(210) Considerations when Using GCaMP6 in Trigeminal Nociceptive Afferents

Abstract

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