RESUMEN
Fear discrimination is critical for survival, while fear generalization is effective for avoiding dangerous situations. Overgeneralized fear is a typical symptom of anxiety disorders, including generalized anxiety disorder and posttraumatic stress disorder (PTSD). Previous research demonstrated that fear discrimination learning is mediated by prefrontal mechanisms. While the prelimbic (PL) and infralimbic (IL) subdivisions of the medial prefrontal cortex (mPFC) are recognized for their excitatory and inhibitory effects on the fear circuit, respectively, the mechanisms driving fear discrimination are unidentified. To obtain insight into the mechanisms underlying context-specific fear discrimination, we investigated prefrontal neuronal ensembles representing distinct experiences associated with learning to disambiguate between dangerous and similar, but not identical, harmless stimuli. Here, we show distinct quantitative activation differences in response to conditioned and generalized fear experiences, as well as modulation of the neuronal ensembles associated with successful acquisition of context-safety contingencies. These findings suggest that prefrontal neuronal ensembles patterns code functional context-danger and context-safety relationships. The PL subdivision of the mPFC monitors context-danger associations to conditioned fear, whereas differential conditioning sparks additional ensembles associated with the inhibition of generalized fear in both the PL and IL subdivisions of the mPFC. Our data suggest that fear discrimination learning is associated with the modulation of prefrontal subpopulations in a subregion- and experience-specific fashion, and the learning of appropriate responses to conditioned and initially generalized fear experiences is driven by gradual updating and rebalancing of the prefrontal memory representations.
Asunto(s)
Condicionamiento Clásico/fisiología , Discriminación en Psicología/fisiología , Miedo/fisiología , Neuronas/fisiología , Corteza Prefrontal/citología , Complejo Relacionado con el SIDA/genética , Complejo Relacionado con el SIDA/metabolismo , Análisis de Varianza , Animales , Doxiciclina/farmacología , Reacción Cataléptica de Congelación/fisiología , Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histonas/genética , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfopiruvato Hidratasa/metabolismo , Factores de TiempoRESUMEN
Prenatal ethanol exposure (PrEE) produces developmental abnormalities in brain and behavior that often persist into adulthood. We have previously reported abnormal cortical gene expression, disorganized neural circuitry along with deficits in sensorimotor function and anxiety in our CD-1 murine model of fetal alcohol spectrum disorders, or FASD (El Shawa et al., 2013; Abbott et al., 2016). We have proposed that these phenotypes may underlie learning, memory, and behavioral deficits in humans with FASD. Here, we evaluate the impact of PrEE on fear memory learning, recall and amygdala development at two adult timepoints. PrEE alters learning and memory of aversive stimuli; specifically, PrEE mice, fear conditioned at postnatal day (P) 50, showed deficits in fear acquisition and memory retrieval when tested at P52 and later at P70-P72. Interestingly, this deficit in fear acquisition observed during young adulthood was not present when PrEE mice were conditioned later, at P80. These mice displayed similar levels of fear expression as controls when tested on fear memory recall. To test whether PrEE alters development of brain circuitry associated with fear conditioning and fear memory recall, we histologically examined subdivisions of the amygdala in PrEE and control mice and found long-term effects of PrEE on fear memory circuitry. Thus, results from this study will provide insight on the neurobiological and behavioral effects of PrEE and provide new information on developmental trajectories of brain dysfunction in people prenatally exposed to ethanol.
RESUMEN
Rubinstein-Taybi syndrome (RSTS) is a rare genetic disorder in humans characterized by growth and psychomotor delay, abnormal gross anatomy, and mild to severe mental retardation (Rubinstein and Taybi, Am J Dis Child 105:588-608, 1963, Hennekam et al., Am J Med Genet Suppl 6:56-64, 1990). RSTS is caused by de novo mutations in epigenetics-associated genes, including the cAMP response element-binding protein (CREBBP), the gene-encoding protein referred to as CBP, and the EP300 gene, which encodes the p300 protein, a CBP homologue. Recent studies of the epigenetic mechanisms underlying cognitive functions in mice provide direct evidence for the involvement of nuclear factors (e.g., CBP) in the control of higher cognitive functions. In fact, a role for CBP in higher cognitive function is suggested by the finding that RSTS is caused by heterozygous mutations at the CBP locus (Petrij et al., Nature 376:348-351, 1995). CBP was demonstrated to possess an intrinsic histone acetyltransferase activity (Ogryzko et al., Cell 87:953-959, 1996) that is required for CREB-mediated gene expression (Korzus et al., Science 279:703-707, 1998). The intrinsic protein acetyltransferase activity in CBP might directly destabilize promoter-bound nucleosomes, facilitating the activation of transcription. Due to the complexity of developmental abnormalities and the possible genetic compensation associated with this congenital disorder, however, it is difficult to establish a direct role for CBP in cognitive function in the adult brain. Although aspects of the clinical presentation in RSTS cases have been extensively studied, a spectrum of symptoms found in RSTS patients can be accessed only after birth, and, thus, prenatal genetic tests for this extremely rare genetic disorder are seldom considered. Even though there has been intensive research on the genetic and epigenetic function of the CREBBP gene in rodents, the etiology of this devastating congenital human disorder is largely unknown.
