Serotonergic Raphe Neurons Research Articles

Single‐unit responses of serotonergic medullary raphe neurons to cardiovascular challenges in freely moving cats

Single-unit activity of serotonergic neurons in the nuclei raphe obscurus (NRO) and raphe pallidus (NRP) were recorded in conjunction with heart rate in freely moving cats in response to systemic administration of vasoactive drugs and to graded haemorrhage. Bolus administration of phenylephrine hydrochloride and sodium nitroprusside (20 microg/kg, i.v.) produced a marked, transient reflex bradycardia (-42 b.p.m.) and tachycardia (+60 b.p.m.), respectively. The activity of NRO/NRP serotonergic neurons remained unchanged after phenylephrine and nitroprusside administration. The administration of hydralazine (1 mg/kg, i.v.), a long-acting vasodilator, produced sustained tachycardia (+60 b.p.m.), which was not accompanied by changes in neuronal activity, despite prolonged reflex activation of the sympathetic nervous system. The initial withdrawal of up to 15% of total blood volume increased heart rate (+12 b.p.m.), whereas the removal of 22.5% of total blood decreased heart rate (-44 b.p.m.). The activity of NRO/NRP serotonergic neurons remained unaltered throughout graded haemorrhage trials, despite the changes in sympathetic outflow. Thus, serotonergic NRO and NRP neurons appear to be insensitive to alterations in blood pressure and baroreceptor activity, and this lack of responsiveness does not support a specific role for these cells in cardiovascular regulation. Furthermore, these neurons do not appear to be involved in physiological mechanisms underlying alterations in autonomic outflow invoked by hypertension and hypotension. Taken within the context of our previous work, the present data suggest that medullary serotonergic neurons may modulate autonomic outflow, but only in relation to their primary role in motor control.

Response to fluoxetine and serotonin 1A receptor (C-1019G) polymorphism in Taiwan Chinese major depressive disorder

Serotonin systems appear to play a key role in the pathogenesis of major depression and the therapeutic mechanisms of antidepressants. The firing rate of dorsal raphe serotonergic neurons is controlled by somatodendritic 5-hydroxytryptamine 1A (HTR1A) autoreceptors, and desensitization of these receptors is implicated in the antidepressant mechanism of selective serotonin reuptake inhibitors. We tested whether a functional polymorphism (C-1019G) in the promoter region of the HTR1A gene and serotonin-related genetic variants are related to fluoxetine antidepressant effect. We genotyped the HTR1A C-1019G polymorphism as well as polymorphisms in the serotonin transporter gene-linked polymorphic region (SERTPR), variable-number tandem-repeat polymorphisms in intron 2 (STin2) of the serotonin transporter gene, serotonin 2A receptor (T102C), tryptophan hydroxylase (A218C), and G-protein beta3 subunit (C825T) in 224 Chinese patients from southern Taiwan with major depression, who accepted 4-week fluoxetine treatment and therapeutic evaluation. Our results demonstrated that the HTR1A -1019C/C carriers (P=0.009) and SERTPR l/l carriers (P<0.001) showed a better response to fluoxetine, while other polymorphisms were not associated with fluoxetine therapeutic response. The major limitation of this study is the lack of a placebo control. Future prospective study with placebo control may help to predict and individualize antidepressant treatment.

