The Journal of Neuroscience, January 1994, 74(l): 263-271 Nitric Oxide-dependent Efflux of cGMP in Rat Cerebellar Cortex: An in viva Microdialysis Study Dasan Luo, Esther Leung, and Steven FL Vincent Kinsmen Laboratory of Neurological Sciences, Department of Psychiatry, The University of British Columbia, Vancouver, British Columbia V6T 123 Canada The stimulation of excitatory amino acid receptors in the [Key words: nitric oxide, cGMP, guanylyl cyclase, micro- cerebellar cortex results in the Ca*+/calmodulin-dependent dialysis, cerebellar cortex, excitatory amino acids, climbing activation of nitric oxide synthase. This leads to an increase fibers, harmaline] in tissue levels of cGMP following the interaction of nitric oxide with soluble guanylyl cyclase. The cerebellar cortex Glutamate receptor activation in cerebellarslices has beenshown has the highest levels of nitric oxide synthase and cGMP in to result in the formation of a diffusible messengerwith prop- the brain; however, the levels of guanylyl cyclase and cGMP- erties similar to those ofthe endothelium-derived relaxing factor phosphodiesterase are remarkably low. Thus, the mecha- (Garthwaite et al., 1988). This has now been shown to corre- nisms regulating cGMP levels in cerebellar cells are unclear. spond to nitric oxide (NO), produced by the Caz+/calmodulin- One report has noted that cGMP can be released from cer- dependent activation of NO synthase(Bredt and Snyder, 1989; ebellar slices. We have therefore used intracerebellar mi- Garthwaite et al., 1989). The releaseof endogenousexcitatory crodialysis in awake, freely moving rats to test the hypoth- amino acids, by activation of the climbing fiber afferents with esis that activation of nitric oxide synthase in the cerebellar harmaline, also evokes NO synthesis(Wood et al., 1990). The cortex results in the release of cGMP. Climbing fibers, which NO formed binds to and activates soluble guanylyl cyclase, release excitatory amino acids in the cerebellum, were ac- producing an increasein the tissuelevels of cGMP. Histochem- tivated with systemic harmaline. This resulted in an imme- ical and biochemical studies of the cerebellum have indicated diate increase in extracellular cGMP, which was blocked by that NO is formed in basket and granule cells, from which it TTX or the removal of extracellular Ca*+, and attenuated by diffuses into other target cells containing the guanylyl cyclase prior lesion of the climbing fibers. Blockade of N-type cal- (Garthwaite, 1991; Vincent and Hope, 1992). Thus, in this sys- cium channels with w-conotoxin also antagonized the har- tem NO appearsto act primarily as an intercellular messenger. maline-induced increase. In contrast, blockade of L-type cal- Although the cerebellum has very high levels of NO synthase, cium channels, or inhibition of anion transport with probenecid and the levels of cGMP in the cerebellum are an order of mag- or bromosulfophthalein, potentiated the increase in cGMP nitude higher than in most brain regions, it has only relatively seen in response to harmaline. Inhibitors of nitric oxide syn- low levels of guanylyl cyclase (Greenberg et al., 1978). The fact thase or guanylyl cyclase prevented the harmaline-induced that the cGMP phosphodiesteraseactivity in the cerebellum is increase in extracellular cGMP, while phosphodiesterase in- the lowest in the brain (Greenberg et al., 1978) may explain the hibitors potentiated the increase. Local application of the high basal cGMP levels, and the dramatic increasesin cGMP NMDA antagonist 2-amino+phosphonopentanoic acid or the levels that can be seen in responseto various drugs. These AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3- observations suggestenzymatic degradation via phosphodies- dione attenuated the effect of harmaline. In the presence of terasemay not play a major role in regulating intracellular cGMP TTX, local application of NMDA, AMPA, or the metabotropic levels in cerebellar cells. Instead, one in vitro study on cerebellar agonist frans-1 -amino-l ,3-cyclopentane-dicarboxylic acid slices found evidence for the release of cGMP that could be (PACPD) elevated extracellular cGMP. Thus, activation of increased by depolarization, glutamate stimulation, or an or- excitatory amino acid receptors results in a nitric oxide- ganic nitroso compound (Tjijrnhammar et al., 1986). This sug- dependent increase in the extracellular levels of cGMP in geststhat cyclic nucleotide releasemay be an important feature the cerebellar cortex. This may indicate the presence of an of the cerebellar cGMP signal transduction system. intercellular metabolic pathway for the regulation of intra- Cyclic nucleotides are the classic intracellular second mes- cellular cGMP levels. It is also possible that extracellular