RESUMEN
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline and amyloid-beta (Aß) depositions generated by the proteolysis of amyloid precursor protein (APP) in the brain. In APPNL-F mice, APP gene was humanized and contains two familial AD mutations, and APP-unlike other mouse models of AD-is driven by the endogenous mouse APP promoter. Similar to people without apparent cognitive dysfunction but with heavy Aß plaque load, we found no significant decline in the working memory of adult APPNL-F mice, but these mice showed decline in the expression of normal anxiety. Using immunohistochemistry and 3D block-face scanning electron microscopy, we found no changes in GABAA receptor positivity and size of somatic and dendritic synapses of hippocampal interneurons. We did not find alterations in the level of expression of perineuronal nets around parvalbumin (PV) interneurons or in the density of PV- or somatostatin-positive hippocampal interneurons. However, in contrast to other investigated cell types, PV interneuron axons were occasionally mildly dystrophic around Aß plaques, and the synapses of PV-positive axon initial segment (AIS)-targeting interneurons were significantly enlarged. Our results suggest that PV interneurons are highly resistant to amyloidosis in APPNL-F mice and amyloid-induced increase in hippocampal pyramidal cell excitability may be compensated by PV-positive AIS-targeting cells. Mechanisms that make PV neurons more resilient could therefore be exploited in the treatment of AD for mitigating Aß-related inflammatory effects on neurons.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Hipocampo/metabolismo , Interneuronas/metabolismo , Mutación , Red Nerviosa/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Animales , Axones/metabolismo , Axones/patología , Hipocampo/patología , Humanos , Interneuronas/patología , Memoria a Corto Plazo , Ratones , Ratones Transgénicos , Red Nerviosa/patología , Fragmentos de Péptidos/genética , Células Piramidales/metabolismo , Células Piramidales/patología , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismoRESUMEN
Hippocampal pyramidal cells encode memory engrams, which guide adaptive behavior. Selection of engram-forming cells is regulated by somatostatin-positive dendrite-targeting interneurons, which inhibit pyramidal cells that are not required for memory formation. Here, we found that γ-aminobutyric acid (GABA)-releasing neurons of the mouse nucleus incertus (NI) selectively inhibit somatostatin-positive interneurons in the hippocampus, both monosynaptically and indirectly through the inhibition of their subcortical excitatory inputs. We demonstrated that NI GABAergic neurons receive monosynaptic inputs from brain areas processing important environmental information, and their hippocampal projections are strongly activated by salient environmental inputs in vivo. Optogenetic manipulations of NI GABAergic neurons can shift hippocampal network state and bidirectionally modify the strength of contextual fear memory formation. Our results indicate that brainstem NI GABAergic cells are essential for controlling contextual memories.
Asunto(s)
Aprendizaje por Asociación/fisiología , Neuronas GABAérgicas/fisiología , Núcleos del Rafe/fisiología , Animales , Femenino , Interneuronas/química , Interneuronas/fisiología , Masculino , Pruebas de Memoria y Aprendizaje , Ratones , Ratones Endogámicos C57BL , Inhibición Neural/fisiología , Células Piramidales/química , Células Piramidales/fisiología , Somatostatina/análisis , Somatostatina/fisiología , Ritmo TetaRESUMEN
The basal forebrain cholinergic system is widely assumed to control cortical functions via non-synaptic transmission of a single neurotransmitter. Yet, we find that mouse hippocampal cholinergic terminals invariably establish GABAergic synapses, and their cholinergic vesicles dock at those synapses only. We demonstrate that these synapses do not co-release but co-transmit GABA and acetylcholine via different vesicles, whose release is triggered by distinct calcium channels. This co-transmission evokes composite postsynaptic potentials, which are mutually cross-regulated by presynaptic autoreceptors. Although postsynaptic cholinergic receptor distribution cannot be investigated, their response latencies suggest a focal, intra- and/or peri-synaptic localisation, while GABAA receptors are detected intra-synaptically. The GABAergic component alone effectively suppresses hippocampal sharp wave-ripples and epileptiform activity. Therefore, the differentially regulated GABAergic and cholinergic co-transmission suggests a hitherto unrecognised level of control over cortical states. This novel model of hippocampal cholinergic neurotransmission may lead to alternative pharmacotherapies after cholinergic deinnervation seen in neurodegenerative disorders.
Asunto(s)
Acetilcolina/fisiología , Hipocampo/fisiología , Receptores de GABA-A/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Calcio/fisiología , Dendritas/fisiología , Femenino , Imagenología Tridimensional , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades Neurodegenerativas/fisiopatología , Neurotransmisores/fisiología , Perfusión , Sinapsis/fisiología , Potenciales Sinápticos , Transmisión Sináptica , Vesículas Sinápticas/fisiologíaRESUMEN
The median raphe region (MRR) is believed to control the fear circuitry indirectly, by influencing the encoding and retrieval of fear memories by amygdala, hippocampus and prefrontal cortex. Here we show that in addition to this established role, MRR stimulation may alone elicit the emergence of remote but not recent fear memories. We substituted electric shocks with optic stimulation of MRR in C57BL/6N male mice in an optogenetic conditioning paradigm and found that stimulations produced agitation, but not fear, during the conditioning trial. Contextual fear, reflected by freezing was not present the next day, but appeared after a 7 days incubation. The optogenetic silencing of MRR during electric shocks ameliorated conditioned fear also seven, but not one day after conditioning. The optogenetic stimulation patterns (50Hz theta burst and 20Hz) used in our tests elicited serotonin release in vitro and lead to activation primarily in the periaqueductal gray examined by c-Fos immunohistochemistry. Earlier studies demonstrated that fear can be induced acutely by stimulation of several subcortical centers, which, however, do not generate persistent fear memories. Here we show that the MRR also elicits fear, but this develops slowly over time, likely by plastic changes induced by the area and its connections. These findings assign a specific role to the MRR in fear learning. Particularly, we suggest that this area is responsible for the durable sensitization of fear circuits towards aversive contexts, and by this, it contributes to the persistence of fear memories. This suggests the existence a bottom-up control of fear circuits by the MRR, which complements the top-down control exerted by the medial prefrontal cortex.
Asunto(s)
Encéfalo/fisiología , Animales , Conducta Animal , Electrochoque , Miedo/fisiología , Halorrodopsinas/metabolismo , Inmunohistoquímica , Masculino , Memoria/fisiología , Ratones , Ratones Endogámicos C57BL , Sustancia Gris Periacueductal/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Serotonina/metabolismo , Grabación en VideoRESUMEN
The median raphe region (MRR) is thought to be serotonergic and plays an important role in the regulation of many cognitive functions. In the hippocampus (HIPP), the MRR exerts a fast excitatory control, partially through glutamatergic transmission, on a subpopulation of GABAergic interneurons that are key regulators of local network activity. However, not all receptors of this connection in the HIPP and in synapses established by MRR in other brain areas are known. Using combined anterograde tracing and immunogold methods, we show that the GluN2A subunit of the NMDA receptor is present in the synapses established by MRR not only in the HIPP, but also in the medial septum (MS) and in the medial prefrontal cortex (mPFC) of the mouse. We estimated similar amounts of NMDA receptors in these synapses established by the MRR and in local adjacent excitatory synapses. Using retrograde tracing and confocal laser scanning microscopy, we found that the majority of the projecting cells of the mouse MRR contain the vesicular glutamate transporter type 3 (vGluT3). Furthermore, using double retrograde tracing, we found that single cells of the MRR can innervate the HIPP and mPFC or the MS and mPFC simultaneously, and these double-projecting cells are also predominantly vGluT3-positive. Our results indicate that the majority of the output of the MRR is glutamatergic and acts through NMDA receptor-containing synapses. This suggests that key forebrain areas receive precisely targeted excitatory input from the MRR, which is able to synchronously modify activity in those regions via individual MRR cells with dual projections.
