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Effects of α-MSH on motivation, vigilance and brain respiration
The Neuropeptides Pharmacology Biochemistry and Behavior. Vol. 5, Suppl. l, pp. 59--64. ANKHO International Inc., 1976.
Effects of =-MSH on Motivation, Vigilance and Brain Respiration J A A K P A N K S E P P . P A T R I C K REII.I,Y, PAL'L BISHOP, RICK B. M E E K E R , T H O M A S R. V I I B E R G
D~7~artment o.f Psychology, Bowling Green State Unil,ersit.l,, Bowling Green, OH 43403 AND ABBA J. KASTIN
l:'ndocrhudogv Secthm o f the Medical Sert'ice, l'eterans AdmhTistration Hospital and Department o f Medicine, Tulane g.#zil'ersitv School o.f :lledicine, A'ew Orleans LA 70122
PANKSEPP. J., P. REILLY, P. BISHOP, R. B. MEFKER, I'. R. VII,BER(; AND A. J. KASTIN. l',~lJ~'ctvo la'-MSH on motivation, r~eilance and hrain re.vpiration. PItARMAC. BIOCIII'*I. BEIIAV. 5: SUPPI.. 1, 59-64, 1976. - In a series of experinlents designed m assess tile effects of a-MStt on ~arit,us motivational processes, it was observed that tile hormone can slightly decrease food intake and increase water consumption during the first hr after administration in rats. a-MSIt also modified avoidance behavior in 1- and 3-day-old chicks, but there were no reliable effects on activity, distress vocalizations and the tonic immobility response, o-MSH appeared to modulate the sleep-waking activity of rats, and tile lllost pronlinent effecl was an increase of slow wave sleep during the 2 3 rd hr after treatment. A possible second effccl WzlS .:1 honlogcnizalioll of sleep patlerns with poor sleepers exhibiting increases of aclivated sleep and good sleeper,; a reduction. Measurement of in ritro brain oxygen consumplion indicated that mice treated with e~-MSH exhibit an 18°,' redt, ction in respiration of the brain stem section which includes the locus coeruleus, bul did not reliahly change respiration in forebrain cortices, c~-MSHalso produced a modest 14'): increase of plasma glucose. These results are discussed in terms of possible modulation by a-MSH of activity in central autononlic cell groups such as the locus coeruleus. a-MSII
Motivation
Vigilance
Sleep
Brain stem
yet materialized. A l t h o u g h it is conceivable that all the behavioral changes may be p r o d u c e d by a unitary underlying change such as increased a t t e n t i o n or certain emotional changes [ 2 0 ] , that is yet to be firmly d e m o n s t r a t e d . The existing evidence does, however, indicate the need for f u r t h e r investigation of the effects o f MSH on basic motivational, e m o t i o n a l and vigilance processes. Accordingly, in the following e x p e r i m e n t s we investigated the effects of this h o r m o n e on a variety o f behaviors and related physiological states.
U N L I K E the p i g m e n t a r y e f f e c t s of m e l a n o c y t e s t i m u l a t i n g h o r m o n e (btSlt) in a m p h i b i a n s , no clear target organ or f u n c t i o n has been f o u n d for the substantial titers o f this h o r m o n e in m a m m a l s . Presently, the most likely possibility is that the brain itself is the ntajor target for the action o f MStl in higher animals. Certainly a large n u m b e r of behaviors can be a f f e c t e d by e x o g e n o u s a d m i n i s t r a t i o n o f the h o r m o n e . E x t i n c t i o n o f b o t h aversive [1] and appetilive tasks [5,22] is increased suggesting that m o t i v a t i o n a l processes which sustain behavioral persistence are intensified. That these effects are not merely due to a decreased capacity to learn new e n v i r o n m e n t a l c o n t i n g e n c i e s is indicated by o b s e r v a t i o n s that rats learn c o m p l e x mazes [2(~] and simple reversal t~,sks [21] faster after MSII than placebo. Surprisingly, however, if the task r e q u i r e m e n t s [24] or relevant cues [18] are c h a n g e d , animals tend to exhibit deficits in reversal learning. MSH also has c o m p l e x e m o t i o n a l e f f e c t s - - increasing dark p r e f e r e n c e and general e m o t i o n a l i t y o f rats u n d e r some c o n d i t i o n s [ 8 , 2 7 ] . t l u m a n studies indicate that MSlt l e n d s to decrease a n x i e t y and increase visual a t t e n t i o n [6, 11, 20]. Still, a definitive f u n c t i o n for MSIt in m a m m a l s has not
EXPERIMEN'I 1: I"EI'IDING AND DRINKING Previous work with appetitive tasks has indicated that hungry rats e x h i b i t delayed e x t i n c t i o n when treated with ~-MSH [22]. This along with the slight increase o f activity e x h i b i t e d by rats after MSH in certain appetitive situations ¢7) may indicate that MSH intensifies hunger in rats. Previous work has failed to note an',' m a j o r e f f e c t s o! ~-MSH on 2 hr food intake o f 22 hr deprived rats, although free water intakes were reliably increased [ 2 2 ] . Similarly. no effect has been observed on daily food intakes o f
"Fhis work was supported by NSH (;rant GB-40150, PHS (;rants AM17157 and NS (17664 and a Research Scientist l)evelopment Award MH-0086 to the senior author. 5q'
6o
P A N K S E P P E'I AL
u n s t a r v e d rats during daily a d m i n i s t r a t i o n of MSH [71. t-lowevcr, if the a p p e t i t e - m o d u l a t o r y effect of 0~-.MSII is to increase h u n g e r over a brief span of time, effects on food intake could easily have been missed in the a b o v e studies. Accordingly, in the following e x p e r i m e n t s wc studied the short- and long-term effects of 0~-MStt on food and w a t e r intake of n o n d e p r i v e d rats. Me th o d Twelve m a t u r e male Long-Evans rats were h o u s e d individually in 14.5 x 7 × 9 cm high wire mcsh cages with free access to Wayne p o w d e r e d l a b o r a t o r y c h o w from spill r e t a r d i n g c o n t a i n e r s and w a t e r from g r a d u a t e d d r i n k i n g tubes. Lighting was o n a 12 hr light-dark cycle and food and w a t e r intakes were r e c o r d e d at 12 hr or s h o r t e r intervals to 0.1 g and 1.0 ml respectively. On the experimental day, all a n i m a l s were injected intrap e r i t o n e a l l y (IP) with 10 ug,,'rat of c¢-MSH ( 1 0 7 ~ / m g ) at the start of the dark and light periods, ltalf the a n i m a l s reclaived the first i n j e c t i o n at the start of each period. 1 o o b t a i n s h o r t - t e r m m e a s u r e s of feeding, i n t a k e s were r e c o r d e d h o u r l y for the first 5 hr of the dark cycle. R e s u l t s ap2d Discussio,1 Daily food and w a t e r intakes are p r e s e n t e d in Table 1. No effect of a-MSH was a p p a r e n t here or in the diurnal d i s t r i b u t i o n s of intakes. The only o b s e r v a t i o n suggestive of a m e a n i n g f u l effect on ingestive b e h a v i o r o c c u r r e d d u r i n g the first hr following I~SH a d m i n i s t r a t i o n , l)uring this h o u r , feeding was reduced 205;- from 1.9 to 1.5 g while w a t e r intake increased 33!; from 3 to 4 ml. A l t h o u g h n e i t h e r o b s e r v a t i o n a l o n e was statistically reliable, clue to the o p p o s i n g t e n d e n c i e s , the f o o d / w a t e r ratio was significantly lower for a n i m a l s receiving oe-MSH ( p < 0 . 0 5 ~ with a n o n p a r a m e t r i c test ( W i l c o x o n ) but not by a p a r a m e t r i c c o m p a r i s o n (t-test). This small difference had d i s a p p e a r e d by the second h o u r of recording. In general, these data c o n f i r m that o~-MSH has no major effect on basic ingestive behavior. Certainly t h e r e was no incrcase in hunger. A c c o r d i n g l y previously r e p o r t e d increases in resistance to e x t i n c t i o n and increased activity in a p p e t i t i v e tasks c a n n o t be ascribed to such a m e c h a n i s m . T h u s it a p p e a r s that a-MSH can be used in learning e x p e r i m e n t s w h i c h e m p l o y food anti w a t e r as r e i n f o r c e r s with little c o n f o u n d i n g by c o n c u r r e n t changes in drive levels.
FIXIJI-I~,IIX,IFIN'I" 2: EMOTIONAl. BEHAVIOR IN Yt)IIN(; (.'lilt'KS Unlike most o t h e r c o m m o n l y used l a b o r a t o r y animals, the y o u n g chick displays a wide variety of s p o n t a n e o u s and easily m e a s u r e d behaviors in response to isolation in an open field. Because MSIt has p r o d u c e d effects w h i c h suggest m o d i f i e d e m o t i o n a l i t y [ 2 4 ] , in the following e x p e r i m e n t we assessed the effect of ct-MSfl on activity. distress vocalization, avoidance b e h a v i o r i, nd tonic i m m o b i l i t y of 1- and 3-day-old chicks. Me th o d In the first e x p e r i m e n t the b e h a v i o r of 22 one- and three-day-old male Leghorn chicks was recorded in a t.~0 x q~0 cm open-field unit ( m a r k e d into t~ equal squares) m a i n t a i n e d at a c o n s t a n t t e m p c r a t u r e of 32 ('. Activity was m e a s u r e d by the n u n t b e r of squares w h i c h the chick entered d u r i n g the 10 rain test session. Distress vocalizations were recorded a u t o m a t i c a l l y by m i c r o p h o n e and a s o u n d activated relay. A v o i d a n c e was m e a s u r e d at the end of the 10 rain o b s e r v a t i o n period by the e x p e r i m e n t e r a p p r o a c h i n g the bird l r o m the f r o n t with an o p e n h a n d and observing the response. A scale from I to 5 was used to rate the a v o i d a n c e response a c c o r d i n g to the following criteria: {1 ) little or no m o v e m e n t away from the h a n d : ~2t slight m o v e m e n t b a c k w a r d from h a n d ( t n a x i m u m of 2 or 3 steps): (3~ c o n t i n u o u s walking or backing away from the a p p r o a c h ing h a n d : ( 4 ) r a p i d walking or r u n n i n g from the h a n d . t5) vigorous u n d i r e c t e d r u n n i n g from the h a n d with wings raised and o f t e n a c c o m p a n i e d by distress vocalizations. E x p e r i m e n t a l animals received 4 ug of 0~-MSll IP 15 rain before testing, and c o n t r o l s received e q u i v o l u m e t r i c doses of the 0.01 M acetic acid .0.t)(:; saline carrie,. In the second e x p e r i m e n t , 14 day old chicks were tested for tonic i m m o b i l i t y after o~-MSIt or vehicle. Initially 2 i n d e p e n d e n t groups of 10 animals were lested, and subseq u e n t l y a n o t h e r l g birds were tested afler b o t h vehicle or MSII (again 4 u g / b i r d ) in a c o u n t e r b a l a n c e d w i t h i n - a n i m a l design, with successive tests being separated by 2 days. 1,11m o b i l i t y testing was c o n d u c t e d in a room m a i n t a i n e d at 24 . I (' on a large table p a r t i t i o n e d into 4 isolated sections. I n d u c t i o n of the i m m o b i l i t y response was a c c o m p a n i e d by restraining the a n i m a l on its back wilt, the h a n d for 15 sec followed by a slow, gentle release. The time t a k e n by the bird to right itself was recorded to the closest sec with a st opwat oh. R~'sttlts atzd Di.~cussiott
TABLE
I
DAILY FOOD AND WATER INTAKES DURING 12 HR INJECTIONS OF CARRIER OR MSH Pre-day Food Intakes (G) Carrier 22.1 ~-+2.2) MSH 21.8t_+.2.5) Water Intakes (MI) CatTier 37 I_+ 4) MSH 36 (_+41 Values are means ± SD.
