- Email: [email protected]
Synaptic organisation of the pigeon's optic tectum: A golgi and current source-density analysis
BRAIN RESEARCH
203
S Y N A P T I C O R G A N I S A T I O N OF T H E P I G E O N ' S OPTIC T E C T U M : A G O L G I A N D C U R R E N T S O U R C E - D E N S I T Y ANALYSIS
JONATHAN STONE* AND JOHN A. FREEMAN** A MA/ERF Institute for Biomedical Research, Chicago, Ill. (U.S.A.)
(Accepted September 14th, 1970)
INTRODUCTION In this study we have investigated the synaptic organisation of the pigeon's optic tectum with particular emphasis on its retinal input. Two principal techniques were used. First, we studied Golgi preparations of the tectum, with particular regard to the structure and spatial distribution of the terminal afferent plexuses and of the tectal cells contacted by them. Two distinct types of afferent terminals are described and a classification of tectal neurones into radial and non-radial types, depending on the orientation of their processes, is suggested. Second, we used a technique of current source-density analysis of field potentials a to plot the flow of net current in the tectum during the response of the tectum to optic nerve stimulation. The results suggest that the radial cells are the principal cells involved in processing afferent activity from the retina. The postsynaptic responses generated in the radial cells by the retinal input are described, and some of the functional implications of these events are discussed. METHODS Adult pigeons (Columba livia, weight approx. 500 g) were used. Golgi material was obtained using a variation of the technique described by Hillman 1°. The pigeons were perfused through the heart with a solution of 1.5 ~o potassium dichromate and 5 ~ glutaraldehyde. The brains were immersed in the same mixture for 5-7 days, then placed in 0.75 ~ silver nitrate for 5 days. They were embedded in celloidin and sectioned at 100/~m. Electrical activity in the optic tectum was recorded with glass micropipettes filled with 2 M NaC1, of resistance 2-6 M~. The animals were anaesthetized with * Present address: Department of Physiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T., Australia. ** Present address: 6570 AMRL (MRBB), Wright-Patterson Air Force Base, Ohio 45433, U.S.A. Brain Research, 27 (1971) 203-221
204
J. S I ( ) N [
A N D J, ~',. t R I c t M A N
sodium pentobarbital, administered first intramuscularly (12.5 rag) it1 tile pectoraJ muscles, then intravenously as necessary. They were paralyzed with i~:travcnou~ Flaxedil (gallamine triethiodide) and artificially respired. The spongy bo~e and dura overlying the lateral part of the optic tectum were removed, and recordings were made from the exposed tectum. Electrode tracks were made in an approximately radial directio~, l-ield potentials were recorded on film and with a LINC-8 computer. The latter was programmed to digitize, average, display and store the potentials and to perform a current source density analysis 6. This analysis is designed to calculate the locations o1" the sources and sinks of current which give rise to the recorded field potentials. The optic nerve was stimulated by a bipolar platinum wire electrode placed on the optic nerve head in the retina. The eye was fixed to a ring and was pierced behind the limbus with a hollow tube, through which the stimulating electrode was pushed to the retina. One pole of the electrode was placed as close as possible to the upper end of the pecten. The other pole was I--2 mm away, in the vitreous humour. The cornea was protected with a plastic contact lens. This had a radius of curvature of 4 mm and base diameter of 7 ram, and provided a good fit to the pigeon cornea. R E S U LTS
(A) Histology of the pigeon's optic lecture Termination of optic afferents The fibres of the optic nerve decussate completely is and spread to form the whitish external layer, the stratum opticum, of the optic tectum. In the stratum opticum the fibres bend, often quite sharply, and pass radially into the tectum. Fibres of the most common 'columnar' type pass without ramifying through the most external layer of cells (Cajal's 3 second layer, sublayer (a) in the description of Cowan et al.4), and continue 100 # m deeper before beginning to ramify. Each fibre forms a densely branched, beaded plexus, roughly columnar in outline, measuring about 150 #m from top to bottom (Figs. 2B, 4a). Each fibre which could be traced from the stratum opticum to its ramification appeared to form just a single plexus. However, individual optic tract axons may branch in the stratum opticum and hence form more than one terminal plexus. The depth to which these plexuses reach is remarkably uniform, as shown in Figs. 4 and 5. They are packed closely together and form a continuous layer of afferent terminals, about 150/zm thick ( l.a.p, in Fig. 2). A second, quite distinct type of terminal was seen rarely. It appeared to pass without branching through the layer of afferent plexuses just described and to form a discrete, loosely branched terminal plexus at a slightly deeper level. An example is drawn in Fig. 4b. The deep limit of the layer of terminal plexuses lies 250 btm, and its superficial limit 100 #m, deep to the internal limit of the stratum opticum. They are an additional 75-250/~m from the tectal surface, depending on the thickness of the stratum opticum (which varies between approx. 75 btm and 250 # m in different areas of the tectum).
Brain Research, 27 ( 1971) 203-221
PIGEON OPTIC TECTUM
205
Superficial stellate cells The inner limit of the stratum opticum is marked by a single layer of small cell bodies, which Cowan et al. 4 called sublayer (a) of the stratum griseum et fibrosum superficiale. The majority of these cells appear to be small stellate cells, like the upper of the two labelled 's' in Fig. 4, although several cells with longer dendrites running 200-300/~m deep to the cell bodies were also seen in this layer (e.g. Fig. 1A, sd). The short dendritic processes of these stellate cells spread about 50/~m downwards and laterally from the cell body. They are presumably not contacted by the afferent plexuses, which are situated 50-100 #m deeper. The only other neuronal structures observed at the level of sublayer (a) were the tips of the dendrites of tectal cells (described below) and optic tract fibres passing down from the stratum opticum to form their plexuses 50-100 #m deeper.
Classification of cells contacted by the afferent terminals A variety of cells was observed whose dendrites extended into the layer of afferent terminals and which could therefore be assumed to be contacted by them. We suggest classifying them into two broad groups: (a) radial ceils, whose processes are predominantly radial in orientation; i.e., they run in the direction normal to the surface of the tectum; and (b) non-radial cells, whose processes show no radial orientation. This classification differs from Cajal's 3 which laid stress on the layer of the rectum in which the somas of the cells were located rather than on the orientation of their processes. However, many of the cell types described here were described or illustrated by Cajal. Considerable variety was apparent within both radial and nonradial groups. This variety is described briefly but, since the various types cannot yet be related to physiological events, they are, for present purposes, considered in just two groups. Radial cells The great majority of cells whose processes extend into the layer of afferent plexuses have radially oriented processes. At least three types can be distinguished. Radial cells of the first type, labelled rl in Figs. 1, 2, 5 and 6, were the most numerous cell type observed in the tectum, and probably constitute most of the cells of the stratum griseum et fibrosum superficiale. They have small, globular bodies from each of which a prominent, apical dendrite runs radially towards the surface of the tectum. These dendrites always reach the layer of afferent plexuses and many continue through the plexuses and reach the layer of superficial stellate cells. Most give a few small branches as they ascend and divide into two or three (still radial) branches in the afferent plexus layer. A small percentage give off prominent laterally spreading branches at a level just deep to the afferent plexuses (Fig. 6, r 0. A minority of the dendrites have spine-like processes on their upper portions (Fig. 6, rl). The bodies of rl cells are distributed over a considerable range of depths. The most superficial are in the sublayer (e) of Cowan et al.'s 4 classification, among the afferent plexuses; the deepest lie some 200 #m deeper, in sublayer (i) (Fig. 5). Usually Brain Research, 27 (1971) 203-221
206
,I. STONE ANi)J. ~,. ~REt[MAN
Fig. 1. Radial and stellate cells, Golgi stain. The dashed lines in A, B, and C mark the superficial and deep limits of the layer of afferent plexuses (labelled I.a.p. in C). The dotted line marks the deep limit of the stratum opticum (str. opt.). A, Two radial cells of the rl type are shown. The small stellate cell "s' is in the layer of afferent plexuses. The cell labelled 'sd' has its soma in sublayer (a). Unlike most of the cells of this layer, which are small stellate cells, the dendrites of this cell pass 300/tin into the deeper layers of the tectum. B, A radial cell of the pyramidal r:~ type. C, A radial cell of the bipolar r2 type. The scale line at lower right represents 200 #m.
PIGEON OPTIC TECTUM
207
their basal dendrites are irregular (i.e., non-radial) in orientation, quite short and much less prominent than the apical dendrite. Occasional cells had a long basal process which passed without branching 100-200 #m deep to the cell body, and which might be portion of an axon. Others gave off axon-like processes from their apical dendrites. as Cajal 3 observed in the sparrow. Radial cells of the second type, labelled r2 in Figs. l, 2 and 5, are bipolar in appearance, having one apical and one basal dendrite which are usually equal in prominence and length, and are strictly radial in orientation. Both dendrites give numerous short, laterally spreading branches. The apical dendrite always reaches the layer of afferent plexuses, in some cases continuing on to reach the superficial stellate layer (Figs. 1, 2 and 5). The cell bodies are fusiform in shape and are found 400-500 # m below the superficial stellate cells, at about the same level as the deepest r~ cells, in the deep part of sublayer (i). Radial cells of the third type, labelled r3 in Figs. 1 and 5, are pyramidal in appearance and their cell bodies are situated about 600-700/~m deep to the superficial stellate cells, in sublayer (j). Each soma gives off a prominent apical dendrite which runs radially to the layer of afferent plexuses. Several large, spiny dendrites arise from the deep side of the cell body and run 100-200/~m lateral and deep to the cell body.
