Role of the solitary tract nucleus and caudal ventrolateral medulla in temperature responses in endotoxemic rats

PI1 LTm24-3205(98)ooS31-1

Life Sciences, Vol. 64, No. 1, pp. 37-43,1999 copyright 0 1998 FJsmier science Inc. Printed in the USA All rights reserved ou243205/99 $19.00 + .oo

ROLE OF TI-IE SOLITARY TRACT NUCLEUS AND CAUDAL VENTROLATERAL MEDULLA IN TEMPERATURE RESPONSES IN ENDOTOXEMIC RATS S.V.Koulchitsky, V.S.Levkovets, D.N.Tchitchkan, V.V.Soltanov, & V.A.Kulchitsky Institute of Physiology, National Academy of Sciences, 28 Skorina Street, Minsk 220072, Belarus (Received in final form October 16, 19!23)

Summary

In experiments on conscious rats it was found that preliminary microinjection of 100 nl 100 pM glutamic acid to the rostral commissural part of the solitary tract nucleus or to the caudal ventrolateral medulla increased a rise in colonic temperature induced by systemically applied endotoxin (3 &kg Escherichia coli lipopolysaccharide, i.p.) as compared to animals with intrabulbar injection of vehicle (control group). Preliminary microinjection of glutamate to the caudal commissural part of the solitary tract nucleus levelled the endotoxininduced temperature response. AtIer glutamate treatment of the caudal vemrolateral medulla there was a significant decrease in the noradrenaline content and decrease in the adrenaline level in the caudal (not significant) and rostral ventrolateral medulla (significant), as well as a small rise in noradrenergic activity at the solitary tract nucleus as compared to control animals. The post-mortem measurement of the optical density of brainstem tissues revealed its significant attenuation at the solitary tract nucleus and caudal ventrolateral medulla after glutamate as compared with these structures after vehicle. The involvement of monoaminergic systems of both structures under study in the initiation and control of temperature responses during endsotoxemia is suggested. Key Words: caudal brainstem, lipopolysaccharide, thermoregulation

A hypothesis has been put forth in the past years, that in endotoxemia, a role in the initiation of systemic responses to endotoxin is played by activated subdiaphragmatic branches of the vagus nerve (1, Z!, 3, 4, 5). Subphrenic transection of the vagus has been demonstrated to level or markedly diminish the temperature response to a relatively small dose of endotoxin (3, 4). Electrical activity of vagal fibres has been shown to increase after application of interleukin-1 (6) which may reflect the activation of vagal terminals during endotoxemia. The primary projection zone of the vagus in the central nervous system is known to be the solitary tract nucleus @II’S, mrcleus tractus sohrii) located near the dorsal medulla (7). NTS neurons are connected with anterior hypothalamic cells (8, 9); thermosensitive neurons of the

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preoptic region of the hypothalamus are key units in the maintenance of temperature homeostasis in homoiothermal animals. Most of these NTS connections are realized via the ventrolateral medulla, namely via cell populations of the caudal ventrolateral medulla (CVLM) (8, 9). There are also monosynaptic projections of the NTS to some anterior hypothalamic nuclei (8). CVLM and NTS neurons are also known to participate in the control of vascular responses, particularly by regulating the release of arginine-vasopressin by neurosecretory cells of the supraoptic and paraventricular hypothalamic nuclei (8, lo), which is a cryogenic factor as well (11, 12, 13, 14). Thus, the primary central afferent projection zone of the vagus, NTS, and the CVLM connected with it are hypothetically capable of performing relay mnctions in neurogenic signalling from activated vagal terminals (e. g., during endotoxemia) to anterior hypothalamic neurons. The present work attempted Gnther elucidate any role of these nuclei in the initiation of temperature responses during experimentally induced fever. Since it is an established fact that NTS neurons (including C2 adrenaline cells and A2 noradrenaline cells) and CVLM neurons (including Al noradrenaline cells) are catecholaminergic, one of the additional tasks of the study was to determine the adrenaline and noradrenaline levels in the NTS and rostral and caudal ventrolateral medulla (RVLM and CVLM) after bilateral application of neurotoxin to the CVLM. The analysis saved an efficiency of the neurotoxin destruction of CVLM neurons. Methods Experiments were performed in Wistar rats weighing 230-280 g. A week-before the experiment the animals were stereotaxically fixed (stereo&c equipment - NY&5; experimental workshops in the Institute of physiology, Kiev) and received 100 nl 100 @I glutamic acid into the rostra1 commissural(3.5 mm posterior to the interaural line, P; 0 mm lateral to the midline, L; and 8 mm down from the skull surface, D) or caudal(4.5 mm P; 0 mm L; and 8 mm D) NTS (n=7 and n=9, respectively) or 100 p.M glutamic acid in the same volume bilaterally to the CVLM (3.5 mm P; X2 mm L; and 9.5 mm D; n=l 1). For control, 100 nl apyrogenic saline was injected into the rostral commissural or caudal NTS or to the CVLM (n=S, n=7, n=6, respectively). The intracerebral injections were made with a nanolitre pump (W-P Instruments, Inc. 1400) in accordance with the stereotaxic atlas coordinates (15). It is known that in certain conditions the excitatory amino acid glutamate is capable of acquiring neurotoxic properties both in vitro and in viva (16, 17). Simultaneously, a silicon catheter for endotoxin application was implanted intraperitoneaily. All operative manipulations were conducted under ketaminexylazine-acepromazine (55.6, 5.5, and 1.1 mglkg, respectively, i. p.) anaesthesia. For 6 days before the experiment the animals were daily handled and habituated to the experimental boxes to minimize stress influences during the experiment. On the experimental day the rats were placed in restraining boxes which were put into a thermostat at 29-3O’C. Humidity in the thermostat was maintained at 50%. Each animal received copper-constantan thermocouples into the rectum and onto the tail skin for recording core (T,). and skin (Tsk) body temperature, respectively (electrothermometer “Physitemp”, USA). Atter adapting the animals for 1.5 h to the experimental conditions the temperatures were measured for an hour with a 30 min interval. Endotoxin 3 ug/kg (lipopolysaccharide, LPS, Escherichiu coli 011 l:B4, List Biological Laboratories, Campbell, CA; lot No LPS-25E) or pyrogen-free saline (PFS, Abbott Laboratories, North Chicago, IL 60064; lot No 18-379-DK) were applied intraperitoneally through the silicon catheter in a volume not exceeding 0.5 ml. T, and T,k were monitored continuously for not less than 7 h after the injection.

