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.

Targeting murine heart and brain: visualisation conditions for multi-pinhole SPECT with (99m)Tc- and (123)I-labelled probes.

PURPOSE: The study serves to optimise conditions for multi-pinhole SPECT small animal imaging of (123)I- and (99m)Tc-labelled radiopharmaceuticals with different distributions in murine heart and brain and to investigate detection and dose range thresholds for verification of differences in tracer uptake. METHODS: A Triad 88/Trionix system with three 6-pinhole collimators was used for investigation of dose requirements for imaging of the dopamine D(2) receptor ligand [(123)I]IBZM and the cerebral perfusion tracer [(99m)Tc]HMPAO (1.2-0.4 MBq/g body weight) in healthy mice. The fatty acid [(123)I]IPPA (0.94 +/- 0.05 MBq/g body weight) and the perfusion tracer [(99m)Tc]sestamibi (3.8 +/- 0.45 MBq/g body weight) were applied to cardiomyopathic mice overexpressing the prostaglandin EP(3) receptor. RESULTS: In vivo imaging and in vitro data revealed 45 kBq total cerebral uptake and 201 kBq cardiac uptake as thresholds for visualisation of striatal [(123)I]IBZM and of cardiac [(99m)Tc]sestamibi using 100 and 150 s acquisition time, respectively. Alterations of maximal cerebral uptake of [(123)I]IBZM by >20% (116 kBq) were verified with the prerequisite of 50% striatal of total uptake. The labelling with [(99m)Tc]sestamibi revealed a 30% lower uptake in cardiomyopathic hearts compared to wild types. [(123)I]IPPA uptake could be visualised at activity doses of 0.8 MBq/g body weight. CONCLUSION: Multi-pinhole SPECT enables detection of alterations of the cerebral uptake of (123)I- and (99m)Tc-labelled tracers in an appropriate dose range in murine models targeting physiological processes in brain and heart. The thresholds of detection for differences in the tracer uptake determined under the conditions of our experiments well reflect distinctions in molar activity and uptake characteristics of the tracers.

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.

P2Y receptors: focus on structural, pharmacological and functional aspects in the brain.

Purine and pyrimidine nucleotides have been identified as potent extracellular signalling molecules, acting at two classes of cell surface receptors, ionotropic P2X and metabotropic P2Y receptor (-R) types. Hitherto eight subtypes of the P2Y-R family have been cloned from mammalian species that exhibit sensitivity to the adenine nucleotides ATP/ADP (P2Y(1,11,12,13)), the uracil nucleotides UTP/UDP (P2Y(2,4,6) or UDP-glucose in the case of P2Y(14)) or both adenine and uracil nucleotides (P2Y(2)). The P2Y-Rs are G protein-coupled receptors activating phospholipase C via Galpha(q/11) protein and stimulating or inhibiting adenylyl cyclase via Galpha(s) and Galpha (i/o) proteins, respectively. These receptors may activate distinct signalling cascades. Although classical models predict that P2Y-Rs exist in the cell membrane as monomers, homo- or heterodimeric assemblies may be generated. Interactions with certain ion channels or ligand-gated receptors as well as the co-localization of several receptor subtypes in the same cell provide the basis for a high functional diversity. The proteins for various P2Y-Rs are expressed early in the embryonic brain and are broadly distributed on both, neurons and astroglial cells. P2Y-R involvement in the regulation of normal physiological processes on the cellular level or in vivo, such as modulation of transmitter release, generation of astroglial Ca(2+) waves, in diverse effects on behavioural functions and in the etiopathology of neurodegenerative diseases, are discussed and own data are presented. However, the exact understanding of the role of individual P2Y-R subtypes is still limited. Concerning the potentially important functions of P2Y-Rs, there is a strong need to develop stable, lipophilic and subtype-selective P2Y-R ligands, which may open new therapeutic strategies.

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.

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.

P2 receptor-mediated effects on the open field behaviour of rats in comparison with behavioural responses induced by the stimulation of dopamine D2-like and by the blockade of ionotrophic glutamate receptors.

