Gian Battista Azzena

Extremely low‐frequency electromagnetic fields promote in vitro neurogenesis via upregulation of Cav1‐channel activity

We previously reported that exposure to extremely low‐frequency electromagnetic fields (ELFEFs) increases the expression and function of voltage‐gated Ca2+ channels and that Ca2+ influx through Cav1 channels plays a key role in promoting the neuronal differentiation of neural stem/progenitor cells (NSCs). The present study was conducted to determine whether ELFEFs influence the neuronal differentiation of NSCs isolated from the brain cortices of newborn mice by modulating Cav1‐channel function. In cultures of differentiating NSCs exposed to ELFEFs (1 mT, 50 Hz), the percentage of cells displaying immunoreactivity for neuronal markers (β‐III‐tubulin, MAP2) and for Cav1.2 and Cav1.3 channels was markedly increased. NSC‐differentiated neurons in ELFEF‐exposed cultures also exhibited significant increases in spontaneous firing, in the percentage of cells exhibiting Ca2+ transients in response to KCl stimulation, in the amplitude of these transients and of Ca2+ currents generated by the activation of Cav1 channels. When the Cav1‐channel blocker nifedipine (5 µM) was added to the culture medium, the neuronal yield of NSC differentiation dropped significantly, and ELFEF exposure no longer produced significant increases in β‐III‐tubulin‐ and MAP2‐immunoreactivity rates. In contrast, the effects of ELFEFs were preserved when NSCs were cultured in the presence of either glutamate receptor antagonists or Cav2.1‐ and Cav2.2‐channel blockers. ELFEF stimulation during the first 24 h of differentiation caused Cav1‐dependent increases in the number of cells displaying CREB phosphorylation. Our data suggest that ELFEF exposure promotes neuronal differentiation of NSCs by upregulating Cav1‐channel expression and function. J. Cell. Physiol. 215: 129–139, 2008.

Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation.

Ca2+ influx through voltage‐gated Ca2+ channels, especially the L‐type (Cav1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca2+ signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca2+ transients induced by membrane depolarization in Fluo 4‐AM‐loaded NSCs using confocal microscopy. Undifferentiated cells (nestin+) exhibited no detectable Ca2+ signals whereas, during 12 days of fetal bovine serum‐induced differentiation, neurons (β‐III‐tubulin+/MAP2+) displayed time‐dependent increases in intracellular Ca2+ transients, with ΔF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch‐clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba2+ current density. These currents were markedly reduced (−77%) by Cav1 channel blockade with 5 µm nifedipine. To determine the influence of Cav1‐mediated Ca2+ influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine (5 µm) or the L‐channel activator Bay K 8644 (10 µm). The latter treatment significantly increased the percentage of β‐III‐tubulin+/MAP2+ cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca2+ signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca2+ transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage‐gated Ca2+ channels, especially the Cav1, and that Ca2+ influx through these channels plays a key role in promoting neuronal differentiation.

Electrophysiological and molecular evidence of L-(Cav1), N- (Cav2.2), and R- (Cav2.3) type Ca2+ channels in rat cortical astrocytes

Changes in intracellular Ca2+ levels are an important signal underlying neuron‐glia cross‐talk, but little is known about the possible role of voltage‐gated Ca2+ channels (VGCCs) in controlling glial cell Ca2+ influx. We investigated the pharmacological and biophysical features of VGCCs in cultured rat cortical astrocytes. In whole‐cell patch‐clamp experiments, L‐channel blockade (5 μM nifedipine) reduced Ba2+ current amplitude by 28% of controls, and further decrease (32%) was produced by N‐channel blockade (3 μM ω‐conotoxin‐GVIA). No significant additional changes were observed after P/Q channel blockade (3 μM ω‐conotoxin‐MVIIC). Residual current (36% of controls) amounted to roughly the same percentage (34%) that was abolished by R‐channel blockade (100 nM SNX‐482). Electrophysiological evidence of L‐, N‐, and R‐channels was associated with RT‐PCR detection of mRNA transcripts for VGCC subunits α1C (L‐type), α1B (N‐type), and α1E (R‐type). In cell‐attached recordings, single‐channel properties (L‐currents: amplitude, −1.21 ± 0.02 pA at 10 mV; slope conductance, 22.0 ± 1.1 pS; mean open time, 5.95 ± 0.24 ms; N‐currents: amplitude, −1.09 ± 0.02 pA at 10 mV; slope conductance, 18.0 ± 1.1 pS; mean open time, 1.14 ± 0.02 ms; R‐currents: amplitude, −0.81 ± 0.01 pA at 20 mV; slope conductance, 10.5 ± 0.3 pS; mean open time, 0.88 ± 0.02 ms) resembled those of corresponding VGCCs in neurons. These novel findings indicate that VGCC expression by cortical astrocytes may be more varied than previously thought, suggesting that these channels may indeed play substantial roles in the regulation of astrocyte Ca2+ influx, which influences neuron‐glia cross‐talk and numerous other calcium‐mediated glial‐cell functions.

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.

