1182

Speeialia

r o o m for 48 h. A t t h i s t i m e t h e y were p l a c e d on m o i s t b l o t t e r p a p e r a n d t h e p e r c e n t a g e h a t c h s u b s e q u e n t l y det e r m i n e d . E a c h e x p e r i m e n t c o n s i s t e d of 6 r e p l i c a t e s of 10 eggs p e r replicate, a n d t h e e x p e r i m e n t was d u p l i c a t e d . T h e t e m p e r a t u r e was m a i n t a i n e d a t 25 + 0.1 ~ Results. The figure s u m m a r i z e s t h e p a t t e r n of loss of w e i g h t (weight loss equals w a t e r loss 1) of eggs e x p o s e d to 48% R H a f t e r 96 h in w a t e r v a p o r or on m o i s t p a p e r . Eggs p r e c o n d i t i o n e d in w a t e r v a p o r d i d n o t lose a s i g n i f i c a n t a m o u n t of w e i g h t until t h e w e i g h i n g a t 24 h ; t h o s e prec o n d i t i o n e d on m o i s t p a p e r lost s i g n i f i c a n t w e i g h t (5% level) in t h e first 15 min. E g g s f r o m m o i s t p a p e r w e i g h e d 85.3 ~g (SD = 3.3 Ezg) a t t h e first w e i g h i n g a n d 37.4 ~zg (SD = 7.7 [~g) a f t e r 48 h ; t h o s e f r o m w a t e r v a p o r w e i g h e d 74.8 [zg (SD = 6.4 ~g) a t t h e s t a r t a n d 52.7 ~g (SD = 6.6 [~g) a t 48 h. T h u s eggs f r o m m o i s t p a p e r w e i g h e d signific a n t l y m o r e (0.5% level) a t t h e o u t s e t of t h e e x p e r i m e n t , b u t t h e y w e i g h e d s i g n i f i c a n t l y less (0.5% level) a t t h e finish. This could be i n t e r p r e t e d t o m e a n t h a t t h e m e a s u r e d loss was s i m p l y e v a p o r a t i o n of w a t e r f r o m t h e outside surface of t h e eggs, b u t it h a s b e e n d e m o n s t r a t e d t h a t , given t h e w e i g h i n g p r o c e d u r e used, superficial w a t e r does n o t c o n t r i b u t e to c h a n g e s in w e i g h t 1. F u r t h e r m o r e , t h e w e i g h t a t t h e e n d of 48 h was s i g n i f i c a n t l y lower in t h e eggs a c c l i m a t e d on m o i s t p a p e r so t h e y h a d lost m o r e weight. W h e n b o t h sets were p l a c e d on m o i s t p a p e r a n d subseq u e n t h a t c h was d e t e r m i n e d , h a t c h was s i g n i f i c a n t l y h i g h e r (70% SD = 13%) for t h e eggs a c c l i m a t e d a t 100% R H t h a n for eggs a c c l i m a t e d on m o i s t p a p e r (10% SD =

10%).

