The present original in vivo data demonstrates that NOCI locally injected in the SN directly affects cell firing as well as DA and 5-HT levels in this brain region. Taken together with the observations from Calo' et al 13 that NOCI is involved in:

(i) Inhibition of glutamate release/anti-epileptic action and disruption of spatial memory; (ii) Inhibition of serotonin release/anxiolytic action; (iii) Inhibition of mesolimbic dopaminergic transmission/anti-rewarding properties; (iv) Modulation of striatal dopamine and glutamate/effects on locomotor activity, the present data further support the wide role of NOCI in the CNS functions as a potent modulator of neurotransmitter activities. In particular, these data support the direct interaction of NOCI with the dopaminergic and the serotoninergic activities in the SN.

Indeed, for what concern dopamine, two parallel ex vivo approaches, dual in situ hybridization (ISH) and neurotoxic lesions of DA neurons by using 6-hydroxydopamine (6-OHDA) were applied in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) by Norton et al. 14. This was done in order to verify whether NOCI acts directly on DA neurons i.e. upon NOCI-receptors (NOP), or indirectly by means of local circuitry, or both. It appears that NOCI mRNA was found largely on non-dopaminergic (i.e., GABA) neurons, whereas NOP mRNA was located on DA neurons. These data indicated that NOCI is in a position to influence DA neuronal activity by means of the NOP located on DA neurons. Accordingly, the present in vivo electrophysiological data support previous in vitro electrophysiological recordings observing that NOCI hyperpolarized the dopaminergic cells of the substantia nigra pars compacta and inhibited their firing activity 6.

Furthermore, in vivo microdialysis studies have shown a large increase of dopamine release (in the order of 300% of control values) in striatum when treating conscious rats with nociceptin at the micromolar concentration 15. This evidence is in accord with the present observation of a large increase of DA levels following local injection of NOCI in SN.

In contrast, intra-cerebroventricular administration of NOCI at a probe concentration of 1 mM but not at 0.1 mM significantly reduced rat nucleus accumbens dialysate DA levels in studies using a dual-probe microdialysis experimental design. Similar data were obtained when NOCI was applied to the ventral tegmental area of anesthetized rats by reverse dialysis while extracellular DA was sampled with a second dialysis probe in the nucleus accumbens 5. The observation that only the 1mM NOCI concentration but not the 0.1 mM concentration is acting upon DA levels may indicate that the technical approach used by these authors is not the most responsive. Nevertheless, this data is in accord with the present detection of reduced levels of DA in SN when the high dose of NOCI is injected (see Figure 2).

It is known that serotoninergic fibers from the raphe system project to the Substancia Nigra (SN) 16 and in particular that serotonin released from terminals in SN, derived from cell bodies in the raphe dorsalis nucleus, decreases the activity of the nigro-striatal dopaminergic system 17. It has been also observed that this modulation is behaviourally relevant 7.

Additionally, the present data showing large decrease of 5-HT levels in SN following local treatment with NOCI and a parallel dose-dependent, rapid decrease of (multi)-cell firing in the SN are in accord with results showing reduction of electrical activity of 5-HT neurons in RDN 18 as well as 5-HT release i.e. in cerebral cortex 19 after NOCI injection. Also, the inhibitory effect of NOCI on the nigral release of 5-HT can be related to the altered motor activity monitored i.e. using the fixed-speed rotarod test 6.

When considering the present data in a whole, it appears that there is a rapid effect of NOCI upon both DA and 5-HT signals in SN. The timing is similar although the effect is the opposite, with a very large increase of DA levels and a parallel large decrease of 5-HT values. However, while DA is rapidly largely significantly affected by NOCI 1nm, 5-HT is modified similarly by NOCI 10nm while is significantly less sensitive to NOCI 1nm (see Figure 2). Based on this observation one may propose that the rapid, large effect of NOCI 1nm on DA release may be possibly the driver of the comparatively slower and reduced change of serotonin levels, indicating that is dopamine that may be primarily influenced by NOCI in the SN.

The interaction between these two aminergic systems has been already reported in such diverse functions as temperature regulation 20, sleep 21, sexual behavior 22 and extrapyramidal function 23 although many results are conflicting 24.

In particular, it has been already reported that electrical stimulation of SN resulted in a rapid increase of the catecholaminergic DPVoltammetric signal followed by a slower decrease of the serotoninergic peak, both recorded simultaneously in the rat striatum 25. Accordingly, Kuhr and coll. 26 observed a rapid rise of DA levels in the caudate after electrical stimulation in SN. A similar and more consistent effect was observed when stimulating the medial forebrain bundle (MFB) and recording DA and 5-HT activities in the striatum 2527.

In conclusion, this work provides further evidence that DPV combined with mCFE is a valuable tool in the study of the in vivo effect of NOCI, an endogenous neuropeptide involved in a number of biological actions 282930. In particular, it proposes a multifaceted implication of this neuropeptide on both dopaminergic and serotoninergic functions in the SN, suggesting a primary influence upon dopamine activity followed by the serotoninergic response. This data may be of importance in the interpretation of the biological functions of NOCI as indeed the NOCI-NOP receptor system is widely represented throughout the CNS 31.

For instance, the influence upon motor behavior may be considered as an implication of both DA and 5-HT in physiological as well as pathological conditions i.e. Parkinson disease (PD) 32.

Furthermore, the involvement of serotonergic mechanisms in the development of Levodopa-induced dyskinesias (LIDs) via aberrant processing of exogenous levodopa and release of dopamine as a false neurotransmitter in PD patients has been recently confirmed 33.

Additionally, the implication of NOCI within the regulation of feeding, body weight homeostasis, stress, the stress-related psychiatric disorders of depression and anxiety, and in drug and alcohol dependence, pathological situations involving both DA and 5-HT, has been described (for a review see 34).

Therefore, the present data proposing a complex interaction between NOCI, DA and 5-HT systems may be of help in the interpretation of physiological as well as pathological states and consequent development of therapeutical approaches.