Giuliano Grignaschi

Glutamate AMPA receptors change in motor neurons of SOD1G93A transgenic mice and their inhibition by a noncompetitive antagonist ameliorates the progression of amytrophic lateral sclerosis-like disease

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder involving the selective degeneration of motor neurons. In a small proportion of patients, ALS is caused by mutations in copper/zinc superoxide dismutase (SOD1), and mice overexpressing SOD1G93A mutant develop a syndrome that closely resembles the human disease. Excitotoxicity mediated by glutamate AMPA receptors has been suggested to be implicated in the selective susceptibility of motor neurons occurring in ALS. In SOD1G93A mice, we found that levels of GluR2 AMPA subunit, which plays a pivotal role in the maintenance of calcium impermeability of AMPA receptors, are decreased in spinal motor neurons before symptom onset in concomitance with a modest increase of GluR3 expression, a calcium‐permeable AMPA subunit. This effect can result in a higher number of calcium‐permeable AMPA receptors on motor neurons of SOD1G93A mice, predisposing these cells to be injured by AMPA‐mediated glutamate firing. In support of this, we showed that treatment with a new noncompetitive AMPA antagonist, ZK 187638, partially protected motor neurons, improved motor function, and prolonged the survival of SOD1G93A mice.

Citalopram‐induced hypophagia is enhanced by blockade of 5‐HT1A receptors: role of 5‐HT2C receptors

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.

Studies on the role of serotonin receptor subtypes in the effect of sibutramine in various feeding paradigms in rats

The effect of the 5‐hydroxytryptamine (5‐HT) and noradrenaline (NA) reuptake inhibitor sibutramine was studied in food deprived, neuropeptide Y (NPY)‐ or muscimol‐injected rats. Sibutramine dose‐dependently reduced feeding caused by food‐deprivation (ED50=5.1±0.8 mg kg−1) or by NPY injection into the paraventricular nucleus of the hypothalamus (ED50=6.0±0.5 mg kg−1). The increase in food intake caused by muscimol injected into the dorsal raphe was not modified by sibutramine (1–10 mg kg−1). The hypophagic effect of 5.1 mg kg−1 sibutramine in food‐deprived rats was studied in rats pretreated with different serotonin receptor antagonists. Metergoline (non‐selective, 0.3 and 1.0 mg kg−1), ritanserin (5‐HT2A/2C, 0.5 and 1.0 mg kg−1) and GR127935 (5‐HT1B/1D, 0.5 and 1.0 mg kg−1) did not modify the hypophagic effect of sibutramine, while SB206553 (5‐HT2B/2C, 5 and 10 mg kg−1) slightly but significantly reduced it (Fint(2.53)=3.4; P<0.05). The reduction in food intake caused by 6.0 mg kg−1 sibutramine in NPY‐injected rats was not modified by GR127935 (1.0 mg kg−1). The results suggest that, with the possible exception of a partial involvement of 5‐HT2B/2C receptors in sibutramines hypophagia in food‐deprived rats, 5‐HT1 and 5‐HT2 receptor subtypes do not play an important role in the hypophagic effect of sibutramine, at least in the first 2 h after injection.

Molecular Signatures of Amyotrophic Lateral Sclerosis Disease Progression in Hind and Forelimb Muscles of an SOD1G93A Mouse Model

