Elsevier

Neurobiology of Disease

Volume 70, October 2014, Pages 21-31
Neurobiology of Disease

Metalloproteinase inhibition prevents inhibitory synapse reorganization and seizure genesis

https://doi.org/10.1016/j.nbd.2014.06.003Get rights and content

Highlights

  • Kindling induced seizures break down perineuronal nets in rat piriform cortex.

  • The breakdown is accompanied by an increase in GABAergic synapses.

  • This “rewiring” is different depending on layer and interneuron subtype.

  • Matrix metalloproteinase inhibition stops perineuronal net breakdown and seizures.

  • Rewiring of the layers is also prevented by matrix metalloproteinase inhibition.

Abstract

The integrity and stability of interneurons in a cortical network are essential for proper network function. Loss of interneuron synaptic stability and precise organization can lead to disruptions in the excitation/inhibition balance, a characteristic of epilepsy. This study aimed to identify alterations to the GABAergic interneuron network in the piriform cortex (PC: a cortical area believed to be involved in the development of seizures) after kindling-induced seizures. Immunohistochemistry was used to mark perineuronal nets (PNNs: structures in the extracellular matrix that provide synaptic stability and restrict reorganization of inhibitory interneurons) and interneuron nerve terminals in control and kindled tissues. We found that PNNs were significantly decreased around parvalbumin-positive interneurons after the induction of experimental epilepsy. Additionally, we found layer-specific increases in GABA release sites originating from calbindin, calretinin, and parvalbumin interneurons, implying that there is a re-wiring of the interneuronal network. This increase in release sites was matched by an increase in GABAergic post-synaptic densities. We hypothesized that the breakdown of the PNN could be due to the activity of matrix metalloproteinases (MMP) and that the prevention of PNN breakdown may reduce the rewiring of interneuronal circuits and suppress seizures. To test this hypothesis we employed doxycycline, a broad spectrum MMP inhibitor, to stabilize PNNs in kindled rats. We found that doxycycline prevented PNN breakdown, re-organization of the inhibitory innervation, and seizure genesis. Our observations indicate that PNN degradation may be necessary for the development of seizures by facilitating interneuron plasticity and increased GABAergic activity.

Introduction

Appropriate brain function relies on highly interconnected and organized networks of interneurons that control the activity of projection (excitatory) neurons that distribute information between neuronal assemblies. Any aberrant re-organization of these networks can and often leads to disorders of brain function. Although epilepsy is a diverse disorder with over 40 recognized types (McCormick and Contreras, 2001), all forms are characterized by highly synchronised and unprovoked bursts of neuronal activity that represent a breakdown in normal neuronal connectivity. Epileptogenesis is the process by which the brain is altered from a normal neural network to a hyper-excitable, epileptic one (McIntyre et al., 2002). It also seems clear that seizures and/or numerous other perturbations like head injury can induce re-wiring exacerbating epileptiform activity. In the case of brain injury seizures may only occur after a latent period lasting months or years (Pitkanen and Lukasiuk, 2011). Thus, it is hypothesized that there are progressive changes in neuronal structure, connections, and functions that ultimately create an imbalance of excitation and inhibition leading to seizures (McNamara, 1994, Morimoto et al., 2004, Racine et al., 2002, Tasker et al., 1996). No matter what the etiology, the prevention and treatment of epilepsy requires an understanding of how the brain mal-adapts, creating “wiring” patterns that support seizures.

The kindling model of epilepsy represents a progressive and permanent development of convulsive motor seizures that arise from daily electrical stimulations of subcortical brain areas, often hippocampus or basal lateral amygdala (Goddard, 1967). The kindling model is not associated with significant cell loss and therefore represents fundamental alterations in network behaviour that may include changes in cellular excitability, synaptogenesis, and/or metabolic activity as well as other outcomes. The same cellular mechanisms that establish and maintain kindling in animals have been suggested to be involved in partial epilepsy in humans (Adamec and Stark-Adamec, 1983, Sato et al., 1998). In particular aberrant plasticity has been postulated to underlie the formation of epileptic foci in the temporal lobe (Wilczynski et al., 2008). Therefore, the identification of mechanisms that are engaged during the kindling procedure that governs this maladaptive plasticity may give clues to a better understanding of the epileptogenesis.

One important process that occurs during brain development is the formation of an extracellular matrix (ECM) that is essential for many processes including proliferation, migration, synaptogenesis, synaptic stability, and cell signaling (Dityatev and Schachner, 2003). The ECM of the nervous system is comprised of a complex mixture of proteoglycans, glycoproteins, tenascin, fibronectin, and hyaluronan (Deepa et al., 2006, Galtrey and Fawcett, 2007). A component of the ECM is perineuronal nets (PNNs), which form structures that surround cell bodies, dendrites, and axon initial segments leaving open “holes” at sites of synaptic contacts (Wang and Fawcett, 2012, Zaremba et al., 1989). PNNs are found in virtually all regions of the central nervous system but predominantly surround inhibitory GABAergic neurons. A high percentage of PNNs are found around interneurons that express the calcium binding protein parvalbumin, as well as being present in other subtypes (Bruckner et al., 1997, Dityatev et al., 2010). Although PNNs are generally thought to be stable structures, various perturbations can cause their breakdown. For example, the breakdown of PNNs has been reported in the kainic acid model of epilepsy (McRae and Porter, 2012, McRae et al., 2012). However, the effect of this breakdown on synaptic wiring or how degradation may be prevented has not been explored. To this end we have investigated whether PNN degradation occurs in the kindling model of epilepsy and if so whether decreased PNNs correlate with changes in inhibitory synapse “wiring patterns”. We also investigated whether the inhibition of matrix metalloprotease (MMP) activity prevents the breakdown of PNNs and subsequent re-wiring.

Section snippets

Animals and surgery

Male Sprague–Dawley rats from Charles River weighing approximately 200 g at the time of initial surgery were used. They were housed in standard plastic cages with free access to food and water under a continuous 12 h/12 h light/dark schedule. For electrode implantation animals were anesthetized with ketamine (100 mg/kg, i.p.)/domitor (10 mg/kg, i.p.) and implanted with two bipolar stimulating/recording electrodes bilaterally in the basolateral amygdala using the following coordinates: 2.6 mm

The expression of perineuronal nets was decreased after kindling

Perineuronal nets (PNNs) may be used as a marker of synaptic stability in the adult central nervous system (CNS) because of their known inhibitory effects on structural rearrangement and axonal growth (Celio and Blumcke, 1994, Frischknecht and Gundelfinger, 2012, Pizzorusso et al., 2002). To determine if experimentally induced epilepsy affected PNN expression after amygdala kindling-induced seizures, PNNs were labeled with either a lectin from Wisteria floribunda agglutinin (WFA) or a primary

Discussion

The goal of this study was to determine if experimentally induced epilepsy (kindling) causes changes in the inhibitory neuron network of the piriform cortex (PC) and what factors may contribute to any changes found. Here we have shown that the expression of perineuronal nets (PNNs) surrounding parvalbumin (PV) interneurons in the PC was significantly decreased after kindling-induced seizures. By contrast only a minority of calbindin (CB) and calretinin (CR) positive interneurons were surrounded

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