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5-HT release from ependymal surface of the caudate nucleus in ‘ence´phale isole´‘ cats
Brain Research, 132 (1977) 575-579 O Elsevier/North-Holland Biomedical Press
575
5-HT release from ependymal surface of the caudate nucleus in 'enc~phale isole' cats
J. P. TERNAUX*, F. HERY, M. HAMON, S. BOURGOIN and J. GLOWINSKI Groupe NB 1NSERM Ul14, Laboratoire de Neurophysiologie, Coll~,e de France, 75231 Paris Cedex 05 (France)
(Accepted May 18th, 1977)
Vogt and Myers were the first to study the 'in vivo' endogenous release of serotonin (5-HT). They used the ventricular perfusion in anesthetized cats2,8,10,14 and the push-pull cannula at hypothalamic levels in unanesthetized cats or monkeys4,7, ~2,13,2a. 5-HT release was estimated using a biological assay or, more recently, spectrofluorimetric methods. In most experiments monoamine oxidase inhibitors were previously injected in the animals. Using a sensitive radioenzymatic assay for 5-HT 6, the release of the amine could be detected in the lateral ventricle of halothane anesthetized rats 2°, and modifications could be observed under various pharmacological conditions. In order to study the release of 5-HT in unanesthetized animals, the same biochemical analysis was used on superfusates collected from the ependymal surface of the caudate nucleus of 'enc6phale isol6 semi-chronique' cats. Such preparations are known to present all the stages of sleep occurring in chronic animals, and allowed the control of various physiological parameters ~5. Forty cats of both sexes, with the spinal cord sectioned at the cervical level (C1-C2), were maintained in physiological conditions under artificial respiration, in non-nociceptive stereotaxic fixationlL Alveolar CO2, blood pressure at the femoral level, heart rate, and body temperature were continuously controlled. In some cases, EEG, activity of the lateral geniculate body, eye movements, and neck muscle tone were recorded. After removing a part of the cerebral cortex on the right hemisphere, a plastic cup designed by Besson et al. 5 was placed on the ependymal surface of the caudate nucleus, a structure known to be rich in serotoninergic terminals17,19. Artificial cerebrospinal fluid (CSF) (in mM: glucose, 6; NaHCOa, 3.57; NaH2PO4, 0.49; MgSO4, 1.15; CaC12, 1.26; KC1, 3.35; NaCl, 139) adjusted to pH 7.4. was introduced in the cup at a flow rate of I ml/10 min and warmed to 37 °C. O2-CO2 bubbling (95 %-5 %) was used to maintain a constant pH in the cup. CSF temperature on the ependymal surface of the caudate nucleus was recorded by means of a microthermo* Present address: INSERM U6 and GR 25 CNRS, 280 Bd Ste Marguerite, 13009 Marseille, France,
576 meter. Fractions of superfusates were collected at 0 °C every 10 min. L-Tryptophan (Merck, 100 mg/kg) and L-5-hydroxytryptophan (Calbiochem, 100 mg/kg) were injected intraperitoneally. KCI (30 mM) and tetrodotoxin (Boehringer, 5 × l0 -7 M) were locally applied on the ependymal surface of the caudate nucleus. Lysergic acid diethylamide (LSD) was injected intravenously (5-20 #g/kg) or added to the CSF (10 -5 M). 5-HT released in the superfusates was estimated by the microradioenzymatic assay developed by Saavedra et al. is, and slightly modified by Boireau et al. 6, for biological fluids. Briefly, 5-HT was first isolated on a Sephadex G10 column (Pharmacia) and then transformed into [aH]melatonin by two enzymatic reactions. The [aH]melatonin extracted was further purified by silica gel plate chromatography. The sensitivity of the whole procedure was of about l0 pg ([3H]melatonin radioactivity was two times the blank value with this minimal amount of 5-HT detectable). In these conditions, 5-HT released during l0 min superfusion was easily detectable. In steady physiological conditions (without important variations of blood pressure, and alveolar CO2, and