RESUMO
Glycine receptors (GlyRs) are the primary mediators of fast inhibitory transmission in the mammalian spinal cord, where they modulate sensory and motor signaling. Mutations in GlyR genes as well as some other genes underlie the hereditary disorder hyperekplexia, characterized by episodic muscle stiffness and exaggerated startle responses. Here, we have investigated pain-related behavior and GlyR expression in the spinal cord of the GlyR deficient mutant mouse spastic (spa). In spastic mice, the GlyR number is reduced due to a ß subunit gene (Glrb) mutation resulting in aberrant splicing of GlyRß transcripts. Via direct physical interaction with the GlyR anchoring protein gephyrin, this subunit is crucially involved in the postsynaptic clustering of heteromeric GlyRs. We show that the mutation differentially affects aspects of the pain-related behavior of homozygous Glrbspa/Glrbspa mice. While response latencies to noxious heat were unchanged, chemically induced pain-related behavior revealed a reduction of the licking time and an increase in flinching in spastic homozygotes during both phases of the formalin test. Mechanically induced nocifensive behavior was reduced in spastic mice, although hind paw inflammation (by zymosan) resulted in allodynia comparable to wild-type mice. Immunohistochemical staining of the spinal cord revealed a massive reduction of dotted GlyRα subunit immunoreactivity in both ventral and dorsal horns, suggesting a reduction of clustered receptors at synaptic sites. Transcripts for all GlyRα subunit variants, however, were not reduced throughout the dorsal horn of spastic mice. These findings suggest that the loss of functional GlyRß subunits and hence synaptically localized GlyRs compromises sensory processing differentially, depending on stimulus modality.
RESUMO
The pharmacological treatment of major depressive disorder with currently available antidepressant drugs is still unsatisfying as response to medication is delayed and in some patients even non-existent. To understand complex psychiatric diseases such as major depressive disorder and their treatment, research focus is shifting from investigating single neurons towards a view of the entire functional and effective neuronal network, because alterations on single synapses through antidepressant drugs may translate to alterations in the entire network. Here, we examined the effects of monoamine reuptake inhibitors on in vitro hippocampal network dynamics using calcium fluorescence imaging and analyzing the data with means of graph theoretical parameters. Hypothesizing that monoamine reuptake inhibitors operate through changes of effective connectivity on micro-scale neuronal networks, we measured the effects of the selective monoamine reuptake inhibitors GBR-12783, Sertraline, Venlafaxine, and Amitriptyline on neuronal networks. We identified a common pattern of effects of the different tested monoamine reuptake inhibitors. After treatment with GBR-12783, Sertraline, and Venlafaxine, the connectivity degree, measuring the number of existing connections in the network, was significantly decreased. All tested substances led to networks with more submodules and a reduced global efficiency. No monoamine reuptake inhibitor did affect network-wide firing rate, the characteristic path length, or the network strength. In our study, we found that monoamine reuptake inhibition in neuronal networks in vitro results in a sharpening of the network structure. These alterations could be the basis for the reorganization of a large-scale miswired network in major depressive disorder.
Assuntos
Transtorno Depressivo Maior , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Transtorno Depressivo Maior/tratamento farmacológico , Hipocampo , Humanos , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Sertralina/farmacologia , Sertralina/uso terapêuticoRESUMO
Analyzing the connectivity of neuronal networks, based on functional brain imaging data, has yielded new insight into brain circuitry, bringing functional and effective networks into the focus of interest for understanding complex neurological and psychiatric disorders. However, the analysis of network changes, based on the activity of individual neurons, is hindered by the lack of suitable meaningful and reproducible methodologies. Here, we used calcium imaging, statistical spike time analysis and a powerful classification model to reconstruct effective networks of primary rat hippocampal neurons in vitro. This method enables the calculation of network parameters, such as propagation probability, path length, and clustering behavior through the measurement of synaptic activity at the single-cell level, thus providing a fuller understanding of how changes at single synapses translate to an entire population of neurons. We demonstrate that our methodology can detect the known effects of drug-induced neuronal inactivity and can be used to investigate the extensive rewiring processes affecting population-wide connectivity patterns after periods of induced neuronal inactivity.
Assuntos
Rede Nervosa/fisiologia , Transmissão Sináptica/fisiologia , Animais , Cálcio/metabolismo , Células Cultivadas , Diagnóstico por Imagem , Estimulação Elétrica/métodos , Hipocampo/citologia , Hipocampo/fisiologia , Ratos , Análise de Célula Única , Sinapses/fisiologia , Tetrodotoxina/farmacologiaRESUMO
[This corrects the article on p. 239 in vol. 6, PMID: 26635717.].
RESUMO
This study was conducted to assess the feasibility and diagnostic accuracy of brain artery territory recognition based on geoprojected two-dimensional maps of diffusion MRI data in stroke patients. In this retrospective study, multiplanar diffusion MRI data of ischemic stroke patients was used to create a two-dimensional map of the entire brain. To guarantee correct representation of the stroke, a computer-aided brain artery territory diagnosis was developed and tested for its diagnostic accuracy. The test recognized the stroke-affected brain artery territory based on the position of the stroke in the map. The performance of the test was evaluated by comparing it to the reference standard of each patient's diagnosed stroke territory on record. This study was designed and conducted according to Standards for Reporting of Diagnostic Accuracy (STARD). The statistical analysis included diagnostic accuracy parameters, cross-validation, and Youden Index optimization. After cross-validation on a cohort of 91 patients, the sensitivity of this territory diagnosis was 81% with a specificity of 87%. With this, the projection of strokes onto a two-dimensional map is accurate for representing the affected stroke territory and can be used to provide a static and printable overview of the diffusion MRI data. The projected map is compatible with other two-dimensional data such as EEG and will serve as a useful visualization tool.
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Effects of the antidepressant fluoxetine on stimulation-dependent synaptic vesicle exocytosis were examined in cultured primary hippocampal neurons. Synaptic vesicles were fluorescently labeled in vitro with FM1-43, washed and subsequently destained in two consecutive cycles. Exocytosis was triggered by electric field stimulation and imaged by fluorescence microscopy. In control preparations, the second staining-destaining cycle caused a significant reduction of relative fluorescence loss, number of active synapses and half-decay time (t(50)). These fatigue effects were largely prevented by short-term administration of 1 microM fluoxetine, which was present before and during the second stimulation cycle. Fluoxetine concentrations above 10 microM inhibited exocytosis almost completely but showed no other toxic effects on neurons. Stressed neurons, grown under hyperosmotic conditions, were even more fatigue-protected by fluoxetine. These observations support the idea that therapeutic concentrations of fluoxetine enhance the recovery of neurotransmission from exhausting stimuli in healthy and in hyperosmotically stressed neurons as well.
Assuntos
Exocitose/efeitos dos fármacos , Fluoxetina/farmacologia , Hipocampo/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Células Cultivadas , Estimulação Elétrica , Exocitose/fisiologia , Hipocampo/metabolismo , Compostos de Piridínio , Compostos de Amônio Quaternário , Ratos , Ratos Wistar , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismoRESUMO
On their way to the synapse and back, neuronal proteins are carried in cargo vesicles along axons and dendrites. Here, we demonstrate that the key parameters of axonal transport, i.e., particle velocities and pausing times can be read out from CCD-camera images automatically. In the present study, this is achieved via cross- and autocorrelation of kymograph columns. The applicability of the method was measured on simulated kymographs and data from axonal transport timeseries of mRFP-labeled synaptophysin. In comparing outcomes of velocity determinations via a performance parameter that is analogous to the signal-to-noise ratio (SNR) definition, we find that outcomes are dependent on sampling, particle numbers and signal to noise of the kymograph. Autocorrelation of individual columns allows exact determination of pausing time populations. In contrast to manual tracking, correlation does not require experience, a priori assumptions or disentangling of individual particle trajectories and can operate at low SNR.
Assuntos
Axônios/metabolismo , Quimografia/métodos , Animais , Cinética , Transporte Proteico , Ratos , Reprodutibilidade dos Testes , Sinaptofisina/metabolismoRESUMO
The detailed analysis of beta-amyloid (Abeta) peptides in human plasma is still hampered by the limited sensitivity of available mass spectrometric methods and the lack of appropiate ELISAs to measure Abeta peptides other than Abeta(1-38), Abeta(1-40), and Abeta(1-42). By combining high-yield Abeta immuno- precipitation (IP), IEF, and urea-based Abeta-SDS-PAGE-immunoblot, at least 30 Abeta-immuno-reactive spots were detected in human plasma samples as small as 1.6 mL. This approach clearly resolved Abeta peptides Abeta(1-40), Abeta(1-42), Abeta(1-37), Abeta(1-38), Abeta(1-39), the N-truncated Abeta(2-40), Abeta(2-42), and, for the first time, also Abeta(1-41). Relative quantification indicated that Abeta(1-40) and Abeta(1-42) accounted for less than 60% of the total amount of Abeta peptides in plasma. All other Abeta peptides appear to be either C-terminally or N-terminally truncated forms or as yet uncharacterized Abeta species which migrated as trains of spots with distinct pIs. The Abeta pattern found in cerebrospinal fluid (CSF) was substantially less complex. This sensitive method (2-D Abeta-WIB) might help clarifying the origin of distinct Abeta species from different tissues, cell types, or intracellular pools as well as their amyloidogenicity. It might further help identifying plasma Abeta species suitable as biomarkers for the diagnosis of Alzheimer's disease (AD).
Assuntos
Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/química , Ureia , Doença de Alzheimer/sangue , Eletroforese em Gel Bidimensional , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The advent of new therapeutic avenues for Alzheimer's disease (AD) calls for an improved early and differential diagnosis. METHODS: With surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), cerebrospinal fluid from patients with AD (n = 10) and nondemented control subjects (n = 9) was studied. RESULTS: Molecular mass signals were observed corresponding to three novel amyloid beta (Abeta) peptides that have not previously been described, in addition to those previously known, with molecular masses of 4525.1 d, 4846.8 d, and 7755.8 d. The signal-to-noise ratios (S/NR) of Abeta(4525.1) and Abeta(7758.8+2H) were significantly decreased in AD [Abeta(4525.1): median 2.2 and 4.3 in AD and control subjects, respectively, p <.01; Abeta(7758.8+2H): median 1.0 and 14.0 in AD and control subjects, respectively, p <.01], whereas the S/NR of Abeta(4846.8) was significantly increased in AD (median 3.6 and 2.5 in AD and control subjects, respectively, p <.05). The S/NR of two known AD biomarkers, Abeta1-42 and Abeta1-40, expectedly turned out to be significantly decreased (p <.01) and unaltered in AD, respectively. A moderate and highly significant correlation was observed between S/NR of Abeta1-42 and Abeta42 concentration as measured with enzyme-linked immunosorbent assay (R =.67, p <.01). CONCLUSIONS: We report evidence of three novel amyloid beta peptides that might play an important role in the diagnosis and pathophysiology of Alzheimer's disease.