Astrocytes and major depression: The purinergic avenue.

Major depressive disorder (MDD) is one of the most prevalent psychiatric illnesses worldwide which impairs the social functioning of the afflicted patients. Astrocytes promote homeostasis of the CNS and provide defense against various types of harmful influences. Increasing evidence suggests that the number, morphology and function of astrocytes are deteriorated in the depressed brain and the malfunction of the astrocytic purinergic system appears to participate in the pathophysiology of MDD. Adenosine 5'-triphosphate (ATP) released from astrocytes modulates depressive-like behavior in animal models and probably also clinical depression in patients. Astrocytes possess purinergic receptors, such as adenosine A2A receptors (Rs), and P2X7, P2Y1, and P2Y11Rs, which mediate neuroinflammation, neuro(glio)transmission, and synaptic plasticity in depression-relevant areas of the brain (e.g. medial prefrontal cortex, hippocampus, amygdala nuclei). By contrast, astrocytic A1Rs are neuroprotective and immunosuppressive. In the present review, we shall discuss the release of purines from astrocytes, and the expression/function of astrocytic purinergic receptors. Subsequently, we shall review in more detail novel evidence indicating that the dysregulation of astrocytic purinergic signaling actively contributes to the pathophysiology of depression and shall discuss possible therapeutic options based on knowledge recently acquired in this field.

Differential effects of P2Y1 deletion on glial activation and survival of photoreceptors and amacrine cells in the ischemic mouse retina.

Gliosis of retinal Müller glial cells may have both beneficial and detrimental effects on neurons. To investigate the role of purinergic signaling in ischemia-induced reactive gliosis, transient retinal ischemia was evoked by elevation of the intraocular pressure in wild-type (Wt) mice and in mice deficient in the glia-specific nucleotide receptor P2Y1 (P2Y1 receptor-deficient (P2Y1R-KO)). While control retinae of P2Y1R-KO mice displayed reduced cell numbers in the ganglion cell and inner nuclear layers, ischemia induced apoptotic death of cells in all retinal layers in both, Wt and P2Y1R-KO mice, but the damage especially on photoreceptors was more pronounced in retinae of P2Y1R-KO mice. In contrast, gene expression profiling and histological data suggest an increased survival of amacrine cells in the postischemic retina of P2Y1R-KO mice. Interestingly, measuring the ischemia-induced downregulation of inwardly rectifying potassium channel (Kir)-mediated K(+) currents as an indicator, reactive Müller cell gliosis was found to be weaker in P2Y1R-KO (current amplitude decreased by 18%) than in Wt mice (decrease by 68%). The inner retina harbors those neurons generating action potentials, which strongly rely on an intact ion homeostasis. This may explain why especially these cells appear to benefit from the preserved Kir4.1 expression in Müller cells, which should allow them to keep up their function in the context of spatial buffering of potassium. Especially under ischemic conditions, maintenance of this Müller cell function may dampen cytotoxic neuronal hyperexcitation and subsequent neuronal cell loss. In sum, we found that purinergic signaling modulates the gliotic activation pattern of Müller glia and lack of P2Y1 has janus-faced effects. In the end, the differential effects of a disrupted P2Y1 signaling onto neuronal survival in the ischemic retina call the putative therapeutical use of P2Y1-antagonists into question.

Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening.

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,ß-methylene ATP (α,ß-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,ß-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.

Electrophysiological classification of P2X7 receptors in rat cultured neocortical astroglia.

BACKGROUND AND PURPOSE: P2X7 receptors are ATP-gated cation channels mediating important functions in microglial cells, such as the release of cytokines and phagocytosis. Electrophysiological evidence that these receptors also occur in CNS astroglia is rare and rather incomplete. EXPERIMENTAL APPROACH: We used whole-cell patch-clamp recordings to search for P2X7 receptors in astroglial-neuronal co-cultures prepared from the cerebral cortex of rats. KEY RESULTS: All the astroglial cells investigated responded to ATP with membrane currents, reversing around 0 mV. These currents could be also detected in isolated outside-out patch vesicles. The results of the experiments with the P2X [alpha,beta-methylene ATP and 2'-3'-O-(4-benzoyl) ATP] and P2Y receptor agonists [adenosine 5'-O-(2-thiodiphosphate), uridine 5'-diphosphate, uridine 5'-triphosphate (UTP) and UDP-glucose] suggested the involvement of P2X receptors in this response. The potentiation of ATP responses in a low divalent cation or alkaline bath, but not by ivermectin, made it likely that a P2X7 receptor is operational. Blockade of the ATP effect by the P2X7 antagonists Brilliant Blue G, calmidazolium and oxidized ATP corroborated this assumption. CONCLUSIONS AND IMPLICATIONS: Rat cultured cortical astroglia possesses functional P2X7 receptors. It is suggested that astrocytic P2X7 receptors respond to high local ATP concentrations during neuronal injury.

