Dendritic Signals Research Articles

Numerical and experimental verification of the single neural adaptive PID real-time inverse method for solving inverse heat conduction problems

For the problems of difficult on-line tuning of PID parameters, weak anti-interference ability and poor self-adaptive ability in the classical proportional-integral-derivative (PID) inverse method, combining single artificial neuron with PID inverse algorithm, a single neural adaptive PID (SNA-PID) inverse method is established to estimate the thermal boundary condition. Compared with the classical PID inverse method, the main difference is that SNA-PID can adaptively adjust the weights of PID parameters. In the SNA-PID inverse method, firstly, the deviation signal between the output of the direct heat conduction model and the measured temperature and its first-order difference and second-order difference are taken as three inputs of a single neuron (dendritic input signal of neuron), and the connection strength of each input signal is determined according to the updated weight of the adaptive learning algorithm, The excitation value of the boundary heat flux increment of the heat conduction system is generated according to the linear truncation function. Finally, the real-time estimation of boundary heat flux of heat conduction system is realized. The unsteady boundary heat flux of one-dimensional heat conduction object is estimated through numerical and experimental tests, which verifies the effectiveness of the proposed SNA-PID inverse method.

JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory Tcell induction.

Dendritic cells (DCs) induce peripheral Tcell tolerance, but cell-intrinsic signaling cascades governing their stable tolerogenesis remain poorly defined. Janus Kinase 1 (JAK1) transduces cytokine-receptor signaling, and JAK inhibitors (Jakinibs), including JAK1-specific filgotinib, break inflammatory cycles in autoimmunity. Here, we report in heterogeneous DC populations of multiple secondary lymphoid organs that JAK1 promotes peripheral Tcell tolerance during experimental autoimmune encephalomyelitis (EAE). Mice harboring DC-specific JAK1 deletion exhibit elevated peripheral CD4+ Tcell expansion, less regulatory Tcells (Tregs), and worse EAE outcomes, whereas adoptive DC transfer ameliorates EAE pathogenesis by inducing peripheral Tregs, programmed cell death ligand 1 (PD-L1) dependently. This tolerogenic program is substantially reduced upon the transfer of JAK1-deficient DCs. DC-intrinsic IFN-γ-JAK1-STAT1 signaling induces PD-L1, which is required for DCs to convert CD4+ Tcells into Tregs invitro and attenuated upon JAK1 deficiency and filgotinib treatment. Thus, DC-intrinsic JAK1 promotes peripheral tolerance, suggesting potential unwarranted DC-mediated effects of Jakinibs in autoimmune diseases.

Extra arginine supplementation during the suckling period alleviates weaning stress through the regulation of dendritic cells and Notch2 signaling in piglets.

This study aims to study the effects of extra arginine (Arg) supplementation during the suckling period on the weaning stress and intestinal barrier function of breastfed piglets. Forty 7-day-old breastfed piglets divided into the control group (CON) and Arg group (Arg) were fed with extra saline or Arg (250 mg per kg per d body weight), respectively. All piglets were weaned when they were 21 days old. Eight piglets from each group were sacrificed before weaning and on the 3rd-day after weaning, respectively. The results showed that Arg improved the average daily weight gain of piglets before weaning (P < 0.01) and decreased the average daily weight loss after weaning (P < 0.05). Weaning decreased the ratio of the villus length versus crypt depth (V/C) in the SI (P < 0.001), while Arg increased the V/C of the jejunum (P < 0.05). Arg increased the levels of immunoglobulins in the serum and SI (P < 0.05), decreased pro-inflammatory cytokines and increased anti-inflammatory cytokines in the SI (P < 0.05). In addition, Arg supplementation increased the numbers of SWC3a+CD40+ (P < 0.01) and SWC3a+SLAII+ DCs (P < 0.05), down-regulated Notch2 expression and up-regulated Jagged1 expression in the ilea of weaning piglets (P < 0.05). In conclusion, Arg supplementation during the suckling period decreased the LDH leakage in the SI, improved the intestinal morphology, down-regulated the contents of pro-inflammatory cytokines, accelerated the accumulation of DC precursors before weaning and increased the number of mature DCs after weaning, and thus improved the growth performance and reduced the weaning stress of piglets, and this might be associated with the regulation of Notch2 signaling.