Non-radial cells with processes in the afferent plexus layer A variety of cell types was seen which fell into this group. Their two common features are that their processes (a) are not radial in orientation, but (b) do extend into the afferent plexus layer, where they presumably are contacted by the afferents. They were much less numerous than the radial cells. As well as being found in sublayer (a), above the afferent terminals, stellate cells were seen 100 150/~m deeper among the afferent plexuses (Figs. IA, 4). They were similar in morphology to the sublayer (a) cells. Similar stellate ceils were also observed at deeper levels, well below the afferent plexuses. A small number of cells were seen whose processes spread laterally, parallel to the tectal surface, rather than radially; an example is shown in Fig. 5. In addition, several cells were observed (labelled 'n' in Fig. 6) whose processes showed no directional orientation, but did extend through most of the stratum griseum et fibrosum superficiale. Ganglion cells The deepest neurones we observed in the rectum were the ganglion cells. Their somata are situated in the periventricular grey matter~ approximately 1200-1400/~m below the tectal surface. Their dendrites spread laterally and up towards the tectal surface, and some come into close relationship with the basal dendrites of radial ceils. Since their dendrites were never seen to reach the layer of afferent terminal plexuses, the ganglion cells are presumably not contacted by the afferent terminals. Several gave rise to axon-like processes. A typical ganglion cell is shown in Fig. 3.
Brain Research, 27 (1971) 203-221
208
J. SIONE AN[) J. ,~, FREEMAN
Fig. 2. Afferent fibre plexuses, radial and stellate cells, Golgi stain. The dashed lines mark the deep and superficial limits of the layer of afferent plexuses (labelled l.a.p.). The arrows mark the deep limit of the stratum opticum (str. opt.), The scale lines on each portion represent 100 pro. A, The soma of an ra radial cell is seen at bottom. From it a prominent dendrite runs radially to come into close relationship with the terminal plexus of an afferent fibre. B, The terminal plexus of an afferent fibre. The fibre descends without branching about 100/~m deep to the deep limit of the stratum opticum. It then arborizes to form a typical terminal plexus. C, Radial cells of the r~ and r2 types are shown. A stellate cell's' can be seen with its soma just deep to the layer of afferent plexuses.
PIGEON OPTIC TECTUM
209
Fig. 3. A ganglion cell (g) lying deep in the tectum. A small portion of the lateral ventricle 'v' is seen at bottom. The scale represents 200/~m. The tectal surface, which is out of the field, runs approximately parallel to the upper border of the picture.
( B) Physiological analysis Field potential profile Fig. 7A shows the field potentials generated at successive depths in the tectum by optic nerve stimulation. The potentials were averaged and displayed by a L I N C - 8 c o m p u t e r . A n electronic stimulus artefact suppression device was used to eliminate the artefact f r o m the waveformsL The time o f s t i m u l a t i o n is indicated in these and subsequent waveforms by dots. The d u r a t i o n o f artefact suppression is indicated by
Brain Research, 27 (1971) 203 221
210
J. STONE AND J. ?',, FRE[!MAN
str. opt. ....
j .........................
$ ,k..
I
100p
Fig. 4. Camera lucida drawings of the most common 'columnar' type of afferent fibre terminal 'a', of the very uncommon second type 'b', which passes without branching through th.e layer of afferent plexuses to come into close relationship with laterally spreading dendrites of a radial cell rl; and of two stellate cells 's'. The more superficial stellate cell lies in subtayer (a) and its processes are entirely above the afferent plexuses. The processes of the deeper stellate cell do extend into the layer of afferent plexuses.
the short length of noise-free baseline which follows the stimulus. The potentials resemble fairly closely those described by Holden n. The following features are important for our subsequent analysis. (a) The waveforms have a prominent negative component (the N-wave n) at superficial locations, which inverts to positive (the P-wave) at deeper locations. The reversal occurs at a depth of approximately 400 #m (the R-zone). (b) The early portion of the N-wave inverts more superficially than the late part. (c) An early deflection, arrowed in Figs. 7A and 8A, is present which, for reasons presented below, we attribute to the presynaptic volley.
Current source-density waveJorms Fig. 7B and C show the current source-density wavetbrms obtained from the field potentials of Fig. 7A (Fig. 7B) and from another similar series. These waveforms show the net local extraneuronat current flowing in the direction normal to the tectal surface at each recording point. At certain depths (e.g. the 200-400 #m depths) the current source-density waveforms resemble the field poteotials. At deeper and more Brain Research, 27 (1971) 203-221
PIGEON OPTIC TECTUM
211
str. opt.
'a
B
7|
r,'
200F
r2
h
% Fig. 5. Camera lucida drawings of radial cells of rl, r2 and r3 types; of afferent fibre terminals 'a' and of stellate ceils 's'. Note the range of depths over which the cell bodies of rl cells were found.
superficial locations they differ quite markedly. The following features were c o m m o n to all sets o f current s o u r c e - d e n s i t y waveforms. (a) A t the 100 # m depth, the current s o u r c e - d e n s i t y w a v e f o r m is flat, indicating t h a t there are no net current sinks or sources in the s t r a t u m opticum. (b) A t the next 3 depths - - 200, 300 and 400 # m - - there are p r o m i n e n t sinks o f current. T h e sinks p e a k at successively later times at the three d e p t h s (2.6 msec, 3.6 msec and 4.7 msec respectively). (c) A t the 500/~m d e p t h there is a sink o f current occurring earlier t h a n the sink at 400 # m . S i m u l t a n e o u s with this sink there are small sources at the 400, 600 and 700 # m depths. D e e p e r t h a n 700/zm there are no p r o m i n e n t sources or sinks. Brain Research, 27 (1971) 203 221
212
J. STONE A N D J. \ .
FREEMAN
f
str. opt.
n
Fig. 6. Camera lucida drawings showing two non-radial cells with long unoriented processes 'n'. The rl cell also shown had spiny dendritic branches. The same general pattern of sources and sinks was observed in all tracks although in some it was displaced upwards or downwards by variations in the thickness of the stratum opticum, and occasionally by incorrect identification of the tectal surface, due to overlying fluid. The superficial (200-400 # m depth) sinks were particularly constant in occurrence. Direction of current flow in tectum The current source-density waveforms presented in Figs. 7B, C and 8B ('Jz') show only the net current flowing in the radial direction. Because of the structural homogeneity of the tectum in any plane parallel to its surface, we assumed that there should be no substantial net current in this plane. Confirming this assumption the potentials recorded at adjacent points (approximately 200/~m apart but at the same depth) were nearly identical. In Fig. 8B the current source-density was calculated for each of 3 mutually orthogonal directions (x, y, z) at depth 250 #m. The top trace ('Jz') shows the net current flowing in the radial direction, and the next two traces ('Jx' and 'Jy') show the net current flowing anteroposteriorly and mediolaterally, parallel to the tectal surface. The bottom trace shows the sum of the three currents. It is clear that the great majority of the net current is flowing in the radial direction. Pre- and postsynaptic components of the tectal response Holden 11 did not describe a presynaptic component in the tectal response but he did conclude that the N-wave is postsynaptic. This is consistent with the observations of KonishP 4 and Sutterlin and Prosser 21 on the configuration of pre- and postsynaptic potentials in the fish rectum and is confirmed by the experiment illusBrain Research, 27 (1971) 203-221
PIGEON OPTIC TECTUM
213
A Fieldpotentials
B Current densities
C Current densities
. . . . . . . .
100 ~
..........
200 ~
..........
300
500
Lvf.
/\
600
700 ~--~.J/~'~,~ , • 800-~]~~--~--900 . - - - - ~ j ~ ~ 1000 ~ .
10msec Source
-
~ m
~
10msec
Sink
+
10msec J lmv Fig. 7. Field potentials and current source density waveforms obtained from the optic tectum after optic nerve stimulation. The waveforms were digitized, averaged and displayed by the LINC-8 computer. An artefact suppression device was used to suppress the stimulus artefact. The time of stimulation is indicated in all traces by the dots. The short lengths of noise-free baseline following the dots indicate the duration of artefact suppression. The depths at which the waveforms were obtained are shown at left, in microns. A, The field potential profile. The presynaptic components are marked by arrows. The presence of two presynaptic components is shown more clearly in Fig. 8A. The superficial potentials are predominantly negative, and invert to positive at about 400 ttm. The early part of the negative wave inverts earlier than the late part. B, Current source-density waveforms obtained from the field potentials in A. Most prominent are the sinks at the 200, 300 and 400 #m depths. C, Current source-density waveforms obtained from another set of field potentials. trated in Fig. 8A, in which the pre- a n d postsynaptic c o m p o n e n t s of the initial tectal response to optic nerve stimulation were separated by tetanic stimulation, at increasing frequencies. A t a stimulus rate of l/sec the response, recorded at a depth of 250/zm below the tectal surface, consists of two small initial negative c o m p o n e n t s (marked by arrows), followed by a larger, long-lasting negative c o m p o n e n t , the N-wave. The peak latency of the N-wave increases and its amplitude decreases with increasing stimulus frequency. A t a stimulus rate of 100/sec the N-wave is almost completely abolished, a n d only the initial two presynaptic c o m p o n e n t s remain. These two c o m p o n e n t s would in fact follow stimulus frequencies of greater t h a n 200/sec. Brain Research, 27 (1971) 203-221
214
J. STONE AND J. A. IRt~EMAN
B
A
Jz
I/see ,. • I ~ ,
~
~
a
10/see - , - . , . ~ ~
e
o
~
Jx
20/see Jy
Jx÷ Jy + Jz
100/sec 4msec
I 1my
4msec Source
Fig. 8. A, Field potentials obtained at depth 250/~m in response to optic nerve stimulation at different rates. At each rate, 32 stimuli were given and only the response to the last stimulus was digitized by the computer. The presynaptic components of the field potentials (marked by arrows) were largely unaffected by high stimulus rates. By contrast the later negative wave (the N-wave) was significantly reduced at 10/sec, and was reduced to a small deflection at 100/sec. B, The current source-density waveforms calculated at 300 /~m depth for net current in the radial direction ('J~'), and in two directions ('Jx' and 'Jy') parallel to the tectal surface. The sum of these 3 net currents is shown at bottom ('Jx + Jy t- Jz'). Clearly most net current flows radially.
