Expression Of Channelrhodopsin-2 Research Articles

Characterization of Optically and Electrically Evoked Dopamine Release in Striatal Slices from Digenic Knock-in Mice with DAT-Driven Expression of Channelrhodopsin.

Fast-scan cyclic voltammetry (FCV) is an established method to monitor increases in extracellular dopamine (DA) concentration ([DA]o) in the striatum, which is densely innervated by DA axons. Ex vivo brain slice preparations provide an opportunity to identify endogenous modulators of DA release. For these experiments, local electrical stimulation is often used to elicit release of DA, as well as other transmitters, in the striatal microcircuitry; changes in evoked increases in [DA]o after application of a pharmacological agent (e.g., a receptor antagonist) indicate a regulatory role for the transmitter system interrogated. Optogenetic methods that allow specific stimulation of DA axons provide a complementary, bottom-up approach for elucidating factors that regulate DA release. To this end, we have characterized DA release evoked by local electrical and optical stimulation in striatal slices from mice that genetically express a variant of channelrhodopsin-2 (ChR2). Evoked increases in [DA]o in the dorsal and ventral striatum (dStr and vStr) were examined in a cross of a Cre-dependent ChR2 line (“Ai32” mice) with a DAT::Cre mouse line. In dStr, repeated optical pulse-train stimulation at the same recording site resulted in rundown of evoked [DA]o using heterozygous mice, which contrasted with the stability seen with electrical stimulation. Similar rundown was seen in the presence of a nicotinic acetylcholine receptor (nAChR) antagonist, implicating the absence of concurrent nAChR activation in DA release instability in slices. Rundown with optical stimulation in dStr could be circumvented by recording from a population of sites, each stimulated only once. Same-site rundown was less pronounced with single-pulse stimulation, and a stable baseline could be attained. In vStr, stable optically evoked increases in [DA]o at single sites could be achieved using heterozygous mice, although with relatively low peak [DA]o. Low release could be overcome by using mice with a second copy of the Ai32 allele, which doubled ChR2 expression. The characteristics reported here should help future practitioners decide which Ai32;DAT::Cre genotype and recording protocol is optimal for the striatal subregion to be examined.

Volumetric Spatial Correlations of Neurovascular Coupling Studied using Single Pulse Opto-fMRI

Neurovascular coupling describes the link between neuronal activity and cerebral blood flow. This relationship has been the subject of intense scrutiny, with most previous work seeking to understand temporal correlations that describe neurovascular coupling. However, to date, the study of spatial correlations has been limited to two-dimensional mapping of neuronal or vascular derived signals emanating from the brain’s surface, using optical imaging techniques. Here, we investigate spatial correlations of neurovascular coupling in three dimensions, by applying a single 10 ms pulse of light to trigger optogenetic activation of cortical neurons transduced to express channelrhodopsin2, with concurrent fMRI. We estimated the spatial extent of increased neuronal activity using a model that takes into the account the scattering and absorption of blue light in brain tissue together with the relative density of channelrhodopsin2 expression across cortical layers. This method allows precise modulation of the volume of activated tissue in the cerebral cortex with concurrent three-dimensional mapping of functional hyperemia. Single pulse opto-fMRI minimizes adaptation, avoids heating artefacts and enables confined recruitment of the neuronal activity. Using this novel method, we present evidence for direct proportionality of volumetric spatial neurovascular coupling in the cerebral cortex.

Direct projections from hypothalamic orexin neurons to brainstem cardiac vagal neurons

Orexin neurons are known to augment the sympathetic control of cardiovascular function, however the role of orexin neurons in parasympathetic cardiac regulation remains unclear. To test the hypothesis that orexin neurons contribute to parasympathetic control we selectively expressed channelrhodopsin-2 (ChR2) in orexin neurons in orexin-Cre transgenic rats and examined postsynaptic currents in cardiac vagal neurons (CVNs) in the dorsal motor nucleus of the vagus (DMV). Simultaneous photostimulation and recording in ChR2-expressing orexin neurons in the lateral hypothalamus resulted in reliable action potential firing as well as large whole-cell currents suggesting a strong expression of ChR2 and reliable optogenetic excitation. Photostimulation of ChR2-expressing fibers in the DMV elicited short-latency (ranging from 3.2ms to 8.5ms) postsynaptic currents in 16 out of 44 CVNs tested. These responses were heterogeneous and included excitatory glutamatergic (63%) and inhibitory GABAergic (37%) postsynaptic currents. The results from this study suggest different sub-population of orexin neurons may exert diverse influences on brainstem CVNs and therefore may play distinct functional roles in parasympathetic control of the heart.

