Circadian fluctuations in pain responsiveness and brain Met-enkephalin-like immunoreactivity in the rat

Pharmacolog~ Bto~hemlstr~ & Behawor, Vol 29, pp 595-599 '~ Pergamon Press plc, 1988 Printed m the U S A

0091-3057/88 $3 00 + 00

Circadian Fluctuations in Pain Responsiveness and Brain Met-Enkephalin-Like Immunoreactivity in the Rat AKEO KEN

KURUMAJI, 1 MIZUO OHI AND KIYOHISA

TAKASHIMA, TAKAHASHI

DlvlSlOn o f M e n t a l Disorder Resear~ h. N a n o n a l Center o f N e u r o l o g y and Psy~ tuatry N a t i o n a l Institute oJ N e u r o s c l e n c e , 4-1-1, Ogawa-htgasht-machl, Kodalra, Tokyo, 187, Japan Received

10 J u n e 1987

KURUMAJI. A , M TAKASHIMA. K OHI AND K TAKAHASHI Ctrtadtanfluctuattons m pam responsiveness and brain Met-enl~ephahn-hl,e tmmunorea~twtt~ m the rat PHARMACOL BIOCHEM BEHAV 29(3) 595-599, 1988--The 24-hour patterns of pare responsiveness and brain Met-enkephahn-hke ,mmunoreactlvlty (MLI) were determined m male W,star rats housed under a 12-hour hght and dark cycle (hghts on from 0700 hr to 1900 hr) A clrcadmn rhythm was observed in latencles to hot plate test (55°C). showing the peak level near the onset of the dark phase (2000 hr) Pretreatment with naloxone (5 mg/kg, subcutaneously) decreased the highest latency (2000 hr), but did not change the lowest latency (1100 hr) In the mesohmb,c area and the stnatum, MLI had a negatwe correlation with the circadian fluctuation m pare sensitivity MLI at 2000 hr was reduced slgmficantly compared to that at 1100 hr m the basal gangha, the frontal cortex and the substantm nlgra These results suggest that the clrcadmn varmUon m hot plate latencles follows a circadian change m the acuv~ty of the endogenous oplmd peptldes system, and that Met-enkephahn may participate m the enhancement of the op~o~d system in the brain Hot plate test

Met-enkephahn

C,rcadmn rhythm

Strmtum

Mesohmb~c area

[2] M o r e o v e r , X u a n et al [14] r e p o r t e d t h a t the lnjecUon o f d - A l a - 2 - L e u - e n k e p h a h n , a n a n a l o g u e o f e n k e p h a l l n , into t h e n u c l e u s a c c u m b e n s o f t h e r a b b i t p r o d u c e s a significant antmoclceptlon In the p r e s e n t s t u d y , we e x a m i n e d 24-hour p a t t e r n s o f l a t e n c l e s to h o t plate t e s t a n d b r a i n M e t - e n k e p h a h n - h k e lmm u n o r e a c t l v l t y ( M L I ) in the rat in o r d e r to clarify the role o f M e t - e n k e p h a h n in t h e c i r c a d i a n r h y t h m in h o t plate latencles

