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Differential effects of cobalt ions on rod and cone synaptic activity in the isolated frog retina
Vwm Res.Vol. 18.pp. 145to ISI. Pmgamon Press1978. PrintedtnGrentBritam
DIFFERENTIAL EFFECTS OF COBALT IONS ON ROD AND CONE SYNAPTIC ACTIVITY IN THE ISOLATED FROG RETINA JUDITHA. *Department
EVANS*, DONALD
C. HOODSand ERICHOLTZMAN*
of Biological Sciences and +Department of Psychology, Columbia University, New York. N.Y. 10027, U.S.A. (Received 28 April 1977)
Abstract-The uptake of horseradish peroxidase (HRP), and electrophysiological techniques, were used to study the effects of cobalt on the synaptic activity of frog rods and cones. In the dark, I or 2.4 mM cobalt produces markedly depressed levels of HRP uptake into rod terminals, while uptake by the
cones is not substantially altered This differential effectresembles that produced by moderate intensities of light HRP uptake by both rods and cones is reduced to very low levels by 5mM cobalt. 1 to 2.4mM cobalt completely eliminates the rod contribution to the ERG b-wave without eliminating the cone contribution. Key Words-horseradish
but it does not affect the aspartate-isolated peroxidase; electrophysiology;
INTRODUCIION In many vertebrate retinas, including the frog, two photoreceptor types exist. The rod receptors are very sensitive to light and dominate vision at low light levels, and the cone receptors are less sensitive to light but dominate at higher light levels. The extent to which these differences arise at the outer segment membrane, or in other parts of the receptor or in the organization of the retina postsynaptic to the receptors has yet to be determined. We have developed a method for monitoring the synaptic activity of the receptors based on the observations made with other neurons (reviewed in Holtzman, 1977) that the levels of uptake of the extracellular tracer horseradish peroxidase (HRP) into synapses are directly related to rates of neurotransmission. With this approach we showed, as predicted from previous physiological studies, that both rods and cones of the frog isolated retina are synaptically active in the dark and that moderate levels of light reduce activity to a much greater extent in rods than in cones (Schacher, Holtzman and Hood, 1974 and 1976). It is known that synaptic activity of many neurons is dramatically affected by divalent cations. Of these, calcium is found to be necessary for transmitter release in several systems (Katz and Miledi, 1965; Llinas and Nicholson, 1975). Its probable involvement in transmission by receptors is strongly suggested by observations such as those on the skate retina, in which magnesium, an ion that often antagonizes the effects of calcium, was shown to inhibit synaptic activity (Dowling and Ripps, 1973; Ripps, Shakib and McDonald, 1976). Cobalt ions, which are known to inhibit synaptic activity in neuromuscular junctions of the frog (Weakly, 1973) also seem to influence the electrical behavior of the retina. Intracellular recordings in Necrurus, turtle and carp retinas (Dacheux and Miller, 1976; Kaneko and Schimazaki, 1975; Schwartz, 1976; Cervetto and Piccolino. 1974) show that l-2 mM cobalt produces a loss of sensi-
receptor potentials.
photoreceptors;
synapses; irog; retina; cobalt.
tivity in bipolars and horizontal cells to flashes of light without dramatically affecting receptor sensitivity. The present study shows that cobalt can be used to manipulate synaptic activity in the rods and cones differentially to produce, in the dark, alterations similar to those engendered by moderate intensities of light. These changes can be monitored with HRP and can also be detected electrophysiologically. A preliminary report of this work has been published (Evans, Hood and Holtzman, 1977).
MATERIALS AND METHODS
Frogs, Ranopipiens. about 3 in. in body length, were dark adapted overnight before each experiment. The retinas were isolated in a dish of Ringers solution at room temperature under a dim red light. Normal Ringers, @H 7.5) contained 111 mM NaCl, 2.3 mM NaHCO,, 2 mM KCI, 0.45 mM C&l, and 5 mM sucrose. Ringers containing 50 mM sodium aspartate or 1, 2.4 or 5 mM Cot& was prepared by making the appropriate adjustments in the NaCl concentration. The retinas used in the HRP experiments were prepared in the dark as described by Schacher et al., 1976. A concentration of 05% HRP (Sigma Chemical Co., St. Louis, MO, Type II) was included in the Ringers solution. All HRP experiments involved 30 min exposures to the tracer in the dark at room temperature. The retinas were then fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2). sectioned at a thickness setting of 4Om on a Smith-Farquhar tissue chopper, incubated to demonstrate peroxidase activity and then prepared for electron microscopy by the procedures routinely used in our laboratory (Schacher et al., 1976). Retinas for electrophysiological studies were similarly isolated in the dark in the appropriate Ringers solution, floated receptor-side down on a cotton pad and placed in a moist oxygenated environment for the duration of the experiment. Mass retinal potentials, electroretinograms (ERGS) were recorded using the extracellular recording system and the optical system described in Hood and Hock. 1975.
