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A group of spirocyclic tropanyl-Δ2-isoxazolines was synthesized exploiting the 1,3-dipolar cycloaddition of nitrile oxides to olefins. Their interaction with the dopamine and serotonin transporters (DAT and SERT, respectively) was evaluated through binding experiments. The majority of the compounds had no inhibitory effects (IC50 >> 10 μM), while some had an IC50 value in the range 5–10 μM (8a–c, 10b and 11c on DAT, 12b on SERT). Unexpectedly, one of the tertiary amines under investigation, that is 3′-methoxy-8-methyl-spiro{8-azabicyclo[3.2.1]octane-3,5′(4′H)-isoxazole 7a, was able to enhance at a concentration of 10 μM both [3H]citalopram and [3H]paroxetine binding to SERT in rat brain homogenate (up to 25%, due to an increase of Bmax) and [3H]serotonin uptake (up to 30%) in cortical synaptosomes. This peculiar pharmacological profile of 7a suggests it binds to an allosteric site on SERT, and positions derivative 7a as a very useful tool to investigate SERT machinery.

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.

Neuronal ceroid lipofuscinoses (NCLs) are a group of hereditary childhood diseases characterized mainly by lipopigment accumulation and a multisystemic pattern of symptoms including mental retardation, seizures, motor impairment, and blindness. The mnd mouse, carrying a mutation in the Cln8 gene, has been proposed as a model of epilepsy with mental retardation (EPMR, ornorthern epilepsy). We recently showed neuronal hyperexcitability and seizure hypersusceptibility in mnd mice. To elucidate the cellular mechanisms related to hippocampal hyperexcitability, the glutamatergic transmission and the expression of postsynaptic glutamate receptors were investigated in hippocampus. A significant increase in either spontaneous or KCl-stimulated overflow of [³H]D-aspartate was found in mnd mice compared with controls. This increase was maintained after DL-threo-β-benzyloxyaspartic acid (TBOA) treatment, suggesting a nonrelevant role for transporter-mediated release and supporting the involvement of exocytotic [³H]D-aspartate release. Accordingly, Ca²⁺-dependent overflow induced by ionomycin was also increased in mnd mice. Levels of glutamate 1-3 AMPA receptor subunits were increased, and levels of the NR2A NMDA receptor subunit were decreased in the hippocampus of mnd mice, suggesting an adaptive response to glutamate overstimulation.

Three different series of 1H-pyrrolopyrimidine-2,4-dione derivatives were designed and synthesized as ligands for the α(1)-adrenergic receptors (α(1)-ARs). A microwave-assisted protocol was developed in order to improve purity and yields of some final products. The majority of the synthesized compounds, tested in binding assays, displayed α(1)-AR affinities in the nanomolar range. Highest affinity values were found in derivatives 10b and 10c (K(i)=1.4 nM for both) whereas compound 10e was endowed with the best profile in term of α(1)-AR affinity (K(i)=2.71 nM) coupled with high selectivity towards 5-HT(1A) receptors (K(i) >10,000). Molecular docking studies were performed on human α(1)-ARs and human 5-HT(1A) receptors in order to rationalize the observed experimental affinity and selectivity; these computational studies helped to clarify molecular requirements for the design of high-selective α(1)-adrenergic ligands.

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.

Background: Professional soccer players are susceptible to amyotrophic lateral sclerosis. Strenuous physical activity has been associated with persistent inflammatory conditions and elevation of systemic cytokine levels, which could contribute to the vulnerability of these athletes. To investigate changes induced by playing soccer in the systemic profiles of growth factors and of the principal cytokines involved in the inflammatory response, we compared the serum concentrations of these factors in Italian professional soccer players and sedentary subjects. We also investigated the effects of the sera on primary cultured motor neurons in relation to their cytokine and growth factor content. Methods: Serum concentrations of cytokines and growth factors were measured by a biochip array analyzer. Neurotoxicity of sera was assessed by immunocytochemical assays in primary motor neuron cultures from mouse embryos. Results: Circulating levels of interleukin-8, tumor necrosis factor-alpha and interleukin-4 were lower in soccer players than controls. However, the viability of primary cultured mouse motor neurons treated with sera from the two groups did not differ significantly. Vascular endothelial growth factor (VEGF) independently emerged as a systemic protective factor for motor neurons. Conclusions: We found significant alterations in circulating pro-inflammatory cytokines in Italian professional soccer players, showing an unbalanced inflammatory condition in these subjects. VEGF was a protective serum factor affecting motor neuron survival.

