Olfactory Bulb Efferents in the Channel Catfish, Lctalurus Punctatus ANDREW H

JOURNAL OF MORPHOLOGY 169:91-lll(1981)

Olfactory Bulb Efferents in the Channel Catfish, lctalurus punctatus ANDREW H. BASS Division of Biological Sciences, Uniuersity of Michigan, Ann Arbor, Michigan 48109

ABSTRACT Autoradiographic, HRP, and Fink-Heimer techniques define olfactory bulb efferents in the channel catfish. The olfactory bulb projects bilaterally to eight targets in the area ventralis telencephali including the preoptic area, five targets in area dorsalis telencephali, and the posterior tuber of the diencephalon. There is additional input to the peripheral margin of the internal cell , layer of the contralateral olfactory bulb. Fibers cross in rostra1 (nervus terminalis and commissure of Goldstein) and caudal components of the anterior commissure and the habenular commissure. HRP techniques reveal the origin of bulb efferents from the internal and mitral cell layers of the olfactory bulb. The olfactory tract is divided into five major components, each with a unique subset of ipsilateral and commissural pathways.

The actinopterygians, or ray-finned fishes, Braford, '80). (4) Comparable experimental are traditionally separated into three superor- data are available for a representative member ders (Romer, '701, namely the Chondrostei, of each of the other groups of actinopterygians Holostei, and Teleostei. The teleosts comprise (Braford and Northcutt, '74; Northcutt and at least 20,000 species and are further divided Braford, '80). into three divisions: I, the eels and eel-like This study is presented within the context fishes; 11, the mormyriforms and osteoglossi- of a cytoarchitectural analysis of the telence- forms; and 111, the bulk of teleost species phalon of the channel catfish, whose nomen- (Greenwood et al., '66). Teleosts display a wide clature is based on a series of normal and ex- range in variation of telencephalic organiza- perimental studies of several actinopterygians tion, which suggests that this is adapted to (Bass, '81a). This is the first experimental re- divergent ecological and behavioral niches. post that utilizes axonal transport methods to This study reveals the olfactory bulb effer- define olfactory bulb efferents in a nonmam- ents in a Division I11 teleost, the channel cat- malian vertebrate. The results offer new data fish, Zctalurus punctatus. The catfishes, order on: (a) the separate projections of the medial Siluriformes, form a major division (about 2,- and lateral olfactory tracts, (b) the number of 000 species) of the superorder Ostariophysi, olfactory bulb targets in the subpallium and which includes the majority of freshwater pallium, (c) olfactory bulb input to the preoptic fishes (5-6,000 species, Greenwood et al., '66). area and the contralateral olfactory bulb, and This demonstration of olfactory bulb efferents (d) the cells of origin of olfactory bulb efferents. is critical to understanding telencephalic or- A portion of these results appeared earlier ganization in catfish, and indeed, other teleosts (Bass, '78). for four reasons: (1) The early anatomists por- tray the telencephalon of teleosts as an olfac- MATERIALS AND METHODS tory-dominated structure (Johnston, '11; Her- rick, '21). (2) The olfactory system is the only Experimental animals sensory system with direct input to the telen- Adult and juvenile specimens of channel cat- cephalon. (3) It is impossible to correlate the fish, Ictalurus punctatus, were collected on the topography of telencephalic olfactory bulb tar- grounds of the Spring Valley Trout Farm, Dex- gets recognized in previous experimental stud- ter, Michigan. Animals were housed in aquaria ies (Scalia and Ebbesson, '71; Ito, '73; Finger, '75) with telencephalic subdivisions identified Andrew H. Bass's present address is Department of Ecology and in the majority of cytoarchitectural studies Behavioral Biology, 108 Zoology Building, 318 Church Street, S.E., (Nieuwenhuys, '63; Elass, '81a; Northcutt and Minneapolis, Minnesota 55455.

