Arginine-selective modulation of the lysosomal transporter PQLC2 through a gate-tuning mechanism.

Lysosomes degrade excess or damaged cellular components and recycle their building blocks through membrane transporters. They also act as nutrient-sensing signaling hubs to coordinate cell responses. The membrane protein PQ-loop repeat-containing protein 2 (PQLC2; "picklock two") is implicated in both functions, as it exports cationic amino acids from lysosomes and serves as a receptor and amino acid sensor to recruit the C9orf72/SMCR8/WDR41 complex to lysosomes upon nutrient starvation. Its transport activity is essential for drug treatment of the rare disease cystinosis. Here, we quantitatively studied PQLC2 transport activity using electrophysiological and biochemical methods. Charge/substrate ratio, intracellular pH, and reversal potential measurements showed that it operates in a uniporter mode. Thus, PQLC2 is uncoupled from the steep lysosomal proton gradient, unlike many lysosomal transporters, enabling bidirectional cationic amino acid transport across the organelle membrane. Surprisingly, the specific presence of arginine, but not other substrates (lysine, histidine), in the discharge ("trans") compartment impaired PQLC2 transport. Kinetic modeling of the uniport cycle recapitulated the paradoxical substrate-yet-inhibitor behavior of arginine, assuming that bound arginine facilitates closing of the transporter's cytosolic gate. Arginine binding may thus tune PQLC2 gating to control its conformation, suggesting a potential mechanism for nutrient signaling by PQLC2 to its interaction partners.

Submillisecond Optogenetic Control of Neuronal Firing with Two-Photon Holographic Photoactivation of Chronos.

Optogenetic neuronal network manipulation promises to unravel a long-standing mystery in neuroscience: how does microcircuit activity relate causally to behavioral and pathological states? The challenge to evoke spikes with high spatial and temporal complexity necessitates further joint development of light-delivery approaches and custom opsins. Two-photon (2P) light-targeting strategies demonstrated in-depth generation of action potentials in photosensitive neurons both in vitro and in vivo, but thus far lack the temporal precision necessary to induce precisely timed spiking events. Here, we show that efficient current integration enabled by 2P holographic amplified laser illumination of Chronos, a highly light-sensitive and fast opsin, can evoke spikes with submillisecond precision and repeated firing up to 100 Hz in brain slices from Swiss male mice. These results pave the way for optogenetic manipulation with the spatial and temporal sophistication necessary to mimic natural microcircuit activity.SIGNIFICANCE STATEMENT To reveal causal links between neuronal activity and behavior, it is necessary to develop experimental strategies to induce spatially and temporally sophisticated perturbation of network microcircuits. Two-photon computer generated holography (2P-CGH) recently demonstrated 3D optogenetic control of selected pools of neurons with single-cell accuracy in depth in the brain. Here, we show that exciting the fast opsin Chronos with amplified laser 2P-CGH enables cellular-resolution targeting with unprecedented temporal control, driving spiking up to 100 Hz with submillisecond onset precision using low laser power densities. This system achieves a unique combination of spatial flexibility and temporal precision needed to pattern optogenetically inputs that mimic natural neuronal network activity patterns.

Computer Generated Holography with Intensity-Graded Patterns.

Computer Generated Holography achieves patterned illumination at the sample plane through phase modulation of the laser beam at the objective back aperture. This is obtained by using liquid crystal-based spatial light modulators (LC-SLMs), which modulate the spatial phase of the incident laser beam. A variety of algorithms is employed to calculate the phase modulation masks addressed to the LC-SLM. These algorithms range from simple gratings-and-lenses to generate multiple diffraction-limited spots, to iterative Fourier-transform algorithms capable of generating arbitrary illumination shapes perfectly tailored on the base of the target contour. Applications for holographic light patterning include multi-trap optical tweezers, patterned voltage imaging and optical control of neuronal excitation using uncaging or optogenetics. These past implementations of computer generated holography used binary input profile to generate binary light distribution at the sample plane. Here we demonstrate that using graded input sources, enables generating intensity graded light patterns and extend the range of application of holographic light illumination. At first, we use intensity-graded holograms to compensate for LC-SLM position dependent diffraction efficiency or sample fluorescence inhomogeneity. Finally we show that intensity-graded holography can be used to equalize photo evoked currents from cells expressing different levels of chanelrhodopsin2 (ChR2), one of the most commonly used optogenetics light gated channels, taking into account the non-linear dependence of channel opening on incident light.

