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Motor skill deficit is a common and invalidating symptom of Rett syndrome (RTT), a rare disease almost exclusively affecting girls during the first/second year of life. Loss-of-function mutations of the methyl-CpG-binding protein2 (MECP2; Mecp2 in rodents) gene is the cause in most patients. We recently found that fluoxetine, a selective serotonin (5-HT) reuptake inhibitor and antidepressant drug, fully rescued motor coordination deficits in Mecp2 heterozygous (Mecp2 HET) mice acting through brain 5-HT. Here, we asked whether fluoxetine could increase MeCP2 expression in the brain of Mecp2 HET mice, under the same schedule of treatment improving motor coordination. Fluoxetine increased the number of MeCP2 immuno-positive (MeCP2+) cells in the prefrontal cortex, M1 and M2 motor cortices, and in dorsal, ventral and lateral striatum. Fluoxetine had no effect in the CA3 region of the hippocampus or in any of the brain regions of WT mice. Inhibition of 5-HT synthesis abolished the fluoxetine-induced rise of MeCP2+ cells. These findings suggest that boosting 5-HT transmission is sufficient to enhance the expression of MeCP2 in several brain regions of Mecp2 HET mice. Fluoxetine-induced rise of MeCP2 could potentially rescue motor coordination and other deficits of RTT.

The aim of this study was to assess cerebrospinal fluid (CSF) concentrations of specific amino acids using a high-performance liquid chromatography system in a sample of patients with functional movement disorders (FMDs) and in a sample of controls. CSF levels of glutamate were significantly lower in patients with FMD than in controls. This finding argues in favor of glutamatergic dysfunction in the pathophysiology of FMD.

In 2020, racemic-fenfluramine was approved in the U.S. and Europe for the treatment of seizures associated with Dravet syndrome, through a restricted/controlled access program aimed at minimizing safety risks. Fenfluramine had been used extensively in the past as an appetite suppressant, but it was withdrawn from the market in 1997 when it was found to cause cardiac valvulopathy. Available evidence indicates that appetite suppression and cardiac valvulopathy are mediated by different serotonergic mechanisms. In particular, appetite suppression can be ascribed mainly to the enantiomers d-fenfluramine and d-norfenfluramine, the primary metabolite of d-fenfluramine, whereas cardiac valvulopathy can be ascribed mainly to d-norfenfluramine. Because of early observations of markedly improved seizure control in some forms of epilepsy, fenfluramine remained available in Belgium through a Royal Decree after 1997 for use in a clinical trial in patients with Dravet syndrome at average dosages lower than those generally prescribed for appetite suppression. More recently, double-blind placebo-controlled trials established its efficacy in the treatment of convulsive seizures associated with Dravet syndrome and of drop seizures associated with Lennox-Gastaut syndrome, at doses up to 0.7 mg/kg/day (maximum 26 mg/day). Although no cardiovascular toxicity has been associated with the use of fenfluramine in epilepsy, the number of patients exposed to date has been limited and only few patients had duration of exposure longer than 3 years. This article analyzes available evidence on the mechanisms involved in fenfluramine-induced appetite suppression, antiseizure effects and cardiovascular toxicity. Despite evidence that stimulation of 5-HT2B receptors (the main mechanism leading to cardiac valvulopathy) is not required for antiseizure activity, there are many critical gaps in understanding fenfluramine's properties which are relevant to its use in epilepsy. Particular emphasis is placed on the remarkable lack of publicly accessible information about the comparative activity of the individual enantiomers of fenfluramine and norfenfluramine in experimental models of seizures and epilepsy, and on receptors systems considered to be involved in antiseizure effects. Preliminary data suggest that l-fenfluramine retains prominent antiseizure effects in a genetic zebrafish model of Dravet syndrome. If these findings are confirmed and extended to other seizure/epilepsy models, there would be an incentive for a chiral switch from racemic-fenfluramine to l-fenfluramine, which could minimize the risk of cardiovascular toxicity and reduce the incidence of adverse effects such as loss of appetite and weight loss.

Significance: Humans cannot synthesize ascorbic acid (AscH2) (vitamin C), so deficiencies in dietary AscH2 cause the life-threatening disease of scurvy and many other diseases. After oral ingestion...

Motor skill is a specific area of disability of Rett syndrome (RTT), a rare disorder occurring almost exclusively in girls, caused by loss-of-function mutations of the X-linked methyl-CpG-binding protein2 (MECP2) gene, encoding the MECP2 protein, a member of the methyl-CpG-binding domain nuclear proteins family. Brain 5-HT, which is defective in RTT patients and Mecp2 mutant mice, regulates motor circuits and SSRIs enhance motor skill learning and plasticity.In the present study, we used heterozygous (Het) Mecp2 female and Mecp2-null male mice to investigate whether fluoxetine, a SSRI with pleiotropic effects on neuronal circuits, rescues motor coordination deficits. Repeated administration of 10 mg/kg fluoxetine fully rescued rotarod deficit in Mecp2 Het mice regardless of age, route of administration or pre-training to rotarod. The motor improvement was confirmed in the beam walking test while no effect was observed in the hanging-wire test, suggesting a preferential action of fluoxetine on motor coordination. Citalopram mimicked the effects of fluoxetine, while the inhibition of 5-HT synthesis abolished the fluoxetine-induced improvement of motor coordination. Mecp2 null mice, which responded poorly to fluoxetine in the rotarod, showed reduced 5-HT synthesis in the prefrontal cortex, hippocampus and striatum, and reduced efficacy of fluoxetine in raising extracellular 5-HT as compared to female mutants. No sex differences were observed in the ability of fluoxetine to desensitize 5-HT1A autoreceptors upon repeated administration. These findings indicate that fluoxetine rescues motor coordination in Mecp2 Het mice through its ability to enhance brain 5-HT and suggest that drugs enhancing 5-HT neurotransmission may have beneficial effects on motor symptoms of RTT.

Increased glucocorticoid concentrations have been shown to favor resilience towards autoimmune phenomena. Here, we addressed whether experimentally induced elevations in circulating glucocorticoids mitigate the abnormalities exhibited by an experimental model of Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS). This is a pathogenic hypothesis linking repeated exposures to Group-A-beta-hemolytic streptococcus (GAS), autoantibodies targeting selected brain nuclei and neurobehavioral abnormalities. To persistently elevate glucocorticoid concentrations, we supplemented lactating SJL/J mice with corticosterone (CORT; 80 mg/L) in the drinking water. Starting in adolescence (postnatal day 28), developing offspring were exposed to four injections - at bi-weekly intervals - of a GAS homogenate and tested for behavioral, immunological, neurochemical and molecular alterations. GAS mice showed increased perseverative behavior, impaired sensorimotor gating, reduced reactivity to a serotonergic agonist and inflammatory infiltrates in the anterior diencephalon. Neonatal CORT persistently increased circulating glucocorticoids concentrations and counteracted these alterations. Additionally, neonatal CORT increased peripheral and CNS concentrations of the anti-inflammatory cytokine IL-9. Further, upstream regulator analysis of differentially expressed genes in the striatum showed that the regulatory effect of estradiol is inhibited in GAS-treated mice and activated in GAS-treated mice exposed to CORT. These data support the hypothesis that elevations in glucocorticoids may promote central immunomodulatory processes.

Variants of tryptophan hydroxylase-2 (Tph2), the gene encoding enzyme responsible for the synthesis of brain serotonin (5-HT), have been associated with neuropsychiatric disorders, substance abuse and addiction. This study assessed the effect of Tph2 gene deletion on motor behavior and found that motor activity induced by 2.5 and 5 mg/kg amphetamine was enhanced in Tph2(-/-) mice. Using the in vivo microdialysis technique we found that the ability of amphetamine to stimulate noradrenaline (NA) release in the striatum was reduced by about 50% in Tph2(-/-) mice while the release of dopamine (DA) was not affected. Tph2 deletion did not affect the release of NA and DA in the prefrontal cortex. The role of endogenous 5-HT in enhancing the effect of amphetamine was confirmed showing that treatment with the 5-HT precursor 5-hydroxytryptophan (10 mg/kg) restored tissue and extracellular levels of brain 5-HT and the effects of amphetamine on striatal NA release and motor activity in Tph2(-/-) mice. Treatment with the NA precursor dihydroxyphenylserine (400 mg/kg) was sufficient to restore the effect of amphetamine on striatal NA release and motor activity in Tph2(-/-) mice. These findings indicate that amphetamine-induced hyperactivity is attenuated by endogenous 5-HT through the inhibition of striatal NA release. Tph2(-/-) mice may be a useful preclinical model to assess the role of 5-HT-dependent mechanisms in the action of psychostimulants. Acute sensitivity to the motor effects of amphetamine has been associated to increased risk of psychostimulant abuse. Here, we show that deletion of Tph2, the gene responsible for brain 5-HT synthesis, enhances the motor effect of amphetamine in mice through the inhibition of striatal NA release. This suggests that Tph2(-/-) mice is a useful preclinical model to assess the role of 5-HT-dependent mechanisms in psychostimulants action. Tph2, tryptophan hydroxylase-2.

