In vivo wide-field voltage imaging in zebrafish with voltage-sensitive dye and genetically encoded voltage indicator.

The brain consists of neural circuits, which are assemblies of various neuron types. For understanding how the brain works, it is essential to identify the functions of each type of neuron and neuronal circuits. Recent advances in our understanding of brain function and its development have been achieved using light to detect neuronal activity. Optical measurement of membrane potentials through voltage imaging is a desirable approach, enabling fast, direct, and simultaneous detection of membrane potentials in a population of neurons. Its high speed and directness can help detect synaptic and action potentials and hyperpolarization, which encode critical information for brain function. Here, we describe in vivo voltage imaging procedures that we have recently established using zebrafish, a powerful animal model in developmental biology and neuroscience. By applying two types of voltage sensors, voltage-sensitive dyes (VSDs, Di-4-ANEPPS) and genetically encoded voltage indicators (GEVIs, ASAP1), spatiotemporal dynamics of voltage signals can be detected in the whole cerebellum and spinal cord in awake fish at single-cell and neuronal population levels. Combining this method with other approaches, such as optogenetics, behavioral analysis, and electrophysiology would facilitate a deeper understanding of the network dynamics of the brain circuitry and its development.

Effects of decreased vitamin D and accumulated uremic toxin on human CYP3A4 activity in patients with end-stage renal disease.

In patients with end-stage renal disease, not only renal clearance but also hepatic clearance is known to be impaired. For instance, the concentration of erythromycin, a substrate of cytochrome P450 3A4 (CYP3A4), has been reported to be elevated in patients with end-stage renal disease. The purpose of this study is to elucidate the reason for the decrease in hepatic clearance in patients with end-stage renal disease. Deproteinized pooled sera were used to assess the effects of low-molecular-weight uremic toxins on CYP3A4 activity in human liver microsomes and human LS180 cells. Four uremic toxins (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, hippuric acid, indole-3-acetic acid, and 3-indoxyl sulfate) present at high concentrations in uremic serum were also studied. Simultaneous treatment of uremic serum (less than 10%) or uremic toxins did not affect testosterone 6ß-hydroxylation in human liver microsomes. On the other hand, pretreatment of each serum activates CYP3A4 in LS180 cells, and the increased CYP3A4 activity in uremic serum-treated cells was smaller than normal serum-treated cells. In addition, CYP3A4 and CYP24A1 mRNA levels also increased in LS180 cells exposed to normal serum, and this effect was reduced in uremic serum-treated cells and in cells exposed to uremic serum added to normal serum. Furthermore, addition of 1,25-dihydroxyvitamin D to uremic serum partially restored the serum effect on CYP3A4 expression. The present study suggests that the decrease of 1,25-dihydroxyvitamin D and the accumulation of uremic toxins contributed to the decreased hepatic clearance of CYP3A4 substrates in patients with end-stage renal disease.