Intracellular injection in fixed slices: obtaining complete dendritic arbors of large cells

Intracellular injection of Lucifer yellow into fixed brain slices is widely used to demonstrate dendritic morphology. A major limitation of this technique is that large dendritic arbors are usually truncated at the cut surfaces. Here we describe modifications that allowed us to obtain complete dendritic arbors of large spiny stellate cells. Lucifer Yellow cadaverine biotin-X (LY-X) was injected into individual neurons within 300-1000 microm thick aldehyde-fixed slices of kitten visual cortex. Subsequently, the LY-X was histochemically reacted using standard ABC methods to obtain a permanent record of the injected cells. Dendrites, studded with a variety of dendritic spines, were darkly labeled and well defined against virtually no background. Somatic spines, dendritic varicosities and growth cones were common in the younger animals. Computer-assisted reconstructions demonstrated that, in older animals, the dendritic arbors of cells injected in 300 microm slices were truncated, whereas the arbors of cells injected deep within thick slices were complete. The modifications described here remove the most critical limitation of intracellular injection in slices, allowing quantitative analysis of even large dendritic arbors.