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Reduction in the death and cytolamination of developing neurons by tetrodotoxin in the axonal target region
94
Developmental Brain Research, ~) (1991) 94-98 © 1991 Elsevier Science Publishers B.V. 0165-3806/91/$03.50 A DONIS 016538069160399P
BRESD 60399
Reduction in the and cytola,'nination of developing neurons by tetrodotoxin in the axonal target region Y. Prquignot and P.G.H. Clarke Institute of Anatomy, University of Lausanne, Lausanne (Switzerland)
(Accepted 15 January 1991) Key words: Centrifugal fiber; Chick embryo; Cytolamination; Isthmo-optic nucleus; Neuronal death; Tetrodotoxin
Tetrodotoxin was injected into one eye of chick embryos so as to block activity in the target territory of the isthmo-optic nucleus (ION) during its period of neuronal death. This markedly reduced the neuronal death and thereby prolonged the survival of some ,aberrantly' projecting neurons which would normally all die. In addition, the cytoarchitecture of the ION developed abnormally. Since these two effects differ markedly in their dose-dependence and in other ways, they cannot both be explained by changes in the strength of a single retrograde signal.
The growth and survival of developing neurons depend on retrograde signals from their axonal territory9'21; these may be regulated by neural activity in the target territory 9'21, since the systemic delivery of neuromuscular synaptic blockers x4aS'2° or tetrodotoxin (TFX) 13 reduces the naturally occurring death of somatic and autonomic motoneurons. But, disturbingly, in the only case so far where the blocking agent (TTX) was restricted approximately to the axonal target region, the parent cell bodies being spared, there was no effect on the naturally occurring loss of presynaptic neurons la. To further elucidate this question, we have injected TFX into the eyes of chick embryos so as to block neural activity preferentially in the retina, and have observed the effects on the isthmo-optic nucleus (ION), virtually all of whose neurons are known to innervate the contralateral retina (Fig. 1) 7. About 55% of the ION neurons die between embryonic day (E) 12 or 13 and E178; the isthmo-optic axons reach their targets in the amacrine sublayer of the retina and start forming synapses at about E1312. We chose to block retinal impulse activity from E13 to E17. Preliminary results have been published in an abstract a9. Electrophysiological experiments involving electrical stimulation of each retina and recording from their contralateral optic tecta indicated that retinal conduction was reliably blocked throughout the chosen period by successive injections of 0.15/,g TFX at E13 and E15, and was partially blocked by doses of 0.05-0.10 /ag TFX,
whereas conduction in the uninjected retina and brain was scarcely affected. In our first series of anatomical experiments we t h e r e f o r e injected 0.15/~g T-FX into the right eye at El3 and El5, and fixed the embryos at El7. Counts of ION neurons were much higher than normal in both IONs (Table I), indicating that naturally occurring neuronal death had been greatly reduced. In addition, the characteristic lamination of the ION, which is known to appear between El3 and E l 4 owing to the reorganization of the dendrites to form a distinct neuropil 5 was almost totally lacking contralateral to the injection, but appeared normal ipsilaterally (Table I, Fig. 2). The retinas appeared quantitatively and qualitatively normal, as will be described in detail in a subsequent paper. The occurrence of many more neurons than normal even in the ipsilateral ION is difficult to explain, but may conceivably have been due to the inevitable slight systemic leakage of "I'TX, even though this did not block conduction completely in the brain. To avoid this uncertainty, we tried to obtain a unilateral effect by repeating the above experiments but with lower doses: 0.09 /~g TTX (close to the threshold for blocking conduction in the injected retina) and 0.05/~g TTX (well below threshold). The 0.09/,g injections (at E13 and E15) affected the ipsilateral ION only slightly, but led to significantly increased neuronal numbers as well as loss of lamination in the contralateral ION (Table I). The 0.05 ag injections had virtually no effect (except in o n e
Correspondence: P.G.H. Clarke, Institute of Anatomy, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland.