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After the end of the experiment an overdose of nembutal was injected and after respiratory arrest the brains were removed for biochemical determination of adrenaline and noradrenaline in the dorsal and ventrolateral medulla (n=9) or for determination of the optical density of sections in lthe same brain parts (n=28). The concentration of catecholamines in bulbar tissues was determined spectrofluorimetrically (18). The fluorescence intensity was measured with a spectrofluorimeter Solar SFL 1211 A (Belarus-Japan). All steps of the biochemical assay were conducted at 0°C. The absolute amount of catecholamines in tissues studied was calculated by comparing the fluorescence intensity value with a calibration curve of standard solutions of noradrenaline and adrenaline and expressed as u@g crude tissue. The optical density (OD) of brain tissue: was integrally evaluated in the texture analysis system for images Leitz-TAS (Germany) within a square 1600 urn’ in brain slices 40 urn thick cut in a freezing microtome and stained with toluidine blue. The OD of nerve cell- free brain tissue was preliminarily measured (OD 1). Then the OD in the NTS (OD2) and CVLM (OD3) was measured. Subsequently the 0D2 to ODl and 0D3 to ODl rations in glutaminic acid-treated animals and solvent-treated animals were compared. Since neurons are stained more intensely than neuronfree brain parts the destruction of nerve cells leads to a decrease in relative optical density. The data were st:atistically treated using Student’s t-test. Results Injection of pyrogen-free

saline to rats did not induce significant

changes in T, and T&,

Intraperitoneal injection of 3 ug/kg lipopolysaccharide to animals whose NTS or CVLM was preliminarily injected with vehicle led to a marked rise in colonic temperature with a maximum of O.SfO.OS’C at 75-90 min (Fig. lA, 1B). At the same time the animals showed a decrease in T,k by 2.3+0.7”C at 60-90 min. No changes in T, and Ta after intraperitoneal application of endotoxin were found in rats whose caudal NTS was pretreated with 100 pM glutamate (Fig. 1A). Conversely, injection of LPS into r,ats which were pretreated with glutamate in the rostra1 commissural NTS, was followed by a marked rise in colonic temperature, which was biphasic and significantly (p
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Gll2amlte(foslIaicolemal~ ---Ghdamtc(dNTs) --w-.- V&icle(rmttaI comnissual NE) --r-Vehicle(cmMNIS) -c-

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Fig. 1 Temperature effect of intraperitoneal injection of lipopolysaccharide (LPS, 3 @kg) in rats which bilaterally received glutamate (100 nl, 100 pM) or vehicle (100 III), into the rostra1 or caudal commissural parts of the solitary tract nucleus (A) or into the caudal ventrolateral medulla (B).

vehicle in CVL glutamate in CVL

vehicle in CVL glutamate in CVL

A

B Fig. 2

Adrenaline (A) and noradrenaline (B) content in the solitary tract nucleus, rostra1 or caudal ventrolateral medulla in rats after bilateral injection of 100 nl 100 pM glutamate or 100 nl vehicle into the caudal ventrolateral medulla.

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Fig. 3 Olptical density of brain tissue in intact (control) and in rats after microinjection of 100 nl 100 PM glutamate or 100 nl vehicle into the solitary tract nucleus (A) and caudal ventrolateral medulla (B).

