97
Neuroscience Letters, 128 (1991) 97-100 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50
ADONIS 0304394091003377 NSL 07855
Tumor necrosis factor-fl specifically inhibits the activity of preoptic warm-sensitive neurons in tissue slices T. Nakashima*, T. Kiyohara* and T. Hori** Department of Physiology, Saga Medical School, Saga (Japan.) (Received 15 February 1991; Revised version received 1 April 1991; Accepted 2 April 1991)
Key words: Tumor necrosis factor; Pyrogen; Thermosensitive neuron; Fever; Brain slice Effects of human tumor necrosis factor-/~ (TNF-fl) were studied on the single neural activities of the preoptic thermosensitive and thermally insensitive neurons in tissue slices of rats. Physiological doses (0.7-40 ng/ml) of TNF-p decreased the firing rate in most of the warm-sensitive neurons, but showed no marked tendency in the response patterns of thermally insensitive neurons. The actions of TNF-p on warm-sensitive neurons were blocked by concurrent application of sodium salicylate. The results suggest that the intracranial TNF may produce fever through local production of prostagiandins.
Tumor necrosis factors (TNF), TNF-at and TNF-fl, which are produced by activated monocytes and lymphocytes, respectively, reveal about 30% identity and 50% homology in the amino acid sequence [10] and share a common receptor [1]. Both proteins have been found to be toxic to many tumor cells and seemed to be nonspecies specific in their actions. Besides tumor necrotic activity, systemic administration of TNF produces fever in rabbits and endotoxin-resistant strain of mice [5, 11]. Now, TNF is believed to be a candidate for pyrogens, i.e. interleukin-1 (IL-1) and interferon-or (IFN-ct) [4]. Recently, it has been reported that microglia and astrocytes stimulated by lipopolysaccharide can induce TNF in the brain [17], and human recombinant TNF when injected into the third ventricle produces fever in rabbits [11]. Now, it is possible that TNF produced in the brain can mediate fever. The preoptic area of the hypothalamus has been generally recognized as the major recepting site of brain temperature, the integrating center of thermoregulation and the primary site of action of pyrogens and antipyretics [3, 12]. We have demonstrated that pyrogens (endotoxin, IFN-0t and IL-lfl) applied locally affect the activi-
*Present address: Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyou-ku, Kyoto 606, Japan. **Present address: Department of Physiology, Faculty of Medicine, Kyushu University 60, Fukuoka 812, Japan. Correspondence: T. Nakashima, Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyou-ku, Kyoto 606, Japan. Fax: (81) 075-702-4404.
ty of preoptic thermosensitive neurons in hypothalamic slices to induce fever [12, 14, 15]. It has also been shown that local synthesis of prostaglandins is essential for the actions of endotoxin and IL-1, although IFN-~ may produce fever with the involvement of the opiate receptor mechanisms. The present study has been performed to see whether TNF affects the activity of thermosensitive neurons in preoptic tissue slices and whether its effect is blocked by antipyretic which is the inhibitor of prostaglandins production. Single neural activities were extracellularly recorded in tissue slices of the preoptic area. Details of procedures for preparing the hypothalamic slice and recording neural activity were described previously [13, 14]. Coronally oriented slices (300-400/2m thick) containing the preoptic area were obtained from 34 male Wistar rats ranging in weight from 120 to 220 g. After pre-incubation in oxygenated (95 % 02 + 5 % CO2) Krebs-Ringer solution (pH 7.4, 300 mOsm/kg) at 37°C, the slice was