Signal flow in the NMDA receptor-dependent phosphoproteome regulates postsynaptic plasticity for aversive learning.

Structural plasticity of dendritic spines in the nucleus accumbens (NAc) is crucial for learning from aversive experiences. Activation of NMDA receptors (NMDARs) stimulates Ca2+-dependent signaling that leads to changes in the actin cytoskeleton, mediated by the Rho family of GTPases, resulting in postsynaptic remodeling essential for learning. We investigated how phosphorylation events downstream of NMDAR activation drive the changes in synaptic morphology that underlie aversive learning. Large-scale phosphoproteomic analyses of protein kinase targets in mouse striatal/accumbal slices revealed that NMDAR activation resulted in the phosphorylation of 194 proteins, including RhoA regulators such as ARHGEF2 and ARHGAP21. Phosphorylation of ARHGEF2 by the Ca2+-dependent protein kinase CaMKII enhanced its RhoGEF activity, thereby activating RhoA and its downstream effector Rho-associated kinase (ROCK/Rho-kinase). Further phosphoproteomic analysis identified 221 ROCK targets, including the postsynaptic scaffolding protein SHANK3, which is crucial for its interaction with NMDARs and other postsynaptic scaffolding proteins. ROCK-mediated phosphorylation of SHANK3 in the NAc was essential for spine growth and aversive learning. These findings demonstrate that NMDAR activation initiates a phosphorylation cascade crucial for learning and memory.

Stress-impaired reward pathway promotes distinct feeding behavior patterns.

Although dietary behaviors are affected by neuropsychiatric disorders, various environmental conditions can have strong effects as well. We found that mice under multiple stresses, including social isolation, intermittent high-fat diet, and physical restraint, developed feeding behavior patterns characterized by a deviated bait approach (fixated feeding). All the tested stressors affected dopamine release at the nucleus accumbens (NAcc) shell and dopamine normalization reversed the feeding defects. Moreover, inhibition of dopaminergic activity in the ventral tegmental area that projects into the NAcc shell caused similar feeding pattern aberrations. Given that the deviations were not consistently accompanied by changes in the amount consumed or metabolic factors, the alterations in feeding behaviors likely reflect perturbations to a critical stress-associated pathway in the mesolimbic dopamine system. Thus, deviations in feeding behavior patterns that reflect reward system abnormalities can be sensitive biomarkers of psychosocial and physical stress.

In Vivo Whole-Cell Recording from the Mouse Brain.

Measuring the membrane potential dynamics of neurons offers a comprehensive understanding of the molecular and cellular mechanisms that form their spiking activity, thus playing a crucial role in unraveling the mechanistic processes governing brain function. Techniques for intracellular recordings of membrane potentials pioneered in the 1940s have witnessed significant advancements since their inception. Among these, whole-cell patch-clamp recording has emerged as a leading method for measuring neuronal membrane potentials due to its high stability and broad applicability ranging from cultured cells to brain slices and even behaving animals. This chapter provides a detailed protocol to acquire stable whole-cell recordings from neurons in the cerebral cortex of awake, head-restrained mice. Significant enhancements to our protocol include implanting a metal head-post using adhesive resin cement and preparing a recording pipette with a long shank for targeting deeper brain regions. This protocol, once implemented, enables whole-cell recordings up to 2.5 mM beneath the cortical surface.

Interareal Synaptic Inputs Underlying Whisking-Related Activity in the Primary Somatosensory Barrel Cortex.

Body movements influence brain-wide neuronal activities. In the sensory cortex, thalamocortical bottom-up inputs and motor-sensory top-down inputs are thought to affect the dynamics of membrane potentials (Vm ) of neurons and change their processing of sensory information during movements. However, direct perturbation of the axons projecting to the sensory cortex from other remote areas during movements has remained unassessed, and therefore the interareal circuits generating motor-related signals in sensory cortices remain unclear. Using a Gi/o -coupled opsin, eOPN3, we here inhibited interareal signals incoming to the whisker primary somatosensory barrel cortex (wS1) of awake male mice and tested their effects on whisking-related changes in neuronal activities in wS1. Spontaneous whisking in air induced the changes in spike rates of a subset of wS1 neurons, which were accompanied by depolarization and substantial reduction of slow-wave oscillatory fluctuations of Vm Despite an extensive innervation, inhibition of inputs from the whisker primary motor cortex (wM1) to wS1 did not alter the spike rates and Vm dynamics of wS1 neurons during whisking. In contrast, inhibition of axons from the whisker-related thalamus (wTLM) and the whisker secondary somatosensory cortex (wS2) to wS1 largely attenuated the whisking-related supra- and sub-threshold Vm dynamics of wS1 neurons. Notably, silencing inputs from wTLM markedly decreased the modulation depth of whisking phase-tuned neurons in wS1, while inhibiting wS2 inputs did not impact the whisking variable tuning of wS1 neurons. Thus, sensorimotor integration in wS1 during spontaneous whisking is predominantly facilitated by direct synaptic inputs from wTLM and wS2 rather than from wM1.

