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Responses of the amacrine cell to optic nerve stimulation in the frog retina
RESPONSES OF THE AMACRINE CELL TO OPTIC STIMULATION IN THE FROG RETINA
NERVE
NOLI~YOSHI MATSCWTO Department of Biophysical Engineering. Faculty of Engineering Science. Osaka University. Toyonaka. Osaka. Japan (Received I December 1973) Abstract-Responses of the frog amacrine cell to optic nerve stimulation were examined. Amacrine cells of “on-off and “off” type responded to optic nerve stimulation in an all or none fashion. When double stimulation was applied the latency to test stimulus was reduced as stimulus interval was shortened (less than about 200 msec). At shorter stimulus intervals (less than several milliseconds). prolongation of the latency was observed. An assumption was proposed that the ganglion cell dendrite makes contact back onto the amacrine cell. and the reduction of latency is caused by facilitation in dendritic invasion of the impulse in the ganglion cell.
INTRODL’CTION
The frog retina has been known to process visual information to highly complexed manner (Lettvin. Maturamt. McCulloch and Pitts. lY5Y: Maturand. Lettvin, McCulloch and Pitts. 1960). Electron microscopic
studies by Dowiing (196X)suggest that the complex information processing is attributed to the complexity of connections in the inner plexiform layer. He found that the number of synapses between bipolar-amacrine. amacrine-ganglion and amacrine-amacrine is considerably high in the frog retina compared to other vertebrates. such as the fish and the primate. These results indicate that the role of the amacrine ceil in the frog retina may be considerably important in the visual information processing. Spike activities in the inner nuclear layer of the frog retina have been reported by some authors (Naka. Inoma. Kosugi and Tong. 1960; Kaneko and Hashimoto. 196X: Nye and Naka. 1971: Matsumoto and Naka. 1972). Matsumoto and Naka (1972) identified most of the neuronal types in the frog retina by Procion dye injection technique. The results revealed that the spike producing cells in the inner nuclear layer are amacrine cells. They also found that the spike activity of the frog amacrine cell is conspicuous compared to that of goldfish (Kaneko, 1970) and mudpuppy (Werblin and Dowling, 1969). In the present study. we inveitigate the responses of amacrine cells to optic nerve stimulation in order to elucidate the function of the amacrine cell in the frog retina. METHODS
The bullfrog (Ram pipiens) was decapitated and the eye was taken out with the optic nerve. which was cut where it enters the cranium. After the lens and vitreous humour were
removed. the eyecup was placed on the stage as shown in Fig. I. The stimulus electrode was attached to the optic nerve just below the bottom of the eyecup. Dissection wus carried out under room light and the preparation was ddrkadapted for more than 30 min before experiment. The experiment was performed at room tempcraturc of 19 2.3 C. A two-channel white light photostimulator was used. One channel gave a spot of light (0.3 mm in diameter) and the other gave an annulus (inner diameter. I.0 mm and outer diameter. 1 mm) which was concentric to the spot. Electrodes were filled with I Y potassium citrate. The resistance of electrodes for intracellular recording ranged from 200 to 500 Ma. Electrodes for extracellular recordine had a resistance of about IO MR. Identification of the ccl1 was made by the criterion ohtained in the dye injecting experiment (Matsumoto and Naka. 19721.
u
ON
Fig. I. Experimental arrangement for electrical stimulation of the optic nerve. The eyecup preparation was placed on the reference electrode (RE) made of a silver plate. A pair of silver wires was attached to the optic nerve (ON) as a stimulating electrode (SE). A recording microelectrode (ME) was connected to a high input impedance pre-amplifier (A).
For optic *idth
nerve stlmulatton.
*asapplied.
a current
In case ofdoubie
nerve. supramaximal
pulse of 0.0~ msec
stimulation
of the optic (a)
intensity was used.
_L
RESL LT
Some morphological and physiological types of amacrine cells in the frog retina were identified in our previous study (Matsumoto and Naka. 1972). Three response types were shown in Fig. 6 of the reference cited above. In this paper. we shall refer to the unit a. h and c in the figure as’type ,-i. B and C amacrine cell. respectively. For central illumination. unit u showed spike potentials superimposed on transient depolarization at “on” and “off’ of light application. unit b showed sustained hyperpolarization and unit c showed spike activity at “off’. In the present study. we shall mainly examine type .4 responses. Examples of type .-I response to light stimulation are shown in Fig. 2 (a. b and c). Spike activity by central illumination (a) is inhibited (c) by simultaneous application of annular illumination (b). Rr.sporrsc~.sf0 opric IIPI’CL’ sfimhfim Figure 2(d) shows the early part of “on” response of the t)pe A amacrine cell. When the recorded amplitude of the response to light stimulation was greater than 20 mV, optic nerve stimulation always evoked an all or none response. Eight such units were found in 111t’.serIes ol’ experiments. Meanwhile. some deteriorated cells did not respond to optic nerve stimulation. An example of the responses is shown in Fig. Z(e). which was recorded from the same unit as (d). Amplitude of the response was typically I&Z0 mV. Time to peak Has about I msec. and the whole time course was B- 100 msec. Conduction velocity of the response was approxitnately I ,3 m/set. Type C (“off’ type) amacrine cell (two cells) also produced an all or none response to optic nerve stimu-
(a)
! L-h’. i : w-i+--
FIN. 2. Intracellul~~r recordmgs ol’amacrme cell IO ltght and electrical stimulation. Recording (a) shows the responx to a spor ol’light. (b) shows the response to annular illuminntion and (c) is the response to simultaneous application of spot and annulus. Recording(d) shows the early part of”on” response IO a spot of light. Recording (e) is the response evoked bl electrical stimulation of the optic nerve. The response is m an all or none hshion. Responses (a). (b) and IC). and (dl and (e) were recorded from different cells.
