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neuropsychological investigation. Brain Lang. 61, 123±144 (1998).

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human auditory steady-state responses. Hearing Res. 101, 62±74 (1996).

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sound examples, see http://www.nsi.edu/users/patel/tone_sequences.

Acknowledgements

We thank L. Kurelowech for technical assistance, R. Srinivasan for advice and discussions,

and S. Makeig, M. Kutas, T. Urbach and S. Hillyard for suggestions. This research was

supported by the Neurosciences Research Foundation as part of its research program on

Music and the Brain at The Neurosciences Institute.

Correspondence and requests for materials should be addressed to A.D.P.

(e-mail: apatel@nsi.edu) or E.B. (e-mail: evan@nsi.edu).

.................................................................

Cannabinoids control spasticity and

tremor in amultiple sclerosis model

David Baker * , Gareth Pryce * , J. Ludovic Croxford * , Peter Brown ² ,

Roger G. Pertwee ³ , John W. Huffman § & Lorna Layward k

* Neuroin¯ammation Group, Department of Neurochemistry, Institute of

Neurology, University College London, 1 Wake®eld Street, London WC1N 1PJ

and the Institute of Ophthalmology, UCL, London EC1V 9EL, UK

² The Medical Research Council Human Movement and Balance Unit,

National Hospital for Neurology and Neurosurgery, Queen Square, London,

WC1N 3BG, UK

³ Department of Biomedical Sciences, Institute of Medical Sciences,

University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK

§ Department of Chemistry, Clemson University, Clemson,

South Carolina 29634-1905, USA

k Multiple Sclerosis Society of Great Britain and Northern Ireland, 25 Ef®e Road,

London SW6 1EE, UK

.............................................................................................................................................

Chronic relapsing experimental allergic encephalomyelitis

(CREAE) is an autoimmune model of multiple sclerosis 1 .

Although both these diseases are typi®ed by relapsing-remitting

paralytic episodes, after CREAE induction by sensitization to

myelin antigens 1 Biozzi ABH mice also develop spasticity and

tremor. These symptoms also occur during multiple sclerosis and

are dif®cult to control. This has prompted some patients to ®nd

alternative medicines, and to perceive bene®t from cannabis use 2 .

Although this bene®t has been backed up by small clinical studies,

mainly with non-quanti®able outcomes 3±7 , the value of cannabis

use in multiple sclerosis remains anecdotal. Here we show that

cannabinoid (CB) receptor agonism using R (+)-WIN 55,212, D 9 -

tetrahydrocannabinol, methanandamide and JWH-133 (ref. 8)

quantitatively ameliorated both tremor and spasticity in diseased

mice. The exacerbation of these signs after antagonism of the CB 1

and CB 2 receptors, notably the CB 1 receptor, using SR141716A

and SR144528 (ref. 8) indicate that the endogenous cannabinoid

system may be tonically active in the control of tremor and

spasticity. This provides a rationale for patients' indications of

the therapeutic potential of cannabis in the control of the

symptoms of multiple sclerosis 2 , and provides a means of evaluating

more selective cannabinoids in the future.

High doses of D 9 -tetrahydrocannabinol THC; (the major psychoactive

component of cannabis) can inhibit the development of

CREAE in rodents 9,10 , but this has been attributed to immunosuppression

preventing the conditions that lead to the development

of paralysis, rather than to a direct effect on the paralysis itself 9,10 .

However, the action of cannabinoids on experimental spasticity and

tremor remains uncertain because there have so far been no

behavioural data on the effects of cannabinoids in animal models

relevant to these symptoms of multiple sclerosis.

It is well established that repeated neurological insults occur

during CREAE; these are associated with increasing primary

demyelination and axonal loss in the central nervous system

(CNS) 1 . However, it was also evident that CREAE animals can

develop additional clinical signs, including unilateral or bilateral

fore- and hindlimb tremor (Fig. 1) and hindlimb spasticity (Fig. 2).

