An increase in force after stretch of diaphragm fibers and myofibrils is accompanied by an increase in sarcomere length nonuniformities and Ca2+ sensitivity.

When muscle fibers from limb muscles are stretched while activated, the force increases to a steady-state level that is higher than that produced during isometric contractions at a corresponding sarcomere length, a phenomenon known as residual force enhancement (RFE). The mechanisms responsible for the RFE are an increased stiffness of titin molecules that may lead to an increased Ca2+ sensitivity of the contractile apparatus, and the development of sarcomere length nonuniformities. RFE is not observed in cardiac myofibrils, which makes this phenomenon specific to certain preparations. The aim of this study was to investigate whether the RFE is present in the diaphragm, and its potential association with an increased Ca2+ sensitivity and the development of sarcomere length nonuniformities. We used two preparations: single intact fibers and myofibrils isolated from the diaphragm of mice. We investigated RFE in a variety of lengths across the force-length relationship. RFE was observed in both preparations at all lengths investigated and was larger with increasing magnitudes of stretch. RFE was accompanied by an increased Ca2+ sensitivity as shown by a change in the force-pCa2+ curve, and increased sarcomere length nonuniformities. Therefore, RFE is a phenomenon commonly observed in skeletal muscles, with mechanisms that are similar across preparations.

GABAergic and glycinergic inputs modulate rhythmogenic mechanisms in the lamprey respiratory network.

We have previously shown that GABA and glycine modulate respiratory activity in the in vitro brainstem preparations of the lamprey and that blockade of GABAA and glycine receptors restores the respiratory rhythm during apnoea caused by blockade of ionotropic glutamate receptors. However, the neural substrates involved in these effects are unknown. To address this issue, the role of GABAA, GABAB and glycine receptors within the paratrigeminal respiratory group (pTRG), the proposed respiratory central pattern generator, and the vagal motoneuron region was investigated both during apnoea induced by blockade of glutamatergic transmission and under basal conditions through microinjections of specific antagonists. The removal of GABAergic, but not glycinergic transmission within the pTRG, causes the resumption of rhythmic respiratory activity during apnoea, and reveals the presence of a modulatory control of the pTRG under basal conditions. A blockade of GABAA and glycine receptors within the vagal region strongly increases the respiratory frequency through disinhibition of neurons projecting to the pTRG from the vagal region. These neurons were retrogradely labelled (neurobiotin) from the pTRG. Intense GABA immunoreactivity is observed both within the pTRG and the vagal area, which corroborates present findings. The results confirm the pTRG as a primary site of respiratory rhythm generation, and suggest that inhibition modulates the activity of rhythm-generating neurons, without any direct role in burst formation and termination mechanisms.

Thalamic projections to the macaque caudal ventrolateral prefrontal areas 45A and 45B.

We studied the sources of thalamic projections to the caudal ventrolateral prefrontal areas 45A and 45B, which display markedly distinct cortical connections [M. Gerbella et al. (2010) Cereb. Cortex, 20, 141-168], and compared them with those to area 8/FEF (frontal eye field). Both areas 45A and 45B were the targets of highly predominant projections from the mediodorsal nucleus (MD) and of additional projections, mostly from the magnocellular ventral anterior and the medial pulvinar nucleus. The projection profiles from different MD subdivisions clearly distinguished these two areas from one another and from area 8/FEF. Area 45A was the target of predominant projections from parvicellular MD and of minor, albeit robust, projections from magnocellular MD. The opposite was true for area 45B: magnocellular MD was the major source of projections and parvicellular MD contributed minor, albeit robust, projections. Furthermore, area 45B, but not area 45A, was targeted by robust projections from multiform MD, the principal thalamic nucleus for area 8/FEF. These results provide further evidence for the distinctiveness of areas 45A and 45B, and support the idea that area 45B is affiliated with the frontal oculomotor system, challenging the proposed homology of this area with part of the human language-related area 45 (rostral part of Broca's region). Furthermore, the present data provide evidence for potentially robust trans-thalamic (via magnocellular MD) afferent, as well as direct and reciprocal, amygdaloid connections of areas 45A and 45B, suggesting the contribution of emotional information to the differential role of these two areas in non-spatial information processing.

Breathing stimulation mediated by 5-HT1A and 5-HT3 receptors within the preBötzinger complex of the adult rabbit.

