Pharmacological studies on the behavioral thermoregulation in the salamander necturus maculosus

J therra BJol Vol 6 pp 331 to 319 198! Printed m Great Britain

0306-4565 81 040331-09502 00 ~ Pergamon Press Led

PHARMACOLOGICAL STUDIES ON THE BEHAVIORAL THERMOREGULATION IN THE SALAMJhNDER, NECTURUS MACULOSUS

V i c t o r H. H u t c h i s o n Department o f Zoology, U n i v e r s i t y o f Oklahoma, Ncrman, OK 73019

ABSTRACT The aquatic salamander Necturus maculosus was tested as a model for znvestigatzons of behavioral thermoregulatory responses to drugs which modify thermoregulatzon in endotherms. Anzmals were acclimatized to 15°C and an LD 12:12 photoperzod and placed in linear thermal gradients (5° to 30-35°C). Drugs were given each day for two days and deep body temperature monitored with trailing thermocouples. Prostaglandin E l produced a pronounced long-lasting behaviorsl hyperthermia. Melatonin and chlorpromazine caused significant falls in mean selected temperature (MST). Oxotremorine and ethanol were wzthout effect on MST, whzle scopolamine treatment resulted in decreased MST on Day 1 and a increase on Day 2. Neurotensin produced hyperthermia on Day 2, but not on Day l, an effect opposite that found wlth mammals. Capsaicln caused a pronounced decrease in MST on Day l, followed by hyperthermia on Day 2, a response simzlar to that observed in mammals. The use of ectothermic animal models for investigation of behavioral thermal responses to those pharmacological agents which influence thermoregulation in endotherms may lead to a better understanding of the evolution of vertebrate temperature regulation.

KEYWORDS Thermoregulation; ectotherms, prostaglandin El; melatonin, chlorpromazine; oxotremorzne; scopolamine; neurotensin, capsaicin; ethanol.

INTRODUCTION During the past two decades the traditional division of animals into "poikilotherms" and "homeotherms", or more accurately, ectotherms and endotherms, has become less distznct (Hutehison, 1976). Among the ectotherms we find true endothermic mechanisms such as heat retention in locomotory muscles of scombrid fishes and lamnid sharks (Carey and colleagues, 1971; Graham, 1975; Dizon and Bri]l, 1979); increased metabolic rates for thermoregulation in brooding Indzan pythons (Hutchison, Dowling and Vinegar, 1966; Vinegar, Hutchison and Dowling, 1970), and heat production and retention in moths and other insects for flight (Bartholomew, 1981), for brooding in bees (Heinrich, 197h), sound production in katydids (Heath and Josephson, 1970), and ball making and rolling in dung beetles (Bartholomew and Casey, 1977). Only recently ha~e investigators begun to recognize that behavioral mechanisms are not only of primary zmportance in endotherms, but also are often more sensitive and effective than autonomic responses in maintainlng homeothermy (Atria and Engel, 1980; Satinoff, 1980). Thus a broad comparative approach to discover both the generalized and the more specialized features of the vertebrate thermoregulatory system is needed (Heller, 1980). Such a pursuit zs likely to lead to answers on many specific questions concerning structure and function of thermoregulatory systems as well as on more general questions on the phylogeny of thermal control. An example of specific problems encountered in mammalian studzes that might be solved through study of ectotherms is the-action of ethanol whlch leads to hypothermia in mammals, a widely held view is that the fall in body temperature after ethanol treatment is due to increased radiant heat loss due to peripheral vasodilation, but other evidence suggests that ethanol has a direct effect on neuronal control of body temperature (Lomax and coworkers, 1980). An example of a more general question, which is likely to be answered through comparative vertebrate studies, zs the degree of independence of behavioral and autonomic thermoregulatory systems: Is there a single integrative system for thermoregulatory responses, as widely believed, or are the neural networks for the two types of responses separate, as suggested by Satinoff (1980)?