95 TABLE I Effect o f T T X on the I O N Embryo
#g T T X
Number o f neurons in ION
Degree of lamination in ION
Contralateral to T T X
Ipsilateral to T T X
Contralateral to T T X
Ipsilateral to T T X
A. TTXblockade E13-EIZfixedat E l 7
1. 2. 3. Mean
0.15pg
20,480 12,761 17,290 16,840"
18,270 13,820 15,200 15,760"
None None None
Normal Normal Reduced
4. 5. 6. 7. Mean
0.09pg
15,090 16,850 14,350 13,470 14,940 *+
12,900 11,970 14,030 11,230 12,530"
None None None None
Normal Normal Reduced? Normal
8. 9. 10. 11. 12. Mean
0.05~g
9,890 10,400 10,880 16,050 12,300 11,900
9,420 10,240 10,780 12,180 11,350 10,740
None None Reduced None Reduced
Normal Normal Normal Normal Normal
13. 14. 15. Mean
0 (but citrate) No injection No injection
10,470
10,660
Normal
Normal Normal Normal
9,360 9,200 9,680
9,740
17,590 16,350 15,430 17,350 14,990 16,350 +
13,220 12,570 12,650 15,250 13,780 13,490
B. TTX-blockadeE12-E14,fixedatE14
16. 17. 18. 19. 20. Mean
0.05#g
21. 22. Mean
Noinjection Noinjection
None None None None None
Normal Normal Normal Normal Normal Normal Normal
13,710 15,510 14,610
* Significantly greater than control (P < 0.05; one-tailed Wilcoxon test). + Significantly greater than ipsilateral side (P < 0.05; one-tailed signed-rank test).
e m b r y o ) on I O N neuronal numbers but r e d u c e d the lamination in the contralateral I O N in all cases (Table I). The retina a p p e a r e d normal. Since one m a j o r p u r p o s e of I O N neuronal death a p p e a r s to be the elimination of neurons projecting to the 'incorrect' (i.e. ipsilateral) retina 6'7'16 or to topographically i n a p p r o p r i a t e parts o f the contralateral retina 3 we have tested for the a b n o r m a l persistence of such neurons. In 8 e m b r y o s injected with 0.15 p g T-I'X at E l 3 and E l 5 and fixed at E l 7 , and in 6 control embryos fixed at E l 7 , we injected the r e t r o g r a d e tracer r h o d a m i n e B isothiocyanate into the vitreous b o d y of the right eye at E l 5 in both groups. In the control embryos, there were on average 0.7 + 0.7 ( S . D . ) labelled neurons within the ipsilateral I O N , as well as 5.8 + 4.1 labelled neurons along its b o r d e r and 22.8 + 10.7 labelled ipsilateral ectopic neurons, which matches the results of O ' L e a r y and Cowan H. But in the T'l'X-injected embryos, 10.5 +_ 3.2 n e u r o n s were labelled in the ipsilateral I O N as well as 7.6
+ 3.2 neurons along its b o r d e r and 25.0 + 7.3 ectopic neurons. T h e striking T T X - i n d u c e d increase in labelled neurons within the I O N is highly significant ( P < 0.001, t-test), whereas the slight increases in the n u m b e r s of b o r d e r neurons and of ectopic neurons were not. In a further 10 such T T X - i n j e c t e d e m b r y o s and 6 controls we placed a small fleck of the fluorescent carbocyanine dye 1,1"-dioctadecyl-3,3,3",3"-tetramethylindocarbocyanine p e r c h l o r a t e (DiI) into the p e r i p h e r y of the v e n t r o t e m p o r a l retinal q u a d r a n t at El53 and studied the p a t t e r n of r e t r o g r a d e labelling in the I O N at E l 7 . T h e r e was manifestly less t o p o g r a p h i c precision in the T l ' X - i n j e c t e d animals than in the controls (Fig. 3). To quantitate the t o p o g r a p h i c imprecision, we fed into a c o m p u t e r the x-, y-coordinates of all diI-iabelled neurons within the I O N and, for each of 3 sections, calculated the n u m b e r of neurons (N), their m o m e n t of inertia (M) about their center of gravity (treating each n e u r o n as a point of unit mass), and the area ( A ) of the ION. We
96 OPTIC TECTUM '\ I
/
,,
""~
TTX
:]
/
'.,'-.
f
--
"J
Fig. 1. Diagram of the experimental situation, showing the main connections of the isthmo-optic nucleus (ION), which projects in topographic order onto the ventral part of the contralateral retina3, and receives its major input from the ipsilateral optic tectum (see ref. 4, for review). The discontinuous lines with arrows indicate the transient occurrence of small, ipsilateral isthmo-optic (until about El7) and retino-tectal projections TM.
defined 2 measures of dispersion (hence topographic imprecision): D t = M/N, D 2 = M/NA. D1 is a direct measure of dispersion: D 2 is n o n - d i m e n s i o n a l , and is a measure of dispersion relative to the size of the ION. The mean of D 1 was 0.032 m m 2 for the T T X - g r o u p a n d 0.0t3 m m 2 for the controls. The m e a n of D 2 was 0.153 for the T T X - g r o u p and 0.054 for the controls. For both D 1 and
Di!
