N/OFQ system in brain areas of nerve-injured mice: its role in different aspects of neuropathic pain.

Several studies showed that chronic pain causes reorganization and functional alterations of supraspinal brain regions. The nociceptin-NOP receptor system is one of the major systems involved in pain control and much evidence also suggested its implication in stress, anxiety and depression. Therefore, we investigated the nociceptin-NOP system alterations in selected brain regions in a neuropathic pain murine model. Fourteen days after the common sciatic nerve ligature, polymerase chain reaction (PCR) analysis indicated a significant decrease of pronociceptin and NOP receptor mRNA levels in the thalamus; these alterations could contribute to the decrease of the thalamic inhibitory function reported in neuropathic pain condition. Nociceptin peptide and NOP mRNA increased in the anterior cingulate cortex (ACC) and not in the somatosensory cortex, suggesting a peculiar involvement of this system in pain regulating circuitry. Similarly to the ACC, an increase of nociceptin peptide levels was observed in the amygdala. Finally, the pronociceptin and NOP mRNAs decrease observed in the hypothalamus reflects the lack of hypothalamus-pituitary-adrenal axis activation, already reported in neuropathic pain models. Our data indicate that neuropathic pain conditions affect the supraspinal nociceptin-NOP system which is also altered in regions known to play a role in emotional aspects of pain.

Human apolipoprotein E4 modulates the expression of Pin1, Sirtuin 1, and Presenilin 1 in brain regions of targeted replacement apoE mice.

The apolipoprotein E4 (apoE4) allele is consistently associated with increased risk for Alzheimer's disease (AD). We investigated the molecular mechanism of this susceptibility by analyzing the levels of genes involved in AD pathogenesis in transgenic mice expressing human apoE3 or apoE4 isoforms. mRNA and protein levels of Pin1, Sirtuin 1 (Sirt1), Presenilin 1 (PS1), and pro-Brain-derived Neurotrophic Factor (BDNF) were analyzed in brain regions affected by neuropathological changes in AD. Pin1 mRNA was significantly higher in the hippocampus of apoE4 mice than in apoE3 controls, whereas lower expression was detected in the entorhinal and parietal cortices. Reduced Pin1 levels may increase neurofibrillary degeneration and amyloidogenic processes, while compensatory mechanisms may take place in the hippocampus to balance spatial memory deficits. Sirt1 levels were significantly reduced in the frontal cortex of apoE4 mice. Sirt1 reduction may hinder its protective role against the formation of plaques and tangles and diminish its anti-inflammatory actions. Sirt1 decrease may also play a role in apoE4-associated memory impairments. Moreover, in apoE4 mice PS1 mRNA levels were lower in the frontal cortex. Lower PS1 expression may hamper γ-secretase function, thus affecting amyloid precursor protein processing. Pro-BDNF mRNA levels did not differ between apoE3 and apoE4 mice in any region analyzed. This study showed dysregulated expression of Pin1, Sirt1, and PS1 genes in different cerebral areas of apoE4 mice, suggesting that these changes may play a role in the mechanism of AD vulnerability.

Alterations of the cortico-cortical network in sensori-motor areas of dystrophin deficient mice.

The dystrophin defective mdx mouse, acknowledged model of Duchenne Muscular Dystrophy (DMD), bears outstanding alterations of the cortical architecture, that could be responsible for the cognitive impairment often accompanying this pathological condition. Using a retrograde tract tracing technique to label neurons in Golgi-like fashion, we investigated the fine anatomical organization of associative cortico-cortical projections in mdx mice. While the absolute number of associative pyramidal neurons was significantly higher in mdx than in control animals, the ratio between the number of supra- and infragranular cortico-cortical cells was substantially unmodified. Basal dendrites of layer 2/3 pyramidal neurons displayed longer terminal branches in mdx compared to controls. Finally, the density of dendritic spines was significantly lower in mdx animals. The anomalies of associative cortico-cortical projections provide potential groundwork on the neurobiological bases of cognitive involvement in DMD and value the role of cortical microcircuitry alterations as possible source of interference with peripheral motor impairment.

Propionate induces pH(i) changes through calcium flux, ERK1/2, p38, and PKC in bovine neutrophils.

