Midbrain Raphe Region Research Articles

Anti-Apoptotic Protein Bcl-xL Expression in the Midbrain Raphe Region Is Sensitive to Stress and Glucocorticoids.

Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synapses as well as for resilience to stress. In order to determine whether stressful events may influence the expression of anti-apoptotic protein Bcl-xL in the midbrain and specifically in the midbrain serotonergic (5-HT) neurons involved in neurobehavioral responses to adverse stimuli, adult male rats were subjected to short-term or chronic forced swim stress. A short-term stress rapidly increased the midbrain bcl-xl mRNA levels and significantly elevated Bcl-xL immunoreactivity in the midbrain 5-HT cells. Stress-induced increase in glucocorticoid secretion was implicated in the observed effect. The levels of bcl-xl mRNA were decreased after stress when glucocorticoid elevation was inhibited by metyrapone (MET, 150 mg/kg), and this decrease was attenuated by glucocorticoid replacement with dexamethasone (DEX; 0.2 mg/kg). Both short-term stress and acute DEX administration, in parallel with Bcl-xL, caused a significant increase in tph2 mRNA levels and slightly enhanced tryptophan hydroxylase immunoreactivity in the midbrain. The increasing effect on the bcl-xl expression was specific to the short-term stress. Forced swim repeated daily for 2 weeks led to a decrease in bcl-xl mRNA in the midbrain without any effects on the Bcl-xL protein expression in the 5-HT neurons. In chronically stressed animals, an increase in tph2 gene expression was not associated with any changes in tryptophan hydroxylase protein levels. Our findings are the first to demonstrate that both short-term stress and acute glucocorticoid exposures induce Bcl-xL protein expression in the midbrain 5-HT neurons concomitantly with the activation of the 5-HT synthesis pathway in these neurons.

P.1.i.031 Deficits in context dependent adaptive coding of reward values in schizophrenia

Animal models revealed that the serotonin-1A (5-HT1A) receptor modulates gray matter structure. However, there is a lack of evidence showing the relationship between 5-HT1A receptor concentration and gray matter in the human brain in vivo. Here, to demonstrate an association between the 5-HT1A receptor binding potential, an index for receptor concentration, and the local gray matter volume (GMV), an index for gray matter structure, we measured 35 healthy subjects with both positron emission tomography (PET) and structural magnetic resonance imaging (MRI). We found that regional heteroreceptor binding was positively associated with GMV in distinctive brain regions such as the hippocampi and the temporal cortices in both hemispheres (R2 values ranged from 0.308 to 0.503, p < 0.05 cluster-level FDR-corrected). Furthermore, autoreceptor binding in the midbrain raphe region was positively associated with GMV in forebrain projection sites (R2 = 0.656, p = 0.001). We also observed a broad range between 5-HT1A receptor binding and GMV. Given the congruence of altered 5-HT1A receptor concentrations and GMV reduction in depression or Alzheimer's disease as reported by numerous studies, these results might provide new insights towards understanding the mechanisms behind GMV alterations observed in these brain disorders.

Serotonin-1A receptor binding is positively associated with gray matter volume — A multimodal neuroimaging study combining PET and structural MRI

Animal models revealed that the serotonin-1A (5-HT(1A)) receptor modulates gray matter structure. However, there is a lack of evidence showing the relationship between 5-HT(1A) receptor concentration and gray matter in the human brain in vivo. Here, to demonstrate an association between the 5-HT(1A) receptor binding potential, an index for receptor concentration, and the local gray matter volume (GMV), an index for gray matter structure, we measured 35 healthy subjects with both positron emission tomography (PET) and structural magnetic resonance imaging (MRI). We found that regional heteroreceptor binding was positively associated with GMV in distinctive brain regions such as the hippocampi and the temporal cortices in both hemispheres (R(2) values ranged from 0.308 to 0.503, p<0.05 cluster-level FDR-corrected). Furthermore, autoreceptor binding in the midbrain raphe region was positively associated with GMV in forebrain projection sites (R(2)=0.656, p=0.001). We also observed a broad range between 5-HT(1A) receptor binding and GMV. Given the congruence of altered 5-HT(1A) receptor concentrations and GMV reduction in depression or Alzheimer's disease as reported by numerous studies, these results might provide new insights towards understanding the mechanisms behind GMV alterations observed in these brain disorders.

