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Neuron

Article

Therapeutic Deep Brain Stimulationin Parkinsonian Rats DirectlyInfluences Motor CortexQian Li,1 Ya Ke,1,* Danny C.W. Chan,1 Zhong-Ming Qian,2 Ken K.L. Yung,3 Ho Ko,1 Gordon W. Arbuthnott,4

and Wing-Ho Yung1,*1School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China2School of Pharmacy, Fudan University, Shanghai 201203, China3Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China4Brain Mechanisms for Behaviour Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa 904-0495, Japan

*Correspondence: [email protected] (Y.K.), [email protected] (W.-H.Y.)

http://dx.doi.org/10.1016/j.neuron.2012.09.032

SUMMARY

Much recent discussion about the origin of Parkin-sonian symptoms has centered around the ideathat they arise with the increase of beta frequencywaves in the EEG. This activitymay be closely relatedto an oscillation between subthalamic nucleus (STN)and globus pallidus. Since STN is the target of deepbrain stimulation, it had been assumed that its actionis on the nucleus itself. By means of simultaneousrecordings of the firing activities from populationsof neurons and the local field potentials in the motorcortex of freely moving Parkinsonian rats, this studycasts doubt on this assumption. Instead, we foundevidence that the corrective action is upon thecortex, where stochastic antidromic spikes origi-nating from the STN directly modify the firing proba-bility of the corticofugal projection neurons, destroythe dominance of beta rhythm, and thus restoremotor control to the subjects, be they patients orrodents.

INTRODUCTION

Deep brain stimulation (DBS) of the subthalamic nucleus (STN)

is now a recognized therapeutic option for Parkinson’s disease

(PD) (Benabid et al., 1994; Benazzouz et al., 1993; Deuschl

et al., 2006; Obeso et al., 2001). However, the exact mechanism

of action of STN-DBS is still not settled (Chang et al., 2008; Gu-

bellini et al., 2009; Montgomery and Gale, 2008). Early studies

favored an inhibitory action of DBS. Functional inactivation, like

depolarizing block and depletion of transmitters, could produce

a lesion-like effect in the STN (Beurrier et al., 2001; Garcia et al.,

2003; Magarinos-Ascone et al., 2002). Later studies suggested

that DBS may exert an excitatory effect on neural elements

and interferes with the abnormal oscillatory and synchronized

activities that are commonly found in the basal ganglia in Parkin-

sonism (Brown and Eusebio, 2008; Eusebio et al., 2011).

1030 Neuron 76, 1030–1041, December 6, 2012 ª2012 Elsevier Inc.

In principle, DBS can directly activate a wide range of neuronal

elements in STN and the surrounding area, including STN

neuronal soma, axons of passage, and also the terminals of de-

scending fibers from the cortex to STN (Deniau et al., 2010; Lee

et al., 2006; Li et al., 2007; McIntyre and Hahn, 2010; Miocinovic

et al., 2006). By activating both afferent and efferent axons, STN-

DBS can potentially generate widespread and heterogeneous

effects at local and distal sites (Hammond et al., 2008; Hashi-

moto et al., 2003;Maurice et al., 2003). In fact, there are a number

of studies in both human and animals suggesting that STN stim-

ulation can evoke or modulate cortical activities, which may be

beneficial to PD symptoms (Dejean et al., 2009; Fraix et al.,

2008; Gradinaru et al., 2009; Kuriakose et al., 2010; Lehmkuhle

et al., 2009). Thus, early experiments in patients undergoing

implantation of STN electrodes demonstrated cortical evoked

potentials that resembled antidromic activation from the elec-

trodes (Ashby et al., 2001;MacKinnon et al., 2005). Later demon-

stration of resonant antidromic cortical circuit activation as

a consequence of STN-DBS in anaesthetized rats (Li et al.,

2007) was followed by demonstration that high frequency sub-

thalamic stimulation in awake rats could release them from the

akinesia that followed application of dopamine antagonists (De-

jean et al., 2009). These studies along with others in different

situations demonstrated that the subthalamic stimulation also

disrupted the beta rhythms in the cortex in akinetic animals (Fraix

et al., 2008; Kuriakose et al., 2010; Lehmkuhle et al., 2009). Along

the same line of reasoning, recent optogenetic experiments sug-

gested that modifying the activity of STN neurons was less effec-

tive than direct cortical stimulation in reversing the movement

deficits following 6-hydroxydopamine (6-OHDA) lesions in mice

(Gradinaru et al., 2009).

Despite these previous studies that suggest the importance of

antidromically activated responses in the cortex in mediating the

beneficial effect of STN-DBS, elucidating the therapeutic mech-

anism of DBS can only rely on direct recordings of the neural

activities during behaviorally effective DBS in freely moving

animals. In this study, we addressed this question by making

recordings of both single-unit activities and local field potentials

in the motor cortex (MI) of freely moving hemi-Parkinsonian

animals before, during, and after STN-DBS. The results not

only better characterize the abnormal activity in single motor

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Neuron

Deep Brain Stimulation Directly Influences Cortex

cortical neurons in Parkinsonism, but also reveal a mechanism

by which STN-DBS directly interferes with the pathological

cortical oscillations characteristic of PD.

