378
Brain Research, 123 (1977) 378-383 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Synaptic activation of facial afferents on the facial neurons of the cat
T. TANAKA Department o]Physiology, School of Medicine, Mie University, Tsu (Japan)
(Accepted December 3rd, 1976)
There have been anatomical and electrophysiological studies indicative of the existence of afferent fibers in the facial nerve. Foley and DuBois 7 showed that 11-15 of the facial nerve of the cat are sensory fibers of small diameter. It was also suggested 1 that such small caliber fibers may be traced to free terminals in the adventitia of blood vessels over the facial area. On the other hand, recent electrophysiological experiments 13 proved the existence of facial afferent fibers which have both excitatory and inhibitory effects on the cat's blink reflex. Furthermore, the afferent fibers were found to project on the facial neurons via the spinal trigeminal nucleus 9. Although the functional nature of the sensory fibers in the facial nerve is not completely agreed upon among investigators, it may be related to pressure and pain ~,4, and proprioceptive sense T M over the face. The aim of the present study was to investigate synaptic actions of the facial afferents on the facial neurons of the cat. Adult cats, weighing 2.0-4.0 kg, were anesthetized with pentobarbital sodium (30 mg/kg, intraperitoneal administration). After surgical procedures, muscular movements were eliminated by intravenous injection of gallamine triethodide and the animal was artificially respired. Methods for recording responses from the facial neurons were the same as those described in a previous paper 17. For stimulation of the peripheral facial nerve, the posterior auricular (PA), the temporal-zygomatico-orbital (TZ) and both the superior and inferior buccolabial (BL) branches were isolated and cut at the point of their entrance into the mimetic muscles. They were stimulated through A g AgC1 electrodes with current pulses of 0.05-0.1 msec in duration. The facial nerve trunk contralateral to the recording side of the facial nucleus was also stimulated at the proximal portion of the cut end just distal to the stylomastoid foramen. In some experiments, the dorsal surface of the medulla oblongata and spinal cord from the obex to C3 segment was exposed by laminectomy. Activities of neurons in or near the spinal trigeminal nucleus and solitary tract nucleus were recorded. To confirm that the recordings were from these nuclei, the infraorbital branch of the ipsilateral trigeminal nerve and cervical vagus nerve were dissected respectively, and the cut end of each nerve was stimulated to induce synaptically activated responses. Microelectrodes for recording were filled with 3 M KC1 and had a DC resistance of 5-15 Mr2. At the end of each experiment, the recording microelectrodes were inserted to the responsive
379
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Fig. 1. A-E: responses to stimulation of the ipsilateral facial nerve branches recorded intracellulady from one (A-C) and another (D, E) facial neurons. Antidromic spike potential and EPSPs were produced in a facial neuron in response to BL stimulation (A). EPSPs evoked by PA stimulation (B). An increase in amplitude of EPSPs due to strong stimuli led to a firing of spike potential (C). Antidromic spike potential followed by EPSPs was produced in another facial neuron by PA stimulation (D). EPSPs evoked by BL stimulation (E). F: afterhyperpolarization recorded from a PA neuron. Stimulus intensity was at threshold so that antidromic activation failed in about half of the trials. G: EPSPs produced in a PA neuron by contralateral facial nerve stimulation. H and I: IPSPs produced in two different BL neurons by PA stimulation. A noticeable small depolarization is indicated by a downward arrow in I. Note that the sweep speeds and amplifications ate different between H and I. Lower traces in each record show extracellular control responses. Stimulus artifacts are on the left of each record. Voltage calibration of 5 mV applies to A-H, 2.5 mV to I. Time scale of 50 msec applies only to H.
