Ablation of Type III Adenylyl Cyclase in Mice Causes Reduced Neuronal Activity, Altered Sleep Pattern, and Depression-like Phenotypes

Abstract

Background: Although major depressive disorder (MDD) has low heritability, a genome-wide association study in humans has recently implicated type 3 adenylyl cyclase (AC3; ADCY3) in MDD. Moreover, the expression level of AC3 in blood has been considered as a MDD biomarker in humans. Nevertheless, there is a lack of supporting evidence from animal studies.

Methods: We employed multiple approaches to experimentally evaluate if AC3 is a contributing factor for major depression using mouse models lacking the Adcy3 gene.

Results: We found that conventional AC3 knockout (KO) mice exhibited phenotypes associated with MDD in behavioral assays. Electroencephalography/electromyography recordings indicated that AC3 KO mice have altered sleep patterns characterized by increased percentage of rapid eye movement sleep. AC3 KO mice also exhibit neuronal atrophy. Furthermore, synaptic activity at cornu ammonis 3-cornu ammonis 1 synapses was significantly lower in AC3 KO mice, and they also exhibited attenuated long-term potentiation as well as deficits in spatial navigation. To confirm that these defects are not secondary responses to anosmia or developmental defects, we generated a conditional AC3 floxed mouse strain. This enabled us to inactivate AC3 function selectively in the forebrain and to inducibly ablate it in adult mice. Both AC3 forebrain-specific and AC3 inducible knockout mice exhibited prodepression phenotypes without anosmia.

Conclusions: This study demonstrates that loss of AC3 in mice leads to decreased neuronal activity, altered sleep pattern, and depression-like behaviors, providing strong evidence supporting AC3 as a contributing factor for MDD.

Keywords: ADCY3); Ciliopathies; Major depressive disorder (MDD); Neuronal activity; Primary cilia; Sleep alteration; Type III adenylyl cyclase (AC3.

Fig. 1

AC3 KO mice (AC3^−/−) exhibit depression-like behaviors. (A) Tail suspension test. The time that AC3 KO mice were immobile was significantly greater than AC3 WT littermates (AC3^+/+). (B) Forced swim test. AC3 KO mice were immobile for greater periods of time than AC3 WT mice during the forced swim test. (C) 3-chamber sociability test. AC3 KO mice spend less time in the chamber with interacting target and more time in chamber without target than AC3 WT mice. (D and E) Novelty-suppressed feeding test. The latency to feed in a novel environment was greater in AC3 KO mice than AC3 WT mice (D). The total feeding time for AC3 KO mice was significantly less than AC3 WT mice (E). (F and G) Novelty-suppressed drinking test. The latency of drinking was greater in AC3 KO mice than AC3 WT mice (F). The total feeding time of AC3 KO mice was significantly less than AC3 WT mice (G). (H) The coat score of AC3 KO mice was lower than WT mice. (I–L) Nesting behavior in home cage (I and J) and in a novel environment (K and L). Representative photos of nests were shown (I and K) and scores of nest at 4 different time points were analyzed (J and L). AC3 KO mice were slow in building nest and made bad nest compared to AC3 WT mice in home cages (J). The nesting behavior of AC3 KO mice was exacerbated in a novel environment (L). For all tests, n = 7–14; * p < 0.05; ** p< 0.01.

Fig. 2

AC3 KO mice exhibit increased amounts of REM sleep and have altered NREM sleep. (A) Percentage time spent awake, in NREM sleep and REM sleep in 24 hours. Inset is a mouse picture during EEG/EMG recording. (B) Time spent awake (accumulated time in 4-h intervals). (C) Time spent in NREM sleep (accumulated time in 4-h intervals). (D) Time spent in REM sleep (accumulated time in 4-h intervals). Nighttime is indicated by a shaded background. ZT0, onset of the light phase; ZT12, onset of dark phase. (E) Representative EEG and EMG sweeps of NREM sleep of AC3 WT and KO mice. (F) Power analysis (in 0.1 Hz bin) of NREM sleep over 24-h. Thin lines are power spectrum from individual mice. Black is for AC3 WT and red for AC3 KO mice. Squares (AC3 WT, black) and circles (AC3 KO, red) are averaged power spectrum from 8–9 mice. (G) Peak power (power at peak frequency) of NREM sleep of AC3 WT and AC3 KO mice. (H) Total delta power (summed power from 0–4.5 Hz frequency, 0.1 Hz bin) of NREM sleep. n = 8–9; **p<0.01.

