Periodic Reporting for period 5 - AXPLAST (Deep brain imaging of cellular mechanisms of sensory processing and learning)
Periodo di rendicontazione: 2024-01-01 al 2024-11-30
Learning and memory are the basis of our behaviour and mental well-being. Understanding the mechanisms of structural and cellular plasticity in defined neuronal circuits in vivo will be crucial to elucidate principles of circuit-specific memory formation and their relation to changes in neuronal ensemble dynamics. Structural plasticity studies were so far technically limited to dorsal cortex, excluding deeper brain areas. Furthermore, these studies mainly focused on the input site (dendritic spines), whilst the plasticity of the axon initial segment, a neuron’s site of output generation, was so far not studied in vivo. The axon initial segment is the site of action potential generation. Length and location of the axon initial segment were found to be plastic in cultured neurons or ex vivo, which strongly affected a neurons spike output. However, it remains unknown if axon initial segment plasticity regulates neuronal activity upon learning in vivo. The objective of this research programme was to combine the expression of live markers of the axon initial segments with novel deep brain imaging techniques via gradient index (GRIN) lenses to investigate how axon initial segment location and length are regulated upon associative learning in brain circuits beyond cortex in vivo. Two-photon time-lapse imaging of the axon initial segment of neurons was combined with associative threat conditioning to track learning-driven axon initial segment location dynamics. It was furthermore important to understand how the structure of the axon initial segment relates to neuronal activity in vivo. This will provide fundamental insights into the cellular mechanisms underlying sensory processing upon learning and relate network level plasticity with the cellular level.
This project allowed us to visualize the site of neuronal signal generation, the axon initial segment, in deep brain areas below the dorsal cortex using a combination of axon initial segment-targeting fluorescent proteins and GRIN-lens-based two-photon imaging. We traced axon initial segments in vivo and found that axon initial segments are plastic in principal neurons during associative learning. This plasticity was dependent on at least two subclasses of axon initial segments: axon initial segments that increased their length and axon initial segments that shortened their length. Furthermore, the length of the axon initial segment was correlated with the strength of spontaneous Ca2+ events in vivo indicating that longer axon initial segments result in stronger neuronal output and that axon initial segment length might be a mechanism to adapt neuronal activity in vivo. Finally, we linked axon initial segment plasticity to the expression of the neuronal activity marker c-fos. C-fos is often used to label neurons that are part of a memory trace, or a so-called engram. Using an ex vivo dual labelling approach, we identified that the shortest and longest axon initial segments are typically linked to the expression of the marker c-fos while these long and short axon initial segments typically not occur in c-fos-negative neurons. These findings support the notion that axon initial segments are plastic during memory formation and these plastic neurons are most likely part of the memory engram.
In summary, in this project we followed and tracked the location of the axon initial segment during learning in vivo. We could furthermore align ex vivo measurements of the length of the axon initial segment with a neuron’s activity in vivo, which has not been achieved before. Finally, we could link axon initial segment length to the expression of immediate early genes in memory engrams upon associative learning and extinction. These findings underline the important role of axon initial segment dynamics in the regulation of neuronal activity in vivo as a novel mechanism of cellular neuronal plasticity in learning and memory. An important next step will be to test if plasticity of the axon initial segment generalizes in vivo across brain areas, learning paradigms and disease states.