regions o f the m e d u l l a o b l o n g a t a a n d left b u r i e d to m a r k a t r a c k t h r o u g h the spinal trigeminal nucleus o r solitary t r a c t nucleus. The histological e x a m i n a t i o n was m a d e with frozen sections stained with 0.1 ~o methylene blue solution after fixation. Fig. 1 illustrates p o t e n t i a l s synaptically activated in the facial n e u r o n s b y stimulation o f the facial nerve branches. In Fig. 1A, s t i m u l a t i o n o f the B L b r a n c h o f the ipsilateral facial nerve elicited a n a n t i d r o m i c spike p o t e n t i a l at a short latency o f 0.6 msec in the facial neuron. The spike was succeeded by an irregular d e p o l a r i z a t i o n which b e g a n at a b o u t 8 msec after the stimulus with a slow rising phase. The irregular d e p o l a r i z a t i o n a p p e a r e d to be synaptically induced. W h e n a stimulus was a p p l i e d to a n o t h e r b r a n c h o f the facial nerve, e.g. the P A branch, it p r o d u c e d EPSPs with a latency o f 7.5 msec in the n e u r o n (Fig. 1B). W i t h an increase in the stimulus intensity, the EPSPs increased in a m p l i t u d e a n d elicited a c t i o n potentials (Fig. 1C). This finding strongly suggests the convergence o f facilitatory actions o f afferent fibers in each b r a n c h o f the facial nerve to a facial neuron. Fig. 1D shows EPSPs p r e c e d e d b y a n a n t i d r o m i c spike p o t e n t i a l in a n o t h e r facial n e u r o n in response to s t i m u l a t i o n o f the P A branch. A s seen in Fig. 1E, the latency o f the EPSPs i n d u c e d in this P A n e u r o n by BL s t i m u l a t i o n was 4.6 msec a n d the d u r a t i o n was a p p r o x i m a t e l y 35 msec. I n 52 facial neurons, the latent times o f the EPSPs were m e a s u r e d on each b r a n c h s t i m u l a t i o n o f the ipsilateral facial nerve. The latencies r a n g e d f r o m 3.8 to 8.0 msec, the m e a n value being 5.6 msec. T h e y are evidently indicative o f a p o l y s y n a p t i c n e u r o n a l connection. I n o r d e r to assess the degree o f i n t e r a c t i o n between m o t o n e u r o n s , the effect o f a n t i d r o m i c impulses on n e i g h b o r i n g m o t o n e u r o n s was e x a m i n e d in the facial nucleus.
380 I n the spinal m o t o r nuclei, such interaction can occur over recurrent axon collateral pathway 5. Fig. 1F shows a sample of a n antidromically activated spike with afterhyperpolarization. PA stimulation straddled the threshold for the axon of the impaled n e u r o n , there being failure of spike initiation in a b o u t half of the trials. Such a stimulus intensity was well below the threshold for producing synaptic potentials evoked by single volleys in the afferent c o m p o n e n t . A synaptic potential superimposed on the afterhyperpolarization was n o t revealed in any of the facial n e u r o n s tested, which indicates a lack of recurrent synaptic action. This is supported by the histological findings of R a m 6 n y Caja115, a n d Falls a n d K i n g 6. Stimulation of the contralateral facial nerve was also capable of producing polysynaptic EPSPs in these facial neurons, although stronger stimuli were required to elicit EPSPs. This is illustrated in Fig. 1G, where the EPSPs began at 8.5 msec after contralateral facial nerve t r u n k stimulation a n d could initiate action potentials. The iatencies, ranging from 6.3 to 10.0 msec (N = 11, m e a n 7.9 msec), were slightly longer t h a n those of the EPSPs evoked by ipsilateral facial nerve stimulation. I n the minority of the facial n e u r o n s (N - - 7), stimulation of the ipsilateral facial nerve b r a n c h produced hyperpolarizing potentials at latencies of 7-10 msec a n d they lasted for 30-60 msec (Fig. 1H a n d I). By electrophoretic injection of chloride ions into the neurons, the hyperpolarization changed to a depolarization, which provided evidence that this response was due to IPSP. Some of these hyperpolarizing responses were preceded by depolarizing potentials at latencies of 4-7 msec (a d o w n w a r d arrow in Fig. 1I).
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Fig. 2. Responses of facial neurons in three different experiments (A-C, D-F, G-K). A : field potential evoked by ipsilateral PA stimulation in the facial nucleus. B and C: field potential (B) and intracellular recording (C) on PA stimulation after intracranial section of the facial nerve in the same preparation as A. D : field potential induced by PA stimulation after intracranial section of the ipsilateral trigeminal nerve. E: no field potentials could be elicited by stimulation of the infraorbital branch of the ipsilateral trigeminal nerve. F: intracellular responses induced by PA stimulation in a PA neuron in the same preparation as D and E. G and H : field potentials elicited by stimulation of PA (G) or BL (H) in the facial nucleus after intracranial section of the vagus nerve. In G, a succeeding slow negative response was abolished. I-K: with intact vagus nerve, PA stimulation elicited antidromic potential followed by synaptically activated action potentials (I). Section of the vagus nerve eliminated synaptic responses on PA stimulation in a PA neuron (J), while the responses remained unchanged on BL stimulation in a BL neuron after section of the vagus nerve (K). Lower traces of C, F, I-K, extracellular control. Voltage calibration of 2.5 mV applies to the records of field potentials. Upward deflection in field potentials indicates negativity of microelectrode.