RESULTS

Alleviation of Motor Deficits by High FrequencySTN-DBS in Hemi-Parkinsonian RatsWe generated the conventional hemi-Parkinsonian model by

unilateral injection of 6-OHDA into the medial forebrain bundle

(MFB) of the adult rat brain. Successful lesion of the nigrostriatal

pathway was confirmed by the apomorphine-induced contralat-

eral rotation test. Then, a stimulating electrode was targeted at

the ipsilateral STN stereotaxically. In some hemi-Parkinsonian

rats, two 16 channel recording arrays were implanted bilaterally

into layer V of the MI (Figure 1A). In this group of animals, two

stimulating electrodes were implanted in the STN bilaterally to

facilitate the identification of layer V MI neurons in both hemi-

spheres (see Experimental Procedures). After all in vivo experi-

ments, correct placements of the stimulating and recording

electrodes were confirmed histologically (Figures S1A and S1B

available online). The dopamine depletion level induced by the

6-OHDA lesion was further evaluated by the tyrosine hydroxy-

lase (TH) immunostaining of the coronal slices at substantia nigra

and striatum (Figures S1C and S1D). In the substantia nigra pars

compacta (SNc), the nigral dopaminergic neuron loss reached

89.5% ± 3.5% (mean ± SEM, 26 rats). In the striatum, the loss

of TH immunoreactivity was 56.8% ± 7.5% (26 rats).

High frequency stimulation (HFS), which consisted of 125 Hz,

60 ms square pulses at an optimal current (see Experimental

Procedures), improved the mobility of the hemi-Parkinsonian

animals in the open arena (Figure 1B). This effect was confirmed

by assessing several parameters in the open field tests, including

the time and number of episodes spent in mobility and freezing,

the averagemobile speed, as well as the time spent in finemove-

ment. For example, as shown in Figure 1C, while the intact

animals (n = 17) spent 48.3% ± 1.7% of time mobile and

10.3% ± 1.6% of time freezing, the hemi-Parkinsonian rats (n =

26) spent significantly less amount of time moving (16.8% ±

2.2%, p < 0.001), but more time freezing (46.7% ± 2.1%,

p < 0.001). When high frequency (125 Hz) STN-DBS was turned

on, the severity of akinesia was largely, though not completely,

reversed. Thus, during DBS, the lesioned animals (n = 26) spent

40.9% ± 2.0% of time mobile (p < 0.001 compared with DBS off;

p < 0.05 compared with intact) and 17.8% ± 1.4% freezing (p <

0.001 compared with DBS off; p < 0.05 compared with intact).

These beneficial effects disappeared immediately when STN-

DBS was turned off. Bradykinesia symptoms, as reflected by

decreased fine movement and reduced mobile speed, were

also evident in the lesioned animals and were similarly alleviated

during the delivery of STN-DBS (Figure 1D). Furthermore, in the

classical apomorphine-induced contralateral rotation test, STN-

DBS resulted in a modest but statistically significant reduction of

the rotation speed, which was measured as the number of turns

per min (pre-DBS: 19.08 ± 0.61/min; DBS: 16.62 ± 0.62/min; p <

0.01, post-DBS: 18.12 ± 0.73/min; n = 26, Figure 1E).

We also characterized the dependence of the therapeutic

effect of the STN-DBS paradigm on the stimulation frequency

N

and pulse width. As summarized in Figure 1F, at constant stim-

ulus width of 60 ms, low frequency (0.2–10 Hz) STN-DBS failed

to alleviate the motor deficit of the hemi-Parkinsonian animals.

However, when the stimulus frequency was 50 Hz and up to

200 Hz, significant improvement was seen in the percentage

of time spent in motion. Among the four effective stimulation

frequencies tested, namely 50, 125, 200, and 250 Hz, the

optimal frequency was 125 Hz, which is in line with those used

in clinical and experimental studies. The efficacy of the DBS

appeared to be less dependent on pulse width. As shown in

Figure 1G, at a constant stimulation frequency of 125 Hz, signif-

icant therapeutic effects could be achieved at pulse width

ranging from 20 to 80 ms. The falling off of efficacy at 100 ms

suggested that the likely target of the stimulation is fibers rather

than cells.

Stimulation of STN Evokes Antidromic Spikes in the MIin Freely Moving PD RatsWe recorded extracellular neuronal activities from the MI layer V

neurons in both intact and 6-OHDA lesioned rats via multi-

channel recording arrays when the animals were awake and

freely moving. Neuronal activities recorded by each channel

were sorted into single units based on the electrophysiological

characteristics of spike waveforms in the principal component

space (Figure S2A). Two major classes of neuronal unit could

be identified. One type of neuron exhibited a relatively long spike

width (�0.5–0.8 ms) and low spontaneous firing rate (<10 Hz),

which were presumed to be pyramidal, projection neurons

(PNs). Compared with the PNs, presumed interneurons (INs)

held shorter spike width (�0.2–0.5 ms), but higher spontaneous

firing rate (�8–45 Hz). Based on the correlation of the firing rate

and spike width (Figure S2B), these two classes of neurons could

be distinguished unambiguously.

