RESUMO
Early postnatal nicotine exposure, a rodent model of smoking during pregnancy, affects hippocampal synaptic plasticity and memory. Here, we investigated the role of α2 nAChR-expressing OLM (α2-OLM) cells in LTP in unexposed and postnatal nicotine-exposed mice. We found that reduced α2 nAChR-dependent activation of OLM cells in α2 heterozygous knockout mice prevented LTP, whereas enhanced α2 nAChR-dependent activation of OLM cells in heterozygous knockin mice expressing hypersensitive α2 nAChRs facilitated LTP. Both optogenetic and chemogenetic activation of α2-OLM cells facilitated LTP as nicotine did. However, in postnatal nicotine-exposed mice, expressing chemogenetic hM3Dq receptors in α2-OLM cells, LTP was facilitated and both nicotinic and chemogenetic activation of α2-OLM cells prevented rather than facilitated LTP. These results demonstrate a critical role of α2-OLM cell activation in LTP as well as altered α2-OLM cell function in postnatal nicotine-exposed mice. To determine whether nicotine-mediated α2 nAChR activation in developing brains causes facilitated LTP and altered nicotinic modulation of LTP in adolescence, we used homozygous knockin mice expressing hypersensitive α2 nAChRs as a way to selectively activate α2-OLM cells. In the knockin mice, postnatal exposure to a low dose of nicotine, which had no effect on LTP in wild-type mice, is sufficient to cause facilitated LTP and altered nicotinic modulation of LTP as found in wild-type mice exposed to a higher dose of nicotine. Thus, the nicotine-mediated activation of α2 nAChRs on OLM cells in developing brains disrupts the α2-OLM cell-mediated control of LTP in adolescence that might be linked to impaired memory.
Assuntos
Nicotina , Receptores Nicotínicos , Animais , Feminino , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Gravidez , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismoRESUMO
In rodent models of smoking during pregnancy, early postnatal nicotine exposure results in impaired hippocampus-dependent memory, but the underlying mechanism remains elusive. Given that hippocampal cholinergic systems modulate memory and rapid development of hippocampal cholinergic systems occurs during nicotine exposure, here we investigated its impacts on cholinergic function. Both nicotinic and muscarinic activation produce transient or long-lasting depression of excitatory synaptic transmission in the hippocampal CA1 region. We found that postnatal nicotine exposure impairs both the induction and nicotinic modulation of NMDAR-dependent long-term depression (LTD). Activation of muscarinic receptors decreases excitatory synaptic transmission and CA1 network activity in both wild-type and α2 knockout mice. These muscarinic effects are still observed in nicotine-exposed mice. M1 muscarinic receptor activity is required for mGluR-dependent LTD. Early postnatal nicotine exposure has no effect on mGluR-dependent LTD induction, suggesting that it has no effect on the function of m1 muscarinic receptors involved in this form of LTD. Our results demonstrate that early postnatal nicotine exposure has more pronounced effects on nicotinic function than muscarinic function in the hippocampal CA1 region. Thus, impaired hippocampus-dependent memory may arise from the developmental disruption of nicotinic cholinergic systems in the hippocampal CA1 region.
Assuntos
Região CA1 Hipocampal/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptor Muscarínico M1/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Região CA1 Hipocampal/crescimento & desenvolvimento , Região CA1 Hipocampal/metabolismo , Fumar Cigarros , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Lactação , Depressão Sináptica de Longo Prazo/fisiologia , Masculino , Exposição Materna , Memória/efeitos dos fármacos , Memória/fisiologia , Camundongos , Camundongos Knockout , Receptor Muscarínico M1/metabolismo , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Nicotínicos/metabolismoRESUMO
Children of mothers who smoked during pregnancy are at significantly greater risk for cognitive impairments including memory deficits, but the mechanisms underlying this effect remain to be understood. In rodent models of smoking during pregnancy, early postnatal nicotine exposure results in impaired long-term hippocampus-dependent memory, functional loss of α2-containing nicotinic acetylcholine receptors (α2∗ nAChRs) in oriens-lacunosum moleculare (OLM) cells, increased CA1 network excitation, and unexpected facilitation of long-term potentiation (LTP) at Schaffer collateral-CA1 synapses. Here we demonstrate that α2 knockout mice show the same pattern of memory impairment as previously observed in wild-type mice exposed to early postnatal nicotine. However, α2 knockout mice and α2 knockout mice exposed to early postnatal nicotine did not share all of the anomalies in hippocampal function observed in wild-type mice treated with nicotine during development. Unlike nicotine-treated wild-type mice, α2 knockout mice and nicotine-exposed α2 knockout mice did not demonstrate increased CA1 network excitation following Schaffer collateral stimulation and facilitated LTP, indicating that the effects are likely adaptive changes caused by activation of α2∗ nAChRs during nicotine exposure and are unlikely related to the associated memory impairment. Thus, the functional loss of α2∗ nAChRs in OLM cells likely plays a critical role in mediating this developmental-nicotine-induced hippocampal memory deficit.
