Experimental setup.

<p>(<b>A</b>) Anatomical landmarks (i.e., sacrum, iliac crest, and spinous processes) for localization of the lumbar spine (L2–S1); (<b>B</b>) Lateral view of anatomical motion analysis markers (1-Lateral iliac crest, 2-Trochanter major, 3-Patella, 4-Lateral malleolus, 5-Fourth metatarsal); (<b>C</b>) Pre-operative computed tomography: Lower thoracic vertebrae, lumbar vertebrae and sacrum; (<b>D</b>) Exposure of the lumbar spine; (<b>E</b>) Laminotomy at L5-left (circle) and epidural electrode (arrow); (<b>F</b>) Exposure of the spinal cord for intraspinal microstimulation; (<b>G</b>) Typical microelectrode implantation in the left hemicord (arrow). Insulating adhesive tape was used for protection of the electrode during forceps-insertion and color-coded to determine insertion depth (e.g., blue tape corresponded to an electrode depth of 6 mm). A reference ruler (centimeters) was used to determine cord dimensions and estimate electrode location.</p

Targeting analysis and functional outcomes.

<p>(<b>A</b>) Epidural electrode placement confirmed by lateral intra-operative fluoroscopy; (<b>B</b>) Posterior-anterior intra-operative fluoroscopy; (<b>C</b>) <i>Ex-vivo</i> axial MRI of spinal cord showing ISMS electrode tract (arrow); (<b>D</b>) Experimental setup for kinematic analysis; (<b>E–F</b>) Typical hip extension evoked by ISMS at L5-segment; (<b>G</b>) Normalized intramuscular EMG amplitude as a function of spinal cord segment evoked by ISMS at 300 µA (n = 3); (<b>H</b>) Kinematic analysis of joint angle change evoked by ISMS as a function of spinal cord segment (n = 3). Abbreviations: G.M. = Gluteus medius, H = Hamstrings, B.F. = Biceps femoris, G = Gastrocnemius, Q.F. = quadriceps femoris.</p

Voltage Dependency of fMRI BOLD signal.

<p>A) Comparison of data from left unilateral NAc stimulation at 3 V 130 Hz 500 µs (i; n = 7) with stimulation at 5 V 130 Hz 500 µs (ii; n = 7). Both voltages showed regions of activation in the prefrontal cortex and insula as well as an area of deactivation in the dorsal region of the thalamus. B) i. Region of interest cluster sizes (mm³) comparing the percent size of areas of activation with 3 V 130 Hz 500 µs (yellow; n = 7) and 5 V 130 Hz 500 µs (red; n = 7), represented by the relative size of the two circles. ii. Event-related time course of percent change in BOLD signal from baseline with 1 V (blue; n = 5), 3 V (yellow; n = 7), and 5 V at 130 Hz (red; n = 7), 500 µs pulse width. C) Unilateral stimulation to the left (left) right (middle) and bilateral (right) NAc (n = 1). Stimulation of the right NAc activated areas corresponding to those of left NAc stimulation, including prefrontal cortex and insula, ipsilateral to the side of stimulation.</p

Significant Clusters in the General Linear Model comparing Stimulation “on” periods to baseline.

<p>I = Ipsilateral, AP = Anterior/Posterior, ML = Medial/Lateral; DV = Dorsal/Ventral.</p><p>Stimulation Parameters = 5 V 130 Hz 500 µs.</p

DBS surgery and Lead Confirmation.

<p>A) Custom designed MRI-compatible head frame. B) Screenshot of MR-image based targeting procedure using modified COMPASS software. C) Surgical introduction of the Medtronic 3389 DBS electrode using the Lexsell stereotactic arc. D) fMRI Experimental Setup. Extension wiring connected the externalized DBS lead with a pulse generator located outside the scan room. E) Representative pre-surgical anatomical MP-RAGE scan. F) Post-surgical CT scan demonstrating electrode location in the left NAc G) MR-CT fusion with atlas overlay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056640#pone.0056640-Felix1" target="_blank">[45]</a>demonstrating the location of the electrode tip in the left NAc. H) Diagram plotting the location of the 0 contact in each animal (red asterisks), as determined by the MR-CT fusion on a stereotaxic pig brain atlas, sagittal slice (lateral 2.00 mm from midline) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056640#pone.0056640-Felix1" target="_blank">[45]</a>.</p

NAc DBS elicits distal network BOLD changes.

