RESUMEN
Deletion of UBE3A causes the neurodevelopmental disorder Angelman syndrome (AS), while duplication or triplication of UBE3A is linked to autism. These genetic findings suggest that the ubiquitin ligase activity of UBE3A must be tightly maintained to promote normal brain development. Here, we found that protein kinase A (PKA) phosphorylates UBE3A in a region outside of the catalytic domain at residue T485 and inhibits UBE3A activity toward itself and other substrates. A de novo autism-linked missense mutation disrupts this phosphorylation site, causing enhanced UBE3A activity in vitro, enhanced substrate turnover in patient-derived cells, and excessive dendritic spine development in the brain. Our study identifies PKA as an upstream regulator of UBE3A activity and shows that an autism-linked mutation disrupts this phosphorylation control. Moreover, our findings implicate excessive UBE3A activity and the resulting synaptic dysfunction to autism pathogenesis.
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Síndrome de Angelman/genética , Trastorno Autístico/genética , Mutación Missense , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Síndrome de Angelman/metabolismo , Animales , Trastorno Autístico/metabolismo , Encéfalo/patología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Espinas Dendríticas/patología , Embrión de Mamíferos/metabolismo , Estabilidad de Enzimas , Femenino , Humanos , Ratones Endogámicos C57BL , Mutagénesis , Fosforilación , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
RAS/MAPK gene dysfunction underlies various cancers and neurocognitive disorders. Although the roles of RAS/MAPK genes have been well studied in cancer, less is known about their function during neurodevelopment. There are many genes that work in concert to regulate RAS/MAPK signaling, suggesting that if common brain phenotypes could be discovered they could have a broad impact on the many other disorders caused by distinct RAS/MAPK genes. We assessed the cellular and molecular consequences of hyperactivating the RAS/MAPK pathway using two distinct genes in a cell type previously implicated in RAS/MAPK-mediated cognitive changes, cortical GABAergic interneurons. We uncovered some GABAergic core programs that are commonly altered in each of the mutants. Notably, hyperactive RAS/MAPK mutants bias developing cortical interneurons towards those that are somatostatin positive. The increase in somatostatin-positive interneurons could also be prevented by pharmacological inhibition of the core RAS/MAPK signaling pathway. Overall, these findings present new insights into how different RAS/MAPK mutations can converge on GABAergic interneurons, which may be important for other RAS/MAPK genes and related disorders.
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Transducción de Señal , Somatostatina , Alelos , Somatostatina/genética , Somatostatina/metabolismo , Transducción de Señal/genética , Sistema de Señalización de MAP Quinasas/genética , Interneuronas/metabolismo , Neuronas GABAérgicas/metabolismoRESUMEN
Premenopausal hysterectomy is associated with a greater relative risk of dementia. We previously demonstrated cognitive impairments in adult rats six weeks after hysterectomy with ovarian conservation compared with intact sham-controls and other gynecological surgery variations. Here, we investigated whether hysterectomy-induced cognitive impairments are transient or persistent. Adult rats received sham-control, ovariectomy (Ovx), hysterectomy, or Ovx-hysterectomy surgery. Spatial working memory, reference memory, and anxiety-like behavior were tested either six-weeks post-surgery, in adulthood; seven-months post-surgery, in early middle-age; or twelve-months post-surgery, in late middle-age. Hysterectomy in adulthood yielded spatial working memory deficits at short-, moderate-, and long-term post-surgery intervals. Serum hormone levels did not differ between ovary-intact, but differed from Ovx, groups. Hysterectomy had no significant impact on healthy ovarian follicle or corpora lutea counts for any post-surgery timepoint compared with intact sham-controls. Frontal cortex, dorsal hippocampus, and entorhinal cortex were assessed for activity-dependent markers. In entorhinal cortex, there were alterations in FOSB and ΔFOSB expression during the early middle-age timepoint, and phosphorylated ERK1/2 levels at the adult timepoint. Collectively, results suggest a primary role for the uterus in regulating cognition, and that memory-related neural pathways may be modified following gynecological surgery. This is the first preclinical report of long-term effects of hysterectomy with and without ovarian conservation on cognition, endocrine, ovarian, and brain assessments, initiating a comprehensive framework of gynecological surgery effects. Translationally, findings underscore critical needs to decipher how gynecological surgeries, especially those involving the uterus, impact the brain and its functions, the ovaries, and overall aging from a systems perspective.
