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1.
Sci Adv ; 10(25): eadj0720, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38896627

RESUMO

Mutations in the transcription factors encoded by PHOX2B or LBX1 correlate with congenital central hypoventilation disorders. These conditions are typically characterized by pronounced hypoventilation, central apnea, and diminished chemoreflexes, particularly to abnormally high levels of arterial PCO2. The dysfunctional neurons causing these respiratory disorders are largely unknown. Here, we show that distinct, and previously undescribed, sets of medullary neurons coexpressing both transcription factors (dB2 neurons) account for specific respiratory functions and phenotypes seen in congenital hypoventilation. By combining intersectional chemogenetics, intersectional labeling, lineage tracing, and conditional mutagenesis, we uncovered subgroups of dB2 neurons with key functions in (i) respiratory tidal volumes, (ii) the hypercarbic reflex, (iii) neonatal respiratory stability, and (iv) neonatal survival. These data provide functional evidence for the critical role of distinct medullary dB2 neurons in neonatal respiratory physiology. In summary, our work identifies distinct subgroups of dB2 neurons regulating breathing homeostasis, dysfunction of which causes respiratory phenotypes associated with congenital hypoventilation.


Assuntos
Proteínas de Homeodomínio , Hipoventilação , Bulbo , Neurônios , Fatores de Transcrição , Hipoventilação/congênito , Hipoventilação/genética , Animais , Neurônios/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Bulbo/metabolismo , Apneia do Sono Tipo Central/genética , Fenótipo , Humanos
2.
Cell Rep ; 42(8): 112934, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37537840

RESUMO

Extracellular potassium [K+]o elevation during synaptic activity retrogradely modifies presynaptic release and astrocytic uptake of glutamate. Hence, local K+ clearance and replenishment mechanisms are crucial regulators of glutamatergic transmission and plasticity. Based on recordings of astrocytic inward rectifier potassium current IKir and K+-sensitive electrodes as sensors of [K+]o as well as on in silico modeling, we demonstrate that the neuronal K+-Cl- co-transporter KCC2 clears local perisynaptic [K+]o during synaptic excitation by operating in an activity-dependent reversed mode. In reverse mode, KCC2 replenishes K+ in dendritic spines and complements clearance of [K+]o, therewith attenuating presynaptic glutamate release and shortening LTP. We thus demonstrate a physiological role of KCC2 in neuron-glial interactions and regulation of synaptic signaling and plasticity through the uptake of postsynaptically released K+.


Assuntos
Potássio , Simportadores , Animais , Glutamatos , Potássio/metabolismo , Sinapses/metabolismo , Cotransportadores de K e Cl-
3.
Front Cell Neurosci ; 16: 913299, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36035261

RESUMO

Interferon-γ (IFN-γ), a cytokine with neuromodulatory properties, has been shown to enhance inhibitory transmission. Because early inhibitory neurotransmission sculpts functional neuronal circuits, its developmental alteration may have grave consequences. Here, we investigated the acute effects of IFN-γ on γ-amino-butyric acid (GABA)ergic currents in layer 5 pyramidal neurons of the somatosensory cortex of rats at the end of the first postnatal week, a period of GABA-dependent cortical maturation. IFN-γ acutely increased the frequency and amplitude of spontaneous/miniature inhibitory postsynaptic currents (s/mIPSC), and this could not be reversed within 30 min. Neither the increase in amplitude nor frequency of IPSCs was due to upregulated interneuron excitability as revealed by current clamp recordings of layer 5 interneurons labeled with VGAT-Venus in transgenic rats. As we previously reported in more mature animals, IPSC amplitude increase upon IFN-γ activity was dependent on postsynaptic protein kinase C (PKC), indicating a similar activating mechanism. Unlike augmented IPSC amplitude, however, we did not consistently observe an increased IPSC frequency in our previous studies on more mature animals. Focusing on increased IPSC frequency, we have now identified a different activating mechanism-one that is independent of postsynaptic PKC but is dependent on inducible nitric oxide synthase (iNOS) and soluble guanylate cyclase (sGC). In addition, IFN-γ shifted short-term synaptic plasticity toward facilitation as revealed by a paired-pulse paradigm. The latter change in presynaptic function was not reproduced by the application of a nitric oxide donor. Functionally, IFN-γ-mediated alterations in GABAergic transmission overall constrained early neocortical activity in a partly nitric oxide-dependent manner as revealed by microelectrode array field recordings in brain slices analyzed with a spike-sorting algorithm. In summary, with IFN-γ-induced, NO-dependent augmentation of spontaneous GABA release, we have here identified a mechanism by which inflammation in the central nervous system (CNS) plausibly modulates neuronal development.