Treatment Day
Post-Day
20.9 (-+2.41 21.1 t_+2.1)
19.5 (_+3.4) 19.8(2.9)
39 1_+6) 41 t~8)
35 (±6) 36 (.+_9)
In birds tested with the vehicle, m e a n activity scores were 38 and 4 crosscs in 1- and 3-day-old chicks. respectively, indicaling the n o r m a l d e v e l o p m e n t of behavioral i n h i b i t i o n d u r i n g these ages. Activity of animals tested with c¢-MSII d r o p p e d trom 37 to 21 crosses, but the difference at 3 days is not naeaningful since it was due exclusively to the e x t r e m e a g i t a t i o n of a single bird. Distress vocalizations were 872 and 340 in 1- and 3-day-old c o n t r o l birds, anti these values were not reliably different frotn the t,'58 and 312 values of chicks tested with o~-MStl. The average a v o i d a n c e rating of 1 day old MSII teated animals t2.5 ± 1.4) was reliably higher than e x h i b i t e d b,,' c o n t r o l s ( I . 2 + 0.7) It = 1.83, d.l = 10, p < 0 . 0 5 ) . At 3 days of age these effects were reversed, a n i m a l s lesled with o¢-MSI-! having average scores of 1.2 + 0.4 anti c o n t r o l s 3.0 +-
MSH A N D B R A I N R E S P I R A T I O N
61
1.1 (t = 3.38, (t/" = 8, p < 0 . 0 1 ) . The c o n t r o l a n i m a l s e x h i b i t e d a clear m a t u r a t i o n a l increase in a v o i d a n c e w h i c h we have typically o b s e r v e d in chicks. T h e reversal of the effects of ~-MSH w i t h age, t h o u g h o u t w a r d l y confusing, may reflect previously observed i n t e r a c t i o n s of MSII t r e a t m e n t with level o f fear. Since there is a m a r k e d d e v e l o p m e n t of fear d u r i n g the first 3 days of lifc in chicks 115], MSH may facilitate a v o i d a n c e at low levels of fear and suppress aw~idance d u r i n g high levels of fear. "lhis would he a n a l o g o u s to the i n t e r a c t i o n of MSH with shock i n t e n s i t y in a v o i d a n c e s i t u a t i o n s with rats [251. Thc average i m m o h i l i t y of birds tested with ~-MSH in lhe e x p e r i m e n t with i n d e p e n d e n t g r o u p s ',,,'as 376 -+ 306 scc, which was twice as long as the 151 ± C,~l sec of c o n t r o l s , but because of the great variability of this b e h a v i o r the d i f f e r e n c e s were not statistically significant. Similarly. in the w i t h i n - g r o u p e x p e r i m e n t , t h r o u g h the t r e n d was in the same d i r e c t i o n ( 4 5 8 sec for MSH and 4 0 8 sec for c o n t r o l birds), the results were again not reliably different.
p o s t e r i o r to bregma and 2 m m lateral to the midline. A bipolar e l e c t r o d e was placed into the dorsal h i p p o c a m p u s for the m e a s u r e m e n t of t h e t a activity. After at least a wcck of recovery, a n i m a l s were h a b i t u a t e d to sound a t t e n u a t e d sleeping c h a m b e r s (30 × 30 × 30 in.) and a prelimim, ry sleep record was collected from each animal to verify the scorability of records. R e c o r d i n g was d o n e with Narco Biosystems PMP-4B p h y s i o g r a p h at a p a p e r speed of 0.5 cm/sec. Records were visually scored into 3 sleep stages a c c o r d i n g to t r a d i t i o n a l criteria. Waking was c h a r a c t e r i z e d by low voltage d e s y n c h r o n i z e d activity in c o r t e x and h i p p o c a m p u s , slow wave sleep (SWS) was c h a r a c t e r i z e d by a high voltage low f r e q u e n c y activity from b o t h recording sites; activated slecp {AS) was c h a r a c t e r i z e d by d c s y n c h r o nized low voltage activity from the c o r t e x and a sustained regular t h e t a activity from h i p p o c a n l p u s . Before each test session, a n i m a l s were placed in the sleep c h a m b e r s with food and water at the start of t h e i r n o r m a l night cycle. Recording was started 12 hr later at the b e g i n n i n g of the light cycle and c o n t i n u e d for 4 c o n s e c u t i v e hr. Food and water were not available d u r i n g this period. All a n i m a l s were tested in a c o u n t e r b a l a n c e d fashion. Vehicle or o~-MSII ( 1 0 u g / r a t ) were injected IP i m m e d i a t e l y at the start of each recording session. T w o a n i m a l s were tested t h r o u g h 3 successive cycles of vehicle and MSH treatment.
I.:XPI,IRIMI.NT 3: SI. EEP-WAKIN(; PA I"I I.:RNS Since cme of the m a j o r h y p o t h e s e s c o n c c r n i n g a c t i o n of MSH is that a t t e n t i o n a l processes arc m o d i f i e d [6, 11, 2 0 ] , a direct analysis of m o d u l a t i o n by MSH of vigilance states is indicated. Such a line of inquiry is also suggested by the o b s e r v a t i o n of increased slow wave cortical activity d u r i n g the first half hr a f t e r a d m i n i s t r a t i o n of MStt in rats [lt~].
I?,esttlls ,'~lc ttl o d
A l t h o u g h overall 4 h o u r values for the vigilance states were not reliably different b e t w e e n the 2 groups, to highlight somc possible effects i m b e d d e d in these results, h o u r l y data for good and p o o r sleepers have been analyzed separately (Fig. 1). Poor sleepers were a n i m a l s which e x h i b i t e d less t h a n 4!;; AS (n = 4) and good slcepers, more
Ten male a l b i n o rats w e i g h i n g 3 1 1 4 2 5 g w c r e s u r g i c a l l y p r e p a r e d with indwelling e l e c t r o d e s for the clcctroe n c c p h a l o g r a p h i c m e a s u r e m e n t of sleep-waking p a t t e r n s . Cortical activity was m o n i t o r e d via 2 stainless-steel screws t h r e a d c d unilaterally over the c o r t e x 2 m m a n t e r i o r and
SWS
WAKE ,o
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Hours 1'1(,. I. thmrly sleep waking measures after lPadminislrationof 1 0 ~ g / r a t o f M S l l or acidified ,,aline carrier. Animal,~werc divided into -ood sleepers ((;.S.) and poor sleepers ILLS.) accordin,e t~ levels of activated sleep cxhibilcd durm,e the control ScssitIIIS.
62
PANKSI"PP k"F AL.
t h a n 6~7, AS (n = 6) u n d e r the vehicle c o n d i t i o n . F r o m this analysis, it is a p p a r e n t t h a t MStt t e n d e d to stabilize the sleep p a t t e r n of the a n i m a l s - p o o r sleepers sleeping m o r e and good sleepers less. This c o n v e r g e n c e was m o s t a p p a r e n t for a c t i v a t e d sleep - t h a t of good sleepers being r e d u c e d by 29% (t = 5.46, dl" = 5, p < 0 . 0 1 ) and t h a t of p o o r sleepers tripled. T h e increase in AS was due to the d o u b l i n g of episodes as well as 75% increase in average episode length. In good sleepers, the n u m b e r o f AS episodes was decreased by 20% and episode length by 18~)'. The only reliable finding for all a n i m a l s c o m b i n e d was the 157~ increase in slow wave sleep d u r i n g the second and third h o u r s o f r e c o r d i n g (t = 2.6, dr "= 9, p < 0 . 0 5 ) . Again, the p o o r sleepers e x h i b i t e d the larger 27U- increase while good sleepers e x h i b i t e d a 9% i n c r e m e n t . T h e 3 successive r e c o r d i n g cycles o b t a i n e d from 2 a n i m a l s which had been categorized as p o o r sleepers indicated t h a t these effects of MSH were not stable across time. In 1 a n i m a l the successive changes in AS t h r o u g h the 3 cycles of t r e a t m e n t w i t h a-MSII were first a 260U increase in AS, t h e n a 67~ decrease, followed by a 10~¢ decrease. In the o t h e r animal, the values were a 4t.~0~.; increase, a 188~2;, increase with a 5% decrease. The o b s e r v e d increase in SWS also d i m i n i s h e d s y s t e m a t i c a l l y with r e p e a t e d testing. This p a t t e r n of results suggests that the effects of o~-MSH in p o o r sleepers was p r o b a b l y due to some aspect of the testing s e q u e n c e , possibly the initial n o v e l t y of the situation. In general, these results indicate that MS!t has relatively little effect on overall sleep-waking activity in rats. Att h o u g h p o o r sleepers a p p e a r e d to rest m o r e and good sleepers less. it s h o u l d be n o t e d that such a t r e n d c o u h t merely be due to statistical regression t o w a r d the mean. In any case, the results suggest that this h o r m o n e has no p r o f o u n d and c o n s i s t e n t effect on the basic m e c h a n i s m s which c o n t r o l e l e c t r o e n c e p h a l o g r a p h i c indices of vigilance. The effects w h i c h were o b s e r v e d m a y be a s e c o n d a r y c o n s e q u e n c e of a variety of e m o t i o n a l changes that m a y have been p r o d u c e d by MSH. EXPERIMI'NT4: BRAIN RESPIRAI'ION MSH has been d e m o n s t r a t e d to have several physiological effects o n the brain which are c o m p a t i b l e with the n o t i o n t h a t the h o r m o n e might selectively increase the capacity for visual processing t h a t has been observed in h u m a n beings [20] and rats [ 2 1 ] . For instance, MSH can increase the p o w e r o u t p u t of occipital c o r t e x [ 11 ]. Also, MS!.! does not reduce b l o o d flow in occipital c o r t e x as in o t h e r p a r i s o f the brain [3] while at the same time a c c u m u l a t i n g m o r e in the occipital cortex than o t h e r f o r e b r a i n areas [ 8 ] . Because of these findings and the changes in e l e c l r o c o r t i c a l activity o b s e r v e d in the previous e x p e r i m e n t , we d e t e r m i n e d w h e t h e r MSH has any effect on the gross m e t a b o l i c activity of s u b r e g i o n s in the brain as m e a s u r e d by in vitro o x y g e n c o n s u m p t i o n .
