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Trigeminal afferent fibers in the trunk of the oculomotor nerve of lambs
EXPERIMENTAL
Trigeminal E.
MANNI, Istituto
Istituto
NEUROLOGY
Afferent R.
465476
50,
Fibers in the Trunk Nerve of Lambs V. E.
BORTOLAMI,
di Fisiologia
(1976)
di Anatomia
Normale
Univevsitd and Veterinaria,
Received
10, 1975;
rrvisio+z
July
Umana,
PETTOROSSI,
of the Oculomotor
AND
Cattolira
CALLEGARI
de1 S. Cuorc,
Universitci received
E.
October
1
Rome
di Bologna,
Italy
10, 1975
In lambs, chronic section of the oculomotor nerve at the base of the skull just proximally to the cavernous sinus induced degeneration of some fibers in the central stump, although the peripheral stump contained some normal fibers. On the other hand, chronic section of the ophthalmic branch just distally to the semilunar ganglion was followed by degeneration of a certain number of medium and large caliber fibers in the ipsilateral oculomotor nerve. The presence of trigeminal afferent fibers in the trunk of an oculomotor nerve is supported by electrophysiologic experiments. Single-shock electrical stimulation of the frontal and nasociliary nerves and of the conjunctiva of the superior and inferior eyelids elicited short-latency evoked potentials, not only in the semilunar ganglion but also in the ipsilateral oculomotor nerve at the base of the skull. Such responses did not appear in those animals in which the ipsilateral ophthalmic and maxillary branches of the trigeminal nerve had been chronically cut. Thus, we can affirm that afferent trigeminal impulses enter the brain stem also through the third nerve. The perikarya of such a pathway are localized in the semilunar ganglion; the peripheral processes attain the conjunctiva of the superior and inferior eyelids through the ophthalmic and maxillary branches, and the central processes enter the oculomotor nerve by anastomoses .between IIIrd and Vth cranial nerves after a short passage in the two trigeminal branches. The trigeminal nature of these fibers is also shown by the fact that electrical stimulation of the central stump of the IIIrd nerve can influence the dorsal neck muscles in the same wa:r as other trigeminal afferents.
INTRODUCTION The oculomotor, abducent, and trochlear nerves are commonly considered as pure motor nerves. However, some investigations have pointed IThis investigation was supported by a grant Miss B. Zannoni and Mr. R. Mancinelli for their work. 465 Copyright 1976 by Academic Press, Inc. All rights o? reproduction in any form reserved.
of CNR. kind help
We are with all
very grateful the experimental
to
466
MANN1
ET
AL.
out the presence also of sensitive fibers (1, 2, G, S, 10-12, 14, 22-25). In fact, after chronic severance of the trigeminal ophthalmic branches of all the mammals studied (8, l&12, 14)) large-diameter, degenerated fibers were found within the ipsilateral IIIrd, IVth, and VIth cranial nerves. In addition to these fibers, further sensitive fibers would have to be taken into consideration, in some species, because of the presence of sensitive ganglion cells located within the oculogyric nerves ( 11, 19-21, 24) ; these ganglion cells, however, are very few and inconstant, both in number and in localization. Only in the calf can they be constantly detected in a very high number (11, 21). Furthermore, it also has been established, after cutting the ophthalmic branch, that the degenerating fibers of the oculomotor nerve revert to the rootlets and enter the mesencephalon (10, 14). Their number seems to be higher in the peripheral stump of the IIIrd nerve than in the central stump. These findings are substantiated by the fact that, after chronic intracranial section of the oculogyric nerves, some degenerated fibers are recognizable in the central stump, whereas some normal fibers are present in the peripheral one (10, 11). However, with regard to the role of the ganglion cells in the IIIrd nerve, we found that after section of this nerve before it enters the cavernous sinus, only a few degenerated muscle spindles could be detected in two calves in which more than 100 ganglion cells were present in the central stump of this nerve (11). The present investigations represent an attempt to state the physiological role of the sensitive fibers in the oculomotor nerve of the lamb in which ganglion cells are very few and inconstant (21). The studies were prompted by the observation that electrical stimulation of the lamb’s central stump of the IIIrd cranial nerve at the base of the skull can influence units of the dorsal neck muscles, as in the case of trigeminal afferents ; in fact, it has been recently shown (3-5, 17) that various trigeminal stimulations can activate or inhibit the ipsilateral neck muscles. Thus, since the sensitive fibers present in the oculomotor nerve are of trigeminal origin, we have investigated the responses of the peripheral stump of the oculomotor nerve to various trigeminal stimulations. METHODS The present research was carried out on lambs weighing 7 to 12 kg. In the first group, five lambs under Nembutal anesthesia (33 mg/kg) and asepsis, underwent chronic section of the left IIIrd nerve just before it entered the cavernous sinus, as described in our previous experiments ( 10, 11) A second group of two lambs underwent chronic section of the left ophthalmic and maxillary branches just distal to the semilunar ganglion. All animals recovered very well, and after 7 to 14 days, they were sub-
