Brain Research, 86 (1975) 121-127
121
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Short Communications
Is the prepositus hypoglossi nucleus the source of another vestibulo-ocular pathway?
R. BAKER AND A. BERTHOZ Division of Neurobiology, Department of Physiology and Biophysics, University o f Iowa, Iowa City, Iowa 52242 (U.S.A.) and Laboratoire de Physiologie du Travail, 41 Rue Gay-Lussac, Paris (France)
Accepted November 25th, 1974)
In spite of their own individual anatomical identity and proximity to the vestibular nucleus, 3 brain stem nuclei (the prepositus hypoglossi, Roller and intercalatus) have always been collectively studied and referred to in the literature as the perihypoglossal nuclei4,s,12,14, is. Since these nuclei surround the hypoglossal nucleus 5,12,1s their functional role was also assumed to be in the motor control of the tongue 4,19,zl. Recently, it has become apparent that neurons in at least one of these nuclei (the prepositus hypoglossi) are more likely to be concerned with eye and head movementl,S,20. A vestibulo-cerebello-ocular role could have been suggested earlier for prepositus neurons based solely on degeneration studies showing connections with the fastigial nucleusa, zl, flocculus2 and interstitial nucleus of Cajal 6,13. In addition, the stimulation and lesion experiments of Hyde and Eliasson 1° would have supported the concept of a hind brain stem area involved with horizontal eye movement. Although little attention was paid to the latter observations, Uemura and Cohen 2° have recently shown distinct changes in the control of eye and head movement following lesions in the dorsal medullary reticular formation including the nucleus prepositus hypoglossi. However, the impetus to re-evaluate the physiological role of the prepositus hypoglossi at the present time was provided by the recent evidence obtained with horseradish peroxidase tracers indicating that some prepositus neurons project to both the cerebellar cortex 1 and the oculomotor complex s. Therefore the intent of our study was to provide electrophysiological correlates for the previously described anatomical observations regarding probable afferent and efferent prepositus pathways 1,2,5,e,s,19,zo. In this paper we present the effect of vestibular nerve stimulation on prepositus hypoglossi neurons which may be identified as anti- or orthodromically activated from stimulation of the oculomotor (Oc) complex and/or cerebellum (Cer). Unexpectedly, the experiments have revealed a strong reciprocal vestibular disynaptic excitatory-inhibitory control of most prepositus hypoglossi neurons projecting to the
122 cerebellum and o c u l o m o t o r area. In addition, a m o n o s y n a p t i c excitation often followed by i n h i b i t i o n has been observed in many prepositus n e u r o n s after either Oc a n d / o r Cer stimulation. These results suggest the p r i m a r y f u n c t i o n of the prepositus hypoglossi nucleus is in the organization of eye and possibly head movement. I n addition, its intimate relationship with the vestibular system provides evidence for a n o t h e r vestibulo-cerebello-oculomotor pathway organized similarly to those so extensively investigated in the last few years 2,s,9,1l.
tl
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Fig. 1. Field potentials recorded in the prepositus hypoglossi nucleus following vestibular, oculomotor and cerebellar stimulation. A-D: field potentials recorded in the left prepositus hypoglossi nucleus with the microelectrode 1 mm from the midline and 0.4 mm from the surface of the brain stem. The recording site is indicated by the circle in F and the lesion in the histological inset. A and B: Vi and Vc stimulation at 2 x vestibular nerve threshold (Thr.). C: Oc stimulation at 2 x Thr. The filled circles in B and C indicate orthodromic activation of a prepositus neuron. D: Cer stimulation at 3 x Thr. Histological controls showed the Oc stimulating electrode to be in the posterior part of the left Oc complex and the Cer electrode in the left juxta-fastigial region. E: horizontal brain stern section depicting the anterior-posterior extent of the prepositus nucleus and its relationship to other nuclei. F: transverse section at the level indicated in E identifying the structures shown in the histological inset (cresyl violet stain). Abbreviations: MLF, medial longitudinal fasciculus; N5, N6 and N10, cranial motor nuclei; Np5, principal sensory nucleus of the 5th; 7N and 10N, 7th and 10th nerve; S, L, M, D, are the superior, lateral, medial and descending vestibular nuclei; ph, prepositus hypoglossi; f and x, vestibular nuclei subgroups; Ncue, accessory cuneate nucleus; Ntrs, nucleus of solitary tract; Ncu, cuneate nucleus; Npr, dorsal group of paramedian reticular nucleus.
