κ-Opioid antagonist strongly attenuates drinking of genetically polydipsic mice

Brain Research, 546 (1991) 1-7 (~) 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391164814

BRES 16481

Research Reports

c-Opioid antagonist strongly attenuates drinking of genetically polydipsic mice Toshihiko Katafuchi, Yukio Hattori, Itsugi Nagatomo and Kiyomi Koizumi Department of Physiology, State University of New York, Health Science Center at Brooklyn, Brooklyn, N Y 11203 (U.S.A.)

(Accepted 30 October 1990) Key words: Genetic polydipsia; Mouse; Opioid receptor; Naltrexone; Opiate; Water metabolism

Effects of opioid antagonists on the genetic polydipsia of the STR/N strain of mice were investigated. Naltrexone (0.5-5.0 mg/kg) injected subcutaneously before dark period attenuated spontaneous drinking for the first 3 h after injection only in the inbred polydipsic mice (STR/N), whose water intake was 5 times that of controls (non-polydipsic mutant, STR/1N, and Swiss/Webster mice). The highest dose (5 mg/kg) of naltrexone administration reduced drinking also during the next 3-6 h period and overnight feeding. Cerebroventricular (i.c.v.) injection of naltrexone, 1.0 and 2.5 pg (per mouse), suppressed drinking only in the polydipsic mice, while the higher dose (5.0/~g) attenuated drinking and feeding of both the polydipsic mice and their controls. However, i.c.v, injection of specific r-receptor antagonist, nor-binaltorphimine (nor-BNI, 0.5-2.5/~g), suppressed drinking only in the polydipsic strain of mice at one-half dose of that needed for naitrexone. Furthermore, even a higher dose of nor-BNI administration was without effect on food intake in all strains. These findings suggest that the central opioid system plays an important role in causing the polydipsia in the STR/N mice, probably through the r-opioid receptor. INTRODUCTION In the late 1950's the first studies z7 were made on an inbred strain of mice, STR/N, having an extreme polydipsia and polyuria with low specific gravity and electrolyte concentration. Hybridization studies suggested that the hereditary polydipsia appeared to have a recessive genetic character 26. Subsequent studies revealed that STR/N mice possess normal plasma osmolarity and sodium concentration, and renal capability of producing a highly concentrated urine under water deprivation and by exogenous vasopressin. No gross histological abnormality was detected in the pituitary, the adrenal, and the salivary glands, and in the supraoptic and paraventricular nucleus of the hypothalamus normally abundant neurosecretory substance in axons and cell bodies was present 28. The polydipsic mice showed high mortality only in males due to hydronephrosis caused by combined presence of polyuria and urethral plugs, which nonpolydipsic mice also had but without causing hydronephrosis. Since these mice survived better when the water intake was restricted and did not die from dehydration, it was concluded that the polydipsia in the STR/N mice was probably due to an abnormally increased appetite for

drinking which was unnecessary for survival 25'27'28. However, the mechanism of polydipsia in this strain of mice has not been studied extensively since the early 1960's. Many studies to clarify mechanisms of drinking behavior have been carried out in the last two decades which have indicated involvement of angiotensin II as an endogenous dipsogenic agent 5'6"14. Recently, we have shown that in the polydipsic mice subcutaneous injection of captopril, an angiotensin-I-converting enzyme inhibitor, reduced drinking to two-thirds of that after vehicle injection without affecting food intake 1~. On the other hand, the endogenous opioids have also been considered to play a role in control of drinking and feeding behavior 4'18"24. Interestingly, involvement of endogenous fl-endorphin in overeating of genetically obese mice (ob/ob) has been suggested 15. In the study presented here, we investigated the effects of opioid antagonists injected subcutaneously (s.c.) or intra-cerebroventricularly (i.c.v.) on the spontaneous drinking and feeding of the polydipsic and control mice to evaluate the contribution of endogenous opioids to the excessive drinking. A preliminary report of this study was previously presented 9.

Correspondence: K. Koizumi, Department of Physiology, Box 31, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203, U.S.A.

injection of nor-BNl (0, I).5, 1.0 and 25 ~tg per mouse: n = 7: for each strain) was the same as that f~r naltrexone i.c.v, injection.

