Olfactory Bulb Projections in the Bullfrog Rana Catesbeiana

Olfactory Bulb Projections in the Bullfrog Rana catesbeiana

R GLENN NORTHCUTT AND G. JAMES ROYCE Department of Zoology, University of Michigan, Ann Arbor, Michigan 481 04, and Department of Anatomy, Albany Medical College, Albany, New York 12208

ABSTRACT The projections of the accessory and main olfactory bulbs of the bullfrog are described as part of a long term analysis of the morphological differences in amphibian and reptilian telencephalons. Unilateral aspiration of the accessory olfactory bulb results in an ipsilateral projection to the pars lateralis of the amygdala via the accessory olfactory tract. Degenerating fibers from the accessory olfactory bulb are tracable into the cell-free zone between the dorsal striatum and the lateral pallium, and projections to these neural populations may also exist. Unilateral lesions of the main olfactory bulb reveal two major secondary pathways: an ipsilateral medial olfactory tract that projects to the rostral ventromedial portion of the medial pallium, the postolfactory eminence and the rostral, lateral and medial septal nuclei; and an ipsilateral lateral olfactory tract that projects to the dorsal striatum, the lateral pallium and the ventral half of the dorsal pallium. Two crossed secondary olfactory pathways to the contralateral telencephalon decussate via the habenular commissure after entering the ipsilateral stria medullaris. A crossed lateral pathway terminates in the dorsal striatum, the caudal, lateral pallium and the ventral portion of the dorsal pallium. A crossed medial pathway terminates in the internal granule layer of the main olfactory bulb.

The transition from amphibians to rep- structures and their interconnections. As tiles marks the beginning of one of the one step in a new analysis we here report major adaptive radiations in vertebrate certain new observations on the normal phylogeny. The emergence of true land anatomy of the telencephalon, describe vertebrates is correlated with striking the secondary olfactory pathways in the morphological changes in the telencepha- bullfrog, and compare them with those of lon (fig. 1). The study of the observed dif- other vertebrates. ferences was pioneered at the turn of the century and resulted in careful descrip- MATERIALS AND METHODS tions of the character and distribution of Twenty-three adult specimens of Rana the neurons in a number of amphibians catesbefana Shaw obtained from Southern and reptiles (P. Ramon, 1896; Gaupp, Biological Supply Company in McKenzie, 1896; Edinger, '08; Rothig, '26; Crosby, Tennessee, were subjected to unilateral '17). While these early workers attempted aspiration, knife-cuts, or electrolytic le- to determine the interconnections of neu- sions of the olfactory nerve (3 specimens), rons, their techniques for tracing long main olfactory bulb (11 specimens), ac- connections were less adequate than the cessory olfactory bulb (6 specimens), or recently developed silver impregnation habenular commissure (3 specimens), un- techniques of Nauta and co-workers (Nau- der ethyl m-aminobenzoate methanesul- ta, '57; Fink and Heimer, '67) for demon- fonate (MS-222) anesthesia (200 mglkg). strating experimentally induced degen- After postoperative survival times ranging erating pathways. Information on neural from 6 to 22 days at 24" to 27"C, the ani- interconnections provides critical data for mals were sacrificed under MS-222 anes- both the recognition of homologous and thesia by transcardial perfusion with 10% functional units in the telencephalon. It formalin. The brains were removed, fixed is impossible to understand the adaptive in 10% formalin for at least one week, significance of the telencephalic differ- and embedded in 25% (W/V) gelatin. ences between amphibians and reptiles Frozen sections were cut at 25 micra, without recognizing homologous neural stored in 2% formalin at 5"C, and proc-

J. MORPH.. 145: 251-268. 25 1 252 R. GLENN NORTHCUTT AND G. JAMES ROYCE essed by several modifications of the Nauta projector, and the degenerating pathways (‘57) and Fink-Heimer (‘67) procedures were charted on these drawings. The for the demonstration of degenerating chartings of the degenerating pathways axoplasm. Appropriate sections were drawn were then transferred to line drawings of with the aid of a Bausch and Lomb micro- photographs taken of a normal brain, cut

Abbreviations ac, anterior commissure m, pars magnocellularis of the act, accessory olfactory tract periventricular preoptic nucleus aot, anterior olfacto-habenular tract ml, mitral layer apl, amygdala, pars lateralis mot, medial olfactory tract apm, amygdala, pars medialis mp, medial pallium bn, bed nucleus of the pallial ms, medial septum commissu c e na, nucleus accumbens D, diencephalon 0, optic nerve dp, dorsal pallium OB, olfactory bulb ds, dorsal striatum OC, optic chiasm dvr, dorsal ventricular ridge ON, olfactory nerve en, entopeduncular nucleus OT, optic tectum epl, extragranular plexiform layer p, periventricular preoptic nucleus gl, glomerular layer pc, pallial commissure hc, habenular commissure pe, postolfactory eminence igl, internal granule layer sm, stria medullaris lct, lateral cortico-habenular tract st, striatum lot, lateral olfactory tract T, telencephalon lp, lateral palliurn vn, vomeronasal nerve Is, lateral septum vs, ventral striatum

