New findings about neuropathological outcomes in the PKU mouse throughout lifespan.

Phenylketonuria (PKU, OMIM 261600) is a genetic disorder caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). If left untreated, PKU leads to systemic phenylalanine (Phe) accumulation, which can result in irreversible brain damage and intellectual disabilities. In the last 60 years, early and strict dietary restriction of phenylalanine (Phe) intake proved to prevent the severe clinical phenotype of untreated PKU. While the specific mechanisms through which phenylalanine causes brain damage are still poorly understood, preclinical models have been deeply explored to characterize the neurotoxic effect of Phe on neurodevelopmental processes. At the same time, that on the aging brain still needs to be explored. In the brain of untreated PAHEnu2(-/-) mouse, we previously reported a reduction of myelin basic protein (MBP) during postnatal development up to 60 PND. Later in the diseased mouse's life, a spontaneous and persistent restoration of MBP was detected. In this present longitudinal study, ranging from 14 to 540 post-natal days (PND) of untreated PAHEnu2(-/-) mice, we further investigated: a) the long-life consistency of two Phe-related brain metabolic alterations, such as large neutral amino acids (LNAA) and biogenic amine neurotransmitters' depletion; b) the outcome of locomotor functions during the same life span; c) the integrity of myelin as assessed ex vivo by central (hippocampus) and peripheral (extensor digitorum longus-sciatic nerve) action potential conduction velocities. In contrast with the results of other studies, brain Leu, Ile, and Val concentrations were not significantly altered in the brain PAHEnu2(-/-) mouse. On the other hand, 3-O-Methyldopa (3-OMD, a biomarker of L-DOPA), serotonin, and its associated metabolites were reduced throughout most of the considered time points, with consistent reductions observed prevalently from 14 to 60 PND. Normal saltatory conduction was restored after 60 PND and remained normal at the last examination at 360 PND, resulting nonetheless in a persistent locomotor impairment throughout a lifetime. These new findings contribute to laying the foundations for the preclinical characterization of aging in PKU, confirming neurotransmitter defects as consistent metabolic traits. LNAAs have a minor role, if any, in brain damage pathogenesis. Transient myelin synthesis failure may impact brain connectivity during postnatal development but not nervous signal conduction.

5HT1AR-FGFR1 Heteroreceptor Complexes Differently Modulate GIRK Currents in the Dorsal Hippocampus and the Dorsal Raphe Serotonin Nucleus of Control Rats and of a Genetic Rat Model of Depression.

The midbrain raphe serotonin (5HT) neurons provide the main ascending serotonergic projection to the forebrain, including hippocampus, which has a role in the pathophysiology of depressive disorder. Serotonin 5HT1A receptor (R) activation at the soma-dendritic level of serotonergic raphe neurons and glutamatergic hippocampal pyramidal neurons leads to a decrease in neuronal firing by activation of G protein-coupled inwardly-rectifying potassium (GIRK) channels. In this raphe-hippocampal serotonin neuron system, the existence of 5HT1AR-FGFR1 heteroreceptor complexes has been proven, but the functional receptor-receptor interactions in the heterocomplexes have only been investigated in CA1 pyramidal neurons of control Sprague Dawley (SD) rats. In the current study, considering the impact of the receptor interplay in developing new antidepressant drugs, the effects of 5HT1AR-FGFR1 complex activation were investigated in hippocampal pyramidal neurons and in midbrain dorsal raphe serotonergic neurons of SD rats and of a genetic rat model of depression (the Flinders Sensitive Line (FSL) rats of SD origin) using an electrophysiological approach. The results showed that in the raphe-hippocampal 5HT system of SD rats, 5HT1AR-FGFR1 heteroreceptor activation by specific agonists reduced the ability of the 5HT1AR protomer to open the GIRK channels through the allosteric inhibitory interplay produced by the activation of the FGFR1 protomer, leading to increased neuronal firing. On the contrary, in FSL rats, FGFR1 agonist-induced inhibitory allosteric action at the 5HT1AR protomer was not able to induce this effect on GIRK channels, except in CA2 neurons where we demonstrated that the functional receptor-receptor interaction is needed for producing the effect on GIRK. In keeping with this evidence, hippocampal plasticity, evaluated as long-term potentiation induction ability in the CA1 field, was impaired by 5HT1AR activation both in SD and in FSL rats, which did not develop after combined 5HT1AR-FGFR1 heterocomplex activation in SD rats. It is therefore proposed that in the genetic FSL model of depression, there is a significant reduction in the allosteric inhibition exerted by the FGFR1 protomer on the 5HT1A protomer-mediated opening of the GIRK channels in the 5HT1AR-FGFR1 heterocomplex located in the raphe-hippocampal serotonin system. This may result in an enhanced inhibition of the dorsal raphe 5HT nerve cell and glutamatergic hippocampal CA1 pyramidal nerve cell firing, which we propose may have a role in depression.