lation. but no recognizable response was recorded from type B (sustained type) amacrine cells (five cells were examined). Several bipolar and horizontal cells were examined. and they did not respond to optic nerve stimulation.
Figure 3 (a and b) shows examples of the response to double stimulation. Traces in (a) are recordings of extracellular unit response recorded just before impalement of the amacrine cell. Traces in (b) are intracellular recordings from the same unit. Remarkable reduction and prolongation of the latency was observed in the response to test stimulus. In Fig. 3(c). the latency of the second response of the same unit as in (b) are plotted against stimulus interval. The latency begins to decrease from the control (broken line) when stimulus
(cl
(b)
+-J-=t
Fig. 2. Extracellular and intracellular responses of an amacrine cell to various intervals of double stimulus are shown in (a) and (b) respectively. Latencies of intracellular responses to test stimulus are plotted against stimulus interval in (c). These points were obtained from the same cell as in (b).
Responses of the amaaine celf mterval becomes shorter than about 200 msec. The amount of reduction in the latecy is maximum (about O-5msec) when the stimulus interval is between 10 and j msec. and the latency incnases in shorter stimulus interval. The curve intersects the control line at about the 4-msec stimulus interval, and the Iatency is prolonged exponentially against decreasing stimufus interval. The second response disappears at about the 2msec stimulus interval. where the amount of proiongation in the latency is about 1 msec. The changes of the latency in the extracellular unit responses [Fig. 3(a)] and the intraceRuar response [Fig. 3(b)] were in good agreement. The same measurements were performed for four cells. and similar resuits were obtained from ail ceils.
In order to examine where such reduction and prolon~tion of the latency occur. we electrically stimulated the optic nerve bundle and recorded responses from intraretinal optic nerve fibers and ganglion .cells Tasaki (1970) identified three distinct optic nerve fiber groups of dierent conduction veIocities; the A, B and C groups. Their average conduction velocities were 3-3. 1.4and O-24m/set within the optic nerve bundle. Intraretinal conduction velocities of the A. B and C fibers were 1-O.O-59and @24 m/set. They conciuded that the group A and 5 fibers are myeI~nated and group C fibers are unmyeiinated. Although we have not measured precise conduction velocities the latency of the response at .amacrine cell indicates that it is driven by group A fiber. In this study. responses of group A fibers were mainly investigated.
When the tip of the eiectrode touc~d on the surface of the retina. field potential was readily recorded which is considered as the response from optic nerve fibers. In our eyecup preptmtions group C responses were not recorded. Group A responses were consistentty recorded. but occasionally group Et responses were not found. In the top trace of Fig. +al a and b show the group A and 5 fiber response respectively. As the eiectrode was advanced further from the surface of the retina, magnitude of the field potential was reduced and then all or none unit response was isotated at a depthof~~~~m from the surface. When the rtectrode was advanced deeper. unit response disappared and the magnitude of the field potential was reduced abruptly. fudging from the depth of the recording. the unit responses are considered to be recorded from the ganglion cell layer. Double stimulation experiments were performed for about ten group A fibers and ten ganglion cel,ls which were driven by group A fibers. Traces in Fig. 4(a) show field potentials from group A and B fibers to various stimulus intervals, Traces in Fig. 4(b) show extracellutar unit responses of gangiion cell of group A. Latency changes of the second response at intraretinal optic nerve fibers (cross)and at a ganglion cell (closed circle) are plotted in Fig. j(c). For comparison. latencies at amacrine ceil are plotted here again (open circie). These points were obtained from the neighbouring pIace. but not by a singIe ~net~tion. In either of optic nerve fibers and ganglion cells, prolongation of latenties was observed and it seemed that the main part of prolongation occurred in the ganglion cell layer rather than in the optic nerve.fib-ers. Reduction of the latency
Fig. 4. Field potentials to double stimulation recorded from the optic nerve layer are shown in (a). Responses ofgroup A and 3 fibers are marked by a and b respectively. Extracellular unit responses of ganglion cell to double st~muiat~on are shown in (bl. In Ic) latency change of the response to test stimulus is plotted against stimulus interval. Late&es of group A fibers and ganglion ceil are shown by a cross f x )
and closed circle (0) respectively. 0pcn circles (0) are the late&es at the amacrine cell, which were brought from fig. 3@. The broken fine indicates the Iatenq to the conditioning stimufus. Upward dtfieotion from the broken line indiates the prolongation of the latency and downward deflection indicates the reduction of the latency.