Assuntos
Região CA1 Hipocampal/fisiologia , Interneurônios/fisiologia , Potenciação de Longa Duração/fisiologia , Transtornos da Memória , Nicotina/efeitos adversos , Agonistas Nicotínicos/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal , Receptores Nicotínicos/fisiologia , Reconhecimento Psicológico/fisiologia , Memória Espacial/fisiologia , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/metabolismo , Modelos Animais de Doenças , Feminino , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Memória Espacial/efeitos dos fármacosRESUMO
Fetal nicotine exposure from smoking during pregnancy causes long-lasting cognitive impairments in offspring, yet little is known about the mechanisms that underlie this effect. Here we demonstrate that early postnatal exposure of mouse pups to nicotine via maternal milk impairs long-term, but not short-term, hippocampus-dependent memory during adolescence. At the Schaffer collateral (SC) pathway, the most widely studied synapses for a cellular correlate of hippocampus-dependent memory, the induction of N-methyl-D-aspartate receptor-dependent transient long-term potentiation (LTP) and protein synthesis-dependent long-lasting LTP are not diminished by nicotine exposure, but rather unexpectedly the threshold for LTP induction becomes lower after nicotine treatment. Using voltage sensitive dye to visualize hippocampal activity, we found that early postnatal nicotine exposure also results in enhanced CA1 depolarization and hyperpolarization after SC stimulation. Furthermore, we show that postnatal nicotine exposure induces pervasive changes to the nicotinic modulation of CA1 activity: activation of nicotinic receptors no longer increases CA1 network depolarization, acute nicotine inhibits rather than facilitates the induction of LTP at the SC pathway by recruiting an additional nicotinic receptor subtype, and acute nicotine no longer blocks LTP induction at the temporoammonic pathway. These findings reflect the pervasive impact of nicotine exposure during hippocampal development, and demonstrate an association of hippocampal memory impairments with altered nicotinic cholinergic modulation of LTP, but not impaired LTP. The implication of our results is that nicotinic cholinergic-dependent plasticity is required for long-term memory formation and that postnatal nicotine exposure disrupts this form of plasticity.
Assuntos
Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Neurônios/efeitos dos fármacos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Fatores Etários , Animais , Ansiedade/induzido quimicamente , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas Nicotínicos/farmacologia , Reconhecimento Psicológico/efeitos dos fármacos , Memória Espacial/efeitos dos fármacosRESUMO
Brain damage causes multiple changes in synaptic function and intrinsic properties of surviving neurons, leading to the development of chronic epilepsy. In the widely used pilocarpine-status epilepticus (SE) rat model of temporal lobe epilepsy (TLE), a major alteration is the marked increase in the fraction of intrinsically bursting CA1 pyramidal cells. Here we have differentiated between two types of bursting phenotypes: 1) bursting in response to threshold-straddling excitatory current pulses (low-threshold bursting) and 2) bursting only in response to suprathreshold stimuli (high-threshold bursting). Low-threshold bursting prevailed in 46.5% of SE-experienced neurons sampled 1-4 wk after pilocarpine-SE, but was rarely seen in control neurons (1.9%). As previously shown, it appeared to be driven predominantly by a T-type Ca(2+) current (I(CaT)) in the apical dendrites. After blocking low-threshold bursting with Ni(2+), the same neurons still manifested a high-threshold bursting phenotype. Another 40.1% of SE-experienced neurons displayed only a high-threshold bursting phenotype and the remaining 13.4% of these neurons were nonbursters. Altogether, high-threshold bursting prevailed in 86.6% of SE-experienced neurons, but only in 33.0% of control neurons. Several lines of evidence indicated that high-threshold bursting is driven by persistent Na(+) current (I(NaP)) at or near the soma. Congruently, I(NaP) was 1.5-fold larger in SE-experienced versus control neurons. We conclude that an increase in I(NaP), conjointly with an increase in I(CaT), strongly contributes to the predominance of bursting phenotypes in CA1 pyramidal cells early after pilocarpine-SE and thus likely plays a role in the development of a chronic epileptic condition in this TLE model.
