Pharmacology Biochem*str)_ & Behavior, Vol 20, pp 875-878, 1984 < Ankho International Inc Pnnted m the U S A 0091-3057/84 $3 00 + 00

Spontaneous Motor Activity Increases After Portacaval Anastomosis in Rats'

A L E X C A M P B E L L * , B E N G T J E P P S S O N ? , J. H O W A R D J A M E S ' , V I N C E N Z O Z I P A R O ? A N D J O S E F E. F I S C H E R ?

*Maihnan Research Center. McLean Hospital, Belmont , MA 02178 ?Department o f Sargeo' , Massachuse t t s General Hospi tal and Harvard Medi~ al St hool

Boston, MA 02114

R e c e i v e d 11 J anua ry 1982

CAMPBELL, A , B. JEPPSSON, J H, JAMES, V. ZIPARO AND J E FISCHER Spontaneous motor a( ttvtty ln¢reases after portacaval anastomosis In rats PHARMACOL BIOCHEM BEHAV 20(6) 875-878, 1984--Spontaneous motor activity was monitored in rats at various times, up to 14 weeks, after portacaval anastomos~s (PCA) or sham operauon Total 24 hour activity scores rose slgmficantly after PCA and remained slgmficantly higher than those of sham operated rats for twelve weeks After PCA, activity during the twelve hour hghted penod was greatly increased, whereas dark period activity was unchanged Increased activity m the hght period was found to be well correlated with the degree of elevatmn m brain concentrations of tryptophan, tyrosme, phenylalanlne and glutamme These results are consistent with prevmusly reported EEG studies and suggest that motor-activity measurement may faclhtate the correlation of biochemical changes with behaviour in rats with portal systemic shunting.

Portacaval anastomosis Hepatic encephalopathy Behavlour after portacaval anastomosis

Locomotor activity Serotonln

THE rat with a surgically created portacaval anastomosis (PCA) is widely used as an experimental model of chronic liver disease with portal-systemic shunting of the circulation. The use of this model has permitted detailed study of several neurochemical consequences of portal-systemic shunting, such as altered ammonia metabolism [3], increased brain serotonin turnover [4,5] and altered blood-brain transport of amino acids [10]. In the rat with PCA, behavioral disturb- ances comparable to human portal-systemic encephalopathy are not obvious Tricklebank reported decreased ambulation in an open field after PCA but no change in other motor activities such as grooming or rearing [18]. He also reported positive correlation between ambulation in an open air field and brain tryptophan concentration [19]. Encephalographic studies in rats after PCA have shown significant reduction in sleep-associated electrical activity of cortical and sub- cortical structures [2,15]. In order to identify behavioral ab- normalities which could be correlated with neurochemical changes, we measured the spontaneous locomotor activity of rats, both before and after PCA or sham-operation as well as brain amino acids at sacrifice. We show here that after PCA, spontaneous motor activity is greatly increased during the normal sleep period and this increase parallels the increase in some brain neutral amino acids.

METHOD

Adult male Sprague-Dawley rats weighing 175-225 grams (Charles River Laboratories, Wilmington, MA) were used.

Experimental animals were anaesthetized with ether and a portacaval anastomosis was performed by a non-suture technique in 15 animals [7]. Using clean, but not sterile technique, the abdominal wall was opened on the mldline; the portal vein was exposed, dissected and hgated. The clamped porta vein was then cut free and fed through a small piece of Teflon tubing previously sculptured to form a button of the type used for vascular anastomoses. The vein was everted over the button and secured with a silk ligature; the vein-button assembly was then anastomosed to the partially clamped vena cava with a purse string suture. After removal of the clamps, the patency of the shunt was checked by the rapid return of normal color to the bowel. Ten sham operated controls received laparotomy and exposure and manipula- tion of the splanchnlc viscera for approximately ten minutes (Experiment 1).

Activity was measured using a Stoelting six-channel elec- tronic activity monitor (EAM, Stoelting Company, Chicago, IL). The six activity sensors were calibrated and standard- ized using a rotating bar magnet. The sensors were housed in an electrically shielded and grounded enclosure to eliminate external electromagnetic artifacts, and were spaced at least 50 cm apart to prevent radio frequency coupling. Monitor gains were set at 0.7 mA and activity levels were set at a level which permitted the recording of locomotion plus a wide range of motor activities such as grooming, but not resting activities such as breathing or spontaneous artifactual discharge from the sensor.

