Epileptiform activity and behavioral arrests in mice overexpressing the calcium channel subunit α2δ-1
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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- 1
Contributed equally to these experiments.