Ropinirole and pramipexole are D3 receptor (D3R)-preferring dopaminergic (DA) agonists used for the symptomatic, “DA-replacement” treatment of Parkinson’s disease. They likewise possess antidepressant properties and, while the exact mechanism of action remains uncertain, a role for restoration of impaired neuroplasticity has been proposed. Since D3R are highly expressed on DA neurons in humans, we evaluated their participation in the influence of ropinirole and pramipexole on structural plasticity using a translational model of human inducible pluripotent stem cells (hiPSCs). Two hiPSC clones from healthy donors were differentiated into midbrain DA neurons. Ropinirole and pramipexole produced dose-dependent increases of dendritic arborization and soma size after 3 days of culture, effects antagonized by the selective D3R antagonists SB277011-A and S33084. Visualization of phosphorylated p70S6 kinase indicated the recruitment of the mTOR pathway, critically involved in neuroplasticity. Phosphorylation of p70S6 kinase and structural plasticity were blocked by the kinase inhibitors LY294002 and rapamycin, confirming involvement of the mTOR pathway. Immunoneutralisation of BDNF, inhibition of TrkB receptors and blockade of MEK-ERK signaling likewise prevented ropinirole-induced structural plasticity. Similar results were obtained with pramipexole. The highly similar profiles of data acquired with DA neurons derived from two hiPSC clones underpins their reliability for characterization of agents acting via dopaminergic mechanisms. The present studies show that BDNF- and mTOR-driven signaling underlie the influence of ropinirole and pramipexole on the neuroplasticity of DA neurons. These actions may play an important role in their influence on DA-dependent functions like mood and motor function, known to be disrupted in several clinical disorders.

Cariprazine is a potential antipsychotic awaiting approval from the US Food and Drug Administration. It is a dopamine D2- and D3-receptor partial agonist, with higher affinity for D3 receptors, as opposed to the D2 antagonism of most older antipsychotic agents. Like most lipophilic antipsychotics, it undergoes extensive hepatic metabolism by cytochrome P450 (CYP), mainly the highly variable 3A4, with the formation of active metabolites. However, the parent compound - particularly its active didesmethyl derivative - is cleared very slowly, with elimination half-lives in schizophrenic patients ranging from 2-5 days for cariprazine to 2-3 weeks for didesmethyl-cariprazine. Exposure to the latter was several times that for cariprazine, although didesmethyl-cariprazine did not reach steady state within the 3 weeks of 12.5 mg/day dosing. Preliminary information on its therapeutic role comes from press releases and a few abstracts presented at scientific meetings. In short-term controlled trials, it was more effective than placebo in reducing positive and negative symptoms of schizophrenia, with an effective dose range of 1.5-12 mg/day. Although cariprazine was associated with a higher incidence of akathisia and extrapyramidal side effects than placebo, it did not cause weight gain, metabolic abnormalities, prolactin increase, or corrected QT prolongation. Similarly, cariprazine's efficacy and tolerability for the treatment of bipolar disorder (manic/mixed and depressive episodes) was established in the dose range of 3-12 mg/day, although again no long-term data are available. Well-designed clinical trials, mainly direct "head-to-head" comparisons with other second-generation antipsychotic agents, are needed to define the therapeutic role and safety profile of cariprazine in schizophrenia and bipolar mania.

Endoplasmic reticulum (ER) and oxidative stress are two related phenomena that have important metabolic consequences. As many skeletal muscle diseases are triggered by oxidative stress, we explored the chain of events linking a hyperoxidized ER (which causes ER and oxidative stress) with skeletal muscle dysfunction. An unbiased exon expression array showed that the combined genetic modulation of the two master ER redox proteins, selenoprotein N (SEPN1) and endoplasmic oxidoreductin 1 (ERO1), led to an SEPN1-related myopathic phenotype due to excessive signalling of transforming growth factor (TGF)-beta. The increased TGF-beta activity in the genetic mutants was caused by accelerated turnover of the ER localized (anti-oxidant) ascorbic acid that affected collagen deposition in the extracellular matrix. In a mouse mutant of SEPN1, which is dependent on exogenous ascorbic acid, a limited intake of ascorbic acid revealed a myopathic phenotype as a consequence of an altered TGF-beta signalling. Indeed, systemic antagonism of TGF-beta re-established skeletal muscle function in SEPN1 mutant mice. In conclusion, this study sheds new light on the molecular mechanism of SEPN1-related myopathies and indicates that the TGF-beta/ERO1/ascorbic acid axis offers potential for their treatment.

Previous studies provided evidence for the alteration of brain cholesterol homeostasis in 129.Mecp2-null mice, an experimental model of Rett syndrome. The efficacy of statins in improving motor symptoms and prolonging survival of mutant mice suggested a potential role of statins in the therapy of Rett syndrome. In the present study, we show that Mecp2 deletion had no effect on brain and reduced serum cholesterol levels and lovastatin (1.5 mg/kg, twice weekly as in the previous study) had no effects on motor deficits and survival when Mecp2 deletion was expressed on a background strain (C57BL/6J; B6) differing from that used in the earlier study. These findings indicate that the effects of statins may be background specific and raise important issues to consider when contemplating clinical trials. The reduction of the brain cholesterol metabolite 24S-hydroxycholesterol (24S-OHC) found in B6.Mecp2-null mice suggests the occurrence of changes in brain cholesterol metabolism and the potential utility of using plasma levels of 24S-OHC as a biomarker of brain cholesterol homeostasis in RTT.

Aldehyde-oxidase-4 (AOX4) is one of the mouse aldehyde oxidase isoenzymes and its physiological function is unknown. The major source of AOX4 is the Harderian-gland, where the enzyme is characterized by daily rhythmic fluctuations. Deletion of the Aox4 gene causes perturbations in the expression of the circadian-rhythms gene pathway, as indicated by transcriptomic analysis. AOX4 inactivation alters the diurnal oscillations in the expression of master clock-genes. Similar effects are observed in other organs devoid of AOX4, such as white adipose tissue, liver and hypothalamus indicating a systemic action. While perturbations of clock-genes is sex-independent in the Harderian-gland and hypothalamus, sex influences this trait in liver and white-adipose-tissue which are characterized by the presence of AOX isoforms other than AOX4. In knock-out animals, perturbations in clock-gene expression are accompanied by reduced locomotor activity, resistance to diet induced obesity and to hepatic steatosis. All these effects are observed in female and male animals. Resistance to obesity is due to diminished fat accumulation resulting from increased energy dissipation, as white-adipocytes undergo trans-differentiation towards brown-adipocytes. Metabolomics and enzymatic data indicate that 5-hydroxyindolacetic acid and tryptophan are novel endogenous AOX4 substrates, potentially involved in AOX4 systemic actions.

Impairment of mitochondrial function might contribute to oxidative stress associated with neurodegeneration in amyotrophic lateral sclerosis (ALS). Glutamate levels in tissues of ALS patients are sometimes altered. In neurons, mitochondrial metabolism of exogenous glutamine is mainly responsible for the net synthesis of glutamate, which is a neurotransmitter, but it is also necessary for the synthesis of glutathione, the main endogenous antioxidant. We investigated glutathione synthesis and glutamine/glutamate metabolism in a motor neuronal model of familial ALS. In standard culture conditions (with glutamine) or restricting glutamine or cystine, the level of glutathione was always lower in the cell line expressing the mutant (G93A) human Cu, Zn superoxide dismutase (G93ASOD1) than in the line expressing wild-type SOD1. With glutamine the difference in glutathione was associated with a lower glutamate and impairment of the glutamine/glutamate metabolism as evidenced by lower glutaminase and cytosolic malate dehydrogenase activity. d-β-hydroxybutyrate, as an alternative to glutamine as energy substrate in addition to glucose, reversed the decreases of cytosolic malate dehydrogenase activity and glutamate and glutathione. However, in the G93ASOD1 cell line, in all culture conditions the expression of pyruvate dehydrogenase kinase l protein, which down-regulates pyruvate dehydrogenase activity, was induced, together with an increase in lactate release in the medium. These findings suggest that the glutathione decrease associated with mutant SOD1 expression is due to mitochondrial dysfunction caused by the reduction of the flow of glucose-derived pyruvate through the TCA cycle; it implies altered glutamate metabolism and depends on the different mitochondrial energy substrates.