I
I
A
I
2 MSEC 11MV +
Fig. 9. A, The potential recorded in the optic tract during optic nerve stimulation. The potential was digitized, averaged and displayed by the LINC-8. Time of stimulation is indicated by the dot; the artefact was electronically suppressed: B, The potential in A differentiated, to emphasize the initial two deflections. The period between these two deflections was 0.6 msec, slightly less than the interval of 0.8 msec between the two presynaptic deflections observed in the tectal response to optic nerve stimulation, suggesting that there are two classes of optic nerve fibres, having different conduction velocities. Brain Research, 27 (1971) 203-221
PIGEON OPTIC TECTUM
215
The presence of two presynaptic deflections (Fig. 8A) suggests that there might be two predominant classes of optic nerve fibres with different conduction velocities, as has been described in other animals2,1L This suggestion is supported by the experiment illustrated in Fig. 9, in which the field potential generated in the optic tract by optic nerve stimulation was recorded with a micropipette placed approximately midway between the optic chiasm and the tectum. Two distinct peaks occur in the response, which are emphasized in the differentiated waveform (Fig. 9B).
Dendritic spikes In tracking through tile optic tectum with a high resistance ( > 10 Mr2) micropipette we commonly observed small spike-like potentials superimposed on the initial phase of the N-wave. They also occurred spontaneously and in response to photic stimulation. Examples are shown in Fig. 10A, C, E, G, 1. The spike-like components are accentuated by the time derivatives shown below each field potential waveform (Fig. 10B, D, F, H, J). They occurred in a restricted zone 200-400 Fm beneath the tectal surface; i.e., in the region of afferent terminal arborizations and radial cell dendrites. They were irregular in shape, but individual components appeared to be all-or-none in character and had clearly differentiable thresholds in response to graded retinal stimulation. They occurred approximately
A
G
U"
c-v- v .
.
.
.
.
.
.
-V-.V |
F
.
.
.
..
+
5msec
I
lmv
5msec
[
lmv
Fig. 10. A, C, E, F, G, I, Tectal responses to optic nerve stimulation, showing spike-like deflections superimposed on the N-wave, which may be dendritic spikes, as described in the text. B, D, F, H, J, The tectal response differentiated to emphasize the spike-like components of the N-wave.
Brain Research, 27 (1971) 203-221
216
,I. STONE AND J. A. FREEMAN
0.5-1.5 msec after the initial afferent volley, superimposed on the rising phase or peak of the N-wave, and would follow stimulus frequencies up to only 50/sec. They were therefore clearly postsynaptic in origin, and because of the region in which they occurred, probably represent spikes or partial spikes produced in the dendrites of the radial cells, as will be discussed presently. The small size of the neuronal processes at these depths and the presence of small arterial pulsations of the tectum precluded obtaining intracellular recordings. DISCUSSION Termination o f afferents to the tectum
In several respects our observations are at variance with earlier work. In our preparations the afferent terminals were (disregarding for the moment the uncommon second type) much more uniform in morphology and depth than Cajal 3 described and gave a much stronger impression of forming a compact layer. This is probably a species or age difference. We studied the adult pigeon tectum, whereas Cajal studied principally the tectum of immature (1-10 day) sparrows. The terminal plexuses are also much simpler than in the frog tectum where Lazar and Szekely (quoted in Szekelyz2) have described four layers of terminal endings. It does seem, however, that there is sufficient variety in the morphology of the tectal neurones to account for the varieties of receptive fields which have been found in the pigeon's optic tectum 13. Cowan et al. 4 reported that after contralateral enucleation degenerating terminals were present between sublayers (a) and (f) of the stratum griseum et fibrosum superficia[e. Our observations confirm that the deepest extent of the terminal arborization is sublayer (f), but revealed no terminals above sublayer (c), 100/~m deeper than sublayer (a). The degenerating material which Cowan et al. observed between sublayers (a) and (c) is probably the remains of axons passing from the stratum opticum to their plexuses 100 ,um deeper, though it is possible that they are terminals of some afferent type which did not stain in our preparations. The depth distribution of the presynaptic component of the tectal response to optic nerve stimulation corresponds well with the depth of the afferent plexuses. In the field potentials, the presynaptic components are prominent above the 400 #m depth, being maximal around 200/~m deep. The two deflections in the presynaptic component of the tectal response (Fig. 8A) almost certainly are not related to the two different types of afferent terminals described above. First, the depth distributions of the two deflections were always very similar, whereas the two afferent types have quite different depth relationships. Second, the two deflections were of almost equal magnitude, whereas one type of afferent (the columnar type) was much more common. It is more likely that the two deflections represent two groups of optic nerve fibres, with different conduction velocities but similar terminal plexuses. The two groups might correspond to the myelinated and unmyelinated groups which Bingelli and Paule I have described in Brain Research, 27 (I 971) 203-221
217
P I G E O N O P T I C TECTUM
the pigeon's optic nerve. However, the velocity difference between myelinated and unmyelinated fibres is much greater (at least in the cat retina and optic nerve 2°) than that indicated by the latencies of the fast and slow groups in our records (I.6 and 2.3 msec respectively). The problem clearly requires further analysis. Anatomical organization of tectal cells Of those cells whose dendrites reach the layer of afferent plexuses (and which are probably therefore contacted by the afferent fibres), the great majority are radial in orientation. Conversely, all radial cells appear to be contacted by retinal afferents. Their somas are distributed over a wide range of depths, from about 400 #m (sublayer (e)) to 800/xm (sublayer (j)) from the tectal surface, and they seem to constitute the majority of the cells of the stratum griseum et fibrosum superficiale. Hence, radial cells are probably the principal cells involved in processing the retinal input. Although the cell bodies of the radial cells appear (in Nissl-stained sections) to be arranged in compact and separate layers, the cells in one layer are not serially connected with cells in the next layer. Rather, their processes run in parallel from the layer of afferent plexuses to varying depths in the tectum. This fact is of importance in interpreting the field potential profile and current source-density waveforms obtained from the tectum. It is also a principal reason for our considering the radial cells as a group despite the different depths and layers at which their cell bodies are located. The electrical activity in these cells is principally radial in direction, a property determined by their dendrites, not by their somas. Only a minority of the ceils which are in a position to be contacted directly by the afferent terminals are non-radial in organization. By contrast, all of the cells which are not in a position to be contacted by the afferent plexuses, being either too superficial or too deep in location, appear to be non-radial in orientation. hlterpretation of superficial (100-400 #m) current source density waveforms In Fig. 7B and C successive sinks occur at the 200, 300 and 400 # m depths. A number of lines of evidence suggest that these depths span the layer of afferent plexuses. First, in the Golgi material described above the layer of afferent plexuses was 150/zm thick. Since shrinkage in celloidin-embedded tissue is 20-25 % of length, this dimension is probably nearer 200 #m in the living brain. Thus, if the stratum opticum were of average thickness, say 100/zm, the layer of afferent plexuses would probably extend from 200 to 400/zm below the tectal surface. Second,the deepest of the 3 sinks (400/~m) is at the same level as the R-zone; i.e., the depth at which the postsynaptic potential reverses from negative to positive 11. Using a lesion technique, Holden it showed that the R-zone corresponds to the depth at which Cowan et al. l found the deepest degenerating terminals after section of the contralateral optic nerve. The successive occurrence of these sinks suggests that there is successive activation of neural elements at the 3 levels. This would result if cells at the 200/~m level excited cells at the 300/,m level, which in turn excited cells at the 400 #m level. Brain Research, 27 (1971) 203 221
218
J. STONE A N D J. A. FREEMAN
Alternatively, it would occur if activity propagated along core conductors extending through these depths. The same two alternatives were proposed by Sutterlin and Prosser 2a to explain their observation that the region of maximum current density during the response of the goldfish optic tectum to an optic nerve volley also shifts down in the tectum with time. They were inclined to the former alternative but in view of the structure of the pigeon's tectum, the latter alternative seems much more likely (at least for the pigeon). There is no evidence o1" 3 serially connected layers of cells in this region, but there are two prominent structures, viz. the afferent plexuses and the dendrites of radial cells, which extend radially through this region and along which activity might be propagated. Dendritic spikes in tectal cells"