381 Results from several experiments which aimed at identifying the peripheral afferent pathway of the facial reflex are illustrated in Fig. 2. Fig. 2A shows a sample of the field potential recorded in the medial portion of the facial nucleus on PA stimulation. The responses consisted of an initial synchronized negative deflection followed by a succeeding prolonged negative wave. The initial response was caused by the antidromic activation of PA neurons, and the second wave could be attributable to depolarization synaptically induced by afferent volleys in the PA branch. In the medial portion of the facial nucleus, the largest antidromic field potentials were produced by PA stimulation (see ref. 11). After these responses had been recorded, the facial nerve was sectioned intracranially at the exit from the brain stem while the recording microelectrode was in the facial nucleus. Then, it was found that PA stimulation could still evoke the second negative wave, even though it failed to produce any antidromic response (Fig. 2B). EPSPs and spike potentials superimposed on the EPSPs could be recorded from neurons in or near the facial nucleus by stimulation of the PA branch in such preparations (Fig. 2C). The latency of the EPSPs was about 5 msec and the duration of the potentials corresponded to the second response of the field potential. These synaptic responses were very similar to those obtained in the animals with intact facial nerve, and they are definitely indicative of polysynaptic EPSPs produced in the facial neurons. Since the intermediate nerve reaches the brain stem along with the facial nerve, the neurons in the geniculate ganglion can be assumed to play a minor role in mediating the afferent inputs involved. In the experiment in which the ipsilateral trigeminal nerve was sectioned intracranially after decerebration, the responses which could be attributed to the action of afferent fibers in the facial nerve remained unchanged, as illustrated in Fig. 2D and F. In 6 other experiments, attempts were made to record extracellular and intracellular responses from facial neurons in animals in which the vagus nerve had been cut intracranially at its entrance into the brain stem; i.e. proximal to the nodosa ganglion. Sample records of field potentials from the facial nucleus are included in Fig. 2G and H. PA stimulation evoked only an initial antidromic potential without eliciting any succeeding reflex response (Fig. 2G), whereas stimulation of the BL branch still resulted in a production of the reflex response preceded by an antidromic one (Fig. 2H). These results were also confirmed by intracellular recordings from individual facial neurons. With the vagus nerve intact, an antidromic action potential was followed by one or two spikes superimposed on EPSPs on PA stimulation (Fig. 21). With the vagus nerve sectioned, PA stimulation elicited no synaptic potentials which could be attributed to the action of afferent volleys, as illustrated in Fig. 2J, where the intensity of stimulus was increased to 5-6 times threshold for the antidromic response. In contrast, severance of the vagus nerve showed no significant effect on the configuration of synaptic potentials produced by BL volleys in a BL neuron, coinciding with the results of the field potential analysis (Fig. 2K, compare with Fig. 2H). It may be anatomically considered that the PA branch of the facial nerve at the level of the stylomastoid foramen is accompanied by the auricular branch of the vagus nerve (Arnold's nerve) which comes to join the PA within the facial canal in the cat 8. It can be assumed that the afferent fibers of the PA branch, carrying volleys from the auricular
382
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Fig. 3. A and B: responses of a single unit evoked by stimulation of the ipsilateral cervical vagus nerve (A) and PA (B) in the vicinity of the solitary tract nucleus. C-E: evoked responses of a unit to stimulation of the infraorbital branch of the ipsilateral trigeminal nerve (C), PA (D) and BL (E) in or near the spinal trigeminal nucleus. Voltage calibration of 2.5 mV applies to A and B, and that of 5 mV to C-E. Negativity upwards. and occipital portions of the face, join the vagus nerve and reach the brain stem via the jugular foramen. Recently, Kubota et al. 12 demonstrated that the facial nerve of the talpoides contains the proprioceptive afferents from the facial muscles, and that the primary neurons of these afferents exist in the glossopharyngeal-vagus ganglion. Although we have at present no available evidence with regard to an afferent pathway of the BL or TZ branch of