Pharmacological characterization of the α_2A-adrenergic receptor inhibiting rat hippocampal CA3 epileptiform activity: comparison of ligand efficacy and potency

Figures & data

Figure 1. EPI reduces epileptiform burst discharge frequency. (A) Generation of hippocampal CA3 activity. Illustrated are pseudo-color images of a single epileptiform burst generated by voltage sensitive dye. (B) Continuous 150 s-long chart recordings of burst discharges are visualized on Axoscope 9.2. Increasing EPI produced a dose-dependent reduction of epileptiform burst activities. (C) Frequency histogram of burst discharges versus time of EPI administration. Each bin represents a 150-s time interval. (D) Concentration–response curve derived from 52 experiments.

Figure 2. Schild regression analysis using a highly selective α₂-AR antagonist. (A) Consecutive EPI concentration–response curves demonstrate a concentration-dependent effect of the selective α₂-AR antagonist RS79948. Pretreatment with 3 nM (○), 10 nM (■), and 30 nM (□) of RS79948 produced consecutive parallel rightward shifts of the curve that were significantly different from control (●) (EC₅₀ = 423 ± 148 nM, 1211 ± 204 nM and 3221 ± 1954 nM, respectively, vs. 95 ± 16 nM for control). (B) Using dose ratios calculated from individual experiments illustrated in (A), a Schild plot was created generating a regression slope equaling 0.95 ± 0.11 and an x-intercept correlating to a pK_b value of 9.00 ± 0.16 (n = 5).

Figure 3. Correlation between experimental and reported pharmacological values for various α-AR ligands at rat or human α_2A-ARs. Using the pK_i values in Table S1, correlation analyses were performed for the rat α_2A- (A), α_2B- (B), and α_2C-AR (C), or using pK_b (D), pEC₅₀ (E), or RE (F) at human α_2A-ARs. The correlation coefficient and slope was 0.99 and 1.02, respectively, for the rat α_2A-AR. A correlation coefficient and slope of 0.91 and 0.98, respectively, when correlating experimental pEC₅₀ with the published human pEC₅₀ for the α_2A-AR. A correlation coefficient and slope of 0.92 and 1.05, respectively, when correlating experimental RE with the published human RE for the α_2A-AR. Individual pK_b and pK_i values can be found in Table S1. Individual pEC₅₀ and relative efficacy values are presented in Table S3. The slope and fit of each correlation are summarized in Tables S2 and S4.

Figure 4. Comparison of various classes of α-AR agonists normalized to percent maximal reduction of NE on epileptiform burst inhibition. (A) The rank order potency for the imidazolines and guanadines is as follows: dexmedetomidine > guanabenz > UK-14304 ≥ oxymetazoline > guanfacine > clonidine ≫ xylazine. (B) The rank order potency for phenethylamines and their derivatives tested is as follows: EPI > 6-FNE ≥ α-methyl-NE > (−)-(R)-NE ≫ (+)-(S)-NE > (−)-Phenylephrine > Isoproterenol.

Figure 5. (A) Effects of age on the EPI-mediated inhibition of epileptiform activity. The average EC₅₀ values for the PND12–30, 60–120, and 121–200 groups were 131 ± 10 nM (n = 140), 160 ± 70 nM (n = 9), and 204 ± 79 nM (n = 8), respectively. The average efficacy values for the three groups were 75 ± 2% (n = 140), 75 ± 5% (n = 9), and 73 ± 9% (n = 7), respectively. (B) Sex does not influence EPI-mediated inhibition of epileptiform activity. Age-matched average EC₅₀ values are 93 ± 31 nM (n = 13) and 86 ± 19 nM (n = 13). Age-matched average efficacies are 75 ± 4.9% (n = 13), and 72 ± 4.9% (n = 13). Paired t-tests indicate no statistical significance in age or gender.

Data availability statement

Supplemental data sets are directly available in this manuscript or by contacting the corresponding author, Dr. Van Doze.