RESUMO
Myelin Basic Protein (MBP) is essential for both elaboration and maintenance of CNS myelin, and its reduced accumulation results in hypomyelination. How different Mbp mRNA levels affect myelin dimensions across the lifespan and how resident glial cells may respond to such changes are unknown. Here, to investigate these questions, we used enhancer-edited mouse lines that accumulate Mbp mRNA levels ranging from 8% to 160% of wild type. In young mice, reduced Mbp mRNA levels resulted in corresponding decreases in Mbp protein accumulation and myelin sheath thickness, confirming the previously demonstrated rate-limiting role of Mbp transcription in the control of initial myelin synthesis. However, despite maintaining lower line specific Mbp mRNA levels into old age, both MBP protein levels and myelin thickness improved or fully normalized at rates defined by the relative Mbp mRNA level. Sheath length, in contrast, was affected only when mRNA levels were very low, demonstrating that sheath thickness and length are not equally coupled to Mbp mRNA level. Striking abnormalities in sheath structure also emerged with reduced mRNA levels. Unexpectedly, an increase in the density of all glial cell types arose in response to reduced Mbp mRNA levels. This investigation extends understanding of the role MBP plays in myelin sheath elaboration, architecture, and plasticity across the mouse lifespan and illuminates a novel axis of glial cell crosstalk.
Assuntos
Proteína Básica da Mielina , Bainha de Mielina , Neuroglia , RNA Mensageiro , Animais , Proteína Básica da Mielina/metabolismo , Proteína Básica da Mielina/genética , Bainha de Mielina/metabolismo , Bainha de Mielina/genética , RNA Mensageiro/metabolismo , Neuroglia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MasculinoRESUMO
Genetic perturbances in translational regulation result in defects in cerebellar motor learning; however, little is known about the role of translational mechanisms in the regulation of cerebellar plasticity. We show that genetic removal of 4E-BP, a translational suppressor and target of mammalian target of rapamycin complex 1, results in a striking change in cerebellar synaptic plasticity. We find that cerebellar long-term depression (LTD) at parallel fiber-Purkinje cell synapses is converted to long-term potentiation in 4E-BP knockout mice. Biochemical and pharmacological experiments suggest that increased phosphatase activity largely accounts for the defects in LTD. Our results point to a model in which translational regulation through the action of 4E-BP plays a critical role in establishing the appropriate kinase/phosphatase balance required for normal synaptic plasticity in the cerebellum.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Potenciação de Longa Duração , Depressão Sináptica de Longo Prazo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Cerebelo/fisiologia , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Mamíferos , Camundongos , Plasticidade Neuronal/fisiologia , Monoéster Fosfórico Hidrolases , Células de Purkinje/fisiologia , Sinapses/fisiologiaRESUMO
Throughout the developing nervous system, considerable synaptic re-organization takes place as postsynaptic neurons extend dendrites and incoming axons refine their synapses, strengthening some and eliminating others. It is well accepted that these processes rely on synaptic activity; however, the mechanisms that lead to this developmental reorganization are not fully understood. Here, we explore the regulation of cap-dependent translation, a mechanism known to play a role in synaptic growth and plasticity. Using sympathetic ganglia in α3 nicotinic acetylcholine receptor (nAChR)-knockout (KO) mice, we establish that electrophysiologically silent synapses between preganglionic axons and postsynaptic sympathetic neurons do not refine, and the growth of dendrites and the targeting of synapses on postsynaptic neurons are impaired. Remarkably, genetically removing 4E-BP, a suppressor of cap-dependent translation, from these α3 nAChR-KO mice largely restores these features. We conclude that synaptic connections can re-organize and refine without postsynaptic activity during post-natal development when 4E-BP-regulated cap-dependent translation is enhanced.
Assuntos
Proteínas de Transporte/genética , Fosfoproteínas/genética , Sinapses/metabolismo , Potenciais Sinápticos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular , Fatores de Iniciação em Eucariotos , Gânglios Simpáticos/citologia , Gânglios Simpáticos/metabolismo , Gânglios Simpáticos/fisiologia , Camundongos , Receptores Nicotínicos/genética , Sinapses/fisiologiaRESUMO
Precise genome editing using CRISPR typically requires delivery of guide RNAs, Cas9 endonuclease, and DNA repair templates. Both microinjection and electroporation effectively deliver these components into mouse zygotes provided the DNA template is an oligonucleotide of only a few hundred base pairs. However, electroporation completely fails with longer double-stranded DNAs leaving microinjection as the only delivery option. Here, we overcome this limitation by first injecting all CRISPR components, including long plasmid-sized DNA templates, into the sub-zona pellucida space. There they are retained, supporting subsequent electroporation. We show that this simple and well-tolerated method achieves intracellular reagent concentrations sufficient to effect precise gene edits.
