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Erythropoietin (EPO) is of great interest as a therapy for many of the central nervous system (CNS) diseases and its administration is protective in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Endogenous EPO is induced by hypoxic/ischemic injury, but little is known about its expression in other CNS diseases. We report here that EPO expression in the spinal cord is induced in mouse models of chronic or relapsing-remitting EAE, and is prominently localized to motoneurons. We found a parallel increase of hypoxia-inducible transcription factor (HIF)-1 alpha, but not HIF-2 alpha, at the mRNA level, suggesting a possible role of non-hypoxic factors in EPO induction. EPO mRNA in the spinal cord was co-expressed with interferon (IFN)-gamma and tumor necrosis factor (TNF), and these cytokines inhibited EPO production in vitro in both neuronal and glial cells. Given the known inhibitory effect of EPO on neuroinflammation, our study indicates that EPO should be viewed as part of the inflammatory/anti-inflammatory network in MS.

Experimentally induced autoimmune encephalomyelitis (EAE) in mice provides an animal model that shares many features with human demyelinating diseases such as multiple sclerosis (MS). To what extent the cerebral cortex is affected by the process of demyelination and how the corollary response of the oligodendrocyte lineage is explicated are still not completely known aspects of EAE. By performing a detailed in situ analysis of expression of myelin and oligodendrocyte markers we have identified areas of subpial demyelination in the cerebral cortex of animals with conventionally induced EAE conditions. On EAE-affected cerebral cortices, the distribution and relative abundance of cells of the oligodendrocyte lineage were assessed and compared with control mouse brains. The analysis demonstrated that A2B5(+) glial restricted progenitors (GRPs) and NG2(+)/PDGFR-α(+) oligodendrocyte precursor cells (OPCs) were increased in number during "early" disease, 20 days post MOG immunization, whereas in the "late" disease, 39 days post-immunization, they were strongly diminished, and there was an accompanying reduction in NG2(+)/O4(+) pre-oligodendrocytes and GST-π mature oligodendrocytes. These results, together with the observed steady-state amount of NG2(-)/O4(+) pre-myelinating oligodendrocytes, suggested that oligodendroglial precursors attempted to compensate for the progressive loss of myelin, although these cells appeared to fail to complete the last step of their differentiation program. Our findings confirm that this chronic model of EAE reproduces the features of neocortex pathology in progressive MS and suggest that, despite the proliferative response of the oligodendroglial precursors, the failure to accomplish final differentiation may be a key contributing factor to the impaired remyelination that characterizes these demyelinating conditions.

J. Neurochem. (2010) 115, 1445–1454. Abstract Hippocampal excitability and the metabolic glial–neuronal interactions were investigated in 22-week-old mice with motor neuron degeneration (mnd), a model of progressive epilepsy with mental retardation. Mnd mice developed spontaneous spikes in the hippocampus and were more susceptible to kainate-induced seizures compared with control mice. Neuronal hyperexcitability in their hippocampus was confirmed by the selective increase of c-Fos positive nuclei. Glial activation and pro-inflammatory cytokines over-expression were observed in the hippocampus of mnd mice, even in the absence of marked hippocampal neurodegeneration, as suggested by unchanged amounts of neuroactive amino acids and N-acetyl aspartate. Concentration of other amino acids, including GABA and glutamate, was not changed as well. However, ex vivo13C magnetic resonance spectroscopy, after simultaneous injection of [1-13C]glucose and [1,2-13C]acetate, followed by decapitation, showed decreased [1,2-13C]GABA formation from hippocampal astrocytic precursors and a marked reduction in [4,5-13C]glutamate derived from glutamine. We suggest that astrocyte dysfunction plays a primary role in the pathology and that mnd mice are of value to investigate early pathogenetic mechanism of progressive epilepsy with mental retardation.

TNF-alpha overexpression may contribute to motor neuron death in amyotrophic lateral sclerosis (ALS). We investigated the intracellular pathway associated with TNF-alpha in the wobbler mouse, a murine model of ALS, at the onset of symptoms. TNF-alpha and TNFR1 overexpression and JNK/p38MAPK phosphorylation occurred in neurons and microglia in early symptomatic mice, suggesting that this activation may contribute to motor neuron damage. The involvement of TNF-alpha was further confirmed by the protective effect of treatment with rhTNF-alpha binding protein (rhTBP-1) from 4 to 9 weeks of age. rhTBP-1 reduced the progression of symptoms, motor neuron loss, gliosis and JNK/p38MAPK phosphorylation in wobbler mice, but did not reduce TNF-alpha and TNFR1 levels. rhTBP-1 might possibly bind TNF-alpha and reduce the downstream phosphorylation of two main effectors of the neuroinflammatory response, p38MAPK and JNK.