IT h a s b e e n d e m o n s t r a t e d t h a t the r e s p o n s i v e n e s s o f mice to n o c l c e p t l v e stimuli, m e a s u r e d b y the h o t plate test, s h o w s a c l r c a d m n r h y t h m , a n d t h a t t r e a t m e n t w i t h n a l o x o n e dec r e a s e s the h o t plate l a t e n c y d u r i n g the late h o u r s in a d a y w h e n the l a t e n c l e s are n a t u r a l l y high [4] A rise m w h o l e m o u s e b r a i n total oplold levels, d e t e r m i n e d b y m o u s e v a s d e f e r e n s b l o a s s a y , was o b s e r v e d in mice sacrificed m late a f t e r n o o n c o m p a r e d to early m o r n i n g [13] A r e c e p t o r binding s t u d y r e v e a l e d t h a t t h e a m o u n t of specifically b o u n d (~rI) naloxone, an antagonist of oplold peptlde, shows a circadian v a r i a t i o n in t h e h g h t - d a r k cycle, with a p e a k n e a r t h e o n s e t o f the d a r k p h a s e [ l l ] A c c o r d i n g l y , it a s s u m e s t h a t the circadian r h y t h m in pain s e n s i t i v i t y is m e d i a t e d b y e n d o g e n o u s oplold p e p t l d e s H o w e v e r , it is n o t c l e a r w h i c h opiold peptide p a r t i c i p a t e s in t h e c i r c a d i a n v a r m t i o n in pain sensitivity M e t - e n k e p h a h n , o n e o f the e n d o g e n o u s oplold p e p t l d e s , is b e l i e v e d to play a role as a n e u r o t r a n s m l t t e r or n e u r o m o d u l a t o r in the c e n t r a l n e r v o u s s y s t e m . This was d e m o n s t r a t e d b y its p r e s e n c e a n d u m q u e d i s t r i b u t i o n In the b r a i n [7,8], its release f r o m n e r v e t e r m i n a l s following d e p o l a r i z a tion [6], a n d its role in the m o d i f i c a t i o n o f pain p e r c e p t i o n

METHOD

Ammals Male W l s t a r rats (220-300 g) were p u r c h a s e d f r o m Sankyo L a b o S e r v i c e C o r p o r a t i o n (Japan) a n d h o u s e d 4 p e r cage u n d e r c o n t r o l l e d c o n d i t i o n s (24_+1°C t e m p e r a t u r e a n d 50% h u m i d i t y ) w i t h a 12 12 light-dark cycle (lights o n at 0700 hr) a n d free a c c e s s to f o o d a n d w a t e r T h e y w e r e u s e d in the following e x p e r i m e n t s a f t e r a c c h m a t i s a t i o n to the e n v i r o n m e n t for t w o w e e k s

~Requests for reprints should be addressed to A Kuramaj1 at present address Wellcome Surgical Institute, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow G61 IQH, Scotland, U K

595

596

K U R U M A J I , T A K A S H I M A , OHI A N D T A K A H A S H I 10-

Hot Plate Te~t To study a 24-hour pattern of pain sensitivity, eight ammals were tested for hot plate latencies at 3-hour Intervals according to the method of Frederickson et al [4], following five days of handhng and succeeding two days of adaption to the apparatus A metal plate (10×23 cm) was electrically heated and thermostatically controlled at a temperature of 55°C A transparent acrylic resin box (40 cm high and open at the top) served to confine the rat to a defined area of the hot plate The t~me in seconds from contact with the plate until a paw hck or an escape jump was recorded as a hot plate latency In each sampling session, one latency was taken from one animal Dunng the dark period, a dim red light was turned on dunng the test Naloxone hydrochlonde (Sigma) dissolved in physiologIcal saline was g~ven subcutaneously to rats at a dose of 5 mg/kg, 15 minutes prior to the test Control animals were treated with an equal volume of the vehicle

