Plant phytochromes perceive red and far-red light to elicit adaptations to the changing environment. Downstream physiological responses revolve around red-light-induced interactions with phytochrome-interacting factors (PIF). Phytochromes double as thermoreceptors, owing to the pronounced temperature dependence of thermal reversion from the light-adapted Pfr to the dark-adapted Pr state. Here, we assess whether thermoreception may extend to the phytochrome:PIF interactions. While the association between Arabidopsis (Arabidopsis thaliana) PHYTOCHROME B (PhyB) and several PHYTOCHROME-INTERACTING FACTOR (PIF) variants moderately accelerates with temperature, the dissociation does more so, thus causing net destabilization of the phytochrome:PIF complex. Markedly different temperature profiles of PIF3 and PIF6 might underlie stratified temperature responses in plants. Accidentally, we identify a photoreception mechanism under strong continuous light, where the extent of phytochrome:PIF complexation decreases with red-light intensity rather than increases. Mathematical modeling rationalizes this attenuation mechanism and ties it to rapid red-light-driven Pr⇄Pfr interconversion and complex dissociation out of Pr. Varying phytochrome abundance, e.g., during diurnal and developmental cycles, and interaction dynamics, e.g., across different PIFs, modify the nature and extent of attenuation, thus permitting light-response profiles more malleable than possible for the phytochrome Pr⇄Pfr interconversion alone. Our data and analyses reveal a photoreception mechanism with implications for plant physiology, optogenetics, and biotechnological applications.

Alteration of synaptic function in the dorsal horn (DH) has been implicated as a cellular substrate for the development of neuropathic pain, but certain details remain unclear. In particular, the lack of information on the types of synapses that undergo functional changes hinders the understanding of disease pathogenesis from a synaptic plasticity perspective. Here, we addressed this issue by using optogenetic and retrograde tracing ex vivo to selectively stimulate first-order nociceptors expressing Nav1.8 (NRsNav1.8) and record the responses of spinothalamic tract neurons in spinal lamina I (L1-STTNs). We found that spared nerve injury (SNI) increased excitatory postsynaptic currents (EPSCs) in L1-STTNs evoked by photostimulation of NRsNav1.8 (referred to as Nav1.8-STTN EPSCs). This effect was accompanied by a significant change in the failure rate and paired-pulse ratio of synaptic transmission from NRsNav1.8 to L1-STTN and in the frequency (not amplitude) of spontaneous EPSCs recorded in L1-STTNs. However, no change was observed in the ratio of AMPA to NMDA receptor-mediated components of Nav1.8-STTN EPSCs or in the amplitude of unitary EPSCs constituting Nav1.8-STTN EPSCs recorded with extracellular Ca2+ replaced by Sr2+ In addition, there was a small increase (approximately 10%) in the number of L1-STTNs showing immunoreactivity for phosphorylated extracellular signal-regulated kinases in mice after SNI compared with sham. Similarly, only a small percentage of L1-STTNs showed a lower action potential threshold after SNI. In conclusion, our results show that SNI induces presynaptic modulation at NRNav1.8 (consisting of both peptidergic and nonpeptidergic nociceptors) synapses on L1-STTNs forming the lateral spinothalamic tract.

Novel stimulation methods are needed to overcome the limitations of contemporary cochlear implants. Optogenetics is a technique that confers light sensitivity to neurons via the genetic introduction of light-sensitive ion channels. By controlling neural activity with light, auditory neurons can be activated with higher spatial precision. Understanding the behaviour of opsins at high stimulation rates is an important step towards their translation. To elucidate this, we compared the temporal characteristics of auditory nerve and inferior colliculus responses to optogenetic, electrical, and combined optogenetic-electrical stimulation in virally transduced mice expressing one of two channelrhodopsins, ChR2-H134R or ChIEF, at stimulation rates up to 400 pulses per second (pps). At 100 pps, optogenetic responses in ChIEF mice demonstrated higher fidelity, less change in latency, and greater response stability compared to responses in ChR2-H134R mice, but not at higher rates. Combined stimulation improved the response characteristics in both cohorts at 400 pps, although there was no consistent facilitation of electrical responses. Despite these results, day-long stimulation (up to 13 h) led to severe and non-recoverable deterioration of the optogenetic responses. The results of this study have significant implications for the translation of optogenetic-only and combined stimulation techniques for hearing loss.

Optogenetics, the method of light-controlled regulation of cellular processes is based on the use of the channelrhodopsins that directly generate photoinduced currents. Most of the channelrhodopsin genes have been identified in the green microalgae Chlorophyta, and the demand for increasing the number of functionally characterized channelrhodopsins and the diversity of their photochemical parameters keeps growing. We performed the expression analysis of cation channelrhodopsin (CCR) genes in natural isolates of microalgae of the genera Haematococcus and Bracteacoccus from the unique Arctic Circle region. The identified full-length CCR transcript of H. lacustris is the product of alternative splicing and encodes the Hl98CCR2 protein with no photochemical activity. The 5\'-partial fragment of the B. aggregatus CCR transcript encodes the Ba34CCR protein containing a conserved TM1-TM7 membrane domain and a short cytosolic fragment. Upon heterologous expression of the TM1-TM7 fragment in CHO-K1 cell culture, light-dependent current generation was observed with the parameters corresponding to those of the CCR. The first discovered functional channelrhodopsin of Bracteacoccus has no close CCR homologues and may be of interest as a candidate for optogenetics.

