders the outer plexiform layer (OPL). B: rod nucleus (rn) with a synaptic pedicle (rp) connected to the cell body via an axon (arrows). HRP-filled Miiller cell processes (Mc) surround the cell. Arrowhead indicates a Miiller cell process invaginating the rod synaptic pedicle. C: section through a rod pedicle (rp) and a cone cell body (cn, top right), arrowheads indicate the membranes that separate them. HRP-filled Miiller cell process (Mc) wraps around the pedicle, but unusually a Miiller cell process also projects out into the outer plexiform layer (OPL) close to a rod ribbon synapse (open arrow).
301
302 Fig. 1A is a light micrograph of a Miiller cell injected with H R P in a transverse section of the retina. Miiller cell processes extend laterally from the main stalk of the cell into the inner plexiform layer (IPL) and around the cell bodies in the inner (INL) and outer nuclear layers (ONL). A single Miiller cell was found to send out processes to partly surround approximately eight photoreceptor cell bodies in the ONL. Chemical synapses between photoreceptors and postsynaptic cells are marked by the presence of synaptic ribbons in the rod and cone pedicles 2J, which generally release transmitter to two or three nearby postsynaptic cells. Although synaptic contacts without synaptic ribbons exist, the chemical nature of these contacts is not certain ]5. In this study we used the synaptic ribbons to define sites of glutamate release, and investigated how close Miiller cell processes come to these sites. Fig. 1B shows several ribbon synapses in a synaptic pedicle (terminal), i.e., the output synapse, of a rod photoreceptor. The cell bodies of the two types of photoreceptors can be distinguished by the position of their nuclei (labelled cn (cone nucleus) and rn (rod nucleus), respectively, in the figures of this paper). The rod cell nuclei are placed more externally (towards the pigment epithelium) while the cone nuclei are located nearer the synaptic region 15. As a result, cone cell bodies are continuous with their pedicles (cp) (Fig. 2A) while the rod cell bodies are linked to their pedicles (rp) by means of an axon (arrows, Fig. 2B).
The electron-dense H R P reaction product can be readily identified in injected Miiller cells (Mc in Figs. 2 and 3). Serial sectioning of two HRP-injected Miiller cells revealed that a single Miiller cell sends out large processes, that fill the space between at least six photoreceptor cell bodies. These processes also project to several more photoreceptors but surround them only in part. Although Miiller cells surround much of the photoreceptor cell m e m b r a n e (Fig. 2B) and also invade the pedicles (Fig. 2B, arrowhead), they seldom project into the outer plexiform layer near the ribbon synapses (Figs. 2A and 3). Fourteen pedicles of rod and cone photoreceptors approached by HRP-injected Mfiller cell processes were sectioned and examined. On only two occasions were minor Miiller cell processes found to be present in the outer plexiform layer close to synaptic ribbons. An example of this is seen in Fig. 2C in which the arrow points to a MiJller cell process in the outer plexiform layer close to a ribbon synapse in a rod pedicle. Further sections through the same pedicle show the presence of many more ribbon synapses (Fig. 3, arrows), but no MiJller cell processes, other than that shown in Fig. 2C, were observed to project into the region postsynaptic to these ribbons in the outer plexiform layer. Fig. 3 illustrates a common arrangement in which up to four rod pedicles cluster around a cone pedicle. In this case two rod pedicles (rp) abut a cone pedicle (cp) (arrowheads indicate the cell m e m b r a n e s that separate
Fig. 3. Clustered photoreceptor synaptic pedicles. Section through a cone cell body (cn) with a synaptic pedicle (cp), which is surrounded by two rod pedicles (rp) (arrowheads indicate the membranes that separate them). The rod pedicle containing synaptic ribbons (arrows) is a section through the same pedicle as shown in Fig. 2C. In contrast to that figure, no MiJller cell processes are present in the outer plexiform layer (OPL). HRP-filled processes (Mc) are seen wrapping around the cone cell body and the group of pedicles without projecting into the cluster.
