Microtubule-associated protein 1a is involved in the early development of the rat spinal cord.

The expression of microtubule-associated protein 1a (MAP1a) in the developing rat spinal cord was studied using the monoclonal antibody BW6. Immunoblots of microtubule preparations revealed the presence of MAP1a in spinal cord tissue of rats aged embryonal day 16 and postnatal day 0. The spinal cord matrix layer, between embryonal days 12-17, displayed a pattern of MAP1a-positive processes, horizontally oriented in between the membrane limitans interna and externa. The mantle layer stained intensely for MAP1a between embryonal day 12 and postnatal day 2. MAP1a was found in neuronal cell bodies, axons and dendrites, located mainly in the ventral and intermediate mantle layer. In the marginal layer, MAP1a-positive axons could be observed between embryonal days 14-18. During further development, the intensity of the MAP1a staining in the spinal columns gradually decreased. These expression patterns indicate an involvement of MAP1a in the proliferation and differentiation of neuroblasts, and the maturation of the long spinal fiber systems, i.e. early events in spinal cord development

Tumor necrosis factor-alpha mediates changes in tissue protein turnover in a rat cancer cachexia model.

Rats bearing the Yoshida AH-130 ascites hepatoma showed enhanced fractional rates of protein degradation in gastrocnemius muscle, heart, and liver, while fractional synthesis rates were similar to those in non-tumor bearing rats. This hypercatabolic pattern was associated with marked perturbations of the hormonal homeostasis and presence of tumor necrosis factor in the circulation. The daily administration of a goat anti-murine TNF IgG to tumor-bearing rats decreased protein degradation rates in skeletal muscle, heart, and liver as compared with tumor-bearing rats receiving a nonimmune goat IgG. The anti-TNF treatment was also effective in attenuating early perturbations in insulin and corticosterone homeostasis. Although these results suggest that tumor necrosis factor plays a significant role in mediating the changes in protein turnover and hormone levels elicited by tumor growth, the inability of such treatment to prevent a reduction in body weight implies that other mediators or tumor-related events were also involved.

Hyperammonemia: regulation of argininosuccinate synthetase and argininosuccinate lyase genes in aggregating cell cultures of fetal rat brain.

Hyperammonemia in the brain leads to poorly understood alterations of nitric oxide (NO) synthesis. Arginine, the substrate of nitric oxide synthases, might be recycled from the citrulline produced with NO by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). The regulation of AS and AL genes during hyperammonemia is unknown in the brain. We used brain cell aggregates cultured from dissociated telencephalic cortex of rat embryos to analyze the regulation of AS and AL genes in hyperammonemia. Using RNase protection assay and non-radioactive in situ hybridization on aggregate cryosections, we show that both AS and AL genes are induced in astrocytes but not in neurons of aggregates exposed to 5 mM NH4Cl. Our work suggests that the hyperammonemic brain might increase its recycling of citrulline to arginine.

Beaming into the rat world: enabling real-time intereaction between rat and human each at their own scale

Immersive virtual reality (IVR) typically generates the illusion in participants that they are in the displayed virtual scene where they can experience and interact in events as if they were really happening. Teleoperator (TO) systems place people at a remote physical destination embodied as a robotic device, and where typically participants have the sensation of being at the destination, with the ability to interact with entities there. In this paper, we show how to combine IVR and TO to allow a new class of application. The participant in the IVR is represented in the destination by a physical robot (TO) and simultaneously the remote place and entities within it are represented to the participant in the IVR. Hence, the IVR participant has a normal virtual reality experience, but where his or her actions and behaviour control the remote robot and can therefore have physical consequences. Here, we show how such a system can be deployed to allow a human and a rat to operate together, but the human interacting with the rat on a human scale, and the rat interacting with the human on the rat scale. The human is represented in a rat arena by a small robot that is slaved to the human"s movements, whereas the tracked rat is represented to the human in the virtual reality by a humanoid avatar. We describe the system and also a study that was designed to test whether humans can successfully play a game with the rat. The results show that the system functioned well and that the humans were able to interact with the rat to fulfil the tasks of the game. This system opens up the possibility of new applications in the life sciences involving participant observation of and interaction with animals but at human scale.

Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding.