A number of observations suggest that if it is propagated activity which generates these successive sinks, it is occurring in the dendrites of the tectal cells rather than in the afferent plexuses. (a) Each sink has the same latency as and about half the duration of the N-wave recorded at the same depth (Fig. 8A and B). The sinks are clearly related to the N-wave and almost certainly generate the N-wave. They must, therefore, like the N-wave, be considered postsynaptic in origin and be related to activity in postsynaptic structures, i.e., in the radial cell dendrites. (b) The delay in occurrence of the sinks from depth to depth suggests a conduction velocity of approximately 10 cm/sec, consistent with dendritic spike conduction velocities reported for other neurones, for example, 10-40 cm/sec in alligator Purkinje cell dendrites16; 15-50 cm/sec in the apical dendrites of rabbit hippocampal cellsV; 20-50 cm/sec in rat and kitten cerebellar cells grown in vitro 9. (c) Small spikes with unusual properties were consistently observed in the region of the tectal cell dendrites. For reasons already discussed they are probably postsynaptic in origin. Because of their irregular shape and long duration (up to 3 msec), they are quite distinct from the sharp presynaptic spikes (duration 0.5 msec) and postsynaptic spikes (duration 1.0 msec) which Holden lz described and Robert and Cu6nod la and we have also seen. Llinhs et al. 16 noted, analogously, that the spikes they recorded in the region of the alligator Purkinje cell dendrites, and interpreted as dendritic spikes, were much longer in duration than spikes recorded from the Purkinje cell somas. Interpretation of the deeper (500-1000 #m) current source-density wave/orms
Perhaps the most important feature of these deeper waveforms is the presence at 500 #m of a sink of current with earlier latency than both the N-wave recorded at the same depth and the more superficial (400 #m) sink of current (Fig. 7B, C). The 500 #m sink clearly cannot contribute to the N-wave and/or be a continuation of the propagated activity which appears to be occurring more superficially. Since 500 #m is well below the deepest afferent plexuses seen in our Golgi material, and Brain Research, 27 (1971) 203-221
P I G E O N O P T I C TECTUM
219
below the deepest terminals observed by Cowan et al. 4, and since no presynaptic volley was recorded at this depth, this sink can best be interpreted as the activation of the cell bodies of the radial cells at this depth by electrotonic spread of current from their dendrites. It has a longer latency than the most superficial (200 /zm) N-wave sink, but the same or shorter latency than the 300 and 400/zm N-wave sinks, and appears therefore to be generated while activity is propagating down the radial cell cendrites. Sources corresponding in latency and time course to the 500 ,urn sink occur at the 600 and 700/zm depths, suggesting that current is supplied to the 500 #m sink from basal processes of the radial cells, such as those illustrated in Figs. 1, 2 and 5. These are the only sources apparent in our current source-density waveforms which correspond in latency and time course to the P-wave. If they do generate the P-wave, the interpretation just stated goes against Holden's 1~ conclusion that the P-wave is produced by passive sources supplying current to the N-wave sinks. It is interesting to note in this regard that Robert and Cu6nod 19 suggested that the P- and N-waves may be differently generated, since the N-wave is selectively inhibited by stimulation of the contralateral optic tectum. O'Leary and Bishop 17 made a similar suggestion based on their observation that the P- and N-waves are differently related to stimulus intensity (in the rectum of duck and goose). It should be noted too that small sources corresponding to the N-wave sinks are apparent at the 200, 300 and 400/~m depths. The N-wave sinks appear to draw current principally from structures immediately superficial and deep to them. This is consistent with the idea that they represent propagated activity, since propagating spikes in the dendrites would draw current from immediately adjacent lengths of dendrite. Functional correlations
(i) We have suggested that the radial cells are the principal cells involved in processing the retinal input. The stellate and horizontally spreading cells most likely are interneurones, though their functions have yet to be studied. Hamdi and Whitteridge 8 plotted the projection of the visual field on the optic tectum. They noted that there was 'little or no change in the localisation of the response obtained from the whole thickness of the stratum griseum et fibrosum superficiale, in spite of the fact that it is sufficiently heterogeneous to be divisible into 7 sublayers.' They speculated that some of these sublayers were involved in other functions, such as colour vision. We suggest that visual localisation is constant along a tectal radius because the retinal input is distributed by radially oriented terminal plexuses synapsing onto radial cells. All the sublayers of the stratum griseum et fibrosum superficiale are involved (except perhaps the most superficial sublayer (a)) because all contain the afferent terminals, or the somas or apical and basal dendrites of radial cells. "l'his radial organisation cuts across the horizontal stratification of cell bodies so prominent in Nissl-stained sections, and appears to predominate functionally. (ii) A volley in the optic nerve generates two systems of sources and sinks, both involving the radial cells. First, propagated activity is generated in the radial cell Brain Research, 27 (1971) 203 221
220
J. STONE AN[) J.
A.
FREEMAN
dendrites. The propagating dendritic sinks draw current principally from adjacent lengths of dendrite, generating the N-wave. Second, this dendritic activity evokes the discharge of tectal cell bodies at 400-600/~m depth. This discharge draws current principally from deeper levels, presumably from the basal dendrites of the radial cells, generating the P-wave. The importance of these two source/sink systems probably lies in the fact that they can be separately inhibited or facilitated, allowing for quite complex control of tectal cell excitability. As already noted Robert and Cu6nod x9 have shown that the intertectal commissural system in the pigeon selectively inhibits the N-wave. (iii) The radial cells send axons out of the tectum 3 and hence provide a monosynaptic output for retinal input to the tectum. The deep-lying ganglion cells, on the other hand, are not contacted directly by the retinal afferents. Their principal synaptic input has not yet been defined, but they may provide a di- or polysynaptic output for retinal input. SUMMARY
This paper presents an analysis of the synaptic organisation of the pigeon's optic tectum, with special reference to its retinal input. The structure of the tectum was studied in Golgi-impregnated material. The manner of termination of the retinal afferent fibres to the tectum and the morphology of the neurones of the tectum are described. The afferent fibres terminate in one or more columnar plexus. The plexuses are closely packed, forming a compact layer of afferent terminals at a constant depth in the tectum. It is suggested that the tectal neurones be classified according to the orientation of their dendritic processes into two broad groups, radial and non-radial cells, and the characteristics of and variations within each group are described. The field potential profile generated in the tectum by optic nerve stimulation was subjected to a current source-density analysis to determine the location and sequence of sources and sinks of current produced by an optic nerve volley. The results suggest that the radial cells are the principal cells involved in processing retinal input. The postsynaptic events generated by an optic nerve volley are described and the functional importance of these events is discussed.
REFERENCES 1 BINGELLI,R. L., AND PAULE,W. J., The pigeon retina: quantitative aspects of the optic nerve and ganglion cell layer, J. comp. Neurol., 137 (1969) 1-18. 2 BisHoP, G. H., CLARE, M. H., AND LANDAU,W. M., Further analysis of fibre groups in the optic tract of the cat, Exp. Neurol., 24 (1969) 386--399. 3 CAJAL,S. RAM6NY, Histologie du Syst~me Nerveux de l'Homme et des Vertdbrds, Tdme 2, Maloine. Paris, 191 l, Ch. X. 4 COWAN,W. M., ADAMSON,L., ANDPOWELL,T. P. S., An experimental study of the avian visual system, J. Anat. (Lond.), 95 (1961) 545-563. 5 FREEMAN, J. A., An electronic stimulus-artefact suppressor, Electroenceph. clin. Neurophysiol., Submitted for publication. Brain Research, 27 (1971) 203-221
PIGEON OPTIC TECTUM
221
6 FREEMAN,J. A., AND STONE, J., A technique for current density analysis of field potentials and its
application to the frog cerebellum. In R. LLIN~S (Ed.), Neurobiology of Cerebellar Evolution and Development, Amer. Med. Ass./Educ. and Res. Found., Chicago, 1969, pp. 421-430. 7 FUJITA, K., AND SAKATA,H., Electrophysiological properties of CA1 and CA2 apical dendrites of rabbit hippocampus, J. Neurophysiol., 25 (1962) 209-222. 8 HAMDI, F. A., AND WHITTERIDGE, D., The representation of the retina on the optic tectum of the pigeon, Quart. J. exp. Physiol., 39 (1954) 111 118. 9 HILO, W., AND TASAKI, I., Morphological and physiological properties of neurones and glial cells in tissue culture, J. Neurophysiol., 23 (1962) 277 304. 10 HICL~AN, D. E., Morphological organisation of the frog cerebellar cortex; a light and electron microscopy study, J. Neurophysiol., 32 (1969) 818 846. 11 HOLDEN, A. L., The field potential profile during activation of the avian optic tectum, J. Physiol. (Lond.), 194 (1968) 75-90. 12 HOLDEN, A. L., Types of unitary response and correlation with the field potential profile during activation of the avian optic rectum, J. Physiol. (Lond.), 194 (1968) 91-104. 13 HOLDEN, A. L., Receptive properties of retinal cells and tectal cells in the pigeon, J. Physiol. (Lond.), 201 (1969) 56 57P. 14 KON~SHI, J., Electric responses of visual centre to optic nerve stimulation in fish, Jap. J. Physiol., 10 (1960) 13-27. 15 LEDERMAN,R. J., ANO NOELC, W. K., Fast-fiber system of rabbit optic nerve, Vision Res., 8 (1968) 1385-1398. 16 LE1N~,S, R., NICHOCSON, C. N., FREEMAN, J. A., AND HIH.MAN, D. E., Dendritic spikes and their inhibition in alligator Purkinje cells, Sc&nce, 160 (1968) 1132-1135. 17 O'LEAR¥, J. L., AND BISHOP, G. H., Analysis of potential sources in the optic lobe of duck and goose, J. cell. comp. Physiol., 22 (1943) 73 87. 18 POLVAK, S., The Vertebrate Visual System, Univ. of Chicago Press, 1957. 19 ROBERT, F., AND CO'NOD, M., Electrophysiology of the intertectal commissures in the pigeon. 1I. Inhibitory interaction, Exp. Brain Res., 9 (1969) 123 136. 20 STONE, J., AND FREEMAN, R. B., Conduction velocity groups in the cat's visual pathway classified according to their retinal origin, In preparation. 21 SUTTERCJN, H. M., AND PROSSER, C. L., Electrical properties of the goldfish optic rectum, J. Neurophyshd., 33 (1970) 36-45. 22 SZEKEt.V, G., Anatomy and synaptology of the optic tectum, in R. JUNG (Ed.), Handbook of Sensory Physiology, Vol. VII, in press.