RESUMO
SUN proteins participate in diverse cellular activities, many of which are connected to the nuclear envelope. Recently, the family member SUN1 has been linked to novel biological activities. These include the regulation of nucleoli, intranuclear compartments that assemble ribosomal subunits. We show that SUN1 associates with nucleoli in several mammalian epithelial cell lines. This nucleolar localization is not shared by all cell types, as SUN1 concentrates at the nuclear envelope in ganglionic neurons and non-neuronal satellite cells. Database analyses and Western blotting emphasize the complexity of SUN1 protein profiles in different mammalian cells. We constructed a STRING network which identifies SUN1-related proteins as part of a larger network that includes several nucleolar proteins. Taken together, the current data highlight the diversity of SUN1 proteins and emphasize the possible links between SUN1 and nucleoli.
RESUMO
Short-term hyperglycemia suppresses superior cervical ganglia neurotransmission. If this ganglionic dysfunction also occurs in the islet sympathetic pathway, sympathetically mediated glucagon responses could be impaired. Our objectives were 1) to test for a suppressive effect of 7 days of streptozotocin (STZ) diabetes on celiac ganglia (CG) activation and on neurotransmitter and glucagon responses to preganglionic nerve stimulation, 2) to isolate the defect in the islet sympathetic pathway to the CG itself, and 3) to test for a protective effect of the WLD(S) mutation. We injected saline or nicotine in nondiabetic and STZ-diabetic rats and measured fos mRNA levels in whole CG. We electrically stimulated the preganglionic or postganglionic nerve trunk of the CG in nondiabetic and STZ-diabetic rats and measured portal venous norepinephrine and glucagon responses. We repeated the nicotine and preganglionic nerve stimulation studies in nondiabetic and STZ-diabetic WLD(S) rats. In STZ-diabetic rats, the CG fos response to nicotine was suppressed, and the norepinephrine and glucagon responses to preganglionic nerve stimulation were impaired. In contrast, the norepinephrine and glucagon responses to postganglionic nerve stimulation were normal. The CG fos response to nicotine, and the norepinephrine and glucagon responses to preganglionic nerve stimulation, were normal in STZ-diabetic WLD(S) rats. In conclusion, short-term hyperglycemia's suppressive effect on nicotinic acetylcholine receptors of the CG impairs sympathetically mediated glucagon responses. WLD(S) rats are protected from this dysfunction. The implication is that this CG dysfunction may contribute to the impaired glucagon response to insulin-induced hypoglycemia seen early in type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Regulação para Baixo , Gânglios Simpáticos/fisiopatologia , Glucagon/metabolismo , Hiperglicemia/etiologia , Ilhotas Pancreáticas/metabolismo , Transmissão Sináptica , Animais , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Regulação para Baixo/efeitos dos fármacos , Estimulação Elétrica , Gânglios Simpáticos/efeitos dos fármacos , Gânglios Simpáticos/metabolismo , Estimulantes Ganglionares/farmacologia , Glucagon/sangue , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/inervação , Masculino , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Agonistas Nicotínicos/farmacologia , Norepinefrina/sangue , Norepinefrina/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos Sprague-Dawley , Ratos Transgênicos , Ratos Wistar , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Degeneração Walleriana/complicaçõesRESUMO
Synapses in autonomic ganglia represent the final output of various CNS structures that regulate the function of the periphery. Normally, these excitatory cholinergic-nicotinic synapses produce large suprathreshold EPSPs on sympathetic and parasympathetic neurons to convey signals from the CNS. However, in certain disease states, synaptic transmission in autonomic ganglia is depressed and the periphery becomes deregulated. For example, previous work demonstrated that hyperglycemia depresses EPSPs on sympathetic neurons and disrupts sympathetic reflexes by causing an ROS-dependent inactivation of the postsynaptic nAChRs. What is not clear, however, is whether some autonomic neurons are more vulnerable to hyperglycemia than others. One possibility is that sympathetic neurons may be more prone than cholinergic parasympathetic neurons to hyperglycemia-induced elevations in cytosolic ROS because sympathetic neurons contain several pro-oxidant molecules involved in noradrenaline metabolism. To test this hypothesis, we recorded synaptic transmission from