RETINA
D2, the difference was highly significant ( P < 0.0001; SAS procedure for hierarchical analysis of variance). We conclude that the death of inappropriately projecting n e u r o n s had been prevented. It had previously been shown that intraocular injection of T T X prevented the
Fig. 2. The effects of tetrodotoxin ('IT'X) on the lamination of the ION, as seen in Nissl-stained coronal paraffin sections. Top: the IONs ipsilaterai (a) and contralaterai (b) to the injected eye, which received 0.15/~g TTX at E13 and again at El5, followed by fixation with Carnoy's medium at El7 (embryo 1 in Table I). Bottom: IONs ipsilateral (c) and contralateral (d) to eye that received 0.05 gg TTX at El2, followed by fixation at El4 (embryo 19 in Table I). Bar = 100/zm. Dorsal is shown up, medial inwards. In each embryo, the lamination is unaffected ipsilaterally (a,c) but is reduced contralaterally (b,d). Note that the ION in (a) contains 18,270 neurons - - almost twice the control number and yet is normally laminated.
-
-
97 TTX
NORMAL
~x x
~
~
x x
f/
- •j
"~, •
200
pm
x
Fig. 3. The effects of the qTX on the elimination of inappropriately projecting ION neurons, as shown in coronal frozen sections from 4 embryos that received a small fleck of diI in the ventrotemporal periphery of the contralateral retina at E15, followed by fixation with 10% formalin in 0.1 M phosphate buffer of pH 7.4 at El7. Filled circles denote diI-labelled neurons within the ION, crosses denote labelled ectopic neurons. In the two normal (uninjected) embryos (left), the labelled neurons are restricted to the topographically appropriate part of the ION, but in the two embryos (right) that received 0.15/~g TFX at E13 and E15 in the eye that also received diI, the topographic precision is clearly reduced. Dorsal is shown up, medial leftwards. Bar = 200/tin.
selective elimination of i n a p p r o p r i a t e l y projecting retinal ganglion cells 18'22 but without affecting the total a m o u n t of cell d e a t h 17. These latter studies differed from ours in that the T T X was p r e s e n t e d to the p e r i k a r y a of the neurons in question rather than to the axon terminals. W e r e the effects of our T F X injections m e d i a t e d by r e t r o g r a d e signals along the isthmo-optic axons or by multistage a n t e r o g r a d e signals via the tectum, whose neurons give rise to most of the synapses in the ION1°? To elucidate this question, we investigated the effects on 1 Angaut, P. and Raffin, J.-P., Embryonic development of the nucleus isthmo-opticus in the chick: a Golgi and electron microscopic study, Arch. Anat. Microsc., 67 (1978) 63-78. 2 Blaser, P.E, Catsicas, S. and Clarke, P.G.H., Retrograde modulation of dendritic geometry in the vertebrate brain during development, Dev. Brain Res., 57 (1990) 139-142. 3 Catsicas, S., Thanos, S. and Clarke, P.G.H., Major role for neuronal death during brain development: refinement of topographical connections, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8165-8168. 4 Clarke, P.G.H., Neuronal death during development in the isthmo-optic nucleus of the chick: sustaining role of afferents from the tectum, J. Comp. Neurol., 234 (1985) 365-379. 5 Clarke, P.G.H. and Caranzano, E, Dendritic development in the isthmo-optic nucleus of chick embryos, Dev. Neurosci., 7 (1985) 161-169. 6 Clarke, P.G.H. and Cowan, W.M., Ectopic neurons and abet-
the I O N of a single intraocular injection of T T X at E12.0 followed by fixation at E l 4 . 0 . A n effect via the tectum is unlikely at this age, since synaptogenesis of the I O N is still only beginning 1, and even gross tectal lesions have no effect on the I O N until later than this 4. The results showed that both the reduction in n e u r o n a l d e a t h and the interference with lamination are a l r e a d y discernible at E14 (Table I, Fig. 2), indicating that b o t h these effects are m e d i a t e d by the r e t r o g r a d e route. O u r interpretation that the genesis of lamination can d e p e n d on a r e t r o g r a d e signal is novel, but is consistent with recent evidence that the generation of specific m o r p h o l o g i c a l features can be similarly regulated 2. O n e of the m o r e intriguing aspects of the above experiments is that the two effects - - r e d u c e d neuronal death and disrupted lamination in the I O N - - were dissociable, since either could occur