Correction of vasopressin deficit in the lateral septum ameliorates social deficits of mouse autism model

Abstract

Intellectual and social disabilities are common comorbidities in adolescents and adults with MAGE family member L2 (MAGEL2) gene deficiency characterizing the Prader-Willi and Schaaf-Yang neurodevelopmental syndromes. The cellular and molecular mechanisms underlying the risk for autism in these syndromes are not understood. We asked whether vasopressin functions are altered by MAGEL2 deficiency and whether a treatment with vasopressin could alleviate the disabilities of social behavior. We used Magel2-knockout mice (adult males) combined with optogenetic or pharmacological tools to characterize disease modifications in the vasopressinergic brain system and monitor its impact on neurophysiological and behavioral functions. We found that the activation of vasopressin neurons and projections in the lateral septum were inappropriate for performing a social habituation/discrimination task. Mechanistically, the lack of vasopressin impeded the deactivation of somatostatin neurons in the lateral septum, which predicted social discrimination deficits. Correction of vasopressin septal content by administration or optogenetic stimulation of projecting axons suppressed the activity of somatostatin neurons and ameliorated social behavior. This preclinical study identified vasopressin in the lateral septum as a key factor in the pathophysiology of Magel2-related neurodevelopmental syndromes.

Trial registration: ClinicalTrials.gov NCT02205034 NCT04283578.

Keywords: Mouse models; Neuroendocrine regulation; Neuroscience; Psychiatric diseases.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Magel2 deficiency impairs electrophysiological and behavioral responses to social stimuli.

(A) Experimental paradigm. Mice are implanted with EEG electrodes on the skull to record electrophysiological activity during exploration of juveniles (novel or familiar) or objects (novel or familiar). (B) Change in EEG power spectrum during social exploration. Data (mean ± SEM) expressed as percentage relative to baseline in n = 12 Magel2+/+, 13 Magel2+/–p. One-way ANOVA: effect of trials on each group F5,125 = 16.47, P < 0.0001; post hoc Sidak test. (C) Time exploring a mouse. Data (mean ± SEM) in n = 14 Magel2+/+, 16 Magel2+/–p mice. Two-way ANOVA: genotype F1,28 = 2.55, P = 0.001; social trials F2,56 = 6.82, P = 0.002; genotype × social trials F2,56 = 4.69, P = 0.01; post hoc Sidak test. (D) Change in EEG power spectrum during nonsocial exploration. Data (mean ± SEM) expressed as a percentage relative to baseline in n = 8 Magel2+/+, 8 Magel2+/–p. One-way ANOVA: effect of trials on each group F5,125 = 5.69, P < 0.0001, post hoc Sidak test. (E) Time exploring an object. Data (mean ± SEM) in n = 9 Magel2+/+, 9 Magel2+/–p mice. Two-way ANOVA: genotype F1,16 = 0.4, P = 0.5; object trials F2,32 = 28.33, P < 0.0001; genotype × object trials F2,32 = 0.09, P = 0.9.

Figure 2. Magel2 deficiency increases c-Fos expression in SST neurons of the dorsal LS during social exploration.

