Spontaneous changes in brain striatal dopamine synthesis and storage dynamics ex vivo reveal end-product feedback-inhibition of tyrosine hydroxylase.

Synaptic events are important to define treatment strategies for brain disorders. In the present paper, freshly obtained rat brain striatal minces were incubated under different times and conditions to determine dopamine biosynthesis, storage, and tyrosine hydroxylase phosphorylation. Remarkably, we found that endogenous dopamine spontaneously accumulated during tissue incubation at 37 °C ex vivo while dopamine synthesis simultaneously decreased. We analyzed whether these changes in brain dopamine biosynthesis and storage were linked to dopamine feedback inhibition of its synthesis-limiting enzyme tyrosine hydroxylase. The aromatic-l-amino-acid decarboxylase inhibitor NSD-1015 prevented both effects. As expected, dopamine accumulation was increased with l-DOPA addition or VMAT2-overexpression, and dopamine synthesis decreased further with added dopamine, the VMAT2 inhibitor tetrabenazine or D2 auto-receptor activation with quinpirole, accordingly to the known synaptic effects of these treatments. Phosphorylation activation and inhibition of tyrosine hydroxylase on Ser31 and Ser40 with okadaic acid, Sp-cAMP and PD98059 also exerted the expected effects. However, no clear-cut association was found between dopamine feedback inhibition of its own biosynthesis and changes of tyrosine hydroxylase phosphorylation, assessed by Western blot and mass spectrometry. The later technique also revealed a new Thr30 phosphorylation in rat tyrosine hydroxylase. Our methodological assessment of brain dopamine synthesis and storage dynamics ex vivo could be applied to predict the in vivo effects of pharmacological interventions in animal models of dopamine-related disorders.

Neurobiology of environmental enrichment in pigs: hanges in monoaminergic neurotransmitters in several brain areas and in the hippocampal proteome.

Environmental enrichment in porcine farms improves animal welfare and leads to better public acceptance. To better understand the neurological mechanisms of the response to environmental enrichment, monoaminergic neurotransmitters were quantified in several brain areas from pigs after eight weeks of housing in barren or enriched conditions. Furthermore, iTRAQ labelling combined with LC-MS/MS was used to identify differentially abundant proteins in the hippocampus. Blood biochemical parameters related with stress and welfare were measured. Pigs under enriched conditions showed a decrease in plasma cortisol and lactate. The decrease in noradrenaline in the prefrontal cortex and amygdala, a general decrease in the dopaminergic system and an increase of serotonin in the striatum indicate a lower response to stress in enriched conditions. In the proteomic analysis, 2304 proteins were identified, of which 56 were differential between housing groups (46 upregulated and 10 downregulated). Bioinformatics analysis revealed that they were mainly related to ribosome, translation, microtubules and metabolic mitochondrial processes, indicating that pigs under enriched environments have higher abundance of proteins related to protein synthesis and neuronal activity. Together with previous behavioural studies, our results suggest that environmental enrichment provides a less stressful environment and that pigs cope better with stress conditions like the slaughterhouse. SIGNIFICANCE: Animal welfare has become an important aspect for the sustainability of animal production. The modification of the environment by enriching it with rooting materials and wider space allowance is known to have a positive effect on pigs' welfare. Searching for the underlying neurobiological mechanisms, we found that housing in an enriched environment increased the abundance of proteins related to protein synthesis, microtubule assembly, vesicle-mediated transport and energy metabolism in the hippocampus of pigs. Likewise, changes in the neurotransmitter profile in several brain areas were compatible with a better response to stress. This study expands the knowledge about the biological basis of animal welfare-promoting actions.

Housing and road transport modify the brain neurotransmitter systems of pigs: Do pigs raised in different conditions cope differently with unknown environments?

