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Effects of neuroactive compounds, noxious and cardiovascular stimuli on the release of amino acids in the rat locus coeruleus
ELSEVIER
Neuroscience Letters 180 (1994) 55 58
HEUROSCIENCE LETTERS
Effects of neuroactive compounds, noxious and cardiovascular stimuli on the release of amino acids in the rat locus coeruleus Nicolas Singewald*, Christoph Schneider, Athineos Philippu Department of Pharmacology and Toxicology, University of lnnsbruck, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria Received 11 July 1994; Revised version received 12 August 1994; Accepted 12 August 1994
Abstract
The release of excitatory amino acids (glutamate, aspartate), inhibitory amino acids (GABA, taurine) and arginine was determined in the locus coeruleus (LC) of anaesthetized rats. The neuronal origin of stimulated amino acid release was verified by superfusion with neuroactive compounds. Electrical stimulation of the sciatic nerve, as well as mechanical footshock, enhanced LC release rates of glutamate and aspartate without influencing those of taurine and arginine. GABA release rate was increased slightly after some delay. Excitatory amino acid release was not influenced by changes in blood pressure. The results provide direct neurochemical evidence that noxious stimuli activate LC neurons via the glutamate and aspartate input into this nucleus. Key words: Locus coeruleus; Blood pressure; Sciatic nerve stimulation; Hindpaw stimulation; Potassium; Veratridine; Tetrodotoxin; Push-pull cannula
The nucleus locus coeruleus (LC) contains the largest group of noradrenergic neurons, with a widespread neuronal network innervating numerous brain areas [4]. LC neurons respond to a variety of stimuli [1]. In the anaesthetized rat, the activity of LC neurons is decreased by blood volume load [6,15] and increased by a fall of blood pressure (BP) elicited by drugs or hypovolaemia [5,18]. Furthermore, it has been shown that peripheral cardiovascular stimuli also alter noradrenergic turnover [17] and noradrenaline release [12,13] within the LC. Noxious stimuli elicited by foot shock enhance vigorously LC activity [3] and modify turnover and metabolism of noradrenaline in the LC, as well as in LC target areas [2,9,16]. Although it is known that the activity of LC cells is influenced by various putative neurotransmitters applied locally [1], the nature of transmitter(s) involved in alteration of LC activity is as yet not fully understood. Recently, we reported changes in the activity of GABAergic LC neurons by disturbances in BP homeostasis [14]. On the other hand, electrophysiologic studies
*Corresponding author. Fax: (43) 512-507-2522. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)00632-6
provide indirect evidence that activation of LC neurons in response to noxious stimuli is mediated by an excitatory amino acid pathway which originates from the nucleus paragigantocellularis [7,8]. By using the push-pull technique, we now investigated directly the effect of noxious stimuli as well as cardiovascular stimuli on the release of the glutamate (GLU), aspartate (ASP), GABA, taurine (TAU) and arginine (ARG) in the LC. Additionally, the effects of neuroactive compounds on the release of amino acids were studied. In male Sprague-Dawley rats (230-280 g) a guide cannula was stereotaxically implanted under ketamine (50 mg/kg, i.p.) and sodium pentobarbital (40 mg/kg, i.p.) anaesthesia, as previously described [11]. The tip of the guide cannula was positioned 2 mm above the right LC. According to the stereotaxic atlas of Paxinos and Watson [10], coordinates of the LC were: AP 0.8 mm posterior to interaural line, 1.3 mm lateral from midline, DV 2.8 mm above the interaural zero plane. The guide cannula was fixed with screws and dental cement. For recording of arterial BP and intravenous infusions of drugs, respectively, the iliolumbar artery and jugular vein were permanently catheterized with PE 50 and
56
N. SingewaM et al./Neuroscience Letters 180 (1994) 55 5~'
o
K+ ( n - 7 - 8 ) 11 -30-:~O-iO
0
. . . . 10
20
30
40
TIME (min) Fig. I. Effect of LC superfusion with a high potassium concentration (50 mM) on the release of amino acids. The mean release rates in the three samples preceding superfusion with the high potassium concentration were taken as 1.0. Horizontal bar denotes superfusion with 50 m M potassium. Mean values _+ S.E.M. *P < 0.05.
