www.elsevier.com/locate/brainres

Brain Research 1011 (2004) 135–138

Short communication

Vagus nerve stimulation inhibits harmaline-induced tremor

Scott E. Krahla,b,*, Fredricka C. Martina, Adrian Handfortha,c

aResearch and Development Service, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USAbDivision of Neurosurgery, University of California, Los Angeles, Los Angeles, CA 90025, USAcNeurology Service, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA

Accepted 23 March 2004

Available online 27 April 2004

Abstract

Excessive olivo-cerebellar burst-firing occurs during harmaline-induced tremor. This system receives rich sensory inputs, including

visceral. We hypothesized that electrical vagus nerve stimulation (VNS) would suppress harmaline tremor, as measured with digitized motion

power in the rat. Cervical vagus nerve stimulation suppressed power in the 8–12-Hz tremor range by 40%, whereas sham stimulation was

ineffective. This study raises the possibility that activation of various sensory modalities, as well as visceral, may reduce tremor.

Published by Elsevier B.V.

Theme: Motor systems and sensorimotor integration

Topic: Control of posture and movement

Keywords: Tremor; Harmaline; Essential tremor; Vagus nerve stimulation; Electrical stimulation

Harmaline-induced tremor is an experimental animal

model that shares features with human essential tremor.

Harmaline tremor has a similar frequency to that of essential

tremor, and occurs with posture and kinesis. Harmaline

tremor can be suppressed by drugs utilized clinically for

essential tremor, including beta-adrenoceptor blockers [17]

and benzodiazepines [12]. Both harmaline tremor and essen-

tial tremor are inhibited by ethanol [7,19], and both display

increased energy metabolism in the cerebellum [2,25].

Evidence suggests an abnormality in inferior olive (IO)

function in essential tremor [5]. Harmaline is believed to

induce tremor experimentally via its effects on IO, especial-

ly the medial accessory IO [4]. These cells normally fire at

0.25–2 Hz; harmaline increases this to 4–10 Hz [4,21] and

induces large cell groups to fire in rhythmic hypersynchrony

[14]. Via climbing fibers, rhythmic firing is propagated to

Purkinje neurons, especially in the vermis [4,14], then to the

deep cerebellar nuclei [1], which in turn drive other portions

of the motor system [1], culminating in tremor [23].

0006-8993/$ - see front matter. Published by Elsevier B.V.

doi:10.1016/j.brainres.2004.03.021

* Corresponding author. VA Greater Los Angeles Healthcare System,

Bldg. 114, Suite 217, 11301 Wilshire Boulevard, Los Angeles, CA 90073,

USA. Tel.: +1-310-268-3352; fax: +1-310-268-4811.

E-mail address: scott.krahl@med.va.gov (S.E. Krahl).

Physiological studies have indicated that the IO and

cerebellum are responsive to visceral sensory stimuli

through the splanchnic nerve [18]. IO cell firing rate is also

affected by afferent vagus nerve activation. Electrical vagus

nerve stimulation (VNS) elicits cerebellar-evoked potentials

via IO climbing fibers [10,22].

Because IO neuronal firing rate is affected by vagal

visceral inputs, it may be expected that vagal activation

may disrupt the olivary hypersynchronous rhythmic firing

that underlies harmaline tremor. Accordingly, we hypothe-

sized that VNS would suppress harmaline-induced tremor in

the rat. In this report, we tested this hypothesis utilizing

digital motion quantitation.

Fifteen adult male 300–350 g Long-Evans rats (Harlan,

San Diego, CA) were housed singly with ad libitum food

and water access in a 12/12-h light/dark cycle. Procedures

were approved by the institutional animal care and use

committee, and conformed to the U.S. Animal Welfare Act.

Under ketamine/xylazine (75:15 mg/kg) anaesthesia, the

left cervical vagus nerve was exposed and a cuff electrode

placed around it. Leads were tunneled subcutaneously to a

connector cemented to the dorsal skull [13].

Two days after surgery, each animal’s VNS leads were

connected to a constant current stimulator (A-M Systems,

Fig. 1. Spectral analysis of movement during pre-harmaline baseline (dark

line) and after harmaline administration (light line) in an example rat.

Motion power, which is directly related to mass and acceleration acting on a

strain gauge, and expressed as mV2, was sampled at 0.03-Hz bandwidths

over 10 min. A moving-average smoothing function was applied to

generate interpretable spectra. Data were collected from 0 to 17 Hz. The

increase in motion power between 8 and 12 Hz corresponds to harmaline-

induced tremor.

Fig. 2. Mean motion power (mV2) in the 8- to 12-Hz bandwidth in six

sham- and seven VNS-stimulated rats during pre-harmaline baseline (Base),

harmaline pre-stimulation (Harm), and harmaline sham- or VNS stimulation

treatment (Treat) conditions; each condition lasted 10 min. MeansF S.E.M.

are shown. *p< 0.05, Student’s t-test as compared to harmaline pre-

stimulation condition.

S.E. Krahl et al. / Brain Research 1011 (2004) 135–138136

Everett, WA). Habituation to the tremor monitor chamber

for 20 min preceded data collection. Pre-harmaline baseline

motion activity was collected for 10 min. Tremor was then

induced with harmaline (30 mg/kg, i.p.), and another 10

min of motion data collected. On completion of this

harmaline tremor pre-stimulation condition, VNS or sham

stimulation was initiated. Continuous VNS consisted of

20-Hz, 0.5-ms charge-balanced biphasic pulses delivered at

a 0.5-mA current intensity. Sham-stimulated animals were

connected in an identical fashion, but did not receive

current. Motion data during VNS or sham stimulation

were collected for 10 min.

