ABSTRACT
The mammalian cortex is a laminar structure containing many areas and cell types that are densely interconnected in complex ways, and for which generalizable principles of organization remain mostly unknown. Here we describe a major expansion of the Allen Mouse Brain Connectivity Atlas resource1, involving around a thousand new tracer experiments in the cortex and its main satellite structure, the thalamus. We used Cre driver lines (mice expressing Cre recombinase) to comprehensively and selectively label brain-wide connections by layer and class of projection neuron. Through observations of axon termination patterns, we have derived a set of generalized anatomical rules to describe corticocortical, thalamocortical and corticothalamic projections. We have built a model to assign connection patterns between areas as either feedforward or feedback, and generated testable predictions of hierarchical positions for individual cortical and thalamic areas and for cortical network modules. Our results show that cell-class-specific connections are organized in a shallow hierarchy within the mouse corticothalamic network.
Subject(s)
Cerebral Cortex/anatomy & histology , Cerebral Cortex/cytology , Neural Pathways/anatomy & histology , Neural Pathways/cytology , Thalamus/anatomy & histology , Thalamus/cytology , Animals , Axons/physiology , Cerebral Cortex/physiology , Female , Integrases/genetics , Integrases/metabolism , Male , Mice , Mice, Inbred C57BL , Neural Pathways/physiology , Thalamus/physiologyABSTRACT
Increasing evidence indicates that the neural circuitry within glomeruli of the olfactory bulb plays a major role in affecting information flow between olfactory sensory neurons (OSNs) and output mitral cells (MCs). Glutamatergic external tufted (ET) cells, located at glomeruli, can act as intermediary cells in excitation between OSNs and MCs, whereas activation of MCs by OSNs is, in turn, suppressed by inhibitory synapses onto ET cells. In this study, we used patch-clamp recordings in rat olfactory bulb slices to examine the function of metabotropic glutamate receptors (mGluRs) in altering these glomerular signaling mechanisms. We found that activation of group II mGluRs profoundly reduced inhibition onto ET cells evoked by OSN stimulation. The mGluRs that mediated disinhibition were located on presynaptic GABAergic periglomerular cells and appeared to be activated by glutamate transients derived from dendrites in glomeruli. In terms of glomerular output, the mGluR-mediated reduction in GABA release led to a robust increase in the number of action potentials evoked by OSN stimulation in both ET cells and MCs. Importantly, however, the enhanced excitation was specific to when a glomerulus was strongly activated by OSN inputs. By being selective for strong vs. weak glomerular activation, mGluR-mediated disinhibition provides a mechanism to enhance the contrast in odor signals that activate OSN inputs into a single glomerulus at varying intensities.
Subject(s)
Action Potentials , Excitatory Postsynaptic Potentials , Olfactory Bulb/metabolism , Receptors, Metabotropic Glutamate/metabolism , Animals , Female , GABAergic Neurons/metabolism , GABAergic Neurons/physiology , Interneurons/metabolism , Interneurons/physiology , Male , Olfactory Bulb/physiology , Rats , Rats, Sprague-Dawley , Sensory Receptor Cells/metabolism , Sensory Receptor Cells/physiology , gamma-Aminobutyric Acid/metabolismABSTRACT
Lateral inhibition between neurons occurs in many different sensory systems, where it can perform such functions as contrast enhancement. In the olfactory bulb, lateral inhibition may occur between odorant receptor-specific glomeruli that are linked anatomically by GABAergic granule cells (GCs) and cells within the glomerular layer, although evidence supporting lateral inhibition at a functional level is modest. Here, we used patch-clamp, imaging, and glutamate uncaging methods in rat olfactory bulb slices to test for the presence of interglomerular lateral inhibition, as well as its underlying mechanisms. We found that a conditioning stimulus applied at one or a small group of glomeruli could suppress stimulus-evoked excitation of output mitral cells (MCs) at another glomerulus for interstimulus intervals of 20-50 ms and glomerular separations of up to 600 µm. The observed lateral inhibition was entirely dependent on circuitry within the glomerular layer, rather than GCs, and it involved GABAergic synaptic inputs that were targeted mainly onto tufted cells, which act as intermediaries in the excitation between olfactory sensory neurons and MCs. The key cell type responsible for mediating lateral interactions between glomeruli were GABAergic short-axon cells. These results suggest a functional segregation of GABAergic cells within the bulb, with one set located in the glomerular layer mediating suppression of MC spiking across glomeruli, and a second set, the GCs, synchronizing different glomeruli.
