Nucleotide‐induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100ngml−1) treated (non‐proliferating) rat microglial cells were recorded by the whole‐cell patch‐clamp technique. Most experiments were carried out on non‐proliferating microglial cells. ATP (100nm–1mm), ADP (10nm–10mm) and UTP (1μm–100mm), but not uridine (100μm–10mm) produced a slow outward current at a holding potential of 0mV. The effect of UTP (1mm) did not depend on the presence of extracellular Mg2+ (1mm). The outward current response to UTP (1mm) was similar in non‐proliferating and proliferating microglia. In non‐proliferating microglial cells, the ATP (10μm)‐induced outward current was antagonized by suramin (300μm) or reactive blue 2 (50μm), whereas 8‐(p‐sulphophenyl)‐theophylline (8‐SPT; 100μm) was inactive. By contrast, the current induced by UTP (1mm) was increased by suramin (300μm) and was not altered by reactive blue 2 (50μm) or 8‐SPT (100μm). The current response to UTP (1mm) disappeared when K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150mm). However, the effect of UTP (1mm) did not change when most Cl− was replaced with an equimolar concentration of gluconate− (145mm). The application of 4‐aminopyridine (1mm) or Cs+ (1mm) to the bath solution failed to alter the UTP (1mm)‐induced current. UTP (1mm) had almost no effect in a nominally Ca2+‐free bath medium, or in the presence of charybdotoxin (0.1μm); the inclusion of U‐73122 (5μm) or heparin (5mgml−1) into the pipette solution also blocked the responses to UTP (1mm). By contrast, the effect of ATP (10μm) persisted under these conditions. I‐V relations were determined by delivering fast voltage ramps before and during the application of UTP (1mm). In the presence of extracellular Cs+ (1mm) and 4‐aminopyridine (1mm) the UTP‐evoked current crossed the zero current level near−75mV. Omission of Ca2+ from the Cs+ (1mm)‐ and 4‐aminopyridine (1mm)‐containing bath medium or replacement of K+ by Cs+ (150mm) in the pipette solution abolished the UTP current. Replacement of GTP (200μm) by GDP‐β‐S (200μm) in the pipette solution abolished the current evoked by UTP (1mm). When the pipette solution contained Cs+ (150mm) instead of K+ and in addition inositol 1,4,5,‐trisphosphate (InsP3; 10μm), an inward current absolutely dependent on extracellular Ca2+ was activated after the establishment of whole‐cell recording conditions. This current had a typical delay, a rather slow time course and did not reverse its amplitude up to 100mV, as measured by fast voltage ramps. A rise of the internal free Ca2+ concentration from 0.01 to 0.5μm on excised inside‐out membrane patches produced single channel activity with a reversal potential of 0mV in a symmetrical K+ solution. The reversal potential was shifted to negative values, when the extracellular K+ concentration was decreased from 144 to 32mm. By contrast, a decrease of the extracellular Cl− concentration from 164 to 38mm did not change the reversal potential. Purine and pyrimidine nucleotides ...

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alpha 2-Adrenoceptor agonists inhibit the firing of locus coeruleus (LC) neurons. It was recently observed that the alpha-adrenoceptor agonists clonidine, rilmenidine and cirazoline, when injected intravenously in anaesthetized rats pretreated with the irreversible alpha 2-adrenoceptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), excite the LC. The effect was attributed to activation of I1 imidazoline receptors. The aim of the present experiments was to characterize the direct effect of alpha 2-adrenoceptor and I1 imidazoline receptor agonists on LC neurons. Electrical activity of LC neurons was extracellularly recorded in midpontine slices prepared from the rat brain. Concentration-response curves were obtained for the alpha 2-agonist noradrenaline and the mixed I1/alpha 2-receptor agonists clonidine, rilmenidine and moxonidine in slices without treatment and in slices treated with 6-chloro-N-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F86466) or EEDQ, alpha 2-adrenoceptor antagonists with low affinity for I1 and I2 imidazoline receptors, respectively. All four agonists concentration-dependently reduced the firing rate of the neurons, with full inhibition at higher concentrations. SK&F86466 shifted the concentration-response curves of the agonists to the right; the calculated antagonist dissociation constants are compatible with an effect of the agonists on alpha 2-adrenoceptors. EEDQ completely prevented the inhibition by the agonists. Neither in SK&F86466- nor in EEDQ-treated slices was an excitation by clonidine, rilmenidine and moxonidine observed. We conclude that the LC neurons do not possess functional I1 (and also no I2) imidazoline receptors. The effects of noradrenaline, clonidine, rilmenidine and moxonidine on the neurons can be fully explained with an interaction with inhibitory alpha 2-adrenoceptors (probably of the alpha 2D subtype). The excitation of the LC by imidazoline receptor agonists under in vivo conditions, hence, is not a direct effect on the neurons of the LC.

The frequency of spontaneous action potentials of locus coeruleus (LC) neurons was recorded extracellularly in pontine slices of the rat brain. Ethanol (1-100 mM) elevated the firing rate in most neurons; this effect was concentration-dependent. (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 0.03-1 microM), kainate (0.1-3 microM), N-methyl-D-aspartate (NMDA; 1-30 microM), substance P (0.01-1 microM), nicotine (0.1-10 microM) and alpha,beta-methylene ATP (alpha,beta-meATP; 0.3-30 microM), all increased the firing. Application of ethanol (10-100 mM) to the superfusion medium for 10 min, reproducibly and concentration-dependently inhibited the facilitatory effect of NMDA (10 microM). However, the inhibitory effect of ethanol (100 mM) decreased during a 30-min superfusion period and after the wash-out of ethanol the sensitivity of LC neurons to NMDA (10 microM) tended to overshoot above their initial level. Although NMDA was more potent in the absence than in the presence of external Mg2+, ethanol (100 mM) continued to depress the facilitatory effect of a low concentration of NMDA (3 microM) in a Mg(2+)-free medium. By contrast, in a medium containing normal Mg2+, ethanol (100 mM) failed to significantly interfere with the increase in firing rate induced by a high concentration of NMDA (30 microM). The effects of kainate (0.5 microM), AMPA (0.3 microM) and nicotine (1 microM) were also depressed by ethanol (100 mM), while the effects of substance P (0.03 microM) and alpha,beta-meATP (30 microM) were not changed.(ABSTRACT TRUNCATED AT 250 WORDS)

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