Glutamatergic CYLD deletion leads to aberrant excitatory activity in the basolateral amygdala: association with enhanced cued fear expression

Abstract

Neuronal activity, synaptic transmission, and molecular changes in the basolateral amygdala play critical roles in fear memory. Cylindromatosis (CYLD) is a deubiquitinase that negatively regulates the nuclear factor kappa-B pathway. CYLD is well studied in non-neuronal cells, yet under-investigated in the brain, where it is highly expressed. Emerging studies have shown involvement of CYLD in the remodeling of glutamatergic synapses, neuroinflammation, fear memory, and anxiety- and autism-like behaviors. However, the precise role of CYLD in glutamatergic neurons is largely unknown. Here, we first proposed involvement of CYLD in cued fear expression. We next constructed transgenic model mice with specific deletion of Cyld from glutamatergic neurons. Our results show that glutamatergic CYLD deficiency exaggerated the expression of cued fear in only male mice. Further, loss of CYLD in glutamatergic neurons resulted in enhanced neuronal activation, impaired excitatory synaptic transmission, and altered levels of glutamate receptors accompanied by over-activation of microglia in the basolateral amygdala of male mice. Altogether, our study suggests a critical role of glutamatergic CYLD in maintaining normal neuronal, synaptic, and microglial activation. This may contribute, at least in part, to cued fear expression.

Introduction

Fear is a conserved, cross-species emotion represented by a series of defensive behavioral responses of an organism to danger or threat. It is a pivotal behavior for animals to adapt and survive in their environment (Johansen et al., 2011; Izquierdo et al., 2016; Koskinen and Hovatta, 2023; Zhao et al., 2024). Disturbances of fear memory can trigger maladaptive responses in psychiatric diseases, for instance, post-traumatic stress disorder (PTSD), phobia, and generalized anxiety (Schmidt et al., 2020; Lee and Jung, 2024). Therefore, better understanding of the neural basis underlying fear memory acquisition and expression may lead to new therapeutic targets. Pavlovian fear conditioning is a generally used paradigm in neurobiological analysis to measure learning and memory as well as emotion of animals (Johansen et al., 2011). Typically, tone-cued fear conditioning (TFC) can be achieved by pairing a conditioned stimulus (CS), usually a neutral auditory tone, with an aversive unconditioned stimulus (US), like a mild foot shock (Sun et al., 2018). The basolateral amygdala (BLA) functions as the central headquarters of fear and is further subdivided into the lateral amygdala and basal amygdala (Ehrlich et al., 2009; Tipps et al., 2018; Zhan et al., 2023). Neuronal activity, Hebbian synaptic plasticity, and molecular changes in the BLA make vital contributions to processes of fear memory such as its formation, storage, and expression (Johansen et al., 2011; Bocchio et al., 2017; Kim and Cho, 2017; Sharp, 2017). The BLA consists of 80%–85% principal neurons (glutamatergic pyramidal output neurons) and 15%–20% γ-aminobutyric acidergic interneurons (Duvarci and Pare, 2014; Sun et al., 2018; Shao et al., 2019). It is clear that the BLA is responsible for tone-cued fear memory, whereas contextual fear memory (CFM) is hippocampal-dependent, and consolidated in the hippocampal subregions CA1 and CA3 (Baldi and Bucherelli, 2015; Chaaya et al., 2018).

Recent studies have shown the involvement of specific deubiquitinases in fear memory, anxiety, neuroinflammation, neurodegeneration, chronic inflammation, and autoimmunity (Guo et al., 2017; Han et al., 2020; Srikanta et al., 2021; Jolly et al., 2022; Chen et al., 2023; Ye et al., 2024). Deubiquitinase cylindromatosis (CYLD) is a negative modulator of the nuclear factor kappa-B (NF-κB) pathway (Kovalenko et al., 2003) and a tumor suppressor protein of familial cylindromatosis, which cleaves non-proteolytic K63 and M1 chains from protein substrates (Lork et al., 2017). Non-neuronal cell studies have shown that CYLD is involved in innate immunity, cell death, cell cycle progression, and DNA damage (Massoumi, 2010; Fernández-Majada et al., 2016; Hrdinka et al., 2016; Bonacci and Emanuele, 2020; Fiil and Gyrd-Hansen, 2021). As shown in a relevant database (BioGPS; www.biogps.org), CYLD is expressed at high levels in multiple brain areas related to emotion, and learning and memory, including the striatum, amygdala, prefrontal cortex, and hippocampus. Surprisingly, only a few reports have noted the physiological functions of CYLD in the brain. CYLD exerts profound effects on synaptic remodeling by interacting with postsynaptic density (PSD) components as well as proteins involved in autophagy signaling (Ma et al., 2017; Colombo et al., 2021; Zajicek et al., 2022; Tan et al., 2023b). Promisingly, and similar to findings from immune cells, CYLD is positively involved in preventing neuroinflammation (Han et al., 2020). We previously reported that reduced expression of cued fear in conventional Cyld knockout mice was accompanied by insufficient neuronal activity and impaired synaptic transmission of BLA glutamatergic neurons (Li et al., 2021a). Of note, existing studies showing a role of CYLD in brain function were conducted in either neuronal cultures or with conventional Cyld knockout mice. The role of glutamatergic CYLD in neuronal activity and fear memory remain unclear.

In the current study, we constructed a transgenic mouse line with specific deletion of Cyld in glutamatergic neurons. To examine the effect of glutamatergic neuron-specific deletion of Cyld on fear memory of mice, electrophysiological activities of BLA pyramidal neurons, and molecular changes in the BLA, we performed immunofluorescence staining of c-Fos positive neurons, electrophysiological recording of BLA neuronal and synaptic activities, immunoblotting of receptor protein levels, and behavioral tests for cued fear memory.

Methods

Experimental animals

Conventional Cyld knockout (Cyld^–/–) mice were previously constructed by Dr. Shao-cong Sun and colleagues (Reiley et al., 2006) (University of Texas MD Anderson Cancer Center, Houston, TX, USA). By intercrossing Cyld^+/– mice of both sexes, male Cyld^+/+ and Cyld^–/– littermates were generated as detailed previously (Zhang et al., 2016; Li et al., 2021a). Male C57BL/6J mice were obtained from the Vital River Laboratory Animal Technology Co., Ltd., (Guangzhou, China, license No. SCXK (Yue) 2022-0063). Vesicular glutamate transporter 2 (Vglut2)-IRES-Cre male mice (Stock No. 028863, RRID: IMSR_JAX:028863) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). The Cyld^flox/flox mice were constructed by our research group at Gempharmatech Co., Ltd., (Nanjing, China). Specifically, CRISPR/Cas9 technology was used to insert loxP sites at both ends of exons 2–4 of the Cyld gene. After mating with mice expressing Cre recombinase, CYLD function was predicted to be disrupted in specific cell types of Cyld^flox/flox mice. Cyld^flox/flox mice were hybridized with Vglut2^Cre+/– to obtain first generation Vglut2^Cre+/–::Cyld^flox/– mice. These were backcrossed with Cyld^flox/flox mice, resulting in homozygous second generation mice of both sexes for further investigations: Vglut2^Cre+/−::Cyld^flox/flox (conditional knockout, cKO) or Cyld^flox/flox (wild-type, WT). Mice were housed in standard laboratory cages (3–5 per cage) and were fed ad libitum. The condition of the feeding room was controlled at 50%–60% humidity and 23–25°C on a 12-hour light/dark cycle. Efforts were made to minimize animal suffering. All of the mice used in this study were 2 to 4 months old and weighed 20–25 g. All animal experiments were approved by the Ethics Committee of Animal Research of South China Normal University (Guangzhou, China) on August 17, 2022 (approval No. SCNU-SLS-2022-022). All experiments were designed and reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (Percie du Sert et al., 2020).

Behavioral tests

The set period of testing time was 9 a.m. to 6 p.m. The operators were uninformed about the genotypes during tests. Before behavioral testing, all experimental mice experienced a 30-minute acclimation period in the testing room. Generally, a 70% ethanol solution was used to clean the behavioral apparatus between tests, except for alternative cleaning agents used to measure cued fear memory. These are described in the corresponding section below.