transferred to a recording chamber which was perfused with the same solution at a flow rate of 2.1-2.3 ml/min. The composition of the Krebs-Ringer solution was (in mM): NaC1 124, KCI 5, KH2PO4 1.24, MgSO4 1.3, CaCI2 2.6, NaHCO3 26, glucose 10. The temperature of the hypothalamic slice was maintained at, or changed to, any temperature within the range of 32--42°C. Every single neural activity was recorded with a glass micropipette filled with Pontamine sky blue acetate (resistance 20-60 MI2). Spontaneous neural activity was amplified, displayed on an oscilloscope and stored as digital signals on floppy disks in a microcomputer. Neurons having ther-
98
mal coefficients of more than 0.6 impulses/s'°C or a QL0 over 2 were considered warm-sensitive. After the thermal responsiveness of each neuron had been studied, the neural response to human TNF-fl (0.770 ng/ml) and heat-inactivated (95°C, 60 min) T N F was observed at a fixed temperature (37-38°C). The T N F was kindly supplied by Hayashihara Biochemistry Institute and had been produced from BALL-1 cells. The biological activity of the T N F which was based on the cytotoxic activity against L929 cells was 4--6 x 108 U/mg protein. The T N F contained a very low concentration ( < 0.1 pg//tg protein) of endotoxin, which was far below threshold dose to alter the neural activity [12]. In some experiments, the responses to T N F were observed during concurrent application of sodium salicylate (SALC, Nakarai Chemical) of which dose (8 x 10 -6 to 3 × 10 -5 M) was lower than effective to neural activity by itself. All the drugs were dissolved in the Krebs-Ringer solution and were injected into the main perfusing line using infusion pumps. It took 24.6+0.4 sec (mean + S.E.M.) for a complete exchange of the medium in the recording chamber with a new solution. The neurons were considered to be responsive after 40 ng/ml or less of T N F was perfused through the medium, on the condition that the frequency of impulses changed by more
than 20 % and lasted over 1 min, over the pre-application period. Next, synaptic blocking experiments were performed after the observation of the responsiveness to T N F in normal Krebs-Ringer solution. For the experiments, Ca2+-free and high-Mg 2+ condition was established by changing the perfusate from normal solution to another solution which contained no CaC12 but 6.5 mM MgC12 [13]. Comparable parameters are described as mean value ___ standard errors of the mean (S.E.M.). Differences between samples were tested for statistical significance by means of a two-tailed Student's t-test with a significance limit of P = 0.01. A total of 42 neurons recorded from 34 preoptic slices were studied for their thermosensitivity and response to bath application of TNF. Of these, 27 neurons were warm-sensitive neurons which remarkably increased their firing rate in response to the rise in local temperature, and 15 neurons were thermally insensitive. Perfusing T N F in doses of less than 40 ng/ml decreased the firing rate in 16 out of 27 (60%) warm-sensitive neurons (Fig. lAa), increased 2 (7%) neurons and did not affect the remaining 9 (33 %) neurons. In contrast to warm-sensitive neurons, a majority of thermally insensitive neurons (8 of 15) did not change in
( • ) a. TNF 12 ng/ml I'~
,_234
39 ng/nd J-'l
}oL
Slice Temp. = 37.0"C
b° Ca 2" free / high Mg 2"
TNF
.o e" 34 L
39 ng/ml
12 ng/ml
~° I 0~
E4 .~P" ~ N3"-
~
TNF 4.6[__lng/ml
39 ng/ml
17
12 ng/ml
1--]
o
¢-i
_E Slice T e m p . - 37.0"C
=5 min =
Fig. 1. Rate-meter records showing the effects of TNF application on preoptic neurons. A: a warm-sensitive neuron. Ab: the effect of a synaptic blockage. B: a thermally insensitive neuron.
