Ultimately, the true test of the cascade hypothesis is to conduct

Ultimately, the true test of the cascade hypothesis is to conduct a true primary prevention trial with anti-Aβ therapy in patients destined to develop AD but prior to the onset of measureable Aβ deposition. Alternatively, one might envision that the hypothesis could be reasonably well tested in secondary prevention (very early intervention) trial in subjects with Aβ pathology only, prior to the neurodegenerative phase (preclinical

stage 1). In the later scenario, the test potentially would be more effective if the therapeutic modality enhanced clearance of Aβ. In order to conduct a primary prevention study with a therapeutic agent selleckchem for any disease, one needs a strong scientific rationale. Although the Aβ aggregate/amyloid hypothesis is not without its critics, it has very strong scientific underpinnings. In the context of trying to move the field toward primary prevention studies with anti-Aβ therapies, it is important to consider the views of those who are skeptical about the role of Aβ in AD.

One valid critique of the hypothesis is that most of the experimental evidence supporting the hypothesis comes from the study of the genetic alterations that cause familial autosomal dominant AD (Selkoe, 2001 and Younkin, 1998). Thus, a key assumption check details is that pathological cascades in sporadic AD are the same as in familial AD. Given that the typical genetic and sporadic forms of AD are very similar with respect to postmortem findings, clinical course, and evidence from the emerging biomarker and imaging cascade in sporadic AD, this seems a reasonable assumption (

Shepherd et al., 2009). Indeed, when examples from other human diseases with genetic and sporadic forms, such as hypercholesterolemia and prion diseases, are considered, this concept is quite tenable ( Brown and Goldstein, 1986 and Prusiner, 1998). Nevertheless, Florfenicol because of the late age of onset, sporadic AD is not a completely uniform clinical or pathological entity: it is complicated by other systemic or CNS illnesses and conditions, and the biological processes of aging. Thus, the underlying brain pathology that results in clinical dementia may represent the convergence of independent processes and not necessarily a single pathologic entity ( Small and Duff, 2008), as thought to be the case in early onset familial AD. Indeed, in the over-80-year-old population who have died with a clinical diagnosis of probable AD dementia, postmortem phenotypes often show not only plaques and tangles pathology characteristic of AD, but other proteinopathies and vascular insults as well ( Dickson, 2001). However, even if this is the case, and multiple pathologies synergize in some subpopulations of those affected with what we define as AD, one might still predict that preventing Aβ deposition would have substantial, but perhaps not complete, efficacy.

In addition, we generated uncorrelated Poissonian spike trains re

In addition, we generated uncorrelated Poissonian spike trains representing background

activity from distant cortical areas. The populations of morphologically reconstructed neurons received a selection of input spike trains from the laminar network and background activity based on the morphology and connectivity of each cell type. In this way each cell in the populations of reconstructed cells had on average Selleckchem Epigenetic inhibitor the same number of incoming synapses as a cell in the laminar network resulting in the same mean synaptic input (see Supplemental Information). Synapses were distributed differently across the dendritic tree of the reconstructed cells depending on the origin of the presynaptic cell type (see Supplemental Information).

This setup produced input correlations cξcξ and correlations between single cell LFP contributions cϕcϕ, that were specific for each population. The resulting Protein Tyrosine Kinase inhibitor input correlations cξcξ between total input currents and LFP correlation cϕcϕ are comparable to our previous simulations (Figure 6D, compare with Figure 4G). The L5 population produced larger LFP correlations cϕcϕ than the L3 and L4 populations even though the input correlation cξcξ was lower, in line with the above results (Figure 4G). We computed the LFP amplitude for three different populations (of the same types as before, Figure 6B) for different cortical depths. Similar to the generic scenarios for the case of uncorrelated synaptic inputs (Figure 3), both the reach and amplitude vary with cortical depth with a minimum reach R∗, and maximum amplitude σ in the soma layer of each population. Further, the level of input correlations provided by the spontaneous spiking activity of the laminar cortical network was sufficient to increase both the reach and amplitude of the LFP for the MycoClean Mycoplasma Removal Kit L3 and L5 populations as compared to the situation where the LFP contributions from different cells would have been uncorrelated ( Figures 6B and 6C, dashed lines; by setting Rc = 0; see Experimental Procedures and Equation 12). The

reach of the LFP from the L4 population was similar to the situation with uncorrelated synaptic input. In Figure 6B, we assumed that each neuron draws its inputs from the same (statistical) ensemble of presynaptic spike trains, resulting in the same input correlation in the whole population. We next calculated the LFP reach in the soma layer for the three different populations assuming that only LFP contributions from cells within a radius Rc were correlated and contributions from cells outside this region were uncorrelated (see Experimental Procedures and Equation 12). Also here we found that for the pyramidal cell populations the LFP reach was largely determined by the spatial scale of correlated activity while the LFP reach for the L4 population was largely unaffected by the spatial scale of correlations ( Figure 6E).