Method O x y g e n c o n s u m p t i o n was m e a s u r e d by the polarographic t e c h n i q u e (Yellow Springs I n s t r u m e n t ('o. Model 53 s y s t e m ) w h i c h m e a s u r e s o x y g e n pressure present in a liquid m e d i u m . F o r t y adult male Swiss-Webster mice were used. !fall were injected with 4 u g / m o u s e ofo~-MSH, and half were injected with equal a m o u n t s of the acidified saline m e d i u m . Fifteen m i n u t e s a f t e r i n j e c t i o n a n i m a l s were
d e c a p i t a t e d , brains were r e m o v e d , and 4 8 mg samples ot occipital c o r t e x and frontal c o r t e x (.just dorsal to the rhinal sulcus) were dissected from each brain. Samples of the dorsal brain stem ( i n c l u d i n g the area of the locus coeruleus) (n = 27), d i a p h r a g m (n = 15), and liver (n = 20~ were o b t a i n e d from smaller subsets of animals. Each tissue sample was weighed to 0.1 m g a n d h o m o g e n i z e d in I m l o f freshly aerated mammalian Ringer's solution. The h o m o g e n a t e plus 2 a d d i t i o n a l ml of aerated Ringer's were i n c u b a t e d at a c o n s t a n t t e m p e r a t u r e of 37' ( ' a n d o x y g e n c o n s u m p t i o n was m o n i t o r e d for 30 rain. At this t e m p e r a ture, the Bunsen coefficient of Ringer's s o l u t i o n is 5 ul of o x y g e n / r o t , yielding a total of 15 ul of oxygen in the 3 ml of m e d i u m . In 13 a d d i t i o n a l animals, the frontal and;'or occipital cortices were sampled before any injections, each sample was divided in half, amt 4 ug of MSI! or the equivalent a m o u n t of vehicle were a d d e d to the i n c u b a t i o n m e d i u m s 30 rain after the start of recording. M e a s u r e m e n t of respiration was c o n t i n u e d for a n o t h e r 30 rains. Because of the changes in brain respiration observed in the a b o v e e x p e r i m e n t s , plasma glucose levels were determ i n e d in 11 a'-MSIt treated (4 u g / a n i m a l ) and 10 vehicle treated mice 15 rain after injection. Blood was collected after d e c a p i l a t i o n and glucose levels were measured p o t e n t i o m e t r i c a l l y in triplicale with a Ycllov,' Springs I n s t r u m e n t s (;lucosc A n a l y z e r IYSI Model 23).
Results and Discussion Tissue 02 c o n s u m p t i o n s after in vivo a d m i n i s t r a t i o n of ~-MSH are s u m m a r i z e d in Table 2. ~-MSI-t bad no reliable effects on tissue respiration except for a significanl l g':; r e d u c t i o n in o x y g e n c o n s u m p t i o n of the dorsal brain stem which i n c l u d e d the area locus c o e r u l e u s (t = 2.14, d.l = 35, p < 0 . 0 5 ) . The d i a p h r a g m also e x h i b i t e d a 2Y.; r e d u c t i o n in respiration, but due to the small n u m b e r of samples and the large variance, the t r e n d was not statistically significant. TABLE 2 OXYGEN CONSUMPTION (ug;mg/30 MINI ()F BRAIN AND PERIPHERAl. TISSUES FOLLOWING IP INJECTIONS OF 4 ug,' MOUSE OF MSH OR ACETIC At'It) CARRIER 15 MINUTES BFFORE SACR[FICtTissue Occipital Corlex Frontal Cortex Dorsal Brain Stem Diaphragm Liver
MSH
0.47 0.45 0.23 0.36 0.23
_+ .24 (20) _+ .17 (20) ± .06 ~ (13) ~- .25 {7) = .06 (101
Control 0.46 0.40 0.28 0.47 0.23
+_ .21 120) _+ .11120) ± .05 114) ± .3618) * .(14110)
Values are Means ± SD. Numbers in parentheses indicale number of observalions. *p <0.05. A d m i n i s t r a t i o n of o~-MSH into tile respiring m e d i m of frontal anti occipital tissues had no effecl on respiration. For occipital cortex, the c o n t r o l value was 0.34 ~. 0 . 2 0 j and the ~-MS]-I o n e 0.33 ~± 0.20) ug 0 2 / r a g wet tissue/30 rain. The c o r r e s p o n d i n g values for frontal cortex were 0.23 (± 0.05) and 0.26 (± 0 . 0 6 ) u g 0 2 / r a g wet tissue/30 rain. Essentially these results indicate that o~-MS]! has ','cry little effect on energy m e t a b o l i s m of the brain. The
MSH A N D BRAIN RESPIRATION r e d u c t i o n of dorsal brain stem r e s p i r a t i o n was p r o b a b l y s e c o n d a r y to changes in b l o o d flow because it was n o t e d d u r i n g tissue dissection t h a t this region as well as the brain generally was m a r k e d l y b l a n c h e d in a n i m a l s t r e a t e d with oe-MSlt. This p r o b a b l y c o r r e s p o n d s to the r e d u c e d b l o o d flow that has b e e n r e p o r t e d for m a n y regions of the brain following o~-MSI-I t r e a t m e n t [ 3 ] . Of course, if this were the only physiological change that resulted from the o~-MSH, o t h e r parts of the brain should have also e x h i b i t e d some r e d u c t i o n in respiration. In fact, the f r o n t a l c o r t e x exh i b i t e d a 12% ( t h o u g h not significant) increase in o x y g e n c o n s u m p t i o n after t r e a t m e n t with o~-MSH. Since this h e i g h t e n e d r e s p i r a t i o n in the p r e s e n c e of decreased b l o o d flow m i g h t have o c c u r r e d if increased s u b s t r a t e s had been m a d e available to the brain we decided to measure plasma gh, cose levels after 0¢-MSH. The results i n d i c a t e d t h a t glucose levels were 14'7~ higher in a n i m a l s t r e a t e d with a~-MSIt t h a n in c o n t r o l s t 143 mgU vs 126 mg%) (t = 1.9, df = I~,L .o<0.05). Since in p r e v i o u s work it has been d e m o n s t r a t e d that e x o g e n o u s a d m i n i s t r a t i o n of glucose can increase the r e s p i r a t o r y activity o f brain tissue [ 1 4 ] , it is suggested that r e d u c e d f o r e b r a i n b l o o d flow may have been c o u n t e r a c t e d by increased e n d o g e n o u s m e t a b o l i c s u b s t r a t e availability.
