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A role of excitatory amino acids in the activation of locus coeruleus neurons following cutaneous thermal stimuli
Brain Research, 521 (1990) 325-328
325
Elsevier BRES 24132
A role of excitatory amino acids in the activation of locus coeruleus neurons following cutaneous thermal stimuli Mihfily Haj6s* and G6ran Engberg Department of Pharmacology, University of GOteborg, GOteborg (Sweden) (Accepted 6 March 1990)
Key words: Locus ceruleus; Excitatory amino acid; Kynurenic acid; C-afferent; Cutaneous thermal sensitivity
Previous electrophysiological experiments have shown that brain noradrenaline neurons in the locus ceruleus are activated by thermal cutaneous stimuli. In the present study a putative involvement of excitatory amino acids (EAA) in cutaneous LC activation was analyzed. Intraventricular administration of kynurenic acid (1 /~mol), a broad spectrum EAA antagonist, or the non-NMDA antagonist 6cyano-7-nitroquinoxaline-2,3-dione (CNQX; 0.1/~mol) as well as subcutaneous administration of the specific NMDA antagonist MK 801 (2 mg/kg) almost totally abolished the response of LC neurons to both non-noxious and noxious cutaneous sensory stimuli. We propose that the activation of LC neurons following thermal cutaneous stimuli is mediated via release of EAA from nerve terminals emanating from nucleus paragigantocellularis (PGi). The nucleus locus ceruleus (LC) constitutes the largest group of noradrenaline neurons in the mammalian brain, with an axonal projection covering almost the entire neuroaxis. This nucleus is suggested to be involved in various sensory and autonomic functions 25, including pain perception and endogenous pain modulatory mechanisms 4'22. For example, previous electrophysiological studies have shown that noxious and non-noxious cutaneous thermal stimulation of the tail is associated with activation of the LC 8, a phenomenon that was shown to be mediated via capsaicin-sensitive C-fiber afferents 16. Recent anatomic and physiologic studies have shown that the major afferent projection to the LC emanates from the nucleus paragigantocellularis (PGi) 3. This innervation is predominantly excitatory and seems to be essentially mediated via release of excitatory amino acids ( E A A ) 11'13. The present electrophysiological study was undertaken in order to analyze a putative role of E A A in the excitatory effect on LC neurons by thermal cutaneous stimulation. Male Sprague-Dawley rats (200-250 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic apparatus. For i.c.v, administration of kynurenic acid (Sigma; 0.05 M, 20 /A, pH 7.2), 6cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris Neuramin; 0.005 M (dissolved in DMSO and diluted to final concentration with saline), 20 /~1, pH 7.2) or saline a plastic cannula was inserted into the right lateral ventri-
cle. The dose of CNQX was calculated on the basis of its relative potency compared to that of kynurenic acid as judged from various in vitro experiments 5"6"19"23'3°. I.c.v. administration of drugs was performed slowly for 1-2 min. A 3 mm burr hole was drilled with its center located approximately 1.1 mm posterior to lambda and 1.1 mm lateral to the midline. For single-barrel experiments a micropipette with a tip diameter of approximately 1-2 /~m and filled with 2 M NaCI saturated with Fast green was lowered by means of a hydraulic microdrive into the region of LC. The in vitro impedance of the electrodes were 3-6 MQ, measured in saline at 135 Hz. For microiontophoretic experiments, a 5-barrel micropipette, broken to obtain a tip diameter of approximately 3-4/~m, was used. Each barrel was filled by direct injection according to the method of Tasaki et al. 27. The central barrel was filled with 2 M NaCI solution saturated with Fast green and was used for recording action potentials. One of the 4 side barrels contained 4 M NaCI solution and was used for automatic current balancing. The remaining barrels were filled with substance P (SP; 1.5 mM) dissolved in 20 mM sodium acetate, pH 4.5, or L-glutamic acid (20 mM, dissolved in 0.2 M NaC1, pH 8.6). The in vitro impedances were typically 2-4 MI2 in the central barrel and 40-90 MI2 in the side barrels. A retaining current of about 5 nA was maintained between ejections to avoid leakage of drugs from the pipette. Ejection of drugs were performed frequently to avoid
* Present address: Department of Physiology, A. Szent-Gy6rgyiMedical University, Szeged, Hungary.
