Oxytocin (OT), a hypothalamic neuropeptide that acts as a neuromodulator in the brain, orchestrates a variety of animal behaviors. However, the relationship between brain OT dynamics and complex animal behaviors remains largely elusive, partly because of the lack of a suitable technique for its real-time recording in vivo. Here, we describe MTRIA_OT, a G-protein-coupled receptor-based green fluorescent OT sensor that has a large dynamic range, suitable affinity, ligand specificity for OT orthologs, minimal effects on downstream signaling and long-term fluorescence stability. By combining viral gene delivery and fiber photometry-mediated fluorescence measurements, we demonstrate the utility of MTRIA_OT for real-time detection of brain OT dynamics in living mice. MTRIA_OT-mediated measurements indicate variability of OT dynamics depending on the behavioral context and physical condition of an animal. MTRIA_OT will likely enable the analysis of OT dynamics in a variety of physiological and pathological processes.

The cochlea of the mammalian inner ear contains an endolymph that exhibits an endocochlear potential (EP) of +80 mV with a [K⁺] of 150 mM. This unusual extracellular solution is maintained by the cochlear lateral wall, a double-layered epithelial-like tissue. Acoustic stimuli allow endolymphatic K⁺ to enter sensory hair cells and excite them. The positive EP accelerates this K⁺ influx, thereby sensitizing hearing. K⁺ exits from hair cells and circulates back to the lateral wall, which unidirectionally transports K⁺ to the endolymph. In vivo electrophysiological assays demonstrated that the EP stems primarily from two K⁺ diffusion potentials yielded by [K⁺] gradients between intracellular and extracellular compartments in the lateral wall. Such gradients seem to be controlled by ion channels and transporters expressed in particular membrane domains of the two layers. Analyses of human deafness genes and genetically modified mice suggested the contribution of these channels and transporters to EP and hearing. A computational model, which reconstitutes unidirectional K⁺ transport by incorporating channels and transporters in the lateral wall and connects this transport to hair cell transcellular K⁺ fluxes, simulates the circulation current flowing between the endolymph and the perilymph. In this model, modulation of the circulation current profile accounts for the processes leading to EP loss under pathological conditions. This article not only summarizes the unique physiological and molecular mechanisms underlying homeostasis of the EP and their pathological relevance but also describes the interplay between EP and circulation current.

Drug interaction with target proteins including ion channels is essential for pharmacological control of various cellular functions, but the majority of its molecular mechanisms is still elusive. We recently found that a series of antidepressants preferentially block astroglial K⁺-buffering inwardly rectifying potassium channel (Kir) 4.1 channels over Kir1.1 channels. Here, using electrophysiological analyses of drug action on mutated Kir4.1 channel as well as computational analyses of three-dimensional (3D) arrangements of the ligands (i.e., bidirectional analyses), we examined the underlying mechanism for the antidepressant-Kir4.1 channel interaction. First, the effects of the selective serotonin reuptake inhibitor fluoxetine and the tricyclic antidepressant nortriptyline on chimeric and site-directed mutants of Kir4.1 expressed in Xenopus laevis oocytes were examined using the two-electrode voltage-clamp technique. Two amino acids, Thr128 and Glu158, on pore and transmembrane domain 2 helices were critical for the drug inhibition of the current. The closed and open conformation models of the Kir4.1 pore suggested that both residues faced the central cavity, and they were positioned within a geometrical range capable of interacting with the drugs. Second, to represent molecular properties of active ligands in geometric terms, a 3D quantitative structure-activity relationship model of antidepressants was generated, which suggested that they share common features bearing a hydrogen bond acceptor and a positively charged moiety. 3D structures and physicochemical features of receptor and ligand were fitted together. Our results strongly suggest that antidepressants interact with Kir4.1 channel pore residues by hydrogen bond and ionic interactions, which account for their preferential inhibitory action on Kir4.1 current. This study may represent a possible general approach for the understanding of the mechanism of ligand-protein interactions.

