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Temperature responses in the rat and cat to cholinomimetic drugs injected into the cerebral ventricles
EUROPEAN JOURNAL OF PHARMACOLOGY 2 t (1973) 203-211. NORTH-HOLLAND PUBLISHING COMPANY
TEMPERATURE CHOLINOMIMETIC
RESPONSES DRUGS
IN THE RAT AND CAT TO
INJECTED
INTO THE CEREBRAL
VENTRICLES Judith BAIRD and W.J. LANG Pharmacology Department, University of Melbourne, Parkville, Victoria 3052, Australia
Accepted 2 November 1972
Received 6 April 1972
J. BAIRD and W.J. LANG, Temperature responses in the rat and cat to cholinomimetic drugs injected into the cerebral ventricles, European J. Pharmacol. 21 (1973) 203-211. Changes in temperature produced by the injection into the cerebral ventricles of cholinomimetic drugs and their antagonists were investigated in cats and rats. In the cat methacholine, 100 #g, caused a rise in temperature which was reversed after pretreatment with atropine, 100 #g. Nicotine, 100 ~g, produced a fall in temperature which was abolished by mecamylamine, 400 ~g. The response to acetylcholine ACh, 200 ~g, was small and variable and was not increased in the presence of physostigmine, 100 #g. Noracetylcholine-12,200 tzg, produced a rise in temperature. In the rat methacholine, 20 ,ug, produced a fall in temperature which was abolished by atropine, 40 ~g. A fall in temperature produced by ACh, 20 ~g, was blocked by atropine, 40 tzg, but was not potentiated by physostigmine, 0.1/~g. The fall in temperature produced by nicotine, 10/zg, was inhibited by mecamylamine, 50 tzg. These results suggest that central cholinergic pathways, both muscarinic and nicotinic, are involved in temperature regulation. In the cat a muscarinic pathway appears to be involved in temperature elevation but in the rat activation of the pathway appears to cause heat loss. These effects are like those of 5-hydroxytryptamine (5-HT) in these species and support suggestions that 5-HT and ACh may be involved together in heat-regulating pathways. Thermoregulation in cats Thermoregulation in rats
lntracerebroventricular injection Cholinergic antagonists
1. Introduction
The role of the biogenic amines in the central regulation of body temperature has been widely studied since Feldberg and Myers (1964) suggested their transmitter function in the hypothalamus. One unusual aspect has been the marked species differences that occur in response to these amines (Feldberg, 1968). Although not as extensively studied, temperature changes also occur in response to cholinergic drugs (see review by Lomax, 1970). In the rat most evidence suggests that cholinergic drugs injected into the cerebral ventricles or rostral hypothalamus produce hypothermia, although some results are controversial. Lomax and Jenden (1966) showed that microinjections of carbachol and o f oxo-
Methacholine Acetytcholine Nicotine
tremorine, a drug which releases acetylcholine, into the hypothalamus, produced hypothermia. Meeter (1971) has also shown that carbachol injected into the cerebral ventricles produced hypothermia. Microinjection o f pilocarpine was also found to produce hypothermia (Lomax et al., 1969). When acetylcholine was injected iontophoretically into the rostral hypothalamus o f the rat over a prolonged period a fall in temperature resulted (Kirkpatrick and Lomax, 1970). In contrast to these findings, however, Avery (1970, 1971, 1972) reported that the cholinergic compound carbachol, injected into the anterior hypothalamus of the rat, produced a rise in temperature. Hulst and de Wied (1967) showed that temperature responses to implantation of carbachol crystals were site-dependent; an implant in the preoptic area result-
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J. Baird, W.J. Lang. Temperature responses to intraventricular cholinomimetic drugs
ing in a fall in body temperature, while implants in the anterior hypothalamic area failed to produce an effect. Implants in the nucleus ventralis thalami caused hyperthermia. Acetylcholine (ACh) alone or with physostigmine, injected into the hypothalamus of conscious monkeys, was shown by Myers and Yaksh (1969) to produce a dose-dependent hyperthermic response. Hyperthermia resulted from injections over diffuse areas of the hypothalamus, but when injections were made in a site at the junction of the posterior hypothalamus and the mesencephalon a fall in temperature was produced suggesting ACh had a dual role in temperature regulation. More recently, Hall and Myers (1971) reported that infusion of an ACh-physostigmine mixture into the lateral cerebral ventricles of the monkey produced a small but significant hypothermic response and that nicotine produced either hypothermia or no effect on temperature. Bligh et al. (1971) studied the effects of ACh, carbachol and physostigmine in several species of animal. In sheep, goats and rabbits, temperature rose when the cholinergic drugs were injected into the cerebral ventricles. These findings with centrally administered cholinergic drugs suggest the occurrence of species differences in temperature response as happens with the biogenic amines. Although there are several reports on the behavioural effects of cholinergic substances injected into the cerebral ventricles of the cat, little attention has been paid to their effects on body temperature in this animal. Rudy and Wolf (1970) reported in an abstract that intrahypothalamic injections of carbachol and ACh with physostigmine produced dose-dependent but variable effects on temperature. It was therefore decided to investigate the effects on body temperature of cholinergic drugs, injected into the cerebral ventricles, in terms of possible muscarinic or nicotinic actions. Because of the possibility of species differences in thermoregulation, the effects of these drugs were compared in the cat and the rat.
2. Materials and methods
Experiments were conducted in male hooded rats
weighing between 270 and 320 g and in male or female cats weighing between 2.0 and 4.0 kg. Rectal temperatures were measured by a thermistor probe inserted about 6 cm into the rectum in rats and about 10cm in cats. The probes were held in position by taping the protruding end to the root of the tail with adhesive tape. Temperatures were monitored continuously on a Rikadenki 2-channel recorder and figures reproduced in this paper are plotted by taking a mean of temperatures of individual animals at 15-min intervals, and plotting these against time. The highest or lowest points of the records obtained in this manner are referred to as maximal responses for each treatment group. Statistical analysis of resuits was performed using Student's t-tests for comparison of mean thermic indices (TI) defined by Jori et al. (1967) and calculated according to the method described by Sprent (1971). The thermic index is a representation of the total cumulative change in body temperature from the original level, recorded throughout the experiment. Thus for each animal, the initial body temperature is subtracted from the body temperature recorded at a particular time and these differences contribute to its thermic index. During experiments rats were housed in their home cages, which were plastic boxes of 35 X 20 X 12 cm; and cats were housed in wire mesh cages of 60 × 30 × 45 cm. Animals were unrestrained throughout. All experiments in cats were conducted at a room temperature of 26-27°C, while experiments in rats were at 23.5-24.5°C. Prior to any drug administration a baseline temperature was recorded for 20 min or until stable. For intraventricular injection in conscious cats a Collison cannula was chronically implanted into the left lateral ventricle, according to the method of Banerjee et al. (1968). The left lateral ventricle was also permanently cannulated for injection of drugs into the conscious rat, the method used being that described by Hayden et al. (1966). Experiments were not commenced until at least 1 week after surgery, and at least 2 days recovery period was allowed to elapse between injections of cholinomimetic drugs alone and after pretreatment. Drugs used were methacholine chloride (Sigma), acetylcholine chloride (Sigma), atropine sulphate (Macfarlan Smith), nicotine hydrogen tartrate (British Drug Houses), mecamylamine hydrochloride (Merck,
205
J. Baird, W.J. Lang, Temperature responses to intraventricular cholinomimetic drugs
Sharp & Dohme (Aust.) Pty. Ltd.) and physostigmine sulphate (Macfarlan Smith). Doses of all drugs used refer to salts. Drugs were administered dissolved in sterile pyrogen-free 0.9% NaCI solution, the volumes for intraventricular injection being kept to a minimum not exceeding 15/al in rats or 0.2 ml in cats. Pyrogen-free, sterile syringes and needles were used for all injections, a Hamilton 50/~1 syringe (No. 705) being used for intraventricular injection in rats.
