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At least 1,531 records · Page 85Linked to original sources
PubMed1998

Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos.

Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mouse embryos were harvested on gestation day 8 or 9 and stained with the vital lysosomal dye, LysoTracker Red. Following incubation in the stain, embryos were fixed in 2% paraformaldehyde overnight, dehydrated in a graded methanol series, and cleared in benzyl alcohol/benzyl benzoate. The resulting embryo is almost transparent and retains specific LysoTracker Red staining. The entire embryo can be optically sectioned and reconstructed in three dimensions to reveal areas of dye staining. To test this approach, the chemotherapeutic drug hydroxyurea was added to day 8 embryos in vitro to induce apoptosis. Our results demonstrated specific regions undergoing programmed cell death in normal development and increased apoptosis in embryos exposed to hydroxyurea. The observed patterns of LysoTracker Red staining correlate well with previous studies of cell death using other lysosomotropic dyes such as Nile blue sulfate, acridine orange, or neutral red. LysoTracker Red has the advantages of being aldehyde-fixable and highly fluorescent (bleaching was not observed even after multiple scans). This procedure allows for the optical imaging of whole day 9 (approximately 22 somites) embryos that were greater than 500 microns thick in the Z-axis.

Animals↗
PubMed1999

Detection of the early negative response in fMRI at 1.5 Tesla.

Recent experimental studies have revealed an initial decrease in magnetic resonance (MR) signal that is consistent with optical imaging results. This initial response, thought to arise from a transient increase in deoxyhemoglobin concentration, is probably more localized to the site of neuronal activation. However, with MR imaging, this early response has only been demonstrated at high fields. In this paper, the observation of the initial response at 1.5 T is reported. Compared with results obtained at 4 T, the present study reveals that the initial response grows more rapidly with the field strength than the late positive response, suggesting a more microvascular origin of the initial response.

Adult↗
PubMed2000

Fundamental GABAergic amacrine cell circuitries in the retina: nested feedback, concatenated inhibition, and axosomatic synapses.

Presynaptic gamma-aminobutyrate-immunoreactive (GABA+) profiles were mapped in the cyprinid retina with overlay microscopy: a fusion of electron and optical imaging affording high-contrast ultrastructural and immunocytochemical visualization. GABA+ synapses, deriving primarily from amacrine cells (ACs), compose 92% of conventional synapses and 98% of the input to bipolar cells (BCs) in the inner plexiform layer. GABA+ AC synapses, the sign-inverting elements of signal processing, are deployed in micronetworks and distinctive synaptic source/target topologies. Nested feedback micronetworks are formed by three types of links (BC --> AC, reciprocal BC <-- AC, and AC --> AC synapses) arranged as nested BC<--> [AC --> AC] loops. Circuits using nested feedback can possess better temporal performance than those using simple reciprocal feedback loops. Concatenated GABA+ micronetworks of AC --> AC and AC --> AC --> AC chains are common and must be key elements for lateral spatial, temporal, and spectral signal processing. Concatenated inhibitions may represent exceptionally stable, low-gain, sign-conserving devices for receptive field construction. Some chain elements are GABA immunonegative (GABA-) and are, thus, likely glycinergic synapses. GABA+ synaptic baskets target the somas of certain GABA+ and GABA- cells, resembling cortical axosomatic synapses. Finally, all myelinated intraretinal profiles are GABA+, suggesting that some efferent systems are sources of GABAergic inhibition in the cyprinid retina and may comprise all axosomatic synapses. These micronetworks are likely the fundamental elements for receptive field shaping in the inner plexiform layer, although few receptive field models incorporate them as functional components. Conversely, simple feedback and feedforward synapses may often be chimeras: the result of an incomplete view of synaptic topology.

Animals↗
PubMed2001

Emergence of ocular dominance columns in cat visual cortex by 2 weeks of age.

Previous anatomic studies of the geniculocortical projection showed that ocular dominance columns emerge by 3 weeks of age in cat visual cortex, but recent optical imaging experiments have revealed a pattern of physiologic eye dominance by the end of the second week of life. We used two methods to search for an anatomic correlate of this early functional ocular dominance pattern. First, retrograde labeling of lateral geniculate nucleus (LGN) inputs to areas of cortex preferentially activated by one eye showed that the geniculocortical projection was already partially segregated by eye at postnatal day 14 (P14). Second, transneuronal label of geniculocortical afferents in flattened sections of cortex after a tracer injection into one eye showed a periodic pattern at P14 but not at P7. In the classic model for the development of ocular dominance columns, initially overlapping geniculocortical afferents segregate by means of an activity-dependent competitive process. Our data are consistent with this model but suggest that ocular dominance column formation begins between P7 and P14, approximately a week earlier than previously believed. The functional and anatomic data also reveal an early developmental bias toward contralateral eye afferents. This initial developmental bias is not consistent with a strictly Hebbian model for geniculocortical afferent segregation. The emergence of ocular dominance columns before the onset of the critical period for visual deprivation also suggests that the mechanisms for ocular dominance column formation may be partially distinct from those mediating plasticity later in life.

Aging↗
PubMed2003

Neuronal assemblies: single cortical neurons are obedient members of a huge orchestra.

Spontaneous cortical activity of single neurons is often either dismissed as noise, or is regarded as carrying no functional significance and hence is ignored. Our findings suggest that such concepts should be revised. We explored the coherent population activity of neuronal assemblies in primary sensory area in the absence of a sensory input. Recent advances in real-time optical imaging based on voltage-sensitive dyes (VSDI) have facilitated exploration of population activity and its intimate relationship to the activity of individual cortical neurons. It has been shown by in vivo intracellular recordings that the dye signal measures the sum of the membrane potential changes in all the neuronal elements in the imaged area, emphasizing subthreshold synaptic potentials and dendritic action potentials in neuronal arborizations originating from neurons in all cortical layers whose dendrites reach the superficial cortical layers. Thus, the VSDI has allowed us to image the rather illusive activity in neuronal dendrites that cannot be readily explored by single unit recordings. Surprisingly, we found that the amplitude of this type of ongoing subthreshold activity is of the same order of magnitude as evoked activity. We also found that this ongoing activity exhibited high synchronization over many millimeters of cortex. We then investigated the influence of ongoing activity on the evoked response, and showed that the two interact strongly. Furthermore, we found that cortical states that were previously associated only with evoked activity can actually be observed also in the absence of stimulation, for example, the cortical representation of a given orientation may appear without any visual input. This demonstration suggests that ongoing activity may also play a major role in other cortical function by providing a neuronal substrate for the dependence of sensory information processing on context, behavior, memory and other aspects of cognitive function.

Action Potentials↗
PubMed2007

Artificial zinc(II) complexes regulate cell cycle and apoptosis-related genes in tumor cell lines.

Various proteins involved in transcriptional regulation possess highly selective DNA-binding domains, known as zinc fingers. However, little is known about small-molecule zinc(II) complexes in the regulation of gene expression and programmed cell death. A new family of zinc(II) complexes is reported, which might be useful against human cancer cells. By using template synthesis and in vitro cell-line screening, a set of zinc(II) complexes has been found to induce apoptosis of cancer cells and display single-reagent in vitro cytotoxicity. The method used to synthesize the molecules resulted in "built-in" luminescent behavior. Confocal optical imaging clearly demonstrated penetration through the cell membrane by these metal complexes. We have discovered that C3, the meso-zinc(II) complex is an extremely efficient regulator of the cell cycle and anti-apoptosis genes bcl-2 and bcl-xL. This study provides a new insight into the development of zinc(II) complexes as potential drugs.

Antineoplastic Agents↗
PubMedDate not supplied

An HPLC/mass spectrometry platform for the development of multimodality contrast agents and targeted therapeutics: prostate-specific membrane antigen small molecule derivatives.

