The extracellular matrix (ECM) plays an important role in regulating use-dependent synaptic plasticity. Distinct aggregates of ECM molecules surround cell bodies and proximal dendrites of some central neurons, forming so-called perineuronal nets (PNNs; Dityatev et al., 2010). These nets are heterogeneous in their structure and composition and are composed of molecules produced by both neurons and astrocytes (Dityatev et al., 2010), such as hyaluronic acid (HA), chondroitin sulfate proteoglycans of the aggrecan family, tenascin-R and link proteins (Bruckner et al., 2000; Galtrey and Fawcett, 2007; Frischknecht and Seidenbecher, 2008). HA is a large, negatively charged, non-branched polymer composed of repeated disaccharides of glucuronic acid and N-acetylglucosamine. Recently, HA has been shown to affect both α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) glutamate receptor mobility and paired-pulse modulation in hippocampal cultures (Frischknecht et al., 2009). Another study established a mechanistic link between HA and long-term potentiation (LTP) by showing that removing HA by hyaluronidase (Hyase) suppresses L-type voltage-dependent Ca²⁺ channel (L-VDCC)-mediated currents in hippocampal slices, reduces Ca²⁺ transients in postsynaptic dendrites and spines, and specifically abolishes an L-VDCC-dependent component of LTP (Kochlamazashvili et al., 2010). Furthermore, the injection of Hyase before contextual fear conditioning impairs the formation/retention of fear memories. A mathematical modeling study highlighted that the ECM may function as a memory substrate by showing that remodeling of ECM may lead to a bistability in which two different stable levels of average firing rates can coexist in a spiking network (Kazantsev et al., 2012).The multiple roles played by the ECM in healthy brain neuroplasticity suggest that it could be an important factor for pathogenic plasticity associated with epileptogenesis (Dityatev, 2010), which results in the transformation of normal neuronal network activity into spontaneous recurrent epileptiform discharges (seizures; Robert et al., 1998). Although accepted to be important, the exact contribution of the multiple structural and functional pathological forms of plasticity in hippocampal circuit alteration during epileptogenesis remains to be elucidated. For most forms of epilepsy, it is unknown how the hippocampus becomes hyperexcitable and how hyperexcitable cells integrate into a pathologically functioning circuit, resulting in the generation of spontaneous recurrent seizures. In recent years, microelectrode array (MEA) and optical imaging technologies have developed rapidly, providing new options to perform long-term, detailed analysis of neural circuitry dynamics.

In this study, we used these technologies to describe the development of epileptiform activity following Hyase treatment in vitro. Our findings revealed that Hyase destructed the ECM of PNNs, surrounding cell bodies and the proximal dendrites of parvalbumin (PV)-expressing interneurons, and induced a slow development of seizure-like activity. The treatment transformed normal network spiking bursts into long-lasting “superbursts” and caused the appearance of neuronal and astrocytic Ca²⁺ “superoscillations.” Seizure-like activity in Hyase-treated cultures persisted for at least 9 days and could be suppressed by an L-VDCC blocker but not by an NMDA receptor antagonist. These results suggest that changes in the expression of HA can be epileptogenic and the underlying mechanisms may involve changes in Ca²⁺ oscillations.

Hyaluronic acid is the backbone of neural ECM, which is enriched in PNNs. To remove HA and study its role in neural network activity, we treated hippocampal cultures with Hyase. Because aggrecan has been shown to be a key component of the ECM of PNNs (Giamanco et al., 2010), we used aggrecan immunostaining to characterize the efficacy of the treatment. Figure 2 shows hippocampal neurons labeled by anti-MAP2 antibodies and PNNs labeled by anti-aggrecan antibody in the control treatment (Figure 2A) and after Hyase treatment (Figures 2B, C). The treatment was done on DIV 17 when the number of PNNs in the control group reached a steady-state level (Figure 2D). Counting the PNNs in the control and Hyase-treated primary hippocampal cultures revealed a strong reduction in the fraction of aggrecan-immunopositive neurons 2 (ANOVA p = 0.001), 3 (ANOVA p < 0.001), and 9 (p = 0.019) days after Hyase treatment (Figure 2D). Between days 3 and 9, however, there was an increase in the fraction of PNNs (ANOVA p = 0.037), suggesting PNN re-formation during this time interval.

