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Evidence that the influence of ganglion cell axons on astrocyte morphology is mediated by action spike activity during development
Developmental Brain Research 110 Ž1998. 177–184
Research report
Evidence that the influence of ganglion cell axons on astrocyte morphology is mediated by action spike activity during development Claudia Gargini
a,d
, Stefania Deplano b, Silvia Bisti
c,d,)
, Jonathan Stone
e
a
e
Istituto Policattedra, Discipline Biologiche, UniÕersita` di Pisa, CNR, Pisa, Italy b Istituto di Anatomia Comparata, UniÕersita` di GenoÕa, GenoÕa, Italy c Dipartimento STB, UniÕersita` di L’Aquila, L’Aquila, Italy d Istituto di Neurofisiologia, CNR, Pisa, Italy NSW Retinal Dystrophy Research Centre, Department of Anatomy and Histology, UniÕersity of Sydney, Sydney, Australia Accepted 23 June 1998
Abstract In many mammal retinas, the morphology of astrocytes is strongly influenced by nearby axons of ganglion cells. Astrocyte processes stretch along the axons, fine extensions of the processes contact node-like specialisation of the axon membrane and the morphology of the adult astrocyte is strongly determined by this relationship. The mechanism which attracts astrocyte processes to contact specific regions of the axon membrane is not known however. This study presents evidence that in the neonatal cat blocking the impulse activity of ganglion cells with the Naq-channel blocker tetrodotoxin ŽTTX. leads to a loss of the axon-related morphology of astrocytes. The morphological change induced in astrocytes by TTX was greater in younger animals and could not be detected in the adult. Conversely, if the TTX block was maintained for 4 postnatal weeks the changes induced in astrocytes persisted at least to 13 weeks. The TTX-induced loss of axon-related morphology in astrocytes suggests that the signal by which axons attract astrocyte processes to contact the axonal membrane in ways which modify astrocyte morphology is released by action spike activity during development. q 1998 Elsevier Science B.V. All rights reserved. Keywords: TTX; Stellation; Macroglia; Cat
1. Introduction The morphology of retinal astrocytes is strongly influenced by attachments made by their processes. In the periphery of the cat retina, where ganglion cell axons are sparse, astrocytes adopt the stellate shape after which they were named ŽFig. 1, see also figure 1 in Karschin et al. w12x and in Karschin et al. w13x and figure 3D in Chan-Ling and Stone w4x.. Many astrocytes in more central regions of the cat’s retina, by contrast, are strongly elongated ŽFig. 1 and see also figures 1A–C in Chan-Ling and Stone w4x., stretched along the axon bundles into a shape similar to that adopted by the other major class of axon-related glia,
)
Corresponding author. Istituto di Neurofisiologia, Via S. Zeno 51, 56127 Pisa, Italy; Fax: q39-50-559725; E-mail: [email protected]
oligodendrocytes w3x. Previous reports w4,12x have considered whether astrocytes so differently shaped are different ‘types’. Because many astrocytes were observed with intermediate morphology and mixed contacts however, differently shaped and committed astrocytes were interpreted as variants of a single class of astrocytes, all competent to adopt relationships with vessels, neurones and Muller ¨ cells. The same studies gave evidence of plasticity in astrocytes. When axons or blood vessels were eliminated retinal astrocytes appeared to reshape to a stellate form, leading ChanLing and Stone w4x to suggest that the stellate form is a ’default’ shape. In the optic nerve w24,26x and spinal cord w17x astrocyte processes contact the membrane of axons at their nodes. The retinal segments of ganglion cell axons in the cat lack myelin but nevertheless astrocyte processes do converge on node-like specialisations of the intraretinal length of these axons w11,21x. Ganglion cell axons may express a
0165-3806r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 1 0 1 - 1
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factor at their nodes which attracts astrocyte processes and causes them to stretch along axons. In the rat, the node-like specialisations found along the intraretinal lengths of axons contain concentrations of Naq channels w10,15x suggesting that, like nodes of myelinated axons, they are the sites of major currents during action potentials. We hypothesised that the astrocyte-trophic signal expressed by axons is expressed at their nodes when action potentials occur. To test this hypothesis, we have examined whether a drug Žtetrodotoxin, TTX. which specifically blocks Naq channels and thereby action spikes will, when applied to the retina by intravitreal injection, result in an adaptive change in the morphology of retinal astrocytes.
2. Materials and methods 2.1. TTX injections Injections were made into the right eye of cats aged P7 to adult, anaesthetised with halothane Ž2–3%. in a 2:1 mixture of nitrous oxide and oxygen. The TTX injections followed published protocols w7,23x. Briefly, the TTX Ž5 mM in phosphate buffered saline ŽPBS.. was injected into the vitreous humour of the right eye. The initial injection was always 1 ml in volume. Subsequent injections Žif made. were made at 3-day intervals and the vol. injected was increased by 5–10% with each injection. Each injection was made through the temporal region of the sclera between the attachment of the iris and the anterior edge of the retina. Close care was taken to make repeat injections through the hole used initially. The pupillary reflex was always abolished after the first injection, confirming the block of electrical activity from the retina.