sengers.However, for many years the releaseof thesemolecules cGMP plays a role in intercellular communication in the cer- from various cell types has also been described (Barber and ebellar cortex. Butcher, 1983).The efflux of CAMP was first noted when pigeon erythrocytes were stimulated by noradrenaline (Davoren and Sutherland, 1963). Shortly thereafter, CAMP releasefrom rabbit Received Mar. 8, 1993; revised June 8, 1993; accepted July 6, 1993. cerebellar sliceswas noted following catecholaminestimulation This research was supported by a grant from the Medical Research Council of (Kakiuchi and Rall, 1968). An efflux of cGMP that parallels the Canada. S.R.V. is an MRC Scientist. Correspondence should be addressed to Steven R. Vincent, Ph.D., at the above increase in tissue levels has since been described in a number address. of tissues,including the pineal and posterior pituitary (O’Dea Copyright 0 1994 Society for Neuroscience 0270-6474/94/140263-09$05.00/O et al., 1978) the isolated perfused rat kidney (Burton et al., 264 Luo et al. * cGMP Release in the Cerebellum 1990; Heuze-Joubert et al., 1992), isolated rat aorta (Schini et In vitro recovery of cGMP by the microdialysisprobes was deter- mined by placing the probe in a beaker containing various concentra- al., 1989), bovine aorta endothelial cells (Schini et al., 1988), tions (1.7-45 nM) of cGMP at the standard perfusion rate of 5 pl/min. pancreatic acinar cells (Kapoor and Krishna, 1977), and he- Additional experiments were also done at various flow rates (l-10 pl/ patocytes (Billiar et al., 1992). Furthermore, there is a substan- min). The cGMP concentrations in the dialysate and in the beaker were tial level of cGMP in the cerebrospinal fluid (CSF) (Trabucchi determined using the same RIA procedure used for the in vivo studies. et al., 1977) and plasma cGMP levels can be influenced by various drug treatments (Brandt and Conrad, 1991; Schuller et Results al., 1992). In the present study, we have used the technique of in vivo microdialysis to determine whether an efflux of cGMP The baseline level of cGMP in the dialysate at a flow rate of 5 is associatedwith the activation of NO production in the cer- pl/min was0.74 f 0.07 pmol/ml (n = 65). The in vitro recovery ebellar cortex following excitatory amino acid receptor stimu- rate at 5 &min was about 1 l%, suggestinga basal extracellular lation. cGMP level in the cerebellar cortex of about 6.7 nM. In vitro experiments indicated that the recovery of cGMP varied in- Materials and Methods versely with the flow rate. In an attempt to determine more accurately the extracellular level of cGMP, animals were per- Microdialysisprobes. Transversemicrodialysis probes were constructed usinga saoonifiedcellulose ester membrane(SCE 135, CordisDow fused overnight at a flow rate of 0.1 Fl/min. This yielded a MedycalB:V., Netherlands).The horizontal probehad an openlength concentration of 4 nM in the dialysate, which at a recovery of 10 mm and an outer diameterof 0.27 mm. Stainlesssteel cannulas estimated to be 55% gave a basal extracellular cGMP concen- wereglued to both endsof the dialysisfiber. tration of 7.3 nM. Animals and surgery. Adult male Wistar rats (250-300 gm) were anesthetizedwith pentobarbitaland stereotaxicallyimplanted with a Climbing fiber activation with harmaline (40 mg/kg, i.p.) transversemicrodialysis probe in the cerebellarcortex (interaural,AP evoked an immediate, fivefold increasein extracellular cGMP -2. I, DV -3.5). The stainlesssteel cannulas were secured with dental levels (Fig. 1). This was prevented by blockade of voltage-de- cementto three anchoringscrews on the top of the skull. The animals pendent sodium channels, and thus action potentials, by the wereallowed to recoverfor 48 hr beforethe experiments. inclusion of 1 PM TTX in the dialysate. TTX had no effect on One groupof animalsreceived injections of the climbingfiber toxin 3-acetylpyridineaccording to the protocolof Llinaset al. (1975)I week basal levels of cGMP. Substitution of Mg2+ for Ca2+ in the prior to implantationof the microdialysisprobe. The animalsreceived perfusate also blocked the harmaline-induced increase,without 75 mg/kgof 3-acetylpyridine,followed 3 hr laterby 15mg/kg harmaline, significantly affecting basal cGMP levels (Fig. 1A). Likewise, and after another 1.5 hr by 300 mg/kg niacinamide. local application of the N-type Ca’+ channel blocker w-cono- Intracerebellar microdialysis. The microdialysisprobes were perfused toxin (100 FM) had no effect on basal cGMP levels, but atten- at 5 &min with artificial CSF,which contained147 mM NaCl, 3.0 mM KCl, I.0 mM MgCl,, 1.3 rnM CaCI,, 1 mM NaPO,, pH 7.4, in awake, uated the increaseevoked by systemic harmaline (Fig. 1B). In freely moving animals.Fractions were collected every 20 min,