To determine the role of gamma-motoneurons in the control of locomotion, we isolated single units from nerves to triceps surae muscles in the premammillary cat. The limb used for recording was largely denervated, except for the muscles of interest, and fixed in place, while the other three limbs walked on a treadmill. One type of gamma-motoneuron (13 units) had a high impulse rate at rest, which changed little on average during walking, but was deeply modulated with each step (phasically modulated gamma-motoneuron or gamma p). Another type (19 units) had a low impulse rate at rest, which increased greatly on average during walking, but was not highly modulated with each step (tonically modulated gamma-motoneuron or gamma t). Peak gamma p rates generally occurred after peak EMG, often near the peak of tension. In contrast, peak gamma t activity generally preceded peak electromyograms (EMG). No significant difference was observed in conduction velocities for the two types of units. At rest all gamma t units were excited by natural stimulation of the fur over a large part of the body surface, whereas 3 of 11 gamma p units were inhibited. During locomotion the same natural stimuli had no observable effect on either type of unit. By recording in continuity from fine branches of the lateral and medial gastrocnemius nerves and stimulating ventral root filaments in continuity, we identified dynamic and static gamma-motoneurons in terms of their effects on muscle spindle afferents. After cutting the nerve branch distally and other ventral root filaments supplying the muscle, the resting discharge of dynamic and static gamma-motoneurons was recorded and found to correspond to that of the gamma p and gamma t units, respectively. Other evidence is presented for a correspondence between phasically and tonically modulated units and dynamic and static gamma-motoneurons, contrary to some suggestions in the literature.
Impulse from soleus muscle afferents were recorded in premammillary cats that were walking on a treadmill. In normal walking the effects of gamma-motoneurons on impulse rates of muscle spindle afferents are confounded by the effects of the large length changes that occur. To isolate the effects of gamma-motoneurons the leg was fixed in place for recording and denervated except for soleus muscle. Because gamma-motoneurons produce marked effects on the stretch sensitivity of muscle afferents, soleus muscle was oscillated about a present length so the stretch sensitivity of its afferents could be determined. The impulse rate of secondary muscle spindle afferents in soleus muscle was generally increased at all phases of the step cycle. The mean rate approximately doubled during walking (82 imp/s), compared with nonwalking (rest) periods (44 imp/s). The sensitivity to sinusoidal length changes was generally reduced throughout the step cycle (mean reduction = 33%). Primary muscle spindle afferents also showed an increased mean rate during walking (47 imp/s) compared with rest (24 imp/s). The impulse rate peaked after the muscle reached its maximum force and often showed a second peak before the maximum electromyogram (EMG) activity. The sensitivity to sinusoidal stretches varied cyclically during locomotion. During the extension phase it sometimes exceeded the resting value, but was greatly reduced during the flexion phase (mean reduction = 49% over whole cycle). Control experiments were carried out in which static and dynamic gamma-motoneurons were stimulated and activity from muscle spindle afferents was recorded in anesthetized cats. With the amplitude and frequency of stretch applied, stimulation of dynamic gamma-motoneurons usually increased and stimulation of static gamma-motoneurons usually decreased the sensitivity of primary muscle spindle afferents to sinusoidal stretch. The patterns observed in muscle spindle afferents suggest a strong, maintained activation of static gamma-motoneurons throughout the step cycle and a phasic activation of dynamic gamma-motoneurons, which is consistent with previous direct recordings from gamma-motoneurons. With this pattern of activating gamma-motoneurons, the secondary muscle spindle afferents will provide a good feedback signal of the large length changes that normally occur in the muscle during locomotion. The changes in sensitivity of primary muscle spindle afferents will complement central changes so the gain of the stretch reflex from extensors is high during extension (when required to help support the weight of the body) and low during flexion (when a high gain would be counterproductive).
Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (KD) of 777 +/- 93 nM for SNA binding to Neu5Ac alpha2,6-LHEB. Transient SPR kinetics confirms the K D value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 microg/mL range with limit of detection of approximately 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time.
We report a microfabrication approach to generate well-defined, addressable, and regenerable lipid membrane arrays in poly(dimethylsiloxane) (PDMS) microchips for label-free analysis of lipid-protein interactions with surface plasmon resonance imaging (SPRi). The multiplexed detection is demonstrated with a tethered bilayer membrane array built in parallel microchannels. These channels allow multiple measurements to be carried out simultaneously, showing low deviations for element-to-element variation in quantifiable signal. Lipid-conjugated receptors were utilized as model systems for protein binding analysis, and the feasibility of regenerating the tethering sublayer after binding was investigated. The results show that the lipid membrane can be removed effectively by nonionic surfactant Triton X-100. The small variance in SPR signal for the buildup process, i.e., <4% RSD for 3 cycles of detection, removal, and regeneration, indicates the sensing interface is highly reproducible. A calibration curve was obtained for cholera toxin using the monosialoganglioside (GM1) receptor, displaying a linear relationship in the 25 to 175 microg/mL range with a limit of detection of 260 nM. In addition, interaction of a phosphatidylinositol (PIP) with its binding protein and biotin/avidin interactions were employed for array measurements. To further enhance the SPR detection signal, a layer-by-layer amplification strategy was demonstrated that uses biotinylated antibody, NeutrAvidin and biotinylated anti-avidin, and the signal for protein binding on the membrane increased by 400%. The tethered membrane array technology, in combination with SPRi, offers an attractive platform for studies of membrane proteins, and can also find a range of applications for rapid screening of drug candidates interacting with proteins embedded in the near-native environment.