Tone-cued fear conditioning

The Panlab Startle and Fear Combined System (Harvard Apparatus, Holliston, MA, USA) were used to perform the TFC. Two distinct contexts were involved in the TFC experiment – Context A: gray chamber walls, metal grid floor, cleaning agent was 70% ethanol; Context B: white chamber walls, covered metal grid floor, cleaning agent was 4% acetic acid solution (had a distinct odor from ethanol in Context A). On the fear training day, an individual mouse was introduced to Context A. The mouse underwent a 3-minute acclimation time followed by five pairings of a tone (CS, 80 dB, 2 kHz, 20 seconds) and shock (US, 0.5 mA, 2 seconds). Specifically, after playing the CS for 18 seconds, the US was delivered to the mouse for 2 seconds with simultaneous presence of the CS for 2 seconds. The interval between the paired deliveries was 60 seconds. Mice stayed in Context A for an additional 2-minute period after fear training and were then taken back to their home cages. The percentage of freezing time of mice (Freezing %) during the 3-minute habituation period and five CS-presented periods were scored as the basal freezing level of habituation and trained freezing level, respectively. At 24 hours after fear training, mice were introduced to Context A again and subjected to the CFM test without CS and US delivery. Freezing % during the 5-minute CFM test was regarded as the CFM level. Four hours after the CFM test, mice were placed in Context B for a 3-minute acclimation and subsequently exposed to 3-minute CS. The pre-tone and tone-cued test freezing levels were defined as the Freezing % of mice during the first 3 minutes and second 3 minutes in Context B, respectively. PACKWIN software (Harvard Apparatus) was used to quantify Freezing % based on sensitive weight transducer of the TFC system.

Open field test

The open field test (OFT) apparatus (Zhenghua Instruments, Hefei, Anhui Province, China) comprised a rectangular chamber (50 × 50 × 40 cm) and an automatic video tracking and data acquisition system. A camera was held 200 cm above the floor of the chamber. Following previously published procedures (Han et al., 2020; Jiang et al., 2023), mice were introduced to the center area of the testing chamber and their locomotor activities were recorded for 10 minutes. The parameter “time spent in the central area” was recorded as a reflection of their anxiety level. Specially, when assessed the motor function of mice, the total distance traveled was recorded for 30 minutes.

Elevated plus maze

The elevated plus maze (EPM) device (Zhenghua Instruments) was held 70 cm above the floor, and included two open arms (30 cm × 7 cm) located opposite one another and two opposing enclosed arms (30 cm × 7 cm × 20 cm). A central platform (7 cm × 7 cm) served as the link between the four arms. Following a previously established protocol (Jiang et al., 2023), mice were placed into the central platform orientated towards the open arms. They were allowed to explore the four arms for a 10-minute period. Data were excluded if the mouse escaped from the device during testing. The time spent in different arms and the number of entries to the arms were quantified using automatic video tracking and data analysis software (Zhenghua Instruments).

Three-chamber social test

The three-chamber social test was performed following a procedure modified from a previous publication (Cao et al., 2018). The testing device (60 cm × 40 cm × 25 cm) comprised three chambers (20-cm wide) separated by a plexiglass divider with entrances for the mice to pass through. Mice were allowed to habituate to the testing device once a day for two consecutive days before the day of testing. The test consisted of three phases. First, mice were introduced into the center chamber for a 10-minute acclimation with two entrances to the chambers on each side closed. In the second phase (sociability test), two wire cups were placed in the chambers on each side, which either contained a novel object (O) or a novel sex- and age-matched non-littermate mouse (Stranger 1, S1). To prevent side chamber preference of the test mouse, the positions of S1 and O were randomly exchanged between tests. Subsequently, mice were allowed to explore the side chambers for a 10-minute period with the two entrances open. The third phase was to test social novelty of the mice. The object in the wire cup was changed to a Stranger 2 mouse (S2). Again, the test mouse was permitted to experience a 10-minute exploration period freely in the three chambers. Both S1 and S2 mice spent a 30-minute habituation period in the wire cup for two consecutive days (once a day, twice in total). The social activities of the test mice were tracked using a real-time video recording and data analysis system (Zhenghua Instruments). Sniffing behavior of mice was defined when the nose of the test mouse touched the wire cup or pointed toward the cup within a 2-cm distance.

Tail suspension test

The experimenter gently fixed the tail of the mouse (about 1 cm from the tip) onto the hook of the height suspension test (TST) device (Xin Soft Computer Technology, Shanghai, China). The nose tip of the mouse was 20 cm away from the floor of the TST device. Xeye animal behavioral analysis software (Xin Soft Computer Technology) recorded immobility of the mouse for 6 minutes. Data were excluded when mice tried to climb back up their tails during the TST.

Forced swimming test

A transparent cylindrical glass container (Xin Soft Computer Technology, 20 cm diameter, 50 cm height) was used for the forced swimming test (FST). During the test, the container was filled to 25 cm depth with fresh 25°C water. After placement in the container, mouse behavior was recorded with the Xeye animal behavioral analysis software (Xin Soft Computer Technology) for 6 minutes. Only immobility over the last 4 minutes was measured. When the mice floated in the absence of any movement (speed ≤ 20 mm/s), they were regarded as being immobile (Jiang et al., 2023).

Marble burying test

Polycarbonate testing cages (27 cm × 16.5 cm × 12.5 cm; Houhuang Animal Experimental Equipment Technology Co., Ltd., Suzhou, Jiangsu, China) evenly lined with 5 cm deep clean bedding were used for the marble burying test. An individual mouse was first introduced into the cage for 30-minutes habituation without any marbles and then removed. The experimenter then evenly distributed 20 dark marbles (diameter 1.6 cm) in the testing cage and the mouse was re-introduced to the cage. After one hour, the number of marbles that were at least 50% covered by bedding was counted (Peng et al., 2021).

Rotarod motor learning

The motor learning test was conducted following a procedure modified from a previous publication (Yang et al., 2012). A rotating drum (Zhenghua Instruments) was used. The test was performed for three consecutive days and there were two trials per day (inter-trial interval was 1 hour). Each trial contained two stages: acclimation and accelerating stage. During the acclimation stage, mice were placed on the stationary rod for 5 minutes and subsequently habituated to the rotating rod (5 r/min) for 5 minutes. Ten minutes later, mice were introduced onto the rotating rod (5 r/min) and subjected to an accelerating rotating test (from 5 r/min to 40 r/min over 5 minutes). Latency to fall was used to evaluate rotarod performance, which was scored as the total time mice spent on the rod minus number of turns when mice made a complete revolution on the rod.

All limb grip strength test

A metal rectangle grid fitted to a strength meter (Zhenghua Instruments) was used for the all limb grip strength test. The experimenter firstly placed mice on the grid and then gently pulled them backward horizontally by their tail. When the value of the strength meter reached a peak, a test trial was finished, and mice were allowed to rest for 2 minutes before being subjected to the next trial. The test included a total three trials, with the mean peak value recorded as the final grip strength parameter (Yang et al., 2012; Roemers et al., 2019).

Wire hang test

A wire cage lid and cage lined with clean bedding were used for the wire hang test. After a 30-second habituation period on the lid, mice were subjected to a rapid reversion of the lid, and the lid was then placed above the cage (40 cm height). The fall latency of mice was tested. The maximum test time was 120 seconds (Yang et al., 2012).

When conducting the behavioral tests described above, the mice were divided into three cohorts. Tests were performed in the order below. Cohort 1: OFT, EPM, three-chamber social test, and TFC; Cohort 2: marble burying test, TST, and FST; Cohort 3: all limb grip strength test, wire hang test, and rotarod motor learning. The resting period between two behavioral tests was 24 hours.