'This investigation supported in part by PHS International Research Fenowshlp 1F05 TW 62575-01 (Vmcenzo Zlparo) And by USPH Grants AM-15347 and AM-19124

2Requests for repnnts should be addressed to Bengt Jeppsson, Department of Surgery, University of Lund, S-221 85 Lund, Sweden,

875

876 C A M P B E L L ET AL

The animals were tested individually in plastic cages ~-, ( 4 2 x 2 0 x 15 cm) with food and water freely available Be- ,~ tween exper iments the animals were housed in groups of four in cages, identical to the testcages. Cont inuous activity scores were automatical ly printed at one hour t ime intervals t, 32

over a 24 hour period and the light cycle was the same as the home cages, 07 00 hour, o n - - 1 9 00 hour, off. Preopera t ive ~ ze recording for each animal was made on three consecut ive days. After either PCA or sham operation, 24 hour recordings "- 24

were made at weekly intervals for one month and then at two week intervals thereafter. In addition, at 19 days postopera- '~,0 ao tlon, recording were made for three consecut ive days in five ~. animals from each group selected at random. ,~

In a separate expenmen t (Exper iment 2) spontaneous ~ ~6 motor actwi ty was correlated with brain neutral amino acids '~ 01 A group of 15 rats underwent PCA and their act ivi ty was moni tored as above When, by the tenth week, the act ivi ty of seven of the 15 rats had returned to within two standard devmtions of the normal mean, all 15 rats were sacrificed and plasma and brains taken for amino acid analyses. Free amino acids of plasma and bram of each animal were analyzed using a Beckman 121-MB automatic amino acid analyzer. Plasma was deprote inized by mixing with an equal volume of 7 5% sulfosahcylic acid (SSA) pH 17. containing an Interval standard. The brains were blotted to remove adhering blood and frozen on dry ice. Each brain was homogenized in three 200o volumes (3 x weight) of 5% SSA, pH 1.4 containing an inter- nal standard.

Data were subjected to analysis of vanance and the Newman-Keu l s test. Analys~s of correla tmn was performed according to Pearson product -moment .

R E S U L T S

Food intake after PCA was depressed for 5-7 days, by the tenth day, food intake achieved or surpassed preopera t ive levels and animals had regained their preopera t ive weight. This depression of food intake was much less m sham oper- ated animals. At sacrifice 16 weeks after operatmn m Exper- iment 1, body weight were larger in sham operated animals than in rats with PCA (395_+23 vs. 370_+30). In Exper iment 2 the body weights were at sacrifice ten weeks postoperat ively 330_+ 18 for sham operated rats and 312_+20 for animals with PCA.

After PCA, total 24 hour spontaneous motor activity scores increased significantly ove r preoperat ive scores (Fig. 1 ). The maximum increase was seen at postoperat ive day 28 Thereaf ter , activity dechned slowly until 98 days after the operat ion, after which t ime no difference be tween rats with PCA and sham-operated rats was observed The level of activity after 98 days approximated the preopera t ive scores.

The daily pattern of activity was invest igated in detail m a group of five PCA animals and five sham-operated controls by making cont inuous hourly recordings for 72 hours. Figure 2 shows the daily activity pattern of these two groups from day 19 to day 21, postoperat ively. Each point on the graph represents the mean hourly activity for five animals for three days. During nine consecut ive hourly intervals (9.00 hour to 18 00 hour), the rats with PCA were more act ive. This period of activity contrasted w~th the behavior of the sham-operated rats, which were relatively reactive during this period. Thus in PCA ammals, the normal day/night activity rhythm was lost, since these animals had up to 50% of their total act lvl ty during the hght period. We have expressed this as the ratio of activity during the light period over the total 24 hour activity

EFFECT OF PCA OR SHAM OPERATION ON 24 hr LOCOMOTOR ACTIVITY

e SHAM OP ] o PCA

i II [ I I I I [ I I 14 28 4 2 56 70 B4 98 1t2

P r e - o p OAYS AFTER PCA O,R SHAM OPERATION

FIG I Cumulanve 24 hr motor activity scores of rats before opera- tion and at various times after portacaval anastomos~s or sham op- eration Each point with error bars represents the mean_+S E M of the activity scores often rats (Experiment I ) *p<0 01, **p<0 001 compared to sham operation

~3

O~

k.

1500

1 0 0 0

500

0 1 i I t 8

L,ghtson~ L,ght~ of!