The STN is one of the innervation targets of the long-range cor-

ticofugal axons (Kita and Kita, 2012). As shown in Figure 2A, we

found evidence of direct activation of corticofugal projection

neurons (CxFn) in layer V of MI by identifying stimulation-evoked

antidromic spikes in these neurons during STN-DBS. The identi-

fication was based on the fact that the spike (1) could be de-

tected after the stimulus pulse with a short and fixed latency

and (2) collided with a spontaneous orthodromic spike gener-

ated by the same neuron within a short time window prior to

the electrical stimulus. On average, an antidromic spike, if any,

occurred at a latency of 0.92 ± 0.07 ms (n = 88) in five intact

animals, 1.05 ± 0.04 ms in unlesioned (n = 98), and 0.96 ±

0.08 ms (n = 115) in 6-OHDA-lesioned side of eight hemi-Parkin-

sonian animals and would be eliminated via the collision with

a spontaneous spike occurring within a short interval (<1.0 ms)

before the electrical stimulation in the STN. Furthermore, anti-

dromic spikes could be identified only in the class of neurons

that exhibited a low firing rate and long spike width, reinforcing

the conclusion that these neurons are the CxFn. Those PNs

that did not show antidromic spikes were considered as either

non-CxFn or CxFn that were not antidromically activated under

the experimental condition. The percentages of these neurons,

CxFn, and INs are summarized in Table 1. These data were ob-

tained from experiments in which the stimulation sites were

confined to the lateral STN.

euron 76, 1030–1041, December 6, 2012 ª2012 Elsevier Inc. 1031

*

* * **** *** *** *** *** ***

+

EP

STN

MI MI

Pre DBSDuring DBSPost DBS

SNSTN

MI

StrGP

STN

Intact Hemi-Parkinsonian Intact Hemi-Parkinsonian Intact Hemi-Parkinsonian

0Tim

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Pre DBS Post Pre DBS Post Pre DBS Post

Stimulation frequency (Hz) Pulse width (μs) Int

act

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Pre DBS Post Pre DBS Post Pre DBS Post

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*** *** *** *** *** ****

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Intact Hemi-Parkinsonian Intact Hemi-Parkinsonian Intact Hemi-Parkinsonian

0

20

NS

Neuron



Neuron


The Frequency of Antidromic Spikes Correlateswith Therapeutic Efficacy of STN-DBSSince we could identify the antidromic spikes in CxFn unambig-

uously, we asked if there was any relationship between the anti-

dromic spikes and the therapeutic action of STN-DBS. It has

been pointed out that antidromic cortical excitation may not be

as reliable as generally assumed (Chomiak and Hu, 2007). So

first, we determined the reliability of antidromic spike generation

by examining its success rates at different stimulation frequen-

cies. In a pool of 115 CxFn from the lesioned side of eight

hemi-Parkinsonian rats, the antidromic spike reliably followed

each pulse at a low stimulation frequency. Over 80% of stimuli

were followed by an antidromic spike when the stimulation

frequency was from 0.2 Hz to 10 Hz. However, the reliability of

an antidromic spike following an electrical stimulus decreased

dramatically as the frequency of stimulation was increased,

dropping to 46.8% ± 1.5% at 50 Hz, 26.9% ± 1.1% at 125 Hz,

16.1% ± 0.8% at 200 Hz, and 9.33% ± 0.43% at 250 Hz (Fig-

ure 2B). This decrease in the reliability of antidromic spike

production with increasing stimulation frequency resulted in

the highest frequency of antidromic spikes being produced at

around 125 Hz stimulation rather than other frequencies (Fig-

ure 2C). Interestingly, within the therapeutic window of STN-

DBS, i.e., 50–250 Hz, a positive correlation (R2 = 0.783) between

the frequency of antidromic spikes and the beneficial effect of

STN-DBS was observed (Figure 2D). In addition, we found that

HFS stimulation confined to the medial STN rather than lateral

STN resulted in a lower percentage of cortical neurons exhibiting

antidromic spikes, which was also correlated with less motor

improvement (Figure S3).

As we have seen, at high frequencies of electrical stimulation,

such as 125 Hz, a large percentage of antidromic spikes failed

to be elicited. What impact did this have on the discharge

pattern of the antidromic spikes produced? At a low frequency

of stimulation (10 Hz), due to the high success rate, the spike

density histogram (SDH) of antidromic spikes fitted well with

a Gaussian distribution, indicating a regular pattern (Figure 2E).

However, at 125 Hz STN-DBS (Figure 2F), the success or failure

of the antidromic invasion became unpredictable, resulting in a

Figure 1. STN-DBS Alleviates Parkinsonian Motor Deficits Induced by

(A) Schematic sections showing the placement of the stimulating electrode at t

channel recording arrays at layer V of motor cortex. MI, primary motor cortex;

nucleus; STN, subthalamic nucleus. See also Figure S1.

(B) An example of the locomotor activity of a hemi-Parkinsonian rat before (black

(C) Quantification of the locomotor activities of the hemi-Parkinsonian rats, includ

time spent in freezing and freezing episodes per min (right panels). Severe akinesi

STN-DBS. *p < 0.05; ***p < 0.001; NS, not significant; ANOVA.

(D) Analysis of the time spent in finemovement aswell as the averagemobile speed

were significantly improved by STN-DBS. *p < 0.05; ***p < 0.001, NS: not signific

(E) Contralateral rotation was induced by administration of a low dose of apomorp

rotation during 2 min of 125 Hz STN-DBS reached a statistical significance when

0.01, paired t test.

