Serotonin Immunoreactivity Research Articles

Analgesic effects of oral Yokukansan on acute postoperative pain and involvement of the serotonin nervous system: a mouse model study.

Yokukansan, a traditional Japanese medicine (Kampo), has been widely used to treat neurosis, dementia, and chronic pain. Previous in vitro studies have suggested that Yokukansan acts as a partial agonist of the 5-HT1A receptor, resulting in amelioration of chronic pain through inhibition of nociceptive neuronal activity. However, its effectiveness for treating postoperative pain remains unknown, although its analgesic mechanism of action has been suggested to involve serotonin and glutamatergic neurotransmission. This study aimed to investigate the effect of Yokukansan on postoperative pain in an animal model. A mouse model of postoperative pain was created by plantar incision, and Yokukansan was administered orally the day after paw incision. Pain thresholds for mechanical and heat stimuli were examined in a behavioral experiment. In addition, to clarify the involvement of the serotonergic nervous system, we examined the analgesic effects of Yokukansan in mice that were serotonin-depleted by para-chlorophenylalanine (PCPA) treatment and intrathecal administration of NAN-190, 5-HT1A receptor antagonist. Orally administered Yokukansan increased the pain threshold dose-dependent in postoperative pain model mice. Pretreatment of para-chlorophenylalanine dramatically suppressed serotonin immunoreactivity in the spinal dorsal horn without changing the pain threshold after the paw incision. The analgesic effect of Yokukansan tended to be attenuated by para-chlorophenylalanine pretreatment and significantly attenuated by intrathecal administration of 2.5µg of NAN-190 compared to that in postoperative pain model mice without para-chlorophenylalanine treatment and NAN-190 administration. This study demonstrated that oral administration of Yokukansan has acute analgesic effects in postoperative pain model mice. Behavioral experiments using serotonin-depleted mice and mice intrathecally administered with a 5-HT1A receptor antagonist suggested that Yokukansan acts as an agonist at the 5-HT1A receptor, one of the serotonin receptors, to produce analgesia.

Vocal and tongue exercise in early to mid-stage Parkinson disease using the Pink1-/- rat

Vocal and swallowing deficits are common in Parkinson disease (PD). Because these impairments are resistant to dopamine replacement therapies, vocal and lingual exercise are the primary treatment, but not all individuals respond to exercise and neural mechanisms of treatment response are unclear. To explore putative mechanisms, we used the progressive Pink1-/- rat model of early to mid-stage PD and employed vocal and lingual exercises at 6- and 10-months of age in male Pink1-/- and wild type (WT) rats. We hypothesized that vocal and lingual exercise would improve vocal and tongue use dynamics and increase serotonin (5HT) immunoreactivity in related brainstem nuclei. Rats were tested at baseline and after 8 weeks of exercise or sham exercise. At early-stage PD (6 months), vocal exercise resulted in increased call complexity, but did not change intensity, while at mid-stage (10 months), vocal exercise no longer influenced vocalization complexity. Lingual exercise increased tongue force generation and reduced relative optical density of 5HT in the hypoglossal nucleus at both time points. The effects of vocal and lingual exercise at these time points are less robust than in prodromal stages observed in previous work, suggesting that early exercise interventions may yield greater benefit. Future work targeting optimization of exercise at later time points may facilitate clinical translation.

Perinatal exposure to bisphenol A or S: Effects on anxiety-related behaviors and serotonergic system

Bisphenols, synthetic organic compounds used in the production of plastics, are an extremely abundant class of Endocrine Disrupting Chemicals, i.e., exogenous chemicals or mixtures of chemicals that can interfere with any aspect of hormone action. Exposure to BPs can lead to a wide range of effects, and it is especially dangerous if it occurs during specific critical periods of life. Focusing on the perinatal exposure to BPA or its largely used substitute BPS, we investigated the effects on anxiety-related behaviors and the serotonergic system, which is highly involved in controlling these behaviors, in adult mice. We treated C57BL/6J dams orally with a dose of 4 μg/kg body weight/day (i.e., EFSA TDI) of BPA or BPS dissolved in corn oil or with vehicle alone, at the onset of mating and continued treatment until the offspring were weaned. Adult offspring of both sexes performed the elevated plus maze and the open field tests. Then, we analyzed the serotonergic system in dorsal (DR) and median (MnR) raphe nuclei by immunohistochemical techniques. Behavioral tests highlighted alterations in BPA- and BPS-treated mice, suggesting different effects of the bisphenols exposure on anxiety-related behavior in males (anxiolytic) and females (anxiogenic). The analysis of the serotonergic system highlighted a sex dimorphism in the DR only, with control females showing higher values of serotonin immunoreactivity (5-HT-ir) than control males. BPA-treated males displayed a significant increase of 5-HT-ir in all analyzed nuclei, whereas BPS-treated males showed an increase in ventral DR only. In females, both bisphenols-treated groups showed a significant increase of 5-HT-ir in dorsal DR compared to the controls, and BPA-treated females also showed a significant increase in MnR.These results provide evidence that exposure during the early phases of life to BPA or BPS alters anxiety and the raphe serotonergic neurons in a sex-dependent manner.

