Characterization of GABAergic marker expression in prefrontal cortex in dexamethasone induced depression/anxiety model

Abstract

Background: The pivotal responsibility of GABAergic interneurons is inhibitory neurotransmission; in this way, their significance lies in regulating the maintenance of excitation/inhibition (E/I) balance in cortical circuits. An abundance of glucocorticoids (GCs) exposure results in a disorder of GABAergic interneurons in the prefrontal cortex (PFC); the relationship between this status and an enhanced vulnerability to neuropsychiatric ailments, like depression and anxiety, has been identified, but this connection is still poorly understood because systematic and comprehensive research is lacking. Here, we aim to investigate the impact of dexamethasone (DEX, a GC receptor agonist) on GABAergic interneurons in the PFC of eight-week-old adult male mice.

Methods: A double-blind study was conducted where thirty-two mice were treated subcutaneously either saline or DEX (0.2 mg/10 ml per kg of body weight) dissolved in saline daily for 21 days. Weight measurements were taken at five-day intervals to assess the emotional changes in mice as well as the response to DEX treatment. Following the 21-day regimen of DEX injections, mice underwent examinations for depression/anxiety-like behaviours and GABAergic marker expression in PFC.

Results: In a depression/anxiety model generated by chronic DEX treatment, we found that our DEX procedure did trigger depression/anxiety-like behaviors in mice. Furthermore, DEX treatment reduced the expression levels of a GABA-synthesizing enzyme (GAD67), Reelin, calcium-binding proteins (parvalbumin and calretinin) and neuropeptides co-expressed in GABAergic neurons (somatostatin, neuropeptide Y and vasoactive intestinal peptide) in the PFC were reduced after 21 days of DEX treatment; these reductions were accompanied by decreases in brain size and cerebral cortex thickness.

Conclusion: Our results indicate that a reduction in the number of GABAergic interneurons may result in deficiencies in cortical inhibitory neurotransmission, potentially causing an E/I imbalance in the PFC; this insight suggests a potential breakthrough strategy for the treatment of depression and anxiety.

Keywords: GABAergic interneurons; GAD67; chronic dexamethasone stress; chronic restraint stress; depression and anxiety; prefrontal cortex; reelin.

Figure 1

DEX treatment led to depression/anxiety-like behaviors. Diagram of the experimental design and timeline (A). b–d The Dex treatment resulted in decreased sucrose preference (B) and increased immobility time in the TST (C), and FST (D). (E, F) The time spent in the light chamber (E) and the times mice shuttled between the two boxes (F) were decreased in the LDB test. (G) DEX resulted in a decreased distance in center zone of the OFT. n = 8 in each group and all the data are presented as mean ± SEM. Data were analyzed using Student’s t-tests (B-G). **p < 0.01 (control mice versus DEX-treated mice). Dex, dexamethasone; FST, forced swim test; OFT, open-field test; SPT, sucrose preference test; TST, tail suspension test; LDBT, light-dark box test.

Figure 2

DEX led to reduced thickness of the cerebral cortex and brain size. (A-C) Nissl staining revealed a significantly reduced cortical thickness at the S1FL region of the neocortex in DEX-treated mice in comparison to control mice. n = 8 for each group. (D, E) The brain size of control and DEX-treated mice at P77; three weeks of DEX treatment induced a mild reduction in brain size. n = 8 in each group. All the data are presented as mean ± SEM. Data were analyzed using Student’s t-tests (C, E). **p < 0.01 (control mice versus DEX-treated mice). DEX, dexamethasone; PFC, prefrontal cortex; S1FL, primary somatosensory cortex of forelimb.

Figure 3

The DEX-treated mice had reduced GAD65 and Reelin expression. (A-F) Coronal sections from P77 brains stained with GAD65 and Reelin markers. (A, B’) GAD65 in situ hybridization showed decreased puncta in DEX-treated mice (E, p<0.01, **) compared with control mice. (C, D’) Reelin puncta were significantly reduced in DEX-treated mice, as shown by in situ hybridization of Reelin (f, p<0.01, **). The boxed area represents the PFC, and the areas in (A-D) are enlarged in (A’-D’), respectively. Scale bar = 100 µm. n = 8 in each group. All the data are presented as mean ± SEM. Data were analyzed using Student’s t-tests. **p<0.01 (control mice versus DEX-treated mice). DEX, dexamethasone; PFC, prefrontal cortex.

Figure 4

The DEX-treated mice had reduced PV and CR expression. (A-F) Representative images of PV and CR immunohistochemistry in the cortex of control and DEX-treated mice. (A, B’) Immunostaining of PV showed decreased puncta in the DEX-treated mice (E, p<0.01, **) compared with the control mice. (C, D’) Similar to the phenotypes observed in PV immunohistochemistry, a decreased number of CR⁺ cells was found in the PFC after stress (f, p<0.01, **). The boxed area represents the PFC, and the areas in (A-D) are enlarged in (A’-D’), respectively. Scale bar = 200 µm. n = 8 in each group. All the data are presented as mean ± SEM. Data were analyzed using Student’s t-tests. **p<0.01 (control mice versus DEX-treated mice). DEX, dexamethasone; PFC, prefrontal cortex.

Figure 5

The DEX-treated mice had reduced neuropeptides specific to GABAergic interneurons. (A-F) Coronal sections from P77 brains stained with various neuropeptides specific to GABAergic interneuron markers. In situ hybridization of SST (A, B’), VIP (C, D’) and NPY (E, F’) is illustrated. (G-I) Quantitative analysis showed that there was an approximately 10% decrease in the number of SST⁺ PFC regions after DEX exposure (G, p<0.01, **). Moreover, VIP and NPY expression detected in the PFC was significantly reduced in DEX-treated mice (H-I, p<0.01, **). The boxed area represents the PFC, and the areas in (A-F) are enlarged in (A’-F’), respectively. Scale bar = 100 µm. n = 8 in each group. All the data are presented as mean ± SEM. Data were analyzed using Student’s t-tests. **p < 0.01 (control mice versus DEX-treated mice). Dex, dexamethasone. PFC, prefrontal cortex.

Figure 6

The possible mechanisms of the depression/anxiety model induced by DEX in mice, with the red arrows indicating upregulation or downregulation.