(F) Based on the time spent in mobility, ANOVA repeated-measures analysis revea

at 50 Hz and a maximum at 125 Hz, with the pulse width set at 60 ms. *p < 0.05,

(G) The efficacy of the STN-DBS paradigm was less dependent on the pulse width

stimulation frequency set at 125 Hz. *p < 0.05, **p < 0.01, ***p < 0.001, compare

Error bars denote SEM.

See also Figures S1, S3, and S4.

N

highly random pattern of SDH that was best fit by the Poisson

distribution. At even higher frequencies of stimulation (i.e.,

200 Hz and 250 Hz), the randomness of the antidromic spikes re-

mained, while the success rate of antidromic invasion decreased

remarkably.

High Frequency STN-DBS Normalizes Firing Rateand Pattern of the CxFnWe then examined the effects of a 6-OHDA lesion and STN stim-

ulation on the firing rates of the layer V CxFn in theMI. To analyze

the firing rate, the antidromic spikes were first removed from the

spike traces (see Experimental Procedures). The average spon-

taneous firing rate of the CxFn was found to be reduced after

6-OHDA treatment (intact: 3.20 ± 0.23 Hz, n = 88, five rats;

lesioned: 2.54 ± 0.17 Hz, n = 115, eight rats, p < 0.05, Figures

3A and 3B). In contrast, in the unlesioned side of theMI, no signif-

icant difference in the CxFn’s mean firing rate was found (3.43 ±

0.26 Hz, n = 98, eight rats, NS compared with intact animals).

During the 2 min of STN-DBS at 125 Hz, a significant increase

in the spontaneous firing of the CxFn in the 6-OHDA-lesioned

hemisphere was observed (3.57 ± 0.19 Hz, n = 115, eight rats,

p < 0.01 compared with DBS off; NS compared with unlesioned

or intact animals). This effect of DBS was absent when the stim-

uluswas delivered at a low frequency of 10 Hz.More importantly,

the 6-OHDA lesion also altered the firing pattern of the CxFn by

increasing episodes of burst firing, as defined by the Legendy

surprise method, which could also be reversed by 125 Hz

STN-DBS, but not at 10 Hz (Figures 3C–3E).

The effects of high frequency STN-DBS on the firing activities

of layer V MI neurons may underlie the motor improvement and

be attributable to the antidromic activation from STN. Since

Degos et al. (2008) showed evidence for a direct STN-cortex

projection, it is important to consider the contribution of ortho-

dromic activation in the MI in mediating the observed behavioral

improvement. However, as shown in Figure S4, unlike the layer V

neurons, 125 Hz STN-DBS did not result in changes in the firing

rates of the layer III/IV neurons, the target of the STN-cortex

orthodromic projection, arguing against a major contribution of

this pathway.

Unilateral 6-OHDA Lesion

he subthalamic nucleus ipsilateral to the 6-OHDA lesion and the bilateral 16-

SN, substantia nigra; Str, striatum; GP, globus pallidus; EP: entopeduncular

line, 1 min), during (red line, 2 min), and after (blue line, 1 min) STN-DBS.

ing the time spent in mobility and mobile episodes per min (left panels) and the

a was induced by 6-OHDA lesion, which was reversed by the delivery of 125 Hz

indicated bradykinesia symptoms in the 6-OHDA-lesioned rats. These deficits

ant, ANOVA.

hine (0.5 mg/kg, subcutaneously). The reduction in the number of contralateral

compared with both pre- (5 min) and post-DBS (5 min) periods. *p < 0.05, **p <

led that the beneficial effect of STN-DBS reached a statistically significant level

**p < 0.01, ***p < 0.001, compared with lesioned condition.

. Significant beneficial effect could be achieved by 20 ms up to 100 ms, with the

d with lesioned condition.


Per

cent

age

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10Hz STN-DBS

125Hz STN-DBS

A B

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Spike density histogram (SDH)

Spike density histogram (SDH)

R2=0.783

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CxFn-1

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Stimulation frequency (Hz)

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

Table 1. Distribution of Interneurons and Projection Neurons

with and without Antidromic Spikes Recorded in MI

Interneurons

(%)

Projection

Neurons without

Antidromic

Spikes (%)

Projection

Neurons with

Antidromic

Spikes (CxFn; %)

Intact (five rats) 52 (24.1%) 76 (35.2%) 88 (40.7%)

Unlesioned

(eight rats)

60 (22.0%) 115 (42.1%) 98 (35.9%)

6-OHDA-lesioned

(eight rats)

71 (24.6%) 103 (35.6%) 115 (39.8%)

See also Figure S2.

Neuron



High Frequency STN-DBS Disrupts PathologicalOscillations and Synchrony of MI Neuronal PopulationsApart from altering the firing rate and pattern of individual CxFn,

dopamine depletion induced pathological activities in the MI at

the population level. Figure 4A shows typical raster plots of 10

CxFn after the 6-OHDA lesion, showing clear synchrony among

most of the neurons, which was confirmed by the cross-correla-

tion analysis between pairs of neurons (Figure 4B). Interestingly,

the degree of correlation was dramatically reduced when the

burst firing was filtered out from analysis, indicating that the syn-

chrony was a reflection of the abnormal burst firing. The pooled

data from the recordings in five intact and eight hemi-PD rats

confirmed this observation (Figure 4B). Power spectrum analysis

of the local field potential (LFP) revealed the appearance of oscil-

latory activity at the beta band (�20–30 Hz) after dopamine

depletion (Figures 4C and 4D). STN-DBS at 125 Hz was highly

effective in removing the abnormal synchrony among the neu-

rons and the beta oscillations (Figures 4B and 4D).

