RESUMEN

We used confocal microscopy and immunohistochemistry (IHC) to look for new cells in the motor cortex of adult macaque monkeys that might form the cellular bases of improved brain function from exercise. Twenty-four female Macaca fascicularis monkeys divided into groups by age (10-12 years, 15-17 years), postexercise survival periods, and controls, received 10 weekly injections of the thymidine analog, bromodeoxyuridine (BrdU) to mark new cells. Sixteen monkeys survived 15 weeks (5 weeks postexercise) and 8 monkeys survived 27 weeks (12 weeks postexercise) after initial BrdU injections. Additionally, five Macaca mulatta female monkeys (∼5.5-7 years) received single injections of BrdU and survived 2 days, 2 weeks, and 6 weeks after BrdU injections. Neural and glial antibodies were used to identify new cell phenotypes and to look for changes in proportions of these cells with respect to time and experimental conditions. No BrdU(+) /DCx(+) cells were found but about 7.5% of new cells were calretinin-positive (Cr(+) ). BrdU(+) /GABA(+) (gamma-aminobutyric acid) cells were also found but no new Cr(+) or GABA(+) cells colabeled with a mature neuron marker, NeuN or chondroitin sulfate antibody, NG2. The proportion of new cells that were NG2(+) was about 85% for short and long survival monkeys of which two, newly described perivascular phenotypes (Pldv and Elu) and a small percentage of pericytes (2.5%) comprised 44% and 51% of the new NG2(+) cells, respectively. Proportions of NG2(+) phenotypes were affected by post-BrdU survival periods, monkey age, and possibly a postexercise sedentary period but no direct effect of exercise was found.

Asunto(s)

Antígenos/metabolismo , Corteza Motora/citología , Neuroglía/fisiología , Neuronas/clasificación , Neuronas/fisiología , Proteoglicanos/metabolismo , Animales , Bromodesoxiuridina/metabolismo , Diferenciación Celular , Femenino , Macaca fascicularis/anatomía & histología , Macaca mulatta/anatomía & histología , Microscopía Confocal , Proteínas del Tejido Nervioso/metabolismo , Organogénesis , Factores de Tiempo

RESUMEN

Motor skill training promotes the formation of parallel fiber multiple-synapse boutons (MSBs) contacting dendritic spine pairs of Purkinje cells in the rat cerebellum. However, the dendritic origin of such spine pairs is unknown. Here, we used three-dimensional electron microscopy reconstruction of synaptic connectivity to demonstrate that motor skill training selectively induced MSBs contacting two spines arising from the same dendrite, consistent with strengthening of local synaptic efficacy. However, excitatory synapses near MSBs were smaller in motor-trained animals, suggesting compensatory depression of MSB-neighbor synapses. Concerted strengthening and weakening of adjacent synapses may enhance synaptic weight differences for information encoding while maintaining stable overall activity levels within local dendritic segments.

Asunto(s)

Aprendizaje/fisiología , Destreza Motora/fisiología , Sinapsis/fisiología , Sinapsis/ultraestructura , Animales , Masculino , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley

RESUMEN

BACKGROUND: Effective treatments for the behavioral and cognitive deficits in children with fetal alcohol spectrum disorders (FASD) are lacking, and translational approaches using animal models can help develop rational interventions. One such model, binge-like alcohol exposure in neonatal rats during the period of brain development comparable with that of the human third trimester, causes structural and functional damage to the cerebellum and disrupts cerebellar-dependent eyeblink classical conditioning. The eyeblink conditioning deficits first demonstrated in this rat model predicted the similar deficits subsequently demonstrated in children with FASD. METHODS: The current study extends this translational approach by testing the hypothesis that rehabilitation training involving 20 days of training on traversal of an obstacle course (complex motor learning) would ameliorate the deficits on classical conditioning of eyeblink responses produced by the neonatal alcohol exposure. We have previously shown that this training stimulates cerebellar synaptic plasticity and improves alcohol-induced deficits on motor coordination tasks. RESULTS: The current studies found that rehabilitation training significantly attenuated alcohol-induced deficits in acquisition of eyeblink conditioning in females but not in males. These results are consistent with normalization of cerebellar-dependent learning, at least in alcohol-exposed females. CONCLUSIONS: These findings extend previous studies in this model suggesting that rehabilitation of adolescents with FASD using training with complex motor learning tasks could be effective in ameliorating functional impairments associated with cerebellar damage. Eyeblink classical conditioning deficits are now well documented in children with FASD and could serve as an evaluation measure to continue to develop therapeutic interventions such as complex motor learning.

