Reconstruction of motoneuronal morphology using two-photon molecular excitation microscopy

Neuronal somato-dendritic morphology is an important determinant of the conduction properties of neurons, and thus the way neurons process information. Current semi-automated methods of neuronal tracing by wide-field light microscopy of diaminobenzidine (DAB) labelled neurons suffer a number of limitations. Firstly, the required fixation introduces a variable spatial distortion. Secondly, under optimal conditions the spatial resolution is at best diffraction limited and these conditions are rarely achieved. Current estimates of dendritic diameter indicate a lower limit of ~0.5 μm [1]. This is close to the limit of resolution, and the diameter of smaller dendrites may therefore have been overestimated. Thirdly, the requirement for extensive, time-consuming user-input limits the practicality of the procedure.

In this study, two-photon molecular excitation microscopy (2PM) was used to reconstruct the morphology of living, cytoplasmically-labelled motoneurons (MNs) from the hypoglossal (XII) nucleus of neonatal mice. Using the improved imaging capabilities of 2PM in light-scattering tissues and combining it with advanced image analysis we overcome the limitations of conventional light microscopy described above. MNs in isolated transverse brainstem slices containing the XII nucleus [2] were visualised using infra-red differential contrast microscopy. Selected MNs were whole-cell voltage-clamped and fluorescent dyes iontophoresed via the patch-pipette. Slices were then transferred to an inverted confocal microscope modified for 2PM, and 3D stacks of images were acquired at diffraction-limited resolution. Custom-written analysis routines were used to assess dendrite diameters at selected points. Fluorescence intensity was measured along a line orthogonal to the dendrite at these points and diameter defined as full width at half maximum intensity. While the resolution of this method is also limited by the wavelength of light, we overcame this limitation by using the fact that in 2PM the signal from structures smaller than the resolution limit (0.4 μm in x-y, 0.8 μm in z) is proportional to the illuminated volume. It is therefore possible to relate the recorded fluorescence intensity to the local diameter of sub resolution dendrites if they are modelled as cylinders [3]. By combining this approach with fully automatic tracing we can quantify the morphology and local diameters of intact functioning MNs and unequivocally clarify the existence of small diameter (<0.5 μm) den-drites.

To date, reconstructions have been completed for the proximal regions of the somato-dendritic tree (<250 μm from the soma). These are comparable in branching pattern, complexity and local diameters (0.8–4 μm) to reconstructions of XII MNs made using wide-field microscopy [1]. Significant progress has also being made towards completion of a fast, robust, fully automatic system for tracing and quantifying the morphology of whole neurons from 2PM volume data.

The functional significance of somato-dendritic complexity is a rapidly expanding research field. Accurate reconstructions of specific somato-dendritic morphologies, and subsequent identification of the generalised principles describing dendritic morphology, is essential to developing models of neuronal behaviour and to elucidating the roles neurons play in information processing.