(a)
(b)
I
20 mV
-%khF-
Fig. 5. EtTectof current on the response of an amacrine cell. Upper traces are controls. middle traces arc responses under I nA of depolarizing current and bottom traces are responses under I nA of hyprrpolarizing current. In lat. sole effect of current is shown. Horizontal line at the bottom shows the nppro\im;tte application of current. In Ib). light stimulation was applied under steady current. Horizontal line at the bottom shows the approximate duration of light application. In (ct. the optic nerve was electrically stimulated under steady current.
was never observed in any response at optic nerve or ganglion cell layer. This finding indicates that reduction of the latency occurs in the region more distal to the optic nerve or the ganglion cell layer.
Effects of current on the responses of amacrine cell are shown in Fig. 5. In each column. upper traces are controls. middle traces are responses under depolarizing current of I nA and bottom traces are responses under hyperpolarizing current of I nA. (a) shows the sole effect ofcurrent. The horizontal line at the bottom indicates the approximate interval of current application. (b) shows the effect of current on the response to light stimulation. The horizontal line at the bottom indicates the approximate duration of light application. (c) shows the effect of current on the response to optic nerve stimulation. Amplitudes of the responses in (b) and (c) becomes greater by applying hyperpolarizing current and smaller by depolarizing current. One nanoamptre of hyperpolarizing current changed the amplitude about two times as much as that of the control. but I nA of depolarizing current did not change the amplitude so much. A depolarizing current greater than I nA made the electrode unstable. DiSCUSSiON
Kaneko and Hashimoto (1968) found spike producing cells in the inner nuclear layer of the frog retina. and they applied optic nerve stimulation to examine the responsiveness. According to their experiment most of the spike producing cells were unresponsive to
optic nerve stimulation. They also found spike producing cells in the inner nuclear layer of the carp retina (Kaneko and Hashimoto. 19691 but these units (other than S-potentials or receptor potentials) did not respond to optic nerve stimulation whether or not it had spike activity. In our present study, however. many of the type A amacrine cells and the type C amacrine cells in the frog retina responded to optic nerve stimulation. Non spike producing units such as bipolar. horizontal or type B amacrine cells in the frog retina did not respond to optic nerve stimulation. We will now discuss the mechanism of how the amacrine cell produces an all or none response to optic nerve stimulation and how the latency changes depends on the stimulus interval. There are some possibilities which might explain the results of our experiments, i.e.: (I) The cell is actually the displaced ganglion cell. (2) An axon collateral of ganglion cell terminates on the amacrine cell. (3) An efferent fiber terminates on the amacrine cell. (4) The dendrite of ganglion cell terminates on the amacrine cell. Displaced lib
yaqlion
cell. a.von collatrral
arld rfjPrrrtt
A displaced ganglion cell in the frog retina has been shown in the Golgi staining by Caja! (19 I I). We successfully injected Procion dye into more than ten cells of “on-off” type in the inner nuclear layer of the frog retina, but no axon was found in any staining (Matsumoto and Naka. 1972 and unpublished data), even when the fine dendrites were well stained. Matsumoto
513
Responsesof the amacrine cell
and Naka (1971) also failed to find a displaced ganglion ceil in their Golgi stainings. These facts indicate that the possibility ofencountering the displaced gangfion cell in the physiological study would also be very low. Axon collaterals in the frog retina have not been described by Cajal (1911) or other authors. Matsumoto and Naka (1972) failed to find axon collateral of the ganglion cell in their Golgi or Procion stainings. Possible existence of efferent fibers have been one of the most interesting problems in the study of visual system of the vertebrate retina. At present, conclusive evidence indicating ihe existence of efferent fibers in the retina is known only in the bird. although some authors suggested inconclusive evidence of efferent fibers in the retina of the frog or some other animals (Branston and Fleming, 1968; Maturana. 1958; cf. Stell 1972). In the pigeon retina; efferent fibers which terminate on the amacrine cell or displaced ganglion cell have been demonstrated in the histological studies (Maturana and Frenk. I965). If we take any of the assumptions (I). (2) or (3). we can explain the origin of the aft or none response to optic nerve stimulation. The reduction in the latency. however, cannot be interpreted by any of these assumptions, as it was not observed at intraretinaf optic nerve layer and ganglion cell layer. Although we have excluded the possibility that the response of the amacrine cell to optic nerve stimulation is caused by an efferent fiber which may be included in the group A fibers it may be likely that group B or C fibers contain efferent fibers.