Diversity and evolution of serotonergic cells in taste buds of elasmobranchs and ancestral actinopterygian fish.

A subset of gustatory cells are serotonin immunoreactive (ir) in the mammalian taste bud. In the taste bud of lamprey, elongated gustatory-like cells are also serotonin-ir. In contrast, flattened serotonin-ir cells are located only in the basal region of the taste buds in the teleosts and amphibians. These serotonin-ir cells are termed as basal cells. To evaluate the evolution and diversity of serotonergic cells in the taste bud of amniote animals, we explored the distribution and morphology of serotonin-ir cells in the taste buds of ancestral actinopterygian fish (spotted gar, sturgeon, Polypterus senegalus) and elasmobranch (stingray). In all examined animals, the taste buds contained serotonin-ir cells in their basal part. The number of serotonin-ir basal cells in each taste bud was different between these fish species. They were highest in the stingray and decreased in the order of the Polypterus, sturgeon, and gar. While serotonin immunoreactivity was observed only in the basal cells in the taste buds of the ancestral actinopterygian fish, some elongated cells were also serotonin-ir in addition to the basal cells in the stingray taste buds. mRNA of tryptophan hydroxylase 1 (tph1), a rate-limiting enzyme of the serotonin synthesis, is expressed in both the elongated and basal cells of stingray taste buds, indicating that these cells synthesize the serotonin by themselves. These results suggest that the serotonin-ir basal cells arose from the ancestor of the cartilaginous fish, and serotonin-ir cells in the elasmobranch taste bud exhibit an intermediate aspect between the lamprey and actinopterygian fish.

Sedative-Hypnotic Effects of Glycine max Merr. Extract and Its Active Ingredient Genistein on Electric-Shock-Induced Sleep Disturbances in Rats.

Glycine max Merr. (GM) is a functional food that provides many beneficial phytochemicals. However, scientific evidence of its antidepressive and sedative activities is scarce. The present study was designed to investigate the antidepressive and calmative effects of GM and its biologically active compound, genistein (GE), using electroencephalography (EEG) analysis in an electric foot shock (EFS)-stressed rat. The underlying neural mechanisms of their beneficial effects were determined by assessing corticotropin-releasing factor (CRF), serotonin (5-HT), and c-Fos immunoreactivity in the brain using immunohistochemical methods. In addition, the 5-HT2C receptor binding assay was performed because it is considered a major target of antidepressants and sleep aids. In the binding assay, GM displayed binding affinity to the 5-HT2C receptor (IC50 value of 14.25 ± 11.02 µg/mL). GE exhibited concentration-dependent binding affinity, resulting in the binding of GE to the 5-HT2C receptor (IC50, 77.28 ± 26.57 mg/mL). Administration of GM (400 mg/kg) increased non-rapid eye movement (NREM) sleep time. Administration of GE (30 mg/kg) decreased wake time and increased rapid eye movement (REM) and NREM sleep in EPS-stressed rats. In addition, treatment with GM and GE significantly decreased c-Fos and CRF expression in the paraventricular nucleus (PVN) and increased 5-HT levels in the dorsal raphe in the brain. Overall, these results suggest that GM and GE have antidepressant-like effects and are effective in sleep maintenance. These results will benefit researchers in developing alternatives to decrease depression and prevent sleep disorders.

Gastroesophageal tube of the Iguana iguana (Iguanidae): histological description, histochemical and immunohistochemical analysis of 5-HT and SS cells.

