Blog

By Arthur Bikbaev, Anna Ciuraszkiewicz-Wojciech, Jennifer Heck, Oliver Klatt, Romy Freund, Jessica Mitlöhner, Sara Enrile Lacalle, Miao Sun, Daniele Repetto, Renato Frischknecht, Cornelia Ablinger, Astrid Rohlmann, Markus Missler, Gerald J. Obermair, Valentina Di Biase and Martin Heine

Excerpt from the article published in Journal of Neuroscience 17 June 2020,  40 (25) 4824-4841; DOI: https://doi.org/10.1523/JNEUROSCI.1707-19.2020

Highlights

• This study provides support for the reported association of the α2δ1 coding region (CACNA2D1) and the α2δ3 coding region (CACNA2D3) genetic aberrations with autism and the high comorbidity of epilepsy in individuals with autism.
• Autism is a pervasive neurodevelopmental disorder diagnosed early in childhood and associated with aberrant brain connectivity. Remarkably, autistic spectrum disorders are accompanied by epilepsy in up to 38% of affected individuals.
• Symptomatic convulsive epilepsy and intellectual disability were also reported in humans with aberration of the CACNA2D1 gene.
• The impact of the α2δ1 subunit becomes prominent later in development and is rather restricted to glutamatergic signaling. One interaction partner for this action could be α-neurexin, which, together with α2δ1, facilitates the trafficking of Ca_V2.1 VGCCs to presynaptic terminals, whereas α2δ3 may play an opposite role. Altered expression of α2δ1 or α2δ3 can therefore cause a chronic imbalance between excitation and inhibition that is rather characteristic for autism spectrum disorders.

Abstract

VGCCs are multisubunit complexes that play a crucial role in neuronal signaling. Auxiliary α2δ subunits of VGCCs modulate trafficking and biophysical properties of the pore-forming α1 subunit and trigger excitatory synaptogenesis. Alterations in the expression level of α2δ subunits were implicated in several syndromes and diseases, including chronic neuropathic pain, autism, and epilepsy. However, the contribution of distinct α2δ subunits to excitatory/inhibitory imbalance and aberrant network connectivity characteristic for these pathologic conditions remains unclear. Here, we show that α2δ1 overexpression enhances spontaneous neuronal network activity in developing and mature cultures of hippocampal neurons. In contrast, overexpression, but not downregulation, of α2δ3 enhances neuronal firing in immature cultures, whereas later in development it suppresses neuronal activity. We found that α2δ1 overexpression increases excitatory synaptic density and selectively enhances presynaptic glutamate release, which is impaired on α2δ1 knockdown. Overexpression of α2δ3 increases the excitatory synaptic density as well but also facilitates spontaneous GABA release and triggers an increase in the density of inhibitory synapses, which is accompanied by enhanced axonaloutgrowth in immature interneurons. Together, our findings demonstrate that α2δ1 and α2δ3 subunits play distinct but complementary roles in driving formation of structural and functional network connectivity during early development. An alteration in α2δ surface expression during critical developmental windows can therefore play a causal role and have a profound impact on the excitatory-to-inhibitory balance and network connectivity.

SIGNIFICANCE STATEMENT The computational capacity of neuronal networks is determined by their connectivity. Chemical synapses are the main interface for transfer of information between individual neurons. The initial formation of network connectivity requires spontaneous electrical activity and the calcium channel-mediated signaling. We found that, in early development, auxiliary α2δ3 subunits of calcium channels foster presynaptic release of GABA, trigger formation of inhibitory synapses, and promote axonal outgrowth in inhibitory interneurons. In contrast, later in development, α2δ1 subunits promote the glutamatergic neurotransmission and synaptogenesis, as well as strongly enhance neuronal network activity. We propose that formation of connectivity in neuronal networks is associated with a concerted interplay of α2δ1 and α2δ3 subunits of calcium channels.

Introduction

The transfer and processing of information in neuronal networks critically depend on structural and functional connections between neurons. Network connectivity is not static but evolves over time and reflects both genetically predetermined factors and the previously processed stimuli. The initial circuitry formation occurs during early development and is associated with the emergence of synaptic contacts, which serve as substrate for functional network interaction. During early development, spontaneous neuronal activity involving transient changes in intracellular calcium is necessary and sufficient for neuronal development, and powerfully drives the establishment of connectivity maps (Ben-Ari, 2001; Spitzer, 2006).

VGCCs (Ca_Vs) on presynaptic boutons play a crucial role in synaptic transmission by mediating the electrochemical conversion of electrical activity into vesicle release. VGCCs are multiunit complexes that consist of a mandatory pore-forming α1 subunit and auxiliary α2δ and β subunits (Catterall, 2000; Arikkath and Campbell, 2003; Zamponi et al., 2015). In mammalian synapses, activation of mainly P/Q-type (Ca_V2.1) and N-type (Ca_V2.2) VGCCs on membrane depolarization results in rapid presynaptic calcium influx that triggers neurotransmitter release (Wheeler et al., 1994; Scholz and Miller, 1995; Cao and Tsien, 2010). Four α2δ isoforms (α2δ1-4) encoded by CACNA2D1-CACNA2D4 genes have been identified, with α2δ1 and α2δ3 being particularly abundant in the cerebral cortex and hippocampus (Klugbauer et al., 1999; Cole et al., 2005; Schlick et al., 2010). Expression of the β and α2δ subunits increases the trafficking of the channel and modulates its biophysical properties at the surface (Arikkath and Campbell, 2003; Dolphin, 2012). For example, overexpression of α2δ subunits triggers synaptic recruitment of VGCCs, enlargement of the presynaptic terminals, and facilitation of presynaptic release (Hoppa et al., 2012; Schneider et al., 2015), whereas downregulation of α2δ subunits decreases the surface expression of α1 subunit and leads to the reduction of presynaptic structures and glutamate release (Dickman et al., 2008; Kurshan et al., 2009; Cordeira et al., 2014). Additionally, α2δ1 and α2δ3 subunits were shown to promote excitatory synaptogenesis in mammalian brain (Eroglu et al., 2009) and in Drosophila (Dickman et al., 2008; Kurshan et al., 2009), respectively.