We report the microfluidic fabrication of robust and fluid tethered bilayer arrays within a poly(dimethylsiloxane) (PDMS) chip, and demonstrate its addressability and biosensing by incorporating the GM1 receptor into the bilayer framework for detection of cholera toxin. Rapid optimization of the experimental conditions is achieved by using nanoglassified surfaces in combination with surface plasmon resonance. The ultrathin glassy film on gold mimics glass surfaces employed in microfluidics, allowing real-time monitoring of multiple assembly steps and therefore permitting rapid prototyping of microfluidic arrays. The tethered bilayer array utilizes a covalently immobilized biotinylated protein for generation of well-defined capture zones where a streptavidin link is employed for the immobilization of biotinylated vesicles. Fusion of captured vesicles is accomplished using a concentrated PEG solution, and the lateral diffusion of the tethered bilayer membrane is characterized by fluorescence recovery after photobleaching methods. The tethered membrane arrays demonstrate marked stability and high mobility, which provide an ideal host environment for membrane-associated proteins and open new avenues for high-throughput analysis of these proteins.
Bioelectronic medicine is driving the need for neuromorphic microcircuits that integrate raw nervous stimuli and respond identically to biological neurons. However, designing such circuits remains a challenge. Here we estimate the parameters of highly nonlinear conductance models and derive the ab initio equations of intracellular currents and membrane voltages embodied in analog solid-state electronics. By configuring individual ion channels of solid-state neurons with parameters estimated from large-scale assimilation of electrophysiological recordings, we successfully transfer the complete dynamics of hippocampal and respiratory neurons in silico. The solid-state neurons are found to respond nearly identically to biological neurons under stimulation by a wide range of current injection protocols. The optimization of nonlinear models demonstrates a powerful method for programming analog electronic circuits. This approach offers a route for repairing diseased biocircuits and emulating their function with biomedical implants that can adapt to biofeedback.
Optical chromatography involves the elegant combination of opposing optical and fluid drag forces on colloidal samples within microfluidic environments to both measure analytical differences and fractionate injected samples. Particles that encounter the focused laser beam are trapped axially along the beam and are pushed upstream from the laser focal point to rest at a point where the optical and fluid forces on the particle balance. In our recent devices particles are pushed into a region of lower microfluidic flow, where they can be retained and fractionated. Because optical and fluid forces on a particle are sensitive to differences in the physical and chemical properties of a sample, separations are possible. An optical chromatography beam focused to completely fill a fluid channel is operated as an optically tunable filter for the separation of inorganic, polymeric, and biological particle samples. We demonstrate this technique coupled with an advanced microfluidic platform and show how it can be used as an effective method to fractionate particles from an injected multicomponent sample. Our advanced three-stage microfluidic design accommodates three lasers simultaneously to effectively create a sequential cascade optical chromatographic separation system.
Supported bilayer membranes (SBMs) formed on solid substrates, in particular glass, provide an ideal cell mimicking model system that has been found to be highly useful for biosensing applications. Although the stability of the membrane structures is known to determine the applicability, the subject has not been extensively investigated, largely because of the lack of convenient methods to monitor changes of membrane properties on glass in real time. This work reports the evaluation of the stability properties of a series of SBMs against chemical and air damage by use of surface plasmon resonance spectroscopy and nanoglassified gold substrates. Seven SBMs composed of phosphatidylcholine and DOPC+, including single-component, mixed, protein-reinforced SBMs (rSBMs) and protein-tethered bilayer membranes (ptBLMs), are studied. The stability properties under various conditions, especially the effects of surfactants, organic solvents, and dehydration damage on the bilayers, are compared. PC membranes are found to be easily removed from the glassy surfaces using relatively low concentrations of the surfactants, while DOPC+ is markedly more stable toward nonionic surfactant. DOPC+ membranes also demonstrated remarkable air stability while PC films exhibited considerable damage from dehydration. Doping of cholesterol does not improve PC's stability against SDS and Triton but changes the lipid membrane packing enough to protect against dehydration damage. Although rSBMs and ptBLMs improve air stability to a certain degree, they are still quite susceptible to significant damage/removal from ionic and nonionic surfactants at lower concentrations. Overall, DOPC+ has noted higher stability on glass, likely due to the favorable electrostatic interaction between the silicate surface and the lipid headgroup, making it a good candidate for application. Nanoglassy SPR proves to be an attractive platform capable of rapidly screening film stability in real-time, providing critical information for future work using supported membranes for sensing applications.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.