Activity-dependent neuronal plasticity is a fundamental mechanism through which the nervous system adapts to sensory experience. Several lines of evidence suggest that parvalbumin (PV+) interneurons are essential in this process, but the molecular mechanisms underlying the influence of experience on interneuron plasticity remain poorly understood. Perineuronal nets (PNNs) enwrapping PV+ cells are long-standing candidates for playing such a role, yet their precise contribution has remained elusive. We show that the PNN protein Brevican is a critical regulator of interneuron plasticity. We find that Brevican simultaneously controls cellular and synaptic forms of plasticity in PV+ cells by regulating the localization of potassium channels and AMPA receptors, respectively. By modulating Brevican levels, experience introduces precise molecular and cellular modifications in PV+ cells that are required for learning and memory. These findings uncover a molecular program through which a PNN protein facilitates appropriate behavioral responses to experience by dynamically gating PV+ interneuron function.

SummaryThe active zone (AZ) matrix of presynaptic terminals coordinates the recruitment of voltage-gated calcium channels (VGCCs) and synaptic vesicles to orchestrate neurotransmitter release. However, the spatial organization of the AZ and how it controls vesicle fusion remain poorly understood. Here, we employ super-resolution microscopy and ratiometric imaging to visualize the AZ structure on the nanoscale, revealing segregation between the AZ matrix, VGCCs, and putative release sites. Long-term blockade of neuronal activity leads to reversible AZ matrix unclustering and presynaptic actin depolymerization, allowing for enrichment of AZ machinery. Conversely, patterned optogenetic stimulation of postsynaptic neurons retrogradely enhanced AZ clustering. In individual synapses, AZ clustering was inversely correlated with local VGCC recruitment and vesicle cycling. Acute actin depolymerization led to rapid (5 min) nanoscale AZ matrix unclustering. We propose a model whereby neuronal activity modulates presynaptic function in a homeostatic manner by altering the clustering state of the AZ matrix.

A diffusion barrier impeding membrane molecule motion between the axon and the somatodendritic compartment develops as neurons mature and the axon initial segment (AIS) is enriched in specific molecules. Albrecht et al. analyze the mobility of lipid-anchored molecules in the AIS using single-particle tracking time course experiments and propose a new mechanistic model for the AIS diffusion barrier.

With the recent development of single-molecule localization-based superresolution microscopy, the imaging of cellular structures at a resolution below the diffraction-limit of light has become a widespread technique. While single fluorescent molecules can be resolved in the nanometer range, the delivery of these molecules to the authentic structure in the cell via traditional antibody-mediated techniques can add substantial error due to the size of the antibodies. Accurate and quantitative labeling of cellular molecules has thus become one of the bottlenecks in the race for highest resolution of target structures. Here we illustrate in detail how to use small, high affinity nanobody binders against GFP and RFP family proteins for highly generic labeling of fusion constructs with bright organic dyes. We provide detailed protocols and examples for their application in superresolution imaging and single particle tracking and demonstrate advantages over conventional labeling approaches.

We report a noninvasive fluorescence microscopy method and demonstrate nanometer resolution along the optical axis. The technique is based on the influence of the microscope slide on the angular intensity distribution of fluorescence. Axial positions are determined by measuring the proportion of light emitted below the critical angle of total internal reflection, which behaves in a classical way, and light emitted above the critical angle, which is exponentially dependent on the distance of the fluorophore from the microscope slide.

The axon initial segment (AIS) is enriched in specific adaptor, cytoskeletal and transmembrane molecules. During AIS establishment, a membrane diffusion barrier is formed between the axon and the somatodendritic domain.Recently, an axonal periodic pattern of actin, spectrin and ankyrin forming 190 nm distanced, ring-like structures has been discovered. However, whether this structure is related to the diffusion barrier function is unclear.Here, we performed single particle tracking timecourse experiments on hippocampal neurons during AIS development. We analyzed the mobility of lipid-anchored molecules by high-speed single particle tracking and correlated positions of membrane molecules with the nanoscopic organization of the AIS cytoskeleton.We observe a strong reduction in mobility early in AIS development.Membrane protein motion in the AIS plasma membrane is confined to a repetitive pattern of ~190 nm spaced segments along the AIS axis as early as DIV4 and this pattern alternates with actin rings. Our data provide a new model for the mechanism of the AIS diffusion barrier.3

Multicolor three-dimensional (3D) superresolution techniques allow important insight into the relative organization of cellular structures. While a number of innovative solutions have emerged, multicolor 3D techniques still face significant technical challenges. In this Letter we provide a straightforward approach to single-molecule localization microscopy imaging in three dimensions and two colors. We combine biplane imaging and spectral-demixing, which eliminates a number of problems, including color cross-talk, chromatic aberration effects, and problems with color registration. We present 3D dual-color images of nanoscopic structures in hippocampal neurons with a 3D compound resolution routinely achieved only in a single color.

The recent development of single-molecule localization-based super-resolution techniques has afforded a resolution in the nanometer range in light microscopy. The ability to resolve biological structures on this scale by multicolor techniques faces significant challenges which have prevented their widespread use. Here, we provide a generic approach for high-quality simultaneous two-color single-molecule localization microscopy imaging of any combination of GFP- and RFP-tagged proteins with the use of nanobodies. Our method addresses a number of common issues related to two-color experiments, including accuracy and density of labeling as well as chromatic aberration and color-crosstalk with only minimal technical requirements. We demonstrate two-color imaging of various nanoscopic structures and show a compound resolution down to the limit routinely achieved only in a single color.