The effects of the P2 receptor ligands 2-methylthio ATP (2-MeSATP; 10 pmol)--as a non-specific agonist--and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10 pmol)--as a non-selective antagonist--after bilateral intra-accumbens injection on the locomotor response were investigated in an open field situation. The P2 receptor-mediated effects on the pattern of locomotor activity were compared with the effects caused by the dopamine D2-like receptor agonist quinpirole (10 pmol) and by the combination of the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 10 pmol) with the alpha-amino-3-hydro-5-methyl-4-isoxazolpropionic acid (AMPA) and kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 30 pmol). The intra-accumbens injection of all tested compounds elicited an increase in the locomotor activity over a test period of 20 min when compared with the controls. No statistically significant differences could be evaluated between the different drug-treated groups. However, a more detailed analysis--using further behavioural parameters such as the number of movement direction changes, the effective running time and the running speed--revealed two basically different patterns of locomotor activity. The locomotor response induced by the injection of 2-MeSATP or quinpirole was characterised by a continuous and consistent locomotion, whereas the enhanced locomotor activity elicited by PPADS or CPP/CNQX was determined by an increased running speed accompanied by more disruptions and more changes of movement direction. The coadministration of 2-MeSATP and quinpirole led to an enhancement of locomotor activity in a limited post-treatment interval. The effects of both compounds could be abolished by the pre-treatment with the D2/D3 receptor antagonist sulpiride (100 pmol). Coadministration of PPADS and CPP/CNQX caused additive effects suggesting that the pathway mediated by P2 and ionotrophic glutamate receptors is different. The stimulation of P2 receptors in the nucleus accumbens (NAc) modulates the locomotion in the direction to be to be longer lasting, more consistent and more goal directed.

Modulation of feeding behaviour by blocking purinergic receptors in the rat nucleus accumbens: a combined microdialysis, electroencephalographic and behavioural study.

The nonspecific P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), the nonspecific P1 receptor antagonist 8-(p-sulphophenyl)-theophylline (8-SPT) and the combination of both were applied by retrograde microdialysis into the nucleus accumbens (NAc) before and during feeding of 18-h food-deprived rats. In addition to the registration of behavioural parameters, such as the amount and duration of food intake, the feeding-induced changes in dopamine (DA) concentration and the concomitant changes of neuronal activity in the NAc and the ventral tegmental area (VTA) were simultaneously determined. The perfusion with PPADS (20 microm) diminished the amount of food intake and the duration of feeding. Furthermore, the P2 receptor antagonist blocked the feeding-induced DA release and prevented the feeding-elicited changes of the electroencephalography (EEG) power distribution which was characterised by an increase in the power of the 8.0-13.0-Hz frequency band in the NAc and the VTA. The effects of PPADS could be completely prevented by the concomitantly perfused adenosine receptor antagonist 8-SPT (100 microm). When given alone, 8-SPT increased the amount of food ingested, the duration of feeding and the EEG power of the higher frequency range, particularly between 19.0 and 30.0 Hz, in both the NAc and the VTA. The feeding-elicited DA release was supplemented to the enhanced DA level caused by the perfusion with 8-SPT in an additive manner. The P2 and P1 receptor antagonists interact antagonistically in the modulation of feeding behaviour and the feeding-induced changes of EEG activity suggesting that both endogenous extracellular ATP and adenosine are involved in the regulation of the feeding-associated mesolimbic neuronal activity in a functionally antagonistic manner.

P2X receptor expression on astrocytes in the nucleus accumbens of rats.

Astrocytes express a variety of neurotransmitter receptors which render them capable of responding to extracellular stimuli, like ATP. Release of ATP, e.g. after brain injury, may initiate reactive gliosis via stimulation of purinergic P2X and P2Y receptors. In the present study, the expression and cellular localization of P2X receptor subtypes on astrocytes in the nucleus accumbens of rats under normal physiological conditions and after stab wound were investigated. Reverse transcription-polymerase chain reaction (RT-PCR) with specific P2X(1-7) primers, and double immunofluorescence with antibodies to glial fibrillary acidic protein (GFAP, a specific marker of fibrous astrocytes) and to different P2X receptor subtypes (P2X(1-4), P2X(7)) were used. The RT-PCR of tissue extracts of the nucleus accumbens of untreated rats revealed the presence of all seven currently known P2X receptor subtype mRNAs indicating the presence of these receptors in this region. A double immunofluorescence approach with confocal laser scanning microscopy showed the localization of P2X(2-4) receptor subtypes on GFAP-labelled astrocytes in untreated rats. Labelling for P2X(1) and P2X(7) receptor subtypes was not found. After mechanical damage all P2X receptor subtypes studied (P2X(1-4), P2X(7)) were observed on the GFAP-labelled reactive astrocytes. A characteristic distribution of the P2X receptors on astrocytic processes and cell bodies as well as an up-regulation of the P2X-immunofluorescence was found. In conclusion, the data show the presence of P2X receptors on rat nucleus accumbens astrocytes and suggest that astrogliosis in vivo is associated with an up-regulation of distinct P2X receptor subtypes.