I23 The results in this study are drawn from 13 experiments carried out in cats anesthetized with pentobarbital (30 mg/kg). Bipolar silver ball electrodes were placed on each VIII nerve extracranially for the purpose of electrical stimulation3, le. A bipolar stimulating electrode was situated in the posterior part of the oculomotor complex as identified physiologically from the field potentials following vestibular nerve stimulation. In some experiments parts of the cerebellar cortex (flocculus, vermis, juxta-fastigial) were stimulated by a bipolar silver wire electrode placed on their surfaces under direct visual control. Following placement of all stimulating electrodes, the animals were paralyzed with gallamine (2 mg/kg/h), artificially respired and a bilateral pneumothorax was performed. In most experiments lobules IX and X were removed to allow recordings throughout the anterior-posterior extent of the prepositus hypoglossus (Fig. 1E). Microelectrodes for intra- and extracellular recordings were filled with either 1.5 M KC1 or 1 M NaC1 (saturated with Fast Green). Since it was possible to place the microelectrode under visual control at a desired location on the surface of the brain stem (surrounding landmarks shown in Fig. 1E, F), extensive histological confirmation of medio-lateral or anterior-posterior tracks were not often required; however, it was important to control for microelectrode depth as Oc or Cer stimulation was found to be effective in producing antidromic and orthodromic responses in neurons throughout the paramedian reticular nucleus. After a few experiments we found the prepositus hypoglossi nucleus could also be identified from the field potential profiles produced by stimulation of either the ipsi- or contralateral vestibular nerve (Vi and Vc, respectively) (Figs. 1A, B and 2F). Although the comments in this paper are likely to reflect the organization of the entire prepositus hypoglossi nucleus most of out
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Fig. 2. Anti- and orthodromic activation of prepositus hypoglossi neurons following oculomotor and cerebellar stimulation. A and B: antidromic invasion of a prepositus neuron following Oc (1 msec) and Cer (0.75 msec) stimulation at a double shock interval of 1.3 msec. The prepositus neuron was ipsilateral to the Oc and Cer stimulation 0.9 mm from the midline and 0.8 mm from the surface of the brain stem. C-E: extra- (C) and intracellular records (D and E) from a left prepositus neuron following left Oc stimulation. The first arrow in C indicates the latency for the orthodromic EPSP (1.0 msec) and the second arrow the antidromic latency (1.4 msec). D and E: the upper record is a high gain AC coupled trace and the lower one a low gain DC coupled trace. The Oc stimulus was 2.5 x Thr in C and D and 1.5 x Thr in E. F: schematic diagram showing the sites stimulated and synaptic connections identified in these experiments.
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Vi-Oc LIVe. lmV]
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Fig. 3. Extra- and intracellular records from prepositus hypoglossi neurons following vestibular and oculomotor stimulation. A-H: extra- (A-E) and intracellular (F-H) records from a left prepositus neuron located 1 mm lateral to the midline and at a depth of 0.5 mm. A: left Oc stimulation at 1.2 × Thr. B: Oc stimulation at 2 x Thr showing antidromic activation followed by orthodromic activation (filled circles). C: preceding Oc by Vi stimulation blocks orthodromic activation. D: Vc stimulation at 2 × Thr activates 2 prepositus neurons; one antidromic (same unit as in B) and the other not identified. E: preceding the Vc by the Vi stimulus blocks orthodromic activation of the antidromically identified prepositus neuron but not activation of the other one. F: intracellular record at 2 x Oc stimulation intensity. The first arrow indicates an EPSP latency of 0.9 msec and the second arrow an antidromic latency of 1.3 msec. G: Vc stimulation at 2 × Thr. Arrow indicates synaptic latency of 1.4 msec when compared to the extracellular trace shown immediately below. H : Vi stimulation at 2 x Thr (upper extracellular trace) shows a latency of 1.6 msec. I and J : intracellular records from a left prepositus neuron located 1.1 mm lateral to the midline and 0.3 mm from the brain stem surface. I: Vi stimulus at 2 x Thr. J: Vc stimulus at 2 × Thr. The immediate extracellular records indicated a latency of 1.6 msec for both IPSPs. The gain of the AC coupled record is 10 x that of the lower DC trace.
