Reduced Excitability and Increased Neurite Complexity of Cortical Interneurons in a Familial Mouse Model of Amyotrophic Lateral Sclerosis

Cortical interneurons play a crucial role in regulating inhibitory-excitatory balance in brain circuits, filtering synaptic information and dictating the activity of pyramidal cells through the release of GABA. In the fatal motor neuron (MN) disease, amyotrophic lateral sclerosis (ALS), an imbalance between excitation and inhibition is an early event in the motor cortex, preceding the development of overt clinical symptoms. Patients with both sporadic and familial forms of the disease exhibit reduced cortical inhibition, including patients with mutations in the copper/zinc superoxide-dismutase-1 (SOD1) gene. In this study, we investigated the influence of the familial disease-causing hSOD1-G93A ALS mutation on cortical interneurons in neuronal networks. We performed whole-cell patch-clamp recordings and neurobiotin tracing from GFP positive interneurons in primary cortical cultures derived from Gad67-GFP::hSOD1G93A mouse embryos. Targeted recordings revealed no overt differences in the passive properties of Gad67-GFP::hSOD1G93A interneurons, however the peak outward current was significantly diminished and cells were less excitable compared to Gad67-GFP::WT controls. Post hoc neurite reconstruction identified a significantly increased morphological complexity of the Gad67-GFP::hSOD1G93A interneuron neurite arbor compared to Gad67-GFP::WT controls. Our results from the SOD1 model suggest that cortical interneurons have electrophysiological and morphological alterations that could contribute to attenuated inhibitory function in the disease. Determining if these phenomena are driven by the network or represent intrinsic alteration of the interneuron may help explain the emergence of inhibitory susceptibility and ultimately disrupted excitability, in ALS.


INTRODUCTION
Amyotrophic lateral sclerosis (ALS) is the most common and severe form of motor neuron (MN) disease. It is clinically characterized by selective loss of the upper and lower MNs in the primary motor cortex and spinal cord, resulting in progressive motor system failure and death within 3-5 years of diagnosis (Talbot, 2014;Brown and Al-Chalabi, 2017). This currently incurable disease is clinically heterogeneous and its etiology remains unknown. However, accumulating evidence from several clinical and experimental studies suggests the disease pathogenesis may center on altered regulation of MN excitability (Turner and Kiernan, 2012;Clark et al., 2015;Geevasinga et al., 2016).
In both sporadic and familial forms of ALS, patients have been found to present with neurophysiological alterations described as hyperexcitability (Vucic and Kiernan, 2006;Vucic et al., 2008;Geevasinga et al., 2015). Originating in the motor cortex and preceding detectable lower MN dysfunction and symptom onset , it is proposed that hyperexcitability enhances the susceptibility of MNs to cell death through glutamatergic excitotoxicity Eisen et al., 2017). In ALS, hyperexcitability likely results from dysfunctional inhibition exerted by GABAergic interneurons, as well as intrinsic changes to sodium (Na + ) and potassium (K + ) channel function on MNs Do-Ha et al., 2018).
Evidence for ion channel dysfunction is highlighted by a convergent hyperexcitability phenotype in patient-derived MNs, including TARDBP, C9ORF72 and superoxide-dismutase-1 (SOD1) mutation carriers (Wainger et al., 2014;Devlin et al., 2015). TDP-43 and SOD1 ALS rodent models identify changes in the excitability of MNs prior to symptoms (Fogarty et al., 2015;Handley et al., 2017). However, more recent studies recognize progressive alterations in the number and excitability of interneurons throughout the disease course in TDP-43 and SOD1 models (Zhang et al., 2016;Clark et al., 2017;Kim et al., 2017). Clinical imaging studies indicate loss of inhibitory activity is a common and early feature of cortical hyperexcitability , and a key determinant of clinical disease progression (Shibuya et al., 2016). As such, there is a growing body of evidence to suggest dysregulated inhibition, presumably mediated by cortical interneurons, may drive an excitatory/inhibitory imbalance in ALS.
Here, we focused on the potential for the familial hSOD1 G93A mutation to influence firing properties and morphology of cortical interneurons in Gad67-GFP::hSOD1 G93A cultures. Structural alteration of pyramidal neurons is demonstrated in hSOD1 G93A studies (Jara et al., 2012;Fogarty et al., 2016;Saba et al., 2016), but few studies report changes to the morphological fine structure of interneurons . Additionally, mutant SOD1 is theorized to mediate non-cell autonomous pathogenicity through perturbed function of multiple cell types early in development (Kuo et al., 2004;van Zundert et al., 2008;Martin et al., 2013;Wainger et al., 2014;Devlin et al., 2015). As such, we hypothesize that the hSOD1 G93A mutation can perturb the excitability and neurite structure of cortical interneurons in culture.