Immunofluorescence staining

Immunofluorescence staining was performed following a previous protocol (Li et al., 2021a). After sedation with urethane (2 g/kg, intraperitoneal injection, Sigma–Aldrich, St. Louis, MO, USA), mice were perfused with 4% paraformaldehyde. The brains were subsequently postfixed with 4% paraformaldehyde for 24 hours and then dehydrated in 30% sucrose. The brains were cut into coronal sections (30 μm thick) using a freezing microtome (Leica CM30505, Wetzlar, Germany). Brain slices were washed three times for 5 minutes each using 1× phosphate buffered saline (PBS) and permeabilized with 0.3% PBST (0.3% Triton X-100 in 1× PBS) and 5% donkey serum at room temperature for 2 hours. Brain slices were then incubated at 4°C with primary antibodies: rabbit anti-ionized calcium binding adapter molecule 1 (Iba-1) (1:800; Wako, Osaka, Japan, Cat# 019-19741, RRID: AB_839504); rabbit anti-c-Fos (1:800; Abcam, Cambridge, UK, Cat# ab190289, RRID: AB_2737414), and rat anti-CD68 (1:200; Bio-Rad, Hercules, CA, USA, Cat# MCA1957, RRID: AB_322219). For c-Fos or Iba-1 labeling, the incubation time was 24 hours, while 48 hours was needed for co-labeling of Iba-1 and CD68. After rinsing in 1× PBS, the brain slices were incubated with goat anti-rat Alexa Fluor 647 (1:1000; Thermo Fisher, Waltham, MA, USA, Cat# A-21247, RRID: AB_141778) and/or goat anti-rabbit Alexa Fluor 594 (1:1000; Thermo Fisher, Cat# A-11012, RRID: AB_2534079) at room temperature for 2 hours. The brain slices were then washed three times for 5 minutes each using 1× PBS and coverslipped with antifade mounting medium with 4,6-diamidino-2-phenylindole (Beyotime, Haimen, Jiangsu, China, Cat# P0131). The same parameters were used between groups for image capturing with a fluorescence microscope (Nikon Eclipse Ni-E, Tokyo, Japan). Three non-consecutive brain slices per mouse were chosen for immunofluorescence staining. Mice used for c-Fos labeling were perfused 90 minutes after tone-cued tests. Cell counts and analyses were conducted using ImageJ software version 1.8.0 (National Institutes of Health, Bethesda, MD, USA; Schneider et al., 2012). Experimenters were blinded to the groups. Images used for measuring CD68 puncta per microglia, CD68 volume, and microglia branch points were acquired (0.2 μm Z-step) using a Zeiss LSM-800 confocal microscope (Zeiss, Jena, Germany). ZEN (Zeiss) and ImageJ software were used for preprocessing of images and Imaris software version 9.0.1 (Oxford Instruments, Oxford, UK) was used for three-dimensional (3D) reconstruction of microglia.

Western blotting

Western blotting was conducted following previously established procedures (Han et al., 2020). BLA tissue from naive WT and cKO mice were quickly disassociated and then homogenized in sodium dodecyl sulfate lysis buffer. Supernatants of samples were collected after 15 minutes (12,000 × g) centrifugation of homogenates at 4°C, and then mixed with loading buffer (Beyotime, Cat# P0015B). Protein denaturation was performed by boiling the mixture for 5 minutes at 95°C in a metal bath. Protein samples (20 µg protein in total per sample) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose (NC) membranes. Tris-buffered saline with Tween-20 (TBST) containing 5% defatted dried milk was used to block NC membranes for 2 hours at room temperature. After rinsing in 1× TBST three times for 10 minutes each, primary antibodies were added for incubation overnight at 4°C on a shaker. Again, NC membranes were washed three times in TBST (10 minutes each). Goat anti-rabbit secondary antibody (1:5000, Beyotime, Cat# A0208, RRID: AB_2892644) in 1× TBST with 5% defatted dried milk was used for incubation of the NC membranes for 1.5 hours at room temperature. BeyoECL Star (Beyotime, Cat# P0018) was used to visualize immunoreactive proteins. Further detection and imaging acquisition of immunoreactive proteins bands were performed by ChemiDoc Touch imaging (Bio-Rad). Quantitation of immunoblotting bands was performed using the Gel-Pro Analysis software (Media Cybernetics, Rockville, MD, USA). The gray value of the target proteins were normalized by an internal loading control – α-tubulin. The primary antibodies used were: rabbit anti-CYLD (1:1000; Proteintech, Chicago, IL, USA, Cat# 11110-1-AP, RRID: AB_10915966), rabbit anti-glutamate receptor 1 (GluA1) (1:1000; Abcam, Cat# ab31232, AB_ 2113447), rabbit anti-glutamate receptor 2 (GluA2) (1:1000; Abcam, Cat# ab206293, RRID: AB_2800401), rabbit anti-N-methyl-D-aspartate receptor 1 (GluN1) (1:1000; Abcam, Cat# ab109182, RRID: AB_10862307), rabbit anti-N-methyl-D-aspartate receptor 2B (GluN2B) (1:1000; Abcam, Cat# ab65783, RRID: AB_1658870), and rabbit anti-α-tubulin (1:1000; Beyotime, Cat# AF0001, RRID: AB_2922414).

Whole-cell patch-clamp recording

Following established procedures (Jiang et al., 2023), BLA slices from naive WT and cKO mice (~320 µm thick) were cut in pre-cooling cutting solution with ice cubes using a vibratome (Leica VT1000S). The cutting solution contained the following (in mM): 93 N-methyl-D-glucamine, 93 HCl, 2.5 KCl, 30 NaHCO₃, 1.2 NaH₂PO₄, 20 N-(2-hydroxyethyl) piperazine-N-2-ethanesulfonic acid (HEPES), 25 D-glucose, 2 thiourea, 5 sodium ascorbate, 3 sodium pyruvate, 10 MgSO₄, and 0.5 CaCl₂. BLA slices were subsequently transferred to a holding solution containing the following (in mM): 92 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 25 D-glucose, 2 thiourea, 5 sodium ascorbate, 3 sodium pyruvate, 2 MgSO₄, and 2 CaCl₂ to recover for at least 60 minutes at room temperature. The whole-cell patch-clamp recording solution comprised (in mM): 124 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 24 NaHCO₃, 5 HEPES, 12.5 D-glucose, 2 MgSO₄, and 2 CaCl₂. During the experiment, 95% O₂ (balanced by 5% CO₂) was used to continuously bubble holding and recording solution. Pipettes (3–5 MΩ) containing a corresponding internal solution were used for recording at room temperature. Briefly, the internal solution for measuring spontaneous excitatory postsynaptic currents (sEPSCs), rheobase, and action potentials comprised (in mM): 110 K-gluconic acid, 10 NaCl, 1 MgCl₂·6H₂O, 10 ethylene glycol-bis (2-aminoethylether)-N,N,N’,N’-tetraacetic acid, 40 HEPES, 2 Mg-ATP, and 0.3 Na-GTP, pH 7.4 (300 mOsm). For determination of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs), the internal solution comprised (in mM): 100 Cs-methanesulfonate, 10 NaCl, 10 TEA-Cl, 1 MgCl₂·6H2O, 10 ethylene glycol-bis (2-aminoethylether)-N,N,N’,N’-tetraacetic acid, 40 HEPES, 2 Mg-ATP, 0.3 Na-GTP, and 4 QX-314, pH 7.40 (310 mOsm).

BLA pyramidal neurons were visually identified under a phase contrast microscope on the basis of their bright pyramidal-shaped soma. Only recordings with series resistance < 30 MΩ were included in data analysis. The first action potential induced by incrementally increasing current injections (start from 0 and end at 260 pA, in 20 pA-steps, 50 ms duration each) was used for measuring intrinsic neuronal properties. When performing the rheobase test, pyramidal cells were subjected to a 500 ms ramp test with continuous current pulses (0–200 pA). Hyperpolarizing current pulses (start from –120 pA and end at 0 pA, in 20 pA-steps) were given to record changes in voltage and calculate membrane input resistance. During sEPSC/mEPSC and mIPSC recordings, the cells were clamped at –60 mV and 0 mV, respectively. While recording mEPSCs and mIPSCs, the recording solution contained 300 nM tetrodotoxin (Sigma–Aldrich). Extra bicuculline (10 µM, Sigma–Aldrich) was added to the recording solution for mEPSCs, while mIPSCs were measured in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (20 µM, Sigma–Aldrich) and D-2-amino-5-phosphonovaleric acid (50 µM, Sigma–Aldrich). Data were acquired with a MultiClamp 700B amplifier (Molecular Devices, San Jose, CA, USA), Digidata 1440A (Molecular Devices), and pClamp10 software (Molecular Devices). Data analysis was performed offline using Mini Analysis (Synaptosoft, Fort Lee, NJ, USA) and Clampfit 10.6 (Molecular Devices).

Biocytin labeling of patched pyramidal neurons

To visualize patched pyramidal cells in the BLA, biocytin (0.1%; Sigma–Aldrich, Cat# B4261) was introduced as a marker material into internal solutions (Tan et al., 2023a). To promote the dispersion of biocytin throughout intact cells, after recording, cells were continuously recorded for an additional 20 minutes in a whole-cell configuration. Slices that contained biocytin-labeled neurons were postfixed with 4% paraformaldehyde at 4°C for 24 hours. After rinsing in 1× PBS three times (each for 10 minutes), slices were permeabilized with 1% PBST (1% Triton X-100 in 1× PBS) and 5% donkey serum at 4°C for 24 hours and incubated with Streptavidin Alexa 594 (1:1000; Thermo Fisher, Cat# S11227) dissolved in 0.3% PBST overnight at 4°C. Finally, slices were coverslipped with antifade mounting medium containing 4′,6-diamidino-2-phenylindole. Images (several patched-up tiles covering the entire labeled pyramidal neurons) were obtained (2 μm Z-steps) using the LSM-800 confocal microscope (Zeiss). ZEN software (Zeiss) was used for image processing.