EXPERIENTIA 33/9

I c o n c l u d e t h e r e f o r e t h a t eggs a c c l i m a t e d in t h e w a t e r v a p o r s y s t e m are less s u s c e p t i b l e t o w a t e r loss t h a n are t h o s e a c c l i m a t e d on m o i s t paper. F u r t h e r m o r e , t h i s difference in w a t e r loss was parallelled b y h i g h e r m o r t a l i t y in t h e g r o u p t h a t lost m o r e water. F o r p u r p o s e s of d e s c r i p t i o n , I h a v e r e f e r r e d to w a t e r s t a t e s as ' v a p o r ' a n d ' m o i s t p a p e r ' . The m o i s t p a p e r s y s t e m is e a s y t o describe : microscopic e x a m i n a t i o n of t h e eggs a n d p a p e r s h o w e d w a t e r on t h e p a p e r fibres on w h i c h t h e eggs rested. The precise c o n d i t i o n of w a t e r in t h e socalled v a p o r s t a t e is u n k n o w n . The difficulty of k n o w i n g t h e a c t u a l s t a t e of w a t e r in a h y g r o s t a t t h a t p u r p o r t s to be a t 100% R H has b e e n reviewed~, 4. As E d n e y 3 p o i n t e d out, it is p r o b a b l y n o t possible to h a v e a h y g r o s t a t w i t h 100% R H w i t h o u t s o m e d r o p l e t s of liquid w a t e r some place t h e r e i n . I did n o t see w a t e r d r o p l e t s on eggs or in c o n t a i n e r s nor on t h e walls of t h e h y g r o s t a t . I a s s u m e t h e r e f o r e t h a t w a t e r was m o r e r e a d i l y available t o eggs on m o i s t p a p e r t h a n to t h o s e in t h e h y g r o s t a t b u t m y o b s e r v a t i o n s do n o t p e r m i t precise d e f i n i t i o n of t h e s t a t e of t h e w a t e r in t h i s w a t e r v a p o r s y s t e m . I c o n c l u d e t h a t in t h i s species, t h e w a t e r c o n s e r v a t i o n m e c h a n i s m of t h e egg is a d a p t a b l e . The i n f o r m a t i o n h a s o b v i o u s significance for l a b o r a t o r y s t u d i e s of egg shell p e r m e a b i l i t y a n d also for e v a l u a t i o n of w a t e r a v a i l a b i l i t y as a m o r t a l i t y f a c t o r in field p o p u l a t i o n s . 3 E. ]3. Edney, in: Water relations of terrestrial arthropods, p. 55. Cambridge Univ. Press, Cambridge 1957. 4 E. Bursell, in: Physiology of insecta, vol. 2, p. 343. Ed. M. Rockstein. Academic Press, New York 1973.

Fluid secretion by isolated cockroach salivary glands R. K. S m i t h a n d C. R. House1

Department o/ Physiology, Royal (Dick) School o/ Veterinary Studies, University o/ Edinburgh, Edinburgh EH9 7QH (Scotland), 13 January 1977 Summary. A n isolated p r e p a r a t i o n of t h e i n n e r v a t e d c o c k r o a c h s a l i v a r y g l a n d has b e e n d e v e l o p e d to s t u d y secretion. This g l a n d secretes a fluid rich in N a a n d Ct in r e s p o n s e to n e r v e s t i m u l a t i o n or b a t h a p p l i c a t i o n of d o p a m i n e . T h e s a l i v a r y g l a n d s of a n u m b e r of insects r e c e i v o an i n n e r v a t i o n w h i c h is p r o b a b l y d o p a m i n e r g i c ~-4. Microe l e c t r o d e r e c o r d i n g f r o m a c i n a r cells in t h e c o c k r o a c h s a l i v a r y g l a n d h a s r e v e a l e d t h a t n e r v e s t i m u l a t i o n causes a h y p e r p o l a r i s a t i o n 5 t h a t can be m i m i c k e d b y b a t h a p p l i c a t i o n s of several biogenic amines, t h e m o s t p o t e n t b e i n g d o p a m i n e ~. I n o r d e r to s t u d y fluid s e c r e t i o n e v o k e d b y n e r v e s t i m u l a t i o n a n d d o p a m i n e in t h i s tissue we h a v e a d a p t e d t h e t e c h n i q u e originally used b y R a m s a y ~ for u r i n e collection f r o m i n s e c t M a l p i g h i a n t u b u l e s . I t is n o t able t h a t p r e v i o u s r e p o r t s of t h e c o m p o s i t i o n of i n s e c t salivas h a v e s h o w n t h a t t h e p r i n c i p a l c a t i o n is p o t a s siumS, 9 while t h a t in v e r t e b r a t e s is sodium. Our r e s u l t s d e m o n s t r a t e t h a t c o c k r o a c h saliva is s o d i u m rich a n d r e s e m b l e s m o r e closely t h a t of c e r t a i n m a m m a l s t h a n of t h e insects s t u d i e d so far. Materials and methods. W h o l e p a i r e d glands, c o n s i s t i n g of reservoirs, r e s e r v o i r d u c t s , acini a n d a c i n a r ducts, were d i s s e c t e d f r o m a d u l t c o c k r o a c h e s ( N a u p h o e t a cinerea) of e i t h e r sex, allowed free access t o food a n d w a t e r . Dissect i o n w a s c a r r i e d o u t u n d e r t h e p e r f u s i o n m e d i u m (pH 7.6) w h i c h h a d t h e following c o m p o s i t i o n (mM): NaC1, 160; KC1, 10; NaH2PO4, 1; NaHCOa, 1; CaCI~, 5; Tris, 5; HC1, 4; glucose, 20. E a c h m a i n acinar d u c t was freed