Asunto(s)
Glutamatos/metabolismo , Neuronas/metabolismo , Prosencéfalo/metabolismo , Núcleos del Rafe/metabolismo , Animales , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Neuronas/citología , Corteza Prefrontal/metabolismo , Prosencéfalo/citología , Núcleos del Rafe/citología , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Three functionally different populations of perisomatic interneurons establish GABAergic synapses on hippocampal pyramidal cells: parvalbumin (PV)-containing basket cells, type 1 cannabinoid receptor (CB1)-positive basket cells both of which target somata, and PV-positive axo-axonic cells that innervate axon initial segments. Using electron microscopic reconstructions, we estimated that a pyramidal cell body receives synapses from about 60 and 140 synaptic terminals in the CA1 and CA3 area, respectively. About 60 % of these terminals were PV positive, whereas 35-40 % of them were CB1 positive. Only about 1 % (CA1) and 4 % (CA3) of the somatic boutons were negative for both markers. Using fluorescent labeling, we showed that most of the CB1-positive terminals expressed vesicular glutamate transporter 3. Reconstruction of somatic boutons revealed that although their volumes are similar, CB1-positive boutons are more flat and the total volume of their mitochondria was smaller than that of PV-positive boutons. Both types of boutons contain dense-core vesicles and frequently formed multiple release sites on their targets and innervated an additional soma or dendrite as well. PV-positive boutons possessed small, macular synapses; whereas the total synaptic area of CB1-positive boutons was larger and formed multiple irregular-shaped synapses. Axo-axonic boutons were smaller than somatic boutons, had only one synapse and their ultrastructural parameters were closer to those of PV-positive somatic boutons. Our results represent the first quantitative measurement-using a highly reliable method-of the contribution of different cell types to the perisomatic innervation of pyramidal neurons, and may help to explain functional differences in their output properties.
Asunto(s)
Hipocampo/ultraestructura , Interneuronas/ultraestructura , Terminales Presinápticos/ultraestructura , Células Piramidales/ultraestructura , Sistemas de Transporte de Aminoácidos Acídicos , Animales , Hipocampo/metabolismo , Interneuronas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/ultraestructura , Parvalbúminas/análisis , Terminales Presinápticos/metabolismo , Células Piramidales/metabolismo , Receptor Cannabinoide CB1/análisisRESUMEN
Neuroligin 2 is a postsynaptic protein that plays a critical role in the maturation and proper function of GABAergic synapses. Previous studies demonstrated that deletion of neuroligin 2 impaired GABAergic synaptic transmission, whereas its overexpression caused increased inhibition, which suggest that its presence strongly influences synaptic function. Interestingly, the overexpressing transgenic mouse line showed increased anxiety-like behavior and other behavioral phenotypes, not easily explained by an otherwise strengthened GABAergic transmission. This suggested that other, non-GABAergic synapses may also express neuroligin 2. Here, we tested the presence of neuroligin 2 at synapses established by cholinergic neurons in the mouse brain using serial electron microscopic sections double labeled for neuroligin 2 and choline acetyltransferase. We found that besides GABAergic synapses, neuroligin 2 is also present in the postsynaptic membrane of cholinergic synapses in all investigated brain areas (including dorsal hippocampus, somatosensory and medial prefrontal cortices, caudate putamen, basolateral amygdala, centrolateral thalamic nucleus, medial septum, vertical- and horizontal limbs of the diagonal band of Broca, substantia innominata and ventral pallidum). In the hippocampus, the density of neuroligin 2 labeling was similar in GABAergic and cholinergic synapses. Moreover, several cholinergic contact sites that were strongly labeled with neuroligin 2 did not resemble typical synapses, suggesting that cholinergic axons form more synaptic connections than it was recognized previously. We showed that cholinergic cells themselves also express neuroligin 2 in a subset of their input synapses. These data indicate that mutations in human neuroligin 2 gene and genetic manipulations of neuroligin 2 levels in rodents will potentially cause alterations in the cholinergic system as well, which may also have a profound effect on the functional properties of brain circuits and behavior.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Neuronas Colinérgicas/metabolismo , Hipocampo/citología , Proteínas del Tejido Nervioso/metabolismo , Sinapsis/metabolismo , Animales , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Neuronas GABAérgicas/metabolismo , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Transporte de ProteínasRESUMEN