Fig. 3

AC3 KO mice demonstrate neuronal atrophy. (A–C) AC3 KO mice have smaller hippocampal volume. The Nissl-stained hippocampus at the comparable location from AC3 WT and AC3 KO mice (A) with a 150-μm grid overlay used for Cavalieri volume estimations. Insets show representative whole hippocampal images with a lower magnification. Scale bar, 200 μm. CA1, closed circles; DG (dentate gyrus), open circles. (B) CA1 volume is smaller in AC3 KO mice than that of AC3 WT mice. (C) The volume of dentate gyrus is smaller than in mice. n = 6 pairs; **, p< 0.01. (D–E) Primarily cultured cortical neurons from AC3 KO mice have fewer dendritic arborization. (D) Representative image of cortical neurons at 12 days in vitro from AC3 WT and AC3 KO mice. Green: anti-GFP staining. Red arrow indicates the stained AC3 in the primary cilium (left, inset), which was absent in AC3 KO mice (right). (E) Sholl analyses revealed the extent of arborization of cultured cortical neurons. Two-way ANOVA, F (1, 1500) = 462, p<0.0001. Cultured neurons were from 5 AC3 WT mice and 4 AC3 KO mice.

Fig. 4

CA3-CA1 synaptic activity is reduced in hippocampal slices from AC3 KO mice. (A–C) Evoked electrical responses in CA3-CA1 synapses of AC3 KO mice were markedly smaller than those from AC3 WT mice. Synaptic responses were electrically elicited with varying stimulation intensity (0–150 μA). (A) Representative synaptic responses (super-imposed) of AC3 WT (left) and KO (right) mice. (B) Slope of field EPSP plotted against stimulus intensity. (C) Field EPSP slope plotted against the amplitude of presynaptic volley. n = 16 for AC3 WTs and n = 19 for AC3 KO mice *p<0.05; **p<0.01. (D–F) Spontaneous excitatory postsynaptic current (sEPSC) recorded from CA1 pyramidal neurons in whole cell patch-clamp recordings. (D) Representative sEPSC recorded from CA1 pyramidal layer; right, AC3 WT; left, AC3 KO. (E and F) Cumulative plot and average values (insets) of sEPSC amplitude (E) and frequency (F). n = 7 for AC3 WT and n = 9 for AC3 KO. *** p< 0.001 by Kolmogorov-Smirnov test, **p< 0.01 by t-test. (G) The resting membrane potential of CA1 pyramidal neuron of AC3 KO mice was more hyperpolarized than that of AC3 WT mice. n = 8 per genotype, * p< 0.05. (H–K) CA1 neurons from AC3 KO mice are less responsive to foot shock stimulation. (H) AC3 WT and AC3 KO mice were injected with AAV1 expressing a calcium indicator GCaMP3 into CA1 region. Representative image of CA1 neurons stained with anti-GFP (green, recognizing the GCaMP3) and anti-AC3 antibodies. Primary cilia marker of AC3 (arrows) was present in AC3 WT mice but absent in AC3 KO mice. (I) Representative full-field images of CA1 region in free-running mice using FFE. (J) 3 panels: zoom-in representative images before, during and after foot shock. Some neurons responded to foot shock (red arrows). (K) Time course of fluorescence intensity of 12 randomly selected cells by FFE calcium imaging. Foot shock induced calcium spikes (changed fluorescence intensity). The increase in the fluorescence in response to foot shock was much smaller in AC3 KO mice (right) compared to WT mice (left). n = 4 pairs of mice. 12–24 neurons from each mouse were analyzed.

Fig. 5

AC3 KO mice exhibit deficits in spatial navigation and suppressed Long-lasting LTP (L-LTP). (A) Learning curve for the hidden platform test in the Morris water maze. Data are averages of 3 trials each day. (B) Time in each quadrant during the probe test. Inset: representative swimming pattern during the probe test. (C) Number of target site crossings during the probe test. (D–E) Suppressed L-LTP in mice. Decremental LTP (D-LTP) was induced with one train of tetanus stimulation (D), while L-LTP was induced by 4 trains of tetanus stimulation (E). Top insets: representative field EPSPs before and at 60 min (D-LTP) or at 150 min (L-LTP) after LTP induction. D-LTP of AC3 KO mice was comparable to AC3 WT mice, (Two-way ANOVA test comparing data points of last 10 min, genotype effect, F (1, 117) = 0.53, p=0.48), while L-LTP was attenuated in AC3 KO mice (Two-way ANOVA test comparing data of last 20 min, genotype effect, F (1, 216) = 101, p<0.0001).