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mitted under Nembutal anesthesia to the same experimental procedures as those which will be described below for the third group. At the end of the experiments the lambs were killed under deep anesthesia, and the IIIrd nerve, the ophthalmic branch, the semilunar ganglion of the left side, and the mesencephalon were removed and fixed in Bouin fluid, embedded in paraffin, sectioned, and stained according to the method of Glees-Marsland-Erikson modified by Winckler for degenerated fibers. A third group of acute experiments was carried out on 12 lambs; seven belonged to the first and second groups, and the others were normal lambs. The animals were anesthetized with Nembutal, tracheotomized, and put in a stereotaxic apparatus. The frontal and the infraorbital nerves on both sides were isolated and protected with warm mineral oil. Subsequently, a craniotomy was performed and most of the cerebral cortex was removed in order to see the base of the skull. The left and right semilunar ganglia were exposed according to the technique in our previous works (7-18). The two oculomotor nerves (only the right one in the lamb of the first group) were cut just before they entered the cavernous sinus. In some cases, the roof of the orbit was removed also and both nasociliary nerves were isolated. Only in a few cases were all the extraocular muscles isolated with their nerve supply, and the eyeballs ablated. Concentric electrodes were introduced in the neck muscles (senzispimzlis capitis, splenius, recks capitis, dorsalis wzaior) and connected with a beam of a 565 dual-beam Tektronix oscilloscope through a conventional preamplifier. Then the central stump of the oculomotor nerve was electrically stimulated by means of bipolar silver electrodes (the tips were 1 mm apart) with single shocks (l/set; 1 msec) or with repetitive stimuli (10-lOO/sec) delivered by a Tektronix 160-161-162 series and the effects on the unitary discharge of ipsi- and contralateral neck muscleswere tested. Because the meningeal stimulation could also influence the neck muscles, care was taken in stimulating the oculomotor nerve to avoid current spreading to the meninx covering the base of the skull: thus a thin sheet of an insulating material was interposed between the meninx and the trunk of the nerve. In the group I lambs, in which the IIIrd nerve had been chronically cut, not only the central stump but also the peripheral one was electrically stimulated at the base of the skull by the same procedure in order to control the effect on the pupil and on the extraocular muscles. Subsequently, the frontal, infraorbital, nasociliary nerves, and conjunctiva of the superior and inferior eyelids were separately stimulated with single shocks by means of bipolar silver electrodes; and responses were recorded at the base of the skull from the previously cut peripheral stump of the ipsilateral IIIrd nerve by means of a tungsten microelectrode (resistance 250 Ka) connected through conventional preamplifier with a 5480 Hewlett-Packard
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analyzer. Other records were taken from the semilunar ganglion or from the ophthalmic and maxillary branches of the trigeminal nerve in the vicinity of the ganglion in order to compare them with responses obtained from the IIIrd nerve. Then the animals were curarized (Tubarine Wellcome 2 mg/kg i.m.) and artificially ventilated ; the stimulations of the trigeminal branches were again tested under the same experimental conditions, after cutting the nasociliary nerve or the ophthalmic and maxillary branches of the Vth nerve. Finally in the lambs of group I which had undergone chronic section of an oculomotor nerve at the base of the skull, the most peripheral branches directed to extraocular muscles were electrically stimulated and the evoked responses were recorded either from the peripheral stump of the IIIrd nerve just proximal to the cavernous sinus or from the ophthalmic branch of the trigeminal nerve. RESULTS Chronic Section of Left Oculomotor Nerve at the Base of the Sk,&. After the operation, the lambs exhibited ipsilateral mydriasis, superior eyelid ptosis, and ophthalmoplegia. The histologic control (Fig. 1) showed the presence of some degenerated fibers of prevalently large diameter in the central stump (Fig. lA), and some normal fibers intermingled with the degenerated ones in the peripheral stump (Fig. lB), in accordance with our previous investigations (8, 10-12, 14). Chronic Section of Left Ophthalmic and Maxillary Branches of the Trigeminal Nerve. The operation was followed by disappearance of the cornea1 reflex and myosis of the ipsilateral pupil; the peripheral stump of the ophthalmic and maxillary branches exhibited a clear-cut degeneration of all their fibers. Degenerated fibers were traced along the central portion of the trunk of the ipsilateral oculomotor nerve and entering the mesencephalon (Fig. 1C). Electrical Stimulation of Central Stump of the IIIrd Cranial Nerve and Neck Muscles. Repetitive or single-shock (one case) electrical stimulation FIG. 1. (A) Lamb 287 killed 7 days after sectioning of left oculomotor nerve at the base of the skull. Longitudinal section of the central stump; arrows indicate degenerated fibers among the normal fibers. Magnification X384. (B) Lamb 289 kihed 10 days after cutting the left oculomotor nerve. Longitudinal section of the peripheral stump. Arrow points to a normal fiber intermingled with the degenerated ones. Magnification X384. (C) Lamb 290 killed 10 days after sectioning of left ophthalmic branch. Longitudinal section of the central portion of the ipsilateral oculomotor nerve trunk showing some degenerated fibers intermingled with the normal were stained according to the MarsIan+ fibers. Magnification X240. (A), (B), (C) Glees-Erikson method modified by Winckler.
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FIG. 2. In (A) (lamb 294) and (C) (lamb 293) activation, and in (B) (lamb 294) inhibition, of unitary discharge of the right neck muscles (upper trace) elicited by electrical stimulation (lower trace) of the central stump of the ipsilateral oculomotor nerve [S V, 1 msec, lOO/sec in (A), (IS), and (D) ; 10 V, 1 msec, 32/set in (C)l. In (A) and (B) the ipsilateral trigeminal root had been previously severed, and in (C) it was intact. In (D) (lamb 293), stimulation of the left oculomotor nerve did not influence the ipsilateral neck muscles since the left ophthalmic and maxillary branches had been chronically cut. Calibration : 1 sec.
