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Cortically induced postsynaptic potentials in hypoglossal motoneurons after axotomy
Neuroscience Vol. 13, No. 3, pp. 855-862, Printed in Great Britain
1984
0306-4522/84
$3.00 + 0.00
PergamonPressLtd IBRO
CORTICALLY INDUCED POSTSYNAPTIC IN HYPOGLOSSAL MOTONEURONS AXOTOMY
POTENTIALS AFTER
M. TAICATAand T. NAGAHAMA Department of Physiology, School of Dentistry, Tokushima University, Kuramoto-cho, Tokushima 770, Japan Abstract-Cortically induced postsynaptic potentials were studied in normal and axotomized cat hypoglossal motoneurons. In normal protruder motoneurons innervating tongue protruder muscles, we have demonstrated that stimulation of the orbital gyrus, at the point optimum for inducing lapping movements of the tongue by repetitive stimuli, produced inhibitory postsynaptic potentials or excitatory postsynaptic potentials followed by predominant inhibitory postsynaptic potentials. The cortically induced excitatory postsynaptic potential in normal protruder motoneurons was composed of ouly the short-latency component. In protruder motoneurons 30, 40, 60 and 80 days after axotomy, we have demonstrated that the number of protruder motoneurons responding with two components of excitatory postsynaptic potentials (the short- and the long-latency component) to cortical stimulation increased in correspondence with the lapse of days after axotomy and that the amplitude of cortically induced inhibitory postsynaptic potentials in axotomized protruder motoneurons was reduced in size as compared with normal protruder motoneurons.
Morphological studies have revealed that the majority of hypoglossal motoneurons exhibited chromatolysis at the end of the first week following axotomy3 and several quantitative studies have shown that by 1 week after axotomy there is a loss of about 50% of the synapses on the hypoglossal motoneurons.‘.‘s’5.23 In axotomized hypoglossal motoneurons, Sumner and SutherlandI and SumnerI have also observed a reduced number of dendritic synapses accompanied with detachment of synapses from the soma membrane. We have also demonstrated that the amplitude of the short- and the long-lasting inhibitory postsynaptic potentials (IPSPs) produced in axotomized hypoglossal motoneurons by stimulation of the lingual nerve was significantly reduced in size as compared with normal hypoglossal motoneurons.‘7.‘9 In the present experiments, we have attempted to demonstrate that excitatory postsynaptic potentials (EPSPs) produced in axotomized hypoglossal motoneurons by cortical stimulation are composed of the short- and the long-latency component. The purpose of the present paper is to investigate whether, in hypoglossal motoneurons, axotomy is followed by the decline of synaptic efficacy of inhibitory synapses for cortically induced IPSPs, while excitation is unaffected. EXPERIMENTALPROCEDURES Adult cats, either male or female and weighing 2.5-3.5 kg, were used. The hypoglossal nerve of the cat divides into the medial and the lateral branches. The fibers of the medial Abbreviahws:
EPSP, excitatory postsynaptic potential; IPSP, inhibitory postsynaptic potential; P-Mn, tongue protruder motoneuron.
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branch innervate the tongue protruder muscles, and those of the lateral branch the tongue retractor muscles. Both the medial and the lateral branch of the right hypoglossal nerves were transected at the point of the posterior belly of the digastric muscle. The central cut end of the right hypoglossal nerves was ligated with silk thread to impede reinnervation. The left hypoglossal nerves were left intact. All surgical procedures were done under sodium pentobarbital anesthesia (Nembutal, 30mg/kg i.p.) with aseptic precautions. Animals were then allowed to survive for intervals of 30, 40, 60 and 80 days. In the terminal experiments, operated animals were anesthetized with sodium pentobarbital (Nembutal, 30 mg/kg i.p.) and a small caudal part of the cerebellum was sucked out in order to enable penetration by a micropipette into the hypoglossal nucleus. A pneumothorax was made, and cats were immobilized by i.v. injection of gallamine triethiodide and respiration was maintained artificially. A sleeve electrode was used to stimulate the cut central end of the previously severed medial branch of the hypoglossal nerves. In a control experiment, the medial branch of either side was isolated for stimulation. The left eye was enucleated and the orbitofrontal area of the cerebral cortex was exposed. The dura was opened and a spring-mounted silver ball electrode (diameter: 1 mm) was placed on the cortical surface and was fixed at the point optimum for inducing lapping movement of the tongue by repetitive stimuli at 50/s, the reference electrode being inserted in temporal muscles. In most cases, monopolar stimulation was done and a single positive pulse (0.2-0.5 ms, 2-10 V) was delivered, but sometimes, a bipolar silver ball electrode (interpolar distance; 2 mm) was placed and a train of three square pulses (500/s, 0.1 ms, below 2 mA) was delivered to the cortical site. The exposed surface of the cortex was covered by warm liquid paraffin and the temperature was kept at 38°C. Body temperature was maintained by means of an electric blanket thermostatically controlled from a rectal thermistor. In the present experiments, micropipettes filled with 2 M potassium citrate were used for recording and for current injection, since an increase of intracellular concentration of chloride ions was found to reverse IPSPs to a depolarizing potentialzO Electrode resistance was between 15and 25 Ma.
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Potentials were recorded with a direct-coupled amplifier. In order to examine the patterns of postsynaptic potentials produced in the hypoglossal motoneurons by cortical stimulation, the responses to ten trials were averaged. In some cases, depolarization of the membrane potential by penetration of a micropipette into a cell masked EPSPs. In such a case, by injecting a constant hyperpolarizing current into a cell, the membrane potential was kept at the resting membrane potential. RESULTS
(open, Type A), a short-latency EPSP followed by predominant IPSPs (hatched, Type C) and an IPSP (dotted, Type D), respectively. It is evident that the major fraction of the total sample of normal P-Mns studied showed the IPSPs and a short-latency EPSP followed by predominant IPSPs. A stimulation site to the cortical surface is demonstrated by a filled dot (arrow) in Fig. 1 (E). The point examined was in the “face area” of Woolsey’s MS I regionz6 as already pointed out by Porter.’