4.
Brain Behav Immun ; 101: 153-164, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34998939

RESUMO

Interferon-γ (IFN-γ), an important mediator of the antiviral immune response, can also act as a neuromodulator. CNS IFN-γ levels rise acutely in response to infection and therapeutically applied IFN-γ provokes CNS related side effects. Moreover, IFN-γ plays a key role in neurophysiological processes and a variety of chronic neurological and neuropsychiatric conditions. To close the gap between basic research, behavioral implications and clinical applicability, knowledge of the mechanism behind IFN-γ related changes in brain function is crucial. Here, we studied the underlying mechanism of acutely augmented neocortical inhibition by IFN-γ (1.000 IU ml-1) in layer 5 pyramidal neurons of male Wistar rats. We demonstrate postsynaptic mediation of IFN-γ augmented inhibition by pressure application of GABA and analysis of paired pulse ratios. IFN-γ increases membrane presence of GABAAR γ2, as quantified by cell surface biotinylation and functional synaptic GABAAR number, as determined by peak-scaled non-stationary noise analysis. The increase in functional receptor number was comparable to the increase in underlying miniature inhibitory postsynaptic current (mIPSC) amplitudes. Blockage of putative intracellular mediators, namely phosphoinositide 3-kinase and protein kinase C (PKC) by Wortmannin and Calphostin C, respectively, revealed PKC-dependency of the pro-inhibitory IFN-γ effect. This was corroborated by increased serine phosphorylation of P-serine PKC motifs on GABAAR γ2 upon IFN-γ application. GABAAR single channel conductance, intracellular chloride levels and GABAAR driving force are unlikely to contribute to the effect, as shown by single channel recordings and chloride imaging. The effect of IFN-γ on mIPSC amplitudes was similar in female and male rats, suggesting a gender-independent mechanism of action. Collectively, these results indicate a novel mechanism for the regulation of inhibition by IFN-γ, which could impact on neocortical function and therewith behavior.


Assuntos
Neocórtex , Receptores de GABA-A , Animais , Cloretos/metabolismo , Feminino , Interferon gama/metabolismo , Interferon gama/farmacologia , Masculino , Neocórtex/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Receptores de GABA-A/metabolismo , Serina/metabolismo , Ácido gama-Aminobutírico/metabolismo
5.
Sci Rep ; 10(1): 13488, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778682

RESUMO

We explored the non-thermal effects of radiofrequency (RF) electromagnetic fields and established a theoretical framework to elucidate their electrophysiological mechanisms. In experiments, we used a preclinical treatment device to treat the human colon cancer cell lines HT-29 and SW480 with either water bath heating (WB-HT) or 13.56 MHz RF hyperthermia (RF-HT) at 42 °C for 60 min and analyzed the proliferation and clonogenicity. We elaborated an electrical model for cell membranes and ion channels and estimated the resulting ion fluxes. The results showed that, for both cell lines, using RF-HT significantly reduced proliferation and clonogenicity compared to WB-HT. According to our model, the RF electric field component was rectified and smoothed in the direction of the channel, which resulted in a DC voltage of ~ 1 µV. This may induce ion fluxes that can potentially cause relevant disequilibrium of most ions. Therefore, RF-HT creates additional non-thermal effects in association with significant ion fluxes. Increasing the understanding of these effects can help improve cancer therapy.

6.
J Neuroinflammation ; 17(1): 69, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32087716

RESUMO

BACKGROUND: Interferon-γ (IFN-γ, a type II IFN) is present in the central nervous system (CNS) under various conditions. Evidence is emerging that, in addition to its immunological role, IFN-γ modulates neuronal morphology, function, and development in several brain regions. Previously, we have shown that raising levels of IFN-ß (a type I IFN) lead to increased neuronal excitability of neocortical layer 5 pyramidal neurons. Because of shared non-canonical signaling pathways of both cytokines, we hypothesized a similar neocortical role of acutely applied IFN-γ. METHODS: We used semi-quantitative RT-PCR, immunoblotting, and immunohistochemistry to analyze neuronal expression of IFN-γ receptors and performed whole-cell patch-clamp recordings in layer 5 pyramidal neurons to investigate sub- and suprathreshold excitability, properties of hyperpolarization-activated cyclic nucleotide-gated current (Ih), and inhibitory neurotransmission under the influence of acutely applied IFN-γ. RESULTS: We show that IFN-γ receptors are present in the membrane of rat's neocortical layer 5 pyramidal neurons. As expected from this and the putative overlap in IFN type I and II alternative signaling pathways, IFN-γ diminished Ih, mirroring the effect of type I IFNs, suggesting a likewise activation of protein kinase C (PKC). In contrast, IFN-γ did neither alter subthreshold nor suprathreshold neuronal excitability, pointing to augmented inhibitory transmission by IFN-γ. Indeed, IFN-γ increased electrically evoked inhibitory postsynaptic currents (IPSCs) on neocortical layer 5 pyramidal neurons. Furthermore, amplitudes of spontaneous IPSCs and miniature IPSCs were elevated by IFN-γ, whereas their frequency remained unchanged. CONCLUSIONS: The expression of IFN-γ receptors on layer 5 neocortical pyramidal neurons together with the acute augmentation of inhibition in the neocortex by direct application of IFN-γ highlights an additional interaction between the CNS and immune system. Our results strengthen our understanding of the role of IFN-γ in neocortical neurotransmission and emphasize its impact beyond its immunological properties, particularly in the pathogenesis of neuropsychiatric disorders.


