RESUMEN
Mounting epidemiologic and scientific evidence indicates that many psychiatric disorders originate from a complex interplay between genetics and early life experiences, particularly in the womb. Despite decades of research, our understanding of the precise prenatal and perinatal experiences that increase susceptibility to neurodevelopmental disorders remains incomplete. Sleep apnea (SA) is increasingly common during pregnancy and is characterized by recurrent partial or complete cessations in breathing during sleep. SA causes pathological drops in blood oxygen levels (intermittent hypoxia, IH), often hundreds of times each night. Although SA is known to cause adverse pregnancy and neonatal outcomes, the long-term consequences of maternal SA during pregnancy on brain-based behavioral outcomes and associated neuronal functioning in the offspring remain unknown. We developed a rat model of maternal SA during pregnancy by exposing dams to IH, a hallmark feature of SA, during gestational days 10 to 21 and investigated the consequences on the offspring's forebrain synaptic structure, synaptic function, and behavioral phenotypes across multiples stages of development. Our findings represent a rare example of prenatal factors causing sexually dimorphic behavioral phenotypes associated with excessive (rather than reduced) synapse numbers and implicate hyperactivity of the mammalian target of rapamycin (mTOR) pathway in contributing to the behavioral aberrations. These findings have implications for neuropsychiatric disorders typified by superfluous synapse maintenance that are believed to result, at least in part, from largely unknown insults to the maternal environment.
Asunto(s)
Conducta Animal , Hipoxia/fisiopatología , Efectos Tardíos de la Exposición Prenatal/etiología , Sinapsis/patología , Animales , Trastorno Autístico/etiología , Modelos Animales de Enfermedad , Femenino , Masculino , Embarazo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Prosencéfalo/crecimiento & desarrollo , Prosencéfalo/fisiopatología , Ratas Sprague-Dawley , Caracteres Sexuales , Síndromes de la Apnea del Sueño , Serina-Treonina Quinasas TORRESUMEN
While research has identified alterations in dorsolateral prefrontal cortical function as a key factor to the etiology of bipolar disorder, few studies have uncovered robust changes in protein signal transduction pathways in this disorder. Given the direct relevance of protein-based expressional alterations to cellular functions and because many of the key regulatory mechanisms for the disease pathogenesis likely include alterations in protein activity rather than changes in expression alone, the identification of alterations in discrete signal transduction pathways in bipolar disorder would have broad implications for understanding the disease pathophysiology. As prior microarray data point to a previously unrecognized involvement of the RhoA network in bipolar disorder, here we investigate the protein expression and activity of key components of a RhoA signal transduction pathway in dorsolateral prefrontal cortical homogenates from subjects with bipolar disorder. The results of this investigation implicate overactivation of prefrontal cortical RhoA signaling in specific subtypes of bipolar disorder. The specificity of these findings is demonstrated by a lack of comparable changes in schizophrenia; however, our findings do identify convergence between both disorders at the level of activity-mediated actin cytoskeletal regulation. These findings have implications for understanding the altered cortical synaptic connectivity of bipolar disorder.
Asunto(s)
Trastorno Bipolar/metabolismo , Corteza Prefrontal/metabolismo , Trastornos Psicóticos/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Adulto , Trastorno Bipolar/complicaciones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trastornos Psicóticos/complicaciones , Transducción de SeñalRESUMEN
Addictive behaviors, including relapse, are thought to depend in part on long-lasting drug-induced adaptations in dendritic spine signaling and morphology in the nucleus accumbens (NAc). While the influence of activity-dependent actin remodeling in these phenomena has been studied extensively, the role of microtubules and associated proteins remains poorly understood. We report that pharmacological inhibition of microtubule polymerization in the NAc inhibited locomotor sensitization to cocaine and contextual reward learning. We then investigated the roles of microtubule end-binding protein 3 (EB3) and SRC kinase in the neuronal and behavioral responses to volitionally administered cocaine. In synaptoneurosomal fractions from the NAc of self-administering male rats, the phosphorylation of SRC at an activating site was induced after 1 d of withdrawal, while EB3 levels were increased only after 30 d of withdrawal. Blocking SRC phosphorylation during early withdrawal by virally overexpressing SRCIN1, a negative regulator of SRC activity known to interact with EB3, abolished the incubation of cocaine craving in both male and female rats. Conversely, mimicking the EB3 increase observed after prolonged withdrawal increased the motivation to consume cocaine in male rats. In mice, the overexpression of either EB3 or SRCIN1 increased dendritic spine density and altered the spine morphology of NAc medium spiny neurons. Finally, a cocaine challenge after prolonged withdrawal recapitulated most of the synaptic protein expression profiles observed at early withdrawal. These findings suggest that microtubule-associated signaling proteins such as EB3 cooperate with actin remodeling pathways, notably SRC kinase activity, to establish and maintain long-lasting cellular and behavioral alterations following cocaine self-administration.SIGNIFICANCE STATEMENT Drug-induced morphological restructuring of dendritic spines of nucleus accumbens neurons is thought to be one of the cellular substrates of long-lasting drug-associated memories. The molecular basis of these persistent changes has remained incompletely understood. Here we implicate for the first time microtubule function in this process, together with key players such as microtubule-bound protein EB3 and synaptic SRC phosphorylation. We propose that microtubule and actin remodeling cooperate during withdrawal to maintain the plastic structural changes initially established by cocaine self-administration. This work opens new translational avenues for further characterization of microtubule-associated regulatory molecules as putative drug targets to tackle relapse to drug taking.