These accumulate with disease duration and activity. Tremor was

associated with voluntary limb movements, but in more severe cases

it was persistent at a frequency of , 40 Hz (Fig. 1e). Although

considerably faster than encountered in humans ( , 6 Hz), this

frequency is consistent with tremor electromyography in mutant

spastic ( Glrb Spa ) mice 11 . These animals develop episodes of rapid

tremor and rigidity of the limb and trunk muscles 12 . However,

unlike the Glrb Spa mouse, spasticity in CREAE mice need not be

triggered by sudden disturbance 12 . The effects of cannabis are

mediated through the CB 1 , CB 2 and putative CB 2 -like receptors 13,14 .

CB 1 is predominant in the CNS and is the main target for

psychoactivity, but it is also expressed at lower levels in many

0 10 20 30 40 50 60

Frequency (Hz)

4

2

0

Power (arbitrary units)

6

a b c

e d

Figure 1 Cannabinoid receptor agonism inhibits tremor in autoimmune

encephalomyelitis 1 . Mice with hindlimb ( a , b ) or fore- and hindlimb ( c , d ) tremor both

before ( a , c ) and after ( b , d ) treatment with 5mg kg - 1 i.p. with R (+)-WIN 55,212. e , Power

spectra of hindlimb tremors recorded with the foot suspended above a strain gauge before

(thick line) and after (thin line) 5mg kg - 1 i.p. R (+)-WIN 55,212 injection. Inset, snapshot of

raw record over 0.5 s.

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NATURE | VOL 404 | 2 MARCH 2000 | www.nature.com 85

peripheral tissues. The CB 2 receptor is expressed at high levels on

leucocytes, but there is also evidence for limited CB 2 receptor

expression in mouse brain 13,4 . The administration of a full CB 1

and CB 2 agonist, R (+)-WIN 55,212 (ref. 8), to post-relapse remission

mice resulted in a rapid (within 1±10 min) amelioration of the

frequency and amplitude of tremor in both the fore- and hindlimbs

of CREAE mice (Fig. 1). This was visually evident at 5mg kg - 1

(Fig. 1a±d; n . 10 = 10) and 1mg kg - 1 intraperitoneal (i.p.)

( n . 6 = 6). In addition, D 9 -THC (10 mg kg - 1 intravenous (i.v.))

also ameliorated this response ( n . 5 = 5). Tremor returned within

hours after treatment. As D 9 -THC was observed to be relatively

ineffective when injected intraperitoneally (i.p.), as seen in other

studies 10 , all subsequent compounds were injected intravenously.

Furthermore, as D 9 -THC is a partial CB 1 agonist but provides more

limited CB 2 agonist activity, these results suggest that the effect on

tremor is mainly mediated by the brain CB 1 receptor 8 .

Pretreatment (10 min) of animals with 5mg kg - 1 i.v. of both

selective CB 1 (SR141716A) (ref. 15) and CB 2 (SR144528) (ref. 16)

receptor antagonists eliminated the capacity of 5 mg kg - 1 i.p. R (+)-

WIN 55,212 to inhibit tremor ( n . 5 = 5). However animals with

residual paresis and mild spasticity became signi®cantly more

spastic after such CB receptor antagonism (Fig. 3). This was

associated with uncontrolled leg crossing (Fig. 3c and d) and

severe tail spasms. These showed gross curling which is atypical of

post-remission animals, in which the tail generally hangs limply

(Fig. 3e). Animals also show hindlimb extension (Fig. 3c), including

a signi®cant ( P , 0 : 0001) increase in resistance to ¯exion (Fig. 3a, f).

This was not observed in vehicle-treated controls (Fig. 3a). These

signs were also not evident in similarly injected normal mice

( n . 0 = 5) or normal-appearing pre-acute EAE animals (hindlimb

resistance to ¯exion 0 : 159 6 0 : 013N compared with

0 : 206 6 0 : 022N in treated mice ( n . 12 limbs, P . 0 : 05) and in

animals with paresis/paralysis without evidence of spasticity

( n . 0 = 5 treated with SR141716A and SR144528, n . 0 = 4 treated

with SR141716A or SR144528 alone). When mildly spastic animals

without tremor were injected with 5mg kg - 1 i.v. CB 1 antagonist, not

only did signi®cant hindlimb ( P , 0 : 001; Fig. 3a) and tail spasticity

( n . 18 = 18, P , 0 : 001) develop compared with vehicle treated

controls ( n . 0 = 6), but forelimb tremor also became evident in 3

out of 10 mice. This suggests a role for CB 1 in the control of tremor.