How housing and transport conditions may affect welfare in porcine production is a leading topic in livestock research. This study investigated whether pigs present a different neurological response to management conditions and to ascertain whether pigs living partially outdoors cope differently with road transport-associated stress. Twenty-four female pigs were divided in two groups: one living indoors (ID, n = 12) and the other housed combining indoor conditions with 4 hours per day of outdoor pasture (OD, n = 12). After one month, one set of animals from each housing condition were driven in a truck to the slaughterhouse in low-stress conditions (5 min drive, no mixing groups, soft management, LS group, n = 12) or high-stress conditions (2 hours drive, mixing groups, harsh management, HS group, n = 12). At the slaughterhouse, blood was collected, and the prefrontal cortex (PFC) and the hippocampus (HC) dissected. OD pigs had lower serum haptoglobin and increased dopaminergic pathway (DA-system) in the PFC, suggesting that living outdoors increases their wellbeing. HS conditions increased serum creatine kinase (CK) and affected several brain pathways: activation of the noradrenergic (NA-system) and DA -system in the PFC and the activation of the DA-system and an increase in c-Fos as well as a decrease in brain-derived neurotrophic factor (BDNF) in the HC. The serotonergic system (5-HT-system) was mildly altered in both areas. There was an interaction between housing and transport in serum NA and the DA-system in the HC, indicating that living conditions affected the response to stress. Multivariate analysis was able to discriminate the four animal groups. In conclusion, this work indicates that housing conditions and road transport markedly modifies the neurophysiology of pigs, and suggests that animals raised partially outdoors respond differently to transport-associated stress than animals raised indoors, indicating that they cope differently with unknown environments.

Effect of handling on neurotransmitter profile in pig brain according to fear related behaviour.

Chemical neurotransmitters (NT) are principal actors in all neuronal networks of animals. The central nervous system plays an important role in stress susceptibility and organizes the response to a stressful situation through the interaction of the dopaminergic and the serotonergic pathways, leading to the activation of the hypothalamus-pituitary-adrenal axis (HPA). This study was designed to investigate: a) the effects of stressful handling of pigs at the slaughterhouse on the neurotransmitter profile in four brain areas: amygdala, prefrontal cortex (PFC), hippocampus and hypothalamus, and b) whether the alterations in the brain NT profile after stressful handling were associated with fear, determined by the tonic immobility (TI) test. In the first place, the characterization of the NT profile allowed to distinguish the four brain areas in a principal component analysis. The most crucial pathway involved in the reaction of pigs to a stressful handling was the serotonergic system, and changes were observed in the amygdala with a decrease in serotonin (5-HT) and total indoleamines, and in the hippocampus, where this pathway was activated. Fearful and non-fearful pigs did not show significant differences in their NT profile in control conditions, but when subjected to a stressful handling in the slaughterhouse, fearful animals showed a significant variation in the serotonin pathway and, in a lesser extent, the dopamine (DA) pathway. In conclusion, the existence of an underlying biological trait - possibly fearfulness - may be involved in the pig's response toward stressful challenges, and the serotonergic system seems to play a central role in this response.

Agonist and antagonist effects of aripiprazole on D2-like receptors controlling rat brain dopamine synthesis depend on the dopaminergic tone.

BACKGROUND: The atypical antipsychotic drug aripiprazole binds with high affinity to a number of G protein coupled receptors, including dopamine D2 receptors, where its degree of efficacy as a partial agonist remains controversial. METHODS: We examined the properties of aripiprazole at D2-like autoreceptors by monitoring the changes of dopamine synthesis in adult rat brain striatal minces incubated ex vivo. The effects of the dopaminergic tone on the properties of aripiprazole were assayed by comparing a basal condition (2 mM K(+), low dopaminergic tone) and a stimulated condition (15 mM K(+), where dopamine release mimics a relatively higher dopaminergic tone). We also used 2 reference compounds: quinpirole showed a clear agonistic activity and preclamol (S-(-)-PPP) showed partial agonism under both basal and stimulated conditions. RESULTS: Aripiprazole under the basal condition acted as an agonist at D2-like autoreceptors and fully activated them at about 10 nM, inhibiting dopamine synthesis similarly to quinpirole. Higher concentrations of aripiprazole had effects not restricted to D2-like autoreceptor activation. Under the stimulated (15 mM K(+)) condition, nanomolar concentrations of aripiprazole failed to decrease dopamine synthesis but could totally block the effect of quinpirole. CONCLUSIONS: Under high dopaminergic tone, aripiprazole acts as a D2-like autoreceptor antagonist rather than as an agonist. These data show that, ex vivo, alteration of dopaminergic tone by depolarization affects the actions of aripiprazole on D2-like autoreceptors. Such unusual effects were not seen with the typical partial agonist preclamol and are consistent with the hypothesis that aripiprazole is a functionally selective D2R ligand.

The pig as an animal model for human pathologies: A proteomics perspective.

Traditional biomedical models are easy to manage in experimental facilities and allow fast and affordable basic genetic studies related to human disorders, but in some cases they do not always represent the complexity of their physiology. Translational medicine demands selected models depending on the particularities of the human disease to be investigated, reproducing as closely as possible the evolution, clinical symptoms and molecular pathways, cells or tissues involved in the dysfunction. Thus, pig models offer an alternative because of their anatomical and physiological similarities to humans and the availability of genomic, transcriptomic and, progressively more, proteomic tools for analysis of this species. Furthermore, there is a wide range of natural, selected and transgenic porcine breeds. The present review provides a summary of the applications of the pig as a model for metabolic, cardiovascular, infectious diseases, xenotransplantation and neurological disorders and an overview of the possibilities that the diverse proteomic techniques offer to study these pathologies in depth.

Transgenic over expression of nicotinic receptor alpha 5, alpha 3, and beta 4 subunit genes reduces ethanol intake in mice.

Abuse of alcohol and smoking are extensively co-morbid. Some studies suggest partial commonality of action of alcohol and nicotine mediated through nicotinic acetylcholine receptors (nAChRs). We tested mice with transgenic over expression of the alpha 5, alpha 3, beta 4 receptor subunit genes, which lie in a cluster on human chromosome 15, that were previously shown to have increased nicotine self-administration, for several responses to ethanol. Transgenic and wild-type mice did not differ in sensitivity to several acute behavioral responses to ethanol. However, transgenic mice drank less ethanol than wild-type in a two-bottle (ethanol vs. water) preference test. These results suggest a complex role for this receptor subunit gene cluster in the modulation of ethanol's as well as nicotine's effects.

Overexpression of the CHRNA5/A3/B4 genomic cluster in mice increases the sensitivity to nicotine and modifies its reinforcing effects.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated pentameric ion channels that account for the effects of nicotine. Recent genetic studies have highlighted the importance of variants of the CHRNA5/A3/B4 genomic cluster in human nicotine dependence. Among these genetic variants those found in non-coding segments of the cluster may contribute to the pathophysiology of tobacco use through alterations in the expression of these genes. To discern the in vivo effects of the cluster, we generated a transgenic mouse overexpressing the human CHRNA5/A3/B4 cluster using a bacterial artificial chromosome. Transgenic mice showed increased functional α3ß4-nAChRs in brain regions where these subunits are highly expressed under normal physiological conditions. Moreover, they exhibited increased sensitivity to the pharmacological effects of nicotine along with higher activation of the medial habenula and reduced activation of dopaminergic neurons in the ventral tegmental area after acute nicotine administration. Importantly, transgenic mice showed increased acquisition of nicotine self-administration (0.015 mg/kg per infusion) and a differential response in the progressive ratio test. Our study provides the first in vivo evidence of the involvement of the CHRNA5/A3/B4 genomic cluster in nicotine addiction through modifying the activity of brain regions responsible for the balance between the rewarding and the aversive properties of this drug.

Activation of caspase-8 by tumour necrosis factor receptor 1 is necessary for caspase-3 activation and apoptosis in oxygen-glucose deprived cultured cortical cells.