Measurement o f MLI Following one week of handling, animals were decapitated at 3-hour intervals over a 24-hour period In the dark phase, a dim red hght was turned on durmg the procedure The brains were qmckly removed in the cold. and stored at -80°C until biochemical analysis The following areas of the brain were dissected out or punched out from senal frozen coronal slices (600 txm) using a mIcroknffe or stainless steel tube (1 5 or 2 0 mm I d ) as previously described [8] The following co-ordinates (atlas of Paxmos and Watson [12]) were used (+ anterior or - posterior to bregma mm) frontal cortex +4 2 - +2 4, mesollmbic area +2 7 - +0 9. striatum + 1 7 - - 0 7, thalamus - 1 8 - - 3 2, amygdalold nuclei and piriform cortex - 2 3 - - 4 1, substantla nigra - 5 3 - - 4 7 The radlolmmunoassay procedure and antibody specificity for Met-enkephalin were described in the previous paper [8] Brain tissues were heated 90°C in 1 N acetic acid containmg 20 mM hydrochloride for 15 minutes, sonlcated and centrifuged 8,800xg at 4°C for 30 minutes The supernatant fired was then neutralised with sodium hydroxide and radlolmmunoassayed for Met-enkephalin Anti-Met-enkephahn antiserum was produced in rabbits which were immunised with Met-enkephalin conjugated to bovine thyroglobuhn using 1-ethyl-3-dimethylamlnopropyl carbodiimide The antiserum so obtained showed no cross-reaction with Leuenkephalln, c~-, /3-. y-endorphln, /3-hpotropm or c~-neoendorphin Standard or brain extract was incubated with antiserum diluted 2,000-fold and trltlated Metenkephahn (20,000 cpm, 39 3 Cl/mmol, New England Nuclear) m 0 5 ml of 0 05 M phosphate buffer (pH 7 4) conraining 0 25% bovine serum albumin and 0 5% 2-mercaptoethanol The incubation was carried out at 4°C for 2 days The trltlated Met-enkephalln bound to the antibody was separated from the free tntlated Met-enkephalin by adding 0 5 ml of 1% charcoal slurry containing 0 5% dextran, and the radioactivity of an aliquot of the supernatant fluid was measured in a liquid scintillation spectrometer Under our conditions, the minimum detection limit was 0 03 pmoles of Met-enkephahn The serial dilution of the extract in each brain region yielded a displacement parallel w~th that of authentic standards MLI in the pooled extract from the strmtum, analysed by high performance liquid chromatography, coeluted with authentic Met-enkephalin Protein was assayed according to the method of Lowry et al [9]

75A

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25

11

14

17

20

23

2

5

8

Tlme of Day FIG 1 A circadian rhythm In pmn sensitivity in the rat The hot plate test was used to evaluate the pain responsiveness The results were expressed as mean with S E M of data obtained from eight animals Lights were off during the time period marked by the hatched bars *p<0 05, **p<0 01 as compared to 1100 hr of day

Stattstl~ al Analysis Results were expressed as mean with S E.M Data from the hot plate test were analysed by one-way A N O V A and followed by multiple comparison test (Duncan's method) MLI data were analysed using two-way A N O V A (region × time of day) and two-tailed Student's t-test Correlation coefficients of linear regression were calculated according to the method of least squares RESULTS

Hot Plate Te~t Hot plate latencles were found to vary significantly over the 24-hour sampling period, F(7,56)=4 26, p < 0 001 The latencles were low during the early part of the light phase, began to rise 6 hours before the dark onset, and fell during the last 5 hours of the dark phase (Fig 1) Effects of pretreatment with naloxone on the hot plate latenoes are shown in Fig 2 The latency at 2000 hr was decreased by the admimstratlon of naloxone (p <0.01), whde the effect of naloxone was not found at 1100 hr The naloxone-reduced latency at 2000 hr did not differ from the latency at 1100 hr In either the saline-treated rats or the naloxone-treated ones The diurnal difference between 2000

PAIN R E S P O N S E A N D M E T - E N K E P H A L I N R H Y T H M 15

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mesolimbic

area

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Circadian variations in MLI are shown in Fig. 3. A twoway (region x time of day) analysis of variance (ANOVA) yielded a slgmficant effect of region, F(1,95)=474 52, p < 0 01, and effect of time, F(7,95)=2 71, p <0 05, but not of interaction between time and reglon, F(7,95)=0.42 Dunng the hght-dark cycle, M L I showed a circadian variation m the mesohmblc area as well as the strlatum, with a nadir between 1700 hr and 2300 hr. The changes in MLI in both areas were found to be parallel (r=0 822, p < 0 02) The MLI content at 2000 hr was lower than that of MLI in 1100 hr in the frontal cortex and the substantla nlgra, but not in the thalamus or the amygdalold nuclei and plrlform cortex (Table 1). Correlation Coefficient The hot plate latencles over the 24-hour period were found to have a negative correlation with MLI not only in the mesohmbic area ( r = - 0 817, p < 0 02, Fig. 4) but also m the stnatum ( r = - 0 . 7 8 7 , p<0.05)

