Laboratory of Neurophysiology, Department of Neurology and Laboratory of Experimental Endocrinology, Department of Medicine ', Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem, Israel
COMPLETE INHIBITION OF ADRENOCORTICAL RESPONSES FOLLOWING SCIATIC NERVE STIMULATION IN RATS WITH HYPOTHALAMIC ISLANDS
By Shaul Feldman,
Nisim
Conforti and Israel Chowers
ABSTRACT Previous studies from this laboratory have demonstrated that in rats with islands the adrenocortical response to photic and acoustic stimulation was partially inhibited indicating that they were at least to a certain degree neurally mediated, though ether stress produced normal adrenocortical responses. With the purpose of determining to what extent afferent somatosensory connections to the hypothalamus participate in the activation of adrenocortical responses following sciatic nerve stimulation, the effects of this stimulus applied through chronically implanted electrodes were studied on the plasma corticosterone levels in pentobarbital anaesthetized intact animals and in rats with hypothalamic islands. Ether stress or sciatic nerve stimulation for 2 min in intact rats produced a rise of plasma corticosterone to 32.1 \m=+-\1.2 and 32.1 \m=+-\1.8 \g=m\g/100 ml, respectively. However, in animals with hypothalamic islands the corresponding values were 29.2 \m=+-\1.8 and 12.4 \m=+-\0.8 \g=m\g/100 ml, respectively. The latter value was not significantly different from the basal corticosterone levels (13.0 \m=+-\1.2 \g=m\g/100ml) found in rats 15 min after pentobarbital anaesthesia, the time when the sciatic stimulus was applied. The present data indicate that the adrenocortical discharge following sciatic nerve stimulation is completely inhibited by hypothalamic deafferentation and therefore depends entirely on the activation of the afferent neural pathways to the
hypothalamic
hypothalamus. Supported by
advancement of Mankind Foundation and by agreement 06-008-1 with the National Institutes of Health, Bethesda, Md. l) Established investigator of the Chief Scientist's Bureau, Ministry of Health.
study of adrenocortical responses to a variety of stressful stimuli in rats hypothalamic islands makes it possible to differentiate between those stresses which act directly on the medial basal hypothalamus and others which are mediated by neural pathways. Thus it was found in our previous experi¬ ments that ether, anoxia and immobilization were systemic stresses, while photic and acoustic stimuli were neurally mediated, since they caused a considerable reduction in the adrenocortical responses in rats with hypothalamic islands (Feldman et al. 1968, 1970). However, in such preparations the in¬ hibition of the response to photic and acoustic stimulation was only partial and hence it is possible that humoral mechanisms may also have played a The with
certain role. Electrical stimulation of the sciatic nerve may be considered as a pure neurogenic stimulus, and the purpose of the present study was to determine to what extent the afferent somatosensory neural connections to the rat hypo¬ thalamus (Same Sc Feldman 1971) participate in the activation of the adreno¬ cortical responses.
MATERIALS AND METHODS
experiments were conducted on male albino rats of the Hebrew University strain, weighing about 250 g. As the sciatic stimulation was performed under pentobarbital anaesthesia, preliminary experiments were done to determine the basal level of plasma corticosterone following the intraperitoneal injection of 45 mg/kg of pentobarbital. The
Adrenocortical responses to ether stress and to sciatic stimulation, as determined by changes in the plasma corticosterone levels analyzed by a micromethod based on fluorescence developed in sulphuric acid (Glick et al. 1964), were studied both in intact animals and in rats with hypothalamic islands (Halasz 8- Pupp 1965), in which all nervous connections were severed from the remainder of the central nervous system. The island extended from behind the optic chiasma to the mammillary region. The protocol consisted of submitting the normal rats and those in which an island was established two weeks previously to ether stress, and only those animals which showed normal adrenocortical responses were implanted two weeks later with a sciatic electrode. The ether stress consisted of exposing the animals to ether for 2 min. Fifteen minutes later a skin incision was made under ether anaesthesia and within 2 min a blood sample was removed for corticosterone determination. The sciatic nerve was exposed under pentobarbital anaesthesia. The chronic sciatic electrodes were made from a silver wire of 9.3 mm diameter and 1.5 cm length, and each electrode consisted of a circular non-insulated loop. Two such electrodes were attached to the sciatic nerve at an interval of 3.5 mm. The stimulating electrodes were embedded and con¬ nected by fine sutures to the adjacent muscle in order to prevent mechanical movement. The remainder of the silver wire was insulated and connected to a stainless steel insulated wire which was passed subcutaneously and attached to a connector on the skull. After 3 days the animals were anaesthetized with pentobarbital and 15 min later bipolar stimulation of the sciatic nerve during 2 min (0.3 mA, 0.1 msec, 10/sec) was performed. Fifteen minutes after beginning of stimulation, blood was withdrawn
from the
jugular
tween 8 and 11
All data
were
vein for corticosterone determination. All stresses were applied be¬ The brains were fixed in formaline and examined histologically. analyzed by the t-test. a.m.