A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain.

BACKGROUND AND PURPOSE: This study was undertaken to compare the analgesic activity of antagonists acting at P2X1, P2X7, and P2Y12 receptors and agonists acting at P2Y1, P2Y2, P2Y4, and P2Y6 receptors in neuropathic, acute, and inflammatory pain. EXPERIMENTAL APPROACH: The effect of the wide spectrum P2 receptor antagonist PPADS, the selective P2X7 receptor antagonist Brilliant Blue G (BBG), the P2X1 receptor antagonist (4,4',4'',4-[carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic acid, octasodium salt (NF449) and (8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis-1,3,5-naphthalene-trisulphonic acid, hexasodium salt (NF023), the P2Y12 receptor antagonist (2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propylester (MRS2395), the selective P2Y1 receptor agonist ([[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS2365), the P2Y2/P2Y4 agonist uridine-5'-triphosphate (UTP), and the P2Y4/P2Y6 agonist uridine-5'-diphosphate (UDP) were examined on mechanical allodynia in the Seltzer model of neuropathic pain, on acute thermal nociception, and on the inflammatory pain and oedema induced by complete Freund's adjuvant (CFA). KEY RESULTS: MRS2365, MRS2395 and UTP, but not the other compounds, significantly alleviated mechanical allodynia in the neuropathic pain model, with the following rank order of minimal effective dose (mED) values: MRS2365 > MRS2395 > UTP. All compounds had a dose-dependent analgesic action in acute pain except BBG, which elicited hyperalgesia at a single dose. The rank order of mED values in acute pain was the following: MRS2365 > MRS2395 > NF449 > NF023 > UDP = UTP > PPADS. MRS2365 and MRS2395 had a profound, while BBG had a mild effect on inflammatory pain, with a following rank order of mED values: MRS2395 > MRS2365 > BBG. None of the tested compounds had significant action on oedema evoked by intraplantar injection of CFA. CONCLUSIONS AND IMPLICATIONS: Our results show that antagonism at P2X1, P2Y12, and P2X7 receptors and agonism at P2Y1 receptors define promising therapeutic strategies in acute, neuropathic, and inflammatory pain respectively.

Increase of intracellular Ca2+ by P2X and P2Y receptor-subtypes in cultured cortical astroglia of the rat.

Astrocytes express purinergic receptors that are involved in glial-neuronal cell communication. Experiments were conducted to characterize the expression of functional P2X/P2Y nucleotide receptors in glial cells of mixed cortical cell cultures of the rat. The vast majority of these cells was immunopositive for glial fibrillary acidic protein (GFAP) and was considered therefore astrocyte-like; for the sake of simplicity they were termed "astroglia" throughout. Astroglia expressed predominantly P2X(4,6,7) as well as P2Y(1,2) receptor-subtypes. Less intensive immunostaining was also found for P2X(5) and P2Y(4,6,13,14) receptors. Pressure application of ATP and a range of agonists selective for certain P2X or P2Y receptor-subtypes caused a concentration-dependent increase of intracellular Ca(2+) ([Ca(2+)](i)). Of the agonists tested, only the P2X(1,3) receptor-selective alpha,beta-methylene ATP was ineffective. Experiments with Ca(2+)-free solution and cyclopiazonic acid, an inhibitor of the endoplasmic Ca(2+)-ATPase, indicated that the [Ca(2+)](i) response to most nucleotides, except for ATP and 2',3'-O-(benzoyl-4-benzoyl)-ATP, was due primarily to the release of Ca(2+) from intracellular stores. A Gprotein-mediated release of Ca(2+) is the typical signaling mechanism of various P2Y receptor-subtypes, whose presence was confirmed also by cross-desensitization experiments and by using selective antagonists. Thus, our results provide direct evidence that astroglia in mixed cortical cell cultures express functional P2Y (P2Y(1,2,6,14) and probably also P2Y(4)) receptors. Several unidentified P2X receptors, including P2X(7), may also be present, although they appear to only moderately participate in the regulation of [Ca(2+)](i). The rise of [Ca(2+)](i) is due in this case to the transmembrane flux of Ca(2+) via the P2X receptor-channel. In conclusion, P2Y rather than P2X receptor-subtypes are involved in modulating [Ca(2+)](i) of cultured astroglia and thereby may play an important role in cell-to-cell signaling.

