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Retinotectal reorganization in goldfish—III. Effect of thyroxine
Neuroscience Vol. 6, No. 8, pp. 1591 to 1600, 1981 Printed in Great Britain
RETINOTECTAL
03oa-4522/s1/0s1591-10~2.~/0 Pergamon Press Ltd 6 1981 IBRO
REORGANIZATION IN GOLDFISH-III. EFFECT OF THYROXINE
L. R. MAROTE, R. F. MARK and J. WYE-DVORAK Department of Behavioural Biology, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra, ACT, Australia Abstract-Reorganization of the goldfish retinotectal projection after ablation of half the tectum is dependent on lighting conditions and season. It occurs in summer but not in winter in normal lighting conditions. Constant light prevents reorganization in summer. Treatment with thyroxine at times when it does not normally occur results in some reorganizatioh of the projection in a majority of cases. This consists of reduplicated receptive fields and partial compression. The ability of thyroxine to induce reorganization is not related to the amount of optic fibre sprouting it produces, suggesting that it may be acting on synapse formation. A hormonal influence on reorganization has been demonstrated and this may underlie the effects of season and lighting conditions, which are both known to be associated with changes in hormone levels in fish.
THE OPTIC tectum of the goldfish receives a topo-
This appears to be related to a loss of ganglion cells
graphic projection from the retina (JACOBSON& GAZE, 1964; SCHWASSMANN & KRUGER, 1965). This projection may reorganize after partial tectal ablation so that the entire visual field again becomes rep resented on the remaining tectum (GAZE & SHARMA, 1970; YOON,1971; SHARMA,1972). In the case of removal of the caudal half of the tectum this reorganization involves the shifiing forward of optic terminals projecting to the rostra1 half which were left intact by the operation and the ingrowth and formation of synapses of those axons cut during the operation. The entire projection is reproduced in the correct retinotopic order, but compressed into half the normal rostrocaudal space on the tectum. Several factors have been found to affect this process of reorganization. YOON(1975) showed that placing the fish in constant light after the operation indefinitely delayed compression after half tectal ablation. Initially, we (MARo~, WYE-DVORAK& MARK, 1977) and others (MEYER& SCOTT,1977; HORDER& MARTIN, 1977) were unable to confirm this. Later work in our laboratory demonstrated that there were effects of season and size of fish, as well as lighting conditions, on retinotectal reorganization (WYE-DVORAK,MAROTTE8z MARK, 1979). Compression of the visual projection onto a rostra1 half tectum occurred in summer but not during winter. Furthermore, the effect of constant light on compression depended on the size of fish. Using fish of similar size (greater than 6cm in length) to those used by YOON(1975) and taking into account the seasonal effect, we were able to confirm his result. Curiously, constant light had the opposite effect in smaller fish in that it caused compression of the retinotectal projection in a proportion of cases.
in the retina projecting to the operated tectum through exposure to constant light (MAROI-IE, WYEDVORAK& MARK, 1979), an effect seen on small but not on large operated fish. We suggested that the seasonal failure of reorganization might point to an hormonal influence (WYEDVORAKet al., 1979). Seasonal hormonal fluctuations in fish are well documented (HOAR, 1957). Furthermore, constant light is known to affect hormone levels (C~ITCHLOW,1963) suggesting that it could also affect reorganization by a hormonal mechanism, rather than by a direct effect on the activity of retinal ganglion cells, thus preventing their rearrangement as suggested by YOON(1975). In this paper, we describe attempts to induce retinotectal reorganization by changing hormonal levels at times when reorganization does not normally occur; during winter in normal lighting conditions and during summer in constant light conditions. Thyroxine was used because of its known influence on the development of the nervous system (see GRAVE,1977) and its known seasonal fluctuations in fish (WHITE & HENDERSON, 1977).
Abbreviations: SFGS, stratum superficiale; SO, stratum opticum.
fibrosum
et griseum
EXPERIMENTAL PROCEDURES The caudal half of the left tectum was removed from 48 goldfish (Carassius auratus) 6.5-9.0cm in body length, under anaesthesia with ethyl m-aminobenzoate methane sulphonate (MS222 Sigma) as described in a previous paper (WYE-DVORAKet al., 1979). Nineteen fish, operated in winter, were kept on a 12 h light/l2 h dark cycle. Ten received 0.006mg of 3,3’,4,5’-tetraiodo-L-thyroxine (sodium salt, pentahydrate, Sigma) per litre of aquarium water, replaced every two to three. days, and nine served as untreated controls. The retinotectal projections were mapped electrophysiologically 35-65 days after operation and the tecta prepared for elec-
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L. R. MAROTTE,R. F. MARK and J. WYE-DVORAK
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tronmicroscopy as previously described (WYE-DVORAKet al., 1979). The controls were mapped within 1 day of corresponding fish treated with thyroxine. Fifteen fish operated in autumn were kept on a light/ dark cycle. Seven received 4 mg/litre of thyroxine replaced fortnightly and eight served as untreated controls. Projections were mapped 41-65 days later, with corresponding controls being mapped within 3 days of the fish treated with thyroxine. In nine cases (seven thyroxine-treated and two untreated fish) the tecta were prepared for electronmicroscopy for the analysis of tectal structure. The five controls not prepared for electronmicroscopy were replaced by controls obtained from a previous series (WYEDVORAKer al., 1979) operated in late summer and with similar post-operative survival times. Fourteen fish were operated in late spring and kept in constant light (luminance of aquaria floors 0.15 Ig ft iamberts and that of the walls 0.24 1 g ft lamberts). Half received 4 mg/litre of thyroxine replaced fortnightly and half served as untreated controls. Projections were mapped 48-99 days later during summer, with controls mapped within two days of the corresponding thyroxine-treated fish. The temperatures of the aquaria ranged from 19-22°C. That immersion of goldfish in water containing thyroxine is effective in elevating plasma thyroxine levels has been demonstrated by HURLBURT(1977). The lower concentration we used was effective in producing behavioural changes in goldfish (HOAR, KEENLEV~IDE& GOODALL, 1955) while the higher concentration, extrapolating from Hurlburt’s data, would produce a large elevation in plasma levels. Analysisof tectal structure The dorsal surface of the operated half tectum was divided into samples from the most rostra1 area and the area adjacent to the cut edge (termed midline in subsequent text and Figures). The unoperated tectum on the opposite side was either similarly divided into such samples, or several samples were taken from the entire dorsal surface of the tectum. The amount of axonal sprouting in the stratum opticum (SO) was measured in 1 pm Araldite sections cut perpendicular to the tectal surface and stained with toluidine blue. A drawing of the SO at a magnification of x 400 was made with the aid of a camera lucida, marking those areas containing myelinated and unmyeiinated axons. The percentage area occupied by unmyelinated axons was measured
using a Kontron MOP-AM03 image analyser. Adjacent ultra-thin sections stained with uranyl acetate and lead citrate were examined in a Hitachi 500 electronmicroscope. The results were analysed statistically using the Wilcoxon Matched-Pairs Signed-Ranks test. RESULTS Electrophysiological analysis
Under conditions in which previous work had shown an inhibition of reorganization of the retinotectal projection, treatment with thyroxine induced some reorganization of the projection in a proportion of cases. The effects observed were partial compressions of the projection and the presence of reduplicated receptive fields for single points on the tectum. The results are summarized in Table 1 and the detailed results for each group are set out below. Low dose thyroxine: light/dark conditions in winter. After removal of the caudal half tectum in winter we have previously reported that there is no reorganization of the retinotectal projection (WYE-DVORAK et al., 1979). We therefore compared pairs of fish which had survived for similar periods after operation, with one of the pair kept under normal conditions and the other under identical conditions except for the addition of thyroxine to the water. As expected, operated fish which received no thyroxine all had uncompressed retinotectal projections. Figure 1 shows three maps of the retinatectal projection made from control fish and one from the treated group. Figure 1A is a normal map of the left visual field onto the right tectum of an unoperated fish. Below, in Figs 1C and 1D, are maps made from a fish that had survived 64 days after operation untreated with thyroxine. Figure 1C shows the projection mapped immediately after ablation of its cau&l half of the right tectum. Comparison with Fig. 1A above shows the absence of representation of the temporal visual field. Figure 1D shows the map made from the left tectum of this fish in which the caudal half tectum had been removed in the same way 64 days previously. The map is a mirror image of that from the normal half tectum, with a scotoma of the
TABLE1. SUMMARYOFTHEEFFECTS OFTHYROXINE ON REORGANIZATIOH OFTHERETIPI’OTECTAL PROJECTION
Season of year and lighting conditions L/D winter 0.006 mg/l Thyroxine L/D au-/winter 4 m0/1 Thyroxine CL summer 4 rnd Thyroxine
Proportion of fish with partial compression or reduplicated fields Operatedand Operated Thyroxine
Time between operation and electrophysiological mapping
8110
OP
35 to 67 days
6/7
218
41 to 65 days
317
017
48 to 99 days
L/D: 12 h Ii&t/12 h dark cycle CL: constant light. Concentrations of thyroxine refer to the amount per litre of aquarium water.
