EXPERIMENTAL
NEUROLOGY
Axonal
49,
246-251 (1975)
Conduction
Velocity
H. W;~SSLE, W. R. LEVICEZ, D. L. Department
of Physiology,
Australian Received
and KIRK,
National April
Perikaryal AND
University,
B. G.
Size CLELAND
Canberra,
1
Australia
28,197s
The recent identification of brisk-transient units with alpha cells in the cat’s retina provided a direct opportunity to test the widely held belief that larger cells give rise to larger axons, and smaller to smaller. Alphacell perikarya increase substantially in size over the first 2 mm from the center of the area centralis. In one cat a map was obtained of the receptivefield centers of all brisk-transient units in the region of the area centralis together with their antidromic latencies to optic tract stimulation. The map was then brought into approximate register with a map of all the alpha cells of the region. The correspondence was controlled by the placement and subsequent identification of electrolytic lesions. Thus perikaryal size could be rather directly compared with axonal conduction latency. The expected relation was verified under singularly uniform circumstances. INTRODUCTION It is widely believed that larger axons come from larger neurons, and smaller from smaller. We report a fairly direct test of this belief as a supplementary result from experiments (6) in which retinal ganglion cells of the physiological brisk-transient type (4) were identified with the morphological alpha-cell class (2). The substantial increase of perikaryal diameter of alpha cells with eccentricity from the center of the area centralis over the first 2 mm (11) provides a useful opportunity to obtain the necessary data under uniform conditions. METHOD
The experiment was conducted on one adult cat in which an extensive series of recordings was made throughout the area centralis with sub1 Dr. H. Wassle was supported by a Fellowship of the Deutsche Forschungsgemeinschaft. His present address is Fachbereich Psychologie, Universitit Konstanz, D-775 Konstanz, Postfach 733, West Germany. Dr. D. L. Kirk was recipient of a Commonwealth Postgraduate Scholarship. 246 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
AXON
CONDUCTION
AND
CELL
SIZE
247
sequent histological examination. The retina had a normal appearance by ophthalmoscopy and microscopy and there was no evidence of the recently described “feline central retinal degeneration” (1) or other abnormality. The correlated physiological and morphological data obtained were typical of numerous observations made separately in other cats. Anesthesia was induced with Z-470 halothane in gas mixture (N,O : 02: CO2 = 70 : 28.5 : 1.5) and continued after surgical procedures with the gas mixture alone. Muscular paralysis was established by continuous infusion (gallamine triethiodide 5 mg/kg.hr + tubocurarine 0.3-0.5 mg/kg.hr). Stimulating bipoles were stereotaxically placed in both optic tracts. The unopenedeye preparation on the left side was used as described previously (3) to obtain recordings from single retinal ganglion cells with tungsten-in-glass electrodes (7). Recognition of Brisk-Transient linits. Cells of this class were diagnosed (4) by their vigorous responses to large (< 2”), rapidly moved, black or white contrast targets. Their average discharge rate could be driven above 120/set by rapidly (lO-30/set) alternating the black and white faces of such a target on the center of the receptive field. Cell recordings were distinguished from fiber recordings by observing the receptive field approximately centered on the electrode tip. Antidromic Activation. All of the brisk-transient units could be activated by electrical stimulation in one or other optic tract. It has been shown that such activation is antidromic (8) and indicates conduction in the ganglion cell’s own axon. The latency was measured from the beginning of the shock artifact to the beginning of the all-or-none deflection produced by the impulse whether the waveform was initially positive-going or negative-going. The latencies ranged from 1.60 to 2.66 msec and were shorter than those of any other physiological type (4, 5) in the part of the retina studied (1 mm around the area centralis). RESULTS Exhaustive Exploration. The microelectrode was moved in small steps (20-30 pm), criss-crossing the area centralis many times in an attempt to record from every brisk-transient unit in the region. Each was characterized by : (i) marking the center of its receptive field on an external tangent screen ; (ii) noting whether on-center or off-center; (iii) noting which optic tract yielded antidromic activation; (iv) measuring the threshold for electrical activation ; and (v) measuring the antidromic latency at 1.4 x threshold. These measures gave a unique signature for each cell and enabled subsequent encounters with the same cell to be recognized. After 3 days of recordings, a map of the positions of the receptive-field centers of
248
W;ISSLE
ET AL.
