Involvement of 5-HT2A receptors in the serotonin (5-HT) syndrome caused by excessive 5-HT efflux in rat brain.

Previous studies have demonstrated that serotonin (5-HT) syndromes, particularly for the malignant cases, can be alleviated by ice water mists, cooling blankets and many other external cooling measures. In this study, we tested the hypothesis that external cooling measures reduce the responsivity of 5-HT(2A) receptors to excessive 5-HT efflux, which may be a possible mechanism underlying the treatment of serotonin syndrome. To test this, rat experiments were carried out in the standard and cool ambient temperature (T(amb) ) by administration of the 5-HT precursor 5-hydroxy-L-tryptophan combined with the monoamine oxidase inhibitor clorgyline. The first set of experiments was to assess severity of the syndromes by measuring body temperature responses. Consistent with the hypothesis, we found that the syndrome was malignant at the standard T(amb) of 22°C but alleviated at 12 or 6°C, these results being similar to those in rats pre-treated with the 5-HT(2A) receptor antagonist ketanserin. The second set of experiments was to utilize microdialysis to determine the relationship between the syndrome severity and 5-HT levels at the above-mentioned T(amb) . We found that excessive 5-HT efflux consisted of primary and secondary components through two distinct mechanisms. Furthermore, the secondary component efflux, which can be ascribed to 5-HT(2A) receptor activation, was proportionally reduced at the cool T(amb) of 12 and 6°C. In conclusion, results of this study support the hypothesis that cooling T(amb) reduces the functional activity of 5-HT(2A) receptors, thus alleviating the malignant syndrome.

mu-Opioids disinhibit and kappa-opioids inhibit serotonin efflux in the dorsal raphe nucleus.

The relative importance of GABAergic and glutamatergic afferents in mediating the effects of mu- and kappa-opioids on serotonin (5-HT) efflux in vivo has not been firmly established. Thus, we used microdialysis in the dorsal raphe nucleus (DRN) of freely behaving rats to study the effect of GABA and glutamate receptor antagonists on opioid-induced changes in 5-HT efflux. Infusing the mu-opioid agonist DAMGO (300 microM) increased extracellular 5-HT in the DRN by approximately 70%. During infusion of the GABA(A) receptor blocker bicuculline (100 microM), extracellular 5-HT increased by approximately 250%, and subsequent infusion of DAMGO decreased 5-HT to approximately 70% above the pre-bicuculline baseline. These data are consistent with the hypothesis that mu-opioids disinhibit 5-HT neurons, an effect attenuated by direct inhibition of 5-HT efflux or inhibition of excitatory influences on 5-HT efflux. To further test this hypothesis, glutamate receptor blockers, AP-5 (1 mM) and DNQX (300 microM), were co-infused with DAMGO. The glutamate receptor antagonists prevented decreases in 5-HT elicited by DAMGO in the presence of bicuculline. This indicates that DAMGO inhibits glutamatergic afferents, which partly offsets the disinhibitory influence of mu-opioids on 5-HT efflux. In contrast, the kappa-opioid agonist, U-50,488 (300 microM), decreased 5-HT by approximately 30% in the DRN. Glutamate and GABA receptor antagonists did not block this effect. In conclusion, mu-opioids inhibit GABAergic and glutamatergic afferents, thereby indirectly affecting 5-HT efflux in the DRN. In contrast, kappa-opioids inhibit 5-HT efflux independent of effects on glutamatergic and GABAergic afferents.

Changes in gamma-aminobutyric acid tone and extracellular serotonin in the dorsal raphe nucleus over the rat estrous cycle.

Gender differences in serotonin (5-HT) metabolism, synthesis, and release suggest that the gonadal steroids estrogen (E) and progesterone (P) influence 5-HT neurotransmission. Based on the effects of ovarian steroids in forebrain sites, this might involve changes in the strength of GABA afferent tone in the dorsal raphe nucleus (DRN). To test this hypothesis, the present study used in vivo microdialysis to measure basal extracellular 5-HT in the rat DRN across the estrous cycle and changes in 5-HT in response to the GABA(A) receptor antagonists bicuculline (50 microM) and picrotoxin (50 microM). During proestrus and estrus, baseline 5-HT levels were significantly higher compared to ovariectomized (OVX) rats. Mean baseline levels across experiments were 8.4 +/- 1.3 pg/ 30 microl in proestrus, 5.2 +/- 0.8 pg/30 mul in estrus, and 3.1 +/- 0.5 pg/30 microl in the DRN of OVX rats. Bicuculline and picrotoxin produced significantly greater increases in 5-HT during proestrus compared to estrus. Moreover, in the DRN of OVX rats, bicuculline and picrotoxin produced negligible increases in 5-HT. These data provide evidence of decreased 5-HT efflux and GABA tone in the rat DRN associated with low circulating E and P.

Effect of fentanyl on 5-HT efflux involves both opioid and 5-HT1A receptors.