P6. This dosage is effective in suppressing ON-responses in the light adapted cat retina for as long as 24 h w9x. 2.3. GFAP, neurofilament immunocytochemistry and lectin histochemistry At selected intervals after TTX injection, animals were euthanised with an overdose of Ketalar. Eyes were immersion-fixed in 4% paraformaldehyde in PBS at pH 7.4 for 2 h. The retinas were then dissected and prepared as wholemounts. Most were labelled with an antibody to glial fibrillary acidic protein ŽGFAP. to demonstrate astrocytes and with the G. simplicifolia isolectin B4 Žconjugated to the fluophore FITC. to demonstrate blood vessels. Briefly, the retinas were washed in PBS and incubated in a rabbit polyclonal anti-GFAP ŽDako, diluted 1:200 in PBS. overnight at 48C, then washed and incubated in goat anti-rabbit IgG conjugated to Cy3 or TRITC ŽSigma, diluted 1:400 in PBS. for 2 h at room temperature. They were then washed, incubated in the isolectin Ždiluted 1:20. overnight at 48C, mounted in glycerol and viewed by confocal microscopy. Selected pieces of wholemounts were labelled for GFAP and for neurofilaments, to demonstrate axons directly. The pieces were washed in PBS and incubated in Triton Ž0.3% in PBS. for 15 min and then in bovine serum albumen Ž2%. for 30 min. They were then incubated for 48h in the rabbit polyclonal anti-GFAP and simultaneously in a monoclonal antibody to neurofilament, both diluted in PBS containing 0.1% Triton. They were then washed in PBS and incubated for 2 h at room temperature in a mixture of FITC-conjugated anti-rabbit IgG ŽVector, 1:75. and TRITC-conjugated anti-mouse IgG ŽSigma, 1:128.. They were then washed and coverslipped.
2.2. APB injection
2.4. HRP histochemistry
In one kitten, the right eye was injected with D,L-2amin-4-phosphonobutyric acid ŽAPB., the glutamate analog which blocks glutamate release by rod bipolar and ON-cone bipolar cells ww18x, reviewed in Ref. w25xx. Four injections Žeach 5 ml of a 100-mM solution. were made at daily intervals from P2 to P5; the animal was euthanised at
In two animals injected with TTX from P7 to P30, horseradish peroxidase ŽHRP. was injected into the dorsal lateral geniculate nucleus ŽdLGN. 2 days prior to euthanasia at P90 and the retinas were prepared as wholemounts and reacted for HRP, using the protocols of Deplano et al. w7x.
Fig. 1. Effect of TTX on astrocyte morphology. The retinas were labelled for astrocytes Žfor GFAP, red. and vessels Žwith the G. simplicifolia lectin, green.. Over the rectangular regions outlined the green signal is suppressed, to demonstrate astrocyte morphology more clearly. The arrows indicate points at which the orientation of astrocytes to vessels can be seen. ŽA,B. Midperipheral regions of retina from the vehicle-injected ŽA. and TTX-injected ŽB. eyes of a P12 kitten, which received injections at ages P8 and P11. ŽC,D. Regions from the edge of the developing inner vasculature of the same P12 kitten. Fig. 2. Effect of TTX on astrocyte morphology. As in Fig. 1, the retinas were labelled for astrocytes Žfor GFAP, red. and vessels Žwith the G. simplicifolia lectin, green.. Over the rectangular regions outlined the green signal is suppressed, to demonstrate astrocyte morphology more clearly. The arrows indicate points at which the orientation of astrocytes to vessels can be seen. ŽA,B. Regions from the midperiphery of the retinas from the uninjected ŽA. and TTX-injected ŽB. eyes of a P27 kitten. The right eye received 7 TTX injections at 3-day intervals from P7–27. ŽC,D. Peripheral regions from the uninjected ŽC. and TTX-injected ŽD. eyes of the same P27 kitten. The regions shown are close the edge of the retina.
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3. Results 3.1. TTX injections change astrocyte morphology Figs. 1 and 2 show our principal findings; the key comparisons are between Fig. 1A and B, between Fig. 1C and D, between Fig. 2A and B and between Fig. 2C and D. 3.1.1. Early neonate Fig. 1 shows areas of retinas from a neonatal cat injected with TTX at P8 and P11, then examined at P12. Fig. 1A and B are from the left and right ŽTTX-injected. retinas respectively and show astrocytes ŽGFAP-labelled, red. and vessels Žlectin-labelled, green. in corresponding regions of mid-peripheral retina. In each of Fig. 1A–D, the lectin labelling Žgreen. of vessels is suppressed over a rectangular window, to show astrocyte morphology more clearly. In all regions of both retinas, astrocytes formed a continuous network at the inner surface of the retina. Within that network, individual astrocytes are distributed in an interactive process of ‘contact spacing’ w4x. In Fig. 1A, some astrocytes can be seen to form the glia limitans of vessels, for example at the sites arrowed, while others course along axon bundles, from upper left to lower right. The apposition of astrocyte processes to axons and vessels confirms many earlier observations w4,12,13,16,20x. In Fig. 1B, the orientation of astrocytes to axons is lost, though astrocyte processes remain oriented to underlying vessels. Fig. 1C and D show regions from the same two retinas, at the edge of the spreading vessels. In Fig. 1C, astrocytes are aligned to underlying vessels Žsmall arrows. and also to axons bundles, which course from right to left. In Fig. 1D, from the TTX-injected retina, the orientation of astrocyte processes to vessels persists Žarrows. but the orientation to axons appears lost.