f r o m its a d h e r e n t r e s e r v o i r d u c t a n d t h e g l a n d s were p l a c e d in a p e r s p e x c h a m b e r (volume 1.5 ml). One of t h e a c i n a r d u c t s was l i g a t u r e d n e a r its c u t e n d w i t h e n a m e l l e d Ag wire a n d pulled i n t o a pool of liquid paraffin, separ a t e d f r o m t h e p e r f u s i o n c h a m b e r b y a p e r f o r a t e d celluloid barrier. The tissue was a n c h o r e d in t h e c h a m b e r b y a ligature r o u n d t h e reservoirs (figure 1). The c h a m b e r was p e r f u s e d a t a r a t e of 2.5 m l / m i n b y a W a t s o n Marlowe flow i n d u c e r ( M H R E 200); t h e r a t e was increased t o 10 m l / m i n w h e n solutions were c h a n g e d .

1 Acknowledgments. We are indebted to the Science Research Council for financial support. 2 N. Klemm, Comp. ]3iochem. Physiol. d3A, 207 (1972). 3 K.P. Bland, C. R. House, B. L. Ginsborg and I. Laszlo, Nature New Biol. 244, 26 (1973). 4 H.A. Robertson, J. exp. ]3iol. 63, 413 (1975). 5 C.R. House, J. exp. Biol. 58, 29 (1973). 6 F. Bowser-Riley and C. R. House, J. exp. Biol. 64, 665 (1976). 7 J . A . Ramsay, _1. exp. Biol. 31, 104 (1954). 8 F.C. Kafatos, J. exp. Biol. 48, 435 (1968). 9 3. L. Oschman and M. J. Berridge, Tissue Cell 2, 281 (1970).

15. 9. 1977

Specialia

Ionic composition of saliva Species

Ionic concentration (mmoles/1) Na K C1

Cockroach (Nauphoeta)* Moth (Antherea) 8 Blowfly (Calliphora) ~, 10 H u m a n parotid n Horse parotid TM Sheep parotid TM

140.9:E4.5 1 20 90 56 170

* Values given are means •

25.7~1.5 190 160 20 15 13

HCO 3

151.7=E6.5 19 180 160 50 60 50 52 11 112

SE.

