Peptidergic and functional delineation of the Edinger-Westphal nucleus.

Many neuronal populations that release fast-acting excitatory and inhibitory neurotransmitters in the brain also contain slower-acting neuropeptides. These facultative peptidergic cell types are common, but it remains uncertain whether neurons that solely release peptides exist. Our fluorescence in situ hybridization, genetically targeted electron microscopy, and electrophysiological characterization suggest that most neurons of the non-cholinergic, centrally projecting Edinger-Westphal nucleus in mice are obligately peptidergic. We further show, using anterograde projection mapping, monosynaptic retrograde tracing, angled-tip fiber photometry, and chemogenetic modulation and genetically targeted ablation in conjunction with canonical assays for anxiety, that this peptidergic population activates in response to loss of motor control and promotes anxiety responses. Together, these findings elucidate an integrative, ethologically relevant role for the Edinger-Westphal nucleus and functionally align the nucleus with the periaqueductal gray, where it resides. This work advances our understanding of peptidergic modulation of anxiety and provides a framework for future investigations of peptidergic systems.

Tracking the movement of discrete gating charges in a voltage-gated potassium channel.

Positively charged amino acids respond to membrane potential changes to drive voltage sensor movement in voltage-gated ion channels, but determining the displacements of voltage sensor gating charges has proven difficult. We optically tracked the movement of the two most extracellular charged residues (R1 and R2) in the Shaker potassium channel voltage sensor using a fluorescent positively charged bimane derivative (qBBr) that is strongly quenched by tryptophan. By individually mutating residues to tryptophan within the putative pathway of gating charges, we observed that the charge motion during activation is a rotation and a tilted translation that differs between R1 and R2. Tryptophan-induced quenching of qBBr also indicates that a crucial residue of the hydrophobic plug is linked to the Cole-Moore shift through its interaction with R1. Finally, we show that this approach extends to additional voltage-sensing membrane proteins using the Ciona intestinalis voltage-sensitive phosphatase (CiVSP).

Oxytocin functions as a spatiotemporal filter for excitatory synaptic inputs to VTA dopamine neurons.

The experience of rewarding or aversive stimuli is encoded by distinct afferents to dopamine (DA) neurons of the ventral tegmental area (VTA). Several neuromodulatory systems including oxytocin regulate DA neuron excitability and synaptic transmission that process socially meaningful stimuli. We and others have recently characterized oxytocinergic modulation of activity in mouse VTA DA neurons, but the mechanisms underlying oxytocinergic modulation of synaptic transmission in DA neurons remain poorly understood. Here, we find that oxytocin application or optogenetic release decrease excitatory synaptic transmission, via long lasting, presynaptic, endocannabinoid-dependent mechanisms. Oxytocin modulation of excitatory transmission alters the magnitude of short and long-term depression. We find that only some glutamatergic projections to DA neurons express CB1 receptors. Optogenetic stimulation of three major VTA inputs demonstrates that oxytocin modulation is limited to projections that show evidence of CB1R transcripts. Thus, oxytocin gates information flow into reward circuits in a temporally selective and pathway-specific manner.

Biased Oxytocinergic Modulation of Midbrain Dopamine Systems.

The release of dopamine (DA) regulates rewarding behavior and motor actions through striatum-targeting efferents from ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Here, we map and functionally characterize axonal projections from oxytocin neurons in the hypothalamic paraventricular nucleus to midbrain DA regions. Electrophysiological recordings of DA neurons reveal that both the application of oxytocin and optogenetic stimulation of oxytocinergic terminals suffice to increase DA neuron activity in the VTA but downregulate it in SNc. This biased modulation is mediated by oxytocin and vasopressin G-protein-coupled receptors. Oxytocin release directly activates DA neurons and indirectly inhibits them through local GABA neurons, but the relative magnitudes of the two mechanisms differ in VTA and SNc. Oxytocin-modulated DA neurons give rise to canonical striatal projections. Since hypothalamic oxytocinergic projections also target the striatum, oxytocin is poised to bias the balance of DA tone through multiple sites in vertebrate reward circuits.

A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor.

G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a voltage sensor. However, the typical domain that serves as a voltage sensor in voltage-gated channels is not present in GPCRs, making the search for the voltage sensor in the latter challenging. Here, we examine the M2R and describe a voltage sensor that is comprised of tyrosine residues. This voltage sensor is crucial for the voltage dependence of agonist binding to the receptor. The tyrosine-based voltage sensor discovered here constitutes a noncanonical by which membrane proteins may sense voltage.

Kv3.1 uses a timely resurgent K(+) current to secure action potential repolarization.