PPARs are members of the nuclear hormone receptor superfamily and are primarily involved in lipid metabolism. The expression patterns of all 3 PPAR isotypes in 22 adult rat organs were analyzed by a quantitative ribonuclease protection assay. The data obtained allowed comparison of the expression of each isotype to the others and provided new insight into the less studied PPAR beta (NR1C2) expression and function. This isotype shows a ubiquitous expression pattern and is the most abundant of the three PPARs in all analyzed tissues except adipose tissue. Its expression is especially high in the digestive tract, in addition to kidney, heart, diaphragm, and esophagus. After an overnight fast, PPAR beta mRNA levels are dramatically down-regulated in liver and kidney by up to 80% and are rapidly restored to control levels upon refeeding. This tight nutritional regulation is independent of the circulating glucocorticoid levels and the presence of PPAR alpha, whose activity is markedly up-regulated in the liver and small intestine during fasting. Finally, PPAR gamma 2 mRNA levels are decreased by 50% during fasting in both white and brown adipose tissue. In conclusion, fasting can strongly influence PPAR expression, but in only a few selected tissues.

Long-term changes in synaptic transmission of the lateral amygdala induced by strong "in vitro" activation

Résumé : L'amygdale latérale (AL) joue un .rôle essentiel dans la plasticité synaptique à la base du conditionnement de la peur. Malgré le faite que la majorité des cellules de l'AL reçoivent les afférentes nécessaires, une potentialisation dans seulement une partie d'entre elles est obligatoire afin que l'apprentissage de la peur ait lieu. Il a été montré que ces cellules expriment la forme active de CREB, et celui-ci a été associé aux cellules dites de type 'nonaccomrnodating' (nAC). Très récemment, une étude a impliqué les circuits récurrents de l'AL dans le conditionnement de la peur. Un lien entre ces deux observations n'a toutefois jamais été établi. t Nous avons utilisé un protocole in vitro de forte activation de l'AL, résultant dans l'induction de 'bursts' provenant de l'hippocampe et se propageant jusqu'à l'AL. Dans l'AL ces 'bursts' atteignent toutes les cellules et se propagent à travers plusieurs chemins. Utilisant ce protocole, nous avons, pour la première fois pu associer dans l'AL, des cellules connectées de manière récurrente avec des cellules de type nAC. Aussi bien dans ces dernières que dans les cellules de type 'accommodating' (AC), une diminution dans la transmission inhibitrice, à la fois exprimée de manière pré synaptique mais également indépendant de la synthèse de protéine a pu être observé. Au contraire, une potentialisation induite et exprimée au niveau pré synaptique ainsi que dépendante de la synthèse de protéine a pu être trouvé uniquement dans les cellules de type nAC. De plus, une hyperexcitabilité, dépendante des récepteurs NMDA a pu être observé, avec une sélection préférentielle des cellules du type nAC dans la génération de bursts. Nous avons également pu démontrer que la transformation d'un certain nombre de cellules de type AC en cellules dites nAC accompagnait cette augmentation générale de l'excitabilité de l'AL. Du faite da la grande quantité d'indices suggérant une connexion entre le système noradrénergique et les états de peur/d'anxiété, les effets d'une forte activation de l'AL sur ce dernier ont été investigués et ont révélés une perte de sa capacité de modulation du 'spiking pattern'. Finalement, des changements au niveau de l'expression d'un certain nombre de gènes, incluant celui codant pour le BDNF, a pu être trouvé à la suite d'une forte activation de l'AL. En raison du lien récemment décrit entre l'expression de la forme active de CREB et des cellules de type nAC ainsi que celui de l'implication des cellules de l'AL connectés de manière récurrente dans l'apprentissage de la peur, nos résultats nous permettent de suggérer un modèle expliquant comment la potentialisation des connections récurrentes entre cellules de type nAC pourrait être à la base de leur recrutement sélectif pendant le conditionnement de la peur. De plus, ils peuvent offrir des indices par rapport aux mécanismes à travers lesquels une sous population de neurones peut être réactivée par une stimulation externe précédemment inefficace, et induire ainsi un signal suffisamment fort pour qu'il soit transmit aux structures efférentes de l'AL. Abstract : The lateral nucleus of the amygdala (LA) is critically involved in the plasticity underlying fear-conditioned learning (Sah et al., 2008). Even though the majority of cells in the LA receive the necessary sensory inputs, potentiation in only a subset is required for fear learning to occur (Repa et al., 2001; Rumpel et al., 2005). These cells express active CREB (CAMP-responsive element-binding protein) (Han et al., 200, and this was related to the non-accommodating (nAC) spiking phenotype (Viosca et al., 2009; Zhou et al., 2009). In addition, a very recent study implicated recurrently connected cells of the LA in fear conditioned learning (Johnson et al., 2008). A link between the two observations has however never been made. In rats, we used an in vitro protocol of strong activation of the LA, resulting in bursting activity, which spread from the hippocampus to the LA. Within the LA, this activity reached all cells and spread via a multitude of pathways. Using this model, we were able to link, for the first time, recurrently connected cells in the LA with cells of the nAC phenotype. While we found a presynaptically expressed, protein synthesis independent decrease in inhibitory synaptic transmission in both nAC and accommodating (AC) cells, only nAC cells underwent a presynaptically induced and expressed, protein synthesis dependent potentiation. Moreover we observed an NMDA dependent hyperexcitability of the LA, with a preferential selection of nAC cells into burst generation. The transformation of a subset of AC cells into nAC cells accompanied this general increase in LA excitability. Given the considerable evidence suggesting a relationship between the central noradrenergic (NA) system and fear/anxiety states (Itoi, 2008), the effects of strong activation of the LA on the noradrenergic system were investigated, which revealed a loss of its modulatory actions on cell spiking patterns. Finally, we found changes in the expression levels of a number of genes; among which the one coding for $DNF, to be induced by strong activation of the LA. In view of the recently described link between nAC cells and expression of pCREB (phosphorylated cAMP-responsive element-binding protein) as well as the involvement of recurrently connected cells of the LA in fear-conditioned learning, our findings may provide a model of how potentiation of recurrent connections between nAC neurons underlies their recruitment into the fear memory trace. Additionally, they may offer clues as to the mechanisms through which a selected subset of neurons can be reactivated by smaller, previously ineffective external stimulations to respond with a sufficiently strong signal, which can be transmitted to downstream targets of the LA.

