Stéphane Dieudonné

Activation and desensitization of N‐methyl‐D‐aspartate receptors in nucleated outside‐out patches from mouse neurones.

1. Activation and desensitization of N‐methyl‐D‐aspartate (NMDA) receptors were studied in large outside‐out patches excised from cultured embryonic neurones dissociated from mouse forebrain. The patches were exposed to rapid changes of NMDA or L‐glutamate concentrations in the presence of glycine at concentrations (10‐20 microM) saturating the modulatory site of the NMDA receptor. 2. Immediately after formation of the patch the responses to NMDA and L‐glutamate showed a slow and small desensitization, even with high concentrations of agonist. During the following hour, the peak response either decreased or remained relatively stable, but in all cases the desensitization increased and accelerated until it stabilized. In this stabilized’ state, the desensitization produced by high concentrations of NMDA (1 mM) or L‐glutamate (300 microM) had an exponential time course, with a time constant of about 30 ms. The ratio of the peak over the steady‐state current was in the order of 40 for NMDA and about 30 for L‐glutamate. 3. Concentration‐response curves were built for the peak and the plateau responses, for NMDA and for L‐glutamate. The comparison of these curves indicated that (i) the EC50 of the peak (K(app) was always higher than the EC50 of the plateau (Kss); (ii) the two EC50 values for NMDA (K(app) and Kss) were higher than those for L‐glutamate; (iii) the Hill coefficient was close to 1.4 for each of the four curves. 4. The application of NMDA or L‐glutamate at a low concentration for 3 s periods reduced the response to a subsequent application of the same agonist at a saturating concentration. The IC50 of this ‘predesensitization’, termed Kpre, was lower than the EC50 of the steady‐state response, Kss. 5. The onset rates of desensitization increased with the concentration of agonist. The EC50 of this relation was close to the value of K(app). 6. The decay of the currents at the end of a 3 s application of agonist was usually well described by the sum of two exponentials both of which were faster for NMDA than for L‐glutamate. 7. The recovery from desensitization after a long (3 s) pulse of agonist was approximately exponential, with a time constant of about 0.5 s for NMDA and about 3.5 s for L‐glutamate.(ABSTRACT TRUNCATED AT 400 WORDS)

Target-Dependent Use of Coreleased Inhibitory Transmitters at Central Synapses

Corelease of GABA and glycine by mixed neurons is a prevalent mode of inhibitory transmission in the vertebrate hindbrain. However, little is known of the functional organization of mixed inhibitory networks. Golgi cells, the main inhibitory interneurons of the cerebellar granular layer, have been shown to contain GABA and glycine. We show here that, in the vestibulocerebellum, Golgi cells contact both granule cells and unipolar brush cells, which are excitatory relay interneurons for vestibular afferences. Whereas IPSCs in granule cells are mediated by GABAA receptors only, Golgi cell inhibition of unipolar brush cells is dominated by glycinergic currents. We further demonstrate that a single Golgi cell can perform pure GABAergic inhibition of granule cells and pure glycinergic inhibition of unipolar brush cells. This specialization results from the differential expression of GABAA and glycine receptors by target cells and not from a segregation of GABA and glycine in presynaptic terminals. Thus, postsynaptic selection of coreleased fast transmitters is used in the CNS to increase the diversity of individual neuronal outputs and achieve target-specific signaling in mixed inhibitory networks.

Submillisecond kinetics and low efficacy of parallel fibre‐Golgi cell synaptic currents in the rat cerebellum

1 The whole‐cell configuration of the patch clamp technique was used to record from Golgi cells in thin slices of the rat cerebellum (P12‐P25). Their active membrane properties and the input that they receive from the parallel fibres were characterized. 2 Most cells were filled with biocytin and morphologically identified by the presence of a large axonal arbor restricted to the granular layer. The morphological parameters of eighteen of the best‐preserved cells were quantified. 3 A slow capacitive current transient, characteristic of the Golgi cell axon, was used to identify Golgi cells whenever their morphology could not be preserved. 4 Golgi cells fire action potentials spontaneously at 3 ± 1.7 Hz (n= 17). Their firing frequency increases linearly with the amplitude of depolarizing current pulses and displays marked adaptation. 5 When hyperpolarized Golgi cells display an anomalous rectification which is blocked by 2 mM CsCl, indicating the presence of an Ih‐like current. 6 Golgi cells receive a spontaneous excitatory input from parallel fibres. This input is composed of small amplitude, mostly monoquantal, EPSCs. Chemical stimulation of granule cells by locally applied kainate evokes tetrodotoxin (TTX)‐dependent events with similar properties. 7 The parallel fibre‐Golgi cell EPSCs have both AMPA and NMDA components. The NMDA component is blocked by 1 mM external magnesium at ‐60 mV and decays with time constants of 31 ± 9 ms and 170 ± 15 ms (at +61 mV in the presence of magnesium). 8 In the presence of 10 μM internal spermine, the AMPA component of the spontaneous EPSCs is markedly slowed (0.96 ± 0.25 ms to 1.86 ± 0.47 ms; n= 4) and reduced in amplitude (49 ± 7 %; n= 4) when depolarizing the cell from ‐70 mV to +61 mV. 9 The decay kinetics of individual AMPA EPSCs were found to be variable, in part because of dendritic filtering. A more detailed analysis reveals that the synaptic AMPA conductances are regulated during development and close faster at days P19‐P25 than at days P13‐P16. 10 These data suggest that the efficacy of the parallel fibre‐Golgi cell input is rather low. This places strong constraints on the conditions in which the inhibitory feedback exerted by the Golgi cell can be operational. 11 The possibility is considered that the Golgi cell‐granule cell circuit shows an oscillatory behaviour. This hypothesis is discussed in relation to the models of Albus and Marr.