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VICTOR H FIbTCHISON

Aquatlc ectothermlc vertebrates offer a slmpler model for the study of thermoregulatory mechanlsms than do terrestrial mammals. Green and Lomax (1976) have ut111zed the escape temperature of flshes to study responses to pharmacological agents which had been shown previously to influence temperature control in endothermlc vertebrates. The mudpuppy, Necturus maculosus, a totally aquatic North American salamander, was selected as an amphlbian model due to availab111ty, ease of maintenance, strong acclimatory response to temperature, and performance in laboratory thermal gradients (Hutchison and Hill, 1976; Hutchison, Black and Ersklne, 1979).

METHODS Adult Necturus were purchased from a dealer in Wisconsin and acclimatized to 15±i°C and a photoperiod of LD 12:12 for at least three weeks before use. All animals were fasted for one week prior to experimentation to remove the possibility of a thermophilic response due to feeding. Animals were removed from acclimatization chambers, place S singly and randomly into thermal gradients (280 cm long, 40 cm wide, 15 cm high, 5° to 30-35~C), and fixed with a 36gauge trailing thermocouple through the cloaca 2-4 cm into the lower intestine. The gradlents have been described in detail (Graham and Hutchison, 1979). Each gradient was lighted uniformly with overhead wide spectrum fluorescent lamps controlled by automatic timers to provide an LD 12:12 photoperiod. Deep body and gradient temperatures were printed each 30 minutes throughout a 48-hour period on an automatic multi-channel data acquisition system (Kaye Model 8000 or Instrulab Model 2000 Datalogger). Necturus displays significant dlel cycles in MST at some seasons of the year (Hutchison and Hill, 1976; Hutchison, Black and Erskine, 1979) and thus require that experiments be conducted over short periods with simultaneous controls. Drugs were adminlstered either by intraperltoneal injection (IP) or by dlrect intraventricular injection into the third ventricle (Ivent) each day between 1230 and 1330 hours. All drugs were diluted in amphibian saline, but additional compounds had to be added to make some of the agents soluble. Prostaglandin El was prepared by the method of Stitt (1973), which required the addition of small amounts of ethanol and sodium carbonate. Melatonin was made soluble by the addition of a small volume of ethanol (Cothran and Hutchison, 1979). Capsaicin was dissolved with the addition of ethanol and tween 80 to the saline (Pabe and colleagues, 1980). Control injections contained the same amount of solvents used for experimentals and were equal in volume to the corresponding experimental injections. Animals glven Ivent injections were placed under cold anesthesia and a hole Just large enough to accommodate a 30 gauge hypodermic needle was made with a dental drill through the skull midline directly over the third ventricle; the site of delivery was verified by injection of dye (3% Allcian blue in 0.5 M sodium acetate, pH 5.8) and dissection of the brain at the termination of the experiment. All glassware and needles used in Ivent injections were made pyrogen free by baking at 200°C for eight hours. Dosages and routes of administration are glven In Table i. An analysis of variance was performed to compare temperatures selected between control and experimental groups each day (Mathur and Silver, 1980). An analysis of covariance, with body weight as a covariate was used to verify that no weight related biases occurred in the mean selected temperatures (MST). The General Linear Model procedure in the Statistical Analysis System (Barr and colleagues, 1976) were used for analysis. Where individual treatment groups differed significantly (P < 0.05) the Duncan (1955) procedure was used to compare means of hourly time periods.

RESULTS AND DISCUSSION Prosta~landln E] (PGE]) There is excellent evidence from m a m ~ I s that fevers initiated by the exogenous pyrogens of pathogenic organisms induce endogenous pyrogens which in turn initiate the synthesis and release of PGE 1 (Kluger, 1979). This compound, which apparently does not easily pass the blood-brain barrier, acts upon the neuronal thermoregulatory center of the preoptic anterior hypothalamus to produce hyperthermia (Lipton, 1980). Necturus injected with PGE! selected significantly elevated temperatures on both Day i and D--~y~ (Table i). The MST on Day I was 4.7°C, and on Day 2, 4.9vC higher in experimental than in untreated controls (Fig. i). The second injection of PGE I on Day 2 did not produce an added dose response, despite the sustained hyperthermia throughout Day i. However, the faster rise in temperature from the cold anesthesia shown by treated animals on Day 2 may have been a response to the added dose (Hutchlson and Erskine, 1981). The behavloral fever in Necturus in response to PGEI is similar to that of another amphibian,