Statistical analysis

Sample sizes were not predetermined using any statistical methods. Nevertheless, sample sizes in this study are similar to those in our previously published works (Han et al., 2020; Feng et al., 2021; Li et al., 2021a). Data are expressed as mean ± standard error of the mean (mean ± SEM). Data plotting and analysis were conducted using GraphPad Prism version 8.0.2 for windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Statistical analysis was performed with the following procedures. First, the data underwent the Shapiro–Wilk test and F test for normality and equal variance assumptions, respectively. If the data showed normal distribution accompanied by equal variance, statistical differences between two groups was evaluated using Student’s t-test. The two-sample t-test with Welch’s correction was used for analyzing data showing a normal distribution but unequal variances. If the data were non-normally distributed, the Mann–Whitney U test was used for a two-group comparison. For multiple comparisons between groups, two-way repeated measures analysis of variance followed by Bonferroni’s post hoc test was used. P < 0.05 was regarded as statistical significant difference between groups.

Results

Tone-cued test reduces CYLD levels in the basolateral amygdala of fear-conditioned mice

We previously reported that conventional Cyld knockout mice exhibited reduced expression of amygdala-dependent tone-cued fear, suggesting a regulatory role of CYLD in tone-cued fear memory (Li et al., 2021a). Thus, to determine whether CYLD in the BLA is indeed involved in tone-cued fear memory, we examined levels of CYLD in the BLA of C57BL/6J mice subjected to classical Pavlovian TFC (Figure 1A). Five CS–US pairings were delivered to fear-conditioned mice, while the CS was presented without the US in the tone-only group. As expected, there was no significant difference between groups in pre-tone freezing level, whereas compared with the tone-only group, fear-conditioned mice showed increased fear behavior in the tone-cued test of the following day (P < 0.0001; Figure 1B). The mice were sacrificed at 24 hours after the tone-cued test and the BLA disassociated for western blotting. A decrease in CYLD levels in the BLA was observed in fear-conditioned mice compared with the tone-only group (P = 0.035; Figure 1C and D). These data suggest that CYLD in the BLA participates in processing of tone-cued fear memory.

Figure 1.

Levels of CYLD in the BLA decreased after cued fear retrieval.

(A) TFC diagram. Tone (CS)–shock (US) pairings with a 60-second inter-trial interval were delivered five times (US, 0.8 mA, 2 seconds) during training in context A. Twenty-four hours later, mice were placed in context B for 3 minutes (pre-tone) and subsequently exposed to the 3-minute tone-cued test. Diagram was created with Adobe Illustrator CC version 21.0.0 (Adobe Systems Incorporated, San Jose, CA, USA). (B) Fear-conditioned (FC) mice, but not tone-only controls, showed fear responses 24 hours after training (means ± SEM, n = 3 mice, per group; two-way analysis of variance, experience effect: F_{(1, 8)} = 193.7, P < 0.0001, period effect: F_{(1, 8)} = 174.4, P < 0.0001, interaction: F_{(1, 8)} = 176.6, P < 0.0001; Bonferroni’s post hoc test, pre-tone: tone-only vs. FC, P > 0.05, tone-cued test: tone-only vs. FC, P < 0.0001, pre-tone, tone-only vs. tone-cued test, tone-only, P > 0.999, pre-tone, FC vs. tone-cued test, FC, P < 0.0001). (C) Representative immunoblots of CYLD from BLA lysates of tone-only and FC mice. (D) Quantification of immunoblots in C revealed that protein levels of CYLD in the BLA were decreased in FC mice compared with tone-only group (means ± SEM, n = 3 mice, per group; Student’s t-test, t₍₄₎ = 3.149, P = 0.035). BLA: Basolateral amygdala; CS: conditioned stimulus; CYLD: cylindromatosis; FC: fear-conditioned; n.s.: not significant; TFC: tone-cued fear conditioning; US: unconditioned stimulus.

Glutamatergic deletion of Cyld leads to exaggerated cued fear in male mice only

BLA glutamatergic neurons play a lead role in cued fear memory (Johansen et al., 2010; Yiu et al., 2014; Kim and Cho, 2017). In view of our earlier findings showing that Cyld ablation affected neuronal activity of BLA pyramidal neurons, which co-occurred with reduced cued fear expression in conventional Cyld knockout mice, we hypothesized that CYLD in glutamatergic neurons was critically involved in the regulation of fear memory. We thus constructed transgenic mice with specific deletion of Cyld in glutamatergic neurons by crossing Cyld^flox/flox mice (exons 2–4 of the Cyld gene flanked by loxP sites) with Vglut2^Cre+/− mice (Figure 2A). Mice with both Cre transgene and floxed alleles, i.e., Vglut2^Cre+/−::Cyld^flox/flox (hereafter referred as cKO mice), and Cyld^flox/flox mice (WT; used as controls) were used for experiments. The construction efficiency of transgenic mice was confirmed by immunoblotting (Figure 2B), which showed that CYLD levels were significantly decreased in the BLA of cKO mice (P = 0.0002; Figure 2C). This result indicated that glutamatergic CYLD accounts for the majority of CYLD (~85%) in the BLA, whereas CYLD in other cell types (including glial cells and γ-aminobutyric acid-ergic interneurons) constitutes only a small portion of the total expression quantity.

Figure 2.

Specific deletion of CYLD in glutamatergic neurons leads to exaggerated cued fear expression in male, but not female, mice.

(A) Schematic of the strategy used to construct mouse line with CYLD specifically deleted in glutamatergic neurons. (B) Representative immunoblots of total protein isolated from the BLA of WT and cKO mice. (C) Quantification of immunoblots from B revealed a significantly reduced protein levels of CYLD in the BLA of cKO mice (means ± SEM, n = 4 mice, per group; Student’s t-test, t₍₆₎ = 8.03, P = 0.0002). (D) CFM was unaffected in CYLD conventional knockout mice (male, means ± SEM, Cyld^+/+: n = 18 mice, Cyld^–/–: n = 21 mice; two-sample t-test with Welch’s correction, t_(34.66) = 0.06, P = 0.952). (E) Lacking CYLD in glutamatergic neurons had no effect on the acquisition phase of fear memory of male cKO mice (means ± SEM, male, WT: n = 14 mice, cKO: n = 15 mice, two-way analysis of variance, genotype effect: F_{(1, 162)} = 0.024, P = 0.878). (F) The CFM was intact in male cKO mice (means ± SEM, male, WT: n = 14 mice, cKO: n = 15 mice, Student’s t-test, t₍₂₇₎ = 0.717, P = 0.479). (G) Glutamatergic neuron-specific ablation of CYLD enhanced cued fear expression of male mice (means ± SEM, WT: n = 14 mice, cKO: n = 15 mice; two-way analysis of variance, genotype effect: F_{(1, 54)} = 13.63, P = 0.0005, period effect: F_{(1, 54)} = 69.01, P < 0.0001, interaction: F_{(1, 54)} = 3.551, P = 0.0649; Bonferroni’s post hoc test, pre-tone: WT vs. cKO, P > 0.05, tone-cued test: WT vs. cKO, P = 0.0014, pre-tone, WT vs tone-cued test, WT, P = 0.0002, pre-tone, cKO vs. tone-cued test, cKO, P < 0.0001). (H) The fear acquisition ability of female cKO mice were unaffected by deleting CYLD in glutamatergic neurons (means ± SEM, female, WT: n = 7 mice, cKO: n = 10 mice, two-way analysis of variance, genotype effect: F_{(1, 90)} = 0.852, P = 0.359). (I) The CFM was unaffected in female cKO mice (means ± SEM, female, WT: n = 7 mice, cKO: n = 10 mice, Student’s t-test, t₍₁₅₎ = 0.5212, P = 0.61). (J) Female cKO mice showed normal level of cued fear expression after tone-cued fear training (means ± SEM, WT: n = 7 mice, cKO: n = 10 mice; two-way analysis of variance, genotype effect: F_{(1, 30)} = 0.843, P = 0.365, period effect: F_{(1, 30)} = 73.06, P < 0.0001, interaction: F_{(1, 30)} = 1.056, P = 0.312; Bonferroni’s post hoc test, pre-tone: WT vs. cKO, P > 0.05, tone-cued test: WT vs. cKO, P > 0.05, pre-tone, WT vs. tone-cued test, WT, P < 0.0001, pre-tone, cKO vs. tone-cued test, cKO, P < 0.0001). BLA: Basolateral amygdala; CFM: contextual fear memory; cKO: conditional knockout; CYLD: cylindromatosis; Hab: habituation; n.s.: not significant; WT: wild-type.

