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Antidromic invasion of ganglion cells in the pigeon retina
ANTIDROMIC INVASION OF GANGLION IN THE PIGEON RETINA
CELLS
A. L. HOLDEN Department of Visual Science. Institute of Ophthalmology.
Judd Street. London WC1 H9QS. England
(Received 21 November 1977: in revised form 25 January 1978)
Abstract-265 single units were recorded in the yellow field of the pigeon retina. Ganglion cell-bodies can be antidromically invaded from the optic tectum. Conduction velocities range from 18 to 1.8 misec extraretinally and from 4.5 to 0.13 m!sec intraretinally.. For most axons conduction slows in the retina by IO-20 times. A minority of ceils are fired antidromically from the isthmo-optic tract, probably resulting from stimulus spread to the dorsolateral thalamus.
lSfRODUCTlON
The experiments reported here examine the antidromic invasion of pigeon ganglion cells. If antidromic invasion can be shown following stimuiation of the central visual pathway, then it is established that the recording is taken from an output cell (ganglion cell or displaced ganglion cell) of the retina. This physiological identification can be ,made with extracetlular recording in a small-cell system, where intracellular recording and dye injection may be difficult. The optic nerve of the pigeon has been examined with the electron microscope (Binggeli and Paule. 1969). The retinotectal system has been examined electrophysiologically (Holden. 1968a; Stone and Freeman. 1971a,b) with mapping techniques (Hamdi and Whitte~dge, 1954; Clarke and Whitteridge, 1976). and with experimental anatomical techniques (McGill. Powell and Cowan, 1966; Lavail and Lavail, 1974; Hayes and Webster, 1975). A previous singleunit study (Holden, 1968af provides an estimate of conduction velocities of retinotectal axons. using orthodromic conduction between the optic nervehead and the lateral tectum. The present experiments extend this evidence by using antidromic invasion to measure axonal conduction times both intraretinally, and between the retina and the tectum. The results show that the majority of spike-generating cells in the central yellow field of the retina can be antidromically invaded from the optic tectum, showing them to be ganglion cells. T’he extraretinal conduction velocities are low, and fal1 into the range of the W-ceil grouping in the cat. Intraretinal velocities are lower, by a factor of 10-20, due to conduction in fine. unmyelinated fibres. METHODS The experiments were carried out on 50 feral pigeons. anaesthetized with urethane. maintained under one-way Row artiticial ventilation. and prepared for intraretinal recording as described in detail in Holden (1977). Three types of preparation were used for electrical stimulation. In each. electrical stimuli were delivered to the exposed surface of the contralateral tectum. This was either the sole site of stimulation, or was combined with stimulation of the optic tract, via the orbit of the contralateral eye, or with stimulation of the isthmo-optic tract intracranialiy,
as described in Holden and Powell (1972). The first preparation was used to accumulate data on the latency of antidromic invasion to tectal stimuli; the second to measure conduction time extraretinally. and the third to assess any possible responses due to orthodromic discharge of the centrifugal system running from isthmo-optic nucleus to retina. Electrical stimuli were delivered isolated from earth, via pairs of steel needles insulated by Araldite epoxy resin PZ 985. The needle tips were separated by 1 mm. and were ground bare before each experiment. An early series of experiments on.19 pigeons was carried out in 1964 in The University Laboratory of Physiology, Oxfoid. Later experiments were carried out in London. In the later experiments an averager (Hewle~-Packard 5480B) was used extensively with an X-Y plotter, both as a quick method of providing permanent records. and as a means of displaying small graded fields close to the baseline noise level. A sweep time of 50 msec was used: with a resolution of loo0 points this corresponds to a sampling rate of 20 kHz. The headstane amplifier was used with 3-pole Butterworth filters providing’ a pass-band bf 0.16 Hz-3 kHz. Calibration signals were obtained from a Brookdeal Signal Source Type 471. Placemenr of stimulating elecrrodes Tecrum. The stimulating electrodes were thrust against the tectal surface, after exposing 1-2 mm of the lateral aspect of the right tectum. In all experiments the head was held in the position of normal walking, with a beak bar 25 mm anterior to, and level with, the ear bars. The projection of the lateral visual field was as used in Holden and Powell (1972) and by Pearlman and Hughes (1976): the point level with the centre of the pupil in the lateral visual field was taken as tlie centre of a poIar projection. When receptive fields of tectal cells in the exposed lateral aspect were recorded. their locations were close to the horizontal meridian, and IO”-25” anterior. Reozptive fields of retinal ganglion cells were clustered round the centre of the projection. Thus there was a partial overlap between receptive fields at the stimulation site and at the recording site. Those retinotectal fibres stimulated in passage at the tectal surface would be travelling anteroposteriorly, thus including fibtes corresponding to the more posterior ganglion cetl receptive field locations. Optic tract. The tract running from left eye to right tecturn was stimulated 9 mm anterior to the lateral tectal electrodes. A pair of stimulating electrodes was advanced through an opening made in the temporal wall of the orbit of the right eye, after displacing the eye forwards. Positioning of the electrodes was completed during the early stages of each experiment by searching for a position which pro-
1357
4. L. HOLDEX
13%
record small. neysttve-gotng sptkes. the htghcr reststdnce electrodes could rssolve and hold dtphastc cell spokes for long periods 10 j-6 hri. but recorded relaavely feu ncgattvs spikes. Thts dtfierencc tn selectivity IS nc71 due to Johnson noise per sz. for the use of averagmg could ea>tlb offset a j-fold dtfference m peak-to-peak notse. Electrodes were adv;Fncrd Into the retma wtth d Trent-
ducsd a rettn31 response matchmg the response to tectal sttmulatton. Thts search was often only partly successful. because of the large and Intractable stimulus arttfacrs produced by sttmulr apphed close to the left eye. However. even If the sttmulus arttfact precluded adequate study of small field potentials. It was always posstble to observe well resolved untt responses. Ivrhmo-opt/c rracr. A patr of stimulatmg electrodes was advanced verttcally downuards through the right forebram. for 9-10 mm. They were placed 2.5 mm lateral to the midline. It was shown in Holden and Powell (1972) that electrodes Inserted hortzontally into the lateral tectum can reach the rsthmo-opttc nucleus. Thus the anterior-postertor location used was one which straddled the level of the dissectton field at the lateral tectum. Electrodes placed III this way UIII excite the isthmo-optic tract and adjacent structures. They excite tectal output cells, in the lateral tectum anttdromically. at a frequency of 3-6 cellstrack. though they do not produce orthodromtc activation of the tectal surface. judged by production of an .Y-wave Thus the! do not produce major activation of the optic tract by current spread; the optic tract electrodes. placed as described above. could always produce a tectal &‘-wave tn addttton to antidromic acttratton of the retina.
Wells stepptng mtcrodrtve Depth readings uerc taken with respect to the mtcrodrive readmg Jt vvhtch the retma was penetrated. Because of tissue drag and dtmpltng they can only provide an approximate guide to rettnal depth. Where the depth dtstrtbutton of ganglton cells :s ssttmated (Ftg. 6). the depth readings were scaled ustng data from an mdependent study where dye marks were used to locahze the proximal negative response (Hayes dnd Holden. 19-Y). In SIX ptgeons Methylene Blue statmng uas used intravttally or suprdvttally to determtne the traJectortes taken by opttc nerve hbres runnmg from the recordmg stte tn the central yellow field to the opttc nerve head. intr3vttreal Injection of 03 ml of OOI”, solutton oi Methylene Blue m saline wrls followed by dtssection of the rettna I hr later Alternattvely. the retina uas dtssected ou: !n avtan Ranger and stamed supravttally
.Ificroelucrrodes
RESC LTS
Mtcroptpettes filled wtth 3 X4 KCI were used throughout In early experiments they were used wtth resistances
Tj,pes
of 5-7 MR. In later Z&70 :\lR Electrodes
spike
experiments with lower
of rtnlt responsr
265 unit
the resistances were resistances tended to
C
b
d
Ib
41
vvere recorded as extracellular fired at fixed latenq bk elcctmal
responses
potentials
ur -
e
I?--4
I
1 20msec
Fig. I. The two types of single unit recorded I” retmal penetrattons. Both ty,pes are fired at tiled latency from the optic tectum. (a) Negative-going spikes. (b-e) Diphastc posrttve-negattve spikes In column c paired stimult are used. The records are of single sweeps. In this and subsequent iigures.
