The molecular mechanism responsible for capturing, sorting, and retrieving vesicle membrane proteins following triggered exocytosis is not understood. Here we image the post-fusion release and then capture of a vesicle membrane protein, the vesicular acetylcholine transporter, from single vesicles in living neuroendocrine cells. We combine these measurements with super-resolution interferometric photo-activation localization microscopy (iPALM), electron microscopy, and modeling to map the nanometer-scale topography and architecture of the structures responsible for the transporter’s capture following exocytosis. We show that after exocytosis, the transporter rapidly diffuses into the plasma membrane, but most travels only a short distance before it is locally captured over a dense network of membrane-resident clathrin-coated structures. We propose that the extreme density of these structures acts as a short-range diffusion trap. They quickly sequester diffusing vesicle material and limit its spread across the membrane. This system could provide a means for clathrin-mediated endocytosis to quickly recycle vesicle proteins in highly excitable cells.

The promotion of structural and functional plasticity by estrogens is a promising approach to enhance central nervous system function in the aged. However, how the sensitivity to estrogens is regulated across brain regions, age and experience is poorly understood. To ask if estradiol treatment impacts structural and functional plasticity in sensory cortices, we examined the acute effect of 17α-Estradiol in adult Long Evans rats following chronic monocular deprivation, a manipulation that reduces the strength and selectivity of deprived eye vision. Chronic monocular deprivation decreased thalamic input from the deprived eye to the binocular visual cortex and accelerated short-term depression of the deprived eye pathway, but did not change the density of excitatory synapses in primary visual cortex. Importantly, we found that the classical estrogen receptors ERα and ERβ were robustly expressed in the adult visual cortex, and that a single dose of 17α-Estradiol reduced the expression of the calcium-binding protein parvalbumin, decreased the integrity of the extracellular matrix and increased the size of excitatory postsynaptic densities. Furthermore, 17α-Estradiol enhanced experience-dependent plasticity in the amblyopic visual cortex, by promoting response potentiation of the pathway served by the non-deprived eye. The promotion of plasticity at synapses serving the non-deprived eye may reflect selectivity for synapses with an initially low probability of neurotransmitter release, and may inform strategies to remap spared inputs around a scotoma or a cortical infarct.

The promotion of structural and functional plasticity by estrogens is a promising therapy to enhance central nervous system function in the aged. However, how the sensitivity to estrogens is regulated across brain regions, age and experience is poorly understood. To ask if estradiol treatment impacts structural and functional plasticity in sensory cortices, we examined the acute effect of 17α Estradiol in adult Long Evans (LE) rats following chronic monocular deprivation, a manipulation that reduces the strength and selectivity of deprived eye vision. Chronic monocular deprivation decreased thalamic input from the deprived eye to the binocular visual cortex and accelerated short-term depression of the deprived eye pathway, without changing the total density of excitatory synapses. Importantly, we found that the classical estrogen receptors ERα and ERβ are robustly expressed in the adult visual cortex, and that a single dose of 17α Estradiol increased the size of excitatory postsynaptic densities, reduced the expression of parvalbumin and decreased the integrity of the extracellular matrix. Furthermore, 17α Estradiol enhanced experience-dependent plasticity in the amblyopic visual cortex, and promoted response potentiation of the pathway served by the non-deprived eye. The promotion of plasticity at synapses serving the non-deprived eye may reflect selectivity for synapses with an initially low probability of neurotransmitter release, and may inform applications to remap spared inputs around a scotoma or a cortical infarct 4 Materials and Methods: Subjects Long Evans (LE) Rats (strain 006, RRID:RGD_2308852) were purchased from Charles River Laboratories (Raleigh, NC). Equal numbers of adult (>postnatal day 180, >P180) males and females were used. Animals were raised in 12/12 hour light/dark cycle and experiments were performed, or subjects were sacrificed, 6 hours into the light phase. Brains from 8-12 week old female ERβ -/rats were provided by M.A. Karim Rumi of the University of Kansas Medical Center 39 . All procedures were approved by the University of Maryland Institutional Animal Care and Use Committee and were carried out in accordance with the Guide for the Care and Use of Laboratory Animals. Monocular deprivationP14 LE rat pups were anesthetized with ketamine/xylazine (100 mg/10 mg/kg, intraperitoneal).

PMT, a 146 kDa bacterial protein, is a potent mitogen and a strong inducer of anchorage‐independent growth for fibroblast and osteoclast cells. We showed that PMT activated the ERK pathway, and that this ERK activation was also detected when quiescent 3T3 cells were treated with conditioned media taken from PMT‐treated cells. These results support the existence of a diffusible factor(s) that mediates ERK activation. Microarray analysis revealed that connective tissue growth factor (CTGF) is the most elevated factor in PMT‐treated cells. CTGF is a 36‐kDa secreted protein and a member of the CCN family. It stimulates cellular proliferation and regulation of angiogenesis and tumorigenesis. Western blot analysis using CTGF antibody revealed a robust elevation of an immunoreactive protein of 36–38 kDa in both cell lysate and conditioned media of PMT‐treated cells. This observed PMT‐induced CTGF elevation was also detected in WT, but not in Gαq/Gα11‐deficient MEF cells. While PMT failed to induce ERK activation in Gαq/Gα11‐deficient MEF cells, the conditioned media can activate ERK in these cells suggesting that PMT upregulates the CTGF protein level and leads to ERK activation.This work was supported, in part, by the Intramural Research Program of the NIH, NHLBI.