TRAF6-mediated ubiquitination of AKT in the nucleus is a critical event underlying the desensitization of G protein-coupled receptors.

BACKGROUND: Desensitization of G protein-coupled receptors (GPCRs) refers to the attenuation of receptor responsiveness by prolonged or intermittent exposure to agonists. The binding of ß-arrestin to the cytoplasmic cavity of the phosphorylated receptor, which competes with the G protein, has been widely accepted as an extensive model for explaining GPCRs desensitization. However, studies on various GPCRs, including dopamine D2-like receptors (D2R, D3R, D4R), have suggested the existence of other desensitization mechanisms. The present study employed D2R/D3R variants with different desensitization properties and utilized loss-of-function approaches to uncover the mechanisms underlying GPCRs homologous desensitization, focusing on the signaling cascade that regulates the ubiquitination of AKT. RESULTS: AKT undergoes K8/14 ubiquitination by TRAF6, which occurs in the nucleus and promotes its membrane recruitment, phosphorylation and activation under receptor desensitization conditions. The nuclear entry of TRAF6 relies on the presence of the importin complex. Src regulates the nuclear entry of TRAF6 by mediating the interaction between TRAF6 and importin ß1. Ubiquitinated AKT translocates to the plasma membrane where it associates with Mdm2 to phosphorylate it at the S166 and S186 residues. Thereafter, phosphorylated Mdm2 is recruited to the nucleus, resulting in the deubiquitination of ß-Arr2. The deubiquitinated ß-Arr2 then forms a complex with Gßγ, which serves as a biomarker for GPCRs desensitization. Like in D3R, ubiquitination of AKT is also involved in the desensitization of ß2 adrenoceptors. CONCLUSION: Our study proposed that the property of a receptor that causes a change in the subcellular localization of TRAF6 from the cytoplasm to the nucleus to mediate AKT ubiquitination could initiate the desensitization of GPCRs.

Switchable rotation of metal nanostructures in an intensity chirality-invariant focus field.

Light-induced rotation is a fundamental motion form that is of great significance for flexible and multifunctional manipulation modes. However, current optical rotation by a single optical field is mostly unidirectional, where switchable rotation manipulation is still challenging. To address this issue, we demonstrate a switchable rotation of non-spherical nanostructures within a single optical focus field. Interestingly, the intensity of the focus field is chiral invariant. The rotation switch is a result of the energy flux reversal in front and behind the focal plane. We quantitatively analyze the optical force exerted on a metal nanorod at different planes, as well as the surrounding energy flux. Our experimental results indicate that the direct switchover of rotational motion is achievable by adjusting the relative position of the nanostructure to the focal plane. This result enriches the basic motion mode of micro-manipulation and is expected to create potential opportunities in many application fields, such as biological cytology and optical micromachining.

All-optically controlled holographic plasmonic vortex array for multiple metallic particles manipulation.

Due to the sub-diffraction-limited size and giant field enhancement, plasmonic tweezers have a natural advantage in trapping metallic particles. However, the strict excitation condition makes it difficult to generate an arbitrary plasmonic field in a controllable manner, thus narrowing its practical applications. Here, we propose an all-optical plasmonic field shaping method based on a digital holographic algorithm and generate plasmonic vortex arrays with controllable spot numbers, spatial location, and topological charge. Our experimental results demonstrate that multiple gold particles can be stably trapped and synchronously rotated in the vortex arrays, and the particles' kinestate can be dynamically switched. The proposed holographic plasmonic vortex tweezers are suitable for a broadband particle trapping, and this method can be generalized to other surface electromagnetic waves like Bloch surface wave.

Generation of discrete higher-order optical vortex lattice at focus.

Higher-order vortices (HOVs) extend the dimensions of optical vortex regulation, which is of great significance in optical communication and optical tweezers. Herein, we demonstrate an alternative scheme to produce a HOV in the focus plane using multiple Laguerre-Gaussian (LG) beam interference, termed a discrete higher-order optical vortex lattice (DHOVL). The modulation depth of the DHOVL exceeds 2π. In this case, the topological charge (TC) of the DHOVL is determined by the difference of the phase period between the innermost and the outermost interference beams. Compared with a conventional HOV (CHOV), the vortex exists in a form of multiple unit singularities sharing a dark core. In addition, the average orbital angular momentum per photon of the DHOVL increases with increasing TC, surpassing that of the CHOV. This work provides a novel, to the best of our knowledge, scheme to produce a HOV, which will facilitate several advanced applications, including optical micromanipulation, optical sensing and imaging, and optical fabrication.

Protocol for modulating the noradrenergic pathway from locus coeruleus to heart to prevent sudden unexpected death in epilepsy in mouse models.

