Periodic Reporting for period 1 - iMAC (Synaptic input mapping during cortical map plasticity)
Periodo di rendicontazione: 2021-04-15 al 2023-04-14
For an individual to form a coherent sensory percept and subsequently exert a relevant behavioral response, various streams of sensory information need to be integrated. The brain’s neocortical neuronal circuits play a crucial role in this process. However, it is still enigmatic how the various information streams are assimilated and represented by individual neurons, let alone by synapses. Primary sensory cortices receive feedforward sensory information from first-order thalamic nuclei and contextual or feedback information from higher-order thalamic nuclei and long-range corticocortical pathways.
Therefore, the pyramidal neurons in the primary sensory cortex integrate mainly two types of excitatory synaptic inputs, one relayed through feedforward thalamocortical and local corticocortical afferents and another relayed through feedback inputs from distinct thalamocortical and long-range corticocortical circuits.
The synaptic connectivity motifs of these inputs and how they rearrange over time remain unknown.
Our main objective was to map higher-order thalamocortical feedback inputs onto cortical pyramidal neurons in vivo using an all-optical approach. The second aim was to characterize the plasticity of sensory-evoked feedback synaptic activity.
Therefore, the pyramidal neurons in the primary sensory cortex integrate mainly two types of excitatory synaptic inputs, one relayed through feedforward thalamocortical and local corticocortical afferents and another relayed through feedback inputs from distinct thalamocortical and long-range corticocortical circuits.
The synaptic connectivity motifs of these inputs and how they rearrange over time remain unknown.
Our main objective was to map higher-order thalamocortical feedback inputs onto cortical pyramidal neurons in vivo using an all-optical approach. The second aim was to characterize the plasticity of sensory-evoked feedback synaptic activity.
The core of the work entailed the establishment of an all-optical approach for mapping higher-order thalamocortical feedback inputs onto cortical pyramidal neurons using the mouse whisker primary somatosensory cortex as a model system.
To this end, we sparsely expressed a genetically-encoded glutamate indicator in cortical pyramidal neurons. We densely expressed the red-light activatable opsin in the higher-order thalamus, followed by cranial window implantation for subsequent in vivo imaging.
We optically stimulated dense axonal projections originating from the higher-order thalamus expressing the light-sensitive opsin through the microscope objective, upon which we near-simultaneously monitored the released glutamate by imaging of the indicator expressed on the post-synaptic pyramidal cells using two-photon laser-scanning microscopy. This identified the location of functional synaptic connections.
We developed an automatized analysis pipeline for high-throughput synaptic mapping analysis. In order to probe functional connectivity longitudinally over several weeks, we refined our imaging and stimulation protocol ensuring neuronal health and integrity. Our findings show that inputs from the higher-order thalamus connect to some dendritic branches of L2/3 pyramidal neurons but not others suggesting a converging organization. Based on this first part, we currently continue investigating the spatial arrangement of other cortical converging pathways. Next, we used our methodology to identify the active pathways in response to sensory-evoked activity. Data from this second part of the project are still being acquired and analyzed.
So far, we have presented this methodology at conferences and included data in two different research grant applications but have not been further disseminated yet.
To this end, we sparsely expressed a genetically-encoded glutamate indicator in cortical pyramidal neurons. We densely expressed the red-light activatable opsin in the higher-order thalamus, followed by cranial window implantation for subsequent in vivo imaging.
We optically stimulated dense axonal projections originating from the higher-order thalamus expressing the light-sensitive opsin through the microscope objective, upon which we near-simultaneously monitored the released glutamate by imaging of the indicator expressed on the post-synaptic pyramidal cells using two-photon laser-scanning microscopy. This identified the location of functional synaptic connections.
We developed an automatized analysis pipeline for high-throughput synaptic mapping analysis. In order to probe functional connectivity longitudinally over several weeks, we refined our imaging and stimulation protocol ensuring neuronal health and integrity. Our findings show that inputs from the higher-order thalamus connect to some dendritic branches of L2/3 pyramidal neurons but not others suggesting a converging organization. Based on this first part, we currently continue investigating the spatial arrangement of other cortical converging pathways. Next, we used our methodology to identify the active pathways in response to sensory-evoked activity. Data from this second part of the project are still being acquired and analyzed.
So far, we have presented this methodology at conferences and included data in two different research grant applications but have not been further disseminated yet.
This project has pushed the current state of the art by extensively using novel tools (optogenetics and glutamate imaging) in an in vivo system through the complementary expertise of the fellowship holder and the supervisor/host institute. To the best of our knowledge, our novel methodological approach has not yet been attempted before. This approach promises to open new ways of understanding synaptic circuit organization and dynamics, governing sensory processing necessary for higher cognitive functions, and provide new leads on how to characterize functional connectivity in other brain areas involving glutamatergic transmission.