Assuntos
Região CA1 Hipocampal/fisiopatologia , Neurônios/fisiologia , Canais de Sódio/fisiologia , Estado Epiléptico/fisiopatologia , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Canais de Cálcio Tipo T/fisiologia , Masculino , Modelos Animais , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Pilocarpina/efeitos adversos , Ratos , Ratos Endogâmicos , Ratos Wistar , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/efeitos dos fármacos , Estado Epiléptico/induzido quimicamente , Tetrodotoxina/farmacologia , Fatores de TempoRESUMO
In many principal brain neurons, the fast, all-or-none Na+ spike initiated at the proximal axon is followed by a slow, graded after depolarization (ADP). The spike ADP is critically important in determining the firing mode of many neurons; large ADPs cause neurons to fire bursts of spikes rather than solitary spikes. Nonetheless, not much is known about how and where spike ADPs are initiated. We addressed these questions in adult CA1 pyramidal cells, which manifest conspicuous somatic spike ADPs and an associated propensity for bursting, using sharp and patch microelectrode recordings in acutely isolated hippocampal slices and single neurons. Voltage-clamp commands mimicking spike waveforms evoked transient Na+ spike currents that declined quickly after the spike but were followed by substantial sustained Na+ spike after currents. Drugs that blocked the persistent Na+ current (INaP), markedly suppressed the sustained Na+ spike after currents, as well as spike ADPs and associated bursting. Ca2+ spike after currents were much smaller, and reducing them had no noticeable effect on the spike ADPs. Truncating the apical dendrites affected neither spike ADPs nor the firing modes of these neurons. Application of INaP blockers to truncated neurons, or their focal application to the somatic region of intact neurons, suppressed spike ADPs and associated bursting, whereas their focal application to distal dendrites did not. We conclude that the somatic spike ADPs are generated predominantly by persistent Na+ channels located at or near the soma. Through this action, proximal INaP critically determines the firing mode and spike output of adult CA1 pyramidal cells.
Assuntos
Potenciais de Ação/fisiologia , Células Piramidais/fisiologia , Canais de Sódio/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Masculino , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Wistar , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
Maternal cigarette smoking during pregnancy and maternal nicotine exposure in animal models are associated with cognitive impairments in offspring. However, the underlying mechanism remains unknown. Oriens-lacunosum moleculare (OLM) cells expressing α2* nicotinic acetylcholine receptors (nAChRs) are an important component of hippocampal circuitry, gating information flow and long-term potentiation (LTP) in the CA1 region. Here we investigated whether early postnatal nicotine exposure alters the normal role of α2*-nAChR-expressing OLM cells during adolescence in rats. We found that early postnatal nicotine exposure significantly decreased not only the number of α2-mRNA-expressing interneurons in the stratum oriens/alveus, but also α2*-nAChR-mediated responses in OLM cells. These effects of nicotine were prevented by co-administration with the nonselective nAChR antagonist mecamylamine, suggesting that nicotine-induced activation, but not desensitization, of nAChRs mediates the effects. α2*-nAChR-mediated depolarization of OLM cells normally triggers action potentials, causing an increase in spontaneous inhibitory postsynaptic currents in synaptically connected pyramidal cells. However, these α2*-nAChR-mediated effects were profoundly reduced after early postnatal nicotine exposure, suggesting altered control of CA1 circuits by α2*-nAChR-expressing OLM cells. Furthermore, these effects were associated with altered excitatory neural activity and LTP as well as the loss of normal α2*-nAChR-mediated control of excitatory neural activity and LTP. These findings suggest the altered function of α2*-nAChR-expressing OLM cells as an important target of further study for identifying the mechanisms underlying the cognitive impairment induced by maternal smoking during pregnancy.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Bicuculina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Antagonistas de Receptores de GABA-A/farmacologia , Hipocampo/citologia , Técnicas In Vitro , Masculino , Antagonistas Nicotínicos/farmacologia , Técnicas de Patch-Clamp , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Valina/análogos & derivados , Valina/farmacologiaRESUMO
The mushroom bodies, bilaterally symmetric regions in the insect brain, play a critical role in olfactory associative learning. Genetic studies in Drosophila suggest that plasticity underlying acquisition and storage of memory occurs at synapses on the dendrites of mushroom body Kenyon cells (Dubnau et al., 2001). Additional exploration of the mechanisms governing synaptic plasticity contributing to these aspects of olfactory associative learning requires identification of the receptors that mediate fast synaptic transmission in Kenyon cells. To this end, we developed a culture system that supports the formation of excitatory and inhibitory synaptic connections between neurons harvested from the central brain region of late-stage Drosophila pupae. Mushroom body Kenyon cells are identified as small-diameter, green fluorescent protein-positive (GFP+) neurons in cultures from OK107-GAL4;UAS-GFP pupae. In GFP+ Kenyon cells, fast EPSCs are mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors (nAChRs). The miniature EPSCs have rapid rise and decay kinetics and a broad, positively skewed amplitude distribution. Fast IPSCs are mediated by picrotoxin-sensitive chloride conducting GABA receptors. The miniature IPSCs also have a rapid rate of rise and decay and a broad amplitude distribution. The vast majority of spontaneous synaptic currents in the cultured Kenyon cells are mediated byalpha-bungarotoxin-sensitive nAChRs or picrotoxin-sensitive GABA receptors. Therefore, these receptors are also likely to mediate synaptic transmission in Kenyon cells in vivo and to contribute to plasticity during olfactory associative learning.