99
their firing activity by application of TNF (Fig. 1B). The remaining 3 neurons were inhibited and 4 neurons were excited by TNF. Regarding Fisher's exact probability test, TNF specifically inhibits the activity of warm-sensitive neurons (P 0.04 and t = 0.24, P > 0.4, respectively). As compared with TNF, the responses produced by IFN-~t in the preoptic neurons appeared after short intervals (1.6 + 0.3 min, n = 32) [14] significantly (t = 4.21, P < 0.01). Either TNF vehicle (human serum albumin, 1.6 ng per 1 ng TNF) or heat inactivated (95°C, 60 min) TNF had no effect on any of the preoptic neurons tested. As shown in Fig. 1Aa, the effect of TNF on preoptic neurons was dose related. The effects of concurrent application of SALC, an antipyretic, with TNF were studied in 3 warm-sensitive and one thermally insensitive neuron which showed the inhibitory responses to TNF. As shown in Fig. 2c, SALC (1.9 x 10 -5 M), which did not affect the neural activity by itself, abolished the inhibitory responses to TNF. SALC also abolished or attenuated the neural responses to TNF in the remaining three neurons. Three warm-sensitive neurons were further examined for their responsiveness to temperature change and TNF application in a Ca2+-free and high-Mg2+ solution where the synaptic transmission was blocked [13]. All of the three neurons did not lose their responsiveness to TNF as well as the thermosensitivity during perfusion of Ca2+-free and high-Mg2+ solution (Fig. lAb). This result indicates that the thermosensitivity and the action of TNF on the neurons are predominantly postsynaptic. The present study has shown (1) that TNF inhibited most of the activities of preoptic warm-sensitive neurons, (2) that TNF-induced changes in the activity of warm-sensitive neurons were effectively blocked by SALC, and (3) that the action of TNF on the preoptic neurons was predominantly postsynaptic. These findings
are very similar to our previous report on the findings concerning IL-lfl actions on the preoptic thermosensitive neurons [15]. Although, unfortunately, no cold-sensitive neuron was recorded in the present work, both TNF and IL-lfl decreased the activity of the majority of warm-sensitive neurons and there was no significant difference between the neural responses to the two cytokines in the onset latency and duration of responses. In addition to this similarity, responses induced by IL-lfl or TNF were abolished or attenuated by concurrent application of SALC. On the other hand, IFN-~ which is also one of pyrogens decreased the activity of most warmsensitive neurons, but the responses appeared after remarkably short onset-latencies [14]. The responses induced by IFN-~ were blocked by naloxone which is an opioid antagonist but not by SALC. These results suggest that there are two mechanisms to produce fever; one is mediated by hypothalamic opioid receptors (IFN-~) and the other involves the mediation of locally produced prostaglandins (TNF and IL-lfl). In the present study, SALC could block the TNFinduced responses in preoptic neurons, suggesting the involvement of cyclooxygenase metabolites of arachidonic acid in the TNF action. This confirms well with the
®
TNF 5.5 ng/ml
-"
2.5 ng/ml
i
,
. i,,
I I 2rr~n
SALC 1.9x10 -5M I TNF 5.5 ng/ml
I
|
Slice Temp. = 37.2°C Fig. 2. Rate-meter records showing the effects of T N F app]ication on a preoptic warm-sensitive neuron with (c) and without (b) concurrent application of SALC.
