Elisa Riccardi

Modulation of LTP at rat hippocampal CA3-CA1 synapses by direct current stimulation

Transcranial direct current stimulation (tDCS) can produce a lasting polarity-specific modulation of cortical excitability in the brain, and it is increasingly used in experimental and clinical settings. Recent studies suggest that the after-effects of tDCS are related to molecular mechanisms of activity-dependent synaptic plasticity. Here we investigated the effect of DCS on the induction of one of the most studied N-methyl-d-aspartate receptor-dependent forms of long-term potentiation (LTP) of synaptic activity at CA3-CA1 synapses in the hippocampus. We show that DCS applied to rat brain slices determines a modulation of LTP that is increased by anodal and reduced by cathodal DCS. Immediate early genes, such as c-fos and zif268 (egr1/NGFI-A/krox24), are rapidly induced following neuronal activation, and a specific role of zif268 in the induction and maintenance of LTP has been demonstrated. We found that both anodal and cathodal DCS produce a marked subregion-specific increase in the expression of zif268 protein in the cornus ammonis (CA) region, whereas the same protocols of stimulation produce a less pronounced increase in c-fos protein expression in the CA and in dentate gyrus regions of the hippocampus. Brain-derived neurotrophic factor expression was also investigated, and it was found to be reduced in cathodal-stimulated slices. The present data demonstrate that it is possible to modulate LTP by using DCS and provide the rationale for the use of DCS in neurological diseases to promote the adaptive and suppress the maladaptive forms of brain plasticity.

Exposure to extremely low-frequency (50 Hz) electromagnetic fields enhances adult hippocampal neurogenesis in C57BL/6 mice

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 Ca(v)1-channel activity. The aim of the present study was to determine whether 50-Hz/1 mT 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. 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 Ca(v)1.2 channel α(1C) subunits. Increased expression of NeuroD1, NeuroD2 and Ca(v)1 channels was also documented by Western blot analysis. Immunofluorescence experiments 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. 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.

P22.22 Exposure to extremely low-frequency (50 Hz) electromagnetic fields enhances adult hippocampal neurogenesis in C57BL/6 mice

Introduction: Paired associative stimulation (PAS) at an interstimulus interval (ISI) of 25 ms produces long term potentiation (LTP)-like effect, but each pair occurs at intervals for producing short afferent inhibition (SAI). This implies that inhibitory mechanisms may play a role in producing LTP-like effects of PAS. Objectives: We assessed the inhibitory synaptic pathways by measuring short-interval intracortical inhibition (SICI). Methods: Twenty-two healthy volunteers (9 females, 34 yrs on average) were recruited. Stimulus intensities were adjusted so that at the start of PAS, the test motor evoked potential (MEP) was suppressed to 60 80% control (SAI). SICI was assessed with a threshold tracking technique using a standard 0.2 mV MEP. Inhibition is expressed as the increase in stimulation intensity needed to maintain 0.2 mV MEP constant in the presence of a conditioning stimulus (CS) of 70% resting motor threshold. Thus high values indicate strong inhibition. Results: MEPs increased by an average of 1.55±0.19 (SE) after PAS, but ranged from 0.54 to 3.67. We divided the subjects into three groups; good responders (1 < PAS effect < 2, n = 11), poor responders (PAS effect < 1, n = 7) and outliers (PAS effect 2, n = 4). Before PAS, good responders had strong SICI at ISI 1.8 to 5 ms compared to poor responders. SICI at ISI 3 ms was 27.3±4.5% in good responders and 4.9±4.7% in poor responders (p = 0.004). SICI was significantly correlated with PAS effect (r = 0.61, p = 0.007). SICI in the outliers (who were musicians) fell out of the 95% confidence interval in this correlation. Conclusions: The relationship between the initial level of SICI and the response to PAS is compatible with the following. The PAS effect relies on increased excitability of late indirect (I)-wave generating mechanisms. SICI has its primary effect on late I-waves. Thus individuals with good SICI may have prominent late I-waves that are readily inhibited by CS; however the same I-waves may be beneficial for PAS.

Dopamine D1-like receptor activation depolarizes medium spiny neurons of the mouse nucleus accumbens by inhibiting inwardly rectifying K+ currents through a cAMP-dependent protein kinase A-independent mechanism

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-38393s 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, BaCl(2). 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 cAMPs 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.

Effects of different amyloid β-protein analogues on synaptic function

Perisynaptic accumulations of amyloid β-protein (Aβ) play a critical role in the synaptic dysfunction underlying the cognitive impairment observed in Alzheimers disease. The methionine residue at position 35 (Met35) in Aβ is highly subject to oxidation in Alzheimers disease brains. In hippocampal brain slices we found that long-term potentiation at CA3-CA1 synapses was significantly inhibited by wild type Aβ42 in which Met35 is reduced, but not by Aβ42 harboring Met35 sulfoxide. Similar differences were observed when basal synaptic transmission was investigated in autaptic hippocampal neurons. The significant decreases in excitatory postsynaptic current amplitude, vesicle release probability and miniature excitatory postsynaptic current frequency caused by 20-minute exposure to wild type Aβ42 were not observed after exposure to Aβ42 harboring Met35 sulfoxide. With longer (24-hour) Aβ treatments, this early impairment of the presynaptic terminal function extended to involve the postsynaptic side as well. The Met35 oxidation also affected Aβ42 negative impact on dendritic spine density and expression of pre- and postsynaptic proteins (synaptophysin and postsynaptic density protein-95). Our findings suggest that oxidation of Met35 is critical for molecular, structural, and functional determinants of Aβ42 synaptotoxicity.

P10.10 Dopamine D1-like receptor activation depolarizes medium spiny neurons of the mouse nucleus accumbens by inhibiting inwardly rectifying K+ currents

Results: Background activity in both control and mutant mice was composed by phases of 1 4 Hz or 6 9 Hz. In mutants, concomitantly with a rising number of seizures, normal background activity progressively got worse by decrease in amplitude, slowing of activity and manifestation of epileptiform abnormalities. Treatment of mutant mice with rapamycin fully reverted the mutant phenotype. Conclusions: Spontaneous epileptic seizures were observed in 100% of mutant mice, that died within day 18 if not treated with rapamycin. Video-EEG proved to be essential to study this model of epilepsy and effects of rapamycin treatment. Moreover, we showed that is possible to perform it also in very young mice.

S7.4 Effects of different forms of amyloid β-peptide on synaptic function

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