37 research outputs found
An investigation into the transport and modulation of synaptophysin positive vesicles
Neuronal function, survival and architecture all critically depend on the precise transport of intracellular proteins to a vast array of synaptic connections. Disrupted intracellular transport leads to deficits in synaptic transmission, irregular cell morphology, misallocated organelles and cell death. In addition, axonal transport deficits have been noted in the early stages of several debilitating neurological conditions, thus, axonal transport deficits may contribute to disease progression. This makes it important that we understand the contribution of axonal transport to both physiological and pathophysiological cellular processes and to the transport of essential organelles.
As such the aims of this project were as follows: to investigate the long-term transport properties of visualised synaptic vesicles, to investigate whether vesicle transport could be modulated by changes in neuronal activity, to examine whether vesicle transport deficits exist in certain disease models and to develop novel assays for focusing the study of vesicle transport to specific neuronal cell types.
To investigate the transport properties of visualised synaptic vesicles we exploited a lentiviral vector to express a fluorescently tagged version of an abundant synaptic vesicle transmembrane protein, synaptophysin. Using synaptophysin-GFP (syp-GFP) as a synaptic vesicle marker we then tracked the movements of synaptic vesicles in the axons of dissociated hippocampal neurons. Synaptophysin-GFP expression revealed two fluorescent vesicle populations, one population that moved in a rapid and bi-directional manner and one population that accumulated into clusters of stationary vesicles at putative presynaptic sites. Each vesicle population was analysed independently. Moving vesicles were termed motile particles, whilst vesicle accumulations were termed vesicle clusters. To investigate potential activity-dependent changes in vesicle transport and vesicle cluster localisation we used acute or co-culture application of the GABAA receptor antagonists bicuculline (bic) (20µM) or Gabazine (gbz) (20µM), which can generate increased neuronal activity or epileptiform-like activity in vitro. As a result of bic treatment we observed a significant decrease in the size of stationary presynaptic vesicle clusters. Under control conditions the average size of vesicle clusters was 14.7±1.67µm2, reducing to 12.1±1.41µm2 following 10 hours of increased neuronal activity (p=0.0042, Wilcoxon-matched pairs test, n=80, 8 experiments). In addition, increased neuronal activity also led to a significant increase in vesicle cluster turnover, which increased from 28±6.89% under control conditions to 44±8.46% as a result of increased neuronal activity (p=0.0261, unpaired student t-test, n=25, 11 experiments). However, these changes were not accompanied by any alteration in vesicle transport, with the speed, the density and the proportion of motile particles remaining unaffected by increased neuronal activity (table 3.1). This suggests that each vesicle population may therefore be differentially modulated by increased neuronal activity.
To probe deeper for potential activity-dependent vesicle transport changes we restricted our study of vesicle transport to a specific axonal subtype, the hippocampal mossy fiber. To visualise mossy fiber vesicle transport, lentivirus expressing syp-GFP was pressure injected directly into the cell body layer of the dentate gyrus (DG) in hippocampal organotypic slice cultures. This revealed syp-GFP positive vesicles occupying both small (2-15µm3) and large (˃15µm3) mossy fiber synaptic terminals, which were found in and along the stratum lucidum. By examining the distribution of vesicle clusters at different time points following gbz or bic treatment (0hrs, 4hrs, 12hrs, 24hrs and 48hrs) we were able to show that epileptiform activity caused a delayed (>12 hours) but significant decrease in the proportion of large vesicle clusters. By 24 and 48 hours there was a significant decrease in the proportion of large vesicle clusters following bic treatment, decreasing from 9.4±1.21% under control conditions (n=11, 5 experiments) to 4.84%±0.72% after 24hrs (n=10, 4 experiments) and to 3.3±0.73% after 48hrs (n=12, 5 experiments), P<0.001, one-way ANOVA. This decrease in the proportion of large vesicle clusters may represent an important pathophysiological change triggered by epileptiform activity. Importantly, we also observed the same decrease in the proportion of large vesicle clusters in a mouse model of Rett syndrome, which models a severe neurodevelopmental disorder caused by a mutation in the gene coding MeCP2. As a consequence of bic treatment we observed a significant decrease in the proportion of large vesicle clusters from 7.2% ±1.78% in control cultures (n=6, 2 experiments), down to 0.9% ±0.6% in 48hr bic treated cultures (n=8, 3 experiments) and recovering to 6.9%±1.5% following bic wash out (n=11, 3 experiments); p<0.0001, one way ANOVA. Interestingly, Mecp2Stop/y hippocampal organotypic slices showed a greater decrease in the proportion of large vesicle clusters following 48hrs of bic treatment. The proportion of large vesicle clusters in 48hr bic treated WT slices was 3.3%±0.73%, whilst in 48rs bic treated Mecp2Stop/y slices it was 0.9%±0.6%, p=0.01, two-way ANOVA. These observations suggest that Mecp2Stop/y hippocampal organotypic slices are more sensitive to epileptiform activity than WT slices and may possess deficits in the vesicle transport system.
