Aims: 

Understanding the dynamic and integrative properties of a neuron requires realistic computer simulations that take into account neuronal morphology and the functional properties of the expressed ion channels. The first step to develop biophysically realistic models involves determining the morphology and passive membrane properties. The standard procedure involves electrophysiological recording and injection of a diffusible tracer. When neurons are electrically coupled via gap junctions, corresponding morphological and functional data can no longer be obtained. Here, we present an alternative workflow where we fill cells with non-gap junction permeable fluorescent dyes during electrophysiological recording and block electrical coupling pharmacologically.

Methods: 

MPE microscopy was used t o obtain fluorescent image stacks of electrophysiologically recorded AII amacrine cells in rat retinal slices, followed by morphological reconstruction and computational modeling.

Results: 

AII amacrines were filled with Alexa Fluor 488 during current-clamp recording with sampling of passive voltage transients. To enable accurate measurement of fast charge distribution, we employed dual somatic recording with separate electrodes for current injection and voltage recording. Meclofenamic acid was added to block gap-junction coupling and electrically isolate the recorded cells. Passive cable properties were estimated by directly fitting the voltage responses of the models evoked by current pulses to the physiologically recorded responses.

Conclusion: 

We have established a method to generate models that can serve as electrical skeletons onto which voltage-gated conductances and synaptic inputs can be added, to build up complete, biophysically realistic computational models.