P2 receptor-types involved in astrogliosis in vivo.

1. 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 (alpha,beta-methylene adenosine 5'-triphosphate; alpha,beta-meATP), P2Y1,12 (adenosine 5'-O-(2-thiodiphosphate; ADP-beta-S) and P2Y2,4,6 receptors (uridine 5'-O-(3-thiotriphosphate; UTP-gamma-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). 2. The agonist-induced changes in comparison to the artificial cerebrospinal fluid (aCSF)-treated control side reveal a strong mitogenic potency of ADP-beta-S and alpha,beta-meATP, whereas UTP-gamma-S was ineffective. The P2 receptor antagonist PPADS decreased the injury-induced proliferation when given alone and in addition inhibited all agonist effects. 3. 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 alpha,beta-meATP or ADP-beta-S. In contrast, UTP-gamma-S failed to increase the GFAP-IR. The morphogenic effects were also inhibited by pre-treatment with PPADS. 4. A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3 and P2Y1 receptors on the GFAP-labelled astrocytes. 5. 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.

Accelerated functional recovery after neuronal injury by P2 receptor blockade.

The effect of the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on changes of the quantitative electroencephalogram (EEG) after injury of rat brain tissue was investigated. PPADS accelerated the functional recovery from microdialysis probe-induced disturbances in the nucleus accumbens by a decrease of the ratio of absolute slow (0.6-4 Hz) to fast (8-30 Hz) power, mainly caused by a decrease in the delta frequency power. These data provide evidence for a possible neuroprotective effect of P2 receptor antagonists in vivo.

Stimulation of P2 receptors in the ventral tegmental area enhances dopaminergic mechanisms in vivo.

It has been shown that endogenous adenosine 5'-triphosphate (ATP) as well as its exogenously applied structural analogue, 2-methylthio ATP (2-MeSATP), facilitate the release of dopamine from axon terminals in the rat nucleus accumbens (NAc) by activating ATP-sensitive P2 receptors. In the present study, reversed microdialysis of 2-MeSATP (10 microM, 100 microM and 1 mM), or its microinjection (0.5, 5.0 and 50 pmol) into the ventral tegmental area (VTA), dose-dependently increased the local extracellular level of dopamine and the locomotion in the open field, respectively. These effects were abolished by the P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). When applied alone, the antagonist decreased the basal dopamine concentration, indicating that endogenous ATP controls the somatodendritic release of dopamine. Repeated microinjections of 2-MeSATP (5 pmol) once daily for 4 days led to a reproducible locomotor stimulation in the open field. Conditioned locomotion was induced by re-exposure to the novel environment on the seventh day. A challenge with amphetamine (1 mg/kg intraperitoneally) on the eighth day enhanced the locomotor activity in the 2-MeSATP-treated group in the sense of a cross-sensitisation, but failed to do so in the control group. Neurons in the VTA were heavily stained with antibodies developed against the P2Y(1) subtype of P2 receptors. Taken together, our data suggest that P2 receptors (probably of the P2Y(1) subtype) are involved in the initiation of somatodendritic dopamine release in the VTA and thereby may have a profound influence on sensitisation and reward-motivated behaviour.

Deafferentation of the septo-hippocampal pathway in rats as a model of the metabolic events in Alzheimer's disease.