Advances in Optogenetic-based Auditory Implants

Advances in Optogenetic-based Auditory Implants

Dopamine Neuron-Specific Optogenetic Stimulation in Rhesus Macaques

SummaryOptogenetic studies in mice have revealed new relationships between well-defined neurons and brain functions. However, there are currently no means to achieve the same cell-type specificity in monkeys, which possess an expanded behavioral repertoire and closer anatomical homology to humans. Here, we present a resource for cell-type-specific channelrhodopsin expression in Rhesus monkeys and apply this technique to modulate dopamine activity and monkey choice behavior. These data show that two viral vectors label dopamine neurons with greater than 95% specificity. Infected neurons were activated by light pulses, indicating functional expression. The addition of optical stimulation to reward outcomes promoted the learning of reward-predicting stimuli at the neuronal and behavioral level. Together, these results demonstrate the feasibility of effective and selective stimulation of dopamine neurons in non-human primates and a resource that could be applied to other cell types in the monkey brain.

Task Learning Promotes Plasticity of Interneuron Connectivity Maps in the Olfactory Bulb.

Elucidating patterns of functional synaptic connectivity and deciphering mechanisms of how plasticity influences such connectivity is essential toward understanding brain function. In the mouse olfactory bulb (OB), principal neurons (mitral/tufted cells) make reciprocal connections with local inhibitory interneurons, including granule cells (GCs) and external plexiform layer (EPL) interneurons. Our current understanding of the functional connectivity between these cell types, as well as their experience-dependent plasticity, remains incomplete. By combining acousto-optic deflector-based scanning microscopy and genetically targeted expression of Channelrhodopsin-2, we mapped connections in a cell-type-specific manner between mitral cells (MCs) and GCs or between MCs and EPL interneurons. We found that EPL interneurons form broad patterns of connectivity with MCs, whereas GCs make more restricted connections with MCs. Using an olfactory associative learning paradigm, we found that these circuits displayed differential features of experience-dependent plasticity. Whereas reciprocal connectivity between MCs and EPL interneurons was nonplastic, the connections between GCs and MCs were dynamic and adaptive. Interestingly, experience-dependent plasticity of GCs occurred only in certain stages of neuronal maturation. We show that different interneuron subtypes form distinct connectivity maps and modes of experience-dependent plasticity in the OB, which may reflect their unique functional roles in information processing. Deducing how specific interneuron subtypes contribute to normal circuit function requires understanding the dynamics of their connections. In the olfactory bulb (OB), diverse interneuron subtypes vastly outnumber principal excitatory cells. By combining acousto-optic deflector-based scanning microscopy, electrophysiology, and genetically targeted expression of Channelrhodopsin-2, we mapped the functional connectivity between mitral cells (MCs) and OB interneurons in a cell-type-specific manner. We found that, whereas external plexiform layer (EPL) interneurons show broadly distributed patterns of stable connectivity with MCs, adult-born granule cells show dynamic and plastic patterns of synaptic connectivity with task learning. Together, these findings reveal the diverse roles for interneuons within sensory circuits toward information learning and processing.

Expression of Channelrhodopsin-2 Using in Suspension Electroporation for Studying the Monosynaptic Transmission in Neuronal Culture

Understanding the mechanism of synaptic transmission and its plasticity is one of the central goals of modern neuroscience. Neuronal culture is a very convenient model for studying mechanisms of synaptic transmission. However, investigation of two or more synaptically connected neurons in culture by conventional methods is a difficult task. In this study, we describe new protocol for studying the synaptic transmission between cultured neurons using in suspension electroporation for the expression of channelrhodopsin-2 (ChR2) which was applied to only certain subpopulation of cultured cells, leaving the majority of neurons completely untreated. We show that this technique allows reliable long-lasting (hours) recording of monosynaptic excitatory postsynaptic potentials (EPSPs) in cultured hippocampal neurons using a repeated light stimulation of neighboring ChR2-expressing neurons.

