Ethanol versus lipopolysaccharide-induced hypothermia: involvement of urocortin

The urocortin1 (Ucn1) neurons of the mid-brain-localized Edinger-Westphal nucleus (EW) are robustly responsive to ethanol (EtOH) administration, and send projections to the dorsal raphe nucleus (DRN), which contains corticotropin-releasing factor type 2 receptors (CRF2) that are responsive to Ucn1. In addition, the DRN has been shown to be involved in regulation of body temperature, a function greatly affected by EtOH administration. The goal of the present study was to identify the role that the urocortinergic projections from the EW to the DRN have in mediating EtOH-induced and lipopolysaccharide (LPS)-induced hypothermia. Male C57BL6/J mice were used. Groups of mice underwent cannulation of the DRN, and then received i.p. injections of EtOH (2g/kg) or LPS (600 microg/kg or 400 microg/kg), followed by intra-DRN injections of artificial cerebrospinal fluid (aCSF) or anti-sauvagine (aSVG) (55 pmol), a CRF2 antagonist. Separate groups of mice received single intra-DRN injections of Ucn1 (20 pmol), CRF (20 pmol) or aCSF. For all experiments, core temperatures were monitored rectally every 30 min for several hours post-injection. Both EtOH and LPS induced hypothermia, and aSVG significantly attenuated this effect after EtOH; however, there was no significant attenuation of hypothermia after either dose of LPS. Ucn1 injection also caused hypothermia, while CRF injection did not. These data demonstrate that EtOH-induced hypothermia, but not LPS-induced hypothermia, may involve Ucn1 from EW acting at CRF2 receptors in the DRN.     

Chronic low dose ovine corticotropin releasing factor or urocortin II into the rostral dorsal raphe alters exploratory behavior and serotonergic gene expression in specific subregions of the dorsal raphe

Corticotropin releasing factor (CRF) family peptides play key roles in integrating neural responses to stress. Both major CRF receptors have been pharmacologically identified in the dorsal raphe nucleus (DRN), a stress sensitive and internally heterogeneous nucleus supplying many forebrain regions with serotonergic input. Despite the involvement of chronic stress and serotonergic dysfunction in human mood and anxiety disorders, little is known about the effects of chronic CRF receptor activation on the DRN. We infused ovine CRF (1 ng/h), urocortin II (UCNII, 1 ng/h), or vehicle alone into rat DRN over 6 days. During infusion, animals were allowed to freely explore an open field for 15 min on each of 2 days, with the addition of a novel object on the second day. Following behavioral testing, 5-HT1A, 5-HT1B, 5-HT transporter (SERT), and tryptophan hydroxylase-2 (Tph2) expression was examined through the DRN by in situ hybridization. Ovine CRF infusion resulted in significantly decreased novel object touches, climbs, as well as increased latency to first novel object contact. UCNII had a similar but less dramatic effect, decreasing only climbing behavior. Both ovine CRF and UCNII blunted the decrease in corner time expected on re-exposure to the open field. Both peptides also produced regionally specific changes in gene expression: 5-HT1A expression was increased 30% in the mid-rostral ventromedial DRN, while SERT was decreased by 30% in the mid-caudal shell dorsomedial DRN. There also appeared to be a shift in the relative level of Tph2 expression between the ventromedial and core dorsomedial DRN at the mid-rostral level. Changes in 5-HT1A, SERT, and relative Tph2 mRNA abundance were correlated with novel object exploration. These findings suggest chronic intra-DRN administration of CRF agonists decreases exploratory behavior, while producing subregionally limited changes in serotonergic gene expression. These studies may be relevant to mechanisms underlying behavioral changes after chronic stress.     