Altered expression of α2δ subunits has been implicated in the pathogenesis of several syndromes and diseases (Geisler et al., 2015; Zamponi et al., 2015). In particular, postinjury overexpression of α2δ1 in sensory neurons is associated with hyperalgesia and chronic neuropathic pain and underlies the antiallodynic efficacy of gabapentinoids (Luo et al., 2001; Bauer et al., 2009; Patel et al., 2013). Null mutation of CACNA2D2 leads to global developmental delay, absence epilepsy, and cerebellar ataxia in mice (Barclay et al., 2001) and humans (Edvardson et al., 2013; Pippucci et al., 2013). Symptomatic convulsive epilepsy and intellectual disability were also reported in humans with aberration of the CACNA2D1 gene (Vergult et al., 2015). Furthermore, analyses of gene-disrupting mutations in individuals with autism highlighted CACNA2D3 among autism susceptibility genes (Iossifov et al., 2012; De Rubeis et al., 2014). Autism is a pervasive neurodevelopmental disorder diagnosed early in childhood and associated with aberrant brain connectivity (Folstein and Rosen-Sheidley, 2001; Freitag, 2007). Remarkably, autistic spectrum disorders are accompanied by epilepsy in up to 38% of affected individuals, which represents manifold higher incidence of epilepsy compared with the population average (Tuchman and Rapin, 2002; Levisohn, 2007).

Thus, converging lines of evidence suggest that α2δ subunits are involved in the establishment and/or modulation of the excitation/inhibition ratio, but little is known about the mechanisms and the contribution of individual α2δ isoforms to network connectivity and activity of central neurons. Therefore, in this study, we used acute upregulation and downregulation of the α2δ subunits to dissect their impact on the formation of structural and functional connectivity, as well as on the balance between excitation and inhibition.

Results

Constitutive KO of the α2δ1 subunit in vivo leads to reduction of excitatory synaptic density

Since α2δ1 and α2δ3 subunits are both abundant in the hippocampus in vivo and in cultured neurons (Klugbauer et al., 1999; Cole et al., 2005; Schlick et al., 2010), we chose to characterize their functional effects on network activity and connectivity in hippocampal neurons as a standard model preparation. Investigations of constitutive α2δ1 KO mice have shown that the chronic loss of α2δ1 subunits has massive impact on structure and density of synapses at least in the cortex (Risher et al., 2018). To first examine whether hippocampal glutamatergic synapses also undergo changes in the constitutive KO model of the α2δ1 subunit, we used transmission electron microscopy. We found changes in both numbers and spine morphology of asymmetric (presumably excitatory) synapses (Fig. 1A–D), with synapse density being reduced by 32% compared with WT (Fig. 1E). Quantitative immunoblotting of brain lysates from WT and α2δ1^−/− mice demonstrated that deletion of the α2δ1 subunit generally did not alter the overall expression levels of various presynaptic marker proteins, including the pore-forming subunit of Ca_V2.1 channels (Fig. 1F). These results confirm and extend recently reported alterations of cortical synapses in the same KO mouse model (Risher et al., 2018). However, the constitutive KOs of α2δ isoforms are associated with severe phenotypes (Striessnig and Koschak, 2008), such as diabetes in the α2δ1 KO mice (Felsted et al., 2017), which might obscure more specific α2δ functions and complicate the distinction between direct and compensatory effects. To brace against this possibility and to be able to alter expression of α2δ1 and α2δ3 at defined time points during development, we mostly used lentivirus-mediated overexpression and knockdown to address the role of these auxiliary subunits in defining the connectivity of neuronal networks.

