A Specialized Neural Circuit Gates Social Vocalizations in the Mouse

Abstract

Vocalizations are fundamental to mammalian communication, but the underlying neural circuits await detailed characterization. Here, we used an intersectional genetic method to label and manipulate neurons in the midbrain periaqueductal gray (PAG) that are transiently active in male mice when they produce ultrasonic courtship vocalizations (USVs). Genetic silencing of PAG-USV neurons rendered males unable to produce USVs and impaired their ability to attract females. Conversely, activating PAG-USV neurons selectively triggered USV production, even in the absence of any female cues. Optogenetic stimulation combined with axonal tracing indicates that PAG-USV neurons gate downstream vocal-patterning circuits. Indeed, activating PAG neurons that innervate the nucleus retroambiguus, but not those innervating the parabrachial nucleus, elicited USVs in both male and female mice. These experiments establish that a dedicated population of PAG neurons gives rise to a descending circuit necessary and sufficient for USV production while also demonstrating the communicative salience of male USVs. VIDEO ABSTRACT.

Keywords: activity-dependent labeling; nucleus retroambiguus; periaqueductal gray; ultrasonic; vocalization.

Figure 1.. The caudolateral PAG contains neurons that are active during USV production.

(A) Spectrogram of USVs produced by a male mouse during a social encounter with a female. Inset shows expanded view. (B) Quantification of number of USVs produced by male mice during encounters with female social partners (black), female urine (blue), or a non-social odor, ethanol (green). (C) Left-most panel shows location of the caudolateral PAG in sagittal and coronal section. Four right-most panels show representative confocal images of Fos expression in the caudolateral PAG following social experience with a female social partner accompanied by USV production, female urine accompanied by USV production, female social partner without USV production, and female urine without USV production (green, Fos; blue, NeuroTrace). Dotted line delineates the boundary dorsal to which PAG Fos+ neurons were quantified in this plane of section (see Methods). (D) (Top) The total number of Fos-positive caudolateral PAG neurons is positively correlated with the number of USVs produced by different males. Quantification is shown for the same plane of section as in (C), and color-coding is as in (B). (Bottom) The strength (R²) and significance (p value) of the linear regression between the number of USVs produced and Fos expression in the caudolateral PAG is plotted across a 1.2 mm extent of the PAG (N = 12 males given female social partner; N = 10 female urine; N = 2 ethanol). (E) Two-color in situ hybridization, showing the overlap of caudolateral PAG neurons expressing c-Fos mRNA (green) following USV production and also either vGluT2 (red, left) or GAD1/2 (red, right) mRNA. Scale bars on insets, 50 um. See also Fig. S1.

Figure 2.. The production of courtship USVs requires PAG-USV neurons and promotes female social affiliation.

(A) Schematic of the CANE method (top) and the experimental time line to permanently express transgenes in PAG-USV neurons using CANE (bottom). (B) Schematic of the viruses injected into the caudolateral PAG of a Fos^TVA male following a vocal encounter with a female to label PAG-USV neurons with GFP. (C) Confocal image showing overlap between CANE-GFP-labeled PAG-USV neurons (green) after USV production toward a female and Fos (red) induced by a subsequent vocal encounter with a female. (D) Quantification of the overlap between CANE-GFP-labeled PAG-USV neurons and Fos expression elicited by USVs (red points) or by wheel running in the home cage (black). (E) Schematic of the viruses injected into the caudolateral PAG of a Fos^TVA male to express tetanus toxin (TeLC) in PAG-USV neurons. (F) Blocking neurotransmitter release from PAG-USV neurons with CANE-driven expression of TeLC abolishes the production of male courtship USVs (N = 12 mice, p < 0.001, Wilcoxon signed-rank test). (G) Blocking neurotransmitter release from PAG-USV neurons has no significant effect on total time spent courting a female (p = 0.32, Wilcoxon signed-rank test). (H) Schematic of the three-chambered test. (I) Vocal output of the control PAG_USV-GFP male is plotted against female preference for that male (> 0.5 is preference for control male)). N = 37 tests from N = 5 PAG_USV-TeLC mice each paired against 1-3 PAG_USV-GFP controls, with each TeLC/GFP pair tested with 2-4 females. The red vertical line indicates where the dataset was split into low vocal rate trials (gray) and high vocal rate trials (black). See also Fig. S2.

Figure 3.. Activating PAG-USV neurons is sufficient to elicit USV production in the absence of social cues

(A) (Left) Schematic of the viruses injected into the caudolateral PAG of a Fos^TVA male to express hM3Dq in PAG-USV neurons. (Middle) Spectrogram showing USVs produced by a CNO-treated PAG_USV-hM3Dq male. (Right) Total USVs produced in a 60 min. solo test period are shown for different groups of males in chemogenetic experiments: control males (no virus, no CNO, black), males not expressing hM3Dq but treated with CNO (gray), and males with CANE-driven expression of hM3Dq in PAG-USV neurons that were treated with saline (i.p.) or CNO (p = 0.02 for PAG_USV-hM3Dq mice saline versus CNO USVs, Wilcoxon signed-rank test). (B) Optogenetic activation of PAG-USV neurons elicits USVs from males in the absence of female cues. Spectrograms comparing optogenetically-elicited USVs (left) and female-directed USVs (right) from the same male. Bottom panels show expanded views. (C) Distributions of 5 acoustic parameters are shown for optogenetically-elicited USVs (blue) and female-directed USVs (gray) for 2 PAG_USV-ChR2 mice. Asterisks indicate acoustic parameters whose median value changed significantly in the same direction for both mice (p < 0.05, Mann Whitney U tests). See also Figs. S3–5.

