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Requirement of Circadian Genes for Cocaine Sensitization in Drosophila

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Science  13 Aug 1999:
Vol. 285, Issue 5430, pp. 1066-1068
DOI: 10.1126/science.285.5430.1066

Abstract

The circadian clock consists of a feedback loop in which clock genes are rhythmically expressed, giving rise to cycling levels of RNA and proteins. Four of the five circadian genes identified to date influence responsiveness to freebase cocaine in the fruit fly,Drosophila melanogaster. Sensitization to repeated cocaine exposures, a phenomenon also seen in humans and animal models and associated with enhanced drug craving, is eliminated in flies mutant for period, clock, cycle, anddoubletime, but not in flies lacking the genetimeless. Flies that do not sensitize owing to lack of these genes do not show the induction of tyrosine decarboxylase normally seen after cocaine exposure. These findings indicate unexpected roles for these genes in regulating cocaine sensitization and indicate that they function as regulators of tyrosine decarboxylase.

In response to exposure to volatilized freebase cocaine, Drosophila perform a set of reflexive behaviors similar to those observed in vertebrate animals, including grooming, proboscis extension, and unusual circling locomotor behaviors (1–3). Additionally, flies can show sensitization after even a single exposure to cocaine provided that the doses are separated by an interval of 6 to 24 hours (1). Sensitization, a process in which repeated exposure to low doses of a drug leads to increased severity of responses, has been linked to the addictive process in humans (4–6) and is potentially involved in the enhanced craving and psychoses that occur after repeated psychostimulant administration.

We have shown circadian variation in the agonist responsiveness ofDrosophila nerve cord dopamine receptors functionally coupled to locomotor output (7). This variation is dependent on the normal functioning of the Drosophila period(per) gene, the founding member of the circadian gene family (8, 9). Because changes in postsynaptic dopamine receptor responsiveness are also seen during cocaine sensitization in vertebrates (10–12), we examined flies mutant in circadian functions for alterations in responsiveness to cocaine.

Wild-type (WT) flies or flies containing a per null mutation, pero , were exposed to 75 μg of cocaine four times over 2 days, and the fraction of flies showing severe responses was quantified after each exposure (Fig. 1A). Whereas WT flies showed sensitization after the initial cocaine exposure,pero flies showed no sensitization either to a normal or increased dose even after repeated exposures. As with WT flies, pero flies showed a dose-dependent increase in the severity of responses, and the normal cocaine-induced types of behaviors were observed (13).

Figure 1

(A)pero flies do not sensitize to repeated cocaine exposures. Wild-type (WT) CantonS(n for each sequential exposure: 105, 95, 30, 17); andpero (n: 81, 60, 61, 57) males were exposed to either 75 or 100 μg of volatilized free-base cocaine twice per day at 6-hour intervals for 2 days, and the behavioral responses were scored during the 5 min after exposure with a behavioral scale (1). Behavioral scores range from 0 (normal behavior) to 7 (death). Behavioral scores of ≥5 indicate rapid twirling, erratic jumping, or paralysis. Significant differences in responses to the first versus subsequent exposures (χ2 test): ** P ≤ 0.01. Error bars are standard deviations calculated for binomial distributions. All behavioral analyses were performed blindly; strains were given drugs in random order by placing a numbered tag in the video field during videotaping. The evaluator assayed behaviors blindly, and was unblinded only after all flies had been scored. (B)pero flies do not modulate quinpirole responsiveness after cocaine exposures. WTOregonR and pero were exposed to 75 μg of volatilized cocaine three times over 2 days and decapitated 4 hours after the last exposure. Flies were decapitated and assayed for locomotion with 2 mM quinpirole as described (24), with modifications (7, 31). Average locomotion ± SEM is shown (n = 30 to 50 flies). Significant differences between sham- and cocaine-treated flies are indicated (* P ≤ 0.05, Student'st test).

per alleles that either shorten or lengthen the circadian periods show distinct patterns of cocaine responsiveness. The short-period mutants perS andperT (14, 15) both showed increased responsiveness to the initial cocaine exposure and weak sensitization to a second 75-μg exposure (Fig. 2A), with only the sensitization ofperS showing statistical significance. Sensitization is not observed in these lines when tested with other cocaine doses (16). The long-period mutantperL1 (17) showed a normal initial cocaine response but no sensitization to a subsequent exposure.