α2δ1 and α2δ3 affect neuronal network activity in distinct developmental windows

To address the central question whether α2δ1 and/or α2δ3 affect synaptogenesis differently and may interfere with the balance between excitation and inhibition, we infected rat hippocampal cultures with lentiviral particles carrying HA-tagged α2δ1 or α2δ3 subunits. The HA-tag was introduced shortly after the N-terminus of the protein (Fig. 2A). The expression, surface delivery (Fig. 2B-D) and impact of tagged α2δ subunits on current properties Ca_V2.1 and Ca_V2.2 channels were tested. Tagged α2δ1 or α2δ3 subunits had no impact on the current density or voltage-dependent inactivation of channels tested by expression of Ca_V2.1 or Ca_V2.2 with the β3 subunit and tagged or untagged α2δ subunits in HEK293T cells (current density: α2δ1, HA 37.2 ± 12.4 pA/pF, n = 14; nontagged 33.0 ± 8.9, n = 15; α2δ3, HA 58.0 ± 15.3, n = 19; nontagged 57.4 ± 14.7, n = 16), or Ca_V2.2 (α2δ1, HA 27.7 ± 7.3 pA/pF, n = 11; nontagged 31.0 ± 3.8, n = 10; α2δ3, HA 140.3 ± 27.7, n = 12; nontagged 115.0 ± 21.5, n = 13; half-maximal steady-state inactivation of Ca_V2.1: α2δ1, HA −26.6 ± 2.3 mV, n = 9; nontagged −30.7 ± 3.1 mV, n = 13; α2δ3, HA −20.8 ± 1.7 mV, n = 13; nontagged −24.0 ± 1.3 mV, n = 15; Ca_V2.2: α2δ1, HA −44.0 ± 1.7 mV, n = 12; nontagged −44.7 ± 1.5 mV, n = 12; α2δ3, HA −37.0 ± 2.3 mV, n = 12; nontagged −37.4 ± 1.2 mV, n= 12). Antibodies against α2δ1 or α2δ3 subunits were suitable for biochemical detection of the proteins in Western blot analysis, but not for evaluation of the surface expression of α2δ subunits in live immunocytochemical experiments (Fig. 2B,C,E,G). Comparison of the α2δ protein levels in infected cultures to the endogenous level in control sister cultures revealed that total expression of α2δ1 or α2δ3 was significantly increased by 36% or 160%, respectively (Fig. 2E–G). These evaluating experiments encouraged us to use the viral expression of the α2δ subunits to probe whether they have a specific impact on neuronal network development and activity.

Because of the default absence of external inputs, the development of cultured neuronal networks is rather stereotypical and culminates in developmental arrest on maturation after ∼28 DIV (van Pelt et al., 2004; Bettencourt et al., 2007; Bikbaev et al., 2015). As a consequence, the spontaneous network activity emerging in neuronal cultures faithfully reflects solely intrinsic formation and maturation of the network connectivity (Fig. 3A–C) without being influenced or masked by external sensory inputs. Therefore, three cohorts of cultures grown on 60-channel MEAs were infected after 7, 14, or 21 DIV, and the spontaneous activity was recorded ∼1 week after infection (Fig. 3D). We found that upregulation of α2δ subunits differentially affected the mean firing rate. Depending on the infection time point, α2δ1 and α2δ3 subunits showed opposite (all p < 0.001, one-way ANOVA; Fig. 3E-I) effects. Upregulation of α2δ3 during second developmental week increased the neuronal firing almost fourfold by DIV14 compared with age-matched control or α2δ1-overexpressing cultures (both p < 0.001, Duncan’s test; Fig. 3E,G). In contrast, α2δ3 overexpression after DIV14 strongly suppressed neuronal firing to 21 ± 4% by DIV21, compared with the mean values in controls (p < 0.001, Duncan’s test). Overexpression of α2δ1 had no impact by DIV14 but consistently increased the mean firing rate after DIV14 compared with corresponding values in controls or α2δ3-overexpressing cultures at DIV21 (p < 0.01 and p < 0.001, respectively; Duncan’s test), with the difference being even more pronounced at DIV28 (both p < 0.001, Duncan’s test; Fig. 3F,G).

Next, we examined the impact of α2δ overexpression on the functional connectivity. For this purpose, we analyzed the occurrence rate of NBs and the burst onset lag, which reflect episodes of functional network interaction between remote neuronal clusters and synchronization of their bursting activity across the network (Bikbaev et al., 2015). At DIV14, we observed no significant change in the mean NB rate on upregulation of α2δ subunits. Intriguingly, functional network interaction at DIV21 was strongly enhanced on α2δ1 overexpression, whereas upregulation of the α2δ3 subunit led to dramatic suppression of NBs (Fig. 3H). Remarkably, the effect of α2δ3 upregulation on the synchronicity of the bursting onset was reversed during the third week in vitro: the burst onset lag was shorter at DIV14, but longer at DIV21 in comparison with respectivevalues in age-matched controls (both p < 0.01, Duncan’s test; Fig. 3I). In α2δ1-overexpressing cultures, the burst onset lag was shorter than in controls at DIV14 and DIV21 (p < 0.01 and p < 0.001, respectively; Duncan’s test).

Thus, we found that overexpression of α2δ1 and α2δ3 differentially changes the spontaneous neuronal firing and network interaction in a development-dependent manner. These results indicate that upregulation of the α2δ subunits indeed alters the excitatory-to-inhibitory balance in developing hippocampal networks and raise the question how upregulation of α2δ1 and α2δ3 affects the transmission in excitatory and inhibitory synapses.