The strength of coupling between spike times and LFP at any

given frequency, known as spike-field coherence, was also

investigated. 6-OHDA lesion caused the coherence value to

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 115CxFn 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.

Firin

g ra

te (s

pike

s/s)

Unlesioned side, 125Hz STN-DBS

Lesioned side, 125Hz STN-DBS

Lesioned side, 10Hz STN-DBS

0 1 2 3 4 5 6 05

1015 1

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Intact Hemi-Parkinsonian Intact Hemi-Parkinsonian

A B

NS **

*NS

NS

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***

******

NS

Intact

SPKC06bSPKC08c

SPKC10bSPKC11a

SPKC08d

6-OHDA lesioned

125Hz STN-DBS

1s

SPKC08aSPKC09aSPKC09bSPKC15aSPKC16a

SPKC06bSPKC08c

SPKC10bSPKC11a

SPKC08d

10Hz STN-DBSSPKC06bSPKC08c

SPKC10bSPKC11a

SPKC08d

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125H

z10

Hz

Unlesio

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125H

z10

Hz

Unlesio

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125H

z10

Hz

Figure 3. High-Frequency STN-DBS Normalizes Firing Rate andPatterns 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.

Neuron


N

increase specifically at the beta band (20–30 Hz; Figures 5A and

5B). Interestingly, the delivery of 125Hz STN-DBS eliminated this

beta band spike-field coherence. In addition, the phase synchro-

nization (or coherence phase) between spikes and LFP at the

beta band was derived and presented as polar histogram (Fig-

ure 5C). It was clear that the 6-OHDA lesion induced a specific

phase-locking phenomenon between spikes and LFP in beta

band (91/115 pairs, eight rats), whereas the pairs from the intact

and unlesioned side showed randomly distributed phases,

ranging from 0� to 360� (intact: 88 pairs from five rats; unle-

sioned: 98 pairs from eight rats). During the delivery of 125 Hz

STN-DBS, the specific phase-locking bias present in the 6-

OHDA-lesioned condition was not notable.

Antidromic Spikes Directly Modulate the FiringProbability of CxFnTo elucidate the possible mechanism underlying antidromic

spikes-mediated beneficial effect, we next asked the crucial

question of whether antidromic spikes can directly modulate

the firing properties of the CxFn. Since the DBS was delivered

at a high frequency of 125 Hz, i.e., with an interstimulus interval

of 8 ms, we analyzed the impact of STN-DBS by selecting and

then aligning all those 8 ms time segments that contained anti-

dromic spikes. A typical example is shown in Figure 6A (left

panel). These time-aligned segments of neuronal activities re-

vealed that the probability of firing of the CxFn was influenced

by the 125 Hz HFS: a complete cessation of firing immediately

following the antidromic spike that lasted for about 1ms followed

by an elevated firing probability in the subsequent 2 ms interval.

The early depression of firing probably represents the refractory

period of the antidromic spikes, while the delayed increase in

firing probability could reflect a change in the intrinsic excitability

of the neurons or functional connectivity within the local circuit as

a consequence of antidromic spikes. In contrast, for those 8 ms

segments that did not contain antidromic spikes, there was

no change in the firing probability (Figure 6A, right panel).

In the unlesioned side, no change in firing probability was

found, whether DBS was turned on or off (Figure 6B). These

results demonstrate that the antidromic spikes directly and

selectively alter the firing probability of the layer V CxFn. On

the other hand, when DBS was delivered at 10 Hz, in addition

(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.


See also Figure S2.


-0.5 0.5 0 -0.5 0.5 0

-0.5 0.5 00

0.1

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Pow

er s

pect

ral

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ity (%

)

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1s200μV

Intact

6-OHDA lesioned

125Hz STN-DBS

Unlesioned

08b vs.12a

Intact Unlesioned 6-OHDA lesioned 125Hz STN-DBS6

B

C

0.4

0

0.1

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ACh01cCh02bCh03aCh05cCh08cCh09cCh11b

Ch14aCh16c

Ch13a

1s

6-OHDA lesioned (-burst) 125Hz STN-DBS

02b vs.14a(-burst)

01c

02b

03a

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08c

09c

11b

14a

16c

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01c02b03a05c08c09c11b

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intact 6-OHDA lesioned

06d08b10a11b12a13c14a

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08b

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02b vs.14a

01c

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Intact Hemi-Parkinsonian

0.3

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*** ***

NS

0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100

Cor

rela

tion

inde

x

***

Correlation index

Figure 4. High Frequency STN-DBS Ameliorates Abnormal Popula-

tion 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

Neuron



to the biphasic changes in firing probability immediately fol-

lowing the antidromic spikes, a slight increase in firing rate at

a much delayed time of around 40–50 ms poststimulation was

observed (Figure S3). This was likely the effect relayed to the

cortex via the basal ganglia circuit under STN-DBS.