Subject(s)
Neural Inhibition/physiology , Neurons/physiology , Olfactory Bulb/physiology , Animals , Female , In Situ Hybridization , Inhibitory Postsynaptic Potentials/physiology , Male , Neurons/cytology , Olfactory Bulb/cytology , Organ Culture Techniques , Patch-Clamp Techniques , Rats , Rats, Sprague-DawleyABSTRACT
Within the olfactory system, information flow from the periphery onto output mitral cells (MCs) of the olfactory bulb (OB) has been thought to be mediated by direct synaptic inputs from olfactory sensory neurons (OSNs). Here, we performed patch-clamp measurements in rat and mouse OB slices to investigate mechanisms of OSN signaling onto MCs, including the assumption of a direct path, using electrical and optogenetic stimulation methods that selectively activated OSNs. We found that MCs are in fact not typically activated by direct OSN inputs and instead require a multistep, diffuse mechanism involving another glutamatergic cell type, the tufted cells. The preference for a multistep mechanism reflects the fact that signals arising from direct OSN inputs are drastically shunted by connexin 36-mediated gap junctions on MCs, but not tufted cells. An OB circuit with tufted cells intermediate between OSNs and MCs suggests that considerable processing of olfactory information occurs before its reaching MCs.
Subject(s)
Excitatory Postsynaptic Potentials/physiology , Olfactory Bulb/cytology , Olfactory Bulb/physiology , Signal Transduction/physiology , Animals , Female , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Rats , Rats, Sprague-DawleyABSTRACT
The Allen Mouse Brain Connectivity Atlas consists of anterograde tracing experiments targeting diverse structures and classes of projecting neurons. Beyond regional anterograde tracing done in C57BL/6 wild-type mice, a large fraction of experiments are performed using transgenic Cre-lines. This allows access to cell-class-specific whole-brain connectivity information, with class defined by the transgenic lines. However, even though the number of experiments is large, it does not come close to covering all existing cell classes in every area where they exist. Here, we study how much we can fill in these gaps and estimate the cell-class-specific connectivity function given the simplifying assumptions that nearby voxels have smoothly varying projections, but that these projection tensors can change sharply depending on the region and class of the projecting cells. This paper describes the conversion of Cre-line tracer experiments into class-specific connectivity matrices representing the connection strengths between source and target structures. We introduce and validate a novel statistical model for creation of connectivity matrices. We extend the Nadaraya-Watson kernel learning method that we previously used to fill in spatial gaps to also fill in gaps in cell-class connectivity information. To do this, we construct a "cell-class space" based on class-specific averaged regionalized projections and combine smoothing in 3D space as well as in this abstract space to share information between similar neuron classes. Using this method, we construct a set of connectivity matrices using multiple levels of resolution at which discontinuities in connectivity are assumed. We show that the connectivities obtained from this model display expected cell-type- and structure-specific connectivities. We also show that the wild-type connectivity matrix can be factored using a sparse set of factors, and analyze the informativeness of this latent variable model.
ABSTRACT
Neurodegenerative tauopathies are hypothesized to propagate via brain networks. This is uncertain because we have lacked precise network resolution of pathology. We therefore developed whole-brain staining methods with anti-p-tau nanobodies and imaged in 3D PS19 tauopathy mice, which have pan-neuronal expression of full-length human tau containing the P301S mutation. We analyzed patterns of p-tau deposition across established brain networks at multiple ages, testing the relationship between structural connectivity and patterns of progressive pathology. We identified core regions with early tau deposition, and used network propagation modeling to determine the link between tau pathology and connectivity strength. We discovered a bias towards retrograde network-based propagation of tau. This novel approach establishes a fundamental role for brain networks in tau propagation, with implications for human disease.