Serotonin (5-HT) has been reported to play excitatory effects on respiration by acting on preBötzinger complex (preBötC) neurons in neonatal or juvenile rodents. However, whether its action is circumscribed to the preBötC and present in other animal species, particularly in adult preparations, is unknown. We investigated the respiratory role of 5-HT within the preBötC and neighbouring respiration-related regions. Experiments were performed on α-chloralose-urethane anesthetized, vagotomized, paralyzed and artificially ventilated rabbits making use of bilateral microinjections (30-50 nl). 5-HT caused excitatory effects on respiratory activity only when applied to the preBötC. These effects were mediated by 5-HT1A and 5-HT3 receptors as shown by microinjections of specific agonists of the different types of 5-HT receptors. Unexpectedly, the blockade of 5-HT1A receptors by methysergide or the specific antagonist (S)-WAY 100135 induced excitatory respiratory effects. Microinjections of the 5-HT3 receptor antagonist ondansetron did not influence respiration, but prevented (S)-WAY 100135-induced responses. The blockade of GABAA receptors by bicuculline within the preBötC prevented the effects of the 5-HT1A receptor agonist 8-OH-DPAT. The involvement of GABAergic inhibition and 5-HT1A receptor-mediated disinhibition is also corroborated by immunohistochemical data. The results show for the first time in an adult animal preparation that 5-HT plays a pivotal role in the modulation of the preBötC activity probably via both presynaptic and postsynaptic mechanisms and highlight the importance of disinhibition phenomena. Present findings may be relevant to some respiratory disorders in which an impairment of central 5-HT mechanisms has been reported, such as sleep apnoea and sudden infant death syndrome.

The unveiling of the Warburg effect and the inscribed innovative approach to a radical non toxic anticancer therapy.

The purpose of this research has been deciphering the Warburg paradox, the biochemical enigma unsolved since 1923. We solved it by demonstrating that its specific character, i.e. the forced aerobic lactate exportation, represents a crucial metabolic device to counteract the cytotoxic effect produced by an excess of pyruvate at the connection of glycolysis with the Krebs cycle. This solution was verified by exposing cancer cells of different histogenesis to pyruvate concentrations higher than the physiological ones, after showing that these concentrations are totally innocuous when injected into mice. The mechanism of the pyruvate cytotoxicity relies on the saturation of the respiratory chain, leading to a negative shift of the cytosolic NADP/NADPH ratio and the consequent restriction of the purine synthesis and the related cell apoptosis. The reducing equivalents generated by glycolysis and by cytosolic metabolism compete each other for their disposal trough the respiratory chain; this makes it that the cytotoxicity of pyruvate is inversely related to the mitochondrial number and efficiency of various cell types. Thus, the cytotoxicity is high in anaplastic cancer stem cells, whose mitochondria are extremely few and immature (cristae-poor); on the contrary, no inhibition is brought about in adult differentiated cells, physiologically rich of mature mitochondria. All this generates the pyruvate anticancer selectivity, together with the lack of a general toxicity, making pyruvate represent an ideal candidate for a radical non toxical anticancer treatment.

Inhibitory control of ascending glutamatergic projections to the lamprey respiratory rhythm generator.

Neurons within the vagal motoneuron region of the lamprey have been shown to modulate respiratory activity via ascending excitatory projections to the paratrigeminal respiratory group (pTRG), the proposed respiratory rhythm generator. The present study was performed on in vitro brainstem preparations of the lamprey to provide a characterization of ascending projections within the whole respiratory motoneuron column with regard to the distribution of neurons projecting to the pTRG and related neurochemical markers. Injections of Neurobiotin were performed into the pTRG and the presence of glutamate, GABA and glycine immunoreactivity was investigated by double-labeling experiments. Interestingly, retrogradely labeled neurons were found not only in the vagal region, but also in the facial and glossopharyngeal motoneuron regions. They were also present within the sensory octavolateral area (OLA). The results show for the first time that neurons projecting to the pTRG are immunoreactive for glutamate, surrounded by GABA-immunoreactive structures and associated with the presence of glycinergic cells. Consistently, GABAA or glycine receptor blockade within the investigated regions increased the respiratory frequency. Furthermore, microinjections of agonists and antagonists of ionotropic glutamate receptors and of the GABAA receptor agonist muscimol showed that OLA neurons do not contribute to respiratory rhythm generation. The results provide evidence that glutamatergic ascending pathways to the pTRG are subject to a potent inhibitory control and suggest that disinhibition is one important mechanism subserving their function. The general characteristics of inhibitory control involved in rhythmic activities, such as respiration, appear to be highly conserved throughout vertebrate evolution.

Extrasynaptic release of GABA and dopamine by retinal dopaminergic neurons.