P2 receptor-mediated stimulation of the PI3-K/Akt-pathway in vivo.

ATP acts as a growth factor as well as a toxic agent by stimulating P2 receptors. The P2 receptor-activated signaling cascades mediating cellular growth and cell survival after injury are only incompletely understood. Therefore, the aim of the present study was to identify the role of the phosphoinositide 3 kinase (PI3-K/Akt) and the mitogen-activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) pathways in P2Y receptor-mediated astrogliosis after traumatic injury and after microinfusion of ADP beta S (P2Y(1,12,13) receptor agonist) into the rat nucleus accumbens (NAc). Mechanical damage and even more the concomitant treatment with ADP beta S, enhanced P2Y(1) receptor-expression in the NAc, which could be reduced by pretreatment with the P2X/Y receptor antagonist PPADS. Quantitative Western blot analysis indicated a significant increase in phosphorylated (p)Akt and pERK1/2 2 h after ADP beta S-microinjection. Pretreatment with PPADS or wortmannin abolished the up-regulation of pAkt by injury alone or ADP beta S-treatment. The ADP beta S-enhanced expression of the early apoptosis marker active caspase 3 was reduced by PPADS and PD98059, but not by wortmannin. Multiple immunofluorescence labeling indicated a time-dependent expression of pAkt and pMAPK on astrocytes and neurons and additionally the colocalization of pAkt, pMAPK, and active caspase 3 with the P2Y(1) receptor especially at astrocytes. In conclusion, the data show for the first time the involvement of PI3-K/Akt-pathway in processes of injury-induced astroglial proliferation and anti-apoptosis via activation of P2Y(1) receptors in vivo, suggesting specific roles of P2 receptors in glial cell pathophysiology in neurodegenerative diseases.

The unusual state-dependent affinity of P2X3 receptors can be explained by an allosteric two-open-state model.

High-affinity desensitization (HAD) by nanomolar agonists was described to shape the ability of P2X(3) receptors for mediating pain sensation. These receptors are activated by micromolar ATP, but nanomolar ATP is sufficient to effectively desensitize them. The mechanism behind HAD is still obscure. It has been suggested ( J Neurosci 25: 7359-7365, 2005 ) that HAD can happen only if the receptor has previously been activated and desensitized by high agonist concentrations. It was not clear, however, whether the high-affinity site was different from the conventional binding site and which mechanism led to its exposure during desensitization. A subsequent article ( Mol Pharmacol 70: 373-382, 2006 ) argued that HAD could also occur without preceding desensitization, because even resting receptors expose high-affinity binding sites. To support this hypothesis, a kinetic model was proposed that could reproduce all major phenomena observed experimentally. We attempted to improve this model and used it to simulate the agonist-induced formation of the high-affinity binding site. We collected electrophysiological data using HEK 293 cells expressing human P2X(3) receptors and fitted simulated currents to experimentally acquired currents. A simple allosteric kinetic model in which only triliganded receptors could open failed to reproduce receptor behavior; introduction of an additional diliganded open state was necessary. Simulation with this model gave results that were in good agreement with experimental data. By using simulations and experiments, we analyzed the process of high-affinity binding site formation upon agonist exposure and propose an explanation, which helps to resolve the apparent conflict regarding the mechanism of HAD.

P2 receptor expression in the dopaminergic system of the rat brain during development.