ABSTRACT
BACKGROUND: Genetic mutations underlying familial Alzheimer's disease (AD) were identified decades ago, but the field is still in search of transformative therapies for patients. While mouse models based on overexpression of mutated transgenes have yielded key insights in mechanisms of disease, those models are subject to artifacts, including random genetic integration of the transgene, ectopic expression and non-physiological protein levels. The genetic engineering of novel mouse models using knock-in approaches addresses some of those limitations. With mounting evidence of the role played by microglia in AD, high-dimensional approaches to phenotype microglia in those models are critical to refine our understanding of the immune response in the brain. METHODS: We engineered a novel App knock-in mouse model (AppSAA) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-ß pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous AppSAA mice at different ages in brain and/ or biofluids. Wild type littermate mice were used as experimental controls. We used in situ imaging technologies to define the whole-brain distribution of amyloid plaques and compare it to other AD mouse models and human brain pathology. To further explore the microglial response to AD relevant pathology, we isolated microglia with fibrillar Aß content from the brain and performed transcriptomics and metabolomics analyses and in vivo brain imaging to measure energy metabolism and microglial response. Finally, we also characterized the mice in various behavioral assays. RESULTS: Leveraging multi-omics approaches, we discovered profound alteration of diverse lipids and metabolites as well as an exacerbated disease-associated transcriptomic response in microglia with high intracellular Aß content. The AppSAA knock-in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered astroglial and microglial responses and elevation of CSF markers of neurodegeneration. Those observations were associated with increased TSPO and FDG-PET brain signals and a hyperactivity phenotype as the animals aged. DISCUSSION: Our findings demonstrate that fibrillar Aß in microglia is associated with lipid dyshomeostasis consistent with lysosomal dysfunction and foam cell phenotypes as well as profound immuno-metabolic perturbations, opening new avenues to further investigate metabolic pathways at play in microglia responding to AD-relevant pathogenesis. The in-depth characterization of pathological hallmarks of AD in this novel and open-access mouse model should serve as a resource for the scientific community to investigate disease-relevant biology.
Subject(s)
Alzheimer Disease , Amyloid beta-Protein Precursor , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Amyloidosis/metabolism , Animals , Brain/metabolism , Disease Models, Animal , Mice , Mice, Transgenic , Microglia/metabolism , Plaque, Amyloid/pathology , Receptors, GABA/metabolismABSTRACT
Estradiol-17beta (E2) is the major regulator of GnRH receptor (GnRHR) gene expression and number during the periovulatory period; however, the mechanisms underlying E2 regulation of the GNRHR gene remain undefined. Herein, we find that E2 conjugated to BSA (E2-BSA) mimics the stimulatory effect of E2 on GnRH binding in primary cultures of ovine pituitary cells. The time course for maximal GnRH analog binding was similar for both E2 and E2-BSA. The ability of E2 and E2-BSA to increase GnRH analog binding was blocked by the estrogen receptor (ER) antagonist ICI 182,780. Also, increased GnRH analog binding in response to E2 and the selective ESR1 agonist propylpyrazole triol was blocked by expression of a dominant-negative form of ESR1 (L540Q). Thus, membrane-associated ESR1 is the likely candidate for mediating E2 activation of the GNRHR gene. As cAMP response element binding protein (CREB) is an established target for E2 activation in gonadotrophs, we next explored a potential role for this protein as an intracellular mediator of the E2 signal. Consistent with this possibility, adenoviral-mediated expression of a dominant-negative form of CREB (A-CREB) completely abolished the ability of E2 to increase GnRH analog binding in primary cultures of ovine pituitary cells. Finally, the presence of membrane-associated E2 binding sites on ovine pituitary cells was demonstrated using a fluorescein isothiocyanate conjugate of E2-BSA. We suggest that E2 regulation of GnRHR number during the preovulatory period reflects a membrane site of action and may proceed through a nonclassical signaling mechanism, specifically a CREB-dependent pathway.
