Search

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.

In this study, we adapted a race-Implicit Association Test (race-IAT) to mouse-tracking (MT) technique to identify the more representative target observed MT-metrics and explore the temporal unfolding of the cognitive conflict emerging during the categorisation task. Participants of Western European descent performed a standard keyboard-response race-IAT (RT-race-IAT) and an MT-race-IAT with the same structure. From a behavioural point of view, our sample showed a typical Congruency Effect, thus a pro-White implicit bias, in the RT-race-IAT. In addition, in the MT-race-IAT, the MT-metrics showed a similar Congruency Effect mirroring the higher attraction of the averaged-trajectories towards the incorrect response button in incongruent than congruent trials. Moreover, these MT-metrics were positively associated with RT-race-IAT scores, strengthening the MT approach’s validity in characterising the implicit bias. Furthermore, the distributional analyses showed that mouse trajectories displayed a smooth profile both in congruent and incongruent trials to indicate that the unfolding of the decision process and the raised conflict is guided by dynamical cognitive processing. This latter continuous competition process was studied using a novel phase-based approach which allowed to temporally dissect an Early, a Mid and a Late phase, each of which may differently reflect the decision conflict between automatic and controlled responses in the evolution of the mouse movement towards the target response. Our results show that the MT approach provides an accurate and finer-grained characterisation of the implicit racial attitude than classical RT-IAT. Finally, our novel phase-based approach can be an effective tool to shed light on the implicit conflict processing emerging in a categorisation task with a promising transferable value in different cognitive and neuropsychological fields.

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.

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.

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.

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.

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.

Adenosine A2A receptors (A2AR) are crucial in facilitating the BDNF action on synaptic transmission in the rat hippocampus primarily upon ageing. Furthermore, it has been suggested that A2AR-Tropomyosin related kinase B receptor (TrkB) crosstalk has a pivotal role in adenosine A2AR-mediated modulation of the BDNF action on hippocampal plasticity. Considering the impact of the above receptors interplay on what concerns BDNF-induced enhancement of synaptic transmission, gaining a better insight into the mechanisms behind this powerful crosstalk becomes of primary interest. Using in situ proximity ligation assay (PLA), the existence of a direct physical interaction between adenosine A2AR and TrkB is demonstrated. The A2AR-TrkB heteroreceptor complexes show a heterogeneous distribution within the rat dorsal hippocampus. High densities of the heteroreceptor complexes were observed in the pyramidal cell layers of CA1-CA3 regions and in the polymorphic layer of the dentate gyrus (DG). The stratum radiatum of the CA1-3 regions showed positive PLA signal in contrast to the oriens region. The molecular and granular layers of the DG also lacked significant densities of PLA positive heteroreceptor complexes, but subgranular zone showed some PLA positive cells. Their allosteric receptor-receptor interactions may significantly modulate BDNF signaling impacting on hippocampal plasticity which is impaired upon ageing.

The heteroreceptor complexes present a novel biological principle for signal integration. These complexes and their allosteric receptor–receptor interactions are bidirectional and novel targets for treatment of CNS diseases including mental diseases. The existence of D2R-5-HT2AR heterocomplexes can help explain the anti-schizophrenic effects of atypical antipsychotic drugs not only based on blockade of 5-HT2AR and of D2R in higher doses but also based on blocking the allosteric enhancement of D2R protomer signaling by 5-HT2AR protomer activation. This research opens a new understanding of the integration of DA and 5-HT signals released from DA and 5-HT nerve terminal networks. The biological principle of forming 5-HT and other heteroreceptor complexes in the brain also help understand the mechanism of action for especially the 5-HT hallucinogens, including putative positive effects of e.g., psilocybin and the indicated prosocial and anti-stress actions of MDMA (ecstasy). The GalR1-GalR2 heterodimer and the putative GalR1-GalR2-5-HT1 heteroreceptor complexes are targets for Galanin N-terminal fragment Gal (1–15), a major modulator of emotional networks in models of mental disease. GPCR-receptor tyrosine kinase (RTK) heteroreceptor complexes can operate through transactivation of FGFR1 via allosteric mechanisms and indirect interactions over GPCR intracellular pathways involving protein kinase Src which produces tyrosine phosphorylation of the RTK. The exciting discovery was made that several antidepressant drugs such as TCAs and SSRIs as well as the fast-acting antidepressant drug ketamine can directly bind to the TrkB receptor and provide a novel mechanism for their antidepressant actions. Understanding the role of astrocytes and their allosteric receptor–receptor interactions in modulating forebrain glutamate synapses with impact on dorsal raphe-forebrain serotonin neurons is also of high relevance for research on major depressive disorder.

