A sensitive and specific genetically-encoded potassium ion biosensor for in vivo applications across the tree of life.

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.

Mapping the Dynamic Recruitment of Spinal Neurons during Fictive Locomotion.

The basic rhythmic activity that underlies stepping is generated by a neural network, situated in the spinal cord, known as the locomotor central pattern generator (CPG). While a series of lesion experiments have demonstrated that the mammalian locomotor CPG is distributed throughout the ventral portion of the caudal spinal cord, the specific transverse distribution of this neural network is unclear. Here we evoke fictive locomotor activity of various frequencies in upright spinal cords prepared from male and female neonatal mice. This preparation enables us to use an imaging approach to identify locomotor-related cells across the transverse plane of the spinal cord. Results indicate that there is a clear shift in the recruitment of cells toward the ventromedial, and away from the ventrolateral, spinal cord as the frequency of fictive locomotion increases. Surprisingly, the analysis of multiple frequencies of fictive locomotion in the same spinal cord indicates that few neurons are involved in locomotor outputs across multiple speeds. Collectively, these experiments allow us to map the transverse distribution of the locomotor CPG and highlight the pattern of dynamic recruitment that occurs within this neural circuit as the frequency is altered. Our findings are consistent with data indicating that there is a speed-dependent recruitment of interneuronal populations during locomotion and suggest that the locomotor CPG is not a static network, but rather the specific cells recruited vary extensively based on demand.SIGNIFICANCE STATEMENT In this article, we use an imaging approach to identify all those cells that are rhythmically active at the same frequency as fictive locomotion recorded from the ventral roots of the isolated spinal cord. These experiments allow us to map the distribution of locomotor-related cells across the transverse plane of the spinal cord and identify the recruitment pattern of these cells as the frequency of locomotor outputs is altered. Our results indicate that there are drastic changes in the specific neurons activated at different frequencies and provide support for the concept that the locomotor central pattern generator is a modular network with speed-dependent recruitment of interneuronal components.

Recent Insights into the Rhythmogenic Core of the Locomotor CPG.

In order for locomotion to occur, a complex pattern of muscle activation is required. For more than a century, it has been known that the timing and pattern of stepping movements in mammals are generated by neural networks known as central pattern generators (CPGs), which comprise multiple interneuron cell types located entirely within the spinal cord. A genetic approach has recently been successful in identifying several populations of spinal neurons that make up this neural network, as well as the specific role they play during stepping. In spite of this progress, the identity of the neurons responsible for generating the locomotor rhythm and the manner in which they are interconnected have yet to be deciphered. In this review, we summarize key features considered to be expressed by locomotor rhythm-generating neurons and describe the different genetically defined classes of interneurons which have been proposed to be involved.

Correction to: A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.

WT1-Expressing Interneurons Regulate Left-Right Alternation during Mammalian Locomotor Activity.

The basic pattern of activity underlying stepping in mammals is generated by a neural network located in the caudal spinal cord. Within this network, the specific circuitry coordinating left-right alternation has been shown to involve several groups of molecularly defined interneurons. Here we characterize a population of spinal neurons that express the Wilms' tumor 1 (WT1) gene and investigate their role during locomotor activity in mice of both sexes. We demonstrate that WT1-expressing cells are located in the ventromedial region of the spinal cord of mice and are also present in the human spinal cord. In the mouse, these cells are inhibitory, project axons to the contralateral spinal cord, terminate in close proximity to other commissural interneuron subtypes, and are essential for appropriate left-right alternation during locomotion. In addition to identifying WT1-expressing interneurons as a key component of the locomotor circuitry, this study provides insight into the manner in which several populations of molecularly defined interneurons are interconnected to generate coordinated motor activity on either side of the body during stepping.SIGNIFICANCE STATEMENT In this study, we characterize WT1-expressing spinal interneurons in mice and demonstrate that they are commissurally projecting and inhibitory. Silencing of this neuronal population during a locomotor task results in a complete breakdown of left-right alternation, whereas flexor-extensor alternation was not significantly affected. Axons of WT1 neurons are shown to terminate nearby commissural interneurons, which coordinate motoneuron activity during locomotion, and presumably regulate their activity. Finally, the WT1 gene is shown to be present in the spinal cord of humans, raising the possibility of functional homology between these species. This study not only identifies a key component of the locomotor circuitry but also begins to unravel the connectivity among the growing number of molecularly defined interneurons that comprise this neural network.

