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This article begins with a discussion of a more-than-life-sized portrait of Jahangir (r. 1605–27) that is surrounded by verses in twenty-six cartouches. These verses are of special interest because they are the connecting link between a series of “allegorical paintings” made for Jahangir and the new concept of sovereignty devised for his father, Akbar, who had ordered the scholar Abuʼl-Fazl to develop a system of legitimization to emancipate him from the endorsement of the chief religious authorities of Islam. The pivotal terms of this new ideology, ṣūrat and maʿnī, are found throughout Abuʼl-Fazl’s Akbarnāma, and they turn up again in the cartouches of Jahangir’s portrait. To understand the meaning of these verses, it is necessary to consider the system of legitimation conceived by Abuʼl-Fazl. The verses then provide valuable information for the interpretation of some portraits of Jahangir and Shah Jahan.

We hypothesize that cortical ATP and ADP accumulating in the extracellular space, eg during prolonged network activity, contribute to a decline in cognitive performance in particular via stimulation of the G protein-coupled P2Y1 receptor (P2Y1R) subtype. Here, we report first evidence on P2Y1R-mediated control of cognitive functioning in rats using bilateral microinfusions of the selective agonist MRS2365 into medial prefrontal cortex (mPFC). MRS2365 attenuated prepulse inhibition of the acoustic startle reflex while having no impact on startle amplitude. Stimulation of P2Y1Rs deteriorated performance accuracy in the delayed non-matching to position task in a delay dependent manner and increased the rate of magazine entries consistent with both working memory disturbances and impaired impulse control. Further, MRS2365 significantly impaired performance in the reversal learning task. These effects might be related to MRS2365-evoked increase of dopamine observed by microdialysis to be short-lasting in mPFC and long-lasting in the nucleus accumbens. P2Y1Rs were identified on pyramidal cells and parvalbumin-positive interneurons, but not on tyrosine hydroxylase-positive fibers, which argues for an indirect activation of dopaminergic afferents in the cortex by MRS2365. Collectively, these results suggest that activation of P2Y1Rs in the mPFC impairs inhibitory control and behavioral flexibility mediated by increased mesocorticolimbic activity and local disinhibition.

The substantia gelatinosa (SG) of the spinal cord processes incoming painful information to ascending projection neurons. Whole-cell patch clamp recordings from SG spinal cord slices documented that in a low Ca(2+) /no Mg(2+) (low X(2+) ) external medium adenosine triphosphate (ATP)/dibenzoyl-ATP, Bz-ATP) caused inward current responses, much larger in amplitude than those recorded in a normal X(2+) -containing bath medium. The effect of Bz-ATP was antagonized by the selective P2X7 receptor antagonist A-438079. Neuronal, but not astrocytic Bz-ATP currents were strongly inhibited by a combination of the ionotropic glutamate receptor antagonists AP-5 and CNQX. In fact, all neurons and some astrocytes responded to NMDA, AMPA, and muscimol with inward current, demonstrating the presence of the respective receptors. The reactive oxygen species H2 O2 potentiated the effect of Bz-ATP at neurons but not at astrocytes. Hippocampal CA1 neurons exhibited a behavior similar to, but not identical with SG neurons. Although a combination of AP-5 and CNQX almost abolished the effect of Bz-ATP, H2 O2 was inactive. A Bz-ATP-dependent and A-438079-antagonizable reactive oxygen species production in SG slices was proven by a microelectrode biosensor. Immunohistochemical investigations showed the colocalization of P2X7-immunoreactivity with microglial (Iba1), but not astrocytic (GFAP, S100β) or neuronal (MAP2) markers in the SG. It is concluded that SG astrocytes possess P2X7 receptors; their activation leads to the release of glutamate, which via NMDA- and AMPA receptor stimulation induces cationic current in the neighboring neurons. P2X7 receptors have a very low density under resting conditions but become functionally upregulated under pathological conditions.

Unveiling the mechanisms participating in the damage and repair of traumatic brain injury (TBI) is fundamental to develop new therapies. The P2Y-like GPR17 receptor has recently emerged as a sensor of damage and a key actor in lesion remodeling/repair in the rodent brain, but its role in humans is totally unknown. Here, we characterized GPR17 expression in brain specimens from seven intensive care unit TBI patients undergoing neurosurgery for contusion removal and from 28 autoptic TBI cases (and 10 control subjects of matched age and gender) of two university hospitals. In both neurosurgery and autoptic samples, GPR17 expression was strong inside the contused core and progressively declined distally according to a spatio-temporal gradient. Inside and around the core, GPR17 labeled dying neurons, reactive astrocytes, and activated microglia/macrophages. In peri-contused parenchyma, GPR17 decorated oligodendrocyte precursor cells (OPCs) some of which had proliferated, indicating re-myelination attempts. In autoptic cases, GPR17 expression positively correlated with death for intracranial complications and negatively correlated with patients’ post-traumatic survival. Data indicate lesion-specific sequential involvement of GPR17 in the (a) death of irreversibly damaged neurons, (b) activation of microglia/macrophages remodeling the lesion, and (c) activation/proliferation of multipotent parenchymal progenitors (both reactive astrocytes and OPCs) starting repair processes. Data validate GPR17 as a target for neurorepair and are particularly relevant to setting up new therapies for TBI patients.

Drug addiction is a chronic, relapsing disease caused by neurochemical and molecular changes in the brain. In this human autopsy study qualitative and quantitative changes of glial fibrillary acidic protein (GFAP)-positive astrocytes in the hippocampus of 26 lethally intoxicated drug addicts and 35 matched controls are described. The morphological characterization of these cells reflected alterations representative for astrogliosis. But, neither quantification of GFAP-positive cells nor the Western blot analysis indicated statistical significant differences between drug fatalities versus controls. However, by semi-quantitative scoring a significant shift towards higher numbers of activated astrocytes in the drug group was detected. To assess morphological changes quantitatively, graph-based representations of astrocyte morphology were obtained from single cell images captured by confocal laser scanning microscopy. Their underlying structures were used to quantify changes in astroglial fibers in an automated fashion. This morphometric analysis yielded significant differences between the investigated groups for four different measures of fiber characteristics (Euclidean distance, graph distance, number of graph elements, fiber skeleton distance), indicating that, e.g., astrocytes in drug addicts on average exhibit significant elongation of fiber structures as well as two-fold increase in GFAP-positive fibers as compared with those in controls. In conclusion, the present data show characteristic differences in morphology of hippocampal astrocytes in drug addicts versus controls and further supports the involvement of astrocytes in human pathophysiology of drug addiction. The automated quantification of astrocyte morphologies provides a novel, testable way to assess the fiber structures in a quantitative manner as opposed to standard, qualitative descriptions.

The development of appropriate models assessing the potential of substances for regeneration of neuronal circuits is of great importance. Here, we present procedures to analyze effects of substances on fiber outgrowth based on organotypic slice co-cultures of the nigrostriatal dopaminergic system in combination with biocytin tracing and tyrosine hydroxylase labeling and subsequent automated image quantification. Selected phosphodiesterase inhibitors (PDE-Is) were studied to identify their potential growth-promoting capacities. Immunohistochemical methods were used to visualize developing fibers in the border region between ventral tegmental area/substantia nigra co-cultivated with the striatum as well as the cellular expression of PDE2A and PDE10. The quantification shows a significant increase of fiber density in the border region induced by PDE2-Is (BAY60-7550; ND7001), comparable with the potential of the nerve growth factor and in contrast to PDE10-I (MP-10). Analysis of tyrosine hydroxylase-positive fibers indicated a significant increase after treatment with BAY60-7550 and nerve growth factor in relation to dimethyl sulfoxide. Additionally, a dose-dependent increase of intracellular cGMP levels in response to the applied PDE2-Is in PDE2-transfected HEK293 cells was found. In summary, our findings show that PDE2-Is are able to significantly promote axonal outgrowth in organotypic slice co-cultures, which are a suitable model to assess growth-related effects in neuro(re)generation.