After injection of 5mg kg - 1 i.v. CB 2 antagonist, some animals

( n . 10 = 14) seemed to show a mild increase in tail spasticity

( P , 0 : 02) and showed a small but signi®cant ( P , 0 : 05) increase

in resistance to hindlimb ¯exion (Fig. 3a). However, when the CB 2

antagonist was injected into animals previously made more spastic

( P , 0 : 01) by CB 1 antagonism, spasticity increased signi®cantly

( P , 0 : 001) compared with animals treated with SR141716A alone,

whereas this was resolved in animals treated with vehicle. This

suggests that both CB receptors may control spasticity (Fig. 3f).

However, it is possible that the effects of SR144528 could be

mediated by CB 2 -like (rather than CB 2 ) receptors as previously

proposed 17 , or that at the dose used, SR144528 may have produced

additional CB 1 antagonism because it has some limited capacity to

bind to CB 1 (ref. 8). These observations may indicate the continual

release of endogenous cannabinoid receptor agonists such as

anandamide and 2-arachidonylglycerol which are present within

the brain and exhibit neurotransmitter function 18 . Alternatively, or

in addition, they may re¯ect the presence of precoupled, constitutively

active cannabinoid receptors, as there is evidence that

SR141716A and SR144528 are both inverse agonists that are capable

of producing inverse cannabimimetic effects by reducing the proportion

of cannabinoid recetors that exist in a precoupled state 8,15,16 .

In comparison to some studies in which the antagonists affected the

exogenous agonists 17 , the actions of the antagonists seen here were

relatively short-lived (Fig. 3f). This may re¯ect the fact that the

animals were attempting to compensate for the antagonist effect,

and would be consistent with tonic control of the endogenous

cannabinoid system. These data provide compelling evidence that

CB receptors are involved in the control of spasticity in an

environment of existing neurological damage, and that exogenous

agonism may be bene®cial.

Indeed, in mice with signi®cant spasticity, 5mg kg - 1 i.p. R (+)-

WIN 55,212 reduced severity both visually ( n . 7 = 7; Fig. 3g, h and i)

and after assessment of resistance to hindlimb ¯exion ( P , 0 : 001)

(Fig. 3a and i). This was also evident with 2.5 mg kg - 1 i.p. R (+)-WIN

55,212 (Resistance of ¯exion of both limbs being reduced

( P , 0 : 05) from 0 : 384 6 0 : 096N to 0 : 276 6 0 : 063N, n . 7,

P , 0 : 05). Similar treatment with 5mg kg - 1 i.p. of the inactive

enantiomer S ( - )-WIN 55,212 failed to signi®cantly affect the

spastic resonse (Fig. 3a). In contrast, 10 mg kg - 1 i.v. D 9 -THC and

5mg kg - 1 i.v. methanandamide (CB 1 -selective; K i for CB 1 < 20nM

and K i for CB 2 < 815 nM) 8 induced a signi®cant ( P , 0 : 001)

amelioration in spasticity (Fig. 3g). Coupled with the observations

using SR141716A, this may suggest further that CB 1 is a main target

for control of spasticity. Currently there are no compounds which

are totally CB 1 or CB 2 receptor speci®c, but the lack of effect after

10 mg kg - 1 i.v. cannabidiol (main non-psychoactive component of

cannabis. K i for CB 1 . 4350 nM) 8 suggested a subthreshold dose for

CB 1 stimulation for treatment of spasticity. Using the CB 2 -selective

agonist JWH-133 (1.5 mg kg - 1 i.v. K i for CB 1 < 680nM and K i for

CB 2 < 3nM . 8,19 spasticity was reduced both 10 min ( P , 0 : 05) and

30 min ( P , 0 : 001) after injection at a time when 0.05 mg kg - 1

i.v. (dose selected to exhibit similar CB 1 activity to JWH-133)