negativity
is displayed
as a downwards
deflectton
Anticlromic invasion of ganglion cells in the pigeon.retma stimulation of the contralateral optic tectum. The spikes were of two kinds: negative-going spikes of low amplitude (43 units). and larger diphasic positivenegative spikes (222 units). Figure la illustrates an unusually well-resolved negative spike cu. 5oOpV in amplitude; often the negative spikes were only SO-1OOpV in amplitude. and could not be readily resolved. The diphasic positive-negative spikes, as illustrated in Fig lb-e were larger in amplitude, and longer in duration. Electrode advance would invariably lose the negative spikes, without a change in conformation, and without injury discharge. In contrast. advance of the electrode in the vicinity of diphasic spikes often resulted in injury discharge, an increase in spike amplitude, and, on rare occasions, the production of “giant” spikes riding from positive-going (depolarizing) potentials. Thus. the diphasic spikes are cell-body recordings. as described in the retina (Fukuda and Stone. 1974) and CNS (Bishop. Burke and Davis. 1962). It follows that the diphasic spikes are derived from ganglion cell bodies. The identification of the negative spikes is less certain: they are probably derived from optic nerve fibres. since they were generally recorded at the retinal surface, prior to obtaining cell body recordings. The receptive fields of diphasic spikes were almost always centred with the PNR (proximal negative response). Some negative spikes had receptive fields centred with the PNR. others had distant, and generally more. posterior receptive fields. Antidromic invasion
Columns c-e of Fig. 1 illustrate further properties of antidromic invasion. To closely paired twicethreshold stimuli, it can be seen that the inflection on the rising phase of the second response is more marked (A-B hesitation); there is occasional delay of the B-spike, and in trace 4 of column c, the B-spike is blocked, leaving the A-spike in isolation. Columns d and e illustrate traces where interaction between antidromic and orthodromic firing was sought. In the third sweep in column d the antidromic response is preceded by an orthodromic spike occurring just outside the collision interval. In the next sweep the orthodromic spike falls within the collision interval, and both the A and the B components of the spike are lost. Collision was also observed for the negative spike in Fig. la In general, every cell examined with the collision test showed deletion of the antidromic spike within the forbidden collision interval (twice the antidromic latency + axonal recovery time). Dispersion and timing of antidromic invasion
Figure 2 a-d illustrates four averaged records taken during a single penetration of the retina. Six diphasic responses were recorded. It is clear that at any one location in the retina there can be a wide range of response latencies (from 9 to 20 msec in this case) and thus there must be a considerable range in the conduction velocity, either intraretinally or extraretinally. In records b and c there are two spikes in each sweep. The two spikes in record b are due to simultaneous recording from two cells: the spike with a 10 msec latency was lost by electrode advance. leaving the later spike in isolation. In sweep c the two spikes
1359
could not be separated by adjusting the tectal voltage. nor were they separated by electrode advance. The first spike could follow repetitive stimuli to 50 Hz. while the second spike failed with A-B block at 10 Hz. Light evoked discharge also consisted of double spikes. It appears likely that the responses represent double spike discharge of one unit. possibIy due to afterdepolarization at the ganglion cell. It should be noted that these are averaged records (unlike the responses in Fig. 1). Since antidromic invasion is time-locked to the stimulating pulse. other responses, close to, or below the baseline noise level of single sweeps, can be distinguished. These field potentials are examined in a following paper. Here, it is apparent that in each trace there are small negative-going spike-like potentials, and that each baseline undergoes a small negative displacement, peaking at a latency of 15-20msec. Recovery
times
Recovery times were measured by applying closely spaced pairs of :wice-threshold stimuli to the tectum, and measuring the delay at which the second spike failed. Most recover) times were in the range of 1-4 msec. When recovery time was plotted against latency, there was a suggestion of a positive relationship; a straight regression line with a slope of 0.24 fitted the data points. The spikes elicited by the second stimulus showed several characteristic features. ‘Commonly, there was pronounced A-B hesitation, and less often A-B fragmentation, as illustrated in Fig. 1. In some cases the diphasic spike disappeared entirely, suggesting axonal refractorines. The latency of the second spike at delays close to the recovery time was often increased, as illustrated in Fig. 2e. Here a simultaneous record was taken from _.__ a negative-going spike fired at a latency of 5 msec. and from a diohasic soike fired at a latencv of _. 20 msec. The figure illustrates the responses to a-pair of stimuli separated by 1.67 msec; this value was selected to be just longer than the recovery time. with both spikes being discharged twice on each trial. The second negative spike is delayed by 1 msec (20% of its latency) and the second diphasic spike is delayed by 1.9 msec (9.4% of its latency). Usually, observation of single antidromic responses was carried out at a sweep repetition rate of l-3 HZ. In some cells simply increasing the rate to 15 Hz resulted in a cumulative increase in latency for a dozen sweeps. with subsequent spike blockage. In other cells responses would continue at 15 Hz at a stable, though increased, latency. The latenp shifts were in the range of IO-15%. When tested with brief trains of 5-7 stimuli. with a train repetition rate of 1 Hz, a wide range of behaviours was observed. Some cells could follow each pulse in the train up to 300-500 Hz. Other cells would block at 50 Hz. Blockage took three forms, involving A-B fragmentation, intermittent spike production, or a graded decline in the whole diphasic spike. Insufficient observations were made to test whether the ability to follow trains was related to response latency. Tectal and isthmo-optic
stimuli
Since the pigeon has a well-developed centrifugal
A
1360
L.
HOLDER
d
r 25 msec
Fig. 1. Averaged records of antidromically fired cells. Each record IS the average of 32 consecutive responses. Records a-d were obtained in a single penetration. e shows the response to closely paired. twice-threshold stimuli. pathway to the retina-the isthmo-optic tract (10-I’) (see Cowan 1970, for a reviewkit is necessary to check that centrifugal responses are not being confused with antidromic activation. Previous experiments have shown that tectal stimuli produce monosynaptic activation of the isthmo-optic nucleus (Holden, 1968b). Therefore, experiments were carried out in which one set of stimulating electrodes was applied to the lateral tectum, and another set to the IO-I. The series was continued until 52 ganglion cells had been investigated. Of these, 51 cells were fired antidromically from the tectum, and one could be fired only from the IOT. Of the 51 cells tied from the tectum, 9 were also tired from the IOT electrodes. In all instances their discharge was judged to be antidromic, by their brief recovery times, fixed latency, and, in some cases, by collision evidence. The responses of a cell fired both from tectum and from the IOT electrodes are shown in Fig 3. The recovery times for paired stimuli delivered to either location was 3 msec. Records a and b show that the
fixed latency spikes to IOT stimulation can be removed by a preceding orthodromic spike occurring within the collision interval. Records e and f show the same phenomenon for the spike elicited by tectal stimulation. Thus activation both from the tectum and from the IOT is shown to be antidromic. Record c shows that a tectal response (with timing illustrated in record d) does not block a succeeding response to IOT stimulation. Record g shows that a spike elicited from the tectum is not blocked by a preceding spike elicited from the IOT. In records c and g block by collision would have to occur if the first spike in each pair were trans-synaptically produced. The only possible interpretations are that both responses are trans-synaptically produced, or that both are antidromic. Trancsynaptic activation would not be expected to show a fixed latency, nor a brief recovery time, nor collision evidence as shown in records a and e. It follows that the cell is antidromically invaded both from the optic tectum and from the electrodes aimed at the isthmo-optic tract. This mode of antidromic discharge probably results
1361
Antidromic mvasion of ganglion cells in the pigeon retina
T-IOT
1
IOT -T
I
l.OmV
25 msec
Fig. 3. Responses to tectal (Tect: T) and tsthmo-optic (IOT) stimulatton. Records a and e are of single sweeps. The other records are the average of 32 responses. (a) Removal of the response to IOT sttmulus. by collision. (b) Response to IOT stimulus. (cl Tectal response (with timing shown m d) precedes the response to IOT stimulus. (et Removal of response to rectal stimulus by collision. ITITiming of response to tectal stimulus. (g) Response to IOT precedes response to tectum. The collision and interaction records show responses to be antidromic.