the mid-periphery of the right Žinjected. eye of the same animal. The eye received 7 injections of TTX at 3-day intervals from P7 to P27, for a total of 12 ml. The astrocytes still show their stellate shape and their processes still invest underlying vessels but their orientation to bundles is lost. Fig. 2C shows a second area from a more peripheral region of the retina from the uninjected Žleft. eye of the same animal and Fig. 2D shows a corresponding region from the retina of the injected Žright. eye. In Fig. 2C, some astrocytes extend along axon bundles Žthough these bundles are sparser than in Fig. 2A.; others spread their processes in a stellate pattern while others wrap blood vessels. In Fig. 2D, many astrocytes are stellate in form and some invest vessels but their orientation to axon bundles appears lost. The continuing astrocytic investment of vessels after TTX injection is seen clearly in the ‘window’ in Fig. 2D within which the vessel labelling is suppressed. 3.1.3. TTX induced changes persist Fig. 3A and B show areas of retina from the vehicle-injected Žleft. eye and of the TTX-injected Žright. eye of a P90 kitten which received 7 injections at 3-day intervals from P7 to P27. The last injection was thus 60 days before the tissue was examined. The absence of astrocyte orientation to axons remains clear ŽFig. 2B.. 3.1.4. TTX does not affect adult astrocytes In one adult cat, the right eye received 10 TTX injections at 3-day intervals. Three days after the last injection the animal was euthanised and the retina examined. No change in astrocyte morphology was detected Ždata not shown.. 3.2. Control obserÕations
3.1.2. Late neonate Fig. 2A shows a region from the mid-periphery of the uninjected Žleft. eye of a kitten aged P29. The astrocytes show more evidence of their characteristic stellate shape and the underlying vessels are finer and less profuse than at P12 ŽFig. 1., both differences evidence of the increasing maturation of the retina. The processes of many astrocytes form the glia limitans of vessels, while others are elongated along axons, which cross the field from upper right to lower left. Fig. 2B shows a corresponding region from
3.2.1. Ganglion cells and their axons persist after TTX injection Astrocytes are known to lose their orientation to axons if the axons are destroyed w12,13x. Previous studies using TTX injected into the vitreous humour to stop action spike activity in ganglion cells reported that ganglion cells survive, although their dendritic morphology may change w27x. Nevertheless, we checked whether TTX might be toxic to ganglion cells in two ways. First, a TTX-injected
Fig. 3. ŽA. Astrocytes from the mid-peripheral region of the uninjected eye a P90 kitten, given 7 injections at 3-day intervals from P7 to P27. ŽB. Astrocytes from the mid-peripheral region of the TTX- injected eye of the same kitten as in ŽA.. ŽC. Area of midperiphery of the same eye as in A labelled for astrocytes Žleft panel. and for the underlying axons Žright panel.. ŽD. Area of midperiphery of the same eye as in ŽB. labelled for astrocytes Žleft panel. and for the underlying axons Žright panel.. ŽE,F. Ganglion cell somas and nearby axons labelled with horseradish peroxidase transported retrogradely from the dLGN, in wholemounts of retinas from the uninjected ŽE. and TTX-injected ŽF. eyes of a P90 kitten. The TTX injections were made at 3-day intervals from P7 to P30; and the HRP injections into the dLGN 2 days prior to euthanasia. ŽG,H. Astrocytes labelled for GFAP in wholemounts of retinas from the uninjected ŽG. and APB-injected ŽH. eyes of a P6 kitten, which received 4 injections at daily intervals from P2–5. ŽI,J. Astrocytes in wholemounts of retinas from the uninjected ŽI. and TTX-injected ŽJ. eyes of P4 kitten which received a single injection at age P2.
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retina showing extensive changes in astrocyte morphology was double-labelled for neurofilament protein as well as GFAP. The normal close relationship of astrocyte processes to axons ŽFig. 3A. is greatly reduced in the TTX-injected retina ŽFig. 3B left panel. but the axons remain Žright panel.. Second, using retrogradely transported HRP, which demonstrates the axons, somas and proximal dendrites of ganglion cells ŽFig. 3C,D., the numbers and morphology of ganglion cells appeared normal in the TTX-injected eyes. 3.2.2. Vehicle injections do not affect astrocyte morphology Fig. 1 shows evidence already described that TTX injected into the vitreous humour causes a loss of axon-related morphology of astrocytes. In that experiment, the other Žleft. eye received the same number and volume of injections, but of the PBS vehicle only. Fig. 3A and B show the loss of axon-related morphology in astrocytes at P90 after TTX was injected into the right eye between P7 and P27. In that case the left eye received no injection. In an additional animal, however, the right eye received the same number and volume of injections of vehicle over the same period ŽP7–27. and the retina was examined at the same age P90. There was no evidence of any loss of axon-related morphology Ždata not shown.. 3.2.3. Pre-ganglion cell block had no effect In one animal, the right eye received four injections of APB at daily intervals from P3 to P5 and was examined at P6. Comparison of Fig. 3E Žuninjected eye, left. with 4B ŽAPB-injected eye, right. suggests that this partial blockage of ganglion cell activity did not affect astrocyte-axon relationships. 3.2.4. Subthreshold injections Fig. 3G,H shows one of two cases in which no change in morphology of astrocytes was apparent in the TTX-injected eye. In each case only a single 1 ml injection of TTX had been given. Fig. 3G and H compare regions at the periphery of the uninjected Žleft. eye ŽFig. 3G. and injected Žright. eye ŽFig. 3H. of a kitten aged P4, after a single injection at P2. In this and the second case, the orientation astrocyte processes to axon bundles was as prominent in the TTX-injected eye as in the control eye.
