Mouse primary somatosensory barrel cortex (wS1) processes whisker sensory information, receiving input from two distinct thalamic nuclei. The first-order ventral posterior medial (VPM) somatosensory thalamic nucleus most densely innervates layer 4 (L4) barrels, whereas the higher-order posterior thalamic nucleus (medial part, POm) most densely innervates L1 and L5A. We optogenetically stimulated VPM or POm axons, and recorded evoked excitatory postsynaptic potentials (EPSPs) in different cell-types across cortical layers in wS1. We found that excitatory neurons and parvalbumin-expressing inhibitory neurons received the largest EPSPs, dominated by VPM input to L4 and POm input to L5A. In contrast, somatostatin-expressing inhibitory neurons received very little input from either pathway in any layer. Vasoactive intestinal peptide-expressing inhibitory neurons received an intermediate level of excitatory input with less apparent layer-specificity. Our data help understand how wS1 neocortical microcircuits might process and integrate sensory and higher-order inputs.
The prospect for spatial imaging with mass spectroscopy at the level of the cell requires new means of cell extraction to conserve molecular structure. To this aim, we demonstrate a new laser extraction process capable of extracting intact biological entities with conserved biological function. The method is based on the recently developed picosecond infrared laser (PIRL), designed specifically to provide matrix-free extraction by selectively exciting the water vibrational modes under the condition of ultrafast desorption by impulsive vibrational excitation (DIVE). The basic concept is to extract the constituent protein structures on the fastest impulsive limit for ablation to avoid excessive thermal heating of the proteins and to use strongly resonant 1-photon conditions to avoid multiphoton ionization and degradation of the sample integrity. With various microscope imaging and biochemical analysis methods, nanoscale single protein molecules, viruses, and cells in the ablation plume are found to be morphologically and functionally identical with their corresponding controls. This method provides a new means to resolve chemical activity within cells and is amenable to subcellular imaging with near-field approaches. The most important finding is the conserved nature of the extracted biological material within the laser ablation plume, which is fully consistent with in vivo structures and characteristics.
The neocortex is composed of layers. Whether layers constitute an essential framework for the formation of functional circuits is not well understood. We investigated the brain-wide input connectivity of vasoactive intestinal polypeptide (VIP) expressing neurons in the reeler mouse. This mutant is characterized by a migration deficit of cortical neurons so that no layers are formed. Still, neurons retain their properties and reeler mice show little cognitive impairment. We focused on VIP neurons because they are known to receive strong long-range inputs and have a typical laminar bias toward upper layers. In reeler, these neurons are more dispersed across the cortex. We mapped the brain-wide inputs of VIP neurons in barrel cortex of wild-type and reeler mice with rabies virus tracing. Innervation by subcortical inputs was not altered in reeler, in contrast to the cortical circuitry. Numbers of long-range ipsilateral cortical inputs were reduced in reeler, while contralateral inputs were strongly increased. Reeler mice had more callosal projection neurons. Hence, the corpus callosum was larger in reeler as shown by structural imaging. We argue that, in the absence of cortical layers, circuits with subcortical structures are maintained but cortical neurons establish a different network that largely preserves cognitive functions.
1The neocortex is composed of layers. Whether layers constitute an essential 2 framework for the formation of functional circuits is not well understood. We 3 investigated if neurons require the layer organization to be embedded into brain-wide 4 circuits using the reeler mouse. This mutant is characterized by a migration deficit of 5 cortical neurons so that no layers are formed. Still, neurons retain their properties and 6 reeler mice show little cognitive impairment. We focused on VIP neurons because 7 they are known to receive strong long-range inputs and have a typical laminar bias 8 towards upper layers. In reeler these neurons are more distributed across the cortex. 9 We mapped the brain-wide inputs of VIP neurons in barrel cortex of wildtype and 10 reeler mice with rabies virus tracing. Innervation by subcortical inputs was not altered 11 in reeler, in contrast to the cortical circuitry. Numbers of long-range ipsilateral cortical 12 inputs were reduced in reeler, while contralateral inputs were strongly increased. 13Reeler mice had more callosal projection neurons. Hence, the corpus callosum was 14 larger in reeler as shown by structural imaging. We argue that in the absence of 15 cortical layers, circuits with subcortical structures are maintained but cortical neurons 16 establish a different network capable to preserve cognitive functions. 17 18 KEYWORDS 19 barrel cortex, corpus callosum, rabies tracing, reelin, VIP neurons 20 29input was increased compared to WT. Reeler mice had more callosal projecting 30 neurons (CPNs) and hence a larger corpus callosum. We argue that the absence of 31 layers has little effect on innervation by subcortical structures but induces a different 32 circuit arrangement within the cortex. 33 5 MATERIAL AND METHODS 1 2 Experimental animals 3 We crossed the reeler line (B6C3Fe a/a-Relnrl/J, The Jackson Laboratory, Bar 4 Harbor, USA) with the VIP-Cre line (VIPtm1(cre)Zjh, The Jackson Laboratory) to 5 breed VIP-Cre/reeler mice heterozygous for reelin mutation and homozygous for Cre. 6These animals were crossed to generate VIP-Cre/reeler mice homozygous for reelin 7 knockout. WT littermates or animals from the VIP-Cre line were used for comparison 8 in tracing experiments. For control experiments to check the quality of our Cre-9 dependent constructs, we used C57BL/6J wildtype mice (The Jackson Laboratory). 10For tracing experiments not requiring Cre expression and for imaging we used WT 11 and homozygous littermate pairs of the reeler line.
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