without the other. This proves that the disruption of lamination was not caused by the reduction in neuronal d e a t h , nor vice versa, and m o r e o v e r suggests the existence of two different r e t r o g r a d e signals that can be differentially affected by T F X . A l t h o u g h the effects in the contralateral I O N might be explained by a single signal-affecting lamination at low threshold but n e u r o n a l n u m b e r only at high threshold, this cannot explain why neuronal numbers were increased ipsilaterally (with the highest dose of TTX) without any change in lamination. T h e simplest interpretation is that a survival-promoting signal is e n h a n c e d by T T X acting on the intraocular portions of the isthmo-optic axons, but also on their extraocular portions following its systemic diffusion, whereas a l a m i n a t i o n - p r o m o t i n g signal is inhibited by T F X acting only within the retina - - p e r h a p s through its effects on the target cells. We thank N. Jeanprrtre and R. Kraftsik for help with statistics and computing, M. Catsicas, S. Clarke and G.M. Innocenti for comments on this manuscript, and C. Vaclavik for typing. This work was supported by Grant 31-9469.88 to P.G.H.C. from the Swiss National Science Foundation.
7
8 9 10
rant connections during neural development, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 4455-4458. Clarke, P.G.H. and Cowan, W.M., The development of the isthmo-optc tract in the chick, with special reference to the occurrence and correction of developmental errors in the location and connections of isthmo-optic neurons, J. Comp. Neurol., 167 (1976) 143-164. Clarke, P.G.H., Rogers, L.A. and Cowan, W.M., The time of origin and the pattern of survival of neurons in the isthmo-optic nucleus of the chick, J. Comp. Neurol., 167 (1976) 125-142. Cowan, W.M., Fawcett, J.W., O'Leary, D.D.M. and Stanfield, B.B., Regressive events in neurogenesis, Science, 225 (1984) 1258-1265. Crossland, W.J., Identification of tectal synaptic terminals in the avian isthmo-optic nucleus. In A.M. Granda and J.H. Maxwell (Eds.), Neural Mechanisms of Behaviour in the Pigeon, Plenum, New York, 1979, pp. 267-286.
98 11 Friedman, S. and Shatz, C.J., The effects of prenatal intracranial infusion of tetrodotoxin on naturally occurring retinal ganglion cell death and optic nerve ultrastructure, Eur. J. Neurosci., 2 (1990) 243-253. 12 Fritzsch, B., Crapon de Caprona, M.-D. and Clarke, EG.H., Development of two morphological types of retinopetal fibers in chick embryos, as shown by diffusion along axons of a carbocyanine dye in the fixed retina, J. Comp. Neurol., 300 (1990) 405-421. 13 Harris, A.J. and McCaig, C.D., Motoneuron death and motor unit size during embryonic development of the rat, J. Neurosci., 4 (1984) 13-24. 14 Laing, N.G. and Prestige, M.C., Prevention of spontaneous motoneurone death in chick embryos, J. Physiol., 282 (1978) 33-34P. 15 Meriney, S.D., Pilar, G., Ogawa, M. and Nunez, R., Differential neuronal survival in the avian ciliary ganglion after chronic acetylcholine receptor blockade, J. Neurosci., 7 (1987) 38403849. 16 O'Leary, D.D.M. and Cowan, W.M., Further studies on the development of the isthmo-optic nucleus with special reference to the occurrence and fate of ectopic and ipsilaterally projecting
neurons, J. Comp. Neurol., 212 (1982) 399-416. 17 O'Leary, D.D.M., Crespo, D., Fawcett, J.W. and Cowan, W.M., The effects of intraocular tetrodotoxin on the postnatal reduction in the numbers of optic nerve axons in the rat, Dev. Brain Res., 30 (1986) 96-103. 18 O'Leary, D.D.M., Fawcett, J.W. and Cowan, W.M., Topographic targeting errors in the retinocollicular projection and their elimination by selective ganglion cell death, J. Neurosci., 6 (1986) 3692-3705. 19 Prquignot, Y. and Clarke, P.G.H., Neuronal death and cytolamination depend on electrical activity in the axonal target area, Eur. J. Neurosci., Suppl. 3 (1990) 285. 20 Pittman, R. and Oppenheim, R.W., Neuromuscular blockade increases motoneurone survival during normal cell death in the chick embryo, Nature, 271 (1978) 364-366. 21 Purves, D., Body and Brain: A T r o p h i c Theory of Neural Connections, Harvard University Press, Cambridge MA, 1988, 231 pp. 22 Thompson, I. and Holt, C., Effects of intraocular tetrodotoxin on the development of the retinocollicular pathway in the Syrian hamster, J. Comp. Neurol., 282 (1989) 371-388.
