Brain Research, 94 (1975) 491-506 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

491

I M P A I R M E N T OF T H E R M O R E G U L A T I O N , FOOD A N D W A T E R INTAKES IN T H E RAT A F T E R H Y P O T H A L A M I C INJECTIONS OF 5,6DI H Y D R O X Y T R Y P T A M I N E

R. D. MYERS

Laboratory of Neuropsychology, Purdue University, Lafayette, Ind. 47907 (U.S.A.) (Accepted March 27th, 1975)

SUMMARY

A bilateral microinjection into the anterior hypothalamus of 5,6-dihydroxytryptamine (5,6-DHT), a substance that lesions serotonin (5-HT)-containing neurons, caused a rise in the body temperature of the rat. The anatomical sites were the same as those at which 5-HT given in the same dose range (l.25-2.5/~g) evoked a similar hyperthermia. When exposed for one hour to a temperature of either 35 °C or 8 °C, the rats were not able to defend against the heat or cold, respectively. The magnitude of this thermoregulatory deficit depended upon the dose of 5,6-DHT given as well as the site of injection. A partial recovery from the warmth deficit was evident 13-17 days following the 5,6-DHT microinjection. Food and water intakes were also suppressed significantly and body weights declined concomitantly. These results provide additional evidence to support the view that a serotonergic mechanism in the hyl~othalamus is involved in both thermoregulation and the control of ingestive behavior.

INTRODUCTION

The monoamine theory of thermoregulation of Feldberg and Myers 19 presupposes that the presynaptic release of serotonin (5-HT) within the anterior hypothalamus activates a heat production pathway originating in this part of the diencephalon '~8 which is thought to be cholinergic in nature 33,35. At present the indoleamine part of this theory is based upon 3 kinds of evidence: pharmacological, physiological and anatomical. First, when 5-HT is applied to the anterior hypothalamus, a rise in body temperature occurs in most species (see review of Myers3°). Second, the release of 5-HT into push-pull perfusates collected from the anterior hypothalamus is enhanced significantly in the primate when the animal is exposed to a cold ambient tempera-

492 ture 31. Third, serotonergic nerve endings terminate in this part of the hypothalamus of the rat e' and cat2L Recently, Baumgarten et al. 4 have reported that the reducing congener of 5-HT, 5,6-dihydroxytryptamine (5,6-DHT), destroys mainly serotonergic neurons after the compound is injected centrally. In view of the postulated role of 5-HT in temperature control, the present investigation was undertaken to determine whether the thermoregulatory capacity of the rat would be altered in any way by the local application of 5,6-DHT. in these experiments, the sites that were selected for the injection of 5,6D H T were those that were found to be sensitive to 5-HT, which evokes a marked hyperthermia when it is microinjected 14. METHODS

Male rats of the Sprague-Dawley strain, weighing from 425 to 522 g were housed in individual cages and fed powdered Wayne Lab Blox and water which were freely available. They were maintained on a 12 h light-dark cycle at a room temperature of 22-24 °C. Food and water intakes as welt as body weights were measured at the same time every day; the amount of food consumed was determined by weighing the spill-proof food vessel and the ml of water taken were calculated using a Kimax drinking tube. Using surgical procedures described previously ~9, 22-gauge stainless steel guide tubes with an overall length of 13 mm were implanted bilaterally so that the beveled tips rested just above the rostral hypothalamus. The intended stereotaxic planes, according to the De Groot 17 coordinates were: AP, 7.0-7.1 ; Lat, 0.9-1.0; Hor, 0.0-0.1. Both of the guides were held in place by cranioplast cement that was packed around the tubes and 3 anchor screws that had been inserted in the calvarium. A post-operative recovery interval of 5-7 days elapsed before the experiments began. Cooling and warming Before an intrahypothalamic injection was given, each animal was tested for its thermoregulatory response to a 1-h challenge of either peripheral cooling or warming given in a random order. Each rat was placed in a specially constructed thermal chamber with a clear plexigtass top for observation. Within the chamber each individual wire mesh compartment was arranged so as to permit the rat to move about in a space measuring 12 in. × 8 in. × 6 in. Warm air from a commercial hair dryer was blown into the chamber so as to raise the ambient temperature to 35 °C ( ± 2 °C) within 3 min. Similarly, cold air from a special cooling apparatus lowered the temperature inside the chamber within 5 rain to 8.0 °C (zk 2.5 °C). The body temperature of each rat was monitored continuously throughout an experiment by a YSt 402 probe which was inserted into the colon to a depth of 6-7 cm and held in place by surgical tape wrapped gently around the base of the rail. Ordinarily, there was a 1-5-h interval between the 1-h periods of warming and cooling, but in some instances 24 h elapsed. A YSI Model 71A Thermistemp temperature controller connected to the