GENERAL DISCUSSION The p r e s e n t results do not lead to an u n a m b i g u o u s u n i t a r y h y p o t h e s i s a b o u t thc role of o~-MStt in the f u n c t i o n i n g of higher animals. All of the positive effects observed in the prcsent e x p e r i m e n t s were weak and in sonle i n s t a n c e s e p h e m e r a l . However, 4 positivc o b s e r v a t i o n s were made, and they may be of some i m p o r t a n c e in d e l i n e a t i n g the n o r m a l biological effect of MSH in n o n a m p i b i a n s . First, the a v o i d a n c e b e h a v i o r o1 y o u n g chicks to an a p p r o a c h i n g object was increased at 1 day of age and decreased at 3 days of age. This finding s u p p o r t s tire c o n s i d e r a b l e evidence linking o~-MStl to the m o d u l a t i o n of fear m o t i v a t e d behaviors [ 1 , 9 ] , and f u r t h e r suggests t h a t the type of effect one observes m a y be d e p e n d e n t on the level of fear. Since e m o t i o n a l b e h a v i o r s of the y o u n g chick are d e v e l o p i n g rapidly d u r i n g the first few days of life, the reversal of the effect of o~-MSI-t m a y not be as p r o b l e m a t i c as it m a y initially seem. If the vigor o f a v o i d a n c e in the chick is an inverted U shaped f u n c t i o n with increasing age, then MSH might shift the a n i m a l up on the curve at a y o u n g age and d o w n at an older age. This result would also be e x p e c t e d if ~-MSI-t i n t e r a c t s with f e a r - m o t i v a t i o n in such a way that it i m p r o v e s behaviors induced by low levcls of fear anti reduces those p r o v o k e d by high levels. Indeed, this kind of i n t e r a c t i o n has been observed in rats w h e n i n t e n s i t y of shock is m a n i p u l a t e d at low shock intensities MSI-! facilitates a v o i d a n c e and at high levels a v o i d a n c e is rcduced
[25t. Second, oe-MSIt a p p e a r s to have subtle effects o n vigilance as m e a s u r e d by c l c c t r o e n c e p h a l o g r a p h i c indices of sleep-waking activity. Slow wave sleep is increased d u r i n g the s e c o n d and third hr a f t e r h o r m o n e a d m i n i s t r a t i o n . This o b s e r v a t i o n is c o m p a t i b l e with early r e p o r t s that some rats tested with /3-MSII e x h i b i t e d behavioral s o m n o l e n c e [17] and also possibly with the high incidence of y a w n i n g and s t r e t c h i n g observed in dogs a f t e r MSH a d m i n i s t r a t i o n 121. This finding m a y also bc c o m p a t i b l e with recent observations that o~-MStl increases the t u r n o v e r of brain norcpi-
63 n e p h r i n e d u r i n g the first hr a f t e r a d m i n i s t r a t i o n followed by a decreased t u r n o v e r for several h o u r s [ 1 0 ] . It n o r e p i n e p h r m e subserves cortical arousal [ 4 , 1 2 ] , a decrease in n o r e p i n e p h r i n c t u r n o v e r m a y have p r o m o t e d the a p p e a r a n c e o f slow wave sleep. If this were the case, we s h o u l d also have observed a period o f increased arousal d u r i n g the b e g i n n i n g of testing. Ilowever, we n o t e d no such effect. The average latency to slow wave sleep in c o n t r o l a n i m a l s was 30.3 min and in animals t r e a t e d with a - M S l t il was 25.7 rain. In fact, 8 of 10 rats studied e n t e r e d slow wave sleep faster a f t e r MSH t h a n after vehicle. ( ' o n v e r s e l y , 8 of the 10 a n i m a l s t o o k longer to e n t e r activated sleep after ~-MStt t h a n vehicle. It might be n o t e d that some a n i m a l s did tend to e x h i b i t initial sleep p a t t e r n s which may have been c h a r a c t e r i z e d by atypically rapid cycling b e l w e e n slow wave sleep and waking, as if sleep were being d i s r u p t e d by an arousal process, but this o b s e r v a t i o n was not at all c o n s i s t e n t across animals. In fact, hall" the a n i m a l s e x h i b i t e d larger average episode lengths of slow wave sleep t h a n controls. A n o t h e r possible effect of a'-MSH was a r e d u c t i o n in the variability of sleep stages across animals, anti this effect was most p r o m i n e n t for activated sleep. This suggests that o~-MSII may have a stabilizing effect on a Brain m e c h a n i s m which c o n t r o l s e l e c t r o c o r t i c a l arousal. This h o m o g e n i z a t i o n of e l e c t r o c o r t i c a l activity may also be n o t e w o r t h y from the perspective that 0~-MStt has previously been n o t e d to reduce the variability of o t h e r behaviors, for instance, the n u m b e r of errors rats m a k e in learning a c o m p l e x maze [ 2 6 ] . ( ' e r t a i n l y it is possible that t r e a t m e n t with o~-MStl stabilizes a physiological s u b s t r a l e which n o r m a l l y e x h i b i t s e i t h e r s u b s t a n t i a l individual or l e m p o r a l variability. Our third m a j o r o b s e r v a t i o n was that MSH decreased in vitro respiration of the dorsal brain stem. This effect was p r o b a b l y due to decreased blood flow to that region, not only because the a n i m a l s treated with ~-MSIt e x h i b i t e d a visually o b v i o u s b l a n c h i n g of brain tissue, but also because MSII has been d e m o n s t r a t e d to reduce blood flow to m a n y regions o f the brain 13]. The fact that respiration was not decreased anti may, in fact, have been increased in f o r e b r a i n areas such as f r o n t a l and occipital cortices, suggested that a n y m e t a b o l i c effects o f reduced blood llow was being h o m e o s t a t i c a l l y c o u n t e r a c t e d by an increase ira energy availability. This idea p r o v o k e d our f o u r t h o b s e r v a t i o n , n a m e l y that o:-MSH p r o d u c e d a reliable t h o u g h m o d e s t increase in blood glucose levels of n o n f a s l c d mice. W h e t h e r this effect was due to a direct a c t i o n on the liver as o p p o s e d to some s e c o n d a r y h o r n l o n a l c h a n g e such as increased secretion of e p i n e p h r i n e , glucagon, or g l u c o c o r t i c o i d s or decreased insulin, c a n n o t be ascertained from the present data. Certainly, from the perspective that MSII is recruited in response to b o t h physical and psychological stressors [ 2 3 ] , a b l o o d glucose increase can be seen to be an adaptive r e s p o n s e in p r e p a r i n g the brain and b o d y to cope with a stressful situation. Still, it should be e m p h a s i z e d lhat the h y p e r g l y c e m i c effect ofo~-MSIl was very small, but it may explain why we observed a slight r e d u c t i o n in feeding and an increase of d r i n k i n g in our first e x p e r i m e n t . The increased awtilability of m e t a b o l i c s u b s t r a t e s may have depressed a p p e t i t e while any increase in b l o o d t o n i c i t y may have i n d u c e d the animal to drink a little more. Also, it is c o n c e i v a b l e that the m e t a b o l i c changes we observett a f t e r 0¢-MStI may have p a r t i c i p a t e d in some of the m o d i f i c a t i o n in sleep waking p a t t e r n s since it is clear that changes in
64
P A N K S E P P ET AL.
glucose m e t a b o l i s m can m o d i f y vigihmce states quite m a r k e d l y [ 1 3]. A l t h o u g h the p r e s e n t results do not provide a conclusive m e c h a n i s m for MSH action, it should be n o t e d that the results are c o n s i s t e n t with the possibility that MSll may have as target organ certain p i g m e n t e d cells of the central a u t o n o m i c nervous system. For instance, it has recently been d e m o n s t r a t e d thal the locus coeruleus c o n t r o l s cerebral b l o o d f l o w [161, and it was in the brain sample c o n t a i n i n g this area that a reliable r e d u c t i o n of tissue respiration was observed. F u r t h e r , since this part of the brain is k n o w n to participate in organizing stress related responses and states of vigilance 14], the changes in sleep-waking activity and e m o t i o n a l behaviors in chicks could have been m e d i a t e d by changes in the activity o f p i g m e n t e d brain stem cell groups such as the locus coeruleus. A l t h o u g h speculative at this stage, the h y p o t h e s i s that MSH may m o d u l a t e central a u t o n o m i c tone could provide a unifa'ing principle which might a c c o u n t for the m a n y e f f e c t s o f MSII that have been described in the literature and the wide variety o f e f f e c t s which were observed in the
present series of e x p e r i m e n t s . If MSH does m o d i f y auton o m i c t o n e in the nervous system, it would be e x p e c t c d that a large variety of behaviors would be a f f e c t e d , but not necessarily to any great e x t e n t , and the hehavioral literature does indicate that the e f f e c t s o f MSH are widespread t h o u g h o f t e n quite weak and sensitive to slight environmental and task changes. Also, from such an a u t o n o m i c action, it would be reasonable thal the behavior of animals in m a n y situations would be stabilized rather than shifted p r o f o u n d l y in any unitary direction, and again it has been occassionally n o t e d that MSH t e n d s to reduce the variability in behavior. F u r t h e r , since rcccnt work suggests that an increased a t t e n t i o n h y p o t h e s i s of MSIt aclion may bc the best general principle available for organizing the diverse behavioral e f f e c t s of the l m r t n o n e [11,201, a r e a s o n a b l e lllode (11' aciion for such it process could be via brain stem cell groups such as the locus coeruleus which have been intplicated in lhe basic m a i n t e n a n c e of corlical arottsal. Finally, a most c o m p e l l i n g fact which is leading us to analyze the role o f such brain areas in MSIt action is their p r o m i n e n t p i g m e n t a t i o n and hence lheir o u t w a r d r e s e m b l a n c e to the epidermal target tissue o f a m p h i h i a n s .
REFERENCES I. de Wied, D. Effects of peptidc hormones in behavior. In: Frontiers #z Neuroen&wrinologv. New York: Oxford University Press, 97-140, 1969. 2. Ferrari, W., G. L. Gessa and L. Vargiu. Strctching activity in dogs intracisternally injected with synthetic melanocytestimulating hexapeptide, k:vperientia 17: 90, 1961. 3. Goldman, H., C. A. Sandman, A. J. Kastin and S. Murphy. MSH affects regional perfusion of the brain. Pharmac. Biochem. Behav. 3 : 6 6 1 - 6 6 4 , 1975. 4. Jouvet, M. "l]le role of monomaines and acelylcholinecontaining neurons in the regt, lation of the sleep-waking cycle. Ergeh. Phy.~'ioL 64: 165--3(17, 1972. 5. Kastin, A. J., G. L. Dempsey, B. LeBhmc, K. Dyster-Aas anti A. V. Schally. Extinction of an appetitive operant response after administration of MStl. Herin. Behav. 5 : 1 3 5 - 139, 1974. 6. Kastin, A. J., L. H. Miller, D. Conzalez-Barcena, W. D. Hawley, K. Dystcr-Aas, A. V. Schall.v, M. L. V. DeParra and M. Velasco. Psycho-physiologic correlates of MSH activity in man. Phv.viol. Behar. 7:893 896, 1971. 7. Kastin, A. J., M. C. Miller, L. Ferrell and A. V. Schally. General activity in intact and hypophysectomized rats after administration of melanocyte-stinmlating hormone (MStt), melantonin, and Pro-Leu-Gly, Nil:. Phys#~l. Behav. tO: 339-401, 1973. 8. Kastin, A. J., C. Nissen, K. Nikolics, K. Medzihradszky, D. t1. Coy, I. l'eplan and A. V. Schall.v. Distribution of ~II-c,-MSIt in rat hrain. Brain Rc~. Bull. I: 19 26, 1976. 9. Kastin, A. J., G. A. Sandman, L. O. Stration, A. V. Schally and L. |t. Miller. Behavioral and Electroenccphalagraphic changes in rats and man after MSH. In: Progres'~" #z Brain Rex'earth. l'oL 42, Amsterdam: Elsevier, 1975, pp. 143 15(I. 10. Kostrzewa, R. M.. A. J. Kastin and MI S. Spirtes. c,-MSH and .MI1--I effects of catecholamine levels ~,nd synthesis in various rat brain areas. Pharmac. Biochcm. Behav. 3: 1017-1023, 1975. I 1. Miller, I. I1., A. J. Kastin, C. A. Sandman, M. Fink and W. J. Van Veen. Polypeptide influences on attention, memory and anxiety in man. Pharmac. Biochem. Behav. 2: 663-668, 1974. 12. Panksepp, J., J. E. Jalowiec, P. J. Morgane, A. J. Zolovick and W. ('. Stern. Noradrenergic pathways and sleep-waking states in cats. Evp/Neuro/. 41:233 245, 1973. 13. Panksepp, J., J. E. Jalowiec, A. J. Zolovick, W. ('. Stern, and P. J. Morgane. Inhihition of glycolytic metabolism and sleepwaking states in cats. Pharmac. Biochem. Behav. I: 117 119, 1973.