methanandamide was not active (Fig. 4). It is possible that sedative

effects may have contributed (though CB 1 receptors) to cannabinoidmediated

effects in these assays, but there was no hypothermia,

indicative of `sedation' after JWH-133 administration (37 : 1 6 0 : 8 C

(baseline), 37 : 2 6 0 : 4 8 C (10 min) 37 : 1 6 0 : 2 8 C (30 min)). That

non-CB 1 receptors may also control spasticity is further indicated

by the transient inhibition of spasticity with the endocannabinoid

palmitoylethanolamide (Fig. 4). This compound has no signi®cant

af®nity for CB 1 but may have activity for CB 2 -like receptors 8 . The

involvement of non-CB 1 receptors may be de®nitively resolved

through the use of CB receptor subtype-speci®c compounds or

CB-receptor-de®cient mice.

Resistance force to flexion of individual hindlimbs (N)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Left Right Left Right Left Right

Non-spastic

remission

Paralysed

relapse

Spastic

remission

0.08 ± 0.01** 0.03 ± 0.01* 0.17 ± 0.10 *,**

Group mean ± s.e.m. resistance to flexion (N)

Leg moved

to full

flexion for

assessment

Spastic Leg

a b

Figure 2 Spasticity develops in autoimmune encephalomyelitis 1 . a , Spastic hindlimb

showing full extension, including the digits. These were pressed against a strain gauge to

measure the force required to bend the leg to full ¯exion. b , Increased resistance to ¯exion

in post-relapse remission animals with spasticity ( n . 12 mice) compared with agematched

mice without evidence of spasticity ( n . 5 mice; asterisk, = P , 0 : 001), or

during active paralytic relapse episodes ( n . 6; two asterisks, = P , 0 : 001).

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86 NATURE | VOL 404 | 2 MARCH 2000 | www.nature.com

Spasticity in patients with multiple sclerosis can be very dif®cult

to control despite the use of oral baclofen, dantrolene, diazepam

and tizanidine, continuous intrathecal baclofen infusion, and selective

injection of botulinum toxin 20 . There is a need for more

effective oral or systemic antispasticity agents. The hydrophobic

nature of cannabinoids allows their rapid access to the CNS.

Although the effects of chronic administration and dose dependency

of CB receptor agonists on experimental spasticity remain to be

investigated further, the data presented here provide evidence for

the rational assessment of cannabinoid derivatives in the control of

spasticity and tremor in multiple sclerosis, in placebo-controlled

trials. The observation that CB 1 appears to be the main therapeutic

target suggests that it may be dif®cult to dissociate the full bene®t

from undesirable psychoactive elements using D 9 -THC or cannabis.

It is also consistent with the unpleasant side effects experienced by

some patients at the doses required for potential therapy by existing

Resistance to flexion of individual limbs (N)

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

R (+)-WIN55,212 S (-)-WIN55,212

CB 1 & CB 2 Agonist

CB 1 & CB 2

Antagonist

Vehicle CB 1

Antagonist

CB 2

Antagonist

0 5 0 5 0 5 0 5 0 10 0 10

Time from injection (min)

0.131 vs 0.154

p<0.001

0.200 vs 0.188

N.S. p>0.05

0.177 vs 0.234

p<0.001

0.165 vs 0.180

p<0.05

0.247 vs 0.174

p<0.001

0.185 vs 0.200

N.S. p>0.05

Group mean resistance to flexion (N) before and after treatment

Mean ± s.e.m. resistance to flexion (N)

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22 CB 2 then CB 1 antagonism

CB 1 then CB 2 antagonism

Baseline After CB 1 or CB 2

antagonism

After Vehicle or

CB 1 and CB 2

antagonism

P< 0.01

P< 0.01 P< 0.001

P< 0.02

P< 0.02

P< 0.001

Time (min)