/ /’ 0
(JD
,d’ 0
0
/ O/ /
00 cy 0
00
p’ OG-
0
0
03 00 ooo,9;1
0
0
/ ‘O /
0
0”
0 0 0
0 0
00 0
O
/
0 @O 0
AXON
CONDUCTION
AND
CELL
SIZE
249
109 unique brisk-transient units had been built up. It was complete in that further searching turned up no new units. At the conclusion of recording, four electrolytic lesions (electrode negative, 1.5 pa for 7 set) were placed around the region recorded. At each position a brisk-sustained cell had been recorded and the position of the center of its receptive field marked on the tangent screen. Since such cells had the smallest receptive fields, the positions so marked were taken to be the projections onto the screen of the subsequently produced lesions. Histological Correlation. The eye was excised and the retina prepared as a whole-mount stained with cresyl violet. The region of recording was verified to cover the area centralis by identifying the lesions in relation to the peak ganglion cell density. The alpha cells were recognized as a sparse population of perikarya distinctly larger than their regional neighbors ( 11). A photomicrograph of the region was enlarged so as to yield optimum registration of the lesions when projected onto the map of receptive fields. A map of the alpha cells was then produced by marking the centers of their perikarya on a transparent sheet. The maximum and minimum diameters of each cell at its optical section were measured directly in a microscope with a focusing micrometer eyepiece. Perikaryal size was recorded as the arithmetic mean of maximum and minimum diameters. The sizes ranged from 14.4-27.2 pm. As described elsewhere (6), the densities of alpha cells and brisk. transient receptive fields approximately agree. Although a 1 : 1 correspondence could be obtained in regions away from the area centralis, the density was sufficiently high in the area centralis to prevent unambiguous assignment of a particular receptive field to a particular alpha cell out of two or three neighboring alternatives (Fig. 1A). However, rearranging the assignments made no essential difference to the result described in the next section. FIG. IA. Superimposed maps of the centers of receptive fields of brisk-transient units (open circles) and of perikarya of alpha cells (solid dots) in the region of the area centralis (histologically estimated center indicated by star). Vertical and horizontal meridians are shown by the intersecting solid lines (S-superior, I-inferior, N-nasal, T-temporal retina). The pairs of larger circles with crosses indicate the projected positions of electrolytic lesions on the receptive field map and the positions of the histological lesions on th’e alpha-cell map respectively. The small relative shift between the members of three of the pairs indicates the adjustment required to produce optimum registration of receptive field and alpha cells judged both by eye and by computer program. The discrepancy of the remaining pair of markers may indicate incorrect projection of the lesion on the receptive field map possibly by axonal rather than cell-body recording of the localizing brisk-sustained unit. B. Scatter diagram of antidromic axonal conduction latency against perikaryal size of the associated alpha cell, selected from the (obliquely running) rectangular region (dot-dashed outline) centered on the vertical meridian.
250
W;iSSLE
ET AL.
Conduction Velocity and Perikaryal Size. Conduction latency, the measure from the electrical study, can be directly related to average conduction velocity only when the paths involved have the same length. To minimize the variation in conduction path, the study was confined to the receptive fields encountered within a narrow band running vertically through the area centralis (Fig. 1A). A scatter diagram of conduction latency against perikaryal size for one particular assignment of receptive fields to neighboring alpha cells is shown in Fig. 1B. Two features are obvious. (i) The general trend of the points reveals the expected inverse relation between conduction latency and perikaryal size. (ii) There is substantial scatter of the points. For example, alpha cells having perikaryal sizes of 16.2 and 26.5 pm each had latencies of about 2.05 msec. The slope of the linear regression of Fig. 1B is -0.045 msec/pm of perikaryal size, although the relation might well be hyperbolic. The latencies can be converted into average conduction velocity on the basis of an approximate path length of 35 mm. The regression of average conduction velocity against perikaryal size had a slope of 0.38 m/sec/ym of perikaryal size. DISCUSSION The above result has been obtained under uniform conditions selected so as to isolate the controlling influence of perikaryal size. Thus the study was restricted to a particular class of cell in one particular cat. A restricted region of retina was chosen to yield approximate equality of the conduction paths and equal balance of myelinated and unmyelinated portions of the axon. The expected qualitative result was confirmed. The numerical values arising from the analysis should be used with caution since the relation of conduction velocity to axon diameter may be different in the case of the unmyelinated and myelinated portions of the axon (9). The scatter in the relation between conduction latency and perikaryal size indicates substantial perturbation of the controlling influence of the latter. The variation is reminiscent of that described (Fig. 4 in Ref. 10) in an investigation of conduction velocity and fiber diameter on individual peripheral nerve fibers of the frog. REFERENCES R. W., and C. A. FISCHER. 1970. Feline central retinal degeneration. J. Amer. Vet. Med. Assoc. 157 : 842-849. 2. BOYCOTT, B. B., and H. WXSSLE. 1974. The morphological types of ganglion cells of the domestic cat’s retina. J. Physiol. (London) 240: 397-419.
