Volume 76, Issue 5, Pages 1030-1041 (December 2012) Therapeutic Deep Brain Stimulation in Parkinsonian Rats Directly Influences Motor Cortex  Qian Li, Ya Ke, Danny C.W. Chan, Zhong-Ming Qian, Ken K.L. Yung, Ho Ko, Gordon W. Arbuthnott, Wing-Ho Yung  Neuron  Volume 76, Issue 5, Pages 1030-1041 (December 2012) DOI: 10.1016/j.neuron.2012.09.032 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 STN-DBS Alleviates Parkinsonian Motor Deficits Induced by Unilateral 6-OHDA Lesion (A) Schematic sections showing the placement of the stimulating electrode at the subthalamic nucleus ipsilateral to the 6-OHDA lesion and the bilateral 16-channel recording arrays at layer V of motor cortex. MI, primary motor cortex; SN, substantia nigra; Str, striatum; GP, globus pallidus; EP: entopeduncular nucleus; STN, subthalamic nucleus. See also Figure S1. (B) An example of the locomotor activity of a hemi-Parkinsonian rat before (black line, 1 min), during (red line, 2 min), and after (blue line, 1 min) STN-DBS. (C) Quantification of the locomotor activities of the hemi-Parkinsonian rats, including the time spent in mobility and mobile episodes per min (left panels) and the time spent in freezing and freezing episodes per min (right panels). Severe akinesia was induced by 6-OHDA lesion, which was reversed by the delivery of 125 Hz STN-DBS. ∗p < 0.05; ∗∗∗p < 0.001; NS, not significant; ANOVA. (D) Analysis of the time spent in fine movement as well as the average mobile speed indicated bradykinesia symptoms in the 6-OHDA-lesioned rats. These deficits were significantly improved by STN-DBS. ∗p < 0.05; ∗∗∗p < 0.001, NS: not significant, ANOVA. (E) Contralateral rotation was induced by administration of a low dose of apomorphine (0.5 mg/kg, subcutaneously). The reduction in the number of contralateral rotation during 2 min of 125 Hz STN-DBS reached a statistical significance when compared with both pre- (5 min) and post-DBS (5 min) periods. ∗p < 0.05, ∗∗p < 0.01, paired t test. (F) Based on the time spent in mobility, ANOVA repeated-measures analysis revealed that the beneficial effect of STN-DBS reached a statistically significant level at 50 Hz and a maximum at 125 Hz, with the pulse width set at 60 μs. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, compared with lesioned condition. (G) The efficacy of the STN-DBS paradigm was less dependent on the pulse width. Significant beneficial effect could be achieved by 20 μs up to 100 μs, with the stimulation frequency set at 125 Hz. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, compared with lesioned condition. Error bars denote SEM. See also Figures S1, S3, and S4. Neuron 2012 76, 1030-1041DOI: (10.1016/j.neuron.2012.09.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Identification and Characterization of the Antidromic Spikes in the CxFn during STN-DBS (A) An example of the identification of antidromic spikes. Shown on the top are the spike templates of two single units picked up by the same recording electrode and discriminated by their spike waveforms. Five overlaid traces are shown for each unit. An antidromic spike appeared following the stimulation artifact (arrows) with a short, fixed latency (i.e., 0.92 ms, top left). Collision occurred when a neuron discharged spontaneously immediately before a stimulus pulse (<1.0 ms) and prevented the propagation of the antidromic response being recorded (top right). However, collision failed to occur if a longer time (i.e., >1.7 ms) elapsed between the spontaneous spike and the stimulus pulse (bottom left). Note that the antidromic spike could not collide with a spontaneous spike generated by another neuron, even within a short time lag (bottom right). See also Figure S2. (B) The success rate of the antidromic spike decreased with the increased frequencies. Data shown here were obtained from 115 CxFn of eight hemi-Parkinsonian rats. (C) The absolute frequency of antidromic spikes increased with the stimulation frequency, reaching a peak that corresponded to 125 Hz stimulation frequency. Data were from 115 CxFn in eight hemi-Parkinsonian rats. In (B) and (C), open circles represent data from an individual rat; closed circles represent overall mean. (D) Within the therapeutic window of STN-DBS frequency (from 50 to 250 Hz, gray shade), there was a positive correlation between the antidromic spike frequency and the time spent by the animal in mobility. (E) At a low stimulation frequency of 10 Hz, highly regular antidromic spikes were produced in two CxFn. The time of occurrence of the stimulus and evoked antidromic spikes are shown on the left, and the resulting SDHs, fit by Gaussian distribution, are shown on the right. (F) In contrast, 125 Hz STN-DBS generated two distinct discharge patterns of the antidromic spikes that were highly random and characterized by Poisson-like SDHs. See also Figures S2 and S3. Neuron 2012 76, 1030-1041DOI: (10.1016/j.neuron.2012.09.