Ž .Brain Research 826 1999 83–94

Research report

Afferent and efferent connections of the torus semicircularis in the sealamprey: an experimental study

Maria Jose Gonzalez a, Julian Yanez a, Ramon Anadon b,)´ ´ ´ ´˜ ´ ´a Department of Cellular and Molecular Biology, UniÕersity of A Coruna, Spain˜

b Department of Fundamental Biology, UniÕersity of Santiago de Compostela, Spain

Accepted 16 February 1999

Abstract

Ž .The afferent and efferent connections of the torus semicircularis TS of larval sea lampreys were studied with horseradish peroxidase,Ž .carbocyanine dye DiI and fluorescein-coupled or Texas-Red-coupled dextran amine tract-tracing methods. Application of tracers to the

TS or to the octavolateral area revealed the presence of bilateral projections from the octavolateral area to the torus semicircularis, mainlyŽ . Žfrom the mechanoreceptive regions medial and ventral octavolateral nuclei though also from the electroreceptive dorsal octavolateral

.nucleus region. The nucleus of the descending root of the trigeminal nerve projects to the contralateral TS, mostly from neurons locatedrostral to the obex. Fairly numerous reticular cells of the rhombencephalon project to the torus semicircularis. In the mesencephalon,scattered cells in the tegmentum, and some in the tectum, have toral projections, mostly ipsilateral. Numerous thalamic neurons, as wellas fairly numerous neurons of the posterior tubercle, hypothalamus and preoptic region, and a few neurons in the ventral telencephalonŽ .striatum, septum , were labeled after tracer application to the TS. The torus semicircularis mainly projects to the thalamus, thehypothalamus and the reticular rhombencephalic nuclei. Our results reveal for the first time a complex pattern of connections of thelamprey TS, which suggests that it is a multisensory center integrating head cutaneous sensitivity with mechano- and electrosensoryinformation from the octavolateral area and with visual information. A number of afferents from the forebrain also appear to contribute toTS function. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Octavolateral center; Mesencephalon; Nerve projection; Tract tracing; Horseradish peroxidase; Carbocyanine; Brain; Agnathan

1. Introduction

Ž .The torus semicircularis TS is the main mesencephalictarget of the octavolateral system in teleosts and anuransw x w x40 , and of the octaval nerve system in sauropsids 76 .Experimental studies in teleosts and amphibians have re-vealed the presence of an octavolateral lemniscus that is

w x Žthe main afferent of the TS 4,21,31,40,48 . In fishes and.in larval and some adult amphibia , the octavolateral sys-

tem consists of the posterior and anterior lateral linenerves, as well as the octaval nerve present in all verte-

Ž .brates. Thus, the octavolateral area OLA of the medullaoblongata receives primary afferents from two special

Žsensory systems, the lateral line involved in electro- and. Žmechanoreception and the octaval nerve concerned with

) Corresponding author. Fax: q 34-981-596904; E-mail:bfanadon@usc.es

.vestibular and auditory capabilities . Comparative studiesin fishes have shown the OLA to have a ‘primitive’

Žpattern, with three regions, namely the dorsal electrore-. Žceptive lateral line area , medial mechanoreceptive lateral

. Ž . w xline area and ventral vestibular nuclei 40 . Some fishes,such as the teleosts, appear to have lost the primitiveelectroreceptive region, which has been functionally re-

w xplaced by newly evolved electrosensory nuclei 40 . Manystudies in adult amphibians have also stressed the impor-

Ž w x.tant role of the TS in audition see Ref. 8 , and similarroles have been reported in other non-mammalian verte-

w xbrates 67 .In lampreys, the OLA is organized into dorsal, medial

w xand ventral OLA nuclei 48 . The dorsal nucleus receiveselectrosensory fibers from the dorsal branch of the anterior

Ž .lateral line nerve ALLN , while the fibers from the poste-Ž .rior lateral line nerve PLLN and the ventral and medial

branches of the ALLN terminate in the medial nucleusw x48,61 . The ventral nucleus receives octaval nerve fibers.