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Histerectomía , Ovario , Femenino , Humanos , Ratas , Animales , Ovariectomía/efectos adversos , Encéfalo , Cognición , Aprendizaje por LaberintoRESUMEN
Many developmental syndromes have been linked to genetic mutations that cause abnormal ERK/MAPK activity; however, the neuropathological effects of hyperactive signaling are not fully understood. Here, we examined whether hyperactivation of MEK1 modifies the development of GABAergic cortical interneurons (CINs), a heterogeneous population of inhibitory neurons necessary for cortical function. We show that GABAergic-neuron specific MEK1 hyperactivation in vivo leads to increased cleaved caspase-3 labeling in a subpopulation of immature neurons in the embryonic subpallial mantle zone. Adult mutants displayed a significant loss of parvalbumin (PV), but not somatostatin, expressing CINs and a reduction in perisomatic inhibitory synapses on excitatory neurons. Surviving mutant PV-CINs maintained a typical fast-spiking phenotype but showed signs of decreased intrinsic excitability that coincided with an increased risk of seizure-like phenotypes. In contrast to other mouse models of PV-CIN loss, we discovered a robust increase in the accumulation of perineuronal nets, an extracellular structure thought to restrict plasticity. Indeed, we found that mutants exhibited a significant impairment in the acquisition of behavioral response inhibition capacity. Overall, our data suggest PV-CIN development is particularly sensitive to hyperactive MEK1 signaling, which may underlie certain neurological deficits frequently observed in ERK/MAPK-linked syndromes.
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Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Neuronas GABAérgicas/metabolismo , Inhibición Psicológica , MAP Quinasa Quinasa 1/metabolismo , Parvalbúminas/metabolismo , Animales , Corteza Cerebral/química , Electroencefalografía/métodos , Desarrollo Embrionario/fisiología , Neuronas GABAérgicas/química , Locomoción/fisiología , MAP Quinasa Quinasa 1/análisis , Ratones , Técnicas de Cultivo de Órganos , Parvalbúminas/análisis , Transducción de Señal/fisiologíaRESUMEN
RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function.
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Corteza Cerebral/metabolismo , Aprendizaje , Mutación , Neuroglía/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/psicología , Proteínas Proto-Oncogénicas c-raf/genética , Animales , Biomarcadores , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Sistema de Señalización de MAP Quinasas , Aprendizaje por Laberinto , Memoria , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Síndrome de Noonan/metabolismo , Oligodendroglía/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismoRESUMEN
Peripheral nerves exhibit robust regenerative capabilities in response to selective injury among amniotes, but the regeneration of entire muscle groups following volumetric muscle loss is limited in birds and mammals. In contrast, lizards possess the remarkable ability to regenerate extensive de novo muscle after tail loss. However, the mechanisms underlying reformation of the entire neuromuscular system in the regenerating lizard tail are not completely understood. We have tested whether the regeneration of the peripheral nerve and neuromuscular junctions (NMJs) recapitulate processes observed during normal neuromuscular development in the green anole, Anolis carolinensis. Our data confirm robust axonal outgrowth during early stages of tail regeneration and subsequent NMJ formation within weeks of autotomy. Interestingly, NMJs are overproduced as evidenced by a persistent increase in NMJ density 120 and 250 days post autotomy (DPA). Substantial Myelin Basic Protein (MBP) expression could also be detected along regenerating nerves indicating that the ability of Schwann cells to myelinate newly formed axons remained intact. Overall, our data suggest that the mechanism of de novo nerve and NMJ reformation parallel, in part, those observed during neuromuscular development. However, the prolonged increase in NMJ number and aberrant muscle differentiation hint at processes specific to the adult response. An examination of the coordinated exchange between peripheral nerves, Schwann cells, and newly synthesized muscle of the regenerating neuromuscular system may assist in the identification of candidate molecules that promote neuromuscular recovery in organisms incapable of a robust regenerative response.