Corticotropin-releasing factor antagonist reduces activation of noradrenalin and serotonin neurons in the locus coeruleus and dorsal raphe in the arousal response accompanied by yawning behavior in rats

We previously reported that intracerebroventricular (icv) administration of corticotropin-releasing factor (CRF) antagonist attenuates the arousal response during yawning behavior in rats. However, the CRF-related pathway involved in the arousal response during yawning is still unclear. In the present study, we assessed the involvement of the CRF-containing pathway from the hypothalamic paraventricular nucleus (PVN) to the locus coeruleus (LC) and the dorsal raphe nucleus (DRN) in the arousal response during frequent spontaneous yawning, which was induced by several microinjections of l-glutamate into the PVN in anesthetized rats, using c-Fos immunohistochemistry. The PVN stimulation showed significant increases in activation of PVN CRF neurons, LC noradrenalin (NA) neurons and DRN serotonin (5-HT) neurons as well as arousal response during yawning. But icv administration of a CRF receptor antagonist, α-helical CRF (9-41), significantly inhibited the activation of both LC NA neurons and DRN 5-HT neurons except the activation of CRF neurons in the PVN, and significantly suppressed the arousal response during yawning. These results suggest that the CRF-containing pathway from PVN CRF neurons to LC NA neurons and DRN 5-HT neurons can be involved in the arousal response during yawning behavior.     

Stress potentiation of morphine-induced dopamine efflux in the nucleus accumbens shell is dependent upon stressor uncontrollability and is mediated by the dorsal raphe nucleus

A single session of uncontrollable (inescapable tailshock, IS), but not controllable (escapable tailshock, ES), stress is known to selectively potentiate subsequent morphine-conditioned place preference in a dorsal raphe nucleus (DRN) serotonin (5-HT) dependent manner. Here, in vivo microdialysis is used to test the hypothesis that prior IS, but not ES, will potentiate morphine-induced dopamine (DA) efflux in the nucleus accumbens (NAc) shell and that this will occur by a pathway involving DRN 5-HT neurons. Male Sprague-Dawley rats were exposed to yoked IS, ES, or no stress. Twenty-four hours later, morphine (3 mg/kg s.c.) or saline was administered during microdialysis. As predicted, prior IS selectively potentiated morphine-induced DA, but not 5-HT, efflux in the NAc. This potentiation was due to morphine's action in the DRN because it was blocked by intra-DRN microinjection of the opioid antagonist naltrexone (10 microg). IS potentiation of morphine-induced DA efflux in the NAc was also dependent upon activation of 5-HT neurons in the DRN because it was blocked by intra-DRN microinjection of the 5-HT1A autoreceptor agonist 8-hydroxy-2-di-n-(propylamino)-tetralin (1 microg). No effect of IS was found on morphine-induced 5-HT or DA efflux in the ventral tegmental area. These results suggest a neural substrate for stress potentiation of morphine reward involving 5-HT neurotransmission in the DRN.     

Nociceptin/orphanin FQ decreases serotonin efflux in the rat brain but in contrast to a kappa-opioid has no antagonistic effect on mu-opioid-induced increases in serotonin efflux

Similar to kappa-opioids, nociceptin/orphanin FQ (OFQ) exerts anti-mu-opioid actions. This may involve interactions within the circuitry controlling 5-HT neurons in the dorsal raphe nucleus (DRN) that project to the nucleus accumbens (NAcc). To test this hypothesis, we compared the effects of OFQ and kappa-opioids on 5-HT efflux in the CNS of freely behaving rats. First, OFQ (30-300 microM) infused into the DRN for 120 min dose-dependently decreased 5-HT efflux in the DRN. The opioid receptor-like 1 (ORL-1) antagonist [Nphe(1)]nociceptin(1-13)NH(2) blocked this effect. Using dual-probe microdialysis we observed that OFQ (300 microM) infused into the DRN for 120 min produced parallel decreases in 5-HT efflux in the DRN and NAcc, suggesting that ORL-1 receptors in the DRN inhibit serotonergic neurons projecting to the NAcc. Also, 5-HT efflux in the NAcc was dose-dependently decreased during OFQ (30-300 microM) infusion into the NAcc. This suggests that OFQ can reduce 5-HT efflux in the NAcc both by inhibiting serotonergic neurons in the DRN and by stimulating ORL-1 receptors in the NAcc. Similar to OFQ, the kappa-opioids U-50,488 (300 microM) and dynorphin A(1-13) (300 microM) infused into the DRN for 120 min decreased 5-HT efflux in the DRN. This effect was blocked only by the kappa-opioid receptor antagonist nor-BNI. Lastly, we compared the ability of OFQ and U-50,488 to block mu-opioid-induced increases in 5-HT. The kappa-opioid U-50,488 (1000 microM) attenuated the increase in 5-HT induced by the mu-opioid agonist endomorphin-1 (300 microM) in the DRN. In contrast, OFQ (300-1000 microM) did not alter mu-opioid-induced increases in 5-HT efflux. In summary, kappa-opioids and OFQ both decreased 5-HT efflux in the CNS. However, in contrast to kappa-opioids, which reversed mu-opioid-induced increases in 5-HT efflux, the anti-mu-opioid effects of OFQ apparently do not involve changes in 5-HT transmission under our experimental conditions.     

