Abstract

Large-scale genome-wide association studies (GWASs) have associated intronic variants in PDE4B, encoding cAMP-specific phosphodiesterase-4B (PDE4B), with increased risk for post-traumatic stress disorder (PTSD), as well as schizophrenia and substance use disorders that are often comorbid with it. However, the pathophysiological mechanisms of genetic risk involving PDE4B are poorly understood. To examine the effects of PDE4B variation on phenotypes with translational relevance to psychiatric disorders, we focused on PDE4B missense variant M220T, which is present in the human genome as rare coding variant rs775201287. When expressed in HEK-293 cells, PDE4B1-M220T exhibited an attenuated response to a forskolin-elicited increase in the intracellular cAMP concentration. In behavioral tests, homozygous Pde4bM220T male mice with a C57BL/6JJcl background exhibited increased reactivity to novel environments, startle hyperreactivity, prepulse inhibition deficits, altered cued fear conditioning, and enhanced spatial memory, accompanied by an increase in cAMP signaling pathway-regulated expression of BDNF in the hippocampus. In response to a traumatic event (10 tone–shock pairings), neuronal activity was decreased in the cortex but enhanced in the amygdala and hippocampus of Pde4bM220T mice. At 24 h post-trauma, Pde4bM220T mice exhibited increased startle hyperreactivity and decreased plasma corticosterone levels, similar to phenotypes exhibited by PTSD patients. Trauma-exposed Pde4bM220T mice also exhibited a slower decay in freezing at 15 and 30 d post-trauma, demonstrating enhanced persistence of traumatic memories, similar to that exhibited by PTSD patients. These findings provide substantive mouse model evidence linking PDE4B variation to PTSD-relevant phenotypes and thus highlight how genetic variation of PDE4B may contribute to PTSD risk.

Significance Statement

Human genetic studies have associated variants in the PDE4B gene, encoding the phosphodiesterase-4B (PDE4B) enzyme, with increased risk for post-traumatic stress disorder (PTSD) and other mental disorders that often occur with it. However, the underlying biological mechanisms of genetic risk involving PDE4B are poorly understood. To examine the effect of PDE4B variation on behaviors relevant to mental disorders, we studied male Pde4bM220T mice that carry a PDE4B variant (M220T), which is also present in humans. Pde4bM220T mice exhibited increased PTSD-like behavior in response to a traumatic event, as well as abnormal neuronal activity in the brain. Our findings provide substantive evidence linking PDE4B variation to PTSD-relevant behaviors and thus highlight how genetic variation of PDE4B may contribute to PTSD risk.

Introduction

Psychiatry has lagged behind other medical fields in mechanistic understanding and the development of improved therapeutics. However, recent large-scale genome-wide association studies (GWASs) are beginning to elucidate the etiology and pathophysiology of psychiatric disorders by identifying genes containing common single nucleotide polymorphisms (SNPs) that increase the risk for disease at genome-wide significance (GWS) levels. PDE4B, encoding cAMP-specific phosphodiesterase-4B (PDE4B), is notable among GWS risk genes by virtue of its pleiotropic effects across diagnostic boundaries.

In a GWAS of post-traumatic stress disorder (PTSD) and other stress-related diagnoses, PDE4B was the only GWS risk locus identified (Meier et al., 2019). A GWAS of re-experiencing symptoms (involuntary retrieval of traumatic memories), a distinctive feature of PTSD, in military veterans identified PDE4B as 1 of 30 genes that reached GWS levels (Gelernter et al., 2019). In a subsequent meta-analysis of 88 PTSD GWAS datasets, PDE4B was among 43 GWS genes prioritized as likely causal (Nievergelt et al., 2024). Transcriptome profiling revealed that PDE4B mRNA expression levels in blood were lower in PTSD patients than those in controls and significantly correlated with the severity of re-experiencing symptoms and trait anxiety and with PDE4B DNA methylation levels (Hori et al., 2024).

