Data on Adiponectin from 2010 to 2020: Therapeutic Target and Prognostic Factor for Liver Diseases?

The review describes the role of adiponectin in liver diseases in the presence and absence of surgery reported in the literature in the last ten years. The most updated therapeutic strategies based on the regulation of adiponectin including pharmacological and surgical interventions and adiponectin knockout rodents, as well as some of the scientific controversies in this field, are described. Whether adiponectin could be a potential therapeutic target for the treatment of liver diseases and patients submitted to hepatic resection or liver transplantation are discussed. Furthermore, preclinical and clinical data on the mechanism of action of adiponectin in different liver diseases (nonalcoholic fatty disease, alcoholic liver disease, nonalcoholic steatohepatitis, liver cirrhosis and hepatocellular carcinoma) in the absence or presence of surgery are evaluated in order to establish potential targets that might be useful for the treatment of liver disease as well as in the practice of liver surgery associated with the hepatic resections of tumors and liver transplantation.

FGF15 improves outcomes after brain dead donor liver transplantation with steatotic and non-steatotic grafts in rats.

BACKGROUND & AIMS: Donation after brain death (DBD) grafts are associated with reduced graft quality and function post liver transplantation (LT). We aimed to elucidate i) the impact of FGF15 levels on DBD grafts; ii) whether this impact resulted from altered intestinal FXR-FGF15; iii) whether administration of FGF15 to donors after brain death could confer a benefit on graft function post LT; and iv) whether FGF15 affects bile acid (BA) accumulation. METHODS: Steatotic and non-steatotic grafts from DBD donors and donors without brain death were transplanted in rats. FGF15 was administered alone or combined with either a BA (cholic acid) or a YAP inhibitor. RESULTS: Brain death induced intestinal damage and downregulation of FXR. The resulting reduced intestinal FGF15 was associated with low hepatic FGF15 levels, liver damage and regenerative failure. Hepatic FGFR4-Klb - the receptor for FGF15 - was downregulated whereas CYP7A1 was overexpressed, resulting in BA accumulation. FGF15 administration to DBD donors increased hepatic FGFR4-Klb, reduced CYP7A1 and normalized BA levels. The benefit of FGF15 on liver damage was reversed by cholic acid, whereas its positive effect on regeneration was maintained. YAP signaling in DBD donors was activated after FGF15 treatment. When a YAP inhibitor was administered, the benefits of FGF15 on regeneration were abolished, whereas its positive effect on hepatic damage remained. Neither the Hippo-YAP-BA nor the BA-IQGAP1-YAP axis was involved in the benefits of FGF15. CONCLUSION: Alterations in the gut-liver axis contribute to the reduced quality of DBD grafts and the associated pathophysiology of LT. FGF15 pre-treatment in DBD donors protected against damage and promoted cell proliferation. LAY SUMMARY: After brain death, potential liver donors have reduced intestinal FXR, which is associated with reduced intestinal, circulatory and hepatic levels of FGF15. A similar reduction in the cell-surface receptor complex Fgfr4/Klb is observed, whereas CYP7A1 is overexpressed; together, these molecular events result in the dangerous accumulation of bile acids, leading to damage and regenerative failure in brain dead donor grafts. Herein, we demonstrate that when such donors receive appropriate doses of FGF15, CYP7A1 levels and hepatic bile acid toxicity are reduced, and liver regeneration is promoted.

Current Knowledge about the Effect of Nutritional Status, Supplemented Nutrition Diet, and Gut Microbiota on Hepatic Ischemia-Reperfusion and Regeneration in Liver Surgery.

Ischemia-reperfusion (I/R) injury is an unresolved problem in liver resection and transplantation. The preexisting nutritional status related to the gut microbial profile might contribute to primary non-function after surgery. Clinical studies evaluating artificial nutrition in liver resection are limited. The optimal nutritional regimen to support regeneration has not yet been exactly defined. However, overnutrition and specific diet factors are crucial for the nonalcoholic or nonalcoholic steatohepatitis liver diseases. Gut-derived microbial products and the activation of innate immunity system and inflammatory response, leading to exacerbation of I/R injury or impaired regeneration after resection. This review summarizes the role of starvation, supplemented nutrition diet, nutritional status, and alterations in microbiota on hepatic I/R and regeneration. We discuss the most updated effects of nutritional interventions, their ability to alter microbiota, some of the controversies, and the suitability of these interventions as potential therapeutic strategies in hepatic resection and transplantation, overall highlighting the relevance of considering the extended criteria liver grafts in the translational liver surgery.