α2δ1 subunit selectively enhances presynaptic release in excitatory and α2δ3 in inhibitory synapses

The enhancement of neuronal firing in α2δ1-overexpressing cultures (Fig. 3G) could potentially reflect a reported earlier increase in glutamate release and synapse structure (Hoppa et al., 2012; Schneider et al., 2015) or be caused by a decreased release of GABA. Similarly, the α2δ3-induced suppression of the network activity after DIV14 indicated a shift inthe excitatory-to-inhibitory balance due to either enhanced GABA release or reduced release of glutamate. To clarify this, we measured mEPSCs and mIPSCs in neurons overexpressing either α2δ1 or α2δ3 subunits. To enable recordings in developing neurons, in the following experiments, the primary hippocampal cultures were infected during first developmental week at DIV2-DIV4. Subsequently, mEPSCs and mIPSCs were recorded in the presence of TTX, APV, and either DNQX or bicuculline, respectively, at three time points between DIV7 and DIV21 (Fig. 4A). No significant effect of α2δ upregulation on miniature currents was observed in 1-week-old cultures. At DIV14 and DIV21, the mean mEPSC frequency was higher in cultures overexpressing the α2δ1, but not α2δ3, compared with control values at corresponding time points (p < 0.01 and p < 0.05, respectively; Dunn’s test; Fig. 4B,C). In striking contrast, upregulation of the α2δ3, but not α2δ1, strongly increased the mean mIPSC frequency at DIV14 and DIV21 compared with age-matched controls (p < 0.001 and p < 0.05, respectively; Dunn’s test; Fig. 4E,F). The amplitude of miniature currents was not affected by α2δ overexpression compared with control values at any time point (Fig. 4D,G). However, the mIPSC amplitude at DIV21 was significantly smaller in α2δ3-overexpressing cultures compared with cultures overexpressing the α2δ1 subunit (p < 0.05; Dunn’s test; Fig. 4G).

The stochastic opening of high VGCCs accounts for ∼50% of mEPSCs and mIPSCs (Goswami et al., 2012; Williams et al., 2012; Ermolyuk et al., 2013). Therefore, the pronounced effect of α2δ overexpression on the mEPSCs and mIPSCs after DIV14 (Fig. 4C,F) strongly suggested a bigger contribution of VGCCs to spontaneous release. Indeed, we found that acute blockade of VGCCs by cadmium (Cd²⁺) strongly decreased the frequency of miniature currents in 2-week-old cultures overexpressing the α2δ1 (mEPSCs: p < 0.001, Mann–Whitney test) or α2δ3 (mIPSCs: p < 0.001) subunit to respective control levels obtained in the presence of Cd²⁺ from noninfected cultures (Fig. 4H).

In central synapses, the neurotransmitter release is triggered predominantly by Ca_V2.1 and Ca_V2.2 (Wheeler et al., 1994; Scholz and Miller, 1995; Cao and Tsien, 2010), but their abundance at excitatory and inhibitory presynaptic terminals may vary (Iwasaki et al., 2000). To clarify whether the elevation of the mEPSC and mIPSC frequency by α2δ1 and α2δ3 subunits involves distinct subpopulations of presynaptic VGCCs, we performed additional patch-clamp recordings in the presence of isoform-specific channel blockers. In α2δ1-overexpressing cultures, the blockade of Ca_V2.2 by ω-conotoxin GVIA did not abolish the increase in the mean mEPSC frequency, but the blockade of Ca_V2.1 by ω-agatoxin IVA reduced the mEPSC frequency (p < 0.001, Dunn’s test) to a level observed in control cultures treated with agatoxin (Fig. 4I). In contrast, we found that the α2δ3 overexpression-induced increase in mIPSC frequency was abolished by conotoxin (p < 0.01, Dunn’s test), but not by agatoxin (Fig. 4J), compared with control values obtained in the presence of respective toxins.

These results revealed a selective impact of the α2δ1and α2δ3 calcium channel subunits on the spontaneous neurotransmitter release in excitatory and inhibitory synapses. Furthermore, we found that facilitation of the spontaneous glutamate release by α2δ1 is predominantly mediated by Ca_V2.1, whereas α2δ3-driven enhancementof GABA release involved mainly Ca_V2.2 calcium channels.

shRNA-mediated knockdown of α2δ1 and α2δ3 subunits mirror the effects of overexpression on neurotransmitter release and network activity

To rule out possible artifacts of overexpression or lentiviral infection and verify that the effects on neurotransmitter release and the neuronal firing are caused by the overexpression of α2δ subunits, we acutely knocked down the α2δ1 and α2δ3 subunits using specific shRNAs.

For the α2δ1 subunit, both live anti-HA labeling of HA-tagged α2δ1 subunits and Western blot analysisof the total α2δ1subunit population demonstrated strong downregulation in neurons (Fig. 5A–C). Since the most pronounced effect of the α2δ1 overexpression on the glutamate release was observed at DIV14 (Fig. 4B,C), we recorded mEPSCs in neurons at DIV14 on α2δ1 downregulation, as well as in neurons infected with GFP-expressing lentiviral particles that served as lentiviral infection control (Fig. 5D). We found that shRNA-induced α2δ1 knockdown markedly reduced the mEPSC frequency compared with noninfected controls (p < 0.05, Mann–Whitney test; Fig. 5E,F). No effect of lentiviral expression of GFP on the mEPSC frequency or amplitude was found (Fig. 5E–H).

Since the strongest effect of α2δ1 overexpression on the network activity was observed at DIV28 (Fig. 3G), in an additional set of 3-week-old cultures, we induced upregulation or downregulation of the α2δ1 subunit and assessed spontaneous neuronal firing 1 week later (Fig. 5I). In control cultures, no significant change of the firing rate was observed between DIV21 and DIV28. The upregulation of α2δ1 enhanced neuronal firing (p < 0.001, Duncan’s test), whereas the α2δ1 knockdown led to suppression of the mean firing rate compared with values in control and α2δ1-overexpressing cultures (p < 0.05 and p < 0.001, respectively; Duncan’s test; Fig. 5J,K).

