The existence of FGFR1-5-HT1A receptor heterocomplexes in midbrain 5-HT neurons of the rat: relevance for neuroplasticity

The ascending midbrain 5-HT neurons to the forebrain may be dysregulated in depression and have a reduced trophic support. With in situ proximity ligation assay (PLA) and supported by coimmunoprecipitation and colocation of the FGFR1 and 5-HT1A immunoreactivities in the midbrain raphe cells, evidence for the existence of FGFR1-5-HT1A receptor heterocomplexes in the dorsal and median raphe nuclei of the Sprague Dawley rat as well as in the rat medullary raphe RN33B cells has been obtained. Especially after combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA clusters was found in the RN33B cells. Similar results were reached with the FRET technique in HEK293T cells, where TM-V of the 5HT1A receptor was found to be part of the receptor interface. The combined treatment with FGF-2 and the 5-HT1A agonist also synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and the RN33B cells as well as their differentiation, as seen from development of the increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and differentiation events were dependent on the receptor interface since they were blocked by incubation with TM-V but not by TM-II. Together, the results indicate that the 5-HT1A autoreceptors by being part of a FGFR1-5-HT1A receptor heterocomplex in the midbrain raphe 5-HT nerve cells appear to have a trophic role in the central 5-HT neuron systems in addition to playing a key role in reducing the firing of these neurons

CB1 knockout mice display impaired functionality of 5-HT1A and 5-HT2A/C receptors

Interaction between brain endocannabinoid (EC) and serotonin (5-HT) systems was investigated by examining 5-HT-dependent behavioural and biochemical responses in CB1 receptor knockout mice. CB1 knockout animals exhibited a significant reduction in the induction of head twitches and paw tremor by the 5-HT2A receptor selective agonist ()DOI, as well as a reduced hypothermic response following administration of the 5-HT1A receptor agonist (±)-8-OH-DPAT. Additionally, exposure to the tail suspension test induced enhanced despair responses in CB1 knockout mice. However, the tricyclic antidepressant imipramine and the 5-HT selective reuptake inhibitor fluoxetine induced similar decreases in the time of immobility in the tail suspension test in CB1 receptor knockout and wild-type mice. No differences were found between both genotypes with regard to 5-HT2A receptor and 5-HT1A receptors levels, measured by autoradiography in different brain areas. However, a significant decrease in the ability of the 5-HT1A receptor agonist (±)-8-OH-DPAT to stimulate 35SGTPS binding was detected in the hippocampal CA1 area of CB1 receptor knockout mice. This study provides evidence that CB1 receptors are involved in the regulation of serotonergic responses mediated by 5-HT2A and 5-HT1A receptors, and suggests that a reduced coupling of 5-HT1A receptors to Gi/o proteins in the hippocampus might be involved in these effects.

The natural hallucinogen 5-MeO-DMT, component of Ayahuasca, disrupts cortical function in rats: reversal by antipsychotic drugs

5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a natural hallucinogen component of Ayahuasca, an Amazonian beverage traditionally used for ritual, religious and healing purposes that is being increasingly used for recreational purposes in US and Europe. 5MeO-DMT is of potential interest for schizophrenia research owing to its hallucinogenic properties. Two other psychotomimetic agents, phencyclidine and 2,5-dimethoxy-4-iodo-phenylisopropylamine (DOI), markedly disrupt neuronal activity and reduce the power of low frequency cortical oscillations (<4 Hz, LFCO) in rodent medial prefrontal cortex (mPFC). Here we examined the effect of 5-MeO-DMT on cortical function and its potential reversal by antipsychotic drugs. Moreover, regional brain activity was assessed by blood-oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI). 5-MeO-DMT disrupted mPFC activity, increasing and decreasing the discharge of 51 and 35% of the recorded pyramidal neurons, and reducing (−31%) the power of LFCO. The latter effect depended on 5-HT1A and 5-HT2A receptor activation and was reversed by haloperidol, clozapine, risperidone, and the mGlu2/3 agonist LY379268. Likewise, 5-MeO-DMT decreased BOLD responses in visual cortex (V1) and mPFC. The disruption of cortical activity induced by 5-MeO-DMT resembles that produced by phencyclidine and DOI. This, together with the reversal by antipsychotic drugs, suggests that the observed cortical alterations are related to the psychotomimetic action of 5-MeO-DMT. Overall, the present model may help to understand the neurobiological basis of hallucinations and to identify new targets in antipsychotic drug development.

