Document mpKjZjjkZzq9wOXByqRv5bY5k

'fCB -<TAS5 712 (Tjpt L*ty " III - 'O Water Deprivation and Trichloroethylene Effect on Hypothalamic Self-Stimulation Anna M. Baetjer, ScD; Zoltan Annau, PhD: and Helen Abbey, ScD, Baltimore Rats with implanted hypothalamic electrodes were trained to press a lever for electrical brain stimulation during a daily 30 minute test period. In rats continuously deprived of water for three days, the rate of lever-pressing declined slightly on the first day but remained at that level during the next two days. Exposure of nondehydrated rats to high concentrations of trichloroethylene during the text periods markedly depressed the rate of pressing on three consecutive days. In rats de prived of water for three days and exposed to trichloroethylene daily, the onset o( depression was delayed during, each test period, and greater tolerance to trichloroethylene developed than in nondehydrated rats. When these rats were later deprived of water for ten days, the rate decreased progressively from days S to 10, which was not characteristic ot control rats. VIOUS experiments in this laboratory demonstrated that general body dehydration produced by deprivation of water for 48 hours increased susceptibility of rats and mice to the acute toxic effects of lead and antimony.1-3 To determine whether this effect was specific for lead and antimony or was a general physiological property ap plicable to all types of toxic chemicals, a Submitted for publication Sept 27, 1969; accepted Jan 20, 1970. From the Department of Environmental Medi cine, School of Hygiene and Public Health, the Johns Hopkins University, Baltimore. Head before, the. 63rd. .annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City, NJ, April 17, 1969. Reprint requests to Department of Environmental Medicine, the Johns Hopkins University School of Hygiene and Public Health, 615 N WoJfe St, Baltimore 21205 (Dr. Baetjer). scries of experiments was undertaken with chemicals of different toxic properties. Trichloroethylene was choeen as a test chemical because, unlike the other com pounds studied, it is insoluble in water, solu ble in lipids, and, in acute doses, exerts its effects on the central nervous system as a narcotizing agent The operant behavior technique of hypothalamic self-stimulation was selected as the measure of central ner vous system function. Although no experiments on the effects of trichloroethylene on self-stimulation have been found in the literature, the role of this compound in other behavioral responses of rats has been studied with varied results. Exposure of rats for three hours to 200 and 800 ppm of trichloroethylene did not modify a conditioned food-motivated, rope-climbing response but stimulated spontaneous climb ing.4 Subjection to 2,500 to 5,500 ppm in creased the latent period and finally inhib ited a food-motivated conditioned response.6 Spontaneous running was lowered during five hours' exposure to 1,600 ppm and swim ming endurance decreased after six hours of exposure at 800 ppm.6 Exploratory behavior in a maze was enhanced after eight hours of exposure at 600 ppm but decreased at 200 and 1,600 ppm.7 At higher concentrations of trichloroethylene, impairment usually pre dominated. -* Studies on hypothalamic self-stimulation during water deprivation have been report ed. Twenty-three hours of water deprivation was without effect in two groups of rats tested at different lever-pressing rates.8 A , Arch Environ Health--Vul 20. June 1970 WATER DEPRIVATION AND TRICHLOROETHYLENE--BAETJER ET AL 713 kTable 1.--Effect of Dehydration and Trichloroethylene on Hypothalamic Self-Stimulation in Hats Group No. of rats Mean ot 3 normal preexposure non* dehydration days Exposure or dehydration* days or both 1 2 3 Recovery days i 2 3 Not Exposed to T rtchlofoethylene Nondehydrated (Controls) De hydrated* Exposed to Trichloroethylene Hieh Concentration Moderate Concentration Non- De dehydrated hydrated* Non- De dehydrated hydrated* 27 36 14 13 19 22 Mean No. of Lever-Presses per 10-Minute Test Period 1,867 2.020 1,955 1 979 1.984 2,119 1.869 1,968 1.889 ... 1,833 1,782 1.S57 ... ... ... 