Leukocyte infiltration is viewed as a pharmacological target in cerebral ischemia. We previously reported that reparixin, a CXCL8 receptor blocker that inhibits neutrophil infiltration, and related molecules can reduce infarct size in a rat model of transient middle cerebral artery occlusion (MCAO). The study aims were to compare the effects of reparixin in transient and permanent MCAO using varied treatment schedules and therapeutic windows to evaluate effects on long-term neurological deficits and late inflammatory response. Reparixin, administered for 1 to 3 days, 3.5 to 6 h after MCAO, ameliorates neurological function recovery and inhibits long-term inflammation. The infarct size reduction at 24 h, evaluated by TTC staining, is more pronounced in transient MCAO. MRI analysis identified a decrease in the progression of infarct size by reparixin that was more evident at 48 h in permanent MCAO, and was associated with a significantly improved recovery from long-term neurological deficits.

Chronic treatment with asialo erythropoietin (ASIALO-EPO) or carbamylated erythropoietin (CEPO) improved motor behavior and reduced motoneuron loss and astrocyte and microglia activation in the cervical spinal cord of wobbler mice, an animal model of amyotrophic lateral sclerosis, but had no effect on hematocrit values. ASIALO-EPO and CEPO, like the parent compound EPO, protected primary motoneuron cultures from kainate-induced death in vitro. Both EPO receptor and the common CD131 beta chain were expressed in cultured motoneurons and in the anterior horn of wobbler mice spinal cord. Our results strongly support a role for the common beta chain CD131 in the protective effect of EPO derivatives on motoneuron degeneration. Thus CEPO, which does not bind to the classical homodimeric EPO receptor and is devoid of hematopoietic activity, could be effective in chronic treatment aimed at reducing motoneuron degeneration.

Experimental studies suggest that the delivery of antiepileptic agents into the seizure focus might be of potential utility for the treatment of focal-onset epilepsies. Botulinum neurotoxin E (BoNT/E) causes a prolonged inhibition of neurotransmitter release after its specific cleavage of the synaptic protein synaptosomal-associated protein of 25 kDa (SNAP-25). Here, we show that BoNT/E injected into the rat hippocampus inhibits glutamate release and blocks spike activity of pyramidal neurons. BoNT/E effects persist for at least 3 weeks, as determined by immunodetection of cleaved SNAP-25 and loss of intact SNAP-25. The delivery of BoNT/E to the rat hippocampus dramatically reduces both focal and generalized kainic acid-induced seizures as documented by behavioral and electrographic analysis. BoNT/E treatment also prevents neuronal loss and long-term cognitive deficits associated with kainic acid seizures. Moreover, BoNT/E-injected rats require 50% more electrical stimulations to reach stage 5 of kindling, thus indicating a delayed epileptogenesis. We conclude that BoNT/E delivery to the hippocampus is both antiictal and antiepileptogenic in experimental models of epilepsy.

This work reports on the synthesis and affinities for the 5-HT(3) versus the 5-HT(4) receptor of new piperazinyl-substituted thienopyrimidine derivatives 20-45 with a view to identify potent and selective ligands for the 5-HT(3) receptor. Some of the new compounds show good affinity for the 5-HT(3) receptor and, notably, do not display any affinity for the 5-HT(4) receptor. 4-(4-Methyl-1-piperazinyl)-2-methylthio-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine 31 exhibits the highest affinity for the 5-HT(3) receptor (Ki = 33 nM) and behaves as noncompetitive antagonist.

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)–expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R–expressing inflammatory cells.

Chewing gum is a common habit, particularly in children, and it is known to stimulate salivary secretion. There are anecdotal claims that chewing gum may be unsafe, particularly in children, due to modifications to gastric fluid volume and pH. This mini-review is aimed at critically analysing the available literature on this specific topic.

We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT1A and 5-HT4 receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[3H]hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) to 5-HT1A receptors was selectively increased by 20% on average (p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average (p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase (p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT4 receptors, which colocalize with 5-HT1A receptors on hippocampal neurons, were not modified in kindled tissue. [3H]5-HT uptake and its release as well as the 5-HT1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 microg kg(-1) 8-OH-DPAT or 1,000 microg kg(-1) WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.