145
JUDITH A. EVANS. DONALD C. HCOD and ERIC HOLTZWS
116
RESULTS
We also confirmed the observanons of Schachc: rt al. (1974, 1976) that illumination by a modera:: intensity light inhibits the uptake of HRP into rod, without inhibiting uptake into cones. No obvious signs of morphological damage WST;’ noted in cobalt-treated retinas. As was true-in our
HRP uptake
With normal Ringers an appreciable proportion of the synaptic vesicles of both rods and cones become labeled with HRP in the dark; the level of labeling is similar to that observed in our previous study
prior work, some multivesicular bodies and short tubules become labeled with HRP. along with ths synaptic vesicles.
(Schacher et a/.. 1976). HRP uptake into rod terminals is markedly depressed by 1 mM cobalt while uptake
by the cones is not significantly altered (Figs. 1 and 2). Basically similar results are obtained with 2.4 mM cobalt, although there is a slight decrease in uptake
Electrophysiolog)t ‘The ERG can be used as a measure of the effectiveness of the receptors in inducing activity in the postsynaptic cells since it is a potential that has a large.
by the cones (Fig. 2). Exposure to 5 mM cobalt depresses HRP uptake by both rod and cone terminals to very low levels (Fig. 2). Rod, <*
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Fig. 2. The frequency distribution of HRP uptake into rod and cone terminals maintained in the dark for 30 min in normal Ringers, and Ringers containing I, 2.4 or 5 mM cobalt Each bar represents the category of terminals with the indicated percentage of labeled vesicks. The bar heights were obtained by averaging the frequency of each category (per cent of the total population of terminals) observed in the number of separate repeat experiments indicated on the figure. The histograms present pooled data; however the results were very similar in each of the experiments.
Fig. I. Rod (R) and cone (C) terminals from a retina exposed to HRP in I mM cobalt Ringers for 30min in the dark. The cone terminal shows numerous HRP-labeled vesicles, while the rod terminal contains very few. N indicates the nucleus of the cone and the arrows. synaptic ribbons. The bar represents 1pm.
149
Differential effects of cobalt ions (a I Dark
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Fig. 3. Sample records of ERG responses elicited by 50 msec flashes of 630 and 500 nm light presented before and after treatment with 1mM cobalt. Intensities and wavelengths were chosen from previously published work (Hood and Hock, 1973 and 1975) according to the following criteria: A 630 nm light was used as a convenient reference. An intensity of SOOnm light (the rod equivalent, R.E.) was selected that would be equally effective to this reference in eliciting a response from the frog’s rods This intensity is about 100 times weaker than the reference 630 light since the rods are very sensitive to 500nm light A second intensity of 5OOnm light (the cone equivalent, C.E.) was selected that would be equally effective to the 630 nm light in eliciting a cone response. This intensity is approximately the same intensity as the reference light, since the cones are relatively less sensitive to 5OOnm light than are the rods The relative dfaaiveness of the two 5OOnm intensities as compared with the 630nm reference permits us to distinguish rod-driven from cone-driven responses of the ERG. (a) Dark adapted retina. &fore cobalt there is no indication of cone activity in the response to flashes of low intensity; the rods dominate the response. Before addition of 1 mM cobalt. the 630 and the 5OOnm R.E. flashes elicit the same size response. As expected, the much more intense 5OOnm C.E. flash elicits a much larger response (note scale change). Addition of 1mM cobalt completely eliminates the rod response. The response to the 630 and 5OOnm C.E. !lashes are now equal in amplitude, as expected for a response dominated by cones. and there is no response at all to the 5OOnm R.E. flash. Notice that in the dark, the cone contribution to the ERG is enhanced by cobalt; the same intensity 630 nm flash which before cobalt did not elicit a detectable cone response (the response illustrated is dominated by the rods), now elicits a sizable response which must be due to the cones. There are many possible explanations for this, such as the existence of interactions between the receptors All flash intensities are expressed relative to the 630 nm fiash intensity which is labeled 0.0 log I and is equal to I.5 log quanta/ scc/micron.z (b) Lighr adapred retina. This retina was illuminated with a light of 0.13 ft-cd which eliminates rod activity. The 0.0 log I value of the 630~1 flash is 2.8 log quanta/sec/micronz: as above, all other intensities are retative to this value. Since cones dominate the ERG in the light adapted retina, the responses to the 630 and 54IOnm C.E. flashes are nearly the same. The much weaker 5OOnm R.E. flash elicits no response; and as expected, addition of 1 mM cobalt has no effect on the cone dominated ERG.
