Next Article in Journal
Preclinical Tumorigenicity Study of an Advanced Therapy Medicinal Product for Diffuse Cartilage Lesions in an Osteoarthritic Environment
Next Article in Special Issue
TRPC6-Mediated Ca2+ Influx Activates MAPK and NFκB Signaling and Elicits Pro-Inflammatory and Catabolic Responses in Human Intervertebral Disc Cells
Previous Article in Journal
Exploring Adipose Tissue Complexity Through Omics Approaches: Implications for Health and Disease
Previous Article in Special Issue
TRPA1 Expressed by Hepatocytes and Liver Macrophages Does Not Mediate Inflammatory Infiltration and Steatosis in a Mouse Model of Chronic Alcohol-Induced Liver Injury
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Cells
Volume 15
Issue 5
10.3390/cells15050428
cells-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Deletion of TRPA1 Ion Channel Modulates the Central Stress Responses in a Mouse Model of Posttraumatic Stress Disorder

by
János Konkoly
1,
Laura Mária Szegner
1,
Tünde Biró-Sütő
1,
Eszter Luspay
1,
Prabhat Kumar
2,
Erika Kvak
2,
Balázs Gaszner
3,
Gergely Berta
4,
Erika Pintér
1,
Dóra Zelena
2,† and
Viktória Kormos
1,*,†
1
Department of Pharmacology and Pharmacotherapy, Medical School, University of Pécs, H-7624 Pécs, Hungary
2
Institute of Physiology, Medical School, University of Pécs, H-7624 Pécs, Hungary
3
Department of Anatomy, Medical School, University of Pécs, H-7624 Pécs, Hungary
4
Department of Medical Biology, Medical School, University of Pécs, H-7624 Pécs, Hungary
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work and shared last authorship.
Cells 2026, 15(5), 428; https://doi.org/10.3390/cells15050428
Submission received: 16 December 2025 / Revised: 11 February 2026 / Accepted: 27 February 2026 / Published: 28 February 2026
(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels and Health and Disease)
Browse Figures
Versions Notes

Highlights

What are the main findings?
  • Genetic deletion of Trpa1 exaggerated TH level in the LC and reduced astrogliosis in PVN in a foot shock-induced mouse model of PTSD.
  • Genetic deletion of Trpa1 did not influence the effects of clonidine treatment on PTSD-related behavior.
What are the implications of the main findings?
  • TRPA1 ion channel may support stress adaptation in PTSD through LC and PVN.
  • This effect is not α2-adrenoceptor-mediated.

Abstract

Background: Posttraumatic stress disorder (PTSD) is a mental illness in which central stress-regulating regions, including locus coeruleus (LC) and paraventricular nucleus of hypothalamus (PVN), play key roles. Clonidine, a central sympatholytic drug, can inhibit LC activity and reduce PTSD-related symptoms, suggesting noradrenergic involvement. Glia-driven immune mechanisms may link LC activity to PVN responses. Since TRPA1 ion channel is implicated in both neuroinflammation and stress adaptation, we aimed to determine whether its presence modulates the function of brain structures contributing to PTSD-related alteration in central stress adaptation. Methods: Foot shock PTSD model was applied to Trpa1 wild-type (WT) and knockout (KO) mice, and outcomes were assessed four weeks later. Immunohistochemistry was used to evaluate tyrosine hydroxylase (TH) levels in the LC and glial activation in the PVN. Behavioral effects of clonidine and circulating corticosterone levels were also examined. Results: Stress increased LC/TH immunoreactivity and PVN glial activation. Trpa1 deletion exaggerated LC/TH responses but reduced PVN astrocyte activation. Clonidine increased freezing and decreased jumping (a hyperarousal marker). KO mice showed enhanced jumping and did not respond to clonidine. Corticosterone levels remained unchanged. Conclusions: TRPA1 may support stress adaptation in PTSD by regulating LC noradrenergic output and PVN neuroinflammation, independently of α2-adrenergic signaling.

1. Introduction

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel involved in diverse patho (physiological) processes of the nervous system including pain sensation and neurodegeneration [1,2,3]. Trpa1 is expressed in several stress sensitive brain regions, such as the piriform cortex, olfactory bulb, hypothalamus and dorsal raphe nucleus [4,5,6]. Its highest mRNA level was found in the urocortinergic neurons of the centrally projecting Edinger-Westphal nucleus (EWcp), an important hub of stress adaptation [7,8,9,10]. The downregulation of Trpa1 mRNA was observed both in the EWcp neurons of mice upon chronic stress, and in the EWcp samples of human suicide victims [11]. In addition, altered stress responses were revealed in Trpa1 gene-deficient mice using diverse stress models [11,12], further supporting the role of this ion channel in stress adaptation.
Posttraumatic stress disorder (PTSD) is a mental illness induced by traumatic events leading to disturbed stress adaptation. The symptoms involve intrusive memories, flashbacks and nightmares remembering the inducing trauma. Generalized anxiety, hyperarousal and severe depression are also commonly associated with PTSD, resulting in social deprivation and precipitating, ultimately, in problems at work and in private life [13,14].
The locus coeruleus (LC) and the paraventricular nucleus of the hypothalamus (PVN) are key brain structures of stress adaptation, and their role is also extensively studied in the pathomechanism of PTSD [15,16,17,18]. LC, the center of the sympatho-adrenomedullary (SAM) system, contains primarily noradrenergic neurons, which are the major regulators of Cannon’s “fight or flight” response together with the adrenal medulla [19,20]. LC is involved in numerous other psychophysiological functions (e.g., arousal, memory formation, pain processing, behavioral flexibility), each of them severely affected by PTSD [20,21]. LC is well positioned to affect PVN function through noradrenergic projections via α2-receptors [22,23]. PVN is the central component of the hypothalamic–pituitary–adrenal (HPA) axis, which is also activated upon stress, leading to the secretion of corticotropin-releasing hormone (CRH). CRH stimulates the anterior pituitary to release adrenocorticotropic hormone, which finally induces the synthesis and secretion of glucocorticoids from the adrenal cortex. Glucocorticoids such as cortisol in humans or corticosterone (CORT) in mice act on various target organs to promote adaptation to stressful situations (e.g., diabetogenic effect, increased metabolism, influence on cognitive functions) and parallelly inhibit further activation of the HPA axis [24,25]. Interestingly, the reduced HPA axis and increased SAM system activity is characteristic for PTSD [15,16], accompanied by elevated CRH levels and catecholamine mobilization, as well as decreased glucocorticoid levels [17,18]. These processes may contribute to the development of vegetative symptoms and hyperarousal, typical signs of PTSD; the lacking anti-inflammatory effect of glucocorticoids may maintain the neuroinflammation [17,18,26].
Indeed, neuroinflammation is the most widely studied process in the pathomechanism of PTSD. Acute severe or chronic stress can directly activate microglia and astrocytes in the central nervous system. These glial cells produce proinflammatory cytokines contributing to the development of neuroinflammation. These glial processes can be influenced by α-adrenergic receptors [27,28]; it is highly plausible that LC-derived noradrenaline modulates PVN glia (particularly astrocytes). On the other hand, the glia cells of the PVN modulate its neuronal output and sympathetic/endocrine function [29,30,31]. Interestingly, TRPA1 ion channels were previously found on astrocytes [32,33,34,35,36] and some of the inflammatory mediators (e.g., unsaturated fatty acids, H2O2) can activate them [2,37,38]. Additionally, the increased cytokine levels may maintain the stress response via promoting the secretion of CRH from the hypothalamus [17,18].
Treatment strategies of PTSD include cognitive psychotherapy, as well as symptomatic treatment using antidepressants for mood disorders and sympatholytic agents such as clonidine (α2 -adrenergic receptor agonist) for vegetative symptoms, nightmares and hyperarousal [39,40,41,42]. Since the therapeutic options described above do not provide causal therapy, it is justified to explore new molecular mechanisms that could provide promising targets.
We hypothesized that the presence of TRPA1 ion channels modulates the function of key brain structures—such as LC and PVN involved in stress adaptation—and PTSD-like behavior is regulated via this. The noradrenergic outflow plays a pivotal role in the interplay between these centers via α2-mediated mechanisms, and we assumed that TRPA1 may indirectly affect this interaction. To test these hypotheses (i) we investigated the regulatory impact of TRPA1 channel on the noradrenergic cells of LC and on the connected neuroinflammatory responses in the PVN using an electric foot shock model of PTSD and a Trpa1 knockout (KO) mice line; then, (ii) the subsequent question was the involvement of α2-adrenoceptors in the process, which was studied by clonidine treatment, behavioral tests and serum CORT outcomes.

2. Materials and Methods

2.1. Animals

The studies were carried out on 3–4 months old male Trpa1+/+ (WT) and Trpa1−/− (KO) mice obtained from Prof. P. Geppetti (University of Florence, Italy) originally generated by Bautista et al. [43]. Animals were bred on C57BL/6J background and crossed back after 10 generations. Offspring were genotyped for Trpa1 gene by PCR (sequences of primers: ASM2: ATC ACC TAC CAG TAA GTT CAT; ASP2: AGC TGC ATG TGT GAA TTA AAT).
Involvement of females from all stages of estrous cycles and/or after ovariectomy with and without hormone replacement would dramatically increase the number of groups involved. Thus, due to ethical, financial and infrastructural considerations we used only males. Animals were kept in standard polycarbonate cages (330 × 160 × 130 mm, 5–7 mice/cage or 330 × 100 × 130 mm, 2–5 mice/cage) in a temperature (20–24 °C) and humidity (50–60%) controlled 12–12 h light–dark cycle environment (lights on at 6 a.m.) at the Department of Pharmacology and Pharmacotherapy of the University of Pécs. Ad libitum standard rodent chow (LT/n, Szinbád LLC., Gödöllő, Hungary) and tap water were provided for the mice. Besides the presence of littermates, paper rolls were placed into the cages to enrich the environment. Microbiological monitoring was performed annually (FELASA “S”). All trials were carried out during the light phase of animals between 9 a.m. and 14 a.m.
All experiments were approved by the Animal Welfare Committee of the University of Pécs and by the National Scientific Ethical Committee on Animal Experimentation in Hungary (permission No: BA02/2000-46/2024), in agreement with the directive of the European Communities Council in 1986, and with the Law of XXCIII in 1998 on Animal Care and Use in Hungary. During the experiments, every effort was made to minimize the number and suffering of the animals. The authors complied with the ARRIVE guidelines.

2.2. Experimental Design

Experiment I investigated the regulatory impact of TRPA1 ion channel on stress-related brain areas involved in the pathomechanism of PTSD (Figure 1A–C). Half of Trpa1 WT and KO mice was exposed to the foot shock protocol (fear conditioning, FC), while the other half of the two genotypes were used as non-stressed controls. After, FC animals were placed back in their original cages for 4 weeks. Then, conditioned fear test (CFT) was applied followed by brain sample collection.
Experiment II analyzed the effect of TRPA1 ion channel on the behavioral responses and serum CORT levels upon the clonidine treatment (Figure 1D,E). All mice underwent FC with foot shock. After 4 weeks resting, half of the WT and KO animals were pretreated intraperitoneally (i.p.) with clonidine (0.05 mg/kg), while the other half received i.p. saline (vehicle); a subsequent CFT was performed 30 min later followed immediately by blood sampling within 5 min. Tail blood samples were collected from a cut under brief isoflurane anesthesia and local heating into heparinized hematocrit capillaries.

