Functional mapping of the neuronal substrates for drug tolerance in Drosophila

Abstract

Physical dependence on alcohol and anesthetics stems from neuroadaptive changes that act to counter the effects of sedation in the brain. In Drosophila, exposure to either alcohol or solvent anesthetics have been shown to induce changes in expression of the BK-type Ca(2+)-activated K(+) channel gene slo. An increase in slo expression produces an adaptive modulation of neural activity that generates resistance to sedation and promotes drug tolerance and dependence. Increased BK channel activity counteracts the sedative effects of these drugs by reducing the neuronal refractory period and enhancing the capacity of neurons for repetitive firing. However, the brain regions or neuronal populations capable of producing inducible resistance or tolerance remain unknown. Here we map the neuronal substrates relevant for the slo-dependent modulation of drug sensitivity. Using spatially-controlled induction of slo expression we identify the mushroom bodies, the ellipsoid body and a subset of the circadian clock neurons as pivotal regions for the control of recovery from sedation.

Figure 1

Temperature-dependent Gal4 induction of UAS-slo expression. A) Map of the UAS-slo construct. Black arrows indicate primers used for PCR amplification from the previously described B52 construct. White boxes indicate untranslated regions, gray boxes indicate protein coding regions. B) Schematic representation of the two-part Gal4/UAS system. A cell- or tissue-specific enhancer drives expression of the Gal4 transcription factor gene. Once translated, GAL4 binds to its DNA binding target UAS to induce expression from the adjacent slo cDNA. GAL4 binding is enhanced at higher temperatures, resulting in higher transcription rates from the UAS-slo cDNA. C) Expression from the UAS-slo transgene in an uninduced state (lacking a Gal4 driver, but otherwise identical) and induced by four independent Gal4 drivers after incubation of female adult flies at 18°C or 30°C for 3 consecutive days. Gal4 drivers were: the cholinergic Cha-Gal4 driver, the dopaminergic and serotonergic Ddc-Gal4 driver, and the OK107-Gal4 and c309-gal4 mushroom body drivers. In all 4 Gal4 lines, induction of UAS-slo was significantly higher in flies incubated at 30°C than those of flies incubated at 18°C. D) Expression of the endogenous neural slo transcript in the four independent UAS-slo/Gal4 lines after incubation at 18°C or 30°C for 3 consecutive days. In all 4 lines, endogenous slo expression was unaffected by the temperature treatment. Error bars show SEM, n=3, * indicates p<0.05 by Student’s t-test.

Figure 2

The temperature induction protocol does not affect recovery from sedation in flies lacking a Gal4 driver. Shown are recovery curves from benzyl alcohol sedation of wild-type CS flies and the UAS-slo line generated for this study. Plotted are averages of the percentage of flies that resumed climbing the walls of the vial over time. A–B) The gray curve represents the recovery of a population incubated at 18°C for 3 consecutive days prior to the benzyl alcohol sedation treatment. The black curve represents the recovery of a population incubated at 30°C for 3 consecutive days prior to the benzyl alcohol sedation treatment. Both, wild-type CS (A) and the UAS-slo transgenic flies lacking a Gal4 driver (B) show no statistical difference between the recovery rates of the groups incubated at the different temperatures. C–D) Capacity to develop tolerance to benzyl alcohol is also unaffected by the presence of the UAS-slo transgene. The gray curve represents the recovery of a population recovering from its 1st exposure to benzyl alcohol. The black curve represents the recovery of a population recovering from its 2nd exposure to benzyl alcohol. Both, wild-type CS (C) and the UAS-slo transgenic flies (D) develop tolerance to benzyl alcohol after a single exposure, as shown by an the more rapid recovery from sedation from their second exposure than from the first. Error bars represent SEM (* indicates p<0.05 by the Log-rank test, n=6).