<p>A–C) Areas of activation with left unilateral NAc stimulation at 5 V 130 Hz 500 µs (n = 7), normalized to a 3D pig brain template <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056640#pone.0056640-Saikali1" target="_blank">[44]</a> Significant activation q(FDR)<0.001 was observed in the anterior and dorsolateral prefrontal (red), insula (brown), parahippocampal (green) and cingulate cortex (blue). Decrease in BOLD signal was observed in the dorsal region of the thalamus (tan). Slice locations are presented in distance (mm) from the posterior commissure. D) Event-related analysis of the average time course for each region of interest was plotted as average percent change in BOLD signal from baseline vs. time (one scan is equal to TR = 3 seconds) using ten frames (30 seconds) prior to stimulation onset as the baseline. Duration of stimulation is marked by the vertical purple lines. In all regions of interest, there is a clear peak in percent change associated with stimulation.</p

Pulse Width dependency of fMRI BOLD signal.

<p>A) Comparison of data from left unilateral NAc stimulation at 5 V 130 Hz 500 µs (i; n = 3) with stimulation at 3 V 130 Hz 100 µs (ii; n = 3). Both pulse widths showed regions of activation in the prefrontal cortex, insula, dorsal anterior cingulate, caudate. There was an additional area of activation in parahippocampal cortex present only with stimulation at 5 V 130 Hz 500 µs. B) i. Region of interest cluster sizes (mm³) comparing the percent size of areas of activation with 5 V 130 Hz 100 µs (yellow; n = 3) and 5 V 130 Hz 500 µs (red; n = 3), represented by the relative size of the two circles. ii. Event-related time course of percent change in BOLD signal from baseline with 100 µs (yellow; n = 3) and 500 µs (red; n = 3) pulse widths at 5 V and 130 Hz.</p

Monitoring In Vivo Changes in Tonic Extracellular Dopamine Level by Charge-Balancing Multiple Waveform Fast-Scan Cyclic Voltammetry

Dopamine (DA) modulates central neuronal activity through both phasic (second to second) and tonic (minutes to hours) terminal release. Conventional fast-scan cyclic voltammetry (FSCV), in combination with carbon fiber microelectrodes, has been used to measure phasic DA release in vivo by adopting a background subtraction procedure to remove background capacitive currents. However, measuring tonic changes in DA concentrations using conventional FSCV has been difficult because background capacitive currents are inherently unstable over long recording periods. To measure tonic changes in DA concentrations over several hours, we applied a novel charge-balancing multiple waveform FSCV (CBM-FSCV), combined with a dual background subtraction technique, to minimize temporal variations in background capacitive currents. Using this method, in vitro, charge variations from a reference time point were nearly zero for 48 h, whereas with conventional background subtraction, charge variations progressively increased. CBM-FSCV also demonstrated a high selectivity against 3,4-dihydroxyphenylacetic acid and ascorbic acid, two major chemical interferents in the brain, yielding a sensitivity of 85.40 ± 14.30 nA/μM and limit of detection of 5.8 ± 0.9 nM for DA while maintaining selectivity. Recorded in vivo by CBM-FSCV, pharmacological inhibition of DA reuptake (nomifensine) resulted in a 235 ± 60 nM increase in tonic extracellular DA concentrations, while inhibition of DA synthesis (α-methyl-dl-tyrosine) resulted in a 72.5 ± 4.8 nM decrease in DA concentrations over a 2 h period. This study showed that CBM-FSCV may serve as a unique voltammetric technique to monitor relatively slow changes in tonic extracellular DA concentrations in vivo over a prolonged time period