1. Holderith et al, 2012 Amelia Moffatt, October 9 2012

2. 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 [Ca 2+ ] transients relate to ultrastructure of hippocampal glutamatergic axon terminals.

3. 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. 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. Morphological Diversity of CA3 pyramidal cell axon terminals Variability in Release probability and short-term plasticity Variability in [Ca 2+ ] in CA3 pyramidal cell axon terminals Contribution of Ca 2+ channel subtypes to [Ca 2+ ] 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. Figure 1 Figure 2

7. Significant correlation between P R 1 and bouton volume and P R 1 and active zone area.

8. Bouton volume and amplitude of [Ca 2+ ] transients= no significant correlation. Amplitude of [Ca 2+ ] transients and active zone area= significant correlation Total amount of Ca 2+ per action potential and active zone area= significant correlation

9. Postsynaptic responses are mediated by voltage gated Ca 2+ 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 Ca 2+ transient was reduced. Cav2.1,Cav2.2 and Cav2.3 subunits mediate postsynaptic responses at hippocampal glutamatergic terminals

10. 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.

12. 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

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

14. Research Questions: How the neurotransmitter release probability and presynaptic [Ca 2+ ] transients relate to ultrastructure of hippocampal glutamatergic axon terminals. Major Conclusions: Positive Correlation between release probability and AZ area Positive Correlation between peak amplitude [Ca 2+ ] 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

15. Inferences: Vesicle docking and release cannot occur anywhere in the active zone Every channel within 0.01µm 2 of AZ will contribute to release of a vesicle Estimate 4 CaV channels in 0.01µm 2 14 presynaptic voltage gated calcium channels facilitating release at the pyramidal cell axon terminal