Assuntos
Interferon gama/metabolismo , Neocórtex/metabolismo , Neuroimunomodulação/fisiologia , Células Piramidais/metabolismo , Receptores de Interferon/metabolismo , Animais , Interferon gama/farmacologia , Masculino , Neocórtex/efeitos dos fármacos , Neocórtex/imunologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/imunologia , Ratos , Ratos Wistar
7.
Neurobiol Dis ; 129: 130-143, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31102767

RESUMO

Congenital microcephaly is highly associated with intellectual disability. Features of autosomal recessive primary microcephaly subtype 3 (MCPH3) also include hyperactivity and seizures. The disease is caused by biallelic mutations in the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the mouse, Cdk5rap2 mutations similar to the human condition result in reduced brain size and a strikingly thin neocortex already at early stages of neurogenesis that persists through adulthood. The microcephaly phenotype in MCPH arises from a neural stem cell proliferation defect. Here, we report a novel role for Cdk5rap2 in the regulation of dendritic development and synaptogenesis of neocortical layer 2/3 pyramidal neurons. Cdk5rap2-deficient murine neurons show poorly branched dendritic arbors and an increased density of immature thin spines and glutamatergic synapses in vivo. Moreover, the excitatory drive is enhanced in ex vivo brain slice preparations of Cdk5rap2 mutant mice. Concurrently, we show that pyramidal neurons receive fewer inhibitory inputs. Together, these findings point towards a shift in the excitation - inhibition balance towards excitation in Cdk5rap2 mutant mice. Thus, MCPH3 is associated not only with a neural progenitor proliferation defect but also with altered function of postmitotic neurons and hence with altered connectivity.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Microcefalia/fisiopatologia , Neocórtex/fisiopatologia , Vias Neurais/fisiopatologia , Neurogênese/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular/fisiologia , Camundongos , Camundongos Mutantes , Microcefalia/genética , Microcefalia/metabolismo , Mutação , Neocórtex/metabolismo , Vias Neurais/metabolismo , Células Piramidais/metabolismo , Células Piramidais/patologia , Transmissão Sináptica/fisiologia
8.
Neuropharmacology ; 144: 58-69, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30315843

RESUMO

Alterations of the hyperpolarization activated nonselective cation current (Ih) are associated with epileptogenesis. Accordingly, the second-generation antiepileptic drug lamotrigine (LTG) enhances Ih in rodent hippocampus. We directly evaluated here whether LTG fails to enhance Ih in neocortical slices from patients with pharmacoresistant epilepsy. With somatic current clamp recordings we observed that LTG depolarized the membrane potential, decreased the input resistance and increased the "sag" in human layer 2/3 neocortical pyramidal neurons when confounding IKir was blocked. In subsequent voltage clamp recordings we confirmed a LTG induced increase of Ih that was qualitatively similar to the one we found in rat neocortical and hippocampal pyramidal neurons. This increase is sufficient to curtail single excitatory postsynaptic potentials (EPSPs) and reduces their temporal summation in human neocortical pyramidal neurons under physiological conditions, i.e. without blocking any other currents, as estimated by sharp microelectrode recordings. Taken together LTG increases Ih and thereby alters neuronal excitability, even in neurons of pharmacoresistant patients. However, whether this increase fully countervails the deficits of Ih in epileptic patients, remains elusive.


