Therefore, the presence of repetitive action potentials is a clear sign that the ion-channels responsible for generating action potentials are properly orchestrated. Repetitive action potentials occur spontaneously or can be evoked by injecting currents. However, the presence of spontaneous action potentials does not necessarily mean that the cells are receiving excitatory synaptic input, as both spontaneous membrane fluctuations as well as various pace-making channels (e.g. hyperpolarization-activated cyclic nucleotide-gated channels) can cause spontaneous firing. The development of passive and active membrane properties is facilitated by but not dependent on the presence of glial cells (Wu et al., 2007). By contrast, however, the formation of functional synapses requires factors secreted by glial cells (Banker, 1980; Eroglu and Barres, 2010). Morphological evidence of synapse formation can be obtained by high-resolution fluorescence microscopy showing a presynaptic vesicle protein such as synapsin, synaptophysin, or synaptotagmin localized in small puncta in close proximity to MAP2-positive dendrites. Electron microscopic analysis and demonstration of a postsynaptic density as well as synaptic vesicles in the presynaptic axonal compartment can provide even stronger evidence. Morphology alone does not prove the existence of functional synapses, but unambiguous evidence of synaptic transmission can be obtained using electrophysiological tools (Regehr and Stevens, 2001).