without non-specific release of 5-HT from blood platelets) the transmitter release was quite stable (Fig. l, top left) during the duration of the superfusion (5-6 h) and the quantities of 5-HT were 214 ~- 16 pg/10 min/ml (mean of 161 fractions from 20 animals) in spite of small individual variations. The local application of KCl (30 mM), that triggers depolarization ofcaudate terminals, was correlated with an increase of 5-HT release, equal to about 25 times the spontaneous value. Such effects were reproducible and the quantities of 5-HT released reached 2500-3000 pg/10 min/ml during the first fraction following KCI infusion into the cup (Fig. l, top right). On the contrary, blockage of Na transport by tetrodotoxin led to a 50% decrease in 5-HT release (Fig. l, bottom left). Both intravenous injection (5-20/~g/kg) and local application (10 -5 M) of LSD produced a significant decrease of 5-HT release (70 %). This effect was longer lasting when LSD was directly applied at the caudate level (Fig. l, bottom right). In order to see if modifications of the rate of 5-HT synthesis in serotoninergic striatal terminals led to variation of the 5-HT release process, the two amino acid precursors L-5-hydroxytryptophan and L-tryptophan were injected intraperitoneally at a dose of 100 mg/kg. Following the injection of L-5-hydroxytryptophan, 5-HT release was immediately enhanced and reached values of about 4-8 rig/l0 min/ml during the second and third hours after the pulse injection. These results were observed in 3 animals (Table I). The injection of L-tryptophan resulted in a slight increase of 5-HT release when compared to that detected after L-5-hydroxytryptophan. Maximal values of 5-HT released were obtained in the first hour following the injection (924 ± 120 pg/l0 min/ml, mean of 6 experiments) and then decreased to spontaneous levels during the fourth and fifth hours (Table l). In these conditions, the time course of the fluctuations of 5-HT release from the ependymal surface of the caudate nucleus was correlated with the changes occurring in the level of free plasma tryptophan. In some experiments, with sleep recording, an increase of 20 ~ of light slow-wave sleep was observed during the 4 h following the injection of L-tryptophan. Nevertheless, this increase was particularly marked during the first hour (33 %).
577
KCl 30raM
KCl 30ram
3000
cm Hg
1500 4,0
f
3
% CO 2
38
~~
2000
100C
ooo
500 o
TTX
LSD I0-5M
510-7M
200
500 100
2
3
4
0
TIME
4
2
3
4
S
hours
Fig. 1. Top left: example of spontaneous release of 5-HT from the ependymal surface of the caudate nucleus in 'enc6phale isol6' cat. 5-HT is expressed in pg/10 min/ml of superfusate, cm Hg: arterial blood pressure. ~ CO2: concentration of alveolar COs. T°C: ©, body temperature and O, cup temperature. Top right: effect of KCI perfusion (30 mM) on 5-HT release from the caudate nucleus. Bottom left: modification of 5-HT release during application of tetrodotoxin (TTX, 5 × 10-7 M ) on the ependymal surface of caudate nucleus. Bottom right: 5-HT release inhibition after local application of LSD (10-5 M). These preliminary results demonstrate that 5-HT is spontaneously released from the ependymal surface of the caudate nucleus in 'enc6phale isol6 semi-chronique' cats. Moreover, the surgical procedure used to drive the cup on the ependymal surface of the caudate nucleus, in order to superfuse this structure, does not disturb the pattern of EEG activity generally observed in this preparation. Stages of slow-wave sleep and episodes of paradoxical sleep occurred spontaneously at the same rate and with identical duration, when compared to animals without the cup. The release of 5-HT seems to be related to the electrical activity of serotoninergic terminals of the caudate nucleus since KC1 and tetrodotoxin are known to modify their membrane potentials. In the case of LSD injections, it is now well demonstrated that this treatment triggers a specific decrease in the firing rate of serotoninergic neurons 1, and this phenomenon can explain the decrease of 5-HT release observed at striatal levels. Inhibition of 5-HT release observed after local application of LSD, was previously detected in 'in vivo'