A previous study reported that Cyld^–/– mice exhibited impaired amygdala-dependent tone-cued fear memory (Li et al., 2021a). Notably, when subjected to a contextual test, Cyld^+/+ and Cyld^–/– mice showed comparable hippocampal-dependent CFM levels (Figure 2D). Hence, we next performed TFC on cKO mice and their littermate controls to determine whether glutamatergic neuron-specific deletion of Cyld has a similar impact on the two types of fear memory. Considerable evidence has shown that fear-related regions (including the hippocampus, prefrontal cortex, and amygdala) are differentially activated in male and female rodents during contextual and cued fear memory (Bauer, 2023). Therefore, male and female cKO mice were investigated separately. Our results showed that fear acquisition was intact in male cKO mice (Figure 2E). Moreover, male WT and cKO mice showed similar CFM levels during the contextual test, indicating a normal CFM (Figure 2F). Interestingly, during the tone-cued test, the freezing level of male cKO mice in response to the CS was significantly higher compared with the WT group (P = 0.0014; Figure 2G), indicating excessive expression of cued fear in male cKO mice. We also performed TFC on female cKO mice and found that the fear acquisition phase and CFM levels were unaffected by glutamatergic Cyld deletion (Figure 2H and I). In particular, unlike the results obtained from male cKO mice, the freezing response of female cKO mice during the tone-cued test was comparable to littermate control mice, showing unaltered cued fear expression of female cKO mice (Figure 2J). As changes in motor function of mice may affect reliability of the fear conditioning test, we performed additional behavioral tests to determine the motor function of male mice in both genotypes. This included the 30 minutes-OFT, all limb grip strength test, wire hang test, and rotarod test. We found no difference in motor function and motor learning between cKO mice and their littermate Cyld^flox/flox mice (Additional Figure 1 ^{(2.1MB, tif)} A–D). As fear usually causes anxiety and vice versa (Izquierdo et al., 2016), we further determined whether male cKO mice showed an anxiety-like phenotype. No significant differences were observed between groups for total distance and duration in the center in the 10 minutes-OFT (Additional Figure 1 ^{(2.1MB, tif)} E–G), showing intact locomotor performance and exploratory activity of cKO mice. In the EPM test, both the number of entries to the open arms and time spent in the open arms were comparable between cKO mice and WT mice (Additional Figure 1 ^{(2.1MB, tif)} H–J), indicating similar anxiety levels for cKO and WT mice. Together, these results indicate that glutamatergic CYLD deficiency increases the expression of cued but not CFM in male mice without affecting their anxiety levels. We then focused on examining male mice.

Glutamatergic CYLD deficiency has no effect on sociability, and repetitive and depression-like behaviors

Previous studies have identified CYLD as a modulator of autophagy at synapses, as well as mouse behaviors including social communication, and repetitive and depression-like behaviors (Colombo et al., 2021; Zajicek et al., 2022). Hence, we performed a battery of behavioral tests to determine whether Cyld deletion in glutamatergic neurons influences mouse behavior. Both cKO and WT mice exhibited a similar social preference for S1 mice (versus object) and S2 mice (versus S1 mice) when subjected to the three-chamber social test, indicating normal sociability and social novelty of male cKO mice (Additional Figure 2 ^{(1.7MB, tif)} A–D). In addition, repetitive behavior was not found in cKO mice, with the number of buried marbles comparable to that in the WT group (Additional Figure 2 ^{(1.7MB, tif)} E). The FST and TST showed that male cKO mice had no aberrancy in immobility duration in either test (Additional Figure 2 ^{(1.7MB, tif)} F and G). Taken together, these results suggest that sociability, and repetitive and depression-like behaviors of male mice were unaffected by specific knockout of Cyld in glutamatergic neurons.

Glutamatergic CYLD deficiency increases the number of c-Fos positive neurons in response to cued fear retrieval and decreases mEPSC amplitudes of pyramidal neurons in the basolateral amygdala

Optically stimulating BLA pyramidal neurons as an US produced tone-cued fear memory, suggesting a key role for the activity of BLA pyramidal neurons in modulating cued fear (Johansen et al., 2010; Yiu et al., 2014). Thus, we next determined whether exaggerated cued fear expression in male cKO mice was related to altered neuronal activation, neuronal excitability, or synaptic transmission in the BLA. Initially, we examined the number of activated cells in the BLA of cKO and WT mice at 90 minutes after fear retrieval by labeling cells with c-Fos (Figure 3A). The results suggested a significant increase in the number of c-Fos⁺ cells in the BLA of cKO mice following the tone-cued test (P = 0.035; Figure 3B). This indicates enhanced neuronal activation in the BLA of cKO mice after cued fear memory recall, and prompted us to further identify any changes in neuronal excitability, intrinsic properties, or synaptic transmission in BLA pyramidal neurons of cKO mice using whole-cell patch-clamp experiments. Biocytin labeling confirmed the pyramidal morphology of patched cells in the BLA (Figure 3C). Pyramidal neurons from cKO mice showed decreased input resistance (P = 0.031; Table 1) and decreased slopes of current–voltage curves (P < 0.0001; Figure 3D and E), implicating higher excitability of BLA pyramidal neurons in cKO mice (Bjorefeldt et al., 2015; Hong et al., 2016; Tonomura and Gu, 2022). Other electrophysiological properties were unchanged (Table 1).

Figure 3.

Glutamatergic CYLD deficiency enhances neuronal activation in the BLA and decreases the slope of current-voltage curve of the BLA pyramidal neurons.

(A) Representative images of c-Fos labeled cells (red, Alexa Fluor 594) in the BLA of WT and cKO mice. The number of c-Fos⁺ cells was significantly increased in the BLA of cKO mice subjected to the tone-cued test. Scale bar: 100 µm. (B) The quantitative analysis of the number of c-Fos⁺ cells in the BLA of WT and cKO mice (means ± SEM, relative c-Fos⁺ cell number, WT: n = 10 mice, cKO: n = 9 mice, Student’s t-test, t₍₁₇₎ = 2.299, P = 0.035). (C) Upper, recording diagram of the whole-cell patch-clamp; lower, representative confocal image of an individual pyramidal neuron filled with biocytin (red, streptavidin Alexa 594) via a patch pipette. Magnification: 20×, scale bar: 50 μm. (D) Representative voltage traces recorded from BLA pyramidal neurons in response to the hyperpolarized current injections ranging from 0 to –120 pA in a step of 20 pA. (E) Pyramidal neurons in the BLA of cKO mice showed a significant decrease in the slope of current-voltage curve (means ± SEM, current–voltage curve, WT: n = 15 cells from 4 mice, cKO: n = 14 cells from 5 mice, two-way analysis of variance, genotype effect: F_{(1, 189)} = 20.3, P < 0.0001, current effect: F_{(6, 189)} = 75.61, P < 0.0001, interaction: F_{(6, 189)} = 0.924, P = 0.479). BLA: Basolateral amygdala; cKO: conditional knockout; CYLD: cylindromatosis; DAPI: 4′,6-diamidino-2-phenylindole; WT: wild-type.

Table 1.

Intrinsic properties of the BLA pyramidal neurons in WT and cKO mice

	WT (n = 15 neurons/4 mice)	cKO (n = 14 neurons/5 mice)
Membrane capacitance (pF)	83.06 ± 5.55	88.22 ± 5.51
Resting membrane potential (mV)	66.87 ± 1.13	–67.76 ± 1.45
AP amplitude (mV)	75.51 ± 1.07	75.13 ± 1.21
AP half-width (ms)	1.13 ± 0.02	1.09 ± 0.04
AP rise time (ms)	0.33 ± 0.01	0.34 ± 0.02
AP decay time (ms)	0.80 ± 0.03	0.73 ± 0.03
After-hyperpolarization (mV)	–5.66 ± 0.87	–6.97 ± 1.39
Input resistance (MΩ)	205.71 ± 11.25	167.20 ± 12.78*
Threshold (mV)	–36.89 ± 0.81	–37.58 ± 1.30
Rheobase (pA)	95.38 ± 7.94	105.9 ± 11.1

Data are presented as the mean ± SEM. *P < 0.05, Student’s t-test. AP: Action potential; BLA: basolateral amygdala; cKO: conditional knockout; WT: wild-type.