MATERIALS AND METHODS

Daily water and food intake The polydipsic STR/N mice and their controls, STR/1N, a non-polydipsic mutant of the STR/N, and Swiss/Webster (S/W) mice of both sexes, 6-12 months of age were used. They were kept in individual cages in a room maintained at 22-24 °C, constant humidity and illuminated from 07.00 h to 19.00 h. Tap water and food (standard laboratory chow) were available ad libitum. Water intake was measured at 10.00 h and 18.00 h daily by weighing a small drinking tube fitted with a stainless steel spout. Food consumption and body weight were measured once a day at 10.(X) h. The measurements were made to the nearest 0.1 g.

Administration of naltrexone: subcutaneous injection Effects on drinking of an opioid antagonist, naltrexone, during dark period under non-deprived conditions were examined in 3 groups of mice (n = 8 for each group). Naltrexone hydrochloride (Sigma) at doses of 0, 0.5, 3.0 or 5.0 mg/kg in 1 ml saline per 100 g body weight was injected subcutaneously between 18.00 and 19.00 h. Animals were immediately returned to the home cage and drinking volume was measured at 3, 6 and 16 h after the injection. Amount of food intake for 16 h until 10.00 h the next morning was also measured. Some animals received two doses of naltrexone with the interval of at least more than one week.

Statistical analysis Water and food intake data were converted to ml and g consumed per 30 g of body weight, respectively. Differences in daily water and food intake as well as light-to-dark (L/D) or water-to-food (W/F) ratios were evaluated by one-way analysis of variance (ANOVA) with repeated measures. Effects of drugs on cumulative water and food intake were analyzed by two-way ANOVA with repeated measures. After ANOVA test, comparison of values across groups was further evaluated by Newman-Keuls' multiple-range test at a significance level of 0.05. RESU EI'S

Daily water and f o o d intake Table I s h o w s the daily a m o u n t of d r i n k i n g a n d f e e d i n g calculated f r o m the a v e r a g e v a l u e for 3 days of e a c h animal,

randomly

control

mice.

sampled

Water

from

intake

of

8 each the

STR/N

polydipsic

and mice

( S T R / N ) d u r i n g b o t h L ( 1 0 . 0 0 - 1 8 . 0 0 h) a n d D ( 1 8 . 0 0 10.00 h) p e r i o d was a p p r o x i m a t e l y 5 t i m e s that o f t h e

lntraventricular injection of naltrexone Under pentobarbital (50 mg/kg) anesthesia, 30 gauge stainless steel cannula was implanted into the right lateral cerebroventricle stereotaxically. The coordinates of the cannula were: A, 3.8; L, 1.0; H, 3.5-4.0, according to the atlas of Montemurro and Dukelow ~7. Successful implantation was confirmed by leakage of cerebrospinal fluid (CSF) from the external tip of the cannula. After the cannula was cemented on the skull, a stainless steel wire (100 /~m in diameter) was inserted and protective polyethylene cap was placed to prevent leakage of CSF and obstruction of the cannula. After one week recovery period from surgery, naltrexone at doses of 0, 1.0, 2.5 and 5.0/~g in 0.5 kd artificial CSF (aCSF) per mouse was injected to the 3 groups of mice (n = 6, for each strain) between 18.00 and 19.00 h. Composition of aCSF was Na ÷, 144 mM; CI , 133 mM; K ÷, 4 mM; Ca 2+, 2.1 mM; Mg 2+, 1.3 mM; HCO 3, 20 mM; phosphate, 1 mM; and glucose, 5 mM. Injection was made within 2 s by microsyringe attached to the ventricular cannula through polyethylene tubing (PE 10) while an animal was restrained by an experimenter's hands. The whole procedure of the injection was done within 30 s. Animals were then returned to the home cage, and drinking volume was measured at 0.5, 1, 1.5, 2, 3, 6 and 16 h after the injection. Amount of food intake for 16 h after the injection (until the next morning) was also measured.

controls ( S T R / 1 N and S/W). T h e r e was no d i f f e r e n c e b e t w e e n the S T R / 1 N a n d S / W ( P > 0.05). W a t e r i n t a k e ratio of light and d a r k p e r i o d s ( L / D ) was n o t d i f f e r e n t a m o n g t h e 3 g r o u p s of m i c e ( F = 1,15; df 2/21, P > 0.05), suggesting that diurnal p a t t e r n was m a i n t a i n e d in t h e STR/N.