Neural mechanisms underlying uninstructed orofacial movements during reward-based learning behaviors.

During reward-based learning tasks, animals make orofacial movements that globally influence brain activity at the timings of reward expectation and acquisition. These orofacial movements are not explicitly instructed and typically appear along with goal-directed behaviors. Here, we show that reinforcing optogenetic stimulation of dopamine neurons in the ventral tegmental area (oDAS) in mice is sufficient to induce orofacial movements in the whiskers and nose without accompanying goal-directed behaviors. Pavlovian conditioning with a sensory cue and oDAS elicited cue-locked and oDAS-aligned orofacial movements, which were distinguishable by a machine-learning model. Inhibition or knockout of dopamine D1 receptors in the nucleus accumbens inhibited oDAS-induced motion but spared cue-locked motion, suggesting differential regulation of these two types of orofacial motions. In contrast, inactivation of the whisker primary motor cortex (wM1) abolished both types of orofacial movements. We found specific neuronal populations in wM1 representing either oDAS-aligned or cue-locked whisker movements. Notably, optogenetic stimulation of wM1 neurons successfully replicated these two types of movements. Our results thus suggest that accumbal D1-receptor-dependent and -independent neuronal signals converge in the wM1 for facilitating distinct uninstructed orofacial movements during a reward-based learning task.

In-situ neutron diffraction study of lattice deformation behaviour of commercially pure titanium at cryogenic temperature.

Titanium has a significant potential for the cryogenic industrial fields such as aerospace and liquefied gas storage and transportation due to its excellent low temperature properties. To develop and advance the technologies in cryogenic industries, it is required to fully understand the underlying deformation mechanisms of Ti under the extreme cryogenic environment. Here, we report a study of the lattice behaviour in grain families of Grade 2 CP-Ti during in-situ neutron diffraction test in tension at temperatures of 15-298 K. Combined with the neutron diffraction intensity analysis, EBSD measurements revealed that the twinning activity was more active at lower temperature, and the behaviour was complicated with decreasing temperature. The deviation of linearity in the lattice strains was caused by the load-redistribution between plastically soft and hard grain families, resulting in the three-stage hardening behaviour. The lattice strain behaviour further deviated from linearity with decreasing temperature, leading to the transition of plastically soft-to-hard or hard-to-soft characteristic of particular grain families at cryogenic temperature. The improvement of ductility can be attributed to the increased twinning activity and a significant change of lattice deformation behaviour at cryogenic temperature.

DNA-double strand breaks enhance the expression of major histocompatibility complex class II through the ATM-NF-κΒ-IRF1-CIITA pathway.

Major histocompatibility complex class II (MHC II) is important for the adaptive immune response because MHC II presents processed antigens to a cluster of differentiation 4 (CD4)-positive T-cells. Conventional doses of chemotherapeutic agents induce tumor cell death by causing DNA double-strand breaks (DSBs). However, cellular responses caused by sub-lethal doses of chemotherapeutic agents are poorly understood. In this study, using low doses of chemotherapeutic agents, we showed that DSBs enhanced the expression of MHC II on cells that originate from antigen-presenting cells (APCs). These agents induced the MHC class II transactivator (CIITA), the master regulator of MHC II, and interferon regulatory factor 1 (IRF1), a transcription factor for CIITA. Short hairpin RNA against IRF1 suppressed chemotherapeutic agent-induced CIITA expression, whereas exogenous expression of IRF1 induced CIITA. Inhibition of ataxia-telangiectasia mutated (ATM), a DSB-activated kinase, suppressed induction of IRF1, CIITA, and MHC II. Similar results were observed by inhibiting NF-κB, a downstream target of ATM. These results suggest that DSBs induce MHC II activity via the ATM-NF-κB-IRF1-CIITA pathway in cells that intrinsically present antigens. Additionally, chemotherapeutic agents induced T-cell regulatory molecules. Our findings suggest that chemotherapeutic agents enhance the antigen presentation activity of APCs for T-cell activation.

Remote Optogenetics Using Up/Down-Conversion Phosphors.

