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Prolonged pentobarbital narcosis in rats with lesions of midbrain raphe
EUROPEAN JOURNAL OF PHARMACOLOGY 6 (1969) 71-73. NORTH-HOLLANDPUBLISHINGCOMPANY, AMSTERDAM
PROLONGED PENTOBARBITAL NARCOSIS IN RATS WITH LESIONS OF MIDBRAIN RAPHE W.KOSTOWSKI * and E.DOLFINI Istituto di Ricerche Farrnacologiche ,,Mano Negrt" Via Eritrea, 62, 20157 Milano, Italy
Received 25 February 1969
Accepted 13 March 1969
W.KOSTOWSKI and E.DOLFINI, Prolonged pentobarbital narcosis in rats with lesions of midbrain raphe, European J. Pharmacol. 6 (1969) 71-73. The mldbrain raphe area is thought to be involved in sleep behaviour; stimulation of this area produces sedation and sleep whereas lesions of the same area elicit marked hypermotflity Pentobarbital increases sleeping time in midbram lesioned rats and the percentage of animals sleeping Concentrations of pentobarbital in brain are also reported. Pentobarbital narcosis Pentobarbital in brain
The midbrain raphe area (MR) contains serotoninergic neurons which project into the forebrain and spinal cord (Dahlstr6m and Fuxe, 1964). It is thought to play a role in the regulation and triggering of sleep behaviour (Mouret, Bobillier and Jouvet, 1967). Low frequency electrical stimulation of this area induces behavioural sleep or drowsiness with associated electroencephalographic changes (Kostowski, Giacalone, Garattini and Valzelli, 1969). Lesions of the MR abolish the slow wave sleep pattern and elicit a marked hypermotility (Kostowski, Giacalone, Garattini and Valzelli, 1968). It was therefore of interest to investigate the effect of a barbiturate in rats with lesions of MR. Female Sprague Dawley rats, weighing 160-200 g were employed. Cerebral lesions were performed by means of a stainless steel electrode of 0.25 mm in diameter using a Stoelting stereotaxic apparatus and the atlas of Krnig and Klippel (1963). The details of the technique have been described elsewhere (Kostow* Visiting Scientist from the Department of Experimental Pharmacology, Medical Academy, Warsaw, Poland (present address).
Midbrain raphe lesion Sleep
ski et al., 1968). Sham operated rats and rats with lesions in lateral midbrain (LM) were also prepared. After surgery, the animals were housed singly in cages at a constant room temperature (22°C) and relative humidity (60%). Biochemical studies were performed 7 days after lesions. Pentobarbital was given intraperitoneally. Sleeping time was evaluated as the time dapsed between loss and regaining of the righting reflex. The concentration of pentobarbital in brain was measured according to Brodie, Burns, Mark, Lief, Bernstein and Papper (1953). 5-Hydroxytryptamine (5HT) and 5-hydroxyindolacetic acid (5HIAA) levels in forebrain were determined in the same sample according to Giacalone and Valzelli (1969). The results obtained are reported in table 1. Lesions of MR, but not of lateral midbrain (LM), increase the sensitivity to pentobarbital in respect to sham lesioned animals. In the group of MR lesioned rats sleeping time was increased and the percentage of sleeping animals increased. This effect was not due to changes in body temperature. Pentobarbital brain concentrations in both groups of lesioned and sham lesioned MR rats showed only minor differences. As shown previously
W KOSTOWSKI and E.DOLFINI
72
Table 1 Effects of pentobarbital in MR lesioned rats Sham lesions 46
Total number of rats Sleeping time (min + S E.) after pentobarbital
20 (mg/kg i.p.) 25 (mg/kg i.p.) 30 (mg/kgi.p.)
62 77 112
+ 14 + 10
(3/6) (7/16) (11/12)
MR lesions 51 81 122 167
+ 10 + 17" -+ 14"
Brain pentobarbltal** (t~g/g -+ S E.)