DISCUSSION

Previous studies demonstrate that STN-DBS can modulate

activities of the cortical motor areas in both PD patients (Cunic

et al., 2002; Dauper et al., 2002; Kuriakose et al., 2010; Limousin

et al., 1997) and in animal models of Parkinsonism (Dejean et al.,

2009; Lehmkuhle et al., 2009; Li et al., 2007). In this study,

making use of multichannel recording arrays implanted into the

MI, we recorded and analyzed single-unit neuronal activities

from populations of the layer V CxFn of freely moving hemi-

Parkinsonian rats during a therapeutically effective STN-DBS

paradigm. This approach allowed us to directly address several

key questions on the involvement of MI in STN-DBS and

provided insight into a mechanism of the therapeutic action

of DBS.

Despite the fact that MI is a major target of the basal ganglia

output and therefore likely transforms patterns of pathological

activities into motor symptoms, there were only very few studies

on characterizing the firing rate and patterns of primary motor

cortical neurons in Parkinsonism at the single cell level (Goldberg

et al., 2002; Pasquereau and Turner, 2011). In fact, single-unit

activities from large populations of CxFn in freely moving PD

rats in the resting state and during STN-DBS had not been

achieved before. Our findings showed that there were dramatic

changes in the neuronal activities of CxFn at both single-cell

and the population level. The increased burst discharge and

oscillatory rhythm at the beta range are similar to the hallmark

events found in human and animal models of PD (Wichmann

and Dostrovsky, 2011) and in line with previous studies on

Parkinsonian primates (Goldberg et al., 2004; Pasquereau and

Turner, 2011) and rodents (Sharott et al., 2005). The origin of

these changes in the motor cortex, like that in the basal ganglia

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-Parkin-

sonian 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 theMI ipsilateral to

6-OHDA lesion, which was absent under both intact and unlesioned condi-

tions. 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.


See also Figure S2.

90°

LFP

Intact 6-OHDA lesioned

400μV

2s

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eren

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alue

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hase

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0°

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A

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Intact Unlesioned 6-OHDA lesioned 125Hz STN-DBS

Intact Unlesioned 6-OHDA lesioned 125Hz STN-DBS

0°

30°60°90°120°

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210°240° 270° 300°

330°

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30°60°90°120°

150°

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210°240° 270° 300°

330°

0°

30°60°120°

150°

180°

210°240° 270° 300°

330°

Figure 5. High Frequency STN-DBS

Disrupts the Beta Band Spike Field Coher-

ence 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-Parkinso-

nian rats, and reversed to random distribution

when 125 Hz STN-DBS was delivered.

See also Figure S2.

Neuron


circuit, remains unknown. However, as the output station of the

motor system, these pathological changes in the CxFn likely

contribute to the symptoms in PD. For example, the pathological

enhancement in beta oscillatory rhythm may underlie abnormal

persistence of the status quo and deterioration of behavioral

control (Engel and Fries, 2010). Furthermore, multiple studies

have shown that a critical effect of STN-DBS is the reduction

of the synchronization of oscillatory activities between the basal

ganglia and cortex (Eusebio et al., 2011; Hammond et al., 2007).

Our data confirmed that the spike firing activities of the CxFn

were highly synchronized under PD condition, especially during

burst discharge. These abnormal firing patterns across the

CxFnwere effectively eliminated by STN-DBS. The simultaneous

recording of spikes and LFP by the same recording channel

also allowed us to study the coherence between them. The

results showed that there was increased coherence level

between spikes and local field potentials at beta band only in

the 6-OHDA-lesioned hemisphere. The significance of the in-

creased coherence is unclear, but may contribute to bradykine-

sia and other movement suppression (Brown and Williams,

2005). Beta-band spike-field coherence may also represent

excessive ‘‘stop’’ signals that underlie akinesia in PD (Swann

et al., 2011).

In this study, we also provided direct evidence of the occur-

rence of antidromic spikes in MI during the DBS paradigm.

This finding supports previous studies using electroencephalo-

gram (EEG) recordings that STN-DBS results in the antidromic

activation of motor cortex (Dejean et al., 2009; Li et al., 2007).

In these studies, evoked wave in the EEG was correlated to

the positive behavioral effects. In a recent study (Gradinaru

Neuron 76, 1030–1041, D

et al., 2009), it was found that, while opto-

genetic stimulation of excitatory nerve

terminals within STN was beneficial in

improving Parkinsonian motor symp-

toms, optical inhibition or excitation

confined to STN neurons was ineffective,

raising the possibility that antidromic activation of the cortico-

STN pathway underlies the therapeutic mechanism. Our finding

that the peak antidromic frequency generated coincided with the

optimal effect of STN-DBS also supports this hypothesis. More

importantly, we showed that an antidromic spike had a strong

effect on the firing probability of the neuron immediately fol-

lowing it, and the increased mean firing rate during DBS was

primarily the effect of antidromic spikes. Our results therefore

provide the neurobiological basis of the recent findings that

highlight the importance of cortex in mediating beneficial effect

of STN-DBS. For example, by using the recorded activity to drive

the stimulation, Rosin et al. (2011) showed that short trains of

stimulation pulses were effective only if they were triggered

from cortical activity, but not from the basal ganglia. Mure

et al. (2012) showed that, in PD patients, the improved sequence

learning with STN-DBS, but not with L-3,4-dihydroxyphenylala-

nine (L-DOPA) treatment, was associated with increases in

activity in supplementary and premotor cortices. In human PD

patients, DBS of the internal globus pallidus (GPi) is also effective

in alleviating Parkinsonian symptoms (Weaver et al., 2012).

