Homeostasis of neuronal avalanches during postnatal cortex development in vitro
Introduction
Neuronal networks are spontaneously active even when maintained isolated from external inputs. This intrinsic activity is crucial for numerous aspects of network development such as neuronal migration, differentiation, synaptogenesis, and synaptic plasticity (Penn et al., 1998, Ben-Ari, 2001, Spitzer, 2006) and typically emerges as synchronized bursts that last for hundreds of milliseconds separated by up to many seconds of quiescence (O’Donovan, 1999). In the cortex, this hallmark of spontaneous activity has been reported since the earliest culture success in vitro (Crain, 1966, Calvet, 1974) and naturally occurs in all isolated cortex preparations thus studied, e.g. in organotypic cultures (Plenz and Aertsen, 1996a, Plenz and Aertsen, 1996b, Gorba et al., 1999, Klostermann and Wahle, 1999, Baker et al., 2006), dissociated cultures (Maeda et al., 1995, Gopal and Gross, 1996, Kamioka et al., 1996, Canepari et al., 1997, Jimbo et al., 2000, Segev et al., 2001, van Pelt et al., 2004, Eytan and Marom, 2006), acute cortex slices (Sanchez-Vives and McCormick, 2000), and acutely isolated cortical slabs in vivo (Steriade et al., 1993, Timofeev et al., 2000). Quantification of these dynamics, however, has been difficult. Widely varying burst rates, burst durations, and levels of synchronization have been reported even within one neuronal preparation (Corner et al., 2005). Rather than confounding classification, we hypothesized that the broad variability of the activity may be itself a quantifiable and defining feature intrinsic to these networks. In such a network state, neurons would spontaneously and transiently synchronize their activities with a large number of other neurons in ever changing constellations thereby exploring a wide variety of different neuronal associations within the network. Furthermore, such a quantitative description would be accompanied by robust and unambiguous quantitative measures.
Cortex slices taken into culture at early postnatal age develop the main features of cortical organization that include all major classes of pyramidal neurons (Cäser et al., 1989, Gutnick et al., 1989, Wolfson et al., 1989, Cäser and Schüz, 1992), interneurons (Götz and Bolz, 1989, Plenz and Aertsen, 1996a, Plenz and Aertsen, 1996b, Gorba et al., 1999, Klostermann and Wahle, 1999), and cortical layers (Götz and Bolz, 1992). Organotypic slice cultures therefore to date represent the most intact culture system for studying cortex function in isolation over the course of many weeks. The typical neuronal activity in this in vitro system has been characterized by brief periods of activity separated by many seconds of quiescence and has been given various labels that highlight different aspects such as the abrupt and brief nature of activity periods (‘bursts’ or ‘network spikes’), their non-oscillatory recurrence (‘irregular’), their overlap in time at different cortical sites (‘synchronous’), or their propagation, i.e. successive initiation at different sites (‘waves’) (Crain, 1966, Calvet, 1974, Dichter, 1978, Gutnick et al., 1989, Wolfson et al., 1989, Plenz and Aertsen, 1996a, Plenz and Aertsen, 1996b, Gorba et al., 1999, Klostermann and Wahle, 1999, Corner et al., 2002, Eytan and Marom, 2006). Using extracellular local field potentials (LFP), which are particularly well suited to study network states as they correlate with synchronized activity of local neuronal populations in vivo (Arieli, 1992, Lampl et al., 1999), we demonstrated recently that the spontaneous LFPs in isolated cortical networks are ordered such that the probability P of a spatiotemporal LFP cluster of size s follows a power law (Beggs and Plenz, 2003, Plenz and Thiagarajan, 2007).This power law quantifies the large diversity of spontaneous bursts with respect to their sizes, which can range from just a few microvolts to many thousands of microvolts. Importantly, the relative probability of bursts occurring with size s1 and s2 = n·s1 is independent of s,In this sense, a power law reveals a particular relationship between different length scales. This organization of LFP activity has been demonstrated in the form of ‘neuronal avalanches’ in mature slice cultures and acute cortex slices taken from adult rats (Beggs and Plenz, 2003, Stewart and Plenz, 2006). A similar organization was demonstrated recently in spontaneous spike bursts of dissociated hippocampus cultures (Mazzoni et al., 2007). The presence of power law organization across spatial scales is suggestive of systems in a critical state which optimizes numerous aspects of information processing (for review see: Plenz and Thiagarajan, 2007, Chialvo, 2007). The critical state fits our hypothesis; the high variability of neuronal synchronization encountered is an intrinsic system feature. Critical states have been reported in a wide range of physical systems with non-linear propagation characteristics (Bak et al., 1987, Bak et al., 2001, Drossel and Schwabl, 1992, Christensen et al., 1993, Paczuski et al., 1996) and have been demonstrated in neuronal network simulations (for review see Plenz and Thiagarajan, 2007).
In the present study, we tested for the robustness of the critical state or neuronal avalanche dynamics using organotypic cortex cultures as an experimental model. These cultures transition through different activity levels during postnatal maturation (Maeda et al., 1995, Kamioka et al., 1996, Corner et al., 2002, Johnson and Buonomano, 2007) and are well suited to identify a common network state that encompasses the variable features of the overall activity. We show that a power law in event sizes with an exponent of α = −1.5, i.e. neuronal avalanches, describes the spatiotemporal dynamics of spontaneous bursts throughout postnatal development despite large changes in neuronal activity levels. These findings suggest that cortical networks homeostatically regulate neuronal avalanches, which are indicative of a critical state, during development.
Section snippets
Preparation of organotypic cultures on the multielectrode arrays (MEA)
Organotypic cultures from slices of rat cortex were prepared in accordance with NIH guidelines (Plenz and Kitai, 1998, Karpiak and Plenz, 2002). In short, coronal sections from rat brains at postnatal day 0–2 (P0–2; Sprague–Dawley, Taconic Farms, MD, USA) were cut at a thickness of 350 μm. A section of the coronal slice that contained dorsal or dorsolateral cortex (∼1.5 mm deep and ∼2–3 mm wide) was placed on a multielectrode array (MEA; MultiChannelSystems, Reutlingen, Germany) with the bottom
Results
Coronal slices of dorsolateral cortex taken from newly born pups at P0–2 were placed on 8 × 8 multielectrode arrays (MEA) and cultured for up to 7 weeks in vitro to study postnatal maturation of the cortex in isolation (n = 9 cultures). Cortical recording locations were obtained and confirmed in three steps. First, during culture preparation, cortical slices were placed on the array with the white matter bordering the bottom row of the electrode grid. Second, pictures taken at 1–2 DIV allowed
Discussion
The present study demonstrates a homeostatically regulated organization of neuronal synchronization that is remarkably robust despite large variability in the rates of activity. This organization is identified by nLFP clusters, i.e. neuronal avalanches, that distribute according to a power law in sizes with slope less than or equal to −3/2 during postnatal maturation. The power law in size distribution, a critical branching parameter of σ ≅ 1, the robustness of the dynamics to large changes in
Acknowledgements
We thank Drs. W. Shew, T. Thiagarajan, T. Petermann, and E.D. Gireesh and T. Bellay for comments on an earlier version of the manuscript. This research was supported by the Intramural Research Program of the NIH/NIMH.
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