Article Figures & Data
Figures
- Figure 1.
VSD imaging of the motor cortical response after electrical stimulation of the VTA. A, Schematic view of VTA stimulation and VSD imaging. The imaged cortical area, as indicated by a red rectangle, covered the motor cortex ipsilateral to the stimulated side. The borders between M1 and adjacent areas were delineated according to the brain atlas. B, A spatiotemporal pattern of neuronal activity resulted from VTA stimulation. Single-pulse stimulation was applied at 0 ms, and the poststimulus time is shown in each image. C, The typical time course of the optical signal from M1. White triangle represents the stimulus onset. Fluorescence decrease is upward; upward or downward deflections of the signal indicate depolarization or hyperpolarization, respectively. D, E, The relationship between stimulus intensity and M1 responses as assessed by the peak amplitude and the half-width duration of the optical signals (average of five trials from one rat). The excitatory or inhibitory components of the optical signal are indicated in red or blue, respectively. D, The peak amplitude of the excitatory signal significantly increased with higher stimulus current. E, The inhibitory signal was gradually prolonged when a stronger stimulus current was applied. Data are mean ± SEM. *p < 0.05. **p < 0.01. A, Anterior; L, lateral. Scale bar: B, 1.0 mm.
- Figure 2.
VTA-evoked neuronal activity spreads from the CFA toward the RFA in the motor cortex. A, Initial activation sites and the direction of neural propagation. Filled circles represent the site of initial activation in each animal, and the tip of the arrow indicates the center of gravity of the activity map 6 ms after the initial activation. B, Motor representations in the M1 as plotted on a single frame of VTA-evoked neural activity. ICMS mapping was conducted immediately after VSD imaging. Colored circles indicate electrode penetration sites and the body parts moved by stimulation. C, Three ROIs selected in the motor cortex: CFA, RFA, and Vib. The position of each ROI was determined by consulting the motor maps from three rats. The red and blue contour lines indicate CFA and RFA representations, respectively. D, E, Peak amplitude and latency of VTA-evoked optical signals in three ROIs. D, The peak amplitudes of the optical signals in both forelimb areas (CFA, RFA) were greater than that in the Vib. To quantify the response amplitude without variation among animals, ΔF/F was normalized to the maximum response value of each animal (ΔF/F)norm. E, The latency-to-peak of the excitatory optical signal was significantly shorter in the CFA compared with the RFA and the Vib. **p < 0.01. Scale bar: B, 1.0 mm.
- Figure 3.
VSD imaging of VTA-evoked neuronal activity in the M1 before and after application of CNQX, bicuculline, or a D1/D2 receptor antagonist to the cortical surface. A, Neuronal activity completely disappeared after CNQX application. Top right panels, The recovered response after CNQX washout. B, Bicuculline completely eliminated inhibitory neuronal activity and greatly enhanced the excitatory response. C, The D1 receptor antagonist SCH 23390 and the D2 receptor antagonist sulpiride had no effect. VSD imaging was performed 1 h after the administration of dopamine antagonists. Traces to the right of the images represent the time course of the recorded optical signals. Scale bar, 1.0 mm.
- Figure 4.
Distribution of TH-positive fibers in the M1 of control rats. A, Photomicrograph of a TH-stained M1 section. B–D, Higher magnification of the regions shown in A. TH-positive fibers were rich in layer 6 and sparsely distributed in the other cortical layers. Scale bar, 100 μm.
- Figure 5.
Stimulation of 6-OHDA-treated VTA fails to evoke neuronal activity in M1. A, Schematic view of the experimental design. Electrical stimulation was applied to the 6-OHDA-treated VTA or the forelimb of the VTA-intact side. B, Micrograph of a TH-stained section, including VTA. An arrow in the 6-OHDA-administered VTA indicates the electrolytic lesion made at the stimulation site. C, Fluorescence image of a TH-stained section of M1 obtained from a 6-OHDA-lesioned rat (top). Cortical layers were delineated using Nissl staining of the same section (bottom). D, VSD imaging of neuronal activity after the stimulation of the VTA (top) or forelimb (bottom). Representative optical signals are shown on the right. No response was observed after stimulation of the 6-OHDA-treated VTA, whereas the forelimb stimulation activated the sensorimotor cortex, including the M1 and S1. The latter result indicates that diminished M1 activity in the case of VTA stimulation was not caused by damage to the imaging cortex. S1, Primary somatosensory cortex. Scale bars: B, 500 μm; C, D, 1.0 mm.
- Figure 6.