A Kalirin missense mutation enhances dendritic RhoA signaling and leads to regression of cortical dendritic arbors across development

Normally, dendritic size is established prior to adolescence and then remains relatively constant into adulthood due to a homeostatic balance between growth and retraction pathways. However, schizophrenia is characterized by accelerated reductions of cerebral cortex gray matter volume and onset of clinical symptoms during adolescence, with reductions in layer 3 pyramidal neuron dendritic length, complexity, and spine density identified in multiple cortical regions postmortem. Nogo receptor 1 (NGR1) activation of the GTPase RhoA is a major pathway restricting dendritic growth in the cerebral cortex. We show that the NGR1 pathway is stimulated by OMGp and requires the Rho guanine nucleotide exchange factor Kalirin-9 (KAL9). Using a genetically encoded RhoA sensor, we demonstrate that a naturally occurring missense mutation in Kalrn, KAL-PT, that was identified in a schizophrenia cohort, confers enhanced RhoA activitation in neuronal dendrites compared to wild-type KAL. In mice containing this missense mutation at the endogenous locus, there is an adolescent-onset reduction in dendritic length and complexity of layer 3 pyramidal neurons in the primary auditory cortex. Spine density per unit length of dendrite is unaffected. Early adult mice with these structural deficits exhibited impaired detection of short gap durations. These findings provide a neuropsychiatric model of disease capturing how a mild genetic vulnerability may interact with normal developmental processes such that pathology only emerges around adolescence. This interplay between genetic susceptibility and normal adolescent development, both of which possess inherent individual variability, may contribute to heterogeneity seen in phenotypes in human neuropsychiatric disease.

Increased Signal Delays and Unaltered Synaptic Input Pattern Recognition in Layer III Neocortical Pyramidal Neurons of the rTg4510 Mouse Model of Tauopathy: A Computer Simulation Study With Passive Membrane.

Abnormal tau proteins are involved in pathology of many neurodegenerative disorders. Transgenic rTg4510 mice express high levels of human tau protein with P301L mutation linked to chromosome 17 that has been associated with frontotemporal dementia with parkinsonism. By 9 months of age, these mice recapitulate key features of human tauopathies, including presence of hyperphosphorylated tau and neurofibrillary tangles (NFTs) in brain tissue, atrophy and loss of neurons and synapses, and hyperexcitability of neurons, as well as cognitive deficiencies. We investigated effects of such human mutant tau protein on neuronal membrane, subthreshold dendritic signaling, and synaptic input pattern recognition/discrimination in layer III frontal transgenic (TG) pyramidal neurons of 9-month-old rTg4510 mice and compared these characteristics to those of wild-type (WT) pyramidal neurons from age-matched control mice. Passive segmental cable models of WT and TG neurons were set up in the NEURON simulator by using three-dimensionally reconstructed morphology and electrophysiological data of these cells. Our computer simulations predict leakage resistance and capacitance of neuronal membrane to be unaffected by the mutant tau protein. Computer models of TG neurons showed only modest alterations in distance dependence of somatopetal voltage and current transfers along dendrites and in rise times and half-widths of somatic Excitatory Postsynaptic Potential (EPSPs) relative to WT control. In contrast, a consistent and statistically significant slowdown was detected in the speed of simulated subthreshold dendritic signal propagation in all regions of the dendritic surface of mutant neurons. Predictors of synaptic input pattern recognition/discrimination remained unaltered in model TG neurons. This suggests that tau pathology is primarily associated with failures/loss in synaptic connections rather than with altered intraneuronal synaptic integration in neurons of affected networks.