the facial nerve in cats, it appears reasonable to conclude that sensory fibers of the facial nerve are distributed widely in the facial area. A further analysis was made to ascertain the destination of facial afferents in the brain stem. Fig. 3A and B illustrate the results obtained by extracellular recording from a unit in the vicinity of the solitary tract nucleus on stimulation of the cervical vagus nerve and the PA branch of the facial nerve. The solitary tract neurons were identified by synaptic invasion of afferent volleys of the vagus nerve 14. On ipsilateral vagus nerve stimulation unitary responses at a latency of around 2.5 msec were recorded at depths of about 1.0-2.2 mm from the dorsal surface of the medulla oblongata at the level of the obex (Fig. 3A). On the other hand, PA stimulation evoked no response in this unit (Fig. 3B). For the sample of units studied here, when the microelectrode was placed in the vicinity of the solitary tract nucleus, unitary responses could not be recorded on PA stimulation. Since it has been suggested that some afferent fibers of both the vagus nerve3, x0 and facial nerveg, 1° project to the spinal trigeminal nucleus, interneurons mediating the pathway from facial afferents are presumed to be located in the region of the spinal trigeminal nucleus. This assumption was tested in several experiments. A single stimulus applied to the infraorbital branch of the trigeminal nerve produced usually a repetitive discharge in units in or near the spinal trigeminal nucleus at the latency of 1.1 msec, which indicated a monosynaptic activation of the units (Fig. 3C). The unit shown in Fig. 3C responded to PA or BL stimulation with repetitive discharges that appeared to begin 2.5-4.5 msec after the stimulus (Fig. 3D and E). In the majority of units in this region, the initial spike had a latency shorter than that of EPSPs produced in the facial neurons. The intensity of stimulus required to elicit discharge of these units was of the same order as that necessary to
383 produce EPSPs in the facial neurons. Therefore, n e u r o n s i n the spinal trigeminal nucleus may be i n t e r n u n c i a l in the reflex pathway. The results presented here indicate that the facial nerve branches c o n t a i n afferent fibers distal to the level of the stylomastoid foramen, a n d afferents in the posterior auricular b r a n c h are considered to be a p o r t i o n of the vagus nerve. The observations strongly suggest that these afferent i n p u t s to the facial m o t o n e u r o n s are relayed by n e u r o n s in or near the spinal trigeminal nucleus. The a u t h o r is i n d e b t e d to Prof. K. Sasaki for kindly reading this m a n u s c r i p t a n d p r o v i d i n g comments. He is also obliged to Dr. Y. Takeuchi for his assistance in histology.
1 Bruesch, S. R., The distribution of myelinated afferent fibers in the branches of the cat's facial nerve, J. comp. NeuroL, 81 (1944) 169-191. 2 Carmichael, E. A. and Woollard, H. H., Some observations on the fifth and seventh cranial nerves, Brain, 56 (1933) 109-125. 3 Cottle, M. K., Degeneration studies of primary afferents of IXth and Xth cranial nerves in the cat, J. comp. Neurol., 122 (1964) 329-345. 4 Davis, L. E., The deep sensibility of the face, Arch. Neurol. Psychiat. (Chic.), 9 (1923) 283-305. 5 Eccles, J. C., Fatt, P. and Koketsu, K., Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurons, J. Physiol. (Lond.), 126 (1954) 524-562. 6 Falls, W. M. and King, J. S., The facial motor nucleus of the opossum: cytology and axosomatic synapses, J. comp. Neurol., 167 (1976) 177-204. 7 Foley, J. O. and DuBois, F. S., An experimental study of the facial nerve, J. comp. Neurol., 79 (1943) 79-105. 8 Huber, E. and Hughson, W., Experimental studies on the voluntary motor innervation of the facial musculature, J. comp. Neurol., 42 (1926) 113-163. 9 Iwata, N., Kitai, S. T. and Olson, S., Afferent component of the facial nerve: its relation to the spinal trigeminal and facial nucleus, Brain Research, 43 (1972) 662-667. 10 Kerr, F. W. L., Facial, vagal and glossopharyngeal nerves in the cat. Afferent connections, Arch. Neurol. (Chic.), 6 (1962) 264-281. 11 Kitai, S. T., Tanaka, T., Tsukahara, N. and Yu, H., The facial nucleus of cat: antidromic and synaptk activation and peripheral representation, Exp. Brain Res., 16 (1972) 161-183. 12 Kubota, K., Masegi, T. and Sato, Y., Location of proprioceptive neurons innervating the muscle spindles of the snout muscles in the talpoides, Exp. Neurol., 48 (1975) 142-151. 13 Lindquist, C. and Mhrtensson, A., Mechanisms involved in the cat's blink reflex, Acta physiol. scand., 80 (1970) 149-159. 14 Porter, R., Unit responses evoked in the medulla oblongata by vagus nerve stimulation, J. Physiol. (Lond.), 168 (1963) 717-735. 15 Ram6n y Cajal, S., Histologie du Systdme Nerveux de l'Homme et des Vertebras, Vol. 1, Maloine, Paris, 1909. 16 Rushworth, G., Observations on the blink reflexes, J. Neurol. Neurosurg. Psychiat., 25 (1962) 93-108. 17 Tanaka, T., Afferent projections in the hypoglossal nerve to the facial neurons of the cat, Brain Research, 99 (1975) 140-144.