The work aims were to describe the histological and histochemical structure of the gastroesophageal tube of Iguana iguana and verify the occurrence and distribution of immunoreactive serotonin (5-HT) and somatostatin (SS) cells. Fragments of the gastrointestinal tract (GIT) of five iguanas were which underwent standard histological and immunohistochemistry technique. Immunoreactive cells for 5-HT and SS were quantified using the STEPanizer. The oesophagus has ciliated columnar pseudostratified epithelium with staining Alcian blue (AB) + and goblet cells highly reactive to periodic acid Schiff (PAS). In the cervical oesophagus, the numerical density of 5-HT cells per unit area (QA [5-HT cells]/µm2) was 4.6x10-2 ± 2.0 and celomatic oesophagus presented QA = 4.0x10-2 ± 1.0. The epithelium of the stomach is simple columnar, PAS and AB +. The cranial and middle regions of the stomach presented (QA [5-HT cells]/µm2) = 6.18x10-2 ± 3.2 and the caudal region, QA = 0.6x10-2 ± 0.2. The SS cells were only observed in the caudal stomach, with numerical density (QA [SS cells]/µm2) = 1.4x10-2 ± 0.9 In I. iguana, variation was observed in terms of the distribution of mucus secretions and the pattern of occurrence of serotonin and somatostatin-secreting enteroendocrine cells in the TGI, which possibly will result in an interspecific adaptive response.

Naringenin modulates Cobalt activities on gut motility through mechanosensors and serotonin signalling

Introduction Cobalt chloride-(CoCl2) exerts beneficial and toxic activities depending on dose however Naringenin-(Nar) a flavonoid, chelates heavy metals. Absorption of ingested heavy metals, or chelates are dependent on gut motility (gastric emptying and intestinal transit time) and mechanosensor regulation. Literature is vague on CoCl2 activities on gut motility and mechanosensor nor probable chelating actions with naringenin which was investigated in this study. Method One hundred male Wistar rats were grouped viz; A to D (25, 62, 150 and 300 mg/kg CoCl2), E to H doses of CoCl2+Nar (50 mg/kg), I-Narigenin and J-Control. Gastric emptying and intestinal transit time were evaluated by day eight, intestinal tissue assayed for biochemical, histological and immunohistochemistry reactivity. Result CoCl2 significantly increased Gastric emptying (150 and 300 mg/kg) and Intestinal transit time unlike Naringenin. CoCl2 (150 mg/kg) significantly increased Catalase and Nitric oxide but ameliorated by Naringenin. ATPase activities significantly increased in 150 mg/kg-CoCl2 but ameliorated by Naringenin. Carbonyl levels increased in all CoCl2+Nar groups. High Enterochromaffin-cell count in 25 and 62 mg/kg-CoCl2 were ameliorated by Naringenin. Serotonin immunoreactivity increased in CoCl2 (25, 62, 300 mg/kg) but reduced in CoCl2+Nar groups. Conclusion Cobalt chloride enhanced gastric motility via increased mechanosensor activities and serotonin expression at low doses. Naringenin ameliorated toxicity of high cobalt chloride via metal-flavonoid chelates.

Neurochemical classification of serotonin-immunoreactive neurons co-innervating motor endplates in the mouse esophagus.

Serotonin immunoreactivity was previously found in myenteric neurons co-innervating motor endplates in the mouse esophagus striated muscle and an involvement in motility control was suggested. However, it is not known if other neuroactive substances are present in these neurons and to what extent they co-localize. First, vasoactive intestinal peptide (VIP) was established as a bona fide marker for putative inhibitory myenteric neurons by evaluating co-localization with neuronal nitric oxide synthase (nNOS) and neuropeptide Y (NPY). Then, co-localization of serotonin and VIP was tested in co-innervating axons on motor endplates, which were visualized with α-bungarotoxin (α-BT) by multilabel immunofluorescence. Myenteric ganglia were also surveyed for co-localization in neuronal perikarya and varicosities. nNOS, NPY, and VIP were completely co-localized in enteric co-innervating nerve terminals on motor endplates. After co-staining with VIP, we found (a) serotonin (5-HT)-positive nerve endings without VIP (44% of 5-HT-positively innervated endplates), (b) 5-HT- and VIP-positive endings without co-localization (35%), and (c) 5-HT- and VIP-positive endings with co-localization (21%). About one-fifth of nerve terminals on motor endplates containing 5-HT originate from putative inhibitory peptidegic nitrergic neurons. However, the majority represents a different population presumably subserving different functions.