This was

already

demonstrated

by d r i n k i n g

p a t t e r n analysis using a d r o p c o u n t e r 1~. T h e r e was n o d i f f e r e n c e in a m o u n t o f f o o d i n t a k e a m o n g t h e s e g r o u p s ( F = 0.35, df 2/21, P > 0.05). T h e r e f o r e , t h e

water-

t o - f o o d i n t a k e ( W / F ) r a t i o o f t h e p o l y d i p s i c strain was a p p r o x i m a t e l y 5 t i m e s that of t h e c o n t r o l s .

Effect o f naltrexone administered subcutaneously In the polydipsic m i c e , w a t e r i n t a k e d u r i n g t h e first 3 h after s.c. i n j e c t i o n o f n a l t r e x o n e h y d r o e h l o r i d e was significantly ( F = 12.90, df 3/28, P < 0 . 0 0 t ) r e d u c e d (Fig. 1, top). N e w m a n - K e u l s '

m u l t i p l e - r a n g e analysis for the

first 3 h i n d i c a t e d that t h e effects of n a l t r e x o n e at all

Intraventricular administration of r-opioid antagonist, nor-BN1 Specific opioid antagonist, nor-binaltorphimine (nor-BNI, Research Biochemicals, Nattick, MA) 21'22'23 was administered to the polydipsic and the control mice. Experimental procedure for i.c.v.

doses w e r e d i f f e r e n t f r o m that o f saline (0,5 m g / k g , P < 0.05; 3.0 m g / k g , P < 0.01; and 5.0 m g / k g , P < 0.01). In a d d i t i o n , t h e effect of 5.0 m g / k g n a l t r e x o n e was d i f f e r e n t

TABLE 1

Daily water and food intake of polydipsic and control mice n = 8 for each strain. Values are ml and g per 30 g body weight and expressed as mean + S.D. L = light period, 10,00-18.00 h; D -- dark period; 18.00-10.00 h; L/D = ratio of water intake during the light period to that in the dark; W/F --- ratio of water to food intake.

Water intake (ml)

STR/N STR/1N S/W

Food intake (g)

L

D

total (W)

L/D

F

W/F

4.0 + 2.1 0.8 + 0.2 0.8 + 0.4

25.8 + 4. l 4.4 + 0.6 5.0 +_ 1.1

29.8 _+ 5.4 5.2 _+0.8 5.8 +_ 1.3

0.16 + 0.07 0.19 + 0.06 0.15 + 0.06

4.0 +_0.7 3.6 +_0.5 3.6 + 0.5

7.9 ___2.9 1.7 _+0.6 1.5 + 0.2

18

from that of 0.5 (P < 0.01) and 3.0 mg/kg ( P < 0.05), as related. During the 3 - 6 h p e r i o d after s.c. naltrexone, water intake was significantly ( F = 8.97, df 3/28, P < 0.01) reduced by 5.0 mg/kg naltrexone ( 3 - 6 h of Fig. 1, top). During the period of 6-16 h after injection no overall difference was observed between the effect due to different dosage ( F = 1.09, df 3/28, P > 0.05), though the amount of water intake a p p e a r e d to be reduced (Fig. 1). Overnight food intake after a dose of 5.0 mg/kg was reduced ( F = 5.42, df 3/28, P < 0.01) c o m p a r e d with that after saline (P < 0.01, multiple-range test) and it was also different c o m p a r e d with the values after 0.5 mg/kg ( P <

18 16 14 12 10 8 6

STR/N

I~ I

SALINE 0.5 mg/kg 3.0 mg~kg 5.O mg/kg

4

2 0 ~-3 hr

3-6

hr

ii

6 - 1 6 hr

LU Nt"

<~ z t'r iii I--

<

/

,

~

14 12 10 8, 6 4

2 0.-2

~ : . ~ " ' ~ ' " ~' '

,v

0

S, O

P<0.001

1

2

3

4

5

6 hr

8 STR/1N

7

E

6

tu v < Iz

5 3

rr

2

4

III

<

4

1

. : ........ 1

3 2

8,

1

7

2

P
4

5

6

hr

o - - o oCSF } ~'- - ~' 1"9 ~ l Naltrexone o - - o z.~/,~ i

S/W

• . . . . * 5.0 ~ j }

6

F.I.

