Stefano Ciuffoli

Morpho-functional characterization of neuronal cells at different stages of maturation in granule cell layer of adult rat dentate gyrus.

Neurogenesis occurs throughout adult life in dentate gyrus of mammal hippocampus. Therefore, neurons at different stages of electrophysiological and morphological maturation and showing various, if any, synaptic inputs co-exist in the adult granule cell layer, as occurs during dentate gyrus development. The knowledge of functional properties of new neurons throughout their maturation can contribute to understanding their role in the hippocampal function. In this study electrophysiological and morphological features of granule layer cells, characterized as immature or mature neurons, without and with synaptic input, were comparatively described in adult rats. The patch-clamp technique was used to perform electrophysiological recordings, the occurrence of synaptic input evoked by medial perforant pathway stimulation was investigated and synaptic input was characterized. Cells were then identified and morphologically described via detection of biocytin injected through the patch pipette. The neuronal phenotype of recorded cells was assessed by immunohistochemistry and single-cell RT-PCR. Cells with very low capacitance, high input resistance, depolarized resting membrane potential and without synaptic activity were found exclusively at the border of the GCL facing hilus; this type of cell expressed the class III beta-tubulin neuronal marker (mRNA and protein) and did not express a glial marker. Immature neuronal cells with progressively increasing capacitance, decreasing input resistance and resting membrane potential getting more hyperpolarized showed only depolarizing GABAergic synaptic input at first and then also glutamatergic synaptic input. Finally, cells showing electrophysiological, synaptic, and morphological features of mature granule, expressing the mature neuron marker NeuN, were identified.

Synaptogenesis in adult-generated hippocampal granule cells is affected by behavioral experiences

Adult‐generated hippocampal immature neurons play a functional role after integration in functional circuits. Previously, we found that hippocampus‐dependent learning in Morris water maze affects survival of immature neurons, even before they are synaptically contacted. Beside learning, this task heavily engages animals in physical activity in form of swimming; physical activity enhances hippocampal neurogenesis. In this article, the effects of training in Morris water maze apparatus on the synapse formation onto new neurons in hippocampus dentate gyrus and on neuronal maturation were investigated in adult rats. Newborn cells were identified using retroviral GFP‐expressing virus infusion. In the first week after virus infusion, rats were trained in Morris water maze apparatus in three different conditions (spatial learning, cue test, and swimming). Properties of immature neurons and their synaptic response to perforant pathway stimulation were electrophysiologically investigated early during neuronal maturation. In controls, newborn cells showing GABAergic and glutamatergic responses were found for the first time at 8 and 10 days after mitosis, respectively; no cell with glutamatergic response only was found. Twelve days after virus infusion almost all GFP‐positive cells showed both synaptic responses. The main result we found was the anticipated appearance of GABAergic synapses at 6 days in learner, cued and swimmer rats, supported also by immunohistochemical result. Swimmer rats showed the highest percentage of GFP‐positive neurons with glutamatergic response at 10 and 12 days postmitosis. Moreover, primary dendrites were more numerous at 7 days in learner, cued and swimmer rats and swimmer rats showed the greatest dendritic tree complexity at 10 days. Finally, voltage‐dependent Ca2+ current was found in a larger number of newborn neurons at 7 days postinfusion in learner, cued and swimmer rats. In conclusion, experiences involving physical activity contextualized in an exploring behavior affect synaptogenesis in adult‐generated cells and their early stages of maturation.