Asunto(s)
Proteína de Unión a CREB/fisiología , Proteína p300 Asociada a E1A/fisiología , Epigénesis Genética/genética , Histona Acetiltransferasas/fisiología , Código de Histonas/fisiología , Proteínas del Tejido Nervioso/fisiología , Procesamiento Proteico-Postraduccional/genética , Síndrome de Rubinstein-Taybi/genética , Acetilación , Animales , Encéfalo/metabolismo , Encéfalo/patología , Proteína de Unión a CREB/deficiencia , Proteína de Unión a CREB/genética , Cognición/fisiología , Modelos Animales de Enfermedad , Proteína p300 Asociada a E1A/deficiencia , Proteína p300 Asociada a E1A/genética , Regulación del Desarrollo de la Expresión Génica , Estudios de Asociación Genética , Histona Acetiltransferasas/deficiencia , Histona Acetiltransferasas/genética , Código de Histonas/genética , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Invertebrados/genética , Invertebrados/fisiología , Mamíferos/genética , Mamíferos/fisiología , Memoria/fisiología , Modelos Neurológicos , Mutación , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , ARN Largo no Codificante/genética , Síndrome de Rubinstein-Taybi/metabolismoRESUMEN
The immune privileged nature of the CNS can make it vulnerable to chronic and latent infections. Little is known about the effects of lifelong brain infections, and thus inflammation, on the neurological health of the host. Toxoplasma gondii is a parasite that can infect any mammalian nucleated cell with average worldwide seroprevalence rates of 30%. Infection by Toxoplasma is characterized by the lifelong presence of parasitic cysts within neurons in the brain, requiring a competent immune system to prevent parasite reactivation and encephalitis. In the immunocompetent individual, Toxoplasma infection is largely asymptomatic, however many recent studies suggest a strong correlation with certain neurodegenerative and psychiatric disorders. Here, we demonstrate a significant reduction in the primary astrocytic glutamate transporter, GLT-1, following infection with Toxoplasma. Using microdialysis of the murine frontal cortex over the course of infection, a significant increase in extracellular concentrations of glutamate is observed. Consistent with glutamate dysregulation, analysis of neurons reveal changes in morphology including a reduction in dendritic spines, VGlut1 and NeuN immunoreactivity. Furthermore, behavioral testing and EEG recordings point to significant changes in neuronal output. Finally, these changes in neuronal connectivity are dependent on infection-induced downregulation of GLT-1 as treatment with the ß-lactam antibiotic ceftriaxone, rescues extracellular glutamate concentrations, neuronal pathology and function. Altogether, these data demonstrate that following an infection with T. gondii, the delicate regulation of glutamate by astrocytes is disrupted and accounts for a range of deficits observed in chronic infection.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/microbiología , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Homeostasis , Neuronas/metabolismo , Toxoplasmosis Cerebral/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/microbiología , Modelos Animales de Enfermedad , Electroencefalografía , Femenino , Homeostasis/fisiología , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microdiálisis , Microscopía Electrónica de Transmisión , Reacción en Cadena en Tiempo Real de la Polimerasa , ToxoplasmaRESUMEN
Normal brain functioning relies critically on the ability to control appropriate behavioral responses to fearful stimuli. Overgeneralized fear is the major symptom of anxiety disorders including posttraumatic stress disorder. This review describes recent data demonstrating that the medial prefrontal cortex (mPFC) plays a critical role in the refining of cues that drive the acquisition of fear response. Recent studies on molecular mechanisms that underlie the role of mPFC in fear discrimination learning are discussed. These studies suggest that prefrontal N-methyl-D-aspartate receptors expressed in excitatory neurons govern fear discrimination learning via a mechanism involving cAMP response element-binding protein-dependent engagement of acetyltransferase.
