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At high levels, extracellular ATP operates as a “danger” molecule under pathologic conditions through purinergic receptors, including the ionotropic P2X7 receptor (P2X7R). Its endogenous activation is associated with neurodevelopmental disorders; however, its function during early embryonic stages remains largely unclear. Our objective was to determine the role of P2X7R in the regulation of neuronal outgrowth. For this purpose, we performed Sholl analysis of dendritic branches on primary hippocampal neurons and in acute hippocampal slices from WT mice and mice with genetic deficiency or pharmacological blockade of P2X7R. Because abnormal dendritic branching is a hallmark of certain neurodevelopmental disorders, such as schizophrenia, a model of maternal immune activation (MIA)-induced schizophrenia, was used for further morphologic investigations. Subsequently, we studied MIA-induced behavioral deficits in young adult mice females and males. Genetic deficiency or pharmacological blockade of P2X7R led to branching deficits under physiological conditions. Moreover, pathologic activation of the receptor led to deficits in dendritic outgrowth on primary neurons from WT mice but not those from P2X7R KO mice exposed to MIA. Likewise, only MIA-exposed WT mice displayed schizophrenia-like behavioral and cognitive deficits. Therefore, we conclude that P2X7R has different roles in the development of hippocampal dendritic arborization under physiological and pathologic conditions.SIGNIFICANCE STATEMENTOur main finding is a novel role for P2X7R in neuronal branching in the early stages of development under physiological conditions. We show how a decrease in the expression of P2X7R during brain development causes the receptor to play pathologic roles in adulthood. Moreover, we studied a neurodevelopmental model of schizophrenia and found that, at higher ATP concentrations, endogenous activation of P2X7R is necessary and sufficient for the development of positive and cognitive symptoms.

Background Immunological markers and related signaling molecules in the blood are altered in schizophrenia mouse models, in acutely relapsed patients with schizophrenia, and in persons at a clinically high risk for subsequently developing psychosis, highlighting their potential as prognostic and theranostic biomarkers. Therefore, we herein aimed to identify novel potential biomarkers in the serum that are associated with purinergic signaling. Methods To our knowledge, this is the first study to assess the correlations among the levels of human serum adenine nucleotides (ATP, ADP), adenosine, P2X7 receptor, and disease activity in patients hospitalized due to an acute relapse of schizophrenia (n = 53) and healthy controls (n = 47). In addition, to validate these findings using a reverse translational approach, we examined the same parameters in an acute phencyclidine-induced schizophrenia mouse model. Results We found consistently elevated levels of ATP, ADP, interleukin (IL)-6, and IL-10 in both schizophrenia groups compared with the controls. The levels of adenosine, IL-1β, IL-12, and C-reactive protein were also increased in the human patient samples. Moreover, ATP and ADP were significantly positively correlated with the Positive and Negative Symptom Scale item “lack of judgment and insight”; IL-1β, IL-12, and tumour necrosis factor alpha were significantly positively correlated with “tension” and “depression”; and “disorientation” and “poor attention” were correlated significantly with IL-6 and IL-8. Conclusions Our study suggests the promising potential of blood purines and inflammatory markers as future prognostic tools.

Background and purpose P2Y12 receptors regulate different forms of pain and inflammation. In this study, we investigated the participation of P2Y12 receptors in an animal model of migraine. Experimental approach We tested the effect of the centrally administered selective P2Y12 antagonist PSB-0739 and P2Y12 receptor gene (P2ry12-/- ) deficiency in acute nitroglycerin-treated mice. Additionally, platelet depletion was used to investigate the role of platelet P2Y12 receptors during migraine-like pain. Key results Nitroglycerin induced sensory hypersensitivity of C57BL/6 wild-type (P2ry12+/+ ) mice accompanied by an increase in c-fos and CGRP expression in the upper cervical spinal cord (C1-C2) and trigeminal nucleus caudalis. Similar changes were also observed in P2Y12 gene-deficient (P2ry12-/- ) mice. Prophylactic intrathecal application of PSB-0739 reversed thermal hyperalgesia and head grooming time in wild-type mice but had no effect in P2ry12-/- mice. Furthermore, PSB-0739 was also effective when applied as a post-treatment. PSB-0739 administration suppressed the expression of c-fos in C1-C2 and trigeminal nucleus caudalis, and decreased the levels of dopamine and 5-hydroxytryptamine in C1-C2 in wild-type mice. Nitroglycerin treatment itself did not change adenosine diphosphate (ADP)-induced platelet activation measured by CD62P up-regulation in wild-type mice. Platelet depletion by anti-mouse CD41 antibody and clopidogrel attenuated nitroglycerin-induced thermal hypersensitivity and head grooming time in mice. Conclusion and implications Our findings show that acute inhibition of P2Y12 receptors alleviates migraine-like pain in mice by modulating the expression of c-fos and that platelet P2Y12 receptors might contribute to this effect. Thus the blockade of P2Y12 receptors may have therapeutic potential against migraine.

International audience; Microglia, the main immunocompetent cells of the brain, regulate neuronal function, but their contribution to cerebral blood flow (CBF) regulation has remained elusive. Here, we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. Microglia establish direct, dynamic purinergic contacts with cells in the neurovascular unit that shape CBF in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in mice, which is reiterated by chemogenetically induced microglial dysfunction associated with impaired ATP sensitivity. Hypercapnia induces rapid microglial calcium changes, P2Y12R-mediated formation of perivascular phylopodia, and microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Microglial actions modulate vascular cyclic GMP levels but are partially independent of nitric oxide. Finally, microglial dysfunction markedly impairs P2Y12R-mediated cerebrovascular adaptation to common carotid artery occlusion resulting in hypoperfusion. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation, with broad implications for common neurological diseases.

P2YWe tested the effect of the centrally administered selective P2YNitroglycerin induced sensory hypersensitivity of C57BL/6 wild-type (P2ry12Our findings show that acute inhibition of P2Y

The release of [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca2+ further increased the release rates of [3H]DA and [3H]NA induced by ischemic conditions. This finding indicated that the Na+/Ca2+ exchanger (NCX), working in reverse in the absence of extracellular Ca2+, fails to trigger the influx of Ca2+ in exchange for Na+ and fails to counteract ischemia by further increasing the intracellular Na+ concentration ([Na+]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca2+ was removed. Hypothermia inhibited the excessive release of [3H]DA in response to ischemia, even in the absence of Ca2+. These findings further indicate that the NCX plays an important role in maintaining a high [Na+]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [3H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [3H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca2+]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na+/Cl−-dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.