Short-Term, Voluntary Exercise Affects Morpho-Functional Maturation of Adult-Generated Neurons in Rat Hippocampus.

Physical exercise is a well-proven neurogenic stimulus, promoting neuronal progenitor proliferation and affecting newborn cell survival. Besides, it has beneficial effects on brain health and cognition. Previously, we found that three days of physical activity in a very precocious period of adult-generated granule cell life is able to antedate the appearance of the first GABAergic synaptic contacts and increase T-type Ca2+ channel expression. Considering the role of GABA and Ca2+ in fostering neuronal maturation, in this study, we used short-term, voluntary exercise on a running wheel to investigate if it is able to induce long-term morphological and synaptic changes in newborn neurons. Using adult male rats, we found that: (i) three days of voluntary physical exercise can definitively influence the morpho-functional maturation process of newborn granule neurons when applied very early during their development; (ii) a significant percentage of new neurons show more mature morphological characteristics far from the end of exercise protocol; (iii) the long-term morphological effects result in enhanced synaptic plasticity. Present findings demonstrate that the morpho-functional changes induced by exercise on very immature adult-generated neurons are permanent, affecting the neuron maturation and integration in hippocampal circuitry. Our data contribute to underpinning the beneficial potential of physical activity on brain health, also performed for short times.

Synthesis and biological characterization of a new fluorescent probe for vesicular trafficking based on polyazamacrocycle derivative.

The fluorescent probes represent an important tool in the biological study, in fact characterization of cellular structures and organelles are an important tool-target for understanding the mechanisms regulating most biological processes. Recently, a series of polyamino-macrocycles based on 1,4,7,10-tetraazacyclododecane was synthesized, bearing one or two NBD units (AJ2NBD·4HCl) useful as sensors for metal cations and halides able to target and to detect apolar environment, as lipid membranes. In this paper, we firstly illustrate the chemical synthesis of the AJ2NBD probe, its electronic absorption spectra and its behavior regarding pH of the environment. Lack of any cellular toxicity and an efficient labelling on fresh, living cells was demonstrated, allowing the use of AJ2NBD in biological studies. In particular, this green fluorescent probe may represent a potential dye for the compartments involved in the endosomal/autophagic pathway. This research's field should benefit from the use of AJ2NBD as a vesicular tracer, however, to ensure the precise nature of vesicles/vacuoles traced by this new probe, other more specific tests are needed.

Melatonin reshapes the mitochondrial network and promotes intercellular mitochondrial transfer via tunneling nanotubes after ischemic-like injury in hippocampal HT22 cells.

Mitochondrial dysfunction is considered one of the hallmarks of ischemia/reperfusion injury. Mitochondria are plastic organelles that undergo continuous biogenesis, fusion, and fission. They can be transferred between cells through tunneling nanotubes (TNTs), dynamic structures that allow the exchange of proteins, soluble molecules, and organelles. Maintaining mitochondrial dynamics is crucial to cell function and survival. The present study aimed to assess the effects of melatonin on mitochondrial dynamics, TNT formation, and mitochondria transfer in HT22 cells exposed to oxygen/glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin treatment during the reoxygenation phase reduced mitochondrial reactive oxygen species (ROS) production, improved cell viability, and increased the expression of PGC1α and SIRT3. Melatonin also preserved the expression of the membrane translocase proteins TOM20 and TIM23, and of the matrix protein HSP60, which are involved in mitochondrial biogenesis. Moreover, it promoted mitochondrial fusion and enhanced the expression of MFN2 and OPA1. Remarkably, melatonin also fostered mitochondrial transfer between injured HT22 cells through TNT connections. These results provide new insights into the effect of melatonin on mitochondrial network reshaping and cell survival. Fostering TNTs formation represents a novel mechanism mediating the protective effect of melatonin in ischemia/reperfusion injury.