IPSC Kinetics at Identified GABAergic and Mixed GABAergic and Glycinergic Synapses onto Cerebellar Golgi Cells

In the rat cerebellum, Golgi cells receive serotonin-evoked inputs from Lugaro cells (L-IPSCs), in addition to spontaneous inhibitory inputs (S-IPSCs). In the present study, we analyze the pharmacology of these IPSCs and show that S-IPSCs are purely GABAergic events occurring at basket and stellate cell synapses, whereas L-IPSCs are mediated by GABA and glycine. Corelease of the two transmitters at Lugaro cell synapses is suggested by the fact that both GABAA and glycine receptors open during individual L-IPSCs. Double immunocytochemical stainings demonstrate that GABAergic and glycinergic markers are coexpressed in Lugaro cell axonal varicosities, together with the mixed vesicular inhibitory amino acid transporter. Lugaro cell varicosities are found apposed to glycine receptor (GlyR) clusters that are localized on Golgi cell dendrites and participate in postsynaptic complexes containing GABAA receptors (GABAARs) and the anchoring protein gephyrin. GABAAR and GlyR/gephyrin appear to form segregated clusters within individual postsynaptic loci. Basket and stellate cell varicosities do not face GlyR clusters. For the first time the characteristics of GABA and glycine cotransmission are compared with those of GABAergic transmission at identified inhibitory synapses converging onto the same postsynaptic neuron. The ratio of the decay times of L-IPSCs and of S-IPSCs is a constant value among Golgi cells. This indicates that, despite a high cell-to-cell variability of the overall IPSC decay kinetics, postsynaptic Golgi cells coregulate the kinetics of their two main inhibitory inputs. The glycinergic component of L-IPSCs is responsible for their slower decay, suggesting that glycinergic transmission plays a role in tuning the IPSC kinetics in neuronal networks.

Electrical Coupling Mediates Tunable Low-Frequency Oscillations and Resonance in the Cerebellar Golgi Cell Network

Tonic motor control involves oscillatory synchronization of activity at low frequency (5-30 Hz) throughout the sensorimotor system, including cerebellar areas. We investigated the mechanisms underpinning cerebellar oscillations. We found that Golgi interneurons, which gate information transfer in the cerebellar cortex input layer, are extensively coupled through electrical synapses. When depolarized in vitro, these neurons displayed low-frequency oscillatory synchronization, imposing rhythmic inhibition onto granule cells. Combining experiments and modeling, we show that electrical transmission of the spike afterhyperpolarization is the essential component for oscillatory population synchronization. Rhythmic firing arises in spite of strong heterogeneities, is frequency tuned by the mean excitatory input to Golgi cells, and displays pronounced resonance when the modeled network is driven by oscillating inputs. In vivo, unitary Golgi cell activity was found to synchronize with low-frequency LFP oscillations occurring during quiet waking. These results suggest a major role for Golgi cells in coordinating cerebellar sensorimotor integration during oscillatory interactions.

Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors

Two-photon scanning microscopy (TPSM) is a powerful tool for imaging deep inside living tissues with sub-cellular resolution. The temporal resolution of TPSM is however strongly limited by the galvanometric mirrors used to steer the laser beam. Fast physiological events can therefore only be followed by scanning repeatedly a single line within the field of view. Because acousto-optic deflectors (AODs) are non-mechanical devices, they allow access at any point within the field of view on a microsecond time scale and are therefore excellent candidates to improve the temporal resolution of TPSM. However, the use of AOD-based scanners with femtosecond pulses raises several technical difficulties. In this paper, we describe an all-digital TPSM setup based on two crossed AODs. It includes in particular an acousto-optic modulator (AOM) placed at 45 degrees with respect to the AODs to pre-compensate for the large spatial distortions of femtosecond pulses occurring in the AODs, in order to optimize the spatial resolution and the fluorescence excitation. Our setup allows recording from freely selectable point-of-interest at high speed (1kHz). By maximizing the time spent on points of interest, random-access TPSM (RA-TPSM) constitutes a promising method for multiunit recordings with millisecond resolution in biological tissues.

