RESUMO
Neuronal differentiation is regulated by neuronal activity. Here, we analyzed dendritic and axonal growth of Basket cells (BCs) and non-Basket cells (non-BCs) using sparse transfection of channelrhodopsin-YFP and repetitive optogenetic stimulation in slice cultures of rat visual cortex. Neocortical interneurons often display axon-carrying dendrites (AcDs). We found that the AcDs of BCs and non-BCs were, on average, the most complex dendrites. Further, the AcD configuration had an influence on BC axonal development. Axons originating from an AcD formed denser arborizations with more terminal endings within the dendritic field of the parent cell. Intriguingly, this occurred already in unstimulated BCs, and complexity was not increased further by optogenetic stimulation. However, optogenetic stimulation exerted a growth-promoting effect on axons emerging from BC somata. The axons of non-BCs neither responded to the AcD configuration nor to the optogenetic stimulation. The results suggest that the formation of locally dense BC plexuses is regulated by spontaneous activity. Moreover, in the AcD configuration, the AcD and the axon it carries mutually support each other's growth.
Assuntos
Axônios , Interneurônios , Animais , Ratos , Células Epiteliais , Células Musculares , DendritosRESUMO
Minipump infusions into visual cortex in vivo at the onset of the critical period have revealed that the proinflammatory cytokine leukemia inhibitory factor (LIF) delays the maturation of thalamocortical projection neurons of the lateral geniculate nucleus, and tecto-thalamic projection neurons of the superior colliculus, and cortical layer IV spiny stellates and layer VI pyramidal neurons. Here, we report that P12-20 LIF infusion inhibits somatic maturation of pyramidal neurons and of all interneuron types in vivo. Likewise, DIV 12-20 LIF treatment in organotypic cultures prevents somatic growth GABA-ergic neurons. Further, while NPY expression is increased in the LIF-infused hemispheres, the expression of parvalbumin mRNA and protein, Kv3.1 mRNA, calbindin D-28k protein, and GAD-65 mRNA, but not of GAD-67 mRNA or calretinin protein is substantially reduced. Also, LIF treatment decreases parvalbumin, Kv3.1, Kv3.2 and GAD-65, but not GAD-67 mRNA expression in OTC. Developing cortical neurons are known to depend on neurotrophins. Indeed, LIF alters neurotrophin mRNA expression, and prevents the growth promoting action of neurotophin-4 in GABA-ergic neurons. The results imply that LIF, by altering neurotrophin expression and/or signaling, could counteract neurotrophin-dependent growth and neurochemical differentiation of cortical neurons.
Assuntos
Fator Inibidor de Leucemia/farmacologia , Neurogênese/efeitos dos fármacos , Córtex Visual/efeitos dos fármacos , Animais , Células Cultivadas , Feminino , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Interneurônios/citologia , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Masculino , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos , Ratos Long-Evans , Córtex Visual/citologia , Córtex Visual/crescimento & desenvolvimentoRESUMO
The ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate glutamate receptors (AMPARs) have been implicated in the establishment of dendritic architecture. The transmembrane AMPA receptor regulatory proteins (TARPs) regulate AMPAR function and trafficking into synaptic membranes. In the current study, we employ type I and type II TARPs to modulate expression levels and function of endogenous AMPARs and investigate in organotypic cultures (OTCs) of rat occipital cortex whether this influences neuronal differentiation. Our results show that in early development [5-10 days in vitro (DIV)] only the type I TARP γ-8 promotes pyramidal cell dendritic growth by increasing spontaneous calcium amplitude and GluA2/3 expression in soma and dendrites. Later in development (10-15 DIV), the type I TARPs γ-2, γ-3 and γ-8 promote dendritic growth, whereas γ-4 reduced dendritic growth. The type II TARPs failed to alter dendritic morphology. The TARP-induced dendritic growth was restricted to the apical dendrites of pyramidal cells and it did not affect interneurons. Moreover, we studied the effects of short hairpin RNA-induced knockdown of endogenous γ-8 and showed a reduction of dendritic complexity and amplitudes of spontaneous calcium transients. In addition, the cytoplasmic tail (CT) of γ-8 was required for dendritic growth. Single-cell calcium imaging showed that the γ-8 CT domain increases amplitude but not frequency of calcium transients, suggesting a regulatory mechanism involving the γ-8 CT domain in the postsynaptic compartment. Indeed, the effect of γ-8 overexpression was reversed by APV, indicating a contribution of NMDA receptors. Our results suggest that selected type I TARPs influence activity-dependent dendritogenesis of immature pyramidal neurons.