records were o b t a i n e d from n e u r o n s 1 m m lateral to the midline at a level near the a n t e r i o r - p o s t e r i o r profile indicated in Fig. 1E a n d F. W h e n the microelectrode is situated in the dorsomedial part of the prepositus hypoglossi 400 # m from the surface of the b r a i n stem, field potential responses shown in Fig. I A - D are found. F r o m either Vi or Vc s t i m u l a t i o n distinct negative-positive presynaptic fields are obvious at a latency of 1 msec. I n the dorsal part of the prepositus hypoglossi nucleus the presynaptic fields are followed by slower positive field potentials. I n the case of Vi s t i m u l a t i o n the large positivity is u n d e r s t a n d a b l e as it could represent the c u r r e n t underlying p r o d u c t i o n o f the IPSP (Fig. 3H) in prepositus neurons. However, genesis of the positivity following Vc s t i m u l a t i o n (Figs. 1B a n d 3G) is n o t clearly correlated with the profile of excitatory synaptic potentials seen in most prepositus neurons. One would expect a negative field potential to be produced by the synaptic a n d action currents u n d e r l y i n g the EPSP a n d action poten-
125 tials in these neurons. Since some prepositus neurons exhibit a Vc IPSP (Fig. 3J) then a combination of (1) active (IPSP) and (2) passive (deeper depolarizations) sources may give rise to the superficial positivity found in the dorsal part of the nucleus. In fact, a Vc induced negative field potential can be recorded in the ventral parts of the prepositus hypoglossi nucleus but it also extends into the underlying brain stem (2-3 mm). In contrast, the Vi positivity is confined to a superficial (1 mm) part of the brain stem. At the superficial and medial microelectrode location, shown in Fig. IF, the field potential produced by Oc stimulation primarily consists of an early sharp negative wave (0.5 msec) followed by a slower negative potential at 1-2 msec with obvious unit activity superimposed (Fig. 1C). The latter negativity corresponds with the antiand orthodromic activation of prepositus neurons while the early short latency one reflects activity in the neighboring MLF. The late negativity becomes increasingly larger as the microelectrode moves deeper into the brain stem and at depths of 1-2 mm (below the prepositus nucleus) the Oc stimulation produces a large anti- and orthodromic negativity. The effects of Oc stimulation were bilateral in the prepositus hypoglossi but detectably stronger on the ipsilateral side. In order to record a distinct negative antidromic field potential from Oc stimulation, several probing microelectrode tracks were often needed; however, the antidromic field produced by Cer stimulation (Fig. 1D) was easily observed throughout the nucleus. The amplitudes of the early sharp negative wave at 0.5 msec and the later positive-negative field always were smaller and larger, respectively, than the ones elicited from Oc stimulation. The antidromic field potentials produced by both Oc and Cer stimulation were mixed with short latency synaptic potentials (EPSPs and IPSPs in Fig. 2D and E), initiated by simultaneous excitation of afferent pathways ending on the prepositus neurons 2,6,s,9,13. Even so, based on its larger antidromic field potential, the cerebellar projection of the prepositus hypoglossi nucleus appears to be more extensive than that to the oculomotor complex. In many cases it was not possible to establish whether a prepositus neuron was responding in an anti- or orthodromic fashion to Oc or Cer stimulation. Although the prepositus neuron shown in Fig. 1B and C was not activated by Cer stimulation, the one shown in Fig. 2 was antidromically excited by both Oc (A) and Cer (B) stimulation. The conduction velocity of axons from prepositus neurons was found to range from 10 to 20 m/sec and is similar to the values determined for vestibular neurons 15,16. Due to the slowly conducting fiber system long latency antidromic activation (1-2 msec) would be expected (Figs. 1C, 2A-C, 3B and F). Close comparison of extra- and intracellular records from the prepositus neuron in Fig. 2C shows on orthodromic EPSP with a latency of 1 msec and distinct antidromic response at 1.3 msec. The EPSP is likely to be produced from the interstitial nucleus of Cajal~, 13 and was found in nearly every prepositus neuron (whether or not antidromic identification was present). Following Oc stimulation inhibition was not frequently detected in prepositus neurons but was always present in neurons more than 1 mm from the brain stem surface. Further comments on these synaptic responses and depth profiles of field potentials will be presented in a more extensive analysis in a later study.