IP ms mS I P Is Is-

na na A B Fig. 1 Representative transverse sections through the left telencephalic hemisphere of Rana (A) and Gekko (B) illustrating the differences in the development of the lateral hemispheric wall and the migration of neuronal populations from a periventricular position. OLFACTORY PATHWAYS IN THE BULLFROG 253

10 mm

Fig. 2 Dorsal view of the brain of the bullfrog. Arrows indicate the level of transverse sections illustrated in figures 3 through 8. in the transverse plane and stained with main olfactory bulbs are fused and all lam- cresyl violet. Additional cresyl violet and inae except the internal granule layer are Bodian stained material of R. catesbeiana continuous between the two bulbs. Beyond cut in the three standard anatomical the most rostral levels of the main bulb, planes was used to study the normal anat- the glomerular layer is restricted to the omy of the prosencephalon. To demon- ventral half of the bulbs (fig. 3). The in- strate cytoarchitectonics selected sections ternal granule layer continues further in the cresyl violet series were photog- caudally than the other bulbar laminae graphed on Kodalith Ortho film, type 3 and is first replaced dorsally by the telen- with a Leitz large-format camera. Degen- cephalic pallial areas then ventrally by erating pathways and terminal fields were the subpallial areas (figs. 3B-5). The transi- photographed on Kodak photomicrography tion from internal granule layer to telen- monochrome film (50-410) with a Leitz cephalic hemispheric areas is sudden and Ortholux I1 microscope. morphologically recognizable by a decrease RESULTS in the packing density and a marked increase in the diameter of the hemispheric Normal anatomy neurons. An anterior olfactory nucleus A dorsal view of the brain of R. cates- conforming to Hoffman’s description as a beiana is shown in figure 2 with indicated portion of the granular layer of the bulb levels of the transverse sections illustrated could not be recognized. in figures 3-8. The nomenclature used in The accessory olfactory bulb occupies the following descriptions is modified from the ventrolateral caudal wall of the main Hoffman (’63), Scalia et al. (‘68), and oIfactory bulb and possesses the same neu- Northcutt (‘74). ral laminae as the main bulb (fig. 4). At Olfactory bulbs. The olfactory bulbs the level of its maximal development, the are formed by evaginations of the rostral accessory bulb occupies most of the bul- telencephalic walls and secondarily form bar wall and the mitral layer of the acces- main and accessory bulbs in anurans. sory bulb is in contact, if not in continuity, The main olfactory bulbs possess a series with the internal granule layer of the main of concentric laminae in the following olfactory bulb. centripetal order: (1) olfactory nerve fibers Subpallium. On the basis of cytoarchi- and glomerular layer, (2) external cellular tectural and histochemical observations layer composed of small granule and larger (Northcutt, ’74) the telencephalon proper mitral cells, (3) extragranular plexiform can be divided into a dorsal pallium and layer composed of secondary olfactory fi- a ventral subpallium. The medial subpal- bers, (4) internal granule layer and (5) lial wall consists of four major nuclei: (1) ependyma (fig. 3). The medial walls of the postolfactory eminence, (2) lateral septa1 254 R. GLENN NORTHCUTT AND G. JAMES ROYCE