Changes in the metabolic activity within the brain of patients suffering from Alzheimer's disease (AD) were investigated and compared with biochemical alterations in the hippocampus induced by fimbria/fornix transection in the rat. The deafferentation of the hippocampus results in a degeneration of cholinergic septo-hippocampal terminals accompanied by a persistent decrease of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) activities similar to the cholinergic malfunction in AD. In the animal model the [3H]-cytochalasin B binding to the glucose transporters was elevated up to the day 7 after surgery as was the activity of the phosphofructokinase (PFK) on day 3. A reactive astrogliosis could be evidenced by the upregulation of glial fibrillary acidic protein (GFAP). An increase of the PFK activity was also found in AD being accompanied by enhanced level of GFAP as well. A higher concentration of mRNA for all three isoenzymes of PFK was shown by reverse transcription (RT)-real time polymerase chain reaction (PCR) amplification. However, the pattern of PFK isoenzyme proteins and mRNAs did neither change in diseased human nor in the lesioned rat brain. The activities of the mitochondrial enzymes pyruvate dehydrogenase complex (PDHC) and cytochrome c oxidase (CO) were diminished in the lesioned rat hippocampus on day 7 as well as in AD brain. Subcellular fractionation showed that the activity of these enzymes was affected in the synaptosomal as well as in the extrasynaptosomal mitochondria indicating a loss of neuronal input and also a vulnerability of intrinsic hippocampal neurons and/or non-neuronal cells. The recovery of the mitochondrial enzyme activity in the animal model at later post lesion intervals may be the result of compensatory responses of surviving cells or of sprouting of other non-affected inputs. It is concluded that common metabolic mechanisms may underlie the concurrent degenerative and repair processes in the denervated hippocampus and the diseased Alzheimer brain.

The purinergic P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2'4'-disulphonic acid prevents both the acute locomotor effects of amphetamine and the behavioural sensitization caused by repeated amphetamine injections in rats.

Repeated administration of amphetamine-like psychostimulants produce a progressive and long-lasting hypersensitivity to their behavioural effects known as behavioural sensitization. Previous studies have shown that administration of the purinergic P2 receptor agonist 2-methylthio ATP into the nucleus accumbens of rats raises the extracellular level of dopamine accompanied with enhanced locomotion in a similar manner. Furthermore, the quantitative EEG after application of 2-methylthio ATP or amphetamine was characterized by an elevation of the alpha1-power. However, purinergic P2 receptor antagonists decreased the basal level of dopamine in the NAc and in addition prevented the effects of 2-methylthio ATP. The purpose of the present study was to investigate, whether endogenous ATP acting via purinergic P2 receptors is involved in the process of amphetamine-induced sensitization. Rats were treated systemically for five successive days with d-amphetamine (1.5 mg/kg) and tested in an open field with respect to their locomotor response. The enhanced locomotor activity after the first injection of amphetamine was diminished by the previous intracerebroventricular application of the purinergic P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2'4'-disulphonic acid (PPADS; 0.6 nmol) (P<0.05). The challenge with a lower dose of amphetamine (0.75 mg/kg) produced an increased locomotion in comparison to the response after the first amphetamine application indicating the expression of a behavioural sensitization. Pretreatment with PPADS prior to each amphetamine administration prevented the increase of locomotor activity after the challenge with amphetamine (P<0.05). In summary, the present study demonstrates that PPADS blocks both the acute locomotor effects of amphetamine and the development of behavioural sensitization to the psychostimulant. We suggest that the activation of purinergic P2 receptors by endogenous ATP is necessary for the expression of these effects.

Mechanisms of adenosine 5'-triphosphate-induced dopamine release in the rat nucleus accumbens in vivo.