Microglia, the main immunocompetent cells of the brain regulate neuronal function in health and disease, but their contribution to cerebral blood flow (CBF) remained elusive. Here we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. We show that microglia establish direct purinergic contacts with cells in the neurovascular unit that shape cerebral perfusion in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in the barrel cortex after whisker stimulation. We also reveal that hypercapnia, which is associated with acidification, induces microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Furthermore, the absence or dysfunction of microglia markedly impairs adaptation to hypoperfusion via P2Y12R after transient unilateral common carotid artery occlusion, which is also influenced by CX3CR1-mediated actions. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation with broad implications for common neurological diseases.

Parkinson’s disease (PD) is a chronic, progressive neurodegenerative condition; characterized with the degeneration of the nigrostriatal dopaminergic pathway and neuroinflammation. During PD progression, microglia, the resident immune cells in the central nervous system (CNS) display altered activity, but their role in maintaining PD development has remained unclear to date. The purinergic P2Y12 receptor (P2Y12R), which is exclusively expressed on the microglia in the CNS has been shown to regulate microglial activity and responses; however, the function of the P2Y12R in PD is unknown. Here we show that while pharmacological or genetic targeting of P2Y12R previous to disease onset augments acute mortality, these interventions protect against neurodegenerative cell loss and the development of neuroinflammation in vivo. Pharmacological inhibition of receptors during disease development reverses the symptoms of PD and halts disease progression. We found that P2Y12R regulate ROCK and p38 MAPK activity and control cytokine production. Understanding protective and detrimental P2Y12 receptor-mediated actions in the CNS may reveal novel approaches to control neuroinflammation and modify disease progression in PD.

Microglia take control Changes in the activity of microglia, the primary immune cells of the central nervous system, are linked with major human diseases, including stroke, epilepsy, psychiatric disorders, and neurodegeneration. Cserép et al. identified a specialized morphofunctional communication site between microglial processes and neuronal cell bodies in the mouse and the human brain (see the Perspective by Nimmerjahn). These junctions are formed at specific areas of the neuronal somatic membranes and possess a distinctive nanoarchitecture and specialized molecular composition linked to mitochondrial signaling. The junctions appear to provide a major site for microglia-neuron communication and may help to mediate the neuroprotective effects of microglia after acute brain injury. Science , this issue p. 528 ; see also p. 510

The addiction-related behavioural effects of drugs of abuse are mediated by the mesocorticolimbic monoamine systems. We investigated the effects of 3,4-methylenedioxymethamphetamine (MDMA), mephedrone, β-phenylethylamine (β-PEA) methylphenidate (MPH) on dopamine release from mouse perfused nucleus accumbens and prefrontal cortex slices. The fractional release of [3H]-dopamine was measured at rest and in response to field stimulation. The distributions of [3H]-dopamine and its metabolites were determined using high-pressure liquid chromatography. The effect of drugs on [3H]-dopamine uptake was measured in synaptosomal P2 preparations from the frontal cortex and striatum. Similar to MDMA, mephedrone β-PEA increased the resting release of [3H]-dopamine from the nucleus accumbens and prefrontal cortex in a [Ca2+]o-independent manner, and the stimulation-evoked release was also augmented. In contrast, MPH failed to affect the resting release but potentiated the release in response to axonal activity. Similar to dopamine transporter antagonist GBR 12909, mephedrone, MDMA and MPH biphasically inhibited the [3H]-dopamine uptake. The administration of GBR 12909 and nisoxetine, or lowering the bath temperature prevented MDMA, mephedrone and β-PEA from enhancing the resting, cytoplasmic release of [3H]-dopamine, indicating the role of transporters in the release process. We conclude that amphetamine-like drugs of abuse and the trace amine β-PEA excessively increase the [Ca2+]o-independent, non-vesicular release of dopamine from the cytoplasm into the extrasynaptic space and inhibit the high-affinity transporters, thereby maintaining a high ambient, non-synaptic concentration of dopamine that may tonically control the activity of neurons equipped with dopamine receptors and is likely involved in the reinforcing effects and abusive potential of amphetamines.

International audience; AimsMedian raphe region (MRR) is an important bottom-up regulatory center for various behaviors as well as vegetative functions, but detailed descriptions and links between the two are still largely unexplored.MethodsPharmacogenetics was used to study the role of MRR in social (sociability, social interaction, resident intruder test) and emotional behavior (forced swim test) parallel with some vegetative changes (biotelemetry: core body temperature). Additionally, to validate pharmacogenetics, the effect of clozapine-N-oxide (CNO), the ligand of the artificial receptor, was studied by measuring (i) serum and brainstem concentrations of CNO and clozapine; (ii) MRR stimulation induced neurotransmitter release in hippocampus; (iii) CNO induced changes in body temperature and locomotor activity.Key findingsMRR stimulation decreased locomotion, increased friendly social behavior in the resident intruder test and enhanced depressive-like behavior. The latter was accompanied by diminished decrease in core body temperature. Thirty minutes after CNO injection clozapine was predominant in the brainstem. Nonetheless, peripheral CNO injection was able to induce glutamate release in the hippocampus. CNO had no immediate (

CART (Cocaine- and Amphetamine-Regulated Transcript) peptide is a neurotransmitter naturally occurring in the CNS and found mostly in nucleus accumbens, ventrotegmental area, ventral pallidum, amygdalae and striatum, brain regions associated with drug addiction. In the nucleus accumbens, known for its significant role in motivation, pleasure, reward and reinforcement learning, CART peptide inhibits cocaine and amphetamine-induced dopamine-mediated increases in locomotor activity and behavior, suggesting a CART peptide interaction with the dopaminergic system. Thus in the present study, we examined the effect of CART (55-102) peptide on the basal, electrical field stimulation-evoked (EFS-evoked) (30V, 2Hz, 120 shocks) and returning basal dopamine (DA) release and on the release of the DA metabolites 3,4-dihydroxyphenyl acetaldehyde (DOPAL), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3,4-dihydroxyphenylethanol (DOPET), 3-methoxytyramine (3-MT) as well as on norepinephrine (NE) and dopamine-o-quinone (Daq) in isolated mouse nucleus accumbens, in a preparation, in which any CART peptide effects on the dendrites or soma of ventral tegmental projection neurons have been excluded. We further extended our study to assess the effect of CART (55-102) peptide on basal cocaine-induced release of dopamine and its metabolites DOPAL, DOPAC, HVA, DOPET and 3-MT as well as on NE and Daq. To analyze the amount of [3H]dopamine, dopamine metabolites, Daq and NE in the nucleus accumbens superfusate, a high-pressure liquid chromatography (HPLC), coupled with electrochemical, UV and radiochemical detections was used. CART (55-102) peptide, 0.1μM, added alone, exerted: (i) a significant decrease in the basal and EFS-evoked levels of extracellular dopamine (ii) a significant increase in the EFS-evoked and returning basal levels of the dopamine metabolites DOPAC and HVA, major products of dopamine degradation and (iii) a significant decrease in the returning basal levels of DOPET. At the same concentration, 0.1μM, CART (55-102) peptide did not have any effect on the release of noradrenaline. In the presence of CART (55-102) peptide, 0.1μM, the effect of cocaine, 30μM, on the basal dopamine release was inhibited and the effect on the basal DOPAC release substantially increased. To our knowledge, our findings are the first to show direct neurochemical evidence that CART (55-102) peptide plays a neuromodulatory role on the dopaminergic reward system by decreasing dopamine in the mouse nucleus accumbens and by attenuating cocaine-induced effects on dopamine release.