Sex-Biased ZRSR2 Mutations in Myeloid Malignancies Impair Plasmacytoid Dendritic Cell Activation and Apoptosis.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive leukemia of plasmacytoid dendritic cells (pDC). BPDCN occurs at least three times more frequently in men than in women, but the reasons for this sex bias are unknown. Here, studying genomics of primary BPDCN and modeling disease-associated mutations, we link acquired alterations in RNA splicing to abnormal pDC development and inflammatory response through Toll-like receptors. Loss-of-function mutations in ZRSR2, an X chromosome gene encoding a splicing factor, are enriched in BPDCN, and nearly all mutations occur in males. ZRSR2 mutation impairs pDC activation and apoptosis after inflammatory stimuli, associated with intron retention and inability to upregulate the transcription factor IRF7. In vivo, BPDCN-associated mutations promote pDC expansion and signatures of decreased activation. These data support a model in which male-biased mutations in hematopoietic progenitors alter pDC function and confer protection from apoptosis, which may impair immunity and predispose to leukemic transformation. SIGNIFICANCE: Sex bias in cancer is well recognized, but the underlying mechanisms are incompletely defined. We connect X chromosome mutations in ZRSR2 to an extremely male-predominant leukemia. Aberrant RNA splicing induced by ZRSR2 mutation impairs dendritic cell inflammatory signaling, interferon production, and apoptosis, revealing a sex- and lineage-related tumor suppressor pathway.This article is highlighted in the In This Issue feature, p. 275.

Neuropeptide Modulation Increases Dendritic Electrical Spread to Restore Neuronal Activity Disrupted by Temperature.

Peptide neuromodulation has been implicated to shield neuronal activity from acute temperature changes that can otherwise lead to loss of motor control or failure of vital behaviors. However, the cellular actions neuropeptides elicit to support temperature-robust activity remain unknown. Here, we find that peptide neuromodulation restores rhythmic bursting in temperature-compromised central pattern generator (CPG) neurons by counteracting membrane shunt and increasing dendritic electrical spread. We show that acutely rising temperatures reduced spike generation and interrupted ongoing rhythmic motor activity in the crustacean gastric mill CPG. Neuronal release and extrinsic application of Cancer borealis tachykinin-related peptide Ia (CabTRP Ia), a substance-P-related peptide, restored rhythmic activity. Warming led to a significant decrease in membrane resistance and a shunting of the dendritic signals in the main gastric mill CPG neuron. Using a combination of fluorescent calcium imaging and electrophysiology, we observed that postsynaptic potentials and antidromic action potentials propagated less far within the dendritic neuropil as the system warmed. In the presence of CabTRP Ia, membrane shunt decreased and both postsynaptic potentials and antidromic action potentials propagated farther. At elevated temperatures, CabTRP Ia restored dendritic electrical spread or extended it beyond that at cold temperatures. Selective introduction of the CabTRP Ia conductance using a dynamic clamp demonstrated that the CabTRP Ia voltage-dependent conductance was sufficient to restore rhythmic bursting. Our findings demonstrate that a substance-P-related neuropeptide can boost dendritic electrical spread to maintain neuronal activity when perturbed and reveals key neurophysiological components of neuropeptide actions that support pattern generation in temperature-compromised conditions.SIGNIFICANCE STATEMENT Changes in body temperature can have detrimental consequences for the well-being of an organism. Temperature-dependent changes in neuronal activity can be especially dangerous if they affect vital behaviors. Understanding how temperature changes disrupt neuronal activity and identifying how to ameliorate such effects is critically important. Our study of a crustacean circuit shows that warming disrupts rhythmic neuronal activity by increasing membrane shunt and reducing dendritic electrical spread in a key circuit neuron. Through the ionic conductance activated by it, substance-P-related peptide modulation restored electrical spread and counteracted the detrimental temperature effects on rhythmic activity. Because neuropeptides are commonly implicated in sustaining neuronal activity during perturbation, our results provide a promising mechanism to support temperature-robust activity.

