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Background Noise exposure impairs outer hair cells (OHCs). The common basis for OHC dysfunction and loss by acoustic over-stimulation is represented by reactive oxygen species (ROS) overload that may affect the membrane structural organization through generation of lipid peroxidation. Methods Here we investigated in OHC different functional zones the mechanisms linking metabolic functional state (NAD(P)H intracellular distribution) to the generation of lipid peroxides and to the physical state of membranes by two photon fluorescence microscopy. Results In OHCs of control animals, a more oxidized NAD(P)H redox state is associated to a less fluid plasma membrane structure. Acoustic trauma induces a topologically differentiated NAD(P)H oxidation in OHC rows, which is damped between 1 and 6 h. Peroxidation occurs after ~ 4 h from noise insult, while ROS are produced in the first 0.2 h and damage cells for a period of time after noise exposure has ended (~ 7.5 h) when a decrease of fluidity of OHC plasma membrane occurs. OHCs belonging to inner rows, characterized by a lower metabolic activity with respect to other rows, show less severe metabolic impairment. Conclusion and significance Our data indicate that plasma membrane fluidity is related to NAD(P)H redox state and lipid peroxidation which may represent key targets for therapeutic rescuing plan from noise insults.

Introduction: Dopamine/cAMP signaling mediates behavioral responses related to drug addiction. It also modulates the plasticity and firing properties of medium spiny neurons (MSNs) in the nucleus accumbens (NAc). Objectives: Our aim was to study the effects of postsynaptic dopamine D1-like receptor (D1R) activation on resting membrane potential (RMP) of NAc MSNs. Methods: In brain slices of C57BL/6 mice we performed whole-cell current- and voltage-clamp recordings from MSNs in the NAc core. Results: Activation of D1Rs by SKF-38393 elicited membrane depolarization and inward currents in MSNs. Similar results were obtained following adenylyl cyclase (AC) stimulation with forskolin or exogenous cAMP application. These effects were unaffected by protein kinase (PK) A or PKC blockade and were not mimicked by the Epac agonist, 8CPT-2Me-cAMP. Forskolin-induced depolarization was associated with increased membrane input resistance. Voltage-clamp experiments revealed that forskolin and SKF-38393 effects were due to inhibition of resting K+ currents exhibiting inward rectification at hyperpolarized potentials and a reversal potential that shifted with the extracellular K+ concentration. The inward rectifier K+ (Kir)-channel blocker, BaCl2, abolished forskolin and D1R agonist effects. Conclusions: These data suggest that postsynaptic D1R stimulation in MSNs of the NAc core causes membrane depolarization by inhibiting Kir currents. This effect is mediated by AC/cAMP signaling but it is independent on PKA, PKC and Epac suggesting that it may stem from cAMP’s direct interaction with Kir channels. D1R/cAMP-mediated excitatory effects may influence the generation of output signals from MSNs by facilitating their transition from the quiescent down-state to the functionally active up-state. Because a specific role in cocaine-seeking behavior has been attributed to the NAc core our findings may contribute to the understanding of pathophysiological mechanisms underlying drug dependence

Introduction: Throughout life, new neurons are continuously generated in the hippocampus, which is therefore a major site of structural plasticity in the adult brain. We recently demonstrated that extremely low-frequency electromagnetic fields (ELFEFs) promote the neuronal differentiation of neural stem cells in vitro by up-regulating Cav1-channel activity. Objectives: Aim of our study was to determine whether 50 Hz/1mT ELFEF stimulation also affects adult hippocampal neurogenesis in vivo, and if so, to identify the molecular mechanisms underlying this action, and its functional impact on synaptic plasticity. Methods: In adult hippocampus of ELFEF- and sham-exposed mice we performed immunoflourescence, molecular and electrophysiological analyses. Results: ELFEF exposure (1 to 7 h/day for 7 days) significantly enhanced neurogenesis in the dentate gyrus (DG) of adult mice, as documented by increased numbers of cells double-labeled for 5-bromo-deoxyuridine(BrdU) and doublecortin. Quantitative RT-PCR analysis of hippocampal extracts revealed significant ELFEF exposure-induced increases in the transcription of pro-neuronal genes (Mash1, NeuroD2, Hes1) and genes encoding Cav1.2 channel alpha1C subunits. Increased expression of NeuroD1, NeuroD2 and Cav1 channels was also documented by Western blot analysis. Immunofluorescence assays showed that, 30 days after ELFEF stimulation, roughly half of the newly generated immature neurons had survived and become mature dentate granule cells (as shown by their immunoreactivity for both BrdU and NeuN) and were integrated into the granule cell layer of the DG. Electrophysiological experiments demonstrated that the new mature neurons influenced hippocampal synaptic plasticity, as reflected by increased long-term potentiation. Conclusions: Our findings show that ELFEF exposure can be an effective tool for increasing in vivo neurogenesis, and they could lead to the development of novel therapeutic approaches in regenerative medicine.