Microneedle (MN)-based electrochemical biosensors hold promising potential for noninvasive continuous monitoring of interstitial fluid biomarkers. However, challenges, such as instability and biofouling, exist. This study proposes a design employing hollow MN to encapsulate a zwitterionic polymer hydrogel sensing layer with excellent biocompatibility and antifouling properties to address these issues. MN shell isolates the internal microporous sensing layer from subcutaneous friction, and the hydrogel filling leverages the MNs' three-dimensional structures, enabling high-dense loading of biorecognition elements. The hollow MNs are successfully fabricated from high-molecular-weight polylactic acid via drawing lithography, exhibiting sufficient strength for effective epidermis penetration. Additionally, a high-performance gold nanoconductive layer is successfully deposited inside the MN hollow channel, establishing a stable electrical connection between the polymer MN and the hydrogel sensing layer. To support the design, numerical simulations of position-based diffusive analyte solutes reveal fast-responsive electrochemical signals attributed to the high diffusion coefficient of the hydrogel and the concentrated structure of the hollow channel encapsulation. Experimental results and numerical simulations underscore the advantages of this design, showcasing rapid response, high sensitivity, long-term stability, and excellent antifouling properties. Fabricated MN sensors exhibited biosafety, feasibility, and effectiveness, with accurate and rapid in vivo glucose monitoring ability. This study emphasizes the significance of rational design, structural utilization, and micro-nanofabrication to unlock the untapped potential of MN biosensors.

Key points: The endocochlear potential (EP) of +80 mV in cochlear endolymph is essential for audition and controlled by K⁺ transport across the lateral cochlear wall composed of two epithelial barrier layers, the syncytium containing the fibrocytes and the marginal cells. The EP depends upon the diffusion potential elicited by a large K⁺ gradient across the apical surface of the syncytium. We examined by electrophysiological approaches an involvement of Na⁺,K⁺‐ATPase, which occurs at the syncytium's basolateral surface comprising the fibrocytes’ membranes and would mediate K⁺ transport across the lateral wall, in maintenance of the EP. We show that the Na⁺,K⁺‐ATPase sustains the syncytium's high [K⁺] that is crucial for the K⁺ gradient across the apical surface of the syncytium. The results help us better understand the mechanism underlying the establishment of the EP as well as the pathophysiological process for deafness induced by dysfunction of the ion transport apparatus. Abstract  The endocochlear potential (EP) of +80 mV in the scala media, which is indispensable for audition, is controlled by K⁺ transport across the lateral cochlear wall. This wall includes two epithelial barriers, the syncytium and the marginal cells. The former contains multiple cell types, such as fibrocytes, which are exposed to perilymph on their basolateral surfaces. The apical surfaces of the marginal cells face endolymph. Between the two barriers lies the intrastrial space (IS), an extracellular space with a low K⁺ concentration ([K⁺]) and a potential similar to the EP. This intrastrial potential (ISP) dominates the EP and represents the sum of the diffusion potential elicited by a large K⁺ gradient across the apical surface of the syncytium and the syncytium's potential, which is slightly positive relative to perilymph. Although a K⁺ transport system in fibrocytes seems to contribute to the EP, the mechanism remains uncertain. We examined the electrochemical properties of the lateral wall of guinea pigs with electrodes sensitive to potential and K⁺ while perfusing into the perilymph of the scala tympani blockers of Na⁺,K⁺‐ATPase, the K⁺ pump thought to be essential to the system. Inhibiting Na⁺,K⁺‐ATPase barely affected [K⁺] in the IS but greatly decreased [K⁺] within the syncytium, reducing the K⁺ gradient across its apical surface. The treatment hyperpolarized the syncytium only moderately. Consequently, both the ISP and the EP declined. Fibrocytes evidently use the Na⁺,K⁺‐ATPase to achieve local K⁺ transport, maintaining the syncytium's high [K⁺] that is crucial for the K⁺ diffusion underlying the positive ISP.