Ncuroendocrinology 18: 192-203 (1975)
Effects of Drugs Influencing Brain Catecholamines on GH Release in Rats with Hypothalamic Surgery1 K. C hihara, Y. K ato, S. O hgo and H. I mura Third Division. Department of Medicine, Kobe University School of Medicine. Kobe
Key Words. Growth hormone • Chlorpromazine • v-Methyl-p-tyrosinc • Hypothalamic deafferentation ■Hypothalamic ablation Abstract. The effect of pretreatment with \-mcthyl-p-tyrosine ( \-MT) on the basal levels of plasma growth hormone (GH) and the responses to chlorpromazine (CPZ) were investigated in urethane-anesthetized rats with either complete hypothalamic deafferenta tion (C.D.), hypothalamic ablation (H.A.) or sham operation (Sham). Basal GH levels were high in C.D. rats, intermediate in H.A. rats, and low in Sham rats without any pretreatment. Pretreatment with a-MT caused a significant increase in basal GH levels in both C.D. and Sham rats, but not in H.A. rats. GH release following the intravenous injection of CPZ, which was observed in C.D. and Sham rats without i-M T pretreatment, was blunted by treatment with a-MT. In H.A. rats CPZ failed to stimulate the secretion of GH regardless of a-MT pretreatment. Neither the injection of l-DOPA nor dl-DOPS affected basal GH levels in non-tx-MT pretreated C.D. rats. However, plasma GH levels significantly decreased following the injection of l-DOPA, but not dl-DOPS. in C.D. rats pretreated with a-MT. These findings suggest that the injection of CPZ causes an enhance ment of GH release by inhibiting the catecholaminergic (dopaminergic) mechanism, which is active within the basal medial hypothalamus (BMH) and plays an inhibitory role in GH secretion. They also suggest that the extrahypothalamic inhibitory neural pathway, which is connected to the BMH and is interrupted by hypothalamic deafferentation, is not catecholaminergic.
It is well known that the neural mechanism plays an important role in regulating the secretion of growth hormone (GH) [M uller, 1973; M artin, 1973]. Our previous reports showed that plasma GH levels were low and 1 Supported in part by grants from the Ministry of Education, Japan.
Downloaded by: Nagoya University 133.6.82.173 - 1/13/2019 6:41:50 AM
Received: January 20th, 1975; revised MS accepted: April 29th, 1975.
C hihara/K ato/O hgo /I mura
193
stable in urethane-anesthetized rats [K ato et al., 1971]. and that serial bleed ing after an intravenous injection of chlorpromazine (CPZ), an anti-dopaminergic agent, caused a significant increase in plasma GH levels in these rats while saline had no significant effect [K ato et al., 1973]. On the basis of these findings, we have suggested that brain catecholamines are involved in the neural regulation of GH secretion. M itch ell el al. [1973] observed a rise in plasma GH in rats following the anterior or complete surgical deafferentation of the basal medial hypothalamus (BMH), and proposed that anterior connections to the BMH inhibit the secretion of GH. This hypothesis was supported by our previous studies demonstrating that the suppressive effect of urethane anesthesia on plasma GH levels is prevented by the anterior or complete deafferenlation, but not by the posterior cut, of the hypothalamus [K ato el al., 1974; C h ihara el al., 1975]. Furthermore, we observed that plasma rat GH responses to CPZ are rather exaggerated after the anterior or complete deafferentation of the hypothalamus but significantly lowered by the posterior cut, and that GH release induced by CPZ is completely abolished by extensive hypothalamic destruction [K ato et al., 1974]. The present studies were undertaken to examine the effects of drugs which are known to modify the levels of brain dopamine and norepinephrine on basal levels of plasma GH and the responses to CPZ in rats with complete hypothalamic deafferentation, hypothalamic ablation and sham operation, in order to clarify the role of catecholamines in the neural regulatory mech anism of GH secretion. Materials anil Methods
Downloaded by: Nagoya University 133.6.82.173 - 1/13/2019 6:41:50 AM
