Effects of Inactivation of the Periaqueductal Gray on Song Production in Testosterone-Treated Male Canaries (Serinus canaria)

Abstract

Male canaries (Serinus canaria) display seasonal changes in the motivation to sing which have been found to be dependent on the action of testosterone (T). During the breeding season when T is high, males sing at a higher rate compared with males with low T. The effect of T on song rate is known to be mediated by the medial preoptic nucleus (POM); however, it is unclear how T signaling in POM impacts song production. One potential mechanism is via modulation of dopaminergic input into song control nuclei by the periaqueductal gray (PAG). In order to test the role of PAG in T-mediated song production, we treated male canaries with peripheral T implants and implanted a guide cannula targeting the PAG. Through this guide cannula, we transiently inactivated PAG with injections of the GABA_A agonist, muscimol. Each bird received multiple infusions of both muscimol and saline with a 48-h washout period between treatments. The order of injection type was randomized and counterbalanced between individuals. Muscimol infusion into the PAG, but not nearby regions, increased the latency to sing post-injection. These results support the hypothesis that PAG is involved in the production of song, potentially mediating the motivation to sing or alternatively interfering with the pre-motor activity of nucleus RA. Other song features were however not affected.

Keywords: motivation; periaqueductal gray; singing behavior; songbird.

Graphical abstract

Figure 1.

Experimental procedures. A, Timeline of experimental procedures (left) and image of cannula trajectory (right) created with BioRender. B, Coronal sections demonstrating final placement of cannula targets after surgery, four in PAG, five in ICo, and five misses. C, Example images of muscimol spread for an individual where cannula placement was on the lateral edge of PAG. Left is a Nissl brightfield photomicrograph showing the location of PAG and right is the corresponding fluorescent photomicrograph.

Figure 2.

Change in latency to begin singing post-infusion. Differences in latency for each individual across trial type. Each line connects the average latency to sing following muscimol infusion to the average latency following saline infusion for an individual bird. Black lines indicate summary statistics (mean and SEM), while colored lines indicate the average latency for each individual bird. Individual points indicate the latency for each single trial. The three sections of the graph represent cannula placement (ICo, miss, or PAG). In order to determine whether an ANOVA was appropriate to determine differences between treatments, we tested the assumption of homoscedasticity (Extended Data Fig. 2-1A). To further assess the differences between groups and treatments, we used bootstrapped estimation statistics of 95% CIs (Extended Data Fig. 2-1B).

Figure 3.

Example song quality measurements. A, The amount of time in minutes spent singing 1 h after singing began. There were no significant differences between treatments or cannula placement. The three sections of the graph represent cannula placement (ICo, miss, or PAG). B, The average duration of songs in seconds. There were no significant differences between treatments or cannula placement. C, Average change in RMS amplitude between treatments (muscimol minus saline). There was a significant interaction between treatment and cannula placement but no main effect. Each line connects the average data following muscimol infusion to the average data following saline infusion. Black lines indicate summary statistics (mean and SEM), while colored lines indicate the average data for each individual bird.

Figure 4.

There were no significant differences in call rate in the hour following infusion. Difference between muscimol and saline trials (average number of calls following muscimol infusion minus average number of calls following saline infusion) for individual birds. Colors represent cannula placement. Each line connects the average call rate following muscimol infusion to the average call rate following saline infusion. Black lines indicate summary statistics (mean and SEM), while colored lines indicate the average data for each individual bird.