Previous studies demonstrated that spontaneous spiking activity appears in high-density dissociated hippocampal cultures after 8–10 days of development on MEAs (Wagenaar et al., 2006). On DIV 16, dissociated culture activity became stabilized. To establish whether the Hyase treatment changes the spontaneous network activity, we analyzed raster plots of electrical spiking activity and spike rate diagrams (Figures 3A1–A4,B1–B4). We revealed significant changes in spontaneous spiking activity 9 days after Hyase treatment. Short network bursts with duration of less than 10 s (Figures 3D1, D2) were transformed into seizure-like superbursts of activity with durations of 15–35s (Figures 3C2, E1, E2). These changes in the spontaneous spiking activity of Hyase-treated cultures appeared on days 5–7 following the treatment and were observed during the next 4 days.

Statistical analysis revealed that the superbursts (Figures 3C1–C4) consist of a large number of network bursts, which are characterized by a low number of spikes per burst and a short interburst interval. Moreover, the general number of network bursts per 10 min increased significantly from day to day after Hyase treatment (19 DIV: 513.3 ± 36; 20 DIV: 712 ± 54; 26 DIV: 827 ± 62, ANOVA; p < 0.05 compared to DIV 17; N = 5), and this parameter was significantly different between the Hyase and control groups on DIV 26 (Control: 257 ± 21, ANOVA p < 0.05; N = 5).

In the control group on the 26th DIV, we recorded short superbursts with a frequency of 1.5 ± 0.3 per 10 min and a mean duration of 10.5 ± 0.5 s. After Hyase treatment, we observed long superbursts with a frequency of 3.3 ± 0.5 per 10 min and a mean duration of 36.5 ± 1.4 s (ANOVA; p < 0.05; N = 5).

Spontaneous Ca²⁺ oscillations appear in dissociated hippocampal cultures beginning on DIV 7 (unpublished observation). On the DIV 17, a large number of neurons have similar patterns of Ca²⁺ oscillations, which are quite distinct from the patterns of Ca²⁺ oscillations in astrocytes (Figures 4A1, B1). Both neuronal and astrocytic patterns of Ca²⁺ activity were fairly stable and did not change significantly up to the 26th DIV (Figures 4A2–A4). The duration of the neuronal oscillations was approximately 6 s (Figures 4C, D) and the duration of the astrocytic Ca²⁺ oscillations was approximately 10 s on DIV 17–26. The Hyase treatment significantly changed the spontaneous Ca²⁺ activity (Figures 4B2–B4, 4C–F), and already on the second day after Hyase application, we observed the appearance of neuronal Ca²⁺ superoscillations with a duration of 94.78 ± 12.33 s (Figure 4C). The superoscillation duration then showed a significant decrease (20 DIV: 50.95 ± 1.25 s; 26 DIV: 28.54 ± 2.71 s; ANOVA p < 0.001 for both days compared to the 19th DIV; N = 5). Additionally, an increase in the duration of the astrocytic Ca²⁺ oscillations was observed after Hyase application (19 DIV: 16.78 ± 1.21 s; 20 DIV: 18.03 ± 2.48 s; 26 DIV: 17.75 ± 2.53 s; ANOVA; p < 0.05 for all days compared to DIV 17; N = 5). We also observed that some astrocytes synchronized their Ca²⁺ activity with that of neurons starting from the 2nd day after the Hyase treatment (Figures 4B2–B4).

Next, we attempted to block the Hyase-induced superbursts of spiking activity and Ca²⁺ superoscillations in the neuronal and glial networks (Figures 5 and 6). Seizure-like elevations in the burst activity of Hyase-treated cultures could be suppressed within 5 min after treatment by the L-VDCC blocker diltiazem (Figures 5B1, C, D) but not by the NMDA receptor antagonist CPP (Figures 5B2, C, D). An AMPA/kainate receptor antagonist, CNQX, also suppressed superbursts. Neuronal Ca²⁺ superoscillations could also be blocked by diltiazem (Figure 6B1, upper curve; Figure 6C) but not by CPP (Figure 6). Diltiazem also significantly reduced the number of astrocytic Ca²⁺ oscillations after the Hyase treatment (Figure 6B1, lower curve; Figure 6D).

Here, we demonstrated that a Hyase treatment, which results in the removal of HA as the backbone of the neural ECM, leads to a slow development of epileptiform activity in cultured hippocampal neurons. These data provide a new in vitro model of epileptogenesis that can be easily induced and studied using MEA technology and optical imaging to characterize the processes underlying epileptogenesis.