PE 20 tubings. Two days after surgery, the rats were anaesthetized with chloralose (40 mg/kg, i.v.) and placed on a feedback-controlled heat pad. In some rats, the sciatic nerve was dissected in the left hindlimb and covered with mineral oil. Before stimulations (see below), the nerve was hooked on the stimulation electrode. The stylet of the guide cannula was replaced by a push-pull cannula (diameters: outer tube 0.6 mm, inner tube 0.2 ram) which was 2 mm longer than the guide cannula thus reaching the LC. The inner tube of the push-pull cannula was retracted 0.3 mm within the outer tube. The inner diameter (0.3 mm) of the outer cannula confined the superfused area. Artificial cerebrospinal fluid (CSF) used for superfusion contained (mM) NaC1 140, KC1 3.0, CaCI 2 1.3, MgC12 1.0, Na2HPO4 1.0, glucose 3,0 and was adjusted to pH 7.2 with NaHzPO4 1.0. The LC was superfused at a rate of 28 ¢tl/min. After an equilibration period of 80 min, superfusate was collected continuously in time periods of 3 min at -50°C. In experiments in which the composition of the CSF was altered by adding potassium, veratridine or tetrodotoxin (TTX), a zero dead volume liquid switch (Carnegie Medicine) was used. Amino acids released in the superfusate were determined by H P L C and fluorimetric detection (MerckHitachi, Tokyo, Japan) after derivatization with ophthaldialdehyde (OPA) as previously described [11]. In
the present study 80 HI of the superfusate were mixed with 20 ~ 1 0 P A reaction mixture and 75/11 were injected. The reproducibility of the derivatization was controlled by addition of S-carboxymethyl-k-cysteine as an internal standard. The sensitivity of measurement of each amino acid was around 50 fmol per sample. Although great care was taken in the preparation of all solutions, traces of ASP and G L U were present in blanc samples. Experiments, in which the ASP blanc value was higher than half the baseline value, were not considered. Because of this, in some experiment series ASP values are not presented. Stimulation of the intact sciatic nerve contralateral to the superfused LC was carried out with bipolar platinum electrodes. Cathodal square-wave pulses (0.5 ms) obtained from a stimulator (H. Sachs, Freiburg, FRG) were applied at a frequency of 5 Hz. The threshold of stimulating intensity required for eliciting muscle twitches was determined in each animal. The stimulus intensity used in the experiment was ten-fold this threshold and amounted usually to 3 5 V. Mechanical stimulation of the hindpaw contralateral to the superfused LC was carried out over 3 min by an appropriate plastic
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.
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-
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• VER (rv=7-9) o VER + TTX (n=5)
- o- o-io 6 ,b
4"0
TIME (min) Fig. 2. Effects of LC superfusion with veratridine (10/aM) and veratridine (10/aM) in the presence of TTX (1/aM) on the release of amino acids, The mean release rates in the three samples preceding superfusion with drugs were taken as 1.0. Horizontal bar denotes superfusion with veratridine (VER). TTX was present throughout. Mean values + S.E.M. ' P = 0.07, *P < 0.05.
N. SingewaM et al./Neuroscience Letters 180 (1994) 55 58
1.4 1.0
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release rates of GABA, TAU, GLU and ASP (Fig. 2). In the presence of the neurotoxin TTX (1/.tM), the releasing effect of veratridine was abolished. Veratridine did not significantly influence the release of arginine. The enhancing effects of potassium and veratridine on the amino acid release rates, as well as the abolition of the veratridine induced increases by TTX indicate that the stimulated amino acid release in the superfusate originates substantially from neuronal sites. Electrical stimulation of the sciatic nerve, as well as mechanical stimulation of the hindpaw, increased slightly the mean arterial BP. Both kinds of stimulation led to a pronounced enhancement in the release rates of the excitatory amino acids GLU and ASP. This effect was due to the noxious stimulus and not to the increased BP, since even a pronounced rise in BP (60 mmHg) did not change the release rates of ASP and GLU (see below). The release of GABA was slightly enhanced in the sample following mechanical stimulation of the hindpaw. These stimuli did not influence significantly the release rates of TAU and ARG (Fig. 3). The effect of electrical stimulation was current-dependent (not shown). Experimentally induced BP changes were elicited by intravenous infusions of phenylephrine or sodium nitroprusside. Neither the pressor response of phenylephrine, nor the fall of BP elicited by sodium nitroprusside influenced the release rates of the excitatory amino acids GLU and ASP in the LC (Fig. 4). Similarly, changes in BP elicited by blood injection (30% hypervolaemia) or
b TIME (min)
Fig. 3. Effects of electrical stimulation of the sciatic nerve or mechanical stimulation of the hindpaw on the release of amino acids in the LC. The mean release rates in the two samples preceding stimulations were taken as 1.0. Horizontal bar denotes stimulations. SN sciatic nerve, HP hindpaw. Mean values + S.E.M. *P < 0.05.