TNF-alpha has been reported to be relevant in stroke-induced neuronal death. However the precise function of TNF-alpha in brain ischemia remains controversial since there are data supporting either a detrimental or a protective effect. Here we show that TNF-alpha is released after oxygen-glucose deprivation (OGD) of cortical cultures and is a major contributor to the apoptotic death observed without affecting the OGD-mediated necrotic cell death. In this paradigm, apoptosis depends on TNF-alpha-induced activation of caspase-8 and -3 without affecting the activation of caspase-9. By using knock-out mice for TNF-alpha receptor 1, we show that the activation of both caspase-3 and -8 by TNF-alpha is mediated by TNF-alpha receptor 1. The pro-apoptotic role of TNF-alpha in OGD is restricted to neurons and microglia, since astrocytes do not express either TNF-alpha or TNF-alpha receptor 1. Altogether, these results show that apoptosis of cortical neurons after OGD is mediated by TNF-alpha/TNF-alpha receptor 1.

Effects of moderate chronic ethanol consumption on hippocampal nicotinic receptors and associative learning.

A number of studies have reported that ethanol exposure induces changes in different brain systems. The hippocampus is a brain region that is very vulnerable to ethanol exposition, which functionally results in impairment of learning and memory processes reported in heavy drinkers. Hippocampal nicotinic receptors are involved in learning and memory. In this study, we determined the effects of ethanol on the main hippocampal subtypes of neural nicotinic receptors (alpha7 and alpha4beta2) in rats non-selected for alcohol consumption, in order to check for possible changes on these receptors that could be linked with alterations in learning acquisition. Binding assays were carried out with [3H]methyllycaconitine ([3H]MLA) to study the alpha7 and [3H]nicotine to study alpha4beta2 receptors. Auto-shaping, continuous ratio and extinction procedures were used as behavioral tests. The results show that moderate chronic ethanol consumption for 10 weeks produces: (a) a decrease of both hippocampal nicotinic receptor subtypes without alterations in affinity; (b) no differences in behavioral performance between control rats and ethanol-drinking rats in auto-shaping and continuous ratio; (c) an improvement of performance of extinction paradigm. These results indicate that chronic ethanol consumption, at moderate levels, induces changes in hippocampal nicotinic receptors but does not impair acquisition and performance of new associative learning and even improves some kind of paradigms. These results may have implications in the biochemical basis of interactions between alcohol and nicotine and the effects of these drugs on behavior.

Ethanol consumption produces changes in behavior and on hippocampal alpha7 and alpha4beta2 nicotinic receptors.

Ethanol consumption produces a wide range of effects on the central nervous system, most of them related to changes in neural receptors. In vitro studies have demonstrated that ethanol increases neural nicotinic acetylcholine receptor (nnAChR) affinity for ACh (Narahashi et al., 1991) and have also reported differences in sensitivity of nnAChRs for ethanol, depending on the subunit composition of the receptor (Cardoso et al., 1999). There is evidence that ethanol induces changes in density of nnAChRs in cellular cultures (Gorbounova et al., 1998). However, there are no clear results concerning the effects of chronic ethanol on nicotinic receptors and on behavior in rats nonselected by their preference to ethanol. A number of studies demonstrate that nnAChRs participate in a variety of functions, including memory and learning processes, neurodegeneration, and neuroprotection (Picciotto et al., 2000). In the present work, we found significant alterations in the Fixed-Interval Behavioral Test, as well as in density and affinity parameters of hippocampal main subtypes of nnAChRs: alpha7 homopentamers and alpha4beta2 heteropentamers of ethanol-drinking rats nonselected by their preference to ethanol.

Contribution of caspase-mediated apoptosis to the cell death caused by oxygen-glucose deprivation in cortical cell cultures.