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.

Glutamate-induced excitotoxicity is suggested to play a central role in the development of amyotrophic lateral sclerosis (ALS), although it is still unclear whether it represents a primary cause in the cascade leading to motor neurone death. We used western blotting, immunocytochemistry and in situ hybridization to examine the expression of GLT-1 in transgenic mice carrying a mutated (G93A) human copper-zinc superoxide dismutase (TgSOD1 G93A), which closely mimic the features of ALS. We observed a progressive decrease in the immunoreactivity of the glial glutamate transporter (GLT-1) in the ventral, but not in the dorsal, horn of lumbar spinal cord. This effect was specifically found in 14- and 18-week-old mice that had motor function impairment, motor neurone loss and reactive astrocytosis. No changes in GLT-1 were observed at 8 weeks of age, before the appearance of clinical symptoms. Decreases in GLT-1 were accompanied by increased glial fibrillary acidic protein (GFAP) levels and no change in the levels of GLAST, another glial glutamate transporter. The glutamate concentration in the cerebrospinal fluid (CSF) of TgSOD1 G93A mice was not modified at any of the time points examined, compared with age-matched controls. These findings indicate that the loss of GLT-1 protein in ALS mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis and it is not associated with increases of glutamate levels in CSF. The lack of changes in GLT-1 at the presymptomatic stage suggests that glial glutamate transporter reduction is not a primary event leading to motor neurone loss.

A series of new compounds containing a benzimidazole, benzothiazole, or benzoxazole nucleus linked to an arylpiperazine by different thioalkyl chains was prepared. They were tested in radioligand binding experiments to evaluate their affinity for 5-HT 1A and 5-HT 2A serotonergic, alpha 1 adrenergic, D1, and D2 dopaminergic receptors. Many of tested compounds showed an interesting binding profile; in particular, 36 displayed very high 5-HT 1A receptor affinity and selectivity over all the other investigated receptors. Selected compounds, evaluated in functional assays, showed antagonistic or partial agonistic activity at 5-HT 1A receptor. An extensive conformational research using both NMR and modeling techniques indicated that extended conformations predominated in vacuum, in solution and during interactions with 5-HT 1A receptor. Finally, the elaborated binding mode of selected compounds at 5-HT 1A receptor was used to explain the influence of spacer length on ligands affinity.

The present report deals with the synthesis of the two couples of enantiomers (+)-(3aS,4R,6aS)-/(−)-(3aR,4S,6aR)-3-hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4-d]isoxazole-4-carboxylic acid [(+)-HIP-A and (−)-HIP-A] and (+)-(3aS,6S,6aS)-/(−)-(3aR,6R,6aR)-3-hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4-d]isoxazole-6-carboxylic acid [(+)-HIP-B and (−)-HIP-B], and the investigation of their inhibitory activity at EAATs. When tested in a [3H] d -aspartate uptake assay on native EAATs present in rat brain synaptosomal fractions, (−)-HIP-A and (+)-HIP-B turned out to be considerably more potent than their enantiomers; in particular, (−)-HIP-A showed an eudismic ratio (ER) higher than 100. Molecular modeling investigations gave clues with regard to the key amino acid residues involved in the binding with our inhibitors and provided explanations for the observed stereoselectivity.

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.

We have tested by [(35)S]-GTPgammaS binding the intrinsic activity of three full agonists (serotonin, 5-methoxytryptamine and 5-methoxy-2-methyl-N,N-dimethyltryptamine) on rat 5-HT(6) receptors cloned in HEK-293 cells, using the scintillation proximity assay. Serotonin and 5-methoxytryptamine are soluble in water, while the agonist 5-methoxy-2-methyl-N,N-dimethyltryptamine is soluble in dimethyl sulfoxide (DMSO). In [(35)S]-GTPgammaS binding 5-HT and 5-methoxytryptamine were able to increase basal binding, while 5-methoxy-2-methyl-N,N-dimethyltryptamine surprisingly showed an inverse agonist activity. So we have tested 5-HT and 5-methoxytryptamine in the presence of DMSO: in this condition the two agonists behaved as antagonists. This interfering effect of DMSO was not observed when GTP-europium filtration binding was used in place of scintillation proximity assay using [(35)S]-GTPgammaS. In addition, DMSO did not affect [(3)H]-5HT binding or cAMP accumulation in cloned HEK-293 cells expressing rat 5-HT(6) receptors. In conclusion, we demonstrated that DMSO, the most common solvent used to dissolve compounds insoluble in water, interferes with the method of scintillation proximity assay using [(35)S]-GTPgammaS. DMSO does not affect basal signal, nor the GTPgammaS binding itself, as indicated by the experiments with GTP-europium. Therefore its interfering effect is likely to occur at the binding of antibodies in the scintillation proximity assay.