4. Discussion This paper describes a change in the morphology of astrocytes in the cat retina which follows the injection into vitreous humour of a Naq channel blocker ŽTTX. in doses sufficient to block impulses in the axons of ganglion cells. The morphological change is seen a loss of orientation of
astrocytes to axon bundles. Control experiments provided evidence that the effects observed are not caused by the process of injection, are dose-related and are not a side-effect of astrocyte death. The change may be caused by the loss of a trophic signal passing from axons to astrocytes, presumably where axon processes contact node-like specialisations of the axon membrane. TTX appears to block the signal, suggesting that its release is spike-mediated. The nature of the signal molecule remains unknown. Possibilities include a polypeptide acting through specific receptors borne by astrocytes, or less specific mechanisms such as the ionic fluxes induced by action spikes. 4.1. TTX effect is restricted to deÕelopmental stages The TTX-induced changes observed appeared stronger in younger neonates ŽP11, Fig. 1. than in older ŽP20, Fig. 2. and were not apparent in adults. Conversely, if induced early and maintained to P90, the changes appear to persist ŽFig. 3A–D.. It thus seems that astrocytes are plastic in the face of particular stimuli during early postnatal life and much more resistant to change after approximately P30. Karschin et al. w12x showed evidence of plasticity in adult astrocytes after very long Ž18 months. survival times. It is possible that if TTX blockade could be maintained over such long times evidence of plasticity of adult astrocytes could be obtained. The mechanisms which control plasticity in astrocytes are not known. 4.2. Relation to preÕious work A shift of astrocyte morphology from an axon-related to a stellate form was shown previously w4,13x in retinas in which ganglion cell axons degenerated following lesions. As Wong et al. w27x demonstrated and Fig. 3C,D confirm, ganglion cells survive long-term exposure to TTX and the morphology of their somas and axons appears normal. The changes observed here have thus occurred in the physical presence of the axons, but in the absence of the normal function of their sodium channels. The importance of the spike activity of retinal ganglion cells for the development of another class of macroglia was shown by Barres et al. w1,2x who used TTX blockade of the retina to gain evidence that a spike-mediated signal from axons controls the proliferation of oligodendrocytes in the optic nerve. The present finding is further evidence of spike-mediated signals passing from neurones to macroglia, in the presence instance to astrocytes. 4.3. Reshaping or death? Our results do not indicate whether the astrocytes affected when TTX is injected die, leaving a population of
C. Gargini et al.r DeÕelopmental Brain Research 110 (1998) 177–184
astrocytes never related to axons; or change shape from an elongated shape to stellate. Both death and reshaping may be involved. Karschin et al. w12x reported a decrease in astrocyte density in the adult cat retina after degeneration of axon bundles, indicating that neurones express a signal essential for the survival of astrocytes. Nevertheless, when ganglion cell axons are totally removed a population of astrocytes persists w4,12x, indicating that many astrocytes can survive without axon-derivedd signals. As in the present study, these axon-independent astrocytes had a highly stellate morphology. 4.4. A direct effect? Retinal astrocytes are known to have TTX-sensitive Naq channels w5,19x, raising the possibility that the changes observed follow an action of TTX directly on the astrocytes. Sontheimer et al. w19x concluded that TTX-blockage of Naq channels in the membrane of astrocytes in vitro leads to their depolarisation, to the slowing of the NaqrKq ATPase activity and in sufficient Žmicromolar. concentrations to death. Present observations do not rule the possibility that TTX affects astrocyte morphology directly, but several speak against it. First, the changes observed here in astrocyte morphology resemble the changes observed when axons degenerate w4,12x. That degeneration presumably removes axon-to-astrocyte communication without any specific action on channels in the astrocyte membrane. Second, the changes observed here are largely restricted to astrocytes related to axons, leaving those initially stellate and those related to vessels relatively unaffected ŽFigs. 1 and 2.. Finally, although there is now a considerable literature on agents which cause reshaping of astrocytes, including cAMP w23x, thrombin w8,22x, phorbol esters w6x, endothelins w14x and hypoxia w28x there is as yet no role suggested for TTX-sensitive Naq Žor other ionotropic. channels. Thus, although the present observations point to a specific axon-to-astrocyte signal occurring when the axons carry action spikes, the nature of the signal remains unkown. The present results add to the known interactions between axons and the glia which invest them, giving evidence that axons induce morphological specialisation in astrocytes by the spike-mediate release of a trophic signal.
Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, the Australian Retinitis Pigmentosa Association, the Sir Zelman Cowen Universities Fund, the Medical Foundation of the University of Sydney, MURST 60% and progetta integrato CNRrUNI.
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Research report
Evidence that the influence of ganglion cell axons on astrocyte morphology is mediated by action spike activity during development Claudia Gargini
a,d
, Stefania Deplano b, Silvia Bisti
c,d,)
, Jonathan Stone
e
a
e
Istituto Policattedra, Discipline Biologiche, UniÕersita` di Pisa, CNR, Pisa, Italy b Istituto di Anatomia Comparata, UniÕersita` di GenoÕa, GenoÕa, Italy c Dipartimento STB, UniÕersita` di L’Aquila, L’Aquila, Italy d Istituto di Neurofisiologia, CNR, Pisa, Italy NSW Retinal Dystrophy Research Centre, Department of Anatomy and Histology, UniÕersity of Sydney, Sydney, Australia Accepted 23 June 1998
Abstract In many mammal retinas, the morphology of astrocytes is strongly influenced by nearby axons of ganglion cells. Astrocyte processes stretch along the axons, fine extensions of the processes contact node-like specialisation of the axon membrane and the morphology of the adult astrocyte is strongly determined by this relationship. The mechanism which attracts astrocyte processes to contact specific regions of the axon membrane is not known however. This study presents evidence that in the neonatal cat blocking the impulse activity of ganglion cells with the Naq-channel blocker tetrodotoxin ŽTTX. leads to a loss of the axon-related morphology of astrocytes. The morphological change induced in astrocytes by TTX was greater in younger animals and could not be detected in the adult. Conversely, if the TTX block was maintained for 4 postnatal weeks the changes induced in astrocytes persisted at least to 13 weeks. The TTX-induced loss of axon-related morphology in astrocytes suggests that the signal by which axons attract astrocyte processes to contact the axonal membrane in ways which modify astrocyte morphology is released by action spike activity during development. q 1998 Elsevier Science B.V. All rights reserved. Keywords: TTX; Stellation; Macroglia; Cat
1. Introduction The morphology of retinal astrocytes is strongly influenced by attachments made by their processes. In the periphery of the cat retina, where ganglion cell axons are sparse, astrocytes adopt the stellate shape after which they were named ŽFig. 1, see also figure 1 in Karschin et al. w12x and in Karschin et al. w13x and figure 3D in Chan-Ling and Stone w4x.. Many astrocytes in more central regions of the cat’s retina, by contrast, are strongly elongated ŽFig. 1 and see also figures 1A–C in Chan-Ling and Stone w4x., stretched along the axon bundles into a shape similar to that adopted by the other major class of axon-related glia,
)
Corresponding author. Istituto di Neurofisiologia, Via S. Zeno 51, 56127 Pisa, Italy; Fax: q39-50-559725; E-mail: [email protected]
oligodendrocytes w3x. Previous reports w4,12x have considered whether astrocytes so differently shaped are different ‘types’. Because many astrocytes were observed with intermediate morphology and mixed contacts however, differently shaped and committed astrocytes were interpreted as variants of a single class of astrocytes, all competent to adopt relationships with vessels, neurones and Muller ¨ cells. The same studies gave evidence of plasticity in astrocytes. When axons or blood vessels were eliminated retinal astrocytes appeared to reshape to a stellate form, leading ChanLing and Stone w4x to suggest that the stellate form is a ’default’ shape. In the optic nerve w24,26x and spinal cord w17x astrocyte processes contact the membrane of axons at their nodes. The retinal segments of ganglion cell axons in the cat lack myelin but nevertheless astrocyte processes do converge on node-like specialisations of the intraretinal length of these axons w11,21x. Ganglion cell axons may express a
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factor at their nodes which attracts astrocyte processes and causes them to stretch along axons. In the rat, the node-like specialisations found along the intraretinal lengths of axons contain concentrations of Naq channels w10,15x suggesting that, like nodes of myelinated axons, they are the sites of major currents during action potentials. We hypothesised that the astrocyte-trophic signal expressed by axons is expressed at their nodes when action potentials occur. To test this hypothesis, we have examined whether a drug Žtetrodotoxin, TTX. which specifically blocks Naq channels and thereby action spikes will, when applied to the retina by intravitreal injection, result in an adaptive change in the morphology of retinal astrocytes.
2. Materials and methods 2.1. TTX injections Injections were made into the right eye of cats aged P7 to adult, anaesthetised with halothane Ž2–3%. in a 2:1 mixture of nitrous oxide and oxygen. The TTX injections followed published protocols w7,23x. Briefly, the TTX Ž5 mM in phosphate buffered saline ŽPBS.. was injected into the vitreous humour of the right eye. The initial injection was always 1 ml in volume. Subsequent injections Žif made. were made at 3-day intervals and the vol. injected was increased by 5–10% with each injection. Each injection was made through the temporal region of the sclera between the attachment of the iris and the anterior edge of the retina. Close care was taken to make repeat injections through the hole used initially. The pupillary reflex was always abolished after the first injection, confirming the block of electrical activity from the retina.