Similar experiments were conducted using shRNA constructs to knock down the α2δ3 subunit. Evaluation of the construct demonstrated a robust suppression of α2δ3 subunit expression by 50%–60% in HEK cells (p < 0.001, Mann–Whitney test; Fig. 6A,B) and primary hippocampal cultured neurons (p < 0.05; Fig. 6C,D). Furthermore, the quantification of the α2δ3 expression level in neuronal cultures demonstrated a significant reduction on shRNA-mediated knockdown both in HEK cells (p < 0.001; Fig. 6E,F) and in neurons (p < 0.05; Fig. 6G–I).

Functional analysis of the α2δ3 knockdown demonstrated that higher frequency of spontaneous GABA release and the enhance neuronal network activity in young α2δ3-overexpressing cultures were indeed caused by upregulation of this auxiliary subunit. We found that the frequency of mIPSCs was markedly decreased on α2δ3 knockdown, but not GFP expression, compared with controls (p < 0.05, Mann–Whitney test; Fig. 6J–L). The amplitudes of mIPSCs where not affected in any of the groups (Fig. 6M,N). Finally, a comparison of the spontaneousactivity recorded under control conditions or on α2δ3 upregulation or downregulation (Fig. 6O) revealed that shRNA-mediated α2δ3 knockdown resulted in suppression of spontaneous neuronal firing compared with values in control or α2δ3-overexpressing cultures (p < 0.05 and p < 0.001, Duncan’s test; Fig. 6P,Q).

So far, these data revealed a selective impact of the α2δ1 as well as α2δ3 calcium channel subunit on the presynaptic neurotransmitter release in excitatory and inhibitory synapses. Given these findings, next we asked whether the elevated frequency of miniature currents on upregulation of α2δ subunits reflects corresponding changes in the number of glutamatergic and/or GABAergic synaptic contacts.

Upregulation of α2δ3 subunit selectively promotes inhibitory synaptogenesis

The α2δ1 subunit was reported earlier to trigger excitatory synaptogenesis in mouse retinal ganglion cells and cortical neurons (Eroglu et al., 2009), but it remained unknown whether α2δ3 plays a similar role in central synapses. To clarify this, we labeled the presynaptic scaffold protein Bassoon and the postsynaptic scaffold protein Homer1 or Gephyrin to identify glutamatergic and GABAergic synapses, respectively. The immunolabeling was conducted in hippocampal cultures 2-3 weeks after infection at DIV14-DIV24 (Fig. 7A). Using colocalization of presynaptic and postsynaptic markers distributed along dendrites (Fig. 7B,C), we evaluated the density of synaptic contacts per µm (for details, see Materials and Methods).

In 2-week-old cultures, we observed a moderate increase of the density of glutamatergic synapses both in α2δ1- and in α2δ3-overexpressing cultures compared with control sister cultures, but the effect was not significant (p = 0.17, Kruskal-Wallis ANOVA). After DIV21, the excitatory synapse number was significantly affected by α2δ overexpression (p < 0.001, Kruskal-Wallis ANOVA), with the synaptic density being higher in the α2δ1- and in the α2δ3-overexpressing cultures compared with control values (both p < 0.001; Fig. 7D). These data confirmed the synaptogenic potential of the α2δ1 subunit (Eroglu et al., 2009) but also showed that α2δ3 upregulation can promote excitatory synaptogenesis. More importantly, we found that overexpression of the α2δ3, but not the α2δ1, subunit significantly increased the GABAergic synapse number already at DIV14 compared with control cultures (p < 0.001, Dunn’s test; Fig. 7E). The effect of α2δ3 overexpression on the inhibitory synaptogenesis was even more pronounced in cultures after DIV21, compared with control or α2δ1-overexpressing cultures (p < 0.001 and p < 0.01, respectively; Dunn’s test). Comparison of the fluorescence intensity of presynaptic and postsynaptic scaffolds in excitatory synapses revealed no difference from the control conditions (tested for Bassoon and Homer). Within inhibitory synapses α2δ1 and α2δ3 expression increased the fluorescence intensity of Bassoon in 2- and 3-week-old cultures compared with age-matched controls (DIV14-DIV17: control 100 ± 5% n = 69, α2δ1 118 ± 5% n = 67, α2δ3 128 ± 5% n = 65/3-week-old (DIV18-DIV24: control 100 ± 5% n = 67, α2δ1 128 ± 8% n = 72, α2δ3 164 ± 11% n = 99; p < 0.01 and p < 0.001, respectively; Kruskal-Wallis ANOVA). The fluorescence of Gephyrin was markedly affected only in 3-week-old cultures (DIV18-DIV24: control 100 ± 4%, α2δ1 92 ± 4%, α2δ3 81 ± 4%; p < 0.001 Kruskal-Wallis ANOVA), with values obtained in α2δ3-overexpressing cultures being smaller than in controls (p < 0.001, Dunn’s test).