Finally. we introduce some other mechanism which can interpret the results of our experiment. Here, we propose the following mechanism. That is. an impulse generated at the optic nerve fiber invades the ganglion cell soma and then the dendrite, which makes synaptic contacts onto the amacrine cell. Some part of the prolongation of the latency occurs in the optic nerve, and some part occurs in IS-SD invasion of the ganglion cell. Reduction of the latency in the double stimulus experiment is interpreted by facilitation of the impulse in the dendrite, i.e. depolarization by the conditioning impulse in the dendrite prolongs some tens of milliseconds or more because of the large time constant of the membrane. and it facilitates the propagation of the following impulses. which reduces the latency at the amacrine cell. As the stimulus interval becomes shorter, prolongation of the latency overcomes the facilitation. In the cortex of a chronically deafferented and a normal cat, parallel fiber stimulation facilitates the response of the Purkinje cell to an antidromic volley at the level of the dendrite (Eccles. Llinas and Sasaki, 1966; Eccles Sasaki and Strata, 1966). As seen in Fig. 5 the effect of current on the responses to optic nerve stimulation is considerably large. We presume that the response of an amacrine
ceil to optic nerve stimulation is generated by synaptictransmissionratherthanbyelect.ricaIcoupIing. It has been known that the granule all in the olfactory bulb resemble the amacrine a11 in the retina. It is interesting to point that Rail. Shepherd Reese and Brightman (1966) suggested the existence of dendrodendritic synaptic pathways between mitral and granule cells of the rat olfactory bulb. According to their analysis the mitral dendrites synapticaIly excite granule dendrites and granule dendrites then synaptically inhibit mitral dendrites. In the frog retina and many other vertebrate retinas. amacrine processes make synapses back onto bipolar terminals (Dowling, 1968; Dowling and Boycott. 1966: Dowling and Werblin. 19691 which are termed reciprocal synapses. It suggests the existence of a feedback interaction between bipolar and amacrine cell. In fact transient depolarization in the amacrine cell may be interpreted by a negative feedback interaction between a bipolar and amacrine cell (Werblin and Dowling. 1969; Toyoda. Hashimoto and Ohtsu, 1973). Baylor, Fuortes and O’Bryan (1971) demonstrated a negative feedback interaction between receptor and horizontal cell in the turtle retina. Conspicuous spike activity which is observed in the amacrine cell of the frog retina may be attributed to the presumed existence of positive feedback interaction between amacrine and ganglion cells. ~.ckrlowlrd~~:qr,nr,lr~-I wish lo Tsukahara for his valuable throughout the investigation. a Grant. Neuroscience 811010. tion.
express my thanks to Dr. N. suggestions and discussions This work was supported by from the Ministry of Educe-
REFERENCES
Baylor D. A.. Fuortes M. G. F. and O’Btyan P. M. (1971) Receptive fields ofcones in the retina of the turtle. J. Phy siof. 2t4,265-294.
Branston N. M. and Fleming D. C. (1968) Efferent fibers in the frog optic nerve. Expl Neurot. 20,61 t-623. Cajal S. R. (1911) ~isru~u~i~ du Syskme Scrveux de l’Homme er des VertGbrds. Maloine. Paris. Dowling J. E. (1968) Synaptic organization of the frog retina: an electron microscopic analysis comparing the retinas of frogs and primates. Proc. R. .Soc. B 170, 201-
228. Dowling J. E. and Boycott B. B. (1966) Organization of the primate retina: electron microscopy. Proc. R. Sot. B 166, 8Clll. Dowling J. E. and Werblin F. S. (1968) Organization of the retina of the mudpuppy, Nrcrurus mnculos~s,I. Synaptic structure. J. NrurophysioL 32, 3 15-338. Eccles J. C.. Llinas R. and Sasaki K. (1966) The action of antidromic impulses on the cerebellar Purkinje cells. J. Physiol. 182. 3 16345. Eccles J. C.. Sasaki K. and Strata P. (1966) The profiles of physiological events produced by a parallel fiber volley in the cerebellar cortex. Expl Brain Rex 2, 18-34. Kaneko A. (1970) Physiologi~i and morphologj~l identification of horizontal, bipolar and amacrine cells in goldfish retina. J. Physiol. 207, 623-633.