Whether a similar antidromic activation of the known cortex-

GPi projection (Naito and Kita, 1994) contributes to the thera-

peutic effect of GPi-DBS remains to be studied.

How could antidromic activation ‘‘jam’’ the synchronized

pathological bursting and beta band oscillatory rhythm in

PD? One important observation in the present study is that, as

the generation of the antidromic spike is not robust, a highly

random pattern of antidromic spikes is produced. As the firing

probability of the neuron is modified by an antidromic spike in

a biphasic manner (i.e., inhibition-excitation), the firing rate and

ecember 6, 2012 ª2012 Elsevier Inc. 1037

6-OHDA lesioned

DBS off

On

Off

Off

0

5

10

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0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8

0 1 2 3 4 5 6 7 8 0

5

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Firin

g ra

te (s

pike

s/s)

DBS on, intervals with antidromic spikes

A

DBS on, intervals without antidromic spikes

Unlesioned B

DBS on, no antidromic spikes

DBS off

Firin

g ra

te (s

pike

s/s)

Time (ms)

Time (ms) Time (ms) Time (ms)

Time (ms) Time (ms)

Time (ms) Time (ms)

a b c

a b c

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Off

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Firin

g ra

te (s

pike

s/s)

Firin

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te (s

pike

s/s)

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DBS offffDBS offff

0 1 2 3 4 5 6 7 80

5

0

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a b c

a b c

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 2min 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 0ms. 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 proba-

bilities 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


rhythm of the neuron would be disrupted. We showed that each

antidromically activated CxFn was influenced by a random but

unique train of antidromic spikes that together would serve as

a powerful means to desynchronize their coherent firing.

Breaking of phase relationship among these CxFn could be

a key to this process. Although Wilson et al. (2011) proposes

that a regular stimulus pattern of DBS causes the desynchroniza-

tion, a randomly generated stimulus could also achieve the

same effect. The idea that the local circuit can be affected by

the antidromic spikes is supported by early studies that a late

response was present in cortical cells that were not antidromi-

cally activated (Phillips, 1959; Porter and Sanderson, 1964; Ste-

fanis and Jasper, 1964). There is also recent evidence from

human studies that STN-DBS has a direct effect on intracortical

neurons, modifying the balance between excitation and inhibi-

tion (Fraix et al., 2008). In fact, our data also show that anti-

dromic activation of the CxFn affected the firing of the interneu-

rons (data not shown). While our results would lend support to

the proposition that the cortex could be a therapeutic target in

PD, epidural or subdural stimulation of cortex in human beings

has been a subject of controversy. While some studies demon-

strated promising results for treating PD patients (Benvenuti

et al., 2006; Drouot et al., 2004), others were less supportive

(Kuriakose et al., 2010; Strafella et al., 2007). Similarly, the results

of transcranial magnetic stimulation were mixed (Benninger


et al., 2011; Eggers et al., 2010; Khedr et al., 2006). It is likely

that the efficacy of cortical stimulation is dependent on the

precise changes imposed on the activity of the cortical neurons,

which in turn depends on the means, locations, and parameters

of stimulation.

It should be pointed out that the observed decrease in reli-

ability of antidromic stimulation at high frequency is a nonclas-

sical observation, in contrast to the three well-accepted criteria

of antidromic spikes: fixed latency, collision, and frequency fol-

lowing (Lemon, 1984). A few factors could contribute to this

phenomenon. First, the success of antidromic invasion to the

neuronal soma in well-myelinated fibers is dependent on the

membrane voltage of the soma, as observed by Chomiak and

Hu (2007). They found that there was an overall sharp decrease

in frequency following from �40mV to �60mV within the fre-

quency range of 30–100 Hz. In the in vivo condition, it is likely

that the membrane potential of the neurons is more hyperpolar-

ized than �40mV, and therefore, one would not expect perfect

fidelity in antidromic activation. In fact, if the membrane potential

is a crucial determinant of the occurrence of antidromic spikes

in the soma, the fluctuation in the membrane potential of the

neurons could be a contributing factor to the random pattern

of antidromic spikes that we observed. Second, frequency fol-

lowing is also dependent on the degree of myelination of the

axons (Chomiak and Hu, 2007; Richardson et al., 2000). As far

Neuron


as we know, although the corticofugal fibers are myelinated and

fast conducting, most of the projection to the subthalamic

nucleus are minor collaterals of corticofugal fibers and are of

unmyelinated type (Afsharpour, 1985; Debanne et al., 2011).

Hence, the branch points of the collaterals could serve as low-

pass filter and increase the difficulty of antidromic invasion.

Also, as mentioned before, recruitment of inhibitory cortical

interneurons may contribute to failure of frequency following.

In conclusion, this study provided evidence that STN-DBS

antidromically activates the layer V corticofugal projection neu-

rons in the MI, which contributes to the disruption of abnormal

neural activities in the MI in PD. The unpredictable nature of anti-

dromic spikesmay hold the key to the process, a hypothesis that

needs to be verified.

EXPERIMENTAL PROCEDURES

Animals

Two groups of adult male Sprague Dawley rats weighing 250–280 gwere used,

including 17 intact and 30 hemi-Parkinsonian rats. All animal handling,

surgical, and behavior testing procedures were carried out in accordance

with university guidelines on animal ethics.