1. BELLHORN,
AXON
COi’GDUCTION
APU’D
CELL
SIZE
251
CLELAND, B. G., M. W. DUBIN, and W. R. LEVICK. 1971. Sustained and transienr neurones in the cat’s retina and lateral geniculate nucleus. J. Physiol. (London) 217 : 473-496. 4. CLELAND, B. G., and W. R. LEVICK. 1974. Brisk and sluggish concentrically organized ganglion cells in the cat’s retina. J. PAssiol. (London) 240: 421-456. 5. CLELAND, B. G., and W. R. LEVICK. 1974. Properties of rarely encountered types of ganglion cells in the cat’s retina and an overall classification. J. 3.
Pkysiol.
(Loadorr.)
240 : 456492.
6. CLELAND, B. G., W. R. LEVICK, and H. WXSSLE. 1975. Physiological identification of a morphological class of cat retinal ganglion cells. J. Pltgsiol. (London) 248: 151-171. 7. LEVICK, W. R. 1972. Another tungsten microelectrode. Med. Electron. Biol. Eng. 10 : 510-51s. 8. LEVICK, W. R., and B. G. CLELAND. 1974. Receptive fields of cat retinal ganglion cells having slowly conducting axons. Brain Res. 74: 156-160. 9. RUSHTON, W. A. H. 1951. A theory of the effects of fibre size in medullated nerve. J. Physiol. (London) 115 : 101-122. 10. TASABI, I., I(. ISHII, and H. ITO. 1943. On the relation between the conduction-rate, the fibre-diameter and the internodal distance of the medullated nerve fibre. Jap. J. Med. Sci. 9: 189-199. 11. W~~SSLE, H., W. R. LEVICK, and B. G. CLELAND. 1975. The distribution of the alpha type of ganglion cell in the cat’s retina. 1. Comb. Nezlrol. 159: 419-438.
NEUROLOGY
Axonal
49,
246-251 (1975)
Conduction
Velocity
H. W;~SSLE, W. R. LEVICEZ, D. L. Department
of Physiology,
Australian Received
and KIRK,
National April
Perikaryal AND
University,
B. G.
Size CLELAND
Canberra,
1
Australia
28,197s
The recent identification of brisk-transient units with alpha cells in the cat’s retina provided a direct opportunity to test the widely held belief that larger cells give rise to larger axons, and smaller to smaller. Alphacell perikarya increase substantially in size over the first 2 mm from the center of the area centralis. In one cat a map was obtained of the receptivefield centers of all brisk-transient units in the region of the area centralis together with their antidromic latencies to optic tract stimulation. The map was then brought into approximate register with a map of all the alpha cells of the region. The correspondence was controlled by the placement and subsequent identification of electrolytic lesions. Thus perikaryal size could be rather directly compared with axonal conduction latency. The expected relation was verified under singularly uniform circumstances. INTRODUCTION It is widely believed that larger axons come from larger neurons, and smaller from smaller. We report a fairly direct test of this belief as a supplementary result from experiments (6) in which retinal ganglion cells of the physiological brisk-transient type (4) were identified with the morphological alpha-cell class (2). The substantial increase of perikaryal diameter of alpha cells with eccentricity from the center of the area centralis over the first 2 mm (11) provides a useful opportunity to obtain the necessary data under uniform conditions. METHOD
The experiment was conducted on one adult cat in which an extensive series of recordings was made throughout the area centralis with sub1 Dr. H. Wassle was supported by a Fellowship of the Deutsche Forschungsgemeinschaft. His present address is Fachbereich Psychologie, Universitit Konstanz, D-775 Konstanz, Postfach 733, West Germany. Dr. D. L. Kirk was recipient of a Commonwealth Postgraduate Scholarship. 246 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
AXON
CONDUCTION
AND
CELL
SIZE
247
sequent histological examination. The retina had a normal appearance by ophthalmoscopy and microscopy and there was no evidence of the recently described “feline central retinal degeneration” (1) or other abnormality. The correlated physiological and morphological data obtained were typical of numerous observations made separately in other cats. Anesthesia was induced with Z-470 halothane in gas mixture (N,O : 02: CO2 = 70 : 28.5 : 1.5) and continued after surgical procedures with the gas mixture alone. Muscular paralysis was established by continuous infusion (gallamine triethiodide 5 mg/kg.hr + tubocurarine 0.3-0.5 mg/kg.hr). Stimulating bipoles were stereotaxically placed in both optic tracts. The unopenedeye preparation on the left side was used as described previously (3) to obtain recordings from single retinal ganglion cells with tungsten-in-glass electrodes (7). Recognition of Brisk-Transient linits. Cells of this class were diagnosed (4) by their vigorous responses to large (< 2”), rapidly moved, black or white contrast targets. Their average discharge rate could be driven above 120/set by rapidly (lO-30/set) alternating the black and white faces of such a target on the center of the