Synthesis and Validation of a Hydroxypyrone-Based, Potent, and Specific Matrix Metalloproteinase-12 Inhibitor with Anti-Inflammatory Activity In Vitro and In Vivo.

A hydroxypyrone-based matrix metalloproteinase (MMP) inhibitor was synthesized and assayed for its inhibitory capacity towards a panel of ten different MMPs. The compound exhibited selective inhibition towards MMP-12. The effects of inhibition of MMP-12 on endotoxemia and inflammation-induced blood-cerebrospinal fluid barrier (BCSFB) disruption were assessed both in vitro and in vivo. Similar to MMP-12 deficient mice, inhibitor-treated mice displayed significantly lower lipopolysaccharide- (LPS-) induced lethality compared to vehicle treated controls. Following LPS injection Mmp-12 mRNA expression was massively upregulated in choroid plexus tissue and a concomitant increase in BCSFB permeability was observed, which was restricted in inhibitor-treated mice. Moreover, an LPS-induced decrease in tight junction permeability of primary choroid plexus epithelial cells was attenuated by inhibitor application in vitro. Taken together, this hydroxypyrone-based inhibitor is selective towards MMP-12 and displays anti-inflammatory activity in vitro and in vivo.

Neuronal Gonadotrophin-Releasing Hormone (GnRH) and Astrocytic Gonadotrophin Inhibitory Hormone (GnIH) Immunoreactivity in the Adult Rat Hippocampus.

Gonadotrophin-releasing hormone (GnRH) and gonadotrophin inhibitory hormone (GnIH) are neuropeptides secreted by the hypothalamus that regulate reproduction. GnRH receptors are not only present in the anterior pituitary, but also are abundantly expressed in the hippocampus of rats, suggesting that GnRH regulates hippocampal function. GnIH inhibits pituitary gonadotrophin secretion and is also expressed in the hippocampus of a songbird; its role outside of the reproductive axis is not well established. In the present study, we employed immunohistochemistry to examine three forms of GnRH [mammalian GnRH-I (mGnRH-I), chicken GnRH-II (cGnRH-II) and lamprey GnRH-III (lGnRH-III)] and GnIH in the adult rat hippocampus. No mGnRH-I and cGnRH-II+ cell bodies were present in the hippocampus. Sparse mGnRH-I and cGnRH-II+ fibres were present within the CA1 and CA3 fields of the hippocampus, along the hippocampal fissure, and within the hilus of the dentate gyrus. No lGnRH-III was present in the rodent hippocampus. GnIH-immunoreactivity was present in the hippocampus in cell bodies that resembled astrocytes. Males had more GnIH+ cells in the hilus of the dentate gyrus than females. To confirm the GnIH+ cell body phenotype, we performed double-label immunofluorescence against GnIH, glial fibrillary acidic protein (GFAP) and NeuN. Immunofluorescence revealed that all GnIH+ cell bodies in the hippocampus also contained GFAP, a marker of astrocytes. Taken together, these data suggest that GnRH does not reach GnRH receptors in the rat hippocampus primarily via synaptic release. By contrast, GnIH might be synthesised locally in the rat hippocampus by astrocytes. These data shed light on the sites of action and possible functions of GnRH and GnIH outside of the hypothalamic-pituitary-gonadal axis.

Linking mass spectrometric imaging and traditional peptidomics: a validation in the obese mouse model.

MALDI mass spectrometry imaging (MSI) is a promising technique in the field of molecular (immuno)histology but is confronted with the problematic large-scale identification of peptides from thin tissue sections. In this study we present a workflow that significantly increased the number of identified peptides in a given MALDI-MSI data set and we evaluated its power concerning relative peptide quantifications. Fourier transform mass spectrometry (FTMS) profiling on matrix-coated thin tissue sections allowed us to align spectra of different MS sources, matching identical peaks in the process, thus linking MSI data to tandem mass spectrometry (MS/MS) on one hand and semiquantitative liquid chromatography (LC)/MS data on the other. Bonanza clustering was applied in order to group MS/MS spectra of structurally related peptides, making it possible to infer the identity of MSI-detected compounds based on identified members within the same cluster, effectively increasing the number of identifications in a single MSI data set. Out of 136 detected peptides with MALDI-MSI, we were able to identify 46 peptides. For 31 of these, a LC/quadrupole time-of-flight (QTOF) counterpart was detected, and we observed similar obese (ob/ob) to wild-type (wt) peak intensity ratios for 18 peptides. This workflow significantly increased the number of identifications of peptide masses detected with MALDI-MSI and evaluated the power of this imaging method for relative quantification of peptide levels between experimental conditions.

Monocular enucleation profoundly reduces secretogranin II expression in adult mouse visual cortex.

Secretogranin II (ScgII), a member of the chromogranin family, is almost exclusively found in large dense core vesicles of a wide variety of endocrine and neuronal tissues, being stored together with many different neurotransmitters, peptide hormones and neuropeptides. In the brain ScgII is almost completely processed to secretoneurin, a peptide involved in neurite outgrowth, neuroprotection and neuronal plasticity. Furthermore, correlations with neurotransmitter release and a variety of neurological diseases were reported. In this study we examined possible changes in ScgII mRNA expression in the visual system of adult mice after removal of one eye. Mice were monocularly deprived of vision and sacrificed 1 day or 1, 3, 5 and 7 weeks after enucleation. Starting 1 day after the deprivation, a marked decrease of ScgII was visible in the contralateral visual cortex. Recovery initiated in the lateral supragranular visual cortex after 5 weeks of enucleation, but was far from complete in the 7 week animals, especially in the monocularly driven medial cortex. By comparison with the immediate early gene zif268, it was proven that ScgII cannot be categorized as an activity marker, but more likely plays a role in visual system plasticity by modulating a range of neurotransmitters and neuropeptides.

Effects of sublethal doses of crop protection agents on honey bee (Apis mellifera) global colony vitality and its potential link with aberrant foraging activity.

Honey bees (Apis mellifera) are the most economically valuable pollinators of fruit crops worldwide. Taking into account bees' contributions to other flowering agricultural crops, about one-third of our total diet comes directly or indirectly from bee-pollinated plants. However, in recent years there increasingly have been worrisome alarm sounds on serious bee mortalities and mysterious disappearance of bees from beehives. Among several environmental factors (e.g. climate and bee pathogens), stress factors arising from agricultural practices can potentially play a role in bee losses. Detailed knowledge on the effects of plant protection products is essential to improve usage with minimal risks. In order to identify potential medium- and long-term effects, we followed up various sublethal contaminated hives during the prolongation of the fruit-growing season. More specifically, a large-scale experiment was conducted in which at four distinct locations (in the Limburg region of Belgium) four different bee colonies (representing three different contaminations -imidacloprid, fenoxycarb, indoxacarb- and a non-contaminated control hive) were thoroughly monitored every 2-7 days. Our observations point towards decays of overall colony vitality for several hives a couple of weeks after treatment, as indicated by a set of carefully assessed parameters including the total amount of active and dead bees, total surface of capped brood and overall colony weight. These outcomes could be linked to subtle differences in foraging activity between distinct hives. The implications of these results are discussed in terms of potential short-term and long-term consequences of disturbed foraging ability triggered by exaggerated exposure to sublethal doses of crop protection chemicals, and its potential impact on colony health.

Amfor expression in the honeybee brain: a trigger mechanism for nurse-forager transition.

The honeybee's colony fitness relies on an optimized age-dependent division of labor. Transition from nursing activities to foraging activities is associated with an increase in the expression of the Amfor gene. Ben-Shahar et al. [Ben-Shahar, Y., Robichon, A., Sokolowski, M.B., Robinson, G.E., 2002. Influence of gene action across different time scales on behavior. Science 296, 741-744] showed that the Amfor transcripts and their gene products are involved in regulating the transition from one task to the next. In this study, we investigated the trajectory of the expression of this gene in the brain over time. The expression pattern could contribute to our understanding of the involvement of Amfor in the transition process. Is there a gradual increase in transcript or a peak in expression triggering a downstream path of multiple differential gene expression? Hereto, bees were sampled from colonies containing marked 1-day-old bees every 2 or 3 days around the expected time of transition from nurse to forager, from day 13 to 25. To quantify Amfor transcript in the brain, we developed a real-time RT-PCR assay, based on Taqman technology, using fluorescent probes. Results revealed a trigger mechanism rather than a continued elevation of Amfor expression. The appearance of an Amfor expression peak suggests that under normal physiological conditions foraging behavior is, at least in part, due to a trigger-effect of Amfor.