Assuntos
Anticonvulsivantes/farmacologia , Epilepsia Resistente a Medicamentos/tratamento farmacológico , Epilepsia do Lobo Temporal/tratamento farmacológico , Lamotrigina/farmacologia , Neocórtex/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Adulto , Animais , Epilepsia Resistente a Medicamentos/fisiopatologia , Epilepsia Resistente a Medicamentos/cirurgia , Epilepsia do Lobo Temporal/fisiopatologia , Epilepsia do Lobo Temporal/cirurgia , Feminino , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neocórtex/fisiopatologia , Células Piramidais/fisiologia , Ratos Wistar , Técnicas de Cultura de Tecidos
9.
Cereb Cortex ; 27(1): 131-145, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27909001

RESUMO

Altered synaptic bioactive lipid signaling has been recently shown to augment neuronal excitation in the hippocampus of adult animals by activation of presynaptic LPA2-receptors leading to increased presynaptic glutamate release. Here, we show that this results in higher postsynaptic Ca2+ levels and in premature onset of spontaneous neuronal activity in the developing entorhinal cortex. Interestingly, increased synchronized neuronal activity led to reduced axon growth velocity of entorhinal neurons which project via the perforant path to the hippocampus. This was due to Ca2+-dependent molecular signaling to the axon affecting stabilization of the actin cytoskeleton. The spontaneous activity affected the entire entorhinal cortical network and thus led to reduced overall axon fiber numbers in the mature perforant path that is known to be important for specific memory functions. Our data show that precise regulation of early cortical activity by bioactive lipids is of critical importance for proper circuit formation.


Assuntos
Axônios/fisiologia , Sinalização do Cálcio/fisiologia , Ácido Glutâmico/metabolismo , Redes e Vias Metabólicas/fisiologia , Crescimento Neuronal/fisiologia , Fosfolipídeos/metabolismo , Transmissão Sináptica/fisiologia , Animais , Axônios/ultraestrutura , Cálcio/metabolismo , Células Cultivadas , Camundongos
10.
Cell Physiol Biochem ; 38(6): 2079-93, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27184742

RESUMO

BACKGROUND/AIMS: Cationic currents (Ih) through the fast activating and relatively cAMP insensitive subtype of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, HCN1, are limited by cytosolic factors in mammalian cells. This cytosolic HCN1 break is boosted by changes in membrane voltage that are not characterized on a biophysical level, yet. METHODS: We overexpressed rat (r)HCN1 in human embryonic kidney cells (HEK293) and recorded pharmacologically isolated Ih in cell-attached or whole-cell mode of the patch-clamp technique. RESULTS: Recurring activation of rHCN1 reduced and slowed Ih in intact HEK293 cells (cell-attached mode). On the contrary, sustained disruption of the intracellular content (whole-cell mode) ceased activity dependence and partially enables voltage dependent hysteresis. The activity induced Ih attenuation in intact cells was independent of the main external cation, depended on the number of previous forced activations and was - at least in part - due to a shift in the voltage dependence of activation towards hyperpolarization as estimated by an adapted tail current analysis. Intracellular elevation of cAMP could not reverse the changes in Ih. CONCLUSION: Reduction of rHCN1 mediated Ih is use dependent and may involve the coupling of voltage sensor and pore.


Assuntos
Células HEK293/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais de Potássio/metabolismo , Animais , Cátions/metabolismo , AMP Cíclico/metabolismo , Células HEK293/citologia , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Técnicas de Patch-Clamp , Canais de Potássio/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sódio/metabolismo , Regulação para Cima
11.
J Neurotrauma ; 32(19): 1465-77, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25923828

RESUMO

Studies of axonal outgrowth and regeneration after spinal cord injury are hampered by the complexity of the events involved. Here, we present a simple and improved in vitro approach to investigate outgrowth, regeneration of the corticospinal tract, and intrinsic parenchymal responses. We prepared organotypic co-cultures using explants from the motor cortex of postnatal donor mice ubiquitously expressing green fluorescent protein and cervical spinal cord from wild type pups of the same age. Our data show that: a) motor-cortical outgrowth is already detectable after 1 d in culture and is source specific; b) treatment with neurotrophin-3 and C3 transferase from Clostridium botulinum significantly enhances axonal outgrowth during the course of cultivation; c) outgrowing axons form synaptic connections, as demonstrated by immunohistochemistry and calcium imaging; and d) migrating cells of motor-cortical origin can be reliably identified without previous tracing and are mostly neural precursors that survive and mature in the spinal cord parenchyma. Thus, our model is suitable for screening for candidate substances that enhance outgrowth and regeneration of the corticospinal tract and for studying the role of endogenous neural precursors after lesion induction.


