Serotonin Development Research Articles

Emerging Targets in Migraine

Migraine is a common and highly disabling neurological disorder. Despite the complexity of its pathophysiology, substantial advances have been achieved over the past 20 years in its understanding, as well as the development of pharmacological treatment options. The development of serotonin 5-HT(1B/1D) receptor agonists ("triptans") substantially improved the acute treatment of migraine attacks. However, many migraineurs do not respond satisfactorily to triptans and cardiovascular co-morbidities limit their use in a significant number of patients. As migraine is increasingly considered to be a disorder of the brain, and preclinical and clinical data indicate that the observed vasodilation is merely an epiphenomenon, research has recently focused on the development of neurally acting compounds that lack vasoconstrictor properties. This review highlights the most important pharmacological targets for which compounds have been developed that are highly likely to enter or have already advanced into clinical trials for the acute and preventive treatment of migraine. In this context, preclinical and clinical data on compounds acting on calcitonin gene-related peptide or its receptor, the 5-HT(1F) receptor, nitric oxide synthase, and acid-sensing ion channel blockers are discussed.

Engrailed Is Required in Maturing Serotonin Neurons to Regulate the Cytoarchitecture and Survival of the Dorsal Raphe Nucleus

Analysis of constitutive Engrailed (En) null mice previously implicated the two En homeobox paralogs in the development of serotonin (5-HT) neurons. An unresolved question is whether En plays intrinsic roles in these neurons. Here, we show that En1 and En2 are expressed in maturing 5-HT neurons that will form the dorsal raphe nucleus (DRN) and part of the median raphe nucleus. Although En1 expression in 5-HT neurons persists postnatally, En2 expression is extinguished by embryonic day 17.5. To investigate intrinsic serotonergic functions for En1/2, we generated compound conditional En mutants with floxed alleles and a cre recombinase line that becomes active in postmitotic fetal 5-HT neurons. We present evidence in support of a requirement for En1/2 in the maturation of DRN cytoarchitecture. The disruption of DRN cytoarchitecture appears to result from a defect in secondary migration of serotonergic cell bodies toward the midline rather than disruption of their primary ventral migration away from the ventricular zone. Furthermore, En1/2 are required for perinatal maintenance of serotonergic identity and postnatal forebrain 5-HT levels. Increased numbers of caspase-3-expressing cells and loss of significant numbers of 5-HT neuron cell bodies, indicative of apoptosis, occurred after loss of serotonergic identity. Analysis of an allelic series of conditional mutants showed that En1 is the predominant functional En paralog in maturing 5-HT neurons, although a small contribution from En2 was reproducibly detected. Together, our findings reveal complex intrinsic functions for En in maturing 5-HT neurons, hence necessitating a reinterpretation of their roles in 5-HT system development.

Peptidergic Agonists of Activity‐Dependent Neurotrophic Factor Protect Against Prenatal Alcohol‐Induced Neural Tube Defects and Serotonin Neuron Loss