The effect of nonventilation during early incubation on the embryonic development of chicks of two commercial broiler strains differing in ascites susceptibility.

Despite thorough selection during the last decade, the incidence of ascites is still high in modern broiler strains. Although ascites occurs mostly at the end of the rearing period, there are indications that the etiology of this problem may have started during embryonic development. Recent studies have shown that the post-hatch performance of the broiler chick might be influenced by changing the environmental conditions in the incubator during embryonic development. This study investigated the effect of increasing incubator CO(2) concentration up to 0.7%, by nonventilation during the first 10 d of incubation, on the embryonic development of 2 commercial broiler strains (Cobb and SAS) differing in their susceptibility for ascites syndrome. The Cobb strain is suspected to be less susceptible than the SAS strain. Overall, the chick embryos of the Cobb strain had a faster development than those of the SAS strain as expressed by their higher BW from embryonic day (ED)10 until ED18. Nonventilation stimulated embryonic development resulting in higher embryonic BW, early hatch, and narrower spread of hatch in both strains. In the SAS strain, nonventilation improved hatchability by more than 10%. Gas composition of the air cell in the egg of the nonventilation groups (both Cobb and SAS) had higher partial pressure of CO(2) and lower partial pressure of O(2) from ED11 until ED14 compared with the ventilation groups. During the entire incubation period, partial pressure of CO(2) was higher in eggs of the Cobb strain compared with the SAS strain. Plasma triiodothyronine, thyroxine, and corticosterone levels were different at the end of the incubation period and during hatching due to nonventilation at the beginning of incubation. It is concluded that nonventilation during the first 10 d of incubation had a stimulatory effect on embryonic development of the 2 broiler strains with no effect of heart weights but with effects on hormone levels, air cell pressures, and hatching parameters.

Behavioural and physiological effects of electrical stimulation in the nucleus accumbens: a review.

Electrical stimulation (ES) in the brain is becoming a new treatment option in patients with treatment-resistant obsessive-compulsive disorder (OCD). A possible brain target might be the nucleus accumbens (NACC). This review aims to summarise the behavioural and physiological effects of ES in the NACC in humans and in animals and to discuss these findings with regard to neuroanatomical, electrophysiological and behavioural insights. The results clearly demonstrate that ES in the NACC has an effect on reward, activity, fight-or-flight, exploratory behaviour and food intake, with evidence for only moderate physiological effects. Seizures were rarely observed. Finally, the results of ES studies in patients with treatment-resistant OCD and in animal models for OCD are promising.

Applications and current challenges of proteomic approaches, focusing on two-dimensional electrophoresis.

Since the formulation of the concept of "proteomics" in 1995, a plethora of proteomic technologies have been developed in order to study proteomes of tissues, cells and organelles. The powerful new technologies enabled by proteomic approaches have lead to the application of these methods to an exponentially increasing variety of biological questions for highly complex protein mixtures. Continuous technical optimization allows for an ever-increasing sensitivity of proteomic techniques. In this review, a brief overview of currently available proteomic techniques and their applications is given, followed by a more detailed description of advantages and technical challenges of two-dimensional electrophoresis (2-DE). Some solutions to circumvent currently encountered technical difficulties for 2-DE analyses are proposed.

Brain-derived neurotrophic factor in the brain of Xenopus laevis may act as a pituitary neurohormone together with mesotocin.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, occurs abundantly in the brain, where it exerts a variety of neural functions. We previously demonstrated that BDNF also exists in the endocrine melanotroph cells in the intermediate lobe of the pituitary gland of the amphibian Xenopus laevis, suggesting that BDNF, in addition to its neural actions within the brain, can act as a hormone. In the present study, we tested whether BDNF, in addition to its neural and hormonal roles, can be released as a neurohormone from the neural pituitary lobe of X. laevis. By light immunocytochemistry, we show that BDNF is present in perikarya, in ventrolaterally projecting axons of the hypothalamic magnocellular nucleus and in the neural lobe of the pituitary gland, and that it coexists in these structures with the amphibian neurohormone, mesotocin. The neural lobe was studied in detail at the ultrastructural level. Two types of neurohaemal axon terminals were observed, occurring intermingled and in similar numbers. Type A is filled with round, moderately electron-dense secretory granules with a mean diameter of approximately 145 nm. Type B terminals contain electron-dense and smaller, ellipsoid granules (long and short diameter approximately 140 and 100 nm, respectively). BDNF is exclusively present in secretory granules of type A axon terminals. Double gold-immunolabelling revealed that BDNF coexists in these granules with mesotocin. Furthermore, we demonstrate in an superfusion study performed in vitro that mesotocin stimulates peptide release from the endocrine melanotroph cells. On the basis of these data, we propose that BDNF can act on these cells as a neurohormone.

Neuroanatomical specificity in the expression of the immediate early gene c-fos following expression of appetitive and consummatory male sexual behaviour in Japanese quail.

We investigated the neural sites related to the occurrence of appetitive (ASB) and consummatory (CSB) aspects of male sexual behaviour in Japanese quail. Castrated males treated with testosterone were exposed for 5 min to one of four experimental conditions: (i) free interaction with a female (CSB group); (ii) expression of rhythmic cloacal sphincter movements in response to the visual presentation of a female (ASB-F group); (iii) or a male (ASB-M group), and (iv) handling as a control manipulation. Brains were collected 90 min after the start of behavioural tests and stained by immunocytochemistry for the FOS protein. An increase in FOS expression was observed throughout the rostro-caudal extent of the medial preoptic nucleus (POM) in CSB males, whereas the view of a female (ASB-F) induced an increased FOS expression in the rostral POM only. In the CSB group, there was also an increase in FOS expression in the bed nucleus striae terminalis, and both the CSB and ASB-F groups exhibited increased FOS expression in aspects of the ventro-lateral thalamus (VLT) related to visual processing. Moreover, both the CSB and ASB-M groups showed increased FOS expression in the lateral septum. These data provide additional support to the idea that there is a partial anatomical dissociation between structures involved in the control of both aspects of male sexual behaviour and independently provide data consistent with a previous lesion study that indicated that the rostral and caudal POM differentially control the expression of ASB and CSB in quail.

Comparison of three lines of broilers differing in ascites susceptibility or growth rate. 2. Egg weight loss, gas pressures, embryonic heat production, and physiological hormone levels.

Ascites is a metabolic disorder that accounts for over 25% of overall mortality in the broiler industry. This disorder is manifested between wk 5 and 6 posthatch, but there are previous indications that predisposition may be identified during embryonic development. In this current study, we determined embryonic physiological and metabolic parameters that may be associated with ascites predisposition. For this purpose, we used broiler eggs from 3 lines that differed in ascites sensitivity. These included an ascites-sensitive dam line (DAS), an ascites-resistant dam line (DAR), and an ascites-sensitive sire line (SASL). Eggs were incubated for 21 d under standard conditions. The following parameters were measured during incubation: egg weights at setting, egg weight losses at 18 d, embryo body weights and embryo heart weights throughout development, air cell partial gas pressures (pCO2 and pO2) levels at d 18 and at internal pipping (IP); plasma triiodothyronine, thyroxine, and corticosterone levels at d 18, IP, and hatch; heat production from d 17 until hatch, hematocrit values at hatch, and posthatch growth rate to 7 d along with hematocrit values. The data obtained revealed that selection for ascites sensitivity or rapid growth rate had no consistent influence on some of these parameters such that they could be wholly associated with ascites sensitivity for predictive purposes. Whereas differences in embryonic developmental patterns were apparent throughout embryonic development, these differences in physiological and metabolic parameters may be due partly to genetic differences unrelated to ascites sensitivity.

Neurotoxicity of polychlorinated biphenyls (PCBs) by disturbance of thyroid hormone-regulated genes.

