A: Thalamocortical slice under DIC at P10. MGN and ACX are indicated. Scale bar=500μm. B: Higher power picture during a recording from a SPN at P9. Note the clear differentiation of the subplate zone (SP) and cortical plate (CP) under DIC. The subplate appears horizontally organized while layer 6 appears darker. Scale bar 200μm. V indicates ventricle C: P5 thalamocortical slice after recording from multiple cells and subsequent biocytin staining. Stimulation site in MGN is indicated (“stim”). Note the stained cells in the subplate in ACX (arrowhead). High power pictures show three of the stained subplate neurons, illustrating diverse morphology. Scale bars=500μm (slice) and 50μm (cells). D: Subplate neurons and layer 6 neurons show different morphologies. Shown are biocytin stained neurons in a 600μm slice counterstained with cresyl violet. The subplate zone (SP) is readily distinguished from the cortical plate in cresyl stains as being located below the transition between radial oriented cells and horizontally oriented cell bodies (future layer 6B). D1 and D2 show subplate neuron. The cell body is located in the subplate and axons project onto the cortical plate while dendrites are in subplate and layer 6. VZ indicates the densely stained ventricular zone. Scale bars: 100μm and 50um. D3 and D4 show pyramidal cells in layers 5/6. Note the prominent apical dendrites (arrows) that reach into superficial cortical layers. Scale bars: 100μm and 50μm. Due to the thickness of the section drying is uneven thus shrinkage of layer 2/3 is more pronounced. E: Current injections cause action potentials at all ages studied. Shown are traces evoked with various current injections at 3 ages. Cells were held at -75mV. F: Average numbers of action potentials during 250 ms current injections. Note that the numbers of action potentials increase between P1/4 and P5/9. G-H: Input resistance (R_m) and resting potential (V_rest) determined from linear fits to I-V curve below action potential threshold. Both R_m and V_rest decrease during the postnatal period. ** indicates P < 0.05.

A: MGN evoked EPSCs with increasing stimulation currents at 3 ages. Traces are shown on the left. Peak EPSC amplitudes as functions of stimulus strength (IO-curve) are shown on right. For comparison, stimulus strength was normalized to the maximum stimulus strength used in a particular cell. Note the limited dynamic range of EPSC amplitudes at young ages. Dashed lines indicate where saturation was reached. Note the occasional disynaptic responses (red traces). B: Graph shows a superposition of the IO curves in A normalized to maximum EPSC and saturation stimulus level. Note the steeper increase in the IO curve from threshold to saturation at younger ages. C: Quantification of EPSC amplitudes. Box plots show EPSC amplitude at threshold (minimal reliably evoked EPSC), maximal evoked EPSC, and the ratio of these values. Box plots show median (line), mean (square) and the 25% and 75% quartiles. The threshold EPSC amplitude does not change (P1/4: 14.6±7.2 pA N=9; P5/9: 19.9±8 pA N=12; P10/13: 17.45±11.8 pA N=8), while maximal EPSC amplitude (P1/4: 42.4±18.1 pA; P5/9: 121±105 pA; P10/13: 184±149 pA) and the ratio (P1/4: 3.1±1.2; P5/9: 4.3±5.6; P10/13: 10.1±6.5) increase from P2/4 to P5/9 and P10/13 (‘*’ indicate P<0.05). D: Latencies of the monosynaptic EPSC (time to peak). Box plots show median (line), mean (square) and the 25% and 75% quartiles. Peak latencies were similar at all ages (P1/4: 11.6 ± 3.2 ms, P5/9: 13.66 ± 5.76 ms, P10/13: 14.74 ± 4.32ms, all P > 0.1).

A: Picture of a slice (600μm thick) from a P5 animal in which both layer 6 and SPns were recorded. Boxes show position of high magnification pictures. Dashed line indicates subplate/layer 6 border. High magnification pictures show differences in morphology between SPNs and layer 6 neuron. Scale bars: 100μm, 50μm. VZ indicates ventricular zone and H hippocampus. B: Traces show response of layer 6 neuron and SPN in A to thalamocortical stimulation at same stimulation site (equal intensity). Note that the SPN but not the layer 6 neuron showed synaptic inputs. C: Picture of a thalamocortical slice (600μm thick) from a P13 animal in which both layer 6 and SPns were recorded. Boxes show position of high magnification pictures. Dashed line indicates subplate/layer 6 border. V indicates ventricle and H hippocampus. High magnification pictures show differences in morphology between SPNs and layer 6 neuron. Arrows show apical dendrite of layer 6 neurons traveling to superficial layers. Scale bars: 1mm, 50μm. D: Traces from SPN and layer 6 neuron in C to electrical stimulation of MGN (multiple traces superimposed). Note the reliable short latency EPSC in the SPN while the layer 6 neuron showed long latency EPSCs on some trials that started when SPNs ceased to receive inputs.

