Raphe-mediated signals control the hippocampal response to SRI antidepressants via miR-16.

Serotonin reuptake inhibitor (SRI) antidepressants such as fluoxetine (Prozac), promote hippocampal neurogenesis. They also increase the levels of the bcl-2 protein, whose overexpression in transgenic mice enhances adult hippocampal neurogenesis. However, the mechanisms underlying SRI-mediated neurogenesis are unclear. Recently, we identified the microRNA miR-16 as an important effector of SRI antidepressant action in serotonergic raphe and noradrenergic locus coeruleus (LC). We show here that miR-16 mediates adult neurogenesis in the mouse hippocampus. Fluoxetine, acting on serotonergic raphe neurons, decreases the amount of miR-16 in the hippocampus, which in turn increases the levels of the serotonin transporter (SERT), the target of SRI, and that of bcl-2 and the number of cells positive for Doublecortin, a marker of neuronal maturation. Neutralization of miR-16 in the hippocampus further exerts an antidepressant-like effect in behavioral tests. The fluoxetine-induced hippocampal response is relayed, in part, by the neurotrophic factor S100ß, secreted by raphe and acting via the LC. Fluoxetine-exposed serotonergic neurons also secrete brain-derived neurotrophic factor, Wnt2 and 15-Deoxy-delta12,14-prostaglandin J2. These molecules are unable to mimic on their own the action of fluoxetine and we show that they act synergistically to regulate miR-16 at the hippocampus. Of note, these signaling molecules are increased in the cerebrospinal fluid of depressed patients upon fluoxetine treatment. Thus, our results demonstrate that miR-16 mediates the action of fluoxetine by acting as a micromanager of hippocampal neurogenesis. They further clarify the signals and the pathways involved in the hippocampal response to fluoxetine, which may help refine therapeutic strategies to alleviate depressive disorders.

Raphé grafts and 3,4-diaminopyridine-evoked overflow of serotonin in the rat hippocampus after 5,7-dihydroxytryptamine lesions: evidence for 5-HT1A autoreceptors.

A first experiment verified that the overflow of 5-HT evoked by 75 microM 3,4-diaminopyridine in superfused hippocampal slices was calcium-dependent, tetrodotoxin-sensitive and modulable by drugs acting on 5-HT autoreceptors. Subsequently, the technique was used in rats to investigate the effects of 5,7-dihydroxytryptamine lesions and intrahippocampal serotonergic grafts. The lesions reduced the accumulation (-81%) and relative evoked overflow (-23%; absolute evoked overflow -86%) of [ H]5-HT, but increased the relative baseline overflow (+23%; absolute baseline overflow -78%). Grafts partially compensated for these effects. In slices from grafted rats, the evoked overflow was reduced by application of a 5-HT receptor agonist (8-OH-DPAT), a response not found in sham-operated and lesion-only rats. Although the graft-induced effects were less marked than in previous studies, they were beneficial and modulated by a mechanism that normally does not operate in the intact hippocampus.

Decrease of serotonin and metabolite in the forebrain and facilitation of lordosis by dorsal raphe nucleus lesions in male rats.

In castrated male rats, a radiofrequency lesion was made in the dorsal raphe nucleus (DRL) and lordosis behavior was observed following treatment with estrogen. After the behavioral test, brains were removed and the contents of 5-HT and 5-HIAA in the forebrain were measured by high pressure liquid chromatography (HPLC). In the results, only 2 of 16 control males without brain surgery showed lordosis, and the mean lordosis quotient (LQ) was extremely low when compared to that in control females. In contrast, all male rats with DRL displayed lordosis and the mean LQ was higher than that of control males without brain surgery but lower than that in control females (P < 0.001). In the DRL males, 5-HT and 5-HIAA contents in the septum (SPT), the preoptic area (POA), the ventromedial hypothalamus (VMH) and the striatum (STM) were lower than those in control male and female groups (P < 0.001). These results suggest that the dorsal raphe nucleus prevents male rats from showing lordosis by serotonergic influence in the forebrain. In addition, HPLC results showed that levels of the 5-HT in the SPT, the POA and the VMH in the female group were higher than those in the control male group (P < 0.05). In female rats, the POA (P < 0.01) and the VMH (P < 0.05) contained larger 5-HT than those in the SPT and the STM, but there were no difference of 5-HT contents in the male rat.