Harmane inhibits serotonergic dorsal raphe neurons in the rat

Harmane and norharmane (two beta-carbolines) are tobacco components or products. The effects of harmane and norharmane on serotonergic raphe neurons remain unknown. Harmane and norharmane are inhibitors of the monoamine oxidases A (MAO-A) and B (MAO-B), respectively. To study the effects of harmane, norharmane, befloxatone (MAOI-A), and selegiline (MAOI-B) on the firing of serotonergic neurons. To compare the effects of these compounds to those of nicotine (whose inhibitory action on serotonergic neurons has been previously described). The effects of cotinine, a metabolite of nicotine known to interact with serotonergic systems, are also tested. In vivo electrophysiological recordings of serotonergic dorsal raphe neurons in the anaesthetized rat. Nicotine, harmane, and befloxatone inhibited serotonergic dorsal raphe neurons. The other compounds had no effects. The inhibitory effect of harmane (rapid and long-lasting inhibition) differed from that of nicotine (short and rapidly reversed inhibition) and from that of befloxatone (slow, progressive, and long-lasting inhibition). The inhibitory effects of harmane and befloxatone were reversed by the 5-HT1A antagonist WAY 100 635. Pretreatment of animals with p-chlorophenylalanine abolished the inhibitory effect of befloxatone, but not that of harmane. Nicotine, harmane, and befloxatone inhibit the activity of raphe serotonergic neurons. Therefore, at least two tobacco compounds, nicotine and harmane, inhibit the activity of serotonergic neurons. The mechanism by which harmane inhibits serotonergic dorsal raphe neurons is likely unrelated to a MAO-A inhibitory effect.

Effect of number of tailshocks on learned helplessness and activation of serotonergic and noradrenergic neurons in the rat

Adult male albino rats were exposed to varying numbers of tailshocks (0, 10, 50 or 100). The following day, their escape latencies in a shuttlebox were measured in order to estimate the degree of learned helplessness (LH) produced by the varying number of shocks. Only the groups exposed to 50 or 100 shocks displayed evidence of LH. In a parallel experiment, c-fos activation was used to determine the degree of activation of raphe serotonergic neurons (FosIR + 5-HT) and locus coeruleus (LC) noradrenergic neurons (FosIR + TH) produced by the same shock conditions. Compared to unhandled cage controls, all shock groups (0 shocks was a restrained group) significantly activated both raphe and LC neurons. The 50 and 100 shock groups had significantly higher degrees of activation of serotonergic neurons in the rostral raphe groups and the LC than the 0 and 10 shock groups. These data are consistent with the hypothesis that activation of rostral raphe serotonergic neurons and LC noradrenergic neurons beyond a certain threshold may be critical for the development of LH. The relevance of these results for elucidating the neural bases of psychopathology is discussed.

The activation of raphe serotonergic neurons in normal and hypoarousal-driven aggression: A double labeling study in rats

The serotonergic system is well known for its aggression lowering effects. It has been shown repeatedly, however, that the serotonergic system is activated during fights, and recent data suggested that it is necessary for the expression of aggressive behavior. We investigated the interaction between serotonergic activation and aggressive behavior by assessing the co-localization of the c-Fos signal (marker of neuronal activation) with tryptophan-hydroxylase activity (marker of serotonin secretion) in the raphe. Control rats were compared with rats exposed to visual and olfactory (but not physical) contacts with opponents (psychosocial stimulation) as well as with rats exposed to aggressive encounters. Fights were accompanied by the activation of the raphe; however, the effect was not aggression-specific, as a similar activation was induced by psychosocial contacts. The lack of behavioral specificity in activation suggests that it was related to social arousal rather than to the execution of fights. The activation of serotonergic raphe neurons showed a negative correlation with aggressive behavior, which is in line with the widespread view that serotonin neurotransmission downregulates aggressive behavior. The activation of serotonergic neurons did not show a correlation with measures of hypoarousal-driven abnormal aggression, which indicates that factors other than the raphe control this behavior. The latter finding may explain the low efficacy of serotonergic treatments in conduct and antisocial personality disorders, in which violence correlates with hypoarousal.