Because local anesthetics are known to inhibit both sodium and potassium channels, and anesthetic properties have been attributed to the former effect, we compared their effects with those of tetrodotoxin (TTX), a selective Na(+) channel inhibitor with anesthetic activity, and 4-aminopyridine (4-AP), a selective potassium channel blocker with convulsive activity, on transmitter release during rest and in response to field (axonal) stimulation using the microvolume perfusion method and isolated prefrontal cortex and spinal cord slice preparations loaded with the radioactive transmitters [(3)H]dopamine ([(3)H]DA) and [(3)H]noradrenaline ([(3)H]NA). It is also known that local anesthetics may exert analgesic effect and, rarely, some adverse effects on the central nervous system (CNS). Neurochemical evidence demonstrated that local anesthetics administered at concentrations ranging from 0.5 to 5mM, which might have been intentionally or accidentally achieved in clinical practice (e.g., during spinal and epidural anesthesia or peripheral nerve block), led to presynaptic failures during neurochemical transmission, including inhibited transmitter release associated with axonal firing and markedly enhanced extraneuronal concentrations of transmitters due to increased resting, [Ca(2+)]o-independent release. Tetrodotoxin, a toxin with selective Na(+) channel-blocking properties, inhibited the stimulation-evoked release but failed to affect the resting release. In contrast, the potassium channel inhibitor 4-AP enhanced both the resting- and action potential-evoked transmitter releases. It is concluded that effects of local anesthetics on resting catecholamine release in the spinal cord may contribute to their action during neuropathic pain relief and spinal analgesia as well as to their side effects in the CNS.

The P2X7R protein, a P2 type purinergic receptor functioning as a non-selective cation channel, is expressed in different cell types of the central nervous system in several regions of the brain. The activation of the P2X7R protein by ATP modulates excitatory neurotransmission and contributes to microglial activation, apoptosis and neuron-glia communication. Zinc is an essential micronutrient that is highly concentrated in the synaptic vesicles of glutamatergic hippocampal neurons where free zinc ions released into the synaptic cleft alter glutamatergic signal transmission. Changes in both P2X7R-mediated signaling and brain zinc homeostasis have been implicated in the pathogenesis of mood disorders. Here, we tested the hypothesis that extracellular zinc regulates P2X7R activity in the hippocampus. We observed that P2X7R is expressed in both neurons and glial cells in primary mouse hippocampal neuron-glia culture. Propidium iodide (PI) uptake through large pores formed by pannexins and P2X7R was dose-dependently inhibited by extracellular zinc ions. Calcium influx mediated by P2X7R in glial cells was also reduced by free zinc ions. Interestingly, no calcium influx was detected in response to ATP or 3'-O-(4-Benzoyl) benzoyl ATP (BzATP) in neurons despite the expression of P2X7R at the plasma membrane. Our results show that free zinc ions can modulate hippocampal glial purinergic signaling, and changes in the activity of P2X7R may contribute to the development of depression-like behaviors associated with zinc deficiency.

The P2X7R protein, a P2 type purinergic receptor functioning as a non-selective cation channel, is expressed in different cell types of the central nervous system in several regions of the brain. The activation of the P2X7R protein by ATP modulates excitatory neurotransmission and contributes to microglial activation, apoptosis and neuron-glia communication. Zinc is an essential micronutrient that is highly concentrated in the synaptic vesicles of glutamatergic hippocampal neurons where free zinc ions released into the synaptic cleft alter glutamatergic signal transmission. Changes in both P2X7R-mediated signaling and brain zinc homeostasis have been implicated in the pathogenesis of mood disorders. Here, we tested the hypothesis that extracellular zinc regulates P2X7R activity in the hippocampus. We observed that P2X7R is expressed in both neurons and glial cells in primary mouse hippocampal neuron-glia culture. Propidium iodide (PI) uptake through large pores formed by pannexins and P2X7R was dose-dependently inhibited by extracellular zinc ions. Calcium influx mediated by P2X7R in glial cells was also reduced by free zinc ions. Interestingly, no calcium influx was detected in response to ATP or 3'-O-(4-Benzoyl) benzoyl ATP (BzATP) in neurons despite the expression of P2X7R at the plasma membrane. Our results show that free zinc ions can modulate hippocampal glial purinergic signaling, and changes in the activity of P2X7R may contribute to the development of depression-like behaviors associated with zinc deficiency.

Thyroid hormone (TH) is present in the systemic circulation and thus should affect all cells similarly in the body. However, tissues have a complex machinery that allows tissue-specific optimization of local TH action that calls for the assessment of TH action in a tissue-specific manner. Here, we report the creation of a TH action indicator (THAI) mouse model to study tissue-specific TH action. The model uses a firefly luciferase reporter readout in the context of an intact transcriptional apparatus and all elements of TH metabolism and transport and signaling. The THAI mouse allows the assessment of the changes of TH signaling in tissue samples or in live animals using bioluminescence, both in hypothyroidism and hyperthyroidism. Beyond pharmacologically manipulated TH levels, the THAI mouse is sufficiently sensitive to detect deiodinase-mediated changes of TH action in the interscapular brown adipose tissue (BAT) that preserves thermal homeostasis during cold stress. The model revealed that in contrast to the cold-induced changes of TH action in the BAT, the TH action in this tissue, at room temperature, is independent of noradrenergic signaling. Our data demonstrate that the THAI mouse can also be used to test TH receptor isoform-specific TH action. Thus, THAI mouse constitutes a unique model to study tissue-specific TH action within a physiological/pathophysiological context and test the performance of thyromimetics. In conclusion, THAI mouse provides an in vivo model to assess a high degree of tissue specificity of TH signaling, allowing alteration of tissue function in health and disease, independently of changes in circulating levels of TH.

