Dendritic Signals Research Articles

Active propagation of dendritic electrical signals in C. elegans

Active propagation of electrical signals in C. elegans neurons requires ion channels capable of regenerating membrane potentials. Here we report regenerative depolarization of a major gustatory sensory neuron, ASEL. Whole-cell patch-clamp recordings in vivo showed supralinear depolarization of ASEL upon current injection. Furthermore, stimulation of animal’s nose with NaCl evoked all-or-none membrane depolarization in ASEL. Mutant analysis showed that EGL-19, the α1 subunit of L-type voltage-gated Ca2+ channels, is essential for regenerative depolarization of ASEL. ASEL-specific knock-down of EGL-19 by RNAi demonstrated that EGL-19 functions in C. elegans chemotaxis along an NaCl gradient. These results demonstrate that a natural substance induces regenerative all-or-none electrical signals in dendrites, and that these signals are essential for activation of sensory neurons for chemotaxis. As in other vertebrate and invertebrate nervous systems, active information processing in dendrites occurs in C. elegans, and is necessary for adaptive behavior.

Reinforcement Learning Recruits Somata and Apical Dendrites across Layers of Primary Sensory Cortex

SUMMARYThe mammalian brain can form associations between behaviorally relevant stimuli in an animal’s environment. While such learning is thought to primarily involve high-order association cortex, even primary sensory areas receive long-range connections carrying information that could contribute to high-level representations. Here, we imaged layer 1 apical dendrites in the barrel cortex of mice performing a whisker-based operant behavior. In addition to sensory-motor events, calcium signals in apical dendrites of layers 2/3 and 5 neurons and in layer 2/3 somata track the delivery of rewards, both choice related and randomly administered. Reward-related tuft-wide dendritic spikes emerge gradually with training and are task specific. Learning recruits cells whose intrinsic activity coincides with the time of reinforcement. Layer 4 largely lacked reward-related signals, suggesting a source other than the primary thalamus. Our results demonstrate that a sensory cortex can acquire a set of associations outside its immediate sensory modality and linked to salient behavioral events.

Abstract A059: Tackling the tumor microenvironment with CD38 blockade to enhance cancer immunotherapy

Abstract The single agent or the combination of anti-PD-1, anti-PD-L1, and anti-CTLA-4 is an effective strategy that is being clinically explored to treat a variety of cancer types. Some patients display primary resistance to the treatment, while others relapse after treatment. Resistance is a major issue that needs to be addressed. Using multiple immunocompetent syngeneic and K-rasLA1/+p53R172HΔg/+ spontaneous animal models of lung cancer, we have explored the mechanisms of resistance to treatments by evaluating the molecular and cellular immune profiles of the tumor microenvironment. We observed that tumor-bearing mice treated with PD-1/PD-L1 blocking antibodies developed resistance through the up-regulation of CD38. We also observed this in the combination therapy of anti-PD-1 and anti-CTLA-4, suggesting that CD38 is a major mechanism of resistance to immune checkpoint inhibitors. In vitro and in vivo studies demonstrated that CD38 impacted CD8+ T-cell function via adenosine receptor signaling and dendritic cell-mediated B7 signaling. Antibody-mediated cell depletion assays were conducted to validate the mechanisms. To determine the applicability to patients, we analyzed 793 lung cancer patients’ specimens with immunohistochemistry staining and assessed biomarker relationships in multiple large independent patient databases (~1,900 tumors). Pathologic analysis revealed positive immunohistochemical staining for CD38 on tumor cells in 15-23% of cases, and bioinformatic analyses revealed a strong correlation between CD38 expression and the immune signature. Lastly, targeting CD38 abolished the treatment resistance by modulating the adenosine levels and thereby enhancing the effector immune cell infiltrates into the tumor microenvironment. Based on our study, CD38 blockade improves the efficacy of single-agent anti-PD-1/PD-L1, or with anti-CTLA-4 combination in lung cancer. CD38 could potentially serve as a novel biomarker of resistance for immune checkpoint inhibition. The data from this study provide a unique target, biomarker, and therapeutic strategy that can be translated into the clinical practice. Citation Format: Limo Chen, Lixia Diao, Xiaohui Yi, Bertha Leticia Rodriguez, Yanli Li, Pamela Villalobos, Tina Cascone, Xi Liu, Lin Tan, Philip Lorenzi, Jared Fradette, David Peng, Ferdinandos Skoulidis, Youhong Fan, Jaime Rodriguez-Canales, Vassiliki Papadimitrakopoulou, Ethan Dmitrovsky, Lauren A Byers, Jing Wang, Ignasio Wistuba, Jim Heymach, Don Gibbons. Tackling the tumor microenvironment with CD38 blockade to enhance cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A059.