759 891 948 1.734 1,868 1,904 1,038 1.040 9S3 1,744 1,871 1,859 1,298 1,704 1,873 1,631 1,765 1,881 1,538 2,069 2,172 2.011 1.943 1,997 * Continuous water deprivation during three days, thus corresponding to 24, 48, and 72 hours' dehydration by first, second, and third exposure day, respectively. few cats and rats increased their lever-pressing rate after 48 hours of water and food deprivation,* probably as a result of food deprivation, which leads to an increase in the rate of self-stimula tion.* Rats deprived of water A^from 23 to 71 hours, ^Bn given a choice of water or self-stimulation as a re ward for pressing a lever, preferred water at low-current levels and self-stimula tion at higher current lev. els.10 Dehydration, even for nine days^was without any measurable effect on the ac tivity of the rats.1112 Since no reports in the literature have been found on the effects of dehydra tion on the reaction to a narcotizing agent such as trichloroethylene, the present studies were undertaken using hypothalamic self-stirauiation in rats as a behav ioral measure of these ef fects. 450 400 350 300 * !*y250 ------- x NORMAL DAYS DEHYDRATION DAYS 50 100 - *---NQRK'Al - VFAN - - DAY ! X---- x DAY 2 A 'ADAr 3 5 DAYS 50 Fig 1.--Effect of water depriva tion for one, two, and three days, respectively, on hypothalamic self stimulation. 0-5 5-10 10-15 15-20 20-25 25-30 MINUTES Arch Environ Health--Vol 20, Jane 1970 SL 033957 -r, wv-r .-- ..T iwn.'i -4] ry -V-t '.'Xj.i'.'i.- -.vV1'H'tf '.r -i*.^.'-TiAkAtow-i.D ---.v"V.:Vv ^ 'IT--: .Y : Is' '> -**.- w*'-*V* ***** F -Y - -y^cyi. 7X4 WATER DEPRIVATION AND TRICHLOROETHYLENE--BAETJER ET AL Methods were fed rat chow pellets and allowed water ad lib except during the test periods and in the Operant Behavior Tests.--Bipolar electrodes dehydration experiments. Hematocrit values were implanted in the lateral hypothalamic were determined in a group of normal rats de area of male rats receiving pentobarbital so hydrated for 48 hours. The rats were weighed dium. Following recovery, the rats were trained daily. in a modified Skinner box to "self-stimulate," The same procedure was followed in each ie, to press a lever which, hy dosing a circuit, experiment. Two groups of rats were tested in delivered a one-fourth second, 60-cps pulse from the self-stimulation chamber for 30 minutes a constant current stimulator to the hypothal daily for a period of nine days. During the first amic area. The rate of lever-pressing could be three days, the normal rate of lever-pressing controlled within limits hy adjusting the was obtained so that each rat could serve as its strength of the current and remained fairly own control. During the next three days, one constant under stable conditions. A normal rate group of rats was continuously deprived of of approximately 70 lever-presses per minute water, thus corresponding to 24, 48, and 72 over a 30-minute test period was used except in hours of dehydration. On each of these three one experiment in which the rate was S5 lever- days this dehydrated group and the second or presses per minute. The number of lever-press nondehydrated group were exposed to trichlo- es was recorded by five-min ute intervals. For the trichloroethylene exposures, the solvent vapor Fig 2.--Effect of high concentration of trichloroethylene vapor on hypothalamic self-stimulation in nondehydrated rats. was added to the air supplied to the test chamber at a rate calculated to produce a con centration of approximately 3,000 ppm for a heavy ex posure and 2.300 ppm for a more moderate exposure. These levels represent the most severe single exposures which do not ,cause death or detectable organic injury in rats in less than five and ten hours, respectively.1- To mini mize the effect of any possible variation in the concentration of vapor or temperature in the test chamber, dehydrated and nondehydrated rats were tested simultaneously in duplicate chambers supplied by the same air-vapor mix ture or were alternated when using the same test chamber. The air flow into the test chambers was the same during the normal preexposure and recovery periods as during the trichloroethylene exposure periods. The chamber temp erature could not be con trolled but was constant on any one day and averaged 83 3 F (28.3 1.6 C) for all experiments. Dehydration was produced in the rats by continuous de privation of water. The rats Arch Environ Health--Vol 20, June 1970 033958 t'.irr:V ... .. ^ .' *'* ET- gg WATER DEPRIVATION AND TRICHLOROETHYLENE--BAETJER ET AL 715 W&&V.T-u ^*S^r-rii^,''r- * -Vi*^.7-.J-*^ a ' NORMAL-MEAN 3 DAYS Dehydration by continu ous water deprivation for 24, 48, or 72 hours produced a body-weight loss of 7%, 12%. and 16.5%, respec tively. Forty-eight hours of 350 water deprivation in a group NORMAL DAYS of normal rats produced a .A. ,x--^ change in the hematocrit value from 49.7% to 57.6%. ,.' .