The Connections of the Trigeminal and Facial Motor Nuclei in the Brain of the Carp (Cyprinus carpio L.) as Revealed by Anterograde and Retrograde Transport of Horseradish Peroxidase P. G. M. LUITEN AND J. N. C. VANDER PERS Department ofZoology, State Unzuersity ofGroningen, Kerklann 30, Haren, the Netherlands
ABSTRACT The connections of the rostra1 and caudal parts of the trigeminal and facial motor nuclei in the carp were studied with the horseradish peroxidase technique. Following ionophoretic peroxidase injections in these motor nuclei, retrogradely labeled cells were observed together with anterogradely labeled motor cell processes. Several cellular areas in thalamus, cerebellum and medulla oblongata were shown to project to the V and VII motor nuclei. Labeled cells were found in the inferior lobe and the glomerular complex of the thalamus. In the medulla oblongata, cells in the descending trigeminal nucleus, reticular nuclei and motor nuclei other than those injected were labeled. Besides these conspicuous projections several smaller connections were also found. These findings are discussed on their significance to respiratory function. Anterogradely labeled cellular processes constitute a relatively simple network of fiber connections between the various motor nuclei and the reticular nuclei of the brainstem. This apparently dendritic system of the bulbar motor complex shows a certain degree of similarity to the structure of the motor system in the spinal cord, and might play a role in the coordinated control of the muscular system. Anatomical investigation of afferent reflex clear areas within the brainstem (Ballintijn connections of respiratory muscles in the and Bamford, '75; Ballintijn and Roberts, '76; carp has led to the hypothesis that the pro- Bamford, '75; Shelton, '61).Obviously some of jection fields of the sensory trigeminal root the recording sites are situated in the bulbar (the nucleus of the descending trigeminal motor systems, since Ballintijn and Bamford root) consists of interneurons that-at least ('75) and Ballintijn and Alink ('77) identified to a considerable degree-project to cells of motor neurons physiologically. Other recordthe brainstem motor column (Luiten, '75a). ings showing the typical character of proprioSuch a cellular organization is thought to re- ceptive elements (Ballintijn and Bamford, sult in a bisynaptic reflex loop carrying '75) could have been recorded from interneusomato-sensory information to the effector rons in the nucleus of the descending trigemisystem thereby allowing appropriate motor nal root (nDV). A third category of recordings response. The crucial question to answer is showing activity in respiratory rhythm, has whether there exists a direct projection from been made from nuclear areas located in the the interneurons in the descending trigeminal bulbar reticular formation as demonstrated nucleus to the motor cells of the trigeminal by Meijer ('77). Other recordings from physioand facial motor nuclei. The interest is logical literature cannot be specified anatomilimited to this part'of the motor column only, cally. since we know that the respiratory muscles The physiological and anatomical properunder study are innervated by cells of the tri- ties of the efferent cells generating the resgeminal and facial motor complexes (Lui- piratory rhythm, the motor cells in the triten, '76). geminal and facial motor nuclei, have been In neurophysiological studies on the res- determined accurately (Ballintijn and Alink, piratory system of teleost fishes single unit '77; Luiten, '76). The aim of the present study activity has been recorded from several nu- then is to establish the structural relationJ. COMP. NEUR.,174: 575-590
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P. G. M. LUITEN AND J. N. C. VAN DER PERS
obtained from hatcheries of the “Organisatie ter Verbetering van de Binnenvisserij.” The animals were anaesthetized in a 1: 20,000 dilution of MS-222 (Sandoz) in water at room temperature. The heads were fixed in a head clamp and a rectangular hole sawed in the neurocranium to expose the brain. The experimental set up is indicated schematically in figure 1. Bipolar electrodes for recordings of electromyograms (EMG) were inserted into four primary respiratory muscles: the adductor mandibulae, the levator hyomandibulae, the dilator operculi and the levator operculi. The brains were explored with glass micropipette electrodes. Immediately before the experiment the micropipettes were filled under pressure with a 5% solution of HRP (Sigma, type VI) in 0.5 M Tris HC1 buffer, pH = 8.2. The filling of the pipette was controlled under the microscope. At the same time, the tip diameter was measured. The glass capillary was fastened in a hydraulic micromanipulator and a chlorided silverwire inserted into the HRP solution in the pipette. The pipette served as both recording and injecting electrode. Another chlorided silverwire was suspended in the water of the tank to serve as the indifferent electrode. The pipette electrode and the muscle electrodes were connected to Grass P16 and P15 amplifiers