578 TABLE I Effects of Lp. infection of L-tryptophan and L-5-hydroxytryptophan on the release o/ 5-HT from the ependymal surface of the eaudate nucleus
Values of 5-HT release are expressed in pg/10 min/ml and are the mean ± S.E.M. of six fractions collected from n animals during the first, second, and third hours following the administration of the amino acid precursor. The spontaneous release determination is the mean ± S.E.M. of the fractions corresponding to two hours of superfusion. 5-HT (pg/lO min/ml) Spontaneous release
L-Tryptophan 100mg/kgi.p. n -- 6 345 ± 52 L-5-Hydroxytryptophan 100mg/kgi.p. n ~ 3 493 i 82
1h
2h
3h
924 ± 120"
777 ± 97*
556 ~ 80
2408 ± 694*
3656 ~ 212"
7002~ 532*
* P < 0.005 when compared to spontaneous value of release. and 'in vitro' conditionsg,16m 1 and this effect is certainly mediated by a local feed-back process linked to the stimulatory action of the drug on 5-HT presynaptic receptors. The increase in the rate of 5-HT synthesis occurring after injection of the two amino acid precursors is correlated with an enhanced release of the transmitter. Nevertheless, as previously observed in the rat using ventricular perfusion, the effect of L-5-hydroxytryptophan upon the release of 5-HT is more important when compared to the tryptophan effect. These differences may be attributed to the non-specific localization of the decarboxylation of L-5-hydroxytryptophan11. On the contrary, tryptophan hydroxylation is specific to serotoninergic neurones, and precursor treatment results in a slight increase of 5-HT release that occurs immediately after the injection. Nevertheless, this fact does not agree with that observed after the same treatment in the rat: no change can be detected during the first hour following the administration of tryptophan. This result was explained as a consequence of an inhibition of the firing rate of raphe neurons due to the tryptophan injectionL Recently Gallager and Aghajanian 8 concluded that this effect on electrical activity of the serotoninergic system is mediated through enzymatic conversion of tryptophan into 5-HT. Probably in the present conditions the quantities of tryptophan metabolized to 5-HT are not sufficient to produce an inhibition of raphe firing, as if the doses injected in the cat were smaller than those used in the rat. An electrophysiological study of raphe neurones under these conditions in the cat would elucidate this mechanism. The increase of light slow-wave sleep after an injection of tryptophan (100 mg/kg) can be compared to the result of Ursin 22 in the chronic cat after administration of 5-HT precursors. Nevertheless, this author does not observe any modification of slow-wave sleep but a slight increase in the awake drowsy stage. The limit between drowsy and light slow-wave sleep is difficult to appreciate, and the differences observed can be due to the nonappropriate definition of drowsy and light slow-wave sleep. In conclusion, the physiological preparation and the biochemical analysis used,