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Lagartos/fisiología , Regeneración/fisiología , Cola (estructura animal)/fisiología , Animales , Axones/fisiología , Bungarotoxinas/farmacología , Colorantes Fluorescentes , Neuronas Motoras/fisiología , Músculo Esquelético/fisiología , Vaina de Mielina/fisiología , Regeneración Nerviosa , Unión Neuromuscular/fisiología , Células de Schwann/fisiología , Cola (estructura animal)/inervaciónRESUMEN
Despite divergent evolutionary origins, the organization of olfactory systems is remarkably similar across phyla. In both insects and mammals, sensory input from receptor cells is initially processed in synaptically dense regions of neuropil called glomeruli, where neural activity is shaped by local inhibition and centrifugal neuromodulation prior to being sent to higher-order brain areas by projection neurons. Here we review both similarities and several key differences in the neuroanatomy of the olfactory system in honey bees, mice, and humans, using a combination of literature review and new primary data. We have focused on the chemical identity and the innervation patterns of neuromodulatory inputs in the primary olfactory system. Our findings show that serotonergic fibers are similarly distributed across glomeruli in all three species. Octopaminergic/tyraminergic fibers in the honey bee also have a similar distribution, and possibly a similar function, to noradrenergic fibers in the mammalian OBs. However, preliminary evidence suggests that human OB may be relatively less organized than its counterparts in honey bee and mouse.
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Neuroanatomía/métodos , Neuroquímica , Neurópilo/citología , Neurópilo/metabolismo , Vías Olfatorias/anatomía & histología , Olfato/fisiología , Animales , Abejas , Humanos , Ratones , Norepinefrina/metabolismo , Octopamina/metabolismo , Vías Olfatorias/citología , Serotonina/metabolismo , Especificidad de la EspecieRESUMEN
We asked whether key morphogenetic signaling pathways interact with 22q11 gene dosage to modulate the severity of cranial or cardiac anomalies in DiGeorge/22q1 deletion syndrome (22q11DS). Sonic hedgehog (Shh) and retinoic acid (RA) signaling is altered in the brain and heart-clinically significant 22q11DS phenotypic sites-in LgDel mouse embryos, an established 22q11DS model. LgDel embryos treated with cyclopamine, an Shh inhibitor, or carrying mutations in Gli3(Xtj), an Shh-signaling effector, have morphogenetic anomalies that are either not seen, or seen at significantly lower frequencies in control or single-mutant embryos. Similarly, RA exposure or genetic loss of RA function via heterozygous mutation of the RA synthetic enzyme Raldh2 induces novel cranial anomalies and enhances cardiovascular phenotypes in LgDel but not other genotypes. These changes are not seen in heterozygous Tbx1 mutant embryos-a 22q11 gene thought to explain much of 22q11DS pathogenesis-in which Shh or RA signaling has been similarly modified. Our results suggest that full dosage of 22q11 genes beyond Tbx1 establish an adaptive range for morphogenetic signaling via Shh and RA. When this adaptive range is constricted by diminished dosage of 22q11 genes, embryos are sensitized to otherwise benign changes in Shh and RA signaling. Such sensitization, in the face of environmental or genetic factors that modify Shh or RA signaling, may explain variability in 22q11DS morphogenetic phenotypes.