126 The primary response pattern found thus far in the prepositus nucleus - namely disynaptic reciprocal, vestibular influence - - is shown extra- and intracellulady for the same cell in Fig. 3. Antidromic activation of this prepositus neuron by threshold and 2 × threshold Oc stimulation is shown in Fig. 3A and B respectively. The powerful excitatory nature of the contralateral vestibular connection is demonstrated by the multiple firing of this neuron (filled circles in Fig. 3D) and by another prepositus neuron (larger spike) not anti- or orthodromically activated in Fig. 3B. In anesthetized as well as unanesthetized animals (in preparation) most prepositus neurons fire in short bursts as opposed to a more tonic type of discharge. Stimulation of the ipsilateral vestibular nerve can completely block the Oc and Vc orthodromic activation of most neurons (the antidromic one in Fig. 3D and E) but only delays Vc activation of other prepositus neurons (large spike in Fig. 3E). Reciprocal vestibular connections are found in a few neurons that cannot be identified as projecting to the ocular complex or to the cerebellar cortex. The opposite pattern - - antidromic activation without any vestibular effect - - is less frequent. Intracellular records (Fig. 3G and H) from the prepositus hypoglossi neuron shown in A - F demonstrate the disynaptic nature of the IPSP (1.6 msec)from ipsilateral vestibular nerve stimulation and the disynaptic nature of the EPSP (1.4 msec) from contralateral vestibular nerve. Another prepositus neuron, not antidromically identified, is shown to have a bilateral inhibition (IPSP) in Fig. 3I and J. From the viewpoint of the polarity of the synaptic potential profile, the reciprocally activated neurons shown in Figs. 1 and 2 would be expected to exhibit type II responses on adequate vestibular stimulation v,16. The prepositus neurons with bilateral inhibition would exhibit a type IV response. We have also obtained neurons that would be likely to show type I and III responses but clearly they occur with a lower frequency than type II or IV. A schematic diagram of the connections illustrated in this paper are shown in Fig. 2F. These results demonstrate an unsuspected, intimate relationship between the vestibular system and the prepositus hypoglossi nucleus. For most prepositus neurons the vestibular connections are primarily reciprocal in nature and in the direction of ipsilateral inhibition and contralateral excitation. It is, in fact, the same profile of synaptic connections (including latency) that each ocular motoneuron receives from the vestibular system 3,9,11. A high percentage of the reciprocally activated prepositus neurons send axons towards the oculomotor complex and the cerebellar cortex. In addition, these prepositus neurons receive afferent information from the cerebellar vermis via the fastigial nucleus (see refs. 4, 5, 21 and this study) and directly from the flocculus (see ref. 2 and this study). There is histological evidence that prepositus neurons are under direct cerebral cortical controP v and this brain stem area has been indicated to be concerned with visual tracking (especially maintenance of eye and head position) 2°. Therefore, it is likely that the prepositus hypoglossi nucleus may not be simply the source of 'another vestibulo-cerebello-ocular pathway' but may be concerned with a very different organization and aspect of eye-head control than the closely related parallel, cerebello-vestibulo-ocular circuit through the vestibular nucleiS,9, al. Whatever its various role(s) may be, the present study serves to demon-
127 strate that the prepositus hypoglossi nucleus contains n e u r o n s exhibiting all the diverse responses previously described for vestibular n e u r o n s 7,11,15,1° a n d has a direct relationship with o c u l o m o t o r a n d cerebellar areas related to eye movement. This research was supported by C.N.R.S., C . N . A . M . a n d Public Health Service G r a n t EY01074-01.
1 ALLEY, K., BAKER, R., AND SIMPSON,J. I., Brain stem afferents to the vestibulo-cerebellum as mapped with horseradish peroxidase tracers, Soc. Neurosci., 4 (1974) I 16. 2 ANGAUT,P., AND BRODAL,A., The projection of the 'vestibulocerebellum' onto the vestibular nuclei in the cat, Arch. ital. Biol., 105 (1967) 441-479. 3 BAKER,R., PRECHT,W., ANDLLIN.~S,R., Cerebellar modulatory action on the vestibulo-trochlear pathway in the cat, Exp. Brain Res., 15 (1972) 364-385. 4 BRODAL,A., Cerebellar afferents from the peri-hypoglossal nuclei. In J. JANSENAND A. BRODAL (Eds.), Aspects of Cerebellar Anatomy, Gunderson, Oslo, 1954, pp. 158-161. 5 BRODAL, A., Experimental demonstration of cerebellar connexions from the peri-hypoglossal nuclei (nucleus intercalatus, nucleus praepositus hypoglossi and nucleus of roller) in the cat, J. Anat. (Lond.), 86 (1952) 110-120. 6 CARPENTER,M. B., HARmSON,J. W., ANDPETER,P., Accessory oculomotor nuclei in the monkey: projections and effects of discrete lesions, J. comp. Neurol., 140 (1970) 131-154. 7 DUENSING, F., UND SCHAEFER,K.P., Die Aktivit~it einzelner Neurone im Bereich der Vestibulariskerne bei Horizontalbeschleunigungen unter besonderer Beriicksichtigung des vestibul/iren Nystagmus, Arch. Psychiat. Nervenkr., 198 (1958) 225-252. 8 GRAYBIEL,A. M., Some afferent connections of the oculomotor complex in the cat, Soc. Neurosci., 4 (1974) 235. 9 HIGHSTEIN,S., ITO, M., AND TSCHUCHIYA,T., Synaptic linkage in the vestibulo-ocular reflex pathway of rabbit, Exp. Brain Res., 18 (1971) 306-326. 