Duchenne muscular dystrophy (DMD) cardiomyopathy patients currently have no therapeutic options. We evaluated catheter-based transendocardial delivery of a recombinant adeno-associated virus (rAAV) expressing a small nuclear U7 RNA (U7smOPT) complementary to specific cis-acting splicing signals. Eliminating specific exons restores the open reading frame resulting in translation of truncated dystrophin protein. To test this approach in a clinically relevant DMD model, golden retriever muscular dystrophy (GRMD) dogs received serotype 6 rAAV-U7smOPT via the intracoronary or transendocardial route. Transendocardial injections were administered with an injection-tipped catheter and fluoroscopic guidance using X-ray fused with magnetic resonance imaging (XFM) roadmaps. Three months after treatment, tissues were analyzed for DNA, RNA, dystrophin protein, and histology. Whereas intracoronary delivery did not result in effective transduction, transendocardial injections, XFM guidance, enabled 30±10 non-overlapping injections per animal. Vector DNA was detectable in all samples tested and ranged from <1 to >3000 vector genome copies per cell. RNA analysis, western blot analysis, and immunohistology demonstrated extensive expression of skipped RNA and dystrophin protein in the treated myocardium. Left ventricular function remained unchanged over a 3-month follow-up. These results demonstrated that effective transendocardial delivery of rAAV-U7smOPT was achieved using XFM. This approach restores an open reading frame for dystrophin in affected dogs and has potential clinical utility.
Asunto(s)
Dependovirus/genética , Distrofina/genética , Imagen por Resonancia Magnética/métodos , Distrofia Muscular de Duchenne/terapia , ARN Nuclear Pequeño/genética , Transducción Genética/métodos , Animales , Secuencia de Bases , Western Blotting , Modelos Animales de Enfermedad , Perros , Distrofina/metabolismo , Exones/genética , Femenino , Expresión Génica , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Miocardio/metabolismo , ARN Nuclear Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Sf9RESUMEN
The two main glutamatergic pathways to the CA1 area, the Schaffer collateral/commissural input and the entorhinal fibers, as well as the local axons of CA1 pyramidal cells innervate both pyramidal cells and interneurons. To determine whether these inputs differ in their weights of activating GABAergic circuits, we have studied the relative proportion of pyramidal cells and interneurons among their postsynaptic targets in serial electron microscopic sections. Local axons of CA1 pyramidal cells, intracellularly labeled in vitro or in vivo, innervated a relatively high proportion of interneuronal postsynaptic targets (65.9 and 53.8%, in vitro and in vivo, respectively) in stratum (str.) oriens and alveus. In contrast, axons of in vitro labeled CA3 pyramidal cells in str. oriens and str. radiatum of the CA1 area made synaptic junctions predominantly with pyramidal cell spines (92.9%). The postsynaptic targets of anterogradely labeled medial entorhinal cortical boutons in CA1 str. lacunosum-moleculare were primarily pyramidal neuron dendritic spines and shafts (90.8%). The alvear group of the entorhinal afferents, traversing str. oriens, str. pyramidale, and str. radiatum showed a higher preference for innervating GABAergic cells (21.3%), particularly in str. oriens/alveus. These data demonstrate that different glutamatergic pathways innervate CA1 GABAergic cells to different extents. The results suggest that the numerically smaller CA1 local axonal inputs together with the alvear part of the entorhinal input preferentially act on GABAergic interneurons in contrast to the CA3, or the entorhinal input in str. lacunosum-moleculare. The results highlight differences in the postsynaptic target selection of the feed-forward versus recurrent glutamatergic inputs to the CA1 and CA3 areas.