1. Fentanyl is a micro -opioid analgesic that might disinhibit 5-HT neurons and thus increase 5-HT efflux. However, fentanyl also binds to 5-HT(1A) receptors, and if it activates 5-HT(1A) somatodendritic autoreceptors, the resultant inhibition might offset opioid-mediated increases in 5-HT efflux. To test this hypothesis, we used microdialysis to study effects of fentanyl on extracellular 5-HT in the dorsal raphe nucleus (DRN) of unanesthetized rats. 2. Systemic administration of fentanyl (0.01-0.2 mg kg(-1), s.c.) increased 5-HT efflux in the DRN. An intermediate dose of fentanyl (0.05 mg kg(-1)) produced the maximum increase in 5-HT to approximately 180% of baseline levels in the DRN. Naltrexone (10 mg kg(-1), s.c.) blocked the increase in response to systemic fentanyl (0.05 mg kg(-1)). 3. In contrast, during infusion into the DRN, fentanyl (10-1000 micro M) induced a dose-dependent decrease in 5-HT. Naltrexone and nor-binaltorphimine failed to block the decrease suggesting that micro - and kappa-opioid receptors did not mediate this effect. 4. Systemic (-)-pindolol (8 mg kg(-1), s.c.) or infusion of WAY-100635 (100 micro M) into the DRN blocked the decrease, and instead 5-HT increased in response to local infusion of fentanyl (100 micro M). WAY-100635 (0.3 mg kg(-1), s.c.) also potentiated the effect of systemic fentanyl (0.2 mg kg(-1), s.c.). (-)-Pindolol and WAY-100635 block 5HT(1A) receptors, indicating that inhibition of 5-HT neuronal activity resulting from fentanyl binding to somatodendritic autoreceptors attenuated opioid-mediated increases in 5-HT efflux. 5. These results provide novel evidence that besides stimulating micro -opioid receptors, fentanyl is a 5-HT(1A) receptor agonist. Possibly, this contributes to lethality of fentanyl overdose.

Effects of selective serotonin and serotonin/noradrenaline reuptake inhibitors on extracellular serotonin in rat diencephalon and frontal cortex.

Some clinical reports suggest that tricyclic antidepressants which block both noradrenaline and serotonin (5-HT) reuptake (SNRIs) are more effective than selective 5-HT reuptake inhibitors (SSRIs) in treating severe depression. Moreover, one neurochemical study reported larger increases in extracellular 5-HT in rat frontal cortex in response to the tricyclic antidepressant imipramine compared to the SSRI fluoxetine. However, imipramine, which blocks both 5-HT and noradrenaline reuptake, also binds with relatively high affinity to receptors for noradrenaline, histamine and acetylcholine. Thus, to test the hypothesis that compounds that inhibit both 5-HT and noradrenaline reuptake produce larger increases in 5-HT efflux, we compared the effects of acute systemic administration of several SNRIs and SSRIs. Extracellular 5-HT was measured using microdialysis probes implanted in the diencephalon and frontal cortex of unanesthetized rats. We tested the SSRIs paroxetine (0.3-10 mg/kg), citalopram (10-20 mg/kg) and fluoxetine (10 mg/kg), the nonselective tricyclic antidepressant imipramine (20 mg/kg) and the more selective SNRIs duloxetine (3-30 mg/kg) and venlafaxine (30-50 mg/kg). During the lights-off period, paroxetine and duloxetine increased 5-HT in the diencephalon approximately 300 and approximately 200%, respectively. During the lights-on period, paroxetine and duloxetine each increased 5-HT approximately 400% in the diencephalon. In the frontal cortex, both paroxetine and duloxetine increased 5-HT approximately 200%. Citalopram and venlafaxine each increased 5-HT in the diencephalon approximately 300%. Fluoxetine and imipramine increased 5-HT in the diencephalon by approximately 125 and approximately 80%, respectively. Thus, these results do not support the hypothesis that compared to SSRIs, compounds which inhibit both 5-HT and noradrenaline reuptake have a larger acute effect on extracellular 5-HT.

Influence of inhibitory and excitatory inputs on serotonin efflux differs in the dorsal and median raphe nuclei.

The dorsal (DRN) and median raphe nuclei (MRN) are two major sources of serotonergic projections to forebrain that are involved in regulation of behavioral state and motor activity, and implicated in affective disorders such as depression and schizophrenia. To investigate afferent influences on serotonergic neurons, this study compared the role of endogenous GABA and glutamate in the DRN and MRN using microdialysis and measurement of locomotor activity in freely behaving rats. Local infusion of the GABA(A) receptor antagonist bicuculline increased serotonin (5-HT) efflux in the DRN but not the MRN. In contrast, infusion of glutamate receptor antagonists produced larger decreases in 5-HT efflux in the MRN compared with the DRN. Moreover, glutamate receptor antagonists attenuated the increase in 5-HT efflux produced by GABA receptor blockade in the DRN. Thus, the disinhibitory effect of GABA blockers could be ascribed in part to an enhanced influence of glutamate. Measurements of locomotor activity indicate that changes in 5-HT were not simply correlated with behavioral activity induced by drug infusion. In summary, the role of inhibitory and excitatory afferents was strikingly different in the DRN and MRN. GABA afferents were the predominant tonic influence on serotonergic neurons in the DRN. In contrast, glutamatergic but not GABAergic afferents had a strong tonic influence on serotonergic neurons in the MRN.

