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Tauopathies, including Alzheimer's disease are the most frequent neurodegenerative disorders in elderly people. Patients develop cognitive and behaviour defects induced by the tauopathy in the forebrain, but most also display early brainstem tauopathy, with oro-pharyngeal and serotoninergic (5-HT) defects. We studied these aspects in Tau.P301L mice, that express human mutant tau protein and develop tauopathy first in hindbrain, with cognitive, motor and upper airway defects from 7 to 8 months onwards, until premature death before age 12 months. Using plethysmography, immunohistochemistry and biochemistry, we examined the respiratory and 5-HT systems of aging Tau.P301L and control mice. At 8 months, Tau.P301L mice developed upper airway dysfunction but retained normal respiratory rhythm and normal respiratory regulations. In the following weeks, Tau.P301L mice entered terminal stages with reduced body weight, progressive limb clasping and lethargy. Compared to age 8 months, terminal Tau.P301L mice showed aggravated upper airway dysfunction, abnormal respiratory rhythm and abnormal respiratory regulations. In addition, they showed severe tauopathy in Kolliker-Fuse, raphé obscurus and raphé magnus nuclei but not in medullary respiratory-related areas. Although the raphé tauopathy concerned mainly non-5-HT neurons, the 5-HT metabolism of terminal Tau.P301L mice was altered. We propose that the progressive raphé tauopathy affects the 5-HT metabolism, which affects the 5-HT modulation of the respiratory network and therefore the breathing pattern. Then, 5-HT deficits contribute to the moribund phenotype of Tau.P301L mice, and possibly in patients suffering from tauopathies, including Alzheimer's disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mouse readiness for gene manipulation allowed the production of mutants with breathing defects reminiscent of breathing syndromes. As C57BL/6J and FVB/N inbred strains were often used as background strains for producing mutants, we compared their breathing pattern from birth onwards. At birth, in vivo and in vitro approaches revealed robust respiratory rhythm in FVB/N, but not C57BL/6J, neonates. With aging, rhythm robustness difference persisted, and interstrain differences in tidal volume, minute ventilation, breathing regulations, and blood-gas parameters were observed. As serotonin affected maturation and function of the medullary respiratory network, we examined the serotoninergic metabolism in the medulla of C57BL/6J and FVB/N neonates and aged mice. Interstrain differences in serotoninergic metabolism were observed at both ages. We conclude that differences in serotoninergic metabolism possibly contribute to differences in breathing phenotype of FVB/N and C57BL/6J mice.

Serotonin (5-HT) is a neuromodulator-transmitter influencing global brain function. Past and present findings illustrate a prominent role for 5-HT in the modulation of ponto-medullary autonomic circuits. 5-HT is also involved in the control of neurotrophic processes during pre- and postnatal development of neural circuits. The functional implications of 5-HT are particularly illustrated in the alterations to the serotonergic system, as seen in a wide range of neurological disorders. This article reviews the role of 5-HT in the development and control of respiratory networks in the ponto-medullary brainstem. The review further examines the role of 5-HT in breathing disorders occurring at different stages of life, in particular, the neonatal neurodevelopmental diseases such as Rett, sudden infant death and Prader-Willi syndromes, adult diseases such as sleep apnoea and mental illness linked to neurodegeneration., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

Sudden infant death syndrome (SIDS) is defined as the sudden and unexpected death of an infant less than 12 months of age that occurs during sleep and remains unexplained after a complete autopsy, death scene investigation, and review of the clinical history. It is the leading cause of postneonatal mortality in the developed world. The cause of SIDS is unknown, but is postulated to involve impairment of brainstem-mediated homeostatic control. Extensive evidence from animal studies indicates that serotonin (5-HT) neurons in the medulla oblongata play a role in the regulation of multiple aspects of respiratory and autonomic function. A subset of SIDS infants have several abnormalities in medullary markers of 5-HT function and genetic polymorphisms impacting the 5-HT system, informing the hypothesis that SIDS results from a defect in 5-HT brainstem-mediated control of respiratory (and autonomic) regulation. Here we review the evidence from postmortem human studies and animal studies to support this hypothesis and discuss how the pathogenesis of SIDS is likely to originate in utero during fetal development.