Synaptic potentials set up in normal hypoglossal motoneurons by cortical stimulation
Synaptic potentials set up in axotomized hypoglossal motoneurons by cortical stimulation
Postsynaptic potentials (PSPs) produced in the tongue protruder motoneurons (P-Mns) by stimulation of the orbital gyrus were explored. The P-Mns were identified from the antidromic spike evoked by stimulation of the medial branch of the hypoglossal nerves. The PSPs produced in normal P-Mns on the right side by stimulation of the orbital gyrus on the left side are shown in Fig. 1. Satisfactory recordings were obtained from 46 P-Mns having an antidromic spike of 6&70mV. The record A, shows an antidromic spike of a P-Mn produced by stimulation of the medial branch of the hypoglossal nerves. In this P-Mn, a single shock delivered to the orbital gyrus evoked an IPSP with a latency of 2.5 ms (A,), and the responses to ten trials were averaged by computer, demonstrated in A,. The record A, shows the recordings of the field potentials after withdrawal of a micropipette to a just extracellular position. The relation between the amplitude of cortically induced hyperpolarizing potentials and the membrane potential displacements was examined and results are shown in A,. Numerals denote the amounts of injected current in nanoamperes (nA). At the resting level (0 nA) cortical stimulation evoked a hyperpolarizing potential. By displacing the mem-
The PSPs produced in axotomized P-Mns on the right side by stimulation of the orbital gyrus on the left side were explored. A stimulating electrode to the cortical surface in operated cats was fixed at the same point as demonstrated in unoperated cats (Fig. 1E). Cortically induced PSPs in P-Mns 30 days after axotomy are illustrated in Fig. 2. In 4 out of 14 explored P-Mns, stimulation of the orbital gyrus produced two components of EPSPs (a short- and a long-latency component) followed by an IPSP (B,). In normal P-Mns, there were no P-Mns responding with two components of EPSPs composed of a shortand a long-latency component to cortical stimulation. Therefore, the P-Mns responding with two components of EPSPs to cortical stimulation were grouped as the Type B. In P-Mns illustrated in AZ, C, and D, of Fig. 2, stimulation of the orbital gyrus evoked an EPSP (Type A), a short-latency EPSP followed by predominant IPSPs (Type C) and an IPSP (Type D), respectively. The relative frequencies of the four types of cortically induced PSPs recorded in 14 P-Mns 30 days after axotomy are shown in the histogram in Fig. 2(E). It is evident that the number of P-Mns responded with two components of EPSPs (filled, Type B) to stimulation of the orbital gyrus increased. The results illustrated in Fig. 3 were obtained from P-Mns 40 days after axotomy. As already reported, 19,*’in many P-Mns 40 days after axotomy a large late depolarization following an antidromic spike was observed and the number of spontaneously firing cells increased. A late depolarization following an antidromic spike evoked in a P-Mn 40 days after axotomy is shown in Fig. 3(A). In spontaneously firing cells, a late depolarization following a spike was also observed (Fig. 3B). In another series of experiments the properties of a late depolarization were examined. A late depolarization following an antidromic spike is shown in Fig. 3(C). In this cell a late depolarization following a spike was produced when an action potential was directly elicited by applying a brief depolarizing pulse through the recording micropipette (C,). On the 40th post-operative day, in 27 out of 68 explored P-Mns two components of EPSPs followed by IPSPs were produced by stimulation of the orbital gyrus (Fig. 3E). Cortically induced EPSPs in these
brane potential toward hyperpolarization, a cortically induced hyperpolarizing potential was decreased in amplitude (- 10 nA) and reversed to a depolarizing potential (- 20 nA). By displacement of the membrane potential to depolarization, this hyperpolarizing potential was increased in amplitude (10 nA). The results indicate that a cortically induced hyperpolarizing potential is an IPSP. In a P-Mn illustrated in Fig. l(B), cortical stimulation produced a mixture of excitatory and inhibitory synaptic potentials. In this group of P-Mns, cortical stimulation generated a short-latency EPSP followed by predominant IPSPs. In 1 out of 46 explored P-Mns, cortical stimulation produced only EPSP and stimuli with three shocks produced a spike on summated EPSPs (Fig. 1C). The PSPs produced in normal P-Mns on the right side by stimulation of the orbital gyrus on the left side are summarized in Fig. l(D). The relative frequency of the three types of cortically induced PSPs recorded in a sample of 46 normal P-Mns are illustrated in histogram form. The three types of cortically induced PSPs are an EPSP
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Fig. 1. Postsynaptic potentials produced in normal protruder motoneurons by stimulation of the orbital gyrus. (A) Cortically induced IPSP. (1) Antidromic spike. (2) Cortically induced IPSP. (3) Extracellular records corresponding to the record (A?). (4) Averaged responses to ten trials. (5) Membrane potential dependence of a cortically induced IPSP. (B) A short-latency EPSP followed by predominant IPSPs. (C) Cortically induced EPSP. (D) Histogram showing the number of P-Mns responded with an EPSP (Type A), a short-latency EPSP followed by predominant IPSPs (Type C) and an IPSP (Type D). (E) Stimulating site to the cortical surface (arrow).