The majority of individuals with PTSD have one or more comorbid psychiatric disorders (Kessler et al., 1995). A cross-trait meta-analysis of GWAS datasets identified PDE4B as one of five GWS risk loci shared by anxiety and stress-related diagnoses (including PTSD) and major depressive disorder (Mei et al., 2022). In a GWAS of schizophrenia, PDE4B was 1 of 106 protein-coding genes prioritized as likely causal (Trubetskoy et al., 2022). PDE4B was also among the top 3 of 42 GWS risk genes identified in a GWAS meta-analysis of general addiction risk derived from substance use disorders (Hatoum et al., 2023).

Finding loci statistically associated with increased risk of psychiatric disorders is merely the start of a long process toward meaningful biological understanding, let alone better treatments. The causal variants that drive the statistical associations, and their biological consequences, are yet to be identified, and thus the pathophysiological mechanisms of genetic risk involving PDE4B are poorly understood. PDE4B is one of four subfamilies of PDE4 enzymes (PDE4A-D) that hydrolyze the phosphodiester bond of cAMP, a key intracellular signaling molecule. Highlighting its physiological importance, PDE4B is classified by the Genome Aggregation Database (gnomAD) as extremely loss-of-function intolerant (Chen et al., 2024).

PDE4B is expressed as multiple isoforms (PDE4B1–5) via mRNA splicing, and each isoform contains a highly conserved C-terminal catalytic domain and either one or two N-terminal regulatory domains termed Upstream Conserved Region 1 and 2 (UCR1 and 2). The long forms PDE4B1, PDE4B3, and PDE4B4 contain both UCR1 and UCR2, the short form PDE4B2 lacks UCR1, and the supershort form PDE4B5 additionally lacks part of UCR2 (Fig. 1A; Cedervall et al., 2015). All five isoforms are present in adult mammalian brain tissue (Bunnage et al., 2015), and single-cell RNA sequencing has detected PDE4B mRNA in nearly all subclasses of GABAergic inhibitory neurons and glutamatergic excitatory neurons and in some types of glial cell (Armstrong et al., 2024). The lead SNPs for stress-related disorders (Meier et al., 2019; Nievergelt et al., 2024) and addiction risk (Hatoum et al., 2023), and the site of DNA methylation (cg14227435) that correlates with PDE4B expression and re-experiencing symptom severity in PTSD patients (Hori et al., 2024), are all located in PDE4B intron 3, thus implicating the long forms over the short forms.

Figure 1.