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01266-4

( )M.J. Gonzalez et al.rBrain Research 826 1999 83–94´84

In lampreys, the PLLN also carries light-sensitive fibers, inw xaddition to the mechanosensory fibers 16 . The primary

projections of the lateral line and octaval nerves, as well asthe organization of the octavolateral nuclei in the OLA,

w xhave been studied experimentally 25,26,32,63 . Knowl-edge of the secondary projections from the OLA nuclei in

Žlampreys is based on studies with classical methods seew x.Refs. 29,34 and various experimental procedures

w x16,27,58,62,64 . The latter studies have focused on partialaspects of these projections, such as the vestibulospinalw x w x62,64 , vestibulo-oculomotor 58 and lateral-line- and

w xvestibulo-reticular secondary projections 16,27 .To date, there have been no published experimental

studies of the connections of the TS in lampreys, with theexception of the occasional reports of anterograde-labeledfibers in the TS after tracer application to certain forebrain

w xsystems 50,57 . The current lack of knowledge about theconnections of the lamprey TS means that there is no firmbasis for comparison the TS of other vertebrates. The aimof the present study was to determine the origin of toropetalfibers and the targets of TS efferents in larvae of the sealamprey, Petromyzon marinus. This knowledge may helpunderstanding of the evolution of the TS in vertebrates.

2. Material and methods

2.1. Animals

Experiments were performed on 39 larval sea lampreysŽ .P. marinus ranging from 80 to 133 mm in length. Theanimals were anesthetized with tricaine methane sulfonateŽ .MS-222, Sigma, St. Louis, MO; 100 mgrl . All experi-ments were conducted in accordance with European Com-munity guidelines on animal care and experimentation.Three different tracing methods were used.

2.2. In ÕiÕo labeling

Ž .For horseradish peroxidase HRP labeling, 13 larvaewere used. After surgical exposure of the brain, HRP pasteŽa concentrated solution of Sigma type-VI HRP in distilled

.water, desiccated on the tip of a 000 insect pin wasapplied to the appropriate brain region for 1–2 min. Appli-

Ž .cations were made to the torus semicircularis ns6 , toŽ . Ž .the ventral ns3 and the medial ns2 octavolateral

nuclei and to the middle reticular rhombencephalic nucleusŽ . Ž w x.ns2 for technical details, see Ref. 27 . The brainswere then washed with Ringer’s solution to remove excesstracer. The larvae were then revived and transferred to

Ž .glass tanks containing fresh cold 48C Ringer’s solutionfor 2–5 days to allow transport of the tracer. After thesurvival period, larvae were reanesthetized and their brainswere fixed in 1% paraformaldehyde and 1% glutaralde-

Ž .hyde in 0.1 M phosphate buffer PB , pH 7.4, for 2–4 h.HRP labeling was revealed in toto by the cobalt–nickel-in-

tensified diaminobenzidine method. Brains were then de-Ž .hydrated, embedded in Spurr’s resin Taab, Berkshire, UK

and transversely sectioned at 30 mm thickness on a slidingmicrotome after warming the block surface. The sectionswere serially mounted on slides with epoxy resin.

ŽIn fluorescein-coupled dextran amine FDA, MW 3.000.Da, Molecular Probes, Eugene, OR and Texas-Red-cou-

Žpled dextran amine TRDA, MW 3.000 Da, Molecular.Probes experiments, a paste of the tracer was applied as in

Ž .HRP experiments to the torus semicircularis ns2 , oc-Ž . Žtavolateral nuclei ns2 , trigeminal descending root ns

. Ž . Ž .2 , dorsal thalamus ns3 , ventral thalamus ns1 andŽ .optic tectum ns2 . After a survival period of 24–65 h,

larvae were reanesthetized and their brains were fixed in4% paraformaldehyde in PB for 24–48 h. Vibratome

Ž .sections 40–50 mm thick were collected on gelatin-coatedŽ .slides, mounted in a glycerol–PB mixture 1:1 , and exam-

ined and photographed with a Nikon fluorescence photo-microscope equipped with fluorescein or rhodamine filtersets, as appropriate.

2.3. Carbocyanine labeling in fixed tissue

For this method, larval lamprey brains were fixed in 4%paraformaldehyde in PB, pH 7.4, for 24–48 h, and thenembedded in 3% agarose. Crystals of 1,1X-dioctadecyl

X X Ž3,3,3 ,3 -tetramethylindocarbocyanine perchlorate DiI,.Molecular Probes, Eugene, OR were applied to the torus

Ž . Ž .semicircularis ns7 , the medial ns2 and the ventralŽ . Ž .ns2 nuclei of the octavolateral area, the dorsal ns1

Ž .and ventral thalamus ns1 and to the optic tectumŽ .ns1 . Brains were left in fixative for 4–40 days at 378Cin the dark for diffusion of the tracer in cell membranes.

Ž .Transverse sections of the brains 40–50 mm thick ob-Žtained on a vibratome Vibroslice, Campden Instruments,

.Cambridge, UK were collected on gelatin-coated slides,Ž .mounted in a glycerol–PB mixture 1:1 , and examined

and photographed using a rhodamine filter set, as above.