Focal laser stimulation of fly nociceptors activates distinct axonal and dendritic Ca2+ signals

Drosophila class IV neurons are polymodal nociceptors that detect noxious mechanical, thermal, optical, and chemical stimuli. Escape behaviors in response to attacks by parasitoid wasps are dependent on class IV cells, whose highly branched dendritic arbors form a fine meshwork that is thought to enable detection of the wasp’s needle-like ovipositor barb. To understand how mechanical stimuli trigger cellular responses, we used a focused 405-nm laser to create highly localized lesions to probe the precise position needed to evoke responses. By imaging calcium signals in dendrites, axons, and soma in response to stimuli of varying positions, intensities, and spatial profiles, we discovered that there are two distinct nociceptive pathways. Direct stimulation to dendrites (the contact pathway) produces calcium responses in axons, dendrites, and the cell body, whereas stimulation adjacent to the dendrite (the noncontact pathway) produces calcium responses in the axons only. We interpret the noncontact pathway as damage to adjacent cells releasing diffusible molecules that act on the dendrites. Axonal responses have higher sensitivities and shorter latencies. In contrast, dendritic responses have lower sensitivities and longer latencies. Stimulation of finer, distal dendrites leads to smaller responses than stimulation of coarser, proximal dendrites, as expected if the contact response depends on the geometric overlap of the laser profile and the dendrite diameter. Because the axon signals to the central nervous system to trigger escape behaviors, we propose that the density of the dendritic meshwork is high not only to enable direct contact with the ovipositor but also to enable neuronal activation via diffusing signals from damaged surrounding cells. Dendritic contact evokes responses throughout the dendritic arbor, even to regions distant and distal from the stimulus. These dendrite-wide calcium signals may facilitate hyperalgesia or cellular morphological changes after dendritic damage.

The Synergistic T-Cell Signal By CD79A/CD40 Costimulatory Endodomain Enhances CD19 Chimeric Antigen Receptor T-Cell Proliferation and Survival

Introduction: Adoptive T-cell therapy of gene modified to express CD19 chimeric antigen receptor (CAR) unveiled promising clinical outcomes, particularly in B-cell hematologic malignancies. Among several studies, CAR T-cell persistence is considered to be a surrogate marker of long-term clinical efficacy of CAR T-cell therapy. Consequently, various CD19 CAR T-cell costimulatory domains have been developed to improve T-cell signal, ultimately enhanced T-cell function and persistence. However, in the context of T-cell activation signaling, most of costimulatory molecules have already investigated, and no novel signaling domains have been reported to be useful recently. Here, we adopted B-cell signal moiety, CD79A, and dendritic cell signal moiety, CD40, and generated a composite costimulatory domain CD79A/CD40 to synergize T-cell signaling in order to improve T cell proliferation and persistence.Methods: The CD79A/CD40 costimulatory domain was developed and inserted in the middle of anti-CD19-CD28TM-CD3z-tEGFR construct. Then the novel CD19.79a40z CAR gene was subsequently cloned into retroviral vector and transduced into human CD3+ cells. The CAR expression was assessed and enriched by anti-EGFR mAb, which resulted in achieving more than 90% purity. Then we expanded CD19 CAR T-cell by coculture with irradiated EBV-LCL. These expanded cells were further used for downstream experiments including 51Cr release assay, coculture assay, intracellular cytokine staining assay (IC-CS), T cell proliferation assay, and T cell phenotype, in order to compare with conventional CD19.28z CAR T-cells. In addition, CD19 CAR-transduced Jurkat-Dual luciferase reporter cells were also used to assess NF-κB pathway activity.Results: After one course of viral transduction and LCL expansion, we obtained similar number of CAR-positive cells between CD19.79a40z and CD19.28z. The CD19.79a40z exhibited similar cytotoxic activities to CD19.28z, against K562-CD19 and two primary B-ALL samples (51Cr release assay). Also with coculture assay, both CD19.79a40z and CD19.28z showed robust cytotoxicity against K562-CD19 at E:T ratio of 1:1 and 1:8 after coculture for 120 hours without IL-2 supplementation. Interestingly, after extended coculture time to 168 hours, a tendency of target cell regrowth in CD19.28z was observed, whereas such regrowth was not seen in CD19.79a40z.Regarding IC-CS assay against K562-CD19, the proportions of IFN-γ+ and IL-2+ responders were comparable between CD19.79a40z and CD19.28z. Similarly IC-CS assay, ELISA revealed that CD19.79a40z and CD19.28z evenly produced IFN-γ, IL-2, and TNF-α upon exposure to K562-CD19.Furthermore, we also assessed the ability of CD19 CAR T-cell proliferation by coculture with K562-CD19, without IL-2 supplementation. Surprisingly, the novel CD19.79a40z unveiled robust T-cell proliferation and persistence throughout 2 weeks of culture when compared to CD19.28z (Figure).We next determined the plausible factors owing to T-cell survival advantage. Firstly, we assessed the surface markers of T-cell subset as well as exhaustion phenotypes, after coculture with K562-CD19 for 10 days. Again, CD19.79a40z showed similar T-cell subset composition and exhaustion markers to CD19.28z. Secondly, we examined the NF-κB activity of CD19 CAR after exposure to target antigens that the novel CD19.79a40z exhibited significantly higher NF-κB activity upon exposure to K562-CD19 and Raji when compared to CD19.28z. [Luciferase activity (units) of CD19.28z vs. CD19.79a.40z: K562-CD19 11306.65±747.63 vs. 21291.93±924.06; Raji 5524.93±321.68 vs. 7537.96±336.16, respectively (p= 0.0001)].Conclusions: The novel CD19.79a40z was successfully transduced into CD3+ cells with exhibited comparable cytotoxicity, cytokine secretion activities, T-cell subset composition, as well as T-cell exhaustion phenotypes to conventional CD19.28z CAR T-cells. On the contrary, the composite signaling domain, CD79A/CD40, astonishingly enhanced CD19 CAR T-cell proliferation and survival comparing to CD19.28z CAR T-cells. In addition, our construct presented higher NF-κB activity upon antigen exposure, which possibly synergized to T-cell signal and led to a proliferation advantage. Collectively, the greater CAR T-cell expansion might improve CAR T-cell persistence in vivo and ultimately improve the efficacy of adoptive CAR T-cell therapy. [Display omitted] DisclosuresKiyoi:FUJIFILM Corporation: Patents & Royalties, Research Funding; MSD K.K.: Research Funding; Eisai Co., Ltd.: Research Funding; Phizer Japan Inc.: Honoraria, Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Meiji Seika Pharma Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis Pharma K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