Brain Research, 123 (1977) 378-383 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Synaptic activation of facial afferents on the facial neurons of the cat
T. TANAKA Department o]Physiology, School of Medicine, Mie University, Tsu (Japan)
(Accepted December 3rd, 1976)
There have been anatomical and electrophysiological studies indicative of the existence of afferent fibers in the facial nerve. Foley and DuBois 7 showed that 11-15 of the facial nerve of the cat are sensory fibers of small diameter. It was also suggested 1 that such small caliber fibers may be traced to free terminals in the adventitia of blood vessels over the facial area. On the other hand, recent electrophysiological experiments 13 proved the existence of facial afferent fibers which have both excitatory and inhibitory effects on the cat's blink reflex. Furthermore, the afferent fibers were found to project on the facial neurons via the spinal trigeminal nucleus 9. Although the functional nature of the sensory fibers in the facial nerve is not completely agreed upon among investigators, it may be related to pressure and pain ~,4, and proprioceptive sense T M over the face. The aim of the present study was to investigate synaptic actions of the facial afferents on the facial neurons of the cat. Adult cats, weighing 2.0-4.0 kg, were anesthetized with pentobarbital sodium (30 mg/kg, intraperitoneal administration). After surgical procedures, muscular movements were eliminated by intravenous injection of gallamine triethodide and the animal was artificially respired. Methods for recording responses from the facial neurons were the same as those described in a previous paper 17. For stimulation of the peripheral facial nerve, the posterior auricular (PA), the temporal-zygomatico-orbital (TZ) and both the superior and inferior buccolabial (BL) branches were isolated and cut at the point of their entrance into the mimetic muscles. They were stimulated through A g AgC1 electrodes with current pulses of 0.05-0.1 msec in duration. The facial nerve trunk contralateral to the recording side of the facial nucleus was also stimulated at the proximal portion of the cut end just distal to the stylomastoid foramen. In some experiments, the dorsal surface of the medulla oblongata and spinal cord from the obex to C3 segment was exposed by laminectomy. Activities of neurons in or near the spinal trigeminal nucleus and solitary tract nucleus were recorded. To confirm that the recordings were from these nuclei, the infraorbital branch of the ipsilateral trigeminal nerve and cervical vagus nerve were dissected respectively, and the cut end of each nerve was stimulated to induce synaptically activated responses. Microelectrodes for recording were filled with 3 M KC1 and had a DC resistance of 5-15 Mr2. At the end of each experiment, the recording microelectrodes were inserted to the responsive
379
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Fig. 1. A-E: responses to stimulation of the ipsilateral facial nerve branches recorded intracellulady from one (A-C) and another (D, E) facial neurons. Antidromic spike potential and EPSPs were produced in a facial neuron in response to BL stimulation (A). EPSPs evoked by PA stimulation (B). An increase in amplitude of EPSPs due to strong stimuli led to a firing of spike potential (C). Antidromic spike potential followed by EPSPs was produced in another facial neuron by PA stimulation (D). EPSPs evoked by BL stimulation (E). F: afterhyperpolarization recorded from a PA neuron. Stimulus intensity was at threshold so that antidromic activation failed in about half of the trials. G: EPSPs produced in a PA neuron by contralateral facial nerve stimulation. H and I: IPSPs produced in two different BL neurons by PA stimulation. A noticeable small depolarization is indicated by a downward arrow in I. Note that the sweep speeds and amplifications ate different between H and I. Lower traces in each record show extracellular control responses. Stimulus artifacts are on the left of each record. Voltage calibration of 5 mV applies to A-H, 2.5 mV to I. Time scale of 50 msec applies only to H.