Figure 3

Induction of slo in the mushroom and ellipsoid bodies causes resistance to sedation. Shown are recovery curves from benzyl alcohol sedation of four different Gal4 lines driving expression of the transgenic slo cDNA and the respective Gal4 lines lacking the UAS-slo transgene as a control. Plotted are averages of the percentage of flies that resumed climbing the walls of the vial over time. The black curve represents the recovery of a population incubated at 30°C for 3 consecutive days prior to the benzyl alcohol sedation treatment (slo-induced). The gray curve represents the recovery of a population incubated at 18°C for 3 consecutive days prior to the benzyl alcohol sedation treatment (slo-uninduced). Temperature-dependent induction of the UAS-slo transgene with 106y-Gal4 (A), c041-Gal4 (C), c309-Gal4 (E), and OK107 (G) enhanced the rate of recovery from benzyl alcohol sedation, while the respective controls (B, D, F and H) did not. Error bars represent SEM (* indicates p<0.05 by the Log-rank test, n=6).

Figure 4

Induction of slo in the lateral ventral neurons causes sensitization to sedation. Shown are recovery curves from benzyl alcohol sedation of three different Gal4 lines driving expression of a transgenic slo cDNA and the respective Gal4 lines lacking the the UAS-slo transgene as a control. Plotted are averages of the percentage of flies that resumed climbing the walls of the vial over time. The black curve represents the recovery of a population incubated at 30°C for 3 consecutive days prior to the benzyl alcohol sedation treatment (slo-induced). The gray curve represents the recovery of a population incubated at 18°C for 3 consecutive days prior to the benzyl alcohol sedation treatment (slo-uninduced). Temperature-dependent induction of the UAS-slo transgene with tim-Gal4 (A), Pdf-Gal4 (C), and 16y-Gal4 (E) causes sensitization to sedation by benzyl alcohol as indicated by the slower recovery rate. Sedation recovery of the respective controls (B, D, and F) remained unaffected by the temperature induction protocol. Error bars represent SEM (* indicates p<0.05 by the Log-rank test, n=6).

Figure 5

Increased expression of slo in distinct neuronal subgroups of the mushroom bodies, ellipsoid bodies and Lateral Neurons differentially affect resistance to sedation. A) Chart shows the change in resistance to sedation after temperature-dependent slo induction in the twenty different Gal4-lines tested. The UAS-slo/+ line lacking a Gal4 Driver is displayed as a reference. Change in resistance was determined by calculating the relative difference in the 50% recovery time between slo-induced and slo-uninduced flies. Error bars are SEM, * denotes a significant difference between the recovery curves of the slo-induced and slo-uninduced flies as calculated by Log-rank test. Three lines expressing slo in the mushroom bodies (Ok107, c309, and 106y) and two lines expressing in the ellipsoid body (106y and c041) induce a significant increase in resistance to sedation. Three lines expressing slo in the lateral neurons (16y, tim and Pdf) induce a significant decrease in resistance to sedation. B) Expression patterns within distinct neuronal subgroups of the mushroom bodies, ellipsoid body and Lateral Neurons (LN) are indicated for each Gal4 line (+). Except for line 238y (gray rectangle), all mushroom body lines expressing in the α′/β′ lobes (Ok107, c309, and 106y) induce a significant increase in resistance to sedation (light-red rectangle). For the ellipsoid body, only those lines expressing in the R1 ring neurons (106y and c041) induce a significant increase in resistance to sedation (dark-red rectangle). All lines expressing in the LN_V neurons (16y, tim and Pdf) induce a significant decrease in resistance to sedation (green rectangle).

Figure 6

Hypothetical model for neuronal control of tolerance to sedation. A) Schematic representation of the fly brain highlighting the structures shown to influence drug resistance in a slo-dependent manner. Induction of slo within the ellipsoid body R1 ring neurons (ebR1/dark red) and mushroom bodies α′/β′ lobes (light red) results in drug resistance, while slo induction in the large and small ventral lateral neurons (lLN_V and sLN_V) causes drug sensitization (green). While there is no neuroanatomical evidence of a direct interaction between the PDF-positive LN_Vs and the ellipsoid body, strong PDF-receptor expression in ellipsoid body neurons suggests this possibility. This hypothetical scenario is marked with a “?”. B) A working model for the regulation of drug resistance is that the ellipsoid body is the central controller of arousal and that the mushroom bodies and ventral lateral clock neurons modulate the activity of the ellipsoid body in opposite ways in response to slo induction. Increased slo expression in the mushroom bodies promotes ellipsoid body-mediated arousal generating benzyl alcohol resistance while increased slo expression in the ventral lateral clock neurons suppress this function of the ellipsoid body leading to a reduction in arousal and sensitization to benzyl alcohol.