303 them). This reflects the arrangement of the photoreceptors in a roughly orthogonal lattice in which four rods surround a cone 2. W h e n the pedicles are clustered like this, Miiller cell processes wrap around the whole group of pedicles without projecting between them or into the outer plexiform layer. The closest approach of Miiller cells to ribbon synapses was measured, for each of the pedicles sectioned, as the distance between the end of the arciform density of the synaptic ribbon (Fig. 1B, arrowhead) nearest to a HRP-filled process and that process. The nearest Miiller cell process lay in the outer plexiform layer for two pedicles, including that of Figure 2C, but more usually occurred to the side of the pedicle (as for the pedicles in Figs. 2A and 3). W h e n this distance was measured as a straight line (an underestimate of the true diffusion distance), the resulting measurements were 1.1 + 0.2 /zm (S.E.M.) for the six rod pedicles, 3.0 + 1.7/xm for the four cone pedicles, and 0.9 + 0.3 /xm for four pedicles that could not be identified as belonging to a rod or a cone. When the diffusion distance from Miiller cell process to the nearest ribbon was measured along the visible extracellular pathway in the plane of section (which probably overestimates the distance glutamate has to diffuse), the resulting measurements were 3.1 + 0.7 ~ m for rod pedicles, 5.8 + 1.5 ~zm for cone pedicles and 1.4 + 0.5 ~ m for unidentified pedicles. The greatest distance that glutamate, released from a photoreceptor, would have to diffuse to a Miiller cell process, would be from a ribbon synapse in the middle of a cone pedicle that is surrounded by several rod pedicles. From our data we estimate that distance to be around 10 tzm. Shrinkage of the tissue during processing was not taken into account in calculating these distances, but is believed to be less than 5% (ref. 10). This study is the first investigation of the spatial relationship between sites of glutamate uptake in glial cells and sites of transmitter release at glutamatergic synapses. Fig. 4 summarizes the results, showing the relationship between a Miiller cell (shaded) and rod and cone photoreceptors and their associated synaptic pedicles. Miiller cell processes surround photoreceptor cell bodies and their synaptic pedicles. However, MiJller cell processes are rarely present in the outer plexiform layer on the side of the pedicles where the synaptic ribbons are, and on the rare occasions where they are present there, they do not invade the spaces between horizontal, bipolar and photoreceptor ceils near the synaptic cleft. For transmission from photoreceptors to horizontal cells in the turtle retina, the time course of the decaying phase of the synaptic impulse response function
photoreceplor outer end Inrler segments
photoreceptor cell b o d l u ONL syneptl© pedlclell wllh ribbon synaINel
procemms of bipolar and horizontal cells
OPL
INL
i ! i
~'~
MQIler cell etalk
Fig. 4. Schematic diagram of Miiller cell processes in the outer layers of the salamander retina. The shaded areas represent HRP-filled Miiller cell processes. Miiller cell processes (shaded) project out from the main stalk and wrap around cell bodies and synaptic pedicles of photoreceptors in the outer nuclear layer (ONL). Only occasionally does a Miiller cell process project out into the outer plexiform layer (OPL) near the synaptic region of the photoreceptor output synapse. Miiller cell processes wrap around cell bodies in the inner nuclear layer (INL). The stalk of the Miiller cell extends to the inner limiting membrane ending in an endfoot in the ganglion cell layer (not shown).