Asunto(s)
Región CA1 Hipocampal/fisiología , Ácido Glutámico/fisiología , Interneuronas/fisiología , Células Piramidales/fisiología , Animales , Región CA1 Hipocampal/ultraestructura , Región CA3 Hipocampal/fisiología , Región CA3 Hipocampal/ultraestructura , Femenino , Interneuronas/ultraestructura , Masculino , Células Piramidales/ultraestructura , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
The available methods for double-labeling preembedding immunoelectron microscopy are highly limited because not only should the ultrastructure be preserved, but also the different antigens should be visualized by reaction end products that can be clearly distinguished in gray-scale images. In these procedures, one antigen is detected with 3,3'-diaminobenzidine (DAB) chromogen, resulting in a homogeneous deposit, whereas the other is labeled with either a gold-tagged immunoreagent, or DAB polymer, on the surface of which metallic silver is precipitated. The detection of the second antigen is usually impeded by the first, leading to false-negative results. The authors aimed to diminish this hindrance by a new silver intensification technique of DAB polymer, which converts the deposit from amorphous to granular. The method includes three major postdevelopmental steps: (1) treatment of nickel-enhanced DAB with sulfide, (2) silver deposition in the presence of hydroquinone under acidic conditions, and (3) precious metal replacement with gold thiocyanate. This new sulfide-silver-gold intensification of DAB (SSGI) allows a subsequent detection of other antigens using DAB. In conclusion, the new technique loads fine gold particles onto the DAB deposit at a very low background level, thereby allowing a reliable discernment between the elements stained for the two antigens at the ultrastructural level.
Asunto(s)
3,3'-Diaminobencidina , Acetatos , Encéfalo/metabolismo , Cloruros , Proteína Ácida Fibrilar de la Glía/metabolismo , Compuestos de Oro , Parvalbúminas/metabolismo , Receptor Cannabinoide CB1/metabolismo , Compuestos de Plata , Animales , Biomarcadores/metabolismo , Encéfalo/ultraestructura , Inmunohistoquímica , Indicadores y Reactivos , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía InmunoelectrónicaRESUMEN
The CXC chemokine receptor 4 (CXCR4) for the chemokine (C-X-C motif) ligand 12/stromal cell-derived factor-1 alpha (CXCL12/SDF-1 alpha) is highly expressed in the postnatal CA1 stratum lacunosum-moleculare. However, both the network events triggered by SDF-1 alpha in this microcircuit and the cellular targets of this chemokine remain virtually unexplored. Here, we have studied SDF-1 alpha-mediated neuromodulation of the stratum lacunosum-moleculare by directly comparing the properties of CXCR4-expressing Cajal-Retzius cells vs. CXCR4-non-expressing interneurons, and by recording the electrophysiological effects caused by application of SDF-1 alpha on either cell type. We demonstrate that SDF-1 alpha dramatically reduces spontaneous firing in Cajal-Retzius cells via hyerpolarization, and that cessation of firing is prevented by the CXCR4-specific antagonist AMD3100. In contrast, no effects on the excitability of interneurons of the same layer were observed following exposure to the chemokine. We also provide evidence that, despite the expression of functional glutamate receptors, Cajal-Retzius cells are integrated in the synaptic network of the stratum lacunosum-moleculare via excitatory GABAergic input. Furthermore, we show that the axons of Cajal-Retzius cells target specifically the stratum lacunosum-moleculare and the dentate gyrus, but lack postsynaptic specializations opposite to their axonal varicosities. These results, taken together with our observation that SDF-1 alpha reduces evoked field responses at the entorhinal cortex-CA1 synapse, suggest that Cajal-Retzius cells produce a diffuse output that may impact information processing of stratum lacunosum-moleculare. We propose that pathological alterations of local levels of SDF-1 alpha or CXCR4 expression may affect the functions of an important hippocampal microcircuit.
Asunto(s)
Potenciales de Acción/fisiología , Moduladores del GABA/metabolismo , Hipocampo/fisiología , Interneuronas/fisiología , Receptores CXCR4/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , RatasRESUMEN
The ability to safely control transgene expression from viral vectors is a long-term goal in the gene therapy field. We have previously reported tight regulation of GFP expression in rat brain using a self-regulating tet-off rAAV vector. The immune responses against tet regulatory elements observed by other groups in nonhuman primates after intramuscular injection of tet-on encoding vectors raise concerns about the clinical value of tet-regulated vectors. However, previous studies have not examined immune responses following injection of AAV vectors into brain. Therefore, rat striatum was injected with tet-off rAAV harboring a therapeutic gene for Parkinson's disease, either hAADC or hGDNF. The expression of each gene was tightly controlled by the tet-off regulatory system. Using an ELISA developed with purified GST-tTA protein, no detectable immunogenicity against tTA was observed in sera of rats that received an intrastriatal injection of either vector. In contrast, sera from rats intradermally injected with an adenovirus containing either tTA or rtTA, as positive controls, had readily detectable antibodies. These observations suggest that tet-off rAAV vectors do not elicit an immune response when injected into rat brain and that these may offer safer vectors for Parkinson's disease than vectors with constitutive expression.
