Characterization of genes involved with classical conditioning that produce differences between bidirectionally selected strains of the blow fly Phormia regina.
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Biomedical subjects
Publications and source records attributed to T Tully.
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To determine whether production of new neurons of a particular type is regulated by the presence of previously differentiated neurons of the same type, we ablated all tyrosine hydroxylase immunoreactive (THIR) cells from larval frog retina with the neurotoxin 6-hydroxydopamine, and examined the retinas in subsequent weeks for newly generated THIR neurons. Three weeks after neurotoxin administration, new THIR cells appeared near the zone of neural proliferation at the ciliary margin at a higher density than that of normal retina, while the densities of other amacrine cell types, serotonin (t-HT) immunoreactive and substance P immunoreactive (SPIR), remained the same as controls. Thus the production of new retinal TRIR cells is selectively up-regulated following ablation of previously differentiated cells of this type.
Guided by genetic recombination experiments that placed the gene for the Drosophila memory mutant amnesiac proximal to forked near carnation, we screened 7 deficiencies of the proximal X in an effort to more precisely localize the amnesiac mutation. After training with a classical conditioning procedure, two deficiency chromosomes, mal8 and mal12, produced amnesiac-like memory deficits in Df/amn flies but not in Df/ + controls. In contrast, the mal13, mal10, mal11, 16-3-22 and DCB1 deficiencies, in combination with either amn or + chromosomes, did not produce amnesiac memory scores. These results indicate that the amnesiac gene lies between the left breakpoint of mal12 (19A1) and the left breakpoint of mal13 (19A1 or A2). This conclusion is supported by the fact that amn males carrying the Y-linked X-chromosome duplication y+ Ymal106 (which spans 18F4-5 to 20A) produce wild-type learning and memory scores. Finally, the data suggest that amnesiac is a complete loss of function (amorphic) mutation, because amn/amn and amn/Df flies have similar learning and memory scores.
Dethier (1957) described an aspect of food-search behavior in Phormia regina as the blow fly dance. A hungry fly walks in relatively straight lines with its proboscis retracted until it encounters food (sucrose). After ingesting even a small amount of sucrose, the fly begins making frequent, tight turns, flexes its front tarsi to bring more chemosensory hairs into contact with the substrate and repeatedly extends and retracts its proboscis. Like the central excitatory state (CES), which causes an increase in proboscis extensions to water when a fly is stimulated with sucrose, the dance lasts longer in hungrier flies or with higher sucrose concentrations. It was considered that dance behavior might be an ethologically relevant manifestation of CES. In order to test this hypothesis, dance duration in lines selected for high- and low-CES levels was measured. As predicted, flies from the high-CES line danced longer than those from the low-CES line, and the CES-dance correlation in individual flies was high. This phenotypic correlation disappeared in the F2 generation of a cross between the high- and low-CES lines, a result indicating that the observed variations in CES and dance duration were not caused by the same set of genes. Further characterization of the underlying genetic system showed that several linked autosomal genes with digenic epistatic interactions and a complex pattern of maternal inheritance were responsible for the difference in dance durations between the high- and low-CES lines.
By changing the conditioned discrimination paradigm of Quinn et al. (1974) from an instrumental procedure to a classical (Pavlovian) one, we have demonstrated strong learning in wildtype flies. About 150 flies were sequestered in a closed chamber and trained by exposing them sequentially to two odors in air currents. Flies received twelve electric shock pulses in the presence of the first odor (CS+) but not in the presence of the second odor (CS-). To test for conditioned avoidance responses, flies were transported to a T-maze choice point, between converging currents of the two odors. Typically, 95% of trained flies avoided the shock-associated odor (CS+). Acquisition of learning was a function of the number of shock pulses received during CS+ presentation and was asymptotic within one training cycle. Conditioned avoidance increased with increasing shock intensity or odor concentration and was very resistant to extinction. Learning was best when CS+ presentations overlap shock (delay conditioning) and then decreased with increasing CS-US interstimulus intervals. Shocking flies immediately before CS+ presentation (backward conditioning) produced no learning. Nonassociative control procedures (CS Alone, US Alone and Explicitly Unpaired) produced slight decreases in avoidance responses, but these affected both odors equally and did not alter our associative learning index (A). Memory in wild-type flies decayed gradually over the first seven hours after training and still was present 24 h later. The mutants amnesiac, rutabaga and dunce showed appreciable learning acquisition, but their memories decayed very rapidly during the first 30 min. After this, the rates of decay slowed sharply; conditioned avoidance still was measureable at least three hours after training.
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With the use of controls not included in previous studies, evidence is presented for the existence of water-induced central excitatory state (w-CES) from analyses of pure-breeding high- and low-CES lines in the blow fly, Phormia regina. The CES lines differed in base-level responsiveness to distilled water (and to saline) as well as in response levels for w-CES and for sucrose-induced CES, which suggests the existence of genetic correlates for several components of the proboscis extension reflex. Recognition of several nonassociative components complicates the interpretation of individual scores in earlier studies of proboscis extension conditioning and of the central excitatory state.
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The requirement for cAMP-dependent protein kinase (PKA) in associative learning of Drosophila was assessed in mutant flies hemizygous for a cold-sensitive allele, X4, of the DC0 gene, which encodes the major catalytic subunit of PKA. DC0X4 hemizygotes died as third-instar larvae at 18 degrees C, the restrictive temperature, but were viable when raised at 25 degrees C. Shifting adult DC0X4 hemizygotes from 25 degrees C to 18 degrees C led to a decrease in PKA activity from 24% to 16% of wild-type without impairing viability. At 25 degrees C, DC0X4 hemizygotes exhibited reduced initial learning relative to controls but normal memory decay in a Pavlovian olfactory learning assay. Shifting the temperature from 25 degrees C to 18 degrees C prior to training reduced initial learning to a similar extent in DC0X4 hemizygotes and controls but resulted in a steeper memory decay curve only in DC0X4 hemizygotes. These observations are suggestive of a role for PKA in medium-term memory formation in addition to its previously established role in initial learning.
The Drosophila mutant turnip (tur) was isolated on the basis of its poor performance in an olfactory learning task, and also has a reduction in protein kinase C (PKC) activity. PKC has been found in the nervous systems of a wide range of organisms and appears to have an important role in learning and memory-related processes. Unfortunately, previous reports documenting the learning defect of tur lacked the controls required to assess the origins of the poor performance of the mutant. We have analyzed the effects of the tur mutation on both associative and nonassociative learning as well as on PKC activity. Additionally, the effects of the mutation on the task-relevant sensorimotor abilities of the flies were assessed. Although we were able to replicate previous behavioral and biochemical results obtained with tur, we discovered that the tur mutation also affected response to electric shock and caused a drastic reduction in the locomotor ability of the flies. Because locomotion is an essential component of the learning assays, this result makes it impossible to conclude that tur specifically affects learning and demonstrates the crucial importance of sensorimotor controls in conditioning experiments.