Elsevier

Neurobiology of Disease

Volume 102, June 2017, Pages 70-80
Neurobiology of Disease

Epileptiform activity and behavioral arrests in mice overexpressing the calcium channel subunit α2δ-1

https://doi.org/10.1016/j.nbd.2017.01.009Get rights and content

Highlights

  • Increased density of neocortical excitatory synapses is present in TG mice overexpressing thrombospondin receptor α2δ-1.

  • Excitatory, but not inhibitory synaptic transmission is increased in α2δ-1 TG mice.

  • Neocortical epileptiform activity and associated behavioral arrests occur in α2δ-1 TG mice.

  • Epileptiform activity in the TG mice is age-dependent, decreased by ethosuximide and provoked by exposure to isoflurane.

  • Overexpression of α2δ-1 thrombospondin receptors in TG mice results in neocortical hyperexcitability and epileptiform events.

Abstract

The alpha2delta-1 subunit (α2δ-1) of voltage-gated calcium channels is a receptor for astrocyte-secreted thrombospondins that promote developmental synaptogenesis. Alpha2delta-1 receptors are upregulated in models of injury-induced peripheral pain and epileptogenic neocortical trauma associated with an enhancement of excitatory synaptic connectivity. These results lead to the hypothesis that overexpression of α2δ-1 alone in neocortex of uninjured transgenic (TG) mice might result in increased excitatory connectivity and consequent cortical hyperexcitability and epileptiform activity. Whole cell recordings from layer V pyramidal neurons in somatosensory cortical slices of TG mice showed increased frequency and amplitude of miniature and spontaneous EPSCs and prolonged bursts of polysynaptic EPSCs. Epileptiform field potentials were evoked in layers II/III and V of brain slices from TG mice, but not controls. Dual immunoreactivity for Vglut-2 and PSD95 showed increased density of close appositions in TG mice compared to controls, suggesting an increased number of excitatory synapses. Video-EEG monitoring showed that 13/13 implanted TG mice aged > P21, but not controls, had frequent abnormal spontaneous epileptiform events, consisting of variable duration, high amplitude bi-hemispheric irregular bursts of delta activity, spikes and sharp waves lasting many seconds, with a variable peak frequency of ~ 1–3 Hz, associated with behavioral arrest. The epileptiform EEG abnormalities and behavioral arrests were reversibly eliminated by treatment with i.p. ethosuximide. Behavioral seizures, consisting of ~ 15–30 s duration episodes of rigid arched tail and head and body extension, followed by loss of balance and falling, frequently occurred in adult TG mice during recovery from isoflurane-induced anesthesia, but were rare in WT mice. Results show that over-expression of α2δ-1 subunits increases cortical excitatory connectivity and leads to neocortical hyperexcitability and epileptiform activity associated with behavioral arrests in adult TG mice. Similar increases in expression of α2δ-1 in models of cortical injury may play an important role in epileptogenesis.

Significance

Binding of astrocytic-secreted thrombospondins to their α2δ-1 receptor facilitates excitatory synapse formation and excitatory transmission during cortical development and after injury. Upregulation of α2δ-1 is present in models of injury-induced pain and epileptogenic cortical trauma, along with many other molecular alterations. Here we show that overexpression of α2δ-1 alone in TG mice can enhance excitatory connectivity in neocortex and lead to neural circuit hyperexcitability and episodes of electrographic epileptiform activity, associated with behavioral arrests in transgenic mice. α2δ-1 is the high-affinity receptor for gabapentinoids and a potential target for prophylactic treatment of posttraumatic epilepsy and other disorders in which excessive aberrant excitatory connectivity is a pathophysiological feature.

Introduction

Thrombospondins are extracellular matrix proteins that have multiple functions including involvement in inflammation, angiogenesis and platelet aggregation (Adams and Lawler, 2004). They also promote excitatory synapse formation in the central nervous system (Christopherson et al., 2005) by interacting with the voltage gated calcium channel subunit α2δ-1, which also binds gabapentin, a widely used anticonvulsant drug (Eroglu et al., 2009). Initially identified as a non-essential calcium channel subunit (Arikkath and Campbell, 2003), the α2δ-1 protein is the product of a single gene (De Jongh et al., 1990) that is post-translationally cleaved into α2 and δ peptides. The α2 subunit is entirely extracellular, whereas δ is mainly a transmembrane protein with a small intracellular portion (Gurnett et al., 1996). The α2δ-1 subunit is expressed in multiple cortical areas (Cole et al., 2005) where it is mainly located in presynaptic terminals (Taylor and Garrido, 2008), as well as in tissues outside the nervous system such as heart and muscle (Gong et al., 2001).

