Activation of G protein-coupled ␣ 2 adrenergic receptors (ARs) inhibits epileptiform activity in the hippocampal CA3 region. The specific mechanism underlying this action is unclear. This study investigated which subtype(s) of ␣ 2 ARs and G proteins (G␣ o or G␣ i ) are involved in this response using recordings of mouse hippocampal CA3 epileptiform bursts. Application of epinephrine (EPI) or norepinephrine (NE) reduced the frequency of bursts in a concentration-dependent manner: (Ϫ)EPI Ͼ (Ϫ)NE ϾϾϾ (ϩ)NE. To identify the ␣ 2 AR subtype involved, equilibrium dissociation constants (pK b ) were determined for the selective ␣AR antagonists atipamezole (8.79), rauwolscine (7.75), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride (WB-4101; 6.87), and prazosin (5.71). Calculated pK b values correlated best with affinities determined previously for the mouse ␣ 2A AR subtype (r ϭ 0.98, slope ϭ 1.07). Furthermore, the inhibitory effects of EPI were lost in hippocampal slices from ␣ 2A AR-but not ␣ 2C AR-knockout mice. Pretreatment with pertussis toxin also reduced the EPImediated inhibition of epileptiform bursts. Finally, using knock-in mice with point mutations that disrupt regulator of G protein signaling (RGS) binding to G␣ subunits to enhance signaling by that G protein, the EPI-mediated inhibition of bursts was significantly more potent in slices from RGS-insensitive G␣ o G184S heterozygous (G␣ o ϩ/GS) mice compared with either G␣ i2 G184S heterozygous (G␣ i2 ϩ/GS) or control mice (EC 50 ϭ 2.5 versus 19 and 23 nM, respectively). Together, these findings indicate that the inhibitory effect of EPI on hippocampal CA3 epileptiform activity uses an ␣ 2A AR/G␣ o protein-mediated pathway under strong inhibitory control by RGS proteins. This suggests a possible role for RGS inhibitors or selective ␣ 2A AR agonists as a novel antiepileptic drug therapy.

Norepinephrine (NE) has potent antiepileptic properties, the pharmacology of which is unclear. The hippocampal CA3 region is vulnerable to over‐excitation (epileptiform burst activity). When GABAergic inhibition is blocked, NE reduces epileptiform activity through alpha‐2 adrenergic receptor (α2 AR) activation on pyramidal cells. We investigated which α2 AR subtype(s) mediate this effect. α2 AR mRNA expression using RT‐PCR in the CA3 region suggested that α2A AR subtype predominates relative to α2C AR and no α2B AR. Using field potential recordings in hippocampal slices we tested CA3 epileptiform activity. With a beta AR blockade, concentration‐response curves for AR agonists suggest that α2A ARs mediate CA3 epileptiform activity inhibition. Equilibrium dissociation constants (Kb) of selective alpha AR antagonists were determined to confirm the specific α2 AR subtype involved. Kb values correlated best with the α2A, but not the α2B and α2C AR subtypes. The results show that under impaired GABAergic inhibition, activation of α2A ARs is primarily responsible for antiepileptic action of NE. These findings increase our understanding of the role NE plays in attenuating epileptogenesis, and may aid the development of a subtype‐selective α2 AR agonist‐based strategy for treating seizures. Supported by NSF ND EPSCoR, NSF CAREER, NSF REU Site, NSF RET, NIH INBRE, EF, APS Explorations Program for Native Americans.

Pharmacological characteristics of the α2 adrenergic receptor (AR) that inhibits epileptiform activity in the hippocampal CA3 region were studied in rat brain slices. The effects of a large number of αAR antagonists on epinephrine (EPI)‐mediated inhibition of epileptiform activity were scrutinized with Schild plot analysis and compared to binding values for the rat and human α1, α2, 5‐HT1A, D2, I1, and I2 receptor subtypes. A perfect correlation and slope was found for the rat α2AAR, but not the other receptors, indicating that α2AARs solely mediate this effect. The actions of different chemical classes of α2AAR agonists at inhibiting epileptiform activity were then determined. While the imidazolines and guanidines exhibited the highest potency, the catecholamines had greater intrinsic activity compared to norepinephrine (NE). Dexmedetomidine, an imidazoline and guanabenz, a guanidine, were the most potent. In contrast, the catecholamines EPI and α‐methyl‐NE were the most efficacious. Furthermore, the α2AAR‐mediated inhibitory actions of EPI on hippocampal CA3 epileptiform burst frequencies were neither sex nor age‐dependent. These findings suggest that the activation of α2AARs could provide a new pharmacotherapeutic strategy for treating epilepsy and highlight the need for selective α2A agonists. Supported by ND EPSCoR, NSF CAREER 0347259, NSF REU Site 0639227, NIH P20 RR016741, APS and ASPET SURF.

Activation of α2A adrenergic receptors by epinephrine (EPI) attenuates seizure activity in the rat hippocampal CA3 region. Questions concerning the specific mechanism of this action are whether the α2A receptors are located pre‐ or post‐synaptically, and what subtype of G protein is coupled to this receptor. Evidence suggests that pre‐synaptic terminals of the recurrent axon collateral on pyramidal cells are involved; we hypothesize that the α2A receptors are located pre‐synaptically. We used transgenic mice with knock‐ins of the inhibitory regulators of G‐protein signaling (RGS)‐insensitive G183S Gnai2 allele for either the pre‐synaptic Gαo or the post‐synaptic Gαi2 protein subtype. EPI's anti‐epileptic effects were assessed with electrophysiological recordings in brain slices from control mice, modified Gαo mice, and modified Gαi2 mice.The EC50 of EPI was not significantly different between age‐matched C57BL/6J control and modified Gαi2 mice. In contrast, EPI was significantly more potent (>7‐fold) in the Gαo mice than the littermate controls. These results suggest that the pre‐synaptic Gαo protein and not the post‐synaptic Gαi2 protein mediates EPI's inhibition of hippocampal CA3 epileptiform activity. These findings could lead to new strategies for treating epilepsy. Supported by ND EPSCoR, NSF CAREER 0347259, NSF REU Site 0639227, NIH 5RO1GM039561, NIH P20 RR016741 from the INBRE Program, APS.

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