I. Identification and characterisation of epigenetically altered tumor supressor genes by proteomic and genomic approches / II. Studies of genes regualted by MeCP2 union to its promoter regions by proteomic and genmomic approches

DNA methylation has an important impact on normal cell physiology, thus any defects in this mechanism may be related to the development of various diseases In this project we are interested in identifying epigeneticaliy modified genes, in general controlled by processes related to the DNA methylation, by means of a new strategy combining protomic and genomic analyses. First, the two Dimensional-Difference Gel Electrophoresis (2-DIGE) protein analyses of extracts obtained from HCT-116 wt and double knockout for DNMT1 and DNMT3b (DKO) cells revealed 34 proteins overexpressed in the condition of DNMTs depletion. From five genes with higher transcript lavels in DKO cells, comparing with HCT-116 wt. oniy AKR1B1, UCHLl and VIM are melhylated in HCT-116. As expected. the DNA methvlation 1s lost in DKO cells. The rneth,vl ation of VIM and UCHLl promoters in some cancer samples has already been repaired, thus further studies has been focused on AKRlBI. AKR1B1 expression due lo DNA methyiaton of promoter region seems to occur specilfically in the colon cancer cell Iines. which was confirmed in the DNA rnethylation status and expression analyses. performed on 32 different cancer cell lines (including colon, breast, lymphoma, leukemia, neuroblastoma, glioma and lung cancer cell Iines) as well as normal colon and normal lymphocytes samples. AKRIBI expression after treatments with DNA demethvlating agent (AZA) was rescued in 5 coloncancer cell lines (including genetic regulation of the candidate gene. The methylation status of the rest of the genes identified in proteomic analysis was checked by methylation specific PCR (MSP) experiment and all appeared to be unmethylated. The similar research has been done also bv means of Mecp2-null mouse model For 14 selected candidate genes the analyses of expression leveis, methylation Status and MeCP2 interaction with promoters are currently being performed.

Lack of CB1 receptor activity impairs serotonergic negative feedback

Serotonergic and endocannabinoid systems are important substrates for the control of emotional behavior and growing evidence show an involvement in the pathophysiology of mood disorders. In the present study, the absence of the activity of the CB1 cannabinoid receptor impaired serotonergic negative feedback in mice. Thus, in vivo microdialysis experiments revealed increased basal 5-HT extracellular levels and attenuated fluoxetine-induced increase of 5-HT extracellular levels in the prefrontal cortex of CB1 knockout compared to wild-type mice. These observations could be related to the significant reduction in the 5-HT transporter binding site density detected in frontal cortex and hippocampus of CB1 knockout mice. The lack of CB1 receptor also altered some 5-HT receptors related to the 5-HT feedback. Extracellular recordings in the dorsal raphe nucleus revealed that the genetic and pharmacological blockade of CB1 receptor induced a 5-HT1A autoreceptor functional desensitization. In situ hybridization studies showed a reduction in the expression of the 5-HT2C receptor within several brain areas related to the control of the emotional responses, such as the dorsal raphe nucleus, the nucleus accumbens and the paraventricular nucleus of the hypothalamus, whereas an overexpression was observed in the CA3 area of the ventral hippocampus. These results reveal that the lack of CB1 receptor induces a facilitation of the activity of serotonergic neurons in the dorsal raphe nucleus by altering different components of the 5-HT feedback as well as an increase in 5-HT extracellular levels in the prefrontal cortex in mice.

On the existence and function of galanin receptor heteromers in the central nervous system