different mouse sympathetic and parasympathetic ganglia, as well as from the adrenal medulla. In addition, we used cellular imaging to measure hyperglycemia-induced changes in cytosolic ROS and whole-cell recordings to measure the use-dependent rundown of ACh-evoked currents. Our results demonstrate that hyperglycemia depresses synaptic transmission on sympathetic neurons and adrenal chromaffin cells and elevates cytosolic ROS. Conversely, hyperglycemia has little effect on synaptic transmission at synapses on parasympathetic neurons. We conclude that sympathetic neurons and adrenal chromaffin cells are more vulnerable to diabetes than parasympathetic neurons, a finding that may have implications for both long-term diabetic autonomic neuropathies and insulin-induced hypoglycemia, a serious complication of diabetes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Gânglios Parassimpáticos/metabolismo , Gânglios Simpáticos/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos , Técnicas de Patch-Clamp , Espécies Reativas de Oxigênio/metabolismoRESUMO
The pH milieu of the central and peripheral nervous systems is an important determinant of neuronal excitability, function, and survival. In mammals, neural acid-base homeostasis is coordinately regulated by ion transporters belonging to the Na(+)/H(+) exchanger (NHE) and bicarbonate transporter gene families. However, the relative contributions of individual isoforms within the respective families are not fully understood. This report focuses on the NHE family, specifically the plasma membrane-type NHE5 which is preferentially transcribed in brain, but the distribution of the native protein has not been extensively characterized. To this end, we generated a rabbit polyclonal antibody that specifically recognizes NHE5. In both central (cortex, hippocampus) and peripheral (superior cervical ganglia, SCG) nervous tissue of mice, NHE5 immunostaining was punctate and highly concentrated in the somas and to lesser amounts in the dendrites of neurons. Very little signal was detected in axons. Similarly, in primary cultures of differentiated SCG neurons, NHE5 localized predominantly to vesicles in the somatodendritic compartment, though some immunostaining was also evident in punctate vesicles along the axons. NHE5 was also detected predominantly in intracellular vesicles of cultured SCG glial cells. Dual immunolabeling of SCG neurons showed that NHE5 did not colocalize with markers for early endosomes (EEA1) or synaptic vesicles (synaptophysin), but did partially colocalize with the transferrin receptor, a marker of recycling endosomes. Collectively, these data suggest that NHE5 partitions into a unique vesicular pool in neurons that shares some characteristics of recycling endosomes where it may serve as an important regulated store of functional transporters required to maintain cytoplasmic pH homeostasis.
Assuntos
Axônios/metabolismo , Encéfalo/metabolismo , Endossomos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Encéfalo/citologia , Células Cultivadas , Endossomos/genética , Concentração de Íons de Hidrogênio , Camundongos , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Coelhos , Trocadores de Sódio-Hidrogênio/genética , Vesículas Sinápticas/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
An intriguing feature of several nicotinic acetylcholine receptors (nAChRs) on neurons is that their subunits contain a highly conserved cysteine residue located near the intracellular mouth of the receptor pore. The work summarized in this review indicates that α3ß4-containing and α4ß2-containing neuronal nAChRs, and possibly other subtypes, are inactivated by elevations in intracellular reactive oxygen species (ROS). This review discusses a model for the molecular mechanisms that underlie this inactivation. In addition, we explore the implications of this mechanism in the context of complications that arise from diabetes. We review the evidence that diabetes elevates cytosolic ROS in sympathetic neurons and inactivates postsynaptic α3ß4-containing nAChRs shortly after the onset of diabetes, leading to a depression of synaptic transmission in sympathetic ganglia, an impairment of sympathetic reflexes. These effects of ROS on nAChR function are due to the highly conserved Cys residues in the receptors: replacing the cysteine residues in α3 allow ganglionic transmission and sympathetic reflexes to function normally in diabetes. This example from diabetes suggests that other diseases involving oxidative stress, such as Parkinson's disease, could lead to the inactivation of nAChRs on neurons and disrupt cholinergic nicotinic signalling.