Developmental Brain Research, ~) (1991) 94-98 © 1991 Elsevier Science Publishers B.V. 0165-3806/91/$03.50 A DONIS 016538069160399P
BRESD 60399
Reduction in the and cytola,'nination of developing neurons by tetrodotoxin in the axonal target region Y. Prquignot and P.G.H. Clarke Institute of Anatomy, University of Lausanne, Lausanne (Switzerland)
(Accepted 15 January 1991) Key words: Centrifugal fiber; Chick embryo; Cytolamination; Isthmo-optic nucleus; Neuronal death; Tetrodotoxin
Tetrodotoxin was injected into one eye of chick embryos so as to block activity in the target territory of the isthmo-optic nucleus (ION) during its period of neuronal death. This markedly reduced the neuronal death and thereby prolonged the survival of some ,aberrantly' projecting neurons which would normally all die. In addition, the cytoarchitecture of the ION developed abnormally. Since these two effects differ markedly in their dose-dependence and in other ways, they cannot both be explained by changes in the strength of a single retrograde signal.
The growth and survival of developing neurons depend on retrograde signals from their axonal territory9'21; these may be regulated by neural activity in the target territory 9'21, since the systemic delivery of neuromuscular synaptic blockers x4aS'2° or tetrodotoxin (TFX) 13 reduces the naturally occurring death of somatic and autonomic motoneurons. But, disturbingly, in the only case so far where the blocking agent (TTX) was restricted approximately to the axonal target region, the parent cell bodies being spared, there was no effect on the naturally occurring loss of presynaptic neurons la. To further elucidate this question, we have injected TFX into the eyes of chick embryos so as to block neural activity preferentially in the retina, and have observed the effects on the isthmo-optic nucleus (ION), virtually all of whose neurons are known to innervate the contralateral retina (Fig. 1) 7. About 55% of the ION neurons die between embryonic day (E) 12 or 13 and E178; the isthmo-optic axons reach their targets in the amacrine sublayer of the retina and start forming synapses at about E1312. We chose to block retinal impulse activity from E13 to E17. Preliminary results have been published in an abstract a9. Electrophysiological experiments involving electrical stimulation of each retina and recording from their contralateral optic tecta indicated that retinal conduction was reliably blocked throughout the chosen period by successive injections of 0.15/,g TFX at E13 and E15, and was partially blocked by doses of 0.05-0.10 /ag TFX,
whereas conduction in the uninjected retina and brain was scarcely affected. In our first series of anatomical experiments we t h e r e f o r e injected 0.15/~g T-FX into the right eye at El3 and El5, and fixed the embryos at El7. Counts of ION neurons were much higher than normal in both IONs (Table I), indicating that naturally occurring neuronal death had been greatly reduced. In addition, the characteristic lamination of the ION, which is known to appear between El3 and E l 4 owing to the reorganization of the dendrites to form a distinct neuropil 5 was almost totally lacking contralateral to the injection, but appeared normal ipsilaterally (Table I, Fig. 2). The retinas appeared quantitatively and qualitatively normal, as will be described in detail in a subsequent paper. The occurrence of many more neurons than normal even in the ipsilateral ION is difficult to explain, but may conceivably have been due to the inevitable slight systemic leakage of "I'TX, even though this did not block conduction completely in the brain. To avoid this uncertainty, we tried to obtain a unilateral effect by repeating the above experiments but with lower doses: 0.09 /~g TTX (close to the threshold for blocking conduction in the injected retina) and 0.05/~g TTX (well below threshold). The 0.09/,g injections (at E13 and E15) affected the ipsilateral ION only slightly, but led to significantly increased neuronal numbers as well as loss of lamination in the contralateral ION (Table I). The 0.05 ag injections had virtually no effect (except in o n e
Correspondence: P.G.H. Clarke, Institute of Anatomy, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland.