Recent results have shown that α2δ-1 is upregulated after nerve and brain injury (Luo et al., 2001), (Luo et al., 2002), (Li et al., 2012). In the neocortical partial isolation (undercut) model of posttraumatic epileptogenesis, gliosis, increases in thrombospondins (TSPs) and α2δ-1, and increased density of excitatory synapses occur in the injured cortex, along with abnormal epileptiform burst discharges (Li et al., 2012). Brief gabapentin treatment reduces the incidence of epileptiform bursts, the density of excitatory synapses and the frequency of excitatory synaptic currents (EPSCs) in this model, suggesting that thrombospondin-induced excitatory synapse formation via interactions with α2δ-1 might contribute to these abnormalities. However cortical injury induces a large number of pathophysiological processes (reviewed in (Prince et al., 2009)) so that it is difficult to determine whether the increases in α2δ-1 play a significant role in development of the epileptogenic activity. Transgenic (TG) mice, overexpressing α2δ-1 subunit proteins, have increased number of excitatory synapses in the visual cortex (Eroglu et al., 2009), and show tactile allodynia without injury (Li et al., 2006) and increased frequency of glutamatergic synaptic currents in spinal cord dorsal horn (Nguyen et al., 2009). Such enhanced excitatory connectivity is one mechanism underlying epileptogenesis and seizures in models of posttraumatic (Jin et al., 2006, Li et al., 2005, Li and Prince, 2002), genetic (Chu et al., 2010) and status epilepticus-induced epilepsy (Esclapez et al., 1999), even in the absence of concurrent decreases in inhibition (Buckmaster and Dudek, 1997a, Buckmaster and Dudek, 1997b, Chu et al., 2010). We hypothesized that increased excitatory synapse formation in uninjured TG mice overexpressing α2δ-1 protein in brain would induce hyperexcitability and epileptiform activity similar to that seen in animal models of epilepsy.

We obtained electrophysiological recordings, immunocytochemical and behavioral data from adult male TG mice that overexpress α2δ1 receptors and from control littermates that have normal complements of α2δ1 (“controls” below). Whole cell recordings were obtained from layer V pyramidal neurons of the somatosensory cortex, an area known for its potential to generate epileptiform activity (Connors, 1984, Graber and Prince, 2004, Hoffman et al., 1994), and video/EEGs were recorded from implanted animals. Results show that an increased density of Vglut2/PSD95 close appositions (putative excitatory synapses), enhanced functional excitatory cortical connectivity and bilaterally synchronous spontaneous paroxysmal electrographic epileptiform activity associated with behavioral arrests that are blocked by ethosuximide, occur in α2δ-1 TG mice.

Section snippets

Materials and methods

All experiments were performed according to the National Institutes of Health guide for the care and use of Laboratory animals and all protocols were approved by the Stanford Institutional Animal Care and Use Committee. TG mice, overexpressing α2δ-1 subunit protein, and control littermates were kindly provided by Z. David Luo (Li et al., 2006) and Ben A. Barres. Mice were housed and bred in the research animal facilities at Stanford University. We used only adult (P > 21; P0 = date of birth) male

Alpha2delta-1 is increased in the TG mice

As expected, the α2δ-1 subunit protein was over-expressed in the TG mice. There was a diffuse increase in IR in comparison to WT mice (Fig. 1A–B). α2δ-1-IR was prominent in neurons and neuropil of deep neocortical laminae and in hippocampus (Fig. 1C, D). The distribution of IR was similar to that previously found with in situ hybridization for α2δ-1 (Z.D. Luo, unpublished). When sections containing biocytin-filled Pyr cells were dual reacted, prominent close appositions of α2δ-1-IR, on

Discussion

Overexpression α2δ-1 protein in TG mice may contribute to increased excitability through independent actions to increase formation of excitatory synapses (Eroglu et al., 2009) and effects on trafficking of calcium channels and modulation of their properties. (Dolphin, 2012) (D'Arco et al., 2015). We hypothesized that network hyperexcitability and epileptiform activity would be a consequence of such effects in TG mice. Results show that epileptiform activity and increased excitatory connectivity

Statement of interest

All authors assert that there is no conflict of interest to declare.

The following are the supplementary data related to this article.

Authors contributions

LCF. FG and DAP planned the experiments, wrote the paper; LCF and FG collected and analyzed electrophysiological data; IP performed and analyzed the ICC data; BB and ZDL provided α2δ-1 TG mice and assisted in interpreting the results.

Conflict of interests

The authors declare no competing financial interests.

Acknowledgements

Supported by NINDS grants NS12151, NS39579 and NS090076 (DAP); NS40135, NS064341 (ZDL) and a postdoctoral fellowship to LCF from the Epilepsy Foundation of America/American Epilepsy Society.

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