Christoph Straub

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk–free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell–derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

A Transmembrane Accessory Subunit that Modulates Kainate-Type Glutamate Receptors

Glutamate receptors play major roles in excitatory transmission in the vertebrate brain. Among ionotropic glutamate receptors (AMPA, kainate, NMDA), AMPA receptors mediate fast synaptic transmission and require TARP auxiliary subunits. NMDA receptors and kainate receptors play roles in synaptic transmission, but it remains uncertain whether these ionotropic glutamate receptors also have essential subunits. Using a proteomic screen, we have identified NETO2, a brain-specific protein of unknown function, as an interactor with kainate-type glutamate receptors. NETO2 modulates the channel properties of recombinant and native kainate receptors without affecting trafficking of the receptors and also modulates kainate-receptor-mediated mEPSCs. Furthermore, we found that kainate receptors regulate the surface expression of NETO2 and that NETO2 protein levels and surface expression are decreased in mice lacking the kainate receptor GluR6. The results show that NETO2 is a kainate receptor subunit with significant effects on glutamate signaling mechanisms in brain.

Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1.

Sensing amino acids at the lysosome The mTORC1 protein kinase is a complex of proteins that functions to regulate growth and metabolism. Activity of mTORC1 is sensitive to the abundance of amino acids, but how the sensing of amino acids is coupled to the control of mTORC1 has been unclear. Wang et al. searched for predicted membrane proteins that interacted with regulators of mTORC1. They identified a protein currently known only as SLC38A9. Interaction of SLC38A9 with mTORC1 regulators was sensitive to the presence of amino acids. SLC38A9 has sequence similarity to amino acid transporters. Effects of modulation of SLC38A9 in cultured human cells indicate that it may be the sensor that connects the abundance of arginine and leucine to mTORC1 activity. Science, this issue p. 188 A possible sensor for detecting and signaling amino acid abundance is identified. [Also see Perspective by Abraham] The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase–dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid–sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

Distinct functions of kainate receptors in the brain are determined by the auxiliary subunit Neto1

Ionotropic glutamate receptors principally mediate fast excitatory transmission in the brain. Among the three classes of ionotropic glutamate receptors, kainate receptors (KARs) have a unique brain distribution, which has been historically defined by 3H-radiolabeled kainate binding. Compared with recombinant KARs expressed in heterologous cells, synaptic KARs exhibit characteristically slow rise-time and decay kinetics. However, the mechanisms responsible for these distinct KAR properties remain unclear. We found that both the high-affinity binding pattern in the mouse brain and the channel properties of native KARs are determined by the KAR auxiliary subunit Neto1. Through modulation of agonist binding affinity and off-kinetics of KARs, but not trafficking of KARs, Neto1 determined both the KAR high-affinity binding pattern and the distinctively slow kinetics of postsynaptic KARs. By regulating KAR excitatory postsynaptic current kinetics, Neto1 can control synaptic temporal summation, spike generation and fidelity.

The regulation of glutamate receptor trafficking and function by TARPs and other transmembrane auxiliary subunits.

At excitatory synapses in the brain, glutamate released from nerve terminals binds to glutamate receptors to mediate signaling between neurons. Glutamate receptors expressed in heterologous cells show ion channel activity. Recently, native glutamate receptors were shown to contain auxiliary subunits that modulate the trafficking and/or channel properties. The AMPA receptor (AMPAR) can contain TARP and CNIHs as the auxiliary subunits, whereas kainate receptor (KAR) can contain the Neto auxiliary subunit. Each of these auxiliary subunits uniquely modulates the glutamate receptors, and determines properties of native glutamate receptors. A thorough elucidation of the properties of native glutamate receptor complexes is indispensable for the understanding of the molecular machinery that regulates glutamate receptors and excitatory synaptic transmission in the brain.