RESULTS
The
plasma corticosterone levels 15 and 30 min following the intraperitoneal injection of pentobarbital were found to be 13.0 ± 1.2 Mg/100 ml and 14.2 ± 2.0 «g/100 ml. respectively. These basal values are close to those obtained in our laboratory in unanaesthetized rats (Feldman et al 1970). The pentobarbital anaesthetized rats were able to react by an adequate adrenocortical discharge 15 min after a stress of bleeding, showing a plasma corticosterone level of 25.2 ± 1.8 /ig/100 ml (n=14). Implantation of the sciatic electrodes 3 days previously did not raise the basal levels of corticosterone in rats anaesthetized with pentobarbital for 30 min (Fig. 1). In intact rats the ether stress produced an elevation of plasma corticosterone to 32.1 ± 1.2 Mg/100 ml and similar results were obtained following sciatic nerve stimulation. Ether stress in rats with a 2 week hypothalamic island produced a normal adrenocortical discharge which was not significantly dif-
Ether
Sc latic
Pentobarbital
stress
sti mulation
a
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A0
I I
intact
E2ZJ island 30Ul
o
z
15
~l
30' sc
o
u
ro
E ui
ns
¬
0
20
20
13
Fig. 1. Adrenocortical responses to ether stress and to sciatic nerve stimulation in intact rats and in animals with hypothalamic islands. The bars on the right demonstrate basal corticosterone levels 15 and 30 min after administration of pentobarbital, including animals with chronic sciatic (sc) electrodes. The numbers at the base of the columns refer to the number of animals in each group.
ferent from that observed in intact rats. However, in the same animals sciatic nerve stimulation had no effect whatsoever, i. e. the plasma corticosterone level being 12.4 ± 0.8 «g/100 ml, since there was no significant difference between the basal levels of corticosterone of rats given pentobarbital anaesthesia for 15 min in which no stimulus was applied, and the values obtained following sciatic stimulation in the rats with hypothalamic islands. However, the dif¬ ference regarding the adrenocortical responses obtained in sciatic stimulated intact rats was highly significant (P
COMPLETE INHIBITION OF ADRENOCORTICAL RESPONSES FOLLOWING SCIATIC NERVE STIMULATION IN RATS WITH HYPOTHALAMIC ISLANDS
By Shaul Feldman,
Nisim
Conforti and Israel Chowers
ABSTRACT Previous studies from this laboratory have demonstrated that in rats with islands the adrenocortical response to photic and acoustic stimulation was partially inhibited indicating that they were at least to a certain degree neurally mediated, though ether stress produced normal adrenocortical responses. With the purpose of determining to what extent afferent somatosensory connections to the hypothalamus participate in the activation of adrenocortical responses following sciatic nerve stimulation, the effects of this stimulus applied through chronically implanted electrodes were studied on the plasma corticosterone levels in pentobarbital anaesthetized intact animals and in rats with hypothalamic islands. Ether stress or sciatic nerve stimulation for 2 min in intact rats produced a rise of plasma corticosterone to 32.1 \m=+-\1.2 and 32.1 \m=+-\1.8 \g=m\g/100 ml, respectively. However, in animals with hypothalamic islands the corresponding values were 29.2 \m=+-\1.8 and 12.4 \m=+-\0.8 \g=m\g/100 ml, respectively. The latter value was not significantly different from the basal corticosterone levels (13.0 \m=+-\1.2 \g=m\g/100ml) found in rats 15 min after pentobarbital anaesthesia, the time when the sciatic stimulus was applied. The present data indicate that the adrenocortical discharge following sciatic nerve stimulation is completely inhibited by hypothalamic deafferentation and therefore depends entirely on the activation of the afferent neural pathways to the
hypothalamic
hypothalamus. Supported by
advancement of Mankind Foundation and by agreement 06-008-1 with the National Institutes of Health, Bethesda, Md. l) Established investigator of the Chief Scientist's Bureau, Ministry of Health.
study of adrenocortical responses to a variety of stressful stimuli in rats hypothalamic islands makes it possible to differentiate between those stresses which act directly on the medial basal hypothalamus and others which are mediated by neural pathways. Thus it was found in our previous experi¬ ments that ether, anoxia and immobilization were systemic stresses, while photic and acoustic stimuli were neurally mediated, since they caused a considerable reduction in the adrenocortical responses in rats with hypothalamic islands (Feldman et al. 1968, 1970). However, in such preparations the in¬ hibition of the response to photic and acoustic stimulation was only partial and hence it is possible that humoral mechanisms may also have played a The with
certain role. Electrical stimulation of the sciatic nerve may be considered as a pure neurogenic stimulus, and the purpose of the present study was to determine to what extent the afferent somatosensory neural connections to the rat hypo¬ thalamus (Same Sc Feldman 1971) participate in the activation of the adreno¬ cortical responses.