, 1 MM

Fig. 3 Transverse sections through middle (A) and caudal (B) levels of the main olfactory bulb in the bullfrog. A Nissl preparation is shown on the right, and the contralateral side is drawn on the left. Degenerated fibers and preterminal degeneration is represented by stippling in figures 3 through 9. The heavy line running obliquely in the left half of A marks the medial extent of the main olfactory bulb lesion in case RC-OB-9. nucleus, (3) medial septal nucleus, and neural population can be recognized, the (4) a pars medialis of the amygdala. pars medialis of the amygdala (fig. 6). The postolfactory eminence is a small This division of the amygdala begins just periventricular nucleus located rostral to rostral to the lamina terminalis and can the septal nuclei in the extreme rostral be distinguished from the lateral septal hemisphere (fig. 4). This nucleus corre- nucleus by its more densely packed neu- sponds to the similarly named nucleus of rons. The pars medialis continues to en- Rothig ('12) and appears to have been de- large as it is traced caudally. Laterally it scribed as part of the primordium hippo- forms a distinct ridge in the floor of the campi by Hoffman ('63). lateral ventricle and medially fuses with The lateral septal nucleus emerges as the contralateral pars medialis across the a caudal continuation of the medial inter- lamina terminalis. Hoffman described nal granule layer (fig. 5) and continues this neural population as a caudal contin- as far caudally as the lamina terminalis uation of his nucleus accumbens. (fig. 6) where it is bounded caudally by Rostrally the medial septal nucleus is the hemispheric commissural system. Ven- first recognized as a migrated cell group tral to the lateral septal nucleus, another medial to the lateral septal nucleus. As OLFACTORY PATHWAYS IN THE BULLFROG 255 this group is traced caudally it is observed periventricular preoptic nucleus of the to form dorsal and ventral divisions (fig. hypothalamus. 5). Hoffman only described the dorsal di- The lateral subpallial wall consists of vision as the medial septal nucleus while a tier of nuclei: a lateral striatum proper he referred to the ventral division as the and a medial nucleus accumbens adjacent nucleus of the diagonal band of Broca. to the septal nuclei (figs. 5, 6). This lat- Scalia et al. ('68) followed the usage of eral wall is composed of a series of almost Rothig ('12) and included both divisions continuous cellular laminae. For this rea- under the medial septal nucleus. That con- son no general agreement exists in the vention is followed in the present report, literature regarding the basic number of but until the connections of these divisions recognizable nuclear units or their homo- are experimentally determined it is im- logues with other vertebrate taxa. At ros- possible to resolve these differences. The tral levels a pars dorsalis of the striatum medial septal nucleus continues caudally can be distinguished from a pars ventralis and finally terminates in the caudal, me- due to differences in the packing density dial pole of the hemisphere (fig. 7). In and the degree of neuronal migration from this region one additional medial cell pop- the ependyma (fig. 5). The pars dorsalis ulation can be recognized, the bed nucleus can be traced caudally to the level of the of the pallial commissure (fig. 7). This nu- lamina terminalis. At this level it is pres- cleus is not included in the list of medial ently impossible to determine whether this subpallial nuclei as preliminary histo- cell group terminates or merges ventrally chemical data suggest that these neurons with the pars ventralis. The pars ventralis are a dorsal and rostral extension of the can be recognized at even more caudal

lot

epl-

A Fig. 4 Transverse sections through the rostral pole of the telencephalon at the level of the accessory olfactory bulb (A). Figure 4B is a line drawing of the contralateral hemisphere at the same telencephalic level as the Nissl preparation illustrating the position of the crossed secondary olfactory pathways. 256 R. GLENN NORTHCUTT AND G. JAMES ROYCE

A 1 MM Fig. 5 Transverse sections through a mid-telencephalic level. Figure 5A illustrates the position of the ipsilateral secondary olfactory pathways, and figure B the position of the crossed secondary pathways at the same telencephalic level. This level marks the most caudal extent of the ipsilateral medial olfactory tract. levels, but it becomes restricted both later- preted as the pars medialis of the amygdala ally and medially and can be traced ven- (fig. 6). trolaterally into the lateral forebrain bun- Pallium. On the basis of cytoarchi- dle where the continuing cell column is tectural and histochemical observations then termed the entopeduncular nucleus (Northcutt, '74) four pallial subdivisions (fig, 7). The medial nucleus accumbens are recognized: dorsal, lateral and medial is defined as a cellular population arising pallial sheets, and a pars lateralis of the immediately caudal to the accessory olfac- amygdala. The pallial sheets replace the tory bulb and replacing the internal gran- internal granule layer dorsally and their ule layer of the main olfactory bulb. It rostral boundary is marked by an increase occupies a subependymal position in the in the size of the neurons. The lateral pal- floor of the lateral ventricle (fig. 5) and lium is the most rostrally occurring pallial its cells are closely packed and stain more component (fig. 3B). The lateral pallium darkly with cresyl violet than the more consists of two components which are rec- lateral cellular populations. Rostrally this ognized on the basis of the differential population corresponds to the population thickness of their compact subependymal similarly labeled by Hoffman ('63) but it zones. The dorsal component possesses a was not traced beyond the rostral lamina compact subependymal zone half the thick- terminalis. The cellular population labeled ness of the ventral component's subepen- as accumbens by Hoffman at levels cau- dymal zone (figs. 3B-5). At present it is not dal to the lamina terminalis is here inter- known whether the anatomical compo- OLFACTORY PATHWAYS IN THE BULLFROG 257 nents of the lateral pallium possess differ- of the distribution of the lateral olfactory ent functions. The medial pallium is the tract and will be discussed after the ex- next pallial element to appear rostrally perimental results of the present study (fig. 4). It is easily recognized by the scat- are presented. tered subependymal layer and the large The pars lateralis of the amygdala is number of migrated neurons throughout first recognized as a ventral continuation its rostrocaudal extent (figs. 4-8). of the lateral pallium (fig. 5). Caudal to The dorsal pallium appears just dorsal the level of the interventricular foramen and caudal to the entry to the medial pal- (fig. 7), the pars lateralis expands to form lium (fig. 4). Traditionally the term dorsal a C-shaped nucleus and more caudally pallium has been restricted to a popula- it becomes oval in outline (fig. 8). Its cau- tion of transitional neurons some 0.38 dal portion has been termed the anterior millimeters in cross-sectional diameter be- entopeduncular nucleus by Frontera ('52). tween the lateral and medial pallia. How- ever, as defined in this study the dorsal Experimental results pallium includes a more lateral population All experimental cases from the short- usually considered to be the dorsal part of est survival time, six days, to the longest the lateral pallium (figs. 5, 6). This deci- survival time, 22 days, showed evidence sion is strongly influenced by the results of degeneration. Cases with survival times

