Altered Socio-Affective Communication and Amygdala Development in mice with Protocadherin10-deficient Interneurons

  1. Tania Aerts
  2. Anneleen Boonen
  3. Lieve Geenen
  4. Anne Stulens
  5. Luca Masin
  6. Anna Pancho
  7. Annick Francis
  8. Frans Van Roy
  9. Markus Wöhr
  10. Eve Seuntjens

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Abstract

Autism Spectrum Disorder (ASD) is a group of neurodevelopmental conditions associated with deficits in social interaction and communication, together with repetitive behaviors. The cell adhesion molecule Protocadherin10 ( Pcdh10 ) has been implicated in the etiology of ASD. Pcdh10 is expressed in the nervous system during embryonic and early postnatal development and has been linked to neural circuit formation. Here, we show strong expression of Pcdh10 in the ganglionic eminences and in the basolateral complex of the amygdala at mid and late embryonic stages, respectively. Both inhibitory and excitatory neurons expressed Pcdh10 in the basolateral complex at perinatal stages and genes linked to vocalization behavior were enriched in Pcdh10- expressing neurons in adult mice. To further investigate the involvement of Pcdh10 in neurodevelopment with relevance to ASD, and to assess the functional and behavioral consequences of loss of Pcdh10 in basolateral amygdala interneurons, we combined a ubiquitous and a conditional Pcdh10 knockout mouse model. Conditional knockout of Pcdh10 reduced the number of interneurons in the basolateral complex. Both models exhibited altered developmental trajectories of socio-affective communication through isolation-induced ultrasonic vocalizations in neonatal pups, characterized by increased emission rates in heterozygous pups. Furthermore, acoustic call features were affected and heterozygous conditional knockout pups emitted calls characterized by reduced peak frequencies but increased frequency modulation. Additionally, we identified distinct clusters of call subtypes with specific developmental trajectories, suggesting the vocalization repertoire is extensive and dynamic during early life. The nuanced alterations in socio-affective communication at the level of call emission rates, acoustic call features, and clustering of call subtypes were primarily seen in heterozygous pups of the conditional knockout and less prominent in the ubiquitous Pcdh10 knockout, suggesting that changes in anxiety levels associated with Gsh2 -lineage interneurons might drive the observed behavioral effects. Together, this demonstrates that loss of Pcdh10 specifically in interneurons contributes to behavioral alterations in socio-affective communication with relevance to ASD.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors conducted a comprehensive investigation into the spatial and temporal expression patterns of Pcdh10 during brain development and employed two deficient mouse models to delve into its neurobehavioral functions, with a specific emphasis on ultrasonic vocalizations (USV). Interestingly, the authors found that heterozygous cKO pups showed an exaggerated effect on USV as compared to the ubiquitous heterozygous KO pups. These observations in general agree with previous studies of Pcdh10's complicated function in ASD-related neurodevelopment, as well as USV, indicating its important function in neurodevelopment. However, several key concerns warrant attention:

    Major comments:

    1. The authors analyzed published scRNAseq adult mouse dataset and found that Pcdh10-expressing cells differentially expressed genes involved in vocalization behaviors, including Foxp2, Cntnap2, Nrxn1 and Nrxn3, as compared to cells that did not express Pcdh10. Given that the authors also performed bulk RNA-seq experiments using the sorted cKO cells, it would be interesting and important to include these data in the analysis and validate whether the above genes are differentially expressed in the animal model in the current study. This may provide additional molecular mechanisms of the behavioral abnormality observed in the animal models.
    2. In Figure 1S, the synapse assembly is one of the most significant GO-terms. Does Pcdh10 deficiency affect neuromophology and synaptic density/assembly? Characterizing the neuroanatomy of the animal model will provide a neuronal explanation for the behavioral abnormalities observed in this study.
    3. Could the authors explain why cHE mice display a stronger phenotype than cKO at P6? Also, why cHE and cKO show stronger phenotypes than whole-body KO? Does this data indicate that loss of Pcdh10 in the inhibitory neurons only resulted in E-I in-balance? The authors should at least discuss the possibilities of this result they observed.

    Minor comments:

    1. It would be interesting and important to know the level of PCDH10 at the adult stage after P7 to learn whether this gene also plays an important role beyond early development.
    2. Besides USV, does Pchd10 deficiency in mice show other autistic phenotypes in adolescence and adulthood, such as social interaction and repetitive behavior?