Our previous findings revealed an essential role of CYLD in regulating excitatory and inhibitory neural networks (Zhang et al., 2016; Li et al., 2021a). Hence, synaptic activity of BLA pyramidal neurons was then measured. We found identical amplitudes and frequencies of sEPSCs in neurons from both cKO and WT mice (Figure 4A–C). Notably, we found a significantly reduced mEPSC amplitude in cKO neurons (P = 0.037; Figure 4D and E), leaving mEPSC frequency unchanged (Figure 4F). However, we found that neither mIPSC frequency nor amplitude differed among genotypes (Figure 4G–I). Overall, these data demonstrate that loss of CYLD in glutamatergic neurons impacts neuronal activation and excitatory synaptic transmission in the BLA, which might be associated with aberrant cued fear expression of male cKO mice.

Figure 4.

Glutamatergic CYLD deficiency results in impaired excitatory synaptic transmission in the BLA.

(A) Representative sEPSC traces recorded from BLA pyramidal neurons. (B, C) Cumulative probability plots for amplitude (B) and inter-event interval (C) of sEPSC. Inset: average sEPSC amplitude and frequency, respectively. Identical sEPSC amplitude and frequency were recorded in neurons from both cKO and WT mice (means ± SEM, n = 14 cells from 4 mice, per group; sEPSC amplitude: Student’s t-test, t₍₂₆₎ = 1.558, P = 0.131. sEPSC frequency: Mann–Whitney U test, U = 91, P = 0.769). (D) Representative mEPSC traces recorded from BLA pyramidal neurons. (E) Cumulative probability plots for mEPSC amplitude. Inset: average mEPSC amplitude. Pyramidal neurons in the BLA of cKO mice showed a significant decrease in mEPSC amplitude (means ± SEM, WT: n = 14 cells from 3 mice, cKO: n = 16 cells from 3 mice; two-sample t-test with Welch’s correction, t_(18.53) = 2.245, P = 0.037). (F) Cumulative probability plots for the inter-event interval of mEPSC. Inset: average mEPSC frequency. The mEPSC frequency was unchanged in BLA pyramidal neurons of cKO mice (means ± SEM, WT: n = 14 cells from 3 mice, cKO: n = 16 cells from 3 mice; two-sample t-test with Welch’s correction, t_(20.16) = 1.297, P = 0.21). (G) Representative mIPSC traces recorded from BLA pyramidal neurons. (H, I) Cumulative probability plots for amplitude (H) and inter-event interval (I) of mIPSC. Inset: average mIPSC amplitude and frequency, respectively. Both amplitude and frequency of mIPSC were unaltered in BLA pyramidal neurons of cKO mice compared with that in WT controls (means ± SEM, WT: n = 12 cells from three mice, cKO: n = 18 cells from three mice; mIPSC amplitude, Student’s t-test, t₍₂₈₎ = 0.632, P = 0.533. mIPSC frequency, Mann–Whitney U test, U = 80, P = 0.248). BLA: Basolateral amygdala; cKO: conditional knockout; CYLD: cylindromatosis; mEPSC: miniature excitatory postsynaptic current; mIPSC: miniature inhibitory postsynaptic current; n.s.: not significant; sEPSC: spontaneous excitatory postsynaptic current; WT: wild-type.

Glutamatergic CYLD deficiency alters protein levels of GluN1 and GluA1 in the basolateral amygdala

Several studies have identified a modulatory effect of CYLD on activity of PSD components including N-methyl-D-aspartate receptors (NMDARs) and -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) (Ma et al., 2017; Zajicek et al., 2022; Tan et al., 2023b). Given that male cKO mice showed a decrease in mEPSC amplitude of BLA pyramidal neurons, we next detected levels of postsynaptic AMPARs and NMDARs relevant to fear memory in BLA homogenates of mice. Western blotting analysis (Figure 5A and B) showed significantly lower levels of GluN1 (P = 0.035) but not GluN2B in the BLA of cKO mice, consistent with weakened excitatory synaptic transmission of BLA pyramidal neurons in cKO mice. Interestingly, although GluA2 protein levels remain unchanged, higher GluA1 levels were observed in BLA homogenates in cKO mice (P = 0.025; Figure 5B). Collectively, these results indicate that glutamatergic CYLD is essential for maintaining normal levels of AMPARs and NMDARs in mouse BLA. These receptors are critical for producing appropriate neuronal activities and synaptic transmission, and thus fear expression (Johansen et al., 2011).

Figure 5.

Glutamatergic CYLD deficiency alters total protein levels of GluN1 and GluA1 in the BLA.

(A) Representative immunoblots of total protein from BLA homogenate of WT and cKO mice. (B) Quantification of immunoblots in A revealed that total protein levels of GluN1 were decreased while GluA1 levels were elevated in the BLA of cKO mice compared with that in WT group (means ± SEM, GluN1: n = 7 mice, per group; Student’s t-test, t₍₁₂₎ = 2.379, P = 0.035; GluN2B: n = 4 mice, per group; two-sample t-test with Welch’s correction, t_(3.174) = 1.501, P = 0.226; GluA1: n = 7 mice, per group; Student’s t-test, t₍₁₂₎ = 2.561, P = 0.025; GluA2: n = 4 mice, per group; Student’s t-test, t₍₆₎ = 0.642, P = 0.545). BLA: Basolateral amygdala; cKO: conditional knockout; CYLD: cylindromatosis; GluA1: glutamate receptor 1; GluA2: glutamate receptor 2; GluN1: N-methyl-D-aspartate receptor 1; GluN2B: N-methyl-D-aspartate receptor 2B; n.s.: not significant; WT: wild-type.

Glutamatergic CYLD deficiency induces over-activation of microglia in the basolateral amygdala

It has become abundantly clear that the function and development of synapses are greatly affected by neural–immune interactions (Zajicek and Yao, 2021). Numerous studies have observed that reactive microglia are critically involved in the functional brain changes and behavioral alterations (reflecting fear memory) associated with PTSD (Hori and Kim, 2019; Li et al., 2021b; Lee et al., 2022; Zhu et al., 2023). Our previous work uncovered a positive function of CYLD in preventing over-activation of microglia and neuroinflammation (Han et al., 2020). Consequently, we sought to identify from a different perspective another mechanism that might account for augmented fear expression of male cKO mice, namely, the regulatory role of CYLD in microglial function.

To gain initial insight into the relationship between CYLD and microglial activation, we determined whether resident microglia in the BLA were activated in naive Cyld^–/– mice. A significant increase in the number of Iba-1⁺ cells was observed (P = 0.009), indicating that microglia in the BLA of male Cyld^–/– mice were in an over-activated state (Figure 6A–D). Consistently, selective deletion of Cyld in glutamatergic neurons also evoked over-activation of microglia in the BLA of cKO mice. Although the intensity of Iba-1 as well as the number and soma size of Iba-1⁺ cells did not change (Figure 6E–H), we observed significantly decreased total process lengths (P = 0.0012; Figure 6I) and branch points of microglia in the BLA of cKO mice (P < 0.0001; Figure 6J). Moreover, we labeled Iba-1⁺ cells with CD68, a maker of activated microglia, and performed 3D reconstructions of microglia. Significant increases in CD68 puncta (P = 0.036; Figure 6K) and volume (P < 0.0001; Figure 6L) per microglia were observed in the BLA of cKO mice, further confirming over-activation of microglia. Notably, in line with unchanged CFM, which is hippocampus-dependent (Baldi and Bucherelli, 2015; Chaaya et al., 2018), no significant difference occurred in microglial morphology in the hippocampus of cKO mice (Figure 7A–J). Altogether, these results show that CYLD is essential to the physiological activities of glutamatergic neurons and microglia, and provides insight into the biological mechanisms of exaggerated cued fear memory.

Figure 6.

Deletion of CYLD in glutamatergic neurons leads to over-activation of microglia in the BLA.

(A) Representative images of Iba-1 labeled microglia (red, Alexa Fluor 594) in the BLA of Cyld^+/+ and Cyld^–/– mice. The Iba-1⁺ cell number was significantly increased in male Cyld^–/– mice compared to that in Cyld^+/+ group. Scale bar: 100 µm. Insets were enlarged images of areas in dotted square. Scale bar: 20 µm. (B–D) Quantitative analysis showed an increase in the Iba-1⁺ cell number but not in the relative Iba-1 intensity and soma area of microglia in male Cyld^–/– mice compared with that in Cyld^+/+ mice (means ± SEM, Cyld^+/+: n = 11 mice, Cyld^–/–: n = 10 mice; relative Iba-1 intensity: Student’s t-test, t₍₁₉₎ = 1.014, P = 0.323. Iba-1⁺ cell number: two-sample t-test with Welch’s correction, t_(10.9) = 3.176, P = 0.009. Soma area of microglia: Student’s t-test, t₍₁₉₎ = 1.642, P = 0.117). (E) Left: representative confocal images of Iba-1-CD68 co-labeled microglia (Iba-1: red, Alexa Fluor 594; CD68: green, far-red for imaging, Alexa Fluor 647) in the BLA of WT and cKO mice. The total process length and branch points of microglia were significantly decreased in the BLA of cKO mice. Scale bar: 20 µm. Right: 3D reconstruction image of the Iba-1-CD68 co-labeled microglia pointed by white arrow in the left image. The CD68 puncta and volume per microglia were significantly increased in male cKO mice. Scale bar: 3 µm. (F–L) Quantitative analysis showed that the total process length and branch points of microglia were significantly decreased, and the CD68 puncta and volume per microglia were significantly increased in male cKO mice compared to that in WT controls, leaving the parameters including relative Iba-1 intensity, Iba-1⁺ cell number and soma area of microglia unchanged (means ± SEM, relative Iba-1 intensity: n = 7 mice, per group, Student’s t-test, t₍₁₂₎ = 0.721, P = 0.485; Iba-1⁺ cell number: n = 7 mice, per group, Student’s t-test, t₍₁₂₎ = 0.825, P = 0.425; the soma area of microglia: n = 7 mice, per group, Student’s t-test, t₍₁₂₎ = 0.797, P = 0.441; total process length: n = 7 mice, per group, Mann–Whitney U test, U = 1, P = 0.0012; microglia branch points: WT: n = 33 cells from four mice, cKO: n = 34 cells from four mice, Mann–Whitney U test, U = 86.5, P < 0.0001; CD68 puncta per microglia: WT: n = 35 cells from four mice, cKO: n = 37 cells from four mice, Student’s t-test, t₍₇₀₎ = 2.136, P = 0.036; CD68 volume per microglia: WT: n = 32 cells from 4 mice, cKO: n = 38 cells from four mice, Mann–Whitney U test, U = 275, P < 0.0001). 3D: Three dimensional; BLA: basolateral amygdala; cKO: conditional knockout; CYLD: cylindromatosis; Iba-1: ionized calcium binding adapter molecule 1; n.s.: not significant; WT: wild-type.

Figure 7.

Glutamatergic neuron-specific knockout of CYLD does not cause microglial activation in CA1 and CA3 regions of the hippocampus.

(A) Representative images of Iba-1 labeled microglia (red, Alexa Fluor 594) in the CA1 regions of the hippocampus in WT and cKO mice. Scale bar: 100 µm. The insets showed magnified images of areas in dotted square. Scale bar: 20 µm. The CA1 microglia of cKO mice was in resting state. (B–E) Quantitative analysis showed that the degree of microglial activation in CA1 regions of cKO mice was comparable to that in WT controls (means ± SEM, CA1, n = 7 mice, per group; total process length: Student’s t-test, t₍₁₂₎ = 1.261, P = 0.231. Relative Iba-1 intensity: Student’s t-test, t₍₁₂₎ = 0.812, P = 0.433. Iba-1⁺ cell number: Student’s t-test, t₍₁₂₎ = 0.342, P = 0.739. Soma area of microglia: Student’s t-test, t₍₁₂₎ = 0.025, P = 0.981). (F) Representative images of Iba-1 labeled microglia (red, Alexa Fluor 594) in the CA3 regions of the hippocampus in WT and cKO mice. Scale bar: 100 µm. The insets showed magnified images of areas in dotted squares. Scale bar: 20 µm. The microglia in the CA3 region of cKO mice were in resting state. (G–J) Quantitative analysis showed that the degree of microglial activation in CA3 regions of cKO mice was comparable to that in WT controls (means ± SEM, CA3, n = 7 mice, per group; total process length: Student’s t-test, t₍₁₂₎ = 1.568, P = 0.143. Relative Iba-1 intensity: Student’s t-test, t₍₁₂₎ = 0.823, P = 0.426. Iba-1⁺ cell number: Mann–Whitney U test, U = 22, P = 0.805. Soma area of microglia: Student’s t-test, t₍₁₂₎ = 0.07, P = 0.945). CYLD: Cylindromatosis; cKO: conditional knockout; Iba-1: ionized calcium binding adapter molecule 1; n.s.: not significant; WT: wild-type.

Discussion

Over the last decade, CYLD has been implicated as an important regulator of synaptic homeostasis (Dosemeci et al., 2013; Ma et al., 2017; Kim et al., 2019; Zajicek and Yao, 2021). In the present study, we found that the levels of CYLD are significantly reduced in the BLA of fear-conditioned mice compared to control (tone-only) mice. We thus generated a cKO mouse model and conducted behavioral analyses in cKO and control mice. Our results showed that CYLD in glutamatergic neurons exerts sex-specific effects on cued fear expression, with the loss of CYLD in glutamatergic neurons resulting in augmented cued fear memory in male but not female mice. Furthermore, excessive neuronal activation after cued fear retrieval and impaired excitatory synaptic transmission were observed in cKO mice, together with changed protein levels of AMPAR and NMDAR subunits in the BLA. Moreover, we found over-activated microglia in the BLA, but not hippocampus, of cKO mice. Thus, we uncovered an essential role of glutamatergic CYLD in the regulation of cued fear expression.

Initially, decreased CYLD levels were found in the BLA of mice undergoing the tone-cued test, indicating involvement of CYLD in cued fear expression. Hebbian processes can activate NMDARs, resulting in calcium elevation in postsynaptic neurons (Dore et al., 2017). Increased intracellular calcium levels promote auto-phosphorylation of calcium/calmodulin-dependent protein kinase II, which is integral to the occurrence of fear memory (Johansen et al., 2011). In neuronal cultures, activation of CYLD relies on calcium/calmodulin-dependent protein kinase II activity, which subsequently enhances the deubiquitinase activity of CYLD (Thein et al., 2014). We thus reasoned that decreased CYLD levels in the BLA after cued fear retrieval might attribute, at least in part, to activity-dependent regulation of CYLD deubiquitinase activity during Hebbian processes.

Conventional knockout of Cyld has a profound influence on neuronal activity and synaptic transmission of BLA pyramidal neurons (Li et al., 2021a). Therefore, we next constructed a cKO mouse line of Cyld deletion in only glutamatergic neurons to identify a neuron-type specific role of CYLD. Previous behavioral testing uncovered abnormalities in sociability, cognition, fear memory, repetitive-like, anxiety-like, and depression-like phenotypes in Cyld^–/– mice (Han et al., 2023). Of note, male mice only or mice of both genders were used in the above studies. By contrast, we specifically addressed the role of glutamatergic CYLD in fear memory of cKO mice of both sexes considering the sexual dimorphism in neural activities of fear memory (Ramikie and Ressler, 2018; Bauer, 2023). We showed that selective deletion of CYLD in glutamatergic neurons led to exaggerated cued fear expression in male mice only, differing from the dampened cued fear expression in Cyld^–/– mice (Li et al., 2021a). Furthermore, sociability, anxiety-, depression-, and repetitive-like behaviors were normal in male cKO mice. These differences might be due to the absence of CYLD in only glutamatergic neurons in cKO compared with Cyld^–/– mice. It is likely that CYLD in other neuronal types may contribute to the behavioral phenotypes of Cyld^–/– mice, which represents a comprehensive representation of conventional knockout of Cyld. Notably, glutamatergic CYLD deficiency exerts sexually dimorphic effects on cued fear expression. This might be due to sexual dimorphism in the structure, neuronal morphology, expression of sex hormone receptors, synaptic plasticity, and intracellular processing during formation and retrieval of fear memory in fear-related regions including the hippocampus, amygdala, and medial prefrontal cortex (Ramikie and Ressler, 2018; Bauer, 2023; Fleischer and Frick, 2023). It was proposed that even subtle sex differences of neural substrates cause a crucial discrepancy in behavioral circuit and molecular recruitment in memory formation and retrieval (Tronson and Keiser, 2019). Interestingly, loss of glutamatergic CYLD did not affect fear acquisition of mice, as we had observed with conventional Cyld knockout mice (Li et al., 2021a). The acquisition phase of cued fear is thought to depend on the activities of existing synaptic proteins, while the consolidation period (24-hour memory processing stage after TFC) is closely related to de novo synthesis of new proteins (Johansen et al., 2011). We propose that CYLD might exert less effect on the function of existing proteins involved in fear acquisition, whereas it plays a vital role in the process of new protein synthesis, which is critical to cued fear expression 24 hours after TFC. In addition, it is worth mentioning that both conventional Cyld knockout and glutamatergic Cyld deletion affected cued fear expression without influencing CFM. During Pavlovian fear conditioning, the hippocampus is responsible for dealing with context information while the BLA merges the auditory cue/context cue with aversive shock stimuli (Chaaya et al., 2018). Together with the fact that CYLD is more abundant in the BLA than the hippocampus (biogps.org; mouse.brain-map.org), CYLD activities might play a critical role in BLA-dependent auditory cue processing but exert little impact on hippocampus-based remembering of the context cue.