Asunto(s)
Cocaína/administración & dosificación , Locomoción/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Síndrome de Abstinencia a Sustancias/metabolismo , Sinapsis/metabolismo , Animales , Trastornos Relacionados con Cocaína/metabolismo , Trastornos Relacionados con Cocaína/patología , Femenino , Locomoción/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/patología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Ratas , Autoadministración , Síndrome de Abstinencia a Sustancias/patología , Sinapsis/efectos de los fármacos , Sinapsis/patologíaRESUMEN
Wiskott-Aldrich syndrome protein (WASP) family verprolin homologous protein 1 (WAVE1) regulates actin-related protein 2/3 (Arp2/3) complex-mediated actin polymerization. Our previous studies have found WAVE1 to be inhibited by Cdk5-mediated phosphorylation in brain and to play a role in the regulation of dendritic spine morphology. Here we report that mice in which WAVE1 was knocked out (KO) in neurons expressing the D1 dopamine receptor (D1-KO), but not mice where WAVE1 was knocked out in neurons expressing the D2 dopamine receptor (D2-KO), exhibited a significant decrease in place preference associated with cocaine. In contrast to wild-type (WT) and WAVE1 D2-KO mice, cocaine-induced sensitized locomotor behavior was not maintained in WAVE1 D1-KO mice. After chronic cocaine administration and following withdrawal, an acute cocaine challenge induced WAVE1 activation in striatum, which was assessed by dephosphorylation. The cocaine-induced WAVE1 dephosphorylation was attenuated by coadministration of either a D1 dopamine receptor or NMDA glutamate receptor antagonist. Upon an acute challenge of cocaine following chronic cocaine exposure and withdrawal, we also observed in WT, but not in WAVE1 D1-KO mice, a decrease in dendritic spine density and a decrease in the frequency of excitatory postsynaptic AMPA receptor currents in medium spiny projection neurons expressing the D1 dopamine receptor (D1-MSNs) in the nucleus accumbens. These results suggest that WAVE1 is involved selectively in D1-MSNs in cocaine-evoked neuronal activity-mediated feedback regulation of glutamatergic synapses.