Experiments using the binding of various ligands for monoamines to rat brain membranes and synaptosomal preparations for studying monoamine uptake and release have shown that d-fenfluramine is more potent than the l isomer in inhibiting 5-HT uptake, whereas d-norfenfluramine preferentially releases 5-HT from a reserpine-insensitive compartment. Studies on brain monoamine metabolism in intact animals have shown that the d and l isomers of fenfluramine at relatively low doses have a specific action on brain 5-HT and catecholamines, respectively. Based on the different ability of metergoline and ritanserin to displace 5-HT2 binding to rat brain membranes and to antagonize d-fenfluramine's anorexia, evidence has been provided that d-fenfluramine preferentially uses 5-HT1 sites in the rat brain to cause anorexia in this animal species. Finally, characteristics, regional distribution, and pharmacological characterization of a high-affinity [3H]d-fenfluramine binding to rat brain membranes have been described. This binding appears to be different from 5-HT uptake sites ([3H]imipramine binding) and 5-HT receptors and is not regionally related to the endogenous levels of 5-HT in the rat brain. It is, however, preferentially displaced by some agents using 5-HT to cause anorexia in rats, raising the possibility that it is somewhat related to 5-HT mechanisms involved in feeding control.

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.

The wobbler mouse is one of the most useful models of motoneuron degeneration, characterized by selective motoneuronal death in the cervical spinal cord. We carried out two parallel studies in wobbler mice, comparing the anti-glutamatergic drug riluzole and the AMPA receptor antagonist RPR119990. Mice were treated with 40 mg/kg/day of riluzole or with 3 mg/kg/day of RPR119990 from the 4th to the 12th week of age. Here, we show that chronic treatment with riluzole improves motor behavior, prevents biceps muscle atrophy and decreases the amount of motoneuron loss in treated wobbler mice. Chronic treatment with the AMPA antagonist RPR119990 is ineffective in improving motor impairment, in reducing motoneuronal loss and muscular atrophy in treated mice. These results, together with the unchanged immunostaining for the AMPA receptor subunit GluR2 in wobbler mice, suggest that AMPA receptor-mediated injury is unlikely to be involved in neurodegeneration in wobbler disease, and that the protective effect of riluzole in wobbler mice seems to be independent of its anti-glutamatergic activity, as suggested in other models of neurodegeneration. Immunostaining of cervical spinal cord sections shows that in riluzole-treated wobbler mice BDNF expression is significantly increased in motoneurons with no changes in the high-affinity receptor Trk-B. Our data confirm that riluzole has beneficial effects in wobbler mice, and suggest that these effects could be associated to the increased levels of the neurotrophic and neuroprotective factor BDNF.

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.

Clinical data indicate that hydroalcoholic extracts of Hypericum perforatum might be as valuable as conventional antidepressants in mild-to-moderate depression, with fewer side effects. One clinical trial using two extracts with different hyperforin contents indicated it as the main active principle responsible for the antidepressant activity. Behavioural models in rodents confirm the antidepressant-like effect of Hypericum extracts and also of pure hyperforin and hypericin. A hydroalcoholic extract lacking hyperforin also lacks the antidepressant-like effect. According to pharmacokinetic data and binding studies, it appears that the antidepressant effect of Hypericum extract is unlikely be due to an interaction of hypericin with central neurotransmitter receptors. The main in vitro effects of hyperforin (at concentrations of 0.1-1 microM) are non-specific presynaptic effects, resulting in the non-selective inhibition of the uptake of many neurotransmitters, and the interaction with dopamine D1 and opioid receptors. However, it is still not clear whether these mechanisms can be activated in vivo, since after administration of Hypericum extract brain concentrations of hyperforin are well below those active in vitro. In the rat, Hypericum extract might indirectly activate sigma receptors in vivo (through the formation of an unknown metabolite or production of an endogenous ligand), suggesting a new target for its antidepressant effects.