The mechanism underlying the serotoninergic neurotoxicity of some amphetamine derivatives, such as p-chloroamphetamine (pCA) and 3,4-methylenedioxymethamphetamine (MDMA), is still debated. Their main acute effect, serotonin (5-HT) release from nerve endings, involves their interaction with 5-HT transporters (SERTs), as substrates. Although this interaction is required for the neurotoxic effects, 5-HT release alone may not be sufficient to induce long-term 5-HT deficits. Some non-neurotoxic compounds, including p-methylthioamphetamine (MTA) and 1-(m-chlorophenyl)piperazine (mCPP), have 5-HT releasing properties in vivo and in brain slices comparable to that of neurotoxic amphetamine derivatives. We measured 5-HT release in superfused rat brain synaptosomes preloaded with [3H]5-HT, a model that distinguishes a releasing effect from reuptake inhibition. MTA and mCPP induced much lower release than pCA and MDMA. The striking difference between our findings in synaptosomes and those obtained in vivo or in brain slices is probably related to a different compartmentalisation of 5-HT in the different experimental models. Studies in synaptosomes, where the vesicular storage of 5-HT is predominant, could therefore bring to light differences between neurotoxic and non-neurotoxic 5-HT releasing agents which cannot be appreciated in other experimental models and might be useful to identify the mechanisms responsible for the neurotoxicity induced by amphetamine derivatives.

New racemic and chiral methyl 2-{[4-(4-chlorophenyl)-4-hydroxypiperi-din-1-yl]methyl}-1-phenylcyclopropanecarboxylate derivatives were synthesized in order to obtain sigma ligands with increased affinity and selectivity compared to (+)-MPCB and haloperidol. The cis-(±)-7 racemic mixture showed a better binding affinity and selectivity than the (±)-8 trans isomers. Between the two cis enantiomers, (+)-7, with configuration (1R,2S), showed a very high affinity and the best selectivity for s1. All compounds synthesized (7­9) showed a reduced or negligible affinity for opioid and dopaminergic D1 and D2 receptors. Nociceptive in vivo test confirms that (+)-7 (namely MR200), such as non-selective antagonist haloperidol, increased the analgesic effect induced by the k opioid selective ligand U50,488H and reversed the inhibiting effect of (+)-pentazocine on analgesia.

In this work we report the affinity of new thienopyrimidinones for 5-HT 1A Rs and the selectivity versus α 1 ARs. The 3-amino-2-[[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]thio]-6-ethyl-thieno[2,3- d ]pyrimidin-4(3 H )-one 27 is the most potent and selective (Ki 0.19 nM, selectivity 115). Compound 31 with the N4 piperazine orthonitrophenyl nucleus instead of the orthomethoxyphenyl also shows a good affinity and selectivity (Ki 1.46 nM, selectivity 84). The results of derivatives 28 , 29 and 30 (Ki 3.28, 12.59 and 4.38 nM; selectivity 24, 4 and 5, respectively), which have, respectively, an ethyl, an allyl and an acetylamino group instead of an N3 amino group, indicate the importance of this last group for the interaction with 5-HT 1A R. Comparison of the results for the superior homologue 53 (Ki 3.72 nM, selectivity 51) and the inferior homologue 52 (5-HT 1A Ki 1 499 nM, α 1 A Ki NA) of 2-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-6,7-dimethyl-8 H -[1,3,4]thiadiazolo[3,2- a ]thieno[2,3- d ]pyrimidin-8-one 57 (Ki 23 nM, selectivity 5) shows how important the length of the chain binding the two heterocyclic systems is in the interaction with 5-HT 1A Rs and α 1 ARs.

This paper reports the synthesis and affinities on the 5-HT1A versus the α1A receptors of new arylpiperazinylalkylthiothienopyrimidine and thiadiazole derivatives 16–24 Download : Download high-res image (79KB) Download : Download full-size image Scheme 1 . Reagents and conditions: (i) CSCl2, CHCl3/H2O, NaHCO3, room temperature; (ii) N2H4.H2O, CHCl3, room temperature; (iii) KOH, EtOH, reflux; (iv) HCl, H2O, room temperature; (v) 1-(3-chloropropyl)-4-(2-methoxyphenyl, 2-nitrophenyl or 2-pyrimidinyl)piperazine, EtOH, reflux. . Arylpiperazines 16–23 show affinities values in the nanomolar range for the 5-HT1A receptor. The compound 16 is highly potent (Ki 0.26 nM, selectivity 28), the derivatives 20 and 21 are less potent, but highly selective (Ki 9.40 and 5.06 nM, selectivity 207 and 73, respectively).

