Acousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial str... more Acousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial structure of light beams, most notably their phase. AOD light modulation of ultra-short laser pulses, however, is not straightforward to implement because of intrinsic chromatic dispersion and non-stationarity of acousto-optic diffraction. While schemes exist to compensate chromatic dispersion, non-stationarity remains an obstacle. In this work we demonstrate an efficient AOD light modulator for stable phase modulation using time-locked generation of frequency-modulated acoustic waves at the full repetition rate of a high power laser pulse amplifier of 80 kHz. We establish the non-local relationship between the optical phase and the generating acoustic frequency function and verify the system for temporal stability, phase accuracy and generation of non-linear two-dimensional phase functions.
Two-photon scanning microscopy (TPSM) is a powerful tool for imaging deep inside living tissues w... more 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 • 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 (1 kHz). 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.
1. Activation and desensitization of N-methyl-D-aspartate (NMDA) receptors were studied in large ... more 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 o...
The cerebellum, like most sensorimotor areas of the brain, receives a serotonergic innervation fr... more The cerebellum, like most sensorimotor areas of the brain, receives a serotonergic innervation from neurons of the reticular formation. It is well established that local application of serotonin modulates the firing rate of cerebellar Purkinje cells in vivo and in vitro, but the mechanisms by which serotonin affects the cerebellar function are still poorly understood. Whereas interactions between serotonin, glutamate, and GABA have been reported to increase or decrease the firing frequency of Purkinje cells, there is little evidence for a modulation of excitatory and inhibitory synapses by serotonin in the cerebellar cortex. Changes in the intrinsic electrical properties of Purkinje cells upon application of serotonin have also been reported, but their impact on Purkinje cell firing is unclear. The recent finding that serotonin specifically modulates the activity of Lugaro cells, a class of inhibitory interneurons of the cerebellar cortex, offers new insights on the action of this neuromodulator. The peculiar axonal projection and specific interneuronal targets of the Lugaro cells suggest that the action of serotonin might occur upstream of Purkinje cells through a resetting of the computational properties of the cerebellar cortex. Understanding the mechanisms of the serotonergic modulation of the cerebellar cortex is of clinical relevance, as abnormal serotonin metabolism has been observed in animal models and pathological cases of motor disorders involving the cerebellum, and as chronic intravenous administration of L-5-hydroxytryptophan (5-HTP), a precursor of serotonin, was the first treatment shown to improve significantly cerebellar symptoms.
Cerebellar unipolar brush cells (UBCs) are glutamatergic interneurons that receive direct input f... more Cerebellar unipolar brush cells (UBCs) are glutamatergic interneurons that receive direct input from vestibular afferents in the form of a unique excitatory synapse on their dendritic brush. UBCs constitute independent relay lines for vestibular signals, and their inherent properties most likely determine how vestibular activity is encoded by the cerebellar cortex. We now demonstrate that UBCs are bimodal cells; they can either fire high-frequency bursts of action potentials when stimulated from hyperpolarized potentials or discharge tonically during sustained depolarizations. The two functional states can be triggered by physiological-like activity of the excitatory input and are encoded by distinct Ca2+-signaling systems. By combining complementary strategies, consisting of molecular and electrophysiological analysis and of ultrafast acousto-optical deflector-based two-photon imaging, we unraveled the identity and the subcellular localization of the Ca2+ conductances activating in each mode. Fast inactivating T-type Ca2+ channels produce low-threshold spikes, which trigger the high-frequency bursts and generate powerful Ca2+ transients in the brush and, to a much lesser extent, in the soma. The tonic firing mode is encoded by a signalization system principally composed of L-type channels. Ca2+ influx during tonic firing produces a linear representation of the spike rate of the cell in the form of a widespread and sustained Ca2+ concentration increase and regulates cellular excitability via BK potassium channels. The bimodal firing pattern of UBCs may underlie different coding strategies of the vestibular input by the cerebellum, thus likely increasing the computational power of this structure.
Proceedings of the National Academy of Sciences, 2013
Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorim... more 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.
Acousto-optic deflectors (AOD) are promising ultrafast scanners for non-linear microscopy. Their ... more Acousto-optic deflectors (AOD) are promising ultrafast scanners for non-linear microscopy. Their use has been limited until now by their small scanning range and by the spatial and temporal dispersions of the laser beam going through the deflectors. We show that the use of AOD of large aperture (13mm) compared to standard deflectors allows accessing much larger field of view while minimizing spatio-temporal distortions. An acousto-optic modulator (AOM) placed at distance of the AOD is used to compensate spatial and temporal dispersions. Fine tuning of the AOM-AOD setup using a frequency-resolved optical gating (GRENOUILLE) allows elimination of pulse front tilt whereas spatial chirp is minimized thanks to the large aperture AOD.
Acousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial str... more Acousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial structure of light beams, most notably their phase. AOD light modulation of ultra-short laser pulses, however, is not straightforward to implement because of intrinsic chromatic dispersion and non-stationarity of acousto-optic diffraction. While schemes exist to compensate chromatic dispersion, non-stationarity remains an obstacle. In this work we demonstrate an efficient AOD light modulator for stable phase modulation using time-locked generation of frequency-modulated acoustic waves at the full repetition rate of a high power laser pulse amplifier of 80 kHz. We establish the non-local relationship between the optical phase and the generating acoustic frequency function and verify the system for temporal stability, phase accuracy and generation of non-linear two-dimensional phase functions.
Two-photon scanning microscopy (TPSM) is a powerful tool for imaging deep inside living tissues w... more 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 • 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 (1 kHz). 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.
1. Activation and desensitization of N-methyl-D-aspartate (NMDA) receptors were studied in large ... more 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 o...
The cerebellum, like most sensorimotor areas of the brain, receives a serotonergic innervation fr... more The cerebellum, like most sensorimotor areas of the brain, receives a serotonergic innervation from neurons of the reticular formation. It is well established that local application of serotonin modulates the firing rate of cerebellar Purkinje cells in vivo and in vitro, but the mechanisms by which serotonin affects the cerebellar function are still poorly understood. Whereas interactions between serotonin, glutamate, and GABA have been reported to increase or decrease the firing frequency of Purkinje cells, there is little evidence for a modulation of excitatory and inhibitory synapses by serotonin in the cerebellar cortex. Changes in the intrinsic electrical properties of Purkinje cells upon application of serotonin have also been reported, but their impact on Purkinje cell firing is unclear. The recent finding that serotonin specifically modulates the activity of Lugaro cells, a class of inhibitory interneurons of the cerebellar cortex, offers new insights on the action of this neuromodulator. The peculiar axonal projection and specific interneuronal targets of the Lugaro cells suggest that the action of serotonin might occur upstream of Purkinje cells through a resetting of the computational properties of the cerebellar cortex. Understanding the mechanisms of the serotonergic modulation of the cerebellar cortex is of clinical relevance, as abnormal serotonin metabolism has been observed in animal models and pathological cases of motor disorders involving the cerebellum, and as chronic intravenous administration of L-5-hydroxytryptophan (5-HTP), a precursor of serotonin, was the first treatment shown to improve significantly cerebellar symptoms.
Cerebellar unipolar brush cells (UBCs) are glutamatergic interneurons that receive direct input f... more Cerebellar unipolar brush cells (UBCs) are glutamatergic interneurons that receive direct input from vestibular afferents in the form of a unique excitatory synapse on their dendritic brush. UBCs constitute independent relay lines for vestibular signals, and their inherent properties most likely determine how vestibular activity is encoded by the cerebellar cortex. We now demonstrate that UBCs are bimodal cells; they can either fire high-frequency bursts of action potentials when stimulated from hyperpolarized potentials or discharge tonically during sustained depolarizations. The two functional states can be triggered by physiological-like activity of the excitatory input and are encoded by distinct Ca2+-signaling systems. By combining complementary strategies, consisting of molecular and electrophysiological analysis and of ultrafast acousto-optical deflector-based two-photon imaging, we unraveled the identity and the subcellular localization of the Ca2+ conductances activating in each mode. Fast inactivating T-type Ca2+ channels produce low-threshold spikes, which trigger the high-frequency bursts and generate powerful Ca2+ transients in the brush and, to a much lesser extent, in the soma. The tonic firing mode is encoded by a signalization system principally composed of L-type channels. Ca2+ influx during tonic firing produces a linear representation of the spike rate of the cell in the form of a widespread and sustained Ca2+ concentration increase and regulates cellular excitability via BK potassium channels. The bimodal firing pattern of UBCs may underlie different coding strategies of the vestibular input by the cerebellum, thus likely increasing the computational power of this structure.
Proceedings of the National Academy of Sciences, 2013
Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorim... more 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.
Acousto-optic deflectors (AOD) are promising ultrafast scanners for non-linear microscopy. Their ... more Acousto-optic deflectors (AOD) are promising ultrafast scanners for non-linear microscopy. Their use has been limited until now by their small scanning range and by the spatial and temporal dispersions of the laser beam going through the deflectors. We show that the use of AOD of large aperture (13mm) compared to standard deflectors allows accessing much larger field of view while minimizing spatio-temporal distortions. An acousto-optic modulator (AOM) placed at distance of the AOD is used to compensate spatial and temporal dispersions. Fine tuning of the AOM-AOD setup using a frequency-resolved optical gating (GRENOUILLE) allows elimination of pulse front tilt whereas spatial chirp is minimized thanks to the large aperture AOD.
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