The Role of Central Serotonin Neurons and 5-HT Heteroreceptor Complexes in the Pathophysiology of Depression: A Historical Perspective and Future Prospects.

Serotonin communication operates mainly in the extracellular space and cerebrospinal fluid (CSF), using volume transmission with serotonin moving from source to target cells (neurons and astroglia) via energy gradients, leading to the diffusion and convection (flow) of serotonin. One emerging concept in depression is that disturbances in the integrative allosteric receptor-receptor interactions in highly vulnerable 5-HT1A heteroreceptor complexes can contribute to causing major depression and become novel targets for the treatment of major depression (MD) and anxiety. For instance, a disruption and/or dysfunction in the 5-HT1A-FGFR1 heteroreceptor complexes in the raphe-hippocampal serotonin neuron systems can contribute to the development of MD. It leads inter alia to reduced neuroplasticity and potential atrophy in the raphe-cortical and raphe-striatal 5-HT pathways and in all its forebrain networks. Reduced 5-HT1A auto-receptor function, increased plasticity and trophic activity in the midbrain raphe 5-HT neurons can develop via agonist activation of allosteric receptor-receptor interactions in the 5-HT1A-FGFR1 heterocomplex. Additionally, the inhibitory allosteric receptor-receptor interactions in the 5-HT1AR-5-HT2AR isoreceptor complex therefore likely have a significant role in modulating mood, involving a reduction of postjunctional 5-HT1AR protomer signaling in the forebrain upon activation of the 5-HT2AR protomer. In addition, oxytocin receptors (OXTRs) play a significant and impressive role in modulating social and cognitive related behaviors like bonding and attachment, reward and motivation. Pathological blunting of the OXTR protomers in 5-HT2AR and especially in 5-HT2CR heteroreceptor complexes can contribute to the development of depression and other types of psychiatric diseases involving disturbances in social behaviors. The 5-HTR heterocomplexes are novel targets for the treatment of MD.

GABAergic Input Affects Intracellular Calcium Levels in Developing Granule Cells of Adult Rat Hippocampus.

In the dentate gyrus (DG) of the mammalian hippocampus, granule neurons are generated from neural stem cells (NSCs) throughout the life span and are integrated into the hippocampal network. Adult DG neurogenesis is regulated by multiple intrinsic and extrinsic factors that control NSC proliferation, maintenance, and differentiation into mature neurons. γ-Aminobutyric acid (GABA), released by local interneurons, regulates the development of neurons born in adulthood by activating extrasynaptic and synaptic GABAA receptors. In the present work, patch-clamp and calcium imaging techniques were used to record very immature granule cells of adult rat dentate gyrus for investigating the actual role of GABAA receptor activation in intracellular calcium level regulation at an early stage of maturation. Our findings highlight a novel molecular and electrophysiological mechanism, involving calcium-activated potassium channels (BK) and T-type voltage-dependent calcium channels, through which GABA fine-tunes intracellular calcium homeostasis in rat adult-born granule neurons early during their maturation. This mechanism might be instrumental in promoting newborn cell survival.

Calsequestrin Deletion Facilitates Hippocampal Synaptic Plasticity and Spatial Learning in Post-Natal Development.

: Experimental evidence highlights the involvement of the endoplasmic reticulum (ER)-mediated Ca2+ signals in modulating synaptic plasticity and spatial memory formation in the hippocampus. Ca2+ release from the ER mainly occurs through two classes of Ca2+ channels, inositol 1,4,5-trisphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs). Calsequestrin (CASQ) and calreticulin (CR) are the most abundant Ca2+-binding proteins allowing ER Ca2+ storage. The hippocampus is one of the brain regions expressing CASQ, but its role in neuronal activity, plasticity, and the learning processes is poorly investigated. Here, we used knockout mice lacking both CASQ type-1 and type-2 isoforms (double (d)CASQ-null mice) to: a) evaluate in adulthood the neuronal electrophysiological properties and synaptic plasticity in the hippocampal Cornu Ammonis 1 (CA1) field and b) study the performance of knockout mice in spatial learning tasks. The ablation of CASQ increased the CA1 neuron excitability and improved the long-term potentiation (LTP) maintenance. Consistently, (d)CASQ-null mice performed significantly better than controls in the Morris Water Maze task, needing a shorter time to develop a spatial preference for the goal. The Ca2+ handling analysis in CA1 pyramidal cells showed a decrement of Ca2+ transient amplitude in (d)CASQ-null mouse neurons, which is consistent with a decrease in afterhyperpolarization improving LTP. Altogether, our findings suggest that CASQ deletion affects activity-dependent ER Ca2+ release, thus facilitating synaptic plasticity and spatial learning in post-natal development.

