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Tetrodotoxin inhibits the formation of refined retinotopography in goldfish
293
Developmental Brain Research, 6 (1983) 293- 298 Elsevier Biomedical Press
Short Communications
Tetrodotoxin inhibits the formation of refined retinotopography in goldfish RONALD L. MEYER
Developmental and Cell Biology, DevelopmentalBiology Center, Universityof California, lrvine, CA 92717(U.S.A.) (Accepted September 7th, 1982)
Key words: goldfish - retinotectal - tetrodotoxin - regeneration - topography
One optic nerve of mature goldfish was crushed in the orbit and allowed to regenerate. During regeneration impulse activity was eliminated by periodic intraocular injections of tetrodotoxin (TTX). At 32-104 days, the retinotopography of the retinotectal projection was measured autoradiographically by intraocular [3H]proline injections simultaneous with either small (10-20 ° sector) or half retinal mapping lesions. TTX had no effect on the time-course and quality of the regeneration of gross topography seen with half retina mapping, but indefinitely inhibited higher order (refined) retinotopography normally seen by 2 months with retina sector mapping.
In development, neurons of the central nervous system (CNS) send out axons to form highly ordered connections onto target cells. Both the nature and n u m b e r of mechanisms which organize these connections is a subject of continuing debate. One candidate is neuronal impulse activity. In several systems, most notably the visual system, reduced or altered sensory stimulation has been reported to lead to a variety of developmental changes 4.5.9,t~.t9. However, the anatomical substrate, interpretation, and generality of these changes remain unclear. For cortical ocular dominance columns for example, there is anatomical evidence for altered connectivity following monocular deprivation 9,~9. However, the rapid recovery of cortical responsivity to the deprived eye such as following enucleation of the undeprived eye ~2or administration of bicuculline~ suggests altered synaptic efficiency rather than connectivity. Also, all the evidence for activity effects in CNS connectivity comes from developing systems. The inherent complexity of developmental interactions and multiplicity of developmental events make it difficult to know which are the relevant ones. Mitosis, cell differentiation and growth, cell death as well as D165-3806/83/0000-0000/$03.00 ©1983 Elsevier Biomedical Press
selective axon growth are often confounded. Finally, there is a serious question whether activity plays a universal role. Some species seem immune to abnormal stimulation 3,m and in susceptible species many parts of the CNS such as the topography of the primary visual projections are unaffected 9.~1. It is possible that the only instructive role played by activity is limited to highly specialized neural systems such as those concerned with stereopsis. In this light, the retinotectal topography formed by regenerating optic fibers in mature goldfish is perhaps an unlikely place to look for activity-dependent changes. Anatomical organization rather than synaptic function is directly measured. Retina and tectum are already differentiated. The projection is a primary sensory one, generally thought to be resistant to activity effects. The system has no known binocular convergence necessary for stereopsis. Constant dark, continuous light and strobe illumination have no reported effect on topography 4.14. In developing urodeles, elimination of impulse activity with tetrodotoxin (TTX) did not prevent optic fibers from forming a retinotopic projection and anatomically normal synapses s. On the other hand, if an effect of activi-
294 ty were to be demonstrated, it would argue ti)r a pervasive and fundamental role for activity in the genesis of orderly connections, would extend such an activity effect to a regenerating system and would offer a valuable model system. There was some hope this would be the case. In the urodele studys, retinotopography was not measured with high precision and in no other study showing negative results was activity entirely eliminated during the period of axon ingrowth. Previous experiments in goldfish and frog had shown that regenerating optic fibers rapidly form a projection that is grossly retinotopic but only later forms one with refined retinotopography v.H. It seemed possible that this later stage might utilize activity-dependent mechanisms and be sensitive to TTX. Common goldfish, 5 7 cm in body length, were maintained in standard aquaria at 19 20°C, Under tricaine methanesulfonate anesthesia one optic nerve was crushed in the orbit as previously described% For chronic blockade, 0.1/,t of 0.3 mM TTX (10 times the minimum acute blocking dose) was injected every 3.5 days by means of a 25 ~m glass pipette inserted through the dorsal limbus. By extracellular recordings in tectum or nerve, this dose was found to block visually evoked action potentials in intact or regenerating nerves, including those injected for 31 90 days and recorded at 3.5 4 days from the last TTX injection (32 different nerve recordings), in large fish injected for 31 90 days (4 cases) weak recovery could be detected as early as 4 days, and in all cases recovery was found at 5 days. An injection of 0.1 t*l of 0.01 mM TTX had no apparent effect and was used as a "subthreshold' control. The anatomical technique used to determine the retinotopography of the projection was identical to that of a previous regeneration study ~4. At the end of each experiment a small area of retina, about 750 1000/xm in diameter and about a third to halfway out from the disc, was lesioned by inserting a needle through the sclera and passing rf current. Both ganglion cells and optic fibers from peripheral retina coursing through this region were destroyed. Tritiated proline (25-50 t*Ci) was immediately injected into the eye followed by autoradiography at
