Article Figures & Data
Figures
- Fig. 1.
Barrels in layer 4 of somatosensory cortex identified by bright-field microscopy correspond to barrels defined by cytochrome C staining. A, Barrels can be visualized in brain slices of rat somatosensory neocortex using bright-field microscopy. A subset of the barrels are shown at higher magnification (A2), and the normalized brightness of pixels is quantified across the barrels in layer 4. The dark barrels are observed to be clearly separated by light septa. B, The same slice as shown above was subsequently stained for cytochrome C, which has been used to define barrels in previous work. A very similar pattern of dark regions separated by light septa within layer 4 of the neocortex is observed. The quantitative analysis of the normalized pixel brightness across the barrels (B2) indicates that there is an excellent correspondence between barrels defined by bright-field microscopy and barrels defined by cytochrome C staining.
- Fig. 2.
Whole-cell recordings from identified neurons in a specific region of a barrel. Barrels within layer 4 can by visualized at high magnification by enhancing contrast through video microscopy, allowing whole-cell recordings to be made from a given region of a barrel. The neuron in this example is located on the right-hand of the barrel as seen with 10× (A) and 40× (B) water immersion lenses. During the electrophysiological recording the cell is filled with biocytin, allowing the neuron to be visualized later in conjunction with a cytochrome C stain (C). These stains confirm the location of the neuron within the barrel and allow the axonal (green) and dendritic (black) arborizations to be reconstructed (D) in relationship to the barrel boundaries (cyan). Scale bar:A, C, D, 100 μm;B, 50 μm.
- Fig. 3.
DiI diffusion is confined to the injected barrel.A, The layer 4 barrels are still visible in these bright-field micrographs (A1,A2) of a slice that has been microinjected with DiI, fixed in paraformaldehyde, and incubated at 35°C for 3 weeks to allow DiI diffusion. The DiI injection was confined to a diameter of 50 μm in the center of the middle barrel outlined in cyan (A2). DiI fluorescence images of the same slice (A3, A4) indicate that although the DiI has spread vertically toward the pia, the diffusion of DiI in the horizontal direction is confined to remain within the barrel boundary. Scale bar, 100 μm. B, The width at half-maximal intensity of the DiI fluorescence is well correlated to the width of the injected barrel (black line shows best linear fit by least squares method).C, The fluorescence intensity versus distance in the horizontal (within layer 4) or vertical (up toward the pia) was fitted by a sigmoidal function and the transition distance from 80% maximal to 20% maximal intensity is shown. At the barrel/septal boundary there is an abrupt decrease in DiI fluorescence, whereas the vertical spread decays slowly.
- Fig. 4.
Axonal and dendritic processes are confined to the home barrel. A, An example of a reconstructed axon (green) and dendrite (black) of an excitatory spiny stellate neuron found at the right edge of a layer 4 barrel (outlined in cyan). Neuronal process are largely confined to the home barrel. Neurons with somata close to the edge of a barrel have axonal and dendritic trees oriented toward the center of the barrel. Scale bar, 100 μm.B, Quantification of the fraction of axonal (hatched bars) and dendritic (open bars) length within layer 4 found in the barrel in which the soma is also located (the home barrel), in the septa between barrels and in the adjacent barrels. Within layer 4 very little axonal and dendritic length is found outside of the home barrel. C, The polarization of axonal (open circles) and dendritic (filled squares) arbors within layer 4 is related to its location in the barrel. The y-axis is normalized so that a neuron that had its entire arbor to the left of the soma would be plotted at zero, whereas a neuron possessing only arborizations to its right would be plotted at unity. Thex-axis is normalized for each barrel such that the left border of the barrel is represented as the left extreme, and the right border of the barrel is also the right extreme of the graph. The graph shows quantitatively that neurons are polarized with respect to their position in the barrel such that their processes avoid entering neighboring barrels through polarization toward the center of the home barrel.
