ii’sirrrr Rex. Vol. IS. pp. I339-1.144. Pergilmon Press 1975. Printed in Great Britain
THE RESPONSES OF THE RETINAL GANGLION CELLS OF THE F’ROG J. D. MORRISON’ Department of Physiology. Medical School. The University. Newcastle-upon-Tyne. NE1 7RU. England
Abstract-Three groups of units were recorded from the retina of Rma esculenta. These were: (a) Units which responded to aare wave modulated lightstimulus and these were of three types: ON units. ON-OFF units and OFF units. Stimulus-response curves for both a stimulating central spot and an annulus were constructed and the effects of increasing the background illumination on these curves were investigated. (b) Movement sensitive neurones. Units were recorded which responded preferentially to slow movement of the stimulus while some units responded to movement of the stimulus along one particular axis only (axis sensitive neurones). (c) Spontaneously discharging units whose discharge frequency was affected by the presence of background illumi~tion. In one group. the discharge frequency fell whilst in the other group, the discharge fnquency increased in the presence of background illumination.
INTRODUCTlON
The receptive fields (RF) of frog retinal ganglion cells
consist of an excitatory cent& area. l*Omm dia (HartIi~. 1938). whiih itself contains a region of high sensitivity, 05 mm dii (Barlow, 1953b).According to Maturana, Lettvin McCulloch and Pitts (1960) who recorded from the terminations of optic tract fibres in the optic tectum, Class III (ON-OFF) units possessed a silent inhibitory surround although Barlow (1953b)was not always able to ~monst~te the presence of this surround. class I (ON) units and Class IV (OFF) units did not possess an inhibitory surround. The diierent types of ganglion cell responses were re-investigated in the present work to see if previous reports could be confirmed and if new findings could be made. Fine glass micropipettes were employed to facilitate single unit recording and to avoid biis towards the recording of responses from larger ganglion cells only, which may occur with relatively coarse tungsten electrodes (Wiesel. 1960; Stone. 1973).Unlike the work of Hartline (1938) and Barlow (1953a, b), the present study involved recording responses from both the central and peripheral retina. instead of just the peripheral retina.
humour which was absorbed by small pieas of filter paper. Then the cornea was carefully excised, the iris was retracted using narrow strips of filter paper and the kns with the vitreous body attached was lifted from the eye cup. Often the iris had to be retraeted further so as to completely expose the retina. The preparation remained viable for periods of up to 8 hr. The relationship between the behaviour of the retina iftsitu and in the intact animal is unknown. However. the same type of response which was recorded from the retina has also been recorded from the terminai arborisations of optic tract fibres in the optic tectum of frogs a~esthet~ed with MS222 (W. T. Catton. personal communication).
Stifmflatioff Two stimulators were employed. one of which projected two stimuli. Figure I shows the structure of the two stimulators. Light from a 12 V, 2*2W tungsten filament bulb (run from a stabilized power supply) was focused onto an aperture A by a condenser lens. The aperture A was one of a series of spots (from O-25 up to 3.0 mm dia) and annuli (with i.d.. I.0 mm and l-5 mm) located on a rotatable disc. One stimulator provided stationary stimuli while the
METHODS The retina of Ranu exulenta was studied in situ and the procedure is described below.
The animal was pithed and pinned to a cork board: a piece of cotton wool moistened with Ringer solution was inserted into the frog’s mouth to partially avert the eyes. Usually the left eye was dissected: this was performed with the room in darkness and using a microscope light whose intensity was 4QIx. Fine forceps and spring scissors were employed in exposing the retina. Both eyelids were removed. The cornea was pierced by a mounted entomological pin and a broad cut was made to release the aqueous
’ Present address: Department of Pharmacy. The University of Aston in Birmingbam. Costa Green. Birmingham. 84 7ET. England. I339
SI
Fig. I. Structure of the optical stimulators. SI. S2 and S3 are the light sources. Other parts are: A. the aperture (spots and annuli of different dimensions); N. neutral density filters; Ml. half silvered mirror; M2 and M3, front silvered mirrors, Further explanation is given in the text.