of the central stump of the acutely severed oculomotor nerve provoked firing of the ipsilateral neck muscle units and inhibition of the contralateral units, in the Iamb under h’embutal (Fig. ZA, C). In some instances, Contraction of the ipsilateral neck muscles was clearly visible. Only
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occasionally, ipsilateral inhibition occurred (Fig. 2B). The latency of the nucal response was lo-12 msec. Since meningeal stimulation could affect neck muscles in a similar manner, control experiments were carried out in two animals in which the ipsilateral trigeminal root was acutely severed : Under the same experimental conditions, the electrical stimulation of the central stump of the IIIrd nerve influenced the extensor muscles of the neck (Fig. 2A, B). It must be pointed out that no response of the neck muscles occurred after stimulation of the central stump of the oculomotor nerve when that nerve or the ipsilateral ophthalmic and maxillary branches (Fig. 2D) had been chronically cut. Evoked PotentiaZs in Trunk of the Oculollzotor Nerve Elicited by Stimulation of Trigeminal Nerve Branches. Single-shock electrical stimulation of the frontal, infraorbital, and nasociliary nerves and of the conjunctiva of the eyelids elicited evoked potentials not only in the ipsilateral ophthalmic and maxillary branches but also in the most aboral portion of the peripheral stump of the ipsilateral oculomotor nerve acutely or chronically sectioned, both in animals under Nembutal and in the curarized animal. In the lamb under Nembutal, the evoked potentials recorded from the ipsilateral oculomotor nerve exhibited an early positive wave (Fig. 3A, C, E) with a latency in the range of 1 msec followed by larger waves with latencies ranging from 6 to 15 msec. Curarization suppressed the late waves which must be attributed to reflex contraction of m. orbicularis oculi. It must be noted that the responses recorded from the peripheral stump of the IIIrd nerve to stimulation of the various trigeminal nerves were different in amplitude and frequency from those which could be recorded from the ipsilateral ophthalmic branch or semilunar ganglion. By reversing the experimental arrangement, electrical stimulation of the IIIrd nerve (peripheral stump just before entering the cavernous sinus) evoked antidromic responses in the medial portion of the ipsilateral semilunar ganglion and in the nasociliary nerve: Even under such experimental conditions, it was possible to note a difference between the semilunar ganglion and the nasociliary nerve recordings (Fig. 4A, B) . In the two chronic animals, in which the ophthalmic and maxillary branches were cut, no response of the oculomotor nerve to electrical stimulation of the frontal, infraorbital, and nasociliary nerves occurred. In the acute experiments, the responses of the oculomotor nerve to stimulation of the ipsilateral frontal nerve also disappeared after sectioning of the nasociliary nerve in the orbit. However, acute sectioning of the ophthalmic and maxillary branches at the base of the skull in some cases completely suppressed the responses, whereas in others they were only decreased; in fact the responses persisted with some modifications probably because of the incompleteness of the sectioning (Fig. 3B, D, F> .
472
MANN1
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FIG. 3. Average of 64 responses recorded in lambs under Nembutal and curare from peripheral stump of the right oculomotor nerve just proximally to the cavernous sinus elicited by electrical stimulation of ipsilateral nasociliary nerve (A) and (B) : lamb 291; (C) and (D) : lamb 294; 1.5 V, 0.1 msec, lO/sec; and of the infraorbital nerve (E), (F) ; lamb 290; 1 V, 0.1 msec, lO/sec; (A), (C), (E) : controls; (B) : after acute sectioning of peripheral ophthalmic branch. No response. (D) : The anastomoses between the oculomotor and the trigeminal nerves in the orbit were completely severed and the two nerves were brought into contact: Only the stimulus artifact is present. (F) : After acute sectioning of the ipsilateral maxillary branch: decrease in amplitude of response. Calibration: 1 msec, 100 pV.
Stimulation Extraocular
of the Orbital
Bt-anches
of the Oculomotor
Nerve
to the
In two lambs which underwent chronic sectioning of the oculomotor nerve at the base of the skull (group I), the branches of the peripheral stump innervating the medial and superior rectus muscles were carefully isolated in the orbit. Electrical stimulation of those branches did not elicit contraction of the above-mentioned muscles, showing that complete degeneration of the motor fibers had occurred. However, under Mzrsclcs.
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FIG. 4. Average of 64 single antidromic responses recorded from the medial portion of the right semilunar ganglion (A) and from the right nasociliary nerve (B) elicited by electrical stimulation (1 mA, 0.05 msec, IO/see) of peripheral stump of the ipsilateral oculomotor nerve just proximally to the cavernous sinus in the curarized lamb 294. Calibration: 1 msec, 100 ,uV = 1 cm.
these experimental conditions, evoked potentials were recorded from the ipsilateral ophthalmic branch although not from the ipsilateral oculomotor trunk at the base of the skull just proximally to the cavernous sinus: this finding is in accordance with the assumption that the afferents from the eye muscle proprioceptors run only in the peripheral branches of the IIIrd nerve and then enter the ophthalmic branch (7-12). DISCUSSION The results of this investigation confirm the presence of trigeminal afferent fibers in the oculomotor nerve (1, 2, 6, 8, 10-12, 14, 22-25) and establish some physiological aspects of these fibers according to the following data : (i) single-shock e1ectrical stimulation of the frontal, nasociliary, and infraorbital nerves and of the conjunctiva of the superior and inferior eyelids induced evoked potentials in the IIIrd nerve at the base of the skull; these responsesexhibited a morphology different from those recorded under the same experimental conditions from the ipsilateral ophthalmic branch or the semilunar ganglion ; and (ii) evoked potentials recorded from the peripheral stump of the IIIrd nerve by stimulation of various trigeminal afferents disappeared after chronic section of the ipsilateral ophthalmic and maxillary branches. The trigeminal course of the afferent fibers contained in the oculomotor nerve is supported also by the possibility of influencing the dorsal neck muscles by electrical stimulation of the oculomotor nerve in the normal animal. Various patterns of activation and inhibition of unitary discharge of neck muscleswere obtained by stimulating the central stump of the IIIrd nerve: the patterns were very similar to those elicited in our previous investigations (17) through stimulation of the frontal and infraorbital nerves, of the ophthalmic branch of the trigeminal nerve, and of the
474
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FIG, 5. Schematic drawing with the probable arrangement of trigeminal fibers contained in the oculomotor nerve. III : oculomotor nerve; 0 : trigeminal ophthalmic branch; Max: trigeminal maxillary branch; S : semilunar ganglion.