about 18 h later. In normal fish without nerve crush, it was previously' reported that silxer grains were normally distributed throughout rectum except for a denervated sector in the tectal region corresponding to the a c u t e lesion ~. In effect, all optic fibers were labeled except for those in the lesioned retinal sector. This procedure will be referred to as "sector mapping'. When done at various times iollowing nerve crush, it was previously l'ound ~" that no zone of denervation was detectable in rectum at up to 40 days postoperatively, indicating that fibers initially lacked a high degree of retinotopography. However, gross topography existed at 40 days as shown by 'hemiretina mapping'., i.e. acute retinal lesions of half of" retina. At 80 days, sector mapping yielded a denervated sector at the appropriate place in the rectum, indicating the retinotopography had become more highly ordered. Some light label remained in the denervated sector and could be attributed to fibers of passage, since many fibers were shown to follow anomalous paths in regeneration. In the present study, nasal mapping lesions (corresponding to posterior tectum) were usually chosen since the fewest fibers of passage are found in posterior rectum. This progressive development of retinotopography has also been seen with etectrophysiological mapping:. Four groups of fish were treated with TTX following crush of the left nerve. The first group (4 fish) was given subthreshold TTX iniections from the time of nerve crush until autoradiography at 80 days. This injection protocol had no dectectable effect on regeneration, that is a denervated sector in posterior rectum was observed and all aspects of innervation were typical of normal regeneration (Fig. la). In the second group, a blocking dose was administered from the time of crush to autoradiography at 32 40 days (6 fish), 66 days (1 fish), 80 days (4 fish), and 90 days (4 fish) (Fig. lb). At 32 40 days, autoradiography was indistinguishable from normal regeneration. Specifically, fibers were within the correct layers and extended to the most posterior regions of tectum where much of the label was string-like in appearance, presumablx. rel]ecting fasciculatioc~ ~ -\xoplasmic
295
b
a
O
,k,,
t
c
"
d
Fig. 1. Autoradiograms of right tectum following sector map lesion to left nasal retina. Left optic nerve was previously crushed. Sections are frontal in orientation (medial to the right) and near the posterior end of tectum. Calibration bar is 400/zm. a: fish from group I in which subthreshold TTX was administered until sector mapping at 80 days after crush• The denervated zone seen in the mediolateral region of the main optic layer is equivalent to fish receiving no ocular injections, b: fish from group 2 in which blocking dose of TTX was given until sector mapping at 90 days. No denervated sector is evident, c: fish from group 3 in which a final blocking dose of TTX was given from day 42 until autoradiography at 80 days. No denervated tectal sector can be seen. d: fish from group 4 in which a blocking dose of TTX was given only from 42 to 81 days followed by autoradiography after an additional 24 days without TTX. A denervated tectal sector is evident.
296 transport (grain density) was distinctly elevated as compared to intact fibers (also labeled as mentioned below), and no denervated sector was detected. At 66 90 days, the overall characteristics of tectal labeling was also typical of normal regeneration. Label was no longer 'fasciculated' but had become homogeneous and axoplasmic transport had returned to nearly normal levels in the 8 ~ 9 0 day fish. Gross topography was demonstrated by nasal hemiretinal mapping (nasal lesion) at 40 days (3 fish), 66 days ( 1 fish), and 90 days (2 fish). In these, dense label was confined to the anterior half of tectum with little or none in the posterior half (Fig. 2a). In the nasal sector-mapped fish, the only apparent difference from normal regeneration was that no denervated sector could be observed with
a
sector mapping. A slight decrease in overall labeling was just detectable in the posterior third of tectum indicating fibers from the lesioned nasal sector were distributed throughout this region. Exactly the same result was observed in a third group (5 fish) in which T r x blockade was initiated at 42 days until autoradiography at 81 days (Fig. lc) . Again, no refined retinotopography could be demonstrated with sector mapping (4 fish) but gross retinotopography was obvious with hemiretina mapping (I fish). Thus, TTX need only be given for 39 days during the period in which refined retinotopography is forming. This argues against a cumulative toxicity in the second (continuous TTX) group, i.e, that toxicity took 40 days or more io develop, a time that just happened to correspond to the period
.
b ".
d
. LLL~LL~_jLIi
~
.............