Assuntos
Axônios/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Tratos Piramidais/crescimento & desenvolvimento , ADP Ribose Transferases/farmacologia , Actinas/genética , Animais , Toxinas Botulínicas/farmacologia , Movimento Celular , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Proteínas de Fluorescência Verde , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Córtex Motor/crescimento & desenvolvimento , Fatores de Crescimento Neural/farmacologia , Técnicas de Cultura de Órgãos , Tratos Piramidais/citologia , Medula Espinal/crescimento & desenvolvimento , Sinapses/efeitos dos fármacos
12.
J Neuroinflammation ; 11: 185, 2014 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-25359459

RESUMO

BACKGROUND: Cytokines are key players in the interactions of the immune and nervous systems. Recently, we showed that such interplay is mediated by type I interferons (IFNs), which elevate the excitability of neocortical pyramidal neurons. A line of indirect evidence suggested that modulation of multiple ion channels underlies the effect. However, which currents are principally involved and how the IFN signaling cascade is linked to the respective ion channels remains elusive. METHODS: We tested several single and combined ionic current modulations using an in silico model of a neocortical layer 5 neuron. Subsequently we investigated resulting predictions by whole-cell patch-clamp recordings in layer 5 neurons of ex vivo neocortical rat brain slices pharmacologically reproducing or prohibiting neuronal IFN effects. RESULTS: The amount and type of modulation necessary to replicate IFN effects in silico suggested protein kinase C (PKC) activation as link between the type I IFN signaling and ion channel modulations. In line with this, PKC activation with 4ß-phorbol 12-myristate 13-acetate (4ß-PMA) or Bryostatin1 augmented the excitability of neocortical layer 5 neurons comparable to IFN-ß in our ex vivo recordings. In detail, both PKC activators attenuated the rheobase and increased the input-output gain as well as the input resistance, thereby augmenting the neuronal excitability. Similar to IFN-ß they also left the threshold of action potential generation unaffected. In further support of PKC mediating type I IFN effects, IFN-ß, 4ß-PMA and Bryostatin1 reduced the amplitude of post-train after-hyperpolarizations in a similar manner. In conjunction with this finding, IFN-ß reduced M-currents, which contribute to after-hyperpolarizations and are modulated by PKC. Finally, blocking PKC activation with GF109203X at the catalytic site or calphostin C at the regulatory site prevented the main excitatory effects of IFN-ß. CONCLUSION: Multiple ion channel modulations underlie the neuromodulatory effect of type I IFNs. PKC activation is both sufficient and necessary for mediating the effect, and links the IFN signaling cascade to the intrinsic ion channels. Therefore, we regard PKC activation as unitary mechanism for the neuromodulatory potential of type I IFNs in neocortical neurons.


Assuntos
Interferon beta/metabolismo , Neocórtex/metabolismo , Neuroimunomodulação/fisiologia , Proteína Quinase C/metabolismo , Células Piramidais/metabolismo , Transdução de Sinais/fisiologia , Animais , Ativação Enzimática/fisiologia , Canais Iônicos/fisiologia , Masculino , Modelos Neurológicos , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar
13.
J Biol Chem ; 289(36): 24956-70, 2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25074937

RESUMO

The transmembrane protein plasticity-related genes 3 and 5 (PRG3 and PRG5) increase filopodial formation in various cell lines, independently of Cdc42. However, information on the effects of PRG5 during neuronal development is sparse. Here, we present several lines of evidence for the involvement of PRG5 in the genesis and stabilization of dendritic spines. First, PRG5 was strongly expressed during mouse brain development from embryonic day 14 (E14), peaked around the time of birth, and remained stable at least until early adult stages (i.e. P30). Second, on a subcellular level, PRG5 expression shifted from an equal distribution along all neurites toward accumulation only along dendrites during hippocampal development in vitro. Third, overexpression of PRG5 in immature hippocampal neurons induced formation of spine-like structures ahead of time. Proper amino acid sequences in the extracellular domains (D1 to D3) of PRG5 were a prerequisite for trafficking and induction of spine-like structures, as shown by mutation analysis. Fourth, at stages when spines are present, knockdown of PRG5 reduced the number but not the length of protrusions. This was accompanied by a decrease in the number of excitatory synapses and, consequently, by a reduction of miniature excitatory postsynaptic current frequencies, although miniature excitatory postsynaptic current amplitudes remained similar. In turn, overexpressing PRG5 in mature neurons not only increased Homer-positive spine numbers but also augmented spine head diameters. Mechanistically, PRG5 interacts with phosphorylated phosphatidylinositols, phospholipids involved in dendritic spine formation by different lipid-protein assays. Taken together, our data propose that PRG5 promotes spine formation.