57
221
q) r~ a)~
1.B ~ 1.4 ('P i.o
E
< 1~
v
0.4
clamp (1 kg force). Localization of the push-pull cannula was verified microscopically in brain slices at the end of the experiment [11]. Experiments with cannula localizations outside the LC were discarded. Data were analyzed statistically by Friedman's test followed by Wilcoxon's signed rank test for paired data. The basal release rates of the amino acids were (pmol/ min): GABA 0.15+0.02, TAU 2.29+0.33, GLU 1.57 + 0.22, ASP 0.55 + 0.08, ARG 1.27 + 0.24 (mean values + S.E.M., n = 22-33). Local perfusion of the LC with CSF containing 50 mM potassium enhanced greatly the release rates of GABA, TAU and GLU during, as well as in the sample following superfusion with the high concentration of potassium (Fig. 1). The release rate of arginine was enhanced slightly after a delay of 10 min. Superfusion with veratridine (10 pM) also enhanced the
I- 8Ol
e .
~ .
~ .
.
.
O.-40 j ,.n O NaCI (n=7)H • PE (n-7-9) o NP (n-13-15) i m
-9
-6 -3
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6
9
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TIME (rain) Fig. 4. Effects of phenylephrine (10/tg/kg/min), physiologic saline (100 /A/min) and sodium nitroprusside (30 pg/kg/min) on arterial BP and release of amino acids in the LC. The mean release rates in the three samples preceding infusion with drugs were taken as 1.0. Horizontal bar denotes infusions with drug. PE, phenylephrine; NP, sodium nitroprusside. Mean values + S.E.M. **P < 0.01.
58
N. Singewaht et aL /Neuroscience Letters 180 (1994) 55 58
controlled haemorrhage (15% hypovolaemia) were ineffective (not shown). Intravenous infusion of physiologic saline did not modify either blood pressure, or release rates of amino acids in the LC. Recently, we reported that increases in BP enhance the release of GABA in the LC. On the other hand, decreases in BP decrease the GABA release rate, thus showing that the release of this inhibitory amino acid in the LC is modified by cardiovascular impulses [14]. In contrast to this, we now observed that BP changes did not modify the release of the excitatory amino acids GLU and ASP in the LC. Thus, GLU and ASP seem to be also excluded as possible excitatory neurotransmitters activating LC neurons elicited by lowering BE as shown in electrophysiologic studies [5,18]. On the other hand, electrical stimulation of the sciatic nerve or mechanical stimulation of the hindpaw enhanced greatly the release rates of the excitatory amino acids GLU and ASP without influencing those of the inhibitory amino acids GABA and TAU. The delayed response of GABA release to the stimulation of the sciatic nerve might point to a secondary enhanced activity of GABAergic neurons. These findings demonstrate that the enhanced activity of LC neurons as a response to noxious sensory stimuli is mediated by the GLU and ASP inputs into the LC. Furthermore, the results show that the excitatory amino acid input into the LC is not involved in central cardiovascular control. This work was supported by the Fonds zur Foerderung der wissenschaftlichen Forschung. [1] Aston-Jones, G., Shipley, M.T., Ennis, M.. Williams, J.T. and Pieribone, V.A., Restricted afferent control of locus coeruleus neurons revealed by anatomic, physiologic and pharmacologic studies. In C.A. Marsden and D.J. Heal (Eds.), The Pharmacology of Noradrenaline in the Central Nervous System, Oxford University Press, Oxford, 1990, pp. 187 247. [2] Brun, P., Suaud-Chagny, M.F., Lachuer, J., Gonon, F. and Buda, M., Catecholamine metabolism in locus coeruleus neurons: a study of its activation by sciatic nerve stimulation in the rat, Eur. J. Neurosci., 3 (1991) 397~,06. [3] Cedarbaum, J.M. and Agbajanian, G.K., Activation of locus coeruleus neurons by peripheral stimuli: modulation by a collateral inhibitory mechanism, Life Sci., 23 (1978) 1383 1392. [4] Dahlstr6m, A. and Fuxe, K., Evidence for the existence of
