Results:
Via in utero electroporation (IUE) of GFP into Grin3a knockout (KO) mice entirely lacking GluN3A vs WT controls and detailed analysis we completed the core focus of the primary objective by identifying a shift in L2/3 callosal axon arbor patterns within the upper layers of the cortex at the S1/S2 border in the absence of GluN3A. We demonstrated a second phenotype of increased targeting to the S2 in Grin3a KOs. Differences in axon distribution of the Grin3a KO were restricted to P13 and P20-22 ages, with mature (P42-44) and younger brains displaying very similar axonal distribution patterns to WT. It appears loss of GluN3A accelerates the developmental trajectory of callosal axon refinement, in line with existing findings. Extensive efforts focused on identifying GluN3A’s location of function in the circuit that influences callosal axons. We showed that specific loss of function from the presynaptic neurons did not cause altered callosal axons compared to controls. Likewise, conditional knockout of Grin3a from SST interneurons did not result in altered callosal axons. Despite technical hurdles preventing us directly showing a postsynaptic location of function, we did characterize dendritic morphology of L2/3 neurons in Grin3a KOs, revealing surprising changes in spine density and dendrite branching, consistent with the axon changes.
We validated by quantitative Western blot three candidates from the RNA-seq list generated prior to the project: CRMP4, KIRREL2 and KCNA1. KIRREL2 presents a particularly interesting candidate given its known roles in axon targeting. Technical issues hindered progress on immunofluorescence experiments so we attempted RNA-seq on FACS isolated neurons to narrow down candidates. Unfortunately this data was not informative due to sample variability. Using CRISPR we then created a knockin mouse line tagging endogenous Grin3a locus with mClover3 that can be used for proteomic experiments to uncover Grin3a interactors/mediators.
Our collaborators in the team of Ramon Reig completed experiments testing for a functional impact of the altered callosal axon connectivity. Spontaneous activity recordings found certain frequency bands were enhanced in L5 and L6, implying disrupted connectivity or functional properties of neurons in Grin3a KOs. Also Grin3a KOs showed longer response duration for callosal mediated sensory evoked activity in L5.
Dissemination overview:
These findings are the basis of a research paper manuscript that will be submitted later this year. A review article on GluN3A advances was published during the reporting period in the Journal of Physiology employing knowledge/insight gained during the project. Data has been disseminated by institute seminars and conference poster presentation, while further sharing of results will take place at the IBRO 2023 and AXON-2023 conferences.