5 4

S~ O ¢o

STR/N

16

well as from saline, indicating that the effect was dose

8

S~ O

7 6 5

3

v

STR/1N

iii

4

4

na

3

"3

2 1 0 0 - 3 hr

3-6

hr

6 - 1 6 hr

F.I.

,v, < l--

z

1:3 o O ii o

8

..; .........

0 0

1

2

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

3

4

.<0.01 5

6 hr

Fig. 2. Cumulative water intake (mean + S.E.M., n = 6, for each group) of polydipsic (STR/N, upper) and control mice (STRJlN, middle and S/W, bottom) for 6 h after i.c.v, injection of naltrexone hydrochloride. In the STR/N, all doses of naltrexone reduced drinking after injection, while in control mice, only a dose of 5.0/~g naltrexone decreased drinking significantly (two- and one-way ANOVA followed by Newman-Keuls' test).

7 6 S 4

S/W 4"

3 2 1

0

0 - 3 hr

3-6

hr

6 - 1 6 hr

lili F,I.

0.05) and 3.0 mg/kg ( P < 0.05) of naltrexone. In the control strains, s.c. injection of naltrexone was without effect on water and food intake (Fig. 1, middle and bottom).

0

Fig. I. Water and food intake (EI.) (mean + S.E.M., n = 8, for each group) of polydipsic (STR/N, upper) and control mice (non-polydipsic mutant, STR/1N, middle; and Swiss Webster, S/W, bottom) after s.c. injection of naltrexone hydrochloride. In STR/N, all doses of naltrexone reduced drinking for the first 3 h after injection. The second 3 h drinking and overnight feeding (16 h) were reduced only at a dose of 5 mg/kg (one-way ANOVA followed by Newman-Keuls' multiple-range test, *P < 0.05 and **P < 0.01). In control mice (STR/1N and S/W), naltrexone was without effect on water and food intake. Note the difference in scales for water intake (ordinates of the top and two lower graphs) in this and all subsequent graphs.

Effects o f i. c.v. administered naltrexone Cumulative water consumptions for each dose of the drug are shown in Fig. 2. Two-way A N O V A indicated that i.c.v, injection of naltrexone significantly reduced water intake for 6 h following the injection in all strains of mice (STR/N, F = 44.35, df 3/120, P < 0.001; STR/1N, F = 26.13, df 3/120, P < 0.001; S/W, F = 16.03, df 3/120, P < 0.001). A multiple-range test indicated that in the polydipsic mice the effect of all doses of naltrexone during 1-6 h after injection differed from that of vehicle

STR/N

18

35 30

16 STR/N

_t~

25 20

,,

4

10

3 2

5

1

2. =,_.-~-, , . . * ........ . .

0 -'"~' • 0 1

0

8

W.I.

F.I.

60"

i,-

3z 2o 1 o O 0 u-

W.I.

S/W

< 1.-z nW p<

lnl/

0

4

4

3 2

3 Z

3 2 1

1

1

0

0

W.I.

0

4

5

6 hr

2 1, 0 1

2

3 a~o

S/W

7 6 5 4

7 ]Naltrexonelt.~.~3 1 0 u,~ I~ 2:5~ 6 /l I m 5.0

P<0.001 6 hr

4

8'

°c,F

5

6 5

F.I.

8]

4

7 STR/1N

O ¢O

uJ

.

8.

s,

STR/1N

. . . 2 3

oCSF

A - - ,, 0 . 5 / a 3 ( n o r - B N l o--o 1.0/a~ I • .... • 2.5/.~y

~--a~ 0

1

n.s. 2

3

4

5

6 hr

F.I.