Fig. 2. A u t o r a d i o g r a m s o f posterior tectum as in Fig. 1. a: fish from group 2 in which a blocking dose o f TTX was given untd nasal h e m i r e t i n a m a p p i n g (actually about 120 ° o f retinal arc) at 90 days. T h e p h o t o m o n t a g e is m a d e from a section through the anterior h a l f o f tectum seen at right and a section through the posterior half seen at left. Note the normal labeling in anterior tectum a n d the relative absence o f label posteriorly, b: fish from group 2 but o f left tectum innervated bv the intact ~uncrushed / fight nerve. T h e fight eye had been given a blocking dose o f T T X until nasal sector m a p p i n g at 90 d a w The denervation is c o m p a r a b l e to that seen previously from intact nerves t3
297 in which refined retinotopography forms. A fourth group of 2 fish was treated identically to the third group but allowed to survive for an additional 24 days without further TTX injections. Sector mapping at 104 days yielded a well-defined denervated sector in posterior tectum (Fig. ld). Apparently, TTX does not eliminate the capacity of fibers to form retinotopography but indefinitely inhibits its expression. In all but 3 of the above fish, the contralateral eye (nerve intact) was also injected with the same dosage and schedule of TTX as the eye with nerve crush and was similarly mapped. Without exception, a clear denervated sector was observed in, and only in, the area oftectum corresponding to the retinal mapping lesion (Fig. 2b). While some encroachment of inappropriate fibers into this sector cannot be ruled out, it was clear that TTX did not lead to a massive degradation of intact retinotopography, nor did the TTX injections produce detectable denervation. There was no hint that TTX adversely affected the autoradiographic mapping technique, particularly the labeling of fibers of passage. In many cases, the retinal mapping corresponded to anterior, lateral or medial tectum through which numerous fibers course. A clear zone of denervation with very light labeling of fibers of passage was observed. This is in line with previous work indicating that TTX has no effect on axoplasmic transport or on synaptogenesis 2,8. Finally, retinal histology on 12 representative eyes revealed no signs of pathology from the injections. The results here demonstrate that the formation of refined retinotopography can be selectively blocked pharmacologically. This implies the involvement of at least 2 cellular processes in the patterning of neural connections: a TTX-in-
sensitive process responsible for gross retinotopography and a TTX-sensitive process responsible for refined retinotopography. Regardless of the mechanism of action of TTX and the precise cellular basis of the inhibited refined retinotopography (e.g. abnormally large but appropriately positioned arbors vs normal sized but inappropriately positioned arbors), a pharmacological distinction between 2 aspects of regeneration is a significant finding and useful probe for further studies. These two aspects may correspond respectively to fiber-tectum chemoaffinity which could produce gross retinotopography by a selective fiber-tectum interaction 21 and to the socalled fiber-fiber interaction where fibers terminate next to their retinal neighbors independently of tectal loci6. The latter may also mediate the formation of ocular dominance columns in cat cortex and goldfish tectum which is also prevented by TTX 16,20. It is highly likely, however, that the inhibition of refined retinotopography is a direct consequence of eliminating impulse activity. TTX binds and blocks the voltage-dependent sodium channel in a highly selective fashion TM. Retinal ganglion cells in goldfish are known to exhibit correlated activity even in the dark t. These and theoretical considerations 22 lead to the suggestion that refined retinotopography is generated according to the rule that fibers that fire together, terminate together. The cellular mechanism of this may be similar to the TTX-sensitive process in the neuromuscular system, synapse elimination '3, in this case one that selects for fibers with correlated activity.