Astrocytes are fundamental for central nervous system (CNS) physiology and are the fulcrum of neurological diseases. Astroglial cells control development of the nervous system, regulate synaptogenesis, maturation, maintenance and plasticity of synapses and are central for nervous system homeostasis. Astroglial reactions determine progression and outcome of many neuropathologies and are critical for regeneration and remodelling of neural circuits following trauma, stroke, ischaemia or neurodegenerative disorders. They secrete multiple neurotransmitters and neurohormones to communicate with neurones, microglia and the vascular walls of capillaries. Signalling through release of ATP is the most widespread mean of communication between astrocytes and other types of neural cells. ATP serves as a fast excitatory neurotransmitter and has pronounced long-term (trophic) roles in cell proliferation, growth, and development. During pathology, ATP is released from damaged cells and acts both as a cytotoxic factor and a proinflammatory mediator, being a universal "danger" signal. In this review, we summarise contemporary knowledge on the role of purinergic receptors (P2Rs) in a variety of diseases in relation to changes of astrocytic functions and nucleotide signalling. We have focussed on the role of the ionotropic P2X and metabotropic P2YRs working alone or in concert to modify the release of neurotransmitters, to activate signalling cascades and to change the expression levels of ion channels and protein kinases. All these effects are of great importance for the initiation, progression and maintenance of astrogliosis-the conserved and ubiquitous glial defensive reaction to CNS pathologies. We highlighted specific aspects of reactive astrogliosis, especially with respect to the involvement of the P2X(7) and P2Y(1)R subtypes. Reactive astrogliosis exerts both beneficial and detrimental effects in a context-specific manner determined by distinct molecular signalling cascades. Understanding the role of purinergic signalling in astrocytes is critical to identifying new therapeutic principles to treat acute and chronic neurological diseases.

Effective therapeutic measures against the development of brain edema, a life-threatening complication of cerebral ischemia, are necessary to improve the functional outcome for the patient. Here, we identified a beneficial role of purinergic receptor P2X7 activation in acute ischemic stroke. Involvement of P2X7 in the development of neurological deficits, infarct size, brain edema, and glial responses after ischemic cerebral infarction has been analyzed. Neurologic evaluation, magnetic resonance imaging, and immunofluorescence assays were used to characterize the receptor's effect on the disease progress during 72 h after transient middle cerebral artery occlusion (tMCAO). Sham-operated animals were included in all experiments for control purposes. We found P2X7-deficient mice to develop a more prominent brain edema with a trend towards more severe neurological deficits 24 h after tMCAO. Infarct sizes, T2 times, and apparent diffusion coefficients did not differ significantly between wild-type and P2X7(-/-) animals. Our results show a characteristic spatial distribution of reactive glia cells with strongly attenuated microglia activation in P2X7(-/-) mice 72 h after tMCAO. Our data indicate that P2X7 exerts a role in limiting the early edema formation, possibly by modulating glial responses, and supports later microglia activation.

Long-term depression (LTD) is a form of synaptic plasticity that may contribute to information storage in the central nervous system. Here we report that LTD can be elicited in layer 5 pyramidal neurons of the rat prefrontal cortex by pairing low frequency stimulation with a modest postsynaptic depolarization. The induction of LTD required the activation of both metabotropic glutamate receptors of the mGlu1 subtype and voltage-sensitive Ca(2+) channels (VSCCs) of the T/R, P/Q and N types, leading to the stimulation of intracellular inositol trisphosphate (IP3) receptors by IP3 and Ca(2+). The subsequent release of Ca(2+) from intracellular stores activated the protein phosphatase cascade involving calcineurin and protein phosphatase 1. The activation of purinergic P2Y(1) receptors blocked LTD. This effect was prevented by P2Y(1) receptor antagonists and was absent in mice lacking P2Y(1) but not P2Y(2) receptors. We also found that activation of P2Y(1) receptors inhibits Ca(2+) transients via VSCCs in the apical dendrites and spines of pyramidal neurons. In addition, we show that the release of ATP under hypoxia is able to inhibit LTD by acting on postsynaptic P2Y(1) receptors. In conclusion, these data suggest that the reduction of Ca(2+) influx via VSCCs caused by the activation of P2Y(1) receptors by ATP is the possible mechanism for the inhibition of LTD in prefrontal cortex.

Nucleotides play an important role in brain development and may exert their action via ligand-gated cationic channels or G protein-coupled receptors. Patch-clamp measurements indicated that in contrast to AMPA, ATP did not induce membrane currents in human midbrain derived neuronal progenitor cells (hmNPCs). Various nucleotide agonists concentration-dependently increased [Ca 2+ ] i as measured by the Fura-2 method, with the rank order of potency ATP > ADP > UTP > UDP. A Ca 2+ -free external medium moderately decreased, whereas a depletion of the intracellular Ca 2+ storage sites by cyclopiazonic acid markedly depressed the [Ca 2+ ] i transients induced by either ATP or UTP. Further, the P2Y 1 receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y 1,2,12 antagonistic suramin only slightly blocked the action of ATP, but strongly inhibited that of UTP. In agreement with this finding, UTP evoked the release of ATP from hmNPCs in a suramin-, but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of P2Y 1,2,4 -immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y 2 receptor-activation and thereby causes [Ca 2+ ] i transients by stimulating a P2Y 1 -like receptor.

The present study investigated the pharmacological properties of excitatory P2X receptors and P2X2 and P2X5 receptor subunit expression in rat-cultured thoracolumbar sympathetic neurons. In patch-clamp recordings, ATP (3–1000 µm; applied for 1 s) induced inward currents in a concentration-dependent manner. Pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS; 30 µm) counteracted the ATP response. In contrast to ATP, α,β-meATP (30 µm; for 1 s) was virtually ineffective. Prolonged application of ATP (100 µm; 10 s) induced receptor desensitization in a significant proportion of sympathetic neurons in a manner typical for P2X2−2 splice variant-mediated responses. Using single-cell RT-PCR, P2X2, P2X2−2 and P2X5 mRNA expression was detectable in individual tyrosine hydroxylase-positive neurons; coexpression of both P2X2 isoforms was not observed. Laser scanning microscopy revealed both P2X2 and P2X5 immunoreactivity in virtually every TH-positive neuron. P2X2 immunoreactivity was largely distributed over the cell body, whereas P2X5 immunoreactivity was most distinctly located close to the nucleus. In summary, the present study demonstrates the expression of P2X2, P2X2−2 and P2X5 receptor subunits in rat thoracolumbar neurons. The functional data in conjunction with a preferential membranous localization of P2X2/P2X2−2 compared with P2X5 suggest that the excitatory P2X responses are mediated by P2X2 and P2X2−2 receptors. Apparently there exist two types of P2X2 receptor-bearing sympathetic neurons: one major population expressing the unspliced isoform and another minor population expressing the P2X2−2 splice variant.

Wild-type human (h) P2X(3) receptors expressed in HEK293 cells responded to the prototypic agonist alpha,beta-methylene ATP (alpha,beta-meATP) with rapidly desensitizing inward currents and an increase in the intracellular Ca(2+) concentration. In contrast to electrophysiological recordings, Ca(2+) microfluorimetry showed a lower maximum of the concentration-response curve of alpha,beta-meATP in the transiently than in the permanently transfected HEK293 cells. However, the concentrations causing 50% of the maximum possible effect (EC(50) values) were identical, when measured with either method. In order to determine the role of certain conserved, positively charged amino acids in the nucleotide binding domains (NBD-1-4) of hP2X(3) receptors for agonist binding, the lysine-63, -65, -176 and -299 as well as the arginine-281 and -295 residues were substituted by the neutral amino acid alanine. We observed no effect of alpha,beta-meATP at the K63A, K176A, R295A, and K299A mutants, and a marked decrease of agonist potency at the K65A and R281A mutants. The P2X(3) receptor antagonist 2',3'-O-trinitrophenyl-ATP (TNP-ATP) blocked the effect of alpha,beta-meATP at the wild-type hP2X(3) receptor with lower affinity than at the mutant K65A, indicating an interference of this mutation with the docking of the antagonist with its binding sites. The use of confocal fluorescence microscopy in conjunction with an antibody raised against the extracellular loop of the hP2X(3) receptor documented the expression of all mutants in the plasma membrane of HEK293 cells. Eventually, we modelled the possible agonist and antagonist binding sites NBD-1-4 of the hP2X(3) subunit by using structural bioinformatics. This model is in complete agreement with the available data and integrates results from mutagenesis studies with geometry optimization of the tertiary structure predictions of the receptor.

Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury, may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased immunoreactivity (IR) for P2X(1,2,4,7) and P2Y(1,2,4,6,12) receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X(7) receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X(7) receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application of the agonists alpha,betameATP, ADPbetaS, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified and activated microglial cells, whereas UTPgammaS appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally applied P2X(7) receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation of P2X(7)-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects.

The present study was aimed to clarify the role of purinergic signalling in the regulation of ingestion behaviour. The ATP/ADP analogues 2-methylthioATP (2-MeSATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPpS) increased the food intake after intracerebroventricular infusion in 18-h food-deprived rats. This effect was abolished by pretreatment with the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) or the selective P2Y 1 receptor antagonist MRS 2179, respectively. The stimulation of food intake mediated by ADPpS was also blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor N W -nitro-L-arginine methylester (L-NAME), as well as with the inhibitor of the soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), suggesting that the orexigenic effect seems to be closely related with the ensuing formation of nitric oxide. The immunohistochemical staining indicating a co-localization of P2Y 1 receptor- and nNOS-immunoreactivities in a population of neurons in the ventromedial hypothalamic nucleus (VMH) agrees with this assumption. Further experiments with the direct local application of these compounds into the VMH and lateral hypothalamic nucleus (LH) show that the stimulation of P2Y 1 receptors in these functionally antagonistic brain regions exerts an increased food intake. Hence, different signal transduction mechanisms may operate in the VMH and LH. Our assumption is supported by distinct effects of the NOS inhibitor L-NAME in these two hypothalamic nuclei. The present data suggest that ATP/ADP, acting as extracellular signal molecules in the rat brain, are involved in the regulation of food intake, possibly depending on P2Yi-receptor-mediated nitric oxide production.

After acute injury of the central nervous system extracellular adenosine 5'-triphosphate (ATP) can reach high concentrations as a result of cell damage and subsequent increase in membrane permeability. Released ATP may act as a toxic agent, which causes cellular degeneration and death, mediated through P2X and P2Y receptors. Mechanisms underlying the various effects of purinoceptor modulators in models of cerebral damage are still uncertain. In the present study the effect of P2 receptor inhibition after permanent middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats was investigated. Rats received either the non-selective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) or artificial cerebrospinal fluid (ACSF) as control by the intracerebroventricular route. First, these treatments were administered 10 min before MCAO and subsequently twice daily for 1 or 7 days after MCAO. The functional recovery of motor and cognitive deficits was tested at an elevated T-labyrinth. The PPADS-treated group showed a significant reduction of paresis-induced sideslips compared with ACSF-treated animals. Infarct volume was reduced in the PPADS group in comparison with the ACSF group. A significant decrease in intermediately and profoundly injured cells in favour of intact cells in the PPADS group was revealed by quantification of celestine blue/acid fuchsin-stained cells in the peri-infarct area. The data provide further evidence for the involvement of P2 receptors in the pathophysiology of cerebral ischaemia in vivo. The inhibition of P2 receptors at least partially reduces functional and morphological deficits after an acute cerebral ischaemic event.

ATP-sensitive P2 receptors are suggested to play an important role in the cerebral signal transduction. We examined the expression of the P2Y1 receptor and the possibly downstream-related neuronal nitric oxide synthase (nNOS) in the hypothalamus of rats food-restricted for 3 or 10 days and rats refed after a restriction of 10 days. The restriction caused a reduction of the body weight and plasma triacylglyceride, an increase of non-esterified fatty acid levels correlating with a decrease of leptin levels and an enhancement of plasma corticosterone. All changes returned to basal levels after refeeding. The restriction induced an enhanced intake within 30 min after food presentation and a reduction in the latency. Interestingly, the latter was not abolished by refeeding. The daily food intake induced by refeeding was enhanced at the first day only. The expression of hypothalamic P2Y1 receptor/nNOS mRNA and protein and of leptin receptor mRNA were enhanced after restricted feeding. These changes were abolished after 3 days of refeeding. Immunofluorescence studies indicated that P2Y1 receptor and nNOS immunoreactivities are present in the dorsomedial, ventromedial and lateral hypothalamus and in the nucleus arcuatus. P2Y1 receptor-positive cells were partially also nNOS-positive. The P2Y1 receptor labeling was restricted to cell bodies of obviously non-glial cells, whereas nNOS labeling could be detected also at cellular processes of these cells. In the nucleus arcuatus, astrocytes were identified, expressing P2Y1 receptors at cell bodies and cellular processes. The data suggest that restricted feeding may enhance the sensitivity of the hypothalamus to extracellular ADP/ATP by regulation of the expression of P2Y1 receptors and possibly of their signal transduction pathway via nitric oxide production.

Extracellular adenosine 5'-triphosphate (ATP) was proposed to be an activity-dependent signaling molecule that regulates glia-glia and glia-neuron communications. ATP is a neurotransmitter of its own right and, in addition, a cotransmitter of other classical transmitters such as glutamate or GABA. The effects of ATP are mediated by two receptor families belonging either to the P2X (ligand-gated cationic channels) or P2Y (G protein-coupled receptors) types. P2X receptors are responsible for rapid synaptic responses, whereas P2Y receptors mediate slow synaptic responses and other types of purinergic signaling involved in neuronal damage/regeneration. ATP may act at pre- and postsynaptic sites and therefore, it may participate in the phenomena of long-term potentiation and long-term depression of excitatory synaptic transmission. The release of ATP into the extracellular space, e.g., by exocytosis, membrane transporters, and connexin hemichannels, is a widespread physiological process. However, ATP may also leave cells through their plasma membrane damaged by inflammation, ischemia, and mechanical injury. Functional responses to the activation of multiple P2 receptors were found in neurons and glial cells under normal and pathophysiological conditions. P2 receptor-activation could either be a cause or a consequence of neuronal cell death/glial activation and may be related to detrimental and/or beneficial effects. The present review aims at demonstrating that purinergic mechanisms correlate with the etiopathology of brain insults, especially because of the massive extracellular release of ATP, adenosine, and other neurotransmitters after brain injury. We will focus in this review on the most important P2 receptor-mediated neurodegenerative and neuroprotective processes and their beneficial modulation by possible therapeutic manipulations.

Extracellular purines act as neuromodulators on transmitter release and may exert toxic effects at higher concentrations. In microdialysis studies, endogenous ATP facilitated the extracellular concentration of glutamate in the nucleus accumbens (NAc) of rats. Additionally, P2 receptors are involved in astrogliosis in vivo after a stab wound injury in the same region, suggesting that these receptors, preferentially the metabotropic P2Y(1) receptor subtype, mediate also trophic responses. Two sets of experimental findings support the involvement of purinergic and glutamatergic mechanisms in the response of brain to mechanical damage. First, in the present studies, the initial time course of extracellular ATP and glutamate was analyzed after a mechanical injury. The concentration of ATP in microdialysates was elevated only in the first 15-min sample whereas glutamate returned to a basal concentration not before a 90-min period had elapsed. We suggest, that the acute injury-evoked stimulation of P2 receptors contributes to glutamate-mediated excitotoxicity. Second, the expression of P2Y(1) receptors and their possible relation to glutamatergic structures, identified by neuronal vesicular glutamate transporters (VGLUTs), were elucidated in non-treated and mechanically injured animals after 4 days. The number of P2Y(1)-positive cells was significantly increased after injury. Furthermore, P2Y(1) receptor-labeled cells do not exhibit immunoreactivity for VGLUT1 and VGLUT2 without and after injury. However, after injury, a co-expression of the P2Y(1) receptor on VGLUT3-immunopositive cells in the NAc was observed. No VGLUT1-, 2- and 3-immunoreactivity was found on P2Y(1)-positive glial fibrillary acidic protein-immunopositive astrocytes at both conditions. Our data suggest that the expression of P2Y(1) receptors at neurons and astrocytes is modulated in response to cerebral injury. It can be assumed, that the enhanced sensitivity of neurons to purinergic signaling may be related directly or indirectly to changes of the glutamatergic transmission.

