Wolfgang Poelchen

Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor

Caffeine is believed to act by blocking adenosine A1 and A2A receptors (A1R, A2AR), indicating that some A1 receptors are tonically activated. We generated mice with a targeted disruption of the second coding exon of the A1R (A1R−/−). These animals bred and gained weight normally and had a normal heart rate, blood pressure, and body temperature. In most behavioral tests they were similar to A1R+/+ mice, but A1R−/− mice showed signs of increased anxiety. Electrophysiological recordings from hippocampal slices revealed that both adenosine-mediated inhibition and theophylline-mediated augmentation of excitatory glutamatergic neurotransmission were abolished in A1R−/− mice. In A1R+/− mice the potency of adenosine was halved, as was the number of A1R. In A1R−/− mice, the analgesic effect of intrathecal adenosine was lost, and thermal hyperalgesia was observed, but the analgesic effect of morphine was intact. The decrease in neuronal activity upon hypoxia was reduced both in hippocampal slices and in brainstem, and functional recovery after hypoxia was attenuated. Thus A1Rs do not play an essential role during development, and although they significantly influence synaptic activity, they play a nonessential role in normal physiology. However, under pathophysiological conditions, including noxious stimulation and oxygen deficiency, they are important.

Role of ATP in fast excitatory synaptic potentials in locus coeruleus neurones of the rat

1 Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). The pressure application of α,β‐methylene ATP (α,β‐meATP) caused reproducible depolarizations which were depressed by suramin (30 μM) and abolished by suramin (100 μM). Pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS; 10, 30 μM) also concentration‐dependently inhibited the α,β‐meATP‐induced depolarization, although with a much slower time‐course than suramin. Almost complete inhibition developed with 30 μM PPADS. Reactive blue 2 (30 μM) did not alter the effect of α,β‐meATP, while reactive blue 2 (100 μM) slightly depressed it. 2 Pressure‐applied (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) also depolarized LC neurones. Kynurenic acid (500 μM) depressed and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 50 μM) abolished the response to AMPA. Suramin (100 μM) potentiated the AMPA effect. 3 Pressure‐applied noradrenaline hyperpolarized LC neurones. Suramin (100 μM) did not alter the effect of noradrenaline. 4 Focal electrical stimulation evoked biphasic synaptic potentials consisting of a fast depolarization (p.s.p.) followed by a slow hyperpolarization (i.p.s.p.). A mixture of D(−)‐2‐amino‐5‐phosphonopentanoic acid (AP‐5; 50 μM), CNQX (50 μM) and picrotoxin (100 μM) depressed both the p.s.p. and the i.p.s.p. Under these conditions suramin (100 μM) markedly inhibited the p.s.p., but did not alter the i.p.s.p. In the combined presence of AP‐5 (50 μM), CNQX (50 μM), picrotoxin (100 μM), strychnine (0.1 μM), tropisetron (0.5 μM) and hexamethonium (100 μM), a high concentration of suramin (300 μM) almost abolished the p.s.p. without changing the i.p.s.p. 5 In the presence of kynurenic acid (500 μM) and picrotoxin (100 μM), PPADS (30 μM) depressed the p.s.p. Moreover, the application of suramin (100 μM) to the PPADS (30 μM)‐containing medium failed to cause any further inhibition. Neither PPADS (30 μM) nor suramin (100 μM) altered the i.p.s.p. 6 It was concluded that the cell somata of LC neurones are endowed with excitatory P2‐purinoceptors. ATP may be released either as the sole transmitter from purinergic neurones terminating at the LC or as a co‐transmitter of noradrenaline from recurrent axon collaterals or dendrites of the LC neurones themselves.

Ethanol-induced inhibition of NMDA receptor channels

Ethanol is a potent inhibitor of the N-methyl-D-aspartate (NMDA)-receptor subtype of glutamate receptor in a number of brain areas. The mechanism of ethanol action has been investigated by means of patch-clamp recording of ionic currents and fura-2 measurement of intracellular Ca2+ concentration in cell culture systems; the subunit composition of NMDA receptors and their influence on the effect of ethanol was determined by molecular biology methods. Ethanol does not appear to interact with NMDA either at the glutamate recognition site of the receptor, or at any of the hitherto known multiple modulatory sites, such as the glycine or polyamine site. Moreover, ethanol does not cause an open channel block by itself and fails to interact with Mg2+ at the site where it causes open channel block. The ability of ethanol to inhibit responses to NMDA is dependent on the subunit combination of NMDA receptors. The NR1/NR2A and NR1/NR2B combinations are preferentially sensitive to ethanol inhibition. Chronic treatment with ethanol leads to an increase of the NMDA receptor number at the transcriptional and posttranscriptional level; the receptor function is also facilitated. This causes withdrawal-type seizures after termination of chronic treatment with ethanol. The inhibition of NMDA receptors by ethanol leads to the depression of excitatory synaptic potentials mediated by this type of excitatory amino acid receptor. Ethanol-induced disturbances in certain regions of the brain, i.e. hippocampus, nucleus accumbens or locus coeruleus may lead to cognitive disorders or drug dependence. Brain slices containing the locus coeruleus may be used as an in vitro test system to investigate the addictive properties of ethanol.