Subject(s)
Estradiol/metabolism , Estrogen Receptor alpha/metabolism , Pituitary Gland, Anterior/metabolism , Receptors, LHRH/metabolism , Signal Transduction , Up-Regulation , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane Permeability , Cells, Cultured , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Estradiol/analogs & derivatives , Estrogen Antagonists/metabolism , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/genetics , Female , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/metabolism , Follicular Phase/metabolism , Gonadotropin-Releasing Hormone/analogs & derivatives , Gonadotropin-Releasing Hormone/metabolism , Kinetics , Mutant Proteins/antagonists & inhibitors , Mutant Proteins/metabolism , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/drug effects , Recombinant Proteins , Serum Albumin, Bovine/metabolism , Sheep, Domestic , Signal Transduction/drug effects , Surface Properties/drug effects , Up-Regulation/drug effectsABSTRACT
The evolutionarily conserved default mode network (DMN) is a distributed set of brain regions coactivated during resting states that is vulnerable to brain disorders. How disease affects the DMN is unknown, but detailed anatomical descriptions could provide clues. Mice offer an opportunity to investigate structural connectivity of the DMN across spatial scales with cell-type resolution. We co-registered maps from functional magnetic resonance imaging and axonal tracing experiments into the 3D Allen mouse brain reference atlas. We find that the mouse DMN consists of preferentially interconnected cortical regions. As a population, DMN layer 2/3 (L2/3) neurons project almost exclusively to other DMN regions, whereas L5 neurons project in and out of the DMN. In the retrosplenial cortex, a core DMN region, we identify two L5 projection types differentiated by in- or out-DMN targets, laminar position, and gene expression. These results provide a multi-scale description of the anatomical correlates of the mouse DMN.
Subject(s)
Brain/diagnostic imaging , Default Mode Network/diagnostic imaging , Nerve Net/diagnostic imaging , Neurons/physiology , Animals , Brain/cytology , Connectome , Default Mode Network/cytology , Magnetic Resonance Imaging , Mice , Nerve Net/cytology , Neurons/cytologyABSTRACT
Fine-grained descriptions of brain connectivity are required to understand how neural information is processed and relayed across spatial scales. Previous investigations of the mouse brain connectome have used discrete anatomical parcellations, limiting spatial resolution and potentially concealing network attributes critical to connectome organization. Here, we provide a voxel-level description of the network and hierarchical structure of the directed mouse connectome, unconstrained by regional partitioning. We report a number of previously unappreciated organizational principles in the mammalian brain, including a directional segregation of hub regions into neural sink and sources, and a strategic wiring of neuromodulatory nuclei as connector hubs and critical orchestrators of network communication. We also find that the mouse cortical connectome is hierarchically organized along two superimposed cortical gradients reflecting unimodal-transmodal functional processing and a modality-specific sensorimotor axis, recapitulating a phylogenetically conserved feature of higher mammals. These findings advance our understanding of the foundational wiring principles of the mammalian connectome.
Subject(s)
Connectome , Animals , Brain/diagnostic imaging , Magnetic Resonance Imaging , Mammals , Mice , Nerve NetABSTRACT
Knowledge of mesoscopic brain connectivity is important for understanding inter- and intraregion information processing. Models of structural connectivity are typically constructed and analyzed with the assumption that regions are homogeneous. We instead use the Allen Mouse Brain Connectivity Atlas to construct a model of whole-brain connectivity at the scale of 100 µm voxels. The data consist of 428 anterograde tracing experiments in wild type C57BL/6J mice, mapping fluorescently labeled neuronal projections brain-wide. Inferring spatial connectivity with this dataset is underdetermined, since the approximately 2 × 105 source voxels outnumber the number of experiments. To address this issue, we assume that connection patterns and strengths vary smoothly across major brain divisions. We model the connectivity at each voxel as a radial basis kernel-weighted average of the projection patterns of nearby injections. The voxel model outperforms a previous regional model in predicting held-out experiments and compared with a human-curated dataset. This voxel-scale model of the mouse connectome permits researchers to extend their previous analyses of structural connectivity to much higher levels of resolution, and it allows for comparison with functional imaging and other datasets.