579 can be a good m o d e l for the study of 5-HT release in different pharmacological situations a n d also in physiological conditions, a n d particularly for the d e t e r m i n a t i o n of release processes d u r i n g the different stages of sleep. 1 Aghajanian, G. K., Influence of drugs on the firing of serotonin containing neurones in brain, Fed. Proc., 31 (1972) 91-96. 2 Ashkenazi, R., Holman, R. B. and Vogt, M., Release of transmitters on stimulation of the nucleus linearis raphe in the cat, J. PhysioL (Lond.), 223 (1972) 225-259. 3 Ashkenazi, R., Holman, R. B. and Vogt, M., Release of transmitters into the perfused third ventricle in the cat, J. Physiol. (Lond.), 233 (1973) 195-209. 4 Beleslin, D. B. and Myers, R. D., The release of acetylcholine and 5-hydroxytryptamine from the mesencephalon of the unanesthetized rhesus monkey, Brain Research, 23 (1970) 437--442. 5 Besson, M. J., Cheramy, A., Gauchy, C. and Glowinski, J., In vivo continuous estimation of aH dopamine release and synthesis in the caudate nucleus: effects of a-meth2~l-p-tyrosineand transection of the nigro striatal pathway, Arch. Pharmacol., 278 (1973) 101-105. 6 Boireau, A., Ternaux, J. P., Bourgoin, S., Hery, F., Glowinski, J. and Flamon, M., The determination of picogram level of 5 HT in biological fluids, J. Neurochem., 26 (1976) 201-204. 7 Felberg, W. and Myers, R. D., Appearance of 5-hydroxytryptamine and an unidentified pharmacologically active lipid acid in effluent from perfused cerebral ventricles, J. Physiol. (Lond.), 184 (1966) 837-855. 8 Gallager, D. W. and Aghajanian, G. K., Inhibition of firing of raphe neurones by tryptophan and 5-hydroxytryptophan: blockage by inhibiting serotinin synthesis with Ro-4-4602, Neuropharmacology, 15 (1976) 149-156. 9 Hamon, M., Bourgoin, S., Jagger, J. and Glowinski, J., Effects of LSD on synthesis and release of 5 HT in rat brain slices, Brain Research, 69 (1974) 265-280. 10 Holman, R. B. and Vogt, M., Release of 5-hydroxytryptamine from caudate nucleus and septum, J. PhysioL (Lond.), 223 (1972) 243-254. 11 Lichtensteiger, W., Mutzner, U. and Langemann, H., Uptake of 5-hydroxytryptamine and 5-hydroxytryptophan by neurones of the central nervous system normally containing catecholamines, J. Neurochem., 14 (1967) 489-497. 12 Myers, R. D. and Beleslin, D. B., Changes in serotinon release in hypothalamus during cooling and warming of the monkey, Amer. J. PhysioL, 220 (1971) 1746-1754. 13 Myers, R. D. and Beleslin, D. B., The spontaneous release of 5-hydroxytryptamine and acetylcholine within the diencephalon of the unanesthetized rhesus monkey, Exp. Brain Res., 11 (1971) 539-552. 14 Portig, P. J. and Vogt, M., Release into the cerebral ventricles of substances with possible transmitter function in the caudate nucleus, J. PhysioL (Lond.), 204 (1969) 687-715. 15 Puizillout, J. J., Ternaux, J. P., Foutz, A. S. et Dell, P., Phases de sommeil b_ondes lentes avec d6charges phasiques. Leur d6clenchement par stimulation vago-aortique, Rev. EEG Neurophysiol., 3 (1973) 21-37. 16 Randic, M. and Padjen, A., Effects of N,N-dimethyltryptamine and D-lysergic diethylamide on the metabolism of brain 5-hydroxytryptamine, Biochem. PharmacoL, 16 (1967) 2011-2021. 17 Reis, D. J., Corvelli, A. and Conners, J., Circadian and ultradian rhythms of serotonin regionally in cat brain, J. Pharmacol. exp. Ther., 167 (1969) 328-333. 18 Saavedra, J. M., Brownstein, M. and Axelrod, J., A specific and sensitive enzymatic isotopic microassay for serotonin in tissues, J. PharmacoL exp. Ther., 186 (1973) 508-515. 19 Sourkes, T. L. and Poirier, L. J., Serotonin and dopamine in the extrapyramidal system, Advanc. Pharmacol., 6A (1968) 335-346. 20 Ternaux, J. P., Boireau, A., Bourgoin, S., Hamon, M., Hery, F. and Glowinski, J., In vivo release of 5 HT in the lateral ventricle of the rat: effects of 5 hydroxytryptophan and tryptophan, Brain Research, 101 (1976) 533-548. 21 Tilson, H. A. and Sparber, S. B., Studies on the concurrent behavioral and neurochemical effects of psychoactive drugs using the push-pull cannula, J. Pharmacol. exp. Ther., 181 (1972) 387-398. 22 Ursin, R., The effects of 5-hydroxytryptophan and L-tryptophan on wakefulness and sleep patterns in the cat, Brain Research, 106 (1976) 105-115. 23 Veale, W. L., Myers, R. D. and Beleslin, D. B., Effects of calcium on the release of serotonin from isolated sites within the diencephalon of the cat, PharmacoL Biochem. Behav., 1 (1973) 259-264.