333

Behav,oral thermoregulauon ,n the salamander

Table I. Effect of drug treatment on mean selected temperatures (MST) of Necturus maculosus in thermal gradients on each of two days. Drugs were given into the third ventricle of the brain (Ivent), intraperltoneally (¿P) or subcutaneously (SC). Exptl - experimental, Ctrl - control group, N number of animals, N - number of observations, a o Treatment

Dose

N

a

N

o

MST (°C -+SEM)

F

P

Prostaglandin E,

(PUE~) Day Day Day Day

I, 1, 2, 2,

Exptl Ctrl Exptl Ctrl

2.5 ~g, sallne, 2.5 ~g, saline,

Ivent Ivent Ivent Ivent

9 9 9 9

783 832 862 803

17.27±.07 12.64±.08 16.75±.12 11.93±.08

4 mg k g - l l P saline, IP 4 mg kg-llP saline, 1P

10 I0 I0 I0

480 480 480 480

9.67±2.63 13.59±2.83 7.42~1.93 13.63~2.79

25 m E kg-IIP saline, IP 25 mg kg-~lP saline, IP

I0 10 10 I0

480 480 480 480

8.71±2.60 13.59±2.83 7.00±2.34 13.63±2.79

10 ~g kg-11vent saline, Ivent i0 ~g kg'llvent sallne, Ivent

10 I0 10 i0

480 480 480 480

15.39±.20 15.50±.17 15.70±.23 16.00±.18

I0 ~g kg-11vent saline, Ivent 10 ~g kg-IIvent sallne, Ivent

I0 i0 10 10

480 480 480 480

14.65±.15 15.81±.22 15.18±.15 14.56±.21

3 mE kg-llP saline, IP 3 mg kg-llP saline, IP

ii ii II ii

479 505 480 503

12.36±.23 12.72±.19 11.46±.19 i0.88t.20

7.5 ~g Ivent sallne 7 . 5 ~g I v e n t saline

6 6 4 4

262 264 186 182

20.02±.29 20.54±.20 20.91±.47 18.30±.24

25 mE kg-~SC sallne, SC 25 mg kg-*SC sallne, SC

I0 11 I0 Ii

472 528 480 527

11.22±0.22 14.31±0.23 14.22±0.23 11.08±0.21

1676

<.0001

1076

<.0001

446

<.0001

1623

<.0001

695

<.0001

1619

<.0001

0.18

N.S.

1.03

N.S.

19.75

<.0001

Melatonin (MT) Day Day Day Day

1, i, 2, 2,

Exptl Ctrl Exptl Ctrl

Chlorpromazine (CPZ) Day Day Day Day

I, 1, 2, 2,

Exptl Ctrl Exptl Ctrl

Oxotremorlne (OT) Day Day Day Day

1, I, 2, 2,

Exptl Ctrl Exptl Ctrl

Scopolamine (SPA) Day Day Day Day

i, l, 2, 2,

Exptl Ctrl Exptl Ctrl

5.84

<.02

Ethanol (ETH) Day Day Day Day

I, i, 2, 2,

Exptl Ctrl Exptl Ctrl

26.01"

N.S.*

*

*

58.91

N.S.

2.18

N.S.

24.66

<.0001

125

<.0001

214

<.0001

Neurotensin (NT) Day Day Day Day

I, 1, 2, 2,

Exptl Ctrl Exptl Ctrl

Capsalcin (CS) Day Day Day Day

i, 1, 2, 2,

Exptl Ctrl Exptl Ctrl

V a l u e f o r ANOV; ANCOVA showed t h a t i n d i v i d u a l v a r i a t i o n amoug a n i m a l s a c c o u n t e d f o r d i f f e r e n c e ; e f f e c t o f drug t r e a U n e n t was n o t s i g n i f i c a n t . Rana e s e u l e n t a , which d i s p l a y e d a h y p e r t h e r m i a o f 5 . 8 ° t o 9.7°C i n a t h e r m a l g r a d i e n t , l a t e n c y i n r e s p o n s e o f o n l y 2 - 3 m/n and a d u r a t i o n o f 25-65 min (Myhre, Cabanac and Myhre, 1977). The increase in MST displayed by Necturus after treatment with PGE I adds caudate amphibians to the rapidly growing list of ectotherms which show similar responses to this endogenous mediator of fever (Reynolds, Casterlin and Covert, 1980).