1 Arnett, D. W., Statistical dependence between neighboring retinal ganglion cells in goldfish, Exp. Brain Res., 32 (1978) 49-53. 2 Anderson, K. E. and Edstrom, A., Effects of nerve blocking agents on fast axonal transport of proteins in frog sciatic nerves in vitro, Brain Res., 50 (1973) 125-134. 3 Chow, K. L. and Spear, P. D., Morphological and functional effects of visual deprivation on the rabbit visual system, Exp. Neurol., 42 (1974) 429-447. 4 Chung, S. H., Gaze, R. M. and Stirling, R. V., Abnormal visual function in Xenopus following stroboscopic illu-
mination, Nature New Biol., 246 (1973) 186-189. 5 Duffy, F. H., Snodgrass, S. R., Burchfel and Conway, J. L., Bicuculline reversal of deprivation ambylopia in the cat, Nature (Lond.), 260 (1976) 256--257. 6 Gaze, R. M:, The Formation of Nerve Connections, Academic Press, New York, 1970. 7 Gaze, R. M. and Keating, M. J., Further studies on the restoration of the contralateral retinotectal projection following regeneration of the optic nerve in the frog, Brain Res., 21 (1970) 183-195. 8 Harris, W. A., The effects of eliminating impulse activity
This work was supported by PHS Grant NS 15381.
298
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on the development of the retinotectal projection in salamanders, J. comp. NeuroL, 194 ( 19801303 317. Hubel, D. H., Wiesel, T. N. and Le Vay, S., Plasticity of ocular dominance columns in monkey striate cortex. Phil Trans. Roy. Soc. B, 278 (1977) 377 -409. Jacobson, M. and Hirsch, H. V. B., Development and maintenance of connectivity in the visual system of the frog. I. The effects of eye rotation and visual deprivation, Brain Res., 49 (1973) 47-- 65, Keating, M. J. and Feldman, J. D., Visual deprivation and intertectal neuronal connections in Xenopus laevis, Proc. roy, Soc. B, 191 (1975) 467 -474. Kratz, K. E., Spear, P. D. and Smith, D. C., Postcritical period reversal of effects of monocular deprivation on striate cortex cells in the cat. J. Neuropt~vsioL, 39 (19761 501--511. Lomo, T. and Janse, J. K. S., Requirements tbr the ti)> mation and maintenance of neuromuscular connections, Curr. Top. Develop. BioL, 16 (19801253 281. Meyer, R. L., Mapping the normal and regenerating retinotectal projection of goldfish with autoradiographic methods, J. cornp. NeuroL, 189 (1980) 273 289. Meyer, R. L., 'Ocular dominance' columns in goldfish,
16 17
18
19
20
21
22
ontogeny and effect of visual envirimment, Soc, Neuro,vci. Abstr., 7(1981)405. Meyer, R. k., Tetrodotoxin blocks the formation of ocular dominance columns in goldfish. Science; in press. Rakic, P., Prenatal development of the visual system in rhesus monkey, Phil Trans. roy. So(. B, 278 (19771 245 260. Ritchie, J, M., A pharmacological approach to sodium channels in myelinated axons. Ann. Rev. Neurosci., 2 (19791 341 362. Shatz, C. J. and Stuker, M. P,. Ocular dominance in layer 1V of the cat's visual cortex and the effects of monocular deprivation, J. Physiol. (Lond.), 28t (19781 267 283. Stryker, M. P., Late segregation ofgeniculate afferents to the cat's visual cortex after recovery from impulse blockade, Soc. Neurosci. A bstr., 7 ( 1981 ) 842. Sperry, R. W., Chemoaffinity in the orderly growth o l nerve fiber patterns and connections. Proc. nat Acad, Sci. U.S.A,, 50(19631703 710. Whitelaw, V. A. and Cowan, J. D., Specificity and plasticity of retinal connections: a computational model, J. Neurosci., l (1981)1369 1387.
Developmental Brain Research, 6 (1983) 293- 298 Elsevier Biomedical Press
Short Communications
Tetrodotoxin inhibits the formation of refined retinotopography in goldfish RONALD L. MEYER
Developmental and Cell Biology, DevelopmentalBiology Center, Universityof California, lrvine, CA 92717(U.S.A.) (Accepted September 7th, 1982)
Key words: goldfish - retinotectal - tetrodotoxin - regeneration - topography
One optic nerve of mature goldfish was crushed in the orbit and allowed to regenerate. During regeneration impulse activity was eliminated by periodic intraocular injections of tetrodotoxin (TTX). At 32-104 days, the retinotopography of the retinotectal projection was measured autoradiographically by intraocular [3H]proline injections simultaneous with either small (10-20 ° sector) or half retinal mapping lesions. TTX had no effect on the time-course and quality of the regeneration of gross topography seen with half retina mapping, but indefinitely inhibited higher order (refined) retinotopography normally seen by 2 months with retina sector mapping.