Dendritic calcium signals in rhesus macaque motor cortex drive an optical brain-computer interface

Calcium imaging is a powerful tool for recording from large populations of neurons in vivo. Imaging in rhesus macaque motor cortex can enable the discovery of fundamental principles of motor cortical function and can inform the design of next generation brain-computer interfaces (BCIs). Surface two-photon imaging, however, cannot presently access somatic calcium signals of neurons from all layers of macaque motor cortex due to photon scattering. Here, we demonstrate an implant and imaging system capable of chronic, motion-stabilized two-photon imaging of neuronal calcium signals from macaques engaged in a motor task. By imaging apical dendrites, we achieved optical access to large populations of deep and superficial cortical neurons across dorsal premotor (PMd) and gyral primary motor (M1) cortices. Dendritic signals from individual neurons displayed tuning for different directions of arm movement. Combining several technical advances, we developed an optical BCI (oBCI) driven by these dendritic signalswhich successfully decoded movement direction online. By fusing two-photon functional imaging with CLARITY volumetric imaging, we verified that many imaged dendrites which contributed to oBCI decoding originated from layer 5 output neurons, including a putative Betz cell. This approach establishes new opportunities for studying motor control and designing BCIs via two photon imaging.

Altered A-Type Potassium Channel Function Impairs Dendritic Spike Initiation and Temporoammonic Long-Term Potentiation in Fragile X Syndrome.

