Fosfomycin Pharmacokinetic Profile in Plasma and Urine and Quantitative Estimation in Prostate and Seminal Vesicles after One and Two Consecutive Doses of Oral Fosfomycin Trometamol in Healthy Male Volunteers.

The present Phase I study investigated, for the first time, fosfomycin pharmacokinetics in humans after two 3 g doses of fosfomycin trometamol administered 27 h apart, according to the dose regimen recommended for the prophylactic indication for transrectal prostate biopsy in adult men. Plasma, urine and seminal plasma concentrations were measured after one and two consecutive doses in 24 healthy men, representative of the target population of the prophylactic indication. Prostate and seminal vesicle concentrations were estimated based on seminal plasma concentrations using a one-step regression method. The exposure to fosfomycin was very similar in rate (Cmax, tmax) after one and two doses. The AUC showed a minimal increment. On average, the apparent volume of distribution was high (>100 L), and the mean clearance had an intermediate value. The total amount and dose fraction of fosfomycin excreted in urine showed a small increment after two doses. The renal clearance was about 5 L/h. The fosfomycin concentration in the prostate and seminal vesicles showed that the antibiotic increased on average after two consecutive doses. This result confirmed the ability of fosfomycin to distribute into the prostate and into seminal vesicles after one single dose and that a two consecutive dose regimen increases the antibiotic availability inside these peripheral tissues.

Dopamine D2 receptor dysfunction is rescued by adenosine A2A receptor antagonism in a model of DYT1 dystonia.

DYT1 dystonia is an inherited disease linked to mutation in the TOR1A gene encoding for the protein torsinA. Although the mechanism by which this genetic alteration leads to dystonia is unclear, multiple lines of clinical evidence suggest a link between dystonia and a reduced dopamine D2 receptor (D2R) availability. Based on this evidence, herein we carried out a comprehensive analysis of electrophysiological, behavioral and signaling correlates of D2R transmission in transgenic mice with the DYT1 dystonia mutation. Electrophysiological recordings from nigral dopaminergic neurons showed a normal responsiveness to D2-autoreceptor function. Conversely, postsynaptic D2R function in hMT mice was impaired, as suggested by the inability of a D2R agonist to re-establish normal corticostriatal synaptic plasticity and supported by the reduced sensitivity to haloperidol-induced catalepsy. Although an in situ hybridization analysis showed normal D1R and D2R mRNA expression levels in the striata of hMT mice, we found a significant decrease of D2R protein, coupled to a reduced ability of D2Rs to activate their cognate Go/i proteins. Of relevance, we found that pharmacological blockade of adenosine A2A receptors (A2ARs) fully restored the impairment of synaptic plasticity observed in hMT mice. Together, our findings demonstrate an important link between torsinA mutation and D2R dysfunction and suggest that A2AR antagonism is able to counteract the deficit in D2R-mediated transmission observed in mutant mice, opening new perspectives for the treatment of this movement disorder.

Impairment of bidirectional synaptic plasticity in the striatum of a mouse model of DYT1 dystonia: role of endogenous acetylcholine.

DYT1 dystonia is a severe form of inherited dystonia, characterized by involuntary twisting movements and abnormal postures. It is linked to a deletion in the dyt1 gene, resulting in a mutated form of the protein torsinA. The penetrance for dystonia is incomplete, but both clinically affected and non-manifesting carriers of the DYT1 mutation exhibit impaired motor learning and evidence of altered motor plasticity. Here, we characterized striatal glutamatergic synaptic plasticity in transgenic mice expressing either the normal human torsinA or its mutant form, in comparison to non-transgenic (NT) control mice. Medium spiny neurons recorded from both NT and normal human torsinA mice exhibited normal long-term depression (LTD), whereas in mutant human torsinA littermates LTD could not be elicited. In addition, although long-term potentiation (LTP) could be induced in all the mice, it was greater in magnitude in mutant human torsinA mice. Low-frequency stimulation (LFS) can revert potentiated synapses to resting levels, a phenomenon termed synaptic depotentiation. LFS induced synaptic depotentiation (SD) both in NT and normal human torsinA mice, but not in mutant human torsinA mice. Since anti-cholinergic drugs are an effective medical therapeutic option for the treatment of human dystonia, we reasoned that an excess in endogenous acetylcholine could underlie the synaptic plasticity impairment. Indeed, both LTD and SD were rescued in mutant human torsinA mice either by lowering endogenous acetylcholine levels or by antagonizing muscarinic M1 receptors. The presence of an enhanced acetylcholine tone was confirmed by the observation that acetylcholinesterase activity was significantly increased in the striatum of mutant human torsinA mice, as compared with both normal human torsinA and NT littermates. Moreover, we found similar alterations of synaptic plasticity in muscarinic M2/M4 receptor knockout mice, in which an increased striatal acetylcholine level has been documented. The loss of LTD and SD on one hand, and the increase in LTP on the other, demonstrate that a 'loss of inhibition' characterizes the impairment of synaptic plasticity in this model of DYT1 dystonia. More importantly, our results indicate that an unbalanced cholinergic transmission plays a pivotal role in these alterations, providing a clue to understand the ability of anticholinergic agents to restore motor deficits in dystonia.