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Fig. 4. Sample records of the aspartate-isolated receptor potentials elicited by 50 mSec flashes of 630 and 500 nm light presented before and after treatment with 1 mM cobalt. The flash intensities were chosen according to the criteria given in Fig. 3; all terminology is the same. (a) Dark adapted retina. The 630 and XI0 nm R.t. flashes elicit the same size response in the dark adapted retina for the reasons explained in Fig. 3; the much more intense 500 nm C.E. elicits a very large response (note scale change). Addition of I mM cobalt does not affect either the response amplitude or its wave form. All flash intensities are expressed relative to the 630nm reference flash which is labeled 0.0 log I and is qua1 to 2.8 log quanta/sec/micron2. (b) Lighr adapted retina Since the cones dominate the response of the light adapted retina, the response to the 630 and 5OOnm C.E. flashes are nearly the same. The weaker 5OOnm R.E. flash elicits no response. The addition of 1 mM cobalt has no effect on the response or its wave form. All flash intensities are expressed relative to the 630 reference flash which is labeled 0.0 log I and is qua1 to
4.5 log quanta/set/micron*. dominant contribution from these cells. Figure 3a shows that the rods control the ERG in the dark and that 1 mM cobalt eliminates this contribution within 1 min after its application. After cobalt treatment, the dark adapted ERG is generated only by the cones as it is in the light adapted retina with or without cobalt (Fig. 3b). The ERG recorded from an aspartate-treated retina is generated by the receptors (reviewed in Hood and Hock, 1975). Using the aspartate-isolated receptor potential and the appropriate selection of lights (Hood and Hock, 1973 and 1975), the sensitivity of the rod and cone receptors can be separately monitored in dark adapted retinas. We found that both rod and cone receptor potentials elicited by light flashes were unaltered when retinas were isolated and incubated in 1 or 2.4mM cobalt and that receptor sensitivity was stable for well over the 30min period used in the cytochemical experiments. However, a concentration of 5mM cobalt drastically decreased the sensitivity of both rods and cones. Figure 4 shows the results of a second type of experiment, in which retinas were initially isolated in cobalt-free aspartate
150
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DOULD
Ringers and then 1 mM cobalt was applied. The addition of 1mhl cobalt does not alter the receptor potentials. DlSCl_SlO~
Both the microscopic and electrophysiological results demonstrate that 1 and 2.4 mM cobalt inhibits synaptic activity in the rods but not in the cones. Both techniques show that the effects produced by these concentrations of cobalt resemble those observed when retinas incubated in normal Ringers are illuminated with moderate intensities of light. Taken by themselves, the HRP results might be thought to reflect an ability of cobalt simply to interfere with endocytosis of the tracer, but the electrophysiological findings strongly suggest that this is not the case and that transmission is indeed affected. One explanation of these results is that cobalt is acting directly at the receptor terminals. In line with this is our finding that 1 or 2.4 mM cobalt has no obvious effect on the aspartate-isolated receptor potentials of either the dark or light adapted retina, which together with the results of others (Dacheux and Miller, 1976; Kaneko and Schimazaki, 1975; Schwartz. 1976; and Cervetto and Piccolino. 1974) suggests that at these concentrations cobalt does not act at the outer segment. However, our results with aspartate cannot yet be directly compared with our other findings since it is possible that cobalt and aspartate may interact in unknown ways. Our results on HRP uptake by receptors with aspartate Ringers, with and without cobalt, have been quite variable and cannot yet be confidently interpreted. We have seen some evidence of enhanced uptake by receptors with aspartate (cf. Ripps et al., 1976) and we know that aspartate can enhance the synaptic activity of cells in the inner plexiform layer (Evans er al., 1977). At 5mM, the receptor potentials are abolished in both the light and dark adapted retinas. One might anticipate that cobalt has an effect on the outer segment membrane, since it is known that calcium applied to the outer segment of the dark adapted receptor causes a hyperpolarization (Hagins and Yoshikami, 1974; Brown and Pinto, 1974). However, we might simply be observing cytotoxic effects. It seems reasonable to speculate that the effects of 1 and 2.4rnM cobalt may be on the mechanism of transmitter release or on those coupling potential changes to synaptic activity. Cobalt inhibits passive calcium infiux associated with the action potential in the barnacle giant muscle fiber (Hagjwara and Takahashi, 1967) and in neurons of the Aplysia visceral ganglion (Geduldig and Junge, 1968); it also inhibits the slow inward calcium current of mammalian cardiac muscle fibers (Kohlhardt, Bauer, Krause and Fleckenstein, 1973). Cobalt could have similar effects on the receptors. The fact that rods and cones differ in sensitivity to the same concentration of cobalt suggests that there might be some differences in the mechanisms governing transmitter release or related phenomena. We do not yet know whether this is simply a difference in concentration dependency or whether rods and cones differ in their permeability to cobalt, or whether a more profound difference exists. These findings do however raise the possibility
C.