Correspondence: G. Engberg, Department of Pharmacology, University of G6teborg, PO Box 33031, 400 33 G6teborg, Sweden 0006-8993/90/$03.50 © •990 Elsevier Science Publishers B.V. (Biomedical Division)
326 d e a d - t i m e artifacts. Single unit potentials were passed through a high input-impedance amplifier and filters. The impulses were discriminated from background noise and fed into a digital counter, which was reset every 10 s, and finally displayed on a storage oscilloscope, an audiomonitor and a strip chart recorder. The neurophysiological characteristics of the cells were identical to those previously described for noradrenergic neurons of the rat LC 1. The body t e m p e r a t u r e of the animals was maintained at 37 °C by means of a heating pad. The position of the electrode was m a r k e d at the end of each experiment by iontophoretic ejection of Fast green. The rats were then perfused with 10% buffered formalin solution and the brains were subjected to conventional histological procedures. Only cells within the LC were included in this study. T h e r m a l cutaneous non-noxious or noxious stimulation was applied to the tail using a plethysmograph connected to two constant t e m p e r a t u r e baths and this allowed stepwise changes in t e m p e r a t u r e 8. The rectal t e m p e r a t u r e of the animal r e m a i n e d constant throughout the experiments. 1
23
_
ca
5 min
1
t
In control rats a slow stepwise (about 4 °C/min), rise in tail skin t e m p e r a t u r e , from 36 °C to 46 °C, was associated with a gradual activation of neuronal activity of the majority of LC neurons tested (Fig. la). The activity returned to the baseline level when the thermal stimulation was terminated. Elevation of tail skin t e m p e r a t u r e to 39 °C, which is defined as a non-noxious warm sensation, was associated with an LC activation of about 22%. Elevation of tail skin t e m p e r a t u r e to 46 °C, defined as noxious heat stimulation, resulted in an LC activation with about 80% (Fig. 2). Following administration of kynurenic acid (1 ktmol i.c.v.) the response of LC neurons to both non-noxious and noxious heat stimulation was practically abolished (Fig. l b ) , including response of LC neurons to toe pinch I~. In fact, the success of a correct i.c.v, injection of kynurenic acid was easily analyzed by the absence of the toe pinch response. Treatment by kynurenic acid also largely r e d u c e d the excitatory effect of microiontophoretically applied glutamate onto the LC neurons (n = 2; 2 cells), but not that of similarly applied substance P (n = 2; 2 cells). A d m i n i s t r a t i o n of kynurenic acid did not significantly change the basal firing rate of the LC neurons; mean firing rate in u n t r e a t e d rats were 2.37 Hz + 0.15 (n = 13 cells) and in rats treated with kynurenic acid 2.51 Hz + 0.34 (n = 13 cells). In addition to kynurenic acid p r e t r e a t m e n t with M K 801 (2 mg/kg, s.c. 10-40 rain), without significantly altering the basal firing rate of the LC neurons, totally blocked the L C excitation following cutaneous stimulation in all cells tested (Fig. 2). l.c.v, administration of C N Q X ( 0 . 1 / t m o l ) significantly reduced the response of LC neurons to cutaneous stimulation (Fig. 2). H o w e v e r , in about 40% of the LC neurons tested the thermal stimulation caused a clear-cut
23
90~
5_._
/~ •
~ I
5 min
Fig. 1. Typical activation of a single noradrenaline-containing neuron in the LC following cutaneous thermal stimulation of the tail in an untreated rat (a) and in the same rat treated with kynurenic acid (b; 1 pmol, i.c.v.). Arrows indicate: 1, start of thermal stimulation; 2, attained tail skin temperature of 45 °C; 3, 46 °C, termination of thermal stimulation. Note that whereas the excitatory response of microiontophoretic application of glutamate is largely reduced following kynurenic acid treatment, the response to similarly applied substance P is unaffected. Horizontal bars indicate periods of iontophoretic ejection and numbers above show the ejection current in nA.
saline kynurenic acid
30"
10' -lo, 3;
3~
4~ tail temperature,
is
4~
"C
Fig. 2. The effect of thermal cutaneous stimulation of the tail on the firing rate of LC noradrenergic neurons in control rats (n = 7; 13 cells) and in rats treated with kynurenic acid (1 ~mol, i.c.v.; n = 7; 13 ceils), MK 801 (2 mg/kg, s.c., n = 5; 18 cells) or CNQX (0,1 #mol, i.c.v, n = 4; 13 cells). The values represents mean and S.E.M. * p < 0.001 according to Mann-Whitney's U-test.