Anchor ligature

Suction electrode with reservoir duct

./

c,n, //

Reservoir

\~/

1183

For electrical stimulation, the reservoir duct (bearing the salivary nerve) on the same side as the ligatured acinar duct was drawn into a suction electrode. Square-wave s t i m u l i (0.5 m s d u r a t i o n ) w e r e d e l i v e r e d e i t h e r b y a G r a s s s t i m u l a t o r ( S D 5) o r b y i s o l a t e d s t i m u l a t o r ( D e v i c e s T y p e 2533) d r i v e n b y a g a t e d p u l s e g e n e r a t o r ( D e v i c e s T y p e 2521) a n d a D i g i t i m e r . Chemical secretagogues were made up as concentrated (1 m g / m l ) s t o c k s o l u t i o n s k e p t a t 4 ~ a n d d i l u t e d i n t o the perfusion medium immediately before use. The secretory rate was determined by removing secreted fluid at suitable intervals to a second paraffin pool with siliconet r e a t e d m i c r o p i p e t t e s w h e r e t h e d i a m e t e r of t h e d r o p l e t was measured optically. Unstimulated tissues were found to secrete at 0-3 nl/min. Secretory responses to nerve stimuli and dopamine were observed for at least 5 h after isolation. Sodium and potassium concentrations in the secreted fluid were estimated by atomic absorption spectrophotometry (Pye Unicam SP90A) and chloride by coulombometric titration (Radiometer CMT10). Results and discussion. F i g u r e 2 s h o w s t y p i c a l s e c r e t o r y responses to nerve stimulation (upper panel) and dopam i n e (lower). W i t h e l e c t r i c a l s t i m u l i t h e s e c r e t o r y r a t e rose to a maximum within 3-5 min and after 10-15 min d e c l i n e d t o w a r d t h e b a s a l v a l u e . T h e m a g n i t u d e of t h e response increased with stimulus voltage over the range 2 - 6 0 V. U s u a l l y s t i m u l i of 30 V w e r e u s e d s i n c e t h e s e g a v e near maximal responses and were thought less likely to e x h a u s t t h e t i s s u e . D o p a m i n e a l s o i n c r e a s e d t h e r a t e of s e c r e t i o n . A b o v e a t h r e s h o l d of a b o u t 3 • 10 .9 M d o p a mine produced secretory responses graded with concent r a t i o n u p t o a m a x i m u m a t 10 .6 M. E a c h s e c r e t o r y re-

duct Fig. 1. Plan view of experimental arrangement for investigating fluid secretion in isolated cockroach salivary glands. Tissues were stimulated either by delivering electrical pulses to the salivary nerve held in the suction electrode or by perfusing the chamber with dopamine solutions.

10 11 12 13

W . T . Prince and M. J. Berridge, J. exp. Biol. 58, 367 (1973). J . H . Thaysen, N. A. Thorn and I. L. Schwartz, Am. J. Physiol. 178, 155 (1954). F. Alexander, J. Physiol., Lond. 784, 646 (1966). R . N . B . Kay, J. Physiol., Lond. 750, 515 (1960).

Nerve 80

40

.E

E

10

20

30

40

50

60

"E

Dopamine

o P

=o

03

8~ ioI 6O

70

N_ 80

90

Time, min

100

110

120

Fig. 2. Typical secretory responses evoked by nerve stimulation and dopamine. Periods of stimulation are indicated by horizontal bars. Electrical stimuli (50 V, 0.5 ms) were delivered at a frequency of 10 Hz. After the secretory responses to nerve stimulation dopamine solutions (2 • 10-~ M) were passed through the chamber for 10 min and subsequently for 25 min. Prolonged nerve stimulation (upper right) was accompanied by a fall in the secretory rate whereas prolonged e x p o s u r e to d o p a m i n e (lower right) was not.

1184

Specialia

sponse reached a peak within about 5 min and during p r o l o n g e d e x p o s u r e t o d o p a m i n e t h e s e c r e t o r y r a t e fell u s u a l l y to a m a i n t a i n e d lower v a l u e a f t e r a b o u t 10 rain. I n t h e s a m e p r e p a r a t i o n t h e a m p l i t u d e s of t h e m a x i m a l r e s p o n s e s to d o p a m i n e a n d n e r v e s t i m u l a t i o n were i d e n t i c a l (figure 2) a n d in tile r a n g e 60-100 n l / m i n . G l a n d s were s t i m u l a t e d for 20-30 m i n p e r i o d s w i t h d o p a m i n e (10-7-10 -" M) a n d t h e secreted fluid collected for e i t h e r chloride (14 samples) or s o d i u m a n d p o t a s s i u m a n a l y s i s (9 Samples). T h e r e s u l t s g i v e n in t a b l e i n d i c a t e t h a t t h e m a j o r c a t i o n in t h e saliva is s o d i u m a n d t h a t