(which probably reflects the time course of glutamate removal from the extracellular space) is 30 ms for glutamate released from cones and 200 ms for glutamate released from rods 7. Similar data are not available for the salamander retina. However, as described in this paper, in the salamander retina there is a difference in shape between rod and cone pedicles (as has also been reported in the turtle retina 14) with the base of cone pedicles, where the ribbon synapses are situated, being much wider than that of rod pedicles. This results in the ribbon synapses in rod pedicles being closer than those in the cone output synapses to processes of Miiller cells. The clustering of rod and cone pedicles seen in the salamander retina, where the rod terminals abut the cone terminals and the Miiller cells wrap around the outside of the group of terminals, makes the average distance between cone ribbon synapses and glutamate uptake sites even longer. Therefore, one would not expect the decaying phase of the impulse response function for rod-horizontal cell transmission to be slower than that for cone-horizontal cell transmission as a result of the time taken for glutamate to diffuse to a Miiller cell. An estimate of the distance, x, glutamate can diffuse in a time, t, is given by the equation8: t = lx2/D, in which D is the diffusion coefficient (about 10 -9 m2/s). This equation suggests it will take 0.6 ms to diffuse 1.1 Izm and 4.5 ms to diffuse 3.0/zm, the direct
304 distances measured for the closest approach of a MiJller cell process to rod and cone pedicles, respectively. However, the actual diffusion distance through the extracellular space is longer due to it being via a tortuous path (see above). Nicholson and Phillips ~9 calculate that, in the rat cerebellum, this tortuosity reduces the effective diffusion coefficient by a factor of 2.4, giving diffusion times of 1.5 ms and 10.8 ms for the direct measured distances given above. These times are both shorter than the decay times of the impulse response functions for transmission from rods and cones to horizontal cells in the turtle retina 7. Thus, although Mfiller cell processes do not come very close to the postsynaptic sites at the photoreceptor output synapse, they apparently come close enough to play a role in terminating the effects of glutamate released from both rod and cone synapses. This work was supported by the Wellcome Trust, the MRC and the Wolfson Foundation. We thank David Attwell for helpful advice. 1 Attwell, D., The photoreceptor output synapse, Progr. Retinal Res., 9 (1990) 337-362. 2 Attwell, D., Wilson, M. and Wu, S.M., A quantitative analysis of interactions between photoreceptors in the salamander (Ambystoma) retina, J. PhysioL, 352 (1984) 703-737. 3 Attwell, D., Mobbs, P., Tessier-Lavigne, M. and Wilson, M., Neurotransmitter-induced currents in retinal bipolar cells of the axolotl, Ambystoma mexicanum, J. Physiol., 387 (1987) 125-161. 4 Ayoub, G.S., Korenbrot, J.I. and Copenhagen, D.R., Release of endogenous glutamate from isolated cone photoreceptors of the lizard, Neurosci. Res. Suppl., 10 (1989) 547-556. 5 Brandon, C. and Lain, D.M.K., L-Glutamic acid: a neurotransmitter candidate for cone photoreceptors in human and rat retinas, Proc. Natl. Acad. Sci. USA, 80 (1983) 5117-5121. 6 Brew, H. and Attwell, D., Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells, Nature, 27 (1987) 707-709. 7 Copenhagen, D.R., Ashmore, J.F. and Schnapf, J.K., Kinetics of synaptic transmission from photoreceptors to horizontal and bipolar cells in turtle retina, Vision Res., 23 (1983) 363-369.