Serotonergic and nonserotonergic dorsal raphe neurons are pharmacologically and electrophysiologically heterogeneous

The dorsal raphe nucleus (DRN) projects serotonergic axons throughout the brain and is involved in a variety of physiological functions. However, it also includes a large population of cells that contain other neurotransmitters. To clarify the physiological and pharmacological differences between the serotonergic and nonserotonergic neurons of the DRN, their postsynaptic responses to 5-hydroxytryptamine (5-HT, serotonin) and to selective activation of 5-HT1A or 5-HT2A/C receptors and their action potential characteristics were determined using in vitro patch-clamp recordings. The slices containing these neurons were then immunostained for tryptophan hydroxylase (TPH), a marker of serotonergic neurons. It was found that subpopulations of both serotonergic and nonserotonergic neurons responded to 5-HT with outward (i.e., inhibitory) and inward (i.e., excitatory) currents, responded to both 5-HT1A and 5-HT2A/C receptor activation with outward and inward currents, respectively, and displayed overlapping action potential characteristics. These findings suggest that serotonergic and nonserotonergic neurons in the DRN are both heterogeneous with respect to their individual pharmacological and electrophysiological characteristics. The findings also suggest that the activity of the different populations of DRN neurons will display heterogeneous changes when the serotonergic tone in the DRN is altered by neurological disorders or by drug treatment.     

Use of peripheral blood transcriptome biomarkers for epilepsy prediction

Fenfluramine reduces hunger and promotes body weight loss by increasing central serotonin (5-HT) signaling. More recently, neuropeptides have been linked to the regulation of feeding behavior, metabolism and body weight. To examine possible interactions between 5-HT and neuropeptides in appetite control, fenfluramine (200 nmol/0.5 μl/side) was administered directly into the hypothalamic paraventricular nuclei (PVN) of male rats. Bilateral fenfluramine produced significant hypophagia and increased expression of PVN corticotropin releasing factor (CRF) mRNA and neuropeptide Y (NPY) mRNA in the arcuate nucleus within the first hour after drug administration. Fenfluramine's effects on feeding behavior and mRNA expression were blocked by PVN injections of a 5-HT(1-2) receptor antagonist, metergoline (15 nmol/0.5 μl/side). These data suggest that 5-HT neurons targeting hypothalamic paraventricular CRF neurons may participate in an appetite control circuit for reducing food intake.     

Unilateral lesion of the nigrostriatal pathway induces an increase of neuronal firing of the midbrain raphe nuclei 5-HT neurons and a decrease of their response to 5-HT1A receptor stimulation in the rat