stlmulatoi proildrd two mdepcndcnt square wave modulated stimuli. These wcrc achieved h!, mountmg a metal flag onto an clcctromagnet so that light fell onto the retina onl! u hen the metal Rag was displaced from its positIon covering the aperture A The electromagnet was actlvatcd b\ a DcGccs Relay L’nit which was driven by a Dcv~ces Digitimcr. The latter controlled the duration of the Ilght esposurc. usuall! I~OOsec.and the cycle period. usualI> 7.00 or 8GOscc. The stimulus was detected by a photodiode (Texas H I I) whose output was monitored by the oscilloscope. After passing through the aperture A. it was rocuscd by another lens onto a front silvered mirror (M2 and M3). In the double beam stimulator. the beams were combined h\ a half silvcrcd mirror Ml. After rcflection from M2 anb M3. the beams were locuscd onto the retina hv a third lens. The light intcnsitics at the retina were measured by a comparison method employing an EEL “Lightmaster” photometcl-. Since the diameter of the photosensitive head grcatl! exceeded that of the light spot. direct measurements could not he made. So the spot was focused onto a circular p~cce of white card placed over the photosensitive head which. itself. was at the level of the frog’s eye. While the beam was being flashed. at a rate of I set on. I set off. the ambient background illummation was adjusted b> moving a 6OW .Anglepoisc lamp. until the flashing spot was just discernible above the background illumination. Then the white card was removed from the photosensitive head and the intensity read on the photometer: recorded values were of the order of 1001x. According to Davson (1972). at background intensity 100 lx, the liminal brightness increment for human vision was 4”,,. So the intensities of the light spots were taken as 4:)” or the measured values. The intensities. without attenuation by neutral density filters. were SI = 1701x. S2 = 4001x and S3 = 5401x. To cheek these measurements. a direct comparison was made between two light spots of equal area. modulated side by side. A 2-in. slide. blacked out by aluminium foil except for a small pin hole at the centre. was placed in a Kodak slide projector and the small spot was focused onto the white card placed on the photoscnsitivc head. Then a small spot from stimulator SI was modulated besides the stationary spot. and its intensity was attenuated in 0.25 log unit steps until the two spots were of equal brightness. The stimulator was turned off and the slide removed from the projector so illuminating the photosensitive head. The recorded intensity was 228 lx which agrees well with the above value for Sl of 170 Ix. The latter value was accepted since the other method was to the nearest 0.25 log units (nearly loo’!;, change). Frequent11 the intensities were attenuated hy neutral density filters (Kodak and Ilford Ltd). The mesopic range of vision cxtcnds from IO-” to IO-’ lx while the photopic range extends from IO-‘Ix upwards. White paper in the mid-day sunlight has an equivalent illuminance of 441x (Talbot and Kuffler. 1952). double
RccorJing Action potentials were recorded using 4 M NaCl micropipettes whose tip impedances were in the range 30- 100 MR. The micropipettes were pulled from Pyrex@ glass tubing. o.d. 2.0 mm and i.d. I.0 mm (Jencons Ltd.) using a modified Palmer horizontal electrode puller (catalo&e number H.104). The micropipettes were first filled with methanol and then with 4 M NaCl by boiling under reduced pressure in both cases. The micro-electrode was connected to the non-inverting input of a d.c. preamplifier which incorporated an Analog Devices 143A operational amplifier. without compensation of Input capacitance. The indifferent lead was a length of silver wire placed in the frog’s mouth: the lead contained a backing-off circuit to annul any standing potential differences. The preamplifier output led to two storage oscillo-
scopes (Tektronix 561 and Telequlpmcnt DS3St and dn audio amplifier. Tracts wcrc stored and then photographed with a Polaroid camera. The mlcroelectrode was advanced manuall! using a Prior micro-manipulator. Smglc unit extracellular recordings were made from ganghon ct’lj bodies. which lasted for up to 2 hi-. and from axons \vhich lasted for 0.5 hr at the most Generally ganglion ccli recordings were positive-going followed hq a small negutive overshoot and of total amplnudc of up to ?Orn\.
.4xon recordings had equal positl\,e and negatllc phascb. of amplitude I m\! maximum and sounded crisp and crackling over the audio amplifier. Further details arc given in Morrison (1974). Single sweeps were recorded l’or each set of stlmulatmg
conditions.
.4Ithough
averaged
rc-
sponscs were not obtained. the consistency of the results was always carefully checked hy repeating either parts or the entire experiment. Latencies were measured on the latest time sweep to an accuracy of i I msec. The spike number was a whole number. usually quite small: so there was probably very little error. Frequencies were not so accurate. usually + 5 impulses/set. since it needed only a small error in the measurement of the duration of the respouse to appreciably affect the frequency.
RESULTS Althqugh histological studies employing glutaraldehyde-fixed tissue and light microscopy failed to reveal
the presence of giant ganglion cells. there was a range of cell body diameters varying from 5 to 10 pm @forrison, unpublished observations). Presumably, the use of fine micropipette electrodes would avoid bias to the recording of larger ganglion cells. Although no measurements of depth were made, the recordings were judged to be from the surface of the retina: no recordings were made from deep within the retina. This suggested that the recordings were from either ganglion cell bodies or from optic nerve fibres. Since there is also the possibility that some recordings were from amacrine cells. which also give spike discharges (Matsumoto and Naka, 1972), the term “unit” has been used throughout this work. When the diameter of the stimulating central spot(of just suprathreshold intensity) was increased from 05 mm, the response increased or remained constant in 75% of ON-OFF and OFF units up to diameters I.0 and 1.5 mm. Above this diameter. the response decreased in the majority of ON-OFF units but in only half of OFF units. This demonstrated the presence of an antagonistic surround. particularly in ONOFF units. Figure 2 shows the behaviour of a typical ON-OFF unit. So the 05mm spot was used to stimulate the RF centre: usually the greatest intensity employed was 17.01~. To stimulate the RF surround, an annulus. i.d. I .O mm, o.d. 2.0 mm and greatest intensity 40.0 lx was employed. Three groups of units were delineated and these are dealt with in turn.
Uttifs which rc3portdcc/ to au irm7fnirrc~fu light srifmfThere were no spontaneous discharges in darkness and the responses evoked by the light stimulus were always transient. Three different types of units were recorded and their proportions were: ON units Iv& OFF units ZY);, ON-OFF units 65%. Barlow (1953a) also reported a dearth of ON units. and. in both instances. they differed from the ON units of lus.
Rcsponsus of retinal ganglion cells
1341
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-x \
fi
I
:
s
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:
i
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x :
X
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i
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: :
I
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I 65
I
I
I
I
I.0
13
20
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Fig. 2. The effects of increasing the diameter of the stimulating spot from Q5 mm up to 2.25 mm in a typical ONOFF unit. Stimulus intensity was I.70 x IO-’ lx: this was just suprathreshold. The Geatest responses occurred at I.0 mm dia: for the ON response this consisted of seven spikes and for the OFF response. five spikes. ON response; ---- OFF response. S is the spike total.