(A) c-Fos induction representative of n = 5 Magel2+/+ mouse interacting with a new conspecific for 5 minutes, euthanized 15 minutes later (left) compared with 5 Magel2+/+ mice interacting with a new object in the same conditions (right). (B) c-Fos induction in the septum representative of the mice making 0 to 5 social trials analyzed in panel C. All mice were euthanized 15 minutes after the last trial. (C) Number of c-Fos+ cells/mm2 in a total of 28,072 Magel2+/+ and 38,534 Magel2+/–p cells. Mean ± SEM of n = 5 controls, 5 novelty, 4 habituation, 4 discrimination Magel2+/+ and 7, 5, 4, 9 Magel2+/–p mice, respectively. Two-way ANOVA for social trials in Magel2+/+F3,56 = 31.7, P < 0.0001; in Magel2+/–pF3,88 = 6.871, P = 0.0003; septal regions in Magel2+/+F3,56 = 2.95, P = 0.04; and in Magel2+/–pF3,56 = 2.39, P = 0.07; post hoc Dunnett test results as indicated. Multiple comparison between genotypes by Wilcoxon unpaired t test at novelty in medial septum (MS) t(8) = 3.5, P = 0.007 and LSV t(8) = 2.8, P = 0.02; at habituation in MS t(6) = 2.76, P = 0.032. (D) Percentage of SST cells with c-Fos expression in the LSD of Magel2+/+ and Magel2+/–p mice (stars mark double-positive cells). Mean ± SEM of n = 7 controls, 6 novelty, 7 habituation, 7 discrimination, 5 novelty + 2 hours for Magel2+/+ and 7, 5, 6, 6, 5 for Magel2+/–p mice, respectively. Two-way ANOVA for genotype F1,52 = 14.05, P = 0.0004; trials F4,52 = 18.36, P < 0.0001; post hoc Sidak test results as indicated. Arrows mark euthanization 15 minutes after the last trial. (E) Percentage of SST cells with c-Fos expression in the LSD of Magel2+/+ mice (stars mark double-positive cells). Mean ± SEM of n = 5 NaCl, 5 AVP, 4 TGOT mice injected in dorsal LS via bilateral cannula and euthanized 15 minutes later. Kruskal-Wallis test, P = 0.0012, post hoc Dunn test results as indicated. LSD, lateral septum dorsal; LSV, lateral septum ventral.

Figure 3. Magel2 deficiency decreases AVP receptor levels in SST neurons of the dorsal LS.

(A) Regional precision of 1 nM [125I]LVA binding without (total) and with (nonspecific) unlabeled AVP in excess on frozen brain sections of Magel2+/+ and Magel2+/–p mice. Data (mean ± SEM) expressed as a percentage relative to Magel2+/+ in n = 8 Magel2+/+ and 7 Magel2+/–p mice. Two-way ANOVA: genotype F1,26 = 2.08, P = 0.16; effect of subregions in LS F1,26 = 0.13, P = 0.71; post hoc Sidak test for comparisons. (B) Cellular precision of 50 μM d[Lys(Alexa Fluor 647)8]VP binding without (total, n = 4 mice) and with (nonspecific, n = 3 mice) 100 μM Manning compound injected directly into the LS of Magel2+/+ mice. OXTR competitor (5 μM TGOT, n = 5 mice) coinjected with d[Lys(Alexa Fluor 647)8]VP to specify AVPR binding sites representative of 3 independent experiments. Scale bar: 1 mm. Binding affinity for AVPR subtypes presented in Supplemental Table 1. (C) Coexpression of AVPR binding sites with the indicated markers. Arrows point to cells with “bright” labeling and arrowheads to cells with “dim” labeling. Scale bars: 25 μm. Percentage of cells coexpressing AVPR and cell markers are indicated. (D) Proportion of cell types with AVPR binding sites for d[Lys(Alexa Fluor 647)8]VP in dorsal LS. Data (mean ± SEM) expressed as percentage relative to Magel2+/+ in n = 6 Magel2+/+ and 6 Magel2+/–p mice. Three-way ANOVA for cell type F2,59 = 37.27, P < 0.0001; AVPR binding F1,59 = 49.85, P < 0.0001; genotype F1,59 = 1.83, P = 0.18; cell type × AVPR binding × genotype F2,59 = 5.35, P = 0.0073; post hoc Sidak test results as indicated.

Figure 4. Magel2 deficiency reduces neuronal response to AVP in the dorsal LS.