Primary dissociated hippocampal cell cultures benefit from being both optically and experimentally accessible but lack a defined cellular arrangement. This hampers both the identification and study of specific cell types and specific synaptic connections. To overcome this limitation we developed a modified dissociated cell culture assay for defining the arrangement of dissociated hippocampal neurons. We cultured purified DG and CA3 cell populations in close opposition using a magnetic barrier, but transduced only DG granule cells with lenti-synaptophysin-GFP in order to visualise vesicle transport specifically in mossy fibers. Immunocytochemistry and vital dyes were used to confirm that specific cell populations could be cultured in close proximity, to confirm that lentiviral transduction was highly selective to DG granule cells and to post-hoc identify that vesicle trafficking was occurring specifically in mossy fibers. Using this method it was possible to image vesicle transport specifically in mossy fibers and to investigate vesicle cluster dynamics at putative MF-CA3 synapses. We conclude that this method is a significant improvement to previous techniques because dissociated cells can be arranged to form physiologically relevant synaptic connections, whilst remaining highly accessible to both live imaging and experimental manipulation
BDNF Expression in Cortical GABAergic Interneurons
Brain-derived neurotrophic factor (BDNF) is a major neuronal growth factor that is widely expressed in the central nervous system. It is synthesized as a glycosylated precursor protein, (pro)BDNF and post-translationally converted to the mature form, (m)BDNF. BDNF is known to be produced and secreted by cortical glutamatergic principal cells (PCs); however, it remains a question whether it can also be synthesized by other neuron types, in particular, GABAergic interneurons (INs). Therefore, we utilized immunocytochemical labeling and reverse transcription quantitative PCR (RT-qPCR) to investigate the cellular distribution of proBDNF and its RNA in glutamatergic and GABAergic neurons of the mouse cortex. Immunofluorescence labeling revealed that mBDNF, as well as proBDNF, localized to both the neuronal populations in the hippocampus. The precursor proBDNF protein showed a perinuclear distribution pattern, overlapping with the rough endoplasmic reticulum (ER), the site of protein synthesis. RT-qPCR of samples obtained using laser capture microdissection (LCM) or fluorescence-activated cell sorting (FACS) of hippocampal and cortical neurons further demonstrated the abundance of BDNF transcripts in both glutamatergic and GABAergic cells. Thus, our data provide compelling evidence that BDNF can be synthesized by both principal cells and INs of the cortex
Differential Dependence of GABAergic and Glutamatergic Neurons on Glia for the Establishment of Synaptic Transmission
In the mammalian cortex, GABAergic and glutamatergic neurons represent 2 major neuronal classes, which establish inhibitory and excitatory synapses, respectively. Despite differences in their anatomy, physiology and developmental origin, both cell types require support from glial cells, particularly astrocytes, for their growth and survival. Recent experiments indicate that glutamatergic neurons also depend on astrocytes for synapse formation. However, it is not clear if the same holds true for GABAergic neurons. By studying highly pure GABAergic cell cultures, established through fluorescent activated cell sorting, we find that purified GABAergic neurons are smaller and have reduced survival, nevertheless they establish robust synaptic transmission in the absence of glia. Support from glial cells reverses morphological and survival deficits, but does little to alter synaptic transmission. In contrast, in cultures of purified glutamatergic neurons, morphological development, survival and synaptic transmission are collectively dependent on glial support. Thus, our results demonstrate a fundamental difference in the way GABAergic and glutamatergic neurons depend on glia for the establishment of synaptic transmission, a finding that has important implications for our understanding of how neuronal networks develop
The Higher Sensitivity of GABAergic Compared to Glutamatergic Neurons to Growth-Promoting C3bot Treatment Is Mediated by Vimentin
The current study investigates the neurotrophic effects of Clostridium botulinum C3 transferase (C3bot) on highly purified, glia-free, GABAergic, and glutamatergic neurons. Incubation with nanomolar concentrations of C3bot promotes dendrite formation as well as dendritic and axonal outgrowth in rat GABAergic neurons. A comparison of C3bot effects on sorted mouse GABAergic and glutamatergic neurons obtained from newly established NexCre;Ai9xVGAT Venus mice revealed a higher sensitivity of GABAergic cells to axonotrophic and dendritic effects of C3bot in terms of process length and branch formation. Protein biochemical analysis of known C3bot binding partners revealed comparable amounts of β1 integrin in both cell types but a higher expression of vimentin in GABAergic neurons. Accordingly, binding of C3bot to GABAergic neurons was stronger than binding to glutamatergic neurons. A combinatory treatment of glutamatergic neurons with C3bot and vimentin raised the amount of bound C3bot to levels comparable to the ones in GABAergic neurons, thereby confirming the specificity of effects. Overall, different surface vimentin levels between GABAergic and glutamatergic neurons exist that mediate neurotrophic C3bot effects
Optically Induced Calcium-Dependent Gene Activation and Labeling of Active Neurons Using CaMPARI and Cal-Light