Asunto(s)
Cocaína/farmacología , Neuronas/metabolismo , Receptores de Dopamina D1/metabolismo , Conducta Espacial/efectos de los fármacos , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Animales , Inhibidores de Captación de Dopamina/farmacología , Fenómenos Electrofisiológicos/genética , Potenciales Postsinápticos Excitadores/genética , Potenciales Postsinápticos Excitadores/fisiología , Ratones Noqueados , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Núcleo Accumbens/metabolismo , Núcleo Accumbens/fisiología , Fosforilación/efectos de los fármacos , Receptores de Dopamina D1/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Synapse degeneration and dendritic spine dysgenesis are believed to be crucial early steps in Alzheimer's disease (AD), and correlate with cognitive deficits in AD patients. Soluble amyloid beta (Aß)-derived oligomers, also termed Aß-derived diffusible ligands (ADDLs), accumulate in the brain of AD patients and play a crucial role in AD pathogenesis. ADDLs bind to mature hippocampal neurons, induce structural changes in dendritic spines and contribute to neuronal death. However, mechanisms underlying structural and toxic effects are not fully understood. Here, we report that ADDLs bind to cultured mature cortical pyramidal neurons and induce spine dysgenesis. ADDL treatment induced the rapid depletion of kalirin-7, a brain-specific guanine-nucleotide exchange factor for the small GTPase Rac1, from spines. Kalirin-7 is a key regulator of dendritic spine morphogenesis and maintenance in forebrain pyramidal neurons and here we show that overexpression of kalirin-7 prevents ADDL-induced spine degeneration. Taken together, our results suggest that kalirin-7 may play a role in the early events leading to synapse degeneration, and its pharmacological activation may prevent or delay synapse pathology in AD.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Espinas Dendríticas/metabolismo , Espinas Dendríticas/patología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Animales , Células Cultivadas , Degeneración Nerviosa , Células Piramidales/metabolismo , Células Piramidales/patología , Ratas , Ratas Sprague-DawleyRESUMEN
Repeated cocaine exposure regulates transcriptional regulation within the nucleus accumbens (NAc), and epigenetic mechanisms-such as histone acetylation and methylation on Lys residues-have been linked to these lasting actions of cocaine. In contrast to Lys methylation, the role of histone Arg (R) methylation remains underexplored in addiction models. Here we show that protein-R-methyltransferase-6 (PRMT6) and its associated histone mark, asymmetric dimethylation of R2 on histone H3 (H3R2me2a), are decreased in the NAc of mice and rats after repeated cocaine exposure, including self-administration, and in the NAc of cocaine-addicted humans. Such PRMT6 down-regulation occurs selectively in NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2-MSNs), with opposite regulation occurring in D1-MSNs, and serves to protect against cocaine-induced addictive-like behavioral abnormalities. Using ChIP-seq, we identified Src kinase signaling inhibitor 1 (Srcin1; also referred to as p140Cap) as a key gene target for reduced H3R2me2a binding, and found that consequent Srcin1 induction in the NAc decreases Src signaling, cocaine reward, and the motivation to self-administer cocaine. Taken together, these findings suggest that suppression of Src signaling in NAc D2-MSNs, via PRMT6 and H3R2me2a down-regulation, functions as a homeostatic brake to restrain cocaine action, and provide novel candidates for the development of treatments for cocaine addiction.
Asunto(s)
Proteínas Portadoras/genética , Trastornos Relacionados con Cocaína/metabolismo , Cocaína/administración & dosificación , Histonas/metabolismo , Núcleo Accumbens/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Animales , Proteínas Portadoras/metabolismo , Trastornos Relacionados con Cocaína/genética , Trastornos Relacionados con Cocaína/patología , Histonas/genética , Humanos , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/patología , Núcleo Accumbens/patología , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismoRESUMEN
The nucleus accumbens (NAc) is a critical brain reward region that mediates the rewarding effects of drugs of abuse, including those of morphine and other opiates. Drugs of abuse induce widespread alterations in gene transcription and dendritic spine morphology in medium spiny neurons (MSNs) of the NAc that ultimately influence NAc excitability and hence reward-related behavioral responses. Growing evidence indicates that within the NAc small GTPases are common intracellular targets of drugs of abuse where these molecules regulate drug-mediated transcriptional and spine morphogenic effects. The RhoA small GTPase is among the most well-characterized members of the Ras superfamily of small GTPases, and recent work highlights an important role for hippocampal RhoA in morphine-facilitated reward behavior. Despite this, it remains unclear how RhoA pathway signaling in the NAc is affected by withdrawal from morphine. To investigate this question, using subcellular fractionation and subsequent protein profiling we examined the expression of key components of the RhoA pathway in NAc nuclear, cytoplasmic, and synaptosomal compartments during multiple withdrawal periods from repeated morphine administration. Furthermore, using in vivo viral-mediated gene transfer, we determined the consequences of revealed RhoA pathway alterations on NAc MSN dendritic spine morphology. Our findings reveal an important role for RhoA signaling cascades in mediating the effects of long-term morphine withdrawal on NAc MSN dendritic spine elimination. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.