In this study the role of P2Y12 receptors (P2Y12R) was explored in rodent models of inflammatory and neuropathic pain and in acute thermal nociception. In correlation with their activity to block the recombinant human P2Y12R, the majority of P2Y12R antagonists alleviated mechanical hyperalgesia dose-dependently, following intraplantar CFA injection, and after partial ligation of the sciatic nerve in rats. They also caused an increase in thermal nociceptive threshold in the hot plate test. Among the six P2Y12R antagonists evaluated in the pain studies, the selective P2Y12 receptor antagonist PSB-0739 was most potent upon intrathecal application. P2Y12R mRNA and IL-1β protein were time-dependently overexpressed in the rat hind paw and lumbar spinal cord following intraplantar CFA injection. This was accompanied by the upregulation of TNF-α, IL-6 and IL-10 in the hind paw. PSB-0739 (0.3 mg/kg i.t.) attenuated CFA-induced expression of cytokines in the hind paw and of IL-1β in the spinal cord. Subdiaphragmatic vagotomy and the α7 nicotinic acetylcholine receptor antagonist MLA occluded the effect of PSB-0739 (i.t.) on pain behavior and peripheral cytokine induction. Denervation of sympathetic nerves by 6-OHDA pretreatment did not affect the action of PSB-0739. PSB-0739, in an analgesic dose, did not influence motor coordination and platelet aggregation. Genetic deletion of the P2Y12R in mice reproduced the effect of P2Y12R antagonists on mechanical hyperalgesia in inflammatory and neuropathic pain models, on acute thermal nociception and on the induction of spinal IL-1β. Here we report the robust involvement of the P2Y12R in inflammatory pain. The anti-hyperalgesic effect of P2Y12R antagonism could be mediated by the inhibition of both central and peripheral cytokine production and involves α7-receptor mediated efferent pathways., Highlights • Pharmacological blockade of P2Y12 receptors alleviates inflammatory and neuropathic pain. • Central inhibition of P2Y12 receptors attenuates cytokine production in the spinal cord. • Central P2Y12 receptor inhibition attenuates cytokine production in the inflamed hind paw. • α7-Receptors mediate the effect of P2Y12 receptor blockade on hyperalgesia and cytokine level. • Genetic deletion of P2Y12 receptors alleviates inflammatory, neuropathic and acute pain.

Maternal immune activation (MIA) is a principal environmental risk factor contributing to autism spectrum disorder (ASD), which compromises fetal brain development at critical periods of pregnancy and might be causally linked to ASD symptoms. We report that endogenous activation of the purinergic ion channel P2X7 (P2rx7) is necessary and sufficient to transduce MIA to autistic phenotype in male offspring. MIA induced by poly(I:C) injections to P2rx7 WT mouse dams elicited an autism-like phenotype in their offspring, and these alterations were not observed in P2rx7-deficient mice, or following maternal treatment with a specific P2rx7 antagonist, JNJ47965567. Genetic deletion and pharmacological inhibition of maternal P2rx7s also counteracted the induction of IL-6 in the maternal plasma and fetal brain, and disrupted brain development, whereas postnatal P2rx7 inhibition alleviated behavioral and morphological alterations in the offspring. Administration of ATP to P2rx7 WT dams also evoked autistic phenotype, but not in KO dams, implying that P2rx7 activation by ATP is sufficient to induce autism-like features in offspring. Our results point to maternal and offspring P2rx7s as potential therapeutic targets for the early prevention and treatment of ASD. SIGNIFICANCE STATEMENT Autism spectrum disorder (ASD) is a neurodevelopmental psychiatric disorder caused by genetic and environmental factors. Recent studies highlighted the importance of perinatal risks, in particular, maternal immune activation (MIA), showing strong association with the later emergence of ASD in the affected children. MIA could be mimicked in animal models via injection of a nonpathogenic agent poly(I:C) during pregnancy. This is the first report showing the key role of a ligand gated ion channel, the purinergic P2X7 receptor in MIA-induced autism-like behavioral and biochemical features. We show that genetic or pharmacological inhibition of both maternal and offspring P2X7 receptors could reverse the compromised brain development and autistic phenotype pointing to new possibilities for prevention and treatment of ASD.

The purpose of this study was to explore how genetic deletion and pharmacological antagonism of the P2X7 receptor (P2rx7) alter mood-related behaviour, gene expression and stress reactivity in the brain. The forced swim test (FST), tail suspension test (TST) and amphetamine-induced hyperlocomotion (AH) tests were used in wild-type (P2rx7+/+) and P2rx7-deficient (P2rx7−/−) mice. Biogenic amine levels were analysed in the amygdala and striatum, adrenocorticotropic hormone (ACTH) and corticosterone levels were measured in the plasma and pituitary after restraint stress. Chimeric mice were generated by bone marrow transplantation. A whole genome microarray analysis with real-time polymerase chain reaction validation was performed on the amygdala. In the absence of P2rx7s decreased behavioural despair in the FST, reduced immobility in the TST and attenuated amphetamine-induced hyperactivity were detected. Basal norepinephrine levels were elevated in the amygdala, whereas stress-induced ACTH and corticosterone responses were alleviated in P2rx7−/− mice. Sub-acute treatment with the selective P2rx7 antagonist, Brilliant Blue G, reproduced the effect of genetic deletion in the TST and AH test in P2rx7+/+ but not P2rx7−/− mice. No change in behavioural phenotype was observed in chimeras lacking the P2rx7 in their haematopoietic compartment. Whole genome microarray analysis indicated a widespread up- and down-regulation of genes crucial for synaptic function and neuroplasticity by genetic deletion. Here, we present evidence that the absence of P2rx7s on non-haematopoietic cells leads to a mood-stabilizing phenotype in several behavioural models and suggest a therapeutic potential of P2rx7 antagonists for the treatment of mood disorders.