Melatonin (MT) and Chlorpromazine (CPZ) Melatonin, one of the major secretory products of the pineal gland, has been implicated as having a major role in both behavioral and physiological thermoregulation in vertebrates,

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VICTOR H HUTCHISON

where mts actlon is generally to lower body temperature (Ralph and coworkers, 1979). However, In the collared lizard, Crotaphytus collarms, exogenous melatonin ramsed body temperature during photophase and lowered it during scotophase (Cothran and Hutchison, 1979). Durlng the season of the year mn which the MT and CPZ experzments were conducted, the control animals showed slgnmficant (P < 0.017) dally cycles in MST wlth hmgher values during the first part of the scotophase (1800-2h00) and lower values during the flrst half of the photophase (0600-1200). Animals injected with MT responded wlth a marked decllne mn MST and disappearance of the diel cycle (Fmg. 2 ~. The animals treated with CPZ produced results very slmllar to those observed wlth melatonin (Table 1). Chlorpromazlne blocks the breakdown of clrculating endogenous melatonln by inhlblting the hepatic enzymes which catabollze the hormone and slows its dlsappearance from the plne~l (0zakl, Lynch and Wurtman, 1976), although a more direct effect of CPZ on the MST can not be ruled out. (L:n, 1979, L1n and colleagues, 1979a). The results of this experiment do suggest, however, that both endogenous and exogenous MT influenced behavioral thermoregulation in amphibians (Hutchison, Black and Ersklne, 1979), since MT and CPZ treatment produced a significant decline in MST compared to controls on both days (Table l, Fig. 2).

0xotremorine (0T) and Scopolamine (SPA) The muscarinlc chollnomimetic OT produced hypothermia in rats (Lom~x and Jenden, 1966) and signlficantly lowered the escape temperature of fish (Chromus chromus, 7.3 to 7.6 g) when added to the aquarium water (1.0 mg 1-i), but not with equal doses of SPA (Green and Lomax, 1976). Atropine and SPA are anticholinergic and highly effective in blocking the action of OT in rats and mice (Cox and Lee, 1977). When injected into Necturus 0T (oxalate form) caused no significant changes in M~T (Table I) even though the dose was ten-fold higher than that which produced a response in fishes (Green and Lomax, 1977). Earlier preliminary experiments with dosages of 1.0 ug also fa11ed to elicit a change MST in Necturus (unpublished observations). Necturus treated only with SPA, however, displayed a significant decrease in MST on Day I and a slight, but significant increase on Day 2 compared to controls (Table i, Fig. 3). The mixed effects of SPA and the absence of a response to OT in Necturus, taken with the failure of SPA to block action of OT and the hypothermic effect of OT in fish (Green and Lomax, 1976), suggest that fundamental differences may exist among the muscarinic thermoregulatory receptors of vertebrates.

Ethanol (ETH) As with central nervous system depressants, ETH lower~body temperature in a dose dependent fashion in mammals (Freund, 1973; Lomax and coworkers, 1980). There has been debate on the question of whether the hypothermic effects of ETH are due to an increased peripheral vasodilation and concimitant radiant heat loss(Gillespie, 1967; Hirvonen, 1979) or to a central neuronal action in the thermoregulatory control system (Cabanac, 1975). No significant dzfferences were found between the MST of untreated Necturus and those given ETH (Table i). These results suggest that the vasodilation and resultant heat loss may be the major cause of hypothermia due to ETH treatment in terrestrial endotherms, since the aquatic Necturus were not stimulated to select lower temperatures after ETH treatment. However, additional experiments with Necturus acclimated to temperatures other than 15°C are suggested, since ambient temperatures, at least in mammals, can influence the response of body temperatures to ETH (Freund, 1973).