    Significance

    The experiments are well designed and the detailed characterization of USV in the animal models would be informative for the audience who are interested in Pcdh10's function in neurodevelopment and Social Communication. However, because previous studies already showed the function of PCDH10 in multiple mouse models, the novelty of this study is limited. The observation that cKO/cHE mice show stronger phenotype than whole-body KO mice could be potentially interesting, it would be nice if the authors could provide some explanation of this result with additional experimental evidence.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    In this paper the authors present a new cKO mouse model for PCDH10-related ASD. This model consists of the ablation of PCDH10 specifically in interneurons of the basolateral complex. Interestingly, the use of this mouse model together with a complete KO adds evidence towards an excitatory/inhibitory imbalance causing the ASD phenotype, rather than the complete ablation of a protein (in this case PCDH10). Firstly, the developmental dynamics of PCDH10 are measured with diverse techniques. The presence of PCDH10 at embryonic stages in different areas of the basal forebrain. Here it is made clear why the specific downregulation of PCDH10 in Gsh2- lineage interneurons. The cKO mouse model is validated with bulk RNA sequencing fluorescence imaging of the eGFP reporter in the telencephalon. The KO mouse model is validated with WB assay showing the gradual decrease of PCDH10 in Het and Ho mice. Secondly, the USV emitted by isolated pups throughout development (P3, P6, P9 and P12) are analysed with different parameters in both mouse models.

    Major Comments

    1. The role of anxiety in the phenotype described in this work should be supported by behavioural experiments in adulthood (Open field/light/dark/Plus Maze test)2-3M for mice to reach the appropriate age + 2 weeks for performing the experiments and extracting results.
    2. The WB in panel F1C&E should be done with non-pooled biological replicates to be informative 3 weeks
    3. The statistical test used for F2B and F3D-I needs to be specified.
    4. The reduced GABA input in the amygdala that is hypothesized to be causing the phenotype could be studied by iPSP analysis through LFP (OPTIONAL) 1M

    Minor comments

    1. The results section should be subdivided in sections corresponding to each figure
    2. Detail the pinhole opening in M&M used for the imaging of the images in panel of Figure 1 M-R
    3. The group size and the power calculation used to determine it should be detailed in M&M
    4. The WB membrane image in Panel 1F has saturated pixels, the image needs to be changed
    5. Instead of asterisks, writing the exact p-value is more informative in the graphs
    6. Figure 1B & D: detail what the Pcdh10 levels are normalised to. In the legend there's a typo "no-way ANOVA"
    7. Figure 1F: specify what it means P17.5 (norm)
    8. Figures 3-4: choose higher contrast colours for an easier readability and more accessibility.
    9. Figure 3B: the WB image needs to be at a higher resolution
    10. Figure 3C: the colour coding for dBFS in the spectrogram needs to be specified in the maximum and minimum number.
    11. Figure 3D-I & 6G: results would be more clear if shown as a ratio of the WT (would be also more evident the mouse model differences). Also the titles of the graphs are misleading, as the graphs are showing data from all the genotypes of the mouse models.
    12. Figure 2B: specify what it is normalised to
    13. Figure 4 E-H: it is not described what the dotted lines correspond to.
    14. In all the figures, the panels are excessively subdivided, the following panels should be grouped in one:
      • a. Figure 1: i. C & E are showing the same data

    ii. G-I are showing the same data

    iii. J-L are showing the same data

    iv. M-R are showing the same data - b. Figure 2: panel 2C-G are showing the same data - c. Figure 3: i. A-B are showing the same data

    ii. D-I are showing the same data - d. Figure 4: i. A-C are showing the same data

    ii. E-H are showing the same data - e. Figure 5: the frequency parameters (A-E) should be all 1 panel f. Figure 6: B-E are showing the same data

    Significance

    The detailed study of the socio-affective communication of these mouse models is accurate and quite informative. There is still a big body of work to do for classifying and using pup USV as biomarkers for mouse model phenotyping, and this thorough work is a step forward. This type of work will be of interest to neurodevelopmental neuroscientists interested in behaviour and mouse model phenotyping. However, the claim of an autistic-like phenotype would be much stronger with additional behavioural assessments in adulthood (as USV in mating behaviour, social novelty recognition and stereotypical behaviours). In addition, the hypothesis of an excitation/inhibition imbalance is interesting, but correlational in this work. Further experiments would need to be done to prove causality.

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    Referee #1

    Evidence, reproducibility and clarity

    This paper provides evidence that Pcdh10 (associated with autism spectrum disorder) is involved in anxiety-like behavior and socio-affective communication in developing mouse pups. Using a specific Cre-driver line targeting Gsh2-lineage interneurons, the authors performed a series of behavioral analyses, including isolation-induced ultrasonic vocalization. Particularly, the authors provided detailed analyses to provide distinct clusters that might correspond to identified call types. This work is excellent and I do not have any further comments, except one minor comment: why did the authors use CD1 line to generate the mouse lines, instead of C57BL/6 substrains?

    Significance

    This is of paramount significance, such that Pcdh10 expressed in Gsh2-lineage interneurons (a subpopulation in the basolateral complex of the amygdala) is required for isolation-induced ultrasonic vocalization.

  5. Version published to 10.1101/2024.01.04.574190v1 on bioRxiv