The locus coeruleus (LC) and noradrenergic neurotransmission are involved in the regulation of sudden unexpected death in epilepsy (SUDEP). Here, we present a protocol for modulating the noradrenergic pathway from LC to heart to prevent SUDEP in acoustic and pentylenetetrazole-induced DBA/1 mouse models of SUDEP. We describe steps for constructing SUDEP models, calcium signal recording, and electrocardiogram monitoring. We then detail measurement of tyrosine hydroxylase content and activity, ß1 and p-ß1-AR content, and destruction of LCNE neurons. For complete details on the use and execution of this protocol, please refer to Lian et al.1.

The States of Different 5-HT Receptors Located in the Dorsal Raphe Nucleus Are Crucial for Regulating the Awakening During General Anesthesia.

General anesthesia is widely used in various clinical practices due to its ability to cause loss of consciousness. However, the exact mechanism of anesthesia-induced unconsciousness remains unclear. It is generally thought that arousal-related brain nuclei are involved. 5-Hydroxytryptamine (5-HT) is closely associated with sleep arousal. Here, we explore the role of the 5-HT system in anesthetic awakening through pharmacological interventions and optogenetic techniques. Our data showed that exogenous administration of 5-hydroxytryptophan (5-HTP) and optogenetic activation of 5-HT neurons in the dorsal raphe nucleus (DR) could significantly shorten the emergence time of sevoflurane anesthesia in mice, suggesting that regulation of the 5-HT system using both endogenous and exogenous approaches could mediate delayed emergence. In addition, we first discovered that the different 5-HT receptors located in the DR, known as 5-HT autoreceptors, are essential for the regulation of general anesthetic awakening, with 5-HT1A and 5-HT2A/C receptors playing a regulatory role. These results can provide a reliable theoretical basis as well as potential targets for clinical intervention to prevent delayed emergence and some postoperative risks.

Ubiquitination of GRK2 Is Required for the ß-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases.

A class-A GPCR dopamine D2 receptor (D2R) plays a critical role in the proper functioning of neuronal circuits through the downstream activation of both G-protein- and ß-arrestin-dependent signaling pathways. Understanding the signaling pathways downstream of D2R is critical for developing effective therapies with which to treat dopamine (DA)-related disorders such as Parkinson's disease and schizophrenia. Extensive studies have focused on the regulation of D2R-mediated extracellular-signal-regulated kinase (ERK) 1/2 signaling; however, the manner in which ERKs are activated upon the stimulation of a specific signaling pathway of D2R remains unclear. The present study conducted a variety of experimental techniques, including loss-of-function experiments, site-directed mutagenesis, and the determination of protein interactions, in order to investigate the mechanisms underlying ß-arrestin-biased signaling-pathway-mediated ERK activation. We found that the stimulation of the D2R ß-arrestin signaling pathway caused Mdm2, an E3 ubiquitin ligase, to move from the nucleus to the cytoplasm and interact with tyrosine phosphorylated G-protein-coupled receptor kinase 2 (GRK2), which was facilitated by Src, a non-receptor tyrosine kinase. This interaction led to the ubiquitination of GRK2, which then moved to the plasma membrane and interacted with activated D2R, followed by the phosphorylation of D2R as well as the mediation of ERK activation. In conclusion, Mdm2-mediated GRK2 ubiquitination, which is selectively triggered by the stimulation of the D2R ß-arrestin signaling pathway, is necessary for GRK2 membrane translocation and its interaction with D2R, which in turn mediates downstream ERK signaling. This study is primarily novel and provides essential information with which to better understand the detailed mechanisms of D2R-dependent signaling.

Protocol for modulation of the serotonergic DR-PBC neural circuit to prevent SUDEP in the acoustic and PTZ-induced DBA/1 mouse models of SUDEP.

The dorsal raphe nucleus (DR) and the pre-Bötzinger complex (PBC) may play an important role in regulating seizure-induced respiratory arrest (S-IRA), the main contributor to sudden unexpected death in epilepsy. Here, we describe pharmacological, optogenetic, and retrograde labeling approaches to specifically modulate the DR to PBC serotonergic pathway. We detail steps for implanting optical fibers and viral infusion into DR and PBC regions and optogenetic techniques for exploring the role of 5-hydroxytryptophan (5-HT) neural circuit of DR-PBC in S-IRA. For complete details on the use and execution of this protocol, please refer to Ma et al. (2022).1.