Prader-Willi syndrome is a neurogenetic disease resulting from the absence of paternal expression of several imprinted genes, including NECDIN. Prader-Willi children and adults have severe breathing defects with irregular rhythm, frequent sleep apneas, and blunted respiratory regulations. For the first time, we show that Prader-Willi infants have sleep apneas already present at birth. In parallel, in wild-type and Necdin-deficient mice, we studied the respiratory system with in vivo plethysmography, in vitro electrophysiology, and pharmacology. Because serotonin is known to contribute to CNS development and to affect maturation and function of the brainstem respiratory network, we also investigated the serotonergic system with HPLC, immunohistochemistry, Rabies virus tracing approaches, and primary culture experiments. We report first that Necdin-deficiency in mice induces central respiratory deficits reminiscent of Prader-Willi syndrome (irregular rhythm, frequent apneas, and blunted respiratory regulations), second that Necdin is expressed by medullary serotonergic neurons, and third that Necdin deficiency alters the serotonergic metabolism, the morphology of serotonin vesicles in medullary serotonergic neurons but not the number of these cells. We also show that Necdin deficiency in neonatal mice alters the serotonergic modulation of the respiratory rhythm generator. Thus, we propose that the lack of Necdin expression induces perinatal serotonergic alterations that affect the maturation and function of the respiratory network, inducing breathing deficits in mice and probably in Prader-Willi patients.

In brainstem-spinal cord preparations of neonatal control C3H and transgenic Tg8 mice where deletion of the gene encoding monoamine oxidase-A results in serotonin (5-hydroxytryptamine (HT)) excess, whole cell recordings of identified phrenic motoneurons (Phr Mns) were performed to study the modulation of their activity by 5-HT. In C3H mice, a dual effect was observed: (i) a facilitation via 5-HT(2A) receptors and (ii) a decrease of the transmission of the central inspiratory drive via 5-HT(1B) receptors. In Tg8 mice, the 5-HT(2A)-mediated facilitation was present but the 5-HT(1B)-mediated decrease was lacking. Therefore, the conservation of the 5-HT(2A) response vs. the loss of the 5-HT(1B) one suggest that the two types of receptors respond differently to 5-HT level changes.

In rodent neonates, the neurotransmitter serotonin (5-HT) modulates the activity of both the medullary respiratory rhythm generator and the cervical phrenic motoneurons. To determine whether 5-HT also contributes to the maturation of the respiratory network, experiments were conducted in vitro on the brainstem-spinal cord preparation of neonatal mice originating from the control strain (C3H) and the monoamine oxidase A-deficient strain, which has a brain perinatal 5-HT excess (Tg8). At birth, the Tg8 respiratory network is unable to generate a respiratory pattern as stable as that produced by the C3H network, and the modulation by 5-HT of the network activity present in C3H neonates is lacking in Tg8 neonates. In addition, the morphology of the phrenic motoneurons is altered in Tg8 neonates; the motoneuron dendritic tree loses the C3H bipolar aspect but exhibits an increased number of spines and varicosities. These abnormalities were prevented in Tg8 neonates by treating pregnant Tg8 dams with the 5-HT synthesis inhibitor p-chlorophenylalanine or a 5-HT(2A) receptor antagonist but were induced in wild-type neonates by treating C3H dams with a 5-HT(2A) receptor agonist. We conclude that 5-HT contributes, probably via 5-HT(2A) receptors, to the normal maturation of the respiratory network but alters it when present in excess. Disorders affecting 5-HT metabolism during gestation may therefore have deleterious effects on newborns.

Developmental changes in levels of serotonin, L-tryptophan and 5-hydroxyindol acetic acid (5-HIAA) were measured by high pressure liquid chromatography (HPLC) in the forebrain, brainstem and cervical cord of fetal, neonatal and adult mice from the wild strain C3H and the transgenic strain Tg8, created from the C3H line by the disruption of the gene encoding monoamine oxidase A. The results indicated that the absence of monoamine oxidase A activity in Tg8 mice results in abnormally high 5-hydroxytryptamine (5-HT) levels in all the central nervous structures and at all the studied developmental ages. Since serotonin levels were 4-5 times larger in Tg8 than in C3H mice at gestational day 20, comparing the central network function at birth of C3H and Tg8 neonates should shed some light on the role of serotonin in prenatal network maturation.

In vitro experiments were conducted on neonatal rat brainstem-spinal cord preparations to test the hypothesis of an inhibitory modulation of phrenic activity by serotonin (5-HT) via non-5-HT2A receptors [Lindsay, A.D. and Feldman, J.L., Modulation of respiratory activity of neonatal rat phrenic motoneurones by serotonin, J. Physiol., 461 (1993) 213-233]. The changes induced by 5-HT and related agents on phrenic root discharges and membrane currents in identified phrenic motoneurons were analysed after blockade of spinal 5-HT2A receptors. Spinal application of 5-HT1B (but not 5-HT1A) receptor agonists depressed the phrenic activity and the effect was prevented by pretreatment with 5-HT1B (but not 5-HT1A, 5-HT2A and 5-HT3) receptor antagonists. Results from phrenic motoneuron whole cell recordings do not reject a presynaptic location of the 5-HT receptors responsible for this depression.