20

23 of

2

5

8

Day

FIG 3 A clrcadmn varmtlon in Met-enkephahn-hke lmmunoreactlVlty m the mesollmblc area and the strlatum Results are expressed as mean with S E M of data obtained from 6 or 7 animals Lights were off during the time period marked by the hatched bars *p<0 05, **p<0 01 as compared to 1100 hr of day (two-taded Student's t-test)

hr and 1100 hr m the salme-treated animals was statistically slgmficant (p<0 05) MLI

17 Time

20

FIG 2 The effect of naloxone on pam sensttw~ty in the rat Naloxone (5 mg/kg, SC) was rejected 15 minutes before the hot plate test Each column indicates the mean with S E M for the number of animals shown in the columns S sahne, N naloxone, 11 1100 hr of day, 20 2000 hr of day *p<0 05, **p<0 01 as compared to the sahne-treated rats at 2000 hr of day

14

DISCUSSION We demonstrated that the hot plate latencles over a 24hour period show a clrcadmn rhythm, and that treatment with naloxone ls effective at the time of the highest latency, but not the lowest latency These results indicate that the elevation of latencles depends on the activity of the endogenous oplo~d peptldes system As the rhythm of hot plate latencles under constant darkness was found to be the same as that under the hght-dark cycle (data not shown), the latency to the hot plate test exhibited an endogenous rhythm Supporting our findings, Fredenckson et al. reported that the responsiveness of mice to noclcept~ve stimuli, using the hot plate tests, shows a circadian rhythm, and that pretreatment with naloxone (3 mg/kg, SC) slgmficantly reduced the hot plate latencles dunng the late hours (1530 hr to 0430 hr) when the latencles are naturally high [4] Other reports have also assumed that naloxone would be expected to have hyperalgeslc effects only when the oplo~d system is activated

[3,101 Our results concerning the concentration of MLI m the d~screte brain areas were m agreement with the report by Hong et al [7], indicating that the MLI content was high in the basal gangha, whereas it was moderate m the thalamus

598

KURUMAJI,

TABLE 1 DIURNAL DIFFERENCE BETWEEN 1100 HOUR AND 2000 HOUR IN BRAIN MET-ENKEPHALIN-LIKE IMMUNOREACTIVITY

TAKASHIMA,

OHI AND TAKAHASHI

o

~p

Met-Enkephahn-Lke ImmunoreactJvlty (pmol/mg prot ) 1100 hr

2000 hr

0 47 _+ 0 04 1 15 _+ 0 06 I 61 _+ 0 18

0 34 _+ 0 02 1 07 _+ 0 07 1 51 _+ 0 08

lFrontal cortex Thalamus Amygdalold Nuclei and Preform Cortex Substantm mgra

p < 0 02 N S N S

fl. o

-r0 81 _+ 0 02

0 56 _+ 0 05

p < 0 01

Results were expressed as mean with S E M of data obtained from 6 or 7 ammals Statlshcal stgmficance was determined by twotaded Student's t-test