Peroxisome proliferator-activated receptor beta/delta activation inhibits hypertrophy in neonatal rat cardiomyocytes.

OBJECTIVE: Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is the predominant PPAR subtype in cardiac cells and plays a prominent role in the regulation of cardiac lipid metabolism. However, the role of PPARbeta/delta activators in cardiac hypertrophy is not yet known. METHODS AND RESULTS: In cultured neonatal rat cardiomyocytes, the selective PPARbeta/delta activator L-165041 (10 micromol/L) inhibited phenylephrine (PE)-induced protein synthesis ([(3)H]leucine uptake), induction of the fetal-type gene atrial natriuretic factor (ANF) and cardiac myocyte size. Induction of cardiac hypertrophy by PE stimulation also led to a reduction in the transcript levels of both muscle-type carnitine palmitoyltransferase (50%, P<0.05) and pyruvatedehydrogenase kinase 4 (30%, P<0.05), and these changes were reversed in the presence of the PPARbeta/delta agonist L-165041. Stimulation of neonatal rat cardiomyocytes with PE and embryonic rat heart-derived H9c2 cells with lipopolysaccharide (LPS) enhanced the expression of the nuclear factor (NF)-kappaB-target gene monocyte chemoattractant protein 1 (MCP-1). The induction of MCP-1 was reduced in the presence of L-165041, suggesting that this compound prevented NF-kappaB activation. Electrophoretic mobility shift assay (EMSA) revealed that L-165041 significantly decreased LPS-stimulated NF-kappaB binding activity in H9c2 myotubes. Finally, coimmunoprecipitation studies showed that L-165041 strongly enhanced the physical interaction between PPARbeta/delta and the p65 subunit of NF-kappaB, suggesting that increased association between these two proteins is the mechanism responsible for antagonizing NF-kappaB activation by PPARbeta/delta activators. CONCLUSION: These results suggest that PPARbeta/delta activation inhibits PE-induced cardiac hypertrophy and LPS-induced NF-kappaB activation.

MRS glucose mapping and PET joining forces: re-evaluation of the lumped constant in the rat brain under isoflurane anaesthesia.

Although numerous positron emission tomography (PET) studies with (18) F-fluoro-deoxyglucose (FDG) have reported quantitative results on cerebral glucose kinetics and consumption, there is a large variation between the absolute values found in the literature. One of the underlying causes is the inconsistent use of the lumped constants (LCs), the derivation of which is often based on multiple assumptions that render absolute numbers imprecise and errors hard to quantify. We combined a kinetic FDG-PET study with magnetic resonance spectroscopic imaging (MRSI) of glucose dynamics in Sprague-Dawley rats to obtain a more comprehensive view of brain glucose kinetics and determine a reliable value for the LC under isoflurane anaesthesia. Maps of Tmax /CMRglc derived from MRSI data and Tmax determined from PET kinetic modelling allowed to obtain an LC-independent CMRglc . The LC was estimated to range from 0.33 ± 0.07 in retrosplenial cortex to 0.44 ± 0.05 in hippocampus, yielding CMRglc between 62 ± 14 and 54 ± 11 μmol/min/100 g, respectively. These newly determined LCs for four distinct areas in the rat brain under isoflurane anaesthesia provide means of comparing the growing amount of FDG-PET data available from translational studies.