100 finding t h a t h u m a n TNF-ct directly stimulates the release o f a r a c h i d o n i c acid f r o m cellular p h o s p h o l i p i d s in hum a n n e u t r o p h i l s [2] a n d t h a t the c y c l o o x y g e n a s e inhibitor, i n d o m e t h a c i n , suppresses T N F - m e d i a t e d m e t a b o l i c a c t i v a t i o n including p r o d u c t i o n o f p r o s t a g l a n d i n E2 in m o u s e m a c r o p h a g e s in vitro [9]. T h e doses o f T N F used in this e x p e r i m e n t r a n g e d f r o m 0.7 to 70 ng/ml. A l t h o u g h the p h y s i o l o g i c a l conc e n t r a t i o n o f T N F released in the b r a i n is n o t k n o w n , i n t r a - c e r e b r o v e n t r i c u l a r injection o f 2-200 ng o f T N F p r o d u c e d fever in r a b b i t s [11] a n d 50-500 ng o f T N F suppressed f o o d i n t a k e in rats [16]. In a d d i t i o n , it has been s h o w n that T N F stimulates p r o l i f e r a t i o n o f h u m a n a s t r o c y t o m a cells [8] a n d the m e t a b o l i s m in m o u s e peritoneal m a c r o p h a g e s [9] in vitro at doses o f 2.5-250 ng/ml a n d 10-200 ng/ml, respectively. Therefore, it seems reas o n a b l e to consider t h a t the range o f doses o f T N F used in this study are physiological. Recently, it was suggested t h a t T N F - f l injected into the third cerebral ventricle increased the activity o f the s y m p a t h e t i c efferent nerves to b r o w n a d i p o s e tissue a n d rectal t e m p e r a t u r e , b u t T N F - ~ decreased the s y m p a t h e t ic efferent activity a n d l o w e r e d b o d y t e m p e r a t u r e in rats [6]. This finding o f T N F - ~ a c t i o n conflicts with the d a t a o f M o r i m o t o et al. which showed febrile response by i n t r a - c e r e b r o v e n t r i c u l a r injection o f T N F - ~ in r a b b i t s [11]. O n e r e a s o n for this d i s c r e p a n c y m a y be due to species differences. A n o t h e r e x p l a n a t i o n m a y be p r o v i d e d by the difference o f site o f T N F - ~ a c t i o n in the h y p o t h a l amus, because T N F facilitated neural activity o f m o s t o f the v e n t r o m e d i a l h y p o t h a l a m i c n e u r o n s (our u n p u b lished o b s e r v a t i o n ) which is k n o w n as a c a r d i n a l center o f thermogenesis at b r o w n a d i p o s e tissue [7]. It is necessary to clarify the effect o f T N F - ~ on the activity o f p r e o p t i c thermosensitive n e u r o n s in rats. This w o r k was s u p p o r t e d in p a r t by G r a n t - i n - A i d from the M o c h i d a M e m o r i a l F o u n d a t i o n for M e d i c a l a n d P h a r m a c e u t i c a l Research a n d f r o m T h e Meiji Life F o u n d a t i o n o f H e a l t h a n d W e l f a r e (T.N.). 1 Aggarwal, B.B., Eessalu, T.E. and Hass, P.E., Characterization of receptors for human tumour necrosis factor and their regulation by ~-interferon, Nature, 318 (I 985) 665~67. 2 Atkinson, Y.H., Murray, A.W., Krilis, S., Vadas, M.A. and Lopez, A.F., Human tumour necrosis factor-alpha (TNF-~) directly stimulates arachidonic acid release in human neutrophils, Immunology, 70 (1990) 8~87.
3 Boulant, J.A., Hypothalamic control of thermoregulation. In P.J. Morgane and J. Panksepp (Eds.), Handbook of Hypothalamus, Dekker, New York, 1980, pp. 1 82. 4 Dinarello, C.A., Interleukins, tumor necrosis factor (cachectin), and interferons as endogenous pyrogens and mediators of fever. In E. Pick and M. Land (Eds.), Lymphokines Vol. 14, Academic Press, New York, 1987, pp. 1 31. 5 Dinarello, C.A., Cannon, J.G., Wolff, S.M., Bernheim, H.A., Beutler, B., Cerami, A., Figari, I.S., Palladino, M.A. Jr. and O'Connor, J.V., Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin-1, J. Exp. Med., 163 (1986) 1433-1450. 6 Holt, S.J., Grimble, R.F. and York, D.A., Tumour necrosis factorct and lymphotoxin have opposite effects on sympathetic efferent nerves to brown adipose tissue by direct action in the central nervous system, Brain Res., 497 (1989) 183-186. 7 Imai-Matsumura, K., Matsumura, K., Tsai, C.L. and Nakayama, T., Thermal responses of ventromedial hypothalamic neurons in vivo and in vitro, Brain Res., 445 (1988) 193-197. 8 Lachman, L.B., Brown, D.C. and Dinarello, C.A., Growth-promoting effect of recombinant interleukin-i and tumor necrosis factor for a human astrocytoma cell line, J. Immunol., 138 (1987) 2913 2916. 9 Lehmmann, V., Benninghoff, B. and Droge, W., Tumor necrosis factor-induced activation of peritoneal macrophages is regulated by prostaglandin E2 and cAMP, J. Immunol., 141 (1988) 587-591. 10 Larrick, J.W. and Kunkel, S.L., The role of tumor necrosis factor and interleukin 1 in the immunoinflammatory response, Pharmaceutical Res., 5 (1988) 129 139. 