An Optogenetic Approach for Investigation of Excitatory and Inhibitory Network GABA Actions in Mice Expressing Channelrhodopsin-2 in GABAergic Neurons.

To investigate excitatory and inhibitory GABA actions in cortical neuronal networks, we present a novel optogenetic approach using a mouse knock-in line with conditional expression of channelrhodopsin-2 (ChR2) in GABAergic interneurons. During whole-cell recordings from hippocampal and neocortical slices from postnatal day (P) 2-P15 mice, photostimulation caused depolarization and excitation of interneurons and evoked barrages of postsynaptic GABAergic currents. Excitatory/inhibitory GABA actions on pyramidal cells were assessed by monitoring the alteration in the frequency of EPSCs during photostimulation of interneurons. We found that in slices from P2-P8 mice, photostimulation evoked an increase in EPSC frequency, whereas in P9-P15 mice the response switched to a reduction in EPSC frequency, indicating a developmental excitatory-to-inhibitory switch in GABA actions on glutamatergic neurons. Using a similar approach in urethane-anesthetized animals in vivo, we found that photostimulation of interneurons reduces EPSC frequency at ages P3-P9. Thus, expression of ChR2 in GABAergic interneurons of mice enables selective photostimulation of interneurons during the early postnatal period, and these mice display a developmental excitatory-to-inhibitory switch in GABA action in cortical slices in vitro, but so far show mainly inhibitory GABA actions on spontaneous EPSCs in the immature hippocampus and neocortex in vivo We report a novel optogenetic approach for investigating excitatory and inhibitory GABA actions in mice with conditional expression of channelrhodopsin-2 in GABAergic interneurons. This approach shows a developmental excitatory-to-inhibitory switch in the actions of GABA on glutamatergic neurons in neocortical and hippocampal slices from neonatal mouse pups in vitro, but also reveals inhibitory GABA actions in the neonatal mouse neocortex and hippocampus in vivo.

Characterization of Channelrhodopsin and Archaerhodopsin in Cholinergic Neurons of Cre-Lox Transgenic Mice

The study of cholinergic signaling in the mammalian CNS has been greatly facilitated by the advent of mouse lines that permit the expression of reporter proteins, such as opsins, in cholinergic neurons. However, the expression of opsins could potentially perturb the physiology of opsin-expressing cholinergic neurons or mouse behavior. Indeed, the published literature includes examples of cellular and behavioral perturbations in preparations designed to drive expression of opsins in cholinergic neurons. Here we investigate expression of opsins, cellular physiology of cholinergic neurons and behavior in two mouse lines, in which channelrhodopsin-2 (ChR2) and archaerhodopsin (Arch) are expressed in cholinergic neurons using the Cre-lox system. The two mouse lines were generated by crossing ChAT-Cre mice with Cre-dependent reporter lines Ai32(ChR2-YFP) and Ai35(Arch-GFP). In most mice from these crosses, we observed expression of ChR2 and Arch in only cholinergic neurons in the basal forebrain and in other putative cholinergic neurons in the forebrain. In small numbers of mice, off-target expression occurred, in which fluorescence did not appear limited to cholinergic neurons. Whole-cell recordings from fluorescently-labeled basal forebrain neurons revealed that both proteins were functional, driving depolarization (ChR2) or hyperpolarization (Arch) upon illumination, with little effect on passive membrane properties, spiking pattern or spike waveform. Finally, performance on a behavioral discrimination task was comparable to that of wild-type mice. Our results indicate that ChAT-Cre x reporter line crosses provide a simple, effective resource for driving indicator and opsin expression in cholinergic neurons with few adverse consequences and are therefore an valuable resource for studying the cholinergic system.

Brief wide-field photostimuli evoke and modulate oscillatory reverberating activity in cortical networks.