*Dipartimento di Scienze Farmaceutiche, Universita di Genova, viale Benedetto XV, 16132 Genoa, Italy Abstract The in-vitro and in-vivo hydrolysis of two benzodiazepine compounds has been studied to evaluate their in-vivo activity in mice. Compounds RL 218 and RL 236, selected as representative examples of N,N-dialkyl-8-chloro-6-phenyl-6H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amines (1) and of their 5-(alkylthio) substituted analogues (2), were rapidly hydrolysed to the corresponding 8-chloro-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5(6H)-one 3 (RL 214) in aqueous acidic solution at pH 1.5. This reaction also occurred extensively in mice when compounds RL 218 and RL 236 were given orally but not intraperitoneally. Both compounds were active against pentylenetetrazole-induced lethal convulsions in mice only when administered orally. After administration of pharmacologically effective oral doses (ED50, the dose protecting 50% of mice), at the time of assessment of the antipentylenetetrazole activity, mean brain concentrations of RL 218 and RL 236 were below the limits of sensitivity of the analytical procedure whereas brain concentrations of their metabolite RL 214 were comparable with that present after an oral equiactive dose of this compound itself. RL 214 but not RL 218 or RL 236 had in-vitro affinity for brain benzodiazepine receptors. These results indicate that the anticonvulsant activity of RL 218 and RL 236 in mice depends essentially on their in-vivo transformation into the common active metabolite RL 214 which most probably arises as a result of acid catalysed hydrolysis in the gastric juice.

1 The mechanism underlying 5-hydroxytryptamine (5-HT) and/or dopamine release induced by (+)-amphetamine ((+)-Amph), 3,4-methylendioxymethamphetamine (MDMA), p-chloroamphetamine (pCA) and (+)-fenfluramine ((+)-Fen) was investigated in rat brain superfused synaptosomes preloaded with the 3H neurotransmitters. 2 Their rank order of potency for [3H]-5-HT-releasing activity was the same as for inhibition of 5-HT uptake (pCAMDMA(+)-Fen>>(+)-Amph). Similarly, their rank order as [3H]-dopamine releasers and dopamine uptake inhibitors was the same ((+)-Amph>>pCA=MDMA>>(+)-Fen). We also confirmed that the release induced by these compounds was prevented by selective transporter inhibitors (indalpine or nomifensine). 3 [3H]-5-HT and/or [3H]-dopamine release induced by all these compounds was partially (31–80%), but significantly Ca2+-dependent. Lack of extracellular Ca2+ did not alter uptake mechanisms nor did it modify the carrier-dependent dopamine-induced [3H]-dopamine release. (+)-Amph-induced [3H]-dopamine release and pCA- and MDMA-induced [3H]-5-HT release were significantly inhibited by ω-agatoxin-IVA, a specific blocker of P-type voltage-operated Ca2+-channels, similar to the previous results on (+)-Fen-induced [3H]-5-HT release. 4 Methiothepin inhibited the Ca2+-dependent component of (+)-Amph-induced [3H]-dopamine release with high potency (70 nM), as previously found with (+)-Fen-induced [3H]-5-HT release. The inhibitory effect of methiothepin was not due to its effects as a transporter inhibitor or Ca2+-channel blocker and is unlikely to be due to its antagonist properties on 5-HT1/2, dopamine or any other extracellular receptor. 5 These results indicate that the release induced by these compounds is both ‘carrier-mediated’ and Ca2+-dependent (possibly exocytotic-like), with the specific carrier allowing the amphetamines to enter the synaptosome. The Ca2+-dependent release is mediated by Ca2+-influx (mainly through P-type Ca2+-channels), possibly triggered by the drug interacting with an unknown intracellular target, affected by methiothepin, common to both 5-HT and dopamine synaptosomes. British Journal of Pharmacology (1997) 121, 1735–1743; doi:10.1038/sj.bjp.0701325

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival., PLoS ONE, 7 (2), ISSN:1932-6203

Sustained inflammatory reactions are common pathological events associated with neuron loss in neurodegenerative diseases. Reported evidence suggests that Toll-like receptor 4 (TLR4) is a key player of neuroinflammation in several neurodegenerative diseases. However, the mechanisms by which TLR4 mediates neurotoxic signals remain poorly understood. We investigated the role of TLR4 in in vitro and in vivo settings of motor neuron degeneration. Using primary cultures from mouse spinal cords, we characterized both the proinflammatory and neurotoxic effects of TLR4 activation with lipopolysaccharide (activation of microglial cells, release of proinflammatory cytokines and motor neuron death) and the protective effects of a cyanobacteria-derived TLR4 antagonist (VB3323). With the use of TLR4-deficient cells, a critical role of the microglial component with functionally active TLR4 emerged in this setting. The in vivo experiments were carried out in a mouse model of spontaneous motor neuron degeneration, the wobbler mouse, where we preliminarily confirmed a protective effect of TLR4 antagonism. Compared with vehicle- and riluzole-treated mice, those chronically treated with VB3323 showed a decrease in microglial activation and morphological alterations of spinal cord neurons and a better performance in the paw abnormality and grip-strength tests. Taken together, our data add new understanding of the role of TLR4 in mediating neurotoxicity in the spinal cord and suggest that TLR4 antagonists could be considered in future studies as candidate protective agents for motor neurons in degenerative diseases.