514
NOWYOSHI
Kaneko A. and Hashimoto H. (1968) Localization of spikeproducing cells in the frog retina. Cisim Rrs. 8, 159-262. Kaneko A. and Hashimoto H. (1969) Electrophysiological study of single neurons in the inner nuclear layer of the carp retina. &ion Res. 9. 37-59. Lettvin J. Y., Maturana H. R., McCulloch W. S. and Pitts W. H. (1959) What the frog’s eye tells the frog’s brain. Proc. Inst. Radio Engrs 41, i94(r 195 I. Matsumoto N. and Naka K. I. (1972) Identifiction of intracellular responses in the frog retina. Brain Rrs. 42. 59-7 I. Maturana H. R. (1958) Etferent fibers in the optic nerve of the toad. J. Anat. 92. 2 l-17. Maturana H. R. and Frenk S. (1965) Synaptic conncetions of the centrifugalfibers in the pigeon retina. Science 150. 359-361. Maturana H. R., Lettvin J. Y.. McCulloch W. S. and Pitts W. H. (1960) Anatomy and physiology of vision in the frog (Rana pipiens). J. gem Physiol. 43, I29- I7 I. Naka K. 1.. lnoma S.. Kosugi Y. and Tong C. (1960) Record-
bhTSUMOT0
ing of action
potentials from single cells in the frog retina,
Jap. J. Physiol.
IO, IX-UZ.
Nye P. W. and Naka K. I. (197 I) The dynamics of inhibitory interaction in a frog receptive field: a paradigm of paracontrast. tirion Rrs. 11. 377-392. Rail W.. Shepherd G. M.. Reese T. S. and Brightman M. W. (1966) Dendrodendritic synaptic pathway for inhibition in the olfactory bulb. E.r& S&l: 14. @56. Stell W. K. (1972) The morphological organization of the vertebrate retina. In Tl~r Harrdhook o/‘Smwr~ Plr~siolmg~ (Edited by Fuortes M. G. F.1. VII I. Springer. Berlin. Tasaki K. (1970) Three fiber groups in the frog optic nerve. J. physiol. Sm. Japarl 32. 566567.
Toyoda J.. Hashimoto H. and Ohtsu K. (1973) Bipolaramacrine transmission in the carp retina. Cisiw~ Rrs. 13, 295-307. Werblin F. S. and Dowlinn
J. E. (1969) Organization of retina of the mudpuppy. .\ccru).i~s rrrcrculos~ts.II. Intracellular recording J. .%‘ricroph~sic~/. 32, 339-355.
NERVE
NOLI~YOSHI MATSCWTO Department of Biophysical Engineering. Faculty of Engineering Science. Osaka University. Toyonaka. Osaka. Japan (Received I December 1973) Abstract-Responses of the frog amacrine cell to optic nerve stimulation were examined. Amacrine cells of “on-off and “off” type responded to optic nerve stimulation in an all or none fashion. When double stimulation was applied the latency to test stimulus was reduced as stimulus interval was shortened (less than about 200 msec). At shorter stimulus intervals (less than several milliseconds). prolongation of the latency was observed. An assumption was proposed that the ganglion cell dendrite makes contact back onto the amacrine cell. and the reduction of latency is caused by facilitation in dendritic invasion of the impulse in the ganglion cell.
INTRODL’CTION
The frog retina has been known to process visual information to highly complexed manner (Lettvin. Maturamt. McCulloch and Pitts. lY5Y: Maturand. Lettvin, McCulloch and Pitts. 1960). Electron microscopic
studies by Dowiing (196X)suggest that the complex information processing is attributed to the complexity of connections in the inner plexiform layer. He found that the number of synapses between bipolar-amacrine. amacrine-ganglion and amacrine-amacrine is considerably high in the frog retina compared to other vertebrates. such as the fish and the primate. These results indicate that the role of the amacrine ceil in the frog retina may be considerably important in the visual information processing. Spike activities in the inner nuclear layer of the frog retina have been reported by some authors (Naka. Inoma. Kosugi and Tong. 1960; Kaneko and Hashimoto. 196X: Nye and Naka. 1971: Matsumoto and Naka. 1972). Matsumoto and Naka (1972) identified most of the neuronal types in the frog retina by Procion dye injection technique. The results revealed that the spike producing cells in the inner nuclear layer are amacrine cells. They also found that the spike activity of the frog amacrine cell is conspicuous compared to that of goldfish (Kaneko, 1970) and mudpuppy (Werblin and Dowling, 1969). In the present study. we inveitigate the responses of amacrine cells to optic nerve stimulation in order to elucidate the function of the amacrine cell in the frog retina. METHODS
The bullfrog (Ram pipiens) was decapitated and the eye was taken out with the optic nerve. which was cut where it enters the cranium. After the lens and vitreous humour were
removed. the eyecup was placed on the stage as shown in Fig. I. The stimulus electrode was attached to the optic nerve just below the bottom of the eyecup. Dissection wus carried out under room light and the preparation was ddrkadapted for more than 30 min before experiment. The experiment was performed at room tempcraturc of 19 2.3 C. A two-channel white light photostimulator was used. One channel gave a spot of light (0.3 mm in diameter) and the other gave an annulus (inner diameter. I.0 mm and outer diameter. 1 mm) which was concentric to the spot. Electrodes were filled with I Y potassium citrate. The resistance of electrodes for intracellular recording ranged from 200 to 500 Ma. Electrodes for extracellular recordine had a resistance of about IO MR. Identification of the ccl1 was made by the criterion ohtained in the dye injecting experiment (Matsumoto and Naka. 19721.
u
ON
Fig. I. Experimental arrangement for electrical stimulation of the optic nerve. The eyecup preparation was placed on the reference electrode (RE) made of a silver plate. A pair of silver wires was attached to the optic nerve (ON) as a stimulating electrode (SE). A recording microelectrode (ME) was connected to a high input impedance pre-amplifier (A).