proprioceptive afferents from the extraocular muscles. The effect of stimulation of the oculomotor nerve central stump on the neck muscles was removed when the lambs had undergone ipsilateral chronic sectioning of the same nerve or of the ophthalmic and maxillary branches. On the other hand, it is to be noted that stimulation of the central stump of the oculomotor nerve was effective on the neck muscles also after acute sectioning of the ipsilateral trigeminal root. This fact shows that the influence on neck muscles could not be attributed to a possible spread of current to the meninx which is innervated by the trigeminal nerve. Thus, we can maintain that impulses along the trigeminal system, in particular from the conjunctiva of the eyelids, abandon the Vth nerve and enter the brain stem through the oculomotor nerve. Since in our histological examinations, no ganglion cells were detected along the IIIrd nerve, an endogenous component of sensitive fibers in the oculomotor nerve can be excluded in our material. Thus, Fig. 5 can be proposed as an attempt to depict the course of trigeminal sensitive fibers present in the IIIrd nerve. The perikarya are located within the semilunar ganglion, in fact, singleshock electrical stimulation of the semilunar ganglion can induce evoked potentials in the oculomotor nerve (10). The peripheral processes reach the conjunctiva of the superior eyelid through the nasociliary and the frontal nerves, whereas they attain the inferior eyelid through the col-
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laterals of the infraorbital nerve. The central processes run in the ophthalmic and maxillary branches, and successively anastomose with the IIIrd nerve at the level of the oral portion of the cavernous sinus, thus reaching the mesencephalon through this nerve. Two equally valuable hypotheses can be formulated with regard to the termination of these trigeminal fibers : (i) they could end in the rhombomesencephalic reticular substance, or (ii) they could run directly to the cervical spinal cord and here make synaptic contacts with motoneurons which supply the extensor muscles of the neck. Further investigations will clarify such a problem. Furthermore, we can exclude a synaptic arch with the oculomotor nerve cells because Bortolami et al. (1, 2) have not found degenerated boutons after cutting the oculomotor nerve. In conclusion, we can establish that the sensitive fibers contained in the oculomotor nerve represent a consistent trigeminal subsidiary pathway which can contribute to the innervation of the eyelid conjunctiva. REFERENCES R., M. L. LUCCHI, E. MARTINI, G. PALMIERI, and E. CALLEGARI. 1975. The acquisition of sensory ganglion cells by the third of ox as an aspect of a wider phenomenon of neuronal migration. 1. Contp. Nezlrol., in press. 2. BORTOLAMI, R., A. VEGGETTI, E. CALLEGARI, M. L. LUCCHI, and G. PALMIERI. 1975. The oculomotor as a mixed nerve: II. On the presence of trigeminal fibers and ganglion cells within the oculomotor nerve in some mammals and man. J. Camp. Neural., in press. 3. EAS-KIN, T. A. 1971a. Inhibition from cat eye muscle stretch. Brain Res. 25: 633-637. 4. EASTON, T. A. 1971b. Patterned inhibition from horizontal eye movement in the cat. Exp. Neural 31: 419-430. 5. EASTON, T. A. 1972. Patterned inhibition from single eye muscle stretch in the cat. Exp. Neural. 34: 497-510. 6. HOGC, I. D. 1964. Observations on the development of the peripheral portion of the oculomotor nerve in man and the albino rat. J. Camp. Neural. 122: 81-112. 7. MANNI, E., R. BORTOLAMI, and P. L. DERIU. 1970. Presence of cells bodies of the afferents from the eye muscle in the semilunar ganglion. Arch. Ital. Biol. 108: 106-120. 8. MANNI, E., R. BORTOLAMI, and P. L. DERIU. 1970. Superior oblique muscle proprioception and the trochlear nerve. Exe. Neural. 26: 543-550. 9. MANNI, E., R. BORTOLAMI, and C. DESOLE. 1966. Eye muscle proprioception and the semilunar ganglion. E.x#J. Nezhrol. 18: 226-236. 10. MANNI, E., R. BORTOLAMI, and C. DESOLE. 1968. Peripheral pathway of the eye muscle proprioception. Exp. Neural. 22: 1-12. 11. MANNI, E., C. DESOLE, and G. PALMIERI. 1970. On whether eye muscle spindles are innervated by ganglion cells located along the oculomotor nerves. Exp. Neural. 28 : 333-343. 12. MANNI, E., G. PALMIERI, and R. MARINI. 1971. Peripheral pathway of the proprioceptive afferents from the lateral rectus muscle of the eye. E.zp. Newel. 30: 46-53. 1. BORTOLAMI,
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13. MANNI, E., G. PALMIERI, and R. MARINI. 1971. Extraocular muscle proprioception and the descending trigeminal nucleus. Exp. Neurol. 33 : 195-204. 14. MANNI, E., G. PALMIERI, and R. MARINI. 1972. Pontine trigeminal terminations of proprioceptive afferents from the eye muscles. Exp. Neural. 36: 210-318. 15. MANNI, E., G. PALMIERI, and R. MARINI. 1972. Mesodiencephalic representation of the eye muscle proprioception. Exp. Neuuol. 37 : 412-421. 16. MANNI, E., G. PALMIERI, and R. MARINI. 1974. Central pathway of the extraocular muscle proprioception. Exp. Nezlrol. 42 : 181-190. 17. MANNI, E., G. PALMIERI, R. MARINI, and V. E. PETTOROSSI. 1975. Trigeminal influences on extensor muscles of the neck. E.zp. NEUYO~., 47 : 330-342. 18. MANNI, E., G. PALMIERI, R. MARINI, and V. E. PETTOROSSI. 1975. New observations on the representation of the eye muscle proprioception in the descending trigeminal nucleus. Expeyimtia, 31 : 944-945. 19. NICHOLLS, G. E. 1915. An occurrence of an intracranial ganglion upon the oculomotor nerve in Sc$liz~ti cartic~lu, with a suggestion as to its bearing upon the question of the segmental value of certain of the cranial nerves. PYOC. RoJ~. Sot. London Ser. B 88: 553-568. 20. NICHOLSON, H. 1924. On the presence of ganglion cells in the third, fourth and sixth nerves of man. J. Camp. Neztyol. 37: 31-36. 21. PALMIERI, G., and A. ASOLE. 1967. Sulla presenza di cellule gangliari nei nervi oculomotori di alcuni mammiferi ed uccelli. Kiv. Biol. 60: 117-137. 22. TAREN, J. A. 1964. An anatomic demonstration of afferent fibers in the IV, V and VI cranial nerves of the AJaacara mzrlatta. Amer. J. Ophthal. 58: 408-412. 23. TORRE, M., and F. R. GRATTAROLA. 1950. Sulla presenza di fihre in degenerazione walleriana nel moncone prossimale dei nervi oculomotori per sezione degli stessi vicino al sistema nervoso centrale. Boll. Sot. it. Biol. spcv. 26: 51-52. 24. TOZER, F. M. 1912. On the presence of ganglion cells in the roots of III, IV and VI cranial nerves. J. Pltj~siol (Lo)ldon) 45: XV-XVI. 25. TOZER, F. M., and C. S. SHERRINGTON. 1910. Receptors and afferents of the third, fourth and sixth cranial nerves. Pyoc. Rag. SOL. Londort Ser. B 82: 450-457.