Fig. 3. Overnight water (W.I.) and food (El.) intake (mean + S.E.M., n = 6, for each group) of polydipsic (STR/N) and control mice (STR/1N and S/W) after i.c.v, injection of naltrexone. In the STR/N, overnight drinking was reduced at doses of 2.5 and 5.0/~g, while in control mice, only a dose of 5.0/~g naltrexone decreased drinking after injection. Overnight feeding was reduced at all doses both in the STR/N and controls (one-way ANOVA followed by Newman-Keuls' test, *P < 0.05 and **P < 0.01).

at a significant level of 0.01 (Fig. 2, top); doses of 2.5 and 5.0/~g showed a different effect from that of 1.0 # g in the cumulative 3 and 6 h drinking, but no overall difference b e t w e e n 5.0 and 2.5/tg was observed. On the other hand, in both the non-polydipsic STR/1N and the S/W, multiple-range test indicated that only the highest dose (5.0 /~g) of naltrexone d e c r e a s e d drinking during 1 - 6 h (Fig. 2, middle and b o t t o m ) (1 and 1.5 h, P < 0.05; 2, 3 and 6 h, P < 0.01 in both strains). Overnight water intake (16 h following injection) in the STR/N was significantly ( F = 8.04, df 3/20, P < 0.01) d e c r e a s e d at doses of 2.5 and 5.0 /~g (P < 0.01, multiple-range test) (Fig. 3, top). F o o d intake in the STR/N was also r e d u c e d ( F = 4.22, df 3/20, P < 0.05),

Fig. 4. Cumulative water intake (mean + S.E.M., n = 7, for each group) of the polydipsic ( S ~ , upper)and control mice (STR/1N; middle and SfW, bottom) after i.c.v, injection of nor,hinaltorphimine (nor-BNI). In the STR/N, doses of 1.0 and 2.5 a g o f nor-BNI reduced drinking from 1 through 6 h. No change occurred in STR/1N and S/W (two- and one,way ANOVA followed by Newman-Keuls' test).

and a multiple-range test indicated that all doses of naltrexone differed from a C S F at a significant level of 0.05. In control strains, only the highest dose (5.0/zg) of naltrexone r e d u c e d overnight drinking significantly (STR/1N, F = 5.28, df 3/20, P < 0.01; S/W, F = 5.09, df 3/20, P < 0.01). O v e r n i g h t f o o d i n t a k e was a l s o reduced (STR/1N, F = 16.95, df 3/20, P < 0.001; S/W, F = 4.15, df 3/20, P < 0.05) at all doses of naltrexone (STR/1N, P < 0.01; S/W, P < 0.05) (Fig. 3. middle and b o t t o m ) .

Effects of i.c.v, administered K-opioid antagonist I.c.v. administration of a r - a n t a g o n i s t , n o r - B N I , reduced water intake in the potydipsic but not in control mice (Fig. 4). T h e r e was overall dose effect ( F = 30.18, df 3/144, P < 0.001) only in the STR/N. A multiple-range

DISCUSSION

35 30 25 STR/N

4

20 15 10

2

5

1

0

0 W.I.

F.I.

T~ 0

7 -~ STR/1N

6

111

uJ

z_ cr

w k-<

W.I.

IM

4 < t--.

3z 2c~ 1 o

o OLL

F.I.

8,

7 nor-BNI 6

S/W

5 4

3. 2. 1

0.5 #g 1.o #g 2.5 #g

1111l ll W.I.

F.I.

Fig. 5. Overnight water (W.l.) and food (F.I.) intake (mean _+ S.E.M., n = 7, for each group) of the polydipsic (STR/N) and control mice (STRJlN and S/W) after i.c.v, injection of nor-BNI. Overnight drinking was significantly reduced at doses of 1.0 and 2.5 /tg, in the STR/N, but not in the controls. Any dose of nor-BNI was without effect on overnight feeding both in STR/N and control mice (one-wayANOVA followed by Newman-Keuls' test, *P < 0.05 and **P < 0.01).

test showed that the effect of 0.5 ~g was not significantly different from that of aCSF throughout the experimental period but 1.0 ~g was effective for 2-6 h (P < 0.05 at each point), and 2.5/~g for 1-6 h (P < 0.01 at each point) (Fig: 4, top). In both control strains cumulative water intake for 6 h was not altered by nor-BNI at all doses. For a 16-h period cumulative water intake in the STR/N mice was also reduced significantly by the r-antagonist at doses of 1.0 and 2.5/tg (F = 6.77, df 3/24, P < 0,01; 1.0 ~tg, P < 0.05 and 2.5 ~g, P < 0.01). In contrast, nor-BNI was without effect on food intake in the STR/N (Fig. 5, top). In the control mice both water and food intake for the 16-h period showed no effect of nor-BNI administration (Fig. 5, middle and bottom).