Noradrenergic pathway from the locus coeruleus to heart is implicated in modulating SUDEP.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death among epilepsy patients. However, the underlying mechanism remains elusive. Seizure-induced respiratory arrest (S-IRA) is recognized as a main cause of SUDEP, but the contribution of other factors such as cardiac arrhythmias cannot be excluded. Here, we found that both the locus coeruleus (LC) and peripheral noradrenergic neurotransmission were involved in S-IRA and the protective effect of atomoxetine in reducing the occurrence of S-IRA and SUDEP could be reversed by esmolol hydrochloride. Moreover, we investigated the connection between the LC and heart implicated in the modulation of SUDEP by fiber photometry. These data suggested that noradrenergic neurons in the LC might regulate the occurrence of SUDEP through ß1-adrenergic receptors on cardiomyocytes. Overall, our findings indicate the involvement of the brain-heart axis in modulating S-IRA and SUDEP and, therefore, will open a new perspective on decoding SUDEP.

Dorsal raphe nucleus to pre-Bötzinger complex serotonergic neural circuit is involved in seizure-induced respiratory arrest.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death among patients with epilepsy. However, the underlying mechanism of SUDEP remains elusive. Previous studies showed seizure-induced respiratory arrest (S-IRA) is the main factor in SUDEP, and that enhancement of serotonin (5-HT) function in the dorsal raphe nucleus (DR) can significantly reduce the incidence of S-IRA in the DBA/1 mouse model of SUDEP. The pre-Bötzinger complex (PBC), known for its role in regulating respiratory rhythm, can express the 5-HT2A receptor (5-HT2AR). Here, using the pharmacological and optogenetic methods, respectively, we observed that the serotonergic neural circuit between DR and PBC was involved in S-IRA evoked by either acoustic stimulation or pentylenetetrazole (PTZ) injection in the DBA/1 mice, and found 5-HT2AR located in PBC plays an important role in it. Our findings will further significantly improve our understanding of SUDEP and provide a promising therapeutic target for SUDEP prevention.

Time-varying orbital angular momentum in tight focusing of ultrafast pulses.

The orbital angular momentum (OAM) of light has important applications in a variety of fields, including optical communication, quantum information, super-resolution microscopic imaging, particle trapping, and others. However, the temporal properties of OAM in ultrafast pulses and in the evolution process of spin-orbit coupling has yet to be revealed. In this work, we theoretically studied the spatiotemporal property of time-varying OAM in the tightly focused field of ultrafast light pulses. The focusing of an incident light pulse composed of two time-delayed femtosecond sub-pulses with the same OAM but orthogonal spin states is investigated, and the ultrafast dynamicsa time delay of OAM variation during the focusing process driven by the spin-orbit coupling is visualized. Temporal properties of three typical examples, including formation, increase, and transformation of topological charge are investigated to reveal the non-uniform evolutions of phase singularities, local topological charges, self-torques, and time-varying OAM per photon. This work could deepen the understanding of spin-orbit coupling in time domain and promote many promising applications such as ultrafast OAM modulation, laser micromachining, high harmonic generation, and manipulation of molecules and nanostructures.

Energy flow inversion in an intensity-invariant focusing field.

Dependence of light intensity on energy flow is the most intuitive presentation of an optical field. This dependence, however, also limits the applications to the interaction of the light field with matter. For further insight into this, we demonstrate a novel case of the optical field, named as the counterintuitive chiral intensity field (CCIF), in the highly focusing situation: the energy flow reverses during the propagation but the intensity distribution pattern is kept approximately invariant. Our results show that, in this process, the mode correlation decreases rapidly while the intensity correlation remains invariant in the focus area. Furthermore, this property is still valid even if the pattern helicity and number of spiral arms are changed. This work deepens the understanding of the relationship between energy flow and field intensity, and it will offer diversified operations in many applications, such as optical micromanipulation, optical fabrication, etc.

Controllable propagation and transformation of chiral intensity field at focus.

As an intrinsic feature of the optical field, chirality could induce many novel phenomena due to the interaction between chiral light and matter. Thus, the generation of optical fields possessing 2D or 3D chiral intensity patterns, called chiral intensity fields, has been widely studied. However, the control of chiral intensity field along the optical axis is still a challenge. Here, we propose a method to manipulate the axial propagation property of a focused chiral intensity field. Two modulation effects are realized: extended chiral intensity field with a focal depth >2λ at 90% mode correlation and tunable transformation of chirality during the axial propagation. This method is simple, stable, and easy to perform and therefore offers broad applications especially in optical tweezers and metamaterial fabrication.

VGLUT2/Cdk5/p25 Signaling Pathway Contributed to Inflammatory Pain by Complete Freund's Adjuvant.