Thyroxine effects on retinotectal reorganization
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the temporal field than would be expected. Points 2 and 6 further rostrally on the tectum also have receptive fields displaced temporally. The most striking feature of the map is the presence of many tectal points with reduplicated receptive fields: that is, points on the tectum which receive an input from two points in the visual field. The reduplicated field was always to the temporal side of and clearly separated from the primary receptive field. The map from the untreated control of this pair showed no signs at all of reorganization of the projection, the size and position of the scotoma being the same as the examples in Fig. 1. Of the remaining seven fish in this group, three had reduplicated receptive fields and four showed partial compression. Signs of reorganization in treated fish were seen at the earliest post-operative time of 35 days, The lack of effect of thyroxine in two of the fish did not appear to be related to the duration of treatment as these two survived 64 and 67 days, the longest post-operative times. High dose thyroxine: light/dark conditions: autumn to winter. Of the untreated operated controls two out
FIG. 1. A. Retinotectal projection of the left visual field onto a normal right tectum. The numbers on the camera lucida drawing of the dorsal surface of the tectum are recording sites and the corresponding numbers on the perimetric chart of the visual field are the locations of the receptive fields at each recording site. B. Retinotectal projection of a fish kept in light/dark conditions and receiving a low dose of thyroxine, showing no reorganization 64 days post-operatively. C and D. Projections of the untreated operated controls for the fish whose projections are shown in B. Maps were made 64 days post-operatively. ‘Midline’ labels the cut edge of the tectum in all maps. C. Projection to the right tectum mapped immediately after removal of the caudal half. D. Projection to the left half tectum. An equivalent area of temporal visual field is missing in maps ED. same size and position indicating that there had been no reorganization of the projection. This may be compared with the map in Fig. 1B above which is from one of two fish in this series in which treatment with thyroxine after operation had no effect on the retinotectal projection. The scotoma is of the same size and shape as in the control of 64 days and the control mapped immediately after operation. With these results as a baseline, eight out of ten fish treated with thyroxine gave maps which clearly indicated some progress towards remodelling of the retinote&al projection. Figure 2 shows on the left a map from one of these made 51 days after ablating the caudal half of the left tectum. For comparison, on the right is the map of an untreated fish kept in the same conditions for 52 days. The map from the treated fish shows partial compression in that points 3 and 7 from the caudal tectum have receptive fields further into
of eight showed signs of retinotectal reorganization, in the form of reduplicated fields (two in one, and four in the other) at 49 and 51 days post-operatively. Fish mapped both before and after these times up to 65 days post-operatively all had uncompressed projections (Fig. 3). In those fish receiving thyroxine postoperatively six out of seven showed some retinotectal reorganization. Four had two reduplicated fields each (Fig. 3). One of these also showed signs of partial compression with points near the cut midline receiving a projection from further temporally in the visual field than normal. Of the two remaining, one had two points on the tectum where instead of the normal receptive field size of about 5”, activity could be evoked from regions 12 and 32” across, the centres of
Wl-
FIG. 2. On the left is the retinotectal projection of a fish in light/dark conditions for 51 days after operation and receiving a low dose of thyroxine. Extensive reorganization in the form of reduplicated fields and partial compression is present. On the right is the projection of its untreated control mapped 52 days post-operatively. The projection remains uncompressed. The half tectum of the fish treated with thyroxine is larger than that of the untreated control in this example. This was not a consistent finding.
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L. R. MAROTTE.R. F. MARK and J. WYE-DVORAK tions with one or two reduplicated fields near the cut midline (Fig. 5). Post-operative survival times of these were 56, 64 and 68 days. Structural analysis. Most of the optic fibres enter in
FIG. 3. On the left is the projection of a fish kept in light/ dark conditions receiving a high dose of thyroxine and mapped 49 days post-operatively. Two reduplicated fields are present. Its untreated control shown on the right and mapped 47 days post-operatively, is uncompressed.
each field being more temporal in the visual field than normal (Fig. 4). The other had four reduplicated fields and one slightly enlarged receptive field of 12”. The only fish in which the projection remained unohanged after thyroxine treatment had the longest post-operative survival of 65 days. Presumably, changes were observed in two of the controls because the operation was done earlier in the season than the previous group. Nevertheless, thyroxine treatment was associated with a higher proportion of reorganizing projections. None of the projections was fully compressed. The effects were no more marked than with the lower dose of thyroxine. High dose thyroxine: constant light conditions in summer. Keeping Iarge fish in the summer in constant
light prevents remodelling of the retinotectal projection after ablation of half the tectum (WYE-DVORAK et al., 1979). As expected, all untreated operated fish had uncompressed retinotectal projections at all postoperative times studied (Fig. 5). Reorganization of retinotectal projections in thyroxine-treated fish in these lighting conditions was less striking, with three out of seven fish having partially compressed projec-
FIG. 4. Projection of fish in light/dark conditions receiving a high dose of thyroxine and mqpal U days postoperatively. It is partially compressed with two extended
^..
recephve tields.