For optic *idth
nerve stlmulatton.
*asapplied.
a current
In case ofdoubie
nerve. supramaximal
pulse of 0.0~ msec
stimulation
of the optic (a)
intensity was used.
_L
RESL LT
Some morphological and physiological types of amacrine cells in the frog retina were identified in our previous study (Matsumoto and Naka. 1972). Three response types were shown in Fig. 6 of the reference cited above. In this paper. we shall refer to the unit a. h and c in the figure as’type ,-i. B and C amacrine cell. respectively. For central illumination. unit u showed spike potentials superimposed on transient depolarization at “on” and “off’ of light application. unit b showed sustained hyperpolarization and unit c showed spike activity at “off’. In the present study. we shall mainly examine type .4 responses. Examples of type .-I response to light stimulation are shown in Fig. 2 (a. b and c). Spike activity by central illumination (a) is inhibited (c) by simultaneous application of annular illumination (b). Rr.sporrsc~.sf0 opric IIPI’CL’ sfimhfim Figure 2(d) shows the early part of “on” response of the t)pe A amacrine cell. When the recorded amplitude of the response to light stimulation was greater than 20 mV, optic nerve stimulation always evoked an all or none response. Eight such units were found in 111t’.serIes ol’ experiments. Meanwhile. some deteriorated cells did not respond to optic nerve stimulation. An example of the responses is shown in Fig. Z(e). which was recorded from the same unit as (d). Amplitude of the response was typically I&Z0 mV. Time to peak Has about I msec. and the whole time course was B- 100 msec. Conduction velocity of the response was approxitnately I ,3 m/set. Type C (“off’ type) amacrine cell (two cells) also produced an all or none response to optic nerve stimu-
(a)
! L-h’. i : w-i+--
FIN. 2. Intracellul~~r recordmgs ol’amacrme cell IO ltght and electrical stimulation. Recording (a) shows the responx to a spor ol’light. (b) shows the response to annular illuminntion and (c) is the response to simultaneous application of spot and annulus. Recording(d) shows the early part of”on” response IO a spot of light. Recording (e) is the response evoked bl electrical stimulation of the optic nerve. The response is m an all or none hshion. Responses (a). (b) and IC). and (dl and (e) were recorded from different cells.
lation. but no recognizable response was recorded from type B (sustained type) amacrine cells (five cells were examined). Several bipolar and horizontal cells were examined. and they did not respond to optic nerve stimulation.
Figure 3 (a and b) shows examples of the response to double stimulation. Traces in (a) are recordings of extracellular unit response recorded just before impalement of the amacrine cell. Traces in (b) are intracellular recordings from the same unit. Remarkable reduction and prolongation of the latency was observed in the response to test stimulus. In Fig. 3(c). the latency of the second response of the same unit as in (b) are plotted against stimulus interval. The latency begins to decrease from the control (broken line) when stimulus
(cl
(b)
+-J-=t
Fig. 2. Extracellular and intracellular responses of an amacrine cell to various intervals of double stimulus are shown in (a) and (b) respectively. Latencies of intracellular responses to test stimulus are plotted against stimulus interval in (c). These points were obtained from the same cell as in (b).
Responses of the amaaine celf mterval becomes shorter than about 200 msec. The amount of reduction in the latecy is maximum (about O-5msec) when the stimulus interval is between 10 and j msec. and the latency incnases in shorter stimulus interval. The curve intersects the control line at about the 4-msec stimulus interval, and the Iatency is prolonged exponentially against decreasing stimufus interval. The second response disappears at about the 2msec stimulus interval. where the amount of proiongation in the latency is about 1 msec. The changes of the latency in the extracellular unit responses [Fig. 3(a)] and the intraceRuar response [Fig. 3(b)] were in good agreement. The same measurements were performed for four cells. and similar resuits were obtained from ail ceils.