respectively. The amplified signals were displayed on an oscilloscope Tektronix 555 and recorded simultaneously on an instrumentation recorder CEC VR-3300. As soon as the micropipette reached one of the motor nuclei of the trigeminal or facial complexes, cellular activity of the motor neurons could be observed in phase with respiration. The recorded signals were cross correlated with the different electromyograms in order to determine whether the observed neurons were motor neurons (Ballintijn and Alink, ’74; ’77). Then the pipette electrode was disconnected from the amplifier and connected to a current generator (custom made). A positive rectangular DC-current was applied to the electrode for 20 minutes total on-time (the generator gave an interrupted current of 1pA 3 seconds on, 1 second off). This resulted in iontophoretic precipitation of HRP a t the electrode tip, giving injection spots of about 200-500 p m depending on the tip diameter of MATERIAL AND METHODS the micropipette: 200 p m when the tip diamThe experiments were carried out on three- eter was 5 pm, 500 p m with tip diameters of year-old carp (Cyprinuscarpio L.) of 25-30 cms 25 p m (figs. 2, 3). After electrophoresis the
ship of the trigeminal and facial motor cells with all other parts of the respiratory control system, notably the respiratory-active cell groups described above. The trigeminal and facial motor complexes were subject of a recent analysis (Luiten, ’76). As is known from other investigators as well, cf. Ariens Kappers et al. (‘361, the two nuclear complexes can be divided into rostral and caudal subnuclei. This nuclear subdivision has been related to functional and topographical properties of the muscles that are innervated by the cell populations of the various subnuclei. Apparently the rostro-caudal nuclear segregation reflects the antagonistic muscular activity in the respiratory cycle that results in the contraction and expansion of the buccal and opercular cavities. The rostral subnuclei of the trigeminal and facial motor systems are concerned with contraction, and the caudal subnuclei with expansion muscles (Luiten, ’76). As such, these subnuclei have a different effector function. An obvious question then is whether the functional differences are also reflected in diferent intracerebra1connections. For that reason this study was performed at the subnuclear level and connections of both rostral and caudal parts of the trigeminal and facial motor nuclei were studied separately. The present study has been undertaken with the horseradish peroxidase (HRP) technique using iontophoretic delivery by means of glass micropipettes. This method offers a number of indispensable advantages: (1) Iontophoresis with glass micropipettes can be combined with electrophysiological recording. The recorded signals are the only possible means to judge whether the pipette is in a motor nucleus, since stereotactic electrode positioning is impractical in the carp brain due to the absence of a fixed relation between position and size of the brain and form and size of the skull, (2) the combination of cellular recording and HRP-iontophoresis at the recording site permits the determination of both neurophysiological and neuro-anatomical characteristics of the cells a t the recording site, (3) the application of a reproducible injection restricted to the motor nuclei can be made small enough to avoid labeling of non-relevant connections.
CONNECTIONS OF V AND VII MOTOR NUCLEI IN CARP
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Fig. 1 Experimental situation. Abbreviations: A, ammeter; Ampl, amplifiers; CG, current generator; IE, indifferent electrode; M, glass micropipette filled with HRP solution serving as recording electrode and for iontophoretic peroxidase injection; ME, muscle electrode; OSC, oscilloscope; REC, instrumentation recorder; S, switch. Explanation see text.
electrode was disconnected and left in situ for a t least five minutes to equalize an increased HRP concentration a t the tip which otherwise might cause loss in the electrode track during retraction of the pipette. The electrode was removed and the skull closed with spongostan and dental cement. The animals were allowed to survive for 10 to 12 days a t 13-17°C. Then they were reanaesthetized in a 1 : 10,000 solution of MS222 and perfused with teleost saline followed by buffered fixative a s described earlier (Luiten, '76). After perfusion-fixation, the brains were removed from the skull, immersed overnight in
fresh fixative a t 4°C and embedded in a 20% gelatin solution in phosphate buffer 0.1 M, pH = 7.5. The gelatin was hardened a t 4°C and then trimmed close to the tissue. The trimmed blocks were transferred to the fixative for another 16 hours. Prior to sectioning the blocks were soaked for one hour in 30% sucrose. Frozen sections were cut a t 40 p m on a cryostat microtome. Every second section was stained for HRP according to the procedure of Graham and Karnovsky ('66). Adjacent sections were stained with Cresyl Fast Violett (CFV). HRP stained sections were studied with darkfield or lightfield illumination.