In development, neurons of the central nervous system (CNS) send out axons to form highly ordered connections onto target cells. Both the nature and n u m b e r of mechanisms which organize these connections is a subject of continuing debate. One candidate is neuronal impulse activity. In several systems, most notably the visual system, reduced or altered sensory stimulation has been reported to lead to a variety of developmental changes 4.5.9,t~.t9. However, the anatomical substrate, interpretation, and generality of these changes remain unclear. For cortical ocular dominance columns for example, there is anatomical evidence for altered connectivity following monocular deprivation 9,~9. However, the rapid recovery of cortical responsivity to the deprived eye such as following enucleation of the undeprived eye ~2or administration of bicuculline~ suggests altered synaptic efficiency rather than connectivity. Also, all the evidence for activity effects in CNS connectivity comes from developing systems. The inherent complexity of developmental interactions and multiplicity of developmental events make it difficult to know which are the relevant ones. Mitosis, cell differentiation and growth, cell death as well as D165-3806/83/0000-0000/$03.00 ©1983 Elsevier Biomedical Press
selective axon growth are often confounded. Finally, there is a serious question whether activity plays a universal role. Some species seem immune to abnormal stimulation 3,m and in susceptible species many parts of the CNS such as the topography of the primary visual projections are unaffected 9.~1. It is possible that the only instructive role played by activity is limited to highly specialized neural systems such as those concerned with stereopsis. In this light, the retinotectal topography formed by regenerating optic fibers in mature goldfish is perhaps an unlikely place to look for activity-dependent changes. Anatomical organization rather than synaptic function is directly measured. Retina and tectum are already differentiated. The projection is a primary sensory one, generally thought to be resistant to activity effects. The system has no known binocular convergence necessary for stereopsis. Constant dark, continuous light and strobe illumination have no reported effect on topography 4.14. In developing urodeles, elimination of impulse activity with tetrodotoxin (TTX) did not prevent optic fibers from forming a retinotopic projection and anatomically normal synapses s. On the other hand, if an effect of activi-
294 ty were to be demonstrated, it would argue ti)r a pervasive and fundamental role for activity in the genesis of orderly connections, would extend such an activity effect to a regenerating system and would offer a valuable model system. There was some hope this would be the case. In the urodele studys, retinotopography was not measured with high precision and in no other study showing negative results was activity entirely eliminated during the period of axon ingrowth. Previous experiments in goldfish and frog had shown that regenerating optic fibers rapidly form a projection that is grossly retinotopic but only later forms one with refined retinotopography v.H. It seemed possible that this later stage might utilize activity-dependent mechanisms and be sensitive to TTX. Common goldfish, 5 7 cm in body length, were maintained in standard aquaria at 19 20°C, Under tricaine methanesulfonate anesthesia one optic nerve was crushed in the orbit as previously described% For chronic blockade, 0.1/,t of 0.3 mM TTX (10 times the minimum acute blocking dose) was injected every 3.5 days by means of a 25 ~m glass pipette inserted through the dorsal limbus. By extracellular recordings in tectum or nerve, this dose was found to block visually evoked action potentials in intact or regenerating nerves, including those injected for 31 90 days and recorded at 3.5 4 days from the last TTX injection (32 different nerve recordings), in large fish injected for 31 90 days (4 cases) weak recovery could be detected as early as 4 days, and in all cases recovery was found at 5 days. An injection of 0.1 t*l of 0.01 mM TTX had no apparent effect and was used as a "subthreshold' control. The anatomical technique used to determine the retinotopography of the projection was identical to that of a previous regeneration study ~4. At the end of each experiment a small area of retina, about 750 1000/xm in diameter and about a third to halfway out from the disc, was lesioned by inserting a needle through the sclera and passing rf current. Both ganglion cells and optic fibers from peripheral retina coursing through this region were destroyed. Tritiated proline (25-50 t*Ci) was immediately injected into the eye followed by autoradiography at