Hartline. which gave sustained responses. ON responses (from ON and ON-OFF units) possessed the following characteristics: latency 7&9Omsec. 4-8 spikes and frequency 200-250 impulses+. No pronounced differences were observed behven the OFF responses of ON-OFF and OFF units. The latency was 70-2lOmsec. spike total 1-22 and frequency I280 impulses/set. Stimulus-response curves (S-log I curues)for the RF centw. These were plotted for an intensity range from 1701x to threshold in log unit steps. Threshold was usually determined to the nearest 05 log units though in later experiments it was to 025 log units: this was taken as the intensity which just evoked a response. After changing the intensity. the fourth sweep was recorded in each case and the consistency of the responses was confirmed by repeating a few or all the intensities. The S-log I curves fell into two groups: (I) The S-log I curve possessed two parts: there was an increase in response for increasing intensities above threshold but a marked fall-off occurred at higher intensities (which nevertheless lay well within the physiological range). This occurred in 70% ON-OFF units and 50% OFF units. The behaviour of an ON response is shown in Fig. 3 which also gives the curves for latency and frequency. In many ON-OFF units the OFF response was abolished at high intensities so that they behaved as ON units. In OFF units. ON responses were never evoked by a central spot. (2) The S-log I curve possessed one phase in the remaining units. When log I was reduced. the response decreased monotonically. Stirnulatior~ 17.~arr ar~ruhrs. Since the stimulating annulus evoked responses at very low intensities (044OIx). it seems unlikely that the responses were evoked by scattered light G4ling upon the RF centre hecause this would be of subthreshold intensity. Fre-
log 1 Fig. 3. The stimulus-response curves of the ON response of a typical ON-OFF unit. The stimulus was a central spot. (35 mm dia. OG log units = 1701x. The curves are: spike total (S). --latency (L). ----frequency (F).
quently, the annulus evoked both ON and Off responses from ON-OFF and OFF units (of a central spot). though sometimes an OFF response only was evoked, especially in OFF units (see Morrison, 1975, Fig. 3). In the S-log 1 curves for the surround in most units, the response decreased monotonically with log 1. The eflects of steady illumination on the S-log I CUIYXS.Steady illumination was provided by a spot 30mm dia which almost covered the entire retina. The effects upon the S-log I curves of progressively increasing the adapting illumination zero up to a maximum of 4-01x
intensity
from
were noted: the results were checked for their consistency by replotting the curves in darkness and in certain adapting
3nl” C_ SOGLC Fig. 4. The etTects of increasing the intensity of steady background illumination on the discharge of a typical ON unit. The stimulus was a central spot. 05 mm dia and of intensity 170 ix. Steady spot was 3-Omm dia. In the trace% upward arrow denotes onset and downward arrow denotes termination of illumination. Horizontal calibration is 50 msec except first trace where it is 20 mscc. (I) No background illumination. ON response only. (2) Background intensity was 1.201~. Double ON response. No OFF response. (3) 12.01~. ON response. (4) I>Oix OFF response. (5) I20 Ix. ON response. (6) I20 lx. OFF response. Vertical calibration 5 mV. The traces have been retouched. _-_.-.-
J. 1). MOKKISO\
Fig. 5. The ctTcctsof increasing the intensity of steady background illumination on the OFF response of an OFF unit. Stimulus was a central spot. @5 mm dia. 0.0 log units = 170 1x. Diameter of stead!, central spot was 30mm. At each background intensity. an S-log I curve was constructed. No background illumination. --@040 Ix. -----@40 Ix. -.-.4.0 lx. ‘.‘.... 12.0 Ix. intensities. With
a few exceptions
which
will
be de-
scribed later. the adapting illumination shifted the Slog 1 curves to the right. and also altered the slope of the curves. For ON-OFF units. the slope on the ON S-log 1 curve was depressed and that of the OFF response was appreciably increased. In Fig. 4. an ON response only was evoked in the absence of steady illumination. When the adapting intensity was increased to 12.0 lx. a powerful OFF response was also evoked. It is noteworthy that this unit also gave a double ON response (Fig. 4, line 2). In contrast to this. ON responses were not evoked. in OFF units. in the presence of adapting illumination; however, the slope of the OFF S-log I curve was always depressed. A small population of 4/44 (4 out of 44) OFF units behaved in a remarkable manner: the S-log 1 curves were shifted to the left by the adapting illumination. Figure 5 shows the behaviour of one unit which displayed a profound tall in response thresboki: a light incre-
behaviour of an OFF unit which responded to movement in the nasal-temporal and dorsal-ventral directions but not in the reverse (null) directions. Furthermore. five units were axis sensitive. i.e. they responded preferentially to movement along one particular axis only. Figure 6 is exceptional for another reason: the unit gave a prolonged response to very slow movement of the stimulus whereas usually, relatively rapid movement (5 mm/set) was required to evoke responses. Only three slow movement detectors were recorded in the present study. Spontarworuly dischurgir~g wits. These numbered only 25/1550 units and were true spontaneously discharging neurones as opposed to those which fired continuously as a result of penetration by the microelectrode. There were two types. For one type, the discharge frequency decreased in the presence of background illumination and Fig. 7(a) shows the beha-
ment of 10m6% was detected at an adapting intensity of 0401x. The possibilities that the results arose as a result of escape of stray light or e vibration when the shutter opened were arefully excluded. Movetrwnt surtsitiw units. Only 61/l 550 units could be classed as pure movement sensitive units i.e. they responded to a moving but not a flashing stimulus. Group one neurones always responded to a moving stimulus as well as a flashing one. In those units whose recording site was determined to be at the ganglion cell body (see Morrison. 1974). responses to movement were evoked only when the edge of the light spot traversed the microelectrode tip and in most cases. the trailing edge evoked a greater response than the leading edge. This finding was a useful means of determining quickly if the recording site was the ganglion cell body or its axon. No evidence of directional sensitivity was displayed by the great majority of units. However. a small group of ten units was directionally sensitive and. of these. six were pure movement sensitive neurones. Figure 6 shows the
Fig. 6. Movement sensitive neurone. Stimulus was 0.5 mm dia and of intensity 17.01~. Responses were evoked by the trailing edge of the moving spot. (a) Refatively rapid movcmcnt (0.5 mm/set). Time calibration: IQOmsec. (I) Movemcnt in the dorsal to ventral direction. (2) Ventral to dorsal. (3) Nasal to temporal. (4) Temporal to nasal. (b) Rclatively slow movcmcnt (0.1 mm/s&. Time calibration: 2COmsec. (5) Dorsal to ventral. (6) Ventral to dorsal. (7) Nasal to temporal. (8) Temporal to nasal. Vertical cafihration = 5 mV. Tracts have been retouched.