We have presented in vivo, for the first time a highly detailed d

We have presented in vivo, for the first time a highly detailed description of the early events following DNA vaccination and this has considerable implications for the rational development, manipulation and application of DNA vaccination. Our data is consistent with the following scenario. Injected DNA vaccines rapidly enter the peripheral blood from the injection site but also reach lymphoid tissues directly as free DNA via the afferent lymphatics. The relatively large molecular size of pDNA probably precludes it from flowing into the

conduits of LNs, and thereby LN resident DCs from sampling Buparlisib concentration it directly, but rather it may be taken up by cells in the subcapsular sinus that then migrate into deeper areas of the LN such as the DC and T cell-containing interfollicular Sirolimus datasheet and paracortical areas. pDNA and/or expressed Ag may then be transferred from these cells to CD11c+ DCs for presentation to naïve T cells. Concomitantly, bloodborne DNA reaches the bone marrow and spleen where it is taken up by CD11b+MHCIIlow cells (monocytes/myeloid DC precursors). The bone marrow may then act as a reservoir for cell-associated pDNA or its presence may induce the maturation and mobilisation of monocytes/myeloid DC precursors into the periphery.

The observation that naïve CD4 T cells in draining and distal LNs and spleen “see” Ag simultaneously, suggests that pMHC complexes are widely distributed and the rapid dissemination Ketanserin of pDNA may be the reason for this. Although we were unable to precisely identify and definitively link the cells acquiring, expressing and presenting DNA-encoded Ag, due to the minute amounts of Ag involved and the rarity of these cells, they are clearly able to initiate DNA vaccine-induced immune responses. This work was supported by a Wellcome Trust

project grant to PG, CMR and TJM Conflict of interest statement: The authors declare no financial conflict of interest. “
“Bacille Calmette-Guerin (BCG), the vaccine for protection against tuberculosis (TB), is currently given to most of the world’s infants as part of the WHO’s Expanded Program on Immunisation (EPI) [1]. Clinical trials of BCG show variable efficacy (0–80%) against pulmonary tuberculosis in adults [2], but high efficacy in infants against the severe forms of childhood tuberculosis [3]. Several new TB vaccines are being tested or are soon to be tested in clinical trials [4]. Some of these would be given as booster vaccines following BCG vaccination, and others are genetically modified BCG vaccines. Biomarkers of protection are urgently required to help assess these new TB vaccines, as without them clinical trials will be lengthy and require very large numbers of study subjects [5]. Studying immune responses to BCG vaccination in the UK, where BCG vaccination has been shown to provide 75% protection, gives us an opportunity to identify biomarkers of protection following successful vaccination against TB.

The joint learning of both mappings in a single-pathway appears t

The joint learning of both mappings in a single-pathway appears to be difficult or impossible. The corollary of these computational insights is that the double dissociations between certain types of aphasia (e.g., conduction aphasia—impaired repetition versus semantic dementia—impaired

Selleck JAK inhibitor comprehension and speaking/naming) reflect these same divisions of labor in the human brain. The simulations also suggest that the division of labor between the two pathways is not absolute or mutually exclusive. The two pathways work together to deliver adult language performance (and aphasic naming and repetition abilities; see Nozari et al. [2010]). This division of labor represents one solution for an intact, fully-resourced computational model. The solution is not fixed, however, and following damage, processing can be reoptimized both within and across the two pathways, thereby mimicking spontaneous recovery observed post stroke (Lambon Ralph, 2010, Leff et al., 2002, Sharp et al., 2010 and Welbourne and Lambon Ralph, 2007). These simulations suggest that this recovery sometimes comes at the cost of Autophagy inhibitor screening library other functions

