Photocycles of Channelrhodopsin‐2

Neurons are sensitive to the relative timing of inputs, both because several inputs must coincide to reach spike threshold and because active dendritic mechanisms can amplify synchronous inputs. To determine if input synchrony can influence behavior, we trained mice to report activation of excitatory neurons in visual cortex using channelrhodopsin-2. We used light pulses that varied in duration from a few milliseconds to 100 ms and measured neuronal responses and animals' detection ability. We found detection performance was well predicted by the total amount of light delivered. Short pulses provided no behavioral advantage, even when they concentrated evoked spikes into an interval a few milliseconds long. Arranging pulses into trains of varying frequency from beta to gamma also produced no behavioral advantage. Light intensities required to drive behavior were low (at low intensities, channelrhodopsin-2 conductance varies linearly with intensity), and the accompanying changes in firing rate were small (over 100 ms, average change: 1.1 spikes per s). Firing rate changes varied linearly with pulse intensity and duration, and behavior was predicted by total spike count independent of temporal arrangement. Thus, animals' detection performance reflected the linear integration of total input over 100 ms. This behavioral linearity despite neurons' nonlinearities can be explained by a population code using noisy neurons. Ongoing background activity creates probabilistic spiking, allowing weak inputs to change spike probability linearly, with little amplification of coincident input. Summing across a population then yields a total spike count that weights inputs equally, regardless of their arrival time.neuronal circuits | population coding | optogenetics | mouse M any neurons in the brain receive thousands of inputs spread over their dendritic trees, and several of those inputs need to be active simultaneously to generate a spike reliably (1, 2). In this way, coincident synaptic inputs can be amplified relative to asynchronous inputs. In addition to this nonlinearity caused by spike threshold, other active processes such as dendritic calcium spikes (3-5) can preferentially amplify synchronous inputs. A variety of ways that timing could impact network function have been explored, including oscillatory synchronization (6, 7), strong cascading effects of individual neurons or synapses (8-11), and information encoding via temporal patterns (12). In the songbird, for example, song neurons receive precisely timed coincident input that recruits active calcium conductances, generating strong, reliable spike bursts that control the song (13, 14). However, synchrony might not always be critical for neuronal processing. Several types of models show that neuronal networks can be insensitive to precise spike timing even though individual neurons are highly sensitive. Most of these models rely on strong (15) or numerous (16-18) inputs and amplification of small perturbations, leading to chaotic network dynamics and noisy single neu...

Section: Resultssupporting

confidence: 62%

Section: Data Inmentioning

confidence: 83%

See 1 more Smart Citation

Cortical neural populations can guide behavior by integrating inputs linearly, independent of synchrony

Histed

Maunsell

2013

“…In contrast, in CrChR2 two conducting states have been recognized by their different relative permeability to H + and Na + (23,24). A model of two interconnected photocycles, each of which contains a closed (nonconducting) state and an open (conducting) state, has been developed for CCRs (25)(26)(27). However, single-turnover photocurrents generated by CrChR2 upon laser excitation decay monoexponentially (28,29), which means that the second conducting state accumulates only under continuous light, indicating that the second state requires secondary photochemistry.…”