monoamine-containing neurons in the central nervous system. 1. Demonstration of monoamines in the cell bodies of brainstem neurons, Acta Physiol. Scand., (Suppl. 232) 62 (1964) 1 55. [5] Elam, M., Svensson, T.H. and Thoren, P., Differentiated cardiovascular afferent regulation of locus coeruleus neurons and sympathetic nerves, Brain Res., 358 (1985t 77 84. [6] Elam, M., Yao, T., Svensson, T.H. and Thoren, P., Regulation of locus coeruleus neurons and splanchnic, sympathetic nerves by cardiovascular afferents, Brain Res., 290 (1984) 281-287. [7] Ennis, M. and Aston-Jones, G., Activation of locus coeruleus from nucleus paragigantocellularis: a new excitatory amino acid pathway in brain, .I. Neurosci., 8 (1986) 3644 3657. [8] Ennis, M., Aston-Jones, G. and Shiekhattar, R., Activation of locus coeruleus neurons by nucleus paragigantocellularis or noxious sensory stimulation is mediated by intracoerulear excitatory amino acid neurotransmission, Brain Res., 598 (1992) 185 195. [9] Korf, J., Aghajanian, G.K. and Roth, R.H., Increased turnover of norepinephrine in the rat cerebral cortex during stress: role of Ihe locus coeruleus, Neuropharmacology, 12 (1973) 933 938. [10] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. [11] Singewald, N., Guo, L.J. and Philippu, A., Release of endogenous GABA in the posterior hypothalamus of the conscious rat; effects of drugs and experimentally induced blood pressure changes, Naunyn-Schmiedeberg's Arch. Pbarmacol., 347 (1993) 402406. [12] Singewald, N. and Philippu, A., Catecholamine release in the locus coeruleus is modified by experimentally induced changes in haemodynamics, Naunyn-Schmiedeberg'sArch. Pharmacol., 347 (1993) 21 27. [13] Singewald, N., Schneider, C and Philippu, A., Effects of blood pressure changes on the catecholamine release in the locus coeruleus of cats anaesthetized with pentobarbital or chloralose, Nannyn-Schmiedeberg's Arch. Pharmacol., 348 (1993) 242 248. [14] Singewald, N., Schneider, C and Philippu, A., Disturbances in blood pressure homeostasis modify GABA release in the locus coeruleus, NeuroReport, 5 (1994) 1709 1712. [15] Svensson, T.H. and Thoren, P., Brain noradrenergic neurons in the locus coeruleus: inhibition by blood volume load through vagal afferents, Brain Res., 172 (1979) 174 178. [16] Thierry, A.M., Javoy, F., GIowinski, J. and Kety, S.S., Effects of stress on the metabolism of norepinephrine, dopamine and serotonin in the central nervous system of the rat. 1. Modifications of norepinephrine turnover, J. Pharmacol. Exp. Ther., 163 (1968) 163 171. [17] Tbrivikraman, K.V., Carlson, D,E. and Gann, D.S., Noradrenergic turnover increases in locus coeruleus after hemorrhage in cats, Am. J. Physiol., 254 (19881 R296 R301. [18] Valentino, R.J., Corticotropin-releasing factor: putative neurotransmitter in the noradrenergic nucleus locus coeruleus, Psychopharmacol. Bull., 25 (1989) 306 311.