(A) Typical firing patterns of dorsal LS neurons in coronal slices at baseline and upon 2-minute bath application of 1 μM AVP (whole cell recordings). Responses representative of more than 5 experiments reverted to baseline after washout. (B) Frequency of action potentials in AVP-excited cells. Mean ± SEM expressed as percentage relative to baseline prior to AVP stimulation (gray bar). Numbers indicate cells excited (blue) and insensitive to AVP (black). Three-way ANOVA for time F30,2259 = 9.5, P < 0.0001; AVP F1,76 = 16.9, P < 0.0001; genotype F1,76 = 1.2, P > 0.2; time × AVP F30,2259 = 8.6, P < 0.0001; time × AVP × genotype F30,2259 = 1.04, P = 0.4; post hoc Tukey test. (C) Frequency of action potentials in AVP-inhibited cells. Mean ± SEM expressed as percentage relative to baseline. Numbers indicate cells excited (blue) and insensitive to AVP (black, same as in panel B). Three-way ANOVA for time F30,2077 = 2.55, P < 0.0001; AVP F1,71 = 4.9, P = 0.03; genotype F1,71 = 0.6, P > 0.4; time × AVP F30,2077 = 3.05, P < 0.0001; time × AVP × genotype F30,2077 = 0.4, P > 0.9; post hoc Tukey test. (D) Proportion of cells categorized according to their responses to AVP on the frequency of action potentials recorded in loose patch (n = 32 Magel2+/+, 26 Magel2+/–p cells) or whole cell (n = 103 Magel2+/+, 45 Magel2+/–p cells) configurations. Comparisons between Magel2+/+ and Magel2+/–p: χ2(2) = 11.47, **P = 0.003 and χ2(2) = 13.22, **P = 0.0013. (E) AVP-inhibited cell representative of at least 2 independent experiments marked with cadaverine dye injected in the patch pipette and labeled with SST antibodies. (F) Effect of AVP on the frequency of synaptic events in dorsal LS neurons from Magel2+/+ mice representative of more than 5 experiments. Effect sizes are presented in Table 1.

Figure 5. Restoration of social discrimination in Magel2+/–p mice by AVP injection in the dorsal LS.

(A) Experimental setup. (B) Change in EEG power spectrum with intraseptal injection of AVP (30 μM for 9 minutes starting 5 minutes before the first social trial) in Magel2+/+ mice. Data (mean ± SEM) of n = 13 NaCl and 10 AVP. Two-way ANOVA for AVP for 10 minutes: F1,650 = 8.13, P = 0.004 and for 60 minutes: F1,650 = 15.5, P < 0.0001; post hoc Sidak test for pairwise comparisons between NaCl and AVP groups as indicated. (C) Time exploring a mouse upon injection of AVP (30 μM for 9 minutes starting 5 minutes before the first social trial) in dorsal LS of Magel2+/–p mice. Data (mean ± SEM) in n = 14 NaCl, 16 AVP-injected mice. Two-way ANOVA for social trials F2,42 = 17.99, P < 0.0001; AVP F1,21 = 0.03, P = 0.8; post hoc Sidak test comparing consecutive trials as indicated. (D) Change in EEG power spectrum with intraseptal injection of AVP during behavior. Data (mean ± SEM) of n = 14 NaCl and 9 AVP Magel2+/–p mice. Two-way ANOVA for AVP at novelty F23,504 = 1.27, P = 0.17 and at discrimination F23,480 = 1.74, P = 0.018; post hoc Sidak test for pairwise comparisons between NaCl and AVP groups as indicated. (E) Time exploring a mouse upon injection of Manning compound (MC, 10 nM for 9 minutes starting 5 minutes before the first social trial) in dorsal LS of Magel2+/+ mice. Data (mean ± SEM) in n = 15 NaCl, 11 MC Magel2+/+ mice. Two-way ANOVA: social trials F2,48 = 11.12, P = 0.0001; MC F1,24 = 2.46, P = 0.12; post hoc Dunnett test for pairwise comparison between consecutive trials as indicated. (F) Change in EEG power spectrum with intraseptal injection of MC during behavior. Data (mean ± SEM) expressed as percentage relative to baseline in n = 17 NaCl, 11 MC. Two-way ANOVA for social novelty F25,650 = 2.83, P < 0.0001; MC F1,650 = 2.56, P = 0.1; social discrimination F25,676 = 0.6, P = 0.9; MC F1,676 = 31.3, P < 0.0001; post hoc Sidak test for pairwise comparison between NaCl and MC groups as indicated.