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Fig. 2. Postsynaptic potentials produced in protruder motoneurons 30 days after axotomy by cortical stimulation. (A) Cortically induced EPSPs. (I) Antidromic spike. (2) Cortically induced EPSP. (B) Cortically induced EPSPs with two components. (I) Two components of EPSPs followed by an IPSP. (C), (D) A short-latency EPSP followed by predominant IPSPs and an IPSP. (A,), (9:). (C,), (D,) Extracellular records corresponding to the record (A?), (B,), (C,) and (D,). (E) Histogram showing the number of P-Mns responded with an EPSP (Type A), EPSPs with a short- and a long-latency component (Type B), a short-latency EPSP followed by predominant IPSPs (Type C) and an IPSP (Type D). axotomized P-Mns were also the composite of a short- and a long-latency component (E,). Responses recorded at a faster sweep speed and low amplification are shown in (E,). The record (EJ shows the field potentials. A P-Mn responded with an EPSP and a short-latency EPSP followed by predominant IPSPs to cortical stimulation are shown in Fig. 3 (D,) and (F,) respectively. In !7 out of 68 explored P-Mns cortical stimulation evoked an IPSP, illustrated in Fig. 3(G,). As shown in the histogram in Fig. 3(H), it is evident that cortical stimulation produced two components of EPSPs followed by IPSPs (filled, Type B) in about 40% of explored P-Mns 40 days after axotomy. In the next studies, the PSPs produced in P-Mns 60 and 80 days after axotomy by cortical stimulation were examined. On the 60th post-operative day, in 11
out of 21 explored P-Mns cortical stimulation produced only EPSPs and the relative frequencies of P-Mns responding with a short-latency EPSP followed by predominant IPSPs was decreased. The results illustrated in Fig. 4 were obtained from 43 P-Mns 80 days after axotomy by stimulation of the orbital gyrus. In the histogram of Fig. 4(E) it is evident that the major fraction (20 of 43 P-Mns, or about 46%) of the total sample of explored P-Mns showed two components of EPSPs followed by IPSPs (filled, Type B). As shown in the record (B,), cortical stimulation produced two components of EPSPs, however, generated PSPs were almost entirely excitatory. A P-Mn responding with only a shortlatency EPSP to cortical stimulation is shown in Fig. 4(A). In (A,), a single shock applied to the orbital gyrus produced only a short-latency EPSP and a
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Fig. 3. Postsynaptic potentials produced in protruder motoneurons 40 days after axotomy by cortical stimu\ation. (A) Late depolarization following an antidromic spike. (B) Late depolarization following a spontane~usj~ firing spike. (Cc)Late depola~zation. (I> tare dep~~~atjon following an antidromie spike. (2) Late depolarization following a spike elicited by injecting a brief depolarizing pulse into a cell. (D) Type A cell. (1) Cortically induced EPSP. (E) Type B cell. (I) Two components of EPSPs followed by an IPSP. (3) Recording of (E,) af a faster sweep speed and low amplification. (F) Type C cell. (I) A short-latency EPSP foliowed by predominant IPSPs, (G} Type D c& (1) Corticatiy induced IPSP. (Q), (E3, (F& (GJ Extracetlukar records corresponding to the record (D,), (E,), (F,) and (G,). (H) Histogram showing the number of P-Mm responded with an EPSP {Type A), EPSPs with a short- and a long-latency component (Type B), a short-latency EPSP followed by predominant IPSPs (Type C) and
latency was measured as 2.Oms. Record (A,) shows an antidtomic spike and (A,) the field potentials. The records (C,) and (ID,,) show a P-Mn responded with a short-latency EPSP foIlowed by IPSPs and an IPSP to cortical stimufation. The relation between the amplitude of cortically induced hyperpolarizing potentials and the membrane potential displacements was examined in a P-Mn 80 days after axotomy, and
the results are i~iustrat~ in (D,). Numerals denote the amounts of injected current in nanoamperes @A). By displacing the membrane potential toward hyperpolarization, a cortically induced hyperpo~arizing potential was reversed to a depolarizing potential (- 10 and -20 nA). By displacement of the membrane potential to depolarization, this potential was increased in size (10 and 20 nA).
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Fig. 4. Postsynaptic potentials produced in protruder motoneurons 80 days after axotomy by cortical stimulation. (A) Cortically induced EPSP. (1) Antidromic spike. (2) Cortically induced EPSP. (B) Cortically induced EPSPs. (1) Two components of EPSPs followed by a small IPSP. (C) Type C cell. (1) A spike (on a short-latency EPSP) followed by predominant IPSPs. (A,), (B,), (C,) Extracellular records corresponding to the record (A,), (B,) and (C,). (D) Type D cell. (a,) Cortically induced IPSP. (az) Extracellular records corresponding to the record (a,). (b) Membrane potential dependence of a cortically induced IPSP. (E) Histogram showing the number of P-Mns responded with an EPSP (Type A), EPSPs with a short- and a long-latency component (Type B), a short-latency EPSP followed by predominant IPSPs (Type C) and an IPSP (Type D).
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DISCUSSION
potentials in normal and axotomized P-Mns. However, such a comparison of PSP pattern demands that Anatomical investigations have settled the presence the membrane potential of the cells being compared of axonal projections from the cerebral cortex to the be within the same range. Therefore, the results are trigeminal sensory nucleus.7~*~22,24,25 Following lesions based on data obtained from normal and axotomized of the orbital and procreate gyri, Wold and Broda124 P-Mns having an antidromic spike of 6&70 mV. By found that the cortical fibers from both gyri termicomparing the peak amplitude of cortically induced nate mainly in the ventral most part of the nucleus IPSPs in normal P-Mns with that in P-Mns 80 days interpolaris. In addition, they have also demonstrated after axotomy, it was found that the average of the that the degenerating fibers from the primary sensopeak amplitude of the IPSP in normal P-Mns rimotor and the primary motor cortical areas were (n = 31) was about 1.5 times greater than that of mainly found contralaterally and appear to end in the P-Mns 80 days after axotomy (n = 7). With respect to rostra1 part of the trigeminal complex, in the nucleus lingually induced PSPS,‘*,~’ we have also demonprincipalis and the rostra1 part of the nucleus oralis. strated that in axotomized P-Mns the amplitude of In addition, there is no anatomical and physiological IPSPs was significantly reduced in size.“,19 From the evidence on monosynaptic projections from the cere- present findings, it is probably safe to assume that in bral cortex or the periphery to the hypoglossal mocortically induced PSPs of axotomized hypoglossal toneurons.7.9.‘6,22In the present experiments, we have motoneurons the effectiveness of inhibitory synapses demonstrated that the latency of cortically induced is diminished, while excitation is unaffected. The fact EPSP and IPSP was about 2.0ms (k 0.2ms) and that two components of EPSPs were produced in the 2.5 ms ( + 0.2 ms), suggesting that cortically induced major fraction of axotomized P-Mns is probably due EPSP and IPSP is probably disynaptic and trito the reduction of numbers of inhibitory synapses synaptic as the shortest pathway. from axotomized P-Mns. However, the changes of In axotomized spinal motoneurons, it was elucisynaptic contacts made by the corticobulbar fibers dated that monosynaptic EPSPs are significantly in the interneurons impinging on hypoglossal reduced in size,2 and inhibitory synapses located on motoneurons are still unknown. the cell body are also blocked, as are the somatic It has earlier been elucidated that hypoglossal excitatory synapses. 