The effect of dorsal raphe nucleus (DRN) lesions on the locomotor activity rhythm in mice.

The study employed electrical lesions of dorsal raphe nucleus (DRN) to determine the functional significance of those nuclei in the regulation of wheel-running activity rhythm in mice in light/dark (LD 12:12), constant light (LL), and constant dark (DD) conditions. The wheel-running records showed that raphe nucleus lesions resulted in few days' decrease in common activity and amplitude in LD. The activity phase was not compact but in fragmentary form, especially in DD condition. In some animals an earlier onset of activity after DRN lesion in LD was observed. In LL extension of the rhythm period occurred. Destruction of DRN only slightly modulates the wheel-running circadian rhythm in mice.

Hormonal regulation of hippocampal spine synapse density involves subcortical mediation.

It is well established that estrogen has positive effects on the density of pyramidal cell spines in the hippocampal CA1 subfield. This study explored whether afferent connections of the hippocampus that come from estrogen-sensitive subcortical structures, including the septal complex, median raphe and supramammillary area, play a role in this estrogen-induced hippocampal synaptic plasticity. These particular subcortical structures have major influences on hippocampal activity, including theta rhythm and long-term potentiation. The latter also promotes the formation of new synapses. All of the rats were ovariectomized; the fimbria/fornix, which contains the majority of subcortical efferents to the hippocampus, was transected unilaterally in each, and half of the animals received estrogen replacement. Using unbiased electron microscopic stereological methods, the CA1 pyramidal cell spine synapse density was calculated. In the estrogen-treated rats, contralateral to the fimbria/fornix transection, the spine density of CA1 pyramidal cells increased dramatically, compared to the spine density values of both the ipsilateral and contralateral hippocampi of non-estrogen-treated animals and to that of the ipsilateral hippocampus of the estrogen replaced rats. These observations indicate that fimbria/fornix transection itself does not considerably influence CA1 area pyramidal cell spine density and, most importantly, that the estrogenic effect on hippocampal morphology, in addition to directly affecting the hippocampus, involves subcortical mediation.

Serotonergic modulation of hippocampal acetylcholine release after long-term neuronal grafting.

Adult female rats sustained aspirative fimbria-fornix lesions and, 2 weeks later, received intrahippocampal grafts of fetal septal or mixed septal-raphe cell suspensions. Twenty-four months later, the extracellular concentration of hippocampal acetylcholine (ACh) was determined by microdialysis. Basal ACh levels (5-65 fmol/5 microl sham-operated rats) were strongly reduced after lesioning (3-7 fmol/5 microl). In septally transplanted and septal-raphe co-transplanted rats, hippocampal ACh concentrations were restored to near-normal levels (15-25 fmol/5 microl), indicating long-term functional survival of hippocampal transplants. After administration of citalopram (100 microM by infusion) and fenfluramine (20 mg/kg i.p.), the hippocampal ACh efflux was increased by 2- to 3-fold in all groups of rats. The relative increase of ACh was highest in co-transplanted rats, an effect which was possibly due to functional interactions between grafted raphe and septal neurons.

Post-lesion up-regulation of 5-HT1B binding sites in the suprachiasmatic nucleus may be reversed after spontaneous or graft-induced serotonin reinnervation.

We have previously reported that selective axotomy of serotoninergic neurons produced by an intraventricular injection of 5, 7-dihydroxytryptamine is followed by an increase in 5-HT1B binding sites in the suprachiasmatic nucleus of the hypothalamus. This post-lesion up-regulation is shown here to be spontaneously reversed after long-term survival in spite of an incomplete reinnervation of the nucleus. Recovery may be accelerated by fetal raphe transplants that produce more rapid reinnervation.

Neurocircuitry of conditioned inhibition of analgesia: effects of amygdala, dorsal raphe, ventral medullary, and spinal cord lesions on antianalgesia in the rat.

