Noradrenaline and Seizures: A Perspective on the Role of Adrenergic Receptors in Limbic Seizures

Background Noradrenergic fibers originating from the locus coeruleus densely innervate limbic structures, including the piriform cortex, which is the limbic structure with the lowest seizure threshold. Noradrenaline (NA) modulates limbic seizures while stimulating autophagy through β2-adrenergic receptors (AR). Since autophagy is related to seizure threshold, this perspective questions whether modulating β2-AR focally within the anterior piriform cortex affects limbic seizures. Objective In this perspective, we analyzed a potential role for β2-AR as an anticonvulsant target within the anterior piriform cortex, area tempestas (AT). Methods We developed this perspective based on current literature on the role of NA in limbic seizures and autophagy. The perspective is also grounded on preliminary data obtained by micro-infusing within AT either a β2-AR agonist (salbutamol) or a β2-AR antagonist (butoxamine) 5 minutes before bicuculline. Results β2-AR stimulation fully prevents limbic seizures induced by bicuculline micro-infusion in AT. Conversely, antagonism at β2-AR worsens bicuculline-induced seizure severity and prolongs seizure duration, leading to self-sustaining status epilepticus. These data indicate a specific role for β2-AR as an anticonvulsant in AT. Conclusion NA counteracts limbic seizures. This relies on various receptors in different brain areas. The anterior piriform cortex plays a key role in patients affected by limbic epilepsy. The anticonvulsant effects of NA through β2-AR may be related to the stimulation of the autophagy pathway. Recent literature and present data draw a perspective where β2-AR stimulation while stimulating autophagy mitigates limbic seizures, focally within AT. The mechanism linking β2-AR to autophagy and seizure modulation should be extensively investigated.

Adult male Sprague Dawley rats weighing 280-320 g were submitted to stereotactic surgery under deep (Cloral Hydrate 400 mg/kg) anesthesia, for placement of a guide 22-gauge cannula.The experiments reported in this perspective have been performed in concomitance with the approved protocol for the experiments outlined by Giorgi et al. (2003).Twenty-four hours after surgery, an injection cannula (27-gauge) allowing infusion of chemoconvulsants was transiently inserted within the guide cannula to reach the anterior extent of left deep piriform cortex (Area Tempestas, AT), whose coordinates were AP=+4 mm from the bregma, ML=+3.2 mm from the midline and DV=−6.5 mm below the dura (according to the atlas of Pellegrino et al. 1979).The injection cannula was connected by polyethylene tube to a Hamilton syringe operated automatically by a Sage infusion pump with an infusion rate of 60 nL/minute.Further details on surgery are reported by Giorgi et al. (2003).
Bicuculline (118 pmol in 120 nL, Sigma Aldrich, Milan, Italy) was microinfused for 2 minutes into the AT.Five minutes before Bicuculline microinfusion rats were microinfused with saline (120 nL) ("Bic"group), or salbutamol (10nmol in 200 nL, Sigma)("Salbut+Bic" group) or butoxamine (10 µmol in 120 nL, Sigma) ("Butox+Bic" group) or both ("Butox+Salbut+Bic" group) (doses inferred and in line with exisiting literature and receptor affinity; Capuano et al., 1992;Waldmeier, 1981;Lazzeri et al., 2021).The amount of each compound to be micro-infused is expressed in number of moles (mol), which corresponds to the number of authentic molecules of each compound, according to the mole X the Avogadro number (moles X 6.022 X 10 23 ).This unit of measure is routinely applied in manuscripts expressing the amount of drugs micro-infused in the piriform cortex, area tempestas, starting from the pioneer paper by Piredda and Gale (1985).
In two groups of rats, animals were microinfused into the AT by either butoxamine or salbutamol, and five minutes later, they were infused with saline (120 nL) rather than bicuculline ("Butox" and "Salbut" groups, respectively).Each microinfusion within the AT was carried out for two minutes.After the end of each infusion, the infusion cannula was left in place for one more minute in order to allow complete delivery of the substance into the AT.Rats were observed individually for up to 1.5 hours (or after the last seizure episode occurrence) after infusion by an operator blinded to treatment; maximum seizure severity was scored using a 0.5-6 score according to Fornai et al., 2005 (Fig. 1).Only rats in which the correct cannula placement into the AT was verified after sacrifice were used for data analysis (N=8 rats for each group).
The following seizure data were compared between groups: a) percentage of rats experiencing at least one seizure episode after bicuculline; b) time from the infusion up to the end of the last seizure episode recorded by behavioral observation; c) maximum seizure score of the seizures experienced by each animal.

Statistical Analysis
Comparisons of the percentage of animals experiencing seizures within each group were performed by Chi Square analysis.Comparisons among the different groups concerning seizure severity (maximum seizure score) and seizure duration (min) were performed by Kruskal-Wallis analysis with Mann-Whitney post-hoc analysis.
Null hypothesis was rejected for P < 0.05.

RELATED REFERENCES
-Capuano CA, Leibowitz SF, Barr GA.The pharmaco-ontogeny of the perifornical lateral hypothalamic beta 2-adrenergic and dopaminergic receptor systems mediating epinephrine-and dopamine-induced suppression of feeding in the rat.