Neuroscience Letters 180 (1994) 55 58
HEUROSCIENCE LETTERS
Effects of neuroactive compounds, noxious and cardiovascular stimuli on the release of amino acids in the rat locus coeruleus Nicolas Singewald*, Christoph Schneider, Athineos Philippu Department of Pharmacology and Toxicology, University of lnnsbruck, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria Received 11 July 1994; Revised version received 12 August 1994; Accepted 12 August 1994
Abstract
The release of excitatory amino acids (glutamate, aspartate), inhibitory amino acids (GABA, taurine) and arginine was determined in the locus coeruleus (LC) of anaesthetized rats. The neuronal origin of stimulated amino acid release was verified by superfusion with neuroactive compounds. Electrical stimulation of the sciatic nerve, as well as mechanical footshock, enhanced LC release rates of glutamate and aspartate without influencing those of taurine and arginine. GABA release rate was increased slightly after some delay. Excitatory amino acid release was not influenced by changes in blood pressure. The results provide direct neurochemical evidence that noxious stimuli activate LC neurons via the glutamate and aspartate input into this nucleus. Key words: Locus coeruleus; Blood pressure; Sciatic nerve stimulation; Hindpaw stimulation; Potassium; Veratridine; Tetrodotoxin; Push-pull cannula
The nucleus locus coeruleus (LC) contains the largest group of noradrenergic neurons, with a widespread neuronal network innervating numerous brain areas [4]. LC neurons respond to a variety of stimuli [1]. In the anaesthetized rat, the activity of LC neurons is decreased by blood volume load [6,15] and increased by a fall of blood pressure (BP) elicited by drugs or hypovolaemia [5,18]. Furthermore, it has been shown that peripheral cardiovascular stimuli also alter noradrenergic turnover [17] and noradrenaline release [12,13] within the LC. Noxious stimuli elicited by foot shock enhance vigorously LC activity [3] and modify turnover and metabolism of noradrenaline in the LC, as well as in LC target areas [2,9,16]. Although it is known that the activity of LC cells is influenced by various putative neurotransmitters applied locally [1], the nature of transmitter(s) involved in alteration of LC activity is as yet not fully understood. Recently, we reported changes in the activity of GABAergic LC neurons by disturbances in BP homeostasis [14]. On the other hand, electrophysiologic studies
*Corresponding author. Fax: (43) 512-507-2522. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)00632-6
provide indirect evidence that activation of LC neurons in response to noxious stimuli is mediated by an excitatory amino acid pathway which originates from the nucleus paragigantocellularis [7,8]. By using the push-pull technique, we now investigated directly the effect of noxious stimuli as well as cardiovascular stimuli on the release of the glutamate (GLU), aspartate (ASP), GABA, taurine (TAU) and arginine (ARG) in the LC. Additionally, the effects of neuroactive compounds on the release of amino acids were studied. In male Sprague-Dawley rats (230-280 g) a guide cannula was stereotaxically implanted under ketamine (50 mg/kg, i.p.) and sodium pentobarbital (40 mg/kg, i.p.) anaesthesia, as previously described [11]. The tip of the guide cannula was positioned 2 mm above the right LC. According to the stereotaxic atlas of Paxinos and Watson [10], coordinates of the LC were: AP 0.8 mm posterior to interaural line, 1.3 mm lateral from midline, DV 2.8 mm above the interaural zero plane. The guide cannula was fixed with screws and dental cement. For recording of arterial BP and intravenous infusions of drugs, respectively, the iliolumbar artery and jugular vein were permanently catheterized with PE 50 and
56
N. SingewaM et al./Neuroscience Letters 180 (1994) 55 5~'
o
K+ ( n - 7 - 8 ) 11 -30-:~O-iO
0
. . . . 10
20
30
40
TIME (min) Fig. I. Effect of LC superfusion with a high potassium concentration (50 mM) on the release of amino acids. The mean release rates in the three samples preceding superfusion with the high potassium concentration were taken as 1.0. Horizontal bar denotes superfusion with 50 m M potassium. Mean values _+ S.E.M. *P < 0.05.
