in Teleost Fishes: A Transneuronal Biocytin Study in Mochokid Catfish

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1 THE JOURNAL OF COMPARATIVE NEUROLOGY 374: (1996) Sonic/Vocal-Acousticolateralis Pathways in Teleost Fishes: A Transneuronal Biocytin Study in Mochokid Catfish FRIEDRICH LADICH AND ANDREW H. BASS Institute of Zoology, University of Vienna, Vienna, Austria (F.L.); Section of Neurobiology and Behavior, Cornell University, Ithaca, New York (A.H.B.) UC Bodega Marine Laboratory, Bodega Bay, California (A.H.B.) ABSTRACT Mochokid catfish have two sound-producing (sonic) organs-a pectoral spine stridulatory apparatus and a swimbladder whose vibration is established by nearby drumming muscles. Dextran-biotin or biocytin application to sonic nerves or muscles identified topographically separated motoneuron pools. Pectoral spine-related motoneurons are located within the ventral motor column whereas swimbladder motoneurons lie just ventral to the central canal or fourth ventricle. Axons of both groups of motoneurons exit the brain and spinal cord via ventral roots of occipital (swimbladder and pectoral) and spinal (swimbladder only) nerves. Transneuronal biocytin transport identified an extensive premotor network only for the swimbladder motor nuclei. Premotoneuron somata are located ipsilaterally in 1) a dorsolateral region of the sonic motor nucleus (SMN); motoneurons were clustered in the ventromedial region of the SMN and 2) the ventromedial medulla at the rostral pole of the SMN. Biocytin-filled fibers and less frequently premotoneuron somata were also found in the contralateral SMN. Biocytin-labeled fibers were continuous farther rostrally with 1) a commissural bundle that terminated bilaterally in the medial reticular formation near the caudal pole of the descending octaval nucleus and 2) a lateral brainstem bundle that terminated ipsilaterally in regions of the medulla and cerebellum considered to subserve acoustic and lateral line functions. Together with other data in distantly related teleost fishes, the results support the hypotheses that 1) central pathways linking sound-generating (sonic or vocal) and acoustic regions of the brain are traits common to both teleosts fishes and tetrapods that actively generate sounds, and 2) sonic/vocal pathways in teleosts have a conserved pattern of organization suggestive of common developmental origins. c 1996 Wiley-Liss, Inc. Indexing terms: brainstem, cerebellum, auditory, lateral-line, vocalization Sound production during intra- and interspecific behavior is known in representatives of families of bony fishes (reviews: Fine et al., 1977; Myrberg, 1981; Ladich, 1997). Teleosts have diverse sound-producing mechanisms, but only catfishes (order Siluriformes) have evolved two sonic organs: a pectoral fin stridulatory apparatus and a swimbladder with drumming muscles. Rubbing of the enlarged first fin ray in a groove of the pectoral girdle causes high-frequency, stridulatory sounds, whereas contractions of drumming muscles cause the swimbladder to vibrate and emit low-frequency sounds (Ladich and Fine, 1994). Stridulation in catfish involves movement of the first enlarged pectoral fin ray by hypertrophied adductor and abductor muscles (Gainer, 1967; Schachner and Schaller, 1981). The origins and insertions of swimbladder drumming muscles vary among different families of catfish (Tavolga, 1962; Abu-Gideiri and Nasr, 1973; Kastberger, 1977; Schachner and Schaller, 1981). In mochokids, ariids, and doradids, a flexible thin bony plate derived from the ventral process of the fourth vertebra is attached to the anterior part of the swimbladder. This so-called elastic spring or ramus Muelleri is the insertion site of protractor muscles; the swimbladder s vibration is thus established by a bony element set into oscillation by the protractor muscle. In ariids and doradids, the protractor muscle originates on postcranial elements (Tavolga, 1962; Kastberger, 19771, whereas in mochokids it originates from dorsolateral musculature (Abu-Gideiri and Nasr, 1973). The first goal of this study was to identify the positions of motoneurons innervat- Accepted May 25,1996 Address reprint requests to Andrew H. Bass, Section of Neurohiology znd Behavior, Cornell University, Seeley G. Mudd Hall, Ithaca, NY ahh3ii cornell.edu o 1996 WILEY-LISS, INC.