(e.g., more of the computation underpinning repetition can be taken up by the ventral pathway but this is only possible for words and not nonwords). Analysis of each layer in the model demonstrated that the internal similarity structure changed gradually across successive regions. In line with found recent neuroimaging results (Scott

et al., 2000 and Visser and Lambon Ralph, 2011), the ventral pathway shifted from coding predominantly acoustic/phonological to predominantly semantic structure. Additional control simulations (comparing this multilayer pathway with a single, larger intermediate layer; see Figure S3) indicated that this gradual shift led to much better performance when extracting the modality-invariant meaning from the time-varying auditory input. Finally, a second key finding from these analyses is that the structure of the representations can change across tasks even within the same region. For example, the aSTG is much more sensitive to semantic similarity during speaking/naming than in comprehension, a fact that might explain recent VSLM data (Schwartz et al., 2009) (see Results). If correct, then this result has clear implications for the limits of the subtraction assumption (Price et al., 1997), commonly utilized in functional neuroimaging. When implementing any cognitive or neural hypothesis in a computational model various assumptions have to be made explicit. In this section we outline our working assumptions and the rationale underlying them. We then provide a summary of implementational details. Copies of the model files are available from the authors upon request.

To achieve this we generated mice carrying a floxed allele of Neu

To achieve this we generated mice carrying a floxed allele of Neurofascin (see Experimental Procedures) CHIR-99021 mw and a transgenic line in which the CreERT2 cassette was driven by the Thy1.2 promoter (TCE) ( Caroni, 1997 and Feil et al., 1997). Using a reporter line, we showed that these TCE mice expressed tamoxifen-inducible Cre robustly in cerebellar Purkinje cells ( Figure S2). To inactivate the Nfasc gene efficiently using

tamoxifen induction of Cre activity, we generated TCE transgenic mice with one floxed and one null allele of the gene (TCE/Nfascfl/−). Western blot analysis of hindbrain homogenates from TCE/Nfascfl/− mice 6 weeks after tamoxifen treatment showed that recombination resulted in a reduction in the level of Nfasc186, whereas the glial isoform (Nfasc155) was unaffected ( Figure 3A). Although we focused our analysis on brains 6 weeks posttamoxifen to ensure complete loss of Nfasc186 at AIS and AIS disruption, the disappearance of Nfasc186 at the AIS was clear at 3 weeks after tamoxifen-induced recombination, a time when the other components of the complex were still present ( Figure 3B). Although there was some reduction in the length of NrCAM staining at 3 weeks, it was not lost completely until 4 weeks posttamoxifen. Between 3 and 4 weeks

posttamoxifen, the kinetics of AnkyrinG, βIV-Spectrin, and NrCAM loss in vivo were rapid and coincident with the KU55933 disappearance of sodium channel immunostaining at the AIS, which was complete by 4 weeks, thus precluding an informative evaluation of the sequence in which these components are lost (data not shown). Nfasc186 was efficiently eliminated at the AIS of Purkinje cells 6 weeks posttamoxifen ( Figure 3C), Vasopressin Receptor since the number of Purkinje cells immunopositive for Nfasc186 was reduced from 99.2% ± 0.8% to 2.5% ± 2.5% (mean values ± SEM, n = 3, 40 cells per animal, p < 0.0001, unpaired Student's t test). Furthermore, and consistent with the results of the cerebellar slice culture experiment with Neurofascin null mice

( Figure 2), loss of Nfasc186 from the AIS abolished the immunofluorescence signal for sodium channels, AnkyrinG, βIV-Spectrin, and NrCAM ( Figure 3C). No demyelination was observed and the levels of myelin proteins, as assessed by western blotting, were unchanged (data not shown). Together, these in vitro and in vivo data suggest a distinct role for Nfasc186 in maintaining the mature configuration of the AIS. Thus, whereas assembly of the AIS appears to involve AnkyrinG acting as a master coordinator (Dzhashiashvili et al., 2007 and Sobotzik et al., 2009) and does not require Nfasc186, maintenance of the AIS, including AnkyrinG localization, appears to require Nfasc186. Because Nfasc186 is also believed to be important for the establishment of inhibitory synaptic input from basket cells onto Purkinje cells (Ango et al.