Prenatal alcohol exposure via maternal liquid diet consumption by C57BL/6 (B6) mice causes conspicuous midline neural tube deficit (dysraphia) and disruption of genesis and development of serotonin (5-HT) neurons in the raphe nuclei, together with brain growth retardation. The current study tested the hypothesis that concurrent treatment with either an activity-dependent neurotrophic factor (ADNF) agonist peptide [SALLRSIPA, (SAL)] or an activity-dependent neurotrophic protein (ADNP) agonist peptide [NAPVSIPQ, (NAP)] would protect against these alcohol-induced deficits in brain development. Timed-pregnant B6 dams consumed alcohol from embryonic day 7 (E7, before the onset of neurulation) until E15. Fetuses were obtained on E15 and brain sections processed for 5-HT immunocytochemistry, for evaluation of morphologic development of the brainstem raphe and its 5-HT neurons. Additional groups were treated either with SAL or NAP daily from E7 to E15 to assess the potential protective effects of these peptides. Measures of incomplete occlusion of the ventral canal and the frequency and extent of the openings in the rhombencephalon were obtained to assess fetal dysraphia. Counts of 5-HT-immunostained neurons were also obtained in the rostral and caudal raphe. Prenatal alcohol exposure resulted in abnormal openings along the midline and delayed closure of ventral canal in the brainstem. This dysraphia was associated with reductions in the number of 5-HT neurons both in the rostral raphe nuclei (that gives rise to ascending 5-HT projections) and in the caudal raphe (that gives rise to the descending 5-HT projections). Concurrent treatment of the alcohol-consuming dams with SAL prevented dysraphia and protected against the alcohol-induced reductions in 5-HT neurons in both the rostral and caudal raphe. NAP was less effective in protecting against dysraphia and did not protect against 5-HT loss in the rostral raphe, but did protect against loss in the caudal raphe. These findings further support the potential usefulness of these peptides for therapeutic interventions in pregnancies at risk for alcohol-induced developmental deficits. Notably, the ascending 5-HT projections of the rostral raphe have profound effects in regulating forebrain development and function, and the descending 5-HT projections of the caudal raphe are critical for regulating respiration. Protection of the rostral 5-HT-system may help prevent structural and functional deficits linked to abnormal forebrain development, and protection of the caudal systems may also reduce the increased risk for sudden infant death syndrome associated with prenatal alcohol exposure.

A Differentially AutoregulatedPet-1Enhancer Region Is a Critical Target of the Transcriptional Cascade That Governs Serotonin Neuron Development

The Pet-1 [pheochromocytoma 12 ETS (E26 transformation-specific)] gene plays a critical role in the development of serotonin (5-HT)-modulated behaviors via its control of embryonic 5-HT neuron differentiation. Pet-1 transcription is induced exclusively in 5-HT neuron postmitotic precursors before the appearance of transmitter, and its restricted expression is maintained in the adult. However, the mechanisms that direct Pet-1 expression to this single CNS neuronal cell type are unknown. Here, we show, using transgenic methods, that genomic sequences upstream, but not downstream or within the Pet-1-coding region, are sufficient for 5-HT neuron-specific transgene expression. Enhancer sequences within a 40 kb upstream fragment directed position-independent lacZ (beta-D-galactosidase) transgene expression to the developing hindbrain before the appearance of 5-HT. Moreover, virtually all of the 5-HT neurons in the adult were lacZ positive in all of the lines examined. Transgene expression in 5-HT neurons was maintained when the 40 kb fragment was truncated on its 5' end to either 12 or 1.8 kb, although position independence was then lost. Analysis of transgene expression in Pet-1 null mice indicated that Pet-1 was required to maintain the activity of the Pet-1 enhancer region in a subset of 5-HT neurons. These findings suggest that a conserved 1.8 kb region immediately flanking the Pet-1-coding region is a critical genomic target of the transcriptional cascade that governs 5-HT neuron development and provide additional evidence for 5-HT neuron heterogeneity at the genetic level. We discuss the potential application of the Pet-1 transgenes reported here to the selective genetic manipulation of 5-HT neurons.

Evidence for serotonin (5-HT) 1B, 5-HT 1D and 5-HT 1F receptor inhibitory effects on trigeminal neurons with craniovascular input

Development of serotonin (5HT 1B/1D) agonists for the acute attack of migraine resulted in considerable interest in their action. The superior sagittal sinus (SSS) was isolated in α-chloralose (60 mg/kg, i.p. and 20 mg/kg i.v.i. supplementary 2 hourly) anaesthetised cats. The SSS was stimulated electrically (100 V, 250 μs duration, 0.3 Hz) and neurons of the trigeminocervical complex monitored using electrophysiological methods. To test 5-HT 1B receptor-mediated activity common carotid blood flow (CCF) was monitored with a transonic flow probe placed around the vessel. Naratriptan (5-HT 1B/1D/1F receptor agonist) and alniditan (5-HT 1B/1D receptor agonist) produced reductions in carotid blood flow of 38±5% and 42±6%, respectively. These effects were attenuated by the 5-HT 1B receptor antagonist SB224289 ( P<0.05). LY344864 (5-HT 1F receptor agonist) had no effect on CCF. Naratriptan inhibited SSS-evoked activity (61±7%), an effect partially inhibited by the 5-HT 1B receptor antagonist SB224289 (30±5%), or by the 5-HT 1D receptor antagonist BRL-15572 (37±6%). There remained an inhibitory effect of naratriptan after both 5-HT 1B and 5-HT 1D receptor blockade (22±5%). Alniditan inhibited SSS-evoked trigeminal activity (53±6%), an effect abolished after 5-HT 1B and 5-HT 1D receptor blockade. LY344864 (5-HT 1F receptor agonist) inhibited SSS-evoked trigeminal activity (28±5%), an effect unaltered by either SB224289 or BRL-15572. It can be concluded that there are inhibitory 5-HT 1B, 5-HT 1D and 5-HT 1F receptors in the trigeminocervical complex of the cat. 5-HT 1B receptor-mediated inhibition is the most potent of the three in terms of inhibition of trigeminovascular nociceptive traffic.

Astrocyte-mediated trophic support of developing serotonin neurons: effects of ethanol, buspirone, and S100B

Previously, this laboratory demonstrated that the development of serotonin (5-HT) neurons and S100B-immunopositive glia proximal to these neurons is impaired in the offspring of ethanol-fed rats. However, maternal treatment with a 5-HT 1A agonist, e.g., buspirone or ipsapirone, between gestational days 13 and 20 prevented most of the ethanol-associated changes to developing 5-HT neurons and S100B-immunopositive glia in offspring. The present in vitro studies examined the hypothesis that the protective effects of a 5-HT 1A agonist on ethanol-exposed, developing 5-HT neurons are mediated in part by astrocyte-produced factors such as S100B. Primary cultures of fetal 5-HT neurons were maintained in conditioned medium (CM) that was obtained from ethanol- and buspirone-treated astrocytes. In order to assess the potential contribution of S100B to the protective effects of buspirone, a mouse monoclonal antibody to S100B was added to the CM to block the biological effects of this protein. These studies demonstrated that CM, obtained from ethanol-treated astrocytes, was unable to support normal development of 5-HT neurons; there was a significant reduction in the number of 5-HT neurons/well. However, CM that was obtained from astrocytes that were co-treated with buspirone and ethanol prevented the ethanol-associated reduction, and the protective effects of buspirone required S100B. We also investigated whether exogenous S100B could protect 5-HT neurons from damage caused by direct exposure to ethanol. Direct exposure of fetal brainstem neurons to ethanol in chemically-defined medium was associated with a significant reduction in the number of 5-HT immunopositive neurons/well. However, exogenous S100B protected 5-HT neurons from the ethanol-associated reduction. Our observations suggest that the protective effects of buspirone on ethanol-exposed, developing 5-HT neurons are mediated in part by the astrocyte-produced factor S100B.

Perinatal supplementation of low doses of ethanol enhances 5-HT restoration in the central nervous system

It has been reported that long-term administration of ethanol has deleterious effects on the central nervous system; the alterations are particularly evident if the exposure occurs during development. Our study shows that rat perinatal administration of 3% and 6% ethanol does not alter development of serotonin (5-HT) pathways in the central nervous system, while their reactive changes triggered by neonatal lesioning are greatly altered. The administration of 5, 7-dihydroxytriptamine (5,7-DHT) within 6 hours from birth causes 5-HT fiber degeneration throughout the central nervous system. The loss of 5-HT is particularly relevant in lumbar spinal cord, occipital cortex, and hippocampus. This early decrease in 5-HT content is followed by a slow and partial recovery. If animals are exposed to 3% ethanol during the perinatal period, there is an enhancement of the 5,7-DHT-induced degeneration that is, however, followed by a faster and greater recovery throughout the central nervous system. Conversely, perinatal exposure to 6% ethanol and 5, 7-DHT administration lead to an irreversible 5-HT loss with no subsequent recovery. The deleterious effects of 6% ethanol are accompanied by a reduced expression of neurotrophin. Thus, our study suggests that chronic exposure to ethanol can influence central nervous system plasticity during development. Low doses may enhance neuronal plasticity and repair perhaps via an increased efficacy of neurotrophic factors, whereas higher doses may negatively affect neural development also by means of the impairment of the expression of neurotrophic factors.

