Serotonin as a regulator of craniofacial morphogenesis: site specific malformations following exposure to serotonin uptake inhibitors.

During craniofacial development in the mouse embryo (days 9-12 of gestation; plug day = day 1), transient expression of serotonin (5-HT) uptake in epithelial structures of this region correlates with critical morphogenetic events (Lauder et al., '88; Shuey, '91; Shuey et al., '89, '92). The purpose of the present investigation was to assess the possible functional significance of these uptake sites by examination of patterns of dysmorphology following exposure of embryos to selective 5-HT uptake inhibitors. Exposure of mouse embryos in whole embryo culture to sertraline, at a concentration (10 microM) which produced no evidence of general embryotoxicity, caused craniofacial malformations consistent with direct action at 5-HT uptake sites. Two other 5-HT uptake inhibitors, fluoxetine and amitriptyline, produced similar defects. The critical period of sertraline exposure occurred on days 10-11. The observed craniofacial defects were associated with decreased proliferation and extensive cell death in mesenchyme located 5-6 cell layers deep from the overlying epithelium. In contrast, the subepithelial mesenchymal layers showed normal or elevated levels of proliferation. From these results it appears that inhibition of 5-HT uptake into craniofacial epithelia may produce developmental defects by interference with serotonergic regulation of epithelial-mesenchymal interactions important for normal craniofacial morphogenesis.     

Differential effects of oestrogen on developing and mature uterine sympathetic nerves

Oestrogen is a key factor in the remodelling of uterine sympathetic nerves during puberty and the oestrous cycle; these nerves are influenced by changes in their target uterine tissue. The magnitude of oestrogen-induced responses might however be influenced by the maturation stage of sympathetic nerve fibres, the age of the neurons and/or the developmental state of the uterus. We have therefore compared the sympathetic innervation of the uterus following chronic oestrogen treatment of infantile/prepubertal and young adult intact and ovariectomised rats. Treatment of infantile/prepubertal rats resulted in the complete loss of intrauterine noradrenaline (NA)-labelled sympathetic nerves and a marked reduction in the total NA content in the uterine horn. Chronic treatment of young adult rats had little effect. To examine whether the age of the neurons or the degree of development of the uterus determined responsiveness of nerves to oestrogen, we assessed the effects of oestrogen on the sympathetic reinnervation of intraocular transplants of young adult uterine myometrium into ovariectomised adult host rats. Early treatment (10 days post-transplantation) resulted in less sympathetic innervation than late treatment (30 days post-transplantation). Measurements of nerve growth factor (NGF) levels in the uterine horn of control rats before and after puberty and following infantile/prepubertal chronic oestrogen treatment and acute oestrogen treatment of young adult rats revealed a coordinated increase between the growth of the uterus and NGF protein levels. Thus, developing and recently regrown sympathetic nerves are more susceptible to oestrogen-induced changes in the uterus than mature nerves, differential susceptibility is not related to the age of the neurons or the developmental state of the uterus and changes in NGF protein do not account for the differential susceptibility of developing and mature uterine sympathetic nerve fibres to oestrogen. Growing sympathetic fibres are more vulnerable to oestrogen than mature fibres and nerve fibres that have been in contact for longer periods with their target become less susceptible to oestrogen.     

Amino acid uptake and incorporation into macromolecules of peripheral nerves

Abstract— The uptake and incorporation of various isotopically labelled amino acids by the amphibian peripheral nerve were analysed within 5 to 24 hr after intraperitoneal injection. Amino acid was accumulated rapidly by nerve and some was incorporated into macromolecules. Nerve separated by transection from its cell bodies showed similar accumulation and incorporation. The uptake by nerve was found to be as high as that for brain and liver. A substantial amount of the isotope was recovered in the protein fraction of peripheral nerve. A lesser, but significant, amount was isolated with aminoacyl-tRNA. The contribution of specific amino acids to the nucleic acid and lipid fractions was also assessed. Finally, the significance of our findings in the understanding of nerve metabolism is discussed.     