Muscle activity prevents the uncoupling of mitochondria from Ca2+ Release Units induced by ageing and disuse.

In fast-twitch fibers from adult mice Ca2+ release units (CRUs, i.e. intracellular junctions of excitation-contraction coupling), and mitochondria are structurally linked to each other by small strands, named tethers. We recently showed that aging causes separation of a fraction of mitochondria from CRUs and a consequent impairment of the Ca2+ signaling between the two organelles. However, whether the uncoupling of mitochondria from CRUs is the result of aging per-se or the consequence of reduced muscle activity remains still unclear. Here we studied the association between mitochondria and CRUs: in a) extensor digitorum longus (EDL) muscles from 2 years old mice, either sedentary or trained for 1 year in wheel cages; and b) denervated EDL muscles from adult mice and rats. We analyzed muscle samples using a combination of structural (confocal and electron microscopy), biochemical (assessment of oxidative stress via western blot), and functional (ex-vivo contractile properties, and mitochondrial Ca2+ uptake) experimental procedures. The results collected in structural studies indicate that: a) ageing and denervation result in partial uncoupling between mitochondria and CRUs; b) exercise either maintains (in old mice) or restores (in transiently denervated rats) the association between the two organelles. Functional studies supported the hypothesis that CRU-mitochondria coupling is important for mitochondrial Ca2+ uptake, optimal force generation, and muscle performance. Taken together our results indicate that muscle activity maintains/improves proper association between CRUs and mitochondria.

Receptor⁻Receptor Interactions in Multiple 5-HT1A Heteroreceptor Complexes in Raphe-Hippocampal 5-HT Transmission and Their Relevance for Depression and Its Treatment.

Due to the binding to a number of proteins to the receptor protomers in receptor heteromers in the brain, the term "heteroreceptor complexes" was introduced. A number of serotonin 5-HT1A heteroreceptor complexes were recently found to be linked to the ascending 5-HT pathways known to have a significant role in depression. The 5-HT1A⁻FGFR1 heteroreceptor complexes were involved in synergistically enhancing neuroplasticity in the hippocampus and in the dorsal raphe 5-HT nerve cells. The 5-HT1A protomer significantly increased FGFR1 protomer signaling in wild-type rats. Disturbances in the 5-HT1A⁻FGFR1 heteroreceptor complexes in the raphe-hippocampal 5-HT system were found in a genetic rat model of depression (Flinders sensitive line (FSL) rats). Deficits in FSL rats were observed in the ability of combined FGFR1 and 5-HT1A agonist cotreatment to produce antidepressant-like effects. It may in part reflect a failure of FGFR1 treatment to uncouple the 5-HT1A postjunctional receptors and autoreceptors from the hippocampal and dorsal raphe GIRK channels, respectively. This may result in maintained inhibition of hippocampal pyramidal nerve cell and dorsal raphe 5-HT nerve cell firing. Also, 5-HT1A⁻5-HT2A isoreceptor complexes were recently demonstrated to exist in the hippocampus and limbic cortex. They may play a role in depression through an ability of 5-HT2A protomer signaling to inhibit the 5-HT1A protomer recognition and signaling. Finally, galanin (1⁻15) was reported to enhance the antidepressant effects of fluoxetine through the putative formation of GalR1⁻GalR2⁻5-HT1A heteroreceptor complexes. Taken together, these novel 5-HT1A receptor complexes offer new targets for treatment of depression.

New insights into the trophic and cytoprotective effects of creatine in in vitro and in vivo models of cell maturation.