Functional consequences of PDE4BM220T variant. A, Schematic diagram depicting domain structure and functions of PDE4B isoforms. The serine residues phosphorylated by PKA (S133; activation), cyclin-dependent kinase 5 (Cdk5; S145; activation), AMP-activated protein kinase (AMPK; S319; activation), and extracellular signal-related kinase (ERK; S659; inhibition) are shown (Baillie et al., 2000; MacKenzie et al., 2002; Plattner et al., 2015; Johanns et al., 2016). Red vertical lines indicate M220T. Black horizontal lines indicate sites for PDE4B dimerization (residues 168–183, 217–235, and 314–316; Richter and Conti, 2002; Bolger et al., 2015; Cedervall et al., 2015), DISC1 binding (residues 212–245, 352–380, and 477–500; Murdoch et al., 2007), and β-amyloid binding (residues 312–313 and 327–328; Sin et al., 2024). B, Sanger sequence chromatograms showing the c.659T>C transition in Pde4b exon 8, which is predicted to convert residue 220 in PDE4B1 from ATG methionine (M) to ACG threonine (T). C, Top, Partial protein sequence alignment of mouse PDE4B1 and orthologs showing conservation in vertebrates of the M220 residue. Human PDE4B1 has a high degree of DNA (89%) and protein (97%) sequence homology with the mouse ortholog. Bottom, Partial protein sequence alignment of mouse PDE4B isoforms and paralogs showing that M220 is restricted to PDE4B1–4. PDE4B4 is not encoded by the human and orangutan genomes (Shepherd et al., 2003; Martin et al., 2023). Amino acid color code: black (nonpolar), green (uncharged polar), red (basic), blue (acidic). Residues disparate between mouse PDE4B1 and orthologs and paralogs have a gray background. D, Left, Structural model of mouse PDE4B1-WT. Right, Structural model of mouse PDE4B1-M220T variant showing cAMP binding pocket conformation altered by a β conformational bend around K282 (black). The conformation of the cAMP binding pocket is altered by the introduction of a β conformational bend around K282 and an internal interaction between E335 and Y358 is abolished. E, cAMP hydrolytic function of eGFP-tagged PDE4B1-WT transfected, eGFP-tagged PDE4B1-M220T transfected, and untransfected HEK-293T cells with (+) and without (−) forskolin treatment (10 μm; 30 min). The cAMP concentration is inversely related to cAMP hydrolysis. F, cAMP hydrolytic function of VSV-G-tagged PDE4B1-WT and M220T constructs expressed in HEK-293 cells (unpaired t test: t = 0.04; p = 0.97). Mean of four experiments. G, Unaltered PDE4B1 protein expression in eGFP-tagged PDE4B1-M220T versus PDE4B1-WT transfected HEK-293T cells. H, Unaltered PDE4B1 protein expression in VSV-G-tagged PDE4B1-M220T versus PDE4B1-WT transfected HEK-293 cells (unpaired t test: t = 2.26; p = 0.08). Mean of three experiments. I, Unaltered PDE4B protein expression in amygdala from Pde4bM220T versus WT mice. J, Unaltered endogenous DISC1 protein expression in VSV-G-tagged PDE4B1-M220T versus PDE4B1-WT transfected HEK-293 cells (unpaired t test: t = −0.66; p = 0.54). K, Unaltered endogenous β-arrestin-1/2 protein expression in VSV-G-tagged PDE4B1-M220T versus PDE4B1-WT transfected HEK-293 cells (unpaired t test: t = 0.57; p = 0.6). L, Unaltered PDE4B1-M220T binding to DISC1 demonstrated by coimmunoprecipitation of endogenous DISC1 in HEK-293 cells expressing VSV-G-tagged PDE4B1-M220T or PDE4B1-WT constructs (unpaired t test: t = −0.32; p = 0.76). VSV-G-tagged PDE4B1-Y358C was included as a positive control for decreased DISC1 binding (McGirr et al., 2016). Mean of three experiments. Data are plotted as mean ± SEM. #p < 0.05; ##p < 0.01 versus untreated within each genotype. PDE, phosphodiesterase; Untrans., untransfected.

To examine the effect of PDE4B variation on phenotypes with translational relevance to psychiatric disorders, we tested mice that replicate human missense variant M220T (rs775201287; 1-66332532-T-C) located in UCR2. Herein, we report that homozygous Pde4bM220T mice exhibit increased PTSD-like behavior in response to trauma, as well as cortical hypoactivity and hyperactivity of the hippocampus and amygdala. Our findings provide substantive biological evidence linking PDE4B variation to PTSD-relevant phenotypes and thus highlight how genetic variation of PDE4B may contribute to PTSD risk.

Materials and MethodsEthical approvals

Mouse experiments were approved by The Centre for Phenogenomics (TCP) Animal Care Committee and were conducted in compliance with the Ontario Animals for Research Act 1971 and Canadian Council on Animal Care guidelines.

Generation of Pde4bM220T mutant

Genomic DNA from 7,502 F1 progeny of ENU-mutagenized C57BL/6JJcl males and untreated DBA/2JJcl females in the RIKEN BioResource Center frozen sperm archive was screened for mutations in Pde4b exon 8 (113 bp), as described previously (Sakuraba et al., 2005). In a single mouse (Pde4bRgsc02383), we detected transition c.659T>C.