In order to determine whether the serotonergic modulation of the central respiratory activity previously reported in neonatal rats occurs in species other than the rat, we performed identical in vitro experiments on the neonatal mouse to those performed on the neonatal rat. The effects of adding serotonin (5-hydroxytryptamine, 5-HT) and related agents to the superfusate suggested that the respiratory rhythm generator undergoes an excitatory modulation via medullary 5-HT1A receptors. Upon applying the drugs to the spinal cord alone, 5-HT was found to have a dual effect on phrenic motoneuron firing: (i) a facilitatory effect mediated by 5-HT2A receptors and (ii) a depressive effect on their inspiratory discharge mediated by non-5-HT1A, non-5-HT2A, non-5-HT3 receptors, possibly of the 5-HT1B subtype. It was therefore concluded that serotonin modulates the neonatal central respiratory activity in mice as well as in rats, and that similar 5-HT receptor subtypes are involved in this process in both species.

The aim of the present work was to know whether the excitatory modulation of the central respiratory rhythm generator by serotonin (5-HT) previously found to occur in the newborn rat, is already functional during the fetal life. Experiments were performed at embryonic day 18 (D18) and 20-21 (D20-21; full-term day 21) on the fetal rat brainstem-spinal cord preparation in which the ability to generate central respiratory activity in vitro persists. Replacing the normal medium which bathed the preparation by a medium containing 5-HT increased the respiratory frequency (RF) within 2-3 min in a dose-dependent manner in both D18 and D20-21 fetuses but the effect was particularly drastic at D18. Applying a medium containing the 5-HT antagonist, methysergide, to block the effect of endogenous 5-HT, if any, reduced the RF within 2-3 min and the reduction was especially drastic at D18 where respiratory arrests occurred for several minutes in most of the experiments. Applying a medium containing either the 5-HT reuptake inhibitor fluoxetine to potentiate the effect of endogenous 5-HT or the 5-HT precursor, L-tryptophan, to activate 5-HT biosynthesis mechanisms, increased the RF. To define the type of 5-HT receptors involved in the modulation of the RF, experiments were conducted with specific 5-HT agonists and antagonists. Both 5-HT1 (8-OH-DPAT, buspirone) and 5-HT2 agonists (DOI, alpha-methyl-5-HT) increased the RF but only the 5-HT1A agonist 8-OH-DPAT was efficient at submicromolar concentrations. Applying the 5-HT1A antagonist NAN-190 alone decreased the RF and even elicited respiratory arrests while the 5-HT2 antagonist ketanserin was inefficient. NAN-190 pre-treatment blocked the increase in the RF due to 8-OH-DPAT and 5-HT. Taken as a whole these results clearly indicate that endogenous 5-HT exerts an excitatory modulation on the respiratory rhythm generator via activation of medullary 5-HT1A receptors well before birth, as soon as D18 where the modulation is particularly potent.

Experiments were conducted on newborn rats in order to study the serotonergic modulation of the central respiratory activity during the neonatal period. In vitro experiments performed on isolated brainstem spinal cord preparations demonstrated that serotonin exerts a permanent excitatory modulation on the central respiratory rhythm generator but depresses the inspiratory motor output to the genioglossus muscle. In vivo experiments confirmed that increasing endogenous serotonin levels decreases the inspiratory activity of the genioglossus and elicits drastic obstructive apneas.