HOOD and ERIC HOL?I.UA>
that the differential involvement of rods and cones in retinal functions at different lighhr intsnsiaes depends not only on differences in their outer segments. but also on differences at the level of the terminals. Our findings that 1-2mM cobalt does not a&c: the receptor potentials of rods or cones agree with the results of prior investigations with intracellular recordings on Necrurus. carp and turtle (Dacheuu and Miller, 1976; Kaneko and Schimazaki. 1975: Schwartz, 1976; and Cervetto and Piccotino. 197-1). However. the reports by the same investigators that cobalt abolishes sensitivity of horizontal and bipolar cells to light flashes could be interpreted as indicating a complete block in synaptic transmission from the photoreceptors. This would differ from our results which demonstrate a differential effect on the two receptor types. If so, the differences might simply refkct variations among species. For example, as judged both electrophysiologically and by tracer uptake (Dowling and Ripps. 1973, and Ripps et al., 1976) high concentrations of magnesium seem to abolish synaptic activity in skate retina, but we have found that concentrations of magnesium as high as 2OmS1 do not similarly inhibit HRP uptake into the photoreceptors of the frog. However, it should be noted that the cytochemical methods provide a more direct evaluation of the behavior of photoreceptor terminals than do the available recording procedures’and the); also permit the study of very large numbers of cells. Inevitably, small sample sizes are involved in intraceilular recording and it is conceivable that the horizontal and bipolar cells studied did not include cells driven by cones. Finally our findings may be useful in further studies on the retina. The fact that we can turn “off rod synaptic activity in the dark without drastically altering the cones and the postsynaptic cells should facilitate future work. Acknowledgements-We thank Fe Reyes for her skiiful technical assistance. The research was supported by NLH grant NS09475 to EH and NIH grant EY01877 to DCH. REFERENCES Brown J. E. and Pinto H. (1974) Ionic mechanism for the
photoreceptor potential of the retina of &fo mntimcf. J. Physiol.. Land. 236. 575-591. Cervetto L. and Piccolino M. (1974) Synaptic transmission between photoreceptors and horizontal cells in the turtle retina. Science 183, 417-419. Dacheux R. and Miller R. (1976) Photoreceptor-bipolar cell transmission in the perfused retina eyecup of the mudpuppy. Science 191, 963-964. Dowling 1. E. and Ripps H. (1973) Effect of magnesium on horizontal cell activity in skate retina. Nature 242. 101-103. Evans J. A.. Hood D. C. and Holtzman E. (1977) Effects of aspartate and cobalt on horseradish peroxidasc (HRP) up&e in frog retina. Assoc. Res. Vis..Oprhalmol.. 1977. (Invest. Oohtkatmol. ADI%. 1977, SUPPI., II. 26.) Giduldig 6. and Jun& D. (1968) sodium and calcium components of action potentials in the Aplysia giant neurone. J. Physiol., Land. 199, 347-365. Hagins W. A. and Yoshikami S. (1974) A role for calcium in excitation of retinal rods and cones. Exp. Eye Res. 18, 29%305.
Differential effects of cobalt ions Hagiwara S. and Takahashi K. (1967) Surface density of. calcium ions and calcium spikes in the barnacle muscle fiber membrane. J. gen. Physiof. SO. 583-601. Holtzman E. (1977) The origin and fate of secretory packages. especially synaptic vesicles. Neuroscience 2. 327-355. Hood D. C. and Hock P. A. (1973) Dark adaptation of the frog’s rods. Vision Res lj. 1943-1951. Hood D. C. and Hock P. A. (1975) Lit&t adaotation of the receptors: Increment threshold Functions for the frog’s rods and cones. Vision Res. 15, 545-553. Kaneko A. and Shimazaki H. (1975) Synaptic transmission from photoreceptors to bipolar and horizontal cells in the carp retina. Cold Spring Harbour Symposium 40. 537-546.
Katz B. and Miledi R. (1965) The effect of calcium on acetylcholine release from motor nerve terminals. Proc. R, Sot. B. 161. 496503. Kohlhardt M.. Batter B., Krause H. and Fleckenstein A. (1973) Selective inhibition of the transmembrane calcium conductivity of mammalian myocardial fibres by Ni. Co
151
and Mn ions. PjKgers Arch. ger Physiol. 338. 115-123. Llinas R. and Nicholson C. (1975) Calcium role in depolarization-secretion coupling: an aequotin study in squid giant synapse. Proc. Mtn. Acad Sci., U.S.A. 72. 187-190. Ripps H., Shakib M. and McDonald E. D. (1976) Peroxidase uptake by photoreceptor terminals of the skate retina. J. Cel[ Biol. 70. 8696. Schacher S.. Holtzman E. and Hood D. C. (1974) Uptake of horseradish peroxidase by frog photoreceptor synapses in the dark and in the light. Nature 249. 261-263. Schacher S.. Hohzman E. and Hood D. C. (1976) Synaptic activity of frog retinal photoreceptors: peroxidase uptake study. J. Cell Biol. 70, 178-192. Schwartz E. A. (1976) Electrical properties of the rod syncytium in the retina of the turtle. J. Physiol., Land. 257, 379-406.