PCBs are known as neurotoxic compounds. Part of this neurotoxicity could be due to an alteration of the expression of TH-regulated genes in brain. To identify such genes, brain protein extracts of hypo- and hyperthyroid as well as PCB-treated embryos were compared by fluorescent 2D-DIGE. In total, we observed 109 differentially expressed proteins, of which 17 differed with both PCB and hypo- or hyperthyroid treatment. It was found that the interaction of PCBs with the expression of TH-regulated genes is congener-specific and that both hyperthyroidism- and hypothyroidism-related effects occur.

Retinotopic map plasticity in adult cat visual cortex is accompanied by changes in Ca2+/calmodulin-dependent protein kinase II alpha autophosphorylation.

In adult cats, the induction of homonymous binocular central retinal lesions causes a dramatic reorganization of the topographic map in the sensory-deprived region of the primary visual cortex. To investigate the possible involvement of the alpha-subunit of the calcium/calmodulin dependent protein kinase type II (alphaCaMKII) in this form of brain plasticity, we performed in situ hybridization and Western blotting experiments to analyze mRNA, protein and autophosphorylation levels of this multifunctional kinase. No differences in the mRNA or protein levels were observed between the central, sensory-deprived and the peripheral, non-deprived regions of area 17 of retinal lesion animals or between corresponding cortical regions of normal control animals. Western blotting with an alphaCaMKII threonine-286 phosphorylation-state specific antiserum consistently showed a small, albeit not significant, increase of alphaCaMKII autophosphorylation in the central versus the peripheral region of cortical area 17, and this both in normal subjects as well as in retinal lesion animals with a 3-day post-lesion survival time. In contrast, a post-lesion survival time of 14 days resulted in a alphaCaMKII autophosphorylation level that was four times higher in visually-deprived area 17 than in the non-deprived cortical region. This increased phosphorylation state is not a direct consequence of the decrease in visual activity in these neurons, because we would have expected to see a similar change at shorter or longer post-lesion survival times or in the visually deprived visual cortex of animals in which the left optic tract and the corpus callosum were surgically cut. No such changes were observed, leading to the conclusion that the phosphorylation changes observed at 14 days are related to a delayed reorganization of the retinotopic map of the striate cortex.

Neurofilament protein: a selective marker for the architectonic parcellation of the visual cortex in adult cat brain.

In this immunocytochemical study, we examined the expression profile of neurofilament protein in the cat visual system. We have used SMI-32, a monoclonal antibody that recognizes a nonphosphorylated epitope on the medium- and high-molecular-weight subunits of neurofilament proteins. This antibody labels primarily the cell body and dendrites of pyramidal neurons in cortical layers III, V, and VI. Neurofilament protein-immunoreactive neurons were prominent in 20 visual cortical areas (areas 17, 18, 19, 20a, 20b, 21a, 21b, and 7; posteromedial lateral, posterolateral lateral, anteromedial lateral, anterolateral lateral, dorsal lateral, ventral lateral, and posterior suprasylvian areas; anterior ectosylvian, the splenial, the cingulate, and insular visual areas; and the anterolateral gyrus area). In addition, we have also found strong immunopositive cells in the A laminae of the dorsal part of the lateral geniculate nucleus (dLGN) and in the medial interlaminar nucleus, but no immunoreactive cells were present in the parvocellular C (1-3) laminae of the dLGN, in the ventral part of the LGN and in the perigeniculate nucleus. This SMI-32 antibody against neurofilament protein revealed a characteristic pattern of immunostaining in each visual area. The size, shape, intensity, and density of neurofilament protein-immunoreactive neurons and their dendritic arborization differed substantially across all visual areas. Moreover, it was also obvious that several visual areas showed differences in laminar distribution and that such profiles may be used to delineate various cortical areas. Therefore, the expression of neurofilament protein can be used as a specific marker to define areal patterns and topographic boundaries in the cat visual system.

Functional striatal hypodopaminergic activity in mice lacking adenosine A(2A) receptors.

