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In JoVE (1)

Other Publications (7)

Articles by Prakash Kara in JoVE

 JoVE Neuroscience

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

1Department of Neuroscience, Medical University of South Carolina


JoVE 50025

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

Other articles by Prakash Kara on PubMed

The Spatial Receptive Field of Thalamic Inputs to Single Cortical Simple Cells Revealed by the Interaction of Visual and Electrical Stimulation

Electrical stimulation of the thalamus has been widely used to test for the existence of monosynaptic input to cortical neurons, typically with stimulation currents that evoke cortical spikes with high probability. We stimulated the lateral geniculate nucleus (LGN) of the thalamus and recorded monosynaptically evoked spikes from layer 4 neurons in visual cortex. We found that with moderate currents, cortical spikes were evoked with low to moderate probability and their occurrence was modulated by ongoing sensory (visual) input. Furthermore, when repeated at 8-12 Hz, electrical stimulation of the thalamic afferents caused such profound inhibition that cortical spiking activity was suppressed, aside from electrically evoked monosynaptic spikes. Visual input to layer 4 cortical cells between electrical stimuli must therefore have derived exclusively from LGN afferents. We used white-noise visual stimuli to make a 2D map of the receptive field of each cortical simple cell during repetitive electrical stimulation in the LGN. The receptive field of electrically evoked monosynaptic spikes (and thus of the thalamic input alone) was significantly elongated. Its primary subfield was comparable to that of the control receptive field, but secondary (flanking) subfields were weaker. These findings extend previous results from intracellular recordings, but also demonstrate the effectiveness of an extracellular method of measuring subthreshold afferent input to cortex.

Role of Subplate Neurons in Functional Maturation of Visual Cortical Columns

The subplate forms a transient circuit required for development of connections between the thalamus and the cerebral cortex. When subplate neurons are ablated, ocular dominance columns do not form in the visual cortex despite the robust presence of thalamic axons in layer 4. We show that subplate ablation also prevents formation of orientation columns. Visual responses are weak and poorly tuned to orientation. Furthermore, thalamocortical synaptic transmission fails to strengthen, whereas intracortical synapses are unaffected. Thus, subplate circuits are essential not only for the anatomical segregation of thalamic inputs but also for key steps in synaptic remodeling and maturation needed to establish the functional architecture of visual cortex.

Efficacy of Retinal Spikes in Driving Cortical Responses

How does a single retinal ganglion cell (RGC) affect the firing of simple cells in the visual cortex? Although much is known of the functional connections between the retina and the lateral geniculate nucleus (LGN) and between LGN and visual cortex, it is hard to infer the effect of disynaptic connections from retina to visual cortex. Most importantly, there is considerable divergence from retina to LGN, so cortical neurons might be influenced by ganglion cells through multiple feedforward pathways. We recorded simultaneously from ganglion cells in the retina and cortical simple cells in the striate cortex with overlapping receptive fields and evaluated disynaptic connections with cross-correlation analysis. In all disynaptically connected pairs, the retinal receptive field center and overlapping cortical subregion always shared the same sign (either both ON or both OFF). Connected pairs were similar in other respects, such as relative position and timing of their receptive fields, and thus obeyed the same rules of connectivity found previously for retinothalamic and thalamocortical connections. We found that a single RGC directly contributed on average to approximately 3% of the activity of its cortical target. The relative timing of pairs of spikes from the retinal cell affected their efficacy in driving the cortical cell. When two retinal spikes were closely spaced (<10 msec), the second spike was several times more likely to drive the cortical target. The relative magnitude of this disynaptic paired spike enhancement was considerably larger than has been found previously for retinogeniculate and geniculocortical connections. The amplified paired spike enhancement from retina to cortex ensures that signal transmission from retina to cortex is particularly effective when the retina fires a series of closely spaced action potentials.

