Isolation, culture optimization and functional characterization of stem cell neurospheres from mouse neonatal olfactory bulb and epithelium.

The olfactory epithelium contains basal cells with stem cell characteristics, which have the capacity to differentiate throughout life into olfactory receptor neurons (ORNs). Here we investigate the in vitro characteristics of stem cells taken from the olfactory bulb (OB) and the olfactory epithelium (OE) of neonatal TIS21 knock-in mice. The major aim of the study was the generation of olfactory neurospheres (ONS) derived from OB and OE of neonatal mice as a tool to further analyze the elementary processes of ORN development. Our data showed that the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) leads to a significant increase in number of ONS derived from OB but not from OE. The differentiation of ONSs led to the formation of different neuronal cell types, in particular to bipolar-shaped cells as well as putative pyramidal-neurons, astrocytes and oligodendrocytes. Immunohistochemical staining confirmed the presence of astrocytes and neurons in both types of ONSs. In order to investigate the functionality of the neurons we performed calcium imaging and patch-clamp experiments. Calcium imaging experiments revealed that the application of high potassium concentration provokes calcium transients. No excitable properties, neither sodium currents nor action potentials, were observed for the bipolar-shaped cells derived from OB and OE neurospheres, which means that these types of cells morphologically defined as putative neuronal cells, were not physiologically active. Interestingly, patch-clamp recordings performed in the pyramidal-shaped cells of OB neurospheres showed sodium and potassium currents as well as action potentials. Our study will help to establish further models in the field of olfactology.

2-Aminoethoxydiphenyl borate activates the mechanically gated human KCNK channels KCNK 2 (TREK-1), KCNK 4 (TRAAK), and KCNK 10 (TREK-2).

Two-pore domain K(+) (KCNK, K2P) channels underlie the "leak" (background) potassium conductance in many types of excitable cells. They oppose membrane depolarization and cell excitability. These channels have been reported to be modulated by several physical and chemical stimuli. The compound 2-aminoethoxydiphenyl borate (2-APB) was originally described as an inhibitor of IP3-induced Ca(2+) release but has been shown to act as either a blocker or an activator for several ion channels. Here, we report the effects of this compound on members of the TREK (TWIK related K(+) channels) subfamily of human KCNK channels. We injected Xenopus laevis oocytes with cRNAs (complementary RNAs) encoding several KCNK channels and measured their response using the two-electrode voltage clamp technique. 2-APB was found to be an effective activator for all members of the TREK subfamily (hKCNK2, hKCNK4, and hKCNK10), with the highest efficacy in hKCNK10. We also found that 2-APB was able to activate these channels in cell-excised patches of HEK293 (human embryonic kidney 293) cell transfected with hKCNK4 or hKCNK10, demonstrating direct activation. TREK channels are widely expressed in the central nervous system and peripheral tissues, where they play roles in several key processes. However, little is known regarding their pharmacology; therefore, the identification of a common, stable and inexpensive agonist should aid further investigations of these channels. Additionally, 2-APB has been used to study native receptors in cell systems that endogenously express members of the TREK subfamily (e.g., rat dorsal root ganglia); our results thus warn against the use of 2-APB at high concentrations in these systems.

Primary hippocampal neurons, which lack four crucial extracellular matrix molecules, display abnormalities of synaptic structure and function and severe deficits in perineuronal net formation.

The extracellular matrix (ECM) of the brain plays crucial roles during the development, maturation, and regeneration of the CNS. In a subpopulation of neurons, the ECM condenses to superstructures called perineuronal nets (PNNs) that surround synapses. Camillo Golgi described PNNs a century ago, yet their biological functions remain elusive. Here, we studied a mouse mutant that lacks four ECM components highly enriched in the developing brain: the glycoproteins tenascin-C and tenascin-R and the chondroitin sulfate proteoglycans brevican and neurocan. Primary embryonic hippocampal neurons and astrocytes were cultivated using a cell insert system that allows for co-culture of distinct cell populations in the absence of direct membrane contacts. The wild-type and knock-out cells were combined in the four possible permutations. Using this approach, neurons cultivated in the presence of mutant astrocytes displayed a transient increase of synapses after 2 weeks. However, after a period of 3 weeks or longer, synapse formation and stabilization were compromised when either neuron or astrocyte cell populations or both were of mutant origin. The development of PNN structures was observed, but their size was substantially reduced on knock-out neurons. The synaptic activity of both wild-type and knock-out neurons was monitored using whole-cell patch clamping. The salient observation was a reduced frequency of IPSCs and EPSCs, whereas the amplitudes were not modified. Remarkably, the knock-out neuron phenotypes could not be rescued by wild-type astrocytes. We conclude that the elimination of four ECM genes compromises neuronal function.

Chondroitin sulfate proteoglycans regulate astrocyte-dependent synaptogenesis and modulate synaptic activity in primary embryonic hippocampal neurons.

It has been shown that astrocyte-derived extracellular matrix (ECM) is important for formation and maintenance of CNS synapses. In order to study the effects of glial-derived ECM on synaptogenesis, E18 rat hippocampal neurons and primary astrocytes were co-cultivated using a cell-insert system. Under these conditions, neurons differentiated under low density conditions (3500 cells/cm(2) ) in defined, serum-free medium and in the absence of direct, membrane-mediated neuron-astrocyte interactions. Astrocytes promoted the formation of structurally intact synapses, as documented by the co-localisation of bassoon- and ProSAP1/Shank2-positive puncta, markers of the pre- and postsynapse, respectively. The development of synapses was paralleled by the emergence of perineuronal net (PNN)-like structures that contained various ECM components such as hyaluronic acid, brevican and neurocan. In order to assess potential functions for synaptogenesis, the ECM was removed by treatment with hyaluronidase or chondroitinase ABC. Both enzymes significantly enhanced the number of synaptic puncta. Whole-cell voltage-clamp recordings of control and enzyme-treated hippocampal neurons revealed that chondroitinase ABC treatment led to a significant decrease in amplitude and a reduced charge of miniature excitatory postsynaptic currents, whereas inhibitory postsynaptic currents were not affected. When the response to the application of glutamate was measured, a reduced sensitivity could be detected and resulted in decreased currents in response to the excitatory neurotransmitter. These findings are consistent with the interpretation that the ECM partakes in the regulation of the density of glutamate receptors in subsynaptic sites.