327 activation of L C neurons (some of them almost within the control range of response), whereas the rest were almost totally insensitive. The present results confirm previous observations 8'16 that a gradual increase in tail skin temperature from 36 °C up to 46 °C is associated with an increase in firing rate of LC noradrenergic neurons. The phenomenon appears to be mediated via activation of polymodal C-fiber afferents 16 and given the involvement of LC in behavioral functions such as attention and arousal reactions 25 it may contribute to the behavioural thermoregulation in animals subjected to warm ambient temperature 16.
ously shown to be mediated via the PGi 7. Thus, thermal cutaneous stimulation may share with systemic administration of nicotine a c o m m o n pathway to affect the firing rate of LC neurons, which include primary C-fiber afferents and E A A containing PGi neurons innervating LC. Based on the present results several subtypes of E A A receptors may be involved in the LC activation following cutaneous stimuli. This view is supported by previous studies 1°'12'2° and by the present finding that the noncompetitive N M D A antagonist M K 801 as well as the n o n - N M D A receptor antagonist C N Q X significantly blocked the LC activation. However, whereas MK 801 is considered as a very specific N M D A receptor antagonist the specificity of C N Q X as a n o n - N M D A receptor antagonist may be questioned 5't7'19'28'29. Thus, in the
The results of the present study show that E A A antagonists, without affecting the basal firing rate, blocked the response of L C neurons to thermal stimuli of both non-noxious and noxious character. Kynurenic acid appears to be an antagonist for several E A A receptors and should not exert any actions on other types of receptors 21'24. In the present study, the action of kynurenic acid showed some specificity since the excitatory response of L C neurons to microiontophoretically applied substance p9,14 was unaffected by the treatment, in contrast to similarly applied glutamate. Altogether the present data show that E A A are involved in the LC activation following thermal stimuli. Given the recently described E A A - m e d i a t e d innervation of the LC from PGi (see Introduction), this pathway appears as a likely candidate to serve as the final link in the response of LC neurons following thermal sensory stimuli. In fact, the response of LC neurons to thermal stimuli resembles that of nicotine-induced excitation of the L C neuronslS,26; both effects are of peripheral origin and blocked by capsaicin treatment as well as i.c.v. administration of kynurenic acid 1°. With respect to the nicotine-induced activation of LC the effect was previ-
present study the antagonism of LC activation observed following administration of C N Q X , which was not quite as complete as that of kynurenic acid or MK 801, may not entirely be a result of the blockade of n o n - N M D A receptors. At any rate, however, our data clearly show that N M D A receptors are involved in the LC activation following cutaneous thermal stimulation. In conclusion, the response of LC neurons to thermal cutaneous stimuli of non-noxious as well as noxious character seems to be mediated via E A A probably released from PGi. Given the implication of LC, e.g. in vigilance reactions to environmental sensory stimuli 2"13 and behavioral thermoregulation 16, the present results point to a functional role of E A A in thermoregulatory mechanisms. Furthermore, the present data emphasize the importance of E A A for peripheral sensory regulation of LC.
