In-Depth Characterization of Layer 5 Output Neurons of the Primary Somatosensory Cortex Innervating the Mouse Dorsal Spinal Cord.

Neuronal circuits of the spinal dorsal horn integrate sensory information from the periphery with inhibitory and facilitating input from higher central nervous system areas. Most previous work focused on projections descending from the hindbrain. Less is known about inputs descending from the cerebral cortex. Here, we identified cholecystokinin (CCK) positive layer 5 pyramidal neurons of the primary somatosensory cortex (CCK + S1-corticospinal tract [CST] neurons) as a major source of input to the spinal dorsal horn. We combined intersectional genetics and virus-mediated gene transfer to characterize CCK+ S1-CST neurons and to define their presynaptic input and postsynaptic target neurons. We found that S1-CST neurons constitute a heterogeneous population that can be subdivided into distinct molecular subgroups. Rabies-based retrograde tracing revealed monosynaptic input from layer 2/3 pyramidal neurons, from parvalbumin positive cortical interneurons, and from thalamic relay neurons in the ventral posterolateral nucleus. Wheat germ agglutinin-based anterograde tracing identified postsynaptic target neurons in dorsal horn laminae III and IV. About 60% of these neurons were inhibitory and about 60% of all spinal target neurons expressed the transcription factor c-Maf. The heterogeneous nature of both S1-CST neurons and their spinal targets suggest complex roles in the fine-tuning of sensory processing.

New variants of inducible Cre recombinase: a novel mutant of Cre-PR fusion protein exhibits enhanced sensitivity and an expanded range of inducibility.

We have developed a novel inducible Cre mutant with enhanced recombinase activity to mediate genetic switching events. The protein, designated Cre*PR, is composed of a new Cre mutant at the N-terminus followed by the ligand-binding domain (LBD) of the progesterone receptor (PR). The response to low doses of inducer is significantly enhanced by elongating the C-terminus of the PR LBD from amino acid 891 to 914. The mutant Cre lacks the first 18 amino acids and contains a Val-->Ala substitution at position 336, thereby destroying a cryptic splice donor at the 3'-end of CRE: The latter mutation reduces unwanted background recombinase activity in the absence of the synthetic ligand RU486 by a factor of at least 10 to an almost undetectable level. Thus, the recombinase activity turns out to be inducible by a factor of >200. We expect Cre*PR to serve as a valuable tool for conditional expression of genes both in vitro and in vivo.

Isotope dilution - high efficiency nebulization-ICP-MS: The coupling of accuracy and sensitivity.

If sample pretreatment, nebulization and method of calibration are suitably adapted to each other the performance of inductively coupled plasma - mass spectrometry ICP-MS can be greatly increased. High accuracy is obtained by high precision and low bias. For a given concentration higher sensitivity means higher count rates and therefore higher precision. Systematic errors are minimized by employing a definitive method of calibration. Increased sensitivity is obtained by introducing higher amounts of sample into the measurement system via high efficiency nebulizers (ultrasonic nebulizer, hydraulic-high pressure nebulizer according to Berndt and concentric high efficiency nebulizer according to Meinhard). Because this means also higher matrix effects a combination of ion chromatographic (IC-TMS) and thermal trace-matrix-separation by aerosol desolvation (T-TMS) is introduced. Isotope dilution (ID) proves to be the calibration most suitable to achieve the highest accuracy. First applications on the analysis of refractory metals (e.g. Ti, V, Nb, Ta) and non-metals (e.g. P, S, As, Se) showed recoveries of 60-105%, an imprecision of the recoveries of 2-50%, but an overall inaccuracy of only 0.1 to 4%.