In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.

Neocortical lamina 6B (L6B) is a largely unexplored layer with a very heterogeneous cellular composition. To date, only little is known about L6B neurons on a systematic and quantitative basis. We investigated the morphological and electrophysiological properties of excitatory L6B neurons in the rat somatosensory barrel cortex using whole-cell patch-clamp recordings and simultaneous biocytin fillings. Subsequent histological processing and computer-assisted 3D reconstructions provided the basis for a classification of excitatory L6B neurons according to their structural and functional characteristics. Three distinct clusters of excitatory L6B neurons were identified: (C1) pyramidal neurons with an apical dendrite pointing towards the pial surface, (C2) neurons with a prominent, “apical”-like dendrite not oriented towards the pia, and (C3) multipolar spiny neurons without any preferential dendritic orientation. The second group could be further subdivided into three categories termed inverted, “tangentially” oriented and “horizontally” oriented neurons. Furthermore, based on the axonal domain two subcategories of L6B pyramidal cells were identified that had either a more barrel-column confined or an extended axonal field. The classification of excitatory L6B neurons provided here may serve as a basis for future studies on the structure, function, and synaptic connectivity of L6B neurons.

The fate of the subplate (SP) is still a matter of debate. The SP and layer 6 (which is ontogenetically the oldest and innermost neocortical lamina) develop coincidentally. Yet, the function of sublamina 6B is largely unknown. It has been suggested that it consists partly of neurons from the transient SP, however, experimental evidence for this hypothesis is still missing. To obtain first insights into the neuronal complement of layer 6B in the somatosensory rat barrel cortex, we used biocytin stainings of SP neurons (aged 0-4 postnatal days, PND) and layer 6B neurons (PND 11-35) obtained during in vitro whole-cell patch-clamp recordings. Neurons were reconstructed for a quantitative characterization of their axonal and dendritic morphology. An unsupervised cluster analysis revealed that the SP and layer 6B consist of heterogeneous but comparable neuronal cell populations. Both contain 5 distinct spine-bearing cell types whose relative fractions change with increasing age. Pyramidal cells were more prominent in layer 6B, whereas non-pyramidal neurons were less frequent. Because of the high morphological similarity of SP and layer 6B neurons, we suggest that layer 6B consists of persistent non-pyramidal neurons from the SP and cortical L6B pyramidal neurons.

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