493 cooling and warming apparatus was used to maintain the chamber temperature at the low or high temperature during the 1-h period.

lntrahypothalamic injections Micro-injections into the hypothalamus were carried out according to procedures described previously 29. A 28-gauge injector needle was connected via PE 50 tubing to a 50 #1 Hamilton syringe mounted on a specially constructed microinfusion pump. After the injector was lowered to a depth of 1.0-2.5 m m below the guide tube, a volume of 0.5 #1 was infused over a 29-sec interval and the needle was kept in place for 60 sec before it was replaced by the permanently indwelling styler. This relatively large volume was selected 26 so that the solution would disperse throughout the entire rostral area of the rat's hypothalamus. To determine each site at which serotonin caused hyperthermia, 0.5, 2.5, or 5.0 #g of serotonin creatinine sulfate (Sigma Chemical) were microinjected unilaterally at successive depths until a rise in temperature occurred of at least 0.5 °C. On the following day, a second series of cooling and warming intervals was carried out in which each animal was exposed to the warm or cold air challenge. Again, a 1-5-h interval intervened between the 1-h exposures. On a subsequent day, 1.25 #g of 5,6-DHT (Regis Chemical) were microinjected bilaterally, again in a volume of 0.5 #1, into the sites at which 5-HT evoked a rise in the rat's temperature. The vehicle for both 5-HT and 5,6-DHT was 0.9 % pyrogen-free saline to which 0.1 mg/ml of ascorbic acid was added to retard the auto-oxidation of the amine. On the day following each injection, each rat was again exposed to the warming and cooling regimen. In instances in which a deficit in thermoregulation was observed, the warming and cooling procedures were repeated two days later or at an even greater interval. In this way the time course of the effect was determined. Depending upon the response of the animal, 5,6-DHT was re-injected at the same sites in a dose of 2.5, or in some animals, 5.0/~g, and another series of warming and cooling exposures was then carried out. The drug solutions were prepared with pyrogen-free glassware immediately prior to an injection. The tubing and syringes were always stored in 70 ~ ethanol and flushed repeatedly with pyrogen-free saline prior to use. The doses are expressed as the amount of free base and calculated on the basis of the amounts suggested by Baumgarten (personal communication) to be efficacious.

Histological analysis Since it was essential to verify histologically the anatomical sites of the 5-HT and 5,6-DHT injections, chemical analyses to determine amine content could not be undertaken. Therefore, at the conclusion of the experiments, each rat was killed with an overdose of pentobarbital sodium. After the heart had been clamped, 10 ~ buffered neutral formalin was perfused through the thoracic aorta. The brain was then cut in the coronal plane at 100 # m on a cryostat and the sections were stained for cells and fibers according to the method of Wolf "14. Histological reconstructions of the sites of injections were made using light microscopy.

494

°C 2.5 ,,

w

n.- 2.0 23

/

,/ ~/

5-HT

'

! ,

!

w

o_ I..iJ I->.. a 0 rn

1.5 t

i 2.5pg

;

! /

t.0 !