Assuntos
Drosophila/fisiologia , Corpos Pedunculados/fisiologia , Neurônios/fisiologia , Receptores de GABA/metabolismo , Receptores Nicotínicos/metabolismo , Transmissão Sináptica/fisiologia , Animais , Bungarotoxinas/farmacologia , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores/fisiologia , Antagonistas GABAérgicos/farmacologia , Proteínas de Fluorescência Verde , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Corpos Pedunculados/citologia , Neurônios/classificação , Neurônios/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Técnicas de Patch-Clamp , Picrotoxina/farmacologia , Pupa , Receptores de GABA/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , TransgenesRESUMO
GABAergic inhibition in Drosophila, as in other insects and mammals, is important for regulation of activity in the CNS. However, the functional properties of synaptic GABA receptors in Drosophila have not been described. Here, we report that spontaneous GABAergic postsynaptic currents (sPSCs) in cultured embryonic Drosophila neurons are mediated by picrotoxin-sensitive chloride-conducting receptors. A rapid increase in spontaneous firing in response to bath application of picrotoxin demonstrates that these GABA receptors mediate inhibition in the neuronal networks formed in culture. Many of the spontaneous GABAergic synaptic currents are sodium action potential independent [miniature IPSCs (mIPSCs)] but are regulated by external calcium levels. The large variation in mIPSC frequency, amplitude, and kinetics properties between neurons suggests heterogeneity in GABA receptor number, location, and/or subtype. A decrease in the mean mIPSC decay time constant between 2 and 5 d, in the absence of a correlated change in rise time, demonstrates that the functional properties of the synaptic GABA receptors are regulated during maturation in vitro. Finally, neurons from the GABA receptor subunit mutant Rdl exhibit reduced sensitivity to picrotoxin blockade of the mIPSCs and resistance to picrotoxin-induced increases in spontaneous firing frequency. This demonstrates that Rdl containing GABA receptors play a direct role in mediating synaptic inhibition in Drosophila neural circuits formed in culture.