Cell assemblies manipulation by optogenetics is pivotal to advance neuroscience and neuroengineering. In in vivo applications, photostimulation often broadly addresses a population of cells simultaneously, leading to feed-forward and to reverberating responses in recurrent microcircuits. The former arise from direct activation of targets downstream, and are straightforward to interpret. The latter are consequence of feedback connectivity and may reflect a variety of time-scales and complex dynamical properties. We investigated wide-field photostimulation in cortical networks in vitro, employing substrate-integrated microelectrode arrays and long-term cultured neuronal networks. We characterized the effect of brief light pulses, while restricting the expression of channelrhodopsin to principal neurons. We evoked robust reverberating responses, oscillating in the physiological gamma frequency range, and found that such a frequency could be reliably manipulated varying the light pulse duration, not its intensity. By pharmacology, mathematical modelling, and intracellular recordings, we conclude that gamma oscillations likely emerge as in vivo from the excitatory-inhibitory interplay and that, unexpectedly, the light stimuli transiently facilitate excitatory synaptic transmission. Of relevance for in vitro models of (dys)functional cortical microcircuitry and in vivo manipulations of cell assemblies, we give for the first time evidence of network-level consequences of the alteration of synaptic physiology by optogenetics.

Cocaine Experience Enhances Thalamo-Accumbens N-Methyl-D-Aspartate Receptor Function

BackgroundExcitatory synaptic transmission in the nucleus accumbens (NAc) is a key biological substrate underlying behavioral responses to psychostimulants and susceptibility to relapse. Studies have demonstrated that cocaine induces changes in glutamatergic signaling at distinct inputs to the NAc. However, consequences of cocaine experience on synaptic transmission from the midline nuclei of the thalamus (mThal) to the NAc have yet to be reported. MethodsTo examine synapses from specific NAc core inputs, we recorded light-evoked excitatory postsynaptic currents following viral-mediated expression of channelrhodopsin-2 in the mThal, prefrontal cortex (PFC), or basolateral amygdala from acute brain slices. To identify NAc medium spiny neuron subtypes, we used mice expressing tdTomato driven by the promoter for dopamine receptor subtype 1 (D1). We recorded N-methyl-D-aspartate receptor (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) properties to evaluate synaptic adaptations induced by cocaine experience, a 5-day cocaine exposure followed by 2 weeks of abstinence. ResultsExcitatory inputs to the NAc core displayed differential NMDAR properties, and cocaine experience uniquely altered AMPAR and NMDAR properties at mThal-D1(+), mThal-D1(−), and PFC-D1(+) synapses, but not at PFC-D1(−) synapses. Finally, at mThal-D1(+) synapses, cocaine enhanced GluN2C/D function and NMDAR-dependent synaptic plasticity. ConclusionsOur results identify contrasting cocaine-induced AMPAR and NMDAR modifications at mThal-NAc and PFC-NAc core synapses. These changes include an enhancement of NMDAR function and plasticity at mThal-D1(+) synapses. Incorporation of GluN2C/D-containing NMDARs most likely underlies these phenomena and represents a potential therapeutic target for psychostimulant use disorders.

Oxytocin neuron activation prevents hypertension that occurs with chronic intermittent hypoxia/hypercapnia in rats.

Hypertension is a common outcome associated with obstructive sleep apnea (OSA), a prevalent yet poorly treated cardiovascular disease. Recent studies showed oxytocin (OXT), released from hypothalamic paraventricular nucleus (PVN) neurons, activates cardiac vagal neurons in the dorsal motor nucleus of the vagus (DMNX) and may blunt cardiovascular responses to stress. This study tests whether the release of OXT from PVN fibers in the DMNX is diminished with chronic intermittent hypoxia-hypercapnia (CIH/H) exposure, an animal model of OSA, and whether activation of PVN OXT neurons restores OXT release in the DMNX and prevents the hypertension resulting from CIH/H. To assess OXT release from PVN fibers, Chinese hamster ovarian (CHO) cells were engineered to be highly sensitive to OXT by stable expression of the human recombinant OXT receptor and the calcium indicator R-GECO1. PVN fibers in the DMNX were selectively photoactivated in vitro by expression of channelrhodopsin. The release of OXT onto CHO cells in the DMNX was blunted in rats exposed to 21 days of CIH/H. Chronic activation of PVN OXT neurons in vivo, using designer receptors exclusively activated by designer drugs, restored the release of OXT onto CHO cells in the DMNX. Chronic PVN OXT neuron activation in vivo also prevented the hypertension that occurred in conscious unrestrained telemetry-equipped sham rats exposed to 3 wk of CIH/H. These results demonstrate that chronic activation of OXT neurons restores the release of OXT from PVN fibers in the DMNX and prevents the hypertension that occurs with 3 wk of CIH/H exposure.

Cerebellar Nuclei Neurons Show Only Small Excitatory Responses to Optogenetic Olivary Stimulation in Transgenic Mice: In Vivo and In Vitro Studies.