I34.J
Responses of retinal ganglion cells
Fig. 7. Spontaneously discharging units. (a) Discharge rate increased by steady illumination. (I. 2) No background illumination. Mean discharge frequency was I.3 impulses/set. (3) Onset of 05 Ix steady illumination. indicated by artefact. (4.5) Steady illumination. Mean discharge frequency was 3G impulscs/sec. (6) Termination of illumination. indicated by artefact. (7) No background illumination. Mean discharge frequency was 1.2 impulses/set. Vertical calibration: 2 mV. Horizontal calibration: I.0 sec. (b) Discharge rate depressed by illumination. (I) No background illumination. Mean discharge frequency was I.6 bursts of spikes/set. (2) Steady illumination (05 lx). No discharge. (3) Termination of illumination, indicated by OFF response. (4) No background illumination. Mean discharge frequency was 1.2 bursts of spikesjsec. In each burst, there were three or four spikes. Vertical calibration 20 mV. Horizontal calibration 05 sec. Both sets of traces have been retouched. viour of a typical unit. The discharge frequency of the second type increased in the presence of background illumination and Fig. 6 illustrates this pattern of behaviour. DISCUSSION
Several types of ganglion cell behaviour have been described here which have not been previously documented for the frog retina. In many ON units. it was possible to evoke an ON-OFF response by either reducing the stimulus intensity or. as previously described for the rabbit retina by Barlow. Hill and Levick (1964). by the presence of background illumination. In agreement with the results of Barlow (1953a). ON-OFF and OFF units were the two main types of units to be encountered. Both types displayed a fall-off in response at high stimulus intensities which lay within the physiological range. This was also described by Hartline (1938) except that the fall-off occurred at much higher intensities: this was probably attributable to the use by Hartline of a very small stimulating spot and so needed a greater intensity. When steady background illumination was projected onto the retina the S-log I curves were not only shifted to the right which was also described for the frequency-log I curves of the frog by Byzov and Kusnezova (1971). but also the slopes of the Slog I curves were markedly affected. Additionally. in a small group of OFF units. the S-log f curves were shifted to the left. These units responded to a light increment of the order IO-” o/0 which contrasts with
an increment of l”,, for the cat retina (Talbot and Kuffler. 1952). Responses were evoked from the RF surround by a stimulating annuius and S-log I curves were plotted over a large range of stimulus intensities. Earlier investigators failed to evoke a response from the RF surround probably because they employed a small stimulating spot (Barlow. 1953b; Maturana cf al.. 1960; and Henn and Griisser. 1969). which would have stimulated only a relatively small number of receptors converging onto the ganglion cell. However. Matsumato and Naka (1972) did find that an annulus was effective in evoking responses from the surround in Rma pipicws. The axis sensitive neurones described in the present study resembled the Class III neurones of Maturana el ~1.. which responded to movement along a particular axis. However they also differed because axis sensitive neurones frequently did not respond to a flashing stimulus whereas Class III units did. The small group of slow movement detector neurones resembled neurones recorded from the visual streak of the rabbit retina by Barlow et al. (1964). but differed from Class I units in that they did not respond to a standing edge. Spontaneously discharging units were recorded infrequently which agrees with Barlow (1953a) and Maturana et al. (1960). Those units whose discharge frequency was depressed by steady background illumination may be comparable with Class V neurones. However those units whose discharge frequency increased have not previously been described for the frog retina although they have been recorded from the cat retina (Spinelli and Weingarten. 1966). To conclude, the present study has confirmed many of the findings reported in earlier investigations. Also. several new findings have been described. This may be attributable to the large number of units sampled and that the sampling was less biased than in previous investigations towards the recording of cells with larger diameters. as a result of using very fine microelectrodes. REFERENCES
Barlow H. B. (1953a) Action potentials from the frog’s retina. J. Physiol.. Lmd. 119. 58-68. Barlow H. B. (1953b) Summation and inhibition in the frog’s retina. J. Physiol.. Lmd. 119. 69-88.
Barlow H. B.. Hill R. M. and,Levick W. R. (1964) Retinal ganglion cells responding selectively to direction and speed of image motion in the rabbit. J. Phpiol.. Loud. 173. 377-407.
Byzov A. L. and Kusnezova L. P. (1971) On the mechanisms of visual adaptation. ti’sion RL’s. (SuppI.) 3. pp. 51-63. Davson H. (1972) 771~Plry.sio/og.v cf r/w Eye. 3rd edition. Churchill-Livingstone. Edinburgh. Hartline H. K. (1938) The responses of single optic nrrve librcs of the vertebrate eye to illumination of the retina. Aat. J. Ph.rsiol. 121. 400-415. Henn V. and Grtisscr 0. J. (1969) The summation of excitation in the receptive fielb of movement scnsitivc neurones of the frog’s retina. I’isiort Rvs. 9. 57-69. Matsumoto N. and Naka K.-I. (1972) Identification of intracellular responses in the frog retina. Bruirl Rcs. 42. 59-71. Maturana H. R.. Lettvin J. Y.. McCulloch W. S. and Pitts W. H. (1960) Anatomy and physiology of vision in the frog (Ranu pi/Gas). J.
