Thermoregulatory effects of carbachol in the posterior hypothalamus of the pigeon

J. therm. Biol. Vol.7. pp. 79 to 82, 1982

0306-4565 82 020079-04$03.000 Copyright ~3 1982 Pergamon Press Ltd

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THERMOREGULATORY EFFECTS OF CARBACHOL IN THE POSTERIOR HYPOTHALAMUS OF THE PIGEON* H. LAHTI, A. PYORNIL.~and R. HISSA Department of Zoology. Zoophysiological Laboratory, University of Oulu. Oulu. Finland (Receired 14 February 1981" accepted 29 October 1981)

Abstract--Thermoregulatory responses to intrahypothalamic injections of carbamylcholine (CCh) were recorded from unanaesthetized pigeons exposed to 20 and 38=C. The drug (0.25-1.0/~g/al- t) was administered into the posterior hypothalamus. 2. At 20~C, CCh in most cases either produced a cessation of shivering and induced hypothermia, or. produced a biphasic response: a slight increase in body temperature was followed by hypothermia. In one pigeon, the injections intensified shivering and an elevation in body temperature was seen. 3. Under ambient heat load (38=C), CCh depressed the thermally induced panting and caused body temperature to rise. A slight elevation in heart rate was also recorded. 4. The results suggest that a cholinergic mechanism also in the posterior hypothalamus may be involved in the temperature regulation of the pigeon. Interaction between cholinergic and serotonergic systems seems possible, but remains to be elucidated.

conditions for several weeks before use. They were maintained singly in cages at 22 _+ 2:C and a photoEXPERIMENTS in this laboratory have indicated that" period of 12L:I2D. Food and water were given od cholinergic mechanisms in the anterior/medial hypo- libitum. thalamus may be involved in temperature regulation Using the operative techniques described earlier of the pigeon (Py6rnil~i et al., 1977, 1979). Regarding (Hissa & Rautenberg, 1974), a guide cannula was imthe direction of temperature change, our results are planted unilaterally into the posterior hypothalamus partly in conflict with those obtained in the pigeon by of 10 pigeons. The coordinates used (Karten & Chawla et al. (1975). The conflicting findings might be Hodos, 1967) were: 4.8-5.5 mm anterior to the interexplained supposing that separate receptor popula- aural line, 1.2-2.0mm lateral to the mid-sagittal tions reached by different routes of administration. suture and 9.0-11.0 mm below the skull surface. The (intrahypothalamic vs intraventricular) also yield dif- experiments were started after a post=operative recovferent responses. This being the case, the participation ery of at least 7 days. The interval between the experiof caudal aspects of the hypothalamus, not reached in mental sessions in each bird was at least 3 days. our experiments (Py~rnil~i et al., 1977, 1979), might be To allow direct comparison of the results with our envisioned. Since it is not known whether the pigeons previous work, the same restraining method as earlier respond to cholinomimetics injected into the pos- was used (PyBrnil~. et al., 1977, 1979). The pigeons terior part of the hypothalamus, the experiments of were placed in the darkened metabolic chamber, the the present work were carried out. Carbamylcholine temperature of which was regulated with a thermosta(CCh) a stable cholinergie agonist was used in these tically controlled water bath at either 20.0 _+ 0.5 C or experiments. The effect of injections was studied at 38.0 + 0.5=C. Relative humidity in the chamber was ambient temperatures of 20 and 38=C. Chawla et al. kept at 40-60,°,;). (1975) have studied the participation of central cholinThe parameters measured at the ambient temperaergic mechanism in thermoregulation and observed ture (T~) of 20=C were: oxygen consumption (V,,:), that CCh induces both hypotherrnic and hyperther- body temperature (Th) from wingpit, foot temperature mic responses at 19-25:C. To make our results com- (Tr) and shivering. Respiratory rate, heart rate, V():. Th parable with them we have performed injections at and Tr were recorded at T~ 38=C. The methods for the same environmental temperatures. these measurements have been described earlier (Py/Srnilii et al., 1979). MATERIALSAND METHODS Carbamylcholine chloride (Carbachol, CCh, Sigma) was dissolved in distilled water and diluted to the Adult pigeons (Columba liria) of either sex, weigh- concentration desired. Doses used (0.25-1.0 #g/A- ') ing 280-330 g, were used. The birds were trapped at refer to the salt. The same CCh-solution was used Helsinki, South Finland, and accustomed to housing maximally for a week. Injections in a volume of 1 #1 * Preliminary data were presented at the XVi Scandina- were delivered through a polyethylene tube using a vian Congress of Physiology and Pharmacology. Oulu. 10 #1 Hamilton syringe and an Agla micrometer. The tip of the injection cannula extended 0.5 mm beyond June 25-28. 1979. INTRODUCTION