Fragile X syndrome (FXS) is the leading monogenetic cause of cognitive impairment and autism spectrum disorder. Area CA1 of the hippocampus receives current information about the external world from the entorhinal cortex via the temporoammonic (TA) pathway. Given its role in learning and memory, it is surprising that little is known about TA long-term potentiation (TA-LTP) in FXS. We found that TA-LTP was impaired in male fmr1 KO mice. Although there were no significant differences in basal synaptic transmission, synaptically evoked dendritic calcium signals were smaller in KO neurons. Using dendritic recording, we found no difference in complex spikes or pharmacologically isolated Ca2+ spikes; however, the threshold for fast, Na+-dependent dendritic spikes was depolarized in fmr1 KO mice. Cell-attached patch-clamp recordings found no difference in Na+ channels between wild-type and fmr1 KO CA1 dendrites. Dendritic spike threshold and TA-LTP were restored by blocking A-type K+ channels with either 150 µm Ba2+ or the more specific toxin AmmTx3. The impairment of TA-LTP shown here, coupled with previously described enhanced Schaffer collateral LTP, may contribute to spatial memory alterations in FXS. Furthermore, as both of these LTP phenotypes are attributed to changes in A-type K+ channels in FXS, our findings provide a potential therapeutic target to treat cognitive impairments in FXS.SIGNIFICANCE STATEMENT Alterations in synaptic function and plasticity are likely contributors to learning and memory impairments in many neurologic disorders. Fragile X syndrome is marked by dysfunctional learning and memory and changes in synaptic structure and function. This study shows a lack of LTP at temporoammonic synapses in CA1 neurons associated with biophysical differences in A-type K+ channels in fmr1 KO CA1 neurons. Our results, along with previous findings on A-type K+ channel effects on Schaffer collateral LTP, reveal differential effects of a single ion channelopathy on LTP at the two major excitatory pathways of CA1 pyramidal neurons. These findings expand our understanding of memory deficits in FXS and provide a potential therapeutic target for the treatment of memory dysfunction in FXS.

Autonomous Purkinje cell activation instructs bidirectional motor learning through evoked dendritic calcium signaling

The signals in cerebellar Purkinje cells sufficient to instruct motor learning have not been systematically determined. Therefore, we applied optogenetics in mice to autonomously excite Purkinje cells and measured the effect of this activity on plasticity induction and adaptive behavior. Ex vivo, excitation of channelrhodopsin-2-expressing Purkinje cells elicits dendritic Ca2+ transients with high-intensity stimuli initiating dendritic spiking that additionally contributes to the Ca2+ response. Channelrhodopsin-2-evoked Ca2+ transients potentiate co-active parallel fiber synapses; depression occurs when Ca2+ responses were enhanced by dendritic spiking. In vivo, optogenetic Purkinje cell activation drives an adaptive decrease in vestibulo-ocular reflex gain when vestibular stimuli are paired with relatively small-magnitude Purkinje cell Ca2+ responses. In contrast, pairing with large-magnitude Ca2+ responses increases vestibulo-ocular reflex gain. Optogenetically induced plasticity and motor adaptation are dependent on endocannabinoid signaling, indicating engagement of this pathway downstream of Purkinje cell Ca2+ elevation. Our results establish a causal relationship among Purkinje cell Ca2+ signal size, opposite-polarity plasticity induction, and bidirectional motor learning.

Ryanodine receptor-mediated Ca2+ release and atlastin-2 GTPase activity contribute to IP3-induced dendritic Ca2+ signals in primary hippocampal neurons

Neuronal Ca2+ signals are fundamental for synaptic transmission and activity-dependent changes in gene expression. Voltage-gated Ca2+ channels and N-methyl-d-aspartate receptors play major roles in mediating external Ca2+ entry during action potential firing and glutamatergic activity. Additionally, the inositol-1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) channels expressed in the endoplasmic reticulum (ER) also contribute to the generation of Ca2+ signals in response to neuronal activity. The ER forms a network that pervades the entire neuronal volume, allowing intracellular Ca2+ release in dendrites, soma and presynaptic boutons. Despite its unique morphological features, the contributions of ER structure and of ER-shaping proteins such as atlastin - an ER enriched GTPase that mediates homotypic ER tubule fusion - to the generation of Ca2+ signals in dendrites remains unreported. Here, we investigated the contribution of RyR-mediated Ca2+ release to IP3-generated Ca2+ signals in dendrites of cultured hippocampal neurons. We also employed GTPase activity-deficient atlastin-2 (ATL2) mutants to evaluate the potential role of atlastin on Ca2+ signaling and ER-resident Ca2+ channel distribution. We found that pharmacological suppression of RyR channel activity increased the rising time and reduced the magnitude and propagation of IP3-induced Ca2+ signals. Additionally, ATL2 mutants induced specific ER morphological alterations, delayed the onset and increased the rising time of IP3-evoked Ca2+ signals, and caused RyR2 and IP3R1 aggregation and RyR2 redistribution. These results indicate that both RyR and ATL2 activity regulate IP3-induced Ca2+ signal dynamics through RyR-mediated Ca2+-induced Ca2+ release, ER shaping and RyR2 distribution.