Receptor dependence of BDNF actions in superficial dorsal horn: relation to central sensitization and actions of macrophage colony stimulating factor 1.

Peripheral nerve injury elicits an enduring increase in the excitability of the spinal dorsal horn. This change, which contributes to the development of neuropathic pain, is a consequence of release and prolonged exposure of dorsal horn neurons to various neurotrophins and cytokines. We have shown in rats that nerve injury increases excitatory synaptic drive to excitatory neurons but decreases drive to inhibitory neurons. Both effects, which contribute to an increase in dorsal horn excitability, appear to be mediated by microglia-derived BDNF. We have used multiphoton Ca2+ imaging and whole cell recording of spontaneous excitatory postsynaptic currents in defined-medium organotypic cultures of GAD67-GFP+ mice spinal cord to determine the receptor dependence of these opposing actions of BDNF. In mice, as in rats, BDNF enhances excitatory transmission onto excitatory neurons. This is mediated via presynaptic TrkB and p75 neurotrophin receptors and exclusively by postsynaptic TrkB. By contrast with findings from rats, in mice BDNF does not decrease excitation of inhibitory neurons. The cytokine macrophage colony-stimulating factor 1 (CSF-1) has also been implicated in the onset of neuropathic pain. Nerve injury provokes its de novo synthesis in primary afferents, its release in spinal cord, and activation of microglia. We now show that CSF-1 increases excitatory drive to excitatory neurons via a BDNF-dependent mechanism and decreases excitatory drive to inhibitory neurons via BDNF-independent processes. Our findings complete missing steps in the cascade of events whereby peripheral nerve injury instigates increased dorsal horn excitability in the context of central sensitization and the onset of neuropathic pain. NEW & NOTEWORTHY Nerve injury provokes synthesis of macrophage colony-stimulating factor 1 (CSF-1) in primary afferents and its release in the dorsal horn. We show that CSF-1 increases excitatory drive to excitatory dorsal horn neurons via BDNF activation of postsynaptic TrkB and presynaptic TrkB and p75 neurotrophin receptors. CSF-1 decreases excitatory drive to inhibitory neurons via a BDNF-independent processes. This completes missing steps in understanding how peripheral injury instigates central sensitization and the onset of neuropathic pain.

A Bioluminescent Ca2+ Indicator Based on a Topological Variant of GCaMP6s.

Fluorescent genetically encoded calcium ion indicators (GECIs) enable Ca2+ dynamics to be monitored in a diverse array of cell types and tissues. One drawback of green fluorescent GECIs, such as the widely used GCaMP6, is that the blue wavelengths of light used to excite the GECI also activate optogenetic actuators such as channelrhodopsins. Accordingly, it is particularly challenging simultaneously to use both optogenetic actuators and GECIs to both control and image cell signaling. Bioluminescence is an alternative imaging modality that circumvents this problem by avoiding the need for illumination for fluorescence excitation. Here, we report the development of a bioluminescent GECI, designated LUCI-GECO1, based on efficient bioluminescent resonance energy transfer (BRET) between the NanoLuc luciferase and a topological variant of GCaMP6s. LUCI-GECO1 is a sensitive ratiometric GECI that retains the highly optimized properties of GCaMP6s, as we demonstrate by imaging of chemically and optogenetically induced Ca2+ concentration changes in cultured cells and neurons.

A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

BACKGROUND: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. RESULTS: Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. CONCLUSION: K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.

Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+ -dependent P2Y1 receptor mechanism.

KEY POINTS: The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre-Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y1 receptor-mediated release of Ca2+ from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure. ABSTRACT: The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady-state phase II) that can be life-threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre-Bötzinger Complex (preBötC) inspiratory rhythm-generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia-induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor-mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+ ]i ) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.