IMM A Fig. 6 Transverse sections at the level of the rostra1 telencephalon medium. Figure 6A illustrates the caudal continuation of the ipsilateral lateral olfactory tract, and figure 6B the course of the crossed secondary pathways at the corresponding contralateral hemispheric level. 258 R. GLENN NORTHCUTT AND G. JAMES ROYCE

A IMM,

Fig. 7 Transverse sections at a telencephalic level immediately caudal to the level of the interventricular foramina. Figure 7A illustrates the position of the ipsilateral lateral olfactory tract at a level immediately rostral to the entry of the decussating fibers into the stria medullaris. Figure 7B illustrates a comparable level in the contralateral hemisphere where the crossed secondary olfactory fibers skirt the forebrain bundles and the preoptic area occupies a medial hemispheric position. of 8 to 12 days showed maximal argyro- sented below are based on density of par- philia of degenerating axoplasm. Both the ticles, proximity of degenerating particles degenerating axons and terminals ap- to cellular groups, and termination of the peared as very fine opaque particles. The degenerating pathways in specific targets argyrophilic pattern usually observed in beyond which the pathways can not be amniotes was not generally apparent. Two traced. factors limit the precision with which we Olfactory nerve. Since several layers can identify the targets of secondary ol- of the main olfactory bulbs in Rana are factory fibers. First the uniformly fine continuous along their medial surfaces, nature of the degenerating particles makes it is possible that each olfactory nerve it difficult, if not impossible, at light mi- could project to both bulbs. In order to croscopic levels to distinguish between examine this possibility three specimens degenerating axons and terminals. Second, were subjected to unilateral transection of much of the degeneration appears outside the left olfactory nerve. A single olfactory the nuclear boundaries suggesting that nerve was exposed by drilling through the most of the terminals are of ~JI axo-den- sphenethmoid bone rostral to the neuro- dritic nature. The conclusions regarding cranium. At this level the olfactory nerves targets of secondary olfactory fibers pre- are separately housed and possible dam- OLFACTORY PATHWAYS IN THE BULLFROG 259 age to the contralateral nerve is mini- gyrophilic particles restricted to the glo- mized. One animal survived eight days merular and mitral layers. The glomerular postoperatively while the remaining two layer of the right olfactory bulbs also con- animals survived 14 days postoperatively, tained dense argyrophilic particles. How- All specimens demonstrated argyrophilic ever, these particles were 2 to 4 times particles in the left olfactory nerve caudal smaller in diameter than those in the left to the transection. The glial elements in bulb, and no change in their diameter or the transected nerve lost their linear ar- density was observed at the longer survival rangement along the individual olfactory time. Thus we believe that each olfactory fascicles and the degenerating fascicles nerve only projects to the ipsilateral olfac- were first characterized by fine almost tory bulb. This conclusion is further re- dust-like particles and subsequently by inforced by the observation that surgical larger argyrophilic particles. The contra- intervention into widely separated neural lateral olfactory nerve retained the order- populations such as the cerebellum, me- ly array of glial elements and very sparse dulla and tectum are almost inevitably argyrophilia was restricted to the circum- associated with the presence of extremely ference of the individual olfactory fasci- fine argyrophilic particles in the accessory cles. This argyrophilia was interpreted as and main olfactory bulbs. restricted to the glial and vascular ele- Main olfactory bulb. The following ments. description is based on case RC-OB-9. This Examination of the left main olfactory specimen sustained a pure unilateral le- bulbs in all specimens revealed dense ar- sion of the main olfactory bulb which