The time-course of dihydropyridine receptor occupancy by lacidipine and its relationship with pharmacological activity has been studied in spontaneously hypertensive rat (SHR), as measured by the inhibition of specific (+)-[3H]PN 200–110 binding in-vivo. After oral administration of doses active in reducing blood pressure, lacidipine did not show tissue target differences in respect to binding sites labelled by (+)-[3H]PN 200–110 in cerebral cortex, heart, ileum, bladder and thoracic aorta. The relative occupancy of receptors in heart 60 min after oral administration of 1 mg kg−1 lacidipine was 75%. After 12 h, when lacidipine was still effective in reducing blood pressure in SHR, a low (15%) but detectable proportion of receptors was still occupied by the drug. The percentage decrease of blood pressure was linear with the percentage of receptor occupancy obtained by different doses of lacidipine; that is, there was a close correspondence between ED25 for decrease in blood pressure (0·33 mg kg−1) and ED25 for inhibition of (+)-[3H]PN 200–110 specific binding in the heart (0·36 mg kg−1). The long-lasting effect of lacidipine on blood pressure might be explained by its selective interaction with dihydropyridine binding sites labelled in-vivo by (+)-[3H]PN 200–110.

In amyotrophic lateral sclerosis (ALS), there is selective degeneration of motor neurons that leads to paralysis and death. Although the etiology of ALS is unclear, its heterogeneity suggests that a combination of factors (endogenous and/or environmental) may induce progressive motor neuron stress that results in the activation of different cell death pathways. Alterations of brain cholesterol homeostasis have recently been considered as possible cofactors in many neurodegenerative disorders, including ALS. The liver X receptor beta (LXRbeta) receptor is involved in lipogenesis and cholesterol metabolism, and we previously found that adult-onset motor neuron pathology occurs in LXRbeta mice. Here, we investigated neuromuscular alterations of LXRbeta mice from ages 3 to 24 months. Increased cholesterol levels, gliosis, and inflammation preceded motor neuron loss and clinical disease onset; the mice showed progressivemotor neuron deficits starting from age 7 months. The numbers ofmotor neurons and neuromuscular junctions were decreased in 24-month-old mice, but neither paralysis nor reduced life span was observed. Moreover, other spinal neurons were also lost in these mice. These results suggest that LXRbeta may inhibit neuroinflammation and maintain cholesterol homeostasis, and that LXRbeta mice represent a potential model for investigating the role of cholesterol in ALS and other neurodegenerative disorders.

The present study was aimed at establishing the importance of brain monoamine uptake and release mechanisms in the anorectic activity of fluoxetine, relating them to the actual brain concentrations of the parent drug and its metabolite norfluoxetine after anorectic doses in rats. Both compounds showed anorectic activity when administered intraperitoneally, norfluoxetine being slightly more active (ED50 = 22·9 μmol kg−1) than fluoxetine (ED50 = 35·0 μmol kg−1) despite the fact that the metabolite is about ten times less potent than the parent drug in inhibiting 5-hydroxytryptamine (5-HT) uptake. Comparing the brain concentrations of norfluoxetine, in terms of maximum concentrations (Cmax) and area under the curve (AUC), after the ED50 of fluoxetine or synthetic norfluoxetine, it also appeared that the metabolite plays a major role in the anorectic effect of the parent drug in rats. Brain Cmax of fluoxetine (48·7 μm) and norfluoxetine (21·7 and 27·3 μm after metabolite and drug, respectively) were several times those blocking 5-HT uptake in-vitro (0·5 μm), making it unlikely that fluoxetine (directly or through its metabolite) reduces food intake by specifically blocking 5-HT neuronal uptake. Brain Cmax of fluoxetine but particularly norfluoxetine were more compatible with those capable in-vitro of affecting catecholaminergic mechanisms, such as inhibition of dopamine and noradrenaline uptake and enhancement of dopamine release. These results together with recent in-vitro findings that the parent compound and its active metabolite induce tritium release from hippocampal synaptosomes previously loaded with [3H]5-HT suggest that mechanisms other than inhibition of 5-HT uptake are involved in the anorectic action of these compounds in rats.