Compartmentalized Dendritic Signaling in the Retina

Compartmentalized Dendritic Signaling in the Retina

NMDA receptors potentiate activity‐dependent dendritic release of neuropeptides from hypothalamic neurons

Using 'sniffer' cell biosensors, we evaluated the effects of specific firing patterns and frequencies on activity-dependent somatodendritic release of vasopressin from paraventricular nucleus neurones. Somatodendritic release of vasopressin was rarely observed during continuous firing but was strengthened by clustered activity. Moreover, release evoked at any given frequency was robustly potentiated by NMDA receptor (NMDAR)-mediated firing. Differently from axonal release, NMDAR activation was necessary for somatodendritic release to occur at physiological firing frequencies, acting thus as a gating mechanism by which activity-dependent release from these two neuronal compartments could be independently regulated. The NMDAR-mediated potentiation was independent of a specific firing pattern and was not accompanied by increased spike broadening, but correlated with higher dendritic Ca2+ levels. Our studies provide fundamental novel information regarding stimulus-secretion coupling at somatodendritic compartments, and shed light into mechanisms by which activity-dependent release of neuronal signals from axonal terminals and dendrites could be regulated in a spatially compartmentalized manner. Dendrites are now recognized to be active transmitting neuronal compartments subserving complex brain functions, including motor behaviours and homeostatic neurohumoral responses. Still, the precise mechanisms underlying activity-dependent release of dendritic signals, and how dendritic release is regulated independently from axonal release, remains largely unknown. We used 'sniffer' biosensor cells to enable the measurement and study of activity-dependent dendritic release of vasopressin (VP) from hypothalamic neurones in brain slices. SnifferVP responses were dose-dependent, with a threshold detection level of 0.5nM for VP, being thus a highly sensitive tool to detect endogenous physiological levels of the neuropeptide. Somatodendritic release of VP was rarely observed in response to a burst of action potentials fired in continuous mode, but was strengthened by clustered firing activity. Moreover, release evoked at any given frequency was robustly potentiated when firing was triggered by NMDA receptor (NMDAR) activation. Differently from axonal release, NMDAR activation was necessary for dendritic release to occur at physiological firing frequencies. Thus, we propose that NMDARs may act as a gating mechanism by which activity-dependent release from these two neuronal compartments can be independently regulated. The NMDAR-mediated potentiation of dendritic release was independent of a particular action potential waveform, firing pattern evoked, or a more pronounced spiked broadening, but correlated with higher dendritic Ca2+ levels. Overall, our studies provide fundamental novel information regarding stimulus-secretion coupling at neuronal dendrites, and shed light into mechanisms by which activity-dependent release of neuronal signals from axonal terminals and dendrites can be regulated in a spatially compartmentalized manner.

Ruxolitinib and Steroid Refractory/Dependent Bronchiolitis Obliterans after Hematopoietic Cell Transplantation: A Steroid Sparing Agent That Also Resulted in Improved Lung Function in Children