EXPERIENTIA 33/9

c h l o r i d e is p r e s e n t a t a c o n c e n t r a t i o n a l m o s t e q u a l to t h e c o m b i n e d c o n c e n t r a t i o n s of s o d i u m a n d p o t a s s i u m . T h e c o m p o s i t i o n of c e r t a i n o t h e r salivas is also s h o w n in tile t a b l e for c o m p a r i s o n . I t c a n b e seen t h a t c o c k r o a c h s a l i v a is d i f f e r e n t f r o m b o t h i n s e c t salivas a n d is s o m e w h a t s i m i l a r to t h e m a m m a l i a n ones. O t h e r e x p e r i m e n t s h a v e s h o w n t h a t s a l i v a r y s e c r e t i o n e v o k e d b y d o p a m i n e in N a u p h o e t a g l a n d s is r e v e r s i b l y a b o l i s h e d in s o d i u m - f r e e m e d i u m . Coupled w i t h t h e a n a l y t i c a l r e s u l t s t h i s s u g g e s t s t h a t fluid s e c r e t i o n in t h i s tissue is d r i v e n b y a c t i v e sodium transport.

The effects of e~metinl]~ydrochloride on!brain/proteLn ~ynthesis and on dark avoidance response in the/goldfish S. Ohi z'z

Department o] Psychology, Faculty o/ Education, Tokyo University o/ Education, Ohtsuka, Bunkyo-ku, Tokyo 7 72 (Japan), 28 January 7977 Summary. E m e t i n e h y d r o c h l o r i d e a t 100 txg i n j e c t e d i n t r a c r a n i a l l y b l o c k e d m o r e t h a n 8 0 % of p r o t e i n s y n t h e s i s of t h e brain go-no tilled agent

i n t h e goldfish for a t l e a s t 4 days, b u t w a s n o t lethal. T e s t i n g t h e effect of e m e t i n e h y d r o c h l o r i d e o n d a r k a v o i d a n c e go learning, t h e fish i n j e c t e d w i t h e m e t i n e h y d r o c h l o r i d e s h o w e d p o o r e r p e r f o r m a n c e t h a n t h o s e i n j e c t e d disw a t e r t h r o u g h o u t t h e e x p e r i m e n t , e x c e p t o n t h e 1st day, s u g g e s t i n g p o t e n t i a l i t i e s of e m e t i n e as a new b l o c k i n g of m e m o r y c o n s o l i d a t i o n .

I t is g e n e r a l l y b e l i e v e d t h a t m e m o r y c o n s o l i d a t i o n is i n h i b i t e d b y t h e b l o c k a g e of p r o t e i n s y n t h e s i s in a n i m a l s 3. T h e m e c h a n i s m s of c h e m i c a l a c t i o n s of p r o t e i n s y n t h e s i s blockers, h o w e v e r , are n e i t h e r simple n o r y e t c o m p l e t e l y clarified. C y c l o h e x i m i d e 4-~ a n d a c e t o x y c y c l o h e x i m i d e s - n , for e x a m p l e , h a v e b e e n said to b l o c k d i r e c t l y m e m o r y c o n s o l i d a t i o n t h r o u g h t h e i n t e r r u p t i o n of p r o t e i n s y n thesis, w h e r e a s t h e i n h i b i t o r y a c t i o n s of p u r o m y c i n z2, la are less clear t h a n t h a t of o t h e r a n t i b i o t i c s . T h u s , t h e r e l a t i o n b e t w e e n p r o t e i n s y n t h e s i s a n d m e m o r y consolid a t i o n h a s b e e n s u g g e s t e d b y m e a n s of o n l y a few specific drugs, b u t is n o t g e n e r a l i z e d u p to now. So, in t h e p r e s e n t e x p e r i m e n t s , e m e t i n e h y d r o c h l o r i d e (E-HC1), w h i c h is a n ipecac a l k a l o i d a n d is r e p o r t e d t o b l o c k tile p r o t e i n s y n t h e s i s for long d u r a t i o n z4, was used for tile first successful a t t e m p t t o c l a r i f y r e l a t i o n s b e t w e e n its i n h i b i t o r y effects on protein synthesis and blocking actions on memory consolidation.