receptive field. Cell recordings were distinguished from fiber recordings by observing the receptive field approximately centered on the electrode tip. Antidromic Activation. All of the brisk-transient units could be activated by electrical stimulation in one or other optic tract. It has been shown that such activation is antidromic (8) and indicates conduction in the ganglion cell’s own axon. The latency was measured from the beginning of the shock artifact to the beginning of the all-or-none deflection produced by the impulse whether the waveform was initially positive-going or negative-going. The latencies ranged from 1.60 to 2.66 msec and were shorter than those of any other physiological type (4, 5) in the part of the retina studied (1 mm around the area centralis). RESULTS Exhaustive Exploration. The microelectrode was moved in small steps (20-30 pm), criss-crossing the area centralis many times in an attempt to record from every brisk-transient unit in the region. Each was characterized by : (i) marking the center of its receptive field on an external tangent screen ; (ii) noting whether on-center or off-center; (iii) noting which optic tract yielded antidromic activation; (iv) measuring the threshold for electrical activation ; and (v) measuring the antidromic latency at 1.4 x threshold. These measures gave a unique signature for each cell and enabled subsequent encounters with the same cell to be recognized. After 3 days of recordings, a map of the positions of the receptive-field centers of
248
W;ISSLE
ET AL.
/ /’ 0
(JD
,d’ 0
0
/ O/ /
00 cy 0
00
p’ OG-
0
0
03 00 ooo,9;1
0
0
/ ‘O /
0
0”
0 0 0
0 0
00 0
O
/
0 @O 0
AXON
CONDUCTION
AND
CELL
SIZE
249
109 unique brisk-transient units had been built up. It was complete in that further searching turned up no new units. At the conclusion of recording, four electrolytic lesions (electrode negative, 1.5 pa for 7 set) were placed around the region recorded. At each position a brisk-sustained cell had been recorded and the position of the center of its receptive field marked on the tangent screen. Since such cells had the smallest receptive fields, the positions so marked were taken to be the projections onto the screen of the subsequently produced lesions. Histological Correlation. The eye was excised and the retina prepared as a whole-mount stained with cresyl violet. The region of recording was verified to cover the area centralis by identifying the lesions in relation to the peak ganglion cell density. The alpha cells were recognized as a sparse population of perikarya distinctly larger than their regional neighbors ( 11). A photomicrograph of the region was enlarged so as to yield optimum registration of the lesions when projected onto the map of receptive fields. A map of the alpha cells was then produced by marking the centers of their perikarya on a transparent sheet. The maximum and minimum diameters of each cell at its optical section were measured directly in a microscope with a focusing micrometer eyepiece. Perikaryal size was recorded as the arithmetic mean of maximum and minimum diameters. The sizes ranged from 14.4-27.2 pm. As described elsewhere (6), the densities of alpha cells and brisk. transient receptive fields approximately agree. Although a 1 : 1 correspondence could be obtained in regions away from the area centralis, the density was sufficiently high in the area centralis to prevent unambiguous assignment of a particular receptive field to a particular alpha cell out of two or three neighboring alternatives (Fig. 1A). However, rearranging the assignments made no essential difference to the result described in the next section. FIG. IA. Superimposed maps of the centers of receptive fields of brisk-transient units (open circles) and of perikarya of alpha cells (solid dots) in the region of the area centralis (histologically estimated center indicated by star). Vertical and horizontal meridians are shown by the intersecting solid lines (S-superior, I-inferior, N-nasal, T-temporal retina). The pairs of larger circles with crosses indicate the projected positions of electrolytic lesions on the receptive field map and the positions of the histological lesions on th’e alpha-cell map respectively. The small relative shift between the members of three of the pairs indicates the adjustment required to produce optimum registration of receptive field and alpha cells judged both by eye and by computer program. The discrepancy of the remaining pair of markers may indicate incorrect projection of the lesion on the receptive field map possibly by axonal rather than cell-body recording of the localizing brisk-sustained unit. B. Scatter diagram of antidromic axonal conduction latency against perikaryal size of the associated alpha cell, selected from the (obliquely running) rectangular region (dot-dashed outline) centered on the vertical meridian.
