Marcello D'Ascenzo

mGluR5 stimulates gliotransmission in the nucleus accumbens

Although metabotropic glutamate receptor 5 (mGluR5) is essential for cocaine self-administration and drug-seeking behavior, there is limited knowledge of the cellular actions of this receptor in the nucleus accumbens (NAc). Although mGluR5 has the potential to regulate neurons directly, recent studies have shown the importance of mGluR5 in regulating Ca2+ signaling in astrocytes and, as a consequence, the Ca2+-dependent release of excitatory transmitters from these glia. In this study, we demonstrate that activation of mGluR5 induces Ca2+ oscillations in NAc astrocytes with the correlated appearance of NMDA receptor-dependent slow inward currents detected in medium spiny neurons (MSNs). Photolysis of caged Ca2+ loaded specifically into astrocytes evoked slow inward currents demonstrating that Ca2+ elevations in astrocytes are responsible for these excitatory events. Pharmacological evaluation of these glial-evoked NMDA currents shows that they are mediated by NR2B-containing NMDA receptors, whereas synaptic NMDA receptors rely on NR2A-containing receptors. Stimulation of glutamatergic afferents activates mGluR5-dependent astrocytic Ca2+ oscillations and gliotransmission that is sustained for minutes beyond the initial stimulus. Because gliotransmission is mediated by NMDA receptors, depolarized membrane potentials exhibited during up-states augment excitation provided by gliotransmission, which drives bursts of MSN action potentials. Because the predominant mGluR5-dependent action of glutamatergic afferents is to cause the sustained activation of astrocytes, which in turn excite MSNs through extrasynaptic NMDA receptors, our results raise the potential for gliotransmission being involved in prolonged mGluR5-dependent adaptation in the NAc.

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.

Nitric oxide inhibits neuroendocrine CaV1 L-channel gating via cGMP-dependent protein kinase in cell-attached patches of bovine chromaffin cells

Nitric oxide (NO) regulates the release of catecholamines from the adrenal medulla but the molecular targets of its action are not yet well identified. Here we show that the NO donor sodium nitroprusside (SNP, 200 μM) causes a marked depression of the single CaV1 L‐channel activity in cell‐attached patches of bovine chromaffin cells. SNP action was complete within 3‐5 min of cell superfusion. In multichannel patches the open probability (NPo) decreased by ∼60 % between 0 and +20 mV. Averaged currents over a number of traces were proportionally reduced and showed no drastic changes to their time course. In single‐channel patches the open probability (Po) at +10 mV decreased by the same amount as that of multichannel patches (∼61 %). Such a reduction was mainly associated with an increased probability of null sweeps and a prolongation of mean shut times, while first latency, mean open time and single‐channel conductance were not significantly affected. Addition of the NO scavenger carboxy‐PTIO or cell treatment with the guanylate cyclase inhibitor ODQ prevented the SNP‐induced inhibition. 8‐Bromo‐cyclicGMP (8‐Br‐cGMP; 400 μM) mimicked the action of the NO donor and the protein kinase G blocker KT‐5823 prevented this effect. The depressive action of SNP was preserved after blocking the cAMP‐dependent up‐regulatory pathway with the protein kinase A inhibitor H89. Similarly, the inhibitory action of 8‐Br‐cGMP proceeded regardless of the elevation of cAMP levels, suggesting that cGMP/PKG and cAMP/PKA act independently on L‐channel gating. The inhibitory action of 8‐Br‐cGMP was also independent of the G protein‐induced inhibition of L‐channels mediated by purinergic and opiodergic autoreceptors. Since Ca2+ channels contribute critically to both the local production of NO and catecholamine release, the NO/PKG‐mediated inhibition of neuroendocrine L‐channels described here may represent an important autocrine signalling mechanism for controlling the rate of neurotransmitter release from adrenal glands.

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.

Reduced D-serine levels in the nucleus accumbens of cocaine-treated rats hinder the induction of NMDA receptor-dependent synaptic plasticity

Cocaine seeking behaviour and relapse have been linked to impaired potentiation and depression at excitatory synapses in the nucleus accumbens, but the mechanism underlying this process is poorly understood. We show that, in the rat nucleus accumbens core, D-serine is the endogenous coagonist of N-methyl-D-aspartate receptors, and its presence is essential for N-methyl-D-aspartate receptor-dependent potentiation and depression of synaptic transmission. Nucleus accumbens core slices obtained from cocaine-treated rats after 1 day of abstinence presented significantly reduced D-serine concentrations, increased expression of the D-serine degrading enzyme, D-amino acid oxidase, and downregulated expression of serine racemase, the enzyme responsible for D-serine synthesis. The D-serine deficit was associated with impairment of potentiation and depression of glutamatergic synaptic transmission, which was restored by slice perfusion with exogenous D-serine. Furthermore, in vivo administration of D-serine directly into the nucleus accumbens core blocked behavioural sensitization to cocaine. These results provide evidence for a critical role of D-serine signalling in synaptic plasticity relevant to cocaine addiction.