Previous studies in our laboratory have shown that the extreme polydipsia of the STR/N strain may involve, at least in part, the angiotensin II (ANG II) system in the brain ~. However, since the reduced water intake after captopril or an ANG II analogue was still larger in the polydipsic mice than in the controls, it was suggested that central mechanisms other than the ANG II system might contribute to this polydipsia. The present results of naltrexone injections (s.c. or i.c.v.) indicate increased sensitivity of the polydipsic mice to administered opioid antagonists and suggest that perturbation of the endogenous opioid system(s) could participate in causing the polydipsia of the STR/N strain. Similar changes were demonstrated in feeding of genetically obese rats and mice J5'23'34. In normal non-polydipsic rats naloxone or naltrexone has been found to reduce both drinking and feeding in deprived and non-deprived states 17"35, and these effects are thought to be mediated by opioid receptors in the central nervous system 2'3'e9. Our results showed that in non-polydipsic controls, the STR/1N and the S/W, only the highest dose (5.0/~g) of i.c.v, naltrexone decreased drinking, while the overnight food intake was reduced at all doses of naltrexone (Figs. 2 and 3), suggesting that the threshold for suppressive effects of the opiate antagonist on feeding is lower than that on drinking. This also implies that the effect of the opiate antagonist on drinking may be separated from their effect on feeding. It has been demonstrated that/~- 5- and ~:-receptors are all involved in the regulation of feeding behavior TM 16.19,30 There is, however, controversy concerning the types of opiate receptors that regulate drinking. Some investigators speculate that the effect of opioid antagonist on drinking is mediated through ~:-receptors 12 while others contend that fl-endorphin, the endogenous/~- and 6-1igand, is more effective in stimulating water intake than the ~¢-ligand, dynorphin 8. In the present study, the specific ~¢-antagonist, nor-BN121'22'23, administered i.c.v. reduced water intake only in the polydipsic mice without affecting food intake (Figs. 4 and 5). In the STR/N mice, an effective dose of nor-BNI for drinking suppression was 1.0/tg which equals 1.24 nmol, while the effective dose of naltrexone was 1.0/~g, 2.65 nmoi. Food intake was affected by i.c.v, naltrexone at 2.65 nmol (1.0/~g) while nor-BNI up to 3.10 nmol (2.5 ~g) had no effect. Moreover, nor-BNI reduced drinking of the polydipsic mice to 25% of pre-injection level or close to that of the control mice during the first 6 h after injection, while it caused no change in water or food intake in the control strains of mice. Although we did not test effects of other ~c-antagonists, nor-BNI is thought to

be the most selective ~¢-antagonist2~'2:, and it is possible

(Kawata, M., Yamashita. H. and Koizumi, K,, unpub-

to suggest that the K-type of opioid receptor mediates most of the polydipsia of the STR/N mice. L e a n d e r and Hynes ~2 suggested that the dipsogenic

lished observations). It is possible that e n d o g e n o u s substances in the brain other than the opioids, such as AII as previously

action of the endogenous ligand for the ~c-receptor,

m e n t i o n e d , may also be involved in this a b n o r m a l

dynorphin-Al_t32°, may be due to its inhibitory effect on vasopressin release from magnocellular cells in the hypothalamic paraventricular and the supraoptic nuclei, where dynorphin coexists with vasopressin 36 and acts on

drinking. A n interaction between opioid and angiotensin II system is also known to exist 3t'~2. Further investigation will be required to clarify mechanisms of this intriguing

an inhibitory autoreceptor ~°. However, the STR/N mice

p h e n o m e n o n of the polydipsia. Such studies will provide better understanding of the central mechanism underly-

could concentrate their urine when water deprived, and they do survive even longer under water restriction 2~.

ing the regulation of thirst and drinking behavior,

Thus, it is unlikely that the release of vasopressin is

Acknowledgements'. We express our appreciation to the National Institute of Health, USPHS for Grant support (No. NS-00847) for this study, to the Japanese Government for Investigator Fellowship (to I.N.); to Dr. Emanuel Silverstein for supply of STR/N and STR/1N mice. to Dr. Alan Ginzler for his help and advice for the experiments, and to Dr. P.S. Portoghese for his advice on the use of nor-BNl. The STR/N and the STR/IN were originally obtained from the National Institute of Health. Bethesda, MD.

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