Adenosine and caffeine modulate locomotor activity and striatal gene expression, partially through the activation and blockade of striatal A(2A) receptors, respectively. The elucidation of the roles of these receptors benefits from the construction of A(2A) receptor-deficient mice (A(2A)-R(-/-)). These mice presented alterations in locomotor behaviour and striatal expression of genes studied so far, which are unexpected regarding the specific expression of A(2A) receptor by striatopallidal neurones. To clarify the functions of A(2A) receptors in the striatum and to identify the mechanisms leading to these unexpected modifications, we studied the basal expression of immediate early and constitutive genes as well as dopamine and glutamate neurotransmission in the striatum. Basal zif268 and arc mRNAs expression was reduced in mutant mice by 60-80%, not only in the striatum but also widespread in the cerebral cortex and hippocampus. Striatal expression of substance P and enkephalin mRNAs was reduced by about 50% and 30%, respectively, whereas the expression of GAD67 and GAD65 mRNAs was slightly increased and unaltered, respectively. In vivo microdialysis in the striatum revealed a 45% decrease in the extracellular dopamine concentration and three-fold increase in extracellular glutamate concentration. This was associated with an up-regulation of D(1) and D(2) dopamine receptors expression but not with changes in ionotropic glutamate receptors. The levels of tyrosine hydroxylase and of striatal and cortical glial glutamate transporters as well as adenosine A(1) receptors expression were indistinguishable between A(2A)-R(-/-) and wild-type mice. Altogether these results pointed out that the lack of A(2A) receptors leads to a functional hypodopaminergic state and demonstrated that A(2A) receptors are necessary to maintain a basal level in immediate early and constitutive genes expression in the striatum and cerebral cortex, possibly via their control of dopamine pathways.

In vivo microdialysis in the visual cortex of awake cat. I: surgery, animal training and sampling.

Sampling and monitoring release of excitatory and inhibitory amino acids in the striate cortex of mammals will provide important information for visual system research. A method allowing repeated microdialysis in the cortical layers of area 17 of the awake cat is described. Under visual control through a surgical microscope and using a stereotactic instrument, four probe guides are permanently implanted in area 17 of one hemisphere of the anesthetized animal and two fixation bars are mounted on the skull to allow fixation of the cat in a stereotactic frame. The implantation of four probe guides in the same hemisphere allows simultaneous sampling from different cortical regions subserving different parts of the visual field. A removable transparent cover protects the probe guides. After recovery from surgery the awake cats are trained to adapt to a fixation in a stereotaxic apparatus. Once adapted to that situation, the cats are used for 5 h in vivo microdialysis experiments without anesthesia.

In vivo microdialysis in the visual cortex of awake cat. II: sample analysis by microbore HPLC-electrochemical detection and capillary electrophoresis-laser-induced fluorescence detection.

Sampling and monitoring release of excitatory and inhibitory amino acids in the striate cortex of mammals will provide important information for visual system research. Two microbore high performance liquid chromatography-electrochemical detection methods and a capillary electrophoresis-laser induced fluorescence detection were developed to determine the inhibitory amino acid, gamma-aminobutyric acid and the excitatory amino acids, glutamate and aspartate in microdialysates of cat striate cortex. In the liquid chromatography method, samples were derivatized using OPA-TBT. Ten microliters of derivatized product was injected onto the microbore column (100 x 1 mm i.d., C8) for quantitative analysis. Electrochemical detection was employed. In the capillary electrophoresis method, samples were derivatized using fluorescein isothiocyanate and separated in borate buffer within 15 min, then detected by a laser-induced fluorescence detector.

In vivo microdialysis in the visual cortex of awake cat. III: histological verification.

In vivo microdialysis sampling extracellular excitatory and inhibitory amino acids from the striate cortex of mammals will provide important information for visual system research. To facilitate the interpretation of microdialysis results, this protocol critically examines: (1) the location of probe implantation in the visual cortex using Nissl staining; (2) the morphological changes after probe implantation by visualization of neurons containing glutamate; (3) the morphological changes after probe implantation by visualization of gliosis using glial fibrillary acidic protein (GFAP) immunocytochemistry; (4) the implantation of the probe in sensory-deprived versus non-deprived cortical regions by visualization of neurons containing c-Fos protein after limited retinal lesion. The histochemical and immunocytochemical methods of Glu, GFAP and c-Fos used are described.