Asunto(s)
Ganglios Basales/inmunología , Dependovirus/inmunología , Terapia Genética , Vectores Genéticos/inmunología , Enfermedad de Parkinson/terapia , Transactivadores/inmunología , Animales , Descarboxilasas de Aminoácido-L-Aromático/genética , Regulación de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Inmunidad Humoral , Masculino , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Elementos de Respuesta/efectos de los fármacos , Tetraciclina/farmacología , Transactivadores/genética , Transgenes/efectos de los fármacosRESUMEN
The morphological properties and connectivity of gamma-aminobutyric acid (GABA)ergic hippocampal cells projecting to the medial septum (HS cells) were examined in the rat. Two types of HS cells are located in different layers of the hippocampus: sparsely-spiny cells are in CA1-3 str. oriens and CA3 str. radiatum, where recurrent axons of pyramidal cells arborize. Densely-spiny HS cells with spiny somata are located in the termination zone of granule cell axons. In the hilus, intermediate morphologies can also be found. HS cells receive GABAergic medial septal afferents in all layers where they occur, thus the connectivity of the septum and the hippocampus is reciprocal at cell level. HS cells receive extremely dense innervation, sparsely-spiny cells are innervated by approximately 19,000 excitatory inputs, while densely-spiny cells get an even larger number (approximately 37,000). While 14% of the inputs are inhibitory for the sparsely-spiny cells, it is only 2.3% in the case of densely-spiny cells. Because a high proportion (up to 54.5% on somata and 27.5% on dendrites) of their GABAergic inputs derived from labelled septal terminals, their predominant inhibitory input probably arises from the medial septum. CA1 area HS cells possessed myelinated projecting axons, as well as local collaterals, which targeted mostly pyramidal cell dendrites and spines in str. oriens and radiatum. The synaptic organization suggests that by sampling the activity of large populations of principal cells HS cells can reliably broadcast hippocampal activity level to the medial septum.
Asunto(s)
Hipocampo/citología , Vías Nerviosas/anatomía & histología , Neuronas/citología , Núcleos Septales/citología , Sinapsis/metabolismo , Animales , Masculino , Modelos Biológicos , Vías Nerviosas/metabolismo , Neuronas/metabolismo , Ratas , Ratas Wistar , Núcleos Septales/metabolismo , Sinapsis/ultraestructuraRESUMEN
The cell line MN9D, a fusion of embryonic ventral mesencephalic and neuroblastoma cells, is extensively used as a model of dopamine (DA) neurons because it expresses tyrosine hydroxylase and synthesizes and releases DA. These cells are also used to test mechanisms and potential therapeutics relevant to the loss of DA neurons in Parkinson's disease. To date, little work has been done to determine whether MN9D cells electrophysiologically resemble mature DA neurons. We examined sodium, calcium and potassium currents in undifferentiated and differentiated MN9D cells, and compared these to those found in acutely dissociated mouse substantia nigra pars compacta DA neurons. It was observed that undifferentiated MN9D cells bore no resemblance to DA neurons. Upon differentiation with butyric acid with or without a prior treatment with glial cell line-derived neurotrophic factor, differentiated MN9D cells produce an electrophysiological profile that more closely resembles substantia nigra pars compacta DA neurons even though the A-type potassium current remains noticeably absent. These observations demonstrate that undifferentiated MN9D cells are not reasonable models of DA neurons. Although differentiated MN9D cells are closer to the mature DA neuronal phenotype, they do not fully mimic DA neurons and are likely to be of questionable value as a model because of their substantive differences, including the lack of the characteristic A-type potassium current. The future use of one or a combination of growth or other factors to differentiate MN9D cells may yield a more useful model system for Parkinson's disease studies in vitro.