1. Experiments were performed on anaesthetized newborn rats (aged 3-10 days) to know whether an increase in central serotonin levels might favour the occurrence of obstructive apnoeas as previously suggested by in vitro results from our group. 2. The levels of serotonin (5-HT), its precursor 5-hydroxytryptophan (5-HTP) and its metabolite, 5-hydroxyindole-acetic acid (5-HIAA), were analysed in cerebrospinal fluid samples collected at the level of the obex prior to and after intraperitoneal injection of L-tryptophan (50 mg kg-1) in sixty-eight anaesthetized newborn rats (control rats prior to injection and injected rats 15, 30 and 45 min after the injection). A significant increase in 5-HT and 5-HTP levels occurred 30 min after the injection, attesting to the activation of 5-HT biosynthesis. 3. The EMG activity of both the genioglossus and the diaphragm was recorded before and after L-tryptophan load (50 mg kg-1) in twenty-two newborn rats. After the injection of L-tryptophan, the amplitude of the integrated genioglossus activity decreased, or was even abolished, either during a few respiratory cycles or for long periods in twenty-one out of twenty-two animals. Recovery of the genioglossus activity occurred within 45-60 min. 4. The thoracic respiratory movements and the resulting upper airway pressure changes were recorded before and after L-tryptophan injection (50 mg kg-1) in sixty-two animals. In some litters (n = 7), most of the animals (21/25) displayed, within 20-40 min of the injection, recurrent episodes of obstructive apnoea often followed by central ones. These respiratory difficulties became severe and drastic, and led in two instances to respiratory distress and death. Lower doses of L-tryptophan (10 mg kg-1) did not induce any respiratory disorders unless these were potentiated by pargyline treatment (50 mg kg-1, n = 7). The obstructive apnoeas liable to occur after an L-tryptophan load (50 mg kg-1) were prevented by blocking the 5-HT receptor with methysergide (50 mg kg-1, n = 5) or by blocking the 5-HT biosynthesis by applying p-chlorophenylalanine (PCPA) pretreatment at birth (300 mg kg-1, n = 7). In other litters (n = 6), none of the eighteen newborn rats tested were affected by L-tryptophan, however, In five young adult rats, L-tryptophan again had no effect.4+ ĕ

In order to investigate the mechanisms through which serotonin (5-HT) modulates the activity of the respiratory rhythm generator, respiratory activity was recorded from cervical ventral roots of the superfused isolated brainstem-spinal cord preparation of the newborn rat. Replacing the normal bathing medium by a medium containing 5-HT (30 microM) increased the respiratory frequency by 70% of the control value. Intact pontomedullary structures are necessary for this effect to take place, however, since the 5-HT-induced increases in respiratory frequency were no longer observed after elimination (section and electrolytic lesion) of the caudal ventro-lateral pons containing the A5 areas. Local applications of 5-HT (dual bath, microdialysis and microinjection experiments) revealed, however, that 5-HT acts at the medullary level and that its effects are not due to a diffuse action on all the neurons of the medullary respiratory centers but to a specific action focusing on structures located in the rostral ventro-lateral medulla.

1. Experiments were performed on the brain stem-spinal cord preparation of newborn rats, in which the phrenic and hypoglossal nerves continue to show rhythmic respiratory activity in vitro, in order to compare the effects of serotonin (5-HT) on both activities and to analyse the mechanisms responsible for the depression by 5-HT of the hypoglossal activity. 2. Under control conditions, simultaneous recordings of the inspiratory discharges of hypoglossal and cervical roots showed that the two bursts did not start simultaneously and had different patterns (time-to-peak and peak values); this suggests that both pools of motoneurons did not share the same central drive(s). 3. Adding 5-HT and related agents to the bathing medium delayed and depressed the hypoglossal inspiratory discharge via activation of 5-HT2 receptors since these effects were elicited by 5-HT2 agonists (alpha-methyl-5-HT and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane-HCl (DOI)) but not by 5-HT1 agonists (RU 24969 and (+/-)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide (8-OH-DPAT)). The 5-HT depression of the hypoglossal discharge was prevented by applying a pretreatment with a specific 5-HT2 antagonist (ketanserin). Parallel to the hypoglossal discharge decrease, 5-HT elicited a permanent cervical root discharge along with a persistent inspiratory bursting. Adding the 5-HT precursor L-tryptophan to the bathing medium depressed the hypoglossal (XII) discharge without affecting the cervical one. 4. Local application of 5-HT within the hypoglossal motor nucleus decreased the hypoglossal output, revealing that the 5-HT depression of the hypoglossal discharge was at least partly mediated by the 5-HT effects at the level of the motoneurons. Local application of 5-HT within the cervical motor nucleus elicited a permanent firing in the cervical root with a persistent inspiratory bursting. 5. Intracellular analysis confirmed the existence of differences in central respiratory drive between cervical and hypoglossal motoneurons under control conditions, as well as differences in response to 5-HT. All the hypoglossal motoneurons became silent under 5-HT bathing, and showed no change in the input membrane resistance, a moderate depolarization, and a delayed central respiratory drive with a decreased amplitude. The cervical motoneurons became more active during inspiration, despite a decrease in the amplitude of the central respiratory drive, which was compensated for by a large depolarization and an increased input membrane resistance. Some cervical motoneurons even fired at a low rate during expiration.(ABSTRACT TRUNCATED AT 400 WORDS)