PE 20 tubings. Two days after surgery, the rats were anaesthetized with chloralose (40 mg/kg, i.v.) and placed on a feedback-controlled heat pad. In some rats, the sciatic nerve was dissected in the left hindlimb and covered with mineral oil. Before stimulations (see below), the nerve was hooked on the stimulation electrode. The stylet of the guide cannula was replaced by a push-pull cannula (diameters: outer tube 0.6 mm, inner tube 0.2 ram) which was 2 mm longer than the guide cannula thus reaching the LC. The inner tube of the push-pull cannula was retracted 0.3 mm within the outer tube. The inner diameter (0.3 mm) of the outer cannula confined the superfused area. Artificial cerebrospinal fluid (CSF) used for superfusion contained (mM) NaC1 140, KC1 3.0, CaCI 2 1.3, MgC12 1.0, Na2HPO4 1.0, glucose 3,0 and was adjusted to pH 7.2 with NaHzPO4 1.0. The LC was superfused at a rate of 28 ¢tl/min. After an equilibration period of 80 min, superfusate was collected continuously in time periods of 3 min at -50°C. In experiments in which the composition of the CSF was altered by adding potassium, veratridine or tetrodotoxin (TTX), a zero dead volume liquid switch (Carnegie Medicine) was used. Amino acids released in the superfusate were determined by H P L C and fluorimetric detection (MerckHitachi, Tokyo, Japan) after derivatization with ophthaldialdehyde (OPA) as previously described [11]. In
the present study 80 HI of the superfusate were mixed with 20 ~ 1 0 P A reaction mixture and 75/11 were injected. The reproducibility of the derivatization was controlled by addition of S-carboxymethyl-k-cysteine as an internal standard. The sensitivity of measurement of each amino acid was around 50 fmol per sample. Although great care was taken in the preparation of all solutions, traces of ASP and G L U were present in blanc samples. Experiments, in which the ASP blanc value was higher than half the baseline value, were not considered. Because of this, in some experiment series ASP values are not presented. Stimulation of the intact sciatic nerve contralateral to the superfused LC was carried out with bipolar platinum electrodes. Cathodal square-wave pulses (0.5 ms) obtained from a stimulator (H. Sachs, Freiburg, FRG) were applied at a frequency of 5 Hz. The threshold of stimulating intensity required for eliciting muscle twitches was determined in each animal. The stimulus intensity used in the experiment was ten-fold this threshold and amounted usually to 3 5 V. Mechanical stimulation of the hindpaw contralateral to the superfused LC was carried out over 3 min by an appropriate plastic
"• "
I'-
2.5
1.0
1 .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
,2.0 0
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1.5 1.0
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1.0
1.5 1.0 0.5 0
-
1
• VER (rv=7-9) o VER + TTX (n=5)
- o- o-io 6 ,b
4"0
TIME (min) Fig. 2. Effects of LC superfusion with veratridine (10/aM) and veratridine (10/aM) in the presence of TTX (1/aM) on the release of amino acids, The mean release rates in the three samples preceding superfusion with drugs were taken as 1.0. Horizontal bar denotes superfusion with veratridine (VER). TTX was present throughout. Mean values + S.E.M. ' P = 0.07, *P < 0.05.
N. SingewaM et al./Neuroscience Letters 180 (1994) 55 58
1.4 1.0
0.8
1.4 I-- 1.0 0.8
.
.
.
.
1.8
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.
.
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.
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,
IE 10t '=
4(,
,
J~ll~l.. _ _
~..~-10 ~
~-2v m
^Jo HP stim. (n--7) • SN stim. (n=7-9)
-'6
-3
release rates of GABA, TAU, GLU and ASP (Fig. 2). In the presence of the neurotoxin TTX (1/.tM), the releasing effect of veratridine was abolished. Veratridine did not significantly influence the release of arginine. The enhancing effects of potassium and veratridine on the amino acid release rates, as well as the abolition of the veratridine induced increases by TTX indicate that the stimulated amino acid release in the superfusate originates substantially from neuronal sites. Electrical stimulation of the sciatic nerve, as well as mechanical stimulation of the hindpaw, increased slightly the mean arterial BP. Both kinds of stimulation led to a pronounced enhancement in the release rates of the excitatory amino acids GLU and ASP. This effect was due to the noxious stimulus and not to the increased BP, since even a pronounced rise in BP (60 mmHg) did not change the release rates of ASP and GLU (see below). The release of GABA was slightly enhanced in the sample following mechanical stimulation of the hindpaw. These stimuli did not influence significantly the release rates of TAU and ARG (Fig. 3). The effect of electrical stimulation was current-dependent (not shown). Experimentally induced BP changes were elicited by intravenous infusions of phenylephrine or sodium nitroprusside. Neither the pressor response of phenylephrine, nor the fall of BP elicited by sodium nitroprusside influenced the release rates of the excitatory amino acids GLU and ASP in the LC (Fig. 4). Similarly, changes in BP elicited by blood injection (30% hypervolaemia) or
b TIME (min)
Fig. 3. Effects of electrical stimulation of the sciatic nerve or mechanical stimulation of the hindpaw on the release of amino acids in the LC. The mean release rates in the two samples preceding stimulations were taken as 1.0. Horizontal bar denotes stimulations. SN sciatic nerve, HP hindpaw. Mean values + S.E.M. *P < 0.05.