about 18 h later. In normal fish without nerve crush, it was previously' reported that silxer grains were normally distributed throughout rectum except for a denervated sector in the tectal region corresponding to the a c u t e lesion ~. In effect, all optic fibers were labeled except for those in the lesioned retinal sector. This procedure will be referred to as "sector mapping'. When done at various times iollowing nerve crush, it was previously l'ound ~" that no zone of denervation was detectable in rectum at up to 40 days postoperatively, indicating that fibers initially lacked a high degree of retinotopography. However, gross topography existed at 40 days as shown by 'hemiretina mapping'., i.e. acute retinal lesions of half of" retina. At 80 days, sector mapping yielded a denervated sector at the appropriate place in the rectum, indicating the retinotopography had become more highly ordered. Some light label remained in the denervated sector and could be attributed to fibers of passage, since many fibers were shown to follow anomalous paths in regeneration. In the present study, nasal mapping lesions (corresponding to posterior tectum) were usually chosen since the fewest fibers of passage are found in posterior rectum. This progressive development of retinotopography has also been seen with etectrophysiological mapping:. Four groups of fish were treated with TTX following crush of the left nerve. The first group (4 fish) was given subthreshold TTX iniections from the time of nerve crush until autoradiography at 80 days. This injection protocol had no dectectable effect on regeneration, that is a denervated sector in posterior rectum was observed and all aspects of innervation were typical of normal regeneration (Fig. la). In the second group, a blocking dose was administered from the time of crush to autoradiography at 32 40 days (6 fish), 66 days (1 fish), 80 days (4 fish), and 90 days (4 fish) (Fig. lb). At 32 40 days, autoradiography was indistinguishable from normal regeneration. Specifically, fibers were within the correct layers and extended to the most posterior regions of tectum where much of the label was string-like in appearance, presumablx. rel]ecting fasciculatioc~ ~ -\xoplasmic
295
b
a
O
,k,,
t
c
"
d
Fig. 1. Autoradiograms of right tectum following sector map lesion to left nasal retina. Left optic nerve was previously crushed. Sections are frontal in orientation (medial to the right) and near the posterior end of tectum. Calibration bar is 400/zm. a: fish from group I in which subthreshold TTX was administered until sector mapping at 80 days after crush• The denervated zone seen in the mediolateral region of the main optic layer is equivalent to fish receiving no ocular injections, b: fish from group 2 in which blocking dose of TTX was given until sector mapping at 90 days. No denervated sector is evident, c: fish from group 3 in which a final blocking dose of TTX was given from day 42 until autoradiography at 80 days. No denervated tectal sector can be seen. d: fish from group 4 in which a blocking dose of TTX was given only from 42 to 81 days followed by autoradiography after an additional 24 days without TTX. A denervated tectal sector is evident.
296 transport (grain density) was distinctly elevated as compared to intact fibers (also labeled as mentioned below), and no denervated sector was detected. At 66 90 days, the overall characteristics of tectal labeling was also typical of normal regeneration. Label was no longer 'fasciculated' but had become homogeneous and axoplasmic transport had returned to nearly normal levels in the 8 ~ 9 0 day fish. Gross topography was demonstrated by nasal hemiretinal mapping (nasal lesion) at 40 days (3 fish), 66 days ( 1 fish), and 90 days (2 fish). In these, dense label was confined to the anterior half of tectum with little or none in the posterior half (Fig. 2a). In the nasal sector-mapped fish, the only apparent difference from normal regeneration was that no denervated sector could be observed with
a
sector mapping. A slight decrease in overall labeling was just detectable in the posterior third of tectum indicating fibers from the lesioned nasal sector were distributed throughout this region. Exactly the same result was observed in a third group (5 fish) in which T r x blockade was initiated at 42 days until autoradiography at 81 days (Fig. lc) . Again, no refined retinotopography could be demonstrated with sector mapping (4 fish) but gross retinotopography was obvious with hemiretina mapping (I fish). Thus, TTX need only be given for 39 days during the period in which refined retinotopography is forming. This argues against a cumulative toxicity in the second (continuous TTX) group, i.e, that toxicity took 40 days or more io develop, a time that just happened to correspond to the period
.
b ".
d
. LLL~LL~_jLIi
~
.............