The overexpression of the bovine neonatal Fc receptor (bFcRn) in transgenic (Tg) mice boosts humoral immune response with increased numbers of antigen-specific spleen cells and a potent humoral immune response against weakly immunogenic targets. One of the interesting questions surrounding this enhanced immune response is whether these Tg mice generate higher number of antigen-specific hybridomas. To address this question, we immunized these Tg mice and wild type (wt) controls with trinitrophenylated proteins, generated hybridomas and analyzed their numbers and specificities. We observed that Tg mice generated a 3-5 fold increase in antigen-specific IgG titers and had significantly larger spleens containing higher number of antigen-specific B cells and plasma cells, analyzed by ELISA and ELISPOT assays. Fusion of the isolated splenocytes with standard mouse myeloma cells (SP2/0-Ag14) resulted in a 2-4 fold elevation of hybridization frequency for the hapten, or carrier-specific IgG positive microcultures, in Tg mice compared to controls. In addition, as augmented immune reactivity leads to autoimmunity in some genetically modified mouse strains, we analyzed autoreactive antibody levels in serum samples derived from elderly bFcRn Tg mice by a protein chip assay. In contrast to the sample from the MRL/lpr mouse suffering from autoimmunity, we did not detect autoantibodies in bFcRn Tg mice or the wt controls. Based on these and our earlier data, we propose that Tg mice that overexpress bFcRn offer major advantages in monoclonal Ab production.

Diclofenac (DCF) is a widely used non-steroidal anti-inflammatory drug, which also act as a mitochondrial toxin. As it is known that selective mitochondrial complex I inhibition combined with mild oxidative stress causes striatal dopaminergic dysfunction, we tested whether DCF also compromise dopaminergic function in the striatum. [3H]Dopamine ([3H]DA) release was measured from rat striatal slices after in vitro (2 h, 10–25 μmol/L) or in vivo (3 mg/kg i.v. for 28 days) DCF treatment. In vitro treatment significantly decreased [3H]DA uptake and dopamine (DA) content of the slices. H2O2 (0.1 mmol/L)-evoked DA release was enhanced. Intracellular reactive oxygen species production was not significantly changed in the presence of DCF. After in vivo DCF treatment no apparent decrease in striatal DA content was observed and the uptake of [3H]DA into slices was increased. The intensity of tyrosine hydroxylase immunoreactivity in the striatum was highly variable, and both decrease and increase were observed in individual rats. The H2O2-evoked [3H]DA release was significantly decreased and the effluent contained a significant amount of [3H]octopamine, [3H]tyramine, and [3H]β-phenylethylamine. The ATP content and adenylate energy charge were decreased. In conclusion, whereas in vitro DCF pre-treatment resembles the effect of the mitochondrial toxin rotenone, in vivo it rather counteracts than aggravates dopaminergic dysfunction. J. Neurochem. (2008) 105, 360–368.

Several lines of recent evidence suggest that endocannabinoids affect behavior by influencing the general patterns of challenge responding. Here, we investigated the brain mechanisms underlying this phenomenon in rats. The anandamide hydrolysis inhibitor URB597 was condensed into the tip of stainless steel cannulae, which were chronically implanted slightly above the prelimbic cortex (PRL) or the basolateral amygdala (BLA), two important regions of coping and endocannabinoid action. Thereafter, we investigated behavioral responsiveness to ambient light level in the elevated plus-maze and conditioned fear tests. URB597 concentration was ~30 μg/mg protein in target areas; local brain anandamide levels increased threefold, without significant changes in 2-arachidonoylglycerol. High levels of illumination halved the time spent by controls in the open arms of the plus-maze. No similar decrease was observed in rats with URB597 implants in the PRL. High light decreased conditioned fear by 30 % in controls, but not in rats with prelimbic URB597 implants. Unresponsiveness to environmental challenges was not attributable to the anxiolytic effects of anandamide enhancement, as implants induced paradoxical anxiogenic-like effects under low light, which could be explained by effects on stimulus responsiveness rather than by effects on anxiety. URB597 implants targeting the BLA did not affect stimulus responsiveness. Our findings show that elevated prelimbic anandamide signaling leads to less environment-dependent (more autonomous) behavioral responses to challenges, which is an attribute of active coping styles. These findings are discussed in light of two emerging concepts of endocannabinoid roles, particularly “emotional homeostasis” and “active coping.”

The present study examined the metabolism of released dopamine from rat striatum upon chronic rotenone exposure. The sample separation was carried out by two-dimensional, reversed-phase and ion pair reversed-phase chromatography using on-line solid phase extraction enrichment. Reduced dopamine content and decreased extracellular level of [(3)H] and endogenous dopamine evoked by electrical stimulation indicated the injury of dopaminergic pathway. Sensitivity of dopaminergic neurons were increased to oxidative stress with enhanced release of dopamine and formation of oxidized metabolite dopamine quinone (DAQ). Utilizing multidimensional detection, EC at -100 mV reduction potential, the method has been applied for identification of DAQ and aminochrome (DAC).

7 pages.-- PMID: 16180729 [PubMed]., For 10 years, the regulatory regions of the mouse and rabbit whey acidic protein gene have been used to express heterologous proteins in the milk of transgenic mice, as well as to produce pharmaceutical proteins, on a large scale, in the milk of transgenic livestock. To date, a broad range of expression levels have been detected, and elucidation of the structure-function relationship in these regulatory regions might help to achieve high levels of expression, reproducibly. An extended 5′ regulatory region (17·6 kb v. 6·3 kb) of the rabbit whey acidic promoter resulted in an increased frequency of rabbit whey acidic protein expression in transgenic mice. However, the expression levels were low compared with the high expression levels achieved in both transgenic mice and rabbits using the heterologous κ-casein in the 6·3 kb rabbit whey acidic protein 5′ regulatory region. These results underline the importance of the 3’ downstream regulatory regions, which still need to be better characterized in the whey acidic protein gene.

It is believed that both mitochondrial dysfunction and oxidative stress play important roles in the pathogenesis of Parkinson's disease (PD). We studied the effect of chronic systemic exposure to the mitochondrial inhibitor rotenone on the uptake, content, and release of striatal neurotransmitters upon neuronal activity and oxidative stress, the latter simulated by H2O2 perfusion. The dopamine content in the rat striatum is decreased simultaneously with the progressive loss of tyrosine hydroxylase (TH) immunoreactivity in response to chronic intravenous rotenone infusion. However, surviving dopaminergic neurons take up and release only a slightly lower amount of dopamine (DA) in response to electrical stimulation. Striatal dopaminergic neurons showed increased susceptibility to oxidative stress by H2O2, responding with enhanced release of DA and with formation of an unidentified metabolite, which is most likely the toxic dopamine quinone (DAQ). In contrast, the uptake of [3H]choline and the electrically induced release of acetylcholine increased, in coincidence with a decline in its D2 receptor-mediated dopaminergic control. Thus, oxidative stress-induced dysregulation of DA release/uptake based on a mitochondrial deficit might underlie the selective vulnerability of dopaminergic transmission in PD, causing a self-amplifying production of reactive oxygen species, and thereby contributing to the progressive degeneration of dopaminergic neurons.