Opioid receptor subtypes differentially modulate serotonin efflux in the rat central nervous system.

Opioid receptor subtypes may have site-specific effects and play different roles in modulating serotonergic neurotransmission in the mammalian central nervous system. To test this hypothesis, we used in vivo microdialysis to measure changes in extracellular serotonin (5-hydroxytryptamine; 5-HT) in response to local infusion of mu-, delta-, and kappa-opioid receptor ligands into the dorsal raphe nucleus (DRN), median raphe nucleus (MRN), and nucleus accumbens (NAcc) of freely behaving rats. The mu-opioids [D-Ala(2)-N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), endomorphin-1, and endomorphin-2 were administered by reverse dialysis infusion into the DRN. In response, extracellular 5-HT was increased in the DRN, an effect that was blocked by the selective mu-receptor antagonist beta-funaltrexamine, but not by the delta-receptor antagonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI-174,864). Infusion of delta-receptor agonists, [D-Ala(2),D-Len(5)]enkephalin (DADLE), [D-Pen(2,5)]enkephalin (DPDPE), and deltophin-II into the DRN also increased extracellular 5-HT, an effect that was blocked by selective delta-receptor antagonists. In contrast to the DRN, local infusion of mu- and delta-opioids had no effect on 5-HT in the MRN or NAcc. These data indicate that mu- and delta-opioid ligands have a selective influence on serotonergic neurons in the DRN. Finally, the kappa-receptor agonist U-50,488 [trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide] produced similar decreases in 5-HT during local infusion into the DRN, MRN, and NAcc. These results provide evidence of differential regulation of 5-HT release by opioid receptor subtypes in the midbrain raphe and forebrain.

GABAergic and glutamatergic afferents in the dorsal raphe nucleus mediate morphine-induced increases in serotonin efflux in the rat central nervous system.

To characterize the effects of morphine on serotonin (5-HT) in the central nervous system, we used microdialysis in freely behaving rats. Subcutaneous injection of morphine sulfate produced a dose-dependent increase in extracellular 5-HT in the dorsal raphe nucleus (DRN) and a forebrain site, the nucleus accumbens (NAcc). To determine the site of action for this effect, the opioid receptor antagonist naltrexone was infused into either the DRN or NAcc. Naltrexone infusion (300 microM) into the DRN but not the NAcc attenuated the increase in 5-HT elicited by systemic morphine (20 mg/kg). This suggests that morphine acts in the DRN to alter the activity of 5-HT neurons that project to NAcc. Consistent with this conclusion, infusion of the GABA(A) receptor antagonist bicuculline (100 microM) into the DRN but not the NAcc also blocked the effect of systemic morphine. Similarly, the effect of systemic morphine was blocked by infusion into the DRN of the GABA(A) receptor agonist muscimol (30 microM) and attenuated by the GABA(B) receptor agonist (+/-)-baclofen (100 microM). This provides evidence that morphine indirectly influences 5-HT release via opioid receptors on GABAergic neurons in the DRN. A new finding is that ionotropic glutamate receptor antagonists [kynurenate or a mixture of (+/-)-2-amino-5-phosphonopentanoic acid and 6,7-dinitro-quinoxaline-2,3-dione] infused in the DRN also attenuated the effect of systemic morphine. These results suggest that morphine acts on GABAergic and glutamatergic afferents to indirectly influence the activity of 5-HT neurons in the DRN. Understanding the details of this neural circuitry may provide new leads for treatment of opiate addiction.

Effects on serotonin in rat hypothalamus of D-fenfluramine, aminorex, phentermine and fluoxetine.

Hypothalamic 5-HT (serotonin) regulates food intake, energy expenditure and bodyweight. Using in vivo microdialysis, we determined the effects of various anorectic drugs on hypothalamic extracellular 5-HT levels during the dark phase when rats predominantly feed. Phentermine and aminorex, which were originally considered to be catecholaminergic drugs, markedly increased 5-HT efflux in rat hypothalamus. Their actions were less profound than D-fenfluramine, but considerably greater than that of the selective 5-HT reuptake inhibitor, fluoxetine. This suggests that enhanced hypothalamic 5-HT function could be involved in their anorectic actions. Pharmacological characterization revealed that D-fenfluramine, aminorex and probably also phentermine potentiate synaptic 5-HT function predominantly by release, whereas fluoxetine acts exclusively by reuptake inhibition. The results also revealed that the combined actions of phentermine and D-fenfluramine on hypothalamic extracellular 5-HT levels were additive, but not synergistic. In contrast, there was a significant negative cooperative effect on extraneuronal 5-HT of combining phentermine with fluoxetine.