In Transgenic Erythropoietin Deficient Mice, an Increase in Respiratory Response to Hypercapnia Parallels Abnormal Distribution of CO2/H+-Activated Cells in the Medulla Oblongata.

Erythropoietin (Epo) and its receptor are expressed in central respiratory areas. We hypothesized that chronic Epo deficiency alters functioning of central respiratory areas and thus the respiratory adaptation to hypercapnia. The hypercapnic ventilatory response (HcVR) was evaluated by whole body plethysmography in wild type (WT) and Epo deficient (Epo-TAgh) adult male mice under 4%CO2. Epo-TAgh mice showed a larger HcVR than WT mice because of an increase in both respiratory frequency and tidal volume, whereas WT mice only increased their tidal volume. A functional histological approach revealed changes in CO2/H+-activated cells between Epo-TAgh and WT mice. First, Epo-TAgh mice showed a smaller increase under hypercapnia in c-FOS-positive number of cells in the retrotrapezoid nucleus/parafacial respiratory group than WT, and this, independently of changes in the number of PHOX2B-expressing cells. Second, we did not observe in Epo-TAgh mice the hypercapnic increase in c-FOS-positive number of cells in the nucleus of the solitary tract present in WT mice. Finally, whereas hypercapnia did not induce an increase in the c-FOS-positive number of cells in medullary raphe nuclei in WT mice, chronic Epo deficiency leads to raphe pallidus and magnus nuclei activation by hyperacpnia, with a significant part of c-FOS positive cells displaying an immunoreactivity for serotonin in the raphe pallidus nucleus. All of these results suggest that chronic Epo-deficiency affects both the pattern of ventilatory response to hypercapnia and associated medullary respiratory network at adult stage with an increase in the sensitivity of 5-HT and non-5-HT neurons of the raphe medullary nuclei leading to stimulation of f R for moderate level of CO2.

Immunohistochemical and ultrastructural study of the immune cell system and epithelial surfaces of the respiratory organs in the bimodally breathing African sharptooth catfish (Clarias gariepinus Burchell, 1822).

Acetylcholine (Ach) represents the old neurotransmitter in central and peripheral nervous system. Its muscarinic and nicotinic receptors (mAChRs and nAChRs) constitute an independent cholinergic system that is found in immune cells and play a key role in the regulation of the immune function and cytokine production. Gas exchanging surfaces of the gills and air-breathing organs (ABOs) of the sharptooth catfish Clarias gariepinus were investigated using ultrastructural and confocal immunofluorescence techniques. This study was predominantly focused on the structure of the immune cell types, the expression of their neurotransmitters, including the antimicrobial peptide piscidin 1, and the functional significance of respiratory gas exchange epithelia. A network of immune cells (monocytes, eosinophils, and mast cells) was observed in the gill and the ABO epithelia. Eosinophils containing 5-hydroxytryptamine (5-HT) immunoreactivity were seen in close association with mast cells expressing acetylcholine (Ach), 5-HT, neuronal nitric oxide synthase, and piscidin 1. A rich and dense cholinergic innervation dispersing across the islet capillaries of the gas exchange barrier and the localization of Ach in the squamous pavement cells covering the capillaries were evidenced byVAChT antibodies. We report for the first time that piscidin 1 (Pis 1)-positive mast cells interact with Pis 1-positive nerves found in the epithelia of the respiratory organs. Pis 1 immunoreactivity was also observed in the covering respiratory epithelium of the ABOs and associated with a role in local mucosal immune defense. The above results anticipate future studies on the neuro-immune interactions at mucosal barrier surfaces, like the gill and the skin of fish, areas densely populated by different immune cells and sensory nerves that constantly sense and adapt to tissue-specific environmental challenges.