Adult male Wistar rats weighing approximately 200 g were used in the experiments. The skulls of the rats were held in a stereotaxic instrument with a toothbar lowered at an angle of 10 inferior to the horizontal zero plane of the D e G root atlas [1959]. Two types of hypothalamic surgery were performed under pentobarbital anesthesia (4 mg/100 g. i.p.): Complete deafferentation. The basal medial hypothalamus (BMH) was completely isolated neurally from the surrounding hypothalamus by a modification of the method described by H alasz and P upp [1965]. A bayonet-shaped knife (vertical 1.5 mm, radius 1.5 mm) was lowered through the midline at a point 1.5 mm posterior to the bregma with the tip facing anteriorly. The knife was then rotated 90 to the left. After the stereotaxic carrier was moved posteriorly 2.0 mm, the knife was rotated peripherally 180 . The stereotaxic carrier was then moved anteriorly 2.0 mm, and the knife rotated 90 to the left and removed from the brain at the site of entry. Hypothalamic ablation. Complete destruction of the BMH was performed by a modi fication of the method described by A rtmura et at. [1972], A stirrup-shaped knife (vertical
194
C hihara/K ato/O hgo/I mura
2.0 mm. diameter 3.0 mm) was lowered through the midlinc to the base of the brain in the same manner as described in ‘complete deafferentation'. Then the knife was rotated 360 several times. The stereotaxic carrier was moved posteriorly 2.0 mm, and again the knife was rotated 360 several times. The BMH isolated by this knife became necrotic due to the interruption of the vascular supply from the ventral surface of the brain. The ‘sham’ operations consisted of lowering the knife through the midlinc to the base of the brain without rotation. Animals subjected to hypothalamic surgery were housed in individual cages at 22 ±2 C with automatic light control (light on at 07.00, off at 21.00) and were fed rat biscuits (Oriental Yeast Co.. Tokyo) and water ad libitum until 2 weeks after surgery, when the experiments were performed on the following schedules: Experiment I. Rats with complete deafferentation, hypothalamic ablation and sham operation received intravenous injections of chlorpromazine (CPZ, 200 jug/100 g b.w.) into the jugular vein under urethane anesthesia (150 mg./lOO g b.w.) after overnight fasting and without any pretreatment. Blood samples of 0.6 ml were withdrawn from the contra lateral jugular vein, immediately before and 10, 20 and 40 min after the injection of CPZ. Details of sampling procedures are described in our previous report [K ato et
Effects of Drugs Influencing Brain Catecholamines on GH Release in Rats with Hypothalamic Surgery1 K. C hihara, Y. K ato, S. O hgo and H. I mura Third Division. Department of Medicine, Kobe University School of Medicine. Kobe
Key Words. Growth hormone • Chlorpromazine • v-Methyl-p-tyrosinc • Hypothalamic deafferentation ■Hypothalamic ablation Abstract. The effect of pretreatment with \-mcthyl-p-tyrosine ( \-MT) on the basal levels of plasma growth hormone (GH) and the responses to chlorpromazine (CPZ) were investigated in urethane-anesthetized rats with either complete hypothalamic deafferenta tion (C.D.), hypothalamic ablation (H.A.) or sham operation (Sham). Basal GH levels were high in C.D. rats, intermediate in H.A. rats, and low in Sham rats without any pretreatment. Pretreatment with a-MT caused a significant increase in basal GH levels in both C.D. and Sham rats, but not in H.A. rats. GH release following the intravenous injection of CPZ, which was observed in C.D. and Sham rats without i-M T pretreatment, was blunted by treatment with a-MT. In H.A. rats CPZ failed to stimulate the secretion of GH regardless of a-MT pretreatment. Neither the injection of l-DOPA nor dl-DOPS affected basal GH levels in non-tx-MT pretreated C.D. rats. However, plasma GH levels significantly decreased following the injection of l-DOPA, but not dl-DOPS. in C.D. rats pretreated with a-MT. These findings suggest that the injection of CPZ causes an enhance ment of GH release by inhibiting the catecholaminergic (dopaminergic) mechanism, which is active within the basal medial hypothalamus (BMH) and plays an inhibitory role in GH secretion. They also suggest that the extrahypothalamic inhibitory neural pathway, which is connected to the BMH and is interrupted by hypothalamic deafferentation, is not catecholaminergic.
It is well known that the neural mechanism plays an important role in regulating the secretion of growth hormone (GH) [M uller, 1973; M artin, 1973]. Our previous reports showed that plasma GH levels were low and 1 Supported in part by grants from the Ministry of Education, Japan.