Coupling of 5‐HT1A autoreceptors to inhibition of mitogen‐activated protein kinase activation via Gβγ subunit signaling

The 5-HT1A receptor is expressed presynaptically as the primary somatodendritic autoreceptor on serotonergic raphe neurons, and postsynaptically in several brain regions. Signaling of the 5-HT1A autoreceptor was studied in RN46A cells, a model of serotonergic raphe neurons that express endogenous 5-HT1A receptors. In undifferentiated RN46A cells stably transfected with the wild-type 5-HT1A receptor, 5-HT1A receptor activation inhibited forskolin-induced cyclic adenosine monophosphate (cAMP) formation (by 50%), increased [Ca2+]i, and induced a novel inhibition (up to 60%) of phospho-p42/p44-mitogen-activated protein kinase (MAPK). Upon differentiation of non-transfected or 5-HT1A-transfected RN46A cells, agonist-mediated inhibition of MAPK was enhanced. These actions were blocked by pretreatment with pertussis toxin indicating mediation via Gi/Go proteins and the calcium response was blocked by preactivation of protein kinase C (PKC). In cells overexpressing the G beta gamma scavenger carboxyl-terminal domain of G protein receptor kinase 2 (GRK-CT), 5-HT1A receptor activation inhibited cAMP formation, but coupling to calcium mobilization and inhibition of MAPK was abolished. The activity of 5-HT1A receptors containing mutations of PKC sites in the second (i2: T149A) or third intracellular loop (i3: T229A/S253G/T343A) was tested. At comparable levels of receptor expression, the signaling of the 5-HT1A i3 mutant was similar to the 5-HT1A wild-type receptor, while the i2 and quadruple (i2/i3) mutants failed to couple to G beta gamma-mediated increase in [Ca2+]i or inhibition of MAPK, but did couple to G alpha i-mediated inhibition of cAMP. Thus, the i2-domain of the 5-HT1A autoreceptor is crucial for coupling to G beta gamma subunits and their subsequent responses (e.g. calcium mobilization and inhibition of MAPK activity).

Effects of brain‐derived neurotrophic factor (BDNF) on glial cells and serotonergic neurones during development

Serotonergic neurones are among the first to develop in the central nervous system. Their survival and maturation is promoted by a variety of factors, including serotonin itself, brain-derived neurotrophic factor (BDNF) and S100beta, an astrocyte-specific Ca(2+) binding protein. Here, we used BDNF-deficient mice and cell cultures of embryonic raphe neurones to determine whether or not BDNF effects on developing serotonergic raphe neurones are influenced by its action on glial cells. In BDNF-/- mice, the number of serotonin-immunoreactive neuronal somata, the amount of the serotonin transporter, the serotonin content in the striatum and the hippocampus, and the content of 5-hydroxyindoleacetic acid in all brain regions analysed were increased. By contrast, reduced immunoreactivity was found for myelin basic protein (MBP) in all brain areas including the raphe and its target region, the hippocampus. Exogenously applied BDNF increased the number of MBP-immunopositive cells in the respective culture systems. The raphe area displayed selectively reduced immunoreactivity for S100beta. Accordingly, S100beta was increased in primary cultures of pure astrocytes by exogenous BDNF. In glia-free neuronal cultures prepared from the embryonic mouse raphe, addition of BDNF supported the survival of serotonergic neurones and increased the number of axon collaterals and primary dendrites. The latter effect was inhibited by the simultaneous addition of S100beta. These results suggest that the presence of BDNF is not a requirement for the survival and maturation of serotonergic neurones in vivo. BDNF is, however, required for the local expression of S100beta and production of MBP. Therefore BDNF might indirectly influence the development of the serotonergic system by stimulating the expression of S100beta in astrocytes and the production MBP in oligodendrocytes.

The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus

Orexin/hypocretin has been well demonstrated to excite the serotonergic neurons in the dorsal raphe nucleus (DRN). We studied the morphological relationships between orexin-containing axon terminals and serotonin- as well as orexin-receptor-containing neurons in the dorsal raphe nucleus. Using immunohistochemical techniques at the light microscopic level, orexin A (OXA)-like immunoreactive neuronal fibers in the DRN were found to make close contact with serotonergic neurons, while some of the serotonergic neurons also expressed the orexin 1 receptor (OX 1R). At the electron microscopic level, double-immunostaining experiments showed that the orexin A-like immunoreactive fibers were present mostly as axon terminals that made synapses on the serotonin- and orexin 1-receptor-containing neurons. While only axodendritic synapses between orexin A-containing axon terminals and serotonergic neurons were detected, the synapses made by orexin A-containing axon terminals on the orexin 1-receptor-containing neurons were both axodendritic and axosomatic. The present study suggests that excitation effect of orexin A on dorsal raphe serotonergic neurons is via synaptic communication through orexin 1 receptor.