Microbial rhodopsins widely used for optogenetics are sensitive to light in the visible spectrum. As visible light is heavily scattered and absorbed by tissue, stimulating light for optogenetic control does not reach deep in the tissue irradiated from outside the subject body. Conventional optogenetics employs fiber optics inserted close to the target, which is highly invasive and poses various problems for researchers. Recent advances in material science integrated with neuroscience have enabled remote optogenetic control of neuronal activities in living animals using up- or down-conversion phosphors. The development of these methodologies has stimulated researchers to test novel strategies for less invasive, wireless control of cellular functions in the brain and other tissues. Here, we review recent reports related to these new technologies and discuss the current limitations and future perspectives toward the establishment of non-invasive optogenetics for clinical applications.

Downstream projection of Barrington's nucleus to the spinal cord in mice.

Barrington's nucleus (Bar), which controls micturition behavior through downstream projections to the spinal cord, contains two types of projection neurons, BarCRH and BarESR1, that have different functions and target different spinal circuitry. Both types of neurons project to the L6-S1 spinal intermediolateral (IML) nucleus, whereas BarESR1 neurons also project to the dorsal commissural nucleus (DCN). To obtain more information about the spinal circuits targeted by Bar, we used patch-clamp recording in spinal slices from adult mice in combination with optogenetic stimulation of Bar terminals. Recording of opto-evoked excitatory postsynaptic currents (oEPSCs) in 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (DiI)-labeled lumbosacral preganglionic neurons (LS-PGNs) revealed that both Bar neuronal populations make strong glutamatergic monosynaptic connections with LS-PGNs, whereas BarESR1 neurons also elicited smaller-amplitude glutamatergic polysynaptic oEPSCs or polysynaptic opto-evoked inhibitory postsynaptic currents (oIPSCs) in some LS-PGNs. Optical stimulation of BarCRH and BarESR1 terminals also elicited monosynaptic oEPSCs and polysynaptic oIPSCs in sacral DCN neurons, some of which must include interneurons projecting to either the IML or ventral horn. Application of capsaicin increased opto-evoked firing during repetitive stimulation of Bar terminals through the modulation of spontaneous postsynaptic currents in LS-PGNs. In conclusion, our experiments have provided insights into the synaptic mechanisms underlying the integration of inputs from Bar to autonomic circuitry in the lumbosacral spinal cord that may control micturition.NEW & NOTEWORTHY Photostimulation of BarCRH or BarESR1 axons in the adult mouse spinal cord elicits excitatory or inhibitory postsynaptic responses in multiple cell types related to the autonomic nervous system including preganglionic neurons (PGNs) in the lumbosacral intermediolateral nucleus and interneurons in the lumbosacral dorsal commissure nucleus. Integration of excitatory inputs from Bar and from visceral primary afferents in PGNs may be important in the regulation of micturition behavior.

Remote control of neural function by X-ray-induced scintillation.

Scintillators emit visible luminescence when irradiated with X-rays. Given the unlimited tissue penetration of X-rays, the employment of scintillators could enable remote optogenetic control of neural functions at any depth of the brain. Here we show that a yellow-emitting inorganic scintillator, Ce-doped Gd3(Al,Ga)5O12 (Ce:GAGG), can effectively activate red-shifted excitatory and inhibitory opsins, ChRmine and GtACR1, respectively. Using injectable Ce:GAGG microparticles, we successfully activated and inhibited midbrain dopamine neurons in freely moving mice by X-ray irradiation, producing bidirectional modulation of place preference behavior. Ce:GAGG microparticles are non-cytotoxic and biocompatible, allowing for chronic implantation. Pulsed X-ray irradiation at a clinical dose level is sufficient to elicit behavioral changes without reducing the number of radiosensitive cells in the brain and bone marrow. Thus, scintillator-mediated optogenetics enables minimally invasive, wireless control of cellular functions at any tissue depth in living animals, expanding X-ray applications to functional studies of biology and medicine.

Frequency-Dependent Block of Excitatory Neurotransmission by Isoflurane via Dual Presynaptic Mechanisms.