Physical exercise and environment exploration affect synaptogenesis in adult-generated neurons in the rat dentate gyrus: Possible role of BDNF

A brief training in a pool maze, with or without cognitive tasks, modifies the synaptogenesis and maturation of newborn neurons in adult rat dentate gyrus. These types of trainings have many aspects, including physical activity and exploration. Therefore, to evaluate whether physical exercise and environment exploration are able to affect synapse formation and the maturation of adult-generated neurons, GFP-retrovirus infusion was performed on rats which, on the fourth day after injection, were housed under running conditions or allowed to explore an enriched environment briefly in the absence of exercise for the following three days. Afterward, at the end of the trainings, electrophysiological and morphological studies were conducted. Considering that neurotrophic factors increase after exercise or environment exploration, hippocampal BDNF levels and TrkB receptor activation were evaluated. In this study, we show that both spontaneous physical activity and enriched environment exploration induced synaptogenesis and T-type voltage-dependent Ca(2+) currents in very immature neurons. Hippocampal BDNF levels and TrkB receptor activation were determined to be increasing following physical activity and exploration. A possible contribution of BDNF signaling in mediating the observed effects was supported by the use of 7-8-dihydroxyflavone, a selective TrkB agonist, and of ANA-12, an inhibitor of TrkB receptors.

Maternal dietary loads of α-tocopherol depress protein kinase C signaling and synaptic plasticity in rat postnatal developing hippocampus and promote permanent deficits in adult offspring.

Vitamin E (α-tocopherol) supplementation has been tested as prophylaxis against gestational disorders associated with oxidative damage. However, recent evidence showing that high maternal α-tocopherol intake can adversely affect offspring development raises concerns on the safety of vitamin E extradosages during pregnancy. Besides acting as an antioxidant, α-tocopherol depresses cell proliferation and modulates cell signaling through inhibiting protein kinase C (PKC), a kinase that is deeply involved in neural maturation and plasticity. Possible effects of α-tocopherol loads in the maturing brain, where PKC dysregulation is associated to developmental dysfunctions, are poorly known. Here, supranutritional doses of α-tocopherol were fed to pregnant and lactating dams to evaluate the effects on PKC signaling and morphofunctional maturation in offspring hippocampus. Results showed that maternal supplementation potentiates hippocampal α-tocopherol incorporation in offspring and leads to marked decrease of PKC phosphorylation throughout postnatal maturation, accompanied by reduced phosphorylation of growth-associated protein-43 and myristoylated alanine-rich C kinase substrate, two PKC substrates involved in neural development and plasticity. Although processes of neuronal maturation, synapse formation and targeting appeared unaffected, offspring of supplemented mothers displayed a marked reduction of long-term synaptic plasticity in juvenile hippocampus. Interestingly, this impairment persisted in adulthood, when a deficit in hippocampus-dependent, long-lasting spatial memory was also revealed. In conclusion, maternal supplementation with elevated doses of α-tocopherol can influence cell signaling and synaptic plasticity in developing hippocampus and promotes permanent adverse effects in adult offspring. The present results emphasize the need to evaluate the safety of supranutritional maternal intake of α-tocopherol in humans.

Dietary supplementation with α-tocopherol reduces neuroinflammation and neuronal degeneration in the rat brain after kainic acid-induced status epilepticus.

Abstract Vitamin E (as α-tocopherol, α-T) is proposed to alleviate glia-mediated inflammation in neurological diseases, but such a role in epilepsy is still elusive. This study investigated the effect of α-T supplementation on glial activation, neuronal cell death and oxidative stress of rat brain exposed to kainate-induced seizures. Animals were fed for 2 weeks with a α-T-enriched diet (estimated intake of 750 mg/kg/day) before undergoing status epilepticus. Compliance to supplementation was demonstrated by the remarkable increase in brain α-T. Four days after seizure, brain α-T returned to baseline and lipid peroxidation markers decreased as compared to non-supplemented rats. Status epilepticus induced a lower up-regulation of astrocytic and microglial antigens (GFAP and MHC II, respectively) and production of pro-inflammatory cytokines (IL-1β and TNF-α) in supplemented than in non-supplemented animals. This anti-inflammatory effect was associated with a lower neuronal cell death. In conclusion, α-T dietary supplementation prevents oxidative stress, neuroglial over-activation and cell death occurring after kainate-induced seizures. This evidence paves the way to an anti-inflammatory and neuroprotective role of α-T interventions in epilepsy.