Loss of muscarinic autoreceptor function impairs long-term depression but not long-term potentiation in the striatum.

Muscarinic autoreceptors regulate cholinergic tone in the striatum. We investigated the functional consequences of genetic deletion of striatal muscarinic autoreceptors by means of electrophysiological recordings from either medium spiny neurons (MSNs) or cholinergic interneurons (ChIs) in slices from single M(4) or double M(2)/M(4) muscarinic acetylcholine receptor (mAChR) knock-out (-/-) mice. In control ChIs, the muscarinic agonist oxotremorine (300 nM) produced a self-inhibitory outward current that was mostly reduced in M(4)(-/-) and abolished in M(2)/M(4)(-/-) mice, suggesting an involvement of both M(2) and M(4) autoreceptors. In MSNs from both M(4)(-/-) and M(2)/M(4)(-/-) mice, muscarine caused a membrane depolarization that was prevented by the M(1) receptor-preferring antagonist pirenzepine (100 nM), suggesting that M(1) receptor function was unaltered. Acetylcholine has been involved in striatal long-term potentiation (LTP) or long-term depression (LTD) induction. Loss of muscarinic autoreceptor function is predicted to affect synaptic plasticity by modifying striatal cholinergic tone. Indeed, high-frequency stimulation of glutamatergic afferents failed to induce LTD in MSNs from both M(4)(-/-) and M(2)/M(4)(-/-) mice, as well as in wild-type mice pretreated with the M(2)/M(4) antagonist AF-DX384 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,1 1-dihydro-6H-pyrido[2,3b][1,4] benzodiazepin-6-one). Interestingly, LTD could be restored by either pirenzepine (100 nM) or hemicholinium-3 (10 microM), a depletor of endogenous ACh. Conversely, LTP induction did not show any difference among the three mouse strains and was prevented by pirenzepine. These results demonstrate that M(2)/M(4) muscarinic autoreceptors regulate ACh release from striatal ChIs. As a consequence, endogenous ACh drives the polarity of bidirectional synaptic plasticity.

Metabotropic glutamate receptor subtype 4 selectively modulates both glutamate and GABA transmission in the striatum: implications for Parkinson's disease treatment.

Alterations of striatal synaptic transmission have been associated with several motor disorders involving the basal ganglia, such as Parkinson's disease. For this reason, we investigated the role of group-III metabotropic glutamate (mGlu) receptors in regulating synaptic transmission in the striatum by electrophysiological recordings and by using our novel orthosteric agonist (3S)-3-[(3-amino-3-carboxypropyl(hydroxy)phosphinyl)-hydroxymethyl]-5-nitrothiophene (LSP1-3081) and l-2-amino-4-phosphonobutanoate (L-AP4). Here, we show that both drugs dose-dependently reduced glutamate- and GABA-mediated post-synaptic potentials, and increased the paired-pulse ratio. Moreover, they decreased the frequency, but not the amplitude, of glutamate and GABA spontaneous and miniature post-synaptic currents. Their inhibitory effect was abolished by (RS)-alpha-cyclopropyl-4-phosphonophenylglycine and was lost in slices from mGlu4 knock-out mice. Furthermore, (S)-3,4-dicarboxyphenylglycine did not affect glutamate and GABA transmission. Finally, intrastriatal LSP1-3081 or L-AP4 injection improved akinesia measured by the cylinder test. These results demonstrate that mGlu4 receptor selectively modulates striatal glutamate and GABA synaptic transmission, suggesting that it could represent an interesting target for selective pharmacological intervention in movement disorders involving basal ganglia circuitry.

Impaired striatal D2 receptor function leads to enhanced GABA transmission in a mouse model of DYT1 dystonia.

DYT1 dystonia is caused by a deletion in a glutamic acid residue in the C-terminus of the protein torsinA, whose function is still largely unknown. Alterations in GABAergic signaling have been involved in the pathogenesis of dystonia. We recorded GABA- and glutamate-mediated synaptic currents from a striatal slice preparation obtained from a mouse model of DYT1 dystonia. In medium spiny neurons (MSNs) from mice expressing human mutant torsinA (hMT), we observed a significantly higher frequency, but not amplitude, of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature currents (mIPSCs), whereas glutamate-dependent spontaneous excitatory synaptic currents (sEPSCs) were normal. No alterations were found in mice overexpressing normal human torsinA (hWT). To identify the possible sources of the increased GABAergic tone, we recorded GABAergic Fast-Spiking (FS) interneurons that exert a feed-forward inhibition on MSNs. However, both sEPSC and sIPSC recorded from hMT FS interneurons were comparable to hWT and non-transgenic (NT) mice. In physiological conditions, dopamine (DA) D2 receptor act presynaptically to reduce striatal GABA release. Of note, application of the D2-like receptor agonist quinpirole failed to reduce the frequency of sIPSCs in MSNs from hMT as compared to hWT and NT mice. Likewise, the inhibitory effect of quinpirole was lost on evoked IPSCs both in MSNs and FS interneurons from hMT mice. Our findings demonstrate a disinhibition of striatal GABAergic synaptic activity, that can be at least partially attributed to a D2 DA receptor dysfunction.

Seletracetam (ucb 44212) inhibits high-voltage-activated Ca2+ currents and intracellular Ca2+ increase in rat cortical neurons in vitro.

PURPOSE: We analyzed the effects of seletracetam (ucb 44212; SEL), a new antiepileptic drug candidate, in an in vitro model of epileptic activity. The activity of SEL was compared to the effects of levetiracetam (LEV; Keppra), in the same assays. METHODS: Combined electrophysiologic and microfluorometric recordings were performed from layer V pyramidal neurons in rat cortical slices to study the effects of SEL on the paroxysmal depolarization shifts (PDSs), and the simultaneous elevations of intracellular Ca(2+) concentration [Ca(2+)](i). Moreover, the involvement of high-voltage activated Ca(2+) currents (HVACCs) was investigated by means of patch-clamp recordings from acutely dissociated pyramidal neurons. RESULTS: SEL significantly reduced both the duration of PDSs (IC(50) = 241.0 +/- 21.7 nm) as well as the number of action potentials per PDS (IC(50) = 82.7 +/- 9.7 nm). In addition, SEL largely decreased the [Ca(2+)](i) rise accompanying PDSs (up to 75% of control values, IC(50) = 345.0 +/- 15.0 nm). Furthermore, SEL significantly reduced HVACCs in pyramidal neurons. This effect was mimicked by omega-conotoxin GVIA and, to a lesser extent, by omega-conotoxin MVIIC, blockers of N- and Q-type HVACC, respectively. The combination of these two toxins occluded the action of SEL, suggesting that N-type Ca(2+) channels, and partly Q-type subtypes are preferentially targeted. CONCLUSIONS: These results demonstrate a powerful inhibitory effect of SEL on epileptiform events in vitro. SEL showed a higher potency than LEV. The effective limitation of [Ca(2+)](i) influx might be relevant for its antiepileptic efficacy and, more broadly, for pathologic processes involving neuronal [Ca(2+)](i) overload.

Endogenous serotonin excites striatal cholinergic interneurons via the activation of 5-HT 2C, 5-HT6, and 5-HT7 serotonin receptors: implications for extrapyramidal side effects of serotonin reuptake inhibitors.