Abnormal brain development of monoamine oxidase mutant zebrafish and impaired social interaction of heterozygous fish

ABSTRACTMonoamine oxidase (MAO) deficiency and imbalanced levels of brain monoamines have been associated with developmental delay, neuropsychiatric disorders and aggressive behavior. Animal models are valuable tools to gain mechanistic insight into outcomes associated with MAO deficiency. Here, we report a novel genetic model to study the effects of mao loss of function in zebrafish. Quantitative PCR, in situ hybridization and immunocytochemistry were used to study neurotransmitter systems and expression of relevant genes for brain development in zebrafish mao mutants. Larval and adult fish behavior was evaluated through different tests. Stronger serotonin immunoreactivity was detected in mao+/− and mao−/− larvae compared with their mao+/+ siblings. mao−/− larvae were hypoactive, and presented decreased reactions to visual and acoustic stimuli. They also had impaired histaminergic and dopaminergic systems, abnormal expression of developmental markers and died within 20 days post-fertilization. mao+/− fish were viable, grew until adulthood, and demonstrated anxiety-like behavior and impaired social interactions compared with adult mao+/+ siblings. Our results indicate that mao−/− and mao+/− mutants could be promising tools to study the roles of MAO in brain development and behavior. This article has an associated First Person interview with the first author of the paper.

Prolongation of absence seizures and changes in serotonergic and dopaminergic neurotransmission by nigrostriatal pathway degeneration in genetic absence epilepsy rats

ObjectiveBasal ganglia structures play an important role in the pathophysiology of absence epilepsy, known as remote control of absence seizures. We examined the role of the nigrostriatal dopaminergic pathway in absence epilepsy through behavioral and electroencephalography (EEG) parameters, immunohistochemical, and biochemical characteristics of dopamine and serotonin in the genetic absence epilepsy rat model. MethodsThe nigrostriatal pathway was degenerated by the injection of chemical 6-hydroxydopamine hydrobromide (6-OHDA) into the medial forebrain bundle (MFB) in Wistar and genetic absence epilepsy rats from Strasbourg (GAERS). On the 21st day after stereotaxic surgery, spike-and-wave discharges (SWDs) on EEG were recorded in GAERS groups. Thereafter, Wistar-Control, GAERS-Control, Wistar-6OHDA, GAERS-6OHDA rats were subjected to the cylinder and apomorphine-induced rotation tests. Dopaminergic or serotonergic immunoreactivity was examined in the cortex, striatum, and substantia nigra pars compacta. High-performance liquid chromatography method was used for biochemical analysis of dopamine and serotonin in the cortex and thalamus. ResultsIn behavioral analysis, the number of rotations in the GAERS-6OHDA group was significantly higher than in Wistar-6OHDA rats. The degeneration of the nigrostriatal dopaminergic pathway produced a significant increase in the cumulative duration of SWDs and the duration of each SWD in GAERS-6OHDA rats. GAERS-Control rats displayed significantly higher cortical and striatal serotonin immunoreactivity and cortical serotonin level compared to Wistar-Control animals. Moreover, cortical and striatal serotonin immunoreactivity and cortical serotonin levels increased in Wistar-6OHDA and GAERS-6OHDA groups compared to their control groups. SignificanceThe effect of 6-OHDA-induced MFB lesion on absence epilepsy was examined for the first time by comparing Wistar and GAERS rats. The nigrostriatal dopaminergic pathway as a part of the remote-control system is likely to participate in the seizure network.

Cortical Serotonergic and Catecholaminergic Denervation in MPTP-Treated Parkinsonian Monkeys.

Decreased cortical serotonergic and catecholaminergic innervation of the frontal cortex has been reported at early stages of Parkinson's disease (PD). However, the limited availability of animal models that exhibit these pathological features has hampered our understanding of the functional significance of these changes during the course of the disease. In the present study, we assessed longitudinal changes in cortical serotonin and catecholamine innervation in motor-symptomatic and asymptomatic monkeys chronically treated with low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Densitometry and unbiased stereological techniques were used to quantify changes in serotonin and tyrosine hydroxylase (TH) immunoreactivity in frontal cortices of 3 control monkeys and 3 groups of MPTP-treated monkeys (motor-asymptomatic [N = 2], mild parkinsonian [N = 3], and moderate parkinsonian [N = 3]). Our findings revealed a significant decrease (P<0.001) in serotonin innervation of motor (Areas 4 and 6), dorsolateral prefrontal (Areas 9 and 46), and limbic (Areas 24 and 25) cortical areas in motor-asymptomatic MPTP-treated monkeys. Both groups of symptomatic MPTP-treated animals displayed further serotonin denervation in these cortical regions (P<0.0001). A significant loss of serotonin-positive dorsal raphe neurons was found in the moderate parkinsonian group. On the other hand, the intensity of cortical TH immunostaining was not significantly affected in motor asymptomatic MPTP-treated monkeys, but underwent a significant reduction in the moderate symptomatic group (P<0.05). Our results indicate that chronic intoxication with MPTP induces early pathology in the corticopetal serotonergic system, which may contribute to early non-motor symptoms in PD.