79

80

H. LAHTIet al. Table 1. Effects of intrahypothalamic injections of CCh on body temperature (baseline range 39.6--41.2°C) in 10 pigeons at T, 20:C Maximum change in body temperature, :C (.~ 4- SE) Biphasic response (6) Hypothermic Hyperthermic Rising Falling response response phase phase (3) ( 1)

Dose (gg) 0.25 0.5 1.0

+ 0.2 + 0.03" +0.3 + 0.07* +0.3 4- 0.06*

-0.8 + 0.41 -1.6 4- 0.26 -2.4 4- 0.54"f"

-2.0 + 0.97 -2.5 + 0.37* -3.7 4- 1.10

+ 1.0 +0.5 +0.6

Number of experiments on equal number of birds is given in parentheses. *Significant difference from preinjection values (P < 0.05 or less). ?Significant difference (P < 0.05) from change induced by the smallest dose (0.25 jug) (Student's t-test).

that of the guide cannula. Pigeons had neither food nor water during the experiments. The location of cannula tips was verified upon completion of the experiments from unstained brains fixed in 10~o formalin and sectioned parasagittally along the cannula tract. RESULTS

Responses at 20°C

The effect of different doses of intrahypothalamic injections of CCh on Th is given in Table 1. Each bird gave consistent results at all doses even though three kinds of responses were observed. In three pigeons the response was unambiguously hypothermic: the injection produced cessation of shivering for 20-60 min and this was accompanied by a decrease of Vo., and Th. Increase in Tr was rarely recorded. In six other pigeons CCh induced a biphasic response: a slight increase of Tb and Vo., within 10-15 rain was followed by a cessation of shivering resulting in a fall of Tb and decreased Vo_,. The maximum fall was reached within 60--70 rain of the injection. The responses of Tr were incompatible. In one pigeon, an increase of T b only was evoked by CCh. Shivering was intensified for 8 min on the average, and at the same time an increase of 0.5--1.0°C of Tb and about 2 0 ~ in Vo., was recorded. In this particular pigeon the responses did not seem to be dose dependent.

Responses at 38°C

The effects of different doses of CCh on Tb, Vo., and respiratory rate are given in Table 2. Representative responses of these parameters and heart rate of one bird are shown in Fig. 1. Intrahypothalamic administration of CCh lowered polypnoeic rate of respiration by an average of 100--250 m i n - t for 5--40 min (considerable variation), the change being significant after the doses of 0.5 and 1.0#g (Table2). Simultaneously with the depression of panting, Tb and Vo: started to rise. but the maximum was not reached until 20-65 min after the injection. A slight (10-60bpm) and short lasting (usually less than 20 min) increase in heart rate was also observed in these experiments. Control injections of 1/ll of distilled water did not induce significant changes in measured variables in either 20 or 38°C. Histology

Figure 2 shows the injection sites portrayed in a mid-sagittal section of the pigeon brain 2.0 mm lateral to the mid-line. The cannula tips are located in the posterior hypothalamus above tractus opticus and close to nucleus ectomamillaris. DISCUSSION

The results of this study demonstrate that activation of cholinergic neuronal pathways in the posterior hypothalamus of the pigeon may give rise to

Table 2. Effects of intrahypothalamic injections of CCh on body temperature, oxygen consumption and respiratory rate in 10 pigeons (6 with the dose of 0.5/ag) at T~ 38:C Body temperature, °C

O2 consumption, ml min- t 100 g-

Respiratory rate, min-l

Dose (/ag)

Preinjection

Maximum change

Preinjection

Maximum change

Preinjection

Minimum rate

0.25 0.5 1.0

42.4+0.15(10) 42.8 4- 0.30 (6) 43.1 4- 0.45 (I0)

+0.6+0.15" + 1.2 + 0.28* + 1.4 + 0.31"?

