Massimiliano Renzi

Synaptic inhibition of Purkinje cells mediates consolidation of vestibulo-cerebellar motor learning

Although feedforward inhibition onto Purkinje cells was first documented 40 years ago, we understand little of how inhibitory interneurons contribute to cerebellar function in behaving animals. Using a mouse line (PC-Δγ2) in which GABAA receptor–mediated synaptic inhibition is selectively removed from Purkinje cells, we examined how feedforward inhibition from molecular layer interneurons regulates adaptation of the vestibulo-ocular reflex. Although impairment of baseline motor performance was relatively mild, the ability to adapt the phase of the vestibulo-ocular reflex and to consolidate gain adaptations was strongly compromised. Purkinje cells showed abnormal patterns of simple spikes, both during and in the absence of evoked compensatory eye movements. On the basis of modeling our experimental data, we propose that feedforward inhibition, by controlling the fine-scale patterns of Purkinje cell activity, enables the induction of plasticity in neurons of the cerebellar and vestibular nuclei.

From synapse to behavior: Rapid modulation of defined neuronal types with engineered GABAA receptors

In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABAA receptor, requires a phenylalanine residue (Phe77) in the γ2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase–induced swapping of the γ2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABAA receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits.

Two different ionotropic receptors are activated by ATP in rat microglia

1 Our aim was to assess whether ATP‐induced inward currents in microglia are due to a single or more than one purinergic receptor. The ATP dose‐response curve showed two components, whose presence might be due to the activation of high and low affinity receptors. 2 The P2Z/P2X7 specific receptor agonist benzoylbenzoyl‐ATP (Bz‐ATP) and some P2 receptor agonists were tested. The rank order of potency was Bz‐ATP >> ATP = 2‐methylthio‐ATP (2‐MeSATP) > α,β‐methylene ATP (α,β‐meATP) ≥ ADP. β,γ‐MethyleneATP (β,γ‐meATP), UTP and adenosine were ineffective. 3 The non‐specific P2 receptor antagonist suramin antagonized by 92 ± 2 % the inward current induced by 100 μm ATP, and by 51 ± 8 and 68 ± 6 % those induced by 3 mM ATP and 100 μm Bz‐ATP, respectively. The P2Z/P2X7 antagonist oxidized ATP (oATP) almost abolished the inward current induced by 3 mM ATP or Bz‐ATP, but was ineffective against 100 μm ATP. 4 Inward currents induced by low ATP concentrations (≤ 100 μm) were generally followed by an almost complete and irreversible desensitization, while those elicited by ATP ≥ 1 mM showed only a partial decline. Interestingly, the inward current induced by 100 μm 2‐MeSATP showed a large desensitization, while that induced by Bz‐ATP did not. 5 In voltage‐ramp experiments, the 100 μm ATP‐induced current exhibited a slight inward rectification more visible at negative potentials, while the 3 mM ATP‐induced current did not. 6 ATP induced a fast and large increase in [Ca2+] that promptly recovered in the continuous presence of low ATP doses, but did not recover in high ATP doses. As with desensitization, the response to Bz‐ATP mimicked that of high doses of ATP. 7 When Ca2+ mobilization due to P2Y receptors was blocked by thapsigargin‐induced Ca2+ depletion or by pertussis toxin treatment, 10 μm ATP was still able to induce a Ca2+ transient, which represented the contribution of the Ca2+ influx induced by P2X receptors 8 In conclusion, the inward currents and a fraction of the Ca2+ transients induced by ATP in microglia are due to at least two ATP‐sensitive receptor channel types, whose different properties and sensitivity to ATP may be associated with different functional roles.

