Materials and Methods: Male Sprague-Dawley rats (n=32) used in the experiments were divided into four groups. Anaesthetized animals were mounted on a stereotaxic apparatus and intracerebroventricularly infused with U-50488H (κ-agonist, 50µg/kg), Nor-BIN (κ-antagonist, 50µg/kg) or U-50488H + Nor-BIN. Control group received saline alone. All animals were decapitated 30 mins after infusions. Hypothalamus, hippocampus and striatum were microdissected and snap frozen. Concentrations of 5-hydroxytryptamin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA, 5-HT metabolite) in these brain regions were determined by HPLC-ECD.

Results: 5-HT and 5-HIAA values in the hypothalamus and hippocampus of U-50488H-treated group were significantly decreased compared to control group (p<0.01 and p<0.05). Indoleamine levels (ng/gr tissue) in the κ-antagonist group were found to be similar to or slightly lower than the control values (p<0.05). Nor-BIN reversed the U-50488H inhibition of 5-HT release in the agonist+antagonist treated group (p<0.001). 5-HT levels in the striatum of all three groups were significantly higher than control group (p<0.01), 5-HIAA concentrations in the same brain region significantly decreased (p<0.001). Ratio of 5-HIAA/5-HT in the hypothalamus, hippocampus and striatum was significantly (p<0.05 and p<0.001, respectively) lowered by the κ-agonist.

Conclusion: These results suggest that κ-opioid receptors modulate serotonergic neurotransmission in the hypothalamus, hippocampus and striatum. ©2006, Fırat Üniversitesi, Tıp Fakültesi

In the literature, most of the reports have examined the role of µ-opioid receptors in the modulation of the central serotonergic neurotransmission ^2,7,8,9. There are relatively few studies on the effects of κ-opioid receptors on the serotonergic system. U-50488H, a selective κ-receptor agonist, has been reported to have no effect on 5-HT release in rat brain slices containing the dorsal raphe nucleus ¹⁰. However, we have previously shown that intracerebroventricular (ICV) administration of another selective κ-opioid agonist, U-69593, significantly reduced concentrations of 5-HT in various nuclei of the hypothalamus in the female rat ⁶. Activation of κ- opioid receptors suppresses electrically evoked excitatory postsynaptic potentials on 5-HT-sensitive neurones in the rat dorsal raphe nucleus in vitro ¹¹. U-50488H has been shown to inhibit K+-evoked 5-HT release in rat cortical synaptosomes ¹². More recently, administration of κ-opioid agonists has been reported to reduce 5-HT content in the rat striatum ¹³.

Existence of κ-opioid receptors, along with the two other major opioid receptor subtypes (µ and δ) has been reported in various brain areas ¹⁴. κ-opioid receptors have been shown in the raphe nucleus and serotonergic axon terminals ¹⁵. Furthermore, gene expression of κ-opioid receptors has been reported in a number of brain regions, including the hypothalamus, hippocampus and striatum ¹⁶.

In the rat brain, several regions including the hypothalamus, hippocampus and striatum are extensively innervated by the serotonergic neurons located in the raphe nucleus ^17,18. In the central nervous system, 5-HT synthesis requires two enzymes: tryptophan hydroxylase and aromatic- L-aminoacid decarboxylase. Presence of these enzymes has also been shown by autoradiography in the rat brain areas containing serotonergic axon terminals ¹⁷.The present study was carried out to examine effects of κ-opioid receptors on the central serotonergic system in the male rat. Site-dependent action of κ-opioids on 5-HT release and turnover was also investigated in the hypothalamus, hippocampus and striatum.

Following chloral hydrate anaesthesia (400 mg/kg I.P.; Botafarma Laboratory, Ankara, Turkey), the rats were mounted on a stereotaxic apparatus and ICV infused with U-50488H (κ- agonist, 50µg/kg/6µl, n=8), Nor-Binaltorphimine (Nor-BIN, κ- antagonist, 50µg/kg/6µl; n=8) or U-50488H + Nor-BIN (n=8). Control group received sterile saline (6µl; n=8) alone. All animals were decapitated 30 mins after ICV manipulations, and their brains rapidly removed on dry ice. Hypothalamus, hippocampus and striatum were microdissected and snap frozen in liquid nitrogen. All experiments were performed in compliance with institutional and international guidelines for laboratory animal care.

Nor-BIN was purchased from Sigma Chem. Co. (St. Louis, USA). U-50488H was provided by Pharmacia-Upjohn (Kalamazoo, Michigan, USA).

Indolamine Analysis: Specific brain regions collected were stored at -80ºC prior to analysis. 600µl of 0.1M HCl was added to the samples, along with another 300µl of HCl containing 2ng of 3,4-dihydroxybenzylamine as an internal standard. The samples were homogenised and centrifuged at 3000rpm, 4ºC for 10 mins. They were filtered through 0.45 µm microfilters (MFS Inc., USA). Aliquots (20µl) of supernatant were injected on to a reverse phase high performance liquid chromatographic (HPLC) column (S5ODS-250A, 5µm, 4.6mm i.d.x25cm, Supelco Inc.) coupled to an electrochemical detector (ECD, Gilson, France). Concentrations of 5-HT and 5- HIAA (5-HT metabolite) in these brain regions were simultaneously detected. The method has been described in our previous report ¹⁹. All brain samples were weighed out on an electronic scale prior to HPLC analysis, and the results were expressed as ng amine/g wet weight tissue.