Vesicular glutamate transporter type 2 (VGLUT2) is known to play an important role in mediating heat hyperalgesia induced by inflammation. However, the underlying mechanism for this activity is poorly understood. Cyclin-dependent kinase 5 (Cdk5), serving as a key regulator in modulating release of glutamate, acted a key player in the formation of heat hyperalgesia of inflammatory pain. However, it remains unknown whether there is a bridge between Cdk5 and VGLUT2 for mediating inflammatory pain. Therefore, we designed the experiment to determine whether VGLUT2 signaling pathway is involved in inflammatory pain mediated by Cdk5 in the inflammatory pain model induced by complete Freund's adjuvant (CFA). Our results showed that the coexpression of Cdk5/VGLUT2 in small- and medium-sized neuronal cells of the dorsal root ganglion (DRG) and spinal cord between days 1 and 3 following subcutaneous injection of CFA was significantly increased. Moreover, our study revealed that the expression of VGLUT2 protein in the DRG and spinal cord was remarkably increased between days 1 and 3 following CFA injection and was significantly reduced by roscovitine, a selective antagonist of Cdk5. Additionally, p25 but not p35, an activator of Cdk5, protein was significantly increased by CFA and reduced by roscovitine. Our findings suggested that VGLUT2/Cdk5 signaling pathway contributes to inflammatory pain mediated by Cdk5/p25.

Grafted optical vortex with controllable orbital angular momentum distribution.

In an optical vortex (OV) field, the orbital angular momentum (OAM) distribution strongly depends on the intensity, which results in difficulty in OAM independent modulation. To overcome this limitation, we propose a grafted optical vortex (GOV) via spiral phase reconstruction of two or more OVs with different topological charges (TCs). To remain the annular shape of the GOV's intensity, the Dirac δ-function is employed to restrict the energy in a ring. Theoretical analysis and manipulation experiments of polystyrene microspheres show that the magnitude and direction of the GOV's local OAM are controllable by modulating the grafted TCs while the intensity remains constant. The results of this work provide an ingenious method to control the local tangential force on the light ring, which will promote potential applications in optical trapping and rotating micro-particles.

Optical vortex shaping via a phase jump factor.

Topological charge (TC) of an optical vortex (OV) is a crucial parameter. We propose two factors, namely, the phase jump factor and the phase gradient factor, to replace the parameter of TC through unwrapping the TC definition integral. Based on these two factors, we report on a novel OV, referred to as the remainder-phase optical vortex (ROV). The properties of the ROV are studied in depth by adjusting these two factors. Results show that the phase gradient factor determines the total orbital angular momentum (OAM), whereas the phase jump factor decides the number of split unit vortices and reshapes the structure of the OAM distribution. This work provides a novel OV with controllable OAM distribution, which will open up new applications such as particle manipulation, beam shaping, and micro-fabrication.

Close-packed optical vortex lattices with controllable structures.

As a spatial structured light field, the optical vortex (OV) has attracted extensive attention in recent years. In practice, the OV lattice (OVL) is an optimal candidate for applications of orbital angular momentum (OAM)-based optical communications, microparticle manipulation, and micro/nanofabrication. However, traditional methods for producing OVLs meet a significant challenge: the OVL structures cannot be adjusted freely and form a close-packed arrangement, simultaneously. To overcome these difficulties, we propose an alternative scheme to produce close-packed OVLs (CPOVLs) with controllable structures. By borrowing the concept of the close-packed lattice from solid-state physics, CPOVLs with versatile structures are produced by using logical operations of expanding OV primitive cells combined with the technique of phase mask generation. Then, the existence of OAM states in the CPOVLs is verified. Furthermore, the energy flow and OAM distribution of the CPOVLs are visualized and analyzed. From a light field physics viewpoint, this work increases the adjustment dimensions and extends the fundamental understanding of the OVL, which will introduce novel applications.

Controllable mode transformation in perfect optical vortices.

We report a novel method to freely transform the modes of a perfect optical vortex (POV). By adjusting the scaling factor of the Bessel-Gauss beam at the object plane, the POV mode transformation can be easily controlled from circle to ellipse with a high mode purity. Combined with the modulation of the cone angle of an axicon, the ellipse mode can be freely adjusted along the two orthogonal directions. The properties of the "perfect vortex" are experimentally verified. Moreover, fractional elliptic POVs with versatile modes are presented, where the number and position of the gaps are controllable. These findings are significant for applications that require the complex structured optical field of the POV.

In situ measurement of the topological charge of a perfect vortex using the phase shift method.

We propose a method to determine the topological charge (TC) of a perfect vortex. With the phase shift technique, the perfect vortex and its conjugate beam exactly overlap and interfere. Consequently, the TC of a perfect vortex is determined by counting the number of interference fringes. This proposed method enables in situ determination of the TC of the perfect vortex without the need for additional optical elements, and it is immune to environmental vibration and parasitic interference.