3. Results

3.1. Organization of the torus semicircularis

The torus semicircularis of larval lampreys extendsalong the ventral surface of the midbrain ventricle, be-tween the optic tectum dorsally and the mesencephalictegmentum ventrally, forming a small protrusion in themesencephalic ventricle. It consists of periventricular graymatter, and is composed of several compact layers of cellsthat are separated from the ependyma by a subependymallayer of fibers, and of a wide lateral neuropil extending to

Ž .the meningeal surface Fig. 1 . A schematic view of thelocation of the torus semicircularis in the brain of a larvallamprey is shown in Fig. 2.

( )M.J. Gonzalez et al.rBrain Research 826 1999 83–94´ 85

Fig. 1. Transverse section through the mesencephalon of a large, premeta-morphic larval lamprey to show the periventricular distribution of

Ž .perikarya in the torus semicircularis between the two arrows and theŽ .wide region of lateral neuropil asterisk in the torus semicircularis. Star,

optic tectum. Hematoxylin-eosin. Scale bars100 mm.

3.2. Afferents to the torus semicircularis

3.2.1. Projections from the OLAApplication of HRP, FDA and DiI to the torus semicir-

cularis labeled both neurons and nerve fibers in several

Ž .brain regions Fig. 3 . In general, HRP and FDA applica-tions produced a Golgi-like labeling of neuronal perikaryaand a more intense labeling of fiber tracts than DiI, thoughthe qualitative results obtained with these procedures weresimilar. Nerve fibers were stained anterogradely, formingbouton-like structures in the terminal fields.

After application of tracers to the torus semicircularis,some cell populations were labeled in the rhomben-cephalon, mesencephalon and diencephalon. In therhombencephalon, most labeled neurons were observed in

Ž .the octavolateral area OLA . Here, most labeled neuronswere located periventricularly, on both the ipsi- and thecontralateral side, though most abundantly on the con-

Ž .tralateral side Figs. 3I–J and 4A–F . Most of these neu-rons were piriform cells with a lateral process extendingand branching into the neuropil associated with the pri-

Žmary lateral line fibers, i.e., medial nucleus cells Fig..4B,D . A thin axon emerges from the opposite pole of the

cell and courses ventrally, joining the internal arcuate fibersystem. These neurons were labeled throughout the entire

Ž .medial octavolateral nucleus Fig. 3I–J . A few labeledcells of the medial OLA nucleus were larger neurons witha thick process running parallel to the ventricular surfaceand giving rise to a number of thinner lateral processesŽ .Fig. 4C .

In the ventral octavolateral nucleus, scattered neuronsŽ .with non-piriform morphologies bipolar or triangular cells

Žwere labeled bilaterally. A few large cells spindle-shaped.cells of the anterior and intermediate octavomotor nuclei

Ž .were sometimes stained Fig. 4E .Ž .In the dorsal electroreceptive octavolateral nucleus, a

few small neurons with monopolar outline were occasion-ally labeled in a subependymal location, mostly contralat-

Ž .erally Figs. 3J and 4A . The neuropil of this nucleus wasalso labeled on the contralateral side.

In order to confirm the termination of secondary oc-tavolateral fibers in the torus semicircularis, DiI or HRPwas applied to the octavolateral nuclei at the level of theVIIIth nerve entrance. This type of application produced

Fig. 2. Schematic drawing of a lateral view of the larval lamprey brain showing the main brain regions and also indicating the levels of the sections of Fig.3. For abbreviations, see legend of Fig. 3.

( )M.J. Gonzalez et al.rBrain Research 826 1999 83–94´86

Ž . Ž .Fig. 3. Schematic drawing of transverse sections of the brain indicating the distribution of perikarya dots and fibers dashes labeled after tracerŽ .application to the torus semicircularis shaded area in G . Only the regions containing neuronal perikarya are indicated at the right of the figures, with the

exception of a few areas of neuropil. Ipsilateral labeling is represented at the left side of the sections, contralateral at the right side. The levels of thesections correspond to those indicated in Fig. 2. DL, dorsolateral pallium; DM, dorsomedial pallium; DO, dorsal octavolateral nucleus; DT, dorsalthalamus; F, interventricular foramen; HL, left habenula; HR, right habenula; HY, hypothalamus; IP, interpeduncular nucleus; IS, nucleus isthmi; IV,trochlear nucleus; LN, lateral neuropil of the torus semicircularis; M, medial pallium; M5, nucleus M5; MO, medial octavolateral nucleus; NH,neurohypophysis; NX, vagal motor nucleus; OB, olfactory bulb; OC, optic chiasma; OM, oculomotor nucleus; OT, optic tectum; P, pineal organ; PC,posterior commissure; PO, preoptic area; PP, parapineal organ; PR, pretectum; PT, posterior tubercle; RA, anterior reticular nucleus; RM, medial reticularnucleus; RP, posterior reticular nucleus; SE, septum; SF, solitary fascicle nucleus; SH, subhippocampal lobe; ST, striatum; TS, torus semicircularis; VD,trigeminal descending nucleus; VIII, octaval nerve; VL, ventrolateral pallium; VM, trigeminal motor nucleus; VO, ventral octavolateral nucleus; VT,ventral thalamus.