Vitamin E Attenuates FasL-Induced Apoptotic Death of Dendritic Cells Through PI3K Signalling

Vitamin E Attenuates FasL-Induced Apoptotic Death of Dendritic Cells Through PI3K Signalling

Freeze-Frame Imaging of Dendritic Calcium Signals With TubuTag.

The extensive dendritic arbor of neurons is thought to be actively involved in the processing of information. Dendrites contain a rich diversity of ligand- and voltage-activated ion channels as well as metabotropic receptors. In addition, they are capable of releasing calcium from intracellular stores. Under specific conditions, large neurons produce calcium spikes that are locally restricted to a dendritic section. To investigate calcium signaling in dendrites, we introduce TubuTag, a genetically encoded ratiometric calcium sensor anchored to the cytoskeleton. TubuTag integrates cytoplasmic calcium signals by irreversible photoconversion from green to red fluorescence when illuminated with violet light. We used a custom two-photon microscope with a large field of view to image pyramidal neurons in CA1 at subcellular resolution. Photoconversion was strongest in the most distal parts of the apical dendrite, suggesting a gradient in the amplitude of dendritic calcium signals. As the read-out of fluorescence can be performed several hours after photoconversion, TubuTag will help investigating dendritic signal integration and calcium homeostasis in large populations of neurons.

Microbial memories: Sex-dependent impact of the gut microbiome on hippocampal plasticity.

Germ‐free rodents, raised in the absence of a measurable gut microbiome, have been a key model to study the microbiome‐gut‐brain axis. Germ‐free mice exhibit marked behavioural and neurochemical differences to their conventionally raised counterparts. It is as yet unclear how these neurochemical differences lead to the behavioural differences. Here, we test the electrophysiological properties of hippocampal plasticity in adult germ‐free mice and compare them to conventionally raised counterparts. Whilst basal synaptic efficacy and pre‐synaptic short‐term plasticity appear normal, we find a striking alteration of hippocampal long‐term potentiation specifically in male germ‐free slices. However, the spike output of these neurons remains normal along with altered input‐output coupling, potentially indicating homeostatic compensatory mechanisms, or an altered excitation/inhibition balance. To our knowledge this is the first time the electrophysiological properties of the hippocampus have been assessed in a microbiome deficient animal. Our data indicate that the absence of a microbiome alters integration of dendritic signalling in the CA1 region in mice.

Dendritic coincidence detection in Purkinje neurons of awake mice.

Dendritic coincidence detection is fundamental to neuronal processing yet remains largely unexplored in awake animals. Specifically, the underlying dendritic voltage-calcium relationship has not been directly addressed. Here, using simultaneous voltage and calcium two-photon imaging of Purkinje neuron spiny dendrites, we show how coincident synaptic inputs and resulting dendritic spikes modulate dendritic calcium signaling during sensory stimulation in awake mice. Sensory stimulation increased the rate of postsynaptic potentials and dendritic calcium spikes evoked by climbing fiber and parallel fiber synaptic input. These inputs are integrated in a time-dependent and nonlinear fashion to enhance the sensory-evoked dendritic calcium signal. Intrinsic supralinear dendritic mechanisms, including voltage-gated calcium channels and metabotropic glutamate receptors, are recruited cooperatively to expand the dynamic range of sensory-evoked dendritic calcium signals. This establishes how dendrites can use multiple interplaying mechanisms to perform coincidence detection, as a fundamental and ongoing feature of dendritic integration in behaving animals.

Lung CD103+dendritic cells and Clec9a signaling are required for neonatal hyperoxia-induced inflammatory responses to rhinovirus infection.