The Role of GLP1 in Rat Steatotic and Non-Steatotic Liver Transplantation from Cardiocirculatory Death Donors.

In liver transplantation (LT), organ shortage has led to the use of steatotic and non-steatotic grafts from donors after cardiocirculatory death (DCD). However, these grafts, especially those with steatosis, exhibit poor post-operative outcomes. To address this problem, we investigated the roles of gut-derived glucagon-like peptide 1 (GLP1) and dipeptidyl peptidase 4 (DPP4), the serine protease that cleaves it, in steatotic and non-steatotic LT from DCDs. Using Zucker rats, liver grafts from DCDs were cold stored and transplanted to recipients. GLP1 was administered to donors. The levels of GLP1 in intestine and of both GLP1 and DDP4 in circulation were unaltered following cardiocirculatory death (CD). In steatotic livers from DCD, increased GLP1 and decreased DPP4 were recorded, and administration of GLP1 caused a rise in hepatic GLP1 and a reduction in DDP4. This protected against inflammation, damage, and proliferation failure. Conversely, low GLP1 and high DDP4 were observed in non-steatotic livers from DCD. The exogenous GLP1 did not modify hepatic DDP4, and the accumulated GLP1 exerted harmful effects, increasing damage, inflammation, and regeneration failure. Herein, we show that there are differences in GLP1/DDP4 regulation depending on the type of liver implanted, suggesting that GLP1 can be used as a novel and effective therapy in steatotic grafts from DCDs but that it is not appropriate for non-steatotic DCDs.

The Effect of Fibroblast Growth Factor 15 Signaling in Non-Steatotic and Steatotic Liver Transplantation from Cardiocirculatory Death.

We elucidate the relevance of fibroblast growth factor 15 (FGF15) in liver transplantation (LT) using rats with both steatotic and non-steatotic organs from donors after cardiocirculatory death (DCD). Compared to LT from non-DCDs, the induction of cardiocirculatory death (CD) increases hepatic damage, proliferation, and intestinal and circulatory FGF15. This is associated with high levels of FGF15, bilirubin and bile acids (BAs), and overexpression of the enzyme involved in the alternative BA synthesis pathway, CYP27A1, in non-steatotic livers. Furthermore, CD activates the proliferative pathway, Hippo/YAP, in these types of liver. Blocking FGF15 action in LT from DCDs does not affect CYP27A1 but causes an overexpression of CYP7A, an enzyme from the classic BA synthesis pathway, and this is related to further accumulation of BAs and exacerbated damage. FGF15 inhibition also impairs proliferation without changing Hippo/YAP. In spite of worse damage, steatosis prevents a proliferative response in livers from DCDs. In steatotic grafts, CD does not modify CYP7A1, CYP27A1, BA, or the Hippo/YAP pathway, and FGF15 is not involved in damage or proliferation. Thus, endogenous FGF15 protects against BA accumulation and damage and promotes regeneration independently of the Hippo/YAP pathway, in non-steatotic LT from DCDs. Herein we show a minor role of FGF15 in steatotic LT from DCDs.

Inflammasome-Mediated Inflammation in Liver Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury is an important cause of liver damage occurring during surgical procedures including hepatic resection and liver transplantation, and represents the main underlying cause of graft dysfunction and liver failure post-transplantation. To date, ischemia-reperfusion injury is an unsolved problem in clinical practice. In this context, inflammasome activation, recently described during ischemia-reperfusion injury, might be a potential therapeutic target to mitigate the clinical problems associated with liver transplantation and hepatic resections. The present review aims to summarize the current knowledge in inflammasome-mediated inflammation, describing the experimental models used to understand the molecular mechanisms of inflammasome in liver ischemia-reperfusion injury. In addition, a clear distinction between steatotic and non-steatotic livers and between warm and cold ischemia-reperfusion injury will be discussed. Finally, the most updated therapeutic strategies, as well as some of the scientific controversies in the field will be described. Such information may be useful to guide the design of better experimental models, as well as the effective therapeutic strategies in liver surgery and transplantation that can succeed in achieving its clinical application.

