Periodic Reporting for period 1 - DEFINE (DEciphering mechanisms of presynaptic reFINEment)
Reporting period: 2020-12-01 to 2022-11-30
Despite extensive research efforts much remains unknown about of how mature brain circuits form and how this is coordinated with sensory experience. Initial wiring is refined during postnatal critical periods. Revealing the roles of genes and timings of circuit refinement is critical to understand how healthy brains form and how disorders such as autism and depression occur which are a major health burden.
A well-known family of synaptic receptors that regulate neuronal connections are NMDA receptors (NMDARs). Classical NMDARs containing GluN1 and GluN2 subunits promote synapse maturation through their biophysical and signalling properties. An alternative subunit called GluN3A confers different properties to receptors. The expression of GluN3A peaks during postnatal stages coinciding with critical refinement periods and absence of GluN3A causes increased numbers of dendritic spines.
Existing work has focused on postsynaptic effects of GluN3A. The limited work on presynaptic roles have focused on functional readouts rather than morphology and targeting. The DEFINE project aimed to address this by analysis of a major cortical circuit, the interhemispheric callosal axons. This circuit is refined during postnatal ages when GluN3A is expressed there.
The primary objective of the project were to assess how global loss of GluN3A affects callosal axonal arborisation and targeting as a readout of presynaptic connectivity. A key objective was to narrow down the cellular location of function within the callosal circuit by genetic loss of Grin3a function in particular cell populations. An ambitious goal was to also test for a role of neuronal activity and sensory experience in mediating any GluN3A axonal effects. A parallel objective was to identify and dissect the molecular mediators of GluN3A´s role in affecting axon refinement and targeting. A further objective was to study potential functional impacts of the altered callosal circuit connectivity by recording neuronal activity in vivo.
Project conclusions
- GluN3A impacts the targeting and refinement of L2/3 callosal axons to ensure appropriate arborization patterns and regional targeting during later postnatal ages.
- These effects do not arise from GluN3A expression in presynaptic cells or SST inhibitory neurons but likely from postsynaptic changes indicated by altered dendritic morphology of L2/3 neurons.
- Extracellular activity recordings found changes in Grin3a KOs consistent with altered local and interhemispheric connectivity that although do not directly correlate to the axonal changes are key progress to understanding the functional effects of GluN3A on the brain.
- Confirmed changes in protein expression of KIRREL2, CRMP4 and KCNA1 provide exciting candidates that may mediate GluN3A effects on the callosal axon circuit.
A well-known family of synaptic receptors that regulate neuronal connections are NMDA receptors (NMDARs). Classical NMDARs containing GluN1 and GluN2 subunits promote synapse maturation through their biophysical and signalling properties. An alternative subunit called GluN3A confers different properties to receptors. The expression of GluN3A peaks during postnatal stages coinciding with critical refinement periods and absence of GluN3A causes increased numbers of dendritic spines.
Existing work has focused on postsynaptic effects of GluN3A. The limited work on presynaptic roles have focused on functional readouts rather than morphology and targeting. The DEFINE project aimed to address this by analysis of a major cortical circuit, the interhemispheric callosal axons. This circuit is refined during postnatal ages when GluN3A is expressed there.
The primary objective of the project were to assess how global loss of GluN3A affects callosal axonal arborisation and targeting as a readout of presynaptic connectivity. A key objective was to narrow down the cellular location of function within the callosal circuit by genetic loss of Grin3a function in particular cell populations. An ambitious goal was to also test for a role of neuronal activity and sensory experience in mediating any GluN3A axonal effects. A parallel objective was to identify and dissect the molecular mediators of GluN3A´s role in affecting axon refinement and targeting. A further objective was to study potential functional impacts of the altered callosal circuit connectivity by recording neuronal activity in vivo.
Project conclusions
- GluN3A impacts the targeting and refinement of L2/3 callosal axons to ensure appropriate arborization patterns and regional targeting during later postnatal ages.
- These effects do not arise from GluN3A expression in presynaptic cells or SST inhibitory neurons but likely from postsynaptic changes indicated by altered dendritic morphology of L2/3 neurons.
- Extracellular activity recordings found changes in Grin3a KOs consistent with altered local and interhemispheric connectivity that although do not directly correlate to the axonal changes are key progress to understanding the functional effects of GluN3A on the brain.
- Confirmed changes in protein expression of KIRREL2, CRMP4 and KCNA1 provide exciting candidates that may mediate GluN3A effects on the callosal axon circuit.
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.
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.
There has been important progress made beyond existing knowledge during the DEFINE project. We showed that loss of the non-canonical GluN3A NMDA receptor subunit disrupts normal arborization and regional targeting of L2/3 callosal axons from the somatosensory cortex during postnatal brain development. In the course of studying GluN3A effects, we have also have furthered the understanding of normal callosal axon development and refinement in the WT brain by detailed analysis of adult brain that was lacking in the literature. Extensive efforts with conditional knockouts together with cutting edge dendritic morphology analysis using Supernova system narrowed the location of GluN3A function to be likely postsynaptic. This suggests postsynaptic synaptic plasticity events controlled by GluN3A NMDARs may trans-synaptically determine callosal axon refinement and targeting. In vivo recordings found altered spontaneous activity and interhemispheric communication in L5 of the cortex in mice lacking GluN3A. This advance in understanding how callosal circuits mature is not only a step towards our understanding of how brains work but could be inform future studies on various neurological disorders, including autism, where callosal circuits are affected.
Another key contribution that arose from the project is the construction of an mClover3-Grin3a knockin mouse that will be a vital tool for advancing objectives of this project but also for various studies in the field.
There are limited direct socio-economic impacts and societal implications for this project as it is basic research and not disease focused but our better understanding of GluN3A´s roles in brain circuit refinement may inform future work on brain disorders.
Another key contribution that arose from the project is the construction of an mClover3-Grin3a knockin mouse that will be a vital tool for advancing objectives of this project but also for various studies in the field.
There are limited direct socio-economic impacts and societal implications for this project as it is basic research and not disease focused but our better understanding of GluN3A´s roles in brain circuit refinement may inform future work on brain disorders.