A Role for the Adenosine ADORA2B Receptor in Midazolam Induced Cognitive Dysfunction

Abstract

Background: We recently reported a role for the circadian rhythm protein Period 2 (PER2) in midazolam induced cognitive dysfunction. Based on previous studies showing a critical role for the adenosine A2B receptor (ADORA2B) in PER2 regulation, we hypothesized that hippocampal ADORA2B is crucial for cognitive function.

Methods: Midazolam treated C57BL/6J mice were analyzed for Adora2b hippocampal mRNA expression levels, and spontaneous T-maze alternation was determined in Adora2b-/- mice. Using the specific ADORA2B agonist BAY-60-6583 in midazolam treated C57BL/6J mice, we analyzed hippocampal Per2 mRNA expression levels and spontaneous T-maze alternation. Finally, Adora2b-/- mice were assessed for mRNA expression of markers for inflammation or cognitive function in the hippocampus.

Results: Midazolam treatment significantly downregulated Adora2b or Per2 mRNA in the hippocampus of C57BL/6J mice, and hippocampal PER2 protein expression or T-maze alternation was significantly reduced in Adora2b-/- mice. ADORA2B agonist BAY-60-6583 restored midazolam mediated reduction in spontaneous alternation in C57BL/6J mice. Analysis of hippocampal Tnf-α or Il-6 mRNA levels in Adora2b-/- mice did not reveal an inflammatory phenotype. However, C-fos, a critical component of hippocampus-dependent learning and memory, was significantly downregulated in the hippocampus of Adora2b-/- mice.

Conclusion: These results suggest a role of ADORA2B in midazolam induced cognitive dysfunction. Further, our data demonstrate that BAY-60-6583 treatment restores midazolam induced cognitive dysfunction, possibly via increases of Per2. Additional mechanistic studies hint towards C-FOS as another potential underlying mechanism of memory impairment in Adora2b-/- mice. These findings suggest the ADORA2B agonist as a potential therapy in patients with midazolam induced cognitive dysfunction.

Keywords: Adora2b; BAY-60-6583; Bdnf; C-fos; Per2; T maze; cognitive function; delirium.

Figure 1.. T-maze spontaneous alternation in Adora2b^−/− mice.

Wildtype mice (C57BL6/J) were injected with midazolam (10mg/kg i.p.) and compared to saline-treated controls. (A) Experimental setup overview. (B) Adora2b transcript levels in the hippocampus. Brain hippocampus tissue was harvested, mRNA was isolated using RNeasy Mini Kit (Qiagen), cDNA was generated using miScript RT II kits (Qiagen), and Adora2b transcript levels were determined by quantitative real-time RT-PCR (iCycler or iCycler IQ; Bio-Rad Laboratories Inc.), n=5, mean±SD; p<0.05. (C) T maze spontaneous alternation test. (D) T-maze alternation in % from Adora2b^−/− mice, n=4–10, mean±SD; p<0.05.

Figure 2.. Hippocampal PER2 levels in Adora2b^−/− mice.

Wildtype (C57BL6/J) or Adora2b^−/− mice were analyzed for hippocampal PER2 protein levels at baseline. (A) Immunoblot and (B) quantification for hippocampal PER2 protein in wildtype and Adora2b^−/− mice, n=4–5, mean±SD; p<0.05.

Figure 3.. BAY-60–6583 reverses midazolam induced cognitive deficits.

Wildtype (C57BL6/J) mice were injected with either saline, midazolam (10mg/kg i.p.), or midazolam + BAY 60–6583 (1 μg/kg i.p.) and compared to saline-treated controls. 2 hours later mice underwent brain PER2 mRNA analysis. 24 hours later, mice underwent behavioral studies using T-maze alternation tests. (A) The molecular formula of BAY 60–6583, a specific ADORA2B agonist. (B) Hippocampal PER2 transcript levels 24hours after midazolam or midazolam + BAY 60–6583 administration (n=5; p<0.05). (C) T-maze alternation in % 24 hours after a single dose of midazolam or midazolam + BAY 60–6583 (n=4–10; p<0.05). All data are presented as mean ± SD.

Figure 4.. Adora2b^−/− mice have less hippocampal C-fos levels.

(A-D) Brain hippocampus tissue was harvested from wildtype (C57BL6/J) or Adora2b^−/− mice, mRNA was isolated using RNeasy Mini Kit (Qiagen), cDNA was generated using miScript RT II kits (Qiagen), and Tnf-α. (A), Il-6 (B), Bdnf (C) and C-fos (D) transcript levels were determined by quantitative real-time RT-PCR (iCycler or iCycler IQ; Bio-Rad Laboratories Inc.), n=4–5, p<0.05, all data are presented as mean ± SD.