Assuntos
Cisteína/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Nicotínicos/metabolismo , Diabetes Mellitus/metabolismo , Gânglios Simpáticos/metabolismo , Humanos , Transmissão SinápticaRESUMO
PURPOSE: As alternatives to autograft become more conventional, clinical outcomes data on their effectiveness in restoring meaningful function is essential. In this study we report on the outcomes from a multicenter study on processed nerve allografts (Avance® Nerve Graft, AxoGen, Inc). PATIENTS AND METHODS: Twelve sites with 25 surgeons contributed data from 132 individual nerve injuries. Data was analyzed to determine the safety and efficacy of the nerve allograft. Sufficient data for efficacy analysis were reported in 76 injuries (49 sensory, 18 mixed, and 9 motor nerves). The mean age was 41 ± 17 (18-86) years. The mean graft length was 22 ± 11 (5-50) mm. Subgroup analysis was performed to determine the relationship to factors known to influence outcomes of nerve repair such as nerve type, gap length, patient age, time to repair, age of injury, and mechanism of injury. RESULTS: Meaningful recovery was reported in 87% of the repairs reporting quantitative data. Subgroup analysis demonstrated consistency, showing no significant differences with regard to recovery outcomes between the groups (P > 0.05 Fisher's Exact Test). No graft related adverse experiences were reported and a 5% revision rate was observed. CONCLUSION: Processed nerve allografts performed well and were found to be safe and effective in sensory, mixed and motor nerve defects between 5 and 50 mm. The outcomes for safety and meaningful recovery observed in this study compare favorably to those reported in the literature for nerve autograft and are higher than those reported for nerve conduits.
Assuntos
Nervos Periféricos/cirurgia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Eletromiografia , Feminino , Humanos , Masculino , Microcirurgia/métodos , Pessoa de Meia-Idade , Procedimentos Neurocirúrgicos/métodos , Nervos Periféricos/transplante , Procedimentos de Cirurgia Plástica , Esterilização , Transplante Homólogo , Adulto JovemRESUMO
Most people with diabetes develop severe complications of the autonomic nervous system; yet, the underlying causes of many diabetic-induced dysautonomias are poorly understood. Here we explore the idea that these dysautonomias results, in part, from a defect in synaptic transmission. To test this idea, we investigated cultured sympathetic neurons and show that hyperglycemia inactivates nAChRs through a mechanism involving an elevation in reactive oxygen species and an interaction with highly conserved cysteine residues located near the intracellular mouth of the nAChR channel. Consistent with this, we show that diabetic mice have depressed ganglionic transmission and reduced sympathetic reflexes, whereas diabetic mice expressing mutant postsynaptic nAChRs that lack the conserved cysteine residues on the alpha3 subunit have normal synaptic transmission in sympathetic ganglia and normal sympathetic reflexes. Our work suggests a new model for diabetic-induced dysautonomias and identifies ganglionic nAChRs as targets of hyperglycemia-induced downstream signals.
Assuntos
Cisteína/metabolismo , Diabetes Mellitus Experimental/patologia , Receptores Nicotínicos/metabolismo , Células Receptoras Sensoriais/fisiologia , Gânglio Cervical Superior/patologia , Transmissão Sináptica/fisiologia , Acetilcolina/farmacologia , Adenoviridae , Fatores Etários , Aldeídos/metabolismo , Animais , Animais Recém-Nascidos , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/fisiologia , Células Cultivadas , Cisteína/genética , Diabetes Mellitus Experimental/induzido quimicamente , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Glucose/farmacologia , Guanidinas/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Leptina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Técnicas de Patch-Clamp , Espécies Reativas de Oxigênio/metabolismo , Receptores para Leptina/genética , Receptores Nicotínicos/deficiência , Transmissão Sináptica/efeitos dos fármacosRESUMO
The purpose of the study was to evaluate safety and feasibility of negative pressure wound therapy (NPWT) during aeromedical evacuation from a combat zone to a regional treatment center. A retrospective review of patients who received NPWT during aeromedical evacuation from Iraq or Afghanistan to Landstuhl Regional Medical Center (LRMC) was performed. Data were collected describing mechanism of injury; anatomic site of NPWT application; number of sites per patient; date and time of NPWT application; date, time, and wound condition on arrival and inspection at LRMC; and complications encountered during aeromedical evacuation. Broad definitions of complications were employed. Any reported malfunction of NPWT devices or need to reinforce NPWT dressings was abstracted. Presence of tissue under the dressing requiring debridement was defined as a minor complication. Major complications were defined as wound sepsis with systemic manifestations. A total of 218 patients who had received NPWT for 298 wounds (1.37 per patient) during aeromedical evacuation were identified. Most wounds were due to high-energy blast or ballistic mechanisms. Average time from NPWT application to removal was 53 hours (range, 18-133 +/- 22 hours). Complications occurred at 14% of NPWT sites and in 19% of patients receiving NPWT. Most recorded complications were minor (95%). Two patients who arrived at LRMC with fever and evidence of wound sepsis improved rapidly after additional operative debridement. In no case was failure of the NPWT device in flight specifically implicated in the genesis of a recorded complication. In-flight device problems were identified in seven cases. Four of these could not be repaired in flight and were clamped. Complications were not increased in this cohort. Use of NPWT during aeromedical evacuation appears safe and feasible in a large cohort of patients with high-energy injuries. Complications were consistent with severity of injury and not related to failure of NPWT.