from axonal branching, one branch running to the tectum, the other to the dorso-lateral complex of the thalamus. The latter region is closer to the isthmooptic electrodes than the more anteriorly located optic tract. It was generally found that activation from the IOT needed stimuli greater than 15 V, while tectal voltage thresholds were often as low as 2 V. A full analysis of branch times by cancellation was not carried out. From the observations on extraretinal conduction made in this paper .it would be expected that these times would be a small fraction of the total antidromic latency. The double-stimulation experiments have a further implication of showing that the majority of ganglion cells were fired from the tectum and not from the IOT. Hence it cannot be argued that responses to tectal stimulation are actually produced via the isthmo-optic loop. Extraretinal and intraretinal conduction
Figure 4 shows a histogram of the latency distribution of antidromically fired cells, following tectal stimulation. The distribution peaks at a latency of
4msec. and has a long tail. with the longest latency being at 35 msec. Antidromic conduction from tectum to retinal ganglion cell takes place in axons of which many are myelinated extraretinally. and unmyelinated intraretinally. To give an estimate of the extraretinal conduction time. experiments were carried out using two sets of stimulating electrodes, placed, respectively. at the lateral tectum and optic tract. Antidromically fired cells were recorded. and the latency to tectal and tract stimulation was noted. For each of 102 units the tectal latency was longer than the tract latency. Tectal latencies ranged from 2 to 16 msec. The lower histogram in Fig. 4 shows the difference between tectal latency and tract latency; this time difference is the time for extraretinal conduction over a distance of 9 mm. It is of interest to give an estimate of the conduction velocities involved, pooled between experiments. The most frequently sampled interval in the histogram is 1 msec. This corresponds to a conduction velocity of 9 m/set. The centres of subsequent bins correspond to conduction velocities of 4.5, 3.0, 2.25 and 1.8 m/set.
1361
It
L HOLDEX
sAmpled latency (2 msec) corresponds to d conduction \eloclt) of 1 m.sec. Umts fired at this and longer latencles (with conductlon \rloanes of I m SK and lower) make up 93”” of the sample. and are probabl! serbed b> unmyelrnated axons In the retina.
The most frtquentlq
Intraretmal and extraretmal conduction \elocltles (10’ and ECV. respectice&) were calculated on the: assumptton of constant conduction velocit> in each segment. The ratlo of ICV ECV uas calculated for each cell. and a frequent! histogram of the ratio is shown m Fig. 5b The maJorit> of cells m the sample 17?“,) are contained within bins 0.05 and 0.1. and hale a intraretmal veloat) that IS slower by a factor of l&-20 than the extraretinal velocit!. A l’ew cells (6”,,. m bin 1.0) have little or no reduction m velocity in the retina. Their conduction velocities ranged from 1’5 to 4.5 m sec. suggestmg that they may & mye_._ lmstcd In both the t&are&al and extraretinal segment of the retinotectal pathway.
FIN. 4 Upper histogram: Latency histogram of responses to tectal stimuli. Ordinate-No. of cells: abscissa-latency in msec. Bin-widths. I msec. Lower Hisrogrnm: Conduction ttmes m 9mm of optic tract. Ordmate--No of cells. abscrssa-time tn msec. Bzn-mldths. I msec
lrmurrtinal
The depths in the retina at whtch antidromlcally mrrtdrd cells are recorded is of interest. for the distributton should peak at the ganglion cell layer tf this IS the chief source of the records. It might be argued that the bagaries
of electrode
selectirlty
ensure
that
condwrm
An estimate of intrarstinal conduction time can be given by scaling the tract conduction time to the distance from tectum to optic nerve-head (1.6cm). and subtracting this time from the tectal antidromic latency. The resultant histogram is shown in Fig. 5a. The distribution peaks at a latency of 2 msec. and its tail extends to 15 msec. While the distribution gives an estimate of intraretinal delay from the optic papilla to the ganglion cell. an intraretinal conduction distance is required in order to estimate intraretinal conduction velocity. The routes taken by optic nerve axons were examined in Methylene Bfue stained retinas. Staining was continued until optic nerve fibre-bundles and a substantial number of ganglion cells had become blue. Fibres stream into the optic nerve-head at rightangles to its long axis. with the fibre bundles travelling in near-parallef straight lines across the entire retinal surface. At the upper and lower ends of the nerve-head the fibre directions fan through 180’ from the nasal to the terriporal retina. (The pattern closely resembles the directions of “opaque nerve fibres” observed ophthalmoscopically by Woods (1917) in the avian fundus). The recording site in the central yellow iietd of the retina is crossed by fibres which run to the upper portion of the optic nerve-head. and enter it after travelling for 2 mm. Conduction velocities corresponding to a conduction distance of 2 mm can be inferred from the histogram in Fig. 5a. They range from 4.5 to 0.13 m/set.
Estlmoted
tntroret’nal
de%Ijs,
msec
FIN 5. (a) Histogram of mtraretmal conducnon times Ordinate--No. of ceils: abscissa-time in msec. Bin%ldLhs, 1 msec. (b) Histogram of ICV ECV (mtraretinal conductlon velocity over extraretmal conduction velocity) Ordinate-ho. of cells: abscissa--ICV/‘ECV. The first bm has B width of 005: subsequent buts are 0.1.
Anudromx
r-l NFL
0
1363
Invasion of ganglion cells In the pigeon retina
a3 NO of OHS
(1968a) and with the field potential study of Mori (1973). Finally, it is of interest that the range of velocities falls within the range of myelinated axons of low diameter (Rushton, 19%; Waxman and Bennett, 1972). and iJ comparable to the T, grouping in the cat optic nerve (Bishop, Clare and Landau, 1969) and to the W-cell grouping in the cat retina (Hoffman. 1973; Fukuda and Stone. 1974). Intrarerinal
and extraretinal
conduction
The intraretinal
Fig. 6. Depths of ganglion cell recordings. Micrometer depth readings were scaled to the depths marked histotogitally by Hayes and Hoiden (1978). The inset shows the thickness of the inner retinal layers in the central yellow field: NFL = nerve fibre layer; GCL = ganglion cell layer: iPL = inner plexiform layer. recordings are made predominantly from the larger, deeper, displaced ganglion cells (Dogiel’s cells). Depth
readings from the digital microdrive were placed in bins of 20~ bin-width. and the depth axis was scaled to be in accord with the mean depths of dye spots placed to mark the PNR (Hayes and Holden, 1978). The scaled distribution in depth is shown as a histogram in Fig 6. There is a considerable scatter in the depth readings. which are centred more or less symmetrically about the ganglion cell Iayer, suggesting that most of the cell records are derived from “conventional” ganglion cells with cell bodies in the gangfion cell layer. DISCUSSION Conduction in the retinotectal
pathway
The conduction times measured in this study suggest that extraretinal conduction velocities range from 18 to 1.8 m/set, with 9 m/set being the most commonly sampled value. This range is in close accord with previous observations of orthodromic conduction from retina to tectum in the pigeon (Holden, 1968a). In the previous study, records were taken from fixed latency negative-going spikes at the tectal surface, probably derived from terminal arborisations. The velocity range was from 16 to 3.2 m/set. with 8 m/set being the most commonly sampled value. However, the latency histograms derived from single unit studies can be used only with caution to infer fibre diameters or a fibre spectrum. A primary reason is that microelectrode selectivity may bias the sample. Further, the short latency fibres, with high conduction velocities, fall into the early bins. If the histogram is used to infer a spectrum, then these bins impose a coarse quantization. This quantization can be avoided if the spectrum is drawn from the actual latency measurements, but in this case great reliance is placed on individual readings. These readings include errors due to utilization time, due to the inexactly specified conduction distance (it is not known where an axon is excited), and to the inaccuracy in measuring latencies, which may be up to 0.25 msec. Thus a latency histogram can only be taken as a rough guide unless the results are in good agreement with independent techniques. The present results are in accord both with the observations of Holden
conduction velocity of individual axons is lower than the extraretinal counterpart by a factor of at least 10-20. This ratio would be expected on the grounds that most axons in the optic nerve are myelinated. while most axons are unmyelinated intraretinally The range of velocities is from 4.5 m/see to 0.13 m/set. with a peak at 1 m/set. These figures are somewhat lower than those derived by Stone and Freeman (1971a.b) for the cat retina. The velocities were calculated from individual latencies on the assumption that conduction velocity is constant in the intraretinal and extraretinal segments. Axonal beading may occur in the retina (Stone and Hollander, 1971), and myelinization may occur in the first few mm of the optic nerve (O’Flaherty, 1971; Cowan, 1970). The latter effect would mean that the extraretina1 conductlon times are under-estimated and the intraretinal conduction times are over-estimated. While the majority of axons have low conduction velocities in the retina, some 6% of the sample show no evidence of intraretinal slowing and conduct at 2.25 to 4.5 m/set. These axons must be myelinated both in the optic nerve and in the retina. There is electron microscopic evidence (B. P. Hayes, personal communication) that some 7% of the axons in the nerve fibre layer in the central yellow field are myelinated. It should be stressed that the measurements of conduction velocity were made primarily from cellbody recordings, and hence characterize conduction from the central yellow field, rather than conduction from the periphery. Repetitive activity
While the present results are not a systematic survey, it is clear that many cells with brief (l-4 msec) recovery times to paired stimuli fail to follow repetitive stimuli above 50Hz for more than a dozen or so sweeps. This seems an anomalous property only if compared with antidromic activation via a fibre system of large myelinated axons. There is evidence that the antidromically activated Rohon-Beard cells in Xenopus block at repetition rates of 1 Hz (Spitzer, 1976). These cells show collision of antidromic and orth~ro~c spikes (the most reliable criterion for antidromic invasion) and have low conduction velocities of 0.6-0.8 m/set. It seems likely that the inability to follow high frequency antidromic stimuli is either an inherent property of fine, unmyelinated fibres, or is due to the accumulation of K,+ in the extracellular space in axon bundles bounded by Miiller processes (cf. Orkand, Nicholls and KuEler, 1966). K,+ accumulation would be expected to depolarize both nerve-axons and Miiller processes, and the former effect could explain the reduction in conduction velocity, and possible
1J6-i
4.
L.
blockage of conduction. lhe inabrlity to follow high frequencres would be less marked for visually evoked tiring. because spoke discharge would not occur in ail elements in a fibre-bundle, since the bursts of evoked discharge are transient. and smce there is little or no spontaneous firing. .-!.uonaf branches to the dorm-iareraf
thalamus
The observations that a minority of ganglion cells can be fired antidromically at high threshold from electrodes aimed at the isthmo-optic tract is probably explained by axonai branching. The projection from retina to thalamus in the pigeon is well established (Cowan. Adamson and Powell. 1961: Karten and Nauta. 1968). The posterior parts of the dorso-lateral complex are only :! mm distant from the isthmo-optic tract. so stimulus spread is likely to occur. It would
be of interest to characterize ganglion cells projecting to the thalamus, tn order to examine the analogy with the mammalian visual system. where the branched axons projecting both to co!!iculus and LGNd are the Y-cells (Hoffman, 1973). Ganglion cells and displaced ganglion cells The present experiments show that the majority of resolved ganglion cells in the yellow ii&d can be
antidromically invaded from the optic tectum. The scaled depth readings suggest that most of these cells are in the ganglion cell layer. This suggestion is in accord with the identification of displaced ganglion cells in the pigeon by Karten, Fite and Brecha (1977). who showed that while conventional ganglion cells project to the optic tectum. the displaced ganglion cells project to the ectomammillary nucleus. However, a similar study by CrandalL Heaton and Brownell (1977) of the chick suggests a differing organization. with numerous displaced ganghon cells projecting to the optic tectum. A further physiological study, combined with depth marking, would be of some interest. The ref~~orecral sysrem in the pigeon and the Wceff system in rke cat
Several observations suggest a close similarity between the retinotectal system in the pigeon and the W-cell system in the cat. Five similanties can be listed: (1) Both systems project predominantly to the optic tectum. (cf. Fukuda and Stone, 1974). (2) Both systems have a similar range of extraretinal and intraretinal conduction velocities. (3) In both systems there is a wide scatter of antidromic latencies at one retinal locus (cf. Stone and Fukuda, 1974). (4) Receptive fields of pigeon ganglion cells resemble those of W-cells more closely than the X- or Y-cells (Holden, 1977). (5) The soma sizes of pigeon ganghon cells are mainly small. in the range of j_lO~~m, like those of W-cells. While the comparison needs to be taken further. with both physiological and morphological techniques, it would be of considerable interest if the W, X, Y ciassification (discussed by Rowe and Stone, 1977) could be shown to extend across vertebrate classes. ,icknowledgements-I am grateful for f!nanaa! The Binstead Fund. and from The Smith. French Foundation.
from
support Kline &
HOLDES
REFERENCES Bmggeh R. L.
J. (1969) The ptgeon rctma. opttc nerve and ganghon ccl! layer. J. camp. &et&. 137. I-18. Bishop P. 0.. Burke W and Davis R. (1962) The tdentification of srngie units m central vrsua! pathways. J. Plqsioi. 162. 409-431. Btshop G. H.. Glare M. H and Landau W. M. (1969) Further analysts of fibre groups m the opttc tract of the cat. ExpI :Veuro/. 24, 386-399. Clarke P. G. H. and Whttteridge D. (1976) The proJection of the retina. mcluding the ‘Red area’ on to the optic tectum of the pigeon. Q. JI e.vp. Phvsrof. 61. 351-358. Cowan W. M. (1970) Centrifugal fibres to the avian retma. Br. med. Bull. 26. 112-I 18. Cowan W. M.. Adamson L. and Powell T. P. S. (1961) An experimental study of the avian visual system. J. Anat. 95. 545-363. Crandalf J. F.. Heaton IM. B and Brownelf W. E. (1977) Tectaf proJectton of disptaced gangtion cells in avian retina. fnrest. ~phtha~. 16, 774-776. Fukuda Y. and Stone J. (1974) Retina! distribution and central proJections of Y, X. and W-cells of the cat’s retina. J. ,Veurophysiol. 37. 749-772. Hamdi F. A. and Whitteridge D. (1954) The representatton of the retina on the optic tectum of the pigeon. Q. JI esg. Phvsiol. 39. I I I-I 19. Hates B. P. and Holden A. L. (1978) Depth-marktng the proxtmal negative response in the ptgeon retina. J camp. .Veurol. 180. 193-202. Hayes B. P. and Webster K. E. (1975) An electron microscope study of the retino-receptive layers of the pigeon optic tectum. J. camp Neural. 162, W7-366. Hoffman K. P (1973) Conduction velocity in pathways from retina to superior co!IicuIus in the cat: a correlation with receptive field properties, J. .V~~rop~~s~o~.36. 409424 Holden A. L. (1968a) Types of Umtary response and correlatton with the field potential profile during activation of the avian opttc tectum. J. Physiol. 194, 91-104. Holden A. L. (1968b) The centrifugal system running to the pigeon retina. .I. Pirpiol. 197, 199-219. Holden A. L. (1977) Concentric receptive fields of pigeon gangtion celis. Vision Res. 17. 545-5.54. Holden A, L. and Powell T. P. S. (1972) The functional organisation of the isthmo-optic nucleus in the pigeon. J. Phpsiol. 223, 419-447. Karten H. J. and Nauta W. J. H. (1968) Organisatton of retinothalmic projections in the pigeon and owl. Anar. Rec. 160. 373. Karten H. J.. Fite K. V. and Brecha N. (1977) Spectfic projection of displaced retinal ganglion cells upon the accessory optic system in the pigeon (Colomba livia). Proc. Sam. Acad. Sci. L’.S.A. 74. 1753-1756. Lavai! J. H. and Lavai! M. M. (1974) The retrograde mtraaxona! transport of horseradish peroxidase in the chick visual system: A light and electron microscope study J. camp. Nacrol. 157, 303-358. McGill J. L. Poweli T. P. S. and Cowan W. M. (t966) The retina1 representation upon the optic tectum and tsthmo-optic nucleus in the ptgeon. J. Anat. 100. 5-33. Mori S. (1973) Analysis of field responses in the optic tecturn of the pigeon. Brain Res. 54, 193-206. O’Flahertv J. J. (1971) The optic nerve of the Mallard Duck: * Fiber-diameter frequency distribution and physiological properties. J. camp. Neural. 143. 17-21. Orkand R. K.. Nicholls J. G. and Kuffler S. W. (I966) EFfect.of nerve impulses upon the membrane potential of ghal cells in the central nervous system of amphrbia. J. Neurophysrol. 29. 788-806. Pearlman A. L. and Hughes C. P. (1976) Functional analy sis of efferents to the avian retina: 1. Analysis of retma! Quantltatiw
and Paule W aspects of the
Antidromic invasion of ganglion ceils in the pigeon retina ganglion cell recepttve fields. J. camp. Neural. 166. 111-12.2. Rowe M. H. and Stone J. (1977) Naming of neurones: Classtfication and naming of cat retinal ganglion cells. Bruin Behac. Ecol. 14. 185-216. Rushton W. A. H. (1951) A theory of the effects of fibre size in medullated nerve. J. Physiol. 115. 101-122. Spitzer N. C. (1976) The ionic basis of the resting potential and a slow depolarising response in Rohon-Beard neurones of Xenopus tadpoles. J. Physiol. 255. 105136. Stone J. and Freeman J. A. (1971a) Synaptic organisation of the pigeon’s optic tectum: a Golgi and current source density analysis. Brain Res. 27. 203-221.