0362-2525/81/1691-0091$06.000 1981 ALAN R. LISS, INC. 92 A.H. BASS for at least 1 week prior to surgery, the water of the olfactory peduncle was transected prior temperature being maintained pre- and post- to injection of proline into the ipsilateral ol- operatively at 24-27°C. factory bulb (as above). These experiments were designed to elucidate the separate pro- Experimental methods jections of the medial (lateral transection) or Olfactory bulb efferents were traced by tran- lateral (medial transection) division of the ol- sections of the olfactory peduncle and olfactory factory tract. bulb injections of tritiated proline or horseradish peroxidase (HRP). Prior to all surgical HRP injections procedures, animals were anesthetized by im- During a subsequent HRP analysis of olfac- mersion in home tank water containing tri- tory bulb afferents, anterograde transport of caine methanesulfonate (MS222). HRP (following olfactory bulb injections) confirmed the trajectory of olfactory bulb effer- Transections ents. Experimental details appear elsewhere In four juvenile (18-10.5 cm, snout-tail (Bass, '81b). length) and four adult (27-28 cm) channel cat- For two juvenile (18-cm) channel catfish, the fish, the olfactory peduncle was transected ros- dorsal surface of the ipsilateral telencephalon tral to the anterior pole of the telencephalon. was exposed. The injection pipette was guided Following survival times of 1-12 days, animals stereotaxically into the rostromedial and cau- were anesthetized with MS222, and perfused dolateral segments of the hemisphere with transcardially with 0.7% saline followed by subsequent injections of 1.0-2.0 pl of HRP over 10%Formalin. All brains were fixed for at least a period of 5-10 min. These experiments were 1 week in 10% Formalin and were embedded designed to reveal the cells of origin of olfac- in 25% gelatin. Frozen sections were cut at 33 tory bulb efferents. Following reanesthetiza- bm in the transverse plane and stored in 2% tion with MS222, animals were perfused tran- Formalin. Sections were stained for degener- scardially with cold phosphate buffer (pH 7.4) ating axons and terminals according to the followed by 2% gluteraldehyde in cold buffer. Wiitanen modification of the Fink-Heimer After removal from the skull, each brain was technique (Wiitanen, '69). washed in 30% sucrose-fixative (2-3 hr) and then 30% sucrose-buffer (12 hr). Brains were Proline injections embedded in gelatin-sucrose and sectioned fro- Eight adult (28- to 42.5-cm) channel catfish zen at 33-40 pm. Staining procedures were received olfactory bulb injections of 0.1-0.5 pl modifications (cf. Bass, '79) of the O-dianisi- of tritiated proline (New England Nuclear dine reactions of Colman et al. ('76) and the NET-323, concentrated to 20 pCi/p,l). All in- tetramethylbenzidine (TMB) reaction of jections were made under pressure via a 1- DeOlmos et al. ('78). lambda pipette (drawn and bevelled to a tip of A distinct advantage of using catfish for the 75 p,m) attached to a stereotaxic device. Injec- study of olfactory bulb efferents with protein tions were made over a 5- to 10-min period. transport methods is that the olfactory bulb is Following postoperative survival times of 1-12 displaced from the rostra1 pole of the telence- days, animals were reanesthetized with phalon along extended olfactory peduncles MS222 and perfused transcardially, with 0.7% (Fig. 1).This condition alleviates the problem saline followed by AFA (see Bass, '81a). The of local diffusion of labeled protein into adja- brains were removed from the skull and placed cent structures (such as the telencephalon in in fixative for at least 1week, with subsequent animals with sessile bulbs), thus hindering the embedding in paraffin. Material was serially interpretation of pathways originating from sectioned at 15 pm, in either the transverse or the olfactory bulb. horizontal plane. The mounted sections were deparaffinized, coated with Kodak NTB2 or 3 RESULTS emulsion, and exposed at 7°C for 28 or 40 days. After development in D-19 or Dektol, sections Methodological notes were counterstained with cresyl violet. Trac- ings of high-contrast photographs from an ear- Silver degeneration lier cytoarchitectural study (Bass '81a) were Optimal terminal degeneration occurred in used to plot the distribution of silver grains. juvenile specimens (13-16 cm), surviving 5-7 In seven adult (31- to 37-cm) channel catfish, days at 26°C. By 9 or 10 days, argyrophilia was a portion of the medial and/or lateral division predominant in the olfactory tracts. Both ax- CATFISH OLFACTORY PATHWAYS 93 onal and terminal argyrophilic debris ap- Fibers appeared as sinuous lines of clumped peared as small- to medium-caliber black gran- reaction product oriented within the plane of ules (Fig. 9). In all cases, degeneration within section. Terminal zones contained a homoge- the lateral olfactory tract and the area dorsalis neous distribution of fine granular precipitate. telencephali was coarser and heavier than in Following telencephalic HRP injections, la- the medial olfactory tract and the area ven- beled cells within the olfactory bulb contain a tralis telencephali. Excepting the preoptic dense homogeneous and/or a granular precip- area, terminal debris occurred in all regions itate (Fig. 11). identified as targets with autoradiographic and HRP material. The trajectory of the medial Secondary olfactory pathways and terminal olfactory tract pars rnedialis could not be ac- fields: Overview curately traced with Fink-Heimer methods. The following description of olfactory bulb One bullhead catfish, Ictalurus natalis, sus- efferents (secondary olfactory pathways) and tained a unilateral olfactory tract cut and sur- terminal fields is based on the results of proline vived 14 days at 26°C. Sparse degeneration injections into the olfactory bulb. Figures 2, 3, appeared in all regions identified as olfactory and 5-7 are representative cross sections targets in I. punctatus, while moderate- to through the forebrain of I. punctatus at the large-caliber fragmented varicosities distin- levels indicated in Figure 1. Corresponding guished fiber pathways. An autoradiographic Nissl-stained sections at the illustrated telen- analysis in a second bullhead confirmed the cephalic levels appear in the previous paper pattern of terminal fields seen in channel cat- (Bass, '81a). Chartings of pathways (dashes, fish. Thus, the bullhead degeneration data Figs. 2,3, 5-7) are based on animals that sur- were taken as representative of the condition vived 5-12 days, while those of terminal fields in channel catfish. Halwever, even in bullheads, (stippling, Figs. 2, 3, 5-7) are based on cases degeneration material did not reveal a clear that survived 1 and 3 days. HRP and Fink- portrayal of the pathways and terminals for Heimer material provided additional confir- the medial components of the medial olfactory mation of pathways and terminal fields. Figure tract. 