regions o f the m e d u l l a o b l o n g a t a a n d left b u r i e d to m a r k a t r a c k t h r o u g h the spinal trigeminal nucleus o r solitary t r a c t nucleus. The histological e x a m i n a t i o n was m a d e with frozen sections stained with 0.1 ~o methylene blue solution after fixation. Fig. 1 illustrates p o t e n t i a l s synaptically activated in the facial n e u r o n s b y stimulation o f the facial nerve branches. In Fig. 1A, s t i m u l a t i o n o f the B L b r a n c h o f the ipsilateral facial nerve elicited a n a n t i d r o m i c spike p o t e n t i a l at a short latency o f 0.6 msec in the facial neuron. The spike was succeeded by an irregular d e p o l a r i z a t i o n which b e g a n at a b o u t 8 msec after the stimulus with a slow rising phase. The irregular d e p o l a r i z a t i o n a p p e a r e d to be synaptically induced. W h e n a stimulus was a p p l i e d to a n o t h e r b r a n c h o f the facial nerve, e.g. the P A branch, it p r o d u c e d EPSPs with a latency o f 7.5 msec in the n e u r o n (Fig. 1B). W i t h an increase in the stimulus intensity, the EPSPs increased in a m p l i t u d e a n d elicited a c t i o n potentials (Fig. 1C). This finding strongly suggests the convergence o f facilitatory actions o f afferent fibers in each b r a n c h o f the facial nerve to a facial neuron. Fig. 1D shows EPSPs p r e c e d e d b y a n a n t i d r o m i c spike p o t e n t i a l in a n o t h e r facial n e u r o n in response to s t i m u l a t i o n o f the P A branch. A s seen in Fig. 1E, the latency o f the EPSPs i n d u c e d in this P A n e u r o n by BL s t i m u l a t i o n was 4.6 msec a n d the d u r a t i o n was a p p r o x i m a t e l y 35 msec. I n 52 facial neurons, the latent times o f the EPSPs were m e a s u r e d on each b r a n c h s t i m u l a t i o n o f the ipsilateral facial nerve. The latencies r a n g e d f r o m 3.8 to 8.0 msec, the m e a n value being 5.6 msec. T h e y are evidently indicative o f a p o l y s y n a p t i c n e u r o n a l connection. I n o r d e r to assess the degree o f i n t e r a c t i o n between m o t o n e u r o n s , the effect o f a n t i d r o m i c impulses on n e i g h b o r i n g m o t o n e u r o n s was e x a m i n e d in the facial nucleus.
380 I n the spinal m o t o r nuclei, such interaction can occur over recurrent axon collateral pathway 5. Fig. 1F shows a sample of a n antidromically activated spike with afterhyperpolarization. PA stimulation straddled the threshold for the axon of the impaled n e u r o n , there being failure of spike initiation in a b o u t half of the trials. Such a stimulus intensity was well below the threshold for producing synaptic potentials evoked by single volleys in the afferent c o m p o n e n t . A synaptic potential superimposed on the afterhyperpolarization was n o t revealed in any of the facial n e u r o n s tested, which indicates a lack of recurrent synaptic action. This is supported by the histological findings of R a m 6 n y Caja115, a n d Falls a n d K i n g 6. Stimulation of the contralateral facial nerve was also capable of producing polysynaptic EPSPs in these facial neurons, although stronger stimuli were required to elicit EPSPs. This is illustrated in Fig. 1G, where the EPSPs began at 8.5 msec after contralateral facial nerve t r u n k stimulation a n d could initiate action potentials. The iatencies, ranging from 6.3 to 10.0 msec (N = 11, m e a n 7.9 msec), were slightly longer t h a n those of the EPSPs evoked by ipsilateral facial nerve stimulation. I n the minority of the facial n e u r o n s (N - - 7), stimulation of the ipsilateral facial nerve b r a n c h produced hyperpolarizing potentials at latencies of 7-10 msec a n d they lasted for 30-60 msec (Fig. 1H a n d I). By electrophoretic injection of chloride ions into the neurons, the hyperpolarization changed to a depolarization, which provided evidence that this response was due to IPSP. Some of these hyperpolarizing responses were preceded by depolarizing potentials at latencies of 4-7 msec (a d o w n w a r d arrow in Fig. 1I).