Asunto(s)

Consumo Excesivo de Bebidas Alcohólicas/fisiopatología , Consumo Excesivo de Bebidas Alcohólicas/rehabilitación , Condicionamiento Clásico/fisiología , Condicionamiento Palpebral/fisiología , Aprendizaje/fisiología , Destreza Motora/fisiología , Animales , Animales Recién Nacidos , Femenino , Masculino , Embarazo , Distribución Aleatoria , Ratas , Ratas Long-Evans

RESUMEN

BACKGROUND: Binge-like alcohol exposure in neonatal rats during the brain growth spurt causes deficits in adult neurogenesis in the hippocampal dentate gyrus (DG). Previous data from our laboratory demonstrated that 12 days of voluntary wheel running (WR) beginning on postnatal day (PD) 30 significantly increased the number of newly generated cells evident in the DG on PD42 in both alcohol-exposed (AE) and control rats, but 30 days later a sustained beneficial effect of WR was evident only in control rats. This study tested the hypothesis that housing rats in environmental complexity (EC) following WR would promote the survival of the newly generated cells stimulated by WR, particularly in AE rats. METHODS: On PD4 to 9, pups were intubated with alcohol in a binge-like manner (5.25 g/kg/d), sham-intubated (SI), or reared normally. In Experiment 1, animals were either assigned to WR during PD30 to 42 or socially housed (SH). On PD42, animals were injected with bromodeoxyuridine (BrdU; 200 mg/kg) and perfused 2 hours later to confirm the WR-induced stimulation of proliferation. In Experiment 2, all animals received WR on PD30 to 42 and were injected with BrdU on the last full day of WR. On PD42, animals were randomly assigned either to EC (WR/EC) or to SH (WR/SH) for 30 days and subsequently perfused and brains were processed for immunohistochemical staining to identify BrdU+-, Ki67+-, and BrdU+/NeuN+-labeled cells in DG. RESULTS: In Experiment 1, WR exposure significantly increased the number of proliferating cells in all 3 postnatal conditions. In Experiment 2, the AE rats given WR/SH had significantly fewer BrdU+ cells compared with control rats given WR/SH. However, WR/EC experience significantly increased the number of surviving BrdU+ cells in both the AE and SI groups compared with WR/SH rats of the same neonatal treatment. Approximately 80% of the surviving BrdU+ cells in the DG across the conditions were colabeled with NeuN. CONCLUSIONS: WR followed by EC could provide a behavioral model for developing interventions in humans to ameliorate hippocampal-dependent impairments associated with fetal alcohol spectrum disorders.

Asunto(s)

Modelos Animales de Enfermedad , Ambiente , Etanol/toxicidad , Hipocampo/fisiología , Vivienda para Animales , Actividad Motora/fisiología , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/patología , Masculino , Actividad Motora/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Neurogénesis/fisiología , Neuronas/efectos de los fármacos , Neuronas/patología , Embarazo , Distribución Aleatoria , Ratas , Ratas Long-Evans

RESUMEN

We studied two groups of adult macaque monkeys to determine the time course of adult neurogenesis in the dentate gyrus of the hippocampus. In the first group, six adult monkeys (Macaca mulatta) received a single injection of the thymidine analog BrdU (75 mg/kg), which is incorporated into replicating DNA and serves as a marker for new cell birth. Brain tissue was collected 48 h, 2 wk, and 6 wk after BrdU injection to examine the initial stages of neurogenesis. Because mature neurons were not evident at 6 wk, we examined tissue collected over a longer time course in a second study. In this study, eight monkeys (Macaca fascicularis) who were subjects in a separate exercise study received 10 weekly injections of BrdU (75 mg/kg), and brain tissue was collected at 16 and 28 wk from the first injection. Based on the timing of expression of neuronal cell markers (ßIII-tubulin, doublecortin, NeuN), the extent of dendritic arborization, and acquisition of mature cell body morphology, we show that granule cell maturation in the dentate gyrus of a nonhuman primate is protracted over a minimum of a 6-mo time period, more than 6 times longer than in rodents. The lengthened time course for new cell maturation in nonhuman primates may be appropriate for preservation of neural plasticity over their longer life span and is relevant to our understanding of antidepressant and other therapies that have been linked to neurogenesis in humans.