, 2009 and Gentet et al , 2010); and (3) the inherent bias in ext

, 2009 and Gentet et al., 2010); and (3) the inherent bias in extracellular recordings which require neurons to fire action potentials before they can be considered in the data set

(cells that do not fire or fire very rarely cannot be detected). Future experiments must directly investigate whether firing rates (and firing correlations) differ depending upon the behavioral conditions, for example running versus stationary (Niell and Stryker, 2010) and/or the complexity of the sensory input and the environment (multiple whiskers contacting textured objects compared to single whisker contacts with simple objects). Under our recording conditions we find a highly skewed distribution of spiking activity in layer 2/3 barrel cortex neurons during active touch, which leads to an interesting unresolved issue of sparse coding regarding the relative importance of the very few neurons that reliably fire many action potentials compared KU-57788 cell line to the very many neurons that fire few action potentials. We found that sparse action potential firing during active touch appeared to be enforced by the hyperpolarized reversal potential of the touch response. Indeed, we found close to linear relationships in individual neurons between PSP amplitude

and precontact membrane potential with reversal potentials usually hyperpolarized with respect to action potential threshold. If the precontact Wnt inhibitor membrane potential is spontaneously depolarized above this reversal potential, then the touch response is hyperpolarizing, therefore in fact playing an inhibitory role

by preventing the membrane potential from reaching action potential threshold. Thymidine kinase Each neuron has its own cell-specific reversal potential for the touch response. Importantly, we found a strong positive correlation of the touch-evoked firing probability with the reversal potential (Figure 5F). Indeed, the only neuron in our study that fired reliably during active touch was also the only neuron with a touch reversal potential above action potential threshold. The generally hyperpolarized reversal potentials suggest a prominent inhibitory GABAergic contribution to the active touch responses, since the reversal potential for glutamatergic excitatory postsynaptic potentials is close to 0 mV and the reversal potential for GABAergic inhibitory postsynaptic potentials is generally estimated between −70 and −90 mV. We find that GABAergic neurons are strongly recruited during active touch and they are therefore likely to contribute to driving the hyperpolarized reversal potential of the touch PSP, thus preventing the membrane potential from crossing action potential threshold for most neurons during active touch. Our results are consistent with the simple idea that active touch for a given cell evokes a well-defined mixture of excitatory and inhibitory conductances, which drive the membrane potential toward a specific reversal potential.

, 1982; Kamyshev et al , 1999; McBride et al , 1999; Siegel and H

, 1982; Kamyshev et al., 1999; McBride et al., 1999; Siegel and Hall, 1979; Tompkins et al., 1983). Memory in this assay is quantified as a learning index (LI), which measures the extent of the courtship suppression ( Experimental Procedures). Males homozygous for orb2+GFP had long-term, 24 hr memory comparable to that of control Canton-S males (2, orb2+GFP, LI = 31.7; 1, Canton-S, LI = 32.6), whereas

orb2ΔQGFP and orb2ΔQGFP/orb2attP males had no long-term memory (3, orb2ΔQGFP, LI = 0.63; 4, orb2ΔQGFP/orb2attP, LI = 3.03) ( Figure 1C; see Table S1 available online). These data are consistent with the lack of long-term memory previously reported for homozygous and hemizygous orb2ΔQ mutants ( Keleman et al., Dasatinib purchase 2007). Short-term, 1 hr memory of all tested genotypes was normal ( Figure 1C; Table S2), as previously reported also for orb2ΔQ mutants ( Keleman et al., 2007). These results validate our general strategy

for introducing targeted modifications at the orb2 locus and confirm that the C-terminal GFP tag does not impair Orb2 function. Accordingly, we also introduced the GFP tag for other modifications to the orb2 locus reported below, although for simplicity it is only indicated in allele or protein names when it is Lapatinib mouse exploited in immunolabeling or biochemistry experiments. We used antibodies against the GFP tag on the endogenous Orb2 protein encoded by orb2+GFP to determine its expression pattern and subcellular localization. At the level of light microscopy, Orb2 appeared to be broadly expressed throughout the nervous system of embryo, larvae, and adult, including the