Mice

Heterozygous N2 backcross progeny of the founder Pde4bM220T heterozygous (DBA/2JJcl × C57BL/6JJcl) F1 male and wild-type (WT) C57BL/6JJcl females were backcrossed to C57BL/6JJcl (CLEA, Tokyo, Japan) for at least 10 generations before heterozygotes were intercrossed to generate homozygous mutant (Pde4bM220T) and WT littermates for phenotypic testing. Mice were genotyped for the M220T variant by the presence of a BsrDI (R0574, New England Biolabs) restriction site. At 3 weeks of age, pups of mixed genotype were weaned and housed in groups of three to five same-sex littermates under controlled temperature (21 ± 1°C), lighting (lights on: 7 A.M.–7 P.M.), and humidity (50–60%). The C57BL/6JJcl congenic Pde4bM220T (Pde4bRgsc02383) line is cryopreserved at TCP, Toronto, Canada.

Structural modeling

The mouse PDE4B1-WT protein and the M220T variant were modeled using the Phyre2 Protein Fold Recognition Server (Kelley et al., 2015).

Mammalian expression constructs

M220T was introduced into ampicillin-resistant constructs pEE7.hCMV>VSV-G-hPDE4B1 (provided courtesy of Kirsty Millar, University of Edinburgh) and pRP[Exp]-EGFP-EF1A>hPDE4B1 (VectorBuilder) using a QuikChange II Site-Directed Mutagenesis Kit (Agilent). The M220T mutation was confirmed by DNA sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).

Phosphodiesterase activity

To measure the cAMP hydrolytic activity of eGFP-tagged PDE4B1-M220T under basal conditions, HEK-293T cells were transfected with eGFP-tagged PDE4B1 constructs. At 48 h post-transfection, some HEK-293T transfectants were exposed to 10 µM forskolin (Enzo Life Sciences) for 30 min before all cells were lysed on ice. The intracellular cAMP concentration in the lysates was measured using a cAMP ELISA Kit (E-EL-0056, Elabscience Biotechnology). The phosphodiesterase activity of lysates from VSV-G-tagged PDE4B1 transfectants was determined using a two-step radioassay procedure, as described previously (Marchmont and Houslay, 1980; McGirr et al., 2016).

Western blotting

Transfected HEK-293 cell lysate Western blots were carried out using mouse monoclonal anti-VSV glycoprotein (V5507, Sigma-Aldrich), mouse monoclonal anti-PDE4B (TA503471, OriGene Technologies), rabbit polyclonal anti-β-Arrestin-1/2 (sc-28869, Santa Cruz Biotechnology), rabbit polyclonal antibody to human DISC1 residues 683–832 (courtesy of Tetsu Akiyama, University of Tokyo; Ogawa et al., 2005), and mouse monoclonal anti-GAPDH (ab8245, Abcam), as described previously (McGirr et al., 2016). Western blotting of amygdala tissue dissected from 16-week-old male Pde4bM220T and WT littermates was carried out using mouse monoclonal anti-PDE4B (1:2,000; TA503471, OriGene Technologies) and mouse monoclonal anti-GAPDH (1:10,000; ab8245, Abcam), as described previously (McGirr et al., 2016).

Coimmunoprecipitation

To measure the effect of M220T on the binding of VSV-G-tagged PDE4B1 to endogenous DISC1, coimmunoprecipitation using mouse monoclonal anti-VSV glycoprotein–agarose antibody (A1970, Millipore) and a rabbit polyclonal antibody to human DISC1 (Ogawa et al., 2005) was conducted as described previously (McGirr et al., 2016). PDE4B1-Y358C was used as a positive control for decreased DISC1 binding (McGirr et al., 2016).

Mouse behavioral testing

All behavioral experiments were conducted using naive male Pde4bM220T and WT littermates at 10–16 weeks of age. Due to budgetary restrictions, female mice were not utilized. Mice were handled for 1 week prior to behavioral testing and transferred to the experimental room 30 min prior to the start of testing. Testing equipment was cleaned with 70% ethanol between each mouse. All experiments were conducted between 9 A.M. and 4 P.M.