Fig. 6

AC3 forebrain KO mice exhibit pro-depression behaviors. From A–K, WT (AC3 control mice): AC3^+/+:Emx1-Cre+; Forebr. KO (AC3 Forebrain KO mice): AC3^fr/fr: Emx1-Cre+. (A) AC3 Forebrain KOs have normal grow rate (left) and EMX1-promoter driven Cre recombinase was expressed specifically in the forebrain as monitored by a Td-Tomato Cre-reported stain (middle and right). (B) Tail-suspension test. The time that AC3 forebrain KO mice were immobile was significantly greater than controls. (C) Forced swim test. AC3 forebrain KO mice were immobile for longer periods of time than controls during the forced swim test. (D) Novelty-suppressed feeding test. The latency to feed in a novel environment was greater in AC3 forebrain KO mice than control mice. (E) Mouse activity during day and during the night. AC3 forebrain KO mice had more activity than control mice at night time. (F) Open field test. The distance moved by AC3 forebrain KO mice in an open field was longer than in control mice. (G) 3-chamber sociability test. AC3 forebrain KO mice and littermate controls mice spent comparable time in each chamber. (H) Elevated plus maze test. AC3 forebrain KO mice and littermate controls spent comparable time in the open arm in the elevated-plus maze test. (I–K) AC3 forebrain KO mice had reduced recognition and spatial memory. (I) Novel objection recognition test. AC3 forebrain KO mice and control mice spent similar time in exploring A1 and A2 during training, but AC3 forebrain KO mice didn’t recognize the familiar object as well as control mice during the test. (J–K) Morris water maze test. (J) Learning curve for the hidden platform test in the Morris water maze. Data are averages of 3 trials each day. AC3 forebrains KO mice were slower to identify the hidden platform than control mice. Two-way ANOVA with Bonferroni post tests, genotype effect, F (1, 92) = 9.99, p=0.004, data were from n=12 control mice and 13 AC3 forebrain KO mice. (K) Time in each quadrant during the probe test. Inset: representative swimming pattern during the probe test. For all tests except section J, n = 6–14; n.s. not significant, * p < 0.05; ** p< 0.01 by Student t test.

Fig. 7

AC3 inducible KO mice demonstrate depression-like phenotypes. From A–K, Ctrl1: AC3^fr/fr: no-Cre, tamoxifen-treated mice; Ctrl2: AC3^fr/fr:Ubc-Cre, vehicle-treated mice; Induci. KO: AC3^fr/fr:Ubc-Cre, tamoxifen-treated mice. (A) AC3 inducible KO mice retained odor-sensing ability. Olfactory habituation/dishabituation test. w1, w2, and w3: the first, second, third water Q-tip exposure; three times of citralva (denote as C in Figure), three times male mouse urine (U), and three times eugenol (E). Q-tips were exposed subsequently. AC3 inducible KO mice and controls spent comparable time to sniff most of odorant Q-tips except that AC3 inducible KO mice sniffed shorter during the citralva exposure. Data were from 8 vehicle-treated control mice and 8 tamoxifen-treated inducible KO mice. (B) The electroolfactogram (EOG) responses in the main olfactory epithelia of AC3 inducible KO mice were similar to controls. Field potential amplitude of EOG responses at different sites in turbinates I, II, and III are shown. D, dorsal; M, middle. Inset shows representative traces of EOG recording (site: middle of turbinate II) from control mice and AC3 inducible KO mice. Data were from 6 vehicle-treated control mice and 7 tamoxifen-treated inducible KO mice. (C) Tail-suspension test. The time that AC3 inducible KO mice were immobile was significantly greater than controls. (D) Forced swim test. AC3 inducible KO mice were immobile for longer periods of time than controls during the forced swim test. (E) Novelty-suppressed feeding test. The latency to feed in a novel environment was slightly longer in AC3 inducible KO mice than control mice. (F) Nesting behavior. The score of nest built by AC3 inducible KO mice was lower than that of their controls. (G) The coat score of AC3 inducible KO mice was lower than controls. (H) Mouse activity during day/night. AC3 inducible KO mice had more activity than control mice during night time. For experiments from section C–H: n = 7–13, * p < 0.05; ** p< 0.01; *** p< 0.001 by one way ANOVA with Tukey post hoc tests. (I–K) AC3 inducible KO mice had altered sleep architecture. (I) Representative normalized EMG power of neck muscle in 3 days/nights. Top, Control mice (AC3^fr/fr:Ubc-Cre, vehicle-treated); bottom, AC3 inducible KO mice (AC3^fr/fr:Ubc-Cre, tamoxifen-treated). (J–K) Accumulated time (in 4 hour intervals) spent awake (J) and NREM (K) were plotted against zeitgeber. Nighttime is indicated by a shaded background. ZT0, onset of the light phase; ZT12, onset of dark phase. * p < 0.05 by Student t-test. EEG/EMG data were from 6 vehicle-treated control mice and 8 tamoxifen-treated inducible KO mice.