Extracellular ATP might act as a trophic factor on growing axons during development of the CNS via P2 receptors. In the present study the postnatal presence of selected P2 receptor subtypes was analyzed and their putative trophic capacity in entorhino-hippocampal slice co-cultures of mouse brain was tested. The effect of the P2 receptor ligands 2-methylthioadenosine-5′-triphosphate (P2X/Y receptor agonist) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (P2X/Y receptor antagonist) on axonal growth and fiber density of biocytin-labeled hippocampal projections was compared both with untreated cultures and with cultures treated with artificial cerebrospinal fluid. After 10 days in vitro , double immunofluorescence labeling revealed the expression of P2X 1 , P2X 2 , P2X 4 as well as P2Y 1 and P2Y 2 receptors in the examined regions of entorhinal fiber termination. Further, quantitative analysis of identified biocytin-traced entorhinal fibers showed a significant increase in fiber density in the dentate gyrus after incubation of the slices with the P2 receptor agonist 2-methylthioadenosine-5′-triphosphate. This neurite outgrowth promoting effect was completely abolished by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid. Our in vitro data indicate that ATP via its P2X and P2Y receptors can shape hippocampal connectivity during development.

Chronic exposure to heroin is known to cause cognitive deficits. However, little is known about the underlying molecular mechanisms. It has been suggested that opiate-induced neurotoxicity as well as impaired plasticity and regeneration may be relevant. One of the target regions where regeneration still can be observed in the adult brain is the hippocampus. Since polysialic acid neural cell adhesion molecule is regarded as one of the key players involved in plasticity and regeneration of neural tissue, we analyzed polysialic acid neural cell adhesion molecule expression in the fascia dentate hilus of the human hippocampus of 29 lethally intoxicated heroin addicts and matched controls. Immunohistochemistry with an antibody directed against polysialic acid neural cell adhesion molecule revealed its expression in differently sized cells which could be identified as neurons and glial cells. We observed an increase in the percentage of polysialic acid neural cell adhesion molecule positive neurons in hippocampal hilus of heroin addicts compared with controls (P = 0.001).Interestingly, we also observed polysialic acid neural cell adhesion molecule expression in glial cells as evidenced by double immunofluorescence with glial fibrillary acidic protein and polysialic acid neural cell adhesion molecule using confocal laser scanning microscopy. The fraction of polysialic acid neural cell adhesion molecule positive glial cells was also higher in heroin addicts compared with controls (P = 0.009). In addition, within the group of addicts morphine blood concentrations showed a positive correlation with the percentage of polysialic acid neural cell adhesion molecule positive neurons (P = 0.04; r = 0.547). In conclusion, we observed an increase in polysialic acid neural cell adhesion molecule positive neurons and glial cells in hippocampi of heroin addicts. This might reflect an attempt to repair cell damage due to heroin exposure.

Neuronally enriched primary cerebrocortical cultures were exposed to glucose-free medium saturated with argon (in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline-associated protein 2 (MAP2)-IR neurons or of glial fibrillary acidic protein (GFAP)-IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP- and 2′-3′-O-(4-benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP)-induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G-sensitive, P2X7 receptor-mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP- and BzATP-induced small inward currents recorded from a subset of non-pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol-induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre-incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non-pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor-mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways.

The presence of ionotropic P2X receptors, targets of ATP in fast synaptic transmission, as well as metabotropic P2Y receptors, known to activate K+ currents in cultured neostriatal neurones, was investigated in medium-spiny neurones and cholinergic interneurones contained in neostriatal brain slices from 5–26-day-old rats. In these cells, adenosine-5′-triphosphate (ATP) (100–1000 μM), 2-methylthioadenosine-5′-triphosphate (2MeSATP), α,β-methyleneadenosine-5′-triphosphate (α,βmeATP, 30–300 μM, each) and adenosine-5′-O-(3-thiotriphosphate (ATPγS) (100 μM) failed to evoke P2X receptor currents even when 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 μM), apyrase (10 U ml−1) or intracellular Cs+ was used to prevent occluding effects of the ATP breakdown product adenosine, desensitisation of P2X receptors by endogenous ATP and an interference with the activation of K+ channels, respectively. P2X receptor agonists were also ineffective in outside-out patches withdrawn from the brain slice tissue. Muscimol (10 μM) evoked GABAA receptor-mediated currents under all these conditions. When used as a control, locus coeruleus neurones responded with P2X receptor-mediated currents to ATP (300 μM), 2MeSATP and α,βmeATP (100 μM, each). ATP and adenosine-5′-diphosphate (ADP) (100 μM, each) did not activate K+ currents in the neostriatal neurones. Despite the observed lack of function, P2X2 and P2Y1 immunofluorescence was found in roughly 50% of the medium-spiny neurones and cholinergic interneurones. A role of ATP in synaptic transmission to striatal medium-spiny neurones and cholinergic interneurones appears unlikely, however, the otherwise silent P2X and P2Y receptors may gain functionality under certain yet unknown conditions. British Journal of Pharmacology (2004) 143, 119–131. doi:10.1038/sj.bjp.0705916

Large amounts of adenosine 5'-triphosphate (ATP) released from cellular sources under pathological conditions such as ischemia may activate purinoceptors of the P2X and P2Y types. In the present study, the expression of the P2X7 receptor-subtype in the brain cortex of spontaneously hypertensive rats was investigated using a permanent focal cerebral ischemia model. Immunocytochemistry with antibodies raised against the intracellular C-terminus of the P2X7 receptor showed a time-dependent upregulation of labeled cells in the peri-infarct region after right middle cerebral artery occlusion (MCAO) in comparison to controls. Double immunofluorescence visualized with confooal laser scanning microscopy indicated the localization of the P2X7 receptor after ischemia on microglial cells (after 1 and 4 days), on tubulin betaIII-labeled neurons (after 4 and 7 days), and on glial fibrillary acidic protein (GFAP)-positive astrocytes (after 4 days). In the following experiments, changes occurring 4 days after MCAO were investigated in detail. Western blot analysis of the cortical tissue around the area of necrosis indicated an increase in the P2X7 receptor protein. Immunoelectron microscopy revealed the receptor localization on synapses (presynaptically), on dendrites, as well as on the nuclear membrane of neurons (postsynaptically) and glial cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling in combination with P2X7 receptor immunocytochemistry indicated a co-expression on the apoptotic cells. Active caspase 3 was especially observed on GFAP-positive astrocytes. In conclusion, the present data demonstrate a postischemic, time-dependent upregulation of the P2X7 receptor-subtype on neurons and glial cells and suggest a role for this receptor in the pathophysiology of cerebral ischemia in vivo.

