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. 2020 Dec 29;9:e63493. doi: 10.7554/eLife.63493

Figure 2. Activating periaqueductal gray (PAG)-projecting POA neurons elicits ultrasonic vocalizations (USVs) in the absence of social cues.

(A) (Left) Viral strategy to express ChR2 in POAPAG neurons. (Right) Example trial showing that optogenetic activation of POAPAG neurons elicits USV production in an isolated animal. (B) (Left) Raster plot shows USVs elicited in many trials in a representative mouse following optogenetic activation of POAPAG neurons. (Middle) Mean USV rate aligned to delivery of blue light pulses plotted for that same mouse. (Right) Mean USV rate plotted for N = 8 mice following optogenetic activation of POAPAG neurons. Please note that one mouse in which USVs were elicited by optogenetic stimuli that did not include the 2s-long, 10 Hz stimulus is excluded from the summary analysis shown in the right-most panel. Gray shading above and below the mean in the middle and right panels represents S.E.M. See also Figure 2—figure supplement 1. (C) Representative confocal image and quantification of in situ hybridization performed on POAPAG neurons (tdTomato, red), showing overlap with Esr1 (white) and VGAT (green). DAPI is blue, N = 2 mice. (D) (Left) Viral strategy used to express ChR2 in Esr1+ POA neurons. (Middle) Raster plot shows USVs elicited in many trials in a representative mouse following optogenetic activation of Esr1+ POA neurons. (Right) Mean USV rate plotted for N = 7 mice following optogenetic activation of Esr1+ POA neurons. Gray shading above and below the mean represents S.E.M. (E) Same as (D), for experiments in which the axon terminals of Esr1+ POA neurons were optogenetically activated within the PAG. Data shown for stimulation with10s-long, 20 Hz blue light pulses. Please note that one mouse in which USVs were elicited by optogenetic stimuli that did not include the 10s-long, 20 Hz stimulus is excluded from the summary analysis shown in the right-most panel. (F) Summary plots show mean number of USVs per second of optogenetic stimulation (left, p=0.0013, one-way ANOVA between all groups, with post-hoc t-tests showing that each experimental condition was significantly different from control conditions at p<0.01), mean number of optogenetic trials with USVs (middle, p=1.8E-6, one-way ANOVA between all groups, with post-hoc t-tests showing that each experimental condition was significantly different from control conditions at p<0.01), and mean latency from onset of optogenetic stimulus to onset of first USV (right) for mice in which optogenetic stimulation was applied to POAPAG neurons (N = 9 mice), Esr1+ POA neurons (N = 7 mice), Esr1+ POA axon terminals within the PAG (N = 5 mice), GFP-expressing Esr1+ POA neurons (N = 5 mice), VGlut+ POA neurons (N = 3 mice), VMHPAG neurons (N = 3), and Esr1+ POA axon terminals within the ventral tegmental area (VTA) (N = 4 mice). See also Figure 2—figure supplements 1–3 and Figure 2—source data 1.

Figure 2—source data 1. Source data for Figure 2C and F.

Figure 2.

Figure 2—figure supplement 1. Additional characterization of ultrasonic vocalizations (USVs) elicited by optogenetic activation of preoptic area (POA) neurons.

Figure 2—figure supplement 1.

(A) Mean USV rate across trials aligned to delivery of blue light pulses plotted for N = 8 individual mice following optogenetic activation of POAPAG neurons. Plots represent the individual animals summarized in the right-most panel of Figure 2B. The example animal represented in the left panels of 2B is mouse number four here. Gray shading above and below the mean in the middle and right panel represents S.E.M. (B) Top: the minimum latency from the onset of optogenetic stimulation to the onset of the first optogenetically elicited USV is plotted for three groups of mice: ChR2 expressed in POAPAG neurons (N = 9 mice, blue points), ChR2 expressed in Esr1+ POA neurons (N = 7 mice, black points), and ChR2 expressed in axon terminals within the PAG of Esr1+ POA neurons (N = 5 mice, red points). Bottom: same plot as above, but with the y range restricted from 0 to 0.5 s. (C) Quantification of the duration of USV bouts elicited by 2 s, 10–20 Hz optogenetic stimulation of POA neurons. Each column represents a different mouse, and each point represents the duration of an USV bout elicited by a 2s-long optogenetic stimulus. Colors as in (B). Please note that five animals shown in (B) had only a small number of 2s-long optogenetic activation trials and were therefore not included in the analysis shown in (C). See also Figure 2—figure supplement 1—source data 1.
Figure 2—figure supplement 1—source data 1. Source data for panels B and C of Figure 2—figure supplement 1.
Figure 2—figure supplement 2. Additional information related to the optogenetic activation of POAPAG, POA-Esr1+ neurons, and AmgC/M-PAG neurons.

Figure 2—figure supplement 2.

(A) Real-time place preference tests were performed in which either POA or AmgC/M neurons were optogenetically activated when mice were in one of two sides of a test chamber (see Materials and methods). (B) The mean speeds of mice were calculated and aligned to the onset of optogenetic activation of POAPAG neurons (top left), POA-Esr1+ neurons (top right), and AmgC/M-PAG neurons (bottom left). (Bottom, right) Distances between male AmgC/M-PAG-ChR2 mice and their female social partners were calculated and aligned to the onset of optogenetic activation of AmgC/M-PAG neurons. Gray shading represents S.E.M. See also Figure 2—figure supplement 2—source data 1.
Figure 2—figure supplement 2—source data 1. Source data for panel A of Figure 2—figure supplement 2.
Figure 2—figure supplement 3. Dual tracing of the axonal projections of POAPAG and AmgC/M-PAG neurons.

Figure 2—figure supplement 3.

POAPAG neurons are labeled with GFP, and AmgC/M-PAG neurons are labeled with tdTomato. (Top left) Plane of section through the preoptic area (POA) shows the beginning of POAPAG cell body labeling in green and axonal projections to the lateral POA from AmgC/M-PAG neurons in red. (Middle left) Plane of section through the AmgC/M-PAG shows axonal projections from POAPAG neurons that overlap with AmgC/M-PAG cell bodies. (Bottom left) Plane of section through the ventral tegmental area (VTA)/SNr. (Top right) Plane of section including the rostral PAG and retrorubral area. (Bottom right) Place of section through the caudal PAG, showing the overlapping terminal fields of POAPAG and AmgC/M-PAG neurons. These representative images are from a female mouse, and we found that POAPAG and AmgC/M-PAG neurons have similar axonal projections in males and females (data not shown).