A growing body of scientific reports indicates that the role of creatine (Cr) in cellular biochemistry and physiology goes beyond its contribution to cell energy. Indeed Cr has been shown to exert multiple effects promoting a wide range of physiological responses in vitro as well as in vivo. Included in these, Cr promotes in vitro neuron and muscle cell differentiation, viability and survival under normal or adverse conditions; anabolic, protective and pro-differentiative effects have also been observed in vivo. For example Cr has been shown to accelerate in vitro differentiation of cultured C2C12 myoblasts into myotubes, where it also induces a slight but significant hypertrophic effect as compared to unsupplemented cultures; Cr also prevents the anti-differentiation effects caused by oxidative stress in the same cells. In trained adults, Cr increases the mRNA expression of relevant myogemic factors, protein synthesis, muscle strength and size, in cooperation with physical exercise. As to neurons and central nervous system, Cr favors the electrophysiological maturation of chick neuroblasts in vitro and protects them from oxidative stress-caused killing; similarly, Cr promotes the survival and differentiation of GABA-ergic neurons in fetal spinal cord cultures in vitro; in vivo, maternal Cr supplementation promotes the morpho-functional development of hippocampal neurons in rat offsprings. This article, which presents also some new experimental data, focuses on the trophic, pro-survival and pro-differentiation effects of Cr and examines the ensuing preventive and therapeutic potential in pathological muscle and brain conditions.

α-Tocopherol and Hippocampal Neural Plasticity in Physiological and Pathological Conditions.

Neuroplasticity is an "umbrella term" referring to the complex, multifaceted physiological processes that mediate the ongoing structural and functional modifications occurring, at various time- and size-scales, in the ever-changing immature and adult brain, and that represent the basis for fundamental neurocognitive behavioral functions; in addition, maladaptive neuroplasticity plays a role in the pathophysiology of neuropsychiatric dysfunctions. Experiential cues and several endogenous and exogenous factors can regulate neuroplasticity; among these, vitamin E, and in particular α-tocopherol (α-T), the isoform with highest bioactivity, exerts potent effects on many plasticity-related events in both the physiological and pathological brain. In this review, the role of vitamin E/α-T in regulating diverse aspects of neuroplasticity is analyzed and discussed, focusing on the hippocampus, a brain structure that remains highly plastic throughout the lifespan and is involved in cognitive functions. Vitamin E-mediated influences on hippocampal synaptic plasticity and related cognitive behavior, on post-natal development and adult hippocampal neurogenesis, as well as on cellular and molecular disruptions in kainate-induced temporal seizures are described. Besides underscoring the relevance of its antioxidant properties, non-antioxidant functions of vitamin E/α-T, mainly involving regulation of cell signaling molecules and their target proteins, have been highlighted to help interpret the possible mechanisms underlying the effects on neuroplasticity.

Developing and testing an Arduino-based microcurrent stimulator to mimic marine electric pollution on benthos.

The lack of economic funds commonly represents a limiting factor in scientific research and prevents scientists from developing brilliant ideas. Indeed, a new project may involve using appropriate scientific instruments and concurrently dealing with the costs before pursuing new research fields. The innovative concept of investigating the effects of electric fields, as a simulation of marine electrical pollution, on benthic organisms such as foraminifera (marine protozoa) has been recently explored by our research group. This pioneering research has resulted in the development of a cost-effective instrument capable of generating customized electric stimulation patterns with accuracy and reliability. Here, we describe the construction of a low-intensity electrical stimulator based on an Arduino programmable board and a few electronic components. The instrument results very stable and precise regarding the stimulation times and the regulation of the current intensity applied to the biological preparation. Moreover, the setup can stimulate the preparation in constant or pulsed direct current. This homemade stimulation apparatus can be improved or modified according to the researchers' needs, as possibilities and fields of application can be innumerable.

Melatonin Modulates Cell Cycle Dynamics and Promotes Hippocampal Cell Proliferation After Ischemic Injury in Neonatal Rats.

Promoting neural cell proliferation may represent an important strategy for enhancing brain repair after developmental brain injury. The present study aimed to assess the effects of melatonin on cell proliferation after an ischemic injury in the developing hippocampus, focusing on cell cycle dynamics. After in vivo neonatal hypoxia-ischemia (HI), hippocampal cell cycle dynamics were assessed by flow cytometry, together with histological evaluation of dentate gyrus cellularity and proliferation. Melatonin significantly increased the number of proliferating cells in the G2/M phase as well as the proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) labeling reduced by HI. In vivo BrdU labeling revealed a higher BrdU-positivity in the dentate gyrus of ischemic rats treated with melatonin, an effect followed by increased cellularity and preserved hippocampal tissue integrity. These results indicate that the protective effect of melatonin after ischemic injury in neonatal rats may rely on the modulation of cell cycle dynamics of newborn hippocampal cells and increased cell proliferation.

Understanding electrical and chemical transmission in the brain.

