Figure 4. Counting synapses in isolated (autaptic) neurons and interconnected neuronal pairs. A, B, The total number of synaptic connections formed by isolated (A1) or pairs (B1) of neurons was determined using superfusion of NH4Cl, herewith unquenching all SypHy-positive synaptic contacts (DIV8; A2, B2). MPTS (red) or Alexa Fluor-568 (blue) was infused during whole-cell recordings to visualize dendritic arborization. Recording electrodes were retracted before imaging, exposing the complete arborization. A3, B3, Manual tracking of the neuronal arborization resulted in dendritic masks of the isolated autaptic neurons and of neuronal pairs. Filled circles represent SypHy-positive puncta that were automatically identified and colocalized with the dendritic mask (Schmitz et al., 2011). A3, SypHy-positive puncta representing autaptic connections (orange dots) based on colocalization with a red dendritic mask of an isolated autaptic neuron. B3, SypHy-positive spots in a pair, representing either autapses or interneuronal synapses on the red neuron (orange dots) or the blue neuron (light blue dots). A small fraction of synapses were allocated to both neurons due to dendritic overlap (purple dots). A4, B4, High-frequency trains (40 Hz, 300 stimulations) were used to determine the number of active GABAergic or glutamatergic synapses. C, The total number of SypHy-positive spots that appear during superfusion with NH4Cl is comparable between glutamatergic and GABAergic isolated neurons. Pairs of neurons establish twice as many synaptic connections, suggesting the number of synaptic connections made per neuron is constant (One-way ANOVA (p < 0.05), followed by Tukey's test for subsequent pairwise comparisons). D, Evoked amplitude is comparable between autaptic and synaptic connections in glutamatergic neurons in isolation and minimal networks (DIV8–9), indicating no strong preference in formation of functional autaptic or synaptic glutamatergic connections (Kruskal–Wallis test, p > 0.05). E, Similarly, evoked amplitude is comparable between autaptic and synaptic connections in GABAergic neurons in isolation and minimal networks (Kruskal–Wallis test, p > 0.05). F, G, Correlation plot between the total number of active synapses (assessed using 40 Hz trains) and the total mEPSC/mIPSC release frequency in all groups (frequencies summed up from both neurons in the case of a pair). F, G, The total mIPSC frequency and GABAergic synapse number correlate in mixed pairs, but the total mEPSC frequency in mixed pairs was lower than expected based on number of glutamatergic synapses. H, I, Correlation plot between the summed eEPSC/eIPSC amplitudes and the total mEPSC/mIPSC release frequency in all groups (frequencies summed up from both neurons in the case of a pair). The mEPSC frequency was much lower than expected based on the summed eEPSC amplitude in mixed pairs, while the mIPSC frequency was more consistent with the eIPSC amplitude found in mixed pairs. Long-term treatment with bicuculline blocks the GABAergic inhibition of mEPSC frequency (Fig. 3B), herewith shifting the mEPSC frequency toward the predicted value (H, gray arrow marked ‘Bic’ and square).