575
5-HT release from ependymal surface of the caudate nucleus in 'enc~phale isole' cats
J. P. TERNAUX*, F. HERY, M. HAMON, S. BOURGOIN and J. GLOWINSKI Groupe NB 1NSERM Ul14, Laboratoire de Neurophysiologie, Coll~,e de France, 75231 Paris Cedex 05 (France)
(Accepted May 18th, 1977)
Vogt and Myers were the first to study the 'in vivo' endogenous release of serotonin (5-HT). They used the ventricular perfusion in anesthetized cats2,8,10,14 and the push-pull cannula at hypothalamic levels in unanesthetized cats or monkeys4,7, ~2,13,2a. 5-HT release was estimated using a biological assay or, more recently, spectrofluorimetric methods. In most experiments monoamine oxidase inhibitors were previously injected in the animals. Using a sensitive radioenzymatic assay for 5-HT 6, the release of the amine could be detected in the lateral ventricle of halothane anesthetized rats 2°, and modifications could be observed under various pharmacological conditions. In order to study the release of 5-HT in unanesthetized animals, the same biochemical analysis was used on superfusates collected from the ependymal surface of the caudate nucleus of 'enc6phale isol6 semi-chronique' cats. Such preparations are known to present all the stages of sleep occurring in chronic animals, and allowed the control of various physiological parameters ~5. Forty cats of both sexes, with the spinal cord sectioned at the cervical level (C1-C2), were maintained in physiological conditions under artificial respiration, in non-nociceptive stereotaxic fixationlL Alveolar CO2, blood pressure at the femoral level, heart rate, and body temperature were continuously controlled. In some cases, EEG, activity of the lateral geniculate body, eye movements, and neck muscle tone were recorded. After removing a part of the cerebral cortex on the right hemisphere, a plastic cup designed by Besson et al. 5 was placed on the ependymal surface of the caudate nucleus, a structure known to be rich in serotoninergic terminals17,19. Artificial cerebrospinal fluid (CSF) (in mM: glucose, 6; NaHCOa, 3.57; NaH2PO4, 0.49; MgSO4, 1.15; CaC12, 1.26; KC1, 3.35; NaCl, 139) adjusted to pH 7.4. was introduced in the cup at a flow rate of I ml/10 min and warmed to 37 °C. O2-CO2 bubbling (95 %-5 %) was used to maintain a constant pH in the cup. CSF temperature on the ependymal surface of the caudate nucleus was recorded by means of a microthermo* Present address: INSERM U6 and GR 25 CNRS, 280 Bd Ste Marguerite, 13009 Marseille, France,
576 meter. Fractions of superfusates were collected at 0 °C every 10 min. L-Tryptophan (Merck, 100 mg/kg) and L-5-hydroxytryptophan (Calbiochem, 100 mg/kg) were injected intraperitoneally. KCI (30 mM) and tetrodotoxin (Boehringer, 5 × l0 -7 M) were locally applied on the ependymal surface of the caudate nucleus. Lysergic acid diethylamide (LSD) was injected intravenously (5-20 #g/kg) or added to the CSF (10 -5 M). 5-HT released in the superfusates was estimated by the microradioenzymatic assay developed by Saavedra et al. is, and slightly modified by Boireau et al. 6, for biological fluids. Briefly, 5-HT was first isolated on a Sephadex G10 column (Pharmacia) and then transformed into [aH]melatonin by two enzymatic reactions. The [aH]melatonin extracted was further purified by silica gel plate chromatography. The sensitivity of the whole procedure was of about l0 pg ([3H]melatonin radioactivity was two times the blank value with this minimal amount of 5-HT detectable). In these conditions, 5-HT released during l0 min superfusion was easily detectable. In steady physiological conditions (without important variations of blood pressure, and alveolar CO2, and without non-specific release of 5-HT from blood platelets) the transmitter release was quite stable (Fig. l, top left) during the duration of the superfusion (5-6 