11 Morimoto, A., Sakata, Y., Watanabe, T. and Murakami, N., Characteristics of fever and acute-phase response induced in rabbits by IL-1 and TNF, Am. J. Physiol., 256 (1989) R35-R41. 12 Nakashima, T., Hori, T., Kiyohara, T. and Shibata, M., Effects of endotoxin and sodium salicylate on the preoptic thermosensitive neurons in tissue slices, Brain Res. Bull., 15 (1985) 459-463. 13 Nakashima, T., Pierau, Fr.-K., Simon, E. and Hori, T., Comparison between hypothalamic thermoresponsive neurons from duck and rat slices, Pflfigers Arch., 409 (1987) 236-243. 14 Nakashima, T., Hori, T., Kuriyama, K. and Matsuda, T., Effects of interferon-~ on the activity of preoptic thermosensitive neurons in tissue slices, Brain Res., 454 (1988) 361-367. 15 Nakashima, T., Hori, T., Mori, T., Kuriyama, K. and Mizuno, K., Recombinant human interleukin-lfl alters the activity of preoptic thermosensitive neurons in vitro, Brain Res. Bull., 23 (1989) 209213. 16 Plata-Salaman, C.R., Oomura, Y. and Kai, Y., Tumor necrosis factor and interleukin-lfl: suppression of food intake by direct action in the central nervous system, Brain Res., 448 (1988) 106-114. 17 Sawada, M., Kondo, N., Suzumura, A. and Marunouchi, T., Production of tumor necrosis factor-alpha by microglia and astrocytes in culture, Brain Res., 491 (1989) 394-397.
Neuroscience Letters, 128 (1991) 97-100 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50
ADONIS 0304394091003377 NSL 07855
Tumor necrosis factor-fl specifically inhibits the activity of preoptic warm-sensitive neurons in tissue slices T. Nakashima*, T. Kiyohara* and T. Hori** Department of Physiology, Saga Medical School, Saga (Japan.) (Received 15 February 1991; Revised version received 1 April 1991; Accepted 2 April 1991)
Key words: Tumor necrosis factor; Pyrogen; Thermosensitive neuron; Fever; Brain slice Effects of human tumor necrosis factor-/~ (TNF-fl) were studied on the single neural activities of the preoptic thermosensitive and thermally insensitive neurons in tissue slices of rats. Physiological doses (0.7-40 ng/ml) of TNF-p decreased the firing rate in most of the warm-sensitive neurons, but showed no marked tendency in the response patterns of thermally insensitive neurons. The actions of TNF-p on warm-sensitive neurons were blocked by concurrent application of sodium salicylate. The results suggest that the intracranial TNF may produce fever through local production of prostagiandins.
Tumor necrosis factors (TNF), TNF-at and TNF-fl, which are produced by activated monocytes and lymphocytes, respectively, reveal about 30% identity and 50% homology in the amino acid sequence [10] and share a common receptor [1]. Both proteins have been found to be toxic to many tumor cells and seemed to be nonspecies specific in their actions. Besides tumor necrotic activity, systemic administration of TNF produces fever in rabbits and endotoxin-resistant strain of mice [5, 11]. Now, TNF is believed to be a candidate for pyrogens, i.e. interleukin-1 (IL-1) and interferon-or (IFN-ct) [4]. Recently, it has been reported that microglia and astrocytes stimulated by lipopolysaccharide can induce TNF in the brain [17], and human recombinant TNF when injected into the third ventricle produces fever in rabbits [11]. Now, it is possible that TNF produced in the brain can mediate fever. The preoptic area of the hypothalamus has been generally recognized as the major recepting site of brain temperature, the integrating center of thermoregulation and the primary site of action of pyrogens and antipyretics [3, 12]. We have demonstrated that pyrogens (endotoxin, IFN-0t and IL-lfl) applied locally affect the activi-
*Present address: Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyou-ku, Kyoto 606, Japan. **Present address: Department of Physiology, Faculty of Medicine, Kyushu University 60, Fukuoka 812, Japan. Correspondence: T. Nakashima, Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyou-ku, Kyoto 606, Japan. Fax: (81) 075-702-4404.