Asunto(s)

Giro Dentado/citología , Macaca/anatomía & histología , Macaca/fisiología , Neurogénesis/fisiología , Neuronas/fisiología , Animales , Biomarcadores/metabolismo , Bromodesoxiuridina/metabolismo , Movimiento Celular , Femenino , Humanos , Neuronas/citología , Factores de Tiempo

RESUMEN

OBJECTIVE: Glutamatergic dysregulation is implicated in the pathophysiology of fragile X syndrome (FXS). Riluzole is hypothesized to have an inhibitory effect on glutamate release, block excitotoxic effects of glutamate, and potentiate postsynaptic GABA(A) receptor function. Extracellular signal-related kinase (ERK) activation is known to be delayed in humans with FXS and knockout animal models of FXS. Correction of delayed ERK activation is a potential biomarker of treatment response in FXS. We conducted a six-week open-label prospective pilot study of riluzole (100 mg/day) in six adults with FXS. METHODS: Riluzole was started at 50mg every evening and then increased to 50mg twice daily at week 2. The dose was kept constant for the final 4 weeks of the trial. Clinical response was determined by a score of 1 "very much improved" or 2 "much improved" on the Clinical Global Impressions Improvement (CGI-I) scale and a≥25% improvement on the Children's Yale-Brown Obsessive Compulsive Scale modified for Pervasive Developmental Disorders. The primary target of treatment in this study was repetitive, compulsive behavior that commonly occurs in persons with FXS. The study incorporated an ERK activation biomarker assay. Potential adverse effects were assessed in a systematic manner at all clinic visits and by phone between visits. RESULTS: Riluzole treatment was associated with clinical response in 1 of 6 subjects (17%). Among a number of secondary outcome measures employed, significant improvement was only noted on the ADHD Rating Scale-IV (became non-significant when corrected for multiple comparisons). Riluzole use was associated with significant correction in ERK activation time in all subjects (mean change from 3.82±0.27 (baseline) to 2.99±0.26 (endpoint) minutes; p=0.007). Riluzole was well tolerated; mean increases in liver function tests occurred but drug discontinuation was not required. CONCLUSION: Overall, riluzole use was not associated with significant clinical improvement despite uniform correction of peripheral ERK activation. Future directions of study include testing of riluzole in animal models of FXS and assessment of psychotropic monotherapy on ERK activation.

Asunto(s)

Trastornos Generalizados del Desarrollo Infantil/tratamiento farmacológico , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Riluzol/administración & dosificación , Niño , Trastornos Generalizados del Desarrollo Infantil/fisiopatología , Trastornos Generalizados del Desarrollo Infantil/psicología , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Antagonistas de Aminoácidos Excitadores/efectos adversos , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/fisiopatología , Humanos , Masculino , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/efectos adversos , Proyectos Piloto , Estudios Prospectivos , Riluzol/efectos adversos , Adulto Joven

RESUMEN

Fragile X syndrome (FXS) is the most common inherited form of mental retardation and is caused by transcriptional inactivation of the X-linked fragile X mental retardation 1 (FMR1) gene. FXS is associated with increased density and abnormal morphology of dendritic spines, the postsynaptic sites of the majority of excitatory synapses. To better understand how lack of the FMR1 gene function affects spine development and plasticity, we examined spine formation and elimination of layer 5 pyramidal neurons in the whisker barrel cortex of Fmr1 KO mice with a transcranial two-photon imaging technique. We found that the rates of spine formation and elimination over days to weeks were significantly higher in both young and adult KO mice compared with littermate controls. The heightened spine turnover in KO mice was due to the existence of a larger pool of "short-lived" new spines in KO mice than in controls. Furthermore, we found that the formation of new spines and the elimination of existing ones were less sensitive to modulation by sensory experience in KO mice. These results indicate that the loss of Fmr1 gene function leads to ongoing overproduction of transient spines in the primary somatosensory cortex. The insensitivity of spine formation and elimination to sensory alterations in Fmr1 KO mice suggest that the developing synaptic circuits may not be properly tuned by sensory stimuli in FXS.

