SummaryNeurons implement a variety of plasticity mechanisms to alter their function over timescales ranging from seconds to days. One powerful means of controlling excitability is to directly modulate the site of spike initiation, the axon initial segment (AIS). However, all plastic structural AIS changes reported thus far have been slow, involving days of neuronal activity perturbation. Here, we show that AIS plasticity can be induced much more rapidly. Just 3 hr of elevated activity significantly shortened the AIS of dentate granule cells in a calcineurin-dependent manner. The functional effects of rapid AIS shortening were offset by dephosphorylation of voltage-gated sodium channels, another calcineurin-dependent mechanism. However, pharmacological separation of these phenomena revealed a significant relationship between AIS length and repetitive firing. The AIS can therefore undergo a rapid form of structural change over timescales that enable interactions with other forms of activity-dependent plasticity in the dynamic control of neuronal excitability.

Highlights d Two-photon glutamate uncaging is used to stimulate clustered excitatory spines d Spine stimulation can trigger inhibitory bouton growth onto the same dendrite d The dendrite triggers presynaptic inhibitory changes via endocannabinoid signaling d This mechanism may ensure local inhibitory control of active excitatory clusters

The most commonly studied form of synaptic plasticity is long-term potentiation (LTP). Over the last 15 years, it has been possible to induce structural and functional LTP in dendritic spines using two-photon glutamate uncaging, allowing for studying the signaling mechanisms of LTP with single synapse resolution. In this review, we compare different stimulation methods to induce single synapse LTP and discuss how LTP is expressed. We summarize the underlying signaling mechanisms that have been studied with high spatiotemporal resolution. Finally, we discuss how LTP in a single synapse can be affected by excitatory and inhibitory synapses nearby. We argue that single synapse LTP is highly dependent on context: the choice of induction method, the history of the dendritic spine and the dendritic vicinity crucially affect signaling pathways and expression of single synapse LTP.

Experience-dependent formation and removal of inhibitory synapses are essential throughout life. For instance, GABAergic synapses are removed to facilitate learning, and strong excitatory activity is accompanied by the formation of inhibitory synapses to maintain coordination between excitation and inhibition. We recently discovered that active dendrites trigger the growth of inhibitory synapses via CB1 receptor-mediated endocannabinoid signaling, but the underlying mechanism remained unclear. Using two-photon microscopy to monitor the formation of individual inhibitory boutons in hippocampal organotypic slices from mice (both sexes), we found that CB1 receptor activation mediated the formation of inhibitory boutons and promoted their subsequent stabilization. Inhibitory bouton formation did not require neuronal activity and was independent of G i/o -protein signaling, but was directly induced by elevating cAMP levels using forskolin and by activating G s -proteins using DREADDs. Blocking PKA activity prevented CB1 receptor-mediated inhibitory bouton formation. Our findings reveal that axonal CB1 receptors signal via unconventional downstream pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels. Our results demonstrate an unexpected role for axonal CB1 receptors in axon-specific, and context-dependent, inhibitory synapse formation.

Single‐molecule fluorescence spectroscopy has become a crucial tool to study the behavior of biomolecules and their roles in complex biological processes. Single‐molecule methods have in particular contributed to our understanding of the mechanism of force generation and motility of motor proteins. Motor proteins are enzymes that convert the chemical free energy obtained from the hydrolysis of adenosine triphosphate (ATP) into mechanical work in order to drive processes such as muscle contraction and intracellular transport. Here, we review how single‐molecule fluorescence spectroscopy can be applied to motor proteins of the kinesin, myosin, and dynein superfamilies and how such studies have advanced our knowledge of the molecular basis of motor protein action.