Section: Discussionmentioning

confidence: 99%

Gating mechanisms of a natural anion channelrhodopsin

Sineshchekov

Govorunova

et al. 2015

102

Anion channelrhodopsins (ACRs) are a class of light-gated channels recently identified in cryptophyte algae that provide unprecedented fast and powerful hyperpolarizing tools for optogenetics. Analysis of photocurrents generated by Guillardia theta ACR 1 (GtACR1) and its mutants in response to laser flashes showed that GtACR1 gating comprises two separate mechanisms with opposite dependencies on the membrane voltage and pH and involving different amino acid residues. The first mechanism, characterized by slow opening and fast closing of the channel, is regulated by Glu-68. Neutralization of this residue (the E68Q mutation) specifically suppressed this first mechanism, but did not eliminate it completely at high pH. Our data indicate the involvement of another, yetunidentified pH-sensitive group X. Introducing a positive charge at the Glu-68 site (the E68R mutation) inverted the channel gating so that it was open in the dark and closed in the light, without altering its ion selectivity. The second mechanism, characterized by fast opening and slow closing of the channel, was not substantially affected by the E68Q mutation, but was controlled by Cys-102. The C102A mutation reduced the rate of channel closing by the second mechanism by ∼100-fold, whereas it had only a twofold effect on the rate of the first. The results show that anion conductance by ACRs has a fundamentally different structural basis than the relatively well studied conductance by cation channelrhodopsins (CCRs), not attributable to simply a modification of the CCR selectivity filter.ion transport | channel gating | channelrhodopsins | optogenetics R ecently we reported a class of rhodopsins from the cryptophyte alga Guillardia theta that act as anion-conducting channels when expressed in cultured animal cells and therefore can be used to suppress neuronal firing by light (1). These proteinsnamed anion channelrhodopsins (ACRs)-show distant sequence homology to cation channelrhodopsins (CCRs) from chlorophyte (green) algae (2), but completely lack permeability for protons and metal cations. This property, as well as large current amplitudes and fast kinetics, makes ACRs superior hyperpolarizing optogenetic tools, compared with proton and chloride pumps (3, 4), or engineered Cl − -conducting CCR variants (5, 6). Two G. theta (Gt) ACRs differ in their spectral sensitivity and channel kinetics (1). GtACR1, with its absorption peak at 515 nm (Fig. S1), has an advantage over a more blue-shifted GtACR2 with maximal efficiency at 470 nm, because it allows using light of longer wavelengths that is less scattered by biological tissue.Because a CCR could be altered to exhibit Cl − channel activity by mutation of a single amino acid residue (5), a fundamental question about ACRs is whether they differ from CCRs only by their selectivity filter. In our search for an answer, we analyzed photocurrents generated by GtACR1 in HEK293 cells under single-turnover conditions in which secondary photochemistry does not complicate the photocycle. We found that GtACR1 cond...

“…Note that the strength of the connections is scaled by the number of neurons that send the afferents. The ChR2 depolarization is modeled by only two states (open and closed) because we are analyzing the slow dynamics of the system (30). Therefore, ChR2 is modeled using I ChR2 ¼ g ChR2 hðtÞðV S − 0 mVÞ, where the reverse potential is set to 0 mV as suggested by Talathi …”

Section: Methodsmentioning

confidence: 99%

Mechanism for Hypocretin-mediated sleep-to-wake transitions

Carter

Brill

Bonnavion³

et al. 2012

248

203

Current models of sleep/wake regulation posit that Hypocretin (Hcrt)-expressing neurons in the lateral hypothalamus promote and stabilize wakefulness by projecting to subcortical arousal centers. However, the critical downstream effectors of Hcrt neurons are unknown. Here we use optogenetic, pharmacological, and computational tools to investigate the functional connectivity between Hcrt neurons and downstream noradrenergic neurons in the locus coeruleus (LC) during nonrapid eye movement (NREM) sleep. We found that photoinhibiting LC neurons during Hcrt stimulation blocked Hcrt-mediated sleep-to-wake transitions. In contrast, when LC neurons were optically stimulated to increase membrane excitability, concomitant photostimulation of Hcrt neurons significantly increased the probability of sleep-to-wake transitions compared with Hcrt stimulation alone. We also built a conductance-based computational model of Hcrt-LC circuitry that recapitulates our behavioral results using LC neurons as the main effectors of Hcrt signaling. These results establish the Hcrt-LC connection as a critical integrator-effector circuit that regulates NREM sleep/wake behavior during the inactive period. This coupling of distinct neuronal systems can be generalized to other hypothalamic integrator nuclei with downstream effector/output populations in the brain.ChR2 | norepinephrine | step function opsin T he neural basis of wakefulness and arousal is thought to depend on subcortical populations of "arousal-promoting" nuclei located in the hypothalamus and brainstem (1, 2). Neurons in the lateral hypothalamus that express hypocretins (Hcrts-also called "orexins"), a pair of neuropeptides produced from the same genetic precursor (3, 4), have been proposed to play a key role in stabilizing wake states by directly projecting throughout the brain to other arousal populations (1,5,6). Electrophysiological recordings of Hcrt neurons show that they are relatively silent during sleep compared with wakefulness, with phasic bursts of activity preceding transitions to wakefulness (7,8). Loss-of-function perturbation of the Hcrts or their receptors causes a narcolepsy phenotype (9-11). When centrally administered, the Hcrts increase the time spent awake and decrease nonrapid eye movement (NREM) and rapid eye movement (REM) sleep (12, 13).Although it is evident that Hcrts promote wakefulness and arousal, it is unknown which downstream structures are necessary and/or sufficient to mediate their effects. Hcrt neurons project diffusely throughout the brain (14), and it is possible that their effects on wakefulness are due to widespread, global postsynaptic targets. Alternatively, Hcrts may affect arousal primarily by projecting to key downstream arousal centers. An intriguing possibility is that Hcrt neurons affect wakefulness by projecting to the locus coeruleus (LC), a noradrenergic structure in the brainstem known to promote wakefulness and arousal (15, 16). Indeed, the LC receives the densest afferent projections from Hcrt neurons (14, 17). Application o...