Asunto(s)
Morfina , Narcóticos , Núcleo Accumbens/metabolismo , Núcleo Accumbens/patología , Síndrome de Abstinencia a Sustancias/metabolismo , Síndrome de Abstinencia a Sustancias/patología , Sinapsis/metabolismo , Sinapsis/patología , Proteínas de Unión al GTP rho/biosíntesis , Animales , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Núcleo Accumbens/efectos de los fármacos , Recompensa , Transducción de Señal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Proteínas de Unión al GTP rho/genética , Proteína de Unión al GTP rhoARESUMEN
Decreased medial prefrontal cortex (mPFC) neuronal activity is associated with social defeat-induced depression- and anxiety-like behaviors in mice. However, the molecular mechanisms underlying the decreased mPFC activity and its prodepressant role remain unknown. We show here that induction of the transcription factor ΔFosB in mPFC, specifically in the prelimbic (PrL) area, mediates susceptibility to stress. ΔFosB induction in PrL occurred selectively in susceptible mice after chronic social defeat stress, and overexpression of ΔFosB in this region, but not in the nearby infralimbic (IL) area, enhanced stress susceptibility. ΔFosB produced these effects partly through induction of the cholecystokinin (CCK)-B receptor: CCKB blockade in mPFC induces a resilient phenotype, whereas CCK administration into mPFC mimics the anxiogenic- and depressant-like effects of social stress. We previously found that optogenetic stimulation of mPFC neurons in susceptible mice reverses several behavioral abnormalities seen after chronic social defeat stress. Therefore, we hypothesized that optogenetic stimulation of cortical projections would rescue the pathological effects of CCK in mPFC. After CCK infusion in mPFC, we optogenetically stimulated mPFC projections to basolateral amygdala or nucleus accumbens, two subcortical structures involved in mood regulation. Stimulation of corticoamygdala projections blocked the anxiogenic effect of CCK, although no effect was observed on other symptoms of social defeat. Conversely, stimulation of corticoaccumbens projections reversed CCK-induced social avoidance and sucrose preference deficits but not anxiogenic-like effects. Together, these results indicate that social stress-induced behavioral deficits are mediated partly by molecular adaptations in mPFC involving ΔFosB and CCK through cortical projections to distinct subcortical targets.
Asunto(s)
Trastornos de Ansiedad/fisiopatología , Colecistoquinina/fisiología , Trastorno Depresivo/fisiopatología , Corteza Prefrontal/fisiología , Proteínas Proto-Oncogénicas c-fos/fisiología , Receptor de Colecistoquinina B/fisiología , Animales , Ansiolíticos/farmacología , Trastornos de Ansiedad/patología , Mapeo Encefálico , Enfermedad Crónica , Trastorno Depresivo/patología , Indoles/farmacología , Sistema Límbico/citología , Sistema Límbico/efectos de los fármacos , Sistema Límbico/fisiología , Masculino , Meglumina/análogos & derivados , Meglumina/farmacología , Ratones , Ratones Endogámicos C57BL , Vías Nerviosas/fisiología , Corteza Prefrontal/citología , Corteza Prefrontal/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/genética , Receptor de Colecistoquinina B/antagonistas & inhibidores , Receptor de Colecistoquinina B/genética , Predominio Social , Estrés Psicológico/patología , Estrés Psicológico/fisiopatologíaRESUMEN
It is well established that behavioral sensitization to cocaine is accompanied by increased spine density and AMPA receptor (AMPAR) transmission in the nucleus accumbens (NAc), but two major questions remain unanswered. Are these adaptations mechanistically coupled? And, given that they can be dissociated from locomotor sensitization, what is their functional significance? We tested the hypothesis that the guanine-nucleotide exchange factor Kalirin-7 (Kal-7) couples cocaine-induced AMPAR and spine upregulation and that these adaptations underlie sensitization of cocaine's incentive-motivational properties-the properties that make it "wanted." Rats received eight daily injections of saline or cocaine. On withdrawal day 14, we found that Kal-7 levels and activation of its downstream effectors Rac-1 and PAK were increased in the NAc of cocaine-sensitized rats. Furthermore, AMPAR surface expression and spine density were increased, as expected. To determine whether these changes require Kal-7, a lentiviral vector expressing Kal-7 shRNA was injected into the NAc core before cocaine exposure. Knocking down Kal-7 abolished the AMPAR and spine upregulation normally seen during cocaine withdrawal. Despite the absence of these adaptations, rats with reduced Kal-7 levels developed locomotor sensitization. However, incentive sensitization, which was assessed by how rapidly rats learned to self-administer a threshold dose of cocaine, was severely impaired. These results identify a signaling pathway coordinating AMPAR and spine upregulation during cocaine withdrawal, demonstrate that locomotor and incentive sensitization involve divergent mechanisms, and link enhanced excitatory transmission in the NAc to incentive sensitization.
