Luigi Avallone

Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs)

The serotonin transporter gene (SLC6A4; synonyms, SERT, 5-HTT) is expressed much more broadly during development than in adulthood. To obtain a full picture of all sites of SERT expression during development we used a new mouse model where Cre recombinase was inserted into the gene encoding the serotonin transporter. Two reporter mouse lines, ROSA26R and the Tau(mGFP), allowed to map all the cells that express SERT at any point during development. Combined LacZ histochemistry and GFP immunolabelling showed neuronal cell bodies and axon fiber tracts. Earliest recombination in embryos was visible in the periphery in the heart and liver by E10.5 followed by recombination in the brain in raphe serotonergic neurons by E12.5. Further, recombination in non-serotonin neurons was visible in the choroid plexus, roof plate, and neural crest derivatives; by E15.5, recombination was found in the dorsal thalamus, cingulate cortex, CA3 field of the hippocampus, retinal ganglion cells, superior olivary nucleus and cochlear nucleus. Postnatally, SERT mediated recombination was visible in the medial prefrontal cortex and layer VI neurons in the isocortex. Recombined cells were co-labelled with Neu-N, but not with GAD67, and were characterized by long range projections (corpus callosum, fornix, thalamocortical). This fate map of serotonin transporter expressing cells emphasizes the broad expression of SERT in non-serotonin neurons during development and clarifies the localization of SERT expression in the hippocampus and limbic cortex. The identification of targets of SSRIs and serotonin releasers during embryonic and early postnatal life helps understanding the very diverse physiological consequences of administration of these drugs during development.

Genetically modified soya bean in rabbit feeding: detection of DNA fragments and evaluation of metabolic effects by enzymatic analysis

The presence of DNA fragments in tissues from rabbits given genetically modified (GM) soya-bean meal (solvent extracted) was investigated by using the polymerase chain reaction (PCR) approach. Moreover, the possible effects on cell metabolism were evaluated by determination of several specific enzymes in serum, heart, skeletal muscle, liver and kidney. The chloroplast sequence for tRNA Leu by using the Clor1/Clor2 primers designed on chloroplast trnL sequence was clearly detected. On the contrary, two couples of species specific primers for conventional (Le1-5/Le 1-3 which amplifies the soya bean lectin gene) and genetically modified (35S1/35S2 which amplifies the 35S CMV promoter that is present in the genomic structure of GM soya bean) soya bean were not found in all samples. No differences in enzyme levels were detected in serum, but a significant increase of lactic dehydrogenase, mainly concerning the LDH1 isoenzyme was found in particular in kidney and heart but not in the muscle, thus suggesting a potential alteration in the local production of the enzyme. Finally, no significant differences were detected concerning body weight, fresh organ weights and no sexual differences were detected.

Cryopreservation of feline epididymal spermatozoa from dead and alive animals and its use in assisted reproduction.

Cryopreservation of gametes is an important tool in assisted reproduction programmes; long-term storage of oocytes or spermatozoa is necessary when in vitro fertilization (IVF) or artificial insemination is to be performed at a future date. Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of endangered populations. The objectives of this work were to: (1) examine sperm motility, viability, abnormality and acrosome integrity of frozen-thawed domestic cat epididymal spermatozoa; and (2) evaluate the same cryopreservation method on wild feline spermatozoa, needed to preserve their genetic resources. Epididymides were collected from 20 domestic cats during routine neutering procedure and from two wild felines at autopsy. The sperm samples, diluted with 4% glycerol/Tris/egg yolk, were loaded into 0.25 ml mini-straws, exposed to nitrogen vapour and stored in liquid nitrogen. After 4 weeks, samples were thawed and re-evaluated. The quality of each fresh and frozen-thawed sperm sample was tested by determining the motility (54.7 +/- 11.3% and 32 +/- 13.1% respectively for cat spermatozoa; 38.3 +/- 18.7% and 21.5 +/- 16.8% respectively for tiger spermatozoa), viability (74.3 +/- 8.6% and 45.2 +/- 9.4% respectively for cat spermatozoa; 42.4 +/- 14.5% and 33.5 +/- 12.9% respectively for wild felid spermatozoa), morphology and acrosomal status. The present study showed that feline epididymal spermatozoa can be frozen in egg-yolk extender with 4.0% glycerol in 0.25 ml straws. The procedure used in the present study for epididymal cat sperm cryopreservation may be applied to bank the genetic resources of wild felid species.