3. Results 3.1. Experiments on cats 3.1.1. Responses to methacholine In 6 cats, with a resting temperature between 38 and 39°C, intraventricular injection of methacholine, 100/lg, produced a maximal rise in temperature of 0.41 (-+ 0.12)°C (fig. 1), this maximal response occurring 30 rain after injection. The mean of the thermic indices (TI), to 90 min after injection, for this response was +1.83 (-+ 0.83). After pretreatment with atropine, 200/ag, intraventricularly, the rise in temperature was converted to a fall, the maximal re-
sponse now being - 0 . 3 4 (+- 0.16)°C, 45 min after methacholine injection (fig. 1). The TI after atropine pretreatment was - 2 . 2 7 (-+ 1.10); this value was significantly different from control response to methacholine (p< 0.001) (see table 1). 3.1.2. Responses to nicotine In 4 cats the maximal response to nicotine, 100/ag, intraventricularly, was a fall in temperature of 0.66 (+--0.21)°C 45 min after injection (fig. 1). The TI to 75 rain after injection, associated with this hypothermia was -3.31 -+ 1.07. When the cats were given mecamylamine, 400/ag, intraventricularly, t0 min beforehand, the maximal temperature change to nicotine was + 0.07 (+ 0.12)°C, 15 rain after injection (fig. 1). The TI after mecamylamine pretreatment was 0.00 (+ 0.66)" this value was significantly different from control response to nicotine (p< 0.01) (see table 1). 3.1.3. Responses to acetylcholine In 11 cats it was found that injection of ACh, 200/Jg, into the cerebral ventricles produced small and variable responses in different individuals. The maximal temperature response to intraventricular ACh was a fall of only 0.14 (+ 0.10)°C, 90 min after Methacholine (6)
] Atr mc
Mch
I
I
i
T
Z
T
T
Z
Acetylcholine(11)
Time
E io
t
Phy
Ach
c
Nicotine(4)
I
l
15 Minutes
Fig. 1. Effects of cholinergic drugs and their antagonists on body temperature in conscious cats. (e) control responses to methacholine, 100 ug (Mch), acetylcholine, 200 ug (ACh), and nicotine, 100 ~g (Nic), injected into the cerebral ventricles. Responses in the presence of: (a) atropine, 200 ug (Atr), injected intraventricularly, (o) physostigmine, 100 ug (Phy), injected intraventricularly, (A) mecamylamine, 400 #g (Mec), injected intraventricularly. Each point represents mean temperature _+S.E.M. (°C) but S.E.M.'s smaller than x 0.05°C were not plotted. Numbers in parentheses represent the number of animals in each treatment group. In all diagrams injections were made at the points indicated by arrows.
206
3". Baird, W.J. Lang, Temperature responses to intraventricular cholinomimetic drugs
Table 1 Thermic indices for cats and rats injected with cholinergic drugs and their antagonists. TI represents thermic index ± standard error of the mean. Figures in parentheses represent times in min to which thermic indices were calculated; p represents significance of difference between thermic indices of responses to cholinomimetic drugs before and after pretreatments, using Student's t-test. Doses used were: methacholine, 100 tag in cat, 20 tag in rat; acetylcholine, 200 tag in cat, 20 tag in rat; nicotine, 100 tag in cat, 10 ta in rat; atropine, 200 tag in cat, 40 tag in rat; physostigmine, 100 tag in cat, 0.1 tag in rat; mecamylamine, 400 tag in cat, 50 tag in rat. Treatment
Cats
Rats
TI • S.E.M.
p
TI -+ S.E.M.
p
Methacholine Methacholine + atropine
1.83 +-0.83 (90) -2.27 +- 1.10 (90)
< 0.001
-2.78 -+ 0.75 (60) -0.62 ± 0.54 (60)
< 0.001
ACh ACh + physostigmine
-0.48 ± 0.57 (105) -1.19 ± 1.06 (105)
> 0.05 NS
-3.80 ± 0.45 (60) -3.63 ± 0.65 (60)
> 0.05 NS
ACh ACh + atropine Atropine
-0.48 ± 0.57 (105) -2.08 ± 0.78 (105) -4.36 ± 2.33 (105)
< 0.001
-2.78 -+0.69 (60) -0.01 ± 0.45 (60) -2.81 ± 1.93 (60)
< 0.001
ACh ACh + mecamylamine Mecamylamine
0.02 ± 0.76 (105) -1.14 ± 1.14 (105) -1.37 ± 0.66 (105)
> 0.05 NS
-1.49 ± 0.27 (60) -1.25 ± 0.43 (60) -0.70 ± 0.88 (60)
> 0.05 NS
Nicotine Nicotine + mecamylamine
-3.31 ± 1.07 (75) 0.00 -+0.66 (75)
injection (fig. 1). The T-] to 105 min, associated with this response was - 0 . 4 8 (-+ 0.57). Pretreatment with physostigmine, 100/~g, intraventricularly, 30 rain prior to ACh, produced marked behavioural responses in all animals tested. The response to ACh after this pretreatment was a maximal fall in temperature of 0.24 (+ 0.16)°C (fig. 1) 30 min after injection, with a T'q of - 1 . 1 9 (+ 1.06). Comparison of the means of the thermic indices showed no significant difference at the p = 0.05 level (table 1). A significant fall in temperature occurred when ACh was injected 10 rain after intraventricular atropine. After pretreatment with atropine, 200/lg, the maximal temperature change to ACh was a fall of 0.36 (-+ 0.10)°C, 75 rain after injection. The TI, 105 min after ACh, in this treatment group was - 2 . 0 8 (+0.78). This value was significantly different from the T-I of - 0 . 4 8 (-+ 0.57), to 105 min, after ACh in the same group of cats ( p < 0.001)(see table 1). After intraventricular mecamylamine the injection of ACh again produced no significant response. The injection of 200/ag of ACh in 6 cats resulted in a maximal temperature change 75 min later of only - 0 . 0 3 (-+ 0.13)°C with an associated TI of +0.02 (+0.76) to 105 min after injection. When ACh was in-
< 0.01
-2.78 ± 0.92 (60) -1.65 ± 1.05 (60)
> 0.05 NS
jected 10 min after mecamylamine, 400/ag, intraventricularly, the maximal temperature change was - 0 . 2 0 (-+ 0.16)°C, 75 min after injection, the Ti to 105 min b e i n g - 1 . 1 4 (+ 1.14). Comparison of these means showed no significant difference at p = 0.05 (table 1). 3.1.4. Control responses to atropine and meeamyl- • amine Intraventricular injection of atropine, 200 gg, in 5~ cats resulted in variable responses in different animals. The maximal response was a fall in temperature of 0.68 (+- 0.31)°C, 1 0 0 m i n after injection. T-i to 105 min was 4 . 3 6 (+ 2.33); this change was not statistically significant ( p > 0.05) (see table 1). Mecamylamine, 400 gg, injected into the cerebral ventricle in 4 cats also failed to produce a statistically significant response. The maximal change in temperature after mecamylamine was a fall of 0.34 (+ O. 14)°C, occurring 45 min after injection. The T-I to 105 min associated with this response was - 1 . 3 7 (-+ 0.66) (see table 1). 3.1.5. Responses to noracetylcholine-12 Injection of noracetylcholine-12, 200/ag, into the
J. Baird, W.J. Lang, Temperature responses to intraventricular cholinomimetic drugs
cerebral ventricles of 5 cats resulted in a maximal rise in temperature ~of 1.40 (-+ 0.22)°C, 165 min after injection. The TI to 150 min after injection, associated with this response was +7.08 (-+ 1.65).