15 min 30 min 90min
18 5 + 1.9 23.3 + 2.8 15 3 + 0 9
24.9 + 2 2° 27.4 + 4 6 15.1 + 1 3
Body temperature ( ° C ~ E.) after pentobarbital**
0 min 30 min 60min 90 min
37 1 + 0.2 35.4 + 0 2 34.6 + 0 2 34.4 + 0.3
38.3 + 0.3 34.9 + 0.3 34.1 + 0 5 34 1 + 0.7
Forebrain*** 5HT (ng/g~.E.) Forebrain*** 5HIAA ( n g / g ~ E.)
364 278
+ 20 + 10
88 82
+- 5* +4"
(5/5) (12/12) (12/12)
LM lesions 23 N.D 80 106
+ 16
(2/5) (6/6)
N.D. N D. 10.8+ 1.2 37 36 36 35
0+0 1 5+04 3 +04 8+02
340 272
-+ 10 + 25
o = p ~ 0 05 m comparison to sham lesioned rats * = p ~ 0.01 in comparison to sham lesioned rats. ** = 30mg/kg 1.p *** = 6 hr after pentobarbltal 30 mg/kg i p. N.D. = not determined. In brackets the ratio between sleeping and non-sleeping animals.
(Kostowski et al., 1968), lesions of MR, but not of LM decreased 5HT and 5HIAA. The increase of pentobarbital narcosis m the MR lesioned rats is of particular interest since only lesions of a few areas are known to increase barbiturate sleeping time, e.g. dorsomedial tegmentum septum and hippocampus (Harvey, Heller, Moore, Hunt and Roth, 1964). The mechanism of the increased pentobarbltal narcosis in MR lesioned rats remains unknown but it is not related to changes in the level of pentobarbital in the whole brain. It is of interest that MR lesioned rats show an increased sensitivity to pentobarbital but a decrease of normal slow-wave sleep (Mouret et al., 1967) and persistent behavioural and electroencephalographic arousal (Kostowskl et al., 1968). ACKNOWLEDGEMENTS This work IS supported by the National Inst. of Health, Bethesda, Md., USA, Contract No. DHEW/PHS/NIH, PH 43-67-83. The technical help of Mr. Sergio Bernasconi is gratefully acknowledged.
REFERENCES Brodie, B.B., J.J.Burns, L.C.Mark, P.A.Lief, E.Bernstein and E.M.Papper, 1953, The fate of pentobarbital in man and dog and a method for its estimation in biological material, J. Pharmacol Exp. Ther. 109, 26 Dahlstr6m, A. and K.Fuxe, 1964, Evidence for the existence of monoamine-contaming neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol Scand. 62, suppl. 232, 1. Giacalone, E. and L.Valzelli, 1969, A spectrofluorometric method for the simultaneous determination of 2-(5hydroxyindol-3-yl) ethylamine (serotonin) and 5-hydroxyindol-3-yl-acetlc acid in the brain, Med. Exp (Basel), in press. Harvey, J.A., A Heller, R Y.Moore, H.F Hunt and L.J.Roth, 1964, Effect of central nervous system lesions on barbiturate sleeping time in the rat, J. Pharmacol. Exp. Ther. 144, 24. Konig, J.F.R and R.A.Klipell, 1963, The rat brain, A stereotaxlc atlas of the forebrain and lower parts of the brain stem (William and Wilkins Co., Baltimore). Kostowski, W., E.Glacalone, S.Garattini and L Valzelli, 1968, Studies on behavioural changes in rats after lesion of midbrain raphe, European J. Pharmacol. 4,371.
MIDBRAIN RAPHE LESIONS AND PENTOBARBITAL NARCOSIS Kostowski, W., E.Giacalone, S.Garattini and L.Valzdli, 1969, Stimulation of various forebrain structures and brain 5HT, 5HIAA and behaviour in rats, European J. Pharmacol., in press.