(6)

o=f 0-20[ *

0.16 0.12 008 i l,s, ,,, f&2~L_.r

00~

t

0

= r

i

3

I

~

2'4

A

72

Hours between emetine and 3H-leucine injections Fig. 1. The time course of protein inhibition in goldfish brain resulting from an intracranial injection of emetine hydrochloride (100 ~.g]10 ~1). Abscissa 0 is the control injection of 5 ~tCi of aH-leucine. Number of fish for each point is shown in parentt/eses. Dots and bars represent, respectively, means and standard errors of P/(P+S).

Materials and methods. T h e s u b j e c t s u s e d were goldfish, 9.5-11.0 c m long, in t h e s e e x p e r i m e n t s . T i m e course of i n h i b i t i o n of p r o t e i n s y n t h e s i s c a u s e d b y 100 txg/10 tzl of E-HC1 was d e t e r m i n e d i n g o l d f i s h b r a i n . A t 1, 3, 24, 48 or 72 h a f t e r t h e i n j e c t i o n of E-HC1, 5 ~Ci/5 ~xl of Lleucine-4, 5-3H (specific a c t i v i t y 58 Ci/mmole, T h e R a d i o c h e m i c a l Center, A m e r s h a m , E n g l a n d : aH-leucine) was injected. F i s h were d e c a p i t a t e d 30 m i n later, t h e w h o l e b r a i n was q u i c k l y r e m o v e d a n d p r o t e i n was i s o l a t e d as t h e t r i c h l o r o a c e t i c acid (TCA) p r e c i p i t a t e fraction. T h e degree of p r o t e i n s y n t h e s i s w a s d e t e r m i n e d b y t h e following formula : P/(P+ S)

cpm in TCA precipitate (P) epm in TCA precipitate (P) + epm in TCA supernate (S)

W h e n 3H-leucine was i n j e c t e d i n t r a c r a n i a l l y , t h e t e m p e r a t u r e of t h e w a t e r in t h e h o m e t a n k w a s 18~ G o l d f i s h were g i v e n d a r k a v o i d a n c e go-no go t r a i n i n g f o r 3 days. F o r i n t r a c r a n i a l i n j e c t i o n s , a s m a l l hole w a s drilled in t h e c e n t r e of t h e skull b e t w e e n t h e 2 eyes, 3 d a y s before t h e b e g i n n i n g of t r a i n i n g t r i a l s 1~. F i s h were n o t fed. O n t h e p r e v i o u s d a y of t r a i n i n g , fish were d i v i d e d r a n d o m l y i n t o e x p e r i m e n t a l a n d c o n t r o l groups, a n d t h e e x p e r i m e n t a l g r o u p was i n j e c t e d w i t h E-HC1 100 ~g/10 txl in distilled w a t e r , while t h e c o n t r o l g r o u p w a s g i v e n 10 ixl of distilled w a t e r . F o r 3 d a y s of t r a i n i n g , a fish was g i v e n 2 sessions of 20 t r i a l s of d a r k a v o i d a n c e go-no g o ' t r a i n i n g in e a c h d a y w h i c h c o n s i s t e d of 10 go t r a i n i n g t i r a l s a n d 10 no-go t r a i n i n g trials, r a n d o m l y o r d e r e d . I n t e r s e s s i o n i n t e r v a l was 2 min. T h e a p p a r a t u s used was a g r a y p l a s t i c s h u t t l e b o x w i t h a trapezoidal barrierIs. The temperature of water in both apparatus and h o m e tanks was 16.0-18.0~ A n electric light bulb (20 W / 1 0 0 V a.c.) was placed over each compartment at 20 c m from surface of the water. These bulbs were ordinarily lighted and the C S was to put off either of t h e 2 b u l b s for 10 sec. W h i c h b u l b w a s p u t off w a s d e t e r m i n e d in a c c o r d a n c e w i t h b o t h t h e sequence of go a n d no-go t r i a l s a n d w h e r e t h e fish was, a n d it was con-