207
ment was reduced to -0.62 ( -+ 0.54) which was significantly different from methacholine control at the p
3.2. Experiments on rats 3.2.1. Responses to methacholine Injection of methacholine, 20 og, into the cerebral ventricles of rats resulted in a fall in temperature, the maximal change being 0.70 (-+ 0.18)°C lOmin after injection (fig. 2). The TI to 60 min after injection was -2.78 (-+ 0.75). Atropine, 40/sg, injected into the cerebral ventricles 10 min prior to methacholine reduced this fall in temperature to a maximum of 0.24 (-+ 0.11)°C (fig. 2). The T-I after atropine pretreatMethacholine(6 ) -
=
Mch
Aeetylcholine(6 )
Time
1
Atr
" Ach
Acetylcholine(6 )
T
Mec
t Ach
i
•
inure;
Fig. 2. Effects of cholinergic drugs and their antagonists on body temperature in conscious rats. (e) Control responses to agonists methacholine, 20t~g (Mch), acetylcholine, 20t~g (ACh), and nicotine, 10 #g (Nic), injected into the cerebral ventricles. Responses in the presence of: (=) atropine, 40 t~g (Atr), injected intraventricularly, (A) mecamylamine, 50 #g (Mec), injected intraventricularly. Each point represents mean temperature _+ S.E.M. (°C) but S.E.M.'s smaller than ± 0.05°C were not plotted. Numbers in parentheses represent the number of animals in each treatment group.
3.2.2. Responses to nicotine Nicotine, 10/lg, injected into the cerebral ventricles of 5 rats resulted in a fall in temperature, the maximal change being -0.76 (+ 0.25)°C, 10 min after injection (fig. 2). Pretreatment with mecamylamine, 50/ag, intraventricularly, 10 min prior to nicotine, reduced this fall in temperature to a value o f - 0 . 3 3 (-+ 0.17)°C, 15 min after nicotine injection (fig. 2). The TI to 60 min after nicotine was -2.78 (-+ 0.92); while after mecamylamine pretreatment in the same rats the TI was reduced to -1.65 (+ 1.05). The difference between these means failed to reach significance at the p = 0.05 level (table 1). 3.2.3. Responses to acetylcholine In contrast to the results obtained in the cat, injection of ACh, 20/2g, into the cerebral ventricles of conscious rats consistently resulted in hypothermia. The maximal response to ACh in 7 rats was a fall in temperature of 0.57 (-+ 0.12)°C, 10rain after injection. The TI to 60 min was -2.63 (+ 0.60). Pretreatment with physostigmine, 5/ag, intraventricularly, produced a mean fall in temperature of 0.56 (+ 0.24)°C during the 10-min period prior to ACh injection. Following this pretreatment the maximal fall after ACh was 1.05 (-+ 0.30)°C occurring 10 rain after ACh injection (fig. 3). The TI to 60 rain after ACh in this group was -4.72 (+- 1.34). Comparison of these means shows a significant difference at the p < 0.01 level (table 1). The dose of physostigmine injected as a pretreatment was then reduced until it failed to cause a fall in temperature. Control responses to ACh in another group of 4 rats was a maximal temperature fall of 0.92 (+ 0.06)°C, 10 min after injection (fig. 3) and an associated TI to 60 rain of -3.80 (+ 0.45). Following pretreatment with physostigmine, 0.1/~g, intraventricularly, the mean temperature fell only 0.07 (+ 0.02)°C and the response to ACh was then a maximal temperature fall of 0.93 (+- 0.13)°C, 10min after injection (fig. 3). The TI for this treatment group was
208
~0 g J~ (J
J'. Baird, W,J. Lang, Temperature responses to intraventricular cholinomimetic drugs
l
Acetylcholine(7 )
Time
Ach
Phy
Acetylcholine¢4 )
15 Minutes'
Fig. 3. Effects of physostigmine pretreatment on acetytcholine-induced hypothermia in rats. Acetylcholine, 20~g (ACh), injected intraventricularly. (e) Control responses to ACh. Upper trace: (o) after physostigmine, 5 ~tg (Phy), injected into the cerebral ventricles. Lower trace: (o) after physostigmine, 0.1 ~g (Phy), injected into the cerebral ventricles. Each point represents mean temperature ± S.E.M. (°C) but S.E.M.'s smaller than 0.05°C were not plotted. Numbers in parentheses represent the number of animals in each treatment group.
- 3 . 6 3 (-+ 0.65), a value not significantly different from ACh control (table 1). Pretreatment with atropine, 40/~g, intraventricularly, caused a fall in temperature in some animals (see 3.2.4.). However, in 6 other animals no response to atropine occurred during the first 10rain after injection. In these 6 animals the maximal fall in temperature observed after ACh, 20/ag, was -0.11 (-+ 0.01)°C (see fig. 2) compared with a control response to ACh of - 0 . 5 5 (-+ 0.14)°C. The T1 to 60 rain, associated with this response was -0.01 (-+ 0.45), a value significantly smaller ( p < 0.001) than the TI o f - 2 . 7 8 (+- 0.69) after ACh alone in this group of animals (see fig. 2 and table 1). Mecamylamine, 50/ag, intraventricularly, 10 min prior to intraventricular ACh, 20/ag, produced no effect on the response to ACh. Prior to mecamylamine pretreatment the maximal response, for a group of 6 rats, given ACh, was - 0 . 3 2 (-+ 0.14)°C (fig. 2). The associated T--I to 60 rain was - 1 . 4 9 (-+ 0.27). After mecamylamine the maximal temperature fall to ACh was 0.37 (+-- 0.09)°C (fig. 2) and the TI was - 1 . 2 5 (+ 0.43). This value was not significantly different, at p = 0.05, from the control response to acetylcholine in this group of animals (table 1).
3.2.4. Responses to atropine and mecamylamine Injection of atropine into the ventricles in 6 rats produced variable effects in different animals. In some animals a marked fall in temperature occurred within rain of injection, while in other animals no response, or a small rise in temperature occurred. The mean maximal response to atropine, 40/ag, was a fall in temperature of 0.51 (+- 0.18)°C occurring 60 min after injection. The TI to 60 min associated with this response was - 2 . 8 1 ( -+ 1.93), a value not significantly different from zero (see table 1). Mecamylamine, injected into the ventricle in 5 rats, did not produce any significant effect on body temperature. The maximal response resulting from injection of mecamylamine, 50,ug, w a s - 0 . 2 2 (-+ 0.20)°C; T-[ being - 0 . 7 0 (-+ 0.88) (see table 1). 3.3. Responses to cholinomimetic drugs injected i.p. In both cats and rats, all drugs tested intraventficularly (except noracetylcholine-12) were also injected i.p. at the same dose level. No change in temperature occurred after i.p. injection, 2 animals being used for each treatment. 3. 4. Control responses to intraventricular saline Injection of sterile 0.9% sodium chloride solution, 0.2 ml, in cats, or 15/al in rats failed to produce any significant change in body temperature. In 6 cats injected with saline the maximal response was a fall in temperature of 0.20 (-+ 0.14)°C, occurring 105 min after injection. The TI to 120 min associated with this response was --0.86 (-+ 1.03). The maximal response to intraventricular saline in 11 rats was a rise in temperature of 0.05 (+- 0.06)°C, 5 min after injection. The T-i-to 60 rain associated with this response was +0.03 (-+ 0.39).
