Concurrent modulation of extracellular levels of noradrenaline and cAMP during stress and by anxiogenic- or anxiolytic-like neuropeptides in the prefrontal cortex of awake rats

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Abstract

The purpose of this study was to examine the effects of stress and the role of locally infused anxiogenic-like neuropeptides galanin, CCK-8, vasopressin, substance P and neurokinin A, and anxiolytic-like peptides NPY, nociceptin/orphanin FQ, somatostatin and neurotensin, on modulation of noradrenaline (NA) and cAMP efflux monitored simultaneously by microdialysis in the medial prefronatal cortex of awake rats. Concentrations of cAMP were determined by a newly developed method based on derivatization of cAMP with 2-chloroacetaldehyde followed by HPLC with fluorescence detection. Local infusion of forskolin (10 and 30 μM) dose-dependently increased the cAMP levels to 417% and 1050% of the control group, respectively. Similarly, local infusion of NA (10 μM) increased the cAMP to the peak level of 168%. A 5-min tail pinch and a 10-min swim stress rapidly increased the NA and cAMP levels to 167% and 203% (NA) and 141% and 161% (cAMP), respectively. Infusion of galanin and CCK-8 (0.5 nmol, and 1.5 nmol/0.5 μl) dose-dependently increased NA to the peak levels of 191% and 179% and cAMP levels to 174% and 166%, respectively. The peak levels following infusions of vasopressin, substance P and neurokinin A were 91%, 135% and 86% for NA and 131%, 83% and 76% for cAMP, respectively. Infusions of anxiolytic-like peptides at highest concentrations significantly increased (NPY, 136%) or decreased (nociceptin, 71%; somatostatin, 86%) the NA levels, whereas neurotensin had no effect. The cAMP levels decreased to 86% (NPY, neurotensin), 78% (nociceptin), somatostatin infusion was without effect. The present findings confirmed a close correlation between the stress-induced increases in prefrontal cortical NA and cAMP levels, as well as, concurrent changes in NA and cAMP levels following infusions of galanin and CCK-8 (increased levels) and nociceptin/orphanin FQ (decreased levels). Infusions of other neuropeptides showed a more complex pattern of NA and cAMP responses.

Highlights

► Novel HPLC fluorescence derivatization method for determination of cAMP. ► Microdialysis monitoring of stress-induced release of noradrenaline and cAMP in rat mPFC. ► Anxiogenic and anxiolytic peptides differentially modulate noradrenaline and cAMP.

Introduction

Microdialysis is a well-established in vivo sampling technique for monitoring presynaptic events including neurotransmitter release, reuptake and metabolism (for review, see Kehr and Yoshitake, 2006). In addition, a number of reports have described a feasibility of using microdialysis also for monitoring the postsynaptic actions including sampling the molecules involved in intracellular signalling cascades and being able to traverse, at least partly, into the extracellular fluid (Egawa et al., 1988, Vallebuona and Raiteri, 1993). There is evidence from both in vitro and in vivo studies that intracellular cyclic AMP (cAMP) is able to leak, egress into the extracellular space and most importantly, that there is a linear relationship between the intracellular cAMP concentrations and the efflux rate of cAMP into the extracellular compartment. Thus, the egress of cAMP in cultured cell lines was shown to account for about 15–18% of the total turnover of cAMP and it was concluded that the efflux process was most likely driven by active, energy-dependent mechanisms rather than just passive diffusion (Barber and Butcher, 1980). Similar findings were reported for tissue slices (Lazareno et al., 1985, Stoof and Kebabian, 1981) and for in vivo experiments carried out first, by use of the push–pull cannula (Korf et al., 1976, Schoener et al., 1979) and then by microdialysis (the year would indicate that it’s not “lately”, I’d use “then” instead) (Egawa et al., 1988). The subsequent microdialysis studies have demonstrated that the increased production of extracellular cAMP in cortical and hippocampal areas was strongly correlated to increased noradrenergic stimulation. Thus, both noradrenaline (NA) and cAMP levels increased in response to stress caused by immobilization or by intraperitoneal injection of saline (Stone and John, 1992) or by pharmacological means (Egawa et al., 1988, Montezinho et al., 2006, Montezinho et al., 2007, Mork, 1993, Mork and Geisler, 1995, Stone and John, 1991). Infusion of NA via the probe implanted in the rat frontal cortex caused a dose-dependent increase in cAMP efflux and this effect was blocked by infusion of the β-adrenoceptor antagonist, timolol, indicating that the NA-induced cAMP efflux is mediated primarily by activation of β-receptors (Egawa et al., 1988). This conclusion was confirmed by the following microdialysis studies showing that stimulation of β-adrenoceptors with isoproterenol or forskolin increased the extracellular cAMP levels in the rat medial prefrontal cortex (mPFC) and these effects were significantly inhibited by mood stabilizers such as/including lithium, carbamazepine, or valproate (Montezinho et al., 2006, Montezinho et al., 2007). These microdialysis data together with a large number of behavioural and pharmacological studies (for review, see Morilak et al., 2005) suggest a role of NA and cAMP in mediating stress and anxiety responses.

