971 resultados para Hippocampal Slices
Resumo:
γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the nervous system and acts via three distinct receptor classes: A, B, and C. GABAC receptors are ionotropic receptors comprising ρ subunits. In this work, we aimed to elucidate the expression of ρ subunits in the postnatal brain, the characteristics of ρ2 homo-oligomeric receptors, and the function of GABAC receptors in the hippocampus. In situ hybridization on rat brain slices showed ρ2 mRNA expression from the newborn in the superficial grey layer of the superior colliculus, from the first postnatal week in the hippocampal CA1 region and the pretectal nucleus of the optic tract, and in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three ρ subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, ρ2 mRNA expression clearly dominated over ρ1 and ρ3. GABAC receptor protein expression was confirmed in the adult hippocampus, superior colliculus, and dorsal lateral geniculate nucleus by immunohistochemistry. From the selective distribution of ρ subunits, GABAC receptors may be hypothesized to be specifically involved in aspects of visual image motion processing in the rat brain. Although previous data had indicated a much higher expression level for ρ2 subunit transcripts than for ρ1 or ρ3 in the brain, previous work done on Xenopus oocytes had suggested that rat ρ2 subunits do not form functional homo-oligomeric GABAC receptors but need ρ1 or ρ3 subunits to form hetero-oligomers. Our results demonstrated, for the first time, that HEK 293 cells transfected with ρ2 cDNA displayed currents in whole-cell patch-clamp recordings. Homomeric rat ρ2 receptors had a decreased sensitivity to, but a high affinity for picrotoxin and a marked sensitivity to the GABAC receptor agonist CACA. Our results suggest that ρ2 subunits may contribute to brain function, also in areas not expressing other ρ subunits. Using extracellular electrophysiological recordings, we aimed to study the effects of the GABAC receptor agonists and antagonists on responses of the hippocampal neurons to electrical stimulation. Activation of GABAC receptors with CACA suppressed postsynaptic excitability and the GABAC receptor antagonist TPMPA inhibited the effects of CACA. Next, we aimed to display the activation of the GABAC receptors by synaptically released GABA using intracellular recordings. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation were prolonged by TPMPA. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABAC receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABAC receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA. Taken together with the restricted expression pattern of GABAC receptors in the brain and their distinctive pharmacological and biophysical properties, our findings supporting extrasynaptic localization of these receptors raise interesting possibilities for novel pharmacological therapies in the treatment of, for example, epilepsy and sleep disorders.
Resumo:
Quality of fresh-cut carambola (Averrhoa carambola L) is related to many chemical and biochemical variables especially those involved with softening and browning, both influenced by storage temperature. To study these effects, a multivariate analysis was used to evaluate slices packaged in vacuum-sealed polyolefin bags, and stored at 2.5 degrees C, 5 degrees C and 10 degrees C, for up to 16 d. The quality of slices at each temperature was correlated with the duration of storage, O(2) and CO(2) concentration in the package, physical chemical constituents, and activity of enzymes involved in softening (PG) and browning (PPO) metabolism. Three quality groups were identified by hierarchical cluster analysis, and the classification of the components within each of these groups was obtained from a principal component analysis (PCA). The characterization of samples by PCA clearly distinguished acceptable and non-acceptable slices. According to PCA, acceptable slices presented higher ascorbic acid content, greater hue angles ((o)h) and final lightness (L-5) in the first principal component (PC1). On the other hand, non-acceptable slices presented higher total pectin content. PPO activity in the PC1. Non-acceptable slices also presented higher soluble pectin content, increased pectin solubilisation and higher CO(2) concentration in the second principal component (PC2) whereas acceptable slices showed lower total sugar content. The hierarchical cluster and PCA analyses were useful for discriminating the quality of slices stored at different temperatures.