N e u r o t e n s i n (NT) The intracisternal administration of .03 to 30 ~g NT into adult (25~) mice induced a highly significant dose-dependent hypothermia at ambient temperatures of h~ and 25~C; ~ given intravenously was without effect on thermoregulation (Lipton and colleagues, 1977). Other endorphins also produce lowered body temperatures in mammals, hut usually only at environmental temperatures below thermoneutrality (Avery, Hawkins and Wunder, 1981; Lin and colleagues, 1979b; Rivier and Brown, 1978; Nemeroff, 1980; Yehuda and Kastin, 1980). The MST of Necturus treated with NT was not significantly different from controls on Day i, but were significantly higher on Day 2 producing an additive dosage capable of a thermoregulatory effect (Fig. h). These results suggest that the endorphins play a role in control of behavioral temperature regulation as well as in physiological control of body temperature. The hyperthermia observed in Necturus contrasts with the hypothermla found in mammalian studies (Yehuda and Kastin, 19B~-?.

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The effect on mean selected temperature (MST) of injections of prostaglandin E l into the third ventricle (Ivent) of Necturus maculosus in a thermal gradient over a 2-day period. Controls were given 2.5 ~i saline (Ivent). Each point is the mean, vertical lines represent the standard errors of the means. Arrows show times of injection and the black bars, the scotophase of an LD 12:12 photoperiod. Dashed lines represent a period of cold anesthesia and injection when animals were removed from the gradients. Dosage and sample sizes are given in Table i. (Modified from Hutchlson and Erskine, 1981).

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The effect of IP injections of melatonin (MY) and chlorpromasine (CPZ) on thermal selection in Necturus. Manner of presentation same as in Fig. I. (From Hutchison, Black and Erskine, 1979). Injections given each day between 1230 and 1330 hours.

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The effect of IF injections of neurotensin (NT) on thermal selection in Necturus. Presentation similar to Fig. i and Fig. 2.

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The effect of SC injections of capsazcin (CS) on thermal selection in Necturus. Presentation slmilar to Fig. 1.

Capsaicin (CS) A subcutaneous dose of CS resulted in an immediate dose-dependent hypothermia in rats. Repeated injections desensitized the animals and the hypothermic effects decreased and were flnally eliminated; at this point the anlmals were incapable of resisting hyperthermia. This effect of CS was ascribed to irreversible damage to neuronal receptors (reviewed by Ob~l and coworkers, 1979). However, Cabanac, Cormareche-Leydier and Poirier (1976) concluded that the hyperthermia of desensitized rats was due to decreased salivary secretion and, presumably a lowered rate of evaporative cooling. The thermoregulatory responses of Necturus to CS treatment is similar to those seen in mammals, with a pronounced fall in MST on Day 1 and a significant increase on Day 2, when the dose was repeated (Table l, Fig. 5). The significant decrease in MST of the control animals from Day 1 to Day 2 is puzzling, but may be due to effects of the tween 80 and ethanol combination. This is apparently the first demonstration of the effect of CS on thermoregulation in an ectotherm. The action of CS on thermoregulatory abilities may be due to the modlfication of brain activlty as measured by EEG and sensory evoked responses (Rabe and colleagues, 1980) and to the depletion of substance P from sensory neurons with a resultant thermal analgesia (Yaksh and coworkers, 1979; Leeman, 1980). The clear change in thermal selection by Necturus suggests that the ability to select different thermal environments'was not impaired by CS.

CONCLUSIONS These preliminary experiments demonstrate that the aquatic urodele Necturus maculosus is a good animal model for studies on thermoregulatory behavior. The combination of such an animal model, together with the Bligh neuronal model (Bllgh, 1973; 1979, 1980) modified to include behavioral as well as physiological output, provides a heuristic coadunation from whlch meaningful questions on the phylogeny of vertebrate thermoregulation may be posed and subsequently answered.

ACKNOWLEDGEMENT Jerry Black, Cindy Southwick, John Egleston and Ninette Hart provided technical assistance. Dale Erskine and Karla Spriestersbach assisted in laboratory studies and with statistical analyses, Zenith Marsh did the illustrations and Janet Johnson assisted with the manuscript. The work was supported in part by an NIH Biomedical Sciences Support Grant to the University of Oklahoma (2 507 RR07078-08).

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V,cvoR H HLTCHISOh

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