Asunto(s)

Espinas Dendríticas/fisiología , Síndrome del Cromosoma X Frágil/fisiopatología , Sinapsis/fisiología , Animales , Espinas Dendríticas/metabolismo , Diagnóstico por Imagen/métodos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados

RESUMEN

Fragile X syndrome is caused by the absence of functional fragile X mental retardation protein (FMRP), an RNA binding protein. The molecular mechanism of aberrant protein synthesis in fmr1 KO mice is closely associated with the role of FMRP in mRNA transport, delivery, and local protein synthesis. We show that GFP-labeled Fmr1 and CaMKIIalpha mRNAs undergo decelerated motion at 0-40 min after group I mGluR stimulation, and later recover at 40-60 min. Then we investigate targeting of mRNAs associated with FMRP after neuronal stimulation. We find that FMRP is synthesized closely adjacent to stimulated mGluR5 receptors. Moreover, in WT neurons, CaMKIIalpha mRNA can be delivered and translated in dendritic spines within 10 min in response to group I mGluR stimulation, whereas KO neurons fail to show this response. These data suggest that FMRP can mediate spatial mRNA delivery for local protein synthesis in response to synaptic stimulation.

Asunto(s)

Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Neuronas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Dendritas/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Hibridación Fluorescente in Situ , Cinética , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Neuronas/citología , Neuronas/efectos de los fármacos , Unión Proteica , Transporte de ARN , ARN Mensajero/genética , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Factores de Tiempo

RESUMEN

Fragile X Syndrome (FXS) is the most common form of inherited mental retardation. The neuroanatomical phenotype of adult FXS patients, as well as adult Fmr1 knockout (KO) mice, includes elevated dendritic spine density and a spine morphology profile in neocortex that resembles younger individuals. Developmental studies in mouse neocortex have revealed a dynamic phenotype that varies with age, especially during the period of synaptic pruning. Here we investigated the hippocampal dentate gyrus to determine if the FXS spine phenotype is similarly tied to periods of maturation and pruning in this brain region. We used high-voltage electron microscopy to characterize Golgi-stained spines along granule cell dendrites in Fmr1 KO and wildtype (WT) mouse dentate gyrus at postnatal days 15, 21, 30, and 60. In contrast to neocortex, dendritic spine density was higher in Fmr1 KO mice across development. Interestingly, neither genotype showed specific phases of synaptogenesis or pruning, potentially explaining the phenotypic differences from neocortex. Similarly, although the KO mice showed a more immature morphological phenotype overall than WT (higher proportion of thin headed spines, lower proportion of mushroom and stubby spines), both genotypes showed gradual development, rather than impairments during specific phases of maturation. Finally, spine length showed a complex developmental pattern that differs from other brain regions examined, suggesting dynamic regulation by FMRP and other brain region-specific proteins. These findings shed new light on FMRP's role in development and highlight the need for new techniques to further understand the mechanisms by which FMRP affects synaptic maturation.

Asunto(s)

Espinas Dendríticas/patología , Giro Dentado/anomalías , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Malformaciones del Sistema Nervioso/metabolismo , Malformaciones del Sistema Nervioso/patología , Animales , Diferenciación Celular/genética , Espinas Dendríticas/metabolismo , Giro Dentado/metabolismo , Modelos Animales de Enfermedad , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Malformaciones del Sistema Nervioso/genética , Distribución Aleatoria

RESUMEN

Fragile X syndrome (FXS), an inherited disorder characterized by mental retardation and autismlike behaviors, is caused by the failure to transcribe the gene for fragile X mental retardation protein (FMRP), a translational regulator and transporter of select mRNAs. FXS model mice (Fmr1 KO mice) exhibit impaired neuropeptide release. Release of biogenic amines does not differ between wild-type (WT) and Fmr1 KO mice. Rab3A, an mRNA cargo of FMRP involved in the recruitment of vesicles, is decreased by ∼50% in synaptoneurosomes of Fmr1 KO mice; however, the number of dense-core vesicles (DCVs) does not differ between WT and Fmr1 KO mice. Therefore, deficits associated with FXS may reflect this aberrant vesicle release, specifically involving docking and fusion of peptidergic DCVs, and may lead to defective maturation/maintenance of synaptic connections.