Abbreviations
Cb, cerebellum FLM, fasciculus longitudinalis medialis L VII, lobus facialis L Inf, lobus inferior LL, lemniscus lateralis n 111, nucleus oculomotorius nDV, nucleus descendens nervi trigemini n Diff, nucleus diffusus lobi inferioris nIF, nucleus intermedius facialis nFM, nucleus funicularis medialis nM Vr, nucleus motorius nervi trigemini rostralis nM Vc, nucleus motorius nervi trigemini caudalis nM VIIr, nucleus motorius nervi facialis rostralis nM VIIc, nucleus motorius nervi facialis caudalis nM IX, nucleus motorius nervi glossopharyngei
nM X, nucleus motorius nervi vagi nPG, nucleus preglomerulosus nPr V, nucleus princeps nervi trigemini nRI, nucleus reticularis inferior nRL, nucleus recessus lateralis nRM, nucleus reticularis medius nRS, nucleus reticularis superior nS V, nucleus spinalis nervi trigemini N V, nervus trigeminus RD V, radix descendens nervi trigemini RS VII, radix sensorius nervi facialis str P, stratum Purkinje cerebelli TGS, tractus secundus gustatorius V IV, fourth ventricle Valv Cb, valvula cerebelli
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Fig. 4 Cellular labeling in the nucleus recessus lateralis following HRP injection in the caudal trigeminal motor nucleus. Bar = 100 Fm.
Fig. 2 Photomicrograph of a n injection spot in the rostral trigeminal motor nucleus. Spot diameter is about 250 pm. Scale bar, 250 p m . Fig. 3 Photomicrograph of a n injection spot in the rostral trigeminal motor nucleus. Spot diameter in this case is about 450 pm. The dark fibers in the upper right of the picture are labeled axons forming the trigeminal motor root. Scale bar, 250 pm. The frames in the drawings t h a t accompany the photomicrographs give the location of these photographs. Fig. 5 Ramifying fibers in the nucleus motorius vagus The description of the results is based on 19 animals on a total of 28 experiments. These 19 anterogradely labeled after a n HRP injection in the caudal facial motor nucleus. The arrow points a t the injection site. animals all had an HRP injection restricted Horizontal section. Scale bar, 100 pm. to one of the motor subnuclei. Of the trigeminal motor nucleus cases, six of the injecRESULTS tions were in the rostral and three in the caudal subnucleus. Of the facial motor complex The site of the injection in the various cases, five were in the rostral and five in the motor nuclei could be recognized easily by a caudal subdivision. concentration of HRP reaction product. Suc-
CONNECTIONS OF V AND VII MOTOR NUCLEI IN CARP
cessful injections always fell within the limits of the nucleus. An interesting feature of these spots was that the enzyme was always distributed in conformity with the elongated shape of the nucleus. As has been pointed out before (Luiten, '75b), both anterograde and retrograde intracellular transport of HRP can be observed in carp material. Retrogradely labeled structures were recognized as fibers which could be followed up to groups of labeled perikarya. In many cases it could be observed that the fiber originated from the cell body (fig. 4). Anterogradely labeled fibers generally ramified distal to the injection site (fig. 5) and split up into very fine branches in what we considered as the terminal areas. These terminal areas usually were found to coincide with well defined nuclei in the CFVstained sections. Injections of HRP in the rostral and caudal subnuclei of the trigeminal motor complex resulted in the labeling of almost identical configurations of fibers and cell bodies. The same holds for the results after injections in the rostral or caudal subnuclei of the facial motor complex. Accordingly the connections of the subnuclei will be described together for both the trigeminal group and the facial group. A. Connections of the trigeminal motor complex (fig. 6) In general all long-distance connections on each side of the brainstem run in two distinct fiber systems connected to each other by a number of fibers crossing the midline at the levels of the various motor nuclei. The lateral fiber system or tract is situated medioventral to the secondary gustatory tract (TGS) and passes along the trigeminal and facial motor column. Rostrally, this fiber system continues as the tractus lobo-bulbaris (Herrick, '05; Woodburne, '36; Beccari, '43) straight forward into the diencephalon. Caudally this tract runs ventral to the glossopharyngeal and vagal motor nuclei, then takes a more medial position and joins a second longitudinal fiber system which courses through the column formed by the reticular nuclei. 1. Retrogradely labeled connections after HRP injections in the trigeminal motor nuclei. As we already pointed out, retrogradely labeled structures are those cellular processes that are continuous with labeled perikarya. Cell bodies labeled in this way after tri-
579
