Experimental Design and timeline

(A) Reference MR scan was obtained using an ex-vivo sample of a 14-day old marmoset. Parasagittal view of the reference scan shows location of injection coordinates targeting the rostral portion of the dorsal ACC (24a and 24b), bilaterally (red). (B) Gas anesthesia was supplied through a custom-made breathing system comprising a facemask fitted with a palate bar with a 0.6mm diameter hole. The palate bar was connected to a vital monitor to accurately detect small tidal end volumes during anesthesia while the animal was secured in the stereotaxic frame. (C) Five-minute vocalization recordings were obtained from infant placed in a softly padded temperature-controlled incubator. (D) Timeline of vocal recordings from postnatal week 2 to postnatal week 6. The ACC lesion was conducted at postnatal week 2 when animals were 14-16 days old. (E) Representative sagittal view of postoperative T2-weighted MR images of a control (left panel) and lesioned (right panel) infant to reveal extent of white hypersignal, which reflects edema due to injections of the excitotoxin and therefore approximate site of the ACC lesion. There was a significant reduction in total ACC volume in the ACC group relative to controls (n=4/per group; F(1,6) = 82.78, p<0.0001). A representative 3-dimensional view of area 24 is presented showing the reduced volume of the ACC (right panel) relative to the normal volume in the control (left panel). (F) Schematic illustration highlights the end point of the experiment involving histological processing and evaluation of cell markers. (G) Longitudinal timeline shows approximate age of animals following vocal recordings, MRI lesion assessments and histological processing.

Lesion verification and impact of early life ACC lesion on vocal downstream

One lesioned animal showed sparing of the lesion in one hemisphere, so the lesion verification was performed in 4 animals only. (A) Left panel shows a sagittal section from the standard marmoset brain depicting the intended ACC lesion shaded in red. The right panel shows schematic lesion reconstructions superimposed on a sagittal and coronal marmoset brain section depicting the extent of the ACC lesion shaded in red. Regions that appear darker indicate greater overlap in the damage present among different animals. (B) Left column shows the unilateral coronal section from the standard marmoset brain depicting area 24 of ACC lesion site. The right column shows magnified images of area 24 stained to visualize myelinated fibers in a representative control and ACC-lesioned animal. The normal radial arrangement of the myelinated fibers is disrupted following the ACC lesion. (C) Histological quantification of mature neurons and glia in representative ACC lesioned animal (top panel) and control (bottom panel). Magnified images show high levels of astrocytes (violet) and microglia/macrophages (white) surrounding the lesion site at the grey and white matter interface in the ACC-lesioned animal relative to the control. (D) Left panel shows grayscale images with anti-NeuN staining depicting divisions of AMY and PAG where relative distribution of GABA-positive immunoreactive expression were quantified. Right graphs show the proportion of GABA expression in each division depicted in the AMY and PAG. Each circle represents one animal (ACC lesioned animal is red, Control animal is blue). Mean expression is represented by black bar. Data for two animals in ACC group overlap for basomedial AMY quantification. GABA-positive immunoreactivity was significantly down in the basomedial AMY and dorsal PAG.

ACC in early life is integral to postnatal development of social contact calls

(A-B) Spectrograms show sample 30-second vocal recordings of a representative control and ACC-lesioned marmosets before (postnatal week 2) and after surgery (postnatal week 6). Before surgery, the infants ‘babbled’ by emitting a wide range of immature concatenated calls, each with its own spectrographic motif illustrated and labeled in boxes. After surgery, at postnatal week 6, calls show reduced variability separated by distinct gaps or inter-call intervals. (C) Both groups show a reduction in the relative call count with increasing age. (D) Animals in both groups were able to emit calls of different call types. Those with ACC lesions made minor calls designated as ‘other’ more frequently than controls but all major call types were produced at equivalent rates. (E) Despite their ability to produce all call types, the proportion of social contact calls comprising phee, twitter and trills, was substantially reduced in animals with early life ACC lesions at postnatal week 6. (F) Chord diagrams show that at postnatal week 6, animals with ACC lesions show a high probability of transitions between all call types with lower frequency of transitions between social contact calls. The chord diagram represents weighted probabilities of transitions and their directionality from each group of call types. Weighted probabilities were used because of the variability in call counts. The size or thickness of arrows/links represents probabilities of call transition and the numbers around each chord diagram represents relative probability value for each call type transition.

ACC lesion alters structural characteristics of long distance social phee calls

(A) Sample spectrograph with examples of three and five syllable phees. (B) The ACC lesion caused a reduction in average phee syllable counts immediately after the ACC lesion (red dots) at PW3 but then normalized to 3-4 syllables thereafter. (C) Phee syllable duration ACC-lesioned group became shorter for multisyllabic phees ≥ 3 especially with increasing age. (D) The effective amplitude for each phee syllable increased for the ACC group until postnatal week 6. (E) Animals with ACC lesions emitted low entropy phees for calls as low as 2-syllables and continued until postnatal week 6. Error bars are confidence intervals. Gray shaded lines or bars represent time of surgery.