5,6 In axotomized hypoglossal motoneurons undergoing chromatolysis have a motoneurons, Sumner I2 showed that boutons with high safety factor for impulse transmission from the spherical vesicles decreased in number after axotomy, initial segment component to the soma-dendritic whereas those with flat vesicles did not, and suggested component and a low threshold for the generation that affinity is lost between excitatory boutons and of a spike in the soma-dendritic membrane,2’ inditheir postsynaptic sites after axotomy, while in- cating that the membrane properties of hypoglossal hibitions is unaffected. In the present experiments, motoneurons are changed after transection of the however, we have demonstrated that cortical stimu- hypoglossal nerves. As the most characteristic signs lation produced two components of EPSPs (a short- of axotomized motoneurons, the generation of the and a long-latency component) followed by IPSPs in late depolarization following an antidromic spike axotomized P-Mns (30, 40 and 46% of explored was reported. 4,2iIn the present experiments, we have P-Mns 30, 40 and 80 days after axotomy, re- demonstrated that when a spike was directly elicited spectively). In normal P-Mns it is evident that the by applying a brief depolarizing pulse through the major fraction of P-Mm studied showed the IPSPs recording micropipette, the late depolarization fol(about 67% of the total sample) and a short-latency lowing a spike was produced, indicating that the EPSP followed by predominant IPSPs (about 30% of generation of the late depolarization is not originated the total sample). In normal P-Mns the long-latency from afferent fibers running along the hypoglossal EPSP is probably depressed by the generation of a nerves. predominant IPSP. Acknowledgements-This work was supported in part by Even when dealing with polysynaptic PSPs, it is Grant-in-Aid for Scientific Research 59480365 from the Japan Ministry of Education, Science and Culture. possible to make qualitative estimates of synaptic
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nerve division of the afferent synapses of axotomized motor neurones. Expl Brain Res. 22, 421425. Eccles J. C., Libet B. and Young R. R. (1959) The behaviour of chromatolysed motoneurones studied by intracellular recording. J. Physiol., Land. 143, 11-40. Flumerfelt B. A. and Lewis P. P. (1975) Chohnesterase activity in the hypoglossal nucleus of the rat and the changes produced by axotomy: a light and electron microscopic study. J. Anat. 119, 309-331. Gustafsson B. (1979) Changes in motoneurone electrical properties following axotomy. J. Physiol., Lond. 293, 197-215. Kuno M. and Llinls R. (1970) Enhancement of synaptic transmission by dendritic potentials in chromatolysed motoneurones of the cat. J. Physiol., Lond. 210, 807-821. Kuno M. and Llinas R. (1970) Alterations of synaptic action in chromatolysed motoneurones of the cat. J. Physiol., Lond. 210, 823-838.
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analysis of corticobulbar connexions to the pons and lower brain stem 7. Kuypers H. G. J. M. (1958) An anatomical in the cat. J. Anat. 92, 198-218. cell groups. An experimental study 8. Kuypers H. G. J. M. (1960) Central cortical projections to motor and somatosensory in the rhesus monkey. Brain 83, 161-184. motoneurones in cats: a proposed role for a common internuncial 9. Porter R. (1967) Cortical actions on hypoglossal cell. J. Physiol., Lond. 193, 295-308. receptors 10. Rotter A., Birdsall N. J. M., Burgen A. S. V., Field P. M., Smolen A. and Raisman G. (1979) Muscarinic in the central nervous system of the rat-IV. A comparison of the effects of axotomy and deafferentation on the binding of [‘Hlpropylbenzilycholine mustard and associated synaptic changes in the hypoglossal and pontine nuclei. Brain Res. Rev. 1, 207-224. study of subsurface cisterns and their relationships in normal and axotomized 11. Sumner B. E. H. (1975) A quantitative hypoglossal neurones. Expl Bruin Res. 22, 175-183. analysis of the responses of presynaptic boutons to postsynaptic motor neuron 12. Sumner B. E. H. (1975) A quantitative axotomy. Expl Neurol. 46, 605-615. analysis of boutons with different types of synapses in normal and injured 13. Sumner B. E. H. (1975) A quantitative hypoglossal nuclei. Expl Neural. 49, 406-418. electron microscopy on the injured hypoglossal nucleus in F. I. (1973) Q uantitative 14. Sumner B. E. H. and Sutherland the rat. J. Neurocytol. 2, 315-328. and expansion of the dendritic tree of motor neurones of adult 15. Sumner B. E. H. and Watson W. E. (1971) Retraction rats induced in oivo. Nature, New Biol. 233, 213-275. J. (1948) Anatomical considerations of monosynaptic reflex arcs. J. Neurophysiol. 11, 445453. 16. Szentitgothai postsynaptic potentials in hypoglossal motoneurons after axotomy. 17. Takata M. (1981) Lingually induced inhibitory Brain Res. 224, 165-169. potentials evoked in hypoglossal motoneurons by lingual nerve stimulation. 18. Takata M. (1982) Inhibitory postsynaptic Expl Neural. 75, 103-l 11. M. and Nagahama T. (1983) Synaptic efficacy of inhibitory synapses in hypoglossal motoneurons after 19. Takata transection of the hypoglossal nerves. Neuroscience 10, 23-29. M. and Ogata K. (1980) Two components of inhibitory postsynaptic potentials evoked in hypoglossal 20. Takata motoneurons by lingual nerve stimulation. Expl Neurol. 69, 299-310. motoneurons undergoing chromatolysis. 21. Takata M., Shohara E. and Fujita S. (1980) The excitability of hypoglossal Neuroscience 5, 413419. 22. Walberg F. (1957) Do the motor nuclei of the cranial nerves receive corticofugal fibers? An experimental study in the cat. Bruin 80, 597-605. study of the incorporation of nucleic acid precursors by neurons and glia 23. Watson W. E. (1965) An autoradiographic during nerve regeneration. J. Physiol., Lond. 180, 740-753. regions onto the trigeminal nucleus in the 24. Wold J. E. and Brodal A. (1973) The projection of cortical sensorimotor cat. An experimental anatomical study. Neurobiology 3, 353-375. 25. Wold J. E. and Brodal A. (1974) The cortical projection of the orbital and procreate gyri to the sensory trigeminal nuclei in the cat. An experimental anatomical study. Bruin Res. 65, 381-395. of somatic sensory and motor areas of the cerebral cortex (eds Harlow H. F. and 26. Woolsey C. N. (1958) Organization Woolsey C. N.), pp. 63-81. The University of Wisconsin Press, Madison. (Accepted
I4 June 1984)
1984
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PergamonPressLtd IBRO
CORTICALLY INDUCED POSTSYNAPTIC IN HYPOGLOSSAL MOTONEURONS AXOTOMY
POTENTIALS AFTER
M. TAICATAand T. NAGAHAMA Department of Physiology, School of Dentistry, Tokushima University, Kuramoto-cho, Tokushima 770, Japan Abstract-Cortically induced postsynaptic potentials were studied in normal and axotomized cat hypoglossal motoneurons. In normal protruder motoneurons innervating tongue protruder muscles, we have demonstrated that stimulation of the orbital gyrus, at the point optimum for inducing lapping movements of the tongue by repetitive stimuli, produced inhibitory postsynaptic potentials or excitatory postsynaptic potentials followed by predominant inhibitory postsynaptic potentials. The cortically induced excitatory postsynaptic potential in normal protruder motoneurons was composed of ouly the short-latency component. In protruder motoneurons 30, 40, 60 and 80 days after axotomy, we have demonstrated that the number of protruder motoneurons responding with two components of excitatory postsynaptic potentials (the short- and the long-latency component) to cortical stimulation increased in correspondence with the lapse of days after axotomy and that the amplitude of cortically induced inhibitory postsynaptic potentials in axotomized protruder motoneurons was reduced in size as compared with normal protruder motoneurons.