With the aim to develop new potent and selective ligands of 5-HT(3)-type serotonin receptors and to acquire more information on their structure-affinity relationships, new thieno[2,3-d]pyrimidine derivatives 32-39 were synthesized and their binding to 5-HT(3) versus 5-HT(4 )receptors was studied. Some of these new compounds exhibit good affinity for cortical 5-HT(3) receptors, but not for 5-HT(4) receptors. Among these derivatives, 6-ethyl-4-(4-methyl-1-piperazinyl)-2-(methylthio)thieno[2,3-d]pyrimidine 32 is the most potent ligand (K(i) = 67 nM); it behaves as a competitive antagonist of the 5-HT(3) receptor function in the guinea pig colon. Its binding interactions with 5-HT(3A )receptors were analysed by using receptor modelling and comparative docking.

We studied two non-neurotoxic amphetamine derivatives (methyl-thioamphetamine, MTA and N,N-dimethylMTA, DMMTA) interacting with serotonin (5-HT) transporters (SERTs) with affinities comparable to that of p-Cl-amphetamine (pCA). The rank order for their maximal effects in inducing both [(3)H]5-HT release from rat brain synaptosomes or hSERT-expressing HEK-293 cells, and currents in hSERT-expressing oocytes, was pCAMTAor = DMMTA. A correlation between drug-induced release and currents is also strengthened by the similar bell shape of the dose-response curves. Release experiments indicated that MTA and DMMTA are SERT substrates although MTA is taken up by HEK-293 cells with a V(max) 40% lower than pCA. The weak effects of MTA and DMMTA in vitro might therefore be due to their properties as 'partial substrates' on the mechanisms, other than translocation, responsible for currents and/or release. After either local or systemic in vivo administration, MTA and DMMTA release 5-HT in a manner comparable to pCA. These findings confirm that the neurotoxic properties of some amphetamine derivatives are independent of their 5-HT-releasing activity in vivo. It is worth noting that only those amphetamine derivatives with high efficiency in inducing 5-HT release and currents in vitro have neurotoxic properties.

Erythropoietin (EPO), a member of the type 1 cytokine superfamily, plays a critical hormonal role regulating erythrocyte production as well as a paracrine/autocrine role in which locally produced EPO protects a wide variety of tissues from diverse injuries. Significantly, these functions are mediated by distinct receptors: hematopoiesis via the EPO receptor homodimer and tissue protection via a heterocomplex composed of the EPO receptor and CD131, the β common receptor. In the present work, we have delimited tissue-protective domains within EPO to short peptide sequences. We demonstrate that helix B (amino acid residues 58–82) of EPO, which faces the aqueous medium when EPO is bound to the receptor homodimer, is both neuroprotective in vitro and tissue protective in vivo in a variety of models, including ischemic stroke, diabetes-induced retinal edema, and peripheral nerve trauma. Remarkably, an 11-aa peptide composed of adjacent amino acids forming the aqueous face of helix B is also tissue protective, as confirmed by its therapeutic benefit in models of ischemic stroke and renal ischemia–reperfusion. Further, this peptide simulating the aqueous surface of helix B also exhibits EPO's trophic effects by accelerating wound healing and augmenting cognitive function in rodents. As anticipated, neither helix B nor the 11-aa peptide is erythropoietic in vitro or in vivo . Thus, the tissue-protective activities of EPO are mimicked by small, nonerythropoietic peptides that simulate a portion of EPO's three-dimensional structure.

Background The localisation of AMPA and NMDA receptor subunits was studied in a model of degeneration of cervical spinal motoneurons, the wobbler mouse. Cervical regions from early or late symptomatic wobbler mice (4 or 12 weeks of age) were compared to lumbar tracts (unaffected) and to those of healthy mice. Results No differences were found in the distribution of AMPA and NMDA receptor subunits at both ages. Western blots analysis showed a trend of reduction in AMPA and NMDA receptor subunits, mainly GluR1 and NR2A, exclusively in the cervical region of late symptomatic mice in the triton-insoluble post-synaptic fraction but not whole homogenates. Colocalisation experiments evidenced the expression of GluR1 and NR2A receptors in activated astrocytes from the cervical spinal cord of wobbler mice, GluR2 did not colocalise with GFAP positive cells. No differences were found in the expression of AMPA and NMDA receptor subunits in the lumbar tract of wobbler mice, where neither motoneuron loss nor reactive gliosis occurs. Conclusion In late symptomatic wobbler mice altered levels of GluR1 and NR2A receptor subunits may be a consequence of motoneuron loss rather than an early feature of motoneuron vulnerability.