Figure 4.. PAG-USV neuronal activity gates USVs and specifies vocal bout duration

(A) (Left) Schematic of the viruses injected into the caudolateral PAG of a Fos^TVA male to express ChR2 in PAG-USV neurons. (Right) The duration of optogenetically-elicited bouts of USVs is similar to the duration of laser stimuli used to activate PAG-USV neurons (mean ± SE).(B) Comparison of the number of USV syllables elicited per second of laser stimulation is shown for 10 Hz versus tonic laser stimuli for PAG-ChR2 mice (blue) and PAG_USV-ChR2 mice (orange; p = 0.03 for difference, Wilcoxon signed-rank test). (C) Same as (B), except comparing mean syllable duration for optogenetically-elicited USVs optogenetically (p = 0.38). (D) Representative portions of two bouts of optogenetically-elicited USVs are shown for a PAG_USV-ChR2 mouse, with breathing shown in the top traces (inspirations are downward deflections in green, expiration is shown in red, see Methods), USVs shown in the spectrograms (middle), and laser stimuli shown in blue (bottom). Gray shading shows the clear alignment between USVs and expiration. (E) (Left, middle) Polar plots showing the distribution of onset times of individual optogenetically-elicited USV syllables relative to the duty cycle of the preceding laser pulse (10 Hz, 50ms on and 50ms off) for PAG-ChR2 mice (left) and PAG_USV-ChR2 mice (middle). Blue line indicates the laser-on portion of the laser duty cycle. Radial values (ranging from 0 to 0.25) represent proportion of total observations at a given time in the laser duty cycle, and total area inside the shaded black line is equal to one. (Right) Polar plot showing the distribution of onset times of individual optogenetically-elicited USV syllables relative to the respiratory cycle. (F) The probability of obtaining an optogenetically-elicited USV (black) and the mean latency from laser onset to USV onset (gray) are plotted in relation to the time that each laser pulse fell within the respiratory cycle (see Methods; green shading indicates inspiration, red shading indicates expiration, n = 1051 laser pulses from N = 1 PAG_USV-ChR2 mouse). (G) Breathing traces are shown for a PAG_USV-ChR2 mouse during optogenetic activation of PAG-USV neurons (top, breathing shown in black, laser in blue, USVs in spectrogram) and during a control period in which the laser was turned on but was disconnected from the optogenetic ferrule (bottom; breathing, black; laser, blue). (H) Optogenetic activation of PAG-USV neurons caused a significant increase in the proportion of the respiratory cycle occupied by expiration (left, blue, p < 0.001, Wilcoxon signed-rank test) and no change in respiration rate (right, blue). When the laser was not connected to the ferrule, laser light alone did not change the expiration/inspiration ratio (left, black, p = 0.42) and caused a significant increase in breathing rate (right, black, p < 0.01), likely due to a startle response (mean ± SE).

Figure 5.. PAG-USV neurons project to downstream vocal-respiratory centers

Representative confocal images show (A) representative CANE-driven GFP labeling of PAG-USV cell bodies and PAG-USV axonal projections to (B) the red nucleus, (C) rostral PAG, (D) the lateral parabrachial nucleus, (E) the pontine reticular formation, (F) the magnocellular reticular formation of the rostral medulla, and (G) nucleus retroambiguus in the caudal medulla. (H) Quantification of axonal projections, presented as projection density (total square pixels of axonal innervation normalized by the total area of each region, organized rostral to caudal, see Methods, N = 4 mice, mean ± SE). ZI, zona incerta; mRT, mesencephalic reticular formation; SC, superior colliculus; PrCnF, precuneiform; CnF, cuneiform; PBn, parabrachial nucleus; Gt, gigantocellular reticular formation; IRt, intermediate reticular formation; GiA/V, magnocellular reticular formation; MdV, ventral medullary reticular formation; MdD, dorsal medullary reticular formation; LPGi, paragigantocellular reticular formation; PCRt, parvocellular reticular formation; RAm, nucleus retroambiguus.

Figure 6.. Activating RAm-projecting PAG neurons elicits USVs in male and female mice

(A) (Left) Schematic showing the viral strategy used to express ChR2 in PAG-RAm neurons. Middle and right panels are confocal images of PAG (middle) and RAm (right), showing ChR2 labeling (green) and Fos expression (red) following USV production. (B) Spectrograms comparing USVs elicited by optogenetic activation of PAG-RAm neurons (left) and female-directed USVs (right) from the same animals. Top row shows example spectrograms from a male mouse, and the spectrograms in the bottom row are from a female. (C) Distributions of 5 acoustic parameters are shown for optogenetically-elicited USVs (blue) and female-directed USVs (gray) for N = 4 male and N = 3 female PAG_RAm-ChR2 mice (N = 2 females excluded from analysis that produced few female-directed USVs). Asterisks indicate acoustic parameters whose median value changed significantly in the same direction for 7/7 mice (p < 0.05, Mann Whitney U tests.) See also Fig. S2 and S6.