In order to examine where such reduction and prolon~tion of the latency occur. we electrically stimulated the optic nerve bundle and recorded responses from intraretinal optic nerve fibers and ganglion .cells Tasaki (1970) identified three distinct optic nerve fiber groups of dierent conduction veIocities; the A, B and C groups. Their average conduction velocities were 3-3. 1.4and O-24m/set within the optic nerve bundle. Intraretinal conduction velocities of the A. B and C fibers were 1-O.O-59and @24 m/set. They conciuded that the group A and 5 fibers are myeI~nated and group C fibers are unmyeiinated. Although we have not measured precise conduction velocities the latency of the response at .amacrine cell indicates that it is driven by group A fiber. In this study. responses of group A fibers were mainly investigated.
When the tip of the eiectrode touc~d on the surface of the retina. field potential was readily recorded which is considered as the response from optic nerve fibers. In our eyecup preptmtions group C responses were not recorded. Group A responses were consistentty recorded. but occasionally group Et responses were not found. In the top trace of Fig. +al a and b show the group A and 5 fiber response respectively. As the eiectrode was advanced further from the surface of the retina, magnitude of the field potential was reduced and then all or none unit response was isotated at a depthof~~~~m from the surface. When the rtectrode was advanced deeper. unit response disappared and the magnitude of the field potential was reduced abruptly. fudging from the depth of the recording. the unit responses are considered to be recorded from the ganglion cell layer. Double stimulation experiments were performed for about ten group A fibers and ten ganglion cel,ls which were driven by group A fibers. Traces in Fig. 4(a) show field potentials from group A and B fibers to various stimulus intervals, Traces in Fig. 4(b) show extracellutar unit responses of gangiion cell of group A. Latency changes of the second response at intraretinal optic nerve fibers (cross)and at a ganglion cell (closed circle) are plotted in Fig. j(c). For comparison. latencies at amacrine ceil are plotted here again (open circie). These points were obtained from the neighbouring pIace. but not by a singIe ~net~tion. In either of optic nerve fibers and ganglion cells, prolongation of latenties was observed and it seemed that the main part of prolongation occurred in the ganglion cell layer rather than in the optic nerve.fib-ers. Reduction of the latency
Fig. 4. Field potentials to double stimulation recorded from the optic nerve layer are shown in (a). Responses ofgroup A and 3 fibers are marked by a and b respectively. Extracellular unit responses of ganglion cell to double st~muiat~on are shown in (bl. In Ic) latency change of the response to test stimulus is plotted against stimulus interval. Late&es of group A fibers and ganglion ceil are shown by a cross f x )
and closed circle (0) respectively. 0pcn circles (0) are the late&es at the amacrine cell, which were brought from fig. 3@. The broken fine indicates the Iatenq to the conditioning stimufus. Upward dtfieotion from the broken line indiates the prolongation of the latency and downward deflection indicates the reduction of the latency.
(a)
(b)
I
20 mV
-%khF-
Fig. 5. EtTectof current on the response of an amacrine cell. Upper traces are controls. middle traces arc responses under I nA of depolarizing current and bottom traces are responses under I nA of hyprrpolarizing current. In lat. sole effect of current is shown. Horizontal line at the bottom shows the nppro\im;tte application of current. In Ib). light stimulation was applied under steady current. Horizontal line at the bottom shows the approximate duration of light application. In (ct. the optic nerve was electrically stimulated under steady current.
was never observed in any response at optic nerve or ganglion cell layer. This finding indicates that reduction of the latency occurs in the region more distal to the optic nerve or the ganglion cell layer.
Effects of current on the responses of amacrine cell are shown in Fig. 5. In each column. upper traces are controls. middle traces are responses under depolarizing current of I nA and bottom traces are responses under hyperpolarizing current of I nA. (a) shows the sole effect ofcurrent. The horizontal line at the bottom indicates the approximate interval of current application. (b) shows the effect of current on the response to light stimulation. The horizontal line at the bottom indicates the approximate duration of light application. (c) shows the effect of current on the response to optic nerve stimulation. Amplitudes of the responses in (b) and (c) becomes greater by applying hyperpolarizing current and smaller by depolarizing current. One nanoamptre of hyperpolarizing current changed the amplitude about two times as much as that of the control. but I nA of depolarizing current did not change the amplitude so much. A depolarizing current greater than I nA made the electrode unstable. DiSCUSSiON
Kaneko and Hashimoto (1968) found spike producing cells in the inner nuclear layer of the frog retina. and they applied optic nerve stimulation to examine the responsiveness. According to their experiment most of the spike producing cells were unresponsive to