4. Discussion
Injection of cholinomimetic drugs into the cerebral ventricles of the cat and the rat indicates that, as with the biogenic amines, there are species differences in their effects on temperature. Methacholine and ACh produced different responses in the rat and cat
J. Baird, W.J. Lang, Temperature responses to intraventricular cholinomimetic drugs
but nicotine in both species caused a fall in body temperature. In the rat, but not in the cat, ACh injected into the cerebral ventricles produced a significant fall in temperature. The hypothermic response in the rat was abolished by intraventricular atropine but not by mecamylamine suggesting that the temperature lowering pathways activated by ACh are muscarinic in nature. This is consistent with the temperature lowering effect of intraventricular methacholine since this drug is claimed to have predominantly muscarinic activity on cholinergic receptors in various tissues (Koelle, 1970). In contrast, intraventricular methacholine in the cat caused a rise in body temperature. After central muscarinic blockade by atropine, ACh, in doses previously producing no change in temperature or small variable effects, consistently produced a fall in temperature in the cat. It may be that central muscarinic pathways in the cat activate temperature elevation, the blockade of which unmasks nicotinic cholinergic pathways leading to a fall in temperature, although variable responses to atropine make interpretation of results difficult. In both the rat and the cat mecamylamine decreases the temperature lowering effect of nicotine. It seems, therefore, that temperature responses in the cat may be modified by opposing cholinergic pathways, muscarinic and nicotinic, occurring within the central nervous system. On the other hand in the rat activation of muscarinic and nicotinic pathways both lead to a lowering of temperature. Previous experiments in rats have produced conflicting results with cholinergic drugs on thermoregulation. Our results support the findings of Lomax et al. (1969); Kirkpatrick and Lomax (1970) and Meeter (1971). Avery (1970, 1971), however, found that carbachol produced a rise in temperature in rats, whilst Hulst and de Wied (1967) showed that temperature responses to carbachol were site dependent. Myers and Yaksh (1968) also reported that ACh in large doses or with physostigmine produced hypothermia although they found variable effects at lower doses. It was argued by Myers and Yaksh (1968) and Avery (1970) that hypothermic responses in rats may be due to restraint of the animals during experimental procedures. Our findings do not support this claim, because in these experiments the rats were not re-
209
strained and received only minimal handling prior to injection. Intraventricular administration of ACh in cats did not produce significant effects on temperature. Pretreatment with physostigmine did not affect the response to ACh suggesting that rapid hydrolysis of ACh was not the factor preventing a response in temperature. Because lack of penetration of ACh across the ventricular wall seemed a possible factor we used the drug noracetylcholine-12. This long carbon chain lipid-soluble derivative of ACh was reported by Nachmansohn (1959) to be able to reproduce the biological action postulated for the physiological role of ACh. According to Nachmansohn (1959) this action of noracetylcholine-12 was not blocked by curare. When noracetylcholine-12 was injected into the ventricles of cats a long lasting hyperthermic response occurred. This may be taken to support the results with methacholine indicating that there exists in the cat a temperature elevating pathway in the central nervous system activated by cholinergic (muscarinic) receptors. Changes in temperature to methacholine showed similar species differences to those reported for 5hydroxytryptamine (5-HT) (Feldberg, 1968). The resuits are consistent with the postulate of Myers and Yaksh (1969) that 5-HT release activates a heat regulating pathway in which ACh is the mediating transmitter. Nicotine, when injected into the cerebral ventricles, produced a fall in temperature in both the cat and the rat. Hall and Myers (1971) reported that the injection of nicotine into the ventricles of monkeys caused either a fall in temperature or no effect. Although we are not aware of other studies with nicotine on temperature regulation the drug carbachol which exhibits a high level of nicotinic activity (Koelle, 1970) has been used in several studies. A possible explanation for the fall in temperature after nicotine is that the drug releases noradrenaline which, in doses above 10/2g intraventricularly, results in hypothermia in the rat (Feldberg and Lotti, 1967). Blockade of the response to nicotine by mecamylamine is not inconsistent with this possibility as the release of noradrenaline by nicotine is prevented by nicotinic antagonists (Burn and Rand, 1965). The results support the concept of a physiological
210
J. Baird, W.J. Lang, Temperature responses to intraventricular cholinomimetic drugs
role of cholinergic pathways in temperature regulation. In the cat it may be that muscarinic and nicotinic pathways have opposing functions. It is of interest that Myers and Yaksh (1969) showed that microinjection of ACh in the monkey caused both hyper- and hypothermia depending on the site of injection. Based on experiments in monkeys and rats Myers (1968) suggested that ACh most likely is the transmitter mediating the effector mechanisms involved in heat production. Our results suggest this may be the case in cats but not rats and that the cholinergic pathway is muscarinic. Furthermore, cholinergic pathways can also cause a fall in temperature. Species differences in the temperature response to centrally administered cholinergic drugs shown here and in other studies (Myers and Yaksh, 1969; Lomax, 1970; Bligh et al., 1971) complicate the physiological role of cholinergic pathways in temperature regulation. Possible interactions between adrenergic and cholinergic agents make interpretation even more difficult (Lomax, 1970). The nature of physiological control of temperature will probably remain essentially speculative until the action of endogenous transmitters and thermoregulatory responses can be related to unit activity of neurones.
Acknowledgements This work was supported by the National Health and Medical Research Council. Judith Baird was supported by a Melbourne University Research Grant. We wish to thank Dr. C. Bell for the supply of noracetylcholine-12.
References Avery, D.D., 1970, Hyperthermia induced by direct injection of carbachol in the anterior hypothalamus, Neuropharmacol. 9, 175. Avery, D.D., 1971, Intrahypothalamic adrenergic and cholinergic injection effects on temperature and ingestive behaviour in the rat, Neuropharmacol. 10, 753. Avery, D.D., 1972, Thermoregulatory effects of intrahypothalamic injections of adrenergic and cholinergic substances at different environmental temperatures, J. Physiol. 220,257. Banerjee, U., W. Feldberg and V.J. Lotti, 1968, Effect on body temperature of morphine and ergotamine injected into the cerebral ventricles of cats, Brit. J. Pharmacol. 32, 523.