2.3. Behavioral Tests

2.3.1. Fear Conditioning (FC)

The mice were put into the plastic FC chamber (internal size: 25.5 (d) × 25.5 (w) × 36 (h) cm; Ugo Basile, Gemonio, Italy) through a circular front door [44] (Figure 1A). The chamber was housed in sound-attenuating cabinets with white noise (60–70 dB) and was equipped with infrared-sensitive cameras. After a stimulus-free habituation period of 150 s, seven 1 s long foot shocks (1.4 mA) with random intervals (40–60 s) were applied together with a 10 s tone (3000 Hz, 85 dB) and increased light intensity to enhance aversiveness. The shock was delivered at the end of the tone/light and co-terminated with that in each case. After 660 s the mice were gently transferred back into their home cages. The box was cleared with tap water and 20% ethanol. Non-stressed control animals were placed into the same chamber without delivering foot shock.

2.3.2. Conditioned Fear Test (CFT)

Animals were reintroduced for 660 s into the FC chamber 4 weeks after the original stress. We used the same background and cleaning materials as those used during the FC phase. Here, none of the mice underwent foot shock; after the habituation period, only the above-mentioned tone and light stimuli were applied in each animal seven times with random intervals. Thus, the first 150 s (baseline) represented the context-dependent, while the stimulus periods showed the cue-related conditioned fear response with interstimulus intervals (ISI) between the cues (context dependency with carry-over effects). The behavior was video recorded and scored later by an experimenter blinded to the treatment groups using computer-based event-recorder software (Solomon coder; version: 17.03.22). We assessed the duration of freezing (time spent immobility for more than one second) [45,46,47] and the frequency of jumping representing an active escape attempt from the threatening situation [47,48,49,50] both associated with PTSD. We measured both the total amount of the listed behavioral parameters over the 11 min period of CFT, and we also separately analyzed the contextual and the cued fear reactions.

2.4. Perfusion and Brain Sample Collection

To exclude the effect of acute stress on the neuromorphological results, perfusion and brain sample collection were performed 24 h after CFT in Exp. I. Mice were deeply anesthetized by i.p. urethane injection (2.4 g/kg) and transcardially perfused with 20 mL ice-cold 0.1 mol/L phosphate-buffered saline (PBS, pH: 7.4). This was followed by fixation with 150 mL of 4% paraformaldehyde (PFA) solution in Millonig buffer (pH 7.4) for 15 min. Subsequently, brains were removed and collected into PFA for 72 h postfixation at 4 °C, then samples were coronally sectioned using a Leica VT1000 S vibratome (Leica Biosystems, Wetzlar, Germany). Three series of 30 μm sections were collected and stored in PBS containing sodium-azide (0.01%) at 4 °C. For long term storage at −20 °C, slices were transferred into antifreeze solution (20% ethylene glycol, 30% glycerol and 0.1 mol/L sodium-phosphate buffer). Brain sections containing the LC (from Bregma −5.34 mm to −5.80 mm) or PVN (from Bregma −0.58 mm to −1.22 mm) were used for histological evaluation [51].

2.5. Immunohistochemistry

2.5.1. Tyrosine Hydroxylase (TH) Immunohistochemistry in Locus Coeruleus (LC)

Sections were first washed for 2 × 15 min with PBS and treated with citrate solution at 90 °C for 10 min. After cooling and washing the samples, 0.5% Triton X-100 (Sigma Chemical, Zwijndrecht, The Netherlands) in PBS was applied for 30 min followed by blocking of non-specific binding sites with 2% normal donkey serum (NDS) in PBS. Then, sections were incubated overnight at room temperature (RT) with a solution containing polyclonal rabbit anti-TH antibody (Abcam, Cambridge, UK, Cat. No.: ab6211) diluted to 1:4000 in PBS with 2% NDS. After 2 × 15 min washes in PBS, sections were incubated with Cy3-conjugated donkey anti-rabbit secondary antibody (Jackson, West Grove, PA, USA, Cat. No: 711-165-152) in 1:500 PBS with 2% NDS for 3 h at RT. After washing the samples, sections were mounted on gelatine-coated glass slides, then covered with glycerol-PBS (1:1) and stored at −20 °C until confocal microscopy. All samples were stained in one session.

2.5.2. Immunohistochemistry on Glial Markers in the Paraventricular Nucleus of the Hypothalamus (PVN)

Glia cells were visualized by double immunostaining using markers specific for astrocytes (glial fibrillary acidic protein; GFAP) and for microglia (ionized calcium binding adaptor molecule 1, IBA1). The pretreatment of sections and the non-specific binding site blocking was carried out as described in Section 2.5.1. Then, monoclonal mouse anti-GFAP (Novocastra, Newcastle upon Tyne, UK, Cat. No.: NCL-GFAP-GA5) and polyclonal rabbit anti-IBA1 (Fuji-Wako, Osaka, Japan, Cat. No.: 019-19741) primary antibodies both diluted to 1:2000 with 2% NDS were used overnight at RT. After washing, Cy3-conjugated donkey anti-mouse (Jackson, Cat. No.: 715-165-150) and Alexa 488-conjugated donkey anti-rabbit (Jackson, Cat. No.: 711-005-152) secondary antibodies both in 1:500 dilution with 2% NDS were applied for 3 h at RT. Sections were mounted, covered and stored according to the protocol mentioned above. All samples were stained in one session.

2.6. Image Analysis

Olympus Fluoview FV-1000 laser scanning confocal microscope (Olympus Europa, Hamburg, Germany) and FluoView FV image acquisition software system (Olympus Europa, Hamburg, Germany) were used for imaging. Digital images were acquired by sequential scanning in analog mode for the corresponding fluorophores to avoid false positive signal resulting from the slight overlap of emission spectra and to reliably quantify the fluorescent signal. The confocal aperture was set to 80 μm. The analog sequential scanning was performed using a 40× objective lens (NA: 0.75). An optical thickness of 3.5 µm was indicated by the software and the resolution was set to 1024 × 1024 pixel. The excitation time was set to 4 µs per pixel. Alexa Fluor 488 was excited at 488 nm and Cy3 at 543 nm. We assigned the virtual color green for Alexa Fluor 488 and red for Cy3. The same microscope settings were used for imaging on all samples deriving from a particular experiment.
The morphometry of LC was performed on non-edited pictures using ImageJ software (version 1.52a) by an experimenter who was blind to the experimental conditions. The intensity of TH-immunofluorescence was measured in all cells visible in the 3.5 µm thick cross-sectional images if the plane of sectioning contained the cell nucleus, (ca. 10–30 cell bodies per LC samples). The region of interest was marked out manually including only the cytoplasmic areas of neurons. The specific signal density (SSD) was determined in arbitrary units (a.u.) and corrected for the background signal. The average of SSDs was calculated in each section. The calculation was performed in four sections per animal, and the average of these four values represented the SSD of one mouse, which was finally subjected to the statistical assessment.
In case of glia activation, we determined the number and the activation score of astrocytes and microglia in the total area of PVN cross-sections with a thickness of 3.5 µm using GFAP and IBA1 immunostaining. We analyzed 60–120 cells per section for astrocytes and 10–20 cells per section for microglia and 4 slices per animal. The degree of glia activation in each counted cell was ranked from 1 (resting) to 5 (severe reactive gliosis) [52]. The main analyzed parameters were the form and enlargement of cell bodies and the complexity and thickness of processes. The glia cells were analyzed on four sections per animal, and the average of the individual scores of each glia cells on the four sections represented the activation score of the mouse.

2.7. Corticosterone Level by Radioimmunoassay

Blood samples were centrifuged at 3000 rpm for 5 min, then plasma was stored at −20 °C until further analysis. Corticosterone levels were analyzed from 10 μL sample using a specific antibody developed by the Institute of Experimental Medicine (Budapest, Hungary) [53]. The intra-assay coefficients of variation were 7.5% and 4.7%, respectively. All samples from a particular experiment were analyzed in one session.

2.8. Statistical Analysis

Data were analyzed by two-way analysis of variance (ANOVA, variables: stress and genotype in Experiment I, as well as treatment and genotype in Experiment II) and repeated measures ANOVA (within-subjects factor: cue presentation), both followed by Tukey’s post hoc test (StatSoft 13.5.0, Tulsa, OK, USA). All datasets showed normal distribution (Kolmogorov–Smirnov) and homogenous variance (Levene). Pearson’s correlation was also conducted. p < 0.05 was considered statistically significant. Data are presented as mean ± SEM. The results of post hoc test are represented in the Figures.

3. Results

3.1. The Genetic Lack of TRPA1 Does Not Influence Stress-Induced Freezing but Enhance Jumping Behavior

Investigating the behavioral responses during the FC phase, increased freezing was detected in both stressed groups compared to the controls (Fstress (1,26) = 1774.46, p < 0.0001; both pWT and pKO = 0.0002), but neither the main effect of the genotype (Fgenotype (1,26) = 1.84, p = 0.19, pcontrol = 0.75; pshock = 0.79) nor an interaction was revealed (Finteraction (1,26) = 0.01, p = 0.92). Similarly, shock-induced jumping behavior in both groups (Fstress (1,26) = 842.27, p < 0.0001; both pWT and pKO = 0.0002) without genotype differences (Fgenotype (1,26) = 0.04, p = 0.85, pcontrol = 1.00; pshock = 0.99; Finteraction (1,26) = 0.04, p = 0.85).
When we reintroduced the animals to the stress-related environment four weeks later, they still showed exaggerated total freezing time (Fstress (1,26) = 914.54, p < 0.0001; both pWT and pKO = 0.0002) independently from genotype (Fgenotype (1,26) = 0.12, p = 0.73, pcontrol = 0.94; pshock = 0.69; Finteraction (1,26) = 1.35, p = 0.26) (Figure 2A). Further analysis confirmed the elevation of both contextual (including the baseline and the ISIs) and cued freezing behavior in stressed groups compared to the controls (Fstress (1,26) = 663.12, p < 0.0001; in each comparison both pWT and pKO < 0.0001) without the main effect of the genotype (Fgenotype (1,26) = 0.02, p = 0.89; Fstress*genotype (1,26) = 0.69, p = 0.41). Cue presentation decreased the freezing behavior of both stressed groups compared to the baseline and to the ISI values over the entire experiment (Fcue presentation (14,364) = 3.50, p < 0.0001, Fstress*cue presentation (14,364) = 6.55, p < 0.0001), whose effect was not influenced by the genotype (Fgenotype*cue presentation (14,364) = 1.21, p = 0.26; Fstress*genotype*cue presentation (14,364) = 0.98, p = 0.48) (Figure 2B).
However, the total amount of jumping behavior (as a sign of hyperarousal) was not only influenced by previous stress (Fstress (1,26) = 31.11, p < 0.0001; pWT = 0.09, while pKO = 0.0002), but also by the genotype (Fgenotype (1,26) = 4.53, p = 0.04; Finteraction (1,26) = 4.53, p = 0.04). More precisely, shocked Trpa1 KO mice jumped more than their WT counterparts (pcontrol = 1.00; pshock = 0.02) (Figure 2C). Interestingly, shocked animals showed a higher jumping rate compared to the controls only until the end of the second cue (Fstress (1,26) = 28.86, p < 0.0001; Fcue presentation (14,364) = 6.45, p < 0.0001; during Cue1: pWT < 0.0001 and pKO = 0.004, while during the baseline and Cue2 pKO < 0.0001), followed by a rapid decline (Fstress*cue presentation (14,364) = 6.45, p < 0.0001). Here, the stress-induced jumping response was also influenced by the genotype (Fgenotype (1,26) = 4.26, p = 0.049), as shocked KOs showed a higher jumping rate during the baseline (p = 0.02) and the second cue presentation (p = 0.002) compared to the WT counterparts (Fstress*genotype (1,26) = 4.67, p = 0.04), with a tendency for interaction between the genotype and cue presentation as well (Fgenotype*cue presentation (14,364) = 1.71, p = 0.051; Fstress*genotype*cue presentation (14,364) = 1.71, p = 0.051) (Figure 2D).
Additionally, Pearson’s test revealed that freezing positively correlated with jumping behavior (r = +0.678, p < 0.001).