The transfection-induced overexpression of α2δ subunits triggers accumulation of presynaptic proteins via increased surface expression of VGCCs (Hoppa et al., 2012; Schneider et al., 2015), which we could not reveal in lentiviral infected cultures. This, in turn, leads to recruitment of presynaptic scaffold components when expressed in combination with the α1 subunit (Davydova et al., 2014; Schneider et al., 2015). To verify that, we assessed the fluorescence intensity of several key presynaptic proteins in hippocampal cultures transfected either with α2δ1-HA or α2δ3-HA. The transfection-induced overexpression allowed us to distinguish the HA-positive transfected synapses and HA-negative puncta of nontransfected neurons embedded into the same network. Indeed, upregulation of either α2δ1 or α2δ3 led to an enhanced accumulation of Bassoon and RIM (Fig. 7F,G; both p < 0.001) that was more pronounced for Bassoon on α2δ1 upregulation (p < 0.001, Bonferroni test). Furthermore, the upregulation of α2δ1 or α2δ3 significantly increased the fluorescence of VGlut1, indicating a structural change of excitatory synapses (Fig. 7G; both p < 0.001). Remarkably, the fluorescence of VGAT, an inhibitory synapse-specific marker, was 38 ± 9% higher only in α2δ3-overexpressing neurons compared with control or α2δ1-overexpressing neurons (p < 0.01 and p < 0.001, respectively; Fig. 7G). Consistent with previous reports (Hoppa et al., 2012; Schneider et al., 2015), upregulation of α2δ1 or α2δ3 subunits increased the synaptic abundance of Ca_V2.1 (both p < 0.001) and Ca_V2.2 (p < 0.001 and p < 0.01, respectively; Fig. 7G). No differences between α2δ1- or α2δ3-overexpressing neurons in the fluorescence intensity of either Ca_V2.1 or Ca_V2.2 were found.

These findings demonstrate that upregulation of α2δ1 or α2δ3 subunits in rat hippocampal neurons triggers the glutamatergic synaptogenesis, hence corroborating previous reports (Dickman et al., 2008; Eroglu et al., 2009; Kurshan et al., 2009). Moreover, we found that upregulation of the α2δ3, but not α2δ1, subunit increases the number of GABAergic synapses in hippocampal cultures already 2 weeks in vitro.

α2δ3 selectively promotes axonal outgrowth and branching in inhibitory neurons

Apart from mediating the synaptic inhibition, GABA is directly involved in a variety of fundamental processes, such as neuronal migration, differentiation, and axonal outgrowth, that take place before the formation of functional synapses (Owens and Kriegstein, 2002; Huang et al., 2007). Given the α2δ3-specific effect on the GABA-dependent inhibitory postsynaptic currents (Fig. 4F) and the inhibitory synaptogenesis (Fig. 7E), next we examined whether upregulation of this subunit is associated with enhanced axonal outgrowth, as it was shown for α2δ2 subunit in the spinal cord (Tedeschi et al., 2016). Therefore, we first looked at rat hippocampal cultures, which were infected with α2δ1-HA or α2δ3-HA subunits at DIV2-DIV4 and additionally transfected at DIV4 with GFP as a volume marker to aid identification of individual neurons and their processes within the network. At DIV9-DIV10, cultures were stained for MAP2 to label the dendritic arbor of individual neurons. Subsequently, the length and branching of axons, which were detected by GFP-positive but MAP2-negative signal, were analyzed using Scholl analysis and Simple Neurite Tracer plug-in for Fiji software (Longair et al., 2011) for semiautomatic reconstruction of cells (for details, see Materials and Methods). We found no significant effect of α2δ1 or α2δ3 upregulation on the mean axonal length, nor were the number of branching points markedly affected. However, individual values obtained in α2δ3-overexpressing neurons were distributed within substantially broader range (length_min-max 15%-394%, mean 121 ± 20%; branches_min-max 32%-402%, mean 124 ± 20%; n = 23 neurons), compared with control (length_min-max 36%-208%, mean 100 ± 11%; branches_min-max 32%-229%, mean 100 ± 12%; n = 21) or α2δ1-overexpressing (length_min-max15%-211%, mean 86 ± 13%; branches_min-max 11%-192%, mean 77 ± 11%; n = 21) neurons. We assumed that such heterogeneity in the dataset might reflect a mixture of values obtained in excitatory and inhibitory neurons. Therefore, we proceeded with the analysis of the axonal outgrowth and branching specifically in interneurons.

In young neurons, GAD67 is a rate-limiting enzyme responsible for up to 90% of GABA synthesis in the brain (Asada et al., 1997). In order to unequivocally identify and quantify individual interneurons, we prepared hippocampal cultures from mice expressing GFP under control of GAD67 promoter (GAD67::GFP). Cultures underwent the infection at DIV2-DIV4 and fixation at DIV9, followed by immunostaining for MAP2 to visualize the dendritic arbor as previously described. Subsequently, the length and the number of axonal branches were quantified exclusively for GFP-positive cells (i.e., for GAD67-positive interneurons) (Fig. 8A-C). In α2δ1-overexpressing interneurons, the mean axon length and the number of branches did not significantly differ from respective values obtained in control noninfected cultures. In contrast, axons of α2δ3-overexpressing interneurons were significantly longer and branched more extensively, compared with controls or α2δ1-overexpressing cultures (both p < 0.001 Dunn’s test; Fig. 8D,E; see Extended Data Fig. 8-1). These data demonstrated that upregulation of auxiliary α2δ3 subunit of calcium channels promotes the axonal outgrowth specifically in inhibitory GABAergic interneurons.