2 494 F. LADICH AND A.H. BASS ing the stridulatory and swimbladder drumming muscles of catfish and the cranial and/or spinal nerves providing that innervation. The central circuitry controlling sound production via swimbladder drumming muscles has been extensively studied in only one order of marine teleost fishes, the Batrachoidiformes (midshipman and toadfish; Bass and Baker, 1991). A recent study in midshipman using biocytin as a transneuronal tracer distinguished eight groups of neurons, including motoneurons, that form a link between sonic/vocal and acoustic regions of the brainstem (Bass et al., 1994). Catfish, unlike batrachoidids, are considered hearing specialists because of accessory structures (Weberian ossicles) that enhance the detection of acoustic signals (review: Popper and Fay, 1993). The neuroanatomical organization of the octavolateralis system of catfish is also among the best studied for teleosts (see Discussion). Hence, a second goal of this investigation was to identify the positions of premotoneurons in the drumming muscle pathway and possible links to the auditory system in a well-known hearing specialist that actively generates sound. Transneuronal biocytin transport was exploited as a neuroanatomical tracing method to identify a vocal motor network in sound-producing catfish. High-molecular-weight dextran-biotin provided verification of the location of motoneurons alone since it is apparently not transported transneuronally (Bass et al., 1994). We focused on mochokid catfishes, the only family so far described in which pectoral and swimbladder-generated sounds are clearly implicated in agonistic and reproductive functions (Abu-Gideiri and Nasr, 1973; also see Hagedorn et al., 1990; Baron et al., 1994). A third, comparative goal of this study was to consider the results within a broader phylogenetic context, namely the evolution of vocal circuitry among vertebrates. The swimbladder-associated drumming muscles of teleosts arise from occipital somites and share a common innervation by ventral occipital nerve roots that are considered homologues of the hypoglossal nerve of tetrapods (review: Bass and Baker, 1997). Bass and Baker (1997) propose that the brainstem circuitry controlling sound production among fishes and tetrapods shares a common descendant circuitry from caudal hindbrain rhombomeres which innervate sonic muscles derived from occipital somites. The latter includes swimbladder drumming muscles of teleosts and the laryngeal and syringeal muscles of tetrapods; hence our use of the term vocal when describing the sonic-swimbladder motor system (also see Bass et al., 1994). As shown here, similarities in the organization of sonicivocal pathways in Batrachoidiformes and Siluriformes now provide support for this hypothesis among distantly related teleosts. A preliminary report of these results has appeared elsewhere (Ladich et al., 1995). MATERIALS AND METHODS This study included 26 Synodontis nigromaculatus ( g) and ten Synodontis nigriventris ( g) purchased from local aquarium shops and maintained at 28 C in aerated and filtered but unplanted aquaria. Gross examination of gonads suggested that all specimens were sexually immature. Surgical and immunohistochemical procedures followed those of Bass et al. (1994). Briefly, following anesthetization with tricaine methanesulfonate (MS 2221, a lateral incision was made through the skin and body wall to expose the ipsilateral pectoral or swimbladder muscle. Portions of the dermo-supraoccipital bone were removed to help expose the nerves innervating the protractor muscle. Crystals of biocytin (Sigma, St. Louis, MO) or dextran-biotin (3 kd, Molecular Probes, Eugene, OR) were directly applied to the sonic muscle using a 00 insect pin. Crystal application to partially or completely severed sonic nerves gave the most complete pattern of biocytin or dextran-biotin labeling. The incisions were sealed shut with a protective layer of parafilm secured with Vetbond (3M, St. Paul, MN); the animal was then returned to its home aquarium for recovery. Following survival times of 1-5 days, specimens were deeply anesthetized with MS 222, perfused transcardially with cold 0.1 M phosphate buffer (PB), followed by cold 4% paraformaldehydei 1% glutaraldehyde dissolved in 0.1 M phosphate buffer. Brains were removed, placed in fixative for 1 hour, and stored overnight in 30% sucrose-pb. The following experiments involved labeling of either a sonic nerve and/or muscle using either biocytin (BC) or dextran-biotin (DB); the neuroanatomical results were Ahhreuiations Ax cg CC or cc CI Cm co CrC DO DS DSO EG EL ELLL EM FL FV GA HM LB LC LL m M axons of swimbladder motoneurons granule cell layer of corpus of cerebellum central canal cleithrum molecular layer of corpus of cerebellum commissural brainstem bundle crista cerebellaris descending octaval nucleus dorsal spine dermosupraoccipital bone eminentia granularis epiotic lamina electroreceptive lateral line lobe epaxial musculature facial lobe ventral fasciculus granule cell population of medial auditory nucleus hypaxial muscle lateral brainstem bundle lobus caudalis lateral lemniscus swimbladder muscle motoneurons medial nucleus MLF MS P NX oc OE OP PE PM PMN PN1,Z PS Pt RM Sb SMN so SP t tf Vorv VL Xm medial longitudinal fasciculus abductor muscle of the dorsal spine neuropil of SMN vagus nerve occipital nerve octavolateralis efferent nucleus operculum nucleus praeeminentialis protractor muscle pectoral muscle motoneurons premotoneuron groups pectoral spine posttemporal hone ramus Miielleri swimbladder sonic motor nucleus supraoccipital hone spinal nerve terminal bouton terminal field in medial reticular formation fourth ventricle vagal lobe vagal motor nucleus

