Commentary: “Comparison of spike parameters from optically identified GABAergic and glutamatergic neurons in sparse cortical cultures”

Citation: Becchetti A and Wanke E (2015) Commentary: " Comparison of spike parameters from optically identified GABAergic and glutamatergic neurons in sparse cortical cultures. " Because of the increasing use of multi-site extracellular recording from neuronal networks, it would be extremely useful to determine reliable ways to distinguish with this method the main neuronal populations in vivo as well as in vitro. In a recent paper, Weir et al. (2015) studied the problem of correctly distinguishing the activity of GABAergic and glutamatergic neurons by multi-electrode array (MEA) recording, in primary neuronal cultures. Because we recently addressed the same problem, obtaining different results (Becchetti et al., 2012), we wish to discuss the main experimental differences between these papers. Discussing the possible reasons underlying the observed discrepancies should be instructive for the researchers attempting to establish culture conditions that approximate the in vivo situation. In both studies, the GAD67-GFP knock-in mouse (Tamamaki et al., 2003) was used to unequivocally identify GABAergic cells. Waveform analysis led to the conclusion that spike width-based criteria are insufficient to reliably assign the recorded units to GABAergic or glutamatergic cells. However, Weir et al. (2015) did not observe significant electrophysiological differences between these neuronal populations, whereas Becchetti et al. (2012) found that some statistics systematically varied between inhibitory (i.e., GAD67+) and excitatory neurons, particularly the Fano factor (FF; the ratio between spike-count variance and mean). Presumably, the discrepancy arises because of the very different experimental conditions applied in the two studies. In order to precisely attribute the spike waveforms to identified GFP+ or GFP− neurons, Weir et al. (2015) chose to plate the cells at the lowest density compatible with viability of neocortical cultures, which caused a low rate of network growth. Moreover, for MEA recording, cells were transferred to artificial cerebrospinal fluid, thus removing the cell-conditioned medium that is rich in regulating factors released by neurons as well as glial cells. According to our experience , these alterations cause not only a low-activity starting point, but also a long-lasting perturbation of the steady-state network activity. In fact, Weir et al. (2015) report an average GABAergic cell density of 115 neurons/mm 2 to be compared with 434 in our conditions and the values reported for brain slices obtained from GAD67-GFP mice. For instance, cell counts approximately twice as