Certain ganglion cells in the retina respond sensitively to differential motion between the receptive field center and surround, as produced by an object moving over the background, but are strongly suppressed by global image motion, as produced by the observer's head or eye movements. We investigated the circuit basis for this object motion sensitive (OMS) response by recording intracellularly from all classes of retinal interneurons while simultaneously recording the spiking output of many ganglion cells. Fast, transient bipolar cells respond linearly to motion in the receptive field center. The synaptic output from their terminals is rectified and then pooled by the OMS ganglion cell. A type of polyaxonal amacrine cell is driven by motion in the surround, again via pooling of rectified inputs, but from a different set of bipolar cell terminals. By direct intracellular current injection, we found that these polyaxonal amacrine cells selectively suppress the synaptic input of OMS ganglion cells. A quantitative model of these circuit elements and their interactions explains how an important visual computation is accomplished by retinal neurons and synapses.
Due to fixational eye movements, the image on the retina is always in motion, even when one views a stationary scene. When an object moves within the scene, the corresponding patch of retina experiences a different motion trajectory than the surrounding region. Certain retinal ganglion cells respond selectively to this condition, when the motion in the cell's receptive field center is different from that in the surround. Here we show that this response is strongest at the very onset of differential motion, followed by gradual adaptation with a time course of several seconds. Different subregions of a ganglion cell's receptive field can adapt independently. The circuitry responsible for differential motion adaptation lies in the inner retina. Several candidate mechanisms were tested, and the adaptation most likely results from synaptic depression at the synapse from bipolar to ganglion cell. Similar circuit mechanisms may act more generally to emphasize novel features of a visual stimulus.
Songbirds learn their songs by trial-and-error experimentation, producing highly variable vocal output as juveniles. By comparing their own sounds to the song of a tutor, young songbirds gradually converge to a stable song that can be a remarkably good copy of the tutor song. Here we show that vocal variability in the learning songbird is induced by a basal-ganglia-related circuit, the output of which projects to the motor pathway via the lateral magnocellular nucleus of the nidopallium (LMAN). We found that pharmacological inactivation of LMAN dramatically reduced acoustic and sequence variability in the songs of juvenile zebra finches, doing so in a rapid and reversible manner. In addition, recordings from LMAN neurons projecting to the motor pathway revealed highly variable spiking activity across song renditions, showing that LMAN may act as a source of variability. Lastly, pharmacological blockade of synaptic inputs from LMAN to its target premotor area also reduced song variability. Our results establish that, in the juvenile songbird, the exploratory motor behavior required to learn a complex motor sequence is dependent on a dedicated neural circuit homologous to cortico-basal ganglia circuits in mammals.
Among eutherian mammals, only primates possess trichromatic color vision. In Old World primates, trichromacy was made possible by a visual pigment gene duplication. In most New World primates, trichromacy is based on polymorphic variation in a single X-linked gene that produces, by random X inactivation, a patchy mosaic of spectrally distinct cone photoreceptors in heterozygous females. In the present work, we have modeled the latter strategy in a nonprimate by replacing the X-linked mouse green pigment gene with one encoding the human red pigment. In the mouse retina, the human red pigment seems to function normally, and heterozygous female mice express the human red and mouse green pigments at levels that vary between animals. Multielectrode array recordings from heterozygous female retinas reveal significant variation in the chromatic sensitivities of retinal ganglion cells. The data are consistent with a model in which these retinal ganglion cells draw their inputs indiscriminately from a coarse-grained mosaic of red and green cones. These observations support the ideas that (i) chromatic signals could arise from stochastic variation in inputs drawn nonselectively from red and green cones and (ii) tissue mosaicism due to X chromosome inactivation could be one mechanism for driving the evolution of CNS diversity.
An important task in vision is to detect objects moving within a stationary scene. During normal viewing this is complicated by the presence of eye movements that continually scan the image across the retina, even during fixation. To detect moving objects, the brain must distinguish local motion within the scene from the global retinal image drift due to fixational eye movements. We have found that this process begins in the retina: a subset of retinal ganglion cells responds to motion in the receptive field centre, but only if the wider surround moves with a different trajectory. This selectivity for differential motion is independent of direction, and can be explained by a model of retinal circuitry that invokes pooling over nonlinear interneurons. The suppression by global image motion is probably mediated by polyaxonal, wide-field amacrine cells with transient responses. We show how a population of ganglion cells selective for differential motion can rapidly flag moving objects, and even segregate multiple moving objects.