Assuntos
Proteínas de Drosophila , Inibição Neural/fisiologia , Neurônios/fisiologia , Receptores de GABA-A/metabolismo , Receptores de GABA/metabolismo , Transmissão Sináptica/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Separação Celular , Células Cultivadas , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Drosophila , Embrião não Mamífero/inervação , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Antagonistas GABAérgicos/farmacologia , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Picrotoxina/farmacologia , Subunidades Proteicas/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Fatores de Tempo , Ácido gama-Aminobutírico/metabolismoRESUMO
A single episode of status epilepticus (SE) causes numerous structural and functional changes in the brain that can lead to the development of a chronic epileptic condition. Most studies of this plasticity have focused on changes in excitatory and inhibitory synaptic properties. However, the intrinsic firing properties that shape the output of the neuron to a given synaptic input may also be persistently affected by SE. Thus, 54% of CA1 pyramidal cells, which normally fire in a regular mode, are persistently converted to a bursting mode after an episode of SE induced by the convulsant pilocarpine. In this model, intrinsic bursts evoked by threshold-straddling depolarizations, and their underlying spike afterdepolarizations (ADPs), were resistant to antagonists of N-, P/Q-, or L-type Ca2+ channels but were readily suppressed by low (30-100 microm) concentrations of Ni2+ known to block T- and R-type Ca2+ channels. The density of T-type Ca2+ currents, but not of other pharmacologically isolated Ca2+ current types, was upregulated in CA1 pyramidal neurons after SE. The augmented T-type currents were sensitive to Ni2+ in the same concentration range that blocked the novel intrinsic bursting in these neurons (IC50 = 27 microm). These data suggest that SE may persistently convert regular firing cells to intrinsic bursters by selectively increasing the density of a Ni2+-sensitive T-type Ca2+ current. This nonsynaptic plasticity considerably amplifies the output of CA1 pyramidal neurons to synaptic inputs and most probably contributes to the development and expression of an epileptic condition after SE.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Hipocampo/fisiopatologia , Neurônios/metabolismo , Estado Epiléptico/fisiopatologia , Regulação para Cima , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Separação Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Estimulação Elétrica , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Níquel/farmacologia , Técnicas de Patch-Clamp , Pilocarpina , Ratos , Ratos Endogâmicos , Ratos Wistar , Estado Epiléptico/induzido quimicamente , Transmissão Sináptica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Mitochondrial dysfunction has recently been implicated as an underlying factor to several common neurodegenerative diseases, including Parkinson's disease, Alzheimer's and amyotrophic lateral sclerosis (ALS). Valosin containing protein (VCP)-associated multisystem proteinopathy is a new hereditary disorder associated with inclusion body myopathy, Paget disease of bone (PDB), frontotemporal dementia (FTD) and ALS. VCP has been implicated in several transduction pathways including autophagy, apoptosis and the PINK1/Parkin cascade of mitophagy. In this report, we characterized VCP patient and mouse fibroblasts/myoblasts to examine their mitochondrial dynamics and bioenergetics. Using the Seahorse XF-24 technology, we discovered decreased spare respiratory capacity (measurement of extra ATP that can be produced by oxidative phosphorylation in stressful conditions) and increased ECAR levels (measurement of glycolysis), and proton leak in VCP human fibroblasts compared with age- and sex-matched unaffected first degree relatives. We found decreased levels of ATP and membrane potential, but higher mitochondrial enzyme complexes II+III and complex IV activities in the patient VCP myoblasts when compared to the values of the control cell lines. These results suggest that mutations in VCP affect the mitochondria's ability to produce ATP, thereby resulting in a compensatory increase in the cells' mitochondrial complex activity levels. Thus, this novel in vitro model may be useful in understanding the pathophysiology and discovering new drug targets of mitochondrial dynamics and physiology to modify the clinical phenotype in VCP and related multisystem proteinopathies (MSP).
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Metabolismo Energético , Mitocôndrias/fisiologia , Doenças Neurodegenerativas/patologia , Deficiências na Proteostase/patologia , Trifosfato de Adenosina/análise , Animais , Modelos Animais de Doenças , Complexo de Proteínas da Cadeia de Transporte de Elétrons/análise , Fibroblastos/metabolismo , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mioblastos/metabolismo , Proteína com ValosinaRESUMO
Prader-Willi syndrome (PWS) is a genetic disorder caused by deficiency of imprinted gene expression from the paternal chromosome 15q11-15q13 and clinically characterized by neonatal hypotonia, short stature, cognitive impairment, hypogonadism, hyperphagia, morbid obesity, and diabetes. Previous clinical studies suggest that a defect in energy metabolism may be involved in the pathogenesis of PWS. We focused our attention on the genes associated with energy metabolism and found that there were 95 and 66 mitochondrial genes differentially expressed in PWS muscle and brain, respectively. Assessment of enzyme activities of mitochondrial oxidative phosphorylation complexes in the brain, heart, liver, and muscle were assessed. We found the enzyme activities of the cardiac mitochondrial complexes II+â«III were up-regulated in the PWS imprinting center deletion mice compared to the wild-type littermates. These studies suggest that differential gene expression, especially of the mitochondrial genes may contribute to the pathophysiology of PWS.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Impressão Genômica/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Síndrome de Prader-Willi/genética , Deleção de Sequência/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Redes Reguladoras de Genes/genética , Genoma/genética , Camundongos , Mitocôndrias/ultraestrutura , Músculos/metabolismo , Músculos/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos TestesRESUMO
Angelman syndrome (AS) is a severe neurological disorder caused by a deficiency of ubiquitin protein ligase E3A (UBE3A), but the pathophysiology of the disease remains unknown. We now report that in the brains of AS mice in which the maternal UBE3A allele is mutated (m-) and the paternal allele is potentially inactivated by imprinting (p+) (UBE3A m-\p+), the mitochondria are abnormal and exhibit a partial oxidative phosphorylation (OXPHOS) defect. Electron microscopy of the hippocampal region of the UBE3A m-\p+ mice (n=6) reveals small, dense mitochondria with altered cristae, relative to wild-type littermates (n=6) and reduced synaptic vesicle density. The specific activity of OXPHOS complex III is reduced in whole brain mitochondria in UBE3A m-\p+ (n=5) mice versus wild-type littermates (n=5). Therefore, mitochondrial dysfunction may contribute to the pathophysiology of Angelman syndrome.