The histochemical Falck-Hillarp method for the localization of dopamine (DA), noradrenaline (NA) and serotonin in the central nervous system (CNS) of rodents was introduced in the 1960s. It supported the existence of chemical neurotransmission in the CNS. The monoamine neurons in the lower brain stem formed monosynaptic ascending systems to the telencephalon and diencephalon and monoamine descending systems to the entire spinal cord. The monoamines were early on suggested to operate via synaptic chemical transmission in the CNS. This chemical transmission reduced the impact of electrical transmission. In 1969 and the 1970s indications were obtained that important modes of chemical monoamine communication in the CNS also took place through the extra-synaptic fluid, the extracellular fluid, and long-distance communication in the cerebrospinal fluid involving diffusion and flow of transmitters like DA, NA and serotonin. In 1986, this type of transmission was named volume transmission (VT) by Agnati and Fuxe and their colleagues, also characterized by transmitter varicosity and receptor mismatches. The short and long-distance VT pathways were characterized by volume fraction, tortuosity and clearance. Electrical transmission also exists in the mammalian CNS, but chemical transmission is in dominance. One electrical mode is represented by electrical synapses formed by gap junctions which represent low resistant passages between nerve cells. It allows for a more rapid passage of action potentials between nerve cells compared to chemical transmission. The second mode is based on the ability of synaptic currents to generate electrical fields to modulate chemical transmission. One aim is to understand how chemical transmission can be integrated with electrical transmission and how putative (aquaporin water channel, dopamine D2R and adenosine A2AR) complexes in astrocytes can significancy participate in the clearance of waste products from the glymphatic system. VT may also help accomplish the operation of the acupuncture meridians essential for Chinese medicine in view of the indicated existence of extracellular VT pathways.

Motor activity affects adult skeletal muscle re-innervation acting via tyrosine kinase receptors.

Recently, muscle expression of brain-derived neurotrophic factor (BDNF) mRNA and protein under activity control has been reported. BDNF is a neurotrophin known to be involved in axon sprouting in the CNS. Hence, we set out to study the effect of chronic treadmill mid-intensity running on adult rat muscle re-innervation, and to explore the involvement of BDNF and tropomyosin-related kinase (Trk) receptors. After nerve crush, muscle re-innervation was evaluated using intracellular recordings, tension recordings, immunostaining and Western blot analyses. An enhanced muscle multiple innervation was found in running rats that was fully reversed to control values blocking Trk receptors or interrupting the running activity. An increase in muscle multiple innervation was also found in sedentary rats treated with a selective TrkB receptor agonist. The expression of TrkB receptors by intramuscular axons was demonstrated, and increased muscle expression of BDNF was found in running animals. The increase in muscle multiple innervation was consistent with the faster muscle re-innervation that we found in running animals. We conclude that, when regenerating axons contact muscle cells, muscle activity progressively increases modulating BDNF and possibly other growth factors, which in turn, acting via Trk receptors, induce axon sprouting to re-innervate skeletal muscle.

Pharmacological doses of melatonin induce alterations in mitochondrial mass and potential, bcl-2 levels and K+ currents in UVB-exposed U937 cells.

Apoptosis is observed in 'actively' dying cells after the exposure to cell stressors such as ultraviolet light irradiation. Since melatonin has been proposed to act under stressful conditions as cell protection factor, in this study we examined the potential of this molecule when used at pharmacological concentrations to control mitochondrial damage and apoptotic signalling of UVB irradiated U937 human leukaemic cells. Moreover, the effect of melatonin treatment on electrophysiological properties and membrane K(+) currents of irradiated U937 cells was investigated as functional aspects relevant to the anti-apoptotic role of melatonin. The general effect is associated with the restoration of mass, number and membrane potential of mitochondria, with a lower caspase activation and bcl-2 upregulation. In the presence of the caspase inhibitor ZVAD-Fmk, melatonin seems to drive UVB stressed cells to follow the mitochondrial intrinsic pathway, interfering just at the mitochondrial level. Moreover, treatment with melatonin, as well as ZVAD-Fmk, prevented the K(+) current reduction observed late following the UVB insult application, by sparing cells from death; this result also indicates that the decrease of K(+) leakage currents could represent a functional feature of apoptotic process in UV-exposed U937 cells.

Evaluation of the Effects of Electrical Stimulation: A Pilot Experiment on the Marine Benthic Foraminiferal Species Amphistegina lessonii.