Studies on serotonin-selective reuptake inhibitors have established that disturbances in the ascending 5-HT neuron systems and their 5-HT receptor subtypes and collateral networks to the forebrain contribute to the etiology of major depression and are targets for treatment. The therapeutic action of serotonin-selective reuptake inhibitors is of proven effectiveness, but the mechanisms underlying their effect are still unclear. There are many 5-HT subtypes involved; some need to be blocked (e.g., 5-HT2A, 5-HT3, and 5-HT7), whereas others need to be activated (e.g., postjunctional 5-HT1A and 5-HT4). These state-of-the-art developments are in line with the hypothesis that the development of major depression can involve an imbalance of the activity between different types of 5-HT isoreceptors. In the current study, using in situ proximity ligation assay (PLA), we report evidence for the existence of brain 5-HT1A–5-HT2A isoreceptor complexes validated in cellular models with bioluminescence resonance energy transfer (BRET2) assay. A high density of PLA-positive clusters visualizing 5-HT1A–5-HT2A isoreceptor complexes was demonstrated in the pyramidal cell layer of the CA1–CA3 regions of the dorsal hippocampus. A marked reduction in the density of PLA-positive clusters was observed in the CA1 and CA2 regions 24 h after a forced swim test session, indicating the dynamics of this 5-HT isoreceptor complex. Using a bioinformatic approach, previous work indicates that receptors forming heterodimers demonstrate triplet amino acid homologies. The receptor interface of the 5-HT1A–5-HT2A isoreceptor dimer was shown to contain the LLG and QNA protriplets in the transmembrane and intracellular domain, respectively. The 5-HT2A agonist TCB2 markedly reduced the affinity of the 5-HT1A agonist ipsapirone for the 5-HT1A agonist binding sites in the frontal lobe using the 5-HT1A radioligand binding assay. This action was blocked by the 5-HT2A antagonist ketanserin. It is proposed that the demonstrated 5-HT1A–5-HT2A isoreceptor complexes may play a role in depression through integration of 5-HT recognition, signaling and trafficking in the plasma membrane in two major 5-HT receptor subtypes known to be involved in depression. Antagonistic allosteric receptor–receptor interactions appear to be involved in this integrative process.

Creatine plays a crucial role in developing the brain, so much that its genetic deficiency results in mental dysfunction and cognitive impairments. Moreover, creatine supplementation is currently under investigation as a preventive measure to protect the fetus against oxidative stress during difficult pregnancies. Although creatine use is considered safe, posing minimal risk to clinical health, we found an alteration in morpho-functional maturation of neurons when male rats were exposed to creatine loads during brain development. In particular, increased excitability and enhanced long-term potentiation (LTP) were observed in the hippocampal pyramidal neurons of weaning pups. Since these effects were observed a long time after creatine treatment had been terminated, long-lasting modifications persisting into adulthood were hypothesized. Such modifications were investigated in the present study using morphological, electrophysiological, and calcium imaging techniques applied to hippocampal Cornu Ammonis 1 (CA1) neurons of adult rats born from dams supplemented with creatine. When compared to age-matched controls, the treated adult offspring were found to retain enhanced neuron excitability and an improved LTP, the best-documented neuronal substrate for memory formation. While translating data from rats to humans does have limitations, our findings suggest that prenatal creatine supplementation could have positive effects on adult cognitive abilities.

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.

The adverse effects of engineered nanomaterials (ENM) in marine environments have recently attracted great attention although their effects on marine benthic organisms such as foraminifera are still largely overlooked. Here we document the effects of three negatively charged ENM, different in size and composition, titanium dioxide (TiO2), polystyrene (PS) and silicon dioxide (SiO2), on a microbial eukaryote (the benthic foraminifera Ammonia parkinsoniana) using multiple approaches. This research clearly shows the presence, within the foraminiferal cytoplasm, of metallic (Ti) and organic (PS) ENM that promote physiological stress. Specifically, marked increases in the accumulation of neutral lipids and enhanced reactive oxygen species production occurred in ENM-treated specimens regardless of ENM type. This study indicates that ENM represent ecotoxicological risks for this microbial eukaryote and presents a new model for the neglected marine benthos by which to assess natural exposure scenarios.

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 μmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.