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.

Metabotropic glutamate receptor 5 and dorsal raphe serotonin release in inflammatory pain in rat

In this study, we evaluated the effects of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective antagonist of metabotropic glutamate subtype 5 receptors (mGlu5), delivered through different paths on dorsal raphe serotonin (5-HT) and on thermoceptive responses in rats with inflammatory pain. Intraplantar formalin and carrageenan increased 5-HT (137±11% and 212±6% of pre-injection baseline, respectively) and reduced nociceptive threshold (23±7% and 19±3% of pre-injection baseline, respectively). MPEP (2 mg/kg i.p.) further enhanced formalin and carrageenan-induced 5-HT increases (180±11% and 260±12% of pre-injection baseline, respectively) and reduced thermal hyperalgesia (71±8% and 80±10% of pre-injection baseline, respectively). MPEP (1 mM) through microdialytic probe into the dorsal raphe did not change formalin- or carrageenan-induced 5-HT increases (147±10% and 189±10% of pre-injection baseline, respectively) and thermal hyperalgesia (35±8% and 25±9% of pre-injection baseline, respectively). Finally, MPEP (30 nmol/rat) into the hind paw reduced the formalin- and carrageenan-induced 5-HT increase (108±3% and 126±7% of pre-injection baseline, respectively) and thermal hyperalgesia (77±6% and 117±7% of pre-injection baseline, respectively). Dorsal raphe serotonergic neurons activity increased following a peripherally induced inflammatory injury. In these conditions, peripheral but not dorsal raphe mGlu5 receptors blockade prevented over activation of dorsal raphe serotonergic neurons and reversed thermal hyperalgesia.

Impaired Repression at a 5-Hydroxytryptamine 1A Receptor Gene Polymorphism Associated with Major Depression and Suicide

Inhibition of serotonergic raphe neurons is mediated by somatodendritic 5-HT1A autoreceptors, which may be increased in depressed patients. We report an association of the C(-1019)G 5-HT1A promoter polymorphism with major depression and suicide in separate cohorts. In depressed patients, the homozygous G(-1019) allele was enriched twofold versus controls (p = 0.0017 and 0.0006 for G/G genotype and G allele distribution, respectively), and in completed suicide cases the G(-1019) allele was enriched fourfold (p = 0.002 and 0.00008 for G/G genotype and G allele distribution, respectively). The C(-1019) allele was part of a 26 bp imperfect palindrome that bound transcription factors nuclear NUDR [nuclear deformed epidermal autoregulatory factor (DEAF-1)]/suppressin and Hairy/Enhancer-of-split-5 (Drosophila) (Hes5) to repress 5-HT1A or heterologous promoters, whereas the G(-1019) allele abolished repression by NUDR, but only partially impaired Hes5-mediated repression. Recombinant NUDR bound specifically to the 26 bp palindrome, and endogenous NUDR was present in the major protein-DNA complex from raphe nuclear extracts. Stable expression of NUDR in raphe cells reduced levels of endogenous 5-HT1A protein and binding. NUDR protein was colocalized with 5-HT1A receptors in serotonergic raphe cells, hippocampal and cortical neurons, and adult brain regions including raphe nuclei, indicating a role in regulating 5-HT1A autoreceptor expression. Our data indicate that NUDR is a repressor of the 5-HT1A receptor in raphe cells the function of which is abrogated by a promoter polymorphism. We suggest a novel transcriptional model in which the G(-1019) allele derepresses 5-HT1A autoreceptor expression to reduce serotonergic neurotransmission, predisposing to depression and suicide.