Propionate is a short-chain fatty acid produced under normal physiological conditions in the rumen of cattle. It is also involved in the inflammatory process and neutrophil function via calcium release, reactive oxygen species and intracellular pH (pH(i)) changes. This study examined the effect of propionate on the pH(i) of bovine neutrophils; specifically if pH(i) changes are controlled by calcium flux, and the mitogen-activated protein kinase (MAPK) pathway. Propionate caused rapid intracellular acidification and sustained alkalinization in bovine neutrophils loaded with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), a fluorescent indicator of pH(i). The acidification phase seems to be controlled by intracellular calcium release and p38 MAPK pathway. The pH recovery phenomenon was mediated by an amiloride-sensitive Na+/H+ exchanger and H+ channel, and was inhibited by UO126 (an ERK1/2 MAPK phosphorylation inhibitor), Gö6850 (a PKC inhibitor) and calcium chelating. Ionomycin, a calcium ionophore, induced intracellular acidification and sustained alkalinization. The intracellular acidification was strongly inhibited by BAPTA-AM (an intracellular calcium chelator) and SB203580 (a p38 MAPK inhibitor). In addition, the intracellular alkalinization was reduced by EGTA (a calcium chelator), UO126, LY294002 (a PI3K inhibitor) and Gö6850. Propionate did not increase superoxide production, however it reduced the superoxide production induced by platelet-activating factor (PAF), and increased the release of superoxide induced by ionomycin. Our results suggest that propionate-induced intracellular acidification is mediated by intracellular calcium release and p38 MAPK activation, and that pH recovery is controlled via ERK1/2 MAPK, PKC and calcium entry in bovine neutrophils.

The organisation of spinal projecting brainstem neurons in an animal model of muscular dystrophy. A retrograde tracing study on mdx mutant mice.

Previous studies we performed on the mdx mouse demonstrated marked central nervous system alterations in this model of human Duchenne muscular dystrophy, such as reduction in number and pathological changes of cortico-spinal neurons. Prompted by these findings we extended the survey of the mdx brain to the major brainstem-descending pathways: the rubro-, vestibulo-, reticulo-, and raphe-spinal projections. Horseradish peroxidase microinjections were performed in the cervical spinal cord of mdx and control mice. The rubro-spinal neurons were found to be significantly reduced in mutants compared to controls. The vestibulo-spinal, reticulo-spinal, and raphe-spinal cell populations, though less numerous in mdx than in control mice, were instead substantially spared. Our data further unveil the selective nature of mdx brain damage indicating a marked and selective involvement of the highest centers for motor control.

c-Fos expression in the auditory pathways related to the significance of acoustic signals in rats performing a sensory-motor task.

Neuronal activity was established in the auditory pathways in relation to behavioural response and cognitive information processing during a sensory-motor acoustic learning. Rats were trained in three consecutive phases. The first phase was an association between an auditory stimulus and a food reward; the second phase a simple discrimination between two sounds of different frequency components, and the third phase a more complex discrimination involving both spectral and spatial sound dimensions. Auditory stimuli were bursts of complex sounds lasting 500 ms. Neuronal activity related to the behaviourally relevant stimuli was established in 20 "learning" rats undergoing this protocol, which were progressively sacrificed at the beginning, middle and end of each phase. For comparison, activity was also established in four "control" rats exposed to the same stimuli delivered pseudo-randomly, thus carrying no behavioural meaning. Neuronal activity was assessed immunocytochemically using the functional marker Fos. To establish a baseline, two rats were unexposed to controlled acoustic stimulation ("unstimulated" rats). In the superior olivary complex (SOC), inferior colliculus (IC) and medial geniculate body (MGB), the number of Fos-like immunopositive cells was comparable in "learning" and "control" animals, but higher than in the "unstimulated" rats. In the auditory cortex (AC), most prominently in the secondary area Te2, the number of Fos-like positive cells differed between "learning" and "control" rats, suggesting that the auditory cortical areas may be involved in the encoding of the behavioural significance of the acoustic stimuli.

Preferential induction of fos-like immunoreactivity in granule cells of the cochlear nucleus by acoustic stimulation in behaving rats.

Neuronal activity in the cochlear nucleus was mapped in relation to acoustic stimuli that signalled a sensory-motor response, using Fos-like immunoreactivity. Rats were trained to associate an acoustic stimulus with a reward and then to discriminate between two sounds ('learning' rats; n = 18). The same stimuli carrying no behavioural significance were pseudo-randomly presented to 'control' rats (n = 4) to differentiate stimulus related- from learning related-activity. To establish a baseline, Fos-like immunoreactivity was determined in rats (n = 2) unexposed to acoustic stimulation. The number of Fos-positive cells was significantly increased in the rats exposed to sounds ('learning' and 'control') as compared to the non-stimulated animals. This stimulus related increase of Fos-like activity in the cochlear nucleus was most prominent in a subpopulation of small neurons, whose spatial distribution corresponds to that of the granule cells. There was also an increase in the number of Fos-positive neurons of larger size, but less prominent than for the small cells. Brief exposure to sounds (30 s) was sufficient to induce Fos-like activity.

A simple pressure microinjecting system for delivery of small substance volumes to the brain: application to the developmental study of thalamo-cortical projections in foetal and neonatal rats.

