Fos-CreER-based genetic mapping of forebrain regions activated by acupuncture.

Acupuncture increasingly is accepted as a potential therapy for many diseases in the Western world. However, the mechanism of acupuncture is not well understood mechanistically. We have established that manual acupuncture (MA) at the Neiguan (P6) acupoint inhibits excitatory cardiovascular reflex responses through modulation of the autonomic nervous system in the brainstem. It is unclear whether P6 MA activates neurons in the brain regions beyond the brainstem. Thus, we mapped P6 specific neural activation by MA in the forebrain using the Fos-CreER; Ai9 mice model, which allows for enhanced sensitivity and efficiency compared to conventional immunohistochemical staining. Compared to sham-MA control without manual stimulation, we find that MA at P6 markedly increases c-Fos positive neurons in a number of the forebrain regions (n = 5 in each group). These activated regions include accumbens nucleus, caudate putamen, claustrum, bed nucleus of the stria terminalis, amygdaloid nucleus, ventral posterior division of the thalamic nucleus, paraventricular hypothalamic nucleus, arcuate hypothalamic nucleus, primary and secondary somatosensory cortex, ectorhinal cortex, and dorsolateral entorhinal cortex. As MA at P6 activates neurons in relatively broad brain networks beyond the brainstem, our data suggest that acupuncture at this acupoint has the potential to influence physiological functions associated with autonomic and non-autonomic nervous systems through its effects on multiple brain regions.

Laminar specificity of functional input to distinct types of inhibitory cortical neurons.

Despite the presence of numerous inhibitory cell types, laminar excitatory input has only been characterized for limited identified types, and it is unknown whether there are differences between cell types in their laminar sources of inhibitory input. In the present study, we characterized sources of local input to nine distinct types of layer 2/3 inhibitory neurons in living slices of mouse somatosensory cortex. Whole-cell recordings from identified cell types, facilitated by use of transgenic mice expressing green fluorescent protein in limited inhibitory neuron populations, were combined with laser scanning photostimulation. We found that each inhibitory cell type received distinct excitatory and inhibitory laminar input patterns. Excitatory inputs could be grouped into three categories. All inhibitory cell types received strong excitation from layer 2/3, and for calretinin (CR)-positive Martinotti cells and burst-spiking interneurons, this was their dominant source of excitatory input. Three other cell types, including fast-spiking basket cells, CR-negative Martinotti cells, and bipolar interneurons, also received strong excitatory input from layer 4. The remaining four inhibitory cell types, including chandelier cells, neurogliaform cells, irregular spiking basket cells, and regular spiking presumptive basket cells, received strong excitatory input from layer 5A and not layer 4. Laminar sources of inhibitory input varied between cell types and could not be predicted from the sources of excitatory input. Thus, there are cell-type specific differences in laminar sources of both excitation and inhibition, and complementary input patterns from layer 4 versus layer 5A suggest cell type differences in their relationships to lemniscal versus paralemniscal pathways.

Enzymatic amplification detection of DNA based on "molecular beacon" biosensors.

We described a novel electrochemical DNA biosensor based on molecular beacon (MB) probe and enzymatic amplification protocol. The MB modified with a thiol at its 5' end and a biotin at its 3' end was immobilized on the gold electrode through mixed self-assembly process. Hybridization events between MB and target DNA cause the conformational change of the MB, triggering the attached biotin group on the electrode surface. Following the specific interaction between the conformation-triggered biotin and streptavidin-horseradish peroxidase (HRP), subsequent quantification of DNA was realized by electrochemical detection of enzymatic product in the presence of substrate. The detection limit is obtained as low as 0.1nM. The presented DNA biosensor has good selectivity, being able to differentiate between a complementary target DNA sequence and one containing G-G single-base mismatches.

Electrochemical detection of point mutation based on surface ligation reaction and biometallization.

A highly sensitive electrochemical method for point mutation detection based on surface enzymatic ligation reaction and biometallization is demonstrated. In this method the surface-immobilized allele-specific probe, complementary to the mutant target, undergoes allele-specific ligation with the 5'-phosphorylated ligation probe in the presence of the mutant oligonucleotide target and E. coli DNA ligase. If there is an allele mismatch, no ligation takes place. After thermal treatment at 90 degrees C, the formed duplex melts apart, which merely allows the ligation product to remain on the electrode surface. Then, biotinylated detection probes hybridize with the ligation product. With the binding of streptavidin-alkaline phosphatase (SA-ALP) to the biotinylated probes, a non-reductive substrate of alkaline phosphatase, ascorbic acid 2-phosphate (AA-P), can be converted into ascorbic acid (AA) at the electrode surface. Silver ions in solution are then reduced by AA, resulting in the deposition of silver metal onto the electrode surface. Linear sweep voltammetry (LSV) is used to detect the amount of deposited silver. The proposed approach has been successfully implemented for the identification of single base mutation in codon 12 of K-ras oncogene target with a detection limit of 80fM, demonstrating that this method provides a highly specific, sensitive and cost-efficient approach for point mutation detection.