RESUMEN
Cannabis continues to be the most accessible and popular illicit recreational drug. Whereas current data link adolescence cannabinoid exposure to increased risk for dependence on other drugs, depression, anxiety disorders and psychosis, the mechanism(s) underlying these adverse effects remains controversial. Here we show in a mouse model of female adolescent cannabinoid exposure deficient endocannabinoid (eCB)-mediated signaling and presynaptic forms of long-term depression at adult central glutamatergic synapses in the prefrontal cortex. Increasing endocannabinoid levels by blockade of monoacylglycerol lipase, the primary enzyme responsible for degrading the endocannabinoid 2-arachidonoylglycerol (2-AG), with the specific inhibitor JZL 184 ameliorates eCB-LTD deficits. The observed deficit in cortical presynaptic signaling may represent a neural maladaptation underlying network instability and abnormal cognitive functioning. Our study suggests that adolescent cannabinoid exposure may permanently impair brain functions, including the brain's intrinsic ability to appropriately adapt to external influences.
Asunto(s)
Potenciación a Largo Plazo/efectos de los fármacos , Abuso de Marihuana/fisiopatología , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/crecimiento & desarrollo , Terminales Presinápticos/efectos de los fármacos , Receptor Cannabinoide CB1/agonistas , Animales , Benzoxazinas/toxicidad , Agonistas de Receptores de Cannabinoides/toxicidad , Trastornos del Conocimiento/inducido químicamente , Trastornos del Conocimiento/metabolismo , Modelos Animales de Enfermedad , Endocannabinoides/metabolismo , Femenino , Potenciación a Largo Plazo/fisiología , Abuso de Marihuana/psicología , Ratones Endogámicos C57BL , Morfolinas/toxicidad , Naftalenos/toxicidad , Corteza Prefrontal/fisiopatología , Terminales Presinápticos/fisiología , Receptor Cannabinoide CB1/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Reconocimiento en Psicología/efectos de los fármacos , Reconocimiento en Psicología/fisiología , Técnicas de Cultivo de TejidosRESUMEN
Recognition of an object's location in space is supported by hippocampus-dependent recollection. Converging evidence strongly suggests that the interplay between the prefrontal cortex and hippocampus is critical for spatial memory. Lesion, pharmacological, and genetic studies have been successful in dissecting the role of plasticity in the hippocampal circuit in a variety of neural processes relevant to spatial memory, including memory for the location of objects. However, prefrontal mechanisms underlying spatial memory are less well understood. Here, we show that an acute hypofunction of the cyclic-AMP regulatory element binding protein (CREB) Binding Protein (CBP) histone acetyltransferase (HAT) in the medial prefrontal cortex (mPFC) results in delay-dependent disruption of object-location memory. These data suggest that mechanisms involving CBP HAT-mediated lysine acetylation of nuclear proteins support selectively long-term encoding in the mPFC circuits. Evidence from the object-location task suggests that long-term memory encoding within the mPFC complements hippocampus-dependent spatial memory mechanisms and may be critical for broader network integration of information necessary for an assessment of subtle spatial differences to guide appropriate behavioral response during retrieval of spatial memories.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Consolidación de la Memoria/fisiología , Corteza Prefrontal/fisiología , Aprendizaje Espacial/fisiología , Memoria Espacial/fisiología , Animales , Proteína de Unión a CREB/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Inmunohistoquímica , Trastornos de la Memoria/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Neuronas/citología , Neuronas/fisiología , Pruebas Neuropsicológicas , Corteza Prefrontal/citología , Proteínas Proto-Oncogénicas c-fos/metabolismoRESUMEN
N-methyl-D-aspartate receptors (NMDARs) are critically involved in various learning mechanisms including modulation of fear memory, brain development and brain disorders. While NMDARs mediate opposite effects on medial prefrontal cortex (mPFC) interneurons and excitatory neurons, NMDAR antagonists trigger profound cortical activation. The objectives of the present study were to determine the involvement of NMDARs expressed specifically in excitatory neurons in mPFC-dependent adaptive behaviors, specifically fear discrimination and fear extinction. To achieve this, we tested mice with locally deleted Grin1 gene encoding the obligatory NR1 subunit of the NMDAR from prefrontal CamKIIα positive neurons for their ability to distinguish frequency modulated (FM) tones in fear discrimination test. We demonstrated that NMDAR-dependent signaling in the mPFC is critical for effective fear discrimination following initial generalization of conditioned fear. While mice with deficient NMDARs in prefrontal excitatory neurons maintain normal responses to a dangerous fear-conditioned stimulus, they exhibit abnormal generalization decrement. These studies provide evidence that NMDAR-dependent neural signaling in the mPFC is a component of a neural mechanism for disambiguating the meaning of fear signals and supports discriminative fear learning by retaining proper gating information, viz. both dangerous and harmless cues. We also found that selective deletion of NMDARs from excitatory neurons in the mPFC leads to a deficit in fear extinction of auditory conditioned stimuli. These studies suggest that prefrontal NMDARs expressed in excitatory neurons are involved in adaptive behavior.
Asunto(s)
Miedo/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Corteza Prefrontal/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Estimulación Acústica , Animales , Percepción Auditiva/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Condicionamiento Psicológico/fisiología , Discriminación en Psicología/fisiología , Extinción Psicológica/fisiología , Femenino , Técnicas de Inactivación de Genes , Generalización de la Respuesta/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
The neural mechanisms underlying the attainment of fear memory accuracy for appropriate discriminative responses to aversive and nonaversive stimuli are unclear. Considerable evidence indicates that coactivator of transcription and histone acetyltransferase cAMP response element binding protein (CREB) binding protein (CBP) is critically required for normal neural function. CBP hypofunction leads to severe psychopathological symptoms in human and cognitive abnormalities in genetic mutant mice with severity dependent on the neural locus and developmental time of the gene inactivation. Here, we showed that an acute hypofunction of CBP in the medial prefrontal cortex (mPFC) results in a disruption of fear memory accuracy in mice. In addition, interruption of CREB function in the mPFC also leads to a deficit in auditory discrimination of fearful stimuli. While mice with deficient CBP/CREB signaling in the mPFC maintain normal responses to aversive stimuli, they exhibit abnormal responses to similar but nonrelevant stimuli when compared to control animals. These data indicate that improvement of fear memory accuracy involves mPFC-dependent suppression of fear responses to nonrelevant stimuli. Evidence from a context discriminatory task and a newly developed task that depends on the ability to distinguish discrete auditory cues indicated that CBP-dependent neural signaling within the mPFC circuitry is an important component of the mechanism for disambiguating the meaning of fear signals with two opposing values: aversive and nonaversive.
Asunto(s)
Percepción Auditiva/fisiología , Discriminación en Psicología/fisiología , Miedo/fisiología , Memoria/fisiología , Corteza Prefrontal/fisiología , Estimulación Acústica , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Condicionamiento Clásico/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Electrochoque , Pie , Ratones Endogámicos C57BL , Actividad Motora/fisiología , Mutación , Pruebas Neuropsicológicas , Transducción de Señal , TransfecciónRESUMEN
In addition to its central role in learning and memory, N-methyl D-aspartate receptor (NMDAR)-dependent signaling regulates central glutamatergic synapse maturation and has been implicated in schizophrenia. We have transiently induced NMDAR hypofunction in infant mice during postnatal days 7-11, followed by testing fear memory specificity and presynaptic plasticity in the prefrontal cortex (PFC) in adult mice. We show that transient NMDAR hypofunction during early brain development, coinciding with the maturation of cortical plasticity results in a loss of an endocannabinoid (eCB)-mediated form of long-term depression (eCB-LTD) at adult central glutamatergic synapses, while another form of presynaptic long-term depression mediated by the metabotropic glutamate receptor 2/3 (mGluR2/3-LTD) remains intact. Mice with this selective impairment of presynaptic plasticity also showed deficits in fear memory specificity. The observed deficit in cortical presynaptic plasticity may represent a neural maladaptation contributing to network instability and abnormal cognitive functioning.