25

Met-enkephalin a n d the a m y g d a l a a n d low in t h e c o r t e x a n d the s u b s t a n t l a nlgra In the m e s o h m b l c a r e a a n d the s t r l a t u m , M L I s h o w e d a c i r c a d i a n f l u c t u a t t o n w i t h m l m m u m c o n t e n t b e t w e e n 1700 h r a n d 2300 h r T h e c o n c e n t r a t i o n of M L I at 2000 h r was f o u n d to b e significantly l o w e r t h a n at 1100 h r in t h e following a r e a s o f t h e b r a i n the b a s a l ganglia, the frontal c o r t e x a n d the s u b s t a n t l a nlgra T h e r e m a y b e t w o f a c t o r s giving rise to the c h a n g e in the b r a i n M L I c o n t e n t O n e f a c t o r is the utilization o f M e t - e n k e p h a h n , a n o t h e r ~s s y n t h e s i s a n d storage T h e c i r c a d i a n v a r i a t i o n in M L I in the b a s a l ganglia was f o u n d to b e a m i r r o r image o f t h a t of pain r e s p o n s i v e n e s s , h a v i n g a high c o r r e l a t i o n c o e f f i c i e n t A t the time o f day w h e n the b r a i n M L I c o n t e n t w a s lowest, the h o t plate l a t e n c l e s w e r e at t h e i r h i g h e s t levels a n d the activity o f t h e oplold p e p t l d e s s y s t e m was e n h a n c e d as s u g g e s t e d b y t h e results o f n a l o x o n e a d m i n i s t r a t i o n In view o f the rapid d e g r a d a t i o n of Mete n k e p h a h n in s y n a p t l c clefts, it is likely t h a t a n i n c r e a s e m the release o f the p e p t l d e r e s u l t e d in the d e c r e a s e in b r a i n M L I c o n t e n t a n d a c t w a t e d the n e u r o n a l acttvlty w h e n the l a t e n c l e s w e r e high Thxs possibility is s u p p o r t e d b y B a y o n et al w h o r e p o r t e d t h a t in VlVO s p o n t a n e o u s r e l e a s e of L e u e n k e p h a h n in t h e g l o b u s p a l h d u s in the rat i n c r e a s e s b e t w e e n n o o n a n d e v e n i n g b y 100% [l] The injection of d-Ala-2-Met-enkephahn, an analogue of M e t - e n k e p h a h n , into the c a u d a t e - p u t a m e n c h a n g e d the motor function, e g, lpslverslve rotahon behavior and bodily a s y m m e t r y [5] X u a n et al has r e c e n t l y r e p o r t e d t h a t M e t - e n k e p h a h n plays a n i m p o r t a n t role in the a n t l n o c l c e p tIon in t h e n u c l e u s a c c u m b e n s o f the rabbit, i n t e r a c t i n g w i t h the s e r o t o n e r g i c n e u r o n [14] T h e p h y s i o l o g i c a l a n d b e h a v Ioral r e s p o n s e s to the h o t plate test, s u c h as n o c l c e p t l o n of h e a t stimuli a n d a p a w h c k or a n e s c a p e j u m p , s e e m to b e

3

35 ( pmol

4 /

mg p r o t

)

FIG 4 The correlation between hot plate latenoes and Metenkephahn-hke lmmunoreactlv~ty m the mesohmb~c area over a 24-hour period A correlahon coefficient of hnear regression was calculated according to the method of least squares ( r = - 0 817, Y = - 2 3 X + 125, p < 0 0 2 N=8)