Trigeminal E.
MANNI, Istituto
Istituto
NEUROLOGY
Afferent R.
465476
50,
Fibers in the Trunk Nerve of Lambs V. E.
BORTOLAMI,
di Fisiologia
(1976)
di Anatomia
Normale
Univevsitd and Veterinaria,
Received
10, 1975;
rrvisio+z
July
Umana,
PETTOROSSI,
of the Oculomotor
AND
Cattolira
CALLEGARI
de1 S. Cuorc,
Universitci received
E.
October
1
Rome
di Bologna,
Italy
10, 1975
In lambs, chronic section of the oculomotor nerve at the base of the skull just proximally to the cavernous sinus induced degeneration of some fibers in the central stump, although the peripheral stump contained some normal fibers. On the other hand, chronic section of the ophthalmic branch just distally to the semilunar ganglion was followed by degeneration of a certain number of medium and large caliber fibers in the ipsilateral oculomotor nerve. The presence of trigeminal afferent fibers in the trunk of an oculomotor nerve is supported by electrophysiologic experiments. Single-shock electrical stimulation of the frontal and nasociliary nerves and of the conjunctiva of the superior and inferior eyelids elicited short-latency evoked potentials, not only in the semilunar ganglion but also in the ipsilateral oculomotor nerve at the base of the skull. Such responses did not appear in those animals in which the ipsilateral ophthalmic and maxillary branches of the trigeminal nerve had been chronically cut. Thus, we can affirm that afferent trigeminal impulses enter the brain stem also through the third nerve. The perikarya of such a pathway are localized in the semilunar ganglion; the peripheral processes attain the conjunctiva of the superior and inferior eyelids through the ophthalmic and maxillary branches, and the central processes enter the oculomotor nerve by anastomoses .between IIIrd and Vth cranial nerves after a short passage in the two trigeminal branches. The trigeminal nature of these fibers is also shown by the fact that electrical stimulation of the central stump of the IIIrd nerve can influence the dorsal neck muscles in the same wa:r as other trigeminal afferents.
INTRODUCTION The oculomotor, abducent, and trochlear nerves are commonly considered as pure motor nerves. However, some investigations have pointed IThis investigation was supported by a grant Miss B. Zannoni and Mr. R. Mancinelli for their work. 465 Copyright 1976 by Academic Press, Inc. All rights o? reproduction in any form reserved.
of CNR. kind help
We are with all
very grateful the experimental
to
466
MANN1
ET
AL.
out the presence also of sensitive fibers (1, 2, G, S, 10-12, 14, 22-25). In fact, after chronic severance of the trigeminal ophthalmic branches of all the mammals studied (8, l&12, 14)) large-diameter, degenerated fibers were found within the ipsilateral IIIrd, IVth, and VIth cranial nerves. In addition to these fibers, further sensitive fibers would have to be taken into consideration, in some species, because of the presence of sensitive ganglion cells located within the oculogyric nerves ( 11, 19-21, 24) ; these ganglion cells, however, are very few and inconstant, both in number and in localization. Only in the calf can they be constantly detected in a very high number (11, 21). Furthermore, it also has been established, after cutting the ophthalmic branch, that the degenerating fibers of the oculomotor nerve revert to the rootlets and enter the mesencephalon (10, 14). Their number seems to be higher in the peripheral stump of the IIIrd nerve than in the central stump. These findings are substantiated by the fact that, after chronic intracranial section of the oculogyric nerves, some degenerated fibers are recognizable in the central stump, whereas some normal fibers are present in the peripheral one (10, 11). However, with regard to the role of the ganglion cells in the IIIrd nerve, we found that after section of this nerve before it enters the cavernous sinus, only a few degenerated muscle spindles could be detected in two calves in which more than 100 ganglion cells were present in the central stump of this nerve (11). The present investigations represent an attempt to state the physiological role of the sensitive fibers in the oculomotor nerve of the lamb in which ganglion cells are very few and inconstant (21). The studies were prompted by the observation that electrical stimulation of the lamb’s central stump of the IIIrd cranial nerve at the base of the skull can influence units of the dorsal neck muscles, as in the case of trigeminal afferents ; in fact, it has been recently shown (3-5, 17) that various trigeminal stimulations can activate or inhibit the ipsilateral neck muscles. Thus, since the sensitive fibers present in the oculomotor nerve are of trigeminal origin, we have investigated the responses of the peripheral stump of the oculomotor nerve to various trigeminal stimulations. METHODS The present research was carried out on lambs weighing 7 to 12 kg. In the first group, five lambs under Nembutal anesthesia (33 mg/kg) and asepsis, underwent chronic section of the left IIIrd nerve just before it entered the cavernous sinus, as described in our previous experiments ( 10, 11) A second group of two lambs underwent chronic section of the left ophthalmic and maxillary branches just distal to the semilunar ganglion. All animals recovered very well, and after 7 to 14 days, they were sub-