Several studies have shown that the 5-hydroxytryptamine (serotonin, 5-HT) system is severely affected after degeneration of nigrostriatal dopaminergic neurons. In the present study, we examined the changes in the firing rate and firing pattern of the dorsal and median raphe nuclei (DRN and MRN) 5-HT neurons, and the effect of the selective 5-HT_1A receptor agonist (R)-(+)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) and antagonist (N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-2-pyridylcyclohexane carboxamide maleate salt (WAY-100635) on the neuronal firing in rats with 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta by using extracellular recording. The unilateral lesion of the nigrostriatal pathway significantly increased the mean firing rate of DRN and MRN 5-HT neurons compared with normal rats, and the firing pattern of these neurons also changed significantly towards a more bursty one. The lower dose of 8-OH-DPAT, 4 μg/kg (cumulative doses, i.v.), completely inhibited the firing activity of all DRN and MRN 5-HT neurons examined in normal and sham rats. In contrast to normal and sham rats, only the higher doses of 8-OH-DPAT, 128 and 64 μg/kg, completely inhibited the firing rate of DRN and MRN 5-HT neurons in 6-OHDA-lesioned rats, respectively. Furthermore, the local application of 8-OH-DPAT, 1.5 μg, in the DRN completely inhibited the firing rate of 5-HT neurons in normal and sham rats, while having no effect on firing rate in the lesioned rats. Altogether, these results indicate that lesion of the nigrostriatal pathway leads to hyperactivity of DRN and MRN 5-HT neurons, suggesting the implication of the DRN and MRN in the pathophysiology of Parkinson's disease, and the decreased response of these 5-HT neurons to 5-HT_1A receptor stimulation, reflecting 5-HT_1A receptor dysfunction in 6-OHDA-lesioned rats.     

Selective activation of 5-HT(2C) receptors stimulates GABA-ergic function in the rat substantia nigra pars reticulata: a combined in vivo electrophysiological and neurochemical study

In vivo electrophysiology and microdialysis were used to investigate the physiological role of 5-HT(2C) receptors in the control of substantia nigra pars reticulata (SNr) function. Extracellular single-unit recordings were performed from putative GABA-containing neurons in the SNr of anesthetized rats, and local GABA release was studied by in vivo microdialysis in the SNr of awake freely-moving rats. Systemic administration of the selective 5-HT(2C) receptor agonist (S)-2-(chloro-5-fluoro-indol-1-yl)-1-methylethylamine 1:1 C(4)H(4)O(4) (RO 60-0175) caused a dose-dependent excitation of about 30% of the SNr neurons recorded. However, the remaining neurons were either inhibited or unaffected by systemic RO 60-0175, in similar proportion. Local application of RO 60-0175 by microiontophoresis caused excitation in the majority of SNr neurons tested (48%), whereas a group of neurons was inhibited (16%) or unaffected (36%). Both the excitatory and the inhibitory effects of systemic and microiontophoretic RO 60-0175 were completely prevented by pretreatment with SB 243213 [5-methyl-1-({2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl}carbamoyl)-6-trifluoromethylindoline], a selective and potent 5-HT(2C) receptor antagonist. Consistent with these electrophysiological data, both systemic and intranigral administration of RO 60-0175 and m-chlorophenylpiperazine (mCPP), a non-selective 5-HT(2C) agonist, markedly increased extracellular GABA levels in the SNr. The stimulatory effect of systemic and local RO 60-0175 on GABA release was completely prevented by systemic administration of SB 243213, whereas local application of SB 243213 into the SNr only partially blocked RO 60-0175-induced GABA release. It is concluded that selective activation of 5-HT(2C) receptors stimulates GABA-ergic function in the SNr, and the clinical relevance of these data is discussed.     

Estrogen receptor beta regulates the expression of tryptophan-hydroxylase 2 mRNA within serotonergic neurons of the rat dorsal raphe nuclei

Dysfunctions of the brain 5-HT system are often associated with affective disorders, such as depression. The raphe nuclei target the limbic system and most forebrain areas and constitute the main source of 5-HT in the brain. All 5-HT neurons express tryptophan hydroxylase-2 (TPH2), the brain specific, rate-limiting enzyme for 5-HT synthesis. Estrogen receptor (ER) beta agonists have been shown to attenuate anxiety- and despair-like behaviors in rodent models. Therefore, we tested the hypothesis that ERbeta may contribute to the regulation of gene expression in 5-HT neurons of the dorsal raphe nuclei (DRN) by examining the effects of systemic and local application of the selective ERbeta agonist diarylpropionitrile (DPN) on tph2 mRNA expression. Ovariectomized (OVX) female rats were injected s.c. with DPN or vehicle once daily for 8 days. In situ hybridization revealed that systemic DPN-treatment elevated basal tph2 mRNA expression in the caudal and mid-dorsal DRN. Behavioral testing of all animals in the open field (OF) and on the elevated plus maze (EPM) on days 6 and 7 of treatment confirmed the anxiolytic nature of ERbeta activation. Another cohort of female OVX rats was stereotaxically implanted bilaterally with hormone-containing wax pellets flanking the DRN. Pellets contained 17-beta-estradiol (E), DPN, or no hormone. Both DPN and E significantly enhanced tph2 mRNA expression in the mid-dorsal DRN. DPN also increased tph2 mRNA in the caudal DRN. DPN- and E-treated rats displayed a more active stress-coping behavior in the forced-swim test (FST). No behavioral differences were found in the OF or on the EPM. These data indicate that ERbeta acts at the level of the rat DRN to modulate tph2 mRNA expression and thereby influence 5-HT synthesis in DRN subregions. Our results also suggest that local activation of ERbeta neurons in the DRN may be sufficient to decrease despair-like behavior, but not anxiolytic behaviors.     

