Narp Mediates Antidepressant-Like Effects of Electroconvulsive Seizures

Abstract

Growing recognition of persistent cognitive defects associated with electroconvulsive therapy (ECT), a highly effective and commonly used antidepressant treatment, has spurred interest in identifying its mechanism of action to guide development of safer treatment options. However, as repeated seizure activity elicits a bewildering array of electrophysiological and biochemical effects, this goal has remained elusive. We have examined whether deletion of Narp, an immediate early gene induced by electroconvulsive seizures (ECS), blocks its antidepressant efficacy. Based on multiple measures, we infer that Narp knockout mice undergo normal seizure activity in this paradigm, yet fail to display antidepressant-like behavioral effects of ECS. Although Narp deletion does not suppress ECS-induced proliferation in the dentate gyrus, it blocks dendritic outgrowth of immature granule cell neurons in the dentate molecular layer induced by ECS. Taken together, these findings indicate that Narp contributes to the antidepressant action of ECT and implicate the ability of ECS to induce dendritic arborization of differentiating granule cells as a relevant step in eliciting this response.

Figure 1

Effect of ECS on the FST (a) and TST (b) in Narp KO and WT mice. The ability of ECS to decrease immobility scores in both the FST and TST is blocked by Narp deletion. Two-way ANOVAs showed a significant interaction between genotype and treatment on immobility in the FST (p<0.05, n=8–12 in each group) and the TST (p<0.01, n=10–11 in each group). Comparison of sham vs ECS-treated WT mice in FST and TST, **p<0.01 and ****p<0.0001, respectively (Fisher’s post hoc test). Bars represent means (±SEM) in this and subsequent figures. ECS, electroconvulsive seizures; FST, forced swim test; TST, tail suspension test; KO, knockout; WT, wild-type; ANOVA, analysis of variance.

Figure 2

Effect of ECS on seizure duration (a), c-Fos expression (b), locomotor activity (c), hippocampal neurogenesis (d) and hippocampal BDNF expression (e–g) in Narp KO and WT mice. (a) Duration of motor seizure activity was measured daily over a course of five ECS treatments. There was neither a significant main effect of genotype nor an interaction between genotype and treatment on seizure duration on a two-way repeated measures ANOVA (n=5 in each group). (b) c-Fos-positive neurons were counted in mice killed 1 h after the fifth daily ECS or sham treatment. Two-way ANOVAs showed a significant main effect of treatment in the BLA, dentate gyrus, NAc (shell), VMH, and mPFC. There was no significant interaction between genotype and treatment on c-Fos expression in any of the regions examined (n=3–6 in each group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 in ECS vs sham-treated mice. (c) Distance traveled in an open field over 30 min measured 48 h after the fifth daily ECS or sham treatment. A two-way ANOVA showed a significant main effect of ECS on locomotor activity (n=5 in each group). No interaction was found between treatment and genotype. (d) Hippocampal neurogenesis (represented as fold-change in BrdU-positive cells in the dentate gyrus vs sham-treated WT mice) following the fifth daily ECT or sham treatment. There was a significant main effect of treatment on BrdU-positive cells using a two-way ANOVA and no interaction between genotype and treatment (n=3–4 for each group). (e) Hippocampal Bdnf total RNA levels are represented as fold change vs sham-treated WT mice. A two-way ANOVA showed a significant main effect of ECS treatment compared to Sham on Bdnf mRNA transcripts. Notably, there was no significant effect of Narp genotype, nor an interaction effect between genotype and treatment (n=4 per group). (f) Hippocampal pro-BDNF levels were monitored by western blot and are represented as fold change vs sham-treated WT mice. A two-way ANOVA showed no significant effect of Narp deletion or ECS treatment on pro-BDNF protein levels (n=4–5 in each group). (g) Hippocampal mature BDNF levels were monitored by western blot and are represented as fold change vs sham-treated WT mice. A two-way ANOVA showed a significant main effect of ECS treatment on mature BDNF protein levels (n=12–13 per group). ****p<0.0001 in ECS vs sham-treated mice. Of note, there was no significant interaction between genotype and treatment in mature BDNF protein levels. ECS, electroconvulsive seizures; KO, knockout; WT, wild-type; ANOVA, analysis of variance; BLA, basolateral amygdala; NAc, nucleus accumbens; VMH, ventromedial hypothalamus; mPFC, medial prefrontal cortex; ECT, electroconvulsive therapy.

Figure 3

Narp modulates ECS-mediated DCX+ dendritic arborization in the molecular layer of the hippocampal dentate gyrus: (a) DCX+ cell count; (b) representative images of immunohistochemistry showing the distribution of DCX+ neurons and dendrites in the dentate gyrus; (c) representative images of inset DCX+ dendrites in the dentate gyrus; (d) DCX+ dendritic branching in the molecular layer of the dentate gyrus. (a) DCX+ cell counts were obtained within the granule layer of the dentate gyrus of WT and Narp KO mice killed 24 h after the fifth daily ECS or sham treatment. Two-way ANOVAs showed no significant main effects of either genotype or treatment (n=6 in each group). (b) Representative histological images of DCX+ immunohistochemistry staining in the hippocampal dentate gyrus for each category of genotype and treatment. Square insets are shown enlarged in (c). (c) Higher resolution representation of DCX+ dendrite distribution in the molecular layer of the hippocampal dentate gyrus. (d) The extent of DCX+ dendrite branching was quantified at the middle of the molecular layer of the hippocampal dentate gyrus (n=12 per group). Two-way ANOVA showed a significant main effect of treatment, as well as a significant interaction effect between genotype and treatment. Of note, post-hoc analysis with Fisher’s LSD showed a significant difference in WT mice treated with either sham or ECS, but no corresponding difference in Narp KO mice. ***p<0.001 in ECS vs sham-treated WT mice. ECS, electroconvulsive seizures; WT, wild-type; KO, knockout; LSD, least significant difference.

Figure 4

Effects of imipramine, fluoxetine, and ketamine on the FST and TST in Narp KO and WT mice. Immobility was scored during the last 4 min of a 6 min FST and over the entire 6 min TST. Imipramine (a,b) was administered at 20 mg/kg daily for 22 days and the TST and FST were performed 60 min following drug administration on days 21 and 22, respectively. Fluoxetine (c,d) was administered at 20 mg/kg daily for 29 days and the TST and FST were performed 60 min following drug administration on days 28 and 29, respectively. Ketamine (e,f) was administered only once at 10 mg/kg, with the TST and FST taking place approximately 23 and 29 h later, respectively. Two-way ANOVAs did not reveal an interaction between genotype and treatment in any of the cohorts (n=9–14 in each group) but showed a main effect of treatment in the TST for mice treated with imipramine and in the FST for mice treated with ketamine. **p<0.01, ****p<0.0001 vs saline-treated mice. FST, forced swim test; TST, tail suspension test; KO, knockout; WT, wild-type.