Unilateral VTA stimulation elicits activation in the bilateral M1. A, Schematic view of the experimental design. After unilateral VTA stimulation, VSD imaging was performed in the bilateral M1. Red rectangles represent the imaged cortical areas. Muscimol was injected into the M1 ipsilateral to the stimulated VTA. B–E, VTA-evoked responses in the ipsilateral and contralateral M1 before muscimol injection. B, In the control, electrical stimulation of unilateral VTA elicited excitatory–inhibitory neuronal activities in the bilateral M1. C, The initial activation sites and the direction of neural propagation in the contralateral M1. The distribution pattern was similar to that in the ipsilateral M1, as shown in Figure 2A. D, Magnified and smoothed traces of the rising phase of the optical signals. The traces are from selected pixels in the area of initial activation. ΔF/F was normalized to the maximum response amplitude of each hemisphere. The contralateral response (blue) was significantly delayed compared with the ipsilateral response (black). E, The VTA-evoked response amplitude was compared between hemispheres. The optical signals were collected from the CFA of the M1, and their peak amplitude was evaluated as the value of ΔF/Fnorm. The contralateral M1 showed reduced activation compared with the ipsilateral, but the difference was not significant (p > 0.05, Mann–Whitney U test). F, VTA-evoked responses after muscimol injection. Neuronal activity in the muscimol-injected M1 (ipsilateral to the stimulated VTA) was completely abolished; however, the activity in the contralateral M1 was mostly unaffected. Arrows (pink) indicate muscimol injection sites. Traces on the right show representative optical signals obtained from the bilateral M1 before (black) and after (red) muscimol injection. Scale bars: B, F, 1.0 mm.
- Figure 7.
CC transection abolishes the contralateral M1 response. A, Schematic illustration of CC transection. A wire knife with a cannula was inserted through a cranial hole drilled above the olfactory bulbs (OBs). B, Nissl-stained coronal section showing that the dorsal part of CC was cut without massive damage to the nearby cortex or underlying brain tissues. Complete transection of CC was not necessary in this experiment. Arrowheads indicate the borders between the M1 and surrounding cortical areas. C, D, Spatiotemporal dynamics of VTA-evoked responses in the bilateral M1 before and after CC transection. C, Stimulation of the right VTA (Rt-VTA) activated both the right M1 (Rt-M1, top) and the left M1 (Lt-M1, bottom) before CC scission. D, After CC section, right VTA (Rt-VTA) stimulation activated the right M1 (Rt-M1, top panels) as usual, but the neural response disappeared in the left M1 (Lt-M1, middle panels). Under this condition, the left M1 (Lt-M1) exhibited typical ipsilateral neuronal activity in response to left VTA (Lt-VTA) stimulation (bottom panels). M2, Secondary motor cortex; S1, primary somatosensory cortex; Rt/Lt, right/left. Scale bars: B, C, D, 1.0 mm.
- Figure 8.
CSD analysis in the bilateral M1. A, Photograph of the 16-channel electrode (left) and an example of a Nissl-stained section showing the needle track and the location of electrode contacts (right). B, C, Depth profiles of LFPs in response to VTA stimulation and corresponding CSD analyses in the ipsilateral and contralateral M1, respectively. In the CSD analyses, the size and the location of current sinks (reddish) and sources (bluish) are color-coded. The white triangle represents stimulus onset.
- Figure 9.
The contralateral M1 response also arises from VTA-DA neurons. A, Schematic view of the experimental design. Unilateral 6-OHDA animals were used. Electrical stimulation was applied to 6-OHDA-treated VTA (right) or intact VTA (left). VSD imaging was performed in the left M1 (VTA-intact side). B, Photomicrograph of a TH-stained section, including electrolytic lesions at the stimulation sites (arrows in bilateral VTAs). C, Stimulation of the 6-OHDA-treated VTA failed to activate the contralateral M1 (top), although the cortex exhibited normal excitatory–inhibitory neuronal activity when the intact VTA was stimulated ipsilaterally (bottom). Traces under the images represent the time courses of the optical signals. Scale bars: B, 500 μm; C, 1.0 mm.
- Figure 10.
The ICMS-evoked EMG response is modulated depending on the timing of conditioning VTA stimulation. A, Schematic view of the experimental design. B, C, EMG of upper limb muscles in response to either VTA or M1 stimulation. Essentially no EMG response was observed after single-pulse stimulation to the VTA (B). Here the intensity of M1 stimulation was below the movement threshold (C). D, When the under-threshold M1 stimulation was conditioned with VTA stimulation, the EMG response was elicited. VTA stimulation preceded the ICMS by 10 ms. E, F, On the contrary, the ICMS-evoked EMG was abolished when paired with the 30 ms preceding VTA stimulation. The M1 stimulation with suprathreshold intensity evoked the EMG (E), but the muscle activity was precluded by the preceding VTA stimulation (F). G–I, The EMG response started to reappear when the interstimulus interval was set to 40 ms (G). As the interval increased, the EMG response recovered gradually and was restored by 200 ms (H, I). Gray lines indicate the EMG traces in each trial, and a red line indicates the mean EMG activity. Black triangles represent the stimulus onset. J, A possible explanation for the time-dependent suppression of EMG. Traces represent the time course of the VTA-evoked optical signal (black, the same trace as in Fig. 1C) and its time derivative (red). The magnified inset on the right represents the initial phase. Differential operations were applied to the smoothened optical signal. The period during which the time derivative signal showed a large negative value (30–50 ms after stimulation, shaded in gray) coincided with the period of strong EMG suppression (F, G).