FC10-05 - Attenuated serotonin transporter association between midbrain and nucleus accumbens in major depression

IntroductionPatients suffering from major depressive disorder (MDD) exhibit alterations in serotonin transporter (5-HTT) binding clinically reflected by lack of hedonic experience. We recently demonstrated the importance to evaluate interregional relationships of neurotransmitter systems, providing important complementary information on a network level.ObjectiveTo identify interregional 5-HTT relationships between the midbrain raphe region and projection areas in MDD patients compared to controls.MethodsEighteen medication-free patients with MDD and 16 healthy subjects underwent PET using [11C] DASB. 5-HTT binding potential (BPND) maps were computed with the multilinear-reference-tissue-model-2 in PMOD3.1. Voxel-wise linear regression was calculated in SPM8 using midbrain 5-HTT BPND as regressor; group comparisons were assessed by ANOVA (p &lt; 0.001).ResultsHealthy subjects showed positive associations between serotonin transporters located in the midbrain and transporters expressed in the nucleus accumbens bilaterally. Importantly, this relationship was significantly decreased in MDD patients as compared to controls (t = 5.41/4.84, right/left hemisphere; see figure).ConclusionsThis study demonstrates for the first time 5-HTT alterations on a network level in MDD patients between the midbrain and a major projection area. Our results complement previous findings emphasizing the importance of the nucleus accumbens in MDD and indicate a disturbed serotonergic regulation in this key area of reward processing.

Interactions of Corticotropin-Releasing Factor, Urocortin and Citalopram in a Primate Model of Stress-Induced Amenorrhea

Background/Aims: We established a cynomolgus macaque model of stress-induced amenorrhea in which the application of combined metabolic and psychosocial stress suppressed ovulation in stress-sensitive (SS) individuals, but not in highly stress-resilient (HSR) individuals. We previously reported that SS monkeys have deficits in global serotonin release and serotonin-related gene expression in the raphe nucleus, and that administration of the selective serotonin reuptake inhibitor S-citalopram increased estrogen and progesterone production in SS monkeys. In this study, we questioned whether there was a difference in corticotropin-releasing factor (CRF) or urocortin (UCN) stress-related peptide systems in the midbrain raphe region when HSR and SS monkeys treated with placebo or S-citalopram are compared. Methods: Monkeys characterized as HSR or SS were administered placebo or S-citalopram for 15 weeks. CRF fibers in the dorsal raphe were detected with an antibody against human CRF. UCN1 fibers were immunostained in an area rostral to the dorsal raphe. The fibers were quantified by stereology and analyzed by two-way ANOVA. UCN1 cell bodies were counted in the supraoculomotor area near the Edinger-Westphal nucleus. Results: S-citalopram significantly decreased the CRF fiber density in SS animals, but not in HSR animals. SS monkeys had a significantly lower UCN1 fiber density compared to HSR monkeys, but S-citalopram treatment did not alter the UCN1 fiber density. SS animals treated with S-citalopram tended to have a higher number of UCN1-positive cell bodies than the other groups. Conclusion: S-citalopram decreased CRF fiber density and appears to increase the production of UCN1 in SS individuals, indicating the likelihood that serotonin is involved in regulating CRF and UCN1 in individuals who are sensitive to the effects of serotonin.

P.3.23 Molecular imaging of early visual cortex by combining pet and fMRI

Animal models revealed that the serotonin-1A (5-HT1A) receptor modulates gray matter structure. However, there is a lack of evidence showing the relationship between 5-HT1A receptor concentration and gray matter in the human brain in vivo. Here, to demonstrate an association between the 5-HT1A receptor binding potential, an index for receptor concentration, and the local gray matter volume (GMV), an index for gray matter structure, we measured 35 healthy subjects with both positron emission tomography (PET) and structural magnetic resonance imaging (MRI). We found that regional heteroreceptor binding was positively associated with GMV in distinctive brain regions such as the hippocampi and the temporal cortices in both hemispheres (R2 values ranged from 0.308 to 0.503, p < 0.05 cluster-level FDR-corrected). Furthermore, autoreceptor binding in the midbrain raphe region was positively associated with GMV in forebrain projection sites (R2 = 0.656, p = 0.001). We also observed a broad range between 5-HT1A receptor binding and GMV. Given the congruence of altered 5-HT1A receptor concentrations and GMV reduction in depression or Alzheimer's disease as reported by numerous studies, these results might provide new insights towards understanding the mechanisms behind GMV alterations observed in these brain disorders.