RESUMEN

Exposure to alcohol during the brain growth spurt results in impaired cognition and learning in adulthood. This impairment is accompanied by permanent structural changes in the hippocampal formation. Exercise improves performance on hippocampal-dependent learning and memory tasks and increases adult neurogenesis in the rat hippocampal dentate gyrus. The present study examined the effects of wheel running during adolescence on dentate gyrus cell proliferation and neurogenesis after postnatal binge-like alcohol exposure. On postnatal days (PD) 4-9, pups were either intubated with alcohol in a binge-like manner, sham intubated, or reared normally. On PD30-42, all animals were randomly assigned to two adolescent conditions: wheel running or inactive control. Animals were injected with BrdU every day between PD32 and PD42 and perfused on PD42 or PD72. In inactive control animals at both PD42 and PD72, cell proliferation and neurogenesis did not differ between postnatal treatment groups. Wheel running significantly increased the number of BrdU-labeled cells on PD42 in all three postnatal treatments. On PD72, only the normal controls showed significant increases in survival of newly generated cells resulting from the wheel running. These results indicate that adolescent wheel running can induce comparable increases in cell proliferation and neurogenesis in alcohol-exposed and control rats, but the long-term survival of those newly generated cells is impaired relative normal controls. Exercise may provide a means to stimulate neurogenesis, with implications for amelioration of hippocampal-dependent learning impairments associated with alcohol exposure. However, benefits requiring long-lasting survival of the newly generated cells will depend on identifying ways to promote survival.

Asunto(s)

Depresores del Sistema Nervioso Central/envenenamiento , Etanol/envenenamiento , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Neurogénesis/efectos de los fármacos , Condicionamiento Físico Animal/fisiología , Animales , Animales Recién Nacidos , Peso Corporal , Bromodesoxiuridina , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Depresores del Sistema Nervioso Central/sangre , Giro Dentado/efectos de los fármacos , Giro Dentado/crecimiento & desarrollo , Giro Dentado/fisiopatología , Modelos Animales de Enfermedad , Etanol/sangre , Hipocampo/fisiopatología , Neurogénesis/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Distribución Aleatoria , Ratas , Ratas Long-Evans

RESUMEN

Myelination is an important process in brain development, and delays or abnormalities in this process have been associated with a number of conditions including autism, developmental delay, attention deficit disorder, and schizophrenia. Myelination can be sensitive to developmental experience; however, although the adult brain remains highly plastic, it is unknown whether myelination continues to be sensitive to experience during adulthood. Male and female rats were socially housed until four months of age, at which time they were moved into either a complex or "enriched" environment (EC) or an isolated condition (IC). Although the area of the splenium (posterior 20% of the callosum, which contains axons from visual cortical neurons) increased by about 10% following two months of EC housing, the area occupied by myelinated axons was not influenced by adult housing condition. Instead, it was the area occupied by glial cell processes and unmyelinated axons which significantly increased following EC housing. Neither the size nor the myelin content of the genu (anterior 15% of the callosum) was sensitive to manipulations of adult housing condition, but males had more area occupied by myelinated axons in both callosal regions. Finally, the inability of two months of complex environment housing during adulthood to impact the number of myelinated axons in the splenium was confirmed in a subset of animals using quantitative electron microscopy. We conclude that the sensitivity of myelination to experience is reduced in adulthood relative to development in both sexes.

Asunto(s)

Cuerpo Calloso/anatomía & histología , Cuerpo Calloso/fisiología , Fibras Nerviosas Mielínicas/fisiología , Medio Social , Análisis de Varianza , Animales , Axones/fisiología , Recuento de Células , Femenino , Masculino , Microscopía Electrónica de Transmisión , Neuroglía/fisiología , Tamaño de los Órganos , Ratas , Ratas Long-Evans , Caracteres Sexuales , Factores Sexuales