Weakly J. (1973) The action of cobalt ions on neuromuscular transmission in the frog. 1. Physiol.. LO~. 2% 597-612.
DIFFERENTIAL EFFECTS OF COBALT IONS ON ROD AND CONE SYNAPTIC ACTIVITY IN THE ISOLATED FROG RETINA JUDITHA. *Department
EVANS*, DONALD
C. HOODSand ERICHOLTZMAN*
of Biological Sciences and +Department of Psychology, Columbia University, New York. N.Y. 10027, U.S.A. (Received 28 April 1977)
Abstract-The uptake of horseradish peroxidase (HRP), and electrophysiological techniques, were used to study the effects of cobalt on the synaptic activity of frog rods and cones. In the dark, I or 2.4 mM cobalt produces markedly depressed levels of HRP uptake into rod terminals, while uptake by the
cones is not substantially altered This differential effectresembles that produced by moderate intensities of light HRP uptake by both rods and cones is reduced to very low levels by 5mM cobalt. 1 to 2.4mM cobalt completely eliminates the rod contribution to the ERG b-wave without eliminating the cone contribution. Key Words-horseradish
but it does not affect the aspartate-isolated peroxidase; electrophysiology;
INTRODUCIION In many vertebrate retinas, including the frog, two photoreceptor types exist. The rod receptors are very sensitive to light and dominate vision at low light levels, and the cone receptors are less sensitive to light but dominate at higher light levels. The extent to which these differences arise at the outer segment membrane, or in other parts of the receptor or in the organization of the retina postsynaptic to the receptors has yet to be determined. We have developed a method for monitoring the synaptic activity of the receptors based on the observations made with other neurons (reviewed in Holtzman, 1977) that the levels of uptake of the extracellular tracer horseradish peroxidase (HRP) into synapses are directly related to rates of neurotransmission. With this approach we showed, as predicted from previous physiological studies, that both rods and cones of the frog isolated retina are synaptically active in the dark and that moderate levels of light reduce activity to a much greater extent in rods than in cones (Schacher, Holtzman and Hood, 1974 and 1976). It is known that synaptic activity of many neurons is dramatically affected by divalent cations. Of these, calcium is found to be necessary for transmitter release in several systems (Katz and Miledi, 1965; Llinas and Nicholson, 1975). Its probable involvement in transmission by receptors is strongly suggested by observations such as those on the skate retina, in which magnesium, an ion that often antagonizes the effects of calcium, was shown to inhibit synaptic activity (Dowling and Ripps, 1973; Ripps, Shakib and McDonald, 1976). Cobalt ions, which are known to inhibit synaptic activity in neuromuscular junctions of the frog (Weakly, 1973) also seem to influence the electrical behavior of the retina. Intracellular recordings in Necrurus, turtle and carp retinas (Dacheux and Miller, 1976; Kaneko and Schimazaki, 1975; Schwartz, 1976; Cervetto and Piccolino. 1974) show that l-2 mM cobalt produces a loss of sensi-
receptor potentials.
photoreceptors;
synapses; irog; retina; cobalt.
tivity in bipolars and horizontal cells to flashes of light without dramatically affecting receptor sensitivity. The present study shows that cobalt can be used to manipulate synaptic activity in the rods and cones differentially to produce, in the dark, alterations similar to those engendered by moderate intensities of light. These changes can be monitored with HRP and can also be detected electrophysiologically. A preliminary report of this work has been published (Evans, Hood and Holtzman, 1977).
MATERIALS AND METHODS
Frogs, Ranopipiens. about 3 in. in body length, were dark adapted overnight before each experiment. The retinas were isolated in a dish of Ringers solution at room temperature under a dim red light. Normal Ringers, @H 7.5) contained 111 mM NaCl, 2.3 mM NaHCO,, 2 mM KCI, 0.45 mM C&l, and 5 mM sucrose. Ringers containing 50 mM sodium aspartate or 1, 2.4 or 5 mM Cot& was prepared by making the appropriate adjustments in the NaCl concentration. The retinas used in the HRP experiments were prepared in the dark as described by Schacher et al., 1976. A concentration of 05% HRP (Sigma Chemical Co., St. Louis, MO, Type II) was included in the Ringers solution. All HRP experiments involved 30 min exposures to the tracer in the dark at room temperature. The retinas were then fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2). sectioned at a thickness setting of 4Om on a Smith-Farquhar tissue chopper, incubated to demonstrate peroxidase activity and then prepared for electron microscopy by the procedures routinely used in our laboratory (Schacher et al., 1976). Retinas for electrophysiological studies were similarly isolated in the dark in the appropriate Ringers solution, floated receptor-side down on a cotton pad and placed in a moist oxygenated environment for the duration of the experiment. Mass retinal potentials, electroretinograms (ERGS) were recorded using the extracellular recording system and the optical system described in Hood and Hock. 1975.