8 Crank, J., The Mathematics of Diffusion, 2nd edn., Clarendon Press, Oxford, 1976, 32 pp. 9 Ehinger, B., Glial and neuronal uptake of GABA, glutamic acid. glutamine and glutathione in the rabbit retina, Exp. Eve Res.. 25 (1977) 221-234. 10 Glauert, A.M., Fixation, Dehydration and Embedding of Biological Specimens, Elsevier, Amsterdam, 1975, 3 pp. 11 Hanker, J.S., Yates, P.E., Metz, C.B. and Rustioni, A., A new specific, sensitive and non-carcinogenic reagent for the demonstration of horseradish peroxidase, Histochem. Z, 9 (1977) 789792. 12 Hirsch, J.G. and Fedorko, M.E., Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and 'postfixation' in uranyl acetate, Z Cell Biol., 38 (1968) 615-627. 13 Kennedy, A.J., Voaden, M.J. and Marshall, J., Glutamate metabolism in the frog retina, Nature, 252 (1974) 50-51. 14 Lasansky, A., Synaptic organization of cone cells in the turtle retina, Phil. Trans. R. Soc. B, 262 (1971) 365-381. 15 Lasansky, A., Organization of the outer synaptic layer in the retina of the larval tiger salamander, Proc. R. Soc. Lond., 265 (1973) 471-489. 16 Marc, R.E. and Lain, D.M.K., Uptake of aspartic and glutamic acid by photoreceptors in goldfish retina, Proc. Natl. Acad. Sci., 78 (1981) 7185-7189. 17 Miller, A.M. and Schwartz, E.A., Evidence for the identification of synaptic transmitters released by photoreceptors in the toad retina, J. PhysioL, 334 (1983) 325-349. 18 Mobbs, P., Brew, H. and Attwell, D., A quantitative analysis of glial cell coupling in the retina of the axolotl (Ambystoma mexicanum), Brain Res., 460 (1988) 235-245. 19 Nicholson, C. and Phillips, J.M., Ion diffusion modified by tortuosity and volume fraction in the extracellular microenvironment of the rat cerebellum, J. Physiol., 321 (1981) 225-257. 20 Sarantis, M. and Attwell, D., Glutamate uptake in mammalian retinal glia is voltage- and potassium-dependent, Brain Res., 516 (1990) 322-325. 21 Sj6strand, F.S., Ultrastructure of retinal rod synapses of the guinea pig eye as revealed by three-dimensional reconstruction from serial sections, J. Ultrastruct. Res., 2 (1958) 122-170. 22 Schnapf, J.L. and Copenhagen, D.R., Differences in the kinetics of rod and cone synaptic transmission, Nature, 296 (1982) 862864. 23 Snow, P.J., Rose, P.K. and Brown, A.G., Tracing axons and axon collaterals of spinal neurons using intracellular injection of horseradish peroxidase, Science, 191 (1976) 312-313. 24 White, R.D. and Neal, M.J., The uptake of L-glutamate by the retina, Brain Res., 111 (1976) 79-93.
Brain Research, 584 (1992) 299-304 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25248
The spatial relationship between MOiler cell processes and the photoreceptor output synapse Monique Sarantis and Peter Mobbs Department of Physiology, University College London, London (UK) (Accepted 31 March 1992)
Key words: Photoreceptor; Ribbon synapse; Miiller cell; Electron microscopy; Horseradish peroxidase; Glutamate; Uptake
Glutamate is the neurotransmitter released by photoreceptors in the retina. The postsynaptic action of glutamate is terminated partly by uptake into glial (Miiller) cells. The anatomical distribution of Miiller cell processes around the synaptic terminals of photoreceptors was investigated electron microscopically in the tiger salamander retina. Miiller cells wrap around the synaptic terminals of both rods and cones and come within 1-3/~m of the sites of glutamate release, close enough to contribute to terminating the synaptic action of glutamate.
Glutamate is a major neurotransmitter in the brain and is thought to be the transmitter released from photoreceptors 3'4'17. Since there are no extracellular enzymes present to inactivate it, the action of glutamate must be terminated either by diffusion or by glutamate being taken up into cells. Even if diffusion of glutamate is initially the predominant mechanism, this is only made possible by glutamate uptake maintaining a low extracellular concentration around the synapse. Despite the importance of uptake for terminating glutamate's synaptic action, there have been no studies of the spatial localization of the uptake process near glutamate-releasing synapses. Studies of the uptake of radiolabelled glutamate in the retina have shown that it is taken up mainly into Miiller cells9A3,24, but also into photoreceptors 5,16. Miiller cells of the salamander 6 and rabbit retina 2° possess a .high-affinity electrogenic uptake mechanism for glutamate. It has been calculated, assuming a spatially uniform extracellular glutamate concentration, that the electrogenic uptake mechanism in salamander Miiller cells can lower the concentration of glutamate in the extracellular space with a time constant of 32 ms t. However, in reality the extracellular glutamate concentration will not be spatially uniform, and it is not known how close MiJller cell processes, that pre-
sumably contain the uptake carriers, come to the sites of glutamate release at the photoreceptor output synapse. Consequently, it is difficult to assess the importance of glutamate uptake by Miiller cells for terminating synaptic action. Copenhagen et al. 7 have calculated (by deconvolving the pre- and postsynaptic light responses to dim flashes) the impulse response function (the calculated form of the voltage response in the postsynaptic horizontal cell for a brief change in voltage in the photoreceptor 22) for transmission from rods and cones. In the turtle retina the decaying phase of this function, which is probably determined by the rate of removal of glutamate from the extracellular space, is 7 times slower for transmission from rods than for transmission from cones to horizontal cells7. This could, in principle, reflect slower removal of glutamate from the rod output synapse, due to a greater diffusion distance to nearby Miiller cell processes. To provide further information on the role of Miiller cells in terminating transmitter action at the photoreceptor output synapse, we have studied the anatomical arrangement of Miiller cell processes near the photoreceptor ribbon synapse, in the outer plexiform layer of the salamander retina. Ribbon synapses are the sites of chemical synaptic contact between photoreceptors and
Correspondence: M. Sarantis, Department of Physiology, University College London, Gower Street, London, WC1E 6BT, London, UK.