Glutamate-mediated excitotoxicity, neuroinflammation, and oxidative stress are common underlying events in neurodegeneration. This pathogenic "triad" characterizes the neurobiology of epilepsy, leading to seizure-induced cell death, increased susceptibility to neuronal synchronization and network alterations. Along with other maladaptive changes, these events pave the way to spontaneous recurrent seizures and progressive degeneration of the interested brain areas. In vivo models of epilepsy are available to explore such epileptogenic mechanisms, also assessing the efficacy of chemoprevention and therapy strategies at the pre-clinical level. The kainic acid model of pharmacological excitotoxicity and epileptogenesis is one of the most investigated mimicking the chronicization profile of temporal lobe epilepsy in humans. Its pathogenic cues include inflammatory and neuronal death pathway activation, mitochondrial disturbances and lipid peroxidation of several regions of the brain, the most vulnerable being the hippocampus. The importance of neuroinflammation and lipid peroxidation as underlying molecular events of brain damage was demonstrated in this model by the possibility to counteract the related maladaptive morphological and functional changes of this organ with vitamin E, the main fat-soluble cellular antioxidant and "conditional" co-factor of enzymatic pathways involved in polyunsaturated lipid metabolism and inflammatory signaling. The present review paper provides an overview of the literature supporting the potential for a timely intervention with vitamin E therapy in clinical management of seizures and epileptogenic processes associated with excitotoxicity, neuroinflammation and lipid peroxidation, i.e. the pathogenic "triad".

In fast-twitch fibers from adult mice Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) uptake, optimal force generation, and muscle performance. Taken together our results indicate that muscle activity maintains/improves proper association between CRUs and mitochondria.

GPCR’s receptosome operates via coordinated changes between the receptor expression, their modifications and interactions between each other. Perturbation in specific heteroreceptor complexes and/or their balance/equilibrium with other heteroreceptor complexes and corresponding homoreceptor complexes is considered to have a role in pathogenic mechanisms. Such mechanisms lead to mental and neurological diseases, including drug addiction, depression, Parkinson’s disease, and schizophrenia. To understand the associations of GPCRs and to unravel the global picture of their receptor–receptor interactions in the brain, different experimental detection techniques for receptor–receptor interactions have been established (e.g., co-immunoprecipitation based approach). However, they have been criticized for not reflecting the cellular situation or the dynamic nature of receptor–receptor interactions. Therefore, the detection and visualization of GPCR homo- and heteroreceptor complexes in the brain remained largely unknown until recent years, when a well-characterized in situ proximity ligation assay (in situ PLA) was adapted to validate the receptor complexes in their native environment. The in situ PLA protocol presented here can be used to visualize GPCR receptor–receptor interactions in cells and tissues in a highly sensitive and specific manner. We have developed a combined method using immunohistochemistry and PLA, particularly aimed to monitor interactions between GPCRs in specific neuronal cell populations. This allows the analysis of homo- and heteroreceptor complexes at a cellular and subcellular level. The method has the advantage that it can be used in clinical specimens, providing localized, quantifiable homo- and heteroreceptor complexes detected in single cells. We compare the advantages and limitations of the methods, underlining recent progress and the growing importance of these techniques in basic research. We discuss also their potential as tools for drug development and diagnostics.

Seizure-triggered maladaptive neural plasticity and neuroinflammation occur during the latent period as a key underlying event in epilepsy chronicization. Previously, we showed that α-tocopherol (α-T) reduces hippocampal neuroglial activation and neurodegeneration in the rat model of kainic acid (KA)-induced status epilepticus (SE). These findings allowed us to postulate an antiepileptogenic potential for α-T in hippocampal excitotoxicity, in line with clinical evidence showing that α-T improves seizure control in drug-resistant patients. To explore neurobiological correlates of the α-T antiepileptogenic role, rats were injected with such vitamin during the latent period starting right after KA-induced SE, and the effects on circuitry excitability, neuroinflammation, neuronal death, and microRNA (miRNA) expression were investigated in the hippocampus. Results show that in α-T-treated epileptic rats, (1) the number of population spikes elicited by pyramidal neurons, as well as the latency to the onset of epileptiform-like network activity recover to control levels; (2) neuronal death is almost prevented; (3) down-regulation of claudin, a blood-brain barrier protein, is fully reversed; (4) neuroinflammation processes are quenched (as indicated by the decrease of TNF-α, IL-1β, GFAP, IBA-1, and increase of IL-6); (5) miR-146a, miR-124, and miR-126 expression is coherently modulated in hippocampus and serum by α-T. These findings support the potential of a timely intervention with α-T in clinical management of SE to reduce epileptogenesis, thus preventing chronic epilepsy development. In addition, we suggest that the analysis of miRNA levels in serum could provide clinicians with a tool to evaluate disease evolution and the efficacy of α-T therapy in SE.