Thiols affect a variety of cell functions, an effect known as redox regulation, largely attributed to modification of transcription factors and intracellular signaling mechanisms. Since exofacial protein thiols are more exposed to redox-acting molecules used in cell culture and may represent sensors of the redox state of the environment, we investigated their susceptibility to redox regulation. Exofacial protein thiols were measured using cell-impermeable Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid), DTNB]. For quantification, we also set up an ELISA assay based on the cell-impermeable biotinylated SH reagent, N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM). Exposure of CHO cells to H(2)O(2) induces oxidation of surface thiols at concentrations not affecting intracellular GSH. Depletion (50%) of GSH decreases surface thiols by 88%. Surface thiols are also highly sensitive to thiol antioxidants, since exposure to 5 mM N-acetyl-L-cysteine (NAC) for 2 h augmented their expression without increasing GSH levels. Using BIAM labeling and two-dimensional gel electrophoresis, we show that this increase in surface thiols is due to the reduction of specific membrane proteins. Peptide mass fingerprinting by MALDI mass spectrometry allowed us to identify two of these proteins as Erp57 and vimentin.

Using rational drug design to develop atypical antipsychotic drug candidates, we generated novel and metabolically stable pyrrolobenzazepines with an optimized pK(i) 5-HT(2A)/D(2) ratio. 5a, obtained by a new palladium-catalyzed three-step synthesis, was selected for further pharmacological and biochemical investigations and showed atypical antipsychotic properties in vivo. 5a was active on conditioned avoidance response at 0.56 mg/kg, it had low cataleptic potential and proved to be better than ST1899, clozapine, and olanzapine, representing a new clinical candidate.

Infiltration of polymorphonuclear neutrophils (PMNs) is thought to play a role in ischemic brain damage. The present study investigated the effect of repertaxin, a new noncompetitive allosteric inhibitor for the receptors of the inflammatory chemokine CXC ligand 8 (CXCL8)/interleukin-8 (IL-8), on PMN infiltration and tissue injury in rats. Cerebral ischemia was induced by permanent or transient occlusion of the middle cerebral artery and myeloperoxidase activity, a marker of PMN infiltration, and infarct volume were evaluated 24 h later. Repertaxin (15 mg/kg) was administered systemically at the time of ischemia and every 2 h for four times. In permanent ischemia repertaxin reduced PMN infiltration by 40% in the brain cortex but did not limit tissue damage. In transient ischemia (90-min ischemia followed by reperfusion), repertaxin inhibited PMN infiltration by 54% and gave 44% protection from tissue damage. Repertaxin had anti-inflammatory and neuroprotective effects also when given at reperfusion and even at 2 h of reperfusion. The protective effect of repertaxin did not interfere with brain levels of the chemokine. Since the PMN infiltration and its inhibition by repertaxin were comparable in the two models we conclude that reperfusion induces PMN activation, and inhibition of CXCL8 by repertaxin might be of pharmacological interest in transient ischemia.

Profound alteration of the oxygen consumption rate (QO2) is present in the cervical spinal cord (CS) of the wobbler mice aged 12 weeks (wr12). Early symptomatic mice at 4 weeks (wr4) show less pronounced changes with decreases of basal QO2 (P < 0.03) and of QO2 through complex I (P < 0.04). Mitochondrial respiratory enzyme activities, measured spectrophotometrically in the CS homogenate, show no difference between wr12 and controls, whereas complex I is reduced in the wr4 CS (P < 0.0003). Complex I activity is lower than normal both in wr12 and wr4 CS when measured in motor neurons by mean of a histochemical technique. Electron microscopy (EM) reveals a mixture of normal and morphologically altered mitochondria in wr4 motor neurons. The wobbler lumbar spinal cord is spared even at 12 weeks. Our results demonstrate the presence of mitochondrial abnormalities in the wobbler CS since the first manifestations of the disease. Thus, chronic mitochondrial dysfunction has a contributory role in motor neuron degeneration in the wobbler disease.