Cued fear memory is highly dependent on appropriate neuronal activities as well as synaptic transmission in the BLA (Johansen et al., 2011; Sun et al., 2018). In the current study, we found that glutamatergic CYLD deficiency resulted in elevated neuronal activation in the BLA of mice following a tone-cued test, which coincided with the exaggerated cued fear expression of cKO mice. Specifically deleting glutamatergic CYLD decreased the amplitude of mEPSCs in BLA pyramidal neurons, indicating reduced miniature excitatory synaptic activity under baseline conditions. This is similar to reduced mEPSC amplitudes of BLA pyramidal neurons in conventional Cyld knockout mice (Li et al., 2021a). Notably, reduced excitatory synaptic transmission was accompanied by reduced GluN1 levels but increased levels of GluA1 in the BLA of cKO mice. This differed from a recent study showing increased levels of GluN1 in the hippocampus of Cyld^–/– mice (Zajicek et al., 2022). Therefore, CYLD may function in a brain region-, neuronal type-, and circuit-specific manner (Colombo et al., 2021). There are two possible mechanisms underlying the regulatory role of CYLD in altering levels of glutamate receptors: 1. CYLD functions as a K63 deubiquitinase of PSD-95, stabilizing the PSD structure and thus affecting the activities of AMPARs (Ma et al., 2017); and 2. CYLD regulates the trafficking of AMPARs via Akt–mTOR autophagy signaling (Colombo et al., 2021; Zajicek et al., 2022). Together with findings that glutamatergic neurons in the amygdala, medial prefrontal cortex, and hippocampus collectively play an essential role in regulating fear expression (Marek et al., 2013; Maroun, 2013; Bocchio et al., 2017; Chen et al., 2017), enhanced neuronal activation, compromised mEPSCs, and altered levels of GluN1 and GluA1 in the BLA might contribute, at least in part, to the aberrant cued fear expression in cKO mice.

Our data suggest that microglia are over-activated in the BLA of both cKO and Cyld^–/– mice. Microglia play critical roles in neuroinflammation, synapse pruning, brain injury, and neurogenesis (Lyman et al., 2014). Microglia-mediated neuroinflammation is a neuropathological feature of many neuropsychiatric diseases including PTSD, depression, and anxiety (Hori and Kim, 2019; Cui et al., 2023; Zhu et al., 2023). Known for participating in innate immune process by regulating the NF-κB pathway (Kovalenko et al., 2003), CYLD appears to act as a positive mediator for preventing over-activation of microglia (Han et al., 2020). Likewise, in the present study, we show that microglia in the BLA are in an over-activated state in both naive Cyld^–/– and cKO mice. Neurotransmitters, cytokines, and the complement system can mediate microglia–neuron cross-talk (Marinelli et al., 2019). Reciprocal interactions between microglia and neurons help to dynamically regulate neuronal function (Shabab et al., 2017; Marinelli et al., 2019). Therefore, over-activated microglia observed in this study may also affect BLA neuronal activity via unknown mechanisms, which deserve future studies.

In summary, we have identified a role for glutamatergic CYLD in cued fear expression and the possible underlying mechanisms. We provide evidence that CYLD deficiency in glutamatergic neurons altered cued fear expression and was associated with elevated neuronal activation, aberrant excitatory synaptic transmission, abnormal levels of GluN1 and GluA1, and excessive microglial activation in the BLA of male mice. Our findings offer insightful patho-mechanisms and potential drug targets for fear-related disorders such as PTSD. As glutamatergic CYLD was non-selectively deleted in the whole brain of cKO mice, loss of glutamatergic CYLD in medial prefrontal cortex may also contribute to aberrant cued fear expression of cKO mice. Selectively deleting BLA glutamatergic CYLD may help to further confirm the results of the current study.

Additional files:

Additional Figure 1 ^{(2.1MB, tif)} : Glutamatergic CYLD deficiency has no effect on motor function and anxiety level of male mice.

Additional Figure 1

Glutamatergic CYLD deficiency has no effect on motor function and anxiety level of male mice.

(A) Basal locomotion activity of male cKO mice was shown normal in 30 minute-OFT (means ± SEM, n = 12 mice; Student’s t-test, t₍₂₂₎ = 0.647, P = 0.524). (B, C) Motor functions did not differ significantly across the genotypes in males (means ± SEM, WT: n = 8 mice, cKO: n = 9 mice; Student’s t-test, all limb grip strength test: t₍₁₅₎ = 0.64, P = 0.532. wire hang test: t₍₁₅₎ = 0.899, P = 0.383). (D) Motor learning of male cKO mice was shown not affected in rotarod test compared with WT groups (means ± SEM, WT: n = 8 mice, cKO: n = 9 mice; two-way analysis of variance, genotype effect: F_{(1, 81)} = 0.526, P = 0.471). (E) Representative traces of 10 minute-OFT. (F, G) Both cKO mice and WT controls showed similar travel distance and time spent in the center in OFT (means ± SEM, n = 16 mice, per group; Student’s t-test, total distance: t₍₃₀₎ = 0.378, P = 0.708, duration in the center: t(30) = 0.675, P = 0.505). (H) Representative traces of EPM. (I, J) There were no differences in number of entries to the open arms and time in the open arms between cKO and WT groups (means ± SEM, WT: n = 12 mice, cKO: n = 13 mice; Student’s t-test, entries to the open arms: t₍₂₃₎ = 0.055, P = 0.957, time in the open arms: t₍₂₃₎ = 0.353, P = 0.727). cKO: Conditional knockout; CYLD: Cylindromatosis; EPM: elevated plus maze; n.s.: not significant; OFT: open field test; WT: wild-type.

Additional Figure 2 ^{(1.7MB, tif)} : Glutamatergic CYLD deficiency has no effect on sociability, repetitive, and depression-like behavior of male mice.

Additional Figure 2

Glutamatergic CYLD deficiency has no effect on sociability, repetitive, and depression-like behavior of male mice.

(A-D) The male cKO mice exhibited normal sociability (A, B) and social novelty (C-D) in three-chamber social test. O, object; S1, stranger 1 mouse; S2, stranger 2 mouse. Sniffing time was refered as time spent sniffing the S1 (A) and the S2 (C) during the social interaction phase (means ± SEM, n = 8 mice, per group; Student’s t-test, (A): t₍₁₄₎ = 0.563, P = 0.583, (B): t₍₁₄₎ = 0.0927, P = 0.927, (C): t₍₁₄₎ = 1.572, P = 0.138, (D): t₍₁₄₎ = 0.137, P = 0.893). (E) The male cKO mice did not show repetitive behavior in marble burying test (means ± SEM, n = 5 mice, per group; Mann–Whitney test, U = 7, P = 0.302). (F, G) Male cKO mice did not show depression-like behaviors (means ± SEM, n = 5 mice, per group; Student’s t-test, FST: t₍₈₎ = 1.1, P = 0.304. TST: t₍₈₎ = 0.311, P = 0.764). cKO: Conditional knockout; CYLD: Cylindromatosis; FST: forced-swimming test; n.s.: not significant; TST: tail suspension test; WT: wild-type.

Funding Statement

Funding: This study was supported by the National Natural Science Foundation of China, Nos. 32371065 (to CL) and 32170950 (to LY); the Natural Science Foundation of the Guangdong Province, No. 2023A1515010899 (to CL); and the Science and Technology Projects in Guangzhou, Nos. 2023A4J0578 and 2024A03J0180 (to CW).

Data availability statement:

All data generated or analyzed during this study are included in this published article and its Additional files.