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Notes: Mice treated with the psychostimulant methamphetamine (MA) showed the appearance of intracellular inclusions in the nucleus of medium sized striatal neurones and cytoplasm of neurones of the substantia nigra pars compacta but not in the frontal cortex. All inclusions contained ubiquitin, the ubiquitin activating enzyme (E1), the ubiquitin protein ligase (E3-like, parkin), low and high molecular weight heat shock proteins (HSP 40 and HSP 70). Inclusions found in nigral neurones stained for α-synuclein, a proteic hallmark of Lewy bodies that are frequently observed in Parkinson's disease and other degenerative disorders. However, differing from classic Lewy bodies, MA-induced neuronal inclusions appeared as multilamellar bodies resembling autophagic granules. Methamphetamine reproduced this effect in cultured PC12 cells, which offered the advantage of a simple cellular model for the study of the molecular determinants of neuronal inclusions. PC12 inclusions, similar to those observed in nigral neurones, were exclusively localized in the cytoplasm and stained for α-synuclein. Time-dependent experiments showed that inclusions underwent a progressive fusion of the external membranes and developed an electrodense core. Inhibition of dopamine synthesis by α-methyl-p-tyrosine (αMpT), or administering the antioxidant S-apomorphine largely attenuated the formation of inclusions in PC12 cells exposed to MA. Inclusions were again observed when αMpT-treated cells were loaded with l-DOPA, which restored intracellular dopamine levels.

Notes: Various studies demonstrated that the neurotransmitter norepinephrine (NE) plays a relevant role in modulating seizures; in particular, a powerful effect consists in delaying the kindling of limbic areas such as the amygdala and hippocampus. Given the rich NE innervation of limbic regions, we selected a sensitive trigger area, the anterior piriform cortex, to test whether previous loss of noradrenergic terminals modifies sporadic seizures in rats. The damage to locus coeruleus terminals was produced by using the selective neurotoxin N-(-2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, 60 mg/kg i.p.). In intact rats, bicuculline (a GABA-A antagonist, 118 pmol) microinfused into this area produced sporadic seizures, while in rats previously injected with DSP-4, bicuculline determined long-lasting self-sustaining status epilepticus. In intact rats, sporadic seizures were accompanied by a marked increase in norepinephrine release in the contralateral piriform cortex, while in locus coeruleus-lesioned rats this phenomenon was attenuated. While bicuculline-induced sporadic seizures were prevented by the focal infusion of amino-7-phosphonoheptanoic acid (AP-7, a selective NMDA antagonist), or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide (NBQX, a selective non-NMDA antagonist), status epilepticus obtained in norepinephrine-lesioned rats was insensitive to AP-7 but was still inhibited by NBQX. By using fluorescent staining for damaged (Fluoro-Jade B) and intact (DAPI) neurons, as well as cresyl violet, we found that rats undergoing status epilepticus developed neuronal loss in various limbic regions. This study demonstrates a powerful effect of noradrenergic terminals in regulating the onset of limbic status epilepticus and its sensitivity to specific glutamate antagonists.

Notes: Abstract The present study investigates the possibility that imidazoline receptors mediate modulation of cholinergic motor functions of the guinea-pig ileum. For this purpose, the effects of a series of compounds with known affinity for α2-adrenoceptors and/or imidazoline recognition sites were examined on the cholinergic twitch contractions evoked by electrical field stimulation (0.1 Hz) of longitudinal muscle-myenteric plexus preparations. Additional experiments were carried out on ileal strips preincubated with [3H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation (1 Hz). The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. α-Methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline and xylazine caused a concentration-dependent inhibition of twitch responses (IC50 from 0.13 to 1.05 µM; Emax from 85.9 to 92.5%). Rilmenidine and agmatine were less potent in reducing the twitch activity, and the latter compound acted also with low intrinsic activity (IC50=44.9 µM; Emax=35.5%). In interaction experiments, the inhibitory action of clonidine on twitch responses was competitively antagonized by RX 821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), idazoxan, rauwolscine, yohimbine and BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)-methyl]-4,5-dihydroimidazoline), whereas prazosin (10 µM), ARC 239 (2-(2,4-(O-methoxy-phenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione; 10 µM) and BRL 41992 (1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazol[1,5-a]azepi-ne; 10 µM) were without effect. Rauwolscine antagonized the inhibitory effects of various agonists on ileal twitch activity in a competitive manner and with similar potency. Agmatine and idazoxan did not significantly modify the twitch contractions when tested in the presence of α2-adrenoceptor blockade by rauwolscine (3 µM) or RX 821002 (1 µM). Linear regression analysis showed that the affinity values of antagonists correlated with their affinity at the α2A and α2D binding sites as well as at previously classified α2A/D adrenoceptor subtypes, whereas no significant correlation was obtained when comparing the potency estimates of agonists and antagonists with the affinity at I1 or I2 binding sites. When tested on the electrically induced outflow of tritium, α-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline, xylazine and rilmenidine yielded inhibitory concentration-response curves which were shifted rightward to a similar extent in the presence of rauwolscine (3 µM). In the absence of further drugs, agmatine significantly reduced the evoked tritium outflow at the highest concentrations tested (10 and 100 µM), whereas idazoxan (up to 100 µM) was without effect. When RX 821002 (1 µM) was added to the superfusion medium, neither agmatine nor idazoxan modified the evoked outflow of radioactivity. The results argue against modulation by imidazoline receptors of acetylcholine release from myenteric plexus nerve terminals. They provide evidence that compounds endowed with imidazoline-like structures affect the cholinergic motor activity of the guinea-pig ileum by interacting with presynaptic α2-adrenoceptors belonging to the α2D subtype.