Assuntos
Síndrome de Angelman/enzimologia , Região CA1 Hipocampal/enzimologia , Modelos Animais de Doenças , Mitocôndrias/enzimologia , Neurônios/enzimologia , Ubiquitina-Proteína Ligases/deficiência , Síndrome de Angelman/genética , Síndrome de Angelman/patologia , Animais , Região CA1 Hipocampal/patologia , Feminino , Genótipo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/patologia , Neurônios/patologia , Neurônios/fisiologia , Células de Purkinje/enzimologia , Células de Purkinje/patologia , Vesículas Sinápticas/genética , Vesículas Sinápticas/patologia , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genéticaRESUMO
Dominant mutations in the valosin containing protein (VCP) gene cause inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD). We have generated a knock-in mouse model with the common R155H mutation. Mice demonstrate progressive muscle weakness starting approximately at the age of 6 months. Histology of mutant muscle showed progressive vacuolization of myofibrils and centrally located nuclei, and immunostaining shows progressive cytoplasmic accumulation of TDP-43 and ubiquitin-positive inclusion bodies in quadriceps myofibrils and brain. Increased LC3-II staining of muscle sections representing increased number of autophagosomes suggested impaired autophagy. Increased apoptosis was demonstrated by elevated caspase-3 activity and increased TUNEL-positive nuclei. X-ray microtomography (uCT) images show radiolucency of distal femurs and proximal tibiae in knock-in mice and uCT morphometrics shows decreased trabecular pattern and increased cortical wall thickness. Bone histology and bone marrow derived macrophage cultures in these mice revealed increased osteoclastogenesis observed by TRAP staining suggestive of Paget bone disease. The VCP(R155H/+) knock-in mice replicate the muscle, bone and brain pathology of inclusion body myopathy, thus representing a useful model for preclinical studies.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Ciclo Celular/fisiologia , Miosite de Corpos de Inclusão/fisiopatologia , Osteíte Deformante/patologia , Animais , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Transgênicos , Proteína com ValosinaRESUMO
Voltage-gated calcium channels containing alpha1 subunits encoded by Ca(v)2 family genes are critical in regulating release of neurotransmitter at chemical synapses. In Drosophila, cac is the only Ca(v)2-type gene. Cacophony (CAC) channels are localized in motor neuron terminals where they have been shown to mediate evoked, but not AP-independent, release of glutamate at the larval neuromuscular junction (NMJ). Cultured embryonic neurons also express CAC channels, but there is no information about the properties of CAC-mediated currents in adult brain nor how these channels regulate transmission in central neural circuits where fast excitatory synaptic transmission is predominantly cholinergic. Here we report that wild-type neurons cultured from late stage pupal brains and antennal lobe projection neurons (PNs) examined in adult brains, express calcium currents with two components: a slow-inactivating current sensitive to the spider toxin Plectreurys toxin II (PLTXII) and a fast-inactivating PLTXII-resistant component. CAC channels are the major contributors to the slow-inactivating PLTXII-sensitive current based on selective reduction of this component in hypomorphic cac mutants (NT27 and TS3). Another characteristic of cac mutant neurons both in culture and in whole brain recordings is a reduced cholinergic miniature excitatory postsynaptic current frequency that is mimicked in wild-type neurons by acute application of PLTXII. These data demonstrate that cac encoded Ca(v)2-type calcium channels regulate action potential (AP)-independent release of neurotransmitter at excitatory cholinergic synapses in the adult brain, a function not predicted from studies at the larval NMJ.