It was recently shown in the olfactory bulb (OB) that the response to olfactory stimulation might be related to local reinforcement mechanisms involved in discrimination of different odors. Therefore, it seemed interesting to study the effects of several drugs of abuse on the release of dopamine (DA) in the OB. Nicotine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"), and cocaine at 37 degrees C increased the release of [3H] DA from olfactory bulb slice preparations of the rats. While nicotine, amphetamine, and MDMA directly evoked DA release, cocaine, by inhibiting the reuptake processes, enhanced the electrical stimulation-evoked release. At low temperature (17 degrees C), a condition in which the transmitter uptake carriers of the plasma membrane in both the normal and reverse mode of operation are inhibited, the nicotine-evoked [3H] DA release was potentiated, whereas those evoked by amphetamine and MDMA were inhibited. At low temperature the field stimulation-evoked [3H] DA release was potentiated, but under this condition cocaine failed to increase the release. Our results show that low temperature (a) increases the concentration of extracellular DA released by Ca(2+)-dependent vesicular exocytosis elicited by nicotine, (b) inhibits the extracellular Ca(2+)-independent amphetamine- and MDMA-induced release of DA that occurs by the reverse operation of membrane carriers transporting DA from the cytoplasm of presynaptic terminals to the extraneuronal space, and (c) does not alter the inhibitory effect of cocaine on DA uptake that increases the concentration of extracellular DA released by field stimulation. The findings that the drugs of abuse tested all enhanced the release of DA in the olfactory bulb suggest that local reinforcing mechanisms may also exist in this brain area. In addition, we also show that lowering the temperature in in vitro experiments is an easy and straightforward method for separating vesicular and cytoplasmic release of transmitters, and is suitable for studying the mechanism of catecholamine release evoked by drugs of abuse. This technique may be applicable in other neurochemical studies that need inhibition of the uptake carriers without the blockade of the ligand-gated ion channels caused by reuptake inhibitor drugs.

Brain ischemia is frequently associated with oxidative stress in the reperfusion period. It is known that noradrenaline (NA) is released in excess under energy deprivation by the sodium-dependent reversal of the monoamine carrier. However, it is not known how oxidative stress affects NA release in the brain alone or in combination with energy deprivation. As a model of oxidative stress, the effect of H(2)O(2) (0.1-1.5 mM) perfusion was investigated in superfused rat hippocampal slices. It elicited a dose-dependent elevation of the release of [(3)H]NA and its tritiated metabolites as well as a simultaneous drop in the tissue energy charge. Mitochondrial inhibitors, i.e. rotenone (10 microM), and oligomycin (10 microM) in combination, also decreased the energy charge, but they had only a mild effect on [(3)H]NA release. However, when H(2)O(2) was added together with oligomycin and rotenone their effect on [(3)H]NA release was greatly exacerbated. H(2)O(2) and mitochondrial inhibitors also induced an increase in [Na(+)](i) in isolated nerve terminals, and their effect was additive. The effect of H(2)O(2) on tritium release was temperature-dependent. It was also attenuated by the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (30 microM) and (+/-)-2-amino-5-phosphonopentanoic acid (10 microM), by the nitric oxide synthase inhibitors, N omega-nitro-L-arginine methyl ester (100 microM), or 7-nitroindazole (50 microM) and by the vesicular uptake inhibitor tetrabenazine (1 microM). Our results suggest that oxidative stress releases glutamate followed by activation of postsynaptic ionotropic glutamate receptors that trigger nitric oxide production and results in a flood of NA from cytoplasmic stores. The massive elevation of extracellular NA under conditions of oxidative stress combined with mitochondrial dysfunction may provide an additional source of highly reactive free radicals thus initiating a self-amplifying cycle leading to neuronal degeneration.

Here, we investigated how nucleotides and nucleosides affect the release of tritiated purines and endogenous adenosine 5′-triphosphate (ATP) from superfused rat hippocampal slices. ATP elicited concentration-dependent [3H]purine efflux from slices preloaded with [3H]adenosine. High-performance liquid chromatography analysis of the effluent showed that the tritium label represented the whole set of adenine nucleotides and nucleosides, and ATP significantly increased the outflow of [3H]ATP. Adenosine 5′-diphosphate, adenosine, uridine, uridine 5′-triphosphate, α,β-methylene-ATP and 3′-O-(4-benzoylbenzoyl)-ATP were also active in eliciting [3H]purine release. Adenosine (300 μM) also evoked endogenous ATP efflux from the hippocampal slices. Reverse transcription-coupled-polymerase chain reaction analysis revealed that mRNAs encoding a variety of P2X and P2Y receptor proteins are expressed in the rat hippocampus. Nevertheless, neither P2 receptor (i.e. pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulphonic acid, 30 μM, suramin, 300 μM and reactive blue 2, 10 μM), nor adenosine receptor (8-cyclopentyl-1,3-dipropylxanthine, 250 nM and dimethyl-1-propargylxanthine, 250 nM) antagonists modified the effect of ATP (300 μM) to evoke [3H]purine release. The nucleoside transport inhibitors, dipyridamole (10 μM), nitrobenzylthioinosine (10 μM) and adenosine deaminase (2–10 U ml−1), but not the ecto-adenylate kinase inhibitor diadenosine pentaphosphate (200 μM) significantly reduced ATP-evoked [3H]purine efflux. In summary, we found that ATP and other nucleotides and nucleosides promote the release of one another and themselves by the nucleoside transport system. This action could have relevance during physiological and pathological elevation of extracellular purine levels high enough to reverse the nucleoside transporter. British Journal of Pharmacology (2003) 139, 623–633. doi:10.1038/sj.bjp.0705285

Acid-precipitated rabbit ‘whole casein’ was digested by trypsin, chymotrypsin, pepsin, and clostripain to screen for possible peptides with antibacterial properties. The peptide fragments were separated by reversed-phase chromatography. The collected fractions were pooled and their antibacterial properties tested against Escherichia coli, Bacillus subtilis and Staphylococcus lentus. Three antibacterial peptide fragments derived from tryptic digestion of rabbit casein were isolated and identified. Their sequences were found as follows: HVEQLLR (residues 50–56 of β-casein), ILPFIQSLFPFAER (residues 64–77 of β-casein), and FHLGHLK (residues 19–25 of αs1-casein). The three peptides were synthesized and found to exert antibacterial effect against Gram positive bacteria only. Proteolytic digestion of rabbit casein by chymotrypsin, pepsin and clostripain yielded several peptide fragments with antibacterial activity. Since antibiotic peptides can be released from casein during the digestion of milk proteins, our results suggest a possible antibacterial function of rabbit caseins. It is conceivable that antibacterial peptides can be generated by endopeptidases of the mammalian gastrointestinal tract possibly providing protection for new-born rabbits against aggression of micro-organisms.