Stereotaxic Surgery

A hemi-Parkinsonian rat was generated by unilateral injection of 6-OHDA

into medial forebrain bundle (0.9% saline vehicle injection into the other

side, named as unlesioned). After two weeks’ recovery, contralateral rotation

behavior was tested for 15 min after subcutaneous injection of apomorphine

(0.5 mg/kg) and those that rotated at least 15 cycles/min were selected for

electrode implantation. Two pairs of stimulating electrodes (STABLOHM

675, CA FineWire, Grover Beach, CA) were implanted into bilateral STN (unilat-

eral in intact rats), targeting at the dorsal-lateral portion of the nucleus, which is

known to receive motor input mainly from the MI and is the site of stimulation

that generates the best motor improvement (Greenhouse et al., 2011; Roma-

nelli et al., 2004). Contralateral muscle contraction at low threshold stimulation

was indicative of the possibility that the electrodewas very near or inserted into

the internal capsule and therefore rejected for further experimentations. To

monitor the extracellular neuronal activities in the layer V of MI, two multi-

channel microwire electrode arrays, each constructed of 16 stainless steel

microwires (Plexon, Dallas, TX), were targeted at MI bilaterally (unilateral in

intact rats, ipsilateral to the stimulating electrode implantation side). The

targeted MI area corresponded to the forelimb territory, and correct loca-

tion was confirmed by epidural stimulation-induced forelimb movement. Elec-

trode placement and dopamine depletion level were confirmed histologically

postmortem.

Behavioral Assessment

The locomotor functions of both intact and hemi-Parkinsonian rats were as-

sessed by open field test two weeks after electrode implantation surgery. A

constant current isolated stimulator (Digitimer, Welwyn Garden City, Hertford-

shire, UK) delivered continuous electrical pulses to the STN electrodes at an

intensity below the threshold for induced movement (50–250 mA). The motor

performance of the hemi-Parkinsonian rat before (5 min), during (2 min), and

after (5 min) STN-DBS were compared with the spontaneous exploratory

movement (5 min) of intact rats (ANY-maze 4.70 software; Stoelting,Wood

Dale, IL). The dependence of the efficacies of DBS-STN on stimulation

frequencies (0.2, 1, 5, 10, 50, 125, 200, and 250 Hz) and pulse width (10, 20,

40, 60, 80, and 100 ms) were studied systematically. While the animals were

performing in the open field test, both extracellular spike trains and the local

field potentials (LFPs) in MI were recorded simultaneously using a 32-channel

electrophysiological data acquisition system (OmniPlex system, Plexon,

Dallas, TX).

In the behavioral assessment, muscle contractions in the contralateral face

and limb could be induced when the stimulation site was located at the lateral

N

STN border (confirmed postmortem) or the stimulation amplitude used was

high (>1 mA). Thus, contralateral muscle contraction at low threshold stimula-

tionwas indicative of the possibility that the electrodewas very near or inserted

into the internal capsule and considered unacceptable. For all other cases, the

stimulation value was set below the threshold of visible muscular contraction,

but at which it could bring behavioral improvement.

Statistics

The t tests were performed to compare the motor performance from different

groups. Paired t tests were performed on the data from hemi-Parkinsonian

rats only, comparing the STN-DBS period to both the ‘‘pre’’ and ‘‘post’’

periods. To study the dependence of behavioral improvement on stimulation

frequency and pulse width in hemi-Parkinsonian rats, an additional ANOVA

repeated-measures analysis (stimulus frequency and pulse width as

repeated-measures, respectively) followed by a LSD post hoc test was also

performed. All these behavioral test results are shown as mean ± SEM.

Electrophysiological Analysis

The stimulus artifact removal and single-unit spike-sorting process were per-

formed in the Off-line Spike Sorter V3 workspace (Plexon, Dallas, TX), using

a combination of automatic and manual sorting techniques. Burst discharge

was quantified by the Legendy surprise method. Cross-correlation analysis

was applied to study the synchronization level among CxFn. The oscillatory

rhythm in MI was measured as the spectrum of LFP using fast Fourier trans-

form at 0.2 Hz resolution. When investigating the coherence phase between

the spikes of each CxFn and the simultaneously recorded LFP, the polar histo-

gram was built by filtering the LFP into beta band (11–30 Hz).

Histology and Immunostaining

Coronal sections were cut at the STN (20 mm), MI (20 mm), SNc (20 mm), and

striatum (200 mm) by freezing microtome. Cresyl violet staining at the level of

STN and MI was performed to confirm the targets of the stimulating and

recording electrode. The sections containing the SNc and striatum were pro-

cessed by TH immunohistochemistry. The numbers of TH-positive neurons in

the SNc were counted manually, and the optical intensity of TH-immunoreac-

tivity in the striatum was quantified with Image J software.

SUPPLEMENTAL INFORMATION

Supplemental Information includes five figures and Supplemental Experi-

mental Procedures and can be found with this article online at http://dx.doi.

org/10.1016/j.neuron.2012.09.032.

ACKNOWLEDGMENTS

This work was supported by the Research Grants Council of Hong

Kong (2900336 and 478308), the NSFC/RGC Joint Research Scheme

(30931160433), and the National 973 Program (2011CB510004).

Accepted: September 17, 2012

Published: December 5, 2012

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