Expression of orexin A and its receptor 1 in the bovine urethroprostatic complex.

Orexin A (oxA) and orexin B are recently discovered peptides derived from the proteolytic cleavage of the common precursor prepro‐orexin. They bind two G protein‐coupled receptors, defined orexin 1 (ox1R) and orexin 2 receptor. Both peptides are highly expressed in the lateral hypothalamic area of the brain and are involved in the regulation of many functions of the body, the best investigated of which is food intake. Recent data described the presence of orexins in peripheral organs such as the adrenal glands, stomach, bowel, pancreas, and testis. Here, we report the detection of oxA and ox1R in the exocrine and endocrine cytotypes of the cattle urethroprostatic complex by using immunohistochemistry. The expression of prepro‐orexin and ox1R mRNA transcripts in the prostatic tissue was assessed by reverse‐transcriptase polymerase chain reaction, while the presence of both the proteins in the tissue was confirmed by Western blotting analysis. Our findings provide the first evidence for the presence of oxA and ox1R in the urethroprostatic complex of the cattle and demonstrate that both proteins are locally synthesized, thus suggesting a role for oxA on both physiological and pathological functioning of the complex. Anat Rec, 291:169–174, 2008.

Serotonin transporter gene deficiency is associated with sudden death of newborn mice through activation of TGF-β1 signalling

The serotonin transporter (SERT) gene has been proposed as a candidate gene responsible for the sudden infant death syndrome (SIDS). In this study, for the first time we obtained a SERT-knockout (KO) mouse model which reproduces SIDS phenotype. SERT-KO mice were generated by mating SERT(Cre/+) heterozygous mice. The SERT-KO mouse embryos at the pre-natal stage E18.5 were lacking of SERT mRNA and protein expression in the heart. A premature death of 75% of SERT-KO mice occurred in the first week after birth. LacZ staining of whole mounts and tissue sections of the heart from SERT(Cre/+);ROSA26R adult mice and E18.5 embryos demonstrated a marked localized expression of SERT in the right ventricle, the conal region, the vasculature, the atrial septum, the ventricular valves, and the sinoatrial node of the conduction system. These data suggest a cardiac phenotype for the sudden death of SERT-KO mice. Histological analysis of heart sections showed that SERT-KO mice develop cardiac fibrosis. Increased collagen accumulation in the myocardium and the valvular and perivascular regions, and enhanced expression of alpha-smooth muscle actin were detected in the heart of SERT-KO mice versus wild-type (WT) mice. Interestingly, higher expression levels of the 5-HT2A receptor and increased levels of phospho-SMAD2/3 and phospho-ERK1/2 were detected in SERT-KO mouse heart versus WT mice. Overall, our findings provide i) new insights into the role of SERT gene in SIDS, and ii) the first in vivo validation of the molecular mechanism involving the activation of TGF-beta1 signalling in the cardiac fibrosis.

Expression of Orexin A and Its Receptor 1 in the Vestibular Glands of the Cattle Genital Tract

The hypothalamic peptide orexin A (oxA) binds specifically the G‐protein–coupled orexin receptor 1 (ox1R). It is involved in many physiological functions including the regulation of food intake, sleep–wake cycle, arterial blood pressure, heart rate, and sexual behavior. The localization of oxA in adrenal glands, stomach, bowel, pancreas, and testis has recently been assessed. Here, we provide the first evidence for the expression of oxA and ox1R in the vestibular glands of mammalian genital tract. Anat Rec, 2009. copy; 2008 Wiley‐Liss, Inc.

Expression of orexin A and its receptor 1 in the rat epididymis

The hypothalamic peptide orexin A (oxA) derives from the proteolytic cleavage of the precursor molecule prepro-orexin. It binds with the high affinity G-protein-coupled orexin receptor 1 (OX1R). Here, we report the detection of oxA and OX1R in the principal cells of the rat caudal epididymis by immunohistochemistry. Both oxA and OX1R immunolabelling showed cytoplasmic supranuclear localization, filling the apical portion of the cells. The expression of prepro-orexin and OX1R mRNA transcripts in the rat epididymis was assessed by reverse-transcriptase polymerase chain reaction, while the presence of both these proteins in the tissue was confirmed by Western blotting analysis. Our findings provide the evidence for the presence of oxA and OX1R in the rat epididymis, and demonstrate that both proteins are locally synthesised, thus suggesting a role for oxA in governing the fertilizing capability of the immature male gamete.