Brain Research, 27 (1971) 203-221
203
S Y N A P T I C O R G A N I S A T I O N OF T H E P I G E O N ' S OPTIC T E C T U M : A G O L G I A N D C U R R E N T S O U R C E - D E N S I T Y ANALYSIS
JONATHAN STONE* AND JOHN A. FREEMAN** A MA/ERF Institute for Biomedical Research, Chicago, Ill. (U.S.A.)
(Accepted September 14th, 1970)
INTRODUCTION In this study we have investigated the synaptic organisation of the pigeon's optic tectum with particular emphasis on its retinal input. Two principal techniques were used. First, we studied Golgi preparations of the tectum, with particular regard to the structure and spatial distribution of the terminal afferent plexuses and of the tectal cells contacted by them. Two distinct types of afferent terminals are described and a classification of tectal neurones into radial and non-radial types, depending on the orientation of their processes, is suggested. Second, we used a technique of current source-density analysis of field potentials a to plot the flow of net current in the tectum during the response of the tectum to optic nerve stimulation. The results suggest that the radial cells are the principal cells involved in processing afferent activity from the retina. The postsynaptic responses generated in the radial cells by the retinal input are described, and some of the functional implications of these events are discussed. METHODS Adult pigeons (Columba livia, weight approx. 500 g) were used. Golgi material was obtained using a variation of the technique described by Hillman 1°. The pigeons were perfused through the heart with a solution of 1.5 ~o potassium dichromate and 5 ~ glutaraldehyde. The brains were immersed in the same mixture for 5-7 days, then placed in 0.75 ~ silver nitrate for 5 days. They were embedded in celloidin and sectioned at 100/~m. Electrical activity in the optic tectum was recorded with glass micropipettes filled with 2 M NaC1, of resistance 2-6 M~. The animals were anaesthetized with * Present address: Department of Physiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T., Australia. ** Present address: 6570 AMRL (MRBB), Wright-Patterson Air Force Base, Ohio 45433, U.S.A. Brain Research, 27 (1971) 203-221
204
J. S I ( ) N [
A N D J, ~',. t R I c t M A N
sodium pentobarbital, administered first intramuscularly (12.5 rag) it1 tile pectoraJ muscles, then intravenously as necessary. They were paralyzed with i~:travcnou~ Flaxedil (gallamine triethiodide) and artificially respired. The spongy bo~e and dura overlying the lateral part of the optic tectum were removed, and recordings were made from the exposed tectum. Electrode tracks were made in an approximately radial directio~, l-ield potentials were recorded on film and with a LINC-8 computer. The latter was programmed to digitize, average, display and store the potentials and to perform a current source density analysis 6. This analysis is designed to calculate the locations o1" the sources and sinks of current which give rise to the recorded field potentials. The optic nerve was stimulated by a bipolar platinum wire electrode placed on the optic nerve head in the retina. The eye was fixed to a ring and was pierced behind the limbus with a hollow tube, through which the stimulating electrode was pushed to the retina. One pole of the electrode was placed as close as possible to the upper end of the pecten. The other pole was I--2 mm away, in the vitreous humour. The cornea was protected with a plastic contact lens. This had a radius of curvature of 4 mm and base diameter of 7 ram, and provided a good fit to the pigeon cornea. R E S U LTS
(A) Histology of the pigeon's optic lecture Termination of optic afferents The fibres of the optic nerve decussate completely is and spread to form the whitish external layer, the stratum opticum, of the optic tectum. In the stratum opticum the fibres bend, often quite sharply, and pass radially into the tectum. Fibres of the most common 'columnar' type pass without ramifying through the most external layer of cells (Cajal's 3 second layer, sublayer (a) in the description of Cowan et al.4), and continue 100 # m deeper before beginning to ramify. Each fibre forms a densely branched, beaded plexus, roughly columnar in outline, measuring about 150 #m from top to bottom (Figs. 2B, 4a). Each fibre which could be traced from the stratum opticum to its ramification appeared to form just a single plexus. However, individual optic tract axons may branch in the stratum opticum and hence form more than one terminal plexus. The depth to which these plexuses reach is remarkably uniform, as shown in Figs. 4 and 5. They are packed closely together and form a continuous layer of afferent terminals, about 150/zm thick ( l.a.p, in Fig. 2). A second, quite distinct type of terminal was seen rarely. It appeared to pass without branching through the layer of afferent plexuses just described and to form a discrete, loosely branched terminal plexus at a slightly deeper level. An example is drawn in Fig. 4b. The deep limit of the layer of terminal plexuses lies 250 btm, and its superficial limit 100 #m, deep to the internal limit of the stratum opticum. They are an additional 75-250/~m from the tectal surface, depending on the thickness of the stratum opticum (which varies between approx. 75 btm and 250 # m in different areas of the tectum).
Brain Research, 27 ( 1971) 203-221
PIGEON OPTIC TECTUM
205
Superficial stellate cells The inner limit of the stratum opticum is marked by a single layer of small cell bodies, which Cowan et al. 4 called sublayer (a) of the stratum griseum et fibrosum superficiale. The majority of these cells appear to be small stellate cells, like the upper of the two labelled 's' in Fig. 4, although several cells with longer dendrites running 200-300/~m deep to the cell bodies were also seen in this layer (e.g. Fig. 1A, sd). The short dendritic processes of these stellate cells spread about 50/~m downwards and laterally from the cell body. They are presumably not contacted by the afferent plexuses, which are situated 50-100 #m deeper. The only other neuronal structures observed at the level of sublayer (a) were the tips of the dendrites of tectal cells (described below) and optic tract fibres passing down from the stratum opticum to form their plexuses 50-100 #m deeper.
Classification of cells contacted by the afferent terminals A variety of cells was observed whose dendrites extended into the layer of afferent terminals and which could therefore be assumed to be contacted by them. We suggest classifying them into two broad groups: (a) radial ceils, whose processes are predominantly radial in orientation; i.e., they run in the direction normal to the surface of the tectum; and (b) non-radial cells, whose processes show no radial orientation. This classification differs from Cajal's 3 which laid stress on the layer of the rectum in which the somas of the cells were located rather than on the orientation of their processes. However, many of the cell types described here were described or illustrated by Cajal. Considerable variety was apparent within both radial and nonradial groups. This variety is described briefly but, since the various types cannot yet be related to physiological events, they are, for present purposes, considered in just two groups. Radial cells The great majority of cells whose processes extend into the layer of afferent plexuses have radially oriented processes. At least three types can be distinguished. Radial cells of the first type, labelled rl in Figs. 1, 2, 5 and 6, were the most numerous cell type observed in the tectum, and probably constitute most of the cells of the stratum griseum et fibrosum superficiale. They have small, globular bodies from each of which a prominent, apical dendrite runs radially towards the surface of the tectum. These dendrites always reach the layer of afferent plexuses and many continue through the plexuses and reach the layer of superficial stellate cells. Most give a few small branches as they ascend and divide into two or three (still radial) branches in the afferent plexus layer. A small percentage give off prominent laterally spreading branches at a level just deep to the afferent plexuses (Fig. 6, r 0. A minority of the dendrites have spine-like processes on their upper portions (Fig. 6, rl). The bodies of rl cells are distributed over a considerable range of depths. The most superficial are in the sublayer (e) of Cowan et al.'s 4 classification, among the afferent plexuses; the deepest lie some 200 #m deeper, in sublayer (i) (Fig. 5). Usually Brain Research, 27 (1971) 203-221
206
,I. STONE ANi)J. ~,. ~REt[MAN
Fig. 1. Radial and stellate cells, Golgi stain. The dashed lines in A, B, and C mark the superficial and deep limits of the layer of afferent plexuses (labelled I.a.p. in C). The dotted line marks the deep limit of the stratum opticum (str. opt.). A, Two radial cells of the rl type are shown. The small stellate cell "s' is in the layer of afferent plexuses. The cell labelled 'sd' has its soma in sublayer (a). Unlike most of the cells of this layer, which are small stellate cells, the dendrites of this cell pass 300/tin into the deeper layers of the tectum. B, A radial cell of the pyramidal r:~ type. C, A radial cell of the bipolar r2 type. The scale line at lower right represents 200 #m.
PIGEON OPTIC TECTUM
207
their basal dendrites are irregular (i.e., non-radial) in orientation, quite short and much less prominent than the apical dendrite. Occasional cells had a long basal process which passed without branching 100-200 #m deep to the cell body, and which might be portion of an axon. Others gave off axon-like processes from their apical dendrites. as Cajal 3 observed in the sparrow. Radial cells of the second type, labelled r2 in Figs. l, 2 and 5, are bipolar in appearance, having one apical and one basal dendrite which are usually equal in prominence and length, and are strictly radial in orientation. Both dendrites give numerous short, laterally spreading branches. The apical dendrite always reaches the layer of afferent plexuses, in some cases continuing on to reach the superficial stellate layer (Figs. 1, 2 and 5). The cell bodies are fusiform in shape and are found 400-500 # m below the superficial stellate cells, at about the same level as the deepest r~ cells, in the deep part of sublayer (i). Radial cells of the third type, labelled r3 in Figs. 1 and 5, are pyramidal in appearance and their cell bodies are situated about 600-700/~m deep to the superficial stellate cells, in sublayer (j). Each soma gives off a prominent apical dendrite which runs radially to the layer of afferent plexuses. Several large, spiny dendrites arise from the deep side of the cell body and run 100-200/~m lateral and deep to the cell body.