1 MM A Fig. 8 Transverse sections at the level of the caudal hemispheric pole. Figure 8A illustrates the entry of the ipsilateral olfactory pathway into the stria medullaris. Figure 8B illustrates a comparable level in the contralateral hemisphere and the distribution of the crossed secondary olfactory fibers. 260 R. GLENN NORTHCUTT AND G. JAMES ROYCE spared the most ventrocaudal portion of era1 olfactory tract (fig. 8B). At this level the bulb. There was no recognizable dam- two separate degenerating pathways were age to the retrobulbar pallium. This ani- traced. A lateral pathway was traced both mal survived ten days postoperatively and dorso-caudally and dorso-rostrally. This displayed what we believe to be the total lateral pathway could not be traced ros- projection pattern of the main olfactory trally beyond a midtelencephalic level (fig. bulb. 5B), and is termed the lateral corticoha- In the caudal bulb degenerating fibers benular tract of Herrick ('48). A second collect in the extragranular plexiform pathway was traced ventrally and medial- layer to form the medial and lateral olfac- ly around the forebrain bundles (fig. 7B) tory tracts (figs. 3B, 10A). The medial where it skirted the periventricular pre- olfactory tract lies in a superficial position optic nucleus (fig. 6B) and then passed in the medial hemispheric wall. Rostrally rostrally in a comparable position to the it passes along the postolfactory eminence, ipsilateral medial olfactory tract (fig. 5B). the ventromedial surface of the medial This crossed medial component was traced pallium, and the lateral and medial septal rostrally to the internal granule and extra- nuclei (figs. 3-5). Ipsilaterally degenerat- granular plexiform layers of the caudal ing fibers in the medial olfactory tract main olfactory bulb (fig. 4B). This crossed were not traced beyond a level just rostra1 pathway corresponds to the anterior olfac- to the lamina terminalis (fig. 5). The me- tohabenular tract of Herrick ('21). dial septal nucleus is the only cellular Since none of the lesions restricted to population throughout the extent of the the main olfactory bulb resulted in defi- medial olfactory tract that demonstrates nite evidence that the crossed olfactory degenerating particles within its confines pathways decussated via the habenular (fig. 5). commissure, three animals were subjected Rostrally degenerating fibers are seen to complete transection of the habenular in the extragranular plexiform and inter- commissure. Case RC-HC-1 survived nine nal granule layers streaming dorsally and days postoperatively and demonstrated laterally to form the lateral olfactory tract bilateral degeneration of the striae medul- (figs. 3B, 10A). This tract continues to add larium, and the lateral and medial olfac- fibers at its ventrolateral edge until the tory tracts along their entire rostro-caudal posterior level of the accessory olfactory extent. Additionally this specimen showed bulb is reached (fig. 4A). As the lateral extensive degenerating particles in the in- olfactory tract is traced caudally it spreads ternal granule layer of both main olfac- over the lateral surface of the hemisphere tory bulbs. (fig. lOC,D) and is confined to the outer Accessory olfactoTy bulb. The following molecular zones of the following cellular description is based on RC-AOB-2. This populations: the dorsal half of the dorsal specimen sustained a unilateral lesion of striatum, the lateral pallium and the ven- the entire left accessory olfactory bulb tral half of the dorsal pallium (figs. 4-8). and survived eight days postoperatively. Degenerating fibers of the lateral olfactory The lesion was inflicted by cannular as- tract were traced far caudally into the pos- piration through a ventral entry in the terior hemispheric pole (fig. 8). braincase by a surgical approach through At the level of the telencephalon me- the roof of the mouth. The lesion included dium (fig. 7), the degenerating fibers in the internal granule layer of the main the ventral edge of the lateral olfactory olfactory bulb immediately medial to the tract turn medially after skirting the pars mitral layer of the accessory olfactory bulb lateralis of the amygdala and enter the (fig. 9). This lesion insures that all layers stria medullaris (fig. 8). A moderate num- of the accessory bulb were destroyed but ber of degenerating particles were seen in analysis is complicated by interruption of the striae medullarium but few particles the secondary olfactory pathways arising were seen in the habenular commissure in the internal granule and extragranular proper. plexiform layers of the main olfactory bulb. Contralateral to the lesioned olfactory However, since the accessory olfactory bulb bulb, degenerating particles were traced does not possess a distinct recognizable in- laterally in the stria medullaris and lat- ternal granule layer separate from that of llmm I Fig. 9 Transverse sections through the left telencephalic hemisphere of the bullfrog illustrating the course and termination of the accessory olfactory tract. The dashed line in figures A through C marks the boundary of the lesion involving the left accessory olfactory bulb. The charted degeneration ventral and dorsal to the accessory bulb and tract is interpreted as interruption of pathways from the main olfactory bulb as these path- A ways are observed following lesions that are restricted to the main olfactory bulb (figs. 3-8). 262 R. GLENN NORTHCUTT AND G. JAMES ROYCE the main olfactory bulb, this analytic com- reveals the existence of a new degenerat- plication cannot be avoided. ing pathway that is first seen collecting in Comparison of the chartings of acces- an area lateral to the strio-pallial boundary wry olfactory bulb ablation (fig. 9) to those (figs. 9C, 10B). This pathway was traced of main olfactory bulb ablation (figs. 3-8) caudally where it terminated in the ipsi-