14. t'anksepp, J. and P. Reilly. Median and lateral hyl~othalamic oxygen consumption as a function of age, starvation and glucose administration in rats. Brain Re.~'. 94:133 140, 1975. 15. Phillips, R. 1-. and P. B. Siegel. Development of fear in chicks of two closely related genetic lines. Animal Behav. 14:84 88, 1966. 16. P,aichle, M. I., B. K. Ilartman..I.O. l'iichling and 1.. (;. Sharpc. ('entral noradrenereic regulation of cerebral blo(~d llov~ and vascular permeability. Prec. hath...h'ad. SoL 72:3726 3730. 1975. 17. Sakanloli. A. llypersensilivily induced In albirlo nlice by melanocyte-stimulating hormone. Nature 2l l: 1370 - 1371, 1966. 18. Sandman, ('. A.. W. Beckwith, M. hi. (;iddis and A. J. Kastin. \lelanocyte-stimulating hormone (MSH) and overtraining effects on extradimensional shift (El)S) learning. Physiol. Behar. 13: 163- 166, 1974. 19. Sandman, ('. A., P. M. l)enman, L. H. Miller, J. R. Knott. A. V. Schally and A. J. Kastin. I-lectroencephalographic measures el melanocyte-stimulaling hormone activity..I, temp. physiol. P.~Tchol. 76:103 109, 1971. 20. Sandman, C. A., J. M. George, J. D. Nolan, l.l. Van Riezen and A. J. Kastin. t'nhancen~ent of attention in m,m with AC'I'H.z MSIt 4 10. Ph.vs'iol. Behar. 15:427 431, 1975. 21. Sandman, C. A., A. J. Kastin and W. 1). Alexander. Net,reendocrine intquences on visual discrimination and reversal learning in tile albino and hooded ral. Phv.siol. Bchav. It: 613 617, 1973. 22. Sandman, ('. A., A. J. Kastin and A. V. Schally. Melanocytestimulatine hormone and learned appetitive behavior. Ek'perient& 25: lll(ll 111112,1969. 23. Sandman, ('. A., A. J. Kastin, A. V. Schally, J. W. Kendall and 1.. H. Miller. Neuroendocrinc responses to physical and psychological stress. J. temp. physiol, lZs'vchol. 84: 386-390, 1973. 24. Stratton, [.. O. and A. J. Kastin. Melanocytc-stimulafing hormone ill learning and extinction of two problems. Phvx'io[. Behal'. tO: 689-692, 1973. 25. Stralt(m, L. O. and A. J. Kaslin. Avoidance learning at two levels of shock in rats receiving MStl. tlormones Behav. 5: 149-.155, 1974. 26. Stratton, L. O. and A..I. Kastin. Increased acquisition uf a complex appetitive task :filer MStl and MII'. Pharmac. Bi(whem. Rehar. 3:901 9(14. 1975. 27. Stratton, I. O., A. J. Kastin and W. p. Coleman. Activity and dark-preference responses of albino and hooded rats receiving MSII. Physiol. Behav. l l: 9(17 .qo9, 1973.
Effects of =-MSH on Motivation, Vigilance and Brain Respiration J A A K P A N K S E P P . P A T R I C K REII.I,Y, PAL'L BISHOP, RICK B. M E E K E R , T H O M A S R. V I I B E R G
D~7~artment o.f Psychology, Bowling Green State Unil,ersit.l,, Bowling Green, OH 43403 AND ABBA J. KASTIN
l:'ndocrhudogv Secthm o f the Medical Sert'ice, l'eterans AdmhTistration Hospital and Department o f Medicine, Tulane g.#zil'ersitv School o.f :lledicine, A'ew Orleans LA 70122
PANKSEPP. J., P. REILLY, P. BISHOP, R. B. MEFKER, I'. R. VII,BER(; AND A. J. KASTIN. l',~lJ~'ctvo la'-MSH on motivation, r~eilance and hrain re.vpiration. PItARMAC. BIOCIII'*I. BEIIAV. 5: SUPPI.. 1, 59-64, 1976. - In a series of experinlents designed m assess tile effects of a-MStt on ~arit,us motivational processes, it was observed that tile hormone can slightly decrease food intake and increase water consumption during the first hr after administration in rats. a-MSIt also modified avoidance behavior in 1- and 3-day-old chicks, but there were no reliable effects on activity, distress vocalizations and the tonic immobility response, o-MSH appeared to modulate the sleep-waking activity of rats, and tile lllost pronlinent effecl was an increase of slow wave sleep during the 2 3 rd hr after treatment. A possible second effccl WzlS .:1 honlogcnizalioll of sleep patlerns with poor sleepers exhibiting increases of aclivated sleep and good sleeper,; a reduction. Measurement of in ritro brain oxygen consumplion indicated that mice treated with e~-MSH exhibit an 18°,' redt, ction in respiration of the brain stem section which includes the locus coeruleus, bul did not reliahly change respiration in forebrain cortices, c~-MSHalso produced a modest 14'): increase of plasma glucose. These results are discussed in terms of possible modulation by a-MSH of activity in central autononlic cell groups such as the locus coeruleus. a-MSII
Motivation
Vigilance
Sleep
Brain stem
yet materialized. A l t h o u g h it is conceivable that all the behavioral changes may be p r o d u c e d by a unitary underlying change such as increased a t t e n t i o n or certain emotional changes [ 2 0 ] , that is yet to be firmly d e m o n s t r a t e d . The existing evidence does, however, indicate the need for f u r t h e r investigation of the effects o f MSH on basic motivational, e m o t i o n a l and vigilance processes. Accordingly, in the following e x p e r i m e n t s we investigated the effects of this h o r m o n e on a variety o f behaviors and related physiological states.
U N L I K E the p i g m e n t a r y e f f e c t s of m e l a n o c y t e s t i m u l a t i n g h o r m o n e (btSlt) in a m p h i b i a n s , no clear target organ or f u n c t i o n has been f o u n d for the substantial titers o f this h o r m o n e in m a m m a l s . Presently, the most likely possibility is that the brain itself is the ntajor target for the action o f MStl in higher animals. Certainly a large n u m b e r of behaviors can be a f f e c t e d by e x o g e n o u s a d m i n i s t r a t i o n o f the h o r m o n e . E x t i n c t i o n o f b o t h aversive [1] and appetilive tasks [5,22] is increased suggesting that m o t i v a t i o n a l processes which sustain behavioral persistence are intensified. That these effects are not merely due to a decreased capacity to learn new e n v i r o n m e n t a l c o n t i n g e n c i e s is indicated by o b s e r v a t i o n s that rats learn c o m p l e x mazes [2(~] and simple reversal t~,sks [21] faster after MSII than placebo. Surprisingly, however, if the task r e q u i r e m e n t s [24] or relevant cues [18] are c h a n g e d , animals tend to exhibit deficits in reversal learning. MSH also has c o m p l e x e m o t i o n a l e f f e c t s - - increasing dark p r e f e r e n c e and general e m o t i o n a l i t y o f rats u n d e r some c o n d i t i o n s [ 8 , 2 7 ] . t l u m a n studies indicate that MSlt l e n d s to decrease a n x i e t y and increase visual a t t e n t i o n [6, 11, 20]. Still, a definitive f u n c t i o n for MSIt in m a m m a l s has not
EXPERIMEN'I 1: I"EI'IDING AND DRINKING Previous work with appetitive tasks has indicated that hungry rats e x h i b i t delayed e x t i n c t i o n when treated with ~-MSH [22]. This along with the slight increase o f activity e x h i b i t e d by rats after MSH in certain appetitive situations ¢7) may indicate that MSH intensifies hunger in rats. Previous work has failed to note an',' m a j o r e f f e c t s o! ~-MSH on 2 hr food intake o f 22 hr deprived rats, although free water intakes were reliably increased [ 2 2 ] . Similarly. no effect has been observed on daily food intakes o f
"Fhis work was supported by NSH (;rant GB-40150, PHS (;rants AM17157 and NS (17664 and a Research Scientist l)evelopment Award MH-0086 to the senior author. 5q'
6o
P A N K S E P P E'I AL
u n s t a r v e d rats during daily a d m i n i s t r a t i o n of MSH [71. t-lowevcr, if the a p p e t i t e - m o d u l a t o r y effect of 0~-.MSII is to increase h u n g e r over a brief span of time, effects on food intake could easily have been missed in the a b o v e studies. Accordingly, in the following e x p e r i m e n t s wc studied the short- and long-term effects of 0~-MStt on food and w a t e r intake of n o n d e p r i v e d rats. Me th o d Twelve m a t u r e male Long-Evans rats were h o u s e d individually in 14.5 x 7 × 9 cm high wire mcsh cages with free access to Wayne p o w d e r e d l a b o r a t o r y c h o w from spill r e t a r d i n g c o n t a i n e r s and w a t e r from g r a d u a t e d d r i n k i n g tubes. Lighting was o n a 12 hr light-dark cycle and food and w a t e r intakes were r e c o r d e d at 12 hr or s h o r t e r intervals to 0.1 g and 1.0 ml respectively. On the experimental day, all a n i m a l s were injected intrap e r i t o n e a l l y (IP) with 10 ug,,'rat of c¢-MSH ( 1 0 7 ~ / m g ) at the start of the dark and light periods, ltalf the a n i m a l s reclaived the first i n j e c t i o n at the start of each period. 1 o o b t a i n s h o r t - t e r m m e a s u r e s of feeding, i n t a k e s were r e c o r d e d h o u r l y for the first 5 hr of the dark cycle. R e s u l t s ap2d Discussio,1 Daily food and w a t e r intakes are p r e s e n t e d in Table 1. No effect of a-MSH was a p p a r e n t here or in the diurnal d i s t r i b u t i o n s of intakes. The only o b s e r v a t i o n suggestive of a m e a n i n g f u l effect on ingestive b e h a v i o r o c c u r r e d d u r i n g the first hr following I~SH a d m i n i s t r a t i o n , l)uring this h o u r , feeding was reduced 205;- from 1.9 to 1.5 g while w a t e r intake increased 33!; from 3 to 4 ml. A l t h o u g h n e i t h e r o b s e r v a t i o n a l o n e was statistically reliable, clue to the o p p o s i n g t e n d e n c i e s , the f o o d / w a t e r ratio was significantly lower for a n i m a l s receiving oe-MSH ( p < 0 . 0 5 ~ with a n o n p a r a m e t r i c test ( W i l c o x o n ) but not by a p a r a m e t r i c c o m p a r i s o n (t-test). This small difference had d i s a p p e a r e d by the second h o u r of recording. In general, these data c o n f i r m that o~-MSH has no major effect on basic ingestive behavior. Certainly t h e r e was no incrcase in hunger. A c c o r d i n g l y previously r e p o r t e d increases in resistance to e x t i n c t i o n and increased activity in a p p e t i t i v e tasks c a n n o t be ascribed to such a m e c h a n i s m . T h u s it a p p e a r s that a-MSH can be used in learning e x p e r i m e n t s w h i c h e m p l o y food anti w a t e r as r e i n f o r c e r s with little c o n f o u n d i n g by c o n c u r r e n t changes in drive levels.