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Fig. 2. Responses of facial neurons in three different experiments (A-C, D-F, G-K). A : field potential evoked by ipsilateral PA stimulation in the facial nucleus. B and C: field potential (B) and intracellular recording (C) on PA stimulation after intracranial section of the facial nerve in the same preparation as A. D : field potential induced by PA stimulation after intracranial section of the ipsilateral trigeminal nerve. E: no field potentials could be elicited by stimulation of the infraorbital branch of the ipsilateral trigeminal nerve. F: intracellular responses induced by PA stimulation in a PA neuron in the same preparation as D and E. G and H : field potentials elicited by stimulation of PA (G) or BL (H) in the facial nucleus after intracranial section of the vagus nerve. In G, a succeeding slow negative response was abolished. I-K: with intact vagus nerve, PA stimulation elicited antidromic potential followed by synaptically activated action potentials (I). Section of the vagus nerve eliminated synaptic responses on PA stimulation in a PA neuron (J), while the responses remained unchanged on BL stimulation in a BL neuron after section of the vagus nerve (K). Lower traces of C, F, I-K, extracellular control. Voltage calibration of 2.5 mV applies to the records of field potentials. Upward deflection in field potentials indicates negativity of microelectrode.
381 Results from several experiments which aimed at identifying the peripheral afferent pathway of the facial reflex are illustrated in Fig. 2. Fig. 2A shows a sample of the field potential recorded in the medial portion of the facial nucleus on PA stimulation. The responses consisted of an initial synchronized negative deflection followed by a succeeding prolonged negative wave. The initial response was caused by the antidromic activation of PA neurons, and the second wave could be attributable to depolarization synaptically induced by afferent volleys in the PA branch. In the medial portion of the facial nucleus, the largest antidromic field potentials were produced by PA stimulation (see ref. 11). After these responses had been recorded, the facial nerve was sectioned intracranially at the exit from the brain stem while the recording microelectrode was in the facial nucleus. Then, it was found that PA stimulation could still evoke the second negative wave, even though it failed to produce any antidromic response (Fig. 2B). EPSPs and spike potentials superimposed on the EPSPs could be recorded from neurons in or near the facial nucleus by stimulation of the PA branch in such preparations (Fig. 2C). The latency of the EPSPs was about 5 msec and the duration of the potentials corresponded to the second response of the field potential. These synaptic responses were very similar to those obtained in the animals with intact facial nerve, and they are definitely indicative of polysynaptic EPSPs produced in the facial neurons. Since the intermediate nerve reaches the brain stem along with the facial nerve, the neurons in the geniculate ganglion can be assumed to play a minor role in mediating the afferent inputs involved. In the experiment in which the ipsilateral trigeminal nerve was sectioned intracranially after decerebration, the responses which could be attributed to the action of afferent fibers in the facial nerve remained unchanged, as illustrated in Fig. 2D and F. In 6 other experiments, attempts were made to record extracellular and intracellular responses from facial neurons in animals in which the vagus nerve had been cut intracranially at its entrance into the brain stem; i.e. proximal to the nodosa ganglion. Sample records of field potentials from the facial nucleus are included in Fig. 2G and H. PA stimulation evoked only an initial antidromic potential without eliciting any succeeding reflex response (Fig. 2G), whereas stimulation of the BL branch still resulted in a production of the reflex response preceded by an antidromic one (Fig. 2H). These results were also confirmed by intracellular recordings from individual facial neurons. With the vagus nerve intact, an antidromic action potential was followed by one or two spikes superimposed on EPSPs on PA stimulation (Fig. 21). With the vagus nerve sectioned, PA stimulation elicited no synaptic potentials which could be attributed to the action of afferent volleys, as illustrated in Fig. 2J, where the intensity