Retinal degeneration is an early and progressive event in many forms of neuronal ceroid lipofuscinoses (NCLs), a heterogeneous group of neurodegenerative disorders with unknown pathogenesis. We here used the mutant motor neuron degeneration (mnd) mouse, a late-infantile NCL variant, to investigate the retinal oxidative state and apoptotic cell death as a function of age and sex. Total superoxide dismutase (SOD) activities and thiobarbituric acid-reactive substance (TBARS) levels revealed progressive increases in retinal oxyradicals and lipid peroxides of mnd mice of both sexes. Female mnd retinas showed a higher oxidation rate and consistently exhibited the 4-hydroxy-2-nonenal (4-HNE)-adducts staining and advanced histopathologic profile when compared to male mnd retinas matched for age. In situ DNA fragmentation (TUNEL staining) appeared in the outer nuclear layer (ONL) as early as 1 month of age. At 4 months, there were more intense and numerous TUNEL-positive cells in the same layer and in the inner nuclear (INL) and ganglion cell (GCL) layers; whereas at 8 months TUNEL staining was restricted to a few scattered cells in the INL and GCL, when a severe retinal cell loss had occurred. Caspase-3 activation confirmed apoptotic demise and its processing turned out to be higher in mnd females than males. These results demonstrate the involvement of oxidation and apoptotic processes in mnd mouse retinopathy and highlight sex-related differences in retinal vulnerability to oxidative stress and damage.

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.

In this study, using the new sigma(1/2) (sigma(1/2)) compound MR200, its parent drug haloperidol and the sigma ligand 1,3-di-o-tolylguanidine (DTG), we have investigated the role of striatal sigma receptors in the control of basal dopamine (DA) outflow, by coupling in vitro binding experiments and in vivo microdialysis in the striatum of halothane-anesthetized rats. MR200 with respect to haloperidol, exhibits high affinity for sigma(1) (1.5 nM) and sigma(2) (21.9 nM) receptors, but only negligible affinity for DA receptors. Compared to DTG, MR200 has similar selectivity across neurotransmitter systems, and 46 times higher affinity for sigma(1) receptors. Intrastriatal application of MR200 at 10, but not 0.1 or 1 microM, elicited a pronounced decrease in striatal DA release (-45% of control values). This inhibitory effect was preceded by a transient increase in DA release (+50% over baseline) after 100 microM MR200 administration. DTG at 100, but not 10 microM, significantly reduced DA release (-40%). Haloperidol, whilst increasing DA release at 1 microM, induced a delayed decrease in DA release after 10 microM application. Finally, haloperidol (10 microM) did not modify the inhibitory effect of 10 microM MR200. These results show that striatal sigma receptors control striatal DA release in resting conditions.

Excessive glutamate release (mediated by reversed uptake) or impaired reuptake contributes to the etiopathology of many neurodegenerative disorders. Thus great effort has been devoted to the discovery of agents that can interfere with high-affinity Na+-dependent glutamate transport, with the aim of finding new therapeutics against neurodegenerative diseases. We developed two different 3D-pharmacophore models for substrate inhibitors and blockers, which led to the rational design of novel and potent glutamate and aspartate analogues that selectively interact with excitatory amino acid transporters (EAAT). Our results indicated that all analysed EAAT ligands share the same orientation of the acidic functions and the protonatable nitrogen, even though the distance between the carboxylic carbons varies from 3.7 to 4.9 A. This distance does not discriminate between substrate inhibitors and blockers, but between glutamate and aspartate derivatives. In contrasts differences in the volume distribution of the rest of the molecule with respect to the axis connecting the two carboxylic groups are responsible for the difference in activity between transportable and nontransportable inhibitors. Thus our 3D receptor interaction model for EAAT substrates and nontransportable inhibitors could lead to the rational design of selective EAAT ligands as possible neuroprotective agents. However, some critical points, such as which glutamate transporter is present on glutamatergic nerve terminals and which glutamate transporter mediates reversed glutamate uptake, still remain to be elucidated.

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.