Altered development in GABA co-release shapes glycinergic synaptic currents in cultured spinal slices of the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

KEY POINTS: Increased environmental risk factors in conjunction with genetic susceptibility have been proposed with respect to the remarkable variations in mortality in amyotrophic lateral sclerosis (ALS). In vitro models allow the investigation of the genetically modified counter-regulator of motoneuron toxicity and may help in addressing ALS therapy. Spinal organotypic slice cultures from a mutant form of human superoxide dismutase 1 (SOD1G93A) mouse model of ALS allow the detection of altered glycinergic inhibition in spinal microcircuits. This altered inhibition improved spinal cord excitability, affecting motor outputs in early SOD1(G93A) pathogenesis. ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurological disease characterized by a progressive degeneration of motoneurons (MNs). In a previous study, we developed organotypic spinal cultures from an ALS mouse model expressing a mutant form of human superoxide dismutase 1 (SOD1(G93A) ). We reported the presence of a significant synaptic rearrangement expressed by these embryonic cultured networks, which may lead to the altered development of spinal synaptic signalling, which is potentially linked to the adult disease phenotype. Recent studies on the same ALS mouse model reported a selective loss of glycinergic innervation in cultured MNs, suggestive of a contribution of synaptic inhibition to MN dysfunction and degeneration. In the present study, we further exploit organotypic cultures from wild-type and SOD1(G93A) mice to investigate the development of glycine-receptor-mediated synaptic currents recorded from the interneurons of the premotor ventral circuits. We performed single cell electrophysiology, immunocytochemistry and confocal microscopy and suggest that GABA co-release may speed the decay of glycine responses altering both temporal precision and signal integration in SOD1(G93A) developing networks at the postsynaptic site. Our hypothesis is supported by the finding of an increased MN bursting activity in immature SOD1(G93A) spinal cords and by immunofluorescence microscopy detection of a longer persistence of GABA in SOD1(G93A) glycinergic terminals in cultured and ex vivo spinal slices.

NMDA Enhances and Glutamate Attenuates Synchrony of Spontaneous Phase-Locked Locus Coeruleus Network Rhythm in Newborn Rat Brain Slices.

Locus coeruleus (LC) neurons are controlled by glutamatergic inputs. Here, we studied in brain slices of neonatal rats NMDA and glutamate effects on phase-locked LC neuron spiking at ~1 Hz summating to ~0.2 s-lasting bell-shaped local field potential (LFP). NMDA: 10 µM accelerated LFP 1.7-fold, whereas 25 and 50 µM, respectively, increased its rate 3.2- and 4.6-fold while merging discrete events into 43 and 56% shorter oscillations. After 4-6 min, LFP oscillations stopped every 6 s for 1 s, resulting in 'oscillation trains'. A dose of 32 µM depolarized neurons by 8.4 mV to cause 7.2-fold accelerated spiking at reduced jitter and enhanced synchrony with the LFP, as evident from cross-correlation. Glutamate: 25-50 µM made rhythm more irregular and the LFP pattern could transform into 2.7-fold longer-lasting multipeak discharge. In 100 µM, LFP amplitude and duration declined. In 25-50 µM, neurons depolarized by 5 mV to cause 3.7-fold acceleration of spiking that was less synchronized with LFP. Both agents: evoked 'post-agonist depression' of LFP that correlated with the amplitude and kinetics of Vm hyperpolarization. The findings show that accelerated spiking during NMDA and glutamate is associated with enhanced or attenuated LC synchrony, respectively, causing distinct LFP pattern transformations. Shaping of LC population discharge dynamics by ionotropic glutamate receptors potentially fine-tunes its influence on brain functions.

TARP mediation of accelerated and more regular locus coeruleus network bursting in neonatal rat brain slices.