12 provides a summary profile of efferents. The initial series of experiments indicated Autoradiographic techniques overlap in the termination patterns of the me- At 1-day survival time, olfactory bulb au- dial and lateral olfactory tracts. Additional toradiographs reveal dense label over all ter- data on their separate pathways was provided minal fields, including the preoptic area, with by proline cases that involved partial transec- sparse grain accumulations over the ipsilat- tions of the olfactory peduncle. era1 and contralateral olfactory tracts. In- The ipsilateral projections of the medial and creased labeling of tracts and terminal fields lateral olfactory tracts are discussed sepa- occurred at longer survival times, with all rately from the contralateral projections and tracts clearly labeled by 5 days (Figs. 4,8,10). the transection experiments. A final section Transneuronal transport of proline should not describes the cells of origin of olfactory bulb have obscured the profile of terminal fields, as efferents. such transport has not been reported for survival times of less than 11 days among non- Ipsilateral projections: Medial olfactory tract mammals (Landreth et al. '75; LA&, '76, In autoradiographs, dense silver grain ac- Reperant et al. '78). cumulations occur throughout the bulb, ex- An advantage of using the autoradiographic tending over cells of the nervus terminalis gan- method is that the experimental material can glion (Fig. 2A,B). The layer of primary be prepared for paraffin histology. This allows olfactory fibers is unlabeled. serial sections to be made of each brain, optim- At far rostral levels, the medial olfactory izing the relationship between terminal fields tract (mt, Fig. 2C) lies ventromedial to area and pathways, and recognized cytoarchitec- dorsomedialis (DM, Fig. 20, and the lateral tural boundaries (Bass, '81a). olfactory tract (llt, lmt; Fig. 2C). Caudally, as the mt expands, sparse label appears over HRP techniques the ventral aspect of nucleus 'h" of area ven- Treatment of HRI? material with the TMB tralis (Vn, Fig. 2D). A rostral segment of the procedure yielded the best visualization of ol- central nucleus (Vc, Fig. 2D) lies adjacent to factory pathways and terminals, with the the mt and probably receives an input, as Golgi heaviest label in the ipsilateral olfactory tract. analysis (not described herein) reveals its proc- 94 A.H. BASS ewes extending into the olfactory tract. of the anterior commissure (ac, Fig. 5A). The As area ventralis expands, the medial olfac- dorsal and medial divisions of the medial ol- tory tract divides into ventromedial (mm, Fig. factory tract converge (mdm, Fig. 5A), their 3A) and dorsolateral (md, Fig. 3A) components, fibers distributing ipsilaterally to area dorsalis terminating densely along the lateral aspect (see below), a supracommissural nucleus (Vs, of Vn and the ventral nucleus of area ventralis Fig. 5A), a rostral segment of a postcommis- (Vv, Fig. 3A), and the medial aspect of Vc. sural nucleus (Vp, Fig. 5A,B), and the contra- Sparse grain accumulations appear over scat- lateral hemisphere (see below). tered elements of Vn. At postcommissural levels, the medial tract Caudally, the dorsal division of the medial continues as a caudal component (mc, Fig. 5B) olfactory tract adjoins a dorsal segment of the coursing ventrolateral to a caudal portion of ventral nucleus (Vv-d; Figs. 3B,C; 8A) and a Vp. Fibers distribute to a migrated interme- ventral segment of the dorsal nucleus of area diate zone of the area ventralis (Vi, Fig. 5C). ventralis, (Vd-v; Figs. 3B,C; 8A). A dense ter- Unlike the supracommissural and postcom- minal field appears lateral to, and over, the missural nuclei, a dense uniform grain accu- cells of Vv-d, while Vv-v continues to receive mulation covers the cells of Vi. At this level, input along its lateral surface, Similarly, a fibers distribute to the most caudal segment dense terminal field surrounds Vd-v, capped of the anterior preoptic nucleus (PPa, Fig. 5C). by a dorsal division of the dorsal nucleus (Vd- Rostrally, these fibers (mp, Fig. 5A) continue d, Figs. 3B,C; 8A). Golgi material shows that as two bundles through the medial preoptic cells within Vv, Vd-v, and Vd-v extend their area. A medial bundle lies near the ventricular processes into adjacent terminal fields. surface and corresponds in position to the trac- A small fiber system, resembling the nervus tus medialis preopticus pars posterior and a terminalis pathway in carp (Sheldon, ’09), more lateral bundle to the tractus medialis branches off the ventromedial component of preopticus pars anterior of carp (Sheldon, ’12: the olfactory tract (nt, Fig. 3B). As the com- Fig. 54). Like the nervus terminalis and the missural ridge expands centrally, a medial commissure of Goldstein, these pathways are branch of the nervus terminalis passes ventral labeled at 1-day survival, but it is difficult to to the ventral nucleus. A second component define a circumscribed input zone. These bun- branches off ventrolaterally and continues dles traverse a zone intermediate to the mag- caudally throughout the lateral preoptic area nocellular and anterior divisions of the preop- to the surface of the brain (Figs. 3B,C; 5A). tic zone, with possible input to either zone. Whether the medial branch terminates be- Finally, the caudal medial olfactory tract neath the fused aspect of the ventral nucleus courses through the medial diencephalon (Fig. or the ventral branch amid the scattered ele- 6A,B) to terminate centrally in the medial nu- ments of the lateral preoptic region is a moot cleus of the posterior tuber (MTP, Figs. 7,8D). point. Both pathways are recognizable at 1-day This terminal field extends ventrally to appose survival, but there are no sudden increases in the most dorsal aspect of the ventricle. Cells grain densities along their trajectories, dem- of the more lateral nucleus of the saccus vas- onstrating a distinct terminal zone. culosus (NSV, Fig. 7) may receive input if their A second branch of the ventromedial olfac- processes extend medially to engage the ter- tory tract crosses a rostral portion of the com- minal zone. missural ridge as the so-called “interbulbar The caudal component of the olfactory tract commissure” of Goldstein (’05). This compo- is often referred to as a hypothalamic branch nent (cg, Fig. 3C) passes ventral to a small (cf. Finger, ’75). I abandon this designation cluster of cells interposed between the sym- because these fibers contribute to telencephalic metrical components of Vv-v. These cells cor- terminal fields and the posterior tuber, a dien- respond topographically to Sheldon’s (’09) me- cephalic zone separate from the hypothalamus dial septa1 nucleus, which he recognized as a proper (Braford and Northcutt, in press). target of the nervus terminalis. In catfish, Ipsilateral projections: Lateral olfactory tract these cells appear more closely associated with the commissure of Goldstein. As with the ner- The lateral olfactory tract, like the medial vus terminalis system, it is difficult to deter- tract, comprises lateral (llt, Figs. 2-6) and mine whether there are terminals in this cen- medial (lmt, Figs. 2, 4) subdivisions. The lat- tral zone. eral division corresponds to both the lateral The most formidable zone of crossed olfac- and intermediate divisions of the olfactory tory fibers appears within the caudal segment tract of carp (Sheldon, ’12). Finger (‘75) ap- CATFISH OLFACTORY PATHWAYS 95 pears to indicate the intermediate division as commissure (Fig. 5A), and (4)the habenular the ventral or medial division of the lateral commissure (Fig. 6B). olfactory tract of bullhead catfish. In channel Both the habenular and anterior commis- catfish, the medial arid lateral divisions of the sural components distribute to the area dor- lateral tract separate at far rostral levels (Figs. salis as described above for the ipsilateral 2D, 41, the medial lying adjacent to the as- tract(s). Rostrally, the crossed component of cending component of the medial olfactory the anterior commissure continues as two bun- tract and the lateral continuing ventral to the dles along the lateral aspect of the medial and pars ventralis of area dorsolateralis (DLv, Fig. dorsal components of the medial tract (Fig. 2C,D). While sparse grain accumulations ap- 3A,B,C).The ventromedial component merges pear dorsal to the lateral division, there is no with the crossed components of the commissure recognizable terminal zone at rostral telence- of Goldstein (Fig. 3C) and the nervus termin- phalic levels. alis (Fig. 3B). The medial and dorsal bundles The lateral division of the lateral olfactory continue as ascending components (ma, Fig. tract distributes densely to rostral and caudal 2C,D) to the contralateral olfactory bulb (Fig. divisions of the dorsal posterior zone (DPr, 2A,B). Within the olfactory peduncle, the me- DPc, respectively; Figs. 3A-C; 5A,B; 8B; 9) and dial bundle occupies a lateral position along sparsely to a caudal remnant of the ventral the medial olfactory tract, while the dorsal division of area dorsolateralis (DLv, Figs. bundle lies ventral within the ascending por- 3A-C; 5A). Within DP, silver grain accumu- tion of the medial tract. The former corre- lations are higher medially and dorsally (Fig. sponds in position to the pars medialis and the 8B). latter to the pars lateralis of the ascending At rostral commissural levels, the lateral component of the olfactory peduncle of carp olfactory tract distributes to the dorsal central [Fig. 22 (Sheldon '1211. At the level of the bulb, zone that lies above DP (DC-3, Fig. 3C). DC-3 the ventral (pars lateralis) component distrib- contains a medial division receiving a dense utes caudal and ventrolateral and the medial olfactory input (Figs. 3C, 5A,B; 8B) and a lat- component rostral and ventromedial; both eral division receiving a sparse input (Figs. components terminate along the outer margin 5A,B). of the internal cell layer (ICL, Fig. 2A,B). At The most caudal telencephalic target of the caudal levels, a few fibers split off the ventral lateral olfactory tract is nucleus taeniae (NT, component and traverse the medial aspect of Figs. 5C, 8C). Just dorsal to nucleus taeniae the lateral olfactory tract (It,Fig. 2B). It is not a sparse terminal field appears over a medial possible to state if elements within both the component of tbe posterior division of area dor- internal and mitral cell layer (MCL, Fig. 2A,B) solateralis (DLp, Figs. 5C, 8C). receive an input, as the boundary between Caudally, the lateral division of the lateral these zones is diffuse (cf. Bass, '81). olfactory tract separates as a distinct bundle just dorsal to the caudal entopeduncular nu- Transections cleus (Ec, Fig. 5C) and continues through the The separate projections of the medial and rostral diencephalon (Fig. 6A) to the habenular lateral olfactory tracts were revealed in spec- commissure (hc, Fig. 6B), where it crosses to imens that sustained partial or whole transec- enter the contralateral component of the lat- tions of, respectively, the lateral or medial eral division of the lateral olfactory tract. tracts. The medial division of the lateral tract con- For autoradiographs of these experimental tributes fibers to a dorsal component of the cases, silver grain accumulations extend over medial tract (Figs. 3, 4). This pathway is con- the entire intact portion of the tract, while they firmed in cases of partial transections of the end at the level of the transection for the le- olfactory tract (see I~elow). sioned portion (Fig. 10). Following total transection of the medial Contralateral projections tract (two cases), all pathways and terminal The contralateral olfactory bulb projections fields are labeled bilaterally, as normal exper- are sparser and topographically overlap the imental cases, except: medial and lateral com- ipsilateral projections (Figs. 2, 3, 5-7). ponents of the medial tract, nervus terminalis There are four possible sources of input to bundles, commissure of Goldstein, and the lat- the contralateral targets: (1)the medial branch eral division of DC-3. Sparse label occurs in of the nervus terminalis (Fig. 3B), (2) the com- the medial preoptic area and lateral to Vv. For missure of Goldstein (Fig. 30, (3) the anterior one additional case involving transection of 96 A.H. BASS only the medial component of the medial tract, components. On the basis of topography, I di- only the newus terminalis pathway is unla- vide the tract into five components, each with beled, suggesting its specific association with a unique subset of ipsilateral and commissural this component. fiber bundles (Table 11, each with a general For cases with medial tract lesions, area ven- subset of olfactory targets. The lateral division tralis receives input from a medial component of the lateral tract, the medial division of the of the lateral olfactory tract (Figs. 2C,D; 3; 4). medial tract, and the ascending division of the Dense silver grain accumulations appear over medial tract appear specialized for input, re- the lateral aspect of the dorsolateral compo- spectively, to area dorsalis, area ventralis (es- nent of the medial tract, the anterior and ha- pecially Vv and the lateral preoptic area), and benular commissures, and the caudal compo- the contralateral olfactory bulb. The dorsal nent of the medial tract; sparse label appears division of the medial tract projects to areas over the ventromedial tract and the medial ventralis and dorsalis; the caudal division to component of the ascending olfactory tract. the caudal telencephalon, the medial preoptic After total transection of the lateral olfac- area, and the posterior tuber of the dience- tory tract (two cases), all pathways and ter- phalon. All