Assuntos
Espinhas Dendríticas/genética , Hipocampo/metabolismo , Proteínas de Membrana/genética , Neurônios/metabolismo , Monoéster Fosfórico Hidrolases/genética , Animais , Astrócitos/metabolismo , Western Blotting , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Células Cultivadas , Espinhas Dendríticas/metabolismo , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Hipocampo/embriologia , Hipocampo/crescimento & desenvolvimento , Humanos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microscopia Confocal , Neurônios/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Gravidez , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia
14.
Cereb Cortex ; 24(1): 199-210, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23042740

RESUMO

Central nervous system (CNS) inflammation involves the generation of inducible cytokines such as interferons (IFNs) and alterations in brain activity, yet the interplay of both is not well understood. Here, we show that in vivo elevation of IFNs by viral brain infection reduced hyperpolarization-activated currents (Ih) in cortical pyramidal neurons. In rodent brain slices directly exposed to type I IFNs, the hyperpolarization-activated cyclic nucleotide (HCN)-gated channel subunit HCN1 was specifically affected. The effect required an intact type I receptor (IFNAR) signaling cascade. Consistent with Ih inhibition, IFNs hyperpolarized the resting membrane potential, shifted the resonance frequency, and increased the membrane impedance. In vivo application of IFN-ß to the rat and to the mouse cerebral cortex reduced the power of higher frequencies in the cortical electroencephalographic activity only in the presence of HCN1. In summary, these findings identify HCN1 channels as a novel neural target for type I IFNs providing the possibility to tune neural responses during the complex event of a CNS inflammation.


Assuntos
Córtex Cerebral/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Interferon Tipo I/fisiologia , Neurônios/fisiologia , Canais de Potássio/fisiologia , Animais , Western Blotting , Córtex Cerebral/citologia , Simulação por Computador , Citocinas/fisiologia , Eletroencefalografia , Fenômenos Eletrofisiológicos/fisiologia , Células HEK293 , Humanos , Imuno-Histoquímica , Interferon Tipo I/biossíntese , Interferon beta/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Neocórtex/citologia , Neocórtex/metabolismo , Neocórtex/fisiologia , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Técnicas de Patch-Clamp , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interferon/fisiologia , Transdução de Sinais/fisiologia , Transfecção
15.
Dev Neurobiol ; 73(10): 785-97, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23821600

RESUMO

The distribution of ion channels in neurons regulates neuronal activity and proper formation of neuronal networks during neuronal development. One of the channels is the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel constituting the molecular substrate of hyperpolarization-activated current (I(h)). Our previous study implied a role for the fastest activating subunit HCN1 in the generation of Ih in rat neonatal cortical plate neurons. To better understand the impact of HCN1 in early neocortical development, we here performed biochemical analysis and whole-cell recordings in neonatal cortical plate and juvenile layer 5 somatosensory neurons of HCN1(-/-) and control HCN1(+/+) mice. Western Blot analysis revealed that HCN1 protein expression in neonatal cortical plate tissue of HCN(+/+) mice amounted to only 3% of the HCN1 in young adult cortex and suggested that in HCN1(-/-) mice other isoforms (particularly HCN4) might be compensatory up-regulated. At the first day after birth, functional ablation of the HCN1 subunit did not affect the proportion of Ih expressing pyramidal cortical plate neurons. Although the contribution of individual subunit proteins remains open, the lack of HCN1 markedly slowed the current activation and deactivation in individual I(h) expressing neurons. However, it did not impair maximal amplitude/density, voltage dependence of activation, and cAMP sensitivity. In conclusion, our data imply that, although expression is relatively low, HCN1 contributes substantially to I(h) properties in individual cortical plate neurons. These properties are significantly changed in HCN1(-/-), either due to the lack of HCN1 itself or due to compensatory mechanisms.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Potenciais da Membrana/fisiologia , Neurônios/metabolismo , Canais de Potássio/metabolismo , Animais , Animais Recém-Nascidos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Feminino , Técnicas de Inativação de Genes , Hipocampo/citologia , Hipocampo/fisiologia , Cinética , Camundongos , Camundongos da Linhagem 129 , Células Piramidais/citologia , Ratos , Ratos Wistar
16.
Cell Physiol Biochem ; 31(4-5): 532-41, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23571312

