Metabolic activity in optic tectum during regeneration of retina in adult goldfish.

AbstractRetinal and visual function returns following retinal destruction by ouabain in adult goldfish (Carassius auratus).Although the precise cellular mechanisms are unclear, the ability to regenerate CNS neurons and connections thatsubsequently sustain visual behavior is remarkable, especially for an adult vertebrate. In this paper, we ask whethervisual stimulation via new retinal cells can activate existing cells in the optic tectum, which normally receives thelargest retinal projection in this species. The right eyes of adult goldfish were injected with ouabain. After 1–18weeks the conscious, freely moving fish were exposed to spatially and temporally varying visual stimuli and theresulting tectal metabolic activity was determined with the autoradiographic deoxyglucose method. In normalcontrols without lesions, visual stimulation produced equally strong metabolic activity in both tectal hemispheres,peaking in the layer where most retinotectal projections terminate (N 5 6). One week after ouabain injection,metabolic activity in the contralateral, deprived tectum was dramatically reduced (N 5 5), closely resembling theeffect of unilateral ocular enucleation (N 5 5). However, 9–18 weeks after ouabain injection, metabolic activityin the deprived tectum recovered to a level that was statistically indistinguishable from normal controls (N 5 6).These findings suggest that, after a comprehensive cytotoxic lesion of the retina, regenerated ganglion cells notonly establish new connections with the preexisting optic tectum, but also effectively transmit visual informationthey receive from newly generated photoreceptors to the “old” tectum.Keywords: Regeneration, Ouabain, Retina, Goldfish, Tectum, DeoxyglucoseIntroductionThe teleost fish retina continues to add new functional neuronsthroughout life (Johns & Easter, 1977; Johns & Fernald, 1981;Falzett et al., 1988; Powers et al., 1988). New neurons are alsoproduced in response to retinal damage (Lombardo, 1968). Forexample, exposure to the cytotoxin ouabain leads to severe neuraldegeneration, yet the retina is capable of completely reconstitutingitself from surviving stem cells (Maier & Wolburg, 1978; Ray-mond & Hitchcock, 1997). Remarkably, the newly regeneratedganglion cells reconnect with the already existing adult optictectum (Stuermer et al., 1985), the photoreceptors’visual pigmentsreturn (Cameron & Powers, 2000), the electroretinogram is re-stored (Mensinger & Powers, 1999), and several visual behaviorsreappear (Lindsey et al., 1995; Mensinger & Powers, 1999).To support visual behavior, regenerated ganglion cell axonsmust develop functional connections with more proximal neuronsin the visual pathway. Here we ask specifically whether a newlyreconstituted goldfish retina establishes functional synapses withits primary projection site, the optic tectum. If not, then connec-tions to other, smaller visual areas must sustain the recovery ofvisual behavior that is known to occur.To address this question, we used the autoradiographic deoxy-glucose method (Sokoloff et al., 1977) to measure tectal metabolicactivity induced by visual stimulation in goldfish with both normaland regenerating retinal projections to separate hemispheres. Pre-liminary findings have appeared elsewhere (Powers & Melzer,1995; Powers et al., 1996).MethodsGoldfish (Carassius auratus 4–6 cm sbl) were maintained understandard laboratory conditions (Mensinger & Powers, 1999), anes-thetized in 0.04% tricaine methanesulfonate solution (MS 222;Sigma Chemical Co., St. Louis, MO), and revived by flowingaerated fresh water over the gills after all surgical survival proce-dures. Eleven fish had 1–2 ml 0.4 mM ouabain (Sigma) injectedinto the vitreal chamber of the right eye through an incision in theventral limbus (intraocular concentration 30 mM; see Mensinger &Powers, 1999). In five additional fish, the right eye was surgicallyremoved and the eye socket was rinsed with Ringer, filled withGelfoam (Upjohn, Kalamazoo MI), and covered with a thin layerof cyanoacrylate cement.