The potential ability to differentiate dedifferentiated adipocytes into a neural lineage is attracting strong interest as an emerging method of producing model cells for the treatment of a variety of neurological diseases. Here, we describe the efficient conversion of dedifferentiated adipocytes into a neural-like cell population. These cells grew in neurosphere-like structures and expressed a high level of the early neuroectodermal marker Nestin. These neurospheres could proliferate and express stemness genes, suggesting that these cells could be committed to the neural lineage. After neural induction, NeuroD1, Sox1, Double Cortin, and Eno2 were not expressed. Patch clamp data did not reveal different electrophysiological properties, indicating the inability of these cells to differentiate into mature neurons. In contrast, the differentiated cells expressed a high level of CLDN11, as demonstrated using molecular method, and stained positively for the glial cell markers CLDN11 and GFAP, as demonstrated using immunocytochemistry. These data were confirmed by quantitative results for glial cell line-derived neurotrophic factor production, which showed a higher secretion level in neurospheres and the differentiated cells compared with the untreated cells. In conclusion, our data demonstrate morphological, molecular, and immunocytochemical evidence of initial neural differentiation of mature adipocytes, committing to a glial lineage.

Myotendinous junction (MTJ) is the highly specialized complex which connects the skeletal muscle to the tendon for transmitting the contractile force between the two tissues. The purpose of this study was to investigate the MTJ development and rat EDL was chosen as a model. 1, 15, 30 day animals were considered and the junctions were analyzed by light and electron microscopy. The MTJ interface architecture increased during the development, extending the interaction between muscle and tendon. 1-day-old rats showed disorganized myofibril bundles, spread cytosol and incomplete rough endoplasmic reticulum, features partially improved in 15-day-old rats, and completely developed in 30-day-old animals. These findings indicate that muscle-tendon interface displays, during rat lifetime, numerically increased and longer tendon interdigitations, correlated with an improved organization of both tissues and with a progressive acquirement of full functionality.

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.

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.

Myotendinous junctions (MTJs) are anatomical regions specialized in transmission of contractile strength from muscle to tendon and, for this reason, a common site where acute injuries occur during sport activities. In this work we investigated the influence of exercise intensity on MTJ plasticity, as well as on the expression of insulin-like growth factor 1 (IGF-1) and transforming growth factor beta (TGF-β) and their receptors in muscle and tendon. Three groups of rats were analyzed: control (CTRL), slow-runner (RUN-S) and fast-runner (RUN-F) trained using a treadmill. Ultrastructural and morphometric analyses of distal MTJs from extensor digitorum longus muscles have been performed. Contractile strength and hypertrophy were investigated by using in vivo tension recordings and muscle cross-sectional area (CSA) analysis, respectively. mRNA levels of PGC-1α, vinculin, IGF-1Ea and TGF-β have been quantified in muscle belly, while IGF-1Ea, TGF-β and their receptors in tendon. Morphometry revealed an increased MTJ complexity and interaction surface between tissues in trained rats according to training intensity. CSA analysis excluded hypertrophy among groups, while muscle strength was found significantly enhanced in exercised rats in comparison to controls. In muscle tissue, we highlighted an increased mRNA expression of PGC-1α and vinculin in both trained conditions and of TGF-β in RUN-F. In tendon, we mainly noted an enhancement of TGF-β mRNA expression only in RUN-F group and a raise of Betaglycan tendon receptor mRNA levels proportional to exercise intensity. In conclusion, MTJ plasticity appears to be related to exercise intensity and molecular analysis suggests a major role played by TGF-β.

Creatine supplementation has been shown to protect neurons from oxidative damage due to its antioxidant and ergogenic functions. These features have led to the hypothesis of creatine supplementation use during pregnancy as prophylactic treatment to prevent CNS damage, such as hypoxic-ischemic encephalopathy. Unfortunately, very little is known on the effects of creatine supplementation during neuron differentiation, while in vitro studies revealed an influence on neuron excitability, leaving the possibility of creatine supplementation during the CNS development an open question. Using a multiple approach, we studied the hippocampal neuron morphological and functional development in neonatal rats born by dams supplemented with 1% creatine in drinking water during pregnancy. CA1 pyramidal neurons of supplemented newborn rats showed enhanced dendritic tree development, increased LTP maintenance, larger evoked-synaptic responses, and higher intrinsic excitability in comparison to controls. Moreover, a faster repolarizing phase of action potential with the appearance of a hyperpolarization were recorded in neurons of the creatine-treated group. Consistently, CA1 neurons of creatine exposed pups exhibited a higher maximum firing frequency than controls. In summary, we found that creatine supplementation during pregnancy positively affects morphological and electrophysiological development of CA1 neurons in offspring rats, increasing neuronal excitability. Altogether, these findings emphasize the need to evaluate the benefits and the safety of maternal intake of creatine in humans.