3 VOCAL-ACOUSTICOLATERALIS PATHWAYS identical regardless of species: 1) drumming muscle: five BC, two DB; 2) drumming muscle nerve: nine BC, six DB; 3) pectoral spine muscles: three BC; 4) pectoral spine muscle nerve: six BC; 5) epaxial muscles: two BC. The saccular or anterior branch of the eighth nerve was labeled with biocytin in three additional specimens to aid in the identification of eighth nerve-recipient nuclei, octavolateralis efferent neurons, and the overall cytoarchitectonic organization of the medulla. Horizontal and frontal sections were cut frozen at 50 pm, stored in phosphate-buffered saline (PBS), and then processed as follows: 1) incubated 30 minutes in 0.4% Triton X-100 in PBS; 2) incubated 3 hours in an avidin-biotinylated horseradish peroxidase complex (Elite Kit, Vector Laboratories, Burlingame, CA); 3) washed twice, 10 minutes each, in 0.1 M PB; 4) incubated 1-2 minutes in 0.05% diaminobenzidine, 0.01% hydrogen peroxide dissolved in 0.1% PB; 5) washed twice in PB; and 6) stored in PB until mounted on chrom-alum-subbed slides. Selected slides were counterstained with cresyl violet; all slides were dehydrated in a graded series of alcohol and then coverslipped. The original research reported here was performed under the guidelines established by the National Institute of Health and the Cornell University Research and Animal Use Committee. RESULTS Pectoral spine innervation An enlarged part of the abductor and adductor muscles of the pectoral fin moves the enlarged pectoral fin spine (PS, Fig. 1). These muscles are attached to and originate at the ventral surface of the coracoid (not shown here, see Abu- Gideiri and Nasr, 1973). Sounds are always produced during abduction of the pectoral spine, but not necessarily during adduction (Schachner and Schaller, 1981; F. Ladich, unpublished observations). Biocytin labeling of the nerve which is part of the brachial plexus and innervates the abductor muscle resulted in extensive labeling of motoneuron somata and their dendrites within the ventral motor column (PMN, Figs. 2A, 3). Retrogradely labeled axons exited via the ventral root of the occipital nerve (OC, Fig. 3C). There was no indication of transneuronal transport. Swimbladder drumming muscle circuitry The sonic-swimbladder mechanism consists of bony plates that originate postcranially and attach to the anterior part of the swimbladder; drumming muscles are never in direct contact with the swimbladder. The elastic spring, the distal part of the ramus muelleri (PM, Fig. 1) is disc-shaped and only touches the swimbladder (Sb, Fig. 1) cranially. The hook-shaped protractor muscle (PM, Fig. 1) originates on epaxial musculature (EM, Fig. 1) ventral to the dorsal spine (DS, Fig. 1) and extends rostral and ventral before inserting via small tendons onto the bony plate of the ramus Muelleri. Biocytin or dextran-biotin labeling of the protractor muscle or its sonic nerve identified paired, sonic motor nuclei (SMN, Fig. 4) extending from the rostral spinal cord into the caudal medulla, lying between the central canal and fourth ventricle (CC, V; Fig. 4) and the medial longitudinal fasciculus (MLF, Fig. 4). Swimbladder motoneurons had a rostrocaudal extent overlapping that of pectoral spine-related motoneurons (see SMN; Figs. 2A, 3). Labeling a single sonic nerve gave extensive labeling of large, densely so PM /, //'/'/ 495 Fig. 1. Line drawing of a dorsolateral view of the postcranial region in Synodontis nigromaculatus showing the elastic spring mechanism and swimbladder protractor muscle (PM). Supraoccipital (SO), dermosupraoccipital (DSO), lateral post-temporal (Pt) bones, and dorsal process of the cleithrum (C1) have been partially removed to reveal the protractor muscle adjacent to the swimbladder (Sb) and elastic spring (ramus Miielleri, RM). Other abbreviations: DS, dorsal spine; EM, epaxial muscle; HM, hypaxial muscle; MS, abducter muscle of the dorsal spine; Op, operculum; PS, pectoral spine. stained somata that were mainly confined to medial and ventral regions of the SMN (m, Fig. 2B-D). Motoneuron somata were contiguous along the midline, except at their far rostral extent (Fig. 2C). Infrequently, a few labeled motoneurons were found laterally adjacent to the MLF. Dextran-biotin labeling of the sonic nerve only filled large somata clustered