Extracellular ATP facilitates the release of dopamine via P2 receptor activation in parts of the mesolimbic system. To characterize P2X/Y receptor subtypes in the developing dopaminergic system, their expression in organotypic slice co-cultures including the ventral tegmental area/substantia nigra (VTA/SN) complex and the prefrontal cortex (PFC) was studied in comparison to the receptor expression in 3-5 day-old and adult rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) with specific primers for the P2X(1,2,3,4,6,7) and P2Y(1) receptors in the tissue extracts of organotypic co-cultures revealed the presence of the P2X and P2Y receptor mRNAs investigated. Multiple immunofluorescence labeling of the P2X/Y receptor protein indicated differences in the regional expression in the organotypic co-cultures after 10 days of cultivation (VTA/SN, P2X(1,2,3,4,6,7), P2Y(1,6,12); PFC, P2X(1,3,4,6,7), P2Y(1,2,4,6,12)). At postnatal days 3-5, an immunofluorescence mostly comparable to that of adult rats was observed (VTA/SN and PFC: P2X(1,2,3,4,6,7), P2Y(1,2,4,6,12)). There was one important exception: the P2X(7) receptor immunocytochemistry was not found in adult tissue, suggesting a potential role of this receptor in the development. Only few P2 receptors (e.g. P2X(1), P2Y(1)) were expressed at fibers interconnecting the dopaminergic VTA/SN with the PFC in the organotypic co-cultures. The treatment of the cultures with the ATP analogues 2-methylthio-ATP and alpha,beta-methylene-ATP induced an increase in axonal outgrowth and fiber density, which could be inhibited by pre-treatment with the P2X/Y receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid. The co-localization of the dopamine-(D1) receptor with the P2X(1) receptor in organotypic slice cultures was evident. In the PFC of the co-cultures, and that of young but not adult rats, a number of tyrosine hydroxylase (TH)-positive cells also possessed P2Y(1)-immunoreactivity (IR). Additionally, a strong P2Y(1)-IR was observed on astrocytes. The present results show a time-, region- and cell type-dependent in vitro and in vivo expression pattern of different P2 receptor subtypes in the dopaminergic system indicating the involvement of ATP and its receptors in neuronal development and growth.

Purinergic modulation of glutamate release under ischemic-like conditions in the hippocampus.

The aim of the present study was to explore whether endogenous activation of different purine receptors by ATP and adenosine contributes to or inhibits excess glutamate release evoked by ischemic-like conditions in rat hippocampal slices. Combined oxygen-glucose deprivation (OGD) elicited a substantial, [Ca(2+)](o)-independent release of [(3)H]glutamate, which was tetrodotoxin (1 microM)-sensitive and temperature-dependent. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 0.1-10 microM), and the selective P2X(7) receptor antagonist Brilliant Blue G (1-100 nM), decreased OGD-evoked [(3)H]glutamate efflux indicating that endogenous ATP facilitates ischemia-evoked glutamate release. The selective A(1)-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1-250 nM) and the selective A(2A) receptor antagonists 4-(2-[7-amino-2-)2-furyl(triazolo-[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385, 0.1-20 nM) and 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261, 2-100 nM) decreased OGD-evoked [(3)H]glutamate efflux, indicating that endogenous adenosine also facilitates glutamate release under these conditions. The effect of DPCPX and ZM241385 was reversed, whereas the action of P2 receptor antagonists was potentiated by the selective ecto-ATPase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL67156, 50 microM). The binding characteristic of the A(2A) ligand [(3)H]CGS21680 to hippocampal membranes did not change significantly in response to OGD. Taken together these data suggest that while A(1) receptors might became desensitized, A(2A) and P2X receptor-mediated facilitation of glutamate release by endogenous ATP and its breakdown product adenosine remains operational under long-term OGD. Therefore the inhibition of P2X/A(2A) receptors rather than the stimulation of A(1) adenosine receptors could be an effective approach to attenuate glutamatergic excitotoxicity and thereby counteract ischemia-induced neurodegeneration.

Purine ionotropic (P2X) receptors.

Purinergic signaling is involved in the proper functioning of virtually all organs of the body. Although in some cases purines have a major influence on physiological functions (e.g. thrombocyte aggregation), more often they are just background modulators contributing to fine tuning of biological events. However, under pathological conditions, when a huge amount of adenosine 5'-triphosphate (ATP) can reach the extracellular space, their significance is increasing. ATP and its various degradation products activate membrane receptors divided into two main classes: the metabotropic P2Y and the ionotropic P2X family. This latter group, the purine ionotropic receptor, is the object of this review. After providing a description about the distribution and functional properties of P2X receptors in the body, their pharmacology will be summarized. In the second part of this review, the role of purines in those organ systems and body functions will be highlighted, where the (patho)physiological role of P2X receptors has been suggested or is even well established. Besides the regulation of organ systems, for instance in the cardiovascular, respiratory, genitourinary or gastrointestinal system, some special issues will also be discussed, such as the role of P2X receptors in pain, tumors, central nervous system (CNS) injury and embryonic development. Several examples will indicate that purine ionotropic receptors might serve as attractive targets for pharmacological interventions in various diseases, and that selective ligands for these receptors will probably constitute important future therapeutic tools in humans.