( )M.J. Gonzalez et al.rBrain Research 826 1999 83–94´ 87

Ž . Ž .Fig. 4. Transverse sections through the rhombencephalon showing labeled cells in the ipsi- right side and contralateral left side octavolateral area afterŽ . Ž . Ž .FDA application to the torus semicircularis TS . A Section at the level of the anterior lateral line nerve showing cells in the dorsal small arrows , medial

Ž . Ž . Ž . Ž . Ž . Ž .thick arrows and ventral long arrow OLA nuclei. B Labeled cells in the contralateral dorsal small arrows and medial thick arrow OLA nuclei. CŽ . Ž .Detail of a large cell labeled in the ipsilateral medial nucleus. D Detail of piriform cells of the ipsilateral medial nucleus. E Two large spindle-shaped

Ž . Ž .cells arrow labeled in the ipsilateral anterior octavomotor nucleus. F Long arrows point to groups of reticular cells, thick arrows to medial nucleus cells.ŽStars, fourth ventricle; d, dorsal octavolateral nucleus; m, medial octavolateral nucleus; v, ventral octavolateral nucleus. Scale barss100 mm for A, B, E

. Ž .and F ; s50 mm for C and D .

the labeling of ascending axons that course ventrally in theipsi- and contralateral rhombencephalon. These axons runthrough the ipsilateral basal plate and accumulate in aposition ventromedial to the descending root of the trigem-inal nerve. Some axons cross the midline as arcuate fibers.At caudal mesencephalic levels, the labeled axons rundorsally and medially to terminate in the inner half of the

Žlateral neuropil of the torus semicircularis results not.shown . These fibers branch and give rise to very thin

beaded fibers, with the appearance of a terminal field. Afew labeled fibers run into the region of the TS perikarya,and a few also course to the ventral part of the optictectum. This type of tracer application also produced thelabeled fibers to additional structures, as reported previ-

Ž w x.ously see Ref. 27 .

3.2.2. Reticular cells projecting to the TSAfter tracer application to the TS, small and medium-

sized multipolar and bipolar cells were observed in associ-ation with the trigeminal and facial motor columns, in the

transition between the basal plate and the OLA and also inmore medial locations of the rhombencephalic basal plateŽ .Figs. 3I–K and 4F . In general, these cells appeared eitherscattered or in small groups, both ipsi- and contralateral tothe application site. Of these cells, the medial neurons maybe considered as small cells of the medial reticular forma-tion, i.e. the medial and posterior rhombencephalic reticu-

Ž w x.lar nuclei see Ref. 47 , while the lateral neurons are partof an ill-defined lateral reticular formation.

3.2.3. Trigeminal and spinal toropetal projectionsTracer application to the TS also labeled neurons of

bipolar and multipolar morphology that were associatedŽ .with the descending trigeminal tract Figs. 3J–K and 5A .

These cells were more abundant caudally, just at theŽcaudal end of the posterior octavomotor nucleus where

.the OLA disappears , but labeled cells were also scatteredventral to the OLA in the region of the octaval nerveentrance and also in the dorsolateral region of the spinalcord. These cells were labeled only contralaterally to the

( )M.J. Gonzalez et al.rBrain Research 826 1999 83–94´88

application site. The axons of these trigeminal neuronscourse transversely from the levels containing perikarya to

cross the midline ventrally, and ascend scattered in theventrolateral region of the brainstem.

( )M.J. Gonzalez et al.rBrain Research 826 1999 83–94´ 89

Application of tracers to the region of the descendingtract of the trigeminal nerve also labeled fibers in thecontralateral torus semicircularis, mainly in the inner halfof the lateral neuropil.

3.2.4. MesencephalonApplication of tracers to the TS retrogradely labeled

neurons in several mesencephalic regions adjacent to theinjection area, although the number of labeled cells varieddepending of the size of the application area. In the optictectum, round periventricular cells were labeled ipsilater-ally in the ventral optic tectum, along the levels of the

Ž .application area Fig. 3F–G . These cells are probablytecto-toral and tecto-tegmental projecting cells. Thus, theapplication of DiI or FDA to the optic tectum produced thelabeling of thin beaded fibers that course in the lateralneuropil of the torus semicircularis and reach the retinopetal

Ž .M5 nucleus Fig. 5B .Periventricular cells of the retinopetal M5 nucleus were

also generally labeled following tracer application to theŽ .TS Fig. 3G . It is known that the axons of M5 cells run in

lateral regions of the TS to the optic nerve and retinaw x11,73 . Whether axons of these cells actually contact TScells could not be determined. Scattered cells also ap-peared labeled in other regions of the mesencephalictegmentum, such as the mesencephalic reticular area, bothipsi- and contralaterally to the injection site. Sometimes,labeled cells were found in the contralateral TS, occupyinga symmetrical position with respect to the HRP injectionsite.