Prenatal diagnosis of a variant of the azygos venous system.

BACKGROUND: The azygos venous system consists of the azygos vein on the right side and the hemiazygos and accessory hemiazygos on the left side. The azygos vein runs through the abdominal cavity along the right side of the vertebral bodies, in a cranial direction, passes through the diaphragm and reaches the mediastinum, where it forms the arch of the azygos which flows into the superior vena cava. Along its course, the azygos vein communicates with the intercostal veins on the right, the hemiazygos vein that collects blood from the left lower intercostal veins, and accessory hemiazygos vein that drains into the left upper intercostal veins. The last two, at the level of the seventh thoracic vertebra, unite and end in the azygos vein. The accessory hemiazygos vein is normally included in the length between T4 and T8. The embryological origin of the accessory hemiazygos vein is the result of an expansion in the direction of the cranial hemiazygos vein, which comes from the left upper sovracardinale vein (Dudiak et al. in Semin Roentgenol 24(1):47-55, 1989; Radiographics 11(2):233-246, 1991; Webb et al. in Am J Roentgenol 139(1):157-161, 1982). FINDINGS: This case report describes a rare variant of azygos vein system identified in prenatal diagnosis and confirmed by postnatal ultrasonography. CONCLUSIONS: The observation of the patient has excluded hemodynamic alterations associated with vascular anomaly.

Action potential-evoked and ryanodine-sensitive spontaneous Ca2+ transients at the presynaptic terminal of a developing CNS inhibitory synapse.

The existence of spontaneous calcium transients (SCaTs) dependent on intracellular store activation has been reported in putative axonal terminals of cerebellar basket interneurons. We used the two-photon imaging technique to optically identify basket terminals in acute cerebellar slices of young rats (11-16 d old) and study the properties of SCaTs unambiguously localized in these regions. The whole-cell recording configuration and preloading technique were alternatively used to load the calcium-dependent dye in the interneuron and compare SCaTs with action potential evoked calcium transients. SCaTs were observed in the basket terminals at frequencies that were significantly increased after bath application of 10 microm ryanodine and did not depend on P/Q- or N-type voltage-dependent calcium channel activation. They originated at specific sites where bursts of events with temporal separation as small as 200 msec could be generated. Their sites of origin were spaced on average 6 microm apart and were preferentially located near axonal endings. SCaTs had amplitudes comparable with those of Ca2+ rises evoked by single action potentials that lead to release of neurotransmitter, as confirmed by parallel recordings of preloaded terminals and evoked IPSCs in the postsynaptic Purkinje cells. These results support the hypothesis that SCaTs at basket terminals underlie the large miniature IPSCs characteristic of Purkinje cells.

Two-photon excitation in scattering media by spatiotemporally shaped beams and their application in optogenetic stimulation.

The use of wavefront shaping to generate extended optical excitation patterns which are confined to a predetermined volume has become commonplace on various microscopy applications. For multiphoton excitation, three-dimensional confinement can be achieved by combining the technique of temporal focusing of ultra-short pulses with different approaches for lateral light shaping, including computer generated holography or generalized phase contrast. Here we present a theoretical and experimental study on the effect of scattering on the propagation of holographic beams with and without temporal focusing. Results from fixed and acute cortical slices show that temporally focused spatial patterns are extremely robust against the effects of scattering and this permits their three-dimensionally confined excitation for depths more than 500 µm. Finally we prove the efficiency of using temporally focused holographic beams in two-photon stimulation of neurons expressing the red-shifted optogenetic channel C1V1.

Enhanced excitatory transmission at cortical synapses as the basis for facilitated spreading depression in Ca(v)2.1 knockin migraine mice.

Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemiplegic migraine type 1: FHM1) is caused by mutations in Ca(V)2.1 (P/Q-type) Ca(2+) channels. Knockin mice carrying a FHM1 mutation show increased neuronal P/Q-type current and facilitation of induction and propagation of cortical spreading depression (CSD), the phenomenon that underlies migraine aura and may activate migraine headache mechanisms. We studied cortical neurotransmission in neuronal microcultures and brain slices of FHM1 mice. We show gain of function of excitatory neurotransmission due to increased action-potential-evoked Ca(2+) influx and increased probability of glutamate release at pyramidal cell synapses but unaltered inhibitory neurotransmission at fast-spiking interneuron synapses. Using an in vitro model of CSD, we show a causative link between enhanced glutamate release and CSD facilitation. The synapse-specific effect of FHM1 mutations points to disruption of excitation-inhibition balance and neuronal hyperactivity as the basis for episodic vulnerability to CSD ignition in migraine.

The high variance of AMPA receptor- and NMDA receptor-mediated responses at single hippocampal synapses: evidence for multiquantal release.

Most of our knowledge about transmission at central synapses has been obtained by studying populations of synapses, but some important properties of synapses can be determined only by studying them individually. An important issue is whether a presynaptic action potential causes, at most, a single vesicle to be released, or whether multiquantal transmission is possible. Previous work in the CA1 region has shown that the response to stimulation of a single axon can be highly variable, apparently because it is composed of a variable number of quantal elements ( approximately 5 pA in amplitude). These quantal events have a low coefficient of variation (CV). Because the number of synaptic contacts involved is not known, the response could be because of uniquantal transmission at a varying number of synapses, or to multliquantal transmission at a single synapse. The former predicts that the CV at individual synapses should be small. We have used optical methods to measure the N-methyl-D-aspartate receptor-mediated Ca(2+) elevation at single active synapses. Our main finding is that the amplitude of nonfailure responses could be highly variable, having a CV as large as 0.63. In one fortuitous experiment, the optically studied synapse was the only active synapse, and we could therefore measure both its N-methyl-D-aspartate (NMDA) receptor- and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated signals. At this synapse, both signals varied over a 10-fold range and were highly correlated. These results strongly suggest that transmission at single CA1 synapses can be multiquantal. Furthermore, the individual quantal response is very far from saturation, allowing the effective summation of many quanta. The existence of multiquantal release has important implications for defining synaptic strength and understanding the mechanisms of synaptic plasticity.

A large sustained Ca2+ elevation occurs in unstimulated spines during the LTP pairing protocol but does not change synaptic strength.

Synapses in the CA1 region of the hippocampus undergo bidirectional synaptic modification in response to different patterns of activity. Postsynaptic Ca2+ elevation can trigger either synaptic strengthening or weakening, depending on the properties of the local Ca2+ signal. During the pairing protocol for long-term potentiation (LTP) induction, the cell is depolarized under voltage-clamp and is given low-frequency synaptic stimulation. As an initial step toward understanding the Ca2+ dynamics during this process, we used confocal microscopy to study the Ca2+ signals in spines evoked by the depolarization itself. This depolarization activates voltage-dependent Ca2+ channels (VDCC), but whether these channels inactivate rapidly or remain functional throughout the long depolarizations used in the pairing protocol remains unknown. Cells were depolarized to 0 mV for 2-3 min. This depolarization led to a large initial elevation of Ca2+ in spines that never decayed back to resting levels. The maintained signal was close to the Kd of the low-affinity (5 microM) Ca2+ dye, Magnesium Green. We attempted to determine the functional role of this elevation, using the Ca2+-channel blocker D-890. The addition of D-890 in the internal solution produced a nearly complete abolition of the Ca2+ elevation during depolarization. Under these conditions, the NMDA conductance was normal, but LTP was almost completely blocked. This might suggest the importance of VDCC in LTP; however, we found that high concentrations of D-890 can directly inhibit calmodulin protein kinase II (CaMKII), an enzyme required for LTP induction. Thus, whereas D-890 is a useful tool for blocking postsynaptic VDCC, it cannot be used to study the contribution of these channels to plasticity. We conclude that the activation of VDCC produces a large and persistent elevation of Ca2+ in all spines, but does not produce either LTP or long-term depression (LTD) in the absence of synaptic stimulation. The possible reasons for this are discussed.