Adiponectin and heme oxygenase-1 suppress TLR4/MyD88-independent signaling in rat Kupffer cells and in mice after chronic ethanol exposure.

Alcoholic liver disease is mediated via activation of TLR4 signaling; MyD88-dependent and -independent signals are important contributors to injury in mouse models. Adiponectin, an anti-inflammatory adipokine, suppresses TLR4/MyD88-dependent responses via induction of heme oxygenase-1 (HO-1). Here we investigated the interactions between chronic ethanol, adiponectin, and HO-1 in regulation of TLR4/MyD88-independent signaling in macrophages and an in vivo mouse model. After chronic ethanol feeding, LPS-stimulated expression of IFN-β and CXCL10 mRNA was increased in primary cultures of Kupffer cells compared with pair-fed control mice. Treatment of Kupffer cells with globular adiponectin (gAcrp) normalized this response. LPS-stimulated IFN-β/CXCL10 mRNA and CXCL10 protein was also reduced in RAW 264.7 macrophages treated with gAcrp or full-length adiponectin. gAcrp and full-length adiponectin acted via adiponectin receptors 1 and 2, respectively. gAcrp decreased TLR4 expression in both Kupffer cells and RAW 264.7 macrophages. Small interfering RNA knockdown of HO-1 or inhibition of HO-1 activity with zinc protoporphyrin blocked these effects of gAcrp. C57BL/6 mice were exposed to chronic ethanol feeding, with or without treatment with cobalt protoporphyrin, to induce HO-1. After chronic ethanol feeding, mice were sensitized to in vivo challenge with LPS, expressing increased IFN-β/CXCL10 mRNA and CXCL10 protein in liver compared with control mice. Pretreatment with cobalt protoporphyrin 24 h before LPS challenge normalized this effect of ethanol. Adiponectin and induction of HO-1 potently suppressed TLR4-dependent/MyD88-independent cytokine expression in primary Kupffer cells from rats and in mouse liver after chronic ethanol exposure. These data suggest that induction of HO-1 may be a useful therapeutic strategy in alcoholic liver disease.

Infectivity of Lactobacillus rhamnosus and Lactobacillus paracasei isolates in a rat model of experimental endocarditis.

The potential pathogenicity of selected (potentially) probiotic and clinical isolates of Lactobacillus rhamnosus and Lactobacillus paracasei was investigated in a rat model of experimental endocarditis. In addition, adhesion properties of the lactobacilli for fibrinogen, fibronectin, collagen and laminin, as well as the killing activity of the platelet-microbicidal proteins fibrinopeptide A (FP-A) and connective tissue activating peptide 3 (CTAP-3), were assessed. The 90 % infective dose (ID(90)) of the L. rhamnosus endocarditis isolates varied between 10(6) and 10(7) c.f.u., whereas four of the six (potentially) probiotic L. rhamnosus isolates showed an ID(90) that was at least 10-fold higher (10(8) c.f.u.) (P<0.001). In contrast, the two other probiotic L. rhamnosus isolates exhibited an ID(90) (10(6) and 10(7) c.f.u.) comparable to the ID(90) of the clinical isolates of this species investigated (P>0.05). Importantly, these two probiotic isolates shared the same fluorescent amplified fragment length polymorphism cluster type as the clinical isolate showing the lowest ID(90) (10(6) c.f.u.). L. paracasei tended to have a lower infectivity than L. rhamnosus (ID(90) of 10(7) to > or =10(8) c.f.u.). All isolates had comparable bacterial counts in cardiac vegetations (P>0.05). Except for one L. paracasei strain adhering to all substrates, all tested lactobacilli adhered only weakly or not at all. The platelet peptide FP-A did not show any microbicidal activity against the tested lactobacilli, whereas CTAP-3 killed the majority of the isolates. In general, these results indicate that probiotic lactobacilli display a lower infectivity in experimental endocarditis compared with true endocarditis pathogens. However, the difference in infectivity between L. rhamnosus endocarditis and (potentially) probiotic isolates could not be explained by differences in adherence or platelet microbicidal protein susceptibility. Other disease-promoting factors may exist in these organisms and warrant further investigation.

Expression of the glucagon-like peptide-1 receptor gene in rat brain.