To study the olivary input to the cerebellar nuclei (CN) we used optogenetic stimulation in transgenic mice expressing channelrhodopsin-2 (ChR2) in olivary neurons. We obtained in vivo extracellular Purkinje cell (PC) and CN recordings in anesthetized mice while stimulating the contralateral inferior olive (IO) with a blue laser (single pulse, 10–50 ms duration). Peri-stimulus histograms (PSTHs) were constructed to show the spike rate changes after optical stimulation. Among 29 CN neurons recorded, 15 showed a decrease in spike rate of variable strength and duration, and only 1 showed a transient spiking response. These results suggest that direct olivary input to CN neurons is usually overridden by stronger PC inhibition triggered by climbing fiber responses. To further investigate the direct input from the climbing fiber collaterals we also conducted whole cell recordings in brain slices, where we used local stimulation with blue light. Due to the expression of ChR2 in PC axons as well as the IO in our transgenic line, strong inhibitory responses could be readily triggered with optical stimulation (13 of 15 neurons). After blocking this inhibition with GABAzine, only in 5 of 13 CN neurons weak excitatory responses were revealed. Therefore our in vitro results support the in vivo findings that the excitatory input to CN neurons from climbing fiber collaterals in adult mice is masked by the inhibition under normal conditions.

Properties of an optogenetic model for olfactory stimulation.

In olfactory research it is difficult to deliver stimuli with defined intensity and duration to olfactory sensory neurons. Expression of channelrhodopsin 2 (ChR2) in olfactory sensory neurons provides a means to activate these neurons with light flashes. Appropriate mouse models are available. The present study explores the suitability of an established olfactory marker protein (OMP)/ChR2-yellow fluorescent protein (YFP) mouse model for ex vivo experimentation. Expression of ChR2 in sensory neurons of the main olfactory epithelium, the septal organ and vomeronasal organ is characterized. Expression pattern of ChR2 in olfactory receptor neurons and the properties of light responses indicate that light stimulation does not impact on signal transduction in the chemosensory cilia. Light-induced electro-olfactograms are characterized with light flashes of different intensities, durations and frequencies. The impact of light-induced afferent stimulation on the olfactory bulb is examined with respect to response amplitude, polarity and low-pass filtering. For the examination of sensory processing, it is helpful to deliver stimuli in precisely defined temporal and spatial patterns with accurate control of stimulus intensity. This is challenging in experiments with the mammalian olfactory system because airborne odorants have to be transported into the intricate sensory structures of the nose and must dissolve in mucus to be detected by sensory neurons. Defined and reproducible activity can be generated in olfactory sensory neurons that express the light-gated ion channel channelrhodopsin 2 (ChR2). The neurons can be stimulated by light flashes in a controlled fashion by this optogenetic approach. Here we examined the application of an olfactory marker protein (OMP)/ChR2-yellow fluorescent protein (YFP) model for ex vivo exploration of the olfactory epithelium and the olfactory bulb of the mouse. We studied the expression patterns of ChR2 in the main olfactory system, the vomeronasal system, and the septal organ, and we found that ChR2 is absent from the sensory cilia of olfactory sensory neurons. In the olfactory epithelium, we characterized light-induced electro-olfactograms with respect to peripheral encoding of stimulus intensity, stimulus duration and stimulus frequency. In acute slices of the olfactory bulb, we identified specific aspects of the ChR2-induced input signal, concerning its dynamic range, its low-pass filter property and its response to prolonged stimulation. Our study describes the performance of the OMP/ChR2-YFP model for ex vivo experimentation on the peripheral olfactory system and documents its versatility and its limitations for olfactory research.

Striatonigral control of movement velocity in mice.

The basal ganglia have long been implicated in action initiation. Using three-dimensional motion capture, we quantified the effects of optogenetic stimulation of the striatonigral (direct) pathway on movement kinematics. We generated transgenic mice with channelrhodopsin-2 expression in striatal neurons that express the D1-like dopamine receptor. With optic fibres placed in the sensorimotor striatum, an area known to contain movement velocity-related single units, photo-stimulation reliably produced movements that could be precisely quantified with our motion capture programme. A single light pulse was sufficient to elicit movements with short latencies (< 30 ms). Increasing stimulation frequency increased movement speed, with a highly linear relationship. These findings support the hypothesis that the sensorimotor striatum is part of a velocity controller that controls rate of change in body configurations.