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Diferenciación Celular/fisiología , Dopamina/metabolismo , Canales Iónicos/metabolismo , Neuronas/metabolismo , Células Madre/metabolismo , Sustancia Negra/embriología , Potenciales de Acción/genética , Animales , Ácido Butírico/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/genética , Canales de Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Hibridomas , Canales Iónicos/efectos de los fármacos , Canales Iónicos/genética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/genética , Canales de Sodio/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Sustancia Negra/citología , Sustancia Negra/metabolismo , Transmisión Sináptica/genéticaRESUMEN
Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are members of the phospholipid growth factor family. A major limitation in the field to date has been a lack of receptor subtype-specific agonists and antagonists. Here, we report that dioctylglycerol pyrophosphate and dioctylphosphatidic acid are selective antagonists of the LPA(1) and LPA(3) receptors, but prefer LPA(3) by an order of magnitude. Neither molecule had an agonistic or antagonistic effect on LPA(2) receptor. Consistent with this receptor subtype selectivity, dioctylglycerol pyrophosphate inhibited cellular responses to LPA in NIH3T3 fibroblasts, HEY ovarian cancer cells, PC12 pheochromocytoma cells, and Xenopus laevis oocytes. Responses elicited by S1P in these cell lines that endogenously express S1P(1), S1P(2), S1P(3), and S1P(5) receptors were unaffected by dioctylglycerol pyrophosphate. Responses evoked by the G protein-coupled receptor ligands acetylcholine, serotonin, ATP, and thrombin receptor-activating peptide were similarly unaffected, suggesting that the short-chain phosphatidates are receptor subtype-specific lysophosphatidate antagonists.
Asunto(s)
Difosfatos/farmacología , Glicerol/análogos & derivados , Glicerol/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores Acoplados a Proteínas G , Células 3T3 , Acetilcolina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Unión Competitiva , Calcio/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Ratones , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Células PC12 , Ratas , Receptores de Superficie Celular/metabolismo , Receptores del Ácido Lisofosfatídico , Receptores Lisofosfolípidos , Serotonina/metabolismo , Trombina/metabolismo , Xenopus laevisRESUMEN
Blood plasma and serum contain factors that activate inwardly rectifying GIRK1/GIRK4 K+ channels in atrial myocytes via one or more non-atropine-sensitive receptors coupled to pertussis-toxin-sensitive G-proteins. This channel is also the target of muscarinic M(2) receptors activated by the physiological release of acetylcholine from parasympathetic nerve endings. By using a combination of HPLC and TLC techniques with matrix-assisted laser desorption ionization-time-of-flight MS, we purified and identified sphingosine 1-phosphate (SPP) and sphingosylphosphocholine (SPC) as the plasma and serum factors responsible for activating the inwardly rectifying K+ channel (I(K)). With the use of MS the concentration of SPC was estimated at 50 nM in plasma and 130 nM in serum; those concentrations exceeded the 1.5 nM EC(50) measured in guinea-pig atrial myocytes. With the use of reverse-transcriptase-mediated PCR and/or Western blot analysis, we detected Edg1, Edg3, Edg5 and Edg8 as well as OGR1 sphingolipid receptor transcripts and/or proteins. In perfused guinea-pig hearts, SPC exerted a negative chronotropic effect with a threshold concentration of 1 microM. SPC was completely removed after perfusion through the coronary circulation at a concentration of 10 microM. On the basis of their constitutive presence in plasma, the expression of specific receptors, and a mechanism of ligand inactivation, we propose that SPP and SPC might have a physiologically relevant role in the regulation of the heart.