ty of preoptic thermosensitive neurons in hypothalamic slices to induce fever [12, 14, 15]. It has also been shown that local synthesis of prostaglandins is essential for the actions of endotoxin and IL-1, although IFN-~ may produce fever with the involvement of the opiate receptor mechanisms. The present study has been performed to see whether TNF affects the activity of thermosensitive neurons in preoptic tissue slices and whether its effect is blocked by antipyretic which is the inhibitor of prostaglandins production. Single neural activities were extracellularly recorded in tissue slices of the preoptic area. Details of procedures for preparing the hypothalamic slice and recording neural activity were described previously [13, 14]. Coronally oriented slices (300-400/2m thick) containing the preoptic area were obtained from 34 male Wistar rats ranging in weight from 120 to 220 g. After pre-incubation in oxygenated (95 % 02 + 5 % CO2) Krebs-Ringer solution (pH 7.4, 300 mOsm/kg) at 37°C, the slice was transferred to a recording chamber which was perfused with the same solution at a flow rate of 2.1-2.3 ml/min. The composition of the Krebs-Ringer solution was (in mM): NaC1 124, KCI 5, KH2PO4 1.24, MgSO4 1.3, CaCI2 2.6, NaHCO3 26, glucose 10. The temperature of the hypothalamic slice was maintained at, or changed to, any temperature within the range of 32--42°C. Every single neural activity was recorded with a glass micropipette filled with Pontamine sky blue acetate (resistance 20-60 MI2). Spontaneous neural activity was amplified, displayed on an oscilloscope and stored as digital signals on floppy disks in a microcomputer. Neurons having ther-
98
mal coefficients of more than 0.6 impulses/s'°C or a QL0 over 2 were considered warm-sensitive. After the thermal responsiveness of each neuron had been studied, the neural response to human TNF-fl (0.770 ng/ml) and heat-inactivated (95°C, 60 min) T N F was observed at a fixed temperature (37-38°C). The T N F was kindly supplied by Hayashihara Biochemistry Institute and had been produced from BALL-1 cells. The biological activity of the T N F which was based on the cytotoxic activity against L929 cells was 4--6 x 108 U/mg protein. The T N F contained a very low concentration ( < 0.1 pg//tg protein) of endotoxin, which was far below threshold dose to alter the neural activity [12]. In some experiments, the responses to T N F were observed during concurrent application of sodium salicylate (SALC, Nakarai Chemical) of which dose (8 x 10 -6 to 3 × 10 -5 M) was lower than effective to neural activity by itself. All the drugs were dissolved in the Krebs-Ringer solution and were injected into the main perfusing line using infusion pumps. It took 24.6+0.4 sec (mean + S.E.M.) for a complete exchange of the medium in the recording chamber with a new solution. The neurons were considered to be responsive after 40 ng/ml or less of T N F was perfused through the medium, on the condition that the frequency of impulses changed by more
than 20 % and lasted over 1 min, over the pre-application period. Next, synaptic blocking experiments were performed after the observation of the responsiveness to T N F in normal Krebs-Ringer solution. For the experiments, Ca2+-free and high-Mg 2+ condition was established by changing the perfusate from normal solution to another solution which contained no CaC12 but 6.5 mM MgC12 [13]. Comparable parameters are described as mean value ___ standard errors of the mean (S.E.M.). Differences between samples were tested for statistical significance by means of a two-tailed Student's t-test with a significance limit of P = 0.01. A total of 42 neurons recorded from 34 preoptic slices were studied for their thermosensitivity and response to bath application of TNF. Of these, 27 neurons were warm-sensitive neurons which remarkably increased their firing rate in response to the rise in local temperature, and 15 neurons were thermally insensitive. Perfusing T N F in doses of less than 40 ng/ml decreased the firing rate in 16 out of 27 (60%) warm-sensitive neurons (Fig. lAa), increased 2 (7%) neurons and did not affect the remaining 9 (33 %) neurons. In contrast to warm-sensitive neurons, a majority of thermally insensitive neurons (8 of 15) did not change in
( • ) a. TNF 12 ng/ml I'~
,_234
39 ng/nd J-'l
}oL
Slice Temp. = 37.0"C
b° Ca 2" free / high Mg 2"
TNF
.o e" 34 L
39 ng/ml
12 ng/ml
~° I 0~
E4 .~P" ~ N3"-
~
TNF 4.6[__lng/ml
39 ng/ml
17
12 ng/ml
1--]
o
¢-i
_E Slice T e m p . - 37.0"C
=5 min =
Fig. 1. Rate-meter records showing the effects of TNF application on preoptic neurons. A: a warm-sensitive neuron. Ab: the effect of a synaptic blockage. B: a thermally insensitive neuron.