Pain inhibition (analgesia) is produced by learned danger signals and inhibited by learned safety signals (antianalgesia). Conditioned analgesia is mediated by brain-to-spinal pathways releasing spinal endogenous opiates. Spinal morphine mimics learned danger signals in producing analgesia, which is inhibited by antianalgesia. The circuitry mediating antianalgesia is unknown. These experiments demonstrate that raphe dorsalis, raphe magnus, and spinal dorsolateral funiculus lesions abolish antianalgesia. Other lesions had no effect on antianalgesia. More important, lesions that blocked development of conditioned analgesia did not block development of antianalgesia. Thus, neural circuitries mediating analgesia and antianalgesia were found to be distinct, and conditioned inhibition of analgesia was found to act by inhibiting the most distal part of the conditioned analgesia circuit, namely, the spinal cord.

Effects of p-chlorophenylalanine on male sexual behavior in female rats with mesencephalic raphe nuclei lesions.

The role of serotonergic neurons in the mesencephalic raphe nuclei in regulating male sexual behavior in female rats was examined. The median or dorsal raphe nucleus lesions (MRL or DRL) were made in ovariectomized rats and behavioral tests were performed after implantation of Silastic tubes containing testosterone and treatment with serotonin synthesis inhibitor p-chlorophenylalanine (pCPA). Half of the animals in each group received 4 100 mg/kg pCPA injections before the behavioral test. As a result, the incidences and frequencies of mounts and intromissive patterns in the MRL and DRL groups were comparable to those in control females without brain surgery. Mount latency in the MRL females was shorter than that in the control females. When pCPA was given, most females with or without brain surgery showed mounts and intromissive patterns, and frequencies were higher than those in females without pCPA. These results suggest that the median raphe nucleus plays an inhibitory role in the onset mechanism for mounting. On the other hand, a stronger inhibitory influence in regulating male sexual behavior exists in other serotonergic neurons than those in the median and dorsal raphe nuclei in female rats.

Effects of chronic lesions in mesencephalic raphe nuclei on induction of pseudopregnancy.

The role of mesencephalic raphe nuclei in the induction of pseudopregnancy was investigated in female rats. The dorsal or median raphe nucleus lesions (DRL or MRL, respectively) were made by means of a radiofrequency lesion generator. Two or 3 weeks after the operation, in order to induce pseudopregnancy, the vagina was stimulated electrically on the day of proestrus or 1 mg/kg b.w. reserpine was injected on the day of diestrus I. Traumatization by passing thread to one uterine horn was performed to induce deciduoma 5 days after vaginal stimulation or 3 days after reserpine injection. As the results, decidual response was seen in most control and sham females in both vaginal stimulation and reserpine-treated groups. In contrast, incidences of deciduoma in DRL females with vaginal stimulation or reserpine-injection were significantly lower than those in control and sham groups. In the MRL females with either vaginal stimulation or reserpine-treatment, incidences of deciduoma were comparable to those of the control and sham operated groups. These results suggest that the dorsal raphe nucleus plays an important role in pseudopregnancy-inducing mechanisms in female rats.

[The effect of lithium oxybutyrate on aggressive animal behavior]. / Vliianie litiia oksibutirata na agressivnoe povedenie zhivotnykh.

The aim of the study was to examine the aggressive behavior of rats after lithium hydroxybutyrate injections (10 mg/kg once a day, at 9 a.m or 7 p.m., for a week). It was found that the drug did not affect the aggressive behavior of isolated rats towards mice placed to them. However, in the evening the drug inhibited aggressive manifestations (attacks, bites) in rats with raphe midbrain lesions. The 5-hydroxytryptophan (20 mg/kg, intraperitoneally, once) augmented the antiaggressive properties of lithium hydroxybutyrate in the evening but weakened them in the morning.

Inhibitory effect of baclofen on lordosis in female and male rats with dorsal raphe nucleus lesion or septal cut.

The inhibitory effect of baclofen, a GABAB receptor agonist, on lordosis was examined in female and male rats with dorsal raphe nucleus lesions (DRL) or cut of the septal fibers (ARD). Both female and male DRL and ARD rats showed higher lordosis quotients (LQ) than corresponding controls without brain surgery. This indicates that the dorsal raphe nucleus and the septum exert lordosis-inhibiting influences in female and male rats. After treatment with 10 mg/kg baclofen, the mean LQs in female control and female ARD groups were significantly lower than those of vehicle-treated control and ARD females. In DRL females, however, LQs did not decrease, even after the injection of baclofen. In males, baclofen also diminished lordosis in ARD rats, but not in DRL rats. These results suggest that the GABAB receptor system plays an inhibitory role in regulating lordosis behavior not only in female but also in male rats. Furthermore, the function of the GABA neurons depends on the inhibitory mechanism of the dorsal raphe nucleus in the regulation of female sexual behavior.