*____ ._____ RECOVERY DAYS The mean number of lever-presses for the 3U-min- ute test period is given for. each group in Table 1 for the normal, dehydration, ex posure, and recovery days, respectively. The number of lever-presses in the rats sub ject to dehydration alone fell significantly below normal on the first dehydration day T',Wr~ - (P = < 0.01) but did not 100 continue to fall as dehydra tion was continued during 50 EXPOSURE DAYS the next two days. The de crease in the rate of leverpressing on the first day occurred chiefly during tire last half of the test period *v5 ,.,.5-J0A WM$-^5-20 ^0-^ 5-30 *15i-r ~ '' '--V' '-*,>**&.--- -, - MI-N- .U.'t-Ta.'iEwLS/^r."7` . - .-v .... (Fig 1). Exposure to a high concentration of trichloro ethylene in both nondehy Fig 3,--Effect of high concentration of trichloroethylene vapor on hypothalamic self-stimulation in dehydrated rats. dra ted and dehydrated rats markedly reduced the total roethylene vapor during the test period. Water was then restored to the dehydrated group. __ Tests were repeated on each group during the L last three days of the experiment to observe the , r&te recovery without trichloroethylene. To determine the effect of dehydration alone, a third group of rats was tested daily on three normal days followed by three deys of dehydra tion. A fourth or control group was tested daily " for six days without any exposure or dehydration. Several months after the end of these experh * ments, all of the rats which survived the trh ' chloroethylene experiments were retrained and ^ tested daily during ten days of continuous J^er deprivation without trichloroethylene ex- Wre- number of lever-presses be low their preexposure levels on each of the three exirosure days (P = < 0.001). The de crease in the rate was not as great in tire dehydrated as in the nondehydrated rats on all three exposure days, but this difference was significant only on the first day (P = 0 05"'. The mte of both the nondehydrated and debydiated rats exposed to trichlo roethylene was markedly lower on all three days than was the rate with dehydra tion alone f P = < 0.005). With tire more moderate concentration of trichloroethylene, the results were similar, although the overall decrement was less. The lever-pressing rate was significantly below the preexposure lev el on the first and second exposure days with the toxic exposure alone (P = < 0.00-5 and Arch Environ Health--Vol 20. June 1970 SL 033959 W- i r- ,w;v .?* y>* <*;* ?'*r"S >-v> {;> .h.'-r,.*vp4--} wK.hr-i-,vi t- .." ';*:. *' ^`"' "'*' "..- k^1'.'' :; 4 / ' - * ';' .' _' >-l. 1' v'/t f*y'r- A Jt* x:lV* > . >. r^V..- r.W ':J2- J-a.v hi. >. x. ->-(a- I ... `rriV-T (Y r- 7i-,./;:. *r-,"r , ^ . .y- \ ;.'.'. , .vr'.'*'.- -r?'''': ..-.N'\.-V-.-;v~-r ;y:; ... ., -r- -r-1*. rm' - w ---- ^>- , .. . " JJ * .^. Ilk T'~"| ^ b*i^m>! *'r^' >-* .Hv' -? i-4-":S,:^-`'-- .V^y-V^ '^-.k- -V .t'V, .' 716 WATER DEPRIVATION AND TRICHLQROETHYLENE--BAETJER ET AL P < 0.05) and on the first exposure day when toxic exposure and dehydration were combined (P *= 0.005 . Again the depression in the rate of lever-pressinc was less in the dehydrated rats than in the nondeh"- drated rats, but the differ ence was not significant. The rats exposed to the moderate concentration of vapor developed a tolerance to the trichloroethylene dur ing the three days of ex posure, ie, the total number of lever-presses was signif icantly higher on the third than on the first exposure day for the nondehydrated rats (P - < 0.05) and the dehydrated rats (P = < 0.01). Rates on the first recovery day were still significantly below preexposure levels in all groups except the nonde- hydrated group exposed to the moderate concentration of trichloroethylene (P = < 0.025). Thus, the effect of exposures persisted at least 24 hours after the exposure ended. Since the rate of lever pressing varied during the 30 minutes of the test, the Fig 4.