New Insights into the Liver-Visceral Adipose Axis During Hepatic Resection and Liver Transplantation.

In the last decade, adipose tissue has emerged as an endocrine organ with a key role in energy homeostasis. In addition, there is close crosstalk between the adipose tissue and the liver, since pro- and anti-inflammatory substances produced at the visceral adipose tissue level directly target the liver through the portal vein. During surgical procedures, including hepatic resection and liver transplantation, ischemia-reperfusion injury induces damage and regenerative failure. It has been suggested that adipose tissue is associated with both pathological or, on the contrary, with protective effects on damage and regenerative response after liver surgery. The present review aims to summarize the current knowledge on the crosstalk between the adipose tissue and the liver during liver surgery. Therapeutic strategies as well as the clinical and scientific controversies in this field are discussed. The different experimental models, such as lipectomy, to evaluate the role of adipose tissue in both steatotic and nonsteatotic livers undergoing surgery, are described. Such information may be useful for the establishment of protective strategies aimed at regulating the liver-visceral adipose tissue axis and improving the postoperative outcomes in clinical liver surgery.

EGF-GH Axis in Rat Steatotic and Non-steatotic Liver Transplantation From Brain-dead Donors.

BACKGROUND: We evaluated the potential dysfunction caused by changes in growth hormone (GH) levels after brain death (BD), and the effects of modulating GH through exogenous epidermal growth factor (EGF) in steatotic and nonsteatotic grafts. METHODS: Steatotic and nonsteatotic grafts from non-BD and BD rat donors were cold stored for 6 hours and transplanted to live rats. Administration of GH and EGF and their underlying mechanisms were characterized in recipients of steatotic and nonsteatotic grafts from BD donors maintained normotensive during the 6 hours before donation. Circulating and hepatic GH and EGF levels, hepatic damage, and regeneration parameters were evaluated. Recipient survival was monitored for 14 days. Somatostatin, ghrelin, and GH-releasing hormones that regulate GH secretion from the anterior pituitary were determined. The survival signaling pathway phosphoinositide-3-kinase/protein kinase B that regulates inflammation (suppressors of cytokine signaling, high-mobility group protein B1, oxidative stress, and neutrophil accumulation) was evaluated. RESULTS: BD reduced circulating GH and increased GH levels only in steatotic livers. GH administration exacerbated adverse BD-associated effects in both types of graft. Exogenous EGF reduced GH in steatotic livers, thus activating cell proliferation and survival signaling pathways, ultimately reducing injury and inflammation. However, EGF increased GH in nonsteatotic grafts, which exacerbated damage. The benefits of EGF for steatotic grafts were associated with increased levels of somatostatin, a GH inhibitor, whereas the deleterious effect on nonsteatotic grafts was exerted through increased amounts of ghrelin, a GH stimulator. CONCLUSIONS: GH treatment is not appropriate in rat liver transplant from BD donors, whereas EGF (throughout GH inhibition) protects only in steatotic grafts.

Relevance of VEGFA in rat livers subjected to partial hepatectomy under ischemia-reperfusion.

We examined the effects of VEGFA on damage and regeneration in steatotic and non-steatotic livers of rats submitted to PH under I/R, and characterized the underlying mechanisms involved. Our results indicated that VEGFA levels were decreased in both steatotic and non-steatotic livers after surgery. The administration of VEGFA increased VEGFA levels in non-steatotic livers, reducing the incidence of post-operative complications following surgery through the VEGFR2-Wnt2 pathway, independently of Id1. Unexpectedly, administration of VEGFA notably reduced VEGFA levels in steatotic livers, exacerbating damage and regenerative failure. After exogenous administration of VEGFA in steatotic animals, circulating VEGFA is sequestered by the high circulating levels of sFlt1 released from adipose tissue. Under such conditions, VEGFA cannot reach the steatotic liver to exert its effects. Consequently, the concomitant administration of VEGFA and an antibody against sFlt1 was required to avoid binding of sFlt1 to VEGFA. This was associated with high VEGFA levels in steatotic livers and protection against damage and regenerative failure, plus improvement in the survival rate via up-regulation of PI3K/Akt independently of the Id1-Wnt2 pathway. The current study highlights the different effects and signaling pathways of VEGFA in liver surgery requiring PH and I/R based in the presence of steatosis. KEY MESSAGES: VEGFA administration improves PH+I/R injury only in non-steatotic livers of Ln animals. VEGFA benefits are exerted through the VEGFR2-Wnt2 pathway in non-steatotic livers. In Ob rats, exogenous VEGFA is sequestered by circulating sFlt1, exacerbating liver damage. Therapeutic combination of VEGFA and anti-sFlt1 is required to protect steatotic livers. VEGFA+anti-sFlt1 treatment protects steatotic livers through a VEGFR2-PI3K/Akt pathway.