Transport of water between the capillary and airspace compartments in lung encounters serial barriers: the alveolar epithelium, interstitium, and capillary endothelium. We previously reported a pleural surface fluorescence method to measure net capillary-to-airspace water transport. To measure the osmotic water permeability across the microvascular endothelial barrier in intact lung, the airspace was filled with a water-immiscible fluorocarbon. The capillaries were perfused via the pulmonary artery with solutions of specified osmolalites containing a high-molecular-weight fluorescent dextran. An increase in perfusate osmolality produced a prompt decrease in surface fluorescence due to dye dilution in the capillaries, followed by a slower return to initial fluorescence as capillary and lung interstitial osmolality equilibrate. A mathematical model was developed to determine the osmotic water permeability coefficient (Pf) of lung microvessels from the time course of pleural surface fluorescence. As predicted, the magnitude of the prompt change in surface fluorescence increased with decreased pulmonary artery perfusion rate and increased osmotic gradient size. With raffinose used to induce the osmotic gradient, Pf was 0.03 cm/s at 23 degrees C and was reduced 54% by 0.5 mM HgCl2. Temperature dependence measurements gave an Arrhenius activation energy (Ea) of 5.4 kcal/mol (12-37 degrees C). The apparent Pf induced by the smaller osmolytes mannitol and glycine was 0.021 and 0.011 cm/s (23 degrees C). Immunoblot analysis showed approximately 1.4 x 10(12) aquaporin-1 water channels/cm2 of capillary surface, which accounted quantitatively for the high Pf. These results establish a novel method for measuring osmotically driven water permeability across microvessels in intact lung. The high Pf, low Ea, and mercurial inhibition indicate the involvement of molecular water channels in water transport across the lung endothelium.
Molecular transport in the aqueous lumen of organelles involves diffusion in a confined compartment with complex geometry. Monte Carlo simulations of particle diffusion in three dimensions were carried out to evaluate the influence of organelle structure on diffusive transport and to relate experimental photobleaching data to intrinsic diffusion coefficients. Two organelle structures were modeled: a mitochondria-like long closed cylinder containing fixed luminal obstructions of variable number and size, and an endoplasmic reticulum-like network of interconnected cylinders of variable diameter and density. Trajectories were computed in each simulation for >10(5) particles, generally for >10(5) time steps. Computed time-dependent concentration profiles agreed quantitatively with analytical solutions of the diffusion equation for simple geometries. For mitochondria-like cylinders, significant slowing of diffusion required large or wide single obstacles, or multiple obstacles. In simulated spot photobleaching experiments, a approximately 25% decrease in apparent diffusive transport rate (defined by the time to 75% fluorescence recovery) was found for a single thin transverse obstacle occluding 93% of lumen area, a single 53%-occluding obstacle of width 16 lattice points (8% of cylinder length), 10 equally spaced 53% obstacles alternately occluding opposite halves of the cylinder lumen, or particle binding to walls (with mean residence time = 10 time steps). Recovery curve shape with obstacles showed long tails indicating anomalous diffusion. Simulations also demonstrated the utility of measurement of fluorescence depletion at a spot distant from the bleach zone. For a reticulum-like network, particle diffusive transport was mildly reduced from that in unobstructed three-dimensional space. In simulated photobleaching experiments, apparent diffusive transport was decreased by 39-60% in reticular structures in which 90-97% of space was occluded. These computations provide an approach to analyzing photobleaching data in terms of microscopic diffusive properties and support the paradigm that organellar barriers must be quite severe to seriously impede solute diffusion.
It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100x objective (0.8-micron spot diam) gave half-times for fluorescence recovery of 15-19 ms with >90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2-3 x 10(-7) cm2/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid beta-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 +/- 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, approximately 0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse.
The decay of evanescent field intensity beyond a dielectric interface depends upon beam incident angle, enabling the 3-d distribution of fluorophores to be deduced from total internal reflection fluorescence microscopy (TIRFM) images obtained at multiple incident angles. Instrumentation was constructed for computer-automated multiple angle-TIRFM (MA-TIRFM) using a right angle F2 glass prism (n(r) 1.632) to create the dielectric interface. A laser beam (488 nm) was attenuated by an acoustooptic modulator and directed onto a specified spot on the prism surface. Beam incident angle was set using three microstepper motors controlling two rotatable mirrors and a rotatable optical flat. TIRFM images were acquired by a cooled CCD camera in approximately 0.5 degree steps for >15 incident angles starting from the critical angle. For cell studies, cells were grown directly on the glass prisms (without refractive index-matching fluid) and positioned in the optical path. Images of the samples were acquired at multiple angles, and corrected for angle-dependent evanescent field intensity using "reference" images acquired with a fluorophore solution replacing the sample. A theory was developed to compute fluorophore z-distribution by inverse Laplace transform of angle-resolved intensity functions. The theory included analysis of multiple layers of different refractive index for cell studies, and the anisotropic emission from fluorophores near a dielectric interface. Instrument performance was validated by mapping the thickness of a film of dihexyloxacarbocyanine in DMSO/water (n(r) 1.463) between the F2 glass prism and a plano-convex silica lens (458 mm radius, n(r) 1.463); the MA-TIRFM map accurately reproduced the lens spherical surface. MA-TIRFM was used to compare with nanometer z-resolution the geometry of cell-substrate contact for BCECF-labeled 3T3 fibroblasts versus MDCK epithelial cells. These studies establish MA-TIRFM for measurement of submicroscopic distances between fluorescent probes and cell membranes.