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARP) increase neuronal excitability. However, it is unknown how TARP affect rhythmic neural network activity. Here we studied TARP effects on local field potential (LFP) bursting, membrane potential and cytosolic Ca2+ (Cai) in locus coeruleus neurons of newborn rat brain slices. LFP bursting was not affected by the unselective competitive ionotropic glutamate receptor antagonist kynurenic acid (2.5 mM). TARP-AMPAR complex activation with 25 µM CNQX accelerated LFP rhythm 2.2-fold and decreased its irregularity score from 63 to 9. Neuronal spiking was correspondingly 2.3-fold accelerated in association with a 2-5 mV depolarization and a modest Cai rise whereas Cai was unchanged in neighboring astrocytes. After blocking rhythmic activities with tetrodotoxin (1 µM), CNQX caused a 5-8 mV depolarization and also the Cai rise persisted. In tetrodotoxin, both responses were abolished by the non-competitive AMPAR antagonist GYKI 53655 (25 µM) which also reversed stimulatory CNQX effects in control solution. The CNQX-evoked Cai rise was blocked by the L-type voltage-activated Ca2+ channel inhibitor nifedipine (100 µM). The findings show that ionotropic glutamate receptor-independent neonatal locus coeruleus network bursting is accelerated and becomes more regular by activating a TARP-AMPAR complex. The associated depolarization-evoked L-type Ca2+ channel-mediated neuronal Cai rise may be pivotal to regulate locus coeruleus activity in cooperation with SK-type K+ channels. In summary, this is the first demonstration of TARP-mediated stimulation of neural network bursting. We hypothesize that TARP-AMPAR stimulation of rhythmic locus coeruleus output serves to fine-tune its control of multiple brain functions thus comprising a target for drug discovery.

Using an upright preparation to identify and characterize locomotor related neurons across the transverse plane of the neonatal mouse spinal cord.

BACKGROUND: The basic rhythmicity underlying stepping in mammals is generated by a neural network, situated in the spinal cord, known as the locomotor central pattern generator (CPG). While a molecular approach has provided information regarding neuronal populations that participate in locomotor activity and their specific function, the distributed nature of the locomotor CPG has made it difficult to identify and characterize the specific neurons belonging to each population that are rhythmically-active during stepping. NEW METHOD: We describe a preparation in which we isolate the spinal cord from a neonatal mouse, section it at a lumbar segment, situate it in an upright orientation under the objective lens of a 2- photon microscope, and evoke fictive locomotion. RESULTS: This preparation allows us to image rhythmic Ca2+ oscillations in spinal neurons, and visually identify those that are involved in fictive locomotor activity. We can then characterize unique features of these neurons. COMPARISON WITH EXISTING METHODS: This builds on existing fictive locomotor preparations and is the first which allows for the visual identification of locomotor related neurons spanning the transverse plane of the spinal cord, facilitating their electrophysiological and anatomical characterization CONCLUSIONS: This approach promises to provide new information regarding the distribution of the locomotor CPG in the transverse plane, the characteristics of its component interneurons, as well as the cellular mechanisms and network properties which underlie rhythm generation. By altering the location of Ca2+ indicator application it can also be used to identify and characterize neurons involved in other facets of sensorimotor processing.

Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration.

Potassium ion (K+) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K+ indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are Förster resonance energy transfer (FRET)-based indicators with a bacterial K+ binding protein (Kbp) inserting between the fluorescent protein FRET pairs mCerulean3/cp173Venus and Clover/mRuby2, respectively. GINKO1 (green indicator of K+ for optical imaging) is a single fluorescent protein-based K+ indicator constructed by insertion of Kbp into enhanced green fluorescent protein (EGFP). These indicators are suitable for detecting K+ at physiologically relevant concentrations in vitro and in cells. KIRIN1 enabled imaging of cytosolic K+ depletion in live cells and K+ efflux and reuptake in cultured neurons. GINKO1, in conjunction with red fluorescent Ca2+ indicator, enable dual-color imaging of K+ and Ca2+ dynamics in neurons and glial cells. These results demonstrate that KIRIN1 and GINKO1 are useful tools for imaging intracellular K+ dynamics.

Suction electrode recording in locus coeruleus of newborn rat brain slices reveals network bursting comprising summated non-synchronous spiking.

