A central question in neuronal network analysis is how the interaction between individual neurons produces behavior and behavioral modifications. This task depends critically on how exactly are signals integrated by individual nerve cells functioning as complex operational units. Regional electrical properties of branching neuronal processes which determine the input-output function of any neuron are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such measurements one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin (synaptic contacts on dendritic spines) and summate at particular locations to influence action potential (AP) initiation. It became possible recently to carry out this type of measurements with sub-millisecond and sub-micrometer resolution using multisite recording of membrane potential changes with intracellular voltage-sensitive dyes (Vm imaging). Our method is based on pioneering work on voltage-sensitive molecular probes that report membrane potential changes in transmission and fluorescence light intensity measurements (Cohen and Salzberg, 1978). Many aspects of the initial technology have been continuously improved over several decades (Canepari et al., 2010). The spatial pattern of Na(+) channel clustering in the axon initial segment (AIS) plays a critical role in tuning neuronal computations, and changes in Na(+) channel distribution have been shown to mediate novel forms of neuronal plasticity in the axon. We took advantage of a critical methodological improvement in the high sensitivity membrane potential imaging technique to directly determine the location and length of the spike trigger zone (TZ) as defined in functional terms. The results show that in mature axons of mouse (postnatal day 18-30) cortical layer 5 pyramidal cells in acute brain slices, action potentials initiate in a region ∼20 μm in length centered between 20 and 40 μm from the soma. From this region, the AP depolarizing wave invades initial nodes of Ranvier within a fraction of a millisecond and propagates in a saltatory fashion into axonal collaterals without failure at all physiologically relevant frequencies. We further demonstrate that, in contrast to the saltatory conduction in mature axons, AP propagation is non-saltatory (monotonic) in immature axons prior to myelination in mouse postnatal day 5-9. The evidence for an important hypothesis that cortical spine morphology might participate in modifying synaptic efficacy that underlies plasticity and possibly learning and memory mechanisms is inconclusive. Both theory and experiments suggest that the transfer of excitatory postsynaptic potential (EPSP) signals from spines to parent dendrites depends on the spine neck morphology and resistance. Furthermore, modeling of signal transfer in the opposite direction predicts that synapses on spine heads are not electrically isolated from voltages in the parent dendrite. To provide direct evidence for the specific question of the transfer of dendritic signals to spine synapses, we further advanced the V(m) imaging technique (Popovic et al. 2012; fig 1) and carried out optical measurements of electrical signals from 4 groups of spines in mouse cortical layer 5 pyramidal cells with different neck length and simultaneously from parent dendrites. The results show that spine neck does not filter membrane potential signals as they spread from the dendrites into the spine heads. The improved Vm imaging technique enabled characterization the transfer of unitary EPSPs from spine synapses across the spine neck to the parent dendrite. A series of measurements showed that the spine/dendrite EPSP amplitude ratio varied from 1 to 4 in different spines.