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Adaptación Biológica , Síndrome de DiGeorge/genética , Síndrome de DiGeorge/metabolismo , Dosificación de Gen , Proteínas Hedgehog/metabolismo , Transducción de Señal , Tretinoina/metabolismo , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Morfogénesis/genética , Tubo Neural/embriología , Tubo Neural/metabolismo , FenotipoRESUMEN
Abnormal extracellular signal-regulated kinase 1/2 (ERK1/2, encoded by Mapk3 and Mapk1, respectively) signaling is linked to multiple neurodevelopmental diseases, especially the RASopathies, which typically exhibit ERK1/2 hyperactivation in neurons and non-neuronal cells. To better understand how excitatory neuron-autonomous ERK1/2 activity regulates forebrain development, we conditionally expressed a hyperactive MEK1 (MAP2K1) mutant, MEK1S217/221E, in cortical excitatory neurons of mice. MEK1S217/221E expression led to persistent hyperactivation of ERK1/2 in cortical axons, but not in soma/nuclei. We noted reduced axonal arborization in multiple target domains in mutant mice and reduced the levels of the activity-dependent protein ARC. These changes did not lead to deficits in voluntary locomotion or accelerating rotarod performance. However, skilled motor learning in a single-pellet retrieval task was significantly diminished in these MEK1S217/221E mutants. Restriction of MEK1S217/221E expression to layer V cortical neurons recapitulated axonal outgrowth deficits but did not affect motor learning. These results suggest that cortical excitatory neuron-autonomous hyperactivation of MEK1 is sufficient to drive deficits in axon outgrowth, which coincide with reduced ARC expression, and deficits in skilled motor learning. Our data indicate that neuron-autonomous decreases in long-range axonal outgrowth may be a key aspect of neuropathogenesis in RASopathies.
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Axones , Corteza Cerebral , MAP Quinasa Quinasa 1 , Neuronas , Animales , Axones/metabolismo , Axones/patología , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 1/genética , Corteza Cerebral/patología , Neuronas/metabolismo , Neuronas/patología , Aprendizaje , Ácido Glutámico/metabolismo , Activación Enzimática , Ratones , Sistema de Señalización de MAP Quinasas , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Mutación/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Actividad Motora , Proteína Quinasa 1 Activada por Mitógenos/metabolismoRESUMEN
Background: The expression of proinflammatory signals at the site of muscle injury are essential for efficient tissue repair and their dysregulation can lead to inflammatory myopathies. Macrophages, neutrophils, and fibroadipogenic progenitor cells residing in the muscle are significant sources of proinflammatory cytokines and chemokines. However, the inducibility of the myogenic satellite cell population and their contribution to proinflammatory signaling is less understood. Methods: Mouse satellite cells were isolated and exposed to lipopolysaccharide (LPS) to mimic sterile skeletal muscle injury and changes in the expression of proinflammatory genes was examined by RT-qPCR and single cell RNA sequencing. Expression patterns were validated in skeletal muscle injured with cardiotoxin by RT-qPCR and immunofluorescence. Results: Satellite cells in culture were able to express Tnfa, Ccl2, and Il6, within 2 h of treatment with LPS. Single cell RNA-Seq revealed seven cell clusters representing the continuum from activation to differentiation. LPS treatment led to a heterogeneous pattern of induction of C-C and C-X-C chemokines (e.g., Ccl2, Ccl5, and Cxcl0) and cytokines (e.g., Tgfb1, Bmp2, Il18, and Il33) associated with innate immune cell recruitment and satellite cell proliferation. One cell cluster was enriched for expression of the antiviral interferon pathway genes under control conditions and LPS treatment. Activation of this pathway in satellite cells was also detectable at the site of cardiotoxin induced muscle injury. Conclusion: These data demonstrate that satellite cells respond to inflammatory signals and secrete chemokines and cytokines. Further, we identified a previously unrecognized subset of satellite cells that may act as sensors for muscle infection or injury using the antiviral interferon pathway.
