Figure 2.

Two Discrete Locations in the Brainstem Affect Vocalizations

(A) Cooling effects on evoked calls. Left: map of the left brainstem surface presenting cooling effects on call numbers. The measures are taken relative to obex (obex: −13.5 mm [posterior] to Bregma and 0 mm lateral). In the sagittal axis, positive coordinates indicate location anterior to obex and negative coordinates indicate location posterior to obex. The heatmap shows the Z score of the effect of cooling on the call numbers following a stimulation, negative values (red) represent a reduction of call numbers. The white areas have not been sampled in this animal. Middle: amplitude of three cooling locations (indicated by the lines) in the same experiment, y axis depicts relative signal strength as a fraction of the detection range of the microphone settings. The observed reduction of amplitude corresponds to a drop of 3.8 dB for the top trace, 0.2 dB for the center trace, and 11.3 dB for the bottom trace, each calculated between the last three trials before cooling and the last three trials during cooling. The red bars indicate onsets and durations of electrical PAG stimulations. The blue backdrop indicates onset and duration of cooling. The yellow and blue bars indicate calls depicted on the right plot. Right: spectrograms of calls before cooling, late cooling, and rewarming as indicated by the respective colors in the waveform plot to the left. Note the strong reduction of call amplitude and duration during cooling of the anterior (top trace) and posterior (bottom trace) locations, but not during cooling of the intermediate location (middle trace).

(B) Map of brainstem stimulation effects from an example animal. Two distinct areas evoked calls upon stimulation. Orientation and scale as in (A). Red indicates locations where calls could be triggered, blue where no calls were triggered. White areas have not been stimulated in this animal.

(C) Average map of the effect of localized brainstem cooling. Depicted is the reduction of evoked calls during the last three stimulation trials compared against the last three trials prior to cooling onset. For each experiment the Z score was calculated and then averaged across experiments. Orientation and scale as in (A).

(D) Mean response to direct brainstem stimulation. Orientation and scale as in (A). Depth data not taken into account. Unsmoothed version of Figures 2C and 2D are depicted in Figure S2.

(E) Top: comparison of different regions on the brainstem with (I) located anterior, (II) medial, and (III) posterior. The grid aligns with the map of (C) and (D). There is a significant difference in the effect of cooling between the three regions (Friedmann, p = 0.0156). The results of the Tukey-Kramer test are detailed in the text. During stimulation, regions differed in the likelihood to evoke calls (Friedmann, p = 0.0006). Bottom: random shuffling of data and comparison against the measured values for each region. Bottom left: the anterior (yellow) and posterior regions (blue) showed a stronger reduction of calls (random shuffling, p = 0.0475 and p = 0.001, respectively). The central (gray) region showed less call number reduction than the randomly shuffled data (random shuffling, p = 0.0406). Bottom right: for stimulation, both the anterior region as well as the posterior region showed a significantly higher rate of evoked calls (random shuffling, p = 0.03 and p = 0.00005, respectively). The center region did not differ from the shuffled data.