Assuntos
Canais de Cálcio/metabolismo , Dendritos/metabolismo , Neocórtex/citologia , Células Piramidais/metabolismo , Animais , Animais Recém-Nascidos , Canais de Cálcio/química , Sinalização do Cálcio/efeitos dos fármacos , Dendritos/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Neocórtex/crescimento & desenvolvimento , Neocórtex/metabolismo , Neurotoxinas/toxicidade , Lobo Occipital/efeitos dos fármacos , Lobo Occipital/metabolismo , Técnicas de Cultura de Órgãos , Estrutura Terciária de Proteína , Células Piramidais/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Long-Evans , Receptores de AMPA/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Glutamatergic transmission converging on calcium signaling plays a key role in dendritic differentiation. In early development, AMPA receptor (AMPAR) transcripts are extensively spliced and edited to generate subunits that differ in their biophysical properties. Whether these subunits have specific roles in the context of structural differentiation is unclear. We have investigated the role of nine GluA variants and revealed a correlation between the expression of flip variants and the period of major dendritic growth. In interneurons, only GluA1(Q)-flip increased dendritic length and branching. In pyramidal cells, GluA2(Q)-flop, GluA2(Q)-flip, GluA3(Q)-flip and calcium-impermeable GluA2(R)-flip promoted dendritic growth, suggesting that flip variants with slower desensitization kinetics are more important than receptors with elevated calcium permeability. Imaging revealed significantly higher calcium signals in pyramidal cells transfected with GluA2(R)-flip as compared with GluA2(R)-flop, suggesting a contribution of voltage-activated calcium channels. Indeed, dendritic growth induced by GluA2(R)-flip in pyramidal cells was prevented by blocking NMDA receptors (NMDARs) or voltage-gated calcium channels (VGCCs), suggesting that they act downstream of AMPARs. Intriguingly, the action of GluA1(Q)-flip in interneurons was also dependent on NMDARs and VGCCs. Cell class-specific effects were not observed for spine formation, as GluA2(Q)-flip and GluA2(Q)-flop increased spine density in pyramidal cells as well as in interneurons. The results suggest that AMPAR variants expressed early in development are important determinants for activity-dependent dendritic growth in a cell type-specific and cell compartment-specific manner.
Assuntos
Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Dendritos/metabolismo , Receptores de AMPA/metabolismo , Processamento Alternativo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Interneurônios/metabolismo , Neurônios/metabolismo , Edição de RNA , Splicing de RNA , Ratos , Ratos Long-Evans , Receptores de N-Metil-D-Aspartato/metabolismo , Fatores de TempoRESUMO
Myelination of the neocortex of altricial species is mostly a postnatal event, and the appearance of myelin has been associated with the end of the critical period for ocular dominance plasticity in rodent visual cortex. Due to their precocality, ungulates may tell a different story. Here, we analyzed the development of PDGFRα positive oligodendrocyte precursor cells and expression of myelin proteins in the laminar compartments of fetal and postnatal porcine cortex from E45 onwards. Precursor cell density initially increased and then decreased but remained present at P90. MAG and MBP staining were detectable at E70 in subventricular zone and deep white matter, ascending into gyral white matter at E85, and into the gray matter and marginal zone at E100 (birth in pig at E114). Protein blots confirmed the declining expression of PDGFRα from E65 onwards, and the increase of MBP and MAG expression from E80 onwards. Somatosensory input elicited by spontaneous activity is considered important for the formation of the body representation. Indeed, PDGFRα, MBP and MAG expression started earlier in somatosensory than in visual cortex. Taken together, myelination proceeded in white and gray matter and marginal zone of pig cortex before birth with an areal-specific time course, and an almost mature pattern was present at P5 in visual cortex.
Assuntos
Bainha de Mielina , Neocórtex , Animais , Suínos , Bainha de Mielina/metabolismo , Neocórtex/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas da Mielina/metabolismo , Sus scrofa , Oligodendroglia/metabolismoRESUMO
Spontaneous or experimentally evoked activity can lead to changes in length and/or branching of neocortical pyramidal cell dendrites. For instance, an early postnatal overexpression of certain AMPA or kainate glutamate receptor subunits leads to larger amplitudes of depolarizing events driven by spontaneous activity, and this increases apical dendritic complexity. Whether stimulation frequency has a role is less clear. In this study, we report that the expression of channelrhodopsin2-eYFP was followed by a 5-day optogenetic stimulation from DIV 5-10 or 11-15 in organotypic cultures of rat visual cortex-evoked dendritic remodeling. Stimulation at 0.05 Hz, at a frequency range of spontaneous calcium oscillations known to occur in the early postnatal neocortex in vivo until eye opening, had no effect. Stimulation with 0.5 Hz, a frequency at which the cortex in vivo adopts after eye opening, unexpectedly caused shorter and somewhat less branched apical dendrites of infragranular pyramidal neurons. The outcome resembles the remodeling of corticothalamic and callosal projection neurons of layers VI and V, which in the adult have apical dendrites no longer terminating in layer I. Exposure to 2.5 Hz, a frequency not occurring naturally during the time windows, evoked dendritic damage. The results suggested that optogenetic stimulation at a biologically meaningful frequency for the selected developmental stage can influence dendrite growth, but contrary to expectation, the optogenetic stimulation decreased dendritic growth.
RESUMO
Y-P30, the 30 amino acid N-terminal peptide of the dermcidin gene, has been found to promote neuronal survival and differentiation. Its early presence in development and import to the fetal brain led to the hypothesis that Y-P30 has an influence on proliferation, differentiation and migration. Neurospheres derived from neural stem cells isolated from E13 mouse cortex and striatal ganglionic eminences were treated with Y-P30, however, the proportion of progenitors, neurons and astrocytes generated in differentiation assays was not altered. A short Y-P30 treatment of undifferentiated striatal and cortical neurospheres failed to alter the proportion of BrdU-positive cells. A longer treatment reduced the percentage of BrdU-positive cells and GABA-immunoreactive neurons only in striatal spheres. The presence of Y-P30 enhanced migration of T24 human bladder carcinoma cells in a wound-healing assay in vitro. Further, Y-P30 enhanced migration of T24 cells, rat primary cortical astrocytes and PC12 cells in chemotactic Boyden chamber assays. Together, these findings suggest that a major function of Y-P30 is to promote migration of neural and non-neural cell types.
Assuntos
Movimento Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Knowledge on cortical development is based mainly on rodents besides primates and carnivores, all being altricial. Here, we analyzed a precocial animal, the pig, looking at dorsoparietal cortex from E45 to P90. At E45, most ionized calcium-binding adapter molecule 1-positive (Iba1+) cells had a macrophage-like morphology and resided in meninges and choroid plexus. Only a few cells were scattered in the ventricular and subventricular zone (VZ and SVZ). At E60/E70, all laminar compartments displayed microglia cells at a low-to-moderate density, being highest in VZ and SVZ followed by intermediate zone/white matter (IZ/WM). The cortical plate and marginal zone displayed only a few Iba1+ cells. Cells were intensely labeled, but still had poorly arborized somata and many resembled ameboid, macrophage-like microglia. Concurrent with a massive increase in cortical volume, microglia cell density increased until E85, and further until E100/E110 (birth at E114) to densities that resemble those seen postnatally. A fraction of microglia colabeled with Ki67 suggesting proliferation in all laminar compartments. Cell-to-cell distance decreased substantially during this time, and the fraction of microglia to all nuclei and to neurons increases in the laminar compartments. Eventually, of all cortical DAPI+ nuclei 7-12% were Iba1+ microglia. From E70 onwards, more and more cells with ramified processes were present in MZ down to IZ/WM, showing, for instance, a close association with NeuN+, NPY+, and GAD65/67+ somata and axon initial segments. These results suggested that the development of microglia cell density and morphology proceeds rapidly from mid-gestation onwards reaching near-adult status already before birth.
Assuntos
Neocórtex , Animais , Feto , Microglia , Neurônios , Sus scrofa , SuínosRESUMO
Electrical activity is considered a key driver for the neurochemical and morphological maturation of neurons and the formation of neuronal networks. Designer receptors exclusively activated by designer drugs (DREADDs) are tools for controlling neuronal activity at the single cell level by triggering specific G protein signaling. Our objective was to investigate if prolonged silencing of differentiating cortical neurons can influence dendritic and axonal maturation. The DREADD hM4Di couples to Gi/o signaling and evokes hyperpolarization via GIRK channels. HM4Di was biolistically transfected into neurons in organotypic slice cultures of rat visual cortex, and activated by clozapine-N-oxide (CNO) dissolved in H2O; controls expressed hM4Di, but were mock-stimulated with H2O. Neurons were analyzed after treatment for two postnatal time periods, DIV 5-10 and 10-20. We found that CNO treatment delays the maturation of apical dendrites of L2/3 pyramidal cells. Further, the number of collaterals arising from the main axon was significantly lower, as was the number of bouton terminaux along pyramidal cell and basket cell axons. The dendritic maturation of L5/6 pyramidal cells and of multipolar interneurons (basket cells and bitufted cells) was not altered by CNO treatment. Returning CNO-treated cultures to CNO-free medium for 7 days was sufficient to recover dendritic and axonal complexity. Our findings add to the view that activity is a key driver in particular of postnatal L2/3 pyramidal cell maturation. Our results further suggest that inhibitory G protein signaling may represent a factor balancing the strong driving force of neurotrophic factors, electrical activity and calcium signaling.
RESUMO
The canonical view of neuronal function is that inputs are received by dendrites and somata, become integrated in the somatodendritic compartment and upon reaching a sufficient threshold, generate axonal output with axons emerging from the cell body. The latter is not necessarily the case. Instead, axons may originate from dendrites. The terms 'axon carrying dendrite' (AcD) and 'AcD neurons' have been coined to describe this feature. In rodent hippocampus, AcD cells are shown to be functionally 'privileged', since inputs here can circumvent somatic integration and lead to immediate action potential initiation in the axon. Here, we report on the diversity of axon origins in neocortical pyramidal cells of rodent, ungulate, carnivore, and primate. Detection methods were Thy-1-EGFP labeling in mouse, retrograde biocytin tracing in rat, cat, ferret, and macaque, SMI-32/ßIV-spectrin immunofluorescence in pig, cat, and macaque, and Golgi staining in macaque and human. We found that in non-primate mammals, 10-21% of pyramidal cells of layers II-VI had an AcD. In marked contrast, in macaque and human, this proportion was lower and was particularly low for supragranular neurons. A comparison of six cortical areas (being sensory, association, and limbic in nature) in three macaques yielded percentages of AcD cells which varied by a factor of 2 between the areas and between the individuals. Unexpectedly, pyramidal cells in the white matter of postnatal cat and aged human cortex exhibit AcDs to much higher percentages. In addition, interneurons assessed in developing cat and adult human cortex had AcDs at type-specific proportions and for some types at much higher percentages than pyramidal cells. Our findings expand the current knowledge regarding the distribution and proportion of AcD cells in neocortex of non-primate taxa, which strikingly differ from primates where these cells are mainly found in deeper layers and white matter.