Many animal models of epilepsy already exist. The majority involve experimental induction of a brain injury through the administration of a chemoconvulsant or electrical stimulation, both of which induce an episode of seizures. Subsequent injury-induced plasticity includes the appearance of mossy fiber sprouting (Parent and Lowenstein, 1997), an increased rate of neurogenesis in the dentate gyrus (Parent et al., 1997), increased activity of microglia, and an induction of reactivity in astrocytes (Wetherington et al., 2008). In addition to cell death and anatomical changes in neurons and glia, seizures induce significant functional plasticity in surviving hippocampal neurons, including alterations in the function of Ca²⁺- and hyperpolarization-activated mixed cationic channels (Shah et al., 2004; Jung et al., 2010) and K⁺ channels (Bernard et al., 2004), changes in neurotransmitter receptors, including GABA_A receptors (Pathak et al., 2007), glutamate receptors (Doherty and Dingledine, 2001), chloride transporters (Pathak et al., 2007) and excitatory amino acid transporters (Crino et al., 2002; Eid et al., 2008).

Thus, multiple mechanisms could underlie epileptogenesis. Because Hyase treatment leads to a disruption of PNNs around PV-expressing GABAergic interneurons, it is plausible to suggest that one of the causes of Hyase-induced epileptiform activity is an imbalance in excitation and inhibition due to impaired PV interneuron function. These cells represent the most frequent group of inhibitory cells, and they are widely distributed across the brain (Karetko and Skangiel-Kramska, 2009). However, PNN removal with another enzyme, chondroitinase ABC, has been found to reduce the firing threshold of these cells in vitro (Dityatev et al., 2007), which would promote rather than deter GABAergic inhibition.

Other possible mechanisms may be related to alterations of the perisynaptic ECM by Hyase. An elegant study by Frischknecht et al. (2009) used Hyase or chondroitinase ABC to remove PNNs from cultured rat hippocampal neurons and revealed that a perisynaptic net-like structure in the ECM influences the mobility of AMPA receptors, creating a barrier for their movement into and out of excitatory synapses. ECM removal impairs paired-pulse depression of excitatory postsynaptic currents (EPSCs) in cultures and thus might promote hyperexcitation in cultured pyramidal cells (Frischknecht et al., 2009).

Our MEA recordings revealed that the spontaneous electrical activity of mature dissociated hippocampal neurons grown on MEAs occurs in the form of synchronized burst discharges, but the pattern of these burst discharges is different from the long-lasting “superburst” discharges induced by Hyase treatment. These “superbursts” became most prominent 9 days after the Hyase treatment. Our Ca²⁺ imaging data demonstrates that the Hyase treatment also transformed the normal neuronal Ca²⁺ oscillations into long-lasting “superoscillations” with mean durations of up to 100 s already on the 2nd day after the Hyase treatment. Thus, dramatic changes in Ca²⁺ oscillations precede changes in spiking activity, suggesting that superoscillations may represent a key event leading to changes in the expression of genes that determine neuronal excitability. In addition, changes in Ca²⁺ signaling in astrocytes were detected after the Hyase treatment, although at a smaller scale than in neurons.

Elevations in burst activity and neuronal Ca²⁺ transients in the Hyase-treated neurons and astrocytes was suppressed by an application of the L-VDCC blocker diltiazem, although not by a NMDA receptor antagonist, whereas the superbursts of spiking activity were additionally suppressed by CNQX, an antagonist of AMPA receptors. The latter is not surprising because CNQX mostly blocks excitatory transmission. The elimination of superbursts and superoscillations by diltiazem is at first glance surprising because HA was reported to support L-VDCC activity and the Hyase treatment leads to an acute deficit in neuronal L-VDCC-mediated currents (Kochlamazashvili et al., 2010). It is plausible to assume that this acute deficit in L-VDCC activity might be later overcompensated by overexpression of L-VDCCs after the Hyase treatment, which might lead to the generation of Ca²⁺ superoscillations and downstream effects on neuronal excitability/excitatory transmission. This hypothesis remains to be verified in follow-up studies. The presented experimental data support the view that the remodeling of ECM may lead to generation of epileptiform activity in vitro in the form of superbursts. Such epileptogenic degradation of HA and ECM remodeling may be due to genetic factors or triggered by insults such as stroke or a brain injury. Thus the described experimental model could be potentially valuable for development of new class of broadly applicable anti-epileptogenic drugs, which would target the HA-based neural ECM and/or ECM-mediated signaling.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.