[Show abstract][Hide abstract] ABSTRACT: The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near −65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor–based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure–function relationships of the light-gated pore.
Full-text · Article · Dec 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The serial ordering of individual movements into sequential patterns is thought to require synaptic plasticity within corticostriatal circuits that route information through the basal ganglia. We used genetically and anatomically targeted manipulations of specific circuit elements in mice to isolate the source and target of a corticostriatal synapse that regulates the performance of a serial order task. This excitatory synapse originates in secondary motor cortex, terminates on direct pathway medium spiny neurons in the dorsolateral striatum, and is strengthened by serial order learning. This experience-dependent and synapse-specific form of plasticity may sculpt the balance of activity in basal ganglia circuits during sequential movements, driving a disparity in striatal output that favors the direct pathway. This disparity is necessary for execution of responses in serial order, even though both direct and indirect pathways are active during movement initiation, suggesting dynamic modulation of corticostriatal circuitry contributes to the choreography of behavioral routines. Many behaviors involve distinct movements performed in a specific serial order. Rothwell et al. show serial order performance is regulated by a monosynaptic pathway linking secondary motor cortex to striatal cells that form the direct pathway through the basal ganglia.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Neurons communicate with each other at specialized contact points called synapses. Presynaptic neurons store chemical neurotransmitters within presynaptic vesicles at the nerve terminal. During synaptic transmission, the presynaptic vesicles fuse with the plasma membrane, releasing their neurotransmitter content into the synaptic cleft to activate postsynaptic receptors. Neurotransmitter release is a multistage process that requires the priming of synaptic vesicles into a readily-releasable pool of vesicles. When an action potential—a transient electrical signal that travels along the neuron—invades a nerve terminal, it promotes the influx of extracellular calcium ions (Ca2+) that, in turn, trigger fusion of primed vesicles, thereby causing neurotransmitter release. Previous studies established that synaptotagmins function as Ca2+ sensors for release and, additionally, inhibit spontaneous fusion of synaptic vesicles in the absence of an action potential. In most neurons of the anterior part of the brain, two synaptotagmins, synaptotagmin-1 and -7, mediate fast and slow neurotransmitter release, respectively. We now show that in addition to their nonoverlapping roles as Ca2+ sensors and fusion clamps, synaptotagmin-1 and -7 perform an essential overlapping function in maintaining the readily-releasable pool of vesicles. This function is redundantly performed by both synaptotagmins; therefore, an impairment of the readily-releasable pool manifests only when both synaptotagmins are deleted. These results extend the functions of synaptotagmins to steps upstream of Ca2+ triggering of release and suggest that synaptotagmins, despite their simple domain structure, perform multiple sequential roles in neurotransmitter release. Thus, synaptotagmins coordinate multiple stages of Ca2+-triggered exocytosis, ensuring fast synaptic transmission for rapid information transfer between neurons at synapses.
[Show abstract][Hide abstract] ABSTRACT: On the anniversary of the Boyden et al. (2005) paper that introduced the use of channelrhodopsin in neurons, Nature Neuroscience asks selected members of the community to comment on the utility, impact and future of this important technique.
Full-text · Article · Aug 2015 · Nature Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Deciphering how neural circuits are anatomically organized with regard to input and output is instrumental in understanding how the brain processes information. For example, locus coeruleus noradrenaline (also known as norepinephrine) (LC-NE) neurons receive input from and send output to broad regions of the brain and spinal cord, and regulate diverse functions including arousal, attention, mood and sensory gating. However, it is unclear how LC-NE neurons divide up their brain-wide projection patterns and whether different LC-NE neurons receive differential input. Here we developed a set of viral-genetic tools to quantitatively analyse the input-output relationship of neural circuits, and applied these tools to dissect the LC-NE circuit in mice. Rabies-virus-based input mapping indicated that LC-NE neurons receive convergent synaptic input from many regions previously identified as sending axons to the locus coeruleus, as well as from newly identified presynaptic partners, including cerebellar Purkinje cells. The 'tracing the relationship between input and output' method (or TRIO method) enables trans-synaptic input tracing from specific subsets of neurons based on their projection and cell type. We found that LC-NE neurons projecting to diverse output regions receive mostly similar input. Projection-based viral labelling revealed that LC-NE neurons projecting to one output region also project to all brain regions we examined. Thus, the LC-NE circuit overall integrates information from, and broadcasts to, many brain regions, consistent with its primary role in regulating brain states. At the same time, we uncovered several levels of specificity in certain LC-NE sub-circuits. These tools for mapping output architecture and input-output relationship are applicable to other neuronal circuits and organisms. More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.
[Show abstract][Hide abstract] ABSTRACT: Postsynaptic remodeling of glutamatergic synapses on ventral striatum (vSTR) medium spiny neurons (MSNs) is critical for shaping stress responses. However, it is unclear which presynaptic inputs are involved. Susceptible mice exhibited increased synaptic strength at intralaminar thalamus (ILT), but not prefrontal cortex (PFC), inputs to vSTR MSNs following chronic social stress. Modulation of ILT-vSTR versus PFC-vSTR neuronal activity differentially regulated dendritic spine plasticity and social avoidance.
Full-text · Article · Jun 2015 · Nature Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Postsynaptic AMPA-type glutamate receptors (AMPARs) are among the major determinants of synaptic strength and can be trafficked into and out of synapses. Neuronal activity regulates AMPAR trafficking during synaptic plasticity to induce long-term changes in synaptic strength, including long-term potentiation (LTP) and long-term depression (LTD). Rab family GTPases regulate most membrane trafficking in eukaryotic cells; particularly, Rab11 and its effectors are implicated in mediating postsynaptic AMPAR insertion during LTP. To explore the synaptic function of Rab11Fip5, a neuronal Rab11 effector and a candidate autism-spectrum disorder gene, we performed shRNA-mediated knock-down and genetic knock-out (KO) studies. Surprisingly, we observed robust shRNA-induced synaptic phenotypes that were rescued by a Rab11Fip5 cDNA but that were nevertheless not observed in conditional KO neurons. Both in cultured neurons and acute slices, KO of Rab11Fip5 had no significant effect on basic parameters of synaptic transmission, indicating that Rab11Fip5 is not required for fundamental synaptic operations, such as neurotransmitter release or postsynaptic AMPAR insertion. KO of Rab11Fip5 did, however, abolish hippocampal LTD as measured both in acute slices or using a chemical LTD protocol in cultured neurons but did not affect hippocampal LTP. The Rab11Fip5 KO mice performed normally in several behavioral tasks, including fear conditioning, but showed enhanced contextual fear extinction. These are the first findings to suggest a requirement for Rab11Fip5, and presumably Rab11, during LTD.
No preview · Article · May 2015 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience