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Hormone release evoked by electrical stimulation of rat neurohypophyses in the absence of action potentials
604
SHORT COMMUNICATIONS
Hormone release evoked by electrical stimulation of rat neurohypophyses in the absence of action potentials Electrical stimuli applied to neurohypophyses incubated in vitro evoke the release of posterior pituitary hormones 2,5; this effect is dependent upon the generation of propagated action potentials4,6, 7, which depolarize the neurosecretory nerve endings and actuate the calcium-dependent release process 8. High frequency electrical stimulation has been shown to be more effective in promoting hormone secretion than low frequency stimulation4,6,7; however, interpretation of this phenomenon, which may be analogous to the process of facilitation observed at certain chemically operated
Fig. 1. Effect of reducing the external Na concentration on the compound action potential recorded from an isolated rat neurohypophysis in response to electrical stimuli (0.05 msec, 2.0 mA) applied to the pituitary stalk. A, in normal Locke's solution. B, abolition of the potential change approximately 5 min after substitution of 154 m M NaC1 by an iso-osmotic amount of sucrose; following substitution, the medium contained no sodium except for 6 m M NaHCOs. C, recovery of the response after re-exposure of the neurohypophysis to normal Locke's solution. Vertical calibration, 50/~V. Time marks, msec.
Brain Research, 45 (1972) 604-607
SHORT COMMUNICATIONS
605
50
7
0 o 40
Z
~ 20 g
Y
/ 0
i
a
i
i
2
4
6
8
i
10
Stimulation intensity (mA)
Fig. 2. Relation between intensity of stimulation and hormone release from rat neurohypophyses kept in a low Na medium (6 mM NaHCOa, 154mM choline chloride). Each point shows hormone output, estimated in a rat milk-ejection assay, during the 10 min period following a 30 sec train of biphasic rectangular electrical stimuli applied at a frequency of 50 Hz to the stalk at the intensity indicated on the abscissa. Points are the means of 4-6 experiments at each stimulation intensity.
synapses, is complicated by possible changes in action potential shape and membrane potential during and after tetanic stimulation. Recently, Douglas and Sorimachi 3 showed that electrical stimulation of neurohypophyses kept in a Na-free medium can also cause an increase in vasopressin secretion. This response was calcium-dependent and was reduced in amplitude by an increase in MgC12, but was unaffected by the addition of tetrodotoxin to the Na-free solution; it was therefore interpreted as being due to a direct, depolarizing action of the current pulses on the neurohypophysial nerve endings. This finding led us to study the effect of trains of electrical stimuli on hormone output from neurohypophyses maintained under conditions where action potentials are no longer generated. Neurohypophyses were isolated from rats following decapitation. Potential changes were recorded in response to 1 msec, monophasic rectangular pulses delivered through silver electrodes placed across the pituitary stalk. A second pair of silver wires were used for recording; they rested on the most distal part of the neurohypophysis, 1-2 mm away from the site of stimulation. The compound action potential recorded in Locke's solution (NaCI 154 mM, KCI 5.6 mM, CaCI2 2.2 mM, MgCI2 1 raM, NaHCOs 6 mM, glucose 10 mM) in one experiment is illustrated in Fig. 1A; it was abolished after replacement of the 154 m M of NaC1 by an equimolar amount of choline chloride or sucrose ('low-Na Locke's solution', Fig. 1B); the effect of partial Na replacement was fully reversible (Fig. 1C). For hormonal release experiments, the cut end of the pituitary stalk of a single neurohypophysis was tied to a platinum wire, and the preparation immersed in 2 ml of 'low-Na Locke's solution' (after an initial 20 min preincubation period in Locke's solution). The solutions were maintained at 37 °C and gassed with 5 ~o CO2-95 ~ 02. Electrical pulses for stimulation were applied between the wire carrying the preparation and a second wire dipping into the incubation medium. During stimulation, the Brain Research, 45 (1972) 604--607
606
SHORT COMMUNICATIONS
6O 100Hz C / I 50 HZ 4o
g 2o
l
~oHZ
I I 10 20 30 40 50
I 100
I 150
Seconds after onset of tetanic stimulation
Fig. 3. Relation between frequency of stimulation and hormone release from rat neurohypophyses kept in a low Na medium (6 mM NaHCOs, 154 mMcholine chloride). Each point shows the hormone output during the I0 min period following a train of electrical (6.0 mA, 2 msec biphasic rectangular) stimuli applied to the stalk at frequencies of 10 (O), 25 (©), 50 ( I ) and 100 Hz (A). Trains were of variable duration so as to contain a total of 500, 1000, 1500 or 2000 (only at 25 Hz) stimuli, respectively. Points are the means of 4-6 experiments, each on a separate neurohypophysis. electrode holding the gland was raised so that the inferior pole of the neurohypophysis remained in contact with the incubation medium by surface tension. The neurohypophysis was incubated for 10 min following the end of electrical stimulation, in order to allow diffusion of the released hormone into the medium. The oxytocin content of the medium was determined against synthetic oxytocin (Sandoz, Basle) using the rat milk-ejection bioassay 1. Fig. 2 illustrates a series of experiments in which neurohypophyses were stimulated at different stimulus intensities (0.25-10.0 m A peak-topeak) with 2 msec long biphasic rectangular pulses at 50 Hz for 30 sec. It is apparent that hormone output is a function of stimulus intensity, and that oxytocin release increases progressively with increases in stimulus intensity up to 6 mA. A non-specific, destructive effect of current on the neuronal membranes seems to be ruled out by the fact that a further increase from 6 to 10 m A does not lead to a higher hormone output. It must be stressed that the actual current flowing through the nerve terminals certainly represents only a small fraction of the total current applied to the preparation. Current pulses did not promote hormone secretion when CaC12 was omitted from the medium; when calcium was present, the [45Ca] content of electrically stimulated neurohypophyses was 3-6 fold larger than in control glands. In another group of experiments, neurohypophyses were stimulated with 6 m A rectangular pulses at frequencies of either 10, 25, 50, or 100 Hz; we chose train durations so that a total of 500, 1000, 1500 or 2000 stimuli were applied. Fig. 3 shows that the amount of hormone released per stimulus is roughly constant during the whole duration of the train at each frequency, but that it varies markedly and systematically with the changes of the stimulation frequency: identical numbers of stimuli release progressively more hormone if delivered at an increased frequency. These results demonstrate a clear-cut dependence of hormone release on frequency of stimulation when the nerve endings are directly stimulated by current pulses in experimental Brain Research, 45 (1972) 604-607
SHORT COMMUNICATIONS
607
conditions precluding action potential generation. In previous studies which had been carried out in normal Locke's solution, the amount of hormone released by identical numbers of stimuli from isolated neurohypophyses reached a maximum with stimulation frequencies of about 35 Hz, and then tended to decline when higher frequencies of stimulation were used 6. Similar findings have been reported by Ishida 6,7. The fact that this depression is not observed in the present study suggests that it was due to sustained changes of the membrane potential of the axonal membrane and of the shape of the action potentials when long duration, high frequency trains of stimulation were applied. This work was supported by Grants from the Swiss National Science Foundation (No. 3.556.71) and the F. Hoffman-La Roche Foundation (No. 117). Department of Physiology, University of Geneva Medical School, Geneva (Switzerland)
J. J. NORDMANN J. J. DREIFUSS
1 BISSET,G. W., CLARK,B. J., HALDAR,J., HARRIS, M. C., LEWIS,G. P., AND ROCHA E SILVA,M., The assay of milk-ejecting activity in the lactating rat, Brit. J. Pharmacol., 31 (1967) 537-549. 2 DOUGLAS,W. W., AND POISNER, k. i . , Stimulus-secretion coupling in a neurosecretory organ: the role of calcium in the release of vasopressin from the neurohypophysis, d. Physiol. (Lond.), 172 (1964) 1-18. 3 DOUGLAS,W. W., AND SORIMACHI,M., Electrically evoked release of vasopressin from isolated neurohypophyses in sodium-free media, Brit. J. PharmacoL, 42 (1971) 647P. 4 DREIFUSS,J. J., KALNINS,I., KELLY,J. S., AND RUF, K. B., Action potentials and release of neurohypophysial hormones in vitro, d. Physiol. ( Lond.), 215 (1971) 805-817. 5 HALLER,E. W., SACHS,H., SPERELAKIS,H., AND SNARE,L., Release of vasopressin from isolated guinea-pig pituitaries, Amer. J. Physiol., 209 (1965) 79-83. 6 ISHIDA,A., The oxytocin release and the compound action potential evoked by electrical stimulation of the isolated neurohypophysis of the rat, ,lap. d. Physiol., 20 (1970) 84-96. 7 ISHIDA,A., The role of action potential on the release of posterior pituitary hormone, Med. J. Osaka Univ., 21 (1971) 101-111. 8 MIKITEN,T. M., AND DOUGLAS,W. W., Effect of calcium and other ions on vasopressin release from rat neurohypophyses stimulated in vitro, Nature (Lond.), 207 (1965) 302. (Accepted August 4th, 1972)
Brain Research, 45 (1972) 604-607
SHORT COMMUNICATIONS
Hormone release evoked by electrical stimulation of rat neurohypophyses in the absence of action potentials Electrical stimuli applied to neurohypophyses incubated in vitro evoke the release of posterior pituitary hormones 2,5; this effect is dependent upon the generation of propagated action potentials4,6, 7, which depolarize the neurosecretory nerve endings and actuate the calcium-dependent release process 8. High frequency electrical stimulation has been shown to be more effective in promoting hormone secretion than low frequency stimulation4,6,7; however, interpretation of this phenomenon, which may be analogous to the process of facilitation observed at certain chemically operated
Fig. 1. Effect of reducing the external Na concentration on the compound action potential recorded from an isolated rat neurohypophysis in response to electrical stimuli (0.05 msec, 2.0 mA) applied to the pituitary stalk. A, in normal Locke's solution. B, abolition of the potential change approximately 5 min after substitution of 154 m M NaC1 by an iso-osmotic amount of sucrose; following substitution, the medium contained no sodium except for 6 m M NaHCOs. C, recovery of the response after re-exposure of the neurohypophysis to normal Locke's solution. Vertical calibration, 50/~V. Time marks, msec.
Brain Research, 45 (1972) 604-607
SHORT COMMUNICATIONS
605
50
7
0 o 40
Z
~ 20 g
Y
/ 0
i
a
i
i
2
4
6
8
i
10
Stimulation intensity (mA)
Fig. 2. Relation between intensity of stimulation and hormone release from rat neurohypophyses kept in a low Na medium (6 mM NaHCOa, 154mM choline chloride). Each point shows hormone output, estimated in a rat milk-ejection assay, during the 10 min period following a 30 sec train of biphasic rectangular electrical stimuli applied at a frequency of 50 Hz to the stalk at the intensity indicated on the abscissa. Points are the means of 4-6 experiments at each stimulation intensity.
synapses, is complicated by possible changes in action potential shape and membrane potential during and after tetanic stimulation. Recently, Douglas and Sorimachi 3 showed that electrical stimulation of neurohypophyses kept in a Na-free medium can also cause an increase in vasopressin secretion. This response was calcium-dependent and was reduced in amplitude by an increase in MgC12, but was unaffected by the addition of tetrodotoxin to the Na-free solution; it was therefore interpreted as being due to a direct, depolarizing action of the current pulses on the neurohypophysial nerve endings. This finding led us to study the effect of trains of electrical stimuli on hormone output from neurohypophyses maintained under conditions where action potentials are no longer generated. Neurohypophyses were isolated from rats following decapitation. Potential changes were recorded in response to 1 msec, monophasic rectangular pulses delivered through silver electrodes placed across the pituitary stalk. A second pair of silver wires were used for recording; they rested on the most distal part of the neurohypophysis, 1-2 mm away from the site of stimulation. The compound action potential recorded in Locke's solution (NaCI 154 mM, KCI 5.6 mM, CaCI2 2.2 mM, MgCI2 1 raM, NaHCOs 6 mM, glucose 10 mM) in one experiment is illustrated in Fig. 1A; it was abolished after replacement of the 154 m M of NaC1 by an equimolar amount of choline chloride or sucrose ('low-Na Locke's solution', Fig. 1B); the effect of partial Na replacement was fully reversible (Fig. 1C). For hormonal release experiments, the cut end of the pituitary stalk of a single neurohypophysis was tied to a platinum wire, and the preparation immersed in 2 ml of 'low-Na Locke's solution' (after an initial 20 min preincubation period in Locke's solution). The solutions were maintained at 37 °C and gassed with 5 ~o CO2-95 ~ 02. Electrical pulses for stimulation were applied between the wire carrying the preparation and a second wire dipping into the incubation medium. During stimulation, the Brain Research, 45 (1972) 604--607
606
SHORT COMMUNICATIONS
6O 100Hz C / I 50 HZ 4o
g 2o
l
~oHZ
I I 10 20 30 40 50
I 100
I 150
Seconds after onset of tetanic stimulation
Fig. 3. Relation between frequency of stimulation and hormone release from rat neurohypophyses kept in a low Na medium (6 mM NaHCOs, 154 mMcholine chloride). Each point shows the hormone output during the I0 min period following a train of electrical (6.0 mA, 2 msec biphasic rectangular) stimuli applied to the stalk at frequencies of 10 (O), 25 (©), 50 ( I ) and 100 Hz (A). Trains were of variable duration so as to contain a total of 500, 1000, 1500 or 2000 (only at 25 Hz) stimuli, respectively. Points are the means of 4-6 experiments, each on a separate neurohypophysis. electrode holding the gland was raised so that the inferior pole of the neurohypophysis remained in contact with the incubation medium by surface tension. The neurohypophysis was incubated for 10 min following the end of electrical stimulation, in order to allow diffusion of the released hormone into the medium. The oxytocin content of the medium was determined against synthetic oxytocin (Sandoz, Basle) using the rat milk-ejection bioassay 1. Fig. 2 illustrates a series of experiments in which neurohypophyses were stimulated at different stimulus intensities (0.25-10.0 m A peak-topeak) with 2 msec long biphasic rectangular pulses at 50 Hz for 30 sec. It is apparent that hormone output is a function of stimulus intensity, and that oxytocin release increases progressively with increases in stimulus intensity up to 6 mA. A non-specific, destructive effect of current on the neuronal membranes seems to be ruled out by the fact that a further increase from 6 to 10 m A does not lead to a higher hormone output. It must be stressed that the actual current flowing through the nerve terminals certainly represents only a small fraction of the total current applied to the preparation. Current pulses did not promote hormone secretion when CaC12 was omitted from the medium; when calcium was present, the [45Ca] content of electrically stimulated neurohypophyses was 3-6 fold larger than in control glands. In another group of experiments, neurohypophyses were stimulated with 6 m A rectangular pulses at frequencies of either 10, 25, 50, or 100 Hz; we chose train durations so that a total of 500, 1000, 1500 or 2000 stimuli were applied. Fig. 3 shows that the amount of hormone released per stimulus is roughly constant during the whole duration of the train at each frequency, but that it varies markedly and systematically with the changes of the stimulation frequency: identical numbers of stimuli release progressively more hormone if delivered at an increased frequency. These results demonstrate a clear-cut dependence of hormone release on frequency of stimulation when the nerve endings are directly stimulated by current pulses in experimental Brain Research, 45 (1972) 604-607
SHORT COMMUNICATIONS
607
conditions precluding action potential generation. In previous studies which had been carried out in normal Locke's solution, the amount of hormone released by identical numbers of stimuli from isolated neurohypophyses reached a maximum with stimulation frequencies of about 35 Hz, and then tended to decline when higher frequencies of stimulation were used 6. Similar findings have been reported by Ishida 6,7. The fact that this depression is not observed in the present study suggests that it was due to sustained changes of the membrane potential of the axonal membrane and of the shape of the action potentials when long duration, high frequency trains of stimulation were applied. This work was supported by Grants from the Swiss National Science Foundation (No. 3.556.71) and the F. Hoffman-La Roche Foundation (No. 117). Department of Physiology, University of Geneva Medical School, Geneva (Switzerland)
J. J. NORDMANN J. J. DREIFUSS
1 BISSET,G. W., CLARK,B. J., HALDAR,J., HARRIS, M. C., LEWIS,G. P., AND ROCHA E SILVA,M., The assay of milk-ejecting activity in the lactating rat, Brit. J. Pharmacol., 31 (1967) 537-549. 2 DOUGLAS,W. W., AND POISNER, k. i . , Stimulus-secretion coupling in a neurosecretory organ: the role of calcium in the release of vasopressin from the neurohypophysis, d. Physiol. (Lond.), 172 (1964) 1-18. 3 DOUGLAS,W. W., AND SORIMACHI,M., Electrically evoked release of vasopressin from isolated neurohypophyses in sodium-free media, Brit. J. PharmacoL, 42 (1971) 647P. 4 DREIFUSS,J. J., KALNINS,I., KELLY,J. S., AND RUF, K. B., Action potentials and release of neurohypophysial hormones in vitro, d. Physiol. ( Lond.), 215 (1971) 805-817. 5 HALLER,E. W., SACHS,H., SPERELAKIS,H., AND SNARE,L., Release of vasopressin from isolated guinea-pig pituitaries, Amer. J. Physiol., 209 (1965) 79-83. 6 ISHIDA,A., The oxytocin release and the compound action potential evoked by electrical stimulation of the isolated neurohypophysis of the rat, ,lap. d. Physiol., 20 (1970) 84-96. 7 ISHIDA,A., The role of action potential on the release of posterior pituitary hormone, Med. J. Osaka Univ., 21 (1971) 101-111. 8 MIKITEN,T. M., AND DOUGLAS,W. W., Effect of calcium and other ions on vasopressin release from rat neurohypophyses stimulated in vitro, Nature (Lond.), 207 (1965) 302. (Accepted August 4th, 1972)
Brain Research, 45 (1972) 604-607