Respiratory activity was recorded in cervical ventral roots during in vitro experiments performed on superfused brainstem-cervical cord preparations from newborn rats aged 0-3 days. The effects of 5-HT agents added to the bathing medium on respiratory frequency were analysed. Adding 5-hydroxytryptamine (5-HT, 30 microM) or 5-HT precursors (either L-tryptophane, L-Trp, 50 microM, or 5-hydroxytryptophane, 5-HTP, 50 microM) elicited increases in respiratory frequency. Precursor effects were blocked by a 5-HT antagonist (methysergide, 70 microM) and significantly reduced by pretreatment with a 5-HT synthesis inhibitor (p-chlorophenylalanine, PCPA, i.p. 300 mg/kg at birth). Adding drugs known to block (methysergide, 40 microM) or to potentiate (fluoxetine, 100 microM) any endogenous 5-HT effects decreased or increased respiratory frequency, respectively. These results suggest that the 5-HT biosynthesis mechanisms remain functional in vitro and that a continuous release of endogenous 5-HT exerts excitatory modulation on the respiratory rhythm generator.

Previous experiments performed in the in vitro newborn rat brainstem-spinal cord preparation reported that the addition of serotonin (5-HT, 30-50 microM) to the bathing medium induced increases in the respiratory frequency and a large tonic discharge on all the cervical ventral roots. The aim of the present work was to define whether the 5-HT-induced tonic discharge involved respiratory or non-respiratory motoneurones. Intracellular recordings demonstrated that cervical (C2) motoneurones (n = 27) were depolarized by 5-HT but that the 5-HT-induced tonic discharge was mainly due to recruitment of silent motoneurones (n = 18) which fired permanently (15/18; 17 +/- 3 Hz) under 5-HT. The respiratory motoneurones (n = 9) retained a phasic inspiratory discharge (5/9), even if some (4/9) occasionally exhibited a few spikes during expiration. Therefore, it is concluded that the 5-HT-induced tonic discharge is unlikely to have functional significance in respiration.

The in vitro brainstem-spinal cord of the newborn rat has been used to study the central effects of serotonin (5-HT) on the brainstem respiratory motor control system. Brainstem superfusion with a medium containing 5-HT (30 microM) induced a short latency increase of respiratory frequency, often (60% of the experiments) followed by delayed tonic activity. Weaker concentrations of 5-HT (10-20 microM) were ineffective but prior application of drugs limiting 5-HT inactivation (pargyline and fluoxetine) revealed 5-HT effects. Changes in respiratory frequency are: (1) completely antagonized by methysergide; (2) not suppressed by 5-HT2 (ketanserine) and 5-HT3 (zacopride, GR3832F) antagonists; and (3) induced by 5-HT1 agonists (RU24969, buspirone). Since 5-HT2 agonists (DOI, alpha-methyl-5-HT) only evoked minor changes in frequency, the central action of 5-HT on the respiratory rhythm generator seems to depend on activation of 5-HT1 receptors. Tonic activity induced by 5-HT is: (1) antagonized by methysergide or ketanserine but not 5-HT3 antagonists; (2) induced by 5-HT2 but not 5-HT1 agonists; (3) still induced in the isolated spinal cord by 5-HT superfusion or 5-HT microinjection in the cervical ventral horn; and (4) sometimes replaced by rhythmic activity at a frequency different from that of respiration. Tonic activity does not involve the central circuitry responsible for respiration but depends on 5-HT2 receptors linked to spinal networks. These results suggest that 5-HT exerts a facilitory modulation on the respiratory rhythm generator through 5-HT1 medullary receptors and on motoneurons through 5-HT2 spinal receptors.

Newborn rat respiratory activity was recorded on hypoglossal nerve and ventral cervical roots during in vitro experiments performed on superfused brainstem spinal cord preparations. The addition of serotonin (5-HT) to the bathing medium increased the respiratory frequency and selectively depressed the hypoglossal activity. Any decreases in the amplitude of cervical recordings were always restricted and reversible, whereas the hypoglossal activity was abolished. Furthermore, on cervical roots, 5-HT induced a tonic activity superimposed on the respiratory one, which was never observed with the hypoglossal nerve. When 5-HT was applied on isolated hemispinal cord, a tonic activity could still be elicited. These results indicate that serotonin (i) modulates the activity of neurons involved in the generation of respiratory rhythm, (ii) depresses the activity of hypoglossal motoneurons, and (iii) evokes tonic activity in cervical motoneurons, probably as the result of direct spinal effects.