High-frequency action potential (AP) transmission is essential for rapid information processing in the central nervous system. Voltage-dependent Kv3 channels play an important role in this process thanks to their high activation threshold and fast closure kinetics, which reduce the neuron's refractory period. However, premature Kv3 channel closure leads to incomplete membrane repolarization, preventing sustainable AP propagation. Here, we demonstrate that Kv3.1b channels solve this problem by producing resurgent K(+) currents during repolarization, thus ensuring enough repolarizing power to terminate each AP. Unlike previously described resurgent Na(+) and K(+) currents, Kv3.1b's resurgent current does not originate from recovery of channel block or inactivation but results from a unique combination of steep voltage-dependent gating kinetics and ultra-fast voltage-sensor relaxation. These distinct properties are readily transferrable onto an orthologue Kv channel by transplanting the voltage-sensor's S3-S4 loop, providing molecular insights into the mechanism by which Kv3 channels contribute to high-frequency AP transmission.

Single-molecule fluorimetry and gating currents inspire an improved optical voltage indicator.

Voltage-sensing domains (VSDs) underlie the movement of voltage-gated ion channels, as well as the voltage-sensitive fluorescent responses observed from a common class of genetically encoded voltage indicators (GEVIs). Despite the widespread use and potential utility of these GEVIs, the biophysical underpinnings of the relationship between VSD movement and fluorophore response remain unclear. We investigated the recently developed GEVI ArcLight, and its close variant Arclight', at both the single-molecule and macroscopic levels to better understand their characteristics and mechanisms of activity. These studies revealed a number of previously unobserved features of ArcLight's behavior, including millisecond-scale fluorescence fluctuations in single molecules as well as a previously unreported delay prior to macroscopic fluorescence onset. Finally, these mechanistic insights allowed us to improve the optical response of ArcLight to fast or repetitive pulses with the development of ArcLightning, a novel GEVI with improved kinetics.

Functional Site-Directed Fluorometry.

Initially developed in the mid-1990s to examine the conformational changes of the canonical Shaker voltage-gated potassium channel, functional site-directed fluorometry has since been expanded to numerous other voltage-gated and ligand-gated ion channels as well as transporters, pumps, and other integral membrane proteins. The power of functional site-directed fluorometry, also known as voltage-clamp fluorometry, lies in its ability to provide information on the conformational changes in a protein in response to changes in its environment with high temporal resolution while simultaneously monitoring the function of that protein. Over time, applications of site-directed fluorometry have expanded to examine the interactions of ion channels with modulators ranging from membrane potential to ligands to accessory protein subunits to lipids. In the future, the range of questions answerable by functional site-directed fluorometry and its interpretive power should continue to improve, making it an even more powerful technique for dissecting the conformational dynamics of ion channels and other membrane proteins.

Real-time imaging of electrical signals with an infrared FDA-approved dye.

Clinical methods used to assess the electrical activity of excitable cells are often limited by their poor spatial resolution or their invasiveness. One promising solution to this problem is to optically measure membrane potential using a voltage-sensitive dye, but thus far, none of these dyes have been available for human use. Here we report that indocyanine green (ICG), an infrared fluorescent dye with FDA approval as an intravenously administered contrast agent, is voltage-sensitive. The fluorescence of ICG can follow action potentials in artificial neurons and cultured rat neurons and cardiomyocytes. ICG also visualized electrical activity induced in living explants of rat brain. In humans, ICG labels excitable cells and is routinely visualized transdermally with high spatial resolution. As an infrared voltage-sensitive dye with a low toxicity profile that can be readily imaged in deep tissues, ICG may have significant utility for clinical and basic research applications previously intractable for potentiometric dyes.

S3-S4 linker length modulates the relaxed state of a voltage-gated potassium channel.

Voltage-sensing domains (VSDs) are membrane protein modules found in ion channels and enzymes that are responsible for a large number of fundamental biological tasks, such as neuronal electrical activity. The VSDs switch from a resting to an active conformation upon membrane depolarization, altering the activity of the protein in response to voltage changes. Interestingly, numerous studies describe the existence of a third distinct state, called the relaxed state, also populated at positive potentials. Although some physiological roles for the relaxed state have been suggested, little is known about the molecular determinants responsible for the development and modulation of VSD relaxation. Several lines of evidence have suggested that the linker (S3-S4 linker) between the third (S3) and fourth (S4) transmembrane segments of the VSD alters the equilibrium between resting and active conformations. By measuring gating currents from the Shaker potassium channel, we demonstrate here that shortening the S3-S4 linker stabilizes the relaxed state, whereas lengthening the linker or splitting it and coinjecting two fragments of the channel have little effect. We propose that natural variations of the length of the S3-S4 linker in various VSD-containing proteins may produce differential VSD relaxation in vivo.

Thermal mechanisms of millimeter wave stimulation of excitable cells.