Volatile anesthetics are widely used for surgery, but neuronal mechanisms of anesthesia remain unidentified. At the calyx of Held in brainstem slices from rats of either sex, isoflurane at clinical doses attenuated EPSCs by decreasing the release probability and the number of readily releasable vesicles. In presynaptic recordings of Ca2+ currents and exocytic capacitance changes, isoflurane attenuated exocytosis by inhibiting Ca2+ currents evoked by a short presynaptic depolarization, whereas it inhibited exocytosis evoked by a prolonged depolarization via directly blocking exocytic machinery downstream of Ca2+ influx. Since the length of presynaptic depolarization can simulate the frequency of synaptic inputs, isoflurane anesthesia is likely mediated by distinct dual mechanisms, depending on input frequencies. In simultaneous presynaptic and postsynaptic action potential recordings, isoflurane impaired the fidelity of repetitive spike transmission, more strongly at higher frequencies. Furthermore, in the cerebrum of adult mice, isoflurane inhibited monosynaptic corticocortical spike transmission, preferentially at a higher frequency. We conclude that dual presynaptic mechanisms operate for the anesthetic action of isoflurane, of which direct inhibition of exocytic machinery plays a low-pass filtering role in spike transmission at central excitatory synapses.SIGNIFICANCE STATEMENT Synaptic mechanisms of general anesthesia remain unidentified. In rat brainstem slices, isoflurane inhibits excitatory transmitter release by blocking presynaptic Ca2+ channels and exocytic machinery, with the latter mechanism predominating in its inhibitory effect on high-frequency transmission. Both in slice and in vivo, isoflurane preferentially inhibits spike transmission induced by high-frequency presynaptic inputs. This low-pass filtering action of isoflurane likely plays a significant role in general anesthesia.

SMARCA4 deficiency-associated heterochromatin induces intrinsic DNA replication stress and susceptibility to ATR inhibition in lung adenocarcinoma.

The SWI/SNF chromatin remodeling complex regulates transcription through the control of chromatin structure and is increasingly thought to play an important role in human cancer. Lung adenocarcinoma (LADC) patients frequently harbor mutations in SMARCA4, a core component of this multisubunit complex. Most of these mutations are loss-of-function mutations, which disrupt critical functions in the regulation of chromatin architecture and can cause DNA replication stress. This study reports that LADC cells deficient in SMARCA4 showed increased DNA replication stress and greater sensitivity to the ATR inhibitor (ATRi) in vitro and in vivo. Mechanistically, loss of SMARCA4 increased heterochromatin formation, resulting in stalled forks, a typical DNA replication stress. In the absence of SMARCA4, severe ATRi-induced single-stranded DNA, which caused replication catastrophe, was generated on nascent DNA near the reversed forks around heterochromatin in an Mre11-dependent manner. Thus, loss of SMARCA4 confers susceptibility to ATRi, both by increasing heterochromatin-associated replication stress and by allowing Mre11 to destabilize reversed forks. These two mechanisms synergistically increase susceptibility of SMARCA4-deficient LADC cells to ATRi. These results provide a preclinical basis for assessing SMARCA4 defects as a biomarker of ATRi efficacy.

[Fanconi anemia].

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure, increased susceptibility to leukemia and cancer, and genomic instabilities. Protein products encoded by 22 FA genes, identified till date, cooperate in a molecular pathway called the FA pathway to repair DNA interstrand cross-links induced by chemotherapeutic agents, such as mitomycin C and cisplatin. An accumulating number of studies have shown several new functional aspects of the FA pathway, particularly in the context of the pathogenesis of bone marrow failure. This review focuses on the following topics: (1) aldehydes as intrinsic interstrand cross-linkers; (2) cytokine-induced hematopoietic stress; (3) increased transforming growth factor-ß signaling; (4) mitochondrial functions of FA proteins. These findings are expected to offer new therapeutic opportunities for bone marrow failure in FA.

Large Timescale Interrogation of Neuronal Function by Fiberless Optogenetics Using Lanthanide Micro-particles.

Optogenetics requires implantation of light-delivering optical fibers, as current light-sensitive opsins are activated by visible light, which cannot effectively penetrate biological tissues. Insertion of optical fibers and subsequent photostimulation inherently damages brain tissue, and fiber tethering can restrict animal behavior. To overcome these technical limitations, we developed minimally invasive "fiberless" optogenetics using lanthanide micro-particles (LMPs), which emit up-conversion luminescence in the visible spectrum in response to irradiation with tissue-penetrating near-infrared light. Depolarizing (C1V1) and hyperpolarizing (ACR1) opsins were strongly activated by up-conversion luminescence from green-emitting LMPs both in vitro and in vivo. Using this technique, we successfully manipulated locomotive behavior of mice by activating and inhibiting neurons in the dorsal striatum, at a depth of 2 mm from the brain surface. LMPs were retained and remained functional for >8 weeks at the injection site. Fiberless optogenetics offers opportunities to control neuronal function over longer time frames using freely behaving animals.

Diverse Long-Range Axonal Projections of Excitatory Layer 2/3 Neurons in Mouse Barrel Cortex.