AIMS This study utilized proteomics, biochemical and enzymatic assays, and bioinformatics tools that characterize protein alterations in hindlimb (gastrocnemius) and forelimb (triceps) muscles in an amyotrophic lateral sclerosis (ALS) (SOD1(G93A)) mouse model. The aim of this study was to identify the key molecular signatures involved in disease progression. RESULTS Both muscle types have in common an early down-regulation of complex I. In the hindlimb, early increases in oxidative metabolism are associated with uncoupling of the respiratory chain, an imbalance of NADH/NAD(+), and an increase in reactive oxygen species (ROS) production. The NADH overflow due to complex I inactivation induces TCA flux perturbations, leading to citrate production, triggering fatty acid synthase (FAS), and lipid peroxidation. These early metabolic changes in the hindlimb followed by sustained and comparatively higher metabolic and cytoskeletal derangements over time precede and may catalyze the progressive muscle wasting in this muscle at the late stage. By contrast, in the forelimb, there is an early down-regulation of complexes I and II that is associated with the reduction of oxidative metabolism, which promotes metabolic homeostasis that is accompanied by a greater cytoskeletal stabilization response. However, these early compensatory systems diminish by a later time point. INNOVATION The identification of potential early- and late-stage disease molecular signatures in an ALS model: muscle albumin, complex I, complex II, citrate synthase, FAS, and phosphoinositide 3-kinase functions as diagnostic markers and peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α), Sema-3A, and Rho-associated protein kinase 1 (ROCK1) play the role of disease progression markers. CONCLUSION The differing pattern of cellular metabolism and cytoskeletal derangements in the hind and forelimb identifies the potential dysmetabolism/hypermetabolism molecular signatures associated with disease progression, which may serve as diagnostic/disease progression markers in ALS patients.

The 5-HT1B receptor mediates the effect of d-fenfluramine on eating caused by intra-hypothalamic injection of neuropeptide Y.

d-Fenfluramine (0.63 mg/kg i.p.), a serotonin (5-hydroxytryptamine, 5-HT) releaser and re-uptake inhibitor, reduced the eating caused by neuropeptide Y (235 pmol) injected into the paraventricular nucleus of the hypothalamus. The 5-HT1 and 5-HT2 receptor antagonist metergoline (1.0 and 2.0 mg/kg i.p.) and the 5-HT1A and 5-HT1B receptor antagonist (+/-)-cyanopindolol (3.0 and 8.0 mg/kg s.c.) significantly antagonized the effect of d-fenfluramine. The 5-HT2A and 5-HT2C receptor antagonist mesulergine (0.1 and 0.3 mg/kg s.c.) and the 5-HT2A receptor antagonist ketanserin (2.5 and 5.0 mg/kg i.p.) did not significantly modify the effect, nor did the 5-HT1A and 5-HT1B receptor antagonist (-)-propranolol (20-40 nmol), injected bilaterally into the paraventricular nucleus of the hypothalamus. The results suggest that d-fenfluramine reduces neuropeptide Ys hyperphagia by indirectly stimulating 5-HT1B receptors outside the paraventricular nucleus of the hypothalamus.

A single high dose of cocaine induces behavioural sensitization and modifies mRNA encoding GluR1 and GAP-43 in rats

Neuroadaptive changes underlying repeated exposure to cocaine‐induced behavioural sensitization have been related to modification in the pattern of synaptic connectivity and excitatory transmission. Remarkably, even a single exposure to abused drugs is sufficient to elicit lasting behavioural sensitization. The present study investigated whether in Sprague–Dawley rats a single, behavioural sensitizing dose of cocaine is sufficient to induce changes in the mRNA levels of growth‐associated protein 43 (GAP‐43), an important protein in mediating experience‐dependent plasticity and synaptic reorganization, and of glutamate receptor 1 (GluR1), a subunit of AMPA glutamate receptors, a protein that is up‐regulated with repeated cocaine. Single exposure to 20, but not 10 mg/kg cocaine induced locomotor sensitization to a second injection of 10 mg/kg cocaine, observed at 24 h, 48 h and 7 days. Single dose of 20 but not 10 mg/kg cocaine 48 h before scheduled death significantly enhanced GluR1 and GAP‐43 mRNA expression in the nucleus accumbens (NAc), both shell and core subregions, and ventral tegmental area (VTA). No changes were found in the levels of mRNA for GluR1 and GAP‐43 in the frontal cortex, caudate putamen, dentate gyrus of hippocampus and basolateral nucleus of the amygdala after the single dose of 20 mg/kg cocaine. These results further strengthen the involvement of NAc and VTA in the behavioural sensitization and suggest a role of GAP‐43 in the synaptic reorganization associated to drug abuse.