1365
Stone J. and Freeman R. B. Jr (197lb) Conduction velocity groups in the cat’s optic nerve classified according to their retina) origin. Expl Brain Res. 13. 489-397. Stone J. and Hollander H. (1971) Optic nerve axon diameters measured in the cat retina: Some functional considerations. Expl Brain Res. 13. 498-503. Stone J. and Fukuda Y. (1974) Properties of cat retinal ganglion cells: A comparison of W-cells with X- and Y-cells. 1. Neurophysiol. 37. 722-748. Waxman S. G. and Eennett M. V. L. (1972) Relative conduction velocities of small myelinated and non-myehnated fibres in the general nervous system. Narure. New Biol. 23& 217-219. Woods C. A. (1917) The Fundus &u/i of Birds. Chicago.
CELLS
A. L. HOLDEN Department of Visual Science. Institute of Ophthalmology.
Judd Street. London WC1 H9QS. England
(Received 21 November 1977: in revised form 25 January 1978)
Abstract-265 single units were recorded in the yellow field of the pigeon retina. Ganglion cell-bodies can be antidromically invaded from the optic tectum. Conduction velocities range from 18 to 1.8 misec extraretinally and from 4.5 to 0.13 m!sec intraretinally.. For most axons conduction slows in the retina by IO-20 times. A minority of ceils are fired antidromically from the isthmo-optic tract, probably resulting from stimulus spread to the dorsolateral thalamus.
lSfRODUCTlON
The experiments reported here examine the antidromic invasion of pigeon ganglion cells. If antidromic invasion can be shown following stimuiation of the central visual pathway, then it is established that the recording is taken from an output cell (ganglion cell or displaced ganglion cell) of the retina. This physiological identification can be ,made with extracetlular recording in a small-cell system, where intracellular recording and dye injection may be difficult. The optic nerve of the pigeon has been examined with the electron microscope (Binggeli and Paule. 1969). The retinotectal system has been examined electrophysiologically (Holden. 1968a; Stone and Freeman. 1971a,b) with mapping techniques (Hamdi and Whitte~dge, 1954; Clarke and Whitteridge, 1976). and with experimental anatomical techniques (McGill. Powell and Cowan, 1966; Lavail and Lavail, 1974; Hayes and Webster, 1975). A previous singleunit study (Holden, 1968af provides an estimate of conduction velocities of retinotectal axons. using orthodromic conduction between the optic nervehead and the lateral tectum. The present experiments extend this evidence by using antidromic invasion to measure axonal conduction times both intraretinally, and between the retina and the tectum. The results show that the majority of spike-generating cells in the central yellow field of the retina can be antidromically invaded from the optic tectum, showing them to be ganglion cells. T’he extraretinal conduction velocities are low, and fal1 into the range of the W-ceil grouping in the cat. Intraretinal velocities are lower, by a factor of 10-20, due to conduction in fine. unmyelinated fibres. METHODS The experiments were carried out on 50 feral pigeons. anaesthetized with urethane. maintained under one-way Row artiticial ventilation. and prepared for intraretinal recording as described in detail in Holden (1977). Three types of preparation were used for electrical stimulation. In each. electrical stimuli were delivered to the exposed surface of the contralateral tectum. This was either the sole site of stimulation, or was combined with stimulation of the optic tract, via the orbit of the contralateral eye, or with stimulation of the isthmo-optic tract intracranialiy,
as described in Holden and Powell (1972). The first preparation was used to accumulate data on the latency of antidromic invasion to tectal stimuli; the second to measure conduction time extraretinally. and the third to assess any possible responses due to orthodromic discharge of the centrifugal system running from isthmo-optic nucleus to retina. Electrical stimuli were delivered isolated from earth, via pairs of steel needles insulated by Araldite epoxy resin PZ 985. The needle tips were separated by 1 mm. and were ground bare before each experiment. An early series of experiments on.19 pigeons was carried out in 1964 in The University Laboratory of Physiology, Oxfoid. Later experiments were carried out in London. In the later experiments an averager (Hewle~-Packard 5480B) was used extensively with an X-Y plotter, both as a quick method of providing permanent records. and as a means of displaying small graded fields close to the baseline noise level. A sweep time of 50 msec was used: with a resolution of loo0 points this corresponds to a sampling rate of 20 kHz. The headstane amplifier was used with 3-pole Butterworth filters providing’ a pass-band bf 0.16 Hz-3 kHz. Calibration signals were obtained from a Brookdeal Signal Source Type 471. Placemenr of stimulating elecrrodes Tecrum. The stimulating electrodes were thrust against the tectal surface, after exposing 1-2 mm of the lateral aspect of the right tectum. In all experiments the head was held in the position of normal walking, with a beak bar 25 mm anterior to, and level with, the ear bars. The projection of the lateral visual field was as used in Holden and Powell (1972) and by Pearlman and Hughes (1976): the point level with the centre of the pupil in the lateral visual field was taken as tlie centre of a poIar projection. When receptive fields of tectal cells in the exposed lateral aspect were recorded. their locations were close to the horizontal meridian, and IO”-25” anterior. Reozptive fields of retinal ganglion cells were clustered round the centre of the projection. Thus there was a partial overlap between receptive fields at the stimulation site and at the recording site. Those retinotectal fibres stimulated in passage at the tectal surface would be travelling anteroposteriorly, thus including fibtes corresponding to the more posterior ganglion cetl receptive field locations. Optic tract. The tract running from left eye to right tecturn was stimulated 9 mm anterior to the lateral tectal electrodes. A pair of stimulating electrodes was advanced through an opening made in the temporal wall of the orbit of the right eye, after displacing the eye forwards. Positioning of the electrodes was completed during the early stages of each experiment by searching for a position which pro-
1357
4. L. HOLDEX
13%
record small. neysttve-gotng sptkes. the htghcr reststdnce electrodes could rssolve and hold dtphastc cell spokes for long periods 10 j-6 hri. but recorded relaavely feu ncgattvs spikes. Thts dtfierencc tn selectivity IS nc71 due to Johnson noise per sz. for the use of averagmg could ea>tlb offset a j-fold dtfference m peak-to-peak notse. Electrodes were adv;Fncrd Into the retma wtth d Trent-
ducsd a rettn31 response matchmg the response to tectal sttmulatton. Thts search was often only partly successful. because of the large and Intractable stimulus arttfacrs produced by sttmulr apphed close to the left eye. However. even If the sttmulus arttfact precluded adequate study of small field potentials. It was always posstble to observe well resolved untt responses. Ivrhmo-opt/c rracr. A patr of stimulatmg electrodes was advanced verttcally downuards through the right forebram. for 9-10 mm. They were placed 2.5 mm lateral to the midline. It was shown in Holden and Powell (1972) that electrodes Inserted hortzontally into the lateral tectum can reach the rsthmo-opttc nucleus. Thus the anterior-postertor location used was one which straddled the level of the dissectton field at the lateral tectum. Electrodes placed III this way UIII excite the isthmo-optic tract and adjacent structures. They excite tectal output cells, in the lateral tectum anttdromically. at a frequency of 3-6 cellstrack. though they do not produce orthodromtc activation of the tectal surface. judged by production of an .Y-wave Thus the! do not produce major activation of the optic tract by current spread; the optic tract electrodes. placed as described above. could always produce a tectal &‘-wave tn addttton to antidromic acttratton of the retina.
