Application of low-frequency alternating current electric fields via interdigitated electrodes: effects on cellular viability, cytoplasmic calcium, and osteogenic differentiation of human adipose-derived stem cells.

Electric stimulation is known to initiate signaling pathways and provides a technique to enhance osteogenic differentiation of stem and/or progenitor cells. There are a variety of in vitro stimulation devices to apply electric fields to such cells. Herein, we describe and highlight the use of interdigitated electrodes to characterize signaling pathways and the effect of electric fields on the proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). The advantage of the interdigitated electrode configuration is that cells can be easily imaged during short-term (acute) stimulation, and this identical configuration can be utilized for long-term (chronic) studies. Acute exposure of hASCs to alternating current (AC) sinusoidal electric fields of 1 Hz induced a dose-dependent increase in cytoplasmic calcium in response to electric field magnitude, as observed by fluorescence microscopy. hASCs that were chronically exposed to AC electric field treatment of 1 V/cm (4 h/day for 14 days, cultured in the osteogenic differentiation medium containing dexamethasone, ascorbic acid, and ß-glycerol phosphate) displayed a significant increase in mineral deposition relative to unstimulated controls. This is the first study to evaluate the effects of sinusoidal AC electric fields on hASCs and to demonstrate that acute and chronic electric field exposure can significantly increase intracellular calcium signaling and the deposition of accreted calcium under osteogenic stimulation, respectively.

A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function.

Understanding the cell-cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate neural cell types. Here we describe methods for the prospective isolation and purification of astrocytes, neurons, and oligodendrocytes from developing and mature mouse forebrain. We used FACS (fluorescent-activated cell sorting) to isolate astrocytes from transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of an S100beta promoter. Using Affymetrix GeneChip Arrays, we then created a transcriptome database of the expression levels of >20,000 genes by gene profiling these three main CNS neural cell types at various postnatal ages between postnatal day 1 (P1) and P30. This database provides a detailed global characterization and comparison of the genes expressed by acutely isolated astrocytes, neurons, and oligodendrocytes. We found that Aldh1L1 is a highly specific antigenic marker for astrocytes with a substantially broader pattern of astrocyte expression than the traditional astrocyte marker GFAP. Astrocytes were enriched in specific metabolic and lipid synthetic pathways, as well as the draper/Megf10 and Mertk/integrin alpha(v)beta5 phagocytic pathways suggesting that astrocytes are professional phagocytes. Our findings call into question the concept of a "glial" cell class as the gene profiles of astrocytes and oligodendrocytes are as dissimilar to each other as they are to neurons. This transcriptome database of acutely isolated purified astrocytes, neurons, and oligodendrocytes provides a resource to the neuroscience community by providing improved cell-type-specific markers and for better understanding of neural development, function, and disease.

Effect of N-acetylaspartylglutamate (NAAG) on non-quantal and spontaneous quantal release of acetylcholine at the neuromuscular synapse of rat.

N-Acetylaspartylglutamate (NAAG), known to be present in rat motor neurons, may participate in neuronal modulation of non-quantal secretion of acetylcholine (ACh) from motor nerve terminals. Non-quantal release of ACh was estimated by the amplitude of the endplate membrane hyperpolarization (H-effect) caused by inhibition of nicotinic receptors by (+)-tubocurarine and acetylcholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Application of exogenous NAAG decreased the H-effect in a dose-dependent manner. The reduction of the H-effect by NAAG was completely removed when N-acetyl-beta-aspartylglutamate (betaNAAG) or 2-(phosphonomethyl)-pentanedioic acid (2-PMPA) was used to inhibit glutamate carboxypeptidase II (GCP II), a presynaptic Schwann cell membrane-associated ectoenzyme that hydrolyzes NAAG to glutamate and N-acetylaspartate. Bath application of glutamate decreased the H-effect similarly to the action of NAAG but N-acetylaspartate was without effect. Inhibition of NMDA receptors by dl-2-amino-5-phosphopentanoic acid, (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), and 7-chlorokynurenic acid or inhibition of muscle nitric oxide synthase (NO synthase) by N(G)-nitro-l-arginine methyl ester and 3-bromo-7-nitroindazole completely prevented the decrease of the H-effect by NAAG. These results suggest that glutamate, produced by enzymatic hydrolysis of bath-applied NAAG, can modulate non-quantal secretion of ACh from the presynaptic terminal of the neuromuscular synapse via activation of postsynaptic NMDA receptors and synthesis of nitric oxide (NO) in muscle fibers. NAAG also increased the frequency of miniature endplate potentials (mEPPs) generated by spontaneous quantal secretion of ACh, whereas the mean amplitude and time constants for rise time and for decay of mEPPs did not change.

Reinnervation of the rat levator ani muscle after neonatal denervation.

After axonal injury on postnatal day 14 (P14), but not P21, motoneurons in the spinal nucleus of the bulbocavernosus (SNB) do not display their normal response to circulating testosterone levels. This could result from a permanent disruption of communication between motoneurons and their testosterone-sensitive target muscles. We assessed the extent of reinnervation of one of these target muscles, the levator ani (LA) muscle, 5 months after the pudendal nerve was cut either on P14 or P21. The number of motoneurons innervating the LA in control and nerve cut animals was determined using retrograde labeling procedures. Functional recovery of the LA muscle was determined via the testing of its in situ contractile properties. Compared to control muscles, reinnervated LA muscles were smaller, had fewer muscle fibers, generated a lower maximum tetanic tension, and were more fatigable. In spite of the fact that fewer motoneurons reinnervated the LA muscle after nerve cut on P14 than on P21, there were no differences in the weight or contractile properties of the LA muscle between these two groups. These data suggest that motoneurons that survived injury on P14 innervated more muscle fibers than normal and exhibited a similar ability to functionally reinnervate the target muscle as those motoneurons that survived injury on P21.

Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.

To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated that express proteins of the green fluorescent protein family under control of transcriptional regulatory sequences of the human S100B gene. Terminal Schwann cells were imaged repetitively in living animals of one of the transgenic lines to show that, except for extension and retraction of short processes, the glial coverings of the adult neuromuscular synapse are stable. In other lines, subsets of Schwann cells were labeled. The distribution of label suggests that Schwann cells at individual synapses are clonally related, a finding with implications for how these cells might be sorted during postnatal development. Other labeling patterns, some present in unique lines, included astrocytes, microglia, and subsets of cerebellar Bergmann glia, spinal motor neurons, macrophages, and dendritic cells. We show that lines with labeled macrophages can be used to follow the accumulation of these cells at sites of injury.