Neuroscience Letters 371 (2004) 12–17 Zaprinast stimulates extracellular adenosine accumulation in rat pontine slices Minou Le a,1 , Yin Lu a,1 , Ya Li a , Robert W. Greene b , Paul M. Epstein c , Paul A. Rosenberg a,∗ a Department of Neurology and Program in Neuroscience, Enders Research Building, Room 349, Children’s Hospital and Harvard Medical School, Longwood Avenue, Boston, MA 02115, USA b Department of Psychiatry and Dallas VAMC, UTSW, Dallas, TX 75216, USA c Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06030, USA Received 11 June 2004; received in revised form 28 July 2004; accepted 28 July 2004 Abstract Adenosine appears to be an endogenous somnogen. The lateral dorsal tegmental/pedunculopontine nucleus (LDT/PPT) located in the mesopontine tegmentum is important in the regulation of arousal. Neurons in this nucleus are strongly hyperpolarized by adenosine and express neuronal nitric oxide synthase. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to inhibit the field excitatory postsynaptic potential. This action could be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by zaprinast. In the present study we tested the effect of zaprinast on extracellular adenosine accumulation in pontine slices containing the LDT. Zaprinast at 10 M evoked an increase in extracellular adenosine concentration. This effect was blocked by impermeant inhibitors of 5 ′ -nucleotidase, indicating that the extracellular adenosine was derived from extracellular AMP. However, inhibitors of cAMP degradation had little or no effect on zaprinast-evoked adenosine accumulation, suggesting that extracellular cAMP was not the source. Removal of extracellular calcium inhibited the effect of zaprinast. These results demonstrate that a pathway exists by which zaprinast stimulates extracellular adenosine accumulation, and the presence of this pathway in the pontine slice suggests the possibility that it may be relevant for the regulation of behavioral state. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Adenosine; Sleep; Zaprinast; cAMP; cGMP; Cyclic nucleotide phosphodiesterase The LDT and PPT nuclei are neighboring anatomically and functionally similar pontine nuclei that contain most of the brainstem cholinergic neurons. Compelling evi- dence associates these nuclei with arousal mechanisms [21,22,33,37,38]. Evidence suggesting an effect of adenosine on behavioral state derives in part from the widely appreciated stimulant effect of caffeine and other methylxanthines that block adenosine receptors [36]. It has been observed using in vivo microdialysis techniques that extracellular adenosine concentrations in the basal forebrain (BF) increased during wakefulness, and decreased during sleep. Furthermore, an ∗ Corresponding author. Tel.: +1 617 355 6962; fax: +1 617 730 0243. E-mail address: paul.rosenberg@tch.harvard.edu (P.A. Rosenberg). 1 These two authors contributed equally. adenosine transport inhibitor introduced unilaterally into the BF produced both significant increases in extracellular adenosine and slow wave sleep [25]. Another site of adeno- sine action that could contribute to changes in behavioral state, and that would be expected to have high efficacy in do- ing so would be the cholinergic neurons of the LDT/PPT that controls thalamocortical activation. In fact, whole-cell and extracellular recordings in an LDT/PPT slice showed that mesopontine cholinergic neurons are under tonic inhibitory control by endogenous adenosine, mediated postsynaptically by the inwardly rectifying potassium conductance and by in- hibition of I H [26]. Therefore, adenosine is a strong candidate for an endogenous sleep regulatory substance with important actions in the BF and the LDT/PPT. In our previous experiments using rat forebrain neuronal cultures, we obtained evidence for a mechanism of extracel- 0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.07.090