99
their firing activity by application of TNF (Fig. 1B). The remaining 3 neurons were inhibited and 4 neurons were excited by TNF. Regarding Fisher's exact probability test, TNF specifically inhibits the activity of warm-sensitive neurons (P 0.04 and t = 0.24, P > 0.4, respectively). As compared with TNF, the responses produced by IFN-~t in the preoptic neurons appeared after short intervals (1.6 + 0.3 min, n = 32) [14] significantly (t = 4.21, P < 0.01). Either TNF vehicle (human serum albumin, 1.6 ng per 1 ng TNF) or heat inactivated (95°C, 60 min) TNF had no effect on any of the preoptic neurons tested. As shown in Fig. 1Aa, the effect of TNF on preoptic neurons was dose related. The effects of concurrent application of SALC, an antipyretic, with TNF were studied in 3 warm-sensitive and one thermally insensitive neuron which showed the inhibitory responses to TNF. As shown in Fig. 2c, SALC (1.9 x 10 -5 M), which did not affect the neural activity by itself, abolished the inhibitory responses to TNF. SALC also abolished or attenuated the neural responses to TNF in the remaining three neurons. Three warm-sensitive neurons were further examined for their responsiveness to temperature change and TNF application in a Ca2+-free and high-Mg2+ solution where the synaptic transmission was blocked [13]. All of the three neurons did not lose their responsiveness to TNF as well as the thermosensitivity during perfusion of Ca2+-free and high-Mg2+ solution (Fig. lAb). This result indicates that the thermosensitivity and the action of TNF on the neurons are predominantly postsynaptic. The present study has shown (1) that TNF inhibited most of the activities of preoptic warm-sensitive neurons, (2) that TNF-induced changes in the activity of warm-sensitive neurons were effectively blocked by SALC, and (3) that the action of TNF on the preoptic neurons was predominantly postsynaptic. These findings
are very similar to our previous report on the findings concerning IL-lfl actions on the preoptic thermosensitive neurons [15]. Although, unfortunately, no cold-sensitive neuron was recorded in the present work, both TNF and IL-lfl decreased the activity of the majority of warm-sensitive neurons and there was no significant difference between the neural responses to the two cytokines in the onset latency and duration of responses. In addition to this similarity, responses induced by IL-lfl or TNF were abolished or attenuated by concurrent application of SALC. On the other hand, IFN-~ which is also one of pyrogens decreased the activity of most warmsensitive neurons, but the responses appeared after remarkably short onset-latencies [14]. The responses induced by IFN-~ were blocked by naloxone which is an opioid antagonist but not by SALC. These results suggest that there are two mechanisms to produce fever; one is mediated by hypothalamic opioid receptors (IFN-~) and the other involves the mediation of locally produced prostaglandins (TNF and IL-lfl). In the present study, SALC could block the TNFinduced responses in preoptic neurons, suggesting the involvement of cyclooxygenase metabolites of arachidonic acid in the TNF action. This confirms well with the
®
TNF 5.5 ng/ml
-"
2.5 ng/ml
i
,
. i,,
I I 2rr~n
SALC 1.9x10 -5M I TNF 5.5 ng/ml
I
|
Slice Temp. = 37.2°C Fig. 2. Rate-meter records showing the effects of T N F app]ication on a preoptic warm-sensitive neuron with (c) and without (b) concurrent application of SALC.