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Aging is accompanied by a host of social and biological changes that correlate with behavior, cognitive health and susceptibility to neurodegenerative disease. To understand trajectories of brain aging in a primate, we generated a multiregion bulk (N = 527 samples) and single-nucleus (N = 24 samples) brain transcriptional dataset encompassing 15 brain regions and both sexes in a unique population of free-ranging, behaviorally phenotyped rhesus macaques. We demonstrate that age-related changes in the level and variance of gene expression occur in genes associated with neural functions and neurological diseases, including Alzheimer's disease. Further, we show that higher social status in females is associated with younger relative transcriptional ages, providing a link between the social environment and aging in the brain. Our findings lend insight into biological mechanisms underlying brain aging in a nonhuman primate model of human behavior, cognition and health.
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Enfermedades Neurodegenerativas , Femenino , Masculino , Humanos , Animales , Macaca mulatta , Transcriptoma , Envejecimiento/genética , Medio Social , Núcleo SolitarioRESUMEN
Nicotine, the primary addictive substance in tobacco, is widely abused. Relapse to cues associated with nicotine results in increased glutamate release within nucleus accumbens core (NAcore), modifying synaptic plasticity of medium spiny neurons (MSNs), which contributes to reinstatement of nicotine seeking. However, the role of cholinergic interneurons (ChIs) within the NAcore in mediating these neurobehavioral processes is unknown. ChIs represent less than 1% of the accumbens neuronal population and are activated during drug seeking and reward-predicting events. Thus, we hypothesized that ChIs may play a significant role in mediating glutamatergic plasticity that underlies nicotine-seeking behavior. Using chemogenetics in transgenic rats expressing Cre under the control of the choline acetyltransferase (ChAT) promoter, ChIs were bidirectionally manipulated before cue-induced reinstatement. Following nicotine self-administration and extinction, ChIs were activated or inhibited before a cue reinstatement session. Following reinstatement, whole-cell electrophysiology from NAcore MSNs was used to assess changes in plasticity, measured via AMPA/NMDA (A/N) ratios. Chemogenetic inhibition of ChIs inhibited cued nicotine seeking and resulted in decreased A/N, relative to control animals, whereas activation of ChIs was unaltered, demonstrating that ChI inhibition may modulate plasticity underlying cue-induced nicotine seeking. These results demonstrate that ChI neurons play an important role in mediating cue-induced nicotine reinstatement and underlying synaptic plasticity within the NAcore.
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Señales (Psicología) , Nicotina , Animales , Colinérgicos , Comportamiento de Búsqueda de Drogas , Extinción Psicológica , Interneuronas , Nicotina/farmacología , Núcleo Accumbens , Ratas , Ratas Sprague-Dawley , AutoadministraciónRESUMEN
Alcohol abuse is a worldwide public health concern, yet the precise molecular targets of alcohol in the brain are still not fully understood. Alcohol may promote its euphoric and motivational effects, in part, by activating the endogenous opioid system. One particular component of this system consists of pro-opiomelanocortin (POMC) -producing neurons in the arcuate nucleus (ArcN) of the hypothalamus, which project to reward-related brain areas. To identify the physiological effects of ethanol on ArcN POMC neurons, we utilized whole cell patch-clamp recordings and bath application of ethanol (5-40 mM) to identify alterations in spontaneous baseline activity, rheobase, spiking characteristics, or intrinsic neuronal properties. We found that 10 mM ethanol increased the number of depolarization-induced spikes in the majority of recorded cells, whereas higher concentrations of ethanol (20-40 mM) decreased the number of spikes. Interestingly, we found that basal firing rates of ArcN POMC neurons may predict physiological responding to ethanol. Rheobase and spontaneous activity, measured by spontaneous excitatory post-synaptic potentials (EPSPs) at rest, were unchanged after exposure to ethanol, regardless of concentration. These results suggest that ethanol has concentration-dependent modulatory effects on ArcN POMC neuronal activity, which may be relevant to treatments for alcohol use disorders that target endogenous opioid systems.