RESUMEN

The long-term effects of binge-like postnatal alcohol exposure on cell proliferation and differentiation in the adolescent rat neocortex were examined. Unlike the hippocampal dentate gyrus, where proliferation of progenitors results primarily in addition of granule cells in adulthood, the vast majority of newly generated cells in the intact mature rodent neocortex appear to be glial cells. The current study examined cytogenesis in the motor cortex of adolescent and adult rats that were exposed to 5.25 g/kg/day of alcohol on postnatal days (PD) 4-9 in a binge manner. Cytogenesis was examined at PD50 (through bromodeoxyuridine [BrdU] labeling) and survival of these newly generated cells was evaluated at PD80. At PD50, significantly more BrdU-positive cells were present in the motor cortex of alcohol-exposed rats than controls. Confocal analysis revealed that the majority (>60%) of these labeled cells also expressed NG2 chondroitin sulfate proteoglycan (NG2 glia). Additionally, survival of these newly generated cortical cells was affected by neonatal alcohol exposure, based on the greater reduction in the number of BrdU-labeled cells from PD50 to PD80 in the alcohol-exposed animals compared to controls. These findings demonstrate that neonatal alcohol exposure triggers an increase in gliogenesis in the adult motor cortex.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Etanol/toxicidad , Corteza Motora/efectos de los fármacos , Neuroglía/efectos de los fármacos , Análisis de Varianza , Animales , Antígenos/metabolismo , Peso Corporal/efectos de los fármacos , Recuento de Células , Supervivencia Celular , Etanol/sangre , Femenino , Masculino , Microscopía Confocal , Corteza Motora/fisiopatología , Neuroglía/fisiología , Proteoglicanos/metabolismo , Ratas , Ratas Long-Evans

RESUMEN

OBJECTIVE: In fragile X syndrome (FXS), it is hypothesized that absence of the fragile X mental retardation protein (FMRP) disrupts regulation of group 1 metabotropic glutamate receptor (mGluR and mGluR5)-dependent translation in dendrites. Lithium reduces mGluR-activated translation and reverses phenotypes in the dfxr mutant fly and fmr1 knockout mouse. This pilot add-on trial was conducted to evaluate safety and efficacy of lithium in humans with FXS. METHODS: Fifteen individuals with FXS, ages 6-23, received lithium titrated to levels of 0.8-1.2 mEq/L. The primary outcome measure, the Aberrant Behavior Checklist --Community Edition (ABC-C) Irritability Subscale, secondary outcome measures (other ABC-C subscales, clinical global improvement scale (CGI), visual analog scale for behavior (VAS), Vineland Adaptive Behavior Scale (VABS)), exploratory cognitive and psychophysiological measures and an extracellular signal-regulated kinase (ERK) activation assay were administered at baseline and 2 months of treatment. Side effects were quantified with a standardized checklist and lithium level, complete blood count (CBC), thyroid stimulating hormone (TSH), and chemistry screen were done at baseline, 2 weeks, 4 weeks and 2 months. RESULTS: The only significant treatment-related side effects were polyuria/polydipsia (n = 7) and elevated TSH (n = 4). Although the ABC-C Irritability Subscale showed only a trend toward improvement, there was significant improvement in the Total ABC-C score (p = 0.005), VAS (p = 0.003), CGI (p = 0.002), VABS Maladaptive Behavior Subscale (p = 0.007), and RBANS List Learning (p = 0.03) and an enhanced ERK activation rate (p = 0.007). Several exploratory tasks proved too difficult for lower-functioning FXS subjects. CONCLUSIONS: Results from this study are consistent with results in mouse and fly models of FXS, and suggest that lithium is well-tolerated and provides functional benefits in FXS, possibly by modifying the underlying neural defect. A placebo-controlled trial of lithium in FXS is warranted.

Asunto(s)

Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Carbonato de Litio/uso terapéutico , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Administración Oral , Adolescente , Adulto , Antidepresivos/administración & dosificación , Antidepresivos/efectos adversos , Antidepresivos/uso terapéutico , Recuento de Células Sanguíneas , Niño , Cognición/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Síndrome del Cromosoma X Frágil/psicología , Humanos , Aprendizaje/efectos de los fármacos , Carbonato de Litio/efectos adversos , Carbonato de Litio/sangre , Proyectos Piloto , Escalas de Valoración Psiquiátrica/estadística & datos numéricos , Pruebas Psicológicas/estadística & datos numéricos , Tirotropina/sangre , Resultado del Tratamiento

RESUMEN

Western blots are used to estimate the relative concentrations of proteins of interest based on staining by specific antibodies. Quantitative measurements are often subject to error due to overloading of the loading control and over-reliance on normalization. We have found that at the protein concentrations normally used to quantify most low-abundance proteins of interest, frequently used single-protein loading controls, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin, do not accurately reflect differences in protein concentration. Two total protein stains, SYPRO Ruby and Amido Black, were compared and found to be acceptable alternatives to single-protein controls. Although we cannot prove that high-abundance loading controls are inaccurate under all possible conditions, we conclude that the burden of proof should lie with the researcher to demonstrate that their loading control is reflective of quantitative differences in protein concentration.