Wells stepptng mtcrodrtve Depth readings uerc taken with respect to the mtcrodrive readmg Jt vvhtch the retma was penetrated. Because of tissue drag and dtmpltng they can only provide an approximate guide to rettnal depth. Where the depth dtstrtbutton of ganglton cells :s ssttmated (Ftg. 6). the depth readings were scaled ustng data from an mdependent study where dye marks were used to locahze the proximal negative response (Hayes dnd Holden. 19-Y). In SIX ptgeons Methylene Blue statmng uas used intravttally or suprdvttally to determtne the traJectortes taken by opttc nerve hbres runnmg from the recordmg stte tn the central yellow field to the opttc nerve head. intr3vttreal Injection of 03 ml of OOI”, solutton oi Methylene Blue m saline wrls followed by dtssection of the rettna I hr later Alternattvely. the retina uas dtssected ou: !n avtan Ranger and stamed supravttally
.Ificroelucrrodes
RESC LTS
Mtcroptpettes filled wtth 3 X4 KCI were used throughout In early experiments they were used wtth resistances
Tj,pes
of 5-7 MR. In later Z&70 :\lR Electrodes
spike
experiments with lower
of rtnlt responsr
265 unit
the resistances were resistances tended to
C
b
d
Ib
41
vvere recorded as extracellular fired at fixed latenq bk elcctmal
responses
potentials
ur -
e
I?--4
I
1 20msec
Fig. I. The two types of single unit recorded I” retmal penetrattons. Both ty,pes are fired at tiled latency from the optic tectum. (a) Negative-going spikes. (b-e) Diphastc posrttve-negattve spikes In column c paired stimult are used. The records are of single sweeps. In this and subsequent iigures.
negativity
is displayed
as a downwards
deflectton
Anticlromic invasion of ganglion cells in the pigeon.retma stimulation of the contralateral optic tectum. The spikes were of two kinds: negative-going spikes of low amplitude (43 units). and larger diphasic positivenegative spikes (222 units). Figure la illustrates an unusually well-resolved negative spike cu. 5oOpV in amplitude; often the negative spikes were only SO-1OOpV in amplitude. and could not be readily resolved. The diphasic positive-negative spikes, as illustrated in Fig lb-e were larger in amplitude, and longer in duration. Electrode advance would invariably lose the negative spikes, without a change in conformation, and without injury discharge. In contrast. advance of the electrode in the vicinity of diphasic spikes often resulted in injury discharge, an increase in spike amplitude, and, on rare occasions, the production of “giant” spikes riding from positive-going (depolarizing) potentials. Thus. the diphasic spikes are cell-body recordings. as described in the retina (Fukuda and Stone. 1974) and CNS (Bishop. Burke and Davis. 1962). It follows that the diphasic spikes are derived from ganglion cell bodies. The identification of the negative spikes is less certain: they are probably derived from optic nerve fibres. since they were generally recorded at the retinal surface, prior to obtaining cell body recordings. The receptive fields of diphasic spikes were almost always centred with the PNR (proximal negative response). Some negative spikes had receptive fields centred with the PNR. others had distant, and generally more. posterior receptive fields. Antidromic invasion
Columns c-e of Fig. 1 illustrate further properties of antidromic invasion. To closely paired twicethreshold stimuli, it can be seen that the inflection on the rising phase of the second response is more marked (A-B hesitation); there is occasional delay of the B-spike, and in trace 4 of column c, the B-spike is blocked, leaving the A-spike in isolation. Columns d and e illustrate traces where interaction between antidromic and orthodromic firing was sought. In the third sweep in column d the antidromic response is preceded by an orthodromic spike occurring just outside the collision interval. In the next sweep the orthodromic spike falls within the collision interval, and both the A and the B components of the spike are lost. Collision was also observed for the negative spike in Fig. la In general, every cell examined with the collision test showed deletion of the antidromic spike within the forbidden collision interval (twice the antidromic latency + axonal recovery time). Dispersion and timing of antidromic invasion
Figure 2 a-d illustrates four averaged records taken during a single penetration of the retina. Six diphasic responses were recorded. It is clear that at any one location in the retina there can be a wide range of response latencies (from 9 to 20 msec in this case) and thus there must be a considerable range in the conduction velocity, either intraretinally or extraretinally. In records b and c there are two spikes in each sweep. The two spikes in record b are due to simultaneous recording from two cells: the spike with a 10 msec latency was lost by electrode advance. leaving the later spike in isolation. In sweep c the two spikes
1359
could not be separated by adjusting the tectal voltage. nor were they separated by electrode advance. The first spike could follow repetitive stimuli to 50 Hz. while the second spike failed with A-B block at 10 Hz. Light evoked discharge also consisted of double spikes. It appears likely that the responses represent double spike discharge of one unit. possibIy due to afterdepolarization at the ganglion cell. It should be noted that these are averaged records (unlike the responses in Fig. 1). Since antidromic invasion is time-locked to the stimulating pulse. other responses, close to, or below the baseline noise level of single sweeps, can be distinguished. These field potentials are examined in a following paper. Here, it is apparent that in each trace there are small negative-going spike-like potentials, and that each baseline undergoes a small negative displacement, peaking at a latency of 15-20msec. Recovery
times
Recovery times were measured by applying closely spaced pairs of :wice-threshold stimuli to the tectum, and measuring the delay at which the second spike failed. Most recover) times were in the range of 1-4 msec. When recovery time was plotted against latency, there was a suggestion of a positive relationship; a straight regression line with a slope of 0.24 fitted the data points. The spikes elicited by the second stimulus showed several characteristic features. ‘Commonly, there was pronounced A-B hesitation, and less often A-B fragmentation, as illustrated in Fig. 1. In some cases the diphasic spike disappeared entirely, suggesting axonal refractorines. The latency of the second spike at delays close to the recovery time was often increased, as illustrated in Fig. 2e. Here a simultaneous record was taken from _.__ a negative-going spike fired at a latency of 5 msec. and from a diohasic soike fired at a latencv of _. 20 msec. The figure illustrates the responses to a-pair of stimuli separated by 1.67 msec; this value was selected to be just longer than the recovery time. with both spikes being discharged twice on each trial. The second negative spike is delayed by 1 msec (20% of its latency) and the second diphasic spike is delayed by 1.9 msec (9.4% of its latency). Usually, observation of single antidromic responses was carried out at a sweep repetition rate of l-3 HZ. In some cells simply increasing the rate to 15 Hz resulted in a cumulative increase in latency for a dozen sweeps. with subsequent spike blockage. In other cells responses would continue at 15 Hz at a stable, though increased, latency. The latenp shifts were in the range of IO-15%. When tested with brief trains of 5-7 stimuli. with a train repetition rate of 1 Hz, a wide range of behaviours was observed. Some cells could follow each pulse in the train up to 300-500 Hz. Other cells would block at 50 Hz. Blockage took three forms, involving A-B fragmentation, intermittent spike production, or a graded decline in the whole diphasic spike. Insufficient observations were made to test whether the ability to follow trains was related to response latency. Tectal and isthmo-optic
stimuli
Since the pigeon has a well-developed centrifugal
A
1360
L.