geminal motor nuclei injections could be detected in various levels in the brain. In the diencephalon, cells were found in the nucleus preglomerulosus (terminology of Schnitzlein, '621, in the nucleus diffusus of the inferior lobe and in the nucleus of the lateral recess (fig. 4). The fibers of these cells run in the lobo-bulbar tract and remain strictly ipsilateral. At the mesencephalic level, Purkinje cells were marked with HRP in the ipsilateral valvula cerebelli. Their axons leave the injection site with the lobo-bulbar tract and run rostrad to the level of the oculomotor nucleus; here they turn sharply dorsad with a rostral inclination up to the Purkinje cell layer. The highest number of labeled cells was found in the myelencephalon. There HRP positive cells were found in the several subdivisions of the nucleus of the descending trigeminal root (nDV), both ipsi- and contralaterally. Cellular labeling was conspicuous in the most rostral part of the nucleus, situated dorsorostral to the nMV. This shows that the nDV is discontinuous and this rostral part apparently should be considered to be the nucleus princeps V (nPrV). The other labeled cells of the nDV were observed dorsal to the facial motor nucleus (fig. 7) and medial and slightly ventral to the glossopharyngeal and vagal efferent complex (nMIX/X) which conforms with the appearance of the nDV as described in an earlier paper (Luiten, '75a). Labeled fibers sprouting from these cells all appear to pass either crossed or uncrossed through the lateral fiber system. No peroxidase deposits were found in the spinal trigeminal nucleus (nSV). Another projection to the trigeminal motor nuclei originates from cells situated ipsilaterally a t the base of the facial lobe. In agreement with Herrick's description ('05) this nucleus is called the nucleus intermedius facialis (nIF). HRP injections in nMV also resulted in cellular marking in the inferior, medial and ipsilateral superior reticular nuclei, a t the vagal, facial and trigeminal root levels respectively (terminology of van Hoevell "111; Ariens Kappers et al. "361; Opdam et al. "761; Smeets and Nieuwenhuys "761.) Thus the trigeminal motor nuclei obviously maintain extensive contacts with the various reticular nuclei on both sides. The fibers of these reticular cells reach the fifth motor complex via the ipsi- and contralateral fiber system through the reticular column.
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CONNECTIONS OF V AND VII MOTOR NUCLEI IN CARP
58 1
of the trigeminal nerve. These fibers-as is well-known by now-swing dorsally over the secondary gustatory tract (fig. 8 ) and join the sensory part of the trigeminal root. The labeled motor root then takes a ventral and ventromedial position within the nerve. The motor axons do not appear to give off any collaterals within their course in the brainstem. Furthermore, various fiber connections were seen ending in the form of very fine branches in various nuclear areas in the brainstem. Rostrally, a fascicle of fibers leaves the ipsilateral lobo-bulbar tract and runs in a medial direction within the ipsi- and
Fig. 7 A cell in t h e nucleus of the descending trigeminal root marked retrogradely with HRP after injection in the caudal facial motor nucleus. Part of the injection spot can be seen in the botton of the photograph. Note that both cell body (arrow) and the fiber sprouting from i t are labeled. Scale bar, 100 Km.
The last group of retrogradely labeled somata is formed by cells of the contralateral counterpart of the nMV. No cellular marking could be detected in the facial and vagal parts of the efferent column. 2. Anterogradely labeled connections after HRP injections in the rostra1 and caudal trigeminal motor nuclei.
The most conspicuous group of fibers of this category are those constituting the motor root
Fig. 8 Injection in the caudal trigeminal motor nucleus with the labeled fibers t h a t form the motor root. This motor root swings dorsally over the secondary gustatory tract. Scale bar, 250 pm.
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.-c
CONNECTIONS OF V AND VII MOTOR NUCLEI IN CARP
583
Fig. 10 Photomicrograph of a horizontal section with a n injection of HRP in the facial motor nucleus. Bundles of labeled fibers running rostrad and caudad can be observed. Scale bar, 250 pm.
contralateral parts of the oculomotor nucleus (fig. 6c). In the myelencephalon a number of fibers running via both the ipsi- and contralateral lobo-bulbar tracts and consequently being of crossed and uncrossed nature could be followed into the trigeminal and facial motor complexes. Hardly any labeled fibers, however, occurred in the efferent nuclei of IX and X after fifth motor nucleus HRP injections. Another conspicuous connection consists of crossed and uncrossed fibers joining the reticular tracts. These fibers could be followed up to the ipsi- and contralateral parts of the superior, medial and inferior reticular nuclei, the largest number running to the latter two nuclear areas.