100 finding t h a t h u m a n TNF-ct directly stimulates the release o f a r a c h i d o n i c acid f r o m cellular p h o s p h o l i p i d s in hum a n n e u t r o p h i l s [2] a n d t h a t the c y c l o o x y g e n a s e inhibitor, i n d o m e t h a c i n , suppresses T N F - m e d i a t e d m e t a b o l i c a c t i v a t i o n including p r o d u c t i o n o f p r o s t a g l a n d i n E2 in m o u s e m a c r o p h a g e s in vitro [9]. T h e doses o f T N F used in this e x p e r i m e n t r a n g e d f r o m 0.7 to 70 ng/ml. A l t h o u g h the p h y s i o l o g i c a l conc e n t r a t i o n o f T N F released in the b r a i n is n o t k n o w n , i n t r a - c e r e b r o v e n t r i c u l a r injection o f 2-200 ng o f T N F p r o d u c e d fever in r a b b i t s [11] a n d 50-500 ng o f T N F suppressed f o o d i n t a k e in rats [16]. In a d d i t i o n , it has been s h o w n that T N F stimulates p r o l i f e r a t i o n o f h u m a n a s t r o c y t o m a cells [8] a n d the m e t a b o l i s m in m o u s e peritoneal m a c r o p h a g e s [9] in vitro at doses o f 2.5-250 ng/ml a n d 10-200 ng/ml, respectively. Therefore, it seems reas o n a b l e to consider t h a t the range o f doses o f T N F used in this study are physiological. Recently, it was suggested t h a t T N F - f l injected into the third cerebral ventricle increased the activity o f the s y m p a t h e t i c efferent nerves to b r o w n a d i p o s e tissue a n d rectal t e m p e r a t u r e , b u t T N F - ~ decreased the s y m p a t h e t ic efferent activity a n d l o w e r e d b o d y t e m p e r a t u r e in rats [6]. This finding o f T N F - ~ a c t i o n conflicts with the d a t a o f M o r i m o t o et al. which showed febrile response by i n t r a - c e r e b r o v e n t r i c u l a r injection o f T N F - ~ in r a b b i t s [11]. O n e r e a s o n for this d i s c r e p a n c y m a y be due to species differences. A n o t h e r e x p l a n a t i o n m a y be p r o v i d e d by the difference o f site o f T N F - ~ a c t i o n in the h y p o t h a l amus, because T N F facilitated neural activity o f m o s t o f the v e n t r o m e d i a l h y p o t h a l a m i c n e u r o n s (our u n p u b lished o b s e r v a t i o n ) which is k n o w n as a c a r d i n a l center o f thermogenesis at b r o w n a d i p o s e tissue [7]. It is necessary to clarify the effect o f T N F - ~ on the activity o f p r e o p t i c thermosensitive n e u r o n s in rats. This w o r k was s u p p o r t e d in p a r t by G r a n t - i n - A i d from the M o c h i d a M e m o r i a l F o u n d a t i o n for M e d i c a l a n d P h a r m a c e u t i c a l Research a n d f r o m T h e Meiji Life F o u n d a t i o n o f H e a l t h a n d W e l f a r e (T.N.). 1 Aggarwal, B.B., Eessalu, T.E. and Hass, P.E., Characterization of receptors for human tumour necrosis factor and their regulation by ~-interferon, Nature, 318 (I 985) 665~67. 2 Atkinson, Y.H., Murray, A.W., Krilis, S., Vadas, M.A. and Lopez, A.F., Human tumour necrosis factor-alpha (TNF-~) directly stimulates arachidonic acid release in human neutrophils, Immunology, 70 (1990) 8~87.
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