Network recruitment of glia-derived fast-spiking interneurons into sensory circuits using 2-photon calcium imaging

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Direct lineage reprogramming has emerged as a powerful strategy for in situ conversion of brain-resident non-neuronal cells into neurons. In addition to repairing acute CNS damage (e.g. following stroke or trauma), developing such technology may unlock treatment options for neurological or neuropsychiatric disorders where loss or hypofunction of specific neuronal subtypes may be involved in pathogenesis. For example, attention has been increasingly paid toward the role of GABAergic cortical interneuron dysfunction in various neuropsychiatric disorders - including ASD and schizophrenia1-5. Thus, generating specific interneuron subtypes and understanding how they functionally integrate into the developing and adult cortex is a crucial goal for the field of cellular and regenerative medicine.Pioneering work demonstrated that postnatal mouse cortical astrocytes could be converted into glutamatergic or GABAergic neuronal identities in vitro 6-9. Since then, interest in the field has exploded with the discovery of a variety of reprogramming factors able to convert a range of starting cell populations into specific neuronal subtypes in vivo, across different disease models and brain regions (recently reviewed in 10). Ascl1 was among the first proneural transcription factors shown to successfully reprogramme glia to neurons6,8. Depending on the starting-cell population, brain region and combination with other reprogramming factors, forced Ascl1 expression can result in various neuronal phenotypes in vivo11-16. For example, the Parmer laboratory found that a 3-factor combination of Ascl1, Lmx1a, and Nurr1 was able to convert NG2-glia in the striatum into parvalbumin (PV) expressing neurons13. Unpublished work by the Berninger lab, has now shown that serine-to-alanine mutations at six Ascl1 phosphorylation sites17-19 not only improves efficiency of in vivo reprogramming of cortical astrocytes but produces a subset of fast-spiking, PV interneurons, without the need for Lmx1a or Nurr1. Moreover, Ascl1SA6 derived induced neurons (iNs) exhibited synaptic currents, suggesting their integration in pre-existing circuits. This strategy utilises a retroviral construct and the anti-ferroptotic factor Bcl2, shown to promote cell-survival during reprogramming20. However, retroviral vectors are only able to transduce dividing cells, reducing the potential number of starting cells for reprogramming. Some remarkable recent success stories in the field of direct reprogramming have employed adeno-associated viruses (AAV) as a gene transfer system11,21,22. This has led to the question of whether it is possible that the delivery system itself could be contributing to greater reprogramming efficiency10,23, for which direct evidence is lacking. Either way, AAVs have some advantages as gene delivery systems, in that they are able to infect non-dividing cells, are less immunogenic and have lower risk of insertional mutagenesis than do retroviral vectors. This is at the expense of a restricted genetic carrying capacity, however. Therefore, it will be important to examine some of these putative benefits using already well-established reprogramming constructs.While cortical glia can be reprogrammed into iNs in vivo, we lack crucial information whether such iNs can be recruited into functional neuronal circuits such as elicited by sensory stimulation. To date, functional characterisation and phenotyping has been conducted in dissected cortical or striatal brain slices. What is currently unknown, is whether and how these induced neurons impact regional cortical network activity in vivo. This is a natural and necessary next step toward better understanding and, ultimately, clinical translation of these technologies.