Experimental design. (A) Montage from photomicrographs taken during an experiment of a somatosensory thalamocortical slice, combined to show the whole slice. The locations of the 2 recording sites and photostimulation are marked by red and yellow stars and a red rectangle, respectively. The division between the ventral posterior medial (VPM) and posterior (PO) nuclei are indicated by white lines. (B) Pattern of stimulation. The stimulation pattern consists of 256 positions arranged in a 16 × 16 square matrix, with 75 μm between adjacent rows and columns. Stimulation of these positions was arranged in a sequence that maximized the distance between consecutive trials. Locations of the first 5 trials are shown.

Spatial specificity of layer 6 photostimulation. Responses of layer 6 neurons to photostimulation at 256 locations, arranged in a 16 × 16 array, are superimposed on a photomicrograph of the cortex. The 200-ms recording immediately after the photostimulation is shown where the slice was stimulated. Selected traces in areas of interest (red, blue, and green rectangles) are shown in a larger scale. (A) Responses of a layer 6 neuron to photostimulation in current clamp recording. Action potentials were evoked only in an area less than 150 μm around the soma. (B) Voltage clamp recordings from another experiment. Photostimulation of layer 6 and layer 5a evoked a mixture of inward and outward currents at −20 mV (B, left) holding potential in this neuron. Direct depolarization from photostimulation was evoked only within a small region within 100 μm of the soma after synaptic responses were abolished using 0.2 mM Ca²⁺/6 mM Mg²⁺ ACSF (B, right). Laser power was 50 mW in (A) and 30 mW in (B).

Photostimulation of the primary somatosensory cortex evokes biphasic EPSC/IPSC responses. (A) Response of a neuron of ventral posterior medial nucleus to cortical photostimulation. The inset displays the photomontage taken during the experiment. The red star and rectangle indicate the site of recording and photostimulation, respectively. Shown are voltage clamp responses of a neuron to photostimulation at 256 locations, arranged in a 16 × 16 array, superimposed on a photomicrograph of the cortex. The 200-ms recording immediately after the photostimulation is shown where the slice was stimulated. Photostimulation at a region in upper layer 6 evoked biphasic EPSC/IPSC responses in this neuron (green square). The response traces in this region are magnified and shown on the right. Three selected traces (numbered 1–3 in [A] and [B]) are shown below in a larger magnification. The 2 red vertical lines indicate timing of the laser pulse. (B) Effects of GABA antagonist. The outward currents were abolished by bath application of a GABA_A antagonist (SR95531). White lines in both figures indicate the approximate separation of cortical layers. The laser power used in both experiments was 21 mW, and the holding potential during recording was −20 mV.

Effects of laser power on the cortical input footprints of the neurons of the ventral posterior medial nucleus. (A, panels 1–7) Pseudocolor plots of the excitatory and inhibitory input footprint obtained at 2 laser powers for 7 experiments. These experiments were performed at holding potentials between −20 and −45 mV so that both EPSCs and IPSCs could be easily detected. The peak inward (EPSCs) and outward (IPSCs) currents evoked at each stimulation position are represented by the intensity of green and red, respectively (see the color scale in lower right). The 2 pseudocolor maps are superimposed, so that the areas where biphasic responses were evoked appear yellowish. The centroids of the layer 6 excitatory and inhibitory responses were calculated as described (see Materials and Methods) and indicated with light-blue (EPSCs) and white triangles (IPSCs); the area where the responses were included in this calculation is encircled with a dotted rectangle in panel 5. Numerical labels in panel 5 indicate approximate location of the cortical layers 4–6. The laser power used for photostimulation is labeled in upper right of the pseudocolor maps. (B) Response traces to photostimulation at selected positions (white squares in panels 1–5). Green vertical lines indicate the timing of the laser pulse. Vertical scale bars and horizontal scale bars equal 50 pA and 50 ms, respectively, for all traces.