Profound Desensitization by Ambient GABA Limits Activation of δ-Containing GABAA Receptors during Spillover

High-affinity extrasynaptic GABAA receptors (GABAARs) are a prominent feature of cerebellar granule neurons and thalamic relay neurons. In both cell types, the presence of synaptic glomeruli would be expected to promote activation of these GABAARs, contributing to phasic spillover-mediated currents and tonic inhibition. However, the precise role of different receptor subtypes in these two phenomena is unclear. To address this question, we made recordings from neurons in acute brain slices from mice, and from tsA201 cells expressing recombinant GABAARs. We found that δ subunit-containing GABAARs of both cerebellar granule neurons and thalamic relay neurons of the lateral geniculate nucleus contributed to tonic conductance caused by ambient GABA but not to spillover-mediated currents. In the presence of a low “ambient” GABA concentration, recombinant “extrasynaptic” δ subunit-containing GABAARs exhibited profound desensitization, rendering them insensitive to brief synaptic- or spillover-like GABA transients. Together, our results demonstrate that phasic spillover and tonic inhibition reflect the activation of distinct receptor populations.

Selective regulation of long-form calcium-permeable AMPA receptors by an atypical TARP, |[gamma]|-5

Although the properties and trafficking of AMPA-type glutamate receptors (AMPARs) depend critically on associated transmembrane AMPAR regulatory proteins (TARPs) such as stargazin (γ-2), no TARP has been described that can specifically regulate the important class of calcium-permeable (CP-) AMPARs. We examined the stargazin-related protein γ-5, which is highly expressed in Bergmann glia, a cell type possessing only CP-AMPARs. γ-5 was previously thought not to be a TARP, and it has been widely used as a negative control. Here we find that, contrary to expectation, γ-5 acts as a TARP and serves this role in Bergmann glia. Whereas γ-5 interacts with all AMPAR subunits, and modifies their behavior to varying extents, its main effect is to regulate the function of AMPAR subunit combinations that lack short-form subunits, which constitute predominantly CP-AMPARs. Our results suggest an important role for γ-5 in regulating the functional contribution of CP-AMPARs.

Climbing-fibre activation of NMDA receptors in Purkinje cells of adult mice

Among principal neurons, adult Purkinje cells have long been considered unusual in lacking functional NMDA receptors. This view has emerged largely from studies on rats, where NMDA receptors are expressed in Purkinje cells of newborn animals, but are lost after 2 weeks. By contrast, immunolabelling data have shown that Purkinje cells from adult mice express multiple NMDA receptor subunits, suggesting a possible species difference. To investigate the presence of functional NMDA receptors in Purkinje cells of mice, and to explore the contribution of different receptor subunits, we made whole‐cell and single‐channel patch‐clamp recordings from Purkinje cells of wild‐type and NR2D−/− mice of different ages. Here we report that multiple NMDA receptor subtypes are indeed expressed in Purkinje cells of young and adult mice; in the adult, both NR2A‐ and NR2B‐containing subtypes are present. Furthermore, we show that NMDA receptor‐mediated EPSCs can be evoked by climbing fibre stimulation, and appear to be mediated mainly by NR2A‐containing receptors.

Bidirectional plasticity of calcium-permeable AMPA receptors in oligodendrocyte lineage cells

Oligodendrocyte precursor cells (OPCs), a major glial cell type that gives rise to myelinating oligodendrocytes in the CNS, express calcium-permeable AMPA receptors (CP-AMPARs). Although CP-AMPARs are important for OPC proliferation and neuron-glia signaling, they render OPCs susceptible to ischemic damage in early development. We identified factors controlling the dynamic regulation of AMPAR subtypes in OPCs from rat optic nerve and mouse cerebellar cortex. We found that activation of group 1 mGluRs drove an increase in the proportion of CP-AMPARs, reflected by an increase in single-channel conductance and inward rectification. This plasticity required the elevation of intracellular calcium and used PI3K, PICK-1 and the JNK pathway. In white matter, neurons and astrocytes release both ATP and glutamate. Unexpectedly, activation of purinergic receptors in OPCs decreased CP-AMPAR expression, suggesting a capacity for homeostatic regulation. Finally, we found that stargazin-related transmembrane AMPAR regulatory proteins, which are critical for AMPAR surface expression in neurons, regulate CP-AMPAR plasticity in OPCs.