The Roles of Different Types of Serotonin Receptors in Activation of the Hypophyseal-Testicular Complex Induced in Mice by the Presence of a Female

Placing of receptive females in the sector of a cage separated by a partition preventing physical contact but allowing sight and olfaction induced increases in blood testosterone levels in male mice. The selective agonist of 5-HT1A receptors 8-OH-DPAT (0.1 mg/kg) and the mixed 5-HT1A/1B receptor agonist eltoprazine (3.0 and 10.0 mg/kg) blocked the activatory effect of presentation of females on the hypothalamohypophyseal-testicular complex (HHTC) in males, while the 5-HT1A receptor antagonist p-MPPI (0.2 mg/kg) prevented the inhibitory effects of 8-OH-DPAT and eltoprazine. The 5-HT1B receptor agonist CGS-12066A (1.0 and 2.0 mg/kg) had no effect, while the mixed 5-HT1B/2C agonist TFMPP (5.0 mg/kg) blocked the increase in blood testosterone in males in response to presentation of females. The 5-HT2A receptor antagonist ketanserin (1.0 and 2.0 mg/kg) prevented the increase in testosterone induced by the presence of females. The 5-HT3 receptor antagonist ondansetron (0.05 and 0.1 mg/kg) increased the initial plasma testosterone level but blocked activation of the HHTC induced by the presence of receptive females. These results led to the conclusion that 5-HT receptors are involved in controlling the sexual activation of males. Different types and even subtypes of the same type of 5-HT receptor had different effects, both inhibitory and activatory, on activation of the HHTC by receptive females. Blockade of HHTC activation induced by the presence of females appears to involve 5-HT1A and 5-HT2C receptors, while activation involves 5-HT2A and 5-HT3 receptors.     

Aggressive encounters alter the activation of serotonergic neurons and the expression of 5-HT1A mRNA in the hamster dorsal raphe nucleus

Serotonergic (5-HT) neurons in the dorsal raphe nucleus (DRN) have been implicated in stress-induced changes in behavior. Previous research indicates that stressful stimuli activate 5-HT neurons in select subregions of the DRN. Uncontrollable stress is thought to sensitize 5-HT neurons in the DRN and allow for an exaggerated 5-HT response to future stimuli. In the current study, we tested the hypothesis that following aggressive encounters, losing male Syrian hamsters would exhibit increased c-Fos immunoreactivity in 5-HT DRN neurons compared to winners or controls. In addition, we tested the hypothesis that losers would have decreased 5-HT1A mRNA levels in the DRN compared to winners or controls. We found that a single 15-min aggressive encounter increased c-Fos expression in 5-HT and non-5-HT neurons in losers compared to winners and controls. The increased c-Fos expression in losers was restricted to ventral regions of the rostral DRN. We also found that four 5-min aggressive encounters reduced total 5-HT1A mRNA levels in the DRN in losers compared to winners and controls, and that differences in mRNA levels were not restricted to specific DRN subregions. These results suggest that social defeat activates neurons in select subregions of the DRN and reduces message for DRN 5-HT1A autoreceptors. Our results support the hypothesis that social stress can activate 5-HT neurons in the DRN, reduce 5-HT1A autoreceptor-mediated inhibition, and lead to hyperactivity of 5-HT neurons.     