THE RESPONSES OF THE RETINAL GANGLION CELLS OF THE F’ROG J. D. MORRISON’ Department of Physiology. Medical School. The University. Newcastle-upon-Tyne. NE1 7RU. England
Abstract-Three groups of units were recorded from the retina of Rma esculenta. These were: (a) Units which responded to aare wave modulated lightstimulus and these were of three types: ON units. ON-OFF units and OFF units. Stimulus-response curves for both a stimulating central spot and an annulus were constructed and the effects of increasing the background illumination on these curves were investigated. (b) Movement sensitive neurones. Units were recorded which responded preferentially to slow movement of the stimulus while some units responded to movement of the stimulus along one particular axis only (axis sensitive neurones). (c) Spontaneously discharging units whose discharge frequency was affected by the presence of background illumi~tion. In one group. the discharge frequency fell whilst in the other group, the discharge fnquency increased in the presence of background illumination.
INTRODUCTlON
The receptive fields (RF) of frog retinal ganglion cells
consist of an excitatory cent& area. l*Omm dia (HartIi~. 1938). whiih itself contains a region of high sensitivity, 05 mm dii (Barlow, 1953b).According to Maturana, Lettvin McCulloch and Pitts (1960) who recorded from the terminations of optic tract fibres in the optic tectum, Class III (ON-OFF) units possessed a silent inhibitory surround although Barlow (1953b)was not always able to ~monst~te the presence of this surround. class I (ON) units and Class IV (OFF) units did not possess an inhibitory surround. The diierent types of ganglion cell responses were re-investigated in the present work to see if previous reports could be confirmed and if new findings could be made. Fine glass micropipettes were employed to facilitate single unit recording and to avoid biis towards the recording of responses from larger ganglion cells only, which may occur with relatively coarse tungsten electrodes (Wiesel. 1960; Stone. 1973).Unlike the work of Hartline (1938) and Barlow (1953a, b), the present study involved recording responses from both the central and peripheral retina. instead of just the peripheral retina.
humour which was absorbed by small pieas of filter paper. Then the cornea was carefully excised, the iris was retracted using narrow strips of filter paper and the kns with the vitreous body attached was lifted from the eye cup. Often the iris had to be retraeted further so as to completely expose the retina. The preparation remained viable for periods of up to 8 hr. The relationship between the behaviour of the retina iftsitu and in the intact animal is unknown. However. the same type of response which was recorded from the retina has also been recorded from the terminai arborisations of optic tract fibres in the optic tectum of frogs a~esthet~ed with MS222 (W. T. Catton. personal communication).
Stifmflatioff Two stimulators were employed. one of which projected two stimuli. Figure I shows the structure of the two stimulators. Light from a 12 V, 2*2W tungsten filament bulb (run from a stabilized power supply) was focused onto an aperture A by a condenser lens. The aperture A was one of a series of spots (from O-25 up to 3.0 mm dia) and annuli (with i.d.. I.0 mm and l-5 mm) located on a rotatable disc. One stimulator provided stationary stimuli while the
METHODS The retina of Ranu exulenta was studied in situ and the procedure is described below.
The animal was pithed and pinned to a cork board: a piece of cotton wool moistened with Ringer solution was inserted into the frog’s mouth to partially avert the eyes. Usually the left eye was dissected: this was performed with the room in darkness and using a microscope light whose intensity was 4QIx. Fine forceps and spring scissors were employed in exposing the retina. Both eyelids were removed. The cornea was pierced by a mounted entomological pin and a broad cut was made to release the aqueous
’ Present address: Department of Pharmacy. The University of Aston in Birmingbam. Costa Green. Birmingham. 84 7ET. England. I339
SI
Fig. I. Structure of the optical stimulators. SI. S2 and S3 are the light sources. Other parts are: A. the aperture (spots and annuli of different dimensions); N. neutral density filters; Ml. half silvered mirror; M2 and M3, front silvered mirrors, Further explanation is given in the text.