L" C0N,.OLSJ

I I , ~ l l l l t i l l l i l l l l I l J 10 ~2 2 4 6 B 10 t2 2 4 6

NOON M{ONIGHT

T / M E

FIG 2 Hourly (60 mm) activity scores of rats with PCA or sham operanon Each point represents the actlvuy dunng the previous 60 minutes of five rats The acttvtty of each rat was momtored on three consecutive days (day 12 to 21 postoperatlon) and the scores for the same t]me of day ~ere averaged Thus, each point with error bar represents the mean_+S E M of 15 acnwty scores (Experiment I ) *p<0 01, **p<0 001 compared to sham operation

(Fig 3) In the dark (normally active) period, there was no slgmficant difference be tween the two groups.

The 15 rats of the separate exper iment (Exper iment 2) all showed increased total ac t iwty with a maximum increase of the ratio of light activity over total ac t lwty at 24 to 26 days (Fig 3) Thereaf ter , the actzvlty slowly decreased and by the hme of sacrifice at day 70 to 72, seven of the animals had regained normal activity patterns. At this t ime, the light period to total activity ratio correlated well with the brain

M O T O R A C T I V I T Y A F T E R PCA 877

Change in Activity Pattern at Times After PCA

4 0 - ,

35

3o

15

m

± ,SD

I0 / / ' I 2 4 - 2 6 70- 'P2

Days After PCA

FIG. 3 Change m activity pattern at times after PCA m 15 rats used for amino acid analyses (Experiment 2 )

Achv,ty and Brain Amino Aods 70-72 Days After PCA

35

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~-~ 2o 4: :z . ~ .

-41~.

15

I0

"• 200 ~ *

• ~ 150

• ~ I0O

$ TNORMAL ~ ~ [

Achwty

[ ] <22%L/T(n :7) [ ] > 22% L/T(n=8)

i TRY PHE TYR

Brain Amino Aods

FIG 4 Activity and brain amino acids 70 to 72 days after PCA Each bar represents the mean_+S E M of brain aromat.c ammo aods of 15 rats. The black points represent normal brain concentra- tions of tryptophan, phenylalamne and tyrosme from Reference 4. (Expenment 2 ) *p<0 001, **p<0 001, compared to L/T<22%

T A B L E 1

CORRELATION BETWEEN PERCENT LIGHT ACTIVITY/ TOTAL ACTIVITY AND BRAIN AMINO ACIDS

Percent L/T Activity Spearman RHO S.gmflcance

Brain Tryptophan 0 652 p <0.01 Brain Tyrosme 0 739 p<0 01 Brain Phenylalanme 0 877 p<0 001 Brain Glutamine 0 906 p<0 001

concentra t ions of t ryptophan, phenylalanine and tyrosme so that animals with increased acuvi ty had the highest brain concent ra t ions of these amino a o d s (Fig 4, Table 1). The act ivi ty ratio also showed an excel lent correlat ion with brain glutamme.

DISCUSSION

These results demonst ra te clearly that rats with PCA were significantly more act ive than control rats during the light period when rats normally sleep. H o w e v e r , no signifi- cant difference in the act ivi ty of the PCA and control rats was observed m these studies during the dark period, which is normally the t ime of maximum activi ty Monmaur showed a significant decrease during daylight hours both in total du- ration of e lec t roencephalographlc slow wave sleep act ivi ty and of paradoxical sleep activity m rats up to 63 days after PCA [ 15]. This is consis tent with the present results since we observed increased motor act ivi ty ove r a comparable post- operat ive period of time. Since the mcrease in act ivi ty oc- curred only during the normal sleep period, it is likely the increased act ivi ty scores ref lected the increased wakefulness demonst ra ted in encephalographic studies [2,15].

Tr icklebank reported decreased ambulat ion in rats after PCA which is in apparent conflict with the present results [18]. It might, however , not be expec ted that our results should agree with these open-field exper iments Their open-field studies in which ambulat ion was moni tored were of shorter durat ion and might not reveal an increase in total motor act ivi ty: i .e., it is possible that our higher act ivi ty levels are due to an increase in small movemen t s such as eating, head movemen t s and grooming, Besides this, in the study by Tricklebank an Animex act ivi ty meter was used and the activity of paired rats were tested. These differences might well account for the different effects o f PCA on gross activ- ity. Fur thermore , both studies agree in as much as the per- cent of total act ivi ty which occurred during the light period was increased. In our studies, the act ivi ty scores of rats with PCA returned to normal levels by the 14th pos toperaUve week. This was consis tent with the biochemical changes in that the normahzat ion o f activity correla ted well with the normalizat ion of brain neutral amino acids. This may reflect a gradual improvement in hepatic function over time due to increased hepatic arterial blood flow or to es tabhshment of compensa to ry collateral circulat ion from the splanchnic vis- cera to the l iver surface. The 24 hour cumulat ive scores of the shamopera ted group increased shghtly over the 112 day testing period. This was probably due to the normal growth of the animal resulting in mcreased electrical impedance and correspondingly higher act ivi ty scores.