Intracellular Accumulation of Amyloid-β (Aβ) Protein Plays a Major Role in Aβ-Induced Alterations of Glutamatergic Synaptic Transmission and Plasticity

Intracellular accumulation of amyloid-β (Aβ) protein has been proposed as an early event in AD pathogenesis. In patients with mild cognitive impairment, intraneuronal Aβ immunoreactivity was found especially in brain regions critically involved in the cognitive deficits of AD. Although a large body of evidence demonstrates that Aβ42 accumulates intraneuronally (inAβ), the action and the role of Aβ42 buildup on synaptic function have been poorly investigated. Here, we demonstrate that basal synaptic transmission and LTP were markedly depressed following Aβ42 injection into the neuron through the patch pipette. Control experiments performed with the reverse peptide (Aβ42-1) allowed us to exclude that the effects of inAβ depended on changes in oncotic pressure. To further investigate inAβ synaptotoxicity we used an Aβ variant harboring oxidized methionine in position 35 that does not cross the neuronal plasma membrane and is not uploaded from the extracellular space. This Aβ42 variant had no effects on synaptic transmission and plasticity when applied extracellularly, but induced synaptic depression and LTP inhibition after patch-pipette dialysis. Finally, the injection of an antibody raised against human Aβ42 (6E10) in CA1 pyramidal neurons of mouse hippocampal brain slices and autaptic microcultures did not, per se, significantly affect LTP and basal synaptic transmission, but it protected against the toxic effects of extracellular Aβ42. Collectively, these findings suggest that Aβ42-induced impairment of glutamatergic synaptic function depends on its internalization and intracellular accumulation thus paving the way to a systemic proteomic analysis of intracellular targets/partners of Aβ42.

Astrocytes control neuronal excitability in the nucleus accumbens.

Though accumulating evidence shows that the metabotropic glutamate receptor 5 (mGluR5) mediates some of the actions of extracellular glutamate after cocaine use, the cellular events underlying this action are poorly understood. In this review, we will discuss recent results showing that mGluR5 receptors are key regulators of astrocyte activity. Synaptic release of glutamate activates mGluR5 expressed in perisynaptic astrocytes and generates intense Ca2+ signaling in these cells. Ca2+ oscillations, in turn, trigger the release from astrocytes of the gliotransmitter glutamate, which modulates neuronal excitability by activating NMDA receptors. By integrating these results with the most recent evidence demonstrating the importance of astrocytes in the regulation of neuronal excitability, we propose that astrocytes are involved in mediating some of the mGluR5-dependent drug-induced behaviors.

Role of methionine 35 in the intracellular Ca2+ homeostasis dysregulation and Ca2+-dependent apoptosis induced by amyloid β-peptide in human neuroblastoma IMR32 cells

Amyloid β‐peptide (Aβ) plays a fundamental role in the pathogenesis of Alzheimer’s disease. We recently reported that the redox state of the methionine residue in position 35 of amyloid β‐peptide (Aβ) 1–42 (Met35) strongly affects the peptide’s ability to trigger apoptosis and is thus a major determinant of its neurotoxicity. Dysregulation of intracellular Ca2+ homeostasis resulting in the activation of pro‐apoptotic pathways has been proposed as a mechanism underlying Aβ toxicity. Therefore, we investigated correlations between the Met35 redox state, Aβ toxicity, and altered intracellular Ca2+ signaling in human neuroblastoma IMR32 cells. Cells incubated for 6–24 h with 10 μM Aβ1–42 exhibited significantly increased KCl‐induced Ca2+ transient amplitudes and resting free Ca2+ concentrations. Nifedipine‐sensitive Ca2+ current densities and Cav1 channel expression were markedly enhanced by Aβ1–42. None of these effects were observed when cells were exposed to Aβ containing oxidized Met35 (Aβ1–42Met35‐Ox). Cell pre‐treatment with the intracellular Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N’,N’‐tetraacetic acid acetoxymethyl ester (1 μM) or the Cav1 channel blocker nifedipine (5 μM) significantly attenuated Aβ1–42‐induced apoptosis but had no effect on Aβ1–42Met35‐Ox toxicity. Collectively, these data suggest that reduced Met35 plays a critical role in Aβ1–42 toxicity by rendering the peptide capable of disrupting intracellular Ca2+ homeostasis and thereby provoking apoptotic cell death.

Intraneuronal Aβ accumulation induces hippocampal neuron hyperexcitability through A-type K+ current inhibition mediated by activation of caspases and GSK-3

Amyloid β-protein (Aβ) pathologies have been linked to dysfunction of excitability in neurons of the hippocampal circuit, but the molecular mechanisms underlying this process are still poorly understood. Here, we applied whole-cell patch-clamp electrophysiology to primary hippocampal neurons and show that intracellular Aβ42 delivery leads to increased spike discharge and action potential broadening through downregulation of A-type K(+) currents. Pharmacologic studies showed that caspases and glycogen synthase kinase 3 (GSK-3) activation are required for these Aβ42-induced effects. Extracellular perfusion and subsequent internalization of Aβ42 increase spike discharge and promote GSK-3-dependent phosphorylation of the Kv4.2 α-subunit, a molecular determinant of A-type K(+) currents, at Ser-616. In acute hippocampal slices derived from an adult triple-transgenic Alzheimers mouse model, characterized by endogenous intracellular accumulation of Aβ42, CA1 pyramidal neurons exhibit hyperexcitability accompanied by increased phosphorylation of Kv4.2 at Ser-616. Collectively, these data suggest that intraneuronal Aβ42 accumulation leads to an intracellular cascade culminating into caspases activation and GSK-3-dependent phosphorylation of Kv4.2 channels. These findings provide new insights into the toxic mechanisms triggered by intracellular Aβ42 and offer potentially new therapeutic targets for Alzheimers disease treatment.