We investigated the role of serotonin(2C) receptor-mediated feedback mechanisms in the response to citalopram in C57BL/6 and DBA/2 mice, which are respectively responders and non-responders to selective serotonin reuptake inhibitors in the forced swimming test. The microdialysis technique was used to assess changes in extracellular serotonin and GABA in the mouse dorsal raphe (DR). Citalopram (1.25-20 mg/kg) raised extracellular serotonin and GABA in the DR of both mouse strains. These effects were abolished by depleting brain serotonin with p-chlorophenylalanine (300 mg/kg × 3). Systemic and/or intra-DR infusion of the serotonin(2C) receptor antagonist 6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline (1 mg/kg and 0.1 μM, respectively) enhanced citalopram's effect on extracellular serotonin in the DR and medial prefrontal cortex and abolished the rise of GABA in the DR of DBA/2 mice but had no effect in C57BL/6 mice. The serotonin(2C) receptor agonist Ro60-0175 (0.03-3.0 mg/kg) reduced extracellular serotonin and raised GABA in the DR of DBA/2 mice but had much less effect in C57BL/6 mice. These findings show that the sensitivity of serotonin(2C) receptors determines the efficacy of augmentation strategies aimed at enhancing the effect of serotonin reuptake inhibitors on extracellular serotonin through the suppression of serotonin(2C) receptor-mediated feedback control of serotonin neurons.

We studied the antidepressant-like effect of paroxetine in strains of mice carrying different isoforms of tryptophan hydroxylase-2 (TPH-2), the enzyme responsible for the synthesis of brain serotonin (5-HT). The effect of paroxetine alone and in combination with pharmacological treatments enhancing or lowering 5-HT synthesis or melatonin was assessed in the forced swimming test in mice carrying allelic variants of TPH-2 (1473C in C57BL/6 and 1473G in DBA/2 and BALB/c). Changes in brain 5-hydroxytryptophan (5-HTP) accumulation and melatonin levels were measured by high-performance liquid chromatography. Paroxetine (2.5 and 5 mg/kg) reduced immobility time in C57BL/6J and C57BL/6N mice but had no such effect in DBA/2J, DBA/2N and BALB/c mice, even at 10 mg/kg. Enhancing 5-HT synthesis with tryptophan reinstated the antidepressant-like effect of paroxetine in DBA/2J, DBA/2N and BALB/c mice whereas inhibition of 5-HT synthesis prevented the effect of paroxetine in C57BL/6N mice. The response to paroxetine was not associated with changes in locomotor activity, brain melatonin or brain levels of the drug measured at the end of the behavioral test. These results support the importance of 5-HT synthesis in the response to SSRIs and suggest that melatonin does not contribute to the ability of tryptophan to rescue the antidepressant-like effect of paroxetine.

Ischemic stroke is the leading cause of disability, but effective therapies are currently widely lacking. Recovery from stroke is very much dependent on the possibility to develop treatments able to both halt the neurodegenerative process as well as to foster adaptive tissue plasticity. Here we show that ischemic mice treated with neural precursor cell (NPC) transplantation had on neurophysiological analysis, early after treatment, reduced presynaptic release of glutamate within the ipsilesional corticospinal tract (CST), and an enhanced NMDA-mediated excitatory transmission in the contralesional CST. Concurrently, NPC-treated mice displayed a reduced CST degeneration, increased axonal rewiring, and augmented dendritic arborization, resulting in long-term functional amelioration persisting up to 60 d after ischemia. The enhanced functional and structural plasticity relied on the capacity of transplanted NPCs to localize in the peri-ischemic and ischemic area, to promote the upregulation of the glial glutamate transporter 1 (GLT-1) on astrocytes and to reduce peri-ischemic extracellular glutamate. The upregulation of GLT-1 induced by transplanted NPCs was found to rely on the secretion of VEGF by NPCs. Blocking VEGF during the first week after stroke reduced GLT-1 upregulation as well as long-term behavioral recovery in NPC-treated mice. Our results show that NPC transplantation, by modulating the excitatory–inhibitory balance and stroke microenvironment, is a promising therapy to ameliorate disability, to promote tissue recovery and plasticity processes after stroke.SIGNIFICANCE STATEMENTTissue damage and loss of function occurring after stroke can be constrained by fostering plasticity processes of the brain. Over the past years, stem cell transplantation for repair of the CNS has received increasing interest, although underlying mechanism remain elusive. We here show that neural stem/precursor cell transplantation after ischemic stroke is able to foster axonal rewiring and dendritic plasticity and to induce long-term functional recovery. The observed therapeutic effect of neural precursor cells seems to underlie their capacity to upregulate the glial glutamate transporter on astrocytes through the vascular endothelial growth factor inducing favorable changes in the electrical and molecular stroke microenvironment. Cell-based approaches able to influence plasticity seem particularly suited to favor poststroke recovery.

The recent discovery of TPH-2, a new isoform of tryptophan hydroxylase, the enzyme that catalyses the transformation of tryptophan into 5-hydroxytryptophan and the rate-limiting step in brain serotonin (5-HT) biosynthesis, has boosted new interest in the many functions of 5-HT in the brain and non-nervous tissues. Recent studies on TPH-2 are reviewed with particular attention to the role of this enzyme in behavior and in response to drugs as assessed by comparing strains of mice carrying a functional polymorphism of TPH-2. Most studies concur to indicate that 5-HT synthesis through TPH-2 influence nervous tissues whereas TPH-1 is responsible for the synthesis and action of 5-HT in peripheral organs. Partial impairment of brain 5-HT synthesis caused by polymorphism of the gene encoding TPH-2 causes reduced release of the neurotransmitter, increased aggressiveness, and alters the response to drugs inhibiting the reuptake of 5-HT. Strain comparison might be a useful strategy to investigate the genotype-dependent alterations of TPH-2.

Repeated exposure to Group-A β-Haemolytic Streptococcus (GAS) may constitute a vulnerability factor in the onset and course of pediatric motor disturbances. GAS infections/colonization can stimulate the production of antibodies, which may cross the blood brain barrier, target selected brain areas (e.g. basal ganglia) and exacerbate motor alterations. Here, we exposed developing SJL male mice to four injections with a GAS homogenate and evaluated the following domains: motor coordination; general locomotion; repetitive behaviors; perseverative responses; and sensorimotor gating (pre-pulse inhibition, PPI). To demonstrate that behavioral changes were associated with immune-mediated brain alterations, we analyzed, in selected brain areas, the presence of infiltrates and microglial activation (immunohistochemistry), monoamines (HPLC) and brain metabolites (in vivo Magnetic Resonance Spectroscopy). GAS-exposed mice showed increased repetitive and perseverative behaviors, impaired PPI and reduced concentrations of serotonin in prefrontal cortex, a brain area linked to the behavioral domains investigated, wherein they also showed remarkable elevations in lactate. Active inflammatory processes were substantiated by the observation of infiltrates and microglial activation in the white matter of the anterior diencephalon. These data support the hypothesis that repeated GAS exposure may elicit inflammatory responses in brain areas involved in motor control and perseverative behavior and result in phenotypic abnormalities.

Substance P receptor antagonists cause antidepressant- and anxiolytic-like effects in rodents that are thought to involve brain monoamines. In the present study, we examined the effects of the NK1 receptor antagonist GR-205,171 on basal and stress-induced rise of extracellular noradrenaline (NA) and dopamine (DA) in the medial prefrontal cortex (mPFC) of conscious rats and gerbils with the in vivo microdialysis technique. GR-205,171 given intraperitoneally to rats (10 and 30 mg/kg) and gerbils (0.3 and 1 mg/kg) did not affect extracellular NA in either species and increased extracellular DA in rats. Forty minutes of immobilization increased extracellular NA and DA by, respectively, 179% and 188% of baseline values in rats and 222% and 316% of baseline values in gerbils. At 10 mg/kg, GR-205,171 attenuated the stress-induced increase of extracellular NA in the rat. At 30 mg/kg, GR-205,171 suppressed the effect of stress on extracellular DA but had no effect on NA. A lower dose (1 mg/kg) attenuated the stress-induced rise of extracellular NA and DA in the mPFC of gerbils. The results show that blockade of NK1 receptors marginally increased basal extracellular DA in rats but had no effect in gerbils, whereas the stress-induced rise of extracellular NA and DA was markedly attenuated in both species. It is suggested that catecholamines may contribute to the functional effects of GR-205,171.

Serotonin (5-HT) receptors are increasingly recognized as major targets for cognitive enhancement in schizophrenia. Several lines of evidence suggest a pathophysiological role for glutamate NMDA receptors in the prefrontal cortex in schizophrenia and associated disorders in attention and executive functioning. We investigated how the interactions between 5-HT1A and 5-HT2A and glutamate NMDA receptor mechanisms in the medial prefrontal cortex (mPFC) contribute to the control of different aspects of attentional performance. Rats were trained on a five-choice serial reaction time (5-CSRT) task, which provides indices of attentional functioning (percentage of correct responses), executive control (measured by anticipatory and perseverative responses), and speed. The competitive NMDA receptor antagonist CPP (50 ng/side) was infused directly into the mPFC 5 min after infusion of either 8-OH-DPAT (30 and 100 ng/side) or M100907 (100 and 300 ng/side) into the same brain area. Impairments in attentional functioning induced by CPP were completely abolished by both doses of 8-OH-DPAT or M100907. In addition, M100907 abolished the CPP-induced anticipatory responding but had no effects on perseverative over-responding, while 8-OH-DPAT reduced the perseverative over-responding but had no effects on anticipatory responding induced by CPP. The selective 5-HT(1A) receptor antagonist WAY100635 (30 ng/side) antagonized the effects of 8-OH-DPAT (100 ng/side). 8-OH-DPAT at 30 ng/side reduced the latency of correct responses in controls and CPP-injected rats and lowered the percentage of omissions in CPP-injected rats. The data show that 5-HT1A and 5-HT2A receptors in the mPFC exert opposing actions on attentional functioning and demonstrate a dissociable contribution of 5-HT1A and 5-HT2A receptors in the mPFC to different aspects of executive control such as impulsivity and compulsive perseveration.

Polymorphism of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of brain serotonin (5-HT), is associated with less synthesis of brain 5-HT in DBA/2J and BALB/c than in C57BL/6J and 129/Sv mice. We selected the forced swimming test, a mouse model used to assess the antidepressant potential of drugs, and neurochemical techniques to study strain differences in the response to citalopram, a selective 5-HT reuptake inhibitor. Citalopram reduced immobility time in C57BL/6J and 129/Sv mice but had no such effect in DBA/2J and BALB/c mice. The drug reduced accumulation of 5-hydroxytryptophan (5-HTP), an indicator of 5-HT synthesis, in C57BL/6J and 129/Sv mice but much less in DBA/2J and BALB/c mice. Pretreatment with tryptophan raised 5-HTP accumulation and reinstated the antidepressant-like effect of citalopram in DBA/2J and BALB/c mice, whereas pharmacological inhibition of 5-HT synthesis prevented the effect of citalopram in C57BL/6J and 129/Sv mice. Because there were no strain differences in catecholamine synthesis, locomotor activity, and brain levels of citalopram at the end of the behavioral test, the results suggest that the failure of citalopram to reduce immobility time in DBA/2J and BALB/c mice is attributable to genotype-dependent impairment of 5-HT synthesis. Interstrain comparisons could probably be a useful strategy for understanding the mechanisms underlying the response to selective serotonin reuptake inhibitors.

Converging evidence implicates glutamate neurotransmission in attention and inhibitory response control. To investigate how the background genotype contributes to glutamate’s effects on attention and response control, we examined how phencyclidine (PCP) affected the performance of a five-choice serial reaction time (5-CSRT) task in two inbred mouse strains, C57BL/6N and DBA/2N. We also tested a potent mGlu2/3 receptor agonist, LY379268, against PCP’s effects. Mice were trained on a 5-CSRT task, which measures visual attention and response control until they reached asymptotic performance. Both strains of mice were then injected intraperitoneally with 0.5, 1.5 or 3.0 mg/kg PCP. Doses of 1.0 and 3.0 mg/kg of LY379268 were injected subcutaneously to vehicle or PCP-treated mice. At asymptotic performance DBA/2N mice were less accurate and made more anticipatory responses than C57BL/6N. PCP impaired accuracy (% correct) and increased perseverative responses of DBA/2N mice at 1.5 mg/kg. However, at doses up to 3.0 mg/kg it had no effect on these measures in C57BL/6N. In DBA/2N mice 1.5 mg/kg PCP increased anticipatory responses far more than 3.0 mg/kg in C57BL/6N mice. No dose of LY379268 prevented the PCP-induced accuracy deficit of DBA/2N mice. The PCP-induced anticipatory and perseverative responding of DBA/2N mice was reduced by 3.0 mg/kg LY379268, while 1.0 and 3.0 mg/kg reduced anticipatory responding in C57BL/6N. The background genotype may determine the effects of PCP on attentional performance and the results confirm the importance of glutamate transmission in some aspects of this performance.

We recently found that intracortical injection of the selective and competitive N-methyl-D-aspartate (NMDA) receptor antagonist 3-(R)-2-carboxypiperazin-4-propyl-1-phosphonic acid (CPP) impaired attentional performance in rats and blockade of 5-hydroxytryptamine (5-HT)2A receptors antagonized this effect. Here, we used the microdialysis technique in conscious rats to study the effect of CPP on extracellular glutamate (GLU) in the medial prefrontal cortex (mPFC) and the regulation of this effect by 5-HT2A receptors. Intraperitoneal injection of 20 mg/kg CPP increased extracellular GLU in the mPFC (201% of basal levels) but had no effect on 5-HT. Intracortical infusion of 100 lM CPP increased extracellular GLU (230% of basal values) and 5-HT (150% of basal values) in the mPFC, whereas 30 lM had no significant effect. The effect of 100 lM CPP on extracellular GLU was abolished by tetrodotoxin, suggesting that neuronal activity is required. Subcutaneous injection of 40 lg/kg M100,907 completely antagonized the effect of 100 lM CPP on extracellular GLU, whereas 10 lg/kg caused only partial attenuation. Likewise, intracortical infusion of 0.1 lM M100,907 completely reversed the increase of extracellular GLU induced by CPP. These findings show that blockade of NMDA receptors in the mPFC is sufficient to increase extracellular GLU locally. The increase of cortical extracellular GLU may contribute to CPP-induced cognitive deficits and blockade of 5-HT2A receptors may provide a molecular mechanism for reversing these deficits caused by dysfunctional glutamatergic transmission in the

Microdialysis was used to study the acute and chronic effects of escitalopram (S-citalopram; ESCIT) and chronic citalopram (CIT), together with the 5-HT1A receptor antagonist WAY100,635 (N-[2-[methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide trihydrochloride) and the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on extracellular 5-hydroxytryptamine (5-HT) levels in the rat prefrontal cortex. Extracellular 5-HT rose to 234 and 298% of basal values after subcutaneous (s.c.) acute doses of 0.15 and 0.63 mg kg−1 ESCIT. No further increase was observed at 2.5 mg kg−1 ESCIT (290%). The effect of 13-day s.c. infusion of 10 mg kg−1day−1 ESCIT on extracellular 5-HT (422% of baseline) was greater than after 2 days (257% of baseline), whereas exposure to ESCIT was similar. In contrast, the increase in extracellular 5-HT induced by the infusion of CIT for 2 (306%) and 13 days (302%) was similar. However, brain and plasma levels of S-citalopram in rats infused with CIT for 13 days were lower than after 2 days. Acute treatment with 2.5 mg kg−1 ESCIT or 5 mg kg−1 CIT raised extracellular 5-HT by 243 and 276%, respectively, in rats given chronic vehicle but had no effect in rats given ESCIT (10 mg kg−1 day−1) or CIT (20 mg kg−1 day−1) for 2 or 13 days, suggesting that the infused doses had maximally increased extracellular 5-HT. WAY100,635 (0.1 mg kg−1 s.c.) increased extracellular 5-HT levels by 168, 174 and 169% of prechallenge values in rats infused with vehicle or ESCIT for 2 or 13 days, respectively. WAY100,635 enhanced extracellular 5-HT levels to 226, 153 and 164% of prechallenge values in rats infused with vehicle or CIT for 2 and 13 days, respectively. 8-OH-DPAT (0.025 mg kg−1) reduced extracellular 5-HT by 54% in control rats, but had no effect in those given ESCIT and CIT for 13 days. This series of experiments led to the conclusion that chronic treatment with ESCIT desensitizes the 5-HT1A receptors, regulating the release of 5-HT in the prefrontal cortex and enhances the effect of the drug on extracellular 5-HT. They also indicate that chronic treatment with ESCIT and CIT did not prevent WAY100,635 from raising extracellular 5-HT. British Journal of Pharmacology (2004) 142, 469–478. doi:10.1038/sj.bjp.0705800