95 TABLE I Effect o f T T X on the I O N Embryo
#g T T X
Number o f neurons in ION
Degree of lamination in ION
Contralateral to T T X
Ipsilateral to T T X
Contralateral to T T X
Ipsilateral to T T X
A. TTXblockade E13-EIZfixedat E l 7
1. 2. 3. Mean
0.15pg
20,480 12,761 17,290 16,840"
18,270 13,820 15,200 15,760"
None None None
Normal Normal Reduced
4. 5. 6. 7. Mean
0.09pg
15,090 16,850 14,350 13,470 14,940 *+
12,900 11,970 14,030 11,230 12,530"
None None None None
Normal Normal Reduced? Normal
8. 9. 10. 11. 12. Mean
0.05~g
9,890 10,400 10,880 16,050 12,300 11,900
9,420 10,240 10,780 12,180 11,350 10,740
None None Reduced None Reduced
Normal Normal Normal Normal Normal
13. 14. 15. Mean
0 (but citrate) No injection No injection
10,470
10,660
Normal
Normal Normal Normal
9,360 9,200 9,680
9,740
17,590 16,350 15,430 17,350 14,990 16,350 +
13,220 12,570 12,650 15,250 13,780 13,490
B. TTX-blockadeE12-E14,fixedatE14
16. 17. 18. 19. 20. Mean
0.05#g
21. 22. Mean
Noinjection Noinjection
None None None None None
Normal Normal Normal Normal Normal Normal Normal
13,710 15,510 14,610
* Significantly greater than control (P < 0.05; one-tailed Wilcoxon test). + Significantly greater than ipsilateral side (P < 0.05; one-tailed signed-rank test).
e m b r y o ) on I O N neuronal numbers but r e d u c e d the lamination in the contralateral I O N in all cases (Table I). The retina a p p e a r e d normal. Since one m a j o r p u r p o s e of I O N neuronal death a p p e a r s to be the elimination of neurons projecting to the 'incorrect' (i.e. ipsilateral) retina 6'7'16 or to topographically i n a p p r o p r i a t e parts o f the contralateral retina 3 we have tested for the a b n o r m a l persistence of such neurons. In 8 e m b r y o s injected with 0.15 p g T-I'X at E l 3 and E l 5 and fixed at E l 7 , and in 6 control embryos fixed at E l 7 , we injected the r e t r o g r a d e tracer r h o d a m i n e B isothiocyanate into the vitreous b o d y of the right eye at E l 5 in both groups. In the control embryos, there were on average 0.7 + 0.7 ( S . D . ) labelled neurons within the ipsilateral I O N , as well as 5.8 + 4.1 labelled neurons along its b o r d e r and 22.8 + 10.7 labelled ipsilateral ectopic neurons, which matches the results of O ' L e a r y and Cowan H. But in the T'l'X-injected embryos, 10.5 +_ 3.2 n e u r o n s were labelled in the ipsilateral I O N as well as 7.6
+ 3.2 neurons along its b o r d e r and 25.0 + 7.3 ectopic neurons. T h e striking T T X - i n d u c e d increase in labelled neurons within the I O N is highly significant ( P < 0.001, t-test), whereas the slight increases in the n u m b e r s of b o r d e r neurons and of ectopic neurons were not. In a further 10 such T T X - i n j e c t e d e m b r y o s and 6 controls we placed a small fleck of the fluorescent carbocyanine dye 1,1"-dioctadecyl-3,3,3",3"-tetramethylindocarbocyanine p e r c h l o r a t e (DiI) into the p e r i p h e r y of the v e n t r o t e m p o r a l retinal q u a d r a n t at El53 and studied the p a t t e r n of r e t r o g r a d e labelling in the I O N at E l 7 . T h e r e was manifestly less t o p o g r a p h i c precision in the T l ' X - i n j e c t e d animals than in the controls (Fig. 3). To quantitate the t o p o g r a p h i c imprecision, we fed into a c o m p u t e r the x-, y-coordinates of all diI-iabelled neurons within the I O N and, for each of 3 sections, calculated the n u m b e r of neurons (N), their m o m e n t of inertia (M) about their center of gravity (treating each n e u r o n as a point of unit mass), and the area ( A ) of the ION. We
96 OPTIC TECTUM '\ I
/
,,
""~
TTX
:]
/
'.,'-.