Patch-clamp recordings from small-diameter rat dorsal root ganglion (DRG) neurons maintained in culture demonstrated preferential inhibition by ATP of high-voltage-activated, but not low-voltage-activated, Ca2+currents (ICa). The rank order of agonist potency was UTP > ADP > ATP. ATP depressed the ω-conotoxin GVIA-sensitive N-type current only. Pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) and 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetraammonium, two P2Y1receptor antagonists, almost abolished the ATP-induced inhibition. Both patch-clamp recordings and immunocytochemistry coupled with confocal laser microscopy indicated a colocalization of functional P2X3and P2Y1receptors on the same DRG neurons. Because the effect of ATP was inhibited by intracellular guanosine 5′-O-(2-thiodiphosphate) or by applying a strongly depolarizing prepulse, P2Y1receptors appear to blockICaby a pathway involving the βγ subunit of a Gq/11protein. Less efficient buffering of the intracellular Ca2+concentration ([Ca2+]i) by reducing the intrapipette EGTA failed to interfere with the ATP effect. Fura-2 microfluorimetry suggested that ATP raised [Ca2+]iby a Gα-mediated release from intracellular pools and simultaneously depressed the high external potassium concentration-induced increase of [Ca2+]iby inhibitingICavia Gβγ. Adenosine 5′-O-(2-thiodiphosphate) inhibited dorsal root-evoked polysynaptic population EPSPs in the hemisected rat spinal cord and prolonged the nociceptive threshold on intrathecal application in the tail-flick assay. These effects were not antagonized by PPADS. Hence, P2Y receptor activation by ADP, which is generated by enzymatic degradation of ATP, may decrease the release of glutamate from DRG terminals in the spinal cord and thereby partly counterbalance the algogenic effect of ATP.

1. N-type Ca(2+) channel modulation by an endogenous P2Y receptor was investigated by the whole-cell patch-clamp method in HEK 293 cells transfected with the functional rabbit N-type calcium channel. 2. The current responses (I(Ca(N))) to depolarizing voltage steps were depressed by ATP in a concentration-dependent manner. Inclusion of either guanosine 5'-O-(3-thiodiphosphate) or pertussis toxin into the pipette solution as well as a strongly depolarizing prepulse abolished the inhibitory action of ATP. 3. In order to identify the P2Y receptor subtype responsible for this effect, several preferential agonists and antagonists were studied. Whereas the concentration-response curves of ADP and adenosine 5'-O-(2-thiodiphosphate) indicated a higher potency of these agonists than that of ATP, alpha,beta-methylene ATP, UTP and UDP were considerably less active. The effect of ATP was abolished by the P2Y receptor antagonists suramin and N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, 2'deoxy-N(6)-methyladenosine-3',5'-diphosphate or 2-methylthio AMP. 4. Using reverse transcription and polymerase chain reaction, mRNA for the P2Y(1), P2Y(4), P2Y(6), P2Y(11) and P2Y(13) receptor subtypes, but not the P2Y(2), and P2Y(12) subtypes, was detected in HEK 293 cells. 5. Immunocytochemistry confirmed the presence of P2Y(1), and to a minor extent that of P2Y(4), but not of P2Y(2) receptors. 6. Hence, it is tempting to speculate that P2Y(13) receptors may inhibit N-type Ca(2+) channels via the betagamma subunits of the activated G(i) protein.

The present study was aimed at characterizing alterations of the nucleotide content and morphological state of rat corticoencephalic cell cultures subjected to metabolic damage and treatment with modulators of mitochondrial ATP-dependent potassium channels (mitoK(ATP)). In a first series of experiments, in vitro ischemic changes of the contents of purine and pyrimidine nucleoside diphosphates and triphosphates were measured by high performance liquid chromatography (HPLC) and the corresponding histological alterations were determined by celestine blue/acid fuchsin staining. As an ischemic stimulus, incubation with a glucose-free medium saturated with argon was used. Ischemia decreased the levels of adenosine, guanine and uridine triphosphate (ATP, GTP, UTP) and increased the levels of the respective dinucleotides ADP and UDP, whereas the GDP content was not changed. Both 5-hydroxydecanoate (5-HD) and diazoxide failed to alter the contents of nucleoside diphosphates and triphosphates, when applied under normoxic conditions. 5-HD (30 microM) prevented the ischemia-induced changes of nucleotide and nucleoside levels. Diazoxide (300 microM), either alone or in combination with 5-hydroxydecanoate (30 microM) was ineffective. Pyruvate (5 mM) partially reversed the effects of ischemia or ischemia plus 2-deoxyglucose (20mM) in the incubation medium. Diazoxide (300 microM) and 5-HD (30 microM) had no effect in the presence of pyruvate (5mM) and 2-deoxyglucose (20mM). Staining the cells with celestine blue/acid fuchsin in order to classify them as intact, reversibly or profoundly injured, revealed a protective effect of 5-HD. When compared with 5-HD, diazoxide, pyruvate and 2-deoxyglucose had similar but less pronounced effects. In conclusion, these results suggest a protective role of 5-hydroxydecanoate on early corticoencephalic nucleotide and cell viability alterations during ischemia.

Changes in glial fibrillary acidic protein (GFAP) are believed to be a sensitive and specific marker for astroglial responses to various kinds of injury. Neurotrophic factors and their receptors involved in the physiological and pathological responses of astrocytes are of special interest. For example, ATP, released from damaged or dying cells, may represent one of the endogenous factors mediating the activation of astrocytes. In the present study, the influence of different ATP analogs on the GFAP-immunoreactivity (IR) and on the expression of P2 receptors on GFAP-labeled cells were studied. Microinjection of the selective P2 receptor agonists α,β-methylene ATP (α,β-meATP; P2X 1,3 ), adenosine 5'-O-(2-thiodiphosphate) (ADP-β-S; P2Y 1,12 ), and uridine 5'-O-(3-thiotriphosphate) (UTP-γ-S; P2Y 2,4,6 ) in the nucleus accumbens (NAc) of rats resulted in an increase of GFAP-IR in comparison to the treatment with artificial cerebrospinal fluid. The agonist-induced morphogenic changes documented a strong potency of α,β-meATP and ADP-β-S. The effect of UTP-γ-S was small. Blockade of P2 receptors with the nonselective antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) decreased the injury-induced elevation of GFAP-IR when given alone and in addition inhibited the agonist effects. Double immunofluorescence measurements confined the localization of P2X 2-4 and P2Y 1 receptor subtypes to GFAP-labeled astrocytes of untreated rats. The receptor subtypes P2X 1,5,7 were not present in the NAc of native rats, but became expressed after mechanical damage. P2X 6 receptor-labeling was absent both with and without mechanical damage. After microinjection of α,β-meATP and ADP-β-S into the NAc, the P2X 1,3 - and P2Y 1 -IR, respectively, appeared to increase. These results show that the stimulation of P2 receptors influences the expression of GFAP-IR in vivo. GFAP-positive astrocytes possess P2X as well as P2Y receptor subtypes and change the expression of these receptors after injury and subsequent long-term activation by ATP.

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency alpha,beta-meATP approximately ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.

Mesencephalic dopaminergic neurons are known to project to the prefrontal cortex (PFC) and the striatum (STR). Organotypic slice co-cultures of the ventral tegmental area/substantia nigra (VTA/SN)-complex and the PFC or STR, respectively, were used to analyze the cytoarchitectural organization of the VTA/SN-complex and the innervation pattern of the target slices by dopaminergic fibers. After 10-28 days of culturing immunocytochemistry with antibodies against tyrosine hydroxylase (TH) was performed. The VTA/SN-complex revealed in vitro an organization of TH-positive cells similar to those observed in rat brains of comparable age. TH-immunoreactive cells exhibited their typical morphology and formed long processes. No TH-immunolabeled elements were found in single cultures of PFC and STR. Tracing of VTA/SN fibers with biocytin as well as TH-immunostaining showed numerous labeled fibers in the co-cultured slices. Extensive fiber crossing was observed in the co-cultures of the VTA/SN-complex and STR but only a sparse fiber bridge in the co-cultured slices of VTA/SN-complex and PFC. The VTA/SN-complex-PFC system obviously retained several of its in vivo characteristics, e.g. the fiber network in the prefrontal cortical subareas. Our results demonstrate that TH-immunoreactive neurons develop their typical innervation pattern in slice co-cultures of VTA/SN-complex and PFC or STR, respectively. This in vitro approach may be useful for investigations of the dopaminergic function in the VTA/SN-prefrontal pathway.