Stimulation-depletion of serotonin and noradrenaline from vesicles of sympathetic nerves in the pineal gland of the rat

Summary The pineal gland of the rat receives a rich nervous supply originating from the superior cervical ganglia. These fibers contain serotonin in addition to their neurotransmitter, noradrenaline. Cytochemical studies at the ultrastructural level have shown that both amines are present in the cores of the granular vesicles that are characteristic of these nerves. It is presently shown that the bilateral electrical stimulation of the preganglionic fibers innervating the ganglia markedly reduces the number of small sites reacting cytochemically for both noradrenaline and serotonin, these sites corresponding to the cores of small granular vesicles, while the larger reactive sites (cores of large vesicles) remain unaltered. The vesicles are retained in nerve terminals after stimulation, as observed in conventionally processed tissues, although with altered sizes and shapes. Apart from these cytochemical and structural changes, nerve stimulation also reduces the endogenous noradrenaline content of the pineal gland. Thus, both noradrenaline and serotonin are released from their storage sites in pineal sympathetic nerves after electrical stimulation in vivo. This suggests the possibility that several substances with presumed transmitter or modulatory functions might be simultaneously released by nerve impulses from a given nerve terminal.     

Mechanical effects of compression of peripheral nerves

Effects of graded compression on nerve function were analyzed in order to evaluate the relative importance of pressure level and duration of compression for functional deterioration. The pressure was applied by means of a small inflatable cuff. The effects of two pressure levels, i.e., 80 mm Hg applied for 2 hr or 400 mm Hg applied for 15 min, were studied in rabbit tibial nerves. The lower pressure tested, which is known to induce ischemia of the compressed nerve segment, also causes some degree of mechanical deformation of the nerve trunk, which leads to incomplete recovery following pressure release. The duration of compression is of importance for the degree of nerve injury even at the higher pressure level tested.     

Effects of novel aggregation inhibitors on serotonin uptake by human blood platelets

Novel aggregation inhibitors blocked serotonin uptake by human blood platelets in concentrations ranging from 0.7 +/- 0.1 microM to 237.5 +/- 35.7 microM; a modified procedure, validated by kinetic analysis, was employed in which pH drift was minimized to 0.03 during the active assay period. Structural features in carbamoylpiperidine and nipecotoylpiperazine derivatives which actually constitute molecular probes, and show remarkable specificity for aggregation-inhibitory target sites, disclosed striking differences between the latter and serotonin receptors or other loci affecting serotonin uptake.     

Mechanism by which uptake inhibitors antagonizep-chloroamphetamine-induced depletion of brain serotonin

The mechanism by which uptake inhibitors antagonizep-chloroamphetamine (PCA)-induced depletion of brain serotonin has been suggested to involve: (1) blockade of PCA uptake into the serotonin neuron by means of the membrane carrier, or (2) blockade of the efflux of released serotonin from the neuron, this efflux also requiring the membrane carrier. According to either mechanism, antagonism of serotonin depletion by PCA would provide a valid index of inhibition of the amine uptake system on serotonin neurons in vivo.     

Morphological changes of sensory CGRP-immunoreactive and sympathetic nerves in peripheral tissues following chronic denervation

Summary The morphological relationship between sensory and sympathetic nerves was studied in tissues of the eye and the oral cavity following chronic sympathetic or sensory denervation. Immunoreactivities for calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH) were used as indexes to assess the changes of the two nerve populations after denervation.Following surgical sympathectomy, a marked increase of CGRP-containing fibres was seen in all tissues studied, while TH-imunoreactive fibres were totally depleated. Conversely, after capsaicin treatment, an increase of TH-immunoreactive nerves was found in the same tissues, concomitant with a sharp decrease of CGRP-immunoreactive nerves. These changes were particularly evident in iridial stroma and around blood vessels in all tissue, where sensory and sympathetic nerves have a closely overlapping distribution pattern.The altered proportion of sensory peptide-and catecholamine-containing nerves following sympathetic and sensory denervation suggest that there is a reciprocal trophic influence between the two nerve subsets, possibly with the intervention of neurotrophic substances such as nerve growth factor. These results indicate a close interaction between sensory peptidergic and sympathetic nervous systems in peripheral organs.     