--Effect of moderate concentration of trichloroethylene vapor on hypothalamic self-stimulation in nondehydrated rats. mean number of lever-presses for each fiveminute interval was compared in the dehy drated and nondehydrated groups. The rate of lever-pressing of control rats either re mained at a constant level or decreased slight ly during a 30-minute test period. Rats not deprived of water when exposed to a high concentration of trichloroethylene vapor ex hibited an immediate and continuous de cline in the rate of pressing during the 30minute test period on all three exposure days (Fig 2). Dehydrated rats similarly exposed showed a decrease below normal in lever-pressing rate during the first five min utes of the test, but, in general, an increase during the second five-minute period, after which their pressing rate declined sharply (Fig 3). Comparison of the change in press ing rate during this five- to ten-minute peri od in the dehydrated and nondehvdrated rats.showed that dehydration significantly delayed the depressing effect of the trichlo roethylene during the early pan of the test period (P -- < 0.01). In the rats exposed to a lower concentration of trichloroethylene, dehydration again delayed, but to a lesser extent, the depressing effect of the trichlo roethylene on the pressing rate (Fig 4 and 5). - - -- - .----- The affect of ten days' continuous water depriva>n on daily hypothalamic self-stim ulation without exposure to trichloroethy lene wife studied in 24 rats which had been used previously in two or three of the Arch Environ Health--Vol 20, June 1970 SL 033960 v> .-\^;"i*": WATER DEPRIVATION AND TRICHLOROETHYLENE--BAETJER ET AL 111 One rat died on the 450 ------------- seventh and one on the tenth day of dehydration. In order to determine if this deter "400' V\ N. ioration with dehydration 'could be bveroome by an in-' crease in the intensity of the brain-stimulating cur jhcv-390- t?*r.v \ 'Kx rent, eight of the surviving rats were retrained several months later at a rate of 85 *V; ""500 ***i/i> lever-presses per minute. They were then subjected to \ dehydration for ten days. No *250 rats died during this peri od, The lever-pressing rate again decreased sharply ^^200 fee;W -, -v ; INKi- )150 life:100 NORMAL "MEAN 3 DAYS *------- DAY I " EXPOSURE a X--------X DAY 2 - DEHYDRATION A......... A DAY 3 . from the fifth to the tenth day of dehydration (Table 2, experiment 2). Comment These experiments demon strated that deprivation of water for 24 hours slightly depressed the rate of self 2R^5o-' stimulation in fats, but con tinued dehydration to 72 hours did not further affect this activity. However, this 40>ill11 5-tO *I0-15 * v^s- * - 20 -20* 25' 25 -30 initial decrease in the rate was statistically significant only in the first experiment, Fig 5.--Effect of moderate concentration of trichloroethylene vapor on "'hypothalamic self-stimulation in dehydrated rats. in which the rates of self stimulation from a large number of rats (36) were - above experiments. Thus, these were older averaged. This decrease in the rate during the -rats in which the electrodes had been im- first 24 hours of dehydration, although pres planted for some months. During this ten- ent, was not significant in the smaller group day period, they lost approximately 35% of of 24 rats dehydrated during the ten-day peri their body weight. These rate showed a de od. No decrement, in hypothalamic self-stimu cline in lever-pressing rate during the 30- lation rate was reported by Mogenson8 during w minute test period on each of the three 23 hours of water deprivation for a group of predehydration days and on each of the ten eight rats in one experiment and 6 of 12 rats dehydration days. The total number of in another experiment, although some de lever-presses per day did not decrease signifi crease was evident in the other six rate of cantly during the first three days of dehydra- the latter group. Thus, it appears that dehy ---tion. as compared with the rate on predehy- dration exerts a minor effect on hypothala =- dration days, but declined sharply from the mic self-stimulation which is evident only ""fifth to the tenth day of dehydration (Table when a large number of rats is used. - 2, experiment 1). The decrease in the lever- Since rate consume less food when de - pressing rate correlated closely with the loss prived of water and since deprivation or - in weight (r = 0.934, P = 0,01). reduction of food intake has been reported Arch Environ Health--Vol 20, June 1970 Si S39 6-Z f*' u, 718 WATER DEPRIVATION AND TRICHLOROETHYLENE--BAET3ER ET AL Table 2.