1 Aghajanian, G.K., Cedarbaum, J.M. and Wang, R.Y., Evidence for norepinephrine-mediated collateral inhibition of locus coeruleus neurons, Brain Research, 131 (1977) 570-577. 2 Aston-Jones, G., Behavioral functions of locus coeruleus derived from cellular attributes, Physiol. Psychol., 13 (1985) 118-126. 3 Aston-Jones, G., Ennis, M., Pieribone, V.A., Nickell, W.T. and Shipley, M.T., The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network, Science, 234 (1986) 734-737. 4 Basbaum, A.I. and Fields, H.L., Endogenous pain control system: brain stem spinal pathways and endorphin circuitry, Annu. Rev. Neurosci., 7 (1984) 309-338. 5 Birch, P.J., Grossman, C.J. and Hayes, A.G., 6,7-Dinitroquinoxaline-2,3-dion and 6-nitro,7-cyano-quinoxaline-2,3-dion antagonise responses to NMDA in the rat spinal cord via an action at the strychnine-insensitive glycine receptor, Eur. J. Pharmacol., 156 (1988) 177-180 6 Bridges, R.J., Stevens, D.R., Kahle, J.S., Nunn, P.B., Kadri, M. and Cotman, C.W., Structure-function studies on Noxalyl-diamino-dicarboxylic acids and excitatory amino acid
receptors: evidence that fl-L-ODAP is a selective non-NMDA agonist, J. Neurosci., 9 (1989) 2073-2079. 7 Chen, Z. and Engberg, G., The rat nucleus paragigantocellularis as a relay station to mediate peripherally induced central effects of nicotine, Neurosci. Lett., 101 (1989) 67-71. 8 Elam, M., Svensson, T.H. and Thor6n, E, Locus coeruleus neurons and sympathetic nerves: activation by cutaneous sensory afferents, Brain Research, 366 (1986) 254-261. 9 Engberg, G., Svensson, T.H., Rosell, S. and Folkers, K., A synthetic peptide as an antagonist of substance P, Nature (Lond.), 293 (1981) 222-223. 10 Engberg, G., Nicotine induced excitation of locus coeruleus neurons is mediated via release of excitatory amino acids, Life Sci., 44 (1989) 1535-1540. 11 Ennis, M. and Aston-Jones, G., A potent excitatory input to the nucleus locus coeruleus from the ventrolateral medulla, Neurosci. Lett., 71 (1986) 299-305. 12 Ennis, M. and Aston-Jones, G., Activation of locus coeruleus neurons from the paragigantocellularis: a new excitatory amino acid pathway in brain, J. Neurosci., 8 (1988) 3644-3657. 13 Foote, S.L., Bloom, EE. and Aston-Jones, G., Nucleus locus
Supported by the Swedish Medical Research Council (Project 7484), 'Loo och Hans Ostermans Fond' and 'Torsten och Ragnar S6derbergs Stiftelser'.
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14
15
16
17
18
19
20
21
coeruleus: new evidence of anatomical and physiological specificity, Physiol. Rev., 63 (1983) 844-914. Guyenet, P.G. and Aghajanian, G.K., Excitation of neurons in the locus coeruleus by substance P and related peptides, Brain Research, 136 (1977) 178-184. Haj6s, M. and Engberg, G., Role of primary sensory neurons in the central effects of nicotine, Ps'ychopharmacology, 94 (1988) 468-470. Haj6s, M., Engberg, G. and Elam, M., Reduced responsiveness of locus coeruleus neurons to cutaneous thermal stimuli in capsaicin-treated rats, Neurosci. Lett., 70 (1986) 382-387. Honor6, T., Davies, S.N., Drejer, J , Fletcher, E.J., Jacobsen, P., Lodge, D. and Nielsen, E, Quinoxalinediones: Potent competitive non-NMDA glutamate receptor antagonists, Science, 241 (1988) 701-703. Korf, J., Bunney, B.S. and Aghajanian, G.K., Noradrenergic neurons: morphine inhibition of spontaneous activity, Eur. J. Pharmacol., 25 (1974) 165-169. Lester, R.A.J., Quarum, M.L., Parker, J.D., Weber, E. and Jahr, C.E., Interaction of 6-cyano-7-nitroquinoxatine-2,3-dione with the N-methyl-D-aspartate receptor-associated glycine binding site, Mol. Pharmacol., 35 (1989) 565-570. Olphe, H.-R., Steinmann, M.W., Brugger, E and Pozza, M.E, Excitatory amino acid receptors in the rat locus coeruleus - an extracellular in vitro study, Naunyn-Schmiedeberg's Arch. Pharmacol.. 339 (1989) 312-314. Perkins, M.N. and Stone, T.W., An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid, Brain Research,
247 (1982) 184-187. 22 Segal, M. and Sandberg, D., Analgesia produced by electrical stimulation of catecholamine nuclei in the rat brain, Brain Research, 123 (1977) 369-372. 23 Stone, T., Comparison of kynurenic acid and 2-APV suppression of epileptiform activity in rat hippocampal slices, Neurosci. Lett., 84 (1988) 234-238. 24 Stone, T.W. and Connick, J.H., Quinolinic acid and other kynurenines in the central nervous system, Neuroscience, 15 (1985) 597-617. 25 Svensson, T.H., Peripheral, autonomic regulation of locus coeruleus noradrenergic neurons in brain: putative implications for psychiatry and psychopharmacology, Psychopharmacology, 92 (1987) 1-7. 26 Svensson,T.H. and Engberg, G., Effect of nicotine on single cell activity in the noradrenergic nucleus locus coeruleus, Acta Physiol. Scand., Suppl. 479 (1980) 31-34. 27 Tasaki, K., Tsukahara, U., Ito, S., Wayner, M.J. and Yu, W.Y., A simple, direct and rapid method for filling microelectrodes, Physiol. Behav., 3 (1968) 1009-1010. 28 Watkins, J.C. and Olverman, H.J., Agonists and antagonists for excitatory amino acids, Trends Neurosci., 10 (1987) 265-272. 29 Wong, E.H.F., Kemp, J.A., Priestley, T., Knight, A.R., Woodruff, G.N and Iversen, L.L., The anticonvulsant MK 801 is a potent N-methyI-D-aspartate antagonist, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7104-710819. 30 Yamada, K.A., Dubinsky, J.M. and Rothman, S.M., Quantitative physiological characterization of a quinoxalinedione nonNMDA receptor antagonist, J. Neurosci,, 9 (1989) 3230-3236.