Assuntos
Traumatismos por Explosões/terapia , Medicina Militar/métodos , Tratamento de Ferimentos com Pressão Negativa , Humanos , Guerra do Iraque 2003-2011 , Transferência de Pacientes , Estudos RetrospectivosRESUMO
A well-accepted view of developing circuits is that synapses must be active to mature and persist, whereas inactive synapses remain immature and are eventually eliminated. We question this long-standing view by investigating nonfunctional cholinergic nicotinic synapses in the superior cervical ganglia (SCG) of mice with a disruption in the alpha3 nicotinic receptor (nAChR) subunit gene, a gene essential for fast synaptic transmission in sympathetic ganglia. Using imaging and electrophysiology, we show that synapses persist for at least 2-3 months without postsynaptic activity; however, the presynaptic terminals lack high-affinity choline transporters (CHTs), and as a result, they are quickly depleted of transmitter. Moreover, we demonstrate with rescue experiments that CHT is induced by signals downstream of postsynaptic activity, converting immature terminals to mature terminals capable of sustaining transmitter release in response to high-frequency or continuous firing. Importantly, postsynaptic neurons must be continually active to maintain CHT in presynaptic terminals.
Assuntos
Acetilcolina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Nicotínicos/genética , Gânglio Cervical Superior/metabolismo , Transmissão Sináptica/genética , Potenciais de Ação/genética , Animais , Diferenciação Celular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terminações Pré-Sinápticas/ultraestrutura , Transdução de Sinais/genética , Gânglio Cervical Superior/citologia , Membranas Sinápticas/genética , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestruturaRESUMO
We report here the construction of a novel knock-in mouse expressing chimeric alpha3 nicotinic acetylcholine receptor (nAChR) subunits with pharmacological sensitivity to alpha-bungarotoxin (alphaBTX). Sensitivity was generated by substituting five amino acids in the loop C (beta9-beta10) region of the mouse alpha3 subunit with the corresponding residues from the alpha1 subunit of the muscle type receptor from Torpedo californica. To demonstrate the utility of the underlying concept, expressed alpha3[5] subunits were characterized in the superior cervical ganglia (SCG) of homozygous knock-in mice, where the synaptic architecture of postsynaptic alpha3-containing nAChR clusters could now, for the first time, be directly visualized and interrogated by live-staining with rhodamine-conjugated alphaBTX. Consistent with the postsynaptic localization of ganglionic nAChRs, the alphaBTX-labeled puncta colocalized with a marker for synaptic varicosities. Following in vivo deafferentation, these puncta persisted but with significant changes in intensity and distribution that varied with the length of the recovery period. Compound action potentials and excitatory postsynaptic potentials recorded from SCG of mice homozygous for alpha3[5] were abolished by 100 nmalphaBTX, even in an alpha7 null background, demonstrating that synaptic throughput in the SCG is completely dependent on the alpha3-subunit. In addition, we observed that the genetic background of various inbred and outbred mouse lines greatly affects the functional expression of alpha3[5]-nAChRs, suggesting a powerful new approach for exploring the molecular mechanisms underlying receptor assembly and trafficking. As alphaBTX-sensitive sequences can be readily introduced into other nicotinic receptor subunits normally insensitive to alphaBTX, the findings described here should be applicable to many other receptors.