Morphological studies have revealed that the majority of hypoglossal motoneurons exhibited chromatolysis at the end of the first week following axotomy3 and several quantitative studies have shown that by 1 week after axotomy there is a loss of about 50% of the synapses on the hypoglossal motoneurons.‘.‘s’5.23 In axotomized hypoglossal motoneurons, Sumner and SutherlandI and SumnerI have also observed a reduced number of dendritic synapses accompanied with detachment of synapses from the soma membrane. We have also demonstrated that the amplitude of the short- and the long-lasting inhibitory postsynaptic potentials (IPSPs) produced in axotomized hypoglossal motoneurons by stimulation of the lingual nerve was significantly reduced in size as compared with normal hypoglossal motoneurons.‘7.‘9 In the present experiments, we have attempted to demonstrate that excitatory postsynaptic potentials (EPSPs) produced in axotomized hypoglossal motoneurons by cortical stimulation are composed of the short- and the long-latency component. The purpose of the present paper is to investigate whether, in hypoglossal motoneurons, axotomy is followed by the decline of synaptic efficacy of inhibitory synapses for cortically induced IPSPs, while excitation is unaffected. EXPERIMENTALPROCEDURES Adult cats, either male or female and weighing 2.5-3.5 kg, were used. The hypoglossal nerve of the cat divides into the medial and the lateral branches. The fibers of the medial Abbreviahws:
EPSP, excitatory postsynaptic potential; IPSP, inhibitory postsynaptic potential; P-Mn, tongue protruder motoneuron.
WC13,3--M
branch innervate the tongue protruder muscles, and those of the lateral branch the tongue retractor muscles. Both the medial and the lateral branch of the right hypoglossal nerves were transected at the point of the posterior belly of the digastric muscle. The central cut end of the right hypoglossal nerves was ligated with silk thread to impede reinnervation. The left hypoglossal nerves were left intact. All surgical procedures were done under sodium pentobarbital anesthesia (Nembutal, 30mg/kg i.p.) with aseptic precautions. Animals were then allowed to survive for intervals of 30, 40, 60 and 80 days. In the terminal experiments, operated animals were anesthetized with sodium pentobarbital (Nembutal, 30 mg/kg i.p.) and a small caudal part of the cerebellum was sucked out in order to enable penetration by a micropipette into the hypoglossal nucleus. A pneumothorax was made, and cats were immobilized by i.v. injection of gallamine triethiodide and respiration was maintained artificially. A sleeve electrode was used to stimulate the cut central end of the previously severed medial branch of the hypoglossal nerves. In a control experiment, the medial branch of either side was isolated for stimulation. The left eye was enucleated and the orbitofrontal area of the cerebral cortex was exposed. The dura was opened and a spring-mounted silver ball electrode (diameter: 1 mm) was placed on the cortical surface and was fixed at the point optimum for inducing lapping movement of the tongue by repetitive stimuli at 50/s, the reference electrode being inserted in temporal muscles. In most cases, monopolar stimulation was done and a single positive pulse (0.2-0.5 ms, 2-10 V) was delivered, but sometimes, a bipolar silver ball electrode (interpolar distance; 2 mm) was placed and a train of three square pulses (500/s, 0.1 ms, below 2 mA) was delivered to the cortical site. The exposed surface of the cortex was covered by warm liquid paraffin and the temperature was kept at 38°C. Body temperature was maintained by means of an electric blanket thermostatically controlled from a rectal thermistor. In the present experiments, micropipettes filled with 2 M potassium citrate were used for recording and for current injection, since an increase of intracellular concentration of chloride ions was found to reverse IPSPs to a depolarizing potentialzO Electrode resistance was between 15and 25 Ma.
855
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Potentials were recorded with a direct-coupled amplifier. In order to examine the patterns of postsynaptic potentials produced in the hypoglossal motoneurons by cortical stimulation, the responses to ten trials were averaged. In some cases, depolarization of the membrane potential by penetration of a micropipette into a cell masked EPSPs. In such a case, by injecting a constant hyperpolarizing current into a cell, the membrane potential was kept at the resting membrane potential. RESULTS
(open, Type A), a short-latency EPSP followed by predominant IPSPs (hatched, Type C) and an IPSP (dotted, Type D), respectively. It is evident that the major fraction of the total sample of normal P-Mns studied showed the IPSPs and a short-latency EPSP followed by predominant IPSPs. A stimulation site to the cortical surface is demonstrated by a filled dot (arrow) in Fig. 1 (E). The point examined was in the “face area” of Woolsey’s MS I regionz6 as already pointed out by Porter.’
Synaptic potentials set up in normal hypoglossal motoneurons by cortical stimulation
Synaptic potentials set up in axotomized hypoglossal motoneurons by cortical stimulation
Postsynaptic potentials (PSPs) produced in the tongue protruder motoneurons (P-Mns) by stimulation of the orbital gyrus were explored. The P-Mns were identified from the antidromic spike evoked by stimulation of the medial branch of the hypoglossal nerves. The PSPs produced in normal P-Mns on the right side by stimulation of the orbital gyrus on the left side are shown in Fig. 1. Satisfactory recordings were obtained from 46 P-Mns having an antidromic spike of 6&70mV. The record A, shows an antidromic spike of a P-Mn produced by stimulation of the medial branch of the hypoglossal nerves. In this P-Mn, a single shock delivered to the orbital gyrus evoked an IPSP with a latency of 2.5 ms (A,), and the responses to ten trials were averaged by computer, demonstrated in A,. The record A, shows the recordings of the field potentials after withdrawal of a micropipette to a just extracellular position. The relation between the amplitude of cortically induced hyperpolarizing potentials and the membrane potential displacements was examined and results are shown in A,. Numerals denote the amounts of injected current in nanoamperes (nA). At the resting level (0 nA) cortical stimulation evoked a hyperpolarizing potential. By displacing the mem-
The PSPs produced in axotomized P-Mns on the right side by stimulation of the orbital gyrus on the left side were explored. A stimulating electrode to the cortical surface in operated cats was fixed at the same point as demonstrated in unoperated cats (Fig. 1E). Cortically induced PSPs in P-Mns 30 days after axotomy are illustrated in Fig. 2. In 4 out of 14 explored P-Mns, stimulation of the orbital gyrus produced two components of EPSPs (a short- and a long-latency component) followed by an IPSP (B,). In normal P-Mns, there were no P-Mns responding with two components of EPSPs composed of a shortand a long-latency component to cortical stimulation. Therefore, the P-Mns responding with two components of EPSPs to cortical stimulation were grouped as the Type B. In P-Mns illustrated in AZ, C, and D, of Fig. 2, stimulation of the orbital gyrus evoked an EPSP (Type A), a short-latency EPSP followed by predominant IPSPs (Type C) and an IPSP (Type D), respectively. The relative frequencies of the four types of cortically induced PSPs recorded in 14 P-Mns 30 days after axotomy are shown in the histogram in Fig. 2(E). It is evident that the number of P-Mns responded with two components of EPSPs (filled, Type B) to stimulation of the orbital gyrus increased. The results illustrated in Fig. 3 were obtained from P-Mns 40 days after axotomy. As already reported, 19,*’in many P-Mns 40 days after axotomy a large late depolarization following an antidromic spike was observed and the number of spontaneously firing cells increased. A late depolarization following an antidromic spike evoked in a P-Mn 40 days after axotomy is shown in Fig. 3(A). In spontaneously firing cells, a late depolarization following a spike was also observed (Fig. 3B). In another series of experiments the properties of a late depolarization were examined. A late depolarization following an antidromic spike is shown in Fig. 3(C). In this cell a late depolarization following a spike was produced when an action potential was directly elicited by applying a brief depolarizing pulse through the recording micropipette (C,). On the 40th post-operative day, in 27 out of 68 explored P-Mns two components of EPSPs followed by IPSPs were produced by stimulation of the orbital gyrus (Fig. 3E). Cortically induced EPSPs in these
brane potential toward hyperpolarization, a cortically induced hyperpolarizing potential was decreased in amplitude (- 10 nA) and reversed to a depolarizing potential (- 20 nA). By displacement of the membrane potential to depolarization, this hyperpolarizing potential was increased in amplitude (10 nA). The results indicate that a cortically induced hyperpolarizing potential is an IPSP. In a P-Mn illustrated in Fig. l(B), cortical stimulation produced a mixture of excitatory and inhibitory synaptic potentials. In this group of P-Mns, cortical stimulation generated a short-latency EPSP followed by predominant IPSPs. In 1 out of 46 explored P-Mns, cortical stimulation produced only EPSP and stimuli with three shocks produced a spike on summated EPSPs (Fig. 1C). The PSPs produced in normal P-Mns on the right side by stimulation of the orbital gyrus on the left side are summarized in Fig. l(D). The relative frequency of the three types of cortically induced PSPs recorded in a sample of 46 normal P-Mns are illustrated in histogram form. The three types of cortically induced PSPs are an EPSP
Postsynaptic potentials in axotomized motoneurons
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Fig. 1. Postsynaptic potentials produced in normal protruder motoneurons by stimulation of the orbital gyrus. (A) Cortically induced IPSP. (1) Antidromic spike. (2) Cortically induced IPSP. (3) Extracellular records corresponding to the record (A?). (4) Averaged responses to ten trials. (5) Membrane potential dependence of a cortically induced IPSP. (B) A short-latency EPSP followed by predominant IPSPs. (C) Cortically induced EPSP. (D) Histogram showing the number of P-Mns responded with an EPSP (Type A), a short-latency EPSP followed by predominant IPSPs (Type C) and an IPSP (Type D). (E) Stimulating site to the cortical surface (arrow).
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Fig. 2. Postsynaptic potentials produced in protruder motoneurons 30 days after axotomy by cortical stimulation. (A) Cortically induced EPSPs. (I) Antidromic spike. (2) Cortically induced EPSP. (B) Cortically induced EPSPs with two components. (I) Two components of EPSPs followed by an IPSP. (C), (D) A short-latency EPSP followed by predominant IPSPs and an IPSP. (A,), (9:). (C,), (D,) Extracellular records corresponding to the record (A?), (B,), (C,) and (D,). (E) Histogram showing the number of P-Mns responded with an EPSP (Type A), EPSPs with a short- and a long-latency component (Type B), a short-latency EPSP followed by predominant IPSPs (Type C) and an IPSP (Type D). axotomized P-Mns were also the composite of a short- and a long-latency component (E,). Responses recorded at a faster sweep speed and low amplification are shown in (E,). The record (EJ shows the field potentials. A P-Mn responded with an EPSP and a short-latency EPSP followed by predominant IPSPs to cortical stimulation are shown in Fig. 3 (D,) and (F,) respectively. In !7 out of 68 explored P-Mns cortical stimulation evoked an IPSP, illustrated in Fig. 3(G,). As shown in the histogram in Fig. 3(H), it is evident that cortical stimulation produced two components of EPSPs followed by IPSPs (filled, Type B) in about 40% of explored P-Mns 40 days after axotomy. In the next studies, the PSPs produced in P-Mns 60 and 80 days after axotomy by cortical stimulation were examined. On the 60th post-operative day, in 11
out of 21 explored P-Mns cortical stimulation produced only EPSPs and the relative frequencies of P-Mns responding with a short-latency EPSP followed by predominant IPSPs was decreased. The results illustrated in Fig. 4 were obtained from 43 P-Mns 80 days after axotomy by stimulation of the orbital gyrus. In the histogram of Fig. 4(E) it is evident that the major fraction (20 of 43 P-Mns, or about 46%) of the total sample of explored P-Mns showed two components of EPSPs followed by IPSPs (filled, Type B). As shown in the record (B,), cortical stimulation produced two components of EPSPs, however, generated PSPs were almost entirely excitatory. A P-Mn responding with only a shortlatency EPSP to cortical stimulation is shown in Fig. 4(A). In (A,), a single shock applied to the orbital gyrus produced only a short-latency EPSP and a
859
Postsynaptic potentials in axotomized motoneurons
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Fig. 3. Postsynaptic potentials produced in protruder motoneurons 40 days after axotomy by cortical stimu\ation. (A) Late depolarization following an antidromic spike. (B) Late depolarization following a spontane~usj~ firing spike. (Cc)Late depola~zation. (I> tare dep~~~atjon following an antidromie spike. (2) Late depolarization following a spike elicited by injecting a brief depolarizing pulse into a cell. (D) Type A cell. (1) Cortically induced EPSP. (E) Type B cell. (I) Two components of EPSPs followed by an IPSP. (3) Recording of (E,) af a faster sweep speed and low amplification. (F) Type C cell. (I) A short-latency EPSP foliowed by predominant IPSPs, (G} Type D c& (1) Corticatiy induced IPSP. (Q), (E3, (F& (GJ Extracetlukar records corresponding to the record (D,), (E,), (F,) and (G,). (H) Histogram showing the number of P-Mm responded with an EPSP {Type A), EPSPs with a short- and a long-latency component (Type B), a short-latency EPSP followed by predominant IPSPs (Type C) and
latency was measured as 2.Oms. Record (A,) shows an antidtomic spike and (A,) the field potentials. The records (C,) and (ID,,) show a P-Mn responded with a short-latency EPSP foIlowed by IPSPs and an IPSP to cortical stimufation. The relation between the amplitude of cortically induced hyperpolarizing potentials and the membrane potential displacements was examined in a P-Mn 80 days after axotomy, and
the results are i~iustrat~ in (D,). Numerals denote the amounts of injected current in nanoamperes @A). By displacing the membrane potential toward hyperpolarization, a cortically induced hyperpo~arizing potential was reversed to a depolarizing potential (- 10 and -20 nA). By displacement of the membrane potential to depolarization, this potential was increased in size (10 and 20 nA).
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Fig. 4. Postsynaptic potentials produced in protruder motoneurons 80 days after axotomy by cortical stimulation. (A) Cortically induced EPSP. (1) Antidromic spike. (2) Cortically induced EPSP. (B) Cortically induced EPSPs. (1) Two components of EPSPs followed by a small IPSP. (C) Type C cell. (1) A spike (on a short-latency EPSP) followed by predominant IPSPs. (A,), (B,), (C,) Extracellular records corresponding to the record (A,), (B,) and (C,). (D) Type D cell. (a,) Cortically induced IPSP. (az) Extracellular records corresponding to the record (a,). (b) Membrane potential dependence of a cortically induced IPSP. (E) Histogram showing the number of P-Mns responded with an EPSP (Type A), EPSPs with a short- and a long-latency component (Type B), a short-latency EPSP followed by predominant IPSPs (Type C) and an IPSP (Type D).
Postsynaptic
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861
DISCUSSION
potentials in normal and axotomized P-Mns. However, such a comparison of PSP pattern demands that Anatomical investigations have settled the presence the membrane potential of the cells being compared of axonal projections from the cerebral cortex to the be within the same range. Therefore, the results are trigeminal sensory nucleus.7~*~22,24,25 Following lesions based on data obtained from normal and axotomized of the orbital and procreate gyri, Wold and Broda124 P-Mns having an antidromic spike of 6&70 mV. By found that the cortical fibers from both gyri termicomparing the peak amplitude of cortically induced nate mainly in the ventral most part of the nucleus IPSPs in normal P-Mns with that in P-Mns 80 days interpolaris. In addition, they have also demonstrated after axotomy, it was found that the average of the that the degenerating fibers from the primary sensopeak amplitude of the IPSP in normal P-Mns rimotor and the primary motor cortical areas were (n = 31) was about 1.5 times greater than that of mainly found contralaterally and appear to end in the P-Mns 80 days after axotomy (n = 7). With respect to rostra1 part of the trigeminal complex, in the nucleus lingually induced PSPS,‘*,~’ we have also demonprincipalis and the rostra1 part of the nucleus oralis. strated that in axotomized P-Mns the amplitude of In addition, there is no anatomical and physiological IPSPs was significantly reduced in size.