325
Elsevier BRES 24132
A role of excitatory amino acids in the activation of locus coeruleus neurons following cutaneous thermal stimuli Mihfily Haj6s* and G6ran Engberg Department of Pharmacology, University of GOteborg, GOteborg (Sweden) (Accepted 6 March 1990)
Key words: Locus ceruleus; Excitatory amino acid; Kynurenic acid; C-afferent; Cutaneous thermal sensitivity
Previous electrophysiological experiments have shown that brain noradrenaline neurons in the locus ceruleus are activated by thermal cutaneous stimuli. In the present study a putative involvement of excitatory amino acids (EAA) in cutaneous LC activation was analyzed. Intraventricular administration of kynurenic acid (1 /~mol), a broad spectrum EAA antagonist, or the non-NMDA antagonist 6cyano-7-nitroquinoxaline-2,3-dione (CNQX; 0.1/~mol) as well as subcutaneous administration of the specific NMDA antagonist MK 801 (2 mg/kg) almost totally abolished the response of LC neurons to both non-noxious and noxious cutaneous sensory stimuli. We propose that the activation of LC neurons following thermal cutaneous stimuli is mediated via release of EAA from nerve terminals emanating from nucleus paragigantocellularis (PGi). The nucleus locus ceruleus (LC) constitutes the largest group of noradrenaline neurons in the mammalian brain, with an axonal projection covering almost the entire neuroaxis. This nucleus is suggested to be involved in various sensory and autonomic functions 25, including pain perception and endogenous pain modulatory mechanisms 4'22. For example, previous electrophysiological studies have shown that noxious and non-noxious cutaneous thermal stimulation of the tail is associated with activation of the LC 8, a phenomenon that was shown to be mediated via capsaicin-sensitive C-fiber afferents 16. Recent anatomic and physiologic studies have shown that the major afferent projection to the LC emanates from the nucleus paragigantocellularis (PGi) 3. This innervation is predominantly excitatory and seems to be essentially mediated via release of excitatory amino acids ( E A A ) 11'13. The present electrophysiological study was undertaken in order to analyze a putative role of E A A in the excitatory effect on LC neurons by thermal cutaneous stimulation. Male Sprague-Dawley rats (200-250 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic apparatus. For i.c.v, administration of kynurenic acid (Sigma; 0.05 M, 20 /A, pH 7.2), 6cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris Neuramin; 0.005 M (dissolved in DMSO and diluted to final concentration with saline), 20 /~1, pH 7.2) or saline a plastic cannula was inserted into the right lateral ventri-
cle. The dose of CNQX was calculated on the basis of its relative potency compared to that of kynurenic acid as judged from various in vitro experiments 5"6"19"23'3°. I.c.v. administration of drugs was performed slowly for 1-2 min. A 3 mm burr hole was drilled with its center located approximately 1.1 mm posterior to lambda and 1.1 mm lateral to the midline. For single-barrel experiments a micropipette with a tip diameter of approximately 1-2 /~m and filled with 2 M NaCI saturated with Fast green was lowered by means of a hydraulic microdrive into the region of LC. The in vitro impedance of the electrodes were 3-6 MQ, measured in saline at 135 Hz. For microiontophoretic experiments, a 5-barrel micropipette, broken to obtain a tip diameter of approximately 3-4/~m, was used. Each barrel was filled by direct injection according to the method of Tasaki et al. 27. The central barrel was filled with 2 M NaCI solution saturated with Fast green and was used for recording action potentials. One of the 4 side barrels contained 4 M NaCI solution and was used for automatic current balancing. The remaining barrels were filled with substance P (SP; 1.5 mM) dissolved in 20 mM sodium acetate, pH 4.5, or L-glutamic acid (20 mM, dissolved in 0.2 M NaC1, pH 8.6). The in vitro impedances were typically 2-4 MI2 in the central barrel and 40-90 MI2 in the side barrels. A retaining current of about 5 nA was maintained between ejections to avoid leakage of drugs from the pipette. Ejection of drugs were performed frequently to avoid
* Present address: Department of Physiology, A. Szent-Gy6rgyiMedical University, Szeged, Hungary.