Optogenetic Control of Mouse Outer Hair Cells

Normal hearing in mammals depends on sound amplification by outer hair cells (OHCs) presumably by their somatic motility and force production. However, the role of OHC force production in cochlear amplification and frequency tuning are not yet fully understood. Currently, available OHC manipulation techniques for physiological or clinical studies are limited by their invasive nature, lack of precision, and poor temporal-spatial resolution. To overcome these limitations, we explored an optogenetic approach based on channelrhodopsin 2 (ChR-2), a direct light-activated nonselective cation channel originally discovered in Chlamydomonas reinhardtii. Three approaches were compared: 1) adeno-associated virus-mediated in utero transfer of the ChR-2 gene into the developing murine otocyst, 2) expression of ChR-2(H134R) in an auditory cell line (HEI-OC1), and 3) expression of ChR-2 in the OHCs of a mouse line carrying a ChR-2 conditional allele. Whole cell recording showed that blue light (470 nm) elicited the typical nonselective cation current of ChR-2 with reversal potential around zero in both mouse OHCs and HEI-OC1 cells and generated depolarization in both cell types. In addition, pulsed light stimulation (10 Hz) elicited a 1:1 repetitive depolarization and ChR-2 currents in mouse OHCs and HEI-OC1 cells, respectively. The time constant of depolarization in OHCs, 1.45 ms, is 10 times faster than HEI-OC1 cells, which allowed light stimulation up to rates of 10/s to elicit corresponding membrane potential changes. Our study demonstrates that ChR-2 can successfully be expressed in mouse OHCs and HEI-OC1 cells and that these present a typical light-sensitive current and depolarization. However, the amount of ChR-2 current induced in our in vivo experiments was insufficient to result in measurable cochlear effects.

Shining light on the role of Parvalbumin interneurons in cortical spreading depression

Shining light on the role of Parvalbumin interneurons in cortical spreading depression

Shining a light on astrocytes and sleep (Commentary on Pelluru etal.).

The idea that glial cells might be important for sleep has a distinguished pedigree. More than 100 years ago, Ramon y Cajal hypothesized that morphological changes in astrocytic coverage of synapses could be the cellular mechanism gating wakefulness and sleep but, after that perhaps prescient insight (Bellesi et al. 2015), the idea languished despite intriguing suggestions and clues over the intervening years. Glial cells, for example, were known to secrete various sleep-promoting substances in vitro which, when introduced in vivo, increased sleep time or indices of sleep intensity. They were also uniquely positioned to sample synaptic and metabolic activity from neurons, and respond in their own ways to create homeostatic control of these processes. Indeed, difficult experiments in anesthetized animals showed that astrocytes buffer extracellular cation concentrations in ways that suggest that these cells influence a classic index of sleep need (EEG slow-wave activity; SWA) (reviewed by Frank, 2013). Direct evidence that glial cells really influence sleep in vivo had to wait for a study by Halassa et al. (2009). This study showed that preventing a form of glial chemical exocytosis (‘gliotransmission’) attenuated signs of sleep drive in vivo. Since that study, there has been a resurgence of interest in the role of glia and sleep. A pubmed search with ‘glial’ and ‘sleep’ in the title or abstract finds only a single study in the decade before 2009 and 59 after 2009. This raises two important questions. What does the future hold, and what have we learned? The future will include the creation of new tools to probe astrocytic function in vivo. Optogenetics is widely used to probe and manipulate neuronal circuits. It involves the expression of light-sensitive ion channels that, when stimulated by specific wavelengths of light, open the channel pore. This has proven to be a highly precise and physiologically relevant tool in neurons because neurons are excitable cells that normally operate via changes in membrane voltage. Several years ago, Deisseroth and colleagues, using an astrocyte-specific viral vector, showed that channelrhodopsin (which passes cations across the membrane) could be expressed in astrocytes in vivo. Upon light stimulation, the pore opened and gliotransmission resulted (Gradinaru et al., 2009). Thus, optogenetics might provide a novel way to interrogate glial function in sleep. This is precisely what Pelluru et al. (2015), decided to do in their study. They expressed channelrhodopsin in astrocytes in a regionally specific manner. The area they selected was the posterior hypothalamus, in astrocytes that envelop histaminergic neurons. These neurons are known to promote wakefulness; thus the idea being tested was that activation of astrocytes would dampen the activity of these neurons and increase sleep or sleep drive, and that is what they found. After verifying that viral expression of channelrhodopsin was limited to astrocytes, light stimulation increased sleep time and also non-rapid-eye movement SWA. These effects were transient, as might be expected if they were physiological as opposed to pathological. This then raises the second question: what have we learned? What we have learned is that astrocytes are capable of influencing sleep under these conditions. What we don't know is whether they do so normally. The channelrhodopsin pore is large and does not normally exist in astrocytes. Opening such pores is akin to suddenly and massively perforating the astrocytic plasma membrane, allowing a rush of cations that may never occur normally (given the normal membrane potential of glial cells). This certainly can result in gliotransmission, but possibly a number of other changes that, perhaps not surprisingly, alter surrounding neuronal activity. The use of optogenetics in astrocytes, therefore, highlights a challenge to the field: creating tools designed for glia based on glial, not neuronal, signaling and biology. Nevertheless, science progresses by such simple elemental steps. The study by Pelluru et al. is important because it pushes the envelope in what we can do to understand the role of glia and sleep. Their results clearly show what might be true. Now, collectively, we must find out whether it is.