Galanin receptor (GalR) subtypes 1-3 linked to central galanin neurons may form heteromers with each other and other types of G protein-coupled receptors in the central nervous system (CNS). These heteromers may be one molecular mechanism for galanin peptides and their N-terminal fragments (gal 1-15) to modulate the function of different types of glia-neuronal networks in the CNS, especially the emotional and the cardiovascular networks. GalR-5-HT1A heteromers likely exist with antagonistic GalR-5-HT1A receptor-receptor interactions in the ascending midbrain raphe 5-HT neuron systems and their target regions. They represent a novel target for antidepressant drugs. Evidence is given for the existence of GalR1-5-HT1A heteromers in cellular models with trans-inhibition of the protomer signaling. A GalR1-GalR2 heteromer is proposed to be a galanin N-terminal fragment preferring receptor (1-15) in the CNS. Furthermore, a GalR1-GalR2-5-HT1A heterotrimer is postulated to explain why only galanin (1-15) but not galanin (1-29) can antagonistically modulate the 5-HT1A receptors in the dorsal hippocampus rich in gal fragment binding sites. The results underline a putative role of different types of GalR-5-HT1A heteroreceptor complexes in depression. GalR antagonists may also have therapeutic actions in depression by blocking the antagonistic GalR-NPYY1 receptor interactions in putative GalR-NPYY1 receptor heteromers in the CNS resulting in increases in NPYY1 transmission and antidepressant effects. In contrast the galanin fragment receptor (a postulated GalR1-GalR2 heteromer) appears to be linked to the NPYY2 receptor enhancing the affinity of the NPYY2 binding sites in a putative GalR1-GalR2-NPYY2 heterotrimer. Finally, putative GalR-α2-adrenoreceptor heteromers with antagonistic receptor-receptor interactions may be a widespread mechanism in the CNS for integration of galanin and noradrenaline signals also of likely relevance for depression

Inhibition of glutathione efflux in the perfused rat liver and isolated hepatocytes by organic anions and bilirubin. Kinetics, sidedness, and molecular forms.

Using isolated, in situ, single-pass perfused rat livers, incubations of freshly isolated hepatocytes, and sinusoidal membrane-enriched vesicles, we and others have shown the saturability of transport (efflux) of hepatic glutathione (GSH). These observations have implicated a carrier mechanism. Our present studies were designed to provide further evidence in support of a carrier mechanism for hepatic GSH efflux by demonstrating competition by liver-specific ligands which are taken up by hepatocytes. Perfusing livers with different substances, we found that: (a) sulfobromophthalein-GSH (BSP-GSH) had a dose-dependent and fully reversible inhibitory effect on GSH efflux, while GSH alone did not have any effect; (b) taurocholate had no inhibitory effect; (c) all of the organic anions studied, i.e., BSP, rose bengal, indocyanine green, and unconjugated bilirubin (UCB), manifested potent, dose-dependent inhibitory effects, with absence of toxic effects and complete reversibility of inhibition in the case of UCB. The inhibitory effects of UCB could be overcome partially by raising (CoCl2-induced) hepatic GSH concentration. Because of the physiological importance of UCB, we conducted a detailed study of its inhibitory kinetics in the isolated hepatocyte model in the range of circulating concentrations of UCB. Studies with Cl- -free media, to inhibit the uptake of UCB by hepatocytes, showed that the inhibition of GSH efflux by UCB is apparently from inside the cell. This point was confirmed by showing that the inhibition is overcome only when bilirubin-loaded cells are cleared of bilirubin (incubation with 5% bovine serum albumin). Using Gunn rat hepatocytes and purified bilirubin mono- and diglucuronides, we found that both UCB and glucuronide forms of bilirubin inhibit GSH efflux in a dose-dependent manner. We conclude that the organic anions, although taken up by a mechanism independent of GSH, may competitively inhibit the carrier for GSH efflux from inside the hepatocyte.

Whole-rat protein content estimation: applicability of the Nx6.25 factor

The amino acid composition of the protein from three strains of rat (Wistar, Zucker lean and Zucker obese), subjected to reference and high-fat diets has been used to determine the mean empirical formula, molecular weight and N content of whole-rat protein. The combined whole protein of the rat was uniform for the six experimental groups, containing an estimate of 17.3% N and a mean aminoacyl residue molecular weight of 103.7. This suggests that the appropriate protein factor for the calculation of rat protein from its N content should be 5.77 instead of the classical 6.25. In addition, an estimate of the size of the non-protein N mass in the whole rat gave a figure in the range of 5.5 % of all N. The combination of the two calculations gives a protein factor of 5.5 for the conversion of total N into rat protein.