the SO in the upper part of the tectum (MAROTTE& MARK, 1975; MURRAY,1976) and comprise the majority of fibres in this layer (MAROTTE& MARK, 1975). Most synapse in the stratum fibrosum et griseum superficiale (SFGS) directly beneath the SO and a very few synapse in the stratum griseum centrale (SHARMA,1972; MAROTTE& MARK, 1975; MURRAY, 1976). In normal unoperated tecta in both light/dark and constant light conditions the percentage area of SO occupied by unmyelinated axons is extremely small, around 4% or less (Fig. 8). This did not change for unoperated tecta in fish treated with thyroxine. On removing the caudal half of the tectum those ganglion cells projecting to the caudal half have their axons cut. There is sprouting of optic fibres throughout the SO and SFGS. Small unmyelinated sprouts appear in the SO and unmyelinated axons bundles, not normally present, are found in the SFGS. All the operated tecta of fish in this study showed these features (Figs 6 and 7) as previously found in fish with reduplicated receptive fields and uncompressed or partially compressed retinotectal projections (MARom et al., 1977; WYE-DVORAKet al., 1979). Near the cut edge of the tectum the SO contained few myelinated axons, the majority being very small unmyelinated axons (Figs 6 and 103). In some fish almost no normal myelinated fibres remained and occasional degenerating myelinated axons and myelin debris within glial cells was present scattered amongst the myelinated fibres (Fig. 6). Sprouting in the SO is seen as an increase in the percentage area occupied by unmyelinated axons (Figs 9-14). The amount of sprouting seen varies, depending on thyroxine treatment, region of tectum sampled and season of year in which the operation was performed. Results were analysed by comparing corresponding tectal regions of thyroxine treated and untreated groups, rostra1 and midline regions within
FIG. 5. Projection of a fish in constant light conditions and treated with a high dose of thyroxine. mapped 68 days post-operatively. It is partially compressed with two reduplicated receptive fitI&. The projection of its untreated control shown on the right is uncompressed after 70 days.
FIG. 6. Stratum opticurn near the cut edge days after operation. The majority of fibres myelinated axons (arrows) and few normal of thyroxine but untreated
of the tectum in are unmyelinated myelinated fibres. fish also showed
a fish kept in light/dark conditions for 41 with some myelin debris and degenerating This fish had been treated with a low dose these features. Bar = 2 pm.
FIG. 7. Stratum fibrosum et griseum superficiale in a fish kept in light/dark conditions for 51 days after operation Bundles of unmyelinated fibres are present in the neuropii. Bar = 2 pm. 1.595
FIG. 8. A. Stratum opticum in an unoperated tectum showing extremely few pale areas of unmyelinated fibres (arrow). Oil immersion. Bar: 20pm for this and Figs 9911. B. Electron micrograph of a normal stratum opticum showing areas of unmyelinated axons amongst myelinated fibres. Part of a glia cell is seen on the lower left. Bar = 2 nm. FIG. 9. A. Stratum opticum rostrally in a fish kept in light/dark conditions for 51 days after operation and receiving a high dose of thyroxine. A small increase in area occupied by unmyelinated axons is seen. In this and subsequent micrographs asterisks mark the larger areas of unmyelinated axons. Two cell bodies are marked by the large arrow. B. Untreated operated control for A. There are more areas of unmyelinated axons. FIG. 10. A. Rostra1 region of stratum opticum 68 days after operation. B. Region near the cut edge in the same fish showing more extensive areas of unmyelinated fibres and fewer myelinated fibres. This fish had been kept in constant light and treated with thyroxine. FIG. 11. A. Rostra1 regton of stratum opttcum m fish operated on m autumn and kept in light/dark conditions for 51 days. Areas of unmyelinated axons are present. B. Rostra1 region of stratum opticum m fish operated on in winter and kept in lightidark for 57 days. Few unmyelinated axons arc present I596
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Thyroxine effects on retinotectal reorganization
FIG. 12. Histograms in this and subsequent figures show percentage area of unmyelinated axons in the stratum opticum of half tecta at various times after operation. Low dose of thyroxine and light/dark conditions. On the left are results for the rostra1 regions and on the right for the midline regions, i.e. areas adjacent to the cut edge, of tecta treated with thyroxine and untreated tecta. There are no differences in sprouting between treated and untreated tecta in either region.
each group and corresponding tectal regions of untreated light/dark groups. In Figs 12,13 and 14, we compare first the effects of thyroxine treatment in rostra1 and midline tectal regions with respective untreated control tectal regions. In those fish kept in post-operative light/dark conditions and receiving the lower dose. of thyroxine, there was no significant difference in the amount of sprouting in the SO in either the rostra1 or the midline regions of operated tecta when compared to their respective untreated operated control tecta (Fig. 12). However, in those fish kept in post-operative light/ dark conditions and treated with the higher dose of thyroxine there was significantly less sprouting (P < 0.02) in the rostra1 region of operated tecta compared with that seen in rostra1 regions of untreated control tecta (Figs 9 and 13). Sprouting in midline regions in treated and untreated fish did not differ significantly (Fig. 13). In fish treated with thyroxine and kept in postoperative constant light conditions there was no significant difference in the amount of sprouting in the SO of either rostra1 or midline tectal regions when compared to corresponding regions of untreated operated controls (Fig. 14).
FIG. 14. Low dose of thyroxine and constant light conditions. On the left are results for rostra1 regions and on the right for the midline regions of treated and untreated tecta. There are no differences in sprouting between treated and untreated tecta in either region.
To summarize, the only significant effect of thyroxine on sprouting in the SO was a decrease in the rostra1 tectum in the group treated with the high dose of thyroxine and kept in light/dark conditions. Regional differences in sprouting in fish under various conditions can also be compared. Comparing the amount of sprouting in the SO in the rostra1 and midline regions within fish, in the group receiving the low dose of thyroxine and post-operative light/dark conditions there was significantly less sprouting rostrally (P < 0.02, Fig. 12, open columns). This was also true of its untreated control group (P < 0.01, Fig. 12, filled columns). In the group treated with the higher dose of thyroxine, there was significantly less sprouting in SO rostrally (P < 0.05, Fig. 13, open columns). Its untreated control group showed no significant difference in sprouting between rostral and midline tectal regions (Fig. 13, filled columns). In the thyroxine treated group kept in constant light conditions, there was significantly less sprouting in the SO rostrally (P < 0.02, Figs 1OA and B, 14, open columns) while in its untreated control group there was no significant difference in sprouting between rostra1 and midline regions (Fig. 14, filled columns). There was also a difference in the amount of sprouting in the SO in the two control groups of fish kept in light/dark conditions and which were operated on at different times of the year (Figs 1IA and B). The rostra1 region of the group operated on in winter (Fig. 12, filled columns) had significantly less sprouting in the SO than the group operated on during late summer and autumn (Fig. 13, filled columns, P < 0.05). Sprouting in midline regions did not differ significantly. DISCUSSION
FIG. 13. High dose of thyroxine and light/dark conditions. On the left are results for the rostra1 regions and on the right for the midline regions of treated and untreated tecta. There is less sprouting rostrally in tecta treated with thyroxine.