Stimulation of 5-HT2A receptors in the paraventricular hypothalamus attenuates neuropeptide Y-induced hyperphagia through activation of corticotropin releasing factor

The effect of 5-HT1 and 5-HT2 receptor agonists administered into the paraventricular hypothalamus was studied on the hyperphagia caused by neuropeptide Y (NPY) injected into the same area. The 5-HT2A/2C receptor agonist DOI (10-20 nmol/0.5 microliter) significantly reduced NPY overeating while the 5-HT1A/1B receptor agonist RU 24969 (3.5-14 nmol/0.5 microliter) and the 5-HT1B/2C receptor agonist mCPP (5-20 nmol/0.5 microliter) had no such effect. The 5-HT2A receptor antagonist spiperone (5 microgram/0.5 microliter) and the corticotropin releasing factor antagonist alpha-helical-CRF9-41 (0.5-1 micrograms/0.5 microliter) completely antagonized the effect of 10 nmol DOI.

The hypophagic effect of restraint stress in rats can be mediated by 5-HT2 receptors in the paraventricular nucleus of the hypothalamus

Ritanserin (0.5 and 1 mg/kg) and ketanserin (2.5 mg/kg), two antagonists with high affinity for 5-HT2 receptors, attenuated restraint stress-induced hypophagia in rats. Two injections of the 5-HT2 receptor antagonist cinanserin (30 nmol/0.5 microliter) in the paraventricular nucleus of the hypothalamus completely reversed the effect of stress on food intake. (+/-)Cyanopindolol (3 and 8 mg/kg), an antagonist at 5-HT1A and 5-HT1B receptors, had no effect whereas 8-hydroxy-2-di-n-propylamino)tetralin (30-300 micrograms/kg), an agonist at 5-HT1A receptors, significantly attenuated the hypophagia. The results suggest that restraint stress-induced hypophagia is mediated by 5-HT2 receptors in the paraventricular nucleus of the hypothalamus. The potential utility of this model in anorexia nervosa is discussed.

Reciprocal interaction of 5-hydroxytryptamine and cholecystokinin in the control of feeding patterns in rats

1 The effect of the CCKA receptor antagonist, devazepide (100 mg kg−1) on meal parameters during the initial phase of the dark period was studied in free‐feeding rats by use of a procedure for continuously monitoring feeding patterns. 2 In a second experiment, the effect of devazepide on the reduction in meal parameters induced by the 5‐hydroxytryptamine (5‐HT) releaser and uptake inhibitor, (+)‐fenfluramine (1.5 mg kg−1) in 4 h food‐deprived rats was examined. 3 The hypophagic effect of an intraperitoneal injection of cholecystokinin (CCK‐8, 4 μg kg−1) was studied in rats treated with the 5‐HT receptor antagonist, metergoline (1 and 2 mg kg−1). 4 Devazepide increased the size of the first meal in free‐feeding, but not in 4 h food‐deprived rats and partially antagonized the effect of (+)‐fenfluramine on the size and duration of the first meal. The reduction in eating rate induced by (+)‐fenfluramine was not modified by devazepide. No changes in (+)‐fenfluramine or (+)‐norfenfluramine levels were found in the brain of rats treated with devazepide. 5 The effect of CCK‐8 on meal size was completely antagonized by 2 mg kg−1 metergoline. A significant interaction was also found between 2 mg kg−1 metergoline and CCK‐8 as regards their effect on the inter‐meal interval. 6 The results suggest a reciprocal interaction between 5‐HT and CCK‐8 in enhancing the satiating effect of food in rats.

Erythropoietin does not preserve motor neurons in a mouse model of familial ALS.

Recombinant human EPO (r‐Hu‐EPO) protects cultured motor neurons from kainate‐ and serum deprivation‐induced toxicity and readily enters the CNS after systemic injection. We examined the effect of rHuEPO in transgenic mice expressing the human Cu/Zn dependent‐superoxide dismutase with G93A mutation (SOD1G93A), a model of familial amyotrophic lateral sclerosis. rHuEPO (4 unit/g BW s.c. three times/week), increased the haematocrit and induced a slight delay in impairment of motor function as measured by the rotating bar test. However, it did not prolong life span or reduce motor neuron loss in lumbar spinal cord. The effect on motor function may be due to the improvement of skeletal muscle oxygenation induced by chronic EPO administration.