Pulmonary vascular effects of serotonin and selective serotonin reuptake inhibitors in the late-gestation ovine fetus

Maternal use of selective serotonin (5-HT) reuptake inhibitors (SSRIs) is associated with an increased risk for persistent pulmonary hypertension of the newborn (PPHN), but little is known about 5-HT signaling in the developing lung. We hypothesize that 5-HT plays a key role in maintaining high pulmonary vascular resistance (PVR) in the fetus and that fetal exposure to SSRIs increases 5-HT activity and causes pulmonary hypertension. We studied the hemodynamic effects of 5-HT, 5-HT receptor antagonists, and SSRIs in chronically prepared fetal sheep. Brief infusions of 5-HT (3-20 μg) increased PVR in a dose-related fashion. Ketanserin, a 5-HT 2A receptor antagonist, caused pulmonary vasodilation and inhibited 5-HT-induced pulmonary vasoconstriction. In contrast, intrapulmonary infusions of GR127945 and SB206553, 5-HT 1B and 5-HT 2B receptor antagonists, respectively, had no effect on basal PVR or 5-HT-induced vasoconstriction. Pretreatment with fasudil, a Rho kinase inhibitor, blunted the effects of 5-HT infusion. Brief infusions of the SSRIs, sertraline and fluoxetine, caused potent and sustained elevations of PVR, which was sustained for over 60 min after the infusion. SSRI-induced pulmonary vasoconstriction was reversed by infusion of ketanserin and did not affect the acute vasodilator effects of acetylcholine. We conclude that 5-HT causes pulmonary vasoconstriction, contributes to maintenance of high PVR in the normal fetus through stimulation of 5-HT 2A receptors and Rho kinase activation, and mediates the hypertensive effects of SSRIs. We speculate that prolonged exposure to SSRIs can induce PPHN through direct effects on the fetal pulmonary circulation.     

Morphological changes of sensory CGRP-immunoreactive and sympathetic nerves in peripheral tissues following chronic denervation

The morphological relationship between sensory and sympathetic nerves was studied in tissues of the eye and the oral cavity following chronic sympathetic or sensory denervation. Immunoreactivities for calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH) were used as indexes to assess the changes of the two nerve populations after denervation. Following surgical sympathectomy, a marked increase of CGRP-containing fibres was seen in all tissues studied, while TH-imunoreactive fibres were totally depleated. Conversely, after capsaicin treatment, an increase of TH-immunoreactive nerves was found in the same tissues, concomitant with a sharp decrease of CGRP-immunoreactive nerves. These changes were particularly evident in iridial stroma and around blood vessels in all tissue, where sensory and sympathetic nerves have a closely overlapping distribution pattern. The altered proportion of sensory peptide- and catecholamine-containing nerves following sympathetic and sensory denervation suggest that there is a reciprocal trophic influence between the two nerve subsets, possibly with the intervention of neurotrophic substances such as nerve growth factor. These results indicate a close interaction between sensory peptidergic and sympathetic nervous systems in peripheral organs.     

Serotonin and morphogenesis. I. Sites of serotonin uptake and -binding protein immunoreactivity in the midgestation mouse embryo