Postmortem measurement of the optical density of the medullary structures revealed its significant decrease in the NTS and CVLM in animals who received 100 @4 glutamate to these brain structures, as compared to the vehicle injected NTS and CVLM (Fig. 3A, 3B).

Discussion Application of glutamate to the caudal NTS was accompanied by a levelling of temperature responses to endotoxin (3 &kg). One of the possible causes of such a reaction may be the b1ockad.e(due to neurotoxin) of signal transfer from peripheral vagal terminals to the primary central projection zone (NT’S) and Guther to the anterior hypothalamus. The caudal NTS is the projection zone for the hepatic branch of the vagus (7) to which particular attention is given in view of its involvement in the initiation of temperature responses during endotoxemia. It is also known that paraganglia in the liver hilus make synaptic contacts with vagal afferents and could be involved in LPS-induced responses (4, 19). How c’an the enhancement of the temperature response to endotoxin in rats pretreated with glutamate into the rostral commissural NTS or CVLM be accounted for? These findings are most likely to support the suggestion that the rostral commissural NTS and CVLM, as well as caudal commissural NTS, are the afferent links of the thermoregulatory system. The CVLM and NTS are also links in the system controlling the release of arginine-vasopressin by neurosecretory cells of the hypothalamus. Additionally, the CVLM and NTS are known to control the release of adrenocorticotropic hormone (20, 21, 22), one of the tinctions of which, like that of vasopressin (11, 12, 13, 14), is cryogenic action (23). We believe that the rostral commissural NTS and CVLM, by regulating the level of arginine-vasopressin and adrenocorticotropic hormone are involved in the mechanism which determine the balance

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between heat production and heat loss processes in certain conditions, including endotoxemia. Hypothetically, destruction of any of these structures may lead to disturbances of normal functioning of the mechanism and development of “uncontrollable” temperature responses. The LPS-induced increase in T, in control animals and in rats pretreated with bilateral injection of neurotoxin into the CVLM was concomitant with a decrease in Tsk, This supposes that in these conditions (ambient temperature 29-30°C; relative humidity 50.0%) the temperature response of this rats to LPS is underlain by the redistribution of blood from superficial to deeper body tissues. The increase of the Tsk in rats pretreated with glutamate into the rostra1 commissural NT’S may be related to that the enhanced temperature response in this animals is underlain by the intense energy exchange processes but not by the redistribution of the blood flow. It is noteworthy that after injection of glutamate to the CVLM the noradrenaline level fell not only in this structure but also in the RVLM which undergoes tonic inhibitory influences from CVLM neurons (24); it is also noteworthy that the NTS showed increased noradrenergic activity in these conditions. These complex transformations of noradrenergic activity in the bulbar brainstem after intracentral injections of glutamate reflect not only the efficacy of the neurotoxin action on noradrenergic neuronal populations but also are one more argument in favour of the assumption that the bulbar links of the aminergic system are involved in temperature homeostasis. The results of the optical density measurements excitatory amino acid in the dose used.

confirm

the neurotoxic

efftcacy

of the

Acknowledgments We are thankful to Mr. A. I. Prudnikovich for his invaluable help in preparation of the manuscript. We are grateful to Prof. V. N. Gourine for his criticism in discussing the design of the work. Special thanks go to Dr. W. W. Blessing, and Dr. A. A. Romanovsky, discussions with whom helped us comprehend anew the results obtained. We are indebted to Dr. I. P. Grigoriev for his excellent consultations on the mechanism of glutamate action. We also thank Mr. V. E. Chelubeyev for his consultations in determination of brain tissue optical density. References 1. C. M. BLATTEIS, and E. SEHIC, News Physiol. Sci. 12 l-9 (1997). 2. A. A. ROMANOVSKY, V. A. KULCHITSKY, C. T. SIMONS, N. SUGIMOTO and M. SZBKELY, Am. J. Physiol. 273 R777-R783 (1997). 3. A. A. ROMANOVSKY, C. T. SIMON& M. SZBKELY and V. A. KULCHITSKY, Am. J. Physiol. 273 R407-R4 13 (1997). 4. L. R. WATKINS, L. E. GOEHLER, J. K. RESTON, N.TARTAGLIA, L. GILBERT, D. MARTIN and S. F. MAIER, Neurosci. Lett. 183 27-3 1 (1995). 5. L. R. WATKINS, L. R. WATKINS and L. E. GOEHLER, Life Sci. 52 101 l-1026 (1995). 6. M. EK, M. KUROSAWA, T. LUNDEBERG, M. HEILIG and A. ERICSSON, Soci. Neurosci. (ABSTRACTS) 231514 (1997). 7. R. NORGEN and G. P. SMITH, J. Comp. Neurol. 273 207-223 (1988).

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