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Proteínas Portadoras/fisiología , Corazón/fisiología , Fosforilcolina/análogos & derivados , Fosforilcolina/sangre , Esfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/sangre , Animales , Western Blotting , Proteínas Portadoras/metabolismo , Atrios Cardíacos/metabolismo , Pruebas de Precipitina , Conejos , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The phospholipid growth-factor (PLGE) terminology is proposed to describe a group of endogenous glycerol- and sphingolipid mediators that regulate cell proliferation through plasma membrane receptors. In addition to LPA and SPP, multiple PLGFs are present in blood plasma and serum. PLGF activity is regulated by its stimulus-coupled production and by endogenous inhibitors. In addition to LPA and SPP, alkenyl-glycerophosphate, cyclic-phosphatidic acid, and sphingosylphosphorylcholine were detected in biological fluids using mass spectrometry. Heterologous desensitization studies indicate the expression of multiple LPA-activated receptors in a variety of cell types, which are differentially activated by the different PLGFs. Northern blot and RT-PCR results reinforce the coexpression of PSP24 alpha and different members of the EDG1-7 receptors in the same cell. Stable heterologous expression of the PSP24 alpha, EDG2, and EDG4 receptors in HEK293 cells show distinct PLGF specificities and dose-response properties for each receptor subtype. Thus, both the controlled availability of the different agonists/inhibitors and the regulated expression of their receptors regulate the biological effects of PLGFs.
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Fosfolípidos/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Animales , Humanos , Ligandos , Receptores de Factores de Crecimiento/efectos de los fármacosRESUMEN
The paper attempts to conceptualise the observations made in therapies of Holocaust survivors. The first case studies of child therapies were published in 1982, demonstrating the induced trauma transference and the three-generational neurosis model, i.e. the transfer of the trauma from the traumatised grandparent through his/her child to the grandchild. The first generation survivor syndrome was first experienced with patients who themselves had suffered deportation or had survived the ghetto, and after several decades, came for treatment to the KUT Psychotherapeutical Clinic. that has been operating for 4 years now.
Asunto(s)
Psiquiatría Infantil/historia , Sueños , Holocausto/historia , Hospitales Psiquiátricos/historia , Terapia Psicoanalítica , Adolescente , Niño , Preescolar , Historia del Siglo XX , Humanos , HungríaAsunto(s)
Lisofosfolípidos/metabolismo , Fosfolípidos/metabolismo , Receptores de Superficie Celular/metabolismo , Células 3T3 , Animales , AMP Cíclico/metabolismo , Femenino , Proteínas de Unión al GTP/metabolismo , Humanos , Ligandos , Lisofosfolípidos/farmacología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Ácidos Fosfatidicos/farmacología , Fosfolípidos/farmacología , Receptores de Superficie Celular/clasificación , XenopusRESUMEN
Lysophosphatidic acid (LPA), plasmalogen-glycerophosphate (alkenyl-GP) and, cyclic-phosphatidic acid (cyclic-PA) are naturally occurring phospholipid growth factors (PLGFs). PLGFs elicit diverse biological effects via the activation of G protein-coupled receptors in a variety of cell types. In NIH3T3 fibroblasts, LPA and alkenyl-GP both induced proliferation, whereas cyclic-PA was antiproliferative. LPA and alkenyl-GP decreased cAMP in a pertussis toxin-sensitive manner, whereas cyclic-PA caused cAMP to increase. LPA and alkenyl-GP both stimulated the activity of the mitogen-actived protein kinases extracellular signal regulated kinases 1 and 2 and c-Jun NH2-terminal kinase, whereas cyclic-PA did not. All three PLGFs induced the formation of stress fibers in NIH3T3 fibroblasts. To determine whether these lipids activated the same or different receptors, heterologous desensitization patterns were established among the three PLGFs by monitoring changes in intracellular Ca2+ in NIH3T3 fibroblasts. LPA cross-desensitized both the alkenyl-GP and cyclic-PA responses. Alkenyl-GP cross-desensitized the cyclic-PA response, but only partially desensitized the LPA response. Cyclic-PA only partially desensitized both the alkenyl-GP and LPA responses. We propose that pharmacologically distinct subsets of PLGF receptors exist that distinguish between cyclic-PA and alkenyl-GP, but are all activated by LPA. We provide evidence that the PSP24 receptor is selective for LPA and not activated by the other two PLGFs. RT-PCR and Northern blot analysis indicate the co-expression of mRNAs encoding the EDG-2, EDG-4, and PSP24 receptors in a variety of cell lines and tissues. However, the lack of mRNA expression for these three receptors in the LPA-responsive Rat-1 and Sp2-O-Ag14 cells suggests that a number of PLGF receptor subtypes remain unidentified.