Corticospinal axons make direct synaptic connections with spinal motoneurons innervating forearm muscles early during postnatal development in the rat.

Direct connections between corticospinal (CS) axons and motoneurons (MNs) appear to be present only in higher primates, where they are essential for discrete movement of the digits. Their presence in adult rodents was once claimed but is now questioned. We report that MNs innervating forearm muscles in infant rats receive monosynaptic input from CS axons, but MNs innervating proximal muscles do not, which is a pattern similar to that in primates. Our experiments were carefully designed to show monosynaptic connections. This entailed selective electrical and optogenetic stimulation of CS axons and recording from MNs identified by retrograde labelling from innervated muscles. Morphological evidence was also obtained for rigorous identification of CS axons and MNs. These connections would be transient and would regress later during development. These results shed light on the development and evolution of direct CS-MN connections, which serve as the basis for dexterity in humans. Recent evidence suggests there is no direct connection between corticospinal (CS) axons and spinal motoneurons (MNs) in adult rodents. We previously showed that CS synapses are present throughout the spinal cord for a time, but are eliminated from the ventral horn during development in rodents. This raises the possibility that CS axons transiently make direct connections with MNs located in the ventral horn of the spinal cord. This was tested in the present study. Using cervical cord slices prepared from rats on postnatal days (P) 7-9, CS axons were stimulated and whole cell recordings were made from MNs retrogradely labelled with fluorescent cholera toxin B subunit (CTB) injected into selected groups of muscles. To selectively activate CS axons, electrical stimulation was carefully limited to the CS tract. In addition we employed optogenetic stimulation after injecting an adeno-associated virus vector encoding channelrhodopsin-2 (ChR2) into the sensorimotor cortex on P0. We were then able to record monosynaptic excitatory postsynaptic currents from MNs innervating forearm muscles, but not from those innervating proximal muscles. We also showed close contacts between CTB-labelled MNs and CS axons labelled through introduction of fluorescent protein-conjugated synaptophysin or the ChR2 expression system. We confirmed that some of these contacts colocalized with postsynaptic density protein 95 in their partner dendrites. It is intriguing from both phylogenetic and ontogenetic viewpoints that direct and putatively transient CS-MN connections were found only on MNs innervating the forearm muscles in infant rats, as this is analogous to the connection pattern seen in adult primates.

Functional Connectome Analysis of Dopamine Neuron Glutamatergic Connections in Forebrain Regions.

Dopamine neurons are important for the control of motivated behavior and are involved in the pathophysiology of several major neuropsychiatric disorders. Recent studies have shown that some ventral midbrain dopamine neurons are capable of glutamate cotransmission. With conditional expression of channelrhodopsin in dopamine neurons, we systematically explored dopamine neuron connections in the forebrain and identified regionally specific dopamine neuron excitatory connections. Establishing that only a subset of forebrain regions receive excitatory connections from dopamine neurons will help to determine the function of dopamine neuron glutamate cotransmission, which likely involves transmission of precise temporal signals and enhancement of the dynamic range of dopamine neuron signals.