Laminar organization of the cortical input footprints of the neurons of the ventral posterior medial nucleus; conventions as in Figure 3. (A) Example where photostimulation of upper layer 6 only evoked biphasic EPSC/IPSC responses (blue square). Photostimulation of layer 5a (red rectangle) and other layers elicited no responses in this neuron. The laser power used in this experiment was 32 mW. (B) Example where photostimulation of both layer 5a (red rectangle) and layer 6 (blue rectangle) elicited responses. The laser power was 30 mW. (C) Example where responses could be evoked from a large area of the cortex. The cell responded to photostimulation in layer 4 (red and purple rectangles), layer 5a (red rectangle), and layer 6 (blue rectangle). The laser power was 25 mW. The holding potential for all 3 experiments was −20 mV.

Relative location of excitatory and inhibitory cortical input footprints; conventions as in Figure 3. (Ai, Bi) Photostimulation of both layer 5a (red rectangles) and layer 6 (blue rectangles) evoked biphasic EPSC/IPSC responses in both neurons. (Aii, Bii) Pseudocolor plots of the peak inward (excitatory) currents. (Aiii, Biii) Pseudocolor plots of the peak outward (inhibitory) currents. The regions where the EPSCs and IPSCs could be elicited in the neuron of (A) were mostly overlapped (laser power was 18 mW and the holding potential was −35 mV). The regions where the EPSCs and IPSCs could be elicited in the neuron of (B) laterally offset (laser power was 32 mW and the holding potential during recording was −45 mV).

Spatial organization of the cortical input footprint. (A, panels 1–29) Pseudocolor plots of the excitatory and inhibitory input footprints for 29 experiments performed at holding potentials between −20 and −45 mV. The peak inward (EPSCs) and outward (IPSCs) currents evoked at each stimulation position are represented in the same conventions as described in Figure 4 and Materials and Methods. The centroids of the layer 6 excitatory and inhibitory responses were calculated as described (see Materials and Methods) and indicated with light-blue (EPSCs) and white triangles (IPSCs); the area where the responses were included is encircled with a dotted rectangle in panel 9. The numerical labels in (A), panel 9 indicate approximate location of the cortical layers 4–6. (A) Panel 1 and panel 27 are the examples shown in Figure 5A,B. For reference, the locations of 2 barrels are indicated in panel 27 with white dotted rectangles. (B) Laminar organization of the cortical input footprints. The histograms represent the average maximum peak inward (left) and outward (right) currents for each row in panels 1–29. Row numbers and the approximate locations of the cortical layers are indicated on the y-axis. (C) Vertical and lateral offsets of the inhibitory input footprints from the excitatory footprints. The lateral offset was larger than 200 μm (the approximate width of a barrel) in 3 experiments. They are indicated with red stars in the graph and in the pseudocolor plots (A, panels 27–29).

Cholinergic modulation of the responses to cortical photostimulation; conventions as in Figure 3. (A) Control. Photostimulation of layer 6 evoked the typical biphasic EPSC/IPSC responses in this neuron of the ventral posterior medial nucleus. (B) Effects of acetyl-β-methylcholine (β-MCh). Bath application of 250 μM of acetyl-β-methylcholine, a muscarinic receptor agonist, abolished the IPSCs without affecting the amplitude of the EPSCs. (C) Blockade of the effects of acetyl-β-methylcholine by atropine. The same slice was washed with normal ACSF for 20 min. After the IPSCs recovered, bath application of acetyl-β-methylcholine together with atropine, a muscarinic antagonist, did not affect the IPSC responses. Laser power used in all 3 maps was 35 mW and the holding potential was −20 mV.

Response of a thalamic reticular neuron to acetyl-β-methylcholine. The thalamic reticular neuron was recorded in current clamp mode in 0.2 mM Ca²⁺/6 mM Mg²⁺ ACSF (to eliminate any possible presynaptic influences of the agonist). Bath application of 250 μM of acetyl-β-methylcholine (gray horizontal line) hyperpolarized the neuron.

Two patterns among others possible for corticothalamic projection from layer 6 to cells of the thalamic reticular nucleus (TRN) and relay cells of the ventral posterior medial nucleus (VPM). (A) Pattern of simple excitation and feed-forward inhibition. (B) More complicated pattern in which activation of a cortical axon can excite some relay cells directly and inhibit others through activation of reticular cells. Further details in text.