2.1+0.19(5) 2.4 ___0.22 (5) 2.3 + 0.25 (6)

+0.3+0.14 + 1.1 ___0.35* +0.6 4- 0.I0"

515 + 24(8) 497 + 30 (6) 550 + 17 (8)

391+80 285 4- 77* 288 + 70*

Values represent .mean + SE. Number of experiments is given in parentheses. * Significant difference from preinjection values (P < 0.05 or less). t Significant difference (P < 0.05) from change induced by the smallest dose (0.25 #g) (Student's t-test).

81

Therrnoregulatory effects of Carbachol in the pigeon Ta 38°C

-,=

600

@ 400

200 Control "[

200

-t- 150

o~

[

o 2

<

-20

0

20 40

60 80 100 120 140 Minutes

Fig. 1. Changes in oxygen uptake (AO2), body temperature (ATe) and respiratory and heart rates recorded from pigeon No. 151 following intrahypothalarnic injections of carbachol (0.25--1.0/~g/~1"t) and distilled water (control) at T, 38:C. Arrow indicates the time of injection.

three different temperature responses at T~ 20°C: (1) hypothermic; (2) hypothermic preceded by a short lasting increase in Tb; or (3) hyperthermic. Under ambient heat load (38°C) the cholinergic stimulation seems to inhibit the heat loss pattern of panting" In those experiments where CCh suppressed shivering, the increase of Tf was only rarely seen. This suggests that activation of this portion of the heat loss avenue plays a minor role in the observed hypothermia. In this respect the response differs from that obtained in the anterior/medial aspects of the pigeon hypothalamus (Pyi~rnilii etal., 1977, 19791 Here the injection of CCh regularly induced vasodilatation in the feet. In a general way, the biphasic response in Tb is in agreement with the results of Chawla et al. (1975) on the pigeon. They showed that CCh injected intraventricularly in doses of 5.0 and 7.5/~g 20 #l-1 produces hyperthermia followed by hypothermia at T, 19-25°C. The rising phase of 10-15 rain in our experiments was, however, considerably shorter than that, about an hour, recorded by Chawla and her coworkers. Thus, although the overall response in most cases was hypothermic, the initial hyperthermic phase is noteworthy. In an attempt to account for this, the possibility of an interaction of different neuronal systems may be considered. Our recent results indicate

that such an action could exist between, for example, the noradrenergic and cholinergic systems in the anterior hypothalamus (Lahti et al., 1980). Accordingly, hyperthermic phase preceding temperature decline might, for example, be accounted for by an interaction of cholinergic and serotonergic neurons (see Butcher & Talbot (1978) for specific examples). Injection of CCh could result in a release of 5-HT effecting rapid activation of heat gain mechanisms. This effect of 5-HT in the posterior hypothalamus has been recently demonstrated in the pigeon (PySrnil~i & Hissa, 1979). The view is further supported by the fact that, both after CCh and 5-HT, the latency to the onset as well as the duration of the hyperthermic effect was short. The following hypothermia would then be effected by an activation of cholinergic receptors driving inhibition of heat production. The sustained duration of hypothermic phase could be explained by the stability of CCh against enzymatic degradation, Alternatively, the successive activation of functionally different cholinergic receptors could yield different temperature responses. This is the presupposition presented also by Chawla etal. (1975) to account for the dual effects recorded in their experiments. In this study, a sole hyperthermic effect at 20°C was recorded in one pigeon only. This is in keeping with the results obtained by Chawla etal. (1975) with small doses of acetylcholine (ACh) and CCh. Although the central cholinergic mechanisms have been reported to mediate also hyperthermic responses in a number of mammalian species (see Cox & Lomax, 1977), no key to the problem of dualistic results seems to exist as yet. The thermoregulatory responses recorded in a hot ambient temperature (38°C) are in agreement with the results obtained in the anterior hypothalamus of the pigeon (Py~rnil~i efal., 1979), or on several mammalian species in similar experiments (Bligh e t a L , 1971 ; Johnson, 1975). This means that, depending on the bird's thermal state, different effector mechanisms