Non-radial cells with processes in the afferent plexus layer A variety of cell types was seen which fell into this group. Their two common features are that their processes (a) are not radial in orientation, but (b) do extend into the afferent plexus layer, where they presumably are contacted by the afferents. They were much less numerous than the radial cells. As well as being found in sublayer (a), above the afferent terminals, stellate cells were seen 100 150/~m deeper among the afferent plexuses (Figs. IA, 4). They were similar in morphology to the sublayer (a) cells. Similar stellate ceils were also observed at deeper levels, well below the afferent plexuses. A small number of cells were seen whose processes spread laterally, parallel to the tectal surface, rather than radially; an example is shown in Fig. 5. In addition, several cells were observed (labelled 'n' in Fig. 6) whose processes showed no directional orientation, but did extend through most of the stratum griseum et fibrosum superficiale. Ganglion cells The deepest neurones we observed in the rectum were the ganglion cells. Their somata are situated in the periventricular grey matter~ approximately 1200-1400/~m below the tectal surface. Their dendrites spread laterally and up towards the tectal surface, and some come into close relationship with the basal dendrites of radial ceils. Since their dendrites were never seen to reach the layer of afferent terminal plexuses, the ganglion cells are presumably not contacted by the afferent terminals. Several gave rise to axon-like processes. A typical ganglion cell is shown in Fig. 3.
Brain Research, 27 (1971) 203-221
208
J. SIONE AN[) J. ,~, FREEMAN
Fig. 2. Afferent fibre plexuses, radial and stellate cells, Golgi stain. The dashed lines mark the deep and superficial limits of the layer of afferent plexuses (labelled l.a.p.). The arrows mark the deep limit of the stratum opticum (str. opt.), The scale lines on each portion represent 100 pro. A, The soma of an ra radial cell is seen at bottom. From it a prominent dendrite runs radially to come into close relationship with the terminal plexus of an afferent fibre. B, The terminal plexus of an afferent fibre. The fibre descends without branching about 100/~m deep to the deep limit of the stratum opticum. It then arborizes to form a typical terminal plexus. C, Radial cells of the r~ and r2 types are shown. A stellate cell's' can be seen with its soma just deep to the layer of afferent plexuses.
PIGEON OPTIC TECTUM
209
Fig. 3. A ganglion cell (g) lying deep in the tectum. A small portion of the lateral ventricle 'v' is seen at bottom. The scale represents 200/~m. The tectal surface, which is out of the field, runs approximately parallel to the upper border of the picture.
( B) Physiological analysis Field potential profile Fig. 7A shows the field potentials generated at successive depths in the tectum by optic nerve stimulation. The potentials were averaged and displayed by a L I N C - 8 c o m p u t e r . A n electronic stimulus artefact suppression device was used to eliminate the artefact f r o m the waveformsL The time o f s t i m u l a t i o n is indicated in these and subsequent waveforms by dots. The d u r a t i o n o f artefact suppression is indicated by
Brain Research, 27 (1971) 203 221
210
J. STONE AND J. ?',, FRE[!MAN
str. opt. ....
j .........................
$ ,k..
I
100p
Fig. 4. Camera lucida drawings of the most common 'columnar' type of afferent fibre terminal 'a', of the very uncommon second type 'b', which passes without branching through th.e layer of afferent plexuses to come into close relationship with laterally spreading dendrites of a radial cell rl; and of two stellate cells 's'. The more superficial stellate cell lies in subtayer (a) and its processes are entirely above the afferent plexuses. The processes of the deeper stellate cell do extend into the layer of afferent plexuses.
the short length of noise-free baseline which follows the stimulus. The potentials resemble fairly closely those described by Holden n. The following features are important for our subsequent analysis. (a) The waveforms have a prominent negative component (the N-wave n) at superficial locations, which inverts to positive (the P-wave) at deeper locations. The reversal occurs at a depth of approximately 400 #m (the R-zone). (b) The early portion of the N-wave inverts more superficially than the late part. (c) An early deflection, arrowed in Figs. 7A and 8A, is present which, for reasons presented below, we attribute to the presynaptic volley.
Current source-density waveJorms Fig. 7B and C show the current source-density wavetbrms obtained from the field potentials of Fig. 7A (Fig. 7B) and from another similar series. These waveforms show the net local extraneuronat current flowing in the direction normal to the tectal surface at each recording point. At certain depths (e.g. the 200-400 #m depths) the current source-density waveforms resemble the field poteotials. At deeper and more Brain Research, 27 (1971) 203-221
PIGEON OPTIC TECTUM
211
str. opt.
'a
B
7|
r,'
200F
r2
h
% Fig. 5. Camera lucida drawings of radial cells of rl, r2 and r3 types; of afferent fibre terminals 'a' and of stellate ceils 's'. Note the range of depths over which the cell bodies of rl cells were found.
superficial locations they differ quite markedly. The following features were c o m m o n to all sets o f current s o u r c e - d e n s i t y waveforms. (a) A t the 100 # m depth, the current s o u r c e - d e n s i t y w a v e f o r m is flat, indicating t h a t there are no net current sinks or sources in the s t r a t u m opticum. (b) A t the next 3 depths - - 200, 300 and 400 # m - - there are p r o m i n e n t sinks o f current. T h e sinks p e a k at successively later times at the three d e p t h s (2.6 msec, 3.6 msec and 4.7 msec respectively). (c) A t the 500/~m d e p t h there is a sink o f current occurring earlier t h a n the sink at 400 # m . S i m u l t a n e o u s with this sink there are small sources at the 400, 600 and 700 # m depths. D e e p e r t h a n 700/zm there are no p r o m i n e n t sources or sinks. Brain Research, 27 (1971) 203 221
212
J. STONE A N D J. \ .
FREEMAN
f
str. opt.
n
Fig. 6. Camera lucida drawings showing two non-radial cells with long unoriented processes 'n'. The rl cell also shown had spiny dendritic branches. The same general pattern of sources and sinks was observed in all tracks although in some it was displaced upwards or downwards by variations in the thickness of the stratum opticum, and occasionally by incorrect identification of the tectal surface, due to overlying fluid. The superficial (200-400 # m depth) sinks were particularly constant in occurrence. Direction of current flow in tectum The current source-density waveforms presented in Figs. 7B, C and 8B ('Jz') show only the net current flowing in the radial direction. Because of the structural homogeneity of the tectum in any plane parallel to its surface, we assumed that there should be no substantial net current in this plane. Confirming this assumption the potentials recorded at adjacent points (approximately 200/~m apart but at the same depth) were nearly identical. In Fig. 8B the current source-density was calculated for each of 3 mutually orthogonal directions (x, y, z) at depth 250 #m. The top trace ('Jz') shows the net current flowing in the radial direction, and the next two traces ('Jx' and 'Jy') show the net current flowing anteroposteriorly and mediolaterally, parallel to the tectal surface. The bottom trace shows the sum of the three currents. It is clear that the great majority of the net current is flowing in the radial direction. Pre- and postsynaptic components of the tectal response Holden 11 did not describe a presynaptic component in the tectal response but he did conclude that the N-wave is postsynaptic. This is consistent with the observations of KonishP 4 and Sutterlin and Prosser 21 on the configuration of pre- and postsynaptic potentials in the fish rectum and is confirmed by the experiment illusBrain Research, 27 (1971) 203-221
PIGEON OPTIC TECTUM
213
A Fieldpotentials
B Current densities
C Current densities
. . . . . . . .
100 ~
..........
200 ~
..........
300
500
Lvf.
/\
600
700 ~--~.J/~'~,~ , • 800-~]~~--~--900 . - - - - ~ j ~ ~ 1000 ~ .
10msec Source
-
~ m
~
10msec
Sink
+
10msec J lmv Fig. 7. Field potentials and current source density waveforms obtained from the optic tectum after optic nerve stimulation. The waveforms were digitized, averaged and displayed by the LINC-8 computer. An artefact suppression device was used to suppress the stimulus artefact. The time of stimulation is indicated in all traces by the dots. The short lengths of noise-free baseline following the dots indicate the duration of artefact suppression. The depths at which the waveforms were obtained are shown at left, in microns. A, The field potential profile. The presynaptic components are marked by arrows. The presence of two presynaptic components is shown more clearly in Fig. 8A. The superficial potentials are predominantly negative, and invert to positive at about 400 ttm. The early part of the negative wave inverts earlier than the late part. B, Current source-density waveforms obtained from the field potentials in A. Most prominent are the sinks at the 200, 300 and 400 #m depths. C, Current source-density waveforms obtained from another set of field potentials. trated in Fig. 8A, in which the pre- a n d postsynaptic c o m p o n e n t s of the initial tectal response to optic nerve stimulation were separated by tetanic stimulation, at increasing frequencies. A t a stimulus rate of l/sec the response, recorded at a depth of 250/zm below the tectal surface, consists of two small initial negative c o m p o n e n t s (marked by arrows), followed by a larger, long-lasting negative c o m p o n e n t , the N-wave. The peak latency of the N-wave increases and its amplitude decreases with increasing stimulus frequency. A t a stimulus rate of 100/sec the N-wave is almost completely abolished, a n d only the initial two presynaptic c o m p o n e n t s remain. These two c o m p o n e n t s would in fact follow stimulus frequencies of greater t h a n 200/sec. Brain Research, 27 (1971) 203-221
214
J. STONE AND J. A. IRt~EMAN
B
A
Jz
I/see ,. • I ~ ,
~
~
a
10/see - , - . , . ~ ~
e
o
~
Jx
20/see Jy
Jx÷ Jy + Jz
100/sec 4msec
I 1my
4msec Source
Fig. 8. A, Field potentials obtained at depth 250/~m in response to optic nerve stimulation at different rates. At each rate, 32 stimuli were given and only the response to the last stimulus was digitized by the computer. The presynaptic components of the field potentials (marked by arrows) were largely unaffected by high stimulus rates. By contrast the later negative wave (the N-wave) was significantly reduced at 10/sec, and was reduced to a small deflection at 100/sec. B, The current source-density waveforms calculated at 300 /~m depth for net current in the radial direction ('J~'), and in two directions ('Jx' and 'Jy') parallel to the tectal surface. The sum of these 3 net currents is shown at bottom ('Jx + Jy t- Jz'). Clearly most net current flows radially.