Interaction between P2Y and NMDA receptors in layer V pyramidal neurons of the rat prefrontal cortex.

In the first part of this study, monosynaptic excitatory postsynaptic potentials (EPSPs) in layer V of the rat prefrontal cortex were evoked by electrical stimulation of layer I. Recordings by intracellular sharp microelectrodes showed that EPSPs were concentration-dependently facilitated by the P2 receptor antagonistic ATP analogue 2-methylthio ATP (2-MeSATP), while ATP itself depressed the synaptic potentials. The inhibitory effect of ATP turned into facilitation in the presence of the adenosine A(1) receptor antagonist DPCPX. The 2-MeSATP-induced potentiation of EPSP amplitudes were prevented by the P2 receptor antagonists PPADS and Suramin. The EPSP was almost abolished by coapplication of the NMDA receptor antagonist AP-5 and the AMPA/kainate receptor antagonist CNQX. After blockade of the NMDA receptor-mediated part of the EPSP by AP-5, the stimulatory effect of 2-MeSATP disappeared. When NMDA or AMPA were pressure-applied onto pyramidal cells, only the NMDA-induced depolarization was potentiated by 2-MeSATP. In the second part of the study, NMDA-induced currents were measured by whole-cell patch-clamp pipettes. ATP, 2-MeSATP, UDP and UTP potentiated the response to NMDA, while ADP-beta-S was inactive. PPADS antagonized the effect of ATP. Synaptic isolation of pyramidal neurons by a Ca(2+)-free medium or tetrodotoxin did not alter the effect of ATP which, however, was markedly depressed when GTP in the micropipette was replaced by GDP-beta-S. These observations suggest that in layer V pyramidal neurons of the prefrontal cortex postsynaptically localized P2Y receptors interact with NMDA receptor-channels.

Inhibition by ethanol of excitatory amino acid receptors in rat locus coeruleus neurons in vitro

Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). In a first series of experiments, various parameters of spontaneous action potentials were evaluated. It turned out that ethanol (100 mM) does not alter the firing rate, the spike amplitude and the afterhyperpolarization following a spike. In subsequent experiments, the generation of action potentials was prevented by passing continuous hyperpolarizing current via the recording electrode. Under these conditions, ethanol (100 mM) had no effect on the membrane potential or input resistance. Pressure-applied N-methyl-D-aspartate (NMDA), (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and α,β-methylene ATP (α,β-meATP) reproducibly depolarized LC neurons. While ethanol (100 mM) depressed the NMDA- and AMPA-induced depolarization to a similar extent, it did not interact with α,β-meATP. Lower concentrations of ethanol (10 and 30 mM) had no effect on depolarizing responses to NMDA or AMPA. Noradrenaline applied by pressure pulses reproducibly hyperpolarized LC cells. These hyperpolarizations were unchanged by ethanol (100 mM). Biphasic synaptic potentials consisting of early depolarizing (PSP) and late hyperpolarizing (IPSP) components were evoked by electrical stimulation. Ethanol (100 mM) depressed the PSP and increased the IPSP. Glutamatergic PSPs recorded in the combined presence of picrotoxin (100 μM) and suramin (100 μM) were also inhibited by ethanol (100 mM). However, IPSPs recorded under these conditions were insensitive to ethanol (100 mM). In conclusion, ethanol may interfere with the AMPA (or NMDA) receptor-mediated fraction of the PSP and slightly facilitate the α2 adrenoceptor-mediated fraction of the IPSP.

In vitro tolerance to inhibition by ethanol of N-methyl-D-aspartate-induced depolarization in locus coeruleus neurons of behaviorally ethanol-tolerant rats.

Intracellular recordings were made in pontine slice preparations of the rat brain containing the locus coeruleus (LC). Ethanol at 100 mM, but not at 10 or 30 mM inhibited depolarizing responses to pressure-applied N-methyl-D-aspartate (NMDA) in LC neurons of ethanol-naive rats. Ethanol (100 mM) had a similar effect in LC neurons of ethanol-naive rats, of rats treated with ethanol for 14 days (3 g/kg daily, i.p.) and of rats treated with equicaloric amounts of saccharose (5 g/kg daily, i.p.). The blood concentration of ethanol was markedly decreased at 4 h, and was below the detection limit at 24 h after the last injection. Behavioral measurements in the open-field system demonstrated the development of tolerance in rats receiving ethanol for 14 days. Moreover, an anxiety-related reaction was shown to develop when the acute effect of the last ethanol injection vanished. Therefore, in subsequent in vitro experiments, ethanol (10 mM) was continuously present in the superfusion medium in order to mimic a steady blood concentration and to prevent a withdrawal-like situation. Under these conditions, ethanol (100 mM) still continued to inhibit the NMDA-induced depolarization in slices of untreated rats, but became ineffective in slices of ethanol-treated rats at 4 h after the last injection. By contrast, a supersensitivity to ethanol developed in brain slices at 24 h after the last ethanol injection. In conclusion, in vitro tolerance between systemically and locally applied ethanol at LC neurons could only be demonstrated when a low concentration of ethanol was added to the superfusion medium to simulate the blood concentration of this compound.