f
--
"J
Fig. 1. Diagram of the experimental situation, showing the main connections of the isthmo-optic nucleus (ION), which projects in topographic order onto the ventral part of the contralateral retina3, and receives its major input from the ipsilateral optic tectum (see ref. 4, for review). The discontinuous lines with arrows indicate the transient occurrence of small, ipsilateral isthmo-optic (until about El7) and retino-tectal projections TM.
defined 2 measures of dispersion (hence topographic imprecision): D t = M/N, D 2 = M/NA. D1 is a direct measure of dispersion: D 2 is n o n - d i m e n s i o n a l , and is a measure of dispersion relative to the size of the ION. The mean of D 1 was 0.032 m m 2 for the T T X - g r o u p a n d 0.0t3 m m 2 for the controls. The m e a n of D 2 was 0.153 for the T T X - g r o u p and 0.054 for the controls. For both D 1 and
Di!
RETINA
D2, the difference was highly significant ( P < 0.0001; SAS procedure for hierarchical analysis of variance). We conclude that the death of inappropriately projecting n e u r o n s had been prevented. It had previously been shown that intraocular injection of T T X prevented the
Fig. 2. The effects of tetrodotoxin ('IT'X) on the lamination of the ION, as seen in Nissl-stained coronal paraffin sections. Top: the IONs ipsilaterai (a) and contralaterai (b) to the injected eye, which received 0.15/~g TTX at E13 and again at El5, followed by fixation with Carnoy's medium at El7 (embryo 1 in Table I). Bottom: IONs ipsilateral (c) and contralateral (d) to eye that received 0.05 gg TTX at El2, followed by fixation at El4 (embryo 19 in Table I). Bar = 100/zm. Dorsal is shown up, medial inwards. In each embryo, the lamination is unaffected ipsilaterally (a,c) but is reduced contralaterally (b,d). Note that the ION in (a) contains 18,270 neurons - - almost twice the control number and yet is normally laminated.
-
-
97 TTX
NORMAL
~x x
~
~
x x
f/
- •j
"~, •
200
pm
x
Fig. 3. The effects of the qTX on the elimination of inappropriately projecting ION neurons, as shown in coronal frozen sections from 4 embryos that received a small fleck of diI in the ventrotemporal periphery of the contralateral retina at E15, followed by fixation with 10% formalin in 0.1 M phosphate buffer of pH 7.4 at El7. Filled circles denote diI-labelled neurons within the ION, crosses denote labelled ectopic neurons. In the two normal (uninjected) embryos (left), the labelled neurons are restricted to the topographically appropriate part of the ION, but in the two embryos (right) that received 0.15/~g TFX at E13 and E15 in the eye that also received diI, the topographic precision is clearly reduced. Dorsal is shown up, medial leftwards. Bar = 200/tin.
selective elimination of i n a p p r o p r i a t e l y projecting retinal ganglion cells 18'22 but without affecting the total a m o u n t of cell d e a t h 17. These latter studies differed from ours in that the T T X was p r e s e n t e d to the p e r i k a r y a of the neurons in question rather than to the axon terminals. W e r e the effects of our T F X injections m e d i a t e d by r e t r o g r a d e signals along the isthmo-optic axons or by multistage a n t e r o g r a d e signals via the tectum, whose neurons give rise to most of the synapses in the ION1°? To elucidate this question, we investigated the effects on 1 Angaut, P. and Raffin, J.-P., Embryonic development of the nucleus isthmo-opticus in the chick: a Golgi and electron microscopic study, Arch. Anat. Microsc., 67 (1978) 63-78. 2 Blaser, P.E, Catsicas, S. and Clarke, P.G.H., Retrograde modulation of dendritic geometry in the vertebrate brain during development, Dev. Brain Res., 57 (1990) 139-142. 3 Catsicas, S., Thanos, S. and Clarke, P.G.H., Major role for neuronal death during brain development: refinement of topographical connections, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8165-8168. 4 Clarke, P.G.H., Neuronal death during development in the isthmo-optic nucleus of the chick: sustaining role of afferents from the tectum, J. Comp. Neurol., 234 (1985) 365-379. 5 Clarke, P.G.H. and Caranzano, E, Dendritic development in the isthmo-optic nucleus of chick embryos, Dev. Neurosci., 7 (1985) 161-169. 6 Clarke, P.G.H. and Cowan, W.M., Ectopic neurons and abet-