Figure 10 OLFACTORY PATHWAYS IN THE BULLFROG 263 lateral pars lateralis of the amygdala (figs. projections of the forebrain are needed to 9D,E, 1OE). The projection included the resolve this problem. entire rostro-caudal extent of this nucleus including the extreme caudal portion la- DISCUSSION beled by Frontera ('52) as the anterior en- Olfactory pathways. We believe that topeduncular nucleus (fig. 10G). This path- each olfactory nerve projects solely to the way is termed the accessory olfactory tract ipsilateral olfactory bulb even though ar- and corresponds in part to the ventrolat- gyrophilic particles are observed in the era1 tract of Herrick ('21). Our material glomerular layer of the contralateral ol- demonstrates that this projection enters factory bulb. These contralateral particles the rostral pole of the pars lateralis of the are much smaller than those observed in amygdala from the external or lateral sur- the ipsilateral bulb, and their density does face of the nucleus rather than the inter- not vary with postoperative survival tirnes nal or ventricular surface as suggested by nor were degenerating fibers ever traced Herrick. While this pathway is clearly the from the ipsilateral to the contralateral major efferent pathway from the accessory olfactory bulb. Hence the contralateral ol- olfactory bulb it may not be the sole one. factory bulb particles do not seem to be Degenerating particles are also seen in associated with anterograde degeneration. the cell-free zone between the dorsal stri- The actual targets of the secondary ol- atum and lateral pallium, as well as, in factory pathways are difficult to determine the deeper portions of the molecular zone due to the fine nature of the degenerating of the lateral pallium (figs. 9B,C, lOB,F). particles and their frequent position out- Degenerating particles were never seen side the cellular boundaries of the neural in these positions following lesions of the populations. The ipsilateral medial olfac- main olfactory bulb. Thus there may be tory tract may project to the rostral ventro- additional projections from the accessory medial portion of the medial pallium, the olfactory bulb, or there may be interrupted postolfactory eminence and the rostral lat- projections from other neural populations eral septum as this pathway courses along on their way to the lateral hemispheric the medial surface of these populations wall. Further experimental studies on the and their dendrites extend into the pathway (Hoffman, '63; Clairambault and Fig. 10 Photomicrographs of degeneration following ablation of the accessory or main olfactory Derer, '68). The medial septa1 nucleus is bulbs in the bullfrog. In all figures except C, the the most caudal target of the ipsilatpral dorsal surface of the brain is toward the top of the medial olfactory tract and is the only rrie- figure. In C the dorsal surface is toward the right dial nucleus that demonstrates degener- of the figure. Both bar scales represent 100 microns. The magnification of A, B and C is identical as is ating particles within its cellular boundary the magnification of D, E, F, and G. A. Degenera- (fig. 5). tion in the ipsilateral lateral olfactory tract in the The ipsilateral olfactory tract probably rostral lateral pallium at the same hemispheric level as figure 4 following ablation of the main projects to the dorsal striatum, the lateral olfactory bulb. B. Degeneration among the cells pallium and the ventral half of the dorsal of the striopallial junction at the same hemispheric pallium as this pathway courses through level as figure 9C following ablation of the acces- the outer half of the molecular zones of sory olfactory bulb. C. Degenerating ipsilateral lateral olfactory tract in the lateral pallium at a mid- all these neural populations (figs. 5, 6). hemispheric level following ablation of the main Additionally the lateral olfactory tract could olfactory bulb. D. Higher magnification of the same project to a restricted part of the pars lat- pallial field as figure 1OC to illustrate the size and eralis of the amygdala since a ventral seg- nature of the degenerating particles seen after ablation of the main olfactory bulb. E. Degeneration in ment of this tract skirts the pars lateralis the pars lateralis of the amygdala following abla- as it enters the stria medullaris (fig. 7). tion of the accessory olfactory bulb. This terminal Rubaschkin ('03) suggested that the den- field is only observable following ablation of the drites of some neurons in this telencephalic accessory bulb. F. Higher magnification of the same ipsilateral strio-pallial junction as figure 10B to region reach the lateral olfactory tract. illustrate degeneration in the accessory olfactory However, such a projection, if present, is tract following ablation of the accessory olfactory minor in comparison to the massive pro- bulb. G. Degeneration in the ipsilateral, caudal jection of the accessory olfactory bulb to pars lateralis of the amygdala following ablation of the accessory olfactory bulb. This field is at the the pars lateralis of the amygdala (figs. same level as figure 9E. 9, 10). 