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1.
Development ; 151(13)2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38856043

RESUMEN

The function of medial entorhinal cortex layer II (MECII) excitatory neurons has been recently explored. MECII dysfunction underlies deficits in spatial navigation and working memory. MECII neurons comprise two major excitatory neuronal populations, pyramidal island and stellate ocean cells, in addition to the inhibitory interneurons. Ocean cells express reelin and surround clusters of island cells that lack reelin expression. The influence of reelin expression by ocean cells and interneurons on their own morphological differentiation and that of MECII island cells has remained unknown. To address this, we used a conditional reelin knockout (RelncKO) mouse to induce reelin deficiency postnatally in vitro and in vivo. Reelin deficiency caused dendritic hypertrophy of ocean cells, interneurons and only proximal dendritic compartments of island cells. Ca2+ recording showed that both cell types exhibited an elevation of calcium frequencies in RelncKO, indicating that the hypertrophic effect is related to excessive Ca2+ signalling. Moreover, pharmacological receptor blockade in RelncKO mouse revealed malfunctioning of GABAB, NMDA and AMPA receptors. Collectively, this study emphasizes the significance of reelin in neuronal growth, and its absence results in dendrite hypertrophy of MECII neurons.


Asunto(s)
Moléculas de Adhesión Celular Neuronal , Dendritas , Corteza Entorrinal , Proteínas de la Matriz Extracelular , Ratones Noqueados , Proteínas del Tejido Nervioso , Proteína Reelina , Serina Endopeptidasas , Animales , Corteza Entorrinal/metabolismo , Dendritas/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Ratones , Interneuronas/metabolismo , Neuronas/metabolismo , Señalización del Calcio
2.
Development ; 148(17)2021 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-34414407

RESUMEN

Reelin is a large secreted glycoprotein that regulates neuronal migration, lamination and establishment of dendritic architecture in the embryonic brain. Reelin expression switches postnatally from Cajal-Retzius cells to interneurons. However, reelin function in interneuron development is still poorly understood. Here, we have investigated the role of reelin in interneuron development in the postnatal neocortex. To preclude early cortical migration defects caused by reelin deficiency, we employed a conditional reelin knockout (RelncKO) mouse to induce postnatal reelin deficiency. Induced reelin deficiency caused dendritic hypertrophy in distal dendritic segments of neuropeptide Y-positive (NPY+) and calretinin-positive (Calr+) interneurons, and in proximal dendritic segments of parvalbumin-positive (Parv+) interneurons. Chronic recombinant Reelin treatment rescued dendritic hypertrophy in Relncko interneurons. Moreover, we provide evidence that RelncKO interneuron hypertrophy is due to presynaptic GABABR dysfunction. Thus, GABABRs in RelncKO interneurons were unable to block N-type (Cav2.2) Ca2+ channels that control neurotransmitter release. Consequently, the excessive Ca2+ influx through AMPA receptors, but not NMDA receptors, caused interneuron dendritic hypertrophy. These findings suggest that reelin acts as a 'stop-growth-signal' for postnatal interneuron maturation.


Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Dendritas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Interneuronas/citología , Neocórtex/crecimiento & desarrollo , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Calbindina 2/metabolismo , Calcio/metabolismo , Moléculas de Adhesión Celular Neuronal/deficiencia , Moléculas de Adhesión Celular Neuronal/farmacología , Dendritas/efectos de los fármacos , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/farmacología , Hipertrofia , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Ratones , Ratones Noqueados , Neocórtex/citología , Neocórtex/efectos de los fármacos , Neocórtex/patología , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/farmacología , Neuropéptido Y/metabolismo , Parvalbúminas/metabolismo , Receptores de GABA-B/metabolismo , Receptores de Glutamato/metabolismo , Proteína Reelina , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/farmacología
3.
J Neurochem ; 156(5): 589-603, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-32083308

RESUMEN

Reelin is a protein that is best known for its role in controlling neuronal layer formation in the developing cortex. Here, we studied its role for post-natal cortical network function, which is poorly explored. To preclude early cortical migration defects caused by Reelin deficiency, we used a conditional Reelin knock-out (RelncKO ) mouse, and induced Reelin deficiency post-natally. Induced Reelin deficiency caused hyperexcitability of the neocortical network in vitro and ex vivo. Blocking Reelin binding to its receptors ApoER2 and VLDLR resulted in a similar effect. Hyperexcitability in RelncKO organotypic slice cultures could be rescued by co-culture with wild-type organotypic slice cultures. Moreover, the GABAB receptor (GABAB R) agonist baclofen failed to activate and the antagonist CGP35348 failed to block GABAB Rs in RelncKO mice. Immunolabeling of RelncKO cortical slices revealed a reduction in GABAB R1 and GABAB R2 surface expression at the plasma membrane and western blot of RelncKO cortical tissue revealed decreased phosphorylation of the GABAB R2 subunit at serine 892 and increased phosphorylation at serine 783, reflecting receptor deactivation and proteolysis. These data show a role of Reelin in controlling early network activity, by modulating GABAB R function. Cover Image for this issue: https://doi.org/10.1111/jnc.15054.


