Brecht Et Al. Single- Cell- Evoked Movements 5

Brecht et al. Single-cell-evoked movements 1 Brecht et al. Single-cell-evoked movements 2 Brecht et al. Single-cell-evoked movements 3 Brecht et al. Single-cell-evoked movements 4 Brecht et al. Single-cell-evoked movements 5 Brecht et al. Single-cell-evoked movements 6 Brecht et al. Single-cell-evoked movements 7 Brecht et al. Single-cell-evoked movements 8 Brecht et al. Single-cell-evoked movements 9 Brecht et al. Single-cell-evoked movements 10 Brecht et al. Single-cell-evoked movements 11 Brecht et al. Single-cell-evoked movements 12

Supplementary material:

Supplementary figure 1 Comparison of L5- and L6-cell stimulation effects. a, Population average of evoked average movements for 10 pyramidal L5 neurons located at sites where extracellular stimulation evoked backwards movements. b, Population average of evoked average movements for nine pyramidal L6 neurons located at sites where extracellular stimulation evoked backwards movements. Movement traces of the individual cells were normalized to the same peak-to-peak amplitude in the poststimulus epoch before averaging (both a and b). The scaling (in parentheses) was chosen such that it matches the peak-to-peak amplitude of the absolute (non-normalized) movement average of the respective cells. c, Plot of peak power frequencies in individual movements against peak power frequencies of averaged evoked movements for L5 and L6 cells. Values were determined from the power spectra of the respective traces. In all 11 identified L6 pyramidal cells, the peak power of the averaged movement occurred at frequencies <1 Hz. The peak power frequencies of averaged movements were significantly different between L5 and L6 cells (unpaired t-test, p<0.001), while the peak power frequencies of individual trials were not (unpaired t-test, p>0.2). d, Rise time of averaged movement traces in L5 and L6. This difference in L5/L6 rise time is significant (t-test, p<0.01). Only those L5 and L6 cells for which poststimulation movement was larger than prestimulation movement in the average of evoked movements (ratio of post- to prestimulation amplitudes >1.2) were included. The dashed lines in a, b indicate the onset of action potential initiation. Brecht et al. Single-cell-evoked movements 13

Supplementary figure 2 Effect of action potential frequency and number on evoked movements: averaged traces. a, Population average of evoked average movements with initiation of 10 action potentials (APs) at 10, 50 or 100 Hz in seven neurons (three L5 pyramidal cells, three L6 pyramidal cells and one unidentified regularly spiking cell). All measurements refer to the best whisker determined by extracellular stimulation, and all cells were located at sites where extracellular stimulation evoked backwards movements. Before averaging, all movement traces of individual cells were normalized to the same poststimulus peak-to-peak amplitude in the maximally effective condition. b, Population average of evoked average movements with initiation of 1, 2, 5 or 10 APs at 50 Hz, in nine neurons (two L5 pyramidal cells, four L6 pyramidal cells and two unidentified regularly spiking cells). All measurements refer to the best whisker determined by extracellular stimulation, and all cells were located at sites where extracellular stimulation evoked backwards movements. Before averaging, all movement traces of individual cells were normalized to the same poststimulus peak-to-peak amplitude in the maximally effective condition. The scaling (in parentheses) was chosen such that it matches the peak-to-peak amplitude of the absolute (non-normalized) movement average of the respective cells. The dashed lines indicate the onset of AP initiation. Brecht et al. Single-cell-evoked movements 14