Setting the Time Course of Inhibitory Synaptic Currents by Mixing Multiple GABAA Receptor α Subunit Isoforms

The kinetics of IPSCs influence many neuronal processes, such as the frequencies of oscillations and the duration of shunting inhibition. The subunit composition of recombinant GABAA receptors (GABAARs) strongly affects the deactivation kinetics of GABA-evoked currents. However, for GABAergic synapses, the relationship between subunit composition and IPSC decay is less clear. Here we addressed this by combining whole-cell recordings of miniature IPSCs (mIPSCs) and quantitative immunolocalization of synaptic GABAAR subunits. In cerebellar stellate, thalamic relay, and main olfactory bulb (MOB) deep short-axon cells of Wistar rats, the only synaptic α subunit was α1, and zolpidem-sensitive mIPSCs had weighted decay time constants (τw) of 4–6 ms. Nucleus reticularis thalami neurons expressed only α3 as the synaptic α subunit and exhibited slow (τw = 28 ms), zolpidem-insensitive mIPSCs. By contrast, MOB external tufted cells contained two α subunit types (α1 and α3) at their synapses. Quantitative analysis of multiple immunolabeled images revealed small within-cell, but large between-cell, variability in synaptic α1/α3 ratios. This corresponded to large cell-to-cell variability in the decay (τw = 3–30 ms) and zolpidem sensitivity of mIPSCs. Currents evoked by rapid application of GABA to patches excised from HEK cells expressing different mixtures of α1 and α3 subunits displayed highly variable deactivation times that correlated with the α1/α3 cDNA ratio. Our results demonstrate that diversity in the decay of IPSCs can be generated by varying the expression of different GABAAR subunits that alone confer different decay kinetics, allowing the time course of inhibition to be tuned to individual cellular requirements.

Fractional Ca2+ current through human neuronal α7 nicotinic acetylcholine receptors

The neuronal alpha7 nicotinic acetylcholine (ACh) receptor is believed to be a highly Ca(2+) permeable ligand-gated receptor-channel. However, the contribution of Ca(2+) to cationic current generated by ACh has not yet been directly measured to date. Simultaneous fluorescence and whole-cell current measurements using the Ca(2+) indicator dye fura-2 were made in GH4C1 pituitary cells stably expressing human alpha7 receptors and the fractional Ca(2+) current (the proportion of whole-cell current carried by Ca(2+); P(f)) was determined. We report that the P(f) value was 11.4+/-1.3%. This value was significantly larger than P(f) of human L248Talpha7 receptor mutant (P(f)=6.3+/-1.0%) and of rat alpha7 receptor (P(f)=8.8+/-1.5%) both determined in transiently transfected GH4C1 cells. In our knowledge, the findings here reported indicate the human alpha7 receptors are the most Ca(2+) conductive homomeric ligand-gated receptor-channels expressed in a heterologous cell system.

Stimulation of chemokine CXC receptor 4 induces synaptic depression of evoked parallel fibers inputs onto Purkinje neurons in mouse cerebellum

In the present work, we studied the effects of the stimulation of the chemokine CXC receptor 4 (CXCR4) by the stromal-derived cell growth factor-1alpha (SDF-1alpha) on the evoked excitatory postsynaptic current. This was generated in Purkinje neurons (PN) from mouse cerebellar slices by the stimulation of parallel fibers. It was found that the amplitude of EPSC was reversibly reduced by SDF-1alpha application. This effect was dose-dependent (IC(50)=0.34 nM) and was abolished by the anti-CXCR4 monoclonal antibody (mAb) 12G5. This SDF-1alpha-induced synaptic depression was caused by a decrease of evoked glutamate release, rather than a decrease in the postsynaptic glutamate receptor (GluR) sensitivity, as the mean amplitude of the spontaneous EPSCs was not influenced by chemokine application. Moreover, NMDA receptors (NMDARs) are involved in EPSC depression being inhibited by the NMDAR blocker 2-amino-5-phosphonopentanoic acid (AP-5). The mechanisms by which SDF-1alpha modulates neurotransmission in the cerebellar cortex are discussed.