Asunto(s)
Cocaína/administración & dosificación , Espinas Dendríticas/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Plasticidad Neuronal/fisiología , Receptores AMPA/metabolismo , Animales , Espinas Dendríticas/efectos de los fármacos , Técnicas de Silenciamiento del Gen/métodos , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Plasticidad Neuronal/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Autoadministración , Síndrome de Abstinencia a Sustancias/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Dendritic spines are protrusions on dendrites forming the postsynaptic aspect of excitatory connections within the brain. Spine morphology is associated with synaptic functional strength and the spatial regulation of protein nanodomains within dendritic spines is an important determinant of spine structure and function. Here, we present a protocol to resolve the nanoscale localization of proteins within dendritic spines using structured illumination microscopy. We describe steps for the structural analysis of dendritic spine parameters, protein localization analysis, and data processing. For complete details on the use and execution of this protocol, please refer to Bjornson et al.1.
Asunto(s)
Espinas Dendríticas , Microscopía , Espinas Dendríticas/metabolismo , Microscopía/métodos , Iluminación , Neuronas/metabolismoRESUMEN
Based on recent genome-wide association studies, it is theorized that altered regulation of autophagy contributes to the pathophysiology of schizophrenia and bipolar disorder. As activity of autophagy-regulatory pathways is controlled by discrete phosphorylation sites on the relevant proteins, phospho-protein profiling is one of the few approaches available for enabling a quantitative assessment of autophagic activity in the brain. Despite this, a comprehensive phospho-protein assessment in the brains of schizophrenia and bipolar disorder subjects is currently lacking. Using this direction, our broad screening identifies an increase in AMP-activated protein kinase (AMPK)-mediated phospho-activation of the pro-autophagy protein beclin-1 solely in the prefrontal cortex of female, but not male, schizophrenia subjects. Using a reverse translational approach, we surprisingly find that this increase in beclin-1 activity facilitates synapse formation and enhances cognition. These findings are interpreted in the context of human studies demonstrating that female schizophrenia subjects have a lower susceptibility to cognitive dysfunction than males.
Asunto(s)
Autofagia , Beclina-1 , Esquizofrenia , Caracteres Sexuales , Esquizofrenia/patología , Esquizofrenia/metabolismo , Esquizofrenia/genética , Humanos , Femenino , Masculino , Beclina-1/metabolismo , Beclina-1/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Fosforilación , Animales , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Sinapsis/metabolismo , Sinapsis/patología , Transducción de Señal , Ratones , Cognición/fisiología , AdultoRESUMEN
The dendritic field of a neuron, which is determined by both dendritic architecture and synaptic strength, defines the synaptic input of a cell. Once established, a neuron's dendritic field is thought to remain relatively stable throughout a cell's lifetime. Perturbations in a dendritic structure or excitatory tone of a cell and thus its dendritic field are cellular alterations thought to be correlated with a number of psychiatric disorders. Although several proteins are known to regulate the development of dendritic arborization, much less is known about the mechanisms that maintain dendritic morphology and synaptic strength. In this study, we find that afadin, a component of N-cadherin·ß-catenin·α-N-catenin adhesion complexes, is required for the maintenance of established dendritic arborization and synapse number. We further demonstrate that afadin directly interacts with AMPA receptors and that loss of this protein reduces the surface expression of GluA1- and GluA2-AMPA receptor subunits. Collectively, these data suggest that afadin is required for the maintenance of dendritic structure and excitatory tone.