Assuntos
Potenciais de Ação/fisiologia , Encéfalo/fisiologia , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/fisiologia , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Neurotransmissores/metabolismo , Sistema Nervoso Parassimpático/metabolismo , Sinapses/metabolismo , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Células Cultivadas , Drosophila , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Microscopia Eletrônica , Atividade Motora/fisiologia , Neurônios/metabolismo , Neurotoxinas/farmacologia , Sistema Nervoso Parassimpático/efeitos dos fármacos , Técnicas de Patch-Clamp , Órgãos dos Sentidos/fisiologia , Sinapses/efeitos dos fármacosRESUMO
Inclusion body myopathy associated with Paget's disease and frontotemporal dementia (IBMPFD) is caused by mutations in the valosin containing protein (VCP) gene. The disease is associated with progressive proximal muscle weakness, inclusions and vacuoles in muscle fibers, malfunction in the bone remodeling process resulting in Paget's disease, and premature frontotemporal dementia. VCP is involved in several cellular processes related to the endoplasmic reticulum associated degradation of proteins. To understand the pathological mechanisms underlying the myopathy in IBMPFD, we have studied the cellular consequences of VCP mutations in human primary myoblasts. Our results revealed that patients' myoblasts accumulate large vacuoles. Lysosomal membrane proteins Lamp1 and Lamp2 show increased molecular weights in patients' myoblasts due to differential N-glycosylation. Additionally, mutant myoblasts show increased autophagy when cultured in the absence of nutrients, as well as defective cell fusion and increased apoptosis. Our results elucidate that VCP mutations result in disturbances in several cellular processes, which will help us in the understanding of the pathological mechanisms resulting in muscle weakness and other features of VCP associated disease.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Autofagia/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mioblastos/fisiologia , Miosite de Corpos de Inclusão/metabolismo , Adulto , Apoptose/genética , Apoptose/fisiologia , Caspase 3/metabolismo , Fusão Celular/métodos , Células Cultivadas , Regulação para Baixo/genética , Regulação para Baixo/fisiologia , Feminino , Demência Frontotemporal/complicações , Humanos , Marcação In Situ das Extremidades Cortadas/métodos , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas de Membrana Lisossomal/genética , Masculino , Microscopia Eletrônica de Transmissão/métodos , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/fisiologia , Mutação/genética , Mioblastos/patologia , Mioblastos/ultraestrutura , Miosite de Corpos de Inclusão/patologia , Osteíte Deformante/complicações , Proteína com ValosinaRESUMO
A single episode of status epilepticus (SE) induced in rodents by the convulsant pilocarpine, produces, after a latent period of > or = 2 weeks, a chronic epileptic condition. During the latent period of epileptogenesis, most CA1 pyramidal cells that normally fire in a regular pattern, acquire low-threshold bursting behaviour, generating high-frequency clusters of 3-5 spikes as their minimal response to depolarizing stimuli. Recruitment of a Ni(2+)- and amiloride-sensitive T-type Ca(2+) current (I(CaT)), shown to be up-regulated after SE, plays a critical role in burst generation in most cases. Several lines of evidence suggest that I(CaT) driving bursting is located in the apical dendrites. Thus, bursting was suppressed by focally applying Ni(2+) to the apical dendrites, but not to the soma. It was also suppressed by applying either tetrodotoxin or the K(V)7/M-type K(+) channel agonist retigabine to the apical dendrites. Severing the distal apical dendrites approximately 150 microm from the pyramidal layer also abolished this activity. Intradendritic recordings indicated that evoked bursts are associated with local Ni(2+)-sensitive slow spikes. Blocking persistent Na(+) current did not modify bursting in most cases. We conclude that SE-induced increase in I(CaT) density in the apical dendrites facilitates their depolarization by the backpropagating somatic spike. The I(CaT)-driven dendritic depolarization, in turn, spreads towards the soma, initiating another backpropagating spike, and so forth, thereby creating a spike burst. The early appearance and predominance of I(CaT)-driven low-threshold bursting in CA1 pyramidal cells that experienced SE most probably contribute to the emergence of abnormal network discharges and may also play a role in the circuitry reorganization associated with epileptogenesis.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Canais de Cálcio Tipo T/metabolismo , Dendritos/metabolismo , Células Piramidais/metabolismo , Estado Epiléptico/metabolismo , Amilorida/farmacologia , Animais , Cálcio/metabolismo , Carbamatos/farmacologia , Dendritos/efeitos dos fármacos , Masculino , Níquel/farmacologia , Fenilenodiaminas/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Células Piramidais/efeitos dos fármacos , Ratos , Recrutamento Neurofisiológico/efeitos dos fármacos , Riluzol/farmacologia , Sódio/metabolismo , Venenos de Aranha/farmacologia , Tetrodotoxina/farmacologiaRESUMO
In Drosophila, nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory synaptic transmission in mushroom body Kenyon cells, a neuronal population involved in generation of complex behaviors, including responses to drugs of abuse. To determine whether activation of nAChRs can induce cellular changes that contribute to functional plasticity in these neurons, we examined nicotine-evoked responses in cells cultured from brains of late stage OK107-GAL4 pupae. Kenyon cells can be identified by expression of green fluorescent protein (GFP+). Nicotine activates alpha-bungarotoxin-sensitive nAChRs, causing a rapid increase in intracellular calcium levels in over 95% of the Kenyon cells. The nicotine-evoked calcium increase has a voltage-gated calcium channel (VGCC) dependent component and a VGCC-independent component that involves calcium influx directly through nAChRs. Thapsigargin treatment reduces the nicotine response consistent with amplification by calcium release from intracellular stores. The response to nicotine is experience-dependent: a short conditioning pulse of nicotine causes a transient 50% reduction in the magnitude of the response to a test pulse of nicotine when the interpulse interval is 4 h. This cellular plasticity is dependent on activation of the VGCC-component of the nicotine response and on cAMP-signaling, but not on protein synthesis. These data demonstrate that activation of nAChRs induces a calcium-dependent plasticity in Kenyon cells that could contribute to adult behaviors involving information processing in the mushroom bodies including responses to nicotine.