Figure 2

Circadian mutants show altered cocaine responses. (A) per mutants. Flies carrying per mutations, as indicated, were exposed twice to 75 μg of volatilized cocaine 6 hours apart. The number of flies assayed, for first and second exposures, is as follows: WTCantonS, n = 105, 95;pero , n = 81, 60;perS , n = 114, 112;perT , n = 88, 52;perL1 , n = 86, 83. (B) Other circadian mutants. As in (A), except that cocaine doses were adjusted to compensate for differences in cocaine responsiveness to the initial dose: WT CantonS exposed to 75 μg of cocaine, n = 105, 95;timo , n = 66, 63. Circadian mutants exposed to 50 μg of cocaine: clock, n =187, 182; and cycle, n = 79, 79.dbt mutants were exposed to 100 μg of cocaine:dbtS , n = 59, 55;dbtL , n = 52, 51. In both (A) and B), significant differences in responses to the first versus second exposures are indicated (* P≤ 0.05, ** P ≤ 0.01; χ2test).

Similarly, other circadian genes showed effects on cocaine sensitization: Both clock and cycle mutants failed to sensitize when given two doses of cocaine (Fig. 2B). Because these mutants showed an increased sensitivity to the first exposure (16), cocaine doses were decreased to 50 μg. The inability of clock and cycle to sensitize is markedly similar to the behavior of pero mutants. The gene product of timeless (tim), TIM, is required for nuclear translocation of PER and its stability in the cytoplasm; in timo mutants, cytoplasmic PER is degraded and per mRNA levels are constant (18–20). Cocaine responses in timo mutant flies were normal (Fig. 2B), both in initial responsiveness and in showing a robust sensitized response to the second exposure.

Recently, a doubletime (dbt) protein with homology to human casein kinase Iɛ was identified and shown to be required for phosphorylation of PER (21). We tested cocaine responses in two viable dbt mutants,dbtS anddbtL , which shorten and lengthen the circadian locomotor period, respectively (22).dbt mutants required a substantially higher cocaine dose to show behaviors normally observed at 75 μg (Fig. 2B), but even at these higher doses dbt flies did not show significant sensitization. If the role of dbt in cocaine responsiveness is analogous to its role in circadian behavior, then PER phosphorylation status may be important in regulating both initial cocaine responsiveness and sensitization.

Modulation of dopamine receptor responsiveness is important in both the sensitization to cocaine in vertebrate animals and in the circadian modulation of locomotion in Drosophila(7, 23). We tested whether cocaine-sensitized flies would show an increase in the responsiveness of the nerve cord dopamine D2-like receptors by using a preparation of behaviorally active decapitated flies that allows direct addition of drugs to the nerve cord (24). After decapitation, cocaine-sensitized WT flies locomoted significantly more than sham-treated controls in response to the dopamine D2-like agonist quinpirole (Fig. 1B). However, there was no increase in quinpirole responsiveness of pero flies that did not sensitize to repeated cocaine exposures. Thus, similar to the inability of pero mutant to modulate receptor responsiveness as a function of the time of day (7), pero is unable to modulate dopamine receptor responsiveness after cocaine exposure. The observation that cocaine sensitization is associated with increased responsiveness of postsynaptic dopamine receptors shows additional similarities between this system and that in higher vertebrates, where a similar relation holds (12, 23).