145
JUDITH A. EVANS. DONALD C. HCOD and ERIC HOLTZWS
116
RESULTS
We also confirmed the observanons of Schachc: rt al. (1974, 1976) that illumination by a modera:: intensity light inhibits the uptake of HRP into rod, without inhibiting uptake into cones. No obvious signs of morphological damage WST;’ noted in cobalt-treated retinas. As was true-in our
HRP uptake
With normal Ringers an appreciable proportion of the synaptic vesicles of both rods and cones become labeled with HRP in the dark; the level of labeling is similar to that observed in our previous study
prior work, some multivesicular bodies and short tubules become labeled with HRP. along with ths synaptic vesicles.
(Schacher et a/.. 1976). HRP uptake into rod terminals is markedly depressed by 1 mM cobalt while uptake
by the cones is not significantly altered (Figs. 1 and 2). Basically similar results are obtained with 2.4 mM cobalt, although there is a slight decrease in uptake
Electrophysiolog)t ‘The ERG can be used as a measure of the effectiveness of the receptors in inducing activity in the postsynaptic cells since it is a potential that has a large.
by the cones (Fig. 2). Exposure to 5 mM cobalt depresses HRP uptake by both rod and cone terminals to very low levels (Fig. 2). Rod, <*
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Fig. 2. The frequency distribution of HRP uptake into rod and cone terminals maintained in the dark for 30 min in normal Ringers, and Ringers containing I, 2.4 or 5 mM cobalt Each bar represents the category of terminals with the indicated percentage of labeled vesicks. The bar heights were obtained by averaging the frequency of each category (per cent of the total population of terminals) observed in the number of separate repeat experiments indicated on the figure. The histograms present pooled data; however the results were very similar in each of the experiments.
Fig. I. Rod (R) and cone (C) terminals from a retina exposed to HRP in I mM cobalt Ringers for 30min in the dark. The cone terminal shows numerous HRP-labeled vesicles, while the rod terminal contains very few. N indicates the nucleus of the cone and the arrows. synaptic ribbons. The bar represents 1pm.
149
Differential effects of cobalt ions (a I Dark
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Fig. 3. Sample records of ERG responses elicited by 50 msec flashes of 630 and 500 nm light presented before and after treatment with 1mM cobalt. Intensities and wavelengths were chosen from previously published work (Hood and Hock, 1973 and 1975) according to the following criteria: A 630 nm light was used as a convenient reference. An intensity of SOOnm light (the rod equivalent, R.E.) was selected that would be equally effective to this reference in eliciting a response from the frog’s rods This intensity is about 100 times weaker than the reference 630 light since the rods are very sensitive to 500nm light A second intensity of 5OOnm light (the cone equivalent, C.E.) was selected that would be equally effective to the 630 nm light in eliciting a cone response. This intensity is approximately the same intensity as the reference light, since the cones are relatively less sensitive to 5OOnm light than are the rods The relative dfaaiveness of the two 5OOnm intensities as compared with the 630nm reference permits us to distinguish rod-driven from cone-driven responses of the ERG. (a) Dark adapted retina. &fore cobalt there is no indication of cone activity in the response to flashes of low intensity; the rods dominate the response. Before addition of 1 mM cobalt. the 630 and the 5OOnm R.E. flashes elicit the same size response. As expected, the much more intense 5OOnm C.E. flash elicits a much larger response (note scale change). Addition of 1mM cobalt completely eliminates the rod response. The response to the 630 and 5OOnm C.E. !lashes are now equal in amplitude, as expected for a response dominated by cones. and there is no response at all to the 5OOnm R.E. flash. Notice that in the dark, the cone contribution to the ERG is enhanced by cobalt; the same intensity 630 nm flash which before cobalt did not elicit a detectable cone response (the response illustrated is dominated by the rods), now elicits a sizable response which must be due to the cones. There are many possible explanations for this, such as the existence of interactions between the receptors All flash intensities are expressed relative to the 630 nm fiash intensity which is labeled 0.0 log I and is equal to I.5 log quanta/ scc/micron.z (b) Lighr adapred retina. This retina was illuminated with a light of 0.13 ft-cd which eliminates rod activity. The 0.0 log I value of the 630~1 flash is 2.8 log quanta/sec/micronz: as above, all other intensities are retative to this value. Since cones dominate the ERG in the light adapted retina, the responses to the 630 and 54IOnm C.E. flashes are nearly the same. The much weaker 5OOnm R.E. flash elicits no response; and as expected, addition of 1 mM cobalt has no effect on the cone dominated ERG.
