Fan-shaped body neurons are involved in period-dependent regulation of long-term courtship memory in Drosophila

Abstract

In addition to its established function in the regulation of circadian rhythms, the Drosophila gene period (per) also plays an important role in processing long-term memory (LTM). Here, we used courtship conditioning as a learning paradigm and revealed that (1) overexpression and knocking down of per in subsets of brain neurons enhance and suppress LTM, respectively, and (2) suppression of synaptic transmission during memory retrieval in the same neuronal subsets leads to defective LTM. Further analysis strongly suggests that the brain region critical for per-dependent LTM regulation is the fan-shaped body, which is involved in sleep-induced enhancement of courtship LTM.

Figure 1.

per overexpression in OK348-positive neurons enhances courtship LTM. In each box plot, the box encompasses the interquartile range. A line is drawn at median and vertical bars corresponding to the 10th and 90th percentiles. Each square within a box is the mean. White and gray boxes show the CIs of naive and conditioned males, respectively. In the labels for the x-axis, “2–4” and “2–3” represent UAS-per2-4 and UAS-per2-3, respectively. (A) 5-d memory after 5-h conditioning in OK348/UAS-per2-4, c232/UAS-per2-4, OK107/UAS-per2-4, and 30Y/UAS-per2-4 males. (P) Probability; (N) sample size; (*) P < 0.05. (B) 5-d memory after 5-h and 7-h conditioning in +/UAS-per2-4 and OK348/+ males; (*) P < 0.05. (C) 5-d memory after 5-h conditioning in +/UAS-per2-3 and OK348/UAS-per2-3 males; (*) P < 0.05. (D) 5-d memory in tub-GAL80^ts/+; OK348/+ and tub-GAL80^ts/UAS-per2-4; OK348/+ males. Males were kept at 21°C. Four hours before conditioning, males were transferred to an environment of 30°C (restrictive temperature), and subsequently conditioned for 5 h at this temperature. Experimental paradigms are indicated above the graphs. The first and second arrows indicate the beginning of the 5-h conditioning and the 10-min test, respectively; (**) P < 0.01. (E) 5-d memory after 5-h conditioning in tub-GAL80^ts/+; OK348/+ and tub-GAL80^ts/UAS-per2-4; OK348/+ males. All experiments were carried out at the permissive temperature (21°C).

Figure 2.

Effect of disruption of synaptic transmission in OK348-positive neurons on courtship LTM. (A–C) 5-d memory after 7-h conditioning. All procedures in the experiments were carried out at the permissive (25°C) or restrictive (30°C) temperature. OK348/UAS-shi^ts1 (A), OK348/+ (B), and +/UAS-shi^ts1 (C) males were used. (P) Probability; (N) sample size; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (D) 5-d memory after 7-h conditioning in OK348/UAS-shi^ts1 males. Experimental paradigms of temperature-shift are indicated above the graphs. The first and second arrows indicate the beginning of 7-h conditioning and 10-min test, respectively; (*) P < 0.05; (**) P < 0.01. (E) 5-d memory after 7-h conditioning in F₁ males between UAS-per RNAi and 30Y, pdf-GAL4, OK348, or Ddc-GAL4; (*) P < 0.05; (***) P < 0.001.

Figure 3.

Confocal images of GAL4-positive neurons in the adult brain. (A–F) Frontal views of the adult brain. F₁ males generated between UAS-mCD8::GFP and OK348 (A), c232 (B), OK107 (C), 30Y (D), pdf-GAL4 (E), or Ddc-GAL4 (F) were used. GFP fluorescence was observed under a confocal microscope (Carl Zeiss LSM710). Z sections were collected at 1-µm intervals and processed to construct projections through an extended depth of focus. Scale bars, 100 μm. (Triangle) Fan-shaped body (A); (arrows) neurons in the lateral region (A,C–E); (arrowheads) DAL neurons (F). (G) GAL4-driven GFP in clusters of the cells in the lateral region of the brain (green) and PDF immunolabeling (magenta). F₁ males generated between UAS-mCD8::GFP and pdf-GAL4, OK348, 30Y, or OK107 were used. Scale bars, 20 μm.