Dan Alin Ganea

The axon initial segment (AIS) is the site of action potential generation and exhibits structural and functional plasticity upon adaptation of neuronal excitability and disease. Performing in vivo longitudinal two-photon imaging of AIS in the medial prefrontal cortex of male mice, we reveal dynamic AIS remodeling during associative fear learning and extinction. These results demonstrate that AIS plasticity is not only crucial for homeostatic adaptation but also a hallmark of memory formation.

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

Summary: During perceptually guided decisions, correlates of choice are found as upstream as in the primary sensory areas. However, how well these choice signals align with early sensory representations, a prerequisite for their interpretation as feedforward substrates of perception, remains an open question. We designed a two alternative forced choice task (2AFC) in which mice compared stimulation frequencies applied to two adjacent vibrissae. The optogenetic silencing of individual columns in the primary somatosensory cortex (wS1) resulted in predicted shifts of psychometric functions, demonstrating that perception depends on focal, early sensory representations. Functional imaging of layer II/III single neurons revealed sensory, choice and engagement coding. From trial to trial, these three varied substantially, but independently from one another. Thus, coding of sensory and non-sensory variables co-exist in orthogonal subspace of the population activity, suggesting that perceptual variability does not originate from wS1 but rather from state or choice fluctuations in downstream areas.

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo , and in vivo , we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.