Despite previous observations on isolated ventricular myocytes, there are still few evidences that angiotensin II induces cardiomyocyte apoptosis in vivo. The possibility that aldosterone, the final hormone of the renin-angiotensin-aldosterone system under Ang II control, can stimulate cardiac apoptosis has not yet been explored. Angiotensin II or aldosterone (1mg/kg each) were infused in adult normotensive rats for different times, and the number of apoptotic ventricular myocyte nuclei was quantified by the TUNEL method, along with caspase-3 activation. The role of angiotensin II type 1 receptor in vivo was assessed by selective blockade with valsartan and ex vivo by binding experiments. In addition, myocytes in primary culture were incubated with Ang II or aldosterone in presence of spironolactone. Continuous infusion of Ang II induced a rapid, AT(1)-mediated increase of apoptotic cardiomyocyte nuclei (from 14+/-9 to 188+/-35 TdT-labeled nuclei/10(6) after 3h, P

The mnd mouse spontaneously develops slowly evolving motoneuron pathology leading to progressive motor impairment. There is strong evidence that a complex interplay between oxidative stress, mitochondria abnormalities and alteration of glutamate neurotransmission plays an important role in the pathogenesis of motor neuron diseases. Therefore, we investigated the presence of mitochondrial dysfunction in frontal, central (comprising the motor area) and occipital regions of the cerebral cortex and in the spinal cord of 35-week-old mnd mice. Lipid peroxide derivatives reacting with thiobarbituric acid (TBARS) were measured in the cervical, thoracic and lumbar spinal cord. In addition biochemical and behavioural analyses were carried out in mnd mice chronically treated with l-carnitine from the 11th to the 34th week of life (mndT mice). Slight but significant alterations of mitochondrial enzyme activities were seen in the mnd cortical regions. The central area was the most affected and both complex I, IV and citrate synthase were decreased with respect to controls. The rate of oxygen consumption (QO2) was markedly decreased in both the upper (cervical + upper portion of the thoracic region) and lower (lumbar + lower portion of the thoracic region) mnd spinal cord. The level of TBARS showed a rostro-caudal trend to increase, being 30% higher in the lumbar tract of mnd mice in comparison with controls. L-carnitine treatment increased the mitochondrial enzyme activities in cortical regions towards control value and was effective in enhancing QO2 and decreasing TBARS levels in the spinal cord of mndT. Behavioural testing showed that L-carnitine significantly delayed the onset of motor behaviour impairment. This beneficial effect was declining at 35 week of age, when the biochemical measurements were performed.

After a single intraperitoneal injection of the irreversible tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA; 300 mg/kg), there was a rapid down-regulation of serotonin (5-HT) transporter mRNA levels in cell bodies. This change was significant at 1 and 2 days after PCPA administration within the ventromedial but not the dorsomedial portion of the dorsal raphe nucleus. Seven days after PCPA treatment, 5-HT transporter mRNA levels were significantly elevated compared with controls in both regions of the dorsal raphe nucleus. PCPA administration produced no change in the [3H]citalopram binding and synaptosomal [3H]5-HT uptake in terminal regions at 2 and 7 days after treatment but significantly reduced both these parameters by approximately 20% in the hippocampus and in cerebral cortex 14 days after PCPA administration. The striatum showed a lower sensitivity to this effect. No significant changes were observed in the levels of [3H]citalopram binding to 5-HT cell bodies in the dorsal raphe nucleus. In the same animals used for 5-HT transporter mRNA level measurements, levels of tryptophan hydroxylase mRNA in neurons of the ventromedial and dorsomedial portions of the dorsal raphe nucleus were increased 2 days after PCPA administration and fell to control levels 7 days after injection in the ventromedial region but not in the dorsomedial portion of the dorsal raphe nucleus, where they remained significantly higher than controls. Altogether, these results show that changes in 5-HT transporter mRNA are not temporally related to changes in 5-HT transporter protein levels. In addition, our results suggest that the 5-HT transporter and tryptophan hydroxylase genes are regulated by different mechanisms. We also provide further evidence that dorsal raphe 5-HT neurons are differentially regulated by drugs, depending on their location.

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.

