210) Considerations when Using GCaMP6 in Trigeminal Nociceptive Afferents

摘要: Without reliable tools to record activity in large populations of neurons, investigators have turned genetically-encoded Ca2+ indicators such as GCaMP, assuming that changes cytosolic Ca2+ ([Ca2+]c) can be used an indirect measure neural activity. Three versions GCaMP6 been tuned for sensitivity (GCaMP6s), speed (GCaMP6f), or a balance between the two (GCaMP6m) by manipulating Ca2+binding kinetics. We recently presented preliminary data suggesting heterogeneity among primary afferents firing frequency-dependent [Ca2+]c regulation does, fact, impact GCaMP6’s ability encode Here, we expanded upon this work assess relationship and [Ca2+]c as well utility GCaMP6s/m/f detect trigeminal sensory neuron subpopulations. To drive expression acutely dissociated rat ganglion AAV9-CAG. Neurons were then stimulated using different protocols frequencies. First, measured infection efficiency AAV9-CAG-GCAMP6(s/m/f). Then, comparing isoforms neuronal subpopulations, assessed (i) single spike, (ii) resolve spikes spike train at frequencies, (iii) dynamic range, (iv) optimal frequency. Using Fura-2, [Ca2+]c in GCaMP6-expressing neurons rest following stimulation. was detected within 6 days, results collected 6-10d post-infection. Infection is >80% with all three All 84-98% However, GCaMP6s's range greater than 6m's 6f's. confirmed our initial observation there coding frequency isoform- subpopulation-dependent, which often below 30 Hz. These suggest care should taken selection, use, interpretation experiments use proxy peripheral