Correspondence: G. Engberg, Department of Pharmacology, University of G6teborg, PO Box 33031, 400 33 G6teborg, Sweden 0006-8993/90/$03.50 © •990 Elsevier Science Publishers B.V. (Biomedical Division)
326 d e a d - t i m e artifacts. Single unit potentials were passed through a high input-impedance amplifier and filters. The impulses were discriminated from background noise and fed into a digital counter, which was reset every 10 s, and finally displayed on a storage oscilloscope, an audiomonitor and a strip chart recorder. The neurophysiological characteristics of the cells were identical to those previously described for noradrenergic neurons of the rat LC 1. The body t e m p e r a t u r e of the animals was maintained at 37 °C by means of a heating pad. The position of the electrode was m a r k e d at the end of each experiment by iontophoretic ejection of Fast green. The rats were then perfused with 10% buffered formalin solution and the brains were subjected to conventional histological procedures. Only cells within the LC were included in this study. T h e r m a l cutaneous non-noxious or noxious stimulation was applied to the tail using a plethysmograph connected to two constant t e m p e r a t u r e baths and this allowed stepwise changes in t e m p e r a t u r e 8. The rectal t e m p e r a t u r e of the animal r e m a i n e d constant throughout the experiments. 1
23
_
ca
5 min
1
t
In control rats a slow stepwise (about 4 °C/min), rise in tail skin t e m p e r a t u r e , from 36 °C to 46 °C, was associated with a gradual activation of neuronal activity of the majority of LC neurons tested (Fig. la). The activity returned to the baseline level when the thermal stimulation was terminated. Elevation of tail skin t e m p e r a t u r e to 39 °C, which is defined as a non-noxious warm sensation, was associated with an LC activation of about 22%. Elevation of tail skin t e m p e r a t u r e to 46 °C, defined as noxious heat stimulation, resulted in an LC activation with about 80% (Fig. 2). Following administration of kynurenic acid (1 ktmol i.c.v.) the response of LC neurons to both non-noxious and noxious heat stimulation was practically abolished (Fig. l b ) , including response of LC neurons to toe pinch I~. In fact, the success of a correct i.c.v, injection of kynurenic acid was easily analyzed by the absence of the toe pinch response. Treatment by kynurenic acid also largely r e d u c e d the excitatory effect of microiontophoretically applied glutamate onto the LC neurons (n = 2; 2 cells), but not that of similarly applied substance P (n = 2; 2 cells). A d m i n i s t r a t i o n of kynurenic acid did not significantly change the basal firing rate of the LC neurons; mean firing rate in u n t r e a t e d rats were 2.37 Hz + 0.15 (n = 13 cells) and in rats treated with kynurenic acid 2.51 Hz + 0.34 (n = 13 cells). In addition to kynurenic acid p r e t r e a t m e n t with M K 801 (2 mg/kg, s.c. 10-40 rain), without significantly altering the basal firing rate of the LC neurons, totally blocked the L C excitation following cutaneous stimulation in all cells tested (Fig. 2). l.c.v, administration of C N Q X ( 0 . 1 / t m o l ) significantly reduced the response of LC neurons to cutaneous stimulation (Fig. 2). H o w e v e r , in about 40% of the LC neurons tested the thermal stimulation caused a clear-cut
23
90~
5_._
/~ •
~ I
5 min
Fig. 1. Typical activation of a single noradrenaline-containing neuron in the LC following cutaneous thermal stimulation of the tail in an untreated rat (a) and in the same rat treated with kynurenic acid (b; 1 pmol, i.c.v.). Arrows indicate: 1, start of thermal stimulation; 2, attained tail skin temperature of 45 °C; 3, 46 °C, termination of thermal stimulation. Note that whereas the excitatory response of microiontophoretic application of glutamate is largely reduced following kynurenic acid treatment, the response to similarly applied substance P is unaffected. Horizontal bars indicate periods of iontophoretic ejection and numbers above show the ejection current in nA.