3.2.5. DiencephalonSome labeled cells were observed in the periventricular

hypothalamic nucleus, between the anterior and posteriorŽ .infundibular recesses Figs. 3D–E and 5C . A subset of

these cells was of cerebrospinal-fluid-contacting type. Mostthese cells were ipsilateral to the injection site. Medium-sized cells with long dendrites were also observed bilater-ally in the nucleus of the posterior tubercle, dorsal to the

Ž .posterior infundibular recess Figs. 3F and 5D .Rostral to the posterior tubercle, groups of ipsilaterally

labeled cells were observed in periventricular positions inŽthe ventral and dorsal thalamic nuclei Figs. 3D–E and

.5E–F . These cells were monopolar with round or oval

bodies, oriented perpendicular to the ventricular surface.Dendrites of these cells extend into the lateral neuropil,and axons course in the lemniscal pathways.

FDA application to the dorsal thalamus led to labelingŽ .of fibers in the neuropil of the TS Fig. 6A–B . In

addition, a few faintly stained neurons could be observedin the ipsilateral TS. This result suggests the presence ofbi-directional connections between the dorsal thalamus andthe TS.

3.2.6. TelencephalonApplication of DiI to the TS led to labeling of some

neurons and fibers in the telencephalon. A few labeledŽcells were observed in the striatum and septum Figs.

.3A–C and 5G–I , and scattered cells were also observedŽ .in the preoptic nucleus Figs. 3C–D and 5C . Labeled

fibers were additionally found coursing into both pallialand subpallial regions. In the lateral pallium, fibers coursed

Žin the ventral, lateral and dorsal portions Figs. 3A–B and.5H . Whether these DiI-labeled fibers originate from telen-

Ž .cephalic neurons i.e., are axon collaterals , from toralneurons or from other cells could not be ascertainedbecause this tracer diffuses in cell membranes in all direc-tions from the bifurcation points of processes.

3.3. Efferents

Application of DiI to the torus semicircularis producedthe labeling of numerous fibers in different regions of the

Ž .brain, from the telencephalon to the spinal cord Fig. 3 .Most of these fibers appear to run ipsilaterally to the

Ž .thalamus Figs. 3D–E and 5E–F and to basal regions ofthe medulla oblongata. Some labeled fibers were observed

Žin the telencephalon lateral pallium, striatum and septal. Ž .region see above , preoptic region, hypothalamus, optic

Žtectum and mesencephalic tegmentum Fig. 3A–G; seealso Fig. 2 for localization of the different brain regions in

.the brain of the larval lamprey . The alar plate of themedulla oblongata does not appear to receive torofugalfibers. In order to confirm the torofugal nature of some ofthese fibers, we made applications of HRP, DiI or FDA tothe brainstem reticular areas, dorsal thalamus and optictectum. We also considered the results of previous experi-ments by our group involving tracer application to other

Ž .Fig. 5. A Section through the region of the vagal nerve entrance showing trigeminal secondary neurons labeled after application of HRP to theŽ . Ž . Ž .contralateral TS. B Anterogradely labeled fibers running in the TS arrow after DiI application to the ipsilateral optic tectum black star . The wide

Ž . Ž .arrow points to the M5 retinopetal nucleus. C Labeled cells in the wall of the anterior infundibular recess thin arrow and the caudal preoptic nucleusŽ . Ž . Ž .thick arrow after DiI application to the TS. D Group of labeled cells of the posterior tubercle arrow after DiI application to the TS. White asterisk,

Ž . Ž .posterior infundibular recess. E Transverse section through the thalamus at the level of the posterior commissure white asterisk showing labeled neuronsŽ . Ž . Ž .in the periventricular region and a large field of fibers black asterisk . F Labeled cells arrow in the ventral thalamus after FDA application to the TS. A

large cell is shown in the contralateral side. Black asterisk, field of labeled fibers; h, neurohypophysis. G, Transverse section through the telencephalonŽ .showing labeled cells in the striatum arrow after DiI application to the TS. Black star, pineal organ; black asterisk, optic chiasma. H, I, transverse section

Ž .through the telencephalon after DiI application to the ipsilateral TS showing labeled perikarya in the septum arrow in H, black arrowheads in I , andŽ . Ž . Ž . Žlabeled fibers in the lateral pallium white asterisk . White star C–F , third ventricle. Scale barss100 mm for C, D, E, F, G and H ; s50 mm for A, B

.and I .