Together, our findings demonstrate that the α2δ1 or α2δ3 calcium channel subunits play an important role in several aspects of early circuitry formation in neuronal networks. The expression of both α2δ1 and α2δ3 favors the formation of synaptic connectivity. However, we found that the impact of the α2δ3 subunit is inhibitory cell type-specific, with α2δ3 upregulation being associated with enhanced GABA release, formation of inhibitory synapses, and axonal outgrowth in interneurons. Furthermore, we found that such synapse type-specific impact of α2δ1 and α2δ3 on the neurotransmitter release is associated with their functional preference for distinct VGCC isoforms.

Discussion

This study characterizes the differential impact of α2δ1 and α2δ3 auxiliary subunits of VGCCs on structural and functional properties of developing hippocampal neurons. To overcome the limitations and side effects of constitutive KO of individual subunits of calcium channels (Striessnig and Koschak, 2008), in this work we used lentiviral overexpression of α2δ subunits in cultured neuronal networks. We found that both α2δ1 and α2δ3 can trigger excitatory synaptogenesis in hippocampal neurons, whereas upregulation of only α2δ3 subunit increases inhibitory synapse number and enhances presynaptic GABA release. Using hippocampal cultures prepared from GAD67::GFP mice, we found that α2δ3 overexpression also promotes the axon outgrowth in young interneurons. Together, these findings shed new light on the earlier reported functional redundancy of α2δ1 and α2δ3 despite pronounced structural differences between these isoforms (Klugbauer et al., 1999; Dolphin, 2013), and show their differential but complementary roles in early circuitry formation.

Throughout the experiments, we implemented two infection protocols. Lentiviral infection at different developmental time points, namely, after first, second, or third week in vitro (Fig. 3D), demonstrated that α2δ subunits alter neuronal firing and network interaction in a development-dependent and subunit-specific manner. Given the isolation of neuronal cultures from external sensory inputs that drive network activity already in the early postnatal period (Khazipov et al., 2004), the suppression of activity on α2δ3 upregulation (Fig. 3G) indicated a prevalence of inhibition over excitation. In contrast, α2δ1 upregulation after the second week in vitro consistently enhanced the network activity and demonstrated a shift toward excitation on the network level. Thus, these results show that α2δ1 and α2δ3 are intimately involved into the establishment and modulation of the excitation/inhibition balance.

To characterize the long-term consequences on neurotransmitter release, in the rest of experiments, the infection wasperformed during the first week in vitro and the data were acquired within the period of DIV7 to DIV24. This protocol revealed that α2δ1 overexpression selectively enhances spontaneous presynaptic glutamate release without affecting the spontaneous release of GABA (Fig. 4C,F), whereas the knockdown of this subunit led to impairment of glutamate release (Fig. 5E,F). Such selectivity of α2δ1 in facilitation of release in excitatory synapses is consistent with previously shown localization of α2δ1 primarily in excitatory presynaptic terminals in the hippocampus (Hill et al., 1993; Bian et al., 2006; Nieto-Rostro et al., 2014) and corroborates recent reports on the positive correlation between surface expression of α2δ1 and the mEPSC frequency (Cordeira et al., 2014; Zhou and Luo, 2015). Notably, higher frequency of spontaneous glutamate release in 2-week-old α2δ1-overexpressing neurons (Fig. 4C) was not accompanied by higher synaptic density (Fig. 7D), suggesting that the elevation of the release probability precedes the synaptogenic function of α2δ1.

One of the central findings of our study is the α2δ3 overexpression-induced increase in the frequency of spontaneous GABA release (Fig. 4E,F), which was accompanied by the higher density of inhibitory synapses (Fig. 7E). Surprisingly, we found that the α2δ3 upregulation also increases the excitatory synapse density in rather mature 3-week-old cultures (Fig. 7D) without affecting the mEPSC frequency (Fig. 4B,C). Electrical activity per se in immature networks is necessary and sufficient for synaptogenesis and early circuitry formation (Ben-Ari, 2001; Spitzer, 2006) and can potently influence the development of GABAergic synapses (Chattopadhyaya et al., 2007). The enhancement of the network activity observed on overexpression (Fig. 3G), but not downregulation (Fig. 6P,Q), after DIV7 in cultures grown on MEAs could therefore indirectly trigger the formation of surplus glutamatergic synapses.