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 High-Frequency STN-DBS Normalizes Firing Rate and Patterns of CxFn (A) Typical examples of spike rate histogram, showing the effect of STN-DBS on the spontaneous firing rates of CxFn. Two minutes of STN-DBS at 125 Hz (gray shade) caused no effect on the CxFn in the unlesioned side, but increased the firing rate of the neurons in the lesioned side. However, STN-DBS delivered at 10 Hz was ineffective in altering the firing rate of the same CxFn in the lesioned side. (B) Statistical analyses revealed that the mean firing rate of the CxFn after the 6-OHDA lesion was reduced significantly, but was normalized by 125 Hz STN-DBS. In these analyses, the antidromic spikes were removed and did not contribute to the counts. Data were from 88 neurons in five intact rats and 98 neurons in eight hemi-Parkinsonian rats. ∗p < 0.05, ∗∗p < 0.01 ANOVA. (C) Typical examples of raster plots showing the neuronal discharge patterns under intact, 6-OHDA-lesioned, 125 Hz, and 10 Hz STN-DBS conditions. Random discharge patterns of CxFn were found in intact rats. In contrast, the firing of the CxFn in the 6-OHDA-lesioned rat was characterized by increased burst firing. The burst discharges, as defined by the Legendy surprise method, were highlighted with red lines. The burst firings of these neurons were clearly reduced under 125 Hz STN-DBS. In contrast, clear burst firing remained in all the neurons at 10 Hz STN-DBS. (D and E) Both the analyses of the % of total number of spikes in burst discharge (D) and the % of time spent in burst discharge (E) were significantly increased after 6-OHDA lesion and were significantly reduced by 125 Hz STN-DBS. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not significant; ANOVA. The pooled data were obtained from 88 CxFn of five intact rats and 98 and 115 CxFn from unlesioned and lesioned sides, respectively, of eight hemi-Parkinsonian rats. Error bars denote SEM. See also Figure S2. Neuron 2012 76, 1030-1041DOI: (10.1016/j.neuron.2012.09.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 High Frequency STN-DBS Ameliorates Abnormal Population Responses in MI (A) Typical raster plots showing the synchronized spikes, especially during burst discharge, across 10 CxFn from the 6-OHDA-lesioned condition. (B) Analysis of cross-correlation between pairs of CxFn in 6-OHDA lesion. The color-coded correlation matrix on the left side of each panel revealed increased correlation in many pairs of CxFn in 6-OHDA, but was absent in intact animals. The correlations were markedly reduced when the burst discharges were filtered out artificially from the record. The strong correlation was abolished when high frequency STN-DBS was delivered. The correlation levels from one pair of neurons in the intact animal and from one pair of neurons in a 6-OHDA rat (indicated by the yellow squares) are shown on the right. Statistical comparison between five intact rats and eight hemi-Parkinsonian rats is shown in the lower panel. ∗∗∗p < 0.001; NS, not significant; ANOVA. (C) Typical raw LFP waveforms recorded in layer V of MI from both intact and hemi-Parkinsonian rats. For each hemi-Parkinsonian rat, recordings were made from both unlesioned and 6-OHDA-lesioned sides and also 125 Hz STN-DBS on the lesioned side. (D) Power spectral distribution of MI-LFPs under the above conditions. A clear increase of power in the beta range (20–30 Hz) was found in the MI ipsilateral to 6-OHDA lesion, which was absent under both intact and unlesioned conditions. STN-DBS of 125 Hz alleviated the dominant beta band oscillatory activities. Data are averages obtained from 61 LFP records in five intact rats and 95 LFP records in eight lesioned rats. Error bars denote SEM. See also Figure S2. Neuron 2012 76, 1030-1041DOI: (10.1016/j.neuron.2012.09.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 High Frequency STN-DBS Disrupts the Beta Band Spike Field Coherence in MI (A) Typical LFP and corresponding spike trains from CxFn recorded simultaneously from intact (left) and 6-OHDA-lesioned (right) hemispheres of a hemi-Parkinsonian rat. (B) Analysis of the spike-field coherence spectrum revealed a prominent peak in beta band (20–30 Hz), which was abolished when high frequency STN-DBS was delivered. (C) Representative results of polar-histogram of the spike-field coherence phase distribution in beta band (20–30 Hz). Phase-locking bias between spikes and LFP was evident after 6-OHDA lesion, which was absent in both the intact rat and unlesioned side of hemi-Parkinsonian rats, and reversed to random distribution when 125 Hz STN-DBS was delivered. See also Figure S2. Neuron 2012 76, 1030-1041DOI: (10.1016/j.neuron.2012.09.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Antidromic Spikes Modulate Firing Probability of CxFn (A) Representative peristimulus rasters and cumulative rate histograms of the firing activities of CxFn in the 6-OHDA-lesioned sides during 2 min of 125 Hz STN-DBS (“on”) and 2 min of “off” period before and after DBS. Poststimulus 8 ms time segments were aligned by setting the onset time of stimulation to be 0 ms. Those segments containing an antidromic spike (left top panel) were separated from those that did not contain an antidromic spike (right top panel). In the left, during the “on” period, the antidromic spikes were concentrated at around 1 ms poststimulus (time window “a”), followed by complete cessation of firing in time window “b,” and increased firing probability in time window “c.” This biphasic change following antidromic spikes was quantified and shown in the cumulative firing rate histogram (middle panel). In contrast, for those time segments that did not contain an antidromic spike (i.e., absence of spike in “a”), there was no change in the firing probabilities in the corresponding time windows “b” and “c” (right panel). The bottom panel shows that the firing rate of the neuron during the DBS “off” period was stable. (B) In the unlesioned side, whether the DBS was “on” or “off,” no change in firing probability was found. Note that the lack of activity in the first ms of the plots was due to masking by the stimulus artifact. See also Figures S2 and S5. Neuron 2012 76, 1030-1041DOI: (10.1016/j.neuron.2012.09.032) Copyright © 2012 Elsevier Inc. Terms and Conditions