Evidence that glucagon-like peptide-1 (GLP-1) (7-36) amide functions as a novel neuropeptide prompted us to study the gene expression of its receptor in rat brain. Northern blot analysis showed transcripts of similar size in RINm5F cells, hypothalamus, and brain-stem. First-strand cDNA was prepared by using RNA from hypothalamus, brainstem, and R1Nm5F cells and subsequently amplified by PCR. Southern blot analysis of the PCR products showed a major 1.4-kb band in all these preparations. PCR products amplified from hypothalamus were cloned, and the nucleotide sequence of one strand was identical to that described in rat pancreatic islets. In situ hybridization studies showed specific labeling in both neurons and glia of the thalamus, hypothalamus, hippocampus, primary olfactory cortex, choroid plexus, and pituitary gland. In the hypothalamus, ventromedial nuclei cells were highly labeled. These findings indicate that GLP-1 receptors are actually synthesized in rat brain. In addition, the colocalization of GLP-1 receptors, glucokinase, and GLUT-2 in the same areas supports the idea that these cells play an important role in glucose sensing in the brain.

The lipid composition of rat brain aggregating cell cultures during development.

The lipid and fatty acid composition of rat brain was studied during its development both in vivo and in an aggregating cell culture system. Although the amount of lipid present in the cultures was very low, the increase in glycolipid content corresponded closely to the period of intense myelin formation. Very long chain fatty acids (hydroxylated and unsubstituted) were present in 41-day cultures. In comparison to the in vivo situation, myelination was delayed in vitro and, after 40 days in culture, cholesterol esters were 5-fold higher than in vivo, indicating that demyelination was occurring.

Fructose Rich Diet-Induced High Plasminogen Activator Inhibitor-1 (PAI-1) Production in the Adult Female Rat: Protective Effect of Progesterone.

The effect of progesterone (P4) on fructose rich diet (FRD) intake-induced metabolic, endocrine and parametrial adipose tissue (PMAT) dysfunctions was studied in the adult female rat. Sixty day-old rats were i.m. treated with oil alone (control, CT) or containing P4 (12 mg/kg). Rats ate Purina chow-diet ad libitum throughout the entire experiment and, between 100 and 120 days of age drank ad libitum tap water alone (normal diet; CT-ND and P4-ND) or containing fructose (10% w/v; CT-FRD and P4-FRD). At age 120 days, animals were subjected to a glucose tolerance test or decapitated. Plasma concentrations of various biomarkers and PMAT gene abundance were monitored. P4-ND (vs. CT-ND) rats showed elevated circulating levels of lipids. CT-FRD rats displayed high (vs. CT-ND) plasma concentrations of lipids, leptin, adiponectin and plasminogen activator inhibitor-1 (PAI-1). Lipidemia and adiponectinemia were high (vs. P4-ND) in P4-FRD rats. Although P4 failed to prevent FRD-induced hyperleptinemia, it was fully protective on FRD-enhanced plasma PAI-1 levels. PMAT leptin and adiponectin mRNAs were high in CT-FRD and P4-FRD rats. While FRD enhanced PMAT PAI-1 mRNA abundance in CT rats, this effect was absent in P4 rats. Our study supports that a preceding P4-enriched milieu prevented the enhanced prothrombotic risk induced by FRD-elicited high PAI-1 production.

Beaming into the rat world: enabling real-time intereaction between rat and human each at their own scale

Immersive virtual reality (IVR) typically generates the illusion in participants that they are in the displayed virtual scene where they can experience and interact in events as if they were really happening. Teleoperator (TO) systems place people at a remote physical destination embodied as a robotic device, and where typically participants have the sensation of being at the destination, with the ability to interact with entities there. In this paper, we show how to combine IVR and TO to allow a new class of application. The participant in the IVR is represented in the destination by a physical robot (TO) and simultaneously the remote place and entities within it are represented to the participant in the IVR. Hence, the IVR participant has a normal virtual reality experience, but where his or her actions and behaviour control the remote robot and can therefore have physical consequences. Here, we show how such a system can be deployed to allow a human and a rat to operate together, but the human interacting with the rat on a human scale, and the rat interacting with the human on the rat scale. The human is represented in a rat arena by a small robot that is slaved to the human"s movements, whereas the tracked rat is represented to the human in the virtual reality by a humanoid avatar. We describe the system and also a study that was designed to test whether humans can successfully play a game with the rat. The results show that the system functioned well and that the humans were able to interact with the rat to fulfil the tasks of the game. This system opens up the possibility of new applications in the life sciences involving participant observation of and interaction with animals but at human scale.