Acute and long-term actions of the antidepressant drug mirtazapine on central 5-HT neurotransmission.

Mirtazapine (ORG 3770, Remeron) is a new alpha 2-adrenoceptor antagonist which has been shown to be an effective antidepressant drug. The aims of the studies were to assess, using an in vivo electrophysiological paradigm in the rat, the effects of acute and long-term treatment with mirtazapine on pre- and postsynaptic alpha 2-adrenoceptors and to determine whether this drug could modulate serotonin (5-HT) neurotransmission. Acute administration of mirtazapine produced a transient increase of the firing activity of dorsal raphe 5-HT neurons. This effect was mediated via norepinephrine (NE) neurons because it was abolished in NE-lesioned rats. In fact, this increased firing rate of 5-HT neurons was due to their activation by the enhanced release of NE resulting from the blockade of alpha 2-adrenergic autoreceptors of locus coeruleus neurons. Furthermore, acute mirtazapine injection transiently enhanced the firing activity of locus coeruleus NE neurons and attenuated the suppressant effect of the alpha 2-adrenoceptor agonist clonidine on these NE neurons. Sustained administration of mirtazapine for 21 days (5 mg/kg/day, s.c., using minipumps) lead to a marked increase in the firing rate of 5-HT neurons (75%) but a more modest increase in the firing rate of NE neurons (30%), as well as to a desensitization of alpha 2-adrenergic heteroreceptors on 5-HT terminals in the hippocampus. The desensitization of these heteroreceptors, resulting from an increased synaptic availability of NE induced by mirtazapine would free 5-HT terminals from the inhibitory influence of NE on 5-HT release. These modifications of 5-HT neurons lead to an increased tonic activation of postsynaptic 5-HT1A receptors. The latter conclusion was based on the capacity of the selective 5-HT1A receptor antagonist WAY 100635 to enhance the firing activity of dorsal hippocampus CA3 pyramidal neurons in mirtazapine-treated rats but not in controls. This enhanced 5-HT neurotransmission may underlie to the antidepressant effect of mirtazapine.     

Stimulation of the parapyramidal region of the neonatal rat brain stem produces locomotor-like activity involving spinal 5-HT7 and 5-HT2A receptors

Locomotion can be induced in rodents by direct application 5-hydroxytryptamine (5-HT) onto the spinal cord. Previous studies suggest important roles for 5-HT7 and 5-HT2A receptors in the locomotor effects of 5-HT. Here we show for the first time that activation of a discrete population of 5-HT neurons in the rodent brain stem produces locomotion and that the evoked locomotion requires 5-HT7 and 5-HT2A receptors. Cells localized in the parapyramidal region (PPR) of the mid-medulla produced locomotor-like activity as a result of either electrical or chemical stimulation, and PPR-evoked locomotor-like activity was blocked by antagonists to 5-HT2A and 5-HT7 receptors located on separate populations of neurons concentrated in different rostro-caudal regions. 5-HT7 receptor antagonists blocked locomotor-like activity when applied above the L3 segment; 5-HT2A receptor antagonists blocked locomotor-like activity only when applied below the L2 segment. 5-HT7 receptor antagonists decreased step cycle duration, consistent with an action on neurons involved in the rhythm-generating function of the central pattern generator (CPG) for locomotion. 5-HT2A antagonists reduced the amplitude of ventral root activity with only small effects on step cycle duration, suggesting an action directly on cells involved in the output stage of the pattern generator for locomotion, including motoneurons and premotor cells. Experiments with selective antagonists show that dopaminergic (D1, D2) and noradrenergic (alpha1, alpha2) receptors are not critical for PPR-evoked locomotor-like activity.     