Bligh, J., W.H. Cottle and M. Maskrey, 1971, Influence of ambient temperature on the thermoregulatory responses to 5-hydroxytryptamine, noradrenaline and acetylcholine injected into the lateral cerebral ventricles of sheep, goats and rabbits, J. Physiol. 212,377. Burn, J.H. and M.]. Rand, 1965, Acetylcholine in adrenergic transmission, Ann. Rev. Pharmacol. 5,163. Feldberg, W., 1968, The monoamines of the hypothalamus as mediators of temperature responses, in: Recent Advances in Pharmacology, 4th ed., eds. J.M. Robson and R.S. Stacey (Churchill, London) p. 349. Feldberg, W. and V.J. Lotti, 1967, Temperature responses to monoamines and an inhibitor of monoamine oxidase injected into the cerebral ventricles of rats, Brit. J. Pharmacol. 31, 152. Feldberg, W. and R.D. Myers, 1964, Effects on temperature of amines injected into the cerebral ventricles. A new concept of temperature regulation, J. Physiol. 173,226. Hall, G.H. and R.D. Myers, 1971, Hypothermia produced by nicotine perfused through the cerebral ventricles of the unanaesthetized monkey, Neuropharmacol. 10, 391. Hayden, J.F., L.R. Johnson and R.P. Maickel, 1966, Construction and implantation of a permanent cannula for making injections into the lateral ventricle of the rat brain, Life Sci. 5, 1509. Hulst, S.G.T. and D. de Wied, 1967, Changes in body temperature and water intake following intracerebrai implantation of carbachol in rats, Physiol. Behav. 2,367. Jori, A., S. Paglialunga and S. Garattini, 1967, Effect of antidepressant and adrenergic blocking drugs on hyperthermia induced by reserpine, Arch. Intern. Pharmacodyn. 165, 384. Kirkpatrick, W.E. and P. Lomax, 1970, Temperature changes following iontophoretic injection of acetylcholine into the rostral hypothalamus of the rat, Neuropharmacol. 9, 195. KoeUe, G.B., 1970, Parasympathomimetic agents, in: The Pharmacological Basis of Therapeutics, 4th ed., eds. L.S. Goodman and A. Gilman (Macmillan, London) p. 467. Lomax, P., 1970, Drugs and body temperature, Intern. Rev. Neurobiol. 12, 1. Lomax, P. and D.J. Jenden, 1966, Hyperthermia following systemic and intracerebral injection of oxotremorine in the rat, Intern. J. Neuropharmacol. 5,353. Lomax, P., R.S. Foster and W.E. Kirkpatrick, 1969, Cholinergic and adrenergic interactions in the thermoregulatory centres of the rat, Brain Research 15,431. Meeter, E., 1971, The mechanism of action of intraventricular carbachol on the body temperature of the rat, Arch. Intern. Pharmacodyn. 194, 318. Myers, R.D. and T.L. Yaksh, 1968, Feeding and temperature responses in the unrestrained rat after injections of cholinergic and aminergic substances into the cerebral ventricles, Physiol. Behav. 3, 917. Myers, R.D. and T.L. Yaksh, 1969, Control of body temperature in the unanaesthetized monkey by cholinergic and aminergic systems in the hypothalamus, J. Physiol. 202, 483.
J. Baird, W.J. Lang, Temperature responses to intraventricular cholinomimetic drugs Nachmansohn, D., 1959, Chemical and Molecular Basis of Nerve Activity (Academic Press, New York) p. 169. Rudy, T.A. and H.H. Wolf, 1970, Cholinergic stimulation of
211
cat hypothalamus - effects on temperature regulation, Federation Proc. 29(2), 779 Abs. Sprent, P., 1971, Analysis of drug effects on body temperature, Biometrics 27,722.
TEMPERATURE CHOLINOMIMETIC
RESPONSES DRUGS
IN THE RAT AND CAT TO
INJECTED
INTO THE CEREBRAL
VENTRICLES Judith BAIRD and W.J. LANG Pharmacology Department, University of Melbourne, Parkville, Victoria 3052, Australia
Accepted 2 November 1972
Received 6 April 1972
J. BAIRD and W.J. LANG, Temperature responses in the rat and cat to cholinomimetic drugs injected into the cerebral ventricles, European J. Pharmacol. 21 (1973) 203-211. Changes in temperature produced by the injection into the cerebral ventricles of cholinomimetic drugs and their antagonists were investigated in cats and rats. In the cat methacholine, 100 #g, caused a rise in temperature which was reversed after pretreatment with atropine, 100 #g. Nicotine, 100 ~g, produced a fall in temperature which was abolished by mecamylamine, 400 ~g. The response to acetylcholine ACh, 200 ~g, was small and variable and was not increased in the presence of physostigmine, 100 #g. Noracetylcholine-12,200 tzg, produced a rise in temperature. In the rat methacholine, 20 ,ug, produced a fall in temperature which was abolished by atropine, 40 ~g. A fall in temperature produced by ACh, 20 ~g, was blocked by atropine, 40 tzg, but was not potentiated by physostigmine, 0.1/~g. The fall in temperature produced by nicotine, 10/zg, was inhibited by mecamylamine, 50 tzg. These results suggest that central cholinergic pathways, both muscarinic and nicotinic, are involved in temperature regulation. In the cat a muscarinic pathway appears to be involved in temperature elevation but in the rat activation of the pathway appears to cause heat loss. These effects are like those of 5-hydroxytryptamine (5-HT) in these species and support suggestions that 5-HT and ACh may be involved together in heat-regulating pathways. Thermoregulation in cats Thermoregulation in rats
lntracerebroventricular injection Cholinergic antagonists
1. Introduction
The role of the biogenic amines in the central regulation of body temperature has been widely studied since Feldberg and Myers (1964) suggested their transmitter function in the hypothalamus. One unusual aspect has been the marked species differences that occur in response to these amines (Feldberg, 1968). Although not as extensively studied, temperature changes also occur in response to cholinergic drugs (see review by Lomax, 1970). In the rat most evidence suggests that cholinergic drugs injected into the cerebral ventricles or rostral hypothalamus produce hypothermia, although some results are controversial. Lomax and Jenden (1966) showed that microinjections of carbachol and o f oxo-
Methacholine Acetytcholine Nicotine
tremorine, a drug which releases acetylcholine, into the hypothalamus, produced hypothermia. Meeter (1971) has also shown that carbachol injected into the cerebral ventricles produced hypothermia. Microinjection o f pilocarpine was also found to produce hypothermia (Lomax et al., 1969). When acetylcholine was injected iontophoretically into the rostral hypothalamus o f the rat over a prolonged period a fall in temperature resulted (Kirkpatrick and Lomax, 1970). In contrast to these findings, however, Avery (1970, 1971, 1972) reported that the cholinergic compound carbachol, injected into the anterior hypothalamus of the rat, produced a rise in temperature. Hulst and de Wied (1967) showed that temperature responses to implantation of carbachol crystals were site-dependent; an implant in the preoptic area result-
204
J. Baird, W.J. Lang. Temperature responses to intraventricular cholinomimetic drugs
ing in a fall in body temperature, while implants in the anterior hypothalamic area failed to produce an effect. Implants in the nucleus ventralis thalami caused hyperthermia. Acetylcholine (ACh) alone or with physostigmine, injected into the hypothalamus of conscious monkeys, was shown by Myers and Yaksh (1969) to produce a dose-dependent hyperthermic response. Hyperthermia resulted from injections over diffuse areas of the hypothalamus, but when injections were made in a site at the junction of the posterior hypothalamus and the mesencephalon a fall in temperature was produced suggesting ACh had a dual role in temperature regulation. More recently, Hall and Myers (1971) reported that infusion of an ACh-physostigmine mixture into the lateral cerebral ventricles of the monkey produced a small but significant hypothermic response and that nicotine produced either hypothermia or no effect on temperature. Bligh et al. (1971) studied the effects of ACh, carbachol and physostigmine in several species of animal. In sheep, goats and rabbits, temperature rose when the cholinergic drugs were injected into the cerebral ventricles. These findings with centrally administered cholinergic drugs suggest the occurrence of species differences in temperature response as happens with the biogenic amines. Although there are several reports on the behavioural effects of cholinergic substances injected into the cerebral ventricles of the cat, little attention has been paid to their effects on body temperature in this animal. Rudy and Wolf (1970) reported in an abstract that intrahypothalamic injections of carbachol and ACh with physostigmine produced dose-dependent but variable effects on temperature. It was therefore decided to investigate the effects on body temperature of cholinergic drugs, injected into the cerebral ventricles, in terms of possible muscarinic or nicotinic actions. Because of the possibility of species differences in thermoregulation, the effects of these drugs were compared in the cat and the rat.