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mitted under Nembutal anesthesia to the same experimental procedures as those which will be described below for the third group. At the end of the experiments the lambs were killed under deep anesthesia, and the IIIrd nerve, the ophthalmic branch, the semilunar ganglion of the left side, and the mesencephalon were removed and fixed in Bouin fluid, embedded in paraffin, sectioned, and stained according to the method of Glees-Marsland-Erikson modified by Winckler for degenerated fibers. A third group of acute experiments was carried out on 12 lambs; seven belonged to the first and second groups, and the others were normal lambs. The animals were anesthetized with Nembutal, tracheotomized, and put in a stereotaxic apparatus. The frontal and the infraorbital nerves on both sides were isolated and protected with warm mineral oil. Subsequently, a craniotomy was performed and most of the cerebral cortex was removed in order to see the base of the skull. The left and right semilunar ganglia were exposed according to the technique in our previous works (7-18). The two oculomotor nerves (only the right one in the lamb of the first group) were cut just before they entered the cavernous sinus. In some cases, the roof of the orbit was removed also and both nasociliary nerves were isolated. Only in a few cases were all the extraocular muscles isolated with their nerve supply, and the eyeballs ablated. Concentric electrodes were introduced in the neck muscles (senzispimzlis capitis, splenius, recks capitis, dorsalis wzaior) and connected with a beam of a 565 dual-beam Tektronix oscilloscope through a conventional preamplifier. Then the central stump of the oculomotor nerve was electrically stimulated by means of bipolar silver electrodes (the tips were 1 mm apart) with single shocks (l/set; 1 msec) or with repetitive stimuli (10-lOO/sec) delivered by a Tektronix 160-161-162 series and the effects on the unitary discharge of ipsi- and contralateral neck muscleswere tested. Because the meningeal stimulation could also influence the neck muscles, care was taken in stimulating the oculomotor nerve to avoid current spreading to the meninx covering the base of the skull: thus a thin sheet of an insulating material was interposed between the meninx and the trunk of the nerve. In the group I lambs, in which the IIIrd nerve had been chronically cut, not only the central stump but also the peripheral one was electrically stimulated at the base of the skull by the same procedure in order to control the effect on the pupil and on the extraocular muscles. Subsequently, the frontal, infraorbital, nasociliary nerves, and conjunctiva of the superior and inferior eyelids were separately stimulated with single shocks by means of bipolar silver electrodes; and responses were recorded at the base of the skull from the previously cut peripheral stump of the ipsilateral IIIrd nerve by means of a tungsten microelectrode (resistance 250 Ka) connected through conventional preamplifier with a 5480 Hewlett-Packard
468
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analyzer. Other records were taken from the semilunar ganglion or from the ophthalmic and maxillary branches of the trigeminal nerve in the vicinity of the ganglion in order to compare them with responses obtained from the IIIrd nerve. Then the animals were curarized (Tubarine Wellcome 2 mg/kg i.m.) and artificially ventilated ; the stimulations of the trigeminal branches were again tested under the same experimental conditions, after cutting the nasociliary nerve or the ophthalmic and maxillary branches of the Vth nerve. Finally in the lambs of group I which had undergone chronic section of an oculomotor nerve at the base of the skull, the most peripheral branches directed to extraocular muscles were electrically stimulated and the evoked responses were recorded either from the peripheral stump of the IIIrd nerve just proximal to the cavernous sinus or from the ophthalmic branch of the trigeminal nerve. RESULTS Chronic Section of Left Oculomotor Nerve at the Base of the Sk,&. After the operation, the lambs exhibited ipsilateral mydriasis, superior eyelid ptosis, and ophthalmoplegia. The histologic control (Fig. 1) showed the presence of some degenerated fibers of prevalently large diameter in the central stump (Fig. lA), and some normal fibers intermingled with the degenerated ones in the peripheral stump (Fig. lB), in accordance with our previous investigations (8, 10-12, 14). Chronic Section of Left Ophthalmic and Maxillary Branches of the Trigeminal Nerve. The operation was followed by disappearance of the cornea1 reflex and myosis of the ipsilateral pupil; the peripheral stump of the ophthalmic and maxillary branches exhibited a clear-cut degeneration of all their fibers. Degenerated fibers were traced along the central portion of the trunk of the ipsilateral oculomotor nerve and entering the mesencephalon (Fig. 1C). Electrical Stimulation of Central Stump of the IIIrd Cranial Nerve and Neck Muscles. Repetitive or single-shock (one case) electrical stimulation FIG. 1. (A) Lamb 287 killed 7 days after sectioning of left oculomotor nerve at the base of the skull. Longitudinal section of the central stump; arrows indicate degenerated fibers among the normal fibers. Magnification X384. (B) Lamb 289 kihed 10 days after cutting the left oculomotor nerve. Longitudinal section of the peripheral stump. Arrow points to a normal fiber intermingled with the degenerated ones. Magnification X384. (C) Lamb 290 killed 10 days after sectioning of left ophthalmic branch. Longitudinal section of the central portion of the ipsilateral oculomotor nerve trunk showing some degenerated fibers intermingled with the normal were stained according to the MarsIan+ fibers. Magnification X240. (A), (B), (C) Glees-Erikson method modified by Winckler.
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FIG. 2. In (A) (lamb 294) and (C) (lamb 293) activation, and in (B) (lamb 294) inhibition, of unitary discharge of the right neck muscles (upper trace) elicited by electrical stimulation (lower trace) of the central stump of the ipsilateral oculomotor nerve [S V, 1 msec, lOO/sec in (A), (IS), and (D) ; 10 V, 1 msec, 32/set in (C)l. In (A) and (B) the ipsilateral trigeminal root had been previously severed, and in (C) it was intact. In (D) (lamb 293), stimulation of the left oculomotor nerve did not influence the ipsilateral neck muscles since the left ophthalmic and maxillary branches had been chronically cut. Calibration : 1 sec.