Open field

Spontaneous locomotor activity was assessed in 1 h duration trials using a VersaMax Animal Activity Monitoring System (Omnitech Electronics), as described previously (Lipina et al., 2013).

Elevated plus maze

The elevated plus maze test was conducted using EthoVision XT video tracking software (Noldus), as described previously (Lipina et al., 2022).

Forced swim test

The forced swim test (FST) was conducted using The Observer 5.0 software (Noldus), as described previously (Lipina et al., 2012).

Three-chamber social approach test

The social approach test was conducted using a three-chambered box (each chamber 40 × 20 cm) and The Observer 5.0 software (Noldus), as described previously (Lipina et al., 2012).

Prepulse inhibition of acoustic startle response

Acoustic startle response (ASR) testing was conducted using a Startle Reflex System (ENV-022s, Med Associates), as described previously (Lipina et al., 2013). Pulse-only trials consisted of a single white noise burst (110 dB, 40 ms). Prepulse + pulse trials consisted of a prepulse of noise (20 ms at 72, 78, 82, or 86 dB) followed 100 ms after prepulse onset by a startling pulse (110 dB, 40 ms). No-stimulus trials consisted of background noise only (65 dB).

Morris water maze

Morris water maze (MWM) testing was conducted using 120-cm-diameter cylindrical tank filled with opaque water (40 cm depth; 24 ± 1°C), as described previously (Lipina et al., 2022). Video output from a camera above the pool center was analyzed using HVS Water 2020 software (HVS Image). Mice were given four training trials for 1 d to a visible platform at the center of the SE (target) quadrant, followed by 20 training trials (four per day) to a now submerged platform (1 cm below water surface) in the SE quadrant. A probe trial with the platform removed from the pool was given 18 h after the last training trial. The maximum duration of each trial was 60 s.

Object location test

The object location test (OLT) was conducted in a transparent acrylic arena (41 × 41 × 31 cm), using The Observer 5.0 software (Noldus), as described previously (Lipina et al., 2013). In training periods lasting for 5 or 15 min, a mouse was placed at the center of the arena and left to explore four identical plastic objects (inverted 100 ml beakers) placed at specific locations near each corner (5 cm from walls).

Y-maze

Y-maze testing was conducted using an apparatus consisting of three arms (40 × 8 × 15 cm; A, B, and C) made of gray opaque polyvinyl plastic placed at 120° from each other, as described previously (Lipina et al., 2013). The percentage of alternations was defined according to the following equation: % alternation = [(number of alternations) / (total arm entries − 2)] * 100.

Puzzle box

The puzzle box test was conducted using an apparatus consisting of a white acrylic arena divided by a removable barrier into two compartments: a brightly lit start zone (58 × 28 cm) and a smaller covered goal zone (15 × 28 cm), as described previously (Lipina et al., 2013).

Fear conditioning

Fear conditioning was conducted using a test chamber (25 × 30 × 25 cm; Med Associates) connected to a personal computer running FreezeFrame software (Actimetrics) that administered two CS–US pairings—auditory tone (3.6 kHz, 75 dB, 30 s) conditioned stimulus (CS) followed by footshock (0.75 mA, 2 s) unconditioned stimulus (US)—delivered 60 s apart, as described previously (Lipina et al., 2013). In the contextual memory test, 24 h after conditioning, the mouse was returned to the chamber and monitored for 8 min. In the cued fear memory test, 48 h after conditioning, the mouse was placed in an altered chamber (novel odor, lighting, background noise, floor, shape, visual cues) and allowed to explore for 3 min before the auditory tone was presented continuously for 8 min.

To model a traumatic event, mice were subject to fear conditioning with 10 CS–US pairings (trauma), 60 s apart, of an auditory tone (3.6 kHz, 75 dB, 30 s) CS immediately followed by a footshock (1 mA, 2 s) US, as described previously with slight modifications (Bliss et al., 2010). At 6, 24 and 48 h after conditioning, cued fear memory was assessed in an altered chamber, as described above. Separate cohorts of mice were assessed for cued fear memory in response to the CS at 3, 15 or 30 d after conditioning.