Mitogen Activated Protein Kinases in Steatotic and Non-Steatotic Livers Submitted to Ischemia-Reperfusion.

: We analyzed the participation of mitogen-activated protein kinases (MAPKs), namely p38, JNK and ERK 1/2 in steatotic and non-steatotic livers undergoing ischemia-reperfusion (I-R), an unresolved problem in clinical practice. Hepatic steatosis is a major risk factor in liver surgery because these types of liver tolerate poorly to I-R injury. Also, a further increase in the prevalence of steatosis in liver surgery is to be expected. The possible therapies based on MAPK regulation aimed at reducing hepatic I-R injury will be discussed. Moreover, we reviewed the relevance of MAPK in ischemic preconditioning (PC) and evaluated whether MAPK regulators could mimic its benefits. Clinical studies indicated that this surgical strategy could be appropriate for liver surgery in both steatotic and non-steatotic livers undergoing I-R. The data presented herein suggest that further investigations are required to elucidate more extensively the mechanisms by which these kinases work in hepatic I-R. Also, further researchers based in the development of drugs that regulate MAPKs selectively are required before such approaches can be translated into clinical liver surgery.

The impact of cortisol in steatotic and non-steatotic liver surgery.

The intent of this study was to examine the effects of regulating cortisol levels on damage and regeneration in livers with and without steatosis subjected to partial hepatectomy under ischaemia-reperfusion. Ultimately, we found that lean animals undergoing liver resection displayed no changes in cortisol, whereas cortisol levels in plasma, liver and adipose tissue were elevated in obese animals undergoing such surgery. Such elevations were attributed to enzymatic upregulation, ensuring cortisol production, and downregulation of enzymes controlling cortisol clearance. In the absence of steatosis, exogenous cortisol administration boosted circulating cortisol, while inducing clearance of hepatic cortisol, thus maintaining low cortisol levels and preventing related hepatocellular harm. In the presence of steatosis, cortisol administration was marked by a substantial rise in intrahepatic availability, thereby exacerbating tissue damage and regenerative failure. The injurious effects of cortisol were linked to high hepatic acethylcholine levels. Upon administering an α7 nicotinic acethylcholine receptor antagonist, no changes in terms of tissue damage or regenerative lapse were apparent in steatotic livers. However, exposure to an M3 muscarinic acetylcholine receptor antagonist protected livers against damage, enhancing parenchymal regeneration and survival rate. These outcomes for the first time provide new mechanistic insight into surgically altered steatotic livers, underscoring the compelling therapeutic potential of cortisol-acetylcholine-M3 muscarinic receptors.

The effect of cortisol in rat steatotic and non-steatotic liver transplantation from brain-dead donors.

In the present study, we examined the effects of cortisol on steatotic and non-steatotic liver grafts from brain-dead donors and characterized the underlying mechanisms involved. Non-steatotic liver grafts showed reduced cortisol and increased cortisone levels in association with up-regulation of enzymes that inactivate cortisol. Conversely, steatotic liver grafts exhibited increased cortisol and reduced cortisone levels. The enzymes involved in cortisol generation were overexpressed, and those involved in cortisol inactivation or clearance were down-regulated in steatotic liver grafts. Exogenous administration of cortisol negatively affected hepatic damage and survival rate in non-steatotic liver transplantation (LT); however, cortisol treatment up-regulated the phosphoinositide 3-kinase (PI3K)-protein kinase C (PKC) pathway, resulting in protection against the deleterious effects of brain-dead donors on damage and inflammatory response in steatotic LT as well as in increased survival of recipients. The present study highlights the differences in the role of cortisol and hepatic mechanisms that regulate cortisol levels based on the type of liver. Our findings suggest that cortisol treatment is a feasible and highly protective strategy to reduce the adverse effects of brain-dead donor livers in order to ultimately improve liver graft quality in the presence of steatosis, whereas cortisol treatment would not be recommended for non-steatotic liver grafts.