of the central stump of the acutely severed oculomotor nerve provoked firing of the ipsilateral neck muscle units and inhibition of the contralateral units, in the Iamb under h’embutal (Fig. ZA, C). In some instances, Contraction of the ipsilateral neck muscles was clearly visible. Only
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occasionally, ipsilateral inhibition occurred (Fig. 2B). The latency of the nucal response was lo-12 msec. Since meningeal stimulation could affect neck muscles in a similar manner, control experiments were carried out in two animals in which the ipsilateral trigeminal root was acutely severed : Under the same experimental conditions, the electrical stimulation of the central stump of the IIIrd nerve influenced the extensor muscles of the neck (Fig. 2A, B). It must be pointed out that no response of the neck muscles occurred after stimulation of the central stump of the oculomotor nerve when that nerve or the ipsilateral ophthalmic and maxillary branches (Fig. 2D) had been chronically cut. Evoked PotentiaZs in Trunk of the Oculollzotor Nerve Elicited by Stimulation of Trigeminal Nerve Branches. Single-shock electrical stimulation of the frontal, infraorbital, and nasociliary nerves and of the conjunctiva of the eyelids elicited evoked potentials not only in the ipsilateral ophthalmic and maxillary branches but also in the most aboral portion of the peripheral stump of the ipsilateral oculomotor nerve acutely or chronically sectioned, both in animals under Nembutal and in the curarized animal. In the lamb under Nembutal, the evoked potentials recorded from the ipsilateral oculomotor nerve exhibited an early positive wave (Fig. 3A, C, E) with a latency in the range of 1 msec followed by larger waves with latencies ranging from 6 to 15 msec. Curarization suppressed the late waves which must be attributed to reflex contraction of m. orbicularis oculi. It must be noted that the responses recorded from the peripheral stump of the IIIrd nerve to stimulation of the various trigeminal nerves were different in amplitude and frequency from those which could be recorded from the ipsilateral ophthalmic branch or semilunar ganglion. By reversing the experimental arrangement, electrical stimulation of the IIIrd nerve (peripheral stump just before entering the cavernous sinus) evoked antidromic responses in the medial portion of the ipsilateral semilunar ganglion and in the nasociliary nerve: Even under such experimental conditions, it was possible to note a difference between the semilunar ganglion and the nasociliary nerve recordings (Fig. 4A, B) . In the two chronic animals, in which the ophthalmic and maxillary branches were cut, no response of the oculomotor nerve to electrical stimulation of the frontal, infraorbital, and nasociliary nerves occurred. In the acute experiments, the responses of the oculomotor nerve to stimulation of the ipsilateral frontal nerve also disappeared after sectioning of the nasociliary nerve in the orbit. However, acute sectioning of the ophthalmic and maxillary branches at the base of the skull in some cases completely suppressed the responses, whereas in others they were only decreased; in fact the responses persisted with some modifications probably because of the incompleteness of the sectioning (Fig. 3B, D, F> .
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FIG. 3. Average of 64 responses recorded in lambs under Nembutal and curare from peripheral stump of the right oculomotor nerve just proximally to the cavernous sinus elicited by electrical stimulation of ipsilateral nasociliary nerve (A) and (B) : lamb 291; (C) and (D) : lamb 294; 1.5 V, 0.1 msec, lO/sec; and of the infraorbital nerve (E), (F) ; lamb 290; 1 V, 0.1 msec, lO/sec; (A), (C), (E) : controls; (B) : after acute sectioning of peripheral ophthalmic branch. No response. (D) : The anastomoses between the oculomotor and the trigeminal nerves in the orbit were completely severed and the two nerves were brought into contact: Only the stimulus artifact is present. (F) : After acute sectioning of the ipsilateral maxillary branch: decrease in amplitude of response. Calibration: 1 msec, 100 pV.
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In two lambs which underwent chronic sectioning of the oculomotor nerve at the base of the skull (group I), the branches of the peripheral stump innervating the medial and superior rectus muscles were carefully isolated in the orbit. Electrical stimulation of those branches did not elicit contraction of the above-mentioned muscles, showing that complete degeneration of the motor fibers had occurred. However, under Mzrsclcs.
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FIG. 4. Average of 64 single antidromic responses recorded from the medial portion of the right semilunar ganglion (A) and from the right nasociliary nerve (B) elicited by electrical stimulation (1 mA, 0.05 msec, IO/see) of peripheral stump of the ipsilateral oculomotor nerve just proximally to the cavernous sinus in the curarized lamb 294. Calibration: 1 msec, 100 ,uV = 1 cm.
these experimental conditions, evoked potentials were recorded from the ipsilateral ophthalmic branch although not from the ipsilateral oculomotor trunk at the base of the skull just proximally to the cavernous sinus: this finding is in accordance with the assumption that the afferents from the eye muscle proprioceptors run only in the peripheral branches of the IIIrd nerve and then enter the ophthalmic branch (7-12). DISCUSSION The results of this investigation confirm the presence of trigeminal afferent fibers in the oculomotor nerve (1, 2, 6, 8, 10-12, 14, 22-25) and establish some physiological aspects of these fibers according to the following data : (i) single-shock e1ectrical stimulation of the frontal, nasociliary, and infraorbital nerves and of the conjunctiva of the superior and inferior eyelids induced evoked potentials in the IIIrd nerve at the base of the skull; these responsesexhibited a morphology different from those recorded under the same experimental conditions from the ipsilateral ophthalmic branch or the semilunar ganglion ; and (ii) evoked potentials recorded from the peripheral stump of the IIIrd nerve by stimulation of various trigeminal afferents disappeared after chronic section of the ipsilateral ophthalmic and maxillary branches. The trigeminal course of the afferent fibers contained in the oculomotor nerve is supported also by the possibility of influencing the dorsal neck muscles by electrical stimulation of the oculomotor nerve in the normal animal. Various patterns of activation and inhibition of unitary discharge of neck muscleswere obtained by stimulating the central stump of the IIIrd nerve: the patterns were very similar to those elicited in our previous investigations (17) through stimulation of the frontal and infraorbital nerves, of the ophthalmic branch of the trigeminal nerve, and of the
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FIG, 5. Schematic drawing with the probable arrangement of trigeminal fibers contained in the oculomotor nerve. III : oculomotor nerve; 0 : trigeminal ophthalmic branch; Max: trigeminal maxillary branch; S : semilunar ganglion.