Introduction: Drugs of abuse usurp learning and memory mechanisms normally related to neuronal rewards producing long-lasting neuroadaptation that underlies drug-seeking behavior and relapse. These alterations involve NMDA-dependent long-term plasticity in neural structure related to addiction such as the nucleus accumbens (NAc). Objectives: Aim of the present study was to investigate whether D-serine, released by astrocytes and/or neurons, may be involved in regulating synaptic activity and plasticity in the NAc. Methods: Experiments have been performed by using electrophysiological and immunohistochemical techniques. Coronal brain slices containing the NAc were prepared from Wistar rats (28 days old). Results: Pre-incubation of slices with D-amino acid oxidase (DAAO; 0.2 U/mL), a D-serine degrading enzyme, induced a significant increase of the AMPA/NMDA ratio at prefrontal cortex-medium spiny neuron synapses (from 1.3±0.2 to 5.8±1.5). This result suggests that D-serine modulates the NMDA component of the EPSCs. Therefore we tested the hypothesis that this amino acid may contribute to the NMDAdependent synaptic plasticity in the NAc. LTP and LTD induction requires the activation of NMDARs; as expected perfusion of the slices with D-AP5, a NMDAR antagonist, abolished these forms of synaptic plasticity. We then compared the amplitude of LTP and LTD in control and in DAAO-treated slices. The results of this set of experiments showed that D-serine degradation strongly reduced LTP and LTD (90.4% and 99.2%, respectively) thus indicating that this amino acid is required for these forms of plasticity. As for the cellular origin of D-serine, preliminary results from immunohistochemical experiments suggest a prevalent astrocytic site of production. Conclusions: These data suggest that astrocytic D-serine regulates NMDARs function and synaptic plasticity in the NAc. Ongoing experiments will address whether this signaling is impaired in cocaine model of drug addiction.

Dopamine/cAMP signaling has been reported to mediate behavioral responses related to drug addiction. It also modulates the plasticity and firing properties of medium spiny neurons (MSNs) in the nucleus accumbens (NAc), although the effects of cAMP signaling on the resting membrane potential (RMP) of MSNs has not been specifically defined. In this study, activation of dopamine D1-like receptors (D1Rs) by SKF-38393 elicited membrane depolarization and inward currents in MSNs from the NAc core of 14 17 day-old mice. Similar results were obtained following stimulation of adenylyl cyclase (AC) activity with forskolin or application of exogenous cAMP. Forskolin occluded SKF-38393 s effects, thus indicating that D1R action is mediated by AC/cAMP signaling. Accordingly, AC blockade by SQ22536 significantly inhibited the responses to SKF-38393. Effects elicited by D1R stimulation or increased cAMP levels were unaffected by protein kinase A (PKA) or protein kinase C (PKC) blockade and were not mimicked by the Epac agonist, 8CPT-2Me-cAMP. Responses to forskolin were also not significantly modified by cyclic nucleotide-gated (CNG) channel blockade. Forskolin-induced membrane depolarization was associated with increased membrane input resistance. Voltage-clamp experiments revealed that forskolin and SKF-38393 effects were due to inhibition of resting K currents exhibiting inward rectification at hyperpolarized potentials and a reversal potential (around 90 mV) that shifted with the extracellular K concentration. Forskolin and D1R agonist effects were abolished by the inward rectifier K (Kir)-channel blocker, BaCl2. Collectively, these data suggest that stimulation of postsynaptic D1Rs in MSNs of the NAc core causes membrane depolarization by inhibiting Kir currents. This effect is mediated by AC/cAMP signaling but it is independent on PKA, PKC, Epac and CNG channel activation, suggesting that it may stem from cAMP s direct interaction with Kir channels. D1R/cAMP-mediated excitat