0 10 20 30 40 50 60

Mean ± s.e.m. resistance to flexion (N)

0.10

0.15

0.20

0.25

0.30

0.35

* *

*

*

R (+)-WIN55, 212 (5mg/kg)

D 9 –THC (10mg/kg)

Cannabidiol (10mg/kg)

Methanandamide (5mg/kg)

*

*

*

a

b c f

g

d e

h i

CB 1 antagonism then Vehicle

Figure 3 Control of spasticity by the cannabinoid system. a , Forces required to ¯ex

individual spastic hindlimbs against a strain gauge before and after injection with vehicle

(0.1 ml i.v., n . 14), SR141716A (5 mg kg - 1 i.v., n . 32), SR144528 (5 kg kg - 1 i.v.,

n . 32), SR141716A and SR144528 ( n . 21 limbs), R (+)-WIN 55,212 (5 mg kg - 1 i.p.,

n . 16) or S ( - )-WIN 55 55,212 (5 mg kg - 1 i.p., n . 19). b ± e , Cannabinoid receptor

antagonism increased spasticity. Before ( b ) and after ( c , e ) SR141716A and SR144528 or

after SR141716A ( d ) administration. c , d , Extension and crossing of limbs; e , spastic tail.

f , Resistance to ¯exion forces 5 min after SR141716A or SR144528 administration.

10 min later, mice were re-injected (5 mg kg - 1 i.v.) with either SR144528 ( n . 10),

vehicle ( n . 15) or SR141716A ( n . 18 limbs) and the resistance to ¯exion assessed

after 5 min. g , Cannabinoid receptor agonism in spastic mice after either R (+)-WIN

55,212 ( n . 16 limbs), D 9 -THC ( n . 18), methanandamide ( n . 23) or cannabidiol

( n . 22). Asterisk, = P , 0 : 001 compared with baseline. h , Spasticity was ameliorated

( i ) by treatment with R (+)-WIN 55,212.

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NATURE | VOL 404 | 2 MARCH 2000 | www.nature.com 87

cannabinoids 3 . The use of selective CB 2 agonists may provide some

symptomatic bene®t without signi®cant psychoactive effects.

Furthermore, it may be possible to upregulate endogenous produced

cannabinoids 18 to mediate therapeutic bene®t. This CREAE

model provides a means of evaluating and controlling the pathophysiology

of spasticity in a chronic in¯ammatory environment

relevant to the control of multiple sclerosis. M

Methods

Induction of CREAE

Biozzi ABH mice, bred at the Institute of Ophthalmology, were injected with 1mg of

mouse spinal cord homogenate emulsi®ed in Freund's complete adjuvant on days 0 and 7

(ref. 1). Animals injected for CREAE, before the onset of acute phase CREAE 1 (usually

occurring 15±20 days post inoculation (p.i.)) were used as normal CREAE controls.

Paralysed CREAE animals were selected during the acute phase or ®rst relapse (typically

occurring 34±45 days p.i.), and remission animals used for the assessment of tremor and

spasticity were used after the second or third relapse 40±80 days p.i.).

Chemicals

R (+)-WIN 55,212, S ( - )-WIN 55,212, D 9 -THC, methanandamide and cannabidiol were

purchased from RBI/Sigma (Poole, UK). Palmitoylethanolamide was purchased from

Tocris Cookson Ltd (Bristol, UK). SR141716A (ref. 15) and SR144528 (ref. 16) were

supplied by M. Mosse and F. Barth (Sano® Research, Montpellier, France). JWH-133

(3-(1 9 1 9 dimethylbutyl)-1-deoxy- D 8 -THC) was synthesised as described 19 . All compounds

were dissolved at 0.5 mg ml - 1 in ethanol containing 1mgml - 1 Tween 80 (Sigma). The

ethanol was removed by vacuum drying, and samples were reconstituted with phosphate

buffered saline to a concentration of 2mgml - 1 . Similar preparations without active drugs

were used as vehicle controls. Suspensions (0.1 ml) were injected either i.v. or i.p. after

CREAE induction.