Metabotropic P2Y receptors inhibit P2X3 receptor-channels via G protein-dependent facilitation of their desensitization.

BACKGROUND AND PURPOSE: The aim of the present study was to investigate whether the endogenous metabotropic P2Y receptors modulate ionotropic P2X(3) receptor-channels. EXPERIMENTAL APPROACH: Whole-cell patch-clamp experiments were carried out on HEK293 cells permanently transfected with human P2X(3) receptors (HEK293-hP2X(3) cells) and rat dorsal root ganglion (DRG) neurons. KEY RESULTS: In both cell types, the P2Y(1,12,13) receptor agonist, ADP-beta-S, inhibited P2X(3) currents evoked by the selective agonist, alpha,beta-methylene ATP (alpha,beta-meATP). This inhibition could be markedly counteracted by replacing in the pipette solution the usual GTP with GDP-beta-S, a procedure known to block all G protein heterotrimers. P2X(3) currents evoked by ATP, activating both P2Y and P2X receptors, caused a smaller peak amplitude and desensitized faster than those currents evoked by the selective P2X(3) receptor agonist alpha,beta-meATP. In the presence of intracellular GDP-beta-S, ATP- and alpha,beta-meATP-induced currents were identical. Recovery from P2X(3) receptor desensitization induced by repetitive ATP application was slower than the recovery from alpha,beta-meATP-induced desensitization. When G proteins were blocked by intracellular GDP-beta-S, the recovery from the ATP- and alpha,beta-meATP-induced desensitization were of comparable speed. CONCLUSIONS AND IMPLICATIONS: Our results suggest that the activation of P2Y receptors G protein-dependently facilitates the desensitization of P2X(3) receptors and suppresses the recovery from the desensitized state. Hence, the concomitant stimulation of P2X(3) and P2Y receptors of DRG neurons by ATP may result both in an algesic effect and a partly counterbalancing analgesic activity.

Involvement of P2X and P2Y receptors in microglial activation in vivo.

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.

Neuroprotective effects of the P2 receptor antagonist PPADS on focal cerebral ischaemia-induced injury in rats.

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.

P2 receptor-stimulation influences axonal outgrowth in the developing hippocampus in vitro.

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.

Increased polysialic acid neural cell adhesion molecule expression in human hippocampus of heroin addicts.

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.

Endocytosis and vesicle trafficking during tip growth of root hairs.

The directional elongation of root hairs, "tip growth", depends on the coordinated and highly regulated trafficking of vesicles which fill the tip cytoplasm and are active in secretion of cell wall material. So far, little is known about the dynamics of endocytosis in living root hairs. We analyzed the motile behaviour of vesicles in the apical region of living root hairs of Arabidopsis thaliana and of Triticum aestivum by live cell microscopy. For direct observation of endocytosis and of the fate of endocytic vesicles, we used the fluorescent endocytosis marker dyes FM 1-43 and FM 4-64. Rapid endocytosis was detected mainly in the tip, where it caused a bright fluorescence of the apical cytoplasm. The internalized membranes proceeded through highly dynamic putative early endosomes in the clear zone to larger endosomal compartments in the subapical region that are excluded from the clear zone. The internalized cargo ended up in the dynamic vacuole by fusion of large endosomal compartments with the tonoplast. Before export to these lytic compartments, putative early endosomes remained in the apical zone, where they most probably recycled to the plasma membrane and back into the cytoplasm for more than 30 min. Endoplasmic reticulum was not involved in trafficking pathways of endosomes. Actin cytoskeleton was needed for the endocytosis itself, as well as for further membrane trafficking. The actin-depolymerizing drug latrunculin B modified the dynamic properties of vesicles and endosomes; they became immobilized and aggregated in the tip. Treatment with brefeldin A inhibited membrane trafficking and caused the disappearance of FM-containing vesicles and putative early endosomes from the clear zone; labelled structures accumulated in motile brefeldin A-induced compartments. These large endocytic compartments redispersed upon removal of the drug. Our results hence prove that endocytosis occurs in growing root hairs. We show the localization of endocytosis in the tip and indicate specific endomembrane compartments and their recycling.