A: SPN at P7. MGN stimulation at increasing intensities results in EPSCs of increasing amplitude. Selected traces (left) and peak EPSC amplitudes and each stimulus level (normalized to the saturating level) (right). B: MGN stimulation at increasing intensities results in EPSPs of increasing amplitude (same cell as in A). Selected traces (left) and peak EPSP amplitudes and each stimulus level (normalized to the saturating level) (right). Note the large maximal depolarization. Cell was held at negative potentials to prevent spiking. C: Another cell. MGN stimulation is able to evoke action potentials. D: MGN stimulation causes both AMPA and NMDA currents in SPNs. Shown is a SPN at P4. The electrode solution contained QX314 to block Na-currents. Currents were separated by holding SPNs at -60mV or +40mV. Trace below shows that no evoked current reversed at 0 mV, suggesting that the synapse is glutamatergic. Note that no outward currents were observed, thus MGN stimulation did not recruit GABAergic neurons.

A: Left panel: Schematic of experimental setup. Recordings are obtained from layer 4 neurons while SPNs are excited by photostimulation. Right panel: Actual setup of photostimulation experiment. Image shows part of the thalamocortical slice at P4. Layer 1 is to the right (1) and white matter (WM) is to the left. Recording of layer 4 neuron through whole-cell pipette (P) while photostimulation fiber (F) and light spot (white) are located in subplate. Note that the elliptical uncaging spot does not extend into overlying cortical plate. The shape of the spot depends on the size of the fiber, its angle and distance to the slice. Imaging artifacts surrounding the fiber are due to DIC illumination. Scale bar 100um. B-D: Recordings from SPNs. B: Photostimulation of SPN at P11. Following a laser pulse (hν), a rapid inward current develops (black trace) that is blocked by NBQX (50 μM; red trace). C, D: Photostimulation of SPNs in current clamp at P4 (C) and P11 (D) reliably evokes action potentials. Occasionally 2 action potentials were observed. E: Spike latency distribution in cell from D. Spike jitter is low: 75% of spikes occurred within 3 ms of each other during photostimulation. Absolute latency was higher in this cell due to hyperpolarized holding potential. F-H: Recordings in layer 4 cells. F: Left panel: pEPSCs in layer 4 neurons elicited by SPN photostimulation at P11. Center panel: the average of 15 pEPSCs (black trace); the pEPSCs were completely blocked by TTX (0.5 μM; red trace). Note prolonged delay after laser pulse (hν) before onset of inward current. Right panel: Histograms of pEPSC latency and amplitude. pEPSC latencies match those of SPN spiking (E). Amplitude histogram shows 2 peaks at ~20pA and at ~40pA possibly indicating the summation of 2 presynaptic inputs. G: Distribution of pEPSC amplitudes in 27 layer 4 neurons. Mean amplitude was 40.6 ± 41.2 pA. H: pEPSP in current clamp in P6 layer 4 neuron (spiking was blocked with the Na channel antagonist, QX314, in the pipette).

A: LSPS stimulation grid (32×32 spots, 50μm spacing) superimposed on IR image. Red dot indicates cell body of recorded neuron, in this case a layer 4 neuron. B: Cell-attached recording from SPN at P13. Traces show action potentials that are evoked in SPN during LSPS. To the right is a DIC image showing the recording pipette. The layer 6/subplate border is identified by large pyramidal cells in layer 6 and horizontal SPNs in upper subplate. The stimulation map is superimposed on the DIC image. Areas that caused action potentials are labeled black. Note that photostimulation can evoke multiple spikes and that region of activation is very small. C: Cell attached recording from a SPN at P9. Image in C1 shows areas that cause an action potential in black. The layer 6/subplate border is identified by large pyramidal cells in layer 6 and horizontal SPNs in upper subplate. Note that this are as confined to subplate. Image in C2 shows, for the same cell as in C1, the latency to first spike of cell in C1 encoded in pseudocolor. Areas closer to the cell body show shorter latencies. D: Whole-cell patch recordings of layer neuron (P7) during LSPS. D1: Photostimulation (blue line) evoked short latency and long latency EPSCs labeled ‘direct’ and ‘evoked’. D2: Traces show post-stimulation currents (100ms) at stimulus locations for this cell. Areas shaded pink gave direct responses and are plotted on a reduced scale (2nA scale). All other traces are plotted on a 100pA scale. Areas shaded yellow gave rise to evoked pEPSCs (see D1). Note the large gap between these two areas. pEPSCs originating from superficial locations partially overlap direct response. D3: pEPSC amplitude (top) and latency (bottom) as function of stimulus xy-position and superimposed on DIC image of slice. Values are encoded in pseudocolor. Locations that led to short latency direct responses are marked in black. Red circle indicates location of cell body. White box indicates stimulated area. E: LSPS of a layer 4 neuron at P13. E1 shows amplitude map. Besides SPN input, this neuron also received intracortical inputs from layers 2/3 and 4. E2: Traces show post-stimulation currents during stimulation of areas overlying layer 6 and subplate (white box in E1 Note discontinuity of stimulation sites in layer 6 that gave responses from those in subplate that gave responses suggesting that these stimulation sites activate distinct populations of cells. F: Cell attached recording from layer 6 neuron. Left image shows areas that cause an action potential. Note that this area is large and includes a wide area in layer 6 and a narrow area in layer 4. Right image shows latency to first spike encoded in pseudocolor. Note the short latency to first spike for somatic stimulation. All scale bars=200μm.

Excitatory SPNs receive input from MGN, while inhibitory SPNs do not receive inputs from MGN but from other unknown sources (‘?’). Excitatory SPNs project to layer 4 and to other SPNs, while the projection targets of inhibitory SPNs are unknown.