Serotonin projection patterns to the cochlear nucleus

The cochlear nucleus is well known as an obligatory relay center for primary auditory nerve fibers. Perhaps not so well known is the neural input to the cochlear nucleus from cells containing serotonin that reside near the midline in the midbrain raphe region. Although the specific locations of the main, if not sole, sources of serotonin within the dorsal cochlear nucleus subdivision are known to be the dorsal and median raphe nuclei, sources of serotonin located within other cochlear nucleus subdivisions are not currently known. Anterograde tract tracing was used to label fibers originating from the dorsal and median raphe nuclei while fluorescence immunohistochemistry was used to simultaneously label specific serotonin fibers in cat. Biotinylated dextran amine was injected into the dorsal and median raphe nuclei and was visualized with Texas Red, while serotonin was visualized with fluorescein. Thus, double-labeled fibers were unequivocally identified as serotoninergic and originating from one of the labeled neurons within the dorsal and median raphe nuclei. Double-labeled fiber segments, typically of fine caliber with oval varicosities, were observed in many areas of the cochlear nucleus. They were found in the molecular layer of the dorsal cochlear nucleus, in the small cell cap region, and in the granule cell and external regions of the cochlear nuclei, bilaterally, of all cats. However, the density of these double-labeled fiber segments varied considerably depending upon the exact region in which they were found. Fiber segments were most dense in the dorsal cochlear nucleus (especially in the molecular layer) and the large spherical cell area of the anteroventral cochlear nucleus; they were moderately dense in the small cell cap region; and fiber segments were least dense in the octopus and multipolar cell regions of the posteroventral cochlear nucleus. Because of the presence of labeled fiber segments in subdivisions of the cochlear nucleus other than the dorsal cochlear nucleus, we concluded that the serotoninergic projection pattern to the cochlear nucleus is divergent and non-specific. Double-labeled fiber segments were also present, but sparse, in the superior olive, localized mainly in periolivary regions; this indicated that the divergence of dorsal and median raphe neurons that extends throughout regions of the cochlear nucleus also extended well beyond the cochlear nucleus to include at least the superior olivary complex as well.

Hippocampal theta activity and facilitated locomotor stepping produced by GABA injections in the midbrain raphe region

Inactivation of neurons in the midbrain raphe region produces increases in locomotor activity, and it appears that they function to suppress locomotion. Inactivation of neurons there also produces hippocampal slow wave (theta) activity and it appears that they also function to inhibit rhythmic activity in the hippocampus. We determined whether the degree of association between the two effects was consistent with the operation of a single mechanism. Stimulation electrodes were implanted into locomotor sites of the hypothalamus of 34 urethane-anesthetized rats. Hindlimb stepping was elicited by 5.12-s trains of perifornical electrical stimulation presented once per minute. Hippocampal theta activity was recorded across the CA1 layer of the dorsal hippocampus. GABA injections were used to locate raphe sites at which neuronal inactivation influenced stepping and hippocampal activity. A glass pipette (80-μm tip) was inserted into the midbrain, and injections of GABA (50–100 mg/0.1–0.2 μl) were made in 70 sites in the midbrain. Injections at 34 sites facilitated stimulation-elicited stepping, and at 17 sites, they also produced intertrial stepping. Facilitating injections, but not ineffective or suppressive injections, increased the mean peak frequency of hippocampal activity, and increased power in the 4–5 Hz band during the period that preceded the stimulation trains, but did not change the 5–6 Hz activity produced during the stimulation trains. Priming locomotor stimulation which also facilitated stepping produced generally similar increases in pre-stimulation peak frequency and 4-Hz power. The magnitudes of the increases in stepping and 4-Hz power were uncorrelated. The increase in 4-Hz power appeared earlier than the increase in stepping in 18 of 34 cases, and later in 11 cases; no increases in 4-Hz power were apparent in five cases. The results indicate that pre-locomotor 4-Hz hippocampal activity in the urethane-anesthetized rat is loosely coupled with facilitated locomotor initiation. Neurons in the midbrain raphe region appear to suppress both processes, but the low degree of association between the magnitudes and onset times of increases in stepping and hippocampal 4-Hz power indicate the operation of multiple mechanisms.

