Paul D. Klinger

Puromycin Effect on Memory Fixation in the Goldfish

Puromycin injected intracranially into the goldfish produces impairment of memory for a shock-avoidance response. Intracranial injection of puromycin aminonucleoside, or of saline has no effect. Puromycin does not afject performance in naive or overtrained goldfish.

Environmental Control of Amnesic Effects of Various Agents in Goldfish

Abstract Fish given a single electroconvulsive shock or intracranial administration of puromycin, acetoxycycloheximide, or KCl immediately following shock avoidance training show amnesia on retraining days later. When the treatments are given 24 hr after training no amnesia develops. But amnesia can be obtained 24 hr after training with KCl, puromycin and acetoxycycloheximide, if the fish are replaced in the intertrial environment for a brief period just prior to injection. Implications of these results to the consolidation hypothesis of memory formation are discussed. Previously unreported results on the amnesic effects of intracranial KCl in goldfish are also described.

Camptothecin Blocks Memory of Conditioned Avoidance in the Goldfish

Intracranial injection of 10 to 75 micrograms of camptothecin, a plant alkaloid that blocks RNA synthesis in eucaryotic cells, blocks incorporation of tritiated uridine into RNA in the goldfish brain. Injection of 10 to 50 micrograms of the drug within 1.5 hours of training results in greatly diminished memory, tested 1 week later. Injection of the drug 5 or 24 hours after training produces no measurable effect on retention of the learned response.

Dissociation of enhanced ornithine decarboxylase activity and optic nerve regeneration in goldfish

The significance of a previously observed increase in retinal ornithine decarboxylase (ODC, EC 4.1.1.17) following optic nerve crush was investigated in goldfish using a specific irreversible inhibitor of ODC, alpha-difluoromethylornithine (DFMO). Retinal ODC activity and putrescine levels were reduced 80% and 40%, respectively, by intraperitoneal injection(s) of DFMO. In fish in which the right optic nerve was crushed and DFMO was injected at the time of crush, at 2 days and at 4 days later, retinal ODC levels were maximally decreased after the third injection and returned to normal levels by 14 days post-crush (PC). Retinal tubulin synthesis was examined at 10 days PC. No difference in the post-crush/normal ratio of tubulin synthesis was observed between saline-injected and DFMO-injected groups. Neuritic extension was also examined in retinal explant cultures from 11-day PC retina. The neuritic growth index of DFMO-treated retinas did not differ from that of control retinas. Behavioral studies revealed no difference in the rate of recovery of vision between the two groups. Similar results were obtained in experiments using fish maintained in a 0.1% DFMO solution. These results suggest that the increase in retinal ODC associated with nerve crush does not play a causal role in restoration of function.

Anterograde and retrograde effects of electroconvulsive shock and of puromycin on memory formation in the goldfish

Electroconvulsive shock [ECS] or puromycin administered prior to training did not significantly impair acquisition of shock-avoidance in goldfish. Significant retention deficits are observed on retraining 72 hr later in groups of fish that received ECS 2.5, 1 or 0.5 hr before training as well as in groups that received ECS 0, 4 or 24 hr after training. Puromycin produces significant retention deficits on retraining when given 24, 16, 8, 4 or 0 hr prior to, or 0 or 0.25 hr following training. A temporal course of development of the retention deficit that has been seen with puromycin was not observed with ECS as the deficit was maximal at the earliest train-retrain interval examined. ECS administered before both training and retraining did not relieve the deficit. Since performance was not diminished in fish retrained just after ECS, it appears that this proactive effect of ECS reflects disruption of memory rather than state-dependent learning.

Cholesterol Synthesis and Nerve Regeneration

Abstract: In this report, we examine the requirement of cholesterol biosynthesis and its axonal transport for goldfish optic nerve regeneration. Cholesterol, labeled by intraocular injection of [3H]mevalonolactone. exhibited a delayed appearance in the optic tectum. Squalene and other minor components were labeled but not transported. Following optic nerve crush, the amount of labeled cholesterol transport was elevated, while retinal labeling was not altered relative to control fish. A requirement for cholesterol biosynthesis is inferred from the inhibition of neurite outgrowth in retinal explants caused by the cholesterol synthesis inhibitor, 20, 25‐diazacholes‐terol. The inhibition of growth could be overcome by addition of mevalonolactone, but not cholesterol, to the medium. Intraperitoneal administration of 200 nmol of dia‐zacholesterol resulted in 92‐98% inhibition of retinal cholesterol synthesis and accumulation of labeled des‐mosterol and other lipids in fish retina and brain which persisted for 2 weeks. Diazacholesterol‐treated fish showed no reduction in the amount of lipid‐soluble radioactivity transported following intraocular injection of [3H]mevalonolactone, but there were alterations in the chromatographic pattern of the transported labeled lipids. In contrast to its effects on neurite outgrowth in vitro, diazacholesterol did not inhibit optic nerve regeneration in vivo, as measured both by arrival of labeled rapidly transported protein at the tectum and by time required for the return of visual function.