3.2. The Genetic Lack of TRPA1 Is Associated with a Greater Increase in LC/TH Immunoreactivity After Foot Shock

Stress increased the TH immunopositivity in the LC of both genotypes (Fstress (1,26) = 40.07, p < 0.0001; pWT = 0.04, pKO = 0.0002) (Figure 3). Although the main genotype effect was also significant (Fgenotype (1,26) = 13.13, p = 0.0012, pcontrol = 0.82; pshock = 0.0011), but this genotype difference became apparent only after foot shock and was not detected among control, non-stressed conditions (Finteraction (1,26) = 5.50, p = 0.03).

3.3. The Lack of TRPA1 Diminished the Astrocyte Activation in the PVN Without Interfering with the Microglia Response in the Mouse Model of PTSD

Stress increased the number of astrocytes in the PVN (Fstress (1,26) = 4.50, p = 0.04) (Figure 4A,C). However, this effect was not visible during post hoc comparisons (pWT = 0.41 and pKO = 0.49) and was independent from the genotype (Fgenotype (1,26) = 0.01, p = 0.92; pcontrol = 1.00 and pshock = 1.00; Finteraction (1,26) = 0.01, p = 0.92).
In addition to the number, the activation of astrocytes was also increased in the PVN of shocked mice (Fstress (1,26) = 17.86, p = 0.0003), which was mainly due to an elevation in the WTs (pWT = 0.0005 and pKO = 0.60) (Figure 4B,C). The significant main genotype effect (Fgenotype (1,26) = 8.49, p = 0.007) was apparent after stress, but not in control, non-shocked individuals (Finteraction (1,26) = 5.99, p = 0.02; pcontrol = 0.99, pshock = 0.004).
In contrast to the astrocytes, we did not find any changes in the number (Fstress (1,26) = 3.46, p = 0.07, pWT = 0.96 and pKO = 0.16; Fgenotype (1,26) = 1.75, p = 0.20; pcontrol = 0.31 and pshock = 1.00; Finteraction (1,26) = 1.41, p = 0.25) (Figure 5A,C) or scoring (Fstress (1,26) = 0.04, p = 0.85, pWT = 0.86 and pKO = 0.96; Fgenotype (1,26) = 0.54, p = 0.47, pcontrol = 1.00 and pshock = 0.66; Finteraction (1,26) = 0.83, p = 0.37) (Figure 5B,C) of the microglial cells in the PVN.

3.4. Correlation Between Behavioral and Morphological Measures

There was a significant positive correlation between LC/TH immunopositivity and both observed behaviors (freezing: r = +0.716, p < 0.001; jumping: r = +0.518, p = 0.003), suggesting the involvement of LC in the development of PTSD-like behavior. However, we did not detect any significant correlation between LC and PVN measures.

3.5. Lack of TRPA1 Did Not Modulate the Effect of Clonidine Treatment on the Behavioral Responses and Serum CORT Levels in a PTSD Model

Clonidine administration significantly increased the total duration of freezing (Ftreatment (1,36) = 12.95, p = 0.001), which was apparent in WT (pWT = 0.045), but not in KO mice (pKO = 0.11). However, major genotype difference was not detected (Fgenotype (1,36) = 2.68, p = 0.11; pvehicle = 0.82, pclonidine = 0.47; Finteraction (1,36) = 0.14, p = 0.72) (Figure 6A). Further analysis of contextual and cued freezing did not reveal significant differences between the vehicle- and clonidine-treated animals (Ftreatment (1,36) = 2.38, p = 0.13) or between the two genotypes (Fgenotype (1,36) = 3.98, p = 0.054; Ftreatment*genotype (1,36) = 1.10, p = 0.30). Though cue presentation decreased the freezing response in each group compared to the baseline and to the ISI values almost throughout the entire experiment (Fcue presentation (14,504) = 18.64, p < 0.0001), but the treatment or the genotype did not influence this effect (Ftreatment*cue presentation (14,504) = 0.92, p = 0.54; Fgenotype*cue presentation (14,504) = 1.22, p = 0.25; Ftreatment*genotype*cue presentation (14,504) = 0.93, p = 0.52). (Figure 6B).
By contrast, we found considerable genotype differences in the total amount of jumping (Fgenotype (1,34) = 12.66, p = 0.001) both in the vehicle- (pvehicle = 0.02) and clonidine-treated groups (pclonidine = 0.02). Clonidine administration decreased the jumping rate (Ftreatment (1,34) = 5.32, p = 0.03) without significant post hoc differences (pWT = 0.14 and pKO = 0.09) or influencing the genotype effect (Finteraction (1,34) = 0.02, p = 0.90) (Figure 6C). The cue presentation increased the jumping response (Fcue presentation (14,504) = 18.32, p < 0.0001), whose effect was influenced by the genotype (Fgenotype (1,36) = 7.25, p = 0.011; Fgenotype*cue presentation (14,504) = 3.12, p < 0.001). More precisely, cue-induced changes in the jumping were observable only in the KOs over the entire experiment, leading to a significant difference between vehicle-treated WT and KO mice during the fourth Cue presentation (pvehicle = 0.049). Though a tendency for reduced jumping was observable in the clonidine-treated mice compared to the vehicle groups (Ftreatment (1,36) = 3.52, p = 0.07), no significant differences were revealed between the treatment groups, and this effect was not influenced by the genotype (Ftreatment*genotype (1,36) = 0.14, p = 0.71) or the cue presentation (Ftreatment*cue presentation (14,504) = 0.72, p = 0.76; Ftreatment*genotype*cue presentation (14,504) = 1.11, p = 0.35) (Figure 6D).
Plasma CORT values at the end of CFT did not reveal any group differences (Ftreatment (1,39) = 0.20, p = 0.66; pWT = 0.98 pKO = 0.73; Fgenotype (1,39) = 0.13, p = 0.72; pvehicle = 0.97 pclonidine = 0.75; Finteraction (1,39) = 1.02, p = 0.32) (Figure 6E).

4. Discussion

Behavioral analysis revealed that foot shock increased freezing and jumping during the FC phase. However, there were no genotype differences, suggesting that the subsequent changes might not be due to altered sensation of the KO animals. In the CFT, the Trpa1 deletion had no effect on shock-induced freezing but exaggerated jumping behavior, as a sign of hyperarousal. These effects were reproducible. The behavioral changes correlated positively with LC/TH immunopositivity. The shock-induced aggravation in LC/TH levels was accompanied by astrocyte activation in the PVN, without microglial activation. The Trpa1 deletion enhanced shock-induced LC/TH elevation and reduced the astrocyte activation. Despite the previously reported positive impact on some PTSD symptoms of clonidine, in our study, manipulating the α2-adrenergic system aggravated the freezing—one of the most frequently used outcome measures of PTSD [45]. However, clonidine had a positive, protective effect on jumping behavior, which does not seem to be transmitted via the HPA axis.
The rate limiting step of noradrenaline biosynthesis is mediated by TH. Thus, the regulation of this enzyme in the LC is an important mechanism upon stress adaptation [54,55]. In line with our findings, elevated TH expression both at the mRNA and protein level was reported upon both single prolonged stress (SPS) and electric foot shock models of PTSD [55,56,57]. These data indicate the involvement of the hyperactive LC-noradrenergic system in the manifestation of PTSD. Importantly, the literature implies that noradrenergic signaling of the LC is deeply implicated in the auditory cued fear conditioning [40,58,59]. The regulatory role of the LC-noradrenergic system in fear memory consolidation depends on the prevailing level of arousal and stress: among high stress conditions its activation facilitates the consolidation and maintenance of cued fear memory, while under low stress levels it promotes fear extinction [40,60,61]. We assume that the role of TRPA1 ion channels become important under high stress conditions (i.e., after foot shock), where it moderates the TH enzyme expression in LC (Figure 3). Therefore, its genetic deletion can contribute to enhanced cue-induced jumping behavior observable in stressed KO mice (Figure 2 and Figure 6) probably via strengthening the noradrenergic outflow from LC as confirmed by positive correlation between jumping and LC/TH. Indeed, the connection between jumping and the noradrenergic system is supported by the diminishing effect of clonidine administration as well (Figure 6). Jumping might be a sign of hyperarousal [47,48,49,50,62], and clonidine is often used for the treatment of this symptom [63].
The observed genotype difference in LC/TH is contradictory in light of the fact that we did not find Trpa1 mRNA in the LC using a highly sensitive RNAscope technique [64]. It is unclear by what mechanism the TRPA1 ion channel would modulate the function of noradrenergic system. One possible explanation is the regulation of LC-input areas. It is well known that the PVN is reciprocally connected with the LC providing positive feedback loops between CRH and noradrenaline [21,40,55,61,65,66,67,68]. TRPA1 might contribute to neuroinflammation [69], especially though modulation of the astrocytes [70,71,72] that are also supported by the lower astrocyte activation in our KO mice (Figure 4). As astrocytes might have anti-inflammatory potential [73,74] and resolve neuroinflammation in the delayed phases of chronic stress [75,76,77], we might assume that their lower activation in the PVN of KO mice can increase local neuroinflammation. This neuroinflammation may elevate the CRH release from the PVN [17,18,25], which may stimulate LC/TH synthesis in KO. In support, Xie et al. reported that astrocyte activation of the hippocampus may reduce the fear responses in PTSD [78]. A further possible mechanism might be mediated through the EWcp. Indeed, we previously demonstrated the high abundance of Trpa1 mRNA in this nucleus, as well as the role of EWcp in modulating the stress response (see Introduction). We might assume that the lacking Trpa1 from the EWcp alter the interpretation of the foot shock, which changes the reactivity of the whole brain, including the LC. This is in line with the James-Lange theory of emotions [79], which states that the reaction of the whole body shapes our emotions, which may manifest itself in divergent activity of different brain areas.
In line with the reciprocal connection, LC may regulate PVN function via neuroinflammation (see Introduction). The previously described cFOS activation in PVN neurons following foot shock suggested its integrative role in FC [80,81,82]. Not only was the role of its neuropeptides (e.g., CRH, oxytocin) described among a wide range of stress conditions [10,83,84,85,86,87,88,89], but there are also studies supporting the role of their glia cells in acute and chronic stress situations [85,88,89,90,91,92,93], with limited data in conditioned fear and PTSD [81]. To our best knowledge, our current study is the first describing the activation of astrocytes in the PVN following foot shock.
The literature data suggest that astrocytes express TRPA1 ion channels (see earlier), and the mediators of oxidative stress produced excessively in PTSD [17,94,95,96,97,98,99] may stimulate TRPA1 [2,37,38] leading to astrocyte activation. Thus, we assume that the TRPA1-mediated astrocyte activation in the PVN of WT mice may play a role in the restoration of neuroendocrine and behavioral functions following stress, while the lacking TRPA1 in KOs accompanied by lacking astrocyte activation may delay recovery. Indeed, our prior study revealed both the exhaustion of CORT secretion and the impaired fear extinction with prolonged fear responses in cases of Trpa1 gene-deficiency [100].
In contrast, microglia of the PVN did not seem to be involved in the presently observed PTSD-related alterations. Interestingly, a prior study reported microglial hyper-ramification and increased soma perimeter in the PVN following contextual FC [81]. Since microglia responses depend on the modality of stressor and the genetic background of animals [81,101], this contradiction may be explained by the different experimental protocols and the diverse strain of mice. However, our previous experiments showed alterations in the microglial morphology of Trpa1 KO mice within several other brain regions implicated in the PTSD pathomechanism (central nucleus of amygdala, hippocampus, medial prefrontal cortex) suggesting the role of this ion channel in microglia-mediated processes upon conditioned fear [100]. Indeed, similar region-specific differences in stress-induced microglia responses have already been reported [102].
Our prior research revealed significantly decreased shock-induced immobility in KO mice compared to the WTs using the SPS protocol of PTSD [12]. One possible explanation is the different stress-level of the two models: the foot shock paradigm is a strong unescapable psychological stressor [45], while the SPS model is based upon mild stressors over a longer period [103,104,105]. Therefore, we suspect that TRPA1 might be important in fine-tuning the behavior to the intensity of stress. The increased jumping rate of KO animals in the foot shock model was found before [100], suggesting the involvement of TRPA1 channel in hyperarousal. However, this effect is not exclusively mediated by the adrenergic system (see ineffectiveness of clonidine). The literature indicates that clonidine may decrease the locomotor activity through presynaptic α2-receptor agonism among others in the LC, which may induce anxiolytic and sedative effects by the reduced central noradrenergic outflow [106,107,108,109,110]. Thus, we suspect that the elevated freezing behavior of clonidine-treated mice represents a decreased locomotion rather than a PTSD-like freezing behavior. This is supported by its jumping reducing effect as well (see reciprocal changes in cued freezing and jumping response, as well as negative correlation between freezing and jumping during Exp II; r = −0.580, p = 0.014).
Though the effect of clonidine on the function of HPA axis is debated depending on the dose and route of administration [111,112,113], a recent study showed that i.p. clonidine treatment may increase the CORT secretion [109]. However, in our case we could not find treatment or genotype difference, which let us to conclude that clonidine-mediated effects may be independent of the HPA axis activation. However, we cannot rule out confounding technical details such as the effect of i.p. injection approx. 40 min before measurement, as well as isoflurane anesthesia during sampling.
This study has several limitations. The selected stress paradigm may constrain translational relevance, while reliance on a single stressor reduces generalizability. Developmental compensation in knockout animals cannot be ruled out. Immunostaining analyses were semiquantitative, and only a limited subset of network components was assessed at discrete time points. Moreover, the observed associations are correlational; functional manipulations are needed to establish causality.