The production of disease-targeted agents requires the covalent conjugation of a targeting molecule with a contrast agent or therapeutic, followed by purification of the product to homogeneity. Typical targeting molecules, such as small molecules and peptides, often have high charge-to-mass ratios and/or hydrophobicity. Contrast agents and therapeutics themselves are also diverse, and include lanthanide chelates for MRI, (99m)Tc chelates for SPECT, (90)Y chelates for radiotherapy, (18)F derivatives for PET, and heptamethine indocyanines for near-infrared fluorescent optical imaging. We have constructed a general-purpose HPLC/mass spectrometry platform capable of purifying virtually any targeted agent for any modality. The analytical sub-system is composed of a single dual-head pump that directs mobile phase to either a hot cell for the purification of radioactive agents or to an ES-TOF MS for the purification of nonradioactive agents. Nonradioactive agents are also monitored during purification by ELSD, absorbance and fluorescence. The preparative sub-system is composed of columns and procedures that permit rapid scaling from the analytical system. To demonstrate the platform's utility, we describe the preparation of five small molecule derivatives specific for prostate-specific membrane antigen (PSMA): a gadolinium derivative for MRI, indium, rhenium and technetium derivatives for SPECT, and an yttrium derivative for radiotherapy. All five compounds are derived from a highly anionic targeting ligand engineered to have a single nucleophile for N-hydroxysuccinimide-based conjugation. We also describe optimized column/mobile phase combinations and mass spectrometry settings for each class of agent, and discuss strategies for purifying molecules with extreme charge and/or hydrophobicity. Taken together, our study should expedite the development of disease-targeted, multimodality diagnostic and therapeutic agents.

Antigens, Surface↗
PubMed2002

In vivo modulation of a cortical functional sensory representation shortly after topical cholinergic agent application.

The aim of the present study was to determine whether cholinergic increase in the size of a functional representation (collective evoked response from a large population of neurons) can be observed shortly (within an hour) after treatment onset and whether nicotinic receptors can participate in this type of modulation. Cholinergic agonist application has been found previously to increase the response of a single cortical neuron to a stimulus. Also, pairing cholinergic basal forebrain stimulation with delivery of a tone has been reported to increase the size of that tone's functional representation. Whereas the increase in a single cortical neuron response can occur within seconds after cholinergic agonist application, to date the increase in the size of a functional representation has only been investigated within one to several weeks after the onset of pairing basal forebrain stimulation with tone delivery. Furthermore, primarily muscarinic receptors have been implicated in these types of changes in cortical activity. By using optical imaging of intrinsic signals in vivo, we found that the size of a whisker's functional representation in the primary somatosensory cortex of the rat increases substantially within 69 or 46 minutes after topical application of either a muscarinic or nicotinic agonist to the exposed cortex, respectively, and decreases within 23 minutes after topical application of a muscarinic antagonist. For each cholinergic agent, we verified that delivery of a cholinergic agent by means of topical application can lead to the agent's successful penetration through the cortical layers in the time allotted to complete an imaging experiment. Furthermore, the time course of penetration for each agent was characterized. Based on the combined imaging/penetration results, we speculate on potential sites of cholinergic action in the cortex. Irrespective of the exact mechanism of action, we demonstrate here that an increase in the size of a functional sensory representation can occur shortly by means of activation of either nicotinic or muscarinic receptors.

Animals↗
PubMed2002

Structural and functional asymmetry in the normal and epileptic rat dentate gyrus.

The rat dentate gyrus is usually described as relatively homogeneous. Here, we present anatomic and physiological data which demonstrate that there are striking differences between the supra- and infrapyramidal blades after status epilepticus and recurrent seizures. These differences appear to be an accentuation of a subtle asymmetry present in normal rats. In both pilocarpine and kainic acid models, there was greater mossy fiber sprouting in the infrapyramidal blade. This occurred primarily in the middle third of the hippocampus. Asymmetric sprouting was evident both with Timm stain as well as antisera to brain-derived neurotrophic factor (BDNF) or neuropeptide Y (NPY). In addition, surviving NPY-immunoreactive hilar neurons were distributed preferentially in the suprapyramidal region of the hilus. Extracellular recordings from infrapyramidal sites in hippocampal slices of pilocarpine-treated rats showed larger population spikes and weaker paired-pulse inhibition in response to perforant path stimulation relative to suprapyramidal recordings. A single stimulus could evoke burst discharges in infrapyramidal granule cells but not suprapyramidal blade neurons. BDNF exposure led to spontaneous epileptiform discharges that were larger in amplitude and longer lasting in the infrapyramidal blade. Stimulation of the infrapyramidal molecular layer evoked larger responses in area CA3 than suprapyramidal stimulation. In slices from the temporal pole, in which anatomic evidence of asymmetry waned, there was little evidence of physiological asymmetry either. Of interest, some normal rats also showed signs of greater evoked responses in the infrapyramidal blade, and this could be detected with both microelectrode recording and optical imaging techniques. Although there were no signs of hyperexcitability in normal rats, the data suggest that there is some asymmetry in the normal dentate gyrus and this asymmetry is enhanced by seizures. Taken together, the results suggest that supra- and infrapyramidal blades of the dentate gyrus could have different circuit functions and that the infrapyramidal blade may play a greater role in activating the hippocampus.

Animals↗
PubMed2004

Local and global chemotopic organization: general features of the glomerular representations of aliphatic odorants differing in carbon number.

To determine whether there is a general strategy used by the olfactory system to represent odorants differing in carbon chain length, rats were exposed to homologous series of straight-chained, saturated aliphatic aldehydes, ethyl esters, acetates, ketones, primary alcohols, and secondary alcohols (32 odorants total). Neural activity across the entire glomerular layer of the olfactory bulb was mapped quantitatively by measuring uptake of [14C]2-deoxyglucose evoked by each odorant. Uptake was observed both in dorsal glomerular modules previously associated with the particular odorant functional groups and in more ventral and posterior modules. Aldehyde-evoked activity patterns were dominated by ventral modules that included the area receiving projections from octanal-responsive sensory neurons expressing the I7 odorant receptor. The dorsal area that has been the focus of optical imaging studies of aldehyde responses contained only minor activity. For all functional groups except for ketones, uptake within functional group-sensitive modules displayed local chemotopy, with longer odorants stimulating more ventral and rostral glomeruli. In more posterior regions, chemotopy was observed for all functional groups, again with uptake shifting ventrally and rostrally with increasing chain length. In addition to these local shifts in activity, correlations analysis of entire activity patterns revealed a global chemotopic organization for all odorant series, with each odorant evoking a pattern most similar to odorants possessing the same functional group but differing by only one carbon in length. Thus, global chemotopy and local modular chemotopy appear to be fundamental principles underlying the representation of odorants differing in carbon chain length.

Animals↗
PubMed2006

Model-based analysis of excitatory lateral connections in the visual cortex.

Excitatory lateral connections within the primary visual cortex are thought to link neurons with similar receptive field properties. Here we studied whether this rule can predict the distribution of excitatory connections in relation to cortical location and orientation preference in the cat visual cortex. To this end, we obtained orientation maps of areas 17 or 18 using optical imaging and injected anatomical tracers into these regions. The distribution of labeled axonal boutons originating from large populations of excitatory neurons was then analyzed and compared with that of individual pyramidal or spiny stellate cells. We demonstrate that the connection patterns of populations of nearby neurons can be reasonably predicted by Gaussian and von Mises distributions as a function of cortical location and orientation, respectively. The connections were best described by superposition of two components: a spatially extended, orientation-specific and a local, orientation-invariant component. We then fitted the same model to the connections of single cells. The composite pattern of nine excitatory neurons (obtained from seven different animals) was consistent with the assumptions of the model. However, model fits to single cell axonal connections were often poorer and their estimated spatial and orientation tuning functions were highly variable. We conclude that the intrinsic excitatory network is biased to similar cortical locations and orientations but it is composed of neurons showing significant deviations from the population connectivity rule.

Animals↗
PubMed2006

Novel aspects of systems biology and clinical cytomics.

The area of Cytomics and Systems Biology became of great impact during the last years. In some fields of the leading cytometric techniques it represents the cutting edge today. Many different applications/variations of multicolor staining were developed for flow- or slide-based cytometric analysis of suspensions and sections to whole animal analysis. Multispectral optical imaging can be used for studying immunological and tumorigenic processes. New methods resulted in the establishment of lipidomics as the systemic research of lipids and their behavior. All of these development push the systemic approach of the analysis of biological specimens to enhance the outcome in the clinic and in drug discovery programs.

Biomedical Research↗
PubMed1998

Fluorescence imaging of light absorption for axial-beam geometry in capillary electrophoresis.

A new method for investigation of axial-beam absorption detection for improved detection limits in microcolumn separations is reported. The method is based on fluorescence imaging of light absorption along a separation capillary. The probing UV light is introduced at one end of the capillary and shows an exponential fall-off along the capillary. As the UV light propagates through the sample peaks, an additional loss in intensity will be observed. In order to view the absorption profile along the capillary, a background fluorophore is added to the buffer. A charge-coupled device (CCD) detector and imaging optics are placed beside the capillary to view the capillary in a direction perpendicular to the capillary. Signal integration is employed for consecutive exposures as well as for neighboring detector pixels in order to increase the signal-to-noise ratio. Measurements for stilbene 3 with sulforhodamine B as a background fluorophore are presented. The characteristics of the detection method and potential improvements are discussed.

Electrophoresis, Capillary↗
PubMed2004

High-resolution computer simulation of the dynamics of isoelectric focusing of proteins.

A dynamic electrophoresis simulator that accepts 150 components and voltage gradients employed in the laboratory was used to provide a detailed description of the focusing process of proteins under conditions that were hitherto inaccessible. High-resolution focusing data of four hemoglobin variants in a convection-free medium are presented for pH 3-10 and pH 5-8 gradients formed with 20 and 40 carrier ampholytes/pH unit, respectively. With 300 V/cm, focusing is shown to occur within 5-10 min, whereas at 600 V/cm separation is predicted to be complete between 2.5 and 5 min. The time interval required for focusing of proteins is demonstrated to be dependent on the input protein charge data and, however less, on the properties of the carrier ampholytes. The simulation data reveal that the number of transient protein boundaries migrating from the two ends of the column towards the focusing positions is equal to the number of sample components. Each protein is being focused via the well-known double-peak approach to equilibrium, a process that is also characteristic for focusing of the carrier ampholytes. The predicted focusing dynamics for the hemoglobin variants in pH 3-10 and pH 5-8 gradients are shown to qualitatively agree well with experimental data obtained by whole-column optical imaging.

Computer Simulation↗
PubMed1998

Magnetic resonance microscopy and histopathology: comparative approach of bromobenzene-induced hepatotoxicity in the rat.

The development of magnetic resonance (MR) microscopy has provided new approaches to histology and histopathology. Recent work has shown the promise of increased sensitivity in animal models of chemically induced hepatotoxicity. However, the field is so new that there is little experience to relate changes seen in MR micrographs to the more traditional optical images stained with hematoxylin and eosin. This work compares the sensitivity and reproducibility of MR microscopy with conventional histopathology in detecting bromobenzene-induced hepatotoxicity in the rat. A time-course study was undertaken to provide a range of histopathologies. Specimens were studied at 24, 48, 72, and 96 hours after exposure to 10% of the median lethal dose of bromobenzene. Using 4 animals per group (a total of 32 rats) added statistical significance to the study and defined a range of interanimal variability over 96 hours. This work shows that MR microscopy, besides being nondestructive and three-dimensional, is at least as sensitive as conventional hematoxylin-eosin staining in detecting bromobenzene-induced centrilobular lesions and recovery of the hepatocellular architecture in the rat. This study further suggests that, as we begin to understand the underlying mechanisms of contrast in MR histology, MR may, in fact, supply even higher specificity than more traditional studies: variations were observed in MR images of treated livers at a given time point that could be not be differentiated based on the grading of necrosis and inflammation on hematoxylin-eosin-stained sections.

Animals↗
PubMed2005

Spatiotemporal visualization of long-term potentiation and depression in the hippocampal CA1 area.

Long-term potentiation (LTP) in the CA1 area of the hippocampus depends critically on the statistical characteristics of its stimulus. The ability of optical imaging to record spatial distribution has made it possible to examine systematically the effect of higher-order statistical characteristics, such as the correlation between successive pairs of inter-stimulus intervals (ISIs) on the induction of LTP. Therefore, the function of frequency (first-order) and temporal pattern (second-order) was examined using this imaging technique. To investigate the dependence of LTP on frequency, periodic stimuli with the same number of pulses were applied at different frequencies (1-10 Hz, n=200) to Schaffer commissural-collateral fibers. While stimulus frequencies from 2-10 Hz induced LTP of varying magnitudes and low-frequency stimuli (1 Hz) induced long-term depression (LTD), spatial distribution remained consistent. These results suggest that induction frequency has a greater effect on the magnitude of LTP than on its spatial distribution. By employing nonperiodic stimuli at the same mean frequency (2 Hz), the effect of varying the temporal structure of a stimulus was also investigated. As the correlation of successive ISIs was increased from negative to positive, not only did the magnitude of LTP increase, there was also a statistically significant change in the spatial distribution of LTP. Interestingly, when a strong negatively correlated stimulus was applied, both LTP and LTD were simultaneously observed in the CA1 area. It was also found that the magnitude of LTP 200-300 mum distal to the cellular layer was larger than that of the LTP induced proximal (<100 microm) to that layer. These results support the hypothesis that the spatial distribution of LTP throughout the hippocampus relies principally on the temporal patterning of input stimulation. This insight into the structure of the CA1 neural network may reveal the importance of stimulus timing events in the spatial encoding of memories.

Action Potentials↗
PubMed2005

Extracellular diffusion is fast and isotropic in the stratum radiatum of hippocampal CA1 region in rat brain slices.

Molecular transport in brain extracellular space (ECS) is hindered by the structure of the tissue. Diffusion analysis of small extracellular markers quantifies tissue hindrance, expressed as tortuosity lambda = (D/D*)(1/2), where D is the free diffusion coefficient and D* is the effective diffusion coefficient in tissue. In healthy brain, lambda is approximately 1.6, but the nature of this parameter is poorly understood. We report that the stratum radiatum of the hippocampal CA1 region in vitro, previously shown to be anisotropic (i.e., different along the x-, y-, and z-axes) in in vivo study, is isotropic like somatosensory neocortex but has a reduced lambda. Diffusion of fluorophore-labeled dextran (f-dex, M(r) 3,000) and tetramethylammonium (TMA(+), M(r) 74) was measured in rat brain slices (400 mum) using integrative optical imaging (IOI) and real-time iontophoresis (RTI), respectively. In the stratum radiatum, diffusion of f-dex was similar along the x-, y-, and z-axes (lambda(x), lambda(y); lambda(z) were 1.55, 1.53, and 1.55), but the tortuosity was significantly lower than in the neocortex, where lambda = 1.81. This finding was confirmed by the RTI method, which measured lambda with TMA(+), a much smaller molecule, and determined volume fraction alpha, the proportion of tissue occupied by the ECS. In stratum radiatum, lambda(x), lambda(y), and lambda(z) were 1.47, 1.44, and 1.46, while in neocortex, lambda was 1.65. The ECS volume fraction was similar (0.24) in both regions. It is proposed that in the hippocampus, low lambda reflects a reduced occurrence of concave extracellular microdomains, referred to as dead spaces, which increase tortuosity by transient trapping of markers. Functionally, a low lambda may permit structural plasticity and facilitate extrasynaptic communication. It may also enhance the spread of neuroactive substances and thus contribute to the sensitivity of the hippocampal CA1 region to ischemia and epilepsy.

Animals↗
PubMed1995

Three-dimensional nuclear magnetic resonance microimaging of trabecular bone.

The conventional approach to measuring structural parameters in trabecular bone rests on stereology from optical images, derived from sections of embedded bone. In order to provide data that are statistically representative of a sufficiently large volume, multiple sections need to be analyzed in each of the three orthogonal planes. In this work, an alternative technique is presented which is based on three-dimensional (3D) volumetric proton nuclear magnetic resonance (NMR) microimaging. The method presented provides from 9 x 9 x 4 mm3 volumes of defatted bone specimens in 15-20 minutes scan time at isotropic resolution corresponding to (78 microm)3 voxel size. Surface-rendered images of bovine and human trabecular bone are shown and an algorithm was developed and implemented for determining the orientation and magnitude of the principle axes of the mean intercept length tensor.

Adult↗