Background: Bronchiolitis obliterans syndrome (BOS), chronic graft versus host (cGVHD) disease of the lungs after hematopoietic cell transplantation, is characterized by fixed airway obstruction, bronchiolar inflammation, and scarring and confers a dismal prognosis. While steroids are the backbone therapy for BOS, prolonged use is not recommended. There is no consensus therapy for patients who are steroid dependent or refractory (SR). Ruxolitinib, a JAK1/2 inhibitor, has potent anti-inflammatory effects by modifying T and dendritic cell signaling. It is FDA approved to treat acute GVHD, though there are also reports of its efficacy in cGVHD. We describe the experience of children with SR BOS treated with ruxolitinib.Methods: IRB approval was obtained for retrospective review of all patients treated with ruxolitinib for SR BOS after allogenic HCT. Patients were identified using departmental records. BOS was defined as SR if patients had worsening pulmonary function tests (PFTs) on therapy or required ≥0.5 mg/kg/day of steroids for greater than 30 days. 2014 NIH consensus conference criteria were used to define PFT diagnostic criteria for BOS and response to therapy. A partial response (PR) was an improvement in FEV1 of 10% and complete response (CR) an FEV1 of ≥80% predicted.Results: Five patients were treated with ruxolitinib for SR BO; 2 patients had no response to steroids and 3 were unable to wean (Figure 1). All patients received an unrelated bone marrow transplant (BMT) and the same aGVHD prophylaxis; methotrexate on days + 1, 3, 6, and 11, cyclosporine, and steroids. 3/6 patients were matched at 10/10 HLA-loci and 2 at 9/10. One patient (#5) received a second BMT for relapsed MDS, all other patients had one BMT. 2/5 patients had acute GVHD that was steroid responsive and resolved prior to BOS diagnosis.BOS developed a median of 285 days after stem cell infusion (range 131-547 days). 4/5 patients presented with dyspnea, and 1 (#4) developed a new oxygen requirement. He was developmentally delayed and could not report symptoms. Patient 4 developed extensive cGVHD 11 months after receiving a donor lymphocyte infusion for relapse of AML 3 months after HCT. Patients 2 and 5 had a concurrent pulmonary fungal infection at the time of diagnosis. Median FEV1% of predicted at diagnosis was 38% (range 23-64%). All patients were treated with corticosteroids and fluticasone, montelukast, azithromycin (FAM) after diagnosis of BOS. Ruxolitinib ranged from the third to fifth line therapy. 2/5 patients had a PR to ruxolitinib with an increase in predicted FEV1% of 10 and 24% (#2 and 3 respectively). One patient (#4) had an increase of FEV1 of 9% and was able to stop steroids. Patient 1 had stable PFTs and was able to wean steroids by 83%. Median time to response was 4 weeks.Patient 5 was treated for 4 months with ruxolitinib and was not able to have any pulmonary function tests (PFTS) during this period because of a pneumothorax. When this resolved, he had new nodules on chest CT, worsened PFTs and was found to have culture confirmed pulmonary Nocaria and Hypodontia palmae. Ruxolitinib was discontinued in the setting of a new infection. Without interim PFTs, his response was considered non-evaluable. He continues to have progressive BOS on corticosteroids and had a third relapse of his MDS. Other than this infection, the other 4/5 patients had no side effects from ruxolitinib. Doses ranged from 2.5 mg three times a week in a patient with end stage renal disease to 10 mg BID. 3/6 patients had extensive cGVHD with multiple organs involved; all other organ systems had a PR or CR to ruxolitinib. All patients are alive and 4/5 are disease free. Median follow up period after BOS diagnosis was 2 years (range 8-32 months).Conclusion: We describe 5 pediatric patients with SR BOS after hematopoietic cell transplantation. In the 4 patients with an evaluable response, 50% (2/4) had a PR to ruxolitinib and an additional patient had an improvement in predicted FEV1 of 9%. All were able to stop steroids or wean the dose by at least 50%. Responses occurred early, and the medication was well tolerated with minimal side effects. This is a small case series, but describes both improvement in pulmonary function testing and the ability to wean steroids after treatment with ruxolitinib in children with SR BOS - two measures notoriously difficult to obtain. Further prospective studies are needed to evaluate the role of ruxolitinib in children with cGVHD, particularly BOS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo.

To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of conditionally Hoxb8-immortalized hematopoietic progenitor cells with CRISPR/Cas9 technology to create a powerful experimental system to investigate DC migration and function. Hematopoietic progenitor cells from the bone marrow of Cas9-transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline-regulated form of the transcription factor Hoxb8 (Cas9-Hoxb8 cells). These cells could be stably cultured for weeks in the presence of doxycycline and puromycin, allowing us to introduce additional genetic modifications applying CRISPR/Cas9 technology. Importantly, modified Cas9-Hoxb8 cells retained their potential to differentiate in vitro into myeloid cells, and GM-CSF-differentiated Cas9-Hoxb8 cells showed the classical phenotype of GM-CSF-differentiated bone marrow-derived dendritic cells. Following intralymphatic delivery Cas9-Hoxb8 DCs entered the lymph node in a CCR7-dependent manner. Finally, we used two-photon microscopy and imaged Cas9-Hoxb8 DCs that expressed the genetic Ca2+ sensor GCaMP6S to visualize in real-time chemokine-induced Ca2+ signaling of lymph-derived DCs entering the LN parenchyma. Altogether, our study not only allows mechanistic insights in DC migration in vivo, but also provides a platform for the immunoengineering of DCs that, in combination with two-photon imaging, can be exploited to further dissect DC dynamics in vivo.