components, excepting the lateral minal fields are labeled bilaterally except: the component of the lateral tract, carry fibers of medial and lateral components of the lateral both the medial and lateral tracts. tract, the habenular commissure, and the dorsal component of the ascending olfactory tract. Origin of olfactory bulb efferents The increased silver grain density along the Following telencephalic HRP injections, dorsomedial border of DP is absent. For such dense label occurs throughout olfactory bulb cases, the medial tract contributes fibers bi- targets of areas dorsalis and ventralis; the laterally at the level of the anterior commis- medial and lateral olfactory tracts are densely sure (Fig. 5A), and ipsilaterally at caudal te- labeled. HRP-filled cells appear bilaterally lencephalic levels (Fig. 5C) to area dorsalis. within the internal and mitral cell layers of Additional data on the projections of the lat- the olfactory bulb. Golgi-like filling of cells eral component of the lateral tract was re- with brown reaction product (buffered DIA vealed in two cases. The first involved tran- procedure) distinguishes a variety of morphol- section of only thi, component (Fig. 10). The ogical types within the mitral cell layer (Fig. lateral component and the habenular commis- 11A). Elements within the internal cell layer sure are unlabeled, while the dorsal grain den- contain a small number of fine-caliber black sity in DP is absent. A stria medullaris com- reaction granules (TMB reaction, Fig. 11B). ponent of the olfactory tract is labeled More detailed accounts of telencephalic HRP bilaterally (Fig. lOF), but label is not contin- experiments will appear in future reports. uous rostrally or caudally with any portion of the olfactory tract. The latter remains unex- plained. DISCUSSION A second case involved total transection of Autoradiographic analysis distinguishes ol- the medial tract and partial transection of the factory bulb input to 15 targets in the forebrain medial component of the lateral tract (thisdata of Zctalurus punctatus. While the data reveal is not included with the above mentioned me- a circumscribed olfactory input to the telen- dial tract lesions). The results match those for cephalon, as earlier degeneration analyses of medial tract lesions with the following addi- teleosts (Scalia and Ebbesson, '71; Ito, '73; Fin- tions: The anterior commissure and all area ger, '75), they emphasize the differentiation of ventralis targets are sparsely labeled; the the olfactory tract and its recipient zones into preoptic area and ascending olfactory tract are multiple subdivisions. unlabeled. All dorsolateral terminal fields are densely labeled (excepting lateral DC-3, as de- Comparisons with earlier teleost studies scribed earlier) as normal cases. The data em- Ito's ('73) study of the secondary olfactory phasize: (a) the contribution of the medial com- pathways in carp, Cyprinus carpio, provides ponent of the lateral tract to area ventralis, little insight into the position and extent of the preoptic area, and the anterior commissure secondary olfactory targets, as terminal fields and (b) the specialization of the lateral com- are indicated by plus symbols on unlabeled line ponent for bilateral input to area dorsalis. drawings. Employing Neiuwenhuys' ('63) ter- The transection experiments underline a minology, Ito ('73) describes ipsilateral input subdivision of the olfactory tract into multiple to area ventralis and posterior dorsolateralis CATFISH OLFACTORY PATHWAYS 97 and contalateral input to area ventralis. Ito’s that between channel and bullhead catfish can study essentially described ultrastructural be accountable to differences in cytoarchitec- profiles of degenerating terminals and axons tural criteria and experimental techniques. following olfactory tract transections. Excepting some variance in the differentiation The more detailed light microscopic analyses of ventral telencephalic nuclei, the same num- of Scalia and Ebbesson (‘71) in the moray eel, ber of olfactory bulb targets are revealed in Gymnothorax funebris, and Finger (‘75) in autoradiographic material for bullhead catfish bullhead catfish, Ictczlurus nebulosus, form a (unpublished observations, see Materials and basis for comparison with the present results Methods). A reexamination of olfactory path- in the channel catfish, I. punctatus. For these ways in the moray eel with the more sensitive species, secondary olfactory input occurs bi- autoradiographic methods might reveal addi- laterally to areas ventralis and dorsalis telen- tional input to area ventralis. cephali, the majority of terminals occurring In both the moray eel and channel catfish, ipsilateral. The ipsilateral olfactory tract the medial terminal zone gets input from both crosses to the contralateral hemisphere via the the medial and lateral olfactory tracts, while anterior commissure in the moray eel and the in bullhead catfish input from only the medial anterior and habenular commissures in cat- olfactory tract is reported. fish. The large number of olfactory targets rec- Olfactory input to the preoptic area is de- ognized in channel catfish makes it desirable scribed only for channel catfish. The precise to frame an extended discussion within the terminal zone($ of this tract remains unclear. context of the three telencephalic input Additional electron microscopical analysis zones-medial, later(a1,and central-posterior- might resolve this issue. Such an input could recognized by Scalia and Ebbesson (‘71) and affect a variety of physiological processes via Finger (’75) and the diencephalic terminal field a preoptic-neurohypophysial tract (review: Pe- described by Finger (’75). ter, ’77). Medial terminal field Lateral terminal field In channel catfish, the medial terminal field The lateral terminal field in channel catfish (area ventralis telencephali) comprises seven comprises three major divisions: rostra1 and divisions. Three precommissural zoneDor- caudal divisions of the dorsal posterior zone sal, ventral, and central nuclei-compare top- and nucleus taeniae. Input to a caudoventral ographically to the medial terminal field re- component of area dorsolateralis comprises a ported for the moray eel and bullhead catfish. minor fourth component. In channel catfish, additional input is recog- In the moray eel, the lateral terminal zone nized to a supracomrnissural nucleus and three ends just caudal to the anterior commissure, postcommissural divisions-postcommissural, while in catfish it extends to the caudal pole intermediate, and preoptic nuclei. While the of the telencephalon. In the moray eel this area difference in the number of ventral targets gets input from the lateral olfactory tract, identified in the channel catfish and the moray while in catfish it gets input from both the eel may represent a real species difference, medial and lateral olfactory tracts.