4-Epidoxycycline: an alternative to doxycycline to control gene expression in conditional mouse models.

Since the pioneering work by Gossen and Bujard in 1992 demonstrating the usefulness of the Escherichia coli derived tet resistance operon for regulating gene expression a large collection of doxycycline-controlled transgenic mice has been established. Gene switching in eukaryotic tissue culture cells or mice requires administration of tetracycline, anhydrotetracycline or doxycycline to efficiently inactivate the transactivator protein tTA (TET-OFF system) or alternatively to activate the reverse transactivator protein rtTA (TET-ON system). However, the antibiotic activity of doxycycline can create an imbalance of the intestinal flora, resulting in diarrhoea and in a smaller number of animals in colitis. Previous studies reported that 4-epidoxycycline (4-ED), a hepatic metabolite of doxycycline, does not function as an antibiotic in mice. This gave us the idea that 4-ED might be useful for controlling gene expression in mice without the unwanted antibiotic side effect. To study the applicability of 4-ED for control of gene expression we used cell lines expressing the oncogene HER2 under control of tTA (TET-OFF) as well as rtTA (TET-ON). 4-ED and doxycycline were similarly efficient in switching on or -off HER2 expression. In vivo we used a conditional mouse model that allows switching off HER2 in tumor tissue. We show that (i) doxycycline, 7.5mg/ml in drinking water (used as a positive control), (ii) 4-ED, 7.5mg/ml in drinking water, (iii) 4-ED, 10mg/kg body weight, s.c., and (iv) anhydrotetracycline, 10mg/kg, s.c. (used as a second positive control), were similarly efficient. Using mice with tumor volumes of 1.6cm(3) all four schedules led to a tumor remission of more than 95% within 7 days. In conclusion, 4-ED is similarly efficient as doxycycline to control gene expression in vitro and in mice. Since 4-ED lacks the antibiotic activity of doxycycline it may help to avoid adverse side effects and selection of resistant bacteria.

P2Y receptor expression on astrocytes in the nucleus accumbens of rats.

The expression of purinoceptor (P2)Y-subtypes on astrocytes in vivo under physiological conditions and after stab wound injury was investigated. Reverse transcriptase-polymerase chain reaction with specific primers for the receptor-subtypes P2Y1,2,4,6,12 in tissue extracts of the nucleus accumbens of untreated rats revealed the presence of all P2Y receptor mRNAs investigated. Double immunofluorescence visualized with laser scanning microscopy indicated the expression of the P2Y1,4 receptors on glial fibrillary acidic protein (GFAP)-labeled astrocytes under physiological conditions. After stab wound injury the additional expression of the P2Y2 and P2Y6 receptors, and an up-regulation of the P2Y1,4 receptor-labeling on astrocytic cell bodies and/or processes was observed. Astrocytes of cortical, in contrast to accumbal areas exhibited P2Y1,2,4,6 receptor-immunoreactivity (IR) under control conditions, which was up-regulated after stab would injury. Labeling for the P2Y12 receptor was not observed on GFAP-positive cortical and accumbal astrocytes under any of the conditions used. For the first time, the co-localization of different P2 receptor-subtypes (e.g. P2Y1 and P2X3) on the same astrocyte was shown immunocytochemically. The up-regulation of P2Y1 receptor-IR on astrocytes and non-glial cells after mechanical injury could be facilitated by microinfusion of the P2Y1,12,13 receptor agonist adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS). Proliferative changes after ADPbetaS-microinjection were characterized by means of double-staining with antibodies against GFAP and 5-bromo-2'-deoxyuridine. The non-selective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, the P2Y1 receptor antagonist N6-methyl-2'-deoxyadenosine 3',5'-bisphosphate and the P2Y1 receptor-antibody itself inhibited the agonist-induced effects. The data indicate the region-specific presence of P2Y receptors on astrocytes in vivo and their up-regulation after injury as well as the co-localization of P2X and P2Y receptor-subtypes on the same astrocyte. The dominant role of P2Y1 receptors in proliferation and the additional stimulation of non-P2Y1 receptors has been demonstrated in vivo suggesting the involvement of this receptor-type in the gliotic response under physiological and pathological conditions.