We describe a reliable and inexpensive method for placing injections of anatomical tracers into the brain of lower mammals. The pressure microinjecting system we developed is specifically designed to deliver very small amount of substances. The injecting portion of the system is relatively easy to assemble and can be repeatedly used for multiple experimental sessions. The system has been validated with experiments of multiple fluorescent retrograde tracing. In these experiments the populations of thalamo-cortical neurons were consistently labeled by the tracers injected bilaterally and symmetrically in the cortex of foetal and neonatal rats.

An optimised procedure for prenatal ethanol exposure with determination of its effects on central nervous system connections.

We describe the protocol set-up to investigate an experimental model of foetal alcohol syndrome in the rat. The protocol has been devised to expose specific cell populations of the central nervous system to ethanol during their neurogenesis and has been applied to the study of diencephalo-telencephalic connections. We were able to demonstrate specific permanent changes of the adult thalamo-cortical circuitry. Our protocol can be applied to study other aspects of central nervous system-ethanol interactions, such as neurotransmitter and receptor patterns. It can also represent a useful tool to test the effects of different diets to prevent nutritional deficiencies and the efficacy of drug treatments to prevent foetal alcohol syndrome. We have shown in fact that ethanol-induced thalamo-cortical alterations are partially prevented by concurrent administration of acetyl-L-carnitine. Finally, the present protocol can be used to investigate the effects of ethanol exposure on the development of different brain structures. To this purpose, the gestational period for ethanol exposure must be chosen according to the peak of neurogenesis for the investigated structure.

Analysis of blink rate patterns in normal subjects.

The present study measured the normal blink rate (BR) variations in relation to behavioral tasks in 150 healthy volunteers (70 males and 80 females; aged 35.9 +/- 17.9 years, range 5-87 years). The subjects were videotaped in a standard setting while performing three different tasks: resting quietly, reading a short passage, talking freely. The mean BR was computed during each task; the data were compared by means of analysis of variance and Student's t tests. Mean BR at rest was 17 blinks/min, during conversation it increased to 26, and it was as low as 4.5 while reading. As compared with rest, BR decreased by -55.08% while reading (p < 1 x 10(-15)) and increased by 99.70% during conversation (p < 1 x 10(-9)). As compared with reading, BR increased during conversation by 577.8% (p < 1 x 10(-17). The distribution curves were highly reproducible in each task. The best curve fit was represented by a log-normal distribution, with the upper tail of each curve having a normal distribution. Eye color and eyeglass wearing did not influence BR. Women had higher BR than men just while reading. No age-related differences were found. The most common BR pattern was conversation > rest > reading, which occurred in 101 subjects (67.3%); 34 subjects (22.7%) had the pattern rest > conversation > reading; 12 (8.0%) had the pattern conversation > reading > rest. This study identified three normal behavioral BR patterns and showed that BR is more influenced by cognitive processes than by age, eye color, or local factors. The present findings provide a normal reference for the analysis of BR in movement disorders such as dystonia or tics.

Functional impairment of nigrostriatal neurons progresses following withdrawal of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

C57 BL/6 mice were rendered severely parkinsonian by exposure to high doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The fluorescent retrograde tracer Fast Blue was injected into the neostriatum one (group A) or five weeks (group B) following exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Neurons located in the substantia nigra pars compacta and in the centre median-parafascicular complex were analysed. There was no variation in the number and distribution of Fast Blue-labelled perikarya located in the centre median-parafascicular complex, which are insensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. No variation was seen in the number of Nissl-stained perikarya located in the substantia nigra pars compacta, indicating that neurons had not degenerated. The number and the density of Fast Blue retrogradely-labelled neurons located in the same region were decreased in group A by 41% and in group B by 55%. Fast Blue labelling provided a measure of functional impairment in viable neurons. The Fast Blue-to-Nissl cell ratio was 55% in controls and declined to 20% in group A and to 17% in group B mice. The present study shows that (1) functional inactivation of viable neurons can be measured by using a fluorescent retrograde tracer following exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and (ii) inactivation of retrograde axonal transport progresses from one to five weeks following withdrawal of the toxin. Fluorescent retrograde probes may be used to measure the anatomical substrate of recovery induced by drugs or by brain grafts in parkinsonian animals.

Crossed thalamo-cortical and cortico-thalamic projections in adult mice.

The crossed thalamo-cortical and cortico-thalamic connections of the mouse are drawn using the tracer wheat germ agglutinin-horseradish peroxidase. After injections in the frontal cortex of the right hemisphere cells labeled retrogradely and axons labeled anterogradely are observed in the thalamus ipsilateral and contralateral to the cortical injections. The retrograde and anterograde labeling in the contralateral thalamus is less intense than ipsilaterally and involves the mediodorsal, ventral medial, central medial, and paracentral nuclei. Crossed fronto-thalamic axons reach more lateral regions than those containing contralateral thalamo-frontal neurons. Our results demonstrate that the thalamo-cortical system of mice has a bilateral component. The functional significance of this pathway and analogies with crossed thalamo-cortical connections in other species are discussed.