Chronic excess of corticosterone increases serotonergic fibre degeneration in aged rats.

Evidence is presented for the potentiating role of corticosterone on axonal degeneration of serotonergic neurones during ageing. Aged rats, 24 months old, were implanted subcutaneously with 2 x 100 mg pellets of corticosterone. Serotonergic and cholinergic (ChAT- and NADPHd-positive) fibre degenerations in the anteroventral thalamic nucleus (AVT) were measured 2 months after corticosterone implantation. Numbers of immunoreactive serotonergic raphe and mesolimbic cholinergic neurones were also quantified. Basal plasma corticosterone and adrenocorticotropin (ACTH) concentrations were assayed at 2, 4, 6, and 8 weeks after implantation in the plasma and at 1, 2, 4 and 6 weeks in urine. The degree of serotonergic fibre aberrations in the AVT increased significantly after corticosterone exposure, while that of ChAT-positive and NADPHd-stained axon aberrations showed a modest but nonsignificant increase. A positive correlation between the magnitudes of serotonergic and cholinergic fibre aberrations appeared in the AVT, but only in the corticosterone-treated rats. The number of serotonin immunopositive neurones in the raphe nuclei after corticosterone decreased marginally, while that of mesopontine ChAT-positive neurones was not influenced. Measurements of basal plasma corticosterone and ACTH, as well as urine corticosterone, revealed that the steroid implantation increased the plasma corticosterone level for at least 4 weeks and decreased ACTH level for at least 6 weeks. By the week 8, the pituitary-adrenal function was apparently restored. However, at sacrifice, both the weight of adrenal glands and that of thymus remained reduced, indicating the long-lasting effects of corticosterone on target tissues. It is concluded that the raphe serotonergic neurones and their projecting fibres are sensitive to corticosterone excess in aged rats and become more vulnerable to degeneration processes than under normal ageing conditions. Cholinergic neurones of brainstem origin, which also express massive NADPHd activity, are more resistant against corticosterone, but their axon degeneration correlates to serotonergic fibre degeneration.

Novel variants of murine serotonin transporter mRNA and the promoter activity of its upstream site

Three variants of murine serotonin transporter (5-HTT) mRNA, which consist of a different exon-one (exon 1a, exon 1b or exon 1c) and the same exon-two to exon-five, were identified. The promoter region for each exon 1 (p1a, p1b and p1c, respectively), ligated to pGL-3 enhancer vector, had activities significantly higher than the empty vector in all cell lines tested except p1c in PC-12, whereas the activity of p1c was significantly lower than the others. Effects of the treatment of dibutyryl-cyclic AMP, human interferon-α or mouse interferon-γ have different profiles among COS-7, PC-12, C6 glioma and immortalized rat serotonergic raphe neurons, RN46A. These three promoter regions may play a role in the transcription of the 5-HTT and could offer a model of the regulation of 5-HTT production in humans and further the pathogenesis of depression.

Freewheel Running Prevents Learned Helplessness/Behavioral Depression: Role of Dorsal Raphe Serotonergic Neurons

Serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) are implicated in mediating learned helplessness (LH) behaviors, such as poor escape responding and expression of exaggerated conditioned fear, induced by acute exposure to uncontrollable stress. DRN 5-HT neurons are hyperactive during uncontrollable stress, resulting in desensitization of 5-HT type 1A (5-HT1A) inhibitory autoreceptors in the DRN. 5-HT1A autoreceptor downregulation is thought to induce transient sensitization of DRN 5-HT neurons, resulting in excessive 5-HT activity in brain areas that control the expression of learned helplessness behaviors. Habitual physical activity has antidepressant/anxiolytic properties and results in dramatic alterations in physiological stress responses, but the neurochemical mediators of these effects are unknown. The current study determined the effects of 6 weeks of voluntary freewheel running on LH behaviors, uncontrollable stress-induced activity of DRN 5-HT neurons, and basal expression of DRN 5-HT1A autoreceptor mRNA. Freewheel running prevented the shuttle box escape deficit and the exaggerated conditioned fear that is induced by uncontrollable tail shock in sedentary rats. Furthermore, double c-Fos/5-HT immunohistochemistry revealed that physical activity attenuated tail shock-induced activity of 5-HT neurons in the rostral-mid DRN. Six weeks of freewheel running also resulted in a basal increase in 5-HT1A inhibitory autoreceptor mRNA in the rostral-mid DRN. Results suggest that freewheel running prevents behavioral depression/LH and attenuates DRN 5-HT neural activity during uncontrollable stress. An increase in 5-HT1A inhibitory autoreceptor expression may contribute to the attenuation of DRN 5-HT activity and the prevention of LH in physically active rats.

Growth/differentiation factor-15 (GDF-15), a novel member of the TGF-beta superfamily, promotes survival of lesioned mesencephalic dopaminergic neurons in vitro and in vivo and is induced in neurons following cortical lesioning.

This review summarizes the evidence that GDF-15, a recently discovered member of the TGF-beta superfamily, is a trophic factor for nigral dopamine neurons, both in vitro and in vivo. Specifically, GDF-15 promotes survival and differentiation of embryonic rat dopaminergic neurons, but not of other neuron populations, with the exception of serotonergic raphe neurons. The neurotrophic effect of GDF-15 seems to be direct and not mediated through glial cells. In the rat 6-hydroxydopamine model of parkinsonism GDF-15 rescues intoxicated dopaminergic neurons and abolishes abnormal turning behavior. The most prominent site of synthesis of GDF-15 within the brain is the choroid plexus, which secretes GDF-15 into the cerebrospinal fluid, from where the molecule can penetrate through the ependymal layer into the parenchyma. Analysis of mouse mutants lacking GDF-15 will reveal whether the endogenous factor also has a role in promoting embryonic and protecting lesioned nigral dopamine neurons.

Arousal systems.

The brain contains autochthonous neural systems that evoke waking from sleep in response to sensory stimuli, prolong or enhance arousal in response to special stimuli, and also generate and maintain wakefulness regardless of sensory stimuli during the active part of the day. Through ascending projections to the cortex, these arousal systems stimulate cortical activation, characterized by high frequency gamma and low frequency rhythmic theta activity, and through descending projections to the spinal cord, they stimulate muscle tonus along with sensory-motor responsiveness and activity. They are comprised of neuronal aggregates within the brainstem reticular formation, thalamus, posterior hypothalamus and basal forebrain, and they utilize multiple different neurotransmitters. Within the brainstem, neurons of the reticular formation, which predominantly utilize glutamate as a neurotransmitter, stimulate cortical activation by exciting the widespread projecting neurons of the nonspecific thalamo-cortical projection system, which similarly utilize glutamate, and neurons of the ventral extra-thalamic relay systems located in the posterior hypothalamus and basal forebrain, many of which also utilize glutamate. In addition, these systems have descending projections by which they can enhance or modulate muscle tonus and activity. Articulating with these are cholinergic neurons of the ponto-mesencephalic tegmentum and basal forebrain that promote cortical activation during waking and also during rapid eye movement sleep (REMS), in association therein with muscle atonia. Dopaminergic ventral mesencephalic neurons stimulate a highly motivated and positively rewarding state during waking and may also do so during REMS. In contrast, noradrenergic locus coeruleus neurons promote an aroused waking state and prevent REMS as well as slow wave sleep (SWS). Serotonergic raphe neurons promote a seemingly quiet or satiated waking state, which though exclusive of REMS, can actually be conducive to SWS. Histaminergic neurons of the posterior hypothalamus act like noradrenergic neurons in enforcing waking and are joined by neurons in the region that contain orexin, a neuropeptide recently shown to maintain waking and in absentia to be responsible for narcolepsy, or the inability to maintain wakefulness. These multiple arousal systems are grossly redundant, since no one system is absolutely necessary for the occurrence of waking; yet they are differentiated, since each plays a special role in waking and sleep. During SWS, they are submitted to an inhibitory influence arising in part at least from particular GABAergic neurons co-distributed with many neurons of the arousal systems and also concentrated within the basal forebrain and adjacent preoptic region.