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Etanol/farmacología , Hipotálamo/efectos de los fármacos , Neuronas/efectos de los fármacos , Proopiomelanocortina/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Ratones , Ratones Transgénicos , Técnicas de Placa-ClampRESUMEN
Macrophage fusion resulting in the formation of multinucleated giant cells (MGCs) is a multistage process that requires many adhesion-dependent steps and involves the rearrangement of the actin cytoskeleton. The diversity of actin-based structures and their role in macrophage fusion is poorly understood. In this study, we revealed hitherto unrecognized actin-based zipper-like structures (ZLSs) that arise between MGCs formed on the surface of implanted biomaterials. We established an in vitro model for the induction of these structures in mouse macrophages undergoing IL-4-mediated fusion. Using this model, we show that over time MGCs develop cell-cell contacts containing ZLSs. Live-cell imaging using macrophages isolated from mRFP- or eGFP-LifeAct mice demonstrated that ZLSs are dynamic formations undergoing continuous assembly and disassembly and that podosomes are precursors of these structures. Immunostaining experiments showed that vinculin, talin, integrin αMß2, and other components of podosomes are present in ZLSs. Macrophages deficient in WASp or Cdc42, two key molecules involved in actin core organization in podosomes, as well as cells treated with the inhibitors of the Arp2/3 complex, failed to form ZLSs. Furthermore, E-cadherin and nectin-2 were found between adjoining membranes, suggesting that the transition of podosomes into ZLSs is induced by bridging plasma membranes by junctional proteins.
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Actinas/metabolismo , Células Gigantes/metabolismo , Podosomas/metabolismo , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Animales , Adhesión Celular/fisiología , Membrana Celular/metabolismo , Movimiento Celular , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Talina/metabolismo , Vinculina/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
5-HT1B receptors (5-HT1BRs) modulate psychostimulant reward and incentive motivation in rodents. Here we investigated the effects of the 5-HT1BR agonist CP94253 (10 mg/kg, IP) on the acquisition and expression of methamphetamine (Meth) conditioned place preference (CPP) in C57BL/6 male mice. We subsequently examined the potential brain regions involved in CP94253 effects using FOS as a marker of neural activity. In the acquisition experiment, mice received the agonist 30 min before each of the Meth injections given during conditioning. In the expression experiment, mice that had acquired Meth-CPP were given either saline or CP94253 and were tested for CPP 30 min later. We found that CP94253 attenuated the expression of Meth-CPP, but had no effect on acquisition. Mice expressing Meth-CPP had elevated numbers of FOS+ cells in the ventral tegmental area (VTA) and basolateral amygdala (BlA) and reduced FOS+ cells in the central amygdala (CeA) compared to saline controls. CP94253 given before the expression test, but not acutely in drug-naive mice, enhanced FOS+ cells in the VTA, the nucleus accumbens (NAc) shell and core, and the dorsomedial striatum and reversed the Meth-conditioned changes in FOS in the BlA and CeA. Approximately 50-70% of FOS+ cells in the NAc and VTA were GABAergic regardless of group. By contrast, we did not observe FOS-labeling in dopamine neurons in the VTA. The findings suggest that CP94253 attenuates the motivational effects of the Meth-associated environment and highlight the amygdala, VTA, NAc, and dorsomedial striatum as potential regions involved in this effect.