Architectural changes of the cortico-spinal system in the dystrophin defective mdx mouse.

The mutant mdx mice which lack the protein dystrophin are an animal model of Duchenne muscular dystrophy. We studied the organization of the cortico-spinal (CS) system in mdx mice using the horseradish peroxidase retrograde tracing technique. Tracer injections were placed in the cervical spinal cord of mutant and control mice. The tangential and radial distribution of CS labeled neurons were similar in mdx and normal mice. Conversely, the absolute number and the cell packing density of labeled CS neurons were considerably lower in mdx than in controls. In mdx, the average size of CS cells was smaller while the perikaryal sizes displayed a normal distribution. In addition, CS neurons of mdx appeared round-shaped compared to the pyramidal cells labeled in control animals. The structural modifications described here should prompt a reconsideration of the involvement of central nervous system in the dystrophin deficient mdx mice.

Acute challenge with apomorphine in Huntington's disease: a double-blind study.

Apomorphine (1.5 or 3 mg) or placebo was acutely administered to choreic patients affected by Huntington's disease in a double-blind fashion. The patients were evaluated before the administration, and at 15-min intervals for 2 h afterward, by means of a rating scale for Huntington's disease. As compared to baseline, the total score improved by 38.54% after 1.5 mg and by 30.41% after 3 mg; no variations were observed after placebo. Several items of the scale improved after the administration of 1.5 mg. An average 35.25% improvement was observed in items measuring the intensity of chorea (at rest, with arms outstretched, during conversation, and voluntary movements of the limbs); in addition, motor impersistence (as measured by tongue protrusion) and the capability to suppress associated movements (as measured by head movements during saccades) improved by an average of 31.46 and 61%, respectively. Some items of the scale improved after the administration of 3 mg. Items measuring the intensity of chorea improved by an average of 30.41%; in addition, the extent of vertical gaze improved by 63.77%. These data indicate that apomorphine brings about a transient symptomatic improvement of chorea and of other associated clinical features in Huntington's disease. The time course observed for the antichoreic activity is only partially consistent with the antiparkinsonian action of apomorphine.

[Changes in left ventricular filling after acute increase of afterload in essential arterial hypertension with or without left ventricular hypertrophy]. / Variazioni del riempimento ventricolare sinistro dopo aumento acuto del postcarico nella ipertensione arteriosa essenziale con e senza ipertrofia ventricolare sinistra.

It is debatable whether a direct causal link exists between left ventricular hypertrophy (LVH) and abnormal left ventricular filling (LVF) in arterial hypertension (AH). To assess if LVH is responsible for peculiar patterns of LVF, we examined 30 selected subjects, similar in age and body surface area, by Doppler echocardiography: 10 hypertensives (SBP: 158 +/- 15; DBP: 105 +/- 7 mmHg) with LVH (IM: 154 +/- 19 mg/m2) (Group 1); 10 hypertensives (SBP: 157 +/- 9; DBP: 101 +/- 5 mmHg) without LVH (IM: 105 +/- 17 mg/m2; Group 2); 10 normotensives without familiar history of AH (Group 3). LVF was analyzed in baseline conditions and during transient afterload increase, obtained by isometric exercise (handgrip, HG, for 5 min at 30% of maximal effort). At rest: 1) A wave peak velocity resulted significantly higher (p less than 0.01) in Group 1 (61 +/- 3 cm/s) versus Group 3 (52 +/- 7 cm/s), and E wave peak velocity was significantly lower in Group 1 (58 +/- 13 cm/s) versus Group 3 (74 +/- 10 cm/s); 2) Group 2 values (E wave: 66 +/- 12; A wave: 58 +/- 15 cm/s) were intermediate between those of groups 1 and 3, and with no significant differences; 3) E/A ratio resulted significantly lower both in Group 1 (1.04 +/- 0.2) and Group 2 (1.21 +/- 0.2) versus Group 3 (1.6 +/- 0.4).(ABSTRACT TRUNCATED AT 250 WORDS)

Botulinum toxin as a treatment for blepharospasm, spasmodic torticollis and hemifacial spasm.

Fifty-two patients affected by focal dystonia or hemifacial spasm were treated with repeated injections of botulinum toxin. A clinical improvement was observed in all patients with blepharospasm; clinical benefit had a mean duration of 10 weeks. Clinical results were less impressive, but also favorable in patients affected by spasmodic torticollis and by hemifacial spasm. In the latter, the incidence of drug-induced paresis was much higher than that observed in patients with blepharospasm, even though the doses of toxin injected were significantly lower.