We investigated whether 5-HT2A receptors contribute to the control of attentional performance by glutamate NMDA receptor mechanisms in the medial prefrontal cortex (mPFC). We examined the effects of NMDA receptor blockade in the mPFC on attentional performance by infusing a competitive glutamate NMDA receptor antagonist, 3-(R)-2-carboxypiperazin-4-propyl-1-phosphonic acid (CPP) into the mPFC of rats performing a task of divided and sustained visual attention. The five-choice serial reaction time task provides indices of attentional functioning (% correct responses), executive control (measured by anticipatory and perseverative responses) and speed. A dose of 10 ng CPP injected bilaterally into the mPFC increased anticipatory and perseverative responding; 50 ng reduced accuracy. Increasing the stimulus duration alleviated the CPP-induced accuracy deficit but did not reduce its effects on anticipatory and perseverative responses. CPP at 50 ng caused motor hyperactivity whereas lower doses had no effect. [R-(+)-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol] (M100907) (M100907), a 5-HT2A receptor antagonist, injected subcutaneously at 10 and 40 microg/kg, had no effect on accuracy but dose dependently reversed the impairment induced by 50 ng CPP. Both doses of M100907 completely abolished CPP-induced anticipatory but not perseverative over-responding. At the dose of 40 microg/kg M100907 reversed CPP-induced motor hyperactivity. This study provides evidence that the prefronto-cortical glutamate NMDA system may make an important contribution to the control of attention and executive functions. It also indicates that 5-HT2A receptors may serve to optimize attentional selectivity and improve some aspects of executive control.

Dysfunction of the ubiquitin-proteasome system (UPS) has been implicated in Parkinson's disease (PD) and other neurodegenerative disorders. We have investigated the effect of UPS inhibition on the metabolism of α-synuclein (SYN) and parkin, two proteins genetically and histopathologically associated to PD. Pharmacological inhibition of proteasome induced accumulation of both parkin and SYN in transfected PC12 cells. We found that this effect was caused by increased protein synthesis rather than impairment of protein degradation, suggesting that inhibition of the UPS might lead to non-specific up-regulation of cytomegalovirus (CMV)-driven transcription. To investigate whether endogenous parkin and SYN can be substrate of the UPS, untransfected PC12 cells and primary mesencephalic neurones were exposed to proteasome inhibitors, and parkin and SYN expression was evaluated at both protein and mRNA level. Under these conditions, we found that proteasome inhibitors did not affect the level of endogenous parkin and SYN. However, we confirmed that dopaminergic neurones were selectively vulnerable to the toxicity of proteasome inhibitors. Our results indicate that studies involving the use of proteasome inhibitors, particularly those in which proteins are expressed from a heterologous promoter, are subjected to potential artefacts that need to be considered for the interpretation of the role of UPS in PD pathogenesis.

Using in vivo intracerebral microdialysis in conscious, freely moving rats, we examined the effect of flibanserin, a potential antidepressant drug with high affinity for human 5-HT1A receptors and four–50-fold lower affinity for 5-HT2A and D4 receptors, on basal extracellular concentrations of serotonin (5-hydroxytryptamine, 5-HT), dopamine (DA) and noradrenaline (NA) in selected regions of the rat brain. Flibanserin at 3 and 10 mg kg−1 significantly reduced extracellular 5-HT in the prefrontal cortex (by 30 and 45%) and dorsal raphe (35 and 44%), but had no effect on extracellular 5-HT in the ventral hippocampus. The 3 and 10 mg kg−1 doses raised extracellular NA to a similar extent in the prefrontal cortex (47 and 50%). In all, 10 mg kg−1 raised extracellular DA in the prefrontal cortex (63%) whereas 3 mg kg−1 had no significant effect. Pretreatment with the selective 5-HT1A receptor antagonist WAY100,635 (0.3 mg kg−1) 30 min before 10 mg kg−1 flibanserin completely antagonized the latter's effects on extracellular 5-HT, DA and NA in the prefrontal cortex. WAY100,635 by itself had no effect on cortical extracellular monoamines. The results show that the stimulation of 5-HT1A receptors plays a major role in the effect of flibanserin on brain extracellular 5-HT, DA and NA. British Journal of Pharmacology (2003) 139, 1281–1288. doi:10.1038/sj.bjp.0705341

Objectives The purpose of this study was to determine the chemical stability and sterility of a diluted, ready-to-use solution of epinephrine for premature neonatal emergencies. Methods A commercially available preparation of epinephrine (1 mg/ml) was diluted with pyrogen-free water under sterile conditions. Diluted solution containing 0.1 mg/ml epinephrine was used to fill Luer-lock syringes which were tightly sealed and stored at room temperature or 2–8°C. A high-performance liquid chromatography method was validated in terms of linearity, accuracy, precision, limits of detection and quantitation, intermediate precision and stability indicating capacity and used to assess epinephrine concentration in syringes stored for up to 24 weeks. Current methods were used to check the sterility of the preparation. Results Diluted epinephrine solution in samples stored at room temperature contained 90%–98% of the freshly prepared dilution. Samples stored at 2–8°C showed no degradation. Sterility was maintained in all samples analysed. Conclusion Diluted epinephrine solution stored at room temperature or at 2–8°C is chemically stable and sterile for 24 weeks. The preparation is fully acceptable for intravenous and endotracheal administration in preterm neonates.

Experiments were performed to confirm that noradrenergic terminals regulate extracellular concentrations of dopamine (DA) in the frontal cortex of rats. The effects of 20 mg/kg 1-[2-[bis(4-fluorphenyl)methoxy]-ethyl]-4-(3- phenylpropyl)piperazine (GBR 12909), a selective inhibitor of DA uptake, and 2.5 mg/kg desipramine (DMI) on the extracellular concentrations of DA in the frontal cortex and striatum were studied in rats given 6-hydroxydopamine (6 micrograms/microliters) bilaterally into the locus coeruleus to destroy noradrenergic terminals. GBR 12909 increased dialysate DA similarly in the striatum of vehicle and 6-hydroxydopamine-treated rats, whereas in the frontal cortex it raised DA concentrations only in lesioned animals. DMI raised extracellular DA concentrations in the frontal cortex but not in the striatum of controls. The effect of DMI on cortical DA was abolished by the 6-hydroxydopamine lesion. GBR 12909, at a subcutaneous dose of 20 mg/kg, further increased cortical dialysate DA in rats given DMI intraperitoneally at 20 mg/kg or through the probe at 10(-5) mol/L. The data support the hypothesis of an important regulation of the extracellular concentrations of DA in the frontal cortex by noradrenergic terminals.

The present study investigated whether 5-HT(2C) receptors in the ventrotegmental area and prefrontal cortex regulate basal and stimulus-evoked dopamine release in the prefrontal cortex. Using the in vivo microdialysis technique in conscious rats, we studied the effect of a selective 5-HT(2C) receptor agonist, Ro60-0175, on basal and immobilization stress-induced dopamine release in the prefrontal cortex. Ro60-0175 intraperitoneally (2.5 mg/kg) and into the ventrotegmental area (10 microg/0.5 microL) completely antagonized the effect of stress on extracellular dopamine without altering basal levels. Infusion of 10 microm Ro60-0175 through the cortical probe had no significant effect on basal and stress-induced dopamine release. SB242084 (10 mg/kg), a selective antagonist of 5-HT(2C) receptors, significantly increased basal extracellular dopamine and completely prevented the effect of intraperitoneal and intraventrotegmental Ro60-0175 on the stress-induced rise of extracellular dopamine, but had no effect itself in stressed rats. The results show that Ro60-0175 suppresses cortical dopamine release induced by immobilization stress through the stimulation of 5-HT(2C) receptors in the ventrotegmental area. While confirming that endogenous 5-HT acting on 5-HT(2C) receptors tonically inhibit basal dopamine release in the prefrontal cortex, the present findings suggest that the stimulation of 5-HT(2C) receptors with an exogenous agonist preferentially inhibit stimulated release.

nized with PLP139-151, a clearly defined relapsing–remitting disease course similar to relapsing–remitting multiple sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease course. To prevent the large infiltration of immune cells from skewing resident CNS cell analysis, we incorporated an antibody against the hematopoietic marker CD45 into the panel and gated out those cells in OLC analysis. Flow cytometry results demonstrated a robust loss of oligodendrocytes during onset of EAE and an early expansion of OPCs. By disease peak the early OPCs expressed more mature markers of OLCs but never fully differentiated into mature myelin-forming oligodendrocytes throughout the rest of the disease course. The early expansion of OPCs was further associated with an increase in the chemotactic protein CD44 in OPCs from the brain. In summary we present here quantification of OLCs and functional global analysis of the myelinproducing cells in EAE mice by flow cytometry. Characterization in mouse models of human CNS disease presents a rapid means to evaluate potential therapeutic interventions in vivo for direct and indirect effects on demyelination and myelin regeneration.