The GABA synthesis and signaling begin already at embryonic stages; thus, GABA acts as a trophic factor influencing fundamental developmental processes before it becomes a principal inhibitory neurotransmitter (Owens and Kriegstein, 2002; Ben-Ari et al., 2007; Huang et al., 2007). Although still debated in the literature, GABA in immature neurons can exert an excitatory action so that binding to GABA_A receptors results in membrane depolarization. In particular, the GABA_A receptor-mediated depolarization in young neurons was shown to be sufficient for VGCC activation (Leinekugel et al., 1995; LoTurco et al., 1995; Ganguly et al., 2001) and required for formation and/or maintenance of GABAergic synapses (Oh et al., 2016). Intriguingly, we observed a dramatic change in the effect of α2δ3, but not α2δ1, overexpression on neuronal firing depending on the developmental stage (Fig. 3G). A reversal from enhancing spontaneous network activity at DIV14 to its suppression at DIV21 likely reflected the switch to hyperpolarizing GABA action and/or formation of functional inhibitory synapses (Fig. 7E), which requires binding of GABA to GABA_Areceptors followed by aggregation of postsynaptic Gephyrin puncta (Oh et al., 2016). In line with increased GABA release (Fig. 4F), we found that the number of Bassoon puncta colocalized with Gephyrin already by the end of second developmental week was bigger in α2δ3-overexpressing cultures than in controls (Fig. 7E). These outcomes corroborate previous reports showing an important role of spontaneous Ca²⁺ transients in regulation of the neurite outgrowth and branching (Ciccolini et al., 2003) and structural maturation of synapses (Choi et al., 2014). Despite our data on the inhibitory synapses number (Fig. 7E) and GABA release (Fig. 4E,F), the finding that upregulation of α2δ3 enhances the axonal growth and branching specifically in interneurons (Fig. 8D,E) provided additional evidence for the α2δ3-specific modulation of GABA-related functions. Importantly, the outgrowth was promoted selectively in interneurons positive for GAD67, which plays major role in GABA synthesis (Asada et al., 1997), as well as in maturation of perisomatic inhibition and elimination of excessive excitatory synapses (Nakayama et al., 2012).

The α2δ subunits are known to support the trafficking of the pore-forming α1 subunit of calcium channels (Dolphin, 2012). Our results, that the elevation of frequency of neurotransmitter release in 2-week-old neurons is abolished by VGCC isoform-specific blockers (Fig. 4I,J), demonstrated a preference of α2δ1 and α2δ3 for trafficking of Cav2.1 in glutamatergic and Cav2.2 in GABAergic synapses, respectively. Agatoxin and gabapentin, but not conotoxin, were reported earlier to induce an identical nonadditive decrease in K⁺-triggered Ca²⁺ influx (Fink et al., 2000), indicating that α2δ1-mediated contribution to calcium signaling is sensitive to Cav2.1 blockade. Furthermore, our finding that α2δ1 modulates the release in excitatory synapses preferentially via P/Q-type channels is in line with strong reduction of spontaneous (Bomben et al., 2016) and evoked (Mallmann et al., 2013) release of glutamate reported in Ca_V2.1 KO mice. Although, P/Q-type channels were shown to induce synaptic recruitment of Bassoon (Davydova et al., 2014), in our study the accumulation of Bassoon was evident only in inhibitory synapses and only on α2δ3 overexpression. Since both evoked and spontaneous GABA release require presynaptic accumulation of VGCCs (Williams et al., 2012), the latter supports previous reports (Hoppa et al., 2012; Schneider et al., 2015) and suggests that both α2δ1 and α2δ3 subunits can serve as rather universal cargos of the pore-forming α1 subunit. The α2δ isoform-specific recruitment of P/Q- or N-type channels can therefore be related to different roles α2δ subunits play in the neuronal network development. Indeed, the dominant role of the α2δ3 subunit in the early and the α2δ1 subunit in the late development matches expression profiles of P/Q- and N-type calcium channels (Scholz and Miller, 1995; Iwasaki et al., 2000; Fedchyshyn and Wang, 2005). Furthermore, the effects of the α2δ1 on the mEPSC frequency and of the α2δ3 on the mIPSC frequency were more pronounced in the presence of conotoxin and agatoxin, respectively, compared with values in respective groups obtained without toxins. The latter finding corroborates the concept of functional competition of VGCC isoforms in presynaptic active zone (Cao and Tsien, 2010; Davydova et al., 2014). In this context, the surface interaction with synaptic adhesion molecules, such as α-neurexin (Missler et al., 2003), which has been suggested in several systems (Tong et al., 2017; Brockhaus et al., 2018), can be an important contributing factor for such specificity of α2δ subunits in network development.

Our data provide support for the reported association of CACNA2D1 and CACNA2D3 genetic aberrations with autism (Iossifov et al., 2012; De Rubeis et al., 2014; Vergult et al., 2015) and the high comorbidity of epilepsy in individuals with autism (Tuchman and Rapin, 2002; Levisohn, 2007). By fostering the GABAergic signaling, the α2δ3 subunit effectively drives the early network activity that is crucial for the initial circuitry formation. The impact of the α2δ1 subunit becomes prominent later in development and is rather restricted to glutamatergic signaling. One interaction partner for this action could be α-neurexin, which, together with α2δ1, facilitates the trafficking of Ca_V2.1 VGCCs to presynaptic terminals (Brockhaus et al., 2018), whereas α2δ3 may play an opposite role (Tong et al., 2017). Altered expression of α2δ1 or α2δ3 can therefore cause a chronic imbalance between excitation and inhibition that is rather characteristic for autism spectrum disorders (Rubenstein and Merzenich, 2003; Nelson and Valakh, 2015). As a consequence, impairment of α2δ-mediated functions during critical developmental periods can trigger in affected individuals devastating maladaptive changes on the network level and potentially lead to global aberrations in the brain connectivity (Baron-Cohen and Belmonte, 2005; Courchesne and Pierce, 2005) and the neural information processing (Belmonte et al., 2004).