within the ventral and medial regions of the ipsilateral nucleus; these obviously represented motoneurons that directly innervate the muscle (Fig. 2D) and were entirely identical in position and morphology with large, biocytin-labeled somata (e.g., Fig. 2C). SMN somata were also obvious in densely stained Nissl sections (m, Fig. 2A). Most biocytin or dextran-biotin labeled axons exiting the SMN entered a ventral occipital nerve root (OC, Figs. 2B, 4D,E). Caudally, labeled axons also exited via a ventral root of the first spinal nerve (SP, Fig. 4A). SMN axons (AX, Fig. 2B) extended laterally along the MLF and ventral fasciculus (FV) before they entered the occipital or spinal root (Fig. 4). The motor branch of the vagus, which exits the brain laterally together with a sensory root, never contained labeled axons (NX, Fig. 4F). Biotin-labeled fibers filled a dorsolateral neuropil region of the ipsilateral SMN (np, Fig. 2B-D) which is also apparent in a Nissl-stained section (Fig. 2A). Labeled fibers were also distinguished contralaterally throughout the SMN only after biocytin labeling (Figs. 2B,C, 5A); motoneuron dendrites were infrequently labeled in the contralateral nucleus in dextran-biotin cases. Biocytin-labeled fibers crossed the midline at three positions at the level of the SMN: 1) ventrally between the MLF and FV (double

4 Figure 2

5 VOCAL-ACOUSTICOLATERALIS PATHWAYS 497 arrows, Fig. 2B, Fig. 4D), 2) at the rostral pole of the SMN (arrow, Figs. 2C, 4F), and 3) dorsal to the central canal (Fig. 4C; arrow, Fig. 5A). Ventrally crossing fibers continued contralaterally just ventral to pectoral motoneurons (LB, Figs. 2B, 4C-GI. Midline crossing fibers were never apparent after dextran-biotin labeling of a sonic nerve. In biocytin cases, neurons of varying size and shape were positioned among densely labeled fibers within the dorsolateral region of the ipsilateral, and infrequently the contralateral, SMN (PN1; Figs. ZC, 4E, 5B-D). These somata are referred to as premotoneurons since they were never found in dextran-biotin cases. At rostral SMN levels, biocytin-labeled processes extended ventrolaterally and were continuous ventromedially with a second group of premotoneurons in the medulla that were only labeled ipsilaterally (PN2, Figs. 4G, 6A). The PN2 neurons were continuous rostrally with a commissural bundle (CO, Figs. 4H, 6B) that formed a dense field of labeled terminals and fibers (tf, Figs. 4H, 6B-D) bilaterally within the medulla at the level of the caudal extent of the descending octaval nucleus (DO, Figs. 4H, 6B,D). The terminal field was formed in part by fibers extending along the lateral brainstem (LB, Fig. 4C-H) that first emerged contralaterally at the level of the SMN (LB, Fig. 2B). Labeled fibers of the commissural-associated terminal field extended toward the descending octaval nucleus (Figs. 4H, 6B,D), where there were sparsely distributed terminals (arrows, Fig. 6E). Farther rostral, biocytin-labeled fibers continued ipsilaterally along the lateral edge of the brainstem (LB, Fig. 7A,B), eventually turning medially. Some fibers terminate in a midline group of neurons identified as the octavolateralis efferent nucleus (OE, Figs. 7A, 8A); these neurons were retrogradely filled with biocytin following saccular nerve labeling (Fig. 8B). Slightly more rostral, the medially coursing bundle of biocytin-labeled fibers terminated in a granule cell population positioned along the fourth ventricle (GA, Figs. 7B, 8C; identified by Finger and Tong, 1984, as a rostral extension of a medial auditory nucleus). Terminals were occasionally found adjacent to the granule cells, but the densest terminal field was over the granule cell population itself. Terminals were also scattered along the trajectory of the medially coursing biocytin-filled fiber. Biocytin-filled fibers and sparse terminals extended lateral to the GA along a cell-poor region continuous with the caudal extent of the eminentia granularis where biocytinfilled fibers formed a dense terminal field (EG, Fig. 7B). Last, biocytin-labeled fibers continued rostrally within the ventral eminentia granularis and terminated along the dorsal and medial aspect of nucleus praeeminentialis (PE, Figs. 7C, 8D). Fig. 2. Photomicrographs of biotin-filled neurons and fibers in S. nigromaculatus after labeling of a single pectoral spine-associated brachial nerve (A, 3-day survival time) or sonic-swimbladder nerve (B-D, 2-day survival). Biocytin-filled pectoral spine motoneurons (PMN) were positioned in the ventrolateral motor column (A). Biocytin (B,C) and dextran-biotin (D) labeled swimbladder motoneurons (m) ipsilaterally. Sonic motor nuclei (SMN) extended along the midline (B-D) and had a rostrocaudal extent including that of pectoral motoneurons (see A). Biocytin-labeled fibers crossed the midline ventrally between the medial longitudinal and ventral fasciculi (double arrows, B; also see Fig. 4D) and at the rostral pole of the SMN (single arrow, C; also see Fig. 4F). Sections were counterstained heavily (A), lightly (B,C), or not at all (D) with cresyl violet. Scale bar = 150 km in A for A and B, 100 pm in C for C and D. Densely filled PN2 neurons were present in only three of the nine cases with biocytin labeling of the sonic nerve; commissural and lateral brainstem bundles were more robustly labeled in these cases. However, biocytin labeling of the commissural bundle and its associated terminal field were not dependent on biocytin-filled PN2 neurons, suggesting that it originates in part from PN1 neurons intrinsic to the sonic motor nucleus (Fig. 5). Biocytin transport to more rostral regions of the medulla and cerebellum via the lateral brainstem bundle (Fig. 7) was apparent only in cases in which PN2 neurons were densely filled, suggesting that they were the source of this ascending pathway. DISCUSSION Catfish, unlike other groups of sound-producing teleosts, have two sound-producing organs-pectoral fins used for stridulation and sonic muscles for swimbladder vibration. Pectoral fins have been adopted for sound production in diverse ways among teleosts including stridulation in catfish (Pfeiffer and Eisenberg, 1965; Gainer, 1967; Schachner and Schaller, 1981), drummingin triggerfish (Salmon et al., 19681, and tendon snapping in croaking gouramis (Kratochvil, 1978). Pectoral fins are also used in catfish, as other teleosts, for swimming and hovering; this correlates with there being little variation among teleosts in the origins and insertions of associated adductor and abductor muscles. Pectoral spine-stridulatory motoneurons in croaking gouramis and catfish are positioned within the ventral motor column lateral to the ventral fasciculus in the caudal brainstem and rostral spinal cord (this report; Ladich and F,ine, 1992, 1994), similar to pectoral fin motoneurons in other teleosts (Finger and Kalil, 1985). Transneuronal biocytin transport did not reveal any overlap in stridulatory and swimbladder motor circuitries, at least at the level of the medulla or spinal cord. Perhaps, midbrain and/or forebrain vocal centers will be the site(s) of origin of descending pathways coordinating the action of stridulatory and swimbladder sonic motor systems. Swimbladder drumming muscles, unlike pectoral fin muscles, have no other known function. Although drumming muscle origins differ among catfish, insertions are generally associated with fixed or moveable processes of the fourth vertebrae (the elastic spring). The hook-like muscle in mochokids usually inserts on the elastic spring. Our findings are in agreement with descriptions of Sorensen (1895) and Abu-Gideiri and Nasr (1973) who originally referred to the sonic muscle as a protractor muscle because the elastic spring is pulled cranially during muscle contractions. Drumming muscles are innervated by ventral roots of the occipital and first spinal nerves in mochokids, pimelodids and ariids (this report; Ladich and Fine, 1994; Ladich et al., 1995). Contrary to a previous nonexperimental report, sonic motor sons do not exit the brain via a motor branch of the vagus nerve in mochokids (Abu-Gideiri and Nasr, 1973). The anatomical demonstration of occipital nerve innervation is consistent with neurophysiological results for a fourth catfish family-doradids; Kastberger (1977) stimulated the ventral root of the occipital nerve in Doras sp. and elicited swimbladder drumming sounds. Although only a series of line drawings is presented by the authors, the motor nucleus which innervates the modified electric organ described by Hagedorn et al. (1990) in Synodontis negrita, obesus, and schoutedeni is identical in position with the swimbladder motor nucleus described

6 498 F. LADICH AND A.H. BASS FV Fig. 3. A-D: Line drawings of transverse sections from 5'. nigromaculatus following injection of biocytin in the abductor muscle that moves the pectoral spine during stridulation (2-day survival). Solid circles and lines represent, respectively, biotin-filled pectoral motoneu- here for mochokids known at least to be sonic. The modified electric organ identified by Hagedorn et al. (1990) and the protractor muscle identified here are also identical in position. The protractor muscle and motoneurons apparently have dual sonic and electromotor functions in weakly electric mochokids (also see Baronet al., 1994). Premotor circuitry The present study has now identified, for mochokid catfish, an anatomical linkage between caudal hindbrain, vocal motor, and rostra1 hindbrain acousticolateralis circuits (fig. 9). Earlier studies demonstrated the use of biocytin as a transneuronal tracer in the sonic-swimbladder motor system of two batrachoidid fishes, the plainfin midshipman, Porichthys notatus, and the Gulf toadfish, Opsanus beta (Bass et al., 1993, 1994; Bass and Baker, 1997). Both mochokids and batrachoidids have paired midline sonic motor nuclei (SMN) whose axons exit the brain via occipital nerves. Despite this similarity, there are three distinct differences in the organization of the motorpremotoneuron circuitry between mochokids and