I
I
A
I
2 MSEC 11MV +
Fig. 9. A, The potential recorded in the optic tract during optic nerve stimulation. The potential was digitized, averaged and displayed by the LINC-8. Time of stimulation is indicated by the dot; the artefact was electronically suppressed: B, The potential in A differentiated, to emphasize the initial two deflections. The period between these two deflections was 0.6 msec, slightly less than the interval of 0.8 msec between the two presynaptic deflections observed in the tectal response to optic nerve stimulation, suggesting that there are two classes of optic nerve fibres, having different conduction velocities. Brain Research, 27 (1971) 203-221
PIGEON OPTIC TECTUM
215
The presence of two presynaptic deflections (Fig. 8A) suggests that there might be two predominant classes of optic nerve fibres with different conduction velocities, as has been described in other animals2,1L This suggestion is supported by the experiment illustrated in Fig. 9, in which the field potential generated in the optic tract by optic nerve stimulation was recorded with a micropipette placed approximately midway between the optic chiasm and the tectum. Two distinct peaks occur in the response, which are emphasized in the differentiated waveform (Fig. 9B).
Dendritic spikes In tracking through tile optic tectum with a high resistance ( > 10 Mr2) micropipette we commonly observed small spike-like potentials superimposed on the initial phase of the N-wave. They also occurred spontaneously and in response to photic stimulation. Examples are shown in Fig. 10A, C, E, G, 1. The spike-like components are accentuated by the time derivatives shown below each field potential waveform (Fig. 10B, D, F, H, J). They occurred in a restricted zone 200-400 Fm beneath the tectal surface; i.e., in the region of afferent terminal arborizations and radial cell dendrites. They were irregular in shape, but individual components appeared to be all-or-none in character and had clearly differentiable thresholds in response to graded retinal stimulation. They occurred approximately
A
G
U"
c-v- v .
.
.
.
.
.
.
-V-.V |
F
.
.
.
..
+
5msec
I
lmv
5msec
[
lmv
Fig. 10. A, C, E, F, G, I, Tectal responses to optic nerve stimulation, showing spike-like deflections superimposed on the N-wave, which may be dendritic spikes, as described in the text. B, D, F, H, J, The tectal response differentiated to emphasize the spike-like components of the N-wave.
Brain Research, 27 (1971) 203-221
216
,I. STONE AND J. A. FREEMAN
0.5-1.5 msec after the initial afferent volley, superimposed on the rising phase or peak of the N-wave, and would follow stimulus frequencies up to only 50/sec. They were therefore clearly postsynaptic in origin, and because of the region in which they occurred, probably represent spikes or partial spikes produced in the dendrites of the radial cells, as will be discussed presently. The small size of the neuronal processes at these depths and the presence of small arterial pulsations of the tectum precluded obtaining intracellular recordings. DISCUSSION Termination o f afferents to the tectum
In several respects our observations are at variance with earlier work. In our preparations the afferent terminals were (disregarding for the moment the uncommon second type) much more uniform in morphology and depth than Cajal 3 described and gave a much stronger impression of forming a compact layer. This is probably a species or age difference. We studied the adult pigeon tectum, whereas Cajal studied principally the tectum of immature (1-10 day) sparrows. The terminal plexuses are also much simpler than in the frog tectum where Lazar and Szekely (quoted in Szekelyz2) have described four layers of terminal endings. It does seem, however, that there is sufficient variety in the morphology of the tectal neurones to account for the varieties of receptive fields which have been found in the pigeon's optic tectum 13. Cowan et al. 4 reported that after contralateral enucleation degenerating terminals were present between sublayers (a) and (f) of the stratum griseum et fibrosum superficia[e. Our observations confirm that the deepest extent of the terminal arborization is sublayer (f), but revealed no terminals above sublayer (c), 100/~m deeper than sublayer (a). The degenerating material which Cowan et al. observed between sublayers (a) and (c) is probably the remains of axons passing from the stratum opticum to their plexuses 100 ,um deeper, though it is possible that they are terminals of some afferent type which did not stain in our preparations. The depth distribution of the presynaptic component of the tectal response to optic nerve stimulation corresponds well with the depth of the afferent plexuses. In the field potentials, the presynaptic components are prominent above the 400 #m depth, being maximal around 200/~m deep. The two deflections in the presynaptic component of the tectal response (Fig. 8A) almost certainly are not related to the two different types of afferent terminals described above. First, the depth distributions of the two deflections were always very similar, whereas the two afferent types have quite different depth relationships. Second, the two deflections were of almost equal magnitude, whereas one type of afferent (the columnar type) was much more common. It is more likely that the two deflections represent two groups of optic nerve fibres, with different conduction velocities but similar terminal plexuses. The two groups might correspond to the myelinated and unmyelinated groups which Bingelli and Paule I have described in Brain Research, 27 (I 971) 203-221
217
P I G E O N O P T I C TECTUM
the pigeon's optic nerve. However, the velocity difference between myelinated and unmyelinated fibres is much greater (at least in the cat retina and optic nerve 2°) than that indicated by the latencies of the fast and slow groups in our records (I.6 and 2.3 msec respectively). The problem clearly requires further analysis. Anatomical organization of tectal cells Of those cells whose dendrites reach the layer of afferent plexuses (and which are probably therefore contacted by the afferent fibres), the great majority are radial in orientation. Conversely, all radial cells appear to be contacted by retinal afferents. Their somas are distributed over a wide range of depths, from about 400 #m (sublayer (e)) to 800/xm (sublayer (j)) from the tectal surface, and they seem to constitute the majority of the cells of the stratum griseum et fibrosum superficiale. Hence, radial cells are probably the principal cells involved in processing the retinal input. Although the cell bodies of the radial cells appear (in Nissl-stained sections) to be arranged in compact and separate layers, the cells in one layer are not serially connected with cells in the next layer. Rather, their processes run in parallel from the layer of afferent plexuses to varying depths in the tectum. This fact is of importance in interpreting the field potential profile and current source-density waveforms obtained from the tectum. It is also a principal reason for our considering the radial cells as a group despite the different depths and layers at which their cell bodies are located. The electrical activity in these cells is principally radial in direction, a property determined by their dendrites, not by their somas. Only a minority of the ceils which are in a position to be contacted directly by the afferent terminals are non-radial in organization. By contrast, all of the cells which are not in a position to be contacted by the afferent plexuses, being either too superficial or too deep in location, appear to be non-radial in orientation. hlterpretation of superficial (100-400 #m) current source density waveforms In Fig. 7B and C successive sinks occur at the 200, 300 and 400 # m depths. A number of lines of evidence suggest that these depths span the layer of afferent plexuses. First, in the Golgi material described above the layer of afferent plexuses was 150/zm thick. Since shrinkage in celloidin-embedded tissue is 20-25 % of length, this dimension is probably nearer 200 #m in the living brain. Thus, if the stratum opticum were of average thickness, say 100/zm, the layer of afferent plexuses would probably extend from 200 to 400/zm below the tectal surface. Second,the deepest of the 3 sinks (400/~m) is at the same level as the R-zone; i.e., the depth at which the postsynaptic potential reverses from negative to positive 11. Using a lesion technique, Holden it showed that the R-zone corresponds to the depth at which Cowan et al. l found the deepest degenerating terminals after section of the contralateral optic nerve. The successive occurrence of these sinks suggests that there is successive activation of neural elements at the 3 levels. This would result if cells at the 200/~m level excited cells at the 300/,m level, which in turn excited cells at the 400 #m level. Brain Research, 27 (1971) 203 221
218
J. STONE A N D J. A. FREEMAN
Alternatively, it would occur if activity propagated along core conductors extending through these depths. The same two alternatives were proposed by Sutterlin and Prosser 2a to explain their observation that the region of maximum current density during the response of the goldfish optic tectum to an optic nerve volley also shifts down in the tectum with time. They were inclined to the former alternative but in view of the structure of the pigeon's tectum, the latter alternative seems much more likely (at least for the pigeon). There is no evidence o1" 3 serially connected layers of cells in this region, but there are two prominent structures, viz. the afferent plexuses and the dendrites of radial cells, which extend radially through this region and along which activity might be propagated. Dendritic spikes in tectal cells"