- Fig. 5.
fEPSPs are attenuated at the barrel border. A, Photograph of layer 4 barrels with glass stimulation pipette in the central barrel. The various positions of the recording electrode are indicated by the white dots. Scale bar, 100 μm. B, The curve is a plot of pixel brightness across the barrels in the photograph above and indicates quantitatively how the barrels are separated by lighter regions of septum. The black dots again represent the locations where fEPSPs were recorded. C, Evoked fEPSPs recorded at the different positions shown above with the left to right order preserved. Relatively small responses are recorded in positions outside of the barrel where the stimulation electrode is located.D, The average brightness of pixels across the region of layer 4 where the field recordings were made defines the barrel border and septum for the entire set of experiments. The border of the barrel is defined as the location where the brightness is half-maximal.E, The amplitudes of field responses recorded from many experiments at many positions were normalized to the value at the edge of the barrel and plotted against distance from the border of the barrel. The superimposed curve is the best fit (least squares method) of a summation of a linear and a sigmoidal function with all parameters held free. The fit suggests that fEPSPs are attenuated at the barrel boundary.
- Fig. 6.
Synaptically connected neurons are located in the same barrel. A, An example of a synaptically unconnected pair of excitatory layer 4 neurons, with somata lying in different barrels. The reconstructed axonal (green) and dendritic (black) processes are shown in relationship to the barrel borders (cyan) (A1). The regular firing pattern (A2) and morphology define these cells as excitatory spiny stellate neurons. Action potentials evoked in the left- or the right-hand cell do not evoke responses in the other cell, showing that they are not synaptically connected.B, An example of a connected pair of excitatory layer 4 spiny stellate neurons with soma in the same barrel (B1) with regular firing patterns (B2). The neurons are bidirectionally coupled, such that an action potential in either neuron evokes an EPSP in the other (B3). Scale bars:A1,B1, 100 μm. Calibration:A2,B2, 50 mV, 100 msec; A3, B3, 50 mV (top trace) or 1 mV (bottom trace), 10 msec.
- Fig. 7.
Neurons are highly connected within a barrel but not with adjacent barrels. A, Photograph of a slice showing barrels divided by a septum. The postsynaptic whole-cell recording pipette is visible, located close to the edge of the left-hand barrel. Square symbols indicate the locations of cells tested using the loose-patch technique that were found to be synaptically connected to the postsynaptic neuron. Open circles indicate the locations of cells that were not connected to the postsynaptic cell. The seven cells that evoked responses in the postsynaptic cell were located either in the same barrel as the postsynaptic cell or in the near half of the septum. Scale bar, 100 μm. B, The responses recorded in the postsynaptic cell in response to loose-patch evoked action potentials of the cells shown above. Traces in bold show the EPSPs evoked by action potentials of the connected neurons, whereas thethin traces show the absence of responses evoked by stimulation of neurons without connections to the postsynaptic cell. An example of an action potential evoked by loose-patch stimulation of a presynaptic cell is shown below. Calibration: 0.2 mV for postsynaptic potentials, 10 mV for presynaptic potentials, 5 msec for horizontal scale bar. C, Summary of locations of cells that were connected (squares) or not (circles) to the postsynaptic cells in the entire set of these experiments. Most of the connected neurons are found to lie inside the same barrel as the location of the postsynaptic cell, which on average was located 25 μm left of the barrel border. D, The fraction of connected cells is strongly dependent on location. Approximately one-third of the cells tested within 300 μm of the postsynaptic cell within the same barrel are connected to a given postsynaptic target cell. Cells from the adjacent barrel make very few synaptic connections. The averaged observed brightness curve defining the home barrel, septum and adjacent barrel in these experiments is superimposed on the connectivity data points. E, The frequency of connection strengths observed for neurons in the same barrel. A wide distribution of EPSP amplitudes is observed with many small amplitude connections and a few large connections. Along with the spatial constraint imposed by the confines of the barrel, this distribution of connection strengths defines the excitatory neuronal network of layer 4. F, A schematic diagram illustrating the connectivity observed in the experiments. Each barrel is shown to have neurons that are synaptically connected to each other within the barrel but not to adjacent barrels.