Chapter 18 P2 receptor-mediated activation of noradrenergic and dopaminergic neurons in the rat brain

Publisher Summary Focal electrical stimulation evokes in the rat locus coeruleus (LC) biphasic synaptic potentials consisting of early depolarizing (p.s.p.) and late hyperpolarizing (i.p.s.p.) components. It has been found that the p.s.p. is because of the release of glutamate from afferent fibres predominantly onto non-N-methyl-D- aspartate (non-NMDA) receptors and of γ-aminobutyric acid (GABA) onto GABA A receptors. In the chapter, the investigations demonstrate the presence of release stimulatory P2 receptors in the central noradrenergic and dopaminergic systems. Hence, exogenously applied or endogenously released ATP may excite noradrenergic neurons of the LC and dopaminergic neurons of the ventral tegmental area (VTA). ATP and its structural analog 2-MeSATP depolarized LC neurons in a slice preparation. In addition, there is strong evidence for the contribution of ATP to excitatory synaptic potentials recorded from the neurons themselves; this ATP appears to be coreleased with noradrenaline from recurrent axon collaterals or dendrites. A combined methodological approach was used to prove the presence of P2 receptors in the VTA and its main projection target in the NAc. Although electrophysiological recordings from VTA neurons in a slice preparation failed to show a depolarizing response to 2-MeSATP, the application of 2-MeSATP into the VTA via a microdialysis probe was a powerful stimulus of dopamine release in vivo .

Tolerance to inhibition by ethanol of N-methyl-d-aspartate-induced depolarization in rat locus coeruleus neurons in vitro

Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus. The pressure application of N-methyl-D-aspartate (NMDA) produced reproducible depolarizations of stable amplitude. Superfusion with ethanol (100 mM) for 15 min inhibited the depolarizing response to NMDA: the effect of ethanol was rapidly reversed on washout. When the superfusion time of ethanol (100 mM) was increased to 60 min, its inhibitory effect disappeared after 50 to 60 min. Moreover, after the subsequent washout of ethanol a withdrawal-like increase in the sensitivity to NMDA became evident. Hence, adaptive mechanisms of locus coeruleus neurons during the long-time contact with ethanol may be modelled in an in vitro system.

Effect of extracellular adenosine 5'-triphosphate on principal neurons of the rat ventral tegmental area.

Intracellular recordings were made in a midbrain slice preparation of the rat brain containing the ventral tegmental area (VTA). Dopaminergic principal cells were identified by their electrophysiological properties and their hyperpolarizing responses to dopamine. Superfusion with dopamine (100 microM) caused hyperpolarization and a decrease of the apparent input resistance. By contrast, two structural analogues of ATP, 2-methylthio ATP (2-MeSATP; 10 microM) and alpha,beta-methylene ATP (alpha, beta-meATP; 30 microM) had no effect, when added to the superfusion medium. Pressure applied dopamine also hyperpolarized the membrane, while both 2-MeSATP and alpha,beta-meATP were ineffective. Hence, dopaminergic principal neurons of the VTA do not possess somatic P2 purinoceptors present on peripheral and central noradrenergic neurons.

Influence of Long-Term Ethanol Treatment on Rat Liver Aniline and p-Nitrophenol Hydroxylation

The present study investigates the influence of long-term ethanol (EtOH) treatment of rats [10% (v/v) for 1, 4, 12, and 36 weeks] on hepatic microsomal cytochrome P450 (P450) content and liver aniline and p-nitrophenol hydroxylation. Total P450 per liver was stimulated after EtOH treatment for 1, 4, and 12 weeks. In the case of longer EtOH treatment no additional stimulation in P450 content was observed. Aniline and p-nitrophenol hydroxylase activity increased in direct relation with the duration of EtOH consumption. The stimulation of both enzymatic activities was different. In comparison to controls, in rats treated with 10% (v/v) EtOH for 1, 4, 12, and 36 weeks, an increase in nitrocatechol formation (1.1-, 1.2-, 2.2-, and 2.8-fold, respectively) was found. In contrast, no effect was observed on the metabolism of aniline after 1 and 4 weeks of EtOH consumption. Aniline hydroxylation increased after 12 and 36 weeks of EtOH treatment only. Addition of EtOH in vitro had an inhibitory effect on both aniline and p-nitrophenol hydroxylation. With liver microsomes from controls as well as EtOH-treated rats the inhibition of p-nitrophenol hydroxylation was competitive in nature (Ki = 5.6 mM and Ki = 5.9 mM). In contrast, there was a competitive inhibition of aniline hydroxylation with liver microsomes from controls only. With microsomes from EtOH-treated rats a mixed inhibition was found.