Comparative Neurotoxicity of Tubulin-Binding Drugs: Inhibition of Goldfish Optic Nerve Regeneration'

Intraperitoneal or intraocular (io) injection of tubulin-binding drugs in goldfish, Carassius auratus L., inhibited axonal regeneration or restoration of functional synapses in optic axons following optic nerve crush. One eye was used to detect effects on regeneration and the other was kept intact to detect effects on maintenance of established optic circuits. Regeneration was assessed by measuring the time to reappearance of a visually evoked branchial suppression response. Three drugs, vincristine, vinblastine, and podophyllotoxin, administered semiweekly by ip injection, each inhibited regeneration at doses that did not impair maintenance of response. Similar results were previously reported for ip colchicine. Vincristine was several times more potent than podophyllotoxin or colchicine and 25 times more potent than vinblastine. Picropodophyllotoxin, an isomer of podophyllotoxin which has low affinity for tubulin, did not inhibit regeneration. The io experiments showed that maintenance of vision was reversibly inhibited by a single injection of 0.05 micrograms/g of colchicine but unaffected by 0.01 microgram/g, and that administration of the lower dose immediately following optic nerve crush inhibited regeneration. Intraocular lumicolchicine, a colchicine photoisomer which has low affinity for tubulin, did not inhibit maintenance or regeneration. In contrast, an io dose of vincristine sufficient to inhibit visual recovery also blocked maintenance of vision. Thus regeneration and maintenance effects could not be dissociated for io vincristine suggesting its mechanism of action on retinal cells is different. A conditioning lesion was shown to decrease the time to reappearance of the visually evoked branchial response following optic nerve crush, which indicates that it is a sensitive index of the rate of axonal outgrowth to the optic tectum.

Temperature-dependent consolidation of puromycin-susceptible memory in the goldfish

Memory of a shock-avoidance task in goldfish ( Carassius auratus ) maintained at 20°C shows a temporal gradient of insusceptibility to post-trial injection of puromycin upon testing 7 days later. Treatment with the antimetabolite 24 hr after training has no effect on retention. There is a significant decrease in the puromycin-induced memory loss if fish are warmed to 30°C for a 90-minute interval between conditioning and injection of puromycin. If fish are cooled to 4.5°C for 24 hr between learning and puromycin injection, a significant block of memory results. There are in addition time-independent effects of the cold treatment on performance. Although temperature increase from 20 to 30°C does not in itself affect retention, it does cause a 3-fold stimulation of incorporation of 3 H-leucine into brain protein. Decrease in temperature from 20 to 4.5°C reduces protein labeling by 86–97 percent.

Nervus terminalis innervation of the goldfish retina and behavioral visual sensitivity

The possibility that axon terminals of the nervus terminalis in the goldfish retina regulate visual sensitivity was examined psychophysically. Fish were classically conditioned to respond in darkness to a diffuse red light conditioned stimulus. Bilateral ablation of the olfactory bulb and telencephalon had no significant effect on response threshold which was measured by a staircase method. Retinopetal nervus terminalis fibres thus appear to play no role in maintaining scotopic photosensitivity.

Spatial discrimination in goldfish following bilateral tectal ablation

Goldfish were classically conditioned to discriminate between right and left or nasal and temporal presentations of a spot-of-light conditioned stimulus (CS). After the conditioning, the fish were administered bilateral optic tectum ablation followed by weekly sessions of conditioning trials to test for retention or relearning of the discrimination response. As their behavioral photosensitivity is greatly decreased, the ablates were dark-adapted prior to each session and trials were administered in darkness. Right x left discrimination was retained postoperatively but the nasal x temporal discrimination was blocked. Sham-operated controls discriminated between the nasal and temporal CS when dark-adapted and tested in darkness. Subsequent transection of the optic nerves obliterated response to the CS, indicating that tectum ablates detect and respond to the CS retinally and not extraretinally. We conclude that memory of visual spatial learning is mediated by non-tectal brain structures and that the ablate can discriminate between right- and left-eye input but sees the CS too diffusely to distinguish its location within the monocular field.