Simultaneous dendritic voltage and calcium imaging and somatic recording from Purkinje neurons in awake mice

Spatiotemporal maps of dendritic signalling and their relationship with somatic output is fundamental to neuronal information processing, yet remain unexplored in awake animals. Here, we combine simultaneous sub-millisecond voltage and calcium two-photon imaging from distal spiny dendrites, with somatic electrical recording from spontaneously active cerebellar Purkinje neurons (PN) in awake mice. We detect discrete 1−2 ms suprathreshold voltage spikelets in the distal spiny dendrites during dendritic complex spikes. Spikelets and their calcium correlates are highly heterogeneous in number, timing and spatial distribution within and between complex spikes. Back-propagating simple spikes are highly attenuated. Highly variable 5–10 ms voltage hotspots are localized to fine dendritic processes and are reduced in size and frequency by lidocaine and CNQX. Hotspots correlated with somatic output but also, at high frequency, trigger purely dendritic calcium spikes. Summarizing, spatiotemporal signalling in PNs is far more complex, dynamic, and fine scaled than anticipated, even in resting animals.

Inhibition gates supralinear Ca2+ signaling in Purkinje cell dendrites during practiced movements.

Motor learning involves neural circuit modifications in the cerebellar cortex, likely through re-weighting of parallel fiber inputs onto Purkinje cells (PCs). Climbing fibers instruct these synaptic modifications when they excite PCs in conjunction with parallel fiber activity, a pairing that enhances climbing fiber-evoked Ca2+ signaling in PC dendrites. In vivo, climbing fibers spike continuously, including during movements when parallel fibers are simultaneously conveying sensorimotor information to PCs. Whether parallel fiber activity enhances climbing fiber Ca2+ signaling during motor behaviors is unknown. In mice, we found that inhibitory molecular layer interneurons (MLIs), activated by parallel fibers during practiced movements, suppressed parallel fiber enhancement of climbing fiber Ca2+ signaling in PCs. Similar results were obtained in acute slices for brief parallel fiber stimuli. Interestingly, more prolonged parallel fiber excitation revealed latent supralinear Ca2+ signaling. Therefore, the balance of parallel fiber and MLI input onto PCs regulates concomitant climbing fiber Ca2+ signaling.

Interleukin-6 secretion is limited by self-signaling in endosomes.

Cells producing cytokines often express the receptor for the same cytokine, which makes them prone to autocrine signaling. How cytokine release and signaling are regulated in the same cell is not understood. In this study, we demonstrate that signaling by exogenous and self-synthesized inflammatory cytokine interleukin-6 (IL-6) within endosomal compartments acts as a cellular brake that limits the synthesis of IL-6. Our data show that IL-6 is internalized by dendritic cells and signals from endosomal compartments containing the IL-6 receptor. Newly synthesized IL-6 also traffics via these endosomal compartments and signals in transit to the plasma membrane. This allows activation of STAT3 which in turn limits toll-like receptor 4 stimulant lipopolysaccharide (LPS) triggered transcription of IL-6. Long-term exposure to LPS removes this brake via inhibition of STAT3 by increased expression of suppressor of cytokine signaling 3 and results in fully fledged IL-6 production. This transient regulation could prevent excessive IL-6 production during early infections.