5. Conclusions

TRPA1 may be involved in stress adaptation during PTSD-related conditioned, especially in cued, fear responses via controlling noradrenergic output from the LC and the neuroinflammation in stress-sensitive brain areas like PVN, most probably not through the α2-adrenergic receptors. We suppose that TRPA1 activation may contribute to the recovery following strong stresses and, therefore, may be a novel drug target in the future therapy of PTSD.

Author Contributions

Conceptualization, D.Z., V.K. and J.K.; Methodology, D.Z., V.K., J.K., P.K., E.K. and T.B.-S.; Investigation, D.Z., V.K., L.M.S., E.L. and J.K.; Validation, D.Z., V.K. and J.K.; Formal Analysis, D.Z., V.K. and J.K.; Visualization, B.G., G.B., V.K. and J.K.; Resources, B.G., E.P. and D.Z.; Supervision, V.K. and D.Z.; Writing—Original Draft Preparation, J.K., E.P. and V.K.; Writing—Review and Editing, D.Z., E.P. and B.G. Funding acquisition, E.P., D.Z., V.K., B.G. and J.K. All authors have read and agreed to the published version of the manuscript.

Funding

J.K. and V.K. was supported by the Research grant of Medical School, University of Pécs (KA-2024-12; 008_2025_PTE_RK/36, 24 January 2025). B.G., E.P., D.Z., J.K. and V.K. were supported by the Thematic Excellence Program 2021 Health Sub-program of the Ministry for Innovation and Technology in Hungary, within the framework of the EGA-16 project of Pécs University (TKP2021-EGA-16); and B.G. by the grant NKFI-146117. This study was supported by the National Brain Research Program (NAP 3.0) of the Hungarian Academy of Sciences, the National Research Development and Innovation Office of Hungary (grant numbers K141934 and K138763).

Institutional Review Board Statement

All procedures were approved by the Animal Welfare Committee at Pécs University, National Scientific Ethical Committee on Animal Experimentation in Hungary (BA02/2000-46/2024, 24 January 2025) in agreement with the directive of the European Communities Council in 1986, and with the Law of XXCIII, in 1998, on Animal Care and Use in Hungary. We have consistently striven to minimize the number of animals used in our studies and to limit their suffering to the greatest extent possible.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

Authors are grateful to Réka Varga for her professional work during the in vivo experiments and for the processing of tail blood samples.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ANOVAanalysis of variance
a.u.arbitrary unit
CFTconditioned fear test
CORTcorticosterone
CRHcorticotropin-releasing hormone
EWcpcentrally projecting Edinger-Westphal nucleus
FCfear conditioning
GFAPglial fibrillary acidic protein
HPAhypothalamic–pituitary–adrenal
IBA1ionized calcium binding adaptor molecule 1
IL-1interleukin-1
IL-6interleukin-6
i.p.intraperitoneal(ly)
ISIinterstimulus interval
KOknockout
LClocus coeruleus
NDSnormal donkey serum
PBSphosphate-buffered saline
PFAparaformaldehyde
PTSDposttraumatic stress disorder
PVNparaventricular nucleus of hypothalamus
RTroom temperature
SAMsympatho-adrenomedullary
SPSsingle prolonged stress
SSDspecific signal density
THtyrosine hydroxylase
Trpa1transient receptor potential ankyrin 1 gene
TRPA1transient receptor potential ankyrin 1 ion channel
TNF-αtumor necrosis factor-α
UCN1urocortin 1
WTwild type

