Visualization of the dendritic arbor of neurons in intact 500 microm thick brain slices

Characterizing the structure and electrophysiological properties of single neurons is essential for understanding how individual cells contribute to the function of neuronal networks. Following intra-cellular recording from neurons in acute brain slices, the structure of the recorded cell has typically been examined by serial sectioning of the tissue slice and then reconstructing the neuron of interest; a labor-intensive and time-consuming process. Here, we have adapted a whole-mount immunohistochemical technique and used it to visualize the dendritic arbor of individual neurons in sections of adult CNS tissue up to 500 microm thick. Permeabilization of the slice and extensive washing allow histochemical reagents to penetrate and be washed from the section, producing limited background staining. Using this method, the cell within the slice can be sectioned optically and reconstructed using the optical sections. We present images of the dendritic trees of neurons in 500 microm thick slices of adult rat entorhinal cortex and hippocampus, labeled either immunohistochemically, or by biocytin injection following whole-cell patch clamp or sharp electrode recordings. The resolution obtained is sufficient to visualize dendritic spines deep within the section. The method is free from artifacts associated with cutting serial sections and is broadly applicable to tasks that require visualization of the fine structure of individual cells in thick slices of CNS tissue.