TABLE 1. Divisions of the olfactory tract‘ Division Fiber bundles Commissural pathway

llt llt hc mm mm cg nt nt lmt ac md ml ac lmt mc-mp ml - mm lmt ma mm - lmt

Abbreviations: ac, anterior commissure;cg, commissure of Goldstein;hc, habenular commissure; lmt, medial component of the lateral tract; llt, lateral component of the lateral tract; ma, ascending component of the medial tract; mc-mp, caudal-preoptic component of the medial tract; ml, lateral Component of the medial tract; mm, medial component of the medial tract; nt, nervus terminalis component of the medial tract. See Figure 12. 98 A.H. BASS

The divisions of the dorsal posterior zone in the presence of secondary olfactory fibers channel catfish are roughly coincident with within this tract. those charted for the lateral terminal field of bullhead catfish. It remains difficult to recon- Olfactory bulb input cile descriptive differences in the absence of In channel catfish, there is input to the con- cytoarchitectural analysis in the bullhead tralateral olfactory bulb, fibers terminating study. Nucleus taeniae, as recognized in bull- predominantly at the interface of the internal head catfish, corresponds to the caudal seg- and mitral cell layers. While degeneration is ment of the dorsal posterior zone of channel described within the contralateral peduncle of catfish. I recognize nucleus taeniae as a sep- bullhead catfish, an input to the contralateral arate caudomedial region of area dorsalis that bulb is reported only for the moray eel; the corresponds to the caudal contingent of Shel- location of terminals is undescribed. don's ('12) nucleus taeniae. Berquist ('32) and In channel catfish, the contralateral olfac- Kallen ('51b) described nucleus taeniae as a tory bulb input arises from the mitral cell layer pallial zone that arises embryonically from the (Bass, '81b). This permits direct modulation of preoptic area and "pars frontalis thalami" contralateral bulb activity by a primary olfac- (forms ventral thalamus). Nucleus taeniae oc- tory target-the mitral cell layer of the ipsi- cupies a comparable position in channel cat- lateral olfactory bulb. fish; it lies adjacent to the intermediate nucleus of area ventralis (Vi) and, along with Vi, Comparisons with other actinopterygians is continuous with the medial preoptic area (Bass '80a). Northcutt and Braford ('80) employ silver- degeneration techniques to identify the secon- Central posterior terminal field dary olfactory targets in a polypteriform, Po- The central-posterior terminal zone in chan- lypterus (see also Braford and Northcutt, '74), nel catfish has two major components: a medial a chondrostean, Schaphirhynchus, and a ho- central zone (medial DC-3) of dense olfactory lostean, Lepisosteus. Intergeneric comparisons input and a lateral central zone (lateral DC-3) are facilitated since a previous cytoarchitec- of sparser input. A third minor component is tural study of the telencephalon of the channel a medial segment of the posterior zone (DLp) catfish (Bass, %la)recognized divisions of the that lies dorsal to nucleus taeniae. areas ventralis and dorsalis telencephali that In moray eels, two components constitute a are comparable, topographically, to those rec- posterior zone-nucleus limitans and area cen- ognized by Northcutt and Braford ('80). tralis. These nuclei lie caudal to the lateral As in catfish, the areas dorsalis and ventralis terminal field, unlike catfish where central- of the forementioned species, with the probable posterior and lateral zones are contiguous. The exception of Polypterus (cf. Braford and North- medial DC-3 component of channel catfish cutt, '741, show bilateral olfactory bulb input. roughly coincides with the central terminal When contralateral projections occur, they are field of bullheads, while the lateral DC-3 com- symmetrical to the denser ipsilateral terminal ponent and possibly medial DLp coincide with fields; fibers cross in both the anterior and ha- the posterior zone. benular commissures. For the series Poly- Both the medial and lateral olfactory tracts pterus-Scaphirhynchus-Lepisosteus-Zctalurus, project to the central-posterior zone in moray the number of targets in area ventralis are, eels and catfish. respectively, 4 [this is a minimum with possible additional targets (see Braford and Diencephalic input Northcutt, '7411, 6, 7, 7; in area dorsalis 1, 1, Olfactory bulb input to the diencephalon is 3,5 (for detailed topographical comparisons see reported only for catfish. Sheldon ('12) referred Bass, '79). There is input to the contralateral to this tract in carp as tractus hypothalamo- olfactory bulb and posterior tuber in all species olfactorius medialis, but rather indicated it as except Polypterus. A preoptic input is recog- an ascending bundle from the posterior tuber nized only for Zctalurus. While the number of nucleus to the corpus precommissurale (area area ventralis targets is relatively constant, ventralis). Ariens-Kappers ('06) referred to a there is a progressive increase in area dorsalis similar bundle as tractus olfacto-lobaris, or targets for the above series. The increased dif- tractus olfacto-hypothalamicus, connecting te- ferentiation of pallial olfactory zones parallels lencephalic olfactory centers with the hypo- that of other dorsal telencephalic regions thalamus. His statements are equivocal as to (Bass, '79; Northcutt and Braford, '80). For Ic- CATFISH OLFACTORY PATHWAYS 99 talurus, parcellation of the pallial olfactory zone into several divisions is associated in part ACKNOWLEDGMENTS with the appearance of a distinct cell popula- I thank Drs. R. Glenn Northcutt, Carl Gans, tion forming reciprocal connections with the Roger Davis, and Stephen Easter and Ms. olfactory bulb (Bass,, '81b). If other divisions Mary Sue Caudle Northcutt for their com- of the olfactory region (or nonolfactory regions) ments on earlier versions of this manuscript. have unique sets of afferents and efferents, it Additional thanks to Dr. Mark R. Braford, Jr. would suggest that subdivision of the pallial for his advice during early stages of this work, olfactory zone (and possibly nonolfactory to Ms. Margaret Ann Marchaterre for assist- zones) is coupled with the emergence of unique ance with the illustrations, and to Ms. Ricka sets of input/output relationships. Robb and Ms. Diane Johnson for typing the final manuscript. This work was supported by a Rackham Dissertation Grant, University of Functional considerations Michigan and NIH Postdoctoral Fellowship 1 For channel catfish, subdivision of the telen- F32 NS06309-01 to A. H. Bass and NIH cephalic olfactory zone is associated with mul- EY02485 to R. Glenn Northcutt. tiple components of the olfactory tract. Olfac- Portions of this work were submitted in par- tory autoradiographs emphasize separation of tial fulfillment of the requirements for a Ph.D. the olfactory tract into five major components. degree at the University of Michigan (Bass, Multiple divisions of the olfactory tract may '79). further relate to a topographical representa- LITERATURE CITED tion of the olfactory bulb, i.e., each component Ariens-Kappers,C.U. (1906) The structure of the teleostean arises from a restricted group of cells. Sheldon and selachian brain. J. Comp. Neur. Psychol. 16:l-112. ('12) documents for the carp, Cyprinus, that the Bass, A.H. (1978) Olfactory pathways in the channel catfish, lateral olfactory tract arises from both mitral Ictalurus punctatus. Neurosci. Abstr. 4:97. cells and the anterioa olfactory nucleus (inter- Bass, A.H. (1979) Telencephalic Afferents and Efferents in the Channel Catfish, Ictalurus punctatus. Ph.D. thesis, nal cell layer of catfish olfactory bulb); the University of Michigan, Ann Arbor, Michigan. medial component arises from mitral cells. Bass. A.H. (1981a) Organization of the telencephalon in the HRP data for channel catfish confirm the ori- channel catfish, Ictuluruspunctatus. J. Morph. 169:71-90. gin of bulb efferents from both the mitral and Bass, A.H. (1981b) Telencephalic efferents in the channel internal cell layers (present study; Bass '81b catfish, Ictalurus punctatus: Projections to the olfactory bulb and optic tectum. Brain, Behav. Evol. (in press). for interbulbar efferents). Transection exper- Berquist, H. (1932) Zur Morphologie des Zwischenhirns bei iments coupled with HRP methodology should niederen Wirbeltieren. Acta Zool. 13~57-304. provide additional topographical data. Bodznick, D. (1978) Characterization of olfactory bulb units Sheldon ('09, '12) further specified a nervus of sockeye salmon with behaviorally relevant stimuli. J. Comp. Physiol. 27:147-155. terminalis pathway that arises from ganglion Braford, M.R. Jr., and R.G. Northcutt (1974) Olfactory bulb cells spread along the ventromedial aspect of projections in the bichir, Polypterus. J. Comp. Neur. the olfactory nerve. While I have identified this 156:165-178. ganglion in channel catfish (Bass, '81a), I have Braford, M.R. Jr., and R.G. Northcutt (1980) The organization of the diencephalon and pretectum of actinopter- not identified an efyerent bundle specifically ygian fishes. In R.G. Nortbcutt and R.E. Davis (eds): Fish associated with these cells. However, the au- Neurobiology and Behavior. Ann Arbor, Mich.: Univer- toradiographic data presented here for channel sity of Michigan Press, in press. catfish decribes a pathway reminiscent of the Colman, D.R., F. Scalia, and E. Cabrales (1976) Light and electron microscopic observations on the anterograde nervus terminalis' a.s described by Sheldon for transport of horseradish peroxidase in the optic pathway carp. in the mouse and rat. Brain Res. 102~156163. If additional experiments demonstrate the DeOlmos, J., H. Hardy, and L. Heimer (1978) The afferent origin of olfactory tract components from cir- connections of the main and the accessory olfactory bulb formations in the rat: An experimental HRP study. J. cumscribed cell groups in the olfactory bulb, Comp. Neur. 181:213-244. such a regional topography might relate to a Doving, K.B., and R. Selset (1980) Behavior patterns in cod map of chemical sensitivity that underlies dis- released by electrical stimulation of olfactory tract bun- crete behavioral functions. While physiologi- dlets. Science 207:559-560. Finger, T.E. (1975) The distribution of the olfactory tracts cal evidence for such a map among teleosts is in the bullhead catfish, Ictalurus nebulosus. J. Comp. inconclusive (Bodznick, '78; Thommesen, '781, Neur. 161:125-142. recent experiments in cod, Gadus, associate Goldstein, K. (1905) Untersuchungen uber das Vorderhirn separate divisions of the olfactory tract with und Zwischenhirn einiger Knochenfische (nebst einigen Beitragen uber Mittelhirn und Kleinhirn derselben). different behavior patterns (Doving and Selset, Arch. Mikr. Anat. 66~135-219. '80). Greenwood, P.H., D.E. Rosen, S.H. Weitzman, and G.S. 100 A.H. BASS