10 HYDE, J. E., AND ELIASSON,S. G., Brainstem induced eye movements in cat, J. comp. Neurol., 108 (1957) 139-172. 11 ITO, M., The control mechanism of cerebellar motor systems. In F. O. SCHMITTAND F. G. WORDEN(Eds.), Neurosciences Third Study Program, MIT Press, Cambridge, Mass., 1974, pp. 293-303. 12 JERMULOWICZ,W., Untersuchungen ~iber die Kerne am Boden der Rauten~ube (Nucleus paramedianus, Nucleus eminentae teretis, Nucleus praepositus hypoglossi, Kappenkern des Facialisknies), Z. Anat. Entwickl.-Gesch., 103 (1934) 290-302. 13 MAaucm, M., AND KUSAMA,T., Mesodienc~phalic projections to the inferior olive and the vestibular and perihypogiossal nuclei, Brain Research, 17 (1970) 133-136. 14 MARBURG,O., Das dorsale L~ingsbundel yon Schutz - - Fasciculus periependymalis - - und seine Beziehungen zu den Kernen des zentralen H6hlengraus, Arb. neurol. Inst. Univ. Wien, 33 (1931) 135-164. 15 MARKHAM,C. H., PRECHT, W., AND SH1MAZU,H., Effect of stimulation of interstitial nucleus of Cajal on vestibular unit activity in the cat, J. Neurophysiol., 29 (1966) 493-507. 16 SHIMAZU,H., AND PRECHr, W., Tonic and kinetic responses of cat's vestibular neurons to horizontal angular acceleration, J. Neurophysiol., 28 (1965) 991-1013. 17 SOUSA-PINTO,A., The cortical projection onto the paramedian reticular and perihypoglossal nuclei (nucleus praepositus hypoglossi, nucleus intercalatus and nucleus of roller) of the medulla oblongata of the cat. An experimental-anatomical study, Brain Research, 18 (1970) 77-91. 18 TAGAKI,J., Studien zur vergleichenden Anatomie des Nucleus vestibularis triangularis. I. Der Nucleus intercalatus und der Nucleus praepositus hypoglossi, Arb. neurol. Inst. Univ. Wien, 27 (1925) 157-188. 19 TORVIK,A., AND BRODAL,A., The cerebellar projection of the peri-hypoglossal nuclei (nucleus intercalatus, nucleus praepositus hypoglossi and nucleus of roller) in the cat, J. Neuropath. exp. Neurol., 13 (1954) 515-527. 20 UEMURA,T., ANt) COHEN, B., Effects of vestibular nuclei lesions on vestibulo-ocular reflexes and posture in monkeys, Acta oto-laryng. (Stockh.J, Suppl. 315 (1973) 1-71. 21 WALBERG,F., Fastigiofugal fibers to the perihypoglossal nuclei in the cat, Exp. Neurol., 3 (1961) 525-541.
121
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Short Communications
Is the prepositus hypoglossi nucleus the source of another vestibulo-ocular pathway?
R. BAKER AND A. BERTHOZ Division of Neurobiology, Department of Physiology and Biophysics, University o f Iowa, Iowa City, Iowa 52242 (U.S.A.) and Laboratoire de Physiologie du Travail, 41 Rue Gay-Lussac, Paris (France)
Accepted November 25th, 1974)
In spite of their own individual anatomical identity and proximity to the vestibular nucleus, 3 brain stem nuclei (the prepositus hypoglossi, Roller and intercalatus) have always been collectively studied and referred to in the literature as the perihypoglossal nuclei4,s,12,14, is. Since these nuclei surround the hypoglossal nucleus 5,12,1s their functional role was also assumed to be in the motor control of the tongue 4,19,zl. Recently, it has become apparent that neurons in at least one of these nuclei (the prepositus hypoglossi) are more likely to be concerned with eye and head movementl,S,20. A vestibulo-cerebello-ocular role could have been suggested earlier for prepositus neurons based solely on degeneration studies showing connections with the fastigial nucleusa, zl, flocculus2 and interstitial nucleus of Cajal 6,13. In addition, the stimulation and lesion experiments of Hyde and Eliasson 1° would have supported the concept of a hind brain stem area involved with horizontal eye movement. Although little attention was paid to the latter observations, Uemura and Cohen 2° have recently shown distinct changes in the control of eye and head movement following lesions in the dorsal medullary reticular formation including the nucleus prepositus hypoglossi. However, the impetus to re-evaluate the physiological role of the prepositus hypoglossi at the present time was provided by the recent evidence obtained with horseradish peroxidase tracers indicating that some prepositus neurons project to both the cerebellar cortex 1 and the oculomotor complex s. Therefore the intent of our study was to provide electrophysiological correlates for the previously described anatomical observations regarding probable afferent and efferent prepositus pathways 1,2,5,e,s,19,zo. In this paper we present the effect of vestibular nerve stimulation on prepositus hypoglossi neurons which may be identified as anti- or orthodromically activated from stimulation of the oculomotor (Oc) complex and/or cerebellum (Cer). Unexpectedly, the experiments have revealed a strong reciprocal vestibular disynaptic excitatory-inhibitory control of most prepositus hypoglossi neurons projecting to the
122 cerebellum and o c u l o m o t o r area. In addition, a m o n o s y n a p t i c excitation often followed by i n h i b i t i o n has been observed in many prepositus n e u r o n s after either Oc a n d / o r Cer stimulation. These results suggest the p r i m a r y f u n c t i o n of the prepositus hypoglossi nucleus is in the organization of eye and possibly head movement. I n addition, its intimate relationship with the vestibular system provides evidence for a n o t h e r vestibulo-cerebello-oculomotor pathway organized similarly to those so extensively investigated in the last few years 2,s,9,1l.