Ultrasonic vocalization

Ultrasonic vocalization (USV) during the last 3 min of cued fear memory testing was recorded using an UltraSoundGate ultrasonic microphone and RECORDER software (Avisoft Bioacoustics). USVs emitted in the 22–35 kHz range, corresponding to fear-related calls (Wöhr and Schwarting, 2013), were analyzed using SASLab Pro software (Avisoft Bioacoustics), as described previously (Mun et al., 2015a).

Hotplate test of pain sensitivity

Pain sensitivity was measured using a Hot Plate Analgesia Meter (Columbus Instruments) set at a constant temperature of 52.5 ± 0.5°C, as described previously (Wilkerson et al., 2018).

Corticosterone measurement

Plasma levels of corticosterone in blood samples collected from naive (nontrauma exposed) mice and trauma-exposed mice at 24 h and 30 d post-trauma, 30 min after re-exposure to the auditory tone (CS), were measured using a Corticosterone ELISA Kit (Cayman Chemical) according to manufacturer's instructions. Each sample was run in triplicate.

Hippocampal BDNF

BDNF levels in the hippocampi dissected from the same mice killed for measurement of plasma corticosterone levels, at baseline (nontrauma exposed) and at 24 h and 30 d post-trauma, 30 min after re-exposure to the auditory tone (CS), were measured using a Mouse BDNF Sandwich ELISA kit (LS-F2404, LifeSpan BioSciences), as described previously (Xian et al., 2019).

Immunohistochemistry staining for c-Fos protein

The number of c-Fos-positive cells in brain sections (50 µm thick) from mice, 90 min after trauma, was measured using rabbit polyclonal anti-c-Fos (1:500; sc-52, Santa Cruz Biotechnology) and Alexa Fluor 488-conjugated goat polyclonal anti-rabbit IgG (1:1,000; ab150077, Abcam), as described previously (Mun et al., 2015b). For quantitative analysis, 6–7 mice per genotype and 4–5 sections per mouse were used.

Statistical analyses

Statistical analyses were performed using Statistica 14.0 (TIBCO Software). Data were tested for normality using the Shapiro–Wilk test and for homoscedasticity using Levene's test. Data passing normality and homoscedasticity assumptions were analyzed using one-way, two-way, or three-way analysis of variance (ANOVA) with repeated measures (RM), as necessary, followed by Tukey’s post hoc tests with statistical significance set at p < 0.05. Pearson's correlation coefficients (r) were calculated to assess relationships between the percentage of freezing in cued fear memory testing and the amplitude of fear-related calls or c-Fos staining and between c-Fos staining in different brain regions. The exact number of samples included is given in each figure legend. Each sample corresponds to an individual dot in the graphs, which were prepared using Prism 8 software (GraphPad Software). Experimenters were blinded to genotype during behavioral testing.

ResultsBiochemical consequences of PDE4BM220T variant

The c.659T>C transition in mutant transcript Pde4bRgsc02383 results in the nonpolar, hydrophobic methionine (M) at position 220 (PDE4B1 numbering) being replaced by a polar, hydrophilic threonine (T) in PDE4B1–4 (Fig. 1A–C). Structural modeling of the mouse PDE4B1-M220T variant predicted subtle changes compared with PDE4B1 wild-type (WT), but not to the extent of disrupting the normal orientation and binding of cAMP (Fig. 1D).

In HEK-293 cells expressing eGFP- or VSV-G-tagged constructs, the enzymatic activity of the PDE4B1-M220T variant was not significantly different compared with PDE4B1-WT under basal conditions (Fig. 1E,F). When challenged with the adenylyl cyclase activator forskolin (10 µm; 30 min), which induces phospho-activation of PDE4 by protein kinase A (PKA; Plattner et al., 2015), untransfected cells showed an expected increase in intracellular cAMP concentration compared with basal conditions. This forskolin-elicited increase was prevented by expression of eGFP-tagged PDE4B1-WT but was only partially attenuated by expression of eGFP-tagged PDE4B1-M220T (Fig. 1E), suggesting that the PDE4B1-M220T variant was less able to respond to a rapid rise in intracellular cAMP.