“,19 From the evidence on monosynaptic projections from the cere- present findings, it is probably safe to assume that in bral cortex or the periphery to the hypoglossal mocortically induced PSPs of axotomized hypoglossal toneurons.7.9.‘6,22In the present experiments, we have motoneurons the effectiveness of inhibitory synapses demonstrated that the latency of cortically induced is diminished, while excitation is unaffected. The fact EPSP and IPSP was about 2.0ms (k 0.2ms) and that two components of EPSPs were produced in the 2.5 ms ( + 0.2 ms), suggesting that cortically induced major fraction of axotomized P-Mns is probably due EPSP and IPSP is probably disynaptic and trito the reduction of numbers of inhibitory synapses synaptic as the shortest pathway. from axotomized P-Mns. However, the changes of In axotomized spinal motoneurons, it was elucisynaptic contacts made by the corticobulbar fibers dated that monosynaptic EPSPs are significantly in the interneurons impinging on hypoglossal reduced in size,2 and inhibitory synapses located on motoneurons are still unknown. the cell body are also blocked, as are the somatic It has earlier been elucidated that hypoglossal excitatory synapses. 5,6 In axotomized hypoglossal motoneurons undergoing chromatolysis have a motoneurons, Sumner I2 showed that boutons with high safety factor for impulse transmission from the spherical vesicles decreased in number after axotomy, initial segment component to the soma-dendritic whereas those with flat vesicles did not, and suggested component and a low threshold for the generation that affinity is lost between excitatory boutons and of a spike in the soma-dendritic membrane,2’ inditheir postsynaptic sites after axotomy, while in- cating that the membrane properties of hypoglossal hibitions is unaffected. In the present experiments, motoneurons are changed after transection of the however, we have demonstrated that cortical stimu- hypoglossal nerves. As the most characteristic signs lation produced two components of EPSPs (a short- of axotomized motoneurons, the generation of the and a long-latency component) followed by IPSPs in late depolarization following an antidromic spike axotomized P-Mns (30, 40 and 46% of explored was reported. 4,2iIn the present experiments, we have P-Mns 30, 40 and 80 days after axotomy, re- demonstrated that when a spike was directly elicited spectively). In normal P-Mns it is evident that the by applying a brief depolarizing pulse through the major fraction of P-Mm studied showed the IPSPs recording micropipette, the late depolarization fol(about 67% of the total sample) and a short-latency lowing a spike was produced, indicating that the EPSP followed by predominant IPSPs (about 30% of generation of the late depolarization is not originated the total sample). In normal P-Mns the long-latency from afferent fibers running along the hypoglossal EPSP is probably depressed by the generation of a nerves. predominant IPSP. Acknowledgements-This work was supported in part by Even when dealing with polysynaptic PSPs, it is Grant-in-Aid for Scientific Research 59480365 from the Japan Ministry of Education, Science and Culture. possible to make qualitative estimates of synaptic
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2. 3. 4.
5. 6.
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and T. Nagahama
analysis of corticobulbar connexions to the pons and lower brain stem 7. Kuypers H. G. J. M. (1958) An anatomical in the cat. J. Anat. 92, 198-218. cell groups. An experimental study 8. Kuypers H. G. J. M. (1960) Central cortical projections to motor and somatosensory in the rhesus monkey. Brain 83, 161-184. motoneurones in cats: a proposed role for a common internuncial 9. Porter R. (1967) Cortical actions on hypoglossal cell. J. Physiol., Lond. 193, 295-308. receptors 10. Rotter A., Birdsall N. J. M., Burgen A. S. V., Field P. M., Smolen A. and Raisman G. (1979) Muscarinic in the central nervous system of the rat-IV. A comparison of the effects of axotomy and deafferentation on the binding of [‘Hlpropylbenzilycholine mustard and associated synaptic changes in the hypoglossal and pontine nuclei. Brain Res. Rev. 1, 207-224. study of subsurface cisterns and their relationships in normal and axotomized 11. Sumner B. E. H. (1975) A quantitative hypoglossal neurones. Expl Bruin Res. 22, 175-183. analysis of the responses of presynaptic boutons to postsynaptic motor neuron 12. Sumner B. E. H. (1975) A quantitative axotomy. Expl Neurol. 46, 605-615. analysis of boutons with different types of synapses in normal and injured 13. Sumner B. E. H. (1975) A quantitative hypoglossal nuclei. Expl Neural. 49, 406-418. electron microscopy on the injured hypoglossal nucleus in F. I. (1973) Q uantitative 14. Sumner B. E. H. and Sutherland the rat. J. Neurocytol. 2, 315-328. and expansion of the dendritic tree of motor neurones of adult 15. Sumner B. E. H. and Watson W. E. (1971) Retraction rats induced in oivo. Nature, New Biol. 233, 213-275. J. (1948) Anatomical considerations of monosynaptic reflex arcs. J. Neurophysiol. 11, 445453. 16. Szentitgothai postsynaptic potentials in hypoglossal motoneurons after axotomy. 17. Takata M. (1981) Lingually induced inhibitory Brain Res. 224, 165-169. potentials evoked in hypoglossal motoneurons by lingual nerve stimulation. 18. Takata M. (1982) Inhibitory postsynaptic Expl Neural. 75, 103-l 11. M. and Nagahama T. (1983) Synaptic efficacy of inhibitory synapses in hypoglossal motoneurons after 19. Takata transection of the hypoglossal nerves. Neuroscience 10, 23-29. M. and Ogata K. (1980) Two components of inhibitory postsynaptic potentials evoked in hypoglossal 20. Takata motoneurons by lingual nerve stimulation. Expl Neurol. 69, 299-310. motoneurons undergoing chromatolysis. 21. Takata M., Shohara E. and Fujita S. (1980) The excitability of hypoglossal Neuroscience 5, 413419. 22. Walberg F. (1957) Do the motor nuclei of the cranial nerves receive corticofugal fibers? An experimental study in the cat. Bruin 80, 597-605. study of the incorporation of nucleic acid precursors by neurons and glia 23. Watson W. E. (1965) An autoradiographic during nerve regeneration. J. Physiol., Lond. 180, 740-753. regions onto the trigeminal nucleus in the 24. Wold J. E. and Brodal A. (1973) The projection of cortical sensorimotor cat. An experimental anatomical study. Neurobiology 3, 353-375. 25. Wold J. E. and Brodal A. (1974) The cortical projection of the orbital and procreate gyri to the sensory trigeminal nuclei in the cat. An experimental anatomical study. Bruin Res. 65, 381-395. of somatic sensory and motor areas of the cerebral cortex (eds Harlow H. F. and 26. Woolsey C. N. (1958) Organization Woolsey C. N.), pp. 63-81. The University of Wisconsin Press, Madison. (Accepted
I4 June 1984)