( )M.J. Gonzalez et al.rBrain Research 826 1999 83–94´90

Ž . Ž .Fig. 6. Neurons of the torus semicircularis arrows labeled after FDA application to the ipsilateral dorsal thalamus A–B and HRP application to theŽ . Ž .ipsilateral medial reticular rhombencephalic nucleus C . Note the presence of thin toropetal fibers arrowheads . The wide arrow in A points the

Ž . Žretinopetal M5 nucleus, the asterisks indicate the optic tectum, and the white star the interpeduncular nucleus. Scale barss100 mm for A ; s50 mm for.B and C .

neuronal systems. HRP and TDA application to the middleand posterior rhombencephalic reticular nuclei producedlabeling of some neurons in the torus semicircularis, ipsi-

Ž .lateral to the application site Fig. 6C . This type ofapplication also produced the labeling of a number offibers in the neuropil of the TS, both ipsi- and contralater-

Ž .ally Fig. 6C . Application of tracers to the region of theoculomotor nucleus also retrogradely labeled some neu-rons in the torus. Application of FDA to the dorsal thala-

Ž .mus led to labeling of some toral neurons Fig. 6A–B .Occasional torus neurons were labeled after tracer applica-tion to the optic tectum. Application of tracers to several

Žother brain regions telencephalon, habenula, pineal com-.plex, and octavolateral area did not result in labeling of

any neurons in the torus semicircularis.The cells of origin of torofugal projections were piri-

form cells with branched dendrites extending in the lateralŽ .neuropil and having coiled or tortuous profiles Fig. 6C .

In cases of tracer application to the rhombencephalic retic-Ž .ular nuclei, thin beaded fibers toropetal could be easily

observed contacting these dendrites. Most labeled perikaryaoccupied outer regions within the cell layers of the torussemicircularis.

4. Discussion

The results of the present study of larval lampreysindicate that a proportion of the neurons projecting to thetorus semicircularis are cells of the octavolateral area, butthat the TS also receives projections from several other

systems. These findings are in accordance with the resultsof early anatomical studies on adult lampreys, which indi-cated that the TS receives most of its afferents from the

w xvestibulo-lateralis lemniscus 29,53,69 , but at the sametime considerably expand our knowledge of the afferentconnections of this structure. Moreover, this experimentalstudy demonstrates, for first time in agnathans, the pres-ence of massive ascending projections of the TS to thethalamus and of descending projections to the ipsilateralbrain stem.

4.1. Afferent connections

The octavolateral area of lampreys can be subdividedŽ .into three nuclei dorsal, medial and ventral , specialized

in the processing of electroreceptive, lateral linew xmechanosensory, and octaval inputs, respectively 48 .

w xBodznick and Northcutt 3 have recorded electrosensoryresponses in the TS of adult lampreys. Octaval and lateralline primary afferents project to separate regions of theOLA, although some overlap has been observed in both

w x w xlarval 25,26 and adult lampreys 32 . Ascending projec-tions to the TS arise from all these regions, although theneurons of the dorsal OLA nucleus are difficult to label inlarvae. This might be a consequence of the poor develop-ment of this electrosensory region during larval stages, in

w xagreement with a previous result 27 . Our results indicateŽthat the octaval region of the lamprey ventral octavolat-

.eral nucleus projects to the TS, in agreement with resultsw xobtained in other vertebrates 12,18,21 . In amphibians and

( )M.J. Gonzalez et al.rBrain Research 826 1999 83–94´ 91

other vertebrates, the octavo-toral projections are auditoryw x8,20,46,75 . The available evidence on hearing in lam-

w xprey, however, is uncertain 35,65 . Some responses tomechanical vibration have been recorded from the labyrinthw x35 , but there are no studies of auditory responses ofcentral neurons in lampreys. Although the presence ofoctavo-toral projections in lampreys suggest they maycarry auditory information, to know whether these projec-tions are auditory andror mechanoreceptive needs electro-physiological studies.