Asunto(s)
Dendritas/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteínas de Microfilamentos/metabolismo , Receptores AMPA/metabolismo , Sinapsis/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Células Cultivadas , Dendritas/genética , Regulación de la Expresión Génica/fisiología , Proteínas con Dominio LIM/genética , Proteínas de Microfilamentos/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores AMPA/genética , Sinapsis/genética , alfa Catenina/genética , alfa Catenina/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
The biological functions of the neuregulin 1 (NRG1) and ERBB4 genes have received much recent attention due to several studies showing associations between these genes and schizophrenia. Moreover, reduced forebrain dendritic spine density is a consistent feature of schizophrenia. It is thus important to understand the mechanisms whereby NRG1 and erbB4 modulate spine morphogenesis. Here, we show that long-term incubation with NRG1 increases both spine size and density in cortical pyramidal neurons. NRG1 also enhances the content of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors in spines. Knockdown of ERBB4 expression prevented the effects of NRG1 on spine size, but not on spine density. The effects of NRG1 and erbB4 on spines were mediated by the RacGEF kalirin, a well-characterized regulator of dendritic spines. Finally, we show that environmental enrichment, known to promote spine growth, robustly enhances the levels of erbB4 protein in the forebrain. These findings provide a mechanistic link between NRG1 signaling and spine morphogenesis
Asunto(s)
Espinas Dendríticas/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Neurregulina-1/fisiología , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/crecimiento & desarrollo , Reactivos de Enlaces Cruzados , Receptores ErbB/genética , Femenino , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Neurregulina-1/farmacología , Plásmidos , Embarazo , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Ratas , Ratas Sprague-Dawley , Receptor ErbB-4 , Receptores AMPA/biosíntesis , Receptores AMPA/efectos de los fármacos , Receptores AMPA/genética , Receptores AMPA/fisiología , Transducción de Señal/fisiología , TransfecciónRESUMEN
The effects of repeated stress on cognitive impairment are thought to be mediated, at least in part, by reductions in the stability of dendritic spines in brain regions critical for proper learning and memory, including the hippocampus. Small GTPases are particularly potent regulators of dendritic spine formation, stability, and morphology in hippocampal neurons. Through the use of small GTPase protein profiling in mice, we identify increased levels of synaptic Rap1 in the hippocampal CA3 region in response to escalating, intermittent stress. We then demonstrate that increased Rap1 in the CA3 is sufficient in and of itself to produce stress-relevant dendritic spine and cognitive phenotypes. Further, using super-resolution imaging, we investigate how the pattern of Rap1 trafficking to synapses likely underlies its effects on the stability of select dendritic spine subtypes. These findings illuminate the involvement of aberrant Rap1 regulation in the hippocampus in contributing to the psychobiological effects of stress.
RESUMEN
The effects of repeated stress on cognitive impairment are thought to be mediated, at least in part, by reductions in the stability of dendritic spines in brain regions critical for proper learning and memory, including the hippocampus. Small GTPases are particularly potent regulators of dendritic spine formation, stability, and morphology in hippocampal neurons. Through the use of small GTPase protein profiling in mice, we identify increased levels of synaptic Rap1 in the hippocampal CA3 region in response to escalating, intermittent stress. We then demonstrate that increased Rap1 in the CA3 is sufficient in and of itself to produce stress-relevant dendritic spine and cognitive phenotypes. Further, using super-resolution imaging, we investigate how the pattern of Rap1 trafficking to synapses likely underlies its effects on the stability of select dendritic spine subtypes. These findings illuminate the involvement of aberrant Rap1 regulation in the hippocampus in contributing to the psychobiological effects of stress.
RESUMEN
The actin cytoskeleton in dendritic spines is organized into microdomains, but how signaling molecules that regulate actin are spatially governed is incompletely understood. Here we examine how the localization of the RacGEF kalirin-7, a well-characterized regulator of actin in spines, varies as a function of post-synaptic density area and spine volume. Using serial section electron microscopy, we find that extrasynaptic, but not synaptic, expression of kalirin-7 varies directly with synapse size and spine volume. Moreover, we find that overall expression levels of kalirin-7 differ in spines bearing perforated and non-perforated synapses, due primarily to extrasynaptic pools of kalirin-7 expression in the former. Overall, our findings indicate that kalirin-7 is differentially compartmentalized in spines as a function of both synapse morphology and spine size.
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Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Factores de Intercambio de Guanina Nucleótido/biosíntesis , Sinapsis/metabolismo , Sinapsis/ultraestructura , Animales , Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Inmunogenética , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Ratas , Transducción de Señal/fisiologíaRESUMEN
Reductions in dendritic arbor length and complexity are among the most consistently replicated changes in neuronal structure in post mortem studies of cerebral cortical samples from subjects with schizophrenia, however, the underlying molecular mechanisms have not been identified. This study is the first to identify an alteration in a regulatory protein which is known to promote both dendritic length and arborization in developing neurons, Kalirin-9. We found Kalirin-9 expression to be paradoxically increased in schizophrenia. We followed up this observation by overexpressing Kalirin-9 in mature primary neuronal cultures, causing reduced dendritic length and complexity. Kalirin-9 overexpression represents a potential mechanism for dendritic changes seen in schizophrenia.