B. Connections of the facial motor nuclei (fig.9) As was the case with the rostral and caudal subnuclei in the trigeminal motor system, the injections of HRP in the rostral and caudal subnuclei of the facialis complex also resulted in labeling of identical sets of connections.
Moreover, these labeled structures fitted remarkably well in the same framework as has been described for the connections of the trigeminal motor system, as presented in A. A condensed description follows below.
1. Retrograde cellular labeling following HRP injections in the rostral and caudal facial motor nuclei Labeled somata could be detected in the following nuclear areas: At the diencephalic level in the ipsilateral nucleus preglomerulosus and nucleus diffusus lobi inferior. In the metencephalon: cells in the Purkinje layer of the ipsilateral transition of valvula and corpus cerebelli. In the myelencephalon: the ipsiand contralateral medial and inferior reticular nuclei and ipsilateral superior reticular nucleus; the ipsilateral and contralateral motor nuclei of the V, VII and IX/X nerves; the various subdivisions of the nucleus of the descending trigeminal tract; and a last group of labeled cells in the nucleus intermedius facialis at the ipsilateral side. In the rostral spinal cord a number of peroxidase-marked
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Fig. 11 A bundle of ramified fibers in the ipsilateral trigeminal motor nucleus following HRP injection in the facial motor nucleus. Thearrow points at the injection site. Horizontal section. The bundle of dark fibers in the upper right of the photograph constitute the facial motor root leaving the brainstem. Scale bar, 100 pm.
somata were observed in the spinal part of the nucleus of the descending trigeminal tract which is called the spinal trigeminal nucleus. In addition we found HRP positive somata in the nucleus recessus lateralis after injections in the rostral facial motor nucleus. After injections in the caudal facial motor nucleus no cellular marking occurred in the nucleus princeps V. The fibers relating to the above mentioned cell groups follow exactly the same course as those labeled after peroxidase injection in the trigeminal system. 2. Anterogradely labeled connections after injections in the rostral and caudal facial motor nuclei
The motor fibers of course make up the facial motor root. From the facial motor nuclei they run dorsomedially to the ventricle wall, then turn sharply rostrad along with the fasciculus longitudinalis medialis. At the level of the caudal trigeminal motor nucleus, the facial motor root swings laterally between
the descending trigeminal root and the secondary gustatory tract. The motor root then leaves the brainstem as an isolated motor nerve and does not mix with the sensory part of the facial nerve a t least up to the ganglion. As was the case with the trigeminal motor axons, no axon collaterals could be detected in the facial motor root. As stated a t the beginning of this section, the pattern formed by the other labeled fiber fascicles after peroxidase injection in the facial motor nuclei closely resembles the fiber system labeled after nucleus motorius V injections. Thus fibers are found running to the ipsilateral oculomotor nucleus, ipsi- and contralateral reticular nuclei and ipsi- and contralateral motor nuclei of the V and VII and also fibers to the IX/X efferent complex. The relatively largest number of fibers lead to the facial and glossopharyngeal/vagal areas (figs. 10, 11). Although a purely quantitative analysis of the results seems hazardous, because we do
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CONNECTIONS OF V AND VII MOTOR NUCLEI IN CARP TABLE
1
Injection site nuc1.mot.V ros t ralis
found in
iucl .mot .V :audalis
ucl.mot.VI1 ostralis
nucl.rnot.VI1 caudalis
n.preglomerulosus ipsi
cells
cells
cells
cells
n.recessus 1at.ipsi
cells
cells
cells
-
n.diffusus lobi inf. ipsi
cells
cells
cells
cells
Mesencephalon
n.oculomotorius
fibers
fibers
fibers
fibers
Metencephalon
Purkinje layer of valv.cereb.
cells
cells
cells
cells
Myelencephaloi
n.princ.V ipsi
cells
cells
cells
-
n.princ.V contra
cells
cells
cells
-
n.desc.V ipsi (level fac.)
cells
cells
cells
cells
n.desc.V contra (level fac.)
cells
cells
cells
cells
n.desc.V ipsi (level vagus)
cells
cells
cells
cells
cells
cells
cells
D iencephalon
n.desc.V contra (level vagus) n.rn0t.V
ipsi
n.mot.V
contra
n.mot.VI1
-injection- -injection- ellscf ibers cells+fibers :ells+fiber
ipsi
n.mot.VI1 contra
fibers
-injection-
-injection-
fibers
fibers
ells+fibers
zells+fibers
fibers
ells+f ibers
cells+fibers
-
ells+f ibers
cells+fibers
contra
n.intermedius fac. ipsi
cells cells+fibers
n.ret.sup.ipsi
.
n. ret sup.contra
fibers
cells fibers fibers?
cells ells+f ibers fibers
cells cells+fibers fibers
cells+fibers :ells+fiber cellscfiber cells+fibers
n.ret.med.ipsi
cells+fibers :ells+fiber
n.ret.med.contra
.
