An investigation into the transport and modulation of synaptophysin positive vesicles

Turko, Paul (2013) An investigation into the transport and modulation of synaptophysin positive vesicles. PhD thesis, University of Glasgow.

Abstract

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.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Synaptophysin, vesicle, vesicle transport, epileptiform activity, activity-dependent, cell culture
• Subjects: Q Science > QP Physiology; Q Science > Q Science (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Psychology & Neuroscience
• Supervisor's Name: Cobb, Dr. Stuart
• Date of Award: 2013
• Depositing User: Mr Paul Turko
• Unique ID: glathesis:2013-3594
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 Jun 2013 15:35
• Last Modified: 10 Jun 2013 15:35
• URI: https://theses.gla.ac.uk/id/eprint/3594