Premature infants, especially those with bronchopulmonary dysplasia (BPD), develop recurrent severe respiratory viral illnesses. We have shown that hyperoxic exposure of immature mice, a model of BPD, increases lung IL-12-producing Clec9a+ CD103+ dendritic cells (DCs), pro-inflammatory responses, and airway hyperreactivity following rhinovirus (RV) infection. However, the requirement for CD103+ DCs and Clec9a, a DAMP receptor that binds necrotic cell cytoskeletal filamentous actin (F-actin), for RV-induced inflammatory responses has not been demonstrated. To test this, 2-day-old C57BL/6J, CD103+ DC-deficient Batf3-/- or Clec9agfp-/- mice were exposed to normoxia or hyperoxia for 14 days. Also, selected mice were treated with neutralizing antibody against CD103. Immediately after hyperoxia, the mice were inoculated with RV intranasally. We found that compared with wild-type mice, hyperoxia-exposed Batf3-/- mice showed reduced levels of IL-12p40, IFN-γ, and TNF-α, fewer IFN-γ-producing CD4+ T cells, and decreased airway responsiveness following RV infection. Similar effects were observed in anti-CD103-treated and Clec9agfp-/- mice. Furthermore, hyperoxia increased airway dead cell number and extracellular F-actin levels. Finally, studies in preterm infants with respiratory distress syndrome showed that tracheal aspirate CLEC9A expression positively correlated with IL12B expression, consistent with the notion that CLEC9A+ cells are responsible for IL-12 production in humans as well as mice. We conclude that CD103+ DCs and Clec9a are required for hyperoxia-induced pro-inflammatory responses to RV infection. In premature infants, Clec9a-mediated activation of CD103+ DCs may promote pro-inflammatory responses to viral infection, thereby driving respiratory morbidity.

Distinct peripheral blood monocyte and neutrophil transcriptional programs following intracerebral hemorrhage and different etiologies of ischemic stroke

Understanding cell-specific transcriptome responses following intracerebral hemorrhage (ICH) and ischemic stroke (IS) will improve knowledge of the immune response to brain injury. Transcriptomic profiles of 141 samples from 48 subjects with ICH, different IS etiologies, and vascular risk factor controls were characterized using RNA-seq in isolated neutrophils, monocytes and whole blood. In both IS and ICH, monocyte genes were down-regulated, whereas neutrophil gene expression changes were generally up-regulated. The monocyte down-regulated response to ICH included innate, adaptive immune, dendritic, NK cell and atherosclerosis signaling. Neutrophil responses to ICH included tRNA charging, mitochondrial dysfunction, and ER stress pathways. Common monocyte and neutrophil responses to ICH included interferon signaling, neuroinflammation, death receptor signaling, and NFAT pathways. Suppressed monocyte responses to IS included interferon and dendritic cell maturation signaling, phagosome formation, and IL-15 signaling. Activated neutrophil responses to IS included oxidative phosphorylation, mTOR, BMP, growth factor signaling, and calpain proteases-mediated blood–brain barrier (BBB) dysfunction. Common monocyte and neutrophil responses to IS included JAK1, JAK3, STAT3, and thrombopoietin signaling. Cell-type and cause-specific approaches will assist the search for future IS and ICH biomarkers and treatments.

Control of GM-CSF-Dependent Dendritic Cell Differentiation and Maturation by DEF6 and SWAP-70.

Although GM-CSF has been widely used in dendritic cell (DC) research, the mechanisms, factors, and signals regulating steady-state differentiation and maturation of GM-CSF-dependent DCs are insufficiently known. We found that the absence, individually or combined, of the related proteins DEF6 and SWAP-70 strongly enhances differentiation of murine GM-CSF-derived DCs. Contrasting SWAP-70, control through DEF6 does not depend on RHOA activation. DEF6 deficiency leads to expression of the DC-specific transcription factor ZBTB46 and prolonged STAT5 activation in GM-CSF cultures. SWAP-70 and DEF6-mediated restriction of DC differentiation converges mechanistically at the NF-κB pathway. DEF6 acts at early stages of DC differentiation in CD115-cKIT+ myeloid DC progenitors, whereas SWAP-70 acts subsequently. SWAP-70 and DEF6 regulate steady-state DC cytokine expression as well as in vivo accumulation in lymphatic tissue of migratory DCs. Our studies thus elucidate previously unknown roles of two closely related factors with distinct and complementary activities in DC differentiation and steady-state DC function.