Authors :
Simon Granak, MD – Massachusetts General Hospital
Lauren Lau, PhD – Massachusetts General Hospital
Volodymyr Dzhala, PhD – Massachusetts General Hospital / Harvard Medical School
Kyle Lillis, PhD – Massachusetts General Hospital
Brian Bacskai, PhD – Massachusetts General Hospital
Maria Sanchez Mico, PhD – Massachusetts General Hospital
Kevin Staley, MD – Massachusetts General Hospital
Presenting Author: Melanie McNally, MD – Massachusetts General Hospital
Rationale: Mitochondria play an important neuroprotective role buffering large increases in cytoplasmic calcium (Ca) during seizures, and mitochondrial Ca dysregulation can contribute to synaptic hyperactivity and excitotoxicity in epilepsy. However, tools to measure mitochondrial Ca handling in living tissue with cellular resolution have been limited. Here, for the first time, we employ novel two-photon (2P) imaging techniques to measure both neuronal cytoplasmic and mitochondrial Ca changes during hippocampal seizures in vitro.
Methods: Organotypic hippocampal slice cultures were prepared from P6-8 C57BL/6J mouse pups of both sexes and transduced with AAV2/9.Syn.NES-jRGECO1a and AAV2/8.hSyn.2mtYC3.6 permitting neuronal co-expression of a red cytoplasmic Ca indicator (jRGECO) and a mitochondrial-matrix-targeted ratiometric Ca sensitive fluorophore (Yellow Cameleon 3.6). Neuronal co-expression was confirmed with immunohistochemistry. Slices develop spontaneous, recurrent seizures within 1 week. Longitudinal 2P imaging of seizing CA1 pyramidal neurons was performed from DIV8 to DIV17 to assess dynamic Ca changes concurrently in neuronal cytosolic and mitochondrial compartments (915nm, 1.27 Hz). All image analyses were carried out using ImageJ, and data analyses were performed using MATLAB, Origine, and Prism.
Results: Neuronal ictal cytoplasmic Ca load increases with longer seizure duration (n = 7 slices, 46 neurons, 24 seizures; linear regression, R
2=0.41,
p< 0.00). At DIV8, there is a trend where ictal mitochondrial Ca buffering increases with longer seizure duration (Figure 1, n=3 slices, 20 neurons, 8 seizures; linear regression, R
2=0.38,
p=0.1). However, at DIV11-13, longer seizures are observed and this relationship is reversed with less ictal mitochondrial Ca buffering during longer seizures (Figure 1, n=2 slices, 15 neurons, 7 seizures; linear regression, R
2=0.58,
p=0.04)
Conclusions: For the first time, we were able to concurrently image mitochondrial and cytoplasmic Ca dynamics during seizures in individual neurons. We found that neuronal somatic mitochondrial Ca buffering of cytoplasmic Ca during seizures varies over time. This may represent normal developmental maturation of mitochondrial Ca handling or emerging mitochondrial dysfunction due to increasing seizure burden or slice age. Whether these changes are adaptive/neuroprotective versus pathological remain to be discovered. With these newly developed tools, we can now test these hypotheses with the necessary temporal and spatial resolution permitting new insights into the role of mitochondrial Ca handling during seizures and epileptogenesis.
Funding:
National Institute of Health grant K08 NS121599-01 (M.A.M.)