Assuntos
Bungarotoxinas/farmacologia , Camundongos Knockout , Neurônios/efeitos dos fármacos , Receptores Nicotínicos/deficiência , Gânglio Cervical Superior/citologia , Acetilcolina/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Denervação Autônoma/métodos , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Bungarotoxinas/metabolismo , Células Cultivadas , Colinérgicos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Neurofilamentos/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Fenótipo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Fatores de Tempo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoAssuntos
Amputação Traumática/cirurgia , Anticoagulantes/uso terapêutico , Reimplante/efeitos adversos , Tromboembolia/prevenção & controle , Feminino , Seguimentos , Humanos , Masculino , Complicações Pós-Operatórias/prevenção & controle , Reimplante/métodos , Medição de Risco , Tromboembolia/etiologia , Resultado do TratamentoRESUMO
Neuronal nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels implicated in a variety of cognitive, motor, and sensory behaviours, are targeted to compartments rich in mitochondria, particularly postsynaptic domains and presynaptic terminals, exposing these receptors to reactive oxygen species (ROS) generated by oxidative phosphorylation. In addition, these receptors can become exposed to ROS during the progression of certain neurodegenerative diseases. Because ROS are known to modify several membrane proteins, including some types of ion channels, it raises the question of whether elevations in cytosolic ROS alter the function of nAChRs. To address this, we elevated ROS in cultured sympathetic neurons, directly by perfusing neurons intracellularly with ROS, indirectly by blocking the mitochondrial electron transport chain, or noninvasively by transient NGF removal; we then simultaneously measured changes in cytosolic ROS levels and whole-cell ACh-evoked currents. In addition, we elevated cytosolic ROS in postganglionic neurons in intact ganglia and measured changes in nerve-evoked EPSPs. Our experiments indicate that mild elevations in cytosolic ROS, including that produced by transient interruption of NGF signaling, induce a use-dependent, long-lasting rundown of ACh-evoked currents on cultured sympathetic neurons and a long-lasting depression of fast nerve-evoked EPSPs. We show that these effects of cytosolic ROS are specific to nAChRs on neurons and do not cause rundown of ACh-evoked currents on muscle. Our results demonstrate that elevations in cytosolic ROS inactivate neuronal nAChRs in a use-dependent manner and suggest that mild oxidative stress impairs mechanisms mediated by cholinergic nicotinic signaling at neuronal-neuronal synapses.
Assuntos
Mitocôndrias/metabolismo , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Nicotínicos/metabolismo , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Citosol/metabolismo , Potenciais Evocados , Camundongos , Camundongos Endogâmicos C57BL , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/metabolismoRESUMO
Eight matched pairs of cadaveric radii were osteotomized by removing a 4-mm dorsal wedge of bone at the level of the sigmoid notch designed to simulate dorsal comminution. They were then fixed with either a volar locking-screw plate or fragment-specific fixation. All constructs underwent biomechanical testing in a custom-designed, custom-fabricated 4-point bending device. No statistically significant difference in stiffness was noted between the groups. Linear displacement and angulation at the osteotomy site were significantly less in the group with fragment-specific fixation at loads expected to be encountered during postoperative rehabilitation. Angulation at the osteotomy site was significantly less in the locking-screw plate group at higher loads.