Medulla spinalis
fibers
fibers
n.mot.IX/X ipsi n.mot.=/X
fibers
cells cells+fibers
cells+fiber cells+fibers cells+fibers
n. ret. inf ipsi
cellssfibers :ells+fiber cells+f iber
n.ret.inf.contra
cells+f ibers :ells+fiber cells+fiber celIs+fibers
n.spinalis V ipsi
l
-
-
cells
cells
The term ”cells” refers to retrograde cellular labeling. “Fibers” refers to anterograde fiber labeling
not know how completely the HRP technique labels all connections, certain conclusions can be drawn from the relative amount of labeled cells and fibers. In our experimental series the density of labeling shows that the number of connections on the ipsilateral side exceeds that on the contralateral side. Based on number of labeled cells, connections from the trigeminal motor nuclei with thalamic areas were more abundant than from the facial motor nuclei. On the same grounds, the caudal parts of the brainstem seem to have much
more fiber relations with the efferent facial system than with the efferent trigeminal nuclei. Some other quantitative facts will be discussed in relation to their possibly functional significance in the next section. An overall survey of the results is given in table 1. DISCUSSION
Experimental data on connections of bulbar motor nuclei in teleosts are extremely scarce. Detailed descriptions of those connections are generally based on normal material. Wood-
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P. G . M. LUITEN AND J. N. C. VAN DER PERS
burne ('361, in a comparative anatomical proprioceptive reflexloops (R. Dale Smith et study on the trigeminal system mentioned al., '68; Rubinson, '70; Azzena and Palmieri, dendritic connections of trigeminal motor '67; Szentagothai, '48). Recently Roberts and cells with the reticular formation; the same Witkovsky ('75) reported monosynaptic contype of connections with t h e secondary nections between t h e mesencephalic trigustatory nucleus are suggested by Ariens geminal nucleus and the trigeminal motor Kappers ('36: p. 357). Furthermore, a rather pool in the dogfish, but doubted the fact that vague direct relation with the cerebellar cor- mesencephalic V cells mediate prioprioceptive pus by the tractus cerebello-motorius is de- information from jaw musculature. A further scribed by Woodburne ('36). This latter type of explanation for the absence of cellular laconnections to trigeminal and facial motor beling in the mesencephalic trigeminal nunuclei has also been found by Tuge ('35) and cleus in this study will not be attempted here. Wallenberg ('07) both using the Marchi tech- A more detailed paper dealing with the mesencephalic V nucleus with regard to a nique in goldfish. Specific knowledge with respect to the fa- proprioceptive reflex loop in the carp is in cial motor nuclei is even more limited. Her- preparation. For some of the other cell groups found to rick ('06) considered the reticular formation to be the main system mediating taste and project on trigeminal and facial motor nuclei touch impulses to the effectory system, thus neurophysiological data from several authors suggesting rather intensive connections with working on the neural control of respiration especially trigeminal and facial motor nuclei. in fishes are available. Usually, however, the Without providing evidence Ariens Kappers anatomical location of respiratory active neue t al. ('36) stated t h a t reflex connections from rons is rather vague in physiological literafacial and vagal lobes to the motor nuclei of ture and reference is only made to undefined the brainstem are present (p. 356). Addi- areas in the central column in the medulla tionally a number of crossing fibers between oblongata (Woldring and Dirken, '51; Hughes the motor nuclei and nuclei of the vestibular and Shelton, '72). Shelton in a 1961 paper and recently Meijer ('77) provide more precise system were described by these authors. The present findings definitely support our information and mention respiratory neurons previous assumption that the descending tri- in the various nuclei of the reticular column. geminal nucleus (nDV) maintains a direct The existence of connections between the reconnection to the trigeminal and facial motor ticular nuclei and the bulbar motor nuclei as neurons. In addition, the nDV cells receive described in the present study are in agreethe central projection from the sensory tri- ment with the fact that respiratory rhythgeminal ganglion cells (Luiten, '75a), and so micity has been found in the reticular nuclei. can form a fast, bisynaptic pathway relaying This clearly demonstrates their important somatosensory information to the motor sys- role in the control of respiration. The reticular tem of the head musculature. Physiological cells possibly supply the efferent system with data on such reflexes in carp have been de- an integrated answer to multifarious inforscribed by Ballintijn and Bamford ('75) and mation obtained from anterior and posterior Ballintijn and Roberts ('76). The