The stimulation lead was connected to a covered relay

switch that was set by one of the investigators to route

current either to the vagus nerve or to a shunt. The

assignment of animals to VNS or sham stimulation was

randomized, with the order of randomization determined

prior to the experiment. The technician performing the

experiment had no knowledge of the relay setting and was

thus blinded to the treatment condition. At the end of each

experiment, the blinded technician judged whether each

animal received active stimulation, based on observations

of the tremor response.

The tremor monitor chamber consisted of a metal plat-

form resting on a strain gauge (Columbus Instruments,

Columbus OH) surrounded by a Plexiglas cage. The strain

gauge was connected to an electrical amplifier (Grass

Instruments, Quincy, MA) that transmitted data to a com-

puter acquisition system (DataWave Technologies, Boulder,

CO). This system digitized data into spectral power analyses

using the fast Fourier transformation (FFT) method. This

method calculates ‘‘power’’, a function of both the frequen-

cy and force of the rats’ movements within the cage.

Previously reported studies [20,24] and our own prelim-

inary data determined that most harmaline-induced tremor is

expressed within the 8–12-Hz frequency range. Rats not

treated with harmaline express low motion power in this

range. Thus, the total power between 8 and 12 Hz was used

as a measure of tremor severity.

VNS suppresses pentylenetetrazol (PTZ)-induced seiz-

ures in rats [13]. This property was employed to assess the

viability of the surgical preparation in each subject. At least

24 h after the tremor experiment, continuous VNS, using the

same parameters described above, was initiated, and PTZ

administered 30 s later (60 mg/kg, i.p.). VNS was continued

another 15 min, during which the highest seizure severity

attained was scored on a 0–6 rating scale [13]. At least 48

h later, rats were given PTZ without VNS. Tremor data were

discarded from two animals that did not demonstrate a

seizure severity reduction of at least 50% with VNS com-

pared to the no-VNS PTZ seizure condition.

Spectral analysis during pre-harmaline baseline demon-

strated relatively uniform moderate motion power in the 0–

17-Hz range, as depicted by the example in Fig. 1. Harma-

line induced a marked increase in the 8–12-Hz range,

corresponding to observed tremor, and contrasting with the

low power in this range during non-tremor baseline (Fig. 1).

VNS reduced harmaline-associated 8–12 Hz motion

power by 40% ( p < 0.05, Student’s t-test), indicating sup-

pression of harmaline-induced tremor by VNS. In contrast,

S.E. Krahl et al. / Brain Research 1011 (2004) 135–138 137

sham stimulation caused no significant change in 8–12-Hz

motion power, indicating that harmaline-induced tremor was

not changed by sham stimulation (Fig. 2). Sham stimulation

resulted in three out of six rats demonstrating increased

tremor and three showing decreased tremor. Of the seven

rats in the treated group, two had increased, and five had

decreased, tremor following VNS.

Detachment of a lead led to unblinding of the observer to

the condition of one sham-stimulated animal. For the

remaining rats, the blinded observer correctly judged the

treatment condition based on tremor change in five of the

seven VNS- and four of the five sham-stimulated animals

( p < 0.05, Chi-square).

In summary, we tested the hypothesis that VNS sup-

presses tremor in the harmaline model by using a random-

ized parallel-group design, with blinding of the observer to

the treatment condition. Digital quantitation of motion

power in the 8–12-Hz tremor range was analyzed. The

viability of the preparation was validated by checking

whether VNS suppressed PTZ-induced seizures.

These results demonstrate that VNS significantly reduces

harmaline-associated 8–12-Hz motion power. The degree of

reduction, 40%, was comparable to the 50% reduction

reported with different methodology [8]. In addition, this

degree of tremor reduction was comparable to the 50%

reduction of accelerometry-measured tremor in a pilot

feasibility clinical study of VNS for essential tremor [9].

Stimulation of vagal afferents presumably desynchro-

nizes the rhythmic hypersynchronous burst-firing within

the olivo-cerebellar system that underlies harmaline tremor.

The anatomic route by which VNS suppresses tremor is not

known, but several anatomic projection systems are candi-

date pathways. Vagal afferents have been reported to project

directly to IO [6]. The main target of vagal afferents is the

solitary nucleus complex, and most of these projection are

bilateral [11]. The lateral solitary subnucleus reportedly

projects to the medial accessory IO [15], although there is

some disagreement on this point [3]. The nucleus of the

solitary tract may also influence the IO and cerebellum

indirectly via effects on brainstem nuclei. For example,

VNS increases locus coeruleus cell firing [16]. The locus

coeruleus has been found to be essential for the anti-seizure

effect of VNS [13]. Electrical stimulation of the locus

coeruleus suppresses harmaline tremor [26]; thus, VNS

may suppress tremor via this mechanism.

Vagal afferents comprise only a small portion of all

sensory afferents to the olivocerebellar system. The partial

efficacy of VNS in reducing tremor raises the interesting

possibility that activation of other sensory modalities may

be as effective or more effective in suppressing tremor.

Acknowledgements

This study was supported by grants from Cyberonics and

the U.S. Department of Veterans Affairs (AH and SEK). The

authors gratefully acknowledge the technical assistance of

Shayani Senanayake.

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