Serotonin inhibits trigeminal nucleus activity evoked by craniovascular stimulation through a 5HT1B/1D receptor: a central action in migraine?

The development of serotonin (5HT1B/1D) agonists as treatments for the acute attack of migraine has resulted in considerable interest in their mechanism of action and, to some extent, renewed interest in the role of serotonin (5-hydroxytryptamine; 5HT) in the disorder. The initial synthesis of this class of compounds was predicated on the clinical observation that intravenous 5HT terminated acute attacks of migraine. In this study the superior sagittal sinus was isolated in the alpha-chloralose (60 mg/kg i.p. and 20 mg/kg i.v. injection supplementary 2 hourly) anesthetized cat. The sinus was stimulated electrically (120V, 250 microsec duration, 0.3 Hz), and neurons of the trigeminocervical complex in the dorsal C2 spinal cord were monitored using electrophysiological methods. After baseline recordings in each animal, 5HT (15 microg/kg/min) was infused for 5 minutes in the presence of either vehicle (group A) or the 5HT1B/1D antagonist GR127935 (100 microg/kg i.v. injection; group B). The baseline probability of cell firing after sagittal sinus stimulation was 0.61 +/- 0.1 at a latency to the fastest peak of 11.1 +/- 0.4 msec. In group A, 5HT infusion alone had a small effect of increasing mean blood pressure (12 +/- 3 mm Hg), which in itself did not alter cell firing. In group A, 5HT alone had an inhibitory effect on evoked trigeminal activity, which developed 15 to 20 minutes after commencement of the infusion. The inhibition of cell firing lasted for 20 minutes, after which the activity returned to baseline. In group B, the combination of 5HT and GR127935 had no effect on trigeminal cell firing, although the small hypertensive effect was still present. These data indicate that 5HT inhibits evoked trigeminal nucleus firing via the 5HT1B/1D receptor at which GR127935 is an antagonist. It is likely that some part of the effect of 5HT in migraine relates to inhibition of trigeminal nucleus activity, just as it is likely that some part of the effect of the triptans is also mediated at this central site and may be complementary to their nonneuronal actions. Moreover, the data highlight the case for describing this class of headache as neurovascular headaches rather than vascular headaches, to recognize the implicit contribution of the trigeminovascular system to their pathophysiology.

Immunohistochemical study on development of serotonin-, substance P-, and enkephalin-positive fibers in the rat spinal motor nucleus.

Developmental changes in the coexistence of serotonin and substance P/enkephalin within single fibers of lamina IX of the rat lumbar spinal cord were examined by the use of a double-labelling immunohistochemical technique. On postnatal day (P) 0, 65.0% of immunoreactive varicosities contained only serotonin, and 21.3% of them had both serotonin and substance P. The coexistent ratio of serotonin and substance P in single fibers increased with development: 61.9% of serotonin positive varicosities co-contained substance P on P28, similar to the ratio found in adult animals (67.4%). The ratios of varicosities containing only substance P remained the same from P0 to adult stage (about 15%). Enkephalin positive immunoreactivity was not co-localized with serotonin positive varicosities at any stage of development. Although numerous serotonin positive fibers were found in lamina IX, only a few substance P and enkephalin positive fibers were observed in the same area on P0. The density of serotonin positive varicosities increased slightly by P28, whereas substance P and enkephalin positive fibers increased considerably by this age. Between P28 and the adult stage, the density of serotonin positive fibers decreased by about 50%. The cross sectional area of axonal varicosities containing serotonin- and substance P-like immunoreactivity was similar in both P0 and adult animals, whereas that of enkephalin positive fibers was different. We also examined the coexistence of serotonin and substance P within single neurons of the caudal raphe nuclei in P7 and adult animals, and found that the coexistent ratio significantly increased with development.