878 C A M P B E L L E T A L

Severa l neurochem~cal abnormal i t i e s have been identif ied m the rat w~th PCA. The inc reased bra in c o n c e n t r a t i o n s of t y ro sme and p h e n y l a l a m n e may resul t m an accumula t i on of so cal led false neu ro t r ansmi t t e r s , e .g. , o c t o p a m m e and B - p h e n y l e t h a n o l a m m e [9] In dogs with P C A and e n c e p h a l o p a t h y deple t ion o f n o r e p m e p h r m e and d o p a m m e has been found in ce reb rosp ina l fired and the brain bes ides the accumula t i on of false n e u r o t r a n s m i t t e r s [6,17]. Bra in t r y p t o p h a n is two to th ree t imes normal in ra ts af ter PCA: s e ro tonm ~s s ignif icant ly e leva ted m the m~dbram and in the medul laJpons region, and brain 5 -HIAA is also s ignif icant ly e leva ted [4] This was also no ted by T r t ck l ebank et al and they found posi t ive cor re la t ion be tween ambula t ion and m- dices of b r am 5 -h yd r oxy t r yp t am l ne me tabo l i sm [19]. The e leva t ion of bra in t y rosme , t r yp t ophan , pheny la lan lne , his- t idme and meth~onme co inc ides w~th m c r e a s e d act iv i ty of the b lood-bra in neut ra l amino acid t r anspo r t sys tem [10].

Bra in a m m o n i a m e t a b o h s m is e n h a n c e d m the ra t with PCA w~th resul t ing accumula t i on in the b ra in of g lu tamlne [3]. Since g lu tamine can compe te with g lu tamate for up take into ne rve endings and be re leased , ~t has been suggested that g lu t amme could func t ion as a false or mact~ve neuro- t r an smi t t e r [1]. Recent ly , h~gb bra in levels of g lu tamine have been sugges ted to raise brain c o n c e n t r a t i o n s of o the r N A A by s t imula t ing exchange t r anspo r t across the b lood-bra in bar r ie r [11] Cereb rosp ina l fluid g lu t amme has p rev ious ly been s h o w n to cor re la te well w~th grade of e n c e p h a l o p a t h y

m pa t ien ts [8], and m th~s s tudy brain g lu tamine toge the r w~th bra in p h e n y l a l a n m e , ty ros ine and t r y p t o p h a n cor re la te well wi th the d i s tu rbance in ac twi ty (Table 1).

P rev ious s tudies have shown tha t a l te ra t ions in s e r o t o n m metabo l i sm can al ter the s ta te of wake fu lness [12,13] How- ever , our resul ts are paradoxica l m that we have s h o w n m- c reased act ivi ty levels at a t ime w h e n brain s e ro tomn is also inc reased We have no exp lana t ion for this contradlct~on H o w e v e r , ra ts with PCA have chronica l ly e l eva ted b ram se ro tonm as well as e leva ted oc toparn ine , g lu t amme and a m m o n m , the in te rac t ion of which may have addi t ional ef- fects on s leep pa t t e rns It ~s of in te res t to note tha t electri- cal ly s t imula t ing the raphe nucle~ of rats at phys~oloDcal f requenc ies increases bra in se ro ton ln and makes the a m m a l s somno len t , howeve r , s t imula t ion of these nucle~ at a fre- quency five to ten t imes th~s ra te p roduces a s ta te of hype ra l e r t ne s s s~mflar to that which fol lows L S D adminis - t ra t ion [13]

Pa t ien t s w~th h v e r c~rrhosis and por ta l - sys temic shunt ing have marked ly e leva ted ce rebrosp ina l fluid t r y p t o p h a n con- centrat~ons [19] and, in the ear ly s tages of hepat ic e n c e p h a l o p a t h y , the sleep pa t t e rns of such pa t ien ts are d~s- tu rbed and the dura t ion of sleep dec reased as well as sens~- twe tests of p s y c h o m o t o r func t ion and v~sual pe rcep t ion [ 14,16] The p resen t resul ts suggest tha t the mo to r act ivi ty of rats w~th PCA could be ut~hzed as a model of sleep and o the r d i s t u rbances in early hepat ic encepha lopa thy

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