proprioceptive afferents from the extraocular muscles. The effect of stimulation of the oculomotor nerve central stump on the neck muscles was removed when the lambs had undergone ipsilateral chronic sectioning of the same nerve or of the ophthalmic and maxillary branches. On the other hand, it is to be noted that stimulation of the central stump of the oculomotor nerve was effective on the neck muscles also after acute sectioning of the ipsilateral trigeminal root. This fact shows that the influence on neck muscles could not be attributed to a possible spread of current to the meninx which is innervated by the trigeminal nerve. Thus, we can maintain that impulses along the trigeminal system, in particular from the conjunctiva of the eyelids, abandon the Vth nerve and enter the brain stem through the oculomotor nerve. Since in our histological examinations, no ganglion cells were detected along the IIIrd nerve, an endogenous component of sensitive fibers in the oculomotor nerve can be excluded in our material. Thus, Fig. 5 can be proposed as an attempt to depict the course of trigeminal sensitive fibers present in the IIIrd nerve. The perikarya are located within the semilunar ganglion, in fact, singleshock electrical stimulation of the semilunar ganglion can induce evoked potentials in the oculomotor nerve (10). The peripheral processes reach the conjunctiva of the superior eyelid through the nasociliary and the frontal nerves, whereas they attain the inferior eyelid through the col-
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laterals of the infraorbital nerve. The central processes run in the ophthalmic and maxillary branches, and successively anastomose with the IIIrd nerve at the level of the oral portion of the cavernous sinus, thus reaching the mesencephalon through this nerve. Two equally valuable hypotheses can be formulated with regard to the termination of these trigeminal fibers : (i) they could end in the rhombomesencephalic reticular substance, or (ii) they could run directly to the cervical spinal cord and here make synaptic contacts with motoneurons which supply the extensor muscles of the neck. Further investigations will clarify such a problem. Furthermore, we can exclude a synaptic arch with the oculomotor nerve cells because Bortolami et al. (1, 2) have not found degenerated boutons after cutting the oculomotor nerve. In conclusion, we can establish that the sensitive fibers contained in the oculomotor nerve represent a consistent trigeminal subsidiary pathway which can contribute to the innervation of the eyelid conjunctiva. REFERENCES R., M. L. LUCCHI, E. MARTINI, G. PALMIERI, and E. CALLEGARI. 1975. The acquisition of sensory ganglion cells by the third of ox as an aspect of a wider phenomenon of neuronal migration. 1. Contp. Nezlrol., in press. 2. BORTOLAMI, R., A. VEGGETTI, E. CALLEGARI, M. L. LUCCHI, and G. PALMIERI. 1975. The oculomotor as a mixed nerve: II. On the presence of trigeminal fibers and ganglion cells within the oculomotor nerve in some mammals and man. J. Camp. Neural., in press. 3. EAS-KIN, T. A. 1971a. Inhibition from cat eye muscle stretch. Brain Res. 25: 633-637. 4. EASTON, T. A. 1971b. Patterned inhibition from horizontal eye movement in the cat. Exp. Neural 31: 419-430. 5. EASTON, T. A. 1972. Patterned inhibition from single eye muscle stretch in the cat. Exp. Neural. 34: 497-510. 6. HOGC, I. D. 1964. Observations on the development of the peripheral portion of the oculomotor nerve in man and the albino rat. J. Camp. Neural. 122: 81-112. 7. MANNI, E., R. BORTOLAMI, and P. L. DERIU. 1970. Presence of cells bodies of the afferents from the eye muscle in the semilunar ganglion. Arch. Ital. Biol. 108: 106-120. 8. MANNI, E., R. BORTOLAMI, and P. L. DERIU. 1970. Superior oblique muscle proprioception and the trochlear nerve. Exe. Neural. 26: 543-550. 9. MANNI, E., R. BORTOLAMI, and C. DESOLE. 1966. Eye muscle proprioception and the semilunar ganglion. E.x#J. Nezhrol. 18: 226-236. 10. MANNI, E., R. BORTOLAMI, and C. DESOLE. 1968. Peripheral pathway of the eye muscle proprioception. Exp. Neural. 22: 1-12. 11. MANNI, E., C. DESOLE, and G. PALMIERI. 1970. On whether eye muscle spindles are innervated by ganglion cells located along the oculomotor nerves. Exp. Neural. 28 : 333-343. 12. MANNI, E., G. PALMIERI, and R. MARINI. 1971. Peripheral pathway of the proprioceptive afferents from the lateral rectus muscle of the eye. E.zp. Newel. 30: 46-53. 1. BORTOLAMI,
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13. MANNI, E., G. PALMIERI, and R. MARINI. 1971. Extraocular muscle proprioception and the descending trigeminal nucleus. Exp. Neurol. 33 : 195-204. 14. MANNI, E., G. PALMIERI, and R. MARINI. 1972. Pontine trigeminal terminations of proprioceptive afferents from the eye muscles. Exp. Neural. 36: 210-318. 15. MANNI, E., G. PALMIERI, and R. MARINI. 1972. Mesodiencephalic representation of the eye muscle proprioception. Exp. Neuuol. 37 : 412-421. 16. MANNI, E., G. PALMIERI, and R. MARINI. 1974. Central pathway of the extraocular muscle proprioception. Exp. Nezlrol. 42 : 181-190. 17. MANNI, E., G. PALMIERI, R. MARINI, and V. E. PETTOROSSI. 1975. Trigeminal influences on extensor muscles of the neck. E.zp. NEUYO~., 47 : 330-342. 18. MANNI, E., G. PALMIERI, R. MARINI, and V. E. PETTOROSSI. 1975. New observations on the representation of the eye muscle proprioception in the descending trigeminal nucleus. Expeyimtia, 31 : 944-945. 19. NICHOLLS, G. E. 1915. An occurrence of an intracranial ganglion upon the oculomotor nerve in Sc$liz~ti cartic~lu, with a suggestion as to its bearing upon the question of the segmental value of certain of the cranial nerves. PYOC. RoJ~. Sot. London Ser. B 88: 553-568. 20. NICHOLSON, H. 1924. On the presence of ganglion cells in the third, fourth and sixth nerves of man. J. Camp. Neztyol. 37: 31-36. 21. PALMIERI, G., and A. ASOLE. 1967. Sulla presenza di cellule gangliari nei nervi oculomotori di alcuni mammiferi ed uccelli. Kiv. Biol. 60: 117-137. 22. TAREN, J. A. 1964. An anatomic demonstration of afferent fibers in the IV, V and VI cranial nerves of the AJaacara mzrlatta. Amer. J. Ophthal. 58: 408-412. 23. TORRE, M., and F. R. GRATTAROLA. 1950. Sulla presenza di fihre in degenerazione walleriana nel moncone prossimale dei nervi oculomotori per sezione degli stessi vicino al sistema nervoso centrale. Boll. Sot. it. Biol. spcv. 26: 51-52. 24. TOZER, F. M. 1912. On the presence of ganglion cells in the roots of III, IV and VI cranial nerves. J. Pltj~siol (Lo)ldon) 45: XV-XVI. 25. TOZER, F. M., and C. S. SHERRINGTON. 1910. Receptors and afferents of the third, fourth and sixth cranial nerves. Pyoc. Rag. SOL. Londort Ser. B 82: 450-457.