Myers (1966) Phyletic studies of teleostean fishes, with cephalon of actinopterygian fishes. In S.O.E. Ebbesson a provisional classification of living forms. Bull. Am. Mus. (ed): Comparative Neurology of the Telencephalon. New Nat. Hist. 131:341455. York: Plenum Press, pp. 41-115. Herrick, C.J. (1921) The origin of the cerebral hemispheres. Peter, R.E. (1977) The preoptic nucleus in fishes: A com- J. Comp. New. 32:429-454. parative discussion of function-activity relationships. Ito, H. (1973) Normal and experimental studies on synaptic Amer. Zool. 17:775-785. patterns in the carp telencephalon, with special reference Reperant, J., J.P. Rio, D. Micelli, and M. Lemire (1978) A to the secondary olfactory termination. J. fur Hirnforsch. radioautographic study of retinal projections in Type I 14r237-253. and Type I1 lizards. Brain Res. 142:401411. Johnston, J.B. (1911) The telencephalon of ganoids and te- Romer, AS. (1970) The Vertebrate Body. Fourth Edition, leosts. J. Comp. Neur. 21t489-591. Philadelphia: W.B. Saunders & Go. Kallh, B. (1951) Embryological studies on the nuclei and Scalia, F., and S.0.E Ebbesson (1971) The central projec- their homologization in the vertebrate forebrain. Kungl. tions of the olfactory bulb in a teleost fish (Gymnothorm Fysiogr. Sallsk. Handl. 62:1-35. funebris). Brain Behav. Evol. 4:37&399. Landreth, G.E., E.A. Neale, J.H. Neale, R.S. Duff, M.R. Sheldon, R.E. (1909) The nervus terminalis in the carp. J. Braford, Jr., R.G. Northcutt, and B.W. Agranoff (1975) Comp. Neur. 19:191-201. Evaluation of (3H) proline for radioautographic tracing Sheldon, R.R. (1912) The olfactory tracts and centers in te- of axonal projections in the teleost visual system. Brain leosta. J. Comp. New. 22:177339. Res. 91:25-42. Thommesen, G. (1978)The spatial distribution of odour in- Lklr, G. (1976) Transneuronal transport in the frog visual duced potentials in the olfactory bulb of char and trout system. Brain Res. 109:623-627. (Salmonidae). Acta Physiol. Scand. 102:205-217. Nieuwenhuys, R. (1963) The comparative anatomy of the Wiitanen, J.T. (1969) Selective impregnation of degener- actinopterygian forebrain. J. Hirnforsch. 6:171-192. ating axons and axon terminals in the CNS of the monkey Northcutt, R.G., and M.R. Braford, Jr. (1980) New obser- (Macaca mulatta). Brain Res. 14:546-548. vations on the organization and evolution of the telen-

Abbreviations

ac anterior F facial lobe mdm fused portion of commissure GL glomerular layer the medial C cerebellum of olfactory and dorsal cg commissure of bulb components of Goldstein HA anterior the medial DC-1, parts of the hypothalamic olfactory tract 2, 3 central zone of nucleus mm medial area dorsalis Hab habenular nuclei component of telencephali hc habenular the medial (D) commissure olfactory tract Dd dorsal zone of D ICL internal cell mP preoptic DL lateral zone of D layer of component of DLd dorsal part of olfactory bulb the medial lateral zone of Ilt lateral olfactory tract D component of mt medial olfactory DLP posterior part of the lateral tract lateral zone of olfactory tract mta ascending D lmt medial component of DLv ventral part of component of the medial lateral zone of the lateral olfactory tract D olfactory tract mtd descending DM medial zone of D lr lateral recess component of DPr rostra1 portion of It lateral olfactory the medial posterior zone tract olfactory tract of D m meninges MTP median nucleus DPc caudal portion of ma ascending of the posterior zone component of posterior tuber of D the medial N nervus e external sulcus olfactory tract terminalis Ec caudal mc caudal ganglion cells entopeduncu- component of nt nervus lar nucleus the medial terminalis Ed dorsal olfactory tract NT nucleus taeniae entopeduncu- MCL mitral cell layer NDIL nucleus diffusus lar nucleus of the of the inferior Ev ventral olfactory bulb lobe entopeduncu- md dorsal NSV nucleus of the lar nucleus component of saccus EP epiphysis the medial vasculosus olfactory tract OB olfactory bulb

(continued) CATFISH OLFACTORY PATHWAYS 101

Abbreviations (continued)