Thyroxine administration is able to induce reorganization of the retinotectal projection at times when, because of seasonal effects or lighting conditiona it does not normally occur. The failure of reorganization does not seem to be due to a failure of sprouting of retinal ganglion cell axons. Teeta with uncompressed retinal projections
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L. R. MAROON,R. F. MARKand J. WYE-DVORAK
always show an increase in the number of very small unmyelinated axons in the SO and bundles of them in the SFGS where they are not normally seen (WYEDVORAKet al., 1979). Although a natural seasonal effect on this sprouting was demonstrated in rostra1 tectum, with more sprouting in untreated fish operated on in late summer or autumn than in winter, this was not correlated with the ability to undergo reorganization of the projection, Neither of these groups showed any significant reorganization. Treatment with thyroxine had no measurable stimu~t~g effect on sprouting. Rather, for those fish receiving the high dose of thyroxine and kept in light/dark conditions there were fewer unmyelinated axons in the rostra1 part of the SO in these fish compared to their untreated operated controls. Similarly, all groups of fish treated with thyroxine had less sprouting in rostral SO than near the cut margin, while two out of three untreated groups showed no significant difference in sprouting in these two regions. Nevertheless, treatment with thyroxine induced electrophysiological signs of reorganization of the retinotectal projections in operated tecta under both conditions in which this normally does not happen, that is in a normal light, dark regime in the winter and in constant light during the summer. In the latter situatian, thyroxine was however less successful, In none of the cases was the reorganization complete with the whole of the visual field represented on the rostra1 half teetum. The most common effect of thyroxine was in producing reduplicated receptive flelds. These are commonly seen during the reorganization process, particularly when the caudal half of the teo turn is removed without crushing or cutting the optic nerve innervating that tectum (GAZE & SHARMA, 1970; HORDW & MARTIN, 1977; M~OTTE et al., 1977). Part of the reason for the failure to induce complete compression may have been that the fish were kept for at most 65 to 99 days after operation. Reorganization under the natural conditions in which it occurs may take longer. The ultr~t~~tur~ analysis shows that althou~ thyroxine is able in other situations to promote sprouting of cut sensory and motor axons (EHRLICH & MARK, 1977) this is not the mechanism of its action on this system. Another level at which it may be exerting its effect is on the formation and maturation of synaptic connections between growing axonal sprouts of retinal ganglion cells and tectal cells. Added thyroxine is known to promote maturation of the brain and synaptogenesis in particular. Excess synapses are produced in the cerebellum of new-born rats given thyroxine (LEORAND,1979) and conversely CRAGG (1970) has demonstrated a reduction in the number of synapses in the visual cortex of hypothyroid rat pups. The physiology of the retinotectal pathway in goldfish is also in3uemxtci by exogermus thyroid hormone. After some nine days of exposure to the hormone, extracellularly-recorded field potentials evoked in the tectum by flashes of light showed
reduced latency and increased amplitude (HARA, UEDA & GORBMAN,1965). Thiourea had opposite effects (HARA, GORBMAN & UEDA, 1966). The possibility that nerve growth factor might be involved in these changes has been raised by the finding of WALKER,WEICHSEL,FISHER, Guo & FISHER (1979) that thyroxine increases the concentration of nerve growth factor in mouse brain. They suggest that thyroid hormone may interact with nerve growth factor to promote axonogenesis and possibly synaptogenesis. Recently nerve growth factor has been shown both’to be present in high concentrations in goldfish tectum and to have an effect on optic nerve regeneration. BENOWITZ& SHASHOUA(1979) demonstrated immunoreactive sites for nerve growth factor in cell bodies in the tectal ependymal layer. Nerve growth factor also accelerates the retinal ganglion cell response to axotomy and increases neurite outgrowth from retinal explants (TURNER,DsLANr3Y& JOHNSON, 1980). Normally there are large daily and seasonal fluctuations in the level of thyroid hormones in Ash blood. SPIELER& NOESKE(1979) using ra~oim~noas~y found a trend of increasing hormone levefs during daylight and decreasing levels at night. WHITE & HENDFR~ (1977) also using radioimmunoassay showed a peak in levels of thyroxine and triiodothyronine in brook trout in spring. Both were at a minimum at spawning time in winter. This cycle corrs sponds to our ob~r~tions on the seasonal ability of the retinotectal system to reorganize and the effect of thyroxine in winter in inducing reorganization. Another possible reason for the lack of success in inducing complete compression might be that daily fluctuating thyroxine levels are necessary. The only accounts of the effects of constant light on thyroid activity in fish that we have found are by SINOH(1967, 1968). He used the freshwater teleost, Mysrus vinatus (Bloch) in tropical conditions. By measuring the uptake of radioiodine, he was able t0 demonstrate a seasonal cycle which was advanced by 2 months when the fish were kept in constant light. These fish may be more responsive to the prevailing wet and dry seasons than to light cycles. A more marked effect of constant light may well be demonstrable in fish such as ours from temperate zones. We propose that reorganization of retinotectal synaptic connections after surgical damage to the goldfish tectum is i~uen~ by thyroxine, exogenously administered or endogenously varied on an annual cycle that may be disrupted by constant light. The effect does not appear to be mediated via stimulation of nerve growth and sprouting but appears to be exerted on the formation of new synaptic connections. YOON(1975), to account for the effects of constant light, proposed that continuous ph~~~~~ activity of ganglion cell Synapses in the techun made them unsusceptible to the developmental s&#&s for growth and reconnection. Our results suggest that the effects of constant light may be hormonally-mediated
Thyroxine effects on retinotectal reorganization
with thyroxine being one, but not necessarily the only, factor involved. Since the first description of reorganization of the retinotectal projection after partial tectal ablation the results have been variable. For example, GAZE & SHARMA(1970) only described partial restoration of the projection with reduplication of receptive fields. Complete compression in their hands only occurred if the optic nerve was cut as well. Following YOON’S (1975) description of the effect of constant light, we have demonstrated that size of fish and season of the
1599
year, as well as lighting conditions, may a&t the reorganization. In this paper, we have shown that hormone levels can also affect the process. It seems likely that, reliably to produce compression of the retinotectal projection, all these factors must be taken into account.
Acknowledgement-We wish to thank I. G. MORGANfor criticism of the manuscript and J. TAFFEfor advice on the statistical analysis.
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JACOB~N M. & GAZE R. M. (1964) Types of visual response from single units in the optic tectum and optic nerve of the goldfish. Q. Jl Exp. Physiol. 49, 199-209. LEGRANDJ. (1979) Morphogenetic actions of thyroid hormones. Trends in Neuroscience, 2, 234-236. MAR~TTEL. R. & MARK R. F. (1975) Ultrastructural localization of synaptic input to the optic lobe of carp (Carassius carassius). Expl Neurol. 49, 772-789. MAROT~E L. R.,. WYE-DVORAKJ. & MARK R. F. (1977) Ultrastructure carp kept in constant light. Neuroscience 2,767-780.
of re-organizing visual projections in half tecta of
MAROTTEL. R.,‘WYE-DVORAKJ. & MARK R. F. (1979) Retinotectal reorganization in goldfish-II. Effects of partial tectal ablation and constant light on the retina. Neuroscience 4, 803-810. MEYERR. L. & Scorr M. Y. (1977) Failure of continuous light to inhibit compression of retinotectal projection in goldfish. Brain Res. 128, 153-157. MURRAY M. (1976) Regeneration of retinal axons into the goldfish optic tectum. J. camp. Neural. 168, 175-195. SCHWASSMANN H. 0. & KRUGERL. (1965) Organization of the visual projection upon the optic tectum of some freshwater fish. J. camp. Neurol. 124, 113-126. SHARMAS. C. (1972) The retinal projections in the goldfish. An experimental study. Brain Res. 39, 213-223. SHARMAS. C. (1972) Reformation of retinotectal projections after various tectal ablations in adult goldfish. Expl Neural. 34, 171-182. SINGH T. P. (1967) Influence of photoperiods on the seasonal fluctuations of TSH content of the pituitary in a freshwater catfish, Mystus oittatus (Btoch). Experientia 23, 1016-1017. SINGHT. P. (1968) Effects of varied photoperiods on rhythmic activity of thyroid gland in a teleost, Mystus vittatus (Bloch). Experientia 24, 93-94. SPIELW R. E. 8s NOESKET. A. (1979) Die1 variations in circulating levels of triiodothyronine and thyroxine in goldfish, Carassius auratus. Can. J. Zool. 57,665-669. TURNW J. E., DELANEYR. K. & JOHNSONJ. E. (1980) Retinal ganglion cell response to nerve growth factor in the regenerating and intact visual system of the goldfish (Carassius auratus). Brain Res. 197, 319-330. WaLxaa P., WEICH~ELM. E., R D. A., Guo S. M. & FISHERD. A. (1979) Thyroxine increases Nerve Growth Factor concentration in adult mouse brain. Science N.Y. 204,427-429.