We studied the expression and distribution of glutamate receptor subtypes in the spinal cord of mnd mice, a model of motor neuron disorders and neuronal ceroid lipofuscinosis, and control mice using immunocytochemistry and in situ hybridization. The constitutive subunit of the NMDA ionotropic glutamate receptor, NMDAR1, was expressed in all neurons of the grey matter and was not modified in the spinal cord of mnd mice in either its normal or phosphorylated form. The immunoreactivity of GluR2, but not its mRNA, was increased mainly in the substantia gelatinosa both in presymptomatic and in 8-month-old symptomatic mice, suggesting compensatory changes aimed at reducing the Ca2+ permeability of the receptor channel. In spinal cord of mnd mice, mRNA, and protein levels of GluR3 were low only at the symptomatic stage, possibly as a consequence of motor neuron dysfunction. This was not due to motoneuron degeneration, because the number of choline acetyltransferase (ChAT) immunopositive lumbar motor neurons and the ChAT activity in the spinal cord and hind leg muscles of symptomatic mnd mice were no different from control mice. GluR4 mRNA was increased throughout the grey matter, presumably in relation to the marked microglia activation reported in the grey matter of the lumbar spinal cord in mnd mice. These changes in ionotropic glutamate receptors may alter glutamatergic neurotransmission and play some role in the pathology of mnd mice. © 2002 Wiley-Liss, Inc.

In recent work we reported that systemically administered erythropoietin (EPO) crosses the blood-brain barrier and has protective effects in animal models of cerebral ischemia, brain trauma and in a rat model of experimental autoimmune encephalomyelitis (EAE). Here we characterize the effect of systemic EPO on the inflammatory component of actively induced, acute EAE in Lewis rats. Administration of EPO at doses of 500-5000 U/kg bw i.p., daily from day 3 after immunization with myelin basic protein (MBP), delayed the onset of EAE and decreased its clinical score at peak time (days 12-13). Immunohistochemical analysis of the spinal cord using anti-glial fibrillary acidic protein (GFAP) and anti-CD11b antibodies showed that EPO markedly diminished inflammation and glial activation/proliferation. EAE induced significant levels of TNF and IL-6 in the spinal cord, where IL-6 was maximum at the onset of the disease (day 10) and TNF at its peak (day 12). EPO delayed the increase of TNF levels, without altering their peak levels, and markedly reduced those of IL-6 suggesting that the decreased inflammation and clinical score may be in part upon attenuation of IL-6. On the other hand, EPO was without effect in a model of adjuvant-induced arthritis in Lewis rats, suggesting a specificity towards autoimmune demyelinating diseases. These data suggest that EPO might act as a protective cytokine in inflammatory pathologies of the CNS.

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.

We evaluated the role of acetyl-L-carnitine (ALCAR) in protecting primary motoneuron cultures exposed to excitotoxic agents or serum-brain derived neurotrophic factor (BDNF) deprived. To exclude that ALCAR works as a metabolic source, we compared its effects with those of L-carnitine (L-CAR), that seems to have no neurotrophic effect. A concentration of 10 mM ALCAR, but not L-CAR, significantly reduced the toxic effect of 50 microM N-methyl-D-aspartate (NMDA, % viability: NMDA 45.4+/-2.80, NMDA+ALCAR 90.8+/-11.8; P

Botanicals in Food Supplements: Criticisms and Novelties from Two Up-Dated Conferences The use of botanicals (herbs and algae) in food supplements is an argument that is receiving more and more attention, as indicated by the number of publications and meetings related to the topic. In Europe there is a substantial presence of plant and plant extracts in dietary supplements, representing, in some Countries, about half of these products. Two recent international conferences, organized by CEC Editore (Milano, I) in occasion of the NUCE meeting (Milano, I, September 24-26) confirmed the interest of the audience in receiving updated information on the matter.

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.

We studied the role of glutamate excitotoxicity in motor neuron degeneration in the wobbler mouse (wr/wr), a model of amyotrophic lateral sclerosis and spinal muscular atrophies. Choline acetyltransferase (ChAT) activity was decreased in the cervical spinal cord and in the muscles innervated by nerves originating in this region of wobbler mice, but no differences were found in the lumbar spinal cord and in the hindleg muscles. Glial fibrillar acid protein (GFAP), a marker of reactive gliosis, was significantly higher in the cervical spinal cord of wobbler mice aged 4 weeks than in controls and the differences were more marked at 12 weeks; no differences were found in the lumbar spinal cord. In spite of this selective degeneration of motor neurons (resulting in strong decrease in the neuronal glutamate transporter EAAC1) and reactive gliosis in the cervical spinal cord, the levels of the glial glutamate transporter proteins GLT-1 and GLAST were similar in wobbler and control mice. Plasma concentrations of excitatory amino acids were no different at any time examined. Our results exclude the involvement of decrease in glutamate GLT 1 transporter in the motor neuron degeneration in wobbler mice.