Human clotting factor VIII is probably the largest protein to be expressed to date in the mammary gland of a transgenic animal, and it requires extensive posttranslational modification to achieve full biological activity. The mammary gland specific construct mWAP-hFVIII-MT-I was injected into the pronuclei of rabbit zygotes, and three transgenic offspring were obtained. Founder 385 showed germ-line transmission of a single integrated copy, and a homozygous line was established from this animal. The rhFVIII was transcribed and translated exclusively in the mammary gland. The activity of rhFVIII in the rabbit milk ranged from 5 to 8% of that found in normal human plasma. Results indicate the suitability of the transgenic rabbit mammary gland for rhFVIII production.

Transmitters and cotransmitters of the sympathetic nervous system are involved in the regulation of a variety of immune cell functions. However, it is not entirely clear what stimuli lead to the release of these molecules in immune organs. In this study, we investigated whether local ischemia can cause the parallel release of norepinephrine and its cotransmitter, ATP, in the spleen. Ischemic-like conditions, simulated by transient (15 min) O(2) and glucose deprivation, elicited a reversible increase in the release of both norepinephrine and purines from superfused spleen strips preloaded with [3H]norepinephrine or [3H]adenosine. HPLC analysis of the released tritium label revealed a net increase in the amount of ATP, ADP, AMP, adenosine, inosine, hypoxanthine and xanthine in response to ischemic-like condition. Selective O(2) or glucose deprivation, and Ca(2+)-free conditions differentially affected the outflow of [3H]norepinephrine and [3H]purines, indicating that they derived from different sources. The ABC transporter inhibitors glibenclamide (100 microM) and verapamil (100 microM) as well as low-temperature inhibited [3H]purine release evoked by ischemic-like conditions. Surgical denervation of the spleen reduced endogenous catecholamine content and [3H]norepinephrine uptake of the spleen, but not that of [3H]adenosine. In summary, these results demonstrate the release of norepinephrine and purines in response to an ischemic-like condition in an immune organ. Although both could provide an important source of extracellular catecholamines and purines involved at various levels of immunomodulation, the source and mechanism of norepinephrine and purine efflux seem different.

In the present study we investigated the effects of NMDA and non-NMDA glutamate receptor antagonists on the ischemia-evoked release of [ 3 H ] noradrenaline from rat spinal cord slices. An in vitro ischemia model (oxygen and glucose deprivation) was used to simulate the ischemic conditions known to cause neuronal injury. Spinal cord slices were loaded with [ 3 H ] noradrenaline and superfused with Krebs solution in a micro-organ bath. Both axonal stimulation and ischemia increased the release of [ 3 H ] noradrenaline, but the release in response to glucose and oxygen deprivation was [Ca2+]o independent. Dizocilpine (MK-801), an NMDA receptor antagonist, suppressed the release of [ 3 H ] noradrenaline produced by ischemia, while it enhanced the release of [ 3 H ] noradrenaline evoked by electrical field stimulation. In contrast, LY300168 (GYKI-53655) [(±)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylene-dioxy-5H-2.3-benzodiazepine] and its (−)isomer LY303070 (GYKI-53784) [(−)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylene-dioxy-5H-2.3-benzodiazepine] AMPA receptor antagonists, had no effect on the release of [ 3 H ] noradrenaline evoked by either electrical stimulation or ischemia. Desipramine, a noradrenaline uptake inhibitor, potentiated the release of [ 3 H ] noradrenaline evoked by ischemia, while in the absence of [Ca2+]o but under conditions when [ 3 H ] noradrenaline release was further increased, it reduced the release. Dizocilpine also decreased glutamate and aspartate release, measured by high performance liquid chromatography, during ischemia. It is concluded that glutamate release and NMDA receptors, but not AMPA receptors, are involved in the acute effect of oxygen and glucose deprivation on the excessive release of noradrenaline and that this release is not related to physiological axonal conduction.

Recent investigations have revealed that the genetic deletion of P2X7 receptors (P2rx7) results in an antidepressant phenotype in mice. However, the link between the deficiency of P2rx7 and changes in behavior has not yet been explored. In the present study, we studied the effect of genetic deletion of P2rx7 on neurochemical changes in the hippocampus that might underlie the antidepressant phenotype. P2X7 receptor deficient mice (P2rx7-/-) displayed decreased immobility in the tail suspension test (TST) and an attenuated anhedonia response in the sucrose preference test (SPT) following bacterial endotoxin (LPS) challenge. The attenuated anhedonia was reproduced through systemic treatments with P2rx7 antagonists. The activation of P2rx7 resulted in the concentration-dependent release of [(3)H]glutamate in P2rx7+/+ but not P2rx7-/- mice, and the NR2B subunit mRNA and protein was upregulated in the hippocampus of P2rx7-/- mice. The brain-derived neurotrophic factor (BDNF) expression was higher in saline but not LPS-treated P2rx7-/- mice; the P2rx7 antagonist Brilliant blue G elevated and the P2rx7 agonist benzoylbenzoyl ATP (BzATP) reduced BDNF level. This effect was dependent on the activation of NMDA and non-NMDA receptors but not on Group I metabotropic glutamate receptors (mGluR1,5). An increased 5-bromo-2-deoxyuridine (BrdU) incorporation was also observed in the dentate gyrus derived from P2rx7-/- mice. Basal level of 5-HT was increased, whereas the 5HIAA/5-HT ratio was lower in the hippocampus of P2rx7-/- mice, which accompanied the increased uptake of [(3)H]5-HT and an elevated number of [(3)H]citalopram binding sites. The LPS-induced elevation of 5-HT level was absent in P2rx7-/- mice. In conclusion there are several potential mechanisms for the antidepressant phenotype of P2rx7-/- mice, such as the absence of P2rx7-mediated glutamate release, elevated basal BDNF production, enhanced neurogenesis and increased 5-HT bioavailability in the hippocampus.