57
221
q) r~ a)~
1.B ~ 1.4 ('P i.o
E
< 1~
v
0.4
clamp (1 kg force). Localization of the push-pull cannula was verified microscopically in brain slices at the end of the experiment [11]. Experiments with cannula localizations outside the LC were discarded. Data were analyzed statistically by Friedman's test followed by Wilcoxon's signed rank test for paired data. The basal release rates of the amino acids were (pmol/ min): GABA 0.15+0.02, TAU 2.29+0.33, GLU 1.57 + 0.22, ASP 0.55 + 0.08, ARG 1.27 + 0.24 (mean values + S.E.M., n = 22-33). Local perfusion of the LC with CSF containing 50 mM potassium enhanced greatly the release rates of GABA, TAU and GLU during, as well as in the sample following superfusion with the high concentration of potassium (Fig. 1). The release rate of arginine was enhanced slightly after a delay of 10 min. Superfusion with veratridine (10 pM) also enhanced the
I- 8Ol
e .
~ .
~ .
.
.
O.-40 j ,.n O NaCI (n=7)H • PE (n-7-9) o NP (n-13-15) i m
-9
-6 -3
0
3
6
9
12
TIME (rain) Fig. 4. Effects of phenylephrine (10/tg/kg/min), physiologic saline (100 /A/min) and sodium nitroprusside (30 pg/kg/min) on arterial BP and release of amino acids in the LC. The mean release rates in the three samples preceding infusion with drugs were taken as 1.0. Horizontal bar denotes infusions with drug. PE, phenylephrine; NP, sodium nitroprusside. Mean values + S.E.M. **P < 0.01.
58
N. Singewaht et aL /Neuroscience Letters 180 (1994) 55 58
controlled haemorrhage (15% hypovolaemia) were ineffective (not shown). Intravenous infusion of physiologic saline did not modify either blood pressure, or release rates of amino acids in the LC. Recently, we reported that increases in BP enhance the release of GABA in the LC. On the other hand, decreases in BP decrease the GABA release rate, thus showing that the release of this inhibitory amino acid in the LC is modified by cardiovascular impulses [14]. In contrast to this, we now observed that BP changes did not modify the release of the excitatory amino acids GLU and ASP in the LC. Thus, GLU and ASP seem to be also excluded as possible excitatory neurotransmitters activating LC neurons elicited by lowering BE as shown in electrophysiologic studies [5,18]. On the other hand, electrical stimulation of the sciatic nerve or mechanical stimulation of the hindpaw enhanced greatly the release rates of the excitatory amino acids GLU and ASP without influencing those of the inhibitory amino acids GABA and TAU. The delayed response of GABA release to the stimulation of the sciatic nerve might point to a secondary enhanced activity of GABAergic neurons. These findings demonstrate that the enhanced activity of LC neurons as a response to noxious sensory stimuli is mediated by the GLU and ASP inputs into the LC. Furthermore, the results show that the excitatory amino acid input into the LC is not involved in central cardiovascular control. This work was supported by the Fonds zur Foerderung der wissenschaftlichen Forschung. [1] Aston-Jones, G., Shipley, M.T., Ennis, M.. Williams, J.T. and Pieribone, V.A., Restricted afferent control of locus coeruleus neurons revealed by anatomic, physiologic and pharmacologic studies. In C.A. Marsden and D.J. Heal (Eds.), The Pharmacology of Noradrenaline in the Central Nervous System, Oxford University Press, Oxford, 1990, pp. 187 247. [2] Brun, P., Suaud-Chagny, M.F., Lachuer, J., Gonon, F. and Buda, M., Catecholamine metabolism in locus coeruleus neurons: a study of its activation by sciatic nerve stimulation in the rat, Eur. J. Neurosci., 3 (1991) 397~,06. [3] Cedarbaum, J.M. and Agbajanian, G.K., Activation of locus coeruleus neurons by peripheral stimuli: modulation by a collateral inhibitory mechanism, Life Sci., 23 (1978) 1383 1392. [4] Dahlstr6m, A. and Fuxe, K., Evidence for the existence of
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