Autoinactivation of Neuronal AMPA Receptors via Glutamate-Regulated TARP Interaction

Neuronal AMPA receptors autoinactivate at high concentrations of glutamate, i.e., the current declines at glutamate concentrations above 10-100 microM. The mechanisms underlying this phenomenon are unclear. Stargazin-like TARPs are AMPA receptor auxiliary subunits that modulate receptor trafficking and channel properties. Here, we found that neuronal AMPA receptors and recombinant AMPA receptors coexpressed with stargazin autoinactivate at high concentrations of glutamate, whereas recombinant AMPA receptors expressed alone do not. The reduction of currents at high glutamate concentrations is not associated with a reduction of AMPA receptor number, but rather with the loss of stargazin-associated allosteric modulation of channel gating. We show that receptor desensitization promotes the dissociation of TARP-AMPA receptor complexes in a few milliseconds. This dissociation mechanism contributes to synaptic short-term modulation. The results demonstrate a mechanism for dynamic regulation of AMPA receptor activity to tune synaptic strength.

CRISPR/Cas9-Mediated Gene Knock-Down in Post-Mitotic Neurons

The prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits.

Multiphasic Modulation of Cholinergic Interneurons by Nigrostriatal Afferents

The motor and learning functions of the striatum are critically dependent on synaptic transmission from midbrain dopamine neurons and striatal cholinergic interneurons (CINs). Both neural populations alter their discharge in vivo in response to salient sensory stimuli, albeit in opposite directions. Whereas midbrain dopamine neurons respond to salient stimuli with a brief burst of activity, CINs exhibit a distinct pause in firing that is often followed by a period of increased excitability. Although this “pause–rebound” sensory response requires dopaminergic signaling, the precise mechanisms underlying the modulation of CIN firing by dopaminergic afferents remain unclear. Here, we show that phasic activation of nigrostriatal afferents in a mouse striatal slice preparation is sufficient to evoke a pause–rebound response in CINs. Using a combination of optogenetic, electrophysiological, and pharmacological approaches, we demonstrate that synaptically released dopamine inhibits CINs through type 2 dopamine receptors, while another unidentified transmitter mediates the delayed excitation. These findings imply that, in addition to their direct effects on striatal projection neurons, midbrain dopamine neurons indirectly modulate striatal output by dynamically controlling cholinergic tone. In addition, our data suggest that phasic dopaminergic activity may directly participate in the characteristic pause–rebound sensory response that CINs exhibit in vivo in response to salient and conditioned stimuli.

Neto2 Modulation of Kainate Receptors with Different Subunit Compositions

Kainate receptors are less well understood than other glutamate receptors, and synaptic kainate receptors display properties that differ from recombinant receptors. In particular, the slow decay of kainate receptor synaptic currents contrasts with the rapid deactivation and desensitization of receptors expressed in heterologous cells. We recently identified Neuropilin and Tolloid like-2 (Neto2) as a novel accessory subunit of kainate receptors and showed that Neto2 modulates the gating kinetics of GluK2 receptors. However, the kainate receptor family consists of five different subunits (GluK1–5) that can form homomeric and heteromeric receptors with different functional properties. Here, we tested whether Neto2 modulation varies with subunit composition. Rapid application techniques were used to apply glutamate to outside–out patches that contained GluK1, GluK1/5, or GluK2/5 kainate receptors. Coexpression of Neto2 slowed desensitization to varying degrees. Responses to 1 ms pulses of glutamate were also slowed by Neto2, especially for receptors containing GluK5, as were postsynaptic currents in neurons expressing recombinant kainate receptors. In addition, Neto2 markedly increased the rate at which some receptors recovered from desensitization. These results suggest that Neto2 modulates the function of most kainate receptors.

Homeostatic control of synaptic transmission by distinct glutamate receptors.

Glutamate is the most abundant excitatory neurotransmitter in the brain, and distinct classes of glutamate receptors coordinate synaptic transmission and spike generation upon various levels of neuronal activity. However, the mechanisms remain unclear. Here, we found that loss of synaptic AMPA receptors increased kainate receptor activity in cerebellar granule cells without changing NMDA receptors. The augmentation of kainate receptor-mediated currents in the absence of AMPA receptor activity is required for spike generation and is mediated by the increased expression of the GluK5 high-affinity kainate receptor subunit. Increase in GluK5 expression is sufficient to enhance kainate receptor activity by modulating receptor channel properties, but not localization. Furthermore, we demonstrate that the combined loss of the AMPA receptor auxiliary TARPγ-2 subunit and the GluK5 subunit leads to early mouse lethality. Our findings reveal mechanisms mediated by distinct classes of postsynaptic glutamate receptors for the homeostatic maintenance of the neuronal activity.