300 postsynaptic horizontal and bipolar ceils 15. They offer the methodological advantage that their precise site of glutamate release is easily visible in the electron microscope (Fig. 1B). Salamanders (Ambystoma tigrinum) were decapitated and their eyes removed. The retina was removed and cut into quarters u n d e r Ringer's solution containing (in raM) NaCl 104, KC1 2.5, CaC12 3, MgCI 2 0.5, glucose 15, H E P E S 5, N a O H 3 (pH 7.3). The pieces of retina were stuck, vitreal surface uppermost, over a window of coverslip-glass inserted into holes cut in a Millipore filter TM. For intracellular recording and injection, microelectrodes (resistance = 200 M J2 in Ringer's) were filled with 1 ~1 5% H R P (horseradish peroxidase, Sigma P-8375) in 0.1 M T r i s / 0 . 2 M KC1 at p H 7.6 (ref. 23), and backfilled with 3 M potassium acetate. Miiller cells were impaled with electrodes from the retina's vitreal surface TM. The identity of cells was confirmed by the m o r e negative resting potential found in Miiller cells (between - 8 0 and - 9 0 mV), than in retinal neurons. H R P was injected into the cell by passing 1 - 2 n A depolarizing current pulses (50% duty cycle) for 1-3 min. T h e H R P was later found to be distributed t h r o u g h o u t the whole of the Miiller cell cytoplasm (as observed in the electron microscope), including the processes near the photoreceptor output synapse (100 ~ m away through the retina from the site of H R P injection). Following injection, the recording c h a m b e r was flooded with primary fixative (1% freshly p r e p a r e d p a r a f o r m a l d e h y d e and 2.5% glutaraldehyde with 3% sucrose in 0.066 M sodium phosphate (S6rensen) buffer at p H 7.4). Tissue was fixed at r o o m t e m p e r a t u r e for 30 min followed by 85 min at 5°C. Tissue which was only going to be examined in the light microscope was fixed in buffered 2.5% glutaraldehyde (Fig. 1A). Tissue which did not contain an H R P - i n j e c t e d cell but which was processed for electron microscopy was fixed in 2.5% g l u t a r a l d e h y d e / l % O s O 4 (ref. 12) in buffer (Fig. 1B). H R P was visualized using a histochemical procedure using H a n k e r - Y a t e s reagent ( p - p h e n y l e n e d i a mine-pyrocatechol (PPD-PC)) 11 in cacodylate buffer. T h e tissue was then processed for examination in the electron microscope by post-fixation in 1% O s O 4, dehydration in graded ethanol solutions and embedding in Epon. E p o n blocks were cut into 30-txm sections and the sections that contained HRP-filled Miiller cells
A
IPL["
GCLE
Fig. 1. A: light micrograph of a 50-tzm transverse section through a tiger salamander retina containing an HRP-injected Miiller cell. The retinal layers are indicated: ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL) and the outer nuclear layer (ONL). Miiller cell processes project out laterally from the stalk into the IPL and project around the cell bodies in the INL and ONL. B: electron micrograph of a rod synaptic pedicle (rp). The pedicle contains several ribbon synapses each surrounded by an array of synaptic vesicles (arrows). Arrow head indicates the arciform density. Postsynaptic processes from horizontal and bipolar cells invaginate the synaptic pedicle of the photoreceptor in the outer plexiform layer (OPL) resulting in a 'dyadic' (d) or 'triadic' (t) arrangement, where a synaptic ribbon releases transmitter to two or three postsynaptic processes (Lasansky, 1973). A cone cell body (cn) and pedicle (cp) are indicated.