stlmulatoi proildrd two mdepcndcnt square wave modulated stimuli. These wcrc achieved h!, mountmg a metal flag onto an clcctromagnet so that light fell onto the retina onl! u hen the metal Rag was displaced from its positIon covering the aperture A The electromagnet was actlvatcd b\ a DcGccs Relay L’nit which was driven by a Dcv~ces Digitimcr. The latter controlled the duration of the Ilght esposurc. usuall! I~OOsec.and the cycle period. usualI> 7.00 or 8GOscc. The stimulus was detected by a photodiode (Texas H I I) whose output was monitored by the oscilloscope. After passing through the aperture A. it was rocuscd by another lens onto a front silvered mirror (M2 and M3). In the double beam stimulator. the beams were combined h\ a half silvcrcd mirror Ml. After rcflection from M2 anb M3. the beams were locuscd onto the retina hv a third lens. The light intcnsitics at the retina were measured by a comparison method employing an EEL “Lightmaster” photometcl-. Since the diameter of the photosensitive head grcatl! exceeded that of the light spot. direct measurements could not he made. So the spot was focused onto a circular p~cce of white card placed over the photosensitive head which. itself. was at the level of the frog’s eye. While the beam was being flashed. at a rate of I set on. I set off. the ambient background illummation was adjusted b> moving a 6OW .Anglepoisc lamp. until the flashing spot was just discernible above the background illumination. Then the white card was removed from the photosensitive head and the intensity read on the photometer: recorded values were of the order of 1001x. According to Davson (1972). at background intensity 100 lx, the liminal brightness increment for human vision was 4”,,. So the intensities of the light spots were taken as 4:)” or the measured values. The intensities. without attenuation by neutral density filters. were SI = 1701x. S2 = 4001x and S3 = 5401x. To cheek these measurements. a direct comparison was made between two light spots of equal area. modulated side by side. A 2-in. slide. blacked out by aluminium foil except for a small pin hole at the centre. was placed in a Kodak slide projector and the small spot was focused onto the white card placed on the photoscnsitivc head. Then a small spot from stimulator SI was modulated besides the stationary spot. and its intensity was attenuated in 0.25 log unit steps until the two spots were of equal brightness. The stimulator was turned off and the slide removed from the projector so illuminating the photosensitive head. The recorded intensity was 228 lx which agrees well with the above value for Sl of 170 Ix. The latter value was accepted since the other method was to the nearest 0.25 log units (nearly loo’!;, change). Frequent11 the intensities were attenuated hy neutral density filters (Kodak and Ilford Ltd). The mesopic range of vision cxtcnds from IO-” to IO-’ lx while the photopic range extends from IO-‘Ix upwards. White paper in the mid-day sunlight has an equivalent illuminance of 441x (Talbot and Kuffler. 1952). double
RccorJing Action potentials were recorded using 4 M NaCl micropipettes whose tip impedances were in the range 30- 100 MR. The micropipettes were pulled from Pyrex@ glass tubing. o.d. 2.0 mm and i.d. I.0 mm (Jencons Ltd.) using a modified Palmer horizontal electrode puller (catalo&e number H.104). The micropipettes were first filled with methanol and then with 4 M NaCl by boiling under reduced pressure in both cases. The micro-electrode was connected to the non-inverting input of a d.c. preamplifier which incorporated an Analog Devices 143A operational amplifier. without compensation of Input capacitance. The indifferent lead was a length of silver wire placed in the frog’s mouth: the lead contained a backing-off circuit to annul any standing potential differences. The preamplifier output led to two storage oscillo-
scopes (Tektronix 561 and Telequlpmcnt DS3St and dn audio amplifier. Tracts wcrc stored and then photographed with a Polaroid camera. The mlcroelectrode was advanced manuall! using a Prior micro-manipulator. Smglc unit extracellular recordings were made from ganghon ct’lj bodies. which lasted for up to 2 hi-. and from axons \vhich lasted for 0.5 hr at the most Generally ganglion ccli recordings were positive-going followed hq a small negutive overshoot and of total amplnudc of up to ?Orn\.
.4xon recordings had equal positl\,e and negatllc phascb. of amplitude I m\! maximum and sounded crisp and crackling over the audio amplifier. Further details arc given in Morrison (1974). Single sweeps were recorded l’or each set of stlmulatmg
conditions.
.4Ithough
averaged
rc-
sponscs were not obtained. the consistency of the results was always carefully checked hy repeating either parts or the entire experiment. Latencies were measured on the latest time sweep to an accuracy of i I msec. The spike number was a whole number. usually quite small: so there was probably very little error. Frequencies were not so accurate. usually + 5 impulses/set. since it needed only a small error in the measurement of the duration of the respouse to appreciably affect the frequency.
RESULTS Althqugh histological studies employing glutaraldehyde-fixed tissue and light microscopy failed to reveal
the presence of giant ganglion cells. there was a range of cell body diameters varying from 5 to 10 pm @forrison, unpublished observations). Presumably, the use of fine micropipette electrodes would avoid bias to the recording of larger ganglion cells. Although no measurements of depth were made, the recordings were judged to be from the surface of the retina: no recordings were made from deep within the retina. This suggested that the recordings were from either ganglion cell bodies or from optic nerve fibres. Since there is also the possibility that some recordings were from amacrine cells. which also give spike discharges (Matsumoto and Naka, 1972), the term “unit” has been used throughout this work. When the diameter of the stimulating central spot(of just suprathreshold intensity) was increased from 05 mm, the response increased or remained constant in 75% of ON-OFF and OFF units up to diameters I.0 and 1.5 mm. Above this diameter. the response decreased in the majority of ON-OFF units but in only half of OFF units. This demonstrated the presence of an antagonistic surround. particularly in ONOFF units. Figure 2 shows the behaviour of a typical ON-OFF unit. So the 05mm spot was used to stimulate the RF centre: usually the greatest intensity employed was 17.01~. To stimulate the RF surround, an annulus. i.d. I .O mm, o.d. 2.0 mm and greatest intensity 40.0 lx was employed. Three groups of units were delineated and these are dealt with in turn.
Uttifs which rc3portdcc/ to au irm7fnirrc~fu light srifmfThere were no spontaneous discharges in darkness and the responses evoked by the light stimulus were always transient. Three different types of units were recorded and their proportions were: ON units Iv& OFF units ZY);, ON-OFF units 65%. Barlow (1953a) also reported a dearth of ON units. and. in both instances. they differed from the ON units of lus.
Rcsponsus of retinal ganglion cells
1341
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Fig. 2. The effects of increasing the diameter of the stimulating spot from Q5 mm up to 2.25 mm in a typical ONOFF unit. Stimulus intensity was I.70 x IO-’ lx: this was just suprathreshold. The Geatest responses occurred at I.0 mm dia: for the ON response this consisted of seven spikes and for the OFF response. five spikes. ON response; ---- OFF response. S is the spike total.