h) and the quantities of 5-HT were 214 ~- 16 pg/10 min/ml (mean of 161 fractions from 20 animals) in spite of small individual variations. The local application of KCl (30 mM), that triggers depolarization ofcaudate terminals, was correlated with an increase of 5-HT release, equal to about 25 times the spontaneous value. Such effects were reproducible and the quantities of 5-HT released reached 2500-3000 pg/10 min/ml during the first fraction following KCI infusion into the cup (Fig. l, top right). On the contrary, blockage of Na transport by tetrodotoxin led to a 50% decrease in 5-HT release (Fig. l, bottom left). Both intravenous injection (5-20/~g/kg) and local application (10 -5 M) of LSD produced a significant decrease of 5-HT release (70 %). This effect was longer lasting when LSD was directly applied at the caudate level (Fig. l, bottom right). In order to see if modifications of the rate of 5-HT synthesis in serotoninergic striatal terminals led to variation of the 5-HT release process, the two amino acid precursors L-5-hydroxytryptophan and L-tryptophan were injected intraperitoneally at a dose of 100 mg/kg. Following the injection of L-5-hydroxytryptophan, 5-HT release was immediately enhanced and reached values of about 4-8 rig/l0 min/ml during the second and third hours after the pulse injection. These results were observed in 3 animals (Table I). The injection of L-tryptophan resulted in a slight increase of 5-HT release when compared to that detected after L-5-hydroxytryptophan. Maximal values of 5-HT released were obtained in the first hour following the injection (924 ± 120 pg/l0 min/ml, mean of 6 experiments) and then decreased to spontaneous levels during the fourth and fifth hours (Table l). In these conditions, the time course of the fluctuations of 5-HT release from the ependymal surface of the caudate nucleus was correlated with the changes occurring in the level of free plasma tryptophan. In some experiments, with sleep recording, an increase of 20 ~ of light slow-wave sleep was observed during the 4 h following the injection of L-tryptophan. Nevertheless, this increase was particularly marked during the first hour (33 %).
577
KCl 30raM
KCl 30ram
3000
cm Hg
1500 4,0
f
3
% CO 2
38
~~
2000
100C
ooo
500 o
TTX
LSD I0-5M
510-7M
200
500 100
2
3
4
0
TIME
4
2
3
4
S
hours
Fig. 1. Top left: example of spontaneous release of 5-HT from the ependymal surface of the caudate nucleus in 'enc6phale isol6' cat. 5-HT is expressed in pg/10 min/ml of superfusate, cm Hg: arterial blood pressure. ~ CO2: concentration of alveolar COs. T°C: ©, body temperature and O, cup temperature. Top right: effect of KCI perfusion (30 mM) on 5-HT release from the caudate nucleus. Bottom left: modification of 5-HT release during application of tetrodotoxin (TTX, 5 × 10-7 M ) on the ependymal surface of caudate nucleus. Bottom right: 5-HT release inhibition after local application of LSD (10-5 M). These preliminary results demonstrate that 5-HT is spontaneously released from the ependymal surface of the caudate nucleus in 'enc6phale isol6 semi-chronique' cats. Moreover, the surgical procedure used to drive the cup on the ependymal surface of the caudate nucleus, in order to superfuse this structure, does not disturb the pattern of EEG activity generally observed in this preparation. Stages of slow-wave sleep and episodes of paradoxical sleep occurred spontaneously at the same rate and with identical duration, when compared to animals without the cup. The release of 5-HT seems to be related to the electrical activity of serotoninergic terminals of the caudate nucleus since KC1 and tetrodotoxin are known to modify their membrane potentials. In the case of LSD injections, it is now well demonstrated that this treatment triggers a specific decrease in the firing rate of serotoninergic neurons 1, and this phenomenon can explain the decrease of 5-HT release observed at striatal levels. Inhibition of 5-HT release observed after local application of LSD, was previously detected in 'in vivo'
578 TABLE I Effects of Lp. infection of L-tryptophan and L-5-hydroxytryptophan on the release o/ 5-HT from the ependymal surface of the eaudate nucleus
Values of 5-HT release are expressed in pg/10 min/ml and are the mean ± S.E.M. of six fractions collected from n animals during the first, second, and third hours following the administration of the amino acid precursor. The spontaneous release determination is the mean ± S.E.M. of the fractions corresponding to two hours of superfusion. 5-HT (pg/lO min/ml) Spontaneous release
L-Tryptophan 100mg/kgi.p. n -- 6 345 ± 52 L-5-Hydroxytryptophan 100mg/kgi.p. n ~ 3 493 i 82
1h
2h
3h
924 ± 120"
777 ± 97*
556 ~ 80
2408 ± 694*
3656 ~ 212"
7002~ 532*
* P < 0.005 when compared to spontaneous value of release. and 'in vitro' conditionsg,16m 1 and this effect is certainly mediated by a local feed-back process linked to the stimulatory action of the drug on 5-HT presynaptic receptors. The increase in the rate of 5-HT synthesis occurring after injection of the two amino acid precursors is correlated with an enhanced release of the transmitter. Nevertheless, as previously observed in the rat using ventricular perfusion, the effect of L-5-hydroxytryptophan upon the release of 5-HT is more important when compared to the tryptophan effect. These differences may be attributed to the non-specific localization of the decarboxylation of L-5-hydroxytryptophan11. On the contrary, tryptophan hydroxylation is specific to serotoninergic neurones, and precursor treatment results in a slight increase of 5-HT release that occurs immediately after the injection. Nevertheless, this fact does not agree with that observed after the same treatment in the rat: no change can be detected during the first hour following the administration of tryptophan. This result was explained as a consequence of an inhibition of the firing rate of raphe neurons due to the tryptophan injectionL Recently Gallager and Aghajanian 8 concluded that this effect on electrical activity of the serotoninergic system is mediated through enzymatic conversion of tryptophan into 5-HT. Probably in the present conditions the quantities of tryptophan metabolized to 5-HT are not sufficient to produce an inhibition of raphe firing, as if the doses injected in the cat were smaller than those used in the rat. An electrophysiological study of raphe neurones under these conditions in the cat would elucidate this mechanism. The increase of light slow-wave sleep after an injection of tryptophan (100 mg/kg) can be compared to the result of Ursin 22 in the chronic cat after administration of 5-HT precursors. Nevertheless, this author does not observe any modification of slow-wave sleep but a slight increase in the awake drowsy stage. The limit between drowsy and light slow-wave sleep is difficult to appreciate, and the differences observed can be due to the nonappropriate definition of drowsy and light slow-wave sleep. In conclusion, the physiological preparation and the biochemical analysis used,
579 can be a good m o d e l for the study of 5-HT release in different pharmacological situations a n d also in physiological conditions, a n d particularly for the d e t e r m i n a t i o n of release processes d u r i n g the different stages of sleep. 1 Aghajanian, G. K., Influence of drugs on the firing of serotonin containing neurones in brain, Fed. Proc., 31 (1972) 91-96. 2 Ashkenazi, R., Holman, R. B. and Vogt, M., Release of transmitters on stimulation of the nucleus linearis raphe in the cat, J. PhysioL (Lond.), 223 (1972) 225-259. 