Estelle Toulmé

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin.

We investigated the properties and regulation of P2X receptors in immortalized C8-B4 cells of cerebellar microglial origin. Resting C8-B4 cells expressed virtually no functional P2X receptors, but largely increased functional expression of P2X4 receptors within 2–6 h of entering the activated state. Using real-time polymerase chain reaction, we found that P2X4 transcripts were increased during the activated state by 2.4-fold, but this increase was not reflected by a parallel increase in total P2X4 proteins. In resting C8-B4 cells, P2X4 subunits were mainly localized within intracellular compartments, including lysosomes. We found that cell surface P2X4 receptor levels increased by ∼3.5-fold during the activated state. This change was accompanied by a decrease in the lysosomal pool of P2X4 proteins. We next exploited our findings with C8-B4 cells to investigate the mechanism by which antidepressants reduce P2X4 responses. We found little evidence to suggest that several antidepressants were antagonists of P2X4 receptors in C8-B4 cells. However, we found that moderate concentrations of the same antidepressants reduced P2X4 responses in activated microglia by affecting lysosomal function, which indirectly reduced cell surface P2X4 levels. In summary, our data suggest that activated C8-B4 cells express P2X4 receptors when the membrane insertion of these proteins by lysosomal secretion exceeds their removal, and that antidepressants indirectly reduce P2X4 responses by interfering with lysosomal trafficking.

Glutamate-Induced AMPA Receptor Desensitization Increases Their Mobility and Modulates Short-Term Plasticity through Unbinding from Stargazin

Short-term plasticity of AMPAR currents during high-frequency stimulation depends not only on presynaptic transmitter release and postsynaptic AMPAR recovery from desensitization, but also on fast AMPAR diffusion. How AMPAR diffusion within the synapse regulates synaptic transmission on the millisecond scale remains mysterious. Using single-molecule tracking, we found that, upon glutamate binding, synaptic AMPAR diffuse faster. Using AMPAR stabilized in different conformational states by point mutations and pharmacology, we show that desensitized receptors bind less stargazin and are less stabilized at the synapse than receptors in opened or closed-resting states. AMPAR mobility-mediated regulation of short-term plasticity is abrogated when the glutamate-dependent loss in AMPAR-stargazin interaction is prevented. We propose that transition from the activated to the desensitized state leads to partial loss in AMPAR-stargazin interaction that increases AMPAR mobility and allows faster recovery from desensitization-mediated synaptic depression, without affecting the overall nano-organization of AMPAR in synapses.

Selective Inactivation of Striatal FosB/ΔFosB-Expressing Neurons Alleviates L-DOPA–Induced Dyskinesia

BACKGROUND ΔFosB is a surrogate marker of L-DOPA-induced dyskinesia (LID), the unavoidable disabling consequence of Parkinsons disease L-DOPA long-term treatment. However, the relationship between the electrical activity of FosB/ΔFosB-expressing neurons and LID manifestation is unknown. METHODS We used the Daun02 prodrug-inactivation method associated with lentiviral expression of β-galactosidase under the control of the FosB promoter to investigate a causal link between the activity of FosB/ΔFosB-expressing neurons and dyskinesia severity in both rat and monkey models of Parkinsons disease and LID. Whole-cell recordings of medium spiny neurons (MSNs) were performed to assess the effects of Daun02 and daunorubicin on neuronal excitability. RESULTS We first show that daunorubicin, the active product of Daun02 metabolism by β-galactosidase, decreases the activity of MSNs in rat brain slices and that Daun02 strongly decreases the excitability of rat MSN primary cultures expressing β-galactosidase upon D1 dopamine receptor stimulation. We then demonstrate that the selective, and reversible, inhibition of FosB/ΔFosB-expressing striatal neurons with Daun02 decreases the severity of LID while improving the beneficial effect of L-DOPA. CONCLUSIONS These results establish that FosB/ΔFosB accumulation ultimately results in altered neuronal electrical properties sustaining maladaptive circuits leading not only to LID but also to a blunted response to L-DOPA. These findings further reveal that targeting dyskinesia can be achieved without reducing the antiparkinsonian properties of L-DOPA when specifically inhibiting FosB/ΔFosB-accumulating neurons.

ATP P2X Receptors Downregulate AMPA Receptor Trafficking and Postsynaptic Efficacy in Hippocampal Neurons.