Repeated Seizure Exposure in the SS Kcnj16‐/‐ Rat Causes Progressive Respiratory Suppression and Associated Brainstem Pathology

Kir5.1 is an inwardly rectifying potassium (Kir) channel subunit highly expressed in the kidney and brain and encoded by the Kcnj16 gene. Knockout of Kcnj16 in a Dahl salt-sensitive rat (SSKcnj16-/-) results in susceptibility to audiogenic, generalized tonic-clonic seizures (GTCSs), which when repeated lead to spontaneous mortality. Patients with uncontrolled epilepsy are at high risk for Sudden Unexplained Death in Epilepsy (SUDEP), hypothesized to be a result of severe cardiorespiratory suppression and/or failure. We hypothesized that repeated audiogenic seizures in SSKcnj16-/- rats lead to progressively worsening cardiorespiratory suppression and associated brainstem pathology. SSKcnj16-/- and wild-type (WT) rats (10-12 weeks of age) were exposed to the seizure-inducing acoustic stimulus 1/day (d) for 10d and breathing measurement were made before, during and after exposure. Solitary GTSCs acutely led to apnea and decreases in breathing frequency for ~5 min postictally, with no change or an increase in total ventilation due to an increase in tidal volume. We also found a significant reduction in breath-to-breath variability throughout the postictal period. After exposure to repeated seizures, postictal frequency suppression became more pronounced and compensatory increases in tidal volume were reduced, such that total ventilation was also significantly reduced. Repeated seizures also further attenuated the ventilatory response to hypercapnia (p=0.0073). To gain insights into the effects of repeated seizures on cardiorespiratory circuits in the brainstem, we used immunofluorescence staining/confocal imaging to label and quantify total serotonin (5-HT) immunoreactivity (ir), serotonergic neurons (tryptophan hydroxylase+, TPH), microglia (Iba1), astrocytes (GFAP), total neurons (NeuN), and total nuclei (DAPI) in respiratory brainstem nuclei of SSkcnj16-/- rats exposed to 0, 3, or 10 seizures (n=5/group). Repeated seizures resulted in reduced 5-HT-ir in multiple cardiorespiratory brainstem nuclei including the pre-Bötzinger Complex (pre-BötC), nucleus ambiguous (NA), and nucleus tractus solitarious (NTS) in addition to a decrease in 5-HT neurons. We also found increased Iba1-ir selectively within the pre-BötC and NA, whereas GFAP-ir was increased after 10 seizures in these same regions. This data suggests that inflammation and/or serotonergic dysregulation in cardiorespiratory brainstem nuclei may mediate progressive respiratory suppression following repeated seizures in this rat model.

The neuromuscular system in flatworms: serotonin and FMRFamide immunoreactivities and musculature in Prodistomum alaskense (Digenea: Lepocreadiidae), an endemic fish parasite of the northwestern Pacific

Using the immunocytochemical method and confocal scanning laser microscopy, we obtained pioneering data on the muscle system organization and presence and localization of biogenic amine serotonin and FMRFamide-related peptides in the nervous system of the trematode Prodistomum alaskense (Ward and Fillingham, 1934) Bray and Merrett 1998 (family Lepocreadiidae). This flatworm is an intestinal parasite of endemic representatives of the marine fauna of the northwestern Pacific Ocean — the prowfish (Zaprora silenus Jordan, 1896) and the lumpfish (Aptocyclus ventricosus (Pallas, 1769)). We provide data of scanning electron microscopy on the tegumental topography of P. alaskense. The body wall musculature of P. alaskense has three layers of muscle fibres — the outer circular, intermediate longitudinal, and inner diagonal. The muscle system elements are well developed in the attachment organs, digestive and reproductive systems, and in the excretory sphincter. Serotonin– and FMRFamide–immunopositive neurons and neurites are found in the head ganglia, circular commissure, longitudinal nerve cords, and in the transversal connective commissures. The innervation of the oral and ventral suckers, pharynx, and the reproductive system compartments by the serotonergic and FMRFamide–immunopositive neurites is revealed. The results discus connection with the published data on the presence and functional roles of the serotonin and FMRFamide-related peptides in Platyhelminthes.