were r e e m b e d d e d . T h e HRP-filled Miiller cells were serially sectioned onto slot grids that were coated with formvar and evaporated carbon. Sections were stained with uranyl acetate and Reynolds lead stain.
Fig. 2. Miiller cell processes in the outer plexiform layer. A: cone nucleus (cn) with synaptic pedicle (cp) containing ribbon synapses (arrows). HRP-filled Miiller cell process is indicated by Mc. The cone cell body borders the outer plexiform layer (OPL). B: rod nucleus (rn) with a synaptic pedicle (rp) connected to the cell body via an axon (arrows). HRP-filled Miiller cell processes (Mc) surround the cell. Arrowhead indicates a Miiller cell process invaginating the rod synaptic pedicle. C: section through a rod pedicle (rp) and a cone cell body (cn, top right), arrowheads indicate the membranes that separate them. HRP-filled Miiller cell process (Mc) wraps around the pedicle, but unusually a Miiller cell process also projects out into the outer plexiform layer (OPL) close to a rod ribbon synapse (open arrow).
301
302 Fig. 1A is a light micrograph of a Miiller cell injected with H R P in a transverse section of the retina. Miiller cell processes extend laterally from the main stalk of the cell into the inner plexiform layer (IPL) and around the cell bodies in the inner (INL) and outer nuclear layers (ONL). A single Miiller cell was found to send out processes to partly surround approximately eight photoreceptor cell bodies in the ONL. Chemical synapses between photoreceptors and postsynaptic cells are marked by the presence of synaptic ribbons in the rod and cone pedicles 2J, which generally release transmitter to two or three nearby postsynaptic cells. Although synaptic contacts without synaptic ribbons exist, the chemical nature of these contacts is not certain ]5. In this study we used the synaptic ribbons to define sites of glutamate release, and investigated how close Miiller cell processes come to these sites. Fig. 1B shows several ribbon synapses in a synaptic pedicle (terminal), i.e., the output synapse, of a rod photoreceptor. The cell bodies of the two types of photoreceptors can be distinguished by the position of their nuclei (labelled cn (cone nucleus) and rn (rod nucleus), respectively, in the figures of this paper). The rod cell nuclei are placed more externally (towards the pigment epithelium) while the cone nuclei are located nearer the synaptic region 15. As a result, cone cell bodies are continuous with their pedicles (cp) (Fig. 2A) while the rod cell bodies are linked to their pedicles (rp) by means of an axon (arrows, Fig. 2B).