saline kynurenic acid
30"
10' -lo, 3;
3~
4~ tail temperature,
is
4~
"C
Fig. 2. The effect of thermal cutaneous stimulation of the tail on the firing rate of LC noradrenergic neurons in control rats (n = 7; 13 cells) and in rats treated with kynurenic acid (1 ~mol, i.c.v.; n = 7; 13 ceils), MK 801 (2 mg/kg, s.c., n = 5; 18 cells) or CNQX (0,1 #mol, i.c.v, n = 4; 13 cells). The values represents mean and S.E.M. * p < 0.001 according to Mann-Whitney's U-test.
327 activation of L C neurons (some of them almost within the control range of response), whereas the rest were almost totally insensitive. The present results confirm previous observations 8'16 that a gradual increase in tail skin temperature from 36 °C up to 46 °C is associated with an increase in firing rate of LC noradrenergic neurons. The phenomenon appears to be mediated via activation of polymodal C-fiber afferents 16 and given the involvement of LC in behavioral functions such as attention and arousal reactions 25 it may contribute to the behavioural thermoregulation in animals subjected to warm ambient temperature 16.
ously shown to be mediated via the PGi 7. Thus, thermal cutaneous stimulation may share with systemic administration of nicotine a c o m m o n pathway to affect the firing rate of LC neurons, which include primary C-fiber afferents and E A A containing PGi neurons innervating LC. Based on the present results several subtypes of E A A receptors may be involved in the LC activation following cutaneous stimuli. This view is supported by previous studies 1°'12'2° and by the present finding that the noncompetitive N M D A antagonist M K 801 as well as the n o n - N M D A receptor antagonist C N Q X significantly blocked the LC activation. However, whereas MK 801 is considered as a very specific N M D A receptor antagonist the specificity of C N Q X as a n o n - N M D A receptor antagonist may be questioned 5't7'19'28'29. Thus, in the
The results of the present study show that E A A antagonists, without affecting the basal firing rate, blocked the response of L C neurons to thermal stimuli of both non-noxious and noxious character. Kynurenic acid appears to be an antagonist for several E A A receptors and should not exert any actions on other types of receptors 21'24. In the present study, the action of kynurenic acid showed some specificity since the excitatory response of L C neurons to microiontophoretically applied substance p9,14 was unaffected by the treatment, in contrast to similarly applied glutamate. Altogether the present data show that E A A are involved in the LC activation following thermal stimuli. Given the recently described E A A - m e d i a t e d innervation of the LC from PGi (see Introduction), this pathway appears as a likely candidate to serve as the final link in the response of LC neurons following thermal sensory stimuli. In fact, the response of LC neurons to thermal stimuli resembles that of nicotine-induced excitation of the L C neuronslS,26; both effects are of peripheral origin and blocked by capsaicin treatment as well as i.c.v. administration of kynurenic acid 1°. With respect to the nicotine-induced activation of LC the effect was previ-
present study the antagonism of LC activation observed following administration of C N Q X , which was not quite as complete as that of kynurenic acid or MK 801, may not entirely be a result of the blockade of n o n - N M D A receptors. At any rate, however, our data clearly show that N M D A receptors are involved in the LC activation following cutaneous thermal stimulation. In conclusion, the response of LC neurons to thermal cutaneous stimuli of non-noxious as well as noxious character seems to be mediated via E A A probably released from PGi. Given the implication of LC, e.g. in vigilance reactions to environmental sensory stimuli 2"13 and behavioral thermoregulation 16, the present results point to a functional role of E A A in thermoregulatory mechanisms. Furthermore, the present data emphasize the importance of E A A for peripheral sensory regulation of LC.
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Supported by the Swedish Medical Research Council (Project 7484), 'Loo och Hans Ostermans Fond' and 'Torsten och Ragnar S6derbergs Stiftelser'.
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