Zygotic Venlafaxine Exposure Impacts Behavioral Programming by Disrupting Brain Serotonin in Zebrafish.

The antidepressant venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is present in surface waters downstream of wastewater treatment plants. We previously showed that zygotic venlafaxine deposition alters larval behavior in zebrafish (Danio rerio), but the mechanisms were unknown. Here we tested the hypothesis that venlafaxine disrupts central serotonergic development, leading to impaired behavioral responses in zebrafish larvae. This was tested by microinjecting embryos with venlafaxine immediately after fertilization and performing spatial distribution of serotonin immunoreactivity, as well as characterizing target genes involved in serotonin turnover in the zebrafish brain. We provide evidence that venlafaxine exposure reduces serotonin immunoreactivity and tyrosine hydroxylase-positive cell populations in specific larval brain regions, and this corresponded with reduced larval activity observed in the drug-exposed group. Lowered serotonin was not due to either reduced synthesis or increased breakdown capacity. However, co-injection of serotonin alongside venlafaxine in embryos recovered brain serotonin immunoreactivity, tyrosine hydroxylase-positive cell populations, and rescued venlafaxine-mediated behavioral changes. Overall, our results demonstrate for the first time that early life exposure to venlafaxine perturbs brain development, which may be due to reduced serotonin, leading to altered larval behavior in zebrafish.

Dorsal Horn of Mouse Lumbar Spinal Cord Imaged with CLARITY.

The organization of the mouse spinal dorsal horn has been delineated in 2D for the six Rexed laminae in our publication Atlas of the Spinal Cord: Mouse, Rat, Rhesus, Marmoset, and Human. In the present study, the tissue clearing technique CLARITY was used to observe the cyto- and chemoarchitecture of the mouse spinal cord in 3D, using a variety of immunohistochemical markers. We confirm prior observations regarding the location of glycine and serotonin immunoreactivities. Novel observations include the demonstration of numerous calcitonin gene-related peptide (CGRP) perikarya, as well as CGRP fibers and terminals in all laminae of the dorsal horn. We also observed sparse choline acetyltransferase (ChAT) immunoreactivity in small perikarya and fibers and terminals in all dorsal horn laminae, while gamma aminobutyric acid (GABA) and glutamate decarboxylase-67 (GAD67) immunoreactivities were found only in small perikarya and fibers. Finally, numerous serotonergic fibers were observed in all laminae of the dorsal horn. In conclusion, CLARITY confirmed the 2D immunohistochemical properties of the spinal cord. Furthermore, we observed novel anatomical characteristics of the spinal cord and demonstrated that CLARITY can be used on spinal cord tissue to examine many proteins of interest.

Nerves innervating copulatory organs show common FMRFamide, FVRIamide, MIP and serotonin immunoreactivity patterns across Dinophilidae (Annelida) indicating their conserved role in copulatory behaviour

BackgroundMales of the microscopic annelid family Dinophilidae use their prominent glandomuscular copulatory organ (penis) to enzymatically dissolve the female’s epidermis and thereafter inject sperm. In order to test for putative conserved copulatory structures and neural orchestration across three dinophilid species, we reconstructed the reproductive myo- and neuroanatomy and mapped immunoreactivity patterns against two specific neurotransmitter markers with reported roles in invertebrate male mating behaviour (FVRIamide, MIP) and three general neural markers (acetylated α-tubulin, serotonin, FMRFamide).ResultsSeminal vesicles (one or two pairs), surrounded by a thin layer of longitudinal and circular muscles and innervated by neurites, are found between testes and copulatory organ in the larger males of Dinophilus vorticoides and Trilobodrilus axi, but are missing in the only 0.05 mm long D. gyrociliatus dwarf males. The midventral copulatory organ is in all species composed of an outer muscular penis sheath and an inner penis cone. Neurites encircle the organ equatorially, either as a ring-shaped circumpenial fibre mass or as dorsal and ventral commissures, which are connected to the ventrolateral nerve cords. All three examined dinophilids show similar immunoreactivity patterns against serotonin, FMRFamide, and FVRIamide in the neurons surrounding the penis, supporting the hypotheses about the general involvement of these neurotransmitters in copulatory behaviour in dinophilids. Immunoreactivity against MIP is restricted to the circumpenial fibre mass in D. gyrociliatus and commissures around the penis in T. axi (but not found in D. vorticoides), indicating its role in controlling the copulatory organ.ConclusionsThe overall myo- and neuroanatomy of the reproductive organs is rather similar in the three studied species, suggesting a common ancestry of the unpaired glandomuscular copulatory organ and its innervation in Dinophilidae. This is furthermore supported by the similar immunoreactivity patterns against the tested neurotransmitters around the penis. Smaller differences in the immunoreactivity patterns around the seminal vesicles and spermioducts might account for additional, individual traits. We thus show morphological support for the putatively conserved role of FMRFamide, FVRIamide, MIP and serotonin in dinophilid copulatory behaviour.