--Effect of Ten Days' Water Deprivation on Hypothalamic Soft-Stimulation Mean No. of Lever Presses per 3Q-M mute Test Period per Day - No. of Rats Normal Day 1 2 3 Dehydration Day 1 2 3 4* 5 6 7 8 9 10 * Tests were omitted on Sundays. Experiment 1 24 ' 1.931 1.842 1,883 1.333 1,759 1,733 1.620 1,631 1,492 1.504 Experiment 2 8 2,783 2.950 3,007 2.845 2.773 2,889 2,731 2.495 2,654 2,238 2.343 2.185 to increase the lever-pressing rate,8 an in crease in rate would have been expected. No measurements were made to relate these behavioral results to the content of water in the brain. However, Jelsma and McQueen14 reported that water deprivation in dogs on a dry diet caused a slight but significant decline in the water content of the brain during the first 24 hours of dehy dration, but no further alteration when con tinued up to 72 hours. If rats resemble dogs in this respect, the slight depression in the Jever-pressing_rate after .24-hours'- dehydra tion could be explained on this basis. Acute exposure of rats to trichloroethy lene vapor, a powerful narcotizing agent, markedly depressed hypothalamic self-stim ulation during a 30-minute test period. In dehydrated rats, the onset of this depressing effect was delayed or preceded by a brief period- of increased self-stimulation. This finding suggests either that the concentra tion of the solvent in the brain cells built up less rapidly in the dehydrated rats or that changes in the brain cells with dehydra tion made them more resistant to the early low concentration of the solvent or both. As the exposure progressed, these differences in responses were no longer apparent. Dehy drated rats also developed greater tolerance to trichloroethylene vapor Lhan did the nondehydrated rats, which indicates that the brains of these animals were more resistant to the chemical. The concentration of trichloroethylene studied in these experiments was much higher but the duration of exposure was much shorter than that used by most pre vious investigators with other types of condi tioned behavioral responses. /The results, however, are comparable. The hypothalamic self-stimulation test used in these experiments has an advantage over ihe food reinforcement behavioral tech niques used in some of the previous studies.4-5 When food reinforcement is used in conjunction with narcotizing agents, the possibility exists that decrements in re sponse are due to-depression of the appeti tive neural mechanism by the narcotizing agent, thus complicating interpretation of the behavioral effects. This factor may be responsible for some of the variations pre viously reported. The activation of the neu ral "reward" circuits, as in self-stimulation, bypasses this problem. Prolonged (ten days) water deprivation pr iduced a progressive decrease in the rate of self-stimulation from the fifth to the tenth day of dehydration- in the-rats which had been subjected several times to trichloro ethylene and dehydration. This result is in contrast to that obtained in other unpub lished studies in this laboratory with ruts which had not been subjected to any pre vious stresses. When these rats were de prived of water for ten days, the lever-pressing rate decreased after..24 hours of dehydra tion but thereafter remained constant. Only In those rats which became too"weak to press the leve*?1did the rate decrease. The dif ference Sf these results suggests the possi- \rch Environ Health--Vol 20. June 1970 5 * `- . ' h. . C ; , , -. - T--4* -j U. i o, '?r *"v ? ,* '*iT i * '. ' "* I WATER DEPRIVATION AND TRICHLOROETHYLENE--BAETJER ET AL 719 bility that repeated exposure to high ooncen- the effect of dehydration on susceptibility to traticnis of trichloroethylene combined with this organic vapor differs, in general, from dehydration may have produced some resid- its effect on acute lead and antimony uai damage. 