Abstract 3609: Differential gene expression is associated with response to chemoradiation and relapse-free and overall survival in rectal adenocarcinoma

Abstract Background: Neoadjuvant chemoradiation is a standard therapy for stage II-III rectal adenocarcinoma, and the degree of pathologic response observed upon resection informs prognosis. However, there is a need to identify novel biomarkers of response to chemoradiation and survival after chemoradiation, particularly using modern next-generation sequencing methods. Methods: We prospectively collected pretreatment endoscopic tumor biopsies from 43 patients with stage II-IV rectal adenocarcinoma prior to neoadjuvant chemoradiation with concurrent fluoropyrimidine. Tumor samples were fresh frozen, and subsequently RNA was extracted, paired end libraries for mRNA sequencing (RNASeq) were prepared using TruSeq RNA Access library prep kits (Illumina), and samples were sequenced on Illumina HiSeq. Differentially expressed genes were determined using DESeq2 and Ingenuity pathway analysis (Qiagen) was performed. Additionally, the association between “claudin-low”-like gene sets established in breast and bladder cancers and clinical outcomes was determined. Results: Among the 36 patients with adequate RNA quality, 7 had a pathologic complete response (pCR) and 29 did not, with 22 differentially expressed genes with false discovery rate (FDR) <0.1 between the two cohorts, including EME2 (2.0-fold change, FDR p-value 0.00627). At 5 years of follow-up, 11 patients had known relapse and 11 were known to be relapse-free, with 33 differentially expressed genes with FDR<0.1 between the two cohorts. Pathway analysis demonstrated that p53 signaling and Wnt/β-catenin signaling pathways were associated with relapse, while dendritic cell maturation and Toll-like receptor signaling pathways were associated with relapse-free survival. Rectal cancers with greater expression of a “claudin-low”-like signature had significantly inferior relapse-free survival (HR 1.79, 95% CI 1.07-3.00) and overall survival (HR 1.91, 95% CI 1.17-3.12) on univariate Cox proportional hazards model. Conclusions: There are multiple differentially expressed genes associated with response to neoadjuvant chemoradiation in rectal adenocarcinoma. EME2, which forms an endonuclease that cleaves stalled replication forks, was one of the most differentially expressed genes overexpressed in patients with pathologic complete response and thus is a rational target for further investigation. Extension of “claudin-low” gene expression signatures to rectal cancers may serve as a new prognostic biomarker. Further investigation into the association of gene expression subtypes and responses to neoadjuvant chemoradiation is warranted. Citation Format: Michael S. Lee, Cheryl Carlson, Benjamin F. Calvo, Bert H. O'Neil, Dante S. Bortone, Benjamin G. Vincent. Differential gene expression is associated with response to chemoradiation and relapse-free and overall survival in rectal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3609.

Immunology of Cryptococcal Infections: Developing a Rational Approach to Patient Therapy.

Cryptococcal meningoencephalitis is responsible for upwards of 15% of HIV-related deaths worldwide and is currently the most common cause of non-viral meningitis in the US, affecting both previously healthy and people with immune suppression caused by cancer chemotherapy, transplantation, and biologic therapies. Despite a continued 30–50% attributable mortality, recommended therapeutic strategies have remained largely unchanged since the 1950s. Recent murine models and human studies examining the role of the immune system in both susceptibility to the infection as well as host damage have begun to influence patient care decisions. The Damage Framework Response, originally proposed in 1999, was recently used to discuss dichotomous etiologies of host damage in cryptococcal disease. These include patients suffering microbiological damage with low host immunity (especially those immunosuppressed with HIV) and those having low (live) microbiological burden but high immune-mediated damage (HIV-related immune reconstitution syndrome and non-HIV-related postinfectious inflammatory response syndrome). Cryptococcal disease in previously healthy hosts, albeit rare, has been known for a long time. Immunophenotyping and dendritic cell-T cell signaling studies on cerebral spinal fluid of these rare patients reveal immune capacity for recognition and T-cell activation pathways including increased levels of HLA-DR and CD56. However, despite effective T-cell signals, brain biopsy and autopsy specimens demonstrated an M2 alternative macrophage polarization and poor phagocytosis of fungal cells. These studies expand the paradigm for cryptococcal disease susceptibility to include a prominent role for immune-mediated damage and suggest a need for careful individual consideration of immune activation during therapy of cryptococcal disease in diverse hosts.