optic nerve stimulation. They also found spike producing cells in the inner nuclear layer of the carp retina (Kaneko and Hashimoto. 19691 but these units (other than S-potentials or receptor potentials) did not respond to optic nerve stimulation whether or not it had spike activity. In our present study, however. many of the type A amacrine cells and the type C amacrine cells in the frog retina responded to optic nerve stimulation. Non spike producing units such as bipolar. horizontal or type B amacrine cells in the frog retina did not respond to optic nerve stimulation. We will now discuss the mechanism of how the amacrine cell produces an all or none response to optic nerve stimulation and how the latency changes depends on the stimulus interval. There are some possibilities which might explain the results of our experiments, i.e.: (I) The cell is actually the displaced ganglion cell. (2) An axon collateral of ganglion cell terminates on the amacrine cell. (3) An efferent fiber terminates on the amacrine cell. (4) The dendrite of ganglion cell terminates on the amacrine cell. Displaced lib
yaqlion
cell. a.von collatrral
arld rfjPrrrtt
A displaced ganglion cell in the frog retina has been shown in the Golgi staining by Caja! (19 I I). We successfully injected Procion dye into more than ten cells of “on-off” type in the inner nuclear layer of the frog retina, but no axon was found in any staining (Matsumoto and Naka. 1972 and unpublished data), even when the fine dendrites were well stained. Matsumoto
513
Responsesof the amacrine cell
and Naka (1971) also failed to find a displaced ganglion ceil in their Golgi stainings. These facts indicate that the possibility ofencountering the displaced gangfion cell in the physiological study would also be very low. Axon collaterals in the frog retina have not been described by Cajal (1911) or other authors. Matsumoto and Naka (1972) failed to find axon collateral of the ganglion cell in their Golgi or Procion stainings. Possible existence of efferent fibers have been one of the most interesting problems in the study of visual system of the vertebrate retina. At present, conclusive evidence indicating ihe existence of efferent fibers in the retina is known only in the bird. although some authors suggested inconclusive evidence of efferent fibers in the retina of the frog or some other animals (Branston and Fleming, 1968; Maturana. 1958; cf. Stell 1972). In the pigeon retina; efferent fibers which terminate on the amacrine cell or displaced ganglion cell have been demonstrated in the histological studies (Maturana and Frenk. I965). If we take any of the assumptions (I). (2) or (3). we can explain the origin of the aft or none response to optic nerve stimulation. The reduction in the latency. however, cannot be interpreted by any of these assumptions, as it was not observed at intraretinaf optic nerve layer and ganglion cell layer. Although we have excluded the possibility that the response of the amacrine cell to optic nerve stimulation is caused by an efferent fiber which may be included in the group A fibers it may be likely that group B or C fibers contain efferent fibers.
Finally. we introduce some other mechanism which can interpret the results of our experiment. Here, we propose the following mechanism. That is. an impulse generated at the optic nerve fiber invades the ganglion cell soma and then the dendrite, which makes synaptic contacts onto the amacrine cell. Some part of the prolongation of the latency occurs in the optic nerve, and some part occurs in IS-SD invasion of the ganglion cell. Reduction of the latency in the double stimulus experiment is interpreted by facilitation of the impulse in the dendrite, i.e. depolarization by the conditioning impulse in the dendrite prolongs some tens of milliseconds or more because of the large time constant of the membrane. and it facilitates the propagation of the following impulses. which reduces the latency at the amacrine cell. As the stimulus interval becomes shorter, prolongation of the latency overcomes the facilitation. In the cortex of a chronically deafferented and a normal cat, parallel fiber stimulation facilitates the response of the Purkinje cell to an antidromic volley at the level of the dendrite (Eccles. Llinas and Sasaki, 1966; Eccles Sasaki and Strata, 1966). As seen in Fig. 5 the effect of current on the responses to optic nerve stimulation is considerably large. We presume that the response of an amacrine
ceil to optic nerve stimulation is generated by synaptictransmissionratherthanbyelect.ricaIcoupIing. It has been known that the granule all in the olfactory bulb resemble the amacrine a11 in the retina. It is interesting to point that Rail. Shepherd Reese and Brightman (1966) suggested the existence of dendrodendritic synaptic pathways between mitral and granule cells of the rat olfactory bulb. According to their analysis the mitral dendrites synapticaIly excite granule dendrites and granule dendrites then synaptically inhibit mitral dendrites. In the frog retina and many other vertebrate retinas. amacrine processes make synapses back onto bipolar terminals (Dowling, 1968; Dowling and Boycott. 1966: Dowling and Werblin. 19691 which are termed reciprocal synapses. It suggests the existence of a feedback interaction between bipolar and amacrine cell. In fact transient depolarization in the amacrine cell may be interpreted by a negative feedback interaction between a bipolar and amacrine cell (Werblin and Dowling. 1969; Toyoda. Hashimoto and Ohtsu, 1973). Baylor, Fuortes and O’Bryan (1971) demonstrated a negative feedback interaction between receptor and horizontal cell in the turtle retina. Conspicuous spike activity which is observed in the amacrine cell of the frog retina may be attributed to the presumed existence of positive feedback interaction between amacrine and ganglion cells. ~.ckrlowlrd~~:qr,nr,lr~-I wish lo Tsukahara for his valuable throughout the investigation. a Grant. Neuroscience 811010. tion.
express my thanks to Dr. N. suggestions and discussions This work was supported by from the Ministry of Educe-
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