2. Materials and methods
Experiments were conducted in male hooded rats
weighing between 270 and 320 g and in male or female cats weighing between 2.0 and 4.0 kg. Rectal temperatures were measured by a thermistor probe inserted about 6 cm into the rectum in rats and about 10cm in cats. The probes were held in position by taping the protruding end to the root of the tail with adhesive tape. Temperatures were monitored continuously on a Rikadenki 2-channel recorder and figures reproduced in this paper are plotted by taking a mean of temperatures of individual animals at 15-min intervals, and plotting these against time. The highest or lowest points of the records obtained in this manner are referred to as maximal responses for each treatment group. Statistical analysis of resuits was performed using Student's t-tests for comparison of mean thermic indices (TI) defined by Jori et al. (1967) and calculated according to the method described by Sprent (1971). The thermic index is a representation of the total cumulative change in body temperature from the original level, recorded throughout the experiment. Thus for each animal, the initial body temperature is subtracted from the body temperature recorded at a particular time and these differences contribute to its thermic index. During experiments rats were housed in their home cages, which were plastic boxes of 35 X 20 X 12 cm; and cats were housed in wire mesh cages of 60 × 30 × 45 cm. Animals were unrestrained throughout. All experiments in cats were conducted at a room temperature of 26-27°C, while experiments in rats were at 23.5-24.5°C. Prior to any drug administration a baseline temperature was recorded for 20 min or until stable. For intraventricular injection in conscious cats a Collison cannula was chronically implanted into the left lateral ventricle, according to the method of Banerjee et al. (1968). The left lateral ventricle was also permanently cannulated for injection of drugs into the conscious rat, the method used being that described by Hayden et al. (1966). Experiments were not commenced until at least 1 week after surgery, and at least 2 days recovery period was allowed to elapse between injections of cholinomimetic drugs alone and after pretreatment. Drugs used were methacholine chloride (Sigma), acetylcholine chloride (Sigma), atropine sulphate (Macfarlan Smith), nicotine hydrogen tartrate (British Drug Houses), mecamylamine hydrochloride (Merck,
205
J. Baird, W.J. Lang, Temperature responses to intraventricular cholinomimetic drugs
Sharp & Dohme (Aust.) Pty. Ltd.) and physostigmine sulphate (Macfarlan Smith). Doses of all drugs used refer to salts. Drugs were administered dissolved in sterile pyrogen-free 0.9% NaCI solution, the volumes for intraventricular injection being kept to a minimum not exceeding 15/al in rats or 0.2 ml in cats. Pyrogen-free, sterile syringes and needles were used for all injections, a Hamilton 50/~1 syringe (No. 705) being used for intraventricular injection in rats.
3. Results 3.1. Experiments on cats 3.1.1. Responses to methacholine In 6 cats, with a resting temperature between 38 and 39°C, intraventricular injection of methacholine, 100/lg, produced a maximal rise in temperature of 0.41 (-+ 0.12)°C (fig. 1), this maximal response occurring 30 rain after injection. The mean of the thermic indices (TI), to 90 min after injection, for this response was +1.83 (-+ 0.83). After pretreatment with atropine, 200/ag, intraventricularly, the rise in temperature was converted to a fall, the maximal re-
sponse now being - 0 . 3 4 (+- 0.16)°C, 45 min after methacholine injection (fig. 1). The TI after atropine pretreatment was - 2 . 2 7 (-+ 1.10); this value was significantly different from control response to methacholine (p< 0.001) (see table 1). 3.1.2. Responses to nicotine In 4 cats the maximal response to nicotine, 100/ag, intraventricularly, was a fall in temperature of 0.66 (+--0.21)°C 45 min after injection (fig. 1). The TI to 75 rain after injection, associated with this hypothermia was -3.31 -+ 1.07. When the cats were given mecamylamine, 400/ag, intraventricularly, t0 min beforehand, the maximal temperature change to nicotine was + 0.07 (+ 0.12)°C, 15 rain after injection (fig. 1). The TI after mecamylamine pretreatment was 0.00 (+ 0.66)" this value was significantly different from control response to nicotine (p< 0.01) (see table 1). 3.1.3. Responses to acetylcholine In 11 cats it was found that injection of ACh, 200/Jg, into the cerebral ventricles produced small and variable responses in different individuals. The maximal temperature response to intraventricular ACh was a fall of only 0.14 (+ 0.10)°C, 90 min after Methacholine (6)
] Atr mc
Mch
I
I
i
T
Z
T
T
Z
Acetylcholine(11)
Time
E io
t
Phy
Ach
c
Nicotine(4)
I
l
15 Minutes
Fig. 1. Effects of cholinergic drugs and their antagonists on body temperature in conscious cats. (e) control responses to methacholine, 100 ug (Mch), acetylcholine, 200 ug (ACh), and nicotine, 100 ~g (Nic), injected into the cerebral ventricles. Responses in the presence of: (a) atropine, 200 ug (Atr), injected intraventricularly, (o) physostigmine, 100 ug (Phy), injected intraventricularly, (A) mecamylamine, 400 #g (Mec), injected intraventricularly. Each point represents mean temperature _+S.E.M. (°C) but S.E.M.'s smaller than x 0.05°C were not plotted. Numbers in parentheses represent the number of animals in each treatment group. In all diagrams injections were made at the points indicated by arrows.
206
3". Baird, W.J. Lang, Temperature responses to intraventricular cholinomimetic drugs
Table 1 Thermic indices for cats and rats injected with cholinergic drugs and their antagonists. TI represents thermic index ± standard error of the mean. Figures in parentheses represent times in min to which thermic indices were calculated; p represents significance of difference between thermic indices of responses to cholinomimetic drugs before and after pretreatments, using Student's t-test. Doses used were: methacholine, 100 tag in cat, 20 tag in rat; acetylcholine, 200 tag in cat, 20 tag in rat; nicotine, 100 tag in cat, 10 ta in rat; atropine, 200 tag in cat, 40 tag in rat; physostigmine, 100 tag in cat, 0.1 tag in rat; mecamylamine, 400 tag in cat, 50 tag in rat. Treatment
Cats
Rats
TI • S.E.M.
p
TI -+ S.E.M.