250
W;iSSLE
ET AL.
Conduction Velocity and Perikaryal Size. Conduction latency, the measure from the electrical study, can be directly related to average conduction velocity only when the paths involved have the same length. To minimize the variation in conduction path, the study was confined to the receptive fields encountered within a narrow band running vertically through the area centralis (Fig. 1A). A scatter diagram of conduction latency against perikaryal size for one particular assignment of receptive fields to neighboring alpha cells is shown in Fig. 1B. Two features are obvious. (i) The general trend of the points reveals the expected inverse relation between conduction latency and perikaryal size. (ii) There is substantial scatter of the points. For example, alpha cells having perikaryal sizes of 16.2 and 26.5 pm each had latencies of about 2.05 msec. The slope of the linear regression of Fig. 1B is -0.045 msec/pm of perikaryal size, although the relation might well be hyperbolic. The latencies can be converted into average conduction velocity on the basis of an approximate path length of 35 mm. The regression of average conduction velocity against perikaryal size had a slope of 0.38 m/sec/ym of perikaryal size. DISCUSSION The above result has been obtained under uniform conditions selected so as to isolate the controlling influence of perikaryal size. Thus the study was restricted to a particular class of cell in one particular cat. A restricted region of retina was chosen to yield approximate equality of the conduction paths and equal balance of myelinated and unmyelinated portions of the axon. The expected qualitative result was confirmed. The numerical values arising from the analysis should be used with caution since the relation of conduction velocity to axon diameter may be different in the case of the unmyelinated and myelinated portions of the axon (9). The scatter in the relation between conduction latency and perikaryal size indicates substantial perturbation of the controlling influence of the latter. The variation is reminiscent of that described (Fig. 4 in Ref. 10) in an investigation of conduction velocity and fiber diameter on individual peripheral nerve fibers of the frog. REFERENCES R. W., and C. A. FISCHER. 1970. Feline central retinal degeneration. J. Amer. Vet. Med. Assoc. 157 : 842-849. 2. BOYCOTT, B. B., and H. WXSSLE. 1974. The morphological types of ganglion cells of the domestic cat’s retina. J. Physiol. (London) 240: 397-419.
1. BELLHORN,
AXON
COi’GDUCTION
APU’D
CELL
SIZE
251
CLELAND, B. G., M. W. DUBIN, and W. R. LEVICK. 1971. Sustained and transienr neurones in the cat’s retina and lateral geniculate nucleus. J. Physiol. (London) 217 : 473-496. 4. CLELAND, B. G., and W. R. LEVICK. 1974. Brisk and sluggish concentrically organized ganglion cells in the cat’s retina. J. PAssiol. (London) 240: 421-456. 5. CLELAND, B. G., and W. R. LEVICK. 1974. Properties of rarely encountered types of ganglion cells in the cat’s retina and an overall classification. J. 3.
Pkysiol.
(Loadorr.)
240 : 456492.
6. CLELAND, B. G., W. R. LEVICK, and H. WXSSLE. 1975. Physiological identification of a morphological class of cat retinal ganglion cells. J. Pltgsiol. (London) 248: 151-171. 7. LEVICK, W. R. 1972. Another tungsten microelectrode. Med. Electron. Biol. Eng. 10 : 510-51s. 8. LEVICK, W. R., and B. G. CLELAND. 1974. Receptive fields of cat retinal ganglion cells having slowly conducting axons. Brain Res. 74: 156-160. 9. RUSHTON, W. A. H. 1951. A theory of the effects of fibre size in medullated nerve. J. Physiol. (London) 115 : 101-122. 10. TASABI, I., I(. ISHII, and H. ITO. 1943. On the relation between the conduction-rate, the fibre-diameter and the internodal distance of the medullated nerve fibre. Jap. J. Med. Sci. 9: 189-199. 11. W~~SSLE, H., W. R. LEVICK, and B. G. CLELAND. 1975. The distribution of the alpha type of ganglion cell in the cat’s retina. 1. Comb. Nezlrol. 159: 419-438.