The beta-site APP-cleaving enzyme (BACE1) is a prerequisite for the generation of β-amyloid peptides, which give rise to cerebrovascular and parenchymal β-amyloid deposits in the brain of Alzheimer's disease patients. BACE1 is neuronally expressed in the brains of humans and experimental animals such as mice and rats. In addition, we have recently shown that BACE1 protein is expressed by reactive astrocytes in close proximity to β-amyloid plaques in the brains of aged transgenic Tg2576 mice that overexpress human amyloid precursor protein carrying the double mutation K670N-M671L. To address the question whether astrocytic BACE1 expression is an event specifically triggered by β-amyloid plaques or whether glial cell activation by other mechanisms also induces BACE1 expression, we used six different experimental strategies to activate brain glial cells acutely or chronically. Brain sections were processed for the expression of BACE1 and glial markers by double immunofluorescence labeling and evaluated by confocal laser scanning microscopy. There was no detectable expression of BACE1 protein by activated microglial cells of the ameboid or ramified phenotype in any of the lesion paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1-immunoreactive astrocytes in proximity to β-amyloid plaques in the brains of aged Tg2576 mice and Alzheimer's disease patients. GLIA 41:169–179, 2003. © 2003 Wiley-Liss, Inc.

ATP and its metabolite adenosine activate membrane receptors, termed P2 and P1, respectively. In the present study, the modulation of the mesolimbic neuronal circuit by ATPergic and adenosinergic mechanisms was investigated by microdialysis in the nucleus accumbens (NAc) and by telemetrically recorded EEG from both the NAc and the ventral tegmental area (VTA) of freely moving rats. The basal extracellular dopamine concentration was enhanced after accumbal perfusion with the ATP analog 2-methylthio ATP (2-MeSATP; 100 μM); by contrast, adenosine (100 μM) caused a reduction of extracellular dopamine. When given alone, the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 20 μM) decreased the concentration of dopamine, whereas the P1 receptor antagonist 8-(p-sulfophenyl)theophylline (8-SPT; 100 μM) increased it. In the same animals, P2 receptor stimulation by 2-MeSATP caused neuronal activation, indicated by an elevation of the absolute power in the EEG of the NAc mainly by enhancement of the relative power in the alpha band (8–13 Hz) of the EEG spectrum. By contrast, adenosine led to a depression of the absolute power in the VTA accompanied by an elevation of the delta-band power (0.4–6 Hz) in the NAc corresponding to a slowing of neuronal activity. When given alone, PPADS reduced the absolute EEG power in the NAc accompanied by a decrease in the high-frequency power, but had no effects on the VTA. 8-SPT on its own enhanced the total power in both the NAc and the VTA, reflected by an enhancement in the slow and the high-frequency bands. Whereas the 8-SPT-evoked changes of EEG pattern as well as of dopamine concentration in the NAc were abolished by the co-application of PPADS, the 8-SPT-induced EEG changes in the VTA persisted under these conditions. In conclusion, the accumbal neuronal output, reflected by both dopamine release and neuronal electrical activity, is modulated in a functionally antagonistic manner by P2 and P1 receptor stimulation. It is suggested that an inhibitory GABAergic feedback projection to the VTA is stimulated by adenosine, either directly or indirectly via glutamate release. Synapse 47:134–142, 2003. © 2002 Wiley-Liss, Inc.

Extracellular purines have multiple functional roles in development, plastic remodelling, and regeneration of the CNS by stimulating certain P2X/Y receptor (R) subtypes. In the present study we elucidated the involvement of P2YRs in neuronal fibre outgrowth in the developing nervous system. We particularly focused on the P2Y1R subtype and the dopaminergic system, respectively. For this purpose, we used organotypic slice co-cultures consisting of the ventral tegmental area/substantia nigra (VTA/SN) and the prefrontal cortex (PFC). After detecting the presence of the P2Y1R in VTA/SN, PFC, and on outgrowing fibres in the border region (e.g. on glial processes) connecting both brain slices, we could show that pharmacological modulation of the receptor influenced neuronal fibre outgrowth. Biocytin-tracing and tyrosine hydroxylase-immunolabelling together with quantitative image analysis revealed a significant increase in fibre growth in the border region of the co-cultures after treatment with ADPβS (P2Y1,12,13R agonist). The observed stimulatory potential of ADPβS was inhibited by pre-treatment with the P2X/YR antagonist PPADS. In P2Y1R knockout (P2Y1R(-/-)) mice, the ADPβS-induced stimulatory effect was absent, while growth was significantly enhanced in the co-cultures of the respective wild-type. This observation was confirmed in entorhino-hippocampal co-cultures, an example of a different projection system, expressing the P2Y1R. Using wortmannin and PD98059 we further showed that PI3K/Akt and MAPK/ERK cascades are involved in the mechanism underlying ADPβS-induced fibre growth. In conclusion, the data of this study clearly indicate that activation of the P2Y1R stimulates fibre growth and thereby emphasises the general role of this particular receptor subtype during development and regeneration.

Acute brain injury and neurodegenerative disorders may result in astroglial activation. Astrocytes are able to determine the progression and outcome of these neuropathologies in a beneficial or detrimental way. Nucleotides, e.g. adenosine 5'-triphosphate (ATP), released after acute or chronic neuronal injury, are important mediators of glial activation and astrogliosis.Acute injury may cause significant changes in ATP balance, resulting in (1) a decline of intracellular ATP levels and (2) an increase in extracellular ATP concentrations via efflux from the intracellular space. The released ATP may have trophic effects, but can also act as a proinflammatory mediator or cytotoxic factor, inducing necrosis/apoptosis as a universal "danger" signal. Furthermore, ATP, primarily released from astrocytes, is a means of communication between neurons, glial cells, and intracerebral blood vessels.Astrocytes express a heterogeneous battery of purinergic ionotropic and metabotropic receptors (P2XRs and P2YRs, respectively) to respond to extracellular nucleotides.In this chapter, we summarize the contemporary knowledge on the pathological potential of P2Rs in relation to changes of astrocytic functions, determined by distinct molecular signaling cascades, in a variety of diseases. We discuss specific aspects of reactive astrogliosis, with respect to the involvement of prominent receptor subtypes, such as the P2X7 and P2Y1/2Rs. Examples of purinergic signaling of microglia, oligodendrocytes, and blood vessels under pathophysiological conditions will also be presented.The understanding of the pathological potential of purinergic signaling in "controlling and fine-tuning" of astrocytic responses is important for identifying possible therapeutic principles to treat acute and chronic central nervous system diseases.

Calcium ions (Ca(2+)) play important roles in neuroplasticity and the regeneration of nerves. Intracellular Ca(2+) concentrations are regulated by Ca(2+) channels, among them L-type voltage-gated Ca(2+) channels, which are inhibited by dihydropyridines like nimodipine. The purpose of this study was to investigate the effect of nimodipine on neurite growth during development and regeneration. As an appropriate model to study neurite growth, we chose organotypic brain slice co-cultures of the mesocortical dopaminergic projection system, consisting of the ventral tegmental area/substantia nigra and the prefrontal cortex from neonatal rat brains. Quantification of the density of the newly built neurites in the border region (region between the two cultivated slices) of the co-cultures revealed a growth promoting effect of nimodipine at concentrations of 0.1μM and 1μM that was even more pronounced than the effect of the growth factor NGF. This beneficial effect was absent when 10μM nimodipine were applied. Toxicological tests revealed that the application of nimodipine at this higher concentration slightly induced caspase 3 activation in the cortical part of the co-cultures, but did neither affect the amount of lactate dehydrogenase release or propidium iodide uptake nor the ratio of bax/bcl-2. Furthermore, the expression levels of different genes were quantified after nimodipine treatment. The expression of Ca(2+) binding proteins, immediate early genes, glial fibrillary acidic protein, and myelin components did not change significantly after treatment, indicating that the regulation of their expression is not primarily involved in the observed nimodipine mediated neurite growth. In summary, this study revealed for the first time a neurite growth promoting effect of nimodipine in the mesocortical dopaminergic projection system that is highly dependent on the applied concentrations.