FIXIJI-I~,IIX,IFIN'I" 2: EMOTIONAl. BEHAVIOR IN Yt)IIN(; (.'lilt'KS Unlike most o t h e r c o m m o n l y used l a b o r a t o r y animals, the y o u n g chick displays a wide variety of s p o n t a n e o u s and easily m e a s u r e d behaviors in response to isolation in an open field. Because MSIt has p r o d u c e d effects w h i c h suggest m o d i f i e d e m o t i o n a l i t y [ 2 4 ] , in the following e x p e r i m e n t we assessed the effect of ct-MSfl on activity. distress vocalization, avoidance b e h a v i o r i, nd tonic i m m o b i l i t y of 1- and 3-day-old chicks. Me th o d In the first e x p e r i m e n t the b e h a v i o r of 22 one- and three-day-old male Leghorn chicks was recorded in a t.~0 x q~0 cm open-field unit ( m a r k e d into t~ equal squares) m a i n t a i n e d at a c o n s t a n t t e m p c r a t u r e of 32 ('. Activity was m e a s u r e d by the n u n t b e r of squares w h i c h the chick entered d u r i n g the 10 rain test session. Distress vocalizations were recorded a u t o m a t i c a l l y by m i c r o p h o n e and a s o u n d activated relay. A v o i d a n c e was m e a s u r e d at the end of the 10 rain o b s e r v a t i o n period by the e x p e r i m e n t e r a p p r o a c h i n g the bird l r o m the f r o n t with an o p e n h a n d and observing the response. A scale from I to 5 was used to rate the a v o i d a n c e response a c c o r d i n g to the following criteria: {1 ) little or no m o v e m e n t away from the h a n d : ~2t slight m o v e m e n t b a c k w a r d from h a n d ( t n a x i m u m of 2 or 3 steps): (3~ c o n t i n u o u s walking or backing away from the a p p r o a c h ing h a n d : ( 4 ) r a p i d walking or r u n n i n g from the h a n d . t5) vigorous u n d i r e c t e d r u n n i n g from the h a n d with wings raised and o f t e n a c c o m p a n i e d by distress vocalizations. E x p e r i m e n t a l animals received 4 ug of 0~-MSll IP 15 rain before testing, and c o n t r o l s received e q u i v o l u m e t r i c doses of the 0.01 M acetic acid .0.t)(:; saline carrie,. In the second e x p e r i m e n t , 14 day old chicks were tested for tonic i m m o b i l i t y after o~-MSIt or vehicle. Initially 2 i n d e p e n d e n t groups of 10 animals were lested, and subseq u e n t l y a n o t h e r l g birds were tested afler b o t h vehicle or MSII (again 4 u g / b i r d ) in a c o u n t e r b a l a n c e d w i t h i n - a n i m a l design, with successive tests being separated by 2 days. 1,11m o b i l i t y testing was c o n d u c t e d in a room m a i n t a i n e d at 24 . I (' on a large table p a r t i t i o n e d into 4 isolated sections. I n d u c t i o n of the i m m o b i l i t y response was a c c o m p a n i e d by restraining the a n i m a l on its back wilt, the h a n d for 15 sec followed by a slow, gentle release. The time t a k e n by the bird to right itself was recorded to the closest sec with a st opwat oh. R~'sttlts atzd Di.~cussiott
TABLE
I
DAILY FOOD AND WATER INTAKES DURING 12 HR INJECTIONS OF CARRIER OR MSH Pre-day Food Intakes (G) Carrier 22.1 ~-+2.2) MSH 21.8t_+.2.5) Water Intakes (MI) CatTier 37 I_+ 4) MSH 36 (_+41 Values are means ± SD.
Treatment Day
Post-Day
20.9 (-+2.41 21.1 t_+2.1)
19.5 (_+3.4) 19.8(2.9)
39 1_+6) 41 t~8)
35 (±6) 36 (.+_9)
In birds tested with the vehicle, m e a n activity scores were 38 and 4 crosscs in 1- and 3-day-old chicks. respectively, indicaling the n o r m a l d e v e l o p m e n t of behavioral i n h i b i t i o n d u r i n g these ages. Activity of animals tested with c¢-MSII d r o p p e d trom 37 to 21 crosses, but the difference at 3 days is not naeaningful since it was due exclusively to the e x t r e m e a g i t a t i o n of a single bird. Distress vocalizations were 872 and 340 in 1- and 3-day-old c o n t r o l birds, anti these values were not reliably different frotn the t,'58 and 312 values of chicks tested with o~-MStl. The average a v o i d a n c e rating of 1 day old MSII teated animals t2.5 ± 1.4) was reliably higher than e x h i b i t e d b,,' c o n t r o l s ( I . 2 + 0.7) It = 1.83, d.l = 10, p < 0 . 0 5 ) . At 3 days of age these effects were reversed, a n i m a l s lesled with o¢-MSI-! having average scores of 1.2 + 0.4 anti c o n t r o l s 3.0 +-
MSH A N D B R A I N R E S P I R A T I O N
61
1.1 (t = 3.38, (t/" = 8, p < 0 . 0 1 ) . The c o n t r o l a n i m a l s e x h i b i t e d a clear m a t u r a t i o n a l increase in a v o i d a n c e w h i c h we have typically o b s e r v e d in chicks. T h e reversal of the effects of ~-MSH w i t h age, t h o u g h o u t w a r d l y confusing, may reflect previously observed i n t e r a c t i o n s of MSII t r e a t m e n t with level o f fear. Since there is a m a r k e d d e v e l o p m e n t of fear d u r i n g the first 3 days of lifc in chicks 115], MSH may facilitate a v o i d a n c e at low levels of fear and suppress aw~idance d u r i n g high levels of fear. "lhis would he a n a l o g o u s to the i n t e r a c t i o n of MSH with shock i n t e n s i t y in a v o i d a n c e s i t u a t i o n s with rats [251. Thc average i m m o h i l i t y of birds tested with ~-MSH in lhe e x p e r i m e n t with i n d e p e n d e n t g r o u p s ',,,'as 376 -+ 306 scc, which was twice as long as the 151 ± C,~l sec of c o n t r o l s , but because of the great variability of this b e h a v i o r the d i f f e r e n c e s were not statistically significant. Similarly. in the w i t h i n - g r o u p e x p e r i m e n t , t h r o u g h the t r e n d was in the same d i r e c t i o n ( 4 5 8 sec for MSH and 4 0 8 sec for c o n t r o l birds), the results were again not reliably different.
p o s t e r i o r to bregma and 2 m m lateral to the midline. A bipolar e l e c t r o d e was placed into the dorsal h i p p o c a m p u s for the m e a s u r e m e n t of t h e t a activity. After at least a wcck of recovery, a n i m a l s were h a b i t u a t e d to sound a t t e n u a t e d sleeping c h a m b e r s (30 × 30 × 30 in.) and a prelimim, ry sleep record was collected from each animal to verify the scorability of records. R e c o r d i n g was d o n e with Narco Biosystems PMP-4B p h y s i o g r a p h at a p a p e r speed of 0.5 cm/sec. Records were visually scored into 3 sleep stages a c c o r d i n g to t r a d i t i o n a l criteria. Waking was c h a r a c t e r i z e d by low voltage d e s y n c h r o n i z e d activity in c o r t e x and h i p p o c a m p u s , slow wave sleep (SWS) was c h a r a c t e r i z e d by a high voltage low f r e q u e n c y activity from b o t h recording sites; activated slecp {AS) was c h a r a c t e r i z e d by d c s y n c h r o nized low voltage activity from the c o r t e x and a sustained regular t h e t a activity from h i p p o c a n l p u s . Before each test session, a n i m a l s were placed in the sleep c h a m b e r s with food and water at the start of t h e i r n o r m a l night cycle. Recording was started 12 hr later at the b e g i n n i n g of the light cycle and c o n t i n u e d for 4 c o n s e c u t i v e hr. Food and water were not available d u r i n g this period. All a n i m a l s were tested in a c o u n t e r b a l a n c e d fashion. Vehicle or o~-MSII ( 1 0 u g / r a t ) were injected IP i m m e d i a t e l y at the start of each recording session. T w o a n i m a l s were tested t h r o u g h 3 successive cycles of vehicle and MSH treatment.
I.:XPI,IRIMI.NT 3: SI. EEP-WAKIN(; PA I"I I.:RNS Since cme of the m a j o r h y p o t h e s e s c o n c c r n i n g a c t i o n of MSH is that a t t e n t i o n a l processes arc m o d i f i e d [6, 11, 2 0 ] , a direct analysis of m o d u l a t i o n by MSH of vigilance states is indicated. Such a line of inquiry is also suggested by the o b s e r v a t i o n of increased slow wave cortical activity d u r i n g the first half hr a f t e r a d m i n i s t r a t i o n of MStt in rats [lt~].