Statistics: One-way analysis of variance (ANOVA) was performed on the data. Level of significance was set at p<0.05.

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Figure 1: 5-HT concentrations (Mean ± SEM) in the hypothalamus of the male rat following ICV administration of κ- opioid agonist, antagonist or their combination. *: p<0.05 compared to the control group values, **: p<0.05 compared to the U-50488H group, One-Way ANOVA.

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Figure 2: 5-HT concentrations (Mean ± SEM) in the hippocampus of the male rat following ICV administration of κ- opioid agonist, antagonist or their combination. *: p<0.05 compared to the control group values, **: p<0.01 compared to the U-50488H group, One-Way ANOVA.

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Figure 3: 5-HT concentrations (Mean ± SEM) in the striatum of the male rat following ICV administration of κ-opioid agonist, antagonist or their combination. *: p<0.01 compared to the control group values, **: p<0.05 compared to the U-50488H group, One-Way ANOVA.

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Table 1: 5-HIAA concentrations (Mean ± SEM) in three brain areas following ICV administration of κ-opioid agonist, antagonist or their combination. a: p<0.01, b: p<0.05, c: p<0.001, d: p<0.005 compared to the control group values, e: p<0.05 compared to the U-50488H group, One-Way ANOVA.

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Table 2: 5-HIAA/5-HT ratio (Mean ± SEM) in three brain areas following ICV administration of κ-opioid agonist, antagonist or their combination. a: p<0.05 b: p<0.001 compared to the control group; c: p<0.01 compared to the U-50488H-treated rats alone, d: p<0.05 compared to the Nor-BIN-treated animals, One-Way ANOVA.

Ratio of 5-HIAA/5-HT in the hypothalamus, hippocampus and striatum was significantly (p<0.05 and p<0.001, respectively) lowered by the κ-agonist. Although administration of Nor-BIN elevated the ratio in all three brain areas, the increase was statistically significant (p<0.05) in only the striatum compared to those rats receiving U-50488H alone. Co-administration of Nor-BIN with U-50488H failed to antagonise the inhibitory effects of this κ-agonist on the 5- HIAA/5-HT ratio in the hypothalamus, hippocampus and striatum.

The hypothalamus receives extensive serotonergic projections originating from the midbrain raphe nuclei ¹⁷. Serotonergic fibres have been visualised in various nuclei of the hypothalamus, and their anatomical distribution overlaps with hypothalamic factor-releasing neurons ^17,20. The highest basal mRNA levels for κ-opioid receptors are found in the hypothalamus in the rat ¹⁶. There have been very few attempts to examine effects of κ-opioid receptors on 5-HT release in the hypothalamus. We have previously shown that ICV administration of U-69593 significantly decreased indolamine levels in the medial preoptic area, suprachiasmatic nucleus, arcuate nucleus and median eminence of the hypothalamus ⁶. Tifluadom, a κ agonist had no significant effect on the indoleamine release and/or turnover in specific hypothalamic areas in the female rat ²¹. In the present experiments, U-50488H significantly reduced concentrations of 5-HT and 5-HIAA in the total hypothalamus in the male rat. The inhibitory effects of the κ-agonist on the indolamine levels were reversed by Nor-BIN following its co-administration of with U-50488H. These findings indicate that κ-opioid receptors modulate the serotonergic release and turnover in the hypothalamus, and their influence is inhibitory.

In the striatum ⁹ and raphe magnus ^22, 5-HT concentrations were increased following administration of morphine. Effects of κ opioids on serotonergic system in the striatum are not well-studied. A recent microdialysis study has shown that systemic administration of salvinorin A, a potent and highly selective κ-opioid agonist had no significant effect on 5-HT concetrations in the nucleus accumbens in the rat ²³. In the present study, 5-HT concentrations were elevated in the striatum by both U-50488H and Nor-BIN or their combination. This is somewhat surprising since 5-HIAA levels and ratio of 5-HIAA/5-HT were significantly lowered by κ-agonist and increased by κ-antagonist alone. It might be that control group neurotransmitter levels rapidly metabolised to 5-HIAA in this brain region.

It has been suggested that effects of morphine on 5-HT synthesis and release are regionally selective ⁹. In order to examine area dependent effects of κ-opioid receptors, indolamine concentrations in three different brain regions were determined in our study. In view of our inconclusive findings in the striatum, the effects of κ-opioids on the serotonergic system may be site-dependent. It appears that the κ-opioid agonist lowers 5-HT synthesis by inhibiting its release from the nerve terminals as both 5-HT and 5-HIAA levels were reduced in parallel in the hypothalamus and hippocampus. This inhibition is probably exerted through their action at presynaptic nerve terminals ¹¹.

In conclusion, our results suggest that κ-opioid receptors modulate serotonergic neurotransmission in the hypothalamus and hippocampus. This effect of κ-opioids may be sitedependent in different brain regions. Furthermore, inhibition of 5-HT synthesis and release in the hypothalamus implicates that κ-opioid receptors may affect secretion profiles of various hypothalamic hormones by serotonergic mediation.

Acknowledgement
The authors would like to thank to the Pharmacia-Upjohn Company (Kalamazoo, Michigan, USA) for generously providing U-50488H.

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