73
Mouret, J., P.Bobillier and M.Jouvet, 1967, Effets de la parachloroph6nylalanine sur ie sommeil du rat, C.R Soc. Biol (Paris) 161, 1600.
PROLONGED PENTOBARBITAL NARCOSIS IN RATS WITH LESIONS OF MIDBRAIN RAPHE W.KOSTOWSKI * and E.DOLFINI Istituto di Ricerche Farrnacologiche ,,Mano Negrt" Via Eritrea, 62, 20157 Milano, Italy
Received 25 February 1969
Accepted 13 March 1969
W.KOSTOWSKI and E.DOLFINI, Prolonged pentobarbital narcosis in rats with lesions of midbrain raphe, European J. Pharmacol. 6 (1969) 71-73. The mldbrain raphe area is thought to be involved in sleep behaviour; stimulation of this area produces sedation and sleep whereas lesions of the same area elicit marked hypermotflity Pentobarbital increases sleeping time in midbram lesioned rats and the percentage of animals sleeping Concentrations of pentobarbital in brain are also reported. Pentobarbital narcosis Pentobarbital in brain
The midbrain raphe area (MR) contains serotoninergic neurons which project into the forebrain and spinal cord (Dahlstr6m and Fuxe, 1964). It is thought to play a role in the regulation and triggering of sleep behaviour (Mouret, Bobillier and Jouvet, 1967). Low frequency electrical stimulation of this area induces behavioural sleep or drowsiness with associated electroencephalographic changes (Kostowski, Giacalone, Garattini and Valzelli, 1969). Lesions of the MR abolish the slow wave sleep pattern and elicit a marked hypermotility (Kostowski, Giacalone, Garattini and Valzelli, 1968). It was therefore of interest to investigate the effect of a barbiturate in rats with lesions of MR. Female Sprague Dawley rats, weighing 160-200 g were employed. Cerebral lesions were performed by means of a stainless steel electrode of 0.25 mm in diameter using a Stoelting stereotaxic apparatus and the atlas of Krnig and Klippel (1963). The details of the technique have been described elsewhere (Kostow* Visiting Scientist from the Department of Experimental Pharmacology, Medical Academy, Warsaw, Poland (present address).
Midbrain raphe lesion Sleep
ski et al., 1968). Sham operated rats and rats with lesions in lateral midbrain (LM) were also prepared. After surgery, the animals were housed singly in cages at a constant room temperature (22°C) and relative humidity (60%). Biochemical studies were performed 7 days after lesions. Pentobarbital was given intraperitoneally. Sleeping time was evaluated as the time dapsed between loss and regaining of the righting reflex. The concentration of pentobarbital in brain was measured according to Brodie, Burns, Mark, Lief, Bernstein and Papper (1953). 5-Hydroxytryptamine (5HT) and 5-hydroxyindolacetic acid (5HIAA) levels in forebrain were determined in the same sample according to Giacalone and Valzelli (1969). The results obtained are reported in table 1. Lesions of MR, but not of lateral midbrain (LM), increase the sensitivity to pentobarbital in respect to sham lesioned animals. In the group of MR lesioned rats sleeping time was increased and the percentage of sleeping animals increased. This effect was not due to changes in body temperature. Pentobarbital brain concentrations in both groups of lesioned and sham lesioned MR rats showed only minor differences. As shown previously
W KOSTOWSKI and E.DOLFINI
72
Table 1 Effects of pentobarbital in MR lesioned rats Sham lesions 46
Total number of rats Sleeping time (min + S E.) after pentobarbital
20 (mg/kg i.p.) 25 (mg/kg i.p.) 30 (mg/kgi.p.)
62 77 112
+ 14 + 10
(3/6) (7/16) (11/12)
MR lesions 51 81 122 167
+ 10 + 17" -+ 14"
Brain pentobarbltal** (t~g/g -+ S E.)