Excitatory projection neurons of the neocortex are thought to play important roles in perceptual and cognitive functions of the brain by directly connecting diverse cortical and subcortical areas. However, many aspects of the anatomical organization of these inter-areal connections are unknown. Here, we studied long-range axonal projections of excitatory layer 2/3 neurons with cell bodies located in mouse primary somatosensory barrel cortex (wS1). As a population, these neurons densely projected to secondary whisker somatosensory cortex (wS2) and primary/secondary whisker motor cortex (wM1/2), with additional axon in the dysgranular zone surrounding the barrel field, perirhinal temporal association cortex and striatum. In three-dimensional reconstructions of 6 individual wS2-projecting neurons and 9 individual wM1/2-projecting neurons, we found that both classes of neurons had extensive local axon in layers 2/3 and 5 of wS1. Neurons projecting to wS2 did not send axon to wM1/2, whereas a small subset of wM1/2-projecting neurons had relatively weak projections to wS2. A small fraction of projection neurons solely targeted wS2 or wM1/2. However, axon collaterals from wS2-projecting and wM1/2-projecting neurons were typically also found in subsets of various additional areas, including the dysgranular zone, perirhinal temporal association cortex and striatum. Our data suggest extensive diversity in the axonal targets selected by individual nearby cortical long-range projection neurons with somata located in layer 2/3 of wS1.

Polη, a Y-family translesion synthesis polymerase, promotes cellular tolerance of Myc-induced replication stress.

Growth of precancerous and cancer cells relies on their tolerance of oncogene-induced replication stress (RS). Translesion synthesis (TLS) plays an essential role in the cellular tolerance of various types of RS and bypasses replication barriers by employing specialized polymerases. However, limited information is available about the role of TLS polymerases in oncogene-induced RS. Here, we report that Polη, a Y-family TLS polymerase, promotes cellular tolerance of Myc-induced RS. Polη was recruited to Myc-induced RS sites, and Polη depletion enhanced the Myc-induced slowing and stalling of replication forks and the subsequent generation of double-strand breaks (DSBs). Overexpression of a catalytically dead Polη also promoted Myc-induced DSB formation. In the absence of Polη, Myc-induced DSB formation depended on MUS81-EME2 (the S-phase-specific endonuclease complex), and concomitant depletion of MUS81-EME2 and Polη enhanced RS and cell death in a synergistic manner. Collectively, these results indicate that Polη facilitates fork progression during Myc-induced RS, thereby helping cells tolerate the resultant deleterious effects. Additionally, the present study highlights the possibility of a synthetic sickness or lethality between Polη and MUS81-EME2 in cells experiencing Myc-induced RS.

Acute HSF1 depletion induces cellular senescence through the MDM2-p53-p21 pathway in human diploid fibroblasts.

Heat shock transcription factor 1 (HSF1) regulates the expression of a wide array of genes, controls the expression of heat shock proteins (HSPs) as well as cell growth. Although acute depletion of HSF1 induces cellular senescence, the underlying mechanisms are poorly understood. Here, we report that HSF1 depletion-induced senescence (HDIS) of human diploid fibroblasts (HDFs) was independent of HSP-mediated proteostasis but dependent on activation of the p53-p21 pathway, partly because of the increased expression of dehydrogenase/reductase 2 (DHRS2), a putative MDM2 inhibitor. We observed that HDIS occurred without decreased levels of major HSPs or increased proteotoxic stress in HDFs. Additionally, VER155008, an inhibitor of HSP70 family proteins, increased proteotoxicity and suppressed cell growth but failed to induce senescence. Importantly, we found that activation of the p53-p21 pathway resulting from reduced MDM2-dependent p53 degradation was required for HDIS. Furthermore, we provide evidence that increased DHRS2 expression contributes to p53 stabilization and HDIS. Collectively, our observations uncovered a molecular pathway in which HSF1 depletion-induced DHRS2 expression leads to activation of the MDM2-p53-p21 pathway required for HDIS.

Sensation during Active Behaviors.

A substantial portion of our sensory experience happens during active behaviors such as walking around or paying attention. How do sensory systems work during such behaviors? Neural processing in sensory systems can be shaped by behavior in multiple ways ranging from a modulation of responsiveness or sharpening of tuning to a dynamic change of response properties or functional connectivity. Here, we review recent findings on the modulation of sensory processing during active behaviors in different systems: insect vision, rodent thalamus, and rodent sensory cortices. We discuss the circuit-level mechanisms that might lead to these modulations and their potential role in sensory function. Finally, we highlight the open questions and future perspectives of this exciting new field.