p
Methacholine Methacholine + atropine
1.83 +-0.83 (90) -2.27 +- 1.10 (90)
< 0.001
-2.78 -+ 0.75 (60) -0.62 ± 0.54 (60)
< 0.001
ACh ACh + physostigmine
-0.48 ± 0.57 (105) -1.19 ± 1.06 (105)
> 0.05 NS
-3.80 ± 0.45 (60) -3.63 ± 0.65 (60)
> 0.05 NS
ACh ACh + atropine Atropine
-0.48 ± 0.57 (105) -2.08 ± 0.78 (105) -4.36 ± 2.33 (105)
< 0.001
-2.78 -+0.69 (60) -0.01 ± 0.45 (60) -2.81 ± 1.93 (60)
< 0.001
ACh ACh + mecamylamine Mecamylamine
0.02 ± 0.76 (105) -1.14 ± 1.14 (105) -1.37 ± 0.66 (105)
> 0.05 NS
-1.49 ± 0.27 (60) -1.25 ± 0.43 (60) -0.70 ± 0.88 (60)
> 0.05 NS
Nicotine Nicotine + mecamylamine
-3.31 ± 1.07 (75) 0.00 -+0.66 (75)
injection (fig. 1). The T-] to 105 min, associated with this response was - 0 . 4 8 (-+ 0.57). Pretreatment with physostigmine, 100/~g, intraventricularly, 30 rain prior to ACh, produced marked behavioural responses in all animals tested. The response to ACh after this pretreatment was a maximal fall in temperature of 0.24 (+ 0.16)°C (fig. 1) 30 min after injection, with a T'q of - 1 . 1 9 (+ 1.06). Comparison of the means of the thermic indices showed no significant difference at the p = 0.05 level (table 1). A significant fall in temperature occurred when ACh was injected 10 rain after intraventricular atropine. After pretreatment with atropine, 200/lg, the maximal temperature change to ACh was a fall of 0.36 (-+ 0.10)°C, 75 rain after injection. The TI, 105 min after ACh, in this treatment group was - 2 . 0 8 (+0.78). This value was significantly different from the T-I of - 0 . 4 8 (-+ 0.57), to 105 min, after ACh in the same group of cats ( p < 0.001)(see table 1). After intraventricular mecamylamine the injection of ACh again produced no significant response. The injection of 200/ag of ACh in 6 cats resulted in a maximal temperature change 75 min later of only - 0 . 0 3 (-+ 0.13)°C with an associated TI of +0.02 (+0.76) to 105 min after injection. When ACh was in-
< 0.01
-2.78 ± 0.92 (60) -1.65 ± 1.05 (60)
> 0.05 NS
jected 10 min after mecamylamine, 400/ag, intraventricularly, the maximal temperature change was - 0 . 2 0 (-+ 0.16)°C, 75 min after injection, the Ti to 105 min b e i n g - 1 . 1 4 (+ 1.14). Comparison of these means showed no significant difference at p = 0.05 (table 1). 3.1.4. Control responses to atropine and meeamyl- • amine Intraventricular injection of atropine, 200 gg, in 5~ cats resulted in variable responses in different animals. The maximal response was a fall in temperature of 0.68 (+- 0.31)°C, 1 0 0 m i n after injection. T-i to 105 min was 4 . 3 6 (+ 2.33); this change was not statistically significant ( p > 0.05) (see table 1). Mecamylamine, 400 gg, injected into the cerebral ventricle in 4 cats also failed to produce a statistically significant response. The maximal change in temperature after mecamylamine was a fall of 0.34 (+ O. 14)°C, occurring 45 min after injection. The T-I to 105 min associated with this response was - 1 . 3 7 (-+ 0.66) (see table 1). 3.1.5. Responses to noracetylcholine-12 Injection of noracetylcholine-12, 200/ag, into the
J. Baird, W.J. Lang, Temperature responses to intraventricular cholinomimetic drugs
cerebral ventricles of 5 cats resulted in a maximal rise in temperature ~of 1.40 (-+ 0.22)°C, 165 min after injection. The TI to 150 min after injection, associated with this response was +7.08 (-+ 1.65).
207
ment was reduced to -0.62 ( -+ 0.54) which was significantly different from methacholine control at the p
3.2. Experiments on rats 3.2.1. Responses to methacholine Injection of methacholine, 20 og, into the cerebral ventricles of rats resulted in a fall in temperature, the maximal change being 0.70 (-+ 0.18)°C lOmin after injection (fig. 2). The TI to 60 min after injection was -2.78 (-+ 0.75). Atropine, 40/sg, injected into the cerebral ventricles 10 min prior to methacholine reduced this fall in temperature to a maximum of 0.24 (-+ 0.11)°C (fig. 2). The T-I after atropine pretreatMethacholine(6 ) -
=
Mch
Aeetylcholine(6 )
Time
1
Atr
" Ach
Acetylcholine(6 )
T
Mec
t Ach
i
•
inure;
Fig. 2. Effects of cholinergic drugs and their antagonists on body temperature in conscious rats. (e) Control responses to agonists methacholine, 20t~g (Mch), acetylcholine, 20t~g (ACh), and nicotine, 10 #g (Nic), injected into the cerebral ventricles. Responses in the presence of: (=) atropine, 40 t~g (Atr), injected intraventricularly, (A) mecamylamine, 50 #g (Mec), injected intraventricularly. Each point represents mean temperature _+ S.E.M. (°C) but S.E.M.'s smaller than ± 0.05°C were not plotted. Numbers in parentheses represent the number of animals in each treatment group.
3.2.2. Responses to nicotine Nicotine, 10/lg, injected into the cerebral ventricles of 5 rats resulted in a fall in temperature, the maximal change being -0.76 (+ 0.25)°C, 10 min after injection (fig. 2). Pretreatment with mecamylamine, 50/ag, intraventricularly, 10 min prior to nicotine, reduced this fall in temperature to a value o f - 0 . 3 3 (-+ 0.17)°C, 15 min after nicotine injection (fig. 2). The TI to 60 min after nicotine was -2.78 (-+ 0.92); while after mecamylamine pretreatment in the same rats the TI was reduced to -1.65 (+ 1.05). The difference between these means failed to reach significance at the p = 0.05 level (table 1). 3.2.3. Responses to acetylcholine In contrast to the results obtained in the cat, injection of ACh, 20/2g, into the cerebral ventricles of conscious rats consistently resulted in hypothermia. The maximal response to ACh in 7 rats was a fall in temperature of 0.57 (-+ 0.12)°C, 10rain after injection. The TI to 60 min was -2.63 (+ 0.60). Pretreatment with physostigmine, 5/ag, intraventricularly, produced a mean fall in temperature of 0.56 (+ 0.24)°C during the 10-min period prior to ACh injection. Following this pretreatment the maximal fall after ACh was 1.05 (-+ 0.30)°C occurring 10 rain after ACh injection (fig. 3). The TI to 60 rain after ACh in this group was -4.72 (+- 1.34). Comparison of these means shows a significant difference at the p < 0.01 level (table 1). The dose of physostigmine injected as a pretreatment was then reduced until it failed to cause a fall in temperature. Control responses to ACh in another group of 4 rats was a maximal temperature fall of 0.92 (+ 0.06)°C, 10 min after injection (fig. 3) and an associated TI to 60 rain of -3.80 (+ 0.45). Following pretreatment with physostigmine, 0.1/~g, intraventricularly, the mean temperature fell only 0.07 (+ 0.02)°C and the response to ACh was then a maximal temperature fall of 0.93 (+- 0.13)°C, 10min after injection (fig. 3). The TI for this treatment group was
208
~0 g J~ (J
J'. Baird, W,J. Lang, Temperature responses to intraventricular cholinomimetic drugs
l
Acetylcholine(7 )
Time
Ach
Phy
Acetylcholine¢4 )
15 Minutes'
Fig. 3. Effects of physostigmine pretreatment on acetytcholine-induced hypothermia in rats. Acetylcholine, 20~g (ACh), injected intraventricularly. (e) Control responses to ACh. Upper trace: (o) after physostigmine, 5 ~tg (Phy), injected into the cerebral ventricles. Lower trace: (o) after physostigmine, 0.1 ~g (Phy), injected into the cerebral ventricles. Each point represents mean temperature ± S.E.M. (°C) but S.E.M.'s smaller than 0.05°C were not plotted. Numbers in parentheses represent the number of animals in each treatment group.
- 3 . 6 3 (-+ 0.65), a value not significantly different from ACh control (table 1). Pretreatment with atropine, 40/~g, intraventricularly, caused a fall in temperature in some animals (see 3.2.4.). However, in 6 other animals no response to atropine occurred during the first 10rain after injection. In these 6 animals the maximal fall in temperature observed after ACh, 20/ag, was -0.11 (-+ 0.01)°C (see fig. 2) compared with a control response to ACh of - 0 . 5 5 (-+ 0.14)°C. The T1 to 60 rain, associated with this response was -0.01 (-+ 0.45), a value significantly smaller ( p < 0.001) than the TI o f - 2 . 7 8 (+- 0.69) after ACh alone in this group of animals (see fig. 2 and table 1). Mecamylamine, 50/ag, intraventricularly, 10 min prior to intraventricular ACh, 20/ag, produced no effect on the response to ACh. Prior to mecamylamine pretreatment the maximal response, for a group of 6 rats, given ACh, was - 0 . 3 2 (-+ 0.14)°C (fig. 2). The associated T--I to 60 rain was - 1 . 4 9 (-+ 0.27). After mecamylamine the maximal temperature fall to ACh was 0.37 (+-- 0.09)°C (fig. 2) and the TI was - 1 . 2 5 (+ 0.43). This value was not significantly different, at p = 0.05, from the control response to acetylcholine in this group of animals (table 1).