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A. & HENDEISONN. E. (1977) Annual variations in the circulating levels of thyroid hormones in the brook trout, Saloelinusjbntinalis, as measured by radioimmunoassay. Can. J. Zoo!. 55, 4755481. WYE-DVORAKJ., MAROTTEL. R. & MARK R. F. (1979) Retinotectal reorganization in goldfish-- 1. Effects of season. lighting conditions and size of fish. Neuroscience 4, 789-802. YOONM. (1971) Reorganization of retinotectal projection following surgical operations on the optic tectum in goldfish. Expl Neural. 33, 395411. YOONM. (1975) Effects of post-operative visual environments on reorganization of retinotectal projection in goldfish. J. Physiol, Lond. 246, 673-694. WHITE B.
(Accepted 16 February 1981)
RETINOTECTAL
03oa-4522/s1/0s1591-10~2.~/0 Pergamon Press Ltd 6 1981 IBRO
REORGANIZATION IN GOLDFISH-III. EFFECT OF THYROXINE
L. R. MAROTE, R. F. MARK and J. WYE-DVORAK Department of Behavioural Biology, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra, ACT, Australia Abstract-Reorganization of the goldfish retinotectal projection after ablation of half the tectum is dependent on lighting conditions and season. It occurs in summer but not in winter in normal lighting conditions. Constant light prevents reorganization in summer. Treatment with thyroxine at times when it does not normally occur results in some reorganizatioh of the projection in a majority of cases. This consists of reduplicated receptive fields and partial compression. The ability of thyroxine to induce reorganization is not related to the amount of optic fibre sprouting it produces, suggesting that it may be acting on synapse formation. A hormonal influence on reorganization has been demonstrated and this may underlie the effects of season and lighting conditions, which are both known to be associated with changes in hormone levels in fish.
THE OPTIC tectum of the goldfish receives a topo-
This appears to be related to a loss of ganglion cells
graphic projection from the retina (JACOBSON& GAZE, 1964; SCHWASSMANN & KRUGER, 1965). This projection may reorganize after partial tectal ablation so that the entire visual field again becomes rep resented on the remaining tectum (GAZE & SHARMA, 1970; YOON,1971; SHARMA,1972). In the case of removal of the caudal half of the tectum this reorganization involves the shifiing forward of optic terminals projecting to the rostra1 half which were left intact by the operation and the ingrowth and formation of synapses of those axons cut during the operation. The entire projection is reproduced in the correct retinotopic order, but compressed into half the normal rostrocaudal space on the tectum. Several factors have been found to affect this process of reorganization. YOON(1975) showed that placing the fish in constant light after the operation indefinitely delayed compression after half tectal ablation. Initially, we (MARo~, WYE-DVORAK& MARK, 1977) and others (MEYER& SCOTT,1977; HORDER& MARTIN, 1977) were unable to confirm this. Later work in our laboratory demonstrated that there were effects of season and size of fish, as well as lighting conditions, on retinotectal reorganization (WYE-DVORAK,MAROTTE8z MARK, 1979). Compression of the visual projection onto a rostra1 half tectum occurred in summer but not during winter. Furthermore, the effect of constant light on compression depended on the size of fish. Using fish of similar size (greater than 6cm in length) to those used by YOON(1975) and taking into account the seasonal effect, we were able to confirm his result. Curiously, constant light had the opposite effect in smaller fish in that it caused compression of the retinotectal projection in a proportion of cases.
in the retina projecting to the operated tectum through exposure to constant light (MAROI-IE, WYEDVORAK& MARK, 1979), an effect seen on small but not on large operated fish. We suggested that the seasonal failure of reorganization might point to an hormonal influence (WYEDVORAKet al., 1979). Seasonal hormonal fluctuations in fish are well documented (HOAR, 1957). Furthermore, constant light is known to affect hormone levels (C~ITCHLOW,1963) suggesting that it could also affect reorganization by a hormonal mechanism, rather than by a direct effect on the activity of retinal ganglion cells, thus preventing their rearrangement as suggested by YOON(1975). In this paper, we describe attempts to induce retinotectal reorganization by changing hormonal levels at times when reorganization does not normally occur; during winter in normal lighting conditions and during summer in constant light conditions. Thyroxine was used because of its known influence on the development of the nervous system (see GRAVE,1977) and its known seasonal fluctuations in fish (WHITE & HENDERSON, 1977).
Abbreviations: SFGS, stratum superficiale; SO, stratum opticum.