New memories are integrated into prior knowledge of the world. But what if consecutive memories exert opposing demands on the host brain network? We report that acquiring a robust (food-context) memory constrains the mouse hippocampus within a population activity space of highly correlated spike trains that prevents subsequent computation of a flexible (object-location) memory. This densely correlated firing structure developed over repeated mnemonic experience, gradually coupling neurons in the superficial sublayer of the CA1 stratum pyramidale to whole-population activity. Applying hippocampal theta-driven closed-loop optogenetic suppression to mitigate this neuronal recruitment during (food-context) memory formation relaxed the topological constraint on hippocampal coactivity and restored subsequent flexible (object-location) memory. These findings uncover an organizational principle for the peer-to-peer coactivity structure of the hippocampal cell population to meet memory demands.

UNASSIGNED: Functional activation of the focal ischemic brain has been reported to improve outcomes by augmenting collateral blood flow. However, functional activation also increases metabolic demand and might thereby worsen outcomes. Indeed, preclinical and clinical reports have been conflicting. Here, we tested the effect of functional activation during acute ischemic stroke using distal middle cerebral artery occlusion in anesthetized mice.
UNASSIGNED: Using transgenic mice expressing channelrhodopsin-2 in neurons, we delivered functional activation using physiological levels of transcranial optogenetic stimulation of the moderately ischemic cortex (ie, penumbra), identified using real-time full-field laser speckle perfusion imaging during a 1-hour distal microvascular clip of the middle cerebral artery. Neuronal activation was confirmed using evoked field potentials, and infarct volumes were measured in tissue slices 48 hours later.
UNASSIGNED: Optogenetic stimulation of the penumbra was associated with more than 2-fold larger infarcts than stimulation of the contralateral homotopic region and the sham stimulation group (n=10, 7, and 9; 11.0±5.6 versus 5.1±4.3 versus 4.1±3.7 mm3; P=0.008, 1-way ANOVA). Identical stimulation in wild-type mice that do not express channelrhodopsin-2 did not have an effect. Optogenetic stimulation was associated with a small increase in penumbral perfusion that did not explain enlarged infarcts.
UNASSIGNED: Our data suggest that increased neuronal activity during acute focal arterial occlusions can be detrimental, presumably due to increased metabolic demand, and may have implications for the clinical management of hyperacute stroke patients.

Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.

Biochemical reaction networks adapt to environmental conditions by sensing chemical or physical stimuli and using tightly controlled mechanisms. While most signals come from molecules, many cells can also sense and respond to light. Among the biomolecular structures that enable light sensing, we selected a light-oxygen-voltage (LOV) domain in a previous study that tested the engineering of novel regulatory mechanisms into a nucleic acid polymerase. In this follow-up study, we studied the activities of previously selected variants in kinetic detail, and we generated additional LOV-polymerase fusion variants based on further insertion criteria. Our results provide mechanistic insights into how LOV domain insertion influences polymerase activity in a light-responsive manner: All active and photoresponsive enzyme variants studied by us to date were partially inhibited (i.e., \"turned off\") after irradiation with blue light at 470 nm, which can be explained by specific obstructions of the polymerase entry or exit structures (substrate entry channels or product exit channels, or both). Although the effects observed are moderate, we anticipate further engineering strategies that could be used to improve the extent of switchability and possibly to develop a \"turn-on mode\" insertion.

Teaching aspects of neuroscience to large undergraduate classes can be difficult in terms of the cost of equipment involved such as microscopes and electrophysiology equipment, the time taken to master techniques such as dissection or intracellular recording, and ethical concerns when using vertebrates. Here, I describe a practical that uses behavioral readouts and optogenetics on Drosophila that can be implemented with minimal cost as well as reduced ethical concerns and uses mostly observational techniques. The practical can be used to teach aspects of genetics and the tools for manipulating neuronal activity for ascribing neuronal function. The practical can be customized to fit different undergraduate levels and learning objectives.

Establishing a highly efficient photoactivatable Cre recombinase PA-Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development. This study examined the blue light-mediated optical regulation of Cre-loxP recombination using PA-Cre3.0 transgenic early mouse pre-implantation embryos. We found that inducing PA-Cre3.0 expression in the heterozygous state did not show detectable recombination activation with blue light. Conversely, in homozygous embryos, DNA recombination by PA-Cre3.0 was successfully induced by blue light and resulted in the activation of the red fluorescent protein reporter gene, while almost no leaks of Cre recombination activity were detected in embryos without light illumination. Thus, we characterize the conditions under which the PA-Cre3.0 system functions efficiently in early mouse embryos. These results are expected to provide a new optogenetic tool for certain biological studies, such as developmental process analysis and lineage tracing in early mouse embryos.