In the mouse retina, dopaminergic amacrine (DA) cells synthesize both dopamine and GABA. Both transmitters are released extrasynaptically and act on neighbouring and distant retinal neurons by volume transmission. In simultaneous recordings of dopamine and GABA release from isolated perikarya of DA cells, a proportion of the events of dopamine and GABA exocytosis were simultaneous, suggesting co-release. In addition, DA cells establish GABAergic synapses onto AII amacrine cells, the neurons that transfer rod bipolar signals to cone bipolars. GABAA but not dopamine receptors are clustered in the postsynaptic membrane. Therefore, dopamine, irrespective of its site of release-synaptic or extrasynaptic-exclusively acts by volume transmission. Dopamine is released upon illumination and sets the gain of retinal neurons for vision in bright light. The GABA released at DA cells' synapses probably prevents signals from the saturated rods from entering the cone pathway when the dark-adapted retina is exposed to bright illumination. The GABA released extrasynaptically by DA and other amacrine cells may set a 'GABAergic tone' in the inner plexiform layer and thus counteract the effects of a spillover of glutamate released at the bipolar cell synapses of adjacent OFF and ON strata, thus preserving segregation of signals between ON and OFF pathways.

Role of nucleus accumbens in neuropathic pain: linked multi-scale evidence in the rat transitioning to neuropathic pain.

Despite recent evidence implicating the nucleus accumbens (NAc) as causally involved in the transition to chronic pain in humans, underlying mechanisms of this involvement remain entirely unknown. Here we elucidate mechanisms of NAc reorganizational properties (longitudinally and cross-sectionally), in an animal model of neuropathic pain (spared nerve injury [SNI]). We observed interrelated changes: (1) In resting-state functional magnetic resonance imaging (fMRI), functional connectivity of the NAc to dorsal striatum and cortex was reduced 28days (but not 5days) after SNI; (2) Contralateral to SNI injury, gene expression of NAc dopamine 1A, 2, and κ-opioid receptors decreased 28days after SNI; (3) In SNI (but not sham), covariance of gene expression was upregulated at 5days and settled to a new state at 28days; and (4) NAc functional connectivity correlated with dopamine receptor gene expression and with tactile allodynia. Moreover, interruption of NAc activity (via lidocaine infusion) reversibly alleviated neuropathic pain in SNI animals. Together, these results demonstrate macroscopic (fMRI) and molecular reorganization of NAc and indicate that NAc neuronal activity is necessary for full expression of neuropathic pain-like behavior.

Synaptic input of ON-bipolar cells onto the dopaminergic neurons of the mouse retina.

In the retina, dopamine fulfills a crucial role in neural adaptation to photopic illumination, but the pathway that carries cone signals to the dopaminergic amacrine (DA) cells was controversial. We identified the site of ON-cone bipolar input onto DA cells in transgenic mice in which both types of catecholaminergic amacrine (CA) cells were labeled with green fluorescent protein or human placental alkaline phosphatase (PLAP). In confocal Z series of retinal whole mounts stained with antibodies to tyrosine hydroxylase (TH), DA cells gave rise to varicose processes that descended obliquely through the scleral half of the inner plexiform layer (IPL) and formed a loose, tangential plexus in the middle of this layer. Comparison with the distribution of the dendrites of type 2 CA cells and examination of neurobiotin-injected DA cells proved that their vitreal processes were situated in stratum S3 of the IPL. Electron microscope demonstration of PLAP activity showed that bipolar cell endings in S3 established ribbon synapses onto a postsynaptic dyad in which one or both processes were labeled by a precipitate of lead phosphate and therefore belonged to DA cells. In places, the postsynaptic DA cell processes returned a reciprocal synapse onto the bipolar endings. Confocal images of sections stained with antibodies to TH, kinesin Kif3a, which labels synaptic ribbons, and glutamate or GABA(A) receptors, confirmed that ribbon-containing endings made glutamatergic synapses onto DA cells processes in S3 and received from them GABAergic synapses. The presynaptic ON-bipolar cells most likely belonged to the CB3 (type 5) variety.

Multimodal architectonic subdivision of the rostral part (area F5) of the macaque ventral premotor cortex.