The concentrations of amino acids in the cerebrospinal fluid (CSF) (n = 20) and serum (n = 20) taken from patients with essential tremor were measured by HPLC and compared with those of controls (n = 10). Reduced concentrations of some amino acids (asparagine, glutamine, glycine, threonine, isoleucine, leucine) were observed in serum taken from patients with tremor. Significant increases were detected in the concentrations of glutamate (p < 0.001) and aspartate (p < 0.01). The general tendency of the changes in CSF and serum was similar; although the highest differences were observed in amino acid concentrations in the serum of patients with essential tremor. Opposite shifts of some amino acids were detected, in the concentrations of aspartate, serine, tyrosine, leucine, and isoleucine, which may indicate the independence of the changes in the serum from those in the CSF. This study raises the possibility that a genetically determined metabolic disorder is involved in the etiology of essential tremor that appears peripherally and, partly, centrally. The slight increase in the concentration of glutamate together with the reduced levels of GABA, glycine, and serine in CSF may form the neurochemical basis of the central oscillation observed in essential tremor.

Release of [3H]dopamine ([3H]DA) from rat striatal slices kept under hypoxic or/and glucose-free conditions was measured using a microvolume perfusion method. The corresponding changes in nucleotide content were determined by reverse-phase high-performance liquid chromatography (RPHPLC). The resting release of [3H]DA was not affected by hypoxia, but under glucose-free conditions massive [Ca2+]o-independent release of [3H]DA was observed. Hypoxia reduced the energy charge (E.C.) and the total purine content from 19.36 +/- 4.15 to 6.98 +/- 1.83 nmol/mg protein. Glucose deprivation by itself, or in combination with hypoxia, markedly reduced the levels of adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The E.C under glucose-free conditions was significantly reduced from 0.73 +/- 0.04 to 0.44 +/- 0.20. When the tissue was exposed to hypoxic and glucose-free conditions for 18 min the level of ATP was reduced to 3.15 +/- 0.11 nmol/mg protein. However, when the exposure time was 30 min the ATP level was further reduced to 1.11 +/- 0.37 nmol/mg protein. The resting release was enhanced in a [Ca2+]o-independent manner, but there was no release in response to stimulation, and tetrodotoxin did not affect the enhanced resting release, indicating that the release was not associated with axonal activity. Similarly, 50 microM ouabain, inhibitor of Na+/K(+)-activated ATPase, enhanced the release of [3H]DA at rest in a [Ca2+]o-independent manner. It seems very likely that the reduced ATP level under glucose-free conditions leads to an inhibition of the activity of Na+/K(+)-ATPase that results in reversal of the uptake processes and in [Ca2+]o-independent [3H]DA release from the axon terminals.

The effects of 5 mM NaF + 10 microM AlCl3, a direct activator of guanine nucleotide-binding proteins (G proteins), on the release of [3H]dopamine ([3H]DA), [3H]gamma-aminobutyric acid ([3H]GABA), and [3H]acethylcholine ([3H]ACh) were investigated in slices of rat striatum. When the tissue was exposed to NaF + AlCl3 the release of [3H]DA, [3H]GABA, and [3H]ACh was enhanced significantly. In a calcium-free solution the release of [3H]GABA and [3H]DA was increased by NaF+AlCl3 much more than in the presence of [Ca2+]o. In slice preparations taken from reserpinized animals, in which the vesicular storage of [3H]DA was therefore prevented, NaF + AlCl3 had no effect on [3H]DA release. HPLC analysis of the radioactivity of the perfusate showed that, in the presence of NaF + AlCl3, the content of dihydroxyphenylacetic acid (DOPAC) in perfusate samples increased significantly, while in pargyline-treated animals only the DA content was increased. Inhibition of DA carriers by nomifensine or low temperature prevented the effect of NaF + AlCl3. N-ethylmaleimide (NEM) preincubation did not modify the effect of NaF + AlCl3 on [3H]DA release Neomycin (0.1 mM), a phospholipase C (PLC) inhibitor, significantly decreased the effect of NaF + AlCl3 on [3H]DA and [3H]GABA release. The internal concentration of Ca2+ in synaptosomes was enhanced by NaF + AlCl3 in normal solution. However, [Ca2+]i was not influenced by NaF + AlCl3 in Ca(2+)-free medium. It is concluded that a non-receptor-mediated activation, by NaF + AlCl3, of the alpha-subunit of a G protein, results in a [Ca2+]o-independent release of DA and GABA, but not that of ACh.

The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits – having one extra copy of the FcRn when hemizygous and two extra copies when homozygous - showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies.

An attempt was made to study the release of acetylcholine (ACh) and noradrenaline and their presynaptic modulation in isolated slice preparations dissected from different subfields of the hippocampus: CA1, CA3 and the dentate gyrus. The slices were perfused and loaded with [3H]choline or with [3H]noradrenaline. The release in response to field stimulation was determined radiochemically and the content of transmitters was assayed by a chemiluminescent method or by HPLC combined with electrochemical detection. After 30 min of loading with [3H]choline there were marked subregional differences in the specific activity of [3H]ACh content. The highest concentration was measured in the dentate gyrus and the lowest in CA3. Evidence was obtained that in all three subfields the cholinergic axon terminals are equipped with inhibitory muscarinic autoreceptors and the noradrenergic terminals with alpha 2-autoreceptors, as indicated by an increase in transmitter release when the tissue was exposed to selective muscarinic or alpha 2-adrenoceptor antagonists. In contrast, the cholinergic boutons are not equipped with alpha 2-adrenoceptors, and noradrenergic terminals do not possess inhibitory muscarinic receptors. It is therefore concluded that while the release of both ACh and noradrenaline is controlled by negative feedback modulation, there is no possibility of establishing a presynaptic inhibitory interaction between the two.

Methods PC12 cells and primary mesencephalic neurons were used in culture, and the striatum and the substantia nigra were prepared from wild-type and P2X7 receptor knockout mice. Rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatments were applied in vitro and in vivo to reproduce neurochemical hallmarks of PD. Receptor expression, cell survival indicators, and endogenous biogenic amine, amino acid, adenine nucleotide and endocannabinoid contents were analyzed.