fibrosum
et griseum
EXPERIMENTAL PROCEDURES The caudal half of the left tectum was removed from 48 goldfish (Carassius auratus) 6.5-9.0cm in body length, under anaesthesia with ethyl m-aminobenzoate methane sulphonate (MS222 Sigma) as described in a previous paper (WYE-DVORAKet al., 1979). Nineteen fish, operated in winter, were kept on a 12 h light/l2 h dark cycle. Ten received 0.006mg of 3,3’,4,5’-tetraiodo-L-thyroxine (sodium salt, pentahydrate, Sigma) per litre of aquarium water, replaced every two to three. days, and nine served as untreated controls. The retinotectal projections were mapped electrophysiologically 35-65 days after operation and the tecta prepared for elec-
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L. R. MAROTTE,R. F. MARK and J. WYE-DVORAK
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tronmicroscopy as previously described (WYE-DVORAKet al., 1979). The controls were mapped within 1 day of corresponding fish treated with thyroxine. Fifteen fish operated in autumn were kept on a light/ dark cycle. Seven received 4 mg/litre of thyroxine replaced fortnightly and eight served as untreated controls. Projections were mapped 41-65 days later, with corresponding controls being mapped within 3 days of the fish treated with thyroxine. In nine cases (seven thyroxine-treated and two untreated fish) the tecta were prepared for electronmicroscopy for the analysis of tectal structure. The five controls not prepared for electronmicroscopy were replaced by controls obtained from a previous series (WYEDVORAKer al., 1979) operated in late summer and with similar post-operative survival times. Fourteen fish were operated in late spring and kept in constant light (luminance of aquaria floors 0.15 Ig ft iamberts and that of the walls 0.24 1 g ft lamberts). Half received 4 mg/litre of thyroxine replaced fortnightly and half served as untreated controls. Projections were mapped 48-99 days later during summer, with controls mapped within two days of the corresponding thyroxine-treated fish. The temperatures of the aquaria ranged from 19-22°C. That immersion of goldfish in water containing thyroxine is effective in elevating plasma thyroxine levels has been demonstrated by HURLBURT(1977). The lower concentration we used was effective in producing behavioural changes in goldfish (HOAR, KEENLEV~IDE& GOODALL, 1955) while the higher concentration, extrapolating from Hurlburt’s data, would produce a large elevation in plasma levels. Analysisof tectal structure The dorsal surface of the operated half tectum was divided into samples from the most rostra1 area and the area adjacent to the cut edge (termed midline in subsequent text and Figures). The unoperated tectum on the opposite side was either similarly divided into such samples, or several samples were taken from the entire dorsal surface of the tectum. The amount of axonal sprouting in the stratum opticum (SO) was measured in 1 pm Araldite sections cut perpendicular to the tectal surface and stained with toluidine blue. A drawing of the SO at a magnification of x 400 was made with the aid of a camera lucida, marking those areas containing myelinated and unmyeiinated axons. The percentage area occupied by unmyelinated axons was measured
using a Kontron MOP-AM03 image analyser. Adjacent ultra-thin sections stained with uranyl acetate and lead citrate were examined in a Hitachi 500 electronmicroscope. The results were analysed statistically using the Wilcoxon Matched-Pairs Signed-Ranks test. RESULTS Electrophysiological analysis
Under conditions in which previous work had shown an inhibition of reorganization of the retinotectal projection, treatment with thyroxine induced some reorganization of the projection in a proportion of cases. The effects observed were partial compressions of the projection and the presence of reduplicated receptive fields for single points on the tectum. The results are summarized in Table 1 and the detailed results for each group are set out below. Low dose thyroxine: light/dark conditions in winter. After removal of the caudal half tectum in winter we have previously reported that there is no reorganization of the retinotectal projection (WYE-DVORAK et al., 1979). We therefore compared pairs of fish which had survived for similar periods after operation, with one of the pair kept under normal conditions and the other under identical conditions except for the addition of thyroxine to the water. As expected, operated fish which received no thyroxine all had uncompressed retinotectal projections. Figure 1 shows three maps of the retinatectal projection made from control fish and one from the treated group. Figure 1A is a normal map of the left visual field onto the right tectum of an unoperated fish. Below, in Figs 1C and 1D, are maps made from a fish that had survived 64 days after operation untreated with thyroxine. Figure 1C shows the projection mapped immediately after ablation of its cau&l half of the right tectum. Comparison with Fig. 1A above shows the absence of representation of the temporal visual field. Figure 1D shows the map made from the left tectum of this fish in which the caudal half tectum had been removed in the same way 64 days previously. The map is a mirror image of that from the normal half tectum, with a scotoma of the
TABLE1. SUMMARYOFTHEEFFECTS OFTHYROXINE ON REORGANIZATIOH OFTHERETIPI’OTECTAL PROJECTION
Season of year and lighting conditions L/D winter 0.006 mg/l Thyroxine L/D au-/winter 4 m0/1 Thyroxine CL summer 4 rnd Thyroxine
Proportion of fish with partial compression or reduplicated fields Operatedand Operated Thyroxine
Time between operation and electrophysiological mapping
8110
OP
35 to 67 days
6/7
218
41 to 65 days
317
017
48 to 99 days
L/D: 12 h Ii&t/12 h dark cycle CL: constant light. Concentrations of thyroxine refer to the amount per litre of aquarium water.
Thyroxine effects on retinotectal reorganization
1593
the temporal field than would be expected. Points 2 and 6 further rostrally on the tectum also have receptive fields displaced temporally. The most striking feature of the map is the presence of many tectal points with reduplicated receptive fields: that is, points on the tectum which receive an input from two points in the visual field. The reduplicated field was always to the temporal side of and clearly separated from the primary receptive field. The map from the untreated control of this pair showed no signs at all of reorganization of the projection, the size and position of the scotoma being the same as the examples in Fig. 1. Of the remaining seven fish in this group, three had reduplicated receptive fields and four showed partial compression. Signs of reorganization in treated fish were seen at the earliest post-operative time of 35 days, The lack of effect of thyroxine in two of the fish did not appear to be related to the duration of treatment as these two survived 64 and 67 days, the longest post-operative times. High dose thyroxine: light/dark conditions: autumn to winter. Of the untreated operated controls two out
FIG. 1. A. Retinotectal projection of the left visual field onto a normal right tectum. The numbers on the camera lucida drawing of the dorsal surface of the tectum are recording sites and the corresponding numbers on the perimetric chart of the visual field are the locations of the receptive fields at each recording site. B. Retinotectal projection of a fish kept in light/dark conditions and receiving a low dose of thyroxine, showing no reorganization 64 days post-operatively. C and D. Projections of the untreated operated controls for the fish whose projections are shown in B. Maps were made 64 days post-operatively. ‘Midline’ labels the cut edge of the tectum in all maps. C. Projection to the right tectum mapped immediately after removal of the caudal half. D. Projection to the left half tectum. An equivalent area of temporal visual field is missing in maps ED. same size and position indicating that there had been no reorganization of the projection. This may be compared with the map in Fig. 1B above which is from one of two fish in this series in which treatment with thyroxine after operation had no effect on the retinotectal projection. The scotoma is of the same size and shape as in the control of 64 days and the control mapped immediately after operation. With these results as a baseline, eight out of ten fish treated with thyroxine gave maps which clearly indicated some progress towards remodelling of the retinote&al projection. Figure 2 shows on the left a map from one of these made 51 days after ablating the caudal half of the left tectum. For comparison, on the right is the map of an untreated fish kept in the same conditions for 52 days. The map from the treated fish shows partial compression in that points 3 and 7 from the caudal tectum have receptive fields further into
of eight showed signs of retinotectal reorganization, in the form of reduplicated fields (two in one, and four in the other) at 49 and 51 days post-operatively. Fish mapped both before and after these times up to 65 days post-operatively all had uncompressed projections (Fig. 3). In those fish receiving thyroxine postoperatively six out of seven showed some retinotectal reorganization. Four had two reduplicated fields each (Fig. 3). One of these also showed signs of partial compression with points near the cut midline receiving a projection from further temporally in the visual field than normal. Of the two remaining, one had two points on the tectum where instead of the normal receptive field size of about 5”, activity could be evoked from regions 12 and 32” across, the centres of
Wl-
FIG. 2. On the left is the retinotectal projection of a fish in light/dark conditions for 51 days after operation and receiving a low dose of thyroxine. Extensive reorganization in the form of reduplicated fields and partial compression is present. On the right is the projection of its untreated control mapped 52 days post-operatively. The projection remains uncompressed. The half tectum of the fish treated with thyroxine is larger than that of the untreated control in this example. This was not a consistent finding.