tl
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Fig. 1. Field potentials recorded in the prepositus hypoglossi nucleus following vestibular, oculomotor and cerebellar stimulation. A-D: field potentials recorded in the left prepositus hypoglossi nucleus with the microelectrode 1 mm from the midline and 0.4 mm from the surface of the brain stem. The recording site is indicated by the circle in F and the lesion in the histological inset. A and B: Vi and Vc stimulation at 2 x vestibular nerve threshold (Thr.). C: Oc stimulation at 2 x Thr. The filled circles in B and C indicate orthodromic activation of a prepositus neuron. D: Cer stimulation at 3 x Thr. Histological controls showed the Oc stimulating electrode to be in the posterior part of the left Oc complex and the Cer electrode in the left juxta-fastigial region. E: horizontal brain stern section depicting the anterior-posterior extent of the prepositus nucleus and its relationship to other nuclei. F: transverse section at the level indicated in E identifying the structures shown in the histological inset (cresyl violet stain). Abbreviations: MLF, medial longitudinal fasciculus; N5, N6 and N10, cranial motor nuclei; Np5, principal sensory nucleus of the 5th; 7N and 10N, 7th and 10th nerve; S, L, M, D, are the superior, lateral, medial and descending vestibular nuclei; ph, prepositus hypoglossi; f and x, vestibular nuclei subgroups; Ncue, accessory cuneate nucleus; Ntrs, nucleus of solitary tract; Ncu, cuneate nucleus; Npr, dorsal group of paramedian reticular nucleus.
I23 The results in this study are drawn from 13 experiments carried out in cats anesthetized with pentobarbital (30 mg/kg). Bipolar silver ball electrodes were placed on each VIII nerve extracranially for the purpose of electrical stimulation3, le. A bipolar stimulating electrode was situated in the posterior part of the oculomotor complex as identified physiologically from the field potentials following vestibular nerve stimulation. In some experiments parts of the cerebellar cortex (flocculus, vermis, juxta-fastigial) were stimulated by a bipolar silver wire electrode placed on their surfaces under direct visual control. Following placement of all stimulating electrodes, the animals were paralyzed with gallamine (2 mg/kg/h), artificially respired and a bilateral pneumothorax was performed. In most experiments lobules IX and X were removed to allow recordings throughout the anterior-posterior extent of the prepositus hypoglossus (Fig. 1E). Microelectrodes for intra- and extracellular recordings were filled with either 1.5 M KC1 or 1 M NaC1 (saturated with Fast Green). Since it was possible to place the microelectrode under visual control at a desired location on the surface of the brain stem (surrounding landmarks shown in Fig. 1E, F), extensive histological confirmation of medio-lateral or anterior-posterior tracks were not often required; however, it was important to control for microelectrode depth as Oc or Cer stimulation was found to be effective in producing antidromic and orthodromic responses in neurons throughout the paramedian reticular nucleus. After a few experiments we found the prepositus hypoglossi nucleus could also be identified from the field potential profiles produced by stimulation of either the ipsi- or contralateral vestibular nerve (Vi and Vc, respectively) (Figs. 1A, B and 2F). Although the comments in this paper are likely to reflect the organization of the entire prepositus hypoglossi nucleus most of out
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Fig. 2. Anti- and orthodromic activation of prepositus hypoglossi neurons following oculomotor and cerebellar stimulation. A and B: antidromic invasion of a prepositus neuron following Oc (1 msec) and Cer (0.75 msec) stimulation at a double shock interval of 1.3 msec. The prepositus neuron was ipsilateral to the Oc and Cer stimulation 0.9 mm from the midline and 0.8 mm from the surface of the brain stem. C-E: extra- (C) and intracellular records (D and E) from a left prepositus neuron following left Oc stimulation. The first arrow in C indicates the latency for the orthodromic EPSP (1.0 msec) and the second arrow the antidromic latency (1.4 msec). D and E: the upper record is a high gain AC coupled trace and the lower one a low gain DC coupled trace. The Oc stimulus was 2.5 x Thr in C and D and 1.5 x Thr in E. F: schematic diagram showing the sites stimulated and synaptic connections identified in these experiments.