ABSTRACT
A variety of Alzheimer's disease (AD) mouse models overexpress mutant forms of human amyloid precursor protein (APP), producing high levels of amyloid ß (Aß) and forming plaques. However, the degree to which these models mimic spatiotemporal patterns of Aß deposition in brains of AD patients is unknown. Here, we mapped the spatial distribution of Aß plaques across age in three APP-overexpression mouse lines (APP/PS1, Tg2576, and hAPP-J20) using in vivo labeling with methoxy-X04, high throughput whole brain imaging, and an automated informatics pipeline. Images were acquired with high resolution serial two-photon tomography and labeled plaques were detected using custom-built segmentation algorithms. Image series were registered to the Allen Mouse Brain Common Coordinate Framework, a 3D reference atlas, enabling automated brain-wide quantification of plaque density, number, and location. In both APP/PS1 and Tg2576 mice, plaques were identified first in isocortex, followed by olfactory, hippocampal, and cortical subplate areas. In hAPP-J20 mice, plaque density was highest in hippocampal areas, followed by isocortex, with little to no involvement of olfactory or cortical subplate areas. Within the major brain divisions, distinct regions were identified with high (or low) plaque accumulation; for example, the lateral visual area within the isocortex of APP/PS1 mice had relatively higher plaque density compared with other cortical areas, while in hAPP-J20 mice, plaques were densest in the ventral retrosplenial cortex. In summary, we show how whole brain imaging of amyloid pathology in mice reveals the extent to which a given model recapitulates the regional Aß deposition patterns described in AD.
Subject(s)
Alzheimer Disease/pathology , Amyloid beta-Peptides , Brain/pathology , Neuroimaging/methods , Animals , Disease Models, Animal , Image Processing, Computer-Assisted , Mice , Mice, TransgenicABSTRACT
Recombinant rabies viral vectors have proven useful for applications including retrograde targeting of projection neurons and monosynaptic tracing, but their cytotoxicity has limited their use to short-term experiments. Here we introduce a new class of double-deletion-mutant rabies viral vectors that left transduced cells alive and healthy indefinitely. Deletion of the viral polymerase gene abolished cytotoxicity and reduced transgene expression to trace levels but left vectors still able to retrogradely infect projection neurons and express recombinases, allowing downstream expression of other transgene products such as fluorophores and calcium indicators. The morphology of retrogradely targeted cells appeared unperturbed at 1 year postinjection. Whole-cell patch-clamp recordings showed no physiological abnormalities at 8 weeks. Longitudinal two-photon structural and functional imaging in vivo, tracking thousands of individual neurons for up to 4 months, showed that transduced neurons did not die but retained stable visual response properties even at the longest time points imaged.
Subject(s)
Cerebral Cortex/physiology , Genetic Vectors/genetics , Neural Pathways/physiology , Neurons/metabolism , Sequence Deletion/genetics , Thalamus/cytology , Action Potentials/physiology , Age Factors , Analysis of Variance , Animals , Female , HEK293 Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Mice , Mice, Transgenic , Optogenetics , Patch-Clamp Techniques , Rats , Rats, Long-Evans , Transduction, GeneticABSTRACT
Ion channel localization to specific cell surface regions is essential for proper neuronal function. The Kv2.1 K+ channel forms large clusters on the plasma membrane of hippocampal neurons and transfected human embryonic kidney (HEK) cells. Using live cell imaging, we address mechanisms underlying this Kv2.1 clustering in both HEK cells and cultured hippocampal neurons. The Kv2.1-containing surface clusters have properties unlike those expected for a scaffolding protein bound channel. After channel is delivered to the plasma membrane via intracellular transport vesicles, it remains localized at the insertion site. Fluorescence recovery after photobleaching (FRAP) and quantum dot tracking experiments indicate that channel within the surface cluster is mobile (FRAP, tau = 14.1 +/- 1.5 and 11.5 +/- 6.1 s in HEK cells and neurons, respectively). The cluster perimeter is not static, because after fusion of adjacent clusters, green fluorescent protein (GFP)-Kv2.1 completely exchanged between the two domains within 60 s. Treatment of hippocampal neurons expressing GFP-Kv2.1 with 5 microM latrunculin A resulted in a significant increase in average cluster size from 0.89 +/- 0.16 microm2 to 12.15 +/- 1.4 microm2 with a concomitant decrease in cluster number. Additionally, Kv2.1 was no longer restricted to the cell body, suggesting a role for cortical actin in both cluster maintenance and localization. Thus, Kv2.1 surface domains likely trap mobile Kv2.1 channels within a well defined, but fluid, perimeter rather than being tightly bound to a scaffolding protein-containing complex. Channel moves directly into these clusters via trafficking vesicles. Such domains allow for efficient trafficking to the cell surface while sequestering channel with signaling proteins.