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Estimulantes del Sistema Nervioso Central/administración & dosificación , Condicionamiento Operante/efectos de los fármacos , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Metanfetamina/administración & dosificación , Neuronas/efectos de los fármacos , Piperidinas/farmacología , Purinas/farmacología , Agonistas del Receptor de Serotonina 5-HT1/farmacología , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Ratones , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptor de Serotonina 5-HT1B/metabolismo , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/metabolismoRESUMEN
During development, motoneurons (MNs) undergo a highly stereotyped, temporally and spatially defined period of programmed cell death (PCD), the result of which is the loss of 40-50% of the original neuronal population. Those MNs that survive are thought to reflect the successful acquisition of limiting amounts of trophic factors from the target. In contrast, maturation of MNs limits the need for target-derived trophic factors, because axotomy of these neurons in adulthood results in minimal neuronal loss. It is unclear whether MNs lose their need for trophic factors altogether or whether, instead, they come to rely on other cell types for nourishment. Astrocytes are known to supply trophic factors to a variety of neuronal populations and thus may nourish MNs in the absence of target-derived factors. We investigated the survival-promoting activities of muscle- and astrocyte-derived secreted factors and found that astrocyte-conditioned media (ACM) was able to save substantially more motoneurons in vitro than muscle-conditioned media (MCM). Our results indicate that both ACM and MCM are significant sources of MN trophic support in vitro and in ovo, but only ACM can rescue MNs after unilateral limb bud removal. Furthermore, we provide evidence suggesting that MCM facilitates the death of a subpopulation of MNs in a p75(NTR) - and caspase-dependent manner; however, maturation in ACM results in MN trophic independence and reduced vulnerability to this negative, pro-apoptotic influence from the target.
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Astrocitos/metabolismo , Neuronas Motoras/fisiología , Músculo Esquelético/metabolismo , Animales , Astrocitos/citología , Supervivencia Celular/fisiología , Células Cultivadas , Embrión de Pollo , Medios de Cultivo Condicionados/farmacocinética , Neuronas Motoras/citología , Músculo Esquelético/citologíaRESUMEN
Vesicle transport in cultured chick motoneurons was studied over a period of 3 days using motion-enhanced differential interference contrast (MEDIC) microscopy, an improved version of video-enhanced DIC. After 3 days in vitro (DIV), the average vesicle velocity was about 30% less than after 1 DIV. In observations at 1, 2 and 3 DIV, larger vesicles moved more slowly than small vesicles, and retrograde vesicles were larger than anterograde vesicles. The number of retrograde vesicles increased relative to anterograde vesicles after 3 DIV, but this fact alone could not explain the decrease in velocity, since the slowing of vesicle transport in maturing motoneurons was observed independently for both anterograde and retrograde vesicles. In order to better understand the slowing trend, the distance vs. time trajectories of individual vesicles were examined at a frame rate of 8.3/s. Qualitatively, these trajectories consisted of short (1-2 s) segments of constant velocity, and the changes in velocity between segments were abrupt (<0.2 s). The trajectories were therefore fit to a series of connected straight lines. Surprisingly, the slopes of theses lines, i.e. the vesicle velocities, were often found to be multiples of ~0.6 mum/s. The velocity histogram showed multiple peaks, which, when fit with Gaussians using a least squares minimization, yielded an average spacing of 0.57 mum/s (taken as the slope of a fit to peak position vs. peak number, R(2)=0.994). We propose that the abrupt velocity changes occur when 1 or 2 motors suddenly begin or cease actively participating in vesicle transport. Under this hypothesis, the decrease in average vesicle velocity observed for maturing motoneurons is due to a decrease in the average number of active motors per vesicle.