Postnatal development of serotonin-, substance p- and enkephalin-like immunoreactive fibers in the motor nucleus of the rat lumbar spinal cord

Postnatal development of serotonin-, substance p- and enkephalin-like immunoreactive fibers in the motor nucleus of the rat lumbar spinal cord

Development of serotonin, substance P and thyrotrophin-releasing hormone in mouse medullary raphe grown in organotypic tissue culture: developmental regulation by serotonin

Substance P (SP) and thyrotrophin-releasing hormone (TRH) are co-localized with serotonin (5-HT) in cells of the medullary raphe nuclei. In order to examine the factors that control development of multiple neurotransmitters within individual brain nuclei, we have grown presumptive raphe nuclei in organotypic tissue culture, an environment in which mammalian embryonic brain is easily accessible and manipulable. Tissue was obtained from E13 mice. A discrete midline segment of the rhombencephalon was dissected intact or was separated into ‘rostral’ (RR) and ‘medullary’ (MR) fragments. Tissue was explanted onto collagen coverslips and grown for up to two weeks in Maximow depression chambers. Tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, was barely detectable at explantation. During the first week in culture, however, TPH activity increased 7-fold. After two weeks, TPH activity increased almost 2.5-fold above the one-week level. Immunocytochemical analysis of the cultures confirmed a widespread distribution of 5-HT-positive cells and fibers throughout the explant. SP, monitored by radioimmunoassay, was detected after two days in culture, and attained a level of111.7 ± 9.8 pg/culture after two weeks. TRH activity was similarly elevated after two weeks in vitro. Therefore, developmental increases in TPH, SP, and TRH occurred in culture, mimicking the condition in vivo. RR and MR fragments, when grown apart on separate coverslips, developed 1.57–2.26 times the TPH activity that developed in the undivided piece. Inclusion of 1 μM pargyline in the fragments restored TPH to control levels. The effect of pargyline was blocked by methiothepin, suggesting autoreceptor-mediated regulation. By contrast, SP activity was additive, the amount of SP present in the whole piece being equal to the sum of the RR and MR fragments. Pargyline did not affect the development of TRH or SP. These data suggest that factors (particularly autoreceptor-mediated interactions) affecting the development of 5-TH in the cultures do not act simultaneously to affect peptide development.

An immunohistochemical study of serotonin development in the opossum cerebellum.

In the present study we have used the indirect antibody peroxidase anti-peroxidase technique to analyze the development of serotonin in the cerebellum of pouch young opossums ranging in age from birth (postnatal day (PD) 1) - to PD 47. The pathways by which serotoninergic axons enter the cerebellum appear to change during development. Between PD 1 and PD 11 varicose serotoninergic fibers course though the continuity between the tectum and the dorsal medial aspect of the cerebellar plate and distribute primarily to the intermediate zone. By PD 11 serotoninergic fibers enter the cerebellum via a connecting band between the cerebellum and the dorsal lateral aspect of the rostral medulla. Fibers entering the cerebellum via this later route course around the lateral recess of the fourth ventricle and initially distribute to ventral and lateral areas of the cerebellum. At later developmental ages (PD 14-PD 47) serotoninergic fibers are present in: 1) the cellular zone of migration between the Purkinje cell layer and deep nuclei; 2) the Purkinje cell layer; 3) the internal granule cell layer; and 4) the deep cerebellar nuclei. The external granule cell and the molecular layers rarely contain serotoninergic fibers. The present study has shown that serotonin is present in the cerebellar anlage on PD 1 (within 13 days of conception). This is prior to the arrival of other major afferent systems (King et al. 1982; Bishop et al. 1983; Martin et al. 1983; Morgan et al. 1983). Further, serotoninergic axons reach the cerebellum via different routes at different stages of development. The presence of serotonin in the intermediate zone early in development supports the hypothesis that it may influence neuronal migration and differentiation (Lauder and Krebs 1976). Axons containing serotonin and arriving later in development may function in synaptic transmission, a role proposed for this indoleamine in the adult (Strahlendorf et al. 1979).