Background and Purpose It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. Experimental Approach A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. Key Results A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,β-methylene ATP (α,β-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,β-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. Conclusions and Implications In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.

Organotypic slice co-cultures are suitable tools to study axonal regeneration and development (growth or regrowth) of different projection systems of the CNS under ex vivo conditions.In this chapter, we describe in detail the reconstruction of the mesocortical and nigrostriatal dopaminergic projection system culturing tissue slices from the ventral tegmental area/substantia nigra (VTA/SN) with the prefrontal cortex (PFC) or the striatum (STR). The protocol includes the detailed slice preparation and incubation. Moreover, different application possibilities of the ex vivo model are mentioned; as an example, the substance treatment procedure and biocytin tracing are described to reveal the effect of applied substances on fiber outgrowth.

The substantia gelatinosa (SG) of the spinal cord processes incoming painful information to ascending projection neurons. Whole-cell patch clamp recordings from SG spinal cord slices documented that in a low Ca(2+) /no Mg(2+) (low X(2+) ) external medium adenosine triphosphate (ATP)/dibenzoyl-ATP, Bz-ATP) caused inward current responses, much larger in amplitude than those recorded in a normal X(2+) -containing bath medium. The effect of Bz-ATP was antagonized by the selective P2X7 receptor antagonist A-438079. Neuronal, but not astrocytic Bz-ATP currents were strongly inhibited by a combination of the ionotropic glutamate receptor antagonists AP-5 and CNQX. In fact, all neurons and some astrocytes responded to NMDA, AMPA, and muscimol with inward current, demonstrating the presence of the respective receptors. The reactive oxygen species H2 O2 potentiated the effect of Bz-ATP at neurons but not at astrocytes. Hippocampal CA1 neurons exhibited a behavior similar to, but not identical with SG neurons. Although a combination of AP-5 and CNQX almost abolished the effect of Bz-ATP, H2 O2 was inactive. A Bz-ATP-dependent and A-438079-antagonizable reactive oxygen species production in SG slices was proven by a microelectrode biosensor. Immunohistochemical investigations showed the colocalization of P2X7-immunoreactivity with microglial (Iba1), but not astrocytic (GFAP, S100β) or neuronal (MAP2) markers in the SG. It is concluded that SG astrocytes possess P2X7 receptors; their activation leads to the release of glutamate, which via NMDA- and AMPA receptor stimulation induces cationic current in the neighboring neurons. P2X7 receptors have a very low density under resting conditions but become functionally upregulated under pathological conditions.

Acute brain injury and neurodegenerative disorders may result in astroglial activation. Astrocytes are able to determine the progression and outcome of these neuropathologies in a beneficial or detrimental way. Nucleotides, e.g. adenosine 5′-triphosphate (ATP), released after acute or chronic neuronal injury, are important mediators of glial activation and astrogliosis.

Organotypic slice co-cultures are suitable tools to study axonal regeneration and development (growth or regrowth) of different projection systems of the CNS under ex vivo conditions.In this chapter, we describe in detail the reconstruction of the mesocortical and nigrostriatal dopaminergic projection system culturing tissue slices from the ventral tegmental area/substantia nigra (VTA/SN) with the prefrontal cortex (PFC) or the striatum (STR). The protocol includes the detailed slice preparation and incubation. Moreover, different application possibilities of the ex vivo model are mentioned; as an example, the substance treatment procedure and biocytin tracing are described to reveal the effect of applied substances on fiber outgrowth.

In the nucleus accumbens (NAc) of rats, the involvement of P2X and P2Y receptors in the generation of astrogliosis in vivo, was investigated by local application of their respective ligands. The agonists used had selectivities for P2X1,3 (α,β-methylene adenosine 5′-triphosphate; α,β-meATP), P2Y1,12 (adenosine 5′-O-(2-thiodiphosphate; ADP-β-S) and P2Y2,4,6 receptors (uridine 5′-O-(3-thiotriphosphate; UTP-γ-S). Pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (PPADS) was used as a non-selective antagonist. The astroglial reaction was studied by means of immunocytochemical double-labelling with antibodies to glial fibrillary acidic protein (GFAP) and 5-bromo-2′-deoxyuridine (BrdU). The agonist-induced changes in comparison to the artificial cerebrospinal fluid (aCSF)-treated control side reveal a strong mitogenic potency of ADP-β-S and α,β-meATP, whereas UTP-γ-S was ineffective. The P2 receptor antagonist PPADS decreased the injury-induced proliferation when given alone and in addition inhibited all agonist effects. The observed morphogenic changes included hypertrophy of astrocytes, elongation of astrocytic processes and up-regulation of GFAP. A significant increase of both GFAP-immunoreactivity (IR) and GFA-protein content (by using Western blotting) was found after microinfusion of α,β-meATP or ADP-β-S. In contrast, UTP-γ-S failed to increase the GFAP-IR. The morphogenic effects were also inhibited by pre-treatment with PPADS. A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3 and P2Y1 receptors on the GFAP-labelled astrocytes. In conclusion, the data suggest that P2Y (P2Y1 or P2Y12) receptor subtypes are involved in the generation of astrogliosis in the NAc of rats, with a possible minor contribution of P2X receptor subtypes. British Journal of Pharmacology (2001) 134, 1180–1189; doi:10.1038/sj.bjp.0704353

The present review summarizes data about the expression of P2 receptors in two types of macroglial cells, i.e., in astrocytes of the nucleus accumbens (NAc) and Muller glial cells of the retina. Changes in the expression of these receptors during pathological conditions may indicate an involvement in the induction and/or maintenance of reactive astrogliosis. In fact, astrocytic proliferation was induced in the NAc by the application of different P2X and P2Y receptor agonists, such as 2-methylthio ATP (2-MeSATP), α,β-methylene adenosine 5'-triphosphate (α,β-meATP) and adenosine 5'-O-(2-thiodiphosphate (ADP-β-S). An increased content of glial fibrillary acidic protein, astrocytic hypertrophy, and proliferation as indicated by enhanced incorporation of 5-bromo-2'-deoxyuridine (BrdU) was found under these conditions; an enhanced BrdU incorporation signalizes a higher rate of DNA synthesis. Muller glial cells of the human retina express both metabotropic P2Y and ionotropic P2X 7 receptors. Application of the P2X 7 receptor agonist benzoyl-benzoyl ATP (BzATP) evoked a cationic conductance increase that led to the depolarization of cells and to Ca 2+ entry from the extracellular space. Long-lasting application of BzATP, however, did not cause the formation of pores permeable for large molecules, excluding the presence of classic P2X 7 receptors. Muller glial cells of the human retina show increased P2X 7 receptor-mediated cationic currents during proliferative gliosis. A role of P2X 7 receptors in glial proliferation is also indicated by the observation that the DNA synthesis rate of cultured Muller cells is enhanced in the presence of BzATP, as shown by the measurement of BrdU incorporation. The data indicate that activation of P2 receptors may exert both short- and long-term effects in macroglial cells, and that these receptors may be involved in glial responses to pathological conditions.

We studied the expression of neuronal (n), endothelial (e) and inducible (i) nitric oxide synthase (NOS) in cell cultures of rat mesencephalic neurons (embryonic day 14), human keratinocytes from juvenile epidermis, human endothelial cells from juvenile coronary arteries, and human osteoblasts. All cell types were cultured for 5, 10 or 15 days. During proliferation (round cells without processes), the intracellular distribution and the intracellular amount of the calcium-dependent NOS isoforms (n- and e-NOS) did not change whereas the calcium-independent i-NOS changed from a cytosolic distribution pattern to compartmentalized distribution. A striking decrease of i-NOS immunoreactivity was measured by means of image analysis. Our results support the opinion that i-NOS acts as a switch between proliferation and differentiation of cells.