Asunto(s)

Western Blotting/métodos , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/química , Neuroquímica/métodos , Coloración y Etiquetado/métodos , Negro de Almidón/química , Secuencia de Aminoácidos , Animales , Química Encefálica/fisiología , Colorantes/química , Homólogo 4 de la Proteína Discs Large , Guanilato-Quinasas , Péptidos y Proteínas de Señalización Intracelular/análisis , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Ratones , Proteína Quinasa 3 Activada por Mitógenos/análisis , Proteína Quinasa 3 Activada por Mitógenos/química , Compuestos Organometálicos/química , Proteómica , Fracciones Subcelulares

RESUMEN

Lack of production of the Fragile X Mental Retardation Protein (FMRP) leads to changes in dendritic morphology and resultant cognitive and behavioral manifestations characteristic of individuals with Fragile X syndrome (FXS). FMRP is an RNA-binding protein that is believed to regulate the translation of a large number (probably over 100) of other proteins, leading to a complex and variable set of symptoms in FXS. In a mouse model of FXS, we previously observed delayed initiation of synaptically localized protein synthesis in response to neurotransmitter stimulation, as compared to wild-type mice. We now likewise have observed delayed early-phase phosphorylation of extracellular-signal regulated kinase (ERK), a nodal point for cell signaling cascades, in both neurons and thymocytes of fmr-1 KO mice. We further report that early-phase kinetics of ERK activation in lymphocytes from human peripheral blood is delayed in a cohort of individuals with FXS, relative to normlal controls, suggesting a potential biomarker to measure metabolic status of disease for individuals with FXS.

Asunto(s)

Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Síndrome del Cromosoma X Frágil/metabolismo , Animales , Biomarcadores/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Humanos , Linfocitos/metabolismo , Linfocitos/patología , Ratones , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Timo/metabolismo , Timo/patología , Factores de Tiempo

RESUMEN

Fragile X syndrome (FXS) has so far resisted efforts to define the basic cellular defects caused by the absence of a single protein, fragile X mental retardation protein (FMRP), because the patients have a wide variety of symptoms of varying severity. Immature-appearing dendritic spines on neurons found in FXS patients and fmr1-KO mice suggest a role for FMRP in modulating production of synaptic structural proteins. We isolated cortical synaptoneurosomes from WT and KO mice and studied MAPK pathway activation after group I metabotropic glutamate receptor (mGluR) stimulation. Here, we show that ERK in KO synaptoneurosomes is rapidly dephosphorylated upon mGluR1/5 stimulation, whereas it is phosphorylated in WT mice, suggesting that aberrant activation of phosphatases occurs in KO synapses in response to synaptic stimulation. In KO synapses, protein phosphatase 2A (PP2A) is overactivated after mGluR1 stimulation, and tyrosine phosphatase is overactivated after mGluR5 stimulation, causing the rapid deactivation of ERK. ERK activation can be restored in KO by pretreatment with phosphatase blockers; blocking of PP2A by okadaic acid could successfully restore normal ERK activation in KO synaptoneurosomes. We propose that overactivation of phosphatases in synapses may be a key deficit in FXS, which affects synaptic translation, transcription, and synaptic receptor regulation.

Asunto(s)

Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Síndrome del Cromosoma X Frágil/enzimología , Neuronas/enzimología , Animales , Activación Enzimática , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sinapsis/metabolismo , Factores de Tiempo