A number of observations suggest that if it is propagated activity which generates these successive sinks, it is occurring in the dendrites of the tectal cells rather than in the afferent plexuses. (a) Each sink has the same latency as and about half the duration of the N-wave recorded at the same depth (Fig. 8A and B). The sinks are clearly related to the N-wave and almost certainly generate the N-wave. They must, therefore, like the N-wave, be considered postsynaptic in origin and be related to activity in postsynaptic structures, i.e., in the radial cell dendrites. (b) The delay in occurrence of the sinks from depth to depth suggests a conduction velocity of approximately 10 cm/sec, consistent with dendritic spike conduction velocities reported for other neurones, for example, 10-40 cm/sec in alligator Purkinje cell dendrites16; 15-50 cm/sec in the apical dendrites of rabbit hippocampal cellsV; 20-50 cm/sec in rat and kitten cerebellar cells grown in vitro 9. (c) Small spikes with unusual properties were consistently observed in the region of the tectal cell dendrites. For reasons already discussed they are probably postsynaptic in origin. Because of their irregular shape and long duration (up to 3 msec), they are quite distinct from the sharp presynaptic spikes (duration 0.5 msec) and postsynaptic spikes (duration 1.0 msec) which Holden lz described and Robert and Cu6nod la and we have also seen. Llinhs et al. 16 noted, analogously, that the spikes they recorded in the region of the alligator Purkinje cell dendrites, and interpreted as dendritic spikes, were much longer in duration than spikes recorded from the Purkinje cell somas. Interpretation of the deeper (500-1000 #m) current source-density wave/orms
Perhaps the most important feature of these deeper waveforms is the presence at 500 #m of a sink of current with earlier latency than both the N-wave recorded at the same depth and the more superficial (400 #m) sink of current (Fig. 7B, C). The 500 #m sink clearly cannot contribute to the N-wave and/or be a continuation of the propagated activity which appears to be occurring more superficially. Since 500 #m is well below the deepest afferent plexuses seen in our Golgi material, and Brain Research, 27 (1971) 203-221
P I G E O N O P T I C TECTUM
219
below the deepest terminals observed by Cowan et al. 4, and since no presynaptic volley was recorded at this depth, this sink can best be interpreted as the activation of the cell bodies of the radial cells at this depth by electrotonic spread of current from their dendrites. It has a longer latency than the most superficial (200 /zm) N-wave sink, but the same or shorter latency than the 300 and 400/zm N-wave sinks, and appears therefore to be generated while activity is propagating down the radial cell cendrites. Sources corresponding in latency and time course to the 500 ,urn sink occur at the 600 and 700/zm depths, suggesting that current is supplied to the 500 #m sink from basal processes of the radial cells, such as those illustrated in Figs. 1, 2 and 5. These are the only sources apparent in our current source-density waveforms which correspond in latency and time course to the P-wave. If they do generate the P-wave, the interpretation just stated goes against Holden's 1~ conclusion that the P-wave is produced by passive sources supplying current to the N-wave sinks. It is interesting to note in this regard that Robert and Cu6nod 19 suggested that the P- and N-waves may be differently generated, since the N-wave is selectively inhibited by stimulation of the contralateral optic tectum. O'Leary and Bishop 17 made a similar suggestion based on their observation that the P- and N-waves are differently related to stimulus intensity (in the rectum of duck and goose). It should be noted too that small sources corresponding to the N-wave sinks are apparent at the 200, 300 and 400/~m depths. The N-wave sinks appear to draw current principally from structures immediately superficial and deep to them. This is consistent with the idea that they represent propagated activity, since propagating spikes in the dendrites would draw current from immediately adjacent lengths of dendrite. Functional correlations
(i) We have suggested that the radial cells are the principal cells involved in processing the retinal input. The stellate and horizontally spreading cells most likely are interneurones, though their functions have yet to be studied. Hamdi and Whitteridge 8 plotted the projection of the visual field on the optic tectum. They noted that there was 'little or no change in the localisation of the response obtained from the whole thickness of the stratum griseum et fibrosum superficiale, in spite of the fact that it is sufficiently heterogeneous to be divisible into 7 sublayers.' They speculated that some of these sublayers were involved in other functions, such as colour vision. We suggest that visual localisation is constant along a tectal radius because the retinal input is distributed by radially oriented terminal plexuses synapsing onto radial cells. All the sublayers of the stratum griseum et fibrosum superficiale are involved (except perhaps the most superficial sublayer (a)) because all contain the afferent terminals, or the somas or apical and basal dendrites of radial cells. "l'his radial organisation cuts across the horizontal stratification of cell bodies so prominent in Nissl-stained sections, and appears to predominate functionally. (ii) A volley in the optic nerve generates two systems of sources and sinks, both involving the radial cells. First, propagated activity is generated in the radial cell Brain Research, 27 (1971) 203 221
220
J. STONE AN[) J.
A.
FREEMAN
dendrites. The propagating dendritic sinks draw current principally from adjacent lengths of dendrite, generating the N-wave. Second, this dendritic activity evokes the discharge of tectal cell bodies at 400-600/~m depth. This discharge draws current principally from deeper levels, presumably from the basal dendrites of the radial cells, generating the P-wave. The importance of these two source/sink systems probably lies in the fact that they can be separately inhibited or facilitated, allowing for quite complex control of tectal cell excitability. As already noted Robert and Cu6nod x9 have shown that the intertectal commissural system in the pigeon selectively inhibits the N-wave. (iii) The radial cells send axons out of the tectum 3 and hence provide a monosynaptic output for retinal input to the tectum. The deep-lying ganglion cells, on the other hand, are not contacted directly by the retinal afferents. Their principal synaptic input has not yet been defined, but they may provide a di- or polysynaptic output for retinal input. SUMMARY
This paper presents an analysis of the synaptic organisation of the pigeon's optic tectum, with special reference to its retinal input. The structure of the tectum was studied in Golgi-impregnated material. The manner of termination of the retinal afferent fibres to the tectum and the morphology of the neurones of the tectum are described. The afferent fibres terminate in one or more columnar plexus. The plexuses are closely packed, forming a compact layer of afferent terminals at a constant depth in the tectum. It is suggested that the tectal neurones be classified according to the orientation of their dendritic processes into two broad groups, radial and non-radial cells, and the characteristics of and variations within each group are described. The field potential profile generated in the tectum by optic nerve stimulation was subjected to a current source-density analysis to determine the location and sequence of sources and sinks of current produced by an optic nerve volley. The results suggest that the radial cells are the principal cells involved in processing retinal input. The postsynaptic events generated by an optic nerve volley are described and the functional importance of these events is discussed.
REFERENCES 1 BINGELLI,R. L., AND PAULE,W. J., The pigeon retina: quantitative aspects of the optic nerve and ganglion cell layer, J. comp. Neurol., 137 (1969) 1-18. 2 BisHoP, G. H., CLARE, M. H., AND LANDAU,W. M., Further analysis of fibre groups in the optic tract of the cat, Exp. Neurol., 24 (1969) 386--399. 3 CAJAL,S. RAM6NY, Histologie du Syst~me Nerveux de l'Homme et des Vertdbrds, Tdme 2, Maloine. Paris, 191 l, Ch. X. 4 COWAN,W. M., ADAMSON,L., ANDPOWELL,T. P. S., An experimental study of the avian visual system, J. Anat. (Lond.), 95 (1961) 545-563. 5 FREEMAN, J. A., An electronic stimulus-artefact suppressor, Electroenceph. clin. Neurophysiol., Submitted for publication. Brain Research, 27 (1971) 203-221
PIGEON OPTIC TECTUM
221
6 FREEMAN,J. A., AND STONE, J., A technique for current density analysis of field potentials and its
application to the frog cerebellum. In R. LLIN~S (Ed.), Neurobiology of Cerebellar Evolution and Development, Amer. Med. Ass./Educ. and Res. Found., Chicago, 1969, pp. 421-430. 7 FUJITA, K., AND SAKATA,H., Electrophysiological properties of CA1 and CA2 apical dendrites of rabbit hippocampus, J. Neurophysiol., 25 (1962) 209-222. 8 HAMDI, F. A., AND WHITTERIDGE, D., The representation of the retina on the optic tectum of the pigeon, Quart. J. exp. Physiol., 39 (1954) 111 118. 9 HILO, W., AND TASAKI, I., Morphological and physiological properties of neurones and glial cells in tissue culture, J. Neurophysiol., 23 (1962) 277 304. 10 HICL~AN, D. E., Morphological organisation of the frog cerebellar cortex; a light and electron microscopy study, J. Neurophysiol., 32 (1969) 818 846. 11 HOLDEN, A. L., The field potential profile during activation of the avian optic tectum, J. Physiol. (Lond.), 194 (1968) 75-90. 12 HOLDEN, A. L., Types of unitary response and correlation with the field potential profile during activation of the avian optic rectum, J. Physiol. (Lond.), 194 (1968) 91-104. 13 HOLDEN, A. L., Receptive properties of retinal cells and tectal cells in the pigeon, J. Physiol. (Lond.), 201 (1969) 56 57P. 14 KON~SHI, J., Electric responses of visual centre to optic nerve stimulation in fish, Jap. J. Physiol., 10 (1960) 13-27. 15 LEDERMAN,R. J., ANO NOELC, W. K., Fast-fiber system of rabbit optic nerve, Vision Res., 8 (1968) 1385-1398. 16 LE1N~,S, R., NICHOCSON, C. N., FREEMAN, J. A., AND HIH.MAN, D. E., Dendritic spikes and their inhibition in alligator Purkinje cells, Sc&nce, 160 (1968) 1132-1135. 17 O'LEAR¥, J. L., AND BISHOP, G. H., Analysis of potential sources in the optic lobe of duck and goose, J. cell. comp. Physiol., 22 (1943) 73 87. 18 POLVAK, S., The Vertebrate Visual System, Univ. of Chicago Press, 1957. 19 ROBERT, F., AND CO'NOD, M., Electrophysiology of the intertectal commissures in the pigeon. 1I. Inhibitory interaction, Exp. Brain Res., 9 (1969) 123 136. 20 STONE, J., AND FREEMAN, R. B., Conduction velocity groups in the cat's visual pathway classified according to their retinal origin, In preparation. 21 SUTTERCJN, H. M., AND PROSSER, C. L., Electrical properties of the goldfish optic rectum, J. Neurophyshd., 33 (1970) 36-45. 22 SZEKEt.V, G., Anatomy and synaptology of the optic tectum, in R. JUNG (Ed.), Handbook of Sensory Physiology, Vol. VII, in press.
Brain Research, 27 (1971) 203-221