References

  1. Julius, D. TRP channels and pain. Annu. Rev. Cell Dev. Biol. 2013, 29, 355–384. [Google Scholar] [CrossRef] [PubMed]
  2. Talavera, K.; Startek, J.B.; Alvarez-Collazo, J.; Boonen, B.; Alpizar, Y.A.; Sanchez, A.; Naert, R.; Nilius, B. Mammalian Transient Receptor Potential TRPA1 Channels: From Structure to Disease. Physiol. Rev. 2020, 100, 725–803. [Google Scholar] [CrossRef]
  3. Paumier, A.; Boisseau, S.; Jacquier-Sarlin, M.; Pernet-Gallay, K.; Buisson, A.; Albrieux, M. Astrocyte-neuron interplay is critical for Alzheimer’s disease pathogenesis and is rescued by TRPA1 channel blockade. Brain 2022, 145, 388–405. [Google Scholar] [CrossRef]
  4. Konkoly, J.; Kormos, V.; Gaszner, B.; Sándor, Z.; Kecskés, A.; Alomari, A.; Szilágyi, A.; Szilágyi, B.; Zelena, D.; Pintér, E. The Role of TRPA1 Channels in the Central Processing of Odours Contributing to the Behavioural Responses of Mice. Pharmaceuticals 2021, 14, 1336. [Google Scholar] [CrossRef]
  5. Olah, E.; Rumbus, Z.; Kormos, V.; Tekus, V.; Pakai, E.; Wilson, H.V.; Fekete, K.; Solymar, M.; Kelava, L.; Keringer, P.; et al. The Hypothermic Effect of Hydrogen Sulfide Is Mediated by the Transient Receptor Potential Ankyrin-1 Channel in Mice. Pharmaceuticals 2021, 14, 992. [Google Scholar] [CrossRef]
  6. Milicic, M.; Nausía Bonet, M.; Lantos, E.; Al-Omari, A.; Csernus, V.; Berta, G.; Pintér, P.; Gaszner, B.; Kormos, V. The role of TRPA1 ion channel in stress regulation in serotonergic and CRH systems. Neurochem. Res. 2025; submitted. [Google Scholar]
  7. Kozicz, T.; Li, M.; Arimura, A. The activation of urocortin immunoreactive neurons in the Edinger-Westphal nucleus following stress in rats. Stress 2001, 4, 85–90. [Google Scholar] [CrossRef] [PubMed]
  8. Kozicz, T.; Bordewin, L.A.; Czéh, B.; Fuchs, E.; Roubos, E.W. Chronic psychosocial stress affects corticotropin-releasing factor in the paraventricular nucleus and central extended amygdala as well as urocortin 1 in the non-preganglionic Edinger-Westphal nucleus of the tree shrew. Psychoneuroendocrinology 2008, 33, 741–754. [Google Scholar] [CrossRef]
  9. Gaszner, B.; Csernus, V.; Kozicz, T. Urocortinergic neurons respond in a differentiated manner to various acute stressors in the Edinger-Westphal nucleus in the rat. J. Comp. Neurol. 2004, 480, 170–179. [Google Scholar] [CrossRef] [PubMed]
  10. Farkas, J.; Kovács, L.Á.; Gáspár, L.; Nafz, A.; Gaszner, T.; Ujvári, B.; Kormos, V.; Csernus, V.; Hashimoto, H.; Reglődi, D.; et al. Construct and face validity of a new model for the three-hit theory of depression using PACAP mutant mice on CD1 background. Neuroscience 2017, 354, 11–29. [Google Scholar] [CrossRef]
  11. Kormos, V.; Kecskés, A.; Farkas, J.; Gaszner, T.; Csernus, V.; Alomari, A.; Hegedüs, D.; Renner, É.; Palkovits, M.; Zelena, D.; et al. Peptidergic neurons of the Edinger-Westphal nucleus express TRPA1 ion channel that is downregulated both upon chronic variable mild stress in male mice and in humans who died by suicide. J. Psychiatry Neurosci. 2022, 47, E162–E175. [Google Scholar] [CrossRef]
  12. Konkoly, J.; Kormos, V.; Gaszner, B.; Correia, P.; Berta, G.; Biró-Sütő, T.; Zelena, D.; Pintér, E. Transient receptor potential ankyrin 1 ion channel expressed by the Edinger-Westphal nucleus contributes to stress adaptation in murine model of posttraumatic stress disorder. Front. Cell Dev. Biol. 2022, 10, 1059073. [Google Scholar] [CrossRef]
  13. Auxéméry, Y. Post-traumatic psychiatric disorders: PTSD is not the only diagnosis. Presse Med. 2018, 47, 423–430. [Google Scholar] [CrossRef]
  14. Maercker, A.; Cloitre, M.; Bachem, R.; Schlumpf, Y.R.; Khoury, B.; Hitchcock, C.; Bohus, M. Complex post-traumatic stress disorder. Lancet 2022, 400, 60–72. [Google Scholar] [CrossRef] [PubMed]
  15. Daskalakis, N.P.; Cohen, H.; Nievergelt, C.M.; Baker, D.G.; Buxbaum, J.D.; Russo, S.J.; Yehuda, R. New translational perspectives for blood-based biomarkers of PTSD: From glucocorticoid to immune mediators of stress susceptibility. Exp. Neurol. 2016, 284, 133–140. [Google Scholar] [CrossRef]
  16. Hendrickson, R.C.; Raskind, M.A. Noradrenergic dysregulation in the pathophysiology of PTSD. Exp. Neurol. 2016, 284, 181–195. [Google Scholar] [CrossRef] [PubMed]
  17. Hori, H.; Kim, Y. Inflammation and post-traumatic stress disorder. Psychiatry Clin. Neurosci. 2019, 73, 143–153. [Google Scholar] [CrossRef] [PubMed]
  18. Lee, D.H.; Lee, J.Y.; Hong, D.Y.; Lee, E.C.; Park, S.W.; Lee, M.R.; Oh, J.S. Neuroinflammation in Post-Traumatic Stress Disorder. Biomedicines 2022, 10, 953. [Google Scholar] [CrossRef]
  19. Ross, J.A.; Van Bockstaele, E.J. The Locus Coeruleus- Norepinephrine System in Stress and Arousal: Unraveling Historical, Current, and Future Perspectives. Front. Psychiatry 2021, 11, 601519. [Google Scholar] [CrossRef]
  20. Reyes, B.A.S. The Locus Coeruleus: Anatomy, Physiology, and Stress-Related Neuropsychiatric Disorders. Eur. J. Neurosci. 2025, 61, e70111. [Google Scholar] [CrossRef]
  21. McCall, J.G.; Al-Hasani, R.; Siuda, E.R.; Hong, D.Y.; Norris, A.J.; Ford, C.P.; Bruchas, M.R. CRH Engagement of the Locus Coeruleus Noradrenergic System Mediates Stress-Induced Anxiety. Neuron 2015, 87, 605–620. [Google Scholar] [CrossRef] [PubMed]
  22. Goldman, C.K.; Marino, L.; Leibowitz, S.F. Postsynaptic alpha 2-noradrenergic receptors mediate feeding induced by paraventricular nucleus injection of norepinephrine and clonidine. Eur. J. Pharmacol. 1985, 115, 11–19. [Google Scholar] [CrossRef]
  23. Ziegler, D.R.; Cass, W.A.; Herman, J.P. Excitatory influence of the locus coeruleus in hypothalamic-pituitary-adrenocortical axis responses to stress. J. Neuroendocr. 1999, 11, 361–369. [Google Scholar] [CrossRef]
  24. Hempelmann, G.; Adams, H.A. Stress und Anästhesie [Stress and anesthesia]. Anasthesiol. Intensiv. Notfallmedizin Schmerzther. 1991, 26, 293. (In German) [Google Scholar] [CrossRef]
  25. Smith, S.M.; Vale, W.W. The role of the hypothalamic-pituitary-adrenal axis in neuroendocrine responses to stress. Dialogues Clin. Neurosci. 2006, 8, 383–395. [Google Scholar] [CrossRef]
  26. Yehuda, R.; Bierer, L.M.; Pratchett, L.C.; Lehrner, A.; Koch, E.C.; Van Manen, J.A.; Flory, J.D.; Makotkine, I.; Hildebrandt, T. Cortisol augmentation of a psychological treatment for warfighters with posttraumatic stress disorder: Randomized trial showing improved treatment retention and outcome. Psychoneuroendocrinology 2015, 51, 589–597. [Google Scholar] [CrossRef]
  27. Bekar, L.K.; He, W.; Nedergaard, M. Locus coeruleus alpha-adrenergic-mediated activation of cortical astrocytes in vivo. Cereb. Cortex 2008, 18, 2789–2795. [Google Scholar] [CrossRef]
  28. Ding, F.; O’Donnell, J.; Thrane, A.S.; Zeppenfeld, D.; Kang, H.; Xie, L.; Wang, F.; Nedergaard, M. α1-Adrenergic receptors mediate coordinated Ca2+ signaling of cortical astrocytes in awake, behaving mice. Cell Calcium 2013, 54, 387–394. [Google Scholar] [CrossRef]
  29. Stern, J.E.; Son, S.; Biancardi, V.C.; Zheng, H.; Sharma, N.; Patel, K.P. Astrocytes Contribute to Angiotensin II Stimulation of Hypothalamic Neuronal Activity and Sympathetic Outflow. Hypertension 2016, 68, 1483–1493. [Google Scholar] [CrossRef] [PubMed]
  30. Moreira, J.D.; Chaudhary, P.; Frame, A.A.; Puleo, F.; Nist, K.M.; Abkin, E.A.; Moore, T.L.; George, J.C.; Wainford, R.D. Inhibition of microglial activation in rats attenuates paraventricular nucleus inflammation in Gαi2 protein-dependent, salt-sensitive hypertension. Exp. Physiol. 2019, 104, 1892–1910. [Google Scholar] [CrossRef] [PubMed]
  31. Han, T.H.; Lee, H.W.; Kang, E.A.; Song, M.S.; Lee, S.Y.; Ryu, P.D. Microglial activation induced by LPS mediates excitation of neurons in the hypothalamic paraventricular nucleus projecting to the rostral ventrolateral medulla. BMB Rep. 2021, 54, 620–625. [Google Scholar] [CrossRef] [PubMed]
  32. Bosson, A.; Paumier, A.; Boisseau, S.; Jacquier-Sarlin, M.; Buisson, A.; Albrieux, M. TRPA1 channels promote astrocytic Ca2+hyperactivity and synaptic dysfunction mediated by oligomeric forms of amyloid-β peptide. Mol. Neurodegener. 2017, 12, 53. [Google Scholar] [CrossRef] [PubMed]
  33. Oh, S.J.; Lee, J.M.; Kim, H.B.; Lee, J.; Han, S.; Bae, J.Y.; Hong, G.S.; Koh, W.; Kwon, J.; Hwang, E.S.; et al. Ultrasonic Neuromodulation via Astrocytic TRPA1. Curr. Biol. 2020, 30, 948. [Google Scholar] [CrossRef]
  34. Cheng, Y.T.; Woo, J.; Luna-Figueroa, E.; Maleki, E.; Harmanci, A.S.; Deneen, B. Social deprivation induces astrocytic TRPA1-GABA suppression of hippocampal circuits. Neuron 2023, 111, 1301–1315.e5. [Google Scholar] [CrossRef] [PubMed]
  35. Kakae, M.; Nakajima, H.; Tobori, S.; Kawashita, A.; Miyanohara, J.; Morishima, M.; Nagayasu, K.; Nakagawa, T.; Shigetomi, E.; Koizumi, S.; et al. The astrocytic TRPA1 channel mediates an intrinsic protective response to vascular cognitive impairment via LIF production. Sci. Adv. 2023, 9, eadh0102. [Google Scholar] [CrossRef]
  36. Wei, G.; Wang, L.; Zhu, J.; Wang, X.; Zang, Y.; Yang, J.; Zheng, C. Astrocytic TRPA1 as a target in reversing social avoidance by low-intensity focused ultrasound stimulation in mice. In Proceedings of the 2024 46th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), Orlando, FL, USA, 15–19 July 2024; pp. 1–4. [Google Scholar] [CrossRef]
  37. Logashina, Y.A.; Korolkova, Y.V.; Kozlov, S.A.; Andreev, Y.A. TRPA1 Channel as a Regulator of Neurogenic Inflammation and Pain: Structure, Function, Role in Pathophysiology, and Therapeutic Potential of Ligands. Biochemistry 2019, 84, 101–118. [Google Scholar] [CrossRef]
  38. Meents, J.E.; Ciotu, C.I.; Fischer, M.J.M. TRPA1: A molecular view. J. Neurophysiol. 2019, 121, 427–443. [Google Scholar] [CrossRef]
  39. Lee, D.J.; Schnitzlein, C.W.; Wolf, J.P.; Vythilingam, M.; Rasmusson, A.M.; Hoge, C.W. Psychotherapy Versus Pharmacotherapy for Posttraumatic Stress Disorder: Systemic Review and Meta-Analyses to Determine First-Line Treatments. Depress. Anxiety 2016, 33, 792–806. [Google Scholar] [CrossRef]
  40. Giustino, T.F.; Maren, S. Noradrenergic Modulation of Fear Conditioning and Extinction. Front. Behav. Neurosci. 2018, 12, 43. [Google Scholar] [CrossRef]
  41. Goodnight, J.R.M.; Ragsdale, K.A.; Rauch, S.A.M.; Rothbaum, B.O. Psychotherapy for PTSD: An evidence-based guide to a theranostic approach to treatment. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 2019, 88, 418–426. [Google Scholar] [CrossRef]
  42. Schrader, C.; Ross, A. A Review of PTSD and Current Treatment Strategies. Mo. Med. 2021, 118, 546–551. [Google Scholar]
  43. Bautista, D.M.; Jordt, S.E.; Nikai, T.; Tsuruda, P.R.; Read, A.J.; Poblete, J.; Yamoah, E.N.; Basbaum, A.I.; Julius, D. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 2006, 124, 1269–1282. [Google Scholar] [CrossRef]
  44. Kumar, P.; Correia, P.; Plangár, I.; Zelena, D. Sweet Relief? Short-Term Post-Traumatic High-Sucrose Intake Attenuates Acute but Not Long-Term Fear Responses in Mice. Biomedicines 2025, 13, 2233. [Google Scholar] [CrossRef]
  45. Török, B.; Sipos, E.; Pivac, N.; Zelena, D. Modelling posttraumatic stress disorders in animals. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 2019, 90, 117–133. [Google Scholar] [CrossRef]
  46. Verbitsky, A.; Dopfel, D.; Zhang, N. Rodent models of post-traumatic stress disorder: Behavioral assessment. Transl. Psychiatry 2020, 10, 132. [Google Scholar] [CrossRef] [PubMed]
  47. Riccardi, E.; Mancini, G.F.; Pisaneschi, A.; Morena, M.; Campolongo, P. Sex differences in fear expression and persistence in an animal model of Post-Traumatic Stress Disorder. Neuroscience 2024, 560, 371–380. [Google Scholar] [CrossRef] [PubMed]
  48. Borkar, C.D.; Fadok, J.P. A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior. J. Vis. Exp. 2021, e61536. [Google Scholar] [CrossRef]
  49. Furuyama, T.; Imayoshi, A.; Iyobe, T.; Ono, M.; Ishikawa, T.; Ozaki, N.; Kato, N.; Yamamoto, R. Multiple factors contribute to flight behaviors during fear conditioning. Sci. Rep. 2023, 13, 10402. [Google Scholar] [CrossRef] [PubMed]
  50. Furuyama, T.; Yamamoto, R.; Kato, N.; Ono, M. Erratum: Modified Fear Conditioning for Inducing Flight Behaviors in Mice. J. Vis. Exp. 2024, e6598. [Google Scholar] [CrossRef]
  51. Paxinos, G.; Franklin, K.B.J. Mouse Brain in Stereotaxic Coordinates, 2nd ed.; Academic Press: San Diego, CA, USA, 2001. [Google Scholar]
  52. Harrison, L.; Pfuhlmann, K.; Schriever, S.C.; Pfluger, P.T. Profound weight loss induces reactive astrogliosis in the arcuate nucleus of obese mice. Mol. Metab. 2019, 24, 149–155. [Google Scholar] [CrossRef]
  53. Zelena, D.; Mergl, Z.; Foldes, A.; Kovács, K.J.; Tóth, Z.; Makara, G.B. Role of hypothalamic inputs in maintaining pituitary-adrenal responsiveness in repeated restraint. Am. J. Physiol. Endocrinol. Metab. 2003, 285, E1110–E1117. [Google Scholar] [CrossRef] [PubMed]
  54. Kvetnansky, R.; Sabban, E.L.; Palkovits, M. Catecholaminergic systems in stress: Structural and molecular genetic approaches. Physiol. Rev. 2009, 89, 535–606. [Google Scholar] [CrossRef] [PubMed]