Insulin-induced hypoglycemia decreases single-unit activity of serotonergic medullary raphe neurons in freely moving cats: relationship to sympathetic and motor output.

Serotonergic single-unit activity during glucoregulatory challenges was studied in the nuclei raphe obscurus (NRO) and raphe pallidus (NRP) of freely moving cats. Systemic insulin administration (2-4 IU/kg, i.v.) suppressed neuronal activity by approximately 40% in direct relationship to blood glucose levels and in inverse relationship to plasma catecholamine levels. NRO and NRP serotonergic neurons displayed a temporary recovery in unit activity in response to i.v. glucose administration (500 mg/kg), which temporarily reversed insulin-induced hypoglycemia. The neuronal responses to insulin and subsequent glucose administration were also directly related to changes in integrated nuchal electromyographic activity. Serotonergic unit activity remained unchanged after glucose loading (500 mg/kg, i.v.), which produced a four-fold increase in blood glucose. Thus, medullary serotonergic neurons appear to be sensitive to reductions, but not increases, in blood glucose. The observed inverse relationship between unit activity and plasma catecholamines does not support a postulated sympathoexcitatory role for these neurons. Instead, the parallel changes in single-unit activity and integrated muscle activity support the hypothesis that the activity of medullary serotonergic neurons is linked to motor output. These neurons may modulate autonomic outflow, but only in relationship to their primary role in motor control. Finally, medullary serotonergic neurons may play a protective role in maintaining glucose homeostasis by disfacilitating the output of the somatomotor system, and hence diminishing muscle energy demands, when peripheral glucose availability is low.

Possible glutamatergic/aspartatergic projections to the supramammillary nucleus and their origins in the rat studied by selective [ 3H] D-aspartate labelling and immunocytochemistry

The supramammillary neurons projecting directly to the hippocampus or indirectly via the septum participate in the regulation of hippocampal theta activity. Inputs to the supramammillary nucleus are only partly specified neurochemically. Glutamate appears to be an excitatory transmitter in this cell group, however, the origin of the glutamatergic afferents is unknown. The present investigations were devoted to study this question. The transmitter-selective [ 3H] D-aspartate retrograde transport method was used injecting the tracer into the lateral subregion of the nucleus. The radioactive tracer was visualized by autoradiography. Non-selective retrograde tracing experiments were also performed for reference injecting wheat germ agglutinin-conjugated colloidal gold into the same supramammillary region. Retrogradely radiolabelled neurons in various numbers were detected in several brain regions including medial septum–diagonal band complex, lateral septum, rostral part of medial and lateral preoptic areas, lateral habenula, ventral premammillary nucleus, apical subregion of interpeduncular nucleus, laterodorsal tegmental nucleus, and dorsal and median raphe nuclei. Radiolabelled neurons in the mentioned raphe nuclei were serotonin-immunonegative. In the non-selective retrograde tracing experiments combined with immunocytochemistry, about 50% of the retrogradely labelled neurons in the raphe nuclei was serotonin-immunonegative, showing that not only serotonergic raphe neurons project to the supramammillary nucleus. The findings indicate that a significant part of the afferents from telencephalic, diencephalic and brainstem regions to the supramammillary nucleus may contain glutamate/aspartate as neurotransmitter. The most important functional implications of these observations concern the role of the supramammillary nucleus in controlling the electrical activity of the hippocampus, and in particular the generation and maintenance of the theta rhythm.