Downloaded by: Nagoya University 133.6.82.173 - 1/13/2019 6:41:50 AM
Received: January 20th, 1975; revised MS accepted: April 29th, 1975.
C hihara/K ato/O hgo /I mura
193
stable in urethane-anesthetized rats [K ato et al., 1971]. and that serial bleed ing after an intravenous injection of chlorpromazine (CPZ), an anti-dopaminergic agent, caused a significant increase in plasma GH levels in these rats while saline had no significant effect [K ato et al., 1973]. On the basis of these findings, we have suggested that brain catecholamines are involved in the neural regulation of GH secretion. M itch ell el al. [1973] observed a rise in plasma GH in rats following the anterior or complete surgical deafferentation of the basal medial hypothalamus (BMH), and proposed that anterior connections to the BMH inhibit the secretion of GH. This hypothesis was supported by our previous studies demonstrating that the suppressive effect of urethane anesthesia on plasma GH levels is prevented by the anterior or complete deafferenlation, but not by the posterior cut, of the hypothalamus [K ato el al., 1974; C h ihara el al., 1975]. Furthermore, we observed that plasma rat GH responses to CPZ are rather exaggerated after the anterior or complete deafferentation of the hypothalamus but significantly lowered by the posterior cut, and that GH release induced by CPZ is completely abolished by extensive hypothalamic destruction [K ato et al., 1974]. The present studies were undertaken to examine the effects of drugs which are known to modify the levels of brain dopamine and norepinephrine on basal levels of plasma GH and the responses to CPZ in rats with complete hypothalamic deafferentation, hypothalamic ablation and sham operation, in order to clarify the role of catecholamines in the neural regulatory mech anism of GH secretion. Materials anil Methods
Downloaded by: Nagoya University 133.6.82.173 - 1/13/2019 6:41:50 AM
Adult male Wistar rats weighing approximately 200 g were used in the experiments. The skulls of the rats were held in a stereotaxic instrument with a toothbar lowered at an angle of 10 inferior to the horizontal zero plane of the D e G root atlas [1959]. Two types of hypothalamic surgery were performed under pentobarbital anesthesia (4 mg/100 g. i.p.): Complete deafferentation. The basal medial hypothalamus (BMH) was completely isolated neurally from the surrounding hypothalamus by a modification of the method described by H alasz and P upp [1965]. A bayonet-shaped knife (vertical 1.5 mm, radius 1.5 mm) was lowered through the midline at a point 1.5 mm posterior to the bregma with the tip facing anteriorly. The knife was then rotated 90 to the left. After the stereotaxic carrier was moved posteriorly 2.0 mm, the knife was rotated peripherally 180 . The stereotaxic carrier was then moved anteriorly 2.0 mm, and the knife rotated 90 to the left and removed from the brain at the site of entry. Hypothalamic ablation. Complete destruction of the BMH was performed by a modi fication of the method described by A rtmura et at. [1972], A stirrup-shaped knife (vertical
194
C hihara/K ato/O hgo/I mura
2.0 mm. diameter 3.0 mm) was lowered through the midlinc to the base of the brain in the same manner as described in ‘complete deafferentation'. Then the knife was rotated 360 several times. The stereotaxic carrier was moved posteriorly 2.0 mm, and again the knife was rotated 360 several times. The BMH isolated by this knife became necrotic due to the interruption of the vascular supply from the ventral surface of the brain. The ‘sham’ operations consisted of lowering the knife through the midlinc to the base of the brain without rotation. Animals subjected to hypothalamic surgery were housed in individual cages at 22 ±2 C with automatic light control (light on at 07.00, off at 21.00) and were fed rat biscuits (Oriental Yeast Co.. Tokyo) and water ad libitum until 2 weeks after surgery, when the experiments were performed on the following schedules: Experiment I. Rats with complete deafferentation, hypothalamic ablation and sham operation received intravenous injections of chlorpromazine (CPZ, 200 jug/100 g b.w.) into the jugular vein under urethane anesthesia (150 mg./lOO g b.w.) after overnight fasting and without any pretreatment. Blood samples of 0.6 ml were withdrawn from the contra lateral jugular vein, immediately before and 10, 20 and 40 min after the injection of CPZ. Details of sampling procedures are described in our previous report [K ato et