presence of parts of the brain via the fasciculus longitudicrossed interneuron connections indicates nalis medialis (Kappers e t al., '36; Hasegathat such a reflex chain acts not merely on one wa, '56). Because of experiments such as from Demside of the brain but provides the possibility for symmetric bilateral responses to sensory ski ('73) and Demski and Knigge ('71) on behavior evoked by electrical brain stimulation stimuli. Surprisingly, after peroxidase injections in in fish, i t can be concluded that the hypothalathe trigeminal motor nucleus, there was never mus plays a n important role in the control of any evidence for retrograde cellular labeling highly programmed neuronal mechanisms rein the so-called mesencephalic trigeminal nu- sulting in aggressive and feeding behavior. cleus. In other vertebrate classes such a An interesting result from our experiments is mesencephalic nucleus consists of primary that the motor nuclei active in food uptake sensory cells concerned with the propriocep- possess a direct connection with hypothalamtion of masticatory muscles. By axon col- ic centers such as the nucleus preglomerulateral connections with trigeminal motor losus, the nucleus diffusus and the nucleus neurons these cells provide monosynaptic recessus lateralis. Besides that, Finger ('76)
58 7
CONNECTIONS OF V AND VII MOTOR NUCLEI IN CARP
root
nDV
Fig. 12 Schematic interpretation in the horizontal plane of the connections of the bulbar motor nuclei exemplified on the rostra1 trigeminal motor nucleus. The interrupted line in the middle of the drawing represents the mldline. For a full explanation see text. “For the sake of clearness the various motor subnuclel are represented as one cell. Consequently the connections should be regarded as nuclear connections rather than connections of one motor cell.”
has demonstrated that neurons in or near the preglomerulosus area maintain connections to the facial lobe. This connection shows once more that the nucleus preglomerulosus and nucleus diffusus are likely a part in the feeding mechanism.
Another interesting result was the finding of a connection from the motor nuclei with the nucleus intermedius facialis of Herrick (’05). He already suggested that taste impulses from the facial lobe do not directly come into contact with the effectory system
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but are relayed by a t least one interneuron system that is situated on the latero-ventral border of the facial lobe. This is in complete agreement with our observations. In conformity with Herrick's view, the anatomical pathway from facial lobe, via nucleus intermedius facialis to nucleus motorius V and VII provides a very rapid pathway for producing a coordinated response of the feeding apparatus to gustatory perception. As has been described in the results, the fibers labeled after HRP injections in the various motor nuclei all together constitute a relatively simple framework of two longitudinal fiber bundles on each side of the brain. These bundles are connected with each other by means of crossing fascicles a t the levels of the various motor nuclei. A considerable part of this fiber system is made up by what we concluded as anterogradely labeled fibers. Theoretically, the possibility exists that anterograde fiber marking as mentioned here is the result of HRP uptake by passing fibers. This possibility is contradicted by a number of facts: 1. HRP injections in different parts of the same motor nucleus always resulted in the same configuration of labeled connections. This indicates that these nuclei are functionally homogeneous structures and contradicts the assumption that part of it is traversed by a passing fiber bundle. 2. I t can also be observed that the trigeminal and facial motor nuclei are rather dense concentrations of efferent somata. Fibers pass around instead of running through these nuclei. Most of the passing fiber tracts such as the lateral lemniscus, the secondary gustatory tract, the ventral tecto-bulbar tract and spino-cerebellar tract are well described. In hardly any experiment, however, could labeling be observed in one of these fiber tracts; thus indicating t h a t labeling indeed was the result of uptake by the cells of the motor nuclei. 3. Tissue damage after iontophoretic injection is always kept minimal (Schubert and Hollander, '75). 4. The main part of the anterogradely labeled neurites of the cells in the injected motor nuclei terminate on the other bulbar motor nuclei. Reciprocally in nearly all cases we found retrogradely labeled somata in t h e motor nuclei other t h a n injected. Among the anterogradely labeled fibers, those forming the efferent root are the most consDicuous. The rest of the anterogradely labeled fibers should be considered asintramed-