The electron-dense H R P reaction product can be readily identified in injected Miiller cells (Mc in Figs. 2 and 3). Serial sectioning of two HRP-injected Miiller cells revealed that a single Miiller cell sends out large processes, that fill the space between at least six photoreceptor cell bodies. These processes also project to several more photoreceptors but surround them only in part. Although Miiller cells surround much of the photoreceptor cell m e m b r a n e (Fig. 2B) and also invade the pedicles (Fig. 2B, arrowhead), they seldom project into the outer plexiform layer near the ribbon synapses (Figs. 2A and 3). Fourteen pedicles of rod and cone photoreceptors approached by HRP-injected Mfiller cell processes were sectioned and examined. On only two occasions were minor Miiller cell processes found to be present in the outer plexiform layer close to synaptic ribbons. An example of this is seen in Fig. 2C in which the arrow points to a MiJller cell process in the outer plexiform layer close to a ribbon synapse in a rod pedicle. Further sections through the same pedicle show the presence of many more ribbon synapses (Fig. 3, arrows), but no MiJller cell processes, other than that shown in Fig. 2C, were observed to project into the region postsynaptic to these ribbons in the outer plexiform layer. Fig. 3 illustrates a common arrangement in which up to four rod pedicles cluster around a cone pedicle. In this case two rod pedicles (rp) abut a cone pedicle (cp) (arrowheads indicate the cell m e m b r a n e s that separate
Fig. 3. Clustered photoreceptor synaptic pedicles. Section through a cone cell body (cn) with a synaptic pedicle (cp), which is surrounded by two rod pedicles (rp) (arrowheads indicate the membranes that separate them). The rod pedicle containing synaptic ribbons (arrows) is a section through the same pedicle as shown in Fig. 2C. In contrast to that figure, no MiJller cell processes are present in the outer plexiform layer (OPL). HRP-filled processes (Mc) are seen wrapping around the cone cell body and the group of pedicles without projecting into the cluster.
303 them). This reflects the arrangement of the photoreceptors in a roughly orthogonal lattice in which four rods surround a cone 2. W h e n the pedicles are clustered like this, Miiller cell processes wrap around the whole group of pedicles without projecting between them or into the outer plexiform layer. The closest approach of Miiller cells to ribbon synapses was measured, for each of the pedicles sectioned, as the distance between the end of the arciform density of the synaptic ribbon (Fig. 1B, arrowhead) nearest to a HRP-filled process and that process. The nearest Miiller cell process lay in the outer plexiform layer for two pedicles, including that of Figure 2C, but more usually occurred to the side of the pedicle (as for the pedicles in Figs. 2A and 3). W h e n this distance was measured as a straight line (an underestimate of the true diffusion distance), the resulting measurements were 1.1 + 0.2 /zm (S.E.M.) for the six rod pedicles, 3.0 + 1.7/xm for the four cone pedicles, and 0.9 + 0.3 /xm for four pedicles that could not be identified as belonging to a rod or a cone. When the diffusion distance from Miiller cell process to the nearest ribbon was measured along the visible extracellular pathway in the plane of section (which probably overestimates the distance glutamate has to diffuse), the resulting measurements were 3.1 + 0.7 ~ m for rod pedicles, 5.8 + 1.5 ~zm for cone pedicles and 1.4 + 0.5 ~ m for unidentified pedicles. The greatest distance that glutamate, released from a photoreceptor, would have to diffuse to a Miiller cell process, would be from a ribbon synapse in the middle of a cone pedicle that is surrounded by several rod pedicles. From our data we estimate that distance to be around 10 tzm. Shrinkage of the tissue during processing was not taken into account in calculating these distances, but is believed to be less than 5% (ref. 10). This study is the first investigation of the spatial relationship between sites of glutamate uptake in glial cells and sites of transmitter release at glutamatergic synapses. Fig. 4 summarizes the results, showing the relationship between a Miiller cell (shaded) and rod and cone photoreceptors and their associated synaptic pedicles. Miiller cell processes surround photoreceptor cell bodies and their synaptic pedicles. However, MiJller cell processes are rarely present in the outer plexiform layer on the side of the pedicles where the synaptic ribbons are, and on the rare occasions where they are present there, they do not invade the spaces between horizontal, bipolar and photoreceptor ceils near the synaptic cleft. For transmission from photoreceptors to horizontal cells in the turtle retina, the time course of the decaying phase of the synaptic impulse response function
photoreceplor outer end Inrler segments
photoreceptor cell b o d l u ONL syneptl© pedlclell wllh ribbon synaINel
procemms of bipolar and horizontal cells
OPL
INL
i ! i
~'~
MQIler cell etalk
Fig. 4. Schematic diagram of Miiller cell processes in the outer layers of the salamander retina. The shaded areas represent HRP-filled Miiller cell processes. Miiller cell processes (shaded) project out from the main stalk and wrap around cell bodies and synaptic pedicles of photoreceptors in the outer nuclear layer (ONL). Only occasionally does a Miiller cell process project out into the outer plexiform layer (OPL) near the synaptic region of the photoreceptor output synapse. Miiller cell processes wrap around cell bodies in the inner nuclear layer (INL). The stalk of the Miiller cell extends to the inner limiting membrane ending in an endfoot in the ganglion cell layer (not shown).
(which probably reflects the time course of glutamate removal from the extracellular space) is 30 ms for glutamate released from cones and 200 ms for glutamate released from rods 7. Similar data are not available for the salamander retina. However, as described in this paper, in the salamander retina there is a difference in shape between rod and cone pedicles (as has also been reported in the turtle retina 14) with the base of cone pedicles, where the ribbon synapses are situated, being much wider than that of rod pedicles. This results in the ribbon synapses in rod pedicles being closer than those in the cone output synapses to processes of Miiller cells. The clustering of rod and cone pedicles seen in the salamander retina, where the rod terminals abut the cone terminals and the Miiller cells wrap around the outside of the group of terminals, makes the average distance between cone ribbon synapses and glutamate uptake sites even longer. Therefore, one would not expect the decaying phase of the impulse response function for rod-horizontal cell transmission to be slower than that for cone-horizontal cell transmission as a result of the time taken for glutamate to diffuse to a Miiller cell. An estimate of the distance, x, glutamate can diffuse in a time, t, is given by the equation8: t = lx2/D, in which D is the diffusion coefficient (about 10 -9 m2/s). This equation suggests it will take 0.6 ms to diffuse 1.1 Izm and 4.5 ms to diffuse 3.0/zm, the direct
304 distances measured for the closest approach of a MiJller cell process to rod and cone pedicles, respectively. However, the actual diffusion distance through the extracellular space is longer due to it being via a tortuous path (see above). Nicholson and Phillips ~9 calculate that, in the rat cerebellum, this tortuosity reduces the effective diffusion coefficient by a factor of 2.4, giving diffusion times of 1.5 ms and 10.8 ms for the direct measured distances given above. These times are both shorter than the decay times of the impulse response functions for transmission from rods and cones to horizontal cells in the turtle retina 7. Thus, although Mfiller cell processes do not come very close to the postsynaptic sites at the photoreceptor output synapse, they apparently come close enough to play a role in terminating the effects of glutamate released from both rod and cone synapses. This work was supported by the Wellcome Trust, the MRC and the Wolfson Foundation. We thank David Attwell for helpful advice. 1 Attwell, D., The photoreceptor output synapse, Progr. Retinal Res., 9 (1990) 337-362. 2 Attwell, D., Wilson, M. and Wu, S.M., A quantitative analysis of interactions between photoreceptors in the salamander (Ambystoma) retina, J. PhysioL, 352 (1984) 703-737. 3 Attwell, D., Mobbs, P., Tessier-Lavigne, M. and Wilson, M., Neurotransmitter-induced currents in retinal bipolar cells of the axolotl, Ambystoma mexicanum, J. Physiol., 387 (1987) 125-161. 4 Ayoub, G.S., Korenbrot, J.I. and Copenhagen, D.R., Release of endogenous glutamate from isolated cone photoreceptors of the lizard, Neurosci. Res. Suppl., 10 (1989) 547-556. 5 Brandon, C. and Lain, D.M.K., L-Glutamic acid: a neurotransmitter candidate for cone photoreceptors in human and rat retinas, Proc. Natl. Acad. Sci. USA, 80 (1983) 5117-5121. 6 Brew, H. and Attwell, D., Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells, Nature, 27 (1987) 707-709. 7 Copenhagen, D.R., Ashmore, J.F. and Schnapf, J.K., Kinetics of synaptic transmission from photoreceptors to horizontal and bipolar cells in turtle retina, Vision Res., 23 (1983) 363-369.
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