Tryptophan hydroxylase and serotonin receptor 1A expression in the retina of the sea lamprey.

The dual development of the retina of lampreys is exceptional among vertebrates and offers an interesting EvoDevo (evolutionary developmental biology) model for understanding the origin and evolution of the vertebrate retina. Only a single type of photoreceptor, ganglion cell and bipolar cell are present in the early-differentiated central retina of lamprey prolarvae. A lateral retina appears later in medium-sized larvae (about 3 years after hatching in the sea lamprey), growing and remaining largely neuroblastic until metamorphosis. In this lateral retina, only ganglion cells and optic fibers differentiate in larvae, whereas differentiation of amacrine, horizontal, photoreceptor and bipolar cells mainly takes place during metamorphosis, which gives rise to the adult retina. Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter found in the retina of vertebrates whose synthesis is mediated by the rate-limiting enzyme tryptophan hydroxylase (TPH). TPH is also the first enzyme in the biosynthetic pathways of melatonin in photoreceptor cells. The serotonin 1A receptor (5-HT1A) is a major determinant of the activity of both serotonergic cells and their targets due to its pre- and post-synaptic location. Here, we report the developmental pattern of expression of tph and 5-ht1a transcripts in the sea lamprey retina by means of in situ hybridization. In larvae, strong tph mRNA signal was observed in photoreceptors and putative ganglion cells of the central retina, and in some neuroblasts of the lateral retina. In adults, strong tph expression was observed in bipolar, amacrine and ganglion cells and in photoreceptors. In the prolarval (central) retina, all the differentiated retinal cells expressed 5-ht1a transcripts, which were not observed in undifferentiated cells. In larvae, photoreceptors, bipolar cells and ganglion cells in the central retina, and neuroblasts in the lateral retina, showed 5-ht1a expression. In the adult retina, expression of 5-ht1a transcript was mainly observed in the myoid region of both short and long photoreceptors, and was also observed in bipolar, amacrine and ganglion cells. Some 5-HT-immunoreactive amacrine cells have already been reported in the adult lamprey retina. Our study supports the serotonergic phenotype of these amacrine cells of lampreys and also suggests that other retinal neurons could synthesize serotonin at levels not detectable by immunohistochemistry. The expression of the tph transcript in retinal photoreceptors of lampreys strongly suggests that they synthesize melatonin and that this pathway appeared early and has been conserved throughout evolution in vertebrates. The expression of tph and 5-ht1a in neuroblasts also indicates that serotonin might be playing developmental roles in the larval lamprey retina.

Full anatomical recovery of the dopaminergic system after a complete spinal cord injury in lampreys.

Following a spinal injury, lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by the regeneration of descending axons from the brain and the production of new neurons in the spinal cord. Here, we aimed to analyse the changes in the dopaminergic system of the sea lamprey after a complete spinal transection by studying the changes in dopaminergic cell numbers and dopaminergic innervation in the spinal cord. Changes in the expression of the D2 receptor were also studied. We report the full anatomical regeneration of the dopaminergic system after an initial decrease in the number of dopaminergic cells and fibres. Numbers of dopaminergic cells were recovered rostrally and caudally to the site of injury. Quantification of dopaminergic profiles revealed the full recovery of the dopaminergic innervation of the spinal cord rostral and caudal to the site of injury. Interestingly, no changes in the expression of the D2 receptor were observed at time points in which a reduced dopaminergic innervation of the spinal cord was observed. Our observations reveal that in lampreys a spinal cord injury is followed by the full anatomical recovery of the dopaminergic system.

The effect of brain death in rat steatotic and non-steatotic liver transplantation with previous ischemic preconditioning.