15 min 30 min 90min
18 5 + 1.9 23.3 + 2.8 15 3 + 0 9
24.9 + 2 2° 27.4 + 4 6 15.1 + 1 3
Body temperature ( ° C ~ E.) after pentobarbital**
0 min 30 min 60min 90 min
37 1 + 0.2 35.4 + 0 2 34.6 + 0 2 34.4 + 0.3
38.3 + 0.3 34.9 + 0.3 34.1 + 0 5 34 1 + 0.7
Forebrain*** 5HT (ng/g~.E.) Forebrain*** 5HIAA ( n g / g ~ E.)
364 278
+ 20 + 10
88 82
+- 5* +4"
(5/5) (12/12) (12/12)
LM lesions 23 N.D 80 106
+ 16
(2/5) (6/6)
N.D. N D. 10.8+ 1.2 37 36 36 35
0+0 1 5+04 3 +04 8+02
340 272
-+ 10 + 25
o = p ~ 0 05 m comparison to sham lesioned rats * = p ~ 0.01 in comparison to sham lesioned rats. ** = 30mg/kg 1.p *** = 6 hr after pentobarbltal 30 mg/kg i p. N.D. = not determined. In brackets the ratio between sleeping and non-sleeping animals.
(Kostowski et al., 1968), lesions of MR, but not of LM decreased 5HT and 5HIAA. The increase of pentobarbital narcosis m the MR lesioned rats is of particular interest since only lesions of a few areas are known to increase barbiturate sleeping time, e.g. dorsomedial tegmentum septum and hippocampus (Harvey, Heller, Moore, Hunt and Roth, 1964). The mechanism of the increased pentobarbltal narcosis in MR lesioned rats remains unknown but it is not related to changes in the level of pentobarbital in the whole brain. It is of interest that MR lesioned rats show an increased sensitivity to pentobarbital but a decrease of normal slow-wave sleep (Mouret et al., 1967) and persistent behavioural and electroencephalographic arousal (Kostowskl et al., 1968). ACKNOWLEDGEMENTS This work IS supported by the National Inst. of Health, Bethesda, Md., USA, Contract No. DHEW/PHS/NIH, PH 43-67-83. The technical help of Mr. Sergio Bernasconi is gratefully acknowledged.
REFERENCES Brodie, B.B., J.J.Burns, L.C.Mark, P.A.Lief, E.Bernstein and E.M.Papper, 1953, The fate of pentobarbital in man and dog and a method for its estimation in biological material, J. Pharmacol Exp. Ther. 109, 26 Dahlstr6m, A. and K.Fuxe, 1964, Evidence for the existence of monoamine-contaming neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol Scand. 62, suppl. 232, 1. Giacalone, E. and L.Valzelli, 1969, A spectrofluorometric method for the simultaneous determination of 2-(5hydroxyindol-3-yl) ethylamine (serotonin) and 5-hydroxyindol-3-yl-acetlc acid in the brain, Med. Exp (Basel), in press. Harvey, J.A., A Heller, R Y.Moore, H.F Hunt and L.J.Roth, 1964, Effect of central nervous system lesions on barbiturate sleeping time in the rat, J. Pharmacol. Exp. Ther. 144, 24. Konig, J.F.R and R.A.Klipell, 1963, The rat brain, A stereotaxlc atlas of the forebrain and lower parts of the brain stem (William and Wilkins Co., Baltimore). Kostowski, W., E.Glacalone, S.Garattini and L Valzelli, 1968, Studies on behavioural changes in rats after lesion of midbrain raphe, European J. Pharmacol. 4,371.
MIDBRAIN RAPHE LESIONS AND PENTOBARBITAL NARCOSIS Kostowski, W., E.Giacalone, S.Garattini and L.Valzdli, 1969, Stimulation of various forebrain structures and brain 5HT, 5HIAA and behaviour in rats, European J. Pharmacol., in press.
73
Mouret, J., P.Bobillier and M.Jouvet, 1967, Effets de la parachloroph6nylalanine sur ie sommeil du rat, C.R Soc. Biol (Paris) 161, 1600.