Transglutaminase Activity Determines Nuclear Localization of Serotonin Immunoreactivity in the Early Embryos of Invertebrates and Vertebrates.

Serotonin (5-HT) is a key player in many physiological processes in both the adult organism and developing embryo. One of the mechanisms for 5-HT-mediated effects is covalent binding of 5-HT to the target proteins catalyzed by transglutaminases (serotonylation). Despite the implication in a variety of physiological processes, the involvement of serotonylation in embryonic development remains unclear. Here we tested the hypothesis that 5-HT serves as a substrate for transglutaminase-mediated transamidation of the nuclear proteins in the early embryos of both vertebrates and invertebrates. For this, we demonstrated that the level of serotonin immunoreactivity (5-HT-ir) in cell nuclei increases upon the elevation of 5-HT concentration in embryos of sea urchins, mollusks, and teleost fish. Consistently, pharmacological inhibition of transglutaminase activity resulted in the reduction of both brightness and nuclear localization of anti-5-HT staining. We identified specific and bright 5-HT-ir within nuclei attributed to a subset of different cell types: ectodermal and endodermal, macro- and micromeres, and blastoderm. Western blot and dot blot confirmed the presence of 5-HT-ir epitopes in the normal embryos of all the species examined. The experimental elevation of 5-HT level led to the enhancement of 5-HT-ir-related signal on blots in a species-specific manner. The obtained results demonstrate that 5-HT is involved in transglutaminase-dependent monoaminylation of nuclear proteins and suggest nuclear serotonylation as a possible regulatory mechanism during early embryonic development. The results reveal that this pathway is conserved in the development of both vertebrates and invertebrates.

Motor cortex and pain control: exploring the descending relay analgesic pathways and spinal nociceptive neurons in healthy conscious rats

Motor cortex stimulation (MCS) is an effective therapy for refractory neuropathic pain. MCS increases the nociceptive threshold in healthy rats via endogenous opioids, inhibiting thalamic nuclei and activating the periaqueductal gray. It remains unclear how the motor cortex induces top-down modulation of pain in the absence of persistent pain. Here, we investigated the main nuclei involved in the descending analgesic pathways and the spinal nociceptive neurons in rats that underwent one session of MCS and were evaluated with the paw pressure nociceptive test. The pattern of neuronal activation in the dorsal raphe nucleus (DRN), nucleus raphe magnus (NRM), locus coeruleus (LC), and dorsal horn of the spinal cord (DHSC) was assessed by immunoreactivity (IR) for Egr-1 (a marker of activated neuronal nuclei). IR for serotonin (5HT) in the DRN and NRM, tyrosine hydroxylase (TH) in the LC, and substance P (SP) and enkephalin (ENK) in the DHSC was also evaluated. MCS increased the nociceptive threshold of the animals; this increase was accompanied by activation of the NRM, while DRN activation was unchanged. However, cortical stimulation induced an increase in 5HT-IR in both serotonergic nuclei. MCS did not change the activation pattern or TH-IR in the LC, and it inhibited neuronal activation in the DHSC without altering SP or ENK-IR. Taken together, our results suggest that MCS induces the activation of serotonergic nuclei as well as the inhibition of spinal neurons, and such effects may contribute to the elevation of the nociceptive threshold in healthy rats. These results allow a better understanding of the circuitry involved in the antinociceptive top-down effect induced by MCS under basal conditions, reinforcing the role of primary motor cortex in pain control.