Western blotting revealed that in vitro PDE4BM220T protein expression was unaltered in PDE4B1-transfected cells (Fig. 1G,H), a finding paralleled in vivo in amygdala tissue from Pde4bM220T mice (Fig. 1I). PDE4B1-M220T-transfected cells also showed unaltered levels of endogenous DISC1 and β-arrestin-1/2 (Fig. 1J,K), known binding partners of PDE4B (Perry et al., 2002; Millar et al., 2005; Cheung et al., 2007; Murdoch et al., 2007). M220 is located within the DISC1 binding site in UCR2, but coimmunoprecipitation of endogenous DISC1 was unaltered in PDE4B1-M220T-transfected cells (Fig. 1L).

Pde4bM220T mice exhibit heightened reactivity to novel environments

Since GWAS data have associated PDE4B with PTSD (Gelernter et al., 2019; Meier et al., 2019; Nievergelt et al., 2024) and schizophrenia (Trubetskoy et al., 2022), which are often comorbid (Dallel et al., 2018), we subjected Pde4bM220T mice to several behavioral tests that examine a range of evolutionary conserved cognitive and behavioral domains. To examine the behavioral response of Pde4bM220T mice to a mildly stressful novel environment, we measured their locomotor activity in an open field (OF) over 1 h. Pde4bM220T mice were more active during the first 20 min, but their locomotor activity was comparable with WT mice thereafter (Fig. 2A), suggesting that the heightened initial activity was in response to the novelty of the environment. Both genotypes showed a similar decline in locomotor activity over time, indicative of unaltered habituation. Pde4bM220T mice exhibited less rearing at 30–40 min and 55–60 min than WT mice (Fig. 2B). Pde4bM220T mice also spent a lower fraction of time at the center versus the periphery of the arena than WT mice (Fig. 2C), demonstrating greater thigmotaxis.

Figure 2.

Behavior of Pde4bM220T mice in open field, EPM, forced swim, and social approach tests. A, OF: locomotor activity quantified as number of horizontal beam breaks in 5 min intervals. Pde4bM220T mice were more active during the first 20 min (RM ANOVA, time: F(11,297) = 82.2, p < 0.0001; genotype × time interaction: F(11,297) = 4.7, p < 0.0001). B, OF: rearing quantified as number of vertical beam breaks. Pde4bM220T mice reared less than WT mice at 30–40 min and 55–60 min (RM ANOVA, time: F(11,297) = 13.6, p < 0.0001; genotype × time interaction: F(11,297) = 2.9, p < 0.001). C, OF: fraction of time in the center versus the periphery in 5 min intervals (RM ANOVA, genotype: F(1,27) = 4.4, p < 0.05; time: F(11,297) = 5.6; p < 0.0001). D, EPM: distance traveled (m). E, EPM: percentage of time in the open arms (F(1,14) = 5.7; p < 0.05), closed arms (F(1,14) = 19.8; p < 0.001), and central square (F(1,14) = 10.7; p < 0.01). F, EPM: number of exploratory head dips. G, FST, percentage of time spent floating. H, Social approach test. Sociability: time (s) spent exploring an empty container versus a novel mouse (stranger 1; RM ANOVA, genotype: F(1,27) = 0.8, p > 0.05; stranger 1: F(1,27) = 17.7, p < 0.01). Social memory: time (s) spent exploring stranger 1 (previously explored mouse) versus a second novel mouse (stranger 2; RM ANOVA, genotype: F(1,27) = 2.5, p > 0.05; stranger 2: F(1,27) = 33.3, p < 0.001). Data are plotted as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 versus WT. ##p < 0.01; ###p < 0.001 versus empty container or stranger 1 within each genotype. Empty, empty cylinder; S1, stranger 1; S2, stranger 2. Additional data are shown in Extended Data Figure 2-1 (EPM) and 2–2 (social approach).