The striatum is richly innervated by serotonergic afferents from the raphe nucleus. We explored the effects of this input on striatal cholinergic interneurons from rat brain slices, by means of both conventional intracellular and whole-cell patch-clamp recordings. Bath-applied serotonin (5-HT, 3-300 microM), induced a dose-dependent membrane depolarization and increased the rate of spiking. This effect was mimicked by the 5-HT reuptake blockers citalopram and fluvoxamine. In voltage-clamped neurons, 5-HT induced an inward current, whose reversal potential was close to the K(+) equilibrium potential. Accordingly, the involvement of K(+) channels was confirmed either by increasing extracellular K(+) concentration and by blockade of K(+) channels with barium. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) profiling demonstrated the presence of 5-HT2C, 5-HT6, and 5-HT7 receptor mRNAs in identified cholinergic interneurons. The depolarization/inward current induced by 5-HT was partially mimicked by the 5-HT2 receptor agonist 2,5-dimethoxy-4-iodoamphetamine and antagonized by both ketanserin and the selective 5-HT2C antagonist RS102221, whereas the selective 5-HT3 and 5-HT4 receptor antagonists tropisetron and RS23597-190 had no effect. The depolarizing response to 5-HT was also reduced by the selective 5-HT6 and 5-HT7 receptor antagonists SB258585 and SB269970, respectively, and mimicked by the 5-HT7 agonist, 5-CT. Accordingly, activation of either 5-HT6 or 5-HT7 receptor induced an inward current. The 5-HT response was attenuated by U73122, blocker of phospholipase C, and by SQ22,536, an inhibitor of adenylyl cyclase. These results suggest that 5-HT released by serotonergic fibers originating in the raphe nuclei has a potent excitatory effect on striatal cholinergic interneurons.

Interaction between vanilloid and glutamate receptors in the central modulation of nociception.

This study investigates the effect of microinjections of capsaicin in the periaqueductal grey matter of rats on nociceptive behaviour and the possible interactions with NMDA and mGlu receptors. Intra-periaqueductal grey microinjection of capsaicin (1-3-6 nmol/rat) increased the latency of the nociceptive reaction in the plantar test. This effect was prevented by pretreatment with capsazepine (6 nmol/rat), which had no effect per se on the latency of the nociceptive reaction. 7-(Hydroxyimino)cyclopropa[b]chromen-1alpha-carboxylate ethyl ester (CPCCOEt, 50 nmol/rat) and 2-Methyl-6-(phenylethynyl)pyridine (MPEP, 50 nmol/rat), antagonists of mGlu(1) and mGlu(5) receptors, respectively, completely blocked the effect of capsaicin. Similarly, pretreatment with DL-2-Amino-5-phosphonovaleric acid (DL-AP5, 5 nmol/rat) and riluzole (4 nmol/rat), an NMDA receptor antagonist and a voltage-dependent Na(+) channels blocker which inhibits glutamate release, respectively, completely antagonized the effect of capsaicin. However, pretreatment with (2S)-alpha-Ethylglutamic acid (30 nmol/rat) and (RS)-alpha-Methylserine-O-phosphate (MSOP, 30 nmol/rat), antagonists of group II and group III mGlu receptors, respectively, had no effects on capsaicin-induced analgesia. Similarly, pretreatment with N-(piperidin-1-yl)-5-(4-chlophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A, 5 pmol/rat), a selective cannabinoid CB(1) receptor antagonist, did not affect the capsaicin-induced antinociception. In conclusion, this study shows that capsaicin might produce antinociception at the periaqueductal grey level by increasing glutamate release, which activates postsynaptic group I mGlu and NMDA receptors.

Centrality of striatal cholinergic transmission in Basal Ganglia function.

Work over the past two decades revealed a previously unexpected role for striatal cholinergic interneurons in the context of basal ganglia function. The recognition that these interneurons are essential in synaptic plasticity and motor learning represents a significant step ahead in deciphering how the striatum processes cortical inputs, and why pathological circumstances cause motor dysfunction. Loss of the reciprocal modulation between dopaminergic inputs and the intrinsic cholinergic innervation within the striatum appears to be the trigger for pathophysiological changes occurring in basal ganglia disorders. Accordingly, there is now compelling evidence showing profound changes in cholinergic markers in these disorders, in particular Parkinson's disease and dystonia. Based on converging experimental and clinical evidence, we provide an overview of the role of striatal cholinergic transmission in physiological and pathological conditions, in the context of the pathogenesis of movement disorders.

Developmental profile of the aberrant dopamine D2 receptor response in striatal cholinergic interneurons in DYT1 dystonia.