The brainstem locus coeruleus (LC) controling behaviors like arousal, sleep, breathing, pain or opioid withdrawal is an established model for spontaneous action potential synchronization. Such synchronous 'spiking' might produce an extracellular field potential (FP) which is a crucial tool for neural network analyses. We found using ≥10 µm tip diameter suction electrodes in newborn rat brainstem slices that the LC generates at ∼1 Hz a robust rhythmic FP (rFP). During distinct rFP phases, LC neurons discharge with a jitter of ±33 ms single spikes that summate to a ∼200 ms-lasting population burst. The rFP is abolished by blocking voltage-gated Na+ channels with tetrodotoxin (TTX, 50 nM) or gap junctions with mefloquine (100 µM) and activating µ-opioid receptors with [D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin (DAMGO, 1 µM). Raising superfusate K+ from 3 to 7 mM either increases rFP rate or transforms its pattern to slower and longer multipeak bursts similar to those during early recovery from DAMGO. The results show that electrical coupling of neonatal LC neurons does not synchronize their spiking as previously proposed. They also indicate that both increased excitability (by elevated K+) and recovery from inhibition (by opioids) can enhance spike desynchronization to transform the population burst pattern. Both observations show that this gap junction-coupled neural network has a more complex connectivity than currently assumed. These new findings along with the inhibitory drug effects that are in line with previous reports based on single neuron recording point out that field potential analysis is pivotal to further the understanding of this brain circuit.

Characterization of Superficial Dorsal Horn Neurons from "Tamamaki" Mice and Stability of their GAD67-EGFP Phenotype in Defined-Medium Organotypic Culture.

Defined medium organotypic cultures (DMOTC) containing spinal dorsal horn neurons are especially useful in studying the etiology and pharmacology of chronic pain. We made whole-cell recordings from neurons in acutely isolated mouse spinal cord slices or from those maintained in DMOTC for up to 6 weeks. In acute slices, neurons in the substantia gelatinosa exhibited 7 different firing patterns in response to 800-ms depolarizing current commands; delay (irregular), delay (tonic), tonic, regular firing, phasic, initial bursting and single spiking. Initial bursting and regular firing neurons are not found in rat substantia gelatinosa. In acute slices from "Tamamaki" mice that express enhanced green fluorescent protein (EGFP) under the control of the glutamic acid decarboxylase 67 (GAD67) promotor, tonic, phasic and regular firing neurons exhibited the strongest GABAergic (GAD67-EGFP+) phenotype. Delay (tonic) and delay (irregular) neurons almost never expressed GAD67 (GAD67-EGFP-) and are likely glutamatergic. All seven phenotypes were preserved in mouse spinal cord neurons in DMOTC prepared from e12 embryos and the GAD67-EGFP+ phenotype continued to associate with phasic and regular firing neurons. Only 3 out of 51 GAD67-EGFP+ neurons exhibited a delay (tonic) firing pattern. Modifications to the mouse genome thus continue to be expressed when embryonic neurons develop in vitro in DMOTC. However, analysis of the amplitude and interevent interval of spontaneous EPSCs (sEPSCs) indicated substantial re-arrangement of synaptic connections within the cultures. Despite this, the characteristics and age-dependence of asynchronous oscillatory activity, as monitored by multiphoton Ca2+ imaging, were similar in acute slices and in DMOTC.

Carbon nanotubes might improve neuronal performance by favouring electrical shortcuts.

Carbon nanotubes have been applied in several areas of nerve tissue engineering to probe and augment cell behaviour, to label and track subcellular components, and to study the growth and organization of neural networks. Recent reports show that nanotubes can sustain and promote neuronal electrical activity in networks of cultured cells, but the ways in which they affect cellular function are still poorly understood. Here, we show, using single-cell electrophysiology techniques, electron microscopy analysis and theoretical modelling, that nanotubes improve the responsiveness of neurons by forming tight contacts with the cell membranes that might favour electrical shortcuts between the proximal and distal compartments of the neuron. We propose the 'electrotonic hypothesis' to explain the physical interactions between the cell and nanotube, and the mechanisms of how carbon nanotubes might affect the collective electrical activity of cultured neuronal networks. These considerations offer a perspective that would allow us to predict or engineer interactions between neurons and carbon nanotubes.