RESUMO

BACKGROUND/AIMS: Hyperpolarization activated cyclic nucleotide gated 1 (HCN1) channels determine neuronal excitability in several brain regions. In contrast to HCN2 and HCN4, HCN1 is less sensitive to cAMP and the number of other known modulators is limited. One of those, the protein kinase C (PKC), showed opposing effects on mouse HCN1 channels expressed in Xenopus oocytes. METHODS: In order to study PKC effects on HCN1 mediated currents in a mammalian environment we expressed rat HCN1 or human HCN1 in human embryonic kidney (HEK293) cells and rat HCN1 in murine neuroblastoma (N1E-115) cells. We recorded the resulting Ih before and during the application of the membrane permeable non-metabolizable PKC-activator 4ßPMA in cell-attached mode of the patch-clamp technique, leaving the intracellular environment intact. RESULTS: 4ßPMA reduced maximal HCN1 mediated currents to about 60-70 % and slowed its activation, but left its voltage sensitivity unchanged. The effect was neither due to species-related differences nor restricted to HEK293 cells, because it was comparable for human and rat HCN1 in HEK293 and for rat HCN1 in N1E-115 cells. However, pre-treatment with the PKC blocker GF109203X abolished 4ßPMA induced Ih changes. Disrupting the intracellular environment by recording in whole-cell mode drastically reduced the 4ßPMA effect. CONCLUSION: PKC activation reduces and slows Ih in non-neuronal and neuronal mammalian cells transfected with rat or human HCN1 if the intracellular content remains intact.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteína Quinase C/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Indóis/farmacologia , Maleimidas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Técnicas de Patch-Clamp , Proteína Quinase C/química , Inibidores de Proteínas Quinases/farmacologia , Ratos , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia
17.
BMC Neurosci ; 14: 36, 2013 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-23506325

RESUMO

BACKGROUND: Recently, we and others proposed plasticity-related gene 3 (PRG3) as a novel molecule in neuritogenesis based on PRG3 overexpression experiments in neuronal and non-neuronal cell lines. However, direct information on PRG3 effects in neuronal development and, in particular, its putative spatio-temporal distribution and conditions of action, is sparse. RESULTS: We demonstrate here that PRG3 induces filopodia formation in HEK293 cells depending on its N-glycosylation status. The PRG3 protein was strongly expressed during mouse brain development in vivo from embryonic day 16 to postnatal day 5 (E16 - P5). From P5 on, expression declined. Furthermore, in early, not yet polarized hippocampal cultured neurons, PRG3 was expressed along the neurite shaft. Knock-down of PRG3 in these neurons led to a decreased number of neurites. This phenotype is rescued by expression of an shRNA-resistant PRG3 construct in PRG3 knock-down neurons. After polarization, endogenous PRG3 expression shifted mainly to axons, specifically to the plasma membrane along the neurite shaft. These PRG3 pattern changes appeared temporally and spatially related to ongoing synaptogenesis. Therefore we tested (i) whether dendritic PRG3 re-enhancement influences synaptic currents and (ii) whether synaptic inputs contribute to the PRG3 shift. Our results rendered both scenarios unlikely: (i) PRG3 over-expression had no influence on miniature excitatory postsynaptic currents (mEPSC) and (ii) blocking of incoming signals did not alter PRG3 distribution dynamics. In addition, PRG3 levels did not interfere with intrinsic neuronal properties. CONCLUSION: Taken together, our data indicate that endogenous PRG3 promotes neurite shaft protrusion and therefore contributes to regulating filopodia formation in immature neurons. PRG3 expression in more mature neurons, however, is predominantly localized in the axon. Changes in PRG3 levels did not influence intrinsic or synaptic neuronal properties.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Neuritos/metabolismo , Neurônios/citologia , Análise de Variância , Animais , Animais Recém-Nascidos , Asparagina/genética , Asparagina/metabolismo , Células Cultivadas , Proteína Básica Maior de Eosinófilos/genética , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Glicosilação , Proteínas de Fluorescência Verde/genética , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/citologia , Técnicas de Patch-Clamp , Mutação Puntual/genética , Gravidez , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Pseudópodes/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transfecção
18.
Biochim Biophys Acta ; 1831(1): 133-8, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23388400