Subject(s)
Membrane Microdomains/chemistry , Membrane Microdomains/metabolism , Shab Potassium Channels/chemistry , Amino Acid Sequence , Animals , Cell Line , Cells, Cultured , Humans , Molecular Sequence Data , Neurons/chemistry , Neurons/metabolism , Protein Transport/physiology , Rats , Shab Potassium Channels/genetics , Shab Potassium Channels/metabolismABSTRACT
The secretion of LH is cued by the hypothalamic neuropeptide, GnRH. After delivery to the anterior pituitary gland via the hypothalamic-pituitary portal vasculature, GnRH binds to specific high-affinity receptors on the surface of gonadotrope cells and stimulates synthesis and secretion of the gonadotropins, FSH, and LH. In the current study, GnRH caused acute and dramatic changes in cellular morphology in the gonadotrope-derived alphaT3-1 cell line, which appeared to be mediated by engagement of the actin cytoskeleton; disruption of actin with jasplakinolide abrogated cell movement and GnRH-induced activation of ERK. In live murine pituitary slices infected with an adenovirus-containing Rous sarcoma virus-green fluorescent protein, selected cells responded to GnRH by altering their cellular movements characterized by both formation and extension of cell processes and, surprisingly, spatial repositioning. Consistent with the latter observation, GnRH stimulation increased the migration of dissociated pituitary cells in transwell chambers. Our data using live pituitary slices are a striking example of neuropeptide-evoked movements of cells outside the central nervous system and in a mature peripheral endocrine organ. These findings call for a fundamental change in the current dogma of simple passive diffusion of LH from gonadotropes to capillaries in the pituitary gland.
Subject(s)
Cell Movement/drug effects , Gonadotropin-Releasing Hormone/pharmacology , Neuronal Plasticity , Neurosecretory Systems/physiology , Pituitary Gland, Anterior/cytology , Animals , Cells, Cultured , Cytoskeleton/drug effects , Gonadotropin-Releasing Hormone/metabolism , Mice , Microscopy, Video , Neuronal Plasticity/drug effects , Neurosecretory Systems/drug effects , Organ Culture Techniques , Pituitary Gland, Anterior/drug effects , SheepABSTRACT
We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3-1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3-1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca(2+) influx via the L-type Ca(2+) channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca(2+) influx through L-type Ca(2+) channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca(2+) influx via L-type channels to facilitate ERK phosphorylation.
Subject(s)
Calcium Channels, L-Type/metabolism , Cortactin/metabolism , Dynamin II/metabolism , Gonadotrophs/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Pituitary Gland/metabolism , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Blotting, Western , Calcium/metabolism , Cell Line , Cell Surface Extensions/metabolism , Dynamins/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Gonadotropin-Releasing Hormone , Immunohistochemistry , Immunoprecipitation , JNK Mitogen-Activated Protein Kinases/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Patch-Clamp Techniques , Phosphorylation , Reverse Transcriptase Polymerase Chain Reaction , Sheep , Sheep, DomesticABSTRACT
GnRH induces marked activation of the actin cytoskeleton in gonadotropes; however, the physiological consequences and cellular mechanisms responsible have yet to be fully elucidated. The current studies focus on the actin scaffolding protein cortactin. Using the gonadotrope-derived αT3-1 cell line, we found that cortactin is phosphorylated at Y(421), S(405), and S(418) in a time-dependent manner in response to the GnRH agonist buserelin (GnRHa). GnRHa induced translocation of cortactin to the leading edge of the plasma membrane where it colocalizes with actin and actin-related protein 3 (Arp3). Incubation of αT3-1 cells with the c-src inhibitor phosphoprotein phosphatase 1, blocked tyrosine phosphorylation of cortactin, reduced cortactin association with Arp3, and blunted actin reorganization in response to GnRHa. Additionally, we used RNA silencing strategies to knock down cortactin in αT3-1 cells. Knockdown of cortactin blocked the ability of αT3-1 cells to generate filopodia, lamellipodia, and membrane ruffles in response to GnRHa. We show that lamellipodia and filopodia are capable of LHß mobilization in primary pituitary culture after GnRHa treatment, and disruption of these structures using jasplakinolide reduces LH secretion. Collectively, our findings suggest that after GnRHa activation, src activity leads to tyrosine phosphorylation of cortactin, which facilitates its association with Arp3 to engage the actin cytoskeleton. The reorganization of actin by cortactin potentially underlies GnRHa-induced secretory events within αT3-1 cells.