Several neuropeptides and their respective receptors are expressed in the central nervous system, including the prefrontal cortex, hippocampus and amygdala, the major anatomical structures implemented in modulation of cognitive functions, as well as affective behaviours (for review, see Ebner et al., 2009, Hökfelt et al., 2000, McGonigle, 2012, Ögren et al., 2010). Thus, neuropeptides including galanin, NPY, nociceptin/orphanin FQ, somatostatin and their receptors are predominantly inhibitory being coupled to Gi/o protein-coupled receptors (GPCRs), whereas cholecystokinin octapeptide (CCK-8) and vasopressin receptors are stimulatory Gs or Gq/11 GPCRs and neurotensin, substance P and neurokinin A receptors are Gq/11 GPCRs (Alexander et al., 2011). A body of evidence exists for the role of these peptides in regulation of stress responses, which in turn are strongly linked to the aetiology of affective disorders including depression, anxiety, panic attack, posttraumatic stress disorder, anorexia or fibromyalgia (Ebner et al., 2009, Engin et al., 2008, Feifel et al., 1999, Fernandez et al., 2004, Goeldner et al., 2012, Heilig et al., 1989, McGonigle, 2012, Ögren et al., 2010). However, how the neuropeptides may modulate NA release and correspondingly, the extracellular cAMP levels in vivo in relevant neuroanatomical structures is yet not fully elucidated. The purpose of the present study was to investigate, by use of microdialysis, whether local infusions of nine representative neuropeptides with proposed anxiogenic or anxiolytic properties to separate groups of rats could affect the extracellular levels of NA and cAMP and how these potential effects could correlate to the stress-induced responses in NA and cAMP efflux in mPFC of awake rats. The extracellular levels of cAMP in the microdialysis samples were determined by a newly developed high-sensitive and selective liquid chromatographic method based of precolumn derivatization with 2-chloroacetaldehyde and fluorescence detection.

Section snippets

Chemicals and solutions

Noradrenaline hydrochloride (NA), adenosine 3′,5′-cyclic monophosphate sodium salt (cAMP), sodium potassium chloride, phosphate monobasic monohydrate, sodium phosphate dibasic were obtained from Sigma (St. Louis, MO, USA). Methanol was purchased from Merck (Darmstadt, Germany), EDTA-2Na was obtained from Dojindo (Kumamoto, Japan). Forskolin was obtained from Wako Pure Chemicals (Osaka, Japan). Galanin (porcine) and nociceptin/orphanin FQ were purchased from Bachem (Bubendorf, Switzerland).

Basal and stimulated levels of cAMP and NA

The initial part of the study was focused on validation of the HPLC method developed for determination of cAMP in the microdialysis samples from the rat mPFC at basal conditions and following local stimulation with forskolin, and NA. Fig. 1A shows typical chromatograms of (a) aCSF blank solution and (b, c) standard solutions containing cAMP at concentrations of 6 fmol (b) and 60 fmol (c) in 10 μl samples derivatized with 10 μl of the 2-chloroacetaldehyde reagent. Other adenine compounds ATP, ADP,

Discussion

The purpose of this study was to examine effects of stress and the role of exogenously infused neuropeptides, galanin, CCK-8, vasopressin, nociceptin/orphanin FQ, somatostatin, neurotensin, NPY, substance P and neurokinin A, on modulation of NA release and cAMP efflux monitored by microdialysis in the mPFC of awake rats. Concentrations of cAMP in the microdialysates were determined by a newly developed sensitive chromatographic method based on derivatization of cAMP with 2-chloroacetaldehyde

Conclusions

In the present study, microdialysis samples were collected from the mPFC of awake rats and the extracellular cAMP and NA levels were determined simultaneously by two separate HPLC systems with fluorescence detection (cAMP) and electrochemical detection (NA). The results confirmed that there is a close correlation between the stress-induced increases in prefrontal cortical NA and cAMP levels. However, monitoring the NA and cAMP levels, following local infusions of some selected neuropeptides

Acknowledgements

The authors have no conflict of interest to declare.

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