Executive functions are an emerging propriety of neuronal processing in circuits encompassing frontal cortex and other cortical and subcortical brain regions such as basal ganglia and thalamus. Glutamate serves as the major neurotrasmitter in these circuits where glutamate receptors of NMDA type play key role. Serotonin and dopamine afferents are in position to modulate intrinsic glutamate neurotransmission along these circuits and in turn to optimise circuit performance for specific aspects of executive control over behaviour. In this review, we focus on the 5-choice serial reaction time task which is able to provide various measures of attention and executive control over performance in rodents and the ability of prefrontocortical and striatal serotonin 5-HT1A, 5-HT2A and 5-HT2C as well as dopamine D1- and D2-like receptors to modulate different aspects of executive and attention disturbances induced by NMDA receptor hypofunction in the prefrontal cortex. These behavioural studies are integrated with findings from microdialysis studies. These studies illustrate the control of attention selectivity by serotonin 5-HT1A, 5-HT2A, 5-HT2C and dopamine D1- but not D2-like receptors and a distinct contribution of these cortical and striatal serotonin and dopamine receptors to the control of different aspects of executive control over performance such as impulsivity and compulsivity. An association between NMDA antagonist-induced increase in glutamate release in the prefrontal cortex and attention is suggested. Collectively, this review highlights the functional interaction of serotonin and dopamine with NMDA dependent glutamate neurotransmission in the cortico-striatal circuitry for specific cognitive demands and may shed some light on how dysregulation of neuronal processing in these circuits may be implicated in specific neuropsychiatric disorders.

1. This study investigated the effect of acute (2 days) and chronic (14 days) treatment with a selective inhibitor of noradrenaline uptake, reboxetine (10 mg kg(-1) day(-1)) by osmotic pumps, on extracellular noradrenaline and the sensitivity of alpha(2)-adrenoceptors in the prefrontal cortex of rats. 2. The effect of continuous infusion of reboxetine for 14 days on cortical extracellular noradrenaline was significantly higher (599% of vehicle levels) than after 2 days (263% of vehicle levels). 3. Brain concentrations of reboxetine after 2 and 14 days of infusion were 37.9+/-17.8 and 37.1+/-7.7 ng g(-1), respectively. 4. Reboxetine infused for 2 and 14 days significantly increased extracellular dopamine in the prefrontal cortex, to a similar extent (257 and 342% of vehicle levels, respectively), whereas extracellular 5-HT was not modified by either treatment. 5. Clonidine (10 and 30 microg kg(-1) i.p.) reduced cortical extracellular noradrenaline similarly in animals treated with reboxetine or vehicle for 2 days whereas the effects in rats infused with reboxetine for 14 days were markedly less than in vehicle-treated animals. 6. Clonidine (0.05 and 0.2 microM), infused through the dialysis probe into the prefrontal cortex, reduced cortical extracellular noradrenaline much less in rats treated with reboxetine for 14 days than in vehicle-treated animals. 7. Reboxetine's effect on extracellular noradrenaline in the prefrontal cortex was greater after chronic treatment and could be associated with desensitization of terminal alpha(2)-adrenoceptors that normally serve to inhibit noradrenaline release.

Missense mutations in the alpha-synuclein gene were associated with a familial Parkinson's disease, and alpha-synuclein is a major component of Lewy bodies, the intracellular inclusions that neuropathologically characterize Parkinson's disease. We investigated the neurotoxic activity of the nonamyloid component (NAC) of senile plaque, the fibrillogenic fragment (61-95) of alpha-synuclein, in vitro and in vivo. Rat primary mesencephalic neurons were exposed for 6 days to low concentrations of preaggregated NAC (0.5-10.0 microM). The number of dopaminergic neurons and dopamine content were both reduced with no effect on the general viability of the cells. At higher concentrations (25-100 microM), the neurotoxic effect of NAC was extended to all neurons. Preaggregated NAC was also toxic on a PC12 dopaminergic cell line differentiated with nerve growth factor. The intracellular localization of NAC has been identified by the exposure of neuronal cells to fluorescent peptide. In vivo application of aggregated NAC in the substantia nigra induced loss of dopaminergic neurons. Our data illustrate the selective neurotoxic effect of NAC for dopaminergic neurons and support the central role of alpha-synuclein in the pathogenesis of Parkinson's disease.

In behavioral and receptor binding studies, 5-(4-methylpiperazin-1-yl)-8-chloro-pyridol[2,3b] [1,5]benzoxazepine (JL13) shows an atypical antipsychotic profile. We used microdialysis in awake rats to study the effects of various intraperitoneal doses of JL13 on extracellular concentrations of dopamine in the prefrontal cortex, nucleus accumbens and striatum. JL13 at 20 mg/kg and 40 mg/kg dose-dependently raised extracellular dopamine (234% and 434% of basal levels at peak, respectively) in the prefrontal cortex whereas lower doses (5 mg/kg and 10 mg/kg) had no effect. Extracellular concentrations of dihydroxyphenylacetic acid and homovanillic acid were also significantly increased in the prefrontal cortex of rats given 40 mg/kg JL13 (310% and 230% of basal levels, respectively). At 20 mg/kg and 40 mg/kg JL13 did not affect the extracellular concentrations of dopamine and its metabolites in the striatum and nucleus accumbens. The mechanisms by which JL13 increases cortical dopamine release and the significance for potential antipsychotic efficacy are discussed.

The effect of reboxetine, a novel antidepressant drug that potently and selectively inhibits neuronal noradrenaline (NA) uptake, on brain extracellular monoamines was studied by microdialysis. Fifteen mg kg−1 i.p. reboxetine raised extracellular NA in the frontal cortex (by 242%) and dorsal hippocampus (by 240%). Idazoxan (1 mg kg−1 s.c.), given 60 min after 15 mg kg−1 reboxetine, markedly potentiated the effect on extracellular NA in the frontal cortex (by 1580%) and dorsal hippocampus (by 1360%), but had no effect by itself. Twenty-four hours after the last injection of a chronic schedule (15 mg kg−1 i.p. once daily for 14 days) reboxetine had no effect on basal extracellular concentrations of NA in the dorsal hippocampus and a challenge dose of reboxetine (15 mg kg−1) raised extracellular NA similarly in rats treated chronically with reboxetine (by 353%) and saline (by 425%). Ten and 20 μg kg−1 i.p. clonidine dose-dependently reduced hippocampal extracellular NA similarly in rats given chronic reboxetine (by 32% and 57%) and saline (by 42% and 56%). Extracellular concentrations of dopamine and 5-HT in the striatum were similar in rats treated chronically with reboxetine and saline. A challenge dose of reboxetine (15 mg kg−1) had no effect on striatal extracellular dopamine and slightly increased striatal extracellular 5-HT to a similar extent in rats treated chronically with reboxetine (by 137%) and saline (by 142%). The results suggest that combining reboxetine with an α2-adrenoceptor antagonist may facilitate its antidepressant activity. Repeated treatment confirmed that reboxetine is fairly selective for the noradrenergic system but provided no evidence of adaptive changes in that system that could facilitate its effect on extracellular NA. British Journal of Pharmacology (1999) 128, 1332–1338; doi:10.1038/sj.bjp.0702926