Functional Coupling between mGluR1 and Cav3.1 T-Type Calcium Channels Contributes to Parallel Fiber-Induced Fast Calcium Signaling within Purkinje Cell Dendritic Spines

T-type voltage-gated calcium channels are expressed in the dendrites of many neurons, although their functional interactions with postsynaptic receptors and contributions to synaptic signaling are not well understood. We combine electrophysiological and ultrafast two-photon calcium imaging to demonstrate that mGluR1 activation potentiates cerebellar Purkinje cell Cav3.1 T-type currents via a G-protein- and tyrosine-phosphatase-dependent pathway. Immunohistochemical and electron microscopic investigations on wild-type and Cav3.1 gene knock-out animals show that Cav3.1 T-type channels are preferentially expressed in Purkinje cell dendritic spines and colocalize with mGluR1s. We further demonstrate that parallel fiber stimulation induces fast subthreshold calcium signaling in dendritic spines and that the synaptic Cav3.1-mediated calcium transients are potentiated by mGluR1 selectively during bursts of excitatory parallel fiber inputs. Our data identify a new fast calcium signaling pathway in Purkinje cell dendritic spines triggered by short burst of parallel fiber inputs and mediated by T-type calcium channels and mGluR1s.

Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (RAMP) microscope

Two-photon microscopy offers the promise of monitoring brain activity at multiple locations within intact tissue. However, serial sampling of voxels has been difficult to reconcile with millisecond timescales characteristic of neuronal activity. This is due to the conflicting constraints of scanning speed and signal amplitude. The recent use of acousto-optic deflector scanning to implement random-access multiphoton microscopy (RAMP) potentially allows to preserve long illumination dwell times while sampling multiple points-of-interest at high rates. However, the real-life abilities of RAMP microscopy regarding sensitivity and phototoxicity issues, which have so far impeded prolonged optical recordings at high frame rates, have not been assessed. Here, we describe the design, implementation and characterisation of an optimised RAMP microscope. We demonstrate the application of the microscope by monitoring calcium transients in Purkinje cells and cortical pyramidal cell dendrites and spines. We quantify the illumination constraints imposed by phototoxicity and show that stable continuous high-rate recordings can be obtained. During these recordings the fluorescence signal is large enough to detect spikes with a temporal resolution limited only by the calcium dye dynamics, improving upon previous techniques by at least an order of magnitude.

Clusters of cerebellar Purkinje cells control their afferent climbing fiber discharge

Significance The inferior olive, one of the major source of inputs to the cerebellum, sends climbing fibers to Purkinje cells, the key processing units of cerebellar-dependent motor control. Using an optogenetic strategy, we demonstrate that Purkinje cells disinhibit their climbing-fiber afferents via a poly-synaptic circuit. These findings identify a functional closed-loop organization in the olivo-cerebellar circuits that is potentially important for cerebellar motor learning. Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorimotor error and clock signals that trigger motor learning by controlling cerebellar Purkinje cell synaptic plasticity and discharge. Purkinje cells target the deep cerebellar nuclei, which are the output of the cerebellum and include an inhibitory GABAergic projection to the inferior olive. This pathway identifies a potential closed loop in the olivo-cortico-nuclear network. Therefore, sets of Purkinje cells may phasically control their own climbing fiber afferents. Here, using in vitro and in vivo recordings, we describe a genetically modified mouse model that allows the specific optogenetic control of Purkinje cell discharge. Tetrode recordings in the cerebellar nuclei demonstrate that focal stimulations of Purkinje cells strongly inhibit spatially restricted sets of cerebellar nuclear neurons. Strikingly, such stimulations trigger delayed climbing-fiber input signals in the stimulated Purkinje cells. Therefore, our results demonstrate that Purkinje cells phasically control the discharge of their own olivary afferents and thus might participate in the regulation of cerebellar motor learning.

Unraveling the cerebellar cortex: Cytology and cellular physiology of large-sized interneurons in the granular layer

Neuronal network behaviors emerge from complex interactions between excitatory relay cells, principal cells and inhibitory interneurons. Therefore, characterizing homogeneous cell types and their properties is an essential step towards understanding information processing in the brain. The cerebellar cortex is generally described as a repetitive circuit composed of only five cell types. However, recent studies have revealed an unexpected diversity in the morphological, neurochemical and electrophysiological properties of the large-sized granular layer interneurons. These data are reviewed here with an emphasis on the synaptic interactions of the different cell types within the cerebellar cortex. The existence of a complex network of excitatory and inhibitory interneurons controlling the spatial and temporal pattern of granule cell firing is documented, providing insights into the cellular and synaptic processes underlying oscillations and synchronization in the cerebellar cortex.