Fig. 2. A u t o r a d i o g r a m s o f posterior tectum as in Fig. 1. a: fish from group 2 in which a blocking dose o f TTX was given untd nasal h e m i r e t i n a m a p p i n g (actually about 120 ° o f retinal arc) at 90 days. T h e p h o t o m o n t a g e is m a d e from a section through the anterior h a l f o f tectum seen at right and a section through the posterior half seen at left. Note the normal labeling in anterior tectum a n d the relative absence o f label posteriorly, b: fish from group 2 but o f left tectum innervated bv the intact ~uncrushed / fight nerve. T h e fight eye had been given a blocking dose o f T T X until nasal sector m a p p i n g at 90 d a w The denervation is c o m p a r a b l e to that seen previously from intact nerves t3
297 in which refined retinotopography forms. A fourth group of 2 fish was treated identically to the third group but allowed to survive for an additional 24 days without further TTX injections. Sector mapping at 104 days yielded a well-defined denervated sector in posterior tectum (Fig. ld). Apparently, TTX does not eliminate the capacity of fibers to form retinotopography but indefinitely inhibits its expression. In all but 3 of the above fish, the contralateral eye (nerve intact) was also injected with the same dosage and schedule of TTX as the eye with nerve crush and was similarly mapped. Without exception, a clear denervated sector was observed in, and only in, the area oftectum corresponding to the retinal mapping lesion (Fig. 2b). While some encroachment of inappropriate fibers into this sector cannot be ruled out, it was clear that TTX did not lead to a massive degradation of intact retinotopography, nor did the TTX injections produce detectable denervation. There was no hint that TTX adversely affected the autoradiographic mapping technique, particularly the labeling of fibers of passage. In many cases, the retinal mapping corresponded to anterior, lateral or medial tectum through which numerous fibers course. A clear zone of denervation with very light labeling of fibers of passage was observed. This is in line with previous work indicating that TTX has no effect on axoplasmic transport or on synaptogenesis 2,8. Finally, retinal histology on 12 representative eyes revealed no signs of pathology from the injections. The results here demonstrate that the formation of refined retinotopography can be selectively blocked pharmacologically. This implies the involvement of at least 2 cellular processes in the patterning of neural connections: a TTX-in-
sensitive process responsible for gross retinotopography and a TTX-sensitive process responsible for refined retinotopography. Regardless of the mechanism of action of TTX and the precise cellular basis of the inhibited refined retinotopography (e.g. abnormally large but appropriately positioned arbors vs normal sized but inappropriately positioned arbors), a pharmacological distinction between 2 aspects of regeneration is a significant finding and useful probe for further studies. These two aspects may correspond respectively to fiber-tectum chemoaffinity which could produce gross retinotopography by a selective fiber-tectum interaction 21 and to the socalled fiber-fiber interaction where fibers terminate next to their retinal neighbors independently of tectal loci6. The latter may also mediate the formation of ocular dominance columns in cat cortex and goldfish tectum which is also prevented by TTX 16,20. It is highly likely, however, that the inhibition of refined retinotopography is a direct consequence of eliminating impulse activity. TTX binds and blocks the voltage-dependent sodium channel in a highly selective fashion TM. Retinal ganglion cells in goldfish are known to exhibit correlated activity even in the dark t. These and theoretical considerations 22 lead to the suggestion that refined retinotopography is generated according to the rule that fibers that fire together, terminate together. The cellular mechanism of this may be similar to the TTX-sensitive process in the neuromuscular system, synapse elimination '3, in this case one that selects for fibers with correlated activity.
1 Arnett, D. W., Statistical dependence between neighboring retinal ganglion cells in goldfish, Exp. Brain Res., 32 (1978) 49-53. 2 Anderson, K. E. and Edstrom, A., Effects of nerve blocking agents on fast axonal transport of proteins in frog sciatic nerves in vitro, Brain Res., 50 (1973) 125-134. 3 Chow, K. L. and Spear, P. D., Morphological and functional effects of visual deprivation on the rabbit visual system, Exp. Neurol., 42 (1974) 429-447. 4 Chung, S. H., Gaze, R. M. and Stirling, R. V., Abnormal visual function in Xenopus following stroboscopic illu-
mination, Nature New Biol., 246 (1973) 186-189. 5 Duffy, F. H., Snodgrass, S. R., Burchfel and Conway, J. L., Bicuculline reversal of deprivation ambylopia in the cat, Nature (Lond.), 260 (1976) 256--257. 6 Gaze, R. M:, The Formation of Nerve Connections, Academic Press, New York, 1970. 7 Gaze, R. M. and Keating, M. J., Further studies on the restoration of the contralateral retinotectal projection following regeneration of the optic nerve in the frog, Brain Res., 21 (1970) 183-195. 8 Harris, W. A., The effects of eliminating impulse activity
This work was supported by PHS Grant NS 15381.
298
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12
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