Figure 6. Magel2 deficiency impairs the number of fibers projecting in the dorsal LS and activation of the PVN AVP neurons by social stimuli.

(A) Neurophysin II (AVP) immunoreactivity and semiautomated fiber tracing in the septum representative of the number of mice analyzed in panel B. (B) Number of AVP fibers in 5 Magel2+/+, 5 Magel2+/–p mice sorted as a function of length. Kolmogorov-Smirnov test, P < 0.0001. (C) Induction of c-Fos in AVP neurons of the paraventricular hypothalamic nucleus (PVN), lateral hypothalamus (LH), supraoptic nucleus (SON), and bed nucleus of stria terminalis (BNST) after social novelty representative of the number of mice analyzed in panel D. Scale bars: 50 μm. (D) Percentage of AVP neurons expressing c-Fos after social tasks (euthanized 15 minutes after trial 5). Mean ± SEM of n = 8 controls, 8 novelty, 12 habituation, 7 discrimination Magel2+/+ and 9, 8, 5, 5 Magel2+/–p mice, respectively. Two-way ANOVA for social trials in Magel2+/+F3,96 = 4.34, P = 0.006 and in Magel2+/–pF3,76 = 2.07, P = 0.11; regional AVP neurons in Magel2+/+F3,96 = 23.76, P < 0.0001 and in Magel2+/–pF3,75 = 1.76, P = 0.16; social trials × regional AVP neurons in Magel2+/+F9,96 = 4.36, P < 0.0001 and in Magel2+/–pF9,75 = 1.17, P = 0.32; post hoc Dunnett test results as indicated. Multiple comparison between Magel2+/+ and Magel2+/–p by Sidak test in the PVN t(8) = 3.05, P = 0.038 and in the BNST t(6) = 4.5, P = 0.0002 at novelty.

Figure 7. Correction of social disabilities in Magel2-deficient mice with optogenetic stimulation of AVP neurons in the BNST/LS pathway correlates with the deactivation of SST neurons in the dorsal LS.

(A) Viral strategy to express ChR2 in AVP neurons of 2 distinct regions with established projections in the LS where optic fibers were implanted to manipulate AVP responses during social behavior. (B) Expression of Cre-dependent ChR2-YFP in AVP neurons in the BNST or in the PVN of Magel2+/–p mice. Percentage (mean ± SEM) of AVP neurons expressing ChR2-YFP in BNST or PVN of 6 Magel2+/–p mice per group. (C) Time exploring a mouse upon stimulation of ChR2 with blue light (473 nm, 20 Hz, 5 ms pulses for 9 minutes during social novelty) in dorsal LS projections from AVP neurons of the BNST or the PVN in Magel2+/–p mice. Mean ± SEM of n = 6 no light, 6 blue light for the BNST/LS groups and 9 no light, 11 blue light for the PVN/LS groups. Two-way ANOVA for the BNST/LS pathway: effect of social trials F2,10 = 17.11, P = 0.0006 and interaction with CHR2 stimulation F2,10 = 3.6, P = 0.06; for the PVN/LS pathway: effect of social trials F2,36 = 9.17, P = 0.0006 and interaction with ChR2 stimulation F2,36 = 4.19, P = 0.02; post hoc Sidak test for pairwise comparisons. (D) Change in EEG power spectrum with intraseptal stimulation of ChR2 during social novelty. Mean ± SEM expressed as percentage relative to baseline in n = 11 no light, 6 blue light. Two-way ANOVA for the effect of blue light on frequency at T1 F1,390 = 18.27, P < 0.0001 and at T5 F25,400 = 1.73, P = 0.016. (E) Percentage of SST neurons expressing c-Fos in dorsal LS 15 minutes after the social discrimination trial (time of euthanization). Stars mark colabeled cells. Mean ± SEM of 6 Magel2+/–p mice per group. Kruskal-Wallis test, P = 0.011; post hoc Dunn test results as indicated.