batrachoidids. First, biocytin or dextran-biotin labeling of the sonic nerve in batrachoidids shows that their motoneurons have rons (PMN) and fibers. Open circles indicate unfilled pectoral and swimbladder neurons (SMN). Distance between sections is 100 pm. Scale bar = 500 pm. extensive dendritic arbors that branch into the contralatera1 motor nucleus, corroborating intracellular staining studies (Bass and Baker, 1990; Bass et al., 1993; Weiser et al., 1985). Motoneuron dendrites appear mostly confined to the ipsilateral nucleus in mochokids. Second, biocytin application to a single sonic nerve in batrachoidids results in extensive transneuronal, bilateral labeling of motoneurons and premotoneurons. In mochokids, motoneuron somata were never labeled contralaterally and premotoneurons only infrequently. Third, in batrachoidids, motoneuron somata are found throughout the SMN, whereas in mochokids there is a concentration of somata in the ventromedial sector of the SMN. This supraordinal difference is correlated with a difference in the primary location of premotoneurons. In batrachoidids, premotor pacemaker neurons are the only known afferent input to motoneurons and are mainly located ventrolateral to the SMN (Bass and Baker, 1990, 1997; Bass et al., 1993). The present results suggest that an analogous population of premotoneurons in mochokids is positioned within the dorsolateral SMN. Although the majority of premotoneurons are found ipsilaterally in mochokids, small somata were occasionally labeled

7 Fig. 4. A-F Line drawings of transverse sections through the caudal medulla and rostral spinal cord of S. nzgromaculatus after application of biocytin to a single swimbladder muscle nerve (2-day survival time). Large black dots represent biocytin-filled motoneurons; small dots are premotoneurons; stippling and lines represent, respectively, terminals and fibers. Sonic motoneurons were only labeled ipsilaterally. Premotoneurons (PN1) intrin- sic to the sonic motor nucleus (SMN) were labeled bdaterally, whereas a second group of premotoneurons (PN2) were labeled only ipsilaterally. Other abbreviations: CO, commissural bundle; tf, terminal field in medial reticular formation. Distance between sections: A,B, 400 Fm; B,C, 300 pm; C,D, 400 Fm; D,E, 100 Fm; E,F, 200 Fm; F,G, 100 pm; G,H, 500 pm, Scale bar = 500 IJ-m.

8 500 F. LADICH AND A.H. BASS Fig. 5. Photomicrographs of labeling intrinsic to the sonic motor nuclei following hiocytin application to a single sonic muscle nerve in S. nzgrornaculatus (2-day survival). Sections were lightly counterstained with cresyl violet. A. Biocytin-labeled fibers crossed the midline (arrow) dorsal to the central canal (cc) to the contralateral motor nucleus (right side of photograph). B-D: Transneuronally filled premotoneurons contralaterally in the dorsolateral SMN, like their homonymow population. Kastberger (1977) stimulated the brainstem of doradid catfish electrically and evoked grunts and one-to-one responses of the drumming muscle. Both sonic muscles contracted synchronously in doradids (Kastberger, 1977) as in batrachoidids (Cohen and Winn, 1967). However, the (PN1) with a variable location, shape, and size in the dorsolateral sector of the ipsilateral sonic motor nucleus. Premotoneurons were embedded in a dense fiber plexus of the ipsilateral nucleus (np region in Fig. 2). Biocytin-filled motoneurons in and out of the plane of focus are also indicated (m); arrows (B-D) point to premotoneurons out of the plane of focus. Scale bar in A = 40 pm for A-D. sonic nerves in doradids could be excited separately by electric stimulation of either the left or right medulla, suggesting weak coupling between both motor nuclei; this has never been found in batrachoidids (Bass and Baker, 1990, 1991). Given that doradid sonic motor and premotoneurons appear to have a similar pattern of organization as that of mochokids (Ladich and Bass, unpublished observa-

9 VOCAL-ACOUSTICOLATERALIS PATHWAYS 501 Fig. 6. A-D: Transneuronal labeling of premotoneuron somata and fibers following application of biocytin to a single swimbladder muscle nerve in S. nigromaculatus (2-day survival time). All sections (transverse) were lightly counterstained with cresyl violet. A: Premotoneurons (PN2) at the rostral pole of the sonic motor nucleus. B: Commissural bundle (CO) and associated terminal field (tf) in the reticular formation at the caudal level of the descendingoctaval nucleus (DO). C: High-magnification view of terminals and fibers in the reticular termi- nal field ipsilateral to the labeled sonic nerve (right side in B). D: Labelled fibers extended from the reticular terminal field toward and sparsely into the ventrolateral extent of DO; the lateral edge of the medulla is indicated (el. E: Higher-magnification view of thin labeled fibers and terminals in DO. Arrows in D and E identify corresponding points. Scale bar in A = 100 pm for A and D, 400 pm for B, 40 pm for C and E. tions), this predicts a similar pattern of neurophysiological organization, namely synchronous firing of the drumming muscles with weak coupling between the two motor nuclei. However, morphological patterns are not always indicative of physiological performance. For example, although the closely related sculpins and sea robins (same order, Scorpaeniformes) have a similar sonic motor nucleus located along the ventrolateral medulla and spinal cord, sculpins have synchronously, and sea robins asynchronously, active sonic motor nuclei (Bass and Baker, 1991). Soniclvocal-acousticolateralis circuitry Transneuronal biocytin transport revealed a number of shared traits in vocal circuitry between catfish and batrachoidids, in addition to both families having midline sonic motor nuclei (see Bass et al., 1994, for midshipman, a batrachoidid). For example, the transneuronally labeled PN2 neurons and commissural-associated terminal field identified in mochokids have a similar rostrocaudal extent as the ventral medullary nucleus of midshipman which extensively links the pacemaker-motoneuron circuit across the midline. As in midshipman, the commissural bundle in mochokids is continuous with a lateral brainstem bundle that links the motoneuron circuitry to regions of the medulla considered to have octaval and lateral line functions. As discussed in more detail below, the latter includes the octavolateralis efferent nucleus, the eminentia granularis and a medial region of the rostral medulla that has been compared to the tetrapod superior olive. Unlike midshipman, there were no transneuronally labeled somata in octaval-recipient regions of the medulla in mochokids after biocytin labeling of a sonic motor nerve. This implies that all transneuronally labeled terminal fields in the rostral medulla and cerebel-

10 502 F. LADICH AND A.H. BASS Fig. 7. Line drawings of selected sections from S. nigromaculatus through the octavolateralis region of the medulla and cerebellum after application of biocytin to a single swimbladder nerve (2-day survival). Biocytin-labeled fibers extended from a lateral brainstem bundle (LB) and terminated on neurons of the octavolateralis efferent nucleus (OE, A), a granule cell population associated with primary and second-order acoustic nuclei (GA, B), the eminentia granularis of the vestibulolateral lobe of the cerebellum (EG; B,C), and the nucleus praeeminentialis (PE, C), a second-order lateral line nucleus. Distance between sections: A,B, 300 pm; B,C, 700 pm. See Bass (1982) and Finger and Tong (1984) for cerebellar terminology. Scale bar = 1 mm. lum of mochokids originated from an ascending pathway formed by premotoneurons of the caudal medulla (PN, Fig. 9). Interspecific differences in the extent of transneuronal labeling of premotoneuron somata may reflect differences in the extent of electrotonic coupling between neurons within the vocal circuit (Bass et al., 1994; also see Pereda et al., 1995). A direct projection of the sonic/vocal motor system upon octavolateralis efferent neurons implies an influence at the level of primary afferents and their receptor organs (e.g., Russell, 1971; Furukawa, 1981; Lin and Faber, 1988). The sonic-recipient region of the eminentia granularis, a part of the cerebellum s vestibulolateral lobe (Bass, 19821, appears to overlap primary octaval and lateral line (electroreceptive and mechanoreceptive) recipient zones of the eminentia granularis (Finger and Tong, 1984). Hence, together with inputs to efferent neurons, the results suggest an influence of vocal pathways on octavolateralis circuitry at both peripheral and central levels. A distinct transneuronally labeled terminal field is also found in the medulla in a granule cell population that is a rostral extension of the medial auditory nucleus (MAN) and which projects to MAN (Finger and Tong, 1984). Portions of MAN in ictalurid catfish have been provisionally compared to the tetrapod superior olive (Finger and Tong, 1984; McCormick and Braford, 1993; McCormick and Hernandez, 1996). MAN is linked to both acoustic and lateral line regions: It has reciprocal connections with a midbrain auditory nucleus (centralis) of the torus semicircularis (homologue of the tetrapod inferior colliculus) and Fig. 8. Photomicrographs illustrating biocytin labeling of the vocal motor circuit at the level of octavolateralis nuclei of S. nigrornaculatus after application of biocytin to a single swimbladder nerve (A,C,D) and the saccular branch of the eighth nerve (B). All sections were lightly counterstained with cresyl violet. Biocytin-filled fibers of the lateral brainstem bundle terminated (t) on neurons within the octavolateralis efferent nucleus (OE, A) whose position was identified following retrograde transport of biocytin from the saccular branch of the eighth nerve (B; arrows point to labelled somata). Sonic-related fibers also terminated (t,c,d) in a granule cell population (GA, C) that is a rostral extension of primary and second order acoustic nuclei (see text), and in the nucleus praeeminentialis (D). Scale bar in A = 40 pm for A, B, D, 100 pm for C.