We used a cyto-, myelo-, and chemoarchitectonic (distribution of SMI-32 and calbindin immunoreactivity) approach to assess whether the rostral histochemical area F5 of the ventral premotor cortex (PMv) comprises architectonically distinct areas, possibly corresponding to functionally different fields. Three areas were identified, occupying different parts of F5. One area, designated as "convexity" F5 (F5c), extends on the postarcuate convexity cortex adjacent to the inferior arcuate sulcus and is characterized, cytoarchitectonically, by a poorly laminated appearance, resulting from an overall cell population rather homogeneous in size and density. The other two areas, designated as "posterior" and "anterior" F5 (F5p and F5a, respectively), lie within the postarcuate bank at different anteroposterior levels. Major cytoarchitectonic features of F5p are a layer III relatively homogeneous in cell size and density, a cell-dense layer Va, and the presence of relatively large pyramids in layer Vb. Major cytoarchitectonic features of F5a are the presence of relatively large pyramids in lowest layer III and a prominent, homogenous layer V. Furthermore, our results showed that F5c and F5p border caudally with a caudal PMv area corresponding to histochemical area F4, providing additional evidence for a general subdivision of the macaque PMv into a caudal and a rostral part, corresponding to F4 and to the F5 complex, respectively. The present data, together with other functional and connectional data, suggest that the three rostral PMv areas F5p, F5a, and F5c correspond to distinct cortical entities, possibly involved in different aspects of motor control and cognitive motor functions.

Expression of circadian clock genes in retinal dopaminergic cells.

The mammalian neural retina contains single or multiple intrinsic circadian oscillators that can be directly entrained by light cycles. Dopaminergic amacrine (DA) cells represent an especially interesting candidate as a site of the retinal oscillator because of the crucial role of dopamine in light adaptation, and the widespread distribution of dopamine receptors in the retina. We hereby show by single-cell, end-point RT-PCR that retinal DA cells contain the transcripts for six core components of the circadian clock: Bmal1, Clock, Cry1, Cry2, Per1, and Per2. Rod photoreceptors represented a negative control, because they did not appear to contain clock transcripts. We finally confirmed that DA cells contain the protein encoded by the Bmal1 gene by comparing immunostaining of the nuclei of DA cells in the retinas of wildtype and Bmal1-/- mice. It is therefore likely that DA cells contain a circadian clock that anticipates predictable variations in retinal illumination.

A multiarchitectonic approach for the definition of functionally distinct areas and domains in the monkey frontal lobe.

Over the last century, anatomical studies have shown that the cerebral cortex can be subdivided into structurally distinct regions, giving rise to a new branch of neuroanatomy: 'architectonics'. Since then, architectonics has been often accused of being overly subjective, and its validity for the definition of functionally different cortical fields has been seriously questioned. Since the late 1980s, however, the problem of localization has become particularly important in functional studies of the primate motor cortex, because of evidence that (1) the primate motor cortex is made up of a mosaic of functionally specialized areas and (2) the human motor cortex shares several general organizational principles with the monkey motor cortex. Studies of the macaque agranular frontal cortex that used a multimodal cyto-, myelo- and immuno-architectonic approach have shown that architectonic borders can be reliably and consistently defined across different individuals, even at a qualitative level of analysis. The validity of this approach has been confirmed by its ability to localize functionally distinct areas precisely and to predict the existence of new functional areas. After more than a century, architectonics as a discipline goes far beyond its original aim of generating cortical maps.

GABAergic synapses made by a retinal dopaminergic neuron.

In the retina, dopaminergic amacrine (interplexiform) cells establish multiple synapses on the perikarya of AII amacrines, the neurons that distribute rod signals to on- and off-cone bipolars. We used triple-label immunocytochemistry and confocal microscopy to identify the receptors contained within the postsynaptic active zone of these synapses in both mouse and rat retinas. We found that at the interface between the dendrites of the dopaminergic neurons and the AII amacrine cell perikarya clusters of postsynaptic gamma-aminobutyric acid type A (GABA(A)) receptors are situated in register with aggregates of presynaptic organelles immunoreactive for GABA, the GABA vesicular transporter, and the vesicular monoamine transporter-2. D1 and D23 dopamine receptors, on the other hand, do not form clusters on the surface of the perikarya of AII amacrine cells. We suggest that the synapses between retinal dopaminergic neurons and AII amacrine cells are GABAergic and that both GABA and dopamine are released by the presynaptic endings. GABA acts on the ionotropic receptors clustered at the postsynaptic active zone, whereas dopamine diffuses to more distant, slower-acting metabotropic receptors.

Postural control in order to prevent chronic locomotor injuries in top level athletes.

Chronic injuries of the locomotor apparatus represent the main cause of drop-out among top level gymnasts. The aim of the present paper was to verify whether the postural control, investigated by using an integrated approach and accordingly optimized, could be an effective tool for the secondary prevention of training-related disorders of the locomotor apparatus, in a cohort of 20 young female athletes practicing rythmic gymnastic at top level. After a preliminary medical consultation all the subjects underwent a static and dynamic baropodometric test, an ophtalmological and a dental screening. Then athletes were given prescriptions based upon the results of the above named examination. After 6 months, symptoms were completely disappeared in 80% and remarkably improved in 20%, and at baropodometric test, the contact duration as well as the contact surface, the max and mean contact pressure were significantly increased in all the athletes. Our data show that the proposed integrated approach is actually an effective tool for the secondary prevention of training related disorders of the locomotor apparatus.

Effects of physical activity on blood pressure monitoring and morphometric parameters of the left ventricle and of the abdominal aorta in healthy elderly subjects.

To investigate the effects of physical activity not only on morphometric left ventricular parameters, but also on the abdominal aorta diameter and on blood pressure, we enrolled 100 healthy subjects, 50 males and 50 females, aged between 63 and 91 years (mean 73.3 +/- s.d. 7.7), each divided into two subgroups, sedentary and non sedentary, come to our observation for a diagnostic screening. For each subject an Echography was performed, by using an Acuson 128XP10 apparatus, equipped with a 2 MHz phased array probe, both for the study of the heart and for the study of the abdominal aorta. Then, all the subjects underwent 24 hours blood pressure monitoring by using a P6 Delmar apparatus in order to get mean systolic and diastolic blood pressure. Diastolic blood pressure was significantly related to physical activity (sedentary > non sedentary, P < 0.01), while systolic blood pressure was not; left ventricle and abdominal aorta diameters were both significantly related to sex (males > females, P < 0.001), but left ventricle diameter was significantly related to diastolic blood pressure (P < 0.01), while abdominal aorta diameter was significantly related to systolic blood pressure (P < 0.001). Our data suggest that physical activity might be strongly recommended to control blood pressure.

Physical activity, heart and ageing: a morpho-functional study by Echo-Color-Doppler in sedentary and non sedentary healthy subjects.

Several papers have shown that in young people sports activity is associated with a higher prevalence of cardiac valves incompetence, that can be detected, though to a lesser extent, even in healthy subjects randomly selected from the population. Aim of the present study was to analyse the effects of physical activity not only in young subjects but even in the elderly, with particular reference to valve competence, by using the Echo-Color-Doppler. The study cohort was represented by 80 healthy young subjects, 40 men and 40 women, aged between 20 and 25 years, each group subdivided into two subgroups, sedentary and non sedentary, and 80 healthy elderly subjects, 40 men and 40 women, aged between 65 and 91 years, again divided into sedentary and non sedentary. Valve incompetence was more frequent in the elderly if compared to young subjects (P<0.001) and in non sedentary if compared to sedentary subjects (P<0.01), while no significant difference was found between males and females. Worth of interest the fact that in young subjects valve incompetence was more frequent in non sedentary if compared to sedentary subjects (P<0.001), while in the elderly no significant difference was found between sedentary and non sedentary subjects. This original datum may be explained both by the natural higher prevalence of valve incompetence in the elderly, and by the kind of physical activity usually performed by the elderly, i.e. endurance activity.

Gene discovery in genetically labeled single dopaminergic neurons of the retina.

In the retina, dopamine plays a central role in neural adaptation to light. Progress in the study of dopaminergic amacrine (DA) cells has been limited because they are very few (450 in each mouse retina, 0.005% of retinal neurons). Here, we applied transgenic technology, single-cell global mRNA amplification, and cDNA microarray screening to identify transcripts present in DA cells. To profile gene expression in single neurons, we developed a method (SMART7) that combines a PCR-based initial step (switching mechanism at the 5' end of the RNA transcript or SMART) with T7 RNA polymerase amplification. Single-cell targets were synthesized from genetically labeled DA cells to screen the RIKEN 19k mouse cDNA microarrays. Seven hundred ninety-five transcripts were identified in DA cells at a high level of confidence, and expression of the most interesting genes was confirmed by immunocytochemistry. Twenty-one previously undescribed proteins were found in DA cells, including a chloride channel, receptors and other membrane glycoproteins, kinases, transcription factors, and secreted neuroactive molecules. Thirty-eight percent of transcripts were ESTs or coding for hypothetical proteins, suggesting that a large portion of the DA cell proteome is still uncharacterized. Because cryptochrome-1 mRNA was found in DA cells, immunocytochemistry was extended to other components of the circadian clock machinery. This analysis showed that DA cells contain the most common clock-related proteins.