Asunto(s)
Dendritas/patología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Esquizofrenia/metabolismo , Esquizofrenia/patología , Adulto , Animales , Corteza Auditiva/metabolismo , Corteza Auditiva/patología , Western Blotting , Dendritas/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Microscopía Confocal , Ratas , Ratas Sprague-DawleyRESUMEN
Regulation of forebrain cellular structure and function by small GTPase pathways is crucial for normal and pathological brain development and function. Kalirin is a brain-specific activator of Rho-like small GTPases implicated in neuropsychiatric disorders. We have recently demonstrated key roles for kalirin in cortical synaptic transmission, dendrite branching, spine density, and working memory. However, little is known about the impact of the complete absence of kalirin on the hippocampus in mice. We thus investigated hippocampal function, structure, and associated behavioral phenotypes in KALRN knockout (KO) mice we have recently generated. Here we show that KALRN KO mice had modest impairments in hippocampal LTP, but normal hippocampal synaptic transmission. In these mice, both context and cue-dependent fear conditioning were impaired. Spine density and dendrite morphology in hippocampal pyramidal neurons were not significantly affected in the KALRN KO mice, but small alterations in the gross morphology of the hippocampus were detected. These data suggest that hippocampal structure and function are more resilient to the complete loss of kalirin, and reveal impairments in fear learning. These studies allow the comparison of the phenotypes of different kalirin mutant mice and shed light on the brain region-specific functions of small GTPase signaling.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Hipocampo/fisiología , Animales , Conducta Animal/fisiología , Células Cultivadas , Miedo/fisiología , Factores de Intercambio de Guanina Nucleótido/genética , Hipocampo/citología , Humanos , Potenciación a Largo Plazo/fisiología , Masculino , Ratones , Ratones Noqueados , Plasticidad Neuronal/fisiología , Fenotipo , Transmisión Sináptica/fisiologíaRESUMEN
Dendritic spine morphogenesis contributes to brain function, cognition, and behavior, and is altered in psychiatric disorders. Kalirin is a brain-specific guanine-nucleotide exchange factor (GEF) for Rac-like GTPases and is a key regulator of spine morphogenesis. Here, we show that KALRN-knockout mice have specific reductions in cortical, but not hippocampal, Rac1 signaling and spine density, and exhibit reduced cortical glutamatergic transmission. These mice exhibit robust deficits in working memory, sociability, and prepulse inhibition, paralleled by locomotor hyperactivity reversible by clozapine in a kalirin-dependent manner. Several of these deficits are delayed and age-dependent. Our study thus links spine morphogenic signaling with age-dependent, delayed, disease-related phenotypes, including cognitive dysfunction.
Asunto(s)
Espinas Dendríticas/fisiología , Lóbulo Frontal/citología , Factores de Intercambio de Guanina Nucleótido/fisiología , Animales , Conducta Animal , Células Cultivadas , Clozapina/farmacología , Cognición , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Noqueados , Morfogénesis , Actividad Motora , Fenotipo , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica , Proteína de Unión al GTP cdc42/fisiologíaRESUMEN
Activity-dependent rapid structural and functional modifications of central excitatory synapses contribute to synapse maturation, experience-dependent plasticity, and learning and memory and are associated with neurodevelopmental and psychiatric disorders. However, the signal transduction mechanisms that link glutamate receptor activation to intracellular effectors that accomplish structural and functional plasticity are not well understood. Here we report that NMDA receptor activation in pyramidal neurons causes CaMKII-dependent phosphorylation of the guanine-nucleotide exchange factor (GEF) kalirin-7 at residue threonine 95, regulating its GEF activity, leading to activation of small GTPase Rac1 and rapid enlargement of existing spines. Kalirin-7 also interacts with AMPA receptors and controls their synaptic expression. By demonstrating that kalirin expression and spine localization are required for activity-dependent spine enlargement and enhancement of AMPAR-mediated synaptic transmission, our study identifies a signaling pathway that controls structural and functional spine plasticity.