~

l

5mm

Fig. 2. A sagitta! section of the pigeon brain 2.0 rnm lateral to the mid-line to show the injection sites. CA, comrnissura anterior; C'b, cerebellum; CP, comrnissura posterior; DLA, nucleus dorsolateralis anterior thalami; EM, nucleus ectornamillaris; OM, tractus ¢¢,cipitornesencephalicus; TrO, tractus opticus; TSM, tractus septomesencephalicus. Symbols denote different responses at T~ 20°C as follows: O, Q, ~7, T, I , x, biphasic; A, A, +, hypotherrnic; E3, hypertherrnic.

82

H. LAHTIet al.

may be affected by the injection of CCh at various locations in the hypothalamus. On the whole, this study suggests that a cholinergic system also in the posterior hypothalamus of the pigeon might function in the thermoregulatory integration between sensors and response mechanisms. The existence here of two cholinergic thermoregulatory centers, one mediating heat gain and the other its inhibition, seems improbable. This has also been ruled out in the rat (Netherton et al., 1977). If the concept of the posterior hypothalamus as being the integrating centre of mechanisms for heat conservation and production is retained (see Bligh, 1973), however. it appears that systems other than cholinergic may also participate in its function.

REFERENCES

BLIGH J. {1973) Temperature Regtdation in Mammals and Other Vertebrates. North-Holland, Amsterdam. BUGH J.. COT'rtE W. H. & MASKrE¥ M. {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.. Lond. 212, 377-392. Bt:'rCHER L. L. & TALaOt K. (19781 Chemical communication processes involving neurons: vocabulary and syntax. In Cholinergic-Motu~amineryic Interactions in the Brain IEdited by Bt"rcHes L. L.k pp. 3-22. Academic Press. New York. CHAWLA N.. JOHRI M. B. L.. SAXENA P. N. & SI.~GrIAL K. C. (1975) Cholincrgic mechanisms in central thermoregulation in pigeons. Br. J. Pharmac. 53, 317-322.

Cox B. & LOMAXP. 11977) Pharmacologic control of temperature regulation. A. Rer. Pharma~. T~ic. 17, 341-353. HISSA R. & RAUTENBERGW. (19741 The influence of centrally applied noradrenaline on shivering and body temperature in the pigeon. J. Physiol., Lond. 238, 421-435. JoHnso~ K. G. (1975)Thermoregulatory changes induced by cholinomimetic substances introduced into the cerebral ventricles of sheep. Br. J. Pharmac. 53, 489-497. KARTEN H. J. & HODOSW. (1967) A stereotaxic Atlas o] the Brain of the Pigeon (Columba livia). Johns Hopkins Press, Baltimore. Lann H. PY6RNIL,~ A. & HISSA R. (1980) The influence of 6-OHDA on hypothermia produced by intrahypothalamically-injected carbachol in the pigeon. Experientia 36, 1188-1189.

NETHI~RTON R. A.. LEE P. S. & OVERSTREETD. H. (1977) Are there 2 cholinergic thermoregulatory centers in rats'? E.\'perientia 33. 1463-1464. PY6R,~IL,~ A. & HISSA R. (1979) Opposing temperature responses to intrahypothalamic injections of 5-hydroxytryptamine in the pigeon exposed to cold. Experientia 35, 59-60. PY6RXIL)~ A., LAHTI H. & HJSSA R. (1977l Thermoregulatory changes induced by intrahypothalamic injections of cholinomimetic substances in the pigeon. Ne,ropharmacolof/y 16, 737-741. PY6rXIL,~ A.. LAHtl H. & HISSA R. (1979) The influence of intrahypothalamically injected cholinergic agents on temperature regulation in the pigeon at ambient temperatures of 20. 32 and 38 C. Neuropharmacology 18, 503-510. Key Word Index--Temperature regulation; carbamylcholine: posterior hypothalamus: pigeon.