P6. This dosage is effective in suppressing ON-responses in the light adapted cat retina for as long as 24 h w9x. 2.3. GFAP, neurofilament immunocytochemistry and lectin histochemistry At selected intervals after TTX injection, animals were euthanised with an overdose of Ketalar. Eyes were immersion-fixed in 4% paraformaldehyde in PBS at pH 7.4 for 2 h. The retinas were then dissected and prepared as wholemounts. Most were labelled with an antibody to glial fibrillary acidic protein ŽGFAP. to demonstrate astrocytes and with the G. simplicifolia isolectin B4 Žconjugated to the fluophore FITC. to demonstrate blood vessels. Briefly, the retinas were washed in PBS and incubated in a rabbit polyclonal anti-GFAP ŽDako, diluted 1:200 in PBS. overnight at 48C, then washed and incubated in goat anti-rabbit IgG conjugated to Cy3 or TRITC ŽSigma, diluted 1:400 in PBS. for 2 h at room temperature. They were then washed, incubated in the isolectin Ždiluted 1:20. overnight at 48C, mounted in glycerol and viewed by confocal microscopy. Selected pieces of wholemounts were labelled for GFAP and for neurofilaments, to demonstrate axons directly. The pieces were washed in PBS and incubated in Triton Ž0.3% in PBS. for 15 min and then in bovine serum albumen Ž2%. for 30 min. They were then incubated for 48h in the rabbit polyclonal anti-GFAP and simultaneously in a monoclonal antibody to neurofilament, both diluted in PBS containing 0.1% Triton. They were then washed in PBS and incubated for 2 h at room temperature in a mixture of FITC-conjugated anti-rabbit IgG ŽVector, 1:75. and TRITC-conjugated anti-mouse IgG ŽSigma, 1:128.. They were then washed and coverslipped.
2.2. APB injection
2.4. HRP histochemistry
In one kitten, the right eye was injected with D,L-2amin-4-phosphonobutyric acid ŽAPB., the glutamate analog which blocks glutamate release by rod bipolar and ON-cone bipolar cells ww18x, reviewed in Ref. w25xx. Four injections Žeach 5 ml of a 100-mM solution. were made at daily intervals from P2 to P5; the animal was euthanised at
In two animals injected with TTX from P7 to P30, horseradish peroxidase ŽHRP. was injected into the dorsal lateral geniculate nucleus ŽdLGN. 2 days prior to euthanasia at P90 and the retinas were prepared as wholemounts and reacted for HRP, using the protocols of Deplano et al. w7x.
Fig. 1. Effect of TTX on astrocyte morphology. The retinas were labelled for astrocytes Žfor GFAP, red. and vessels Žwith the G. simplicifolia lectin, green.. Over the rectangular regions outlined the green signal is suppressed, to demonstrate astrocyte morphology more clearly. The arrows indicate points at which the orientation of astrocytes to vessels can be seen. ŽA,B. Midperipheral regions of retina from the vehicle-injected ŽA. and TTX-injected ŽB. eyes of a P12 kitten, which received injections at ages P8 and P11. ŽC,D. Regions from the edge of the developing inner vasculature of the same P12 kitten. Fig. 2. Effect of TTX on astrocyte morphology. As in Fig. 1, the retinas were labelled for astrocytes Žfor GFAP, red. and vessels Žwith the G. simplicifolia lectin, green.. Over the rectangular regions outlined the green signal is suppressed, to demonstrate astrocyte morphology more clearly. The arrows indicate points at which the orientation of astrocytes to vessels can be seen. ŽA,B. Regions from the midperiphery of the retinas from the uninjected ŽA. and TTX-injected ŽB. eyes of a P27 kitten. The right eye received 7 TTX injections at 3-day intervals from P7–27. ŽC,D. Peripheral regions from the uninjected ŽC. and TTX-injected ŽD. eyes of the same P27 kitten. The regions shown are close the edge of the retina.
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3. Results 3.1. TTX injections change astrocyte morphology Figs. 1 and 2 show our principal findings; the key comparisons are between Fig. 1A and B, between Fig. 1C and D, between Fig. 2A and B and between Fig. 2C and D. 3.1.1. Early neonate Fig. 1 shows areas of retinas from a neonatal cat injected with TTX at P8 and P11, then examined at P12. Fig. 1A and B are from the left and right ŽTTX-injected. retinas respectively and show astrocytes ŽGFAP-labelled, red. and vessels Žlectin-labelled, green. in corresponding regions of mid-peripheral retina. In each of Fig. 1A–D, the lectin labelling Žgreen. of vessels is suppressed over a rectangular window, to show astrocyte morphology more clearly. In all regions of both retinas, astrocytes formed a continuous network at the inner surface of the retina. Within that network, individual astrocytes are distributed in an interactive process of ‘contact spacing’ w4x. In Fig. 1A, some astrocytes can be seen to form the glia limitans of vessels, for example at the sites arrowed, while others course along axon bundles, from upper left to lower right. The apposition of astrocyte processes to axons and vessels confirms many earlier observations w4,12,13,16,20x. In Fig. 1B, the orientation of astrocytes to axons is lost, though astrocyte processes remain oriented to underlying vessels. Fig. 1C and D show regions from the same two retinas, at the edge of the spreading vessels. In Fig. 1C, astrocytes are aligned to underlying vessels Žsmall arrows. and also to axons bundles, which course from right to left. In Fig. 1D, from the TTX-injected retina, the orientation of astrocyte processes to vessels persists Žarrows. but the orientation to axons appears lost.