Neurons contain a high number of mitochondria, these neuronal cells produce elevated levels of oxidative stress and live for a long time without proliferation; therefore, mitochondrial homeostasis is crucial to their health. Investigations have recently focused on mitochondrial dynamics revealing the ability of these organelles to change their distribution and morphology. It is known that mitochondrial fission is necessary for the transmission of mitochondria to daughter cells during mitosis and mitochondrial fragmentation has been used as an indicator of cell death and mitochondrial dysfunction. Oxidative stress is a trigger able to induce changes in the mitochondrial network. The aim of the present study was to determine the effects of melatonin on the mitochondrial network in HT22 serum-deprived cells. Our results showed that serum deprivation increased reactive oxygen species (ROS) content, promoted the activation of plasma membrane voltage-dependent anion channels (VDACs) and affected the expression of pDRP1 and DRP1 fission proteins. Moreover, parallel increases in apoptotic and autophagic features were found. Damaged and dysfunctional mitochondria are deleterious to the cell; hence, the degradation of such mitochondria through mitophagy is crucial to cell survival. Our results suggest that melatonin supplementation reduces cell death and restores mitochondrial function through the regulation of autophagy.

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.

During muscle tissue differentiation, in particular in the formation of myotubes from the myoblasts, plasma membrane changes its morpho-functional characteristics. In this study, muscle cell membrane behaviour has been studied along the differentiation of C2C12, a mouse myoblastic adherent cell line. Flat undifferentiated cells, cultured for 3-4 days in the differentiation medium, progressively become thick, long and multinucleated myotubes covered with microvilli. They lose stress fibers and adhesion to the underlying substrate evidentiating an actin redistribution, followed by the spatial organization of thick and thin myofilaments. Sarcomeres and myofibrils occasionally appear, even if a certain percentage of "myosacs" containing randomly oriented filaments can be identified all along the differentiation. M-cadherin, a molecule involved in cell-cell adhesion, also appears in the early differentiation stage, during myoblast fusion. Occasional focal contractions can also be observed in myotubes, which prompt an electrophysiological membrane analysis. When studied by means of patch clamp technique, resting membrane potential appears to undergo a transient depolarization, while input resistance increases until day 5 after differentiation induction, then successively decreases. Capacitance declines until day 5, later appearing enhanced. Moreover, with the induction of differentiation, the pattern of functional voltage-dependent ion channels changes. Therefore, during myogenesis, cell maturation is coupled with changes in cell membrane morphological features and functional characteristics.

Thyroid hormones play a crucial role in new neuron production and maturation during brain development. However, the knowledge about the involvement of these hormones on adult neurogenesis is still incomplete. Hippocampus is an anatomical region where neurogenesis occurs throughout adulthood and where high levels of thyroid hormone receptors have been found. In this work the possible involvement of thyroid hormones in the regulation of adult neurogenesis in the granule cell layer of rat hippocampus dentate gyrus was investigated using an experimental model of adult-onset pharmacologically-induced hypothyroidism. Neurogenesis was assessed by means of the thymidine analogue 5-bromo-2′-deoxyuridine 24 h and 30 days after its last administration in order to study neural precursor proliferation and newborn cell survival, respectively. Mitotic activity of the neural precursors was not affected by thyroid hormone deficiency; on the contrary, newborn cell survival dramatically decreased under these conditions when compared with controls, leading to a lower number of immature neurons being added to the granule cell layer. Moreover, in conditions of hypothyroidism, new neurons exhibit a delay in neuronal differentiation showing a prolonged expression of the neuritogenesis-associated immature neuron marker TUC-4 and a very immature morphology. Finally, the total number and size of granule cells, and granule cell layer volume decreased in hypothyroid rats. These results suggest that thyroid hormones play a role in regulating new neuron production during adult life in dentate gyrus of rat hippocampus.

Neuron production carries on throughout adult life in granule cell layer of mammalian dentate gyrus. The acquisition of a hippocampus-dependent task enhances newborn cell survival in granule cell layer (GCL) during the period in which several newborn cells die. In this paper the effect of learning, occurring after the maximal period of newborn cell death, on newborn cells was investigated. Rats were trained for hippocampus-dependent learning in Morris water maze. 5-bromo-2'-deoxyuridine was administered 8-10 days before training beginning and labeled cells were counted after training. Learning decreased BrdU-labeled cell density in GCL, and increased TUNEL-positive cells. Moreover, learning diminished immature neuron density prevalently in the internal blade of GCL. Therefore, a different effect of learning on immature neuron survival, depending on the time elapsing from mitosis to learning, is suggested.