Figure 2-1

Behavior of Pde4bM220T and WT mice in the elevated plus maze. Pde4bM220T mice made more entries to the open arms, closed arms and center, and more passages between the open arms, than WT mice. *p < 0.05, **p < 0.01, ***p < 0.001 versus WT. ANOVA, analysis of variance; No., number of; WT, wild-type. Download Figure 2-1, DOCX file.

To probe the response of Pde4bM220T mice to novel environments further, we examined their behavior in an elevated plus maze (EPM), consisting of two wall-enclosed arms intersected by two open arms. Over the 5 min test, Pde4bM220T mice ambulated further, spent a greater percentage of time in the open arms, and made more exploratory head dips (Fig. 2D–F) and passages between the open arms (Extended Data Fig. 2-1) than WT mice. Pde4bM220T mice thus displayed exaggerated locomotor activity responses to two different novel environments, indicating heightened reactivity to environmental novelty. Pde4bM220T mice did not display differences in the FST and the social approach test (Fig. 2G,H; Extended Data Fig. 2-2).

Figure 2-2

Behavior of Pde4bM220T and WT mice in the three-chamber social approach test. Pde4bM220T mice did not display differences in habituation, sociability or social memory. No., number of; WT, wild-type. Download Figure 2-2, DOCX file.

Pde4bM220T mice exhibit increased startle reactivity and decreased prepulse inhibition

The acoustic startle reflex is a coordinated contraction of the skeletal musculature in response to a loud and unexpected noise. An exaggerated startle reflex is one of the clinical symptoms of PTSD (Jovanovic et al., 2009; American Psychiatric Association, 2013). When presented with a startling pulse of sound (110 dB, 40 ms), Pde4bM220T mice displayed a greater acoustic startle response (ASR) than WT mice (Fig. 3A). Prepulse inhibition (PPI) refers to the amygdala-modulated attenuation of the ASR when a brief low-intensity prepulse shortly precedes the startle-eliciting pulse (Cano et al., 2021). When presented with prepulses of 82 and 86 dB before the 110 dB startle pulse, Pde4bM220T mice displayed less PPI than WT mice, but PPI with prepulses of 72 and 78 dB was not significantly different between genotypes (Fig. 3B).

Figure 3.

Acoustic startle response and prepulse inhibition in Pde4bM220T mice. A, Amplitude of acoustic startle response (ASR) to a startle stimulus (110 dB, 40 ms; RM ANOVA, genotype: F(1,22) = 4.6; p < 0.05). B, Prepulse inhibition of the ASR expressed as the percent reduction in startle amplitude at prepulse levels of 72, 78, 82, and 86 dB (RM ANOVA, genotype: F(1,22) = 5.1, p < 0.05; prepulse: F(3,66) = 71.8, p < 0.0001). Data are plotted as mean ± SEM. *p < 0.05 versus WT. dB, decibels.

Pde4bM220T mice exhibit enhanced hippocampus-dependent spatial memory

In the visible platform (nonspatial) version of the MWM, latency to reach a visible escape platform was not significantly different between genotypes (Fig. 4A). In the hippocampus-dependent hidden platform (spatial) version of the MWM, latency and path length to reach a submerged (hidden) escape platform and swimming speed over 5 d of training were not significantly different between genotypes (Fig. 4A–C), thus demonstrating unaltered spatial learning in Pde4bM220T mice. During a probe trial with the platform removed, 18 h after the last training trial, the number of crossings of the former platform location was not significantly different between genotypes (Pde4bM220T: 8.2 ± 0.6; WT: 8.3 ± 0.8). However, Pde4bM220T mice spent more time than WT mice in the target quadrant (Fig. 4D), demonstrating a more focused search of the pool.

Figure 4.

Hippocampus-dependent spatial memory in Pde4bM220T mice. A, MWM training trials: escape latency (s) at a block of four training trials with a visibl