Functional Imaging with Cellular Resolution Reveals Precise Micro-architecture in Visual Cortex

Neurons in the cerebral cortex are organized into anatomical columns, with ensembles of cells arranged from the surface to the white matter. Within a column, neurons often share functional properties, such as selectivity for stimulus orientation; columns with distinct properties, such as different preferred orientations, tile the cortical surface in orderly patterns. This functional architecture was discovered with the relatively sparse sampling of microelectrode recordings. Optical imaging of membrane voltage or metabolic activity elucidated the overall geometry of functional maps, but is averaged over many cells (resolution >100 microm). Consequently, the purity of functional domains and the precision of the borders between them could not be resolved. Here, we labelled thousands of neurons of the visual cortex with a calcium-sensitive indicator in vivo. We then imaged the activity of neuronal populations at single-cell resolution with two-photon microscopy up to a depth of 400 microm. In rat primary visual cortex, neurons had robust orientation selectivity but there was no discernible local structure; neighbouring neurons often responded to different orientations. In area 18 of cat visual cortex, functional maps were organized at a fine scale. Neurons with opposite preferences for stimulus direction were segregated with extraordinary spatial precision in three dimensions, with columnar borders one to two cells wide. These results indicate that cortical maps can be built with single-cell precision.

Highly Ordered Arrangement of Single Neurons in Orientation Pinwheels

In the visual cortex of higher mammals, neurons are arranged across the cortical surface in an orderly map of preferred stimulus orientations. This map contains 'orientation pinwheels', structures that are arranged like the spokes of a wheel such that orientation changes continuously around a centre. Conventional optical imaging first demonstrated these pinwheels, but the technique lacked the spatial resolution to determine the response properties and arrangement of cells near pinwheel centres. Electrophysiological recordings later demonstrated sharply selective neurons near pinwheel centres, but it remained unclear whether they were arranged randomly or in an orderly fashion. Here we use two-photon calcium imaging in vivo to determine the microstructure of pinwheel centres in cat visual cortex with single-cell resolution. We find that pinwheel centres are highly ordered: neurons selective to different orientations are clearly segregated even in the very centre. Thus, pinwheel centres truly represent singularities in the cortical map. This highly ordered arrangement at the level of single cells suggests great precision in the development of cortical circuits underlying orientation selectivity.

A Micro-architecture for Binocular Disparity and Ocular Dominance in Visual Cortex

In invertebrate predators such as the praying mantis and vertebrate predators such as wild cats the ability to detect small differences in inter-ocular retinal disparities is a critical means for accurately determining the depth of moving objects such as prey. In mammals, the first neurons along the visual pathway that encode binocular disparities are found in the visual cortex. However, a precise functional architecture for binocular disparity has never been demonstrated in any species, and coarse maps for disparity have been found in only one primate species. Moreover, the dominant approach for assaying the developmental plasticity of binocular cortical neurons used monocular tests of ocular dominance to infer binocular function. The few studies that examined the relationship between ocular dominance and binocular disparity of individual cells used single-unit recordings and have provided conflicting results regarding whether ocular dominance can predict the selectivity or sensitivity to binocular disparity. We used two-photon calcium imaging to sample the response to monocular and binocular visual stimuli from nearly every adjacent neuron in a small region of the cat visual cortex, area 18. Here we show that local circuits for ocular dominance always have smooth and graded transitions from one apparently monocular functional domain to an adjacent binocular region. Most unexpectedly, we discovered a new map in the cat visual cortex that had a precise functional micro-architecture for binocular disparity selectivity. At the level of single cells, ocular dominance was unrelated to binocular disparity selectivity or sensitivity. When the local maps for ocular dominance and binocular disparity both had measurable gradients at a given cortical site, the two gradient directions were orthogonal to each other. Together, these results indicate that, from the perspective of the spiking activity of individual neurons, ocular dominance cannot predict binocular disparity tuning. However, the precise local arrangement of ocular dominance and binocular disparity maps provide new clues regarding how monocular and binocular depth cues may be combined and decoded.

An Artery-specific Fluorescent Dye for Studying Neurovascular Coupling

We demonstrate that Alexa Fluor 633 hydrazide (Alexa Fluor 633) selectively labels neocortical arteries and arterioles by binding to elastin fibers. We measured sensory stimulus-evoked arteriole dilation dynamics in mouse, rat and cat visual cortex using Alexa Fluor 633 together with neuronal activity using calcium indicators or blood flow using fluorescein dextran. Arteriole dilation decreased fluorescence recorded from immediately underlying neurons, representing a potential artifact during neuronal functional imaging experiments.

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