Animals
All procedures were approved by the Animal Ethics Committee of the University of Tasmania (#A0013586) and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Gad67-GFP knock-in transgenic mice (Tamamaki et al., 2003) express green fluorescent protein (GFP) under the interneuron-specific Gad67 promoter. High copy number hSOD1 G93A mice were maintained and fully backcrossed to the C57BL/6 background (Gurney et al., 1994; B6.Cg-Tg(SOD1 G93A )1Gur.J 1 , Jackson Laboratories, Bar Harbor, ME, USA).

Cortical Culture Electrophysiology
Primary cortical cultures were prepared by carefully dissecting individual mouse embryo neocortices to enrich for neurons as previously described (Brizuela et al., 2015(Brizuela et al., , 2017, with some modifications. Cells were plated at 3.5 × 10 4 cells/mm 2 for up to 12 days and genotyped for GFP (Brizuela et al., 2015) and the hSOD1 G93A mutation (Leitner et al., 2009). Whole-cell voltage and current clamp recordings were performed as previously described (Brizuela et al., 2015), with minor modifications. Glass capillaries (impedance 7-9 Ω) were filled with intracellular solution containing neurobiotin for post hoc cell identification. Voltage responses to current injection were recorded from the cell's resting potential (applying 25 pA steps for 200 ms from −100 pA to 500 pA). Recordings were filtered at 5 kHz and sampled at 10 kHz and terminated when access resistance was ≥15 MΩ. Voltage-gated sodium and potassium currents were investigated as previously described (Brizuela et al., 2015). Data was analyzed using the programs Igor (WaveMetrics, USA) and Axograph (Axograph Scientific, Australia).

Statistical Analysis
All statistical analysis was performed in GraphPad Prism (Version 6.0c, GraphPad Software La Jolla, CA, USA). Unless otherwise stated, comparisons utilized Mann-Whitney tests, after applying d'Agostino and Pearson's normality test, results expressed as median with interquartile range. Sholl analysis and current-frequency relationships used two-way ANOVA. Data was considered significant at * p < 0.05.

RESULTS
Membrane Properties and Reduced Excitability in Cultured Gad67-GFP::hSOD1 G93A Interneurons Previous studies have demonstrated that mutant hSOD1 G93A can perturb neuronal excitability during development, which may contribute to cellular vulnerability in disease (Kuo et al., 2004;van Zundert et al., 2008;Martin et al., 2013;Wainger et al., 2014;Devlin et al., 2015). Here, we focus on investigating cortical interneuron excitability in neuronal culture by examining firing patterns and membrane properties of interneurons in Gad67-GFP::hSOD1 G93A cultures.