264 R. GLENN NORTHCUTT AND G. JAMES ROYCE Our results also indicate the existence This pathway could also form connections of crossed secondary olfactory pathways with the periventricular preoptic nucleus to the contralateral telencephalon. We be- as it skirts this formation laterally and/or lieve two pathways exist which decussate the septal nuclei through which is passes through the habenular commissure after on its way to the main olfactory bulb. While entering the ipsilateral stria medullaris. such connections can not be ruled out, it While lesions of the main olfactory bulb will require electron microscopic evidence did not yield definitive evidence that these to decide if such connections exist. pathways cross in the habenular commis- A major difference was seen in the ros- sure as evinced by the clear presence of tral extent of the degeneration in the con- degenerating particles, degenerating par- tralateral lateral olfactory tract following ticles were observed in both medullar striae habenular commissural transection when and in the contralateral lateral and me- compared to olfactory bulb ablation. Fol- dial olfactory tracts. At least two other al- lowing transection of the habenular com- ternate interpretations of these results are missure, degenerating fibers in the lateral possible: (1) the contralateral degeneration olfactory tracts were traced rostrally into is due to damage to both olfactory bulbs, both olfactory bulbs. However, following and (2) the contralateral degenerating ablation of the main olfactory bulb, degen- pathways decussate in the interbulbar erating fibers were observed only in the bridge formed by the continuation of the caudal half of the contralateral hemi- outer layers of the two main olfactory bulbs sphere. This difference in degeneration (fig. 3). If either of the alternate inter- might be the result of retrograde changes pretations is correct, degenerating fibers in the axons of cells located in the bulb, should be observed in the interbulbar or more likely be due to interruption of an bridge and the degenerating pathways in additional class of neurons that also pro- the contralateral hemisphere should be in ject via the commissure but whose cell bod- continuity with the main olfactory bulbs. ies are located in the retrobulbar hemi- In all experimental cases of lesions involv- sphere. ing the main olfactory bulb, no degener- After lesions of the accessory olfactory ating fibers were ever observed crossing bulb, a tract of degenerating fibers can be the interbulbar bridge and the degenera- traced caudally to terminate massively in tion in the contralateral lateral pallium the ipsilateral pars lateralis of the amyg- was always restricted to the caudal half dala (fig. 9). While projections to other of the telencephalon (figs. 5-8). The most targets (such as the dorsal striatum and compelling evidence that the contralateral lateral pallium) can not be ruled out since olfactory pathways decussate in the ha- the dendrites of cells in these regions ex- benular commissure results from transec- tend into the pathway, the main if not sole tion of the habenular commissure. In these target of the accessory bulb appears to be preparations, degenerating particles were the pars lateralis of the amygdala. followed bilaterally from the stria medul- Our experimental results are in general laris into the lateral olfactory tracts of the agreement with those of Scalia et al. ('68) telencephalon, as well as, from the stria and Scalia ('72) on the olfactory projec- medullaris into tracts which run ventrally tions in Rana pipiens. In addition to cor- and medially around the forebrain bundles roborating their earlier results on leopard to turn rostrally in the medial hemispheric frogs and extending these observations to wall just medial to the septal nuclei where bullfrogs, we recognize an additional these tracts terminate in the internal gran- crossed olfactory path, the anterior olfac- ule layer of the main olfactory bulbs. The tohabenular tract, which projects to the crossed lateral pathway, the lateral cor- internal granule layer of the contralateral ticohabenular tract, appears to terminate main olfactory bulb. This difference in in a ventral portion of the dorsal pallium, reported observations is most likely due to the lateral pallium and a restricted dorsal differences in interpreting criteria for rec- portion of the dorsal striatum. The crossed ognizing degenerating fields or to differ- medial pathway, the anterior olfactoha- ences in staining techniques rather than benular tract, terminates in the internal to species differences. Their earlier work granule layer of the main olfactory bulb. relied on the original Nauta (Nauta, '57) OLFACTORY PATHWAYS IN THE BULLFROG 265 and modified Cajal (Romeis, '48) tech- systems in which the genetic changes niques rather than the later developed only reprogram ipsicontralateral differences Fink-Heimer modifications (Fink and in already existing targets rather than Heimer, '67). changes that utilize new targets for an Pyhlogenetic problems. Lateral and expanding system. medial crossed olfactory pathways, similar Telencephalic evolution. Experimental to those here demonstrated, have been re- studies on the main olfactory bulb of rep- ported in reptiles (Gamble, '56; Heimer, tiles reveal that the primary target is the '69; Scalia et al., '69; Northcutt, '70), but lateral pallium (Gamble, '56; Heimer, '69; not in birds (Rieke and Wenzel, '73) or Northcutt, '70; Scalia et al., '69; Halpern, in mammals (Scalia, '68). Thus it is pos- '73). In frogs a comparable topographical sible that amphibians and reptiles have region of the telencephalon also is the pri- independently evolved crossed olfactory mary target of the main olfactory bulb pathways, or that ancestral land verte- (fig. 1A). However, in frogs the position of brates possessed such pathways which the lateral olfactory tract does not coincide were independently lost in birds and mam- with cytoarchitectonic boundaries as it does mals. Information on other anamniotic in reptiles. The anuran lateral olfactory olfactory projections is presently too frag- tract