Assuntos
Sinalização do Cálcio/fisiologia , Drosophila/metabolismo , Corpos Pedunculados/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Bungarotoxinas/farmacologia , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Feminino , Masculino , Corpos Pedunculados/citologia , Corpos Pedunculados/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Tapsigargina/farmacologiaRESUMO
Agrin, through its interaction with the receptor tyrosine kinase MuSK, mediates accumulation of acetylcholine receptors (AChR) at the developing neuromuscular junction. Agrin has also been implicated in several functions in brain. However, the mechanism by which agrin exerts its effects in neural tissue is unknown. Here we present biochemical evidence that agrin binds to the alpha3 subunit of the Na+/K+-ATPase (NKA) in CNS neurons. Colocalization with agrin binding sites at synapses supports the hypothesis that the alpha3NKA is a neuronal agrin receptor. Agrin inhibition of alpha3NKA activity results in membrane depolarization and increased action potential frequency in cortical neurons in culture and acute slice. An agrin fragment that acts as a competitive antagonist depresses action potential frequency, showing that endogenous agrin regulates native alpha3NKA function. These data demonstrate that, through its interaction with the alpha3NKA, agrin regulates activity-dependent processes in neurons, providing a molecular framework for agrin action in the CNS.
Assuntos
Agrina/metabolismo , Neurônios/metabolismo , Subunidades Proteicas/metabolismo , Receptores de Superfície Celular/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Potenciais de Ação/fisiologia , Agrina/genética , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Corantes Fluorescentes/metabolismo , Camundongos , Neurônios/citologia , Técnicas de Patch-Clamp , Subunidades Proteicas/genética , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Sinapses/metabolismo , Tirosina/metabolismoRESUMO
Spontaneous calcium oscillations in mushroom bodies of late stage pupal and adult Drosophila brains have been implicated in memory consolidation during olfactory associative learning. This study explores the cellular mechanisms regulating calcium dynamics in Kenyon cells, principal neurons in mushroom bodies. Fura-2 imaging shows that Kenyon cells cultured from late stage Drosophila pupae generate spontaneous calcium transients in a cell autonomous fashion, at a frequency similar to calcium oscillations in vivo (10-20/h). The expression of calcium transients is up regulated during pupal development. Although the ability to generate transients is a property intrinsic to Kenyon cells, transients can be modulated by bath application of nicotine and GABA. Calcium transients are blocked, and baseline calcium levels reduced, by removal of external calcium, addition of cobalt, or addition of Plectreurys toxin (PLTX), an insect-specific calcium channel antagonist. Transients do not require calcium release from intracellular stores. Whole cell recordings reveal that the majority of voltage-gated calcium channels in Kenyon cells are PLTX-sensitive. Together these data show that influx of calcium through PLTX-sensitive voltage-gated calcium channels mediates spontaneous calcium transients and regulates basal calcium levels in cultured Kenyon cells. The data also suggest that these calcium transients represent cellular events underlying calcium oscillations in the intact mushroom bodies. However, spontaneous calcium transients are not unique to Kenyon cells as they are present in approximately 60% of all cultured central brain neurons. This suggests the calcium transients play a more general role in maturation or function of adult brain neurons.