The possible involvement of the neurotransmitter serotonin (5-HT) in morphogenesis of the craniofacial region in the mouse embryo has been investigated using the method of whole-embryo culture. Day-12 embryos were incubated for 3-4 h in the presence of 5-HT or its precursors L-tryptophan (L-TRP) or 5-hydroxytryptophan (5-HTP), followed by fixation, sectioning and staining with a specific antiserum to 5-HT. Sites of 5-HT immunoreactivity were found in a variety of locations in tissues of the head and neck, which are either epithelia derived from the non-neural ectoderm or are non-neuronal midline brain structures. These sites include the surface epithelia of the head, face, nasal prominences, branchial arches, oral cavity and associated parts of the nasal epithelium, the epithelium covering the eye, parts of the otic vesicle, the epiphysis and roof of the diencephalon. With the exception of the oral cavity, sites of immunoreactivity for serotonin-binding protein were identified in the mesenchyme adjacent to these sites. This mesenchyme consists of ectodermally derived neural crest cells, which are known to receive inductive influences from the epithelia with which they interact during their migration through the craniofacial region. The presence of 5-HT uptake sites in epithelia and adjacent sites of SBP in the underlying mesenchyme raises the possibility that 5-HT might be involved in those epithelial-mesenchymal interactions known to be important for the development of structures in the craniofacial region.     

Vascular effects of diphenylmethoxypiperidine-derived dopamine uptake inhibitors

Vascular effects of 4-aryl methoxypiperidinol compounds previously shown to share with cocaine the ability to inhibit the dopamine transporter are described. All the compounds tested inhibit KCl-induced and noradrenaline-dependent contractions in mesenteric arteries ex vivo. Thus, diphenylpyraline and its analogs may have a role as therapeutic options for the treatment of some of the cardiotoxic effects of cocaine intoxications.     

Anorectic activities of serotonin uptake inhibitors: correlation with their potencies at inhibiting serotonin uptake in vivo and 3H-mazindol binding in vitro

The mechanism of anorectic action of several serotonin uptake inhibitors was investigated by comparing their anorectic potencies with several biochemical and pharmacological properties and in reference to the novel compound SL 81.0385. The anorectic effect of the potent serotonin uptake inhibitor SL 81.0385 (ED50 = 4 mg/kg, i.p.) was potentiated by pretreatment with 5-hydroxytryptophan and blocked by the serotonin receptor antagonist metergoline. A good correlation (r = 0.98, p less than 0.01) was obtained between the ED50 values of anorectic action and the ED50 values of serotonin uptake inhibition in vivo (but not in vitro) for several specific serotonin uptake inhibitors. Most of the drugs tested displaced [3H]-mazindol from its binding to the anorectic recognition site in the hypothalamus, except the pro-drug zimelidine which was inactive (IC50 greater than 100 microM). Excluding zimelidine, a good correlation (r = 0.835, p less than 0.01) was obtained between the affinities of these drugs for [3H]-mazindol binding and their anorectic action indicating that their anorectic activity may be associated with an effect mediated through this site. Taken together these results suggest that the anorectic action of serotonin uptake inhibitors is directly associated to their ability to inhibit serotonin uptake and thus increasing the synaptic levels of serotonin. The interactions of these drugs with the anorectic recognition site labelled with [3H]-mazindol is discussed in connection with the serotonergic regulation of carbohydrate intake.     

Peptide YY reduces effects of sympathetic nerves and neuropeptide Y on cardiac vagal action.

In anesthetized dogs intravenous injection of neuropeptide Y (NPY) or stimulation of the cardiac sympathetic nerve is followed by a period of attenuation of vagal action at the heart lasting from many minutes to over an hour. Peptide YY (PYY), a related peptide (but one not reported to occur in the heart or its autonomic innervation), also inhibits cardiac vagal action but is more powerful and has a longer duration action. In 5 of 9 dogs, cardiac sympathetic nerve stimulation inhibited vagal action on the heart in control conditions, but relieved preexisting inhibition when repeated in the presence of PYY. In 3 dogs, exogenous NPY inhibited cardiac vagal action in control conditions, but failed to augment preexisting inhibition in the presence of PYY. An explanation offered for these results is that when PYY is occupying receptors on vagal nerve terminals, nerve-released NPY or exogenous NPY is either unable to produce an effect, because it cannot gain access to the receptors, or displaces PYY from at least some receptors and, being less powerful than PYY in its inhibitory action, lessens the preexisting vagal attenuation. The results reported are consistent with the proposal that the factor released from the sympathetic nerves following their stimulation and which is responsible for cardiac vagal inhibition is NPY.