RESUMEN

BACKGROUND: Prenatal alcohol exposure can cause damage to the developing fetus with outcomes including growth deficiency, facial dysmorphology, brain damage, and cognitive and behavioral deficits. Smaller brains in children with FASD have been linked both with reduced cell proliferation in the developing CNS and with apoptotic cell loss of postmitotic neurons. Prenatal alcohol exposure in rodents during the period of brain development comparable to that of the first and second trimesters of human pregnancy persistently alters adult neurogenesis. Long-term effects of alcohol exposure during the third trimester equivalent, which occurs postnatally in the rat, on adult neurogenesis have not been previously reported. The goal of this study was to examine the effect of postnatal binge-like alcohol exposure on cell proliferation and neurogenesis in hippocampal dentate gyrus during adolescence and young adulthood. METHODS: Male Long-Evans rat pups were assigned to 3 groups: alcohol-exposed (AE), sham-intubated (SI) or suckle control (SC). AE pups received ethanol in a milk formula in a binge manner (2 feedings, 2 hours apart, total dose 5.25 g/kg/day) on postnatal days (PD) 4-9. BrdU was injected every other day on PD30-50. Animals were perfused either on PD50 to examine cytogenesis and neurogenesis in hippocampal dentate gyrus at the end of BrdU injections or on PD80 to evaluate new cell survival. Dorsal hippocampal sections were immunostained for BrdU, a marker for proliferating cells, Ki67, endogenous marker of proliferation, and NeuN, a marker for mature neurons. RESULTS: Binge-like alcohol exposure on PD4-9 significantly reduced the number of mature neurons in adult hippocampal dentate gyrus (DG) both on PD50 and PD80, without altering cumulative cytogenesis on PD50. In addition, the number of new neurons, that were generated between PD30 and 50, was further reduced after 30 days of survival in all 3 groups (SC, SI, and AE). CONCLUSIONS: These observations suggest that early postnatal binge alcohol exposure results in long-term deficits of adult hippocampal neurogenesis, providing a potential basis for the deficits of hippocampus-dependent behaviors reported for this model.

Asunto(s)

División Celular/fisiología , Giro Dentado/patología , Etanol/toxicidad , Trastornos del Espectro Alcohólico Fetal/patología , Hipocampo/patología , Neuronas/patología , Factores de Edad , Animales , Animales Recién Nacidos , Recuento de Células , Etanol/sangre , Femenino , Masculino , Embarazo , Ratas , Ratas Long-Evans

RESUMEN

Motor skill learning, but not mere motor activity, is associated with an increase in both synapse number and glial cell volume within the cerebellar cortex. The increase in synapse number has been shown to persist for at least 4 weeks in the absence of continued training. The present experiment similarly examined how a prolonged interruption in training affects the training-induced increase in astrocytic volume. Adult female rats were randomly allocated to either an acrobatic motor learning condition (AC) or a motor control condition (MC). The AC animals were trained to traverse a complex series of obstacles and each AC animal was pair matched with an MC animal that traversed an obstacle-free runway. These groups were further assigned to one of three training conditions. Animals in the early condition were trained for 10 consecutive days, animals in the delay condition received the same 10 days of training followed by a 28-day period without training, and animals in the continuous condition were trained for the entire 38 days. Unbiased stereological techniques were used to determine that AC animals had a significantly greater volume of astrocytes per Purkinje cell in the cerebellar paramedian lobule than the MC animals, a difference which was reduced (and not statistically detectable) among animals in the delay condition. These findings demonstrate that learning triggers the hypertrophy of astrocytic processes and furthermore that, unlike learning-induced synaptogenesis, astrocytic growth is reduced in the absence of continued training.

Asunto(s)

Aprendizaje por Asociación/fisiología , Astrocitos/citología , Corteza Cerebelosa/citología , Destreza Motora/fisiología , Condicionamiento Físico Animal/fisiología , Adaptación Fisiológica , Animales , Aumento de la Célula , Tamaño de la Célula , Femenino , Distribución Aleatoria , Ratas , Ratas Long-Evans , Sinapsis/fisiología

RESUMEN

The peptide content of individual mammalian cells is profiled using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Both enzymatic and nonenzymatic procedures, including a glycerol cell stabilization method, are reported for the isolation of individual mammalian cells in a manner compatible with MALDI MS measurements. Guided microdeposition of MALDI matrix allows samples to be created with suitable analyte-to-matrix ratios. More than 15 peptides are observed in individual rat intermediate pituitary cells. The combination of accurate mass data, expected cleavages by proteolytic enzymes, and postsource decay sequencing allows identification of 14 of these peptides as pro-opiomelanocortin prohormone-derived molecules. These protocols permit the classification of individual mammalian cells by peptide profile, the elucidation of cell-specific prohormone processing, and the discovery of new signaling peptides on a cell-to-cell basis in a wide variety of mammalian cell types.

Asunto(s)

Células/metabolismo , Espectrometría de Masas/métodos , Proteínas/análisis , Proteínas/química , Transducción de Señal , Animales , Femenino , Hormonas/análisis , Hormonas/química , Hormonas/metabolismo , Neuronas/química , Hipófisis/citología , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Ratas , Ratas Long-Evans