BACKGROUND: DYT1 dystonia, a severe form of genetically determined human dystonia, exhibits reduced penetrance among carriers and begins usually during adolescence. The reasons for such age dependence and variability remain unclear. METHODS AND RESULTS: We characterized the alterations in D2 dopamine receptor (D2R) signalling in striatal cholinergic interneurons at different ages in mice overexpressing human mutant torsinA (hMT). An abnormal excitatory response to the D2R agonist quinpirole was recorded at postnatal day 14, consisting of a membrane depolarization coupled to an increase in spiking frequency, and persisted unchanged at 3 and 9 months in hMT mice, compared to mice expressing wild-type human torsinA and non-transgenic mice. This response was blocked by the D2R antagonist sulpiride and depended upon G-proteins, as it was prevented by intrapipette GDP-ß-S. Patch-clamp recordings from dissociated interneurons revealed a significant increase in the Cav2.2-mediated current fraction at all ages examined. Consistently, chelation of intracellular calcium abolished the paradoxical response to quinpirole. Finally, no gross morphological changes were observed during development. CONCLUSIONS: These results suggest that an imbalanced striatal dopaminergic/cholinergic signaling occurs early in DYT1 dystonia and persists along development, representing a susceptibility factor for symptom generation.

Functional and ultrastructural analysis of group I mGluR in striatal fast-spiking interneurons.

Striatal parvalbumin-containing fast-spiking (FS) interneurons provide a powerful feedforward GABAergic inhibition on spiny projection neurons, through a widespread arborization and electrical coupling. Modulation of FS interneuron activity might therefore strongly affect striatal output. Metabotropic glutamate receptors (mGluRs) exert a modulatory action at various levels in the striatum. We performed electrophysiological recordings from a rat striatal slice preparation to investigate the effects of group I mGluR activation on both the intrinsic and synaptic properties of FS interneurons. Bath-application of the group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (3,5-DHPG), caused a dose-dependent depolarizing response. Both (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), selective mGluR1 antagonists, significantly reduced the amplitude of the membrane depolarization caused by 3,5-DHPG application. Conversely, mGluR5 antagonists, 2-methyl-6-(phenylethylnyl)pyridine hydrochloride (MPEP) and 6-methyl-2-(phenylazo)-3-pyridinol (SIB1757), were unable to affect the response to 3,5-DHPG, suggesting that only mGluR1 contributes to the 3,5-DHPG-mediated excitatory action on FS interneurons. Furthermore, mGluR1 blockade significantly decreased the amplitude of the glutamatergic postsynaptic potentials, whereas the mGluR5 antagonist application produced a small nonsignificant inhibitory effect. Surprisingly, our electron microscopic data demonstrate that the immunoreactivity for both mGluR1a and mGluR5 is expressed extrasynaptically on the plasma membrane of parvalbumin-immunoreactive dendrites of FS interneurons. Together, these results suggest that despite a common pattern of distribution, mGluR1 and mGluR5 exert distinct functions in the modulation of FS interneuron activity.

Inhibition of persistent sodium current fraction and voltage-gated L-type calcium current by propofol in cortical neurons: implications for its antiepileptic activity.

PURPOSE: Although it is widely used in clinical practice, the mechanisms of action of 2,6-di-isopropylphenol (propofol) are not completely understood. We examined the electrophysiologic effects of propofol on an in vitro model of epileptic activity obtained from a slice preparation. METHODS: The effects of propofol were tested both on membrane properties and on epileptiform events consisting of long-lasting, paroxysmal depolarization shifts (PDSs) induced by reducing the magnesium concentration from the solution and by adding bicuculline and 4-aminopyridine. These results were integrated with a patch-clamp analysis of Na(+) and high-voltage activated (HVA) calcium (Ca(2+)) currents from isolated cortical neurons. RESULTS: In bicuculline, to avoid any interference by gamma-aminobutyric acid (GABA)-A receptors, propofol (3-100 microM) did not cause significant changes in the current-evoked, sodium (Na(+))-dependent action-potential discharge. However, propofol reduced both the duration and the number of spikes of PDSs recorded from cortical neurons. Interestingly, relatively low concentrations of propofol [half-maximal inhibitory concentration (IC(50)), 3.9 microM) consistently inhibited the "persistent" fraction of Na(+) currents, whereas even high doses (< or =300 microM) had negligible effects on the "fast" component of Na(+) currents. HVA Ca(2+) currents were significantly reduced by propofol, and the pharmacologic analysis of this effect showed that propofol selectively reduced L-type HVA Ca(2+) currents, without affecting N or P/Q-type channels. CONCLUSIONS: These results suggest that propofol modulates neuronal excitability by selectively suppressing persistent Na(+) currents and L-type HVA Ca(2+) conductances in cortical neurons. These effects might cooperate with the opening of GABA-A-gated chloride channels, to achieve depression of cortical activity during both anesthesia and status epilepticus.