RESUMO

Plasticity-related genes (PRGs, Lipid phosphate phosphatase-related proteins LPPRs) are a defined as a subclass of the lipid phosphate phosphatase (LPP) superfamily, comprising so far five brain- and vertebrate-specific membrane-spanning proteins. LPPs interfere with lipid phosphate signaling and are thereby involved in mediating the extracellular concentration and signal transduction of lipid phosphate esters such as lysophosphatidate (LPA) and spingosine-1 phosphate (S1P). LPPs dephosphorylate their substrates through extracellular catalytic domains, thus making them ecto-phosphatases. PRGs/LPPRs are structurally similar to the other LPP family members in general. They are predominantly expressed in the CNS in a subtype specific pattern rather than having a wide tissue distribution. In contrast to LPPs, PRGs/LPPRs may act by modifying bioactive lipids and their signaling pathways, rather than possessing an ecto-phosphatase activity. However, the exact functional roles of PRGs/LPPRs have just begun to be explored. Here, we discuss new findings on the neuron-specific transcriptional regulation of PRG1/LPPR4 and new insights into protein-protein interaction and signaling pathway regulation. Further, we start to shed light on the subcellular localization and the resulting functional modulatory influence of PRG1/LPPR4 expression in excitatory synaptic transmission to the established neural effects such as promotion of filopodia formation, neurite extension, axonal sprouting and reorganization after lesion. This range of effects suggests an involvement in the pathogenesis and/or reparation attempts in disease. Therefore, we summarize available data on the association of PRGs/LPPRs with several neurological and other diseases in humans and experimental animals. Finally we highlight important open questions and emerging future directions of research. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.


Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Monoéster Fosfórico Hidrolases/genética , Animais , Encéfalo/patologia , Humanos , Neoplasias/genética , Neoplasias/patologia , Proteínas do Tecido Nervoso/metabolismo , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/fisiopatologia , Monoéster Fosfórico Hidrolases/metabolismo
19.
Neural Dev ; 7: 21, 2012 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-22694806

RESUMO

BACKGROUND: During neocortical development, multiple voltage- and ligand-gated ion channels are differentially expressed in neurons thereby shaping their intrinsic electrical properties. One of these voltage-gated ion channels, the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel and its current I(h), is an important regulator of neuronal excitability. Thus far, studies on an early I(h) appearance in rodent neocortex are missing or conflicting. Therefore, we focused our study on perinatal neocortical I(h) and its properties. RESULTS: In the perinatal rat neocortex we observed a rapid increase in the number of neurons exhibiting I(h). Perinatal I(h) had unique properties: first, a pronounced cAMP sensitivity resulting in a marked shift of the voltage sufficient for half-maximum activation of the current towards depolarized voltages and second, an up to 10 times slower deactivation at physiological membrane potentials when compared to the one at postnatal day 30. The combination of these features was sufficient to suppress membrane resonance in our in silico and in vitro experiments. Although all four HCN subunits were present on the mRNA level we only detected HCN4, HCN3 and HCN1 on the protein level at P0. HCN1 protein at P0, however, appeared incompletely processed. At P30 glycosilated HCN1 and HCN2 dominated. By in silico simulations and heterologous co-expression experiments of a 'slow' and a 'fast' I(h) conducting HCN channel subunit in HEK293 cells, we mimicked most characteristics of the native current, pointing to a functional combination of subunit homo- or heteromeres. CONCLUSION: Taken together, these data indicate a HCN subunit shift initiated in the first 24 hours after birth and implicate a prominent perinatal role of the phylogenetically older HCN3 and/or HCN4 subunits in the developing neocortex.


Assuntos
Córtex Cerebral/citologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Biofísica , Cálcio/metabolismo , Linhagem Celular Transformada , Simulação por Computador , AMP Cíclico/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/antagonistas & inibidores , Canais de Cátion Regulados por Nucleotídeos Cíclicos/classificação , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Estimulação Elétrica , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Modelos Neurológicos , Mutação/genética , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Gravidez , Pirimidinas/farmacologia , Pirrolidinonas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transfecção
20.
Parkinsons Dis ; 2011: 414682, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21603182

RESUMO

Deep brain stimulation (DBS) has become a treatment for a growing number of neurological and psychiatric disorders, especially for therapy-refractory Parkinson's disease (PD). However, not all of the symptoms of PD are sufficiently improved in all patients, and side effects may occur. Further progress depends on a deeper insight into the mechanisms of action of DBS in the context of disturbed brain circuits. For this, optimized animal models have to be developed. We review not only charge transfer mechanisms at the electrode/tissue interface and strategies to increase the stimulation's energy-efficiency but also the electrochemical, electrophysiological, biochemical and functional effects of DBS. We introduce a hemi-Parkinsonian rat model for long-term experiments with chronically instrumented rats carrying a backpack stimulator and implanted platinum/iridium electrodes. This model is suitable for (1) elucidating the electrochemical processes at the electrode/tissue interface, (2) analyzing the molecular, cellular and behavioral stimulation effects, (3) testing new target regions for DBS, (4) screening for potential neuroprotective DBS effects, and (5) improving the efficacy and safety of the method. An outlook is given on further developments of experimental DBS, including the use of transgenic animals and the testing of closed-loop systems for the direct on-demand application of electric stimulation.

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