i n f l u e n c e d by the e n k e p h a h n e r g l c n e u r o n a l activity in the basal g a n g h a O u r findings s e e m to b e c o n t r a d i c t e d b y t h o s e of W e s c h e a n d F r e d e r l c k s o n . w h o r e p o r t e d t h a t a n i n c r e a s e in w h o l e b r a i n total oplold levels, d e t e r m i n e d by m o u s e vas d e f e r e n s b l o a s s a y , is o b s e r v e d in mice scarlficed m late a f t e r n o o n ( w h e n t h e y are least r e s p o n s i v e to pain) c o m p a r e d to early m o r n i n g ( w h e n t h e y are m o s t r e s p o n s i v e to pain) H o w e v e r , t h e y could not find a n y e l e v a t i o n in the levels o f Met- and L e u - e n k e p h a h n m e a s u r e d b y r a d l o i m m u n o a s s a y [13] T h i s d i s c r e p a n c y m~ght be due to the different kind of species, or the m e t h o d of s a m p l e p r e p a r a t i o n o r the specificity o f the a n t i s e r u m for the M e t - e n k e p h a h n a s s a y W e d i s s e c t e d out e a c h rat brain a r e a b y t h e precise p r o c e d u r e d e s c r i b e d in t h e m e t h o d s e c t i o n , w h e r e a s t h e y used the whole m o u s e b r a i n for the b i o a s s a y a n d the r a d l o l m m u n o a s s a y s C o n c e r n i n g t h e specificity of a n t i s e r u m for M e t - e n k e p h a l i n , we c o n f i r m e d it w i t h high p e r f o r m a n c e liquid c h r o m a t o g r a p h y M L I In the p o o l e d e x t r a c t f r o m the s t r l a t u m c o e l u t e d with a u t h e n t i c Met-enkephahn In c o n c l u s i o n , the p r e s e n t p a p e r i n d i c a t e s t h a t the c h a n g e in the n e u r o n a l activity o f b r a i n M e t - e n k e p h a h n m a y play an i m p o r t a n t role m t h e c i r c a d i a n r h y t h m m pain s e n s i t i v i t y o f the rat It will be n e c e s s a r y to d e t e r m i n e b r a i n regions in w h i c h the peptlde r e g u l a t e s the c i r c a d i a n v a r i a t i o n in pain threshold

REFERENCES 1 Bayon, A and B Anton Dmrnal rhythm of the m vlvo release of enkephahn from the globus palhdus of the rat Regul Pept 15 63-70, 1986 2 Belluzz~, J D , N Grant, V Garsky, D Sarantakls. C D Wise and L Stem Analgesia reduced m vlvo by central administration of enkephahn m rat Nature 260: 625-626, 1976

3 EI-Sobky, A , J O Dostrovsky and P D Wall Lack of effect ofnaloxone on pain perception in humans Nature 263" 783-784, 1976 4 Frederlckson, R C A , V Burgls and J D Edwards Hyperalgesla reduced by naloxone follows dmrnal rhythm m responslvlhy to painful stlmuh ~¢ tern e 198: 756-758, 1977

PAIN RESPONSE AND MET-ENKEPHALIN

RHYTHM

5 Geula, C and D Asodounan Asymmetric behavior reduced by enkephalmerglc agents in the basal ganglia Pharrnaco! B l o c h e m B e h a v 23: 207-213, 1985 6 Henderson, G , J Hughes and H W Kosterhtz In VlVOrelease of Leu- and Met-enkephahn from the corpus stnatum N a t u r e 271: 677-679, 1978 7 Hong, J S , H - Y Yang, W Fratta and E Costa Determination of methlonme enkephalm in discrete regions of rat brain Bram R e s 134: 383-386, 1977 8 Kurumaj1, A , M Takashima and H Shibuya Cold and immobilization stress-induced changes in pain responsiveness and brain Met-enkephahn-hke tmmunoreactwlty in the rat Peptldes 8: 355-359, 1987 9 Lowry, O H , N J Rosebrough, A L Farr and R J Randall Protein measurement with the fohn phenol reagent J Btol Chern 193: 265-275, 1951

599

10 Madden, J , IV, H Akll, R L Patrick and J D Barchas Stress-reduced parallel changes in central opiold levels and pare responsiveness in the rat N a t u r e 265: 358-360, 1977 11 Naber, D , A Wlrz-Justice and M S Kafka Circadian rhythm in rat brain opiate receptor N e u r o s u Lett 21: 45-50, 1981 12 Paxlnos, G and C Watson The R a t Bram in S t e r e o t a x w Coordmates Sydney Academic Press, 1982 13 Wesche, D L and R C A Fredenckson Dmrnaldlfferences m op~md pept~de levels correlated with nocicept~ve senslUv~ty Life Sol 24. 1861-1868, 1979 14 Xuan, Y T , Y S Shl, Z F Z h o u a n d J S Han Studles on the mesohmblc loop of antmoclceptlon-II A serotonm-enkephahn interaction in the nucleus accumbens Neurosc~ence 19: 403409, 1986