The selective 5-hydroxytryptamine reuptake inhibitor citalopram (10 and 20 mg kg−1, i.p.) significantly reduced food intake in male rats (CD-COBS) habituated to eat their daily food during a 4-h period. The 5-HT1A receptor antagonist WAY100635 (0.3 mg kg−1) administered systemically did not modify feeding but significantly potentiated the reduction in food intake caused by 10 mg kg−1 i.p. citalopram. The dose of 5 mg kg−1 i.p. citalopram was not active in animals pretreated with vehicle but significantly reduced feeding in animals pretreated with WAY100635. WAY100635 (0.1 μg 0.5 μl−1) injected into the dorsal raphe significantly potentiated the hypophagic effect of 10 mg kg−1 citalopram. WAY100635 (1.0 μg 0.5 μl−1) injected into the median raphe did not modify feeding or the hypophagic effect of 10 mg kg−1 citalopram. The 5-HT2B/2C receptor antagonist SB206553 (10 mg kg−1, p.o.) slightly reduced feeding by itself but partially antagonized the effect of WAY100635 administered systemically (0.3 mg kg−1, s.c.) or into the dorsal raphe (0.1 μg 0.5 μl−1) in combination with 10 mg kg−1 i.p. citalopram. The hypophagic effect of 10 mg kg−1 i.p. citalopram alone was not significantly modified by SB206553. Brain concentrations of citalopram and its metabolite desmethylcitalopram in rats pretreated with SB206553, WAY100635 and their combination were comparable to those of vehicle-pretreated rats, 90 min after citalopram injection. The hypophagic effect of citalopram was potentiated by blocking 5-HT1A receptors. Only the effect of the WAY100635/citalopram combination seemed to be partially mediated by central 5-HT2C receptors. British Journal of Pharmacology (1998) 124, 1781–1787; doi:10.1038/sj.bjp.0702028

Background: Dopaminergic degeneration is a major finding in brains of patients with Parkinson's disease (PD), together with Lewy bodies, intraneuronal inclusions mainly composed of the fibrillogenic protein α-synuclein (α-syn). The familial-PD-related protein DJ-1 was reported to reduce dopaminergic degeneration triggered by α-syn or by the dopaminergic-selective neurotoxin 6-hydroxydopamine (6-OHDA). Objective: The aim was to further investigate the role of DJ-1 in dopaminergic degeneration and to see whether a cell-permeable recombinant form of DJ-1 (TAT-DJ-1) could restore dopamine depletion in vivo, thus representing an innovative therapeutic approach. Methods: We developed in vitro (PC12/TetOn cells and mouse primary mesencephalic neurons) and in vivo models [including DJ-1 knockout (-/-) mice] to investigate DJ-1 in dopaminergic degeneration. Results: We found that in PC12/TetOn cells overexpressing α-syn with the familial-PD linked mutation A30P, DJ-1 silencing increased α-syn (A30P) toxicity. Primary mesencephalic neurons from DJ-1 (-/-) mice were more vulnerable to a cell-permeable form of α-syn (TAT-α-syn) and to 6-OHDA. Intrastriatally administered TAT-DJ-1 reduced 6-OHDA toxicity in vivo in C57BL/6 mice. Finally, when we injected TAT-α-syn (A30P) in the striatum of DJ-1 (-/-) animals, dopamine was depleted more than in the control strain. Conclusion: DJ-1 appears to have a protective role against dopaminergic degeneration triggered by α-syn or 6-OHDA, reinforcing the possible therapeutic importance of this protein in PD.

Crinoid echinoderms can completely and rapidly regenerate arms lost following self-induced or traumatic amputation. Arm regeneration in these animals therefore provides a valuable experimental model for studying all aspects of regenerative processes, particularly with respect to the nervous system and its specific contribution to regenerative phenomena. Taking into account the primary role of the nervous system in regeneration in other invertebrates, we have investigated the specific involvement of neural factors, viz. the monoamine neurotransmitters dopamine and serotonin, in arm regeneration of Antedon mediterranea. In the present work, the presence of classical monoamines has been revealed by employing specific immunocytochemical and histofluorescence tests in association with biochemical detection by means of high pressure liquid chromatography. The distribution pattern of these neurohumoral molecules at standard regenerative stages has been compared with that of normal non-regenerating arms. Results indicate that both dopamine and serotonin dramatically change in both their distribution and concentration during the repair and regenerative processes. Their remarkably enhanced pattern during regeneration and widespread presence at the level of both nervous and non-nervous tissues indicates that they are important neural growth-promoting factors in crinoid arm regeneration.

Citalopram, 1 and 10 mg/kg i.p., that in a previous study using identical treatment and dialysis conditions had little or no effect on dialysate serotonin (5-HT) in the frontal cortex, dose-dependently raised the extracellular concentrations of 5-HT in the dorsal hippocampus, by 70% and 205% respectively at the peak. In animals given 10 mg/kg citalopram twice daily for 14 days, intraperitoneal doses of 1 and 10 mg/kg or infusion of 10(-8) - 10(-6) M through the hippocampal probe raised dialysate 5-HT in the dorsal hippocampus similarly in animals treated chronically with citalopram or saline. A dose of 100 micrograms/kg 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), an agonist at 5-HT1A receptors, reduced hippocampal extracellular 5-HT concentrations to the same extent in rats repeatedly given saline or citalopram. Half this dose had no such effect in either group. The effect of citalopram and the sensitivity of autoreceptors controlling 5-HT release in the dorsal hippocampus were unaffected by a chronic treatment known to facilitate the drug's effect on dialysate 5-HT and to reduce the sensitivity of 5-HT1A receptors controlling 5-HT release in the frontal cortex. The effects of acute and chronic treatment with citalopram on dialysate 5-HT in the rat brain thus appear to differ in at least two brain regions: the frontal cortex and the dorsal hippocampus.

The effect of chronic treatment with (3-alpha-tropanyl)1H-benzimidazolone-3-carboxamide chloride (DAU 6215; 15 micrograms/kg s.c. twice daily for 21 days), a serotonin3 receptor antagonist, on the extracellular concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was studied by intracerebral dialysis in the striatum, nucleus accumbens and frontal cortex of conscious rats. Twenty-four hours after the last injection, the basal extracellular concentrations of DA in the nucleus accumbens of rats given DAU 6215 were significantly lower than in saline-treated rats. DA output in the dorsolateral striatum or frontal cortex was not significantly different between the DAU 6215 and saline-treated rats. Chronic DAU 6215 significantly reduced the extracellular concentrations of DOPAC and HVA in the frontal cortex but had no effect in the other brain regions. A subcutaneous challenge dose of DAU 6215 (15 micrograms/kg) did not significantly modify the extracellular concentrations of DA and its metabolites in either DAU 6215 or saline treated rats in any of the brain regions examined. The present investigation is the first on the effect of chronic administration of a 5-HT3 receptor antagonist on basal extracellular DA in the rat brain. The results provide evidence of an association between the electrophysiological and biochemical effects of chronic treatment with a serotonin3 receptor antagonist on the activity of the mesolimbic DA system. In line with the theory that hyperactivity of the mesolimbic dopaminergic system is involved in psychosis, the results suggest that DAU 6215 may be useful in the treatment of psychotic disorders, possibly with limited extrapyramidal effects.

The effect of chronic oral treatment with clozapine (20 mg/kg daily for 21 days) on the extracellular concentrations of dopamine, dihydroxyphenylacetic acid and homovanillic acid in the dorsolateral anterior striatum, nucleus accumbens and frontal cortex was studied by microdialysis in conscious rats. Basal levels of dopamine and its metabolites in the three brain regions of rats treated chronically with clozapine were not significantly different from those of vehicle-treated rats. A subcutaneous challenge dose of clozapine (20 mg/kg) significantly increased the extracellular concentrations of dopamine and its metabolites in the three brain regions, with no differences between chronic vehicle- and clozapine-treated rats.

We investigated the interaction between the corticostriatal glutamatergic afferents and dopamine D1-like and D2-like receptors in the dorsomedial striatum (dm-STR) in attention and executive response control in the five-choice serial reaction time (5-CSRT) task. The competitive NMDA receptor antagonist 3-(R)-2-carboxypiperazin-4-propyl-1-phosphonic acid (CPP) injected in the mPFC impaired accuracy and increased premature and perseverative responding, raising GLU, DA, and GABA release in the dm-STR. The D1-like antagonist SCH23390 injected in the dm-STR reversed the CPP-induced accuracy deficit but did not affect the increase in perseverative responding. In contrast, the D2-like antagonist haloperidol injected in the dm-STR reduced the CPP-induced increase in perseverative responding but not the accuracy deficit. The different roles of dorsal striatal D1-like and D2-like receptor were further supported by the finding that activation of D1-like receptor in the dm-STR by SKF38393 impaired accuracy but not perseverative responding while the D2-like agonist quinpirole injected in the dm-STR increased perseverative responding but did not affect accuracy. These findings suggest that integration of cortical information by D1-like receptors in the dm-STR is a key mechanism of the input selection process of attention while the integration of corticostriatal signals by D2-like receptors preserves the ability to switch from one act/response to the next in a complex motor sequence, thus providing for behavioral flexibility.