In other fishes and in amphibians, mechanosensory,electrosensory and auditory systems form independent andparallel lemniscal pathways to morphologically specialized

w x w xsubnuclei of the TS 4,12,21,38,39 . Knudsen 31 , in astudy of Ictalurus, was the first to show subdivisions ofthe TS of a teleost, and described two lateral subnuclei thatreceived lateral line mechano- and electroreceptive inputsand a medial nucleus that received acoustic information.Similar results have also been obtained in chondrichthyansw x w x4,9 , other teleosts 2,18,21,28 and amphibiansw x1,23,77,83 . These studies have indicated that the terminalareas of mechanosensory, electrosensory and octaval fibersremain segregated within the TS. With the procedures usedhere, a morphological subdivision of the TS of the larvallamprey into subnuclei was not evident. However, whetherthe different octavolateral sensory modalities are segre-gated in the lamprey TS needs further study.

In many anamniote vertebrates, a proportion of theauditory projections to the TS are relayed via the nucleusof the lateral lemniscus and the superior olivary nucleusw x w x2,4,5,9,12,18,21,41,44,48 54,68,75,78 . Although in lam-preys these nuclei have not yet been described, there is afairly number of tegmental neurons in the region of theoctaval nerve that project to the TS. Some of these neuronsmight be a lamprey relative to the lemniscal and olivarypopulations found in other vertebrates.

Our results in lamprey indicate the presence of a con-tralateral projection to the TS originating from neuronsassociated with the trigeminal descending root, mostlylocated in the medulla oblongata between the levels of theoctaval and vagal nerve entrance. A few spinal neuronswere also observed to project to the TS. These resultssuggest the presence in lampreys of a somatosensory pro-jection to the TS, mediated mainly by secondary trigeminalneurons. The presence of secondary trigeminal projectionsto the torus semicircularis has been reported in a teleostw x44 . A contralateral projection to the torus from perisoli-

w xtary cells has also been reported in frogs 75 , and it ispossible that they form part of the same system. Thetoropetal medullary and spinal projections of lamprey arealso reminiscent of those of the dorsal column nucleus andthe ‘ventral quadrant system’ of the spinal cord of amphib-

w xians, respectively 42,43 . Convergence of somatosensoryŽ .and octaval auditory information in the TS has been

w x w xdemonstrated in reptiles 33 and pigeons 76 . Thus, re-sults in vertebrates as different as lampreys and sauropsids

suggest a shared convergence of somatosensory and oc-Ž .taval octavolateral systems in the torus semicircularis.

Other toral afferents in larval lamprey originate fromnuclei in the subpallial telencephalon. Specifically, somecells in the striatum, septum and preoptic region project tothe TS. Studies with anterograde tracers in adult lampreyhave found a few fibers in the TS after tracer application

w xto the medial pallium 50,57 , but not after tracer applica-tion to the lateral pallium. These results are consistent withour results in larvae showing that striatal and septal cells,but not pallial cells, are retrogradely labeled by tracerapplication to the TS. In teleosts, some telencephalic neu-

w xrons appear to project to the TS 18,44 , although these arecells of the dorsal telencephalic area, generally consideredto be homologous to the lamprey pallium. A telencephalicprojection from the anterior entopeduncular nucleus to the

w xTS has also been reported in the bullfrog 75 .The absence of retrogradely labeled neurons in the

pineal organ after tracer application to the TS is in dis-agreement with the reported presence of pineal projections

w xto the TS in adult 60 . The results of a previous study ofw xlarval lampreys 82 , together with present results, suggest

instead that the pinealo-tegmental fibers ‘avoid’ the TSregion and project mainly to the retinopetal nuclei andother regions of the tegmentum. A considerable number ofneurons were labeled in the walls of the preoptic andinfundibular recesses, including a conspicuous group in theposterior tubercle. In these regions, many neurons containbiogenic amines andror a variety of neuropeptides. Im-munocytochemical studies in larval lamprey have shownthe presence of dopaminergic and serotoninergic fibers in

w xthe torus semicircularis 81 , and similar patterns are foundw xin adults 55,56,59 . The cells labeled in the posterior

tubercle have a location and morphology similar to that ofthe large tyrosine–hydroxylase-immunoreactive neurons

w xobserved in this region 56,81 , and indeed these neuronsmight be the origin of a portion of the dopaminergic fibersobserved in the TS. Other neurons labeled in the walls ofthe infundibulum and preoptic recess might be the originof other aminergic or peptidergic fibers observed in the TSw x81 . Our results thus indicate that the lamprey torussemicircularis may be regulated by a variety of neuroactivesubstances produced by hypothalamic cells. Immunocyto-chemical studies have also demonstrated the presence of a

w xvariety of neuroactive substances in the TS of teleosts 10 .In a teleost, some retrograde-labeled neurons have alsobeen reported in the periventricular hypothalamus after DiI

w xapplication to the TS 70 , suggesting that hypothalamo-toral projections are present in other fishes. The amphibiantorus semicircularis receives a rich monoaminergic inner-

w xvation 24 , and tracer studies in frog also indicate thatcells of the caudal preoptic area, hypothalamus and poste-

w xrior tubercle occasionally project to the torus 75 , inagreement with our results in larval lamprey.