Hartline. which gave sustained responses. ON responses (from ON and ON-OFF units) possessed the following characteristics: latency 7&9Omsec. 4-8 spikes and frequency 200-250 impulses+. No pronounced differences were observed behven the OFF responses of ON-OFF and OFF units. The latency was 70-2lOmsec. spike total 1-22 and frequency I280 impulses/set. Stimulus-response curves (S-log I curues)for the RF centw. These were plotted for an intensity range from 1701x to threshold in log unit steps. Threshold was usually determined to the nearest 05 log units though in later experiments it was to 025 log units: this was taken as the intensity which just evoked a response. After changing the intensity. the fourth sweep was recorded in each case and the consistency of the responses was confirmed by repeating a few or all the intensities. The S-log I curves fell into two groups: (I) The S-log I curve possessed two parts: there was an increase in response for increasing intensities above threshold but a marked fall-off occurred at higher intensities (which nevertheless lay well within the physiological range). This occurred in 70% ON-OFF units and 50% OFF units. The behaviour of an ON response is shown in Fig. 3 which also gives the curves for latency and frequency. In many ON-OFF units the OFF response was abolished at high intensities so that they behaved as ON units. In OFF units. ON responses were never evoked by a central spot. (2) The S-log I curve possessed one phase in the remaining units. When log I was reduced. the response decreased monotonically. Stirnulatior~ 17.~arr ar~ruhrs. Since the stimulating annulus evoked responses at very low intensities (044OIx). it seems unlikely that the responses were evoked by scattered light G4ling upon the RF centre hecause this would be of subthreshold intensity. Fre-
log 1 Fig. 3. The stimulus-response curves of the ON response of a typical ON-OFF unit. The stimulus was a central spot. (35 mm dia. OG log units = 1701x. The curves are: spike total (S). --latency (L). ----frequency (F).
quently, the annulus evoked both ON and Off responses from ON-OFF and OFF units (of a central spot). though sometimes an OFF response only was evoked, especially in OFF units (see Morrison, 1975, Fig. 3). In the S-log 1 curves for the surround in most units, the response decreased monotonically with log 1. The eflects of steady illumination on the S-log I CUIYXS.Steady illumination was provided by a spot 30mm dia which almost covered the entire retina. The effects upon the S-log I curves of progressively increasing the adapting illumination zero up to a maximum of 4-01x
intensity
from
were noted: the results were checked for their consistency by replotting the curves in darkness and in certain adapting
3nl” C_ SOGLC Fig. 4. The etTects of increasing the intensity of steady background illumination on the discharge of a typical ON unit. The stimulus was a central spot. 05 mm dia and of intensity 170 ix. Steady spot was 3-Omm dia. In the trace% upward arrow denotes onset and downward arrow denotes termination of illumination. Horizontal calibration is 50 msec except first trace where it is 20 mscc. (I) No background illumination. ON response only. (2) Background intensity was 1.201~. Double ON response. No OFF response. (3) 12.01~. ON response. (4) I>Oix OFF response. (5) I20 Ix. ON response. (6) I20 lx. OFF response. Vertical calibration 5 mV. The traces have been retouched. _-_.-.-
J. 1). MOKKISO\
Fig. 5. The ctTcctsof increasing the intensity of steady background illumination on the OFF response of an OFF unit. Stimulus was a central spot. @5 mm dia. 0.0 log units = 170 1x. Diameter of stead!, central spot was 30mm. At each background intensity. an S-log I curve was constructed. No background illumination. --@040 Ix. -----@40 Ix. -.-.4.0 lx. ‘.‘.... 12.0 Ix. intensities. With
a few exceptions
which
will
be de-
scribed later. the adapting illumination shifted the Slog 1 curves to the right. and also altered the slope of the curves. For ON-OFF units. the slope on the ON S-log 1 curve was depressed and that of the OFF response was appreciably increased. In Fig. 4. an ON response only was evoked in the absence of steady illumination. When the adapting intensity was increased to 12.0 lx. a powerful OFF response was also evoked. It is noteworthy that this unit also gave a double ON response (Fig. 4, line 2). In contrast to this. ON responses were not evoked. in OFF units. in the presence of adapting illumination; however, the slope of the OFF S-log I curve was always depressed. A small population of 4/44 (4 out of 44) OFF units behaved in a remarkable manner: the S-log 1 curves were shifted to the left by the adapting illumination. Figure 5 shows the behaviour of one unit which displayed a profound tall in response thresboki: a light incre-
behaviour of an OFF unit which responded to movement in the nasal-temporal and dorsal-ventral directions but not in the reverse (null) directions. Furthermore. five units were axis sensitive. i.e. they responded preferentially to movement along one particular axis only. Figure 6 is exceptional for another reason: the unit gave a prolonged response to very slow movement of the stimulus whereas usually, relatively rapid movement (5 mm/set) was required to evoke responses. Only three slow movement detectors were recorded in the present study. Spontarworuly dischurgir~g wits. These numbered only 25/1550 units and were true spontaneously discharging neurones as opposed to those which fired continuously as a result of penetration by the microelectrode. There were two types. For one type, the discharge frequency decreased in the presence of background illumination and Fig. 7(a) shows the beha-
ment of 10m6% was detected at an adapting intensity of 0401x. The possibilities that the results arose as a result of escape of stray light or e vibration when the shutter opened were arefully excluded. Movetrwnt surtsitiw units. Only 61/l 550 units could be classed as pure movement sensitive units i.e. they responded to a moving but not a flashing stimulus. Group one neurones always responded to a moving stimulus as well as a flashing one. In those units whose recording site was determined to be at the ganglion cell body (see Morrison. 1974). responses to movement were evoked only when the edge of the light spot traversed the microelectrode tip and in most cases. the trailing edge evoked a greater response than the leading edge. This finding was a useful means of determining quickly if the recording site was the ganglion cell body or its axon. No evidence of directional sensitivity was displayed by the great majority of units. However. a small group of ten units was directionally sensitive and. of these. six were pure movement sensitive neurones. Figure 6 shows the
Fig. 6. Movement sensitive neurone. Stimulus was 0.5 mm dia and of intensity 17.01~. Responses were evoked by the trailing edge of the moving spot. (a) Refatively rapid movcmcnt (0.5 mm/set). Time calibration: IQOmsec. (I) Movemcnt in the dorsal to ventral direction. (2) Ventral to dorsal. (3) Nasal to temporal. (4) Temporal to nasal. (b) Rclatively slow movcmcnt (0.1 mm/s&. Time calibration: 2COmsec. (5) Dorsal to ventral. (6) Ventral to dorsal. (7) Nasal to temporal. (8) Temporal to nasal. Vertical cafihration = 5 mV. Tracts have been retouched.