HSV-1 promotes Ca2+-mediated APP phosphorylation and Aβ accumulation in rat cortical neurons

Epidemiological and experimental findings suggest that chronic infection with Herpes simplex virus type 1 (HSV-1) may be a risk factor for Alzheimers disease (AD), but the molecular mechanisms underlying this association have not been fully identified. We investigated the effects of HSV-1 on excitability and intracellular calcium signaling in rat cortical neurons and the impact of these effects on amyloid precursor protein (APP) processing and the production of amyloid-β peptide (Aβ). Membrane depolarization triggering firing rate increases was observed shortly after neurons were challenged with HSV-1 and was still evident 12 hours postinfection. These effects depended on persistent sodium current activation and potassium current inhibition. The virally induced hyperexcitability triggered intracellular Ca(2+) signals that significantly increased intraneuronal Ca(2+) levels. It also enhanced activity- and Ca(2+)-dependent APP phosphorylation and intracellular accumulation of Aβ42. These findings indicate that HSV-1 causes functional changes in cortical neurons that promote APP processing and Aβ production, and they are compatible with the co-factorial role for HSV-1 in the pathogenesis of AD suggested by previous findings.

Generation of human auditory steady-state responses (SSRs). I: Stimulus rate effects

Auditory evoked responses were recorded in 16 normally hearing subjects in order to investigate the mechanisms underlying the generation of the 40 Hz steady-state response (SSR). In the first part of our study, auditory potentials were evoked by 0.1 ms clicks presented at 105 dB p.e. SPL with repetition rates of 7.9 (to obtain middle latency response, MLR), 20, 30, 40, 50, 60 Hz. In each subject predictions of the responses recorded at stimulus repetition rates of 30, 40, 50, 60 Hz were synthesized by superimposing MLRs at suitable time intervals. The calculated mean amplitude/rate and phase/rate functions behaved similarly for the recorded and predicted curves, showing the highest amplitude at 40 Hz and a linear increase of phase values when increasing the stimulus rate. Nevertheless the synthetic curves closely predicted amplitude and phase values of the recorded responses only at 40 Hz. At frequencies below 40 Hz, the mean amplitude of the predicted curve was lower than that of the recorded one while at frequencies above 40 Hz the mean amplitude was higher. Predicted phase values were found lagging at 30 Hz, and leading at 50 Hz and 60 Hz in comparison to phase values calculated on the recorded responses. Our findings suggest that a model based on the linear addition of transient MLRs is not able to adequately predict steady-state responses at stimulus rates other than at 40 Hz. Other mechanisms related to the recovery cycle of the activated system come into play in the steady-state response generation causing a decrease in amplitude and an increase in phase lag when increasing the stimulus repetition rate.

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.

Dysregulation of intracellular calcium homeostasis is responsible for neuronal death in an experimental model of selective hippocampal degeneration induced by trimethyltin

Trimethyltin (TMT) intoxication is considered a suitable experimental model to study the molecular basis of selective hippocampal neurodegeneration as that occurring in several neurodegenerative diseases. We have previously shown that rat hippocampal neurons expressing the Ca2+‐binding protein calretinin (CR) are spared by the neurotoxic action of TMT hypothetically owing to their ability to buffer intracellular Ca2+ overload. The present study was aimed at determining whether intracellular Ca2+ homeostasis dysregulation is involved in the TMT‐induced neurodegeneration and if intracellular Ca2+‐buffering mechanisms may exert a protective action in this experimental model of neurodegeneration. In cultured rat hippocampal neurons, TMT produced time‐ and concentration‐dependent [Ca2+]i increases that were primarily due to Ca2+ release from intracellular stores although Ca2+ entry through Cav1 channels also contributed to [Ca2+]i increases in the early phase of TMT action. Cell pre‐treatment with the Ca2+ chelator, 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetrakis(acetoxymethyl ester) (2 μM) significantly reduced the TMT‐induced neuronal death. Moreover, CR+ neurons responded to TMT with smaller [Ca2+]i increases. Collectively, these data suggest that the neurotoxic action of TMT is mediated by Ca2+ homeostasis dysregulation, and the resistance of hippocampal neurons to TMT (including CR+ neurons) is not homogeneous among different neuron populations and is related to their ability to buffer intracellular Ca2+ overload.

Nitric oxide regenerates the normal colonic peristaltic activity in mdx dystrophic mouse

We demonstrated in vitro that the colonic peristaltic activity is modified in dystrophin-deficient mdx mouse indicating a defect in the enteric nervous system (ENS). Since nitric oxide (NO) has been proposed as a putative inhibitory mediator of ENS, here we have examined the effects of both L-Arginine (L-Arg) and Nomega-nitro-L-arginine methyl ester (L-NAME) on the peristaltic activity of mdx mouse distal colon. The motor pattern of colonic segment showed irregular peristaltic waves. L-Arg (10(-7) - 10(-5) M) induced the peristaltic activity to slow down. At a concentration of 10(-5) M, L-Arg produced hypomotility, characterised by a decrease in amplitude, frequency and ejected fluid volume. Conversely, L-NAME elicited hypermotility, this effect being reversed once again by the subsequent addition of L-Arg. Interestingly the addition of 10(-5) M L-Arg to the organ bath led to the normal progression, in an oral to aboral direction, of 90% of the peristaltic waves. This last result strongly suggests that exogenous application of L-Arg restores the integrative circuits of the ENS responsible for programming and co-ordinating peristaltic activity in the distal colon of mdx mouse.