Resumo:
We propose a method to encode a 3D magnetic resonance image data and a decoder in such way that fast access to any 2D image is possible by decoding only the corresponding information from each subband image and thus provides minimum decoding time. This will be of immense use for medical community, because most of the PET and MRI data are volumetric data. Preprocessing is carried out at every level before wavelet transformation, to enable easier identification of coefficients from each subband image. Inclusion of special characters in the bit stream facilitates access to corresponding information from the encoded data. Results are taken by performing Daub4 along x (row), y (column) direction and Haar along z (slice) direction. Comparable results are achieved with the existing technique. In addition to that decoding time is reduced by 1.98 times. Arithmetic coding is used to encode corresponding information independently
Resumo:
Study of hypersynchronous activity is of prime importance for combating epilepsy. Studies on network structure typically reconstruct the network by measuring various aspects of the interaction between neurons and subsequently measure the properties of the reconstructed network. In sub-sampled networks such methods lead to significant errors in reconstruction. Using rat hippocampal neurons cultured on a multi-electrode array dish and a glutamate injury model of epilepsy in vitro, we studied synchronous activity in neuronal networks. Using the first spike latencies in various neurons during a network burst, we extract various recurring spatio-temporal onset patterns in the networks. Comparing the patterns seen in control and injured networks, we observe that injured networks express a wide diversity in their foci (origin) and activation pattern, while control networks show limited diversity. Furthermore, we note that onset patterns in glutamate injured networks show a positive correlation between synchronization delay and physical distance between neurons, while control networks do not.
Resumo:
Study of hypersynchronous activity is of prime importance for combating epilepsy. Studies on network structure typically reconstruct the network by measuring various aspects of the interaction between neurons and subsequently measure the properties of the reconstructed network. In sub-sampled networks such methods lead to significant errors in reconstruction. Using rat hippocampal neurons cultured on a multi-electrode array dish and a glutamate injury model of epilepsy in vitro, we studied synchronous activity in neuronal networks. Using the first spike latencies in various neurons during a network burst, we extract various recurring spatio-temporal onset patterns in the networks. Comparing the patterns seen in control and injured networks, we observe that injured networks express a wide diversity in their foci (origin) and activation pattern, while control networks show limited diversity. Furthermore, we note that onset patterns in glutamate injured networks show a positive correlation between synchronization delay and physical distance between neurons, while control networks do not.
Resumo:
How does the presence of plastic active dendrites in a pyramidal neuron alter its spike initiation dynamics? To answer this question, we measured the spike-triggered average (STA) from experimentally constrained, conductance-based hippocampal neuronal models of various morphological complexities. We transformed the STA computed from these models to the spectral and the spectrotemporal domains and found that the spike initiation dynamics exhibited temporally localized selectivity to a characteristic frequency. In the presence of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, the STA characteristic frequency strongly correlated with the subthreshold resonance frequency in the theta frequency range. Increases in HCN channel density or in input variance increased the STA characteristic frequency and its selectivity strength. In the absence of HCN channels, the STA exhibited weak delta frequency selectivity and the characteristic frequency was related to the repolarization dynamics of the action potentials and the recovery kinetics of sodium channels from inactivation. Comparison of STA obtained with inputs at various dendritic locations revealed that nonspiking and spiking dendrites increased and reduced the spectrotemporal integration window of the STA with increasing distance from the soma as direct consequences of passive filtering and dendritic spike initiation, respectively. Finally, the presence of HCN channels set the STA characteristic frequency in the theta range across the somatodendritic arbor and specific STA measurements were strongly related to equivalent transfer-impedance-related measurements. Our results identify explicit roles for plastic active dendrites in neural coding and strongly recommend a dynamically reconfigurable multi-STA model to characterize location-dependent input feature selectivity in pyramidal neurons.
Resumo:
The growth of axons is an intricately regulated process involving intracellular signaling cascades and gene transcription. We had previously shown that the stimulus-dependent transcription factor, serum response factor (SRF), plays a critical role in regulating axon growth in the mammalian brain. However, the molecular mechanisms underlying SRF-dependent axon growth remains unknown. Here we report that SRF is phosphorylated and activated by GSK-3 to promote axon outgrowth in mouse hippocampal neurons. GSK-3 binds to and directly phosphorylates SRF on a highly conserved serine residue. This serine phosphorylation is necessary for SRF activity and for its interaction with MKL-family cofactors, MKL1 and MKL2, but not with TCF-family cofactor, ELK-1. Axonal growth deficits caused by GSK-3 inhibition could be rescued by expression of a constitutively active SRF. The SRF target gene and actin-binding protein, vinculin, is sufficient to overcome the axonal growth deficits of SRF-deficient and GSK-3-inhibited neurons. Furthermore, short hairpin RNA-mediated knockdown of vinculin also attenuated axonal growth. Thus, our findings reveal a novel phosphorylation and activation of SRF by GSK-3 that is critical for SRF-dependent axon growth in mammalian central neurons.
Resumo:
A gradient in the density of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels is necessary for the emergence of several functional maps within hippocampal pyramidal neurons. Here, we systematically analyzed the impact of dendritic atrophy on nine such functional maps, related to input resistance and local/transfer impedance properties, using conductance-based models of hippocampal pyramidal neurons. We introduced progressive dendritic atrophy in a CA1 pyramidal neuron reconstruction through a pruning algorithm, measured all functional maps in each pruned reconstruction, and arrived at functional forms for the dependence of underlying measurements on dendritic length. We found that, across frequencies, atrophied neurons responded with higher efficiency to incoming inputs, and the transfer of signals across the dendritic tree was more effective in an atrophied reconstruction. Importantly, despite the presence of identical HCN-channel density gradients, spatial gradients in input resistance, local/transfer resonance frequencies and impedance profiles were significantly constricted in reconstructions with dendrite atrophy, where these physiological measurements across dendritic locations converged to similar values. These results revealed that, in atrophied dendritic structures, the presence of an ion channel density gradient alone was insufficient to sustain homologous functional maps along the same neuronal topograph. We assessed the biophysical basis for these conclusions and found that this atrophy-induced constriction of functional maps was mediated by an enhanced spatial spread of the influence of an HCN-channel cluster in atrophied trees. These results demonstrated that the influence fields of ion channel conductances need to be localized for channel gradients to express themselves as homologous functional maps, suggesting that ion channel gradients are necessary but not sufficient for the emergence of functional maps within single neurons.
Resumo:
Lithium is an effective mood stabilizer but its use is associated with many side effects. Electrophysiological recordings of miniature excitatory postsynaptic currents (mEPSCs) mediated by glutamate receptor AMPA-subtype (AMPARs) in hippocampal pyramidal neurons revealed that CLi (therapeutic concentration of 1 mM lithium, from days in vitro 4-10) decreased the mean amplitude and mean rectification index (RI) of AMPAR mEPSCs. Lowered mean RI indicate that contribution of Ca2+-permeable AMPARs in synaptic events is higher in CLi neurons (supported by experiments sensitive to Ca2+-permeable AMPAR modulation). Co-inhibiting PKA, GSK-3 beta and glutamate reuptake was necessary to bring about changes in AMPAR mEPSCs similar to that seen in CLi neurons. FM1-43 experiments revealed that recycling pool size was affected in CLi cultures. Results from minimum loading, chlorpromazine treatment and hyperosmotic treatment experiments indicate that endocytosis in CLi is affected while not much difference is seen in modes of exocytosis. CLi cultures did not show the high KCl associated presynaptic potentiation observed in control cultures. This study, by calling attention to long-term lithium-exposure-induced synaptic changes, might have implications in understanding the side effects such as CNS complications occurring in perinatally exposed babies and cognitive dulling seen in patients on lithium treatment.
Resumo:
The synaptic plasticity literature has focused on establishing necessity and sufficiency as two essential and distinct features in causally relating a signaling molecule to plasticity induction, an approach that has been surprisingly lacking in the intrinsic plasticity literature. In this study, we complemented the recently established necessity of inositol trisphosphate (InsP(3)) receptors (InsP(3)R) in a form of intrinsic plasticity by asking if InsP(3)R activation was sufficient to induce intrinsic plasticity in hippocampal neurons. Specifically, incorporation of D-myo-InsP(3) in the recording pipette reduced input resistance, maximal impedance amplitude, and temporal summation but increased resonance frequency, resonance strength, sag ratio, and impedance phase lead. Strikingly, the magnitude of plasticity in all these measurements was dependent on InsP 3 concentration, emphasizing the graded dependence of such plasticity on InsP(3)R activation. Mechanistically, we found that this InsP(3)-induced plasticity depended on hyperpolarization-activated cyclic nucleotide-gated channels. Moreover, this calcium-dependent form of plasticity was critically reliant on the release of calcium through InsP(3)Rs, the influx of calcium through N-methyl-D-aspartate receptors and voltage-gated calcium channels, and on the protein kinase A pathway. Our results delineate a causal role for InsP(3)Rs in graded adaptation of neuronal response dynamics, revealing novel regulatory roles for the endoplasmic reticulum in neural coding and homeostasis.
Resumo:
Hippocampal pyramidal neurons exhibit gamma-phase preference in their spikes, selectively route inputs through gamma frequency multiplexing and are considered part of gamma-bound cell assemblies. How do these neurons exhibit gamma-frequency coincidence detection capabilities, a feature that is essential for the expression of these physiological observations, despite their slow membrane time constant? In this conductance-based modelling study, we developed quantitative metrics for the temporal window of integration/coincidence detection based on the spike-triggered average (STA) of the neuronal compartment. We employed these metrics in conjunction with quantitative measures for spike initiation dynamics to assess the emergence and dependence of coincidence detection and STA spectral selectivity on various ion channel combinations. We found that the presence of resonating conductances (hyperpolarization-activated cyclic nucleotide-gated or T-type calcium), either independently or synergistically when expressed together, led to the emergence of spectral selectivity in the spike initiation dynamics and a significant reduction in the coincidence detection window (CDW). The presence of A-type potassium channels, along with resonating conductances, reduced the STA characteristic frequency and broadened the CDW, but persistent sodium channels sharpened the CDW by strengthening the spectral selectivity in the STA. Finally, in a morphologically precise model endowed with experimentally constrained channel gradients, we found that somatodendritic compartments expressed functional maps of strong theta-frequency selectivity in spike initiation dynamics and gamma-range CDW. Our results reveal the heavy expression of resonating and spike-generating conductances as the mechanism underlying the robust emergence of stratified gamma-range coincidence detection in the dendrites of hippocampal and cortical pyramidal neurons.
Resumo:
Hippocampal pyramidal neurons express an intraneuronal map of spectral tuning mediated by hyperpolarization-activated cyclic-nucleotide-gated nonspecific-cation channels. Modeling studies have predicted a critical regulatory role for A-type potassium (KA) channels towards augmenting functional robustness of this map. To test this, we performed patch-clamp recordings from soma and dendrites of rat hippocampal pyramidal neurons, and measured spectral tuning before and after blocking KA channels using two structurally distinct pharmacological agents. Consistent with computational predictions, we found that blocking KA channels resulted in a significant reduction in resonance frequency and significant increases in input resistance, impedance amplitude and action-potential firing frequency across the somato-apical trunk. Furthermore, across all measured locations, blocking KA channels enhanced temporal summation of postsynaptic potentials and critically altered the impedance phase profile, resulting in a significant reduction in total inductive phase. Finally, pair-wise correlations between intraneuronal percentage changes (after blocking KA channels) in different measurements were mostly weak, suggesting differential regulation of different physiological properties by KA channels. Our results unveil a pivotal role for fast transient channels in regulating theta-frequency spectral tuning and intrinsic phase response, and suggest that degeneracy with reference to several coexisting functional maps is mediated by cross-channel interactions across the active dendritic arbor.
Resumo:
This paper provides a numerical approach on achieving the limit equilibrium method for 3D slope stability analysis proposed in the theoretical part of the previous paper. Some programming techniques are presented to ensure the maneuverability of the method. Three examples are introduced to illustrate the use of this method. The results are given in detail such as the local factor of safety and local potential sliding direction for a slope. As the method is an extension of 2D Janbu's generalized procedure of slices (GPS), the results obtained by GPS for the longitudinal sections of a slope are also given for comparison with the 3D results. A practical landslide in Yunyang, the Three Gorges, of China, is also analyzed by the present method. Moreover, the proposed method has the advantages and disadvantages of GPS. The problem frequently encountered in calculation process is still about the convergency, especially in analyzing the stability of a cutting corner. Some advice on discretization is given to ensure convergence when the present method is used. However, the problem about convergency still needs to be further explored based on the rigorous theoretical background.
Resumo:
We report here the patterning of primary rat neurons and astrocytes from the postnatal hippocampus on ultra-thin parylene-C deposited on a silicon dioxide substrate, following observations of neuronal, astrocytic and nuclear coverage on strips of different lengths, widths and thicknesses. Neuronal and glial growth was characterized 'on', 'adjacent to' and 'away from' the parylene strips. In addition, the article reports how the same material combination can be used to isolate single cells along thin tracks of parylene-C. This is demonstrated with a series of high magnification images of the experimental observations for varying parylene strip widths and thicknesses. Thus, the findings demonstrate the possibility to culture cells on ultra-thin layers of parylene-C and localize single cells on thin strips. Such work is of interest and significance to the Neuroengineering and Multi-Electrode Array (MEA) communities, as it provides an alternative insulating material in the fabrication of embedded micro-electrodes, which can be used to facilitate single cell stimulation and recording in capacitive coupling mode. © 2010 Elsevier Ltd.