BACKGROUND & AIMS: Most liver grafts undergoing transplantation derive from brain dead donors, which may also show hepatic steatosis, being both characteristic risk factors in liver transplantation. Ischemic preconditioning shows benefits when applied in non-brain dead clinical situations like hepatectomies, whereas it has been less promising in the transplantation from brain dead patients. This study examined how brain death affects preconditioned steatotic and non-steatotic liver grafts undergoing transplantation. METHODS: Steatotic and non-steatotic grafts from non-brain dead and brain dead-donors were cold stored for 6h and then transplanted. After 2, 4, and 16 h of reperfusion, hepatic damage was analysed. In addition, two therapeutic strategies, ischemic preconditioning and/or acetylcholine pre-treatment, and their underlying mechanisms were characterized. RESULTS: Preconditioning benefits in non-brain dead donors were associated with nitric oxide and acetylcholine generation. In brain dead donors, preconditioning generated nitric oxide but did not promote acetylcholine upregulation, and this resulted in inflammation and damage. Acetylcholine treatment in brain dead donors, through PKC, increased antioxidants and reduced lipid peroxidation, nitrotyrosines and neutrophil accumulation, altogether protecting against damage. The combination of acetylcholine and preconditioning conferred stronger protection against damage, oxidative stress and neutrophil accumulation than acetylcholine treatment alone. These superior beneficial effects were due to a selective preconditioning-mediated generation of nitric oxide and regulation of PPAR and TLR4 pathways, which were not observed when acetylcholine was administered alone. CONCLUSIONS: Our findings propose the combination of acetylcholine+preconditioning as a feasible and highly protective strategy to reduce the adverse effects of brain death and to ultimately improve liver graft quality.

Traumatic injury induces changes in the expression of the serotonin 1A receptor in the spinal cord of lampreys.

After spinal cord injury (SCI) in mammals, the loss of serotonin coming from the brainstem reduces the excitability of motor neurons and leads to a compensatory overexpression of serotonin receptors. Despite the key role of the serotonin receptor 1a in the control of locomotion, little attention has been put in the study of this receptor after SCI. In contrast to mammals, lampreys recover locomotion after a complete SCI, so, studies in this specie could help to understand events that lead to recovery of function. Here, we showed that in lampreys there is an acute increase in the expression of the serotonin 1A receptor transcript (5-ht1a) after SCI and a few weeks later expression levels go back to normal rostrally and caudally to the lesion. Overexpression of the 5-ht1a in rostral levels after SCI has not been reported in mammals, suggesting that this could be part of the plastic events that lead to the recovery of function in lampreys. The analysis of changes in 5-ht1a expression by zones (periventricular region and horizontally extended grey matter) showed that they followed the same pattern of changes detected in the spinal cord as a whole, with the exception of the caudal periventricular layer, where no significant differences were observed between control and experimental animals at any time post lesion. This suggests that different molecular signals act on the periventricular cells of the rostral and caudal regions to injury site and thus affecting their response to the injury in terms of expression of the 5-ht1a.

Role of Slit and Robo proteins in the development of dopaminergic neurons.

Dopamine plays a number of important roles in the nervous system and the dopaminergic system is affected in several brain disorders. It is therefore of great interest to study the axonal guidance systems that specifically participate in the correct establishment of dopaminergic projections during development and possibly during regenerative processes. In recent years, several reports have shown that Slits and their Robo receptors control the growth of longitudinal (both ascending and descending) mesodiencephalic dopaminergic axons to their appropriate target areas. In vitro studies have shown that Slit1, 2 and 3 are potent repellents of dopamine neurite extension. In vivo studies using both mice and zebrafish mutants for Slits and Robos have shown that Slits and Robos control the lateral and dorsoventral positioning of dopaminergic longitudinal projections during early development. In the present review, we aimed to compile the existing knowledge from both in vitro and in vivo studies on the role of Slit and Robo proteins in the development of dopaminergic neurons as a basis for future studies.

The sea lamprey tryptophan hydroxylase: new insight into the evolution of the serotonergic system of vertebrates.