ullary neurites from nMV and nMVII cells. The functional character of these neurons, however, is not very clear. From a motor nuclei analysis we know that all cells in the nMV and nMVII are primary neurons, directly innervating the head muscles (Luiten, '76). Ballintijn and Bamford ('75), Ballintijn and Roberts ('76) and Meijer (personal communication) found cells located in or adjacent to the nMV and nMVII that are active in more than one phase of the respiratory cycle and reacted like proprioceptive neurons. As such, the anterogradely labeled fibers of nMV and nMVII cells could be regarded as axons of primary sensory neurons carrying proprioceptive information from respiratory muscles. Its connections with the other motor nuclei then should be interpreted as monosynaptic reflex circuits analogous to mesencephalic V connections in higher vertebrates. On the other hand, if one assumes that all cells forming the motor nuclei are motor cells indeed, then the dendritic extent of these motoneurons must be concluded to be considerable (figs. 11, 12). The following view is one possible functional meaning for extensive dendritic contacts between the various motor nuclei. A coordinated and rather complex movement like that of the respiratory mechanism requires a considerable degree of interaction between the neuronal elements involved in its performance. A dense network provides an organization with a close functional relationship between the constituent parts. A hypothesis based on the present data is that the dendritic network between the motor neurons in the various motor nuclei serves to maintain an intensive exchange of information between those nuclei, and thus coordinates respiratory movements. In this regard, Ballintijn and Alink ('77) found electrophysiological evidence in carp for interconnections between the motor pools of respiratory muscles. The general problem of intercellular contact by dendritic processes received the attention of several investigators. Anatomical studies revealed the extensive ramifications of dendritic branches of spinal motor neurons in fish and amphibians and a variety of dendro-dendritic contacts have been described or suggested like dendrodendritic synapses, tight junctions and gap junctions. (Anderson e t al., '71; Sotelo and Taxi, '70; Szekely and Kosaras, '76; Stensaas, '7 1; Nieuwenhuys, ;64). Furthermore Grinnel
CONNECTIONS OF V AND VII MOTOR NUCLEI IN CARP
('66) described short-latency interaction between motor neurons in the frog spinal cord, probably by means of electrotonic coupling of overlapping dendrites of neighboring motor cells. Some authors speculate t h a t such interneuron mechanisms control the sequential activation of agonist and antagonist muscle groups as in spinal segments in the cat (Szekely, '76; Scheibel and Scheibel, '73). By neurophysiological experiments and Golgi analysis as currently performed in this laboratory we hope to provide more evidence for the existence of a n interaction system a s described above. AKNOWLEDGMENTS
This study was supported by the Foundation for Fundamental Biological Research (BION), a n organization subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). The authors thank Doctor C . M. Ballintijn for his helpful advice and criticism. We are also grateful to Doctor J. L. Dubbeldam and Doctor M. Duijm for their valuable comments on the manuscript; and Mrs. J. Poelstra-Hiddinga for secretarial aid. LITERATURE CITED Anderson, W., M. W. Stromberg and E. J. Hinsman 1976 Morphological characteristics of dendrite bundles in the lumbar spinal cord of the rat. Brain Res., 110: 215-228. Ariens Kappers, C. U., G. C. Huber and E. Crobsy 1936 The effectory system. In: The Comparative Anatomy of the Nervous System of Vertebrates, Including Man. Hafner Publishers, New York, pp. 516-551. Azzena, G. B., and G. Palmieri 1967 A trigeminal monosynaptic reflex in birds. Exp. Neurol., 18: 184-193. Ballintijn, C. M., and G. M. Alink 1974 Connections of teleost respiratory motorneurons. Proc. 26th Congr. Physiol. Sci., New Delhi. 1977 Interconnections between the motorneurons of different respiratory muscles in the carp. Brain Res., in press. Ballintijn, C. M., and 0. S.Bamford 1975 Proprioceptive motor control in fish respiration. J. Exp. Biol., 62:99-114. Ballintijn, C. M., and J. L. Roberts 1976 Neural control and proprioceptive load matching in reflex respiratory movements of fishes. Fed. Proc., 35: 1983-1991. Bamford, 0. S. 1975 Respiratory neurons in rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol., 48A: 77-83. Beccari, N. 1943 -Neurologia comparata, anatomo-funcionale dei vertebrati, compreso l'homo. Sunsoni Firenze. Dale Smith, R., H. Q. Marcarian and W. T. Niemer 1968 Direct projections from the masseteric nerve to the mesencephalic nucleus. J. Comp. Neur., 133: 495-502. Demski, L. S. 1973 Feeding and aggressive behavior evoked by hypothalamic stimulation in a cichlid fish. Comp. Biochem. Physiol., 44A: 685-692. Demski, L. S.,and R. M. Knigge 1971 The telencephalon and hypothalamus of the bluegill (Lepornis rnacrochirus):
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