Asunto(s)
Moléculas de Adhesión Celular Neuronal/deficiencia , Proteínas de la Matriz Extracelular/deficiencia , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Receptores de GABA-B/fisiología , Serina Endopeptidasas/deficiencia , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Femenino , Agonistas de Receptores GABA-B/farmacología , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Técnicas de Cultivo de Órganos , Proteína Reelina , Serina Endopeptidasas/genética , Transducción de Señal/efectos de los fármacos
4.
Biomolecules ; 14(5)2024 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-38786001

RESUMEN

During the first and second stages of postnatal development, neocortical neurons exhibit a wide range of spontaneous synchronous activity (SSA). Towards the end of the second postnatal week, the SSA is replaced by a more sparse and desynchronized firing pattern. The developmental desynchronization of neocortical spontaneous neuronal activity is thought to be intrinsically generated, since sensory deprivation from the periphery does not affect the time course of this transition. The extracellular protein reelin controls various aspects of neuronal development through multimodular signaling. However, so far it is unclear whether reelin contributes to the developmental desynchronization transition of neocortical neurons. The present study aims to investigate the role of reelin in postnatal cortical developmental desynchronization using a conditional reelin knockout (RelncKO) mouse model. Conditional reelin deficiency was induced during early postnatal development, and Ca2+ recordings were conducted from organotypic cultures (OTCs) of the somatosensory cortex. Our results show that both wild type (wt) and RelncKO exhibited an SSA pattern during the early postnatal week. However, at the end of the second postnatal week, wt OTCs underwent a transition to a desynchronized network activity pattern, while RelncKO activity remained synchronous. This changing activity pattern suggests that reelin is involved in regulating the developmental desynchronization of cortical neuronal network activity. Moreover, the developmental desynchronization impairment observed in RelncKO was rescued when RelncKO OTCs were co-cultured with wt OTCs. Finally, we show that the developmental transition to a desynchronized state at the end of the second postnatal week is not dependent on glutamatergic signaling. Instead, the transition is dependent on GABAAR and GABABR signaling. The results suggest that reelin controls developmental desynchronization through GABAAR and GABABR signaling.


Asunto(s)
Proteínas de la Matriz Extracelular , Ratones Noqueados , Neocórtex , Proteínas del Tejido Nervioso , Proteína Reelina , Serina Endopeptidasas , Animales , Ratones , Neocórtex/metabolismo , Neocórtex/crecimiento & desarrollo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Neuronas/metabolismo , Red Nerviosa/metabolismo , Red Nerviosa/crecimiento & desarrollo , Corteza Somatosensorial/metabolismo , Corteza Somatosensorial/crecimiento & desarrollo
5.
J Neurosci Methods ; 337: 108666, 2020 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-32119875

RESUMEN

BACKGROUND: Biolistic gene gun transfection has been used to transfect organotypic cultures (OTCs) or dissociated cultures in vitro. Here, we modified this technique to allow successful transfection of acute brain slices, followed by measurement of neuronal activity within a few hours. NEW METHOD: We established biolistic transfection of murine acute cortical slices to measure calcium signals. Acute slices are mounted on plasma/thrombin coagulate and transfected with a calcium sensor. Imaging can be performed within 4 h post transfection without affecting cell viability. RESULTS: Four hours after GCaMP6s transfection, acute slices display remarkable fluorescent protein expression level allowing to study spontaneous activity and receptor pharmacology. While optimal gas pressure (150 psi) and gold particle size used (1 µm) confirm previously published protocols, the amount of 5 µg DNA was found to be optimal for particle coating. COMPARISON WITH EXISTING METHODS: The major advantage of this technique is the rapid disposition of acute slices for calcium imaging. No transgenic GECI expressing animals or OTC for long periods are required. In acute slices, network interaction and connectivity are preserved. The method allows to obtain physiological readouts within 4 h, before functional tissue modifications might come into effect. Limitations of this technique are random transfection, low expression efficiency when using specific promotors, and preclusion or genetic manipulations that require a prolonged time before physiological changes become measurable, such as expression of recombinant proteins that require transport to distant subcellular localizations. CONCLUSION: The method is optimal for short-time investigation of calcium signals in acute slices.


Asunto(s)
Biolística , Neuronas , Animales , ADN , Técnicas de Transferencia de Gen , Ratones , Transfección
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