Recent research has shown that at least two tryptophan hydroxylase (Tph) genes are present in gnathostome vertebrates, but it is not known when the duplication of the ancestral Tph gene took place during evolution. By their position as an out-group of gnathostomes, lampreys (agnathans) are key models to understand molecular evolution in vertebrates. Here, we report the cloning of a Tph cDNA of the sea lamprey and the pattern of Tph mRNA expression in larval and postmetamorphic (young adult) sea lampreys using in situ hybridization. Phylogenetic analysis indicated that the lamprey Tph is an orthologue of Tphs of other vertebrates and suggested that the duplication of the ancestral Tph gene occurred before the separation of agnathans and gnathostomes, although alternative hypothesis are also discussed in the present study. In the sea lamprey brain, the Tph transcript was expressed in perikarya of the pineal organ, the retina, the diencephalic and rhombencephalic nuclei reported previously with serotonin immunohistochemistry and in small cells of the spinal cord, with a pattern similar to that observed with anti-serotonin antibodies. This suggests that expression of this Tph gene is shared by all lamprey serotonergic brain populations, unlike that reported in zebrafish and mammals for their different Tph genes. However, no Tph expression was observed in peripheral serotonergic cells, which, unlike in other vertebrates, are widely distributed in lampreys. Our results suggest that the selection of Tph2 to be expressed in raphe neurons may have occurred along the line leading to gnathostomes.

Serotonin 1A receptor (5-HT1A) of the sea lamprey: cDNA cloning and expression in the central nervous system.

Serotonergic cells are among the earliest neurons to be born in the developing central nervous system and serotonin is known to regulate the development of the nervous system. One of the major targets of the activity of serotonergic cells is the serotonin 1A receptor (5-HT1A), an ancestral archetypical serotonin receptor. In this study, we cloned and characterized the 3D structure of the sea lamprey 5-HT1A, and studied the expression of its transcript in the central nervous system by means of in situ hybridization. In phylogenetic analyses, the sea lamprey 5-HT1A sequence clustered together with 5-HT1A sequences of vertebrates and emerged as an outgroup to all gnathostome sequences. In situ hybridization analysis during prolarval, larval and adult stages showed a widespread expression of the lamprey 5-ht1a transcript. In P1 prolarvae 5-ht1a mRNA expression was observed in diencephalic nuclei, the rhombencephalon and rostral spinal cord. At P2 prolarval stage the 5-ht1a expression extended to other brain areas including telencephalic regions. 5-ht1a expression in larvae was observed throughout almost all the main brain regions with the strongest expression in the olfactory bulbs, lateral pallium, striatum, preoptic region, habenula, prethalamus, thalamus, pretectum, hypothalamus, rhombencephalic reticular area, dorsal column nucleus and rostral spinal cord. In adults, the 5-ht1a transcript was also observed in cells of the subcommissural organ. Comparison of the expression of 5-ht1a between the sea lamprey and other vertebrates reveals a conserved pattern in most of the brain regions, likely reflecting the ancestral vertebrate condition.

Spontaneous regeneration of the serotonergic descending innervation in the sea lamprey after spinal cord injury.

In contrast to mammals, lampreys are capable of recovering apparently normal locomotion after complete spinal cord transection, and the spinal axons regenerate selectively in their correct paths. Descending serotonergic projections to the spinal cord play a role in the modulation of locomotion at spinal levels in both mammals and lampreys. In this study, we used combined immunofluorescence and tract-tracing techniques to show that in the sea lamprey, serotonergic descending neurons of the caudal rhombencephalon (vagal nucleus) regenerate their axons across the lesion site after complete spinal cord transection. The spinal cord of mature larval sea lampreys was transected at the level of the fifth gill, then after a recovery period of 5 months, the spinal cord was exposed again, 1 mm caudal to the injury site, and the tracer Neurobiotin(™) was applied. Double-labeled cells were observed in the caudal portion of the serotonin-immunoreactive vagal nucleus of the caudal rhombencephalon. In order to investigate whether the reinnervation was due to sprouting from axons above the injury site or to regeneration of axotomized axons, the experiments were performed again, but the tracer Fluoro-Gold(™) was applied at the time of transection. Triple-labeled cells were observed in the vagal nucleus, indicating that at least part of the reinnervation corresponds to true regeneration. This study provides a new and interesting model for investigating the intrinsic molecular mechanisms involved in regeneration of the serotonergic descending axons in vertebrates. Use of this model may provide valuable information for proposing new therapies for patients with spinal cord injury.