3H]Paroxetine binding and serotonin content of rat and rabbit cortical areas, hippocampus, neostriatum, ventral mesencephalic tegmentum, and midbrain raphe nuclei region

The high-affinity binding of [3H]paroxetine to membranes was measured in different regions of the rat and rabbit brain: cingulate, frontal, parietal, piriform, entorhinal, and visual cortical areas; dorsal and ventral hippocampus; rostral and caudal halves of neostriatum (rat) or caudate nucleus and putamen (rabbit); ventral mesencephalic tegmentum; and midbrain raphe nuclei region. The tissue concentrations of serotonin (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxy-l-tryptophan (5-HTP) were also determined by high-performance liquid chromatography (HPLC) in the same brain samples. The regional density of [3H]paroxetine binding varied in both species; the highest values (Bmax) were found in the midbrain raphe region and ventral mesencephalic tegmentum. The cortical values ranged from moderate to low, with a significantly higher density in the cingulate cortex of the rat compared with rabbit. In the rat, there was also a higher density in the ventral than dorsal hippocampus, and the caudal than rostral neostriatum. In the rabbit, the hippocampal and neostriatal values were generally lower and more uniform. In both species, there was an excellent correlation between regional 5-HT levels and specific [3H]paroxetine binding (r = 0.87 in the rat and 0.96 in the rabbit). Considering the available quantitative data on the number of 5-HT nerve cell bodies and axon terminals in different regions of the rat brain, it appears likely that the high amount of [3H]paroxetine binding in the midbrain raphe region and ventral mesencephalic tegmentum reflects the presence of 5-HT uptake sites on 5-HT nerve cell bodies and dendrites as well as axon terminals. In other brain regions, the heterogeneous distribution of [3H]paroxetine binding parallels that of the number of 5-HT axon terminals, emphasizing the potential usefulness of this radioligand as a marker of 5-HT innervation density.

Assessment of blood platelets as a model for CNS response: Comparative effects of caffeine on 5-HT uptake and release mechanisms in rat platelets and rat brain serotonin neurons

We have previously reported that caffeine significantly enhanced 5-HT uptake and reduced 5-HT release from crude synaptosomal fractions obtained from rat cerebral cortex and from midbrain raphe region. Blood platelets, as reported by many laboratories and also demonstrated in our own labs, have a very active mechanism for 5-HT uptake and storage. In this regard platelets bear a high degree of similarity to brain serotonin neurons. The present experiments were, therefore, carried out to investigate the effects of caffeine on 5-HT uptake and release from rat platelets in an attempt to assess the possibility of using platelets as a model for studying the CNS effects of caffeine. Platelet rich plasma was prepared from the trunk blood of decapitated rats. Effects of caffeine were investigated at 10 −7, 10 −6, 10 −5 and 10 −4M, on both the high affinity 3H-5-HT uptake and the spontaneous 5-HT release from 3H-5-HT preloaded platelets. The results show that caffeine did not change 5-HT uptake into platelets. In brain synaptosomes the same concentration of caffeine, however, increased 5-HT uptake dose-dependently. The results also revealed that caffeine increased 5-HT release from rat platelets in a concentration-dependent manner. The concentrations 10 −6, 10 −5, and 10 −4M increased release significantly compared to control. This finding is also in contrast to that observed in synaptosomes of brain serotonin neurons where caffeine decreased 5-HT release. It is concluded, therefore, that the rat blood platelet is not a suitable model for studying these CNS actions of caffeine. Furthermore, our observations imply that rat platelet serotonin uptake and release mechanisms are not identical to those mechanisms in brain serotonin neurons.