the mid-periphery of the right Žinjected. eye of the same animal. The eye received 7 injections of TTX at 3-day intervals from P7 to P27, for a total of 12 ml. The astrocytes still show their stellate shape and their processes still invest underlying vessels but their orientation to bundles is lost. Fig. 2C shows a second area from a more peripheral region of the retina from the uninjected Žleft. eye of the same animal and Fig. 2D shows a corresponding region from the retina of the injected Žright. eye. In Fig. 2C, some astrocytes extend along axon bundles Žthough these bundles are sparser than in Fig. 2A.; others spread their processes in a stellate pattern while others wrap blood vessels. In Fig. 2D, many astrocytes are stellate in form and some invest vessels but their orientation to axon bundles appears lost. The continuing astrocytic investment of vessels after TTX injection is seen clearly in the ‘window’ in Fig. 2D within which the vessel labelling is suppressed. 3.1.3. TTX induced changes persist Fig. 3A and B show areas of retina from the vehicle-injected Žleft. eye and of the TTX-injected Žright. eye of a P90 kitten which received 7 injections at 3-day intervals from P7 to P27. The last injection was thus 60 days before the tissue was examined. The absence of astrocyte orientation to axons remains clear ŽFig. 2B.. 3.1.4. TTX does not affect adult astrocytes In one adult cat, the right eye received 10 TTX injections at 3-day intervals. Three days after the last injection the animal was euthanised and the retina examined. No change in astrocyte morphology was detected Ždata not shown.. 3.2. Control obserÕations
3.1.2. Late neonate Fig. 2A shows a region from the mid-periphery of the uninjected Žleft. eye of a kitten aged P29. The astrocytes show more evidence of their characteristic stellate shape and the underlying vessels are finer and less profuse than at P12 ŽFig. 1., both differences evidence of the increasing maturation of the retina. The processes of many astrocytes form the glia limitans of vessels, while others are elongated along axons, which cross the field from upper right to lower left. Fig. 2B shows a corresponding region from
3.2.1. Ganglion cells and their axons persist after TTX injection Astrocytes are known to lose their orientation to axons if the axons are destroyed w12,13x. Previous studies using TTX injected into the vitreous humour to stop action spike activity in ganglion cells reported that ganglion cells survive, although their dendritic morphology may change w27x. Nevertheless, we checked whether TTX might be toxic to ganglion cells in two ways. First, a TTX-injected
Fig. 3. ŽA. Astrocytes from the mid-peripheral region of the uninjected eye a P90 kitten, given 7 injections at 3-day intervals from P7 to P27. ŽB. Astrocytes from the mid-peripheral region of the TTX- injected eye of the same kitten as in ŽA.. ŽC. Area of midperiphery of the same eye as in A labelled for astrocytes Žleft panel. and for the underlying axons Žright panel.. ŽD. Area of midperiphery of the same eye as in ŽB. labelled for astrocytes Žleft panel. and for the underlying axons Žright panel.. ŽE,F. Ganglion cell somas and nearby axons labelled with horseradish peroxidase transported retrogradely from the dLGN, in wholemounts of retinas from the uninjected ŽE. and TTX-injected ŽF. eyes of a P90 kitten. The TTX injections were made at 3-day intervals from P7 to P30; and the HRP injections into the dLGN 2 days prior to euthanasia. ŽG,H. Astrocytes labelled for GFAP in wholemounts of retinas from the uninjected ŽG. and APB-injected ŽH. eyes of a P6 kitten, which received 4 injections at daily intervals from P2–5. ŽI,J. Astrocytes in wholemounts of retinas from the uninjected ŽI. and TTX-injected ŽJ. eyes of P4 kitten which received a single injection at age P2.
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retina showing extensive changes in astrocyte morphology was double-labelled for neurofilament protein as well as GFAP. The normal close relationship of astrocyte processes to axons ŽFig. 3A. is greatly reduced in the TTX-injected retina ŽFig. 3B left panel. but the axons remain Žright panel.. Second, using retrogradely transported HRP, which demonstrates the axons, somas and proximal dendrites of ganglion cells ŽFig. 3C,D., the numbers and morphology of ganglion cells appeared normal in the TTX-injected eyes. 3.2.2. Vehicle injections do not affect astrocyte morphology Fig. 1 shows evidence already described that TTX injected into the vitreous humour causes a loss of axon-related morphology of astrocytes. In that experiment, the other Žleft. eye received the same number and volume of injections, but of the PBS vehicle only. Fig. 3A and B show the loss of axon-related morphology in astrocytes at P90 after TTX was injected into the right eye between P7 and P27. In that case the left eye received no injection. In an additional animal, however, the right eye received the same number and volume of injections of vehicle over the same period ŽP7–27. and the retina was examined at the same age P90. There was no evidence of any loss of axon-related morphology Ždata not shown.. 3.2.3. Pre-ganglion cell block had no effect In one animal, the right eye received four injections of APB at daily intervals from P3 to P5 and was examined at P6. Comparison of Fig. 3E Žuninjected eye, left. with 4B ŽAPB-injected eye, right. suggests that this partial blockage of ganglion cell activity did not affect astrocyte-axon relationships. 3.2.4. Subthreshold injections Fig. 3G,H shows one of two cases in which no change in morphology of astrocytes was apparent in the TTX-injected eye. In each case only a single 1 ml injection of TTX had been given. Fig. 3G and H compare regions at the periphery of the uninjected Žleft. eye ŽFig. 3G. and injected Žright. eye ŽFig. 3H. of a kitten aged P4, after a single injection at P2. In this and the second case, the orientation astrocyte processes to axon bundles was as prominent in the TTX-injected eye as in the control eye.
