The Temporal Association Cortex Plays a Key Role in Auditory-Driven Maternal Plasticity

Abstract

Mother-infant bonding develops rapidly following parturition and is accompanied by changes in sensory perception and behavior. Here, we study how ultrasonic vocalizations (USVs) are represented in the brain of mothers. Using a mouse line that allows temporally controlled genetic access to active neurons, we find that the temporal association cortex (TeA) in mothers exhibits robust USV responses. Rabies tracing from USV-responsive neurons reveals extensive subcortical and cortical inputs into TeA. A particularly dominant cortical source of inputs is the primary auditory cortex (A1), suggesting strong A1-to-TeA connectivity. Chemogenetic silencing of USV-responsive neurons in TeA impairs auditory-driven maternal preference in a pup-retrieval assay. Furthermore, dense extracellular recordings from awake mice reveal changes of both single-neuron and population responses to USVs in TeA, improving discriminability of pup calls in mothers compared with naive females. These data indicate that TeA plays a key role in encoding and perceiving pup cries during motherhood.

Keywords: TRAP; auditory cortex; motherhood; neuropixels; plasticity; rabies tracing; temporal association cortex; ultra sonic vocalizations.

Figure 1.. USVs recruit neurons in TeA

(A) Schematic of the TRAP x TB system. (B) The experimental protocol used for TRAPing in mothers. Top - no stimulation (NS-TRAP); Bottom- USV stimulation (USV-TRAP). (C) Representative fluorescent micrographs of coronal brain slices stained for TRAPed cells (Myc). Slices are from a region containing A1 and TeA, corresponding to Bregma – 2.92 mm in the Brain Atlas (Paxinos and Franklin, 2004). Scale bar, 500 Pm. (D) Quantification of the fold induction of TRAPed cells in A1 and TeA as compared to S1 (relative density), normalized to the ‘No Stim (NS-TRAP)’ condition (mean ± SEM; NS-TRAP, N = 6 mice; USV-TRAP, N = 6 mice; **, p < 0.01, Mann-Whitney U-test with Bonferroni correction, all statistical tests and results are listed in Table S3).

Figure 2.. TeA has a particularly rich presynaptic landscape

(A) Overview of the TRAP-rabies monosynaptic retrograde tracing components. TRAP1 and TVA^66T were used for tracing from A1 and TRAP2 and TVA were used for tracing from TeA. (B) The experimental protocol for TRAP-rabies. (C) Schematic of the TRAP-rabies experiments from two different targets – A1 (left) and TeA (right). Yellow cells indicate the location of starter cells. Green cells indicate the monosynaptic inputs to the starter cells. (D) Representative fluorescent micrographs from the injection sites in A1 (left) or TeA (right). Zoomed-in micrographs (middle) show starter cells indicated by white arrows. Scale bar, 200 Pm. (E and F) Representative micrographs from select input regions into A1 (E) and TeA (F). Scale bar, 200 Pm. (G and H) A schematic map of selected-long range monosynaptic inputs into A1 (G) and TeA (H) (A1, N = 4 mice; TeA, N = 5 mice). Not all but regions received more than 0.2 % of total input fraction are shown (see text and Table S1 and S2 for full lists and abbreviations of regions). The colors indicate the proportion of each region out of the total inputs. Injection sites are shown by a red circle. The values of inputs into A1 from MGv and TeA from A1 are indicated separately as their value exceeds the color bar scale.

Figure 3.. USV responsive neurons in TeA receive more long range inputs

(A) Representative micrographs from TeA of TRAP-rabies injected mice. Left: NS-TRAP, Middle: USV-TRAP, Right: WC-TRAP. Scale bar, 200 Pm. (B-C) The total number of starter cells from NS-, USV-, and WC-TRAP mice are not different (B) (NS-TRAP, N = 4 mice; USV-TRAP, N = 5 mice; WC-TRAP, N = 5 mice; ns, not significant, Mann-Whitney U test), and also spread equally in the TeA and adjacent regions (C) (ns, not significant, Mann-Whitney U test). (D) Convergence index (CI) from all regions projecting into TeA for the NS-, USV-, and WC-TRAP groups. USV-TRAP had larger CI than NS- and WC-TRAP (*, p < 0.05, Mann-Whitney U test with Bonferroni correction). (E) CI from A1 into TeA for indicated groups. USV-TRAP had the larger number of inputs from A1 to TeA than NS- and WC-TRAP (*, p < 0.05, Mann-Whitney U test with Bonferroni correction). (F) A differential input map from TeA (left) and A1 (right) comparing USV-TRAP neurons versus the inputs of NS-TRAP neurons. Color indicates the d-prime evaluated by comparing the CI of the indicated regions comparing NS- and USV-TRAP animals. White colored regions show roughly similar CIs (d’ < 1). d-prime was calculated as $\text{d}’=\frac{\left|{\mathit{\mu }}_{\mathit{U}\mathit{S}\mathit{V}-\mathit{T}\mathit{R}\mathit{A}\mathit{P}}-{\mathit{\mu }}_{\mathit{N}\mathit{S}-\mathit{T}\mathit{R}\mathit{A}\mathit{P}}\right|}{\sqrt{\frac{\mathbf{1}}{\mathbf{2}}\left({\mathit{\sigma }}_{\mathit{U}\mathit{S}\mathit{V}-\mathit{T}\mathit{R}\mathit{A}\mathit{P}}{}^{\mathbf{2}}+{\mathit{\sigma }}_{\mathit{N}\mathit{S}-\mathit{T}\mathit{R}\mathit{A}\mathit{P}}{}^{\mathbf{2}}\right)}}$. See Table S2 for abbreviations of indicated regions.