In Drosophila, sensitization requires the trace amine tyramine because the mutant inactive, which is defective in sensitization, shows both reduced tyramine and reduced levels of the enzyme involved in tyramine synthesis, tyrosine decarboxylase (TDC) (25). An active role for tyramine in sensitization is indicated because TDC enzyme activity is induced after a single cocaine exposure, with a time course consistent with that for the development of sensitization (25). To test if the correlation between induction of TDC activity and behavioral sensitization holds for the circadian mutants, we measured TDC activity in the circadian mutant flies after a single exposure of cocaine (Fig. 3). In contrast to WT flies, in which TDC activity was induced after cocaine exposure, thepero , cycle, andclock lines that are defective in sensitization showed no such induction; only timo , which showed normal sensitization, induced TDC activity. It thus seems likely that the transcriptional regulator PER, presumably in conjunction with CLOCK and CYCLE, is a direct or indirect regulator of TDC after exposure to cocaine.

Figure 3

(A)pero flies and (B)cycle and clock flies do not induce TDC activity after cocaine exposure. Assays within each panel were carried out with the same batch of TDC assay mix because different batches can show small variations in activity. TDC assays on dissected adult brains were performed as described (25, 32). Wild-typeCantonS, pero , andtimo flies were exposed to 75 μg of volatilized cocaine or a sham treatment, and brains were dissected 6 hours later. clock and cycle flies were exposed to 50 μg of cocaine to compensate for the increased cocaine responsiveness of these lines. The hypomorphicdbtL mutant shows marginal induction of TDC that does not reach statistical significance, consistent with the weak effects of this allele on cocaine sensitization (16). The number of independent samples assayed for each strain is as follows: (A) WT: n = 9;pero : n = 9. (B) WT:n = 3; timo : n =5; cycle: n = 3; clock:n = 3. Pairwise comparisons were performed between sham and cocaine values by Student's t test (* P ≤ 0.05).

The sensitization defects in inactive andper mutant flies can be distinguished by differences in the ability of tyramine feeding to restore sensitization. The locomotor and cocaine sensitization defects in inactive mutant flies can be rescued by feeding tyramine to adults (25), but the sensitization defect in pero flies is distinct, because feeding tyramine topero adults did not rescue sensitization (Fig. 4). We presume that tyramine from the food can enter tyramine nerve terminals in inactiveflies, where it is still subject to a cocaine-stimulated release mechanism that mediates sensitization. The failure of tyramine feeding to rescue sensitization in pero flies is most readily understood if the per gene product is required for this regulated release.

Figure 4

Tyramine feeding increases initial cocaine responsiveness of pero flies but does not restore sensitization. pero flies were fed on instant food (Carolina Biologicals, Burlington, NC) with or without tyramine (20 mg/ml) for 2 days (0 mg/ml tyramine, n = 81, 73; 20 mg/ml tyramine, n = 73, 57). Flies were exposed to the indicated amounts of volatilized cocaine and assayed as in Fig. 3.

Similar to inactive (25), tyramine increases initial cocaine responsiveness inpero flies. Exposure of tyramine-fedpero flies to 35 μg of cocaine induced behaviors normally seen in control flies exposed to 75 μg (Fig. 4). Thus, although long-term increase of tyramine levels can affect initial cocaine responsiveness, it is not sufficient for sensitization in flies lacking normal per function.

A unifying feature of most genes that regulate circadian rhythmicity inDrosophila and vertebrates is the PAS dimerization domain, common to a subset of basic helix-loop-helix transcription factors (26, 27). Within the circadian cycle, CLOCK/CYCLE heterodimers activate per transcription, whereas PER/TIM heterodimers inhibit the activity of CLOCK/CYCLE (28–30). We find that mutations inper, clock, and cycle share the same cocaine phenotype: a deficiency in the ability to sensitize after one or more drug exposures. This similarity leads us to suspect that as in circadian behaviors, these genes are functioning in a common pathway.

In contrast to the above mentioned genes, thetimo mutant showed normal cocaine responses. The implication of this finding is twofold. First, there must be an as yet unidentified PER binding partner that is specifically involved in regulation of drug responsiveness. Second, drug responsiveness is likely regulated by per expression in a set of cells distinct from those involved in circadian function. Intimo mutants, PER levels are constitutively low (19, 20); if the same TIM-containing cells were involved in circadian and cocaine responses,timo flies should not sensitize.

  • * To whom correspondence should be addressed. E-mail: jh6u{at}virginia.edu

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