3 Ashkenazi, R., Holman, R. B. and Vogt, M., Release of transmitters into the perfused third ventricle in the cat, J. Physiol. (Lond.), 233 (1973) 195-209. 4 Beleslin, D. B. and Myers, R. D., The release of acetylcholine and 5-hydroxytryptamine from the mesencephalon of the unanesthetized rhesus monkey, Brain Research, 23 (1970) 437--442. 5 Besson, M. J., Cheramy, A., Gauchy, C. and Glowinski, J., In vivo continuous estimation of aH dopamine release and synthesis in the caudate nucleus: effects of a-meth2~l-p-tyrosineand transection of the nigro striatal pathway, Arch. Pharmacol., 278 (1973) 101-105. 6 Boireau, A., Ternaux, J. P., Bourgoin, S., Hery, F., Glowinski, J. and Flamon, M., The determination of picogram level of 5 HT in biological fluids, J. Neurochem., 26 (1976) 201-204. 7 Felberg, W. and Myers, R. D., Appearance of 5-hydroxytryptamine and an unidentified pharmacologically active lipid acid in effluent from perfused cerebral ventricles, J. Physiol. (Lond.), 184 (1966) 837-855. 8 Gallager, D. W. and Aghajanian, G. K., Inhibition of firing of raphe neurones by tryptophan and 5-hydroxytryptophan: blockage by inhibiting serotinin synthesis with Ro-4-4602, Neuropharmacology, 15 (1976) 149-156. 9 Hamon, M., Bourgoin, S., Jagger, J. and Glowinski, J., Effects of LSD on synthesis and release of 5 HT in rat brain slices, Brain Research, 69 (1974) 265-280. 10 Holman, R. B. and Vogt, M., Release of 5-hydroxytryptamine from caudate nucleus and septum, J. PhysioL (Lond.), 223 (1972) 243-254. 11 Lichtensteiger, W., Mutzner, U. and Langemann, H., Uptake of 5-hydroxytryptamine and 5-hydroxytryptophan by neurones of the central nervous system normally containing catecholamines, J. Neurochem., 14 (1967) 489-497. 12 Myers, R. D. and Beleslin, D. B., Changes in serotinon release in hypothalamus during cooling and warming of the monkey, Amer. J. PhysioL, 220 (1971) 1746-1754. 13 Myers, R. D. and Beleslin, D. B., The spontaneous release of 5-hydroxytryptamine and acetylcholine within the diencephalon of the unanesthetized rhesus monkey, Exp. Brain Res., 11 (1971) 539-552. 14 Portig, P. J. and Vogt, M., Release into the cerebral ventricles of substances with possible transmitter function in the caudate nucleus, J. PhysioL (Lond.), 204 (1969) 687-715. 15 Puizillout, J. J., Ternaux, J. P., Foutz, A. S. et Dell, P., Phases de sommeil b_ondes lentes avec d6charges phasiques. Leur d6clenchement par stimulation vago-aortique, Rev. EEG Neurophysiol., 3 (1973) 21-37. 16 Randic, M. and Padjen, A., Effects of N,N-dimethyltryptamine and D-lysergic diethylamide on the metabolism of brain 5-hydroxytryptamine, Biochem. PharmacoL, 16 (1967) 2011-2021. 17 Reis, D. J., Corvelli, A. and Conners, J., Circadian and ultradian rhythms of serotonin regionally in cat brain, J. Pharmacol. exp. Ther., 167 (1969) 328-333. 18 Saavedra, J. M., Brownstein, M. and Axelrod, J., A specific and sensitive enzymatic isotopic microassay for serotonin in tissues, J. PharmacoL exp. Ther., 186 (1973) 508-515. 19 Sourkes, T. L. and Poirier, L. J., Serotonin and dopamine in the extrapyramidal system, Advanc. Pharmacol., 6A (1968) 335-346. 20 Ternaux, J. P., Boireau, A., Bourgoin, S., Hamon, M., Hery, F. and Glowinski, J., In vivo release of 5 HT in the lateral ventricle of the rat: effects of 5 hydroxytryptophan and tryptophan, Brain Research, 101 (1976) 533-548. 21 Tilson, H. A. and Sparber, S. B., Studies on the concurrent behavioral and neurochemical effects of psychoactive drugs using the push-pull cannula, J. Pharmacol. exp. Ther., 181 (1972) 387-398. 22 Ursin, R., The effects of 5-hydroxytryptophan and L-tryptophan on wakefulness and sleep patterns in the cat, Brain Research, 106 (1976) 105-115. 23 Veale, W. L., Myers, R. D. and Beleslin, D. B., Effects of calcium on the release of serotonin from isolated sites within the diencephalon of the cat, PharmacoL Biochem. Behav., 1 (1973) 259-264.