Involvement of glutamate neurotransmission and N-methyl-d-aspartate receptor in the activation of midbrain dopamine neurons by 5-HT1A receptor agonists: an electrophysiological study in the rat

Systemic administration of selective 5-HT1A agonists, such as 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OHDPAT), stimulates the electrical activity of ventral tegmental area (VTA) dopamine neurons by a mechanism which remains unknown. We have examined if this activation is dependent on glutamatergic, serotonergic and GABAergic neurotransmission and if 5-HT1A receptors located within the VTA or within the prefrontal cortex (PFC) could contribute. In vivo electrophysiological recordings were obtained from VTA dopamine neurons from anesthetized rats. The i.v. administration of the 5-HT1A agonist 8-OHDPAT induced a strong stimulation of burst and firing activity of dopamine neurons. This activation remained unchanged in rats pre-treated with the 5-HT depleting agent parachlorophenylalanine. However, pre-administration of the GABAB receptor antagonist phaclophen, but not of the GABAA antagonist picrotoxin, significantly reduced the 8-OHDPAT-induced activation. The N-methyl-d-aspartate (NMDA) antagonist MK 801 (dizocilpine), but not the AMPA/kainate antagonist [1,2,3,4-tetrahydro-7-morpholinyl-2,3-dioxo-6-(fluoromethyl)quinoxalin-1-yl] methyl-phosphonate (ZK 200775), partially prevented or reversed the effects of 8-OHDPAT. However, only the combined pre-administration of the two glutamate antagonists did completely prevent the activatory response to 8-OHDPAT and even converted the effect of 8-OHDPAT into an inhibition, in half of the dopamine neurons tested. Inactivation of the local 5-HT1A receptors by the microinfusion within the VTA of the selective 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY 100,635), or of pertussis toxin, reduced the ability of 8-OHDPAT to stimulate the firing of dopamine neurons but not their burst activity. On the other hand, burst activation elicited by 8-OHDPAT was strongly reduced following the inactivation of prefrontal 5-HT1A receptors achieved by the microinfusion of WAY 100,635 within the PFC. These results show that activation of midbrain dopamine neurons by the systemic administration of 5-HT1A agonists does involve the inactivation of a tonic GABAergic tone, involving mainly the GABAB receptors, probably leading to the stimulation of a glutamatergic excitatory drive from the PFC to the VTA and an increase in glutamate release. This will excite dopamine neurons, preferentially through NMDA receptors. Furthermore, our results suggest that some 5-HT1A receptors located within the VTA may also participate in this activation.     

Serotonin type II receptor activation facilitates synaptic plasticity via N-methyl-D-aspartate-mediated mechanism in the rat basolateral amygdala

The modulation of synaptic plasticity by serotonin type II (5-hydroxytryptamine type II (5-HT(2)))-receptor stimulation was explored using intracellular, field potential and Fura-2 fluorescence image recordings in a rat amygdala slice preparation. Bath application of 5HT(2) receptor agonist 1-(2,5)-dimethoxy-4-iodophen-2-aminopropane (DOI) transformed theta-burst-stimulated (TBS) synaptic plasticity from short-term potentiation to long-term potentiation. DOI enhanced N-methyl-D-aspartate (NMDA) receptor-mediated potentials and calcium influx without affecting the resting membrane potential or input resistance of the neurons. In contrast, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor-mediated excitatory synaptic responses were unaffected by DOI. The facilitating effects of DOI were blocked by the 5-HT(2) receptor antagonist, ketanserin, and by the 5-HT(2C)-receptor selective antagonist, RS102221. These results indicate that 5-HT(2)-receptor activation enhances NMDA receptor-mediated synaptic function in the basolateral amygdala (BLA).     

Opioidergic, GABAergic and serotonergic neurotransmission in the dorsal raphe nucleus modulates tonic immobility in guinea pigs