'However, the difference could "-poisoning.1-3 Thk-result-may be due to a have been due to the-prolonged implantation ? of the electrodes. Further experiments are fc required to determine the cause of the decre* ment in performance of these rats, k These experiments demonstrated that susb-ceptibHity of rate to trichloroethylene as ^measured by the hypothalamic self-stimulat tion technique is not increased by dehydra* difference in the -chemical and physical properties of these materials, such as waterlipid solubility. On the other hand, the brain may be more adequately protected against dehydration than the heart, which is the principal site of acute antimony damage, and the various tissues which are affected by lead. iHion. Other unpublished studies in this labo- k- ratory have shown that deprivation of water Jfor 48 hours did not affect either the mortal#ity or average survival time of rats. Thus, This investigation was supported by public Health Service grant 5 R 01 ES 00144. Technical assistance was provided by Mary Jo George, and Euward L. Brewster, MSc, Naval Ar chitecture, assisted in these experiments. References fl. Baetjer AM, Joerdar SND, McQuary WA: tioned and spontaneous behavior of the rat. J Effect of environmental temperature end humidity Physiol 53:353-354, 1961. tr on lead poisoning in animals. Arch Environ Health 8. Mogenson GJ: Water deprivation and exces ja 1:463-477,1960. sive water intake during self-stimulation. Physiol * 2. Baetjer AM, Horiguehi S: Effects of environ- Behav 4:393-397, 1969. fcmentai temperature and. dehydration on lead poi- 9. Brady JV, et. al: Effect of food and water ie Boning in laboratory animals. Congress series 62, deprivation upon intracranial self-stimulation. J Z^^^pedings of the 14th International Congress on Comp Physiol Psychol 50:194 137. 1957. t^^mpational Health, Madrid, Sept 16-21, 1963. Am- 10. Morgan CM, Mogenson GJ: Preference of "nffian, Excerpt* Medics Foundation, 1963, pp 795- water-deprived rats for stimulation of the lateral 797. hypothalamus rather than water. P$\chonomic Sci 3. Baetjer AM: Effects of dehydration and enviTonmental temperature on antimony toxicity. Arch & Environ Health 19:784-792, 1969. 4. Grandjean-E: Trichloroethylene effects on ani^inal-behavior. Arch Environ Health 1:106-103, I960. V 5. Formanek J, Horvath M, quoted by Grandjean |E: Trichloroethylene effects on animal behavior. feArch Environ Health 1:106-108,1960. 6:337-338, 1966. 11. Campbell BA, Cicala GA: Studies on water deprivation in rats as a function of age. J Como Physiol Psychol 55:763-708, 1962. 12. Bolles PC: Effects of deprivation 'conditions upon the rat's borne cage behavior. J Comp Physiol Psychol 60:244-248, 1965. 13. Adams EM, et al: Vapor toxicity of trichlo roethylene determined by experiments on laboratory 6. Grandjean E: The effects of short exposures to animals Arch Indus.4* Il\g Occtip Med 4:469-493, ^trichloroethylene on swimming performances and 1951. - motor activity at rats. Amer Indusfr H\g Assoc J ^"24:376-379,1963. 14. Jclsma EF, McQueen JD: Effect of experi ment?! water restriction on brain water. J NVuro- r 7. Grandjean E: Effects of a narcotic on condi surg 26:35-40, 1567, )' f f !SJm*f)T crv-> MORALITY Mankind's interest in moral values, paiticularly as they ariect his, relationship to his fellow man, his society, and his God, stretches back at least as far as historical records can carry ns. The great minds of every age have sought answers about the nature, universality, and permanence of moral principles. Yet it often seems that we are no closer to definitive answers than was Socrates when he admitted to Meno that he did not know how virtue is acquired, whether it can be taught, or even what it really tS---The Acquisition and Development of Values, Perspective on Research. Report of a Conference, May 15-17, 1963, Washington, DC, National Institute of Child Health and 3#Human Development, Bethesda, Md. Arch Environ Health--Vol 20 June 1270 I* SL 033963 t*