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Fig. 4. Sample records of the aspartate-isolated receptor potentials elicited by 50 mSec flashes of 630 and 500 nm light presented before and after treatment with 1 mM cobalt. The flash intensities were chosen according to the criteria given in Fig. 3; all terminology is the same. (a) Dark adapted retina. The 630 and XI0 nm R.t. flashes elicit the same size response in the dark adapted retina for the reasons explained in Fig. 3; the much more intense 500 nm C.E. elicits a very large response (note scale change). Addition of I mM cobalt does not affect either the response amplitude or its wave form. All flash intensities are expressed relative to the 630nm reference flash which is labeled 0.0 log I and is qua1 to 2.8 log quanta/sec/micron2. (b) Lighr adapted retina Since the cones dominate the response of the light adapted retina, the response to the 630 and 5OOnm C.E. flashes are nearly the same. The weaker 5OOnm R.E. flash elicits no response. The addition of 1 mM cobalt has no effect on the response or its wave form. All flash intensities are expressed relative to the 630 reference flash which is labeled 0.0 log I and is qua1 to
4.5 log quanta/set/micron*. dominant contribution from these cells. Figure 3a shows that the rods control the ERG in the dark and that 1 mM cobalt eliminates this contribution within 1 min after its application. After cobalt treatment, the dark adapted ERG is generated only by the cones as it is in the light adapted retina with or without cobalt (Fig. 3b). The ERG recorded from an aspartate-treated retina is generated by the receptors (reviewed in Hood and Hock, 1975). Using the aspartate-isolated receptor potential and the appropriate selection of lights (Hood and Hock, 1973 and 1975), the sensitivity of the rod and cone receptors can be separately monitored in dark adapted retinas. We found that both rod and cone receptor potentials elicited by light flashes were unaltered when retinas were isolated and incubated in 1 or 2.4mM cobalt and that receptor sensitivity was stable for well over the 30min period used in the cytochemical experiments. However, a concentration of 5mM cobalt drastically decreased the sensitivity of both rods and cones. Figure 4 shows the results of a second type of experiment, in which retinas were initially isolated in cobalt-free aspartate
150
JLD~TH .A. Euus.
DOULD
Ringers and then 1 mM cobalt was applied. The addition of 1mhl cobalt does not alter the receptor potentials. DlSCl_SlO~
Both the microscopic and electrophysiological results demonstrate that 1 and 2.4 mM cobalt inhibits synaptic activity in the rods but not in the cones. Both techniques show that the effects produced by these concentrations of cobalt resemble those observed when retinas incubated in normal Ringers are illuminated with moderate intensities of light. Taken by themselves, the HRP results might be thought to reflect an ability of cobalt simply to interfere with endocytosis of the tracer, but the electrophysiological findings strongly suggest that this is not the case and that transmission is indeed affected. One explanation of these results is that cobalt is acting directly at the receptor terminals. In line with this is our finding that 1 or 2.4 mM cobalt has no obvious effect on the aspartate-isolated receptor potentials of either the dark or light adapted retina, which together with the results of others (Dacheux and Miller, 1976; Kaneko and Schimazaki, 1975; Schwartz. 1976; and Cervetto and Piccolino. 1974) suggests that at these concentrations cobalt does not act at the outer segment. However, our results with aspartate cannot yet be directly compared with our other findings since it is possible that cobalt and aspartate may interact in unknown ways. Our results on HRP uptake by receptors with aspartate Ringers, with and without cobalt, have been quite variable and cannot yet be confidently interpreted. We have seen some evidence of enhanced uptake by receptors with aspartate (cf. Ripps et al., 1976) and we know that aspartate can enhance the synaptic activity of cells in the inner plexiform layer (Evans er al., 1977). At 5mM, the receptor potentials are abolished in both the light and dark adapted retinas. One might anticipate that cobalt has an effect on the outer segment membrane, since it is known that calcium applied to the outer segment of the dark adapted receptor causes a hyperpolarization (Hagins and Yoshikami, 1974; Brown and Pinto, 1974). However, we might simply be observing cytotoxic effects. It seems reasonable to speculate that the effects of 1 and 2.4rnM cobalt may be on the mechanism of transmitter release or on those coupling potential changes to synaptic activity. Cobalt inhibits passive calcium infiux associated with the action potential in the barnacle giant muscle fiber (Hagjwara and Takahashi, 1967) and in neurons of the Aplysia visceral ganglion (Geduldig and Junge, 1968); it also inhibits the slow inward calcium current of mammalian cardiac muscle fibers (Kohlhardt, Bauer, Krause and Fleckenstein, 1973). Cobalt could have similar effects on the receptors. The fact that rods and cones differ in sensitivity to the same concentration of cobalt suggests that there might be some differences in the mechanisms governing transmitter release or related phenomena. We do not yet know whether this is simply a difference in concentration dependency or whether rods and cones differ in their permeability to cobalt, or whether a more profound difference exists. These findings do however raise the possibility
C.