ventral nerve cord (VNC) and the brain. In the adult brain Orb2 appeared to be widely expressed throughout various regions including the lobes, calyces, and soma of the mushroom bodies (MB), a center for olfactory memory formation in insect brains ( Heisenberg, 2003; Figure 2A). Previous studies in other species have variously placed CPEBs at either pre- or postsynaptic sites. Mouse CPEB3, for example, was reported to be present in postsynaptic densities, whereas Aplysia CPEB was shown to localize in presynaptic compartments ( Huang et al., 2003, 2006; Liu Sclareol and Schwartz, 2003; Wu et al., 1998). To examine the subcellular localization of Drosophila Orb2, we examined the calyx (input) region of the MB (see Experimental Procedures for details). Using immuno-electron microscopy, we detected Orb2 both in the presynaptic compartment of the extrinsic MB neurons, characterized by the presence of presynaptic specializations such as electron-dense active zones, synaptic vesicles and occasionally T bars, and the postsynaptic compartment likely to be the termini of the Kenyon cells (KCs) in the calyx, characterized by the presence of close membrane alignments with the presumptive presynaptic region ( Figure 2B). Furthermore, consistent with the reported role for Orb2 during development ( Hafer et al., 2011; Keleman et al.

, 2005) Three to four fMRI time series (1,125 measurements each

, 2005). Three to four fMRI time series (1,125 measurements each series) were acquired in each scan session, during which the monkey rested in the dark (lights off in the scanner and console room). The monkey’s eye position at the MRI scanner was monitored using a 60 Hz Docetaxel mouse long-range optics system (Model LRO, Applied Science Laboratories) to determine the periods when the eyes were stable. At the end of the awake fMRI scans, we anesthetized the animals (ketamine, 2–10 mg/kg, i.m.) to collect the field map

and structural images. Fixation Task. Monkey BU participated in a third fMRI experiment that required him to fixate on a central fixation point. A single fMRI time series was acquired (2,250 measurements) per scan session while the monkey performed a simple fixation task. A juice reward was provided at regular 2 s intervals as long as the monkey fixated on a central fixation point (0.50° diameter) within an invisible 4° this website square window ( Pinsk et al., 2005). This small gray fixation point on a black background was projected from a single-lamp, three-chip LCD projector (Christie LX650; Christie Digital Systems) outside the scanner room onto a translucent screen located at the end of the scanner bore at an ∼60 cm viewing distance. We synchronized

the display, eye position recordings, reward delivery, and the beginning of each scan via a computer running Presentation software (Neurobehavioral Systems). A total of five fMRI time series was acquired over five scan sessions. We acquired structural MRI and fMRI images on a 3 T head-dedicated scanner (Magnetom Allegra; Siemens) using a 12 cm transmit-receive surface coil (model NMSC-023; Nova Medical). fMRI images for the anesthesia condition, resting-state, and fixation task scanning sessions were acquired

with a gradient echo, echo planar sequence (field of view [FOV] = until 95 × 95 mm; matrix = 64 × 64; number of slices = 24; slice orientation = transverse; slice thickness = 1.5 mm; interslice gap = 0.5 mm; repetition time [TR] = 1,600 ms; echo time [TE] = 26 ms; flip angle = 66°; in-plane resolution = 1.5 mm2). Matching in-plane gradient echo field map and magnitude images were acquired to perform geometric unwarping of the echo planar imaging (EPI) images (TR = 500 ms, TE = 4.17/6.63 ms, flip angle = 55°) as well as T1-weighted structural images for coregistration of the fMRI data (magnetization-prepared rapid gradient echo; FOV = 128 × 128 mm; matrix = 256 × 256; number of slices = 160; slice thickness = 1.0 mm; TR = 2,500 ms; TE = 4.38 ms; flip angle = 8°; inversion time [TI] = 1,100 ms; in-plane resolution = 0.5 mm2). Details of the imaging parameters used for retinotopic mapping sessions are described in Arcaro et al. (2011). fMRI Data Preprocessing.

, 1994, Faraci and Breese, 1993, Lindauer

et al , 1999, N

, 1994, Faraci and Breese, 1993, Lindauer

et al., 1999, Niwa et al., 2000a and Peng et al., 2002). In addition, the vasculature-targeted information is conveyed by anatomically discrete local interneurons see more (Figure 1D), which either can be activated locally by presynaptically released glutamate, as in stellate neurons of the cerebellum (Rancillac et al., 2006 and Yang et al., 2000), or can act as relays for remote brainstem nuclei, such as the cholinergic basal forebrain nucleus or the serotonergic raphe nuclei (Cauli et al., 2004). In sum, although there is a close relationship between oxygen consumption and functional hyperemia (Hoge et al., 1999, Lin et al., 2010 and Offenhauser et al., 2005), metabolic byproducts do