Interactions between millimeter waves (MMWs) and biological systems have received increasing attention due to the growing use of MMW radiation in technologies ranging from experimental medical devices to telecommunications and airport security. Studies have shown that MMW exposure alters cellular function, especially in neurons and muscles. However, the biophysical mechanisms underlying such effects are still poorly understood. Due to the high aqueous absorbance of MMW, thermal mechanisms are likely. However, nonthermal mechanisms based on resonance effects have also been postulated. We studied MMW stimulation in a simplified preparation comprising Xenopus laevis oocytes expressing proteins that underlie membrane excitability. Using electrophysiological recordings simultaneously with 60 GHz stimulation, we observed changes in the kinetics and activity levels of voltage-gated potassium and sodium channels and a sodium-potassium pump that are consistent with a thermal mechanism. Furthermore, we showed that MMW stimulation significantly increased the action potential firing rate in oocytes coexpressing voltage-gated sodium and potassium channels, as predicted by thermal terms in the Hodgkin-Huxley model of neurons. Our results suggest that MMW stimulation produces significant thermally mediated effects on excitable cells via basic thermodynamic mechanisms that must be taken into account in the study and use of MMW radiation in biological systems.

Depolarization induces a conformational change in the binding site region of the M2 muscarinic receptor.

G protein-coupled receptors play a central role in signal transduction and were only known to be activated by agonists. Recently it has been shown that membrane potential also affects the activity of G protein-coupled receptors. For the M(2) muscarinic receptor, it was further shown that depolarization induces charge movement. A tight correlation was found between the voltage-dependence of the charge movement and the voltage-dependence of the agonist binding. Here we examine whether depolarization-induced charge movement causes a conformational change in the M(2) receptor that may be responsible for the voltage-dependence of agonist binding. Using site-directed fluorescence labeling we show a voltage-dependent fluorescence signal, reflecting a conformational change, which correlates with the voltage-dependent charge movement. We further show that selected mutations in the orthosteric site abolish the fluorescence signal and concomitantly, the voltage-dependence of the agonist binding. Surprisingly, mutations in the allosteric site also abolished the voltage-dependence of agonist binding but did not reduce the fluorescence signal. Finally, we show that treatments, which reduced the charge movement or hindered the coupling between the charge movement and the voltage-dependent binding, also reduced the fluorescence signal. Our results demonstrate that depolarization-induced conformational changes in the orthosteric binding site underlie the voltage-dependence of agonist binding. Our results are also unique in suggesting that the allosteric site is also involved in controlling the voltage-dependent agonist binding.

The molecular phylogeny of the white blister rust genus Pustula reveals a case of underestimated biodiversity with several undescribed species on ornamentals and crop plants.

Despite their economic importance, the knowledge of the biodiversity of many plant pathogens is still fragmentary. In this study we show that this is true also for the white blister rust genus Pustula that is parasitic on several genera in the asterids, including sunflower and the gentian, Eustoma. It is revealed that several distinct species exist in Pustula, suggesting that species are mostly host genus specific. No geographic patterns were observed in the occurrence of Pustula, the host range of which includes the Araliaceae, Asteraceae, Gentianaceae, and Goodeniaceae. Evidence points to these becoming hosts as a result of jumps from the Asteraceae, with subsequent host-specific adaptation and speciation. Among the undescribed species are pathogens of economic importance, e.g. the white blister rusts of sunflower, or with still restricted geographical ranges, e.g. Pustula centaurii, which could potentially spread with international seed trade, if no quarantine restrictions are implemented.

Fungal endophytes of native Gossypium species in Australia.

Fungal endophytes of 17 genera were found in stems of four native Gossypium species (G. australe, G. bickii, G. nelsonii, G. sturtianum) collected from inland areas in Queensland, the Northern Territory, and South Australia in 2001. Phoma, Alternaria, Fusarium, Botryosphaeria, Dichomera, and Phomopsis were common, accounting for 58, 18, 11, 3, 1, and 1 % of the 281 recovered isolates, respectively, and occurring in 47, 29, 19, 5, 5, and 4 % of the 79 sampled populations. Among the four Gossypium species in Queensland and the Northern Territory, Alternaria spp. and Fusarium spp. had the greatest recovery frequency in G. bickii stems. The recovery frequencies of Phoma spp. and Alternaria spp. were significantly greater in the G. sturtianum stems collected from South Australia than in those from Queensland and the Northern Territory. Pathogenicity of 42 representative isolates was tested on cultivated cotton (G. hirsutum). All isolates caused some localized discoloration in stem tissue when inoculation was conducted with the stem puncturing method, but none of the isolates could induce any foliar symptoms during the five-week experimental period by either inoculation method (root dipping or stem puncturing), suggesting that the endophytic fungi of native Gossypium species are unlikely sources of cotton pathogens.

Phaeohyphomycotic soft tissue infections caused by the coelomycetous fungus Microsphaeropsis arundinis.

Microsphaeropsis arundinis is an anamorphic fungal plant inhabitant belonging to the form class Coelomycetes. We describe two cases of M. arundinis soft tissue infections in immunosuppressed patients. This organism has not previously been described as causing disease in humans. It was identified on the basis of its typical ostiolate pycnidial conidiomata, ampulliform conidiogenous cells, and small, smooth-walled, brown, cylindrical conidia.