OE olfactory rh rhinocoele Vd-d dorsal division of epithelium S spinal cord the dorsal ON olfactory nerve sc suprachiasmatic nucleus of V OP olfactory nucleus Vd-v ventral division peduncle SD saccus dorsalis of the dorsal OT optic tract slt sulcus limitans nucleus of V PG nucleus telencephali Vi intermediate preglomer- SOF secondary nucleus of V ulosus olfactory fiber v1 lateral nucleus PLL posterior lateral layer of the of v line lobe olfactory bulb Vn 'nother nucleus PM magnocellular sv saccus of v preoptic vasculosus VP postcommissural nucleus SY sulcus nucleus of V PP periventricular ypsiliformis vs supracommis- preoptic T telencephalon sural nucleus nucleus tC tela chorioidea of v PPa anterior segment TeO optic tectum vv ventral nucleus of the ToL tONS of v parvocellular longitudinalis Vv-d dorsal division of part of PP V vagal lobe vv PPP posterior vc central nucleus vv-v ventral division segment of thie of area of vv parvocellular ventralis z sulcus z part of PP telencephali preoptic recess (V)

Fig. 1. Dorsal view of the brain oflctulurus punctafus. The numbered lines indicate the levels of the transverse sections in Figures 2, 3, 5-7. 102 A.H. BASS

A B

Fig. 2. This figure and Figures 3 and 5-7 are line draw- ures 2, 3, and 5-7 is 1 mm. (A) Transverse section through ings illustrating the position and extent of the secondary the olfactory bulb. (B) Transverse section through the caudal olfactory projections. The chartings are baaed on animals pole of the olfactory bulb. Cross-hatching in A and B indi- that received intrabulbar injections of tritiated proline. cates extent of silver grain labeling within olfactory bulb. Drawings to the left lie ipsilateral to the injection site, (C)Transverse section through the rostral telencephalon. whereas drawings to the right lie contralateral to the in- (D)Transverse section through the telencephalon at the jection site. Olfactory bulb axons are indicated by dashed rostral pole of Vn. lines and terminal fields by stippling. The bar scale in Fig CATFISH OLFACTORY PATHWAYS 103

C - Fig. 3. (A) Transverse section through caudal Vn. Note the medial and dorsal branches of the medial olfactory tract. (B) Transverse section through rostra1 Vd. Note the medial and ventral branches of the nervus terminals. (C) Transverse section through the commlissure of Goldstein (cg). Bar scale is 1 mm. 104 A.H. BASS

Fig. 4. Photomicrographs of horizontal sections through medial (lmt) and lateral (llt) components of the lateral the olfactory tract as demonstrated by dark-field illumina- olfactory tract. (B) A horizontal section caudal and dorsal tion of autoradiographs following an intrabulbar injection to that in Figure 4A, illustrating the joining of lmt and the of tritiated proline. The rostra1 (r)-caudal (c) axis is indi- lateral component ofthe medial tract (ml).The llt continues cated. The bar scale represents 0.1 mm. for A and B. (A) as a separate component to caudal olfactory targets (see Ipsilateral olfactory tract illustrating the divergence of the text). CATFISH OLFACTORY PATHWAYS 105

Fig. 5. (A) Transverse section through the caudal pole of the anterior commissure. Note the fusion of the medial and dorsal branches of the medial olfactory tract. (B) Transverse section through the telencephalon. Note the dense input of the caudal DP-DC zone. (C) Transverse section through the caudal pole of the telencephalon. Bar scale is 1 mm. 106 A.H. BASS

TeO

Fig. 6. (A) Transverse section through the rostra1 thalamus as the lateral components of the lateral olfactory tract (llt)course toward the habenular commissure and the caudal component of the medial olfactory tract (mc) courses toward the posterior tuber. (B) Transverse section at the level of the habenular commissure, indicating the crossed fibers of the lateral component of the lateral olfactory tract (llt). Bar scale is 1 mm. 107

Fig. 7. Transverse section through the posterior tuber. A Nissl preparation appears on the right. To the left is a line drawing indicating the position and extent of the olfactory projection to the median posterior tuber nucleus (MTP). Bar scale is 1 mm.

Fig. 8. Photomicrographisof transverse sectionsof the sec- Figure 14B.(B) Ipsilateral terminal field of the caudal DP- ondary olfactory projections as demonstrated by darkfieldil- DC zone at the level of Figure 5B. (C) Ipsilateral terminal lumination of autoradiographs following intrabulbar injec- field over nucleus taeniae at the level of Figure 5C. (D) tions of tritiated proline. Bar scale represents 0.01 mm for Terminal field over the median posterior tuber nucleus at A-D. (A)Ipsilateral terminal fieldoftheventral (Vv)anddor- the level of Figure 7. sal (Vd)nuclei of area ventralis at about the level indicated in 108 A.H. BASS

Fig. 9. Photomicrograph of terminal degeneration within the caudal DP-DC zone. Bar scale represents 0.05 mm. CATFISH OLFACTORY PATHWAYS 109

Fig. 10. PhotomicrographsA-D, F, and G are transverse medial tract). (B) Caudal aspect of the transection of the sections through the olfactory tract as demonstrated by llt. (C) Rostra1 telencephalon illustrating labeled mt and dark-field illumination of autoradiographs. A-C and F are medial component of the lateral tract (lmt). Note absence from a specimen that sustained transection of the lateral of label over llt. (D) Label over llt and lmt from specimen component of the lateral olfactory tract (llt). D and G (as with intact peduncle. (El Transverse section through the photomicrograph E) are from a specimen that sustained no habenular nuclei (Habl at the level of the habenular com- transections. Bar scale represents 0.2 mm. for A-G. (A) In- missure (hc). (F) Restricted label near hc from specimen tact descending medial (mt) and lateral (llt, lmt) olfactory with llt transection. (G)Label over hc from specimen with tracts rostra1 to transection (ma is ascending component to intact peduncle. 110 A.H. BASS

Fig. 11. Transverse sections through the olfactory bulb mm. Arrow points to cell illustrated in A' (bar scale rep- following HRP injections of the ipsilateral telencephalon. resents 0.05 mm). (B) Dark-field illumination of HRP-la- (A) HRP-labeled cells in the mitral cell layer (MCL). Buff- beled cells in internal (ICL) and mitral (MCL) layers. Te- ered dianisidine (brown) reaction. Bar scale represents 0.1 tramethylbenzidine reaction. Bar scale represents 0.1 mm. CATFISH OLFACTORY PATHWAYS 111

MTP Fig. 12. Summary diagram in the horizontal plane il- tory bulb. The rostra1 (rlkaudal (c) axis is indicated. See lustrating the trajectory of the lateral (solid-black)and me- text for details. dial (hatched-black)olfactory tracts of the ipsilateral olfac-