Tonic immobility (TI) is an innate defensive behavior that can be elicited by physical restriction and postural inversion and is characterized by a profound and temporary state of akinesis. Our previous studies demonstrated that the stimulation of serotonin receptors in the dorsal raphe nucleus (DRN) appears to be biphasic during TI responses in guinea pigs (Cavia porcellus). Serotonin released by the DRN modulates behavioral responses and its release can occur through the action of different neurotransmitter systems, including the opioidergic and GABAergic systems. This study examines the role of opioidergic, GABAergic and serotonergic signaling in the DRN in TI defensive behavioral responses in guinea pigs. Microinjection of morphine (1.1 nmol) or bicuculline (0.5 nmol) into the DRN increased the duration of TI. The effect of morphine (1.1 nmol) was antagonized by pretreatment with naloxone (0.7 nmol), suggesting that the activation of μ opioid receptors in the DRN facilitates the TI response. By contrast, microinjection of muscimol (0.5 nmol) into the DRN decreased the duration of TI. However, a dose of muscimol (0.26 nmol) that alone did not affect TI, was sufficient to inhibit the effect of morphine (1.1 nmol) on TI, indicating that GABAergic and enkephalinergic neurons interact in the DRN. Microinjection of alpha-methyl-5-HT (1.6 nmol), a 5-HT(2) agonist, into the DRN also increased TI. This effect was inhibited by the prior administration of naloxone (0.7 nmol). Microinjection of 8-OH-DPAT (1.3 nmol) also blocked the increase of TI promoted by morphine (1.1 nmol). Our results indicate that the opioidergic, GABAergic and serotonergic systems in the DRN are important for modulation of defensive behavioral responses of TI. Therefore, we suggest that opioid inhibition of GABAergic neurons results in disinhibition of serotonergic neurons and this is the mechanism by which opioids could enhance TI. Conversely, a decrease in TI could occur through the activation of GABAergic interneurons.     

Involvement of 5-hydroxytryptamine(1A) receptors in the descending anti-nociceptive pathway from periaqueductal gray to the spinal dorsal horn in intact rats,rats with nerve injury and rats with inflammation

Studies have shown that 5-hydroxytryptamine (5-HT) plays an important role in the descending pathway of pain modulation from brainstem to the spinal cord. Using selective 5-HT receptor antagonists, the present study investigated which type of 5-HT receptor(s) in the spinal cord was involved in the morphine-induced anti-nociception in intact rats, in rats with nerve injury and in rats with inflammation. The hindpaw withdrawal latencies decreased significantly after sciatic nerve injury and hindpaw inflammation compared with intact rats. Intrathecal administration of 25 or 10 microg of the selective 5-HT(1A) recepter antagonist spiroxatrine, but not 1 microg of spiroxatrine, significantly blocked the increased hindpaw withdrawal latencies to thermal and mechanical stimulation induced by intra-periaqueductal gray injection of 1 microg of morphine in intact rats. Intrathecal injection of the 5-HT(2) receptor antagonist RS 102221 and the 5-HT(3) receptor antagonist MDL 72222 had no significant effects on the increased hindpaw withdrawal latencies to both noxious stimulations induced by intra-periaqueductal gray injection of morphine. Furthermore, intrathecal administration of spiroxatrine, but not RS 102221 nor MDL 72222, significantly attenuated the increased hindpaw withdrawal latencies induced by intra-periaqueductal gray administration of morphine in rats with nerve injury and in rats with inflammation.The results demonstrate that the 5-HT(1A) receptor, not 5-HT(2) nor 5-HT(3) receptor, plays an important role in the descending pathway of anti-nociception from the brainstem to the spinal cord in intact rats, in rats with nerve injury and in rats with inflammation.     

Somatosensory regulation of serotonin release in the central nucleus of the amygdala is mediated via corticotropin releasing factor and gamma-aminobutyric acid in the dorsal raphe nucleus

Noxious cutaneous stimulation increases, whereas innocuous cutaneous stimulation decreases serotonin (5-HT) release in the central nucleus of the amygdala (CeA) in anesthetized rats. In the present study, we investigated the contribution of corticotropin releasing factor (CRF) receptors and gamma-aminobutyric acid (GABA) receptors in the dorsal raphe nucleus (DRN) to those responses. Release of 5-HT in the CeA was monitored by microdialysis before and after 10-min stimulation by pinching or stroking. Increased 5-HT release in the CeA in response to pinching was abolished by CRF₂ receptor antagonism in the DRN. Decreased 5-HT release in the CeA in response to stroking was abolished by either CRF₁ receptor antagonism or GABA_A receptor antagonism in the DRN. These results suggest that opposite responses of 5-HT release in the CeA to noxious versus innocuous stimulation of the skin are due to separate contributions of CRF₂, CRF₁ and GABA_A receptors in the DRN.