the I O N of a single intraocular injection of T T X at E12.0 followed by fixation at E l 4 . 0 . A n effect via the tectum is unlikely at this age, since synaptogenesis of the I O N is still only beginning 1, and even gross tectal lesions have no effect on the I O N until later than this 4. The results showed that both the reduction in n e u r o n a l d e a t h and the interference with lamination are a l r e a d y discernible at E14 (Table I, Fig. 2), indicating that b o t h these effects are m e d i a t e d by the r e t r o g r a d e route. O u r interpretation that the genesis of lamination can d e p e n d on a r e t r o g r a d e signal is novel, but is consistent with recent evidence that the generation of specific m o r p h o l o g i c a l features can be similarly regulated 2. O n e of the m o r e intriguing aspects of the above experiments is that the two effects - - r e d u c e d neuronal death and disrupted lamination in the I O N - - were dissociable, since either could occur without the other. This proves that the disruption of lamination was not caused by the reduction in neuronal d e a t h , nor vice versa, and m o r e o v e r suggests the existence of two different r e t r o g r a d e signals that can be differentially affected by T F X . A l t h o u g h the effects in the contralateral I O N might be explained by a single signal-affecting lamination at low threshold but n e u r o n a l n u m b e r only at high threshold, this cannot explain why neuronal numbers were increased ipsilaterally (with the highest dose of TTX) without any change in lamination. T h e simplest interpretation is that a survival-promoting signal is e n h a n c e d by T T X acting on the intraocular portions of the isthmo-optic axons, but also on their extraocular portions following its systemic diffusion, whereas a l a m i n a t i o n - p r o m o t i n g signal is inhibited by T F X acting only within the retina - - p e r h a p s through its effects on the target cells. We thank N. Jeanprrtre and R. Kraftsik for help with statistics and computing, M. Catsicas, S. Clarke and G.M. Innocenti for comments on this manuscript, and C. Vaclavik for typing. This work was supported by Grant 31-9469.88 to P.G.H.C. from the Swiss National Science Foundation.
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rant connections during neural development, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 4455-4458. Clarke, P.G.H. and Cowan, W.M., The development of the isthmo-optc tract in the chick, with special reference to the occurrence and correction of developmental errors in the location and connections of isthmo-optic neurons, J. Comp. Neurol., 167 (1976) 143-164. Clarke, P.G.H., Rogers, L.A. and Cowan, W.M., The time of origin and the pattern of survival of neurons in the isthmo-optic nucleus of the chick, J. Comp. Neurol., 167 (1976) 125-142. Cowan, W.M., Fawcett, J.W., O'Leary, D.D.M. and Stanfield, B.B., Regressive events in neurogenesis, Science, 225 (1984) 1258-1265. Crossland, W.J., Identification of tectal synaptic terminals in the avian isthmo-optic nucleus. In A.M. Granda and J.H. Maxwell (Eds.), Neural Mechanisms of Behaviour in the Pigeon, Plenum, New York, 1979, pp. 267-286.
98 11 Friedman, S. and Shatz, C.J., The effects of prenatal intracranial infusion of tetrodotoxin on naturally occurring retinal ganglion cell death and optic nerve ultrastructure, Eur. J. Neurosci., 2 (1990) 243-253. 12 Fritzsch, B., Crapon de Caprona, M.-D. and Clarke, EG.H., Development of two morphological types of retinopetal fibers in chick embryos, as shown by diffusion along axons of a carbocyanine dye in the fixed retina, J. Comp. Neurol., 300 (1990) 405-421. 13 Harris, A.J. and McCaig, C.D., Motoneuron death and motor unit size during embryonic development of the rat, J. Neurosci., 4 (1984) 13-24. 14 Laing, N.G. and Prestige, M.C., Prevention of spontaneous motoneurone death in chick embryos, J. Physiol., 282 (1978) 33-34P. 15 Meriney, S.D., Pilar, G., Ogawa, M. and Nunez, R., Differential neuronal survival in the avian ciliary ganglion after chronic acetylcholine receptor blockade, J. Neurosci., 7 (1987) 38403849. 16 O'Leary, D.D.M. and Cowan, W.M., Further studies on the development of the isthmo-optic nucleus with special reference to the occurrence and fate of ectopic and ipsilaterally projecting
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