Holderith et al, 2012

Amelia Moffatt, October 9 2012

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

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.

Introduction

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?

Previous Research:

**PSD= Postsynaptic density

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)

Methods:

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).

Results:

Morphological Diversity of terminals:

Figure 1 Figure 2

Significant correlation between PR1 and

bouton volume and PR1 and active zone area.

Release probability and short term plasticity:

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

Variability of [Ca2+] in axon terminals:

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

Contribution of Ca2+ channel subtypes to [Ca2+] in boutons:

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.

Cav2.1 subunits in the presynaptic active zone

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-25SynaptobrevinCav2.1 subunit

Not randomly distributed! Rim1/2

Cav2.1 and Rim1/2 have uniform densities in active zones

Cav2.1 and Rim1/2: Positive correlation between number of gold

particles and active zone area.

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 areaCav2.1 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

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 vesicleEstimate 4 CaV channels in 0.01µm2

14 presynaptic voltage gated calcium channels facilitating release at the pyramidal cell axon terminal

Conclusion: