The synthesis-diffusion-degradation model explains Bicoid gradient formation in unfertilized eggs.

Precise formation of morphogen gradients is essential to the establishment of reproducible pattern in development. Mechanisms proposed for obtaining the requisite precision range from simple models with few parameters to more complex models involving many regulated quantities. The synthesis-diffusion-degradation (SDD) model is a relatively simple model explaining the formation of the Bicoid gradient in Drosophila melanogaster, in which the steady-state characteristic length of the gradient is determined solely by the rates of diffusion and degradation of the morphogen. In this work, we test the SDD model in unfertilized D. melanogaster eggs, which contain a single female pronucleus and lack the nuclear division cycles and other zygotic regulatory processes seen in fertilized eggs. Using two-photon live imaging as well as a novel method for quantitative imaging based on decorrelation of photoswitching waveforms, we find that the Bicoid gradient is longer and shallower in unfertilized eggs as compared to the gradient at the same time points in fertilized eggs. Using a means of measuring the Bicoid lifetime by conjugation to a photoconvertible fluorophore, we find that the lifetime is correspondingly longer in unfertilized eggs, providing qualitative and quantitative agreement with the predictions of the SDD model.

Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints.

Measurements of lateral molecular diffusion on blebs formed on the surfaces of isolated muscle cells and myoblasts are reported. These blebbed membranes retain integral proteins but apparently separate from the detectable cytoskeleton. On blebs, acetylcholine receptors, concanavalin A receptors, and stearoyldextran extrinsic model receptor molecules are free to diffuse with a diffusion coefficient (D) approximately 3 x 10(-9) cm2/s, which is close to the value predicted for hydrodynamic drag in the lipid membrane. In contrast, diffusion of these typical receptors in intact cell membranes is constrained to D approximately less than 10(-10) cm2/s with substantial fractions virtually nondiffusible (D less than 10(-12) cm2/s). Lipid analog diffusion is also slightly enhanced in blebs as expected of evanescent lipid protein interaction. This strong enhancement of membrane protein diffusion is attributed to release from unidentified natural constraints that is induced in some way by detachment of the bleb membrane.

Electric field-induced redistribution and postfield relaxation of low density lipoprotein receptors on cultured human fibroblasts.

The lateral mobility of unliganded low density lipoprotein-receptor (LDL-R) on the surface of human fibroblasts has been investigated by studying the generation and relaxation of concentration differences induced by exposure of the cultured cells to steady electric fields. The topographic distribution of receptors was determined by fluorescence microscopy of cells labeled with the intensely fluorescent, biologically active LDL derivative dioctadecylindolcarbocyanine LDL (dil(3)-LDL), or with native LDL and anti-LDL indirect immunofluorescence. Exposure of the LDL-receptor-internalization defective J. D. cells (GM2408A) to an electric field of 10 V/cm for 1 h at 22 degrees C causes greater than 80% of the cells to have an asymmetric distribution of LDL-R; receptors accumulate at the more negative pole of the cell. In contrast, only 20% of LDL-internalization normal GM3348 cells exposed to identical conditions have asymmetrical distributions. Phase micrographs taken during electric-field exposure rule out cell movement as the responsible mechanism for the effect. In both cell types, postfield labeling with the F-actin-specific fluorescent probe nitrobenzoxadiazole-phallacidin shows that no topographic alteration of the actin cytoskeleton accompanies the redistribution of cell surface LDL-Rs, and indirect immunofluorescence labeling of the coat protein clathrin shows that coated pits do not redistribute asymmetrically. Measurements of the postfield relaxation in the percentage of GM2408A cells showing an asymmetric distribution allow an estimate of the effective postfield diffusion coefficient of the unliganded LDL-R. At 37 degrees C, D = 2.0 X 10(-9) cm2/s, decreasing to 1.1 X 10(-9) cm2/s at 22 degrees C, and D = 3.5 X 10(-10) cm2/s at 10 degrees C. These values are substantially larger than those measured by photobleaching methods for the LDL-R complexed with dil(3)-LDL on intact cells, but are comparable to those measured on membrane blebs, and are consistent with diffusion coefficients measured for other unliganded integral membrane receptor proteins by postfield-relaxation methods.

Direct measurement of coupling between dendritic spines and shafts.

Characterization of the diffusional and electrotonic coupling of spines to the dendritic shaft is crucial to understanding neuronal integration and synaptic plasticity. Two-photon photobleaching and photorelease of fluorescein dextran were used to generate concentration gradients between spines and shafts in rat CA1 pyramidal neurons. Diffusional reequilibration was monitored with two-photon fluorescence imaging. The time course of reequilibration was exponential, with time constants in the range of 20 to 100 milliseconds, demonstrating chemical compartmentalization on such time scales. These values imply that electrical spine neck resistances are unlikely to exceed 150 megohms and more likely range from 4 to 50 megohms.

Computing with neural circuits: a model.

A new conceptual framework and a minimization principle together provide an understanding of computation in model neural circuits. The circuits consist of nonlinear graded-response model neurons organized into networks with effectively symmetric synaptic connections. The neurons represent an approximation to biological neurons in which a simplified set of important computational properties is retained. Complex circuits solving problems similar to those essential in biology can be analyzed and understood without the need to follow the circuit dynamics in detail. Implementation of the model with electronic devices will provide a class of electronic circuits of novel form and function.

In vivo Ca2+ dynamics in a cricket auditory neuron: an example of chemical computation.

Fura-2 calcium imaging in the cricket omega neuron revealed increased intracellular free calcium ion concentration in response to simulated cricket calling songs and other sound stimuli. The time course of the increase and decrease in intracellular calcium coincided with the time course of forward masking, a time-dependent modulation of auditory sensitivity. The buffering of calcium transients with high concentrations of a kinetically fast calcium buffer eliminated the post-stimulus hyperpolarization associated with forward masking, whereas the uncaging of calcium inside the neuron produced a hyperpolarization. The results suggest that sound-stimulated intracellular calcium accumulation acts by means of a calcium-activated hyperpolarizing current to produce forward masking. These findings underscore the importance of chemical dynamics in neural computation by demonstrating a behaviorally relevant role of calcium dynamics in vivo.

Spatially resolved calcium dynamics of mammalian Purkinje cells in cerebellar slice.

Microfluorometric imaging was used to study the correlation of intracellular calcium concentration with voltage-dependent electrical activity in guinea pig cerebellar Purkinje cells. The spatiotemporal dynamics of intracellular calcium concentration are demonstrated during spontaneous and evoked activity. The results are in agreement with hypotheses of dendritic segregation of calcium conductances suggested by electrophysiological experiments. These in vitro slice fluorescence imaging methods are applicable to a wide range of problems in central nervous system biochemical and electrophysiological functions.

The maintenance of LTP at hippocampal mossy fiber synapses is independent of sustained presynaptic calcium.

We have examined the role of presynaptic residual calcium in maintaining long-term changes in synaptic efficacy observed at mossy fiber synapses between hippocampal dentate granule cells and CA3 pyramidal cells. Calcium concentrations in individual mossy fiber terminals in hippocampal slice were optically measured with the calcium indicator fura-2 while stimulating the mossy fiber pathway and recording excitatory postsynaptic potentials extracellularly. Short-term synaptic enhancement was accompanied by increased presynaptic residual calcium concentration. A 2-fold enhancement of transmitter release was accompanied by a 10-30 nM increase in residual calcium. Following induction of mossy fiber LTP, transiently elevated presynaptic calcium decayed to prestimulus levels, whereas enhancement of synaptic transmission persisted. Our results demonstrate that, despite an apparent strong sensitivity of synaptic enhancement to presynaptic residual calcium levels, sustained increases in presynaptic residual calcium levels are not responsible for the maintained synaptic enhancement observed during mossy fiber LTP.

A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals.

Two-photon microscopy has enabled anatomical and functional fluorescence imaging in the intact brain of rats. Here, we extend two-photon imaging from anesthetized, head-stabilized to awake, freely moving animals by using a miniaturized head-mounted microscope. Excitation light is conducted to the microscope in a single-mode optical fiber, and images are scanned using vibrations of the fiber tip. Microscope performance was first characterized in the neocortex of anesthetized rats. We readily obtained images of vasculature filled with fluorescently labeled blood and of layer 2/3 pyramidal neurons filled with a calcium indicator. Capillary blood flow and dendritic calcium transients were measured with high time resolution using line scans. In awake, freely moving rats, stable imaging was possible except during sudden head movements.

Ca2+ accumulations in dendrites of neocortical pyramidal neurons: an apical band and evidence for two functional compartments.

Apical dendrites constitute a prominent feature of the microcircuitry in the neocortex, yet their function is poorly understood. Using fura-2 imaging of layer 5 pyramidal neurons from slices of rat somatosensory cortex, we have investigated the Ca2+ influx into dendrites under intracellular, antidromic, synaptic, and receptor-agonist stimulation. We find three spatial patterns of Ca2+ accumulations: an apical band in the apical dendrite approximately 500 microns from the soma, an accumulation restricted to the basal dendrites, soma, and proximal apical dendrite, and a combination of both of these. We show that the apical band can be activated antidromically and synaptically and that, under blocked Na+ and K+ conductances, it generates Ca2+ spikes. Thus, the apical band may serve as a dendritic trigger zone for regenerative Ca2+ spikes or as a current amplifier for distal synaptic events. Our results suggest that the distal apical dendrite should be considered a separate functional compartment from the rest of the cell.

Stability of the memory of eye position in a recurrent network of conductance-based model neurons.

Studies of the neural correlates of short-term memory in a wide variety of brain areas have found that transient inputs can cause persistent changes in rates of action potential firing, through a mechanism that remains unknown. In a premotor area that is responsible for holding the eyes still during fixation, persistent neural firing encodes the angular position of the eyes in a characteristic manner: below a threshold position the neuron is silent, and above it the firing rate is linearly related to position. Both the threshold and linear slope vary from neuron to neuron. We have reproduced this behavior in a biophysically plausible network model. Persistence depends on precise tuning of the strength of synaptic feedback, and a relatively long synaptic time constant improves the robustness to mistuning.

In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons.

Dendritic Ca2+ action potentials in neocortical pyramidal neurons have been characterized in brain slices, but their presence and role in the intact neocortex remain unclear. Here we used two-photon microscopy to demonstrate Ca2+ electrogenesis in apical dendrites of deep-layer pyramidal neurons of rat barrel cortex in vivo. During whisker stimulation, complex spikes recorded intracellularly from distal dendrites and sharp waves in the electrocorticogram were accompanied by large dendritic [Ca2+ ] transients; these also occurred during bursts of action potentials recorded from somata of identified layer 5 neurons. The amplitude of the [Ca 2+] transients was largest proximal to the main bifurcation, where sodium action potentials produced little Ca2+ influx. In some cases, synaptic stimulation evoked [Ca2+] transients without a concomitant action potential burst, suggesting variable coupling between dendrite and soma.

Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo.

In layer 2/3 pyramidal neurons of barrel cortex in vivo, calcium ion concentration ([Ca2+]) transients in apical dendrites evoked by sodium action potentials are limited to regions close to the soma. To study the mechanisms underlying this restricted pattern of calcium influx, we combined two-photon imaging of dendritic [Ca2+] dynamics with dendritic membrane potential measurements. We found that sodium action potentials attenuated and broadened rapidly with distance from the soma. However, dendrites of layer 2/3 cells were electrically excitable, and direct current injections could evoke large [Ca2+] transients. The restricted pattern of dendritic [Ca2+] transients is therefore due to a failure of sodium action-potential propagation into dendrites. Also, stimulating subcortical activating systems by tail pinch can enhance dendritic [Ca2+] influx induced by a sensory stimulus by increasing cellular excitability, consistent with the importance of these systems in plasticity and learning.

In vivo intracellular recording and perturbation of persistent activity in a neural integrator.

To investigate the mechanisms of persistent neural activity, we obtained in vivo intracellular recordings from neurons in an oculomotor neural integrator of the goldfish during spontaneous saccades and fixations. Persistent changes in firing rate following saccades were associated with step changes in interspike membrane potential that were correlated with changes in eye position. Perturbation of persistent activity with brief intracellular current pulses designed to mimic saccadic input only induced transient changes of firing rate and membrane potential. When neurons were hyperpolarized below action potential threshold, position-correlated step changes in membrane potential remained. Membrane potential fluctuations were greater during more depolarized steps. These results suggest that sustained changes in firing rate are supported not by either membrane multistability or changes in pacemaker currents, but rather by persistent changes in the rate or amplitude of synaptic inputs.

Dendritic calcium dynamics.

Further characterization of the biochemical components that contribute to calcium handling, together with advances in optical imaging of ion concentration, are providing quantitative information on the dynamics of calcium in the dendrites of neurons in tissue culture, brain slices and in vivo. It has recently been demonstrated that strong spatial gradients and transient calcium elevations result from the geometry and membrane properties of dendrites. These studies are adding to our understanding of calcium's role in synaptic plasticity and in shaping the electrophysiological properties of neurons.

Imaging calcium dynamics in dendritic spines.

Recent advances in optical imaging technology have enabled the measurement of Ca2+ dynamics in individual synaptic spines with high time resolution. Results from work using this new technology have confirmed the view that individual synaptic spines can act as functional chemical compartments with independent dynamics of second-messenger concentration. In particular, the ability of Ca2+ to directly mediate Hebbian coincidence detection has been confirmed.

Molecular mobility on the cell surface.

Many measurements of lateral diffusion of proteins and lipids on cell membranes and lipid model membranes have become available through application of fluorescence photobleaching recovery methods. A puzzling aspect of these results is slow diffusion and partial immobilization of protein molecules on the cell surface. Observed protein diffusion coefficients on vertebrate structural tissue cells are consistently D less than or equal to 10(-10) cm2/s, while lipid analogues diffuse with D approx. 10(-8) cm2/s. Substantial fractions of the cell membrane proteins are not diffusible. In a pure viscous membrane, theoretical fluid dynamics has suggested only small differences between lipid and protein diffusion coefficients. Measurements of protein diffusion in model membranes recently showed D less than or equal to 10(-9) cm2/s, as expected. Recent experiments on cell membranes show that uncoupling of the membrane from the cytoskeleton by formation of blebs releases the membrane protein molecules so that diffusion is enhanced to D greater than or equal to 10(-9) cm2/s and the non-diffusible fraction is eliminated.

Functional brain mapping using magnetic resonance imaging. Signal changes accompanying visual stimulation.

Easily detectable (5%-20%) transient increases in the intensity of water proton magnetic resonance (MR) signals in human primary visual cortex were observed during visual stimulation in gradient echo images at 4-T field strength. The signal intensity increases were predominantly restricted to areas containing gray matter and were used to produce high-spatial-resolution human functional brain maps. Time dependence of the functional brain maps also was monitored during visual stimulation using images acquired every approximately 5 seconds; these images with high spatial and temporal resolution demonstrated that photic stimulation first resulted in signal increases in a large area of the visual cortex followed by a reduction in the size of the area, and that signal intensity increases in the gray matter were time dependent. Reducing the image acquisition echo times reduced the amplitude of the fractional signal change, suggesting that it is produced by a change in T2 or T2*. The amplitude, sign, and echo time dependence of these intrinsic signal changes are consistent with the idea that neural activation increases regional cerebral with the idea that neural activation increases regional cerebral blood flow (rCBF) with a concomitant increase in venous blood oxygenation.

Presynaptic calcium in transmitter release and posttetanic potentiation.

This review gives some indication of the progress that has been made in understanding synaptic transmission by use of new methods for measuring and controlling presynaptic [Ca2+]i. Many unsolved problems remain. We still do not have a clear idea of the exact relationship between [Ca2+]i and transmitter release and whether this relationship is the same under all circumstances. The apparently different [Ca2+]i-dependence of evoked transmitter release and of PTP suggest multiple molecular sites of calcium action that remain to be identified. A complete and comprehensive model of transmitter release has yet to be devised, and questions raised by our experiments may indicate that it is still too early to try to construct a precise model. We also do not know just how serotonin acts to modulate transmitter release, only that it does not appear to alter either resting or entering calcium. Some of these questions may be approachable with the techniques described here; others are not and require different methods for their resolution. The work continues.

Human primary visual cortex and lateral geniculate nucleus activation during visual imagery.

The functional magnetic resonance (fMRI) technique can be robustly used to map functional activation of the visual pathway including the primary visual cortex (V1), the lateral geniculate nucleus (LGN), and other nuclei of humans during visual perception stimulation. One of the major controversies in visual neuroscience is whether lower-order visual areas involve the visual imagery process. This issue was examined using fMRI at high magnetic field. It was demonstrated for the first time that the LGN was activated during visual imagery process in the human brain together with V1 and other activation. There was a tight coupling of the activation between V1 and the LGN during visual imagery.