11 m Figure 2

12 504 CoTf OCT CBL I CBL OCT + SMN r-i I Biocytin Label I I protractor I protractor "drumming" I "drumming" muscle I muscle I M 1 d I i n e Fig. 9. Summary diagram of proposed pattern of organization for the sonicivocal-acousticolateralis pathways in mochokid catfish. Biocytin application to a single sonic nerve that innervates the ipsilateral protractor muscle results in biocytin-filled sonic motor (SMN, ipsilateral only) and premotor neurons (PN, bilateral). Biocytin-labeled terminal fields mainly occur 1) bilaterally in a commissural region at the level of the caudal pole of the descending octaval nucleus (CoTf), 2) ipsilaterally in octavolateralis-related (OCT) nuclei (octavolateralis efferent nucleus, granule cell division of the medial auditory nucleus, nucleus praeeminentalis), and 3) ipsilaterally in the eminentia granularis and granule cell layer of the corpus of the cerebellum (CBL). See text for details. also receives input from the eighth nerve-recipient descending octaval nucleus (Tong and Finger, 1983). MAN also terminates sparsely in a region between the lateral lemniscus and nucleus praeeminentialis, a second-order lateral line nucleus (Finger and Tong, 1984); the ascending vocal pathway apparently overlaps this region as well as extending into the nucleus praeeminentialis proper. Portions of MAN may receive direct saccular nerve input (Fritzsch et al., 1990; Bleckmann et al., 1991; McCormick and Braford, 1993; McCormick and Hernandez, 1996). An understanding of the organization of a superior olive homologue or analogue in teleosts indeed requires additional anatomical and neurophysiological studies in a variety of teleosts. Nevertheless, the available data indicate an extensive circuitry linking the sonic/vocal system to acoustic and lateral line regions of the medulla and cerebellum in teleosts that actively generate sound, including both hearing specialists (e.g., siluriforms) and nonspecialists (batrachoidids) (see Popper and Fay, 1993). Evolutionary considerations Ontogenetic studies of the drumming muscles of batrachoidiforms (toadfish and midshipman) indicate that they originate from an occipital somite and then migrate together with occipital nerve axons to attach to the lateral walls of the swimbladder (Tracy, 1959; Lindholm and Bass, 1993). The common innervation of the drumming muscle by an occipital nerve in batrachoidiforms and siluriforms implies a common origin for the muscle from occipital F. LADICH AND A.H. BASS somites (this study; Bass and Baker, 1991; Ladich and Fine, 1994). The same conclusion could be made for other teleosts with swimbladder-associated drumming muscles innervated by occipital nerves, for example, sea robins, sculpin, and squirrelfish (Bass and Baker, 1991; Gainer et al., 1965). Bass and Baker (1997) propose that hindbrain sonic/vocal circuitry across the vertebrates shares common embryonic origins inclusive of sonic musculature (swimbladder drumming, laryngeal, syringeal) from occipital somites and motor and premotoneurons from the most caudal hindbrain segments (rhombomeres 7 and 8). The similarity in vocal-acoustic circuitry between such distantly related teleosts as siluriforms and batrachodiforms provides further support for shared mechanical and developmental factors favoring common origins of vocal circuitry from rhombomeres 7 and 8. This by no means implies that the vocal circuitry between catfishes and other teleosts, or for that matter other vertebrates, are equivalent cell populations. It merely proposes that homologous regions of the embryonic hindbrain give rise to the premotor-motoneuron circuitry establishing sound-producing behaviors in a variety of vertebrates, irrespective of the motoneuron pools, that innervate the diversity of vocal-related muscles which include swimbladder drumming, syringeal, and laryngeal. It is currently not possible to integrate pectoral-spine stridulatory mechanisms into this general framework until more information is available regarding its premotor circuitry. ACKNOWLEDGMENTS The authors thank Margaret Ann Marchaterre and Lee Goldstein for assistance with the biocytin experiments and Heidemarie Grillitsch for the line drawings. Research support was from the Austrian Science Foundation (FWF grant to F.L.) and the U.S. National Science Foundation (IBN to A.H.B.). LITERATURE CITED Abu-Gideiri, J.B., and D.H. Nasr (1973) Sound production by Synodontis schall (Bloch-Schneider). Hydrobiologia 43t Baron, V.D., K.S. Morshnev, V.M. 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