P2X receptors (P2XRs) are ATP-gated cation channels widely expressed in the brain where they mediate action of extracellular ATP released by neurons or glia. Although purinergic signaling has multiple effects on synaptic transmission and plasticity, P2XR function at brain synapses remains to be established. Here, we show that activation of postsynaptic P2XRs by exogenous ATP or noradrenaline-dependent glial release of endogenous ATP decreases the amplitude of miniature excitatory postsynaptic currents and AMPA-evoked currents in cultured hippocampal neurons. We also observed a P2X-mediated depression of field potentials recorded in CA1 region from brain slices. P2X2Rs trigger dynamin-dependent internalization of AMPA receptors (AMPARs), leading to reduced surface AMPARs in dendrites and at synapses. AMPAR alteration required calcium influx through opened ATP-gated channels and phosphatase or CamKII activities. These findings indicate that postsynaptic P2XRs play a critical role in regulating the surface expression of AMPARs and thereby regulate the synaptic strength.

An intracellular motif of P2X(3) receptors is required for functional cross-talk with GABA(A) receptors in nociceptive DRG neurons.

Functional cross‐talk between structurally unrelated P2X ATP receptors and members of the ‘cys‐loop’ receptor‐channel superfamily represents a recently‐discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross‐talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross‐inhibition between P2X subtypes and GABAA receptors, suggesting a P2X subunit‐dependent mechanism. We show here that cross‐inhibition between neuronal P2X3 or P2X2+3 and GABAA receptors does not depend on chloride and calcium ions. We identified an intracellular QST386–388 motif in P2X3 subunits which is required for the functional coupling with GABAA receptors. Moreover the cross‐inhibition between native P2X3 and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABAAβ3 subunits. We provide evidence that P2X3 and GABAA receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X3‐GABAA subunit interactions underlie a pre‐synaptic cross‐talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.

Cross-talk between P2X4 and γ-aminobutyric acid, type A receptors determines synaptic efficacy at a central synapse

The essence of neuronal function is to generate outputs in response to synaptic potentials. Synaptic integration at postsynaptic sites determines neuronal outputs in the CNS. Using immunohistochemical and electrophysiological approaches, we first reveal that steroidogenic factor 1 (SF-1) green fluorescent protein (GFP)-positive neurons in the ventromedial nucleus of the hypothalamus express P2X4 subunits that are activated by exogenous ATP. Increased membrane expression of P2X4 channels by using a peptide competing with P2X4 intracellular endocytosis motif enhances neuronal excitability of SF-1 GFP-positive neurons. This increased excitability is inhibited by a P2X receptor antagonist. Furthermore, increased surface P2X4 receptor expression significantly decreases the frequency and the amplitude of GABAergic postsynaptic currents of SF-1 GFP-positive neurons. Co-immunopurification and pulldown assays reveal that P2X4 receptors complex with aminobutyric acid, type A (GABAA) receptors and demonstrate that two amino acids in the carboxyl tail of the P2X4 subunit are crucial for its physical association with GABAA receptors. Mutation of these two residues prevents the physical association, thereby blocking cross-inhibition between P2X4 and GABAA receptors. Moreover, disruption of the physical coupling using competitive peptides containing the identified motif abolishes current inhibition between P2X4 and GABAA receptors in recombinant system and P2X4 receptor-mediated GABAergic depression in SF-1 GFP-positive neurons. Our present work thus provides evidence for cross-talk between excitatory and inhibitory receptors that appears to be crucial in determining GABAergic synaptic strength at a central synapse.

Imaging P2X4 receptor lateral mobility in microglia: regulation by calcium and p38 MAPK

Background: P2X4 receptors are ATP-gated ion channels that are expressed in microglia. Results: P2X4 receptors in microglial processes display distinct types of lateral mobility that is regulated by ATP and by the activated state of microglia. Conclusion: P2X4 receptor lateral mobility is a regulated process. Significance: Regulation of P2X4 lateral mobility may be important in neuropathic pain, when microglia become activated. ATP-gated ionotropic P2X4 receptors are up-regulated in activated microglia and are critical for the development of neuropathic pain, a microglia-associated disorder. However, the nature of how plasma membrane P2X4 receptors are regulated in microglia is not fully understood. We used single-molecule imaging to track quantum dot-labeled P2X4 receptors to explore P2X4 receptor mobility in the processes of resting and activated microglia. We find that plasma membrane P2X4 receptor lateral mobility in resting microglial processes is largely random, consisting of mobile and slowly mobile receptors. Moreover, lateral mobility is P2X subunit- and cell-specific, increased in an ATP activation and calcium-dependent manner, and enhanced in activated microglia by the p38 MAPK pathway that selectively regulates slowly mobile receptors. Thus, our data indicate that P2X4 receptors are dynamically regulated mobile ATP sensors, sampling more of the plasma membrane in response to ATP and during the activated state of microglia that is associated with nervous system dysfunction.