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Vc
Fig. 3. Extra- and intracellular records from prepositus hypoglossi neurons following vestibular and oculomotor stimulation. A-H: extra- (A-E) and intracellular (F-H) records from a left prepositus neuron located 1 mm lateral to the midline and at a depth of 0.5 mm. A: left Oc stimulation at 1.2 × Thr. B: Oc stimulation at 2 x Thr showing antidromic activation followed by orthodromic activation (filled circles). C: preceding Oc by Vi stimulation blocks orthodromic activation. D: Vc stimulation at 2 × Thr activates 2 prepositus neurons; one antidromic (same unit as in B) and the other not identified. E: preceding the Vc by the Vi stimulus blocks orthodromic activation of the antidromically identified prepositus neuron but not activation of the other one. F: intracellular record at 2 x Oc stimulation intensity. The first arrow indicates an EPSP latency of 0.9 msec and the second arrow an antidromic latency of 1.3 msec. G: Vc stimulation at 2 × Thr. Arrow indicates synaptic latency of 1.4 msec when compared to the extracellular trace shown immediately below. H : Vi stimulation at 2 x Thr (upper extracellular trace) shows a latency of 1.6 msec. I and J : intracellular records from a left prepositus neuron located 1.1 mm lateral to the midline and 0.3 mm from the brain stem surface. I: Vi stimulus at 2 x Thr. J: Vc stimulus at 2 × Thr. The immediate extracellular records indicated a latency of 1.6 msec for both IPSPs. The gain of the AC coupled record is 10 x that of the lower DC trace.
records were o b t a i n e d from n e u r o n s 1 m m lateral to the midline at a level near the a n t e r i o r - p o s t e r i o r profile indicated in Fig. 1E a n d F. W h e n the microelectrode is situated in the dorsomedial part of the prepositus hypoglossi 400 # m from the surface of the b r a i n stem, field potential responses shown in Fig. I A - D are found. F r o m either Vi or Vc s t i m u l a t i o n distinct negative-positive presynaptic fields are obvious at a latency of 1 msec. I n the dorsal part of the prepositus hypoglossi nucleus the presynaptic fields are followed by slower positive field potentials. I n the case of Vi s t i m u l a t i o n the large positivity is u n d e r s t a n d a b l e as it could represent the c u r r e n t underlying p r o d u c t i o n o f the IPSP (Fig. 3H) in prepositus neurons. However, genesis of the positivity following Vc s t i m u l a t i o n (Figs. 1B a n d 3G) is n o t clearly correlated with the profile of excitatory synaptic potentials seen in most prepositus neurons. One would expect a negative field potential to be produced by the synaptic a n d action currents u n d e r l y i n g the EPSP a n d action poten-
125 tials in these neurons. Since some prepositus neurons exhibit a Vc IPSP (Fig. 3J) then a combination of (1) active (IPSP) and (2) passive (deeper depolarizations) sources may give rise to the superficial positivity found in the dorsal part of the nucleus. In fact, a Vc induced negative field potential can be recorded in the ventral parts of the prepositus hypoglossi nucleus but it also extends into the underlying brain stem (2-3 mm). In contrast, the Vi positivity is confined to a superficial (1 mm) part of the brain stem. At the superficial and medial microelectrode location, shown in Fig. IF, the field potential produced by Oc stimulation primarily consists of an early sharp negative wave (0.5 msec) followed by a slower negative potential at 1-2 msec with obvious unit activity superimposed (Fig. 1C). The latter negativity corresponds with the antiand orthodromic activation of prepositus neurons while the early short latency one reflects activity in the neighboring MLF. The late negativity becomes increasingly larger as the microelectrode moves deeper into the brain stem and at depths of 1-2 mm (below the prepositus nucleus) the Oc stimulation produces a large anti- and orthodromic negativity. The effects of Oc stimulation were bilateral in the prepositus hypoglossi but detectably stronger on the ipsilateral side. In order to record a distinct negative antidromic field potential from Oc stimulation, several probing microelectrode tracks were often needed; however, the antidromic field produced by Cer stimulation (Fig. 1D) was easily observed throughout the nucleus. The amplitudes of the early sharp negative wave at 0.5 msec and the later positive-negative field always were smaller and larger, respectively, than the ones elicited from Oc stimulation. The antidromic field potentials produced by both Oc and Cer stimulation were mixed with short latency synaptic potentials (EPSPs and IPSPs in Fig. 2D and E), initiated by simultaneous excitation of afferent pathways ending on the prepositus neurons 2,6,s,9,13. Even so, based on its larger antidromic field potential, the cerebellar projection of the prepositus hypoglossi nucleus appears to be more extensive than that to the oculomotor complex. In many cases it was not possible to establish whether a prepositus neuron was responding in an anti- or orthodromic fashion to Oc or Cer stimulation. Although the prepositus neuron shown in Fig. 1B and C was not activated by Cer stimulation, the one shown in Fig. 2 was antidromically excited by both Oc (A) and Cer (B) stimulation. The conduction velocity of axons from prepositus neurons was found to range from 10 to 20 m/sec and is similar to the values determined for vestibular neurons 15,16. Due to the slowly conducting fiber system long latency antidromic activation (1-2 msec) would be expected (Figs. 1C, 2A-C, 3B and F). Close comparison of extra- and intracellular records from the prepositus neuron in Fig. 2C shows on orthodromic EPSP with a latency of 1 msec and distinct antidromic response at 1.3 msec. The EPSP is likely to be produced from the interstitial nucleus of Cajal~, 13 and was found in nearly every prepositus neuron (whether or not antidromic identification was present). Following Oc stimulation inhibition was not frequently detected in prepositus neurons but was always present in neurons more than 1 mm from the brain stem surface. Further comments on these synaptic responses and depth profiles of field potentials will be presented in a more extensive analysis in a later study.