  55. Sabban, E.L.; Serova, L.I.; Newman, E.; Aisenberg, N.; Akirav, I. Changes in Gene Expression in the Locus Coeruleus-Amygdala Circuitry in Inhibitory Avoidance PTSD Model. Cell. Mol. Neurobiol. 2018, 38, 273–280. [Google Scholar] [CrossRef]
  56. George, S.A.; Knox, D.; Curtis, A.L.; Aldridge, J.W.; Valentino, R.J.; Liberzon, I. Altered locus coeruleus-norepinephrine function following single prolonged stress. Eur. J. Neurosci. 2013, 37, 901–909. [Google Scholar] [CrossRef]
  57. Sabban, E.L.; Laukova, M.; Alaluf, L.G.; Olsson, E.; Serova, L.I. Locus coeruleus response to single-prolonged stress and early intervention with intranasal neuropeptide Y. J. Neurochem. 2015, 135, 975–986. [Google Scholar] [CrossRef] [PubMed]
  58. Dębiec, J.; Bush, D.E.; LeDoux, J.E. Noradrenergic enhancement of reconsolidation in the amygdala impairs extinction of conditioned fear in rats—A possible mechanism for the persistence of traumatic memories in PTSD. Depress. Anxiety 2011, 28, 186–193. [Google Scholar] [CrossRef]
  59. Soya, S.; Shoji, H.; Hasegawa, E.; Hondo, M.; Miyakawa, T.; Yanagisawa, M.; Mieda, M.; Sakurai, T. Orexin receptor-1 in the locus coeruleus plays an important role in cue-dependent fear memory consolidation. J. Neurosci. 2013, 33, 14549–14557. [Google Scholar] [CrossRef]
  60. Giustino, T.F.; Ramanathan, K.R.; Totty, M.S.; Miles, O.W.; Maren, S. Locus Coeruleus Norepinephrine Drives Stress-Induced Increases in Basolateral Amygdala Firing and Impairs Extinction Learning. J. Neurosci. 2020, 40, 907–916. [Google Scholar] [CrossRef]
  61. Maren, S. Unrelenting Fear Under Stress: Neural Circuits and Mechanisms for the Immediate Extinction Deficit. Front. Syst. Neurosci. 2022, 16, 888461. [Google Scholar] [CrossRef]
  62. Costa, B.M.; Hines, D.; Phillip, N.; Boehringer, S.C.; Anandakrishnan, R.; Council-Troche, M.; Davis, J.L. Preliminary pharmacokinetics and in vivo studies indicate analgesic and stress mitigation effects of a novel NMDA receptor modulator. J. Pharmacol. Exp. Ther. 2025, 392, 103401. [Google Scholar] [CrossRef]
  63. Ziegenhorn, M.A.; Hildebrand, J.A.; Oleson, E.M.; Baird, R.W.; Wiggins, S.M.; Baumann-Pickering, S. Odontocete spatial patterns and temporal drivers of detection at sites in the Hawaiian islands. Ecol. Evol. 2023, 13, e9688. [Google Scholar] [CrossRef]
  64. Milicic, M.; Gaszner, B.; Berta, G.; Pintér, E.; Kormos, V. The Lack of TRPA1 Ion Channel Does Not Affect the Chronic Stress-Induced Activation of the Locus Ceruleus. Int. J. Mol. Sci. 2024, 25, 1765. [Google Scholar] [CrossRef] [PubMed]
  65. Prouty, E.W.; Waterhouse, B.D.; Chandler, D.J. Corticotropin releasing factor dose-dependently modulates excitatory synaptic transmission in the noradrenergic nucleus locus coeruleus. Eur. J. Neurosci. 2017, 45, 712–722. [Google Scholar] [CrossRef] [PubMed]
  66. Beverly, J.L.; de Vries, M.G.; Beverly, M.F.; Arseneau, L.M. Norepinephrine mediates glucoprivic-induced increase in GABA in the ventromedial hypothalamus of rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2000, 279, R990–R996. [Google Scholar] [CrossRef]
  67. Samuels, E.R.; Szabadi, E. Functional neuroanatomy of the noradrenergic locus coeruleus: Its roles in the regulation of arousal and autonomic function part II: Physiological and pharmacological manipulations and pathological alterations of locus coeruleus activity in humans. Curr. Neuropharmacol. 2008, 6, 254–285. [Google Scholar] [CrossRef]
  68. Kubota, N.; Amemiya, S.; Motoki, C.; Otsuka, T.; Nishijima, T.; Kita, I. Corticotropin-releasing factor antagonist reduces activation of noradrenalin and serotonin neurons in the locus coeruleus and dorsal raphe in the arousal response accompanied by yawning behavior in rats. Neurosci. Res. 2012, 72, 316–323. [Google Scholar] [CrossRef] [PubMed]
  69. Szabó, K.; Makkai, G.; Konkoly, J.; Kormos, V.; Gaszner, B.; Berki, T.; Pintér, E. TRPA1 Covalent Ligand JT010 Modifies T Lymphocyte Activation. Biomolecules 2024, 14, 632. [Google Scholar] [CrossRef]
  70. Shigetomi, E.; Tong, X.; Kwan, K.Y.; Corey, D.P.; Khakh, B.S. TRPA1 channels regulate astrocyte resting calcium and inhibitory synapse efficacy through GAT-3. Nat. Neurosci. 2011, 15, 70–80. [Google Scholar] [CrossRef]
  71. Kimura, Y.; Mikami, Y.; Osumi, K.; Tsugane, M.; Oka, J.; Kimura, H. Polysulfides are possible H2S-derived signaling molecules in rat brain. FASEB J. 2013, 27, 2451–2457. [Google Scholar] [CrossRef]
  72. Verkhratsky, A.; Reyes, R.C.; Parpura, V. TRP channels coordinate ion signalling in astroglia. In Reviews of Physiology, Biochemistry and Pharmacology; Springer: Cham, Switzerland, 2014; Volume 166, pp. 1–22. [Google Scholar] [CrossRef]
  73. Sofroniew, M.V. Astrocyte barriers to neurotoxic inflammation. Nat. Rev. Neurosci. 2015, 16, 249–263, Erratum in Nat. Rev. Neurosci. 2015, 16, 372.. [Google Scholar] [CrossRef]
  74. Li, T.; Chen, X.; Zhang, C.; Zhang, Y.; Yao, W. An update on reactive astrocytes in chronic pain. J. Neuroinflammation 2019, 16, 140. [Google Scholar] [CrossRef] [PubMed]
  75. Bian, Y.; Pan, Z.; Hou, Z.; Huang, C.; Li, W.; Zhao, B. Learning, memory, and glial cell changes following recovery from chronic unpredictable stress. Brain Res. Bull. 2012, 88, 471–476. [Google Scholar] [CrossRef] [PubMed]
  76. Norden, D.M.; Trojanowski, P.J.; Walker, F.R.; Godbout, J.P. Insensitivity of astrocytes to interleukin 10 signaling following peripheral immune challenge results in prolonged microglial activation in the aged brain. Neurobiol. Aging 2016, 44, 22–41. [Google Scholar] [CrossRef] [PubMed]
  77. Kwon, H.S.; Koh, S.H. Neuroinflammation in neurodegenerative disorders: The roles of microglia and astrocytes. Transl. Neurodegener. 2020, 9, 42. [Google Scholar] [CrossRef]
  78. Xia, L.; Zhai, M.; Wang, L.; Miao, D.; Zhu, X.; Wang, W. FGF2 blocks PTSD symptoms via an astrocyte-based mechanism. Behav. Brain Res. 2013, 256, 472–480. [Google Scholar] [CrossRef]
  79. Dalgleish, T. Cognitive approaches to posttraumatic stress disorder: The evolution of multirepresentational theorizing. Psychol. Bull. 2004, 130, 228–260. [Google Scholar] [CrossRef]
  80. Pezzone, M.A.; Lee, W.S.; Hoffman, G.E.; Rabin, B.S. Induction of c-Fos immunoreactivity in the rat forebrain by conditioned and unconditioned aversive stimuli. Brain Res. 1992, 597, 41–50. [Google Scholar] [CrossRef]
  81. Schubert, I.; Ahlbrand, R.; Winter, A.; Vollmer, L.; Lewkowich, I.; Sah, R. Enhanced fear and altered neuronal activation in forebrain limbic regions of CX3CR1-deficient mice. Brain Behav. Immun. 2018, 68, 34–43. [Google Scholar] [CrossRef]
  82. Li, M.; Wang, J.; Liu, Y.; Wu, J.; Li, F.; Feng, S.; Du, D.; Chen, F. Central changes in the Kv10.2 potassium channel in stress-induced hypertension rats. Neuroreport 2019, 30, 637–644. [Google Scholar] [CrossRef]
  83. Kormos, V.; Gáspár, L.; Kovács, L.Á.; Farkas, J.; Gaszner, T.; Csernus, V.; Balogh, A.; Hashimoto, H.; Reglődi, D.; Helyes, Z.; et al. Reduced response to chronic mild stress in PACAP mutant mice is associated with blunted FosB expression in limbic forebrain and brainstem centers. Neuroscience 2016, 330, 335–358. [Google Scholar] [CrossRef]
  84. Jiang, Z.; Rajamanickam, S.; Justice, N.J. CRF signaling between neurons in the paraventricular nucleus of the hypothalamus (PVN) coordinates stress responses. Neurobiol. Stress 2019, 11, 100192. [Google Scholar] [CrossRef]
  85. Daviu, N.; Füzesi, T.; Rosenegger, D.G.; Rasiah, N.P.; Sterley, T.L.; Peringod, G.; Bains, J.S. Paraventricular nucleus CRH neurons encode stress controllability and regulate defensive behavior selection. Nat. Neurosci. 2020, 23, 398–410. [Google Scholar] [CrossRef]
  86. Kovács, L.Á.; Berta, G.; Csernus, V.; Ujvári, B.; Füredi, N.; Gaszner, B. Corticotropin-Releasing Factor-Producing Cells in the Paraventricular Nucleus of the Hypothalamus and Extended Amygdala Show Age-Dependent FOS and FOSB/DeltaFOSB Immunoreactivity in Acute and Chronic Stress Models in the Rat. Front. Aging Neurosci. 2019, 11, 274. [Google Scholar] [CrossRef]
  87. Kovács, L.Á.; Füredi, N.; Ujvári, B.; Golgol, A.; Gaszner, B. Age-Dependent FOSB/ΔFOSB Response to Acute and Chronic Stress in the Extended Amygdala, Hypothalamic Paraventricular, Habenular, Centrally-Projecting Edinger-Westphal, and Dorsal Raphe Nuclei in Male Rats. Front. Aging Neurosci. 2022, 14, 862098. [Google Scholar] [CrossRef]
  88. Sandoval, K.C.; Rychlik, J.; Choe, K.Y. Calcium Dynamics in Hypothalamic Paraventricular Oxytocin Neurons and Astrocytes Associated with Social and Stress Stimuli. eNeuro 2025, 12, ENEURO.0196-24.2025. [Google Scholar] [CrossRef]
  89. Tillinger, A.; Zvozilová, A.; Mach, M.; Horváthová, Ľ.; Dziewiczová, L.; Osacká, J. Single Intranasal Administration of Ucn3 Affects the Development of PTSD Symptoms in an Animal Model. Int. J. Mol. Sci. 2024, 25, 11908. [Google Scholar] [CrossRef]
  90. Chen, C.; Jiang, Z.; Fu, X.; Yu, D.; Huang, H.; Tasker, J.G. Astrocytes Amplify Neuronal Dendritic Volume Transmission Stimulated by Norepinephrine. Cell Rep. 2019, 29, 4349–4361.e4. [Google Scholar] [CrossRef] [PubMed]
  91. Sugama, S.; Fujita, M.; Hashimoto, M.; Conti, B. Stress induced morphological microglial activation in the rodent brain: Involvement of interleukin-18. Neuroscience 2007, 146, 1388–1399. [Google Scholar] [CrossRef]
  92. Wohleb, E.S.; Hanke, M.L.; Corona, A.W.; Powell, N.D.; Stiner, L.M.; Bailey, M.T.; Nelson, R.J.; Godbout, J.P.; Sheridan, J.F. β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat. J. Neurosci. 2011, 31, 6277–6288. [Google Scholar] [CrossRef] [PubMed]
  93. Pohl, R.; Botscharow, J.; Böckelmann, I.; Thielmann, B. Stress and strain among veterinarians: A scoping review. Ir. Vet. J. 2022, 75, 15. [Google Scholar] [CrossRef] [PubMed]
  94. Ozdemir, O.; Selvi, Y.; Ozkol, H.; Tuluce, Y.; Besiroglu, L.; Aydin, A. Comparison of superoxide dismutase, glutathione peroxidase and adenosine deaminase activities between respiratory and nocturnal subtypes of patients with panic disorder. Neuropsychobiology 2012, 66, 244–251. [Google Scholar] [CrossRef] [PubMed]
  95. Wilson, C.B.; Ebenezer, P.J.; McLaughlin, L.D.; Francis, J. Predator exposure/psychosocial stress animal model of post-traumatic stress disorder modulates neurotransmitters in the rat hippocampus and prefrontal cortex. PLoS ONE 2014, 9, e89104. [Google Scholar] [CrossRef] [PubMed]
  96. Miller, M.W.; Wolf, E.J.; Sadeh, N.; Logue, M.; Spielberg, J.M.; Hayes, J.P.; Sperbeck, E.; Schichman, S.A.; Stone, A.; Carter, W.C.; et al. A novel locus in the oxidative stress-related gene ALOX12 moderates the association between PTSD and thickness of the prefrontal cortex. Psychoneuroendocrinology 2015, 62, 359–365. [Google Scholar] [CrossRef] [PubMed]
  97. Atli, A.; Bulut, M.; Bez, Y.; Kaplan, İ.; Özdemir, P.G.; Uysal, C.; Selçuk, H.; Sir, A. Altered lipid peroxidation markers are related to post-traumatic stress disorder (PTSD) and not trauma itself in earthquake survivors. Eur. Arch. Psychiatry Clin. Neurosci. 2016, 266, 329–336. [Google Scholar] [CrossRef]
  98. Miller, M.W.; Lin, A.P.; Wolf, E.J.; Miller, D.R. Oxidative Stress, Inflammation, and Neuroprogression in Chronic PTSD. Harv. Rev. Psychiatry 2018, 26, 57–69. [Google Scholar] [CrossRef]
  99. Oroian, B.A.; Ciobica, A.; Timofte, D.; Stefanescu, C.; Serban, I.L. New Metabolic, Digestive, and Oxidative Stress-Related Manifestations Associated with Posttraumatic Stress Disorder. Oxidative Med. Cell. Longev. 2021, 2021, 5599265. [Google Scholar] [CrossRef]
  100. Konkoly, J.; Kormos, V.; Szegner, L.M.; Schram, F.; Finsnes, M.A.; Bakány, A.; Luspay, E.; Kumar, P.; Kvak, E.; Gaszner, B.; et al. Transient receptor potential ankyrin 1 ion channels may control the stress adaptation and fear conditioning in the mouse model of posttraumatic stress disorder. Prog. Neurobiol. 2025; submitted. [Google Scholar]
  101. Kopp, B.L.; Wick, D.; Herman, J.P. Differential effects of homotypic vs. heterotypic chronic stress regimens on microglial activation in the prefrontal cortex. Physiol. Behav. 2013, 122, 246–252. [Google Scholar] [CrossRef]
  102. Tynan, R.J.; Naicker, S.; Hinwood, M.; Nalivaiko, E.; Buller, K.M.; Pow, D.V.; Day, T.A.; Walker, F.R. Chronic stress alters the density and morphology of microglia in a subset of stress-responsive brain regions. Brain Behav. Immun. 2010, 24, 1058–1068. [Google Scholar] [CrossRef]
  103. Yamamoto, S.; Morinobu, S.; Fuchikami, M.; Kurata, A.; Kozuru, T.; Yamawaki, S. Effects of single prolonged stress and D-cycloserine on contextual fear extinction and hippocampal NMDA receptor expression in a rat model of PTSD. Neuropsychopharmacology 2008, 33, 2108–2116. [Google Scholar] [CrossRef]
  104. Ganon-Elazar, E.; Akirav, I. Cannabinoids and traumatic stress modulation of contextual fear extinction and GR expression in the amygdala-hippocampal-prefrontal circuit. Psychoneuroendocrinology 2013, 38, 1675–1687. [Google Scholar] [CrossRef]
  105. Perrine, S.A.; Eagle, A.L.; George, S.A.; Mulo, K.; Kohler, R.J.; Gerard, J.; Harutyunyan, A.; Hool, S.M.; Susick, L.L.; Schneider, B.L.; et al. Severe, multimodal stress exposure induces PTSD-like characteristics in a mouse model of single prolonged stress. Behav. Brain Res. 2016, 303, 228–237. [Google Scholar] [CrossRef]
  106. Maj, J.; Sowińska, H.; Baran, L.; Kapturkiewicz, Z. The effect of clonidine on locomotor activity in mice. Life Sci. 1972, 11, 483–491. [Google Scholar] [CrossRef]
  107. Parale, M.P.; Kulkarni, S.K. Clonidine—Induced behavioural despair in mice: Reversal by antidepressants. Psychopharmacology 1986, 89, 171–174. [Google Scholar] [CrossRef] [PubMed]
  108. Capasso, A.; Loizzo, A. Clonidine-induced antinociception and locomotor hypoactivity are reduced by dexamethasone in mice. J. Pharm. Pharmacol. 2001, 53, 351–360. [Google Scholar] [CrossRef]
  109. Wickramasekara, R.N.; Bockman, C.; Hanke, J.; Schwegler, H.; McGee, J.; Walsh, E.; Yilmazer-Hanke, D. Alpha2-adrenergic dysregulation in congenic DxH recombinant inbred mice selectively bred for a high fear-sensitized (H-FSS) startle response. Pharmacol. Biochem. Behav. 2020, 188, 172835. [Google Scholar] [CrossRef] [PubMed]
  110. Bamgbade, O.A.; Tai-Osagbemi, J.; Bamgbade, D.O.; Murphy-Akpieyi, O.; Fadire, A.; Soni, N.K.; Mumporeze, L. Clonidine is better than zopiclone for insomnia treatment in chronic pain patients. J. Clin. Sleep Med. 2022, 18, 1565–1571. [Google Scholar] [CrossRef] [PubMed]
  111. Lanes, R.; Herrera, A.; Palacios, A.; Moncada, G. Decreased secretion of cortisol and ACTH after oral clonidine administration in normal adults. Metabolism 1983, 32, 568–570. [Google Scholar] [CrossRef]
  112. Bugajski, J. Central metabolic and pituitary-adrenocortical stimulatory action of histamine and clonidine. Pol. J. Pharmacol. Pharm. 1984, 36, 159–176. [Google Scholar]
  113. Bugajski, J.; Gadek, A. The effect of adrenergic and cholinergic antagonists on central histaminergic stimulation of pituitary-adrenocortical response under stress in rats. Neuroendocrinology 1984, 38, 447–452. [Google Scholar] [CrossRef]
Cells 15 00428 g001
Figure 1. Schematic representation of the experimental settings. (A) In Experiment I, stressed Trpa1 wild type (WT) and knockout (KO) mice were exposed to electric foot shock, while the controls were placed into the shocking chamber without delivery of shock (fear conditioning, FC). (B) Number of animals in Experiment I. (C) Time course of Experiment I: 4 weeks after FC all animals were placed back in shocking chamber without further foot shock (conditioned fear test, CFT). Mice were sacrificed 24 h later by transcardial perfusion and brain samples were collected for morphological analysis. (D) Time course of Experiment II: during FC each experimental group was exposed to foot shock. After 4 weeks, half of WT and KO animals were pretreated with clonidine (0.05 mg/kg; i.p.), while the other half received saline. Subsequently, 30 min after the i.p. injection, CFT (without foot shock) was performed followed by blood sampling from the tail within 5 min. (E) Number of animals in the groups of Experiment II.
Figure 1. Schematic representation of the experimental settings. (A) In Experiment I, stressed Trpa1 wild type (WT) and knockout (KO) mice were exposed to electric foot shock, while the controls were placed into the shocking chamber without delivery of shock (fear conditioning, FC). (B) Number of animals in Experiment I. (C) Time course of Experiment I: 4 weeks after FC all animals were placed back in shocking chamber without further foot shock (conditioned fear test, CFT). Mice were sacrificed 24 h later by transcardial perfusion and brain samples were collected for morphological analysis. (D) Time course of Experiment II: during FC each experimental group was exposed to foot shock. After 4 weeks, half of WT and KO animals were pretreated with clonidine (0.05 mg/kg; i.p.), while the other half received saline. Subsequently, 30 min after the i.p. injection, CFT (without foot shock) was performed followed by blood sampling from the tail within 5 min. (E) Number of animals in the groups of Experiment II.
Cells 15 00428 g001
Cells 15 00428 g002
Figure 2. Effect of electric foot shock on the behavioral responses of Trpa1 wild type (WT) and (knockout) KO mice 4 weeks later. Shock increased freezing without genotype influence, while jumping was influenced both by stress and genotype. (A,C) Bar charts represent the summarized behavioral responses over the 11 min period of the conditioned fear test. Circles demonstrate individual values. Two-way ANOVA followed by Tukey’s post hoc test was conducted; * p < 0.05; *** p < 0.001; n = 7–8/group. (B,D) Line graphs represent the changes in behavioral responses over cue presentation. Repeated measures ANOVA was conducted, followed by Tukey’s post hoc test. Colored star (*) symbols were used for showing significant differences between control and shocked animals (blue * between WT groups, red * between KO groups). Hashtags (#) demonstrate significant differences within the same group over cue presentation (teal # within shocked WT and orange # within shocked KO). Cross (×) symbols represent the significant differences between WT and KO shocked animals. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001; × p < 0.05, ×× p < 0.01; n = 7–8/group. PTSD: posttraumatic stress disorder. ISI: interstimulus interval.
Figure 2. Effect of electric foot shock on the behavioral responses of Trpa1 wild type (WT) and (knockout) KO mice 4 weeks later. Shock increased freezing without genotype influence, while jumping was influenced both by stress and genotype. (A,C) Bar charts represent the summarized behavioral responses over the 11 min period of the conditioned fear test. Circles demonstrate individual values. Two-way ANOVA followed by Tukey’s post hoc test was conducted; * p < 0.05; *** p < 0.001; n = 7–8/group. (B,D) Line graphs represent the changes in behavioral responses over cue presentation. Repeated measures ANOVA was conducted, followed by Tukey’s post hoc test. Colored star (*) symbols were used for showing significant differences between control and shocked animals (blue * between WT groups, red * between KO groups). Hashtags (#) demonstrate significant differences within the same group over cue presentation (teal # within shocked WT and orange # within shocked KO). Cross (×) symbols represent the significant differences between WT and KO shocked animals. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001; × p < 0.05, ×× p < 0.01; n = 7–8/group. PTSD: posttraumatic stress disorder. ISI: interstimulus interval.
Cells 15 00428 g002
Cells 15 00428 g003
Figure 3. Effect of electric foot shock on the tyrosine hydroxylase (TH) immunoreactivity of noradrenergic cells in the locus coeruleus (LC) of Trpa1 wild type (WT) and knockout (KO) mice. (A) Columns represent the average of the specific signal density of TH immunoreactivity per group. Circles demonstrate individual values. Two-way ANOVA followed by Tukey’s post hoc test was conducted; * p < 0.05, ** p < 0.01, *** p < 0.001; n = 7–8/group. (B) Representative confocal images of the TH (red) immunostaining. Scale bars: 25 µm. a.u.: arbitrary unit.
Figure 3. Effect of electric foot shock on the tyrosine hydroxylase (TH) immunoreactivity of noradrenergic cells in the locus coeruleus (LC) of Trpa1 wild type (WT) and knockout (KO) mice. (A) Columns represent the average of the specific signal density of TH immunoreactivity per group. Circles demonstrate individual values. Two-way ANOVA followed by Tukey’s post hoc test was conducted; * p < 0.05, ** p < 0.01, *** p < 0.001; n = 7–8/group. (B) Representative confocal images of the TH (red) immunostaining. Scale bars: 25 µm. a.u.: arbitrary unit.
Cells 15 00428 g003
Cells 15 00428 g004
Figure 4. Effect of electric foot shock on the astrocyte activation in the paraventricular nucleus of hypothalamus (PVN) in wild type (WT) and knockout (KO) animals using glial fibrillary acidic protein (GFAP) immunostaining. Columns represent (A) the number of glial cells and (B) average of the astrocyte activation score. Circles demonstrate individual values. Two-way ANOVA was conducted, followed by Tukey’s post hoc test; ** p < 0.01; *** p < 0.001 n = 7–8/group. (C) Representative confocal images of the GFAP (red) immunostaining. Dashed lines mark the borders of PVN. 3rd refers to the third ventricle. Scale bars: 25 µm. a.u.: arbitrary unit.
Figure 4. Effect of electric foot shock on the astrocyte activation in the paraventricular nucleus of hypothalamus (PVN) in wild type (WT) and knockout (KO) animals using glial fibrillary acidic protein (GFAP) immunostaining. Columns represent (A) the number of glial cells and (B) average of the astrocyte activation score. Circles demonstrate individual values. Two-way ANOVA was conducted, followed by Tukey’s post hoc test; ** p < 0.01; *** p < 0.001 n = 7–8/group. (C) Representative confocal images of the GFAP (red) immunostaining. Dashed lines mark the borders of PVN. 3rd refers to the third ventricle. Scale bars: 25 µm. a.u.: arbitrary unit.
Cells 15 00428 g004
Cells 15 00428 g005
Figure 5. Effect of electric foot shock on the microglia activation in the paraventricular nucleus of hypothalamus (PVN) in wild type (WT) and knockout (KO) animals using ionized calcium binding adaptor molecule 1 (IBA1) immunofluorescence. Columns represent (A) the average of the microglia number and (B) activation score. Circles demonstrate individual values. Two-way ANOVA followed by Tukey’s post hoc test was conducted; n = 7–8/group. (C) Representative confocal images of the IBA1 (green) immunostaining. Dashed lines mark the borders of PVN. 3rd refers to the third ventricle. Scale bars: 25 µm. a.u.: arbitrary unit.
Figure 5. Effect of electric foot shock on the microglia activation in the paraventricular nucleus of hypothalamus (PVN) in wild type (WT) and knockout (KO) animals using ionized calcium binding adaptor molecule 1 (IBA1) immunofluorescence. Columns represent (A) the average of the microglia number and (B) activation score. Circles demonstrate individual values. Two-way ANOVA followed by Tukey’s post hoc test was conducted; n = 7–8/group. (C) Representative confocal images of the IBA1 (green) immunostaining. Dashed lines mark the borders of PVN. 3rd refers to the third ventricle. Scale bars: 25 µm. a.u.: arbitrary unit.
Cells 15 00428 g005
Cells 15 00428 g006
Figure 6. Effect of clonidine pretreatment (0.05 mg/kg; i.p.) on freezing and jumping behavior during conditioned fear test, as well as on serum corticosterone (CORT) levels in shocked Trpa1 wild type (WT) and knockout (KO) animals. (A,C) Bar charts represent the summarized behavioral responses over the 11 min period of the conditioned fear test and (E) the plasma CORT levels. Circles demonstrate individual values. Two-way ANOVA followed by Tukey’s post hoc test was conducted; * p < 0.05; n = 9–11/group. (B,D) Line graphs represent the changes in behavioral responses over cue presentation. Repeated measures ANOVA followed by Tukey’s post hoc test was conducted. Colored hashtag (#) symbols demonstrate significant differences within the same group over cue presentation (red # within vehicle-treated KO, teal # within clonidine-treated WT and orange # within clonidine-treated KO). Cross (×) symbol represents the significant difference between vehicle-treated WT and KO animals during the fourth cue presentation. # p < 0.05; ## p < 0.01; ### p < 0.001, #### p < 0.0001; × p < 0.05; n = 9–11/group. PTSD: posttraumatic stress disorder. ISI: interstimulus interval.
Figure 6. Effect of clonidine pretreatment (0.05 mg/kg; i.p.) on freezing and jumping behavior during conditioned fear test, as well as on serum corticosterone (CORT) levels in shocked Trpa1 wild type (WT) and knockout (KO) animals. (A,C) Bar charts represent the summarized behavioral responses over the 11 min period of the conditioned fear test and (E) the plasma CORT levels. Circles demonstrate individual values. Two-way ANOVA followed by Tukey’s post hoc test was conducted; * p < 0.05; n = 9–11/group. (B,D) Line graphs represent the changes in behavioral responses over cue presentation. Repeated measures ANOVA followed by Tukey’s post hoc test was conducted. Colored hashtag (#) symbols demonstrate significant differences within the same group over cue presentation (red # within vehicle-treated KO, teal # within clonidine-treated WT and orange # within clonidine-treated KO). Cross (×) symbol represents the significant difference between vehicle-treated WT and KO animals during the fourth cue presentation. # p < 0.05; ## p < 0.01; ### p < 0.001, #### p < 0.0001; × p < 0.05; n = 9–11/group. PTSD: posttraumatic stress disorder. ISI: interstimulus interval.
Cells 15 00428 g006
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Konkoly, J.; Szegner, L.M.; Biró-Sütő, T.; Luspay, E.; Kumar, P.; Kvak, E.; Gaszner, B.; Berta, G.; Pintér, E.; Zelena, D.; et al. Deletion of TRPA1 Ion Channel Modulates the Central Stress Responses in a Mouse Model of Posttraumatic Stress Disorder. Cells 2026, 15, 428. https://doi.org/10.3390/cells15050428

AMA Style

Konkoly J, Szegner LM, Biró-Sütő T, Luspay E, Kumar P, Kvak E, Gaszner B, Berta G, Pintér E, Zelena D, et al. Deletion of TRPA1 Ion Channel Modulates the Central Stress Responses in a Mouse Model of Posttraumatic Stress Disorder. Cells. 2026; 15(5):428. https://doi.org/10.3390/cells15050428

Chicago/Turabian Style

Konkoly, János, Laura Mária Szegner, Tünde Biró-Sütő, Eszter Luspay, Prabhat Kumar, Erika Kvak, Balázs Gaszner, Gergely Berta, Erika Pintér, Dóra Zelena, and et al. 2026. "Deletion of TRPA1 Ion Channel Modulates the Central Stress Responses in a Mouse Model of Posttraumatic Stress Disorder" Cells 15, no. 5: 428. https://doi.org/10.3390/cells15050428

APA Style

Konkoly, J., Szegner, L. M., Biró-Sütő, T., Luspay, E., Kumar, P., Kvak, E., Gaszner, B., Berta, G., Pintér, E., Zelena, D., & Kormos, V. (2026). Deletion of TRPA1 Ion Channel Modulates the Central Stress Responses in a Mouse Model of Posttraumatic Stress Disorder. Cells, 15(5), 428. https://doi.org/10.3390/cells15050428

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop