RESUMO
Mutations in methyl-CpG binding protein 2 (MeCP2), such as the T158M, P152R, R294X, and R306C mutations, are responsible for most Rett syndrome (RTT) cases. These mutations often result in altered protein expression that appears to correlate with changes in the nuclear size; however, the molecular details of these observations are poorly understood. Using a C2C12 cellular system expressing human MeCP2-E1 isoform as well as mouse models expressing these mutations, we show that T158M and P152R result in a decrease in MeCP2 protein, whereas R306C has a milder variation, and R294X resulted in an overall 2.5 to 3 fold increase. We also explored the potential involvement of the MeCP2 PEST domains in the proteasome-mediated regulation of MeCP2. Finally, we used the R294X mutant to gain further insight into the controversial competition between MeCP2 and histone H1 in the chromatin context. Interestingly, in R294X, MeCP2 E1 and E2 isoforms were differently affected, where the E1 isoform contributes to much of the overall protein increase observed, while E2 decreases by half. The modes of MeCP2 regulation, thus, appear to be differently regulated in the two isoforms.
RESUMO
We identified three types of monosynaptic cholinergic inputs spatially arranged onto medial substantia nigra dopaminergic neurons in male and female mice: cotransmitted acetylcholine (ACh)/GABA, GABA-only, and ACh only. There was a predominant GABA-only conductance along lateral dendrites and soma-centered ACh/GABA cotransmission. In response to repeated stimulation, the GABA conductance found on lateral dendrites decremented less than the proximally located GABA conductance, and was more effective at inhibiting action potentials. While soma-localized ACh/GABA cotransmission showed depression of the GABA component with repeated stimulation, ACh-mediated nicotinic responses were largely maintained. We investigated whether this differential change in inhibitory/excitatory inputs leads to altered neuronal excitability. We found that a depolarizing current or glutamate preceded by cotransmitted ACh/GABA was more effective in eliciting an action potential compared with current, glutamate, or ACh/GABA alone. This enhanced excitability was abolished with nicotinic receptor inhibitors, and modulated by T- and L-type calcium channels, thus establishing that activity of multiple classes of ion channels integrates to shape neuronal excitability.SIGNIFICANCE STATEMENT Our laboratory has previously discovered a population of substantia nigra dopaminegic neurons (DA) that receive cotransmitted ACh and GABA. This study used subcellular optogenetic stimulation of cholinergic presynaptic terminals to map the functional ACh and GABA synaptic inputs across the somatodendritic extent of substantia nigra DA neurons. We determined spatially clustered GABA-only inputs on the lateral dendrites while cotransmitted ACh and GABA clustered close to the soma. We have shown that the action of GABA and ACh in cotransmission spatially clustered near the soma play a critical role in enhancing glutamate-mediated neuronal excitability through the activation of T- and L-type voltage-gated calcium channels.
Assuntos
Acetilcolina , Neurônios Dopaminérgicos , Masculino , Feminino , Camundongos , Animais , Acetilcolina/farmacologia , Ácido Glutâmico/fisiologia , Colinérgicos , Ácido gama-Aminobutírico , Transmissão Sináptica/fisiologiaRESUMO
PURPOSE/AIM: Rett (RTT) syndrome, a neurodevelopmental disorder, results from loss-of-function mutations in methyl-CpG-binding protein 2. We studied activity-dependent plasticity induced by sensory deprivation via whisker trimming in early symptomatic male mutant mice to assess neural rewiring capability. METHODS: One whisker was trimmed for 0-14 days and intrinsic optical imaging of the transient reduction of brain blood oxygenation resulting from neural activation by 1 second of wiggling of the whisker stump was compared to that of an untrimmed control whisker. RESULTS: Cortical evoked responses to wiggling a non-trimmed whisker were constant for 14 days, reduced for a trimmed whisker by 49.0 ± 4.3% in wild type (n = 14) but by only 22.7 ± 4.6% in mutant (n = 18, p = 0.001). CONCLUSION: As the reduction in neural activation following sensory deprivation in whisker barrel cortex is known to be dependent upon evoked and basal neural activity, impairment of cortical re-wiring following whisker trimming provides a paradigm suitable to explore mechanisms underlying deficiencies in the establishment and maintenance of synapses in RTT, which can be potentially targeted by therapeutics.