Asunto(s)
Endocannabinoides/deficiencia , Miedo/fisiología , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Trastornos de la Memoria/metabolismo , Aminoácidos/farmacología , Animales , Animales Recién Nacidos , Encéfalo/patología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Condicionamiento Psicológico/efectos de los fármacos , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/toxicidad , Miedo/efectos de los fármacos , Femenino , Potenciación a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Masculino , Trastornos de la Memoria/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Fenciclidina/toxicidad , Receptores de N-Metil-D-Aspartato/deficiencia , Transducción de Señal/efectos de los fármacosAsunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Epigénesis Genética/fisiología , Sistema Nervioso , Factores de Transcripción/metabolismo , Animales , Metilación de ADN , Ambiente , Silenciador del Gen , Genoma , Histonas/metabolismo , Modelos Moleculares , Neuronas , Trastornos Relacionados con Sustancias/genéticaRESUMEN
The neuronal response to a Ca2+ stimulus is a complex process involving direct Ca2+/calmodulin (CaM) actions as well as secondary activation of multiple signaling pathways such as cAMP and ERK (extracellular signal-regulated kinase). These signals can act in both the cytoplasm and the nucleus to control gene expression. To dissect the role of nuclear from cytoplasmic Ca2+/CaM signaling in memory formation, we generated transgenic mice that express a dominant inhibitor of Ca2+/CaM selectively in the nuclei of forebrain neurons and only after the animals reach adulthood. These mice showed diminished neuronal activity-induced phosphorylation of cAMP response element-binding protein, reduced expression of activity-induced genes, altered maximum levels of hippocampal long-term potentiation, and severely impaired formation of long-term, but not short-term, memory. Our results demonstrate that nuclear Ca2+/CaM signaling plays a critical role in memory consolidation in the mouse.
Asunto(s)
Señalización del Calcio , Calcio/fisiología , Calmodulina/fisiología , Memoria/fisiología , Transporte Activo de Núcleo Celular , Animales , Reacción de Prevención/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas de Unión a Calmodulina/genética , Núcleo Celular/fisiología , Condicionamiento Operante , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Doxiciclina/farmacología , Conducta Exploratoria , Femenino , Reacción Cataléptica de Congelación/fisiología , Regulación de la Expresión Génica , Genes fos , Hipocampo/metabolismo , Potenciación a Largo Plazo/fisiología , Masculino , Aprendizaje por Laberinto , Trastornos de la Memoria/genética , Trastornos de la Memoria/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora , Fosforilación , Regiones Promotoras Genéticas/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Reconocimiento en Psicología/fisiología , Convulsiones/metabolismoRESUMEN
The stabilization of learned information into long-term memories requires new gene expression. CREB binding protein (CBP) is a coactivator of transcription that can be independently regulated in neurons. CBP functions both as a platform for recruiting other required components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. To dissect the chromatin remodeling versus platform function of CBP or the developmental versus adult role of this gene, we generated transgenic mice that express CBP in which HAT activity is eliminated. Acquisition of new information and short-term memory is spared in these mice, while the stabilization of short-term memory into long-term memory is impaired. The behavioral phenotype is due to an acute requirement for CBP HAT activity in the adult as it is rescued by both suppression of transgene expression or by administration of the histone deacetylase inhibitor Trichostatin A (TSA) in adult animals.
Asunto(s)
Acetiltransferasas/fisiología , Trastornos de la Memoria/fisiopatología , Memoria/fisiología , Proteínas Nucleares/fisiología , Transactivadores/fisiología , Acetiltransferasas/genética , Animales , Conducta Animal , Proteína de Unión a CREB , Condicionamiento Psicológico/efectos de los fármacos , Miedo/efectos de los fármacos , Miedo/fisiología , Expresión Génica/genética , Genes fos/fisiología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Histona Acetiltransferasas , Ácidos Hidroxámicos/farmacología , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/genética , Ratones , Ratones Mutantes , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Nucleares/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Desempeño Psicomotor/efectos de los fármacos , Desempeño Psicomotor/fisiología , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transactivadores/genéticaRESUMEN
A distinction between short-term memories lasting minutes to hours and long-term memories lasting for many days is that the formation of long-term memories requires new gene expression. In this review, the focus is on the current understanding of the relation of transcription to memory consolidation based on the data collected from behavioral studies performed primarily on genetically altered animals. Studies in Drosophila and Aplysia indicate that the transcription factor cAMP/Ca(2+) response element binding protein (CREB) is critical in mediating the conversion from short- to long-term memory. More recent genetic studies in mice also demonstrated CREB and inducible transcription factor Zif268 involvement in information storage processes. Transcription seems to play essential role in memory formation but the mechanisms for activation of transcription and downstream processes during memory consolidation remain unclear.