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Transporte Axonal/fisiología , Senescencia Celular/fisiología , Neuronas Motoras/fisiología , Vesículas Sinápticas/fisiología , Animales , Embrión de Pollo , Procesamiento de Imagen Asistido por Computador , Cinética , Microscopía de Interferencia , Percepción de Movimiento/fisiología , Neuronas Motoras/ultraestructura , Neuritas/fisiología , Neuritas/ultraestructura , Médula Espinal/citología , Vesículas Sinápticas/ultraestructura , Factores de Tiempo , ViscosidadRESUMEN
The dependence of developing spinal motoneuron survival on a soluble factor(s) from their target, muscle tissue is well established both in vivo and in vitro. Considering this apparent dependence, we examined whether a specific component of the stress response mediates motoneuron survival in trophic factor-deprived environments. We demonstrate that, although endogenous expression of heat shock protein 70 (HSP70) did not change during trophic factor deprivation, application of e-rhHsp70 (exogenous recombinant human Hsp70) promoted motoneuron survival. Conversely, depletion of HSP70 from chick muscle extract (MEx) potently reduces the survival-promoting activity of MEx. Additionally, exogenous treatment with or spinal cord overexpression of Hsp70 enhances motoneuron survival in vivo during the period of naturally occurring cell death [programmed cell death (PCD)]. Hindlimb muscle cells and lumbar spinal astrocytes readily secrete HSP70 in vitro, suggesting potential physiological sources of extracellular Hsp70 for motoneurons. However, in contrast to exogenous treatment with or overexpression of Hsp70 in vivo, muscle-targeted injections of this factor in an ex vivo preparation fail to attenuate motoneuron PCD. These data (1) suggest that motoneuron survival requirements may extend beyond classical trophic factors to include HSP70, (2) indicate that the source of this factor is instrumental in determining its trophic function, and (3) may therefore influence therapeutic strategies designed to increase motoneuron Hsp70 signaling during disease or injury.
Asunto(s)
Supervivencia Celular/fisiología , Líquido Extracelular/citología , Líquido Extracelular/fisiología , Proteínas HSP70 de Choque Térmico/fisiología , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Animales , Muerte Celular/fisiología , Células Cultivadas , Embrión de PolloRESUMEN
Aberrant signaling through the Raf/MEK/ERK (ERK/MAPK) pathway causes pathology in a family of neurodevelopmental disorders known as 'RASopathies' and is implicated in autism pathogenesis. Here, we have determined the functions of ERK/MAPK signaling in developing neocortical excitatory neurons. Our data reveal a critical requirement for ERK/MAPK signaling in the morphological development and survival of large Ctip2(+) neurons in layer 5. Loss of Map2k1/2 (Mek1/2) led to deficits in corticospinal tract formation and subsequent corticospinal neuron apoptosis. ERK/MAPK hyperactivation also led to reduced corticospinal axon elongation, but was associated with enhanced arborization. ERK/MAPK signaling was dispensable for axonal outgrowth of layer 2/3 callosal neurons. However, Map2k1/2 deletion led to reduced expression of Arc and enhanced intrinsic excitability in both layers 2/3 and 5, in addition to imbalanced synaptic excitation and inhibition. These data demonstrate selective requirements for ERK/MAPK signaling in layer 5 circuit development and general effects on cortical pyramidal neuron excitability.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Neocórtex/embriología , Neuronas/fisiología , Animales , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Ratones Transgénicos , NeurogénesisRESUMEN
Early life stress (ELS) is highly related to the development of psychiatric illnesses in adulthood, including substance use disorders. A recent body of literature suggests that long-lasting changes in the epigenome may be a mechanism by which experiences early in life can alter neurobiological and behavioral phenotypes in adulthood. In this study, we replicate our previous findings that ELS, in the form of prolonged maternal separation, increases adult methamphetamine self-administration (SA) in male rats as compared with handled controls. In addition, we show new evidence that both ELS and methamphetamine SA alter the expression of the epigenetic regulator methyl CpG-binding protein 2 (MeCP2) in key brain reward regions, particularly in the nucleus accumbens (NAc) core. In turn, viral-mediated knockdown of MeCP2 expression in the NAc core reduces methamphetamine SA, as well as saccharin intake. Furthermore, NAc core MeCP2 knockdown reduces methamphetamine, but not saccharin, SA on a progressive ratio schedule of reinforcement. These data suggest that NAc core MeCP2 may be recruited by both ELS and methamphetamine SA and promote the development of certain aspects of drug abuse-related behavior. Taken together, functional interactions between ELS, methamphetamine SA, and the expression of MeCP2 in the NAc may represent novel mechanisms that can ultimately be targeted for intervention in individuals with adverse early life experiences who are at risk for developing substance use disorders.