I?,esttlls ,'~lc ttl o d
A l t h o u g h overall 4 h o u r values for the vigilance states were not reliably different b e t w e e n the 2 groups, to highlight somc possible effects i m b e d d e d in these results, h o u r l y data for good and p o o r sleepers have been analyzed separately (Fig. 1). Poor sleepers were a n i m a l s which e x h i b i t e d less t h a n 4!;; AS (n = 4) and good slcepers, more
Ten male a l b i n o rats w e i g h i n g 3 1 1 4 2 5 g w c r e s u r g i c a l l y p r e p a r e d with indwelling e l e c t r o d e s for the clcctroe n c c p h a l o g r a p h i c m e a s u r e m e n t of sleep-waking p a t t e r n s . Cortical activity was m o n i t o r e d via 2 stainless-steel screws t h r e a d c d unilaterally over the c o r t e x 2 m m a n t e r i o r and
SWS
WAKE ,o
~
6o
AS
"" Q~
0 . - - 0 G.S.- Control
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~
i
so
T
I I
I
2
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3
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P.S.- Control
2o 0--0
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,,
,,
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Hours 1'1(,. I. thmrly sleep waking measures after lPadminislrationof 1 0 ~ g / r a t o f M S l l or acidified ,,aline carrier. Animal,~werc divided into -ood sleepers ((;.S.) and poor sleepers ILLS.) accordin,e t~ levels of activated sleep cxhibilcd durm,e the control ScssitIIIS.
62
PANKSI"PP k"F AL.
t h a n 6~7, AS (n = 6) u n d e r the vehicle c o n d i t i o n . F r o m this analysis, it is a p p a r e n t t h a t MStt t e n d e d to stabilize the sleep p a t t e r n of the a n i m a l s - p o o r sleepers sleeping m o r e and good sleepers less. This c o n v e r g e n c e was m o s t a p p a r e n t for a c t i v a t e d sleep - t h a t of good sleepers being r e d u c e d by 29% (t = 5.46, dl" = 5, p < 0 . 0 1 ) and t h a t of p o o r sleepers tripled. T h e increase in AS was due to the d o u b l i n g of episodes as well as 75% increase in average episode length. In good sleepers, the n u m b e r o f AS episodes was decreased by 20% and episode length by 18~)'. The only reliable finding for all a n i m a l s c o m b i n e d was the 157~ increase in slow wave sleep d u r i n g the second and third h o u r s o f r e c o r d i n g (t = 2.6, dr "= 9, p < 0 . 0 5 ) . Again, the p o o r sleepers e x h i b i t e d the larger 27U- increase while good sleepers e x h i b i t e d a 9% i n c r e m e n t . T h e 3 successive r e c o r d i n g cycles o b t a i n e d from 2 a n i m a l s which had been categorized as p o o r sleepers indicated t h a t these effects of MSH were not stable across time. In 1 a n i m a l the successive changes in AS t h r o u g h the 3 cycles of t r e a t m e n t w i t h a-MSII were first a 260U increase in AS, t h e n a 67~ decrease, followed by a 10~¢ decrease. In the o t h e r animal, the values were a 4t.~0~.; increase, a 188~2;, increase with a 5% decrease. The o b s e r v e d increase in SWS also d i m i n i s h e d s y s t e m a t i c a l l y with r e p e a t e d testing. This p a t t e r n of results suggests that the effects of o~-MSH in p o o r sleepers was p r o b a b l y due to some aspect of the testing s e q u e n c e , possibly the initial n o v e l t y of the situation. In general, these results indicate that MS!t has relatively little effect on overall sleep-waking activity in rats. Att h o u g h p o o r sleepers a p p e a r e d to rest m o r e and good sleepers less. it s h o u l d be n o t e d that such a t r e n d c o u h t merely be due to statistical regression t o w a r d the mean. In any case, the results suggest that this h o r m o n e has no p r o f o u n d and c o n s i s t e n t effect on the basic m e c h a n i s m s which c o n t r o l e l e c t r o e n c e p h a l o g r a p h i c indices of vigilance. The effects w h i c h were o b s e r v e d m a y be a s e c o n d a r y c o n s e q u e n c e of a variety of e m o t i o n a l changes that m a y have been p r o d u c e d by MSH. EXPERIMI'NT4: BRAIN RESPIRAI'ION MSH has been d e m o n s t r a t e d to have several physiological effects o n the brain which are c o m p a t i b l e with the n o t i o n t h a t the h o r m o n e might selectively increase the capacity for visual processing t h a t has been observed in h u m a n beings [20] and rats [ 2 1 ] . For instance, MSH can increase the p o w e r o u t p u t of occipital c o r t e x [ 11 ]. Also, MS!.! does not reduce b l o o d flow in occipital c o r t e x as in o t h e r p a r i s o f the brain [3] while at the same time a c c u m u l a t i n g m o r e in the occipital cortex than o t h e r f o r e b r a i n areas [ 8 ] . Because of these findings and the changes in e l e c l r o c o r t i c a l activity o b s e r v e d in the previous e x p e r i m e n t , we d e t e r m i n e d w h e t h e r MSH has any effect on the gross m e t a b o l i c activity of s u b r e g i o n s in the brain as m e a s u r e d by in vitro o x y g e n c o n s u m p t i o n .
Method O x y g e n c o n s u m p t i o n was m e a s u r e d by the polarographic t e c h n i q u e (Yellow Springs I n s t r u m e n t ('o. Model 53 s y s t e m ) w h i c h m e a s u r e s o x y g e n pressure present in a liquid m e d i u m . F o r t y adult male Swiss-Webster mice were used. !fall were injected with 4 u g / m o u s e ofo~-MSH, and half were injected with equal a m o u n t s of the acidified saline m e d i u m . Fifteen m i n u t e s a f t e r i n j e c t i o n a n i m a l s were
d e c a p i t a t e d , brains were r e m o v e d , and 4 8 mg samples ot occipital c o r t e x and frontal c o r t e x (.just dorsal to the rhinal sulcus) were dissected from each brain. Samples of the dorsal brain stem ( i n c l u d i n g the area of the locus coeruleus) (n = 27), d i a p h r a g m (n = 15), and liver (n = 20~ were o b t a i n e d from smaller subsets of animals. Each tissue sample was weighed to 0.1 m g a n d h o m o g e n i z e d in I m l o f freshly aerated mammalian Ringer's solution. The h o m o g e n a t e plus 2 a d d i t i o n a l ml of aerated Ringer's were i n c u b a t e d at a c o n s t a n t t e m p e r a t u r e of 37' ( ' a n d o x y g e n c o n s u m p t i o n was m o n i t o r e d for 30 rain. At this t e m p e r a ture, the Bunsen coefficient of Ringer's s o l u t i o n is 5 ul of o x y g e n / r o t , yielding a total of 15 ul of oxygen in the 3 ml of m e d i u m . In 13 a d d i t i o n a l animals, the frontal and;'or occipital cortices were sampled before any injections, each sample was divided in half, amt 4 ug of MSI! or the equivalent a m o u n t of vehicle were a d d e d to the i n c u b a t i o n m e d i u m s 30 rain after the start of recording. M e a s u r e m e n t of respiration was c o n t i n u e d for a n o t h e r 30 rains. Because of the changes in brain respiration observed in the a b o v e e x p e r i m e n t s , plasma glucose levels were determ i n e d in 11 a'-MSIt treated (4 u g / a n i m a l ) and 10 vehicle treated mice 15 rain after injection. Blood was collected after d e c a p i l a t i o n and glucose levels were measured p o t e n t i o m e t r i c a l l y in triplicale with a Ycllov,' Springs I n s t r u m e n t s (;lucosc A n a l y z e r IYSI Model 23).
Results and Discussion Tissue 02 c o n s u m p t i o n s after in vivo a d m i n i s t r a t i o n of ~-MSH are s u m m a r i z e d in Table 2. ~-MSI-t bad no reliable effects on tissue respiration except for a significanl l g':; r e d u c t i o n in o x y g e n c o n s u m p t i o n of the dorsal brain stem which i n c l u d e d the area locus c o e r u l e u s (t = 2.14, d.l = 35, p < 0 . 0 5 ) . The d i a p h r a g m also e x h i b i t e d a 2Y.; r e d u c t i o n in respiration, but due to the small n u m b e r of samples and the large variance, the t r e n d was not statistically significant. TABLE 2 OXYGEN CONSUMPTION (ug;mg/30 MINI ()F BRAIN AND PERIPHERAl. TISSUES FOLLOWING IP INJECTIONS OF 4 ug,' MOUSE OF MSH OR ACETIC At'It) CARRIER 15 MINUTES BFFORE SACR[FICtTissue Occipital Corlex Frontal Cortex Dorsal Brain Stem Diaphragm Liver
MSH
0.47 0.45 0.23 0.36 0.23
_+ .24 (20) _+ .17 (20) ± .06 ~ (13) ~- .25 {7) = .06 (101
Control 0.46 0.40 0.28 0.47 0.23
+_ .21 120) _+ .11120) ± .05 114) ± .3618) * .(14110)
Values are Means ± SD. Numbers in parentheses indicale number of observalions. *p <0.05. A d m i n i s t r a t i o n of o~-MSH into tile respiring m e d i m of frontal anti occipital tissues had no effecl on respiration. For occipital cortex, the c o n t r o l value was 0.34 ~. 0 . 2 0 j and the ~-MS]-I o n e 0.33 ~± 0.20) ug 0 2 / r a g wet tissue/30 rain. The c o r r e s p o n d i n g values for frontal cortex were 0.23 (± 0.05) and 0.26 (± 0 . 0 6 ) u g 0 2 / r a g wet tissue/30 rain. Essentially these results indicate that o~-MS]! has ','cry little effect on energy m e t a b o l i s m of the brain. The
MSH A N D BRAIN RESPIRATION r e d u c t i o n of dorsal brain stem r e s p i r a t i o n was p r o b a b l y s e c o n d a r y to changes in b l o o d flow because it was n o t e d d u r i n g tissue dissection t h a t this region as well as the brain generally was m a r k e d l y b l a n c h e d in a n i m a l s t r e a t e d with oe-MSlt. This p r o b a b l y c o r r e s p o n d s to the r e d u c e d b l o o d flow that has b e e n r e p o r t e d for m a n y regions of the brain following o~-MSI-I t r e a t m e n t [ 3 ] . Of course, if this were the only physiological change that resulted from the o~-MSH, o t h e r parts of the brain should have also e x h i b i t e d some r e d u c t i o n in respiration. In fact, the f r o n t a l c o r t e x exh i b i t e d a 12% ( t h o u g h not significant) increase in o x y g e n c o n s u m p t i o n after t r e a t m e n t with o~-MSH. Since this h e i g h t e n e d r e s p i r a t i o n in the p r e s e n c e of decreased b l o o d flow m i g h t have o c c u r r e d if increased s u b s t r a t e s had been m a d e available to the brain we decided to measure plasma gh, cose levels after 0¢-MSH. The results i n d i c a t e d t h a t glucose levels were 14'7~ higher in a n i m a l s t r e a t e d with a~-MSIt t h a n in c o n t r o l s t 143 mgU vs 126 mg%) (t = 1.9, df = I~,L .o<0.05). Since in p r e v i o u s work it has been d e m o n s t r a t e d that e x o g e n o u s a d m i n i s t r a t i o n of glucose can increase the r e s p i r a t o r y activity o f brain tissue [ 1 4 ] , it is suggested that r e d u c e d f o r e b r a i n b l o o d flow may have been c o u n t e r a c t e d by increased e n d o g e n o u s m e t a b o l i c s u b s t r a t e availability.