This work reports the synthesis and the binding tests on the 5-HT(3) and 5-HT(4) receptors of new thienopyrimidopiperazine and piperazinylacylaminodimethylthiophene derivatives, in order to identify potent and selective ligands for each receptor. The compound with higher affinity and selectivity for the 5-HT(3) over the 5-HT(4) receptor was the 3-amino-2-(4-benzyl-1-piperazinyl)-5,6-dimethyl-thieno[2,3-d]pyrimidin-4(3H)-one 28 (5-HT(3) K(i)=3.92 nM, 5-HT(4) not active), the compound with higher affinity and selectivity for the 5-HT(4) over the 5-HT(3) receptor was the 2-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butanoylamino]-4,5-dimethyl-3-thiophenecarboxylic acid ethyl ester 41 (5-HT(4) K(i)=81.3 nM, 5-HT(3) not active). Conformational analyses were carried out on the compounds of the piperazinylacylaminodimethylthiophene series (39-42) taking compound 41 as the template.

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).

The hydroalcoholic extract of Hypericum perforatum L. is an effective antidepressant, although its mechanism of action is still unknown. It inhibits the synaptosomal uptake of serotonin (5-HT), dopamine and noradrenaline, suggesting a biochemical mechanism similar to the synthetic standard antidepressants. In the present study, further investigating this hypothesis, we confirmed that a hydromethanolic extract of H. perforatum inhibited [3H]5-HT accumulation in rat brain cortical synaptosomes with an IC50 value of 7.9 microg/ml. The IC50 of pure hyperforin was 1.8 microg/ml, so the activity of the total extract is not related only to its hyperforin content (5%). This inhibitory effect, however, is not due to a direct interaction with, and blockade of, the 5-HT transporters since the extract, like hyperforin, did not inhibit [3H]citalopram binding (IC50100 microg/ml and 10 microg/ml, respectively). We also found that 3-10 microg/ml of the extract, or 0.3-1 microg/ml hyperforin, induced marked tritium release from superfused synaptosomes previously loaded with [3H]5-HT. The releasing effect of the extract resembles the releasing effect of a reserpine-like compound (Ro 04-1284), i.e. it was slightly delayed and was 5-HT carrier- and calcium-independent. These data suggest that the hydromethanolic extract of H. peforatum, similarly to Ro 04-1284, rapidly depletes storage vesicles, raising the cytoplasmic concentration of 5-HT, and this increase is presumably responsible for the apparent inhibition of [3H]5-HT uptake. Therefore, our in vitro data do not confirm that the hydromethanolic extract of H. perforatum acts as a classical 5-HT uptake inhibitor but indicate reserpine-like properties. However, the concentrations of the active component(s) effective in vitro as reserpine-like agent(s) (i.e. corresponding toor =3 microg/ml of the hydromethanolic extract) do not seem to be achieved in the brain after pharmacologically effective doses of the extract, as indicated by the finding that there were no significant changes of rat brain 5-HT and 5-hydroxyindoleacetic acid levels after a schedule of treatment (3 x 300 mg/kgday, orally) active in an animal model predictive of antidepressant-like activity. These data also suggest that the antidepressant effect of H. perforatum extracts is unlikely to be associated with interaction with GABA, benzodiazepine and 5-HT1 receptors since, in receptor binding studies, we found IC50 values higher than 5 microg/ml. Therefore other, still unknown, mechanisms are possibly involved in H. perforatum antidepressant effects.

We studied ionotropic glutamate receptor subtypes and the effect of chronic treatment with NBQX [6-nitro-7-sulphamoyl-benzo(F)quinoxaline-2,3-dione], a selective ( rs)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist, in the spinal cord of mnd mice. NBQX (8 mg/kg daily i.p. for 3 weeks starting from 24 weeks old) significantly improved the behavioural scores (hind leg extension reflex, cage rung grasping and gait) in mnd mice, measured after the last drug injection, and increased the number of mice with ‘normal’ gait (from 50% to 90%, P

*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.