not directly trigger blood flow changes. Instead, the same neurotransmitters that mediate neuron-to-neuron information exchange also initiate polysynaptic signaling Sirolimus clinical trial pathways that ultimately trigger functional hyperemia. The intraparenchymal vasculature is extensively covered by astrocytic endfeet (Mathiisen et al., 2010 and McCaslin et al., 2011) (Figure 2A and 2B), which may serve as functional intermediaries between neurons and blood vessels. This intimate anatomical relationship between astrocytes and blood vessels was already noted in some of the earliest descriptions of astrocytic morphology by Cajal and Golgi (Golgi, 1886 and Ramon y Cajal, 1895). Recent analyses of astrocytic morphology have

revealed that the vascular external surface is almost completely covered by astrocytic endfeet (Mathiisen et al., 2010, Nielsen et al., 1997, Petzold et al., 2008 and Simard et al., 2003). Moreover, perivascular astrocytic endfeet (Figure 2B) are important and highly specialized cellular compartments that are enriched in astrocyte-specific proteins such as aquaporin-4, connexin 43, purinergic receptors, and potassium channels (Price et al., 2002 and Simard et al., 2003). Finally, at the ultrastructural level, the processes of many vasoactive interneurons, ADP ribosylation factor in particular those expressing noradrenaline, synapse onto astrocytes rather than directly onto blood vessels (Hamel, 2006). These morphological and functional data indicate that, with the possible exception of gaseous transmitters, all signaling molecules targeted to the vasculature must first act on or pass through astrocytes in order to reach the smooth muscle cells in the vessel wall (Figure 1D). The organization of astrocytes into separate domains (Halassa et al., 2007) (Figure 2C) and the very close anatomical and functional relationship between astrocytes and neuronal synapses (Barres, 2008 and Haydon, 2001) (Figure 2D) make these cells ideal candidates to convey changes in neuronal activity levels to the vasculature and to be common executors of neurovascular pathways.

Our findings suggest that a small recycling pool supports neurotr

Our findings suggest that a small recycling pool supports neurotransmission in native central synapses and that the physical position of recycling vesicles in the terminal is an important factor in GDC-0973 solubility dmso their favored stimulus-driven fusion. To label functional vesicle pools in native hippocampal tissue, we prepared acute slices from rat brain and activated CA3 axons while the styryl dye FM1-43 (Betz and Bewick, 1992; Gaffield and Betz, 2006; Ryan et al., 1993) was applied to a target region in CA1 (Zakharenko et al., 2001) (Figure 1A). Confocal imaging revealed clear punctate

fluorescent staining (Figure 1B), the intensity of which was stimulus dependent (0–2,400 action potentials [APs]), consistent with the loading of synaptic vesicles in presynaptic terminals (Figure 1C). Labeling intensity reached saturation when electrical stimulation exceeded 600 APs and this

maximal load was not significantly different from the intensity of synapses labeled with hyperkalemic stimulation (Figure 1C, bottom, see figure legend for statistics). Next, we tested whether labeled terminals were release competent by monitoring fluorescence intensity during a further round of stimulation. Synapses readily underwent activity-evoked destaining consistent with exocytosis and dye loss (Figures 1D and 1E). Across the synaptic population, the timecourse OSI-906 of destaining became faster as the stimulation frequency increased but was highly variable between terminals (Figures 1E and 1F), reflecting substantial heterogeneity in individual synaptic release properties similar to previous findings in cultured hippocampal neurons (Branco et al., 2008; Murthy et al., 1997; Waters and Smith, 2002; Welzel et al., 2011). To establish that the recycling pool accessed during these destaining experiments had the same composition as the pool that was dye marked

during the loading protocol—in other words that it was preferentially Chlormezanone reused—we compared our experimental dye loss profiles to simulated destaining curves based on the reuse of varying fractions (0%–100%) of the recycling pool (see Experimental Procedures). The experimental data were best described by the simulated destaining profile corresponding to ∼90% vesicle reuse (see Experimental Procedures), implying that the recycling pool was essentially immutable over the timecourse of our experiments. These results demonstrate the robust stimulus-driven FM dye labeling and subsequent reuse of functionally recycling synaptic vesicles in native hippocampal slice. Next, we used an experimental approach that allows dye-labeled functional vesicle pools to be visualized at ultrastructural level.