The advent of optogenetic methods has made it possible to use endogeneously produced molecules to image and manipulate cellular, subcellular, and synaptic activity. It has also led to the development of photoactivatable calcium-dependent indicators that mark active synapses, neurons, and circuits. Furthermore, calcium-dependent photoactivation can be used to trigger gene expression in active neurons. Here we describe two sets of protocols, one using CaMPARI and a second one using Cal-Light. CaMPARI, a calcium-modulated photoactivatable ratiometric integrator, enables rapid network-wide, tunable, all-optical functional circuit mapping. Cal-Light, a photoactivatable calcium sensor, while slower to respond than CaMPARI, has the capacity to trigger the expression of genes, including effectors, activators, indicators, or other constructs. Here we describe the rationale and provide procedures for using these two calcium-dependent constructs (1) in vitro in dissociated primary neuronal cell cultures (CaMPARI & Cal-Light); (2) in vitro in acute brain slices for circuit mapping (CaMPARI); (3) in vivo for triggering photoconversion or gene expression (CaMPARI & Cal-Light); and finally, (4) for recovering photoconverted neurons post-fixation with immunocytochemistry (CaMPARI). The approaches and protocols we describe are examples of the potential uses of both CaMPARI & Cal-Light. The ability to mark and manipulate neurons that are active during specific epochs of behavior has a vast unexplored experimental potential
Pion emission from the T2K replica target: method, results and application
The T2K long-baseline neutrino oscillation experiment in Japan needs precise
predictions of the initial neutrino flux. The highest precision can be reached
based on detailed measurements of hadron emission from the same target as used
by T2K exposed to a proton beam of the same kinetic energy of 30 GeV. The
corresponding data were recorded in 2007-2010 by the NA61/SHINE experiment at
the CERN SPS using a replica of the T2K graphite target. In this paper details
of the experiment, data taking, data analysis method and results from the 2007
pilot run are presented. Furthermore, the application of the NA61/SHINE
measurements to the predictions of the T2K initial neutrino flux is described
and discussed.Comment: updated version as published by NIM
Measurement of negatively charged pion spectra in inelastic p+p interactions at = 20, 31, 40, 80 and 158 GeV/c
We present experimental results on inclusive spectra and mean multiplicities
of negatively charged pions produced in inelastic p+p interactions at incident
projectile momenta of 20, 31, 40, 80 and 158 GeV/c ( 6.3, 7.7,
8.8, 12.3 and 17.3 GeV, respectively). The measurements were performed using
the large acceptance NA61/SHINE hadron spectrometer at the CERN Super Proton
Synchrotron.
Two-dimensional spectra are determined in terms of rapidity and transverse
momentum. Their properties such as the width of rapidity distributions and the
inverse slope parameter of transverse mass spectra are extracted and their
collision energy dependences are presented. The results on inelastic p+p
interactions are compared with the corresponding data on central Pb+Pb
collisions measured by the NA49 experiment at the CERN SPS.
The results presented in this paper are part of the NA61/SHINE ion program
devoted to the study of the properties of the onset of deconfinement and search
for the critical point of strongly interacting matter. They are required for
interpretation of results on nucleus-nucleus and proton-nucleus collisions.Comment: Numerical results available at: https://edms.cern.ch/document/1314605
Updates in v3: Updated version, as accepted for publicatio
Measurement of Production Properties of Positively Charged Kaons in Proton-Carbon Interactions at 31 GeV/c
Spectra of positively charged kaons in p+C interactions at 31 GeV/c were
measured with the NA61/SHINE spectrometer at the CERN SPS. The analysis is
based on the full set of data collected in 2007 with a graphite target with a
thickness of 4% of a nuclear interaction length. Interaction cross sections and
charged pion spectra were already measured using the same set of data. These
new measurements in combination with the published ones are required to improve
predictions of the neutrino flux for the T2K long baseline neutrino oscillation
experiment in Japan. In particular, the knowledge of kaon production is crucial
for precisely predicting the intrinsic electron neutrino component and the high
energy tail of the T2K beam. The results are presented as a function of
laboratory momentum in 2 intervals of the laboratory polar angle covering the
range from 20 up to 240 mrad. The kaon spectra are compared with predictions of
several hadron production models. Using the published pion results and the new
kaon data, the K+/\pi+ ratios are computed.Comment: 10 pages, 11 figure
NA61/SHINE facility at the CERN SPS: beams and detector system
NA61/SHINE (SPS Heavy Ion and Neutrino Experiment) is a multi-purpose
experimental facility to study hadron production in hadron-proton,
hadron-nucleus and nucleus-nucleus collisions at the CERN Super Proton
Synchrotron. It recorded the first physics data with hadron beams in 2009 and
with ion beams (secondary 7Be beams) in 2011.
NA61/SHINE has greatly profited from the long development of the CERN proton
and ion sources and the accelerator chain as well as the H2 beamline of the
CERN North Area. The latter has recently been modified to also serve as a
fragment separator as needed to produce the Be beams for NA61/SHINE. Numerous
components of the NA61/SHINE set-up were inherited from its predecessors, in
particular, the last one, the NA49 experiment. Important new detectors and
upgrades of the legacy equipment were introduced by the NA61/SHINE
Collaboration.
This paper describes the state of the NA61/SHINE facility - the beams and the
detector system - before the CERN Long Shutdown I, which started in March 2013