Assuntos
Placas Ósseas , Parafusos Ósseos , Fixação Interna de Fraturas , Fixadores Internos , Fraturas do Rádio/cirurgia , Fenômenos Biomecânicos , Humanos , Técnicas In Vitro , Rádio (Anatomia)/cirurgia , Fraturas do Rádio/fisiopatologia , Articulação do Punho/fisiopatologiaRESUMO
Neuronal synapse formation is a multistep process regulated by several pre- and postsynaptic adhesion and signaling proteins. Recently, we found that agrin acts as one such synaptogenic factor at neuronal synapses in the PNS by demonstrating that structural synapse formation is impaired in the superior cervical ganglia (SCG) of z+ agrin-deficient mice and in SCG cultures derived from those animals. Here, we tested whether synaptic function is defective in agrin-null (AGD-/-) ganglia and began to define agrin's mechanism of action. Our electrophysiological recordings of compound action potentials showed that presynaptic stimulation evoked action potentials in approximately 40% of AGD-/- ganglionic neurons compared to 90% of wild-type neurons; moreover, transmission could not be potentiated as in wild-type or z+ agrin-deficient ganglia. Intracellular recordings also showed that nerve-evoked excitatory postsynaptic potentials in AGD-/- neurons were only 1/3 the size of those in wild-type neurons and mostly subthreshold. Consistent with these defects in transmission, we found an approximately 40-50% decrease in synapse number in AGD-/- ganglia and cultures, and decreased levels of differentiation at the residual synapses in culture. Furthermore, surface levels of acetylcholine receptors (AChRs) were equivalent in cultured AGD-/- and wild-type neurons, and depolarization reduced the synaptic localization of AChRs in AGD-/- but not wild-type neurons. These findings provide the first direct demonstration that agrin is required for proper structural and functional development of an interneuronal synapse in vivo. Moreover, they suggest a novel role for agrin, in stabilizing the postsynaptic density of nAChR at nascent neuronal synapses.
Assuntos
Agrina/fisiologia , Sistema Nervoso Autônomo/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Agrina/genética , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Espaço Extracelular/fisiologia , Gânglios Autônomos/fisiologia , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Receptores Colinérgicos/genética , Receptores Colinérgicos/fisiologia , Gânglio Cervical Superior/fisiologia , Transmissão Sináptica/genéticaRESUMO
In vertebrates, synaptic activity exerts an important influence on the formation of neural circuits, yet our understanding of its role in directing presynaptic and postsynaptic differentiation during synaptogenesis is incomplete. This study investigates how activity influences synaptic differentiation as synapses mature during early postnatal life. Specifically, we ask what happens to presynaptic terminals when synapses develop without functional postsynaptic receptors and without fast synaptic transmission. To address this issue, we investigated cholinergic nicotinic synapses in sympathetic ganglia of mice with a null mutation for the alpha3 nicotinic ACh receptor gene. Disrupting the alpha3 gene completely eliminates fast excitatory synaptic potentials on postganglionic sympathetic neurons, establishing a crucial role for alpha3-containing postsynaptic receptors in synaptic transmission. Interestingly, the preganglionic nerve terminals form morphologically normal synapses with sympathetic neurons, and these synapses persist without activity in postnatal animals. Surprisingly, when stimulating the preganglionic nerve at physiological rates, we discovered a significant decrease in ACh output from the presynaptic terminals in these alpha3(-/-) sympathetic ganglia. We show that this decrease in ACh output from the presynaptic terminals results, in part, from a lack of functional high-affinity choline transporters. We conclude the following: (1) fast synaptic transmission in mammalian SCG requires alpha3 expression; (2) in the absence of activity, the preganglionic nerve forms synapses that appear morphologically normal and persist for several weeks; and (3) to sustain transmitter release, developing presynaptic terminals require an activity-dependent retrograde signal.
Assuntos
Axônios/fisiologia , Gânglios Simpáticos/fisiologia , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/fisiologia , Animais , Modelos Animais de Doenças , Genótipo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Receptores Nicotínicos/genética , Degeneração RetrógradaRESUMO
The developing nervous system adapts to a wide array of stimuli, in part, by evoking activity-dependent mechanisms that signal to the nucleus and induce long-term modifications in neuronal function. It is well established that one such stimulus is strong synaptic activity. Our interest, however, is whether weak activity generated at developing synapses also signals to the nucleus and if so, can these signals be modulated by extrinsic factors. Using cultured hippocampal neurons and a highly sensitive readout of CRE-mediated gene expression, we demonstrate that weak synaptic transmission, including non-evoked, spontaneous transmitter release, induces ongoing gene expression. These weak synaptic stimuli, acting through NMDA receptors, signal to the nucleus through a MAPK pathway, without a significant contribution of L-type Ca2+ channels. In addition, we show that BDNF, a molecule that has clear effects on synaptic plasticity, enhances this CRE-dependent gene expression by acting upstream of NMDA receptors. On the other hand, low levels of nicotine, which also effects synaptic plasticity, suppress ongoing CRE-mediated gene expression indirectly by acting on GABAergic neurons; this indirect action on gene expression suggests an alternative mechanism for how nicotine produces long-lasting changes.