Trace amines (TAs) such as β-phenylethylamine, p-tyramine, or tryptamine are biogenic amines found in the brain at low concentrations that have been implicated in various neuropsychiatric disorders like schizophrenia, depression, or attention deficit hyperactivity disorder. TAs are ligands for the recently identified trace amine-associated receptor 1 (TAAR1), an important modulator of monoamine neurotransmission. Here, we sought to investigate the consequences of TAAR1 hypersignaling by generating a transgenic mouse line overexpressing Taar1 specifically in neurons. Taar1 transgenic mice did not show overt behavioral abnormalities under baseline conditions, despite augmented extracellular levels of dopamine and noradrenaline in the accumbens nucleus (Acb) and of serotonin in the medial prefrontal cortex. In vitro, this was correlated with an elevated spontaneous firing rate of monoaminergic neurons in the ventral tegmental area, dorsal raphe nucleus, and locus coeruleus as the result of ectopic TAAR1 expression. Furthermore, Taar1 transgenic mice were hyposensitive to the psychostimulant effects of amphetamine, as it produced only a weak locomotor activation and failed to alter catecholamine release in the Acb. Attenuating TAAR1 activity with the selective partial agonist RO5073012 restored the stimulating effects of amphetamine on locomotion. Overall, these data show that Taar1 brain overexpression causes hyposensitivity to amphetamine and alterations of monoaminergic neurotransmission. These observations confirm the modulatory role of TAAR1 on monoamine activity and suggest that in vivo the receptor is either constitutively active and/or tonically activated by ambient levels of endogenous agonist(s).

Complex microenvironmental stimuli influence neural cell properties. To study this, we developed a three-dimensional (3-D) neural culture system, composed of different populations including neurons, astrocytes, and neural stem cells (NSCs). In particular, these last-mentioned cells represent a source potentially exploitable to test drugs, to study neurodevelopment and cell-therapies for neuroregenerations. On seeding on matrigel in a medium supplemented with serum and mitogens, cells obtained from human fetal brain tissue formed 3-D self-organizing neural architectures. Immunocytochemical analysis demonstrated the presence of undifferentiated nestin+ and CD133+ cells, surrounded by β-tub-III+ and GFAP+ cells, suggesting the formation of niches containing potential human NSCs (hNSCs). The presence of hNSCs was confirmed by both neurosphere assay and RT-PCR, and their multipotentiality was demonstrated by both immunofluorescent staining and RT-PCR. Flow cytometry analysis revealed that neurosphere forming cells originating from at least two different subsets expressing, respectively, CD133 and CD146 markers were endowed with different proliferative and differentiation potential. Our data implicate that the complexity of environment within niches and aggregates of heterogeneous neural cell subsets may represent an innovative platform for neurobiological and neurodevelopmental investigations and a reservoir for a rapid expansion of hNSCs.

Rationale Disruption in cognition is characteristic of psychiatric illnesses such as schizophrenia. Studies of drugs that improve cognition might provide a better insight into the mechanisms underlying cognitive deficits. Objectives We compared the effects of the antipsychotic drugs aripiprazole, olanzapine, and haloperidol on performance deficit in a test of divided and sustained visual attention, the five-choice serial reaction time task (5-CSRTT), which provides information on attentional functioning (accuracy of visual discrimination), response control (measured by anticipatory and perseverative responses) and speed. Methods The cognitive deficit was induced by infusion of the competitive NMDA receptor antagonist 3-(R)-2-carboxypiperazin-4-propyl-1-phosphonic acid (CPP) in the rat medial prefrontal cortex (mPFC). In vivo microdialysis was used to compare the effects of aripiprazole, olanzapine and haloperidol on CPP-induced glutamate (GLU) and serotonin (5-HT) release in the mPFC of conscious rats. Results Oral aripiprazole (1.0 and 3.0 mg/kg) and olanzapine (0.3 and 1.0 mg/kg), but not haloperidol (0.1 mg/kg), abolished the CPP-induced accuracy deficit and GLU release. Haloperidol and aripiprazole, but not olanzapine, reduced perseverative over-responding, while anticipatory responding was best controlled by olanzapine. However, these effects were not associated with changes in GLU release. No association was found between the effects of these antipsychotics on CPP-induced attentional performance deficits in the 5-CSRTT and 5-HT efflux. Conclusions The data confirm that excessive GLU release in the mPFC is associated with attentional deficits. Thus, suppression of GLU release may be a target for the development of novel antipsychotic drugs with greater effect on some aspects of cognitive deficits.

The cyclic-adenosine monophosphate response element-binding protein (CREB) family of transcription factors has been implicated in numerous forms of behavioral plasticity. We investigated CREB phosphorylation along some nodes of corticostriatal circuitry such as frontal cortex (FC) and dorsal (caudate–putamen, CPu) and ventral (nucleus accumbens, NAC) striatum in response to the contingent or non-contingent performance of the five-choice serial reaction time task (5-CSRTT) used to assess visuospatial attention. Three experimental manipulations were used; an attentional performance group (contingent, “master”), a group trained previously on the task but for whom the instrumental contingency coupling responding with stimulus detection and reward was abolished (non-contingent, “yoked”) and a control group matched for food deprivation and exposure to the test apparatus (untrained). Rats trained on the 5-CSRTT (both master and yoked) had higher levels of CREB protein in the FC, CPu, and NAC compared to untrained controls. Despite the divergent behavior of “master” and “yoked” rats CREB activity in the FC was not substantially different. In rats performing the 5-CSRTT (“master”), CREB activity was completely abolished in the CPu whereas in the NAC it remained unchanged. In contrast, CREB phosphorylation in CPu and NAC increased only when the contingency changed from goal-dependent to goal-independent reinforcement (“yoked”). The present results indicate that up-regulation of CREB protein expression across cortical and striatal regions possibly reflects the extensive instrumental learning and performance whereas increased CREB activity in striatal regions may signal the unexpected change in the relationship between instrumental action and reinforcement.

The hypothesis that attention deficits induced by the hypofunction of N-methyl d-aspartate (NMDA) receptors in the prefrontal cortex (PFC) might be associated with increased glutamate release and changes in the phosphorylation of the cyclic adenosine monophosphate response element-binding protein on serine 133 (p-S(133)CREB) was investigated in this study. Infusion of 50 ng/side 3-(R)-2-carboxypiperazin-4-propyl-1-phosphonic acid ((R)-CPP), a competitive glutamate NMDA receptor antagonist, into the medial prefrontal cortex (mPFC) of rats performing the five-choice serial reaction time (5-CSRT) task, reduced accuracy of visual discrimination (measured by % correct responses) and enhanced impulsivity (measured by the number of premature responses) and compulsivity (measured by the number of perseverative responses). The mGluR2/3 receptor agonist, LY379268, injected s.c. at 0.1 mg/kg, reduced (R)-CPP-induced impairment in attentional functioning (accuracy) and impulsivity but not compulsive perseveration. In parallel studies using microdialysis technique and Western blot analysis we found that (R)-CPP (100 μM) infused in the medial prefrontal cortex increased glutamate efflux whereas injected in the medial prefrontal cortex at a dose causing impairments in attentional performance (50 ng/side) increased p-S(133)CREB in the frontal cortex (FC), decreased it in the caudate-putamen (CPu) and was without effect in the nucleus accumbens (NAC). LY379268 at the dose effective in reducing (R)-CPP-induced behavioral deficit reduced both the (R)-CPP-induced rise in glutamate efflux in the prefrontal cortex and the increase in p-S(133)CREB in the frontal cortex but was without effect on the decrease in p-S(133)CREB in the caudate-putamen. The data provide evidence that enhanced glutamate release and phosphorylation of cAMP response element binding protein (CREB) on serine 133 may be associated to attention deficit and loss of impulse control. Furthermore they suggest that mGluR2/3 agonists have a therapeutic potential for cognitive deficits.