Environmental disturbances resulting from anthropogenic energy pollution are intensely growing and represent a concern for the marine environment. Benthic organisms are the significant fauna exposed to this kind of pollution; among them, foraminifera are largely used as pollution bioindicators in marine environments, but studies on the effects induced by electrical stimulation are not documented. In the present research, we evaluated the effects of short-term different electric current densities on the viability of benthic foraminiferal species Amphistegina lessonii by checking the pseudopodial activity and defined the threshold electrical density range. After 3 days of treatment, A. lessonii stimulated with a constant current showed pseudopodial activity at a lower electric current density (0.29, 0.86 µA/cm2) up to 24 h. With increasing stimulation time, the percentages of pseudopodial activity decreased. The pseudopodial activity was absent at high current densities (5.71, 8.57 µA/cm2). The viability of A. lessonii exposed to a pulsed current was higher at a low and middle electric current density (from 0.29 to 5.71 µA/cm2) than at a high electric current density (from 11.43 to 20 µA/cm2). Based on these preliminary results, the selected benthic foraminiferal species seems to better stand pulsed currents than constant ones. These first experiments might provide useful information for the definition of the appropriate electrical density threshold to avoid side effects on a part of the benthic community.

Melatonin Attenuates Ischemic-like Cell Injury by Promoting Autophagosome Maturation via the Sirt1/FoxO1/Rab7 Axis in Hippocampal HT22 Cells and in Organotypic Cultures.

Dysfunctional autophagy is linked to neuronal damage in ischemia/reperfusion injury. The Ras-related protein 7 (Rab7), a member of the Rab family of small GTPases, appears crucial for the progression of the autophagic flux, and its activity is strictly interconnected with the histone deacetylase Silent information regulator 1 (Sirt1) and transcription factor Forkhead box class O1 (FoxO1). The present study assessed the neuroprotective role of melatonin in the modulation of the Sirt1/FoxO1/Rab7 axis in HT22 cells and organotypic hippocampal cultures exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin re-established physiological levels of autophagy and reduced propidium iodide-positive cells, speeding up autophagosome (AP) maturation and increasing lysosomal activity. Our study revealed that melatonin modulates autophagic pathways, increasing the expression of both Rab7 and FoxO1 and restoring the Sirt1 expression affected by OGD/R. In addition, the Sirt1 inhibitor EX-527 significantly reduced Rab7, Sirt1, and FoxO1 expression, as well as autolysosomes formation, and blocked the neuroprotective effect of melatonin. Overall, our findings provide, for the first time, new insights into the neuroprotective role of melatonin against ischemic injury through the activation of the Sirt1/FoxO1/Rab7 axis.

The impact of different exercise protocols on rat soleus muscle reinnervation and recovery following peripheral nerve lesion and regeneration.

Background: Incomplete functional recovery following traumatic peripheral nerve injury is common, mainly because not all axons successfully regenerate and reinnervate target muscles. Exercise can improve functional outcomes increasing the terminal sprouting during the muscle reinnervation. However, exercise is not a panacea per se. Indeed, the type of exercise adopted dramatically impacts the outcomes of rehabilitation therapy. To gain insight into the therapeutic effects of different exercise regimens on reinnervation following traumatic nerve lesion, we evaluated the impact of different clinically transferable exercise protocols (EPs) on metabolic and functional muscle recovery following nerve crush. Methods: The reinnervation of soleus muscle in adult nerve-crushed rats was studied following 6 days of different patterns (continuous or intermittent) and intensities (slow, mid, and fast) of treadmill running EPs. The effects of EPs on muscle fiber multiple innervation, contractile properties, metabolic adaptations, atrophy, and autophagy were assessed using functional and biochemical approaches. Results: Results showed that an intermittent mid-intensity treadmill EP improves soleus muscle reinnervation, whereas a slow continuous running EP worsens the functional outcome. However, the mid-intensity intermittent EP neither enhanced the critical mediators of exercise-induced metabolic adaptations, namely, PGC-1α, nor improved muscle atrophy. Conversely, the autophagy-related marker LC3 increased exclusively in the mid-intensity intermittent EP group. Conclusion: Our results demonstrated that an EP characterized by a mid-intensity intermittent activity enhances the functional muscle recovery upon a nerve crush, thus representing a promising clinically transferable exercise paradigm to improve recovery in humans following peripheral nerve injuries.