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Inner pillar cells express Fgfr which may contribute to thei
Inner pillar cells express Fgfr3, which may contribute to their capacity for regeneration (Jacques et al., 2007). Supporting cells in the chicken basilar papilla express Fgfr3, which is transiently downregulated during hair cell regeneration (Bermingham-McDonogh et al., 2001). Fgfr3−/− mice lack inner pillar cells in the apical and middle regions of the cochlea but exhibit an extra row of OHCs in these areas (Hayashi et al., 2007; Puligilla et al., 2007). Expression of both Lgr5 and Fgfr3 may be important for the transdifferentiation of cochlear cells after damage. We did not find any evidence of transdifferentiation in supporting cells that did not express Lgr5, suggesting that signaling through Lgr5 or Fgfr3 may be necessary to generate new hair cells with Notch inhibition. These studies therefore confirm the identification of Lgr5-expressing supporting cells as hair cell progenitors and show that damage to the newborn cochlea results in hair cell regeneration initiated by these Lgr5-positive cells.
Experimental Procedures
Acknowledgments
Introduction
Spontaneous neuronal activity refers to the ability of an individual neuron or an entire neuronal network to be electrophysiologically active without receiving external stimulation. Such spontaneous activity is present in both the developing and the adult NVP-TNKS656 cost (Blankenship and Feller, 2010; de Pasquale et al., 2010; Liu et al., 2010). Also, isolated brain slices (i.e., segments of brain that are deprived of input from other brain regions) show spontaneous neuronal activities (Darbon et al., 2002a; Le Bon-Jego and Yuste, 2007; Shew et al., 2010), and dissociated neuronal cells self-organize into spontaneously spiking or synchronously bursting neuronal networks in vitro (Arnold et al., 2005; Streit et al., 2001; Van Pelt et al., 2004). Autonomous network activities rely on neurons, which are able to be spontaneously active even in conditions where fast synaptic communication (FSC) has been silenced. These neurons have therefore been termed intrinsically active neurons (IANs). IANs can be found in nearly all brain regions that have been investigated to date (Atherton and Bevan, 2005; Atherton et al., 2008; Beatty et al., 2012; Mao et al., 2001; Streit et al., 2001; Tazerart et al., 2008), where they act as pacemaker neurons in regulating oscillatory activities in neuronal assemblies (Beatty et al., 2012; Feldman and Del Negro, 2006; Le Bon-Jego and Yuste, 2007; Tazerart et al., 2008; Tscherter et al., 2001). Thus, IANs represent key neuronal elements that spontaneously induce activities in other neurons, and may regulate different types of orchestrated activities in neuronal assemblies. Moreover, the ability of neurons to self-organize into functional neuronal networks generating oscillatory neuronal activities is crucial for proper cognitive as well as motor functions.
Multielectrode array (MEA) recordings and calcium imaging of human and murine pluripotent stem cell (PSC)-derived neurons have revealed the capacity of these neurons to self-organize into spontaneous active neuronal networks, generating concerted activities that are measured as synchronous bursting (Eiraku et al., 2008; Heikkilä et al., 2009; Illes et al., 2007, 2009). Therefore, PSC-neuronal networks display activities that in principle resemble those observed during brain function (Gullo et al., 2010a; Heikkilä et al., 2009; Illes et al., 2007, 2009; Nimmrich et al., 2005); however, the underlying mechanisms of the genesis of spontaneous activity and synchronicity have yet to be deciphered. This is crucial for understanding brain development and function, as well as for determining the proper use of PSC neurons in future transplantation approaches (Daadi et al., 2008; Eiraku et al., 2008; McDonald et al., 1999). Here, we hypothesize that IANs are responsible for the autonomous behavior of PSC-neuronal assemblies, i.e., spontaneous and synchronous neuronal network activities. Therefore, we tested for the presence of neurons that became active after pharmacological inhibition of FSC, a cardinal feature of IANs, and determined whether these putative IANs were functionally integrated into the neuronal network. We further tested whether such FSC-independent neuronal activities relied on persistent sodium currents, another cardinal characteristic of IANs. Finally, we classified the putative IANs and discriminated between neurons that contributed to population bursts (PBs) and those that had pacemaker properties, based on a comparative analysis of network-wide PBs.