I594
L. R. MAROTTE.R. F. MARK and J. WYE-DVORAK tions with one or two reduplicated fields near the cut midline (Fig. 5). Post-operative survival times of these were 56, 64 and 68 days. Structural analysis. Most of the optic fibres enter in
FIG. 3. On the left is the projection of a fish kept in light/ dark conditions receiving a high dose of thyroxine and mapped 49 days post-operatively. Two reduplicated fields are present. Its untreated control shown on the right and mapped 47 days post-operatively, is uncompressed.
each field being more temporal in the visual field than normal (Fig. 4). The other had four reduplicated fields and one slightly enlarged receptive field of 12”. The only fish in which the projection remained unohanged after thyroxine treatment had the longest post-operative survival of 65 days. Presumably, changes were observed in two of the controls because the operation was done earlier in the season than the previous group. Nevertheless, thyroxine treatment was associated with a higher proportion of reorganizing projections. None of the projections was fully compressed. The effects were no more marked than with the lower dose of thyroxine. High dose thyroxine: constant light conditions in summer. Keeping Iarge fish in the summer in constant
light prevents remodelling of the retinotectal projection after ablation of half the tectum (WYE-DVORAK et al., 1979). As expected, all untreated operated fish had uncompressed retinotectal projections at all postoperative times studied (Fig. 5). Reorganization of retinotectal projections in thyroxine-treated fish in these lighting conditions was less striking, with three out of seven fish having partially compressed projec-
FIG. 4. Projection of fish in light/dark conditions receiving a high dose of thyroxine and mqpal U days postoperatively. It is partially compressed with two extended
^..
recephve tields.
the SO in the upper part of the tectum (MAROTTE& MARK, 1975; MURRAY,1976) and comprise the majority of fibres in this layer (MAROTTE& MARK, 1975). Most synapse in the stratum fibrosum et griseum superficiale (SFGS) directly beneath the SO and a very few synapse in the stratum griseum centrale (SHARMA,1972; MAROTTE& MARK, 1975; MURRAY, 1976). In normal unoperated tecta in both light/dark and constant light conditions the percentage area of SO occupied by unmyelinated axons is extremely small, around 4% or less (Fig. 8). This did not change for unoperated tecta in fish treated with thyroxine. On removing the caudal half of the tectum those ganglion cells projecting to the caudal half have their axons cut. There is sprouting of optic fibres throughout the SO and SFGS. Small unmyelinated sprouts appear in the SO and unmyelinated axons bundles, not normally present, are found in the SFGS. All the operated tecta of fish in this study showed these features (Figs 6 and 7) as previously found in fish with reduplicated receptive fields and uncompressed or partially compressed retinotectal projections (MARom et al., 1977; WYE-DVORAKet al., 1979). Near the cut edge of the tectum the SO contained few myelinated axons, the majority being very small unmyelinated axons (Figs 6 and 103). In some fish almost no normal myelinated fibres remained and occasional degenerating myelinated axons and myelin debris within glial cells was present scattered amongst the myelinated fibres (Fig. 6). Sprouting in the SO is seen as an increase in the percentage area occupied by unmyelinated axons (Figs 9-14). The amount of sprouting seen varies, depending on thyroxine treatment, region of tectum sampled and season of year in which the operation was performed. Results were analysed by comparing corresponding tectal regions of thyroxine treated and untreated groups, rostra1 and midline regions within
FIG. 5. Projection of a fish in constant light conditions and treated with a high dose of thyroxine. mapped 68 days post-operatively. It is partially compressed with two reduplicated receptive fitI&. The projection of its untreated control shown on the right is uncompressed after 70 days.
FIG. 6. Stratum opticurn near the cut edge days after operation. The majority of fibres myelinated axons (arrows) and few normal of thyroxine but untreated
of the tectum in are unmyelinated myelinated fibres. fish also showed
a fish kept in light/dark conditions for 41 with some myelin debris and degenerating This fish had been treated with a low dose these features. Bar = 2 pm.
FIG. 7. Stratum fibrosum et griseum superficiale in a fish kept in light/dark conditions for 51 days after operation Bundles of unmyelinated fibres are present in the neuropii. Bar = 2 pm. 1.595
FIG. 8. A. Stratum opticum in an unoperated tectum showing extremely few pale areas of unmyelinated fibres (arrow). Oil immersion. Bar: 20pm for this and Figs 9911. B. Electron micrograph of a normal stratum opticum showing areas of unmyelinated axons amongst myelinated fibres. Part of a glia cell is seen on the lower left. Bar = 2 nm. FIG. 9. A. Stratum opticum rostrally in a fish kept in light/dark conditions for 51 days after operation and receiving a high dose of thyroxine. A small increase in area occupied by unmyelinated axons is seen. In this and subsequent micrographs asterisks mark the larger areas of unmyelinated axons. Two cell bodies are marked by the large arrow. B. Untreated operated control for A. There are more areas of unmyelinated axons. FIG. 10. A. Rostra1 region of stratum opticum 68 days after operation. B. Region near the cut edge in the same fish showing more extensive areas of unmyelinated fibres and fewer myelinated fibres. This fish had been kept in constant light and treated with thyroxine. FIG. 11. A. Rostra1 regton of stratum opttcum m fish operated on m autumn and kept in light/dark conditions for 51 days. Areas of unmyelinated axons are present. B. Rostra1 region of stratum opticum m fish operated on in winter and kept in lightidark for 57 days. Few unmyelinated axons arc present I596
1597
Thyroxine effects on retinotectal reorganization
FIG. 12. Histograms in this and subsequent figures show percentage area of unmyelinated axons in the stratum opticum of half tecta at various times after operation. Low dose of thyroxine and light/dark conditions. On the left are results for the rostra1 regions and on the right for the midline regions, i.e. areas adjacent to the cut edge, of tecta treated with thyroxine and untreated tecta. There are no differences in sprouting between treated and untreated tecta in either region.
each group and corresponding tectal regions of untreated light/dark groups. In Figs 12,13 and 14, we compare first the effects of thyroxine treatment in rostra1 and midline tectal regions with respective untreated control tectal regions. In those fish kept in post-operative light/dark conditions and receiving the lower dose. of thyroxine, there was no significant difference in the amount of sprouting in the SO in either the rostra1 or the midline regions of operated tecta when compared to their respective untreated operated control tecta (Fig. 12). However, in those fish kept in post-operative light/ dark conditions and treated with the higher dose of thyroxine there was significantly less sprouting (P < 0.02) in the rostra1 region of operated tecta compared with that seen in rostra1 regions of untreated control tecta (Figs 9 and 13). Sprouting in midline regions in treated and untreated fish did not differ significantly (Fig. 13). In fish treated with thyroxine and kept in postoperative constant light conditions there was no significant difference in the amount of sprouting in the SO of either rostra1 or midline tectal regions when compared to corresponding regions of untreated operated controls (Fig. 14).
FIG. 14. Low dose of thyroxine and constant light conditions. On the left are results for rostra1 regions and on the right for the midline regions of treated and untreated tecta. There are no differences in sprouting between treated and untreated tecta in either region.