Toxicity of allyl esters in insect cell lines and in Spodoptera littoralis larvae

We investigated the effects of five allyl esters, two aromatic (allyl cinnamate and allyl 2-furoate) and three aliphatic (allyl hexanoate, allyl heptanoate, and allyl octanoate) in established insect cell lines derived from different species and tissues. We studied embryonic cells of the fruit fly Drosophila melanogaster (S2) (Diptera) and the beet armyworm Spodoptera exigua (Se4) (Lepidoptera), fat body cells of the Colorado potato beetle Leptinotarsa decemlineata (CPB) (Coleoptera), ovarian cells of the silkmoth Bombyx mori (Bm5), and midgut cells of the spruce budworm Choristoneura fumiferana (CF203) (Lepidoptera). Cytotoxicity was determined with use of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and trypan blue. In addition, we tested the entomotoxic action of allyl cinnamate against the cotton leafworm Spodoptera littoralis .The median (50%) cytotoxic concentrations (EC50s) of the five allyl esters in the MTT bioassays ranged between 0.25 and 27 mM with significant differences among allyl esters (P = 0.0012), cell lines (P < 0.0001), and the allyl estercell line interaction (P < 0.0001). Allyl cinnamate was the most active product, and CF203 the most sensitive cell line. In the trypan blue bioassays, cytotoxicity was produced rapidly and followed the same trend observed in the MTT bioassay. In first instars of S. littoralis, allyl cinnamate killed all larvae at 0.25% in the diet after 1 day, while this happened in third instars after 5 days. The LC50 in first instars was 0.08%. In addition, larval weight gain was reduced (P < 0.05) after 1 day of feeding on diet with 0.05%. In conclusion, the data provide evidence of the significant but differential cytotoxicity among allyl esters in insect cells of different species and tissues. Midgut cells show high sensitivity, indicating the insect midgut as a primary target tissue. Allyl cinnamate caused rapid toxic effects in S. littoralis larvae at low concentrations, suggesting further potential for use in pest control.

Effects of prolonged ethanol intake and malnutrition on rat pancreas

Nutritional factors, especially the protein and fat content of the diet, may change pancreatic morphology after ethanol induced injury. This study was performed to delineate the combined effects of a low fat diet and longterm ethanol ingestion on the rat pancreas. Male Sprague-Dawley rats were maintained with five different diets for 12 weeks and the pancreas removed on the day they were killed. Rats fed a very low fat diet without ethanol (5% of total calories as lipid) developed malnutrition, pancreatic steatosis, and reduction in zymogen granules content. Animals fed a 35% lipid diet with ethanol also developed pancreatic steatosis but changes in zymogen granules content were not detected. Both malnutrition and longterm ethanol consumption increased pancreatic cholesterol ester content, and their effects were additive. Pancreatic steatosis was accompanied with hypercholesterolaemia. Amylase, lipase, and cholesterol esterase content were reduced in malnourished rats; but longterm ethanol ingestion, regardless of the nutritional state, increased lipase content and decreased amylase. It is suggested that high serum cholesterol concentrations and increased pancreatic lipase activity could cause accumulation of cholesterol esters in acinar cells. Fat accumulation in the pancreas has been reported as the earliest histopathological feature in alcoholic patients and may be responsible for cytotoxic effects on the acinar cells at the level of the cell membrane. Although it is difficult to extrapolate results in this animal study to the human situation, the results presented in this work might explain the higher incidence of pancreatitis is malnourished populations as well as in alcoholic subjects that is reported in dietary surveys.

Effects of prolonged ethanol intake and malnutrition on rat pancreas

Nutritional factors, especially the protein and fat content of the diet, may change pancreatic morphology after ethanol induced injury. This study was performed to delineate the combined effects of a low fat diet and longterm ethanol ingestion on the rat pancreas. Male Sprague-Dawley rats were maintained with five different diets for 12 weeks and the pancreas removed on the day they were killed. Rats fed a very low fat diet without ethanol (5% of total calories as lipid) developed malnutrition, pancreatic steatosis, and reduction in zymogen granules content. Animals fed a 35% lipid diet with ethanol also developed pancreatic steatosis but changes in zymogen granules content were not detected. Both malnutrition and longterm ethanol consumption increased pancreatic cholesterol ester content, and their effects were additive. Pancreatic steatosis was accompanied with hypercholesterolaemia. Amylase, lipase, and cholesterol esterase content were reduced in malnourished rats; but longterm ethanol ingestion, regardless of the nutritional state, increased lipase content and decreased amylase. It is suggested that high serum cholesterol concentrations and increased pancreatic lipase activity could cause accumulation of cholesterol esters in acinar cells. Fat accumulation in the pancreas has been reported as the earliest histopathological feature in alcoholic patients and may be responsible for cytotoxic effects on the acinar cells at the level of the cell membrane. Although it is difficult to extrapolate results in this animal study to the human situation, the results presented in this work might explain the higher incidence of pancreatitis is malnourished populations as well as in alcoholic subjects that is reported in dietary surveys.