Several evidences suggest that cell death after cerebral ischemia involves both necrosis and apoptosis. However, it is still unknown which is the relative contribution of both types of cell death. Exposing rat cortical cultures to oxygen-glucose deprivation (OGD), we show the simultaneous presence of necrotic and apoptotic cells. The relative contribution of necrosis and apoptosis was dependent on the duration of the OGD. OGD-mediated apoptotic cell death is caspase-dependent because the addition of a pan-caspase inhibitor specifically blocked the apoptotic component of the OGD-mediated cell death. Moreover, we observed the activation of caspase-3, -7, and -9 after OGD in neurons and microglial cells. No activation of these caspases was observed in GFAP positive cells. Our results also show that calpain is related to OGD-mediated proteolysis of caspase-3 and -9 but not of caspase-7. These data suggest that different pathways could be involved in OGD-mediated caspase activation.

Histamine H2-receptor antagonist ranitidine protects against neural death induced by oxygen-glucose deprivation.

BACKGROUND AND PURPOSE: Administration of histamine receptor antagonists has been reported to produce contradictory results, either reducing or increasing neural damage induced by ischemia. In this study, we investigated the neuroprotective effects of histamine H2-receptor antagonists in an "in vitro" model of ischemia. METHODS: Cultured rat brain cortical neurons were exposed to oxygen-glucose deprivation (OGD) in the presence or absence of different histaminergic drugs. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay. Necrosis and apoptosis were quantified by staining cells with propidium iodide and Hoechst 33258. Caspase 3 activation was determined by immunocytochemistry and Western blot. RESULTS: Pretreatment with H2 antagonists effectively reduced neuronal cell death induced by OGD. Ranitidine decreased the number of necrotic and apoptotic cells. Caspase 3 activation and alteration of the neuronal cytoskeleton were also prevented by ranitidine pretreatment. The neuroprotective effect of ranitidine was still evident when added 6 hours after OGD. CONCLUSIONS: H2-receptor antagonists protected against OGD-induced neuronal death. Ranitidine attenuated cell death even when administered after OGD. These data suggest that this drug, which is currently used for the treatment of gastric ulcers, may be useful in promoting recovery after ischemia.

Retinoic acid-induced differentiation into astrocytes and glutamatergic neurons is associated with expression of functional and activable phospholipase D.

Phospholipase D (PLD) activity in mammalian cells has been associated with cell proliferation and differentiation. Here, we investigated the expression of PLD during differentiation of pluripotent embryonal carcinoma cells (P19) into astrocytes and neurons. Retinoic acid (RA)-induced differentiation increased PLD1 and PLD2 mRNA levels and PLD activity that was responsive to phorbol myristate acetate. Various agonists of membrane receptors activated PLD in RA-differentiated cells. Glutamate was a potent activator of PLD in neurons but not in astrocytes, whereas noradrenaline and carbachol increased PLD activity only in astrocytes. P19 neurons but not astrocytes released glutamate in response to a depolarizing stimulus, confirming the glutamatergic phenotype of these neurons. These results indicate upregulation of PLD gene expression associated with RA-induced neural differentiation.

Release of neurotransmitters from rat brain nerve terminals after chronic ethanol ingestion: differential effects in cortex and hippocampus.

To gain a better insight into the alterations of brain function after chronic ethanol, we measured the release of various neurotransmitters from nerve terminals of cortex and hippocampus isolated fm rats chronically fed with ethanol. The K+-evoked release of [3H]acetylcholine (ACh), f[H]dopamine (DA), [3H] glutamate(Glu) and [3H]noradrenaline (NA) was determined in superfused synaptosomes of brain cortex and hippocampus from rats exposed to the Lieber-DeCarli alcohol liquid diet for 5 weeks. In cortical synaptosomes, chronic ethanol administration did not affect the release of ACh and of DA, while significantly decreasing the release of Glu and NA. The endogenous levels of NA, DA and their metabolites were unchanged. In hippocampal synaptosomes the only effect of chronic alcohol was an increased release of Glu. It can be concluded that at presynaptic level chronic ethanol alters brain neurotransmitter systems selectively. Glutamatergic and noradrenergic nerve terminals from cortex are more vulnerable than those from hippocampus.