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Release probability of hippocampal glutamatergic terminals scales with the size of the active zone

Release probability of hippocampal glutamatergic terminals scales with the size of the active zone. Holderith et al, 2012 Amelia Moffatt, October 9 2012 . Introduction . Synapses are diverse in their structure and functioning

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Release probability of hippocampal glutamatergic terminals scales with the size of the active zone

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  1. Release probability of hippocampal glutamatergic terminals scales with the size of the active zone Holderith et al, 2012 Amelia Moffatt, October 9 2012

  2. Introduction • Synapses are diverse in their structure and functioning • Probability of neurotransmitter release, size of synaptic vesicles, plasticity at the synapse, and size of post-synaptic response. • To what extent can the morphological features of cortical glutamatergic terminals predict their functional properties? • How the neurotransmitter release probability and presynaptic [Ca2+] transients relate to ultrastructure of hippocampal glutamatergic axon terminals.

  3. Previous Research: • Postsynaptically • Postsynaptic glutamate receptors (AMPA and NMDA), area of PSD and amplitude of postsynaptic response are all correlated. • Presynaptically • Release probability correlates with number of readily releasable vesicles=number docked at presynaptic active zones (AZ). • Number of docked vesicles proportional to area of AZ and volume of terminal. • Larger the presynaptic AZ area=higher the release probability • Challenges of such a clear correlation? **PSD= Postsynaptic density

  4. Methods: • Subject: All brain slices were taken from male Wistar rats (14-17 days old) • Area of Study: CA3 pyramidal cells of the hippocampus- WHY? • Techniques: • In vitro patch clamp electrophoresis • Two-photon fluorescence Ca2+ imaging (learning Issue) • Post-hoc electron-microscopy 3D reconstruction of the imaged pre and post synaptic structures • Freeze fracture immugold labeling (localization of presynaptic proteins) (Learning Issue)

  5. Results: • Morphological Diversity of CA3 pyramidal cell axon terminals • Variability in Release probability and short-term plasticity • Variability in [Ca2+] in CA3 pyramidal cell axon terminals • Contribution of Ca2+ channel subtypes to [Ca2+] in boutons. • Cav2.1 subunit is confined to the presynaptic active zone • Rim1/2 and Cav2.1 have uniform densities in active zones (are not randomly distributed).

  6. Morphological Diversity of terminals: Figure 2 Figure 1

  7. Release probability and short term plasticity: • Significant correlation between PR1 and bouton volume and PR1 and active zone area.

  8. Variability of [Ca2+] in axon terminals: • Bouton volume and amplitude of [Ca2+] transients= no significant correlation. • Amplitude of [Ca2+] transients and active zone area= significant correlation • Total amount of Ca2+ per action potential and active zone area= significant correlation

  9. Contribution of Ca2+ channel subtypes to [Ca2+] in boutons: • Postsynaptic responses are mediated by voltage gated Ca2+ channels (cav)- Learning Issue • Cav2.1 (P/Q-type) • Cav2.2 (N-type) • Cav2.3 (R-type) • When a blocker was used the peak amplitude of Ca2+ transient was reduced. • Cav2.1,Cav2.2 and Cav2.3 subunits mediate postsynaptic responses at hippocampal glutamatergic terminals

  10. Cav2.1 subunits in the presynaptic active zone • Assumption: Number of Cav channels scales linearly with AZ area. • Freeze fractioning and electron microscopy immunogold labeling. • Cav2.1 subunit was confined to the active zone of presynaptic axon terminals of the hippocampal CA3 area. • Tested this by performing a double immunogold label for Rim1/2 and AMPA-type glutamate receptors.

  11. Cav2.1 and Rim1/2 have uniform densities in active zones • Found that release probability of neurotransmitters scales with size of presynaptic active zone. • Leads us to predict that presynaptic AZ has multiple neurotransmitter release sites. • Number of presynaptic AZ proteins should scale linearly with AZ area. • Analyzed: • SNAP-25 • Synaptobrevin • Cav2.1 subunit • Not randomly distributed! • Rim1/2

  12. Cav2.1 and Rim1/2: • Positive correlation between number of gold particles and active zone area.

  13. Conclusion • Research Questions: • How the neurotransmitter release probability and presynaptic [Ca2+] transients relate to ultrastructure of hippocampal glutamatergic axon terminals. • Major Conclusions: • Positive Correlation between release probability and AZ area • Positive Correlation between peak amplitude [Ca2+] transients and AZ area • Cav2.1 subunits and AZ protein Rim1/2 are confined to the AZ, and their numbers scale linearly with AZ area. • Cav2.1 subunits are clustered ‘nonrandomly’ within the AZ

  14. Conclusion: • Inferences: • Vesicle docking and release cannot occur anywhere in the active zone • Every channel within 0.01µm2 of AZ will contribute to release of a vesicle • Estimate 4 CaV channels in 0.01µm2 • 14 presynaptic voltage gated calcium channels facilitating release at the pyramidal cell axon terminal

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