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Privação Sensorial , Vibrissas , Camundongos , Animais , Masculino , Privação Sensorial/fisiologia , Vibrissas/fisiologia , Córtex Somatossensorial/fisiologiaRESUMO
The crayfish claw opener neuromuscular junction is a biological model for studying presynaptic neuromodulation by serotonin (5HT) and synaptic vesicle recycling. It has been hypothesized that 5HT enhances release by recruiting a population of either previously nonrecycling or "reluctant" vesicles to increase the readily releasable pool. To determine if 5HT activates a distinct population of synaptic vesicles, recycling membranes were labeled with the membrane dye, FM1-43. Unloading (destaining) protocols could not resolve a population of vesicles that were only releasable in the presence of 5HT. Instead, we conclude synaptic vesicles change behavior in axon terminals independent of 5HT, becoming less likely to exocytose and unload dye over periods of >1 hr after recycling. We hypothesized this to be due to the slow conversion of a portion of recycled vesicles to a difficult to release state. The possibility that vesicles in these pools were spatially separated within the terminal was tested using photoconversion of FM1-43 and transmission electron microscopy. The location of FM1-43-labeled vesicles fixed 2 min following 3 min of 20-Hz stimulation did not reveal preferential localization of recycling vesicles specifically near release sites and the distribution of labeled vesicles was not significantly different between early (2 min) and late (180 min) time points. Terminals fixed 30 s following stimulation contained a significant proportion of vesicular structures equivalent in diameter to 2-5 regular vesicles, with multivesicular bodies and calveoli rarely seen, suggesting that endocytosis during sustained release at crayfish terminals occurs via multiple routes, most commonly through large "vesicle" intermediates.
Assuntos
Exocitose , Junção Neuromuscular/metabolismo , Serotonina/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Astacoidea , Junção Neuromuscular/fisiologia , Potenciais Sinápticos , Vesículas Sinápticas/ultraestruturaRESUMO
Mitral cells express low-voltage activated Cav3.3 channels on their distal apical tuft dendrites (McKay et al., 2006; Johnston and Delaney, 2010). They also discharge Na(+)-dependent dendritic action potentials and release glutamate from these dendrites. Around resting membrane potentials, between -65 and -50 mV, Cav3.x channels are a primary determinant of cytoplasmic [Ca(2+)]. In this study using C57 mice, we present evidence that subthreshold Cav3.x-mediated Ca(2+) influx modulates action potential evoked transmitter release and directly drives asynchronous release from distal tuft dendrites. Presynaptic hyperpolarization and selective block of Cav3.x channels with Z941 (Tringham et al., 2012) reduce mitral-to-mitral EPSP amplitude, increase the coefficient of variation of EPSPs, and increase paired-pulse ratios, consistent with a reduced probability of transmitter release. Both hyperpolarization and Cav3.x channel blockade reduce steady-state cytoplasmic [Ca(2+)] in the tuft dendrite without reducing action potential evoked Ca(2+) influx, suggesting that background [Ca(2+)] modulates evoked release. We demonstrate that Cav3.x-mediated Ca(2+) influx from even one mitral cell at membrane potentials between -65 and -50 mV is sufficient to produce feedback inhibition from periglomerular neurons. Deinactivation of Cav3.x channels by hyperpolarization increases T-type Ca(2+) influx upon repolarization and increases feedback inhibition to produce subthreshold modulation of the mitral-periglomerular reciprocal circuit.
Assuntos
Canais de Cálcio Tipo T/fisiologia , Caveolina 3/fisiologia , Dendritos/fisiologia , Bulbo Olfatório/fisiologia , Condutos Olfatórios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Potenciais de Ação/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Inibição Neural/fisiologia , Técnicas de Cultura de ÓrgãosRESUMO
BACKGROUND: The explanted, developing rodent retina provides an efficient and accessible preparation for use in gene transfer and pharmacological experimentation. Many of the features of normal development are retained in the explanted retina, including retinal progenitor cell proliferation, heterochronic cell production, interkinetic nuclear migration, and connectivity. To date, live imaging in the developing retina has been reported in non-mammalian and mammalian whole-mount samples. An integrated approach to rodent retinal culture/transfection, live imaging, cell tracking, and analysis in structurally intact explants greatly improves our ability to assess the kinetics of cell production. RESULTS: In this report, we describe the assembly and maintenance of an in vitro, CO2-independent, live mouse retinal preparation that is accessible by both upright and inverted, 2-photon or confocal microscopes. The optics of this preparation permit high-quality and multi-channel imaging of retinal cells expressing fluorescent reporters for up to 48h. Tracking of interkinetic nuclear migration within individual cells, and changes in retinal progenitor cell morphology are described. Follow-up, hierarchical cluster screening revealed that several different dependent variable measures can be used to identify and group movement kinetics in experimental and control samples. CONCLUSIONS: Collectively, these methods provide a robust approach to assay multiple features of rodent retinal development using live imaging.
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Retina/crescimento & desenvolvimento , Animais , Cinética , Camundongos , Retina/citologiaRESUMO
The corneal epithelium is continuously subjected to external stimuli that results in varying degrees of cellular damage. The use of live-cell imaging approaches has facilitated understanding of the cellular and molecular mechanisms underlying the corneal epithelial wound healing process. Here, we describe a live, ex vivo, whole-eye approach using laser scanning confocal microscopy to simultaneously induce and visualize short-term cellular responses following microdamage to the corneal epithelium. Live-cell imaging of corneal cell layers was enabled using the lipophilic fluorescent dyes, SGC5 or FM4-64, which, when injected into the anterior chamber of enucleated eyes, readily penetrated and labelled cell membranes. Necrotic microdamage to a defined region (30 µm x 30 µm) through the central plane of the corneal basal epithelium was induced by continuously scanning for at least one minute using high laser power and was dependent on the presence of lipophilic fluorescent dye. This whole-mount live-cell imaging and microdamage approach was used to examine the behavior of Cx3cr1:GFP-expressing resident corneal stromal macrophages (RCSMs). In undamaged corneas, RCSMs remained stationary, but exhibited a constant extension and retraction of short (~5 µm) semicircular, pseudopodia-like processes reminiscent of what has previously been reported in corneal dendritic cells. Within minutes of microdamage, nearby anterior RCSMs became highly polarized and extended projections towards the damaged region. The extension of the processes plateaued after about 30 minutes and remained stable over the course of 2-3 hours of imaging. Retrospective immunolabeling showed that these responding RCSMs were MHC class II+. This study adds to existing knowledge of immune cell behavior in response to corneal damage and introduces a simple corneal epithelial microdamage and wound healing paradigm.
Assuntos
Epitélio Corneano , Estudos Retrospectivos , Córnea , Macrófagos , Corantes Fluorescentes , LasersAssuntos
Hipocampo , Síndrome de Rett , Animais , Endossomos , Proteína 2 de Ligação a Metil-CpG , Camundongos , Plasticidade Neuronal , NeurôniosRESUMO
Stroke profoundly disrupts cortical excitability which impedes recovery, but how it affects the function of specific inhibitory interneurons, or subpopulations therein, is poorly understood. Interneurons expressing vasoactive intestinal peptide (VIP) represent an intriguing stroke target because they can regulate cortical excitability through disinhibition. Here we chemogenetically augmented VIP interneuron excitability in a murine model of photothrombotic stroke and show that it enhances somatosensory responses and improves recovery of paw function. Using longitudinal calcium imaging, we discovered that stroke primarily disrupts the fidelity (fraction of responsive trials) and predictability of sensory responses within a subset of highly active VIP neurons. Partial recovery of responses occurred largely within these active neurons and was not accompanied by the recruitment of minimally active neurons. Importantly, chemogenetic stimulation preserved sensory response fidelity and predictability in highly active neurons. These findings provide a new depth of understanding into how stroke and prospective therapies (chemogenetics), can influence subpopulations of inhibitory interneurons.
Assuntos
Interneurônios/fisiologia , Acidente Vascular Cerebral/terapia , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Clozapina/análogos & derivados , Clozapina/uso terapêutico , Humanos , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Camundongos , Inibição Neural/efeitos dos fármacos , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Recuperação de Função Fisiológica , Córtex Somatossensorial/citologia , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/fisiologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Mitral cells are the primary output of the olfactory bulb, projecting to many higher brain areas. Understanding how mitral cells process and transmit information is key to understanding olfactory perception. Mitral dendrites possess high densities of voltage-gated channels, are able to initiate and propagate orthodromic and antidromic action potentials, and release neurotransmitter. We show that mitral cells also possess a low-voltage-activated T-type Ca(2+) current. Immunohistochemistry shows strong Cav3.3 labeling in the primary dendrite and apical tuft with weaker staining in basal dendrites and no staining in somata. A low-voltage-activated Ca(2+) current activates from -68 mV, is blocked by 500 microM Ni(2+) and 50 microM NNC 55-0396, but is insensitive to 50 microM Ni(2+) and 500 microM isradipine. 2-photon Ca(2+) imaging shows that T channels are functionally expressed in the primary dendrite where their activity determines the resting [Ca(2+)] and are responsible for subthreshold voltage-dependent Ca(2+) changes previously observed in vivo. Application of the group 1 mGluR agonist dihydroxyphenylglycine (DHPG) (50 microM) robustly upregulates T-channel current in the primary and apical tuft dendrite. Olfactory nerve stimulation generates a long-lasting depolarization, and we show that mGluRs recruit T channels to contribute approximately 36% of the voltage integral of this depolarization. The long-lasting depolarization results in sustained firing and block of T channels decreased action potential firing by 84.1 +/- 4.6%. Therefore upregulation of T channels by mGluRs is required for prolonged firing in response to olfactory nerve input.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Dendritos/fisiologia , Neurônios/fisiologia , Bulbo Olfatório/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Dendritos/efeitos dos fármacos , Imuno-Histoquímica , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Bulbo Olfatório/efeitos dos fármacos , Nervo Olfatório/fisiologia , Técnicas de Patch-Clamp , Receptores de Glutamato Metabotrópico/agonistas , Sinapses/efeitos dos fármacosRESUMO
Recent studies indicate that the precise timing and location of excitation and inhibition (E/I) within active dendritic trees can significantly impact neuronal function. How synaptic inputs are functionally organized at the subcellular level in intact circuits remains unclear. To address this issue, we took advantage of the retinal direction-selective ganglion cell circuit, where directionally tuned inhibition is known to shape non-directional excitatory signals. We combined two-photon calcium imaging with genetic, pharmacological, and single-cell ablation methods to examine the extent to which inhibition 'vetoes' excitation at the level of individual dendrites of direction-selective ganglion cells. We demonstrate that inhibition shapes direction selectivity independently within small dendritic segments (<10µm) with remarkable accuracy. The data suggest that the parallel processing schemes proposed for direction encoding could be more fine-grained than previously envisioned.
Assuntos
Dendritos/fisiologia , Inibição Neural/fisiologia , Células Ganglionares da Retina/fisiologia , Potenciais de Ação , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutation in the X-linked MECP2 gene. Random X-inactivation produces a mosaic of mutant (MT) and wild-type (WT) neurons in female Mecp2+/- (het) mice. Many RTT symptoms are alleviated by increasing activity in medial prefrontal cortex (mPFC) in RTT model mice (Howell et al., 2017). Using a GFP-MeCP2 fusion protein to distinguish WT from MT pyramidal neurons in mPFC we found cell autonomous (cell genotype specific) and non-autonomous effects of MeCP2 deficiency on spontaneous excitatory/inhibitory balance, nicotinic acetylcholine receptor (nAChR) currents and evoked activity. MT Layer 5 and 6 (L5, L6) neurons of male nulls, and MT L6 of het mice had reduced spontaneous excitatory synaptic input compared to WT in wild-type male (WTm), female (WTf) and het mice. Inhibitory synaptic charge in MT L6 equaled WT in 2-4-month hets. At 6-7â¯months inhibitory charge in WT in het slices was increased compared to both MT in het and WT in WTf; however, in hets the excitatory/inhibitory charge ratio was still greater in WT compared to MT. nAChR currents were reduced in L6 of nulls and MT L6 in het slices compared to WT neurons of het, WTm and WTf. At 2-4â¯months, ACh perfusion increased frequency of inhibitory currents to L6 neurons equally in all genotypes but increased excitatory inputs to MT and WT in hets less than WT in WTfs. Unexpectedly ACh perfusion evoked greater sustained IPSC and EPSC input to L5 neurons of nulls compared to WTm.
Assuntos
Proteína 2 de Ligação a Metil-CpG/genética , Córtex Pré-Frontal/metabolismo , Células Piramidais/fisiologia , Receptores Nicotínicos/metabolismo , Síndrome de Rett/genética , Acetilcolina/farmacologia , Animais , Modelos Animais de Doenças , Feminino , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Camundongos Knockout , Mutação , Córtex Pré-Frontal/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Receptores Nicotínicos/genética , Síndrome de Rett/metabolismoRESUMO
Recovery of function after stroke is thought to be dependent on the reorganization of adjacent, surviving areas of the brain. Macroscopic imaging studies (functional magnetic resonance imaging, optical imaging) have shown that peri-infarct regions adopt new functional roles to compensate for damage caused by stroke. To better understand the process by which these regions reorganize, we used in vivo two-photon imaging to examine changes in dendritic and vascular structure in cortical regions recovering from stroke. In adult control mice, dendritic arbors were relatively stable with very low levels of spine turnover (<0.5% turnover over 6 h). After stroke, however, the organization of dendritic arbors in peri-infarct cortex was fundamentally altered with both apical dendrites and blood vessels radiating in parallel from the lesion. On a finer scale, peri-infarct dendrites were exceptionally plastic, manifested by a dramatic increase in the rate of spine formation that was maximal at 1-2 weeks (5-8-fold increase), and still evident 6 weeks after stroke. These changes were selective given that turnover rates were not significantly altered in ipsilateral cortical regions more distant to the lesion (>1.5 mm). These data provide a structural framework for understanding functional and behavioral changes that accompany brain injury and suggest new targets that could be exploited by future therapies to rebuild and rewire neuronal circuits lost to stroke.
Assuntos
Córtex Cerebral/ultraestrutura , Espinhas Dendríticas/ultraestrutura , Plasticidade Neuronal , Recuperação de Função Fisiológica , Acidente Vascular Cerebral/patologia , Animais , Córtex Cerebral/patologia , Córtex Cerebral/fisiologia , Infarto Cerebral/patologia , Infarto Cerebral/fisiopatologia , Espinhas Dendríticas/patologia , Espinhas Dendríticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasticidade Neuronal/fisiologia , Recuperação de Função Fisiológica/fisiologia , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Purpose: Despite numerous studies associating Visual System Homeobox 1 (VSX1), with posterior polymorphous corneal dystrophy and keratoconus, its role in these diseases is unclear. Here we examine the pathogenicity of VSX1 missense mutations in vitro and in a mouse genetic model. Methods: Vsx1 transcriptional repressor activity, protein stability, and subcellular localization activity, was examined using luciferase reporter-based assays, western blotting and immunolabeling, respectively, in transfected human embryonic kidney 293T cells. A genetic model for VSX1 p.P247R was generated to investigate pathogenicity of the mutation, in vivo. A wholemount confocal imaging approach on unfixed intact eyes was developed to examine corneal morphology, curvature, and thickness. Immunolabeling and electroretinography was used to examine retinal phenotype. Results: A mutation corresponding to human VSX1 p.P247R led to enhanced transcriptional repressor activity, in vitro. A mouse model for VSX1 p.P247R did not have any observable corneal defect, but did exhibit an abnormal electroretinogram response characterized by a more prominent ON as opposed to OFF panretinal responsiveness. In vitro analysis of additional VSX1 missense mutations showed that they either enhanced repressor activity or did not alter activity. Conclusions: Our results indicate that although VSX1 sequence variants can alter transcriptional activity, in the context of a mouse genetic model, at least one of these changes does not lead to corneal abnormalities. While we cannot exclude a role for VSX1 as a risk factor for corneal disease, on its own, it does not appear to play a major causative role.
Assuntos
Distrofias Hereditárias da Córnea/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Ceratocone/genética , Mutação de Sentido Incorreto/genética , Animais , Distrofias Hereditárias da Córnea/diagnóstico , Distrofias Hereditárias da Córnea/fisiopatologia , Modelos Animais de Doenças , Eletrorretinografia , Feminino , Células HEK293 , Humanos , Ceratocone/diagnóstico , Ceratocone/fisiopatologia , Masculino , Camundongos , Plasmídeos , Retina/fisiopatologia , Ativação Transcricional/fisiologiaRESUMO
Neurotransmitter release is triggered by the cooperative action of approximately five Ca2+ ions entering the presynaptic terminal through Ca2+ channels. Depending on the organization of the active zone (AZ), influx through one or many channels may be needed to cause fusion of a vesicle. Using a combination of experiments and modeling, we examined the number of channels that contribute Ca2+ for fusion of a single vesicle in a frog neuromuscular AZ. We compared Ca2+ influx to neurotransmitter release by measuring presynaptic action potential-evoked (AP-evoked) Ca2+ transients simultaneously with postsynaptic potentials. Ca2+ influx was manipulated by changing extracellular [Ca2+] (Ca(ext)) to alter the flux per channel or by reducing the number of open Ca2+ channels with omega-conotoxin GVIA (omega-CTX). When Ca(ext) was reduced, the exponent of the power relationship relating release to Ca2+ influx was 4.16 +/- 0.62 (SD; n = 4), consistent with a biochemical cooperativity of approximately 5. In contrast, reducing influx with omega-CTX yielded a power relationship of 1.7 +/- 0.44 (n = 5) for Ca(ext) of 1.8 mM and 2.12 +/- 0.44 for Ca(ext) of 0.45 mM (n = 5). Using geometrically realistic Monte Carlo simulations, we tracked Ca2+ ions as they entered through each channel and diffused in the terminal. Experimental and modeling data were consistent with two to six channel openings per AZ per AP; the Ca2+ that causes fusion of a single vesicle originates from one or two channels. Channel cooperativity depends mainly on the physical relationship between channels and vesicles and is insensitive to changes in the non-geometrical parameters of our model.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/fisiologia , Fusão de Membrana/fisiologia , Junção Neuromuscular/fisiologia , Vesículas Sinápticas/fisiologia , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio/metabolismo , Junção Neuromuscular/metabolismo , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Rana pipiens , Vesículas Sinápticas/metabolismoRESUMO
Two-photon laser scanning microscopy was used to correlate electrical events detected with whole-cell somatic recordings to Ca2+ transients in dendrites of olfactory bulb granule cells. A subset of spontaneous subthreshold depolarizing events recorded at the soma were shown to correspond to suprathreshold dendritic, Na-dependent action potentials [APs; dendritic spikes (D-spikes)]. These potentials were blocked by intracellular QX-314 (lidocaine N-ethyl bromide), hyperpolarizing current injection at the soma, and by partial inhibition of AMPA/kainate receptors with 0.75 microM DNQX. They were affected only slightly by 100 microM NiCl2. The majority of D-spikes recorded at the soma had a time to peak of <4 ms, comparable with somatic APs, a nonexponential decay, and amplitudes between 3 and 21 mV. Somatically recorded APs produced Ca2+ transients that were observed in spines and dendrites in all parts of the cell. Ca2+ transients from D-spikes were restricted to subsets of distal dendrites and their associated spines but were absent from the soma and dendrite within approximately 50-80 microm of the soma. Ca2+ transients in different branches could be correlated with different-sized D-spikes. D-spike and backpropagating AP-induced Ca2+ transients summed in dendrites, provided the interval between them was >5-6 ms. Generation of a D-spike in a particular dendrite <5-6 ms before a somatic AP blocked backpropagation of the somatic AP into that dendrite. The temporally specific interplay between D-spikes and backpropagating APs may play a role in regulating feedback and feedforward inhibition of groups of mitral cells synapsing on different granule cell dendrites.
Assuntos
Potenciais de Ação/fisiologia , Sinalização do Cálcio/fisiologia , Dendritos/fisiologia , Bulbo Olfatório/citologia , Bulbo Olfatório/fisiologia , Sódio/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Cátions Bivalentes/metabolismo , Dendritos/efeitos dos fármacos , Técnicas In Vitro , Bulbo Olfatório/efeitos dos fármacos , Neurônios Receptores Olfatórios/efeitos dos fármacos , Neurônios Receptores Olfatórios/fisiologia , Quinoxalinas/farmacologia , Rana pipiens , Sódio/farmacologiaRESUMO
Synaptic interactions between the dendrites of mitral/tufted (MT) and granule cells (GCs) in the olfactory bulb are important for the determination of spatiotemporal firing patterns of MTs, which form an odor representation passed to higher brain centers. These synapses are subject to modulation from several sources originating both within and outside the bulb. We show that dopamine, presumably released by TH-positive local interneurons, reduces synaptic transmission from MTs to GCs. MT neurons express D2-like receptors (D2Rs), and both dopamine and the D2 agonist quinpirole decrease EPSC amplitude at the MT--> GC synapse. D2R activation also increases paired pulse facilitation and decreases the frequency of action potential-independent spontaneous miniature EPSCs in GCs, consistent with an effect on MT glutamate release downstream from Ca2+ influx. Analysis of spike-evoked Ca2+ transients in MT lateral dendrites additionally shows that quinpirole reduces Ca2+ influx preferentially at distal locations, possibly by reducing dendritic excitability via increased transient K+ channel availability. When the OB is activated physiologically by using odor stimuli, blocking D2Rs increases the power of GABA(A)-dependent oscillations in the local field potential. This demonstrates a functional role for the dopaminergic circuit during normal odor-evoked responses and for the modulation of dendritic release and excitability in neuronal circuit function. Regulation of spike invasion of lateral dendrites by transient K+ currents also may provide a mechanism for local outputs of MTs to be controlled dynamically via other neuromodulators or by postsynaptic potentials.
Assuntos
Dopamina/farmacologia , Neurônios/efeitos dos fármacos , Bulbo Olfatório/citologia , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , 4-Aminopiridina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Dendritos/fisiologia , Depressão Química , Agonistas de Dopamina/farmacologia , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Retroalimentação Fisiológica , Ácido Glutâmico/metabolismo , Transporte de Íons/efeitos dos fármacos , Masculino , Mamíferos/fisiologia , Neurônios/classificação , Neurônios/fisiologia , Odorantes , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio/metabolismo , Quimpirol/farmacologia , Rana pipiens , Receptores de Dopamina D2/agonistas , Receptores de GABA-A/fisiologia , Olfato/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
Developing hippocampal neurons in microisland culture undergo rapid and extensive transmitter release-dependent depression of evoked (phasic) excitatory synaptic activity in response to 1 sec trains of 20 Hz stimulation. Although evoked phasic release was attenuated by repeated stimuli, asynchronous (miniature like) release continued at a high rate equivalent to approximately 2.8 readily releasable pools (RRPs) of quanta/sec. Asynchronous release reflected the recovery and immediate release of quanta because it was resistant to sucrose-induced depletion of the RRP. Asynchronous and phasic release appeared to compete for a common limited supply of release-ready quanta because agents that block asynchronous release, such as EGTA-AM, led to enhanced steady-state phasic release, whereas prolongation of the asynchronous release time course by LiCl delayed recovery of phasic release from depression. Modeling suggested that the resistance of asynchronous release to depression was associated with its ability to out-compete phasic release for recovered quanta attributable to its relatively low release rate (up to 0.04/msec per vesicle) stimulated by bulk intracellular Ca2+ concentration ([Ca2+]i) that could function over prolonged intervals between successive stimuli. Although phasic release was associated with a considerably higher peak rate of release (0.4/msec per vesicle), the [Ca2+]i microdomains that trigger it are brief (1 msec), and with asynchronous release present, relatively few quanta can accumulate within the RRP to be available for phasic release. We conclude that despite depression of phasic release during train stimulation, transmission can be maintained at a near-maximal rate by switching to an asynchronous mode that takes advantage of a bulk presynaptic [Ca2+]i.
Assuntos
Hipocampo/fisiologia , Sinapses/fisiologia , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Animais , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores , Hipocampo/crescimento & desenvolvimento , Cinética , Depressão Sináptica de Longo Prazo , Modelos Neurológicos , Técnicas de Patch-Clamp , Periodicidade , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Ratos , Sinapses/metabolismoRESUMO
Rett syndrome (RTT) is a progressive neurological disorder primarily caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). The heterozygous female brain consists of mosaic of neurons containing both wild-type MeCP2 (MeCP2+) and mutant MeCP2 (MeCP2-). Three-dimensional morphological analysis was performed on individually genotyped layer V pyramidal neurons in the primary motor cortex of heterozygous (Mecp2(+/-) ) and wild-type (Mecp2(+/+) ) female mice ( > 6 mo.) from the Mecp2(tm1.1Jae) line. Comparing basal dendrite morphology, soma and nuclear size of MeCP2+ to MeCP2- neurons reveals a significant cell autonomous, genotype specific effect of Mecp2. MeCP2- neurons have 15% less total basal dendritic length, predominantly in the region 70-130 µm from the cell body and on average three fewer branch points, specifically loss in the second and third branch orders. Soma and nuclear areas of neurons of mice were analyzed across a range of ages (5-21 mo.) and X-chromosome inactivation (XCI) ratios (12-56%). On average, MeCP2- somata and nuclei were 15 and 13% smaller than MeCP2+ neurons respectively. In most respects branching morphology of neurons in wild-type brains (MeCP2 WT) was not distinguishable from MeCP2+ but somata and nuclei of MeCP2 WT neurons were larger than those of MeCP2+ neurons. These data reveal cell autonomous effects of Mecp2 mutation on dendritic morphology, but also suggest non-cell autonomous effects with respect to cell size. MeCP2+ and MeCP2- neuron sizes were not correlated with age, but were correlated with XCI ratio. Unexpectedly the MeCP2- neurons were smallest in brains where the XCI ratio was highly skewed toward MeCP2+, i.e., wild-type. This raises the possibility of cell non-autonomous effects that act through mechanisms other than globally secreted factors; perhaps competition for synaptic connections influences cell size and morphology in the genotypically mosaic brain of RTT model mice.
RESUMO
We studied tyrosine hydroxylase (TH)-immunoreactive neurons and neuropil in the olfactory bulb of the leopard frog, Rana pipiens, and in the clawed frog, Xenopus laevis. In both frogs, TH processes in the main olfactory bulb showed a trilaminar organization, with a densely stained external glomerular layer (GL), a moderately stained middle mitral cell layer (MCL), and internally a weakly stained internal plexiform layer (IPL) and granule cell layer (GRL). TH-positive cells in the MCL and IPL could be divided into two types. Type 1 cells had one or two thick dendrites that arborized within glomeruli in the GL and often had a thin "axon-like" process that exited the cell on the internal surface, with a recurrent collateral that ascended into the GL. Type 2 cells had beaded dendrites arborizing in the MCL and no discernible axons. Both type 1 and type 2 cells were numerous in the MCL and IPL of Rana, whereas only type 2 cells were common in the MCL and IPL of Xenopus. In the GL, labeled cells were numerous in Xenopus but rare in Rana. Mitral cells were stained retrogradely by tracer injection into the lateral olfactory tract and by local injection into the bulb. In no case was double labeling for TH observed, suggesting that TH-positive cells in frog olfactory bulb are likely to be interneurons. Double labeling with an anti-gamma-aminobutyric acid (GABA) antibody showed that the TH-positive cells formed a population separate from the GABA-containing interneurons.