MATERIALS AND METHODS
experiments were conducted on male albino rats of the Hebrew University strain, weighing about 250 g. As the sciatic stimulation was performed under pentobarbital anaesthesia, preliminary experiments were done to determine the basal level of plasma corticosterone following the intraperitoneal injection of 45 mg/kg of pentobarbital. The
Adrenocortical responses to ether stress and to sciatic stimulation, as determined by changes in the plasma corticosterone levels analyzed by a micromethod based on fluorescence developed in sulphuric acid (Glick et al. 1964), were studied both in intact animals and in rats with hypothalamic islands (Halasz 8- Pupp 1965), in which all nervous connections were severed from the remainder of the central nervous system. The island extended from behind the optic chiasma to the mammillary region. The protocol consisted of submitting the normal rats and those in which an island was established two weeks previously to ether stress, and only those animals which showed normal adrenocortical responses were implanted two weeks later with a sciatic electrode. The ether stress consisted of exposing the animals to ether for 2 min. Fifteen minutes later a skin incision was made under ether anaesthesia and within 2 min a blood sample was removed for corticosterone determination. The sciatic nerve was exposed under pentobarbital anaesthesia. The chronic sciatic electrodes were made from a silver wire of 9.3 mm diameter and 1.5 cm length, and each electrode consisted of a circular non-insulated loop. Two such electrodes were attached to the sciatic nerve at an interval of 3.5 mm. The stimulating electrodes were embedded and con¬ nected by fine sutures to the adjacent muscle in order to prevent mechanical movement. The remainder of the silver wire was insulated and connected to a stainless steel insulated wire which was passed subcutaneously and attached to a connector on the skull. After 3 days the animals were anaesthetized with pentobarbital and 15 min later bipolar stimulation of the sciatic nerve during 2 min (0.3 mA, 0.1 msec, 10/sec) was performed. Fifteen minutes after beginning of stimulation, blood was withdrawn
from the
jugular
tween 8 and 11
All data
were
vein for corticosterone determination. All stresses were applied be¬ The brains were fixed in formaline and examined histologically. analyzed by the t-test. a.m.
RESULTS
The
plasma corticosterone levels 15 and 30 min following the intraperitoneal injection of pentobarbital were found to be 13.0 ± 1.2 Mg/100 ml and 14.2 ± 2.0 «g/100 ml. respectively. These basal values are close to those obtained in our laboratory in unanaesthetized rats (Feldman et al 1970). The pentobarbital anaesthetized rats were able to react by an adequate adrenocortical discharge 15 min after a stress of bleeding, showing a plasma corticosterone level of 25.2 ± 1.8 /ig/100 ml (n=14). Implantation of the sciatic electrodes 3 days previously did not raise the basal levels of corticosterone in rats anaesthetized with pentobarbital for 30 min (Fig. 1). In intact rats the ether stress produced an elevation of plasma corticosterone to 32.1 ± 1.2 Mg/100 ml and similar results were obtained following sciatic nerve stimulation. Ether stress in rats with a 2 week hypothalamic island produced a normal adrenocortical discharge which was not significantly dif-
Ether
Sc latic
Pentobarbital
stress
sti mulation
a
nest hesia
A0
I I
intact
E2ZJ island 30Ul
o
z
15
~l
30' sc
o
u
ro
E ui
ns
¬
0
20
20
13
Fig. 1. Adrenocortical responses to ether stress and to sciatic nerve stimulation in intact rats and in animals with hypothalamic islands. The bars on the right demonstrate basal corticosterone levels 15 and 30 min after administration of pentobarbital, including animals with chronic sciatic (sc) electrodes. The numbers at the base of the columns refer to the number of animals in each group.
ferent from that observed in intact rats. However, in the same animals sciatic nerve stimulation had no effect whatsoever, i. e. the plasma corticosterone level being 12.4 ± 0.8 «g/100 ml, since there was no significant difference between the basal levels of corticosterone of rats given pentobarbital anaesthesia for 15 min in which no stimulus was applied, and the values obtained following sciatic stimulation in the rats with hypothalamic islands. However, the dif¬ ference regarding the adrenocortical responses obtained in sciatic stimulated intact rats was highly significant (P