Transcytosis of TrkA leads to diversification of dendritic signaling endosomes

The development of the peripheral nervous system relies on long-distance signaling from target organs back to the soma. In sympathetic neurons, this long-distance signaling is mediated by target derived Nerve Growth Factor (NGF) interacting with its axonal receptor, TrkA. This ligand receptor complex internalizes into what is commonly referred to as the signaling endosome which is transported retrogradely to the soma and dendrites to mediate survival signaling and synapse formation, respectively. The molecular identity of signaling endosomes in dendrites has not yet been determined. Here, we perform a detailed analysis of TrkA endosomal compartments and trafficking patterns. We find that signaling endosomes are not uniform but molecularly diversified into Rab7 (late endosome) and Rab11 (recycling endosome) populations in axons and dendrites in vitro and in the soma in vivo. Surprisingly, TrkA-NGF signaling endosomes in dendrites undergo dynamic trafficking events, including putative fusion and fission. Overall, we find that signaling endosomes do not remain as a singular endosomal subtype but instead exist in multiple populations that undergo dynamic endosomal trafficking events. These dynamic events might drive functional diversification of the signaling endosome.

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

Glucocorticoids promote apoptosis of proinflammatory monocytes by inhibiting ERK activity

Glucocorticoids (GCs) are potent anti-inflammatory drugs whose mode of action is complex and still debatable. One likely cellular target of GCs are monocytes/macrophages. The role of GCs in monocyte survival is also debated. Although both granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) are important regulators of macrophage lineage functions including their survival, the former is often associated with proinflammatory functions while the latter is important in lineage homeostasis. We report here that the GC, dexamethasone, induces apoptosis in GM-CSF-treated human monocytes while having no impact on M-CSF-induced monocyte survival. To understand how GCs, GM-CSF, and M-CSF are regulating monocyte survival and other functions during inflammation, we firstly examined the transcriptomic changes elicited by these three agents in human monocytes, either acting alone or in combination. Transcriptomic and Ingenuity pathway analyses found that dexamethasone differentially modulated dendritic cell maturation and TREM1 signaling pathways in GM-CSF-treated and M-CSF-treated monocytes, two pathways known to be regulated by ERK1/2 activity. These analyses led us to provide evidence that the GC inhibits ERK1/2 activity selectively in GM-CSF-treated monocytes to induce apoptosis. It is proposed that this inhibition of ERK1/2 activity leads to inactivation of p90 ribosomal-S6 kinase and Bad dephosphorylation leading in turn to enhanced caspase-3 activity and subsequent apoptosis. Furthermore, pharmacological inhibition of GC receptor activity restored the ERK1/2 signaling and prevented the GC-induced apoptosis in GM-CSF-treated monocytes. Increased tissue macrophage numbers, possibly from enhanced survival due to mediators such as GM-CSF, can correlate with inflammatory disease severity; also reduction in these numbers can correlate with the therapeutic benefit of a number of agents, including GCs. We propose that the ERK1/2 signaling pathway promotes survival of GM-CSF-treated proinflammatory monocytes, which can be selectively targeted by GCs as a novel mechanism to reduce local monocyte/macrophage numbers and hence inflammation.

Tuning the Lateral Range of L-Type Calcium Channel-Dependent Calcium Signals in Dendrites of Hippocampal Neurons

In cultured hippocampal neurons, voltage-gated L-type Ca2+ channel (LTCC) activity is subject to potentiation by protein kinase A (PKA) and to feedback inhibition by STIM1, a sensor of endoplasmic reticulum (ER) [Ca2+]. Feedback by STIM1 is engaged following NMDA receptor-dependent LTCC activation: LTCCs allow influx of Ca2+ that induces Ca2+ release (CICR) from ER stores, and CICR-driven stores depletion activates STIM1, which is able to span the gap between cortical ER and plasma membrane to contact and inhibit LTCCs. PKA potentiation of LTCC activity relies upon anchoring of the kinase to LTCCs by the scaffolding protein AKAP150; AKAP150 co-anchoring of the phosphatase calcineurin (CaN) provides a counterbalance on LTCC activity. Uncaging of MNI-glutamate adjacent to a dendritic spine triggers a rise in [Ca2+]cytosol and a drop in [Ca2+]ER. A substantial component of each of these changes in [Ca2+] is dependent upon LTCC activity, as measured by sensitivity to the LTCC antagonist nimodipine. For a stimulated spine, the nimodipine-sensitive component of both changes in [Ca2+]cytosol and [Ca2+]ER extends in ∼exponential fashion along the adjoining dendrite, with length constants of ∼5 µm. AKAP150 knockdown and replacement with AKAP150 lacking its PKA binding domain reduces the spread of both the cytosolic and ER Ca2+ changes, whereas knockdown and replacement with an AKAP150 mutant lacking the CaN site increases the lateral range of Ca2+ signaling. The effects on [Ca2+]cytosol and [Ca2+]ER of delocalizing PKA or CaN are attributable, respectively, to depotentiation of LTCC Ca2+ influx and hence decreased CICR or to unopposed potentiation and increased CICR. These results suggest that the spatial extent of LTCC-dependent Ca2+ signals in dendrites may be tuned in accord with the local balance of PKA and CaN activity.