Some thalamic neurons project to the lamprey TS. Inteleosts, experimental studies have found one or two thala-

( )M.J. Gonzalez et al.rBrain Research 826 1999 83–94´92

w xmic nuclei that project to the TS 18,44,70 , which isroughly in agreement with our observations in lamprey. Infrogs, some dorsal and ventral thalamic neurons also pro-

w xject to the torus semicircularis 20,37 .w xTectal neurons project to the TS in holosteans 49 and

w xteleosts 36,44,66,80 , a result similar to that obtained inlampreys, in which a sparse projection has been observedŽ .present results . However, similar tecto-toral projections

w xhave not been reported in frogs 20 . Electrophysiologicalstudies have shown the responsiveness of toral units toboth visual and auditory stimuli, although the visual re-

w xsponses of the toral units are poor 67 . Other tegmentalregions adjacent to the torus also appear to give rise to

Ž .toral projections in lamprey present results and frogsw x20 .

Finally, another important source of afferents to thelamprey TS arose from scattered neurons in the rhomben-cephalic reticular formation. DiI application to the TSresulted in ipsilateral and contralateral labeling of neuronsin the middle and posterior rhombencephalic reticular nu-clei, between the entrance levels of the Vth and Xthnerves, and also of more lateral cells here considered as apart of a lateral reticular region. The finding of a reticulo-

w xtoral projection is similar to that obtained in a teleost 44 .Reticulo-toral projections have also been described in frogsw x w x75 , but not in pigeon 76 .

4.2. Efferent connections

The TS gives rise to ascending and descending projec-tions. The ascending toral projections of lampreys appearto be roughly similar to those of other vertebrates. Anumber of labeled fibers were observed in hypothalamic,thalamic and telencephalic regions of lamprey after tracerapplication to the TS. The labeled fibers in the thalamusand hypothalamus of lamprey are reminiscent of the termi-nation fields observed in the anterior tuberal nucleus andcentral thalamic nucleus after DiI application to the TS of

w xthe channel catfish 70 . Similar ascending projectionsfrom the TS to the thalamus have been also reported in

w x w xfrogs 20,46 and reptiles 6,22,30 . In the telencephalon oflamprey, numerous fibers were labeled after DiI applica-tion to the TS. However, the absence of labeled neurons inthe TS after tracer application to pallial regions in adult

w xlampreys 50,57 argues against the existence of directtoro-pallial projections, although direct toro-telencephalic

w xprojections have been reported in a frog 74 . On the basisof the present results, the possibility that a subset of these

Žlabeled fibers especially those observed in the lateral.pallium and subpallium are collaterals of descending te-

lencephalic fibers cannot be ruled out.The main descending efferent projection of the lamprey

TS was the toro-reticular projection. This result is inagreement with recent observations using the cobaltous-

w xlysine method in lampreys 84 . Descending toro-medullaryand toro-spinal efferents have been observed in teleosts

w x w x w x12 , amphibians 20,45,75 and reptiles 7,71,79 , mainlyipsilaterally. However, the TS does not appear to project to

w xthe spinal cord in lampreys 17 .The reticulospinal neurons in lampreys form the main

descending system from the brain to the spinal cord,making monosynaptic excitatory connections with spinal

w xinterneurons and motoneurons 51 . The reticular formationof lampreys receives information from different sensorysystems, such as the lateral line and vestibular centers and

w xthe visual system 13–16,27,52,64,72 . Recently, it hasalso been shown that the reticular formation receives affer-ents from higher centers such as the thalamus, pretectum,

w xoptic tectum and mesencephalic tegmentum 19,84 . Pre-sent results also indicate that the TS is a source of affer-ents to the reticular formation, suggesting that this centermight have a role in the control of body movements.

In conclusion, the torus semicircularis of larval lam-preys receives projections from several brain centers in-

Žcluding some types of secondary sensory fibers lateral.line, vestibular and general somatosensory fibers , proba-

bly representing a multisensory integration center. Thepresence of efferent projections to the thalamus, hypothala-mus and the rhombencephalic reticular area indicates thatthe TS intervenes in control of ascending sensory path-ways and in premotor circuits. Finally, the marked similar-ity of TS connections in lampreys and jawed vertebratessuggests that the TS system evolved before the separationof lampreys from the gnathostome line.

Acknowledgements

This study was supported by grants from the SpanishŽ .Education Ministry PB96-0945-C02 and the Xunta de

Ž .Galicia XUGA20002B97 .

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