Intraocular co-grafts of fetal dorsal raphe nucleus and suprachiasmatic nucleus.

Serotonergic neurons in the fetal dorsal raphe nucleus were grafted together with fetal anterior hypothalamic tissue including the suprachiasmatic nucleus (SCN) to the anterior eye chamber of adult rats. After 6 weeks transplantation, the double grafts were immunocytochemically examined using antisera against serotonin, arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). The raphe grafts contained a large number of serotonin-immunoreactive neurons and fibers, but only a few AVP-immunoreactive fibers and VIP-immunoreactive neurons and fibers. On the other hand, numerous AVP- and VIP-immunoreactive neurons and fibers were found in the SCN of the anterior hypothalamic graft. Outgrowing serotonin-immunoreactive fibers from the raphe tissue were densely distributed in the anterior hypothalamic graft. In the SCN, however, only a few fibers were detected. The results demonstrate that the isolated anterior hypothalamic grafts can be innervated by the serotonergic neurons from the raphe grafts, but the innervation pattern of these fibers was quite different from the normal rat. The present results indicate that the isolated SCN has an inhibitory influence on the growth of serotonergic fibers.

[Effects of electrical cauterization of nucleus raphe magnus on gastric acid output and serum gastrin level in rats].

Medullary raphe nucleus complex plays an important role in the regulation of visceral function. The effects of electrical damage of the nucleus raphe magnus on gastric acid output and serum gastrin level in anesthetized rats were observed. The experiments showed that damage of nucleus raphe magnus increased gastric acid output and serum gastrin level, which could be prevented by vagotomy but not by coeliac and superior mesenteric ganglionectomy.

Effect of median raphe nucleus electrocoagulation and serotonin synthesis blockade on oestrogen receptor content of rat uterus.

The number of oestrogen receptors (OR) in rat uterus changes in relation to the cyclic function of the ovary. In the dioestrus phase the OR content of rat uterus is significantly greater than in the oestrus phase. Electrocoagulation of the median raphe nucleus causing in rats increased release of gonadotrophins and consequent disturbances in the cyclic ovarian function is associated also with changes in OR content of the uterus. These changes are, however, not analogous to those observed in the normal sexual cycle. In the oestrus phase a very high accumulation of OR is found in the uterus, while in the dioestrus phase OR are barely detectable. Serotonin synthesis blockade with p-CPA, which is associated with an increase in the number of maturating and mature follicles in the ovary and dioestrus phase prolongation leads to a striking increase in the number of OR in the uterus.

Effect of brain serotonin level on induced hippocampal paroxysmal activity in rats.

Experiments were performed to study the involvement of brain 5-HT in an experimental model of epilepsy induced by repeated electrical stimulation of the dorsal hippocampus of rats. The experiments included: (1) systemic injections of 5-hydroxytryptophan (5-HTP) and p-chlorophenylalanine (pCPA) and (2) electrolytic lesions of the midbrain raphe nuclei. The pCPA group showed a significant increase while animals which received systemic injections of 5-HTP showed a great reduction in the electrographic seizure activity. Although several reports have shown that midbrain raphe lesions do not modify the epileptic susceptibility, we observed a clear enhancement in the epileptiform activity in lesioned animals. The results presented here support the view that serotonergic systems may exert a tonic inhibitory effect on hippocampal epileptic activity.

[Sleep organisation in the kitten following early lesions to the structures involved in the regulation of alertness (author's transl)]. / Organisation du sommeil chez le chaton après lésions précoces des structures impliquées dans la régulation des états de vigilance.

Electrolytic lesions were used to destroy the anterior raphe nuclei and the loci caeruleus and subcaeruleus in kittens at varying stages of maturation. No lesion performed in the first week after birth led to changes in the characteristics of sleep in the 8 post-operative days. On the other hand, lesions performed in kittens aged 1-2 months had the same effect as is described as occurring in the adult animal. The structures which are responsible for states of alertness thus appear to start acting during the post-natal period.