Z

~

o - 0.5 Z

1.25pg

I< w .Q

0 saline -0.5

!

-I

i

|

0

I

|

2

|

i

3

4

HOURS Fig. 1. Changes from the base-line temperature(°C) of a representative rat following a0.5/~linjection of 5-HT at zero hour. The two doses indicated and the saline control were given at weekly intervals. Sites of injection are designated by the dots on the histological map. Abbreviations: A, anterior hypothalamic area; GP, globus pallidus; LH, lateral hypothalamus; O, optic chiasm.

RESULTS

Neither the successive microinjections of 5-HT nor the control injections of saline had an appreciable effect on the rat's capacity to thermoregulate; in fact, some of the animals not unexpectedly exhibited evidence o f acclimatization to the thermal stress. That is, prior to the microinjections the control response to cooling showed a mean fall of 0.38 °C ( ~ 0.07); to warming, the mean rise was 0.8 °C ( + 0.18). Following saline and the series of 5-HT injections prior to 5,6-DHT, the second control tests to cooling showed a mean fall of 0.23 °C ( ± 0.07) and to warming a mean rise of 0.47 °C ( ± 0.17). These latter changes were used as the control values f o r the comparison with the responses to cooling and warming after 5,6-DHT was administered (i.e., Fig. 3). At the onset of warming or cooling the rat often became very active, groomed intensively, searched or explored its chamber and even gnawed at the tape holding the thermistor cable in place.

5-HT and 5,6-DHT hyperthermia O f the 18 rats tested, 8 demonstrated a clear-cut and reliable elevation in temperature following the injection of 5-HT and 5,6-DHT to the anterior hypothalamus.

495

°C 2.5 IJJ cr


-0

m

LO

Z Z

o_

0.5

ILl

saline

HOURS

Fig. 2. Changes from the base-line temperature (°C) of a representative rat following a 0.5/~1 injection of saline and 3 doses of 5,6-DHT. The microgram doses of 5,6-DHT in brackets were given at weekly intervals in the order as indicated. The sites of injection are designated by the dots on the histological map. Abbreviations are as in Fig. 1 plus: C, anterior commissure; P, preoptic area; PU, caudateputamen complex; S, septum pellucidum.

An anatomical analysis, based on the histological reconstructions revealed morphological specificity in that those sites at which the indoleamines evoked hyperthermia were distributed in the area ventral to the anterior commissure and dorsal to the optic chiasm. In every case, at least one of the sites of microinjection impinged upon this region of m a x i m u m sensitivity to both of the indoleamines. The remaining 12 animals, which showed no temperature response to intrahypothalamic 5-HT or the saline vehicle, were not given 5,6-DHT because of the morphological concordance of loci sensitive to 5-HT and 5,6-DHT 42,43. The distribution of sites unresponsive to the indoleamine extended rostrally beyond the tractus diagonalis and caudally in the lateral, dorsal and ventromedial regions of the hypothalamus. The rise in temperature following a microinjection of either 5 - H T or 5,6-DHT into the rostral hypothalamus was dependent upon the dose contained in the 0.5 ffl volume. Fig. 1 shows that the hyperthermia was more intense with the lower dose of 1.25 #g. As in the primate 2v, the higher doses (e.g., 5 #g) often caused an initial transient fall in the rat's body temperature and, therefore, was not used. The sites of the bilateral microinjections in the anterior hypothalamus are illustrated on the inset map in Fig. 1.

496 control pre-5,6-DHT

5,6-DHT post 1.25 pg

5,6-DHT post 2.5 pg

recovery

o 1.0 UJ

Impairment of thermoregulation, food and water intakes in the rat after hypothalamic injections of 5,6-dihydroxytryptamine.

A bilateral microinjection into the anterior hypothalamus of 5,6-dihydroxytryptamin (5,6-DHT), a substance that lesions serotonin (5-HT)-containing ne...
886KB Sizes 0 Downloads 0 Views