Acamprosate has recently been introduced in relapse prophylaxis in weaned alcoholics. Using fura-2 microfluorimetry, the present study investigates whether acamprosate affects N-methyl-D-aspartate (NMDA) or K+-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) in rat cultured mesencephalic neurones. Both application of NMDA (plus glycine) and elevation of extracellular K+ induced rapid increases in [Ca2+]i which respectively were insensitive and sensitive to omega-conotoxin (omega-CTX) MVIIC, a blocker of voltage-dependent Ca2+ channels (VDCCs). Acamprosate (100 microM and 300 microM) significantly attenuated the response induced by NMDA as well as that induced by K+ in a concentration-dependent manner. Concurrent application of omega-CTX MVIIC and acamprosate impaired the K+-induced increase in [Ca2+]i to the same extent as omega-CTX MVIIC alone. The present data suggest that acamprosate inhibits Ca2+ influx through both NMDA receptors and VDCCs.

Whole-cell patch clamp experiments were used to investigate the transduction mechanism of adenosine A(2A) receptors in modulating N-methyl-D-aspartate (NMDA)-induced currents in rat striatal brain slices. The A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) inhibited the NMDA, but not the (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current in a subset of striatal neurons. Lucifer yellow-filled pipettes in combination with immunostaining of A(2A) receptors were used to identify CGS 21680-sensitive cells as typical medium spiny striatal neurons. Dibutyryl cyclic AMP and the protein kinase A activator Sp-cyclic AMPs, but not the protein kinase A inhibitors Rp-cyclic AMPS or PKI(14 - 24)amide abolished the inhibitory effect of CGS 21680. The phospholipase C inhibitor U-73122, but not the inactive structural analogue U-73343 also interfered with CGS 21680. The activation of protein kinase C by phorbol 12-myristate 13-acetate or the blockade of this enzyme by staurosporine did not alter the effect of CGS 21680. Heparin, an antagonist of inositol 1, 4,5-trisphosphate (InsP(3)) and a more efficient buffering of intracellular Ca(2+) by BAPTA instead of EGTA in the pipette solution, abolished the CGS 21680-induced inhibition. The calmodulin antagonist W-7 and cytochalasin B which enhances actin depolymerization also prevented the effect of CGS 21680; the calmodulin kinase II inhibitors CaM kinase II(281 - 309) and KN-93 but not the inactive structural analogue KN-92 were also effective. The calcineurin inhibitor deltamethrin did not interfere with CGS 21680. It is suggested that the transduction mechanism of A(2A) receptors to inhibit NMDA receptor channels is the phospholipase C/InsP(3)/calmodulin and calmodulin kinase II pathway. The adenylate cyclase/protein kinase A and phospholipase C/protein kinase C pathways do not appear to be involved.

Morphological changes induced by 30 min of hypoxia (incubation in medium saturated with 95% N 2 –5% CO 2 instead of the normal 95% O 2 –5% CO 2 ) were investigated in neurons (layers II/III of the parietal cortex) of rat neocortical brain slices. The cells were identified as intact, reversibly or irreversibly injured. As expected, hypoxia decreased the number of intact cells and increased the number of irreversibly injured cells. Pretreatment of slices with diazoxide (300 μM), an agonist of ATP-dependent potassium (K ATP ) channels completely prevented the morphological damage induced by hypoxia, whereas tolbutamide (300 μM), an antagonist of K ATP channels, was ineffective when given alone. However, tolbutamide (300 μM) co-applied with diazoxide (300 μM), partly reversed the neuroprotective effect of this agonist during hypoxia. In conclusion, K ATP channels appear to be present on neocortical neurons and their opening counteracts hypoxia-induced cell injury.

Unveiling the roles of distinct cell types in brain response to insults is a partially unsolved challenge and a key issue for new neuroreparative approaches. In vivo models are not able to dissect the contribution of residential microglia and infiltrating blood-borne monocytes/macrophages, which are fundamentally undistinguishable; conversely, cultured cells lack original tissue anatomical and functional complexity, which profoundly alters reactivity. Here, we tested whether rodent organotypic co-cultures from mesencephalic ventral tegmental area/substantia nigra and prefrontal cortex (VTA/SN-PFC) represent a suitable model to study changes induced by oxygen/glucose deprivation and reperfusion (OGD/R). OGD/R induced cytotoxicity to both VTA/SN and PFC slices, with higher VTA/SN susceptibility. Neurons were highly affected, with astrocytes and oligodendrocytes undergoing very mild damage. Marked reactive astrogliosis was also evident. Notably, OGD/R triggered the activation of CD68-expressing microglia and increased expression of Ym1 and Arg1, two markers of "alternatively" activated beneficial microglia. Treatment with two well-known neuroprotective drugs, the anticonvulsant agent valproic acid and the purinergic P2-antagonist PPADS, prevented neuronal damage. Thus, VTA/SN-PFC cultures are an integrated model to investigate OGD/R-induced effects on distinct cells and easily screen neuroprotective agents. The model is particularly adequate to dissect the microglia phenotypic shift in the lack of a functional vascular compartment.

The adenosine 5'-triphosphate (ATP)-gated P2X7 receptor is a membrane-bound, non-selective cation channel, expressed in a variety of cell types. The P2X7 senses high extracellular ATP concentrations and seems to be implicated in a wide range of cellular functions as well as pathophysiological processes, including immune responses and inflammation, release of gliotransmitters and cytokines, cancer cell growth or development of neurodegenerative diseases. In the present study, we identified natural compounds and analogues that can block or sensitize the ATP (1 mM)-induced Ca(2+) response using a HEK293 cell line stably expressing human P2X7 and fluorometric imaging plate reader technology. For instance, teniposide potently blocked the human P2X7 at sub-miromolar concentrations, but not human P2X4 or rat P2X2. A marked block of ATP-induced Ca(2+) entry and Yo-Pro-1 uptake was also observed in human A375 melanoma cells and mouse microglial cells, both expressing P2X7. On the other hand, agelasine (AGL) and garcinolic acid (GA) facilitated the P2X7 response to ATP in all three cell populations. GA also enhanced the YO-PRO-1 uptake, whereas AGL did not affect the ATP-stimulated intracellular accumulation of this dye. According to the pathophysiological role of P2X7 in various diseases, selective modulators may have potential for further development, e.g. as neuroprotective or antineoplastic drugs.

Postmortem analysis of relevant biomarkers might aid in characterizing causes of death and survival times in legal medicine. However, there are still no sufficiently established results of practical postmortem biochemical investigations in cases of traumatic brain injury (TBI). The two biomarkers--S100 protein subunit B (S100B) and neuronal specific enolase (NSE)--could be of special interest. Therefore, the aim of the present study was to investigate changes in their postmortem levels for further determination of brain damage in TBI. In 17 cases of TBI (average age, 58 years) and in 23 controls with different causes of death (average age, 59 years), serum and cerebrospinal fluid (CSF) samples were analyzed with a chemiluminescence immunoassay for marker expression. An increase in serum S100B, as well as a subsequent decrease after survival times4 days, were detected in TBI cases (p0.01). CSF NSE values6,000 ng/mL and CSF S100B levels10,000 ng/mL seem to indicate a TBI survival time of at least 15 min (p0.01). It is of particular interest that CSF S100B levels (p0.01) and serum S100B levels (p0.05) as well as CSF NSE values (p0.01) were significantly higher in TBI cases in comparison to the controls, especially when compared with fatal non-head injuries. In conclusion, the present findings emphasize that S100B and NSE are useful markers in postmortem biochemistry in cases of suspected TBI. Further, S100B may be helpful to estimate the survival time of fatal injuries in legal medicine.