To summarize, the only significant effect of thyroxine on sprouting in the SO was a decrease in the rostra1 tectum in the group treated with the high dose of thyroxine and kept in light/dark conditions. Regional differences in sprouting in fish under various conditions can also be compared. Comparing the amount of sprouting in the SO in the rostra1 and midline regions within fish, in the group receiving the low dose of thyroxine and post-operative light/dark conditions there was significantly less sprouting rostrally (P < 0.02, Fig. 12, open columns). This was also true of its untreated control group (P < 0.01, Fig. 12, filled columns). In the group treated with the higher dose of thyroxine, there was significantly less sprouting in SO rostrally (P < 0.05, Fig. 13, open columns). Its untreated control group showed no significant difference in sprouting between rostral and midline tectal regions (Fig. 13, filled columns). In the thyroxine treated group kept in constant light conditions, there was significantly less sprouting in the SO rostrally (P < 0.02, Figs 1OA and B, 14, open columns) while in its untreated control group there was no significant difference in sprouting between rostra1 and midline regions (Fig. 14, filled columns). There was also a difference in the amount of sprouting in the SO in the two control groups of fish kept in light/dark conditions and which were operated on at different times of the year (Figs 1IA and B). The rostra1 region of the group operated on in winter (Fig. 12, filled columns) had significantly less sprouting in the SO than the group operated on during late summer and autumn (Fig. 13, filled columns, P < 0.05). Sprouting in midline regions did not differ significantly. DISCUSSION
FIG. 13. High dose of thyroxine and light/dark conditions. On the left are results for the rostra1 regions and on the right for the midline regions of treated and untreated tecta. There is less sprouting rostrally in tecta treated with thyroxine.
Thyroxine administration is able to induce reorganization of the retinotectal projection at times when, because of seasonal effects or lighting conditiona it does not normally occur. The failure of reorganization does not seem to be due to a failure of sprouting of retinal ganglion cell axons. Teeta with uncompressed retinal projections
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L. R. MAROON,R. F. MARKand J. WYE-DVORAK
always show an increase in the number of very small unmyelinated axons in the SO and bundles of them in the SFGS where they are not normally seen (WYEDVORAKet al., 1979). Although a natural seasonal effect on this sprouting was demonstrated in rostra1 tectum, with more sprouting in untreated fish operated on in late summer or autumn than in winter, this was not correlated with the ability to undergo reorganization of the projection, Neither of these groups showed any significant reorganization. Treatment with thyroxine had no measurable stimu~t~g effect on sprouting. Rather, for those fish receiving the high dose of thyroxine and kept in light/dark conditions there were fewer unmyelinated axons in the rostra1 part of the SO in these fish compared to their untreated operated controls. Similarly, all groups of fish treated with thyroxine had less sprouting in rostral SO than near the cut margin, while two out of three untreated groups showed no significant difference in sprouting in these two regions. Nevertheless, treatment with thyroxine induced electrophysiological signs of reorganization of the retinotectal projections in operated tecta under both conditions in which this normally does not happen, that is in a normal light, dark regime in the winter and in constant light during the summer. In the latter situatian, thyroxine was however less successful, In none of the cases was the reorganization complete with the whole of the visual field represented on the rostra1 half teetum. The most common effect of thyroxine was in producing reduplicated receptive flelds. These are commonly seen during the reorganization process, particularly when the caudal half of the teo turn is removed without crushing or cutting the optic nerve innervating that tectum (GAZE & SHARMA, 1970; HORDW & MARTIN, 1977; M~OTTE et al., 1977). Part of the reason for the failure to induce complete compression may have been that the fish were kept for at most 65 to 99 days after operation. Reorganization under the natural conditions in which it occurs may take longer. The ultr~t~~tur~ analysis shows that althou~ thyroxine is able in other situations to promote sprouting of cut sensory and motor axons (EHRLICH & MARK, 1977) this is not the mechanism of its action on this system. Another level at which it may be exerting its effect is on the formation and maturation of synaptic connections between growing axonal sprouts of retinal ganglion cells and tectal cells. Added thyroxine is known to promote maturation of the brain and synaptogenesis in particular. Excess synapses are produced in the cerebellum of new-born rats given thyroxine (LEORAND,1979) and conversely CRAGG (1970) has demonstrated a reduction in the number of synapses in the visual cortex of hypothyroid rat pups. The physiology of the retinotectal pathway in goldfish is also in3uemxtci by exogermus thyroid hormone. After some nine days of exposure to the hormone, extracellularly-recorded field potentials evoked in the tectum by flashes of light showed
reduced latency and increased amplitude (HARA, UEDA & GORBMAN,1965). Thiourea had opposite effects (HARA, GORBMAN & UEDA, 1966). The possibility that nerve growth factor might be involved in these changes has been raised by the finding of WALKER,WEICHSEL,FISHER, Guo & FISHER (1979) that thyroxine increases the concentration of nerve growth factor in mouse brain. They suggest that thyroid hormone may interact with nerve growth factor to promote axonogenesis and possibly synaptogenesis. Recently nerve growth factor has been shown both’to be present in high concentrations in goldfish tectum and to have an effect on optic nerve regeneration. BENOWITZ& SHASHOUA(1979) demonstrated immunoreactive sites for nerve growth factor in cell bodies in the tectal ependymal layer. Nerve growth factor also accelerates the retinal ganglion cell response to axotomy and increases neurite outgrowth from retinal explants (TURNER,DsLANr3Y& JOHNSON, 1980). Normally there are large daily and seasonal fluctuations in the level of thyroid hormones in Ash blood. SPIELER& NOESKE(1979) using ra~oim~noas~y found a trend of increasing hormone levefs during daylight and decreasing levels at night. WHITE & HENDFR~ (1977) also using radioimmunoassay showed a peak in levels of thyroxine and triiodothyronine in brook trout in spring. Both were at a minimum at spawning time in winter. This cycle corrs sponds to our ob~r~tions on the seasonal ability of the retinotectal system to reorganize and the effect of thyroxine in winter in inducing reorganization. Another possible reason for the lack of success in inducing complete compression might be that daily fluctuating thyroxine levels are necessary. The only accounts of the effects of constant light on thyroid activity in fish that we have found are by SINOH(1967, 1968). He used the freshwater teleost, Mysrus vinatus (Bloch) in tropical conditions. By measuring the uptake of radioiodine, he was able t0 demonstrate a seasonal cycle which was advanced by 2 months when the fish were kept in constant light. These fish may be more responsive to the prevailing wet and dry seasons than to light cycles. A more marked effect of constant light may well be demonstrable in fish such as ours from temperate zones. We propose that reorganization of retinotectal synaptic connections after surgical damage to the goldfish tectum is i~uen~ by thyroxine, exogenously administered or endogenously varied on an annual cycle that may be disrupted by constant light. The effect does not appear to be mediated via stimulation of nerve growth and sprouting but appears to be exerted on the formation of new synaptic connections. YOON(1975), to account for the effects of constant light, proposed that continuous ph~~~~~ activity of ganglion cell Synapses in the techun made them unsusceptible to the developmental s&#&s for growth and reconnection. Our results suggest that the effects of constant light may be hormonally-mediated
Thyroxine effects on retinotectal reorganization
with thyroxine being one, but not necessarily the only, factor involved. Since the first description of reorganization of the retinotectal projection after partial tectal ablation the results have been variable. For example, GAZE & SHARMA(1970) only described partial restoration of the projection with reduplication of receptive fields. Complete compression in their hands only occurred if the optic nerve was cut as well. Following YOON’S (1975) description of the effect of constant light, we have demonstrated that size of fish and season of the
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year, as well as lighting conditions, may a&t the reorganization. In this paper, we have shown that hormone levels can also affect the process. It seems likely that, reliably to produce compression of the retinotectal projection, all these factors must be taken into account.
Acknowledgement-We wish to thank I. G. MORGANfor criticism of the manuscript and J. TAFFEfor advice on the statistical analysis.
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(Accepted 16 February 1981)