Presynaptic Ca2+ evokes exocytosis, endocytosis, and synaptic plasticity. However, Ca2+ flux and interactions at presynaptic molecular targets are difficult to quantify because fluorescence imaging has limited resolution. We have focused on two presynaptic models of calcium signaling - firstly a simple  vertebrate model, the lamprey, which enables extraordinary experimental access to presynaptic terminals. Highlights of this work is indicated below and our most recent work recording and imaging presynaptic calcium channels has enabled us to quantify their activity with unprecedented accuracy.

Cochilla, A.J. And Alford, S. (1998) Metabotropic glutamate receptor-mediated control of neurotransmitter release. Neuron. 20:1007-1016

Cochilla, A.J. And Alford, S (1999). NMDA receptor-mediated control of presynaptic calcium and neurotransmitter release. Journal of Neuroscience 19:193-205

Photowala H, Freed R, Alford S. (2005) Location and function of vesicle clusters, active zones and Ca2+ channels in the lamprey presynaptic terminal. Journal of Physiology (London). 569:119-135

S Ramachandran, S Rodriguez, M Potcoava, S Alford (2022) Single calcium channel nanodomains drive presynaptic calcium entry at lamprey reticulospinal presynaptic terminals. Journal of Neuroscience 42 (12), 2385-2403.

In mammalian systems we have also quantified calcium entry during action potential firing and have used computer simulations derived from these data to further constrain properties of presynaptic calcium signaling. This work along with collaborations with Dr Leon Tai on the effects of APOE on presynaptic homeostasis, is the core of work we are now performing to understand how aging, sex and APOE genotype alter both signaling and calcium homeostasis in presynaptic terminals

S Zaldua, FC Damen, R Pisharody, R Thomas, KD Fan, GK Ekkurthi, SB Scheinman, S Alahmadi, FM Marottoli, S Alford, K Cai, LM Tai. (2020) Epidermal growth factor treatment of female mice that express APOE4 at an age of advanced pathology mitigates behavioral and cerebrovascular dysfunction. Heliyon 6 (5), e03919

Scheinman SB, Tseng KY, Alford S and Tai LM (2023) Higher Neuronal Facilitation and Potentiation with APOE4 Suppressed by Angiotensin II. Molecular Neurobiology (in press)

 In rats of either sex, we measured single varicosity presynaptic Ca2+ using Ca2+ dyes as buffers, and constructed models of Ca2+ dispersal. Action potentials evoked Ca2+ transients  with little variation when measured with low-affinity dye within 2 ms of stimulation. We have quantified endogenous Ca2+ buffering capacity, action potential-evoked free [Ca2+]i and total Ca2+ amounts entering terminals by using Ca2+dyes as buffers. We have focused on two presynaptic models of calcium signing - firstly a vertebrate model, the lamprey, which enables extraordinary experimental access to presynaptic terminals. Highlight of this work is indicated below:

These data constrained Monte Carlo (MCell) simulations of Ca2+ entry, buffering, and removal. Simulations of experimentally-determined Ca2+ fluxes, buffered by simulated calbindin28K well fit data, and are consistent with clustered Ca2+ entry followed within 4 ms by diffusion throughout the varicosity. Repetitive stimulation caused free varicosity Ca2+ to sum. However, simulated in nanometer domains, its removal by pumps and buffering was negligible, while local diffusion dominated. Thus, Ca2+ within tens of nanometers of entry, did not accumulate.  A model of synaptotagmin1 (syt1)-Ca2+ binding indicates that even with 10 µM free varicosity evoked Ca2+, syt1 must be within tens of nanometers of channels to ensure occupation of all its Ca2+ binding sites. Repetitive stimulation, evoking short-term synaptic enhancement, does not modify probabilities of Ca2+ fully occupying syt1’s C2 domains, suggesting that enhancement is not mediated by Ca2+-syt1 interactions. We conclude that at spatio-temporal scales of fusion machines, Ca2+ necessary for their activation is diffusion dominated.

Hamid E, Church E, Wells CA, Zurawski Z, Hamm HE, Alford S. (2014) Modulation of Neurotransmission by GPCRs Is Dependent upon the Microarchitecture of the Primed Vesicle Complex. Journal of Neuroscience. 34(1):260-274.

E Hamid, E Church, S Alford (2019) Quantitation and simulation of single action potential-evoked Ca2+ signals in CA1 pyramidal neuron presynaptic terminals Eneuro 6 (5). 

E Church, E Hamid, Z Zurawski, M Potcoava, E Flores-Barrera, A Caballero, KY Tseng, S Alford (2022) Synaptic Integration of Subquantal Neurotransmission by Colocalized G Protein-Coupled Receptors in Presynaptic Terminals. Journal of Neuroscience 42 (6), 980-1000