2026 ECNP Congress

PS04-4023

The role of vitamin D in anxiety-like behavior and stress-response signaling (Tlr4/Nr3c1/Tgfb1) in rats

Zsolt Gáll — N. Miklós, E. Simon, P. Salamon · George Emil Palade University of Medicine- Pharmacy- Science and Technology of Targu Mures, Pharmacology and Clinical Pharmacy, Tirgu Mures, Romania

2026 ECNP Congress

Introduction

Vitamin D has a crucial role in bone and mineral homestasis, but it also acts as a neurosteroid in several brain regions regulating mood and anxiety [1]. The hippocampus, prefrontal and cingulate cortices express vitamin D receptors, which directly influences neurotrophic factors, neurotransmission, and neuroinflammation [2]. Clinical evidence supports that low serum 25-hydroxy vitamin D is associated with higher anxiety scores and greater odds of mood disorders in adults; however, the causality remains to be determined [3]. Rodent models showed that vitamin D deficiency (VDD) induced anxiety-like behaviours and altered performance in anxiety tests, while supplementation can improve it [4]. VDD was shown to disrupt immune-brain and neuroendocrine balance, which could be involved in the pathophysiology of anxiety disorders [5]. The aim of this study was to evaluate the behavioural features and altered gene expression profile of key elements of the stress-response in transient vitamin D deficiency.

Methods

Adult Wistar rats (n=36) were injected intraperitoneally with paricalcitol (6 doses of 32 ng) to induce transient VDD, which was maintained with specific “zero-cholecalciferol” diet. Three treatment groups were constituted: (1) control group (supplemented with 1,000 IU cholecalciferol per kg food to overcome deficiency), (2) VDD group, (3) vitamin deficient but supplemented group (using high dose 10,000 IU cholecalciferol per kg food, VDS). Behavioural assessment included an open-field test, focusing on exploration, novelty induced stress and self-grooming. All parameters were standardized using z‑scores, following the integrated behavioural z‑scoring approach. Higher z‑scores indicate less anxiety‑like behaviour. Brain tissue was sampled using RNA fixing solution and gene expression analysis was performed for Tlr4, Nr3c1, and Tgf‑β1 by RT-qPCR.

Results

Behaviorally, control rats showed the greatest exploratory activity, with higher supported rearing, longer stretch-attend posture, and lower velocity in the center zone of the OF compared with both VDD and VDS groups. There was a significant effect of group on the composite anxiety index (F(2,33)=4.14, p = 0.025), driven by higher z‑scores in the control group compared with both VDD and VDS groups. On the other hand, both VDD and VDS groups had increased expression of Tlr4, Tgfb1 and Nr3c1 genes relative controls animals. Tlr4 expression was increased ~5.7‑fold in VDD and ~33.9‑fold in VDS groups, while Nr3c1 was elevated ~4.4‑ and ~8.8‑fold, respectively. Relative to control rats, TGF‑β1 expression was increased ~6.8‑fold in VDD animals and ~54‑fold in VDS animals.

Conclusion

Together, these data suggest a U‑shaped effect of vitamin D on immune–brain interactions, both vitamin D deficiency and high dose supplementation were associated with heightened Tlr4/Tgfb1/Nr3c1 gene expression and reduced exploratory behaviour. Vitamin D deficiency plausibly contributes to anxiety via immune–stress pathways. The VDD group combines higher pro‑/regulatory inflammatory gene expression (Tlr‑4, Tgfb1) and elevated Nr3c1 with reduced exploratory behaviours relative to controls, aligning with evidence that low vitamin D status is linked to increased inflammation, altered hypothalamus-pituitary-adrenal axis signalling and higher anxiety risk.

References

  1. Casseb, G.A.S., Kaster, M.P. & Rodrigues, A.L.S., 2019. Potential Role of Vitamin D for the Management of Depression and Anxiety. CNS Drugs 33, 619–637.
  2. Gáll, Z., Székely, O, 2021. Role of Vitamin D in Cognitive Dysfunction: New Molecular Concepts and Discrepancies between Animal and Human Findings. Nutrients 13, 3672.
  3. Dionisie, V., Gaman, M.A., Anghele C., Manea, M.C., Puiu, M.G., Stanescu-Spinu, I.I., Baiu, O.I., Antonescu, F., Manea, M., Ciobanu, A.M. 2025. Vitamin D and depression in adults: A systematic review. Biomol Biomed. 25(10):2171-2196
  4. Al-Ramadhan, F. R., Abulmeaty, M. M. A., Alquraishi, M., Razak, S., & Alhussain, M. H. Effect of Vitamin D3 on Depressive Behaviors of Rats Exposed to Chronic Unpredictable Mild Stress. Biomedicines, 11(8), 2112.
  5. Kouba, B.R., Camargo, A., Gil-Mohapel, J., Rodrigues, A.L.S., 2022. Molecular basis underlying the therapeutic potential of vitamin D for the treatment of depression and anxiety. IJMS 23,13: 7077