Dovgan A. Molecular mechanisms of calcium-dependent hippocalcin translocation in dendritic tree of rat hippocampal neurons

Hippocalcin is a Ca2+-binding protein that belongs to a family of neuronal Ca2+sensors and is a key mediator of many cellularfunctions including synaptic plasticity and learning. However, the molecular mechanisms involved in hippocalcin signalling remainillusive. Here we studied the role of glutamatereceptors and voltage-operated calcium channels activation in hippocalcin translocation during electric activity in hippocampal neurons. Also we studied biophysical properties of such signaling - i.e. how different calcium transients "transform" into certain hippocalcin translocations. At all, we tried to understand how hippocalcin discriminate calcium signals, associated with it physiological functions - such as LTD and sAHP. Intrinsic network bursting(current clamp mode in investigated cell) produced hippocalcin-YFP translocation to a set ofdendritic sites and spines. Translocation in spines was restricted to spine heads and even closely (within 1-2 lm) located spines on thesame dendritic branch signalled independently. Translocation in spines was observed during intrinsic network bursting(voltage clamp mode in investigated cell) at Vh= -70 mV and 0 mM Mg in external solution- so no any Mg2+ block of NMDAr. No tranlocation to spines was observed in presence DL-APV(NMDAr-blocker) at 2 mM Mg(current clamp mode in investigated cell)- but translocation in dendrite didn't vanish .So, thetranslocation to spines during intrinsic network bursting required Ca2+influx via synaptic NMDA receptors. Local brief iontophoretic glutamate application leads to hippocalcin translocation in dendrite. Cyclopiazonic acid(specific inhibitor of Ca2+-ATPase in the intracellular Ca2+ storage sites) application didn't change hippocalcin translocation on glutamate application in dendrite , but completely blocked hippocalcin translocation in soma. So, Ca influx from intracellular Ca2+ storage sites plays curtail role in hippocalcin translocation in soma on glutamate receptors activation, but don'influence on hippocalcin translocations in dendrite. External stimulation of questioned neuron's innervated axons didn't lead to hippocalcin translocation at Vh= -70 mV (voltage clamp mode in investigated cell), but at Vh=-40mV we observed hippocalcin translocation on such stimulation. Therefore activation of synaptic NMDAr leads to HPCA tanslocation to spines, and activation only synaptic AMPAr and mGluR doesn't lead to any hippocalcin translocation. Activation of VOCCs by long depolarisation(0.5-2 s) in voltage clamp and 20-100 bAPs induced by current steps leads to hippocalcin translocation in dendrit- but not spines. We observed hippocalcin translocation in the same sites during strong spontaneous synaptic activity in current clamp mode. Conseqently, hippocalcin translocation in dendrit- but not spines - associated mostly with VOCCs activation due to depolarization and bAPs induced by synaptic glutamate receptors activation during intrinsic network bursting. To investigate how different calcium transients "transform" to hippocalcin translocation in dendrite, we registrated Ca concentration during current step's induced bAPs with different number of bAPs - and made the same registrations for hippocalcin translocation in dendrit. Surprisingly, such registrations showed that dependence of hippocalcin translocation's amplitude from number of bAPs differs a lot from the same dependence for calcium transient's amplitude - for example at a level of 50 bAPs calcium transient's amplitude tend to saturation - but translocation's amplitudes are still linear. Hippocalcin presents in hippocampal neurons at high concentration - so hippocalcin plays role not only a calcium sensor ptotein, but can be attributed to be a main calcium buffer in hippocampal neurons. So, in this case hippocalcin activation is determined by whole amount of Ca injected in cell during stimulation - but not by free calcium concentration. To investigate this hypothesis , we evaluated whole Ca influx from our Ca transients registration - as an integral from whole calcium registration. Dependence of whole Ca influx from number of bAPs was linear till at list 100 bAPs - and be in good accordance with the same dependence for hippocalcin translocation. So, hippocalcin activation depends on integrated calcium signal over long period of time (probably about characteristic time for hippocalcin translocation recovery) rather than free calcium concentration. Another question is if translocation in spines was related with different calcium signals in spines and dendrit . To answer the question, we registrated calcium concentration during intrinsic network bursting in investigated neuron in spines and dendrite - and at the same time - hippocalcin translocation. We observed higher calcium concentration in spines than in dendrite during hippocalcin translocation in this spines. Thus, we conclude that hippocalcin may differentially decode various spatiotemporalpatterns of glutamate receptor activation into site- and time-specific translocation to its targets. Hippocalcin also possesses an abilityto produce local signalling at the single synaptic level providing a molecular mechanism for homosynaptic plasticity.