HOOD and ERIC HOL?I.UA>
that the differential involvement of rods and cones in retinal functions at different lighhr intsnsiaes depends not only on differences in their outer segments. but also on differences at the level of the terminals. Our findings that 1-2mM cobalt does not a&c: the receptor potentials of rods or cones agree with the results of prior investigations with intracellular recordings on Necrurus. carp and turtle (Dacheuu and Miller, 1976; Kaneko and Schimazaki. 1975: Schwartz, 1976; and Cervetto and Piccotino. 197-1). However. the reports by the same investigators that cobalt abolishes sensitivity of horizontal and bipolar cells to light flashes could be interpreted as indicating a complete block in synaptic transmission from the photoreceptors. This would differ from our results which demonstrate a differential effect on the two receptor types. If so, the differences might simply refkct variations among species. For example, as judged both electrophysiologically and by tracer uptake (Dowling and Ripps. 1973, and Ripps et al., 1976) high concentrations of magnesium seem to abolish synaptic activity in skate retina, but we have found that concentrations of magnesium as high as 2OmS1 do not similarly inhibit HRP uptake into the photoreceptors of the frog. However, it should be noted that the cytochemical methods provide a more direct evaluation of the behavior of photoreceptor terminals than do the available recording procedures’and the); also permit the study of very large numbers of cells. Inevitably, small sample sizes are involved in intraceilular recording and it is conceivable that the horizontal and bipolar cells studied did not include cells driven by cones. Finally our findings may be useful in further studies on the retina. The fact that we can turn “off rod synaptic activity in the dark without drastically altering the cones and the postsynaptic cells should facilitate future work. Acknowledgements-We thank Fe Reyes for her skiiful technical assistance. The research was supported by NLH grant NS09475 to EH and NIH grant EY01877 to DCH. REFERENCES Brown J. E. and Pinto H. (1974) Ionic mechanism for the
photoreceptor potential of the retina of &fo mntimcf. J. Physiol.. Land. 236. 575-591. Cervetto L. and Piccolino M. (1974) Synaptic transmission between photoreceptors and horizontal cells in the turtle retina. Science 183, 417-419. Dacheux R. and Miller R. (1976) Photoreceptor-bipolar cell transmission in the perfused retina eyecup of the mudpuppy. Science 191, 963-964. Dowling 1. E. and Ripps H. (1973) Effect of magnesium on horizontal cell activity in skate retina. Nature 242. 101-103. Evans J. A.. Hood D. C. and Holtzman E. (1977) Effects of aspartate and cobalt on horseradish peroxidasc (HRP) up&e in frog retina. Assoc. Res. Vis..Oprhalmol.. 1977. (Invest. Oohtkatmol. ADI%. 1977, SUPPI., II. 26.) Giduldig 6. and Jun& D. (1968) sodium and calcium components of action potentials in the Aplysia giant neurone. J. Physiol., Land. 199, 347-365. Hagins W. A. and Yoshikami S. (1974) A role for calcium in excitation of retinal rods and cones. Exp. Eye Res. 18, 29%305.
Differential effects of cobalt ions Hagiwara S. and Takahashi K. (1967) Surface density of. calcium ions and calcium spikes in the barnacle muscle fiber membrane. J. gen. Physiof. SO. 583-601. Holtzman E. (1977) The origin and fate of secretory packages. especially synaptic vesicles. Neuroscience 2. 327-355. Hood D. C. and Hock P. A. (1973) Dark adaptation of the frog’s rods. Vision Res lj. 1943-1951. Hood D. C. and Hock P. A. (1975) Lit&t adaotation of the receptors: Increment threshold Functions for the frog’s rods and cones. Vision Res. 15, 545-553. Kaneko A. and Shimazaki H. (1975) Synaptic transmission from photoreceptors to bipolar and horizontal cells in the carp retina. Cold Spring Harbour Symposium 40. 537-546.
Katz B. and Miledi R. (1965) The effect of calcium on acetylcholine release from motor nerve terminals. Proc. R, Sot. B. 161. 496503. Kohlhardt M.. Batter B., Krause H. and Fleckenstein A. (1973) Selective inhibition of the transmembrane calcium conductivity of mammalian myocardial fibres by Ni. Co
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and Mn ions. PjKgers Arch. ger Physiol. 338. 115-123. Llinas R. and Nicholson C. (1975) Calcium role in depolarization-secretion coupling: an aequotin study in squid giant synapse. Proc. Mtn. Acad Sci., U.S.A. 72. 187-190. Ripps H., Shakib M. and McDonald E. D. (1976) Peroxidase uptake by photoreceptor terminals of the skate retina. J. Cel[ Biol. 70. 8696. Schacher S.. Holtzman E. and Hood D. C. (1974) Uptake of horseradish peroxidase by frog photoreceptor synapses in the dark and in the light. Nature 249. 261-263. Schacher S.. Hohzman E. and Hood D. C. (1976) Synaptic activity of frog retinal photoreceptors: peroxidase uptake study. J. Cell Biol. 70, 178-192. Schwartz E. A. (1976) Electrical properties of the rod syncytium in the retina of the turtle. J. Physiol., Land. 257, 379-406.
Weakly J. (1973) The action of cobalt ions on neuromuscular transmission in the frog. 1. Physiol.. LO~. 2% 597-612.