126 The primary response pattern found thus far in the prepositus nucleus - namely disynaptic reciprocal, vestibular influence - - is shown extra- and intracellulady for the same cell in Fig. 3. Antidromic activation of this prepositus neuron by threshold and 2 × threshold Oc stimulation is shown in Fig. 3A and B respectively. The powerful excitatory nature of the contralateral vestibular connection is demonstrated by the multiple firing of this neuron (filled circles in Fig. 3D) and by another prepositus neuron (larger spike) not anti- or orthodromically activated in Fig. 3B. In anesthetized as well as unanesthetized animals (in preparation) most prepositus neurons fire in short bursts as opposed to a more tonic type of discharge. Stimulation of the ipsilateral vestibular nerve can completely block the Oc and Vc orthodromic activation of most neurons (the antidromic one in Fig. 3D and E) but only delays Vc activation of other prepositus neurons (large spike in Fig. 3E). Reciprocal vestibular connections are found in a few neurons that cannot be identified as projecting to the ocular complex or to the cerebellar cortex. The opposite pattern - - antidromic activation without any vestibular effect - - is less frequent. Intracellular records (Fig. 3G and H) from the prepositus hypoglossi neuron shown in A - F demonstrate the disynaptic nature of the IPSP (1.6 msec)from ipsilateral vestibular nerve stimulation and the disynaptic nature of the EPSP (1.4 msec) from contralateral vestibular nerve. Another prepositus neuron, not antidromically identified, is shown to have a bilateral inhibition (IPSP) in Fig. 3I and J. From the viewpoint of the polarity of the synaptic potential profile, the reciprocally activated neurons shown in Figs. 1 and 2 would be expected to exhibit type II responses on adequate vestibular stimulation v,16. The prepositus neurons with bilateral inhibition would exhibit a type IV response. We have also obtained neurons that would be likely to show type I and III responses but clearly they occur with a lower frequency than type II or IV. A schematic diagram of the connections illustrated in this paper are shown in Fig. 2F. These results demonstrate an unsuspected, intimate relationship between the vestibular system and the prepositus hypoglossi nucleus. For most prepositus neurons the vestibular connections are primarily reciprocal in nature and in the direction of ipsilateral inhibition and contralateral excitation. It is, in fact, the same profile of synaptic connections (including latency) that each ocular motoneuron receives from the vestibular system 3,9,11. A high percentage of the reciprocally activated prepositus neurons send axons towards the oculomotor complex and the cerebellar cortex. In addition, these prepositus neurons receive afferent information from the cerebellar vermis via the fastigial nucleus (see refs. 4, 5, 21 and this study) and directly from the flocculus (see ref. 2 and this study). There is histological evidence that prepositus neurons are under direct cerebral cortical controP v and this brain stem area has been indicated to be concerned with visual tracking (especially maintenance of eye and head position) 2°. Therefore, it is likely that the prepositus hypoglossi nucleus may not be simply the source of 'another vestibulo-cerebello-ocular pathway' but may be concerned with a very different organization and aspect of eye-head control than the closely related parallel, cerebello-vestibulo-ocular circuit through the vestibular nucleiS,9, al. Whatever its various role(s) may be, the present study serves to demon-
127 strate that the prepositus hypoglossi nucleus contains n e u r o n s exhibiting all the diverse responses previously described for vestibular n e u r o n s 7,11,15,1° a n d has a direct relationship with o c u l o m o t o r a n d cerebellar areas related to eye movement. This research was supported by C.N.R.S., C . N . A . M . a n d Public Health Service G r a n t EY01074-01.
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