HOLDER
d
r 25 msec
Fig. 1. Averaged records of antidromically fired cells. Each record IS the average of 32 consecutive responses. Records a-d were obtained in a single penetration. e shows the response to closely paired. twice-threshold stimuli. pathway to the retina-the isthmo-optic tract (10-I’) (see Cowan 1970, for a reviewkit is necessary to check that centrifugal responses are not being confused with antidromic activation. Previous experiments have shown that tectal stimuli produce monosynaptic activation of the isthmo-optic nucleus (Holden, 1968b). Therefore, experiments were carried out in which one set of stimulating electrodes was applied to the lateral tectum, and another set to the IO-I. The series was continued until 52 ganglion cells had been investigated. Of these, 51 cells were fired antidromically from the tectum, and one could be fired only from the IOT. Of the 51 cells tied from the tectum, 9 were also tired from the IOT electrodes. In all instances their discharge was judged to be antidromic, by their brief recovery times, fixed latency, and, in some cases, by collision evidence. The responses of a cell fired both from tectum and from the IOT electrodes are shown in Fig 3. The recovery times for paired stimuli delivered to either location was 3 msec. Records a and b show that the
fixed latency spikes to IOT stimulation can be removed by a preceding orthodromic spike occurring within the collision interval. Records e and f show the same phenomenon for the spike elicited by tectal stimulation. Thus activation both from the tectum and from the IOT is shown to be antidromic. Record c shows that a tectal response (with timing illustrated in record d) does not block a succeeding response to IOT stimulation. Record g shows that a spike elicited from the tectum is not blocked by a preceding spike elicited from the IOT. In records c and g block by collision would have to occur if the first spike in each pair were trans-synaptically produced. The only possible interpretations are that both responses are trans-synaptically produced, or that both are antidromic. Trancsynaptic activation would not be expected to show a fixed latency, nor a brief recovery time, nor collision evidence as shown in records a and e. It follows that the cell is antidromically invaded both from the optic tectum and from the electrodes aimed at the isthmo-optic tract. This mode of antidromic discharge probably results
1361
Antidromic mvasion of ganglion cells in the pigeon retina
T-IOT
1
IOT -T
I
l.OmV
25 msec
Fig. 3. Responses to tectal (Tect: T) and tsthmo-optic (IOT) stimulatton. Records a and e are of single sweeps. The other records are the average of 32 responses. (a) Removal of the response to IOT sttmulus. by collision. (b) Response to IOT stimulus. (cl Tectal response (with timing shown m d) precedes the response to IOT stimulus. (et Removal of response to rectal stimulus by collision. ITITiming of response to tectal stimulus. (g) Response to IOT precedes response to tectum. The collision and interaction records show responses to be antidromic.
from axonal branching, one branch running to the tectum, the other to the dorso-lateral complex of the thalamus. The latter region is closer to the isthmooptic electrodes than the more anteriorly located optic tract. It was generally found that activation from the IOT needed stimuli greater than 15 V, while tectal voltage thresholds were often as low as 2 V. A full analysis of branch times by cancellation was not carried out. From the observations on extraretinal conduction made in this paper .it would be expected that these times would be a small fraction of the total antidromic latency. The double-stimulation experiments have a further implication of showing that the majority of ganglion cells were fired from the tectum and not from the IOT. Hence it cannot be argued that responses to tectal stimulation are actually produced via the isthmo-optic loop. Extraretinal and intraretinal conduction
Figure 4 shows a histogram of the latency distribution of antidromically fired cells, following tectal stimulation. The distribution peaks at a latency of
4msec. and has a long tail. with the longest latency being at 35 msec. Antidromic conduction from tectum to retinal ganglion cell takes place in axons of which many are myelinated extraretinally. and unmyelinated intraretinally. To give an estimate of the extraretinal conduction time. experiments were carried out using two sets of stimulating electrodes, placed, respectively. at the lateral tectum and optic tract. Antidromically fired cells were recorded. and the latency to tectal and tract stimulation was noted. For each of 102 units the tectal latency was longer than the tract latency. Tectal latencies ranged from 2 to 16 msec. The lower histogram in Fig. 4 shows the difference between tectal latency and tract latency; this time difference is the time for extraretinal conduction over a distance of 9 mm. It is of interest to give an estimate of the conduction velocities involved, pooled between experiments. The most frequently sampled interval in the histogram is 1 msec. This corresponds to a conduction velocity of 9 m/set. The centres of subsequent bins correspond to conduction velocities of 4.5, 3.0, 2.25 and 1.8 m/set.
1361
It
L HOLDEX
sAmpled latency (2 msec) corresponds to d conduction \eloclt) of 1 m.sec. Umts fired at this and longer latencles (with conductlon \rloanes of I m SK and lower) make up 93”” of the sample. and are probabl! serbed b> unmyelrnated axons In the retina.
The most frtquentlq
Intraretmal and extraretmal conduction \elocltles (10’ and ECV. respectice&) were calculated on the: assumptton of constant conduction velocit> in each segment. The ratlo of ICV ECV uas calculated for each cell. and a frequent! histogram of the ratio is shown m Fig. 5b The maJorit> of cells m the sample 17?“,) are contained within bins 0.05 and 0.1. and hale a intraretmal veloat) that IS slower by a factor of l&-20 than the extraretinal velocit!. A l’ew cells (6”,,. m bin 1.0) have little or no reduction m velocity in the retina. Their conduction velocities ranged from 1’5 to 4.5 m sec. suggestmg that they may & mye_._ lmstcd In both the t&are&al and extraretinal segment of the retinotectal pathway.
FIN. 4 Upper histogram: Latency histogram of responses to tectal stimuli. Ordinate-No. of cells: abscissa-latency in msec. Bin-widths. I msec. Lower Hisrogrnm: Conduction ttmes m 9mm of optic tract. Ordmate--No of cells. abscrssa-time tn msec. Bzn-mldths. I msec
lrmurrtinal
The depths in the retina at whtch antidromlcally mrrtdrd cells are recorded is of interest. for the distributton should peak at the ganglion cell layer tf this IS the chief source of the records. It might be argued that the bagaries
of electrode
selectirlty
ensure
that
condwrm
An estimate of intrarstinal conduction time can be given by scaling the tract conduction time to the distance from tectum to optic nerve-head (1.6cm). and subtracting this time from the tectal antidromic latency. The resultant histogram is shown in Fig. 5a. The distribution peaks at a latency of 2 msec. and its tail extends to 15 msec. While the distribution gives an estimate of intraretinal delay from the optic papilla to the ganglion cell. an intraretinal conduction distance is required in order to estimate intraretinal conduction velocity. The routes taken by optic nerve axons were examined in Methylene Bfue stained retinas. Staining was continued until optic nerve fibre-bundles and a substantial number of ganglion cells had become blue. Fibres stream into the optic nerve-head at rightangles to its long axis. with the fibre bundles travelling in near-parallef straight lines across the entire retinal surface. At the upper and lower ends of the nerve-head the fibre directions fan through 180’ from the nasal to the terriporal retina. (The pattern closely resembles the directions of “opaque nerve fibres” observed ophthalmoscopically by Woods (1917) in the avian fundus). The recording site in the central yellow iietd of the retina is crossed by fibres which run to the upper portion of the optic nerve-head. and enter it after travelling for 2 mm. Conduction velocities corresponding to a conduction distance of 2 mm can be inferred from the histogram in Fig. 5a. They range from 4.5 to 0.13 m/set.