3.2.4. Responses to atropine and mecamylamine Injection of atropine into the ventricles in 6 rats produced variable effects in different animals. In some animals a marked fall in temperature occurred within rain of injection, while in other animals no response, or a small rise in temperature occurred. The mean maximal response to atropine, 40/ag, was a fall in temperature of 0.51 (+- 0.18)°C occurring 60 min after injection. The TI to 60 min associated with this response was - 2 . 8 1 ( -+ 1.93), a value not significantly different from zero (see table 1). Mecamylamine, injected into the ventricle in 5 rats, did not produce any significant effect on body temperature. The maximal response resulting from injection of mecamylamine, 50,ug, w a s - 0 . 2 2 (-+ 0.20)°C; T-[ being - 0 . 7 0 (-+ 0.88) (see table 1). 3.3. Responses to cholinomimetic drugs injected i.p. In both cats and rats, all drugs tested intraventficularly (except noracetylcholine-12) were also injected i.p. at the same dose level. No change in temperature occurred after i.p. injection, 2 animals being used for each treatment. 3. 4. Control responses to intraventricular saline Injection of sterile 0.9% sodium chloride solution, 0.2 ml, in cats, or 15/al in rats failed to produce any significant change in body temperature. In 6 cats injected with saline the maximal response was a fall in temperature of 0.20 (-+ 0.14)°C, occurring 105 min after injection. The TI to 120 min associated with this response was --0.86 (-+ 1.03). The maximal response to intraventricular saline in 11 rats was a rise in temperature of 0.05 (+- 0.06)°C, 5 min after injection. The T-i-to 60 rain associated with this response was +0.03 (-+ 0.39).
4. Discussion
Injection of cholinomimetic drugs into the cerebral ventricles of the cat and the rat indicates that, as with the biogenic amines, there are species differences in their effects on temperature. Methacholine and ACh produced different responses in the rat and cat
J. Baird, W.J. Lang, Temperature responses to intraventricular cholinomimetic drugs
but nicotine in both species caused a fall in body temperature. In the rat, but not in the cat, ACh injected into the cerebral ventricles produced a significant fall in temperature. The hypothermic response in the rat was abolished by intraventricular atropine but not by mecamylamine suggesting that the temperature lowering pathways activated by ACh are muscarinic in nature. This is consistent with the temperature lowering effect of intraventricular methacholine since this drug is claimed to have predominantly muscarinic activity on cholinergic receptors in various tissues (Koelle, 1970). In contrast, intraventricular methacholine in the cat caused a rise in body temperature. After central muscarinic blockade by atropine, ACh, in doses previously producing no change in temperature or small variable effects, consistently produced a fall in temperature in the cat. It may be that central muscarinic pathways in the cat activate temperature elevation, the blockade of which unmasks nicotinic cholinergic pathways leading to a fall in temperature, although variable responses to atropine make interpretation of results difficult. In both the rat and the cat mecamylamine decreases the temperature lowering effect of nicotine. It seems, therefore, that temperature responses in the cat may be modified by opposing cholinergic pathways, muscarinic and nicotinic, occurring within the central nervous system. On the other hand in the rat activation of muscarinic and nicotinic pathways both lead to a lowering of temperature. Previous experiments in rats have produced conflicting results with cholinergic drugs on thermoregulation. Our results support the findings of Lomax et al. (1969); Kirkpatrick and Lomax (1970) and Meeter (1971). Avery (1970, 1971), however, found that carbachol produced a rise in temperature in rats, whilst Hulst and de Wied (1967) showed that temperature responses to carbachol were site dependent. Myers and Yaksh (1968) also reported that ACh in large doses or with physostigmine produced hypothermia although they found variable effects at lower doses. It was argued by Myers and Yaksh (1968) and Avery (1970) that hypothermic responses in rats may be due to restraint of the animals during experimental procedures. Our findings do not support this claim, because in these experiments the rats were not re-
209
strained and received only minimal handling prior to injection. Intraventricular administration of ACh in cats did not produce significant effects on temperature. Pretreatment with physostigmine did not affect the response to ACh suggesting that rapid hydrolysis of ACh was not the factor preventing a response in temperature. Because lack of penetration of ACh across the ventricular wall seemed a possible factor we used the drug noracetylcholine-12. This long carbon chain lipid-soluble derivative of ACh was reported by Nachmansohn (1959) to be able to reproduce the biological action postulated for the physiological role of ACh. According to Nachmansohn (1959) this action of noracetylcholine-12 was not blocked by curare. When noracetylcholine-12 was injected into the ventricles of cats a long lasting hyperthermic response occurred. This may be taken to support the results with methacholine indicating that there exists in the cat a temperature elevating pathway in the central nervous system activated by cholinergic (muscarinic) receptors. Changes in temperature to methacholine showed similar species differences to those reported for 5hydroxytryptamine (5-HT) (Feldberg, 1968). The resuits are consistent with the postulate of Myers and Yaksh (1969) that 5-HT release activates a heat regulating pathway in which ACh is the mediating transmitter. Nicotine, when injected into the cerebral ventricles, produced a fall in temperature in both the cat and the rat. Hall and Myers (1971) reported that the injection of nicotine into the ventricles of monkeys caused either a fall in temperature or no effect. Although we are not aware of other studies with nicotine on temperature regulation the drug carbachol which exhibits a high level of nicotinic activity (Koelle, 1970) has been used in several studies. A possible explanation for the fall in temperature after nicotine is that the drug releases noradrenaline which, in doses above 10/2g intraventricularly, results in hypothermia in the rat (Feldberg and Lotti, 1967). Blockade of the response to nicotine by mecamylamine is not inconsistent with this possibility as the release of noradrenaline by nicotine is prevented by nicotinic antagonists (Burn and Rand, 1965). The results support the concept of a physiological
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J. Baird, W.J. Lang, Temperature responses to intraventricular cholinomimetic drugs
role of cholinergic pathways in temperature regulation. In the cat it may be that muscarinic and nicotinic pathways have opposing functions. It is of interest that Myers and Yaksh (1969) showed that microinjection of ACh in the monkey caused both hyper- and hypothermia depending on the site of injection. Based on experiments in monkeys and rats Myers (1968) suggested that ACh most likely is the transmitter mediating the effector mechanisms involved in heat production. Our results suggest this may be the case in cats but not rats and that the cholinergic pathway is muscarinic. Furthermore, cholinergic pathways can also cause a fall in temperature. Species differences in the temperature response to centrally administered cholinergic drugs shown here and in other studies (Myers and Yaksh, 1969; Lomax, 1970; Bligh et al., 1971) complicate the physiological role of cholinergic pathways in temperature regulation. Possible interactions between adrenergic and cholinergic agents make interpretation even more difficult (Lomax, 1970). The nature of physiological control of temperature will probably remain essentially speculative until the action of endogenous transmitters and thermoregulatory responses can be related to unit activity of neurones.
Acknowledgements This work was supported by the National Health and Medical Research Council. Judith Baird was supported by a Melbourne University Research Grant. We wish to thank Dr. C. Bell for the supply of noracetylcholine-12.
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