GENERAL DISCUSSION The p r e s e n t results do not lead to an u n a m b i g u o u s u n i t a r y h y p o t h e s i s a b o u t thc role of o~-MStt in the f u n c t i o n i n g of higher animals. All of the positive effects observed in the prcsent e x p e r i m e n t s were weak and in sonle i n s t a n c e s e p h e m e r a l . However, 4 positivc o b s e r v a t i o n s were made, and they may be of some i m p o r t a n c e in d e l i n e a t i n g the n o r m a l biological effect of MSH in n o n a m p i b i a n s . First, the a v o i d a n c e b e h a v i o r o1 y o u n g chicks to an a p p r o a c h i n g object was increased at 1 day of age and decreased at 3 days of age. This finding s u p p o r t s tire c o n s i d e r a b l e evidence linking o~-MStl to the m o d u l a t i o n of fear m o t i v a t e d behaviors [ 1 , 9 ] , and f u r t h e r suggests t h a t the type of effect one observes m a y be d e p e n d e n t on the level of fear. Since e m o t i o n a l b e h a v i o r s of the y o u n g chick are d e v e l o p i n g rapidly d u r i n g the first few days of life, the reversal of the effect of o~-MSI-t m a y not be as p r o b l e m a t i c as it m a y initially seem. If the vigor o f a v o i d a n c e in the chick is an inverted U shaped f u n c t i o n with increasing age, then MSH might shift the a n i m a l up on the curve at a y o u n g age and d o w n at an older age. This result would also be e x p e c t e d if ~-MSI-t i n t e r a c t s with f e a r - m o t i v a t i o n in such a way that it i m p r o v e s behaviors induced by low levcls of fear anti reduces those p r o v o k e d by high levels. Indeed, this kind of i n t e r a c t i o n has been observed in rats w h e n i n t e n s i t y of shock is m a n i p u l a t e d at low shock intensities MSI-! facilitates a v o i d a n c e and at high levels a v o i d a n c e is rcduced
[25t. Second, oe-MSIt a p p e a r s to have subtle effects o n vigilance as m e a s u r e d by c l c c t r o e n c e p h a l o g r a p h i c indices of sleep-waking activity. Slow wave sleep is increased d u r i n g the s e c o n d and third hr a f t e r h o r m o n e a d m i n i s t r a t i o n . This o b s e r v a t i o n is c o m p a t i b l e with early r e p o r t s that some rats tested with /3-MSII e x h i b i t e d behavioral s o m n o l e n c e [17] and also possibly with the high incidence of y a w n i n g and s t r e t c h i n g observed in dogs a f t e r MSH a d m i n i s t r a t i o n 121. This finding m a y also bc c o m p a t i b l e with recent observations that o~-MStl increases the t u r n o v e r of brain norcpi-
63 n e p h r i n e d u r i n g the first hr a f t e r a d m i n i s t r a t i o n followed by a decreased t u r n o v e r for several h o u r s [ 1 0 ] . It n o r e p i n e p h r m e subserves cortical arousal [ 4 , 1 2 ] , a decrease in n o r e p i n e p h r i n c t u r n o v e r m a y have p r o m o t e d the a p p e a r a n c e o f slow wave sleep. If this were the case, we s h o u l d also have observed a period o f increased arousal d u r i n g the b e g i n n i n g of testing. Ilowever, we n o t e d no such effect. The average latency to slow wave sleep in c o n t r o l a n i m a l s was 30.3 min and in animals t r e a t e d with a - M S l t il was 25.7 rain. In fact, 8 of 10 rats studied e n t e r e d slow wave sleep faster a f t e r MSH t h a n after vehicle. ( ' o n v e r s e l y , 8 of the 10 a n i m a l s t o o k longer to e n t e r activated sleep after ~-MStt t h a n vehicle. It might be n o t e d that some a n i m a l s did tend to e x h i b i t initial sleep p a t t e r n s which may have been c h a r a c t e r i z e d by atypically rapid cycling b e l w e e n slow wave sleep and waking, as if sleep were being d i s r u p t e d by an arousal process, but this o b s e r v a t i o n was not at all c o n s i s t e n t across animals. In fact, hall" the a n i m a l s e x h i b i t e d larger average episode lengths of slow wave sleep t h a n controls. A n o t h e r possible effect of a'-MSH was a r e d u c t i o n in the variability of sleep stages across animals, anti this effect was most p r o m i n e n t for activated sleep. This suggests that o~-MSII may have a stabilizing effect on a Brain m e c h a n i s m which c o n t r o l s e l e c t r o c o r t i c a l arousal. This h o m o g e n i z a t i o n of e l e c t r o c o r t i c a l activity may also be n o t e w o r t h y from the perspective that 0~-MStt has previously been n o t e d to reduce the variability of o t h e r behaviors, for instance, the n u m b e r of errors rats m a k e in learning a c o m p l e x maze [ 2 6 ] . ( ' e r t a i n l y it is possible that t r e a t m e n t with o~-MStl stabilizes a physiological s u b s t r a l e which n o r m a l l y e x h i b i t s e i t h e r s u b s t a n t i a l individual or l e m p o r a l variability. Our third m a j o r o b s e r v a t i o n was that MSH decreased in vitro respiration of the dorsal brain stem. This effect was p r o b a b l y due to decreased blood flow to that region, not only because the a n i m a l s treated with ~-MSIt e x h i b i t e d a visually o b v i o u s b l a n c h i n g of brain tissue, but also because MSII has been d e m o n s t r a t e d to reduce blood flow to m a n y regions o f the brain 13]. The fact that respiration was not decreased anti may, in fact, have been increased in f o r e b r a i n areas such as f r o n t a l and occipital cortices, suggested that a n y m e t a b o l i c effects o f reduced blood llow was being h o m e o s t a t i c a l l y c o u n t e r a c t e d by an increase ira energy availability. This idea p r o v o k e d our f o u r t h o b s e r v a t i o n , n a m e l y that o:-MSH p r o d u c e d a reliable t h o u g h m o d e s t increase in blood glucose levels of n o n f a s l c d mice. W h e t h e r this effect was due to a direct a c t i o n on the liver as o p p o s e d to some s e c o n d a r y h o r n l o n a l c h a n g e such as increased secretion of e p i n e p h r i n e , glucagon, or g l u c o c o r t i c o i d s or decreased insulin, c a n n o t be ascertained from the present data. Certainly, from the perspective that MSII is recruited in response to b o t h physical and psychological stressors [ 2 3 ] , a b l o o d glucose increase can be seen to be an adaptive r e s p o n s e in p r e p a r i n g the brain and b o d y to cope with a stressful situation. Still, it should be e m p h a s i z e d lhat the h y p e r g l y c e m i c effect ofo~-MSIl was very small, but it may explain why we observed a slight r e d u c t i o n in feeding and an increase of d r i n k i n g in our first e x p e r i m e n t . The increased awtilability of m e t a b o l i c s u b s t r a t e s may have depressed a p p e t i t e while any increase in b l o o d t o n i c i t y may have i n d u c e d the animal to drink a little more. Also, it is c o n c e i v a b l e that the m e t a b o l i c changes we observett a f t e r 0¢-MStI may have p a r t i c i p a t e d in some of the m o d i f i c a t i o n in sleep waking p a t t e r n s since it is clear that changes in
64
P A N K S E P P ET AL.
glucose m e t a b o l i s m can m o d i f y vigihmce states quite m a r k e d l y [ 1 3]. A l t h o u g h the p r e s e n t results do not provide a conclusive m e c h a n i s m for MSH action, it should be n o t e d that the results are c o n s i s t e n t with the possibility that MSll may have as target organ certain p i g m e n t e d cells of the central a u t o n o m i c nervous system. For instance, it has recently been d e m o n s t r a t e d thal the locus coeruleus c o n t r o l s cerebral b l o o d f l o w [161, and it was in the brain sample c o n t a i n i n g this area that a reliable r e d u c t i o n of tissue respiration was observed. F u r t h e r , since this part of the brain is k n o w n to participate in organizing stress related responses and states of vigilance 14], the changes in sleep-waking activity and e m o t i o n a l behaviors in chicks could have been m e d i a t e d by changes in the activity o f p i g m e n t e d brain stem cell groups such as the locus coeruleus. A l t h o u g h speculative at this stage, the h y p o t h e s i s that MSH may m o d u l a t e central a u t o n o m i c tone could provide a unifa'ing principle which might a c c o u n t for the m a n y e f f e c t s o f MSII that have been described in the literature and the wide variety o f e f f e c t s which were observed in the
present series of e x p e r i m e n t s . If MSH does m o d i f y auton o m i c t o n e in the nervous system, it would be e x p e c t c d that a large variety of behaviors would be a f f e c t e d , but not necessarily to any great e x t e n t , and the hehavioral literature does indicate that the e f f e c t s o f MSH are widespread t h o u g h o f t e n quite weak and sensitive to slight environmental and task changes. Also, from such an a u t o n o m i c action, it would be reasonable thal the behavior of animals in m a n y situations would be stabilized rather than shifted p r o f o u n d l y in any unitary direction, and again it has been occassionally n o t e d that MSH t e n d s to reduce the variability in behavior. F u r t h e r , since rcccnt work suggests that an increased a t t e n t i o n h y p o t h e s i s of MSIt aclion may bc the best general principle available for organizing the diverse behavioral e f f e c t s of the l m r t n o n e [11,201, a r e a s o n a b l e lllode (11' aciion for such it process could be via brain stem cell groups such as the locus coeruleus which have been intplicated in lhe basic m a i n t e n a n c e of corlical arottsal. Finally, a most c o m p e l l i n g fact which is leading us to analyze the role o f such brain areas in MSIt action is their p r o m i n e n t p i g m e n t a t i o n and hence lheir o u t w a r d r e s e m b l a n c e to the epidermal target tissue o f a m p h i h i a n s .
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