Estlmoted
tntroret’nal
de%Ijs,
msec
FIN 5. (a) Histogram of mtraretmal conducnon times Ordinate--No. of ceils: abscissa-time in msec. Bin%ldLhs, 1 msec. (b) Histogram of ICV ECV (mtraretinal conductlon velocity over extraretmal conduction velocity) Ordinate-ho. of cells: abscissa--ICV/‘ECV. The first bm has B width of 005: subsequent buts are 0.1.
Anudromx
r-l NFL
0
1363
Invasion of ganglion cells In the pigeon retina
a3 NO of OHS
(1968a) and with the field potential study of Mori (1973). Finally, it is of interest that the range of velocities falls within the range of myelinated axons of low diameter (Rushton, 19%; Waxman and Bennett, 1972). and iJ comparable to the T, grouping in the cat optic nerve (Bishop, Clare and Landau, 1969) and to the W-cell grouping in the cat retina (Hoffman. 1973; Fukuda and Stone. 1974). Intrarerinal
and extraretinal
conduction
The intraretinal
Fig. 6. Depths of ganglion cell recordings. Micrometer depth readings were scaled to the depths marked histotogitally by Hayes and Hoiden (1978). The inset shows the thickness of the inner retinal layers in the central yellow field: NFL = nerve fibre layer; GCL = ganglion cell layer: iPL = inner plexiform layer. recordings are made predominantly from the larger, deeper, displaced ganglion cells (Dogiel’s cells). Depth
readings from the digital microdrive were placed in bins of 20~ bin-width. and the depth axis was scaled to be in accord with the mean depths of dye spots placed to mark the PNR (Hayes and Holden, 1978). The scaled distribution in depth is shown as a histogram in Fig 6. There is a considerable scatter in the depth readings. which are centred more or less symmetrically about the ganglion cell Iayer, suggesting that most of the cell records are derived from “conventional” ganglion cells with cell bodies in the gangfion cell layer. DISCUSSION Conduction in the retinotectal
pathway
The conduction times measured in this study suggest that extraretinal conduction velocities range from 18 to 1.8 m/set, with 9 m/set being the most commonly sampled value. This range is in close accord with previous observations of orthodromic conduction from retina to tectum in the pigeon (Holden, 1968a). In the previous study, records were taken from fixed latency negative-going spikes at the tectal surface, probably derived from terminal arborisations. The velocity range was from 16 to 3.2 m/set. with 8 m/set being the most commonly sampled value. However, the latency histograms derived from single unit studies can be used only with caution to infer fibre diameters or a fibre spectrum. A primary reason is that microelectrode selectivity may bias the sample. Further, the short latency fibres, with high conduction velocities, fall into the early bins. If the histogram is used to infer a spectrum, then these bins impose a coarse quantization. This quantization can be avoided if the spectrum is drawn from the actual latency measurements, but in this case great reliance is placed on individual readings. These readings include errors due to utilization time, due to the inexactly specified conduction distance (it is not known where an axon is excited), and to the inaccuracy in measuring latencies, which may be up to 0.25 msec. Thus a latency histogram can only be taken as a rough guide unless the results are in good agreement with independent techniques. The present results are in accord both with the observations of Holden
conduction velocity of individual axons is lower than the extraretinal counterpart by a factor of at least 10-20. This ratio would be expected on the grounds that most axons in the optic nerve are myelinated. while most axons are unmyelinated intraretinally The range of velocities is from 4.5 m/see to 0.13 m/set. with a peak at 1 m/set. These figures are somewhat lower than those derived by Stone and Freeman (1971a.b) for the cat retina. The velocities were calculated from individual latencies on the assumption that conduction velocity is constant in the intraretinal and extraretinal segments. Axonal beading may occur in the retina (Stone and Hollander, 1971), and myelinization may occur in the first few mm of the optic nerve (O’Flaherty, 1971; Cowan, 1970). The latter effect would mean that the extraretina1 conductlon times are under-estimated and the intraretinal conduction times are over-estimated. While the majority of axons have low conduction velocities in the retina, some 6% of the sample show no evidence of intraretinal slowing and conduct at 2.25 to 4.5 m/set. These axons must be myelinated both in the optic nerve and in the retina. There is electron microscopic evidence (B. P. Hayes, personal communication) that some 7% of the axons in the nerve fibre layer in the central yellow field are myelinated. It should be stressed that the measurements of conduction velocity were made primarily from cellbody recordings, and hence characterize conduction from the central yellow field, rather than conduction from the periphery. Repetitive activity
While the present results are not a systematic survey, it is clear that many cells with brief (l-4 msec) recovery times to paired stimuli fail to follow repetitive stimuli above 50Hz for more than a dozen or so sweeps. This seems an anomalous property only if compared with antidromic activation via a fibre system of large myelinated axons. There is evidence that the antidromically activated Rohon-Beard cells in Xenopus block at repetition rates of 1 Hz (Spitzer, 1976). These cells show collision of antidromic and orth~ro~c spikes (the most reliable criterion for antidromic invasion) and have low conduction velocities of 0.6-0.8 m/set. It seems likely that the inability to follow high frequency antidromic stimuli is either an inherent property of fine, unmyelinated fibres, or is due to the accumulation of K,+ in the extracellular space in axon bundles bounded by Miiller processes (cf. Orkand, Nicholls and KuEler, 1966). K,+ accumulation would be expected to depolarize both nerve-axons and Miiller processes, and the former effect could explain the reduction in conduction velocity, and possible
1J6-i
4.
L.
blockage of conduction. lhe inabrlity to follow high frequencres would be less marked for visually evoked tiring. because spoke discharge would not occur in ail elements in a fibre-bundle, since the bursts of evoked discharge are transient. and smce there is little or no spontaneous firing. .-!.uonaf branches to the dorm-iareraf
thalamus
The observations that a minority of ganglion cells can be fired antidromically at high threshold from electrodes aimed at the isthmo-optic tract is probably explained by axonai branching. The projection from retina to thalamus in the pigeon is well established (Cowan. Adamson and Powell. 1961: Karten and Nauta. 1968). The posterior parts of the dorso-lateral complex are only :! mm distant from the isthmo-optic tract. so stimulus spread is likely to occur. It would
be of interest to characterize ganglion cells projecting to the thalamus, tn order to examine the analogy with the mammalian visual system. where the branched axons projecting both to co!!iculus and LGNd are the Y-cells (Hoffman, 1973). Ganglion cells and displaced ganglion cells The present experiments show that the majority of resolved ganglion cells in the yellow ii&d can be
antidromically invaded from the optic tectum. The scaled depth readings suggest that most of these cells are in the ganglion cell layer. This suggestion is in accord with the identification of displaced ganglion cells in the pigeon by Karten, Fite and Brecha (1977). who showed that while conventional ganglion cells project to the optic tectum. the displaced ganglion cells project to the ectomammillary nucleus. However, a similar study by CrandalL Heaton and Brownell (1977) of the chick suggests a differing organization. with numerous displaced ganghon cells projecting to the optic tectum. A further physiological study, combined with depth marking, would be of some interest. The ref~~orecral sysrem in the pigeon and the Wceff system in rke cat
Several observations suggest a close similarity between the retinotectal system in the pigeon and the W-cell system in the cat. Five similanties can be listed: (1) Both systems project predominantly to the optic tectum. (cf. Fukuda and Stone, 1974). (2) Both systems have a similar range of extraretinal and intraretinal conduction velocities. (3) In both systems there is a wide scatter of antidromic latencies at one retinal locus (cf. Stone and Fukuda, 1974). (4) Receptive fields of pigeon ganglion cells resemble those of W-cells more closely than the X- or Y-cells (Holden, 1977). (5) The soma sizes of pigeon ganghon cells are mainly small. in the range of j_lO~~m, like those of W-cells. While the comparison needs to be taken further. with both physiological and morphological techniques, it would be of considerable interest if the W, X, Y ciassification (discussed by Rowe and Stone, 1977) could be shown to extend across vertebrate classes. ,icknowledgements-I am grateful for f!nanaa! The Binstead Fund. and from The Smith. French Foundation.
from
support Kline &
HOLDES
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