Subject(s)
Actins/metabolism , Cortactin/metabolism , Cytoskeleton/metabolism , Pituitary Gland/metabolism , Actin-Related Protein 3/metabolism , Animals , Cell Line , Cytoskeleton/drug effects , Gonadotropin-Releasing Hormone/pharmacology , Male , Mice , Phosphoprotein Phosphatases/pharmacology , Phosphorylation/drug effects , Pituitary Gland/cytology , Pituitary Gland/drug effects , Proto-Oncogene Proteins pp60(c-src)/metabolism , SheepABSTRACT
In recordings from anterior piriform cortex in awake behaving mice, we found that neuronal firing early in the olfactory pathway simultaneously conveyed fundamentally different information: odor value (is the odor rewarded?) and identity (what is the smell?). Thus, this sensory system performs early multiplexing of information reflecting stimulus-specific characteristics with that used for decision-making.
Subject(s)
Odorants , Olfactory Pathways/physiology , Olfactory Perception/physiology , Recognition, Psychology/physiology , Action Potentials , Animals , Decision Making/physiology , Electrodes, Implanted , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred C57BL , Neurons/physiology , Olfactory Bulb/physiology , Reinforcement, Psychology , RewardABSTRACT
Animals that depend on smell for communication and survival extract multiple pieces of information from a single complex odor. Mice can collect information on sex, genotype, health and dietary status from urine scent marks, a stimulus made up of hundreds of molecules. This ability is all the more remarkable considering that natural odors are encountered against varying olfactory backgrounds; the olfactory system must therefore provide some mechanism for extracting the most relevant information. Here we discuss recent data indicating that the readout of olfactory input by mitral cells in the olfactory bulb can be modified by behavioral context. We speculate that the olfactory cortex plays a key role in tuning the readout of olfactory information from the olfactory bulb.
Subject(s)
Odorants , Olfactory Bulb/anatomy & histology , Olfactory Pathways/physiology , Olfactory Perception/physiology , Olfactory Receptor Neurons/physiology , Action Potentials , Animals , Learning/physiology , Mice , Models, Neurological , Neural Inhibition , Olfactory Bulb/physiology , Sensation/physiologyABSTRACT
The delayed-rectifier voltage-gated K(+) channel (Kv) 2.1 underlies the cardiac slow K(+) current in the rodent heart and is particularly interesting in that both its function and localization are regulated by many stimuli in neuronal systems. However, standard immunolocalization approaches do not detect cardiac Kv2.1; therefore, little is known regarding its localization in the heart. In the present study, we used recombinant adenovirus to determine the subcellular localization and lateral mobility of green fluorescent protein (GFP)-Kv2.1 and yellow fluorescent protein-Kv1.4 in atrial and ventricular myocytes. In atrial myocytes, Kv2.1 formed large clusters on the cell surface similar to those observed in hippocampal neurons, whereas Kv1.4 was evenly distributed over both the peripheral sarcolemma and the transverse tubules. However, fluorescence recovery after photobleach (FRAP) experiments indicate that atrial Kv2.1 was immobile, whereas Kv1.4 was mobile (tau = 252 +/- 42 s). In ventricular myocytes, Kv2.1 did not form clusters and was localized primarily in the transverse-axial tubules and sarcolemma. In contrast, Kv1.4 was found only in transverse tubules and sarcolemma. FRAP studies revealed that Kv2.1 has a higher mobility in ventricular myocytes (tau = 479 +/- 178 s), although its mobility is slower than Kv1.4 (tau(1) = 18.9 +/- 2.3 s; tau(2) = 305 +/- 55 s). We also observed the movement of small, intracellular transport vesicles containing GFP-Kv2.1 within ventricular myocytes. These data are the first evidence of Kv2.1 localization in living myocytes and indicate that Kv2.1 may have distinct physiological roles in atrial and ventricular myocytes.