Assessment of Clinical Signs

Spasticity and tremor were initially assessed by blinded analysis of video recordings.

Digital images were sampled from video at 0.04 s. Signs of tail spasticity (¯icking and

curling) were assessed visually as being either present or absent. Spasticity was con®rmed

by assessing limb spasticity against a small purpose-build strain gauge. Limbs of animals

without clinical evidence of spasticity (propensity to full extend the limb after tension on

the leg) or the propensity to cross were not examined in drug studies. The analogue signal

was ampli®ed and digitally converted using an Amplicon card (Brighton, UK). This was

captured using dacquire V10 software (D. Buckwell,MRC HMBU, Institute of Neurology)

and analysed using Spike 2 software (Cambridge Electronic Design, UK). The hindlimbs

were fully extended twice then moved to full ¯exion against the strain gauge. Each

hindlimb was individually assessed by a blinded operator. The mean of 4±8 individual

readings per limb was taken. Tremor frequency and severity were also recorded by holding

the limb , 5mmabove the strain gauge. Tremor lead to the foot knocking the strain gauge.

The strain gauge output was notch ®ltered at 50 Hz. The device had a resonance frequency

of 95 Hz. The frequency of limb tremor was also con®rmed using a lightweight

unidirectional accelerometer (EGA XT-50, Entrain, UK) mounted over the foot.

Statistical Analysis

Results are expressed as means of individual feet or animals 6 s : e : m : per group. The data

were assessed using either a t -test, paired t -test for ¯exion data or nonparametric Mann±

Whitney U -test using SigmaStat 2.0 software (Jandel Corp, San Rafael, California, USA).

Received 18 August 1999; accepted 20 January 2000.

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20. Noth, J. Trends in the pathophysiology and pharmacotherapy of spasticity. J. Neurol. 238, 131±139

(1991).

Acknowledgements

The authors would like to thank the Multiple Sclerosis Society of Great Britain and

Northern Ireland, the Medical Research Council, the National Institute on Drug Abuse

and the Wellcome Trust for their ®nancial support.

Correspondence and requests for materials should be addressed to D.B.

(e-mail: D.Baker@ion.ucl.ac.uk).

Time (min)

0 10 20 30 40 50 60

Resistance to flexion (N)

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

Methanandamide (0.05 mg kg –1 )

JWH-133 (1.5 mg kg –1 )

Palmitoylethanolamide (10 mg kg –1 )

**

* *

*

**

* *

Figure 4 Treatment of spasticity in autoimmune encephalomyelitis 1 with non-CB 1

receptor agonists. Forces (mean 6 s : e : m : ) required to ¯ex individual spastic hindlimbs

against a strain gauge after i.v. injection with either low-dose methanandamide ( n . 9

limbs), JWH-133 ( n . 9) or palmitoylethanolamide ( n . 14). Asterisk, P , 0 : 05; two

asterisks, P , 0 : 001 compared with baseline.

.................................................................

Light acts directly on organs

and cells in culture to set

the vertebrate circadian clock

David Whitmore * , Nicholas S. Foulkes * & Paolo Sassone-Corsi

Institut de GeÂneÂtique et de Biologie MoleÂculaire et Cellulaire,

CNRS-INSERM-ULP, 1 rue Laurent Fries, 67404 Illkirch CeÂdex,

CU de Strasbourg, France

* These authors contributed equally to this work

.............................................................................................................................................

The expression of clock genes in vertebrates is widespread and not

restricted to classical clock structures 1,2 . The expression of the

Clock gene in zebra®sh shows a strong circadian oscillation in

many tissues in vivo and in culture, showing that endogenous

oscillators exist in peripheral organs 3 . A de®ning feature of

circadian clocks is that they can be set or entrained to local

time, usually by the environmental light±dark cycle 4,5 . An important

question is whether peripheral oscillators are entrained to

local time by signals from central pacemakers such as the eyes or

are themselves directly light-responsive. Here we show that the

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