4. Discussion This paper describes a change in the morphology of astrocytes in the cat retina which follows the injection into vitreous humour of a Naq channel blocker ŽTTX. in doses sufficient to block impulses in the axons of ganglion cells. The morphological change is seen a loss of orientation of
astrocytes to axon bundles. Control experiments provided evidence that the effects observed are not caused by the process of injection, are dose-related and are not a side-effect of astrocyte death. The change may be caused by the loss of a trophic signal passing from axons to astrocytes, presumably where axon processes contact node-like specialisations of the axon membrane. TTX appears to block the signal, suggesting that its release is spike-mediated. The nature of the signal molecule remains unknown. Possibilities include a polypeptide acting through specific receptors borne by astrocytes, or less specific mechanisms such as the ionic fluxes induced by action spikes. 4.1. TTX effect is restricted to deÕelopmental stages The TTX-induced changes observed appeared stronger in younger neonates ŽP11, Fig. 1. than in older ŽP20, Fig. 2. and were not apparent in adults. Conversely, if induced early and maintained to P90, the changes appear to persist ŽFig. 3A–D.. It thus seems that astrocytes are plastic in the face of particular stimuli during early postnatal life and much more resistant to change after approximately P30. Karschin et al. w12x showed evidence of plasticity in adult astrocytes after very long Ž18 months. survival times. It is possible that if TTX blockade could be maintained over such long times evidence of plasticity of adult astrocytes could be obtained. The mechanisms which control plasticity in astrocytes are not known. 4.2. Relation to preÕious work A shift of astrocyte morphology from an axon-related to a stellate form was shown previously w4,13x in retinas in which ganglion cell axons degenerated following lesions. As Wong et al. w27x demonstrated and Fig. 3C,D confirm, ganglion cells survive long-term exposure to TTX and the morphology of their somas and axons appears normal. The changes observed here have thus occurred in the physical presence of the axons, but in the absence of the normal function of their sodium channels. The importance of the spike activity of retinal ganglion cells for the development of another class of macroglia was shown by Barres et al. w1,2x who used TTX blockade of the retina to gain evidence that a spike-mediated signal from axons controls the proliferation of oligodendrocytes in the optic nerve. The present finding is further evidence of spike-mediated signals passing from neurones to macroglia, in the presence instance to astrocytes. 4.3. Reshaping or death? Our results do not indicate whether the astrocytes affected when TTX is injected die, leaving a population of
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astrocytes never related to axons; or change shape from an elongated shape to stellate. Both death and reshaping may be involved. Karschin et al. w12x reported a decrease in astrocyte density in the adult cat retina after degeneration of axon bundles, indicating that neurones express a signal essential for the survival of astrocytes. Nevertheless, when ganglion cell axons are totally removed a population of astrocytes persists w4,12x, indicating that many astrocytes can survive without axon-derivedd signals. As in the present study, these axon-independent astrocytes had a highly stellate morphology. 4.4. A direct effect? Retinal astrocytes are known to have TTX-sensitive Naq channels w5,19x, raising the possibility that the changes observed follow an action of TTX directly on the astrocytes. Sontheimer et al. w19x concluded that TTX-blockage of Naq channels in the membrane of astrocytes in vitro leads to their depolarisation, to the slowing of the NaqrKq ATPase activity and in sufficient Žmicromolar. concentrations to death. Present observations do not rule the possibility that TTX affects astrocyte morphology directly, but several speak against it. First, the changes observed here in astrocyte morphology resemble the changes observed when axons degenerate w4,12x. That degeneration presumably removes axon-to-astrocyte communication without any specific action on channels in the astrocyte membrane. Second, the changes observed here are largely restricted to astrocytes related to axons, leaving those initially stellate and those related to vessels relatively unaffected ŽFigs. 1 and 2.. Finally, although there is now a considerable literature on agents which cause reshaping of astrocytes, including cAMP w23x, thrombin w8,22x, phorbol esters w6x, endothelins w14x and hypoxia w28x there is as yet no role suggested for TTX-sensitive Naq Žor other ionotropic. channels. Thus, although the present observations point to a specific axon-to-astrocyte signal occurring when the axons carry action spikes, the nature of the signal remains unkown. The present results add to the known interactions between axons and the glia which invest them, giving evidence that axons induce morphological specialisation in astrocytes by the spike-mediate release of a trophic signal.
Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, the Australian Retinitis Pigmentosa Association, the Sir Zelman Cowen Universities Fund, the Medical Foundation of the University of Sydney, MURST 60% and progetta integrato CNRrUNI.
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