of stimulus was increased to 5-6 times threshold for the antidromic response. In contrast, severance of the vagus nerve showed no significant effect on the configuration of synaptic potentials produced by BL volleys in a BL neuron, coinciding with the results of the field potential analysis (Fig. 2K, compare with Fig. 2H). It may be anatomically considered that the PA branch of the facial nerve at the level of the stylomastoid foramen is accompanied by the auricular branch of the vagus nerve (Arnold's nerve) which comes to join the PA within the facial canal in the cat 8. It can be assumed that the afferent fibers of the PA branch, carrying volleys from the auricular
382
2.5mV]5mV
, iE 10msec
'
Fig. 3. A and B: responses of a single unit evoked by stimulation of the ipsilateral cervical vagus nerve (A) and PA (B) in the vicinity of the solitary tract nucleus. C-E: evoked responses of a unit to stimulation of the infraorbital branch of the ipsilateral trigeminal nerve (C), PA (D) and BL (E) in or near the spinal trigeminal nucleus. Voltage calibration of 2.5 mV applies to A and B, and that of 5 mV to C-E. Negativity upwards. and occipital portions of the face, join the vagus nerve and reach the brain stem via the jugular foramen. Recently, Kubota et al. 12 demonstrated that the facial nerve of the talpoides contains the proprioceptive afferents from the facial muscles, and that the primary neurons of these afferents exist in the glossopharyngeal-vagus ganglion. Although we have at present no available evidence with regard to an afferent pathway of the BL or TZ branch of the facial nerve in cats, it appears reasonable to conclude that sensory fibers of the facial nerve are distributed widely in the facial area. A further analysis was made to ascertain the destination of facial afferents in the brain stem. Fig. 3A and B illustrate the results obtained by extracellular recording from a unit in the vicinity of the solitary tract nucleus on stimulation of the cervical vagus nerve and the PA branch of the facial nerve. The solitary tract neurons were identified by synaptic invasion of afferent volleys of the vagus nerve 14. On ipsilateral vagus nerve stimulation unitary responses at a latency of around 2.5 msec were recorded at depths of about 1.0-2.2 mm from the dorsal surface of the medulla oblongata at the level of the obex (Fig. 3A). On the other hand, PA stimulation evoked no response in this unit (Fig. 3B). For the sample of units studied here, when the microelectrode was placed in the vicinity of the solitary tract nucleus, unitary responses could not be recorded on PA stimulation. Since it has been suggested that some afferent fibers of both the vagus nerve3, x0 and facial nerveg, 1° project to the spinal trigeminal nucleus, interneurons mediating the pathway from facial afferents are presumed to be located in the region of the spinal trigeminal nucleus. This assumption was tested in several experiments. A single stimulus applied to the infraorbital branch of the trigeminal nerve produced usually a repetitive discharge in units in or near the spinal trigeminal nucleus at the latency of 1.1 msec, which indicated a monosynaptic activation of the units (Fig. 3C). The unit shown in Fig. 3C responded to PA or BL stimulation with repetitive discharges that appeared to begin 2.5-4.5 msec after the stimulus (Fig. 3D and E). In the majority of units in this region, the initial spike had a latency shorter than that of EPSPs produced in the facial neurons. The intensity of stimulus required to elicit discharge of these units was of the same order as that necessary to
383 produce EPSPs in the facial neurons. Therefore, n e u r o n s i n the spinal trigeminal nucleus may be i n t e r n u n c i a l in the reflex pathway. The results presented here indicate that the facial nerve branches c o n t a i n afferent fibers distal to the level of the stylomastoid foramen, a n d afferents in the posterior auricular b r a n c h are considered to be a p o r t i o n of the vagus nerve. The observations strongly suggest that these afferent i n p u t s to the facial m o t o n e u r o n s are relayed by n e u r o n s in or near the spinal trigeminal nucleus. The a u t h o r is i n d e b t e d to Prof. K. Sasaki for kindly reading this m a n u s c r i p t a n d p r o v i d i n g comments. He is also obliged to Dr. Y. Takeuchi for his assistance in histology.
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