The Morphology of Cortical Interneurons Is
Changed in Gad67-GFP::hSOD1 G93A Cultures GABAergic interneurons have dynamic axonal structures that can alter in response to changes in activity, to modify post-synaptic targets (Flores and Méndez, 2014). Given the role of interneuron morphology in shaping inhibition during development (Huang, 2009; Le Magueresse and Monyer, 2013), we determined whether there was evidence of changes to the interneuron neurite arbor in Gad67-GFP::hSOD1 G93A cultures.
Interneuron morphology was characterized by tracing the three-dimensional structure of neurobiotin-filled GFP-positive interneuron processes in WT and hSOD1 G93A cultures. Initial examination revealed a distinct increase in the complexity of hSOD1 G93A interneurons (Figure 2A). Assessing the characteristics of the interneuron neurite field with sholl analysis (intersections per concentric shell placed at 10 µm radiating outward from the soma), we identified a significant increase in the number of processes on hSOD1 G93A interneurons at 110-150 µm from the cell soma compared to WT (p < 0.05, two-way ANOVA, Bonferroni post hoc; F (1,849) = 24.77, p = 0.0001, main effect of genotype in two-way ANOVA; see Figure 2B). The distance from the soma also independently influenced the neurite arbor complexity (F (40,849) = 10.58, p = 0.0001, main effect of distance in two-way ANOVA). To investigate the increased complexity of hSOD1 G93A interneurons we next used branched structural analyses. As shown in Figure 2C, the path length was significantly increased in hSOD1 G93A interneurons (2363 µm, 1914-3020 for WT (n = 20) v 3380, 2673-4929 for hSOD1 G93A (n = 11), p < 0.05). However, there was also a significant increase in total branch numbers ( Figure 2D; 91.00, 76.65-121.0 for WT (n = 20) v 117.0, 105.1-166.7 for hSOD1 G93A (n = 11), p < 0.05) and the highest order of branches on hSOD1 G93A interneurons (Figure 2E; 10. 00, for hSOD1 G93A (n = 11), p < 0.05). These results suggest that the presence of the hSOD1 G93A mutation can result in early inhibitory arbor structural remodeling in cortical networks.

DISCUSSION
This study is the first investigation of changes in the excitability and neurite arborization of cortical interneurons in neocortical embryonic cultures from a SOD1 rodent model. The principal finding is that excitability of cultured cortical interneurons may be altered by the hSOD1 G93A mutation, and that changes to the complexity of the neurite structure accompany this effect in vitro. Specifically, attenuation of excitability is supported by differences in the current-voltage relationship between WT and hSOD1 G93A mouse interneurons, and by the observation of reduced peak outward currents in hSOD1 G93A interneurons. Interestingly, outward potassium currents are affected in MNs derived from SOD1 ALS patients (Wainger et al., 2014), and by mutant SOD1 oligomers (Zhang et al., 2017), which could account for the changes in the hSOD1 G93A cortical interneurons. However, a number of studies highlight increased excitability of cortical pyramidal neuron populations both in vivo and in vitro (Pieri et al., 2009;Fogarty et al., 2015), which could drive changes in the morphological development of cortical interneurons. The inhibitory neurite arbor can undergo activity-dependent structural remodeling (Bartolini et al., 2013;Babij and De Marco Garcia, 2016) and the level of network activity can produce subtle but significant changes to interneuron morphology (Schuemann et al., 2013). Importantly, in networks deprived of activity, cortical interneurons have been shown to extend collaterals beyond their normal projection range (Marik et al., 2010). While the data presented here are correlational not causal, altered structural and electrophysiological properties of cortical interneurons provide evidence that hSOD1 G93A could disturb the inhibitory/excitatory balance in developing neuronal networks, early in the disease pathogenesis.
Previous studies highlight progressive and differential interneuron involvement in the hSOD1 G93A mouse model Kim et al., 2017). In particular, Kim et al support altered interneuron excitability in hSOD1 G93A models, finding progressive hyperexcitability with disease progression in vivo (Kim et al., 2017). In the current study we find evidence for intrinsically hypoexcitability. These data support a role for the hSOD1 G93A mutation in the early perturbation of inhibitory neuron populations in the disease. However, to determine the effect on functional inhibition, future studies should establish if synaptic excitability is changed to compensate for abnormal firing properties which could normalize firing rates (Turrigiano, 2008). In addition, further experimentation will be required to delineate if this is the cause or consequence of perturbed excitatory neuron excitability in the disease.

DATA AVAILABILITY
All data generated or analyzed in this study are available from the corresponding author upon request.

RC
performed cortical culturing, genotyping, immunocytochemistry, data analysis and was a major contributor in writing the manuscript. MB performed electrophysiology and data analysis. CB, MB and TD contributed to experimental design, drafting and editing of manuscript. All authors read and approved the final manuscript.