Serotonergic Raphe Neurons Express TASK Channel Transcripts and a TASK-Like pH- and Halothane-Sensitive K+Conductance

The recently described two-pore-domain K+ channels, TASK-1 and TASK-3, generate currents with a unique set of properties; specifically, the channels produce instantaneous open-rectifier (i.e., "leak") K+ currents that are modulated by extracellular pH and by clinically useful anesthetics. In this study, we used histochemical and in vitro electrophysiological approaches to determine that TASK channels are expressed in serotonergic raphe neurons and to show that they confer a pH and anesthetic sensitivity to these neurons. By combining in situ hybridization for TASK-1 or TASK-3 with immunohistochemical localization of tryptophan hydroxylase, we found that a majority of serotonergic neurons in both dorsal and caudal raphe cell groups contain TASK channel transcripts (approximately 70-90%). Whole-cell voltage-clamp recordings were obtained from raphe cells that responded to 5-HT in a manner characteristic of serotonergic neurons (i.e., with activation of an inwardly rectifying K+ current). In those cells, we isolated an endogenous K+ conductance that had properties expected of TASK channel currents; raphe neurons expressed a joint pH- and halothane-sensitive open-rectifier K+ current. The pH sensitivity of this current (pK approximately 7.0) was intermediate between that of TASK-1 and TASK-3, consistent with functional expression of both channel types. Together, these data indicate that TASK-1 and TASK-3 are expressed and functional in serotonergic raphe neurons. The pH-dependent inhibition of TASK channels in raphe neurons may contribute to ventilatory and arousal reflexes associated with extracellular acidosis; on the other hand, activation of raphe neuronal TASK channels by volatile anesthetics could play a role in their immobilizing and sedative-hypnotic effects.

Control of Serotonergic Function in Medial Prefrontal Cortex by Serotonin-2A Receptors through a Glutamate-Dependent Mechanism

We examined the in vivo effects of the hallucinogen 4-iodo-2,5-dimethoxyamphetamine (DOI). DOI suppressed the firing rate of 7 of 12 dorsal raphe (DR) serotonergic (5-HT) neurons and partially inhibited the rest (ED(50) = 20 microg/kg, i.v.), an effect reversed by M100907 (5-HT(2A) antagonist) and picrotoxinin (GABA(A) antagonist). DOI (1 mg/kg, s.c.) reduced the 5-HT release in medial prefrontal cortex (mPFC) to 33 +/- 8% of baseline, an effect also antagonized by M100907. However, the local application of DOI in the mPFC increased 5-HT release (164 +/- 6% at 100 microm), an effect antagonized by tetrodotoxin, M100907, and BAY x 3702 (5-HT(1A) agonist) but not by SB 242084 (5-HT(2C) antagonist). The 5-HT increase was also reversed by NBQX (AMPA-KA antagonist) and 1S,3S-ACPD (mGluR 2/3 agonist) but not by MK-801 (NMDA antagonist). AMPA mimicked the 5-HT elevation produced by DOI. Likewise, the electrical-chemical stimulation of thalamocortical afferents and the local inhibition of glutamate uptake increased the 5-HT release through AMPA receptors. DOI application in mPFC increased the firing rate of a subgroup of 5-HT neurons (5 of 10), indicating an enhanced output of pyramidal neurons. Dual-label fluorescence confocal microscopic studies demonstrated colocalization of 5-HT(1A) and 5-HT(2A) receptors on individual cortical pyramidal neurons. Thus, DOI reduces the activity of ascending 5-HT neurons through a DR-based action and enhances serotonergic and glutamatergic transmission in mPFC through 5-HT(2A) and AMPA receptors. Because pyramidal neurons coexpress 5-HT(1A) and 5-HT(2A) receptors, DOI disrupts the balance between excitatory and inhibitory inputs and leads to an increased activity that may mediate its hallucinogenic action.