projects to the ventral half of a more mentary to relate to tetrapod olfactory pro- dorsally located compact zone of cells rec- jections. Phylogenetic questions of this ognized as the dorsal pallium (fig. 1A). At nature may be answered by continued ex- present little is known regarding other pos- amination of variation among closely re- sible projections to this dorsal region. His- lated radiations of recent animals. If future tochemical study of the dorsal pallium studies reveal crossed olfactory pathways reveals a distinct band of succinate dehy- in lungfish (Dipnoi) andlor generalized ac- drogenase activity in the outer plexiform tinopterygian fishes, the argument for the zone (Northcutt, '74). Such activity is fre- presence of ancestral crossed pathways quently associated with areas of high syn- that were subsequently lost in birds and aptic density such as sensory projection mammals would be greatly strengthened. areas (Friede, '60). Based on the present An alternative approach to this type of available evidence, the dorsolateral hemi- phylogenetic question involves investiga- spheric wall in anurans consists of two re- tion of the functional and adaptive sig- gions on either cytological or connectional nificance of crossed olfactory pathways in criteria. However, the boundary between different taxa. Such an analysis may pin- these two areas differs depending on the point the selective pressures operating for criterion used. Final recognition of a crossed projections. If these pressures can boundary should be facilitated by further be inferred to have existed in the ancestral experimental studies. population then we are probably dealing The most striking difference between the with retention of an ancestral character. telencephalon of amphibians and reptiles On the other hand, if these pressures are is the presence of a dorsal ventricular ridge restricted to descendant populations which in reptiles (fig. 1B). This ridge consists of have entered similar adaptive zones then a rostra1 division containing the targets of we are probably dealing with the indepen- tharamic nuclei concerned with vision and dent evolution of similar characters due to audition (Hall and Ebner, '70; Pritz, '72; a common genome. Unfortunately, the Butler and Ebner, '72), and a caudal divi- functional significance of crossed olfactory sion containing a cup-shaped nucleus, projections is at present unknown. nucleus sphaericus, which is the target The multiple evolution of new connec- of the accessory olfactory bulb (Heimer, tions between cerebral hemispheres or '69; Halpern, '73). their loss may seem unlikely, particularly Does the anuran telencephalon possess if we assume that such characters are a neuronal field homologous to the reptilian polygenic. However, Guillery et al. ('73) dorsal ventricular ridge and if so, what is has recently shown similar changes in the its topographical position? If the telen- bilaterality of retinal projections that are cephalon of modern amphibians is com- due to a single allele. This may be par- parable to that of the ancestral amphibians ticularly true when we are dealing with that gave rise to reptiles, then two possi- 266 R. GLENN NORTHCUTT AND G. JAMES ROYCE bilities must be considered. The dorsal ven- changed their biochemical properties. It is tricular ridge could arise by migration of possible to argue that the connections of cells from either the striatum or the lateral the anuran and reptilian accessory olfac- pallium. At present three lines of evidence tory bulb evolved independently and do suggest that the reptilian dorsal ventricu- not project to homologous targets. It is lar ridge is homologous to a field of cells possible to argue that due to yet unknown in the amphibian lateral pallium: (1) simi- selective pressures reptilian embryos de- larities in the histochemistry of the anuran velop their telencephalon in a manner that lateral pallium and the reptilian dorsal spuriously correlates with a hypothesis ventricular ridge; (2) cellular continuity regarding probable phylogenetic develop- of the anuran pars lateralis of the amyg- ment. Yet evolutionary hypotheses and dala with the lateral pallium and the prob- recognition of homologies are based on able homology of the anuran pars lateralis the number and degree of character simi- with the reptilian nucleus sphaericus; and larities. The most parsimonious interpre- (3) embryological evidence in reptiles that tation is that the anuran lateral pallium the lateral pallium and dorsal ventricular and pars lateralis of the amygdala are ridge represent subsequent migrations of field homologues of the reptilian dorsal the same matrix zone. ventricular ridge and lateral cortex. In both amphibians and reptiles the areas labeled as striatum in figure 1 con- ACKNOWLEDGMENTS tain high concentrations of acetylcholin- The authors are pleased to thank Dr. esterase and biogenic monoamines while Mark Braford who provided helpful sug- neither the anuran lateral pallium nor rep- gestions, and Mr. Louis Martonyi €or his tilian dorsal ventricular ridge demonstrate expert assistance with the photography. such concentrations (Shen et al., '55; Histological preparations were made by Braak, '70; Parent and Oliver, '70; Kusu- Mrs. Neena N. Shah and Mr. Homer Fer- noki, '71; Parent, '71, '73; Northcutt, '74). guson. This study was supported by Na- In amphibians the accessory olfactory tional Institutes of Health Grant number bulb projects to the pars lateralis of the R01 N508417 to R. G. N. and PHS Grant amygdala and in reptiles to nucleus FR-5494 to G. J. R. sphaericus. 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