The endogenous mechanisms modulating ATP-induced dopamine release in the nucleus accumbens (NAc) were studied by microdialysis in freely moving rats. The ATP analog 2-Methylthio ATP (2-MeSATP) facilitated the release of dopamine in a manner sensitive to pertussis toxin and tetrodotoxin. It is suggested that G-protein-coupled P2Y receptors and voltage-gated sodium channels are involved in this process. N-methyl-D-aspartate (NMDA) applied in a concentration of 100 microM decreased the extracellular dopamine level, whereas 1 and 10 mM NMDA enhanced it. The endogenous agonist glutamate (10 microM) inhibited the basal and facilitated release of dopamine. Infusion with a combination of the ionotropic glutamate receptor antagonists (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), as well as with the metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG) increased the basal level of dopamine and potentiated the 2-MeSATP-facilitated dopamine release, suggesting an ATP-mediated glutamate release. The GABA(A) receptor antagonist bicuculline infused into the NAc also enhanced the basal level of dopamine; however, the application of 2-MeSATP in the presence of bicuculline caused an early decrease and a subsequent increase of dopamine release. The facilitatory phase of the 2-MeSATP effect was comparable with that measured in the absence of bicuculline. By contrast, when bicuculline was infused into the ventral tegmental area (VTA) it elevated the accumbal basal dopamine level and in addition facilitated the 2-MeSATP- and the glutamate-induced dopamine release above that measured in the absence of bicuculline. These results suggest that ATP in the NAc has a physiologically relevant function in modulating dopaminergic transmission depending on the mesolimbic neuronal activity. The first component of the ATP effect involves a direct stimulation of the terminals of VTA neurons, while the second inhibitory component involves a sequential activation of glutamate and, finally, via ionotropic and metabotropic glutamate receptors, of GABA neurons projecting to the VTA.

Suppression of feeding-evoked dopamine release in the rat nucleus accumbens by the blockade of P(2) purinoceptors.

In order to investigate whether endogenous adenosine 5'-triphosphate (ATP) is involved in the regulation of feeding, the influence of the P(2) receptor antagonist pyridoxalphospate-6-azophenyl-2', 4'-disulphonic acid (PPADS) infused into the rat nucleus accumbens on 18-h food-deprived feeding was tested. PPADS suppressed the feeding-induced dopamine release and reduced the amount of food consumed as well as the time of feeding. These results indicate that activation of P(2) purinoceptors by endogenous ATP facilitates feeding behaviour and contributes to the feeding-associated dopamine release.

Effects of intra-accumbens injection of 2-methylthio ATP: a combined open field and electroencephalographic study in rats.

RATIONALE: Previous experiments have shown that P2 receptor activation increases the release of dopamine in the mesolimbic mesocortical system. OBJECTIVE: In order to investigate the functional correlates of dopaminergic stimulation, EEG and behavioural responses to injection of the P2 receptor agonist 2-methylthio ATP (2-MeSATP) into the nucleus accumbens (NAc) of rats were investigated. METHODS: EEG electrodes were positioned into the NAc together with the guide cannula for intracerebral injection. Behavioural analysis was performed in an open field cage and was evaluated by a video activity measurement system. Rats were assigned to separate groups that were given artificial cerebrospinal fluid (aCSF) or drug treatment. RESULTS: 2-MeSATP significantly extended the period of locomotor activity in the novel environment. The quantitative EEG was characterized by an elevation of the power in the alpha-range and a decrease in power in the delta range. The P2 receptor antagonists reactive blue 2 but not pyridoxalphosphate-6-azophenyl-2'4'-disulphonic acid (PPADS) also enhanced locomotion when given alone, and elevated the alpha-1 and beta-2 bands. Both antagonists abolished the locomotor and EEG responses to 2-MeSATP. The dopamine D1 receptor antagonist SCH 23390 and the D2/D3 receptor antagonist sulpiride did not alter locomotor activity when given either alone or in combination. Only sulpiride and especially sulpiride in combination with SCH 23390 prevented the effect of 2-MeSATP. Sulpiride produced a selective increase in the alpha-1 band of the power spectrum whereas SCH 23390 elevated the power of the alpha-1, alpha-2 and beta-1 activities. Neither antagonist inhibited the effect of 2-MeSATP on the EEG when applied separately; however, the co-administration of SCH 23390 and sulpiride abolished the 2-MeSATP-induced alteration of power distribution. After a 6-hydroxydopamine (6-OHDA)-induced lesion of the accumbal dopaminergic terminals, 2-MeSATP failed to enhance the locomotor activity and to induce the characteristic EEG changes. CONCLUSIONS: The observed alterations in open field behaviour and quantitative EEG after injection of 2-MeSATP into the NAc may be mostly due to P2 receptor-mediated dopamine release and subsequent receptor activation.