Figure 4.. USV responsive neurons in TeA receive preferential input from USV-responsive neurons in A1

(A) Schematic of the analysis used to quantify the number of presynaptic cells onto TeA TRAPed neurons that are also TRAPed in A1 (yellow cells). (B) Representative micrographs from the two TRAP-rabies experiments, injected in TeA and analyzed in A1. In A1, TRAP cells are in red (stained with anti-Myc) and green cells are presynaptic to TeA. Scale bars, 200 Pm. (C) Quantitative analysis of functional connectivity between A1 and TeA in the four experimental groups. Black curve shows the Poisson distribution estimated from the expected number of double-labeled cells as lambda. The dotted color line and an arrow indicate the observed number of double-labeled cells for each group (NS-TRAP, n = 12 cells; USV-TRAP, n = 132 cells; WC-TRAP, n = 74 cells; NBN-TRAP, n = 5 cells). USV- and WC-TRAP but not NS- and NBN-TRAP neurons in A1 have significantly higher probability to connect to TRAPed neurons in TeA (NS-TRAP, N = 4 mice; USV-TRAP, N = 5 mice; WC-TRAP, N = 5 mice; NBN-TRAP, N = 4 mice; *, p < 0.05, **, p < 0.01, extreme upper tail probability computed by a Poisson cumulative distribution function).

Figure 5.. TeA is causally related to auditory driven maternal preference

(A) Schematic of the behavioral test. (B) Overview of the TRAP-chemogenetics components. (C) Experimental protocol for TRAP-chemogenetics to test auditory-driven maternal behavior. (D) Representative micrographs from DREADD injected mice bilaterally into TeA. Left: left hemisphere; Right: right hemisphere. Scale bar, 200 Pm. (E) Quantification of the maternal preference to a chamber playing USVs over NBN. Chance level is 50% (Control, N = 12 mice; USV-TeA, N = 13 mice; WC-TeA, N = 11 mice; UPTTeA, N = 9 mice; **, p < 0.01; ns, not significant, Mann-Whitney U test with Bonferroni correction).

Figure 6.. Distinct plasticity of USV coding at single cell level in A1 and TeA

(A) Schematic of the Neuropixels probe trajectory. Scale bar, 1 mm. (B-C) Evoked vs spontaneous spike rates of single neurons (mean ± SEM) in response to USV, NBN, or UPT in A1 (B) and TeA (C) (naïve-A1, n = 238 cells, mother-A1, n = 182 cells, naïve-TeA, n = 69 cells, mother-TeA, n = 132 cells; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant, Mann-Whitney U-test with Bonferroni correction). (D) Raster and PSTHs from three neurons, which show different preference indices (Top/middle: raster plots in response to USV/NBN; Bottom: PSTHs). 3 different sound intensities were presented 20 times for each and thus each raster shows 60 trials in total. The gray bars indicate the position of syllables in the sound. (E-F) Plots of the preference index of USV over NBN (E) and USV over UPT (F) in A1 and TeA (*, p < 0.05, **, p < 0.01; ns, not significant, Mann-Whitney U-test).

Figure 7.. Distinct plasticity of population coding in A1 and TeA

(A) Raster plots from one Neuropixels probe recording simultaneously A1 and TeA of a mother (Cell#1–19; A1, Cell#20–33; TeA). (B) Cumulative plots of pairwise PSTH correlation within regions (‘A1-A1’ naives, n = 3302 pairs [r = 0.10 ± 0.20]; mothers, 1613 pairs [r = 0.09 ± 0.21]. ‘TeA-TeA’ naives, n = 409 pairs [r = 0.17 ± 0.21]; mothers, 844 pairs [r = 0.12 ± 0.19]). Dotted lines indicate shuffled PSTHs (**, p < 0.01; ns, not significant, Kolmogorov-Smirnov test). All values listed here are [mean ± SD]. (C) Cumulative plots of pairwise noise correlation (A1-A1: naives, n = 3302 pairs [r = 0.03 ± 0.08]; mothers: 1613 pairs [r = 0.04 ± 0.08]. TeA-TeA: naives, n = 409 pairs [r = 0.04 ± 0.33]; mothers, 844 pairs [r = 0.04 ± 0.21]). Dotted lines indicate shuffled PSTHs (***, p < 0.001; ns, not significant, Kolmogorov-Smirnov test). All values listed here are [mean ± SD]. (D) Trajectories of PCA components from representative animals. (E) Mean eucledian distance of PCA components between USV and NBN (red, mothers; blue, naives). (F) Cumulative distance of PCA components between USV and NBN. (G) Classification performance of a SVM decoder trained by the dataset of A1 neurons. The decoder was tested for its accuracy to differentiate USV from NBN or UPT. The decoder was trained with first five syllables. The graphs indicate the mean value of accuracy across 1000 iterations (‘USV vs NBN’ mothers, 88.0 %; naives, 85.5 %, ‘USV vs UPT’ mothers, 88.6 %; naives, 91.3 %) (H) Same as (G) but in TeA (‘USV vs NBN’ mothers, 73.9 %; naives, 65.3 %, ‘USV vs UPT’ mothers, 89.8 %; naives, 61.7 %).