Regional differences in Purkinje cell morphology in the cerebellar vermis of male mice.

Regional differences in dendritic architecture can influence connectivity and dendritic signal integration, with possible consequences for neuronal computation. In the cerebellum, analyses of Purkinje cells (PCs), which are functionally critical as they provide the sole output of the cerebellar cortex, have suggested that the cerebellar cortex is not uniform in structure as traditionally assumed. However, the limitations of traditional staining methods and microscopy capabilities have presented difficulties in investigating possible local variations in PC morphology. To address this question, we used male mice expressing green fluorescent protein selectively in PCs. Using Neurolucida 360 with confocal image stacks, we reconstructed dendritic arbors of PCs residing in lobule V (anterior) and lobule IX (posterior) of the vermis. We then analyzed morphologies of individual arbors and the structure of the assembled "jungle," comparing these features across anatomical locations and age groups. Strikingly, we found that in lobule IX, half of the reconstructed PCs had two primary dendrites emanating from their soma, whereas fewer than a quarter showed this characteristic in lobule V. Furthermore, PCs in lobule V showed more efficient spatial occupancy compared to lobule IX, as well as greater packing density and increased arbor overlap in the adult. When analyzing complete ensembles of PC arbors, we also observed "hot spots" of increased dendritic density in lobule V, whereas lobule IX showed a more homogeneous spread of dendrites. These differences suggest that input patterns and/or physiology of PCs could likewise differ along the vermis, with possible implications for cerebellar function.

Role of presynaptic calcium stores for neural network dysfunction in Alzheimer's disease.

Role of presynaptic calcium stores for neural network dysfunction in Alzheimer's disease.

The Early Dendritic Cell Signaling Induced by Virulent Francisella tularensis Strain Occurs in Phases and Involves the Activation of Extracellular Signal-Regulated Kinases (ERKs) and p38 In the Later Stage

Dendritic cells (DCs) infected by Francisella tularensis are poorly activated and do not undergo classical maturation process. Although reasons of such unresponsiveness are not fully understood, their impact on the priming of immunity is well appreciated. Previous attempts to explain the behavior of Francisella-infected DCs were hypothesis-driven and focused on events at later stages of infection. Here, we took an alternative unbiased approach by applying methods of global phosphoproteomics to analyze the dynamics of cell signaling in primary DCs during the first hour of infection by Francisella tularensis Presented results show that the early response of DCs to Francisella occurs in phases and that ERK and p38 signaling modules induced at the later stage are differentially regulated by virulent and attenuated ΔdsbA strain. These findings imply that the temporal orchestration of host proinflammatory pathways represents the integral part of Francisella life-cycle inside hijacked DCs.

Heterosynaptic Plasticity Determines the Set-Point for Cortical Excitatory-Inhibitory Balance

Excitation in neural circuits must be carefully controlled by inhibition to regulate information processing and network excitability. During development, inhibitory and excitatory inputs in the cerebral cortex are initially mismatched but become co-tuned or ‘balanced’ with experience. However, little is known about the set-points for excitatory-inhibitory balance or the mechanisms for establishing or maintaining this balance. Here we show how coordinated long-term synaptic modifications calibrate populations of excitatory and inhibitory inputs onto mouse auditory cortical pyramidal neurons. Pairing pre- and postsynaptic activity induced plasticity at paired inputs and different forms of heterosynaptic plasticity at the strongest unpaired synapses, which required minutes of activity and dendritic Ca2 signaling to be computed. Theoretical analyses demonstrated how the relative amount of heterosynaptic plasticity could normalize and stabilize synaptic strengths to achieve any possible excitatory-inhibitory correlation. Thus excitatory-inhibitory balance is dynamic and cell-specific, determined by distinct plasticity rules across multiple excitatory and inhibitory synapses.