HGF/c-MET Axis in Tumor Microenvironment and Metastasis Formation

Tumor metastases are responsible for approximately 90% of all cancer-related deaths. Metastasis formation is a multistep process that requires acquisition by tumor cells of a malignant phenotype that allows them to escape from the primary tumor site and invade other organs. Each step of this mechanism involves a deep crosstalk between tumor cells and their microenvironment where the host cells play a key role in influencing metastatic behavior through the release of many secreted factors. Among these signaling molecules, Hepatocyte Growth Factor (HGF) is released by many cell types of the tumor microenvironment to target its receptor c-MET within the cells of the primary tumor. Many studies reveal that HGF/c-MET axis is implicated in various human cancers, and genetic and epigenetic gain of functions of this signaling contributes to cancer development through a variety of mechanisms. In this review, we describe the specific types of cells in the tumor microenvironment that release HGF in order to promote the metastatic outgrowth through the activation of extracellular matrix remodeling, inflammation, migration, angiogenesis, and invasion. We dissect the potential use of new molecules that interfere with the HGF/c-MET axis as therapeutic targets for future clinical trials in cancer disease.

Heart valve cardiomyocytes of mouse embryos express the serotonin transporter SERT

Multiple evidence demonstrate a role for serotonin and its transporter SERT in heart valve development and disease. By utilizing a Cre/loxP system driven by SERT gene expression, we recently demonstrated a regionally restricted distribution of SERT-expressing cells in developing mouse heart. In order to characterize the cell types exhibiting SERT expression within the mouse heart valves at early developmental stages, in this study we performed immunohistochemistry for Islet1 (Isl1) and connexin-43 (Cx-43) on heart sections from SERT(Cre/+);ROSA26R embryos previously stained with X-gal. We observed the co-localization of LacZ staining with Isl1 labelling in the outflow tract, the right ventricle and the conal region of E11.5 mouse heart. Cx-43 labelled cells co-localized with LacZ stained cells in the forming atrioventricular valves. These results demonstrate the cardiomyocyte phenotype of SERT-expressing cells in heart valves of the developing mouse heart, thus suggesting an active role of SERT in early heart valve development.

Expression of orexin A and its receptor 1 in the choroid plexuses from buffalo brain

The hypothalamic peptide orexin A, deriving from the proteolytic cleavage of the precursor molecule prepro-orexin, has a wide range of physiological effects including the regulation of feeding behaviour, neuroendocrine functions, sleep-wake cycle, and energy homeostasis. Lowered excretion of orexin A into the cerebrospinal fluid (CSF) plays a pathological role in animal and human narcolepsy. Altered levels of orexin A into the CSF have been also found in numerous disorders of the central nervous system, including Parkinsons and Huntingtons disease, dementia, and depressive disorders. While the localization of orexin A and its receptor 1, OX(1), has been elicited in many regions of the mammalian brain and in peripheral organs, there are no information on the expression of the neuropeptide and its receptor 1 in the choroid plexuses (CPs) producing the CSF. In this study, we investigated the expression of orexin A and OX(1) in the CPs from the brain of an adult mammalian species, Bubalis bubalis, by immunogold-labelling in scanning electron microscopy. Both orexin A and OX(1) immuno-reactivity appeared to be widely distributed on the surface of choroid epithelium. Interestingly, a marked orexin A labelling was detected in the areas surrounding the CP blood capillaries. The expression of prepro-orexin and OX(1) mRNA transcripts of 200 and 300 bp, respectively, was assessed in the CPs by reverse-transcription polymerase chain reaction, while Western blotting analysis confirmed the presence of these two proteins in the tissue. Our findings provide the first evidence for orexin A and OX(1) expression in the CPs from mammalian brain, and suggest that the levels of orexin A into the CSF are probably regulated by CP activity.