Assuntos
Neocórtex , Idoso , Animais , Axônios/fisiologia , Dendritos/fisiologia , Furões , Haplorrinos , Humanos , Camundongos , Células Piramidais , Ratos , SuínosRESUMO
A battery of genetically encoded calcium indicators (GECIs) with different binding kinetics and calcium affinities was developed over the recent years to permit long-term calcium imaging. GECIs are calcium buffers and therefore, expression of GECIs may interfere with calcium homeostasis and signaling pathways important for neuronal differentiation and survival. Our objective was to investigate if the biolistically induced expression of five commonly used GECIs at two postnatal time points (days 14 and 22-25) could affect the morphological maturation of cortical neurons in organotypic slice cultures of rat visual cortex. Expression of GCaMP3 in both time windows, and of GCaMP5G and TN-XXL in the later time window impaired apical and /or basal dendrite growth of pyramidal neurons. With time, the proportion of GECI transfectants with nuclear filling increased, but an only prolonged expression of TN-XXL caused higher levels of neurodegeneration. In multipolar interneurons, only GCaMP3 evoked a transient growth delay during the early time window. GCaMP6m and GCaMP6m-XC were quite "neuron-friendly." Since growth-impaired neurons might not have the physiological responses typical of age-matched wildtype neurons the results obtained after prolonged developmental expression of certain GECIs might need to be interpreted with caution.
RESUMO
NMDA receptors are important players for neuronal differentiation. We previously reported that antagonizing NMDA receptors with APV blocked the growth-promoting effects evoked by the overexpression of specific calcium-permeable or flip-spliced AMPA receptor subunits and of type I transmembrane AMPA receptor regulatory proteins which both exclusively modify apical dendritic length and branching of cortical pyramidal neurons. These findings led us to characterize the role of GluN2B and GluN2A for dendritogenesis using organotypic cultures of rat visual cortex. Antagonizing GluN2B with ifenprodil and Ro25-6981 strongly impaired basal dendritic growth of supra- and infragranular pyramidal cells at DIV 5-10, but no longer at DIV 15-20. Growth recovered after washout, and protein blots revealed an increase of synaptic GluN2B-containing receptors as indicated by a enhanced phosphorylation of the tyrosine 1472 residue. Antagonizing GluN2A with TCN201 and NVP-AAM077 was ineffective at both ages. Dendrite growth of non-pyramidal interneurons was not altered. We attempted to overexpress GluN2A and GluN2B. However, although the constructs delivered currents in HEK cells, there were neither effects on dendrite morphology nor an enhanced sensitivity to NMDA. Further, co-expressing GluN1-1a and GluN2B did not alter dendritic growth. Visualization of overexpressed, tagged GluN2 proteins was successful after immunofluorescence for the tag which delivered rather weak staining in HEK cells as well as in neurons. This suggested that the level of overexpression is too weak to modify dendrite growth. In summary, endogenous GluN2B, but not GluN2A is important for pyramidal cell basal dendritic growth during an early postnatal time window.
RESUMO
GABA(C) receptors are enriched in the upper grey layers of the mammalian superior colliculus and contribute to synaptic processing. Electrophysiological data suggested that the GABA(C) receptor ρ subunits are expressed by GABAergic interneurons which represent about half of the neurons in the stratum griseum superficiale (SGS). Combining in situ hybridization for ρ2 receptor mRNA and the glutamic acid decarboxylase GAD-65 mRNA confirmed this assumption. A majority of ρ-labeled neurons in SGS and pretectum are GABAergic. Combining in situ hybridization with immunohistochemistry for the two projection neuron markers calbindin and parvalbumin revealed that a few ρ2 mRNA expressing cells coexpressed calbindin, but not parvalbumin. In visual cortex, ρ2 mRNA was present in pyramidal neurons and parvalbumin-containing interneurons. The results show that in the SGS primarily GABAergic neurons express GABA(C) receptors whereas the majority of tectothalamic calbindin neurons and intrinsically projecting parvalbumin neurons do not.
Assuntos
Neurônios/metabolismo , Receptores de GABA/biossíntese , Colículos Superiores/metabolismo , Córtex Visual/metabolismo , Ácido gama-Aminobutírico/fisiologia , Animais , Regulação da Expressão Gênica/fisiologia , Neurônios/fisiologia , RNA Mensageiro/biossíntese , Ratos , Ratos Long-Evans , Receptores de GABA/genética , Colículos Superiores/citologia , Córtex Visual/citologiaRESUMO
The synRas transgenic mice express constitutively activated Valin12-Harvey Ras in postnatal neocortical pyramidal neurons. This leads to somatodendritic hypertrophy, higher densities of spines and synapses, and an enhancement of synaptic long-term potentiation associated with an increased glutamate receptor-mediated activity. It was less clear how the interneurons respond to these alterations, and this prompted the quantitative assessment of interneuron neurochemistry. Interneurons rarely expressed the transgene, however, several interneuron types displayed a transient somatic hypertrophy. Furthermore, NPY mRNA expression was persistently increased as were the laminar percentages of labeled neurons. The expression of parvalbumin and voltage-gated potassium channels Kv3.1b/3.2 was unchanged. A significant decline of GAD-67, but not GAD-65, mRNA expressing neurons was observed in layer VI in animals older than P60. This suggested that subtle deficits in inhibition and enhanced excitation evoke the interneuronal changes in the synRastransgenic mouse cortex.
Assuntos
Regulação da Expressão Gênica , Interneurônios/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Córtex Visual/metabolismo , Fatores Etários , Animais , Crescimento Celular , Dendritos/genética , Dendritos/metabolismo , Inibidores do Crescimento/genética , Interneurônios/citologia , Interneurônios/patologia , Camundongos , Camundongos Transgênicos , Inibição Neural/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Córtex Visual/citologia , Córtex Visual/patologiaRESUMO
During neuronal development, AMPA receptors (AMPARs) and NMDA receptors (NMDARs) are important for neuronal differentiation. Kainate receptors (KARs) are closely related to AMPARs and involved in the regulation of cortical network activity. However, their role for neurite growth and differentiation of cortical neurons is unclear. Here, we used KAR agonists and overexpression of selected KAR subunits and their auxiliary neuropilin and tolloid-like proteins, NETOs, to investigate their influence on dendritic growth and network activity in organotypic cultures of rat visual cortex. Kainate at 500 nM enhanced network activity and promoted development of dendrites in layer II/III pyramidal cells, but not interneurons. GluK2 overexpression promoted dendritic growth in pyramidal cells and interneurons. GluK2 transfectants were highly active and acted as drivers for network activity. GluK1 and NETO1 specifically promoted dendritic growth of interneurons. Our study provides new insights for the roles of KARs and NETOs in the morphological and physiological development of the visual cortex.
Assuntos
Dendritos/fisiologia , Interneurônios/fisiologia , Células Piramidais/fisiologia , Receptores de Ácido Caínico/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Animais Recém-Nascidos , Dendritos/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Ácido Caínico/farmacologia , Técnicas de Cultura de Órgãos , Organogênese/efeitos dos fármacos , Organogênese/fisiologia , Subunidades Proteicas/agonistas , Subunidades Proteicas/fisiologia , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Long-Evans , Receptores de Ácido Caínico/agonistas , Córtex Visual/efeitos dos fármacos , Córtex Visual/crescimento & desenvolvimento , Receptor de GluK2 CainatoRESUMO
The signal specificity of G protein-coupled receptors (GPCRs) including serotonin receptors (5-HT-R) depends on the trafficking and localization of the GPCR within its subcellular signaling domain. Visualizing traffic-dependent GPCR signals in neurons is difficult, but important to understand the contribution of GPCRs to synaptic plasticity. We engineered CaMello (Ca2+-melanopsin-local-sensor) and CaMello-5HT2A for visualization of traffic-dependent Ca2+ signals in 5-HT2A-R domains. These constructs consist of the light-activated Gq/11 coupled melanopsin, mCherry and GCaMP6m for visualization of Ca2+ signals and receptor trafficking, and the 5-HT2A C-terminus for targeting into 5-HT2A-R domains. We show that the specific localization of the GPCR to its receptor domain drastically alters the dynamics and localization of the intracellular Ca2+ signals in different neuronal populations in vitro and in vivo. The CaMello method may be extended to every GPCR coupling to the Gq/11 pathway to help unravel new receptor-specific functions in respect to synaptic plasticity and GPCR localization.
Assuntos
Técnicas Biossensoriais , Cálcio/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Optogenética/métodos , Receptor 5-HT2A de Serotonina/genética , Opsinas de Bastonetes/genética , Animais , Cerebelo/citologia , Cerebelo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Eletrodos Implantados , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Transporte Proteico , Ratos , Ratos Long-Evans , Receptor 5-HT2A de Serotonina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Opsinas de Bastonetes/metabolismo , Técnicas EstereotáxicasRESUMO
The 30-amino acid peptide Y-P30 corresponds to the N-terminus of the primate-specific, sweat gland-derived dermcidin prepropeptide. Previous work has revealed that Y-P30 enhances the interaction of pleiotrophin and syndecans-2/3, and thus represents a natural ligand to study this signaling pathway. In immature neurons, Y-P30 activates the c-Src and p42/44 ERK kinase pathway, increases the amount of F-actin in axonal growth cones, and promotes neuronal survival, cell migration and axonal elongation. The action of Y-P30 on axonal growth requires syndecan-3 and heparan sulfate side chains. Whether Y-P30 has the potential to influence dendrites and dendritic protrusions has not been explored. The latter is suggested by the observations that syndecan-2 expression increases during postnatal development, that syndecan-2 becomes enriched in dendritic spines, and that overexpression of syndecan-2 in immature neurons results in a premature morphological maturation of dendritic spines. Here, analysing rat cortical pyramidal and non-pyramidal neurons in organotypic cultures, we show that Y-P30 does not alter the development of the dendritic arborization patterns. However, Y-P30 treatment decreases the density of apical, but not basal dendritic protrusions at the expense of the filopodia. Analysis of spine morphology revealed an unchanged mushroom/stubby-to-thin spine ratio and a shortening of the longest decile of dendritic protrusions. Whole-cell recordings from cortical principal neurons in dissociated cultures grown in the presence of Y-P30 demonstrated a decrease in the frequency of glutamatergic mEPSCs. Despite these differences in protrusion morphology and synaptic transmission, the latter likely attributable to presynaptic effects, calcium event rate and amplitude recorded in pyramidal neurons in organotypic cultures were not altered by Y-P30 treatment. Together, our data suggest that Y-P30 has the capacity to decelerate spinogenesis and to promote morphological, but not synaptic, maturation of dendritic protrusions.
Assuntos
Espinhas Dendríticas/metabolismo , Neocórtex/citologia , Peptídeos/metabolismo , Animais , Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Neocórtex/metabolismo , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Ratos , Sindecana-2/metabolismoRESUMO
Knowledge on cortical development is based mainly on small rodents besides primates and carnivores, all being altricial nestlings. Ungulates are precocial and born with nearly mature sensory and motor systems. Almost no information is available on ungulate brain development. Here, we analyzed European wild boar cortex development, focusing on the neuropeptide Y immunoreactive (NPY-ir) neuron system in dorsoparietal cortex from E35 to P30. Transient NPY-ir neuron types including archaic cells of the cortical plate and axonal loop cells of the subplate which appear by E60 concurrent with the establishment of the ungulate brain basic sulcal pattern. From E70, NPY-ir axons have an axon initial segment which elongates and shifts closer towards the axon's point of origin until P30. From E85 onwards (birth at E114), NPY-ir neurons in cortical layers form basket cell-like local and Martinotti cell-like ascending axonal projections. The mature NPY-ir pattern is recognizable at E110. Together, morphologies are conserved across species, but timing is not: in pig, the adult pattern largely forms prenatally.
Assuntos
Neocórtex/embriologia , Neurônios/fisiologia , Neuropeptídeo Y/metabolismo , Animais , Axônios , Feminino , Masculino , Neocórtex/citologia , Neurônios/citologia , Neurônios/metabolismo , Sus scrofa/embriologiaRESUMO
A multitude of 18 iGluR receptor subunits, many of which are diversified by splicing and RNA editing, localize to >20 excitatory and inhibitory neocortical neuron types defined by physiology, morphology, and transcriptome in addition to various types of glial, endothelial, and blood cells. Here we have compiled the published expression of iGluR subunits in the areas and cell types of developing and adult cortex of rat, mouse, carnivore, bovine, monkey, and human as determined with antibody- and mRNA-based techniques. iGluRs are differentially expressed in the cortical areas and in the species, and all have a unique developmental pattern. Differences are quantitative rather than a mere absence/presence of expression. iGluR are too ubiquitously expressed and of limited use as markers for areas or layers. A focus has been the iGluR profile of cortical interneuron types. For instance, GluK1 and GluN3A are enriched in, but not specific for, interneurons; moreover, the interneurons expressing these subunits belong to different types. Adressing the types is still a major hurdle because type-specific markers are lacking, and the frequently used neuropeptide/CaBP signatures are subject to regulation by age and activity and vary as well between species and areas. RNA-seq reveals almost all subunits in the two morphofunctionally characterized interneuron types of adult cortical layer I, suggesting a fairly broad expression at the RNA level. It remains to be determined whether all proteins are synthesized, to which pre- or postsynaptic subdomains in a given neuron type they localize, and whether all are involved in synaptic transmission. J. Comp. Neurol. 525:976-1033, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Neocórtex/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Animais , Humanos , MamíferosRESUMO
The microdomain that orchestrates action potential initiation in neurons is the axon initial segment (AIS). It has long been considered to be a rather homogeneous domain at the very proximal axon hillock with relatively stable length, particularly in cortical pyramidal cells. However, studies in other brain regions paint a different picture. In hippocampal CA1, up to 50% of axons emerge from basal dendrites. Further, in about 30% of thick-tufted layer V pyramidal neurons in rat somatosensory cortex, axons have a dendritic origin. Consequently, the AIS is separated from the soma. Recent in vitro and in vivo studies have shown that cellular excitability is a function of AIS length/position and somatodendritic morphology, undermining a potentially significant impact of AIS heterogeneity for neuronal function. We therefore investigated neocortical axon morphology and AIS composition, hypothesizing that the initial observation of seemingly homogeneous AIS is inadequate and needs to take into account neuronal cell types. Here, we biolistically transfected cortical neurons in organotypic cultures to visualize the entire neuron and classify cell types in combination with immunolabeling against AIS markers. Using confocal microscopy and morphometric analysis, we investigated axon origin, AIS position, length, diameter as well as distance to the soma. We find a substantial AIS heterogeneity in visual cortical neurons, classified into three groups: (I) axons with somatic origin with proximal AIS at the axon hillock; (II) axons with somatic origin with distal AIS, with a discernible gap between the AIS and the soma; and (III) axons with dendritic origin (axon-carrying dendrite cell, AcD cell) and an AIS either starting directly at the axon origin or more distal to that point. Pyramidal cells have significantly longer AIS than interneurons. Interneurons with vertical columnar axonal projections have significantly more distal AIS locations than all other cells with their prevailing phenotype as an AcD cell. In contrast, neurons with perisomatic terminations display most often an axon originating from the soma. Our data contribute to the emerging understanding that AIS morphology is highly variable, and potentially a function of the cell type.