I34.J
Responses of retinal ganglion cells
Fig. 7. Spontaneously discharging units. (a) Discharge rate increased by steady illumination. (I. 2) No background illumination. Mean discharge frequency was I.3 impulses/set. (3) Onset of 05 Ix steady illumination. indicated by artefact. (4.5) Steady illumination. Mean discharge frequency was 3G impulscs/sec. (6) Termination of illumination. indicated by artefact. (7) No background illumination. Mean discharge frequency was 1.2 impulses/set. Vertical calibration: 2 mV. Horizontal calibration: I.0 sec. (b) Discharge rate depressed by illumination. (I) No background illumination. Mean discharge frequency was I.6 bursts of spikes/set. (2) Steady illumination (05 lx). No discharge. (3) Termination of illumination, indicated by OFF response. (4) No background illumination. Mean discharge frequency was 1.2 bursts of spikesjsec. In each burst, there were three or four spikes. Vertical calibration 20 mV. Horizontal calibration 05 sec. Both sets of traces have been retouched. viour of a typical unit. The discharge frequency of the second type increased in the presence of background illumination and Fig. 6 illustrates this pattern of behaviour. DISCUSSION
Several types of ganglion cell behaviour have been described here which have not been previously documented for the frog retina. In many ON units. it was possible to evoke an ON-OFF response by either reducing the stimulus intensity or. as previously described for the rabbit retina by Barlow. Hill and Levick (1964). by the presence of background illumination. In agreement with the results of Barlow (1953a). ON-OFF and OFF units were the two main types of units to be encountered. Both types displayed a fall-off in response at high stimulus intensities which lay within the physiological range. This was also described by Hartline (1938) except that the fall-off occurred at much higher intensities: this was probably attributable to the use by Hartline of a very small stimulating spot and so needed a greater intensity. When steady background illumination was projected onto the retina the S-log I curves were not only shifted to the right which was also described for the frequency-log I curves of the frog by Byzov and Kusnezova (1971). but also the slopes of the Slog I curves were markedly affected. Additionally. in a small group of OFF units. the S-log f curves were shifted to the left. These units responded to a light increment of the order IO-” o/0 which contrasts with
an increment of l”,, for the cat retina (Talbot and Kuffler. 1952). Responses were evoked from the RF surround by a stimulating annuius and S-log I curves were plotted over a large range of stimulus intensities. Earlier investigators failed to evoke a response from the RF surround probably because they employed a small stimulating spot (Barlow. 1953b; Maturana cf al.. 1960; and Henn and Griisser. 1969). which would have stimulated only a relatively small number of receptors converging onto the ganglion cell. However. Matsumato and Naka (1972) did find that an annulus was effective in evoking responses from the surround in Rma pipicws. The axis sensitive neurones described in the present study resembled the Class III neurones of Maturana el ~1.. which responded to movement along a particular axis. However they also differed because axis sensitive neurones frequently did not respond to a flashing stimulus whereas Class III units did. The small group of slow movement detector neurones resembled neurones recorded from the visual streak of the rabbit retina by Barlow et al. (1964). but differed from Class I units in that they did not respond to a standing edge. Spontaneously discharging units were recorded infrequently which agrees with Barlow (1953a) and Maturana et al. (1960). Those units whose discharge frequency was depressed by steady background illumination may be comparable with Class V neurones. However those units whose discharge frequency increased have not previously been described for the frog retina although they have been recorded from the cat retina (Spinelli and Weingarten. 1966). To conclude, the present study has confirmed many of the findings reported in earlier investigations. Also. several new findings have been described. This may be attributable to the large number of units sampled and that the sampling was less biased than in previous investigations towards the recording of cells with larger diameters. as a result of using very fine microelectrodes. REFERENCES
Barlow H. B. (1953a) Action potentials from the frog’s retina. J. Physiol.. Lmd. 119. 58-68. Barlow H. B. (1953b) Summation and inhibition in the frog’s retina. J. Physiol.. Lmd. 119. 69-88.
Barlow H. B.. Hill R. M. and,Levick W. R. (1964) Retinal ganglion cells responding selectively to direction and speed of image motion in the rabbit. J. Phpiol.. Loud. 173. 377-407.
Byzov A. L. and Kusnezova L. P. (1971) On the mechanisms of visual adaptation. ti’sion RL’s. (SuppI.) 3. pp. 51-63. Davson H. (1972) 771~Plry.sio/og.v cf r/w Eye. 3rd edition. Churchill-Livingstone. Edinburgh. Hartline H. K. (1938) The responses of single optic nrrve librcs of the vertebrate eye to illumination of the retina. Aat. J. Ph.rsiol. 121. 400-415. Henn V. and Grtisscr 0. J. (1969) The summation of excitation in the receptive fielb of movement scnsitivc neurones of the frog’s retina. I’isiort Rvs. 9. 57-69. Matsumoto N. and Naka K.-I. (1972) Identification of intracellular responses in the frog retina. Bruirl Rcs. 42. 59-71. Maturana H. R.. Lettvin J. Y.. McCulloch W. S. and Pitts W. H. (1960) Anatomy and physiology of vision in the frog (Ranu pi/Gas). J.