Mice homozygous for the spontaneous motor neuron degeneration mutation (mnd) show at the age of 8 months a marked impairment of the motor function and accumulation of lipofuscin granules in the cytoplasm of almost all neurons of the central nervous system. We previously reported a significant increase in GFAP protein levels in the lumbar spinal cord homogenates by western blot analysis and upregulation of TNF, a proinflammatory cytokine, in the motor neurons of lumbar spinal cord of mnd mice, already in a presymptomatic stage (4 months of age). In the present study, using immunohistochemical analysis, we performed a time course in mnd mice (1, 4 and 9 months of age) evaluating the expression and the distribution of astroglial and microglial cells and the expression of both TNF receptors, TNFR-I and TNFR-II. We observed a marked increase in astroglial and microglial cells and in TNFR-I immunoreactivity already at the 4th month. Since motor neuron dysfunction occurs in mnd mice in the absence of evident loss of spinal motor neurons, the present results indicate that the activation of microglial cells and astrocytes is independent from neuronal degeneration. The role of TNF and TNFR-I on motor neurons is still to be demonstrated.

S100B is a calcium-binding protein that, in the nervous system, is mainly concentrated in glial cells. Although its biological role is still unclear, the protein is hypothesized, at high concentrations, to act in the pathogenesis of neurodegenerative processes, possibly through oxidative stress mechanisms. To investigate this hypothesis we studied the spinal cord of wobbler mice, an animal model of motor neuron degeneration. Using immunocytochemistry, we detected an overexpression of S100B in astrocytes of the cervical spinal cord of these animals. We also confirmed this finding by reverse transcriptase polymerase chain reaction. In the same spinal cord regions, scattered neurons appeared to be immunostained for 4-hydroxynonenal-modified proteins, an indicator of lipid peroxidation. This finding constitutes a sign of oxidative stress-induced neurodegeneration.

Dietary nitrate NO3 intake through a diet rich in vegetables has been reported to exert several health effects, due to its transformation into the biological messenger nitric oxide (NO), which induces smooth muscle relaxation leading to haemodynamic effects like vasodilatation, increased blood flow and reduced blood pressure. The healthy effects of dietary nitrate have also been tested in sport, like cycling, walking and running, due to the reduction of O2 uptake during exercise. Dietary nitrate intake can be greatly enhanced making appropriate selections of high nitrate-containing foods or dietary supplements. A recent study [1], done on 10 healthy men, who ingested the supplement 2.5 h prior to moderate-intensity and severity-intensity cycle exercise tests, reported that dietary supplementation with beetroot juice (BRJ), containing 0.06 nmol/ml (about 4 mg/ml) nitrate concentration, altered the physiological responses to exercise, with 70 ml (4.2 nmol) having no effects, while 140 (8.4 nmol) and 280 ml (16.8 nmol) reduced the steady-state oxygen uptake during moderate-intensity exercise by 1.7% (p=0.06) and 3% (p=0.05), respectively. The time-totask failure was extended by 14% and 12% (both p=0.05), respectively. Although plasma nitrite (NO2) levels were increased dose dependently, with peak occurring at 2–3 h, there is no additional improvement in exercise tolerance after ingestion of 8.4 or 16.8 nmol NO3. The positive effect of 140 ml/day of BRJ was confirmed also after repeated ingestion [2]. In this randomised, double-bind, crossover study, nine healthy, physically active subjects were assigned to receive BRJ or placebo for 6 days. On Days 4, 5 and 6, the subjects completed a double-step exercise followed immediately by moderateto severe-intensity exercise. BRJ elevated plasma nitrite concentration compared to placebo (348 ± 170 and 65 ± 32 nM respectively, p

Erythropoietin (EPO) promotes neuronal survival after hypoxia and other metabolic insults by largely unknown mechanisms. Apoptosis and necrosis have been proposed as mechanisms of cellular demise, and either could be the target of actions of EPO. This study evaluates whether antiapoptotic mechanisms can account for the neuroprotective actions of EPO. Systemic administration of EPO (5,000 units/kg of body weight, i.p.) after middle-cerebral artery occlusion in rats dramatically reduces the volume of infarction 24 h later, in concert with an almost complete reduction in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling of neurons within the ischemic penumbra. In both pure and mixed neuronal cultures, EPO (0.1–10 units/ml) also inhibits apoptosis induced by serum deprivation or kainic acid exposure. Protection requires pretreatment, consistent with the induction of a gene expression program, and is sustained for 3 days without the continued presence of EPO. EPO (0.3 units/ml) also protects hippocampal neurons against hypoxia-induced neuronal death through activation of extracellular signal-regulated kinases and protein kinase Akt-1/protein kinase B. The action of EPO is not limited to directly promoting cell survival, as EPO is trophic but not mitogenic in cultured neuronal cells. These data suggest that inhibition of neuronal apoptosis underlies short latency protective effects of EPO after cerebral ischemia and other brain injuries. The neurotrophic actions suggest there may be longer-latency effects as well. Evaluation of EPO, a compound established as clinically safe, as neuroprotective therapy in acute brain injury is further supported.