Supplementary figure 3 Interaction of initiated action potentials with cortical up states and down states. a, Top: topographic position of the stimulated cell with dendritic (red) and axonal (blue) arbors of the stimulated neuron, which was situated at the lower boundary of L5. The cell was located at a site where extracellular stimulation evoked backwards movements of multiple whisker rows, with maximal deflection observed for whisker C1. b, Top: both the local field potential recording (upper trace) and the membrane potential of a nearby recorded cell (lower trace) fluctuated between two states: more depolarized membrane potentials (up states) and less depolarized (down states), leading to a bimodal distribution of membrane potential values over time (histogram at right). Bottom: action potential (AP) initiation during up states (left) or down states (right); APs have been clipped. c, Movement traces sorted according to up states (left) and down states (right). 1: average movement of whisker C1 for up-state (left) and down-state (right) initiation trials. 2: movements associated with up states and down states in the absence of AP initiation. Movement traces were sorted according to the presence of an up state or a down state in the cortical cell at a random time point between stimulation trials and were subsequently averaged. 3: net movement contribution of AP initiation in up states (left) and down states (right) computed by subtracting trace 2 from trace 1. d, 1: population average of evoked average movements with initiation of 10 APs at 50 Hz in up states (left) and down states (right) in nine regularly spiking neurons. All cells were located at sites where intracortical microstimulation evoked backwards movements. Before averaging, all movement traces of individual cells were normalized to the same poststimulus peak-to-peak amplitude in the maximally effective condition. 2: movements associated with up states and down states in the absence of AP initiation. Movement traces were sorted according to the presence of an up state or a down state in the cortical cell at a random time point between stimulation trials and were subsequently averaged. 3: net movement contribution of AP injection in up states (left) and down states (right) computed by subtracting trace 2 from trace 1. The dashed lines in c and d indicate the onset of AP initiation. Brecht et al. Single-cell-evoked movements 15

Location of cells.

All cells intracellularly labelled at extracellular stimulation sites that evoked whisker movements were found in L5 and L6 of a distinct part of the rat dorsomedial frontal cortex, the ‘precentral medial area’45. This area is distinguished from the neighbouring motor cortex by a variety of cytoarchitectonic differences such as a thin L3 and dorsally extended

46 L5. It has also been referred to as agranular medial area (AGm) , as precentral area three (Prc3)47 or as frontal area 2 (Fr2)48. Consistent with Neafsey49, but in contrast to other reports46, no whisker-movement specific cells were identified lateral from this area. According to our own observations, and in agreement with the stereotaxic coordinates of numerous published motor maps26,50–52, this area forms a whisker-movement specific subpart of rat primary motor cortex. Our data on the partitioning of rat M1 will be discussed in detail elsewhere (Brecht et al. in preparation).

Movie 1 Whisker movements evoked by intracellular stimulation of an L6 cell.

Movie 2 Whisker movements evoked by intracellular stimulation of an L5 cell. Brecht et al. Single-cell-evoked movements 16

45. Krettek, J. E. & Price, J. L. The cortical projections of the mediodorsal nucleus and adjacent thalamic nuclei in the rat. J. Comp. Neurol. 171,157–191 (1977).

46. Donoghue, J. P. & Wise, S. P. The motor cortex of the rat: cytoarchitecture and microstimulation mapping. J. Comp. Neurol. 212, 76–88 (1982).

47. Zilles, K., Zilles, B. & Schleicher, A. A quantitative approach to cytoarchitectonics. VI. The areal pattern of the cortex of the albino rat. Anat. Embryol. (Berl.) 159, 335–360 (1980).

48. Zilles, K. & Wree, A. Cortex: areal and laminar structure. In The Rat Nervous System 2nd edn (ed. Paxinos, G.) pp. 649–685 (Academic, London, 1995).

49. Neafsey, E. J. The complete Ratunculus: output organization of layer V of the cerebral cortex. In The Cerebral Cortex of the Rat (ed. Kolb, B. & Tees, R. C.) pp 197–212 (MIT Press, Cambridge, 1990).

50. Gioanni, Y. & Lamarche, M. A reappraisal of rat motor cortex organization by intracortical microstimulation. Brain Res. 344, 49–61(1985).

51. Miyashita, E., Keller, A. & Asanuma, H. Input–output organization of the rat vibrissal motor cortex. Exp. Brain Res. 99, 223–232 (1994).

52. Huntley, G. W. Differential effects of abnormal tactile experience on shaping representation patterns in developing and adult motor cortex. J. Neurosci. 17, 9220–9232 (1997).