Effects of prolonged ethanol intake and malnutrition on rat pancreas

Nutritional factors, especially the protein and fat content of the diet, may change pancreatic morphology after ethanol induced injury. This study was performed to delineate the combined effects of a low fat diet and longterm ethanol ingestion on the rat pancreas. Male Sprague-Dawley rats were maintained with five different diets for 12 weeks and the pancreas removed on the day they were killed. Rats fed a very low fat diet without ethanol (5% of total calories as lipid) developed malnutrition, pancreatic steatosis, and reduction in zymogen granules content. Animals fed a 35% lipid diet with ethanol also developed pancreatic steatosis but changes in zymogen granules content were not detected. Both malnutrition and longterm ethanol consumption increased pancreatic cholesterol ester content, and their effects were additive. Pancreatic steatosis was accompanied with hypercholesterolaemia. Amylase, lipase, and cholesterol esterase content were reduced in malnourished rats; but longterm ethanol ingestion, regardless of the nutritional state, increased lipase content and decreased amylase. It is suggested that high serum cholesterol concentrations and increased pancreatic lipase activity could cause accumulation of cholesterol esters in acinar cells. Fat accumulation in the pancreas has been reported as the earliest histopathological feature in alcoholic patients and may be responsible for cytotoxic effects on the acinar cells at the level of the cell membrane. Although it is difficult to extrapolate results in this animal study to the human situation, the results presented in this work might explain the higher incidence of pancreatitis is malnourished populations as well as in alcoholic subjects that is reported in dietary surveys.

Effects of prolonged ethanol intake and malnutrition on rat pancreas

Nutritional factors, especially the protein and fat content of the diet, may change pancreatic morphology after ethanol induced injury. This study was performed to delineate the combined effects of a low fat diet and longterm ethanol ingestion on the rat pancreas. Male Sprague-Dawley rats were maintained with five different diets for 12 weeks and the pancreas removed on the day they were killed. Rats fed a very low fat diet without ethanol (5% of total calories as lipid) developed malnutrition, pancreatic steatosis, and reduction in zymogen granules content. Animals fed a 35% lipid diet with ethanol also developed pancreatic steatosis but changes in zymogen granules content were not detected. Both malnutrition and longterm ethanol consumption increased pancreatic cholesterol ester content, and their effects were additive. Pancreatic steatosis was accompanied with hypercholesterolaemia. Amylase, lipase, and cholesterol esterase content were reduced in malnourished rats; but longterm ethanol ingestion, regardless of the nutritional state, increased lipase content and decreased amylase. It is suggested that high serum cholesterol concentrations and increased pancreatic lipase activity could cause accumulation of cholesterol esters in acinar cells. Fat accumulation in the pancreas has been reported as the earliest histopathological feature in alcoholic patients and may be responsible for cytotoxic effects on the acinar cells at the level of the cell membrane. Although it is difficult to extrapolate results in this animal study to the human situation, the results presented in this work might explain the higher incidence of pancreatitis is malnourished populations as well as in alcoholic subjects that is reported in dietary surveys.

Contribution of femoral and proximal sciatic nerve branches to the sensory innervation of hindlimb digits in the rat

The present study was performed to investigate the possibility of 'aberrant' innervation of the tips of the hindlimb digits in the rat, i.e., from other sources than the femoral and the main sciatic branches (tibial, peroneal, sural). Cutaneous injections of fluorescent tracers in the digits were combined with either selective nerve transections to restrict afferent routes followed by detection of labeled neurons in dorsal root ganglia (DRGs), or by a delayed application of a second tracer to afferent nerves under study to detect double labeled neurons in DRGs. The results show that the tips of the digits were represented in DRGs L3-6. The femoral nerve afferents from digits 1 and 2 projected primarily to DRG L3 and to a smaller extent to DRG L4. A small number of neurons from primarily medial digits 1 and 2, but also from lateral digits 3-5, were found to project to DRGs L4 and L5 via a proximal branch that leaves the sciatic nerve near the sciatic notch and runs distally in the posterior part of the thigh, here called the musculocutaneous nerve of the hindlimb. We also have some evidence indicating innervation of the tips of the digits from the posterior cutaneous nerve of the thigh. Aberrant innervation such as that described here might contribute to remaining and perhaps abnormal sensibility after nerve injury and is of interest for the interpretation of results in experimental studies of collateral and regenerative sprouting after such injury