Creatine affects in vitro electrophysiological maturation of neuroblasts and protects them from oxidative stress.

Creatine (Cr) is a very popular ergogenic molecule that has recently been shown to have antioxidant properties. The effectiveness of Cr supplementation in treating neurological diseases and Cr deficiency syndromes has been demonstrated, and experimental reports suggest that it plays an important role in CNS development. In spite of this body of evidence, the role of Cr in functional and structural neuronal differentiation is still poorly understood. Here we used electrophysiological, morphological, and biochemical approaches to study the effects of Cr supplementation on in vitro differentiation of spinal neuroblasts under standard conditions or subjected to oxidative stress, a status closely related to perinatal hypoxia‐ischemia, a severe condition for developing brain. Cr supplementation (10 and 20 mM) completely prevented the viability decrease and neurite development impairment induced by radical attack, as well as nonprotein sulphydryl antioxidant pool depletion. Similar results were obtained using the antioxidant trolox. Furthermore, Cr supplementation induced a significant and dose‐dependent anticipation of Na+ and K+ current expression during the period of in vitro network building. Consistently with the latter finding, higher excitability, expressed as number of spikes following depolarization, was found in supplemented neuroblasts. All effects were dependent on the cytosolic fraction of Cr, as shown using a membrane Cr‐transporter blocker. Our results indicate that Cr protects differentiating neuroblasts against oxidative insults and, moreover, affects their in vitro electrophysiological maturation, suggesting possibly relevant effects of dietary Cr supplementation on developing CNS.

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.

Maternal dietary loads of α-tocopherol differentially influence fear conditioning and spatial learning in adult offspring

α-Tocopherol, the main component of vitamin E, is well known to be a radical scavenger, so an increased intake of vitamin E is recommended in complicated pregnancy, to prevent possible fetus damage by free radical. In a previous work, we found that maternal α-tocopherol supplementation affects PKC-mediated cellular signaling and hippocampal synaptic plasticity in developing brain; the latter effect persists in adulthood. Here, adult rats maternally exposed to supranutritional doses of α-tocopherol were evaluated for Contextual Fear Conditioning and spatial learning in Morris Water Maze, two different hippocampus-dependent learning tasks. Moreover, anxiety, spontaneous activity, and explorative drive were also evaluated as factors potentially affecting learning performance. Treated rats showed a different behavior with respect to controls: performance in Contextual Fear Conditioning was improved, while spatial learning tested in Morris Water Maze, was impaired. The improvement of fear response was not ascribable to differences in anxiety level and/or spontaneous activity; thus it appears to be a specific effect of α-tocopherol overloading during brain development. On the contrary, the impaired performance in Morris Water Maze exhibited by treated rats can be in part explained by their enhanced explorative drive. Although extrapolation from rats to humans is difficult, a caveat in assuming supranutritional doses of vitamin E in pregnancy arises from this study.

FGF2 modulates the voltage-dependent K+ current and changes excitability of rat dentate gyrus granule cells

Fibroblast growth factor 2 (FGF2) is involved in hippocampus-dependent learning. In this study, the effects of FGF2 on the excitability were investigated in granule cells of rat dentate gyrus. Hippocampal slices were used to perform patch clamp recordings in granule cells. Extracellularly applied FGF2 early quenched the depolarization-induced repetitive firing, suggesting a decreased excitability under these conditions. Consistently, transient and sustained voltage-gated K(+) currents decreased in a dose-dependent manner, repolarization phase of action potential was slowed down, afterhyperpolarization was reduced, and membrane resistance was decreased. These effects were not mediated by tyrosine kinase FGF2 receptors. Moreover, an involvement of G protein signaling was ruled out, as well as an intracellular action of FGF2. Considering the relationship between FGF2 and hippocampal functions, the modulation of neuron excitability by activity-driven FGF2 release may be regarded as a part of a homeostatic mechanism of self-regulation of hippocampal activity.

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.