Differential actions of diazepam on the release of [ 3H]5-hydroxytryptamine from cortical and midbrain raphe slices in the rat

The possible interaction of the benzodiazepine diazepam with synaptic mechanisms of the central neurotransmitter 5-hydroxytryptamine (5-HT) has been studied in the rat using the superfusion in vitro release technique. The effect of diazepam (1–100 μM) on the spontaneous and potassium-induced release of radioactivity from superfused slices preloaded with [ 3H]5-HT prepared from cerebral cortex (fronto-parietal) and midbrain raphe region was monitored and compared. The results demonstrate a differential action of diazepam on the release of cortical and raphe 5-HT. In cerebral cortex diazepam significantly reduces both spontaneous and potassium-evoked release of [ 3H]5-HT. Conversely in raphe slices diazepam significantly enhances both spontaneous and potassium-evoked release of [ 3H]5-HT. In both tissues the effect of diazepam on K +-stimulated neurotransmitter release was abolished in the presence of picrotoxin. The experiments suggest that the effects of benzodiazepines on 5-HT mechanisms may be manifested at least in part through γ-aminobutyric acid receptors.

Temperature-responsive neurones in the midbrain raphe region of rabbits

To evaluate the thermoregulatory role of the temperature-responsive neurones in the midbrain of rabbits, unit responses in the raphe region to changes in midbrain and cutaneous temperature were explored in rabbits. It was revealed that the concentrated pool of warm-responsive neurones was located in the raphe region of rabbits as it is known from rats and cats, and that they were influenced remarkably by skin temperature and mostly also sensitive to lysergic acid diethylamide (LSD).

The development of monamine-containing neurons in the brain and spinal cord of the salamander, Ambystoma mexicanum.

The distribution of monoamine-containing neurons in the CNS of the developing and adult axolotl, Ambystoma mexicanum, has been investigated using the histochemical fluorescence technique of Falck and Hillarp combined with microspectrofluorimetry. The earliest catecholamine-containing neurons to be detected are located in the ventral ependymal zone of the spinal cord at the time of hatching (Stage 41). Between stages 43 and 46, catecholamine fluorescence can be detected in neurons in the following regions: nucleus preopticus, the hypothalamic-infundibular region, and the brain stem reticular formation. 5-HT-containing neurons are only observed in the midbrain raphe region and are first detected at stage 44. In contrast to these early monoamine fluorescing groups, catecholamine-containing neurons are not routinely detectable in the nucleus interpeduncularis until six months of age. All monoamine-containing neuronal groups detected in developing axolotls are also present in both sexes of the adult. However, the fluorescence intensity is less in monoamine-containing neurons observed in adults than in early developing subjects. All catecholamine-containing neuronal groups, with the exception of those located in the midbrain region (nucleus interpeduncularis, reticular zone) have fluorescent processes that contact the cerebrospinal fluid (CSF). The presence of CSF-contacting processes in the hypothalamic and spinal cord regions suggest that the CSF may act as a medium through which bioactive substances are transported from one brain region to another. Intense catecholamine fluorescence is observed in cells of the notochord prior to the detection of the monoamine-containing neurons in the CNS. A possible involvement of catecholamines in the inductive effects of the notochord during development is discussed.

Neural Release of Brain Serotonin and Body Temperature

FELDBERG1,2 has suggested that brain amines are directly involved in the hypothalamic control of body temperature. In cats and dogs serotonin was found to cause a rise in temperature and norepinephrine a fall, whereas in the rabbit3 and sheep4 there is a fall with serotonin and a rise with norepinephrine. Moreover, serotonin antagonizes the normal decrease of temperature of animals under anaesthesia. All these results, however, have been obtained using exogenous amines. We have shown that there is a neurally mediated release of endogenous serotonin in rat brain when the caudal midbrain raphe region is stimulated electrically and we have found that a specific behavioural consequence of such stimulation is a failure of the normal physiological habituation to repetitive sensory stimuli5. We have also measured the effect on body temperature of neurally released endogenous serotonin both in awake, unrestrained and in anaesthetized rats.